Snake Venomics of Bothriechis nigroviridis Reveals Extreme

Snake Venomics of Bothriechis nigroviridis Reveals Extreme Variability among Palm Pitviper Venoms: Different Evolutionary Solutions for the Same Trophic Purpose Juliań Fernańdez,† Bruno Lomonte,† Libia Sanz,‡ Yamileth Angulo,† Jose´ Marıá Gutie´rrez,† and Juan J. Calvete*,‡ Instituto Clodomiro Picado, Facultad de Microbiologıá, Universidad de Costa Rica, San Jose´, Costa Rica, and Instituto de Biomedicina de Valencia, C.S.I.C., Jaume Roig 11, 46010 Valencia, Spain Received June 2, 2010

We report the proteomic characterization and biological activities of the venom of the black-speckled palm pitviper, Bothriechis nigroviridis, a neotropical arboreal pitviper from Costa Rica. In marked contrast to other Bothriechis species investigated, the venom of B. nigroviridis does not possess detectable Zn2+-dependent metalloproteinases, and is uniquely characterized by a high content of crotoxin-like PLA2 and vasoactive peptides. These data suggest that different evolutionary solutions have evolved within the arboreal genus Bothriechis for the same trophic purpose. The venom from B. nigroviridis is devoid of hemorrhagic activity, has low edematogenic and coagulant effects, presents modest myotoxic and phospholipase A2 activities, but has higher lethality than the venoms of other Bothriechis species. Neutralization of its lethal activity by an anti-Crotalus durissus terrificus antivenom confirmed the major role of crotoxin-like PLA2 in B. nigroviridis venom-induced lethality. Keywords: Bothriechis nigroviridis • black-speckled palm pitviper • snake venom proteomics • venomics • N-terminal sequencing • mass spectrometry

Introduction The genus Bothriechis comprises 9 species (B. aurifer, B. bicolor, B. lateralis, B. marchi, B. nigroviridis, B. rowleyi, B. schlegelii, B. supraciliaris, B. thalassinus) of relatively slender to medially robust, arboreal, prehensile-tailed, New World pitvipers.1 Except for B. schlegelii, which ranges in humid lowlands and foothills from southern Mexico through Pacific Ecuador to Peru ´ and western Venezuela, Bothriechis species are confined to montane regions between the Isthmus of Tehuantepec in southern Mexico and Central Panama´.1 B. nigroviridis,2 also termed black-speckled palm pitviper, black-spotted palm viper or yellow-spotted palm viper, is a relatively rare, small (most adults commonly averaging 60-80 cm in length) venomous pitviper.1 The specific name is derived from the Latin nigro (black) and viridis (green) in reference to its distinctive color pattern, which may represent an arboreal adaptation providing camouflage to avoid detection (Figure 1). According to Campbell and Lamar,1 this species inhabits subtropical rainforests and temperate forests at medium to high elevations, from 1150 to over 3000 m, on both the Atlantic and Pacific slopes of the Cordillera Tilarań (highlands of Monteverde) and Cordillera Volcańica Central in the southeastern Alajuela province in the central valley of Costa Rica, southeastward through the Cordillera de Talamanca to Chiriquı´ * To whom correspondence should be addressed. Juan J. Calvete, Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia (Spain). Phone: +34 96 339 1778. Fax: +34 96 369 0800. E-mail: [email protected]. † Universidad de Costa Rica. ‡ Instituto de Biomedicina de Valencia.

4234 Journal of Proteome Research 2010, 9, 4234–4241 Published on Web 06/30/2010

province in Panama´.1 Like the other Bothriechis species, B. nigroviridis is arboreal, although it has been also found on the ground or at the base of trees and shrubs, where the dense foliage may offer camouflage and in addition represents a supportive ecosystem to capture prey. Solo´rzano reported that B. nigroviridis is mostly nocturnal. Adults feed primarily on small rodents, lizards, and frogs, although occasionally they capture small birds.3 Although Bothriechis venoms investigated seem to be of moderate toxicity, bites may have dire consequences due to the arboreal nature of these snakes which results in many of the bites being inflicted in the head, neck, and shoulder regions.1,3 B. schlegelii (eyelash pit viper), a relatively small species that rarely exceeds 75 cm in length found in mesic forest at elevations almost from sea level to 2640 m altitude in Central and South America,3 causes a number of envenomations in Costa Rica.4 Documentation of human accidents by Bothriechis snakebites is scarce. Indeed, we were unable to find any report on the pathophysiology of human envenomation by B. lateralis. On the other hand, initial symptoms of B. schlegelii snakebite include localized pain, progressive hemorrhagic edema, and in some cases, hemorrhagic blisters or hives, ecchymoses, and necrosis.5-7 It has also been shown that the venom of B. schlegelii induces significant myonecrosis in experimental animal models.8,9 Gutie´rrez and Chaves8 studied the venoms of 10 Costa Rican species of pitvipers and found that those of B. schlegelii (and Bothrops asper) had the greatest myonecrotic activity, whereas the venoms of B. lateralis (and Cerrophidion godmani) showed the strongest proteolytic activ10.1021/pr100545d

 2010 American Chemical Society

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Bothriechis nigroviridis Venom Proteome

(44%), respectively. Their different venom toxin compositions provided clues for rationalizing the distinct signs of envenomation in experimental animals caused by B. schlegelii and B. lateralis.10 Here we sought to investigate the composition and biological activities of the venom of B. nigroviridis, a third species of the arboreal genus Bothriechis found in Costa Rica. Strikingly, the venom proteome of this species does not possess detectable Zn2+-dependent metalloproteinases, and is uniquely characterized by a high content of crotoxin-like PLA2 subunit chains and vasoactive peptides. Each of these groups of toxins represents as much as 38% of total venom proteins. These data suggest that different evolutionary solutions have evolved within the arboreal genus Bothriechis for the same trophic purpose, and underscore the versatility of venoms as adaptive traits in these viperid snakes. Figure 1. Reverse-phase HPLC separation of the venom proteins from B. nigroviridis. Two milligrams of B. nigroviridis venom was applied to a Lichrosphere RP100 C18 column, which was then developed with the following chromatographic conditions: isocratically (5% B) for 10 min, followed by 5-15% B for 20 min, 15-45% B for 120 min, and 45-70% B for 20 min. Fractions were collected manually and characterized by N-terminal sequencing, SDS-PAGE, and ESI mass spectrometry. Inset, SDS-PAGE showing the protein composition of the reverse-phase HPLC separated venom protein fractions run under nonreduced (upper panels) and reduced (lower panels) conditions. Molecular mass markers (in kDa) are indicated at the left of each gel. Protein bands were excised and subjected to in-gel digestion, tryptic peptide mass fingerprinting, and CID-MS/MS of selected doubly or triply charged peptide ions. The results are shown in Table 1. The picture of B. nigroviridis was taken at the Instituto Clodomiro Picado by J.F. and corresponds to one of the specimens used to obtain venom for this study.

ity toward casein. Campbell and Lamar cite that B. nigroviridis has been implicated in human fatalities, and bites are reported to cause intense pain, nausea, and asphyxia.1 However, clinical literature on envenomations by B. nigroviridis is scarce, and we found no single reference to this species in the literature (PubMed). In a previous work, we have reported the proteomic characterization of B. schlegelii and B. lateralis venoms.10 The venom proteomes of B. lateralis and B. schlegelii comprise similar number of distinct proteins belonging, respectively, to 8 and 7 protein families. The two Bothriechis venoms contain bradykinin-potentiating peptides (BPPs), and proteins from the phospholipase A2 (PLA2), serine proteinase, L-amino acid oxidase (LAO), cysteine-rich secretory protein (CRISP), and Zn2+-dependent metalloproteinase (SVMP) families, albeit each species exhibits different relative abundances. Moreover, these venoms also contain unique components, for example, svVEGF and C-type lectin-like molecules in B. lateralis, and Kazal-type serine proteinase inhibitor-like proteins in B. schlegelii.10 B. schlegelii represents the sister taxon to all other Bothriechis species, whereas B. lateralis segregates with B. bicolor and B. marchi in the most recently isolated clade from a common ancestor.11 Using a similarity coefficient, we estimated that the similarity of the venom proteins between these two Bothriechis taxa may be 38% of the

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Table 1. Assignment of the Reverse-Phase Fractions from the Venoms of B. nigroviridis, Isolated as in Figure 1, to Protein/Peptide Classes by N-Terminal Edman Sequencing, Mass Spectrometry, and Collision-Induced Fragmentation by nESI-MS/MS of Selected Peptide Ions from In-Gel Digested Protein Bands Separated by SDS-PAGE (inset in Figure 1)a peptide ion

HPLC fraction

N-terminal sequence

molecular mass

m/z

z

1007.5 989.4 1513.6 1839.4 1291.4 1275.7 2303.6 2288.2 1244.8 1163.2 1235.4 9 kDa1

504.2 495.2 757.3 920.2 646.2 638.2 768.5 1144.1 622.8 582.1 618.2

2 2 2 2 2 2 3 2 2 2 2

SPPAGPDGGPR SPPAGP (483.3) SPPAGDPDGGPRGA(282.1) SPPAGDPDGGPRDS(535.1) (249.1)WSXGHHIPP (249.1)WSPGHHIPP

14092

871.8

2

GTWCEEQICECDR

∼Crotoxin-like basic chain [P62022] ∼Mojave toxin-like basic chain [P62023]

685.3 569.7 1190.4 1571.3 1443.5 1456.5 595.2 763.8 617.8 837.8 929.6

2 2 1 1 1 1 2 2 3 3 1

ZVMPFMEVYSR SVDFDSESPR XMGWGTXSPTK BVXNEDEQTRDPK VXNEDEQTRDPK SXPSNPPSVGSVCR WDBDIMXXR IIGGDECNINEHR XAPXSXPSNPPSVGSVCR (214.3)XPDVPHCANXNXXDYEVCR LQFGLHSK

svVEGF CRISP Serine proteinase

782.8 488.3 696.4 919.4 860.3 795.2 1289.7 757.9 1651.0 1281.8 514.8 788.0

2 2 2 2 2 2 1 2 1

CAPXNXXDYAVCR ETYPNVPR AAYPWW(370.2)TK XAPXSXPSSPPS(257.2)VCR ETPVXSNPGTNSAEFR XXAXGHSGFFEDQR ZVPVVQAYAFGK ETDYEEFLEIAR NDKEGWYANLGPMR SAGQLYEESLGK XHYTXXXR (189.0)PTSVPPRPVAPXR

1 2 3 6

np SPPAGPDGGPR ND ND

7 8 9

ND Blocked ND

10 11

Blocked ND

12, 13

15, 16 17 18

a SPENCQGESQPC b GCYCDAEGQGWPQDA c EENGDIVCGEXTPC NLLQFNRMIKLETKKNAV PFYAFYGCYCGWGGQGQ PKDATDRCCXEHDCCYG KLTKCNTKWDLY Blocked SVDFDSESPRKPEIQ V(I/V)GGDECNINEHR(S/F)L

13 kDa1 23 kDa1 36 kDa1

19

IIGGDECNINEHRSL

38 kDa1

20 21

VIGGDECNINEHRSL VVGGDECNINEHRSL

33 kDa1 31 kDa1

22

ND

72 kDa1

23

ADTRNPLEECFRETD

66 kDa1

24

ND

110 kDa1

14

2

MS/MS-derived sequence

ZKDWPPPISPP PP(756.1)PP PP(828.1)PP

protein/peptide class

Bradykinin-inhibitory peptide Bradykinin-inhibitory peptide Bradykinin-inhibitory peptide Bradykinin-inhibitory peptide Bradykinin-potentiating peptide Bradykinin-potentiating peptide Glycopeptide Glycopeptide Bradykinin-potentiating peptide Bradykinin-potentiating peptide Bradykinin-potentiating peptide ∼Crotoxin-like acidic chain [P08878] ∼Mojave toxin-like acidic chain [P18998]

Serine proteinase

Serine proteinase Serine proteinase

5′-nucleotidase

L-amino acid oxidase

Unknown

a In MS/MS-derived sequences, X ) Ile or Leu; Z, pyrrolidone carboxylic acid; B, Lys or Gln. Unless otherwise stated, for MS/MS analyses, cysteine residues were carbamidomethylated. Molecular masses of native proteins were determined by electrospray-ionization ((0.02%) or MALDI-TOF ((0.2%) mass spectrometry. Apparent molecular masses were determined by SDS-PAGE of nonreduced (9) and reduced (1) samples. np, nonpeptidic material found. Underlined residues in the N-terminal sequences of the crotoxin-like acidic and basic chains depart from the homologue crotoxin/Mojave toxin amino acid sequences.

toxin proteome. Crotoxin is a heterodimeric PLA2 molecule exhibiting presynaptic β-neurotoxicity, but also inducing systemic myotoxicity and other deleterious effects,38-40 which was first isolated in 1938 by Slotta and Fraenkel-Conrat41 from the venom of the tropical rattlesnake (C. d. terrificus). A homologue of South American crotoxin, Mojave toxin,42 is present in venoms of certain populations of North American Crotalus species, including the Mojave rattlesnake (Crotalus scutulatus scutulatus), the midget-faded rattlesnake (Crotalus oreganus concolor), the southern Pacific rattlesnake (Crotalus helleri), and the tiger rattlesnake (Crotalus tigris).43,44 Crotoxin and Mojave toxin are responsible for the characteristic systemic neuro- and myotoxic effects observed in envenomations by these taxa.7 To our knowledge, crotoxin or crotoxin-like proteins have not been previously found in the venoms of New World pit vipers other

than rattlesnakes, that is, Crotalus and Sistrurus spp, and are certainly absent from the venom proteomes of B. lateralis and B. schlegelii.10 Serine proteinases comprise 18% of venom toxins and represent the third more abundant protein family in B. nigroviridis venom (Tables 1 and 2, Figure 2). Members of this toxin class contribute to viperid venom toxicity by affecting platelet aggregation, blood coagulation, fibrinolysis, the complement system, blood pressure, and the nervous system.45 Internal peptide sequences gathered from the serine proteinases recovered in fractions 18 and 21 display highest similarity (76-100%) with thrombin-like enzymes from a number of snake species (i.e., ABB76280 from B. asper). The tryptic peptides sequenced from the serine proteinase eluted in fraction 19 exhibit 90-100% identity with plasminogen activaJournal of Proteome Research • Vol. 9, No. 8, 2010 4237

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Table 2. Overview of the Relative Occurrence of Protein/ Peptides (in Percentage of the Total HPLC-Separated Components) of the Different Classes in the Venoms of B. nigroviridisa protein/ peptide class

PLA2 - myotoxin - Crotoxin-like Vasoactive peptides - BIP - BPP Serine proteinase svVEGF CRISP L-amino acid oxidase 5′ nucleotidase unknown Kazal-type inhibitor C-type lectin-like SVMP - PIII - PI

% of total venom toxins B. nigroviridis

B. lateralis

B. schlegelii

38.3 38.3 37.0 10.1 26.9 18.4 2.8 2.1 0.5 0.5 0.4 -

8.7 8.7 11.1 0.1 11.0 11.3 0.5 6.5 6.1 -

43.8 43.8 13.4 0.1 13.3 5.8 2.1 8.9 -

0.9 55.1 54.5 0.6

8.3 17.7 17.0 0.7

a BIP and BPP, bradykinin-inhibitory and bradykinin-potentiating peptides, respectively. For comparison, the compositions of the venom proteomes of B. lateralis and B. schlegelii10 are also displayed. Major toxins are highlighted in boldface in each taxa.

Figure 2. Overall composition of B. nigroviridis venom. Pie chart of the relative abundance (in % of total venom proteins) of the different toxin classes found in the pooled venom of B. nigroviridis. CRISP, cysteine-rich secretory protein; LAO, L-amino acid oxidase; svVEGF, snake venom vascular endothelial growth factor. Details of the individual proteins are shown in Table 1 and the percentages of the different toxin classes in the venoms are listed in Table 2.

tor AAD01623 from the short-tailed Mamushi (Gloydius blomhoffi brevicaudus).46 Minor toxin groups found in B. nigroviridis venom include snake venom vascular endothelial growth factor (VEGF) isoforms (fractions 15 and 16), a cysteine-rich secretory protein (CRISP, fraction 17), a 5′-nucleotidase (peak 22), an L-amino acid oxidase (LAO, fraction 23), and an unknown protein of molecular mass 110 kDa (fraction 24). Each of these protein families comprises 8 µg 79 ( 6 µg 198 ( 17 U/µg No hemorrhagic activity up to a dose of 100 µg of venom

a Detemined in 16-18 g mice; 95% confidence limits are included in parentheses. b Myotoxicity is expressed as the plasma CK activity (in U/L) 3 h after im injection of 50 µg of venom. Control mice injected with PBS alone showed a plasma CK activity of 212 ( 93 U/L. c Minimum Edema-forming Dose: dose of venom inducing 30% edema after subcutaneous injection. d Minimum Coagulant Dose (MCD) is the lowest venom dose that induced clotting of citrated human plasma in 60 s. e One unit of PLA2 activity was defined as a change of 0.001 in absorbance per minute.

cyclic nucleotide-gated ion channels;48 the pharmacological effects of LAO include induction of platelet aggregation, apoptosis of cells, and cytotoxicity.49 The pathophysiological relevance of the minor B. nigroviridis venom toxins deserves detailed investigation, but we suspect that their effects may be completely overshadowed by the hypotensive and neurotoxic symptoms provoked by BPPs and crotoxin-like PLA2, respectively. Rapid immobilization due to BPP-induced hypotension and crotoxin-induced paralytic effects causing progressive paralysis may represent an adaptation of B. nigroviridis, a “sit-and-wait” predator, for outweighing the threat of holding large, dangerous prey, and for restraining it from escaping during the swallowing process. Toxicological Profile of B. nigroviridis Venom. Table 3 displays the toxic and enzymatic activities characterized in B. nigroviridis venom. The most striking feature is the absence of hemorrhagic activity up to a dose of 100 µg of the venom, which is in evident contrast with all other Central American pit viper venoms previously studied and whose Minimum Hemorrhagic Doses ranged between 0.5 and 5.1 µg.27 This is in agreement with the lack of SVMPs in the venom proteome of this species, and is also in marked contrast to the proteomic and toxicological profiles of the venoms of other Bothriechis taxa, B. lateralis and B. schlegelii,10 in which SVMPs play a very important role in the pathophysiology of envenomation, particularly in B. lateralis.8,9,50 The venom from B. nigroviridis had also low edematogenic and coagulant activities. The Minimun Edema-forming Dose (MED) was higher than 8 µg (this dose only induced 24% edema after an hour), while the Minimum Coagulant Dose (79.2 ( 5.7 µg) is very low compared with that of other viperid venoms.29 This venom also presented modest myotoxic and phospholipase A2 activities (Table 3). The mild in vitro coagulant effect and the lack of hemorrhagic activity of B. nigroviridis venom suggests that the role of its serine proteinases may not be associated with coagulopathy and bleeding, at least in the mouse, and may instead be linked to other effects, such as hypotension.

Bothriechis nigroviridis Venom Proteome The intraperitoneal Median Lethal Dose (LD50) of B. nigroviridis venom was 30 µg/mouse, that is, 1.76 mg/kg. This activity is slightly more potent than those reported for other Costa Rican snake venoms.51 The fact that the ICP polyvalent antivenom, prepared against the crotoxin-negative venoms of B. asper, C. simus, and L. stenophrys, was unable to neutralize the lethal activity of B. nigroviridis up to a level of 2000 µL antivenom/mg venom (Table 3) pointed to the crotoxin-like PLA2 as the major contributor to B. nigroviridis venom toxicity. This hypothesis is supported by neutralization assays using the crotalic antivenom, produced at Instituto Butantan by the immunization of horses with venom of C. d. terrificus, which contains high amounts of crotoxin.22 This antivenom fully neutralized the lethal activity of B. nigroviridis venom at a ratio of 2000 µL antivenom/mg venom. The scarcity of venom obtained for the present work precluded determining the complete amino acid sequence of the crotoxin-like component of B. nigroviridis, a task that might provide valuable insights into the evolution and biological significance of this particular type of toxin, intensively studied over many years. It is noteworthy, however, that the toxicity of this venom is not particularly high, especially when compared to crotoxin/Mojave toxin-positive Crotalus venoms (i.e., C. d. terrificus, 0.13 mg/kg; Crotalus horridus, 1.0 mg/kg; C. s. scutulatus, 0.2 mg/kg; C. tigris, 0.07 mg/kg; C. o. concolor, 0.46 mg/kg).51 This suggests that the relative abundance and structural differences between the crotoxin-like component of B. nigroviridis and the highly neurotoxic crotoxin/Mojave toxin molecules may account for their different toxic potentials to mice. In line with this hypothesis, the amino acid sequence of crotoxin [P62022] and Mojave toxin [P62023] basic chains are 100% identical whereas the partial sequence (77 residues) gathered for the B. nigroviridis homologue protein (HPLC fraction 14, Table 1) exhibits 10 different positions (13% divergence). Similarly, the acidic chain sequences of B. nigroviridis crotoxin-like molecule (fraction 13, Table 1) depart in 7 positions (17% divergence) from crotoxin and Mojave toxin. For comparison, the amino acid sequences of the acidic chains of crotoxin [P0887] and Mojave toxin [P18998] differ in just 3 positions (3% divergence). Comparative venomics of the Central American rattlesnake C. simus and the South American C. durissus complex points to neurotoxicity and lethal venom activities to rodents, associated to an increased concentration of neurotoxins crotoxin and crotamine, as an adaptive paedomorphic trend along Crotalus dispersal in South America.22 Whether the high concentration of crotoxin-like toxin endows B. nigroviridis with increased (neuro)toxicity to different prey (i.e., lizard or frog) deserves detailed investigation. Extreme Variability among Bothriechis Venoms: Multiple Toxin Formulations for the Same Trophic Purpose? Venom lethal toxicity and venom metalloproteinase activity appear to be negatively associated. This observation has been generalized to rattlesnakes (Crotalus, Sistrurus) as an entire clade:44,52 type I venoms show high metalloproteinase activity and low toxicity, while type II venoms are very toxic and have low to very low levels of SVMPs. High toxicity is commonly associated to neurotoxic PLA2s. Powell and Lieb53 have predicted that the extremely high neurotoxicity exhibited by North American rattlesnakes represents a transitory populational phenomenon associated with novel prey bases. Comparative venomics of Central and South American rattlesnakes pointed to neurotoxicity and lethal venom activities to rodents, associated to an increased concentration of crotoxin, as an adaptive paedomor-

research articles phic trend along Crotalus dispersal in South America.22 Paedomorphosis, the retention of juvenile traits in adult forms, has been also invoked to explain the occurrence of type I versus type II venoms in C. oreganus/viridis.52,54 The occurrence of high concentration of crotoxin-like and the absence of SVMPs (Table 2) indicate that B. nigroviridis venom belongs to the type II class, although the toxicity of this venom is not as high as that of other crotoxin-containing venoms. Whether this venom composition results from a paedomorphic trait deserves further investigations. Venoms represent trophic adaptations, and thus, a deep insight into their toxin composition may provide clues for rationalizing their biological effects and for reconstructing the natural history of the organisms that produce them. Unraveling the composition of B. nigroviridis venom offered us the opportunity to compare the venom phenotypes of three adult Bothriechis taxa (Table 2). The marked compositional differences between the venoms of B. schlegelli, B. lateralis, and B. nigroviridis are puzzling. Hence, although a small but increasing number of studies strongly support the view that a high degree of differentiation in the venom proteome among congeneric taxa may reflect adaptation for differential utilization of distinct prey types,55-59 diet/composition relationship is likely rather complex,60 and this notion remains controversial.61,62 In addition, as discussed below, the three Bothriechis species alluded exhibit similar patterns of diet. Being arboreal, Bothriechis snakes are essentially “sit-andwait” predators, which passively wait for prey at strategic hunting sites, likely selected through chemosensory searching.63 General questions related to arboreality and the constraints or opportunities of arboreal habitats have been recently addressed by Lillywhite and Henderson.64 For example, the arboreal snakes may feed on smaller, less aggressive prey, such as frogs and lizards, and do not release prey during the feeding process, avoiding the inconvenience of tracing the released prey from a twig. Feeding on large rodents may be dangerous to the snake if not released immediately after the strike. Campbell and Solo´rzano65 mentioned that arboreal species such as Bothriechis spp. almost invariably seize and hold their prey. B. lateralis preys primarily on mice and sometimes on small birds and bats.3 Although its diet is relatively unknown, adult B. nigroviridis appear to feed on rodents (mice), lizards, and frogs, but occasionally they also capture small birds.3 The diet of B. schlegelii is composed of rodents, lizards (genera Dactyloa and Norops), frogs (genera Hyla and Eleuterodactylus), bats, and occasionally small birds.3 Despite exhibiting similar patterns of diet, comparison of venom proteomes reveals a remarkable compositional diversification among the three Bothriechis species (Table 2), mirrored by their distinct toxic effects. B. schlegelii venom inflicts significant myonecrosis in experimental animal models,8,9 whereas rabbits injected with B. lateralis venom developed a conspicuous edema and hemorrhage in the muscle where venom was administered, without histological evidence of myonecrosis.50 These results suggest that different venom formulations have evolved in different taxa for the same trophic purpose. In line with the above outlined trend among rattlesnake venoms,44,52 in mice, the type I venom of B. schlegelii showed a higher toxicity than the type II venom of B. lateralis (intravenous LD50 of 2 mg/kg for the former and 4.8 mg/kg for the latter),51 and the venom of B. nigroviridis has a higher toxicity than that of the other Bothriechis species. Results on the proteomics of Bothriechis venoms illustrate the versatility of venoms as a system to achieve the purpose of prey immobilization through different strategies. Journal of Proteome Research • Vol. 9, No. 8, 2010 4239

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Concluding Remarks Elucidation of the venom proteomes of three palm pitviper species found in Costa Rica, B. nigroviridis (present work), B. lateralis, and B. schlegelii,10 clearly evidenced that, despite their common arboreal habitats and similarities in diet, markedly different adaptations in terms of toxin composition have evolved in each case. In particular, B. nigroviridis venom presents two outstanding features when compared to that of other pitvipers, including the abundant presence of a crotoxinlike PLA2, only typical of rattlesnakes, and the complete lack of SVMPs, well-known to be main venom components in many viperid snake species. Our comparative venomic analyses of three arboreal pitviper species of the same genus illustrate how quite distinct toxicological strategies, based upon highly variable biochemical formulations of their venoms, provide successful evolutive solutions to the same trophic purpose. To fully understand the biological implications of our findings, further work needs to be carried out on the feeding behavior, diet, and other biological aspects of these arboreal species.

Acknowledgment. This work has been financed by grants BFU2007-61563 and BFU2010-17373 from the Ministerio de Ciencia e Innovacioń, Madrid, Spain, projects from the Vicerrectorıá de Investigacioń, Universidad de Costa Rica (741-A7-611), CRUSA-CSIC (2007CR0004), and CYTED (206AC0281). Proteomic studies performed at the Proteomics Laboratory of Instituto Clodomiro Picado were supported by CONARE and Vicerrectorıá de Investigacioń, Universidad de Costa Rica. Travelling between Spain and Costa Rica was financed by Acciones Integradas 2006CR0010 between CSIC and the University of Costa Rica (UCR). References (1) Campbell, J. A.; Lamar, W. W. The Venomous Reptiles of the Western Hemisphere; Comstock Publishing Associates: Ithaca, New York, and London, 2004. ¨ . Dr. Hoffmann in Costa Rica gesam(2) Peters, W. die von Hrn, U melten und an das Ko¨nigl. In Zoologische Museum gesandten Schlangen. Monatsber. Ko¨nigl. Preuss Akad. Wiss.: Berlin, 1859; pp 275-278. (3) Solo´rzano, A. Serpientes de Costa Rica. Editorial INBio: San Jose´, Costa Rica, 2004. (4) Bolan ˜ os, R. Las serpientes venenosas de Centroame´rica y el problema del ofidismo. Rev. Cost. Cienc. Med. 1982, 3, 165–184. (5) Bolan ˜ os, R. Serpientes, Venenos y Ofidismo en Centroame´rica; Editorial Universidad de Costa Rica: San Jose´, Costa Rica, 1984. (6) Gutie´rrez, J. M.; Lomonte, B. Local tissue damage induced by Bothrops snake venoms. A review. Mem. Inst. Butantan 1989, 51, 211–223. (7) Warrell, D. Snakebites in Central and South America: Epidemiology, Clinical Features, and Clinical Management. In The Venomous Reptiles of the Western Hemisphere; Campbell, J. A., Lamar, W. W., Eds.; Comstock Publishing Associates: Ithaca, New York, and London, 2004; pp 709-762. (8) Gutie´rrez, J. M.; Chaves, F. Proteolytic, hemorrhagic and myonecrotic effects of the venoms of Costa Rican snakes from the genera Bothrops, Crotalus and Lachesis. Toxicon 1980, 18, 315–321. (9) Tu, A. T.; Homma, M. Toxicologic study of snake venoms from Costa Rica. Toxicol. Appl. Pharmacol. 1970, 16, 73–78. (10) Lomonte, B.; Escolano, J.; Fernańdez, J.; Sanz, L.; Angulo, Y.; Gutie´rrez, J. M.; Calvete, J. J. Snake venomics and antivenomics of the arboreal neotropical pitvipers Bothriechis lateralis and Bothriehis schlegelii. J. Proteome Res. 2008, 7, 2445–2457. (11) Crother, B. I.; Campbell, J. A.; Hillis, D. M. Phylogeny and historical biogeography of the palm pitvipers, genus Bothriechis: biochemical and morphological evidence. In Biology of the Pitvipers; Campbell, J. A., Brodie, E. D., Jr., Eds.; Tyler: Selva, TX, 1992; pp 1-19. (12) Gutie´rrez, J. M.; Sanz, L.; Escolano, J.; Fernańdez, J.; Lomonte, B.; Angulo, Y.; Rucavado, A.; Warrell, D. A.; Calvete, J. J. Snake venomic of the Lesser Antillean pit vipers Bothrops caribbaeus and Bothrops

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