Inferring species-level phylogenies and taxonomic distinctiveness using multilocus data in Sistrurus rattlesnakes

Phylogenetic relationships and taxonomic distinctiveness of closely related species and subspecies are most accurately inferred from data derived from multiple independent loci. Here, we apply several approaches for understanding species-level relationships using data from 18 nuclear DNA loci and 1 mitochondrial DNA locus within currently described species and subspecies of Sistrurus rattlesnakes. Collectively, these methods provide evidence that a currently described species, the massasauga rattlesnake (Sistrurus catenatus), consists of two well-supported clades, one composed of the two western subspecies (S. c. tergeminus and S. c. edwardsii) and the other the eastern subspecies (S. c. catenatus). Within pigmy rattlesnakes (S. miliarius), however, there is not strong support across methods for any particular grouping at the subspecific level. Monophyly based tests for taxonomic distinctiveness show evidence for distinctiveness of all subspecies but this support is strongest by far for the S. c. catenatus clade. Because support for the distinctiveness of S. c. catenatus is both strong and consistent across methods, and due to its morphological distinctiveness and allopatric distribution, we suggest that this subspecies be elevated to full species status, which has significant conservation implications. Finally, most divergence time estimates based upon a fossil-calibrated species tree are > 50% younger than those from a concatenated gene tree analysis and suggest that an active period of speciation within Sistrurus occurred within the late Pliocene/Pleistocene eras.     

Proteomic analysis of ontogenetic and diet-related changes in venom composition of juvenile and adult Dusky Pigmy rattlesnakes (Sistrurus miliarius barbouri)

Snake venom proteins show high levels of variation at the level of the individual yet the environmental and molecular mechanisms that generate this diversity remain unclear. Here we report the results of a controlled feeding experiment combined with proteomic analyses of periodically collected venom samples to assess the roles of ontogenetic and diet-related effects on venom composition of captive juvenile and adult Dusky Pigmy rattlesnakes (Sistrurus miliarius barbouri). Juvenile snakes fed from birth with mice, lizards, or frogs showed little evidence for an ontogenetic shift in venom composition from 5 to 26 months in terms of substantial changes in the relative abundance of major classes of venom toxins. However, there were fine-scale changes in the relative abundance of D49-PLA? 15, PI-SVMPs, and PIII-SVMP 28, and a decline in the abundance of other PIII-SVMPs. Although juveniles raised on different prey exhibited distinct relative toxin compositional change rates, at 26 months old, their venoms showed similar patterns of protein composition suggesting little effect of diet on the overall make-up of venom in snakes this age or younger. In contrast, adult females raised on different prey over a 26 month period showed prey-related changes in the relative abundance of major protein families from initial to final samples. Specifically, mouse-fed females showed substantial increases in the relative abundance of total PLA?s and serine proteinases of 95% and >100%, respectively, whereas comparable values for lizard- (42% and -22%) and frog-fed females (2% and 11%) were distinctly smaller in magnitude. Venom from adult snakes fed on different prey also showed distinct changes in the abundance of PLA? molecules 15, 19a, and 19, which were, respectively, (>100%, 33%, 63%), (>100%, 0%, 35%), and (71%, 20%, -4%) for the mice-, lizard-, and frog-diet. Venom from snakes raised on frogs contained a small (1.1%) but consistent amount of a PLA? molecule (15a) not present in snakes fed on mice or lizards. This work provides evidence that venom composition is somewhat plastic in both juvenile and adult S. m. barbouri and that, at least in adults, prey consumed may influence the relative abundance of possibly functionally-distinct classes of venom proteins.     

Snake venomics of Crotalus tigris: the minimalist toxin arsenal of the deadliest Neartic rattlesnake venom. Evolutionary Clues for generating a pan-specific antivenom against crotalid type II venoms

We report the proteomic and antivenomic characterization of Crotalus tigris venom. This venom exhibits the highest lethality for mice among rattlesnakes and the simplest toxin proteome reported to date. The venom proteome of C. tigris comprises 7-8 gene products from 6 toxin families; the presynaptic β-neurotoxic heterodimeric PLA(2), Mojave toxin, and two serine proteinases comprise, respectively, 66 and 27% of the C. tigris toxin arsenal, whereas a VEGF-like protein, a CRISP molecule, a medium-sized disintegrin, and 1-2 PIII-SVMPs each represent 0.1-5% of the total venom proteome. This toxin profile really explains the systemic neuro- and myotoxic effects observed in envenomated animals. In addition, we found that venom lethality of C. tigris and other North American rattlesnake type II venoms correlates with the concentration of Mojave toxin A-subunit, supporting the view that the neurotoxic venom phenotype of crotalid type II venoms may be described as a single-allele adaptation. Our data suggest that the evolutionary trend toward neurotoxicity, which has been also reported for the South American rattlesnakes, may have resulted by pedomorphism. The ability of an experimental antivenom to effectively immunodeplete proteins from the type II venoms of C. tigris, Crotalus horridus , Crotalus oreganus helleri, Crotalus scutulatus scutulatus, and Sistrurus catenatus catenatus indicated the feasibility of generating a pan-American anti-Crotalus type II antivenom, suggested by the identification of shared evolutionary trends among South and North American Crotalus species.     

Snake venomics: characterization of protein families in Sistrurus barbouri venom by cysteine mapping, N-terminal sequencing, and tandem mass spectrometry analysis

The protein composition of the crude venom of Sistrurus barbouri was analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were separated by reversed phase high-performance liquid chromatography and characterized by N-terminal sequence analysis. The molecular mass and number of cysteine residues of the purified proteins were determined by matrix-associated laser desorption/ionization-time of flight mass spectrometry. Selected protein bands were subjected to in-gel tryptic digestion and peptide mass fingerprinting. Analysis of the tandem mass spectrometry spectra of selected doubly-charged peptide ions was done by collision-induced dissociation in a quadrupole-linear ion trap instrument. Our results show that the venom proteome of the pigmy rattlesnake S. barbouri is composed of proteins belonging to a few protein families, which can be structurally characterized by their disulfide bond contents.     

Comparative analysis of the venom proteomes of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis

The venom proteomics of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis, snakes of public health significance and the most poisonous reptiles in Europe, were analyzed by FPLC, 2-D electrophoresis, sequence analysis, and MS/MS. FPLC analysis showed the presence of l-amino acid oxidase, monomeric and heterodimeric phospholipases A2, C-type lectin protein, and proteinases in the venom of V. a. ammodytes. Representatives of the same protein families were found in the venom of the other subspecies, V. a. meridionalis. N-terminally identical PLA2 neurotoxins were identified in both venoms. Difference in the PLA2 compositions of the venoms was also observed: a monomeric protein with phospholipase A2 activity, identical in the first 20 amino acid residues to the catalitically inactive acidic component of the heterodimeric PLA2 present in both venoms, was found only in that of V. a. meridionalis. Probably, this protein represents an intermediate form of the two components of the heterodimer. 2-D electrophoresis and MS/MS analysis showed that the two venoms shared a number of protein families: monomeric and heterodimeric Group II PLA2s, serine proteinases, Group I, II, and III metalloproteinases, l-amino acid oxidases (LAAOs), cysteine-rich secretory proteins, disintegrins, and growth factors. Totally, 38 venom components of the V. a. ammodytes, belonging to 9 protein families, and 67 components of the V. a. meridionalis venom belonging to 8 protein families were identified. The venom proteome of V. a. ammodytes shows larger diversity of proteins (139) in comparison to that of V. a. meridionalis (104 proteins). Most of the proteins are homologues of known representatives of the respective protein families. The protein compositions explain clinical effects of the V. ammodytes snakebites, such as difficulties in the breathing, paralysis, apoptosis, cloting disorders, hemorrhage, and tissue necrosis. The lists of secreted proteins by the two vipers can be used for further study of structure-function relationships in the toxins and for prediction and treatment of snakebite consequences.     

Snake venomics and antivenomics of the arboreal neotropical pitvipers Bothriechis lateralis and Bothriechis schlegelii

We report the comparative proteomic characterization of the venoms of two related neotropical arboreal pitvipers from Costa Rica of the genus Bothriechis, B. lateralis (side-striped palm pit viper) and B. schlegelii (eyelash pit viper). The crude venoms were fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venom proteomes of B. lateralis and B. schlegelii comprise similar number of distinct proteins belonging, respectively, to 8 and 7 protein families. The two Bothriechis venoms contain bradykinin-potentiating peptides (BPPs), and proteins from the phospholipase A 2 (PLA 2), serine proteinase, l-amino acid oxidase (LAO), cysteine-rich secretory protein (CRISP), and Zn (2+)-dependent metalloproteinase (SVMP) families, albeit each species exhibit different relative abundances. Each venom also contains unique components, for example, snake venom vascular endothelial growth factor (svVEGF) and C-type lectin-like molecules in B. lateralis, and Kazal-type serine proteinase inhibitor-like proteins in B. schlegelii. Using a similarity coefficient, we estimate that the similarity of the venom proteins between the two Bothriechis taxa may be <10%, indicating a high divergence in their venom compositions, in spite of the fact that both species have evolved to adapt to arboreal habits. The major toxin families of B. lateralis and B. schlegelii are SVMP (55% of the total venom proteins) and PLA 2 (44%), respectively. Their different venom toxin compositions provide clues for rationalizing the distinct signs of envenomation caused by B. schlegelii and B. lateralis. An antivenomic study of the immunoreactivity of the Instituto Clodomiro Picado (ICP) polyvalent antivenom toward Bothriechis venoms revealed that l-amino acid oxidase and SVMPs represent the major antigenic protein species in both venoms. Our results provide a ground for rationalizing the reported protection of the ICP polyvalent antivenom against the hemorrhagic, coagulant, defibrinating, caseinolytic and fibrin(ogen)olytic activities of Bothriechis ( schlegelii, lateralis) venoms. However, these analyses also evidenced the limited recognition capability of the polyvalent antivenom toward a number of Bothriechis venom components, predominantly BPPs, svVEGF, Kazal-type inhibitors, some PLA 2 proteins, some serine proteinases, and CRISP molecules.     

Phylogeny-Based Comparative Analysis of Venom Proteome Variation in a Clade of Rattlesnakes (Sistrurus sp.)

A long-standing question in evolutionary studies of snake venoms is the extent to which phylogenetic divergence and diet can account for between-species differences in venom composition. Here we apply phylogeny-based comparative methods to address this question. We use data on venom variation generated using proteomic techniques for all members of a small clade of rattlesnakes (Sistrurus sp.) and two outgroups for which phylogenetic and diet information is available. We first complete the characterization of venom variation for all members of this clade with a “venomic” analysis of pooled venoms from two members of this genus, S. milarius streckeri and S. m. milarius. These venoms exhibit the same general classes of proteins as those found in other Sistrurus species but differ in their relative abundances of specific protein families. We then test whether there is significant phylogenetic signal in the relative abundances of major venom proteins across species and if diet (measured as percent mammals and lizards among all prey consumed) covaries with venom composition after phylogenetic divergence is accounted for. We found no evidence for significant phylogenetic signal in venom variation: K values for seven snake venom proteins and two composite venom variables [PC 1 and 2]) were all nonsignificant and lower (mean = 0.11+0.06 sd) than mean K values (>0.35) previously reported for a wide range of morphological, life history, physiological and behavioral traits from other species. Finally, analyses based on Phylogenetic Generalized Least Squares (PGLS) methods reveal that variation in abundance of some venom proteins, most strongly CRISP is significantly related to snake diet. Our results demonstrate that venom variation in these snakes is evolutionarily a highly labile trait even among very closely-related taxa and that natural selection acting through diet variation may play a role in molding the relative abundance of specific venom proteins.     

Venomic analysis and evaluation of antivenom cross-reactivity of South American Micrurus species

Coral snakes from Micrurus genus are the main representatives of the Elapidae family in South America. However, biochemical and pharmacological features regarding their venom constituents remain poorly investigated. Here, venomic analyses were carried out aiming at a deeper understanding on the composition of M. frontalis, M. ibiboboca, and M. lemniscatus venoms. In the three venoms investigated, proteins ranging from 6 to 8 kDa (3FTx) and 12 to 14 kDa (PLA(2)) were found to be the most abundant. Also, the N-terminal sequences of four new proteins, purified from the M. lemniscatus venom, similar to 3FTx, PLA(2) and Kunitz-type protease inhibitor from other Micrurus and elapid venoms are reported. Cross-reactivity among different Micrurus venoms and homologous or heterologous antivenoms was carried out by means of 2D-electrophoresis and immunoblotting. As, expected, the heterologous anti-Elapid venom displayed the highest degree of cross-reactivity. Conversely, anti-M. corallinus reacted weakly against the tested venoms. In gel digestions, followed by mass spectrometry sequencing and similarity searching, revealed the most immunogenic protein families as similar to short and long neurotoxins, weak neurotoxins, PLA(2), β-bungarotoxin, venom protein E2, frontoxin III, LAO and C-type lectin. The implications of our results for the production of Micrurus antivenoms are discussed.     

Barbourin. A GPIIb-IIIa-specific integrin antagonist from the venom of Sistrurus m. barbouri

Sixty-two snake venoms were screened to identify those which specifically inhibit the adhesive protein binding function of the glycoprotein (GP) IIb-IIIa complex, the receptor-mediating platelet aggregation. Although 52 of these venoms inhibited GPIIb-IIIa, only one of these, from the southeastern pigmy rattlesnake, Sistrurus m. barbouri, was specific for GPIIb-IIIa versus other integrins. The peptide responsible for this activity, termed barbourin, was sequenced and found to be highly homologous to other peptides of the viper venom GPIIb-IIIa antagonist family but was the first member which did not contain the Arg-Gly-Asp (RGD) amino acid sequence, believed to be required for inhibition of receptor function. Instead, barbourin contains the sequence, Lys-Gly-Asp (KGD). The conservative Lys for Arg substitution appears to be the sole structural feature which imparts integrin specificity to barbourin, since venom peptide analogs with Lys substitutions were also specific for GPIIb-IIIa. Thus, barbourin represents a new structural model useful for designing potent and GPIIb-IIIa-specific compounds that may have therapeutic value as platelet aggregation inhibitors.     

Snake venomics of Central American pitvipers: clues for rationalizing the distinct envenomation profiles of Atropoides nummifer and Atropoides picadoi

We report the proteomic characterization of the Central American pitvipers Atropoides nummifer and Atropoides picadoi. The crude venoms were fractionated by reverse-phase high-performance liquid chromatography (HPLC), followed by analysis of each chromatographic fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequencing, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass fingerprinting, and collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) of tryptic peptides. Each venom contained a number of bradykinin-potentiating peptides and around 25-27 proteins of molecular masses in the range of 7-112 kDa, belonging to only nine different toxin families (disintegrin, DC fragment, snake venom vascular endothelial growth factor, phospholipases A2, serine protease, cysteine-rich secretory proteins, C-type lectins, L-amino acid oxidase, and Zn2+-dependent metalloproteases), albeit distinctly distributed among the two Atropoides species. In addition, A. nummifer expresses low amounts of a three-finger toxin not detected in the venom of A. picadoi. The major toxins of A. nummifer belong to the PLA2 (relative abundance, 36.5%) and the serine proteinase (22%) families, whereas the most abundant A. picadoi toxins are Zn2+-dependent metalloproteinases (66.4%). We estimate that the similarity of venom proteins between the two Atropoides taxa may be around 14-16%. The high degree of differentiation in the venom proteome among congeneric taxa emphasizes unique aspects of venom composition of related species of Atropoides snakes and points to a strong role for adaptive diversification via natural selection as a cause of this distinctiveness. On the other hand, their distinct venom toxin compositions provide clues for rationalizing the low hemorrhagic, coagulant, and defibrinating activities and the high myotoxic and proteolytic effects evoked by A. nummifer snakebite in comparison to other crotaline snake venoms and the high hemorrhagic activity of A. picadoi.     

Proteomic and biological characterization of the venom of the redtail coral snake, Micrurus mipartitus (Elapidae), from Colombia and Costa Rica

Venoms of the redtail coral snake Micrurus mipartitus from Colombia and Costa Rica were analyzed by "venomics", a proteomic strategy to determine their composition. Proteins were separated by RP-HPLC, followed by SDS-PAGE, in-gel tryptic digestion, identification by MALDI or ESI tandem mass spectrometry, and assignment to known protein families by similarity. These analyses were complemented with a characterization of venom activities in vitro and in vivo. Proteins belonging to seven families were found in Colombian M. mipartitus venom, including abundant three-finger toxins (3FTx; ~60% of total proteins) and phospholipases A(2) (PLA(2); ~30%), with the remaining ~10% distributed among l-amino acid oxidase, P-III metalloproteinase, Kunitz-type inhibitor, serine proteinase, and C-type lectin-like families. The venoms of two M. mipartitus specimens from Costa Rica, also referred to as M. multifasciatus in some taxonomic classifications, were also analyzed. Both samples were highly similar to each other, and partially resembled the chromatographic and identity profiles of M. mipartitus from Colombia, although presenting a markedly higher proportion of 3FTxs (~83.0%) in relation to PLA(2)s (~8.2%), and a small amount of acetylcholinesterase, not detected in the venom from Colombia. An equine antivenom against the Central American coral snake, M. nigrocinctus, did not recognize venom components of M. mipartitus from Colombia or Costa Rica by enzyme-immunoassay. Four major components of Colombian M. mipartitus venom were isolated and partially characterized. Venomics of Micrurus species may provide a valuable platform for the rational design of immunizing cocktails to obtain polyspecific antivenoms for this highly diverse group of American elapids.     

Snake venomics of the lancehead pitviper Bothrops asper: geographic, individual, and ontogenetic variations

We report the comparative proteomic characterization of the venoms of adult and newborn specimens of the lancehead pitviper Bothrops asper from two geographically isolated populations from the Caribbean and the Pacific versants of Costa Rica. The crude venoms were fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The two B. asper populations, separated since the late Miocene or early Pliocene (8-5 mya) by the Guanacaste Mountain Range, Central Mountain Range, and Talamanca Mountain Range, contain both identical and different (iso)enzymes from the PLA 2, serine proteinase, and SVMP families. Using a similarity coefficient, we estimate that the similarity of venom proteins between the two B. asper populations may be around 52%. Compositional differences between venoms among different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. To investigate venom variability among specimens from the two B. asper populations, the reverse-phase HPLC protein profiles of 15 venoms from Caribbean specimens and 11 venoms from snakes from Pacific regions were compared. Within each B. asper geographic populations, all major venom protein families appeared to be subjected to individual variations. The occurrence of intraspecific individual allopatric variability highlights the concept that a species, B. asper in our case, should be considered as a group of metapopulations. Analysis of pooled venoms of neonate specimens from Caribbean and Pacific regions with those of adult snakes from the same geographical habitat revealed prominent ontogenetic changes in both geographical populations. Major ontogenetic changes appear to be a shift from a PIII-SVMP-rich to a PI-SVMP-rich venom and the secretion in adults of a distinct set of PLA 2 molecules than in the neonates. In addition, the ontogenetic venom composition shift results in increasing venom complexity, indicating that the requirement for the venom to immobilize prey and initiate digestion may change with the size (age) of the snake. Besides ecological and taxonomical implications, the geographical venom variability reported here may have an impact in the treatment of bite victims and in the selection of specimens for antivenom production. The occurrence of intraspecies variability in the biochemical composition and symptomatology after envenomation by snakes from different geographical location and age has long been appreciated by herpetologist and toxinologists, though detailed comparative proteomic analysis are scarce. Our study represents the first detailed characterization of individual and ontogenetic venom protein profile variations in two geographical isolated B. asper populations, and highlights the necessity of using pooled venoms as a statistically representative venom for antivenom production.     

Comparative proteomic analysis of the venom of the taipan snake, Oxyuranus scutellatus, from Papua New Guinea and Australia: role of neurotoxic and procoagulant effects in venom toxicity

The venom proteomes of populations of the highly venomous taipan snake, Oxyuranus scutellatus, from Australia and Papua New Guinea (PNG), were characterized by reverse-phase HPLC fractionation, followed by analysis of chromatographic fractions by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. Proteins belonging to the following seven protein families were identified in the two venoms: phospholipase A(2) (PLA(2)), Kunitz-type inhibitor, metalloproteinase (SVMP), three-finger toxin (3FTx), serine proteinase, cysteine-rich secretory proteins (CRISP), and coagulation factor V-like protein. In addition, C-type lectin/lectin-like protein and venom natriuretic peptide were identified in the venom of specimens from PNG. PLA(2)s comprised more than 65% of the venoms of these two populations. Antivenoms generated against the venoms of these populations showed a pattern of cross-neutralization, corroborating the immunological kinship of these venoms. Toxicity experiments performed in mice suggest that, at low venom doses, neurotoxicity leading to respiratory paralysis represents the predominant mechanism of prey immobilization and death. However, at high doses, such as those injected in natural bites, intravascular thrombosis due to the action of the prothrombin activator may constitute a potent and very rapid mechanism for killing prey.     

Venom from spiders of the genus Hippasa: biochemical and pharmacological studies

The venoms from female spiders of the genus Hippasa namely H. partita, H. agelenoides and H. lycosina are compared for biochemical and pharmacological properties. SDS-PAGE pattern revealed varied protein composition. Marked variability is seen with casein hydrolyzing enzymes in SDS-PAGE zymogram. H. partita venom was the only venom that hydrolyzed gelatin while the other two venoms did not. The venoms shared similar hyaluronidase activity, showing a single activity band in SDS-PAGE zymogram. The PLA2 activity varied as H. partita>H. agelenoides>H. lycosina venoms. Marked differences were noted in the ability to induce edema, cytotoxicity, myotoxicity and neurotoxicity, while hemorrhage was associated exclusively with H. partita venom.     

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

The ability to detect biochemical diversity in animal venoms has wide-ranging implications for a diverse array of scientific disciplines. Matrix-assisted laser desorption time-of-flight mass spectrometry (and, for comparative purposes, isoelectric focusing) were used to characterize venoms from a geographically diverse sample of Trimeresurus stejnegeri ( n < 229) from Taiwan. Previously unrealized levels of heterogeneity were detected in venom phospholipase A(2) isoforms (PLA(2)) and in whole venom profiles. Geographic variation in venom was primarily between Taiwan and two Pacific islets. Despite the common assumption that venom variation is a product of neutral molecular evolution, statistical testing failed to link venom variation with phylogenetic descent convincingly. Instead, pronounced differences in venom composition may be the product of natural selection for regional diets or of independent founder effects. More data are required on the functional differences between the isoforms to distinguish between these alternatives.     

Evaluation of individual variability in the composition of Agkistrodon contortrix contortrix venom by means of HPLC and two-dimensional PAGE

Qualitative and quantitative differences of coagulant enzymes in venom samples from individuals of the Agkistrodon c.c. were studied by means of 2D-PAGE and high performance anion-exchange chromatography. A great diversity was found among individual venoms. The two most similar venoms had an identical composition in only about 90%, the least similar ones in about 45% spots. All venoms studied contained more than two fractions with fibrinolytic activity. In three out of the nine analysed venoms two different thrombic proteases were present, one venom contained only one of these enzymes, whereas in five venoms no thrombic activity was detectable.