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Click here for details about the volume and how to order it, or send e-mail to @llu.edu Pp. 495-510 in W. K. Hayes, K. R. Beaman, M. D. Cardwell, and S. P. Bush (eds.), The Biology of Rattlesnakes. Loma Linda University Press, Loma Linda, California. 495

Venom Composition in Rattlesnakes: Trends and Biological Significance

Stephen P. Mackessy1,2

1School of Biological Sciences, University of Northern Colorado, 501 20th St., CB 92, Greeley, Colorado 80639-0017 USA

Abstract.— glands of rattlesnakes have a largely conserved morphology, and protein products of the gland are often highly homologous across many species. As with generally, a primary role of venoms is to incapacitate prey remotely and to facilitate prey handling. However, even within a single species (e.g., o. oreganus and C. o. concolor), very prominent differences in absolute composition are observed, suggesting that closely related taxa are utilizing different trophic “strategies.” Using SDS-PAGE, enzyme assays, and toxicity studies, venoms of 27 rattlesnake taxa were analyzed and compared with the intent of placing venom biochemistry into a broader biological context. Venom composition is genetically determined, and therefore is related to phylogeny; however, within specific lineages, venom composition varies considerably. Within the constraints of relatedness of taxa, there is some variability in absolute venom composition, which appears to be related primarily to diet and secondarily to environment. Venom often (but not always) varies ontogenetically, and this variation commonly tracks changes in dominant preferred prey type. For example, both ontogenetically and between subspecies/species, highly toxic venoms lack high concentrations of metalloproteases, enzymes responsible for much of the structural damage resulting from envenomation. Conversely, those with potent metalloprotease activity generally lack presynaptic neurotoxins characteristic of the most toxic venoms. Toxicity versus tissue-damaging effects of rattlesnake venoms follows a predictable inverse trend. The thrombin-like serine proteases and a homologs also vary ontogenetically in some species, and higher levels in venoms appear to be directed toward mammalian prey. Further, maintenance of venom complexity is likely driven by adaptive detoxification responses of prey, and the ensuing arms race has led to apparent multiplicity of similar activities and well over 100 distinct molecular entities in a single venom. However, many unanswered questions remain, particularly concerning many Mexican rattlesnake taxa and with regard to comparative toxicity of venom component isoforms to native prey species. Within the rattlesnakes, one finds venom compositional “strategies” which are observed broadly among venomous colubroid , and thus rattlesnakes represent an excellent group for studying venom evolution in squamates.

Introduction Venoms, therefore, serve numerous well-documented trophic roles for snakes, but a major unanswered question is Produced within the venom gland of snakes is a com- why venoms are so complex biochemically, and in particu- plex secretion containing dozens of enzymes, , pep- lar, why apparent multiplicity of components within specific tides, small organic molecules, and inorganic components protein families (e.g., phospholipases A2, serine proteases, (Mackessy and Baxter, 2006). Upon envenomation, ho- metalloproteases, etc.) exists in venom from a single indi- meostatic mechanisms in a myriad of systems are affected vidual. An adaptive response among prey toward detoxifi- rapidly, and in prey , the result of envenomation is cation of venom components has been proposed (Mackessy rapid incapacitation leading to death and initiation (in many et al., 2003), and an ensuing “arms race” between predator cases) of digestion. Among colubrid snakes, there has been and prey could drive the selection for dynamic evolution some debate about venom occurrence and action in many of venom composition. Prey-driven evolution of venom is rear-fanged species, but the recent demonstration of taxon- strongly suggested by differential resistance to Crotalus o. specific effects (Mackessy et al., 2006) suggests that, de- oreganus venoms observed among populations of Califor- pending on the model chosen for toxicity assays, ef- nia Ground Squirrels (Spermophilus beecheyi; Poran et al., fects vary tremendously; differential sensitivity of specific 1987; Biardi et al., 2000, 2005; Biardi, this volume) and the taxa may have profound implications for the biological roles high level of resistance to C. atrox venom among woodrats of these venoms. In rattlesnakes (Chiszar et al., 1999), and (Perez et al., 1978a,b). Additionally, many rattlesnake spe- likely among pit vipers in general (e.g., Stiles et al., 2002; cies feed on a variety of prey (Klauber, 1956; Wallace and Greenbaum et al., 2003), venom also serves to “tag,” or Diller, 1990; Holycross and Mackessy, 2002; Holycross et change the odor of, envenomated prey, allowing the snake to al., 2002), some characteristically at different life stages relocate prey via strike-induced chemosensory searching. (Mackessy, 1988; Mackessy et al., 2003), and so venoms ______may include toxins or other components which act differ- 2 Correspondence e-mail: [email protected] entially toward specific prey classes. Rattlesnake venom © 2008 Loma Linda University Press composition often varies with snake age (Minton, 1967; 496 S. P. Mackessy

Reid and Theakston, 1978; Lomonte et al., 1983; Mackessy, oms of several species (Aird and Kaiser, 1985; Aird et al., 1988; Mackessy et al., 2003), and a recent proteomic study 1985; Bieber et al., 1990; Weinstein and Smith, 1990; see of the venom of a crotaline viperid (Bothrops atrox) demon- also the chapters in this volume by Powell et al., Powell and strated a larger number of proteins in venoms from juvenile Lieb, and Werman), including the Tropical Rattlesnake (C. snakes (Guércio et al., 2006). durissus terrificus), the Mohave Rattlesnake (C. s. scutula- In a broad sense, venom composition variation does tus), the Midget-faded Rattlesnake (C. oreganus concolor), follow phylogenetic trends, particularly at the familial level and the (C. tigris). Rattlesnake venoms among the front-fanged snakes (e.g., Tan and Ponnadurai, also contain specific non-enzymatic peptides, typically with 1990, 1991). For example, α-neurotoxins (three-finger tox- potent biological activities, such as the , disinteg- ins) are typical and abundant components of elapid venoms rins, and bradykinin-potentiating peptides, and a variety of (Nirthanan and Gwee, 2004), and although they have re- less well characterized smaller organic and inorganic com- cently been documented in the venom gland genome of one pounds. Many of these common venom components and species (Lachesis muta; Junqueira-de-Azevedo et al., 2006), their likely biological roles are listed in Table 1. they have not yet been demonstrated to be present in secreted Venom composition may vary ontogenetically, some- viperid venoms. Conversely, viperid venoms are rich sourc- times with profound effects on the overall pharmacology of es of hydrolytic enzymes, such as multiple metalloproteases the venom. Previously, we have demonstrated distinct onto- (Bjarnason and Tu, 1978; Bjarnason and Fox, 1989, 1995; genetic changes in venom composition, which appear to be Mackessy, 1985, 1993a,b, 1996), many of which are either related to concomitant changes in diet (C. o. oreganus and C. absent or at low concentrations in elapid venoms. However, o. helleri: Mackessy, 1988). Toxicity and metalloprotease ac- patterns within lower level taxa (such as genera) are less tivity levels were inversely related, with neonate venoms more clear and have not been analyzed extensively. Rattlesnakes, toxic and adult venom containing much higher levels of metal- by virtue of their wide distribution, varied ecology, relatively loproteases. However, in a related subspecies (C. o. concolor), large venom yields, and robust venom toxinology database, these shifts in venom composition were not seen, and adult represent an ideal group to investigate venom composition venoms were very toxic and showed low metalloprotease ac- variation and its relation to phylogeny, ecology, and natural tivity, in spite of similar ontogenetic shifts in diet (Mackessy history of the animals (c.f. Beaman and Hayes, this volume). et al., 2003). These two rather disparate “venom composition One limitation is that, although the venom composition of strategies” appear to be a broader reflection of trends in rattle- some species is very well known (e.g., C. atrox; Fox and snake venoms of different species, as well as among venom- Serrano, 2005; Serrano et al., 2005; Sistrurus: Sanz et al., ous snakes in general, and the observations from these studies 2006), we have little or no information for many other spe- have led to the investigations detailed below. In this paper, cies, particularly for the many species occupying the Mexi- venoms which show higher levels of metalloprotease activ- can highlands (see Campbell and Lamar, 2004). ity and lower toxicity (>1.0 μg/g mouse body weight) will be Although rattlesnake venoms are known to vary as a referred to as type I venoms, whereas those with low metal- function of numerous intrinsic and extrinsic factors, many loprotease activity and higher toxicity (<1.0 μg/g mouse body components are common to venoms of most species, and a weight) will be referred to as type II venoms. high degree of sequence homology exists among specific The goal of the present study is to begin to identify components, such as the phospholipases (Kini, 1997) and patterns of protein component occurrence in different rat- small myotoxins (Mebs and Ownby, 1990). However, ac- tlesnake venoms and to evaluate trends in venom composi- tivity levels of many enzymes (and therefore, presumably, tion variation. New data are presented and integrated with content levels) may vary tremendously. Common rattle- data collected from the literature. Because several phyloge- components include a variety of enzymes, in- netic hypotheses concerning relationships among the rattle- cluding several different nucleases, hyaluronidase, L-amino snakes exist (e.g., Murphy et al., 2002; Wüster et al., 2005; acid oxidase, several to many metalloproteases, serine pro- Castoe and Parkinson, 2006), one can begin to set these teases, phospholipases, and other less abundant enzymes. trends in venom composition in a phylogenetic context and These catalytic proteins may have both specific and gen- ask whether or not trends observed follow evolutionary eralized effects, as demonstrated by the activity of several lineages. This paper does not attempt to define absolutely metalloproteases toward numerous protein components of the composition of rattlesnake venoms in general; rather, it the basement membrane defining the capillary endothelium. should be seen as a step toward understanding the complex Hydrolysis of this layer results in loss of capillary integrity, interactions between composition, phylogeny, and biologi- producing hemorrhage (Fox and Serrano, 2005). Metallo- cal roles of rattlesnake venoms. proteases also hydrolyze various other structural proteins (such as collagen), and so are important to tissue degrada- Materials and Methods tion (Gutiérrez and Rucavado, 2000), one of the “prediges- tion” effects of venoms. One subclass of the phospholipases Reagents.—NuPage 12% acrylamide gels, Novex Mark has evolved from hydrolytic enzymes into potent presynap- 12 standards, and related supplies were obtained from In- tic β-neurotoxins responsible for the high toxicity of ven- vitrogen, Inc. (San Diego, California, USA). All other bio- Venom composition in rattlesnakes 497

Table 1. Common components of rattlesnake venoms and general characteristics. Approximate ______Component Name Mass (kDa) Function Biological Activity References Enzymes Phosphodiesterase 94-140 Hydrolysis of nucleic acids Depletion of cyclic, di- and Mackessy, 1998; and nucleotides tri-nucleotides; Aird, 2002 hypotension/shock (?) 5’-nucleotidase 53-82 Hydrolysis of 5’-nucleotides Nucleoside liberation Rael, 1998; Aird, 2002 Alkaline 90-110 Hydrolysis of Uncertain Rael, 1998 phosphomonoesterase phosphomonoester bonds Hyaluronidase 73 Hydrolysis of interstitial Decreased interstitial Tu and Kudo, 2001 hyaluronan viscosity – diffusion of venom components L-amino acid oxidase 85-150 Oxidative deamination of Induction of apoptosis, cell Tan, 1998 (homodimer) L-amino acids damage Snake venom metallo- proteases: M12 repro- lysins P-IV 48-85 Hydrolysis of many Hemorrhage, myonecrosis, Fox and Serrano, 2005 P-III 43-60 structural proteins, including prey digestion P-II 25-30 basal lamina components, P-I 20-24 fibrinogen, etc. Serine proteases: Thrombin-like 31-36 Catalysis of fibrinogen Rapid depletion of fibrinogen; Markland,1998; Swensen hydrolysis hemostasis disruption and Markland, 2005 Kallikrein-like 27-34 Release of bradykinin from Induces rapid fall in blood Nikai and Komori, HMW kininogen; hydrolysis pressure; prey 1998 of angiotensin immobilization “Arginine esterase” 25-36 Peptidase and esterase activity Uncertain; predigestion of Schwartz and Bieber, prey (?) 1985 2+ Phospholipase A2 13-15 Ca -dependent hydrolysis of Myotoxicity, myonecrosis, Kini, 1997, 2003 enzymes 2-acyl groups in 3-sn- lipid membrane damage phosphoglycerides Non-enzymatic proteins/peptides Cysteine-rich secretory 21-29 Possibly block cNTP-gated Induced hypothermia; prey Yamazaki and proteins (CRiSPs)/ channels immobilization (?) Morita, 2004 helveprins

PLA2-based presynaptic 24 Blocks release of Potent neurotoxicity; prey Aird et al., 1985; neurotoxins (2 subunits, acetylcholine from axon immobilization Ducancel et al., 1988; acidic and basic) terminus Faure et al., 1994 C-type lectins 27-29 Binds to platelet and Anticoagulant, platelet- Leduc and Bon, 1998 collagen receptor modulator Disintegrins 5.2-15 Inhibit binding of integrins to Platelet inhibition; promotes Calvete et al., 2005 receptors hemorrhage Myotoxins – non- 4-5.3 Modifies voltage-sensitive Myonecrosis, analgesia; Laure, 1975; Fox et

PLA2 Na channels; interacts with prey immobilization al., 1979; Bieber and lipid membranes Nedelhov, 1997 Smaller peptides Bradykinin- 1.0-1.5 Increases potency of Pain, hypotension; prey Wermelinger et al., potentiating peptides bradykinin immobilization 2005 Tripeptide inhibitors 0.43-0.4 Inhibit venom Stabilization of venom Francis and Kaiser, metalloproteases and other components 1993; Munekiyo and enzymes Mackessy, 2005 Smaller organic compounds Purines and pyrimidines AMP = 0.347 Broad effects on multiple Hypotension, paralysis, Aird, 2002, 2004 (AMP, Hypoxanthine, cell types (?) apoptosis, necrosis (?); Inosine) prey immobilization Citrate 0.192 Inhibition of venom enzymes Stabilization of venom Francis et al., 1992; Freitas et al., 1992 Mass in kilodaltons (kDa). Note that this list is not all-inclusive and that masses, functions, and activities do not apply to all compounds isolated from all rattlesnake venoms. Specific rattlesnake venoms may not contain all components. (?) – indicates hypothetical function and/or activity. 498 S. P. Mackessy Florida Arizona: Cochise Co. SW Brazil Quintana Roo Mexico: Baja California Sur Mexico: Arkansas York New Co. Verde Val Texas: Arizona: Cochise Co. Co. California: Riverside Arizona: Cochise Co. Co. Obispo Luis San California: Co. Sweetwater Wyoming: Angeles Co. California: Los Jalisco Mexico: Arizona: Cochise Co. Michoacan Mexico: Co. California: Riverside Arizona: Cochise Co. Arizona: Pima Co. Co. Weld Colorado: Wisconsin Colorado: Lincoln Co. Kansas: Barton Co. Florida Puebla Mexico: General Locality

, μg/g) 2.0 3.5 2.8 2.5 2.8 1.0 3.0 2.5 2.7 2.8 1.5 3.4 3.8 0.2 1.8 2.0 1.2 0.9 2.6 4.5 3.2 0.13 1.55 0.46 1.25 0.07 1.35 2.51 0.65 50 (Mouse Toxicity LD ), phosphodiesterase (PDE), and L-amino acid oxidase 2 4.0 7.8 49.7 31.8 23.0 69.4 37.6 24.9 70.8 65.2 61.9 22.4 26.4 34.3 34.4 43.2 33.8 12.2 50.2 49.2 1.95 49.2 36.5 20.5 62.1 88.5 86.9 56.0 19.7 62.6 12.5 , (PLA 2 LAAO (nmol/min/mg) 1.36 1.04 0.82 1.55 1.34 0.71 0.36 2.10 0.38 0.45 1.31 0.36 0.62 0.69 0.39 0.64 0.05 0.74 0.29 1.04 0.16 0.60 0.77 0.51 PDE 0.134 0.257 0.137 0.078 0.063 0.134 0.034 min/mg) ( Δ A400 nm/ 2 5.6 5.80 5.40 19.5 41.7 14.9 16.1 38.8 31.2 28.5 22.60 18.01 52.37 20.71 33.65 42.66 27.84 61.50 79.30 21.79 13.11 15.05 29.27 22.76 27.15 70.10 24.70 36.76 28.77 41.30 31.84 PLA (nmol/min/mg) 1.1 1.06 0.69 0.21 1.32 1.02 0.19 MPr 0.333 1.341 2.522 0.071 1.496 0.825 0.285 1.315 1.070 1.320 1.242 0.924 1.572 0.135 1.460 1.379 1.180 1.575 2.263 0.021 0.052 0.900 0.853 0.901 min/mg) ( Δ A342 nm/

253 Kal 90.4 69.0 18.9 1000 902.7 937.6 796.4 145.4 221.9 849.1 983.4 781.6 602.5 621.8 551.8 841.5 918.7 919.8 781.8 971.8 695.4 359.9 428.5 533.4 537.7 550.5 123.7 1032.6 1034.1 1226.8 (nmol/min/mg)

97.4 671.9 898.1 211.0 972.9 764.5 897.4 971.7 315.9 657.3 981.7 621.6 847.4 985.4 960.6 670.9 860.9 968.4 981.4 853.9 252.3 593.9 396.3 642.8 676.4 999.7 661.8 1012.2 1073.4 1194.4 1267.4 Thr and toxicity of rattlesnake venoms. and toxicity of rattlesnake a (nmol/min/mg)

Enzyme activities: thrombin-like (Thr), kallikrein-like (Kal), caseinolytic metalloprotease (MPr), phospholipase A

Species (LAAO). Table 2. Enzyme activities Table Crotalus C. adamanteus C. atrox C. basiliscus C. durissus terrificus C. (durissus) tzabcan C. enyo C. horridus (atricaudatus) C. horridus C. lepidus C. lepidus klauberi pyrrhus C. mitchelli C. molussus molossus oreganus C. oreganus concolor C. oreganus helleri C. oreganus C. polystictus C. pricei C. pusillus C. ruber C. scutulatus C. tigris C. viridis Average 1 SE Sistrurus S. catenatus S. catenatus edwardsii S. catenatus tergeminus S. miliarius barbouri (Sistrurus) ravus Crotalus Average 1 SE a Venom composition in rattlesnakes 499

chemicals (analytical grade or better) were obtained from et al., 2006). Potential relationships between venom toxicity Sigma Chemical Co. (St. Louis, Missouri, USA). and enzyme activities were analyzed using Spearman rank Venoms.—Snakes were collected from a variety of correlations. Significant differences were determined at the locations in the western United States under permits from level of P < 0.05. Identification of electrophoretic bands state and federal authorities (see acknowledgements), and was accomplished by comparative electrophoretic analysis several were received as donations or from law enforcement with previously purified venom proteins (e.g., Mackessy, confiscations. Venoms were obtained via manual extraction 1993a,b, 1996; Sanz et al., 2006) and by comparison with as described previously (Mackessy, 1988), and were cen- published masses of specific venom components (see also trifuged (to remove solids), frozen, lyophilized, and stored ExPaSy Proteomics Server, http://us.expasy.org/). frozen until used (this material is referred to as crude ven- As an initial analysis of the relationship between phy- om). All venoms were initially reconstituted in nanopure logeny and venom composition, venom type (I or II) was water (Millipore Ultrafiltration system; Millipore Corpora- mapped onto the relevant portion of the Bayesian MCMC tion, Billerica, Massachusetts, USA). Venoms from several phylogenetic analysis of Castoe and Parkinson (2006: Fig. species were gifts from C. Ownby (C. h. horridus, C. d. 3). This hypothesis was chosen because it contains a large terrificus) and the late S. Minton (C. polystictus, C. pusil- number of the taxa analyzed in the present study and because lus, C. ravus). All venoms analyzed in this study were from their analysis of phylogenetic data is particularly robust. individual adult snakes. Electrophoresis.—NuPage 12% polyacrylamide gels Results (17 lane, 1.0 mm thick) were run as suggested by the man- ufacturer (Invitrogen). Briefly, venom samples at a final Rattlesnake venoms contained large amounts of pro- concentration of 2.0 mg/ml were prepared in sample buf- tein (~225-250 mg/mL), and protein concentration of lyo- fer containing 15 mM DTT (final concentration). Samples philized venoms was 90-93%. Venom yields for all species were heated at 70ºC for 10 min, and 12 µL was added to increased exponentially with size, as has been noted in each lane. Electrophoresis was conducted using denatur- larger series of samples from individual species (Mackessy, ing MES (morpholino-ethane sulfonic acid) buffer (50 mM 1988; Mackessy et al., 2003). Venoms of most species were MES, 50 mM Tris-HCl, 0.1% SDS, 1.0 mM EDTA, pH 7.3) clear yellow; a notable exception was venom from C. tigris, for approximately 40 min at 200 V. Gels were then stained which was colorless. Species sampled and general localities in 0.1% Coomassie Brilliant Blue R-250 overnight and are listed in Table 2. destained. Gels were photographed using an Alpha Imager Electrophoresis.—One-dimensional electrophoresis (Alpha Inotech, San Leandro, California, USA), and clar- demonstrated that venoms contained 20-30 proteins (Figs. ity was optimized using Adobe Photoshop software (Adobe 1-3); however, this is an underestimate of the total number Systems Incorporated, San Jose, California, USA). Indi- of proteins, as all molecules with similar masses (± 10%) vidual bands were not digitally quantitated, and qualitative and carbohydrate modifications may migrate on gels as ap- comparisons were based on presence/absence and relative parently single entities. Regardless, relative positions of band intensity of a given molecular mass class of proteins. bands allowed identification of specific protein families Enzyme assays.—Crude venoms were assayed for six in individual samples, in turn providing a comparison of common enzyme activities, including thrombin-like (Thr functional components present in various species’ venoms. act), kallikrein-like (Kal act), caseinolytic metalloprotease Eight groups of functionally important activities are high- lighted on gels (Figs. 1-3). It should be noted, however, that (MPr act), phospholipase A2, (PLA2 act), phosphodiesterase (PDE act), and L-amino acid oxidase (LAAO act), as de- within each of the designated functional groups, additional scribed previously (Munekiyo and Mackessy, 1998). All en- non-related proteins likely exist. zymes were assayed in triplicate and corrected for substrate Levels of metalloproteases appeared to vary consid- blanks. Protein concentration was determined (in triplicate) erably between species, based on relative band intensity using BioRad Coomassie brilliant blue reagent as described differences (large dense bands contain greater amounts of by the manufacturer. Enzyme activities were expressed as protein than thin, more diffuse bands); structural classifica- specific activities (nanomoles product formed per minute tions of venom metalloproteases (P-I through P-IV) refer to per milligram protein, nmol/min/mg) or as a change in ab- increasing domain complexity (Bjarnason and Fox, 1995; sorbance per min per milligram protein (ΔAU/min/mg). Fox and Serrano, 2005). High molecular mass metallopro- Toxicity and other venom properties.—Toxicities teases (PIII), which are typically hemorrhagic and have masses of 52-55 kDa, were present in most venoms, but (mouse LD50) of venoms were compiled from published reports (Glenn and Straight, 1982, 1985; Minton and Wein- were very faint in those from C. d. terrificus, C. o. concolor, stein, 1984; Weinstein and Smith, 1990; Khole, 1991); sev- C. s. scutulatus, and C. tigris. Another distinctive class of metalloproteases (PI), with a mass of approximately 21-23 eral species for which reliable LD50 values were not available (Sistrurus catenatus edwardsii, S. c. tergeminus, C. lepidus kDa, were present in many venoms, but again were con- klauberi, C. p. pricei) were determined using intravenous spicuously absent from C. d. terrificus, C. o. concolor, C. (caudal vein) delivery and standard methods (e.g., Mackessy s. scutulatus, and C. tigris venoms, as well as from those 500 S. P. Mackessy

Figure 1. SDS-PAGE analysis of venoms from twelve taxa of

Crotalus. Approximate molecu-

lar masses (Mr in kiloDaltons) are given on the left, and protein

classes of major constituents, and their major actions, are given on the right. All venoms showed

MW Stds C. h. horridus C. h. atricaudatus C. mitchelli pyrrhus C. molossus molossus C. scutulatus scutulatus C. basciliscus C. tigris “ C. durissus terrificus “ C. tzabcan C. enyo enyo “ C. polystictus C. pusillus MW Stds considerable complexity, but note that PIII metalloprotease 116.4 97.4 – levels are very low in C. scutu- 66.3 – Nucleases, LAAO latus, C. tigris, and C. durissus 55.6 – terrificus venoms, and PI met- Hemorrhagic metalloproteases (PIII) alloproteases are absent from these and C. horridus atricauda- 36.6 – Serine Proteases -

31.0 coagulopathy tus and C. enyo venoms. CRISP, CRISP - unknown cysteine-rich secretory protein; 21.0 – Hemorrhagic LAAO, L-amino acid oxidase. metalloproteases (PI)

14.2 – Phospholipases A2 – neurotoxins, membrane damage, etc Disintegrins – platelet inhibition 6.0 – Myotoxins – myonecrosis and 3.5 – paralysis

Figure 2. SDS-PAGE analy- sis (composite gel) of venoms from four taxa of Sistrurus and

Figure 1. eight taxa of Crotalus. Relevant portions of individual gels were

aligned using protein standards

run on each gel, and labels are as in Fig. 1. It is apparent that many proteins are shared between Sis- trurus and Crotalus.

MW Stds Sistrurus catenatus edwardsii “ tergeminusS. c. catenatusS. c. S. miliarius barbouri C. pricei C. lepidus klauberi C. o. oreganus C. tigris C. o. helleri C. adamanteus C. atrox C. ruber MW Stds

116.4

97.4 – 22 66.3 – Nucleases, LAAO 55.6 – Hemorrhagic metalloproteases (PIII) 36.6 – Serine Proteases - 31.0 coagulopathy CRISP - unknown 21.0 – Hemorrhagic metalloproteases (PI)

14.2 – Phospholipases A2 – neurotoxins, membrane damage, etc

Disintegrins – platelet inhibition

Myotoxins – 6.0 – myonecrosis and paralysis 3.5 –

Figure 2.

23 Venom composition in rattlesnakes 501 of C. enyo, C. l. klauberi, C. pricei, and S. c. edwardsii; presynaptic β-neurotoxins (Bieber et al., 1987, 1990), which in rattlesnakes, only PI metalloprotease(s) are observed at are highly homologous with crotoxin (isolated originally this position in gels. All venoms contained at least one band from the venom of C. d. terrificus). This also appears of ~14 kD, and phospholipases A2 are the most common in venoms from some populations of species which typical- proteins of this mass in venoms. Variation in number and in- ly produce venom of lower toxicity (C. lepidus: Rael et al., tensity of bands was apparent for many other components, 1992; C. o. helleri: French et al., 2004), and the presence of particularly serine proteases, disintegrins, and myotoxins. crotoxin homologs greatly increases toxicity (see Wooldridge Myotoxins were apparently absent from venoms of C. enyo, et al., 2001). Among rattlesnakes, production of highly toxic C. l. klauberi, C. pricei, C. tzabcan, and some S. c. edward- venoms is apparently dependent upon expression of the cro- sii. Myotoxin levels also varied ontogenetically in C. o. toxin homolog gene in the venom proteome. However, myo- concolor venoms (Fig. 3). toxin a and homologues have an important role in rapid prey Enzyme assays.—Venoms from all species showed ac- immobilization (Ownby et al., 1988), and this small toxin is tivity in all assays (Table 2), though several species varied also typically present in the most toxic venoms. notably from average values. Thrombin-like activity was The four most toxic venoms also showed the lowest met- greatest in C. pricei venom and lowest in C. d. terrificus alloprotease activity, and venom toxicity for all data showed 2 venom. Kallikrein-like activity was greatest in C. pricei an inverse correlation (rs = 0.65; P = 0.005) with metal- venom and lowest in C. tigris venom. Metalloprotease ac- loprotease activity (Fig. 5). However, this correlation was tivity was greatest in C. basiliscus venom and lowest in C. driven by the high toxicity venoms, and when type I and type tigris venom. Phospholipase A2 activity was greatest in C. II venoms are analyzed separately, the corresponding cor- l. klauberi venom and lowest in C. ruber venom. Venom relation of toxicity with metalloprotease activity was 0.15 (P phosphodiesterase activity was greatest in C. h. horridus = 0.55) for 19 type I venoms and 0.82 (P = 0.04) for the six venom and lowest in C. (Sistrurus) ravus venom. L-amino type II venoms. None of the other enzyme activities showed acid oxidase activity was greatest in S. c. edwardsii venom an apparent relationship with toxicity (data not shown). and lowest in C. tigris venom. The most toxic venoms tend- Occurrence of type I and type II venoms and ed to have lower enzyme activities, but with the exception phylogeny.—The occurrence of type I or type II venom of metalloprotease activity (see below), no relationships among rattlesnake species investigated was mapped onto (concordance or discordance) among relative enzyme ac- the phylogenetic hypothesis of Castoe and Parkinson (2006; tivity levels were apparent. Fig. 6). Both venom types occur in single species clades

Toxicity.—Mouse LD50 data for venoms from adult C. l. (e.g., C. mitchellii, C. oreganus, C. scutulatus) and among klauberi, C. pricei, and S. c. tergeminus were determined in species groups (e.g., C. durissus and related species), and the author’s lab using NSA mice; all others are from literature both venom types occur in basal (Sistrurus) and the most de- reports (Table 2). Venom toxicity varies considerably with rived lineages. Based on the broad occurrence of both type I species, but there are two basic groups: species with venom and type II venoms, neither venom type appears to be a basal

LD50s below 1.0 μg/g, and those with LD50s of ~1-5 μg/g (Fig. vs. a derived trait, suggesting that this apparent dichotomy is 4). All high toxicity venoms contain phospholipase-based related to other factors affecting venom composition.

Figure 3. SDS-PAGE analysis of

venoms from adult and neonate Mr adult neonate Mr Crotalus oreganus concolor, a 97.4 – subspecies with type II venom Nucleases, LAAO (Mackessy et al., 2003). For 55.6 – Hemorrhagic metalloproteases (PIII) – this subspecies, PIII metallopro- predigestion teases are very faint, and all of 36.6 – these venoms appear to lack the Serine proteases - PI metalloprotease(s) found in 31.0 - coagulopathy many rattlesnake venoms. Myo-

CRISP - unknown toxins are one of the components which vary ontogenetically, and 21.0 – Hemorrhagic levels in neonate venoms are metalloproteases (PI) very low (note very faint bands predigestion relative to adult venoms). Phospholipases A2 – neurotoxins, 14.2 – membrane damage, etc

Disintegrins – platelet inhibition 6.0 – Myotoxins – myonecrosis and paralysis

Figure 3.

24 502 S. P. Mackessy

Discussion (~14-140 kDa) than lower molecular weight non-enzymatic toxins (<15 kDa), while elapid venoms generally show the Rattlesnake venoms are complex glandular secretions opposite trend. These components also comprise the ma- with a diversity of activities, both enzymatic and non-enzy- jority of venom mass, respectively. Many components are matic (e.g., Table 1). Like most viperids, rattlesnake venoms common to most species of rattlesnakes, likely a reflection have a greater number of higher molecular mass enzymes of their common evolutionary history, but relative amounts vary widely, and some venom proteins, such as the crotoxin 10 homologs, appear to be expressed only in a limited number of lineages or in particular populations of a single species/ subspecies (c.f. Werman, this volume). This differential ex- pression of the venom genome (including post-translational modifications) leads to much of the variation observed in

, 24 hr) the venom proteome of rattlesnakes, which in turn yields 50 venoms with very different biological properties and effects 1 (Sanz et al., 2006; Pahari et al., 2007). Venoms are trophic adaptations which allow for a chem- ical (rather than a mechanical) means of dispatching prey. This chemical mechanism, envenomation, involves a com-

Toxicity (LD plex series of biochemical and physiological events result- ing from the disruption of many homeostatic mechanisms 0.1 of prey. Because venom proteins are injected at very high concentrations in relatively large amounts, these disruptions

C.atrox happen very rapidly, leading to prey incapacitation and death. C. enyo C. C. tigris Average C. r. ruber C. C. p. pricei

C. v. viridis Although this process involves many components which are C. o. helleri C. (S.) C. (S.) ravus C. basiliscus C. C. l. klauberi C. d. tzabcan C.d.terrificus C. polystictus C.o. concolor C. m. pyrrhus S.c. catenatus C.s.scutulatus C. h. horridus S.c. edwardsii S. m. barbouri C.h.atricaudat C. m. mitchelli S.c.tergeminus C. C. o. oreganus C. adamanteus C. m. molossus injected simultaneously, there are some compounds which act very rapidly and others which have a slower onset of Species gross effects. One can therefore envision a “diffusion wave”

Figure 4. Lethal toxicity of crude venoms toward inbred mice. emanating from the injected venom bolus, with smaller tox- The horizontal line at 1.0 arbitrarily divides venoms with an LD50 ins generally among the fast-acting components and larger value (dose lethal to 50% of test animals within 24 h) less than and enzymes among the slower-onset components (Fig. 7). It greater than 1.0 μg/g mouse body weight. The large dot near the should be stressed that, although this hypothesis is consis- center represents the average value for all 26 taxa. tent with diffusion rates as a function of particle size, it has not been experimentally confirmed, so this figure should be Figure 4. 5 viewed as a conceptual representation of envenomation. 2 LD50 < 1.0 - r = 0.82 General trends in venom composition among 2 g/g) LD50 > 1.0 - r = 0.15 µ µ µ µ rattlesnakes.—Patterns of occurrence and levels of various

, 4

50 venom components vary considerably between groups and even within species, but one basic trend is apparent: venoms 3 which25 are characteristically high in metalloprotease activity typically are less toxic (type I), and those with high toxicity typically show low levels of metalloprotease activity (type 2 II). It is important to keep in mind that these venom types refer to general features of adult rattlesnake venoms, as 1 there may also be ontogenetic shifts in composition which further complicate this general dichotomy. Toxicity (Mouse24 hr LD Type I venoms are characteristic of many of the larger- 0 0.0 0.5 1.0 1.5 2.0 2.5 bodied rattlesnakes, and this venom type contains high to very high metalloprotease activity and moderate lethal tox- Metalloprotease Activity (∆ A342 nm/min/mg protein) icity, commonly showing an LD50 of 2-5 μg/g body mass

Figure 5. Relationship between toxicity and metalloprotease ac- (inbred mice). This venom type appears to be more com- tivity of crude venoms of 25 taxa of rattlesnakes. The horizon- mon among rattlesnakes, and species with type I venom in- tal line indicates the arbitrary distinction between venoms with clude C. atrox, C. basiliscus, C. cerastes, C. d. durissus, C. mouse LD50 values <1.0 μg/g body weight (solid black circles) h. horridus, C. mitchellii pyrrhus, C. molossus molossus, C. and >1.0 μg/g body weight (open circles). The moderate correla- o. oreganus, C. r. ruber, C. tzabcan, C. v. viridis (possibly tion (r 2 = 0.65) observed between toxicity and metalloprotease s also C. polystictus and C. pusillus), S. c. tergeminus, and S. for all venoms is driven by the high correlation for the most toxic 2 miliarius barbouri. venoms (rs =0.82).

Figure 5.

26 Venom composition in rattlesnakes 503

Type II venom is characterized by high to very high in neonate venoms and quite high in venoms from adult toxicity (less than 1.0 μg/g body mass, inbred mice) and snakes (Mackessy et al., 2003). Therefore, the type I/type very low to non-detectable metalloprotease activity. This II dichotomy probably oversimplifies the actual underlying venom type appears to be less broadly distributed among complex patterns of variation. rattlesnakes, and because of the gross similarity to venoms So what is the functional significance of these general of juvenile snakes which show ontogenetic shifts in compo- patterns of venom composition? One difference is the differ- sition (see below), it has been suggested that type II venom ential effect on prey. If venoms are of high toxicity, prey will represents a paedomorphic trait (Mackessy et al., 2003). be killed rapidly and the bulk of the venom bolus, contain- Species showing type II venom include C. d. terrificus, C. ing the myriad enzymes found in many venoms, will remain h. atricaudatus, C. l. klauberi (some), C. o. concolor, C. localized. For small snakes feeding on prey with a relatively o. helleri (some), C. s. scutulatus A, C. tigris, S. c. catena- high surface-to-volume ratio, a low predigestion quality to tus, and perhaps S. c. edwardsii. Species not analyzed in venom may not be important, because stomach enzyme ac- this study which also show Type II patterns include C. d. tivity will lead to rapid penetration of the prey peritoneum collilineatus (Lennon and Kaiser, 1990) and C. vegrandis and neutralization of gut flora (see Mackessy, 1988, for a (Kaiser and Aird, 1987). fuller discussion). In particular, if prey abundance is limited, Note that within several well-defined species clades, such as C. durissus and C. oreganus, both venom types oc- - I or II cur, suggesting that this aspect of venom composition is - I independent of phylogeny. The presence of type I or type - I II venoms, when mapped on a recent phylogenetic hypoth- - I esis of relationships among rattlesnake species (Castoe and - I - I? Parkinson, 2006), again shows no apparent trend with phy- logeny (see Fig. 6; c.f. Werman, this volume). Even among different populations of the same subspecies (C. l. klauberi: - I or II Rael et al., 1992; C. o. helleri: French et al., 2004; C. s. scutulatus: Glenn and Straight, 1978, 1989; Glenn et al., 1983), striking differences in toxicity were noted, indicat- - II? ing that type I or II variants can occur within a single sub- species. At present, the functional significance of this pop- - II ulation-level variation is unknown, but it may be related to - I other important aspects of the snakes’ biology (see below). Venom ontogeny.—In addition to the trends in adult rattlesnake venom composition discussed above, many venoms, perhaps most, show some aspects of ontogenet- ic shifts in venom composition and/or toxicity (Minton, - I or II - II 1967; Fiero et al., 1972; Reid and Theakston, 1978; Lo- - I or II monte et al., 1983; Minton and Weinstein, 1986; Mackessy, - I or II 1988; Gutiérrez et al., 1991; Mackessy et al., 2003). Many - I of these ontogenetic patterns of variation also appear to follow the type I/type II dichotomy discussed above. For type I species, neonate and juvenile snakes produce a more toxic venom with low metalloprotease activity, low ser- - I - I ine protease (thrombin-like, kallikrein-like, plasmin-like) - I activity, and often high phospholipase A2 activity. Con- - I versely, venoms from adult snakes are less toxic but have - II much higher levels of metalloproteases and serine pro- - I or II teases (Mackessy, 1993a,b). Venoms from type II species - II have been suggested to be paedomorphic because both the neonate/juveniles and the adults have highly toxic venoms Figure 6. Adult rattlesnake venom type mapped onto the rattle- with low metalloprotease activity, similar to the venoms of snake portion of the phylogenetic hypothesis of Castoe and Par- 27 the neonate/juveniles of type I species (but not the adult kinson (2006, Fig. 3). All symbols are from the original figure: snakes). However, venoms from type II species also show posterior probability estimates, numerals, and black boxes; circles indicate posterior probability support of 100%. Venom type does some aspects of venom ontogenetic shifts in composition: not appear to be related to phylogeny, as neither basal nor derived activities of several of the serine proteases (thrombin-like, taxa within any clade groupings show one type preferentially. plasmin-like) and levels of the non-enzymatic myotoxins, Both types are also seen several times in subspecies of a single potent inducers of immobilization in rodents, are very low species (C. durissus, C. oreganus, C. mitchellii). 504 S. P. Mackessy the most important selective advantage conferred by venom mum prey size possible is directly related to gape, but is to immobilize/kill prey rapidly and with certainty. How- physical and mechanical limits are likely only part of the ever, for large snakes feeding on large prey, typically mam- defining factors. If large prey cannot be digested efficiently malian or avian, putrefaction of prey via gut flora activity is and sufficiently, then capacity to ingest large prey isfu- a potential problem (e.g., Thomas and Pough, 1979), and the tile. Initiation of chemical degradation of prey before swal- preponderance of metalloproteases in venoms of Type I spe- lowing is a major biological role for metalloproteases, and cies obviates this concern. Venoms of these adult snakes are these increase with increasing gape size for Type I species. somewhat less toxic than venoms of juvenile snakes, but the A prediction which follows from this hypothesis is that a high levels of kallikrein-like proteases (Mackessy, 1993b), constraint on maximum prey size typically consumed by which promote vascular tone collapse via liberation of bra- species with Type II venoms should be observed, and prey dykinin, and the common occurrence of myotoxin a homo- on average should be significantly smaller than maximum logues (Bober et al., 1988), assure that prey will be rapidly gape size would permit. Some supportive evidence has immobilized. Extended survival of prey while immobilized been provided by diet and venom data for C. o. concolor can allow for venom components to be delocalized from the (Mackessy et al., 2003), which most often consumes rela- site of injection, increasing systemic effects of lytic compo- tively small prey, whereas other subspecies of C. oreganus, nents such as metalloproteases. such as C. o. oreganus and C. o. helleri, may consume very A second significant effect may be freedom from con- large prey (Klauber, 1956; Mackessy, 1988). straints on prey size. Large bulky prey species which are A third functionally significant effect of venom com- covered by inert epidermal/dermal derivatives are difficult positional variation may relate to relaxation of constraints to digest whole, and forced egestion (due to prey putrefac- on foraging activities. Ectotherm digestive rates will fluc- tion) appears to be poorly tolerated by many species, par- tuate with ambient temperature (cf. Dorcas et al., 1997, ticularly among neonates (unpubl. obs.). Conversely, the 2004), which at higher latitude or altitude may be signifi- caloric value of a single large meal is significant (Greene, cantly variable within the span of a single day. It has been 1997). Rattlesnakes are gape-limited predators, and maxi- suggested that venomous snakes generally will ingest prey

Enzyme-mediated: Hyaluronidase – decreased interstitial viscosity  diffusion of other venom components Kallikrein-like – rapid fall in BP  hypotension Toxin mediated: Myotoxin – disruption of sodium ion gradients  muscle dysfunction, immobilization PLA2-based -neurotoxin – inhibition of ACh vesicle fusion  neuromuscular paralysis Immediate events Enzyme-mediated: Thrombin-like – hydrolysis of fibrinogen  coagulopathy Injected Slower onset Hemorrhagic MPr – hydrolysis of basal lamina of venom events endothelium  hemorrhage bolus Toxin mediated: Disintegrins – interfere with platelet-fibrinogen binding  promote hemorrhage Long term events Enzyme-mediated: LAAO – generation of peroxide radicals  generalized apoptotic events Phosphodiesterase – hydrolysis of cNMPs  depletion of 2nd messengers & disruption of cNMP-gated channels Phospholipase A2 – hydrolysis of PLs  hemolysis, nephrotoxicity etc. Metalloproteases – hydrolysis of structural proteins  inflammation, necrosis, predigestion

Figure 7. Hypothetical sequence of events following envenomation. Note that all activities of all enzymes and toxins are not included. “Immediate events” are most likely responsible for rapid incapacitation of prey, whereas “long-term events” contribute significantly to the predigestion roles of venom. Abbreviations: ACh, acetylcholine; BP, blood pressure; cNMP, cyclic nucleotide monophosphate; LAAO, L-amino acid oxidase; MPr, metalloprotease; PLA , phospholipase A ; PL, phospholipid. 2 2

Figure 7.

28 Venom composition in rattlesnakes 505 killed by venoms regardless of the absolute composition B. Tomberlin. The assistance of many students at UNC is of those venoms (i.e., dead is dead: Mebs, 1999), and ob- also gratefully acknowledged: A. Boswell, C. Csizmadi, servations of scavenging by rattlesnakes (summarized in B. Heyborne, J. Honea, A. Kaye, J. Kreps, R. Malecki, A. DeVault and Krochmal, 2002) demonstrate that snakes Malone, R. Milne, S. Munekiyo, N. Sixberry, H. Sommers, do not have to envenomate prey in order to digest it ef- A. Wang, A. Wastell, and K. Williams. Scientific collecting ficiently. It is possible that under optimal conditions (e.g., permits for snakes or/and venom samples were granted by low daily temperature variation, approximately 20-32ºC), the Arizona Game and Fish Department (MCKSY000221), metalloproteases may not confer a significant selective ad- Colorado Division of Wildlife (0456, 06HP456), Kansas vantage of increasing digestion efficiency (McCue, 2007). Wildlife and Parks (SC-147-96), and the New Mexico However, animals rarely operate under optimal conditions, Game and Fish Department (1984). Financial support for and particularly at extremes of latitude and/or altitude, en- projects which contributed to this work was provided by the vironmental thermal conditions can be quite variable, and Colorado Division of Wildlife and by the UNC Sponsored the advantage of highly degrading venoms under these Programs and Academic Research Center. conditions may be critically important. Vipers gener- ally are among the few heavy-bodied snakes to occur at Literature Cited high elevations and/or latitudes (e.g., Thomas and Pough, 1979; Carlsson and Tegelström, 2002; Campbell and La- Aird, S. D. 2002. Ophidian envenomation strategies and mar, 2004), and for the few which have been investigated, the role of purines. Toxicon 40:335-393. venom shows type I characteristics (Mackessy, 1988). It ———. 2005. Taxonomic distribution and quantitative anal- has been hypothesized that, particularly in these extreme ysis of free purine and pyrimidine nucleosides in snake environments, the selective advantage conferred by tissue- venoms. Comp. Biochem. Physiol. B 140:109-126. degrading metalloproteases is greatest, and these abundant ———, and I. I. Kaiser. 1985. Comparative studies on venom components may permit broader ranges of foraging three rattlesnake toxins. Toxicon 23:361-374. (and therefore digesting) temperatures. ———, ———, R. V. Lewis, and W. G. Kruggel. 1985. Advances in genomics and proteomics are greatly ac- Rattlesnake presynaptic neurotoxins: primary structure celerating the rate at which one can analyze venoms, and and evolutionary origin of the acidic subunit. Biochem- techniques in these areas promise to provide near-complete istry 24:7054-7058. compositional data in the near future (c.f. Serrano et al., Biardi, J. E., R. G. Coss, and D. G. Smith. 2000. Cali- 2005; Sanz et al., 2006). However, for many species, detailed fornia Ground Squirrel (Spermophilus beecheyi) blood aspects of the natural history and ecology, such as diet, for- sera inhibits crotalid venom proteolytic activity. Toxi- aging, and general activity patterns, and even distributional con 38:713–721. limits, are incompletely known. These data are critical for ———, D. C. Chien, and R. G. Coss. 2005. California understanding relationships between venom composition Ground Squirrel (Spermophilus beecheyi) defenses and the selective impetus favoring particular compositional against rattlesnake venom digestive and hemostatic trends, such as the type I/type II trends noted above. The toxins. J. Chem. Ecol. 31:2501-18. tremendous number of molecular tools which can be ap- Bieber, A. L., and D. Nedelhov. 1997. Structural, bio- plied to evolutionary and ecological questions have largely logical and biochemical studies of myotoxin a and ho- overshadowed many other techniques and methods, but ro- mologous myotoxins. J. Toxicol.-Toxin Rev. 16:33-52. bust natural history data are required to provide a meaning- ———, R. H. McParland, and R. R. Becker. 1987. ful biological context to the biochemical data (e.g., Jorge da Amino acid sequences of myotoxins from Crotalus Silva and Aird, 2001). There is a paucity of data available viridis concolor venom. Toxicon 25:677-680. on the venoms of many Mexican species of rattlesnakes, ———, J. P. Mills, Jr., C. Ziolkowski, and J. Harris. particularly the smaller montane species, and an integrative 1990. Rattlesnake neurotoxins: biochemical and bio- approach toward these species and their venoms, currently logical aspects. J. Toxicol.-Toxin Rev. 9:285-306. in progress, should provide important information concern- Bjarnason, J. B., and J. W. Fox. 1989. Hemorrhagic toxins ing the evolution of venoms in rattlesnakes. from snake venoms. J. Toxicol.-Toxin Rev. 7:121-209. ———, and ———. 1995. Snake venom metalloendopep- Acknowledgments tidases: reprolysins. Pp. 345-368 in A. J. Barrett (ed.), Methods in Enzymology. Proteolytic Enzymes: Aspar- Many friends and colleagues contributed venom sam- tic and Metallo Peptidases, Vol. 248. Academic Press, ples, helped collect snakes and venoms, and/or allowed New York, New York. access to animals in their care; their help was critical to ———, and A. T. Tu. 1978. Hemorrhagic toxins from the results presented here and is greatly appreciated. These Western Diamondback Rattlesnake (Crotalus atrox) contributors include J. Badman, D. Chiszar, A. Holycross, venom: isolation and characterization of five toxins B. Jennings, C. Montgomery, S. Minton, T. Moisi, C. and the role of zinc in hemorrhagic toxin e. Biochem- Ownby, R. Reiserer, R. Seigel, B. Starrett, S. Sweet, and istry 17:3395-3404. 506 S. P. Mackessy

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