Asian Pacific Journal of Tropical Biomedicine

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1 S sian ac J Trop Biomed 0; (Suppl ): S-S sian acific Journal of Tropical Biomedicine journal homepage: Document heading doi:0.0/jtb..0 襃 0 by the sian acific Journal of Tropical Biomedicine. ll rights reserved. Revised morphological identification key to the larval anopheline (Diptera: ulicidae) of Sri Lanka Nayana Gunathilaka, Thilan Fernando, enaka Hapugoda, Wimaladharma beyewickreme *, Rajitha Wickremasinghe olecular edicine Unit, Faculty of edicine, University of Kelaniya, Sri Lanka Department of arasitology, Faculty of edicine, University of Kelaniya, Sri Lanka Department of ublic Health, Faculty of edicine, University of Kelaniya, Sri Lanka EER REVIEW eer reviewer Dr. Diawo Diallo, edical Entomology Unit, Institut asteur de Dakar, v. asteur Dakar, B.. 0, Dakar, Senegal. Tel: 00 diawod@yahoo.com omments This study is very important because the identification of mosquito species is always the first step in all surveillance and control of malaria strategies. It describes an updated and easy way to use illustrated key that will be useful for Sri Lankan malaria control personnel and researchers interested in local anopheline fauna. Details on age S BSTRT bjective: To revise morphological identification keys to the anophelines in Sri Lanka. ethod: Samples were collected from selected entomological sites in different districts in the country. Stage I and IV larvae were identified under a light microscope with an objective ( 伊 0) using standard larval keys developed for Sri Lankan anophelines. Key larval characters were recorded for each species based on original observations and previous usage in literature. Results: This manuscript describes an illustrated key for the identification of of mosquitoes which are currently recognized as local anopheline species in Sri Lanka, as a guide to workers engaged in malaria surveillance and control in the country. onclusions: Revised morphological keys to the larval of these species may be helpful in easy and accurate identification at the field level. KEYWRDS nopheline, Immature, osquitoes, ontrol, Keys. Introduction The identification keys to the immature stages of nopheles mosquitoes have long been a necessity for entomologists dealing with malaria vectors. any of the illustrated keys to the nopheles of Sri Lanka are of limited value[,], as these were published more than 0 years ago and significant advances in our knowledge of the nopheles mosquitoes have occurred in the intervening years. The number of anopheline species has not changed substantially from arter s checklist; there have been many changes in the identity of the species actually listed, as evidenced by the checklist of Jayasekera and helliah[]. hanges subsequently to this checklist include the invalidation of one species record and description of nopheles jeyporiensis (n. jeyporiensis ). This paper presents an updated illustrated key for the identification of larval anophelines occurring in the country. The purpose of the keys presented here is to assist field surveillance teams to identify the larval stages of nopheles mosquitoes. The keys can be used initially to identify specimens to species group and then to species. Discriminating characteristics are highlighted in drawings wherever possible. *orresponding author: rof. Wimaladharma beyewickrem, Department of arasitology, Faculty of edicine, University of Kelaniya,.. Box, Thalagolla Road, Ragama 00, Sri Lanka. Tel: + Fax: wabeyewickreme@yahoo.com Foundation roject: Supported by Global Fund for ids, Tuberculosis and alaria (GFT) (Round ) through TEDH (Grant No. SRL0G). rticle history: Received Dec 0 Received in revised form 0 Dec, nd revised form Dec, rd revised form Jan 0 ccepted 0 Feb 0 vailable online ar 0

2 Nayana Gunathilaka et al./sian ac J Trop Biomed 0; (Suppl ): S-S S. aterials and methods.. ollecting samples Samples were collected from entomological sites in mpara, Batticaloa, annar, Trincomalee and Killinochchi districts as part of the malaria elimination program of Tropical and Environmental Diseases and Health ssociates (TEDH) vt. Ltd. Further, wet zone specimens were obtained by the entomological teams attached to the nti alaria ampaign... Identification of field samples Stage I and IV larvae were placed individually in microscopic slides and identified under a light microscope with an objective ( 伊 0) using standard larval keys developed for Sri Lankan anophelines[]. Key larval characters were recorded for each species. Further, larval species identification was reconfirmed through adult identification[]... Revising larval identification key for Sri Lankan nopheles The morphological characters used here are based on original observations and previous use in the literature. The following were referred to during the construction of this key: marasinghe, [], hristophers, [], olless, [], Reid, [], Harrison, 0[], Harrison and Scanlon, [], Linton et al., 00[], Junkum et al., 00[0], Linton et al., 00[], Rattanarithikul et al., 00[], Sallum et al., 00[] and Rattanarithikul et al., 00[]. Taxonomic characteristics were cross checked in relation to Sri Lankan specimens by examining the reference materials archived at the olecular edicine Unit, Faculty of edicine, University of Kelaniya, Sri Lanka. Species nomenclature follows that proposed by Knight and Stone[], and abbreviations used in the text follow that used by Reinert[,]. orphological terminology and chaetotaxy follow that used by Harbach and Knight[]. bdominal segments are numbered by roman numerals. Twenty two Sri Lankan anopheline species have been included into this key. The species considered include: Subgenus nopheles aitkenii James 0 (n. aitkenii), nopheles barbirostris Van der Wulp (n. barbirostris), nopheles barbumbrosus Strickland and houdhury (n. barbumbrosus), nopheles gigas var. refutans lcock, nopheles interruptus uri (n. interruptus), nopheles nigerrimus Giles 00 (n. nigerrimus), nopheles peditaeniatus (Leicester) 0 (n. peditaeniatus), nopheles peytoni Kulasekera (n. peytoni), Harrison and merasinghe. Subgenus ellia: nopheles aconitus Donitz 0 (n. aconitus), nopheles annularis Vander Wulp, nopheles culicifacies Giles 0 (n. culicifacies), nopheles elegans (James) 0 (n. elegans), nopheles jamesii Theobald 0, n. jeyporiensis (James) 0, nopheles karwari (James) 0, nopheles maculatus Theobald 0, nopheles pallidus Theobald 0, nopheles pseudojamesi Strickland and howdhury, nopheles ramsayi ovell, nopheles subpictus Grassi (n. subpictus), nopheles tessellatus Theobald 0 (n. tessellatus), nopheles vagus Donitz 0 (n. vagus), nopheles varuna Iyengar (Figure ). Thorax T Dorsal Ventral 0 0 I 0 IV 0 V 0 VI 0 Head -x 0 T Tropacic pleural groups V 0 VI - S Figure. Larval morphology. Head: =ntenna, =ranium head capsule; Thorax: =ro-thorax, =esothorax, T=eta-thorax, -=setae on designated areas (seta, setae -T); bdomen: I-V, X=bdominal segments, S=Spiracular apparatus, -= Setae on designated areas (seta -I, seta -IV).. Results The revised larval morphological key is shown below. This key presents an illustrated key for the identification of larvae of of currently recognized local anopheline species in Sri Lanka. orphological features noted in different regions of the body were recorded: head (antennal hairs, inner clypeal hairs, outer clypeal hairs, frontal hairs and sutural hairs), thorax (thoracic palmate hairs, shoulder hairs, pro, meso and meta thoracic hairs), abdomen (abdominal tergal plates, palmate hairs in abdominal segments, lateral hairs in the abdominal segments). The inclusion of n. jeyporiensis is based on the reported collection during the TEDH alaria Elimination rogram in 0. This species was included in this key as local workers may encounter the species and may difficulty in its identification. Generally, members of subgenus nopheles are characterized by branched seta - and closely situated setae -. However, exceptions such as n. aitkenii sens. str., with widely spaced -[] and n. interruptus, with

3 S Nayana Gunathilaka et al./sian ac J Trop Biomed 0; (Suppl ): S-S simple - and closely spaced -[,,] have been reported in Sri Lanka. Figure in the key has been worded to avoid initial misidentification of larvae.. Discussion The key to the Sri Lankan anopheline larvae has been revised according to the currently accepted classification of sections, series and groups in the nophelinae. Brief notes are provided at each step in the identification process along with illustrative diagrams. It is emphasized that taxonomic keys are only a rapid and convenient guide to identification based on the examination of a few important characters. In case of doubt, it is essential to consult published literature with detailed descriptions of species characters... Subgenus nopheles... yzorhynchus series The yzorhynchus series can be separated from other Sri Lankan representatives of subgenus nopheles by the outer clypeal hair (-) being strongly branched. Two species groups are present in Sri Lanka: the Hyrcanus and Siphon absent; abdominal seta palmate ntennal hair (-) branched Inner clypeal (-) hairs close together, & frontal hairs (-, - & -) long & branches ntennal hair (-) simple Inner clypeal (-) hairs well separated, & frontal hairs (-, - & -) short, simple or with few branches Subgenus nopheles Subgenus ellia uter clypeal (-) hair simple or few branches uter clypeal (-) hair strongly branched bdominal tergal plates very large bdominal tergal plates small, transverse Figure a Figure Thoracic palmate hair well developed Thoracic palmate hair rudimentary IV-V Inner clypeal (-) hair with - short lateral barbs. ost clypeal (-) with - branches. Inner clypeal (-) hair with - short lateral barbs. ost clypeal (-) simple or bifid rarely. n. gigas n. aconitus n. varuna IV-V Figure b Figure. Sri Lankan anopheline mosquito - key to the fourth instar larvae. Barbirostris Group Inner shoulder (-) hair branched from the base almate hairs in the abdominal segment I- usually pigmented (Except n. barbumbrosus) Hycanus Group Inner shoulder (-) hair simple or split at the tip almate hairs in the abdominal segment I- un-pigmented - - uter clypeal (-) hair with thin attenuated branches usually loose and spread out. uter clypeal (-) hair with more and thick branches (-), usually stiff and broom like Inner sutural (-) hair with - branches uter sutural (-) hairs with branches Inner sutural (-) hair with - branches. uter sutural (-) hairs with - branches n. barbumbrosus n. barbirostris n. peditaeniatus n. nigerrimus Figure. uter clypeal hair strongly branched.

4 Nayana Gunathilaka et al./sian ac J Trop Biomed 0; (Suppl ): S-S S Barbirostris group (Figure ).... Hyrcanus group The Hyrcanus group can be separated from the Barbirostris group by the morphological characters given in the key. This group bears a superficial resemblance to members of the Hyrcanus group, all of which have pale or un-pigmented I ; the separation of n. peditaeniatus and n. nigerrimus needs care. The definitive identifying feature is the basally branched, sinuous seta - of n. peditaeniatus which is small and often difficult to observe. This may be confused with - resulting in misidentification as n. nigerrimus. In this instance, branching of setae - and - are useful in confirmation. The correct identification of n. nigerrimus is important as it is a suspected vector of malaria in Sri Lanka while n. peditaeniatus is considered a non-vector (Figure ).... Barbirostris group Within the Barbirostris group of subgenus nopheles, the orientation of the inner shoulder hair (-) can be used as the primary character: seta with or more branches Figure a Figure b Inner clypeal (-) hair long stout & simple uter clypeal (-) hair simple or bifid n. peytoni Inner clypeal (-) hair branched ost clypeal hair (-) branched from the base n. aitkenii Inner clypeal hair simple, outer clypeal () hair branched. & branched n. interruptus Inner (-) & outer (-) clypeal hairs with inconspicuous fraying Inner (-) & outer (-) clypeal hairs simple Inner sutural (-) hair branched from the base Inner (-) & outer (-) clypeal hairs strongly branched Inner sutural (-) hair simple or split at the tip almate hair on abdominal segment I & rudimentary eso () & etathoracic (T) plural hairs simple Inner shoulder (-) hair weak with - Inner shoulder (-) hair well developed, branches. Root small and pale with many branches. Root large and dark Figure n. pallidus almate hair on abdominal segment I, with well developed leaflets almate hair on abdominal segment I rudimentary without obvious leaflet n. tessellatus n. elegans n. jeyporiensis n. annularis Figure. Thoracic palmate hair and abdominal tergal plates. a: Thoracic palmate hair well developed; b: bdominal tergal plates small, transverse. ost clypeal hair (-) with - branches Jamesii Group Figure almate hair on abdominal segment I rudimentary esothoracic leural hairs not both simple; one branched almate hairs on abdominal segment no I well developed; almate hairs with sharp pointed filaments bdominal seta in segment V, VI with - branches almate hairs with blunt filaments bdominal seta in segment V, VI with - branches V V Thoracic palmate hair rudimentary; roots of shoulder () hairs small & pale - uter (-) and post (-) clypeal hairs very short, situated posteriorly between the inner clypeals (-) Base of the post clypeal (-) hairs wide apart & length of the outer clypeal (-) hair is about / of the length of inner clypeal hair (-) - Thoracic palmate hair moderately developed; roots of shoulder () hairs strong and dark n. culicifacies uter (-) and post (-) clypeal hairs longer, situated posteriorly below and outside the inner clypeals (-) Base of the post clypeal (-) hairs close together & length of the outer clypeal (-) hair is about / of the length of inner clypeal hair (-) n. maculatus n. karwari n. vagus n. subpictus Figure. almate hairs on abdominal segments. n abdominal segment always well developed; hairs on segment I sometimes rudimentary; eso and etathoracic leural hairs not all simple; one or more branched.

5 S Nayana Gunathilaka et al./sian ac J Trop Biomed 0; (Suppl ): S-S is characteristic of this group. This character should be checked carefully during identification. The pigmentation of seta I- is an useful secondary character in separating barbirostris from barbumbrosus (pale or colorless seta). In addition, seta orientation of the outer clyepeal hair (-) can be used to separate these two species as the optional character in order to confirm the identification. It can be described as follows; - of n. barbumbrosus has - thin attenuated branches usually loose and separated out, whereas - of n. barbirostris has more branches which are thick (-), usually stiff and broom like (Figure ). The larval morphology of n. reidi has still not been reported.... Lophoscelomyia series n. interruptus is the only species in this series reported in the country. It can be distinguished by the simple inner clypeal hair (-), and branched outer clypeal hair (-), - and -. In the previous key for Sri Lankan anopheline larvae, the identification was confirmed through the presence of reduced setae, and -, some or all of which were short and a few-branched or simple; seta - simple or branched only in distal half[]. These characters are rather difficult in identification and controversial. Therefore, the new character which has been included in this key is useful to minimize misidentification of this species (Figure ).... nopheles series There are three species in the country, namely: nopheles gigas, n. aitkenii and nopheles peytoni (n. peytoni). n. peytoni is an endemic species in Sri Lanka and had been confused with n. insulaeflorum; the larvae of n. peytoni has bases of setae - nearly as wide apart as - and - on one side which is different from the larval morphology of n. insulaeflorum. In this key the following characters have been included to confirm identification of n. peytoni: - long and simple, - simple or bifid. These features facilitate the separation of n. aitkenii which has branched - and - (Figure )... Subgenus ellia... yzomyia series Separating nopheles varuna and n. aconitus in the yzomyia series of subgenus ellia is sometimes difficult when the barbed nature of setae -, - and branching of - in n. aconitus are observed by drift or the larval mouth brushes. It is more difficult to distinguish n. jeyporiensis as it separates only by having a small tergal plate and - branches in - (Figure ). n. culicifacies, the main vector of malaria in Sri Lanka can be separated very easily among the members in yzomyia series of subgenus ellia by the presence of small tergal plates, unbranched - and - and moderately developed thoracic palmate hairs (Figure ).... yretophorus series n. subpictus and n. vagus are the only two recorded species in this series in Sri Lanka. These two species can be easily separated from n. culicifacies by the moderately developed thoracic palmate hair and strong and dark roots of shoulder hairs. n. vagus has very short - and -, - is situated between - with the base of - being wide apart, and the length of - being about / of the length of -; n. subpictus has longer - and -, and - seta is situated below and outside -; the base of the - seta and length of the - is about ½ of the length of - (Figure ).... Neocellia series Six species of Sri Lankan Neocellia can be divided into those with bushy setae - (nopheles jamesii, nopheles annularis, nopheles pallidus) and those with simple or barbed - (nopheles maculatus, nopheles kawari, nopheles pseudojamesi). further character that separates the two groups is the length of the filaments in abdominal palmate setae IV V, these being equal to or greater than ½ the length of the blades in the former and /rd or less in the latter (Figure ). Inner (-) & outer (-) clypeal hairs has short barbs Inner clypeal (-) hair with short barbs & outer clypeal (-) hair with brush like branches n. pseudojamesi n. jamesii Figure. almate hair on abdominal segment I rudimentary without obvious leaflet.... Neomyzomyia series Two species, n. tessellatus and n. elegans (the latter belonging to the leucosphyrus group) have been reported in Sri Lanka. n. elegans is an endemic species in the country. Identification of n. tessellatus is important in malaria vector surveillance programmes in the Sri Lanka as it has been incriminated as a secondary vector of malaria transmission in the country. Identification of this species can be confirmed by the presence of - branches in the inner shoulder hair (-) with small and pale roots. Individuals with opposite morphological features can be regarded as n. elegans (Figure ). Sri Lankan anopheline larvae can be differentiated easily by observing basic characters. However, this may become a difficult task when processing whole larvae in routine malaria entomological surveys.

6 Nayana Gunathilaka et al./sian ac J Trop Biomed 0; (Suppl ): S-S S onflict of interest statement We declare that we have no conflict of interest. cknowledgements I would like to thank Dr. anduka Wijeyarathne, the rogram Director of the alaria Elimination rogram of Tropical and Environmental Diseases and Health ssociates (TEDH) vt. Ltd for his encouraging support and financial assistance from the Global Fund for ids, Tuberculosis and alaria (GFT) (Round ) through TEDH (Grant No. SRL0G). omments Background orphological taxonomic keys are indispensable tools in malaria vectors surveillance and control. They help field operators to easily and accurately identify anopheline species in the field. In Sri Lanka, the published keys are outdated and need to be revised as knowledge on anophelines have advanced. Research frontiers This paper is carried out to revise a larval morphological identification key of the anopheline species of Sri Lanka. Related reports This key has reported one more species than the guide to the identification of anopheline mosquitoes of Sri Lanka published by marasinghe in. Innovations and breakthroughs This paper contains the first illustrated key that allows the identification of the immature of anopheline species currently known in Sri Lanka. pplications This key will assist field workers involved in malaria surveillance and control in Sri Lanka to firmly identify the anopheline species currently known in this island. eer review This study is very important because the identification of mosquito species is always the first step in all surveillance and control of malaria strategies. It describes an updated and easy way to use illustrated key that will be useful for Sri Lankan malaria control personnel and researchers interested in local anopheline fauna. References [] arter HF. eylon mosquitoes list species and names of mosquitoes recorded from eylon. eylon J Sci Sect B Zool 0; : -. [] marasinghe F. guide to the identification of anopheline mosquitoes (Diptera; ulicidae) of Sri Lanka.. Larvae. eylon J Sci ; : -. [] marasinghe F. guide to the identification of anopheline mosquitoes (Diptera; ulicidae) of Sri Lanka. I. dult females. eylon J Sci 0; : -. [] hristophers SR. The fauna of British India, including eylon and Burma. London: Taylor & Francis;, p.. [] olless DH. Further notes on the systematic of the nopheles leucosphyrus group (Diptera: ulicidae). roc R Entomol Soc London ; : -. [] Reid J. nopheles mosquitoes of alaya and Borneo. England: Staples rinters Limited;, p.. [] Harrison B. edical entomology studies XI. The yzomyia series of nopheles (ellia). In Thiland, with emphasis on intrainterspecific variations (Diptera: ulicidae). ontrib m Entomol Inst 0; (): -. [] Harrison B, Scanlon JE. The subgenus nopheles in Thailand (Diptera: ulicidae). ontrib m Entomol Inst ; (): -0. [] Linton Y, Harbach RE, hang S, nthony TG, atusop. orphological and molecular identity of nopheles (ellia) sundaicus (Diptera: ulicidae), the nominotypical member of a malaria vector species complex in Southeast sia. Syst Entomol 00; : -. [0] Junkum, Komalamisra N, Jitpakdi, Jariyapan N, in GS, ark H, et al. Evidence to support two conspecific cytological races of nopheles aconitus in Thailand. J Vector Ecol 00; 0: -. [] Linton Y, Dusfour I, Howard T, Ruiz LF, Duc anh N, Ho Dinh T, et al. nopheles (ellia) epiroticus (Diptera: ulicidae), a new malaria vector species in the Southeast sian Sundaicus omplex. Bull Entomol Res 00; : -. [] Rattanarithikul R, Harrison B, anthusiri, oleman RE. Illustrated keys to the mosquitoes of Thailand I. Background; geographic distributions; list of genera, subgenera, and species; and a key to genera. Southeast sian J Trop ed ublic Health 00; (Suppl ): -0. [] Sallum, eyton EL, Wilkerson R. Six new species of the nopheles leucosphyrus group, reinterpretation of nopheles elegans and vector implications. ed Vet Entomol 00; : -. [] Rattanarithikul R, Harrison B, Harbach RE, anthusiri, oleman RE. Illustrated keys to the mosquitoes of Thailand IV nopheles. Southeast sian J of Trop ed & ub Health. 00; (Suppl ): -. [] Knight KL, Stone. catalog of the mosquitoes of the world (Diptera: ulicidae). nd ed. aryland: Entomology Society of merica;, p. -. [] Reinert JF. osquito generic and subgeneric abbreviations (Diptera: ulicidae). osquito Systematics ; : 0-0. [] Reinert JF. Revised list of abrreviations for genera and subgenera of ulicidae (Diptera) and notes on generic and subgeneric changes. J m osq ontrol ssoc 00; : -. [] Harbach RE, Knight KL. Taxonomists glossary of mosquito anatomy. arlton: lexus ublishing; 0, p. -0.