Available online at www.journalijmrr.com

INTERNATIONAL JOURNAL OF MODERN RESEARCH AND REVIEWS

Int. J. Modn. Res. Revs. IJMRR Volume 4, Issue 12, pp 1425-1430, December, 2016 ISSN: 2347-8314

ORIGINAL ARTICLE

SPERM MORPHOLOGY OF PUMPKIN , foveicollis AND (COLEOPTERA:CHRYSOMELIDAE)

*1S.Sethuraman, 2T.Vivekananthan and 1T.Ramesh Kumar

*1Department of Zoology, Annamalai University, Annamalai Nagar, Tamil Nadu-India. 2Assistant Professor, Thiru Kolanchiyappar Govt. Arts and Science College, Vriddhachalam

Article History: Received 14th October ,2016, Accepted 30th November,2016, Published 1st December,2016

ABSTRACT Spermatozoa morphology has, for some years, been used to help answer some phylogenetic questions for Coleoptera. In the present study describing spermatozoa morphology of an Aulacophora species was observed. Aulacophora foveicollis spermatozoa overall length measured 636±30.1µm and head length was 30.8±2.3µm. The ratio of the head length and tail length ~ 32.1.Spermatozoa head portion of the nucleus appears star like structure exposed by DAPI. In Aulacophora nigripennis spermatozoa overall length measured 355±21µm and the head length of the single sperm measured 11.3±1.1µm. The ratio of the head length and tail length was ~ 25.1. Spermatozoa nucleus appears kite like structure exposed by DAPI. Flagellar length of A.foveicollis spermatozoa was long and strongly attached in sperm head. In A.nigripennis appeared short and weakly breakup from the sperm head. The length of sperm heads was not always proportional to the total lengths of the sperm. The similarity of the A.foveicollis and A.nigripennis sperms appear rope like structure and the characters with those of Coleopteran indicates a clear phylogenetic relationship of Chrysomeloidea.

Keywords: Sperm morphology, Aulacophora, Chrysomelidae, DAPI

1.INTRODUCTION

sperm morphology commonly observed, both within species In Coleoptera, spermatozoa are produced in the testes largely and within males (Morrow and Gage, 2000; Pitnick et al., during late larval and pupal stages, and the adult male 2009a). Consequently explaining the enormous variation emerges with a more or less complete complement of sperm. typically observe in sperm morphology is a major challenge However, before these sperm can be used, they must descend for evolutionary biologists (Morrow and Gage, 2001; Pitnick from the testes into and along the length of the male et al., 2009a; Fitzpatrick and Baer, 2011) reproductive tract. Prior to ejaculation, they are stored, the location of sperm storage within the tract varying among Spermatozoan morphology has been used to help answer species. At the time of mating, the sperm must be transferred some phylogenetic questions in Coleoptera (Quicke et al., into a spermatophore, formed during copulation by the male 1992; Jamieson et al., 1999). The sperm structure and within the female’s bursa copulatrix. spermatogenesis of thrips have been well studied recently

(Paccagnini et al., 2006, 2007, 2009). Sperm length can vary considerably, in some species sperms are relatively short while in other they can reach gigantic The morphology of the spermatozoa of various groups of proportions. Sperm and pollen morphology remains a Coleoptera has been described, providing useful conundrum for evolutionary biologist (Birkhead and Moller, information for phylogenetic studies. Burrini et al., 1998; Birkhead et al., 2009). The variation in sperm (1988), for instance, realized an extensive work with morphology is assumed to evolve in response to variation in spermatozoa of different curculionoid families similarly, postcopulatory sexual selection (Snook, 2005; Birkhead et al., (Baccetti and Daccordi, 1988) investigated sperm structure 2009) either through sperm competition (Parker, 1970). In in the family Chrysomelidae and (Dallai et al., 1998) contract, the evolutionary significance of the variation in described the sperm structure of Bruchidae, Chrysomelidae and several Curculionoids. However the *C orresponding author: Mr S.Sethuraman, Department of Zoology, morphological description of the spermatozoa of Annamalai University, Annamalai Nagar, 608 002,Tamil Nadu, India. Chrysomelidae remains practically unknown, limited to

1425

S.Sethuraman et al., 2016 only a short description by Baker, (2005) who examined diamidino-2-phenylindole (DAPI, 1 mg/mL distilled water) to the species Leptinotarsa decemlineata. The aim of the this permeabilization buffer. All nuclei label in cells that are present study is to describe the sperm morphology of the exposed to DAPI. Squashes of testes were mounted in 70% pumpkin beetles, Aulacophora foveicollis (Lucas) and glycerin (v/v) in 0.1 M Tris (pH 9.0). Specimens were Aulacophora nigripennis (Motulansky). imaged with a Cannon F600 using a combination of fluorescent and Sperm dimensions were measured using 2.MATERIALS AND METHODS Image Pro software (Nardi et al., 2013).

Field collection of beetles 3.RESULTS

The adult pumpkin beetles were collected in the vicinity of Measurement data of sperm cells in A.foveicollis

Annamalai university, Chidambaram, Tamil Nadu. Spermatozoa of A.foveicollis as seen under the Flurocense Collections were made in the early morning by hand picking microscope appear as rope like, long and thin with an overall because the beetles are very sluggish so all the are length 636±30.1µm (Table. 1; Fig.1). They are divided is to collected before 11a.m. The beetles were brought to the head and flagellum regions. Staining the sperm nucleus with laboratory and reared in plastics cages, having a dimension DAPI reveals the head piece which can otherwise hardly be 30X2 cm, at a laboratory temperature of 29 ± 1°C with 12 distinguished from the flagellum (Fig.2). The length of the hours light and 12 hours dark photoperiod. The floor of the sperm head is 30.8±2.3µm, the ratio of the total length and cage was covered with fine sand, moderately moistened with head length is ~32.1 (Table.1). Situated in front of the water daily in order to maintain the humidity of the cage. The nucleus there is the acrosome, the spermatozoa taken from the insects were fed daily with fresh leaves of testis are aggregated in bundles (Fig.3). The more than (Cucumis sativus), bottle gourd (Lagenaria siceraria), hundreds of sperms are arranged in a group of cells. The Water melon (Citrullus lanatus), Muskmelon (Cucumis flagellum is connected to the head portion of the sperm. The melo) and Round gourd belongs to same family sperm heads were exposed to DAPI stain. In single . The dissection made under binocular spermatozoa the sperm head exposed star like structure microscope because the is very tiny. (Fig.1).

Preparation of Sperm Whole Mounts Measurement data of sperm cells in A.nigripennis

Whole testes used for fluorescent labeling were always fixed Spermatozoa of A.nigripennis as seen under Flurocense in 100% methanol and stored at -200C until prepared as microscope appear like spiral, long and thin structure, with an squashes. From 100% methanol, tissues were transferred to overall length 355.1±21.3µm (Table.1; Fig.4). The DAPI 50% acetic acid for at least 30 min. After exposure to acetic staining reveals sperm nucleus and head piece which can be acid, testes were placed on a glass slide and a cover glass was distinguished from the flagellum (Fig.5). The length of the positioned over the testis. By applying gentle pressure to the sperm head is 11.3±1.1µm. The ratio of the total length and cover glass, cells of the testis were dispersed from a three- head length is ~25.1 (Table.1). The spermatozoa are arranged dimensional configuration into a two-dimensional array. in bundle and the single sperm head was exposed kite like Excess liquid was removed from the edge of the cover glass, structure (Fig.6). The flagellum directly connected at the and the slide was placed upside down on dry ice for at least sperm heads. However, the connection is not strong and two minutes. The cover glass was quickly flipped off the bundles released in buffer tend to break up when agitated by frozen and squashed testis with the edge of a razor blade. gently stirring the buffer. At higher magnification the sperm Tissue was further permeabilized for at least 30 min by tails reveals two major structures, one straightly blended and addition of a permeabilization buffer (PBS + 10% normal another one arranged in deeply coil (Fig.6). The goat serum + 0.1%Triton X-100). The cells were measurements of 4-10 times the sperm head and tail length subsequently stained by addition of a 1:1,000 dilution of 4',6- are shown in Table.1.

Table.1. Spermatozoan morphometric data of two species

Order Family Species Total length ±SD (µm) Head length ±SD (µm) Ratio of tail length: Head length

Coleoptera Chrysomelidae Aulacophora 636±30.1 (n=4) 30.8±2.3 (n=10) 32:1 foveicollis

Coleoptera Chrysomelidae Aulacophora 355.1±21.3 (n=8) 11.3±1.1 (n=7) 25:1 nigripennis

1426

Volume 4, Issue 12, pp 1425-1430, December, 2016

Figures. 1-3. Flurocense Microscopic view of the spermatozoa of A.foveicollis Sperm were measured whole mounts squashes prepared from testes. Nuclei have been labeled with the fluorescent probe DAPI. (1) Single sperm cell of A.foveicollis overall length 636±30.1µm. (2). The length of the sperm head is 30.8±2.3µm. (3). Spermatozoa taken from the testis are aggregated in bundles.

1427

S.Sethuraman et al., 2016

Figures. 4-6. Flurocense microscopic view of the spermatozoa of A.nigripennis. Sperm were measured whole mounts squashes prepared from testes. Nuclei have been labeled with the fluorescent probe DAPI. 4. Flurocense microscope appear A.nigripennis rope like, lo ng and thin structure with an overall length 355.1±21.3µm.5. Sperm head distinguished from the flagellum 6. Sperm head were exposed kite like structure.

1428

Volume 4, Issue 12, pp 1425-1430, December, 2016

4. DISCUSSION triangulum, Strongylogaster distans, Dolerus tejoniensis, Tenthredo xantha, Cimbex americanum reported by Nathan et A number of studies have shown sperm length to be al. (2001). The present observation indicate solid evidence positively associated with sperm competition intensity (Gage that variation in sperm length between the two Chrysomelidae 1994; Briskie et al., 1997; LaMunyon and Ward 1998, 1999, species, and they have to compare the sperm measurement and structure of present investigation as well as earlier 100µm 2002; Morrow and Gage 2000). However the present study demonstrated that the sperm head and tail length of pumpkin reports, have support for a phylogenetic relationship and beetle A.foveicollis overall length is 636±30.1µm and head transformation of species among the different families of length is 11.3±1.1µm. The ratio of the head length and tail Coleopterans.

length is 32.1. In A.nigripennis overall length 355.1±21.3µm 5.ACKNOWLEDGEMENTS and the head length is 11.3±1.1µm. The ratio of head length and tail length 25.1. Substantial variation in length of short The authors thankful to the UGC for funding the financial and long sperm were found across a large sample of both assistance and also thank to the SRM University, butterflies (short length 106-883µm and long length 345-1545 Kattankulathur for Flurocense image analysis. µm) and moth (short length 106-883 µm, long length 110- 12675 µm) (Morrow and Gage, 2000). Consequently, many 6.REFERENCE authors generally recognize two sperm length morphs. Drosophila in the observe group display more variation in the Baccetti, B., Daccordi, M., 1988. Sperm structure and length of the long sperm (139-1875 µm) than the short sperm phylogeny of the Chrysomelidae. In: Jolivet, P., morph (56-299µm) (Pasini et al., 1996). Many reports are Petitpierre, E., Hsiao, T.H. (Eds.), Biology of deals with histology and morphology of reproductive organs Chysomelidae. Kluwer Academic Press, Dordrecht. such as testes, vas deference and male accessory reproductive 357–378. glands. Morphology of spermatozoa in chrysomelidae no Baker, M.B., Andrei Alyokhin, Shana R. Dastur, Adam H. records are found. Porter, Anddavid N. Ferro. 2005. Sperm Precedence in Overwintered Colorado Potato Beetle (Coleoptera: Approximate ratios of sperm flagellar length to sperm head Chrysomelidae) and Its Implications for Insecticide length for all strepsipterans examined are all smaller than 10 Resistance Management. Ann. Entomol. Soc. Am. to 1: 5:1 reported in Halictophagus chilensis 98( 6): 989-995. (Carcupino et al., 1993), 3:1 in Xenos vesparum (Dallai et al., Bernasconi, G., Hellriegel B., Heyland A., Ward P.I. 2002. 2003), and 2:1 in Eoxenos laboulbenei (Nardi et al., 2013). Sperm survival in the female reproductive tract in the Each of these species represents a different family of fly Scathophaga Stercoraria (L.). Journal of Strepsiptera. H. chilensis and X. vesparum represent the Physiology. 48(2); 197-203. suborder Stylopidia, and E. laboulbenei represents one of the Birkhead, T.R., Hosken, D.J., Pitnick, S. 2009. Sperm two extant families in its suborder Mengenillidia. Flurecense Biology: An EvolutionaryPerspective. Burlington, MA: image analysis in other order of insects were recorded such as Academic Press. Albanycada albigera, Platypleura capensis, Azanocada Birkhead, T.R., Moller A.P., 1998. Sperm Competition and zuluensis, Platypleura hirtipennis with long and short sperm Sexual Selection. San Diego, CA: Academic Press. heads reported by Chawanji et al. (2007). Sperm survival in Briskie, J.V., Montgomerie, R. & Birkhead, T.R. 1997. The the female reproductive tract of Scathophaga stercoraria (L.) evolution of sperm size in birds. Evolution, 51, 937– reported by Bernasconi, (2002). Flurocense image of the 945. starvation on male insects in Tribolium castaneum reported Burrini, A.G., Magnano, L., Magnano, A.R., Scala, C., by Sonja et al. (2011); sperm head shaped and conjugation in Baccetti, B., 1988. Spermatozoa and phylogeny of diving beetles Lyius oblitus, Neoporus undulates, N.undulatus Curculionoidea (Coleoptera). Int. J. Insect Morphol. rouleau, Hydatius bimarginatus, Ilybius larsoni, Derovatellus Embryol. 17:1–50 bimarginatus reported by Higgison et al. (2011). The types of Carcupino, M.; Mazzini, M.; Olmi, M.; Kathirithamby, J. sperm conjugates with greater total length than the sperm they 1993. The spermatozoon of Halictophagus chilensis contain up to three times longer in Graphoderus liberus, Hofmann (Strepsiptera, Halictophagidae). Boll. Zool. Ilybius oblitus, Uvarus lacustris, Neoporus undulates, 60, 361±365 Hygrotus sayi reported by Higgison et al. (2012). In the Chawanji, A.S., Hodgson, N.A., Villet, M.H., Sanbornand, present study demonstrated that overall sperm length is short A.F. and Phillips, P.K. 2007. Spermiogenesis in three in A.nigripennis (355.1±21.3) compared to A.foveicollis species of cicadas (Heteroptera: Cicadidae). Acta Zool. (636±30.7) and the flagellum is distinguished from the head 88,337-348. of the sperm cells. Flagellums are strongly connected with Dallai, R., Afzelius, B.A., Lupetti, P., Osella, G., 1998. sperm head in A.foveicollis. In A.nigripennis the flagellum is Sperm structure of some Curculionoidea and their weakly breakup from the sperm head. Almost similar type of relationship with Chrysomeloidea. Mus. Reg. Sci. results was observed by Nardi et al. (2013) in different group Nat. Torino 16,27–50 of Coleopteran insects, sperm overall length such as (Zonitis Dallai, R.; Beani, L.; Kathirithamby, J.; Lupetti, P.; Afzelius, flava (365.1±22.3), ~Hyleus scutellatus (362.5±13.4), Mylaris B.A. 2003. New findings on sperm ultrastructure of Variabilis (603.3±77.1), and Macrosiagon triscuspidata Xenos vesparum(Rossi) (Strepsiptera, Insecta).Tissue (1,236.3±38.1) and also noted, a similar hierarchy of ratios of Cell. 35, 19-27. length of sperm flagellum and length of sperm head ~27:1, Fitzpatrickand Boris Baer, J.L. 2011. Polyandry reduces ~19:1, ~19:1 and ~ 37:1 respectively. The spermatozoa are sperm length variation in social insects. The Society for DAPI stained in the sawflies of Acordulecera sp, Trichiosoma the Study of Evolution. Evolution 65-10: 3006–3012.

1429

S.Sethuraman et al., 2016

Gage, M.J.G. (1994) Associations between body size, mating Nathan Schiff, Anthony flemminc J., and Donald L., Quicke pattern, testis size and sperm lengths across butterflies. J. 2001. Spermatodesmata of the sawflies Proceedings of the Royal Society of London, Series B, (Hymenoptera: Symphyta): Evidence for multiple Biological Sciences, 258, 247–254 increases in sperm bundle size. Journal of hymenoptera Higginson M.D., Kelly Miller, Kari segraves A., and Scott research. 10(2); 119-125. pitnick. 2012. Female reproductive tract form drives the Paccagnini, E., De Marzo, L., Giusti, F., Dallai, R., 2006. The evolution of complex sperm morphology. Journal of aberrant spermatogenesis of the Haplothrips simplex PNAS. 109(12); 4538-4543 (Buffa) (Thysanoptera): ultrastructural study. Tissue Higginson, M.D., Kelly Miller, Kari segraves A., and Scott Cell. 38, 177–180. pitnick. 2011. Convergence, Recurrence and Paccagnini, E., Lupetti, P., Afzelius, B.A., Dallai, R., 2009. diversification of complex sperm traits in diving beetles New findings on sperm ultrastructure in thrips (DYTISCIDAE). The society for the study of evolution. (Thysanoptera, Insecta). Struct. Develop. 66 (5); 1650-1661. 38,70–83. Jamieson, B.G.M., Dallai, R., Afzelius, B.A., 1999. Insects: Paccagnini, E., Mencarelli, C., Mercati, D., Afzelius, B.A., Their Spermatozoa and Phylogeny. Science Publishers, Dallai, R., 2007. Ultrastructural analysis of the aberrant Enfield, NH, p. 555. axoneme morphogenesis in thrips (Thysanoptera, LaMunyon, C.W. & Ward, S. 1998. Larger sperm outcompete Insecta). Cell Motil. Cytoskeleton 64, 645–661. smaller sperm in the nematode Caenorhabditis elegans. Parker, G.A. 1970. Sperm competition and its evolutionary Proceedings of the Royal Society of London, Series B, consequences in the insects. Biol Rev. 45:525 – 567 Biological Sciences, 265, 1997–2002 Pasini, M.E., Redi, C.A., Cavaglia, O. and Perotti M.E. 1996. LaMunyon, C.W. & Ward, S. 1999. Evolution of sperm size Ultrastructural and cytochemical analysis of sperm in nematodes: sperm competition favours larger sperm. dimorphism in Drosophila subobscura. Tissue Cell 28: Proceedings of the Royal Society of London, Series B, 165–175. Biological Sciences, 266, 263–267. Pitnick, S., Hosken, D.J., Birkhead, T.R. (2009a). Sperm LaMunyon, C.W. & Ward, S. 2002. Evolution of larger sperm morphological diversity. In: Birkhead TR, Hosken DJ, in response to experimentally increased sperm Pitnick S (eds) Sperm biology: an evolutionary competition in Caenorhabditis elegans. Proceedings of perspective. Academic, Amsterdam, 69–149. the Royal Society of London, Series B, Biological Quicke, D.J.L., Ingram, S.N., Baillie, H.S., Gaitens, P.V., Sciences, 269, 1125 –1128 1992. Sperm structure and ultra structure in the Morrow, E.H. and Gage, M.J.G. 2000. The evolution of Hymenoptera (Insecta). Zool. Scr. 21, 381– 402. sperm length in moths.Proceedings of the Royal Society Snook, R.R. 2005. Sperm in competition: not playing by of London, Series B, Biological Sciences, 267, 307–313 the numbers.Trends Ecol Evol. 20(1):46-53. Nardi, B.J., Juan Delgado A., Francisco Collantes, Lou Ann Sonja Sbilordo, H., Vera grazer M., Marcodemont and Oliver Miller, Charles Bee M.B., and Jeyaraney martin Y. 2011. Impacts of starvation on male Karthirithamby. 2013. Sperm cells of a Promitive reproductive success in Tribolium castaneum. Strepsiteran. Insects 4, 463-473. Evolutionary Ecology Research 13: 347-359.

*****

1430