Musculature in three species of Proales (Monogononta, Rotifera) stained with phalloidin-labeled fluorescent dye

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1 Zoomorphology (2005) 124: DOI /s ORIGINAL ARTICLE Martin V. Sørensen Musculature in three species of Proales (Monogononta, Rotifera) stained with phalloidin-labeled fluorescent dye Received: 3 January 2004 / Accepted: 10 September 2004 / Published online: 1 February 2005 Ó Springer-Verlag 2005 Abstract The musculature in the rotifer species Proales daphnicola, P. reinhardti and P. fallaciosa was stained with phalloidin-labeled fluorescent dye and compared using confocal laser scanning microscopy. All three species share several homologous muscle systems, but each system s detailed morphology varies among the species both concerning appearance, number and location. The obtained results were compared with data from other rotifers and it was concluded that the muscles pars coronalis and the corona sphincter probably represent conditions in Ploima or Monogononta, while incomplete circular muscles and dorsal and ventral trunk retractors might be part of the eurotatorian ground pattern. Keywords Confocal microscopy Æ Morphology Æ Muscles Æ Proales Æ Rotifera Introduction Staining with fluorescently tagged phalloidin was recently introduced in the study of rotifer morphology, and within the past few years different investigations of rotifer musculature using epifluorescence or confocal laser scanning microscopy (CLSM) have been published (Hochberg and Litvaitis 2000; Kotikova et al. 2001; Sørensen et al. 2003; Sørensen 2005). However, data on the musculature of Rotifera are still scarce and restricted to a few more or less randomly selected taxa, and the conclusions in the most recently published studies have therefore been focused on functional aspects related to the investigated species, whereas the potential phylogenetic signals in the musculature have been hampered by M. V. Sørensen Department of Evolutionary Biology, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark mvsorensen@bi.ku.dk Tel.: the inadequate taxon sampling. Our knowledge of the rotifer phylogeny is extremely limited and the need for detailed and consistent information on morphological traits that could be potential characters is obvious. Sørensen (2002) recently presented a hypothesis about the rotifer relationship that mostly dealt with characters related to the pharyngeal apparatus (the mastax), and information from other systems, such as the muscles, would therefore be extremely valuable. Hence, the necessity for compiling more data is obvious. The aim of the present paper is to contribute more information about the rotifer musculature. It gives a detailed description of the musculature in three species of the genus Proales; Proales daphnicola Thompson 1892, P. fallaciosa Wulfert 1937, and P. reinhardti (Ehrenberg 1834). Proales was chosen because thorough descriptions of the musculature had not been previously presented for this genus. Furthermore, a revision of the genus, or in fact of the whole taxon Proalidae, is highly needed (De Smet 1996), and any kind of information on the morphology in Proales is therefore valuable. Materials and methods Proales fallaciosa was collected from submerged mosses on 11 October 2002, in a pond named Mossø in Rold Forest, Jutland, Denmark ( N, E). P. reinhardti was collected on 27 March 2003, from sandy sediment mixed with detritus at 9-m depth in the Kattegat, North of Elsinore, Zealand, Denmark ( N, E). P. daphnicola was collected on 4 July 2003, from a small pond in the large peat bog Store Vildmose in Jutland, Denmark ( N, E). Specimens for CLSM were placed in a drop of freshwater and relaxed by adding a few cocaine crystals to the drop. The anesthetized specimens were fixed for 1 h in phosphate-buffered 4% formaldehyde, rinsed in PBS, made permeable by exposure to 0.1% Triton X-100 in PBS for 1 h, stained for 6 24 h in Alexa-488-

2 48 phalloidin, rinsed in PBS, and then mounted in Fluoromount-G, on a cover slip. Images under blue-excitation wavelength were obtained with an Olympus BX50WI light microscope equipped with an Ultra View LCI confocal imaging system. The obtained images series were analyzed with Volocity visualization software. It was not possible to resolve the arrangement of the single fibers in the densely packed pharyngeal musculature. Hence these will be omitted in the following descriptions. Results Proales daphnicola Six specimens were investigated. All were positioned on their lateral side; hence, the dorsoventral orientation of the muscles is based on computerized reconstructions. The trunk musculature constitutes five incomplete circular muscles that run from a ventrolateral position and meet on the dorsal side (Figs. 1, 2). Each circular muscle consists of six fibers. A pair of broad, ventral trunk retractors, positioned close to the ventral midline, run from the neck to the toes (Figs. 1, 2a, c). Each muscle consists of three fibers. Anteriorly, the muscle trifurcates and in the trunk, posterior to the second circular muscle, a conspicuous muscular appendage is present (Figs. 1, 2a, c). A pair of ventrolateral trunk retractors runs from the neck to the tip of the toes (Figs. 1, 2a). These muscles are thinner than the ventral trunk retractors and consist of two fibers. A pair of lateral trunk retractors runs from the most apical part of the head and anchors laterally in the trunk posterior to the second circular muscle (Figs. 1, 2a, c). More dorsally, a pair of dorsolateral trunk retractors extends from the neck and anchors laterally, posterior to the fourth circular muscle (Figs. 1, 2a, b). Middorsally, a pair of strong dorsal retractors is present. These muscles Fig. 1 a, b Line drawing of the musculature in Proales daphnicola. a Dorsal view. b Lateral view. ap appendage on vr, cs corona sphincter, dlr dorsolateral trunk retractor, dhr dorsal head retractor, dr dorsal trunk retractor, ic incomplete circular muscle, fe foot extensor, lcr lateral corona retractor, lhr lateral head retractor, lr lateral trunk retractor, pc pars coronalis, vfr ventral foot retractor, vlr ventrolateral trunk retractor, vr ventral trunk retractor

3 Fig. 2 a c Confocal laser scanning microscopy photos of Proales daphnicola and d e Proales fallaciosa. a P. daphnicola, Z-projection through specimen, dorsoventral view. b P. daphnicola, single dorsal section. c P. daphnicola, single ventral section. d P. fallaciosa, Z-projection through specimen, dorsoventral view. e P. fallaciosa, Z-projection through specimen, lateral view. ap appendage on vr, cs corona sphincter, dlr dorsolateral trunk retractor, dhr dorsal head retractor, dr dorsal trunk retractor, ic incomplete circular muscle, fe foot extensor, lhr lateral head retractor, lr lateral trunk retractor, ma mastax, pc pars coronalis, vfr ventral foot retractor, vlr ventrolateral trunk retractor, vr ventral trunk retractor 49

4 50 are the broadest and most conspicuous in P. daphnicola (Figs. 1, 2a, b). The muscles are bipartite anteriorly and run from the most apical part of the head. Behind the neck, they make a distinct twist towards the dorsal side and anchor dorsally in the trunk, behind the third circular muscle. Three pairs of longitudinal retractors, the dorsal and the lateral head retractors, and the lateral corona retractors control the movements in the head. The latter extend anteriorly from a position in the neck and attach in the corona area with their bifurcate terminals (Fig. 1). Furthermore, the head contains two transverse muscles: the thin, dorsal pars coronalis (Figs. 1, 2a, b) near the rostrum, and the very broad and conspicuous coronal sphincter (Figs. 1, 2). The latter is not a true sphincter, since it is clearly composed of a dorsal and a ventral muscle that are not joined. However, it certainly functions the same way as the coronal sphincters in other rotifers. The movement of the foot is partly controlled by the ventral and lateroventral trunk retractors, but it furthermore has a pair of ventral foot retractors and foot extensors (Figs. 1, 2a). The ventral foot retractors are relatively thin and extend from the tip of the toes to a position near the second circular muscle. The extensors attach in the dorsal part of the toes and extend to a medial position in the trunk. Near their apical ends, each muscle splits into two separate terminals (Fig. 2b). Fig. 3 a, b Line drawing of the musculature in Proales fallaciosa. a Dorsal view. b Lateral view. dhr dorsal head retractor, dr dorsal trunk retractor, ic incomplete circular muscle, lr lateral trunk retractor, pc pars coronalis, vr ventral trunk retractor

5 51 Proales fallaciosa Two specimens were investigated. The trunk musculature consists of three double pairs of longitudinal retractors and four incomplete circular muscles (Figs. 2d e, 3). The circular muscles consist of two fibers that each runs from a ventrolateral position and meets on the dorsal side. The longitudinal muscles form three paired units that each consists of two closely set muscle bands that all run from the head to the foot terminal (Fig. 3). The ventral and the lateral trunk retractors are composed of three fibers that join in the head and near the foot. The dorsal retractors are composed of a broad muscle with an apical trifurcation, and a thinner muscle that runs from the neck to the foot. The head musculature consists of a dorsal head retractor and an apical pars coronalis. A corona sphincter is not present. Proales reinhardti Five specimens were investigated. The trunk musculature consists of longitudinal retractors and four incomplete circular muscles (Figs. 4, 5a c). The circular muscles are only present laterally and do not meet dorsally. The longitudinal muscles constitute a pair of Fig. 4 a, b Line drawing of the musculature in Proales reinhardti. a Dorsal view. b Lateral view. cs corona sphincter, dhr dorsal head retractor, dfm dorsal foot muscle, dlr dorsolateral trunk retractor, dr dorsal trunk retractor, fe foot extensor, ic incomplete circular muscle, lhr lateral head retractor, pc pars coronalis, ta toe abductor, tf toe flexor, ttm transverse toe muscle, vfr ventral foot retractor, vlr ventrolateral trunk retractor, vr ventral trunk retractor

6 52 dorsal, ventral, dorsolateral and ventrolateral retractors (Figs. 4, 5a, b). The ventral retractors are a pair of broad muscles with tripartite anterior ends that run from the neck region to a point in the middle of the trunk. Very close to the ventral retractors runs another pair, namely the ventrolateral retractors. These muscles are considerably thinner and run from the neck to the posterior part of the trunk (Figs. 4, 5c). The dorsal trunk retractors run from a more apical position in the head to a point in the middle of the trunk. At their posterior ends the muscles are slightly bifurcated and tend to wrap around the thinner dorsolateral retractors, which run through the entire animal (Figs. 4a, 5c). Fig. 5 a d Confocal laser scanning microscopy photos of Proales reinhardti. a Z-projection of whole specimen, dorsoventral view. b e Computerized 3D-reconstructions. b Dorsal view. c Trunk, ventral view. d Foot, dorsal view. e Foot, caudal view. dfm dorsal foot muscle, dlr dorsolateral trunk retractor, dr dorsal trunk retractor, fe foot extensor, ic incomplete circular muscle, ma mastax, pc pars coronalis, ta toe abductor, tf toe flexor, ttm transverse toe muscle, vfr ventral foot retractor, vfr ventral foot retractor, vm visceral musculature, vlr ventrolateral trunk retractor, vr ventral trunk retractor The head musculature consists of two pairs of longitudinal fibers: the dorsal and the lateral head retractors (Fig. 4). There is furthermore a dorsal pars coronalis and a corona sphincter. The latter is U-shaped and positioned ventrally (Fig. 4). Besides the dorsolateral trunk retractors, the foot contains a pair of large foot extensors and two strong ventral foot retractors (Figs. 4, 5a b, d e). The foot extensors run from the dorsal side of the trunk and attach ventrally in the proximal foot pseudosegment. The ventral foot retractors anchor in the posterior part of the trunk and extend caudally to the distal part of the foot. The ventral foot muscles are composed of three joined parts, and the most caudal part is composed of two fibers that join the single fiber in the medial part of the retractors (Fig. 5d). Furthermore a pair of very thin dorsal foot muscles is located dorsally in the most distal foot pseudosegment. The dorsal foot muscles are joined anteriorly so that together they are V-shaped (Fig. 5d e). Posteriorly they join with the large transverse toe muscle. A complex of very fine muscles is associated with the toes (Fig. 5d e). The transverse toe muscle is unpaired and located most distally in the foot. Two paired muscles extend into the toes from the trans-

7 53 verse toe muscle. One pair, the toe abductors, is located in the outer lateral sides of the toes. Contraction of these muscles will draw each toe outwards. Another pair, the toe flexors, is located more dorsally and runs from the transverse toe muscle to the tip of the toes. Contraction of these muscles will pull the tip of the toes in a dorsal direction. Discussion Comparison and homology hypotheses Basically, with a few exceptions, the three investigated species seem to possess muscular systems that can be considered homologues, but with different specific modifications. The incomplete circular muscles are certainly homologues, but the pattern of the muscles differs in each species. In P. reinhardti the circular muscles are only present laterally, whereas P. daphnicola and P. fallaciosa have circular muscles on both the dorsal and lateral sides. Furthermore, each circular in P. daphnicola is composed of six fibers while the circulars in P. fallaciosa are composed of only two fibers that join dorsally. All three species possess dorsal and ventral trunk retractors. Lateral trunk retractors are present in P. daphnicola and P. fallaciosa but lacking in P. reinhardti, whereas ventrolateral retractors only are present in P. daphnicola and P. reinhardti (Table 1). The location of these muscles supports the concept that they can be considered as homologous, but they vary at several points concerning appearance and precise location. P. daphnicola and P. reinhardti both have a pair of strong ventral retractors with a conspicuous trifurcation at their anterior ends; but, whereas the ventral retractors in P. daphnicola run throughout the entire body, they stop in the middle of the trunk in P. reinhardti. The ventral retractors in P. daphnicola are furthermore composed of three fibers and have a conspicuous appendage one third from the anterior end. In P. fallaciosa each ventral retractor is composed of two broad, closely set muscles, and none of them are trifurcated at the tip. The morphology of the dorsal trunk retractors also varies. P. daphnicola has very strong dorsal retractors that are bifurcated at both ends, and make two conspicuous twists before they anchor on the dorsal wall in the trunk. In P. reinhardti the dorsal retractors run from the head to a point in the middle of the trunk. At their posterior ends they form a small hinge that appears to wrap around the thinner dorsolateral retractor. P. fallaciosa has one thick and one much thinner dorsal retractor and both of them run through the entire body. Both pairs are certainly located dorsally, which suggest that they both are homologues with the dorsal retractors in the two other species and have been doubled like the ventral retractors. However, whereas the ventral retractors are extremely close-set and appear identical, the double dorsal retractors are rather different displaying one thick and apically trifurcated muscle and a thinner one without trifurcation that extends from the neck. This contradicts that they both origin from the same muscle system, and a reinterpretation of the muscles would suggest that only the thickest of the muscles, based on its apical trifurcation, should be considered homologous with the dorsal trunk retractor and that the thinner one then is a dorsolateral retractor. Hence, at this point the nature of these muscles must still be considered uncertain. The dorsolateral and ventrolateral retractors in P. daphnicola and P. reinhardti are also difficult to homologize because in P. reinhardti, they have moved to a position very close to the dorsal and ventral retractors, respectively. The double lateral retractors that protrude through the whole body in P. fallaciosa also differ much from their single and much shorter homologues in P. daphnicola. In their head regions, the three species share the presence of a dorsal head retractor and a pars coronalis. P. daphnicola and P. reinhardti furthermore have a strong corona sphincter, but in the latter species only its ventral part is present. In P. fallaciosa there is no trace of this huge muscle. Proales daphnicola and P. reinhardti both have differentiated foot muscles, whereas they are lacking in P. fallaciosa. The presence of foot retractors in P. daphnicola and P. reinhardti is probably correlated with the presence of an offset and movable foot. Differentiated toe muscles were only found in P. reinhardti. Hence, in summary the three investigated species share the following homologous muscles: Pars coronalis, dorsal head retractors, incomplete circular trunk muscles, dorsal and ventral trunk retractors, and maybe dorsolateral trunk retractors, depending on how the musculature in P. fallaciosa is interpreted. Furthermore, P. daphnicola and P. reinhardti share the following muscles: corona sphincter, lateral head retractors, ventrolateral trunk retractors, foot extensor and ventral foot retractor (see also Table 1). Comparison within the Rotifera and evolutionary evaluation In general, comparison of the different rotifer taxa is still impeded by the limited amount of information on the rotiferan musculature. However, it is possible to identify some of the above mentioned muscle systems in other taxa. The presence of pars coronalis, corona sphincter, incomplete circular trunk muscles and dorsal and ventral longitudinal trunk retractors have been reported from species of different taxa such as Epiphanes, Rhinoglena, Synchaeta and Brachionus, Notholca (Martini 1912; Stoszberg 1932; Peters 1931; Sørensen et al. 2003). Dorsal and ventral trunk retractors and incomplete circulars are furthermore

8 54 Table 1 Comparative table showing the muscle systems in the three investigated species Proales daphnicola Proales fallaciosa Proales reinhardti Head musculature Pars coronalis 1 dorsal band with 2 fibers 1 circular band 1 dorsal band with 2 fibers Corona sphincter 1 dorsal and 1 ventral fiber N/A 1 ventral fiber Dorsal head retractor 1 pair 1 pair 1 pair Lateral head retractors 1 pair N/A 1 pair Lateral corona retractors 1 pair, apically bifurcated N/A N/A Trunk musculature Incomplete circulars muscles 5 dorsal and lateral bands, each with 6 fibers Ventral retractors 1 pair, each with 3 fibers, from neck to toes, apically trifurcated Dorsal retractors 1 pair, each with 1 fiber, from head to mid of trunk, bifurcated apically and caudally Lateral retractors 1 pair, each with 1 fiber, from head to mid of trunk Ventrolateral retractors 1 pair, each with 2 fibers, from neck to toes Dorsolateral retractors 1 pair, each with 1 fiber, from neck to mid of trunk Foot and toe musculature Ventral foot retractor 1 pair, each with 1 fiber, from mid of trunk to toes Foot extensor 1 pair, each with 1 fiber, apically bifurcated 4 dorsal and lateral bands, each with 2 fibers 2 pairs, each with 3 fibers, from head to foot 1 or 2 pairs, each with 2 fibers, from head/neck to foot, one pair is apically bifurcated 2 pairs, each with 3 fibers, from head to foot 4 lateral pairs, each with 1 fiber 1 pair, each with 1 fiber, from neck to mid of trunk, apically trifurcated 1 pair, each with 1 fiber, from head to mid of trunk N/A N/A 1 pair, each with 1 fiber, from neck to foot 1 pair, each with 2 fibers, from neck to toes? 1 pair, each with 2 fibers, from head to toes N/A 1 pair, each with 3 fibers, from caudal part of trunk to toes N/A 1 pair, each with two fibers Dorsal foot muscles N/A N/A 1 apically joined pair Transverse toe muscle N/A N/A One broad transverse muscle Toe abductors N/A N/A One muscle lateral in each toe Toe flexors N/A N/A One muscle dorsal in each toe

9 55 found in bdelloid rotifers (Remane ; Hochberg and Litvaitis, 2000), and a corona sphincter has also been reported from species of the flosculariacean genera Hexarthra and Conochilus (Remane ), but it is apparently not present in the Testudinella species (Seehaus, 1930; Sørensen 2005). These distributions suggest that the pars coronalis and corona sphincter probably can be considered part of the ground pattern of Ploima. Whether these muscles evolved earlier also were present in the stem species of the Monogononta is still uncertain due to the incongruent observations from, on the one hand, Atrochus and Hexarthra species and on the other, Testudinella species. Incomplete circulars and dorsal and ventral trunk retractors are much more basal and were probably already evolved at the base of the Eurotatoria. Acknowledgements I thank the Marine Biological Laboratory in Elsinore for providing all the microscopical facilities and for letting me use their research vessel to get the marine material. I am indebted to Michael Ku hl for his help in operating the CLSM and his introduction to Volocity; to Stine Elle for producing the line art illustration; and Mary E. Petersen for correcting the language in the manuscript. This work was supported by the Carlsberg Foundation (Grants no. ANS-0178/20 and ANS-0724/20) and the Danish Natural Science Research Council (Grant no ). The CLSM was financed by the Danish Natural Science Research Council (Grants no and ). References De Smet WH (1996) The Proalidae (Monogononta). In: Dumont HJF (ed.) Guides to the identification of the microinvertebrates of the continental waters of the world, vol 9. SPB Academic, Amsterdam, pp Hochberg R, Litvaitis MK (2000). Functional morphology of the muscles in Philodina sp. (Rotifera: Bdelloidea). Hydrobiologia 432:57 64 Kotikova EA, Raikova OI, Flyatchinskaya LP, Reuter M, Gustafsson MKS (2001). Rotifer muscles as revealed by phalloidin-tritc staining and confocal scanning laser microscopy. Acta Zool 82:1 9 Martini E (1912). Studien u ber die Konstanz histologischer Elemente III. Hydatina senta. Z Wiss Zool 102: Remane A ( ). Rotatoria. In: Bronn HG (ed) Klassen und Ordnungen des Tier-Reichs, vol. 4. Vermes. Akademische Verlagsgesellschaft, Leipzig, pp Sørensen MV (2002). On the evolution and morphology of the rotiferan trophi, with a cladistic analysis of Rotifera. J Zool Syst Evol Res 40: Sørensen MV (2005). The musculature of Testudinella patina (Rotifera: Flosculariacea), revealed with CLSM. Hydrobiologia (in press) Sørensen MV, Funch P, Hooge M, Tyler S (2003) Musculature of Notholca acuminata (Rotifera: Ploima: Brachionidae) revealed by confocal scanning laser microscopy. Invert Biol 122: Seehaus W (1930). Zur Morphologie der Ra dertiergattung Testudinella Bory de St. Vincent (= Pterodina Ehrenberg). Z Wiss Zool 137: Stoszberg K (1932) Zur Morphologie der Rädertiergattungen Euchlanis, Brachionus und Rhinoglena. Z Wiss Zool 142: