Snake Venomics of the Lancehead Pitviper Bothrops asper: Geographic, Individual, and Ontogenetic Variations

Snake Venomics of the Lancehead Pitviper Bothrops asper:

Geographic, Individual, and Ontogenetic Variations

Alberto Alape-Giron,†,‡ Libia Sanz,§ Jose Escolano,§ Marietta Flores-Dıaz,† Marvin Madrigal,†

Mahmood Sasa,† and Juan J. Calvete*,§

Instituto Clodomiro Picado, Universidad de Costa Rica, San Jose, Costa Rica, Departamento de Bioquımica,Escuela de Medicina, 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 April 30, 2008

We report the comparative proteomic characterization of the venoms of adult and newborn specimens ofthe lancehead pitviper Bothrops asper from two geographically isolated populations from the Caribbeanand the Pacific versants of Costa Rica. The crude venoms were fractionated by reverse-phase HPLC, followedby analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF massfingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The twoB. asper populations, separated since the late Miocene or early Pliocene (8-5 mya) by the GuanacasteMountain Range, Central Mountain Range, and Talamanca Mountain Range, contain both identical anddifferent (iso)enzymes from the PLA2, serine proteinase, and SVMP families. Using a similarity coefficient,we estimate that the similarity of venom proteins between the two B. asper populations may be around52%. Compositional differences between venoms among different geographic regions may be due toevolutionary environmental pressure acting on isolated populations. To investigate venom variability amongspecimens from the two B. asper populations, the reverse-phase HPLC protein profiles of 15 venoms fromCaribbean specimens and 11 venoms from snakes from Pacific regions were compared. Within each B.asper geographic populations, all major venom protein families appeared to be subjected to individualvariations. The occurrence of intraspecific individual allopatric variability highlights the concept that a species,B. asper in our case, should be considered as a group of metapopulations. Analysis of pooled venoms ofneonate specimens from Caribbean and Pacific regions with those of adult snakes from the samegeographical habitat revealed prominent ontogenetic changes in both geographical populations. Majorontogenetic changes appear to be a shift from a PIII-SVMP-rich to a PI-SVMP-rich venom and the secretionin adults of a distinct set of PLA2 molecules than in the neonates. In addition, the ontogenetic venomcomposition shift results in increasing venom complexity, indicating that the requirement for the venomto immobilize prey and initiate digestion may change with the size (age) of the snake. Besides ecologicaland taxonomical implications, the geographical venom variability reported here may have an impact inthe treatment of bite victims and in the selection of specimens for antivenom production. The occurrenceof intraspecies variability in the biochemical composition and symptomatology after envenomation bysnakes from different gegraphical location and age has long been apreciated by herpetologist andtoxinologists, though detailed comparative proteomic analysis are scarce. Our study represents the firstdetailed characterization of individual and ontogenetic venom protein profile variations in two geographicalisolated B. asper populations, and highlights the necessity of using pooled venoms as a statisticallyrepresentative venom for antivenom production.

Keywords: Snake venomics • Bothrops asper • snake venom protein families • proteomics • viperidtoxins • N-terminal sequencing • mass spectrometry • geographical venom variation • individual venomvariation • ontogenetic shift

Introduction

The genus Bothrops (subfamily Crotalinae of Viperidae) com-prises 32 (http://www.reptile-database.org) or 37 species1 ofpitvipers, commonly referred as lanceheads, which are widelydistributed in tropical Latin America, from northeastern Mexico

to Argentina, and the southern parts of the lower Caribbeanislands.1 The species of this genus are responsible for the vastmajority of snakebites in Central and South America.2 In thisregard, the most important species are Bothrops asper (CentralAmerica and northern South America), Bothrops atrox (tropicallowlands of northern South America east of the Andes) andBothrops jararaca (southern Brazil, Paraguay and northern Ar-gentina). Without treatment, the fatality rate is estimated to beabout 7%, but with an appropriate antivenom therapy, it can bereduced to 0.5-3%.1 Nevertheless, many victims of B. aspersnakebite suffer from life threatening sequelae due to the tissue-damaging effects of the venom.2,3

* Address correspondence to: Juan J. Calvete, Instituto de Biomedicinade Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain. Phone, +34 96339 1778; fax, +34 96 369 0800; e-mail, [email protected].

† Instituto Clodomiro Picado, Universidad de Costa Rica.‡ Departamento de Bioquımica, Escuela de Medicina, Universidad de

Costa Rica.§ Instituto de Biomedicina de Valencia.

3556 Journal of Proteome Research 2008, 7, 3556–3571 10.1021/pr800332p CCC: $40.75 2008 American Chemical SocietyPublished on Web 06/17/2008

During the period 1990-2000, a total of 5550 snakebiteaccidents were reported in health centers in Costa Rica.4 Mostof the snakebites occurred in the Caribbean, South and CentralPacific, and North regions of the country, where B. asper, theonly Bothrops species present in Central America, is verycommon.4 In Costa Rica, B. asper (commonly named ”tercio-pelo”) is responsible for almost half of all snakebite accidents.5

The victims of B. asper bites show conspicuous local tissuedamage characterized by dermonecrosis, blistering, edema,local hemorrhage and myonecrosis,6,7 and, in severe cases,defibrin(ogen)ation, thrombocytopenia, platelet hypoaggrega-tion, bleeding distant from the bite site, disseminated intra-vascular coagulation, cardiovascular shock and acute renalfailure.6–8

Venoms represent the critical innovation in ophidian evolu-tion that allowed advanced snakes to transition from a me-chanical (constriction) to a chemical (venom) means of sub-duing and digesting prey and represents a key adaptation thathas played an important function in the diversification of theseanimals. Venom toxins likely evolved from a reduced set ofproteins with normal physiological functions which wererecruited into the venom proteome before the diversificationof the advanced snakes.9–12 Venoms produced by snakes of thefamily Viperidae (vipers and pit vipers) contain proteins thatinterfere with the coagulation cascade, the normal hemostaticsystem and tissue repair.13,14 Snake venom proteins belong toonly a few major protein families, including enzymes (serineproteinases, Zn2+-metalloproteinases, L-amino acid oxidase,group II PLA2) and proteins without enzymatic activity (disin-tegrins, C-type lectins, natriuretic peptides, myotoxins, CRISPtoxins, nerve and vascular endothelium growth factors, cystatinand Kunitz-type proteinase inhibitors). However, viperid ven-oms depart from each other in the composition and the relativeabundance of toxins.15

Snake venom composition may retain information on itsevolutionary history, and may thus have a potential taxonomi-cal value.16 In addition to understanding how venoms evolve,characterization of the protein/peptide content of snake ven-oms also has a number of potential benefits for basic research,clinical diagnosis, development of new research tools and drugsof potential clinical use, and for antivenom production strate-gies.17 To explore the putative venom components, severallaboratories have carried out transcriptomic analyses of thevenom glands of viperid (Bitis gabonica,18 Bothrops insularis,19

Bothrops jararacussu,20 B. jararaca,21 Agkistrodon acutus,22,23

Echis ocellatus,24 Lachesis muta,25 and Sistrurus catenatusedwardsii26), elapid (Oxyuramus scutellatus27), and colubrid(Philodryas olfersii28) snake species. Transcriptomic investiga-tions provide catalogues of partial and full-length transcriptsthat are synthesized by the venom gland. However, transcrip-tomes include translated and nontranslated mRNAs, transcriptsencoding nonsecreted proteins, housekeeping and cellular, inaddition to toxin precursor genes. Moreover, the transcriptomedoes not reflect within-species ontogenetic,29,39 individual andgeographic31 heterogeneity of venoms, which may account fordifferences in the clinical symptoms observed in accidentalenvenomations.

Geographic variability concerning the toxic and enzymaticactivites of B. asper venoms from the Caribbean versus thePacific regions of Costa Rica has been documented.32–34 Venomof specimens from the Caribbean region exhibited enhancedprocoagulant hemorrhagic and myonecrotic effects, whereasthe venom of specimens from the Pacific versant displayed a

higher proteolytic activity. Variations in the pattern of PLA2

isoforms in relation to geographic origin have been alsoreported.35,36

Prominent ontogenetic changes in the toxic and enzymaticactivites of B. asper venoms from Costa Rica34,37 and Colom-bia38 have also been noticed. This phenomenon appears to belinked to a shift in the feeding habits of juvenile versus adultsnakes, from cold-blooded (frogs and lizards) to warm-blooded(mammals) prey.38 Clinical reports indicated that envenoma-tion by juvenile B. asper specimens is associated to prominentblood coagulation alterations and hemorrhagic symptoms,39

despite the low amount of venom that a small size specimensmay inject in a bite. In line with the clinical observations, thevenom from newborns appeared to be more procoagulant invitro and, in experimental envenomations, showed a higherdefibrinating and hemorrhagic activities and a lower myotox-icity than the venom from adult specimens.34,37 On the otherhand, the latter displayed higher phospholipase A2 activity andhad a higher number of PLA2 isoforms.35 Besides its intrinsicbiological relevance, the characterization of the ontogeneticvariability of B. asper venom also has implications to under-stand the characteristics of envenomations in humans.

To address the need for detailed proteomic studies of snakevenoms, we have initiated a snake venomics project whoselong-term goal is the in-depth analysis of viperid venomproteomes. To date, we have reported the protein compositionof the venoms from the North American rattlesnakes Sistrurusmiliarius barbouri,40,41 Sistrurus catenatus (subspecies catena-tus, tergeminus and edwardsii),41 the Tunisian vipers Cerastescerastes, Cerastes vipera and Macrovipera lebetina,42 the Afro-tropical species Bitis arietans (Ghana),43 B. gabonica gabonica,44

Bitis gabonica rhinoceros, Bitis nasicornis, and Bitis caudalis,16

and the Central and South American pitvipers Atropoidesnummifer,45 Atropoides picadoi,45 L. muta,31 Lachesisstenophrys,31 Bothriechis lateralis,46 and Bothriechis schlegeli.46

Here, we describe the proteomes of the venoms of B. asperadult and neonate speciments from the Caribbean and thePacific versants of Costa Rica. Geographic, individual, andontogenetic venom composition variations are reported.

Experimental Section

Venom Samples. Venom samples were obtained from B.asper specimens collected in the Caribbean (Distrito Quesada,San Carlos, Alajuela province) and the Pacific (Distrito deSabanillas, Acosta, San Jose province) regions of Costa Rica(Figure 1, Table 1) and kept in captivity at the Serpentarium ofInstituto Clodomiro Picado (Universidad de Costa Rica, SanJose). In both cases, the collection areas comprised 145-150Km2. Venoms from adult specimens (15 from the Caribbeanand 11 from the Pacific regions) and from 6-7 weeks old snakes(at least 20 from each versant) were collected by snake bitingon a parafilm-wrapped jar. Crude venoms were centrifuged atlow speed to remove cells and debris, lyophilized, weighed ona microbalance, and stored at -20 °C until used. Venom poolswere prepared by mixing equal amounts of samples from atleast 11 specimens from both sexes from the Caribbean or fromthe Pacific regions.

Isolation and Proteomic Characterization of VenomProteins. Proteins from 2-5 mg of crude, lyophilized venomswere separated by reverse-phase HPLC using an ETTAN LCHPLC system (Amersham Biosciences) and a LichrosphereRP100 C18 column (250 × 4 mm, 5 µm particle size) asdescribed.15,16,31,41,44–46 The relative abundances (% of the total

Proteomics of Bothrops asper research articles

Journal of Proteome Research • Vol. 7, No. 8, 2008 3557

venom proteins) of the different protein families in the venomswere estimated from the relation of the sum of the areas ofthe reverse-phase chromatographic peaks containing proteinsfrom the same family to the total area of venom protein peaks.

Isolated protein fractions were subjected to N-terminalsequence analysis using a Procise instrument (Applied Biosys-tems, Foster City, CA). Amino acid sequence similarity searcheswere performed against the available databanks using theBLAST program47 at http://www.bork.embl-heidelberg.de. Themolecular masses of the purified proteins were determined bySDS-PAGE (on 12-15% polyacrylamide gels) and by electro-spray ionization (ESI) mass spectrometry using an AppliedBiosystems QTrap 2000 mass spectrometer48 operated inEnhanced Multiple Charge mode in the range m/z 600-1700.

Protein bands of interest were excised from a CoomassieBrilliant Blue-stained SDS-PAGE and subjected to automated

in-gel digestion, mass fingerprinting, and CID-MS/MS asdescribed.15,16,31,41,44–46 CID spectra were interpreted manuallyor using a licensed version of the MASCOT program (http://www.matrixscience.com) against a private database containing927 viperid protein sequences deposited in the Swiss-Prot/TrEMBL database (Knowledgebase Release 12 of July 2007;http://us.expasy.org/sprot/; 212 in Swiss-Prot, 715 in TrEMBL)plus the previously assigned peptide ion sequences from snakevenomics projects carried out in our laboratory.15,16,31,40–46

Variation in venom protein composition between taxa wasestimated using a Protein Similarity Coefficient [PSCab ) [2 ×(no. of proteins shared between a and b)/(total number ofdistinct proteins in a + total number of distinct proteins in b)]× 100] based on bandsharing coefficient used to compareindividual genetic profiles based on multilocus DNA finger-prints,49 and previously described criteria.15,16,31,40–46

Figure 1. Geographic origin of the venoms. Physical map of Costa Rica showing the geographical origin of the B. asper venoms fromthe Pacific, Bas(P), and the Caribbean, Bas(C), versants of Costa Rica (Table 1), whose proteomic characterization is reported in thiswork. Collection areas comprised 145-150 Km2. The Guanacaste, Tilaran, Central Volcanic and Talamanca, mountainous chains extendingthe entire length of the country from the Northwest to the Southeast and dividing the Caribbean and the Pacific regions, are labeled.

Table 1. Phenotypic Characteristics of the B. asper Specimens from the Caribbean and Pacific Versants of Costa Rica (Figure 1)Whose Venoms Are Described in This Work

Caribbean versant Pacific versant

(Distrito Quesada, Canton de San Carlos, Provincia de Alajuela) (Distrito de Sabanillas, Canton de Acosta, Provincia de San Jose)

specimen no. sex size specimen no. sex size

472 Female 76 cm 389 Female 104cm545 Female 139cm 497 Female 120cm355 Female 139cm 341 Female 123cm384 Female 146cm 496 Female 132cm733 Female 147cm 499 Female 144cm734 Female 152cm 429 Female 152cm544 Female 161cm 735 Female 154cm446 Female 164cm 580 Male 146cm449 Male 82 cm 607 Male 129cm447 Male 110cm 611 Male 117cm406 Male 113cm 736 Male 134cm560 Male 124cm473 Male 136cm489 Male 140cm404 Male 146cm

research articles Alape-Giron et al.

3558 Journal of Proteome Research • Vol. 7, No. 8, 2008

2-D SDS-PAGE. The proteins of the venom pools wereseparated by 2-D SDS-PAGE using an IPGphor (AmershamBioscience, Uppsala, Sweden) instrument. For isolectric focus-ing, 200 µg of total venom proteins (in 250 µL of 8 M urea, 4%CHAPS and 0.5% IPG buffer) was loaded on a 13 cm IPG strip(pH range 3-11) and the following focusing conditions wereused: 30 V for 6 h, 60 V for 6 h, 500 V for 1 h, 1000 V for 1 h,and 8000 V for 2 h. SDS-PAGE was done in a 16 cm 12%polyacrylamide gel. Coomassie blue was employed for proteinstaining.

Results and Discusion

Geographical Variation between the Venom Proteomesof B. asper Specimens from the Caribbean and PacificRegions of Costa Rica. Previous studies have shown thatvenoms from adult B. asper specimens from the Caribbeanversant of Costa Rica are more hemorrhagic and myonecrotic,whereas those from Pacific regions are more proteolytic, havingsimilar lethality, edema-forming activity, and hemolytic effect.34

For the characterization of their overall protein compositionand gaining a deeper insight into geographic and individualvariations, the venoms of 15 (from Caribbean regions) and 11(from Pacific regions) (Figure 1, Table 1) adult B. asperspecimens were analyzed, both as geographic pools andindividually. Pooled crude venoms (herein called Bas(C) andBas(P) according to their Caribbean or Pacific origin) werefractionated by reverse-phase HPLC (Figures 2 and 4), followedby analysis of each chromatographic fraction by SDS-PAGE(Figures 3 and 5), N-terminal sequencing, and MALDI-TOFmass spectrometry (Table 2). Protein fractions showing singleelectrophoretic band, molecular mass, and N-terminal se-quence were straightforwardly assigned by BLAST analysis(http://www.ncbi.nlm.nih.gov/BLAST) to a known protein fam-ily. Protein fractions showing heterogeneous or blocked N-termini were analyzed by SDS-PAGE and the bands of interestwere subjected to automated reduction, carbamidomethylation,

and in-gel tryptic digestion. The resulting tryptic peptides werethen analyzed by MALDI-TOF mass fingerprinting followed byamino acid sequence determination of selected doubly andtriply charged peptide ions by collision-induced dissociationtandem mass spectrometry. Despite its medical relevance, theSwiss-Prot/TrEMBL UniProt Knowledgebase (release of 15January 2008) contains only 6 full-length B. asper venom toxinsequences: four PLA2 myotoxins (P20474, P24605, Q9PVE3, andP0C616), a serine proteinase (Q072L6), and one PI (P83512 andQ072L4) and one PII (Q072L5) metalloproteinases. Thus, exceptfor these proteins, all of which were found in the pooledvenoms (Table 2), the peptide mass fingerprinting approachalone was unable to identify any protein in the databases. Inaddition, as expected from the rapid amino acid sequencedivergence of venom proteins evolving under acceleratedevolution,50–56 with a few exceptions, the product ion spectradid not match any known protein. Hence, mass spectra weremanually interpreted for de novo sequencing and the CID-MS/MS-deduced peptide ion sequences (Table 2) were submittedto BLAST similarity searches. High-quality MS/MS peptide ionfragmentation spectra yielded sufficient amino acid sequenceinformation derived from almost complete series of sequence-specific b- and/or y-ions to unambiguously identify a homo-logue protein in the current databases. All the identifiedproteins displayed strong similarity with entries from Bothropsspecies, highlighting the close phylogenetic relationship of B.asper with the New World South American bothopoid genera.57

Closest homologues to B. asper proteins were venom proteinsfrom B. atrox (medium-sized disintegrin P18618), B. jararacussu(serine proteinases AAB30013 and P81824; PIII-SVMPs Q7T1T5;PLA2 Q8AXY1; LAO Q6TGQ9), and B. jararaca (C-type lectin-like proteins P22028, P22029, and AAB47092; PIII-SVMPAAG48931), supporting the allocation of B. asper within theclade that includes B. atrox, B. jararaca and B. jararacussu.57

The 30-31 fractions isolated by reverse-phase HPLC fromthe Bas(A) (Figures 2 and 3) and Bas(P) (Figures 4 and 5)venoms comprised, respectively, about 30 and 27 differentproteins (Table 2), which in both cases belong to 8 differentgroups of toxins, distributed into 4 major (SVMP, PLA2, serineprotease, and L-amino acid oxidase (LAO)) and 4 minor(disintegrin, DC-fragment, C-type lectin-like, and cysteine-richsecretory protein (CRISP)) protein families (Figure 6). However,the venoms from the two B. asper populations exhibited distinctrelative protein family abundances, which are listed in Table3. Specifically, venom pooled from Caribbean specimenscontained higher content of serine proteinases (410%), LAO(200%), and disintegrin (160%) than venom pooled from Pacificsnakes, whereas the latter was enriched in PLA2s (160%)compared to the Caribbean specimens (Figure 7). The two B.asper populations also depart in the relative proportion of PIand PIII SVMPs. In addition to their different overall venomcompositions, the venoms from each geographic populationalso showed distinct protein expression profiles, as judged byboth reverse-phase HPLC (Figures 2 and 4) and 2D-SDS-PAGE(Figure 6). Knowledge of the within geographic locality venomvariability is essential for isolating pharmacologically activefractions from crude venoms. Thus, except for the LAOmolecule(s), which appears to be highly conserved in venomsfrom both B. asper populations, each protein family compriseddifferent complements of molecules (Tables 2 and 3). Forinstance, the disintegrin found in the venom of B. asper(Caribbean) appears to be identical to medium-sized RGD-disintegrins from other Bothrops species, as B. atrox [P18618],

Figure 2. Reverse-phase HPLC separation of the proteins from apool of venoms of B. asper adult specimens from the Caribbeanregion of Costa Rica. Two milligrams of total venom proteinswas applied 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,ESI mass spectrometry, tryptic peptide mass fingerprinting, andCID-MS/MS of selected doubly or triply charged peptide ions.The results are shown in Table 2.



B. insularis [AY736107], and B. jararacussu [DQ408681], butdeparts in residues 10GT11 from bothrasperin (10DA11) [Q072L5]secreted into the venom of B. asper from the Pacific versant.Moreover, B. asper (Caribbean) DC-fragment shows similarityto the DC-domains of B. jararacussu metalloprotease BOJUMETII [AY255004], whereas the N-terminal sequence of the DC-fragment from B. asper (Pacific) is conserved in the DC-domains from a variety of PIII-metalloproteases from speciesof the genera Bothrops, Agkistrodon, Trimeresurus, Gloydius,Crotalus, Echis.

The two B. asper populations contain both identical anddifferent (iso)enzymes from the PLA2, serine proteinase, andSVMP families (Table 2). Our results confirm and extendprevious studies by Moreno and co-workers35 and Lomonteand Carmona36 showing clear differences in the PLA2 isoformelectrophoregrams from venoms of B. asper specimens fromthe Caribbean and Pacific regions of Costa Rica. Similarly,among the major proteins found in Sistrurus venoms, PLA2

proteins appear to be exceptionally divergent at both the intra-and the interspecific level,41 suggesting that they have beenthe subject of strong balancing selection58 within, and diver-sifying selection between, taxa. Other studies have also shownthat PLA2 genes show high levels of divergence between speciesand high levels of variation in the composition of myotoxicPLA2 molecules in different geographical populations of severalpitvipers, including L. muta,31 Trimeresurus flavoviridis,59,60

Trimeresurus stejneri,61 and Bothrops neuwedi.62

Using a similarity coefficient, we estimate that the similarityof venom proteins between the two B. asper populations maybe around 52%. Compositional differences between venomsamong different geographic regions may be due to evolutionaryenvironmental pressure acting on isolated populations. Theuplift of the mountains of lower Central America, including theGuanacaste Mountain Range, Central Mountain Range, andTalamanca Mountain Range which presently separates theCaribbean and Pacific regions of Costa Rica (Figure 1), occurredin the late Miocene or early Pliocene (8-5 Mya) and culminatedin the Pliocene with the closure of the Panamanian Portal.63

This uplift may have fragmented the original homogeneouslowland Costa Rican herpetofauna into allopatric Caribbean

and Pacific populations.64,65 In Costa Rica, B. asper occurs inlowlands up to an elevation of about 1500 m above sea levelin both the Pacific and the Caribbean versants. These two snakepopulations, separated by the high mountain ridge rangingfrom approximately 1000 to 2000 m, and extending diagonallyacross the center the entire length of the country, representreproductive isolated communities. The formation of such aphysical barrier may have promoted (sub)speciation66 of thetwo separated Costa Rican B. asper populations. A subspeciesis a taxonomic subdivision that ranks just below a species andcomprises a geographically separated group of geneticallydistinct individuals whose members can interbreed. Subspeciesusually arise as a consequence of geographical isolation withina species. In this sense, although our comparative proteomicanalysis would support the classification of B. asper Caribbeanand Pacific populations as subspecies, additional venomicanalyses from areas of sympatry (i.e., Central Panama, whereboth forms might converge) are needed. In addition, detailedgenomic analyses are also required to address this point andto assess whether the distinct venom protein profiles ofCaribbean and Pacific B. asper populations arise from expres-sion of population-specific genes or from distinct translationpatterns of the same genome.

Individual Variations among the Venoms of B. asperSpecimens from Caribbean and Pacific Regions of CostaRica. The use of pooled venoms stems from a requirement fora statistically representative venom, that is, for antivenomproduction, but identification of individual variations is re-moved from the researcher’s control. To investigate the degreeof variability among specimens from the two B. asper popula-tions, the reverse-phase HPLC protein profiles of 15 venomsfrom Caribbean specimens and 11 venoms from snakes fromPacific regions (Figure 1) were compared. Individual venomsfrom each population consistenly exhibited the characteristicCaribbean or Pacific protein profile, clearly showing thatreverse-phase HPLC can be employed for unambiguouslytracing the geographic origin of Costa Rican B. asper snakes.Nevertheless, the concentration of specific components variedbetween specimens from each of the two B. asper populations.Major differences between individuals from the Caribbean and

Figure 3. SDS-PAGE of reverse-phase separated fractions from the venom of adult B. asper from the Caribbean region of Costa Rica.SDS-PAGE showing the protein composition of the reverse-phase HPLC separated venom protein fractions displayed in Figure 2 andrun under nonreduced (upper panels) and reduced (lower panels) conditions. Molecular mass markers (in kDa) are indicated at the leftof each gel. Protein bands were excised and characterized by mass fingerprinting and CID-MS/MS. The results are shown in Table 2.



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the Pacific regions are highlighted in panels A and B of Figure8, respectively. The variability at this level provides supportiveevidence to venom composition being under genetic control.Among Caribbean B. asper venoms, PLA2 molecules (BasC9-15),serine proteinases (BasC18-20), SVMPs (BasC26 and 27), andto a minor extent LAO (BasC23 and 24), displayed the largestvariability (Figure 8A). A similar trend was observed in venomsfrom the Pacific B. asper population, with PLA2s (BasP9-13),serine proteinases (BasP14 and 17), LAO (BasP20,21) and theSVMPs (BasP18, 25-30) exhibiting the most noticeable expres-sion variation (Figure 8B). Hence, within both B. asper geo-graphic populations, all major venom protein families appearedto be subjected to individual variations. The sources of suchvariability are not obvious. On one hand, no obvious gender-specific trend could be established, and venom componentsappear to vary independently. On the other hand, individualvenom variation seems to be a stochastic phenomenon, at leastconcerning ecological niches, because venoms from specimensfrom close geographic locations do not follow related venomcomposition variability. The occurrence of intraspecific indi-vidual variability highlights the concept that a species, B. asperin our case, should be considered as a group of metapopula-tions.67 The fact that venoms exhibit larger compositionalvariation between the two isolated geographic populations(Caribbean vs Pacific) than among specimens from the samegeographic range may be the consequence of allopatric spe-ciation of the two B. asper subpopulations isolated by mountainbarriers. Evidence from mitochondrial gene sequences indicatesthat the two Costa Rican B. asper populations split duringPleistocene, 3-3.5 mya ago (Saldariaga and Sasa, unpublishedresults). The Modern Synthesis of Evolution emphasizes theimportance of populations as the units of evolution. Localadaptation within a restricted population can occur if thestrength of selection exceeds the rate of gene flow.68 Hence,Natural Selection acting on individual venom variations mayendow specimens within a reproductive community the neces-sary versatility to adapt to the changing environments of ageographical range.

Individual venom variation is well-documented in the lit-erature and appears to be a general feature of venoms.69 In

particular, Taborska and Kornalik70 reported considerableindividual variability in both pathophysiological and enzymaticactivities between parents and siblings of a family of B. aspersnakes. The disparity of symptoms in victims of the samespecies of snake has alerted clinicians to the requirement formore specific antivenoms.69,71 Thus, knowledge of the geo-graphical and the individual variability in venoms, as reportedhere, could be relevant for antivenom production.

Defining Ontogenetic Changes in the Venom Proteomesamong B. asper Specimens from Caribbean and PacificRegions of Costa Rica. In line with a previous paper byGutierrez and colleagues50 who reported marked differencesin electrophoretic and immunoelectrophoretic patterns be-tween newborn and adult venoms from two Costa Ricanpopulations (San Carlos in the Caribbean versant and Puriscalin the Pacific), comparison of the HPLC separation profiles ofpooled venoms of neonate specimens from Caribbean andPacific regions with those of adult snakes from the samegeographical habitat (Figure 9 vs 2 and Figure 10 vs 4) revealedprominent protein expression changes. Thus, among Caribbeansnakes, neonate venoms (Figure 9) express neither the majorK49-PLA2 molecules Bas(C)-9-13 nor the abundant PI-SVMPsBas(C)-21 and -26 characterized in adult venoms (Figure 2).On the other hand, neonate venoms contained a larger propor-tion of D49-PLA2s 15 and 16, and of PIII-SVMPs, including atleast three molecules (numbered 32-34 in Figure 9 and labeledwith asterisks) not observed in adults. The N-terminal sequenceof Bas(C)_neo-32, AFTAEQRRYLNTRKY, shows extensive simi-larity with the region 147-160 of PIII-metalloprotease HF2precursor from B. jararaca [P30431]. Bas(C)_neo-33 and 34contained blocked N-termini and were identified by in-geltryptic digestion and ESI-MS/MS as PIII-SVMPs. These neonate-specific PIII-SVMPs account for about 14% of the total venomproteins (32, 3%; 33, 8.3%; 34, 2.7%).

Comparison of the protein profiles of pooled venoms fromPacific neonates and adult specimens (Figure 10 vs Figure 4)indicated the occurrence of a similar ontogenetic trend thanin Caribbean snakes. Thus, Pacific neonates express largeramounts of D49-(PLA2 Bas(P)-12 and 31) than K49-PLA2s(Bas(P)-10 and 11), though the total amount of PLA2 moleculesis only 40% than that of adults (Table 3). The concentration ofPI-SVMPs 18 and 19 is also lower in neonate versus adultvenoms, and the secreted complement of PIII-SVMPs also showage-dependent qualitative and quantitative variations. Proteinpeaks labeled with asterisks in Figure 10 represent neonate-specific toxins not found in adult venoms. Bas(P)_ neo-31-34were characterized by N-terminal sequencing, respectively, asa D49-PLA2 molecule, a 38 kDa serine proteinase, and PIII-SVMPs apparently identical to Bas(C)_neo-32 and -34 (Table2). Bas(P)_neo-35-38 contain PIII-SVMPs (48-72 kDa), whichshare at least ion 670.3(2+), (263.2)EXVXVADYR, (R�)2 C-typelectin-like proteins, and a PI-SVMP (23 kDa) highly similar oridentical to Bas(P)-27 (Table 2).

The overall protein composition of pooled venoms fromneonate B. asper snakes from Caribbean and Pacific versantsare displayed in Figure 6C,D and listed in Table 3. Majorontogenetic changes appear to be a shift from a PIII-SVMP-rich to a PI-SVMP-rich venom and the secretion in adults of adistinct set of PLA2 molecules than in the neonates. The age-dependent P-III to PI SVMP ontogenetic variation has also beenreported in B. atrox.29 However, whether it represents a generalor a genus-specific phenomenon requires detailed analysis ina higher number of species.

Figure 4. Reverse-phase HPLC separation of the proteins from apool of venoms of B. asper adult specimens from the Pacificregion of Costa Rica. Two milligrams of total venom proteinswas applied to a Lichrosphere RP100 C18 column, which was thendeveloped as in Figure 2. Fractions were collected manually andcharacterized by N-terminal sequencing, ESI mass spectrometry,tryptic peptide mass fingerprinting, and CID-MS/MS of selecteddoubly or triply charged peptide ions. The results are shown inTable 2.



Interestingly, in neonate B. asper venoms, the ratio K49/D49PLA2s is about 80%/20%, adult Caribbean and Pacific venomscontain, respectively, about 65% of K49- and 35% of D49-PLA2s

and 80% of K49- and 20% of D49-PLA2s (Figure 6C,D, Table3). D49-PLA2s are Ca2+-dependent esterases (E.C. 3.1.1.4) thatcatalyze the hydrolysis of the ester bond in the sn-2 positionof sn-3 glycerophospholipids to release lysophospholipids andfatty acids, whereas K49-PLA2 molecules are devoid of enzy-matic activity.72–74 Substitutions at the Ca2+-binding site(Asp49), such as Lys49, render toxins independent of phos-pholipid-hydrolysis to exert their functions. Snake venom PLA2scan be subdivided into acidic and basic molecules. Acidic D49-PLA2s display, in general, stronger enzymatic activity andweaker myotoxic effects than basic D49- and K49-PLA2s, whosetoxic activities include potent local myotoxicity, anticoagulationand edema formation.71–73 The major PLA2 molecules in bothCaribbean and Pacific neonate venoms are acidic proteins(Figure 10), though the latter snakes also express basic PLA2(s)(Figure 10B; consult also ref 75). These results may help torationalize the outcome of a previous comparative studyshowing that venoms from newborn specimens (from both theCaribbean and the Pacific versants of Costa Rica) are moreproteolytic, hemorrhagic, edema-forming and lethal, whereas

Figure 5. SDS-PAGE of reverse-phase separated fractions from the venom of adult B. asper from the Pacific region of Costa Rica.SDS-PAGE showing the protein composition of the reverse-phase HPLC separated venom fractions (see Figure 4) run under nonreduced(upper panels) and reduced (lower panels) conditions. Molecular mass markers (in kDa) are indicated at the left of each gel. Proteinbands were excised and characterized by mass fingerprinting and CID-MS/MS. The results are shown in Table 2.

Figure 6. Overall protein compositions of B. asper venoms.Comparison of the protein composition of the pooled venomsof adult B. asper from the Caribbean region (A) and from thePacific region of Costa Rica (B), and of the pooled venoms fromneonate specimens from the Caribbean region (C) and from thePacific region (D). DC, disintegrin/cysteine-rich fragment from PIIIsnake venom metalloproteinase (SVMPs); C-lectin, C-type lectin-like protein; PLA2, phospholipase A2; CRISP, cysteine-rich secre-tory protein; LAO, L-amino acid oxidase; SerProt, serine protein-ase. Details of the individual proteins characterized in adultvenoms are shown in Table 2 (but see also Table 3).

Table 3. Overview of the Relative Occurrence of Proteins (inPercentage of the Total HPLC-Separated Proteins) of theDifferent Families in the Venoms of B. asper Populations fromthe Caribbean and the Pacific Versants of Costa Rica

% of total venom proteins

Caribbean Pacific

protein family adult neonate adult neonate

Medium-sized disintegrin 2.1 1.6 1.4 0.6DC-fragments <0.1 - <0.1 -PLA2 28.8 23.7 45.1 27.7(- K49 18.8 2.1 36.0 4.2)

(- D49 10.0 21.6 9.1 23.5.CRISP 0.1 2.5 0.1 <0.1Serine proteinase 18.2 6.7 4.4 2.6L-amino acid oxidase 9.2 2.5 4.6 3.4C-type lectin-like 0.5 <0.1 0.5 0.2Zn2+-metalloproteinase 41.0 63.0 44.0 65.5

(- PI-SVMPs 32.2 2.9 30.5 7.1)(- PIII-SVMPs 8.8 61.0 13.3 58.2)



those of adult specimens are more hemolytic and induce astronger myonecrotic action, characterized by a myolytic typeof necrosis.50

The rationale for the observed ontogenetic changes remainsobscure. In addition to their distinct protein profiles, theontogenetic venom composition shift results in increasingvenom complexity (compare Figures 2 and 4 with 9 and 10),indicating that the requirement for the venom to immobilizeprey and initiate digestion may change with the size of thesnake. At this respect, it has been documented that venomsfrom neonate snakes are more toxic to lizards and inbred micethan adult venoms.77 Young Bothrops snakes preferentially eatamphibians, lizards, birds, and shift to mammals when theybecome adults.1 The qualitative and quantitative adjustmentsin the composition of the venom proteome linked to thedevelopment of B. asper are likely related to the survival of thesnake by prey adaptation.78–80 However, the notion thatevolutionary interactions between snakes and their prey maybe responsible for variation in venom composition has beenquestioned.81,82

Concluding Remarks. Intraspecific geographical venomvariations represents a well-known phenomenon since morethan 70 years ago,83,84 and numerous authors have describeddifference in symptomatology after envenomation by snakesfrom the same species from different geographical origin.69

However, the molecular player, mechanisms, and evolutionaryforces that underlie intraspecific venom variation within snake

populations is an important yet largely unrealized goal inevolutionary biology. The present work, which reports the firstdetailed characterization of individual and ontogenetic venomprotein profile variations in two geographical isolated B. asperpopulations, represents an effort in that direction. Our studyshows that, despite within population individual venom varia-tion, B. asper snakes from the Caribbean versant of Costa Ricacan be distinguished from those from the Pacific zones by theirdistinctly different venom protein profiles. Venom patternsamong snakes from the same area were so similar that theconsistency between geographic origin of the snakes andvenom properties could be utilized to identify the zone of originof the animals. Besides ecological and taxonomical implica-tions, the geographical venom variability reported here mayhave an impact in the treatment of bite victims and in theselection of specimens for antivenom production. Our studyhighlights the necessity of using pooled venoms as a statisticallyrepresentative venom for antivenom production.

Figure 7. 2D-SDS-PAGE of venom proteins from adult B. asperfrom the Caribbean (A) and the Pacific (B) regions of Costa Rica.A total of 200 µg of total proteins from pooled venoms wereisoelectrically focused (pI range 3-11) followed by separationby SDS-PAGE and Coomassie blue staining. Distinctly expressedprotein spots between the two geographic population are labeledand were characterized by tryptic peptide mass fingerprintingand CID-MS/MS (Table 2). Molecular mass markers (in kDa) areindicated at the left of each gel.

Figure 8. Intraspecific individual variation in the composition ofthe venom of adult B. asper from the Caribbean (A) and thePacific (B) regions of Costa Rica. Details of the reverse-phaseHPLC chromatograms of the venoms from B. asper from Carib-bean regions (473, 404, 489, 560, 384, 472, 544, 355) and fromPacific regions (341, 735, 497, 736, 607, 611, 499, 496), showingrepresentative differences in the relative concentrations of venomcomponents labeled as in Figures 2 (Caribbean) and 4 (Pacific).



If we assume a link between structural and functionalvariation in terms of effectiveness at killing and processingdifferent prey, then our results have implications for howvenom has evolved as an adaptation in these snakes. A majordifference between the two B. asper populations sampled liesin their distinct PLA2 content. Snake venom PLA2s represent arapidly evolving gene family,53,85,86 suggesting that functionaldifferences due to structural changes in PLA2 molecules amongB. asper Caribbean and Pacific snakes may have been a

phenotypic hallmark during adaptation of diverging snakepopulations to new ecological niches, or competition forresources in existing ones.

Although the occurrence of intraspecies variability in thebiochemical composition and symptomatology after enveno-mation by snakes from different age has long been appreciatedby herpetologist and toxinologists through electrophoresisWestern blotting, and zymography (see refs 69, 76 and refer-ences cited), detailed comparative proteomic analysis arescarce. Indeed, the identity of stage-specific venom proteinsdifferentially during ontogenetic development has only beenreported for B. atrox.29 In this species, P-III class metallopro-teinases and serine proteinases appear to be more abundantin juvenile specimens, while metalloproteinases from class P-Iexhibit higher concentration in adult venoms.29 Our results,reported here, showing similar PIII-to-PI ontogenetic shift inB. asper raise the question of whether this phenomenonrepresents a botropoid-specific or a higher order taxon-specificphenotypic trait. Detailed investigations in species across thewhole phylogenetic tree of Viperidae are needed to answer thisquestion. In addition, the adaptive fitness advantage for a snakepopulation of the observed ontogenetic changes remainsobscure. In several species of rattlesnakes, ontogenetic shift inactivity appears to be associated with a decrease in venomtoxicity and a reliance on larger prey by adults.30,77 Besides preyimmobilization, venoms play a digestive role. This is particu-larly important when a large prey (in relation to the snake’sdigestive apparatus size) is ingested. The high amounts of

Figure 9. Reverse-phase HPLC separation of the proteins from apool of venoms of neonate B. asper specimens from the Carib-bean region of Costa Rica. Two milligrams of total venomproteins was applied to a Lichrosphere RP100 C18 column, whichwas then developed as described in the legend of Figure 2.Fractions were collected manually and characterized by N-terminal sequencing, ESI mass spectrometry, tryptic peptidemass fingerprinting, and CID-MS/MS of selected doubly or triplycharged peptide ions. Peaks are numbered as in Figure 4. Arrowsindicate protein fractions present in adults but not in the neonatesnakes. Peaks 32, 33 and 34 (labeled with asterisks) werecharacterized as PIII-SVMPs distinctly secreted into neonatevenom. Inset, SDS-PAGE of fractions 15 (PLA2), 16 (CRISP andPLA2), and the neonate-specific proteins 32-34.

Figure 10. Reverse-phase HPLC separation of the proteins froma pool of venoms of neonate B. asper specimens from the Pacificregion of Costa Rica. Two milligrams of total venom proteinswas applied to a Lichrosphere RP100 C18 column, which was thendeveloped as described in the legend of Figure 4. Fractions werecollected and processed as in Figure 9. Peaks are numbered asin Figure 4. Arrows indicate protein fractions present in adultsbut not in the neonate snakes. Peaks 31-38 (labeled withasterisks) correspond to proteins distinctly secreted into neonatevenom. Inset, SDS-PAGE of fractions 12 (PLA2) and the neonate-specific proteins 31 (PLA2), 32 (serine proteinase), and 33-38,SVMPs.

Figure 11. 2D-SDS-PAGE of venom proteins from neonate B.asper from the Caribbean (A) and the Pacific (B) regions of CostaRica. A total of 200 µg of total proteins from pooled venoms wereisoelectrically focused (pI range 3-11) followed by separationby SDS-PAGE and Coomassie blue staining. Spots correspondingto PLA2 molecules are encircled and numbered as in Table 2.Molecular mass markers (in kDa) are indicated at the left of eachgel.



histolytic enzymes secreted into the venoms of newborn snakesmay serve to breakdown the bolus, avoiding putrefaction ofthe ingested prey. On the other hand, the ontogenetic shifttoward a more complex toxin composition points to a strongrole for adaptive diversification via natural selection. Venomcomplexity, along with individual variation, suggests an im-portant role for balancing selection58 in maintaining high levelsof functional variation in venom proteins of adult sit-and-waitpredators encountersing different types of prey, each of whichis most efficiently subdued with different venom proteins.

Abbreviations: DC, fragment, disintegrin/cysteine-rich frag-ment from PIII-SVMPs; SVMP, snake venom metalloproteinase;CRISP, cysteine-rich secretory protein; LAO, L-amino acidoxidase; PLA2; phospholipase A2.

Acknowledgment. This study has been financed bygrants from the Ministerio de Educacion y Ciencia, Madrid,Spain (BFU2004-01432/BMC and BFU2007-61563), CRUSA-CSIC (2007CR0004), and Vicerrectorıa de Investigacion (741-A7-611) from the University of Costa Rica.

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