Snake Venomics of Bothriechis nigroviridis Reveals Extreme

Variability among Palm Pitviper Venoms: Different Evolutionary

Solutions for the Same Trophic Purpose

Julian Fernandez,† Bruno Lomonte,† Libia Sanz,‡ Yamileth Angulo,† Jose Marıa Gutierrez,† andJuan J. Calvete*,‡

Instituto Clodomiro Picado, Facultad de Microbiologıa, Universidad de Costa Rica, San Jose, Costa Rica, andInstituto 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-speckledpalm pitviper, Bothriechis nigroviridis, a neotropical arboreal pitviper from Costa Rica. In marked contrastto other Bothriechis species investigated, the venom of B. nigroviridis does not possess detectableZn2+-dependent metalloproteinases, and is uniquely characterized by a high content of crotoxin-likePLA2 and vasoactive peptides. These data suggest that different evolutionary solutions have evolvedwithin the arboreal genus Bothriechis for the same trophic purpose. The venom from B. nigroviridis isdevoid of hemorrhagic activity, has low edematogenic and coagulant effects, presents modest myotoxicand 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 majorrole 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 slenderto medially robust, arboreal, prehensile-tailed, New Worldpitvipers.1 Except for B. schlegelii, which ranges in humidlowlands and foothills from southern Mexico through PacificEcuador to Peru and western Venezuela, Bothriechis speciesare confined to montane regions between the Isthmus ofTehuantepec 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 arelatively rare, small (most adults commonly averaging 60-80cm in length) venomous pitviper.1 The specific name is derivedfrom the Latin nigro (black) and viridis (green) in reference toits distinctive color pattern, which may represent an arborealadaptation providing camouflage to avoid detection (Figure 1).According to Campbell and Lamar,1 this species inhabitssubtropical rainforests and temperate forests at medium to highelevations, from 1150 to over 3000 m, on both the Atlantic andPacific slopes of the Cordillera Tilaran (highlands of Mon-teverde) and Cordillera Volcanica Central in the southeasternAlajuela province in the central valley of Costa Rica, south-eastward through the Cordillera de Talamanca to Chiriquı

province in Panama.1 Like the other Bothriechis species, B.nigroviridis is arboreal, although it has been also found on theground or at the base of trees and shrubs, where the densefoliage may offer camouflage and in addition represents asupportive ecosystem to capture prey. Solorzano reported thatB. nigroviridis is mostly nocturnal. Adults feed primarily onsmall rodents, lizards, and frogs, although occasionally theycapture small birds.3

Although Bothriechis venoms investigated seem to be ofmoderate toxicity, bites may have dire consequences due tothe arboreal nature of these snakes which results in many ofthe bites being inflicted in the head, neck, and shoulderregions.1,3 B. schlegelii (eyelash pit viper), a relatively smallspecies that rarely exceeds 75 cm in length found in mesicforest at elevations almost from sea level to 2640 m altitude inCentral and South America,3 causes a number of envenoma-tions in Costa Rica.4 Documentation of human accidents byBothriechis snakebites is scarce. Indeed, we were unable to findany report on the pathophysiology of human envenomationby B. lateralis. On the other hand, initial symptoms of B.schlegelii snakebite include localized pain, progressive hemor-rhagic edema, and in some cases, hemorrhagic blisters or hives,ecchymoses, and necrosis.5-7 It has also been shown that thevenom of B. schlegelii induces significant myonecrosis inexperimental animal models.8,9 Gutierrez and Chaves8 studiedthe venoms of 10 Costa Rican species of pitvipers and foundthat those of B. schlegelii (and Bothrops asper) had the greatestmyonecrotic activity, whereas the venoms of B. lateralis (andCerrophidion godmani) showed the strongest proteolytic activ-

* To whom correspondence should be addressed. Juan J. Calvete, Institutode 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: jcalvete@ibv.csic.es.

† Universidad de Costa Rica.‡ Instituto de Biomedicina de Valencia.

4234 Journal of Proteome Research 2010, 9, 4234–4241 10.1021/pr100545d 2010 American Chemical SocietyPublished on Web 06/30/2010

ity toward casein. Campbell and Lamar cite that B. nigroviridishas been implicated in human fatalities, and bites are reportedto cause intense pain, nausea, and asphyxia.1 However, clinicalliterature on envenomations by B. nigroviridis is scarce, andwe found no single reference to this species in the literature(PubMed).

In a previous work, we have reported the proteomic char-acterization of B. schlegelii and B. lateralis venoms.10 Thevenom proteomes of B. lateralis and B. schlegelii comprisesimilar number of distinct proteins belonging, respectively, to8 and 7 protein families. The two Bothriechis venoms containbradykinin-potentiating peptides (BPPs), and proteins from thephospholipase A2 (PLA2), serine proteinase, L-amino acidoxidase (LAO), cysteine-rich secretory protein (CRISP), andZn2+-dependent metalloproteinase (SVMP) families, albeit eachspecies exhibits different relative abundances. Moreover, thesevenoms also contain unique components, for example, svVEGFand C-type lectin-like molecules in B. lateralis, and Kazal-typeserine proteinase inhibitor-like proteins in B. schlegelii.10

B. schlegelii represents the sister taxon to all other Bothriechisspecies, whereas B. lateralis segregates with B. bicolor and B.marchi in the most recently isolated clade from a commonancestor.11 Using a similarity coefficient, we estimated that thesimilarity of the venom proteins between these two Bothriechistaxa may be <10%, indicating a high divergence in their venomcompositions in spite of the fact that both species have evolvedto adapt to arboreal habits and seem to have similar generalist-type diets. The major toxin families of B. lateralis and B.schlegelii venoms are SVMP (55% of the total proteins) and PLA2

(44%), respectively. Their different venom toxin compositionsprovided clues for rationalizing the distinct signs of enveno-mation in experimental animals caused by B. schlegelii and B.lateralis.10 Here we sought to investigate the composition andbiological activities of the venom of B. nigroviridis, a thirdspecies of the arboreal genus Bothriechis found in Costa Rica.Strikingly, the venom proteome of this species does not possessdetectable Zn2+-dependent metalloproteinases, and is uniquelycharacterized by a high content of crotoxin-like PLA2 subunitchains and vasoactive peptides. Each of these groups of toxinsrepresents as much as 38% of total venom proteins. These datasuggest that different evolutionary solutions have evolvedwithin the arboreal genus Bothriechis for the same trophicpurpose, and underscore the versatility of venoms as adaptivetraits in these viperid snakes.

Experimental Section

Isolation and Characterization of Venom Proteins. Venomwas milked from three adult B. nigroviridis specimens collectedin Vara Blanca, province of Heredia (one specimen), andCascajal, Coronado, province of San Jose (two specimens), andkept at the serpentarium of the Instituto Clodomiro Picado(University of Costa Rica). Proteins from pooled venom wereseparated by reverse-phase HPLC as described.10,12-14 Isolatedfractions were subjected to N-terminal sequence analysis (usinga Procise instrument, Applied Biosystems, Foster City, CA)following the manufacturer’s instructions. Amino acid sequencesimilarity searches were performed against the available data-banks using the BLAST program15 implemented in the WU-BLAST2 search engine at http://www.bork.embl-heidelberg.de.The molecular masses of the purified proteins were determinedby SDS-PAGE (on 12 or 15% polyacrylamide gels), and byelectrospray ionization (ESI) mass spectrometry using anApplied Biosystems QTrap 2000 mass spectrometer16 operatedin Enhanced Multiple Charge mode in the range m/z 600-1700.Protein bands of interest were excised from Coomassie BrilliantBlue-stained SDS-PAGE gels and subjected to automatedreduction, alkylation, and in-gel digestion with sequencinggrade porcine pancreas trypsin (Promega). Doubly or triplycharged ions of selected peptides from mass fingerprint spectra(recorded with Applied Biosystems Voyager-DE Pro and 4800mass analyzers) were sequenced by CID-MS/MS using AppliedBiosystem’s TOF-TOF 4800 and QTrap 2000 and 3200 instru-ments. Production spectra were interpreted manually or usingthe online form of the MASCOT program at http://www.matrixscience.com against a private database containing vi-perid protein sequences deposited in the SwissProt/TrEMBLdatabase plus the previously assigned peptide ion sequencesfrom snake venomics projects carried out in our laborato-ries.17-25 MS/MS mass tolerance was set to (0.6 Da. Carba-midomethyl cysteine and oxidation of methionine were fixedand variable modifications, respectively.

Studies of the Toxic and Enzymatic Activities of B.nigroviridis Venom. Lethal Activity and Its Neutralizationby Two Antivenoms. Different amounts of venom were injectedintraperitoneally (ip) in a volume of 0.5 mL phosphate-bufferedsaline (PBS; 0.12 M NaCl, 40 mM sodium phosphate, pH 7.2).Groups of five mice (CD-1; 16-18 g body weight) were usedfor each dose tested. Control animals received the same volumeof PBS alone. Deaths occurring within 48 h were recorded andthe Median Lethal Dose (LD50) was estimated by probits.Neutralization of lethality was assessed by ip route. Forassessing the neutralization ability of antivenoms, groups of

Figure 1. Reverse-phase HPLC separation of the venom proteinsfrom B. nigroviridis. Two milligrams of B. nigroviridis venom wasapplied to a Lichrosphere RP100 C18 column, which was thendeveloped with the following chromatographic conditions: iso-cratically (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 werecollected manually and characterized by N-terminal sequencing,SDS-PAGE, and ESI mass spectrometry. Inset, SDS-PAGE show-ing the protein composition of the reverse-phase HPLC separatedvenom 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 wereexcised and subjected to in-gel digestion, tryptic peptide massfingerprinting, and CID-MS/MS of selected doubly or triplycharged peptide ions. The results are shown in Table 1. Thepicture of B. nigroviridis was taken at the Instituto ClodomiroPicado by J.F. and corresponds to one of the specimens used toobtain venom for this study.

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five mice (16-18 g) were injected with either 4 LD50 of thevenom dissolved in 0.5 mL of PBS, or with the same amountof venom which had been previously incubated for 30 min at37 °C with the antivenom, at a ratio of 2000 µL antivenom/mgvenom. The antivenoms tested were the polyvalent antivenomfrom Instituto Clodomiro Picado, Costa Rica, and the crotalicantivenom from Instituto Butantan, Brazil. The former isproduced by immunizing horses with equal amounts of thevenoms of B. asper, Crotalus simus, and Lachesis stenophrys,26

whereas the latter is generated by immunization with a mixtureof equal amounts of Crotalus durissus terrificus and Crotalusdurissus collilineatus venoms collected in Southeastern andMidwestern Brazil, in the states of Sao Paulo, Mato Grosso, andMinas Gerais (Marisa Maria Teixeira da Rocha, InstitutoButantan, personal communication). Deaths occurring within48 h were recorded. All procedures involving animals used inthis study were approved by the Institutional Committee forthe Care and Use of Laboratory Animals (CICUA) of Univer-sidad de Costa Rica.

Hemorrhagic Activity. A group of five mice (18-20 g) wasinjected intradermally (id), in the ventral abdominal region,with a dose of 100 µg of venom dissolved in 100 µL of PBS.After 2 h, mice were sacrificed by CO2 inhalation, and theirskins were removed to determine the diameter of hemorrhagicspots in the inner side of the skin.27

Myotoxic Activity. A group of four mice (18-20 g) receivedan intramuscular (im) injection with 50 µg of venom dissolvedin 50 µL of PBS. Control animals (four mice with the sameweight) were injected with 50 µL of PBS alone. Three hoursafter injection, mice were bled, plasma was separated, and thecreatine kinase (CK) activity in plasma was determined usingthe CK-NAc kit (Biocon Diagnostik, Germany), and expressedas Units/L.

Coagulant Activity. Various amounts (80, 100, and 120 µg)of venom, dissolved in 100 µL of PBS, were added to 200 µL ofcitrated human plasma that had been previously incubated for5 min at 37 °C. Controls included the addition of 100 µL ofPBS to plasma samples. Clotting times were recorded and theMinimum Coagulant Dose (MCD), defined as the lowest venomdose that induced clotting in 60 s, was determined.28,29 Assayswere performed in triplicate.

Phospholipase A2 Activity. The phospholipase A2 activity ofthe venom was determined on micellar phosphatidylcholineaccording to the phenol red-based colorimetric method.30

Briefly, aliquots of 10 µL, containing varying amounts of venom(125, 250, 500, 1000, 2000, and 4000 ng), were added to 1 mLof substrate in a thermoregulated cuvette at 30 °C and, after alag period of 20 s, the decrease in absorbance at 558 nm wascontinuously monitored for 2 min. One unit of PLA2 activitywas defined as a change of 0.001 in absorbance per minute.

Edema-Forming Activity. Various amounts (1, 2, 4, and 8µg) of venom, dissolved in 50 µL of PBS, were injectedsubcutaneously (sc) in the right footpad of mice (18-20 g; n )4 per group). The same animals received an injection of 50 µLof PBS alone in the left foot pad. After 30, 60, 180, and 360min, the thickness of both footpads was measured with a low-pressure spring caliper. Edema was expressed as the percentageincrease in thickness of the footpad injected with venom, ascompared to that injected with PBS. The Minimum Edema-forming Dose (MED) corresponds to the dose of venom thatinduced 30% edema.

Statistical Analysis. Results are presented as mean ( SD,with the exception of lethality assays in which variability was

expressed as 95% confidence limits. Comparisons betweenmeans of two groups were performed with the Student’s ttest.

Results and Discussion

Characterization of the Venom Proteome of B. nigroviridis.The protein composition of the venom of B. nigroviridis wasinvestigated using our snake venomics approach,23-25 whichincludes fractionation by reverse-phase HPLC (Figure 1),analysis of each chromatographic fraction by SDS-PAGE (Figure1, inset), N-terminal sequencing (Table 1), and MS/MS analysisof tryptic peptides recovered after in-gel digestion of electro-phoretic protein bands (Table 1).

HPLC fractions 2-11 contained a number of vasoactive, bothbradykinin-inhibitory and bradykinin-potentiating, peptides.Bradykinin-inhibitory peptides (BIP) have been reported in onlya few other New World pitviper venoms, for example, from theMexican moccasin (Agkistrodon bilineatus), the prairie rattle-snake (Crotalus viridis viridis), the western diamondbackrattlesnake (Crotalus atrox), and the South American bush-master (Lachesis muta).31,32 BIPs are proteolytically releasedfrom larger precursors, which also encode an N-terminalbradykinin-potentiating peptides (BPP) and a C-terminal C-type natriuretic peptide (NAP).31,32 By contrast, the vasoactivepeptide precursors commonly found in Bothrops species, thatis, Bothrops jararaca [Q6LEM5], Bothrops jararacussu [Q7T1M3],and Bothrops insularis [P68515], contain several N-terminalBPPs and the C-terminal NAP, but lack BIP, indicating adifferent evolutionary trend in this group of snakes, which haveevolved hypotensive venoms. Mapping the different vasoactivepeptide precursor structures onto a cladogram of Crotalinaesuggested that duplications of the N-terminal region bearingthe BPPs occurred in the common ancestor of Lachesis andBothrops, and that BIP was subsequently lost in Bothrops.32

Our finding of the occurrence of BIP in Bothriechis suggeststhat rearrangements in the BPP-BIP-NAP ancestor gene thatgave rise to the (BPP)n-NAP type of gene occurred after thedivergence of the Middle American Bothriechis and the SouthAmerican Bothrops clades during the early middle Miocene,13-14 Mya.33

A synthetic replicate of the BIP TPPAGPDVGPR was foundto antagonize the vasodilatory actions of bradykinin at the B2receptor in a rat-tail artery preparation.31 Although BIP ap-peared to be a less potent vasoconstrictor than sarafotoxins,34

a group of 21-residue cardiotoxic peptides isolated from Africansnake venoms of genus Atractaspis that induce severe coronaryvasoconstriction leading to bradycardia and vasospasm,34,35 BIPmay disrupt the functioning of the cardiovascular system,supplementing the overall toxic effect of the snake venom. Onthe other hand, bradykinin-potentiating peptides are inhibitorsof the angiotensin I-converting enzyme, which enhance thehypotensive effect of bradykinin causing a vascular shock inthe snake’s prey.36,37 The combination of bradykinin-potentiat-ing and -inhibitory peptides in the same venom is somehowpuzzling, owing to the antagonistic effects induced by thesefamilies of peptides. However, it can be speculated that eachof these groups may have a predominant effect in differenttypes of prey. Owing to the varied diet of this species, this mightprovide versatility in affecting the cardiovascular performanceof different prey items.

Crotoxin-like acidic and basic chains, eluted in fractions12-14 (Figure 1, Table 1), represent highly abundant toxincomponents of B. nigroviridis venom, comprising >38% of the

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toxin proteome. Crotoxin is a heterodimeric PLA2 moleculeexhibiting presynaptic �-neurotoxicity, but also inducing sys-temic myotoxicity and other deleterious effects,38-40 which wasfirst isolated in 1938 by Slotta and Fraenkel-Conrat41 from thevenom of the tropical rattlesnake (C. d. terrificus). A homologueof South American crotoxin, Mojave toxin,42 is present invenoms of certain populations of North American Crotalusspecies, including the Mojave rattlesnake (Crotalus scutulatusscutulatus), the midget-faded rattlesnake (Crotalus oreganusconcolor), the southern Pacific rattlesnake (Crotalus helleri), andthe tiger rattlesnake (Crotalus tigris).43,44 Crotoxin and Mojavetoxin are responsible for the characteristic systemic neuro- andmyotoxic effects observed in envenomations by these taxa.7 Toour knowledge, crotoxin or crotoxin-like proteins have not beenpreviously found in the venoms of New World pit vipers other

than rattlesnakes, that is, Crotalus and Sistrurus spp, and arecertainly absent from the venom proteomes of B. lateralis andB. schlegelii.10

Serine proteinases comprise 18% of venom toxins andrepresent the third more abundant protein family in B. ni-groviridis venom (Tables 1 and 2, Figure 2). Members of thistoxin class contribute to viperid venom toxicity by affectingplatelet aggregation, blood coagulation, fibrinolysis, the comple-ment system, blood pressure, and the nervous system.45

Internal peptide sequences gathered from the serine protein-ases recovered in fractions 18 and 21 display highest similarity(76-100%) with thrombin-like enzymes from a number ofsnake species (i.e., ABB76280 from B. asper). The trypticpeptides sequenced from the serine proteinase eluted infraction 19 exhibit 90-100% identity with plasminogen activa-

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

peptide ionHPLC

fraction N-terminal sequence molecular mass m/z z MS/MS-derived sequence protein/peptide class

1 np2 SPPAGPDGGPR 1007.5 504.2 2 SPPAGPDGGPR Bradykinin-inhibitory peptide3 ND 989.4 495.2 2 SPPAGP (483.3) Bradykinin-inhibitory peptide6 ND 1513.6 757.3 2 SPPAGDPDGGPRGA(282.1) Bradykinin-inhibitory peptide

1839.4 920.2 2 SPPAGDPDGGPRDS(535.1) Bradykinin-inhibitory peptide7 ND 1291.4 646.2 2 (249.1)WSXGHHIPP Bradykinin-potentiating peptide8 Blocked 1275.7 638.2 2 (249.1)WSPGHHIPP Bradykinin-potentiating peptide9 ND 2303.6 768.5 3 Glycopeptide

2288.2 1144.1 2 Glycopeptide10 Blocked 1244.8 622.8 2 ZKDWPPPISPP Bradykinin-potentiating peptide11 ND 1163.2 582.1 2 PP(756.1)PP Bradykinin-potentiating peptide

1235.4 618.2 2 PP(828.1)PP Bradykinin-potentiating peptide12, 13 a SPENCQGESQPC 9 kDa1 ∼Crotoxin-like acidic chain [P08878]

b GCYCDAEGQGWPQDA ∼Mojave toxin-like acidic chain [P18998]c EENGDIVCGEXTPC

14 NLLQFNRMIKLETKKNAV 14092 871.8 2 GTWCEEQICECDR ∼Crotoxin-like basic chain [P62022]PFYAFYGCYCGWGGQGQ ∼Mojave toxin-like basic chain [P62023]PKDATDRCCXEHDCCYGKLTKCNTKWDLY

15, 16 Blocked 13 kDa1 685.3 2 ZVMPFMEVYSR svVEGF17 SVDFDSESPRKPEIQ 23 kDa1 569.7 2 SVDFDSESPR CRISP18 V(I/V)GGDECNINEHR(S/F)L 36 kDa1 1190.4 1 XMGWGTXSPTK Serine proteinase

1571.3 1 BVXNEDEQTRDPK1443.5 1 VXNEDEQTRDPK1456.5 1 SXPSNPPSVGSVCR

19 IIGGDECNINEHRSL 38 kDa1 595.2 2 WDBDIMXXR Serine proteinase763.8 2 IIGGDECNINEHR617.8 3 XAPXSXPSNPPSVGSVCR837.8 3 (214.3)XPDVPHCANXNXXDYEVCR929.6 1 LQFGLHSK

20 VIGGDECNINEHRSL 33 kDa1 Serine proteinase21 VVGGDECNINEHRSL 31 kDa1 782.8 2 CAPXNXXDYAVCR Serine proteinase

488.3 2 ETYPNVPR696.4 2 AAYPWW(370.2)TK919.4 2 XAPXSXPSSPPS(257.2)VCR

22 ND 72 kDa1 860.3 2 ETPVXSNPGTNSAEFR 5′-nucleotidase795.2 2 XXAXGHSGFFEDQR

1289.7 1 ZVPVVQAYAFGK23 ADTRNPLEECFRETD 66 kDa1 757.9 2 ETDYEEFLEIAR L-amino acid oxidase

1651.0 1 NDKEGWYANLGPMR1281.8 SAGQLYEESLGK

24 ND 110 kDa1 514.8 2 XHYTXXXR Unknown788.0 (189.0)PTSVPPRPVAPXR

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, cysteineresidues 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 materialfound. Underlined residues in the N-terminal sequences of the crotoxin-like acidic and basic chains depart from the homologue crotoxin/Mojave toxinamino acid sequences.

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tor AAD01623 from the short-tailed Mamushi (Gloydius blom-hoffi brevicaudus).46

Minor toxin groups found in B. nigroviridis venom includesnake venom vascular endothelial growth factor (VEGF) iso-forms (fractions 15 and 16), a cysteine-rich secretory protein(CRISP, fraction 17), a 5′-nucleotidase (peak 22), an L-aminoacid oxidase (LAO, fraction 23), and an unknown protein ofmolecular mass 110 kDa (fraction 24). Each of these proteinfamilies comprises <3% of the total venom proteins (Table 2).The biological activities of these toxins isolated from a varietyof snake venoms have been investigated in different in vitrosystems, but their role in the pathology of envenomationremains elusive. Snake venom VEGFs induce endothelial cellproliferation, increment in vascular permeability, and hypoten-sion;47 CRISP molecules inhibit smooth muscle contraction and

cyclic nucleotide-gated ion channels;48 the pharmacologicaleffects of LAO include induction of platelet aggregation, apo-ptosis of cells, and cytotoxicity.49 The pathophysiologicalrelevance of the minor B. nigroviridis venom toxins deservesdetailed investigation, but we suspect that their effects maybe completely overshadowed by the hypotensive and neuro-toxic symptoms provoked by BPPs and crotoxin-like PLA2,respectively. Rapid immobilization due to BPP-induced hy-potension and crotoxin-induced paralytic effects causing pro-gressive paralysis may represent an adaptation of B. nigrovir-idis, a “sit-and-wait” predator, for outweighing the threat ofholding large, dangerous prey, and for restraining it fromescaping during the swallowing process.

Toxicological Profile of B. nigroviridis Venom. Table 3displays the toxic and enzymatic activities characterized in B.nigroviridis venom. The most striking feature is the absenceof hemorrhagic activity up to a dose of 100 µg of the venom,which is in evident contrast with all other Central Americanpit viper venoms previously studied and whose MinimumHemorrhagic Doses ranged between 0.5 and 5.1 µg.27 This isin agreement with the lack of SVMPs in the venom proteomeof this species, and is also in marked contrast to the proteomicand toxicological profiles of the venoms of other Bothriechistaxa, B. lateralis and B. schlegelii,10 in which SVMPs play a veryimportant role in the pathophysiology of envenomation, par-ticularly in B. lateralis.8,9,50 The venom from B. nigroviridis hadalso low edematogenic and coagulant activities. The MinimunEdema-forming Dose (MED) was higher than 8 µg (this doseonly induced 24% edema after an hour), while the MinimumCoagulant Dose (79.2 ( 5.7 µg) is very low compared with thatof other viperid venoms.29 This venom also presented modestmyotoxic and phospholipase A2 activities (Table 3). The mildin vitro coagulant effect and the lack of hemorrhagic activityof B. nigroviridis venom suggests that the role of its serineproteinases may not be associated with coagulopathy andbleeding, at least in the mouse, and may instead be linked toother effects, such as hypotension.

Table 2. Overview of the Relative Occurrence of Protein/Peptides (in Percentage of the Total HPLC-SeparatedComponents) of the Different Classes in the Venoms of B.nigroviridisa

% of total venom toxinsprotein/

peptide class B. nigroviridis B. lateralis B. schlegelii

PLA2 38.3 8.7 43.8- myotoxin - 8.7 43.8

- Crotoxin-like 38.3 - -Vasoactive peptides 37.0 11.1 13.4

- BIP 10.1 0.1 0.1- BPP 26.9 11.0 13.3

Serine proteinase 18.4 11.3 5.8svVEGF 2.8 0.5 -CRISP 2.1 6.5 2.1L-amino acid oxidase 0.5 6.1 8.95′ nucleotidase 0.5 - -unknown 0.4Kazal-type inhibitor - - 8.3C-type lectin-like - - 0.9 -SVMP - 55.1 17.7

- PIII 54.5 17.0- PI 0.6 0.7

a BIP and BPP, bradykinin-inhibitory and bradykinin-potentiatingpeptides, respectively. For comparison, the compositions of the venomproteomes of B. lateralis and B. schlegelii10 are also displayed. Majortoxins are highlighted in boldface in each taxa.

Figure 2. Overall composition of B. nigroviridis venom. Pie chartof the relative abundance (in % of total venom proteins) of thedifferent toxin classes found in the pooled venom of B. nigroviri-dis. CRISP, cysteine-rich secretory protein; LAO, L-amino acidoxidase; svVEGF, snake venom vascular endothelial growthfactor. Details of the individual proteins are shown in Table 1and the percentages of the different toxin classes in the venomsare listed in Table 2.

Table 3. Toxic and Enzymatic Activities of B. nigroviridisVenom and Neutralization of Its Lethal Activity

biological activity result

Median Lethal Dose,ip (LD50%)a

30.3 (10.8-44.9) µg

Neutralization of lethalityby the Costa Ricanpolyvalent antivenom

No neutralization upto 2000 µLantivenom/mg venom

Neutralization of lethalityby the crotalic antivenomfrom Instituto Butantan

All mice survived atthe level of 2000 µLantivenom/mg venom

Myotoxicityb 608 ( 230 U/LEdema (MED)c >8 µgCoagulant (MCD)d 79 ( 6 µgPhospholipase A2

e 198 ( 17 U/µgHemorrhage No hemorrhagic activity

up to a dose of100 µg of venom

a Detemined in 16-18 g mice; 95% confidence limits are included inparentheses. 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 PBSalone showed a plasma CK activity of 212 ( 93 U/L. c MinimumEdema-forming Dose: dose of venom inducing 30% edema aftersubcutaneous injection. d Minimum Coagulant Dose (MCD) is the lowestvenom 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 inabsorbance per minute.

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The intraperitoneal Median Lethal Dose (LD50) of B. ni-groviridis venom was 30 µg/mouse, that is, 1.76 mg/kg. Thisactivity is slightly more potent than those reported for otherCosta Rican snake venoms.51 The fact that the ICP polyvalentantivenom, prepared against the crotoxin-negative venoms ofB. asper, C. simus, and L. stenophrys, was unable to neutralizethe lethal activity of B. nigroviridis up to a level of 2000 µLantivenom/mg venom (Table 3) pointed to the crotoxin-likePLA2 as the major contributor to B. nigroviridis venom toxicity.This hypothesis is supported by neutralization assays using thecrotalic antivenom, produced at Instituto Butantan by theimmunization of horses with venom of C. d. terrificus, whichcontains high amounts of crotoxin.22 This antivenom fullyneutralized the lethal activity of B. nigroviridis venom at a ratioof 2000 µL antivenom/mg venom.

The scarcity of venom obtained for the present workprecluded determining the complete amino acid sequence ofthe crotoxin-like component of B. nigroviridis, a task that mightprovide valuable insights into the evolution and biologicalsignificance of this particular type of toxin, intensively studiedover many years. It is noteworthy, however, that the toxicityof this venom is not particularly high, especially when com-pared 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, 0.46mg/kg).51 This suggests that the relative abundance andstructural differences between the crotoxin-like component ofB. nigroviridis and the highly neurotoxic crotoxin/Mojave toxinmolecules may account for their different toxic potentials tomice. In line with this hypothesis, the amino acid sequence ofcrotoxin [P62022] and Mojave toxin [P62023] basic chains are100% identical whereas the partial sequence (77 residues)gathered for the B. nigroviridis homologue protein (HPLCfraction 14, Table 1) exhibits 10 different positions (13%divergence). Similarly, the acidic chain sequences of B. ni-groviridis crotoxin-like molecule (fraction 13, Table 1) departin 7 positions (17% divergence) from crotoxin and Mojave toxin.For comparison, the amino acid sequences of the acidic chainsof crotoxin [P0887] and Mojave toxin [P18998] differ in just 3positions (3% divergence). Comparative venomics of the Cen-tral American rattlesnake C. simus and the South American C.durissus complex points to neurotoxicity and lethal venomactivities to rodents, associated to an increased concentrationof neurotoxins crotoxin and crotamine, as an adaptive paedo-morphic trend along Crotalus dispersal in South America.22

Whether the high concentration of crotoxin-like toxin endowsB. nigroviridis with increased (neuro)toxicity to different prey(i.e., lizard or frog) deserves detailed investigation.

Extreme Variability among Bothriechis Venoms: MultipleToxin Formulations for the Same Trophic Purpose? Venomlethal toxicity and venom metalloproteinase activity appear tobe negatively associated. This observation has been generalizedto rattlesnakes (Crotalus, Sistrurus) as an entire clade:44,52 typeI venoms show high metalloproteinase activity and low toxicity,while type II venoms are very toxic and have low to very lowlevels of SVMPs. High toxicity is commonly associated toneurotoxic PLA2s. Powell and Lieb53 have predicted that theextremely high neurotoxicity exhibited by North Americanrattlesnakes represents a transitory populational phenomenonassociated with novel prey bases. Comparative venomics ofCentral and South American rattlesnakes pointed to neurotox-icity and lethal venom activities to rodents, associated to anincreased concentration of crotoxin, as an adaptive paedomor-

phic trend along Crotalus dispersal in South America.22 Pae-domorphosis, the retention of juvenile traits in adult forms,has been also invoked to explain the occurrence of type I versustype II venoms in C. oreganus/viridis.52,54 The occurrence ofhigh concentration of crotoxin-like and the absence of SVMPs(Table 2) indicate that B. nigroviridis venom belongs to the typeII class, although the toxicity of this venom is not as high asthat of other crotoxin-containing venoms. Whether this venomcomposition results from a paedomorphic trait deserves furtherinvestigations.

Venoms represent trophic adaptations, and thus, a deep insightinto their toxin composition may provide clues for rationalizingtheir biological effects and for reconstructing the natural historyof the organisms that produce them. Unraveling the compositionof B. nigroviridis venom offered us the opportunity to comparethe venom phenotypes of three adult Bothriechis taxa (Table 2).The marked compositional differences between the venoms ofB. schlegelli, B. lateralis, and B. nigroviridis are puzzling. Hence,although a small but increasing number of studies stronglysupport the view that a high degree of differentiation in the venomproteome among congeneric taxa may reflect adaptation fordifferential utilization of distinct prey types,55-59 diet/compositionrelationship is likely rather complex,60 and this notion remainscontroversial.61,62 In addition, as discussed below, the threeBothriechis species alluded exhibit similar patterns of diet.

Being arboreal, Bothriechis snakes are essentially “sit-and-wait” predators, which passively wait for prey at strategichunting sites, likely selected through chemosensory searching.63

General questions related to arboreality and the constraints oropportunities of arboreal habitats have been recently addressedby Lillywhite and Henderson.64 For example, the arborealsnakes may feed on smaller, less aggressive prey, such as frogsand lizards, and do not release prey during the feeding process,avoiding the inconvenience of tracing the released prey froma twig. Feeding on large rodents may be dangerous to the snakeif not released immediately after the strike. Campbell andSolorzano65 mentioned that arboreal species such as Both-riechis spp. almost invariably seize and hold their prey. B.lateralis preys primarily on mice and sometimes on small birdsand 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 andNorops), frogs (genera Hyla and Eleuterodactylus), bats, andoccasionally small birds.3 Despite exhibiting similar patternsof diet, comparison of venom proteomes reveals a remarkablecompositional diversification among the three Bothriechisspecies (Table 2), mirrored by their distinct toxic effects. B.schlegelii venom inflicts significant myonecrosis in experimen-tal animal models,8,9 whereas rabbits injected with B. lateralisvenom developed a conspicuous edema and hemorrhage inthe muscle where venom was administered, without histologi-cal evidence of myonecrosis.50 These results suggest thatdifferent venom formulations have evolved in different taxa forthe same trophic purpose. In line with the above outlined trendamong rattlesnake venoms,44,52 in mice, the type I venom ofB. schlegelii showed a higher toxicity than the type II venomof B. lateralis (intravenous LD50 of 2 mg/kg for the former and4.8 mg/kg for the latter),51 and the venom of B. nigroviridishas a higher toxicity than that of the other Bothriechis species.Results on the proteomics of Bothriechis venoms illustrate theversatility of venoms as a system to achieve the purpose of preyimmobilization through different strategies.

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Concluding Remarks

Elucidation of the venom proteomes of three palm pitviperspecies found in Costa Rica, B. nigroviridis (present work), B.lateralis, and B. schlegelii,10 clearly evidenced that, despite theircommon arboreal habitats and similarities in diet, markedlydifferent adaptations in terms of toxin composition haveevolved in each case. In particular, B. nigroviridis venompresents two outstanding features when compared to that ofother pitvipers, including the abundant presence of a crotoxin-like PLA2, only typical of rattlesnakes, and the complete lackof SVMPs, well-known to be main venom components in manyviperid snake species. Our comparative venomic analyses ofthree arboreal pitviper species of the same genus illustrate howquite distinct toxicological strategies, based upon highly vari-able biochemical formulations of their venoms, provide suc-cessful evolutive solutions to the same trophic purpose. To fullyunderstand the biological implications of our findings, furtherwork needs to be carried out on the feeding behavior, diet, andother biological aspects of these arboreal species.

Acknowledgment. This work has been financed bygrants BFU2007-61563 and BFU2010-17373 from theMinisterio de Ciencia e Innovacion, Madrid, Spain, projectsfrom the Vicerrectorıa de Investigacion, Universidad deCosta Rica (741-A7-611), CRUSA-CSIC (2007CR0004), andCYTED (206AC0281). Proteomic studies performed at theProteomics Laboratory of Instituto Clodomiro Picado weresupported by CONARE and Vicerrectorıa de Investigacion,Universidad de Costa Rica. Travelling between Spain andCosta Rica was financed by Acciones Integradas 2006CR0010between CSIC and the University of Costa Rica (UCR).

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