Herpetologica. 55(2), 1999, 200-204 @ 1999 hy The Herpetologists' League, Inc. RELATIONSHIP OF VENOM ONTOGENY AND DIET IN BOTHROPS DENIS V ANDRADE AND AUGUSTO S. ABE Departamento de Zoologia, Universidade Estadual Paulista, c. p. 199, 13506-900, Rio Claro, São Paulo, Brazil ABSTRACT: We studied ontogenetic changes in venom toxicity of the pitvipers Bothrops jararaca and B. altematus in order to evaluate the relationship between venom action and diet. Toxicity tests (LD",,) were performed for the venoms of adult and juvenile snakes on mice and bullfrog froglets, which represented endothermic and ectothermic prey respectively. The venom of juveniles of B. jararaca, but not of B. altematus, had a higher toxicity on anurans than that of adults. This finding is consistent with the feeding habits of these two species, because juveniles of B. jararaca feed mainly on small anurans and lizards, shifting to endothermic prey at maturity, whereas B. altematus preys mainly on endotherms throughout its life. Venom toxicity in endotherms was higher for adults of B. jararaca compared to juveniles, a feature not observed for B. altematus. It is proposed that prey death!immobilization is the main function of the venom of juvenile snakes. As the snake grows, the digestive role of venom may become increasingly important, because adults prey upon large and bulky prey. The importance of adult venoms in prey digestion is reflected in their higher proteolytic activity. Key words: Bothrops; Electrophoresis; Venom ontogeny; Venom specificity; Viperidae SNAKESare strictly camivorous and al- ways ingest their prey whole, and for many species feeding episodes occur sporadical- ly on relatively large animals (Greene, 1983, 1997). Juvenile and adult snakes of- ten show differences in body size and oth- er behavioral, morphological, and physio- logical characteristics that may lead to an ontogenetic shift in diet (Mushinsky, 1987). In a number of snakes, such dietary shifts are characterized by juveniles prey- ing primarily upon ectotherms while adults feed mainly on endotherms (Mushinsky, 1987; Saint-Girons, 1980; Sexton, 1956- 1957). Diet shifts are widespread among viper- ids, a group of snakes in which prey cap- ture is largely dependent on a parenteral venom injection (Greene, 1992). Viperid venoms are among the most complex and variable substances secreted by vertebrates (Gans and Elliot, 1968), varying specifical- ly (Bemadsky et al., 1986; Tan and Pon- nudurai, 1990a,b; Wüster and Thorpe, 1991), geographically (Daltry et al., 1996; Glenn et al., 1983; Jayanthi and Gowda, 1988), seasonally (Gubensek et al., 1974; Ishii et al., 1970), and ontogenetically (Dempfle et al., 1990; Lomonte et al., 1983; Meier, 1986). It has been proposed that ontogenetic variation could be related to differences between the feeding habits of juvenile and adult snakes (Gans and EI- liot, 1968; Sazima, 1991). In this study, we tested the association between venom on- togeny and diet for two species df the pit- viper genus Bothrops. This genus is com- posed of species that change their diet during growth (Martins and Gordo, 1993; Sazima, 1991, 1992; Sexton, 1956-1957) and that feed mainly on endotherms throughout their life (Andrade, 1995; M. Martins, personal communication), mak- ing it possible to examine the relationship between the ontogeny of venom and diet. To evaluate the possible correlations be- tween venom toxicity and prey type, we tested venoms of juveniles and adults of Bothrops for their effectiveness in frogs and mice, which represent ectothermic and endothermic prey, respectively. Among Bothrops exhibiting ontogenetic shifts in diet, we studied the venom of B. jararaca, a medium-sized (approximately 100 cm snout-vent length: SVL), relatively slender pitviper widespread in southeast- em Brazil (see Sazima, 1992, for natural history). Among Bothrops that feed on en- dotherms throughout life, we investigated the venom of B. altematus, a large, heavy- 200 June 1999] HERPETOLOG1CA 201 bodied snake growing up to 160 em SVL (Andrade, 1995; Campbell and Lamar, 1989). MATER1ALS AND METHODS Snakes and Venom Samples Individuals of B. jararaca were eolleeted at several loealities in São Paulo State, southeastem BraziI. Speeimens of B. alter- natus were eolleeted in Botueatu, São Pau- lo. Venoms from juveniles of B. jararaca and B. altematus were obtained mainly from the offspring of females kept in eap- tivity. Venom samples of B.jararaca were eol- leeted from juveniles «45 em SVL), sub- adults (45-80 em SVL), and adults (>80 em SVL). These age classes refleet ap- proximately the size at which diet ehanges in this speeies, with juveniles feeding mainly on eetotherms, adults on endo- therms, and sub-adults making the transi- tion between both diets (Andrade et al., 1996). For eomparative purposes, the same size-based separation was applied to B. altematus, regardless that its diet does not ehange. Venoms were extraeted manually, pooled, and immediately vaeuum dried and stored at -20 C. Storage never exeeed 10 mo after eolleetion. For alI toxicity tests, venom solutions were prepared im- mediately before use. Toxicity Tests We determined lethal toxicity of the venoms in outbred Swiss-Webster miee (18-22 g) and in juvenile bullfrogs (Rana catesbeiana, 5-10 g). We used five dose levels in eaeh toxieity assay. Dried venom was diluted in 0.9% saline and injeeted in- traperitoneally (i.p.) in six animals at eaeh dose leveI. The volume injeeted was 0.5 ml for miee, but in frogs it was adjusted to individual masses (0.025 ml of venom so- lution for 1 g of frog) due to their larger range of masses. Toxieity was expressed as lethal dose 50% (LDso) estimated using the probit analysis following Finney (1971). We eonsidered mortality reeorded up to 48 h treatment for miee and 72 h for frogs. AlI experiments with frogs were performed in a elimatie ehamber at 25 C TABLE 1.-Toxicity (LD.;o) of the venoms of adults and juveniles of Bothrops jararaca and B. altematus in mice and frogs (mglkg); 95% confidence limits in parentheses. Mice Frogs Bothrops jararaca Juvenile venom 5.88 (4.6-7.31) 1.74 (1.26-2.2) 53.64 (44.04-65.99) 91.44 (73.lI-lI4.79) Adult venom Bothrops altematus Juvenile venom 4.54 (3.56-5.86) 4.69 (3.49-6.44) 79. II (65.03-99.2) 77.53 (63.73-97.22) Adult venom to prevent temperature effeets on meta- bolie rate (Witford, 1973) which eould af- feet the aetion of the venom. RESULTS Venoms from adults ofboth B.jararaca and B. altematus as well as juveniles of B. altematus did not differ markedly in their LDso values in frogs (Table 1). Conversely, the toxicity of juvenile venom from B. ja- raraca in frogs was nearly twiee that ob- served for the venom of adults. For mice, the venom of the adults of B. jararaca was about 3.4 times more lethal than that of juveniles, whereas for B. altematus, the toxieity of the venoms was similar in ju- veniles and adults. D1SCUSSION Toxieity data indieate that the venom of juveniles of B. jararaca is especially effi- eient on anurans. As the snake grows and its diet ehanges, the venom of this species loses about 70% of its toxieity upon the preferred prey of the juvenile phase. Sim- ilarly, in B. moojeni, which also ehanges its diet from eetotherms to endotherms dur- ing ontogeny, the toxicity of the juvenile venom is about 86% higher for eetotherms than that of adults (Andrade et al., 1996). Thus, in B. jararaca (this study), B. moo- jeni (Andrade et al., 1996), and possibly other species of Bothrops that possess an ontogenetie shift in diet, the toxieity of the venom of juveniles eorrelates with their feeding habits. In eontrast, in B. altema- tus, which feeds on endotherms at any 202 HERPETOLOGICA [VoZ. 55, No. 2 size, the venom of juveniles lacks an in- creased toxicity on ectothermic organisms. Further investigation may reveal that this pattem extends to other heavy-bodied spe- cies of Bothrops, like B. cotiara and B.fon- secai, possessing ontogenetically flxed di- ets. For miee, the venom toxicity of adults of B. jararaca is 2.3 times higher than that of juveniles, reflecting a diet change to en- dothermic prey. For B. altematus, which feeds on endotherms both as juvenile and adult, the venom of juveniles is expected to have a higher toxicity on endotherms to compensate for its smaller volume. How- ever, we found no difference in the toxicity of juvenile venom from B. altematus and that of adults. Therefore, a clear relation- ship between venom toxicity in endo- therms and feeding habits is not apparent in the species of Bothrops presently stud- ied. In general, the amount of venom spent by a snake to capture an endother- mie prey item is small (Hayes, 1991; Hayes et al., 1995) compared to the quantity pro- duced by an adult or even a juvenile snake (Furtado et al., 1991). Thus, it is possible that, in endotherms, a clear adaptive re- lationship between the prey capture role of the venom and diet may be disguised by functions other than prey killing. In this regard, altemative functions for venom (discussed below) may acquire increasing importance as the snake grows. Juvenile snakes are constrained by body size to feed on small prey (in absolute terms), which are not as difficult to digest as the larger prey of adults (Andrade et al., 1997). Juveniles, however, may have lim- ited resources to withstand long food dep- rivation periods, such as aestivation (Hirth, 1966). Therefore, venoms of juvenile snakes are thought to be under strong se- lective pressure to ensure prey capture (Mackessy, 1988), which may explain the high toxicity of juvenile venoms of B. ja- raraca (this study) and B. moojeni (Andra- de et al., 1996) on anurans. In the case of juveniles of B. altematus, a high toxic specificity on their preferred prey could be redundant, because the venom already has a high toxicity for endotherms. As the snakes grow, larger mammalian prey are taken (Greene, 1992; Sazima, 1992), and digestion becomes more diffi- cult due to the greatly reduced surface to volume ratio of the prey (Pough, 1983; Pough and Groves, 1983). At this point, the selective pressures acting on venom composition may shift, and its function in prey digestion may become increasingly important. Accordingly, increased prey di- gestive resistance leads to a concurrent in- crease in venom proteolytic activity (An- drade et al., 1996; Mackessy, 1988), a fea- ture also observed for B. jararaca and B. altematus (Furtado et al., 1991). As the venom is injected into the prey's body, higher proteolytic activity associated with injection of a greater volume (Hayes et al., 1995) could accelerate the rupture of tis- sues, increasing the prey surface area ex- posed to stomach acids and enzymes (Thomas and Pough, 1979). Enhancement of prey digestion may reduce time and en- ergy allocation, shortening the period in which snakes have their capacity for loco- motion and defense decreased (Ford and Shuttlesworth, 1986), and lowering the high cost of prey digestion observed for sit-and-wait foraging snakes such as Both- rops (Andrade et al., 1997). Therefore, venom changes in Bothrops are consistent with an optimization of the venom action towards specific prey at specific stages of life. It appears that venom from Bothrops is specialized to kill prey for juveniles, and to aid in prey digestion for adults. This conclusion reinforces the ecological inter- pretation of venom ontogeny first envis- aged by Kardong (1986) and further ex- perimentally confirmed by Mackessy (1988) in Crotalus viridis. The difference in toxicity between the venoms of adults of B. moojeni (Andrade et al., 1996) and B. jararaca in endother- mic prey may also involve the prey capture role of venom. Although an ontogenetic shift in diet is observed in both species, adults of B. jararaca have a specialized diet preying exclusively upon endotherms (Sazima, 1992; Sazima and G. Puorto, un- published data), while adults of B. moojeni include a larger variety of prey items in their diet, occasionally preying upon ec- totherms (A. S. Abe and P. R. Manzani, June 1999] HERPETOLOGICA 203 unpublished data). Thus, the increased toxicity of adult venom of B. jararaca in mice may reflect, at least partially, the spe- cialized feeding habits of adults, while the more generalist adults of B. rrwojeni have a venom able to act on a broader spectrum of prey, but with lower specificity. Snake venoms have long been a subject of great research interest; however, due to their medical importance, emphasis has been devoted mainly to the biochemical and pharmacological properties of the ven- oms. The contribution of such approaches to the understanding of venom system evolution and its relevance to snakes in their natural environment is limited and may even be misleading (Kardong, 1996). On the other hand, a few studies have ex- amined snake venoms in an ecologicallevo- lutionary framework seeking to understand the functional significance of their com- plexity, diversity, and variability to snake's survival. 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