ISSN: 2347-3215 Volume 2 Number 10 (October-2014) pp. 17-24 www.ijcrar.com

Comparision of Male and Female Genitalia of species with Scanning Electron Microscopy (SEM)

Jagbir Singh Kirti, Shipali* and Jasneet Kaur

Department of Zoology and Environmental Sciences, Punjabi University, Patiala- 147002, *Corresponding author KEYWORDS A B S T R A C T

SEM, genitalia, The genitalic structure of male and female mosquitoes of two species belonging to genus Culex i.e Culex (Culex) vishnui Theobald and Culex and Culex (Culex) pseudovishnui Colless is studied morphologically and illustrated with pseudovishnui. the aid of Scanning Electron Microscope (SEM) from Punjab.

Introduction The species or different forms of it, has a pseudovishnui and , wide distribution throughout African its significance in natural transmission of continent including even the Central Sahara disease has not yet been clearly established and it also occurs in Madagascar. It is Miyagi et al. (1992). In , (Cates and widely distributed throughout the Detels, 1969) found vishnui to be only Mediterranean region from Punjab virus positive (Edwards, 1941; Mattingly, 1954). Culex and concluded that it was the vishnui has been confused with Culex important vector of this disease. Regarding pseudovishnui in the past due to their close breeding sites both the species having resemblance; they are definitely distinct similar habitat like open cultivated lands species, which are easily distinguished by such as rice fields, stagnant pools, pits and larval and genitalic characters. The larvae of edges of swamps. Their preferred hosts Culex vishnui, and include cows, buffaloes, pigs etc. They also Culex tritaeniorhynchus were common in attack on man occasion. These species has rice fields where they aggregated near the been found to be involved in cycle of West edges of paddy fields during early rice Nile virus in Egypt and becoming probably growing season, April to May. In addition to infected from birds (Taylor et al., 1956). Culex tritaeniorhynchus, Culex vishnui is The Southeast Asian Culex vishnui complex possibly one of the important vectors of has attracted much attention because of its Japanese Encephalitis virus in Southeast importance as potential vectors of Japanese Asia, because of difficulty in separating the Encephalitis virus and related arboviruses adults from sympatric sibling species, Culex (Sirivanakarn, 1976).

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Work done on the above said species by important for species identification. Female Barruad (1934), Bram (1967), Colless genitalia are important to point out that (1957), Delfinado (1966), Hara (1957), Jupp spermatheca located inside abdomen and its and Brown (1967), LaCasse and Yamaguti morphological architecture as well as its (1950), Matsuo (1959, 1968 & 1970), number is used as taxonomic characters. Matsuo and Ramalingam (1971& 1972), (Alexander- Pires, 2010). Matsuo and Iwaki (1972), Miyagi (1971), Miyagi et al. (1992), Reuben (1969, 1971a Male genitalia & 1971b), Sagandeep and Kirti (2006), Sirivanakarn (1975, 1976), Theobald (1901), Culex vishnui: Dististyle arcuate and Yamamoto (1969). flanged laterally along with two minute papillionated hair and subapical claw Material and methods bilobed at apex (Fig. 7); basistyle conical and abruptly tapered beyond the subapical Adult mosquitoes were procured with the lobe with long setae on lateral side and small help of oral aspirators, test tubes, torch and setae on dorsal surface (Fig. 1); subapical collecting nets from gardens, cattle lobe with (a-h) setae all of varying size and sheds, hay stacks, nurseries, forests and length (Fig. 6). human dwellings from the different districts of Punjab. For the identification of species Seta a very elongated and bent downwardly, keys given by Darsie and Pradhan (1990), b long with sharp apex, d leaf like, e and f Sirivanakarn (1976), Huang (1979), Reinert rod like, g acuminate apically in the present et al. (2004) and Reuda (2004) and studies but it is of axe shaped according to terminology given by Harbach and Knight Sagandeep and Kirti (2006) and h small in (1980) and for SEM studies protocol given size; inner division of lateral plate of by Chaudhry and Gupta (2004) and Lee and phallosome having nine finger like Craig (1983) was followed. For genitalic projections (Fig. 4). Out of nine projections, studies, the last two abdominal segments of five long and well developed, other four adult male and female mosquitoes were short and poorly developed (Sagandeep and snipped off with fine forceps from their Kirti, 2006). body. However, these finger like projections give These abdominal tips were boiled in 10% palmate like appearance with varying length KOH solution until their clearance. i.e. first two longest, three medium and 4-5 Dissected material washed several times shorter with their apices reaching beyond with water and dehydrated by passing apical margin of spiculate sternal process; through ascending grades of alcohol. The rest four on other side comparatively specimens were placed on stubs in dorsal shorter; inner sternal spiculate process not positions after air drying on filter paper and completely separated from outer division coated with gold. After that images were bears numerous spines (Sagandeep and observed under JSM-6610LV SEM. Kirti, 2006); outer division with strong mesal spine with bifid apex and a strong Result and Discussion sternal spine with pointed apex; basal sternal process (Fig. 3) dark, stout, long, curved and Regarding genitalia, several characters i.e. well developed; cercal setae (Fig. 2) two in basistyle, dististyle, subapical lobe, number on proximal region of paraproct. paraproct, aeadegus, phallosome etc. are 18

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Culex pseudovishnui: Basistyle (Fig. 12) crown of setae at tip (Fig. 13); inner division having elongated dististyle (Fig. 16) with of lateral plate of phallosome having eight bilobed claw at tip; subapical lobe bearing finger like projections (Fig. 15) four on one various types of (a h) setae (Fig. 17). Seta a side and four other on the another side, prominent, b elongated, c flattened at apex giving appearance of palmate process; inner and having spatualate appearance, d, e and f sternal spiculate portion not completely long, thin and rod like with curved apices, g separated from outer division having broad, acuminate or rounded apically, h hair numerous spines (Fig. 18); 1-2 pair of cercal like in appearance; lateral plate of setae observed at the proximal and distal end phallosome has both inner and outer of paraproct (Fig. 13); basal sternal process divisions; paraproct bearing numerous well developed, long and stout (Fig. 14).

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Female genitalia family Culicidae, tribe Megarhinini and Culicini. Taylor and Francis, London. Culex vishnui: Cerci short with numerous Bram, R.A. 1967. Contribution to the mosquito setae on the dorsal surface (Fig. 8 & 9); fauna of Southeast Asia- II. The Genus tergum IX slightly curved with minute row Culex in (Diptera: Culicidae). Contrib. Am. Entomol. Inst., 2: 1 296. of setae (Fig. 10 & 11). Cates, M.D., Detels, R. 1969. Japanese Encephalitis virus in Taiwan: Preliminary Culex pseudovishnui: Cerci moderately evidence for Culex annulus Theobald as a smaller in size with numerous large setae; vector. J. Med. Entomol., 6: 327 328. postgenital lobe (Fig. 20 & 21) bears five Chaudhry, S., Gupta, S. 2004. Scanning electron pair of setae on its margin; tergum IX microscopic studies on the egg architecture forming a U- shaped structure with 6-7 setae of Anopheles (Cellia) stephensi Liston (Fig. 22). (Diptera: Culicidae). Proc. Zool. I. Soc. Calcutta., 57(1): 1 4. Culex pseudovishnui species has been most Colless, D.H. 1957. Notes on the Culicine frequently misidentified as vishnui. During mosquitoes of - II. The Culex vishnui group (Diptera: Culicidae) with this study an attempt was made to correlate description of two new species. Ann. Trop. the differences between two species with Med. Parasit., 51: 87 101. varying genitalic attributes. Although Darsie, R.E., Pradhan, S.P. 1990. The genitalia of Culex vishnui was studied earlier mosquitoes of : their identification, by Sagandeep and Kirti (2006). In the distribution and biology. Mosq. Syst., 22: present research work, genitalia of above 69 130. said species is compared with the previous Delfinado, M.D. 1966. The Culicine mosquitoes one and try to find out some new and of the . Tribe Culicini (Diptera: additional taxonomic attributes. The work Culicidae). Mem. Am. Ent. Inst., 7: 252. on genitalia of Culex pseudovishnui of male Edwards, F.W. 1941. Mosquitoes of Ethopian and female mosquitoes is done for the first region III. Culicine adults and pupae. Lond,: British Museum (Nat. Hist.). time in Punjab. Hara, J. 1957. Studies on female terminalia of Japanese Mosquitoes. Jap. J. Exp. Med., Abbreviations 27(1-2): 45 91. Harbach, R.E., Knight, K.L. 1980. Taxonomist s Bs (Basistyle), Ce (Cerci), CSe (Cercal glossary of mosquito anatomy. Plexus Seta), Ds (Dististyle), PGL (Post Genital Publishing, Marlton, NJ. Pp. 1 413. Lobe), PpC (Paraproct Crown), Ppr Huang, Y.M. 1979. Medical Entomology (Paraproct) and IX-Te (IX- Tergum). studies- XI. The subgenus Stegomyia of Aedes in the Oriental region with keys to References the species (Diptera: Culicidae). Contrib. Am. Entomol. Inst., 15(6): 1 79. Jupp, P.G., Brown, R.G. 1967. The laboratory Alexander-Pires, G., Ramilo, D., Diaz, S., colonization of Culex (Culex) univittatus Meireles, J., Boinas, F., Fonseca, I.P.D. Theobald (Diptera: Culicidae) from 2010. Investigating morphological material collected in the Highveld region of structures of Culicoides from obsoletus South Africa. J. Ent. Soc. Sth. Afr., 30: 34 complex by using scanning electron 39. microscopy and compressed optical LaCasse, W.J., Yamaguti, S. 1950. Mosquito microscopy. Formatax, 792 802. fauna of and Korea. Off. Surgeon, 8th Barruad, P.J. 1934. The fauna of British India U.S. Army, Kyoto. including Ceylon and Burma. Diptera 5, Lee, R.M.K.W., Craig, D.A. 1983. Cibarial

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sensilla and armature in mosquito adults tritaeniorhynchus Giles, from Southern (Diptera: Culicidae). Can. J. Zool., 61(3): India. Bull. Ent. Res., 58: 643 652. 633 646. Reuben, R., 1971a. Studies on the mosquitoes of Matsuo, K. 1959. Taxonomic studies on Culex North Arcot district, Madras State, India. tritaeniorhynchus Giles and Culex vishnui Part IV. Breeding places of Culex vishnui Theobald. J. Kyoto Pref. Med. Univ., 65: group of species. J. Med. Entomol., 8: 363 581 621. 367. Matsuo, K., 1968. Morphological studies on Reuben, R. 1971b. Studies on the mosquitoes of Culex vishnui group of Japan. Abstract of North Arcot district, Madras State, India. the 61st Annual Meeting of Formosan Part VI. Seasonal prevalence of Culex Medical Association. vishnui group of species. J. Med. Entomol., Matsuo, K. 1970. Morphological and ecological 8: 367 371. studies of Culex tritaeniorhynchus and Reuda, L.M. 2004. Pictorial keys for the Culex pseudovishnui adults. Abstract of the identification of mosquitoes (Diptera: 25th Regional Meeting of Japanese Journal Culicidae) associated with Dengue Virus of Sanitary Zoology. Transmission. Zootaxa., 589: 1 60. Matsuo, K., Ramalingam, S. 1971. On Culex Sagandeep, K., Kirti, J.S. 2006. Current tritaeniorhynchus and Culex analysis of sensilla and other structures on pseudovishnui. X. Morphological the body appendages of three Indian Culex characters of Culex pseudovishnui larvae species: 193 211. In: Sharma, V.P. and from . The 23rd Annual Meeting of Kirti, J.S. (Eds), Vector Biology. National Japanese Sanitary Zoology. Academy of sciences India, Allahabad. Pp. Matsuo, K., Ramalingam, S. 1972. 256. Morphological characters of Culex Sirivanakarn, S. 1975. The systematic of Culex pseudovishnui from Malaysia, Japan and vishnui complex in Southeast Asia with the Formosa. Southeast Asian J. Trop. Med. diagnosis of three common species Pub. Hlth., 3: 55 61. (Diptera: Culicidae). Mosq. Syst., 7: 69 85. Matsuo, K., Iwaki, M. 1972. Morphological Sirivanakarn, S. 1976. A revision of the differences betweem Culex subgenus Culex in the Oriental region tritaeniorhynchus and Culex pseudovishnui (Diptera: Culicidae). Contrib. Am. Entomol. adults from Japan. J. Kyoto Pref. Univ. Inst., 12: 1 186. Med., 81(7): 349 357. Taylor, R.M., Work, T.H., Hurlbut, H.S., Rizk, Mattingly, P.F. 1954. The distribution of some F. 1956. A study of the ecology of West African mosquitoes. Pro. Linn. Soc. Lond., Nile virus in Egypt. Amer. J. Trop. Med. Pp. 1 165. Hyg., 5: 579 620. Miyagi, I. 1971. Culex vishnui group from Theobald, F.V. 1901. A monograph of the Philippines. The 23rd Annual Meeting of Culicidae or mosquitoes I. London: Brit. Japanese Sanitary Zoology. Mus. (Nat. Hist.). Miyagi, I., Toma, T., Hasegawa, H., Tadano, M., Yamamoto, H. 1969. Culex tritaeniorhynchus Fukunaga, T. 1992. Occurrence of Culex and Culex pseudovishnui as the vector of (Culex) vishnui Theobald on Ishigakijima, Japanese Encephalitis. Jap. J. Zool., 20: Ryukyu Archipelago. Jpn. J. Sanit. Zool., 117. 43(3): 259 262. Reinert, J.F., Harbach, R.E., and Kitching, I. 2004. Phylogeny and classification of Aedinii (Diptera: Culicidae) based on morphological characters of all life stages. Zool. J. Linn. Soc., 142: 289 368. Reuben, R. 1969. A redescription of Culex vishnui Theobald with notes on Culex pseudovishnui Colless and Culex

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