Combined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two Bothrops jararaca populations from geographic isolated regions within the Brazilian Atlantic rainforest.

JPROT-02141; No of Pages 17 Journal of Proteomics xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Journal of Proteomics journal homepage: www.elsevier.com/locate/jprot

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Article history: Received 25 March 2015 Received in revised form 21 April 2015 Accepted 28 April 2015 Available online xxxx

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Keywords: Snake venom proteomics Venom gland transcriptomics Antivenomics Geographic venom variation Bothrops jararaca

Instituto de Bioquímica Médica, Programa de Biologia Estrutural, Laboratório de Hemostase e Venenos, Universidade Federal do Rio de Janeiro (UFRJ), Brazil Instituto Nacional de Biologia Estrutural e Bioimagem, Rede Proteomica do Rio de Janeiro, Universidade Federal do Rio de Janeiro (UFRJ), Brazil Laboratorio de Venómica Estructural y Funcional, Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain d Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará (UFC), Fortaleza 60430-270, Brazil e Fundação Zoobotânica do Rio Grande do Sul, Museu de Ciências Naturais, Núcleo Regional de Ofiologia de Porto Alegre, RS, Brazil f Laboratorio de Herpetologia, Departamento de Zoologia, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçãlves 9500, Agronomia, 91501-970 Porto Alegre, RS, Brazil g Hygeia Biotecnologia Aplicada S.A., Fundação Bio-Rio, Rio de Janeiro, Brazil h Laboratorio de Herpetologia, Instituto Butantan, Avenida Vital Brazil 1500, São Paulo 05503-900, SP, Brazil i Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica j Instituto Vital Brazil, Niterói, Rio de Janeiro, Brazil b

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Larissa Gonçalves-Machado a,b,c,1, Davinia Pla c,⁎,1, Libia Sanz c, Roberta Jeane B. Jorge d, Moema Leitão-De-Araújo e, Maria Lúcia M. Alves e, Diego Janisch Alvares f, Joari De Miranda g, Jenifer Nowatzki g, Karen de Morais-Zani h, Wilson Fernandes h, Anita Mitico Tanaka-Azevedo h, Julián Fernández i, Russolina B. Zingali a,b,⁎⁎, José María Gutiérrez i, Carlos Corrêa-Netto a,b,j,⁎⁎, Juan J. Calvete c,⁎

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Bothrops jararaca is a slender and semi-arboreal medically relevant pit viper species endemic to tropical and subtropical forests in southern Brazil, Paraguay, and northern Argentina (Misiones). Within its geographic range, it is often abundant and is an important cause of snakebite. Although no subspecies are currently recognized, geographic analyses have revealed the existence of two well-supported B. jararaca clades that diverged during the Pliocene ~ 3.8 million years ago and currently display a southeastern (SE) and a southern (S) Atlantic rainforest (Mata Atlântica) distribution. The spectrum, geographic variability, and ontogenetic changes of the venom proteomes of snakes from these two B. jararaca phylogroups were investigated applying a combined venom gland transcriptomic and venomic analysis. Comparisons of the venom proteomes and transcriptomes of B. jararaca from the SE and S geographic regions revealed notable interpopulational variability that may be due to the different levels of population-specific transcriptional regulation, including, in the case of the southern population, a marked ontogenetic venom compositional change involving the upregulation of the myotoxic PLA2 homolog, bothropstoxin-I. This population-specific marker can be used to estimate the proportion of venom from the southern population present in the B. jararaca venom pool used for the Brazilian soro antibotrópico (SAB) antivenom production. On the other hand, the southeastern population-specific D49-PLA2 molecules, BinTX-I and BinTX-II, lend support to the notion that the mainland ancestor of Bothrops insularis was originated within the same population that gave rise to the current SE B. jararaca phylogroup, and that this insular species endemic to Queimada Grande Island (Brazil) expresses a pedomorphic venom phenotype. Mirroring their compositional divergence, the two geographic B. jararaca venom pools showed distinct bioactivity profiles. However, the SAB antivenom manufactured in Vital Brazil Institute neutralized the lethal effect of both venoms to a similar extent. In addition, immobilized SAB antivenom immunocaptured most of the venom components of the venoms of both B. jararaca populations, but did not show immunoreactivity against vasoactive peptides. The Costa Rican bothropic–crotalic–lachesic (BCL) antivenom showed the same lack of reactivity against vasoactive peptides but, in addition, was less efficient immunocapturing PI- and PIII-SVMPs from the SE venom, and bothropstoxin-I, a CRISP molecule, and a D49-PLA2 from the venom of the southern B. jararaca

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Combined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two Bothrops jararaca populations from geographic isolated regions within the Brazilian Atlantic rainforest☆

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☆ This article is part of a Special Issue entitled: Omics Evolutionary Ecolog. ⁎ Corresponding authors at: Laboratorio de Venómica Estructural y Funcional, Instituto de Biomedicina de Valencia, CSIC, Jaime Roig 11, 46010 Valencia, Spain. ⁎⁎ Corresponding authors at: Instituto de Bioquímica Médica, Universidade Federal de Rio de Janeiro, Avenida Carlos Chagas Filho, 373, Rio de Janeiro, RJ, Brazil. E-mail addresses: [email protected] (D. Pla), [email protected] (R.B. Zingali), [email protected] (C. Corrêa-Netto), [email protected] (J.J. Calvete). 1 These authors contributed equally and should be considered “first authors”.

http://dx.doi.org/10.1016/j.jprot.2015.04.029 1874-3919/© 2015 Published by Elsevier B.V.

Please cite this article as: L. Gonçalves-Machado, et al., Combined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two Bothrops jararaca populations..., J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.04.029

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phylogroup. The remarkable paraspecificity exhibited by the Brazilian and the Costa Rican antivenoms indicates large immunoreactive epitope conservation across the natural history of Bothrops, a genus that has its roots in the middle Miocene. This article is part of a Special Issue entitled: Omics Evolutionary Ecolog. © 2015 Published by Elsevier B.V.

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Bothrops jararaca (Wied-Neuwied, 1824) is a well-studied semiarboreal venomous pit viper species endemic to southern Brazil (S Bahia, Espirito Santo, Rio de Janeiro, Minas Gerais, São Paulo, Paraná, Santa Catarina, Rio Grande do Sul), northeastern Paraguay, and northern Argentina (Misiones), where it occurs in deciduous tropical forests and semitropical upland forests, from near sea level to over 1000 m altitude [1–4] (Fig. 1). B. jararaca preys mainly on small vertebrates and exhibits an ontogenetic shift in feeding habit: adult snakes prey mainly on medium-size rodents, whereas food items of juvenile snakes consist mainly of frogs [2]. The specific name, jararaca, is derived from the Tupi words yarará and ca, which mean “large snake”. B. jararaca is a slender lancehead that can grow to a maximum total length of 160 cm, although the average length is considerable less [1,2]. B. jararaca is abundant in many parts of its broad range of distribution and represents an important cause of snakebite, particularly in heavily populated areas of southeastern Brazil, where it is responsible for most accidental envenomings [5–8]. On average, B. jararaca produces

about 25 mg, with a maximum of 300 mg, of highly toxic venom, whose median lethal dose (LD50) for mice is 1.4 mg/kg intraperitoneal (1.2 mg/kg intravenous, 3 mg/kg subcutaneous) [9]. In humans, the typical clinical picture of envenoming includes local swelling, petechiae, bruising and blistering of the affected limb, spontaneous systemic bleeding in various organs, subconjunctival hemorrhage and clotting disturbances [8,10,11]. The systemic symptoms can potentially be fatal and may involve hemostatic disorders, intracranial hemorrhage, shock and renal failure [8,10,11]. From a natural history perspective, venom represents an adaptive trophic trait in the evolution of advanced snakes [12–14]. A deep understanding of the composition of venoms and of the principles governing the evolution of venomous systems is of applied importance for exploring the enormous potential of venoms as sources of chemical and pharmacological novelty, but also to fight the dire consequences of snakebite envenomings. In this regard, the last decade has witnessed the development of techniques and strategies for assessing the toxin composition of snake venoms, “venomics”, directly (through proteomic-centered approaches) and indirectly (via venom gland transcriptomic and

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Fig. 1. Geographic distribution of the B. jararaca populations investigated. Panel A, physical map of Brazil showing the collection sites of the southeastern (SE) and the southern (S) populations within the Atlantic forest. The number of specimens milked in each locality is indicated in the Materials and methods section. The range of B. jararaca in Southern Brazil, including the States of Bahia (BA); Espirito Santo (ES); Rio de Janeiro (RJ); Minas Gerais (MG); São Paulo (SP); Paraná (PR); Santa Catarina (SC); and Rio Grande do Sul (RS) is highlighted in pale orange. The broken line indicates the main genetic barrier between the SE and the S B. jararaca phylogroups [3]. Panels B and C, respectively, pictures of juvenile (© Tyelli Ramos) and adult (© Claudio Machado) specimens of B. jararaca from the SE population. Panels D and E, respectively, pictures of juvenile and adult specimens of B. jararaca from the S population (© Sergio Bavaresco).


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Venoms of B. jararaca from the southeastern clade population were collected from a number (n) of adult and juvenile (b 1 year) specimens captured in the following localities and States of Brazil (Fig. 1) and maintained at the serpentarium of Vital Brazil Institute (Niterói, Rio de Janeiro, Brazil) under register of IBAMA no. 26354-9: Búzios, Rio de Janeiro (1), Espirito Santo (1), Guapimirim, RJ (1), Mendes, RJ (1), Nova Friburgo, RJ (1), Maricá, RJ (1), Duque de Caxias, RJ (1), Caxambu, Minas Gerais (1), Areal, RJ (1), Rio de Janeiro, RJ (2), Niterói, RJ (5), Seropédica (1), and Petrópolis, RJ (3). Venoms of B. jararaca from the southern clade population were collected from adult and juvenile (b1 year) specimens captured in the following localities of Rio Grande do Sul (RS) and kept in captivity in the serpentarium of Nucleo de Ofiologia de Porto Alegre, Rio Grande do Sul, Brazil, under register of IBAMA no. 1/43/1999/000764-9: Veranópolis (1), São José do Sul (6), Ivoti (1), Nova Petrópolis (6), Novo Hamburgo (1), Dom Pedro de Alcântara (1), São Franscisco de Paula (2), and Salvador do Sul (1), Dois Irmâos (2), and Sapucaica do Sul (2) (Fig. 1). Venoms were manually milked by gland massage and collect into a jar. Crude venoms were centrifuged at low speed to remove cells and debris, lyophilized and stored at −20 °C until used. Venoms from B. jararaca used in Instituto Butantan for SAB (soro antibotrópico) antivenom production during the period 1963–2008

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2.1. Venoms and antivenoms

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2.2. cDNA synthesis and 454 sequencing

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Two specimens of Bothrops jararaca were collected from the south and southeastern populations and kept at the serpentarium of Vital Brazil Institute, Niterói, Rio de Janeiro. Venom glands from both specimens were obtained 4 days after manually milking the venom, when transcription is maximal [43], from anesthetized snakes using fine forceps. Following gland extraction, the animals were sacrificed with CO2 and cooling. Animal handling was conducted in agreement to Ethical Principles in Animal Research and approved by the Ethical Committee for Animal Experimentation from Center of Health and Science of the Federal University of Rio de Janeiro (no. 077/10). The venom glands were immediately placed in RNAlater™ solution (Qiagen) and placed at −70 °C until gland RNA extraction. Glands were disrupted and homogenized using a rotor–stator homogenizer, and RNA was extracted using RNAeasy kit according to the manufacturer's (Qiagen) instructions. mRNA was isolated from total RNA pool with Dynabeads® mRNA Purification kit (Invitrogen), and cleaved by ZnCl2 at 70 °C for 30 s to generate fragments of about 700 bp. Random primers were used to synthesize the first strand of cDNA using a standard cDNA Synthesis System (Roche Diagnostics). Adaptor-ligated population-specific cDNA libraries were constructed using the cDNA Rapid Library Prep kit following the manufacturer's (Roche Diagnostics) instructions. For emulsion PCR amplification of the libraries, the cDNA fragments were conjugated onto magnetic beads. Clonally amplified cDNA library beads were selected and were deposited on a picotiter plate for 454 pyrosequencing [44] by Hygeia Biotecnologia Aplicada S.A., Fundação Bio-Rio, Rio de Janeiro, Brasil, using Sequencing Titanium chemistry following the manufacturer's (Roche Diagnostics) instructions. Sequencing was outsourced to Helixxa Ltda and Scylla Bioinformática, who performed the first draft. Raw 454 reads were cleaned with SeqClean to remove low complexity and contaminant sequences and submitted to de novo assembly using Newbler 2.5 (454 Life Sciences) and/or iAssembler (http:// bioinfo.bti.cornell.edu/tool/iAssembler). For functional annotation, assembled contig sequences were translated into the 6 possible forward and reverse reading frames using Søren W. Rasmussen's Seqtools (http://www.seqtools.dk), and blasted against the non-redundant NCBI database (http://blast.ncbi.nlm.nih.gov) and the UniProtKB/ Swiss-Prot Toxin Annotation Program database (http://us.expasy.org/ sprot/tox-prot), using BlastX and BlastN [45] algorithms, specifying a

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were pooled from the following number of extractions from a nonspecified number of individuals from the southeastern and southern populations: 18,755 (1963); 10,961 (1973); 51,035 (1977–1988); 1841 (1997); 888 (2008). The antibothropic polyvalent antivenom, SAB, used in this study was kindly provided by Instituto Vital Brazil (Niterói, RJ; http://www.ivb.rj. gov.br). This antivenom was raised in horses by conventional immunization schedules against a pool of venoms from B. jararaca (50%), Bothrops jararacussu (12.5%), Bothrops moojeni (12.5%), Bothrops alternatus (12.5%) and Bothrops neuwiedi (12.5%). The final formulation consists of purified F(ab′)2 fragments generated by digestion with pepsin of ammonium sulfate-precipitated IgG molecules [40]. A vial of SAB (10 mL, 18.7 mg F(ab′)2/mL) neutralizes 65 mg of B. jararaca reference venom. The polyvalent (Crotalinae) antibothropic–crotalic–lachesic (BCL) antivenom produced at Instituto Clodomiro Picado, Universidad de Costa Rica (http://icp.ucr.ac.cr), is produced by immunizing horses with a mixture of equal amounts of the venoms of Bothrops asper, Crotalus simus simus, and Lachesis stenophrys obtained from adult specimens kept in captivity at the ICP serpentarium [41]. It consists of whole immunoglobulins purified by caprylic acid precipitation [42]. Each vial of BCL antivenom used in this study (10 mL, 57 mg IgG/mL) neutralizes ≥30 mg of B. asper venom, ≥20 mg of C. s. simus venom, and ≥30 mg of L. stenophrys venom.

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bioinformatic analysis) [15]. Very recently, the first genome sequencing of a venomous snake, the king cobra (Ophiophagus hannah), has been reported [16], and others are in the pipeline [17,18]. Comparative squamate genomics is expected to reshape our conceptual understanding of the origin of venoms and the regulation of the venom toxin genes. A number of peptidomic, proteomic and transcriptomic studies on the venom [19–27] and venom gland [28,29], respectively, of adult and newborn B. jararaca have been conducted, chiefly by researchers at Instituto Butantan. While these studies revealed a qualitative picture of the repertoire, and the degree of intraspecific ontogenetic variability, of the toxin classes that constitute the venom proteome of B. jararaca, a quantitative overview of the venom proteome is pending. Moreover, phylogenetic and network analyses of a large-scale survey of the genetic variation at the mitochondrial cytochrome b gene have revealed the existence of two well-supported B. jararaca clades, exhibiting a southern and a southeastern Atlantic rainforest (Mata Atlântica) distribution, which diverged during the Pliocene ~ 3.8 million years ago, when the tropical rain forest was fragmented by large climatic changes [3]. However, despite evidence of intraspecific morphological and population structure [3 and references cited therein], venomic studies have not been conducted in these two B. jararaca phylogroups. Intraspecific geographic variation in venom composition has long been appreciated by herpetologists and toxinologists as a common feature of highly adaptable and widely distributed snake species [30–32], and may result in a different symptomatology after envenomation, suggesting that they may require variations in the clinical management [15,32–35]. To bridge this gap, we have performed combined venom gland transcriptomic and venom proteomic analyses of the two isolated populations of B. jararaca distributed in extreme regions of the species range, in current southeastern and southern regions within the Brazilian Atlantic rainforest. In addition, as the assessment of venom variation is of outmost importance for quality control of venoms used in the generation of antivenoms [36–38], we have applied neutralization assays and a second generation antivenomic approach [39] to investigate the immunoreactivity of the pentabothropic antivenom produced at Instituto Vital Brazil (Niterói, RJ, Br) [40] and the polyvalent (Crotalinae) antivenom manufactured by Instituto Clodomiro Picado (San José, CR) [41,42] towards the venom proteins of the two geographic isolated populations of B. jararaca.

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2.4. Characterization of the venom peptidome and proteome

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Chromatographic fractions containing peptides (m/z ≤ 1700) were loaded in a nanospray capillary column and submitted to peptide sequencing using a QTrap™ 2000 mass spectrometer (Applied Biosystems) as described [49]. Protein bands were excised from Coomassie Brilliant Bluestained SDS-PAGE gels and subjected to in-gel tryptic digestion and nanoAcquity-LC–MS/MS analysis as described [49]. Fragmentation spectra were interpreted i) manually (de novo sequencing), ii) using the on-line form of the MASCOT program at http://www. matrixscience.com against the NCBI non-redundant database, and iii) processed in Waters Corporation's ProteinLynx Global SERVER 2013 version 2.5.2. (with Expression version 2.0) against the UniProtKB/TrEMBL and NCBI non-redundant databases, and against the B. jararaca population-specific 454 contig datasets translated in all six possible reading frames. MS/MS mass tolerance was set to ± 0.6 Da. Carbamidomethyl cysteine and oxidation of methionine were selected as fixed and variable modifications, respectively. The relative abundances (expressed as percentage of the total venom proteins) of the different protein families were calculated as the ratio of the sum of the areas of the reverse-phase chromatographic peaks containing proteins from the same family to the total area of venom protein peaks in the reverse-phase chromatogram [50,51]. The relative contributions of proteins from different protein families eluting in the same chromatographic fraction were estimated by densitometric analysis of Coomassie Blue-stained SDS-polyacrylamide gels. On the other hand, the relative abundances of different proteins contained in the same SDS-PAGE band were computed according to the relative ion intensities averaged for the three more abundant peptide ions associated with each protein by MS/MS analysis.

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Experimental proteomic data were used to verify or correct transcriptomics assembly. To this end, proteomic gathered data were searched against a 6-frame translation of the population-specific transcriptomic sequences. Transcripts encoding MS/MS-derived peptide sequences found in the tryptic digests of the SDS-PAGE-excised

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2.6.1. Lethality Groups of four CD-1 mice (18–20 g) were injected intravenously in the caudal vein with various doses of each, SE and S, adult B. jararaca venom dissolved in 100 μL of 0.14 M NaCl, 0.04 M phosphates, pH 7.2 (PBS). Deaths occurring during 24 h were recorded and the median lethal dose (LD50) was estimated by probits. The protocols of all experiments involving the use of mice were approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUA) of Universidad de Costa Rica.

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Proteins from 2 mg of crude, lyophilized venoms were dissolved in 190 μL of water containing 0.1% trifluoroacetic acid (TFA) and 5% acetonitrile, centrifuged to remove debris, and separated by reverse-phase HPLC as described [49]. Molecular masses of the purified proteins were estimated by SDS-PAGE (on 15% polyacrylamide gels), or determined by electrospray ionization (ESI) mass spectrometry using an Applied Biosystems QTrap™ 2000 mass spectrometer operated in Enhanced Multiple Charge mode in the range m/z 600–1700, or a Waters SYNAPT G2 High Definition Mass Spectrometry System. Chromatographic fractions were submitted to SDS-PAGE analysis in 15% polyacrylamide gels, under non-reducing and reducing conditions, and protein bands were stained with Coomassie Brilliant Blue G-250.

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protein bands, were blasted against the UniProtKB/TrEMBL and NCBI non-redundant databases without taxonomy restriction. Amino acid sequence contained in transcripts translated in any reading frame and exhibiting similarity to a known snake venom protein were aligned onto a prototypic full-length sequence of the closest homolog toxin family member. The aligned peptide sequence strings were again submitted to BLAST analysis against a restricted database comprising the 23,843 UniProtKB snake venom protein hits. A toxin-class specific multiple sequence alignment was then used as a template to manually refine the boundaries between the protein parts gathered from transcripts translated in different reading frames. The occurrence of the refined protein sequence was also checked in both population-specific transcriptomic databases. Whenever possible, proteotranscriptomicproposed full-length protein sequences were validated by measuring the isotope-averaged native molecular mass in an Applied Biosystems' QTrap™ 2000 mass spectrometer or a Waters SYNAPT G2 High Definition Mass Spectrometry System.

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2.6.2. Hemorrhagic activity Groups of four CD-1 mice (18–20 g) were injected intradermally in the ventral abdominal region with various doses of each venom, dissolved in 100 μL PBS. After 2 h, mice were sacrificed by CO2 inhalation, their skins were removed, and the hemorrhagic area in the inner side of the skin was measured [52]. Hemorrhagic activity was expressed as the Minimum Hemorrhagic Dose (MHD), corresponding to the dose of venom that induces a hemorrhagic area of 10 mm diameter [52].

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2.6.3. In vitro coagulant activity Citrated plasma was obtained from a healthy human volunteer. Aliquots of 200 μL of plasma were incubated at 37 °C for several minutes. Then, 50 μL containing various doses of each venom, dissolved in PBS, were added to the plasma, and clotting times were determined. The Minimum Coagulant Dose (MCD) was defined as the dose of venom that induced coagulation of plasma in 60 s [53,54].

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2.6.4. Phospholipase A2 activity The phospholipase A2 (PLA2) activity was determined based on the assay developed by Holzer and Mackessy [55], using the monodisperse synthetic substrate 4-nitro-3-octanoyloxy-benzoic acid (NOBA). Different amounts of venom (from 3.125 to 200 μg of each venom) dissolved in water, were added and mixed in 200 μL of 10 mM Tris–HCl, 10 mM CaCl2, 0.1 M NaCl, pH 8.0, in triplicate wells of a 96 well microplate. Then, 25 μL of NOBA (1 mg/mL in acetonitrile) was added (final substrate concentration of 0.32 mM). After incubating for 60 min at 37 °C, the absorbance at 405 nm was recorded. PLA2 activity was expressed as the change in absorbance in comparison to a blank containing all the reagents except venom. One unit of PLA2 activity was defined as the change of 0.001 in absorbance at 405 nm after 60 min.

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2.6.5. Proteinase activity An azocasein substrate was used to measure proteinase activity [56]. Aliquots (20 μL) of venoms from adult B. jararaca (from the S or SE population) containing 62.5 ng to 256 μg of venom (dissolved in

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cut-off E-value b e−03 and BLOSUM62 as scoring matrix. Results were imported to software Blast2GO [46] to assign gene ontology terms. The relative expression of a given toxin protein family was calculated according to the RPKM (Reads Per Kilobase of exon per Million mapped reads) standard procedure described by Mortazavi and coworkers [47]. The relative expression of a given toxin protein family (mol%) was calculated as the number of reads assigned to this protein family (Ri ) normalized by the length (in nucleotides) of the reference transcript sequence (ntREF) and expressed as the % of total reads in the snake transcriptome (∑ Reads): mol% toxin family “i” = %[(Ri / ntREF) / ∑ Reads] [48].

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2.6.7. Antivenomics The second-generation affinity chromatography-based antivenomic protocol described by Pla et al. [39] was employed. To this end, 400 μL of NHS-activated Sepharose 4 Fast Flow (GE Healthcare) was packed in a column and washed with 10–15 matrix volumes of cold 1 mM HCl, followed by two matrix volumes of coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3) to adjust the pH of the column between 7.0 and 8.0. Then, 51 mg of F(ab′)2 of pentavalent antibothropic antivenom (SAB, Vital Brazil Institute, Niterói, RJ, Br) and 57.8 mg of polyspecific antibothropic, anti-crotalic, and anti-lachesic (BCL) from Instituto Clodomiro Picado (Costa Rica) in 300 μL of coupling buffer were incubated with the matrix in an orbital shaker overnight at 4 °C. Antivenom F(ab′)2 (SAB) or IgG (BCL) molecules coupled to the matrices were quantified indirectly, by densitometry of the SDS-PAGE-separated F(ab ′)2 or IgG bands in the non-bound fraction using MetaMorph software (MDS Analytical Technologies). Different amounts of a stock solution of pre-coupled antivenom (quantified using an extinction coefficient at 280 nm of 1.36 for 1 mg/mL, [57]) were used for plotting the calibration curve. Coupling yields were 13.1 mg of SAB and 13.6 mg of BCL. Non-reacted NHS-matrix functional groups were blocked by incubation with 400 μL of 0.1 M Tris–HCl, pH 8.5, overnight at 4 °C in an orbital shaker. The affinity columns were washed alternately at high and low pH, with three volumes of 0.1 M acetate buffer, 0.5 M NaCl, pH 4.0–5.0 and three volumes of 0.1 M Tris–HCl buffer, pH 8.5. This treatment was repeated six times. Before incubation with the crude venoms, the matrix was equilibrated with five matrix volumes of binding buffer (PBS). For the immunoaffinity assay, 300 μg of B. jararaca crude venom, representing a venom:(SAB or BCL) antivenom mass ratio of 1:45 were dissolved in 1/2 matrix volume of PBS and incubated 3 h with the matrix at room temperature (~25 °C) using an orbital shaker. As specificity controls, 300 μL of Sepharose 4 Fast Flow matrix, without or with 13.5 mg of immobilized control IgGs (from non-immunized horses), were incubated with venom and the columns developed in parallel to the immunoaffinity experiment. After elution of the non-binding fraction, columns were washed two times with PBS, and the immunocaptured venom proteins were eluted with 2.5 matrix volumes of elution buffer (0.1 M glycine-HCl, pH 2.0), and neutralized with neutralization buffer (1 M Tris–HCl, pH 9.0). The nonretained and the immunocaptured venom fractions were fractionated by reverse-phase HPLC and the venom components identified by LC–MS/MS and quantified as described [49]. 2.6.8. Western blot analysis Reverse-phase HPLC chromatographic fractions were analyzed by SDS-PAGE on 15% polyacrylamide gels under reduced conditions, followed by electrotransfer to Hybond-P PVDF membrane (GE-

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2.6.9. Statistical analysis The significance of differences between the means of two experimental groups was determined by Student's t-test using the Prism® software (GraphPad), where p b 0.05 was considered significant.

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3.1. Southeastern and southern B. jararaca populations express distinct 431 venom proteomes 432

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Reverse-phase HPLC protein profiles of venoms from 21 individual adult specimens from each the southeastern (SE) and the southern (S) populations of B. jararaca sampled (Fig. 1), revealed highly conserved intrapopulational venom proteomes (Supplementary Figs. S1 and S2). Within population venom variability mainly involved different relative abundances of the same proteins rather than the occurrence of different proteins in the venoms of sympatric conspecific specimens (Supplementary Figs. S1 and S2). On the other hand, the proteomes of venoms pooled from each of these two B. jararaca geographic regions exhibited notable interpopulational variation (Fig. 2). Thus, although both venom proteomes comprise a similar number of chromatographic fractions and electrophoretic bands (Fig. 2), it must also be noted the presence of major peaks (and associated protein bands) 33 (25 kDa) and 36 (46 kDa) in the venom of the SE population (Fig. 2A), and peaks 15 (16 kDa) and 28 (23 and 26 kDa) in the venom from the southern B. jararaca population (Fig. 2B). These peaks account for 10.5%, 15.2%, 13.8% and 7.3% of their respective venom proteome. In order to assess the extent and possible causes of this venom variability, we investigated the venom gland transcriptomic and venom proteomic profiles of the southeastern and southern populations of B. jararaca. 454 pyrosequencing of venom gland cDNA libraries from southeastern (Rio de Janeiro) and southern (Rio Grande do Sul) B. jararaca specimens yielded, respectively, 205,449 and 281,569 reads of average sizes 198 and 309 nucleotides. These datasets were assembled into 16,186 (SE)/21,892 (S) singletons and 14,246 (SE)/12,240 (S) contigs, of which 334 (SE) and 579 (S) coded for putative toxins of known protein families. The expression levels of these putative venom toxin classes were qualitatively and quantitatively similar in both transcriptomes, except for SVMPs and galactose-binding proteins (Gal) (compare panels A and B of Fig. 3). In decreasing order of relative abundance, putative toxin transcripts coded for SVMPs N serine proteinases N C-type lectin-like proteins N L -amino acid oxidase N PLA2 molecules N precursors of vasoactive peptides N snake venom growth factors N CRISPs N (phospholipase B, glutaminyl cyclase, phosphodiesterase, 5′-nucleotidase) N serine proteinase Kunitz-type inhibitor (compare Figs. 3A and 3B). The nonrefined population-specific transcriptomics datasets were six-frame translated and implemented in ProteinLynx Global SERVER as search databases to achieve locus-resolution matching of the MS/MS spectra generated in the venom proteomic analysis. Excepting for 91 and 60 MS/MS spectra from SE and S venom tryptic digests, respectively,

D

362 363

2.6.6. Neutralization of lethal activity by antivenom Mixtures containing a fixed amount of venom and variable dilutions of antibothropic antivenom of Instituto Vital Brazil were prepared and incubated at 37 °C for 30 min. Controls included venom incubated with PBS instead of antivenom. Then, aliquots of 100 μL of the mixtures, containing 4 LD50s of venom, were injected i.v. into groups of four mice (18–20 g). Deaths occurring during 24 h were recorded in order to assess the neutralizing efficacy of the antivenom.

E

360 361

Healthcare) using a Bio-Rad Semi-Dry mini-transfer cell at 200 mA during 90 min. Unoccupied sites in the membranes were blocked by incubation in blocking buffer (5% (w/v) skimmed milk in 20 mM phosphate, 135 mM NaCl, pH 7.3) at 4 °C overnight in an orbital shaker. Membranes were then incubated for 1.5 h at room temperature with a 1:500 (v/v) dilution in blocking buffer of a stock solution (28 mg/mL) of the SAB antivenom, or a 1:400 (v/v) dilution of a 22.2 mg/mL BCL stock solution. After 3 × washings (5 min each) with PBS-Tween buffer (20 mM phosphate, 135 mM NaCl, pH 7.3, containing 0.1% Tween-20), the membrane was incubated for 1.5 h at room temperature with alkaline phosphatase-conjugated with rabbit anti-horse IgG (Sigma) diluted 1:5000 (v/v) in the same buffer, washed 4 times as above, and developed using the ECL Prime Western Blotting Detection Reagent (Amersham), and the chemiluminescent signal digitalized in an Image Quant LAS 4000 mini.

T

358 359

C

357

E

355 356

R

354

R

352 353

N C O

350 351

50 mM Tris–HCl, 150 mM NaCl, 5 mM CaCl2, pH 8) were added to 100 μL of a substrate solution that contained 10 mg/mL of azocasein dissolved in the same buffer. After incubation for 90 min at 37 °C, the reaction was stopped by the addition of 200 μL of 5% trichloroacetic acid. The tubes were then centrifuged at 1500 rpm for 5 min, 150 μL of the supernatant was mixed with 150 μL of 0.5 M NaOH in a 96 well microplate, and the absorbance at 450 nm was measured. One unit of proteolytic activity was defined as a change of 0.2 in absorbance per minute. Reactions were performed in triplicates. Blanks contained buffer instead of venom solutions.

U

348 349

5


433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472


O

R

R

E

C

T

E

D

P

R O

O

F

6

474 475 476 477 478 479 480 481 482 483 484 485 486 487

which were manually (de novo) interpreted or matched to snake venom toxin sequences available in the UniProtKB/TrEMBL and NCBI nonredundant databases, all the 638 and 998 peptides analyzed in tryptic digests from venom toxin spots from the SE and the S B. jararaca populations, respectively, were found in the homologous transcriptomic database (Supplementary Tables S1 and S2). Experimental proteomic data were used to verify, and eventually correct, the transcriptome assemblies. In many cases, tryptic peptides recovered from the same electrophoretic band digest matched different (generally two, but also three) translations of the same transcript, clearly indicating one or more shifts in the reading frame (Figs. 4A and 4B). BLAST analysis was used to delimit the boundaries of the toxin-coding sequences encoded in the different translations and align them onto a close homolog template sequence (Figs. 4A and 4B). Where possible, the accuracy of the assembly was verified by measuring the mass of

U

473

N

C

Fig. 2. Chromatographic separations of the proteins of venoms pooled from adult B. jararaca specimens. Panels A and B display, respectively, reverse-phase HPLC separations of the proteins of 2 mg of B. jararaca venom pools from specimens collected in the Southeastern (SE) Brazilian States of Espirito Santo (ES), Minas Gerais (MG), and Rio de Janeiro (RJ), and the Southern (S) State of Rio Grande do Sul (RS) (Fig. 1). Fractions were collected manually and analyzed by SDS-PAGE (inserts) under non-reduced (upper panels) and reduced (lower panels) conditions. Protein bands were excised and characterized by LC–nESI-MS/MS collision-induced dissociation of doubly- or triply-charged tryptic peptide ions (Supplementary Tables S1 and S2).

the intact protein (Fig. 4A). Twenty and 35 full-length venom protein sequences, and a larger number of partial sequences, were identified in the respective proteotranscriptomics-guided venom proteomes of the southeastern and southern populations of B. jararaca, including the C-type galactose-binding protein BiL [Q6QX33] displayed in Fig. 4B (Supplementary Tables S3 and S4, respectively). Panels C and D of Fig. 3 display the overall relative abundances of the 11 and 16 protein families found, respectively, in the venom proteomes of the southeastern and southern populations of B. jararaca. As expected from their different chromatographic separation profiles (Fig. 2), but in contrast to their overall venom gland transcriptional activity (compare panels A and B of Fig. 3), the venom proteomes of the SE (Fig. 3C) and S (Fig. 3D) populations of B. jararaca markedly depart in their relative abundances of major protein families, most notably SVMP, PLA2, disintegrin, and serine proteinase. In addition, some minor proteins


488 489 490 491 492 493 494 495 496 497 498 499 500 501 502

7

E

D

P

R O

O

F


507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528

R

R

505 506

(svNGF, hyaluronidase, glutaminyl cyclase, phosphodiesterase, and 5′-nucleotidase) were only found in the southern population, whereas svVEGF exhibited a SE population-specific expression. These proteins may exhibit a population-specific transcription pattern, representing thus alleles unevenly distributed in the population. Alternatively, detection of very scarce component found in only one population may have been missed in the other population.

N C O

504

3.2. Transcriptional, post-transcriptional, and ontogenetic regulation underlie population-specific venom variation Venom is a polygenic, complex trait. Venom phenotypic patterns generated by spatially varying selection have long been observed [32]. Geographic venom variability may result from local adaptation in response to geographically variable selection (i.e., regional diet) but also from independent founder effects. Local adaptation may contribute to the maintenance of genetic variation, be a stepping stone to ecological speciation, and facilitate species range expansion [58,59]. Comparative analyses of full-length protein/transcript sequence datasets (Supplementary Tables S3 and S4) allowed us to study patterns of population-level venom variation between the southeastern and southern B. jararaca phylogroups. Except for a few venom proteins (disintegrin jararacin [P31989], C-type lectin-like (CTL) BiL [Q6QX33]), orthologous protein sequences had amino acid changes, which varied from a single mutation in CRISP (221 residues) to typically ~ 3% in serine proteinases, CTLs, and SVMPs. Allopatric venom variability represents a source of chemical innovation at genomic level, that allows vicariant genes to evolve over time distinctly different characteristics. However, although new variation is

U

503

E

C

T

Fig. 3. Comparison of the overall toxin composition of proteome and transcriptome of the SE and S populations of B. jararaca. Pie charts in panels A and B display, respectively, the relative abundances of putative toxin-coding transcripts sequenced in the cDNA libraries of venom glands from the B. jararaca populations from Southeastern (SE) and Southern (S). Pie charts in panels C and D display, respectively, the relative occurrence (in percentage of total venom proteins) of toxins from different protein families in the venom pools of B. jararaca populations from Southeastern (SE) and Southern (S) Atlantic forest regions (Fig. 1). PI- and PIII-SVMP, snake venom Zn2+-metalloproteinases of PI and PIII class, respectively; Disi, disintegrin; VAP, vasoactive peptides (including BPPs, bradykinin potentiating peptides); DC, disintegrin-like/cysteine-rich domain; svVEGF, snake venom vascular endothelial growth factor; PLA2, phospholipase A2; CRISP, cysteine-rich secretory protein; Ser-Prot, serine proteinase; PLB, phospholipase B; CTL, C-type lectin-like molecule; Gal, galactose-binding lectin; LAO, L-amino acid oxidase; 5′-NT, 5′-nucleotidase; PDE, phosphodiesterase; GC, glutaminyl cyclotransferase; Hyal, hyaluronidase; svNGF, snake venom nerve growth factor, SerPro Inhib, serine proteinase Kunitz-type inhibitor; NI, not identified.

continually introduced via mutation, and genetic variation is the raw material of evolution, it is also a weak evolutionary force. Thus, although mutation is crucial for the evolutionary process, changes in gene frequencies caused by accumulation of mutations occur at an imperceptibly slow rate, typically on the order of 1 mutation per gene per 105 meiotic events [60]. In addition to genomic divergence, variation in venom composition is also dictated by distinct postgenomic mechanisms [48,61]. Hence, differentially expressed venom proteins across the two B. jararaca geographic groups studied exhibited a shared (SE and S) or a populationspecific (SE or S) transcriptional pattern (Table 1). Population-specific translation of transcripts shared in both transcriptomic datasets (i.e., those encoding svVEGF [Q90X23], svNGF [Q90W38], sv 5′-NT, PDE, Hyal, and GC [Q9YIB5]) (Table 1) suggests the action of a posttranscriptional regulatory mechanisms. Posttranscriptional modulation of the venom transcriptome may contribute to venom evolvability without large-scale alterations of the underlying genomic structure and gene expression machinery. On the other hand, the differential expression of proteins, such as disintegrin jarastatin [Q0NZX5]; the PLA2 molecules bothropstoxin-I [Q90249], BjSE-1, BinTX-I [Q8QG87], and acid PLA2 BjS-1; and the metalloproteases PIII-SVMP BjS-1, PI-SVMP [Q98SP2], PII- and P-I MP_IIa [ADO21511], and PIII-SVMP jararhagin [P30431], whose coding messages exhibited population-specific transcriptional activity (Table 1) suggests regulation of gene transcription or enhanced transcript degradation. Furthermore, there is some evidence that genome-level effects, i.e., the presence or absence of toxin genes in the genome can dictate major shifts in venom composition [62–64]. The possibility that the lack of transcription of the population


529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 Q13 548 549 550 551 552 553 554 555 556


U

N

C

O

R

R

E

C

T

E

D

P

R O

O

F

8

Fig. 4. Proteotranscriptomics. Panel A, MS/MS-derived peptide sequences from the 14 kDa protein band eluted in RP-HPLC peak 15 of B. jararaca venom from the S population (Fig. 2B) matched different translations of the same transcript (a). The length and relative order of the toxin-coding region of the +2 and the +1 frame-translated transcript 1838 were resolved using BLAST (b). This analysis identified the protein encoded in transcript 1838 as the precursor of basic PLA2 bothropstoxin-I characterized in the venom of B. jararacussu [Q90249]. The signal peptide sequence is underlined. The perfect matching of experimentally determined and the calculated monoisotopic molecular masses for the mature protein [17–137, with all the 14 cysteines involved in disulfide linkages] (c) validated the proteotranscriptomic-guided protein assembly. (d) ESI-MS spectrum of RP-HPLC peak 15 of B. jararaca venom from the SE population showing the isotope-resolved (M + 9H)9+ ion cluster. Panel B, a) MS/MS-derived peptide sequences from the 28 (non-reduced)/14 (reduced) kDa protein band found in RP-HPLC peaks 20–23 of B. jararaca venom from the S population (Fig. 2B) matched 3 different translations of transcript 2110 (frames −1, −2, and −3) and the frame +2-translated read 1895. b) BLAST analysis was then used to delimit the boundaries of the toxin-coding sequences encoded in these four reads, and to align them onto a template sequence. The protein sequence reconstructed in this way was identified as the precursor (1–23, signal peptide; 24–158, mature polypeptide chain) of a close homolog (only one conservative substitution (D/E) in position 49 of the precursor) of B. insularis C-type lectin molecule BiL [Q6QX33].


9

U

N C O

R

R

E

C

T

E

D

P

R O

O

F


557 558 559 560 561 562 563 564 565 566 567 568

Fig. 4 (continued).

protein markers listed in Table 1 reflects the pseudogenization or the loss of the corresponding coding genes deserves further detailed investigation. The above results revealed that different levels of regulation are responsible for generating compositionally distinct venom proteomes in allopatric populations of B. jararaca. Phenotypic venom variation across geographically isolated conspecific populations often involves ontogenetic shifts in venom protein expression [reviewed in ref. 15]. Ontogenetic variation in venom composition has long been appreciated by herpetologists as a mechanism generating intraspecific stagedependent venom diversity [32], although age-dependent proteomic transitions have only recently been unveiled [48,65,66]. To investigate

whether the southeastern and southern B. jararaca venoms could be related by an ontogenetic/pedomorphic phenotypic duality, as described in other Bothrops species [35,65], the venom proteomes of juvenile specimens from both populations were explored (Fig. 5). Although changes between juveniles and adults from each population were found, they were radically different in each phylogroup. Thus, while in the SE population the ontogenetic change involves the downregulation of reverse-phase peak 40* (Fig. 5A), in the southern population upregulation of peaks 15* (along with some rearrangements of minor components, such as serine proteinase 18, CTLs 29 and 30, and SVMP 36) was observed (Fig. 5B). SDS-PAGE of peaks 40* and 15*, which account for 1.88% and 0.85% of their respective total venom proteomes (Table 2),


569 570 571 572 573 574 575 576 577 578 579 580

Table 1 Venom transcripts/proteins showing population-specific venom gland transcription and/or translation into the venom proteome. Reverse-phase peak numbering as in Figs. 2 and 5. *, Proteins exclusively found in juvenile venoms. N, non-transcribed; Y, transcribed.

10,11

t1:9 t1:10 t1:11

14

t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23

14 15 15* 15,16 19,20 27 30 32 33 34 35 36

t1:24

36

t1:25

40*

592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618

S

Y

N

Y Y N

Y Y Y

N Y Y N Y N Y Y N N Y Y

N N N Y Y Y Y N Y Y Y Y

Y

N

N

N

T

C

E

R

590 591

R

588 589

O

586 587

C

584 585

SE

showed major 10–15 kDa bands and minor multimeric forms of them (Fig. 5B, inset). ESI-MS analysis revealed that the proteins eluted in peaks 40* and 15* had isotope-averaged molecular masses 13,791.8 Da and (13,713.6 + 13,649.4 Da), respectively. Proteomic analysis (Table 2) identified the 13,791.8 and 13,713.6 as acidic D49-PLA2 BinTX-II (B. insularis P84397) [67] and basic K49-PLA2 BthTx-I (B. jararacussu Q90249), respectively. These PLA2 molecules represent, respectively, 1.88% and 0.59% of the total venom proteomes of neonates of B. jararaca from the SE and S populations (Table 2). MS/MS-derived internal tryptic peptide sequences from the molecule co-eluted in peak 15*, which comprised 0.26% of the venom proteome of neonate B. jararaca from the S population (Table 2), were identical to svVEGF Q90X24 from B. insularis [68]. However the molecular mass of the B. jararaca protein, 13,649.4 Da, differed from the calculated isotope-averaged molecular mass of svVEGF Q90X24 (25Z–V146: 13,818.8 Da), indicating that these molecules may not be identical. Neither PLA2 BinTX-II (P84397) nor svVEGF (Q90X24) transcripts were found in the homolog adult S and SE transcriptomes (Table 1). Although comparative age-dependent venom gland transcriptomic studies of snakes from both regions are required, these data strongly suggest that PLA2 BinTX-II (P84397) and svVEGF (Q90X24) are subjected to ontogenetic regulation of gene transcription or transcript degradation, and rule out that the adult venom proteomes represent different translation profiles of unique population-specific transcriptomes. Overall, we conclude that the geographic variability of B. jararaca venom stems from the integration of genomic and post-genomic mechanisms including, in the case of the southern population, a marked ontogenetic venom compositional change. In addition, the expression in the venom of the B. jararaca S population of a basic K49-PLA2 molecule identical to B. jararacussu BthTX-I (Q90249) raise the yet untested hypothesis of the occurrence of gene flow (through crossbreeding) between these two congeneric species, which often co-occur throughout portions of their geographical ranges [1,69]. Alternatively, bothropstoxin-I would represent an extraordinary case of evolutionary conservation across the phylogeny of Bothrops. However, against this hypothesis, there is no support for B. jararacussu being a sister species of B. jararaca [70–72].

N

582 583

Disintegrin jarastatin, B. jararaca [Q0NZX5] svVEGF, B. jararaca [Q90X23] svNGF, B. jararacussu [Q90W38] Basic PLA2 bothropstoxin-I, B. jararacussu [Q90249] sv VEGF, B. insularis [Q90X24] PLA2 BjSE-1 PLA2 BITP01A, B. insularis [Q8QG87] Acid PLA2 BjS-1 sv 5′-nucleotidase (sv 5′-NT) PIII-SVMP BjS-1 Phosphodiesterase (PDE) PI-SVMP B. jararaca [Q98SP2] PII-SVMP MP_IIa, B. neuwiedi [ADO21511] PI-SVMP MP_IIa, B. neuwiedi [ADO21511] Hyaluronidase (Hyal) Glutaminyl-cyclotransferase (GC), B. jararaca [Q9YIB5] PIII-SVMP jararhagin, B. jararaca [P30431] Acidic D49-PLA2 BinTX-II, B. insularis [P84397]

U

581

S

Venoms of adult B. jararaca from the southeastern and southern populations showed undistinguishable i.v. LD50s (Table 3). MHDs were also identical (p b 0.05) for the venoms from the southeastern and the southern populations (Table 3). However, a significant difference was observed for the coagulant activity of these venoms. Venom of specimens from the southeastern population had a MCD of 39 ± 1 μg (n = 3), whereas the venom of the southern population did not clot human citrated plasma even at a dose of 100 μg (Table 3). Hence, in these experimental conditions the venom of the southern population was devoid of in vitro coagulant activity. Metal chelators EDTA and EGTA impaired the thrombin-generating activity of venom pooled from specimens of B. jararaca snakes born in captivity at the Laboratory of Herpetology, Institute Butantan [73], indicating that the coagulant activity of B. jararaca venom from the SE population is mainly due to procoagulant metalloproteinases. Bothrojaractivase, a prothrombin-activating PI-SVMP that cleaves coagulation factors II and X, has been isolated from the venom of B. jararaca from Instituto Butantan [74]. However, only partial amino acid sequences of four internal tryptic peptides are available in the NCBI database [P0C7A9] [74], thus preventing an unambiguous matching to the SE and S venom proteomic and transcriptomic data listed in the Supplementary Tables S1–S4. On the other hand, the SE venom also contains the serine proteinase bothrombin [P81661] (fractions 18–25, Fig. 2A, Supplementary Table S1), for which no evidence was found in the venom of B. jararaca from the southern population. Bothrombin is a thrombin-like snake venom serine protease that clots fibrinogen in vitro by releasing fibrinopeptide A from the α-chain of fibrinogen (FGA), induces platelet aggregation through its interaction with platelet glycoprotein GPIb, and activates coagulation factor VIII [75]. The distinct set of metalloproteinases and serine proteinases present in the SE and S B. jararaca venoms may account for the different coagulant activities of these regional venom pools. In this regard, although addressing this medically relevant issue requires further detailed investigation, the differences observed in in vitro coagulant activity of these venoms suggest that the incidence of coagulopathy might be higher in envenomings caused by the specimens of the southeastern population than by those of the southern population, a hypothesis that awaits comparative clinical observations. Venoms of B. jararaca from the southeastern and southern populations also exhibited statistically significant differences in phospholipase activity, being higher in the southern venom, whereas the southeastern population venom shows higher proteinase activity (Table 3). The higher phospholipase A2 activity exhibited by the southern B. jararaca venom is consistent with the greater concentration of D49-PLA2 in this venom than in the venom of the SE population. Thus, the D49-PLA2 P81243 [76] recovered in fraction 27 of the southern venom (Fig. 2B) comprises 5.7% of the total venom proteins (Supplementary Table S1), whereas D49-PLA2 [=B. insularis BinTX-I, Q8QG87] (peaks 19 and in Fig. 2A) [67] accounts for 1.6% of the southeastern venom proteome (Table S1). Although no comparative clinical studies of the effects of envenomings by B. jararaca from the SE and S populations are available in the literature, the abundant expression (13% of the venom proteome) of the K49 myotoxic PLA2 homolog, bothropstoxin-I [Q90249] [77], in the venom of the southern B. jararaca population (peak 15, Fig. 2B), and its absence in the venom of the SE population, indicates that the pathophysiological effects attributed to this toxin, such as muscle necrosis, and edema [78–80], are likely to be responsible for differential clinical manifestations between the envenomings inflicted by the two geographic phylogroups of B. jararaca. In this regard, the prediction would be that venom of adult specimens of the southern population would induce a higher increment in plasma creatine kinase (CK) activity, a marker of acute muscle necrosis.

F

SE

t1:8

Transcriptome (adult)

O

t1:7

Differentially expressed proteins

R O

RP-HPLC fraction

P

t1:5 t1:6

3.3. B. jararaca venoms from the southeastern and southern populations 619 exhibit similarities and differences in their bioactivities 620

D

t1:1 t1:2 Q1 t1:3 t1:4


E

10


621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683

11

R

R

E

C

T

E

D

P

R O

O

F


685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700

The venoms from the two populations of B. jararaca have also a significant difference in proteinase activity in vitro. There is a higher proteolytic activity in the venom of B. jararaca from the southeast (Table 3), which can be explained by its larger percentage of metalloproteinases in comparison to the venom of B. jararaca from the southern population (Fig. 3). P-I metalloproteinases generally have higher proteolytic activity than other venom components, so the greater percentage of P-I metalloproteinases in the venom of B. jararaca from the southeastern phylogroup may also explain its higher proteinase activity.

U

684

N C O

Fig. 5. Chromatographic separations of the proteins of venoms pooled from juvenile specimens of B. jararaca. Panels A and B display, respectively, reverse-phase HPLC separations of the proteins of juvenile B. jararaca specimens from the Southeastern (SE) and Southern (S) populations. Numbering indicates chromatographic identity with peaks separated from the venom pool of adult specimens from the same population (Fig. 2). Peaks containing differentially expressed proteins between juvenile and adult from the same geographic population are highlighted and labeled with an asterisk. The insert in panel B shows an SDS-PAGE analysis of the PLA2 molecules 40* and 15* from the SE and S populations of B. jararaca. Table 2 lists the proteins found in each SDS-PAGE protein band.

3.4. BthTx-I, a population-specific marker useful for standardizing the composition of B. jararaca reference venom pools Venom of B. jararaca comprises 50% of the pool of venoms used to generate the Brazilian antibothropic antivenom [40]. However, although the institutions manufacturing the SAB antivenom for the Brazilian Ministry of Health (Instituto Butantan, São Paulo, SP; Fundação Ezequiel Dias-FUNED, Belo Horizonte, MG; Institute Vital Brazil,

Niterói, RJ; and Centro de Produção e Pesquisas de Immunobiológicos, Piraquara, PR) prepare their antivenom using virtually identical techniques, and supply an equivalently efficient final product (1 mL of antivenom neutralizes the lethality of 5 mg of standard B. jararaca venom), each antivenom producer uses its own immunizing venom mixture, which may vary in the geographic distribution of the snakes from which the venom is milked and pooled along the years. The geographic differences in the composition and biological activities of the venom of B. jararaca here reported, highlight the need to take into account this variability to generate a well-defined reference venom pool for antivenom production. In this regard, the relative content of bothropstoxin-I (BthTx-I) can be used as a marker to estimate the proportion (P) of venom from the southern population present in a B. jararaca venom pool “i” as: P = (% BthTx-I)i / 0.13. Fig. 6 illustrates this point. B. jararaca venom pools used in Instituto Butantan for antivenom production and research during the period 1963–2008 contained the following relative amounts of BthTx-I (ESI-MS, 13,711.4 Da): 3.43% (1963), 1.61% (1973), 1.40% (1977), 1.74% (1997), and 0.94% (2008). These figures clearly reveal a trend towards increasing the relative


701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719

12

Table 2 Assignment by nESI-MS/MS collision-induced dissociation of tryptic peptide ions generated by in-gel digestion of the protein bands separated by SDS-PAGE (Fig. 5B, inset) of the RP-HPLC separated differentially expressed venom proteins between neonates and adult B. jararaca specimens from the SE and S populations.

720

1.75

S-15*

ESI-MS (ave)

13,791.8

0.85 0.1

SDS-PAGE (kDa)

m/z

z

Peptide sequence

Score

Best NCBI match

Protein/protein family

28▼

460,7 501,8 490,7 501,8 460,8 761,3 507,8 825,9 550,9 583,3 596.6

2 2 2 2 2 2 3 2 3 2 3

NLWQFGR LDLYTYSK QLCECDR LDLYTYSK NLWQFGR CCYVHDCCYGK CCYVHDCCYGK ETGDLVCGGDDPCQK ETGDLVCGGDDPCQK VAALCFQDNK VAALCFQDNKDTYDK

71

B. insularis P84397

Acidic D49-PLA2 BinTX-II

266

B. insularis P84397

Acidic D49-PLA2 BinTX-II

702,8 767,3 579,6 443,9 767,3 579,6 707,4 784,4 643,7 549,7

2 2 3 3 2 3 3 4 2 4

TIVCGENNPCLK SYGAYGCNCGVLGR ELCECDKAVAICLR MILQETGKNPAK SYGAYGCNCGVLGR ELCECDKAVAICLR SLFELGKMoxILQETGKNPAK ETLVSILEEHPDEVSHIFRPSCVTALR CGGCCTDESLK TEVMQFTEHTDCECRPR

236

B. jararacussu Q90249

261

B. jararacussu Q90249

14

▼

28▼

0.49

13,712.6

14▼

0.26

13,649.4

10▼

724

3.5. BinTX-I and BinTX-II PLA2s: biogeographical markers

725 726

740

Bothrops insularis, commonly known as the golden lancehead pit viper, is a venomous snake endemic to the small (43 ha) Ilha da Queimada Grande, located 40 miles off the coast of Itanhaém, São Paulo State (24.4833° S, 46.6833° W) (Fig. 1) [1]. It is thought that the ancestor of B. insularis became isolated in this island very recently, during a late Pleistocene or Holocene sea level oscillation (around 11.000 years ago), when the sea level rose enough to isolate the island from mainland Brazil, causing the species of snakes that lived on the island to evolve on a different path than their mainland B. jararaca brethren [81]. Proteomic mapping of the distribution of taxa-specific toxins between populations of closely-related snakes provides clues for tracing dispersal routes that account for the current biogeographic distribution of the species [35]. Our finding that venoms from juvenile snakes of the SE population of B. jararaca and from adult B. insularis contain identical D49-PLA2 molecules, BinTX-I (13,791.8 Da) and BinTX-II (13,974.6 Da), indicates that the mainland ancestor of

t3:1 t3:2 Q4

Table 3 Toxic and enzymatic activities of the venoms from the two populations of B. jararaca.

736 737 738 739

B. insularis Q90X24

Basic K49-PLA2 BthTx-I

svVEGF

B. insularis originated within the same population that gave rise to the current SE B. jararaca phylogroup. This view is in line with a phylogeographical study showing that haplotypes of B. insularis built up a strongly supported group with specimens from inland São Paulo State and samples of areas near the city of São Paulo [3]. In addition, our results also suggest that B. insularis expresses a pedomorphic venom phenotype. Retention of juvenile characters in another endemic and offshore island snake of southeastern Brazil, B. alcatraz, isolated in the Arquipélago dos Alcatrazes (24°06′S, 45°42′W), 35 km off the Atlantic coast of São Paulo State (Fig. 1), since approximately 9000 years ago [3], has been associated with a diet based on ectotherms (mainly centipedes), owing to the absence of small mammalian prey on the islands [4]. Similarly, adult golden lanceheads are arboreal snakes whose diet is predominantly based on migrant passerine birds from the adjacent Atlantic forests on the mainland, whereas newborns and juveniles prey primarily on invertebrates [82–84]. Geographical variation in venom composition could reflect the effects of natural selection pressure towards adaptation of an ecologically isolated snake population to differences in availability of local prey [85]. PLA2 BinTX-I has been reported to produce presynaptic neuromuscular blockade in chick biventer cervicis preparations and muscle fiber damage [67,86]. However, defining the biological effects of pedomorphic BinTX-II [P84397] that confer selective advantage to an essentially monophagous organism, such as B. insularis, requires detailed investigations.

741

3.6. Neutralization of lethality and antivenomics

765

All mice injected with 4 LD50s of venom incubated with PBS instead of antivenom died. Two ratios of mg venom/mL antivenom were tested for each venom (2 mg/mL and 4 mg/mL). The SAB manufactured in Vital Brazil Institute neutralized the lethal effect of both venoms to a similar extent. In the case of B. jararaca venom from the southern population, one out of four mice died at both venom/antivenom ratios. In the case of venom from the southeastern population, one out of four mice and two out of four mice died at the ratios of 2 mg/mL and 4 mg/mL, respectively. Hence, at the ratio of 4 mg/mL, antivenom protected at least half of the population of injected mice for the two venoms tested. The immunological reactivity of the therapeutic antivenoms manufactured by Instituto Vital Brazil (Niterói, RJ) and Instituto Clodomiro Picado (San José, CR) towards the venom toxins of B. jararaca was assessed by Western blotting and immunoaffinity-

766

T

C

E

R

R

734 735

O

732 733

C

730 731

N

728 729

U

727

229

Basic K49-PLA2 BthTx-I

E

723

content of venoms from the SE population in the B. jararaca venom pools in the 45-year period investigated, in which the ratios of SE to S venom were: 74/26 (1963), 88/12 (1973), 90/10 (1977), 87/13 (1997), and 93/7 (2008).

721 722

F

1.88 0.13

O

%

SE-40*

R O

Spot ID

t2:5 t2:6 Q3 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24 t2:25 t2:26 t2:27

P

t2:4

D

t2:1 t2:2 Q2 t2:3


t3:3

Activity

t3:4 t3:5 t3:6 t3:7 t3:8

Lethal (LD50, μg)a Hemorrhagic (MHD, μg)b Coagulant (MCD, μg)c Proteolytic (U/mg)d Phospholipase A2 (U/mg)e

t3:9 t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16 t3:17 t3:18

a

B. jararaca (SE)

Bn jararaca (S)

21.2 (13.3–30.1) 1.10 ± 0.10 39 ± 1⁎ 8.54 ± 0.35⁎ 2180 ± 280⁎

23.1 (16.1–31.2) 1.12 ± 0.13 N100 1.51 ± 0.19 3140 ± 400

LD50: Median lethal dose by the i.v. route, in μg venom per mouse (18–20 g). b MHD: Minimum Hemorrhagic Dose: Amount of venom that induces a hemorrhagic halo in the skin of 10 mm diameter 2 h after venom injection. c MCD: Minimum Coagulant Dose: Amount of venom that induces clotting of citrated human plasma in 60 s. d Proteolytic activity: One unit corresponds to a change in absorbance of 0.2 per min at 450 nm. e Phospholipase A2 activity: One unit corresponds to a change in absorbance of 0.001 at 405 nm after 60 min. ⁎ p b 0.05.


742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764

767 768 769 770 771 772 773 774 775 776 777 778 779

13

N C O

R

R

E

C

T

E

D

P

R O

O

F


780 781 782 783 784 785 786 787 788 789 790 791 792

U

Fig. 6. Reverse-phase HPLC comparison of B. jararaca reference pools. Panel A, from top to bottom, chromatographic separations of the venom components of B. jararaca reference pools used at Instituto Butantan for antivenom production the specified year. The bothropstoxin-I peak is labeled as BthTX-I and highlighted in the box. Panel B, the isotope-averaged molecular mass of BthTX-I was determined as 13,712.4 Da by electrospray-ionization mass spectrometry.

based antivenomics [39]. Both antivenoms displayed similar patterns of immunorecognition of electroblotted proteins from the SE (Fig. 7, panels C and E) and S venoms (Fig. 7, panels H and J). The F(ab′)2 SAB antivenom column immunocaptured most of the venom components of the venoms of both B. jararaca populations (Fig. 7, panels B and G), but did not show immunoreactivity against the vasoactive peptides eluting in fractions 5, 6, 11, and 12 (Fig. 7, panels C and H). On the other hand, the Costa Rican polyvalent (Crotalinae) BCL antivenom showed the same lack of reactivity against vasoactive peptides but, in addition, was less efficient immunocapturing PI-SVMP 33 and PIII-SVMP 36 from the Southeastern venom (Fig. 7E); and K49-PLA2 BthTx-I, CRISP 17, and D49-PLA2 27 from the venom of the Southern B. jararaca phylogroup (Fig. 7J).

Our results are in general concordance with previous studies [87,88] in which the pentabothropic antivenom from Instituto Butantan was evaluated for its ability to neutralize the lethal and toxic activities of the venoms of nineteen bothropic snakes, including B. jararaca and B. jararacussu. The study, conducted by researchers from the Immunochemistry and the Herpetology laboratories of Instituto Butantan in 2008 [87], may have used B. jararaca venom exhibiting a “SE phenotype”. The results here reported show that the antibothropic antivenom produced by IVB, prepared using virtually identical protocols as the Butantan antivenom [40,89], is highly effective immunocapturing the proteins from both the southeastern and the southern B. jararaca populations. This indicates that homologous proteins present in these venoms share extensive cross-immunoreactivity. On the other hand,


793 794 795 796 797 798 799 800 801 802 803 804 805


D

P

R O

O

F

14

806

R

E

C

T

E

Fig. 7. Western blotting and second generation antivenomic analyses of B. jararaca venoms. Panel A, RP-HPLC separations of the venom proteins of B. jararaca from the Southeastern population. Protein peaks are labeled as in Fig. 2A. 300 μg of venom was incubated with 400 μL of Sepharose-immobilized antivenom. Panels B and C display, respectively, reversephase separations of the immunocaptured and the non-bound fraction from the SAB F(ab′)2 affinity column. Panels D and E show, respectively, reverse-phase HPLC separations of the venom components recovered in the bound and the flow-through fractions of the Costa Rican BCL IgG affinity column. Panel F, RP-HPLC separations of the venom proteins of B. jararaca from the southern population. Protein peaks are labeled as in Fig. 2B. 300 μg of venom was incubated with 400 μL of Sepharose-immobilized antivenom. Panels G and H display, respectively, reverse-phase separations of the immunocaptured and the non-bound fraction from the SAB F(ab′)2 affinity column. Panels I and J show, respectively, reverse-phase HPLC separations of the venom components recovered in the bound and the flow-through fractions of the Costa Rican BCL IgG affinity column. Column eluates were monitored at 215 nm and quantified by comparing the areas of homologous peaks in the two fractions [39]. Major components are labeled with acronyms described in the legend of Fig. 3. Inserted in panels C and H, and E and J, Western blot analyses showing, respectively, the immunoreactivity pattern of the SAB and the BCL antivenoms towards reverse-phase HPLC fractions isolated from the venom of the SE and the S B. jararaca phylogroups. Non-labeled peaks in panels D and E are autodegradation products from the SE venom. The autoproteolytic action of B. jararaca venom from the SE population upon incubation for 48 h at acidic (pH 5.0) or alkaline (8.5) pH has been previously reported by Sousa and co-workers [112].

814

4. Concluding remarks

815

The fact of having evolved diverse life-history strategies [90] makes snakes emerging model organisms in ecology [91]. Past interspecific interactions may have played a role in snake co-evolution and evolutionary divergence [92,93], and habitat partition modeling has been applied to evaluate interspecific interactions that help explain occupancy patterns between sympatric snakes [93–95]. However, models, per se, do not rule out alternative interpretations [96] and thus a judicious hypothesis requires integrating ecological and evolutionary constraints. In this respect, molecular evidence gathered by omic technologies is increasingly used to address a wide range of evolutionary questions [97]. Omic-driven strategies complement field and biogeographic research and provide useful insights into the migration patterns and the molecular footprints of local adaptations. The population-specific markers identified here through a combined transcriptomics and proteomic approach inform that the ancestor of B. insularis originated in the southeastern population of B. jararaca, and that within the southern

816 817 818 819 820 821 822 823 824 825 826 827 828 829 830

O

C

N

811 812

U

809 810

R

813

the ability of the IVB antivenom to quantitatively immunocapture the southern population-specific marker, PLA2 bothropstoxin-I, may be ascribed to the presence in the immunization mixture of 12.5% of B. jararacussu venom, which contains a high concentration of this protein. This observation opens up the possibility of generating a broad-spectrum antibothropic antivenom by carefully selecting a small pool of venoms containing all the classes of medically relevant toxins.

807 808

population B. jararaca and B. jararacussu may have exchanged venom toxin genes. The existence of geographical structure in the morphology of B. jararaca prompted Salomão et al. [98] to conclude that this taxon may represent a complex of several species. Data from Grazziotin and coworkers also indicated that the genetic diversity found in the B. jararaca complex is relatively high when compared to those observed in other Bothrops species [3]. To explain the clinal pattern detected by autocorrelation analyses within the southern B. jararaca phylogroup, these authors suggested the occurrence of founder effects accompanied by range expansion with gene flow. The here reported differences in venomics data gathered from populations assigned to well-differentiated clades of the same B. jararaca species suggest the existence of separate cryptic sibling evolutionary lineages caught in the act of speciation. Several species of forest vertebrates show well-defined phylogeographic breaks in southern São Paulo, all dating back to the Early Pliocene or Late Pleistocene. Species distribution models, phylogeographic analyses, and cytogenetic data provided evidence of hybridization between phylogroups of the litter frog Proceratophrys boiei in Iperó [99], a location in central São Paulo that closely match B. jararaca's SE and S split. Congruent patterns of phylogeographical breaks in the southern Atlantic forest fauna may indicate common biological responses to landscape changes [100]. The use of molecular tools has become a major approach to recognize cryptic species [101], including tropical pit vipers [102–104]. Future detailed omic studies are needed to test the hypothesis of the occurrence of gene exchange between B. jararaca and B. jararacussu.


831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857


858 859

(Fundação Zoobotânica do Rio Grande do Sul) for generously providing 920 the pictures of juvenile and adult B. jararaca specimens from SE and S, 921 respectively, displayed in Fig. 1. 922

C

891 Q16 5. Data accessibility 892 893

898

Acknowledgments

R

R

N C O

894 895

E

896 897

DNA sequences: NCBI SRA: SRP051157 (venom gland transcriptome of B. jararaca from the southeastern population: SRS791307; venom gland transcriptome of B. jararaca from the southern population: SRS791308). Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jprot.2015.04.029.

899 Q17 This study was supported by grants BFU2010-17373 (until 31 August 900 2014) from the former Ministerio de Ciencia é Innovación, Madrid; 901 902

U

BFU2013-42833-P from the Ministerio de Economía y Competitividad, Madrid; i-LINK0950 (Programa CSIC i-LINK+); CYTED project BIOTOX 903 P211RT0412; project 741-B2-652 (Vicerrectoría de Investigación, 904 Q18 UCR); FEES-CONARE (Costa Rica); project Genoprot 560931/2010-7 905 (CNPq); and grant Pensa-Rio E-26/110.319/2010 (FAPERJ, Brazil). 906 Larissa Gonçalves-Machado gratefully acknowledges CNPq (Conselho 907 Nacional de Desenvolvimento Científico e Tecnológico, Brasilia DF, 908 Brasil) and INBEB (Instituto Nacional de Biologia Estrutural e 909 Bioimagem) for providing a 1-year scholarship from Programa Ciência 910 sem Fronteiras to perform this study at IBV-CSIC. Roberta Jeane B. Jorge 911 and Carlos Corrêa-Netto gratefully acknowledge CAPES (Coordenação 912 de Aperfeiçoamento de Pessoal de Nivel Superior, Brazilian Ministry of 913 Education) for providing a 1-year scholarship to perform this study at 914 IBV-CSIC. The authors wish to thank Alicia Pérez and Yania Rodríguez 915 (Instituto de Biomedicina de Valencia, IBV) for their excellent technical 916 assistance, and Jordi Durban (IBV) for helping with the 6-frame transla917 tion of the transcriptomes. The authors wish to thank Claudio Machado 918 (Laboratorio de Herpetologia, Instituto Vital Brazil) and Tyelli Ramos 919 (Bioterium of Micrurus, Instituto Vital Brazil), and Sergio Bavaresco

923

References

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[1] J.A. Campbell, W.W. Lamar, The Venomous Reptiles of the Western Hemisphere, Comstock (Cornell University Press), Ithaca NY, 2004, ISBN 0-8014-4141-2. (962 pp.). [2] I. Sazima, Natural history of the jararaca pitviper, Bothrops jararaca, in Southeastern Brazil, in: J.A. Campbell, E.D. Brodie Jr. (Eds.), Biology of the Pitvipers, Tyler, Selva, Texas 1992, pp. 199–216. [3] F.G. Grazziotin, M. Monzel, S. Echeverrigaray, S.L. Bonatto, Phylogeography of the Bothrops jararaca complex (Serpentes: Viperidae): past fragmentation and island colonization in the Brazilian Atlantic Forest, Mol Ecol 15 (2006) 3969–3982. [4] M. Martins, O.A.V. Marques, I. Sazima, Ecological and phylogenetic correlates of feeding habits in Neotropical pitvipers of the genus Bothrops, in: G.W. Schuett, M.E. Douglas, M. Höggren, H.W. Greene (Eds.), Biology of the Vipers, Eagle Mountain Publishing, Eagle Mountain, UT 2002, pp. 1–22. [5] J.L.C. Cardoso, Bothropic accidents, Mem Inst Butantan 52 (1990) 43–44. [6] Ministério da Saúde do Brasil, Normas de Produção e Controle de Qualidade de Soros Antiofıdicos, Diário Oficial da União1996. 23491–23512. [7] Ministério da Saúde do Brasil/FUNASA (Fundação Nacional de Saúde)., Manual de diagnóstico e tratamento de acidentes por animais peçonhentos. COMED/ASPLAN/ FNS, Brasília (1998) (131 pp.). [8] D.A. Warrell, Snakebites in Central and South America: epidemiology, clinical features, and clinical management, in: J.A. Campbell, W.W. Lamar (Eds.),The Venomous Reptiles of the Western Hemisphere 2004, pp. 709–761 (Ithaca NY). [9] J.H. Brown, Toxicology and Pharmacology of Venoms From Poisonous Snakes, Thomas, Springfield, IL, 1973, ISBN 0-398-02808-7. [10] G. Rosenfeld, Symptomatology, pathology and treatment of snake bites in South America, in: W. Burcherl, W.W. Buckley, V. Deulofeu (Eds.), Venomous Animals and Their Venoms, 1971, Academic Press, New York 1971, pp. 345–841. [11] F.O.S. França, C.M.S. Malaque, Acidente botrópico, in: J.L.C. Cardoso, F.O.S. França, F.-H. Wen, C.M.S. Malaque, V. Haddad jr. (Eds.), Animais peçonhentos no Brasil: biologia, clínica e terapêutica dos acidentes, Sarvier, São Paulo, ISBN: 85-7378133-5 2003, pp. 81–95. [12] H.L. Gibbs, S.P. Mackessy, Functional basis of a molecular adaptation: prey-specific toxic effects of venom from Sistrurus rattlesnakes, Toxicon 53 (2009) 672–679. [13] B.G. Fry, N.R. Casewell, W. Wüster, N. Vidal, B. Young, T.N. Jackson, The structural and functional diversification of the Toxicofera reptile venom system, Toxicon 60 (2012) 434–448. [14] N.R. Casewell, W. Wüster, F.J. Vonk, R.A. Harrison, B.G. Fry, Complex cocktails: the evolutionary novelty of venoms, Trends Ecol Evol 28 (2013) 219–229. [15] J.J. Calvete, Snake venomics: from the inventory of toxins to biology, Toxicon 75 (2013) 44–62. [16] F.J. Vonk, N.R. Casewell, C.V. Henkel, A.M. Heimberg, H.J. Jansen, R.J. McCleary, et al., The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system, Proc Natl Acad Sci U S A 110 (2013) 20651–20656. [17] D.R. Schield, D.C. Card, J. Reyes-Velasco, A.L. Andrew, C.A. Modahl, S.P. Mackessy, et al., A role for genomics in rattlesnake research — current knowledge and future potential, in: G.W. Schuett, R.S. Reiserer, C.F. Smith (Eds.), Rattlesnakes of Arizona, Eco Books, 2015. [18] K.J. Shaney, D.R. Schield, D.C. Card, R.P. Ruggiero, D.D. Pollock, S.P. Mackessy, et al., Squamate reptile genomics and evolution, in: P. Gopalakrishnakone, J.J. Calvete (Eds.), Handbook of Toxinology: Venom Genomics and Proteomics, Springer Reference Press, New York, NY, 2015. [19] J.W. Fox, L. Ma, K. Nelson, N.E. Sherman, S.M. Serrano, Comparison of indirect and direct approaches using ion-trap and Fourier transform ion cyclotron resonance mass spectrometry for exploring viperid venom proteomes, Toxicon 47 (2006) 700–714. [20] M.C. Menezes, M.F. Furtado, S.R. Travaglia-Cardoso, A.C. Camargo, S.M. Serrano, Sex-based individual variation of snake venom proteome among eighteen Bothrops jararaca siblings, Toxicon 47 (2006) 304–312. [21] D.C. Pimenta, B.C. Prezoto, K. Konno, R.L. Melo, M.F. Furtado, A.C. Camargo, et al., Mass spectrometric analysis of the individual variability of Bothrops jararaca venom peptide fraction. Evidence for sex-based variation among the bradykininpotentiating peptides, Rapid Commun Mass Spectrom 21 (2007) 1034–1042. [22] A. Zelanis, A.K. Tashima, M.M. Rocha, M.F. Furtado, A.C. Camargo, P.L. Ho, et al., Analysis of the ontogenetic variation in the venom proteome/peptidome of Bothrops jararaca reveals different strategies to deal with prey, J Proteome Res 9 (2010) 2278–2291. [23] A. Zelanis, A.K. Tashima, A.F. Pinto, A.F. Paes Leme, D.R. Stuginski, M.F. Furtado, et al., Bothrops jararaca venom proteome rearrangement upon neonate to adult transition, Proteomics 11 (2011) 4218–4228. [24] A. Zelanis, S.M. Serrano, V.N. Reinhold, N-glycome profiling of Bothrops jararaca newborn and adult venoms, J Proteomics 75 (2012) 774–782. [25] A.K. Tashima, A. Zelanis, E.S. Kitano, D. Ianzer, R.L. Melo, V. Rioli, et al., Peptidomics of three Bothrops snake venoms: insights into the molecular diversification of proteomes and peptidomes, Mol Cell Proteomics 11 (2012) 1245–1262. [26] G.S. Dias, E.S. Kitano, A.H. Pagotto, S.S. Sant'anna, M.M. Rocha, A. Zelanis, et al., Individual variability in the venom proteome of juvenile Bothrops jararaca specimens, J Proteome Res 12 (2013) 4585–4598.

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Identifying and inferring geographical patterns in the distribution of cryptic species should not be underestimated for an appropriate treat860 ment of snakebites. Here we sought to investigate the neutralizing capa861 bility of two commercial antivenoms against the venom of the SE and 862 the S populations of B. jararaca. We chose the antivenom manufactured 863 in Instituto Clodomiro Picado because a number of studies investigating 864 the preclinical neutralizing ability against several toxic and enzymatic 865 activities of viperid snake venoms have shown a widespread pattern 866 of immunological reactivity of this polyspecific Costa Rican antivenom 867 Q14 against homologous (B. asper) and heterologous venoms, including 868 Bothrops lanceolatus, Bothrops caribbaeus, Bothrops atrox, Bothrops 869 colombiensis, and Bothrops erythromelas [35,49,105–107]. These studies 870 complemented a previous comparative investigation of the ability of the 871 Costa Rican BCL antivenom and the antibothropic antivenom produced 872 in Instituto Butantan to neutralize the lethal, hemorrhagic and coagu873 lant activities of the venoms of 16 Central and South American viperids. 874 The study showed that both antivenoms neutralized these activities 875 of different Bothrops venoms, albeit with different ED50s [108]. The 876 study included the venoms of B. asper, B. jararaca, B. jararacussu, 877 Q15 B. alternatus, B. atrox, Bothrops pradoi, Bothrops cotiara, B. insularis, 878 B. moojeni, B. erythromelas, and B. neuwiedi, which exhibit i.p. LD50s in 879 the range of 37–114 μg/18–22 g mouse [108]. 880 The remarkable paraspecificity exhibited by the Brazilian SAB and 881 the Costa Rican BCL antivenom against venom toxins from B. jararaca, 882 and against a range of Central and South American Bothrops venoms, in883 dicates large immunoreactive epitope conservation across the natural 884 history Bothrops, a genus that has its roots in the middle Miocene 885 14.07 Mya (CI95% 16.37–11.75 Mya) [109–111]. This, and previous 886 works reviewed in [15], highlight that the roadmap to achieve the 887 ambitious goal of generating antibothropic antivenoms of wide neutral888 izing and immunoreactive coverage must include systematic investiga889 tions integrating omic techniques, biological assays, biogeographical, 890 and natural history studies.

15


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[58] P. Tiffin, J. Ross-Ibarra, Advances and limits of using population genetics to understand local adaptation, Trends Ecol Evol 29 (2014) 673–680. [59] O. Savolainen, M. Lascoux, J. Merilä, Ecological genomics of local adaptation, Nat Rev Genet 14 (2013) 807–820. [60] M. Lynch, Evolution of the mutation rate, Trends Genet 26 (2010) 345–352. [61] N.R. Casewell, S.C. Wagstaff, W. Wüster, D.A. Cook, F.M. Bolton, S.I. King, et al., Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms, Proc Natl Acad Sci U S A 111 (2014) 9205–9210. [62] S. Schenberg, Geographical pattern of crotamine distribution in the same rattlesnake subspecies, Science 129 (1959) 1361–1363. [63] B.J. Wooldridge, G. Pineda, J.J. Banuelas-Ornelas, R.K. Dagda, S.E. Gasanov, E.D. Rael, et al., Mojave rattlesnakes (Crotalus scutulatus scutulatus) lacking the acidic subunit DNA sequence lack Mojave toxin in their venom, Comp Biochem Physiol B Biochem Mol Biol 130 (2001) 169–179. [64] Y.M. Wang, J. Parmelee, Y.W. Guo, I.H. Tsai, Absence of phospholipase A2 in most Crotalus horridus venom due to translation blockage: comparison with Crotalus horridus atricaudatus venom, Toxicon 56 (2010) 93–100. [65] A. Alape-Girón, L. Sanz, J. Escolano, M. Flores-Díaz, M. Madrigal, M. Sasa, et al., Snake venomics of the lancehead pitviper Bothrops asper. Geographic, individual and ontogenetic variations, J Proteome Res 7 (2008) 3556–3571. [66] M. Madrigal, L. Sanz, M. Flores-Diaz, M. Sasa, V. Núñez, A. Alape-Girón, et al., Snake venomics across genus Lachesis Ontogenetic changes in the venom composition of L. stenophrys and comparative proteomics of the venoms of adult L. melanocephala and L. acrochorda, J. Proteomics 77 (2012) 280–297. [67] J.C. Cogo, S. Lilla, G.H. Souza, S. Hyslop, G. de Nucci, Purification, sequencing and structural analysis of two acidic phospholipases A2 from the venom of Bothrops insularis (jararaca ilhoa), Biochimie 88 (2006) 1947–1959. [68] Junqueira de Azevedo IL, S.H. Farsky, M.L. Oliveira, P.L. Ho, Molecular cloning and expression of a functional snake venom vascular endothelium growth factor (VEGF) from the Bothrops insularis pit viper. A new member of the VEGF family of proteins, J Biol Chem 276 (2001) 39836–39842. [69] M.C. Rocha, P.A. Hartmann, G.R. Winck, S.Z. Cechin, Seasonal, daily activity, and habitat use by three sympatric pit vipers (Serpentes, Viperidae) from southern Brazil, An Acad Bras Cienc 86 (2014) 695–705. [70] M.G. Salomão, W. Wüster, R.S. Thorpe, BBBSP, mtDNA phylogeny of neotropical pitvipers of the genus Bothrops (Squamata: Serpentes: Viperidae), Kaupia 8 (1999) 127–134. [71] A.M. Fenwick, R.L. Gutberlet jr., J.A. Evans, C.L. Parkinson, Morphological and molecular evidence for phylogeny and classification of South American pitvipers, genera Bothrops, Bothriopsis, and Bothrocophias (Serpentes: Viperidae), Zool J Linn Soc 156 (2009) 617–640. [72] P.A. Carrasco, C.I. Mattoni, G.C. Laynaud, G.J. Scrocchi, Morphology, phylogeny and taxonomy of South American bothropoid pitvipers (Serpentes, Viperidae), Zool Scr 41 (2012) 109–124. [73] T.C. Antunes, K.M. Yamashita, K.C. Barbaro, M. Saiki, M.L. Santoro, Comparative analysis of newborn and adult Bothrops jararaca snake venoms, Toxicon 56 (2010) 1443–1458. [74] M. Berger, A.F. Pinto, J.A. Guimarães, Purification and functional characterization of bothrojaractivase, a prothrombin-activating metalloproteinase isolated from Bothrops jararaca snake venom, Toxicon 51 (2008) 488–501. [75] S. Nishida, Y. Fujimura, S. Miura, Y. Ozaki, Y. Usami, M. Suzuki, et al., Purification and characterization of bothrombin, a fibrinogen-clotting serine protease from the venom of Bothrops jararaca, Biochemistry 33 (1994) 1843–1849. [76] S.M. Serrano, A.P. Reichl, R. Mentele, E.A. Auerswald, M.L. Santoro, C.A. Sampaio, et al., A novel phospholipase A2, BJ-PLA2, from the venom of the snake Bothrops jararaca: purification, primary structure analysis, and its characterization as a platelet aggregation-inhibiting factor, Arch Biochem Biophys 367 (1999) 26–32. [77] M.I. Homsi-Brandeburgo, L.S. Queiroz, H. Santo-Neto, L. Rodrigues-Simioni, J.R. Giglio, Fractionation of Bothrops jararacussu snake venom: partial chemical characterization and biological activity of bothropstoxin, Toxicon 26 (1988) 615–627. [78] S.H. Andrião-Escarso, A.M. Soares, V.M. Rodrigues, Y. Angulo, C. Díaz, B. Lomonte, et al., Myotoxic phospholipases A2 in Bothrops snake venoms: effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu, Biochimie 82 (2000) 755–763. [79] H.S. Neto, M.J. Marques, Microvessel damage by B. jararacussu snake venom: pathogenesis and influence on muscle regeneration, Toxicon 46 (2005) 814–819. [80] Y. Oshima-Franco, G.B. Leite, C.A. Belo, S. Hyslop, J. Prado-Franceschi, A.C. Cintra, et al., The presynaptic activity of bothropstoxin-I, a myotoxin from Bothrops jararacussu snake venom, Basic Clin Pharmacol Toxicol 95 (2004) 175–182. [81] O.A.V. Marques, M. Martins, I. Sazima, A new insular species of pitviper from Brazil, with comments on evolutionary biology and conservation of the Bothrops jararaca group (Serpentes, Viperidae), Herpetologica 58 (2002) 303–312. [82] W. Wüster, M.R. Duarte, M.G. Salomão, Morphological correlates of incipient arboreality and ornithophagy in island pitvipers, and the phylogenetic position of Bothrops insularis, J Zool 266 (2005) 1–10. [83] M.R. Duarte, G. Puorto, F.L. Franco, A biological survey of the pitviper Bothrops insularis Amaral (Serpentes: Viperidae): an endemic and threatened offshore island snake of Southeastern Brazil, Stud Neotropical Fauna Environ 30 (1995) 1–13. [84] O.A.V. Marques, M. Martins, P.F. Develey, A. Macarrão, I. Sazima, The Golden Lancehead Bothrops insularis (Serpentes: Viperidae) relies on two seasonally plentiful bird species visiting its island habitat, J Nat Hist 46 (2012) 885–895. [85] J.C. Daltry, W. Wuster, R.S. Thorpe, Diet and snake venom evolution, Nature 379 (1996) 537–540. [86] J.C. Cogo, J. Prado-Franceschi, J.R. Giglio, A.P. Corrado, M.A. Cruz-Höfling, J.L. Donato, et al., An unusual presynaptic action of Bothrops insularis snake venom mediated by phospholipase A2 fraction, Toxicon 36 (1998) 1323–1332.

N

C

O

R

R

E

C

T

[27] L.F1. Sousa, C.A. Nicolau, P.S. Peixoto, J.L. Bernardoni, S.S. Oliveira, J.A. Portes-Junior, et al., Comparison of phylogeny, venom composition and neutralization by antivenom in diverse species of bothrops complex, PLoS Negl Trop Dis 7 (2013) e2442. [28] D.A. Cidade, T.A. Simão, A.M. Dávila, G. Wagner, I.L. Junqueira-de-Azevedo, P.L. Ho, et al., Bothrops jararaca venom gland transcriptome: analysis of the gene expression pattern, Toxicon 48 (2006) 437–461. [29] A. Zelanis, D. Andrade-Silva, M.M. Rocha, M.F. Furtado, S.M. Serrano, I.L. Junqueirade-Azevedo, et al., A transcriptomic view of the proteome variability of newborn and adult Bothrops jararaca snake venoms, PLoS Negl Trop Dis 6 (2012) e1554. [30] J. Vellard, Variation géographique du venin de Bothrops atrox, C. R Acad Sci 204 (1937) 1369–1371. [31] J. Vellard, Variation géographique du venin de Crotalus terrificus, C R Soc Biol 130 (1939) 463–464. [32] J.-P. Chippaux, V. Williams, J. White, Snake venom variability: methods of study, results and interpretation, Toxicon 29 (1991) 1279–1303. [33] J.L. Glenn, R.C. Straight, Mojave rattlesnake Crotalus scutulatus scutulatus venom: variation in toxicity with geographical origin, Toxicon 16 (1978) 81–84. [34] D.J. Massey, J.J. Calvete, E.E. Sánchez, L. Sanz, K. Richards, R. Curtis, et al., Venom variability and envenoming severity outcomes of the Crotalus scutulatus scutulatus (Mojave rattlesnake) from Southern Arizona, J Proteomics 75 (2012) 2576–2587. [35] J.J. Calvete, L. Sanz, A. Pérez, A. Borges, A.M. Vargas, B. Lomonte, et al., Snake population venomics and antivenomics of Bothrops atrox: paedomorphism along its transamazonian dispersal and implications of geographic venom variability on snakebite management, J Proteomics 74 (2011) 510–527. [36] M.F.D. Furtado, W. Dias da Silva, G.M.D.D. Colleto, Controle de qualidade dos venenos animais e dos correspondentes antivenenos. Padronização dos métodos de ensaio das atividades bioquímicas e farmacológicas dos venenos de algumas espécies do gênero Bothrops e Crotalus usando amostras secas a temperatura ambiente ou liofilizadas, Mem Inst Butantan 53 (1991) 149–159. [37] J.J. Calvete, L. Sanz, D. Pla, B. Lomonte, J.M. Gutiérrez, Omics meets biology: application to the design and preclinical assessment of antivenoms, Toxins 6 (2014) 3388–3405. [38] WHO, Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins, World Health Organization, Geneva, 2010. (Available from: http:// www.who.int/bloodproducts/snake_antivenoms/snakeantivenomguide/en/). [39] D. Pla, J.M. Gutiérrez, J.J. Calvete, Second generation snake antivenomics: comparing immunoaffinity and immunodepletion protocols, Toxicon 60 (2012) 688–699. [40] I. Raw, R. Guidolin, H.G. Higashi, E.M.A. Kelen, Antivenins in Brazil: preparation, in: A. Tu (Ed.), Handbook of Natural Toxins, Marcel Dekker, New York 1991, pp. 557–811. [41] Y. Angulo, R. Estrada, J.M. Gutiérrez, Clinical and laboratory alterations in horses during immunization with snake venoms for the production of polyvalent (Crotalinae) antivenom, Toxicon 35 (1997) 81–90. [42] G. Rojas, J.M. Jiménez, J.M. Gutiérrez, Caprylic acid fractionation of hyperimmune horse plasma: description of a simple procedure for antivenom production, Toxicon 32 (1994) 59–67. [43] M.J. Paine, H.P. Desmond, R.D.G. Theakston, J.M. Crampton, Gene expression in Echis carinatus (carpet viper) venom glands following milking, Toxicon 30 (1992) 379–386. [44] M. Margulies, M. Egholm, W.E. Altman, S. Attiya, J.S. Bader, L.A. Bemben, et al., Genome sequencing in microfabricated high-density picolitre reactors, Nature 437 (2005) 376–380. [45] S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, Basic local alignment search tool, J Mol Biol 215 (1990) 403–410. [46] A. Conesa, S. Götz, J.M. García-Gómez, J. Terol, M. Talón, M. Robles, Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research, Bioinformatics 21 (2005) 3674–3676. [47] A. Mortazavi, B.A. Williams, K. McCue, L. Schaeffer, B. Wold, Mapping and quantifying mammalian transcriptomes by RNA-Seq, Nat Methods 5 (2008) 621–628. [48] J. Durban, A. Pérez, L. Sanz, A. Gómez, F. Bonilla, S. Rodríguez, et al., Integrated “omics” profiling indicates that miRNAs are modulators of the ontogenetic venom composition shift in the Central American rattlesnake Crotalus simus simus, BMC Genomics 14 (2013) 234. [49] R.J.B. Jorge, H.S.A. Monteiro, J. Gonçalves-Machado, M.C. Guarnieri, R.M. Ximenes, D.M. Borjes-Nojosa, et al., Venomics and antivenomics of Bothrops erythromelas from five geographic populations within the Caatinga ecoregion of northeastern Brazil, J Proteomics 114 (2015) 93–114. [50] J.J. Calvete, Proteomic tools against the neglected pathology of snake bite envenoming, Expert Rev Proteomics 8 (2011) 739–758. [51] J.J. Calvete, Next-generation snake venomics: protein-locus resolution through venom proteome decomplexation, Expert Rev Proteomics 11 (2014) 315–329. [52] J.M. Gutiérrez, J.A. Gené, G. Rojas, L. Cerdas, Neutralization of proteolytic and hemorrhagic activities of Costa Rican snake venoms by a polyvalent antivenom, Toxicon 23 (1985) 887–893. [53] R.D.G. Theakston, H.A. Reid, Development of simple standard procedures for the characterisation of snake venom, Bull World Health Organ 61 (1983) 949–956. [54] J.A. Gené, A. Roy, G. Rojas, J.M. Gutiérrez, L. Cerdas, Comparative study on coagulant, defibrinating, fibrinolytic and fibrinogenolytic activities of Costa Rican crotaline snake venoms and their neutralization by a polyvalent antivenom, Toxicon 27 (1989) 841–848. [55] M. Holzer, S.P. Mackessy, An aqueous endpoint assay of snake venom phospholipase A2, Toxicon 34 (1996) 1149–1155. [56] W.-J. Wang, C.-H. Shih, T.-F. Huang, A novel P-I class metalloproteinase with broad substrate-cleaving activity, agkislysin, from Agkistrodon acutus venom, Biochem Biophys Res Commun 324 (2004) 224–230. [57] A. Johnstone, R. Thorpe, Immunochemistry in Practice, 2nd ed. Blackwell Scientific Publications, Oxford, 1987.

U

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E

D

P

R O

O

F

[101] D. Bickford, D.J. Lohman, N.S. Sodhi, P.K. Ng, R. Meier, K. Winker, et al., Cryptic species as a window on diversity and conservation, Trends Ecol Evol 22 (2007) 148–155. [102] A. Malhotra, R.S. Thorpe, Maximising information in systematic revisions: a combined molecular and morphological analysis of a cryptic green pitviper complex (Trimeresurus stejnegeri), Biol J Linn Soc 82 (2004) 219–235. [103] K.L. Sanders, A. Malhotra, R.S. Thorpe, Combining molecular, morphological and ecological data to infer species boundaries in a cryptic tropical pitviper, Biol J Linn Soc 87 (2006) 343–364. [104] A. Malhotra, K. Dawson, P. Guo, R.S. Thorpe, Phylogenetic structure and species boundaries in the mountain pitviper Ovophis monticola (Serpentes: Viperidae: Crotalinae) in Asia, Mol Phylogenet Evol 59 (2011) 444–457. [105] J.M. Gutiérrez, L. Sanz, J. Escolano, J. Fernández, B. Lomonte, Y. Angulo, et al., Snake venomics of the Lesser Antillean pit vipers Bothrops caribbaeus and Bothrops lanceolatus: correlation with toxicological activities and immunoreactivity of a heterologous antivenom, J Proteome Res 7 (2008) 4396–4408. [106] J.J. Calvete, A. Borges, A. Segura, M. Flores-Díaz, A. Alape-Girón, J.M. Gutiérrez, et al., Snake venomics and antivenomics of Bothrops colombiensis, a medically important pitviper of the Bothrops atrox-asper complex endemic to Venezuela: contributing to its taxonomy and snakebite management, J Proteomics 72 (2009) 227–240. [107] J.M. Gutiérrez, B. Lomonte, L. Sanz, J.J. Calvete, D. Pla, Immunological profile of antivenoms: preclinical analysis of the efficacy of a polyspecific antivenom through antivenomics and neutralization assays, J Proteomics 105 (2014) 340–350. [108] G. Bogarín, J.F. Morais, I.K. Yamaguchi, M.A. Stephano, J.R. Marcelino, A.K. Nishikawa, et al., Neutralization of crotaline snake venoms from Central and South America by antivenoms produced in Brazil and Costa Rica, Toxicon 38 (2000) 1429–1441. [109] W. Wüster, L. Peppin, C.E. Pook, D.E. Walker, A nesting of vipers: phylogeny and historical phylogeography of the Viperidae (Squamata: Serpentes), Mol Phylogenet Evol 49 (2008) 445–459. [110] T.A. Castoe, C.L. Parkinson, Bayesian mixed models and the phylogeny of pitvipers (Viperidae: Serpentes), Mol Phylogenet Evol 39 (2006) 91–110. [111] T. Machado, V.X. Silva, M.J. Silva, Phylogenetic relationships within Bothrops neuwiedi group (Serpentes, Squamata): geographically highly-structured lineages, evidence of introgressive hybridization and Neogene/Quaternary diversification, Mol Phylogenet Evol 71 (2014) 1–14. [112] J.R. Sousa, R.Q. Monteiro, H.C. Castro, R.B. Zingali, Proteolytic action of Bothrops jararaca venom upon its own constituents, Toxicon 39 (2001) 787–792.

N C O

R

R

E

C

T

[87] G.P. Queiroz, L.A. Pessoa, F.C. Portaro, M.F. Furtado, D.V. Tambourgi, Interspecific variation in venom composition and toxicity of Brazilian snakes from Bothrops genus, Toxicon 52 (2008) 842–851. [88] Y. Oshima-Franco, S. Hyslop, A.C. Cintra, J.R. Giglio, M.A. da Cruz-Höfling, L. RodriguesSimioni, Neutralizing capacity of commercial bothropic antivenom against Bothrops jararacussu venom and bothropstoxin-I, Muscle Nerve 23 (2000) 1832–1839. [89] J.L.C. Cardoso, H.W. Fan, F.O.S. França, M.T. Jorge, R.P. Leite, S.A. Nishioka, et al., Randomized comparative trial of three antivenoms in the treatment of envenoming by lance-headed vipers (Bothrops jararaca) in São Paulo Brazil, Q J Med 86 (1993) 315–325. [90] H.W. Greene, Snakes: The Evolution and Mystery in Nature, University of California Press, California, 1997, ISBN 0520224876. (351 pages). [91] R. Shine, X. Bonnet, Snakes: a new ‘model organism’ in ecological research? Trends Ecol Evol 15 (2000) 221–222. [92] J.H. Connell, Diversity and the coevolution of competitors, or the ghost of competition past, Oikos 35 (1980) 131–138. [93] D.A. Steen, C.J. McClure, J.C. Brock, D. Craig Rudolph, J.B. Pierce, J.R. Lee, et al., Snake co-occurrence patterns are best explained by habitat and hypothesized effects of interspecific interactions, J Anim Ecol 83 (2014) 286–295. [94] H.K. Reinert, Habitat separation between sympatric snake populations, Ecology 65 (1984) 478–486. [95] L. Luiselli, Resource partitioning and interspecific competition in snakes: the search for general geographical and guild patterns, Oikos 114 (2006) 193–211. [96] A. Sih, P. Crowley, M. McPeek, J. Petranka, K. Strohmeier, Predation, competition, and prey communities: a review of field experiments, Annu Rev Ecol Syst 16 (1985) 269–311. [97] A.P. Diz, M. Martínez-Fernández, E. Rolán-Alvarez, Proteomics in evolutionary ecology: linking the genotype with the phenotype, Mol Ecol 21 (2012) 1060–1080. [98] M.G. Salomão, W. Wüster, R.S. Thorpe, J.M. Touzet, BBBSP, A. Malhotra, DNA evolution of South American pitvipers of the genus Bothrops (Reptilia: Serpentes: Viperidae), in: R.S. Thorpe, W. Wüster (Eds.), Venomous Snakes: Ecology, Evolution and Snakebite, Oxford University Press, New York, ISBN: 978-0198549864 1996, pp. 89–98. [99] R.C. Amaro, M.T. Rodrigues, Y. Yonenaga-Yassuda, A.C. Carnaval, Demographic processes in the montane Atlantic rainforest: molecular and cytogenetic evidence from the endemic frog Proceratophrys boiei, Mol Phylogenet Evol 62 (2012) 880–888. [100] A.C. Carnaval, M.J. Hickerson, C.F. Haddad, M.T. Rodrigues, C. Moritz, Stability predicts genetic diversity in the Brazilian Atlantic forest hotspot, Science 323 (2009) 785–789.

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Purification and characterization of two fibrinolytic enzymes from Bothrops jararaca (jararaca) venom.

Activation of Bothrops jararaca snake venom gland and venom production: a proteomic approach.

Snake Venomics and Antivenomics of Bothrops diporus, a Medically Important Pitviper in Northeastern Argentina.

Interaction of Bothrops jararaca venom metalloproteinases with protein inhibitors.

Combined venom gland cDNA sequencing and venomics of the New Guinea small-eyed snake, Micropechis ikaheka.

Coralsnake Venomics: Analyses of Venom Gland Transcriptomes and Proteomes of Six Brazilian Taxa.

Isolation and chemical characterization of two structurally and functionally distinct forms of botrocetin, the platelet coagglutinin isolated from the venom of Bothrops jararaca.

Molecular cloning of a hyaluronidase from Bothrops pauloensis venom gland.

Jellyfish venomics and venom gland transcriptomics analysis of Stomolophus meleagris to reveal the toxins associated with sting.

Systemic haemorrhage in rats induced by a haemorrhagic fraction from Bothrops jararaca venom.

Appraisal of antiophidic potential of marine sponges against Bothrops jararaca and Lachesis muta venom.

Bothrops jararaca venom metalloproteinases are essential for coagulopathy and increase plasma tissue factor levels during envenomation.

Circulating vasotocin in the snake Bothrops jararaca.

Factor X converting and thrombin-like activities of Bothrops jararaca snake venom.

Neutralization of the oedematogenic activity of Bothrops jararaca venom on the mouse paw by an antibothropic fraction isolated from opossum (Didelphis marsupialis) serum.

Neutralization of different activities of venoms from nine species of Bothrops snakes by Bothrops jararaca antivenom.

Effects of catecholamines on the isolated aorta of the snake Bothrops jararaca.

An alternative micromethod to access the procoagulant activity of Bothrops jararaca venom and the efficacy of antivenom.

The vasopressor action of angiotensin in the snake Bothrops jararaca.

Erratum to: Angiotensin-converting enzyme inhibitors of Bothrops jararaca snake venom affect the structure of mice seminiferous epithelium.

Geographical and altitudinal distribution of Brachycephalus (Anura: Brachycephalidae) endemic to the Brazilian Atlantic Rainforest.

Secondary amines isolated from venom gland of dolichoderine antTechnomyrmex albipes.

Bothrops jararaca envenomation: Pathogenesis of hemostatic disturbances and intravascular hemolysis.

Aqueous leaf extract of Jatropha gossypiifolia L. (Euphorbiaceae) inhibits enzymatic and biological actions of Bothrops jararaca snake venom.