Latin American Journal of Aquatic Research ISSN X

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2 Latin American Journal of Aquatic Research ISSN X CHIEF EDITOR Sergio Palma Pontificia Universidad Católica de Valparaíso, Chile ASSOCIATE EDITORS Cristian Aldea Centro de Estudios del Cuaternario (CEQUA) Punta Arenas, Chile Patricio Arana Pontifícia Universidad Católica de Valparaíso Chile Patricio Dantagnan Escuela de Acuicultura Universidad Católica de Temuco, Temuco, Chile Walter Helbling Estación de Fotobiología Playa Unión, Argentina Ricardo Prego Instituto de Investigaciones Marinas (CSIC), España Nelson Silva Pontificia Universidad Católica de Valparaíso, Chile José Angel Alvarez Perez Universidade do Vale do Itajaí, Brasil Claudia S. Bremec Instituto de Investigación y Desarrollo Pesquero Argentina Enrique Dupré Universidad Católica del Norte, Chile Guido Plaza Pontificia Universidad Católica de Valparaíso, Chile Erich Rudolph Universidad de Los Lagos, Chile Oscar Sosa-Nishizaki Centro de Investigación Científica y Educación Superior de Ensenada, México Ingo Wehrtmann Universidad de Costa Rica, Costa Rica Financiamiento parcial de CONICYT obtenido en el Concurso Fondo de Publicación de Revistas Científicas año 2013 Escuela de Ciencias del Mar, Pontificia Universidad Católica de Valparaíso Casilla 1020, Valparaíso, Chile - Fax: (56-32) , lajar@ucv.cl

3 LATIN AMERICAN JOURNAL OF AQUATIC RESEARCH Lat. Am. J. Aquat. Res., 41(2) 2013 CONTENTS Research Articles Irene Cardoso On some rare Oplophoridae (Caridea, Decapoda) from the South Mid-Atlantic Ridge. Sobre algunos Oplophoridae (Caridea, Decapoda) raros de la Cordillera Meso-Atlántica Sur Raimunda N.F. Carvalho-Neta & Ana L. Abreu-Silva Glutathione S-Transferase as biomarker in Sciades herzbergii (Siluriformes: Ariidae) for environmental monitoring: the case study of São Marcos Bay, Maranhão, Brazil. Glutatión S-Transferasa como biomarcador en Sciades herzbergii (Siluriformes: Ariidae) para el monitoreo ambiental: el caso de estudio de la bahía de São Marcos, Maranhão, Brasil Mauricio Cerda, Conceição Denise Nunes-Barboza, Camila Nunes Scali-Carvalho, Kelly de Andrade-Jandre & Aguinaldo- Nepomuceno Marques Jr. Nutrient budgets in the Piratininga-Itaipu lagoon system (southeastern Brazil): effects of sea-exchange management. Balance de nutrientes del sistema lagunar Piratininga-Itaipu (sudeste de Brasil): efectos del manejo del sistema a través del intercambio con el agua de mar Jana Menegassi del Favero & June Ferraz-Dias Spatio-temporal variation in surf zone fish communities at Ilha do Cardoso State Park, São Paulo, Brazil. Variación espacio temporal de la ictiofauna del Parque Estatal Ilha do Cardoso, São Paulo, Brasil Graciela S. Diniz, Elisabete Barbarino, João Oiano-Neto, Sidney Pacheco & Sergio O. Lourenço Gross chemical profile and calculation of nitrogen-to-protein conversion factors for nine species of fishes from coastal waters of Brazil. Perfil químico bruto y cálculo de los factores de conversión de nitrógeno a proteína en nueve especies de peces de aguas costeras de Brasil Antonio C. Marques, Aline dos Santos Klôh, Alvaro Esteves Migotto, Ana C. Cabral, Ana P. Ravedutti Rigo, Ariane Lima Bettim, Emanuel L. Razzolini, Helena Matthews Cascon, Juliana Bardi, Laura Pioli Kremer, Leandro Manzoni Vieira, Luis E. Arruda Bezerra, Maria A. Haddad, Ronaldo Ruy de Oliveira Filho, Silvia M. Millan Gutierre, Thaís Pires Miranda, Wilson Franklin Jr. & Rosana Moreira da Rocha Rapid assessment survey for exotic benthic species in the São Sebastião Channel, Brazil. Estudio de evaluación rápida de especies bentónicas exóticas en São Sebastião, Brasil Thaís Pires-Miranda, Amanda Ferreira-Cunha & Antonio C. Marques Taxonomic position of Lovenella gracilis (Clarke, 1882) (Lovenellidae, Hydrozoa): new evidences of microanatomy justify its maintenance in the genus Lovenella (Hincks, 1868). Posición taxonómica de Lovenella gracilis (Clarke, 1882) (Lovenellidae, Hydrozoa): nuevas evidencias de microanatomía justifican su permanencia en el género Lovenella (Hincks, 1868) Fredy A. Ortiz-Ramírez, Magui Aparecida-Vallim, Diana Negrão-Cavalcanti & Valéria Laneuville-Teixeira Effects of the secondary metabolites from Canistrocarpus cervicornis (Dictyotales, Phaeophyceae) on fertilization and early development of the sea urchin Lytechinus variegatus. Efectos de los metabolitos secundarios de Canistrocarpus cervicornis (Dictyotales, Phaeophyceae) sobre la fertilización y desarrollo embrionario temprano del erizo del mar Lytechinus variegatus Débora Batista Pinheiro-Sousa, Zafira da Silva de Almeida & Raimunda Nonata Fortes Carvalho-Neta Integrated analysis of two biomarkers in Sciades herzbergii (Ariidae, Siluriformes), to assess the environmental impact at São Marcos Bay, Maranhão, Brazil. Análisis integrado de dos biomarcadores en Sciades herzbergii (Ariidae, Siluriformes) para evaluar el impacto ambiental en la Bahía de San Marcos, Maranhão, Brasil

4 Rodrigo Tardin, Carine Galvão, Mariana Espécie & Sheila Simão Group structure of Guiana dolphins, Sotalia guianensis (Cetacea, Delphinidae) in Ilha Grande Bay, Rio de Janeiro, southeastern Brazil. Estructura grupal en delfines Guyana, Sotalia guianensis (Cetacea, Delphinidae), en la bahía de Ilha Grande, Río de Janeiro, sureste de Brasil Bruna Tovar-Faro, Michele Leocádio & Paulo Cesar de Paiva Distribution of Iospilidae (Annelida) along the eastern Brazilian coast (from Bahia to Rio de Janeiro). Distribución de Iospilidae (Annelida) a lo largo de la costa oriental brasileña (de Bahía a Río de Janeiro) Paula C. Jimenez, Elthon G. Ferreira, Luana A. Araújo, Larissa A. Guimarães, Thiciana S. Sousa, Otília Deusdenia L. Pessoa, Tito M. C. Lotufo & Letícia V. Costa-Lotufo Cytotoxicity of actinomycetes associated with the ascidian Eudistoma vannamei (Millar, 1977), endemic of northeastern coast of Brazil. Citotoxicidad de actinomicetos asociada a la ascidia Eudistoma vannamei (Millar, 1977), endémica de la costa noreste de Brasil Beatriz N. Torrano-Silva, Carlos E. Amancio & Eurico C. Oliveira Seaweeds in ornamental aquaria in Brazil: anticipating introductions. Algas de acuarios ornamentales en Brasil: previsión de las introducciones Ricardo J. Miranda, Igor C.S. Cruz & Zelinda M.A.N. Leão Coral bleaching in the Caramuanas reef (Todos os Santos Bay, Brazil) during the 2010 El Niño event. Blanqueamiento de corales en el arrecife de Caramuanas (Bahía de Todos los Santos, Brasil) durante el evento El Niño Felipe Bezerra Ribeiro, Helena Matthews Cascon & Luis Ernesto Arruda Bezerra Morphometric sexual maturity and allometric growth of the crab Sesarma rectum Randall, 1840 (Crustacea: Sesarmidae) in an impacted tropical mangrove in northeast Brazil. Madurez sexual morfométrica y crecimiento alométrico del cangrejo Sesarma rectum Randall, 1840 (Crustacea: Sesarmidae) en un manglar tropical impactado en el noreste de Brasil

5 Marine Biology in Brazil: a promising and challenging future The current issue of the Latin American Journal of Aquatic Research (LAJAR) gathers full papers of studies presented at the 3 rd Brazilian Congress of Marine Biology (3 rd CBBM, abbreviation in Portuguese), hosted at the Praiamar Hotel Convention Center, Natal, Rio Grande do Norte State, Brazil, from 15 th to 19 th May, The event has been held by the Brazilian Association of Marine Biology (ABBM), which organizes its congresses every two years. Studies of Marine Biology in Brazil seem to be more important than ever. The country, well known by its unique terrestrial biodiversity, apparently has assumed its historical debt with the sea. Interest and exploitation of marine resources is raising fast, as well as the responsibility to achieve its sustainable use. In this respect, Brazilian marine scientists play an important role consolidating a sounding scientific background to support society s demand. The 3 rd CBBM had the attendance of 1,470 delegates, who presented 558 studies as posters and 70 talk presentations. Twenty-nine plenary lectures (six of them presented by invited international speakers) and four round-tables were presented in the event. Many other important activities were organized, such as the ABBM Award for Students (in two categories: undergraduate and graduate students), the photography contest, parallel scientific meetings, film exhibitions, and the special award for outstanding researchers on Marine Biology (Medalha Biologia Marinha Brasil), among others. A key-activity of the 3 rd CBBM was the publication of full papers presented at the Congress. This excellent alternative has only been possible due the fundamental support of the Latin American Journal of Aquatic Research. The manuscripts were evaluated through the regular peer review process and the current issue consists in a representative summary of the research on Marine Biology in Brazil nowadays. The Brazilian Association of Marine Biology takes this opportunity to express its profound gratitude to the LAJAR, which made a very important activity of the 3 rd CBBM to be possible. Supporting the Congress, the LAJAR strengthened the event and reinforced its essential role to the development of Sea Sciences in Latin America. A special acknowledgement is due to the members of the editorial committee of this issue of the LAJAR: Leticia Veras Costa Lotufo (Universidade Federal do Ceará), Sigrid Newmann Leitão (Universidade Federal de Pernambuco), Paulo Cesar de Paiva (Universidade Federal do Rio de Janeiro) and Paulo de Tarso Cunha Chaves (Universidade Federal do Paraná). Their work was essential to accomplish this endeavor. We extend our gratitude to the anonymous reviewers of the manuscripts of this issue. The activities of the 3 rd CBBM had the financial support of several sponsors, such as CNPq, CAPES, Petrobras, CFBio, Aquatec, and Technical Books. In addition, the event was supported by the volunteer and enthusiastic work of several professionals and students. We thank all sponsors and volunteers for their contributions that made possible the accomplishment of the 3 rd Brazilian Congress of Marine Biology. Antonio Carlos Marques Universidade de São Paulo, Co-Editor of the Special Editorial Committee Elisabete Barbarino Universidade Federal Fluminense, President of the Brazilian Association of Marine Biology

6 Lat. Am. J. Aquat. Res., 41(2): , 2013 Oplophoridae from south Mid-Atlantic Ridge Proceedings of the 3 rd Brazilian Congress of Marine Biology A.C. Marques, L.V.C. Lotufo, P.C. Paiva, P.T.C. Chaves & S.N. Leitão (Guest Editors) DOI: /vol41-issue2-fulltext Research Article On some rare Oplophoridae (Caridea, Decapoda) from the South Mid-Atlantic Ridge Irene Cardoso 1 1 Setor de Carcinologia, Museu Nacional, Quinta da Boa Vista São Cristóvão, Rio de Janeiro, , Brazil ABSTRACT. The Mid-Atlantic Ridge (MAR) divides the Atlantic Ocean longitudinally into two halves, each with a series of major basins delimited by secondary, more or less transverse ridges. Recent biological investigations in this area were carried out within the framework of the international project Mar-Eco (Patterns and Processes of the Ecosystems of the Mid-Atlantic Ridge). In 2009 (from October, 25 to November, 29) 12 benthic sampling events were conducted on the R/V Akademik Ioffe, during the first oceanographic cruise of South Atlantic Mar-Eco. As a result we report some rare Oplophoridae species collected during the cruise. This family includes 73 species occurring strictly on the meso- and bathypelagic zones of the oceans. Five Oplophoridae species were sampled: Acanthephyra acanthitelsonis Bate, 1888; A. quadrispinosa Kemp, 1939; Heterogenys monnioti Crosnier, 1987; Hymenodora glacialis (Buchholz, 1874) and Kemphyra corallina (A. Milne-Edwards, 1883). Among these, H. monnioti and K. corallina are considered extremely rare, both with very few records. Of the sampled species, only A. quadrispinosa and H. glacialis were previously recorded to southwestern Atlantic, so the Oplophoridae fauna of the South MAR seems more related with the fauna from the eastern Atlantic and Indian oceans. Keywords: Oplophoridae, Caridea, Decapoda, systematics, Mid-Atlantic Ridge. Sobre algunos Oplophoridae (Caridea, Decapoda) raros de la Cordillera Meso-Atlántica Sur RESUMEN. La Cordillera Meso-Atlántica (CMA) divide el Océano Atlántico en dos partes, cada una con varias cuencas delimitadas por cadenas de montañas transversales. Recientemente se han efectuado investigaciones biológicas en esta región en el marco del proyecto internacional Mar-Eco (Patterns and Processes of the Ecosystems of the Mid-Atlantic Ridge). Entre octubre y noviembre de 2009 se obtuvo 12 muestras bentónicas durante el primer crucero oceanográfico del Mar-Eco del Atlántico Sur. Durante este crucero se encontraron algunas especies raras de la familia Oplophoridae. Esta familia incluye 73 especies que ocurren estrictamente en las zonas meso- y batipelágicas de los océanos. Se colectaron cinco especies de Oplophoridae: Acanthephyra acanthitelsonis Bate, 1888; A. quadrispinosa Kemp, 1939; Heterogenys monnioti Crosnier, 1987; Hymenodora glacialis (Buchholz, 1874) y Kemphyra corallina (A. Milne-Edwards, 1883). De éstas, H. monnioti y K. corallina son consideradas muy raras, ambas con escasos registros. Solamente A. quadrispinosa y H. glacialis se han registrado previamente en el Atlántico sudoccidental; así la fauna de Oplophoridae de la CMA Sur parece más relacionada con la fauna del Atlántico este y océano Índico. Palabras clave: Oplophoridae, Caridea, Decapoda, sistemática, Cordillera Meso-Atlántica. Corresponding author: Irene Cardoso (irenecardoso@mn.ufrj.br)

7 210 Latin American Journal of Aquatic Research INTRODUCTION The seamounts are biologically distinctive habitats of the open ocean exhibiting a number of unique features (Rogers, 1994). One of the most well-known oceanographic effects of seamounts is the formation of eddies of water associated with upwelling of nutrient rich waters, leading to increasing productivity. Due to restricted food supplies in open-oceans, seamounts and the water column above them serve as important habitats, feeding grounds and sites of reproduction for many open-ocean and deep-sea species (Rogers, 1994; Probert, 1999). In general, seamounts appear to support a highly diverse fauna, but these effects were never studied in seamounts chains. The Mid-Atlantic Ridge (MAR) is a seamount chain that divides the Atlantic Ocean longitudinally into two halves, each with a series of major basins, delimited by secondary more or less transverse ridges. The Ridge extends above the 2000 m contour along most of its length and has a major influence on the circulation of near-bottom water masses (Levin & Gooday, 2003). The most important transverse ridges are, the Walvis Ridge on the southeastern MAR and the Rio Grande Rise, on the opposite side of the MAR. The Walvis Ridge extends for thousands of miles, rising from oceanic depths of more than 5 km to reach peak depths of less than 1 km (Linden, 1980). The ridge stretches southwest to northeast between the MAR and the continental margin of south-west Africa and separates the Angola basin in the north from the Cape Basin in the south (Linden, 1980). The Walvis Ridge and the Rio Grande Rise, are commonly treated as a combination, probably originated from hotspot volcanism. However, they are morphologically completely different and their origin is still controversial (Linden, 1980). The whole region, with such a diverse and peculiar nature, probably nests a diverse and interesting deepsea fauna. This expectation has prompted recent biological investigations on the southern Mid Atlantic Ridge, including the Walvis Ridge area, within the framework of the international project Mar-Eco. One of the most important goals of the South Atlantic Mar- Eco is to relate the Western and Eastern Atlantic MAR faunas, evaluating the effects of the Walvis Ridge and the Rio Grande Rise as bridges. The present study reports on some rare species of Oplophoridae Dana, The family includes 73 species distributed in 10 genera (Fransen & De Grave, 2009). The members of this family are considered pelagic and occur strictly on the meso- and bathypelagic zones of the oceans. Morphological adaptations for pelagic life such as natatory exopods, which occur in all species, and lightly calcified, almost membranous exoskeleton of some genera are evidence of the habitat they occupy (Bauer, 2004). Among the papers dealing with oplophorids, many deal with the southwestern Atlantic (Bate, 1888; Barnard, 1950; Crosnier & Forest, 1967, 1973) and few of them with the south-western Atlantic (Cardoso & Young, 2005; Cardoso, 2006). However none of them has ever considered the South Mid Atlantic Ridge oplophorids. MATERIALS AND METHODS The Mar Eco project (Patterns and Processes of the Ecosystems of the Northern Mid-Atlantic) is part of the Census of Marine Life which aims to assess and explain the diversity, distribution, and abundance of marine life. Mar Eco objectives aims to enhance our understanding on the occurrence, distribution and ecology of animals and animal communities along the Mid-Atlantic Ridge, being supported by the Sloan Foundation ( data/page/ 805/ Finalscienceplan.pdf). Sampling was done during the first oceanographic cruise of Mar Eco South from 25 October to 29 November of 2009 on the R/V Akademik Ioffe. A total of 12 benthic sampling sites, using a Sigsbee trawl, were conducted, five in the South Equatorial MAR Sector (SEMS), two in the Tropical MAR Sector (TMS) and five in the Walvis Ridge Sector (WRS). Despite the pelagic habit of Oplophoridae they were caught probably during the trawl movements in the water column. The specimens were fixed in ethanol 70%, identified, drawn, redescribed and deposited at the Crustacea collection of Museu Nacional/Universidade Federal do Rio de Janeiro (MNRJ). To identify one species comparative material from National Museum of Natural History (NMNH) was examined. Measurements presented are from carapace length. RESULTS Family Oplophoridae Dana, 1852 Genus Acanthephyra A. Milne-Edwards, 1881 Acanthephyra acanthitelsonis Bate, 1888 (Fig. 1) Acanthephyra acanthitelsonis Bate, 1888: 745, pl. 125, fig. 3; Kemp, 1939: 574; Barnard, 1950: 668; Chace, 1947: 16; 1986: 9; Crosnier & Forest, 1967: 1129; 1973: 31, figs.7a-7b.

8 Oplophoridae from south Mid-Atlantic Ridge 211 Figure 1. Acanthephyra acanthitelsonis Bate, 1888, male (16.1 mm), MNRJ a) Carapace, lateral view; b) telson and uropods, lateral view; ovigerous female (13.58 mm), USNM ; c) carapace, lateral view; male (21.12 mm), USNM ; d) telson and uropods, lateral view. Acanthephyra pelagica (Risso, 1816), female (11.73 mm), USNM ; e) carapace, lateral view; f) telson and uropods, lateral view. Material examined: superstation 3, 04 o S, 12 o W, SEMS, 3342 m, 1 male (16.1 mm), MNRJ Comparative material: 09 o 03 N, 49 o 16 W, north Atlantic, 490 m, coll. Atlantis II, 2 ovigerous females ( mm), id. Judkins, USNM ; 15 o 59 S, 02 o 02 E, south Atlantic, 290 m, coll. Atlantis, 3 males ( mm), 21 females ( mm), USNM Diagnosis: rostrum in adults not reaching scaphocerite end, shorter than carapace (brooked at south Atlantic examined specimen); branchiostegal spine strong, flared outwards, with distinct carina extending backwards on to carapace for three times the spine length (Figs. 1a and 1c). Abdomen dorsally carinated on all somites, except on somite 1; somites 3-6 with a posteromesial tooth, that of somite 3 longer than any other. Telson sulcate on dorsal midline, with (18 at south Atlantic examined specimen) pairs of dorsolateral spines and two pairs of distal spines (Figs. 1b and 1d) (modified from Kemp, 1939). Distribution: restrict to central and south Atlantic from 14 o N to 28 o S: Western: Bahamas; Central: South Equatorial MAR (first record) (Fig. 2); Eastern: Serra Leoa, Gabon, Congo, Angola. From m. Remarks: this species is included in A. purpurea species group (Kemp, 1939) with more than seven species that can be identified mainly by the number of dorsolateral spines of the telson. In this group, the rostrum shape and dentition are of minor taxonomic importance. The material sampled at south Mid Atlantic Ridge agrees with Kemp (1939) diagnosis in all aspects. At 24 o S, Acanthephyra acanthitelsonis begins to be replaced by a form with 7-11 dorsolateral spines at telson: A. pelagica (Risso, 1816). These two species are close related, so comparative material of A. acanthitelsonis (Figs. 1c-1d) and also from A. pelagica (Figs. 1e-1f) from National Museum of Natural History (NMNH) was examined and figured to facilitate the identification of the south MAR specimens and the future identifications of these species. All the material of A. acanthitelsonis herein examined, from north and south Atlantic, do not vary in the characters mentioned in the identification key of Crosnier & Forest (1973) presenting: smooth dorsal margin at abdominal somites 3-5; abdominal somite 2 without dorsal carina; long and slender rostrum; and pairs of dorsolateral spines at telson. From the 26 specimens of A. acanthitelsonis examined at NMNH, two were sampled at north Atlantic and 24 at south Atlantic. The telson was entire in 23 from the 24 south Atlantic specimens and in the two north Atlantic specimens. The number of dorsolateral spines at telson in these 25 specimens was within the range established by Kemp (1939) for this species (13-19). In the south Atlantic material, two males with 15 and one male with 13 pairs of dorsolateral spines; two females with 13, two with 14, two with 15, seven with 16, six with 17 and one with 19 pairs of dorsolateral spines; were observed, and in the north Atlantic material, one ovigerous female with 16 and one with 17 pairs of dorsolateral spines were observed. Acanthephyra quadrispinosa Kemp, 1939 Acanthephyra batei Stebbing, 1905: 107, pl. 24B (not A. batei Faxon, 1895). Acanthephyra quadrispinosa Kemp, 1939: 576; Barnard, 1950: 668, fig. 124g; Chace, 1986: 26, figs.

9 212 Latin American Journal of Aquatic Research Figure 2. Distribution map of Oplophoridae sampled by the first South Atlantic Mar-Eco cruise. 3h, 4t, 7g, 10c, 14; Kensley, 1987: 284; Cardoso & Young, 2005: 21, figs Material examined: superstation 9, 32 o S, 01 o E, WRS, 1107 m, local station 201, Mar Eco 1040, 1 male (11.6 mm), MNRJ Diagnosis: rostrum usually as long as carapace, overreaching scaphocerite, ventral margin straight, with six teeth, dorsal margin with eight teeth; antennal spine present; branchiostegal spine present with distinct carina extending twice the length of the spine. Abdomen dorsally carinated on all somites, except on somite 1; somites 3 to 6 with posteromesial tooth, the one of somite 3 distinctly strong. Telson sulcate on dorsal midline, with four pairs of dorsolateral spines and two pairs of distal spines (modified from Cardoso & Young, 2005). Distribution: south Atlantic from 32 o S to 45 o S: Western: Brazil (Espírito Santo and Rio de Janeiro); Eastern: Cape Point, Walvis Ridge (first record) (Fig. 2). Indian and Pacific oceans: from eastern south Africa to 163 o W, and from 25 o N to 44 o S. From 250 to 1700 m. Remarks: this species, as well as A. acanthitel-sonis, is included in A. purpurea species group (Kemp, 1939), but it is easily identifiable by the telson with four pairs of dorsolateral spines and the fourth abdominal somite with posteromesial tooth (Cardoso & Young, 2005).

10 Oplophoridae from south Mid-Atlantic Ridge 213 Genus Heterogenys Chace, 1986 Heterogenys monnioti Crosnier, 1987 (Fig. 3a) Heterogenys monnioti Crosnier, 1987: 704, fig. 3. Material examined: superstation 5, 18 o S, 13 o W, TMS, 2663 m, local station 201, Mar Eco 1023, 1 female (5.6 mm), MNRJ 22562; Superstation 7, 29 o S, 01 o E, WRS, 3721 m, local station 201, Mar Eco 1030, 4 females ( mm), MNRJ Diagnosis: rostrum directed anterodorsally, large at base tapering to tip, short, reaching a maximum of ¾ scaphocerite length, dorsal margin slightly concave, with 7-8 teeth at all rostrum length, ventral margin sinuous with or without 0-3 sub-distal teeth; antennal spine present, branchiostegal spine absent, pterygostomian spine present without carina; hepatic groove present; cervical groove well developed; branchiostegal suture not well developed; eyes with cornea weakly developed, more slender than the ocular peduncle; scaphocerite with short distal spine not overreaching blade; stylocerite short, not overreaching the antennular peduncle first segment distal margin (Fig. 3a). Abdominal somite 3 with long and slender posteromesial tooth overreaching a half of abdominal somite 4 length (Fig. 3a). Telson with four pairs of dorsolateral spines and two pairs of distal spines (modified from Crosnier, 1987). Distribution: occidental Indian Ocean: from 29 o 50,9 S, 48 o 35,5 E to 30 o 40,2`S, 48º14,1 E; south Atlantic Ocean: Central: Tropical Mid Atlantic Ridge (Fig. 2); Eastern: Walvis Ridge (Fig. 2) (first records in bold). From m. Remarks: Chace (1986) created Heterogenys as a monotypic genus based on the presence of a posterodorsal tooth on abdominal somite 3 that overreaches abdominal somite 4, abdominal somites 5-6 without posterodorsal tooth, cornea little more than ½ as wide as maximum width of eyestalk and the mandibles with few blunt teeth on the incisor process. All these features were observed in the material examined herein. With the description of H. monnioti by Crosnier (1987) this genus is no longer monotypic. The main difference between this species and H. microphthalma (Smith, 1885) is in the rostrum length and shape (Chace, 1985; Crosnier, 1987). The rostrum at H. microphthalma is as long as carapace, overreaching the scaphocerite, with 3 dorsal and 8-9 ventral teeth, it is slender and curved upwards (Chace, 1985). At H. monnioti, and also at the material from south Atlantic examined herein (Fig. 3a), the rostrum is short, not overreaching sacphocerite, with a wide base tapering to the tip and with 7 dorsal teeth (Crosnier, 1987). The material herein examined agrees in all features with the original description of H. monnioti but all pereopods and telson extremity were lost and could not be checked. The main differences between the material from the south Atlantic and that from the Indian Ocean are a not well marked branchiostegal suture (Fig. 3a), observed only herein, and the rostrum ventral margin without teeth at south Atlantic material (Fig. 3a). In this way, the diagnosis of this species was modified to include these variations. Since its first record at occidental Indian Ocean, when two specimens (a male and a female) were sampled (Crosnier, 1987), H. monnioti have never been recorded anymore. So the present record of five females, the second for this species, is very important, expanding its known distribution to the south Atlantic Ocean. Chace (1986) stated that the long posteromesial tooth on third abdominal somite of H. microphthalma could constrain its swimming habit, but analysis of the foregut contents indicate that it is not confined to the neighborhood of the bottom (Wasmer, 1972). The posteromesial tooth on third abdominal somite of H. monnioti probably also constrains the total extension of abdomen but there are no data on foregut contents of this species. Genus Hymenodora Sars, 1877 Hymenodora glacialis (Buchholz, 1874) Hymenodora mollicutis Bate, 1888: 848, pl. 137, fig. 2. Hymenodora glacialis Sivertsen & Holthuis, 1956: 15, figs. 11, 12; Crosnier & Forest, 1973: 84, fig. 25b; Wasmer, 1986: 48, figs. 10a, 11. Material examined: superstation 7, 29 o S, 01 o E, WRS, 3721 m, local station 201, Mar Eco 1030, 02 specimens without pleopods (6.6, 13.1 mm), MNRJ Diagnosis: rostrum short, not overreaching eyes, dorsal and lateral margins swollen, dorsal margin with 6 teeth, ventral margin strongly convex and unarmed; carapace with anteriorly convex groove connecting suprabranchial groove to a groove extending dorsoposteriorly from near mid length of hepatic groove. Epipod of second maxilliped without podobranch. Anterior margin of second segment of antennular peduncle forming broadly and evenly rounded lobe over outer basal part of scaphocerite (modified from Wasmer, 1986). Distribution: Arctic Ocean. North and south Atlantic Ocean; Western: Argentina; Central: Tristan da Cunha; Eastern: Canary Islands, Sierra Leone, Walvis Ridge (first record) (Fig. 2). Indian Ocean: 9 o 06 N,

11 214 Latin American Journal of Aquatic Research Figure 3. Heterogenys monnioti Crosnier, 1987, female (7.4 mm), MNRJ a) lateral view; Kemphyra corallina (A. Milne-Edwards, 1883), male (14.5 mm), MNRJ 22565; b) lateral view. 53 o 41 E. North Pacific: Bering Sea, gulf of Panama. Southern Ocean: Antarctic Region at Antarctic Polar Front and slightly crossing it. Rarely taken above 2000 m, maximum depth reported of 3900 m. Remarks: according Sivertsen & Holthuis (1956) specimens of Hymenodora glacialis and H. gracilis Smith, 1886 have very similar morphology and have been considered by several authors to be one species until around 1939 when Dr. Stanley Kemp studied the genus and came to the conclusion that the two species are really distinct. His results, however, were published only in 1956 by Sivertsen & Holthuis and accordingly them Dr. Kemp stated that the best character to distinguish both species is a groove that connects the suprabranchial groove to a groove that extends dorsoposteriorly from near mid length of hepatic groove, that is present in H. glacialis and absent in H. gracilis. Yet, according to Sivertsen & Holthuis (1956) the character mentioned by Smith (1886) in the original description of H. gracilis, namely the presence of a podobranch on the second maxilliped in this species and its absence in H. glacialis, is also reliable to distinguish these species. There are also some differences in the rostrum shape that are used to distinguish these species, but they are not so clear. The presence of the specific groove on carapace and the absence of podobranch in the second maxilliped has been used by several authors to identify H. glacialis (Crosnier & Forest, 1973; Wasmer, 1986). The material examined herein was identified as H. glacialis due to the presence of the groove that connects suprabranchial groove to a groove that extends dorsoposteriorly from near mid length of hepatic groove, and the absence of podobranch in the second maxilliped. Other features mentioned by Wasmer (1986) (as the rostrum and second segment of antennular peduncle shape) also agree with that observed at the material examined. Due to this past confusion between H. gracilis and H. glacialis many of the older references to H. glacialis, mainly before Sivertsen & Holthuis, (1956); cannot be trusted (Sivertsen & Holthuis, 1956; Wasmer, 1986). According Sivertsen & Holthuis (1956) its distribution includes the Arctic region down to the Atlantic as far south as 30 o N and the gulf of Panama. Crosnier & Forest (1973) examined the types of H. mollicutis Bate, 1888 (a synonym junior of H. glacialis) at British Museum, and the material of H. glacialis (from Valdivia Expedition - published by Balls, 1925) and H. mollicutis (from Challenger Expedition - published by Bate, 1888) at the Zoological Museum of Berlin and assigned this species to the Atlantic (Canary Islands, Sierra Leone, Argentina and Tristan da Cunha) and Indian oceans. Wasmer (1986) confirmed the data above and also added records for the North Pacific from the gulf of Bering to the gulf of Panama and at the Southern Ocean. Genus Kemphyra Chace, 1986 Kemphyra corallina (A. Milne-Edwards, 1883) (Fig. 3b) Notostomus corallinus A. Milne-Edwards, 1883: pl. 32. Acanthephyra valdiviae Balss, 1914: 595; 1925: 260. Acantephyra corallina Chace, 1936: 27; Kensley, 1968: 314, figs Kemphyra corallina Chace, 1986: 46, fig. 25. Material examined: superstation 9, 32 o S, 01 o E, WRS, 1107 m, local station 201, Mar Eco 1040, 3 males ( mm), MNRJ

12 Oplophoridae from south Mid-Atlantic Ridge 215 Diagnosis: rostrum overreaching scaphocerite, slightly curved upwards, with lateral carina overreaching orbits; dorsal and 3-4 ventral teeth, 7-8 of them posterior to the orbits (Fig. 3b). Carapace with dorsal carina notched at two-thirds of its length; hepatic spine strong, at base of a not well marked cervical groove and of a well defined suprabranchial ridge; antennal spine small; bran-chiostegal spine stout, flared outwards, with a branchiostegal carina extending to the hepatic spine region, followed by a lateral carina that extends to carapace end and marks the lower border of the branchial region (Fig. 3b). Eyes slightly wider than eyestalk. Abdominal somites dorsally carinated, somites 3-6 with a distal tooth (Fig. 3b). Exopod of uropod as long as telson, with two spines on posterior half of outer margin; telson with 4-5 pairs of dorsolateral spines and one pair of distal spines (Fig. 3b) (modified from Kensley, 1968). Distribution: Central Indian Ocean. Eastern Atlan-tic: north: off northern Portugal; south: off Cape of Good Hope; Walvis Ridge (first records) (Fig. 2). From 1000 to 2782 m depth. Remarks: Kemphyra corallina is a rare species, restricted to the Atlantic Ocean, with less than 20 specimens deposited in zoological collections around the world. The presence of an hepatic spine on its carapace distinguishes this species from the remaining Oplophoridae. Described originally as Notostomus corallinus (A. Milne-Edwards, 1883), posteriorly it was included in the enigmatic genus Acanthephyra by Chace (1936) and Kensley (1968). In 1985, Chace used this clear species autapomorphy (presence of hepatic spine) to define a monotypic genus (Kemphyra) and accommodate this species. The only difference noticed between the material herein examined and Kensley s (1968) description is the number of dorsolateral spines on telson (4-5 in MAR material and 4 in South African material). DISCUSSION The oplophorid fauna of south Mid-Atlantic Ridge is so far poorly investigated; these are the first records of these species at this area. Interestingly, some rare species were sampled, as K. corallina and mainly H. moniotti (second record of the species), showing how the MAR is a special and unknown environment. There is some discussion about the rarity of deep-sea species. As some authors mentioned, rare in respect to abyssopelagial fauna could be replaced by seldom found till now (Tiefenbacher, 2001). But, if we compare the frequency and abundance of some deep sea Oplophoridae we can conclude that some species are effectively rare and others are common, in terms of how many specimens were historically sampled. In this way H. monnioti is extremely rare, K. corallina is rare, and the three remaining MAR species are relatively common. All Oplophoridae species sampled occur at southeastern Atlantic and at Indian Ocean, except H. monnioti that is restricted to Indian Ocean (Crosnier, 1987). Only two of them occur also at southwestern Atlantic (A. quadrispinosa and H. glacialis) (Wasmer, 1986; Cardoso & Young, 2005). So, the South MAR Oplophoridae fauna looks to be more related with that of southeastern Atlantic and Indian Ocean than to that of southwestern Atlantic. Probably, the Walvis Ridge is acting as a bridge between the MAR and the east Atlantic. A strong sample effort is needed at Rio Grande Rise region, to get a more satisfactory observation of the relations between the MAR fauna and the western Atlantic. The MAR fauna is also weakly related with that of Antarctic waters, which actually is poor in Decapoda species. Only H. glacialis is shared between MAR and Antarctic waters. ACKNOWLEDGEMENTS The author would like to acknowledge the south Atlantic Mar-Eco team for sampling and sorting the samples analyzed herein, and the Sloan Foundation for financial support. REFERENCES Balss, H Diagnosen neuer Macruren der Valdivia Expedition. Zool. Anz., 44: Barnard, K.H Descriptive catalogue of South African Decapod Crustacea. Ann. S. Afr. Mus., 38: Bate, C.S Report on the Crustacea Macrura collected by the Challenger during the years Rep. Voy. Challenger, 24: Bauer, R Remarkable shrimps, adaptations and natural history of the carideans. University of Oklahoma Press, Oklahoma, 282 pp. Cardoso, I.A. & P.S. Young Deep-sea Oplophoridae (Crustacea Caridea) from the southwestern Brazil. Zootaxa, 1031: Cardoso, I.A New record of Meningodora vesca (Smith, 1887) (Caridea, Oplophoridae) to the southwestern Atlantic. Nauplius, 14(1): 1-7. Chace, F.A Revision of the bathypelagic prawns of the family Acanthephyridae, with notes on a new

13 216 Latin American Journal of Aquatic Research family, Gomphotidae. J. Wash. Acad. Sci., 26(1): Chace, F.A The deep-sea prawns of the family Oplophoridae in the Bingham Oceanographic collection. Bull. Bingham Oceanogr. Coll., 11(1): Chace, F.A The Caridean shrimps (Crustacea: Decapoda) of the Albatross Philippine Expedition, , part 4: families Oplophoridae and Nematocarcinidae. Smith. Contr. Zool., 432: Crosnier, A Oplophoridae (Crustacea Decapoda) récoltés de 1971 à 1982 par les navires français dans l ócean Indien occidental sud. Bull. Mus. Hist. Nat, Paris, Ser. 4, 9(3): Crosnier, A. & J. Forest Note préliminaire sur les carides recueillis par l'ombango au large du plateau continental, du Gabon à l'angola (Crustacea, Decapoda, Natantia). Bull. Mus. Hist. Nat., Paris, Ser. 2, 39(6): Crosnier, A. & J. Forest Les crevettes profondes de l'atlantique oriental tropical. Faune Tropicale, 19: Fransen, C.H. & S. De Grave Evolution and radiation of shrimp-like decapods: an overview. In: J.W. Martin, K.A. Crandall & D.L. Felder (eds.). Decapod crustacean phylogenetics. crustacean issues. S. Koenemann, Vol. 18. CRC Press, Taylor & Francis Group, Boca Raton, pp Kemp, S.W On Acanthephyra purpurea and its allies (Crustacea Decapoda: Hoplophoridae). Ann. Mag. Nat. Hist., Ser. 11, 4: Received: 16 May 2011; Accepted: 22 October 2012 Kensley, B Deep-sea decapod Crustacea from west of Cape Point, South Africa. Ann. S. Afr. Mus., 50(12): Kensley, B Deepwater Decapod Crustacea from eastern Australia (Penaeidea and Caridea). Rec. Austr. Mus., 39: Levin, L.A. & A.J. Gooday The deep Atlantic Ocean. In: Ecosystems of the World: The Deep-Sea. Elsevier, Amsterdam, pp Linden, W Walvis Ridge, a piece of Africa? Geology, 8: Milne-Edwards, A Recueil de figures de crustacés nouveaux ou peu connus, Paris. 3 pp, 44 plates. Probert, K Seamounts, sanctuaries and sustainnability: moving towards deep-sea conservation. Aquat. Conserv. Mar. Freshw. Ecosyst., 9: Rogers, A.D The biology of seamounts. Adv. Mar. Biol., 30: Sivertsen, E. & L.B. Holthuis Crustacea Decapoda (the Penaeida and Stenopodidea Excepted). Report on the Scientific Results of the Michael Sars North Atlantic. Deep-Sea Exped., 5(12): Stebbing, T.R South African Crustacea: Part III. Mar. Invest. S. Afr., 4: Tiefenbacher, L Recent samples of mainly rare decapod Crustacea taken from the deep-sea floor of the southern west Europe Basin. Hydrobiologia, 449: Wasmer, R.A Pelagic shrimps of the Family Oplophoridae (Crustacea Decapoda) from the Pacific sector of the Southern Ocean: USNS Eltanin Cruises 10, 11, 14-16, 19-21, 24 and 25. In: L. Kornicker (ed.). Biology of the Antarctic Seas XVII. Antarc. Res. Ser., 44:

14 Lat. Am. J. Aquat. Res., 41(2): , 2013 GST as biomarker in Sciades herzbergii of São Marcos Bay Proceedings of the 3 rd Brazilian Congress of Marine Biology A.C. Marques, L.V.C. Lotufo, P.C. Paiva, P.T.C. Chaves & S.N. Leitão (Guest Editors) DOI: /vol41-issue2-fulltext Research Article Glutathione S-Transferase as biomarker in Sciades herzbergii (Siluriformes: Ariidae) for environmental monitoring: the case study of São Marcos Bay, Maranhão, Brazil Raimunda N.F. Carvalho-Neta 1 & Ana L. Abreu-Silva 2 1 Departamento de Química e Biologia, Laboratório de Pesca, Biodiversidade e Dinâmica Populacional de Peixes, Universidade Estadual do Maranhão, Campus Paulo VI, Tirirical São Luís-MA, , Brazil 2 Departamento de Patologia, Laboratório de Anatomopatologia Programa de Pós-Graduação em Biotecnologia-Rede Nordeste de Biotecnologia (RENORBIO) Universidade Estadual do Maranhão, Campus Paulo VI, Tirirical, São Luís-MA, , Brazil ABSTRACT. The Glutathione S-Transferase (GST) activity has been proposed as a biomarker of susceptibility to the presence of potentially damaging xenobiotics in aquatic organisms. The aim of this work was to measure GST activity in the liver of Sciades herzbergii (catfish) in order to evaluate the biochemical effects of pollutants. The catfish samples were collected along known pollution gradients areas (A1) and from areas regarded as relatively free of anthropogenic input (A2), in São Marcos Bay, São Luis de Maranhão, Brazil. The variables analyzed in fish were: length, weight, gonadal stages, gonadosomatic index and GST activity. The databases from this analysis were compiled, and generalized linear models were used to analyze the dependence of enzyme activity on the areas of sampling and on selected biological parameters of fish. A significant difference was observed in GST activity in the liver of S. herzbergii in the comparison between fish from the contaminated site and those from the reference site (P < 0.05). Morphometric (length and weight) parameters and gonadosomatic index of collected fish were significant in the linear model of GST activity only in the reference site. These results may be due to the activity pattern of the enzyme, which increases with the sexual maturity of the animals in healthy environments. In the contaminated area (A1) these correlations do not exist, probably as a result of the energy used in the biotransformation of the various contaminants. Keywords: biomonitoring, gonadosomatic index, catfish, São Marcos Bay, Brazil. Glutatión S-Transferasa como biomarcador en Sciades herzbergii (Siluriformes: Ariidae) para el monitoreo ambiental: el caso de estudio de la bahía de São Marcos, Maranhão, Brasil RESUMEN. La actividad de Glutatión S-Transferasa (GST) ha sido propuesta como un biomarcador de susceptibilidad a la presencia de xenobióticos potencialmente perjudiciales en los organismos acuáticos. El objetivo de este trabajo fue medir la actividad de GST en el hígado del bagre Sciades herzbergii para evaluar los efectos bioquímicos de los contaminantes. Las muestras de bagres fueron colectadas a lo largo de aéreas con gradientes de contaminación conocidas (A1) y en áreas consideradas relativamente libres de intervención antropogénica (A2), en la bahía de São Marcos, São Luis de Maranhão, Brasil. Las variables analizadas en los peces fueron: longitud, peso, estado gonadal (madurez sexual), índice gonodosomático y actividad de la enzima GST. Las bases de datos de los análisis fueron compiladas y se utilizaron modelos generales lineales y no lineales para analizar la dependencia entre la actividad de la enzima en peces colectados en las aéreas de muestreo A1 y A2. Una diferencia significativa en la actividad de la GST fue observada en peces colectados en el área contaminada (A1) comparada con la actividad enzimática de aquellos colectados en el sitio de referencia (A2) (P < 0,05). El peso, longitud, estados de madurez sexual y el índice gonodosomático fueron significativos en el modelo lineal de actividad de la GST en A2, pero no en A1. Estos resultados pueden ser debidos a los patrones de actividad enzimática que aumenta con la madurez sexual de los peces en ambientes

15 218 Latin American Journal of Aquatic Research saludables (A2). En el área contaminada (A1) estas correlaciones no existen, probablemente como resultado de la energía usada en la biotransformación de los contaminantes. Palabras clave: biomonitoreo, índice gonodosomático, bagre, bahía de São Marcos, Brasil. Corresponding author: Raimunda Carvalho-Neta INTRODUCTION The use of biomarkers has become relevant in (eco) toxicological assessments since it allows the early detection of overall effects of contaminants (Passino, 1984; Livingstone, 1993; Ahmad et al., 2006; Amado et al., 2006; Camargo & Martinez, 2006; Umbuzeiro et al., 2006; Zanette et al., 2006). The International Council for the Exploration of the Sea (ICES) has proposed the evaluation of antioxidant and biotransformation enzymes as biomarkers of exposure to xenobiotics. The main antioxidant defence enzymes in aquatic organisms are the superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and the phase II biotransformation enzyme glutathione-s-transferase (Pavlović et al., 2004). Glutathione-S-Transferases (GSTs) are a multigene family of dimeric, mainly cytosolic enzymes, which play an important role in the biotransformation and detoxification of a number of electropholic compounds (Rao, 2006). The alterations in the GST activities directly reflect the metabolic disturbances and cell damage in specific organs of fish (Livingstone et al., 2001). Previous field studies using catfish (Sciades herzbergii) as a model on harbour water exposure under natural environmental condition showed that GST is a useful biomarker of pollution in São Marcos Bay, Brazil (Carvalho-Neta & Abreu-Silva, 2010). The S. herzbergii is a benthic euryhaline catfish widely distributed along the São Marcos Bay, an important fishing location and it has the most important port in the Northeastern Brazil. In the last decade, chemical contamination of this Bay due to sewage discharge, the nutrient runoff from pesticides, and industrial waste, have all induced environmental impact. The main contaminants that can be found in this area, such as heavy metals, derive from industrial activities through their respective marine terminals (Itaqui, VALE and ALUMAR/ALCOA) located in São Luís Island (Carvalho-Neta & Abreu-Silva, 2010). Other non-polluted areas are also found in São Marcos Bay. An example is the Caranguejos (Crab) Island, which is part of the Environmental Protected Area. The aim of the present study was to investigate if the contaminants present in the marine terminals located in São Luís Island are generating biological responses by comparing catfishes from this site with those collected in the Caranguejos Island, a nonpolluted site. To accomplish this objective, GST activity was measured in tissue samples from catfishes collected in these two regions, in rainy and dry periods. MATERIALS AND METHODS Study area The studied areas are located along the Brazilian coast: São Luís Island and Caranguejos (Crab) Island (São Marcos Bay, Maranhão, Northeastern Brazil). São Marcos Bay is an estuarine system located in the Gulf of Maranhão (Fig. 1). To evaluate the impact of human activities in the areas, two sampling sites were chosen in each studied region. The first site (A1) is located near the Itaqui port (2º42 18 S, 44º22 33 W) which receives agricultural, industrial and domestic sewage effluents, as well as harbor related residuals. The reference site (A2) was located in Caranguejos (Crab) Island, a protected natural reserve (3º2 18 S, 44º40 25 W). Itaqui port is located to the west coast of São Luis Island. The infrastructure of this port has an extension 1,616 m of quaysides. Fuel, iron, ore, manganese, grains, fertilizers, petroleum, bauxite and alumina are shipped in this port (ANTAQ, 2010). The Caranguejos (Crab) Island, located at a distance of 30 km from the Itaqui port in São Marcos Bay is an Environmental Protection Area created by the government of the Maranhão. This region is uninhabited and occupies an area of km 2 within a perimeter of km, containing the largest continuous stretch of mangroves in Maranhão (Carvalho-Neta & Castro, 2008). Sample collection Specimens of S. herzbergii were collected from three streams, about a kilometer from each other at each site (A1 and A2). The capture of fish was made during the rainy period (June 2010) and the dry period (November 2010). The catfish were collected in their natural habitats using gill nets. Fish were anesthetized by placing them

16 GST as biomarker in Sciades herzbergii of São Marcos Bay 219 Figure 1. Map showing São Marcos Bay with details of its sampling sites (A1: contaminated, A2: reference). for 10 min in benzocaine hydrochloride and then sacrificed by cervical section in order to remove the liver. Livers were frozen in liquid nitrogen and then stored at -80 o C until GST activity assays. At each sampling-site, water temperature, salinity, turbidity, dissolved oxygen (DO), conductivity and ph were measured. Water and sediment samples collected from sampling-sites along São Marcos Bay were analyzed for dissolved aluminum, total cadmium, dissolved iron, manganese, lead, total mercury, benzene, phenols, tributyltin and polychlorinated biphenyls. Metal determinations were performed by flame atomic absorption spectrophotometry (AAS), modified, with a nitrous oxide-acetylene flame reading (Abollino et al., 1995). Organic analysis in water and sediment samples was performed by Electrochemical (EC) Methods. Tissue processing and biochemical measurements To determine glutathione-s-transferase (GST) activity, liver samples were processed according to previously established protocols (Livingstone, 1988). They were homogenized with 1:4 vol. of buffer (Tris-HCl 50 mm, 0.15 M KCl, ph 7.4), and centrifuged at 9,000 g for 30 min at 4ºC. A fraction of the supernatant was centrifuged at 37,000 g for 60 min at 4ºC, to obtain the cytosolic fraction. This fraction was used to analyze the enzymatic activity of the GST. Protein concentration of the supernatant was determined in accordance with the modified method of Lowry et al. (Peterson, 1977), using bovine serum albumin as a standard. GST activity was assessed following the method described by Habig & Jakoby (1981). This method is based on the conjugation of 1 mm glutathione (Sigma)

17 220 Latin American Journal of Aquatic Research with 1 mm of 1-chloro-2,4-dinitro-benzene (CDNB; Sigma). Enzyme activity was measured as absorbance increments at 340 nm and it was expressed in GST units, where one unit is the amount of enzyme necessary to conjugate 1 µmol de CDNB/min/mg protein, at 25 o C and ph The GST activity is expressed as µmol min -1 mg -1 protein. Biometric data and gonadosomatic index For all the fish whose livers were removed biometric data were recorded: total length (Lt), standard length (Ls), furcal length (Lf), total weight (Wt) and gonad weight (Wg). The macroscopic classification of the gonadal stage (GS) was also undertaken: immature (GS1), maturing (GS2), mature (GS3) and spent (GS4), following the scale given by Vazoller (1996). The gonadosomatic index (GSI) was calculated as gonad wet weight expressed as a percentage of body wet weight (total weight). Statistical analysis Results were expressed as mean ± standard deviation. For each variable studied differences observed among the sampling-sites (on the potentially contaminated and the reference site) were tested for significance by single-factor ANOVA or the Kruskal-Wallis test, depending on whether the data followed a normal distribution and showed homogeneity of variance. Significant differences between groups (A1 and A2) were verified using the Student s t-test. Confidence level adopted was 95%. Linear correlation (Pearson s coefficient) between GST activity and biometric data (Lt, Ls, Lf, Wt, Wg and GSI) were calculated using the mean values observed for each parameter. RESULTS Water and sediment chemistry measured at the moment of fish collection is listed in Tables 1 and 2. The concentrations of Al, Cd, Pb, Cr, Fe, Hg, benzene, total phenols, tributyltin and polychlorinated biphenyls in potentially contaminated site were higher than the acceptable limit by national standards (CONAMA, 2005). In the sediment (A1: potentially contaminated) these values were even higher (30 to 100%), confirming the character of chemical contamination of the marine terminal (São Luís Island). The average water surface temperatures were constant during both the periods analyzed. Salinity was uniform in both areas sampled, being lower during the rainy season in both areas. Dissolved oxygen and the saturation of dissolved oxygen were always lower in the contaminated area. The values for ph and turbidity were constants for both areas, demonstrating the homogeneity of these abiotic factors in both areas studied. Morphometric parameters (total length, standard length, furcal length, total weight and gonad weight) of collected fish are showed in Table 3. At the nonpolluted site, catfishes were bigger (P < 0.05) than those from the polluted area. The gonadosomatic index (GSI) was also higher in the reference site than the one from the polluted site in both periods (Fig. 2). The GSI in fish from the contaminated site was significantly lower (P < 0.05) than in control fish during all the phases of the gonadal cycle. In each period, differences (t-test; P < 0.05) in GST activity were found between catfishes from the polluted and non-polluted sites (Tables 4 and 5). When comparing GST values among females and males, activity did not appear to be sex-dependent (P > 0.05). In the contaminated area the greatest values for the GST activity (2.82 ± 0.33 µmol mg -1 of protein) were registered in juveniles females (GS1). However, in the reference area the values for enzymatic activity increased progressively with the gonads stages (GS1-GS2-GS3-GS4) in males and females (t-test; P < 0.05). On the other hand, seasonal variations were not observed in the GST activity of catfishes from the A1 and A2 (P > 0.05). A significant linear correlation was established among GST activity and all the biometric data of the reference area, but not with those of the contaminated area in any of the periods analyzed (Tables 6 and 7). DISCUSSION In the present study, GST activity showed a different pattern in catfishes from both polluted and nonpolluted sites. In a similar study carried out by Carvalho-Neta & Abreu-Silva (2010), samples of Sciades herzbergii (Ariidae) caught at São Marcos Bay showed significantly higher levels of GST only in polluted areas (compared to Caranguejos Crab Island), which confirms that São Marcos Bay is a site with high exposure risks for some contaminants. São Marcos Bay receives contaminant input from hundreds of industrial and domestic sources in the São Luís (Maranhão, Brazil) metropolitan area. Benzene, phenols, TBT and the PCB measured in the contaminated area of São Marcos Bay were considerably greater than the largest tolerable value prescribed by Brazilian Environmental legislation (Conselho Nacional de Meio Ambiente, 2005) and can affect the detoxifying responses and biometric data of many aquatic species, especially fish (Oruç et al., 2004).

18 GST as biomarker in Sciades herzbergii of São Marcos Bay 221 Table 1. Physical and chemical analysis of water in rainy and dry periods at the polluted and non-polluted sites of the São Marcos Bay (Brazil). Rainy period: June 2010, dry period: November Contaminated (A1) Reference (A2) Parameters Rainy period Dry period Rainy period Dry period Point Point Point Point Point Point Point Point Point Point Point Point Temperature ( o C) Salinity (UPS) ph DO (ml D2 L -1 ) % satur. OD Turbidity (NTU) Aluminum (mg L -1 ) Cadmium (mg L -1 ) Lead (mg L -1 ) Chromium (mg L -1 ) Iron (mg L -1 ) Mercury (mg L -1 ) Benzene (µm L -1 ) Total phenols (mg L -1 ) TBT (µm L -1 ) Total PCB (µm L -1 )

19 222 Latin American Journal of Aquatic Research Table 2. Physical and chemical analysis of sediment in rainy and dry periods at the polluted and non-polluted sites of the São Marcos Bay (Brazil). Rainy period: June 2010, dry period: November Contaminated (A1) Reference (A2) Parameters Point 1 Rainy period Dry period Rainy period Dry period Point 2 Point 3 Point 1 Point 2 Point 3 Point Point Point Point Point Point Temperature ( o C) Salinity (UPS) ph DO (mg L -1 O2) Aluminum (mg L -1 ) Cadmium (mg L -1 ) Lead (mg L -1 ) Chromium (mg L -1 ) Iron (mg L -1 ) Mercury (mg L -1 ) Benzene (µm L -1 ) Total phenols (mg L -1 ) TBT (µm L -1 ) Total PCB (µm L -1 )

20 GST as biomarker in Sciades herzbergii of São Marcos Bay 223 Table 3. Morphometric data of catfish (Sciades herzbergii) as mean values (± standard deviation) collected in rainy and dry period in the polluted and non-polluted sites of the São Marcos Bay (Brazil). Parameters Contaminated (A1) Reference (A2) Females Males Females Males Total length (cm) ± ± ± ± 1.67 Furcal length (cm) 23.2 ± ± ± ± 1.60 Total weight (g) ± ± ± ± Gonad weight (g) 2.01 ± ± ± ± 0.16 Figura 2. Gonadosomatic index of males and females of Sciades herzbergii collected in São Marcos Bay in the rainy (June 2010) and dry period (November 2010). (A1: contaminated, A2: reference). Water and sediment chemistry parameters (temperature, salinity and oxygen) showed no seasonal variations, being similar in both studied sites (A1 and A2). However, a heavy metal concentration (Al, Cd, Pb, Cr, Fe and Hg) in surface waters and in sediments has much higher values for the contaminated area than for the area of reference. The heavy metals, such as Cd, Ni, Cr, Pb and Hg are especially toxic to aquatic organisms due to their oxidative potential, whereas other metals such as Fe, Zn, Cu, Se and Mn are essential for their metabolism but, could become toxic in excessive concentrations (Chang et al., 1996). Our results have shown a decrease in the gonadosomatic index (GSI) in fish from the contaminated site (São Marcos Bay). Several in situ studies have demonstrated a decrease in the GSI in fish collected from polluted areas (Carvalho-Neta & Abreu-Silva, 2010; Noaksson et al., 2001). This decrease in the index can result in abnormal gonadal development in the form of delayed maturation, high levels of atresia or intersexuality (Kime, 1998). The specimens of S. herzbergii of the contaminated area of São Marcos Bay had a significantly higher level of GST activity than that found in the organisms of the reference area. Previous studies using Sciades herzbergii caught in São Marcos Bay on the Maranhão coast showed a possible susceptibility of the youngest and sexually immature fish to oxidative stress in the studied area (Carvalho-Neta & Abreu-Silva, 2010). Increased GST activity in fish liver has been demonstrated in various fish species as the result of exposure to PCBs (Gadagbui & Goksoyr, 2001), PAHs and pesticides (Bello et al., 2001). The GST activity has grown linearly proportional to the phase of maturity of S. herzbergii only in the reference area. The same type of response was found by Winston & Di Giulio (1991), who demonstrated that the activity of detoxifying enzymes differs

21 224 Latin American Journal of Aquatic Research between the various organs and tissues of fish, depending on their stage of gonadal development in health environments. GST is an important enzyme responsible for the hepatic degradation of sex steroids and marked differences observed in GST could be connected with hormone alterations (Gallagher et al., 2001). However, exposure to xenobiotics usually causes changes both in enzyme expression and in reproductive endocrine system in fishes, initiating degenerative processes (Vargas et al., 2001). GST activity showed a similar pattern in S. herbergii from both rainy and dry periods. Amado et al. (2006) in their study about biomarkers in croakers Micropogonias furnieri (Sciaenidae) from polluted and nonpolluted areas from the Patos Lagoon estuary (southern Brazil) indicated a clear seasonal variation pattern for GST activity (enzymatic activity was higher in the warmer season). The results suggest that the seasonal adjustments in the antioxidant defence of fish from northern Brazil are different from those of southern Brazil. In southern Brazil the climate is subtropical with a wide water temperature range (up to 32ºC in summer and below 9ºC in some winter), but in northern Brazil the climate is tropical (Pell et al., 2007). The temperature influences a great variety of biological processes of fish species (Bicego et al., 2007). Consequently, significant changes in water temperature may cause a serious challenge to the maintenance of physiological function in fish (Garcia et al., 2008). The linear correlation among the GST activity when related to all the biometric data of the catfish is significant in the reference area, but not in the contaminated area, probably due to the pattern of enzyme activity which increases with age and sexual maturity of the animals in environments free of contaminants. In the port area, this correlation does not exist, probably as a result of the energy used in the biotransformation of the various contaminants in the water and in the sediment. The fact that the polluted site is characterized by higher levels of metals like Al, Cd, Pb, Cr, Fe, Hg, that can generate oxidative stress in fish, gives support to our hypothesis. This is a significant result because the morphometric differences between two groups of the population of the same fish species indicate that these populations are subjected to different selection processes (Shibatta & Hoffmann, 2005). Ecological and biological factors should be taken into account to explain variations in enzymatic biotransformation activities in fish (Mayon et al., 2006), but the use of GST in this study enhances our understanding of the Sciades herzbergii in situ as demonstrating its great suitability in the monitoring of polluted and non-polluted areas. REFERENCES Abollino, O., M. Aceto, G. Saccchero, C. Sarzanini & E. Mentasti Determination of copper, cadmium, iron, manganese, nickel and zinc in Antarctic sea water. Comparison of electrochemical and spectroscopic procedures. Analyt. Chim. Acta, 305: Agência Nacional de Transportes Aquaviários (AN- TAQ) Principais portos do Brasil. [ antaq.gov.br/portal/anuarios]. Reviewed: 20 August Ahmad, I., M. Pacheco & M.A. Santos Anguilla anguilla L. oxidative stress biomarkers: an in situ study of freshwater wetland ecosystem (Pateira de Fermentelos, Portugal). Chemosphere, 65: Amado, L.L., C.E. da Rosa, A.M. Leite, L. Moraes, W.V. Pires, G.L. Leães Pinho, C.M.G. Martins, R.B. Robaldo, L.E.M. Nery & J.M. Monserrat Biomarkers in croakers Micropogonias furnieri (Teleostei: Sciaenidae) from polluted and nonpolluted areas from the Patos Lagoon estuary (southern Brazil): evidences of genotoxic and immunological effects. Mar. Pol. Bull., 52: Bello, S.M., D.G. Franks, J.J. Stegeman & M.E. Hahn Acquired resistance to Ah receptor agonists in a population of Atlantic killifish (Fundulus heteroclitus) inhabiting a marine superfund site: in vivo and in vitro studies on the inducibility of xenobiotic metabolizing enzymes. Toxicol. Sci., 60: Bicego, K.C., R.C.H. Barros & L.G.S. Branco Physiology of temperature regulation: comparative aspects. Comp. Biochem. Physiol., A, 147: Camargo, M.M.P. & C.B.R. Martínez Biochemical and physiological biomarkers in Prochilodus lineatus submitted to in situ tests in an urban stream in southern Brazil. Environ. Toxicol. Pharm., 21: Carvalho-Neta, R.N.F. & A.L. Abreu-Silva Sciades herzbergii oxidative stress biomarkers: an in situ study of estuarine ecosystem (São Marcos Bay, Maranhão, Brazil). Braz. J. Oceanogr., 58: Carvalho-Neta, R.N.F. & A.C.L. de Castro Diversidade das assembléias de peixes estuarinos da Ilha dos Caranguejos, Maranhão. Arq. Ciên. Mar., 41: Chang, L.W., L. Magos & T. Suzuki Toxicology of metals. CRC Lewis, Boca Raton, pp

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23 Lat. Am. J. Aquat. Res., 41(2): , 2013 Nutrient budgets in the Itaipu-Piratininga lagoon system Proceedings of the 3 rd Brazilian Congress of Marine Biology A.C. Marques, L.V.C. Lotufo, P.C. Paiva, P.T.C. Chaves & S.N. Leitão (Guest Editors) DOI: /vol41-issue2-fulltext Research Article Nutrient budgets in the Piratininga-Itaipu lagoon system (southeastern Brazil): effects of sea-exchange management Mauricio Cerda 1, Conceição Denise Nunes-Barboza 1, Camila Nunes Scali-Carvalho 1 Kelly de Andrade-Jandre 1 & Aguinaldo-Nepomuceno Marques Jr 1 1 Departamento de Biologia Marinha, Universidade Federal Fluminense Outeiro São João Batista s/n, Centro, Niterói, , Brazil ABSTRACT. The Piratininga-Itaipu Lagoon System and its drainage basin, located on the southeastern coast of Brazil, Niterói (RJ), has been the target of an intense process of urbanization over the last four decades. As a result of this process, both the lagoon and the adjacent sea area have shown signs of eutrophication due to the release of large quantities of domestic sewage semi (or not) treated. In an attempt to minimize these effects, in 2008 the government re-established a connection between the Piratininga lagoon and the sea. To evaluate changes and variations in the balance of dissolved inorganic nutrients, before and after interventions, the geochemical model of two stoichiometric mass balance boxes of the "LOICZ Program (Land-Ocean Interactions in the Coastal Zone) was used. The salt and water balance showed different water residence times in the Piratininga lagoon for the two periods: (before the opening of the connection between the Piratininga lagoon and the sea) and (after the opening of the connection with the sea). The first period showed a value about two times higher than that obtained for , τ = 83 and 39 days respectively. The water residence time of the Itaipu lagoon did not show great variations between the two periods (τ = 9 and 8 days). For the period of , before the opening of the connection with the sea, both the lagoons were autotrophic ( DIP > 0) and nitrogen loss was predominant ( DIN < 0). The second period ( ) was characterized by changes only in the trophic state of the Piratininga lagoon, which became heterotrophic ( DIP > 0). Keywords: nutrient balance, DIP, DIN, LOICZ, Piratininga-Itaipu, Brazil. Balance de nutrientes del sistema lagunar Piratininga-Itaipu (sudeste de Brasil): efectos del manejo del sistema a través del intercambio con el agua de mar RESUMEN. El Sistema Lagunar y su cuenca de drenaje, localizada en la costa sudeste de Brasil, Niterói (RJ), han sido objeto de un intenso proceso de urbanización en las últimas cuatro décadas. Como resultado, tanto el área lagunar como el área costera adyacente han mostrado signos de eutrofización debido a la liberación de aguas residuales domésticas tratadas y no tratadas. En un intento por minimizar estos efectos, el gobierno restableció en el 2008 la conección, que une la laguna de Piratininga con el mar. Para evaluar los cambios y las variaciones de los balances de nutrientes inorgánicos disueltos, antes y después de las intervenciones, se utilizó el modelo geoquímico LOICZ. El balance de agua y sal mostró tiempos de residencia de agua diferenciados para los dos períodos simulados. Para el periodo en Piratininga, mostró un valor casi dos veces superior al obtenido después de la apertura de la conexión con el mar, τ = 83 y 39 días respectivamente, la laguna de Itaipu no reveló gran variación entre los dos períodos (τ = 9 y 8 días). Para el periodo , antes de la apertura de la conección con el mar, ambas lagunas fueron autotróficas ( DIP > 0) y con procesos de pérdida de nitrógeno ( DIN < 0). El segundo periodo ( ) fue caracterizado por cambios en el estado trófico solo en la laguna de Piratininga que pasó a estado heterotrófico ( DIP > 0).

24 227 Latin American Journal of Aquatic Research Palabras clave: balance de nutrientes, DIP, DIN, LOICZ, Piratininga-Itaipu, Brasil. Corresponding author: Mauricio Cerda INTRODUCTION Coastal lagoons are among the most productive ecosystems in the world (Vollenweider et al., 1992; Bianchi, 2007). High levels of primary production, frequently observed in these systems, are directly associated with the large supply of dissolved inorganic nutrients from land, either of natural origin or anthropogenic sources or both (Zhang & Liu, 1994; Abreu et al., 1995; Souza et al., 2003, 2009). The input of nutrients in the lagoon systems can be from river discharge, streams, marshes, human activities and organic matter decomposition in water and sediments. Thus, knowledge about the nutrient flux from these sources is fundamental for understanding the functioning of lagoon systems, and improving management of such ecosystems (Bormann & Likens, 1967; Andrews et al., 1998; Micheli, 1999). The Piratininga-Itaipu Lagoon System and its drainage basin, located on the southeastern coast of Brazil, Niterói (RJ), has been the target of an intense process of urbanization over the last four decades. In the 70s, urbanization was accelerated by the construction of the Rio-Niterói Bridge. At that time, the opening of a permanent channel between the Piratininga Lagoon and the sea was also performed and caused drastic changes in the lagoon system (Barroso et al., 2000). Urbanization processes followed by high human occupation in drainage basin area increased the organic load to the lagoons. In addition, fresh water from rivers carried large quantities of domestic sewage, which were stored in the form of macroalgal biomass in banks. In the 80 s, macroalgal biomass reached 60-70% of the system surface area of the Piratininga Lagoon (Barroso et al., 2000). As a result, both the lagoon system and the adjacent sea area have shown signs of eutrophication due to the release of large quantities of semi-treated or raw domestic sewage. The eutrophication process was more intense in the Piratininga Lagoon, which had its old channel of communication with the sea closed during the 80 s. This led to an increase in water residence time and organic matter accumulation in the lagoon, creating a hypertrophic environment. In an attempt to minimize these effects, the local government re-established in 2008, the connection of the Piratininga Lagoon with the sea. Within the same period, a domestic wastewater treatment station for the region was constructed to reduce sewage loads into the lagoons. This study aimed to evaluate the effects of these interventions in the metabolism of the lagoon system by calculating the mass balance, considering conservative (salinity) and non-conservative elements (N, P). We performed calculations using the LOICZ Program (Land-Ocean Interactions in the Coastal Zone), by applying a two box geochemical model. The time series data was obtained from two years of the monitoring, and These periods were characterized by the absence and presence of the canal that linked the Piratininga Lagoon with the sea. MATERIALS AND METHODS Study area The Piratininga-Itaipu Lagoon System and its adjacent coastal area are located in Niterói City (22 55' 'S and 43 07'-43 03'W), Rio de Janeiro (Brazil), covering an area of 34.1 km 2. The system is formed by two shallow lagoons communicating with each other through the Camboata Channel. Mean depth in the lagoons is about 1 m and freshwater input comes from the Arrozal, Jacaré and João Mendes rivers (Knoppers & Kjerfve, 1999). The drainage basin, with 45.5 km 2, is limited by the peaks of surrounding hills (Viração, Proventório, Sapezal, Santos Ignacio) and mountains (Cantagalo and Jacaré). The lagoons are separated from the sea by a sand barrier system with three sandy beaches: Piratininga, Camboinhas and Itaipu (Fig.1). The area is subjected to a seasonal meteorological cycle characterized by a rainy summer and a dry winter. Tidal regime is semi-diurnal, with a short time-scale dynamics and 1.0 m tidal height (Knoppers & Kjerfve, 1999). Sampling Water samples were collected during ten expeditions on the Piratininga-Itaipu Lagoon System and Itaipu Inlet, between June 2005 and November 2006 (before the opening of the channel connecting Piratininga Lagoon with the sea) and twenty three expeditions between April 2009 and March 2010 (after the opening). Sampling was carried out in six sites located downstream of the three rivers (sites 2, 3 and 4), in the two lagoons (sites 1 and 5) and in the Itaipu Inlet (site 6) (Fig. 1). Lagoons and inlet water samples were collected 0.5 m deep and river samples at the surface

25 Nutrient budgets in the Itaipu-Piratininga lagoon system 228 Figure 1. The Piratininga-Itaipu Coastal Lagoons System and Itaipu Inlet (adjacent coastal area).*1-*2-*3-*4-*5-*6 = sampling points, Piratininga Beach (PB); Camboinhas Beach (CB) and Itaipu Beach (IB). (0.1 m). The sampling periods included both high and low tides. Water samples and in situ data were collected using a 2 L Van Dorn Bottle. At each station, ph, temperature and salinity were measured using a WTW 330 phmeter and an YSI 30 probe. Water sub samples were taken to analyze nutrients in laboratory. About 1 L of water was filtered through a pre-combusted GF/F Whattman glass fiber filter in order to separate the dissolved and the particulate fractions. Samples were stored in 1 L polyethylene bottles pre-washed with HCl 1:1, rinsed with distilled water and then with water from the sample. After collection, samples were kept on ice during transport and frozen until dissolved inorganic nutrients determination. Sampling period included both high and low tides. Analytical methods Dissolved inorganic nitrogen species (DIN): ammonium (N-NH 4 + ), nitrite (N-NO 2 - ), nitrate (N- NO 3 - ), dissolved reactive phosphate (P-PO 4-3 ) (DIP), particulate organic nitrogen (PON) and particulate organic phosphorus (POP) of water samples were measured spectrophotometrically in the laboratory (Grasshoff et al., 1983). Absorbance was measured using a Shimadzu mod. UV 1601 PC spectrophotometer. Detection limits (µm) were as follows (standard deviations of replicates in brackets): N-NO 2 -, 0.01 (±3%); N-NO 3 -, 0.05 (±3%); N-NH 4 +, 0.1 (±5%); P-PO 4-3, 0.03 (±5%). Principles of the LOICZ approach To understand the functioning of the Piratininga-Itaipu Lagoon System, before and after the opening of the connecting channel between the sea and Piratininga Lagoon, we adopted the simple mass balance modeling procedure described in the LOICZ guidelines (Gordon et al., 1996). The LOICZ is a budgeting procedure describing the rate material delivered to the system (inputs), the rate of material removed from de system (outputs) and the rate of changes of material within the system (internal sources or sink).in the LOICZ approach, four sequential budgets are established: (i) water balance, (ii) salt balance, (iii) balance of non-conservative materials N:P, and (iv) stoichiometric relationships among non-conservative balance. To construct the mass balance model, the system was divided into two boxes (Piratininga and Itaipu lagoons). Fresh water inflows (V q ), precipitation (V p ), and sewage discharge (V o ) into this lagoon system were considered equivalent to the outflow and their difference with evaporation (V e ) represents the residual flow (V r ). In this study, measured data for river discharge (V q ), direct precipitation (V p ), direct evaporation (V e ) and sewage (V o ), are available in Tables 1 and 2.

26 229 Latin American Journal of Aquatic Research Table 1. Summary of annual values, quantification of the parameters and data source, used for two-box models LOICZ, for the Piratininga-Itaipu Coastal Lagoons System. DIP: disolved inorganic phosphorus, DIN: disolved inorganic nitrogen. Lagoons-Itaipu inlet Parameters Code Quantity Value Data source Piratininga-Itaipu lagoons Evaporation V e mm yr Piratininga-Itaipu Lagoons Precipitation V p mm yr Average annual evaporation, 54-year time series ( ) Climatological Atlas (Niteroi) Average annual evaporation, 54-year time series ( ) Climatological Atlas (Niteroi) Piratininga lagoon Surface runoff (Rivers) Arrozal V q m 3 s Annual cycle (Knoppers et al., 1999) Piratininga lagoon Surface runoff (Rivers) Jacaré V q m 3 s Annual cycle (Knoppers et al., 1999) Itaipu lagoon Surface runoff (Rivers) Joao Mendes V q m 3 s Annual cycle (Knoppers et al., 1999) Lagoon System Sewage (DIN) V o x(din) 10 3 mol yr Annual cycle (Couto et al., 2000) Lagoon System Sewage (DIP) V o x(dip) 10 3 mol yr -1 3 Annual cycle (Couto et al., 2000) Piratininga lagoon Area km 2 3 Knopper et al (1999) Itaipu lagoon Area km 2 2 Knopper et al (1999) Piratininga lagoon Salt ( ) S sist 18.3 This study average annual (n=10) Itaipu lagoon Salt ( ) S sist 30.4 This study average annual (n=10) Itaipu inlet Salt ( ) S sea 33.2 This study average annual (n=10) Piratininga lagoon DIP ( ) DIP 1 mm 11.7 This study average annual (n=10) Itaipu lagoon DIP ( ) DIP 2 mm 2.1 This study average annual (n=10) Itaipu inlet DIP ( ) DIP ocn mm 0.97 This study average annual (n=10) Piratininga lagoon DIN ( ) DIN 1 mm 21.8 This study average annual (n=10) Itaipu lagoon DIN ( ) DIN 2 mm 12.1 This study average annual (n=10) Itaipu inlet DIN ( ) DIN ocn mm 4.3 This study average annual (n=10) Piratininga lagoon NOP:POP ( ) N:P 19 This study average annual (n=10) Itaipu lagoon NOP:POP ( ) N:P 8 This study average annual (n=10) Piratininga lagoon Salt ( ) S sist 26 This study average annual (n=23) Itaipu lagoon Salt ( ) S sist 31.3 This study average annual (n=23) Itaipu inlet Salt ( ) S sea 34 This study average annual (n=23) Piratininga lagoon DIP ( ) DIP 1 mm 2.4 This study average annual (n=23) Itaipu lagoon DIP ( ) DIP 2 mm 2.1 This study average annual (n=23) Itaipu inlet DIP ( ) DIPsea mm 0.6 This study average annual (n=23) Piratininga lagoon DIN ( ) DIN 1 mm 17.7 This study average annual (n=23) Itaipu lagoon DIN ( ) DIN2 mm 15 This study average annual (n=23) Piratininga lagoon NOP:POP ( ) N:P 18 This study average annual (n=23) Itaipu lagoon NOP:POP ( ) N:P 13 This study average annual (n=23) Itaipu inlet DIN ( ) DIN ocn mm 4.2 This study average annual (n=23)

27 Nutrient budgets in the Itaipu-Piratininga lagoon system 230 Table 2. Summary of dissolved inorganic nutrients in the Piratininga and Itaipu lagoons (mean ±SD, Minimum- Maximum). Location Piratininga Lagoon Itaipu Lagoon Itaipu Inlet Sampling period NO 3 -N (mm) NO 2 -N (mm) NH 4 -N (mm) PO 4 -P (mm) 1.24 ± (0.22) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± We used the estimated sewage discharge presented by Couto et al. (2000), for the same lagoon system. Because of the difficulty to obtain data flow of groundwater, this input was not included in the model. Camboata Channel flow was not considered for the calculations done for LOICZ model since a bidirectional flow was deduced based on temporal series of salinity measured in a fixed sample station in the channel. Mean and standard deviation obtained were 14.8 ± 6.9 and 24.5 ± 5.2 for the and series, respectively. These results indicate an active water exchange between the two lagoons. Based on this information, residual flow (V r ) was calculated according to the following equation (1): Vr = ( Vq + Vp + Vo + Ve) (1) In this equation the numerical value of V e is negative, implying that water leaves the lagoons system by evaporation. V q, V p and V o are positive, implying that water enters the system by river discharge, precipitation and sewage flow. If V r is negative, a net outflow from the lagoon system to the adjacent area takes place (export). To conserve salt in the system, the amount of salt leaving the lagoon system with residual flow (V r ) is balanced by an amount of salt entering the system with mixing flow (V x ) caused by winds, tides or estuarine flow, leading to equation (2): Vx = ( VrSr) /( Ssea Ssyst) (2) where S syst and S sea are salt concentrations of system and sea, respectively. The ratio of the volume of the system (V syst ) and the sum of volume of mixture (V x ) with the absolute value of residual volume ( V r ), is expressed in units of time, mean residence time of freshwater (τ), or hydraulic residence time of the system following equation (3). τ = ( Vsyst ) /( Vx + Vr ) (3) The balance of non-conservative materials (N and P) is formulated as equation (4): [ Source Sink] = inputs outputs= y (4) Inputs and outputs are calculated as products of water input or output (V) and appropriate concentrations of nutriens (Y) following equation (5): y = Vy (5) So, Y is given asequation (6): Y = VqYq + VpYp + Vx( Ysea Ysyst) + VrYr (6) where Y sea is the concentration of nutriens in the sea, and Y syst the concentration in the system. In case of conservative behaviour, Y should be zero ( Y = 0). A positive Y indicates that the system is releasing material (r) and a negative Y indicates that it is uptaking.

28 231 Latin American Journal of Aquatic Research Stoichiometric relationships among non-conservative balance The calculations are based on the stoichiometric model of the C:N:P ratio of 106:16:1 described by Redfield (1934), where the content of these elements in organic matter (OM) has a molar ratio similar to production of the phytoplankton (equation 7). Whereas the basis of primary production is phytoplankton, the production processes and mineralization of organic matter are described by equation (7): 106 CO2 + 16HNO3 + H 3PO H 2O ( CH 2O) 106( NH 3) 16( H 3PO4) + 138O 2 (7) where: the reaction, from the left to the right, is set to produce organic matter (p) and the reverse reaction shows mineralization (r). So, p-r is a measure of net metabolism ecosystem (NEM). Therefore, to obtain the concentration of an element of the equation above and to define the stoichiometric relationship between these elements, the rest of the equation can be theoretically inferred. Whereas non-conservative flow of dissolved inorganic phosphorus (DIP) is an approximation of net metabolism of the ecosystem (photosynthesis and respiration) and the various reactions that occur with phosphorus are no less complex than those that occur with the nitrogen and carbon, the net metabolism of the system is defined by equation (8): [ p r] = DIP( C : P part DIC = ) (8) A system that shows a positive DIP ( DIP > 0) is interpreted as a DIC (dissolved inorganic carbon) producer mainly via respiration (P-r < 0; respiration > photosynthesis). If a system presents a negative DIP ( DIP < 0), it seems to be a primary producer of organic matter (P-r > 0; photosynthesis > respiration). Assuming that the N:P ratio of particulate matter in a system is known, (N:P) part = 16 (Redfield ratio), the flux of DIN associated with the production and decomposition of the particulate matter from the flow of phosphorus dissolved ( P = DIP) can be estimated when multiplied by 16 (equation 9). In this study we used the N:P Redfield ratio and the N:P ratio obtained from our results for the suspended particulate matter (PON = particulate organic nitrogen, and POP = particulate organic phosphorus). Thus, Nfixation-denitrification can be estimated as the difference between the measure of flow of DIN ( DIN = NO 2 + NO 3 + NH 4 ) and flow of DIN expected for the production and decomposition of organic matter ( N exp ) (equation 9). The difference between the N observed and the N expected is indicative of other processes that can alter the concentration of N in the system, besides the production and mineralization of organic matter. When positive, the values obtained represent the fixed nitrogen in the system, whereas negative values represent nitrogen lost (equation 9). ( Nfix Ndenit) = DINobs DIP exp (9) ( Nfix Ndenit) = DINobs DIPx16 where: nitrogen fixation (N fix ) and nitrogen denitrification (N denit ) RESULTS Water and salt balance To calculate the mass balance is important to quantify the input and output flows of conservative materials in the aquatic system. The water and salt balances for the Piratininga-Itaipu Lagoon System are represented by two compartments (box 1 = Piratininga, box 2 = Itaipu; Fig. 1 and Figs. 2a-2b) and by the adjacent Itaipu inlet, representing the coastal area. The salinity obtained during the first period ( ) for the Piratininga-Itaipu Lagoon System showed that the residual flow was negative (V r ), indicating a water volume export of 2.9x10 6 m 3 yr -1 from the Piratininga to the Itaipu lagoon, and of 3.1x10 6 m 3 yr -1 from the Itaipu lagoon to the Itaipu inlet (Table 3). The values in the volume of mixture (V x1 = 5.9 and V x2 = 37.8) were higher than the flow of fresh water and residual water, but enough to ensure that a salinity gradient could be observed (Table 3). The water residence time was 83 days for Piratininga and 9 days for Itaipu (Fig. 2a). The salinity observed for the second period ( ) showed a similar pattern, with negative values for V r1 = 2.9;V r2 = 3.2 and greater positive values for V x1 = 15; V x2 = 39.6 (Table 3). For both periods the export volume was higher for fresh water inflow and small tributary streams, that support the Piratininga-Itaipu Lagoon System. The entry of groundwater probably also contributes to this balance. However, the water volume that enters the ecosystem from this source was not quantified for this study. In 2008, the opening of the channel connecting Piratininga lagoon to the sea increased salinity in 58% (Piratininga = 26.0), and decreased significantly the water residence time in the lagoon (31%; Piratininga = 39 days; Fig. 2b). Nutrient balance The balance of dissolved inorganic nutrients, was obtained from the temporal variation of the

29 Nutrient budgets in the Itaipu-Piratininga lagoon system 232 Figure 2. Water and salt budget for Piratininga-Itaipu Coastal Lagoons System = a) , and b) = Water flux in 10 6 m 3 yr -1, salt flux in 10 6 m 3 yr -1. Table 3. Summary of the results of the balance of salt and water of the conservative elements to the Piratininga-Itaipu Coastal Lagoon System. V q : Volume of the rivers; V p : precipitation volume; V e : evaporation volume; V r : residual volume; V x : mixture volume; S sist : salinity system; S sea : salinity of the external system; S r : residual salinity. Location Year Vq 10 6 (m 3 yr -1 ) Vp 10 6 (m 3 yr -1 ) Ve 10 6 (m 3 yr -1 ) Vr 10 6 (m 3 yr -1 ) Vx 10 6 (m 3 yr -1 ) Piratininga Lagoon Itaipu Lagoon Piratininga Lagoon Itaipu Lagoon S sist salt S sea salt S r salt concentrations of these constituents in the water of the lagoons and the adjacent coastal waters (Table 2). As mentioned above, the model was composed by two boxes representing (1) the Piratininga lagoon, and (2) the Itaipu lagoon with adjacent coastal area. The exchange of nutrients between compartments (input and output) allows to identify if the system and the individual compartments act as retainers (sinks) or exporters (sources) of nutrients. The balance of dissolved inorganic phosphorus during showed a unidirectional flux between the compartments (Piratininga Lagoon Itaipu Lagoon Itaipu Inlet) in all conditions (Fig. 3a). Taking into account the annual flux for the whole system, the results suggest a net production of DIP (Piratininga +20x10 3 mol yr -1 and Itaipu +18x10 3 mol yr -1 ). Thus, the behaviour of Piratininga-Itaipu Lagoon System characterizes itself as an exporter ( syst = +38x10 3 mol yr -1 ) (Fig. 3a). During the period, after the opening of the channel, the phosphorus concentrations decreased in 65% (Piratininga = 2.4 µm), and the annual flow became negative of DIP (Piratininga = -45x10 3 mol yr -1 ). On the other hand, the non-conservative flux in Itaipu lagoon was positive, with an increase of 32% on phosphorus concentration and an export of +35x10 3 mol P yr -1 to the Itaipu Inlet. The behavior of the whole system revealed a net uptake of phosphorus ( syst = -10x10 3 mol yr -1 ) (Fig. 3b; Table 4). The balance for dissolved inorganic nitrogen forms in showed a non-conservative DIN flux ( DIN), with a negative value (Piratininga = -1200x10 3

30 233 Latin American Journal of Aquatic Research Figure 3. Dissolved inorganic phosphorus fluxes in the Piratininga-Itaipu System a) , and b) Fluxes in 10 3 mol yr -1. Table 4. Summary of the results of the balance of non-conservative material elements, phosphorus (DIP), dissolved inorganic nitrogen forms (NID), net ecosystem metabolism (p-r) and nitrogen fixation minus denitrification (nfix-denit) to the Piratininga-Itaipu Coastal Lagoon System. D DIP obs D DIN obs D DIN exp (nfix-denit) (nfix-denit) *1 (p-r) Location Year 10 3 (mol yr -1 ) 10 3 (mol yr -1 ) 10 3 (mol yr -1 ) 10 3 (mol yr -1 ) 10 3 (mol yr -1 ) 10 3 (mol yr -1 ) Piratininga Lagoon Itaipu Lagoon Piratininga Lagoon Itaipu Lagoon mol N yr -1 and Itaipu= -892x10 3 mol N yr -1 ) indicating that the lagoon system acts as a non-conservative DIN exporter. During , it was observed that DIN concentrations decreased in 10% in Piratininga Lagoon, reaching 17.1 µm. In opposite, DIN concentrations in the Itaipu lagoon increased approximately 10%, presenting concentrations up to 15.0 µm. Negative flows were observed for the whole system (Piratininga = -1180x10 3 mol N yr -1 and Itaipu -1370x10 3 mol N yr -1 ). For both cases, the Piratininga- Itaipu Lagoon System seems to be a nitrogen exporter showing a syst = -2092x10 3 mol yr -1 (Fig. 4a) during , and a sys =-2550x10 3 mol yr -1 (Fig. 4b, Table 4) during Stoichiometric balance Annual average values showed that the total liquid metabolism of the whole Piratininga-Itaipu Lagoon

31 Nutrient budgets in the Itaipu-Piratininga lagoon system 234 Figure 4. Dissolved inorganic nitrogen fluxes in the Piratininga-Itaipu System a) , and b) Fluxes in 10 3 mol yr -1. System was heterotrophic during , presenting organic matter degradation. The production minus respiration (P-r) parameter was negative in both Piratininga and Itaipu lagoons (p-r Piratininga = -2100; p- r Itaipu = -1910) (Fig. 3a). In the system changed from an heterotrophic to an autotrophic condition, showing positive values of (P-r) for Piratininga (+4800) and Itaipu (+822) (Fig. 3b). Despite these differences in the (p-r) parameter, the stoichiometric balance results revealed that nitrogen loss exceeded nitrogen fixation in both periods, i.e., before and after the opening of the connection of the Piratininga Lagoon with the sea in The nitrogen balances (nfix-denit) obtained for and ranged from to and -321 to -457 for the Piratininga Lagoon, and from to and to for the Itaipu lagoon in both periods, respectively (Table 4). DISCUSSION Coastal lagoons are similar to lakes since they are naturally prone to eutrophication, but they are more dinamyc systems once they are also submitted to daily variations of water movement due to tides. Althoug the Piratininga-Itaipu Lagoon System is considered as a choking lagoon (Knoppers & Kjerfve, 1999), the water residence time differs between them. These characteristics were highlighted by the water and salt balance modeled with LOICZ (Fig. 2a). A higher water residence time was observed in Piratininga lagoon, but it was most evident during the first period ( ). These considerable differences resulted from the low mixing and renewal of Piratininga waters due to the reduced capacity of the Camboata Channel (channel that connects the two lagoons) in transporting water. Couto et al. (2000) reported a similar pattern

32 235 Latin American Journal of Aquatic Research for Piratininga and Itaipu lagoons (Piratininga = 46 days and Itaipu = 6 days) during 1980 s, with a lower water residence time for Piratininga. Carneiro et al. (1990) estimated the water residence time for the two lagoons. For Piratininga the water residence time was 46 days in summer and 995 days in winter. For Itaipu, the water residence time was estimated to vary from 14 days in summer to 289 days in winter. This variation is attributed to the worsening of the low circulation of the system and mainly to the energy of tidal waves (ranging between 0.8 and 1.0 m) that regulates the circulation and dynamics of the system. The opening of the connection between the Piratininga Lagoon and the sea in 2008 had a significant effect on water renewal of this lagoon. As expected, the great differences in the salt and water balances were observed in Piratininga, with a decrease of 31% in the water residence time in this lagoon (39 days). However, this intervention affected both Piratininga and Itaipu lagoons by increasing salinity in the whole system and improving the hydrodynamics (water and salt balances, as observed in ). Therefore, the Piratininga-Itaipu Lagoon System changed from choking to restricted (Carneiro et al., 1990). The water residence time is a crucial factor associated to biological productivity in aquatic systems. Environments with high water residence time tend to produce more nutrients, while systems with higher water renewal tend to be conservative in relation to the variation of nutrients along a salinity gradient (Grelowski et al., 2000). The highest DIP fluxes ( DIP) observed in the period before the opening of the channel ( ) resulted from the high phosphate concentrations on waters in this period. Fluxes were positive between all compartments: Piratininga Lagoon Itaipu Lagoon and Itaipu Lagoon Itaipu Inlet (the adjacent coastal area). The system exported phosphorous and the water balance was positive. A similar pattern was observed for Paranaguá Bay estuary (Marone et al., 2005), Mhathuze and Mvoti estuary (Wepener, 2007) and for Piratininga-Itaipu Lagoon System in 1980 s (Couto et al., 2000). After the opening of the channel ( ), the water residence time and DIP concentrations decreased in Piratininga and DIP became negative. Therefore, the outputs became smaller than the inputs meaning that primary productivity prevailed over mineralization and/or respiration processes making the system autotrophic (Talaue-McManus et al., 2003). In contrast, DIP values increased after 2008 in the Itaipu Lagoon, making the lagoon more heteretrofic than before, as also observed in other study carried out in the Piratininga-Itaipu System (Couto et al., 2000) and in other lagoons (Wattayakorn et al., 2001). The phosphorus has a complex dynamics in lagoon systems (Lillebo et al., 2004) and these results should be interpreted with caution since phosphorous concentrations are influenced both by its biological uptake and by physical adsorption-desorption processes in sediments. A DIN exportation was verified for both studied periods, like it was observed for other estuarine systems such as Mhathuze and Mvoti estuary (Wepener, 2007). The Piratininga-Itaipu Lagoon System has also shown a denitrification capacity (nitrogen loss) in comparison to N assimilation (fixation). This pattern was observed for the whole system during both periods ( and ) and for the two lagoons separately. Indeed, both systems presented a certain degree of anoxia (Couto et al., 2000) and denitrification was also observed for the upper Piauí Estuary (PI) during short periods of anoxia (Souza et al., 2003). In general, the majority of estuaries are considered denitrificators, and may cause a nitrogen loss from 10 to 20% to the atmosphere, and in extreme cases, up to 30% (Seitzinger, 1988; Knoppers & Kjerfve, 1999; Crossland et al., 2005; Capone et al., 2008). Of course other processes may also account for these losses of nitrogen, such as loss of ammonia to the atmosphere (Guimarães & Mello, 2006). The LOICZ model results, carried out for Brazilian estuarine areas, revealed that these ecosystems work in autotrophic conditions and act as nitrogen-fixing and denitrifying systems, like Guaratuva Bay, Paranaguá Bay, Conceição Lagoon and Maricá Lagoon (Couto et al., 2000; Souza et al., 2003; Marone et al., 2005; Brandini, 2008). Some exceptions were the Piratininga-Itaipu Lagoon System during (our data) and the upstream sector of Piaui River Estuary, with NEM heterotrophic denitrifying system (Souza et al., 2003) (Table 5). All systems have a different behavior compared to net ecosystem metabolism, depending on the different characteristics of each system. NEM Piratininga Lagoon behavior is similar to that presented by Paranaguá Bay in dry periods. On the other hand, Itaipu Lagoon behavior is similar to Guarapina Lagoon. Although the values of (p-r) in the Piratininga-Itaipu System are relatively low and, comparable to other systems, the Itaipu Lagoon increases its capacity of exporting DIP to adjacent coastal areas by increasing the exchange of this element. As results of the opening of the connection between Piratininga Lagoon and coastal waters, a decrease on the water residence time in the lagoon and an increase of the DIP loading to the Itaipu Lagoon were observed. Furthermore, the system in both

33 Nutrient budgets in the Itaipu-Piratininga lagoon system 236 Table 5. Balance of NIP and DIN in some Brazilian coastal ecosystems. τ: Residence time; DIPobs and DINobs: observed; 1*: (N:P) assuming particulate detritus from plankton to be 106; 2*= (N:P) assuming particulate detritus from plankton to be 16. Estuary Periods Section t DDIPobs (10 3 molp yr -1 ) (p-r) 1 * (mmol cm 2 yr -1 ) Process NEM DDINobs (10 3 moln yr -1 ) nfix-denit 2 * (molnm 2 yr -1 ) Process NEM Baia de Guaratuva 1 annual Total autotrophic fixation Baia de Paranaguá 2 Dry Total autotrophic to denitrication Rainy Total to heterotrophic to fixation Estuary Piaui River 3 annual total heterotrophic fixation Lagoa Conceicao 4 June 1982 North autotrophic denitrication South autotrophic denitrication Central autotrophic denitrication Maricá and Guarpina 5 Maricá autotrophic fixation Guarapina heterotrophic denitrication Piratininga Lagoon 6 annual ( ) Total heterotrophic denitrication Itaipu Lagoon 6 annual ( ) Total heterotrophic denitrication Piratininga Lagoon 6 annual ( ) Total autotrophic denitrication Itaipu Lagoon 6 annual ( ) Total heterotrophic denitrication Source: 1 Marone et al. (2005) 2 Brandini (2008) 3 Souza et al. (2009) 4 Souza & Knoppers (2002) 6 This study

34 237 Latin American Journal of Aquatic Research periods always showed an exportation of N, where denitrification is higher than N fixation (nfix-denit "negative") in contrast to the lower values observed in the Guarapina Lagoon System (Table 5). Lacerda et al. (1992) estimated for Piratininga that anoxia extends throughout the water column and reaches very low Eh values (-320 to +130 mv), whereas in Itaipu, anoxia is restricted to bottom waters (-66 to + 20 mv). This scenario is undergoing changes due to the positive effects generated by the construction of the channel that connects the system and coastal waters, decreasing the values of denitrification in Piratininga. Otherwise, as negative effects, an increasing on the denitrification values in Itaipu was observed. One should bear in mind, however, that LOICZ model has limitations that may be important, especially when applied for shallow coastal ecosystems such as the Piratininga-Itaipu Lagoon System. These environments are affected by the high variability of physical phenomena such as change in weather conditions related to the entry of cold fronts (with frequency of 7-8 days) which end ups adding variability to the tidal waves force, directly influencing the sedimentary dynamics of the system. CONCLUSIONS Based on the water and salt balances and dissolved nutrients (DIP-DIN), this study shows that, the engineering work for the Piratininga and Itaipu lagoons (the construction of the channel) has direct effects on the system, resulting in lower water residence times, specifically for Piratininga Lagoon, increasing and enhancing the system hydrodynamics. Associated to the lower water residence time, the whole system works like a producer of organic mater (autotrophic). Separately, Piratininga and Itaipu lagoons showed different patterns: Piratininga was autotrophic and Itaipu was heterotrophic, exporting material to the adjacent Itaipu Inlet System. The Piratininga-Itaipu System is a denitrifying system, despite the fact that the presence of benthic algae on the smaller mud flats could also account for nitrogen fixation. In general, despite of all mitigation measures and changes in the system, there are still sporadic dystrophic episodes and fish mortality induced by nutrient inputs, showing that measures are not yet sufficient. ACKNOWLEDGEMENTS The authors thank to the scholarship granted by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), to Laboratory of Biogeochemistry of Aquatic Environments (Marine Biology Department, UFF). We also thank Dr. Cassiano Monteiro- Neto for comments and manuscript review, and Dr. Marcelo Correa Bernardes for assistance in the field work. REFERENCES Abreu, P.C., C. Hartmann & C. Odebrecht Nutrient-rich saltwater and its influence on the phytoplankton of the patos lagoon estuary, southern Brazil. Estuar. Coast. Shelf Sci., 40: Andrews, J.E., A.M. Greenaway & P.F. Dennis Combined carbon isotope and C/N ratios as indicators of source and fate of organic matter in a poorly flushed, tropical estuary: HuntsBay, Kingston Harbour, Jamaica. Estuar. Coast. Shelf Sci., 46: Barroso, L., R.S. Medina, P. Moreira-Turcq & M. Bernardes A pesca nas lagoas costeiras fluminenses. IBAMA, Brasília, 50 pp. Bianchi, T.S Biogeochemistry of estuaries. Oxford University Press, New York, 720 pp. Bormann, F.H. & G.E. Likens Nutrient cycling. Science, 155: Brandini, N Biogeoquímica da Baía de Guaratuba, Paraná, Brasil: origem, metabolismo, balanço de massa e destino da matéria biogênica. Pós- Graduação em Geociências. Universidade Federal Fluminense, Niteroi, 274 pp. Capone, D.G., D.A. Bronk, M.R. Mulholland & E.J. Carpenter Nitrogen in the marine environment. Elsevier, Amsterdam, 1705 pp. Carneiro, M.E.R., N.M. Ramalho, L.S. Valentim, C. Azevedo & B. Knoppers Distribuição e comportamento dos nutrientes na bacia de drenagem do sistema lagunar de Piratininga-Itaipu, Niterói, RJ., Simpósio de Ecossistemas da Costa Sul e Sudeste Brasileira: estrutura, função e manejo aguas de Lindóia, pp Couto, C.G., A.C. Zyngier & M.F. Landim-de-Souza Piratininga-Itaipú coastal lagoons, Rio de Janeiro State-and studies estuarine system of the South American regions carbon nitrogen and phosphorus fluxes. LOICZ Reports & Studies Nº5. Institute for Sea Research, Texel, 87 pp. Crossland, C.J., H.H. Kremer, H.J. Lindeboom, J.I. Crossland-Marshall & M.D.A. Le Tissier Coastal fluxes in the Anthropocene: the land-ocean interactions in the coastal zone project of the International Geosphere-Biosphere Programme. Springer-Verlag, Berlin Heidelberg, 231 pp. Gordon, D.C., P.R. Boudreau, K.H. Mann, J.-E. Ong, W.L. Silvert, S.V. Smith, G. Wattayakorn, F. Wulff

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36 Lat. Am. J. Aquat. Res., 41(2): , 2013 Fish community at Ilha do Cardoso State Park, Brazil Proceedings of the 3 rd Brazilian Congress of Marine Biology A.C. Marques, L.V.C. Lotufo, P.C. Paiva, P.T.C. Chaves & S.N. Leitão (Guest Editors) DOI: /vol41-issue2-fulltext Research Article Spatio-temporal variation in surf zone fish communities at Ilha do Cardoso State Park, São Paulo, Brazil Jana Menegassi del Favero 1 & June Ferraz Dias 1 1 Instituto Oceanográfico, Universidade de São Paulo, Pça do Oceanográfico, 191 São Paulo, Brazil ABSTRACT. In order to analyze the time-space variation of the fish fauna in the surf zone fish communities at Ilha do Cardoso State Park, São Paulo, Brazil, four consecutive hauls were done over a year on three beaches with different degrees of exposure, at low and high tide. To evaluate the influence of each abiotic variable over the fish community, a Canonical Correspondence Analysis was conducted. We identified 7,286 individuals belonging to 20 families and 47 species, most specimens collected were juveniles. At low tide, the highest diversity and richness values were calculated while the highest dominance was obtained at high tide. As for the number of species collected at the three beaches, stood out for the lower values the cooler months, between June and September. Abiotic variables explained 41.3% of the variability of biological data, where 11.4% corresponds to the spatial variation. Meanwhile the temporal variables accounted for 31.9% of the variation in abundance, where 26.3% of the variance explained nycthemeral variation. Additionally two groups were clearly observed between months with low and high temperature. However in this variable, the tidal variation, excluding the seasonal effect, explained 6.2%, while seasonality, excluding tide effect, explained 26.3%. Although the main measurable seasonal changes were related to temperature, water temperature showed a low percentage of explanation in the fish fauna variability (2.7%). Finally, it is emphasized that the seasonal changes in surf zone fish community primarily reflect patterns of recruitment determined by the reproductive activity and coastal circulation. Keywords: ichthyofauna, surf zone, seasonality, environmental variability, southeastern Brazil. Variación espacio temporal de la ictiofauna del Parque Estatal Ilha do Cardoso, São Paulo, Brasil RESUMEN. Para analizar la variación espacio-temporal de la ictiofauna en las playas del Parque Estatal Ilha do Cardoso São Paulo-Brasil; se realizaron, durante un año, cuatro muestreos en tres playas con diferentes grados de exposición. Para evaluar la influencia de cada variable abiótica sobre la comunidad íctica, se efectuó un Análisis de Correspondencia Canonica. Se identificaron individuos pertenecientes a 20 familias y 47 especies, la mayoría de especímenes colectados fueron juveniles. Durante la marea baja se encontraron los mayores valores de diversidad y riqueza, y durante la marea alta los valores altos fueron los de dominancia. En cuanto al número de especies colectadas en las tres playas, se destacaron por presentar los menores valores los meses más fríos, entre junio y septiembre. Las variables abióticas evaluadas explicaron el 41,3% de la variabilidad de los datos biológicos, donde el 11,4% correspondió a la variación espacial. Por su parte las variables temporales explicaron 31,9% de la variación de la abundancia, donde 26,3% explicó la variación nictimeral. Adicionalmente, se observaron claramente dos grupos entre los meses con temperatura baja y alta. Sin embargo, la temperatura, a pesar de ser significativa, registró un bajo porcentaje de influencia en la variación de la ictiofauna (2,7%). Finalmente, cabe resaltar que los cambios estacionales en la comunidad de peces fueron causados principalmente por los patrones de reclutamiento determinados por las actividades reproductivas y de circulación costera. Palabras clave: ictiofauna, zona de surf, estacionalidad, variabilidad ambiental, sudeste de Brasil. Corresponding author: Jana M. del Favero (janamdf@usp.br)

37 240 Latin American Journal of Aquatic Research INTRODUCTION Beach surf zones are considered feeding and growth areas for a large number of fish species at juvenile and larval stage due to the turbidity, turbulence and shallowness that characterized this habitat. These same characteristics also inhibit use by large-sized fish, thus offering the young fish protection against predators (Lasiak, 1981; McLachlan et al., 1981; Gaelzer & Zalmon, 2003). Although the primary production in situ is not high, the tidal effect distributes the nutrients and minerals through the surf zone community (Carter, 1988), favoring the phyto and zooplankton bloom in the surf zone (Spring & Woodbum, 1960; Ferreira et al., 2010). This can be used as food resources for many fish species. Beaches adjacent to estuaries also serve as migration routes for various fish at larval or juvenile stage, that spend one or more stages of their life cycle within estuaries (Cowley et al., 2001; Watt-Pringle & Strydom, 2003). Various environmental factors influence the surf zone fish community structure. Low diversity and high dominance of a few fish species are explained mainly by the extreme beach hydrodynamics (Clark et al., 1996a, 1997). Most fish species present in such environments are classified as non-resident, and occur in the surf zone only at certain times of the year (Brown & McLachlan, 1990; Félix et al., 2007a), or stages of their life cycle (Modde, 1980; Layman, 2000). Several fish communities have been described mainly based on the spatio-temporal variations, indicating some patterns: as the level of the beach exposure increases it is observed an increase in dominance and decrease in the fish abundance and richness (Romer, 1990; Teixeira & Almeida, 1998; Félix et al., 2007b; Vasconcellos et al., 2007); and a greater fish diversity and richness during the warmer months (Bennett, 1989; Gianinni & Paiva-Filho, 1995; Clark, 1996b; Godefroid et al., 2003; Araújo et al., 2008; Lima & Vieira, 2009). Studies in Brazilian beaches have mainly examined the structure and spatio-temporal variation on fish communities (Paiva-Filho & Toscano, 1987; Giannini & Paiva-Filho, 1995; Saul & Cunningham, 1995; Gaelzer & Zalmon, 2003; Gomes et al., 2003; Araújo et al., 2008; Oliveira-Silva et al., 2008; Lima & Vieira, 2009), the day and night fish composition and structure variability (Pessanha & Araújo, 2003; Gaelzer & Zalmon, 2008a), the beach dynamics and morphology influence upon fish communities (Félix et al., 2007a; Vasconcellos et al., 2007), the tidal influence (Godefroid et al., 1998; Gaelzer & Zalmon, 2008b; Félix et al., 2010), and the trophic aspects (Stefanoni, 2008). There are few studies about surf zone fish communities in the São Paulo State coast (Paiva- Filho & Toscano, 1987; Giannini & Paiva-Filho, 1995; Saul & Cunningham, 1995), and none in the State Park. Thus, the objective of this study is to analyze the spatio-temporal variation in abundance and structure of fish communities at Ilha do Cardoso State Park, São Paulo State, Brazil. MATERIALS AND METHODS Sampling methods The beaches studied are located in the Ilha do Cardoso State Park, south of São Paulo coast, Brazil (Fig. 1). They were named as Sheltered, Moderate and Exposed, according to their exposure level. The beach exposure was classified based mainly on their geographical location. Although no studies were found that characterize the morphology of these beaches, it could be observed, during the surveys, that the more into the channel, smaller wave heights and more silty sediments. The slope of the beaches can be used as an index of exposure level, and was calculated from a transect perpendicular to the shore down to 5 m isobath, using the nautical chart number 175. Fishes were sampled monthly over one year, from February 2009 to January On each beach and tide, four consecutive hauls, of approximately 30 m each, were made using a beach seine net, 9 m long and 1.5 m height, with a stretched mesh size of 5 mm, totaling 24 fish samples per month. All samples were collected at low and high spring tide. Low tide was sampled at its morning peak while the high tide was usually sampled at the beginning of the afternoon, at a time close to its peak. At the start of the first haul and at the end of the last one, on each beach, the water temperature was measured with a mercury thermometer and the water salinity with a refractometer. All fish collected were identified following Figueiredo & Menezes (1978, 1980, 2000); Menezes & Figueiredo (1980, 1985) and Richards (2006). Due to the difficulty in identifying juvenile Mugilidae and the lack of adequate bibliography for the specific distinction within this family in Brazilian southeast coast, all mugilids collected were separated based on Vieira (1991). The following nomenclatures were used: Mugil hospes (previously Mugil gaimardianus), Mugil liza (previously Mugil platanus) according to Menezes et al. (2010), Mugil 1 for mugilids that were identified by their anal fin having 13 elements (two

38 Fish community at Ilha do Cardoso State Park, Brazil 241 Figure 1. Location of the three sample beaches (S: Sheltered, M: Moderate, E: Exposed) at Ilha do Cardoso State Park, southeast Brazil. spines and 11 rays) and Mugil 2 with two spines and eight rays on the anal fin. These fish were then measured to the nearest 1 mm (standard length) and weighted (g), except when samples were too large. In these occasions, measurements were restricted to a subsample of 50 individuals per species, done at random. The excess was weighted, counted and incorporated as weight and number counts. In addition, sex (male, female or nonidentified) and maturity stages were documented for the subsample through direct observation, according to Vazzoler (1996) and Dias et al. (1998). Juvenile fish were separated from larvae by the presence of scales. Data analysis Analysis of variance (one way ANOVA) was used to test the significance differences between the abiotic data of water temperature and salinity, when calculated monthly, per beach and per tide. Tukey post-hoc tests were conducted to evaluate betweenmean differences. The analysis of non-metric multidimensional scaling (nmds) was used to identify possible patterns among samples, in terms of water temperature and salinity. Groupings found were tested by the analysis of similarity (ANOSIM). The abiotic data was transformed by log (x+1), and Euclidean distance was used. Fish numerical abundance was used to calculate ecological indexes: dominance, Shannon diversity, Margalef richness, and evenness according to Begon et al. (2006). Only the occurrence constancy (C) was calculated according to Dajoz (1983), who classifies the species as: constant C 50, accessory 50< C >25 and accidental C 25. The differences among indexes were tested using the Bootstrap method, with 95% confidence. The influence of each abiotic variable upon the fish community was assessed by Canonic Correspondence Analysis (CCA) (Legendre & Legendre, 1998), and the distribution of the species in relation to the significant abiotic variables was determined by CANOCO. Rare species, those with less than 0.2% relative abundance, were eliminated from the biotic matrix that contained the species numerical abundance in each sample. The abiotic data, after a first analysis using a single matrix, were divided into three matrices: environmental (salinity and temperature), temporal (high tide, low tide and months) and spatio (sheltered, moderate and exposed beach). In all

39 242 Latin American Journal of Aquatic Research analyses the biological data were transformed by log (x+1), and low weight was given to rare species. The percentage of explanation of each abiotic variable, their interaction and the non-explainable, was calculated according to Borcard et al. (1992). RESULTS Environmental data Water temperature varied over the sampling period following a seasonal pattern. Lowest values occurred between May-October and highest from November to April (Fig. 2a). Water temperature did not change among beaches (Fig. 3a), and among different tides (Fig. 4a). The maximum temperature was 30 C in April and the minimum was 18 C in August. There was no significant difference among the water salinity over the months (Fig. 2b). The highest water salinity was obtained on the Exposed beach (maximum 36) followed by the moderate and then by the sheltered one (minimum 10) (Fig. 3b). Highest water salinity was measured at high tide (Fig. 4b). The nmds analysis enabled the visualization of two abiotic sample groups, being the cooler months correlated positively with the second axis, while those with greater salinity correlated negatively with the first axis (Fig. 5). The ANOSIM routine separated the two groups (R = 0.63, P < ). The beach slope was higher at sheltered (8.2%), than at moderate (5.4%), and exposed (1.9%). Species composition A total of 7,286 fish from 20 families and 47 species was collected. The families Mugilidae (37.0%), Carangidae (23.0%), Gerreidae (15.1%), Atherinopsidae (9.3%), Engraulidae (8.3%), Sciaenidae (3.6 %) and Clupeidae (2.3%) contributed with 98.6% of total catch. Mugil curema (17.6%), Gerreidae larvae (16.7%), Mugil hospes (15.7%), Trachinotus carolinus (15.5%), Atherinella brasiliensis (9.2%) and Anchoa tricolor (4.6%) were the six most representative species. A predominance of accidental species occurrence was observed, only T. carolinus and Trachinotus goodei were classified as constant, and A. brasiliensis, Oligoplites saliens, M. hospes, Mugil liza, M. curema, Menticirrhus littoralis, Engraulidae and Gerreidae larvae were considered as accessory (Table 1). Total catch weight was 14, g. Atherinopsidae (35.5%) and Carangidae (32.2%) equaled 67.6% of biomass. A. brasilensis (35.3%), T. goodei (11.6%), O. saliens (9.5%) and T. carolinus (9.1%) were the species with greatest abundance in terms of Figure 2. a) Monthly variation of water temperature, and b) water salinity, recorded in the surf zone of Ilha do Cardoso State Park, from February 2009 to January 2010 (Horizontal line: median; box: standard deviation; I: standard error). biomass. Strongylura timucu, Paralichthys orbignyanus and Sphoeroides testudineus represented the species with greatest standard length. The smaller individuals belonged to T. carolinus, Oligoplites sp., M. littoralis and Gerreidae larvae. Among the specimens with greater range in standard length, more than 100 mm, were S. timucu, T. goodei, M. littoralis, O. saliens and A. brasiliensis. 78% of the specimens measured between 4 and 60 mm, and the standard length modal class predominance was 21 to 40 mm, totaling 34%. In most individuals, the sex could not be identified due to their small standard length,or the gonads were not located or were very small, disabling macroscopic classification. These individuals totaled 88.2% of the total sampled, with only 7.8% and 4.0% of females and males, respectively. Among females, there was little presence of mature and spent gonads (5.5% and 1.3% respectively). No hydrated gonad was observed. The immature (39.7%) and maturing (53.4%) stages were the most abundant. Individuals that could not be classified due to their small standard length were considered as immature and/or juvenile.

40 Fish community at Ilha do Cardoso State Park, Brazil 243 Figure 3. a) Water temperature, and b) water salinity recorded in the surf zone of three different beaches in Ilha do Cardoso State Park (Horizontal line: median; box: standard deviation; I: standard error). Figure 4. a) Water temperature, and b) water salinity recorded in the surf zone during High and Low tide (Horizontal line: median; box: standard deviation; I: standard error). Atherinella brasiliensis was the only species represented by adult (N = 176), juvenile (N = 497) and larvae stage (N = 1). S. testudineus and S. timucu were represented byadults (N=2) andjuveniles (N=9). P. orbignyanus was represented by a single adult individual. M. littoralis, M. americanus and the Engraulidae were sampled in their juvenile and larvae stages, while Porichthys porosissimus, Elops saurus, Micropogonias furnieri and all the Gerreidae were collected only in larval stage. Spatio-temporal variation Although the greater quantity of specimens was collected on the Exposed beach, it presented the least richness among the beaches (Table 2). There was no significant difference in richness values between the Sheltered and Moderate beaches, but the latter presented the smallest number of individuals obtained. The Moderate beach was more diverse than the Sheltered followed by the Exposed one, which in turn showed the highest dominant values (Table 2). The dominant species at Exposed beach were M. hospes and T. carolinus. The Gerreidae larvae and A. brasiliensis were abundant at the Sheltered beach, while M. curema and T. carolinus were abundant at the Moderate beach. Highest diversity and richness values were obtained at low tide, in addition to a greater quantity of individuals. There was no significant difference in the evenness value, and at high tide dominance was greater than at low tide (Table 2). The Gerreidae larvae and T. carolinus dominated the sampling during high tide. The greatest quantity of individuals was collected in months with higher water temperatures. The Margalef richness, Shannon diversity and Pielou s evenness indexes varied similarly, with October being the month with the highest values of the three indexes, in spite of the small quantity of specimens collected at that time. After October, March and January presented high diversity and richness values, while the evenness was followed by May and March. The least rich and diverse months were August, September and November, and the latter had, in addition, the least evenness. November and September showed high dominance due to the large capture of gerreids and A. brasiliensis, respectively (Fig. 6).

41 244 Latin American Journal of Aquatic Research Figure 5. Non-metric multidimensional scaling (MDS) of abiotic samples using the water temperature and water salinity values as attributes. Labels = month + beach (S: Sheltered, M: Moderate, E: Exposed) + tide (H: high, L: Low). T. carolinus, T. goodei and A. brasiliensis were collected during the entire year. O. saliens only did not occur in August, and M. littoralis in February. The Gerreidae larvae and M. hospes did not occur during low water temperatures months. Nineteen and nine species occurred, respectively, in only one or two months during the year (Table 1). The abiotic variables explained 41.3% of the variability on biological data, being only water salinity not significant (P < 0.05), by Monte Carlo permutation test. The first two axes were responsible for 54.5% of the biological data variation. The axis 1 was positively correlated with Exposed beach and high tide, and negatively correlated with the sheltered beach. The axis 2 was negatively correlated with water temperature. The species-environment correlation presented high values with the first (0.84) and second (0.82) axis (Table 3). The canonic axes were significantly different by the Monte Carlo permutation test (F = 3.16; P = ). Figure 7 represents the distribution of the species in relation to the significant abiotic variables. Water temperature explained 13.93% of biological data variation (sum of all canonic eigenvalues = 0.321). Excluding water temperature influence from the spatio-temporal interaction, the explanation percentage decreased to 2.70%. Gerreidae larvae, T. carolinus and M. hospes showed preference for warmer waters, while M. liza and A. brasiliensis preferred cooler waters (Fig. 7). The different beach types explained 11.41% of biological data variation (sum of all canonic eigenvalues = 0.263). Removing the interaction with temporal variables, the percentage of explanation decreased approximately 1%. The following species were abundant on the most sheltered beach A. brasiliensis, A. tricolor, S. timucu, A. lepidentostole and Engraulidae larvae, while M. hospes, T. carolinus, T. goodei and M. littoralis were abundant on the most exposed one (Fig. 7). The temporal variables explained 31.9% of biological data variation (sum of all canonic eigenvalues = 0.734). Two influences were observed on this variable, one related to the different tide amplitudes and the other with the months sampled. Only tidal variation, excluding the seasonal effect, explained 6.1%, while seasonality, without the tidal effect, explained 26.3%. At high tide T. carolinus and M. littoralis were sampled in abundance, while at low tide A. tricolor, A. brasiliensis and S. timucu were abundant. DISCUSSION The fish community studied was characterized by the dominance of few species, a pattern described in several studies on surf zone fish communities (Brown & McLachlan, 1990; Godefroid et al., 2003; Pessanha & Araújo, 2003; Félix et al., 2007b; Stefanoni, 2008).

42 Fish community at Ilha do Cardoso State Park, Brazil 245 Table 1. Relative frequency (%), total contribution and occurrence constancy (C) and standard length (SL) of fish sampled in the surf zone of Ilha do Cardoso State Park. *larvae, **larvae and juveniles, *** larvae, juveniles and adults. Family/Species Elopidae Relative frequency (%) SL (mm) Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Total C Min Max Elops saurus* Engraulidae Anchoa januaria Anchoa lyolepis Anchoa tricolor Anchoviella lepidentostole Cetengraulis edentulus Non identified larvae Lycengraulis grossidens < Clupeidae Harengula clupeola Opisthonema oglinum < Platanichthys platana <0.1 < Sardinella brasiliensis Synodontidae Synodus foetens 0.7 < Belonidae Strongylura marina <0.1 < Strongylura timucu Batrachoididae Porichthys porosissimus* 0.4 <

43 246 Latin American Journal of Aquatic Research (continuation) Relative frequency (%) Family/Species SL (mm) Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Total C Min Max Mugilidae Mugil curema Mugil hospes Mugil liza Mugil Mugil < Atherinopsidae Atherinella brasiliensis*** Odontesthes argentinensis 0.2 < Hemiramphidae Hemiramphus spp. 0.2 < Carangidae Caranx latus Choloroscombrus chrysurus Oligoplites saliens Oligoplites spp. 0.3 < Selene vomer 0.6 < Trachinotus carolinus Trachinotus falcatus < Trachinotus goodei Syngnathidae Syngnathus folletti 0.1 < Lobotidae Lobotes surinamensis 0.2 < Gerreidae Non-identified larvae

44 Fish community at Ilha do Cardoso State Park, Brazil 247 (continuation) Relative frequency (%) Family/Specie SL (mm) Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Total C Min Max Haemulidae Pomadasys corvinaeformis 0.6 < Polynemidae Polydactylus oligodon 0.4 < Sciaenidae Menticirrhus americanus ** Menticirrhus littoralis** Micropogonias furnieri* 0.4 < Umbrina coroides 0.4 < Pomatomidae Pomatomus saltatrix <0.1 < Paralichthyidae Etropus crossotus Paralichthys orbignyanus 0.4 < Tetraodontidae Sphoeroides greeleyi Sphoeroides testudineus < Diodontidae Chilomycterus spp. 0.1 < Individuals number Species number Family number

45 248 Latin American Journal of Aquatic Research Table 2. Ecological indexes calculated using fish data collected at different beaches and tides. In indicated the significatives differences. Beach Tide Sheltered Moderate Exposed High Low Species number Individuals number Dominance Diversity Richness Equitability Figure 6. Monthly variation of ecological indexes calculated using the fish data collected in the surf zone of Ilha do Cardoso State Park from February 2009 to January Species of Trachinotus, Mugil, Atherinella and Anchoa genera were the most abundant. Several studies carried out in the Paranaguá Coastal System at Paraná coast, an ecosystem contiguous to the area of this study, indicated the importance of these genera in structuring the surf zone fish community, even when sampled at different beaches and years (Godefroid et al., 1998, 2003; Spach et al., 2004; Félix et al., 2006, 2007b, Stefanoni, 2008). These genera are also representative on south Brazilian beaches, except for Anchoa (Lima & Vieira, 2009). At Rio de Janeiro state coast, it was observed that the relative abundance of Mugilidae decreased (Gaelzer & Zalmon, 2003; Gomes et al., 2003; Pessanha & Araújo, 2003; Vasconcellos, 2007). In a beach in Espírito Santo state, mugilids were not observed and the most abundant species were Lutjanus synagris, Achosargus romboidalis, Eucinostomus lefroyi, Paralonchurus brasiliensis (Araújo et al., 2008). On beaches at Todos os Santos Bay, Bahia State, L. synaris, Larimus breviceps, Chaetodipterus faber, Polydactylus virginicus, Ophioscion punctatissimus and Conodon nobilis were dominant (Oliveira-Silva, 2008). In Pernambuco state, approximately 40% of the total species were represented by O. punctatissimus (Lira & Teixeira, 2008). Thus, it is possible to observe a greater difference in the species composition of the studied beaches than in the ones in Espírito Santo state along the northeast. It is known that the southeast and southern Brazilian coast have subtropical characteristics, and is commonly considered as transition area to a temperate fauna. (Floeter et al., 2006). This fact probably explains the difference of the surf zone ichthyofauna observed in the southeast and southern Brazilian coast compared to the northeast, which has tropical characteristics. Comparing the surf zone fish composition with the fish species collected in hauls at Cananéia Estuary

46 Fish community at Ilha do Cardoso State Park, Brazil 249 Table 3. Summary of the CCA performed on abundance of 22 most numerous fish species. Axes Correlation of environmental variables December January June November October Sheltered beach Moderate beach Exposed beach High tide Low tide Water temperature Summary statistics for ordination axes Eigenvalues Species-environment correlations Cumulative percentage variance: of species data of species-environment relation Sum of all eigenvalues Sum of all canonical eigenvalues Figure 7. Ordination diagram (biplot) from CCA including fish species and significant environmental variables (represented by vectors). Species coded by the first two letters of genus and the species scientific name (e.g. TRCA= Trachinotus carolinus).

47 250 Latin American Journal of Aquatic Research Complex and in beaches at Bom Abrigo Island, located on the platform adjacent to the estuary (Zani- Teixeira, 1983; Saul & Cunningham, 1995; Maciel, 2001), it is possible to notice that a low number of species were exclusive to the studied beaches, indicating connectivity of the surf zone fish community with other habitats, both inside and outside the Cananéia-Iguape Coastal System. The great abundance of sampled specimens at juvenile and larval stages corroborated the importance of the studied area for the initial ontogenetic stages. Godefroid et al. (2003), Félix et al. (2007a), Inoue et al. (2008) and Stefanoni (2008) also reported a high proportion of juvenile and/or larval individuals in surf zone fish communities. The beach exposure is considered one of the main surf zone fish community structuring factors (Romer, 1990; Clark, 1996b; Gaelzer & Zalmon, 2003; Vasconcellos et al., 2007). However, the beach exposure influence on fish community composition may be misunderstood, mainly due to the interconnection between this variable and others, such as macroalgae abundance and/or organic matter decomposing, water salinity and water transparency (Clark, 1997). In the present study, the beach exposure explained only 11.41% of biological data variation. In several studies there was an increase in the species richness and diversity as the beach shelter increased, while the most exposed beaches were dominated by few species (Romer, 1990; Gaelzer & Zalmon, 2003; Vasconcellos et al., 2007). This pattern was also observed in the present study, but, as in Stefanoni (2008), the study area may not have been appropriate to test this hypothesis because there were interactions with other variables related to the estuary presence. Beaches considered as Sheltered and Moderate in the present study were influenced by estuary waters, while the Exposed beach had marine waters influence. The greater species richness and fish diversity on sheltered beaches may be due to the greater food availability and accessibility compared to more exposed beaches. The turbulence generated by waves may reduce the food ingestion rate due to the continuous need to adjust the body position in the water column and a decrease of the visual field (Clark, 1997). T. goodei and M. littoralis were associated with the high energy environments where the greatest salinity was registered as a reflex of greater exposure. This was also observed by Félix et al. (2007a), Vasconcellos et al. (2007) and Stefanoni (2008). Water salinity can be a structuring factor in estuaries (Barletta et al., 2005), but at exposed beaches this factor does not satisfactorily explain the biological data variability. The species that are correlated with the more protected beaches were also associated with estuary regions (Zani-Teixeira, 1983; Maciel, 2001; Peres-Rios, 2001; Ramos & Vieira, 2001). The highest water salinity measured at high tide probably was due to the fact that, at low tide, the beaches suffered greater influence from inland waters. Temporal variations had the greatest influence on fish community composition and structuring by approximately 30%. Within the temporal variations, a small relevance was due to tidal variation, with seasonal variation the most important variable. Although the main seasonal alterations measured were related to water temperature, this variable alone showed a low percentage of explained biological data variation. Thus, it is emphasized that, as already reported by Ross et al. (1987) and Félix et al. (2007a), seasonal changes in the surf zone fish community are mainly due to recruitment patterns determined by reproductive activity and coastal circulation. As in the present study, several studies as Bennett (1989), Giannini & Paiva-Filho (1995), Clark (1996b), Godefroid et al. (2003); Félix et al. (2006) and Araújo et al. (2008), reported the highest diversities and abundance values during warmer months, coinciding with the reproductive period of many fish species. High water temperatures also favor phytoplankton and zooplankton bloom, increasing the food available for larvae and juvenile fish, and consequently, their chances of survival (Nybakken & Bertness, 2004). However, a problem concerning fish population studies on dynamic and fish reproductive aspects is the sampling frequency. Thus, relating warmer months (end of spring or summer) and capture of juvenile specimens in the same period is not very enlightening. The fish length and their growth rate should be investigated and related with the hatching period and not with the reproductive period. Studies on growth rates of the most abundant surf zone species were not available, and therefore might restrict interpretation of the results. ACKNOWLEDGEMENTS The authors would like to thank all the volunteers and employees involved in the fieldwork and fish identification. We also thank CNPq, which has conceded the author s postgraduate scholarship. REFERENCES Araújo, C.C.V., D.M. Rosa, J.M. Fernandes, L.V. Ripoli & W. Kroling Composição e estrutura da

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50 Fish community at Ilha do Cardoso State Park, Brazil 253 Teixeira, R.L. & G.I. Almeida Composição da ictiofauna de três praias arenosas de Maceió, AL- Brasil. Bol. Mus. Biol. Mello Leitão, 8: Vasconcellos, R.M., J.N. Santos, M.A. Silva & F.G. Araújo Efeito do grau de exposição às ondas sobre a comunidade de peixes juvenis em praias arenosas do Município do Rio de Janeiro, Brasil. Biota Neotrop., 7(1): Vazzoler, A.E.A.M Biologia da reprodução de peixes teleósteos: teoria e prática. Eduem, Maringá, 169 pp. Received: 16 May 2011; Accepted: 22 October 2012 Vieira, J.P Juvenile mullets (Pisces: Mugilidae) in the estuary of Lagoa dos Patos, RS, Brazil. Copeia, 2: Watt-Pringle, P. & N.A. Strydom Habitat use by larval fishes in a temperate South African surf zone. Estuar. Coast. Shelf Sci., 58: Zani-Teixeira, M.L Contribuição ao conhecimento da ictiofauna da Baía do Trapandé, Complexo Estuarino Lagunar de Cananéia, São Paulo. Dissertação Mestrado em Oceanografia Biológica, Instituto Oceanográfico, Universidade de São Paulo, São Paulo, 254 pp.

51 Lat. Am. J. Aquat. Res., 41(2): , 2013 Gross chemical profile Proceedings of the 3 rd Brazilian Congress of Marine Biology A.C. Marques, L.V.C. Lotufo, P.C. Paiva, P.T.C. Chaves & S.N. Leitão (Guest Editors) DOI: /vol41-issue2-fulltext Research Article Gross chemical profile and calculation of nitrogen-to-protein conversion factors for nine species of fishes from coastal waters of Brazil Graciela S. Diniz 1,3, Elisabete Barbarino 1, João Oiano-Neto 2,4 Sidney Pacheco 2 & Sergio O. Lourenço 1 1 Universidade Federal Fluminense, Departamento de Biologia Marinha P.O. Box , CEP , Niterói, RJ, Brazil 2 Embrapa Agroindústria de Alimentos, Laboratório de Cromatografia Líquida Avenida das Américas, 29501, CEP , Rio de Janeiro, RJ, Brazil 3 Universidade Federal do Rio de Janeiro, Instituto Virtual Internacional de Mudanças Globais UFRJ/IVIG Rua Pedro Calmon s/nº, CEP Cidade Universitária Rio de Janeiro, RJ, Brazil 4 Embrapa Pecuária Sudeste, Rodovia Washington Luiz, km 234 P.O. Box 339, CEP , São Carlos, SP, Brazil ABSTRACT. The amino acid composition and contents of nitrogen, phosphorus, lipid, carbohydrate and protein were determined in muscles of Dactylopterus volitans, Genypterus brasiliensis, Mullus argentinae, Paralichthys patagonicus, Percophis brasiliensis, Pinguipes brasilianus, Rhizoprionodon lalandii, Rhizoprionodon porosus, and Urophycis cirrata. The samples showed low carbohydrate content (<3.5% dry weight in all species) and were rich in protein (>66% dry weight in all species). The percentage of total lipid varied widely among species, and M. argentinae showed the highest concentrations (16%). The percentage of nitrogen and phosphorus was high and similar among species, with overall average values of 13.3% and 1.2%, respectively. The amino acids composition was similar among the animals, with glutamic acid and lisine as the most abundant amino acid and histidine in low concentrations. Among species, the content of proteinaceous nitrogen was high, with an average of 96.8% of the total nitrogen. From data of total amino acid and total nitrogen, specific nitrogen-to-protein conversion factors were calculated for each species. The nitrogen-toprotein conversion factors ranged from 5.39 to 5.98, with an overall average of These findings showed that the traditional conversion factor of 6.25 overestimates the actual protein content and should be avoided. Keywords: nitrogen-to-protein conversion factors, protein, amino acid, fish, tropical environment. Perfil químico bruto y cálculo de los factores de conversión de nitrógeno a proteína en nueve especies de peces de aguas costeras de Brasil RESUMEN. Se determinó la composición de aminoácidos y el contenido de nitrógeno, fósforo, lípidos, carbohidratos y proteínas en los músculos de Dactylopterus volitans, Genypterus brasiliensis, Mullus argentinae, Paralichthys patagonicus, Percophis brasiliensis, Pinguipes brasilianus, Rhizoprionodon lalandii, Rhizoprionodon porosus y Urophycis cirrata. Las muestras mostraron un contenido bajo en carbohidratos (<3,5% en peso seco para todas las especies) y alto en proteínas (>66% en peso seco para todas las especies). El porcentaje de lípidos totales varió ampliamente entre las especies, y M. argentinae presentó las mayores concentraciones (16% en peso seco). El porcentaje de nitrógeno y fósforo fue alto y similar entre las especies, con valores promedios de 13,3% y 1,2%, respectivamente. La composición de aminoácidos fue similar entre los animales, los aminoácidos más abundantes fueron ácido glutámico y lisina e histidina la de menor concentración. Entre las especies, el contenido de nitrógeno proteico fue alto, con un promedio de 96,8% de nitrógeno total. En consecuencia, los peces mostraron una concentración muy baja de nitrógeno no proteico. A partir de los datos de aminoácidos totales y nitrógeno total para cada especie, se calcularon factores de conversión de nitrógeno a proteína específicos para cada especie de pez. Los factores de conversión de nitrógeno a proteína variaron de 5,39 hasta 5,98, con un promedio general de 5,71. Estos hallazgos muestran

52 255 Latin American Journal of Aquatic Research que el factor de conversión tradicional de 6,25 sobreestima el contenido de proteína real de peces y debería ser evitado. Palabras clave: factores de conversión de nitrógeno a proteína, proteínas, aminoácidos, peces, ambiente tropical. Corresponding author: Sergio O. Lourenço (solourenco@id.uff.br) INTRODUCTION Studies on the chemical composition of marine organisms can offer important subsidies for the development of research in the fields of physiology, biochemistry, ecology, and conservation of the species, for example (Barbarino & Lourenço, 2009). These approaches are particularly uncommon in marine biology. Studies on the biochemical composition of wild fish populations are rarely undertaken, possibly because others are more traditional in fish studies, such as ecological research and fisheries. The knowledge on chemical composition of fish species has fundamental importance in the application of different technological processes. The chemical composition of the organisms, in general, can be influenced by many factors, such as physiological characteristics, habitat and life cycle, in addition to environmental characteristics. The chemical composition of heterotrophic organisms is also influenced by the food that they ingest, age and reproductive traits (Childress et al., 1990; Zaboukas et al., 2006). Fish is an important component in the diet, not only as a source of protein of high nutritional quality, but as a significant source of polyunsaturated fatty acids of the omega-3 series (ω-3). These substances are thought to be very beneficial to human health (Venugopal & Shahidi, 1996; Ramos Filho et al., 2008). Studies with focus on the chemical composition of fish are predominantly found in food science, and in general the results are available in fresh weight basis. According to Ogawa & Maia (1999), the moisture in muscles reaches typically 80% of the weight, but Ramos-Filho et al. (2008) recorded values for moisture from 60 to 80%. The use of fresh weight makes comparisons and interpretations on chemical composition among different studies more difficult. Particularly, protein data of marine fishes present many applications, involving both basic and applied research. However, comparisons of protein content among species are difficult because of methodological differences. The most common methods used for protein determination are Lowry et al. (1951), Bradford (1976) and the use of conversion factor of nitrogen-protein 6.25 (Jones, 1931). These two first methods are subject to interferences from many factors (Stoscheck, 1990), which are a consequence of the protein extraction and effects of some substances on specific amino acids. This happens bacause the chemical reactions which produce the protein quantification depends on the reactivity of the amino acid side groups (Legler et al., 1985). Although the problems with protein analyses have been known for many decades, they are very hard to avoid. In general, adaptations of the traditional techniques to specific species are necessary to run the analysis (Barbarino & Lourenço, 2005). The total nitrogen (TN) analysis is relatively simple and easy to perform, and nitrogen-to-protein conversion factors (N-Prot factors) can be used to estimate crude protein content. The use of N-Prot factors to determine protein content has some important advantages if compared to other methodologies. Total nitrogen analysis, carried out by Kjeldhal s method (AOAC, 1990), Hach techniques (Hach et al., 1987) or CHN analysis, eliminates the necessity of extracting the protein content of the sample to be analyzed. The use of specific N-Prot factors is widely recommended in order to get more accurate estimates of protein content (Sosulski & Imafidon, 1990). The nitrogen: protein ratio does vary according to the source considered (Mariotti et al., 2008). The use of N-Prot factors is particularly wide in food science, such as those calculated for certain cereals (e.g for rise, 5.47 for wheat; Fujihara et al., 2008), legumes (e.g for cassava root; Yeoh & Truong, 1996), mushrooms (4.70; Mattila et al., 2002), Cheddar cheese (6.38; Rouch et al., 2008) and fish and fish s products (4.94; Salo-Väänänen & Koivistoinen, 1996), among other products. The use of N-Prot factors is still uncommon in sea science, possibly because most of the scientific community ignores this methodological alternative. A few studies using N-Prot factors in sea science were performed with seaweeds (Aitken et al., 1991; Lourenço et al., 2002) and microalgae (Lourenço et al., 2004), which yielded specific N-Prot factors lower than 5.0. The use of the factor 6.25, in case of high concentration of non-protein nitrogen (NPN), tends to

53 Gross chemical profile 256 overestimate the protein data, since NPN and protein- N can not be distinguished in TN analysis. Despite this, several authors continue to use the factor 6.25 to estimate the content of fish protein (Polvi & Ackman, 1992; Maia et al., 1999; Undeland et al., 1999; Zaboukas et al., 2006; Ramos- Filho et al., 2008). Except for a short list of specific N-Prot factors available for marine organisms, the factor 6.25 calculated by Jones (1931) is still used for most plant and animal sources from the sea. Studies in this field are needed for fishes, since very limited information is available. The purpose of our study was to determine specific N-Prot factors for nine species of marine fishes from coastal areas, based on the ratio of amino acid composition to total nitrogen (TN) content. In addition, we also characterized and compared the fish species regarding carbohydrate, lipid, nitrogen and phosphorus contents. MATERIALS AND METHODS Fishes In this study nine fish species were analyzed. The identification of the species was carried with experts supervision. Dactylopterus volitans (Linnaeus, Dactylopteridae; common name: flying gurnard ), Genypterus brasiliensis (Regan, Ophidiidae; cusk eels ), Mullus argentinae (Hubbs & Marini, Mulidae; Argentine goatfish ), Paralichthys patagonicus (Jordan, Paralichthydae; Patagonia flounder ), Percophis brasiliensis (Quay & Gaimard, Percophidae; Brazilian flathead ), Pinguipes brasilianus (Cuvier, Pinguipedidae; Brazilian sandperch ), Rhizoprionodon lalandii (Muller & Henle, Carcharhinidae; Brazilian sharpnose shark ), Rhizoprionodon porosus (Poey, 1861 Carcharhinidae; Caribbean sharpnose shark ), and Urophycis cirrata (Goode & Bean, Phycidae; gulf hake ). These species were selected given their ecological importance and abundance in the field. Sampling For each analysis, the samples were obtained from different individuals, randomly selected. Six to ten individuals were sampled, but only four of them (n = 4), were chemically analyzed for carbohydrate, protein, lipid, nitrogen, and phosphorus. Due the high cost of the amino acid analysis using the method employed here, only three replicates were analyzed for each fish species (n = 3), a widely accepted procedure. D. volitans, R. lalandii, and R. porosus were collected in May 2004 at Niterói (Itaipu Beach, S, W). M. argentinae, P. patagonicus, P. brasiliensis, P. brasilianus, U. cirrata, and G. brasiliensis were sampled in June 2007 at Arraial do Cabo (22 57 S, W). Both sites are located in Rio de Janeiro State, southeastern Brazil (Fig. 1). Not-sexed adult fishes (with similar body sizes) were collected with bottom trawls. The animals were packed in plastic bags and kept on ice until returned to the laboratory. In the laboratory, samples were cut and the muscles were frozen at -18 C. Subsequently, the samples were freeze dried in a Terroni Fauvel, model LB1500TT device. The dried material was powdered manually using a mortar and pestle, and it was kept in desiccators containing silica-gel, under vacuum and at room temperature, until the chemical analyses were carried out. Tissue analysis Total carbohydrate was extracted with 80% H 2 SO 4, according to Myklestad & Haug (1972). The carbohydrate concentration was determined spectrophotometrically at 485 nm, 30 min after the start of the chemical reaction, by the phenol-sulfuric acid method (Dubois et al., 1956), using glucose as a standard. Total lipids was extracted according to Folch et al. (1957), and determined gravimetrically after solvent (chloroform) evaporation. Total nitrogen and phosphorus were determined in muscle after peroxymonosulphuric acid digestion, using a Hach digestor (Digesdhal, Hach Co.) (Hach et al., 1987). Samples were digested with concentrated sulfuric acid at 440ºC and treated with 30% hydrogen peroxide. Total nitrogen and phosphorus contents in the samples were determined spectrophotometrically after specific chemical reactions. See Lourenço et al. (2005) for analytical details. Total amino acid was determined by high performance liquid chromatography with pre-column derivatization with AccQ.Fluor reagent (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate), reverse phase column C 18 AccQ.Tag Nova-Pak (150x3.9 mm; 4 µm), ternary mobile phase in gradient elution composed by sodium acetate 140 mm + TEA 17 mm ph 5.05 (solvent A), acetonitrile (solvent B) and water (solvent C), flow 1 ml min -1 (Cohen & De Antonis, 1994). A Waters, model Alliance 2695 chromatograph was used, equipped with a fluorescence detector Waters 2475 (µ ex. 250 nm, µ em. 395 nm). Analytical conditions were suitable to determine all amino acids, except tryptophan, cysteine + cistine and methionine. The percent of nitrogen in each amino acid was used to calculate nitrogen recovered from total amino acid analysis. Aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, isoleucine, leucine,

54 257 Latin American Journal of Aquatic Research Figure 1. Map showing the sampling sites in Rio de Janeiro State, Brazil. 1: Arraial do Cabo, 2: Niterói. tyrosine, phenylalanine, histidine, lysine, and arginine contents were multiplied by 0.106, 0.118, 0.134, 0.096, 0.123, 0.188, 0.158, 0.120, 0.108, 0.108, 0.078, 0.085, 0.271, 0.193, and 0.322, respectively (Diniz et al., 2011). Calculation of N-Prot factors N-Prot factors were determined for each species by the ratio of amino acid residues (AA-Res) to total nitrogen (TN) of the sample: N-Prot factor = AA-Res / TN. Thus, for a 100 g (dry weight) sample having g of amino acid residues and 3.48 g of TN, a N-Prot factor of 4.66 is calculated. The amino acid residues of the samples was calculated by summing up the amino acid masses retrieved after acid hydrolysis (total amino acids), minus the water mass (18 H 2 O mol -1 of amino acid) incorporated into each amino acid after the disruption of the peptide bonds (Mossé, 1990). Statistical analysis The results were analyzed by one-way analysis of variance (One Way ANOVA) with significance level α = 0.05 (Zar, 1996), followed, where applicable, with a Tukey s multiple comparison test. The raw data were tested for normality and homoscedasticity and no transformation was needed. RESULTS Carbohydrate was the less abundant organic substance measured in all species, ranging from 1.03% (M. argentinae) to 3.45% (D. volitans) of the dry weight The value of D. volitans was significantly higher than the other species (P < 0.001). The percentage of total lipid showed wide variations among species, ranging from 4.40% (R. porosus) to 16.1% (D. volitans). The lowest lipid concentrations were recorded in elasmobranch fishes (R. porosus and R. lalandii). M. argentinae showed significantly higher lipid concentration than other species (P < 0.001) (Table 1). The fishes showed high values of total nitrogen, ranging from 11.6% (M. argetinae) to 14.9% (R. porosus) of the dry weight. Elasmobranch fishes showed the highest concentrations of total nitrogen in the muscles (Table 1). The concentrations of phosphorus also varied widely among species. P. brasiliensis showed the highest value (1.45%, P < 0.001) and D. volitans showed the lowest concentrations (0.94%, P < 0.001). Following the trends described for nitrogen concentration, the muscles showed high values of hydrosoluble protein. The values ranging from 45.7% (R. porosus) to 56% (G. brasiliensis). The higher concentrations were recorded in G. brasiliensis and

55 Gross chemical profile 258 Table 1. Gross chemical composition of nine species of fishes sampled in tropical sites of Brazil. Values are expressed as percentage of the dry mass and represent the mean of four replicates ± standard deviation (n = 4) #. Species Total nitrogen Total phosphorus Total carbohydrate Total lipid Hydrosoluble protein *** *** *** *** *** Dactylopterus volitans 11.7 ± 0.23 b 0.94 ± 0.05 d 3.45 ± 0.25 a 7.81 ± 0.84 b 47.1 ± 1.32 b Genypterus brasiliensis 14.0 ± 0.67 a 1.29 ± 0.08 ab 1.11 ± 0.03 de 8.28 ± 0.72 b 56.0 ± 1.79 a Mullus argentinae 11.6 ± 0.72 b 1.12 ± 0.08 c 1.03 ± 0.10 e 16.1 ± 3.68 a 47.7 ± 2.24 b Paralichthys patagonicus 13.4 ± 0.33 a 1.21 ± 0.06 bc 1.38 ± 0.09 bc 5.62 ± 0.55 bc 53.9 ± 0.75 a Percophis brasiliensis 13.7 ± 0.08 a 1.45 ± 0.10 a 1.15 ± 0.07 cde 5.50 ± 1.42 bc 55.9 ± 1.23 a Pinguipes brasilianus 12.6 ± 0.53 b 1.30 ± 0.10 ab 1.46 ± 0.21 b 8.03 ± 1.19 b 53.3 ± 3.11 a Rhizoprionodon lalandii 14.4 ± 0.41 a 1.15 ± 0.04 bc 1.40 ± 0.09 bc 4.40 ± 0.37 c 48.2 ± 1.39 b Rhizoprionodon porosus 14.9 ± 0.28 a 1.10 ± 0.01 cd 1.17 ± 0.04 cde 5.28 ± 0.33 bc 45.7 ± 0.30 b Urophycis cirrata 13.8 ± 0.67 a 1.41 ± 0.09 a 1.36 ± 0.10 bcd 5.54 ± 1.30 bc 55.5 ± 1.62 a # Mean values significantly different: ***P < 0.001, a > b > c > d > e. Identical superscript letters (a, a; b, b) or absence of letters indicate that mean values are not significantly different P. brasiliensis. The lower concentrations of hydrosoluble protein were observed in the elasmobranch fishes R. porosus and R. lalandii and to bone fishes M. argentinae and D. volitans. The amino acid profiles of the samples were very similar and are presented in Table 2. Glutamic acid was the most abundant amino acid in all species. The highest concentration of glutamic acid (14% of total amino acid) was found in D. volitans, while M. argentinae had the lowest (12.5%) concentrations. The fish muscles were also rich in lysine. These values varied from 9.63% (D. volitans) to 11.1% (P. patagonicus). The sum of the most abundant amino acids (glutamic acid and lysine) represented more than 20% of total amino acid. The percentage of histidine was the lowest in all species, with an average value of 2.12%. The higher concentration of arginine was recorded in D. volitans. Percentages of the other amino acids were similar among all species. The total protein content of the samples is showed in Table 3 as total amino acid residues. The marine fish muscles showed high protein concentration, varying from 66.2% (M. argentinae) to 81.5% (G. brasiliensis) of the dry weight. Nitrogen mass within total amino acid ranged from 11.4% (M. argentinae) to 13.9% (G. brasiliensis). The relative percentage of protein nitrogen was estimated as the ratio of nitrogen recovered from amino acid to total nitrogen (Table 1). High percentages of protein nitrogen were estimated, ranging from 91.4% (R. porosus) to 101.4% (D. volitans). The elasmobranch fishes showed the higher percentages of NPN (average = 7.85%, estimated from Table 3, as [100% - protein- N]). From the ratio of the mass of amino acid residues to total nitrogen we calculated specific N-Prot factors for the species. The N-Prot factors ranged from 5.39 (R. porosus) to 5.98 (D. volitans). The elasmobranch species (R. porosus and R. lalandii) recorded the lowest values of N-Prot factors. An overall average N- Prot factor = 5.71 was calculated from the data for all species (Table 3). DISCUSSION Despite the remarkable importance of marine fishes, these organisms use to be chemically studied only in food science, covering species of commercial importance as food. The present study is also potentially useful in food science, but it includes ecologically important fish species, independent of their possible use as food. In addition, despite the low number of replicates used, the interpretation of the results is strengthened by the low dispersion of the data, as shown in Tables 1-3. All fish species analyzed showed low content of carbohydrate. According to Ogawa & Maia (1999), in general, the presence of glycogen in the muscles is low, varying from 1.5 to 5% of carbohydrate (values converted into a dry mass basis). There is a remarkable lack of data in the literature on carbohydrates in fishes, probably due the small importance of carbohydrate for the nutritional value of fishes. The majority of the chemical studies on fishes are focused on both protein and lipid composition (Puwastien et al., 1999; Vila-Nova et al., 2005; Simões et al., 2007).

56 259 Latin American Journal of Aquatic Research Table 2. Total amino acid composition of nine species of fishes. Results are expressed as grams of amino acid measured in 100 g of fish protein and represent the actual recovery of amino acids after acid hydrolysis. Values are the mean of three replicates ± SD (n = 3). Amino acids Dactylopterus volitans Genypterus brasiliensis Mullus argentinae Paralichthys patagonicus Percophis brasiliensis Pinguipes brasilianus Rhizoprionodon lalandii Rhizoprionodon porosus Urophycis cirrata Aspartic acid 8.41 ± ± ± ± ± ± ± ± ± 2.23 Threonine 5.26 ± ± ± ± ± ± ± ± ± 0.17 Serine 4.26 ± ± ± ± ± ± ± ± ± 0.70 Glutamic acid 14.0 ± ± ± ± ± ± ± ± ± 2.29 Proline 4.49 ± ± ± ± ± ± ± ± ± 0.26 Glycine 3.89 ± ± ± ± ± ± ± ± ± 0.25 Alanine 6.45 ± ± ± ± ± ± ± ± ± 0.26 Valine 6.34 ± ± ± ± ± ± ± ± ± 0.52 Isoleucine 5.85 ± ± ± ± ± ± ± ± ± 0.63 Leucine 9.62 ± ± ± ± ± ± ± ± ± 0.92 Tyrosine 3.69 ± ± ± ± ± ± ± ± ± 0.17 Phenylalanine 5.42 ± ± ± ± ± ± ± ± ± 2.07 Histidine 1.13 ± ± ± ± ± ± ± ± ± 0.26 Lysine 9.63 ± ± ± ± ± ± ± ± ± 0.35 Arginine 11.0 ± ± ± ± ± ± ± ± ± 0.08 Total 99.5 ± ± ± ± ± ± ± ± ± 11.1

57 Gross chemical profile 260 Table 3. Calculation of nitrogen-to-protein conversion factors for nine species of fishes based on the amino acid residues to total nitrogen ratio. Values are expressed as percentage of the dry matter, except for the nitrogen-to-protein factors (no units). Results represent the mean of three replicates ± SD (n = 3). Species Total amino acid Amino acid residues Amino acid-n Protein-N N-Prot factor Dactylopterus volitans 81.5 ± ± ± ± ± 0.67 Genypterus brasiliensis 94.7 ± ± ± ± ± 0.12 Mullus argentinae 76.9 ± ± ± ± ± 0.11 Paralichthys patagonicus 91.7 ± ± ± ± ± 0.46 Percophis brasiliensis 91.0 ± ± ± ± ± 0.08 Pinguipes brasilianus 86.0 ± ± ± ± ± 0.08 Rhizoprionodon lalandii 92.3 ± ± ± ± ± 0.07 Rhizoprionodon porosus 93.6 ± ± ± ± ± 0.65 Urophycis cirrata 91.0 ± ± ± ± ± 0.50 The lipid concentrations were higher than the carbohydrates for all species. This trend suggests that the animals concentrations store energy as fat content, converting the excess of sugar in fatty tissues. In general, data for lipid showed wider variations among the species. According to Mathew et al. (1999), Zaboukas et al. (2006) and Özogul & Özogul (2007), the lipid percentages are more influenced by the environmental conditions, physiological conditions and feeding than the carbohydrate and protein percentages. Henderson & Tocher (1987), Ramos- Filho et al. (2008) and Mathew et al. (1999) state that the concentration of lipids in the muscles vary from 0.3 to 20% fresh weight. In the present study, the average value for the lipid content of the nine species was 6.3% (dry weight). Only M. argentinae showed a high lipid concentration (16.1% dry weight), which possibly reflects species-specific physiological traits. The lowest concentrations of lipid were recorded in elasmobranch fishes (R. lalandii and R. porosus), which may be related to the fact that these fishes store fat mainly in the liver (Ogawa & Maia, 1999). According to Pigott & Tucker (1990), fish can be classified by their percentage of fat (fresh weight) as fish with low (< 2% fresh weight), moderate (from 2 to 5%), and high (> 5%) content of fat. Following this classification, the species analyzed in the present study would be of low fat content, except M. argentinae, with moderate content of fat. Analysis of the phosphorus content in the organisms may be useful to interpret metabolic speed. High concentrations of phosphorus could be related to the characteristics of muscle tissues. Muscles of vertebrates have storage of high potential phosphorus in the form of creatinine-phosphate, which can quickly transfer its phosphate group to ATP (Stryer, 1996). Ogawa & Maia (1999) state that the animals that move fast spend more energy and consequently use more ATP, requiring a greater supply of phosphorus in the muscles than animals of more limited movements. The use of muscles in our analysis explains the typical high content of phosphorus found for all fish species analyzed here. Our data indicate high protein concentrations in the species, and this agrees with the typical muscle characteristics. Concentrations of total protein were similar to the results reported by Zaboukas et al. (2006) and Simões et al. (2007), who state that total protein in fish fluctuates ca. 75% of dry weight. The lowest protein concentration was found in M. argentinae, which can be related to the high lipid content recorded in this species. According to Ogawa & Maia (1999) there is an inverse relationship between lipid and protein contents in fishes. The protein quantification, by the method of Lowry et al. (1951), and by the sum of the amino acid residues, showed remarkable differences. For all species, the values for protein measured by the method of Lowry et al. (1951) were lower (ca. 35%) than results estimated by the sum of AA-Res. The possible cause for these differences could be related to the more difficult extraction of protein from freeze-dried samples according to Barbarino & Lourenço (2005). This could suggest a lower efficiency on extraction with the Lowry method. On the other hand, total amino acid analysis involves the acidic hydrolysis of the samples, which eliminates problems with protein extraction. It is widely accepted in the literature that the best estimation of protein content is the sum of AA-Res, which represents the actual protein in each sample (Sosulski & Imafidon, 1990; Yeoh & Truong, 1996; Fujihara et al., 2008; Diniz et al., 2011).

58 261 Latin American Journal of Aquatic Research Proteins are composed of one or more chains of amino acids and the nutritional quality of a protein is basically determined by their content, proportion and availability of amino acids (Taboada et al., 2010). The main findings of amino acid composition of fish proteins described here are in agreement with previous studies (Uhe et al., 1991; Ogawa & Maia, 1999). All fish samples exhibited similar amino acid patterns. In general, all species are rich in glutamic acid and lysine, and poor in histidine. The high nitrogen concentration observed in the muscles can be related with the high proportion of protein in the tissues, and by the presence of OTMA (oxydeoftrimethylamine), a nitrogenous substance widely found in marine animals (Ogawa & Maia, 1999). Sterner & George (2000) and Dantas & Attayde (2006) found similar values of TN in freshwater fishes compared to those recorded in the present study, with values fluctuating from 9.5% to 10.35% of dry weight. More than 90% of the total nitrogen present in fish muscles are in proteins. In cartilaginous fishes, a smaller concentration of protein nitrogen (PN) was found, which may be related to the high concentration of OTMA. This substance is associated to urea in the control of the osmotic pressure; a high concentration of urea can be found in the muscles, achieving up to 2% (Ogawa & Maia, 1999). As a consequence of a high NPN, the budget of nitrogen is particularly affected in these species. According to Puwastein et al. (1999), the quantity of NPN in muscles can vary from 6 to 14% of the total nitrogen. NPN in animals is present in the constitution of several substances, such as OTMA, amines, guanidines, nucleotides and their degrading products such as urea and ammonia. Other non-proteinaceous, substances with nitrogen that can be present are glycilbetaine, carnitine and homarine (Ogawa & Maia, 1999). The flavor of seafoods depends on the species, the fat content, and the presence as well as the type of nonprotein nitrogenous compounds (Venugopal & Shahidi, 1996). In most studies of the protein content of fishes N- Prot factors were used. However, the factor used in these studies was the traditional factor 6.25 calculated by Jones (1931), from animal muscles. The use of the factor 6.25 is based on the assumption that samples contain protein with 16% nitrogen and an insignificant amount of non-protein nitrogen (Coklin-Brittain et al., 1999). However, the amino acid composition varies from one protein source to another, existing different N content in each amino acid. Moreover, this assumption is invalid for organisms that contain high concentrations of other nitrogenous compounds, such as nucleic acids, amines, urea, inorganic intracellular nitrogen (ammonium, nitrate and nitrite), vitamins and alkaloids (Fujihara et al., 2001). The contribution of non-protein nitrogenous compounds to the total crude protein content of different kinds of seafood depends on the composition (species) of the raw material, and ranges from 10 to 40% (Venugopal & Shahidi, 1996). There are different ways to calculate N-Prot conversion factors. Several studies calculate the N- Prot factors as the ratio between AA-Res and NT (Levey et al., 2000; Matilla et al., 2002; Fujihara et al., 2008), such as it has been done in the present study. On the other hand, many studies determined conversion factors taking into account the proportion between total amino acid and the recovery of nitrogen from the amino acids (AA-N) (Mossé, 1990; Sosulski & Imafidon, 1990; Yeoh & Wee, 1994). Salo- Väänänen & Koivistoinen (1996) calculated a conversion factor of 4.94 for fish and fish products based on AA-N. However, the application of the conversion factor calculated only by AA-N can overestimate the actual protein content of species with high NPN. This happens because the factor would multiply the total nitrogen content to calculate the percentage of protein: it is not possible to distinguish protein nitrogen and NPN in total nitrogen. For a more accurate determination of protein using conversion factors, the quantity of TN should be corrected according to the NPN. Thus, the use of conversion factors based on AA-N does not have a practical value. The total amino acid content represents not only amino acids derived from proteins but also those in free form. Thus. the presence of free amino acids contributes to an overestimation of the total protein. However, according to Mossé (1990), the use of data of total amino acid, without determination of free amino acids, is a widely accepted procedure to estimate protein, since in acid hydrolysis some amino acids are partially or totally destroyed (e.g. tryptophan, cystine, methionine and serine). The loss during acid hydrolysis might compensate for the influence of free amino acids in the quantification of protein by the sum of the total amino acid residues The overall mean N-Prot factors calculated in this report was Conversion factors calculated in this study were very similar among the species. Values for the elasmobranch fishes were smaller, achieving 5.39 and 5.5, in R. porosus e R. lalandii, respectively. This is possibly related to the higher concentration of NPN than in the other species. The high N-Prot factors calculated in this study reflect the low concentrations of NPN in the muscles. Despite the high concentrations of PN, all conversion factors were lower than the traditional factor 6.25.

59 Gross chemical profile 262 The best estimate of the protein content is the sum of AA-Res, representing the actual protein content in the samples. A comparison of the values obtained using the N-Prot factor and the values obtained by the sum of AA-Res shows clearly that the factor 6.25 overestimates the protein content in fish samples in ca. 9% (14% for elasmobranchs). Such differences are not negligible, which means that the presence of nonproteinaceous substances with nitrogen affects the use of nitrogen-to-protein conversion factors in fish samples. ACKNOWLEDGMENTS Authors are indebted to Brazil s National Council for Scientific and Technological Development (CNPq), and Research Support Foundation of Rio de Janeiro State (FAPERJ) for the financial support of this study. GSD thanks Coordination of Improvement of Higher Education Personnel (CAPES) for her scholarship. Authors thank Dr. Renato Crespo Pereira (UFF) for the use of laboratory facilities and to Dr. Cassiano Monteiro Neto and his team (UFF) for confirming the identification of the fishes. REFERENCES Aitken, K.A., L.D. Melton & M.T. Brown Seasonal protein variation in the New Zealand seaweeds Porphyra columbina Mont. & Porphyrasubtumens J. Ag. (Rhodophyceae). Jap. J. Phycol., 39: Association of Official Analytical Chemists (AOAC) Official Methods of Analysis. Washington, D.C., 556 pp. Barbarino, E. & S.O. Lourenço An evaluation of methods for extraction and quantification of protein from marine macro- and microalgae. J. Appl. Phycol., 17: Barbarino, E. & S.O. Lourenço Comparison of CHN analysis and Hach acid digestion to quantify total nitrogen in marine organisms. Limnol. Oceanogr: Methods, 7: Bradford, M A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal. Biochem., 72: Childress, J.J., M.H. Price, J. Favuzzi & D. Cowles Chemical composition of midwater fishes as a function of depth of occurrence off the Hawaiian Islands: food availability as a selective factor? Mar. Biol., 105: Cohen, S.A. & K.M. De Antonis Applications of amino acid derivatization with 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate. Analysis of feed grains, intravenous solutions and glycoproteins. J. Chromatogr., 661: Coklin-Brittain, N.L., E.S. Dierenfeld, R.W. Wranghan, M. Norconk & S.C. Silver Chemical protein analysis: a comparasion of Kjeldhal crude protein and total ninhydrin protein from wild, tropical vegetation. J. Chem. Ecol., 25: Dantas, M.C. & J.L. Attayde Nitrogen and phosphorus content of some temperate and tropical freshwater fishes. J. Fish Biol., 70: Diniz, G.S., E. Barbarino, J. Oiano-Neto, S. Pacheco & S.O. Lourenço Gross chemical profile and calculation of nitrogen-to-protein conversion factors for five tropical seaweeds. Am. J. Plant Sci., 2: Dubois, M., K.A. Gilles, J.K. Hamilton, P.A. Reberts & F. Smith Colorimetric method for determination of sugars and related substances. Anal. Chem., 28: Folch, J., M. Lees & G.H. Sloanne-Stanley A simple method for the isolation and purification of total lipid from animal tissue. J. Biol. Chem., 226: Fujihara, S., A. Kasuga & Y. Aoyagi Nitrogen-toprotein conversion factors for common vegetables in Japan. J. Food Sci., 66(3): Fujihara, S., H. Sasaki, Y. Aoyagi & T. Sugahara Nitrogen-to-protein conversion factors for some cereal products in Japan. J. Food Sci., 73: Hach, C.C., B.K. Bowden, A.B. Kopelove & S.T. Brayton More powerful peroxide Kjeldhal digestion method. J. Assoc. Anal. Chem., 70: Henderson, R.J. & D.R. Tocher The lipid composition and biochemistry of fresh water fish. Prog. Lipid Res., 26(4): Jones, D.B Factors for converting percentages of nitrogen in foods and feeds into percentages of protein. USDA Circ., 183: Legler, G., C.M. Müller-Platz, M. Mentges-Hettkamp, G. Pflieger & E. Jülich On the chemical basis of the Lowry protein determination. Anal. Biochem., 150: Levey, D.J., H.A. Bissell & S.F. O'Keefe Conversion of nitrogen to protein and amino acids in wild fruits. J. Chem. Ecol., 26: Lourenço, S.O., E. Barbarino, A. Nascimento & R. Paranhos Seasonal variations in tissue nitrogen

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62 Lat. Am. J. Aquat. Res., 41(2): , 2013 Exotic benthic species in São Sebastião, Brazil Proceedings of the 3 rd Brazilian Congress of Marine Biology A.C. Marques, L.V.C. Lotufo, P.C. Paiva, P.T.C. Chaves & S.N. Leitão (Guest Editors) DOI: /vol41-issue2-fulltext Research Article Rapid assessment survey for exotic benthic species in the São Sebastião Channel, Brazil Antonio C. Marques 1, Aline dos Santos Klôh 2, Alvaro Esteves Migotto 3, Ana C. Cabral 2 Ana P. Ravedutti Rigo 2, Ariane Lima Bettim 2, Emanuel L. Razzolini 2, Helena Matthews Cascon 4 Juliana Bardi 1, Laura Pioli Kremer 2, Leandro Manzoni Vieira 1, Luis E. Arruda Bezerra 5 Maria A. Haddad 2, Ronaldo Ruy de Oliveira Filho 6, Silvia M. Millan Gutierre 7 Thaís Pires Miranda 1, Wilson Franklin Jr. 6 & Rosana Moreira da Rocha 2 1 Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo R. Matão, Trav. 14, , São Paulo, Brazil 2 Departamento de Zoologia, Universidade Federal do Paraná CP 19020, , Curitiba, Brazil 3 Centro de Biologia Marinha, Universidade de São Paulo, Rodovia Manoel Hypólito do Rego 131,5 km, Cabelo Gordo, , São Sebastião, Brazil 4 Departamento de Biologia, Centro de Ciências, Universidade Federal do Ceará Campus do Pici, Departamento de Biologia Bloco, 906, Pici, , Fortaleza, Brazil 5 Departamento de Oceanografia, Universidade Federal de Pernambuco, Av. da Arquitetural s/n Cidade Universitária, , Recife, Brazil 6 Instituto de Ciências do Mar, Universidade Federal do Ceará Av. Abolição, 3207, Meireles, , Fortaleza, Brazil 7 Departamento de Fisiologia, Universidade Federal do Paraná CP 19031, , Curitiba, Brazil ABSTRACT. The study of biological invasions can be roughly divided into three parts: detection, monitoring, mitigation. Here, our objectives were to describe the marine fauna of the area of the port of São Sebastião (on the northern coast of the state of São Paulo, in the São Sebastião Channel, SSC) to detect introduced species. Descriptions of the faunal community of the SSC with respect to native and allochthonous (invasive or potentially so) diversity are lacking for all invertebrate groups. Sampling was carried out by specialists within each taxonomic group, in December 2009, following the protocol of the Rapid Assessment Survey (RAS) in three areas with artificial structures as substrates. A total of 142 species were identified (61 native, 15 introduced, 62 cryptogenic, 4 not classified), of which 17 were Polychaeta (12, 1, 1, 3), 24 Ascidiacea (3, 6, 15, 0), 36 Bryozoa (17, 0, 18, 1), 27 Cnidaria (2, 1, 24, 0), 20 Crustacea (11, 4, 5, 0), 2 Entoprocta (native), 16 Mollusca (13, 3, 0, 0). Twelve species are new occurrences for the SSC. Among the introduced taxa, two are new for coastal Brazil. Estimates of introduced taxa are conservative as the results of molecular studies suggest that some species previously considered cryptogenic are indeed introduced. We emphasize that the large number of cryptogenic species illustrates the need for a long-term monitoring program, especially in areas most susceptible to bioinvasion. We conclude that rapid assessment studies, even in relatively wellknown regions, can be very useful for the detection of introduced species and we recommend that they be carried out on a larger scale in all ports with heavy ship traffic. Keywords: bioinvasion, fouling, artificial structures, port, São Sebastião, Brazil, southwest Atlantic. Estudio de evaluación rápida de especies bentónicas exóticas en São Sebastião, Brasil RESUMEN. El estudio de invasiones biológicas puede ser dividido en tres partes: detección, supervisión y atenuación. El objetivo fue describir la fauna marina del puerto de São Sebastião (costa norte del estado de

63 266 Latin American Journal of Aquatic Research São Paulo, en el canal de São Sebastião, SSC) para detectar las especies introducidas. No existen descripciones de la comunidad faunística del SSC en relación a la diversidad nativa y alóctona (invasiva o potencialmente) de todos los grupos de invertebrados. El muestreo se efectuó por especialistas de cada grupo taxonómico en diciembre de 2009, siguiendo el protocolo de Estudio de Evaluación Rápida (EER) en tres zonas con sustratos artificiales. Se identificaron 142 especies (61 nativas, 15 introducidas, 62 criptogénicas y cuatro no determinadas), de las cuales 17 correspondieron a poliquetos (12, 1, 1, 3), 24 a ascidias (3, 6, 15, 0), 36 a briozoarios (17, 0, 18, 1), 27 a cnidarios (2, 1, 24, 0), 20 a crustáceos (11, 4, 5, 0), 2 a entoproctos (nativo) y 16 a moluscos (13, 3, 0, 0). Doce especies constituyen nuevos registros para el SSC. Entre los taxa introducidos, dos son nuevos para la costa de Brasil. Las estimaciones de los taxa introducidos son conservativas dado que los resultados obtenidos en estudios moleculares sugieren que algunas especies anteriormente consideradas criptogénicas son introducidas. Se destaca que el gran número de especies criptogénicas refleja la necesidad de un programa de monitoreo a largo plazo, especialmente en las zonas más susceptibles a la invasión biológica. Se concluye que los estudios de evaluación rápida, incluso en las regiones relativamente bien conocidas, pueden ser útiles para detectar especies introducidas y se recomienda expandir este tipo de estudios en todos los puertos con tráfico marítimo. Palabras clave: bioinvasión, incrustaciones, sustratos artificiales, puerto, São Sebastião, Brasil, Atlántico suroccidental. Corresponding author: Rosana Moreira da Rocha (rmrocha@ufpr.br) INTRODUCTION Bioinvasions are biological phenomena with ecological and evolutionary consequences to local biota. The study of biological invasions can be roughly divided into detection, monitoring, and remediation/mitigation, and all of them are better informed by basic (e.g., biogeographically, ecological, ecophysiological constraints) and applied knowledge (e.g., prevention, management, eradication, conservation). Biological invasions are ubiquitous, continental and marine, in all biomes and ecosystems. The introduction of species is the successful establishment of a species in a region where it did not occur before (Elton, 1958). This process can happen naturally by the expanding population of a species, but can also be caused by human activities, that occur more frequently in recent decades (Carlton, 1996). Becoming invasive, introduced species may compete with native species (including those of commercial or cultural importance) and displace or prevail over native species, change trophic relationships in the food chain, introduce new diseases or toxic substances that affect native organisms and human populations. Thus, biological invasions have obvious negative consequences for biodiversity and public health, cultural and economical issues for the affected regions. In the marine realm, biological invasion vectors are fundamentally associated with economic activities such as commerce and tourism, both of which are associated with ports. Consequently, records of invasive species in ports around the world are increasing (Carlton, 1989; Ruiz et al., 2000), often seriously endangering natural habitats. Transportation of potential invaders may be by hull fouling and its associated species, or ballast water (Fofonoff et al., 2003). Both have reached alarming proportions as ships become more numerous, faster and larger, thereby requiring more ballast (Cohen & Carlton, 1998). Despite of the impact of marine bioinvasions, the marine realm has historically received less attention compared to terrestrial and freshwater habitats (Carlton, 1989). In port areas, the availability of hard artificial substrates (such as buoys, ropes, concrete walls, and marinas) provides many opportunities for settlement and metamorphosis for larvae of introduced species (Stachowicz et al., 1999; Tyrrel & Byers, 2007). After recruitment and establishment, a following stage of the invasion process comprises expansion of the geographical distribution of the species or populations. Virtually any marine taxon may include invasive lineages, such as algae (Mathieson et al., 2003) and Chordata (Castilla et al., 2004). Detecting invasive species depends on monitoring and adequate knowledge of the native fauna, including genetic, taxonomic, biological and ecological data. Invasion and establishment of a species is frequently not recorded and very seldom observed (Carlton, 2009). Ideally, the control of exotic species will be more efficient with early detection, when populations are small and more easily eradicated or controlled (Bax et al., 2001). The lack of previous information about the species makes it difficult to determine its invasive status, in which case the species is of uncertain origin and is classified as cryptogenic (Carlton, 1996).

64 Exotic benthic species in São Sebastião, Brazil 267 Strategies for monitoring species in port areas include Rapid Assessment Survey (RAS, see for instance Cohen et al., 2005; Pederson et al., 2005), a procedure with standardized field sampling effort carried out by specialists in taxonomy of target groups, in which the goal is to sample the maximum number of taxa possible in a short time period. The only previous large scale RAS to detect introduced species in Brazil was in Sepetiba Bay, with three ports, as a demonstration study of the Globallast program of the International Maritime Organization IMO (Clarke et al., 2004). Few other surveys have been carried out and are only available for the port of Paranaguá, southern Brazil (Neves et al., 2007), Ilha Grande Bay, southeastern Brazil (Ignacio et al., 2010), and the port of Recife, northeastern Brazil (Amaral et al., 2010; Lira et al., 2010). Nonetheless, the literature on introduced species identified in specific taxonomic groups is growing, such as those for ascidians (Rocha & Kremer, 2005; Rocha et al., 2009; Marins et al., 2010); cnidarians (Calder & Maÿal, 1998; Paula & Creed, 2004; Nogueira Jr. & Oliveira, 2006; Bardi & Marques, 2009); crustaceans (Tavares, 2004; Farrapeira, 2010a); mollusks (Domaneschi & Martins, 2002; Souza et al., 2003; Silveira et al., 2006; Breves et al., 2010; Lopez et al., 2010) and bryozoans (Gordon et al., 2006; Farrapeira, 2010b). General overviews were given by Lopes et al. (2009) and Ferreira et al. (2009). Unfortunately, comprehensive surveys in Brazil of the benthos, including those not focusing on bioinvasion, are also rare, even for the better known areas of the country (Migotto & Marques, 2006). Here we attempt to detect potential bioinvasion of benthic species in the São Sebastião Channel, an important Brazilian port area, using a Rapid Assessment Survey technique. MATERIALS AND METHODS Study area The São Sebastião Channel (SSC), on the northern coast of the state of São Paulo, Brazil, comprises several areas of preservation, and among the best known biodiversity in the coast of Brazil, due to the many extensive floral and faunal surveys carried out under the auspices of the Center of Marine Biology of the University of São Paulo. The SSC also includes the large Port of São Sebastião, in use for more than 50 years, and through which passes a total load of 50 million tons year -1. An important part of the port is the Terminal Marítimo Almirante Barroso (TEBAR- Transpetro/Petrobras), responsible for intense traffic of ships importing and exporting petroleum products both coastal and long-distance ( portes. gov.br/-bit/portos/ssebastiao/deposaosebastiao. htm). Rapid Assessment Survey (RAS) Surveys were carried out in three sites in the vicinity of the port of São Sebastião: a) Ilhabela Yacht Club (IYC, 23 o 46'27''S, 45 o 21'20''W), a floating marina located on São Sebastião Island, on 11 December 2009; b) Petrobras Pier (PP, 23 o 48'07''S, 45 o 23'27''W), a concrete structure with large columns on 12 December 2009; and c) Pontal da Cruz Pier (PCP, 23 o 46'53''S, 45 o 23'48''W), a cement structure, on 12 December IYC and PCP were examined for two hours by 17 people including specialists in Annelida Polychaeta, Ascidiacea, Bryozoa, Cnidaria, Crustacea Cirripedia, Entoprocta and Mollusca that defined the taxonomical scope of the survey. PP was surveyed to a maximum depth of 8.5 m for an hour by four scuba divers. Benthic animals were collected by hand on artificial substrates, including floats and pier columns, during low tide at PCP and by snorkeling in shallow waters at IYC and PCP. When necessary, samples were kept separately in individualized jars in the field, and larger samples were packed to be sorted in the laboratory. Material was brought to the laboratory and maintained in vivo to be examined by specialists. Dissecting and compound microscopes were used when necessary. Samples were identified while alive to the lowest taxonomic level possible. Part of the material was anesthetized (using magnesium chloride, menthol or M-aminobenzoate ethyl) and fixed (in formalin at 4% prepared with saltwater or in ethanol 70%-100%) following standard procedures for each taxon. The material sampled was deposited in the scientific collections of the Federal University of Ceará, Federal University of Paraná, and University of São Paulo. RESULTS A total of 142 taxa were identified, twelve of which are new records for the SSC, but may not all be introduced (Table 1). IYC had 89 taxa, PCP had 81 and PP had 70. The taxa comprise Annelida Polychaeta (17), Ascidiacea (24), Bryozoa (36), Cnidaria (27), Crustacea (20), Entoprocta (2) and Mollusca (16). The majority of taxa were classified as either cryptogenic (69), native (53) or introduced (15), and five were not classified because they were not identified at the species level. Most taxa were found in only one site (50.7%), with 30.3% in two sites and 19.0% in all three sites

65 268 Latin American Journal of Aquatic Research Table 1. List of species found in São Sebastião Channel (SSC) in three sites: Ilhabela Yatch Club (IYC), Petrobras Pier (PP) and Pontal da Cruz Pier (PCP) in December Family Species IYC PP PCP Status Previous records for SSC Records for Brazilian states First record for Brazil Known distribution Observation Annelida Polychaeta Palpata Aciculata Eunicida Dorvilleidae Chamberlin, 1919 Dorvillea sociabilis (Webster, 1879) x Cryptogenic Morgado & Amaral (1981a) Eunicidae Berthold, 1827 Eunice rubra Grube, 1856 x Native Treadwell (1932); Morgado & Amaral (1981b) Oenonidae Kinberg, 1865 Oenone fulgida (Savigny, 1818) Annelida Polychaeta Palpata Aciculata Phyllodocida x Cryptogenic Treadwell (1932); Morgado & Amaral (1981a) SP Amaral (1977) Gulf of Mexico, Caribbean Sea RN-RS Treadwell (1932) Gulf of Mexico, Caribbean Sea RN BA RJ SP Treadwell (1932) Gulf of Mexico, Caribbean Sea, Mozambique, Madagascar, Red Sea, New Zealand Hesionidae Grube, 1850 Hesione picta Müller, 1858 x Cryptogenic first record AL SE SP Nonato & Luna (1970) Nereididae Johnston, 1865 Perinereis anderssoni Kinberg, 1866 Pseudonereis palpata (Treadwell, 1923) Syllidae Grube, 1850 Haplosyllis sp. x -- x Cryptogenic Amaral et al. (2003); Omena & Amaral (2003) PA MA PI CE RN SP PR x x Native Amaral et al. (2010b) SP Santos & Steiner; (2006) Gulf of Mexico, Caribbean Sea Nonato (1981) Gulf of Mexico, Caribbean Sea Brazil Reported to S. Sebastião as Oenone diphyllidia Schmarda, 1861 Syllis variegata (Grube, 1860) x Cryptogenic Duarte & Nalesso (1996) RN-PR Nonato & Luna (1970) Caribbean Sea, United Kingdom Annelida Polychaeta Palpata Canalipalpata Sabellida Sabellidae Latreille, 1825 Branchiomma patriota Nogueira, Silva & Rossi, 2006 Branchiomma luctuosum (Grube, 1869) Sabellastarte sp. x -- x x x Native Rossi ( 2008) SP Nogueira et al. (2006) x x x Introduced Amaral et al. (2010b) SP Rossi (2008) Europe, Mediterranean Sea Brazil Previously reported to Brazil as B. nigromaculatum (Baird, 1865) Previously reported to Brazil as B nigromaculatum (Baird, 1865) Serpulidae Rafinesque, 1815 Hydroides sp. x -- Annelida Polychaeta Palpata Canalipalpata Spionida Chaetopterus sp. x -- Chaetopteridae Audouin & Milne-Edwards, 1833d Spionidae Grube, 1850 Polydora colonia Moore, 1907 x Cryptogenic first record SP PR Neves et al. (2007) W Atlantic, Mediterranean Sea, South Africa

66 Exotic benthic species in São Sebastião, Brazil 269 (continuation) Family Species IYC PP PCP Status Previous records for SSC Records for Brazilian states First record for Brazil Known distribution Observation Annelida Polychaeta Palpata Canalipalpata Terebellida Cirratulidae Carus, 1863 Cirriformia punctata (Grube, 1859) Terebellidae Malmgren, 1867 Annelida Polychaeta Scolecida Nicolea uspiana (Nogueira, 2003) x Cryptogenic first record SP Souza (1989) Gulf of Mexico, Caribbean Sea, Mozambique, South Africa x x Native Alves ( 2008) SP Nogueira (2003) Brazil Orbiniidae Hartman, 1942 Naineris laevigata (Grube, 1855) Ascidiacea Aplousobranchia Clavelinidae Forbes & Hanley, 1848 Polyclinidae Milne Edwards, 1842 Clavelina oblonga Savigny, 1816 Aplidium accarense (Millar, 1953) Polyclinum constellatum Savigny, 1816 x Cryptogenic first record BA SP Rullier & Amoureux (1979) x x x Introduced Rodrigues (1962) CE ES RJ SP PR SC Cosmopolitan Hartmeyer (1912) Caribbean Sea x x x Cryptogenic Rocha & Bonnet (2009) SP SC Rocha et al. (2005) Atlantic x x Cryptogenic Rodrigues (1962) CE ES RJ SP SC Michaelsen (1923) Circuntropical Aplidiopsis sp. x Introduced first record First record Pacific Holozoidae Berrill, 1950 Distaplia bermudensis Van Name, 1902 Distaplia stylifera (Kowalewsky, 1874) Didemnidae Giard, 1872 Didemnum perlucidum Monniot, 1983 Diplosoma listerianum (Milne-Edwards, 1841) Lissoclinum fragile (Van Name, 1902) Trididemnum orbiculatum (Van Name, 1902) x x x Native Rodrigues & Rocha (1993) PA CE BA ES RJ SP PR SC Millar (1958) W. Atlantic x Introduced first record SP first record Circuntropical D. stylifera previously reported (Rodrigues et al. 1998) is a new species x x Cryptogenic Rocha & Monniot (1995) BA RJ SP SC Rocha & Monniot (1995) Circuntropical x Cryptogenic Van Name (1945) RN-SC Van Name (1945) Cosmopolitan x Cryptogenic Rodrigues et al. (1998) CE PE RJ SP PR SC x x Native Rodrigues & Rocha (1993) CE BA RJ SP PR SC Rodrigues et al. (1998) Rodrigues & Rocha (1993) Circuntropical W Atlantic Ascidiacea Phlebobranchia Ascidiidae Adams, 1858 Phallusia nigra Savigny, 1816 Ascidia cf. multitentaculata (Hartmeyer, 1912) Ascidia sydneiensis Stimpson, 1855 x x Cryptogenic Van Name (1945) CE AL BA RJ SP Van Name (1945) Atlantic, Mediterranean, Red Sea x Cryptogenic Bonnet & Rocha (2011) CE BA ES SP Millar (1977) Brazil, South Africa x Introduced Millar (1958) CE ES RJ SP PR SC Millar (1958) Cosmopolitan Ascidiacea Stolidobranchia Styelidae Sluiter, 1895 Botrylloides giganteum (Pérès, 1949) x Cryptogenic Rodrigues & Rocha (1993) ES RJ SP SC Rodrigues & Rocha (1993) Senegal, South Africa, Brazil

67 270 Latin American Journal of Aquatic Research (continuation) Family Species IYC PP PCP Status Previous records for SSC Records for Brazilian states First record for Brazil Known distribution Observation Pyuridae Hartmeyer, 1908 Bryozoa Gymnolaemata Cheilostomata Botrylloides nigrum (Herdman, 1886) Symplegma brakenhielmi (Michaelsen, 1904) Symplegma rubra Monniot, 1972 x x Cryptogenic Rodrigues (1962) PB AL BA ES RJ SP PR SC Rodrigues (1962) Circuntropical x x Cryptogenic Rodrigues (1962) PA- SC Millar (1958) Circuntropical x x x Native Rodrigues & Rocha (1993) ES RJ SP PR SC Rodrigues & Rocha (1993) Atlantic Eusynstyela sp. x x Introduced Rodrigues et al. (1998) SP Rodrigues et al. (1998) Van Name (1945) Circuntropical Polyandrocarpa anguinea (Sluiter, 1898) Polyandrocarpa zorritensis (Van Name, 1931) Styela canopus (Savigny, 1816) Styela plicata (Lesueur, 1823) Herdmania pallida (Heller, 1878) Microscosmus exasperatus Heller, 1878 x Cryptogenic Van Name (1945) ES RJ SP PR SC x Cryptogenic Rodrigues (1962) BA ES RJ SP SC x x Cryptogenic Rodrigues et al. (1998) RN PE BA RJ SP PR SC x x Introduced Rodrigues (1962) BA RJ SP PR SC Rodrigues (1962) Atlantic, Pacific, Mediterranean, Japan Monniot (1969/70) Cosmopolitan Millar (1958) Cosmopolitan x x x Cryptogenic Rodrigues (1962) AL BA RJ SP Van Name (1945) Cosmopolitan x x Cryptogenic Rodrigues (1962) CE - SC Van Name (1945) Aeteidae Smitt, 1868 Aetea anguina (Linnaeus, 1758) x Cryptogenic Amaral et al. (2010b) PE ES RJ SP PR Marcus (1937) Circuntropical Aetea anguina is a complex of species (SP specimens are A. australis Jullien from Patagonia, but not from Australia) Aetea sp. x x Native Migotto, Vieira & Winston, unpublished data SP Marcus (1938) Atlantic Not Aetea truncata Marcus 1938 or Aetea curta Hastings 1943 Aetea ligulata Busk, 1852 x x Cryptogenic Amaral et al. (2010b) SP Marcus (1937) Cosmopolitan Electridae Stach, 1937 Electra tenella (Hincks, 1881) Membraniporidae Busk, 1852 Biflustra arborescens Canu & Bassler, 1928 Biflustra denticulata (Busk, 1856) Catenicellidae Busk, 1852 Catenicella uberrima (Harmer, 1957) x Cryptogenic Amaral et al. (2010b) SP Marcus (1937) Europe and W. Atlantic x x x Cryptogenic Migotto et al. (2011) RJ SP PR SC Marcus (1937) W. Atlantic (Long Island to Brazil, Caribbean and Gulf of Mexico) x Cryptogenic Amaral et al. (2010b) ES SP PR SC Marcus (1937) W. Atlantic and Pacific Biflustra sp. x Native Migotto et al. (2011) RJ SP PR Marcus (1937) as Acanthodesia savartii Savignyella lafontii (Audouin, 1826) W. Atlantic x x Cryptogenic Amaral et al. (2010b) AL SP Busk (1884) Circuntropical x x x Cryptogenic Amaral et al. (2010b) AL SP Marcus (1937) Circuntropical

68 Exotic benthic species in São Sebastião, Brazil 271 (continuation) Family Species IYC PP PCP Status Previous records for SSC Records for Brazilian states First record for Brazil Known distribution Observation Hippopodinidae Levinsen, 1909 Microporellidae Hincks, 1879 Schizoporellidae Jullien, 1883 Hippopodina feegeensis (Busk, 1884) x Cryptogenic Migotto et al. (2011) PE SP Marcus (1937) Circuntropical Microporella sp. x Native Migotto et al. (2011) SP Marcus (1937) as Microporella ciliata Schizoporella pungens (Canu & Bassler, 1928) Brazil not Pallas (1766) x x x Cryptogenic Amaral et al. (2010b) RJ SP D'Orbigny (1842) Brazil, widespread in port areas Schizoporella sp. x Native Migotto et al. (2011) PE RJ SP PR Marcus (1937) as S. unicornis Smittinidae Levinsen, 1909 Parasmittina sp. x x Native Amaral et al. (2010b) SP Marcus (1937) Brazil Brazil errata-pungensisabelleana complex (isabelleana described from Rio de Janeiro) Watersiporidae Vigneaux, 1949 Antroporidae Vigneaux, 1949 Watersipora subtorquata (D'Orbigny, 1852) Antropora leycocypha (Marcus, 1937) Bugulidae Gray, 1848 Bugula neritina (Linnaeus, 1758) Bugula stolonifera Ryland, 1960 Candidae D'Orbigny, 1851 Scrupocellaria aff. diadema Busk, 1852 Epistomiidae Gregory, 1893 Quadricellariidae Gordon, 1984 Bryozoa Gymnolaemata Ctenostomata Alcyonidiidae Johnston, 1838 x Cryptogenic Amaral et al. (2010b) ES RJ SP D'Orbigny (1842) W. Atlantic and Australia x x Native Amaral et al. (2010b) SP PR Marcus (1937) Brazil x x x Cryptogenic Amaral et al. (2010b) RJ SP PR D'Orbigny (1841) Widespread in port areas x Cryptogenic Amaral et al. (2010b) RJ SP Marcus (1937) Widespread in port areas x x Cryptogenic first record RJ Ramalho et al. (2005) Scrupocellaria sp. x x x Native Amaral et al. (2010b) RJ SP Marcus (1937) as Scrupocellaria cornigera Synnotum aegyptiacum (Audouin, 1826) Aeverriliidae Jebram, 1973 Aeverrillia setigera (Hincks, 1887) Arachnidiidae Hincks, 1880 Vesiculariidae Hincks, 1880 Pacific (=S. diadema) diadema is a widespread complex species. The Brazilian specimens require investigation. Brazil x Cryptogenic Migotto et al. (2011) PE AL ES SP Kirkpatrick (1888) Circuntropical Nellia oculata Busk, 1852 x Cryptogenic Amaral et al. (2010b) PE BA Busk (1884) Circuntropical Alcyonidium sp. x Native Migotto et al. (2011) ES SP PR Marcus (1937) as A. polyoum Arachnoidella evelinae (Marcus, 1937) Amathia brasiliensis Busk, 1886 Amathia distans Busk, 1886 Brazil x Cryptogenic Migotto et al. (2011) SP Marcus (1937) W. Atlantic and Pacific x Native Migotto et al. (2011) SP Marcus (1937) Brazil x x x Native Fehlauer-Ale et al. (2011) x Native Amaral et al. (2010b) AL BA ES RJ SP PR ES RJ SP PR Marcus (1937) W. Atlantic Amathia sp. x -- Amaral et al. (2010b) AL SP Rocha (1995) as A. vidovici Busk (1886) W. Atlantic Some records of this species in port areas represent distinct species Circuntropical in port areas

69 272 Latin American Journal of Aquatic Research (continuation) Family Species IYC PP PCP Status Previous records for SSC Records for Brazilian states First record for Brazil Known distribution Bowerbankia maxima Winston, 1982 Zoobotryon verticillatum (Delle Chiaje, 1828) Nolellidae Harmer, 1915 Anguinella palmata van Beneden, 1845 Sundanellidae Jebram, 1973 Nolella sawayai Marcus, 1938 x x Native Amaral et al. (2010b) RJ SP PR Marcus (1937) W. Atlantic x x x Cryptogenic Amaral et al. (2010b) RJ SP Marcus (1955) Widespread in port areas x Cryptogenic Migotto et al. (2011) SP PR Marcus (1937) Widespread in port areas x Native Migotto et al. (2011) SP Marcus (1937) Brazil Nolella sp. x x Native Amaral et al. (2010b) PE AL ES SP Marcus (1937) as N. gigantea Sundanella sp. x Native Amaral et al. (2010b) RJ SP PR Marcus (1937) as V. sibogae Victorellidae Hincks, 1880 Victorella sp. x Native Migotto et al. (2011) RJ Marcus (1955) as V. pavida Brazil Brazil Brazil Bryozoa Stenolaemata Cyclostomata Crisiidae Johnston, 1838 Crisia pseudosolena (Marcus, 1937) x x x Native Amaral et al. (2010b) PE RJ SP PR Marcus (1937) Brazil Cnidaria Anthozoa Hexacorallia Actiniaria Actiniidae Gosse 1858 Bunodosoma caissarum Corrêa in Belém, 1987 x x Native Oliveira et al. (2004) PE, ES, RJ, SP, PR, SC Correa1(964) Brazil Cnidaria Anthozoa Hexacorallia Scleractinia Rhizangiidae D'Orbigny, 1851 Cnidaria Anthozoa Octocorallia Telestacea Astrangia sp. x x Native first record PE-SC Laborel (1969) Brazil, Uruguay, Puerto Rico Telestidae Milne-Edwards & Haime, 1857 Carijoa riisei (Duchassaing & Michelotti, 1860) x x x Introduced Silveira (1986) PA MA RN PE BA ES RJ SP SC Deichmann (1936) as Telesto rupicola Atlantic, Pacific Cnidaria Hydrozoa Anthoathecata Bougainvilliidae Lütken, 1850 Eudendriidae L. Agassiz, 1862 Oceaniidae Eschscholtz, 1829 Pennariidae McCrady, 1859b Tubulariidae Fleming, 1828 Bougainvillia muscus (Allman, 1863) Eudendrium caraiuru Marques & Oliveira, 2003 Eudendrium carneum Clarke, 1882 Corydendrium parasiticum (Linnaeus, 1767) Turritopsis nutricula (McCrady, 1859a) Pennaria disticha Goldfuss, 1820 Acharadria crocea (L. Agassiz, 1862) x x Cryptogenic Vannucci & Rees (1961) x x Native Marques & Oliveira (2003) AL PR SC Vannucci & Rees (1961) CE RJ SP Migotto (1996) Brazil Atlantic, Indian, W. Pacific x Cryptogenic Marques (2001) CE - SC Vannucci (1954) Atlantic, Indian, E. Pacific x Cryptogenic Migotto (1996) PE RJ Migotto (1996) Atlantic, Indian, Pacific x x Cryptogenic Migotto (1996) PE - RS Migotto (1996) Atlantic, Indian, Pacific x x x Cryptogenic Migotto (1996) CE - SC Vannucci (1950) Atlantic, Indian, Pacific x Cryptogenic Migotto (1996) ES RJ SP PR SC RS Migotto & Silveira (1987) as Ectopleura warrani Atlantic, Indian Observation

70 Exotic benthic species in São Sebastião, Brazil 273 (continuation) Family Species IYC PP PCP Status Previous records for SSC Records for Brazilian states First record for Brazil Known distribution Zyzzyzus warreni Calder, 1888 x Cryptogenic Migotto (1996) PE AL SP SC Migotto & Silveira (1987) Atlantic, Indian Cnidaria Hydrozoa Leptothecata Aglaopheniidae Marktanner-Turneretscher, 1890 Campanulariidae Johnston, 1836 Aglaophenia latecarinata Allman, 1877 Macrorhynchia philippina Kirchenpauer, 1872 Clytia gracilis (M. Sars, 1851) Obelia bidentata Clark, 1875 Obelia dichotoma (Linnaeus, 1758) Obelia geniculata (Linnaeus, 1758) Lafoeina amirantensis (Millard & Bouillon, 1973) Haleciidae Hincks, 1868 Halecium?tenellum Hincks, 1861 Halopterididae Millard, 1962 Nemalecium lighti (Hargitt, 1924) Halopteris diaphana (Heller, 1868) Hebellidae Fraser, 1912 Hebella furax Millard, 1957 Plumulariidae McCrady, 1859b Sertulariidae Lamouroux, 1812 Plumularia strictocarpa Pictet, 1893 Dynamena disticha (Bosc, 1802) Idiellana pristis (Lamouroux, 1816) Sertularia marginata (Kirchenpauer, 1864) Sertularia turbinata (Lamouroux, 1816) x Cryptogenic Migotto (1996) MA - SC Ritchie (1909) Atlantic, Indian, W. Pacific x Cryptogenic Migotto (1996) PE AL BA ES RJ SP SC Nutting (1900) Atlantic, Indian, Pacific x x Cryptogenic Migotto (1996) CE - PR Vannucci & Mendes Atlantic, Indian, (1946) Pacific x x x Cryptogenic Migotto (1996) PE SE BA RJ Jäderholm (1903) Atlantic, Indian, SP SC Pacific x x x Cryptogenic Migotto (1996) CE - RS Stechow (1919) Atlantic, Indian, Pacific x Cryptogenic Migotto (1996) AL ES SP PR SC RS x Cryptogenic Migotto & Cabral (2005) Vannucci & Mendes (1946) PE ES RJ SP SC Nogueira et al. (1997) Atlantic, Indian, Pacific Atlantic, Indian, Pacific, Mediterranean x Cryptogenic Migotto (1996) PE SP SC Migotto (1996) Atlantic, Indian, Pacific x Cryptogenic Migotto (1996) ES RJ SP Migotto (1996) Indian, W Pacific x Cryptogenic Migotto, 1996 CE AL ES RJ SP x Cryptogenic Migotto & Andrade (2000) x Cryptogenic Migotto (1996) AL BA ES RJ SP Vannucci & Mendes (1946) RJ SP Migotto & Andrade, 2000 Atlantic, Indian, Pacific Atlantic, Indian Vannucci (1949) Atlantic, Indian, W. Pacific x Cryptogenic Migotto (1996) CE PE BA-RS Ritchie (1909) Atlantic, Indian, Pacific x x Cryptogenic Migotto (1996) AL BA SP Allman (1888) Circumtropical x x Cryptogenic Migotto (1996) CE - SC Allman (1888) Atlantic, Indian, Pacific x Cryptogenic Migotto(1996) Vannucci & Mendes (1946) Atlantic, Indian, Pacific Crustacea Cirripedia Balanidae Leach, 1817 Amphibalanus amphitrite (Darwin, 1854) Amphibalanus improvisus (Darwin, 1854) Amphibalanus reticulatus (Utinomi, 1967) Balanus trigonus Darwin, 1854 x x Introduced Young (1994) AP - RS Oliveira (1941) Cosmopolitan x x Cryptogenic? MA - RS Darwin (1854) Cosmopolitan x x x Introduced first record MA RN PB PE AL BA RJ PR SC Young (1989) Cosmopolitan x x x Introduced Young (1994) AP - RS Darwin (1854) Cosmopolitan Observation

71 274 Latin American Journal of Aquatic Research (continuation) Family Species IYC PP PCP Status Previous records for SSC Records for Brazilian states First record for Brazil Known distribution Observation Chthamalidae Darwin, 1854 Megabalanus coccopoma (Darwin, 1854) Megabalanus tintinnabulum (Linnaeus, 1758) Chthamalus proteus Dando & Southward, 1980 Tetraclitidae Gruvel, 1903 Newmanella radiata (Bruguière, 1789) Tetraclita stalactifera (Lamarck, 1818) x x x Introduced Young (1994) RN ES RJ SP PR RS SC Lacombe & Monteiro (1974) W. Atlantic, Indo- Pacific x x x Cryptogenic Luederwaldt (1929) MA - RS Linnaeus (1758) Cosmopolitan x Native? MA - RS Dando & Southward (1980) W. Atlantic x x Cryptogenic? PE BA RJ SP Lacombe (1977) Cosmopolitan x Native Luederwaldt (1929) MA - RS Darwin (1854) W. Atlantic Crustacea Decapoda Pleocyemata Caridea Palaemonidae Rafinesque, 1815 Periclemenes longicaudatus (Stimpson, 1860) Hippolytidae Bate, 1888 Thor manningi Chace, 1972 Alpheidae Rafinesque, 1815 Synalpheus sp. x x Cryptogenic x x Native? AP - SC Rathbun (1900) as Urocaris longicauda x x Native Christoffersen (1980) PB, BA, SP Fausto-Filho (1970 ) as T. floridanus W. Atlantic W. Atlantic, Central Atlantic, E. Pacific Commonly found on grass flats from the tide line to a depth of at least 11 m Crustacea Decapoda Pleocyemata Anomura Porcellanidae Haworth, 1825 Pachycheles monilifer (Dana, 1852) Pisidia brasiliensis Haig in Rodrigues da Costa, 1968 x x x Native Melo (1999) CE - SC Cano (1889) as Pachycheles moniliferus x x Native Melo (1999) PA - SP Coelho (1964) as Megalobrachium poeyi W. Atlantic, E. Pacific From shallow waters to 40 m W. Atlantic (Brazil) Intertidal Crustacea Decapoda Pleocyemata Brachyura Inachidae MacLeay, 1838 Stenorhynchus seticornis (Herbst, 1788) Majidae Salmouelle, 1819 Microphrys bicornutus (Latreille, 1825) Epialtidae MacLeay, 1838 Epialtus bituberculatus H. Mil i ne - Edwards, 1834 Pilumnidae Salmouelle, 1819 Pilumnus quoii H. Milne- Edwards, 1834 Pilumnus dasypodus Kingsley, 1879 Grapsidae MacLeay, 1838 Pachygrapsus transversus (Gibbes, 1850) x x Native Melo (1996) AP - RS Miers (1886) as Leptodia sagittaria x Native Melo (1996) MA - RS Smith (1869) as Milnia bicornuta x x Native Melo (1996) CE, PB, PE, BA, ES, RJ, SP x x x Native Melo (1996) as P. quogi W. Atlantic From shallow waters to great depths W. Atlantic From shallow waters to 70 m Rathbun (1894) W. Atlantic Shallow water species living on algae and seagrass meadows, on hard bottoms, and tidal pools AP - SP Milne-Edwards (1834) W. Atlantic From shallow waters to 100 m x x x Native Melo (1996) CE - SC Rathbun (1900) W. Atlantic From shallow waters to 30 m x x x Cryptogenic Melo (1996) MA - RS Rathbun (1898) W, E. Atlantic, Mediterranean, E. Pacific Shallow waters

72 Exotic benthic species in São Sebastião, Brazil 275 ( c ontinuation) Family Species IYC PP PCP Status Previous records for SSC Records for Brazilian states First record for Brazil Known distribution Observation Entoprocta Barentsidae Hincks, 1880 Barentsia capitata Calvet, 1904 Pedicellinidae Johnston, 1847 Sangavella vineta Marcus, 1957 Mollusca Bivalvia Lamellibranchia Pteroida Mytilidae Rafinesque, 1815 Isognomonidae Woodring, 1925 Perna perna (Linnaeus, 1758) Brachidontes exustus (Linnaeus, 1758) Brachidontes solisianus (D'Orbigny, 1846) Myoforceps aristatus (Dillwyn, 1817) Isognomon bicolor (C.B. Adams, 1845) Pteriidae Gray, 1847 Pteria hirundo (Linnaeus, 1758) Pinctata imbricata Roding, 1798 Mollusca Bivalvia Lamellibranchia Myoida Myidae Lamarck, 1809 Sphenia antillensis Dall & Simpson, 1901 Mollusca Gastropoda Eogastropoda Patellogastropoda Lottiidae Gray, 1840 Collisella subrugosa (D'Orbigny, 1846) x x Native first record RJ Marcus (1940) SW. Atlantic x Native first record SP Marcus (1957) Brazil x x Introduced Migotto et al. (1993) ES RJ SP PR SC RS x Native? CE RN BA RJ SP Atlantic From intertidal to 10 m depth Atlantic Shallow waters x Native Migotto et al. (1993) AP - RS Atlantic Shallow waters x x Introduced first record RJ SP Simone & Gonçalves (2006) x x Introduced Rios (2009) CE RN RJ SP SC Domaneschi & Martins (2002) Atlantic, Pacific From intertidal to 5 m depth Atlantic From intertidal to 7 m depth x Native first record AP - RS Atlantic Shallow waters x Native Rios (1975) PA - SC Atlantic Shallow waters x Native Migotto et al. (1993) CE - SC Atlantic Shallow waters x x Native Migotto et al. (1993) CE - RS Atlantic Shallow waters Mollusca Gastropoda Orthogastropoda Vetigastropoda Fissurellidae Fleming, 1822 Diodora dysoni (Reeve, 1850) Fissurella clenchi Farfante, 1943 Mollusca Gastropoda Orthogastropoda Caenogastropoda Littorinidae Gray, 1840 Littorina ziczac (Gmelin, 1791) Columbellidae Swainson, 1840 Muricidae Rafinesque, 1815 Littorina flava King & Broderip, 1832 Anachis sertulariarum D Orbigny, 1841 Mitrella dichroa Sowerby, 1844 Stramonita brasiliensis Claremont & Reid, 2011 x x Native first record RN - SC Atlantic Shallow waters x Native Migotto et al. (1993) PA - RS Atlantic Shallow waters x Native Migotto et al AP - RS Atlantic From intertidal to 5 m x x Native Rios (1975) MA - RS Atlantic From intertidal to 6 m x Native Rios (1975) CE AL BA ES RJ SP PR x Native Duarte & Nalesso (1996) AL RJ SP PR SC Atlantic Shallow waters Atlantic Shallow waters x Native Salvador et al. (1998) AP - RS Atlantic Shallow waters

73 276 Latin American Journal of Aquatic Research (Table 2). By taxon status, half of the native and more than half of the cryptogenic species were in only one site, while only 20% of the introduced species were from one site and 40% in two or three sites (Table 3). The fifteen introduced species are Polychaetes (Branchiomma luctuosum), Ascidiacea (Clavelina oblonga, Distaplia stylifera, Aplidiopsis sp., Ascidia sydneiensis, Styela plicata, Eusynstyela sp.), Cnidaria (Carijoa riisei), Crustacea Cirripedia (Amphibalanus amphitrite, Amphibalanus reticulatus, Megabalanus coccopoma, Balanus trigonus), and Mollusca Bivalvia (Myoforceps aristatus, Isognomon bicolor, Perna perna). Three introduced species are reported here for the first time in Brazilian waters (D. stylifera, Aplidiopsis sp., Eusynstyela sp.). DISCUSSION Although the SSC may have the best known marine fauna in Brazil (Migotto & Marques, 2006), at least 14 species found, 9.8% of the total, are new records (four polychaetes, two ascidians, one cnidarian, one bryozoan, one barnacle, two entoprocts and three mollusks). Clearly, more taxonomical studies are necessary even for the well-known regions. Of the 15 recognized introductions, only eight were previously listed in Lopes (2009), with the remaining seven either not included in that publication (C. oblonga, C. riisei), considered cryptogenic (A. amphritite, B. trigonus), or are new introductions (D. stylifera, Aplidiopsis sp., Eusystyela sp). All the 10 introductions recorded in Ilha Grande Bay (Ignacio et al., 2010) were also found in SSC, evidence of the established condition of these species in the coast of Brazil. Among them, one bryozoan species reported by Ignacio et al. (2010), Schizoporella errata, is herein identified as Schizoporella pungens. Winston (2005) suggested S. errata to be part of a species complex that includes S. pungens, described from the Caribbean, and Schizoporella isabelleana (D Orbigny, 1842), described from Rio de Janeiro. The similarity among the species in the complex, the necessity of molecular techniques to confirm the identity of Brazilian specimens (as suggested by Tompsett et al., 2009) and the absence of additional studies on biogeography, led us to give a cryptogenic status for S. pungens. We also found Scrupocelaria aff. diadema but this is another complex of species in need of revision and without knowing which species is actually in Brazil we also prefered to give a cryptogenic status to the complex. It is important to note that most taxa were found in only one site (Table 2). Of the introduced taxa, most were in two or three sites (80%), and only 20% in one site (Table 3). Thus, distribution patterns of the introduced taxa are quite different than those of the native and cryptogenic taxa, and may indicate that the introduced are already widespread at the SSC, in the process of successful establishment. It is therefore urgent that monitoring in the region (as well as other major ports) be swiftly carried out to better understand whether those species are also successfully colonizing natural habitats and threatening the native species elsewhere. The study in Ilha Grande Bay showed that most introduced species also occurred on natural substrata in that region (Ignacio et al., 2010). Although the sites were not surveyed with the same effort, the number of species in each site was not very different (IYC = 89, PP = 70, PCP = 81). Subtidal areas are usually richer in species than intertidal, which is the case for IYC and PP, but effort in PP was much less then IYC and PCP. PCP survey was mainly intertidal. The fact the most species were found in only one site shows that sites were complementary in species composition and that the RAS should include different types of habitats to comprehensively survey a region. In this study, Ascidiacea has the greatest number of introduced species, followed by Cirripedia, and Bivalvia. However, if we base ranking on the proportion of introduced, relative to the total number per taxa, the order changes to Cirripedia, Bivalvia, and Ascidiacea (Table 4). In the SSC, Cirripedia is a critical case in which most taxa are introduced, and all introduced taxa are widespread (three species in three sites, one in two sites). These introduced species have been reported elsewhere along the Brazilian coast (Farrapeira, 2010a) and some are very old introductions (Carlton et al., 2011). The taxon Cirripedia certainly deserves attention, especially because of its difficult taxonomy and the few taxonomists that address biodiversity inventories (which may have caused other introduced species to be overlooked). The three introduced species of bivalves reported here were also recorded elsewhere in Brazil. Perna perna was probably introduced during intensive ship traffic between Africa and Brazil during the 1800 and 1900s; earlier sambaquis (coastal Indian fossil deposits) do not contain shells of this species (Souza et al., 2003). It is now established throughout the region between Espírito Santo and Santa Catarina and is cultivated for food. Myoforceps aristatus, widely distributed in the Atlantic Ocean, is a borer that lives in hard substrates and shells of other mollusks. It was first recorded in southeastern Brazil in 2006 (Simone & Gonçalves, 2006). Isognomon bicolor, from the Caribbean, has been seen on rocky coasts in Brazil

74 Exotic benthic species in São Sebastião, Brazil 277 Table 2. Number of species (percentage) present in one, two or three sites in the São Sebastião Channel Sum Annelida - Polychaeta 13 (76.5) 2 (11.8) 2 (11.8) 17 Ascidiacea 10 (41.7) 9 (39.1) 5 (21.7) 24 Bryozoa 20 (55.6) 8 (22.2) 8 (22.2) 36 Cnidaria 15 (55.6) 8 (30.8) 4 (15.4) 27 Crustacea - Cirripedia 2 (22.2) 3 (33.3) 4 (44.4) 9 Crustacea - Decapoda 1 (9.1) 6 (54.5) 4 (36.4) 11 Entoprocta 1 (50) 1 (50) 0 (0) 2 Mollusca - Bivalvia 5 (62.5) 3 (37.5) 0 (0) 8 Mollusca - Gastropoda 5 (62.5) 3 (37.5) 0 (0) 8 Total 72 (50.7) 44 (30.3) 26 (19.0) 142 Table 3. Number of species (percentage) by status present in one, two or three sites in the São Sebastião Channel Sum Native 24 (45.3) 20 (37.8) 9 (17.0) 53 Cryptogenic 40 (58.0) 18 (26.1) 11 (15.9) 69 Introduced 3 (20.0) 6 (40.0) 6 (40.0) 15 Not classified 5 (100) 0 (0) 0 (0) 5 Total 72 (50.7) 44 (30.3) 26 (19.0) 142 Table 4. Number of species (percentage) by status in the São Sebastião Channel. N: native, I: Introduced, C: Cryptogenic. N I C Not classified Annelida - Polychaeta 4 (23.5) 1 (5.9) 8 (47.1) 4 (23.5) Ascidiacea 3 (12.5) 6 (25.0) 15 (62.5) 0 (0) Bryozoa 17 (47.2) 0 (0) 18 (50) 1 (2.8) Cnidaria 3 (11.0) 1 (3.7) 23 (85.2) 0 (0) Crustacea - Cirripedia 2 (22.2) 4 (44.4) 3 (33.3) 0 (0) Crustacea - Decapoda 9 (81.8) 0 (0) 2 (18.2) 0 (0) Entoprocta 2 (100) 0 (0) 0 (0) 0 (0) Mollusca - Bivalvia 5 (62.5) 3 (37.5) 0 (0) 0 (0) Mollusca - Gastropoda 8 (100) 0 (0) 0 (0) 0 (0) Total 53 (37.3) 15 (10.6) 69 (48.6) 5 (3.5) since the 1990s and was first recorded in 1989 in Santa Catarina (Domaneschi & Martins, 2002). It is believed that this invasive bivalve was accidentally introduced between 1970 and 1980 by petroleum platforms, boat hulls or by ballast water of ships. Their rapid population growth must have occurred during the 1990s. The species has no commercial value but competes for the same habitat with other commercially valuable species, causing economic losses. In 2002/2003 it has reached very dense populations of more than 800 individuals 100 cm -2 (Breves-Ramos et al., 2010) in Rio de Janeiro, causing

75 278 Latin American Journal of Aquatic Research serious changes to the natural hard bottom benthic community, but suffered great mortality in this region in 2006 (Lopes et al., 2009). Ascidiacea, with the greatest number of introduced species in our study, includes well-known important and aggressive invasive species (McKindsey et al., 2007). Globally, the distributions of many introduced ascidians are restricted to artificial substrates in ports or marinas (Lambert & Lambert, 2003), but little is known about their impact on natural habitats. The cryptogenic Didemnum perlucidum, Diplosoma listerianum, Styela canopus, Microcosmus exasperatus, Herdmania pallida are members of this group. Styela plicata and Ascidia sydneiensis, are well known introduced species, both invading the bivalve commercial cultures in the state of Santa Catarina (Rocha et al., 2009). Eusynstyela sp. (previously identified as Eusynstyela floridana, Rodrigues et al., 1998) may be a new species that was probably introduced in Brazil during the 1990s. Distaplia stylifera was introduced during the last ten years, while the species previously identified as D. stylifera by Rodrigues et al. (1998) is another new and also introduced species. Clavelina oblonga was classified as cryptogenic, but a recent molecular study suggested that it is introduced (Rocha et al., 2012). The genus Aplidiopsis was not found in Brazil until this RAS. We were unable to identify it to species due to the lack of reproductive structures. Two species of polychaetes, Branchiomma (B. patriota and B. luctuosum) were previously identified as B. nigromaculatum (Baird, 1865) which was reported in coastal Brazil (Rullier & Amoureux, 1979; Duarte & Nalesso, 1996), including at SSC. Because of this erroneous identification, B. patriota was described only recently by Nogueira et al. (2006). According to these authors, B. luctuosum is an introduced species known in the SSC at least since 2009 (Amaral et al., 2010b), while its type locality is in the Red Sea and it is known to be an invasive species in Italy. The cryptogenic species of Spionidae, Polydora colonia, was first recorded in 2001 at Ilha do Mel, Paranaguá Bay, in the state of Paraná (Neves, 2006; Neves & Rocha, 2008). Its records in coastal North and Central America raise doubts as to whether it is an introduced species and hence we consider it to be cryptogenic. Many other species of polychaetes were classified as cryptogenic because of lack of knowledge about their life history and dispersal patterns. Eunice rubra has a wide distribution along the Brazilian coast, in addition to Gulf of Mexico and Caribbean Sea, what led us to consider the hypothesis that its natural dispersal area is throughout tropical and subtropical Western Atlantic, and thus it was classified as native. The other native species are those reported only to the Brazilian coast: Pseudonereis palpata, Nicolea uspiana and Branchiomma patriota. The cnidarian Carijoa riisei was considered native to the Caribbean until a recent molecular analysis showed that this species is actually Indo-Pacific in origin and the Atlantic records are therefore introductions (Concepcion et al., 2010). It is now very widespread along the Brazilian coast and can be considered naturalized. Decapod crustaceans, entoprocts, polychaetes and gastropods all have high proportions of native species (over 70%, see Table 4). Pachigrapsus transversus was previously known to occur on both sides of the Atlantic and in the eastern Pacific (Manning & Holthuis, 1981; Hendrickx, 1995; Melo, 1996; Poupin et al., 2005). Recently, Schubart et al. (2005), using morphological and genetic differences (16S mt DNA sequences), revalidated the species P. socius Stimpson, 1871 for the eastern Pacific, limiting the occurrence of P. transversus to the Atlantic Ocean. However, we maintained the status of the species as cryptogenic because it has been proposed that the different populations on both sides of Atlantic may be introduced in some areas. If so, to determine which populations are introduced will require further molecular studies. On the other hand, the decapod Pachycheles monilifera, native to Brazil, has been introduced in Ecuador, in the eastern Pacific (Veloso & Melo, 1993). In this study, Bryozoa has the greatest number of native species (17). Of these species, both Amathia distans and A. brasiliensis were reported as widespread in warm tropical waters, although a recent study suggested a restricted distribution of these taxa in the western Atlantic (Fehlauer-Ale et al., 2011). In addition, the four widespread species in the western Atlantic found in our study (A. distans, A. brasiliensis, Bowerbankia maxima and Biflustra sp.) were also found in pelagic algae and may be dispersed by algal rafting, as reported for other bryozoans (Taylor & Monks, 1997; Vieira et al., 2010). It is remarkable that while taxa with a longer history of surveys such as Crustacea Decapoda and Mollusca have a very small proportion of cryptogenic species, a large proportion of species of ascidians, bryozoans and cnidarians, are still considered cryptogenic. These taxa are typically found in small colonies that may have been overlooked in previous faunal studies, and they often comprise many species with wide geographic distribution. Thus the uncertainty of their status, also illustrating the need for periodic monitoring of areas sensitive to bioinvasion, comprehensive surveys of natural areas, and molecular

76 Exotic benthic species in São Sebastião, Brazil 279 studies to understand their geographical distribution. Also, some widespread bryozoans have been reported to be quite variable morphologically in disjoint areas, which suggest that, in species with short-lived larvae, a complex of cryptic species and hidden endemism may be common (Vieira et al., 2010). With the use of molecular tools, populations of a given species have been shown to be introduced in other areas (e.g., populations of the Atlantic Clavelina lepadiformis introduced in the Mediterranean Turon et al., 2003) and species previously considered widely distributed have been split in one or more new species with narrower geographical ranges (e.g., Thais haemastoma Claremont et al., 2011; Pachigrapsus transversus Schubart et al., 2005; Botryllus schlosseri Bock et al., 2012). We suggest that this kind of genetic monitoring is also important for the study of marine bioinvasions, and such studies are being conducted by our research group for ascidians, bryozoans, and cnidarians. Previous results from these molecular studies show that some species of these groups, now considered cryptogenic, may be instead introduced species or introduced populations of haplotypes. Therefore, the estimated number of introduced taxa should be thought to be very conservative and with continued study, many more species will be shown to be introduced. Here we demonstrate that RASs, even for reasonably well-known regions of Brazil, such as the São Sebastião Channel, are useful strategies to monitor and detect introduced species. We recommend that RASs be replicated on a large scale in all ports with moderate to heavy ship traffic. ACKNOWLEDGMENTS We thank the Center of Marine Biology of the University of São Paulo for hosting us during the field studies, the administration of the Ilhabela Yacht Club for allowing us to sample in the marina; Marcos Barbeitos and Carlos Rocha for their field help; Vasily Radashevsky for helping with the identification of the Spionidae Polydora colonia; James Roper for the English text review; María de Los Angeles Becerril for the Spanish abstract; Julia Beneti for information on Anthozoa. This study was an initiative of and primarily supported by the project CAPES-PROCAD 2007/150 Benthos in port areas along the Brazilian coast: biodiversity, phylogeography and aspects of bioinvasion by biofouling. ACM, JB, and TPM have support from FAPESP (2004/ ; 2006/ ; 2010/ ), CAPES (Pró-Equipamentos and Prodoc projects) and Boticário Foundation. RMR received a research grant from CNPq; APRR, ASK received a master scholarship from CAPES; LMV received a doctoral scholarship from FAPESP (2008/ ); LEAB received a post-doctoral scholarship from PNPD/CAPES and FACEPE (BCT /10). This contribution has the participation of several scientists of NP-BioMar, USP. REFERENCES Allman, G.J Report on the Hydroida dredged by H.M.S. Challenger during the years Part II. The Tubularinae, Corymorphinae, Campanularinae, Sertularinae, and Thalamophora. Rep. Scient. Res. Voy. Challenger Zool., 23: Alves, T.M Contribuição ao conhecimento taxonômico de Terebellidae e Trichobranchidae (Annelida: Polychaeta) da região sudeste-sul do Brasil. Dissertação do grau de Mestre. Instituto de Biociências, Universidade de São Paulo, 176 pp. Amaral, A.C.Z Anelídeos poliquetos do infralitoral em duas enseadas da região de Ubatuba - Aspectos Ecológicos. Tese do grau de Doutor, Universidade de São Paulo, São Paulo, 137 pp. Amaral, A.C.Z., S.A.H. Nallin & T.M. Steiner. 2010a. Catálogo das espécies de Annelida Polychaeta do Brasil. [ bentos_marinho\prod_cien-\texto_poli.pdf]. Reviewed: 20 January Amaral, A.C.Z., M.R. Denadai, A. Turra & A.E. Rizzo Intertidal macrofauna in Brazilian subtropical tide-dominate sandy beaches. J. Coastal Res., 35: Amaral, A.C.Z., A.E. Migotto, A. Turra & Y. Schaeffer- Novelli. 2010b. Araçá: biodiversity, impacts and threats. Biota Neotrop., 10(1): Amaral, F.M.D., C.M.R. Farrapeira, S.M.A. Lira & C.A.C. Ramos Benthic macrofauna inventory of two shipwrecks from Pernambuco coast, northeastern of Brazil. Rev. Nordest. Zool., 4(1): Bardi, J. & A.C. Marques The invasive hydromedusa Blackfordia virginica (Cnidaria: Blackfordiidae) in southern Brazil, with comments on taxonomy and distribution of the genus Blackfordia. Zootaxa, 2198: Bax, N., J.T. Carlton, A. Mathews-Amos, R.L. Haedrich, F.G. Howarth, J.E. Purcell, A. Riese & A. Gray The control of biological invasions in the world s ocean. Conserv. Biol., 15(5): Bock, D.G., H.J. MacIsaac & M.E. Cristescu Multilocus genetic analyses differentiate between widespread and spatially restricted cryptic species in

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83 Lat. Am. J. Aquat. Res., 41(2): , 2013 Taxonomical position of Lovenella gracilis Clarke, 1882 Proceedings of the 3 rd Brazilian Congress of Marine Biology A.C. Marques, L.V.C. Lotufo, P.C. Paiva, P.T.C. Chaves & S.N. Leitão (Guest Editors) DOI: /vol41-issue2-fulltext Research Article Taxonomic position of Lovenella gracilis Clarke, 1882 (Lovenellidae, Hydrozoa): new evidences of microanatomy justify its maintenance in the genus Lovenella Hincks, 1868 Thaís Pires Miranda 1, Amanda Ferreira Cunha 1 & Antonio C. Marques 1 1 Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo Rua do Matão Trav. 14, 101, , São Paulo, Brazil ABSTRACT. Lovenella gracilis Clarke, 1882 is one of the 15 nominal species referred to the genus Lovenella Hincks, 1868, a genus characterized by the presence of a basal line demarcating the separation between operculum and hydrotheca. However, Lovenella gracilis apparently does not have the demarcating line under light microscopy therefore, the resurrection of the genus Dipleuron Brooks, 1882 was proposed to accommodate this species. The goal of this study is to redescribe the polyp of L. gracilis trying to resolve this doubtful taxonomical status. Fertile colonies were collected in the intertidal zone of Rio Grande do Norte and Santa Catarina States, representing the first record of the species for the South Atlantic. Scanning electron microcopy of L. gracilis has shown a tenuous demarcation between operculum and hydrotheca, corroborating its position in the genus Lovenella. Considering the new evidences presented, we propose the maintenance of the species L. gracilis in the genus Lovenella, and corroborate the synonymy of Dipleuron and Lovenella. Keywords: Lovenella, Dipleuron, operculum, taxonomy, South Atlantic, Brazil. Posición taxonómica de Lovenella gracilis Clarke, 1882 (Lovenellidae, Hydrozoa): nuevas evidencias de microanatomía justifican su permanencia en el género Lovenella Hincks, 1868 RESUMEN. Lovenella gracilis Clarke, 1882 es una de las 15 especies nominales referidas al género Lovenella Hincks, 1868, un género caracterizado por la presencia de una línea basal separando el opérculo de la hidroteca. Sin embargo, la microscopia de luz reveló la ausencia de una demarcación entre el opérculo y la hidroteca de Lovenella gracilis, y por eso se propuso su transferencia al género Dipleuron Brooks, 1882, que resurgió para acomodar esta única especie. Este trabajo tiene como finalidad la redescripción del pólipo de L. gracilis con la intención de resolver este status taxonómico dudoso. Las colonias fértiles fueron colectadas en el intermareal de las provincias de Rio Grande do Norte y Santa Catarina y representan el primer registro de la especie para el Atlántico sur. Electromicrografias evidencian una discreta demarcación entre el opérculo y la hidroteca, corroborando la posición de L. gracilis en el género Lovenella. Con respecto a las nuevas evidencias de la morfología opercular presentadas, se propone la permanencia de L. gracilis en el género Lovenella, y se corrobora la sinonimia de Dipleuron y Lovenella. Palabras clave: Lovenella, Dipleuron, opérculo, taxonomía, Atlántico sur, Brasil. Corresponding author: Antonio C. Marques (marques@ib.usp.br) INTRODUCTION Leptothecate hydrozoans of the family Lovenellidae Russell, 1953 have a troubled taxonomical history (Calder, 1991; Bouillon et al., 2004). They have a metagenetic life cycle and, like in many other hydroid taxa, the parallel and independent use of the morphological characters of polyps and medusae

84 287 Latin American Journal of Aquatic Research eventually generated a dual classification, with different understandings and diagnoses for the, presumably, same genera. The genus Lovenella was proposed by Hincks (1868), based on the polyp stage, and assigned to the family Campanulariidae. Russell (1953), based on the medusa stage, proposed the family Lovenellidae including the genera Eucheilota McCrady, 1859 and Lovenella Hincks, The family Lovenellidae, as described by Russell (1953), includes medusae without marginal cirri, excretory pores or peduncle, with gonads on four simple radial canals, and with lateral cirri. Kramp (1959) proposed the genus Cirrholovenia as a third genus for the Lovenellidae, based on the presence of marginal cirri in the medusa, amending the original diagnosis of the family. Other disputable arrangements have also been proposed for the family, even comprising genera traditionally included in the family Haleciidae Hincks, 1868, such as Campalecium Torrey, 1902 and Hydranthea Hincks, The genus Lovenella Hincks, 1868, type species Lovenella clausa (Lóven, 1836), comprises 14 nominal species (Tables 1 and 2) distributed worldwide (Figs. 1 and 2). Only L. chiquitita Millard, 1957 and L. corrugata Thornely, 1908 were recorded for the South Atlantic hitherto. The main diagnostic characters of Lovenella are the medusae with indefinite number of statocysts and polyp with hydrotheca well demarcated by a basal line, separating it from the operculum (Fraser, 1944; Kramp, 1959; Bouillon et al., 2004). Oddly, the polyp of Lovenella gracilis Clarke, 1882 is defined by the operculum being a continuation of the hydrothecal wall, therefore lacking a basal line separating operculum and hydrotheca (Calder, 1971, 1975). Based on this unique character, Calder (1991) proposed the resurrection of the genus Dipleuron Brooks, 1882 in order to encompass L. gracilis. Bouillon & Boero (2000) and Bouillon et al. (2004) did not agree with this proposal, arguing that the medusa of L. gracilis presents the typical characters of the genus and that the diagnostic characters of the polyps of lovenellid are puzzling, since the opercular structure can be variable within the family, and even within the same genus. No other addition was made to the knowledge of the morphology of L. gracilis, and the taxonomical status of the species remains doubtful. The aim of this study is to redescribe in detailed morphology the polyp of L. gracilis, based on the first material of the species sampled for the South Atlantic, and reveal new data corroborating its maintenance in the genus Lovenella. MATERIALS AND METHODS The material studied was collected in the intertidal zone of Tibau Beach (Tibau, State of Rio Grande do Norte, Brazil) and Bombas Beach (Bombinhas, State of Santa Catarina, Brazil). The colonies were fixed in 92.5% ethanol and 4% formaldehyde solution. We have studied the morphology, morphometry and cnidome of all specimens. Morphological details were studied in scanning electronic microscopy (SEM), following routine protocol (Migotto & Marques, 1999). The cnidome was studied with squashed preparations of the fixed material, in light microscopy. Studied material has been deposited in the Cnidarian Collection of the Museu de Zoologia of the Universidade de São Paulo (MZUSP), São Paulo, Brazil. RESULTS Lovenella gracilis Clarke, 1882 (Figs. 3a-3d; 4a-4f). Lovenella gracilis Clarke, 1882, p. 139, pl. 9, fig ; Fraser, 1944, p. 174, pl. 31, fig. 147; Calder, 1971, p. 61, pl. 4, fig. h, pl. 8, fig. b-c; 1975, p. 298, fig. 3c. Dipleuron parvum Brooks, 1882, p. 135, Lovenella clausa Fraser, 1910, p. 364, fig. 26a-d; 1912, p. 45 [non Lovenella clausa (Lóven, 1836)]. Dipleuron gracilis Huvé, 1952, p. 389, fig. 1a-b, 2ab; Calder, 1991, p. 3. Material examined. Santa Catarina, Bombinhas, Bombas Beach (27.131ºS ºW, 2 m, 3.xii.2006) MZUSP4242, in formaldehyde 4%, without gonophores, on rock and Sargassum sp.; MZUSP4260, in ethanol 92.8%, without gonophores, on rock; MZUSP4263, in ethanol 92.8%, with gonophores, on rock; MZUSP4266, in formaldehyde 4%, with gonophores, on rock and Sargassum sp. Rio Grande do Norte, Tibau, Tibau Beach (4.835ºS, ºW, intertidal zone, on Donax striatus, in ethanol 92.8%) MZUSP5356, with gonophores, 5.vi.2004; MZUSP 5357, MZUSP5358, with gonophores, 15.ix.2004; MZUSP5359, with gonophores, 9.iii Description. Colonies stolonal or erect, up to 19 mm (n = 10) in height, arising directly from creeping hydrorhiza µm (n = 10) in diameter. Hydrocaulus monosiphonic, with 0-6 annulations (n = 10) at the proximal region, branched or unbranched, divided into internodes by transverse septa at more or less regular intervals. Perisarc of main stem moderately thick, thinner at secondary branches and pedicels. Internode length µm (n = 10), diameter

85 Taxonomical position of Lovenella gracilis Clarke, Table 1. Comparison of the diagnostic characters of species of Lovenella with polyp stage recorded in literature. Species Shape of colony Growth Septa per internode Annulations on pedicel Hydrothecae Frequency of hydrothecae regeneration Shape of operculum valves Number of operculum valves Diaphragm Arising of Nematophores Gonothecae gonothecae References L. chiquitita Millard, 1957 Stolonal or erect Sympodial Deepcampanulate - Throughout Occasionally Triangular with rounded base elongated, widening distally 8-10 Oblique Absent Smooth, From hydrorhiza Millard (1957, 1975) L. clausa (Lóven, 1836) Stolonal or erect 2-5 on proximal and distal Sympodial - regions Cylindrical, widening distally 2-3 times Triangular with rounded base Smooth, 8-10 Transversal Absent elongated, widening distally Axillary from pedicel García Corrales et al. (1979); Cornelius (1995) L. corrugata Thornely, 1908 Erect Sympodial on proximal and distal regions Deepcampanulate, corrugated proximally 2-4 times Triangular with rounded base Ringed, 8-12 Oblique Absent spindle shaped Axillary from pedicel Vervoort (1959); Millard (1980) L. gracilis Clarke, 1882 L. grandis Nutting, 1901 Erect Sympodial on proximal and distal regions Erect Sympodial - Not specified Campanulate - Conical 8 Transversal Absent Smooth, clavate Cylindrical, widening distally - Triangular with rounded base Absent Smooth, elongated, widening distally Axillary from pedicel Axillary from pedicel Clarke (1882); Calder (1971, 1975) Nutting (1901); Fraser (1941) L. nodosa Fraser, 1938 Erect Sympodial on distal region Cylindrical, widening distally - Triangular with rounded base Smooth, 8 - Absent tubular, widening distally Axillary from pedicel Fraser (1938) L. paniculata (G.O. Sars, 1874) Erect Sympodial - On proximal region Cylindrical, widening distally - Conical - Oblique Absent - - Sars (1874) L. producta (G.O. Sars, 1874) Stolonal Monopodial on proximal and distal regions, sometimes, in the middle Cylindrical, widening distally - Conical Up to 12 Transversal Present - - Sars (1874); Cornelius (1995) L. rugosa Fraser, 1938 Stolonal or erect Sympodial - Throughout Deepcampanulate - Triangular with rounded base Smooth, 9-10 Oblique Absent elongated, widening distally From hydrorhiza Fraser (1938)

86 289 Latin American Journal of Aquatic Research Table 2. Comparison of diagnostic characters of species of Lovenella with medusa stage recorded in literature. Species Umbrella Velum Manubrium Number of gonads Position of gonads Morphology of gonads Number of tentacles Number of statocysts Number of marginal vesicles Number of marginal bulbs Number of cirri Position of cirri References L. annae (von Lendenfeld, 1884) Broader than high - Small, globular, with 4 interradial spots 4 Near radial canals Oval clusters On each side of tentacles Kramp (1961) L. assimilis (Browne, 1905) Broader than high Narrow Short 4 On distal end of radial canals Oval clusters On each side of tentacles Browne (1905); Hirano & Yamada (1985) L. bermudensis (Fewkes, 1883) Wide, low, without raised apex - Short and wide 4 On stomach Spherical On each side of tentacles Fewkes (1883); Kramp (1959) L. chiquitita Millard, 1957 Higher than wide - Short Millard (1975) L. clausa (Lóven, 1836) ca. ¼ of the bell radius Short and small 4 On distal end of radial canals Oval and longitudinally divided On each side of tentacles Kramp (1959); Cornelius (1995) L. gracilis Clarke, 1882 Hemispherical Hemispherical Wide Short 4 On midway of 2 radial canals Spherical Clarke (1882); Calder (1971) L. haichangensis Xu & Huang, 1983 Hemispherical - Short 4 On 2/3 of the distal portion of radial canals Slender linear pairs On each side of tentacles and marginal bulbs Xu & Huang (1983) L. sinuosa Lin, Xu, Huang & Wang, 2009 Flatter than hemispherical On proximal portion of manubrium Linear, longitudinally divided pairs On each side of tentacles Lin et al. (2009)

87 Taxonomical position of Lovenella gracilis Clarke, Figure 1. Distribution of the polyp stage of the species of the genus Lovenella Hincks, : Millard (1957, 1975); García Corrales et al. (1979); 2: Hincks (1868); García Corrales et al. (1979); Cornelius (1995); 3: Thornely (1908); Jäderholm (1920); Vervoort (1959); Millard (1980); 4: Clarke (1882); Huvé (1952); Calder (1971, 1975); Bandel & Wedler (1987); Manning & Lindquist (2003); Dougherty & Russell (2005); Calder & Cairns (2009); 5: Nutting (1901); Fraser (1941, 1944); 6: Fraser (1938); Lees (1986); Calder et al. (2009); 7: Sars (1874); Picard (1955); 8: Fraser (1937, 1938, 1944); Vervoort (1985); Cornelius (1995); Schuchert (2000); 9: Fraser (1938, 1939, 1948); Calder et al. (2009). The symbol? refers to the doubtful record of Picard (1955) concerning L. (?)paniculata. Figure 2. Distribution of the medusa stage of the species of the genus Lovenella Hincks, : von Lendenfeld (1887); Kramp (1961); 2: Browne (1905); Kramp (1959, 1961); Bouillon (1984, 1995); Hirano & Yamada (1985); Navas-Pereira & Vannucci (1991); Xu et al. (2008); Xu (2009); 3: Fewkes (1883); Kramp (1959); 4: Millard (1975); 5: Hincks (1871); Russell (1936a, 1936b, 1953); Kramp (1959, 1961); 6: Clarke (1882); Brooks (1882); Calder (1971); 7: Xu & Huang, (1983); 8: Lin et al. (2009).

88 291 Latin American Journal of Aquatic Research Figure 3. Light microscopy of Lovenella gracilis Clarke, a) General aspect of the colony from Rio Grande do Norte (Scale: 1 mm), b) detail of the hydrothecae (Scale: 200 µm), c) general aspect of a portion of the hydrocaulus of the colony of Santa Catarina, with internodes and hydrothecal pedicel arising from distal apophysis (Scale: 200 µm), d) detail of the hydrothecae and hydrocaulus (Scale: 100 µm) µm (n = 10), with 1-7 septa (n = 10), supporting hydrothecal pedicel arising from distal apophysis. Apophyses alternate; branches or additional pedicels, when present, arising laterally to the apophysis. Pedicels either annulated throughout or with 2-11 (n = 10) distal annulations, length µm (n = 10), diameter µm (n = 10). Hydrotheca campanulate, µm (n = 10) deep from rim to base, µm (n = 10) wide at margin, µm (n = 10) wide at diaphragm; diaphragm thin, transversal; operculum with 8-11 triangular to pentagonal valves (n = 10), apparently as folded continuation of hydrothecal wall, but with discrete line demarcating operculum from hydrotheca (only in SEM). Gonothecae inverted cone-shaped, length µm (n = 10), diameter at margin µm (n = 10), diameter at base µm (n = 10); walls smooth, distal region of gonothecae deepened, with a central aperture. Gonothecal pedicels short, length µm (n = 10), diameter µm (n = 10), with 2-8 annulations (n = 10) throughout, arising near base of hydrothecal pedicels or directly from hydrorhiza; several medusa buds in each gonotheca, but some gonothecae empty. Nematocysts of one type: small microbasic mastigophores, dimensions 6-7 µm X µm (n = 10, undischarged capsules). Distributional range. North Atlantic (Clarke, 1882; Brooks, 1882; Fraser, 1910, 1912, 1944; Calder, 1971,

89 Taxonomical position of Lovenella gracilis Clarke, ; Manning & Lindquist, 2003; Bouillon et al., 2004; Dougherty & Russell, 2005), Gulf of Mexico (Calder & Cairns, 2009), Caribbean Sea (Bandel & Wedler, 1987), Mediterranean Sea (Huvé, 1952; Picard, 1958; Bouillon et al., 2004). DISCUSSION Clarke (1882: 139) described the polyp and medusa of Lovenella gracilis for Chesapeake Bay, uncertain of its relationships and systematic position when compared to L. clausa (Lóven, 1836). Indeed, Fraser (1910, 1912) mistakenly assigned North Carolina and Massachusetts specimens of L. gracilis to L. clausa; but he corrected himself after examining further material, noting that both species are distinct and that the European species L. clausa has not been observed in the Western Atlantic (Fraser, 1944: 174). Concomitantly to Clarke s description of L. gracilis, Brooks (1882) described the new genus Dipleuron, and its type-species D. parvum, based on a medusa found at North Carolina coast. Huvé (1952), based on Mediterranean material, considered L. gracilis and D. parvum similar, adopting the name Dipleuron gracilis because Lovenella would not be a valid genus since the type species L. clausa was linked to the medusa of Eucheilota hartlaubi by Russell (1936a). However, as explained by Calder (1971: 64) Eucheilota and Lovenella are not congeneric, and the medusa E. hartlaubi has since been shown to be a Lovenella [ ]. Life cycle studies of L. gracilis eventually revealed that its medusa stage is indistinguishable from D. parvum as described by Brooks (1882) (Calder, 1971). Then, Dipleuron was reaffirmed as junior synonym of Lovenella, with the actual name L. gracilis Clarke, Figure 4. Scanning electron microscopy of Lovenella gracilis Clarke, a) General aspect of the colony (Scale: 200 µm), (b-d) detail of the hydrothecae (Scales: b, c, 100 µm; d, 50 µm), e) detail of the demarcation between hydrotheca and operculum (Scale: 50 µm). f) gonotheca arising from hydrocaulus (Scale: 100 µm).

90 293 Latin American Journal of Aquatic Research 1882 having priority over Dipleuron parvum Brooks, However, Calder (1991) reconsidered this synonymy when referring to L. gracilis, arguing that Dipleuron and Lovenella would be distinct because of differences in the morphology of their opercula (Calder, 1991: 3). Under light microscopy, the operculum of L. gracilis is a continuation of the hydrothecal wall, without demarcation (cf. Calder, 1971, 1975; Bouillon et al., 2004). The original definition of the genus Lovenella has no mention to a basal line demarcating the operculum (Hincks, 1868), therefore potentially accommodating L. gracilis. Amending diagnoses, however, have defined Lovenella by the presence of this line demarcating the operculum (Calder, 1991; Cornelius, 1995), a notable characteristic of most of the species of the genus (Millard, 1957; Cornelius, 1995). Based on this pattern, the absence of the demarcation in L. gracilis justified its transference to Dipleuron (Calder, 1991). A refinement of the morphological study was necessary. We have found specimens representing the first record of L. gracilis for the South Atlantic and Brazilian coast [cf. Migotto et al., 2002; even though Stechow (1914) recorded Gonothyraea (?)nodosa, a disputable and inconclusive similar hydroid for Rio de Janeiro coast, to which we prefer not to make inferences about its taxonomic status]. Scanning electron microscopy of this Brazilian L. gracilis revealed the presence of a tenuous line separating the operculum from the hydrotheca (Fig. 4), making it clear it is a Lovenella species. Therefore, considering the troubled taxonomy of the family Lovenellidae and the new evidence presented herein, we propose the maintenance of the genus Dipleuron Brooks, 1882 as a junior synonym of Lovenella Hincks, ACKNOWLEDGEMENTS We would like to thank Dr. Helena Matthews-Cascon (Universidade Federal do Ceará) for providing material from Rio Grande do Norte, Dr. Peter Schuchert (Muséum d Histoire Naturelle) and Dr. Yayoi Hirano (Chiba University) for the help with literature, and Enio Mattos (Universidade de São Paulo) for technical support. This study was supported by CAPES Procad, Prodoc e Pró-Equipamentos 1887/ 2007, CNPq (Proc /2006-8, /2009-7, /2009-7, /2010-6, /2010-7) and FAPESP (Proc. 2004/ , 2006/ , 2010/ ). This is a contribution of NP-BioMar, USP. REFERENCES Bandel, K. & E. Wedler Hydroid, amphineuran and gastropod zonation in the littoral of the Caribbean Sea, Colombia. Senck. Marit., 19(1-2): Bouillon, J Hydroméduses de la mer de Bismarck (Papouasie Nouvelle-Guinée). Partie IV: Leptomedusae (Hydrozoa-Cnidaria). Indo-Malayan Zool., 1: Bouillon, J Hydromedusae of the New Zealand Oceanographic Institute (Hydrozoa, Cnidaria). N.Z. J. Zool., 22: Bouillon, J. & F. Boero Synopsis of the families and genera of the hydromedusae of the world, with a list of the worldwide species. Thalassia Salentina, 24: Bouillon, J., M.D. Medel, F. Pagès, J.M. Gili, F. Boero & C. Gravili Fauna of the Mediterranean Hydrozoa. Sci. Mar., 68(Suppl. 2): Brooks, W.K List of medusae found at Beaufort, N.C., during the summers of 1880 and Studies Biol. Lab., Johns Hopkins Univ., 2: Browne, E.T Report on the Medusae (Hydromedusae, Scyphomedusae and Ctenophora) collected by Prof. Herdman at Ceylon in Rep. Government of Ceylon on Pearl Oyster Fisheries of the Gulf of Manaar, 4(27): Calder, D.R Hydroids and Hydromedusae of southern Chesapeake Bay. Virginia Inst. Mar. Sci., Spec. Pap. Mar. Sci., 1: Calder, D.R Biotic census of Cape Cod bay: hydroids. Biol. Bull., 149(2): Calder, D.R Shallow-water hydroids of Bermuda. The thecatae, exclusive of Plumularioidea. Life Sci. Contr. R. Ontario Mus., 154: Calder, D.R. & S.D. Cairns Hydroids (Cnidaria: Hydrozoa) of the Gulf of Mexico. In: J.W. Tunnell, D.L. Felder & S.A. Earle (eds.). Gulf of Mexico origin, waters, and biota. Texas A&M University Press, USA, pp Clarke, S.F New and interesting hydroids from Chesapeake Bay. Mem. Boston Soc. Natur. Hist., 3: Cornelius, P.F.S North-west European thecate hydroids and their medusa. Part 1. In: R.S.K. Barnes & J.H. Crothers (eds.). Synopses of the British fauna. The Linnean Society of London and The Estuarine and Coastal Sciences Association, Shrewsbury, 347 pp. Dougherty, J.R. & M.P. Russell The association between the coquina clam Donax fossor Say and its epibiotic hydroid Lovenella gracilis Clarke. J. Shellfish Res., 24(1):

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93 Lat. Am. J. Aquat. Res., 41(2): , 2013 Effects of the brown algae metabolites on sea urchin Proceedings of the 3 rd Brazilian Congress of Marine Biology A.C. Marques, L.V.C. Lotufo, P.C. Paiva, P.T.C. Chaves & S.N. Leitão (Guest Editors) DOI: /vol41-issue2-fulltext Research Article Effects of the secondary metabolites from Canistrocarpus cervicornis (Dictyotales, Phaeophyceae) on fertilization and early development of the sea urchin Lytechinus variegatus Fredy A. Ortiz-Ramírez 1, Magui Aparecida Vallim 1 Diana Negrão Cavalcanti 1 & Valéria Laneuville Teixeira 1 1 Programa de Pós-Graduação em Biologia Marinha, Departamento de Biologia Marinha Instituto de Biologia, Universidade Federal Fluminense CP , Niterói, RJ, , Brazil ABSTRACT. Marine organisms are rich sources of natural products that, among other activities, help to maintain the species equilibrium. Samples of the marine brown alga Canistrocarpus cervicornis (Kützing) De Paula & De Clerck were collected in Búzios, Rio de Janeiro, Brazil, in June The extract was obtained in CH 2 Cl 2 and subjected to fractionation by chromatographic methods in order to isolate and purify the compound (4R, 7R, 14S)-4α, 7α-diacethoxy-14-hydroxydolastane-1(15), 8-dien. Then, the effects of the extract and the dolastane diterpene on zygotes and gametes of the sea urchin Lytechinus variegatus were evaluated. The exposure of male and female gametes to the C. cervicornis extract promoted, respectively, a reduction of 10-30% in fertilization and a 20 to 70% decrease in the number of eggs. Furthermore, the exposure of zygotes to the extract inhibited their development up to 86.7 ± 1.6% (at a concentration of 250 µg ml -1 ), as well as generating abnormalities in 39-50% of zygotes. The results of the dolastane diterpene showed no evidence of inhibition in the zygotes development, thought it was proved to induce anomalies. At higher concentrations (25 and 50 µg ml -1 ), it was possible to observe cell lyses. Keywords: natural products, diterpenes, brown seaweed, dolastanes, Lytechinus variegatus, Brazil. Efectos de los metabolitos secundarios de Canistrocarpus cervicornis (Dictyotales, Phaeophyceae) sobre la fertilización y desarrollo embrionario temprano del erizo del mar Lytechinus variegatus RESUMEN. Los organismos marinos son fuentes ricas en productos naturales que, entre otras actividades, ayudan a mantener el equilibrio entre las especies. Se colectaron muestras de Canistrocarpus cervicornis (Kützing) De Paula & De Clerck en Armação de Buzios, Estado de Rio de Janeiro, Brasil, durante junio de 2006 y se sometieron a extracción en CH 2 Cl 2 hasta obtener un extracto bruto. Mediante técnicas tradicionales de fitoquímica se aisló y purificó el producto natural mayoritario dolastano (4R, 7R, 14S)-4α, 7α-diacetoxi-14- hidroxidolasta-1(15),8-dieno. Posteriormente, se evaluaron los efectos en diferentes concentraciones del extracto bruto en gametos y del extracto bruto y dolastano en cigotos del erizo de mar Lytechinus variegatus. Los resultados sugieren que cuando los gametos son expuestos al extracto bruto se promueve una disminución en las tasas de fecundación, 10 a 30% (espermatozoides) y de 20 a 70% (óvulos). Además, cuando los cigotos fueron expuestos al extracto bruto se inhibió el desarrollo en un 86,7% ± 1,6 (250 µg ml -1 ) y se generaron cigotos anormales que variaron entre 39 y 50%. Los resultados de los experimentos con el dolastano no mostraron evidencias de inhibición del desarrollo embrionario. Sin embargo, se constató la inducción de anomalías y en las mayores concentraciones (25 y 50 µg ml -1 ) se registró el evento de lisis en los cigotos. Palabras clave: productos naturales, dolastanos, algas pardas, diterpenos, Lytechinus variegatus, Brasil. Corresponding author: Fredy A. Ortiz-Ramírez (faortizr@gmail.com)

94 297 Latin American Journal of Aquatic Research INTRODUCTION In the course of evolution, marine organisms have developed different strategies to ensure their survival. As a consequence, many of these species are rich sources of natural products that can help keep the balance among species (Harper et al., 2001). Such substances have also biotechnological potential as antibiotic, antiviral, cytotoxic and antitumor (Blunt et al., 2010). The brown algae (Phaeophyceae, Ochrophyte) are very abundant in the tropical region, especially the Fucales and the Dictyotales. More than 1,140 secondary metabolites were reported for them. Among these, there are rich sources of terpenes, especially diterpenes, belonging to the order Dictyotales (Maschek & Baker, 2008), and around 300 diterpene shave been isolated from at least 35 species of the family Dictyotaceae collected worldwide (Vallim et al., 2005). These algae are chemically characterized by the production of dolastane and secodolastane diterpenes which are obtained from the cyclization the precursor geranilgeraniol between positions 1 and 11 (Teixeira et al., 1986a, 1986b; Teixeira & Kelecom, 1988; De Paula, 2001; Vallim et al., 2005; De Paula et al., 2007). Canistrocarpus cervicornis is known for exhibiting a chemical profile composed by dolastane and the secodolastane diterpenes (Teixeira et al., 1986a, 1986b; Teixeira & Kelecom, 1988; Oliveira et al., 2008). The studies revealed that some of these secondary metabolites inhibit the sodium potassium ATPase (Garcia et al., 2009), as well as present antiviral (Vallim et al., 2010), antifouling (Bianco et al., 2009), and anti-herbivore (Pereira et al., 2002; Bianco et al., 2010) activities. Interdisciplinary studies have shown that the algae are not passive participants in the biological interactions between producers and consumers. Given the development of new methods of chemical analysis and adequate experimental drawings, it is widely accepted that algae have chemical defense mechanisms against their consumers and also against threats caused by their intra-specific relations, which makes them important agents in the structure of marine communities (Potin et al., 2002). Thus, the capacity of chemical defense of some algae (Coilodesme californica (Ruprecht) Kjellman, Dictyota flabellata (F.S. Collins) Setchell & N.L. Gardner, Laurencia obtusa (Hudson) J.V. Lamouroux, etc.) against herbivores has been shown (Steinberg, 1985; Hay & Fenical, 1988; Pereira et al., 2003). In fact, the ecological relations mediated by natural products from macro algae have been widely studied. Therefore, numerous types of diterpenes were pointed out as chemical defenses capable to inhibit the herbivory (Pereira et al., 2004; Vallim et al., 2007; Pereira & Da Gama, 2008; Bianco et al., 2010). Also, diterpene alcohols found in algae from the genus Dictyota reduce the growth of herbivorous fishes (Hay et al., 1988), as well as decreasing the survival, growth or reproduction of many species of amphipod (Cruz-Rivera & Hay, 2003). However, the consequences of their effects on the mechanisms of fecundation and embryonic development to population control of herbivores have been little studied. In this context, C. cervicornis has also shown strategies of chemical defense against the gastropod Astraea latispina (Pereira et al., 2002) and the sea urchin Lytechinus variegatus (Bianco et al., 2010). However, ecological studies relating the chemical components of C. cervicornis and its effects on gametes and early life stages of L. variegates are not yet known. The aim of the present work was, thus, to evaluate the effect of the crude extract in dichloromethane (CH 2 Cl 2 ) from C. cervicornis and its major diterpene (4R, 7R, 14S)-4α, 7α-diacethoxy-14-hydroxydolastane-1(15), 8-dien on the gametes and early life stages in vitro of the sea urchin L. variegatus. MATERIALS AND METHODS Algal material Canistrocarpus cervicornis was collected in June 2006 at Praia do Forno, Armação de Búzios, Rio de Janeiro State ( S, W) by snorkeling. Intact samples of alga were cleaned from epiphytic organisms and washed with sea water. The specimens were deposited at the Herbarium of the Universidade do Rio de Janeiro (HRJ10754). Extraction and isolation The air-dried algae were extracted five times with 100% CH 2 Cl 2 at room temperature (25 C) for 24 h. The solvent was evaporated under reduced pressure, yielding a brownish residue (14 g). Crude extract (5 g) was subjected to silica gel chromatography (4 x 40 cm) and eluted with a gradient of n-hexane to methanol in order to obtain 228 fractions. Fractions F 97 to F 99 (771 mg) were combined and subjected to silica gel column chromatography and eluted with n- hexane and ethyl acetate (4:6), affording the pure compound (4R, 7R, 14S)-4α, 7α-diacethoxy-14- hydroxydolastane-1(15), 8-dien (Fig. 1). The diterpene was identified by comparison of physical and spectroscopic data ( 13 C and 1 H NMR data) with reported values (Sun et al., 1981).

95 Effects of the brown algae metabolites on sea urchin 298 OAc OH OAc Figure 1. Structure of the major dolastane diterpene of Canistrocarpus cervicornis. All solvents were HPLC grade. Analytical thinlayer chromatography (TLC) separations were carried out on Merck silica gel 60 F-254 (0.2 mm) percolated aluminum plates. Once developed, plates were visualized by spraying 2% ceric sulphate in sulfuric acid, followed by gentle heating. Silica gel 60 (Merck, and mesh) was used for column chromatography. NMR spectra were recorded in CDCl3 (100% Aldrich) on a Varian Unity Plus 300 spectrometer using TMS as internal standard. Collection of gametes and storage Lytechinus variegatus were collected in Itaipú Beach, Niterói, Rio de Janeiro, Brasil ( S, W) by snorkeling. Eggs and sperm had been obtained by previous spawning induction with injections of 1-3 ml KCl 0.5M. Eggs stocks (16,000 eggs ml -1 ) were prepared in artificial seawater Sea Red (ASW) and the salinity was adjusted to 32 psu adding ultra-pure water (Milli-Q ). Sperm were obtained by direct extraction with a pipette Pasteur and stock solutions (1:9 ASW) were preserved at refrigeration in Eppendorf (2-5ºC). Crude extract and dolastane solutions Stock solution of the crude extract (500 µg ml -1 in DMSO) were diluted at 10 ml ASW and it was utilized for preparing sequential dilutions of 250, 125, 62.5 and µg ml. Stock solution of the dolastane (100 µg ml -1 in DMSO) was diluted at 10 ml ASW and it was utilized for preparing sequential dilutions of 50, 25, 12.5 and 6.25 µg ml -1. Lytechinus variegatus embryonic development One ml of stock solution of motile sperm were added to the egg suspension and carefully stirred for 30s to allow fertilization. Afterwards, fertilization was confirmed by observation of the fertilization envelope in at least 80% of the eggs after 5 min. 1 ml of fertilized eggs were deposited in plates of 3 ml in triplicate for both treatment and control. Then, we added 1 ml of the solutions of crude extract and dolastane isolated from C. cervicornis to treatment, whereas in control we used 1 ml of ASW. Eggs were incubated at a final volume of 2 ml a 25 ± 2 C. After two hours, we added 2 drops of 10% formaldehyde. This time was sufficient for zygotes reach to cleavage IV. Finally, the unfertilized eggs, fertilized not divided, eggs cleaved in I, II, III, IV stages and abnormal zygotes (A) were counted from aliquots of 1 ml of each sample in camera Sedgwick-Rafter, on 10 random points. This method was adapted from tests used in cytotoxicity, ecotoxicity, and preliminary studies of pharmacological activity (Lera et al., 2006; Semenova et al., 2006; Kiselyov et al., 2010; Magalhães et al., 2010). Eggs pre-fertilization tests 1 ml of eggs and 1 ml of each dilution of the extract of C. cervicornis were deposited in plaques of 3 ml capacity. After 10 min, a solution with 20 µl of sperm was added and maintained at mild agitation for 30 sec. Each experiment was performed in triplicate and 1 ml of ASW was added to control. The incubation period, the fixation and counting of zygotes followed the same procedure previously described. Sperm pre-fertilization tests Twenty µl of sperm solution and 1 ml of each dilution of the extract of C. cervicornis were deposited in plaques of 3 ml capacity. After 10 min, a solution with 1 ml of eggs was added and maintained at mild agitation for 30 sec following the same procedure previously described. Statistical analysis The results of the experiments with crude extract and dolastane were analyzed by nonparametric Kruskal- Wallis. In the pre-fertilization tests, multiple comparisons were performed using the statistical analysis Infostat 2009 (Di Rienzo et al., 2009), and then analyzed by Wilcoxon (Mann-Whitney U) when significant differences were recorded. All statistical tests were conducted according to Zar (1996), adopting the significance level α < RESULTS Lytechinus variegatus embryonic development The embryonic development experiments started after that the elevation of the fertilization membrane was confirmed in at least 80% of the eggs (Fig. 2b). The unfertilized eggs, the ones fertilized without cleavage, the zygotes in cleavages I-IV and the anomalous (A)

96 299 Latin American Journal of Aquatic Research were evaluated in each experiment and typical examples of these categories can be found in Fig. 2. The results demonstrated that, at all concentrations, the number of fertilized eggs not cleaved increased in proportion to the concentration of the crude extract. Besides, most of the few eggs that reached cleavage generated anomalous, which resulted in impracticability of the development up to cleavage IV. Indeed, cleavages III and IV presented significant differences (P < ) when compared to control, since we observed merely between zero and 1% of individuals on cleavage III and, consequently, no eggs on cleavage IV. The effect of the addition of the crude extract at the smallest concentration caused an increase in the percentage of anomalous, whereas the greatest concentrations caused a very strong inhibition of cleavages, even though it was not accompanied by a rise in the percentage of anomalous. The results of the tests with the dolastane revealed that there were no significant differences (P > 0.05) between treatment and control, except for the variables III, IV and A. The differences found in cleavages III and IV correspond to the greatest concentrations (25 y 50 µg ml -1 ), which the value of proportions was overestimated due to the lyses of zygotes. Therefore, the proportion of 9.85 ± 8.8% was registered in cleavage IV for the concentration of 25 µg ml -1 (dolastane), while a 0.8 ± 1.4% rate was registered for the concentration of 50 µg ml -1 (dolastane). The results of anomalous zygotes were established differently for the crude extract and dolastane. The response caused by the crude extract was inversely proportional to the concentration; in other words, the smallest concentration caused the highest anomaly rates. On the other hand, the response caused by different dilutions of dolastane did not have significant difference in values of anomalous zygotes (P > 0.05). However, at all dilutions tested, the presence of anomalous was found with significant difference compared to the control. This indicates that this secondary metabolite can be responsible for the anomalies observed in the experiments with crude Figure 2. Early stages of the embryonic development of Lytechinus variegatus and events registered during experiments. a) Unfertilized egg, b) egg fertilized without cleavage, c) first cleavage, two blastomeres, d) second cleavage, four blastomeres, e) third cleavage, eight blastomeres, f) fourth cleavage, 16 blastomeres, g) abnormal zygotes, and h) lyses.

97 Effects of the brown algae metabolites on sea urchin 300 extract, especially in the smallest concentrations of the extract ( µg ml -1 ) (Table 1). Pre-fertilization tests In our experiments, the male gametes showed less resistance to the compounds of C. cervicornis than the female at the smallest concentration (15.62 µg ml -1 ). The increase of the concentration of the crude extract in the treatment of gametes before fecundation promoted a rise in the number of unfertilized eggs. Indeed, the sperm were susceptible to the exposure to crude extract, since the percentage of unfertilized eggs was superior to 70% in all concentrations tested. No significant difference was noticed among these concentrations (P > 0.05). On the other hand, when the feminine gametes were submitted to different concentrations of crude extract, the rates of unfertilized ova was considerably higher than in control, ranging between 30 and 86%, except for the smallest concentration (15.62 µg ml -1 ), in which this percentage was similar to control (Table 2). In all experiments, both pre-fertilization and zygote, our results showed that both crude extract and dolastane induced the appearance of anomalous zygotes, characterized by asymmetric plans of cleavage and atypical proliferations even at low concentrations (Fig. 2g and Fig. 3). DISCUSSION Our results indicate that the gametes have become unviable for the process of syngamy during the prefertilization experiments, reducing the fertilization rates of 10 to 30% in the tests with sperm and of 20 to 70% in the tests with eggs. Indeed, some of the substances extracted from marine algae have shown to affect fertilization. The fucoidans, for instance, inhibit the penetration of the male gametes into the human eggs (Oehninger et al., 1991; Patankar et al., 1993). Nonetheless, the octadecapentanoic acid, a polyunsaturated fatty acid uncommon in algae, has not demonstrated any capacity of derailing the gametes of the sea urchin Paracentrotus lividus in the fertilization processes, yet it has proved to inhibit the embryonic development in the first cleavage (Fériel et al., 2000). In our experiments, the male gametes showed less resistance to the compounds of C. cervicornis than the female. It is relevant to notice that the sperm of the sea urchin are motionless when inside the gonads, only when they get in contact with saline is the motion activated as the intracellular ph is alkalinized and the ATPase, enzyme that promotes the mobility of zygotes in response to the mitochondrial respiration, is activated. Taking this into consideration, it is possible to explain the impossibility of the gametes to realize the fertilization processes since two dolastanes Table 1. Percentage of zygotes according to development stages in the experiments with raw extract (CE) and dolastane (D): EWC: eggs without cleavage, I IV: cleavage, and A: abnormal. CE (µg ml -1 ) EWC (%) I (%) II (%) III (%) IV (%) A (%) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± v ± 4.1 Control 27.9 ± ± ± ± ± ± 0.0 D EWC I II III IV A ± ± ± v 12.6* 0.8 ± 1.4* 9.1 ± ± ± ± ± 7.9* 9.8 ± 8.8* 12.9 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 9.9 Control 6.5 ± ± ± ± ± ± 0.0 *Proportion overestimated due to the lyses of zygotes.

98 301 Latin American Journal of Aquatic Research Table 2. Percentage of unfertilized eggs in the prefertilization experiments with raw extract. Average ± standard deviation. Abnormal zygotes (%) Treatment (µg ml -1 ) Experiment Sperm (%) Ovule (%) ± ± ± ± ± ± ± ± ± ± ± ± 2.2 Control Code of the concentration Zygote (D) zygote (CE) sperm (CE) ovule (CE) Figure 3. Percentages of abnormal zygotes in experiments with raw extract (CE) and dolastane (D): the code of the concentration 1 to 5 represent at dilutions detailed on material and methods, from less concentration to high concentration. isolated from C. cervicornis inhibited Na + K + -ATPase (García et al., 2009). In the experiments to evaluate the effects of crude extract and dolastane on the embryonic development, the crude extract inhibited the mitotic cycles of the zygotes of L. variegatus even at the smallest concentration, while dolastane showed no clear evidences of inhibition. In fact, extracts in acetone from the marine algae Botryocladia occidentalis, Ulva fasciata, Gracilaria lemaneiformis and Hypnea musciformis have demonstrated the ability to inhibit the embryonic development of L. variegatus in more than 50% at the concentration of 100 µg ml -1 (Torres et al., 2005). Therefore, our results indicate that C. cervicornis presents a greater inhibitory effect than other algae, even at lower concentrations. Given the synchronous divisions and the uniformity at the first cleavages, the tests with zygotes of sea urchins are sufficiently sensitive to identify and differentiate the toxic effects of the antimitotic compounds (Jacobs et al., 1981). In addition, the presence of clear points in the cytoplasm is attributed to the blocking of the coupling of microtubules (Jacobs & Wilson, 1986). In spite of the inhibitory effects of the crude extract on the mitotic cycle, the cytoplasm of the zygotes revealed a homogeneous aspect, which presented no clear points in the cytoplasm. Therefore, the inhibitory action of crude extract can be related to a different mechanism than the blocking of the coupling of microtubules. On the other hand, both crude extract and dolastane induced the appearance of anomalous zygotes, characterized by asymmetric plans of cleavage and atypical proliferations even at low concentrations (Fig. 2g and Fig. 3). When the gametes were exposed to the highest concentration of crude extract, the inhibition of the cleavages caused an increase in the number of fertilized eggs without cleavage (EWC, Fig. 2b), providing a deceiving impression that the anomalies were reduced. It was at the highest concentration (250 µg ml -1 ), that the most significant inhibition occurred, more than 80% of the fertilized eggs did not reach cleavage and the cleaved ones became anomalous. In the tests with dolastane, as previously mentioned, it was possible to notice a similar formation of anomalous at all the concentrations tested. Nevertheless, the greatest abnormality rates were found in the experiments with crude extract (Table 1). During their development, embryos and larvae from marine invertebrates have a more accelerated metabolism when compared to adults. Their metabolic rates might increase according an order of magnitude before the metamorphosis or settlement (Hoegh- Guldberg & Manahan, 1995). In addition, experiments in vitro with L. pictus revealed that the activity of the enzyme Na + K + -ATPase gradually raises in the early life stages of this organism, reaching a peak of 80% (prisma stage) (Leong & Mahanan, 1999). If the inhibition of this enzyme may affect the transportation of ions, the action of C. cervicornis on the activity of Na + K + -ATPase could provoke disorders in the electrochemical gradient compromising all the cellular processes that depend on ions. Considering that an antimitotic agent blocks the mitosis whereas a cytotoxic agent damages the cells exposed to it, our results indicate that the crude extract of C. cervicornis presents both antimitotic and cytotoxic effects, although the latter was more evident to the tests with dolastane due to the registration of lyses in the highest concentrations (Table 1).

99 Effects of the brown algae metabolites on sea urchin 302 The alterations on the operation of the Na + K + pump might explain the lyses of the embryos, verified when the zygotes were submitted to the highest concentration of the dolastane. As the pump Na + K + is considered the main active transport system in the majority of animal cells, its inhibition causes conditions that favor the intracellular accumulation of Na + (Aizman et al., 2001). This fact may create a hypertonic environment that leads to lyses. Still, other experiments are necessary in order to achieve a deeper comprehension of these events. One of the main challenges in marine biology and ecology is the demographic understanding of the organisms that present planktonic and benthic stages (Pechenik, 1991) and the influence of biological interactions on the size of the population. In this sense, it has recently been discovered that chemical signs of the alga Delisea pulcra (Williamson et al., 2000) induce the metamorphosis and the recruiting of the larvae of invertebrates. The same way, it was demonstrated that some species of Dictyota present chemical defenses that can facilitate their perpetuation on reefs by competition for space, showing that the early life stages of some corals, may be vulnerable to allelopathic effects (Paul et al., 2011). Nevertheless, the question that remains is: are the capable of avoiding or defending themselves against the new young recruits of their consumers? However, complementary studies must be conducted to elucidate this question. Our results in vitro indicated, therefore, that the compounds of the crude extract of C. cervicornis act on the sea urchin L. variegatus in two ways: 1) reducing fertilization rates, and 2) inhibiting embryonic development. ACKNOWLEDGEMENTS FAO-R thanks CAPES for providing a PhD fellowship. DNC thanks CNPq for post-doctoral degree. We are grateful to CNPq and FAPERJ for financial support and Productivity Fellowships to VLT and finally, we all thank to Dr. Joel Campos de Paula for his help in the identification of the marine alga material. REFERENCES Aizman, O., P. Ulhen, M. Lal, H. Brismar & A. Asperia Ouabain, a steroid hormone that signals with slow calcium oscillations. Proc. Nat. Acad. Sci., 98(23): Bianco, E.M., R. Rogers, V.L. Teixeira & R.C. Pereira Antifoulant diterpenes produced by the brown seaweed Canistrocarpus cervicornis. J. Appl. Phycol., 21: Bianco, E.M., V.L. Teixeira & R.C. Pereira Chemical defenses of the tropical marine seaweed Canistrocarpus cervicornis against herbivory by sea urchin. Braz. J. Oceanogr., 58: Blunt, J.W., B.R. Copp, M.H.G. Munro, P.T. Northcote & M.R. Prinsep Marine natural products. Nat. Prod. Rep., 27: Cruz-Rivera, E. & M.E. Hay Prey nutritional quality interacts with chemical defenses to affect consumer feeding and fitness. Ecol. Monogr., 73: De Paula, J.C., A.G. Pedrini, M.D. Pinheiro, R.C. Pereira & V.L. Teixeira Chemical similarity between the brown alga Dictyota cervicornis and D. pardalis (Dictyotales, Phaeophyta). Biochem. Syst. Ecol., 29: De Paula, J.C., V. Cassano, Y. Yoneshigue-Valentin & V.L. Teixeira Diterpenes from Brazilian brown alga Dictyota crispata (Dictyotaceae, Phaeophyta). Nat. Prod. Commun., 2: Di Rienzo, J.A., F. Casanoves, M.G. Balzarini, L. Gonzalez, M. Tablada & C.W. Robledo Grupo InfoStat, FCA, Universidad Nacional de Córdoba, Argentina. Fériel, S., D. Pesando, G. Bodennec, A. El Abed & J.-P. Girard Toxic effects of Gymnodinium cf. mikimotoi unsaturated fatty acids to gametes and embryos of the sea urchin Paracentrotus lividus. Wat. Res., 34(2): Garcia, D.G., E.M. Bianco, M.C. Santos, R.C. Pereira, M.V. Faria & V.L. Teixeira Inhibition of mammal Na + + K + -ATPase by diterpenes extracted from the Brazilian brown alga Dictyota cervicornis. Phytother. Res., 23: Harper, M.K., T.S. Bugni, B.R. Copp, R.D. James, B.S. Lindsay, A.D. Richardson, P.C. Schnabel, D. Tasdemir, R.M. Van Wagoner, S.M. Verbitski & C.M. Ireland Introduction to the chemical ecology of marine natural products. In: J.B. McClintock & B.J. Baker (eds.). Marine chemical ecology. CRC Press, Boca Raton, pp Hay, M.E & W. Fenical Marine plant-herbivore interactions: the ecology of chemical defense. Ann. Rev. Ecol. Syst., 19: Hoegh-Guldberg, O. & D.T. Manahan Coulometric measurement of oxygen consumption during development of marine invertebrate embryos and larvae. J. Exp. Biol., 198: Jacobs, R.S. & L. Wilson Fertilized sea urchin egg as a model for detecting cell division inhibitors. In: A. Aszalor & M. Dekker (eds.). Modern analysis of antibiotics. New York, pp

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102 Lat. Am. J. Aquat. Res., 41(2): , 2013 Analysis of two biomarkers in Sciades herzbergii Proceedings of the 3 rd Brazilian Congress of Marine Biology A.C. Marques, L.V.C. Lotufo, P.C. Paiva, P.T.C. Chaves & S.N. Leitão (Guest Editors) DOI: /vol41-issue2-fulltext Research Article Integrated analysis of two biomarkers in Sciades herzbergii (Ariidae, Siluriformes), to assess the environmental impact at São Marcos Bay, Maranhão, Brazil Débora Batista Pinheiro-Sousa 1, Zafira da Silva de Almeida 1 & Raimunda Nonata Fortes Carvalho-Neta 1 1 State University of Maranhão, Department of Chemistry and Biology, Laboratory of Fishing Biodiversity and Population Dynamics of Fish, Campus Paulo VI, University City P.O. Box 09, Tirirical, São Luís, Maranhão, Brazil ABSTRACT. Guribu catfish (Sciades herzbergii) is a resident species in estuaries of Maranhão, Brazil. The aim of this work was to determine the feasibility of integrated analysis of the branchial lesions and gonadosomatic index of Sciades herzbergii in order to evaluate the effects of pollutants in São Marcos' Bay. The first site (S1) is located near the Ilha dos Caranguejos and was used as a reference area for being an environmental protection area. The second site (S2) is located near the ALUMAR/ALCOA port, and was used as a potentially polluted area. Fish were collected at each site, forty-eight in S1 and forty in S2. Gills were fixed in 10% formalin, and usual histological techniques were used in the first right gill arch, with inclusion in paraffin and sections of 5 µm thickness. There were no histopathological changes in animals captured at the reference site. However, in those catfish collected in the potentially contaminated area it was observed several branchial lesions, such as lifting of the lamellar epithelium, fusion of some secondary lamellae, hypertrophy of epithelial cells and lamellar aneurysm. The analysis using the gonadosomatic index (GSI) showed significant differences, being higher in fish analyzed from the reference area (P < 0.05). The branchial lesions and GSI were sensitive for monitoring environmental impacts of different locations at São Marcos Bay, Maranhão, Brazil. Keywords: biomonitoring, branchial lesions, GSI, catfish, Ilha dos Caranguejos, Brazil. Análisis integrado de dos biomarcadores en Sciades herzbergii (Ariidae, Siluriformes) para evaluar el impacto ambiental en la Bahía de San Marcos, Maranhão, Brasil RESUMEN. El bagre guribú (Sciades herzbergii) es una especie residente en los estuarios del Maranhão, Brasil. El objetivo de este trabajo fue determinar la viabilidad de un análisis integrado de las lesiones branquiales y el índice gonadosomático de Sciades herzbergii, para evaluar los efectos de los contaminantes en la bahía de San Marcos. El primer sitio (S1), situado cerca de la Ilha dos Caranguejos, fue utilizado como zona de referencia, por ser un área de protección ambiental. El segundo sitio (S2) situado cerca del puerto ALUMAR/ALCOA, fue utilizado como una zona potencialmente contaminada. En cada sitio se recogieron peces, cuarenta y ocho en S1 y cuarenta en S2. Las branquias fueron fijadas en formol al 10% y las técnicas histológicas habituales fueron empleadas en el primer arco branquial derecho, con inclusión en parafina y secciones de 5 µm de espesor. En los peces capturados en el sitio de referencia no hubo ningún cambio histopatológico. Sin embargo, en los bagres de la región contaminada se encontró un amplio rango de alteraciones branquiales, como por ejemplo, el desprendimiento del epitelio, fusión y plegamiento lamelar, hipertrofia del epitelio y aneurisma lamelar. El análisis del índice gonadosomático (GSI), mostró diferencias significativas, siendo mayor en los peces analizados en el área de referencia (P < 0,05). Las lesiones branquiales y el GSI fueron sensibles para el monitoreo del impacto ambiental de diferentes lugares en la bahía de San Marcos, Maranhão, Brasil.

103 306 Latin American Journal of Aquatic Research Palabras clave: biomonitoreo, lesiones branquiales, GSI, bagre, Ilha dos Caranguejos, Brasil. Corresponding author: Débora Batista INTRODUCTION The monitoring of aquatic ecosystems with biomarkers is responsible for identifying impacts and biologic changes in organisms (Mozeto & Zagato, 2006). Biomarkers are defined as cellular changes, biochemical, molecular or physiological, which are measured in cells, body fluids, tissues or organs within an organism, and are indicative of exposure and doses of xenobiotics that lead to biological effects (Lan & Gray, 2001). The contaminants effects in fish can be express in various levels of biological organization, including physiological dysfunction, structural changes in organs and tissues and behavioral alteration that lead to impaired growth and reproduction (Adams, 1990). The gill lesions are used as sensitive biomarkers of environmental impacts on fish (Stentiford et al., 2003), and it has been recognized, by many researchers, that histopathological examination is a valuable tool for assessment of environmental impacts on fish populations (Teh et al., 1997). Those morphologic alterations could occur because the gill of the fish is in permanent contact with the environment (Heath, 1995). The detection of early warning signals through branchial lesions is ecologically relevant, economic and faster, and it has the potential to be used as a type of biomarker. In the south and southeast of Brazil, there are already some studies using different types of biomarkers (Amado et al., 2006; Camargo & Martinez 2006; Umbuzeiro et al., 2006; Zanette et al., 2006; Valdez-Domingos et al., 2007). These researches indicate the need for biomarkers to diagnose the key impacts in aquatic ecosystems. In this context, some biomarkers have been frequently used in programs for evaluating the impact on aquatic ecosystems, because they have well-founded methodology, generating answers in a short time, with low cost of analysis and highly sensitive (Freire et al., 2008). In São Luís (Maranhão), a region that has the largest port with cargo movement in Brazil, studies using biomarkers in Sciades herzbergii from São Marcos Bay has indicated the necessity of continuing this type of analysis (Carvalho-Neta & Abreu-Silva, 2010). The economic importance of this species and the pollution of the port are factors that suggest the need for biomonitoring this bay. Thus, the aim of this work was to determine the feasibility of integrated analysis of the branchial lesions and gonadosomatic index of Sciades herzbergii in order to evaluate the effects of pollutants in São Marcos' Bay. MATERIALS AND METHODS Site description and sample collection Two samples were collected in the seasonal period from August 2010 to April 2011 in two distinct sites in São Marcos Bay. The first site (S1) is located near the Crabs Island ( S, W) and was used as a reference area for being an environmental protection area. The second site (S2), located near the ALUMAR/ALCOA port (02º43 14 S, 44º23 35 W), was used as a potentially impacted area (Fig. 1). The catfish were captured in their natural habitat in three points of each area (S1 and S2) using gill nets, approximately twenty-four hours in each sampled area. We collected forty fish in the potentially impacted area and forty-eight fish in the reference area. The collected animals at each sampling location were placed in plastic bags properly labeled, identified and sealed, placed in coolers containing ice and transported to the laboratory, about 40 km. Fish were dissected and their gills were fixed immediately in 10% formalin. Water chemistry parameters (salinity, ph, temperature, dissolved oxygen, dissolved oxygen saturation and turbidity) were measured directly in the field. Analysis of biometric data The total length (LT), fork length (LF), total weight (WT) and gonad weight (WG) were recorded. After measured and weighed, the specimens of fish were opened for macroscopic observation and classification of the gonads, considering the scale of gonadal stages of development given by Vazoller (1996) and modified by Carvalho-Neta & Castro (2008): EG1 (immature), EG2 (in maturation our repose), EG3 (mature) and EG4 (exhausted). Gonadosomatic index (GSI) was calculated as follows: (gonad weight 100) / total weight (Vazzoler, 1996). Histopathological analysis In the laboratory, the gills were fixed in 10% formalin and kept in 70% alcohol until histological processing. For this, the first right gill arch was dehydrated in ascending series of alcohols, cleared in xylene,

104 Analysis of two biomarkers in Sciades herzbergii 307 Atlantic Ocean Figure 1. Sampling locations for Sciades herzbergii in São Marcos Bay, indicating the reference area (S1), and the potentially contaminated site (S2). impregnated and embedded in paraffin. The tissue sections were stained with hematoxylin-eosin. Four tissue sections from each fish were examined by Zeiss light photomicroscope. Histopathological lesions were classified according to the diagnostic criteria of Bernet et al. (1999). Statistical analysis The analysis of environmental parameters was made by comparing seasonal data (dry season and rainy season) in the potentially contaminated area with the reference site. The results of the fish biometric data were expressed as mean ± standard deviation for males and females, and compared using the Student t- test. The level of significance was RESULTS Average values of abiotic variables registered from São Marcos Bay during the two collections were grouped into dry season and rainy season, as is shown in Table 1. The salinity was found to be uniform in both sampling sites, decreasing during the rainy season. The dissolved oxygen and oxygen saturation were always lower at the potentially contaminated area (S2). The values for ph and turbidity were constants for both areas, demonstrating the homogeneity of these abiotic factors in both areas. Results of the statistical analysis of the biometric data for males and females of Sciades herzbergii, during the dry (August 2010) and rainy (April 2011) seasons, in the two sites (S1 and S2) in São Marcos Bay, can be seen in Tables 2 and 3 respectively. The data indicate that total and fork length of fish caught in the potentially contaminated site (S2) were significantly lower (P < 0.05) than those of the reference site (S1). However, the gonadosomatic index (GSI) showed significant differences between the two fish groups. The GSI in fish from the contaminated site was significantly lower (P < 0.05) than in control fish during all phases of the gonadal cycle. The results of gonadal stages of fish captured during the rainy and dry season are shown in Table 4. The data showed fish from the reference area in all gonadal stages, but in potentially contaminated site juveniles (EG1) were not found. The histopathological analysis in Sciades herzbergii sampled during the dry (August 2010) and rainy (April 2011) seasons from Ilha dos Caranguejos (reference area) showed no morphological changes in gills of the catfish (Fig. 2). However, individuals caught in the potentially contaminated area showed several histopathological changes (Fig. 3). The most important change found in the gills of S. herzbergii was lamellar narrowing and epithelial lifting of the primary lamella (Table 5). Histopathological seasonal variation was not detected in the potentially contaminated site (P < 0.05). DISCUSSION The environmental parameters of the two sample sites indicated that waters of the São Marcos Bay are uniform in terms of salinity and ph. This characteristic pattern of the salinity and ph was found by Lopes (2005) in the same region. The lowest concentrations of dissolved oxygen were found in the area of influence of the port. The low levels of dissolved oxygen are considered unsuitable for estuarine waters according to the Brazilian Agency for Water Quality Legislation (CONAMA,

105 308 Latin American Journal of Aquatic Research Table 1. Environmental parameters analyzed at collection site in each region of São Marcos Bay, Maranhão, during dry (August 2010) and rainy (April 2011) seasons. Parameter Reference Potentially contaminated Dry season Rainy season Dry season Rainy season Temperature ( C) Salinity (UPS) ph Dissolved oxygen (ml L -1 ) % Saturation of dissolved oxygen Turbidity (NTU) Table 2. Biometric data of males and females of Sciades herzbergii collected in reference area and potentially contaminated area of São Marcos Bay during dry (August 2010) season. Parameters Mean ± Standard deviation Reference Potentially contaminated (Dry season) (Dry season) Females Males Females Males LT (cm) ± 6.64* ± ± ± 2.54 LF (cm) ± 5.70* ± ± ± 2.36 WT (g) ± 45.10* ± 70.19* ± ± Wg (g) 6.25 ± ± ± ± 0.95 GSI 2.46 ± 1.28* 1.80 ± 0.09* 0.37 ± ± 0.07 *indicates significant difference relative to the contaminated site (P < 0.05). Total number of animals: 88. Number of females in: S1: 18; S2: 25. Number of males in: S1: 22; S2: 23. Biometric data: LT: total length; LF: fork length; WT: total weight; Wg: gonad weight, and GSI: gonadosomatic index. Table 3. Biometric data of males and females of Sciades herzbergii collected in reference area and potentially contaminated area of São Marcos Bay during rainy (April 2011) season. Parameter Reference (Rainy season) Mean ± Standard deviation Potentially contaminated (Rainy season) Females Males Females Males LT (cm) ± 2.4* ± ± ± 6.87 LF (cm) ± 2.04* ± ± ± 6.06 WT (g) ± 36.10* ± 14.17* ± ± 3.35 Wg (g) ± 1.95* 9.14 ± 4.04* 5.41 ± ± 1.59 GSI 1.53 ± 0.86* 1.16 ± 0.11* 0.56 ± ± 0.42 * Indicates significant difference in relation to the contaminated site (P < 0.05). Total number of animals: 88; Number of females in: S1: 18; S2: 25; Number of males in: S1: 22; S2: 23. Biometric data: LT: total length; LF: fork length; WT: total weight; Wg: gonad weight, and GSI: gonadosomatic index.

106 Analysis of two biomarkers in Sciades herzbergii 309 Table 4. Gonad stage (males and females) of Sciades herzbergii captured São Marcos Bay during dry (August 2010) and rainy (April 2011) season. Reference Potentially contamined Gonad stage Dry season Rainy season Dry season Rainy season %F %M %F %M %F %M %F %M EG EG EG EG Total number of animals: 88. Number of females in: S1: 18; S2: 25. Number of males in: S1: 22; S2: 23. Gonadal stages: EG1: immature, EG2: in maturation our repose, EG3: mature, and EG4: exhausted, F: females; M: males. Figure 2. a) Photomicrograph of the gill of Sciades herzbergii sampled during the dry season (August 2010) and rainy season (April 2011), in São Marcos Bay with no morphological changes (arrow), b) detail of gill filaments showing one primary (lp) and secondary lamellae (ls). Scale bar = 20 µm. 2005). In the Ilha dos Caranguejos values were registered inside of the limits considered normal in an estuary (CONAMA, 2005). The abiotic data, such as water temperature, conductivity, ph, dissolved oxygen and turbidity can change the fish richness and assemblage composition (Fialho et al., 2008). These can also be affected by anthropogenic impacts (Penczac et al., 1994). The similar data for turbidity, salinity and ph recorded for the two analyzed areas indicate a dynamic region, where the winds, tides and river discharges determine a high load of particulate matter. On the other hand, previous studies on sediment and water in the potentially contaminated area showed significantly higher levels of mercury and chrome which confirms that port area in São Marcos Bay is a site with high exposure risks for some contaminants (Carvalho-Neta et al., 2012). The results of the biometric data of S. herzbergii in São Marcos Bay showed significant differences between the potentially contaminated site (S2) and the reference area (S1). In the same period, both males and females of S1 were higher in total weight, total length, and fork length, when compared with individuals of S2 (P < 0.05). The gonadosomatic index (GSI) was also higher in the S1 than the one from the S2 in both periods analyzed. These results indicate a greater reproductive activity in S1, since higher values of GSI expressed appropriate maturation of the gonads. In a similar study carried out by Carvalho-Neta & Abreu-Silva (2010), the GSI in S. herzbergii, from the potentially contaminated site (São Marcos Bay), was significantly lower than in control fish during all phases of the gonadal cycle. Several studies have showed a decrease in the gonadosomatic index of fish from the contaminated site (Mayon et al., 2006). Xenobiotics cause problems to the endocrine and reproductive system of fish, directly affecting the development of gametes and their viability (Kime, 2000). Intersex and atresia in fish have been

107 310 Latin American Journal of Aquatic Research Figure 3. Photomicrographs of the gill of Sciades herzbergii caged in São Marcos Bay with branchial lesions. a) Lamellar narrowing (arrow), b) epithelial lifting of the primary lamella (arrow), c) fusion of secondary lamellae (arrow), d) lamellar aneurysm (arrow). Scale bar = 20 µm. Table 5. Occurrence of lesions in gills of S. herzbergii sampled during dry (August 2010) and rainy (April 2011) seasons from São Marcos Bay. Lesions Reference Potentially contamined Dry season Rainy season Dry season Rainy season Lamellar narrowing % 62% Epithelial lifting of the primary lamella % 25% Fusion of secondary lamella 0 0 4% 7% Lamellar aneurysm 0 0 4% 6% Total % 100% investigated in terms of chemical exposures (Blazer, 2002). The gills of catfish collected from the reference site in São Marcos Bay showed no histopathological changes. However, the animals collected in the potentially contaminated site showed severe histopathological changes, such as narrowing of the lamellae, epithelial lifting, fusion of lamellae and lamellar aneurism. Kim et al. (2001), has emphasized that histopathological changes in fish tissues has been important biomarkers of exposure to toxic substances, which reflect changes in biochemical functions. The histopathological examination performed in the gill epithelium of catfish could clearly differentiate the region of Ilha dos Caranguejos (reference area) and the harbor site (potentially contaminated). The great number of severe branchial lesions indicates that fish of the harbor are stressed by the pollutants. Branchial lesions like epithelial lifting, hypertrophy of the epithelial cells, and fusion of some secondary lamellae are examples of defense mechanisms (Camargo & Martinez, 2007; Fernandes & Mazon, 2003). Histopathological lesions may occur earlier than reproductive changes and they are more responsive than the patterns of growth and reproduction of organisms. When used as an integrated parameter, this

108 Analysis of two biomarkers in Sciades herzbergii 311 biomarker allows a better assessment of health status of fish than a simple biochemical parameter (Fontaínhas-Fernandes, 2006). In São Marcos' Bay, the integrated analysis of branchial lesions and GSI was useful because gills alterations were detected only in fish of the potentially contaminated site. Furthermore, in that area GSI was very low and juvenile fish were not found. Carvalho-Neta et al. (2012), suggested more studies to validate the use of branchial lesions and enzymes correlated with GSI as biomarkers of aquatic contamination in the same region. Those results reinforce the importance of using different methods of biomonitoring of the estuarine ecosystems. The use of biomarkers on only one level of biological organization (e.g., branchial lesions = histological) would not accurately represent the impact to environment (Moore et al., 2004). In this study, the method based on branchial lesions and GSI proved to be sensitive for the monitoring of the environmental impacts with relatively low cost and speed. ACKNOWLEDGEMENTS We would like to acknowledge the Laboratory of Fishing, biodiversity and population dynamics of fish for the supply of the fish, and Technological Development CNPq/FAPEMA, for financial support. REFERENCES Adams, S.M Application of bioindicators in assessing the health of fish populations experiencing contaminant stress. In: J.F. Mccarthy & L.R. Shugart (eds.). Biomarkers of environmental contamination. Lewis Publishers, Boca Raton, pp Amado, L.L., C.E. da Rosa, A.M. Leite, L. Moraes, W.V. Pires, G.L. Leães Pinho, C.M.G. Martins, R.B. Robaldo, L.E.M. Nery & J.M. Monserrat Biomarkers in croakers Micropogonias furnieri (Teleostei: Sciaenidae) from polluted and nonpolluted areas from the Patos Lagoon estuary (Southern Brazil): evidences of genotoxic and immunological effects. Mar. Poll. Bull., 52(2): Bernet, D., H. Schimidt, W. Meier & T. Whali Histopatology in fish: proposal for a protocol to assess aquatic pollution. J. Fish Dis., 22: Blazer, V.S Histopathological assessment of gonadal tissue in wild fishes. Fish Physiol. Biochem., 26: Camargo, M.M.P. & C.B.R. Martínez Biochemical and physiological biomarkers in Prochilodus lineatus submitted to in situ tests in an urban stream in southern Brazil. Environ. Toxicol. Phar., 21: Camargo, M.M.P. & C.B.R. Martínez Histopathology of gills, kidney and liver of a Neotropical fish caged in an urban stream. Neotrop. Ichthyol., 5(3): Carvalho-Neta, R.N.F. & A.L. Abreu-Silva Sciades herzbergii oxidative stress biomarkers: an in situ study of estuarine ecosystem (São Marcos Bay, Maranhão, Brazil). Braz. J. Oceanogr., 58: Carvalho-Neta, R.N.F & A.C.L. Castro Diversidade das assembléias de peixes estuarinas na Ilha dos Caranguejos, Maranhão. Arq. Cienc. Mar, 41: Carvalho-Neta, R.N.F., A.R. Torres Jr. & A.L. Abreu- Silva Biomarkers in catfish Sciades herzbergii (Teleostei: Ariidae) from polluted and non-polluted areas (São Marcos Bay, Northeastern Brazil). Appl. Biochem. Biotech., 166(1): Congreso Nacional de Medio Ambiente (CONAMA), Resolução Nº357. Diário Oficial da República Federativa do Brasil, Brasília, DF, 17 Mar. Seção 1, pp Fernandes, M.N. & A.F. Mazon Environmental pollution and fish gill morphology. In: A.L. Val & B.G. Kapoor (eds.). Fish adaptations. Science Publishers, Enfield, pp Fialho, A.P., L.G. Oliveira, F.L. Tejerina-Garro & B. Mérona Fish-habitat relationship in a tropical river under anthropogenic influences. Hydrobiologia, 598: Freire, M.M., V.G. Santos, I.S.F Ginuino & A.R.L. Arias Biomarcadores na avaliação da saúde ambiental dos ecossistemas aquáticos. Oecol. Bras., 12(3): Fontaínhas-Fernandes, A O uso de biomarcadores em toxicologia aquática. Série didáctica- Ciências Aplicadas, UTAD, Porto, 34 pp. Heath, A.G Water pollution and fish physiology. Lewis Publishers, Virginia, 359 pp. Kim, S., R.L. Lochmiller, E.L. Stair, J.W. Lish, D.P Rafferty & C.W. Qualls Jr Efficacy of histopathology in detecting petrochemical-induced toxicity in wild cotton rats (Sigmodon hispidus). Environ. Pollut., 113: Kime, D.E A strategy for assessing the effects of xenobiotics on fish reproduction. Kluwer Academy Publishers, New York, pp

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110 Lat. Am. J. Aquat. Res., 41(2): , 2013 Group structure of Guiana dolphins Proceedings of the 3 rd Brazilian Congress of Marine Biology A.C. Marques, L.V.C. Lotufo, P.C. Paiva, P.T.C. Chaves & S.N. Leitão (Guest Editors) DOI: /vol41-issue2-fulltext Research Article Group structure of Guiana dolphins, Sotalia guianensis (Cetacea, Delphinidae) in Ilha Grande Bay, Rio de Janeiro, southeastern Brazil Rodrigo Tardin 1,2, Carine Galvão 1, Mariana Espécie 1,3 & Sheila Simão 1 1 Universidade Federal Rural do Rio de Janeiro, BR 465, km 7 Rio de Janeiro, RJ, , Brazil 2 Programa de Pós Graduação em Ecologia e Evolução, Universidade do Estado do Rio de Janeiro R. São Francisco Xavier, Maracanã, Rio de Janeiro, RJ, , Brasil 3 Programa de Pós-Graduação em Biologia Animal, Universidade Federal Rural do Rio de Janeiro BR 465, km 7, Seropédica, RJ, , Brasil ABSTRACT. Cetaceans present a group structure of great complexity and display a wide behavioral plasticity. Many efforts have been made to understand the group structures of the various species, however, this type of information is still lacking for some species. Therefore, our objectives were to 1) characterize the structure of the Sotalia guianensis groups in Ilha Grande Bay, Rio de Janeiro, Brazil, and 2) investigate how both behavior and season influence the group structure of this population. This species is considered data deficient by the IUCN. We conducted 28 boat trips using group focal procedures, and a total of 1,314 groups were observed. Of these groups, 1,268 (94.4%) contained calves, the largest percentage ever reported for the species. Groups with calves were larger than those without them, suggesting a strategy to protect these individuals with underdeveloped physiology. The mean group sizes reached 17.6 ± 18.3 individuals. Within these groups, we observed that both behavior (H = 112.5, d.f. = 2, P < 0.05) and season (number of simulations: 10,000; sample size of fall-winter = 544; sample size of spring-summer = 684; P < 0.05), demonstrated a statistically significant influence. The most common degree of cohesion was mixed, and cohesion also varied with behavior (χ² = 10.1, P < 0.05) and season (χ² = 31.0, P < 0.05). This paper contributes towards understanding the highly variable nature of S. guianensis group dynamics. These data may be important in understanding the structure of groups in a site that is being increasingly impacted by different human activities. Moreover, this area contains the largest aggregation ever observed for this species and may therefore represent an important source of genetic diversity for the species as a whole. Keywords: Sotalia guianensis, Guiana dolphin, group structure, Ilha Grande Bay, southeastern Brazil. Estructura grupal en delfines Guyana, Sotalia guianensis (Cetacea, Delphinidae), en la bahía de Ilha Grande, Río de Janeiro, sureste de Brasil RESUMEN. Los cetáceos muestran una complejidad en la estructura de grupos, caracterizado por una gran plasticidad en su comportamiento. Se han efectuado numerosos esfuerzos para comprender las estructuras de grupos de muchas especies, pero para algunas estos datos son desconocidos. Los objetivos de este estudio fueron: 1) caracterizar la estructura de grupos de Sotalia guianensis en la Baía da Ilha Grande, Rio de Janeiro, Brasil, y 2) investigar como el comportamiento y las estaciones del año afectan la estructura de grupos de esta población. Esta especie es considerada con datos deficientes por la IUCN. Se efectuaron 28 salidas en bote en la Baía da Ilha Grande, usando el procedimiento de grupo focal y se observó un total de grupos. De estos grupos, (94,4%) contenían infantes, el mayor porcentaje registrado para esta especie. Los grupos con infantes fueron más grandes que los sin infantes, sugiriendo una estrategia para proteger estos individuos con fisiología menos desarrollada. El tamaño medio de los grupos observados fue de 17,6 ± 18,3 individuos. Dentro de estos grupos, se observó que ambos, comportamiento (H = 112,5; d.f. = 2; P < 0,05) y estación del año (número de simulaciones: ; tamaño de muestra en otoño-invierno = 544; tamaño de muestra en primavera-verano = 684; P < 0,05), fueron estadísticamente significativos. El grado más común de cohesión fue mixto y varió con el comportamiento (χ² = 10,1; P < 0,05) y estaciones del año (χ² = 31,0; P < 0,05). Este

111 314 Latin American Journal of Aquatic Research estudio contribuye a la comprensión de la naturaleza altamente variable de la dinámica de grupos de S. guianensis. Estos datos pueden ser importantes para comprender la estructura de grupos en un área que ha sido impactada crecientemente por diferentes actividades humanas. Además, esta área contiene la mayor agregación de individuos observada para esta especie y representa una importante fuente de su diversidad genética. Palabras clave: Sotalia guianensis, delfín de Guyana, estructura de grupo, bahía da Ilha Grande, sureste de Brasil. Corresponding author: Rodrigo Tardin (rhtardin@gmail.com) INTRODUCTION In general, most vertebrates show some degree of gregariousness. They tend to form different levels of group structure, such as shoals, flocks, herds, packs, parties or colonies of conspecific individuals (Reicziegel et al., 2008). Conspecifics may influence different aspects of their lives, such as foraging success, sexual selection, predation pressure and aggression (Krause & Ruxton, 2002). Consequently, a wide range of behavioral, morphological and life history traits may be affected by sociality (Krause & Ruxton, 2002). The group structure of cetaceans presents a wide behavioral plasticity, in which some species may form stable pods (Baird, 2000), fission-fusion groups (Connor, 2000), mother-calf groups (Clapham et al., 2000) or groups formed only by males (Whitehead & Weilgart, 2000). Despite the great effort of various researchers, the group structure of cetaceans is not fully understood, especially for some species like the Guiana dolphin (Sotalia guianensis), which are considered data deficient by the IUCN (Van Bénéden, 1864). The Guiana dolphin is a small delphinid, which can be found from northern Honduras to southern Brazil, and inhabits estuaries and bays (Simões-Lopes, 1998; Carr & Bonde, 2000). The diet of Guiana dolphins along the Brazilian coast has been investigated in detail and has revealed an opportunistic and generalist feeding habit in which their main prey tends to form small or large schools with pelagic or demersal habits (Di Beneditto & Ramos, 2004). Distribution studies on the S. guianensis social structure have shown a variety of relationships between biological and environmental components. These studies have demonstrated a lack of influence of the seasons in Guanabara Bay, RJ (Azevedo et al., 2005), heterogeneity in group size during the day in Baia Norte, SC (Daura-Jorge et al., 2005), differences in group size based on behavior in Sepetiba Bay, RJ (Nery et al., 2010) and on different habitats in Paranaguá Estuarine Complex, SP (Santos et al., 2010). However, several aspects (e.g., the relationship between composition and group size, cohesion and seasonality), of Guiana dolphin behavior, remain unknown, as is the case in Ilha Grande Bay. Our first objective was to characterize the structure of S. guianensis groups in Ilha Grande Bay, RJ, Brazil. Nery et al. (2010) has shown that the S. guianensis population in Sepetiba Bay, a similar and adjacent area to Ilha Grande Bay, demonstrates large group sizes and a high percentage of calf group members. Our hypothesis is that these components of group structure will show the same pattern in Ilha Grande Bay. Our second objective was to investigate how behavior and season influence the group structure of this population. Our hypothesis is that behavior and season will be the main factors in determining S. guianensis group size, as the number of dolphins engaged in each group may be a better indicator of ecological variability because fish schools may consequently be responding to specific habitat features and to differences in seasons. MATERIALS AND METHODS Study area Ilha Grande Bay (Fig. 1) is a large area encompassing 653 km², and is divided into the eastern, central and western zones. The western zone, where we conducted boat trips (23º02 S, 44º26 W), is of shallower depth (<10 m), contains approximately 31 islands and has a sandy/muddy bottom (Lodi, 2003b). The central zone of the bay behaves as a transition area between the land and sea (Nogara, 2000), receiving organic matter from river drainage and mangrove production (Signorini, 1980). From the sea, this bay receives deep waters rich in nutrients derived from the South Atlantic Central Waters (SACW) (Signorini, 1980). This zone is preferentially used by the S. guianensis population (Lodi, 2003b), and is surrounded by an outer region with depths between 20 and 40 m, and a smaller proportion of land mass than the inner region (DHN, 2011). The S. guianensis population in Ilha Grande Bay represents the largest aggregation of individuals ever seen at once (approximately 450

112 Group structure of Guiana dolphins 315 Figure 1. Study area, located in Ilha Grande Bay, southeastern Brazil. animals) (Lodi & Hetzel, 1998), the highest abundance ever reported [1,311 individuals (95% CI 1,232-1,389 individuals) (Espécie, 2011) and fluid degree of residence patterns with individuals presenting different degrees of residence to the area (Espécie et al., 2010). Procedures We carried out focal-group observations with continuous sampling (Lehner, 1996), from May 2007 to March 2010, onboard a 7.5 m vessel. A digital Sony DCRTRV 120 handycam was used to capture spatiotemporal behavior, which increased the analysis efficiency, as observations of dolphins in the wild are brief and many details can be lost. A group was defined according to the 10 m chain rule of Smolker et al. (1992), calves and juveniles were defined according to Geise et al. (1999) and the definition of cohesion was the same as that described by Shane (1990). We collected data for the three behavioral states of feeding, traveling as defined by Karczmarski et al. (2000) and socializing as defined by Slooten (1994). Table 1 summarizes all the definitions used in this work. Every time we spotted a group of dolphins, we recorded their behavior until they disappeared. Like some other delphinids, such as the bottlenose dolphin (Tursiops truncatus), S. guianensis presents fission-fusion dynamics (Connor, 2000) with groups changing in composition and members over short periods of time. Therefore, we believe that pseudoreplication is not an issue. For the purpose of analysis, we divided our data into two sampling periods: fall-winter (20 th March-22 th September) and spring-summer (23 th September-19 th March) (CPTEC-INPE, 2011). To reach our first objective, we counted the number of groups and individuals in each group, the degree of cohesion between them and quantified the number of calves. For the second objective, we correlated all these variables with the behavior and sampling period. Statistical analyses To determine if the number of groups displaying a behavior category was more than expected, we ran a chi-square test for one sample. To test for differences between this variable and the sampling periods, we created the following index: G i = N/n*100 where G i = standardized proportion of the number of groups observed in sampling period I; N = number of

113 316 Latin American Journal of Aquatic Research Table 1. Definitions of all variables considered in this paper. Variable Definition Group size Dolphins 10 m apart from each other Smolker et al. (1992). Age classes Feeding Calves were individuals presenting 1/4 of adults body size and juveniles 1/2 to 2/3 of adults body size Geise et al. (1999). Dolphins presented an absence of directional movements, diving frequently in asynchronous fashion Karcsmarski et al. (2000). Travelling Dolphins presented directional and constant movements Karcsmarski et al. (2000). Socializing Dolphins interacted with each others, displaying socio-sexual behavior. It was usual to observe sexual interactions such as the belly-to-belly position, in which there is a joint union of dolphins genital region Slooten et al. (1994). groups observed in sampling period i; and n = number of seconds of observation for each sampling period. This index was created to standardize the differences in the amount of observation time for each sampling period. To investigate differences between group size and behavior, we used a Kruskal-Wallis test. For group size and sampling periods, we used the bootstrap t-test for two samples as suggested by Reiczigel et al. (2008) using ten thousand simulations. As the group size data almost never reach parametric assumptions, the data were log transformed for this purpose. Due to the heterogeneity and the typical nonnormal group size distributions, we used bootstrap bias corrected and accelerated procedure to estimate the confidence interval using Flocker 1.1 as described in Efron & Tibishirani (1993). To determine if there were differences between age classes and group size, we used the Kruskal-Wallis test. When batching all age classes in only one category (offspring), we used the Mood s median test to quantify differences in group size between groups with offspring and groups without offspring. To investigate differences between the composition and behavior, we used a partition chi-square test. For the composition and sampling periods, we used a chisquared test for two samples. To evaluate the influence of behavior and sampling period on the degree of cohesion, we performed a partition chisquare test. RESULTS Twenty-eight boat trips were conducted for a total of 42.4 h of direct observation (75.5 h of effort). No solitary individual was spotted during the entire study period. Number of groups We observed a total of groups. A chi-squared test showed significant differences for behavioral influences (N feeding = 783; N traveling = 470; N socializing = 61; χ² = 589.2; P < 0.05). The Gi values were higher for the spring-summer period (Gi = 3.08) than for the fall-winter period (Gi = 1.31). Group size The mean group size was 17.6 ± 18.3 individuals (modal value = 15), and the group size ranged from 2 to 200 individuals. The bootstrap bias corrected and accelerated confidence interval ranged from 16.7 to The most common group size was 2 to 10 animals (44.8%). Other group sizes included 11 to 20 (28.0%), 21 to 30 (17.2%), 31 to 40 (2.8%), 41 to 50 (2.2%) and more than 50 animals (5.0%). Traveling groups ranged from 2 to 200 individuals (mean = 21.1 ± 24.7, BCa CI = 19.4 to 23.8), feeding groups ranged from to 2 to 100 individuals (mean = 14.9 ± 13.5, BCa CI = 14.1 to 15.8) and socializing groups ranged from 2 to 15 individuals (mean = 5.7 ± 3.1, BCa CI = 5.2 to 6.6) (Fig. 2). The Kruskal-Wallis test showed significant differences between group size and behavior (N = 1,345, H = 112.5, d.f. = 2, P < 0.05). The post-hoc multiple comparison of mean ranks test showed that the differences found occur among all behaviors (P < 0.05). Group sizes were larger in the spring-summer period (24.0 ± 21.6) than in the fall-winter period (14.4 ± 16.9). The bootstrap t-test showed significant differences between mean group size and sampling

114 Group structure of Guiana dolphins 317 Figure 2. Variation of mean group size according to different behaviors. Figure 3. Mean group size variation according to different age class groups. period (number of simulations: 10,000; sample size of fall-winter = 544; sample size of spring-summer = 684; P < 0.05). Group composition Of all the groups observed, 1,268 of them contained offspring members (94.4%). After discriminating between age classes, 61.6% (N = 781) of the groups contained only adults and calves as their members, 34.1% (N = 433) contained calves, juveniles and adults as their members and 4.3% (N = 54) contained only adults and juveniles as their members. Groups with only calves and adults ranged from 2 to 200 individuals (mean = 13.0 ± 14.5, BCa CI = 11.5 to 13.6), groups with only juveniles and adults ranged from 2 to 15 individuals (mean = 5.6 ± 3.4, BCa CI = 4.8 to 6.5), groups with calves, juveniles and adults ranged from 3 to 200 individuals (mean = 16.7 ± 17.9, BCa CI = 15.2 to 18.5) and groups with only adults ranged from 2 to 20 individuals (mean = 7.1 ± 3.5, BCa CI = 6.6 to 7.8). The Kruskal-Wallis test showed a statistically significant difference between group size and different age classes (H = 78.6, N = 1,268, d.f. = 2, P < 0.05) (Fig. 3). When grouping all age classes into a single category (e.g., offspring), the Mood s median test also showed statistical significance (χ² = 78.1, d.f. = 1, P < 0.05). When analyzing the influence of the annual period on group structure, the chi-squared test for two samples showed no significant difference (χ² = 0.98; P > 0.05). Cohesion patterns The most commonly observed cohesion pattern was mixed (Table 2). Chi-square tests showed significant differences between cohesion and behavior (χ² = 10.1; mixed vs tight P < 0.05; mixed+tight vs loosely P > 0.05; mixed+tight+loosely vs widely dispersed (P > 0.05); total: P < 0.05), and cohesion and sampling periods (χ² = 31.0; mixed vs tight P < 0.05; mixed+tight vs loosely P < 0.05; mixed+tight+loosely vs widely dispersed P < 0.05; total: P < 0.05). DISCUSSION The structure and organization of groups of small cetaceans can be a result of several factors, which include biological (e.g., predation risk, abundance, distribution of prey and reproduction) and environmental (e.g., water depth, sea surface temperature, salinity, and bottom topography) components (Gygax, 2002). The group sizes, observed in Ilha Grande Bay, were among the largest in the entire distribution. The large aggregation of individuals in this location may be a result of the ecological functions of Ilha Grande Bay, including a site with shallow and protected waters for rearing calves, and an seasonable site for social learning, especially coordinated with feeding behavior (Tardin et al., 2011). In the Paranaguá Estuarine Complex (PR), Santos et al. (2010) reported that Guiana dolphin group sizes ranged from 2 to 90 individuals with mean group size of 11 individuals. In Guanabara Bay, the group size ranged from 1 to 40 individuals with a mean group size of 13 individuals (Azevedo et al., 2005). In Baía Norte (SC), the group

115 318 Latin American Journal of Aquatic Research Table 2. Degrees of cohesion in different behaviors and sampling periods. Numbers inside cells are the number of groups observed. Behavior Feeding Travelling Autumn-Winter Spring-Summer Tight Mixed Loosely Widely dispersed size ranged from 1 to 59 individuals with a mean group size of 29 individuals. However, in Sepetiba Bay, an adjacent area of Ilha Grande Bay, the group size ranged from 2 to more than 50 individuals with a mean group size of 16 individuals (Nery et al., 2010). It is interesting to note that the number of individuals sighted, in a single day in Ilha Grande Bay, was the highest of all other estuaries, except Sepetiba Bay. Both bays are very similar in terms of oceanographic conditions, such as input of freshwater and the availability and seasonality of prey, which could be possible explanations for the high abundance of these two populations [Sepetiba abundance: 1,043 individuals (95% CI: ) (Nery, 2008); Ilha Grande Bay abundance: 1,311 individuals (95% CI 1,232-1,389 individuals) (Espécie, 2011)]. Therefore, the Sepetiba-Ilha Grande complex seems to be an important location for the species as it contains the two largest populations that demonstrate the largest group sizes. With regard to the environmental influences S. guianensis group formation, some studies have shown that environmental data do not influence group formation (e.g., Azevedo et al., 2005 (group size vs water depth), Santos et al., 2010 (group size vs temperature, water depth, transparency and salinity). However, others authors did find a relationship, e.g., dos Santos et al., 2010 (group size vs water depth, tide and depth), Cremer et al (group size vs season), Daura-Jorge et al., 2005 (group size vs daylight h). Our study area contains similar conditions throughout (i.e., sandy/muddy bottom, large number of islands, and input of freshwater and similar depth); therefore, there is no large environmental variation. This study did not tested whether group structure varied with environmental data. However, our results show that implementation of future research about how group structure changes with non-biological components can be important to better understand the population dynamic of S. guianensis in Ilha Grande bay. Our data showed that the number of groups was higher during the fall-winter period than during the spring-summer period. However, the number of individuals in each group during the fall-winter period was smaller than compared with the spring-summer period. This result may suggest a differential distribution of food resources during the spring-summer period that could support larger groups without enhancing the competition between individuals. As a result, these larger groups would enhance prey capture success. In fact, the Social Foraging Theory suggests that animals should maximize individual intake and thus aggregate to exploit available food resources (Giraldeau & Caraco, 2000). During the springsummer period in Ilha Grande Bay, fish that form large schools such as Sardinella brasiliensis and Harengula clupeola are observed and reported to spawn (Matsuura, 1978). These fishes are part of the S. guianensis diet (Di Beneditto & Ramos, 2004) and may therefore be driving the fission-fusion dynamics of dolphins to adapt to prey schooling patterns. Interestingly, Tardin et al. (2011) reported that the number of individuals engaged in coordinated feeding tactics in this population was higher during the springsummer period than during the fall-winter period. The mean group size was greater in traveling groups than in groups performing other behaviors. This result can be explained by the observation that the groups were often traveling toward the outer part of the bay, which represents deeper and unprotected waters, requiring the gathering of more individuals (group sizes of individuals). As most of the groups had offspring among their members, the aggregation of individuals while traveling to the outer areas of the bay may represent a strategy to protect the offspring from predators. During feeding, group size might be limited by intraspecific competition, during which individuals would aggregate in larger groups only when the net individual intake would be higher than when feeding in smaller groups. In terms of socializing, this study analyzed only socio-sexual behavior, and the observed group sizes were smaller given that only a few individuals were performing sexual activities. This behavior, of a very intense nature, occurs in a smaller percentage of the

116 Group structure of Guiana dolphins 319 population than traveling or feeding behavior (Tardin, pers. comm.). During this time, it is easy to observe individuals constantly slapping water and each other, lifting water, and swimming belly-to-belly. Different behavioral influences on group size may be found in the literature. While observing the same species, Daura-Jorge et al. (2005) in Baía Norte (SC) reported that the largest group sizes were seen during feeding behavior. In Sepetiba Bay (RJ), Nery et al. (2010), reported that the largest group sizes occurred during socializing behavior. One hypothesis, which may be derived from these interpopulation comparisons, is that these differences represent different ecological conditions that each habitat provides for each population. The high behavioral plasticity of the Delphinidae family may be reflected in an interspecies comparison. May-Collado & Ramirez (2005), reported that the spotted dolphin (Stenella attenuata) aggregated in larger groups during feeding, whereas the Atlantic white-sided dolphin (Lagenorhynchus acutus) in New England (Weinrich et al., 2007), and the Dusky dolphin (Lagenorhynchus obscurus) in Golfo Nuevo, Argentina (Degrati et al., 2008), demonstrated the largest group sizes when in traveling behavior. To our knowledge, Ilha Grande Bay has the highest percentage of S. guianensis offspring ever reported in its entire distribution. These data reinforce the important ecological function that this bay may have for this population, especially containing shallow and warm waters that seems to be free of predators (W. Alcantara do Carmo, pers. comm.). These conditions seem to favor the aggregation of many groups with offspring. These groups provide the opportunity for both better physiological development and social learning for the offspring. Other S. guianensis habitats along its distribution may also serve as important sites for the care for offspring. For instance, in the Paranaguá Estuarine Complex, Santos et al. (2010), reported that groups with offspring represented 84.6% of all groups. These two ecologically similar areas may provide interesting insight about the costs and benefits of group formation in S. guianensis. Both areas are mainly shallow with a sandy/muddy bottom and a large number of microhabitats, such as islands, rocky coasts and mangroves, which increase biological diversity and can therefore minimize the individual costs of living in large groups, such as intraspecific competition. Groups with offspring contained a larger number of individuals compared to groups without offspring. Our hypothesis is that mothers with offspring aggregate to protect against other conspecifics, which allows them to benefit from the dilution effect (Landeau & Terborgh, 1986), decreasing the calves chances to be attacked and killed. In fact, a record of infanticide has been reported in the adjacent population of Sepetiba Bay (Nery & Simão, 2009) and therefore may be a source of injury to the offspring. Moreover, studies on the bottlenose dolphin demonstrated that offspring have an underdeveloped physiology, including less muscle mass (Dearolf et al., 2000) and limited aerobic (Noren et al., 2001) and anaerobic (Noren, 2004) capacities. Therefore, they are easily targeted by predators or conspecifics. When discriminating between age classes, our data showed that groups with only adults and juveniles demonstrated similar characteristics to groups with only adults (i.e., few groups with a smaller number of individuals). Groups with only adults and calves had up to 200 individuals and demonstrated higher mean values, possibly reinforcing the dilution effect for this specific age class. While working with Lagenorhynchus acutus in New England and L. obscurus in Golfo Nuevo, Argentina, Weinrich et al. (2007) and Degrati et al. (2008), respectively, reported that groups containing offspring members were larger than those that did not, indicating a possible common strategy to avoid injuries to offspring. Our data on group cohesion indicated that a mixed formation was most commonly observed. In this formation, individuals were spatially separated from each other to varying degrees. This result suggests that the social bonds connecting individuals may be different, and this difference is observed in the various degrees of cohesion that individuals share within the same group. For example, individuals with stronger bonds may stay closer to each other by spacing themselves one body length apart from each other, whereas others that may be casual acquaintances may distance themselves further apart. In Guanabara Bay, Azevedo et al. (2005) found that no degree of cohesion (called spatial geometry by the author) was predominant; however, group sizes were higher in the mixed formation. Feeding and traveling behaviors were most commonly observed to occur in mixed formation. The coordinated feeding behavior displayed by this population represents random and fluidic movements, which seems to overcome prey defenses (Tardin et al., 2011). In mixed formations, individuals are spaced at different degrees and may better coordinate their actions to herd prey from multiple locations. When traveling, social bonds may be the driving force causing individuals to space themselves at different degrees, as spatial proximity may influence the hydrodynamics of each individual. Interestingly, during socio-sexual behavior, all the

117 320 Latin American Journal of Aquatic Research observed groups were tightly spaced, which describes the nature of this behavior in which individuals engage in sexual and aggressive activities and are in constant physical contact. These groups contained only adult members, in contrast to the traveling and feeding groups, which contained both calves and adults. This relationship between cohesion and group composition may indicate that groups with and without calves may be a force that influences cohesion and, consequently, behavior. This paper contributes to understand the highly variable nature of S. guianensis group dynamics. These data may be important in understanding the structure of groups in a site increasingly impacted by different human activities. Moreover, this area contains the largest aggregation of this species ever seen (Lodi & Hetzel, 1998) and, therefore, may represent an important source of genetic and social diversity for the species as a whole. ACKNOWLEDGMENTS We thank Sergio Moreira from Aqualie Institute, Dona Elza, Walcir Alcantara do Carmo (Tico) and Gilberto for their valuable help. Personnel for this study were partially supported by the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) (R.H.O. Tardin, Grant number E-26/ /2007 y E-26/ /2011); Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) (M.A. Espécie, Grant Number /2008-6), and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (R.H.O. Tardin, and M.A. Espécie). REFERENCES Azevedo, A.F., S.C. Viana, A.M. Oliveira & M. Van Sluys Group characteristics of marine tucuxis (Sotalia fluviatilis) (Cetacea: Delphinidae) in Guanabara Bay, south-eastern Brazil. J. Mar. Biol. Assoc. U.K., 85(1): Baird, R.W The killer whale: foraging specializations and group hunting. In: J. Mann, R.C. Connor, P. Tyack & H. Whitehead (eds.). Cetacean societies: field studies of dolphins and whales. The University of Chicago Press, Chicago, pp Carr, T. & R.K. Bonde Tucuxi (Sotalia fluviatilis) occurs in Nicaragua, 800 km north of its previously known range. Mar. Mammal. Sci., 16(2): Clapham, P The humpback whale: seasonal feeding and breeding in a baleen whale. In: J. Mann, R.C. Connor, P. Tyack & H. Whitehead (eds.). Cetacean societies: field studies of dolphins and whales. The University of Chicago Press, Chicago, pp Connor, R.C Group living in whales and dolphins. In: J. Mann, R.C. Connor, P. Tyack & H. Whitehead (eds.). Cetacean societies: field studies of dolphins and whales. The University of Chicago Press, Chicago, pp Daura-Jorge, F.G., L.L. Wedekin, V.Q. Piacentini & P.C. Simões-Lopes Seasonal and daily patterns of group size, cohesion and activity of the estuarine dolphin, Sotalia guianensis (P.J. Van Bénéden) (Cetacea, Delphinidae), in southern Brazil. Rev. Bras. Zool., 22(4): Dearolf, J.L., W.A. McLellan, R.M. Dillaman, D. Frierson Jr. & D.A. Pabst Precocial development of axial locomotor muscle in bottlenose dolphins (Tursiops truncatus). J. Morphol., 244(3): Degrati, M., S.I. Dans, S.N. Pedraza, E.A. Crespo & G.V. Garaffo Diurnal behavior of dusky dolphins, Lagenorhynchus obscurus, in golfo Nuevo, Argentina. J. Mammal., 89(5): Di Beneditto, A.P.M. & R.M.A. Ramos Biology of the marine Tucuxi dolphin (Sotalia fluviatilis) in south-eastern Brazil. J. Mar. Biol. Assoc. U.K., 84(6): Diretoria de Hidrografia e Navegação (DHN) Cartas Nauticas. cartas/cartas.htmlartas/cartas.html]. Reviewed: 16 April Efron, B. & R. Tibshirani An introduction to the bootstrap. Chapman & Hall, London, pp Espécie, M.A Tamanho populacional e estimativa da sobrevivência relativa de Sotalia guianensis (Cetacea, Delphinidae) na parte oeste da Baía da Ilha Grande, RJ. M.Sc. Thesis, Universidade Federal Rural do Rio de Janeiro, Seropédica, 52 pp. Espécie, M.A., R.H.O. Tardin & S.M. Simão Degrees of residence of Guiana dolphins (Sotalia guianensis) in Ilha Grande Bay, south-eastern Brazil: a preliminary assessment. J. Mar. Biol. Assoc. U.K., 90(8): Figueiredo, L.D. & S.M. Simão Possible occurrence of signature whistles in a population of Sotalia guianensis (Cetacea, Delphinidae) living in Sepetiba Bay, Brazil. J. Acoust. Soc. Am., 126(3): Filla, G.F. & E.L.A. Monteiro-Filho Monitoring tourism schooners observing estuarine dolphins (Sotalia guianensis) in the Estuarine Complex of Cananéia, south-east Brazil. Aquat. Conserv., 19(7):

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119 322 Latin American Journal of Aquatic Research usage by the estuarine dolphin, Sotalia guianensis (Van Bénéden, 1864) (Cetacea; Delphinidae) at its southern limit of distribution. Braz. J. Biol., 67(1): 1-8. Weinrich, M.T., C.R. Belt & M. Orin Behavior and ecology of the Atlantic white-sided dolphin (Lagenorhynchus acutus) in coastal New England waters. Mar. Mammal. Sci., 17(2): Weir, C.R., K.A. Stockin & G.J. Pierce Spatial and temporal trends in the distribution of harbor porpoises, white-beaked dolphins and minke whales off Aberdeenshire (UK), north-western North Sea. J. Mar. Biol. Assoc. U.K., 87(1): Whitehead, H. & L. Weilgart The sperm whale: social females and roving males. In: J. Mann, R.C. Connor, P. Tyack & H. Whitehead (eds.). Cetacean societies: field studies of dolphins and whales. The University of Chicago Press, Chicago, pp Wilson, E.O Sociobiology: the new synthesis. The Belknap Press of Harvard University Press, Massachusetts, 697 pp. Received: 16 May 2011; Accepted: 22 October 2012

120 Lat. Am. J. Aquat. Res., 41(2): , 2013 Iospilidae from the eastern Brazilian coast Proceedings of the 3 rd Brazilian Congress of Marine Biology A.C. Marques, L.V.C. Lotufo, P.C. Paiva, P.T.C. Chaves & S.N. Leitão (Guest Editors) DOI: /vol41-issue2-fulltext Research Article Distribution of Iospilidae (Annelida) along the eastern Brazilian coast (from Bahia to Rio de Janeiro) Bruna Tovar-Faro 1,2, Michele Leocádio 2 & Paulo Cesar de Paiva 1,2 1 Programa de Pós Graduação em Biologia Marinha, Departamento de Biologia Marinha Universidade Federal Fluminense 2 Laboratório de Polychaeta, Departamento de Zoologia, Universidade Federal do Rio de Janeiro Av. Carlos Chagas Filho, 373 Bloco A - Sala A Edifício do Centro de Ciências da Saúde (CCS) Ilha do Fundão, Cidade Universitária, Rio de Janeiro, , Brazil ABSTRACT. We present the spatial distribution and abundance of the holoplanktonic family Iospilidae (Annelida, Polychaeta), along part of the eastern Brazilian coast, and its relation to environmental variables in the region. Samples were obtained from two collections made in 1998 and 2000 between S, and W, on the Brazilian coast, between the Bay of Todos os Santos (BA) to Cape São Tomé (RJ). 216 stations were selected, covering the continental shelf, slope and oceanic regions, where plankton samples were collected for water and nutrient analysis. We analyzed environmental variables: temperature, salinity, dissolved oxygen, ph, ammonia, nitrite, nitrate, phosphate, silicate and chlorophyll-a. 363 individuals were collected, identifying two species, viz., Phalacrophorus uniformis and Phalacrophorus pictus, the first being the most abundant, with 354 individuals, while only nine specimens of P. pictus were found. Both species are mainly distributed in the oceanic region stations. The distribution of P. uniformis was related to the concentration of phosphate and nitrate. Significant differences between samples and between sectors of the continental shelf and oceanic region were found. Keywords: pelagic polychaetes, zooplankton, taxonomy, plankton ecology, oceanographic features. Distribución de Iospilidae (Annelida) a lo largo de la costa oriental brasileña (de Bahía a Río de Janeiro) RESUMEN. Se presenta la distribución espacial y abundancia de la familia holoplanctónica Iospilidae (Annelida, Polychaeta), a lo largo de la costa oriental brasileña, y su relación con variables ambientales de la región. Las muestras fueron obtenidas de dos muestreos realizados en 1998 y 2000 entre S, y W, en la costa brasileña, entre Bahía de Todos los Santos (BA) y el cabo de São Tomé (RJ). Se seleccionaron 216 estaciones, cubriendo las regiones de plataforma continental, talud y oceánica, donde se colectaron muestras de plancton y de agua para el análisis de nutrientes. Se analizaron las variables ambientales de temperatura, salinidad, oxigeno disuelto, ph, amonio, nitrito, nitrato, fosfato, silicato y clorofila-a. Se recolectaron 363 individuos, identificandose dos especies, viz., Phalacrophorus uniformis y Phalacrophorus pictus, siendo la primera la más abundante, con 354 individuos, y la segunda con sólo nueve especímenes Ambas especies se distribuyeron principalmente en las estaciones de la región oceánica. La distribución de P. uniformis estuvo relacionada con la concentración de fosfato y nitrato. Se encontraron diferencias significativas entre los muestreos, así como entre los sectores de plataforma continental y la región oceánica. Palabras clave: poliquetos pelágicos, zooplancton, taxonomía, ecología de plancton, características océanográficas. Corresponding author: Bruna Tovar-Faro (brunafaro@gmail.com)

121 324 Latin American Journal of Aquatic Research INTRODUCTION Iospilids are holoplanktonic polychaetes with small and delicate bodies. They are cosmopolitan and relatively common from surface waters down to a depth of 200 m, mainly in the Southern Ocean near the Antarctic Peninsula (Halanych et al., 2007). Nevertheless, even though widely distributed geographically, their records are scarce and scattered. According to Dales (1957), Tebble (1962) and Fernández-Álamo (2009), this is due to the small size and fragility of the animal s body susceptible to easy breakage during tows. Thus, iospilids fragments, although often collected in plankton samples, are commonly overlooked. The specimens of this family have elongated bodies, with a rounded prostomium carrying two small palps located near the mouth. The eversible pharynx is armed with a pair of jaws in one genera, and unarmed in the other. Two tentacular segments are fused bearing two pairs of tentacular cirri, and chaetae present along the second pair. The first 2 to 10 parapodia are reduced. More posterior parapodia are uniramous, with small dorsal and ventral cirri. Parapodial lobes are longer than the cirri, and with spinigerous chaetae. The chaetae are always compound, with elongated distal part. Antennae absent. The family Iospilidae (Bergström, 1914), previously named Iospilinae, was considered by Ushakov (1972) as a subfamily of Phyllodocidae. Nevertheless, Day (1967), Dales & Peter (1972), Orensanz & Ramirez (1973), Fauchald (1977) and Fernández- Álamo (2009) adopted the family status for Iospilidae. This status will be followed in this work, as well. There are only two recognized genera in the family: Iospilus Viguier, 1886 and Phalacrophorus Greeff, The main difference between the two is the presence or absence of jaws on the proboscis. In Iospilus, (unarmed proboscis), there is only one species, Iospilus phalacroides Viguier, 1886, whereas in Phalacrophorus two species are known, Phalacrophorus uniformis Reibisch, 1895 and Phalacrophorus pictus Greeff, Study area The present study focused on the Brazilian coast between S and W, from Todos os Santos Bay (BA) to Cabo de São Tomé (RJ), all told approximately 1,100 km in coastal line extent. It also includes the area surrounding the Vitória-Trindade chain of submarine banks and the Trindade and Martin Vaz islands, accounting a total study area of approximately 800,000 km 2. The region is dominated by the Brazil Current (BC), which carries the Tropical Water (TW) in the first 200 m of water column and the South Atlantic Central Water (SACW), down from 200 to approximately 700 m depth. Thermohaline characteristics of TW are temperatures above 20 C and salinities higher than 36 psu, whereas for SACW, temperatures range from 6 to 20 C and salinities from 34.6 to 36 psu. Below SACW, there are two further water masses, the Antarctic Intermediate Water (AIW) and the North Atlantic Deep Water (NADW) (Da Silveira et al., 2001). Only TW and SACW will be discussed in this paper. One of the most relevant oceanographic phenomena in the region is the presence of cyclonic eddies, formed by the meandering course of the Brazil Current. The meandering is caused by the geographic barrier of the Abrolhos banks and the Vitória-Trindade chain that cause a deviation in the current-flow. These eddies occasionally increase regional productivity by inducing SACW-outcropping (rich in nutrients), into the surface layers and shelf-break upwellings (Campos et al., 2000). Despite the wide extent of the study area, there are still few studies on polychaetes in this region (e.g., Attolini, 1997; Zanol et al., 2000; Paiva, 2006), all of which referring to benthic polychaetes, with no published studies of local holoplanktonic forms. The aim here is to report on the distribution of species from the family Iospilidae, along the eastern Brazilian coast, describe their morphology, and relate their distribution to the environmental variables of the area. MATERIALS AND METHODS The plankton samples analyzed for this study were collected during two surveys as part of a Brazilian government research program to study the biota of the country's exclusive economic zone, named REVIZEE. The first survey, Central III (CIII), was held in spring 1998, from October 26 to December 12, and the second, Central IV (CIV), during autumn 2000, from March 28 to May 1. Samples were collected along the eastern Brazilian coast in 108 stations for each survey, distributed in neritic and oceanic areas, 216 in total (Fig. 1). Plankton samples were collected with 200 µmmesh cylindrical-conical nets in vertical tows in the first 200 m of the water column. Samples were first fixed in 4% formaldehyde diluted in ocean water, and then preserved in 4% formaldehyde diluted in distilled water. In laboratory, stereomicroscopic analysis was

122 Iospilidae from the eastern Brazilian coast 325 Figure 1. Study area showing the main oceanographic features (isobaths, Abrolhos ridge and Vitória-Trindade chain) and the sampling points. X: shows the sampling stations of Central III and the cross refers to sampling points in Central IV. Many stations are coincident in both surveys. applied to separate polychaetes from other planktonic organisms and a compound microscope was used for pelagic polychaete identification at the species level. In each station, water was collected in Niskin bottles. Temperature, salinity, dissolved oxygen (DO) and ph measures were obtained for each station at the moment of sampling. Temperature ( C) and salinity (S) were measured using a CTD. Dissolved oxygen (mg L -1 ) was analyzed by the Winkler titration method, whereas ph was obtained with a ph meter. The nutrients, ammonia, nitrite, nitrate, silicate and phosphate concentration (µm), as well as chlorophylla, were analyzed in the laboratory, by the usual methods employed in oceanographic studies (Grasshoff et al., 1983; Parsons et al., 1984). Software Surfer 8.0 was applied to plot iospilid distribution and abundance. The relative percentage of each species was calculated. For assessment of spatial distribution, stations were grouped into five regions according to their oceanographic and topographic features: Bahia (BAH), Abrolhos (ABR), Vitória (VIT), the Vitória-Trindade chain (VTC) and Cabo de São Tomé (CST), as shown in Fig. 2. In order to investigate relationships between hydrochemical variables and species density, a Regression Model was applied by means of a General Linear Model (GLM), using quasi Poisson error terms and a log-link function. The use of quasi Poisson instead of regular Poisson error terms was due to the over-dispersion of residuals (Logan, 2010). The differences between the surveys and spatial patterns among the different regions (mentioned above) and sectors (shelf, slope and offshore) were also assessed by the GLM model, using quasi Poisson errors (Analysis of Deviance). Owing to the rare occurrence of P. pictus, both analyses were applied only to P. uniformis data. RESULTS From a total of 363 specimens examined in the two surveys, two species, both belonging to the genus Phalacrophorus, were identified, viz., Phalacrophorus pictus Greeff, 1879 and Phalacrophorus uniformis Reibisch, Both are very similar, except for the number of rudimentary parapodia on the anterior part of the body. P. uniformis has 8 to 10 (Fig. 3c), whereas P. pictus has only 2 to 3 (Fig. 3b). Phalacrophorus pictus Greeff, 1879 Phalacrophorus pictus Greeff, 1879:249; Fauvel, 1923:196; Dales, 1957:109; Ushakov, 1972:184; Tebble, 1962: ; Day, 1967:171; Dales & Peter, 1972:62; Orensanz & Ramírez, 1973:33. Phalacrophorus maculatus Treadwell, 1943:34 Phalacrophorus borealis Reibisch, 1895:15

123 326 Latin American Journal of Aquatic Research Figure 2. Study area showing the five main regions (the sampling stations were grouped): triangles: Bahia; crosses: Abrolhos; white circle: Vitória; black square: Vitória-Trindade chain, and black dot: Cabo de São Tomé. Material examined. A total of nine specimens were analyzed from the CIII survey. CIII: C9 (1); C18 (2); C26 (1); C37 (1); C51 (1); C55 (2); C108 (1) (Appendix 1). Description. Posterior part of body lacking in all specimens. Fragments with about 10 mm and chaetigers. Body cylindrical. Prostomium rounded with pair of short palps and two small eyes (present in all specimens). Pharynx thick, strong, with two falcate chitinous mandibles; not everted in any of the specimens analyzed, but observable through transparency (Fig. 3b). First three parapodia reduced, with two or three spinigerous chaetae and no cirri (Fig. 3d). Other parapodia developed, with small dorsal and ventral cirri. Parapodia along body gradually increase in size and number of chaetae (Fig. 3b). Chaetae compound, numerous, with long distal part. Remarks. Through being extremely fragile, these animals are often found broken in plankton samples. Figure 3. a) Phalacrophorus uniformis, entire body on dorsal view with pharynx everted, b) P. pictus anterior end on dorsal view, c) P. uniformis, anterior end on dorsal view, d) compound chaeta, common to both species, e) anterior end of P. uniformis on dorsal view with pharynx everted. Phalacrophorus uniformis Reibisch, 1895 Phalacrophorus uniformis Reibisch, 1895:15-16; Fauvel, 1923:196; Dales, 1957:109; Ushakov, 1972: 184; Tebble, 1962: ; Day, 1967:171; Dales & Peter, 1972:62; Orensanz & Ramírez, 1973:33. Iospilopsis antillensis Augener, 1922:41 Phalacrophorus attenuatus Treadwell 1943:34

124 Iospilidae from the eastern Brazilian coast 327 Material examined. 354 specimens were examined, 133 from the CIII survey and 221 from the CIV. CIII: C3 (2); C4 (1); C6 (1); C11 (1); C17 (1); C18 (25); C21 (10); C22 (4); C25 (1); C27 (1); C33 (8); C35 (1); C37 (4); C38 (2); C39 (2); C42 (13); C43 (2); C46 (10); C50 (3); C51 (1); C52 (6); C55 (4); C58 (2); C59 (1); C60 (2); C65 (1); C66 (4); C67 (3); C70 (1); C77 (3); C78 (2); C80 (1); C82 (1); C86 (5); C93 (1); C94 (1); C95 (2); CIV: C13 (4); C9 (3);C10 (1); C11 (1); C20 (3); C21 (1); C24 (1); C25 (2); C26 (1); C30 (1); C31 (4); C34 (6); C36 (9); C37 (5); C38 (2); C43 (6); C44 (7); C46 (3); C49 (1); C50 (25); C52 (2); C60 (3); C62 (4); C67 (2); C68 (6); C72 (4); C73 (7); C74 (2); C76 (1); C79 (8); C80 (1); C81 (1); C83 (1); C84 (4); C87 (5); C89 (9); C91 (1); C92 (3); C95 (7); C96 (2); C97 (1); C100 (6); C102 (4); C103 (1); C104 (4); C106 (5); C108 (9); C109 (3); C110 (5); C112 (1); C117 (1); C119 (4); C121 (1); C122 (8) (Appendix 2). Description. Few specimens complete. Posterior end lacking in most specimens. Body cylindrical. Prostomium rounded, with pair of short digital palps, two small eyes, no antennae. First 2 to 10 parapodia reduced, with small spinigerous chaetae, no cirri. Those following, well-developed, increasing in size along body, until reaching maximum by mid-portion, whence there is gradual decrease until posterior end (Fig. 3a). Parapodia, with dorsal and ventral cirri, smaller than parapodial lobe. Chaetae compound, numerous, with long distal part. Some specimens with everted pharynx opening into two falcate chitinous jaws (Fig. 3e). Pharynx muscles thick and strong, well-developed jaws dark, easily observable through transparency. Small parapodia and chaeta close to pigidium. Pygidium without appendages. Remarks. This species is similar to P. pictus, the main difference being in the anterior end. P. uniformis, has the first 2 to 10 parapodia reduced, while P. pictus has the first 3 parapodia reduced. The pharynx of P. uniformis, through being long and fragile, requires care on manipulation to avoid breakage. It is easily observable, when not everted, through transparency, in the anterior part of the body. Distribution. The most abundant species in both surveys was P. uniformis, representing 94% of Iospilidae abundance in CIII and 100% in CIV. P. pictus was encountered only in CIII, with 9 specimens, thus representing 6% of the total. In CIII, 133 specimens of P. uniformis were identified, against 221 in CIV. Both species were distributed throughout the study area (Fig. 4). Data from sampling stations are presented in Tables 1 and 2, respectively. The two species occurred mainly in offshore stations, i.e., those with local depths of over 250 m. In the CIII survey, P. uniformis was most abundant in ABR region (Figs. 2, 4), and in CIV survey, in CST region (Figs. 2, 4). Both regions are known as highly productive, ABR due to the coral reefs, and CST due to SACW upwelling, which induces an input of colder and nutrient richer water than the normally warmer and oligotrophic surface layers. This was confirmed by Analysis of Deviance (Table 1), which revealed a significant difference in P. uniformis abundance, not only between the two surveys, but also among sectors (shelf, slope and offshore), and the five different regions. Statistical tests showed the greater abundance of P. uniformis in the CIV survey, as well as significant differences between the ABR and CST regions, and between ABR and the VTC, but not among the other regions, which were considered intermediate (Fig. 5). This is probably due to the high abundance in ABR in both surveys, in contrast to the lowest abundance encountered in the VTC in the CIII survey and in CST in the CIV. In ABR, BAH and VIT, abundance was high in both surveys, whereas in the VTC, this ranged from the lowest in CIII to almost the highest in CIV, and in CST, low abundance in both surveys (Fig. 6). On considering sectors, offshore and shelf were significantly mutually different (P < 0.01), whereas the continental slope did not differ significantly from either shelf or offshore (Fig. 5). Significant interaction (P < 0.01) was found only between surveys and regions, owing to the VTC, the only region where abundance from CIII to CIV increased sharply (Fig. 6). The model selected for multiple regression showed that due to the high abundance in ABR, P. uniformis, distribution is related to offshore waters (higher local depth) at lower latitudes, characterized by lower temperatures and higher ph levels. Regarding nutrients, the relationship to nitrates is positive and negative to phosphates and chlorophyll-a (Table 2). These results reflect differences in the spatial distribution of nutrients and P. uniformis abundance in the CIII and CIV surveys. In CIII, the highest concentrations of chlorophyll-a were detected in the CST region, and of phosphate in the VTC. In both regions, abundance was low. On the other hand, the high nitrate concentrations in the ABR region coincided with species abundance. The contrary occurred in the CIV survey. Concentrations of chlorophyll-a and phosphate were high in the ABR region, and of nitrate, high in the CST region. In this survey, although P. uniformis

125 328 Latin American Journal of Aquatic Research Figure 4. Distribution and abundance of P. uniformis and P. pictus along the study area in both surveys. Table 1. Analysis of deviance table (GLM model). DF: degrees of freedom, DR: deviance residual, F: F statistics, P: probability of Type I error. Factor DF DR F P Survey Area Sector <0.001 Survey Area Survey Sector Area Sector Survey Area Sector Residuals abundance was also high in Abrolhos, the highest abundance was detected in one station in CST, thus leading to the positive relationship with nitrate content as selected by the regression model. DISCUSSION Overall, there are few studies of the taxonomy, biology and ecology of pelagic polychaetes, even less Figure 5. Plot of the design showing significant differences. OF: offshore sector; SL: slope sector; SH: shelf sector; ABR: Abrolhos region; BAH: Bahia region; VIT: Vitoria region; CVT: Vitoria Trindade Chain region; CST: Cabo de São Tomé region. Shaded polygons cover with homogeneous means in Tukey test. in the southern hemisphere. In this study, P. pictus occurred in all the study area, although in very low

126 Iospilidae from the eastern Brazilian coast 329 Figure 6. Box plots of P. uniformis abundance (in log (x+1)) in each region, showing median values (black dots), the interval of 25-75% of the data (box), the maximum value (dashed line) and the outliers (white dots). Abr: Abrolhos region; Bah: Bahia region; Vit: Vitória region; VTC: Vitória-Trindade Chain region; CST: Cabo de São Tomé region. abundances. Notwithstanding, this species has been reported in literature to all oceans. P. pictus was found throughout the Pacific Ocean, off Japan and along the coasts of California and Peru (Treadwell, 1943); from the South Atlantic to the Antarctic region (Støp- Bowitz, 1951), for the Mediterranean Sea and Atlantic Ocean (Dales, 1957) and to the North Pacific Ocean, where it was frequent in the Sub-Arctic Zone and considered as related to cold waters (Tebble, 1962). According to Orensanz & Ramirez (1973), P. pictus is cosmopolitan, occurring from the Arctic to the Antarctic through the tropics, and a population differentiation is possibly related to geographic dispersion, due to intra-specific morphological differences. P. uniformis was reported in several papers, from the South and Equatorial Pacific (Dales, 1957), as well as North Pacific Ocean (Tebble, 1962), the Atlantic, Pacific and Indic Oceans (Orensanz & Ramirez, 1973). Tebble (1960) studied the distribution of holoplanktonic polychaetes from the South Atlantic Ocean, including both Brazilian and African latitudes, but made no mention of Iospilidae. However, P. uniformis was reported for the African coast (Day, 1967) and now, from the Brazilian coast aswell. According to Orensanz & Ramirez (1973) and Fernández-Álamo (1996), this species is associated to surface tropical and subtropical waters. Although the two species are considered cosmopolitan, having been reported worldwide, the difference in their distribution appears to be according to climate zones. P. pictus is more abundant and frequent in cold waters of higher latitudes and P. uniformis is abundant and frequent in warmer waters of lower latitudes. If true, this might explain the dominance of P. uniformis in this study where water masses are warmer, and thus, their greater abundance in a gradient from tropical (ABR) to subtropical (CST) climate. Fernández-Álamo & Färber-Lorda (2006) reported higher abundance and frequency of P. uniformis in the Baja California region (Eastern Pacific), in lower latitudes than polar zones, where P. pictus is more abundant. The authors also reported P. pictus as a rare species in Baja California, the same pattern observed in this study. Near Madagascar, P. pictus was reported as being more common in neritic than oceanic waters (Day, 1975). Nevertheless, in the present study, this species occurred exclusively in oceanic stations with a local depth of 1000 m or over (except in one at 270 m). Despite the very low abundance, and except for the BAH region, distribution was widespread. There were differences in P. uniformis abundance, both between surveys and among regions along the coast. The higher abundance in ABR is probably related to the intense regional biological activity, through regenerated primary production, supported by microbial loop-activity (Tovar-Faro, 2005). Along the Brazilian coast, the ABR region is known for its high productivity (Gaeta et al., 1999; Susini-Ribeiro, 1999) preceded by small phytoplankton species (Prochlorococus) (Zubkov et al., 1998, 2000; Susini-Ribeiro, 1999), and based on regenerate primary production, firmly dependent on the microbial loop, as observed in tropical oligotrophic oceans (Fernández-Álamo & Färber- Lorda, 2006). The close relationship between primary production, grazing and zooplankton excre-tion, as well as the importance of this excretion as a nutrient supply, have already been shown (Eppley et al., 1973). High primary productivity, both new and regenerate, causes an increase in secondary production at all levels of the web chain, this including planktonic top predators, supposedly the case of P. uniformis and P. pictus, on considering their buccal apparatus morphology (Fernández-Álamo, 2009). Thus, the spatial distribution of P. uniformis seems to be related to primary production, with the consequential increase in prey-availability, once chlorophyll concentration is an indirect measure of primary production, and it was

127 330 Latin American Journal of Aquatic Research Table 2. Variables selected by Regression Analysis (GLM). F: F statistics, P: probability of Type I error. Variables Coefficient Deviance F P ph <0.001 Latitude <0.001 Chlorophyll-a Temperature Phosphate Nitrate high in the same regions where P. uniformis was abundant. Furthermore, the relationship between plankton distribution and water-mass flow has been welldocumented for other regions (e.g., Fernández-Álamo & Färber-Lorda, 2006). Along the Brazilian coast, the ABR banks (and further south, the Vitoria-Trindade chain) form a natural geographical barrier to the BC, with the consequential occurrence of eddies formed by BC meandering. These eddies introduce nutrients into the euphotic zone, thereby contributing to an increase in primary production. The origin of this input is the SACW, which flow into BC under the TW. Besides being colder and richer in nutrients, SACW is less salty than TW (Da Silveira et al., 2001). This probably explains the correlation found between P. uniformis and lower temperatures and ph, both water-mass markers. Furthermore, there are seasonal variations in BC flow, as the entrance of SACW into the continental shelf causes coastal upwelling (Campos et al., 1995, 2000; Castro & Miranda, 1998), possibly the reason for the significant difference found between the two surveys, since they occurred at different seasons (spring and autumn). Nevertheless, one must consider that observed variability could also be due to processes both during and between the years, since surveys took place not only in different seasons, but also in distinct years (1998 and 2000). Thus, further surveys are required, in order to assess actual temporal patterns. ACKOWLEDGEMENTS We are grateful to CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for a scholarship to BTF, to CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for a fellowship to PCP and to CNPq/UFRJ/PIBIC for a scholarship to ML. We also thank to MSc. Nury Eunice Riascos for the Spanish version of the abstract and legends, to LIZI-UFRJ, especially, Dr. Sérgio Bonecker and Dra. Ana Bonecker, for providing us the plankton samples, to laboratory of Hidrobiology- UFRJ, for environmental variables analysis and to ABBM (Associação Brasileira de Biologia Marinha) for this special issue. We also thank two anonymous referees for providing valuable suggestions. The work was supported by grants from Brazilian government through MMA (Ministério do Meio Ambiente), SECIRM (Secretaria da Comissão Interministerial para os Recursos do Mar) and all members of the executive committee of the REVIZEE program. REFERENCES Attolini, F.S Composição e distribuição dos anelídeos poliquetas na plataforma continental da região da Bacia de Campos, RJ, Brasil. Dissertação (Mestrado em Oceanografia Biológica) Universidade de São Paulo, São Paulo, 122 pp. Augener, H Üeber litorale polychaeten von Westindien. Sber Ges. naturf. Freunde, Berl., pp Campos, E.J.D., J.E. Gonçalves & Y. Ikeda Water mass structure and geostrophic circulation in the South Brazil Bight-Summer of J. Geophys. Res., 100(C9): Campos, E.J.D., D. Velhote & I.C.A. da Silveira Shelf breaks upwelling driven by Brazil Current cyclonic meanders. Geophys. Res. Let., 27(6): Castro, B.M. & L.B. Miranda Physical oceanography of the western Atlantic continental shelf located between 4ºN and 34ºS. In: A.R. Robinson & K.H. Brink (eds.). The sea. John Wiley & Sons, New York, 11: Da Silveira, I.C.A., A.C.K. Schmidt, E.J. Campos, S.S. Godoi & Y. Ikeda A corrente do Brasil ao largo da costa leste brasileira. Braz. J. Oceanogr., 48:

128 Iospilidae from the eastern Brazilian coast 331 Dales, R.P Pelagic polychaetes of the Pacific Ocean. Bull. Scripps. Inst. Oceanogr. Univ. Calif., 7: Dales, R.P. & G. Peter A synopsis of the pelagic Polychaeta. J. Nat. Hist., 6: Day, J.H A monograph on the Polychaeta of southern Africa. Part 1 Errantia. Brit. Mus. (Nat. Hist.) Publ., 656: Day, J.H The biology of planktonic Polychaeta near Nosy-Bé, Madagascar. Cah. ORSTOM., sér. Océanogr., 13(3): Eppley, R.W., E.H. Renger, E.L. Venrick & M.M. Mullin A study of plankton dynamics and nutrient cycling in the central gyre of the North Pacific Ocean. Limnol. Oceanogr., 18(4) Fauchald, K The Polychaete worms. Definitions and keys to the orders, families and genera. Nat. Hist. Mus. L.A. Count. Sci. Ser., 28: Fauvel, P Polychétes errantes. Faune de France 5. Le Chevalier, Paris, 488 pp. Fernández-Álamo, M.A Holoplanktonic polychaetes off the southwestern coast of Baja California, Mexico, in March, An. Inst. Biol. Univ. Nac. Autón. México, Ser. Zool., 67(1): Fernández-Álamo, M.A Iospilidae Bergström, In: J.A. de Leon-González, J.R. Bastida- Zavala, L.F. Carrera-Parra, M.E. García-Garza, A. Peña-Rivera, S.I. Salazar-Vallejo & V. Solís-Weiss (eds.). Poliquetos (Annelida: Polychaeta) de México y América Tropical. Universidad Autónoma de Nuevo León, Monterrey, pp Fernández-Álamo, M.A. & J. Färber-Lorda Zooplankton and the oceanography of the eastern tropical Pacific: a review. Prog. Oceanogr., 69(2-4): Gaeta, S.A., J.A. Lorenzetti, L.B. Miranda, S.M.M. Susini-Ribeiro, M. Pompeu & C.E.S. de Araujo The Vitória eddy and its relation to the phytoplankton biomass and primary productivity during the austral fall of Arch. Fish. Mar. Res., 47(2/3): Grasshoff, K. A., M. Ehrhardt & K. Kremling Methods of seawater analysis. Verlag Chemie, Weinheim, 419 pp. Greeff, R Uber pelagische Anneliden von der Kuste der canarischen Insela. Z. teiss. Zool., 32: Halanych, K.M., L.N. Cox & T.H. Struck A brief review of holopelagic annelids. Integr. Comp. Biol., 47(6): Logan, M Biostatistical design and analysis using R. A practical guide. Wiley-Blackwell, Oxford, 546 pp. Orensanz, J.M. & F.C. Ramírez Taxonomia y distribución de los poliquetos pelágicos del Atlántico Sudoccidental. Bol. Inst. Biol. Mar. Mar del Plata, 21: Paiva, P.C Filo Annelida - Classe Polychaeta. In: H.P. Lavrado & B.L. Ignácio (eds.). Biodiversidade bentônica da região central da zona econômica exclusiva brasileira. Série Livros 18. Museu Nacional, Rio de Janeiro, pp Parsons, T.R., M. Takahashi & B. Hargrave Biological oceanographic processes. Pergamon Press, Oxford, 330 pp. Reibisch, J.G.F Die pelagischer Phyllodocien und Typhloscoleciden der Plankton-Expedition. Ergbn. Der Plankton-Exped. der Humboldt-Stitung, 2, Hc: Støp-Bowitz, C Polychètes pèlagiques de l expédition suédoise Antarctique Results Swed. Antarct. Exped., 4(7): Susini-Ribeiro, S.M.M., Biomass distribution of pico-, nano- and microplankton on the continental shelf of Abrolhos, East Brazil. Arch. Fish. Mar. Res., 47: Tebble, N Distribution of pelagic polychaetes in the South Atlantic Ocean. Discovery Rep., 30: Tebble, N The distribution of pelagic polychaetes across the North Pacific Ocean. Bull. Br. Mus. Nat. Hist. (Zool.), 7(9): Tovar-Faro, B.C.M Abundância e atividade das bactérias heterotróficas no Oceano Atlântico Sudoeste entre 13 e 25 S e 28 e 42 o W. Dissertação Mestrado em Biologia, Marinha). Universidade Federal Fluminense, Niterói, 79 pp. Treadwell, A.L Scientific results of cruise VII of the Carnegie during under Command of Captain J.P. Ault. Biology, 4. Biological results of the last cruise of Carnegie: Polychaetous annelids. Carnegie Institution, Washington, 555: Ushakov, P.V Polychaeta I Polychaetes of the suborder Phyllodociforma of the Polar Basin and the north-western part of the Pacific. Akad Nauk SSR Zool. Inst. Fauna USSR, 102: (Translated from de Russian by the Israel Program for Scientific Translation, Jerusalem, 1974). Zanol, J., P.C. Paiva & F.S. Atollini Eunice and Palola (Eunicidae: Polychaeta) from the eastern Brazilian coast (13º00'-22º30'S). Bull. Mar. Sci., 67(1): Zubkov, M.V., M.A. Sleigh, G.A. Tarran, P.H. Burkill & R.J.G. Leakey Picoplanktonic community structure on an Atlantic transect from 50 o N to 50 o S. Deep-Sea Res. I, 45:

129 332 Latin American Journal of Aquatic Research Zubkov, M.V., M.A. Sleigh, P.H. Burkill & R.J.G. Leakey Picoplankton community structure on the Atlantic meridional transect: a comparison between seasons. Prog. Oceanogr., 45: Received: 16 May 2011; Accepted: 22 October 2012 Appendix 1. Data of the sampling stations where Phalacrophorus pictus occurred. Station Long ( W) Lat ( S) Date Local depth Number of (m) specimens C /10/ C /11/ C /11/ C /11/ C /11/ C /11/ C /12/ Appendix 2. Data of the sampling stations where Phalacrophorus uniformis occurred. Survey Station Long ( W) Lat ( S) Date Local depth Number of (m) specimens CIII C /10/ CIII C /10/ CIII C /10/ CIII C /10/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/

130 Iospilidae from the eastern Brazilian coast 333 continuation Survey Station Long ( W) Lat ( S) Date Local depth Number of (m) specimens CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /11/ CIII C /12/ CIII C /12/ CIII C /12/ CIII C /12/ CIII C /12/ CIII C /12/ CIII C /12/ CIII C /12/ CIV C /03/ CIV C /03/ CIV C /03/ CIV C /03/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/

131 334 Latin American Journal of Aquatic Research continuation Survey Station Long ( W) Lat ( S) Date Local depth Number of (m) specimens CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /05/ CIV C /04/ CIV C /04/ CIV C /04/ CIV C /04/

132 Lat. Am. J. Aquat. Res., 41(2): , 2013 Cytotoxicity of actinomycetes associated with tunicate Proceedings of the 3 rd Brazilian Congress of Marine Biology A.C. Marques, L.V.C. Lotufo, P.C. Paiva, P.T.C. Chaves & S.N. Leitão (Guest Editors) DOI: /vol41-issue2-fulltext Research Article Cytotoxicity of actinomycetes associated with the ascidian Eudistoma vannamei (Millar, 1977), endemic of northeastern coast of Brazil Paula C. Jimenez 1,2, Elthon G. Ferreira 1,2, Luana A. Araújo 1, Larissa A. Guimarães 1, Thiciana S. Sousa 3 Otília Deusdenia L. Pessoa 3, Tito M. C. Lotufo 1 & Letícia V. Costa-Lotufo 1,2 1 Instituto de Ciências do Mar (LABOMAR), Universidade Federal do Ceará Av. Abolição, 3207, , Fortaleza, Brazil 2 Departamento de Fisiologia e Farmacologia, Faculdade de Medicina Universidade Federal do Ceará, Av. da Universidade, 2853 Benfica Fortaleza, CE, , Brazil 3 Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará Av. da Universidade, 2853, Benfica, Fortaleza, CE, , Brazil ABSTRACT. Previous studies demonstrated that the crude extract of the ascidian Eudistoma vannamei, endemic from northeasttern Brazil, strongly hinders growth of tumor cells in vitro by inducing apoptosis due to tryptophan derivatives, which are commonly found in bacteria. This study presents a bioactivity-guided screening among actinomycetes, associated with E. vannamei, aiming at recognizing active principles with biological relevance. Twenty strains of actinomycetes, designated as EVA 0101 through 0120, were isolated from colonies of E. vannamei among which 11 were selected for cytotoxicity evaluation. The extracts from EVA 0102, 0103, 0106, 0109 and 0113 were the most active, and were further studied for IC 50 determination and chemical analysis by 1 H NMR. IC 50 values obtained ranged from 3.62 µg ml -1 (for EVA 0109 in leukemia cells) to µg/ml (for EVA 0106 in melanoma cells). All active extracts exhibited the same TLC and spectroscopic profiles, suggesting the presence of quinones and other related secondary metabolites. Furthermore, these strains were identified and compared based on their respective 16S rrna sequences. The results herein identified the five strains as Micromonospora spp. while phylogenetic analysis suggests that they are possibly two different Micromonospora species producing the cytotoxic compounds. Keywords: marine biotechnology, Eudistoma vannamei, marine microorganisms, Micromonospora sp., Brazil. Citotoxicidad de actinomicetos asociada a la ascidia Eudistoma vannamei (Millar, 1977), endémica de la costa noreste de Brasil RESUMEN. Estudios previos demostraron que el extracto crudo de la ascidia Eudistoma vannamei, endémica de la costa noreste de Brasil, obstaculiza fuertemente el crecimiento de células tumorales in vitro por inducir apoptosis. El análisis químico del extracto sugirió la presencia de derivados de triptófano, comúnmente encontrados en bacterias. El estudio presenta un screening de citotoxicidad en actinomicetos asociados con E. vannamei, para reconocer principios activos biológicamente relevantes. Veinte cepas de actinomicetos, designados como EVA 0101 hasta 0120, fueron aisladas de colonias de E. vannamei y 11 fueron seleccionadas para la evaluación de citotoxicidad. Los extractos de EVA 0102, 0103, 0106, 0109 y 0113 resultaron las más activas y fueron estudiadas para determinación de la CL 50 y perfiles de CCF y 1 H RMN. Las CL 50 obtenidas oscilaron entre 3,62 (EVA 0109 en las células de leucemia) y 84,65 µg ml -1 (EVA 0106 en las células de melanoma). Los extractos activos presentan el mismo perfil de CCF y espectroscópico, lo que sugiere la presencia de quinonas y metabolitos secundarios relacionados. Además, las cinco cepas fueron identificadas y comparadas sobre la base de sus secuencias de 16S ARNr. Las que se identificaron como Micromonospora spp. y el análisis filogenético sugiere que sean por lo menos dos especies de Micromonospora las que producen los compuestos citotóxicos.

133 336 Latin American Journal of Aquatic Research Palabras clave: biotecnología marina, Eudistoma vannamei, microorganismos marinos, Micromonospora sp., Brasil. Corresponding author: Letícia Lotufo INTRODUCTION Colorful soft-bodied sessile marine invertebrates have been a rich source of natural products with biomedical properties for the past 60 years (Baily, 2009; Molinski et al., 2009; Mayer et al., 2010). Among them, the tunicates are one of the most prolific in terms of novel drug prototypes for the anticancer pharmaceutical arsenal (Blunt et al., 2010, 2011). For instance, trabectedin, a tetrahydroisoquinoline alkaloid obtained from Ecteinascidia turbinata, was recently approved for the treatment of soft tissue sarcoma, with the commercial name Yondelis (Cuevas & Francesh, 2009; D Incalci & Galmarini, 2010). Additionally, plitidepsin, a cyclodepsipeptide isolated from Aplidium albicans is in advanced trial phase for the therapy of multiple myeloma and other malignant tumors, but has already received orphan drug status for the treatment of multiple myeloma and acute lymphoblastic leukemia (Ocio et al., 2008; Singh et al., 2008). There are mounting evidences that the associated microorganisms are the authentic suppliers for most of the relevant compounds (Zhang et al., 2005; Imhoff et al., 2011). For the ascidians, in particular, there is a compelling amount of information pointing into this direction (Schmidt & Donia, 2010). Despite the oceans are acknowledged as a rich and highly complex microbiological environment, they have only shared their wealth as a natural drug source for the past 10 years. Thus, the study of marine microorganisms as sources of new drugs is still at its youth (Jensen et al., 2005; Fenical & Jensen, 2006; Liu et al., 2010). This study takes part in a pioneer venture aiming to the exploration of the biotechnological potentials of microorganisms, mainly actinomycetes, from the coast of Ceará. The actinomycetes have proven to be a prolific source of new and bioactive compounds (Fenical & Jensen, 2006). The purpose of the present investigation is to find biologically active strains of actinomycetes and evaluate their respective cytotoxicity, while gathering information on the microbiota associated with Eudistoma vannamei. This species is endemic from the northeastern coast of Brazil and is the most abundant ascidian recorded for the state of Ceará. Previous studies have revealed that its crude extract strongly hinders growth of tumor cells in vitro, promoting further apoptosis (Jimenez et al., 2003, 2008). MATERIALS AND METHODS Sampling and isolation of bacteria strains Specimens of E. vannamei were hand collected at Taiba Beach (03 30'21.23 S, '40.16 W), located on the northeastern coast of Brazil. Sampling was carried out with the slightest contamination possible, as the material was gathered and stored with sterile utensils. For isolation of microorganisms, five colonies were homogenized in sterile seawater 1:5 (m/v), and aliquots of 20 L were stroked onto Petri dishes containing different agar based media, which may favor actinomycetes growth (modified from Jensen et al., 1991). The following agar-based media were used herein, all added with cycloheximide 0.1 mg ml -1 : SCA (Starch Casein Agar, HIMEDIA, prepared with distilled H 2 O), AIA (Actinomycetes Isolation Agar, HIMEDIA, prepared in 75% seawater), SWA (Sea-water Agar, 1.8% agar in 75% seawater) and GYM (glucose 0.4%, yeast extract 0.4%, malt extract 1%, CaCO 3 0.2%, 1.8% agar in 75% seawater). Purity of strains were resolved by sequential restrikes onto brand new agar plates and pure strains were inoculated in liquid A1 medium (10% soluble starch, 4% yeast extract and 2% peptone in 75% seawater) for culture maintenance and up-scaled growth (Jensen et al., 1991). Grown media for each pure strain was sampled into cryovials and supplemented with 25% glycerol for storage at -70 o C. Up-scaled growth and extraction Initially, pure cultures were inoculated in 500 ml Erlenmeyer flasks filled with 100 ml of liquid A1 medium. Flasks were kept at 28 o C and 200 rpm for 5 days. Next, 10 ml of grown media were added to newly assembled Erlenmeyer flasks with 100 ml of fresh media and kept at 28 o C and 200 rpm agitation for 10 days. The resulting media was spun down to separate the bacterial biomass from the broth. Biomass was extracted with MeOH 1:1 (m/v) while broth was extracted in EtOAc 1:1 (v/v). The crude extracts were evaporated under vacuum, washed with MeOH to

134 Cytotoxicity of actinomycetes associated with tunicate 337 remove excessive salt, and fully dried under compressed air, yielding the crude extracts. At this point, extracts were resuspended in DMSO to a final concentration of 10 µg ml -1 and were ready to be evaluated for their biological activity. Screening for cytotoxicity-mtt assay Cytotoxic activity of extracts was evaluated against four human tumor cell lines obtained from the National Cancer Institute (Bethesda, MD, USA): HL- 60 (promyelocytic leukemia), MDA-MB-435 (melanoma), SF-295 (glioblastoma) and HCT-8 (colon carcinoma). Cells were grown in RPMI-1640 medium supplemented with 2 mm glutamine, 10% fetal calf serum, 100 µg ml -1 strep-tomycin and 100 U ml -1 penicillin and housed at 37ºC with a 5% CO 2 atmosphere. The cell cultures were regularly split to keep them in a logarithmic phase of growth. Firstly, in a qualitative approach, cell lines were exposed to 100 µg ml -1 of the extracts for 72 h and the effect on cell proliferation was evaluated in vitro using the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2h-tetrazolium bromide] assay, as described by Mosmann (1983). The extracts that reduced over 80% of cell growth, when compared to non-treated cells, were tested again, in a quantitative manner, however, at concentrations varying serially from 0.02 to 100 µg ml -1 to determine the IC 50 (the concentration that inhibits growth in 50%), which was calculated, along with the respective 95% CI (confidence interval), by a non-linear regression using the software GraphPad Prism 5.0. Preliminary chemical analysis The active extracts (EVA 0102, EVA 0106, EVA 0109 and EVA 0113) were submitted to a preliminary chemical characterization. Initially, these extracts were compared by thin layer chromatography (TLC) on precoated silica gel aluminum sheets (Kieselgel 60 F 254, 0.20 mm, Merck) using two different solvent systems (CHCl 3 and CHCl 3 : EtOAc 1:1) and the compounds were detected by exposure to UV (312 and 365 nm) light and by spraying with vanillin/ perchloric acid/etoh solution followed by heating at 100 C. The extracts were also analyzed by 1 H NMR spectroscopy. The 1 H NMR spectra were acquired on a Bruker Avance DRX-500 spectrometer equipped with 5 mm inverse detection z-gradient probe. The chemical shifts, given on the δ scale, were referenced to the residual undeuterated CHCl 3 (δ H 7.27) fraction. The methodology was based in a previous work published by Jimenez et al. (2008). Molecular identification: nucleic acid extraction, 16S rrna gene amplification, and sequencing The selected microorganisms were characterized using a molecular approach based on the protocol described by Gontang et al. (2007) with slight modifications. Genomic DNA was extracted from a fresh, 3 to 5 day grown, bacterial culture following the DNeasy protocol (QIAGEN Inc., USA). Amplification of the 16S rrna gene was carried out with Ilustra PureTaq Ready-To-Go PCR beads (GE Healthcare, UK) using the universal eubacterial primers 27F (5 - AGAGTTTGATCCTGGCTCAG-3 ) and 1492R (5 - TACGGCTACCTTGTTACGACTT-3 ). PCR products were purified with MiniElute PCR purification kit (QIAGEN Inc., USA). DNA sequencing was performed by Macrogen Inc. (Seoul, Korea) using the ABI PRISM BigDye TM Terminator Cycle Sequencing kit (Applied Biosystems, USA) and the same primers used for amplification, following the protocols supplied by the manufacturer. The nucleotide sequences were assembled, analyzed and manually edited using the BioEdit Sequence Alignment Editor (Hall, 1999) and compared to sequences within the NCBI database ( using the Basic Local Alignment Search Tool (BLAST). The identity of the strain was confirmed after an evaluation of the phylogenetic relationships with the most similar sequences obtained from the EzTaxon server (Chun et al., 2007). The sequences were aligned using Muscle ver. 3.6, and the reconstruction was performed using a combined bootstrapping and maximum likelihood approach through RAxML (Stamakis, 2006) on the CIPRES portal (Miller et al., 2010). The tree was produced using Archaeopterix ver (Han & Zmasek, 2009). After the phylogenetic analysis, the sequences were submitted to NCBI GenBank. RESULTS Isolation and cytotoxicity screening of bacteria associated with E. vannamei Twenty colonies were isolated from E. vannamei respecting their actinomycete-like distinctiveness. Out of those, 11 were elected for cytotoxicity evaluation on tumor cells. In the single concentration screening, extracts obtained from strains EVA 0102, EVA 0103, EVA 0106, EVA 0109 and EVA 0113 were the most active, reducing proliferation of HCT-8 and SF-295 cells in over 75% (Fig. 1).

135 338 Latin American Journal of Aquatic Research Eva 0101 HCT-8 cell growth inhibition (%) MDA MB-435 cell growth inhibition (%) SF-295 cell growth inhibition (%) Eva 0102 Eva 0103 Eva 0105 Eva 0106 Eva 0109 Eva 0110 Eva 0111 Eva 0113 Eva 0114 Eva 0118 Figure 1. Qualitative cytotoxicity screening of EtOAc extracts from liquid medium (dark gray) and MeOH extracts from biomass (light gray) of bacterial strains isolated from E. vannamei. Extracts were tested (100 µg ml -1 ) against HCT-8, MDA-MB-435 and SF-295 cell lines by the MTT assay after 72 h incubation. Data were obtained from two independent experiments and presented as a percentage of growth inhibition relative to untreated cells. Table 1. Cytotoxic activity of EtOAc extracts obtained from bacterial strains isolated from Eudistoma vannamei evaluated by the MTT assay after 72 h incubation. IC 50 values and their respective 95% confidence intervals were obtained by nonlinear regression on the GraphPad Prism 5.0 software (Intuitive Software for Science, San Diego, CA). N.T.: not tested. Strain EVA 0102 EVA 0106 EVA 0109 EVA 0113 Isolation media SCA AIA GYM SWA IC 50 (µg ml -1 ) 95% CI HL-60 HCT-8 MDA MB-435 SF N.T The IC 50 was determined for the extracts obtained from EVA 0102, EVA 0106, EVA 0109 and EVA 0113, since the yield of the extract from EVA 0103 was insufficient for further analyses. The tested extracts showed a similar activity profile, with little variation in potency. As shown on Table 1, EVA 0109 yielded the most cytotoxic extract, with IC 50 values varying from 3.62 to µg ml -1. EVA 0113 followed, with IC 50 ranging from 9.97 to µg ml -1. Chemical analysis of active extracts The extracts from EVA 0102, EVA 0106, EVA 0109 and EVA 0113 were similar on TLC and, as expected, showed the same spectroscopic profile by 1 H NMR. For this reason, just one 1 H NMR spectrum was

136 Cytotoxicity of actinomycetes associated with tunicate 339 depicted (Fig. 2). After a detailed analysis, it was clear the presence of chelated hydroxyl groups (δ ), aromatic and/or olefine hydrogens (δ ), hydrogens bound to oxygenated carbon (δ ), as well as methylene, methyne and methyl protons (δ ) (Silverstein et al., 1991). The aforementioned data were compatible with the presence of quinones or related secondary metabolites. Identification and phylogenetic analyses The analysis of the 16S rrna from the 5 EVA bacterial strains revealed a high degree of similarity among them (Fig. 3). When included in a reconstruction of the phylogeny along with the most similar sequences found on Genbank, the EVA clade came out as the sister group of a clade formed by Micromonospora echinospora, M. marina, M. aurantiaca, M. halophytica, M. humi, M. purpureochromogenes, M. coxensis, M. chalcea and M. tulbagihiae. The EVA clade was formed with a relatively high support value and EVA 0109, 0113, 0102 and 0106 showed a higher degree of overall similarity. For this clade a single contig was then generated and deposited on NCBI GenBank under accession number JN The sequence from EVA 0103 is slightly different, so it was deposited with a separate accession number (JN797619), despite its 16S rrna sequence is still more than 99.9% similar to the other EVA strains. DISCUSSION The study herein began with a screening for cytotoxicity among actinomycetes isolated from the tunicate E. vannamei. The active extracts showed identical 1 H NMR profiles and the respective strains were identified based on a molecular biology approach, where all five isolates were identified within the genus Micromonospora. The results pointed Figure 2. 1 H NMR spectrum (500 MHz, CDCl 3 ) of the EtOAc extract from EVA 0102.

137 340 Latin American Journal of Aquatic Research Micromonospora auratinigra (AB159779) Dactylosporangium aurantiacum (X93191) Micromonospora coerulea (X92598) Micromonospora citrea (X92617) 71 Micromonospora echinofusca (X92625) 78 Micromonospora nigra (X92609) Micromonospora viridifaciens (X92623) Micromonospora echinaurantiaca (X92618) Micromonospora eburnea (AB107231) Micromonospora narathiwatensis (AB193559) Micromonospora pallida (X92608) Micromonospora inyonensis (X92629) Micromonospora sagamiensis (X92624) Micromonospora fulviviridis (X92620) Micromonospora rosaria (X92631) Micromonospora chersina (X92628) 85 Micromonospora endolithica (AJ560635) Micromonospora coriariae (AJ784008) Micromonospora inositola (X92610) Micromonospora chaiyaphumensis (AB196710) 81 Micromonospora rhizosphaerae (FJ261956) Micromonospora echinospora (U58532) 94 Micromonospora marina (AB196712) Micromonospora aurantiaca (ADBZ ) Micromonospora halophytica (X92601) Micromonospora humi (U459068) Micromonospora purpureochromogenes (X92611) Micromonospora coxensis (AB241455) Micromonospora chalcea (X92594) Micromonospora tulbaghiae (U196562) EVA0109 EVA EVA0102 EVA0106 EVA0103 Micromonospora peucetia (X92603) Actinoplanes auranticolor (AB047491) Figure 3. Maximum likelihood tree of the Micromonospora lineages isolated from E. vannamei and most similar species. Dactylosporangium and Actinoplanes were used as outgroups. to at least two species of Micromonospora producing cytotoxic compounds associated with E. vannamei. Micromonospora are a well-known group of Grampositive, spore forming microbes, and are considered the most abundant actinobacteria, along with Rhodococcus and Streptomyces in marine environments (Maldonado et al., 2005). Additionally, they are long accepted as an important source of antibiotics and other substances with commercial value. For instance, some of the best-known antibiotics, such as the aminoglycosides amikacin, gentamicin and netamicin, were initially isolated from these microorganisms (Bérdy, 2005). Furthermore, Micromonospora species with marine occurrence have

138 Cytotoxicity of actinomycetes associated with tunicate 341 yielded many compounds with biomedical relevance. Thiocoraline is produced by a strain of M. marina isolated from a soft coral collected near the coast of Mozambique (Romero et al., 1997). This cyclic thiodepsipeptide showed strong cytotoxic activity against various tumor cell lines, with IC 50 around 2nM. Moreover, thiocoraline induced cell cycle perturbations due to inhibition of DNA-polymerase activity (Pérez-Baz et al., 1997; Romero et al., 1997; Erba et al., 1999). Other example is diazepinomicin, a dibenzodiazepine alkaloid with antimicrobial properties isolated from a Micromonospora strain recovered from Didemnum proliferum, a tunicate collected off the Japanese coast (Charan et al., 2004). In fact, associations of Micromonospora and other actinomycetes with tunicates have been documented elsewhere (Menezes et al., 2010). The Fijian tunicate Polysyncraton lithostrotum is the host to the then unidentified species M. lomaivitiensis, the producer of the potent anticancer compounds lomaivitins A and B (He et al., 2001). These molecules also have antimicrobial activity, which is thought to scare off other bacteria (He et al., 2001). The results presented here, showing the cytotoxicity of a Micromonospora strain and descri-bing its association with the Brazilian endemic ascidian E. vannamei, highlight their potential as producers of relevant molecules. The chemical profile of Micromonospora extracts was different from the one observed by Jimenez et al. (2008) for E. vannamei extracts. While the active compounds presented in ascidian extracts correspond to tryptophan derivatives, spectra data generated for the bacterial extracts herein hinted on the presence of quinoid-like compounds, such as anthracyclines or anthracyclinones (Pretsch et al., 2000; Laatsch & Fotso, 2008). This data suggested that the isolated Micromonospora strain is not responsible for the production of ascidian bioactive compounds. Nonetheless, the activity was similar for both ascidian extract (IC 50 ranging from bellow 2.00 to µg ml -1, Jimenez et al., 2008) and bacterial ones (IC 50 ranging from 3.62 to µg ml -1 ). The occurrence of anthracyclines or related compounds withholding some sort of bioactivity is rather common within the Micromonospora genus. M. lupini yielded the anti-cell invasion anthraquinones lupinacidins A, B and C (Igarashi et al., 2007, 2011). The anthracyclines micromonomycin (Yang et al., 2004) and spartamicins A and B (Nair et al., 1992), produced by different strains of Micromonospora, have antimicrobial and antifungal properties, respectively. Moreover, the previously mentioned lomaivitins are hybrid molecules flanked with naphtoquinone moieties (He et al., 2001). Finally, it can be concluded that acnitomycetes isolated from the Brazilian endemic ascidian E. vannamei are a promising source of bioactive compounds. Therefore, additional studies are in progress to resolve the active principles and to characterize the means by which they may exert cytotoxicity. ACKNOWLEDGMENTS This work has been supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP) and International Foundation for Science (IFS). The authors also wish to thank Silvana França, for technical assistance with the cell cultures, and M.Sc. José Gustavo de A. Lima for generating and editing the 1 H NMR spectra. REFERENCES Baily, C Ready for a comeback of natural products in oncology. Biochem. Pharmacol., 77(9): Bérdy, J Bioactive microbial metabolites: a personal view. J. Antibiot., 58: Blunt, J.W., B.R. Copp, W.P. Hu, M.H. Munro, P.T. Northcote & M.R. Prinsep Marine natural products. Nat. Prod. Rep., 27: Blunt, J.W., B.R. Copp, W.P. Hu, M.H. Munro, P.T. Northcote & M.R. Prinsep Marine natural products. Nat. Prod. Rep., 28: Charan, R.D., G. Schlingmann, J. Janso, V. Bernan, X. Feng, & G.T. Carter Diazepinomicin, a new antimicrobial alkaloid from a marine Micromonospora sp. J. Nat. Prod., 67(8): Chun, J., J.H. Lee, Y. Jung, M. Kim, S. Kim, B.K. Kim & Y.W. Lim EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int. J. Syst. Evol. Microbiol., 57: Cuevas, C. & A. Francesch Development of Yondelis (trabectedin, ET-743). A semisynthetic process solves the supply problem. Nat. Prod. Rep., 26(3): D'Incalci, M. & C.M. Galmarini A review of trabectedin (ET-743): a unique mechanism of action. Mol. Cancer Ther., 9(8): Erba, E., D. Bergamaschi, S. Ronzoni, M. Faretta, S. Taverna, M. Bonfanti, C.V. Catapano, G. Faircloth, J. Jimeno & M. D'Incalci Mode of action of thiocoraline, a natural marine compound with antitumour activity. Braz. J. Cancer, 80:

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141 Lat. Am. J. Aquat. Res., 41(2): , 2013 Cytotoxicity of actinomycetes associated with tunicate Proceedings of the 3 rd Brazilian Congress of Marine Biology A.C. Marques, L.V.C. Lotufo, P.C. Paiva, P.T.C. Chaves & S.N. Leitão (Guest Editors) DOI: /vol41-issue2-fulltext Research Article Cytotoxicity of actinomycetes associated with the ascidian Eudistoma vannamei (Millar, 1977), endemic of northeastern coast of Brazil Paula C. Jimenez 1,2, Elthon G. Ferreira 1,2, Luana A. Araújo 1, Larissa A. Guimarães 1, Thiciana S. Sousa 3 Otília Deusdenia L. Pessoa 3, Tito M. C. Lotufo 1 & Letícia V. Costa-Lotufo 1,2 1 Instituto de Ciências do Mar (LABOMAR), Universidade Federal do Ceará Av. Abolição, 3207, , Fortaleza, Brazil 2 Departamento de Fisiologia e Farmacologia, Faculdade de Medicina Universidade Federal do Ceará, Av. da Universidade, 2853 Benfica Fortaleza, CE, , Brazil 3 Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará Av. da Universidade, 2853, Benfica, Fortaleza, CE, , Brazil ABSTRACT. Previous studies demonstrated that the crude extract of the ascidian Eudistoma vannamei, endemic from northeasttern Brazil, strongly hinders growth of tumor cells in vitro by inducing apoptosis due to tryptophan derivatives, which are commonly found in bacteria. This study presents a bioactivity-guided screening among actinomycetes, associated with E. vannamei, aiming at recognizing active principles with biological relevance. Twenty strains of actinomycetes, designated as EVA 0101 through 0120, were isolated from colonies of E. vannamei among which 11 were selected for cytotoxicity evaluation. The extracts from EVA 0102, 0103, 0106, 0109 and 0113 were the most active, and were further studied for IC 50 determination and chemical analysis by 1 H NMR. IC 50 values obtained ranged from 3.62 µg ml -1 (for EVA 0109 in leukemia cells) to µg/ml (for EVA 0106 in melanoma cells). All active extracts exhibited the same TLC and spectroscopic profiles, suggesting the presence of quinones and other related secondary metabolites. Furthermore, these strains were identified and compared based on their respective 16S rrna sequences. The results herein identified the five strains as Micromonospora spp. while phylogenetic analysis suggests that they are possibly two different Micromonospora species producing the cytotoxic compounds. Keywords: marine biotechnology, Eudistoma vannamei, marine microorganisms, Micromonospora sp., Brazil. Citotoxicidad de actinomicetos asociada a la ascidia Eudistoma vannamei (Millar, 1977), endémica de la costa noreste de Brasil RESUMEN. Estudios previos demostraron que el extracto crudo de la ascidia Eudistoma vannamei, endémica de la costa noreste de Brasil, obstaculiza fuertemente el crecimiento de células tumorales in vitro por inducir apoptosis. El análisis químico del extracto sugirió la presencia de derivados de triptófano, comúnmente encontrados en bacterias. El estudio presenta un screening de citotoxicidad en actinomicetos asociados con E. vannamei, para reconocer principios activos biológicamente relevantes. Veinte cepas de actinomicetos, designados como EVA 0101 hasta 0120, fueron aisladas de colonias de E. vannamei y 11 fueron seleccionadas para la evaluación de citotoxicidad. Los extractos de EVA 0102, 0103, 0106, 0109 y 0113 resultaron las más activas y fueron estudiadas para determinación de la CL 50 y perfiles de CCF y 1 H RMN. Las CL 50 obtenidas oscilaron entre 3,62 (EVA 0109 en las células de leucemia) y 84,65 µg ml -1 (EVA 0106 en las células de melanoma). Los extractos activos presentan el mismo perfil de CCF y espectroscópico, lo que sugiere la presencia de quinonas y metabolitos secundarios relacionados. Además, las cinco cepas fueron identificadas y comparadas sobre la base de sus secuencias de 16S ARNr. Las que se identificaron como Micromonospora spp. y el análisis filogenético sugiere que sean por lo menos dos especies de Micromonospora las que producen los compuestos citotóxicos.

142 336 Latin American Journal of Aquatic Research Palabras clave: biotecnología marina, Eudistoma vannamei, microorganismos marinos, Micromonospora sp., Brasil. Corresponding author: Letícia Lotufo INTRODUCTION Colorful soft-bodied sessile marine invertebrates have been a rich source of natural products with biomedical properties for the past 60 years (Baily, 2009; Molinski et al., 2009; Mayer et al., 2010). Among them, the tunicates are one of the most prolific in terms of novel drug prototypes for the anticancer pharmaceutical arsenal (Blunt et al., 2010, 2011). For instance, trabectedin, a tetrahydroisoquinoline alkaloid obtained from Ecteinascidia turbinata, was recently approved for the treatment of soft tissue sarcoma, with the commercial name Yondelis (Cuevas & Francesh, 2009; D Incalci & Galmarini, 2010). Additionally, plitidepsin, a cyclodepsipeptide isolated from Aplidium albicans is in advanced trial phase for the therapy of multiple myeloma and other malignant tumors, but has already received orphan drug status for the treatment of multiple myeloma and acute lymphoblastic leukemia (Ocio et al., 2008; Singh et al., 2008). There are mounting evidences that the associated microorganisms are the authentic suppliers for most of the relevant compounds (Zhang et al., 2005; Imhoff et al., 2011). For the ascidians, in particular, there is a compelling amount of information pointing into this direction (Schmidt & Donia, 2010). Despite the oceans are acknowledged as a rich and highly complex microbiological environment, they have only shared their wealth as a natural drug source for the past 10 years. Thus, the study of marine microorganisms as sources of new drugs is still at its youth (Jensen et al., 2005; Fenical & Jensen, 2006; Liu et al., 2010). This study takes part in a pioneer venture aiming to the exploration of the biotechnological potentials of microorganisms, mainly actinomycetes, from the coast of Ceará. The actinomycetes have proven to be a prolific source of new and bioactive compounds (Fenical & Jensen, 2006). The purpose of the present investigation is to find biologically active strains of actinomycetes and evaluate their respective cytotoxicity, while gathering information on the microbiota associated with Eudistoma vannamei. This species is endemic from the northeastern coast of Brazil and is the most abundant ascidian recorded for the state of Ceará. Previous studies have revealed that its crude extract strongly hinders growth of tumor cells in vitro, promoting further apoptosis (Jimenez et al., 2003, 2008). MATERIALS AND METHODS Sampling and isolation of bacteria strains Specimens of E. vannamei were hand collected at Taiba Beach (03 30'21.23 S, '40.16 W), located on the northeastern coast of Brazil. Sampling was carried out with the slightest contamination possible, as the material was gathered and stored with sterile utensils. For isolation of microorganisms, five colonies were homogenized in sterile seawater 1:5 (m/v), and aliquots of 20 L were stroked onto Petri dishes containing different agar based media, which may favor actinomycetes growth (modified from Jensen et al., 1991). The following agar-based media were used herein, all added with cycloheximide 0.1 mg ml -1 : SCA (Starch Casein Agar, HIMEDIA, prepared with distilled H 2 O), AIA (Actinomycetes Isolation Agar, HIMEDIA, prepared in 75% seawater), SWA (Sea-water Agar, 1.8% agar in 75% seawater) and GYM (glucose 0.4%, yeast extract 0.4%, malt extract 1%, CaCO 3 0.2%, 1.8% agar in 75% seawater). Purity of strains were resolved by sequential restrikes onto brand new agar plates and pure strains were inoculated in liquid A1 medium (10% soluble starch, 4% yeast extract and 2% peptone in 75% seawater) for culture maintenance and up-scaled growth (Jensen et al., 1991). Grown media for each pure strain was sampled into cryovials and supplemented with 25% glycerol for storage at -70 o C. Up-scaled growth and extraction Initially, pure cultures were inoculated in 500 ml Erlenmeyer flasks filled with 100 ml of liquid A1 medium. Flasks were kept at 28 o C and 200 rpm for 5 days. Next, 10 ml of grown media were added to newly assembled Erlenmeyer flasks with 100 ml of fresh media and kept at 28 o C and 200 rpm agitation for 10 days. The resulting media was spun down to separate the bacterial biomass from the broth. Biomass was extracted with MeOH 1:1 (m/v) while broth was extracted in EtOAc 1:1 (v/v). The crude extracts were evaporated under vacuum, washed with MeOH to

143 Cytotoxicity of actinomycetes associated with tunicate 337 remove excessive salt, and fully dried under compressed air, yielding the crude extracts. At this point, extracts were resuspended in DMSO to a final concentration of 10 µg ml -1 and were ready to be evaluated for their biological activity. Screening for cytotoxicity-mtt assay Cytotoxic activity of extracts was evaluated against four human tumor cell lines obtained from the National Cancer Institute (Bethesda, MD, USA): HL- 60 (promyelocytic leukemia), MDA-MB-435 (melanoma), SF-295 (glioblastoma) and HCT-8 (colon carcinoma). Cells were grown in RPMI-1640 medium supplemented with 2 mm glutamine, 10% fetal calf serum, 100 µg ml -1 strep-tomycin and 100 U ml -1 penicillin and housed at 37ºC with a 5% CO 2 atmosphere. The cell cultures were regularly split to keep them in a logarithmic phase of growth. Firstly, in a qualitative approach, cell lines were exposed to 100 µg ml -1 of the extracts for 72 h and the effect on cell proliferation was evaluated in vitro using the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2h-tetrazolium bromide] assay, as described by Mosmann (1983). The extracts that reduced over 80% of cell growth, when compared to non-treated cells, were tested again, in a quantitative manner, however, at concentrations varying serially from 0.02 to 100 µg ml -1 to determine the IC 50 (the concentration that inhibits growth in 50%), which was calculated, along with the respective 95% CI (confidence interval), by a non-linear regression using the software GraphPad Prism 5.0. Preliminary chemical analysis The active extracts (EVA 0102, EVA 0106, EVA 0109 and EVA 0113) were submitted to a preliminary chemical characterization. Initially, these extracts were compared by thin layer chromatography (TLC) on precoated silica gel aluminum sheets (Kieselgel 60 F 254, 0.20 mm, Merck) using two different solvent systems (CHCl 3 and CHCl 3 : EtOAc 1:1) and the compounds were detected by exposure to UV (312 and 365 nm) light and by spraying with vanillin/ perchloric acid/etoh solution followed by heating at 100 C. The extracts were also analyzed by 1 H NMR spectroscopy. The 1 H NMR spectra were acquired on a Bruker Avance DRX-500 spectrometer equipped with 5 mm inverse detection z-gradient probe. The chemical shifts, given on the δ scale, were referenced to the residual undeuterated CHCl 3 (δ H 7.27) fraction. The methodology was based in a previous work published by Jimenez et al. (2008). Molecular identification: nucleic acid extraction, 16S rrna gene amplification, and sequencing The selected microorganisms were characterized using a molecular approach based on the protocol described by Gontang et al. (2007) with slight modifications. Genomic DNA was extracted from a fresh, 3 to 5 day grown, bacterial culture following the DNeasy protocol (QIAGEN Inc., USA). Amplification of the 16S rrna gene was carried out with Ilustra PureTaq Ready-To-Go PCR beads (GE Healthcare, UK) using the universal eubacterial primers 27F (5 - AGAGTTTGATCCTGGCTCAG-3 ) and 1492R (5 - TACGGCTACCTTGTTACGACTT-3 ). PCR products were purified with MiniElute PCR purification kit (QIAGEN Inc., USA). DNA sequencing was performed by Macrogen Inc. (Seoul, Korea) using the ABI PRISM BigDye TM Terminator Cycle Sequencing kit (Applied Biosystems, USA) and the same primers used for amplification, following the protocols supplied by the manufacturer. The nucleotide sequences were assembled, analyzed and manually edited using the BioEdit Sequence Alignment Editor (Hall, 1999) and compared to sequences within the NCBI database ( using the Basic Local Alignment Search Tool (BLAST). The identity of the strain was confirmed after an evaluation of the phylogenetic relationships with the most similar sequences obtained from the EzTaxon server (Chun et al., 2007). The sequences were aligned using Muscle ver. 3.6, and the reconstruction was performed using a combined bootstrapping and maximum likelihood approach through RAxML (Stamakis, 2006) on the CIPRES portal (Miller et al., 2010). The tree was produced using Archaeopterix ver (Han & Zmasek, 2009). After the phylogenetic analysis, the sequences were submitted to NCBI GenBank. RESULTS Isolation and cytotoxicity screening of bacteria associated with E. vannamei Twenty colonies were isolated from E. vannamei respecting their actinomycete-like distinctiveness. Out of those, 11 were elected for cytotoxicity evaluation on tumor cells. In the single concentration screening, extracts obtained from strains EVA 0102, EVA 0103, EVA 0106, EVA 0109 and EVA 0113 were the most active, reducing proliferation of HCT-8 and SF-295 cells in over 75% (Fig. 1).

144 338 Latin American Journal of Aquatic Research Eva 0101 HCT-8 cell growth inhibition (%) MDA MB-435 cell growth inhibition (%) SF-295 cell growth inhibition (%) Eva 0102 Eva 0103 Eva 0105 Eva 0106 Eva 0109 Eva 0110 Eva 0111 Eva 0113 Eva 0114 Eva 0118 Figure 1. Qualitative cytotoxicity screening of EtOAc extracts from liquid medium (dark gray) and MeOH extracts from biomass (light gray) of bacterial strains isolated from E. vannamei. Extracts were tested (100 µg ml -1 ) against HCT-8, MDA-MB-435 and SF-295 cell lines by the MTT assay after 72 h incubation. Data were obtained from two independent experiments and presented as a percentage of growth inhibition relative to untreated cells. Table 1. Cytotoxic activity of EtOAc extracts obtained from bacterial strains isolated from Eudistoma vannamei evaluated by the MTT assay after 72 h incubation. IC 50 values and their respective 95% confidence intervals were obtained by nonlinear regression on the GraphPad Prism 5.0 software (Intuitive Software for Science, San Diego, CA). N.T.: not tested. Strain EVA 0102 EVA 0106 EVA 0109 EVA 0113 Isolation media SCA AIA GYM SWA IC 50 (µg ml -1 ) 95% CI HL-60 HCT-8 MDA MB-435 SF N.T The IC 50 was determined for the extracts obtained from EVA 0102, EVA 0106, EVA 0109 and EVA 0113, since the yield of the extract from EVA 0103 was insufficient for further analyses. The tested extracts showed a similar activity profile, with little variation in potency. As shown on Table 1, EVA 0109 yielded the most cytotoxic extract, with IC 50 values varying from 3.62 to µg ml -1. EVA 0113 followed, with IC 50 ranging from 9.97 to µg ml -1. Chemical analysis of active extracts The extracts from EVA 0102, EVA 0106, EVA 0109 and EVA 0113 were similar on TLC and, as expected, showed the same spectroscopic profile by 1 H NMR. For this reason, just one 1 H NMR spectrum was

145 Cytotoxicity of actinomycetes associated with tunicate 339 depicted (Fig. 2). After a detailed analysis, it was clear the presence of chelated hydroxyl groups (δ ), aromatic and/or olefine hydrogens (δ ), hydrogens bound to oxygenated carbon (δ ), as well as methylene, methyne and methyl protons (δ ) (Silverstein et al., 1991). The aforementioned data were compatible with the presence of quinones or related secondary metabolites. Identification and phylogenetic analyses The analysis of the 16S rrna from the 5 EVA bacterial strains revealed a high degree of similarity among them (Fig. 3). When included in a reconstruction of the phylogeny along with the most similar sequences found on Genbank, the EVA clade came out as the sister group of a clade formed by Micromonospora echinospora, M. marina, M. aurantiaca, M. halophytica, M. humi, M. purpureochromogenes, M. coxensis, M. chalcea and M. tulbagihiae. The EVA clade was formed with a relatively high support value and EVA 0109, 0113, 0102 and 0106 showed a higher degree of overall similarity. For this clade a single contig was then generated and deposited on NCBI GenBank under accession number JN The sequence from EVA 0103 is slightly different, so it was deposited with a separate accession number (JN797619), despite its 16S rrna sequence is still more than 99.9% similar to the other EVA strains. DISCUSSION The study herein began with a screening for cytotoxicity among actinomycetes isolated from the tunicate E. vannamei. The active extracts showed identical 1 H NMR profiles and the respective strains were identified based on a molecular biology approach, where all five isolates were identified within the genus Micromonospora. The results pointed Figure 2. 1 H NMR spectrum (500 MHz, CDCl 3 ) of the EtOAc extract from EVA 0102.

146 340 Latin American Journal of Aquatic Research Micromonospora auratinigra (AB159779) Dactylosporangium aurantiacum (X93191) Micromonospora coerulea (X92598) Micromonospora citrea (X92617) 71 Micromonospora echinofusca (X92625) 78 Micromonospora nigra (X92609) Micromonospora viridifaciens (X92623) Micromonospora echinaurantiaca (X92618) Micromonospora eburnea (AB107231) Micromonospora narathiwatensis (AB193559) Micromonospora pallida (X92608) Micromonospora inyonensis (X92629) Micromonospora sagamiensis (X92624) Micromonospora fulviviridis (X92620) Micromonospora rosaria (X92631) Micromonospora chersina (X92628) 85 Micromonospora endolithica (AJ560635) Micromonospora coriariae (AJ784008) Micromonospora inositola (X92610) Micromonospora chaiyaphumensis (AB196710) 81 Micromonospora rhizosphaerae (FJ261956) Micromonospora echinospora (U58532) 94 Micromonospora marina (AB196712) Micromonospora aurantiaca (ADBZ ) Micromonospora halophytica (X92601) Micromonospora humi (U459068) Micromonospora purpureochromogenes (X92611) Micromonospora coxensis (AB241455) Micromonospora chalcea (X92594) Micromonospora tulbaghiae (U196562) EVA0109 EVA EVA0102 EVA0106 EVA0103 Micromonospora peucetia (X92603) Actinoplanes auranticolor (AB047491) Figure 3. Maximum likelihood tree of the Micromonospora lineages isolated from E. vannamei and most similar species. Dactylosporangium and Actinoplanes were used as outgroups. to at least two species of Micromonospora producing cytotoxic compounds associated with E. vannamei. Micromonospora are a well-known group of Grampositive, spore forming microbes, and are considered the most abundant actinobacteria, along with Rhodococcus and Streptomyces in marine environments (Maldonado et al., 2005). Additionally, they are long accepted as an important source of antibiotics and other substances with commercial value. For instance, some of the best-known antibiotics, such as the aminoglycosides amikacin, gentamicin and netamicin, were initially isolated from these microorganisms (Bérdy, 2005). Furthermore, Micromonospora species with marine occurrence have

147 Cytotoxicity of actinomycetes associated with tunicate 341 yielded many compounds with biomedical relevance. Thiocoraline is produced by a strain of M. marina isolated from a soft coral collected near the coast of Mozambique (Romero et al., 1997). This cyclic thiodepsipeptide showed strong cytotoxic activity against various tumor cell lines, with IC 50 around 2nM. Moreover, thiocoraline induced cell cycle perturbations due to inhibition of DNA-polymerase activity (Pérez-Baz et al., 1997; Romero et al., 1997; Erba et al., 1999). Other example is diazepinomicin, a dibenzodiazepine alkaloid with antimicrobial properties isolated from a Micromonospora strain recovered from Didemnum proliferum, a tunicate collected off the Japanese coast (Charan et al., 2004). In fact, associations of Micromonospora and other actinomycetes with tunicates have been documented elsewhere (Menezes et al., 2010). The Fijian tunicate Polysyncraton lithostrotum is the host to the then unidentified species M. lomaivitiensis, the producer of the potent anticancer compounds lomaivitins A and B (He et al., 2001). These molecules also have antimicrobial activity, which is thought to scare off other bacteria (He et al., 2001). The results presented here, showing the cytotoxicity of a Micromonospora strain and descri-bing its association with the Brazilian endemic ascidian E. vannamei, highlight their potential as producers of relevant molecules. The chemical profile of Micromonospora extracts was different from the one observed by Jimenez et al. (2008) for E. vannamei extracts. While the active compounds presented in ascidian extracts correspond to tryptophan derivatives, spectra data generated for the bacterial extracts herein hinted on the presence of quinoid-like compounds, such as anthracyclines or anthracyclinones (Pretsch et al., 2000; Laatsch & Fotso, 2008). This data suggested that the isolated Micromonospora strain is not responsible for the production of ascidian bioactive compounds. Nonetheless, the activity was similar for both ascidian extract (IC 50 ranging from bellow 2.00 to µg ml -1, Jimenez et al., 2008) and bacterial ones (IC 50 ranging from 3.62 to µg ml -1 ). The occurrence of anthracyclines or related compounds withholding some sort of bioactivity is rather common within the Micromonospora genus. M. lupini yielded the anti-cell invasion anthraquinones lupinacidins A, B and C (Igarashi et al., 2007, 2011). The anthracyclines micromonomycin (Yang et al., 2004) and spartamicins A and B (Nair et al., 1992), produced by different strains of Micromonospora, have antimicrobial and antifungal properties, respectively. Moreover, the previously mentioned lomaivitins are hybrid molecules flanked with naphtoquinone moieties (He et al., 2001). Finally, it can be concluded that acnitomycetes isolated from the Brazilian endemic ascidian E. vannamei are a promising source of bioactive compounds. Therefore, additional studies are in progress to resolve the active principles and to characterize the means by which they may exert cytotoxicity. ACKNOWLEDGMENTS This work has been supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP) and International Foundation for Science (IFS). The authors also wish to thank Silvana França, for technical assistance with the cell cultures, and M.Sc. José Gustavo de A. Lima for generating and editing the 1 H NMR spectra. REFERENCES Baily, C Ready for a comeback of natural products in oncology. Biochem. Pharmacol., 77(9): Bérdy, J Bioactive microbial metabolites: a personal view. J. Antibiot., 58: Blunt, J.W., B.R. Copp, W.P. Hu, M.H. Munro, P.T. Northcote & M.R. Prinsep Marine natural products. Nat. Prod. Rep., 27: Blunt, J.W., B.R. Copp, W.P. Hu, M.H. Munro, P.T. Northcote & M.R. Prinsep Marine natural products. Nat. Prod. Rep., 28: Charan, R.D., G. Schlingmann, J. Janso, V. Bernan, X. Feng, & G.T. Carter Diazepinomicin, a new antimicrobial alkaloid from a marine Micromonospora sp. J. Nat. Prod., 67(8): Chun, J., J.H. Lee, Y. Jung, M. Kim, S. Kim, B.K. Kim & Y.W. Lim EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int. J. Syst. Evol. Microbiol., 57: Cuevas, C. & A. Francesch Development of Yondelis (trabectedin, ET-743). A semisynthetic process solves the supply problem. Nat. Prod. Rep., 26(3): D'Incalci, M. & C.M. Galmarini A review of trabectedin (ET-743): a unique mechanism of action. Mol. Cancer Ther., 9(8): Erba, E., D. Bergamaschi, S. Ronzoni, M. Faretta, S. Taverna, M. Bonfanti, C.V. Catapano, G. Faircloth, J. Jimeno & M. D'Incalci Mode of action of thiocoraline, a natural marine compound with antitumour activity. Braz. J. Cancer, 80:

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150 Lat. Am. J. Aquat. Res., 41(2): , 2013 Seaweeds in ornamental aquaria in Brazil Proceedings of the 3 rd Brazilian Congress of Marine Biology A.C. Marques, L.V.C. Lotufo, P.C. Paiva, P.T.C. Chaves & S.N. Leitão (Guest Editors) DOI: /vol41-issue2-fulltext Research Article Seaweeds in ornamental aquaria in Brazil: anticipating introductions Beatriz N. Torrano-Silva 1, Carlos E. Amancio 1 & Eurico C. Oliveira 1,2 1 Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo CEP , São Paulo, SP, Brazil 2 Departamento de Botânica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina CEP , Santa Catarina, Brazil ABSTRACT. Home and public sea aquaria are becoming more and more popular worldwide. Aquaria species are selected for their aesthetic appeal in terms of color, shape or behavior. In Brazil, most species are imported from remote places, usually tropical areas around the world. Water discarded from aquaria often includes organisms, or their propagula, that may become locally established. There have been extensive discussions concerning the vectors of species dispersion from their native areas, which includes ornamental aquaria. In order to evaluate the potential of local aquaria as a source of non native macro algae, we surveyed sea-aquaria shops and exhibition aquaria in São Paulo State. Thirty eight species of seaweeds were isolated in the aquaria surveyed. Among them, six have not been recorded from Brazil and nineteen were not reported for São Paulo littoral, including Caulerpa scalpelliformis var. denticulata the only species that so far deserves being designated as invasive in Brazil. The present paper should alert environmental agencies and aquaria shops about the risk of involuntarily introducing non-native species through aquariophyly. Keywords: aquariophyly, Caulerpa scalpelliformis, exotic species, invasive macroalgae, species introduction, Brazil. Algas de acuarios ornamentales en Brasil: previsión de las introducciones RESUMEN. Por todo público cada vez son más populares los acuarios marinos, sean caseros o de exhibición. Las especies vivientes escogidas generalmente tienen algún atractivo tal como su color, aspecto general o comportamiento de interés. Se sabe que muchos organismos utilizados para esta actividad en Brasil vienen de sitios lejanos, generalmente tropicales. El agua descartada de los acuarios suele contener organismos vivos o sus propágulos, los cuales se pueden establecer localmente en una comunidad natural. La comunidad científica ha realizado investigaciones y discusiones al respecto de los vectores de dispersión de especies, los que incluyen a los acuarios ornamentales. Para evaluar el potencial de los acuarios brasileños como fuentes dispersoras de macroalgas no-nativas, se investigaron en tiendas de acuariofilia y en establecimientos de exposición al público. Treinta y ocho especies de algas marinas fueron encontradas; entre estas, seis no eran conocidas en Brasil y diecinueve no habían sido reportadas para el litoral de São Paulo, incluyendo Caulerpa scalpelliformis var. denticulata, la única especie conocida hasta el momento como macroalga invasora en Brasil. El presente trabajo debe alertar a los sectores ambientales y establecimientos comerciales ligados a la acuariofilia en cuanto al riesgo de introducciones involuntarias de organismos no-nativos por esta actividad. Palabras clave: acuariofilia, Caulerpa scalpelliformis, especies exóticas, macroalgas invasoras, introducción de especies, Brasil. Corresponding author: Beatriz Torrano-Silva (biatorrano@yahoo.com.br) INTRODUCTION Aquaria business is growing 14% annually worldwide, and it is credited as a source of around 150 species of marine organisms that have reached and become established in natural ecosystems; they comprise one third of the worst recorded aquatic cases (Padilla & Williams, 2004). The most publicized algal introduction is the killer alga, Caulerpa taxifolia (Vahl) C. Agardh in the Mediterranean, which spread

151 345 Latin American Journal of Aquatic Research within a short time, displacing many native species (Meinesz et al., 1993, 2001). Everything indicates that the original source of propagula of C. taxifolia originated from discarded water from the Monaco Aquarium (Jousson et al., 1998; Meusnier et al., 2001; Wiedenmann et al., 2001). Exporting countries of a variety of marine aquarium specimens include Vietnam, Hong Kong, Taiwan, Thailand, Australia, New Zealand, Sri Lanka, Ethiopia, Saudi Arabia, Egypt, Kenya, Madagascar, Mauritius, Polynesia, Micronesia, Melanesia, Mexico, Honduras, Costa Rica, Venezuela, Colombia, several Caribbean islands, and Brazil (Lubbock & Polunin, 1975; Padilla & Williams, 2004). Although ballast water discharge and biofouling has long been seen as the major marine introducing vector, aquarium trade could carry out selectively resistant species, tolerant to stressing conditions during collection and transportation (Padilla & Williams, 2004). Although the introduction of non-native aquatic species is a recent concern in Brazil, the issue has reached popular media in the last decade. The concern is probably due to the dissemination of news about some dramatic ecological problem originated from invasive introduced species, as well as negative economic impacts from recent local introductions. Among those, we can mention the case of the golden mussel, Limnoperna fortunei (Dunker), which invaded freshwater ecosystems, resulting in numerous events of clogging water pipes and pumps, from hydroelectric reservoirs to industrial and domestic use (Mansur et al., 1999, 2003). Another case, is the bivalve Isognomon bicolor (C.B. Adams), which was probably introduced around the 1980 s and is now one of the most pressing concerns among marine organisms. This invasive species is now rapidly expanding on rocky coasts from southeastern Brazil (Fernandes et al., 2004). The introduction of non-native seaweeds in Brazil has been documented by many authors, but approaches were made usually on commercial species or involuntary introductions via ballast water (Horta & Oliveira, 2000; Bellorin & Oliveira, 2001; Oliveira & Paula, 2003; Paula & Oliveira, 2004). A national review was recently summarized (Lopes, 2009; Silva et al., 2010), including lists of species, as well as procedures, to avoid and restrict those events. Although the federal government, worried about the introduction of exotic organisms, has been organizing special forums to discuss this matter, since 2005, the emphasis is on agricultural pests and freshwater organisms. So far, only one case of invasive seaweed is confirmed for Brazil: Caulerpa scalpelli-formis (R. Br. ex Turner) C. Agardh var. denticulata (Decaisne) Weber-van Bosse. Considering that this seaweed is often used in aquaria and the potential that this activity has as a vector for invasive species, motivated us to carry on this investigation, aiming to: 1) disclose what kind of seaweed specimens are present in ornamental aquaria in Brazil; 2) provide a baseline to look for the sudden appearance of one of these taxa on the coast of the State of São Paulo; 3) alert local regulatory agencies about the risk of species introduction via aquaria; and 4) publicize this risk. MATERIALS AND METHODS In order to analyze the potential of aquaria as a vector of macro algal introductions in Brazil we have conducted the first survey of macro algae in public and commercial establishments within the country. Our sampling was concentrated to São Paulo, the main commercial city in the country and known to be the main port of entry and distribution of sea organisms in Brazil. Samples of all the algae visible to the naked eye were collected in two exhibition aquaria and five shops, of which two are also national distributors. All shops are located in São Paulo city. The exhibition aquaria are located in the coastal cities of Guarujá and Ubatuba. In an attempt to identify the macro algal species, live samples were scraped from the walls or removed with a forceps from various substrata and transported to the laboratory for identification. Information about the origin of the seaweeds, if known, and animals, usually fish and corals present in the aquaria, the source of seawater and substrata, was recorded (Table 1). In cases where specimens were not fully developed or reproductive structures were necessary for identification, the samples were cultivated in vitro, in sterilized seawater enriched with von Stosch medium, according to the conditions described in Oliveira et al. (1995). The medium was renewed weekly, aerated intermittently for 30 min and kept at 25 ± 1 C, salinity 33 psu, and photoperiod of 14 h per day. RESULTS We isolated 38 macroalgae species (Table 2), seven of which could not be identified. Six of the species identified were not previously reported from Brazil: Caulerpa nummularia, Chaetomorpha spiralis, Cladophora submarina, Derbesia tenuissima, D. turbinata and Halimeda goreaui. Of the total list, 18 species have not been reported for the coast of the State of São Paulo, although reported northward in the

152 Seaweeds in ornamental aquaria in Brazil 346 Table 1. Conditions find in tanks during sampling. The names of establishments have been abbreviated shops: EM, EA and AI; shop and distributor: ON and AI; exhibition aquaria: AM. Tanks from aquaria of Ubatuba are not computed due to the absence of seaweeds. Tanks with no seaweed entries are related to fail on isolating specimens. Tank Establishments Date N of seaweed spp. Water temp. ( C) Salinity (psu) Water source Substrata source Animals Animals source A EM 02/25/ ,9 Artificial Brazil (northeastern coast) fish Unknown B EM 02/25/ ,9 Artificial Brazil (northeastern coast) fish, corals, crustaceans Unknown C EA 03/02/ ,8 33,5 São Paulo coast Artificial corals Hawaii, Indonesia D EA 03/02/ ,8 33,5 São Paulo coast Artificial fish, corals, molluscs Hawaii, Indonesia E EA 03/02/ ,8 33,5 São Paulo coast Artificial fish Hawaii, Indonesia F EA 05/11/ ,8 33,5 São Paulo coast Artificial fish, coral, sea anemones Brazil G EA 05/11/ ,8 33,5 São Paulo coast Artificial corals Indonesia H EA 05/11/ ,8 33,5 São Paulo coast Artificial fish, corals, Indonesia I EP 08/06/ ,5 35,6 Artificial Artificial fish, corals, sea anemones, polychaetes Indonesia, Fiji, Australia, Saudi Arabia; Brazil (northeastern coast) J AM 01/03/ ,5 São Paulo coast Brazil (northeastern coast) fish, Octopus Brazil (northeastern coast), other distributors in São Paulo K AM 01/03/ ,5 35,5 São Paulo coast Brazil (northeastern coast) fish, sea anemones Unknown (other distributors in São Paulo) L AM 01/03/ São Paulo coast Brazil (northeastern coast) fish Unknown (other distributors in São Paulo) M AI 01/21/ ,5 Artificial Artificial fish, corals Asia, Australia (directly or from other distributor) N AI 01/21/ ,6 Artificial Brazil (northeastern coast) fish, polychaetes Asia, Australia (directly or from other distributor) O ON 01/24/ ,5 Artificial Natural substrata of unknown origin fish, corals Red Sea, Caribbean, Indonesia, Falklands, Sri Lanka; Brazil (northeastern coast)

153 347 Latin American Journal of Aquatic Research Table 2. List of species collected from various evaluated aquaria. The non-native column indicates species unknown from the São Paulo State coast (SP) or from Brazil (BR). The list of tanks with its corresponding conditions is presented in Table 1. Abundance was visually estimated. Species Non-native Tanks Abundance Chlorophyta Anadyomene stellata (Wulfen) C. Agardh SP N low Avrainvillea sp. SP O low Bryopsis pennata J.V. Lamouroux I, O high Bryopsis plumosa (Hudson) C. Agardh G high Caulerpa nummularia Harvey ex J. Agardh BR G low Caulerpa racemosa (Forsskål) J. Agardh C high Caulerpa scalpelliformis var. denticulata (Decaisne) Weber-van Bosse SP B, I, N high Caulerpa webbiana Montagne SP O low Chaetomorpha spiralis Okamura BR N low Cladophora coelothrix Kützing E high Cladophora prolifera (Roth) Kützing N high Cladophora submarina P.L. Crouan & H.M. Crouan BR L high Derbesia marina (Lyngbye) Kjellman G medium Derbesia tenuissima (Moris & De Notaris) P.L. Crouan & H.M. Crouan BR I medium Derbesia turbinata M. Howe & Hoyt BR C, N medium Halimeda goreaui W.R. Taylor BR N low Halimeda tuna (J. Ellis & Solander) J.V. Lamouroux SP C, N high Ulva flexuosa Wulfen O low Ulva rigida C. Agardh SP O, N low Valonia aegagropila C. Agardh SP A, N medium Valonia ventricosa (J. Agardh) J.L. Olsen & J.A. West SP H, O low Phaeophyceae Canistrocarpus cervicornis (Kützing) De Paula & De Clerck G high Dictyota bartayresiana J.V. Lamouroux C, I high Dictyota mertensii (Martius) Kützing SP I high Sargassum vulgare C. Agardh O low Rhodophyta Acanthophora spicifera (M. Vahl) Børgesen A low Amansia multifida J.V. Lamouroux SP N low Amphiroa sp. N, O low Digenea simplex (Wulfen) C. Agardh SP N medium Gelidiaceae (two species) A, G, I, J, K medium Gracilaria birdiae Palastino & Oliveira SP N low Gracilaria spp. (three underproductive species) K low Hypnea spinella (C. Agardh) Kützing I medium Palisada gemmifera (Harvey) K.W. Nam SP I high Peyssonelia sp. N low

154 Seaweeds in ornamental aquaria in Brazil 348 country (Horta et al., 2001; brasilis.ccb.ufsc.br). DISCUSSION The brown algae were the least represented group within the sampled aquaria. Such a pattern was expected as the Phaeophyceae are less diversified than other seaweed groups on Brazilian coast, and because they are usually rather bulky and difficult to maintain within aquaria. Green algae were the most diverse group, despite the fact that red algae are the most diverse group in Brazil (Horta et al., 2001). One hypothesis to explain the larger diversity of green algae in marine aquaria comes from their higher tolerance to a broader variation of light, temperature and salinity (Davison & Pearson, 1996; Taylor et al., 2001). Although we did not measure nutrient contents in the sampled aquaria, it may well be the case that their water is rather eutrophic due to a relatively large proportion of animals. Such conditions would favor opportunistic species with short life cycles, as are the ones we found. In fact, aquarium hobbyists need to actively control the nutrients equilibrium and adopt other procedures to avoid dissemination of blue-green algae. Temperatures ranged from 25 to 31 C and salinities from 30 to 36 psu (Table 1), which were adequate for tropical organisms. The algal species found in aquaria stores are kept as ornamental species or on the assumption that they would contribute to the health of the mesocosm, mainly by the absorption of soluble phosphorus and nitrogen from animal excreta. Among the sampled aquaria, the preferred species for those purposes were two green algae, Caulerpa scalpelliformis var. denticulata and Halimeda tuna. Most of the other species we found are considered as a nuisance, and are periodically removed and discarded. Many were collected from large acclimatization and quarantine tanks for newly arrived material. Such species probably arrive as contaminating propagules, especially on rhodoliths, that are locally known as living-rocks. Most aquaria owners use rhodoliths as an obligate component of marine aquaria for aesthetic purposes and to balance the carbonate system. Most particulate calcareous substrates at the bottom are also biogenic and made out of rhodoliths, and dead Halimeda fragments, collected at the coast of the states of Espírito Santo and Pernambuco. They are usually not sterilized before being dumped in aquaria. Such a usage pattern could explain why the large majority of the species we found are also present on Espírito Santo and on Pernambuco coasts. On the other hand, sea animals come from various distant places such as the Red Sea, the Caribbean, Indonesia, Hawaii and Australia. It is relevant to register that we did not find any seaweed in the public aquaria of Ubatuba, which documents the effectiveness of their water treatment, including the usage of ozone. Other treatments commonly adopted, such as filtration and decanting, proved to help, but did not eliminate micro propagules from water. On the other hand the use of artificial marine water was not completely efficient against seaweed contamination due to the utilization of liverocks, certainly a source of propagules and plantlets. The case of Caulerpa scalpelliformis var. denticulata is emblematic because this species, known to occur from the State of Espírito Santo to the north and found recently in the State of Rio de Janeiro, is spreading and displacing other species (Falcão & Széchy, 2005). As the occurrence spot (Ilha Grande Bay), is a popular tourist area, biofouling on boats hulls has been considered as the introduction vector. However, the hypothesis of an introduction via aquariophily cannot be discarded, considering its common presence in aquaria, due to its beautiful color and shape (Oliveira et al., 2009). Nevertheless, the disseminated use of Caulerpa species in aquaria in Brazil is a motive of concern. Therefore, our observations contribute to disseminate the idea that aquaria may effectively work as a vector for species introduction not only of seaweeds, the matter of our concern here, but also of other organisms including microorganisms, as have already been remarked by Corrêa et al. (1980) and Stewart (1991). Although it is well known that the effective establishment of a species in a new locality depends on many factors, such as tolerance to changes in various local environmental parameters, absence of predators, competitiveness vis a vis to other local organisms that utilize a similar ecological niche, and other factors. There is a rich literature supporting successful establishment of transplanted species (e.g., Bellorín & Oliveira, 2001; Oliveira & Paula, 2003). Nevertheless, caution should be taken, and educational procedures established, concerning the discharging of various substrata, seawater and live or dead organisms, especially in locations close to the sea. Such steps should be enforced in order to avoid the introduction of undesired organisms that could become pests and are impossible to eradicate. Introductions from aquaria can be avoided with simple measures, once there is an understanding and acceptance from aquarists to suggestions that contribute to the ecological safety of their hobby (Weigle et al., 2005).

155 349 Latin American Journal of Aquatic Research AKNOWLEDGEMENTS We thank the aquaria establishments that collaborated with this survey, Prof. Robert Okasaki for improving the English and the Conselho Nacional para o Desenvolvimento Científico e Tecnológico (CNPq) for supporting this work. REFERENCES Bellorín, A. & E.C. Oliveira Introducción de especies exóticas de algas marinas: situación en América Latina. In: K. Alveal & T. Antezana (eds.). Sustentabilidad de la biodiversidad. Universidad de Concepción, Concepción, pp Corrêa, L.L., M.O.A. Corrêa, J.F. Vaz, M.I.P.G. Silva, R.M. Silva & M.T. Yamanaka Importância das plantas ornamentais dos aquários como veículos de propagação de vetores de Schistosoma mansoni. Rev. Inst. Adolfo Lutz, 40: Davison, I.R. & G.A. Pearson Stress tolerance in intertidal seaweeds. J. Phycol., 32: Falcão, C. & M.T.M. Széchy Changes in shallow phytobenthic assemblages in southeastern Brazil, following the replacement of Sargassum vulgare (Phaeophyta) by Caulerpa scalpelliformis (Chlorophyta). Bot. Mar., 48: Fernandes, F.C., L.C. Rapagnã & G.B.D. Bueno Estudo da população do bivalve exótico Isognomon bicolor (C.B. Adams, 1845) (Bivalvia, Isonomonidae) na Ponta da Fortaleza em Arraial do Cabo - RJ. In: J.S.V. Silva & R.C.C.L. Souza (eds.). Água de lastro e bioinvasão. Interciência, Rio de Janeiro, pp Horta, P.A., C.E. Amancio, C.S. Coimbra & E.C. Oliveira Considerações sobre a distribuição e origem da flora de macroalgas marinhas brasileiras. Hoehnea, 28: Horta, P.A. & E.C. Oliveira Morphology and reproduction of Anotrichium yagii (Ceramiales, Rhodophyta): a new invader seaweed in the American Atlantic? Phycologia, 39: Jousson, O., J. Pawlowsli, L. Zaninetti, A. Meinesz & C.F. Boudouresque Molecular evidence for the aquarium origin of the green alga Caulerpa taxifolia introduced on the Mediterranean Sea. Mar. Ecol. Prog. Ser., 172: Lopes, R.M Informe sobre espécies exóticas invasoras marinhas no Brasil. Ministério do Meio Ambiente, Secretaria de Biodiversidade e Florestas, Brasília, 440 pp. Lubbock, H.R. & N.V.C. Polunin Conservation and the tropical marine aquarium trade. Environ. Conserv., 2: Mansur, M.C.D., L.M.Z. Richinitti & C.P. Santos Limnoperna fortunei (Dunker, 1857): molusco bivalve invasor na bacia do Guaíba, Rio Grande do Sul, Brasil. Biociências, 7: Mansur, M.C.D., C.P. Santos, G. Darrigran, I. Heydrich, C.T. Callil & F.R. Cardoso Primeiros dados quali-quantitativos do mexilhão-dourado, Limnoperna fortunei (Dunker), no Delta do Jacuí, no Lago Guaíba e na Laguna dos Patos, Rio Grande do Sul, Brasil e alguns aspectos de sua invasão no novo ambiente. Rev. Bras. Zool., 20: Meinesz, A.J.V., B. Hesse & X. Mari Spread of the introduced tropical green alga Caulerpa taxifolia in northern Mediterranean waters. J. Appl. Phycol., 5: Meinesz, A., T. Belsher, T. Thibaut, B. Antolic, K.B. Mustapha, C.F. Boudouresque, D. Chiaverini, F. Cinelli, J.M. Cottalorda, A. Djellouli, A.E. Abed, C. Orestano, A.M. Grau, L. Ivesa, A. Jaklin, H. Langar, E. Massuti-Pascual, A. Peirano, L. Tunesi, J. Vaugelas, N. Zavodnik & A. Zuljevic The introduced green alga Caulerpa taxifolia continues to spread in the Mediterranean. Biol. Invasions, 3: Meusnier, I., J.L. Olsen, W.T. Stam, C. Destombe & M. Valero Phylogenetic analyses of Caulerpa taxifolia (Chlorophyta) and of its associated bacterial microflora provide clues to the origin of the Mediterranean introduction. Mol. Ecol., 10: Oliveira, E.C. & E.J. Paula Exotic seaweeds: friends or foes? In: A.R.O.C. Chapman, R.J. Anderson, V. Vreeland & I.R. Davison (eds.). Proceedings of 17th International of Seaweed Symposium, Oxford University Press, Oxford, pp Oliveira, E.C., B.N.T. Silva & C.E. Amancio Fitobentos (Macroalgas). In: R.M. Lopes (ed.). Informe sobre espécies exóticas invasoras marinhas no Brasil. Ministério do Meio Ambiente, Secretaria de Biodiversidade e Florestas, Brasília, pp Oliveira, E.C., E.J. Paula, R. Petti & E.M. Plastino Metodologias para el cultivo no axénico de macroalgas marinhas in vitro. In: K. Alveal, M.E. Ferrario, E.C. Oliveira & E. Sar (eds.). Manual de métodos ficológicos. Universidad de Concepción, Concepción, pp Padilla, D.K. & S.L. Williams Beyond ballast water: aquarium and ornamental trades as sources of invasive species in aquatic ecosystems. Front. Ecol. Environ., 2: Paula, E.J. & E.C. Oliveira Macroalgas exóticas no Brasil com ênfase à introdução de espécies visando à maricultura. In: J.S.V. Silva & R.C.C.L. Souza (eds.). Água de lastro e bioinvasão. Interciência, Rio de Janeiro, pp

156 Seaweeds in ornamental aquaria in Brazil 350 Silva, B.N.T., C.E. Amancio & E.C. Oliveira Exotic marine macroalgae on the Brazilian coast: a revision. Oecol. Australis, 14: Stewart, J.E Introductions as factors in diseases of fish and aquatic invertebrates. Can. J. Fish. Aquat. Sci., 48(Suppl. 1): Taylor, R., R.L. Fletcher & J.A. Raven Preliminary studies on the growth of selected green tide algae in laboratory culture: effects of irradiance, temperature, salinity and nutrients on growth rate. Bot. Mar., 44: Weigle, S.M., L.D. Smith, J.T. Carlton & A.J. Pederson Assessing the risk of introducing exotic species via the live marine species trade. Cons. Biol., 19: Wiedenmann, J., A. Baumstark, T.L. Pillen, A. Meinesz & W. Vogel DNA fingerprints of Caulerpa taxifolia provide evidence for the introduction of an aquarium strain into the Mediterranean Sea and its close relationship to an Australian population. Mar. Biol., 138: Received: 16 May 2011; Accepted: 22 October 2012

157 Lat. Am. J. Aquat. Res., 41(2): , 2013 Analysis of two biomarkers in Sciades herzbergii Proceedings of the 3 rd Brazilian Congress of Marine Biology A.C. Marques, L.V.C. Lotufo, P.C. Paiva, P.T.C. Chaves & S.N. Leitão (Guest Editors) DOI: /vol41-issue2-fulltext Research Article Coral bleaching in the Caramuanas reef (Todos os Santos Bay, Brazil) during the 2010 El Niño event Ricardo J. Miranda 1,2, Igor C.S. Cruz 3 & Zelinda M.A.N. Leão 4 1 União Metropolitana de Educação e Cultura, Av. Luis Tarquínio Pontes 600, Centro, Lauro de Freitas, Brazil 2 Organização Sócioambientalista Pró-Mar, Sede Mar Grande, Av. Beira Mar 13 Ilhota, , Vera Cruz, Brazil 3 Universidade Estadual do Rio de Janeiro, Programa de Pós-Graduação em Ecologia e Evolução Rua São Francisco Xavier 524, PHLC Sala 220, Maracanã, Rio de Janeiro, Brazil 4 Instituto de Geociências, Universidade Federal da Bahia, Rua Barão de Jeremoabo Campus Universitário de Ondina, , Salvador, Brazil ABSTRACT. Episodes of coral bleaching related to El Niño events have been increasing in frequency and severity. This phenomenon is cited as a major cause of degradation of coral reefs. This study evaluates the effects of coral bleaching on the Caramuanas reef community, which occurred during the southern hemisphere summer of 2009/2010. Within this period the sea surface temperature of 31 C and thermal anomalies up to almost 1 C were recorded. During and after this El Niño event, frequency and severity of bleaching, live coral cover, number of colonies, class size, disease occurrence, and mortality rate were monitored on corals larger than 20 cm in diameter. The samples were taken at twelve fixed transects, in three reef stations. Statistical analysis showed that the severity of bleaching was different between the two periods, during and after the 2010 ENSO event. The Caramuanas reef showed sublethal bleaching effects indicating that this reef is tolerant to bleaching when the temperature anomalies do not exceed 0.75 C within one week. Keywords: coral bleaching, sublethal effects, thermal anomaly, thermal tolerance, eastern coast of Brazil. Blanqueamiento de corales en el arrecife de Caramuanas (Bahía de Todos los Santos, Brasil) durante el evento El Niño 2010 RESUMEN. Los episodios de blanqueamientos de corales relacionados con los eventos de El Niño han ido aumentando en frecuencia e intensidad durante los últimos años. Estos fenómenos se citan como una de las principales causas de la degradación de los arrecifes de coral. Este estudio evaluó los efectos del blanqueamiento del arrecife de coral en la comunidad de Caramuanas, que se produjeron durante el verano austral de 2009/2010. En este período se registró temperaturas de la superficie del mar de hasta 31 C y anomalías térmicas del orden de 1 C. Durante y después de este episodio El Niño, se analizó la frecuencia y severidad del blanqueamiento, cobertura de coral vivo, número de colonias, clases de tamaño, aparición de enfermedades y tasa de mortalidad en los corales mayores de 20 cm de diámetro. Las muestras fueron tomadas en doce transectas fijas, divididas en tres estaciones de arrecife. El análisis estadístico mostró que la severidad del blanqueamiento fue diferente entre los dos períodos, durante y después del evento. El arrecife de Caramuanas mostró efectos subletales de blanqueamiento que probó que este arrecife es tolerante a los fenómenos de blanqueamiento cuando las anomalías de temperatura no exceden 0,75 C durante una semana. Palabras clave: blanqueamientos de corales, efectos subletales, anomalías térmicas, tolerancia térmica, costa oriental de Brasil. Corresponding author: Ricardo J. Miranda (ricardojdemiranda@gmail.com)

158 352 Latin American Journal of Aquatic Research INTRODUCTION Bleaching is a major cause of coral mortality and represents one of the major threats to corals worldwide (Hoegh-Guldberg, 1999). This phenomenon has become more frequent and is responsible for the loss of biodiversity and fishing resources in the reef ecosystems (Bellwood et al., 2004). Coral bleaching occurs in response to environmental stress and disrupts the symbiotic relationship established between corals and the zooxanthellae (dinoflagellates). These microalgae live in the coral tissue and provide a considerable portion of the energy required by the coral to survive. Under stress the coral tissue becomes transparent upon the expulsion of the zooxanthellae and/or the loss of their photosynthetic pigments; once transparent, the coral appears to be white due to the color of its subjacent calcareous skeleton (Douglas, 2003; Weis, 2008). Bleaching episodes of large intensity and scale (lethal or mass bleaching) have been attributed to abnormal increases in oceanic water temperature, and these phenomena occur frequently during El Niño Southern Oscillation (ENSO) periods (Glynn, 1988; Bruno et al., 2001). This suggests that mass bleaching is related to global climate changes (Oxenford et al., 2008; Wilkinson, 2008; Hoegh-Guldberg & Bruno, 2011). And this fact also explains the large number of studies conducted in the last decades that have recorded lethal effects of bleaching, for example, the mass mortality of some coral species (Wilkinson et al., 1999; Wilkinson & Souter, 2008; Oxenford et al., 2010; Eakin et al., 2011). Sub-lethal bleaching effects have been observed, recently, but they have not been extensively studied (Suggett & Smith, 2011). They occur when corals do not experience mortality after bleaching, but rather undergo a temporary loss of zooxanthellae and/or of their photosynthetic pigments, with later recovery. This potential for recovery suggests that corals have an adaptive capacity or resistance to seasonal changes in environmental conditions (Buddemeier & Fautin, 1993; Hennige et al., 2010; Hughes et al., 2011). Thermal anomalies on the Brazilian coast have been monitored by NOAA satellite imaging since 1998, and there have been many reports of bleaching events occurring in association with ocean warming events (Migotto, 1997; Castro & Pires, 1999; Dutra et al., 2000; Costa et al., 2001, 2004; Costa & Amaral, 2002; Kikuchi et al., 2003; Leão et al., 2003, 2008; Ferreira & Maida, 2006). None-theless, only Dutra et al. (2000), reported observations of the reefs after the occurrence of the bleaching phenomenon. To help bridge this gap in knowledge, this study was designed to evaluate the effect of bleaching in the corals from Caramuanas reef by comparing their communities during and immediately after the thermal anomalies related to ENSO MATERIALS AND METHODS Study area The Caramuanas reef is located in the entrance of Todos os Santos Bay, on the State of Bahia, at the eastern coast of Brazil. In this region there are the largest and the richest coral reefs of the southwestern Atlantic ocean (Laborel, 1970; Leão et al., 2003). The Caramuanas reef comprise three main flat reef banks (13 07'S-38 43'W, 13 07'S-38 44'W and 13 08'S 'W) (Fig. 1). These banks are distributed within an area of approximately 4 km in length and 50 m in width, at a depth of less than 6 m. The reef is exposed during low spring tides, and is exploited by several different types of fishing activities. The thermal anomalies, along the coast of Bahia, were recorded from the National Environmental Satellite Data and Information Service (NESDIS) database, provided by National Oceanic Atmospheric Administration (NOAA), and the temperature of the ocean surface water was generated by processing satellite images collected from NASA and publically available online. It was used data from January to April 2010, which correspond to the end of summer and beginning of fall in the southern hemisphere. This also corresponds to the period of highest ocean surface water temperatures off the Brazilian coast (Leão et al., 2008). The data were acquired every three days using the hot spot maps for coral bleaching from The water temperature data were generated by the MODIS sensor on the NASA AQUA satellite and obtained by processing remote images taken during and after the occurrence of the thermal anomalies using GIOVANNI software ( gov/giovanni). Field data collection and statistical analysis The field data in the Caramuanas reef were collected in three stations, located as follows: one on the northern part of the reef (north station), one on its center (central station), and one on the southern portion (south station) (Fig. 1). At each station, four fixed transects were marked with rebar. Each station was studied during the thermal anomaly period (March 2010), and after the anomaly period (October and/or November 2010), with a total of six visits to the reef.

159 Analysis of two biomarkers in Sciades herzbergii 353 ATLANTIC OCEAN Figure 1. Location of studied reef stations. Caramuanas reef, Todos os Santos Bay, eastern Brazilian coast. Following the BLAGRRA protocol (Bleaching Atlantic and Gulf Rapid Reef Assessment, Lang et al., 2010), a visual census of corals along transects was performed during scuba diving. The original protocol was modified by increasing the length of transects. Instead of six transects 10 m long, in five reef stations, as it is in the original protocol, we used four transects 20 m long in three reef stations. A total of twelve replicates were made at each time point, i.e., four replicates per station. Along these 12 fixed transects the percent live coral cover, determined as the extension of the live surface of each coral colony present under the transect line, was measured. For each colony larger than 20 cm in diameter present in a distance up to 50 cm on each side of the transect line (a band of 1 m in width), the following parameters were recorded: number and name of the coral species, length, width and height of the colony, percentage of the colony area bleached, bleaching intensity (how pale the coral was), presence of disease, extension (%) and time (recent, intermediate or old) of mortality of the colony surface area. The bleaching intensity (weak or strong) was assigned accordingly to the tonality of the colony (pale or white), and recorded as a percentage of the colony affected area. The extension of mortality was the percentage of the dead colony area. The time of mortality (recent, intermediate or old) was assigned to the colony dead area. Recent mortality was characterized by a white skeleton that had recently been exposed due to death of living tissue covering it (i.e., a complete, intact skeletal structure). Colonies were classified as having intermediate mortality when the skeleton was slightly eroded or covered by a fine layer of sediment, filamentous algae, diatoms or cyanobacteria, and the structure of the corallite could still be observed. Colonies were classified as having old mortality when the corallites were covered by organisms that were not easily removed, such as macroalgae and/or invertebrates. All types of mortality, as designated in the protocol, were recorded as the percentage of the affected area of the colony surface. The Kruskal-Wallis (KW) test was used to evaluate differences between the investigated time periods of the following parameters: total bleaching, bleaching severity, bleaching pattern among species, live coral cover, number of colonies, and mortality, which did not have a normal distribution. In the overall, 13 statistical tests were performed, leading to the use of the Bonferroni for correcting the α = 0.05 to α = Analysis of variance (ANOVA) was used to verify differences in the colony sizes among the most abundant coral species. RESULTS ENSO intensity and bleaching severity Starting in February, the thermal anomalies that affected the TSB in 2010 lasted for eight weeks, reaching a maximum value of 0.75 C in March, completely dissipating in April (Fig. 2). During the thermal anomaly period, the water temperature reached 31 C, which is the highest value recorded in the last ten years for this region. Total and weak bleaching showed significant differences during and after thermal anomalies within the study period (KW total bleaching: P < 0.004; weak bleaching: P < 0.004; strong bleaching: P > 0.004, Fig. 3). During the thermal anomalies, 133 coral colonies were measured and 57.1% were affected to

160 354 Latin American Journal of Aquatic Research Temperature Figure 2. Thermal anomalies in Todos os Santos Bay in Figure 3. Graph depicting the total bleaching and the degree of bleaching intensity (weak, strong) in each evaluated period, during and after the thermal anomalies. some degree of bleaching most of them with weak bleaching. After the occurrence of the thermal anomalies, when a total of 118 colonies were recorded, only 5.1% still had some signal of weak bleaching (Table 1). Fifteen coral colonies were not found during the second survey, although both assessments were performed along fixed transects. Coral bleaching on the assessed coral community Five scleractinian coral species and one hydrocoral were found on the studied reef sites. The most abundant species were Siderastraea spp., followed by Montastraea cavernosa, Mussismilia hispida, Millepora alcicornis, Mussismilia braziliensis and Mussismilia harttii. They presented different percentages of the area affected by bleaching, as well as different intensity of bleaching (weak and/or strong). Of the six species recorded only Mussismilia harttii did not suffer bleaching (Table 1). Siderastraea spp., the most abundant coral during both surveys, had 60.3% of their colonies affected by bleaching, during the occurrence of the thermal anomalies. Of the 37% of the colony area affected, 34% was by weak bleaching and only 3% by strong bleaching. After the period of occurrence of the ENSO event, only 5.8% colonies still presented signal of bleaching, with less than 1% of the colony area affected by weak and/or strong bleaching. Montastraea cavernosa was the coral specie the most affected during the occurrence of warming waters, with 81% of their colonies presenting some degree of bleaching (weak or strong). 58% of their

161 Analysis of two biomarkers in Sciades herzbergii 355 Table 1. Number of colonies recorded, number of bleached colonies and average (± SD) of the colony area affected by different intensity of bleaching (weak, strong) for the coral species and the calcareous hydroid Millepora alcicornis in the Caramuanas reef during and after the 2010 thermal anomalies. NC: number of colonies, NBC: number of bleached colonies, TB: total bleaching, WB: weak bleaching, SB: strong bleaching, SD: standard deviation. During thermal anomalies After thermal anomalies NC NBC TB WB SB NC NBC TB WB SB Species (#) (#) (%) (average ± SD) (average ± SD) (average ± SD) (#) (#) (%) (average ± SD) (average ± SD) (average ± SD) Siderastraea spp ± ± ± ± ± ± 0.00 Montastraea cavernosa ± ± ± ± ± ± 0.00 Mussismilia hispida ± ± ± ± ± ± 0.00 Mussismilia braziliensis ± ± ± ± ± ± 0.00 Mussismilia harttii ± ± ± ± ± ± 0.00 Millepora alcicornis ± ± ± ± ± ± 0.00 Total surface area was bleached, being the highest value observed among all studied species, also, with the highest percentage affected by strong discoloration (38%). After the anomalies, only two colonies still presented some signal of weak bleaching. Mussismilia hispida the third coral specie in abundance, in both surveys, had 28.6% of its colonies bleached during the occurrence of the 2010 ENSO event. Only 12.1% of the colonies area was affected by bleaching, being circa 8% by strong bleaching. After the thermal anomalies no colony was found bleached. Mussismilia braziliensis was the specie with the smallest number of colonies recorded during the first survey (two colonies) and none was affected by bleaching. After the anomalies, of the total of 11 recorded colonies, only one showed circa 7% of its surface area affected by weak bleaching. The hydrocoral Millepora alcicornis had 57.1% of its colonies bleached during the occurrence of the 2010 ENSO event, mostly by strong bleaching (37% of the total affected surface area of 46%). After the thermal anomalies no colony of this specie was found bleached. The results of the statistical analysis showed that differences among bleached colonies, during and after the thermal anomalies, were significant only between the species Montastraea cavernosa and Mussismilia hispida (KW test, P < 0.004; Fig. 4). Coral parameters measured Three measured parameters of the studied coral community are related with the vital condition of the reef: the percent of live coral cover, the number of live colonies and the rate of coral mortality. The records found for all three parameters, from both surveys (during and after the occurrence of the thermal anomalies), show that statistically there are no significant differences (KW test, P > 0.004) between the two evaluations. The percent of live coral cover varied from 2.9 ± 1.3% during the anomalies, to 3.6 ± 0.45% after the event of warming waters (Fig. 5). From a total of 133 live coral colonies that were recorded during the first survey (warmer time), only 15 live colonies were not found during the survey performed after the 2010 ENSO event (Fig. 6). The values of the mortality rates (recent, intermediate and old) were not significantly different between the thermal anomaly period and the subsequent period of thermal normalcy (Table 2, Fig. 7). Table 3 shows, for the most abundant coral species, Siderastraea spp., Montastraea cavernosa and Mussismilia hispida, what were the most common

162 356 Latin American Journal of Aquatic Research Figure 4. Total bleaching pattern among species, during thermal anomalies. Ma: Millepora alcicornis, Mc: Montastraea cavernosa, Mb: Mussismilia braziliensis, Mha: M. harttii, Mhi: M. hispida, Ssp: Siderastraea spp. Figure 5. Mean (± SD) live coral cover during each evaluated period, during and after the thermal anomalies. colony size classes in both surveys. In the three coral species, predominated the smallest colonies, the size class cm diameter, either during or after the thermal anomalies. Statistically there was no change detected in the structure of this evaluated parameter (ANOVA, P > 0.004; Figs. 8a-8c). About the occurrence of coral disease, only one colony was recorded affected, disabling any statistical test. DISCUSSION Of the 57% coral colonies that were affected by bleaching in the study area during the thermal anomalies of the 2010 ENSO, the great majority recovered, which indicate that the effects were sublethal for the Caramuanas corals. The 5% of the colonies that still exhibited signs of bleaching after the anomalies could represent normal episodes in shallow reefs exposed to daily thermal variations, as it is suggested by Leão et al. (2008) for the coastal reefs along the coast of the state of Bahia. After this bleaching event, neither the rate of mortality nor the number of colonies with disease increased; the size class structure of the most abundant species did not vary; and the number of live colonies and live coral cover also remained the same. Therefore, the reef showed certain resilience to the perturbations caused by the 2010 ENSO event. Among the most common coral species analyzed, Montastraea cavernosa exhibited the highest susceptibility to bleaching, and its colonies had the highest relative bleaching values. However, this specie exhibited great capacity for recovery after the Figure 6. Number of live colonies in each evaluated period, during and after the thermal anomalies. anomalies ended. Similarly, most Siderastraea spp. colonies were pale during the anomalies but later recovered completely. The three species of Mussismilia, M. hispida, M. braziliensis and M. harttii, were the least affected, exhibiting low bleaching rates and complete recovery. These findings are consistent with the literature, suggesting that variations in the susceptibility to bleaching are common and that bleaching affects different species in different ways (McClanahan et al., 2005). Susceptibility and response to bleaching are thought to be influenced by colony morphology, colony growth rate, metabolic rate and size, as well as the lineage of the symbiotic algae, and thickness of coral polyps (Brandt, 2009). The water depth and/or the zonation of the corals within the same reef may also affect the

163 Analysis of two biomarkers in Sciades herzbergii 357 Table 2. Number of colonies of the most abundant coral species registered in each size class analyzed during and after the period of thermal anomalies. Mortality types During After (Average ± SD) (Average ± SD) New 0.31 ± ± Intermediate 0.43 ± ± Old ± ± Total ± ± bleaching process (Marshall & Baird, 2000; Williams et al., 2010). The hydrocoral Millepora alcicornis showed a rather high susceptibility to bleaching, and besides of being the second species with the highest relative bleaching, it also had the highest percent of dead colonies after the occurrence of the bleaching event. The high sensitivity of this calcareous hydroid has been described in other bleaching events (Glynn & De Weerdt, 1991; Stafford-Smith et al., 1993). According to these authors, after the bleaching occurs, several colonies with dead areas become covered with crustose coralline algae, as was seen in the Millepora alcicornis colonies. The sub-lethal effects of bleaching in Brazilian corals have been observed previously. In 1998, the northern littoral of Bahia experienced a bleaching event that affected up to 60% of the coral community, which after one year have completely recovered (Dutra et al., 2000). At this same period, the blea- Figure 7. Average total mortality and mortality times (recent, intermediate and old) between the two evaluated periods, during and after the occurrence of thermal anomalies.

164 358 Latin American Journal of Aquatic Research Table 3. Average (± SD) of total mortality and mortality time (recent, intermediate and old) during and after the 2010 thermal anomaly events. Coral species Period of thermal anomaly Number of colonies Siderastraea spp. During After Montastraea cavernosa During After Mussismilia hispida During After Size classes (cm) Figure 8. Colonies averages by size class in 10 m 2 of the Caramuanas reef for: a) Siderastraea spp. (ANOVA, F = , P = ); b) Montastraea cavernosa (ANOVA, F = , P = ); c) Mussismilia hispida (ANOVA, F = , P = ). ching associated with ENSO caused the death of approximately 16% of reefs worldwide, particularly those from the Indian and Pacific oceans (Wilkinson & Souter, 2008). In all other occasions of bleaching events in Brazilian reefs, there were no mass deaths reported for the coral fauna associated with thermal perturbations due to ENSO events (Migotto, 1997; Castro & Pires, 1999; Leão et al., 2003, 2008; Dutra et al., 2000; Costa et al., 2001, 2004; Costa & Amaral, 2002; Kikuchi et al., 2003; Ferreira & Maida, 2006). The Brazilian reefs seem to be resilient to bleaching and resistant to mortality, or they may be functionally adapted to the environmental stresses of the Brazilian waters, such as high turbidity and

165 Analysis of two biomarkers in Sciades herzbergii 359 elevated rates of sedimentation (Leão & Ginsburg, 1997). The Brazilian zooxanthellate coral fauna is characterized by endemic species, with some reminiscent of a Tertiary coral fauna that may be adapted to these inhospitable environment conditions (Leão et al., 2003). Examples are the species of the genus Mussismilia, which showed the lowest percent of bleached colonies and those that bleached completely recovered during this investigation. The results found in this work indicate that the coral fauna of the Caramuanas reef can become tolerant to the effects of bleaching when the thermal anomaly does not exceed 0.75 C over a period of one week. The high degree of endemism of the Brazilian coral fauna (Laborel, 1970) is one important reason for choosing them as targets for conservation. The fact that the Caramuanas reef was shown to be resilient for bleaching events from its first report, in 1998, is a further evidence of its importance. Eliminating or reducing anthropogenic effects on this reef may increase its resistance and resilience to bleaching, allowing its maintenance. The Caramuanas reef could than acts as a reserve of species and genes for this geographic region. ACKNOWLEDGEMENTS This work received support from the Project Maré Global granted by the GDK SA enterprise. R.J.M. is grateful to the RECOR - Group of Studies of Coral Reefs from the Federal University of Bahia, and to Rafael Carvalho, Robin Faillettaz and Mariana Coxey for their helpful support. ICSC and ZMANL benefits from CNPq research grants. Suggestions from anonymous reviewers greatly improved the final version of the manuscript. REFERENCES Bellwood, D.R., T.P. Hughes, C. Folke & M. Nystrom Confronting the coral reef crisis. Nature, 429: Brandt, M.E The effect of species and colony size on the bleaching response of reef-building corals in the Florida Keys during the 2005 mass bleaching event. Coral Reefs, 28(4): Bruno, J.F., C.E. Siddon, J.D. Witman, P.L. Colin & M.A. Toscano El Niño related coral bleaching in Palau, western Caroline Islands. Coral Reefs, 20: Buddemeier, R.W. & D.G. Fautin Coral bleaching as an adaptive mechanism. Bioscience, 43(5): Castro, C.C. & D.O. Pires Brazilian coral reefs: what we already know and what is still missing. Bull. Mar. Sci., 69: Costa, C.F., F.M.D. Amaral & R. Sassi Branqueamento em Siderastraea stellata (Cnidaria, Scleractinia) da praia de Gaibu, Pernambuco, Brasil. Rev. Nordestina Biol., 15(1): Costa, C.F. & F.M.D. Amaral Density and size differences in zooxanthellae from five reef-building coral species from Brazil. Proceedings of International Coral Reef Symposium Indonesian Institute of Sciences, Bali, 1: Costa, C.F., C.S. Coutinho, R. Sassi & L.A.C. Brito Microsymbionts of Siderastraea stellata (Cnidaria, Scleractinia) in coastal reefs of Cabo Branco, State of Paraíba, northeastern Brazil. Trop. Oceanogr., 32(2): Douglas, A.E Coral bleaching-how and why? Mar. Pollut. Bull., 46: Dutra, L.X.C., R.K.P. Kikuchi & Z.M.A.N. Leão Thirteen months monitoring coral bleaching on Bahia s north coast, Brazil. Proceedings of International Coral Reef Symposium Indonesian Institute of Sciences, Bali, pp Eakin, C.M., J.A. Morgan, S.F. Heron, T.B. Smith, G. Liu, L. Alvarez-Smith, B. Baca, E. Bartels, C. Bastidas, C. Bouchon, M. Brandt, A.W. Bruckner, L. Bunkley-Williams, A. Cameron, B.D. Causey, M. Chiappone, T.R.L. Christensen, M.J.C. Crabbe, O. Day, E. de la Guardia, G. Diaz-Pulido, D. DiResta, D.L. Gil-Agudelo, D.S. Gilliam, R.N. Ginsgurg, S. Gore, H.M. Guzmán, J.C. Hendee, E.A. Hernandez- Delgado, E. Husain, C.F.G. Jeffrey, R.J. Jones, E. Jordan-Dahlgren, L.S. Kaufman, D.I. Kline, P.A. Kramer, J.C. Lang, D. Lirman, J. Mallela, C. Manfrino, J.P. Marechal, K. Marks, J. Mihaly, W.J. Miller, E.M. Mueller, E.M. Muller, C.A. O. Toro, H.A. Oxenford, D. Ponce-Taylor, N. Quinn, K.B. Ritchie, S. Rodriguez, A. Rodriguez-Ramirez, S. Romano, J.F. Samhouri, J.A. Sanchez, G.P. Schmahl, B.V. Shank, W.J. Skirving, S.C.C. Steiner, E. Villamizar, S.M. Walsh, C. Walter, E. Weil, E.H. Williams, K.W. Roberson & Y. Yusuf Caribbean corals in crisis: record thermal stress, bleaching, and mortality in PLoS ONE, 5(11): e Ferreira, B.P. & M. Maida Monitoramento dos recifes de coral do Brasil-situação atual e perspectivas. Ministério do Meio Ambiente, Secretaria de Biodiversidade e Florestas, Brasília, 116 pp. Glynn, P.W El Niño-southern Oscillation : nearshore population, community, and ecosystem responses. Ann. Rev. Ecol. Syst., 19:

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167 Lat. Am. J. Aquat. Res., 41(2): , Morphometric 2013 sexual maturity and allometry of Sesarma rectum Proceedings of the 3 rd Brazilian Congress of Marine Biology A.C. Marques, L.V.C. Lotufo, P.C. Paiva, P.T.C. Chaves & S.N. Leitão (Guest Editors) DOI: /vol41-issue2-fulltext Research Article Morphometric sexual maturity and allometric growth of the crab Sesarma rectum Randall, 1840 (Crustacea: Sesarmidae) in an impacted tropical mangrove in northeast Brazil Felipe Bezerra Ribeiro 1,2, Helena Matthews Cascon 1,2 & Luis Ernesto Arruda Bezerra 2,3 ¹Laboratório de Invertebrados Marinhos do Ceará (LIMCE), Departamento de Biologia Universidade Federal do Ceará, Av. Mister Hull, s/n, Campus do Pici, Fortaleza, Brazil 2 Programa de Pós-Graduação em Ciências Marinhas Tropicais, Instituto de Ciências do Mar (LABOMAR) Universidade Federal do Ceará, Av. da Abolição, 3207, Meireles, Fortaleza, Brazil 3 Universidade Federal Rural do Semi-Árido, Departamento de Ciências Animais & Programa de Pós-Graduação em Ecologia e Conservação, Av. Francisco Mota, 572, , Mossoró, RN, Brazil ABSTRACT. The size at sexual maturity and the allometric growth of the semi-terrestrial crab Sesarma rectum were studied in an impacted tropical mangrove in northeast Brazil. Crabs were monthly collected during spring low-tide periods, from October 2009 through September A catch-per-unit-effort (CPUE) technique was used to sample the crab population, with two-hour sampling periods, by two people. A total of 492 crabs were obtained, being 262 males and 230 females. The specimens were measured at carapace width (CW), the left and right propodus length and height (RPL, RPH, LPL and LPH), and the gonopod length of males (GL), and abdomen width (AW) of females. In males, the inflection point was at mm CW in the relationship between CW and the length of right propodus (LRP), considering the morphological size at the onset of maturity. Based on the relationship between CW and AW, the size at sexual maturity in females was mm. In spite of living in an impacted area, this population attained the maturity onset at a bigger size than other localities. Keywords: Sesarma rectum, sexual maturity, allometric growth, tropical mangrove, northeast Brazil. Madurez sexual morfométrica y crecimiento alométrico del cangrejo Sesarma rectum Randall, 1840 (Crustacea: Sesarmidae) en un manglar tropical impactado en el noreste de Brasil RESUMEN. La madurez sexual morfométrica y el crecimiento alométrico del cangrejo semiterrestre Sesarma rectum fueron estudiados en un manglar tropical modificado en el noreste de Brasil. Los cangrejos fueron colectados mensualmente durante los períodos de marea baja desde octubre 2009 a septiembre Para obtener una muestra representativa de la población se utilizó la técnica de captura por unidad de esfuerzo (CPUE), con períodos de dos horas de captura realizada por dos personas. Se obtuvo un total de 492 cangrejos: 262 machos y 230 hembras. Se realizaron mediciones del ancho del caparazón (AC), longitud y altura del propodio izquierdo y derecho, longitud del gonopodo de los machos (LPI, API, LPD, APD e LG), y ancho del abdomen (AW) de las hembras. En los machos, el punto de inflexión se determinó en 27,14 mm CW en la relación entre CW y la longitud del propodio derecho (LPD), considerado el tamaño morfológico al inicio de la madurez. Basado en la relación entre el CW y AW, el tamaño de madurez sexual de las hembras fue de 22,97 mm. A pesar de vivir en una zona afectada, esta población alcanza el inicio de la madurez sexual a un tamaño mayor que en otras localidades. Palabras clave: Sesarma rectum, madurez sexual, crecimiento alométrico, manglar tropical, noreste de Brasil. Corresponding author: Felipe Ribeiro (fbribeiro.ufc@gmail.com)

168 362 Latin American Journal of Aquatic Research INTRODUCTION Growth can be expressed by the increase of size, volume, wet weight or dry weight over time. Organisms that do not have an exoskeleton present a continuous growth, but in Crustacea, which have a rigid and inextensible exoskeleton, growth becomes an essentially discontinuous process. There is a succession of molts or ecdyses, separated by an intermolt period (Hartnoll, 1982). According to Huxley (1932), the allometric equation is the most utilized method for analysis of growth during the ontogeny. The relationship between the size of a body part (y) compared with another body part (x), generally the carapace width (CW), can be expressed by the equation y = a.x b, where the exponent b is the measure of the different rates of growth of the two body parts. To estimate the coefficients of allometric equation, the data are usually logarithmized and a linear regression is fitted, and represented by the equation log y = log a + b log x, where the allometric exponent b is the slope of the resulting linear equation (Teissier, 1960). In crustaceans, the allometric relationships between body size and various organs are used to estimate the sexual maturity, assuming that the secondary sexual characteristics appear and grow at different rates in mature and immature stages (Leme, 2005). The transitional phase in Brachyura involves morphological changes that can be detected by inflections or discontinuities in a series of linear or curvilinear relationships using a bivariate analysis (Haefner Jr., 1990). Studies of relative growth in crustaceans allow to define the type of allometry in the growth of different body parts, such as chelae, locomotor appendages, abdomen and pleopods, and to relate them to their specific functions. One responsible factor for these changes in the allometric growth is the sexual maturity (González-Gurriarán & Freire, 1994). The allometric relationships are powerful tools used by taxonomists and ecologists in the analysis of intraspecific and interspecific variation among different populations and to estimate the average size at sexual maturity, also related to environmental conditions (Costa & Soares-Gomes, 2008). Several studies related to allometry in Brachyura have been conducted, mainly in Brazil and especially regarding mangrove crabs of the genus Uca (Negreiros-Fransozo et al., 2003; Benetti & Negreiros-Fransozo, 2004; Masunari et al., 2008). Sesarmid crabs are very abundant in mangrove forests (Priyadarshani et al., 2008), and the species Sesarma rectum Randall, 1840, that builds burrows in shaded areas or along the edge of the mangrove, and feeds on detritus and leaves (Prado, 1999), is very numerous in these environments. There are few studies about sexual maturity and allometric growth of sesarmid crabs (Leme, 2005; Castiglioni et al., 2011). The human impacts, such as residential development in mangrove or estuarine areas drastically change this environment, preventing the establishment of certain organisms like crabs (Silva et al., 2007). The present contribution aimed to elucidate some aspects of the growth and sexual maturity of S. rectum, in an impacted tropical mangrove in the State of Ceará, northeast Brazil, contributing to generate information for the conservation of this area. MATERIALS AND METHODS The study site is an impacted mangrove situated in the Cocó River Ecological Park in the municipality of Fortaleza, Ceará ( S, W) (Fig. 1). The Cocó River Ecological Park is the largest urban park in South America, with an area of 379 hectares. Almost the entire park shows advanced degradation of its ecosystem, with major consequences especially from city expansion promoted by real-estate speculation, and also from slum areas around the park (SEMACE, 2006). Monthly sampling (CPUE, 2 people for 2 hours), took place during spring low-tide periods, from October 2009 through September Specimens were obtained manually, some exposed on the surface of the mangrove substrate, and others captured by digging. In the laboratory, specimens were identified and sexed according to secondary sexual characters (abdomen morphology and number of pleopods). The following variables were measured in the laboratory to the nearest 0.1 mm using a vernier caliper: carapace width (CW), carapace length (CL), the left and right propodus length and height (RPL, RPH, LPL and LPH), and gonopod length of males (GL), and abdomen width (AW) of females (Fig. 2). The morphometric relationships CW/RPL for males and CW/AW for females were tested to estimate the size at sexual maturity, based in changes of these structures. The examined variables were subjected to a regression analysis, considering two ranges of amplitude of CW corresponding to the two stages of reproductive development (immature and mature) in both sexes, based solely on morphology. The allometric technique was employed to distinct adults (matures) from juvenile crabs (immatures) and

169 Morphometric sexual maturity and allometry of Sesarma rectum 363 Figure 1. Sampling area in the Cocó River Ecological Park, municipality of Fortaleza, Ceará State, Northeast Brazil. Figure 2. Sesarma rectum. Body measurements. a) Carapace in dorsal view, CW: carapace width; CL: carapace length, b) male chelipod, PL: propodus length; PH: propodus height, c) female abdomen CW: abdomen width, d) male gonopod, GL: gonopod length (12.77 mm). Scale = 1 mm.