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THE SOUTHWESTERN NATURALIST 44(4):478-483 DECEMBER 1999

VENOM COMPOSITION AND DIET OF THE CANTIL BILINEATUS HOWARDGLOYDI (SERPENTES: )

ALEJANDRO SOLORZANO, MARJORIE ROMERO, JOSE MARIA GUTIERREZ, AND MAHMOOD SASA*

Serpentario Nacional, calles 9-11, Ave 1, San Josi, Costa Rica (AS) Instituto Clodomiro Picado, Facultad de Microbiologia, Universidad de Costa Rica, San Josi, Costa Rica, Jgutierr@cariari. ucrac. cr (MR, JMG) Department of Biology, University of Texas at Arlington, Arlington, TX 76019, [email protected] (MS)

ABSTRACT-The southernmost subspecies of the cantil, Agkistrodon bilineatus howardgloydi, occurs in almost all terrestrial habitats of the Sector Santa Rosa, in Guanacaste Conservation Area, Costa Rica. We obtained 21 specimens during several visits to the park from 1993 to 1996. Neonates were observed only during late May through July, despite searching in other months, suggesting that birth in this subspecies occurs at the beginning of the rainy season. Fecal analyses revealed differences in diet composition between juveniles and adults. Juveniles prey primarily on lizards and various of frogs, whereas adults eat rodents. This ontogenetic change in diet does not seem to trigger any corresponding change in venom composition with age. Venom of this subspecies is highly toxic (LD50 = 1.25 ?ig/g), and has elevated hemorrhagic, hemolytic, and myotoxic activities. Extreme deforestation of dry forest habitats in the Pacific lowlands of Central America has made A. bilineatus a rare species over most of its range. Sector Santa Rosa in Costa Rica is the only area within its distributional range where the species still can be regularly observed.

RESUMEN-La subespecie mis austral del cantil, Agkistrodon bilineatus howardgloydi, ocupa casi todos los habitats terrestres del Sector Santa Rosa, Area de Conservaci6n Guanacaste, Costa Rica. Se obtuvieron 21 especimenes durante varias visitas al area desde 1993 a 1996. Neonatos fueron observados s61o desde fines de mayo hasta julio, aunque se buscaron en otros meses. Esto sugiere que los nacimientos de esta subespecie coinciden con el inicio de la temporada de lluvias. Un anilisis de restos fecales revel6 diferencias en la composici6n de dieta entre juveniles y adultos. Los juveniles depredan principalmente lagartijas y varias especies de ranas, mientras que los ad- ultos comen roedores. Esta variaci6n en dieta no parece estar relacionada con cambios en la composici6n de los venenos con la edad de la serpiente. En esta subespecie, el veneno es altamente t6xico (DL50 = 1.25 pLg/g) y tiene elevada actividad hemorrigica, hemolitica y miot6xica. La destrucci6n acelerada de los bosques xericos en las tierras bajas del Pacifico Centroamericano ha convertido A. bilineatus en una especie rara en la mayoria de su rango de distribuci6n. El Sector Santa Rosa, en Costa Rica, es la finica ;irea dentro de su rango de distribuci6n donde la especie a6in puede ser observada con regularidad.

The cantil, Agkistrodon bilineatus, has received the Agkistrodon complex. They provided infor- considerable attention in terms of distribution mation from more than four decades of re- (Campbell and Lamar, 1989), diagnosis (Bur-search on this and related genera (Gloyd and ger and Robertson, 1951; Gloyd, 1969), Conant,mor- 1990). Despite these efforts, most as- phological variation (Gloyd, 1969), and pectsphy- of the natural history of the southern- logenetic relationships (Knight et al., 1992; most subspecies of the cantil, A. b. howardgloydi, Parkinson et al., 1997). Most of our knowledge are poorly known. on this species was summarized by Gloyd and Agkistrodon bilineatus howardgloydi is the only Conant (1990) in their monumental review of member of the genus occurring south of Hon- duras. It inhabits dry forest and its various suc- *Corresponding author cessional stages, including tropical deciduous

This content downloaded from 163.178.114.52 on Tue, 06 Sep 2016 14:57:06 UTC All use subject to http://about.jstor.org/terms December 1999 Solorzano et al.--Venom composition and diet of the cantil 479 forest, thorn forest, and savannas. Agkistrodon loydi is not encountered frequently (Sasa and Sol6r- b. howardgloydi is distributed from zano,the 1996).lowlands of Golfo de Fonseca (Departments of TwoValle of usand (A. S. and M. S.), periodically visited the GCA to record activity patterns, habitat selection Choluteca) in , along the Pacific and behavior. Specimens were weighed, sexed, and coast of , including the Departments their lengths measured to the nearest 0.5 cm. Cap- of Granada and Jinotega (according to J. Villa, tured individuals were maintained in individual cited in Gloyd and Conant, 1990), and then aquaria for fecal analysis, venom extraction, and fur- south to Golfo de Papagayo (Guanacaste Prov- ther studies of reproductive biology. Representative ince) in northwestern Costa Rica. The species preserved specimens were cataloged in the Collec- was first reported in Costa Rica by Bolafios and tion of Vertebrates, University of Texas at Arlington Montero (1970) from a specimen collected 5 (UTA R-42837-39) or the Museo de Zoologia, Univ- km E of Playa Naranjo (Golfo de Papagayo), ersidad de Costa Rica (UCR 8062, 13071, 13496-99). Venom and Antivenom--Venom was extracted from in what is today Sector Santa Rosa of the Guan- all specimens at Instituto Clodomiro Picado, Univ- acaste Conservation Area (GCA). Subsequent- ersidad de Costa Rica. Sufficient venom was collect- ly, Conant (1984) examined a specimen col- ed from juvenile for electrophoretic analysis, lected from the same place ("near Mirador but only adult venom was used for the other analys- Cafi6n del Tigre in Santa Rosa National Park, es. Venoms were lyophilized and stored at -20'C. Guanacaste, Costa Rica"), and recognized it as The polyvalent (Crotalinae) antivenom (batch a new subspecies, geographically isolated from 2571294 LQ) produced at Instituto Clodomiro Pi- other populations. Because it is a snake that is cado, according to Bolafios and Cerdas (1980), was rarely encountered in most of its range, infor- used in neutralization experiments. Ontogenetic variation in venom was analyzed by mation pertaining to natural history, toxicolo- conducting electrophoresis (SDS-PAGE) of individ- gy of the venom, and snake bite risk is sparse. ual venom samples, performed under reducing con- We suspect that bites from this species proba- ditions on 12% polyacrylamide gels. Proteins were bly are capable of causing human fatalities; stained with Coomasie Blue G-250. however, envenomation by A. bilineatus ho- Median lethal dose (LD50) was estimated by the wardgloydi has not been reported in detail. intraperitoneal route in 16 to 18 g Swiss Webster Here, we summarize field observations on the mice. Groups of six mice were injected with various natural history of this subspecies in the region amounts of venom dissolved in 0.5 ml of phosphate- of its type locality in Sector Santa Rosa of the buffered saline solution, pH 7.2 (PBS). Deaths were Guanacaste Conservation Area and provide a recorded after 48 h and the LD50 was estimated by the Spearman-Kaber method (WHO, 1981). Neu- characterization of the composition and effects tralization experiments were performed by prepar- of its venom. ing mixtures of various ratios of venom and antiv- enom. Mixtures were incubated at 370C for 30 min MATERIALS AND METHODS-Specimens and Study and 0.5-ml aliquots (containing 4 LD50s of venom) Site-Twenty-one specimens of Agkistrodon bilineatus were injected intraperitoneally into groups of six howardgloydi were collected in Sector Santa Rosa mice of (16 to 18 g). A group of control mice received the GCA between June 1993 and January 1996. venom Sec- and no antivenom. Deaths were recorded af- tor Santa Rosa of the GCA, formerly known as Santater 48 h and the effective dose 50% (ED50) was es- Rosa National Park, lies between the Interamerican timated. ED50 is defined as ml volume of antivenom Highway and Playa Naranjo, 30-35 km north of Li- required to neutralize 1 mg of venom. beria, Guanacaste Province, Costa Rica. Two life Hemorrhagic activity was studied using a skin test zones are included in this area: humid Premontane (Kondo et al., 1969), as modified by Gutierrez et al. Tropical Forest and Dry Tropical Forest (Tosi, (1985). Groups of four mice (18 to 20 g) were in- 1969). These life zones are similar climatically, re- jected intradermally with various amounts of venom ceiving an annual precipitation of 1,500 to 2,000 mm dissolved in 100 pl1 of PBS. Two hours after injection, that is strongly seasonal (Hartshorn, 1983). Eight mice were sacrificed, their skin removed and the vegetational associations are recognized from this area of the hemorrhagic spot on the inner side of area: riparian vegetation, mangroves, Prosopis the skin measured. The minimum hemorrhagic dose swamps, semi-deciduous alluvial forest, deciduous (MHD) was the amount of venom that induced a forest, evergreen forest, Quercus forest, and jaragua hemorrhagic area of 10 mm diameter. For neutrali- grasslands (Hyparrhenia rufa). The GCA is 120,000 zation experiments, mixtures containing various ra- ha of conserved dry forest and associated wetter and tios of venom and antivenom were prepared and in- marine habitats. In Sector Santa Rosa, A. b. howardg- cubated at 37oC for 30 min. Then, hemorrhagic ac-

This content downloaded from 163.178.114.52 on Tue, 06 Sep 2016 14:57:06 UTC All use subject to http://about.jstor.org/terms 480 The Southwestern Naturalist vol. 44, no. 4

TABLE 1-Prey items recorded for 13 A. b. howardgloydi from Sector Santa Rosa, Guanacaste Conservation Area, Costa Rica. Numbers of prey items are presented by snake age category. Each snake contained just one prey item.

Age category Newborn Juveniles Adults Prey (<380 mm) (380 to 500 mm) (>500 mm)

Mabuya unimarginata 2 Ameiva undulata 1 Ctenosaura similis - 1 2 Hypopachus variolosus 1 Leptodactylus poecilochilus 1 Liomys salvinii 1 3 Sigmodon hispidus 1

tivity was estimated b. howardgloydi were found as in various described. macro- A control group of mice received venom alone. Neutralization of venom habitats in Sector Santa Rosa. From our data activity for hemorrhagic activity was expressed and as the ef- confirmed observations of this species fective dose 50% (ED50), defined as the ratio of ml by resident biologists at the ACG, it seems that antivenom/mg venom in which the activity of venom this snake occurs in all sorts of habitats. No was reduced 50%. The agarose-erythrocyte-egg yolk assay described association with temporary or permanent wa- by Gutierrez et al. (1988) was used to detect indirect ter was recorded and snakes were found from hemolytic activity. Various amounts of venom, rockydis- ridges to the banks of dry temporary solved in PBS, were applied to wells on agarose streams.gels containing sheep erythrocytes and egg yolk. After Elevena specimens maintained in captivity 20 h incubation period at 370C, diameters of the showedhe- similar striking behaviour. In all cases, molytic halos were measured. The MHD was the they bit, injected venom, and released the of- amount of venom that induced a hemolytic halo of fered prey (mostly white mice). No gravid fe- 20 mm diameter. For neutralization experiments, mixtures with various ratios of venom and antiven- males were encountered during our visits to om were prepared and incubated at 37?C for the 30 min.park. However, neonates a few months old Aliquots of each mixture were then assayed (aged for in-by body size and the presence of an um- direct hemolysis as described above. A control bilical con- scar) were observed from late May to taining venom and no antivenom was included, July, aswhich are the first 3 months of the rainy well as a control of PBS alone. After 20 h of incu- season. bation at 37'C, hemolysis was quantified as Fecalde- samples collected early in the rainy scribed. Neutralizing ability of antivenom was ex- season in 1994 indicated age-related variation pressed as ED50, defined as the ratio of ml antiven- in diet of these snakes (Table 1). Neonates and om/mg venom in which the diameter of the hemo- lytic halo was reduced by 50% when compared juveniles to preyed on small lizards (Mabuya uni- the halo induced by venom alone. marginata, Ameiva undulata) that are common Myotoxic activity of the venom of A. b. howardgloydi in the leaf litter, and on several frog species was evaluated histologically. Venom (50 ?pg dissolved that are particularly abundant during the rainy in 50 p1 PBS) was injected intramuscularly season in the (Hypopachus variolosus, Leptodactylus sp.). right gastrocnemius of mice (18 to 20 g). A Specimens group with lengths greater than 500 mm of control mice received PBS alone. Three and 24 h (adults) contained rodents (Liomys salvinii, Sig- after injection, groups of mice were sacrificed and a sample of injected muscle was obtained, fixed modon in hispidus) and large lizards (Ctenosaura Karnovsky's fixative and processed routinely forsimilis). his- tological evaluation (Gutihrrez et al., 1991). Activities of the Venom and Neutralization by An- tivenom---A similar, but not identical, electro- RESULTs-Habitat, Behavior and Diet-Almost phoretic pattern was found in venom proteins all snakes were found at night, except for two of adults and juveniles (Fig. 1). In both cases, found at noon and late afternoon. Agkistrodon nine bands were observed, with molecular

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polymorphonuclear leukocytes and macro- I..!: . ?~:: d~ ? :i i:..: .Ir..: ?.. ...c?.:B ? : ?rli; phages.

.gal

.i. . . I~i?'~. DIscussIoN-Ag*istrodon b. howardgloydi oc- curs in many types of dry forest, especially along the ecotonal edge with tropical scrub ...... :~: ?l~i1- :,.:g habitat. Contrary to the general belief [as stat- ed in Villa (1983) ] that this snake is semiaquat- ::''I :.'?.?? 6~:ij~3 ~e _i~Efp;~ ??51: ??;: ?.ic Im Y ic, we found it in a variety of terrestrial habi- in.. . -0 1;-!?. tats, never associated with water. The northern *? u r '";-A Z;i

mmrs*~~vr C'?. subspecies, A. b. taylori, also seemed to prefer the ecotonal area between tropical scrub and FIG. 1-Gel electrophoresis of the venom of Agkis- semi-deciduous forest, and not the riparian trodon bilineatus. Electrophoresis was run in 12% habitats (Burchfield, 1982). polyacrylamide gels under reducing conditions. 1) In Santa Rosa, births in A. b. howardgloydi ap- Adult A. b. bilineatus from ; 2) Adult A. b. parently occur between late May and August, howardgloydi from Costa Rica; 3) Juvenile A. b. ho- coincident with the first half of the rainy sea- wardgloydi from Costa Rica; 4) Molecular weight son. A similar reproductive cycle was described markers. for Crotalus durissus in that region (Sol6rzano and Cerdas, 1988), with copulation taking place early in the dry season (December to weights of 95, 90, 82, 67, 42, 28, 23, 20, and February) 16 and births occurring early in the kDa. Along with these common bands, venom rainy season. Burchfield (1982) observed cop- of adults had an additional band (26 kDa) that ulations of captive A. b. taylori from Tamaulipas was not present in juveniles. The 28 kDa band in late January and February. In this subspe- was brighter in venom of adults than in juve- cies, litters of four to nine neonates were born niles. The electrophoretic pattern of venom of from late June to October, at the beginning of adults of A. b. howardgloydi from Costa Rica was the cold rainy season in that region. identical to the pattern in A. b. bilineatus from Fecal analyses indicated that the cantil preys Guatemala (Fig. 1). on a diverse group of taxa (Table 1). This ob- Lethal dose (LD50 by the intraperitoneal servation is further supported by previous ob- route) of venom of adult A. b. howardgloydi was servations summarized by Gloyd and Conant 1.25 ttg/g mouse. Antivenom neutralized the (1990) that diets of cantils include snakes (Im- lethal effect of venom with an ED50 of 0.98 ml antodes gracillimus [= gemmistratus], Thamnophis antivenom/mg venom. Venom had a strong sp.), rodents (Liomys pictus, L. irroratus, Mus hemorrhagic activity with a MHD of 0.12 gig. musculus, Peromyscus leucopus, Oryzomys palus- Antivenom also neutralized this activity with an tris), frogs (Hyla squirella), and grasshoppers. ED50 of 1.06 ml antivenom/mg venom. Indi- Moreover, our data indicate that a change in rect hemolytic activity was observed, and the diet appears to occur as the snakes mature. MHD was estimated at 0.20 ptg. Antivenom Ontogenetic shifts in diet from frogs and liz- neutralized this activity, with an ED50 of 2.17 ards to mammals also have been reported for ml antivenom/mg venom. Histological analysis other pitvipers (i.e., aspe, B. moojeni, of mouse skeletal muscle injected with A. b. ho- C. viridis, see also Greene [1992]), and usually wardgloydi venom showed evidence of conspic- are related to changes in venom composition uous myonecrosis with muscle fibers having hy- within different age groups (Mackessy, 1988). percontracted masses of myofibrils in the cel- In Cerrophidion godmani (Sasa, 1997), no age- lular space. This pattern was qualitatively sim- related changes in diet or differences in the ilar to that induced by the venom of Bothrops venom composition of adult and juveniles are asper (Guti&rrez et al., 1984). In addition, hem- evident. Similarly, dietary shift in Agkistrodon orrhage was evidenced by abundant erythro- bilineatus may not be related to a change in cytes in the interstitial space. In samples col- toxin composition, as suggested by the similar- lected 24 h after injection, a prominent inflam- ities of venom electrophoretic patterns of ju- matory infiltrate was observed with abundant veniles and adults. However, the low amount

This content downloaded from 163.178.114.52 on Tue, 06 Sep 2016 14:57:06 UTC All use subject to http://about.jstor.org/terms 482 The Southwestern Naturalist vol. 44, no. 4 of venom collected range. Recentfrom efforts juvenilemade by one of us specimens(M. precluded other analyses S.) to find specimens and, in the placestherefore, where it is it is not clear if biological known to activitiesoccur, including the dryof valleys their of venom differ from those Rio ofLagarteros adults (Department as ofreported Huehueten- here. Whether ontogenetic ango) and Rio changes Chixoy (Department in of venomAlta and diet composition Verapaz)reflect in Guatemala, a phylogenetic and in several localities trend or are influenced in byGolfo de selectiveFonseca (Honduras) and Granadaforces acting in an ecological context (Nicaragua), is yielded still no specimens. unclear. Further- Lethal doses in mice more, it waswere noted that mostslightly locals in these lower than those reported by regions Bolafios were not aware of(1972) the presence offor this two spec- imens of A. b. howardgloydi species there, thus confirming collected that low popu- in north- ern Costa Rica (1.70 lation densitiesxg/g (or localI.P.), extinction) and may be by Burch- field (1982) for A. the trendb. intaylori most of the range (2.30 of the species. [g/g). Ven- om of Agkistrodon Unfortunately, b. howardgloydi large portions of habitat for has A. highly le- thal, hemorrhagic, bilineatus haveindirect been destroyed for agriculturalhemolytic and myotoxic activities. purposes Almost within the last fewidentical centuries. Habitat lethal and hemorrhagic effects disturbance is thewere main reason describedthat "the cantil, for the venom of A. b. bilineatus except in still remote from areas or where Guatemala it is pro- (Ro- jas et al., 1987). tected,It isas in thelikely Parque Nacional that Santa Rosa, this venom would induce prominent Costa Rica, is now almost local certainly atissue rare damage in humans. A hemorrhagic snake," (Conant, 1992:29). metalloproteinase, named bilitoxin, was isolated from this venom (Ownby et al., 1990), and a myotoxic phospho- We are particularly indebted to R. Conant for lipase A2 also was purified (Mebs and Sameji- sharing some of his vast knowledge and experiences ma, 1986). On the other hand, indirect he- on the biology of the cantil. We thank R. Blanco for molytic effect is due to the action of phospho- collecting permits, and V. Nielsen, P. Hurtado and the workers at Guanacaste Conservation Area for as- lipases A2 on egg yolk phospholipids with the sistance in the field. We are very grateful to those consequent release of lysolecithin, which in individuals who helped us collect snakes in Santa turn induces lysis of erythrocytes by detergent Rosa. We also thank J. Nufiez, R. Aymerich, and J. action (Tu, 1977). Sanabria for their technical support in the labora- No well-documented clinical reports of en- tory. M. Sasa is deeply grateful to D. Janzen for his venomation by this species in Costa Rica are criticisms and improving the English in an earlier available, although it is likely that they would draft of the manuscript. J. A. Campbell, W. W. La- be characterized by local and systemic alter- mar, A. Knight, and an anonymous reviewer read the ations similar to that reported for other North manuscript and provided important suggestions. American species of this genus (G6mez and This material is based in part on work supported by the North Texas Herpetological Society, and the Dart, 1995). The polyvalent antivenom pro- Texas Advanced Research Program under grant duced at Instituto Clodomiro Picado was effec- 003656-001 to J. A. Campbell. tive in the neutralization of lethal, hemorrhag- ic and indirect hemolytic activities in our ex- LITERATURE CITED periments, despite the fact that A. bilineatus is not included in the immunizing mixture BoLA&os, used R. 1972. Toxicity of Costa Rican snake ven- for production of this antivenom (Bolafios oms and for the white mouse. American Journal of Cerdas, 1980). Lomonte et al. (1987) observed Tropical Medicine and Hygiene 23:3. that antibodies to the lancehead, Bothrops BoLAIos, asper R., AND L. CERDAS. 1980. Producci6n y con- myotoxins cross react against proteins in trolthe de sueros antiofidicos en Costa Rica. Boletin de la Oficina Sanitaria Panamericana 88:184- venom of A. bilineatus. Thus, in agreement with 196. an earlier study (Rojas et al., 1987), our results BoLAlJos, R., AND J. R. MONTERO. 1970. Agkistrodon strongly suggest that this polyvalent antivenom bilineatus Gunter from Costa Rica. Revista de Biol- is likely to be effective in the treatment ofogia en- Tropical 16:277-279. venomation by A. bilineatus in Central America. BURCHFIELD, P. M. 1982. Additions to the natural his- Despite the extensive distribution of the torycan- of the crotaline snake Agkistrodon bilineatus til, it is difficult to find in most parts of taylori. its Journal of Herpetology 16:376-382.

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BURGER, W. L., AND W. B. ROBERSON. 1951. A new KONDO, H., S. KONDO, I. IKEZAWA, R. MUROTA, AND subspecies of the Mexican moccasin, Agkistrodon A. OHSAKA. 1969. Studies of the quantitative bilineatus. University of Kansas Science Bulletin method for the determination of hemorrhagic 34:213-218. activity of Habu snake venom. Japanese Journal CAMPBELL, J. A., AND W. W. LAMAR. 1989. The of ven- Medical Science Biology 13:43-51. omous of Latin America. Comstock. LOMONTE, Cor- B., E. MORENO, AND J. M. GUTIItRREZ. 1987. nell University Press, Ithaca, New York. Detection of proteins antigenically related to CONANT, R. 1984. A new subspecies of the pitviper myotoxin in crotaline snake ven- Agkistrodon bilineatus (Reptilia: Viperidae) fromoms. Toxicon 25:947-55. Central America. Proceedings of the Biological MACKESSY, S. P. 1988. Venom ontogeny in the Pacific Society of Washington 97:135-141. rattlesnakes Crotalus viridis helleri and C. v. oregan- CONANT, R. 1992. Comments on the survival status us. Copeia 1988:92-101. of members of the Agkistrodon complex. Cincin- MEBS, D., AND Y. SAMEJIMA. 1986. Isolation and char- nati Herpetological Society Contributions in Her- acterization of myotoxic phospholipases A2 from petology 1992:29-33. crotalid venoms. Toxicon 24:161-168. GLOYD, H. K. 1969. Two additional subspecies of OWNBY, C. L., T. NIKAI, K. IMAI, AND H. SUGIHARA. North American crotalid snakes, genus Agkistro- 1990. Pathogenesis of hemorrhage induced by don. Proceedings of the Biological Society of bilitoxin, a hemorrhagic toxin isolated from the Washington 84:219-232. venom of the common cantil (Agkistrodon bilinea- GLOYD, H. K., AND R. CONANT. 1990. Snakes of the tus bilineatus). Toxicon 28:837-846. Agkistrodon complex: a monographic review. Con- PARKINSON, C. L., M. M. SCOTT, AND J: E. AHLQUIST. tributions to Herpetology 6:1-614. 1997. Phylogenetic relationships of the Agkistro- GOMEZ, H. F., AND R. C. DART. 1995. Clinical toxi- don complex based on mitochondrial DNA se- cology of snakebite in North America. In: Meier, quence data. In: Thorpe, R. S., W. Wuster, and A. J., and J. White, editors. Handbook of clinical Malhotra, editors. Venomous snakes: ecology, toxicology of venoms and poisons. CRC evolution and snakebite. Clarendon Press, Ox- Press, Boca Raton, Florida. Pp. 619-644. ford, United Kingdom. Pp. 63-78. GREENE, H. W. 1992. The ecological and behavioral ROJAs, G., J. M. GUTItRREZ, J. A. GENt, M. G6OMEZ, AND L. CERDAS. 1987. Neutralizaci6n de las activ- context for pitviper evolution. In: Campbell, J. A., and E. D. Brodie Jr., editors. Biology of the pit- idades t6xicas y enzimiticas de cuatro venenos de vipers. Selva Press, Tyler, Texas. Pp. 107-117. serpientes de Guatemala y Honduras por el an- GUTIERREZ, J. M., C. L. OWNBY, AND G. V. ODELL. tiveneno polivalente producido en Costa Rica. 1984. Pathogenesis of mionecrosis induced by Revista de Biologia Tropical 35:59-67. crude venom and a myotoxin of Bothrops asper SASA, M. 1997. Cerrophidion godmani in Costa Rica: A Experimental and Molecular Pathology 40:367- case of extremely low allozyme variation? Journal 379. of Herpetology 31:569-572. GUTIERREZ, J. M., C. AVILA, E. RojAs, AND L. CERDAS. SASA, M., AND A. SOLORZANO. 1996. The reptiles and 1988. An alternative in vitro method for testing amphibians of Santa Rosa National Park, Costa Rica, with comments about the herpetofauna of the potency of the polyvalent antivenom pro- duced in Costa Rica. Toxicon 26:411-413. xerophytic areas. Herpetological Natural History 3:113-126. GUTIERREZ,J. M.,J. A. GENt, G. ROJAS, AND L. CERDAS. SOLORZANO, A., AND L. CERDAS. 1988. Biologia re- 1985. Neutralization of proteolytic and hemor- productiva de la cascabel centroamericana Cro- rhagic activities of Costa Rican snake venoms by talus durissus durissus (Serpentes: Viperidae) en a polyvalent antivenom. Toxicon 23:887-893. Costa Rica. Revista de Biologia Tropical 21:383- GUTIIRREZ, J. M., J. NUrEZ, C. DiAZ, A. C. O. CINTRA, 397. M. I. HOMSI-BRADEBURGO, AND J. R. GIGLIO. 1991. TosI, J. A., JR. 1969. Mapa ecol6gico de Costa Rica. Skeletal muscle degeneration and regeneration Centro Cientifico Tropical, San Jos6, Costa Rica. after injection of bothropstoxin II, a phospholi- Tu, A. T. 1977. Venoms: chemistry and molecular pase A2 isolated from the venom of the biology.snake John Wiley & Sons, New York. Bothrops jararacussu. Experimental and Molecular VILLA, J. 1983. Peces, anfibios y reptiles Nicaragilen- Pathology 55:217-229. ses: lista y bibliografia. Escuela de Ecologia y Re- HARTSHORN, G. S. 1983. Plants. In: Janzen, D.cursos H., Naturales, Universidad Centroamericana, editor. Costa Rica natural history. University Managua, of Nicaragua. Chicago Press, Chicago, Illinois. Pp. 118-157. WORLD HEALTH ORGANIZATION. 1981. Progress in the KNIGHT, A., L. D. DENSMORE III, AND E. D. RAEL. characterization of venoms and standardization 1992. Molecular systematics of the Agkistrodon of antivenoms. W.H.O., Geneva, Switzerland. complex. In: Campbell, J. A., and E. D. Brodie, Jr., editors. Biology of the pitvipers. Selva Press, Submitted 14 April 1998. Accepted 10 February 1999. Tyler, Texas. Pp. 49-69. Associate Editor was Stanley F Fox.

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