Crotoxina en cascabeles de Latinoamérica: Caracterización bioquímica en Crotalus durissus cumanensis y análisis evolutivo de la subunidad B

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2025
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Universidad de Caldas
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Repositorio Institucional U. Caldas
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spa
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oai:repositorio.ucaldas.edu.co:ucaldas/22712
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https://repositorio.ucaldas.edu.co/handle/ucaldas/22712
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570 - Biología
1. Ciencias Naturales
Cotalus
Veneno
Crotoxina
Caracterización bioquímica
Reconstrucción filogenética
Caracteres ancestrales
Ecología evolutiva
Biología
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id REPOUCALDA_ae0957085483df9a3918f292e1bf0bcf
oai_identifier_str oai:repositorio.ucaldas.edu.co:ucaldas/22712
network_acronym_str REPOUCALDA
network_name_str Repositorio Institucional U. Caldas
repository_id_str
dc.title.none.fl_str_mv Crotoxina en cascabeles de Latinoamérica: Caracterización bioquímica en Crotalus durissus cumanensis y análisis evolutivo de la subunidad B
title Crotoxina en cascabeles de Latinoamérica: Caracterización bioquímica en Crotalus durissus cumanensis y análisis evolutivo de la subunidad B
spellingShingle Crotoxina en cascabeles de Latinoamérica: Caracterización bioquímica en Crotalus durissus cumanensis y análisis evolutivo de la subunidad B
570 - Biología
1. Ciencias Naturales
Cotalus
Veneno
Crotoxina
Caracterización bioquímica
Reconstrucción filogenética
Caracteres ancestrales
Ecología evolutiva
Biología
title_short Crotoxina en cascabeles de Latinoamérica: Caracterización bioquímica en Crotalus durissus cumanensis y análisis evolutivo de la subunidad B
title_full Crotoxina en cascabeles de Latinoamérica: Caracterización bioquímica en Crotalus durissus cumanensis y análisis evolutivo de la subunidad B
title_fullStr Crotoxina en cascabeles de Latinoamérica: Caracterización bioquímica en Crotalus durissus cumanensis y análisis evolutivo de la subunidad B
title_full_unstemmed Crotoxina en cascabeles de Latinoamérica: Caracterización bioquímica en Crotalus durissus cumanensis y análisis evolutivo de la subunidad B
title_sort Crotoxina en cascabeles de Latinoamérica: Caracterización bioquímica en Crotalus durissus cumanensis y análisis evolutivo de la subunidad B
dc.contributor.none.fl_str_mv Rodríguez-Rey, Ghennie Tatiana
Franco Vásquez, Adrián Marcelo
Mejía-Sanchez Miguel Ángel
Arreguín-Espinosa Roberto
Grupo de Ecología y Diversidad de Anfibios y Reptiles (Categoría B)
dc.subject.none.fl_str_mv 570 - Biología
1. Ciencias Naturales
Cotalus
Veneno
Crotoxina
Caracterización bioquímica
Reconstrucción filogenética
Caracteres ancestrales
Ecología evolutiva
Biología
topic 570 - Biología
1. Ciencias Naturales
Cotalus
Veneno
Crotoxina
Caracterización bioquímica
Reconstrucción filogenética
Caracteres ancestrales
Ecología evolutiva
Biología
description Figuras, tablas, estadísticas
publishDate 2025
dc.date.none.fl_str_mv 2025-09-11T16:17:47Z
2025-09-11T16:17:47Z
2025-09-10
2055-12-31
dc.type.none.fl_str_mv Trabajo de grado - Pregrado
http://purl.org/coar/resource_type/c_7a1f
Text
info:eu-repo/semantics/bachelorThesis
dc.identifier.none.fl_str_mv https://repositorio.ucaldas.edu.co/handle/ucaldas/22712
Universidad de Caldas
Repositorio Institucional Universidad de Caldas
repositorio.ucaldas.edu.co
url https://repositorio.ucaldas.edu.co/handle/ucaldas/22712
identifier_str_mv Universidad de Caldas
Repositorio Institucional Universidad de Caldas
repositorio.ucaldas.edu.co
dc.language.none.fl_str_mv spa
language spa
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dc.publisher.none.fl_str_mv Universidad de Caldas
Facultad de Ciencias Exactas y Naturales
Manizales, Caldas, Colombia
Biología
publisher.none.fl_str_mv Universidad de Caldas
Facultad de Ciencias Exactas y Naturales
Manizales, Caldas, Colombia
Biología
institution Universidad de Caldas
repository.name.fl_str_mv
repository.mail.fl_str_mv
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spelling Crotoxina en cascabeles de Latinoamérica: Caracterización bioquímica en Crotalus durissus cumanensis y análisis evolutivo de la subunidad B570 - Biología1. Ciencias NaturalesCotalusVenenoCrotoxinaCaracterización bioquímicaReconstrucción filogenéticaCaracteres ancestralesEcología evolutivaBiologíaFiguras, tablas, estadísticasResumen. Los elementos proteicos del veneno pueden variar de forma intra o interespecífica por factores geográficos, ecológicos, genéticos, sexuales y estacionales. Sin embargo, existen pocos estudios sobre la variación intraespecífica en componentes particulares y que consideren un marco evolutivo. La subunidad B de la crotoxina (CB), una fosfolipasa A2 básica, con actividad neurotóxica y letal, constituye un componente principal del veneno en algunas especies del género Crotalus. Su caracterización permite predecir cuadros clínicos y comprender su papel en la toxicidad del veneno, además, su estudio en un contexto evolutivo permite entender mejor la trayectoria de la crotoxina en el género. El objetivo de este estudio fue caracterizar bioquímicamente la variación intraespecífica de la CB en Crotalus durissus cumanensis de tres ecorregiones colombianas: Llanos Orientales (Orn), Magdalena Medio (And) y Caribe (Car), y reconstruir sus estados ancestrales de la presencia, abundancia y masa, con el fin de explorar su evolución en el género Crotalus. Para ello, se analizó la composición y variación de los venenos por técnicas electroforéticas (SDS-PAGE) y cromatográficas (RP-HPLC), y se determinó la presencia de la CB mediante ensayos inmunoquímicos como Western-blot y ELISA. La CB fue aislada por RP-HPLC, su identidad confirmada mediante espectrometría de masas (MALDI-TOF y LC-MS/MS), y su secuencia primaria fue obtenida por digestión proteolítica. Posteriormente, se probó su actividad enzimática in vitro y letalidad in vivo. Las relaciones filogenéticas de las especies de Crotalus se reconstruyeron empleando máxima verosimilitud e inferencia bayesiana a partir de secuencias de dos genes mitocondriales y uno nuclear. Finalmente, la reconstrucción de caracteres ancestrales se realizó mediante mapeo estocástico de caracteres para la presencia, mientras que la abundancia y la masa fueron modeladas bajo un proceso de evolución Browniana. Los resultados confirman la variación bioquímica y funcional entre ecorregiones, siendo el veneno de la ecorregión Orn el más letal de los tres. En cuanto a la CB, las proteínas de las ecorregiones Car y And comparten tanto el mismo peso molecular como secuencia de aminoácidos, mientras que la de Orn difiere en ambos aspectos; asimismo, fue la única en presentar diferencias con una mayor actividad enzimática respecto al grupo control, y el análisis de secuencias confirmó que es la más divergente, tanto a nivel genético como funcional. Finalmente, la reconstrucción ancestral indicó que el ancestro común del género ya presentaba CB con un peso molecular comparable al actual y que esta proteína se ha perdido independientemente en múltiples ocasiones a lo largo de la historia evolutiva de Crotalus. En conjunto, estos hallazgos muestran que la variación geográfica influye tanto en la composición y actividad de la CB como en la letalidad del veneno, además, se evidenció una clara separación de estas características en concordancia con las relaciones filogenéticas entre las tres ecorregiones, lo que sugiere que la CB ha sido un componente clave en la diversificación de Crotalus, aportando nuevas perspectivas sobre la ecología evolutiva y la adaptación de la especie a diferentes entornos.Abstract. The protein elements of venom can differ intra- or interspecifically due to geographic, ecological, genetic, sexual, and seasonal factors. However, there are few studies on intraspecific variation in particular components that consider an evolutionary framework. The B subunit of crotoxin (CB), a basic phospholipase A2 with neurotoxic and lethal activity, is a major component of the venom in some species of the genus Crotalus. Its characterization allows us to predict clinical patterns and understand its role in venom toxicity. Furthermore, its study in an evolutionary context allows us to better understand the trajectory of crotoxin in the genus. The objective of this study was to biochemically characterize the intraspecific variation of CB in Crotalus durissus cumanensis from three Colombian ecoregions: Llanos Orientales (Orn), Magdalena Medio (And), and Caribe (Car), and to reconstruct their ancestral states of presence, abundance, and mass in order to explore their evolution in the genus Crotalus. To this purpose, the composition and variation of the venoms were analyzed by electrophoretic (SDS-PAGE) and chromatographic (RP-HPLC) techniques, and the presence of CB was determined by immunochemical assays such as Western blot and ELISA. CB was isolated by RP-HPLC, their identity confirmed by mass spectrometry (MALDI-TOF and LC-MS/MS), and their primary sequence obtained by proteolytic digestion. Subsequently, their enzymatic activity was tested in vitro and their lethality in vivo. The phylogenetic relationships of Crotalus species were reconstructed using maximal likelihood and Bayesian inference from sequences of two mitochondrial genes and one nuclear gene. Finally, ancestral characters were reconstructed using stochastic character mapping for presence, while abundance and mass were modeled under a Brownian evolution process. The results confirm biochemical and functional variation between ecoregions, the venom from the Orn ecoregion being the most lethal of the three. In terms of CB, the proteins from the Car and And ecoregions both share the same molecular weight and amino acid sequence, while that from Orn differs in both respects; it was also the only one to show differences with higher enzymatic activity compared to the control group, and sequence analysis confirmed that it is the most divergent, both genetically and functionally. Finally, ancestral reconstruction indicated that the common ancestor of the genus already had CB with a molecular weight comparable to the current one and that this protein has been lost independently on multiple occasions throughout the evolutionary history of Crotalus. Altogether, these findings show that geographic variation influences both the composition and activity of CB and the lethality of the venom. Furthermore, a clear separation of these characteristics was evident in accordance with the phylogenetic relationships between the three ecoregions, suggesting that CB has been a key component in the diversification of Crotalus, providing new insights into the evolutionary ecology and adaptation of the species to different environments.Introducción -- Materiales y métodos -- Obtención del material biológico -- Caracterización bioquímica del veneno de Crotalus durissus cumanensis -- Cuantificación de proteínas -- Electroforesis en gel de poliacrilamida (SDS-PAGE) -- Purificación del veneno y aislamiento de la crotoxina de Crotalus durissus cumanensis -- Identificación inmunoquímica de CB -- Pruebas de actividad -- Letalidad (DL50) -- Espectrometría de masas -- Secuenciación de aminoácidos -- Análisis filogenéticos y reconstrucción de caracteres ancestrales -- Resultados -- Electroforesis SDS-PAGE veneno crudo C. d. cumanensis -- Aislamiento, purificación y cuantificación de la crotoxina subunidad B (CB) por cromatografía de fase reversa RP-HPLC y electroforesis SDS-PAGE -- Electroforesis SDS-PAGE crotoxina subunidad B (CB) aislada del veneno crudo de C. d. cumanensis -- Reconocimiento de la crotoxina por el anticuerpo anti-subunidad B -- Actividad PLA₂ en veneno crudo y crotoxina B purificada -- Letalidad (DL50) -- Secuencia de aminoácidos -- Análisis filogenéticos -- Reconstrucción de caracteres ancestrales -- Discusión -- Conclusiones -- Referencias bibliográficas.PregradoSe trabajó con cuatro pools de veneno de tres ecorregiones de Colombia de la especie C. d. cumanensis, con un total de 10 mg cada uno. Las ecorregiones fueron: (i) Magdalena medio, con muestras del municipio de Armero Guayabal, Tolima (And1), y del municipio de Villavieja, Huila (And3); (ii) Caribe, con una muestra del municipio de Valledupar, Cesar (Car); y (iii) Llanos Orientales, con una muestra del municipio de Puerto Gaitán, Meta (Orn) (Figura 1). La ecorregión Andina se dividió en dos muestras con el fin de evaluar si la distancia geográfica entre departamentos, aun perteneciendo a la misma ecorregión, era suficiente para detectar diferencias bioquímicas y funcionales en la CB.Biólogo(a)ProteómicaUniversidad de CaldasFacultad de Ciencias Exactas y NaturalesManizales, Caldas, ColombiaBiologíaRodríguez-Rey, Ghennie TatianaFranco Vásquez, Adrián MarceloMejía-Sanchez Miguel ÁngelArreguín-Espinosa RobertoGrupo de Ecología y Diversidad de Anfibios y Reptiles (Categoría B)Correa, EstefaníaMarínOspina, Milton David2025-09-11T16:17:47Z2055-12-312025-09-11T16:17:47Z2025-09-10Trabajo de grado - Pregradohttp://purl.org/coar/resource_type/c_7a1fTextinfo:eu-repo/semantics/bachelorThesis59 páginasapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttps://repositorio.ucaldas.edu.co/handle/ucaldas/22712Universidad de CaldasRepositorio Institucional Universidad de Caldasrepositorio.ucaldas.edu.cospaAguilar, I., Guerrero, B., Maria Salazar, A., Girón, M. 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Toxins, 8(6), 188.https://creativecommons.org/licenses/by/4.0/Atribución 4.0 Internacional (CC BY 4.0)http://purl.org/coar/access_right/c_f1cfoai:repositorio.ucaldas.edu.co:ucaldas/227122025-09-12T08:01:28Z

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