Skip to main content
Log in

Deconstructing a Complex Molecular Phenotype: Population-Level Variation in Individual Venom Proteins in Eastern Massasauga Rattlesnakes (Sistrurus c. catenatus)

  • Published:
Journal of Molecular Evolution Aims and scope Submit manuscript

Abstract

Identifying the molecular basis for complex adaptations such as the toxic proteins used by venomous snakes to subdue and digest prey is an important step in understanding the evolutionary and functional basis for such traits. Recent proteomics-based analyses have made possible the identification of all constituent proteins in whole venom samples. Here we exploit this advance to study patterns of population-level variation in venom proteins from 254 adult eastern massasauga rattlesnakes (Sistrurus c. catenatus) collected from 10 populations. Analysis of presence–absence variation in specific proteins from 1D PAGE gels shows that: (1) The frequency spectra for individual protein bands is U-shaped with a large number of specific proteins either being consistently “common” or “rare” across populations possibly reflecting functional differences. (2) Multivariate axes which summarize whole venom variation consist of bands from all major types of proteins implying the integration of functionally distinct components within the overall venom phenotype. (3) There is significant differentiation in venom proteins across populations and the specific classes of proteins contributing to this differentiation have been identified. (4) Levels of population differentiation in venom proteins are not correlated with levels of neutral genetic differentiation, or genetically effective population sizes which argues that patterns of venom variation are not simply a consequence of population structure but leaves open the role of selection in generating population differences in venom. Our results identify particular classes of venom proteins and their associated genes as being fruitful targets for future studies of the molecular and functional basis for this complex adaptive phenotype.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Aguilar I, Guerrero B, Salazar AM et al (2007) Individual venom variability in the South American rattlesnake, Crotalus durissus cumanensis. Toxicon 50:214–224

    Article  PubMed  CAS  Google Scholar 

  • Barlow A, Pook CE, Harrison RA et al (2009) Co-evolution of diet and prey-specific venom activity supports the role of selection in snake venom evolution. Proc R Soc B 276:2443–2449

    Article  PubMed  CAS  Google Scholar 

  • Beerli P (2009) MIGRATE-N: estimation of population sizes and gene flow using the coalescent. http://popgen.scs.fsu.edu/Migrate-n.html

  • Benfey PN, Mitchell-Olds T (2008) From genotype to phenotype: systems biology meets natural variation. Science 320:495–497

    Article  PubMed  CAS  Google Scholar 

  • Bonnet E, Van de Peer Y (2002) zt: a software tool for simple and partial Mantel tests. J Stat Softw 7:1–12

    Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Calvete JJ (2009) Venomics: digging into the evolution of venomous systems and learning to twist nature to fight pathology. J Proteomics 72:121–126

    Article  PubMed  CAS  Google Scholar 

  • Calvete JJ, Juarez P, Sanz L (2007) Snake venomics: strategy and applications. J Mass Spectrom 42:1405–1414

    Article  PubMed  CAS  Google Scholar 

  • Campbell JA, Lamar W (2004) The venomous reptiles of the western hemisphere. Cornell University Press, Ithaca, NY

    Google Scholar 

  • Chaim-Matyas A, Borkow G, Ovadia M (1995) Synergism between cytotoxin P4 from the snake venom of Naja nigricollis nigricollis and various phospholipases. Comp Biochem Physiol B: Biochem Mol Biol 110:83–89

    Article  Google Scholar 

  • Chippaux J-P, Williams V, White J (1991) Snake venom variability: methods of study, results and interpretation. Toxicon 29:1279–1303

    Article  PubMed  CAS  Google Scholar 

  • Chiucchi J, Gibbs HL (2010) Similarity of contemporary and historical levels of gene flow among highly-fragmented populations of a rattlesnake. Mol Ecol 19:5345–5358

    Article  PubMed  Google Scholar 

  • Daltry JC, Wuster W, Thrope RS (1996) Diet and snake venom evolution. Nature 379:537–540

    Article  PubMed  CAS  Google Scholar 

  • Duda TF Jr, Lee T (2009) Ecological release and venom evolution of a predatory marine snail at Easter Island. PLoS ONE 4:e5558

    Article  PubMed  Google Scholar 

  • Dufrêne M, Legendre P (1997) Species assemblages and indicator species: the need for a flexible asymmetrical approach. Ecol Monogr 67:345–366

    Google Scholar 

  • Forstner MRJ, Hilsenbeck RA, Scudday JF (1997) Geographic variation in whole venom profiles from the mottled rock rattlesnake (Crotalus l. lepidus) (Kennicott) in Texas. J Herpetol 31:277–285

    Article  Google Scholar 

  • Fuerst PA, Chakraborty R, Nei M (1977) Statistical studies on protein polymorphism in natural populations. I. Distribution of single locus heterozygosity. Genetics 86:455–483

    PubMed  CAS  Google Scholar 

  • Gibbs HL, Rossiter W (2008) Rapid evolution by positive selection and gene gain and loss: PLA2 venom genes in closely related Sistrurus rattlesnakes with divergent diets. J Mol Evol 66:151–166

    Article  PubMed  CAS  Google Scholar 

  • Gibbs HL, Sanz L, Calvete JJ (2009) Snake population venomics: proteomics-based analyses of individual variation reveals gene regulation effects on venom protein expression in Sistrurus rattlesnakes. J Mol Evol 68:113–125

    Article  PubMed  CAS  Google Scholar 

  • Gregory-Dwyer VM, Egen NB, Bosisio AB et al (1986) An isolectric focusing study of seasonal variation in rattlesnake venom proteins. Toxicon 24:995–1000

    Article  PubMed  CAS  Google Scholar 

  • Huang P, Mackessy SP (2004) Biochemical characterization of phospholipase A2 (trimorphin) from the venom of the Sonoran lyre snake Trimorphodon biscutatus lambda (family Colubridae). Toxicon 44:25–34

    Article  Google Scholar 

  • Legendre P, Fortin M-J (2010) Comparison of the Mantel test and alternative approaches for detecting complex multivariate relationships in the spatial analysis of genetic data. Mol Ecol Resour 10:831–844

    Article  Google Scholar 

  • Mackessy SP (1988) Venom ontogeny in the Pacific rattlesnakes, Crotalus viridis helleri and C. viridis oreganus. Copeia 1988:92–101

    Article  Google Scholar 

  • Mackessy SP (2009) Venom composition in rattlesnakes: trends and biological significance. In: Hayes WK, Beaman KR, Cardwell MD, Bush SP (eds) The biology of rattlesnakes. Loma Linda University Press, Loma Linda, CA, pp 495–510

    Google Scholar 

  • Mackessy SP, Sixberry NM, Heyborne WH et al (2006) Venom of the Brown Treesnake, Boiga irregularis: ontogenetic shifts and taxa-specific toxicity. Toxicon 47:537–548

    Article  PubMed  CAS  Google Scholar 

  • Mather PM (1976) Computational methods of multivariate analysis in physical geography. Wiley, Chichester

    Google Scholar 

  • McCune B, Grace JB (2002) Analysis of ecological communities. MjM Software, Gleneden Beach, Oregon, USA, 304 pp. (www.pcord.com)

  • Mielke PW (1984) Meteorological applications of permutation techniques based on distance functions. In: Krishnaiah PR, Sen PK (eds) Handbook of statistics, vol 4. North-Holland, Amsterdam, pp 813–830

    Google Scholar 

  • Mitchell-Olds T, Willis J, Goldstein D (2007) Which evolutionary processes influence natural genetic variation for phenotypic traits? Nat Genet 8:845–856

    Article  CAS  Google Scholar 

  • Nachman MW (2006) Detecting selection at the molecular level. In: Fox CW, Wolf JB (eds) Evolutionary genetics case concepts studies. Oxford University Press, Oxford

    Google Scholar 

  • Narum S (2006) Beyond Bonferroni: less conservative analyses for conservation genetics. Conserv Genet 7:783–787

    Article  CAS  Google Scholar 

  • Nei M (1987) Molecular evolutionary genetics. Columbia University Press, New York

    Google Scholar 

  • Salazar AM, Rodriguez-Acosta A, Giron ME et al (2007) A comparative analysis of the clotting and fibrinolytic activities of the mapanare (Bothrops atrox) snake venom from different geographical areas in Venezuela. Thromb Res 120:95–104

    Article  PubMed  CAS  Google Scholar 

  • Sanz L, Gibbs HL, Mackessy SP et al (2006) Venom proteomes of closely-related Sistrurus rattlesnakes with divergent diets. J Proteome Res 5:2098–2112

    Article  PubMed  CAS  Google Scholar 

  • Serrano SMT, Shannon JD, Wang D et al (2005) A multifaceted analysis of viperid snake venoms by two-dimensional gel electrophoresis: an approach to understanding venom proteomics. Proteomics 5:501–510

    Article  PubMed  CAS  Google Scholar 

  • Urton EJM, Hobson KA (2005) Intrapopulation variation in gray wolf isotope (15N and 13C) profiles: implications for the ecology of individuals. Oecologia 145:317–326

    Article  PubMed  Google Scholar 

  • Warrington JA, Nair A, Mahadevappa M (2000) Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes. Physiol Genomics 2:143–147

    PubMed  CAS  Google Scholar 

  • Weatherhead PJ, Knox JM, Harvey DS et al (2009) Diet of Sistrurus catenatus in Ontario and Ohio: effects of body size and habitat. J Herpetol 43:693–697

    Article  Google Scholar 

  • Williams V, White J, Schwaner TD et al (1988) Variation in venom proteins from isolated populations of tiger snakes (Notechis ater niger, N. scutatus) in south Australia. Toxicon 26:1067–1075

    Article  PubMed  CAS  Google Scholar 

  • Wooldridge BJ, Pineda G, Banuelas-Ornelas JJ et al (2001) Mojave rattlesnakes (Crotalus scutulatus scutulatus) lacking the acidic subunit DNA sequence lack Mojave toxin in their venom. Comp Biochem Physiol Part B 130:169–179

    Article  CAS  Google Scholar 

  • Wuster W (1999) Testing causal hypotheses for venom variation in snakes. Toxicon 37:289

    Article  Google Scholar 

Download references

Acknowledgments

Most importantly, we thank Jeff Davis, Mike Dreslik, Dan Harvey, Matt Kowalski, Greg Lipps, Kevin Shoemaker, and Doug Wynn for their generous help with collecting samples. Without their assistance this study would not have been possible. Steve Mackessy, Jose Diaz, and Andrea Doseff provided important assistance with lab procedures, Stuart Ludsin, and Laura Kubatko gave advice on statistical analyses, and the Gibbs lab, Paul Fuerst and an anonymous reviewer provided comments. This work was supported by funds from Ohio State University and the Ohio Division of Wildlife.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to H. Lisle Gibbs.

Appendix

Appendix

See Tables 6 and 7.

Table 6 Venom protein bands used in S. c. catenatus analyses
Table 7 P values for post-hoc comparisons of NMS scores between populations

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lisle Gibbs, H., Chiucchi, J.E. Deconstructing a Complex Molecular Phenotype: Population-Level Variation in Individual Venom Proteins in Eastern Massasauga Rattlesnakes (Sistrurus c. catenatus). J Mol Evol 72, 383–397 (2011). https://doi.org/10.1007/s00239-011-9437-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00239-011-9437-4

Keywords

Navigation