Journal of Molecular Evolution

, Volume 68, Issue 2, pp 113–125 | Cite as

Snake Population Venomics: Proteomics-Based Analyses of Individual Variation Reveals Significant Gene Regulation Effects on Venom Protein Expression in Sistrurus Rattlesnakes

  • H. Lisle Gibbs
  • Libia Sanz
  • Juan J. Calvete


Studies of the molecular basis of adaptations seek to understand the relative importance of structural changes in proteins versus gene regulation effects as determinants of phenotype. Amino acid substitutions in gene coding sequences are well documented as causes of variation in snake venom proteins, whereas the importance of gene regulation effects on venom protein abundance and composition is less well known. Here, we use a proteomics-based approach to infer the effects of gene regulation on protein expression by comparing the relative abundance of specific, known venom proteins among different individuals in each of two species of Sistrurus rattlesnakes. Variation in the presence or absence, and in the relative amounts, of proteins was high in both species across all major protein families. Based on our empirical criteria for inferring regulatory effects (presence-absence of specific proteins and/or more than threefold variation in abundance) between 51% and 83% of S. catenatus individuals and between 40% and 63% of S. miliarius individuals showed evidence for gene regulation across the four most abundant proteins (disintegrins, phospholipase A2’s, serine proteinases, and snake venom metalloproteases). Thus, the effects of gene regulation should be considered an important cause of variation in the composition of whole venoms at the intraspecific level. They also suggest the need for testing the adaptive hypothesis for venom plasticity in relation to prey consumed by adult snakes. Finally, the venom variability reported may have an impact in the treatment of bite victims, highlighting the necessity of using pooled venoms as a substrate for antivenom production.


Snake venom proteins Sistrurus rattlesnakes Proteomics analyses Gene regulation 



We thank Paulo Nuin for provision of a preliminary version of his metaComps program; Jimmy Chiucchi, Michael Dreslik, Terry Farrell, Dan Harvey, and Doug Wynn for help in obtaining venom samples; and Erich Grotewald and Laura Kubatko for discussion. H.L.G. also thanks John Pérez and Elda Sánchez for their generous advice and assistance at the start of his work on venomous snakes. This study was financed by Grant BFU2007-61563 from the Ministerio de Ciencia e Innovación, Madrid, Spain, and by funds from the Ohio State University.


  1. Alape-Girón A, Sanz L, Escolano J et al (2008) Snake venomics of the lancehead pitviper Bothrops asper: geographic, individual, and ontogenetic variations. J Proteome Res 7:3556–3571PubMedCrossRefGoogle Scholar
  2. Andrade DV, Abe AS (1999) Relationship of venom ontogeny and diet in Bothrops. Herpetologica 55:200–204Google Scholar
  3. Burstin J, de Vienne D, Dubreuil P et al (1994) Molecular markers and protein quantities as genetic descriptors in maize. I. Genetic diversity among 21 inbred lines. Theor Appl Genet 89:943–950CrossRefGoogle Scholar
  4. Calvete JJ, Juárez P, Sanz L (2007) Snake venomics. Strategy and applications. J Mass Spectrom 42:1405–1414PubMedCrossRefGoogle Scholar
  5. Cavalieri D, Townsend JP, Hartl DL (2000) Manifold anomalies in gene expression in a vineyard isolate of Saccharomyces cerevisiae revealed by DNA microarray analysis. Proc Natl Acad Sci USA 97:12369–12374PubMedCrossRefGoogle Scholar
  6. Chang L-S (2007) Genetic diversity in snake venom three-finger proteins and phospholipase A2 enzymes. Toxin Reviews 26:143–167CrossRefGoogle Scholar
  7. Chang L-S, Lin SR, Wang JJ et al (2000) Structure function studies on Taiwan cobra long neurotoxin homolog. Biochem Biophys Res Commun 219:116–121CrossRefGoogle Scholar
  8. Chippaux J-P, Boche J, Courtois B (1982) Electrophoretic patterns of the venoms from a litter of Bitis gabonica snakes. Toxicon 27:1397–1399Google Scholar
  9. Chippaux J-P, Williams V, White J (1991) Snake venom variability:methods of study, results and interpretation. Toxicon 29:1279–1303PubMedCrossRefGoogle Scholar
  10. Chijiwa T, Deshimaru M, Nobuhisha I et al (2000) Regional evolution of venom-gland phospholipase A2 isoenzymes of Trimeresurus flavoviridis snakes in the southwestern islands of Japan. Biochem J 347:491–499PubMedCrossRefGoogle Scholar
  11. Chijiwa T, Yamaguchi Y, Ogawa T et al (2003) Interisland evolution of Trimeresurus flavoviridis venom phospholipase A2 isozymes. J Mol Evol 56:286–293PubMedCrossRefGoogle Scholar
  12. Colossimo PF, Hoemann KE, Balabhadra S et al (2005) Widespread parallel evolution in sticklebacks by repeated fixation of ecotdysplasin alleles. Science 307:1928–1933CrossRefGoogle Scholar
  13. Conant R, Collins JT (1998) Reptiles and amphibians of eastern and central North America. Houghton Mifflin, New YorkGoogle Scholar
  14. Costa P, Plomion C (1999) Genetic analysis of needle proteins in maritime pine. 2. Variation in protein accumulation. Silvae Genet 48:146–150Google Scholar
  15. Creer S, Malhotra A, Thorpe RS et al (2003) Genetic and ecological correlates of intraspecific variation in pitviper venom composition detected using matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and isoelectric focusing. J Mol Evol 56:317–329PubMedCrossRefGoogle Scholar
  16. Daltry JC, Wüster W, Thorpe RS (1996a) Diet and snake venom evolution. Nature 379:537–540PubMedCrossRefGoogle Scholar
  17. Daltry JC, Ponnudurai G, Shin CK et al (1996b) Electrophoretic profiles and biological activities: intraspecific variation in the venom of the Malayan pit viper (Calloselasma rhodostoma). Toxicon 34:67–79PubMedCrossRefGoogle Scholar
  18. Damerval C, Maurice A, Josse AJM et al (1994) Quantitative trait loci underlying gene product variation: a novel perspective for analyzing regulation of genome expression. Genetics 137:289–301PubMedGoogle Scholar
  19. De Vienne D, Bost B, Fieveta J et al (2001) Genetic variability of proteome expression and metabolic control. Plant Physiol Biochem 39:271–283CrossRefGoogle Scholar
  20. Deshimaru M, Ogawa T, Nakashima K et al (1996) Accelerated evolution of crotalinae snake venom gland serine proteases. FEBS Lett 397:83–88PubMedCrossRefGoogle Scholar
  21. Deyrup S, Farrell T, Niclas D (2000) Venom ontogeny in dusky pigmy rattlesnakes (Sistrurus miliarius barbouri). Fla Acad Sci 63:23Google Scholar
  22. Duda TF Jr, Palumbi SR (2004) Gene expression and feeding ecology:evolution of piscivory in the venomous gastropod genus Conus. Proc R Soc London B Biol Sci 271:1165–1174CrossRefGoogle Scholar
  23. Enard W, Khaitovich P, Klose J et al (2002) Intra- and interspecific variation in primate gene expression patterns. Science 296:340–343PubMedCrossRefGoogle Scholar
  24. Fry BG (2005) From genome to ‘venome’: molecular origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences and related body proteins. Genome Res 15:403–420PubMedCrossRefGoogle Scholar
  25. Fry BG, Wüster W (2004) Assembling an arsenal: origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences. Mol Biol Evol 21:870–883PubMedCrossRefGoogle Scholar
  26. Gerber S, Fabre F, Planchon C (2000) Genetics of seed quality in soybean analysed by capillary gel electrophoresis. Plant Sci 152:181–189CrossRefGoogle Scholar
  27. 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 66:151–166PubMedCrossRefGoogle Scholar
  28. Gloyd HK (1940) The rattlesnakes, genera Crotalus and Sistrurus: a study in zoogeography and evolution. Special Publication No. 4. Chicago Academy of Science, ChicagoGoogle Scholar
  29. Golding GB, Dean AM (1998) The structural basis of molecular adaptation. Mol Biol Evol 15:355–369PubMedGoogle Scholar
  30. Gottlieb LD, de Vienne D (1998) Assessment of pleiotropic effects of a gene substitution in pea by two-dimensional polyacrylamide gel electrophoresis. Genetics 119:705–710Google Scholar
  31. Greene HW (1983) Dietary correlates of the origin and radiation of snakes. Am Zool 23:431–441Google Scholar
  32. Gregory-Dwyer VM, Egen NB, Bosisio AB et al (1986) An isolectric focusing study of seasonal variation in rattlesnake venom proteins. Toxicon 24:995–1000PubMedCrossRefGoogle Scholar
  33. Guercio RAP, Shevchenko A, Shevchenko A et al (2006) Ontogenetic variations in the venom proteome of the Amazonian snake, Bothrops atrox. Proteome Sci 4:11PubMedCrossRefGoogle Scholar
  34. Gutierrez JM, Avila C, Camancho Z et al (1990) Ontogenic changes in venom of the snake Lachesis muta stenophrys (Bushmaster) from Costa Rica. Toxicon 28:419–426PubMedCrossRefGoogle Scholar
  35. Gygi SP, Corthals GL, Zhang Y et al (2000) Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. Proc Natl Acad Sci USA 97:9390–9395PubMedCrossRefGoogle Scholar
  36. Haygood R, Fedrigo R, Hanson B et al (2007) Promoter regions of many neural- and nutrition-related genes have experienced positive selection during human evolution. Nature Genetics 39:1141–1144CrossRefGoogle Scholar
  37. Hoekstra H, Coyne J (2007) The locus of evolution: evo-devo and the genetics of adaptation. Evolution 61(5):995–1016PubMedCrossRefGoogle Scholar
  38. Holycross AT, Mackessy SP (2002) Variation in the diet of Sistrurus catenatus (massasauga) with emphasis on Sistrurus catenatus edwardsii (desert massasauga). J Herpetol 36:454–464Google Scholar
  39. Jiménez-Porras JM (1964) Intraspecific variations in composition of venom of the jumping viper, Bothrops nummifera. Toxicon 2:187–195CrossRefGoogle Scholar
  40. Juárez P, Sanz L, Calvete JJ (2004) Snake venomics: characterization of protein families in Sistrurus barbouri venom by cysteine mapping, N-terminal sequencing, and tandem mass spectrometry analysis. Proteomics 4:327–338PubMedCrossRefGoogle Scholar
  41. Junqueira-de-Azevedo ILM, Ho P (2002) A survey of gene expression and diversity in the venom glands of the pitviper snake Bothrops insularis through the generation of expressed sequence tags (ESTs). Gene 299:279–291CrossRefGoogle Scholar
  42. Junqueira-de-Azevedo ILM, Ching ATC, Carvalho E et al (2006) Lachesis muta (Viperidae) cDNAs reveal emerging pit viper molecules and scaffolds typical of cobra (Elapidae) venoms: implications for snake toxin repertoire evolution. Genetics 173:877–879PubMedCrossRefGoogle Scholar
  43. Klose J, Nock C, Herrmann M et al (2002) Genetic analysis of the mouse brain proteome. Nat Genet 30:385–393PubMedCrossRefGoogle Scholar
  44. Kordiš D, Gubenšek F (2000) Adaptive evolution of animal toxin multigene families. Gene 261:43–52PubMedCrossRefGoogle Scholar
  45. Lewontin RC (1974) The genetic basis of evolutionary change. Columbia University Press, New YorkGoogle Scholar
  46. Ma DH, Armugam A, Jeyaseelan K (2001) Expression of cardiotoxin-2 gene. Cloning, characterization, and deletion of the promoter. Eur J Biochem 268:1844–1850PubMedCrossRefGoogle Scholar
  47. Ma DH, Armugam A, Jeyaseelan K (2002) Alpha-neurotoxin gene expression in Naja sputatrix: identification of a silencer element in the promotor region. Arch Biochem Biophys 404:98–105PubMedCrossRefGoogle Scholar
  48. Mackessy SP (1988) Venom ontogeny in the Pacific rattlesnakes, Crotalus viridis helleri and C. viridis oreganus. Copeia 1988:92–101CrossRefGoogle Scholar
  49. Mackessy SP, Sixberry NM, Heyborne WH et al (2006) Venom of the brown treesnake, Boiga irregularis: ontogenetic shifts and taxa-specific toxicity. Toxicon 47:537–548PubMedCrossRefGoogle Scholar
  50. Markland FS (1998) Snake venoms and the hemostatic system. Toxicon 36:1749–1800PubMedCrossRefGoogle Scholar
  51. Mebs D, Kornalik F (1984) Intraspecific variation in content of a basic venom in eastern diamondback rattlesnake (Crotalus adamanteus) venom. Toxicon 22:831–833PubMedCrossRefGoogle Scholar
  52. Meier J, Stocker KF (1995) Biology and distribution of venomous snakes of medical importance and the composition of snake venoms. In: Meier J, White J (eds) Handbook of clinical toxicology of animal venoms and proteins. CRC Press, Boca Raton, FL, pp 367–412Google Scholar
  53. Ménez A (ed) (2002) Perspectives in molecular toxinology. J Wiley and Sons, Chichester, UKGoogle Scholar
  54. Nuin PAS (2008) metaComps, metabolomics comparison software. Program distributed by the author. Department of Pathology and Molecular Medicine, Queen’s University, CanadaGoogle Scholar
  55. Ohno M, Chijiwa T, Oda-Ueda N et al (2003) Molecular evolution of myotoxic phospholipases A2 from snake venom. Toxicon 42:841–854PubMedCrossRefGoogle Scholar
  56. Orr HA, Coyne JA (1992) The genetics of adaptation—a reassessment. Am Nat 140:725–742CrossRefGoogle Scholar
  57. Pahari S, Bickford D, Fry BG et al (2007) Expression pattern of three-finger toxin and phospholipase A2 genes in the venom glands of two sea snakes, Lapemis curtus and Acalyptophis peronii: comparison of evolution of these toxins in land snakes, sea kraits and sea snakes. BMC Evol Biol 7:175PubMedCrossRefGoogle Scholar
  58. Sanz L, Gibbs HL, Mackessy SP et al (2006) Venom proteomes of closely related Sistrurus rattlesnakes with divergent diets. J Proteome Res 5:2098–2112PubMedCrossRefGoogle Scholar
  59. Sanz L, Escolano J, Ferretti M et al (2008) Snake venomics of the South and Central American bushmasters. Comparison of the toxin composition of Lachesis muta gathered from proteomic versus transcriptomic analysis. J Proteom 71:46–60CrossRefGoogle Scholar
  60. Serrano SM, 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 5:501–510PubMedCrossRefGoogle Scholar
  61. Shapiro MD, Marks ME, Peichel CL et al (2004) Genetic and developmental basis of evolutionary pelvic reduction in threespine sticklebacks. Nature 428:717–723PubMedCrossRefGoogle Scholar
  62. Shapiro MD, Bell MA, Kingsley DM (2006) Parallel origins of pelvic reduction in vertebrates. Proc Natl Acad Sci USA 103:3753–3758Google Scholar
  63. Taborska E, Kornalik F (1985) Individual variability of Bothrops asper venom. Toxicon 23:612Google Scholar
  64. Tsai I-H, Chen YH, Wang YM et al (2001) Differential expression and geographic variation of venom phospholipase A2 of Callosellasma rhodostoma and Trimeresurus muscrosquamatus. Arch Biochem Biophys 387:257–264PubMedCrossRefGoogle Scholar
  65. Tsai I-H, Wang Y-M, Chen Y-H et al (2003) Geographic variations, cloning and functional analyses of the venom acidic phospholipases A2 of Crotalus viridis viridis. Arch Biochem Biophys 411:289–296Google Scholar
  66. Warrington JA, Nair A, Mahadevappa M (2000) Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes. Physiol Genom 2:143–147Google Scholar
  67. Whitehead A, Crawford DL (2006) Variation within and among species in gene expression: raw material for evolution. Mol Ecol 15:1197–1211PubMedCrossRefGoogle Scholar
  68. Whitney AR, Diehn M, Popper SJ et al (2003) Individuality and variation in gene expression patterns in human blood. Proc Natl Acad Sci USA 100:1896–1901PubMedCrossRefGoogle Scholar
  69. Winter EE, Goodstadt L, Ponting CP (2004) Elevated rates of protein secretion, evolution, and disease among tissue-specific genes. Genome Res 2004 14:54–61Google Scholar
  70. 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 130:169–179CrossRefGoogle Scholar
  71. Wray GA, Hahn MW, Abouheif E et al (2003) The evolution of transcriptional regulation in eukaryotes. Mol Biol Evol 20:1377–1419PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Department of Evolution, Ecology and Organismal BiologyOhio State UniversityColumbusUSA
  2. 2.Instituto de Biomedicina de Valencia, CSICValenciaSpain

Personalised recommendations