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Joumal of Huptlology, Vol.30,No. 2. pp. 285-288,1996Copyrighl 1996 Society for lhe 5tudy of Amphibians and Reptiles

Is the Venom Related to Diet and

Tail Colo r During Bothrops moojeniOntogeny?

DENIS v. ANDRADE, AUGUSTO S. ABEAND MARtA C. DOS

SANTOS, Department of Zoology, Universidade EstadualPaulista, c p 199, Rio Claro 13506-900, São Paulo, Brazil.

Snakes are strict1y carnivorous reptiles, and manyof them feed upon large prey which they swallowwhole. Juveniles and adults, however, may exhibit asignificant difference in body size, which can lead toan ontogenetic dietary shift (see Mushinsky, 1987).In this regard, many species of snakes feed on anuransand lizards while juveniles, and on birds and mam-maIs as adults (e.g., Sexton, 1956-1957; Saint Girons,1980). Moreover, juveniles of some snakes use theconspicuous tip of the tail to lure ectothermic prey(Greene and Campbell, 1972; Heatwole and Davison,1976), losing this feature with growth, when the dietchanges to endotherms (NeiU, 1960; Henderson, 1970;Murphy et aI., 1978; Jackson and Martin, 1980). Nev-ertheless, some sub-adult and adult snakes, alwaysmales, retain the conspicuous color of the tail (seeBurger and Smith, 1950).

The occurrence of ontogenetic changes in diet andcaudal luring are well documented for the familyViperidae, a group of snakes in which venom has animportant role in prey capture (e.g., Mushinsky, 1987;Meier and Stocker, 1991). Because venom propertiesmay vary ontogenetically (Fiero, 1972; Theakston andReid, 1978; Gutiérrez et aI., 1980; Lomonte et aI., 1983;Meier, 1986; Furtado et aI., 1991) it has been proposedthat such variation could be caused by differences inthe feeding habits of juveniles and adults (Gans andElliot, 1968; Sazima, 1991).

To evaluate the possible specificity between venomand prey, we investigated the toxicity of juvenile andadult Bothrops moojeni venom in frogs and mice, which

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TABLE1. Toxicity (LOso)of the venom of adult andjuvenile B. moojeni in mice and frogs (mg/kg). 95%Confidence limits in parenthesis.

represent the preferred prey of juveniles and adults,respectively. Bothropsmoojeniis a large neotropical pitviper, reaching 130 cm in snout-vent length, in cen-tral and southern Brazil (Campbell and Lamar, 1989).Although dietary information is scant, field data re-vealed amphibians and reptiles in gut content of ju-veniles, while mammals and birds are more oftenfound in adults (A. S. Abe and P. R. Manzani, unpubl.obs.). Furthermore, B. moojeni belongs to a genus inwhich an ontogenetic shift of diet from ectothermsto endotherms is well documented (e.g., Sexton, 1956-1957; Sazima, 1991; Sazima, 1992;Gasparini et aI., 1993;Martins and Gordo, 1993). Juvenile B. moojeni alsohave conspicuous tail tips (Leloup, 1975) employedto lure ectothermic prey (pers. obs.). Thus, the rela-tionship between venom composition, accessed byelectrophoresis, and change in tail color was also in-vestigated.

Snakes were collected at the municipalities of Ara-çatuba (21°38'5; 50"25'W) and Pereira Barreto (20"38'5;51"06'W) São Paulo, Brazil. Three size classes weredefined: juveniles «450 mm SVL), sub-adults (450-840 mm SVL), and adults (>840 mm SVL) (see Leloup,1975). Venoms of 48 juveniles and 8 adults were man-ual1y extracted, pooled, and immediately vacuum driedand stored at - 20 C for later use in the toxicity tests.For electrophoresis, fresh samples of individual ven-oms (four juveniles, six sub-adults, and four adults)were collected in glass capillaries and stored frozen.

Toxicity of venoms from juvenile and adult B. moo-jeni was determined in outbred Swiss-Webster mice(18-22 g) and juvenile bullfrogs (Rana catesbeiana, 5to 10 g). For mice, five dose levels (2.4, 3.6, 5.4, 8.1and 12.15 mg venom per kg body weight), were uti-lized in the toxicity tests, both for juvenile and adultvenoms. In frogs, the dose levels employed were: 17.5,26.25,39.37,59.06 and 88.59 mg venom per kg bodyweight for juvenile venoms, and 35, 49, 68.6, 96.04and 134.45 mg venom per kg body weight for venomsof adult snakes. Venom was diluted in 0.9% salineand injected intra-peritoneally in six animais at eachdose leveI. The volume injected was 0.5 ml for miceand from 0.125 to 0.25 ml for frogs, according to theirweight (0.025 ml of venom solution for 1 g of frog).Toxicity was expressed as lethal dose 50% (LOso) es-timated using the probit analysis following Finney(1971). We considered mortality recorded up to 48 hfollowing treatment for mice and 72 h for frogs. Theexperiments on frogs were done in a climatic chamberat 25 C to prevent temperature effects on metabolicrate (Witford, 1973) which could affect the action ofthe venom.

For electrophoresis 10 Jtl of venom solutions (10 Jtlof fresh individual venom per ml of deionized water)

was diluted in equal volume of 80 mM Tris Buffer,pH 7.5, and subjected to 10% SOS-PAGE (Laemmli,1970). Molecular weight calibration markers (MW 50S70L: SIGMA) were also run with the venom. Proteinswere stained with Comassie Blue and the gels de-stained with a solution containing 30%methanol, and7% acetic acid in distilled water. Electropherogrampatterns of the venoms were analyzed for snake size,sex, and tail color.

The results of toxicity tests are shown in Table 1.LOsovalues for adult and juvenile venoms were sim-ilar in mice and the adult/juvenile ratio was 1.06. Onthe other hand, a marked difference in LOso valueswas found between adult and juvenile venoms infrogs. The juvenile venom toxicity was near1y twicethat of adults with an adult/juvenile ratio of 1.86.Electropherograms revealed qualitative differences inthe venom of snakes of different sizes (see Furtado etaI., 1991). There was a trend towards reduction in thenumber of bands with an increase in body size, par-ticular1y bands of high molecular weight (above 66,000M. W.). Electropherograms of sub-adult venoms wererather variable and exhibited a transitional patternbetween juveniles and adults: some samples contain-ing fractions of juvenile venoms, while other elec-tropherograms were closer to the adult pattern.

The electrophoretic pattern of a venom was notassociated with either sex or tail color. The greatervariation of the pattern reported for sub-adult sam-pies is likely to be only due to transitional stage ofthis size group. Therefore, the electrophoretic changeof B. moojenivenom seems to be related only to thestage of development of the animal, and is indepen-dent of tail color. The 1055of high molecular weightfractions of the venom during ontogeny was previ-ously reported for others Bothropsspecies(Meier, 1986;Furtado et aI., 1991). In B. moojeni this feature wasrelated with the 1055 of high toxicity of venom onanurans. Therefore, high molecular weight fractionscould be responsible for the specific toxicity of ju-venile venom on anurans.

Bothropsmoojenihas a wide geographic distributionin a seasonal climate (Campbell and Lamar, 1989).Recruitment in this species occurs during the warmand rainy season (Leloup, 1975), which is also favor-able for breeding activities of tropical anurans (e.g.,Ouelman and Trueb, 1985).Bothropsmoojenirecruit-ment appears to be synchronized with high food(frogs) availability (see Seigel and Ford, 1987, Sazima,1992, for similar cases), whose predation is favoredby tailluring. Such strategies may reduce both costand risk of active foraging for small and vulnerablesnakes (Mushinsky, 1987).The overall result of thesynergistic interaction of tailluring, food availability,time of birth, and high venom toxicity in anuransseems to maximize the capture of prey by juveniles.Thus, among the two main biological roles of thevenom, initiation of digestion and immobilizationldeath of prey (e.g., Gans and Elliot, 1968), killing preyappears to be the most important role in juvenilesnakes; since they possess limited resources to with-stand long fasting periods, such as aestivation (seeHirth, 1966). The capture of a prey once located byjuveniles could be also ensured by holding the preyafter the strike (Mackessy, 1988), a strategy that wehave observed in captivity for juvenile B. moojeni feed-ing on anurans and newborn mice. On the other hand,adult snakes that handle potentially retaliative prey

Mice Frogs

Juvenile 4.54 45.02venom (3.46-5.75) (32.94-66.36)

Adult 4.8 83.7venom (3.65-6.19) (68.15-108.57)

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commonly release them after rapid strike, and thenlocate the dead prey by visual, olfactory, and vome-ronasal cues (Radcliffe et aI., 1980; Kardong, 1986).

The toxieity of adult and juvenile B.moojenivenoms,in mice, did not change substantially with snakes'growth and ontogenetic change of diet. Adult Bo-throps had greater amount of venom in their glands(Furtado et aI., 1991), and injected a greater volumeof it into their prey (Puorto et aI., 1993), a fact thatmay improve prey capture. As the snakes grow largermammalian prey are taken (see Greene, 1992; Sazima,1992), and digestion became more troublesome be-cause of the greatly reduced surface to volume ratio(Pough, 1983; Pough and Groves, 1983). Such an in-crease in prey digestive resistance is accompanied bya concurrent increase in venom proteolytic activityin B. moojeni (Furtado et aI., 1991), which speeds therupture of visceral tissues and increases the prey sur-face area available to stomach enzymes (Gans, 1961;Thomas and Pough, 1979; Pough and Groves, 1983).The improvement of prey digestion may reduce thetime in which snakes suffer reduced locomotion anddefense capabilities (Ford and Shuttlesworth, 1986)and may allow them to spend more time in otheractivities such as foraging and reproduction. Diges-tion by juveniles is believed not to be problematicbecause they take small prey (in absolute terms) whichare readily digested (Mackessy, 1988).

Bothropsmoojenivenom changes are consistent withan optimization of the venom effects toward a speeificprey in a speeific stage of life. Lethality could be moreimportant (selective) for juveniles than adults, whilefor adults the pressure could arise from problems indigesting large prey (see Mackessy, 1988, for a similarcase in Crotalus viridis). Furthermore, the wide occur-rence of conspicuous tail color in Bothrops, frequentlyassoeiated with tailluring, and the ontogenetic shiftin diet (Sazima, 1992) suggests that the sort of venomvariation reported here for B. moojeni could be a wide-spread feature among the genus Bothrops.

Acknowledgments.-We wish to thank R. Wirz andD. Fontanello from Instituto de Pesca, who kindlysupplied the froglets used in this study. An earlierversion of this manuscript was largely improved bythe comments of David L. Hardy, Sr. and an anony-mous reviewer. D. V. A. and A. S. A. were supportedby CNPq grant.

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Accepted: 25 January 1996.