Ontogeny of intersexual head shape and prey selection in the pitviper Agkistrodon piscivorus

Different animal intraspecific classes commonly differ in their prey selection. Such differences in feeding ecology are thought to reduce resource competition between classes, but other factors (i.e. behavioural, morphological, and physiological differences) also contribute to this widespread phenomenon. Although several studies have correlated the size of the feeding apparatus with prey selection in many animals, few studies have examined how the shape of the feeding apparatus is related to prey selection. Furthermore, even though the dietary regimen of many animals changes during ontogeny, few studies have examined how shape changes in the feeding apparatus may be related to these ontogenetic dietary shifts. Here we address these issues by examining how head shape, head size and prey selection change over ontogeny in adult males, adult females and juveniles of the cottonmouth snake Agkistrodon piscivorus . Our scaling data for head characteristics showed that all head measurements in adult male and female A. piscivorus scaled with significant negative allometry, whereas juvenile head measurements typically scaled isometrically, except for head volume (positive) and head length (negative). Thus, juveniles have relatively broad and high, but short, heads. Large adult male and female A. piscivorus have relatively small head dimensions overall. Thus, juveniles appear to undergo a rapid change in head volume, which subsequently slows considerably as sexual maturity is achieved. However, our multivariate analysis of size-adjusted head dimensions showed that juveniles differed only slightly in their head shape compared with adult male and female A. piscivorus . In general, prey size increased with snake size across all age and sex groups, but an ontogenetic shift in prey type was not detected in either males or females.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 81 , 151–159.     

Beta-fibrinogenase from the venom of Agkistrodon p. piscivorus

1. Beta-fibrinogenase was isolated from the venom of Agkistrodon p. piscivorus by column chromatography on Sephadex G-100, DEAE-Sephacel and by chromatofocusing, with a yield of 2.5 mg of purified enzyme from 1 g of crude venom. 2. The enzyme was homogeneous by SDS and non-SDS disc electrophoresis on polyacrylamide gel at pH 8.3. 3. Beta-fibrinogenase is a glycoprotein possessing both TAME hydrolase and kinin-releasing activities. 4. A mol. wt of approximately 33,500 and an isoelectric point 4.5 was determined. 5. The enzyme is stable to heat treatment and to a pH range of 2-10. 6. Beta-fibrinogenase activity is inactivated by DFP, suggesting that serine is involved in the enzymatic activity. 7. The Michaelis constant (Km) of this enzyme for TAME and inhibition constant (Ki) for DFP were found to be 7.04 X 10(-3) and 4.13 X 10(-3) M, respectively.     

Changes in Ca2+ affinity upon activation of Agkistrodon piscivorus piscivorus phospholipase A2

Changes in the affinity of calcium for phospholipase A2 from Agkistrodon piscivorus piscivorus during activation of the enzyme on the surface of phosphatidylcholine vesicles have been investigated by site-directed mutagenesis and fluorescence spectroscopy. Changes in fluorescence that occur during lipid binding and subsequent activation have been ascribed to each of the three individual Trp residues in the protein. This was accomplished by generating a panel of mutant proteins, each of which lacks one or more Trp residues. Both Trp21, which is found in the interfacial binding region, and Trp119 show changes in fluorescence upon protein binding to small unilamellar zwitterionic vesicles or large unilamellar vesicles containing sufficient anionic lipid. Trp31, which is near the Ca2+ binding loop, exhibits little change in fluorescence upon lipid bilayer binding. A change in the fluorescence of the protein also occurs during activation of the enzyme. These changes arise from residue Trp31 as well as residues Trp21 and Trp119. The calcium dependence of the fluorescence change of Trp31 indicates that the affinity of the enzyme for calcium increases at least 3 orders of magnitude upon activation. These studies suggest either that a change in conformation of the enzyme occurs upon activation or that the increase in calcium affinity reflects formation of a ternary complex of calcium, enzyme, and substrate.     

Kallikrein-like enzyme from the venom of Agkistrodon p. piscivorus

1. A kallikrein-like enzyme was isolated from Agkistrodon p. piscivorus venom by Sephadex G-100, DEAE-Sephacel and S-Sepharose column chromatographies. 2. A kallikrein-like enzyme was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodiffusion. 3. Its molecular weight is approx. 29,000 with an isoelectric point of 7.8. 4. A kallikrein-like enzyme is able to cleave a kininogen analog to release bradykinin, and the B beta chain of fibrinogen. These proteolytic and tosyl-L-arginine methyl ester hydrolytic activities were inhibited by diisopropyl fluorophosphate, suggesting that the serine hydroxyl group is involved in enzymatic activities.     

Crystallization and preliminary diffraction studies of the Lys-49 phospholipase A2 from Agkistrodon piscivorus piscivorus

Previous chemical and structural studies have proposed a major role for Asp-49 in the calcium-mediated activation of phospholipases A2. Recently, a new class of phospholipases A2 has been characterized with a lysine in the place of aspartate at position 49 (Maraganore, J. M., Merutka, G., Cho, W., Welches, W., Kézdy, F. J., and Heinrikson, R. L. (1984) J. Biol. Chem. 259, 13839-13843; Maraganore, J. M., and Heinrikson, R. L. (1986) J. Biol. Chem. 261, 4797-4804). Although both the Lys-49 and Asp-49 phospholipases require calcium for enzymatic activity, the Lys-49 enzymes appear to be unique in their ability to bind phospholipids prior to undergoing calcium-mediated activation. We have successfully crystallized the Lys-49 phospholipase A2 from the venom of the American cottonmouth water moccasin (Agkistrodon piscivorus piscivorus). The crystals are tetragonal, the space group being P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = b = 71.05 A, and c = 57.76 A. There is only one molecule in the asymmetric unit and the crystals provide good quality diffraction data to 2.2 A.     

Thermodynamic and kinetic studies of the interaction of vesicular dipalmitoylphosphatidylcholine with Agkistrodon piscivorus piscivorus phospholipase A2

The tryptophan fluorescence emission intensity at 340 nm of monomeric phospholipase A2 from Agkistrodon piscivorus piscivorus increased about 70% upon addition of dipalmitoylphosphatidylcholine small unilamellar vesicles (DPPC SUV) at 25 degrees C. The emission spectrum was also blue-shifted 6-8 nm, suggesting that the environment of 1 or more tryptophan residues had become less polar. This effect of SUV on the phospholipase A2 fluorescence was independent of Ca2+ at 25 degrees C, and the apparent association constant for the interaction was approximately 1.7 x 10(4) M-1. The apparent Km for hydrolysis of DPPC SUV was equal to the inverse of the estimated association constant. In the absence of Ca2+, the change in fluorescence intensity decreased with increasing temperature. Thermodynamic analysis of this reversible, temperature-dependent fluorescence change indicated that the A. p. piscivorus monomer phospholipase A2 interacts only with SUV in the true gel phase existing below the pretransition of gel to "ripple" phase lipid in the absence of Ca2+. In contrast, the fluorescence intensity change upon addition of SUV in the presence of Ca2+ was independent of temperature over the range of 25-48 degrees C. Under these conditions, hydrolysis of the lipid occurred concomitantly with the change in fluorescence which could not be reversed by the addition of EDTA. With a nonhydrolyzable analog of DPPC, however, the fluorescence changes upon mixing of SUV, Ca2+, and phospholipase A2 were reversible and temperature-dependent. Thus, the apparent irreversibility of the change in fluorescence observed with Ca2+ and DPPC SUV was correlated with hydrolysis of the vesicles. These results indicate that the magnitude of the initial interaction of enzyme with substrate is reversible, is Ca2+-independent, depends upon the lipid state, and is quantitatively correlated to the maximum rate of hydrolysis.     

Ontogeny of Anti-Predator Behavioral Habituation in Cottonmouths (Agkistrodon piscivorus)

Few studies of venomous snakes have addressed how anti‐predator behavior may be affected by experience with a potential predator. Because defensive strikes may be costly to snakes, individuals with the ability to learn to discriminate among potentially harmful and non‐harmful predatory stimuli should be favored by natural selection. In large venomous snakes, adults are capable of successfully defending themselves against most potential predators, whereas neonates suffer higher predator‐induced mortality and are faced with a large diversity of predators. Consequently, we hypothesized that the relative costs of habituation to potential predatory stimuli should vary ontogenetically. This hypothesis predicts that adults should habituate rapidly to non‐harmful predatory stimuli, whereas neonates should consistently employ active defensive displays (e.g. striking) because they are at higher risk. To test this prediction, we examined daily changes in the defensive behavior of adult and neonate cottonmouths (Agkistrodon piscivorus) towards a standardized non‐harmful predatory stimulus. As predicted, adults and neonates differed in their tendencies to habituate: adults decreased defensiveness over days while neonates did not. Adults showed habituation of striking components but not of warning displays. Our results support the hypothesis that there may be ontogenetic differences in predator perception.     

HPLC-based two-step purification of fibrinolytic enzymes from the venom of Agkistrodon contortrix contortrix and Agkistrodon piscivorus conanti

In investigations aimed at characterizing snake venom blood clot-dissolving enzymes, we have developed a rapid two-step high-performance chromatography method for the isolation of these fibrinolytic enzymes from the venoms of Agkistrodon contortrix contortrix and Agkistrodon piscivorus conanti. The first step consisted of hydrophobic interaction chromatography on a propyl-aspartamide column. Fractions containing the fibrinolytic activity were then concentrated and applied to a hydroxylapatite column. The resulting preparation, assessed for purity by reverse-phase chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was homogeneous. The molecular weight of both venom fibrinolytic enzymes was approximately 23,000 and amino acid analysis, immunological cross-reaction, cyanogen bromide, and tryptic digestion indicate a significant degree of structural similarity. However, the general proteolytic activity of the A. p. conanti venom enzyme was significantly lower than the corresponding activity of the A. c. contortrix venom, whereas their fibrinolytic activities were quite similar.     

Variation in cottonmouth (Agkistrodon piscivorus leucostoma) resting metabolic rates

The study of intra- and inter-individual variation in the metabolic response to environmental variation can provide mechanistic explanations to large-scale ecological and evolutionary patterns. In a study of range-limiting factors, variation in resting metabolic rates of cottonmouths (Agkistrodon piscivorus leucostoma) was investigated along a latitudinal gradient in southern populations and in populations near and at the northern range limit. CO(2) production rates of 53 snakes were measured in response to body mass, temperature, time of day, latitude of origin, and sex. The within-subjects effects were similar to those reported for other pit vipers. Metabolic cold adaptation appears to exist, with cottonmouths from northern populations having higher low temperature metabolic rates. Calculations suggest that Arkansas cottonmouths allocate almost twice as much energy to resting metabolism during non-feeding periods (brumation) as Louisiana cottonmouths. While maintenance metabolism alone during brumation is more costly near the northern range limit, it is most likely not a limiting factor in geographic distribution and may be used to fuel important processes other than activity metabolism.     

Ultrastructure of spermiogenesis in the Cottonmouth,Agkistrodon piscivorus (Squamata: Viperidae: Crotalinae)

To date multiple studies exist that examine the morphology of spermatozoa. However, there are limited numbers of data detailing the ontogenic characters of spermiogenesis within squamates. Testicular tissues were collected from Cottonmouths (Agkistrodon piscivorus) and tissues from spermiogenically active months were analyzed ultrastructurally to detail the cellular changes that occur during spermiogenesis. The major events of spermiogenesis (acrosome formation, nuclear elongation/DNA condensation, and flagellar development) resemble that of other squamates; however, specific ultrastructural differences can be observed between Cottonmouths and other squamates studied to date. During acrosome formation vesicles from the Golgi apparatus fuse at the apical surface of the nuclear membrane prior to making nuclear contact. At this stage, the acrosome granule can be observed in a centralized location within the vesicle. As elongation commences the acrosome complex becomes highly compartmentalized and migrates laterally along the nucleus. Parallel and circum‐cylindrical microtubules (components of the manchette) are observed with parallel microtubules outnumbering the circum‐cylindrical microtubules. Flagella, displaying the conserved 9 + 2 microtubule arrangement, sit in nuclear fossae that have electron lucent shoulders juxtaposed on either side of the spermatids basal plates. This study aims to provide developmental characters for squamates in the subfamily Crotalinae, family Viperidae, which may be useful for histopathological studies on spermatogenesis in semi‐aquatic species exposed to pesticides. Furthermore, these data in the near future may provide morphological characters for spermiogenesis that can be added to morphological data matrices that may be used in phylogenetic analyses. J. Morphol. 2010. © 2009 Wiley‐Liss, Inc.     

Phylogeography of the pitviper clade Agkistrodon: historical ecology, species status, and conservation of cantils

We used mitochondrial DNA sequences from three gene regions and two tRNAs (ND4, tRNA-HIS-SER, 12S, and 16S rDNA) to investigate the historical ecology of the New World pitviper clade Agkistrodon, with emphasis on the disjunct subspecies of the cantil, A. bilineatus. We found strong evidence that the copperhead (A. contortrix) is basal to its congeners, and that the cottonmouth (A. piscivorus) is basal to cantils. Phylogeography and natural history of the living terminal taxa imply that Agkistrodon primitively occupied relatively temperate habitats, with subsequent evolution of tropicality in ancestral A. bilineatus. Our best supported phylogeny rejects three gulf arc scenarios for the biogeography of A. bilineatus. We find significant statistical support for an initial divergence between populations on the east and west coasts of México and subsequent occupancy of the Yucatán Peninsula, by way of subhumid corridors in northern Central America. Based on phylogenetic relationships, morphological and molecular divergence, and allopatry we elevate A. b. taylori of northeastern México to species status. Taylor's cantil is likely threatened by habitat destruction and small geographical range, and we offer recommendations for its conservation and management.     

Phylogenetic Analysis of Bacterial Communities in Different Regions of the Gastrointestinal Tract of Agkistrodon piscivorus,the Cottonmouth Snake

Vertebrates are metagenomic organisms in that they are composed not only of their own genes but also those of their associated microbial cells. The majority of these associated microorganisms are found in the gastrointestinal tract (GIT) and presumably assist in processes such as energy and nutrient acquisition. Few studies have investigated the associated gut bacterial communities of non-mammalian vertebrates, and most rely on captive animals and/or fecal samples only. Here we investigate the gut bacterial community composition of a squamate reptile, the cottonmouth snake, Agkistrodon piscivorus through pyrosequencing of the bacterial 16S rRNA gene. We characterize the bacterial communities present in the small intestine, large intestine and cloaca. Many bacterial lineages present have been reported by other vertebrate gut community studies, but we also recovered unexpected bacteria that may be unique to squamate gut communities. Bacterial communities were not phylogenetically clustered according to GIT region, but there were statistically significant differences in community composition between regions. Additionally we demonstrate the utility of using cloacal swabs as a method for sampling snake gut bacterial communities.     

Climate change and evolution of the New World pitviper genus Agkistrodon (Viperidae)

Aim We derived phylogenies, phylogeographies, and population demographies for two North American pitvipers, Agkistrodon contortrix (Linnaeus, 1766) and A. piscivorus (Lacépède, 1789) (Viperidae: Crotalinae), as a mechanism to evaluate the impact of rapid climatic change on these taxa. Location Midwestern and eastern North America. Methods We reconstructed maximum parsimony (MP) and maximum likelihood (ML) relationships based on 846 base pairs of mitochondrial DNA (mtDNA) ATPase 8 and ATPase 6 genes sequenced over 178 individuals. We quantified range expansions, demographic histories, divergence dates and potential size differences among clades since their last period of rapid expansion. We used the Shimodaira–Hasegawa (SH) test to compare our ML tree against three biogeographical hypotheses. Results A significant SH test supported diversification of A. contortrix from northeastern Mexico into midwestern–eastern North America, where its trajectory was sundered by two vicariant events. The first (c. 5.1 Ma) segregated clades at 3.1% sequence divergence (SD) along a continental east–west moisture gradient. The second (c. 1.4 Ma) segregated clades at 2.4% SD along the Mississippi River, coincident with the formation of the modern Ohio River as a major meltwater tributary. A single glacial refugium was detected within the Apalachicola region of southeastern North America. Significant support was also found for a hypothesis of trans‐Gulf rafting by the common ancestor of A. piscivorus from eastern Mexico (possibly the Yucatan Peninsula) to northern Florida. There, a Mid–Late Pliocene marine transgression separated it at 4.8% SD from mainland North America. Significant range expansions followed compressive glacial effects in three (of four) A. contortrix clades and in two (of three) A. piscivorus clades, with the Florida A. piscivorus clade exhibiting significant distributional stasis. Main conclusions Pliocene glaciations, rapidly developing western aridity, and Pleistocene glacial meltwaters seemingly led to the diversification of A. contortrix and A. piscivorus in North America. Both species were pushed southwards by Pleistocene climate change, with subsequent northward expansions uninhibited topographically. The subspecific taxonomy used for A. contortrix and A. piscivorus today, however, appear non‐representative. The monophyletic Florida subspecies of A. piscivorus may be a distinct species (at 4.8% SD), whereas two western subspecies of A. contortrix also appear to constitute a single distinct species, pending additional analyses. We conclude that both species of Agkistrodon can be used as suitable ectothermic models to gauge impacts of future climate change.     

Mother Cottonmouths (Agkistrodon piscivorus) Alter Their Antipredator Behavior in the Presence of Neonates

Neonate‐directed care is rare in non‐avian reptiles, but female pitvipers attend their young for a period of time after birth. One of the primary functions of parental care is the protection of offspring from predators, and parents of diverse taxa are able to modulate their antipredator behavior in the presence of offspring. To test the hypothesis that the antipredator behavior of post‐parturient pitvipers is altered during neonate attendance, we conducted behavioral trials on female cottonmouths (Agkistrodon piscivorus) in which we measured female response to a simulated predator encounter. Cottonmouths were divided into three treatment groups: (1) post‐parturient, attending neonates; (2) post‐parturient, not attending neonates; and (3) non‐reproductive. All females were subjected to a second trial approximately 3 wk later, when females in Group 1 were no longer attending neonates. When mothers were attending offspring, they were more hesitant to engage the predator and exhibited more warning than aggressive behaviors once they did, relative to non‐attending and non‐reproductive females. When these same mothers were no longer attending offspring, they significantly increased their antipredator behavior by engaging the predator quickly and displaying more aggressive than warning behaviors. This change in behavior was not observed in post‐parturient females who did not have neonates present during either trial, nor was it observed in non‐reproductive females, indicating that the presence of neonates directly affected the antipredator behavior of attending females. We discuss hypotheses concerning the possible adaptive value of reduced antipredator behavior in female pitvipers attending neonates.     

Size-based variation in antipredator behavior within a snake (Agkistrodon piscivorus) population

Variation in an animal's response to a predator likely reflectsthe complex interaction of factors that influence predationrisk. Due to their high degree of behavioral variation and simplifiedbauplan, snakes offer a unique model for investigating the influenceof sex and body size on antipredator behavior. We examined variationin antipredator behavior within a cottonmouth (Agkistrodon piscivorusleucostoma) population. Behavioral responses to human-inducedpredation risk were compared across a continuous scale of bodysize. Defensive responses significantly declined with increasingbody size. After controlling for body size, no differences betweenthe sexes were detected. Although this study suggests that variationin antipredator behavior is, in part, related to body size,some studies on snakes have not found this relationship. Likewise,some studies have demonstrated differences between sexes. Suchdisparate patterns of variation indicate a need for future comparativestudies examining the complex interaction of factors that mayinfluence predator–prey relationships.     

The temporal sequence of events in the activation of phospholipase A2 by lipid vesicles. Studies with the monomeric enzyme from Agkistrodon piscivorus piscivorus

The substrate dependence of the time courses of hydrolysis of both small and large unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC) by Agkistrodon piscivorus piscivorus monomeric phospholipase A2 is consistent with an activation process involving enzyme aggregation on the vesicle surface. The time course of hydrolysis of large unilamellar vesicles is particularly complex; a slow initial rate of hydrolysis is followed by an extremely abrupt increase in enzyme activity. The length of this slow phase is a minimum at the phase transition temperature of the vesicles. The intrinsic fluorescence intensity of the phospholipase A2 also abruptly increases (50-60%) after a latency period revealing a strong temporal correlation between enzyme activity and the increase in fluorescence intensity. The length of the latency period before the sudden increase in fluorescence intensity is directly proportional to substrate concentration at DPPC concentrations above 20-100 microM. At lower concentrations, the length of the latency period is inversely proportional to the DPPC concentration. Such biphasic substrate dependence is predicted by a previously proposed enzyme activation model involving dimerization on the surface vesicle. Simultaneous monitoring of the protein fluorescence and hydrolysis demonstrates that the magnitude of the fluorescence change and the rate of hydrolysis are in exact temporal correlation. Furthermore, simultaneous monitoring of the fluorescence of the protein and that of a lipid probe, trimethylammonium diphenylhexatriene, indicates a change in lipid vesicle structure prior to, or coincident with, the abrupt change in protein activation. These results are consistent with the hypothesis that the monomeric phospholipase A2 from A. piscivorus piscivorus initially possesses a low level of intrinsic activity toward large unilamellar DPPC vesicles and that the enzyme slowly becomes further activated on the vesicle surface via dimerization. Eventually, the vesicles undergo an abrupt transition in internal structure leading to sudden rapid activation of the enzyme.     

Purification and characterization of a new RGD/KGD-containing dimeric disintegrin, piscivostatin, from the venom of Agkistrodon piscivorus piscivorus: the unique effect of piscivostatin on platelet aggregation

Piscivostatin, a novel dimeric disintegrin containing Arg-Gly-Asp (RGD) and Lys-Gly-Asp (KGD) sequences, was isolated from the venom of Agkistrodon piscivorus piscivorus. The molecule consisted of two chains designated as the alpha and beta chains, comprising 65 and 68 amino acid residues, respectively. Piscivostatin had two binding motifs recognized by platelet glycoprotein IIb/IIIa (GPIIb/IIIa), and the biological activity of dimeric disintegrin piscivostatin toward platelet aggregation differed from those of other monomeric disintegrins such as trimestatin and echistatin. We measured platelet aggregation by the laser light scattering method during the process of ADP-induced platelet aggregation. Both dimeric and monomeric disintegrins inhibited the formation of small (9 to 25 microm in diameter), medium-sized and large aggregates (25 to 70 microm in diameter) in a dose-dependent manner. The platelet aggregates disaggregated after reaching a maximal number on either treatment with ADP alone or monomeric disintegrin/ADP. However, the small aggregates did not disaggregate on treatment with piscivostatin/ADP even when applied over time. When washed platelets were incubated with an anti-GPIIb/IIIa monoclonal antibody, PT25-2, which induces conformational changes of GPIIb/IIIa to a form accessible to fibrinogen and other adhesion proteins without platelet activation, piscivostatin induced a platelet shape change alone with no aggregate formation. The present study indicated that piscivostatin has two unique contradictory activities; acting as a double inhibitor of platelet aggregation and platelet aggregate dissociation.