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

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 sub...

Descripción completa

Detalles Bibliográficos
Autores: Gonçalves Machado, Larissa, Pla Ferrer, Davinia, Sanz, Libia, Jorge, Roberta Jeane B., Leitão De Araújo, Moema, Alves, Maria Lúcia M., Alvares, Diego Janisch, De Miranda, Joari, Nowatzki, Jenifer, de Morais Zani, Karen, Fernandes, Wilson, Tanaka Azevedo, Anita Mitico, Fernández Ulate, Julián, Zingali, Russolina B., Gutiérrez, José María, Corrêa Netto, Carlos, Calvete Chornet, Juan José
Tipo de recurso: artículo
Fecha de publicación:2016
País:Costa Rica
Institución:Universidad de Costa Rica
Repositorio:Kérwá
OAI Identifier:oai:kerwa.ucr.ac.cr:10669/30169
Acceso en línea:http://www.sciencedirect.com/science/article/pii/S1874391915002213
https://hdl.handle.net/10669/30169
Access Level:acceso embargado
Palabra clave:Venom gland transcriptomics
Antivenomics
Geographic venom variation
Bothrops jararaca
Snake venom
Proteomics
id CR_11b9dbc028f8425969965302febb4e2c
oai_identifier_str oai:kerwa.ucr.ac.cr:10669/30169
network_acronym_str CR
network_name_str Costa Rica
repository_id_str
dc.title.es_ES.fl_str_mv Combined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two Bothrops jararaca populations from geographic isolated regions within the Brazilian Atlantic rainforest
title Combined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two Bothrops jararaca populations from geographic isolated regions within the Brazilian Atlantic rainforest
spellingShingle Combined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two Bothrops jararaca populations from geographic isolated regions within the Brazilian Atlantic rainforest
Gonçalves Machado, Larissa
Venom gland transcriptomics
Antivenomics
Geographic venom variation
Bothrops jararaca
Snake venom
Proteomics
title_short Combined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two Bothrops jararaca populations from geographic isolated regions within the Brazilian Atlantic rainforest
title_full Combined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two Bothrops jararaca populations from geographic isolated regions within the Brazilian Atlantic rainforest
title_fullStr Combined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two Bothrops jararaca populations from geographic isolated regions within the Brazilian Atlantic rainforest
title_full_unstemmed Combined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two Bothrops jararaca populations from geographic isolated regions within the Brazilian Atlantic rainforest
title_sort Combined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two Bothrops jararaca populations from geographic isolated regions within the Brazilian Atlantic rainforest
dc.creator.none.fl_str_mv Gonçalves Machado, Larissa
Pla Ferrer, Davinia
Sanz, Libia
Jorge, Roberta Jeane B.
Leitão De Araújo, Moema
Alves, Maria Lúcia M.
Alvares, Diego Janisch
De Miranda, Joari
Nowatzki, Jenifer
de Morais Zani, Karen
Fernandes, Wilson
Tanaka Azevedo, Anita Mitico
Fernández Ulate, Julián
Zingali, Russolina B.
Gutiérrez, José María
Corrêa Netto, Carlos
Calvete Chornet, Juan José
author Gonçalves Machado, Larissa
author_facet Gonçalves Machado, Larissa
Pla Ferrer, Davinia
Sanz, Libia
Jorge, Roberta Jeane B.
Leitão De Araújo, Moema
Alves, Maria Lúcia M.
Alvares, Diego Janisch
De Miranda, Joari
Nowatzki, Jenifer
de Morais Zani, Karen
Fernandes, Wilson
Tanaka Azevedo, Anita Mitico
Fernández Ulate, Julián
Zingali, Russolina B.
Gutiérrez, José María
Corrêa Netto, Carlos
Calvete Chornet, Juan José
author_role author
author2 Pla Ferrer, Davinia
Sanz, Libia
Jorge, Roberta Jeane B.
Leitão De Araújo, Moema
Alves, Maria Lúcia M.
Alvares, Diego Janisch
De Miranda, Joari
Nowatzki, Jenifer
de Morais Zani, Karen
Fernandes, Wilson
Tanaka Azevedo, Anita Mitico
Fernández Ulate, Julián
Zingali, Russolina B.
Gutiérrez, José María
Corrêa Netto, Carlos
Calvete Chornet, Juan José
author2_role author
author
author
author
author
author
author
author
author
author
author
author
author
author
author
author
dc.subject.es_ES.fl_str_mv Venom gland transcriptomics
Antivenomics
Geographic venom variation
Bothrops jararaca
Snake venom
Proteomics
topic Venom gland transcriptomics
Antivenomics
Geographic venom variation
Bothrops jararaca
Snake venom
Proteomics
description 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 Mya 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 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
publishDate 2016
dc.date.issued.none.fl_str_mv 2016-03-01
dc.date.accessioned.none.fl_str_mv 2017-07-03T16:15:12Z
dc.date.available.none.fl_str_mv 2017-07-03T16:15:12Z
dc.type.none.fl_str_mv artículo original
http://purl.org/coar/resource_type/c_2df8fbb1
info:eu-repo/semantics/article
format article
dc.identifier.citation.none.fl_str_mv http://www.sciencedirect.com/science/article/pii/S1874391915002213
dc.identifier.issn.none.fl_str_mv 1874-3919
dc.identifier.uri.none.fl_str_mv https://hdl.handle.net/10669/30169
dc.identifier.doi.none.fl_str_mv 10.1016/j.jprot.2015.04.029
dc.identifier.pmid.none.fl_str_mv 25968638
url http://www.sciencedirect.com/science/article/pii/S1874391915002213
https://hdl.handle.net/10669/30169
identifier_str_mv 1874-3919
10.1016/j.jprot.2015.04.029
25968638
dc.language.iso.es_ES.fl_str_mv en_US
language_invalid_str_mv en_US
dc.rights.none.fl_str_mv acceso embargado
http://purl.org/coar/access_right/c_f1cf
info:eu-repo/semantics/embargoedAccess
rights_invalid_str_mv acceso embargado
http://purl.org/coar/access_right/c_f1cf
eu_rights_str_mv embargoedAccess
dc.source.es_ES.fl_str_mv Journal of Proteomics; Volumen 135. 2016
dc.source.none.fl_str_mv reponame:Kérwá
instname:Universidad de Costa Rica
instacron:UCR
instname_str Universidad de Costa Rica
instacron_str UCR
institution UCR
reponame_str Kérwá
collection Kérwá
bitstream.url.fl_str_mv https://www.kerwa.ucr.ac.cr/bitstreams/a38802fb-bc6f-4191-9085-6c520ea9f975/download
https://www.kerwa.ucr.ac.cr/bitstreams/f23d66bb-23f2-4616-b966-8d7f731ef8bf/download
https://www.kerwa.ucr.ac.cr/bitstreams/f4cf07fe-4114-4b45-af9d-a212a843fad1/download
https://www.kerwa.ucr.ac.cr/bitstreams/2433fef8-5b89-41de-a351-184358f1a73e/download
bitstream.checksum.fl_str_mv f7e10844a567239cadfc38eab3042122
23d1cca7e51c71d1256464ad30bf7b3b
f390d89ef134b2cf93d74a0f1594d4ae
403c5dc98e1a7dc96b2f967712cca44b
bitstream.checksumAlgorithm.fl_str_mv MD5
MD5
MD5
MD5
repository.name.fl_str_mv Kérwá - Universidad de Costa Rica
repository.mail.fl_str_mv meilyn.garro@ucr.ac.cr
_version_ 1849325532184838144
spelling Gonçalves Machado, Larissa431b7585-c85c-41ca-aa0a-8c408de65032600Pla Ferrer, Davinia4dd10ec7-e6df-49e1-b953-079d9993b8ce600Sanz, Libiaa71fb767-584b-409f-b843-18c1977e6747600Jorge, Roberta Jeane B.2f40604a-6cad-4bb6-9c72-7e2b48d642d5600Leitão De Araújo, Moema7e2dc410-f316-4662-a26d-f3c75e4c807b-1Alves, Maria Lúcia M.bffed115-e994-4648-a769-aa0be9c534c6-1Alvares, Diego Janischb04179c7-0b16-4a74-b8c8-bc0c7cb57c33-1De Miranda, Joari94e8bcb4-7ed3-4526-8fef-b1291725259c-1Nowatzki, Jeniferb0da55a9-5b94-4f18-abab-2c1b70619d84-1de Morais Zani, Karen10039d27-05c6-4f93-9e24-a3864b067c59-1Fernandes, Wilson51dd09ca-c310-4c8c-b3bb-97f749a511ea-1Tanaka Azevedo, Anita Miticob8734f03-04d1-47f6-a3d2-6dddf45393a4600Fernández Ulate, Juliándc5bee91-8304-4496-9bd9-957f59389841600Zingali, Russolina B.66bff9c8-0392-47e9-879e-908634f75234600Gutiérrez, José María6a9bac5f-130f-4977-8ddf-ab97fcafacc4600Corrêa Netto, Carlos5fd09a2c-665f-4f99-ae3f-6ee5d497e69b600Calvete Chornet, Juan José03718753-da20-4f77-b16a-f5867f73b0db6002017-07-03T16:15:12Z2017-07-03T16:15:12Z2016-03-01http://www.sciencedirect.com/science/article/pii/S18743919150022131874-3919https://hdl.handle.net/10669/3016910.1016/j.jprot.2015.04.02925968638Bothrops 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 Mya 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 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 EcologMinisterio de Ciencia é Innovación/[BFU2010-17373]//EspañaMinisterio de Economía y Competitividad/[BFU2013-42833-P]//EspañaPrograma iberoamericano de ciencia y tecnología para el Desarrollo/[BIOTOX P211RT0412]/CYTED/EspañaUniversidad de Costa Rica/[741-B2-652]/UCR/Costa RicaConselho Nacional de Desenvolvimento Científico e Tecnológico/[Genoprot 560931/2010-7]/CNPq/BrasilUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)en_USacceso embargadohttp://purl.org/coar/access_right/c_f1cfinfo:eu-repo/semantics/embargoedAccessJournal of Proteomics; Volumen 135. 2016reponame:Kérwáinstname:Universidad de Costa Ricainstacron:UCRVenom gland transcriptomicsAntivenomicsGeographic venom variationBothrops jararacaSnake venomProteomicsCombined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two Bothrops jararaca populations from geographic isolated regions within the Brazilian Atlantic rainforestartículo originalhttp://purl.org/coar/resource_type/c_2df8fbb1info:eu-repo/semantics/articleTHUMBNAIL412_2016_J.Proteomics_Goncalves_B.jararaca_populations.pdf.jpg412_2016_J.Proteomics_Goncalves_B.jararaca_populations.pdf.jpgGenerated Thumbnailimage/jpeg4711https://www.kerwa.ucr.ac.cr/bitstreams/a38802fb-bc6f-4191-9085-6c520ea9f975/downloadf7e10844a567239cadfc38eab3042122MD54ORIGINAL412_2016_J.Proteomics_Goncalves_B.jararaca_populations.pdf412_2016_J.Proteomics_Goncalves_B.jararaca_populations.pdfVersión Finalapplication/pdf3075132https://www.kerwa.ucr.ac.cr/bitstreams/f23d66bb-23f2-4616-b966-8d7f731ef8bf/download23d1cca7e51c71d1256464ad30bf7b3bMD51LICENSElicense.txtlicense.txttext/plain; charset=utf-82437https://www.kerwa.ucr.ac.cr/bitstreams/f4cf07fe-4114-4b45-af9d-a212a843fad1/downloadf390d89ef134b2cf93d74a0f1594d4aeMD52TEXT412_2016_J.Proteomics_Goncalves_B.jararaca_populations.pdf.txt412_2016_J.Proteomics_Goncalves_B.jararaca_populations.pdf.txtExtracted texttext/plain98527https://www.kerwa.ucr.ac.cr/bitstreams/2433fef8-5b89-41de-a351-184358f1a73e/download403c5dc98e1a7dc96b2f967712cca44bMD5310669/301692021-04-12 09:24:31.243embargo2096-01-01oai:kerwa.ucr.ac.cr:10669/30169https://www.kerwa.ucr.ac.crInstitucionalhttp://www.kerwa.ucr.ac.crUniversidadhttp://www.ucr.ac.crhttps://kerwa.ucr.ac.cr/oai/requestmeilyn.garro@ucr.ac.crCosta RicaNo aplicaNo aplicaNo aplicaopendoar:18712025-03-29T18:07:02.027Kérwá - Universidad de Costa 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
score 15,228081

Similares en LA Referencia