Herpetologica. 55(2), 1999, 200-204@ 1999 hy The Herpetologists' League, Inc.

RELATIONSHIP OF VENOM ONTOGENY ANDDIET 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 jararacaand 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 findingis consistent with the feeding habits of these two species, because juveniles of B. jararaca feedmainly on small anurans and lizards, shifting to endothermic prey at maturity, whereas B. altematuspreys mainly on endotherms throughout its life. Venom toxicity in endotherms was higher for adultsof B. jararaca compared to juveniles, a feature not observed for B. altematus. It is proposed thatprey 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 largeand bulky prey. The importance of adult venoms in prey digestion is reflected in their higherproteolytic activity.

Key words: Bothrops; Electrophoresis; Venom ontogeny; Venom specificity; Viperidae

SNAKESare strictly camivorous and al-ways ingest their prey whole, and for manyspecies 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 anontogenetic shift in diet (Mushinsky,1987). In a number of snakes, such dietaryshifts are characterized by juveniles prey-ing primarily upon ectotherms while adultsfeed 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 parenteralvenom injection (Greene, 1992). Viperidvenoms are among the most complex andvariable 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 relatedto differences between the feeding habitsof juvenile and adult snakes (Gans and EI-liot, 1968; Sazima, 1991). In this study, wetested 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 dietduring growth (Martins and Gordo, 1993;Sazima, 1991, 1992; Sexton, 1956-1957)and that feed mainly on endothermsthroughout their life (Andrade, 1995; M.Martins, personal communication), mak-ing it possible to examine the relationshipbetween the ontogeny of venom and diet.

To evaluate the possible correlations be-tween venom toxicity and prey type, wetested venoms of juveniles and adults ofBothrops for their effectiveness in frogsand mice, which represent ectothermicand endothermic prey, respectively.Among Bothrops exhibiting ontogeneticshifts in diet, we studied the venom of B.

jararaca, a medium-sized (approximately100 cm snout-vent length: SVL), relativelyslender pitviper widespread in southeast-em Brazil (see Sazima, 1992, for naturalhistory). Among Bothrops that feed on en-dotherms throughout life, we investigatedthe venom of B. altematus, a large, heavy-

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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 eolleetedat 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. jararacaand B. altematus were obtained mainlyfrom 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 (>80em SVL). These age classes refleet ap-proximately the size at which diet ehangesin this speeies, with juveniles feedingmainly on eetotherms, adults on endo-therms, and sub-adults making the transi-tion between both diets (Andrade et al.,1996). For eomparative purposes, thesame size-based separation was applied toB. altematus, regardless that its diet doesnot ehange.

Venoms were extraeted manually,pooled, and immediately vaeuum driedand stored at -20 C. Storage never exeeed10 mo after eolleetion. For alI toxicitytests, venom solutions were prepared im-mediately before use.

Toxicity TestsWe determined lethal toxicity of the

venoms in outbred Swiss-Webster miee(18-22 g) and in juvenile bullfrogs (Ranacatesbeiana, 5-10 g). We used five doselevels in eaeh toxieity assay. Dried venomwas diluted in 0.9% saline and injeeted in-traperitoneally (i.p.) in six animals at eaehdose leveI. The volume injeeted was 0.5 mlfor miee, but in frogs it was adjusted toindividual masses (0.025 ml of venom so-lution for 1 g of frog) due to their largerrange of masses. Toxieity was expressed aslethal dose 50% (LDso) estimated usingthe probit analysis following Finney(1971). We eonsidered mortality reeordedup to 48 h treatment for miee and 72 hfor frogs. AlI experiments with frogs wereperformed in a elimatie ehamber at 25 C

TABLE 1.-Toxicity (LD.;o) of the venoms of adultsand juveniles of Bothrops jararaca and B. altematusin 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.jararacaand B. altematus as well as juveniles of B.altematus did not differ markedly in theirLDso 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 wasabout 3.4 times more lethal than that ofjuveniles, whereas for B. altematus, thetoxieity of the venoms was similar in ju-veniles and adults.

D1SCUSSION

Toxieity data indieate that the venom ofjuveniles of B. jararaca is especially effi-eient on anurans. As the snake grows andits diet ehanges, the venom of this speciesloses about 70% of its toxieity upon thepreferred prey of the juvenile phase. Sim-ilarly, in B. moojeni, which also ehanges itsdiet from eetotherms to endotherms dur-ing ontogeny, the toxicity of the juvenilevenom is about 86% higher for eetothermsthan that of adults (Andrade et al., 1996).Thus, in B. jararaca (this study), B. moo-

jeni (Andrade et al., 1996), and possiblyother species of Bothrops that possess anontogenetie shift in diet, the toxieity of thevenom of juveniles eorrelates with theirfeeding 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 thispattem 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 adultsof B. jararaca is 2.3 times higher than thatof juveniles, reflecting a diet change to en-dothermic prey. For B. altematus, whichfeeds on endotherms both as juvenile andadult, the venom of juveniles is expectedto have a higher toxicity on endotherms tocompensate for its smaller volume. How-ever, we found no difference in the toxicityof juvenile venom from B. altematus andthat of adults. Therefore, a clear relation-ship between venom toxicity in endo-therms and feeding habits is not apparentin the species of Bothrops presently stud-ied. In general, the amount of venomspent by a snake to capture an endother-mie prey item is small (Hayes, 1991; Hayeset al., 1995) compared to the quantity pro-duced by an adult or even a juvenile snake(Furtado et al., 1991). Thus, it is possiblethat, in endotherms, a clear adaptive re-lationship between the prey capture roleof the venom and diet may be disguisedby functions other than prey killing. In thisregard, altemative functions for venom(discussed below) may acquire increasingimportance as the snake grows.

Juvenile snakes are constrained by bodysize to feed on small prey (in absoluteterms), which are not as difficult to digestas 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 juvenilesnakes are thought to be under strong se-lective pressure to ensure prey capture(Mackessy, 1988), which may explain thehigh toxicity of juvenile venoms of B. ja-raraca (this study) and B. moojeni (Andra-de et al., 1996) on anurans. In the case ofjuveniles of B. altematus, a high toxicspecificity on their preferred prey could beredundant, because the venom already hasa 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 tovolume ratio of the prey (Pough, 1983;Pough and Groves, 1983). At this point,the selective pressures acting on venomcomposition may shift, and its function inprey digestion may become increasinglyimportant. 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 thevenom is injected into the prey's body,higher proteolytic activity associated withinjection 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). Enhancementof prey digestion may reduce time and en-ergy allocation, shortening the period inwhich snakes have their capacity for loco-motion and defense decreased (Ford andShuttlesworth, 1986), and lowering thehigh cost of prey digestion observed forsit-and-wait foraging snakes such as Both-rops (Andrade et al., 1997). Therefore,venom changes in Bothrops are consistentwith an optimization of the venom actiontowards specific prey at specific stages oflife. It appears that venom from Bothropsis specialized to kill prey for juveniles, andto aid in prey digestion for adults. Thisconclusion 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 thevenoms of adults of B. moojeni (Andradeet al., 1996) and B. jararaca in endother-mic prey may also involve the prey capturerole of venom. Although an ontogeneticshift in diet is observed in both species,adults of B. jararaca have a specializeddiet preying exclusively upon endotherms(Sazima, 1992; Sazima and G. Puorto, un-published data), while adults of B. moojeniinclude a larger variety of prey items intheir diet, occasionally preying upon ec-totherms (A. S. Abe and P. R. Manzani,

June 1999] HERPETOLOGICA 203

unpublished data). Thus, the increasedtoxicity of adult venom of B. jararaca inmice may reflect, at least partially, the spe-cialized feeding habits of adults, while themore generalist adults of B. rrwojeni havea venom able to act on a broader spectrumof prey, but with lower specificity.

Snake venoms have long been a subjectof great research interest; however, due totheir medical importance, emphasis hasbeen devoted mainly to the biochemicaland pharmacological properties of the ven-oms. The contribution of such approachesto the understanding of venom systemevolution and its relevance to snakes intheir natural environment is limited andmay even be misleading (Kardong, 1996).On the other hand, a few studies have ex-amined snake venoms in an ecologicallevo-lutionary framework seeking to understandthe functional significance of their com-plexity, diversity, and variability to snake'ssurvival. In this context, tests of venomtoxicity on specific prey should be consid-ered as a promising tool to examine fur-ther the biological role of venoms andtheir importance in the ecology and evo-lution of venomous snakes. Nonetheless, itis clear that snake venoms serve diverseroles (e.g., digestion, defense), such thatadaptive relationships wiil require an in-tegrated view of venom function.

Acknowledgments.-This study was a part of theM.Sc. thesis by D. V. Andrade, who thanks his thesiscommittee, L Sazima and M. F. D. Furtado, for theirvaluable suggestions. An early version of this manu-script was largely improved by the comments of M.Martins and L Sazima, both of whom also providedunpublished information about natural history ofBothrops. We thank R. Wirz and D. FontanelIo fromthe Instituto de Pesca for the donation of the froglets,J. Jim from the Centro de Estudo de Venenos e An-imais Peçonhentos (CEVAP), and W Femandes andM. F. D. Furtado from the Instituto Butantan forsnakes and venom donation. The first author was sup-ported by a Graduate Research Grant from Coorden-ação de Aperfeiçoamento de Pessoal de Nível Supe-rior (CAPES).

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Accepted: 15 March 1998Associate Editor: Allison Alberts