Proteomic and functional characterization of the venom of two species of snakes from the genus Bothrops submitted to diet change

As peçonhas de serpentes são um dos venenos animais mais estudados. Sua composição é complexa, sendo compostas majoritariamente por proteínas, que correspondem a aproximadamente 90% de seu peso seco. As proteínas do veneno estão sujeitas à evolução Darwiniana e a variação na composição do veneno é observada em diferentes níveis taxonômicos. Estudos demonstram de forma consistente que há grandes variações na composição dos venenos de serpentes entre diferentes espécies e mesmo dentro de uma mesma espécie. Essas diferenças podem ser decorrentes de fatores variados, incluindo sexo, idade, distribuição geográfica e dieta. A variação da composição do veneno devido à dieta pode estar relacionada à especialização da serpente a diferentes presas. Entretanto, a relação entre a dieta e a composição e atividades do veneno ainda é um tema controverso e pouco estudado. Logo, o objetivo deste trabalho é investigar a influência da dieta na composição e nas atividades bioquímicas e biológicas do veneno das serpentes das espécies Bothrops moojeni e B. alternatus. Para isso, as serpentes de ambas as espécies tiveram sua dieta alterada de mamíferos (camundongos ou ratos), o padrão utilizado na manutenção das serpentes do plantel do Laboratório de Herpetologia do Instituto Butantan, para anfíbios (Lithobates catesbeianus) por um ano e, depois, retornaram à dieta com mamíferos. Com relação às serpentes, notou-se que a espécie B. moojeni apresentou menos rejeição à alimentação, sem episódios de regurgitação ou morte devido à dieta. Já as B. alternatus apresentaram muitos episódios de regurgitação, além de problemas bucais graves, incluindo estomatite com presença de cáseos. Observaram-se poucas diferenças no perfil eletroforético e cromatográfico de B. moojeni, porém as serpentes da espécie B. alternatus apresentaram uma banda de ~15 kDa mais intensa nos venenos finais, mas que se manteve após o retorno à alimentação. Além disso, houve o surgimento de novos picos cromatográficos na cromatografia líquida de alta eficiência (HPLC) em especial no veneno das fêmeas, o que não foi observado em B. moojeni. Com relação às atividades enzimáticas, as diferenças mais notáveis foram na atividade de LAAO e PLA2 das serpentes B. alternatus, enquanto o veneno de B. moojeni demonstrou poucas alterações. Os ensaios in vivo demonstraram uma diminuição da atividade letal em camundongos e um aumento da letalidade em anfíbios para ambas as espécies. As atividades hemorrágica e edematogênica apresentaram uma diminuição com a alimentação baseada em anfíbios em ambas as espécies, mas houve retorno à atividade inicial apenas na espécie B. moojeni. Sendo assim, os resultados corroboram a influência da dieta na composição e função do veneno, indicando sua plasticidade fenotípica.Snake venoms are the most studied animal toxins. Its composition is complex, being composed mainly of proteins, which correspond to approximately 90% of its dry weight. The venom proteins are subject to Darwinian evolution and the variation in the composition of the venom is observed at different taxonomic levels. Studies consistently demonstrate that there are large variations in the composition of snake venoms between different species and even within the same species. These differences can be due to different factors, including sex, age, geographic distribution and diet. The variation in the venom composition due to diet may be related to the snake\'s specialization to different prey. However, the correlation between diet and venom composition and activities is still a controversial topic. Therefore, the objective of this work is to investigate the influence of the diet on the composition, and biochemical and biological activities of the venom from snakes of the species Bothrops moojeni and B. alternatus. For this, the snakes of both species had their diet changed from mammals (mice or rats), the standard diet used in the maintenance of the snakes at the Laboratory of Herpetology of the Butantan Institute, to amphibians (Lithobates catesbeianus) for one year and then, returned to the diet based on mammals. Regarding snakes, it was noted that the species B. moojeni showed less rejection of feeding, without episodes of regurgitation or death due to the diet. B. alternatus, on the other hand, had many episodes of regurgitation, in addition to severe oral problems, including stomatitis with the presence of pus. Few differences were observed in the electrophoretic and chromatographic profile of B. moojeni, but the snakes of the species B. alternatus showed a more intense protein band of ~15 kDa in the final venom, but which remained after returning to the diet base on mammals. In addition, there were new chromatographic peaks in high performance liquid chromatography (HPLC), especially in female venoms, which was not observed in B. moojeni. Regarding the enzymatic activities, the most notable differences were in the LAAO and PLA2 activities of the B. alternatus snakes, whereas the B. moojeni venom showed few changes. In vivo assays demonstrated a decrease in lethal activity in mice and an increase in amphibians for both species. Hemorrhagic and edematogenic activities showed a decrease with amphibian-based feeding in both species, but there was a return to the initial activity only in B. moojeni species. Thus, the results of this work corroborate the influence of the diet on the composition and function of the snake venom, indicating a phenotypic plasticity

Comparative compositional and functional analyses of Bothrops moojeni specimens reveal several individual variations.

Snake venoms are complex protein mixtures with different biological activities that can act in both their preys and human victims. Many of these proteins play a role in prey capture and in the digestive process of these animals. It is known that some snakes are resistant to the toxicity of their own venom by mechanisms not yet fully elucidated. However, it was observed in the Laboratory of Herpetology of Instituto Butantan that some Bothrops moojeni individuals injured by the same snake species showed mortalities caused by envenoming effects. This study analyzed the biochemical composition of 13 venom and plasma samples from Bothrops moojeni specimens to assess differences in their protein composition. Application of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed distinct venom protein profiles, but very homogeneous plasma profiles. Western Blotting (WB) was performed with plasma samples, which were submitted to incubation with the respective venom. Some individuals showed an immunorecognized band zone around 25 kDa, indicating interaction between the same individual plasma and venom proteins. Crossed-WB assay using non-self-plasma and venom showed that this variability is due to venom protein composition instead of plasma composition. These venoms presented higher caseinolytic, collagenolytic and coagulant activities than the venoms without these regions recognized by WB. Mass spectrometry analyses performed on two individuals revealed that these individuals present, in addition to higher protein concentrations, other exclusive proteins in their composition. When these same two samples were tested in vivo, the results also showed higher lethality in these venoms, but lower hemorrhagic activity than in the venoms without these regions recognized by WB. In conclusion, some Bothrops moojeni specimens differ in venom composition, which may have implications in envenomation. Moreover, the high individual venom variability found in this species demonstrates the importance to work with individual analyses in studies involving intraspecific venom variability and venom evolution