Sexual dimorphism in development and venom production of the insular threatened pit viper Bothrops insularis (Serpentes: Viperidae) of Queimada Grande Island, Brazil
Silvia Regina Travaglia-Cardoso 1, André Zelanis 2 & Maria de Fátima Domingues Furtado 3
1,3 Laboratório de Herpetologia, Instituto Butantan - SP, Av. Vital Brazil, 1500, 05503-900, São Paulo-SP, Brazil
2 Laboratório Especial de Toxinologia Aplicada (CAT/CEPID), Instituto Butantan-SP, Brazil
Email: 1 email@example.com, 2 firstname.lastname@example.org, 3 email@example.com
Sexual dimorphism is an important morphological feature among snakes, often related to differences in ecology and behavior between males and females in many families (Shine 1994), including differences in vulnerability to predation, choice of prey items, and reproductive success (Shine 1994). Sexual dimorphism may vary in intensity during an animal’s ontogeny (Beaupre et al. 1998) and may appear in neonates or only be expressed after sexual maturity (King et al. 1999). In snakes sexual dimorphism may be apparent in such characteristics as length and mass of the body (Shine 1990; Forsman 1991; Madsen & Shine 1993; King et al. 1999; Bertona & Chiaraviglio 2003), tail length (King et al. 1999) length and shape of the head (Shine 1990; King et al. 1999; Rodriguez-Robles 2002). These morphometric variations are usually related to sexual and ontogenetic variations in diet and may strongly influence the snake’s reproductive strategies (Vanzolini 1986; Mushinsky 1987; Shine 1988; Sazima 1992; Marques et al. 2002; Shine et al. 2002; Shine 2003).
Bothrops insularis (Image 1) is a Critically Endangered snake endemic to Queimada Grande Island, southern coast of São Paulo State, Brazil (Marques et al. 2004). This species was first described by Afrânio do Amaral (Amaral 1921) as having peculiar characteristics such as a semi-arboreal habit, and both diurnal and nocturnal activity. The diet is composed of migratory birds and occasionally lizards. Chilopods are food items of juvenile snakes (Hoge et al. 1959; Martins et al. 2002). Hoge et al. (1959) showed sexual abnormalities within the B. insularis population, with true females, true males and intersexes all occurring. Intersexes are genetically females, with functional ovaries, but with a unilateral or bilateral non-functional hemipenis (Hoge et al. 1959). According to cytogenetic studies made by Beçak et al. (1990) this phenomenon could be due either to inbreeding or to mutations. Recently, studies concerning several aspects of the reproductive biology of B. insularis showed that both females and intersexes have hemipenis retractor muscles, and now are designated as “females” (Kasperoviczus 2009).
To date, studies on this species mainly have dealt with biological/ecolocical characteristics (Hoge et al. 1959; De Biasi et al. 1986; Federsoni et al. 1986 a,b; Duarte et al. 1995; Martins et al. 2002; Duarte & Garrubo 2003), systematics (Salomão et al. 1999; Martins et al. 2001; Wuster et al. 2005), with few data available on reproductive strategies (Almeida-Santos & Salomão 2002) or sexual dimorphism (Hoge et al. 1959), and none related to developmental characteristics. The lack of such data denotes the importance of studies on this species, which has been designated as Critically Endangered (CR) in the IUCN Red List of Threatened Species (Marques et al 2004). Martins et al. (2008), showed a decrease in the population size of B. insularis at Queimada Grande Island, a main factor reinforcing the need for conservation efforts. This work provides biological data on sexual dimorphism in the development and venom production of B. insularis specimens in captivity.
Materials and methods
Queimada Grande Island is located approximately 34km from the southern coastline of São Paulo State (24029’S & 46041’W). It has been proposed that this island was isolated from the mainland about 11,000 years ago, after the last glaciation and elevation of sea level during the late Pleistocene (Vanzolini 1973). The Island is not inhabited by humans and comprises a total area of 430,000m2, with the highest point at 200m. There are no sandy beaches and the vegetation is of the general Atlantic Forest pattern, with grasslands and bushy areas (Hoge 1959; Vanzolini 1973). The density of snakes is one of the highest in the world; the species represented are the endemic pit viper Bothrops insularis (Amaral 1921; Hoge et al. 1959; Duarte et al. 1995) and Dipsas albifrons cavalheiroi (Duarte et al. 1995).
Snakes and their maintenance in captivity
Gravid Bothrops insularis specimens were collected on Queimada Grande Island - SP and three clutches were obtained. Snout vent length (SVL), tail length (TL), and body mass was measured and sex determined. The animals were maintained at the Herpetology Laboratory of Instituto Butantan, São Paulo. They were placed in individual cages, in rooms with controlled temperature (24 ± 3 0C). All the animals received mice (Mus musculus) and rats (Rattus norvegicus) for food, fortnightly, until 12 months of age, and thereafter at monthly intervals. Mice of different weights were fed to the snakes, according to the snakes’ length.
The small number of specimens used is because. B. insularis is difficult to obtain and to maintain in captivity. This Critically Endangered snake has small litter sizes (mean 6.5) (Hoge et al. 1959). Nevertheless, the results demonstrated important ontogenetic and individual shifts.
Biometric data and extraction of venom
Snout-vent length (SVL), tail length (TL) and mass were obtained over intervals of three months at which time venom was extracted from individiual snakes. All the correlation data were obtained taking into account the multiple measurements on the same animal at different ages. After extraction the venom was lyophilized and weighted.
Biometric data and development in captivity
Twelve animals (6 males and 6 females) from three litters were born in summer (February and March). Masses of the neonates varied from 7.8 to 10 g, and SVL varied from 205 to 235 mm. Four animals that reached adulthood and which were analyzed in this paper, were named “male”, “female 1”, “female 2” and “female 3”. The neonates did not show significant differences of mass, SVL or TL. The analysis of the specimens that reached adulthood showed that females had higher SVL and mass than did males, although the latter had higher relative tail length. The animals showed similar patterns of growth (mass and length) up to two years of age, after which males and females diverged (Fig. 1A). Females showed an accelerated growth up to four years of age and they kept this pattern until they reached their seventh year; on the other hand; the male continued to grow up to four years of age but thereafter showed no significant changes in body size. The patterns for mass were similar, with females becoming heavier over time up to seven years but males stabilizing weight after four years of age (Fig. 1B).
Taking into account the multiple measurements on the same animal at different ages, neonates did not show any correlation between TL and SVL or between TL and mass. On the other hand, among adults there was a strong correlation between TL and SVL (r2 = 0.97 for males, and r2 = 0.96, r2 = 0.98 and r2 = 0.99, for females 1, 2 and 3, respectively) (Fig. 2A, Table 1). Correlation between mass and total length was found both for the male (r2 = 0.90), and for the females (r2 = 0.85; 0.85 and 0.86, respectively) (Fig. 2B, Table 1).
Head length was not followed during animal’s ontogeny, but in adults, females had larger relative length of the heads in comparison to the male (females: 3.2 to 3.8% of body length and male 3.6% of body length).
We verified significantly larger amounts of venom produced by females in comparison to males of the same age. Venom yield (here shown as the median per year) diverged between the sexes after two years of age, with females having a higher production of venom in comparison to the male. At the third year females’ venom yield varied (73.9 to 94.3 mg) and the male’s yield was 34.1mg. This difference becomes increasingly significant, until at the age of seven years female 2 showed a median venom yield of 334.5mg, while that of the male was only 51.9 mg (Fig. 3). Taking into account the multiple measurements on the same animal at different ages, venom yield and TL were correlated in females (r2 = 0.81; r2 = 0.71 and r2 = 0.81), but only weak so in the male (r2 = 0.33) (Fig.4A, Table 1). The same results were found in relation to venom yield and body mass, in which a strong correlation was observed for females (r2 = 0.90; r2 = 0.91 and r2 = 0.90), and a weak one for the male (r2 = 0.43) (Fig. 4B, Table 1).
Almeida-Santos, S.M. & M.G. Salomão (2002). Reproduction in neotropical pitvipers, with emphasis on species of the genus Bothrops, pp. 445-462. In: Schuett, G.W. M. Hoggren, M.E. Douglas & H.W. Greene (eds.). Biology of The Vipers. Eagle Mountain Publishing, Carmel, Indiana USA, 580pp.
Almeida, M.T. & M. Martins (1999). Historia natural de Bothrops neuwiedi pubescens (Serpentes, Viperidae). Abstracts of the 5th Congress of Herpetology of Latin American – Uruguay, 26pp.
Amaral, A. (1921). Contribuição para o conhecimento dos ofídeos do Brasil. A Parte I. Biologia de uma nova espécie, Lachesis insularis. Anexos Memória do Instituto Butantan 1 (1): 18-37.
Beaupre, S.J., D. Duvall & J. O’Leile (1998). Ontogenetic variation in growth and sexual size dimorphism in a Central Arizona population of the Western Diamondback Rattlesnake (Crotalus atrox). Copeia 1998: 40-47.
Beçak, M.L., M.N. Rabello-Gay, W. Beçak, M. Soma, R.F. Batistic & I. Trajtengertz (1990). The W chromossome during the evolution and in sex abnormalities of snakes. DNA content, C-banding, pp. 221-240. In: Olmo, E. (ed). Cytogenetics of Amphibians and Reptiles. Birkhauser Verlag, Basel.
Bertona, M. & M. Chiaraviglio (2003). Reproductive biology, mating aggregations, and sexual dimorphism of the argentine Boa constrictor (Boa constrictor occidentalis). Journal of Herpetology 37(3): 510-516.
Cundal, D. (2002). Envenomation strategies, head form, and feeding ecology in vipers, pp. 149-161. In: Schuett, G.W. M. Hoggren, M.E. Douglas & H.W. Greene (eds.). Biology of The Vipers. Eagle Mountain Publishing, Carmel, Indiana USA, 580pp.
De Biasi, P., P.A. Federsoni Jr, M.A. Buononato & G. Puorto (1986). Comportamento de Bothrops insularis (Amaral) – Ilha da Queimada Grande – São Paulo – Brasil (Serpentes-Viperidae-Crotalinae). Resumos do XIII Congresso Brasileiro de Zoologia – Cuiabá-MT.
Duarte, M.R. & P.S. Garrubo (2003). Bothrops insularis (Golden Lancehead). Diet. Herpetological Review 34(2): 148.
Duarte, M.R., G. Puorto & F.L. Franco (1995). A biological survey of the pitviper Bothrops insularis Amaral (Serpentes, Viperidae): an endemic and threatened offshore island snake of southeastern Brazil. Studies on Neotropical Fauna and Environment 30(1): 1-13.
Federsoni Jr, P.A., M.A. Buonoato, N. Vitiello & E. Zolcsak (1986a). Observações sobre alimentação de ninhadas de Bothrops insularis, no biotério do Museu do Instituto Butantan (Serpentes-Viperidae-Crotalinae). Resumos do XIII Congresso Brasileiro de Zoologia – Cuiabá-MT: 138.
Federsoni Jr, P.A., M.A. Buonoato, G. Puorto & P. De Biasi (1986b). Exame de conteúdo estomacal de um jovem (Serpentes-Viperidae-Crotalinae). Resumos do XIII Congresso Brasileiro de Zoologia – Cuiabá-MT: 138.
Forsman, A. (1991). Variation in sexual size dimorphism and maximum body size among adder populations: effects of prey size. Journal of Animal Ecology 60: 253-267.
Forsman, A. (1994). Growth rate and survival in relation to relative head size in Vipera berus.Journal of Herpetology 28(2): 231-238.
Furtado, M.F.D., S.R.T. Travaglia-Cardoso & M.M.T. Rocha (2006). Sexual dimorphism in venom of Bothrops jararaca (Serpentes: Viperidae). Toxicon 48: 401-410.
Hoge, A.R., H.E. Belluomini, G. Schreiber & A.M. Penha (1959). Sexual abnormalities in Bothrops insularis (Amaral) 1921 (Serpentes). Mem.Inst. Butantan 29: 17-88.
Holycross, A.T. & S.P. Mackessy (2002). Variation in the diet of Sistrurus catenatus (Massasauga), with emphasis on Sistrurus catenatus edwardsii (Desert Massasauga). Journal of Herpetology 36(3): 454-464.
Kasperoviczus, K.N. (2009). Biologia reprodutiva da jararaca ilhoa, Bothrops insularis (Serpentes:Viperidae) da Ilha da Queimada Grande, São Paulo. Msc Thesis. Biotechnology and Biodiversity Program (USP/Butantan Institute/IPT), São Paulo University, 124pp.
King, R.B., T.D. Bittner, A. Queral-Regil & J.H. Cline (1999). Sexual dimorphism in neonate and adult snakes. Journal Zool. London 247: 19-28.
Lillywhite, H.B. (1987). Circulatory adaptations of snakes to gravity. Animal Zoology 27: 81-95.
Martins, M., O.A.V. Marques & I. Sazima (2002). Ecological and phylogenetic correlates of feeding habits in neotropical pitvipers of the genus Bothrops, pp. 307-328. In: Schuett, G.W. M. Hoggren, M.E. Douglas & H.W. Greene (eds.). Biology of The Vipers. Eagle Mountain Publishing, Carmel, Indiana USA, 580pp.
Martins, M., R.J. Sawaya, O.A.V. Marques (2008). A first estimate of the population of the critically endangered lancehead, Bothrops insularis. South American Journal Herpetology 3: 168-174.
Mackessy, S.P., K. Williams & K.G. Ashton (2003). Ontogenetic variation in venom composition and diet of Crotalus oreganos concolor. A case of venom paedomorphosis? Copeia 2003: 769-782.
Mackessy, S.P., N.M. Sixberry, W.H. Heyborne & T. Fritts (2006). Venom of the brown treesnake, Boiga irregularis: ontogenetic shifts and taxa-specific toxicity. Toxicon 47: 537-548.
Madsen, T. & R. Shine (1993). Phenotipic plasticity in body sizes and sexual dimorphism in european grass snakes. Evolution 46(1): 321-325.
Marques O.A.V., M. Martins & I. Sazima (2002). A new insular species of pitviper from Brazil, with comments on evolutionary biology and conservation of the Bothrops jararaca group (Serpentes:Viperidae). Herpetologica 58: 303-312.
Marques O.A.V., M. Martins & I. Sazima (2004). Bothrops insularis, IUCN 2004. 2004 IUCN Red List of Threatened Species. <www.iucnredlist.org>. Downloaded on 23 September 2010.
Martins, M., M.S. Araujo, R.J. Sawaia & R. Nunes (2001). Diversity and evolution of macrohabitat use, body size and morphology in a monophyletic group of Neotropical pitvipers (Bothrops). Journal of Zoology, London 254: 529-538.
Menezes, M.C., M.F.D. Furtado, S.R. Travaglia-Cardoso, A.C.M. Camargo & S.M.T. Serrano (2006). Sex-based individual variation of snake venom proteome among eighteen Bothrops jararaca siblings. Toxicon 47: 304-312.
Mushinsky, H.R. (1987). Foraging Ecology, pp. 302-334. In: Seigel, R.A., J.T. Collins & S.S. Novak (eds.). Snakes: Ecology and Evolutionary Biology. MacMillan Publishing Company, New York, 529pp.
Nogueira, C., R.J. Sawaya & M. Martins (2003). Ecology of the Pitviper, Bothrops moojeni, in the Brazilian Cerrado. Journal of Herpetology 37: 653-659.
Rodriguez–Robles, J.A. (2002). Feeding ecology of North American gopher snakes (Pituophis catenifer, Colubridae). Biological Journal of the Linnean Society 77: 165-183.
Salomão, M.G., W. Wüster & R.S. Thorpe (1999). MtDNA Phylogeny of neotropical pitvipers of the genus Bothrops (Squamata: Serpentes: Viperidae). Kaupia 8:127-134.
Sazima, I. (1992). Natural history of the jararaca pitviper, Bothrops jararaca, in southeastern Brazil, pp. 199-216. In: Campbell, J.A. & E.D. Brodie (eds.). Biology of the Pitvipers. Selva Publishing, Tyler, Texas,USA, 467pp.
Seigel, R.A. & N.B. Ford (1987). Reproductive ecology, pp. 210-252. In: Seigel, R.A., J.T. Collins & S.S. Novak (eds.). Snakes: Ecology and Evolutionary Biology. MacMillan Publishing Company, New York, 529pp.
Shine, R. (1988). Constraints on reproductive investment: a comparison between aquatic and terrestrial snakes. Evolution 42: 17-27.
Shine, R. (1990). Proximate determination of sexual differences in adult body size. American Naturalist 135(2): 278-283.
Shine, R. (1991). Intersexual dietary divergence and the evolution of sexual dimorphism in snakes. American Naturalist 138: 103-122.
Shine, R. (1993). Sexual dimorphism in snakes, pp. 49-86. In: Seigel, R.A. & J.T. Collins (eds.). Snakes: Ecology & Behavior. McGraw-Hill, New York, USA, 414pp.
Shine, R. (1994). Sexual size dimorphism in snakes revisited. Copeia 1994: 326-346.
Shine, R. (2003). Reproductive strategies in snakes. Proceedings of the Royal Society of London, Series B. 270: 995-1004.
Shine, R., L. Sun, M. Kearney & M. Fitzgerald (2002). Why do juvenile chinese pit-vipers (Gloydius shedaoensis) select arboreal ambush sites?. Ethology 108: 897-910.
Vanzolini, P.E. (1973). Distribution and differentiation of animals along the coast and continental island of the state of São Paulo, Brasil I. Introduction to the area and problems. Papéis Avulsos de Zoologia 16: 281-294.
Vanzolini, P.E. (1986). Levantamento herpetológico da área do Estado de Rondônia sob a influência da rodovia BR 364. Polonoroeste. Ecologia Animal. DF, Rel. Pesq. 1, CNPQ, 50pp.
Vanzolini, P.E. (1991). A biometrical note on Bothrops moojeni Hoge, 1966 (Serpentes:Viperidae). Anais Academia Brasileira de Ciências 63(4): 389- 401.
Vincent, S.E., A. Herrel & D.J. Irschick (2004). Ontogeny of intersexual head shape and prey selection in the pitviper Agkistrodon piscivorus. Biological Journal of the Linnean Society 81: 151-159.
Wüster, W., M.R. Duarte & M.G. Salomão (2005). Morphological correlates of incipient arboreality and ornithophagy in island pitvipers, and the phylogenetic position of Bothrops insularis. Journal of Zoology, London 266: 1-10.
Zelanis, A., J.S. Ventura, A.M. Chudzinski-Tavassi & M.F.D. Furtado (2007). Variability in expression of Bothrops insularis snake venom proteases: An ontogenetic approach. Comparative Biochemistry and Physiology 145: 601-609.