Elsevier

Toxicon

Volume 47, Issue 3, 1 March 2006, Pages 304-312
Toxicon

Sex-based individual variation of snake venom proteome among eighteen Bothrops jararaca siblings

https://doi.org/10.1016/j.toxicon.2005.11.007Get rights and content

Abstract

Variation of venom proteome is relevant to basic research, to management of envenoming, and to studies on the evolution of poisonous snakes. In this study, we explored the venom proteomes of eighteen Bothrops jararaca specimens of a single litter born and raised in laboratory. Using electrophoretic techniques and various protocols for measuring the proteolytic activities of these venoms we have detected individual variability and highlighted sex-specific proteomic similarities and differences among sibling snakes. SDS-polyacrylamide gel electrophoresis under non-reducing conditions showed protein bands of ∼100 kDa specific of male venoms. 2D-electrophoresis showed regions with varying spot complexity between pooled female and male venoms as well as spots that were gender specific. Gelatin zymography showed that female venoms contained proteinases of ∼25 kDa absent from male venoms. Female venoms were more active than male venoms in degrading fibrinogen whereas on fibrin no significant differences were detected. Among various chromogenic peptide substrates tested, male venoms showed higher amidolytic activity than female venoms on D-Val-Leu-Lys-pNA and D-Phe-Pip-Arg-pNA. Taken together, these results show sex-based differences in the venom proteome of sibling snakes of a single litter raised under controlled conditions which seem to be genetically inherited and imposed by evolutionary forces.

Introduction

Snake venoms are complex mixtures of hundreds of proteins and peptides that function to immobilize or to kill the prey as well as to assist in the digestion of the prey. The toxic effects exerted by snake venoms are complex because their different components have distinct actions and may also act in concert with other venom toxins in a synergistic fashion to enhance their activities. Venom variability has long been appreciated by investigators and it is a well documented phenomenon. It occurs at several levels including interfamily, intergenus, interspecies, intersubspecies and intraspecies variation (Chippaux et al., 1991). Moreover, venom composition may be influenced by the geographical origin and habitat of the snake (Chippaux et al., 1991). Intraspecific venom variation occurs between individual specimens, and also in individual specimens, due to seasonal variation, diet, habitat, age, and sexual dimorphism (Chippaux et al., 1991).

Venom variation is relevant to both basic venom research as well as to the management of snake envenomation. In the former case, considerations of the variation in venom composition are relevant for the choice of snake donors that are used for venom production and in the latter case the selection of snake donors for antivenom production and subsequently the selection of anti-sera for therapeutic use (Chippaux et al., 1991, Theakston et al., 2003). Moreover, venom proteome variation is also an important issue of studies on the evolutionary aspects of venomous snakes.

At the level of intraspecific variation the individual contribution to the venom composition is important but the effects contributed by environmental conditions, age and feeding habits also influence the proteome picture exhibited by each specimen. Studies of captive-bred snakes indicated that the intraspecific variation in venom is genetically inherited rather than environmentally induced (Daltry et al., 1996a, Daltry et al., 1996b). However, according to Sasa (1999), microevolutionary forces other than selection for local prey should also be considered as a source of the high variation of venom components among populations.

Previous studies on the venoms from 21 individuals of Echis carinatus from a climatically, geographically and nutritionally homogeneous habitat showed both qualitative and quantitative variability among the venom electrophoretic profiles while enzymatic activity showed similarity in some cases (phosphodiesterase, 5′-nucleotidase and caseinase) and individual variation in phospholipase A2 and l-amino acid oxidase activities (Taborska, 1971). The venom analysis from over 100 specimens of Calloselasma rhodostoma using isoelectric focusing revealed significant geographic variation which was further correlated with variation in the biological activities of the venom (Daltry et al., 1996a). Venoms of 30 young from a single litter of Bitis gabonica were analyzed by electrophoresis and showed individual variation however no sex-based variation was noticed (Chippaux et al., 1982).

The pit viper Bothrops jararaca is of major clinical importance as a leading cause of snake bite in Brazil. Although B. jararaca venom has been the focus of extensive studies, the degree to which the venom proteome varies between individuals is poorly understood. Early studies by Schenberg and colleagues showed variability in the enzymatic content of B. jararaca individuals with reduction due to milking frequency (Schenberg et al., 1970). Recently, a multifaceted analysis of the B. jararaca venom proteome was conducted by 2D-electrophoresis and mass spectrometry analysis showing the subpopulations of toxins present in this venom (Serrano et al., 2005). At the individual level, the variation of B. jararaca venom composition was also evaluated by comparing the proteomes of six specimens by 2D-electrophoresis (Fox et al., 2002). The 2D-gel images showed notable differences between the proteomes of these individual snakes collected from geographically distinct regions in São Paulo State, Brazil (Fox et al., 2002).

The objective of this investigation was to analyse venom samples from eighteen B. jararaca sibling snakes, which were born in a single litter at the Laboratory of Herpetology (Instituto Butantan) and kept under controlled laboratory conditions, in order to evaluate sex-based differences in venom composition. Using electrophoretic techniques and various protocols for measuring the proteolytic activities of the individual venoms we have highlighted sex-specific proteomic similarities and differences among sibling snakes.

Section snippets

Venoms

Venom samples were from eighteen B. jararaca snakes that were born in a single litter from a snake that was captured pregnant from the wild in the area of Ibiuna (São Paulo State, Brazil) and kept in the Laboratory of Herpetology of Instituto Butantan (São Paulo, Brazil) under controlled conditions. The siblings (eleven female and seven male snakes) were raised in the same laboratory and venom for this study was milked when the snakes were 36 months old.

Protein determination

Protein concentrations were determined

Comparison of individual venom samples by SDS-polyacrylamide gel electrophoresis

In order to get a thorough understanding of a venom proteome, both reducing and non-reducing SDS-PAGE are best used as complementary approaches to understand the nature of venom complexity. The analysis of individual venom samples from the single litter of B. jararaca snakes by SDS-PAGE under reducing conditions showed variation of protein bands among the eighteen siblings irrespective of the gender of the animal (Fig. 1(A)). Female venoms showed a more homogenous electrophoretic profile than

Acknowledgements

This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), grants 98/14307-9 and 04/14524-2.

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