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Evolution of venom variation in the Florida cottonmouth, Agkistrodon piscivorus conanti

Permanent Link: http://ufdc.ufl.edu/UFE0041197/00001

Material Information

Title: Evolution of venom variation in the Florida cottonmouth, Agkistrodon piscivorus conanti
Physical Description: 1 online resource (207 p.)
Language: english
Creator: Mccleary, Ryan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: cottonmouth, diet, drift, evolution, genetics, selection, snake, venom
Zoology -- Dissertations, Academic -- UF
Genre: Zoology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Populations of organisms evolve when they are genetically isolated from each other, have variation within phenotypic characters, and exist in different environments. The evolution of venom in snakes has been examined for many reasons including antivenom research, development of medical research tools, and pharmaceutical drug discovery. Previous studies have disagreed on the manner by which snake venom evolves, however. In this study, we tried to determine whether snake venom is more influenced by natural selection, gene flow, or genetic drift. To do this, we first developed a safe, standardized, and effective method of venom extraction using Propofol anesthetic and an electric nerve stimulator. We then validated the use of previously-published microsatellite primers for use in the Florida cottonmouth, Agkistrodon piscivorus conanti, in order to determine population genetic relationships. Next, we collected 76 venom and 129 deoxyribonucleic acid (DNA) samples from cottonmouths in Florida. The venom samples were collected from live snakes from three different populations, one of which was the focal population of Seahorse Key (SHK). The DNA samples were collected from both live and road-killed specimens from four populations, including SHK. We analyzed the venom for activities of four enzymes: protease, hyaluronidase, L-amino acid oxidase (LAAO), and phospholipase A2 (PLA2). Because the SHK population primarily feeds on fish dropped or regurgitated by colonially nesting seabirds, and because it is located on an island separated from the mainland by a salt water corridor, we expected the population to be genetically isolated and be evolving through genetic drift. Because the other populations are found on the mainland in diverse habitats, and because the species is known to be generalist in prey preference, we expected that venom enzyme activity would be correlated with local prey, indicating natural selection. We found all four populations to be genetically distinct, but with SHK being very different from the rest and likely inbred. We also found some differences in venom enzyme activity with SHK having higher LAAO and intermediate protease and PLA2. These results support the idea that the SHK population is under different evolutionary constraints than the mainland populations.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Ryan Mccleary.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Lillywhite, Harvey B.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041197:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041197/00001

Material Information

Title: Evolution of venom variation in the Florida cottonmouth, Agkistrodon piscivorus conanti
Physical Description: 1 online resource (207 p.)
Language: english
Creator: Mccleary, Ryan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: cottonmouth, diet, drift, evolution, genetics, selection, snake, venom
Zoology -- Dissertations, Academic -- UF
Genre: Zoology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Populations of organisms evolve when they are genetically isolated from each other, have variation within phenotypic characters, and exist in different environments. The evolution of venom in snakes has been examined for many reasons including antivenom research, development of medical research tools, and pharmaceutical drug discovery. Previous studies have disagreed on the manner by which snake venom evolves, however. In this study, we tried to determine whether snake venom is more influenced by natural selection, gene flow, or genetic drift. To do this, we first developed a safe, standardized, and effective method of venom extraction using Propofol anesthetic and an electric nerve stimulator. We then validated the use of previously-published microsatellite primers for use in the Florida cottonmouth, Agkistrodon piscivorus conanti, in order to determine population genetic relationships. Next, we collected 76 venom and 129 deoxyribonucleic acid (DNA) samples from cottonmouths in Florida. The venom samples were collected from live snakes from three different populations, one of which was the focal population of Seahorse Key (SHK). The DNA samples were collected from both live and road-killed specimens from four populations, including SHK. We analyzed the venom for activities of four enzymes: protease, hyaluronidase, L-amino acid oxidase (LAAO), and phospholipase A2 (PLA2). Because the SHK population primarily feeds on fish dropped or regurgitated by colonially nesting seabirds, and because it is located on an island separated from the mainland by a salt water corridor, we expected the population to be genetically isolated and be evolving through genetic drift. Because the other populations are found on the mainland in diverse habitats, and because the species is known to be generalist in prey preference, we expected that venom enzyme activity would be correlated with local prey, indicating natural selection. We found all four populations to be genetically distinct, but with SHK being very different from the rest and likely inbred. We also found some differences in venom enzyme activity with SHK having higher LAAO and intermediate protease and PLA2. These results support the idea that the SHK population is under different evolutionary constraints than the mainland populations.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Ryan Mccleary.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Lillywhite, Harvey B.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041197:00001


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1 EVOLUTION OF VENOM VARIATION IN THE FLORIDA COTTONMOUTH, Agkistrodon piscivorus conanti By RYAN JAMES ROBERT MCCLEARY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Ryan James Robert McCleary

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3 To my parents, Karen Rogers, Dr. Ken Mc Cleary, and Dr. Pamela Weaver, without whose support none of this would have been possible

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4 ACKNOWLEDGMENTS I thank my parents and extended family, w ho supported me throughout this long endeavor. I also wish to thank all those intrepid individuals who forayed into the wild with me to search for venomous snakes, w hether during hot, steamy Florida days, or pitch black, hot, insect-infested Florida ni ghts. Special thanks go to Leslie Babonis for absolutely (almost) everything, Dr. Ke lly Hyndman for unfailing encouragement and numerous reviews, and Kevin Neal for ex treme field support and continued fascination with venom. I would further like to thank all the collaborators and mentors on the various projects that came out of this work, including my major professor, Dr. Harvey Lillywhite; committee members Drs. Martin Cohn, Marta Wayne, David Steadman, and Darryl Heard; all the staff (past and present) and the University of Florida College of Veterinary Medicine, including Dr. Ro lando Quesada and Dahlone ga Peck; A. M. “Ginger” Clark and everyone at the UF Inte rdisciplinary Center for Biotechnology Research (including Dr. D. “G igi” Ostrow, Angela Gomez, Dr Margaret Kellogg, and Dr. Kimberly Pause); Irvy Quit myer and the UF Zooarcheol ogy section of the Florida Museum of Natural History (FLMNH); Drs. Kenney Krysko and Max Nickerson of the Herpetology Department at FLMNH; Dr. Rob Robins of the FLMNH Ichthyology Department; Drs. David Julian, Kent Vliet, and Charlie Baer; my wonderful undergraduate students throughout t he years, especially Vanessa Trujillo and Kristy Staudenmaier; Dr. Stephen Mackessy of the Univer sity of Northern Colorado; Dr. NgetHong Tan of the University of Malaya; vari ous and diverse individuals with whom I have interacted at scientific meetings; and last, but definitely not least, all of the excellent people in the Departments of Zoology and Biol ogy, including the staff and faculty, but especially graduate students, both past and present.

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5 TABLE OF CONTENTS page ACKNOWLEDG MENTS..................................................................................................4 LIST OF TABLES............................................................................................................8 LIST OF FI GURES..........................................................................................................9 ABSTRACT ...................................................................................................................11 CHAPTER 1 INTRODUC TION....................................................................................................13 Venoms, Poisons, and Toxi ns................................................................................13 Snake Ve noms.......................................................................................................14 Venom Delivery Systems........................................................................................23 Variables That May Affect Snake Venom Ev olutio n................................................28 Studies of Venom Va riation—Ge neral ....................................................................29 Studies of Venom Variation— Evolutionary Im plicati ons.........................................30 The Florida Cottonmouth—A Model fo r Study of Venom Evolut ion........................33 2 THE VENOM OF TH E COTTONM OUTH...............................................................36 Introducti on.............................................................................................................36 Human Envenomations by Cottonm ouths...............................................................38 Demographics of Hum an Envenoma tion..........................................................38 General Physiological Effects (Human) ............................................................39 Specific Physiological Effects (Com parativ e)...................................................40 Epithelial tissue..........................................................................................40 Subdermal ti ssue.......................................................................................40 Blood—hemo lysis......................................................................................40 Blood—coagul ation....................................................................................42 Blood—anti-c oagulatio n.............................................................................43 Muscle .......................................................................................................44 Nervous ti ssue...........................................................................................45 Respirator y system....................................................................................46 Circulatory system.....................................................................................46 Immune sy stem..........................................................................................48 Lymphatic system......................................................................................49 Excretory system.......................................................................................49 Treatment of Co ttonmouth Bite........................................................................50 Medical tr eatment ......................................................................................50 Antivenom re search...................................................................................50 Venom-Associat ed Anatom y...................................................................................52 Venom Characte ristics............................................................................................53

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6 Basic Descri ption..............................................................................................53 Volume .......................................................................................................53 Specific gr avity...........................................................................................54 Protein co ntent...........................................................................................54 Glycoprotein content ..................................................................................54 Dry mass....................................................................................................54 Spectrophoto metry.....................................................................................55 Stabili ty......................................................................................................55 Variatio n.....................................................................................................56 Toxici ty.......................................................................................................56 Separation te chniques ...............................................................................58 Specific Co mponents.......................................................................................61 Enzymes....................................................................................................61 Other enzymatic activiti es..........................................................................76 Nonenzymatic prot eins/pept ides................................................................80 Small organic compou nds..........................................................................82 Toxins ........................................................................................................83 Vascular endothelial growth factor.............................................................83 Potential Co mponents ................................................................................84 Venom St udies .......................................................................................................84 Human Disease Clinic al Res earch...................................................................84 Immunol ogy...................................................................................................... 86 Conclusi on..............................................................................................................88 3 VENOM EXTRACTION FROM ANESTHE TIZED FLORIDA COTTONMOUTHS...93 Introducti on.............................................................................................................93 Materials and Methods............................................................................................95 Animal s.............................................................................................................95 Anesthes ia........................................................................................................96 Venom Extr action.............................................................................................97 Demographi c Data...........................................................................................98 Result s....................................................................................................................98 Discussio n..............................................................................................................99 4 CROSS SUBFAMILY AMPLIFIC ATION: A WORKING SET OF MICROSATELLITE PRIMERS FOR USE IN THE FLORIDA COTTONMOUTH..105 Introducti on...........................................................................................................105 Materials and Methods..........................................................................................106 Results ..................................................................................................................107 Discussio n............................................................................................................108 5 POPULATION GENETIC ANALYSI S OF ONE INSULAR AND THREE MAINLAND POPULATIONS OF TH E FLORIDA COTTO NMOUTH.....................113 Introducti on...........................................................................................................113

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7 Materials and Methods..........................................................................................116 Animals ...........................................................................................................116 Analysis ..........................................................................................................117 Results ..................................................................................................................118 Discussio n............................................................................................................119 6 ENZYMATIC ACTIVITY IN THE VENOM OF THE FLORIDA COTTONMOUTH: A POPULATION-LEVEL COMPARIS ON..............................................................127 Introducti on...........................................................................................................127 Materials and Methods..........................................................................................130 Animals ...........................................................................................................130 Venom Preparat ion........................................................................................130 Enzyme Assa ys..............................................................................................131 Results ..................................................................................................................133 Electrophores is...............................................................................................133 Protease .........................................................................................................134 Hyaluronid ase................................................................................................134 L-Amino Acid Ox idase....................................................................................134 Phospholipase A2...........................................................................................134 Venom Correla tions........................................................................................135 Discussio n............................................................................................................135 7 A POPULATION-LEVEL COMPARISON OF THE DIET OF THE FLORIDA COTTONMOUTH, Agkistrodon piscivorus conanti ...............................................144 Introducti on...........................................................................................................144 Materials and Methods..........................................................................................147 Results ..................................................................................................................149 Discussio n............................................................................................................150 8 EVOLUTIONARY IMPLICAT IONS AND SU MMARY............................................156 Introducti on...........................................................................................................156 Quantitative Tr ait Loci...........................................................................................160 Conclusi on............................................................................................................162 LIST OF REFE RENCES.............................................................................................165 BIOGRAPHICAL SKETCH ..........................................................................................207

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8 LIST OF TABLES Table page 2-1 Published records of cottonmouth bites and bite-caused fatalities.....................89 2-2 Published toxicity values for cottonm outh venom ...............................................90 2-3 Reported minimum and mean lethal doses for cottonm outh venom...................92 4-1 Utility of 31 microsatellite pr imers for use with cottonmouths ( Agkistrodon piscivorus )........................................................................................................110 4-2 Information for micr osatellite loci used with Agkstrodon piscivorus conanti DNA.................................................................................................................. 112 5-1 Specifics of microsat ellite amplific ation............................................................122 5-2 Locusand population-specific dat a for microsatellite analysis of cottonmouths ....................................................................................................123

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9 LIST OF FIGURES Figure page 1-1 Phylogenetic relationships am ong venomous snak e familie s.............................35 3-1 Anesthetized Fl orida cottonmouth ( Agkistrodon piscivorus conanti ) in tube with only head prot ruding.. ...............................................................................102 3-2 Anesthetized Fl orida cottonmouth ( Agkistrodon piscivorus conanti ) showing placement of electrodes on the skin surface across the venom gland..............103 3-3 Anesthesia induction in Florida cottonmouths, Agkistrodon piscivorus conanti ..............................................................................................................103 3-4 Mass-specific effects of propofol on induction time in Florida cottonmouths, Agkistrodon piscivorus conanti .........................................................................104 3-5 Mass-specific venom yiel d from Florida cottonmouths, Agkistrodon piscivorus conanti by electrical st imulation. ......................................................................104 5-1 Locations of cottonmout h populations st udied..................................................124 5-2 STRUCTURE population identity for individual cottonmouths in Florida...............125 5-3 TESS Population identity for individual cottonmouths in Florid a........................125 5-4 Spatial and genetic relationships of individual Florida cottonmouths................126 6-1 Map of Florida with populat ion locations in dicated...........................................138 6-2 Electrophoresis banding patterns for i ndividual cottonmouth venom samples. Individual samples were run on 12% acrylamide gels with 2-( N -morpholino)ethanesulfonic ac id running bu ffer.........................................139 6-3 Comparison of mean protease activi ties for three populations of Florida cottonmout h......................................................................................................140 6-4 Comparison of mean hyal uronidase activities for th ree populations of Florida cottonmouth. .....................................................................................................141 6-5 Comparison of mean L-amino acid oxidase (LAAO ) activities for three populations of Flori da cottonmout h...................................................................142 6-6 Comparison of mean phospholipase A2 activities for three populations of Florida cott onmouth. .........................................................................................143 7-1 Four-category diet composition com parison for three populations of Florida cottonmouth. .....................................................................................................153

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10 7-2 Five-category diet composition com parison for three populations of Florida cottonmouth. .....................................................................................................154 7-3 Stable isotope analysis of scale ti ssue from Florida cottonmouths...................155 8-1 QST/FST comparison of enzyme activities for each of three populations of Florida cottonm ouths........................................................................................164

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11 Abstract of Dissertation Pr esented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Doctor of Philosophy EVOLUTION OF VENOM VARIATION IN THE FLORIDA COTTONMOUTH, Agkistrodon piscivorus conanti By Ryan James Robert McCleary December 2009 Chair: Harvey B. Lillywhite Major: Zoology Populations of organisms evolve when they are genetically isolated from each other, have variation within phenotypic characte rs, and exist in different environments. The evolution of venom in snakes has been examined for many reasons including antivenom research, development of medica l research tools, and pharmaceutical drug discovery. Previous studies have dis agreed on the manner by which snake venom evolves, however. In this study, we tried to determine w hether snake venom is more influenced by natural selection, gene flow, or genetic dri ft. To do this, we first developed a safe, standardized, and effective met hod of venom extraction using Propofol anesthetic and an electric nerve stimulator. We then validated the use of previously-published microsatellite primers for us e in the Florida cottonmouth, Agkistrodon piscivorus conanti in order to determine population genetic rela tionships. Next, we collected 76 venom and 129 deoxyribonucleic acid (DNA) samples from cottonmouths in Florida. The venom samples were collected from live snak es from three different populations, one of which was the focal population of Seahorse Key (SHK). The DNA samples were collected from both live and roadkilled specimens from four populations, including SHK.

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12 We analyzed the venom for activities of f our enzymes: proteas e, hyaluronidase, Lamino acid oxidase (L AAO), and phospholipase A2 (PLA2). Because the SHK population primarily f eeds on fish dropped or regurgitated by colonially nesting seabirds, and because it is located on an island separated from the mainland by a salt water corridor, we expec ted the population to be genetically isolated and be evolving through genetic drift. Bec ause the other populations are found on the mainland in diverse habitats, and because the s pecies is known to be generalist in prey preference, we expected that venom enzym e activity would be correlated with local prey, indicating natural selection. We found all four populations to be genetically distinct, but with SHK being very different from the rest and lik ely inbred. We also found some differences in venom enzyme acti vity with SHK having higher LAAO and intermediate protease and PLA2. These results support the idea that the SHK population is under different evolutionary c onstraints than the mainland populations.

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13 CHAPTER 1 INTRODUCTION Venoms, Poisons, and Toxins Toxic secretions of different forms ar e produced by various animal taxa and may be used for acquisition of prey, digestion of prey, and/or defense against potential. Venoms are potentially toxic se cretions produced by one organi sm to the detriment of another, delivered through some form of specia lized morphological adaptation (e.g., fang, stinger, and nematocyst). Poisons, on the other hand, must be ingested (via gastrointestinal tract or skin) to affect the target organism. Both venoms and poisons tend to work in a dose-dependent manner (M ebs, 2002), the main difference between them being the m ode of delivery. Venoms are often confused with toxins, whic h are chemicals of biological origin that can adversely affect the physiology of another organism. Generally, venoms and poisons both contain toxins, but toxins are pure substances, whereas venoms and poisons are mixtures that may include other bi ologically active and inert substances. For a toxic secretion to be considered veno m, there must be an assumption of an ecological function (Kardong 1996). While su ch function is definitely known for the chemical secretions of some animals, it has not been shown for all of them. As an example, the secretion of the cone snail ( Conus catus ) is used to paralyze fish that are then consumed by the snail (Jakubowski et al., 2005). This indicates, at the very least, that the secretion is used for prey acquisiti on and can be considered venom. It does not indicate, however, whether the venom is al so used for prey digestion or predator deterrence. As a further example, m any snakes (such as the cottonmouth, Agkistrodon piscivorus ) use oral chemical secretions from specialized glands to immobilize prey—

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14 this indicates that these oral secretions ar e venoms. These same oral secretions have not been analyzed in many species of snakes (such as the watersnake, Nerodia fasciata ), so there is debate as to whether th ey should be considered venoms (Kardong, 2002). Recent publications have championed t he opinion that all oral secretions with biologically-active compounds developed in specialized glands should be considered venoms (Fry et al 2003a), regardless of other knowle dge as to specific effects. Snake Venoms Among venomous animals, snakes are of part icular interest for many reasons. Approximately 2700 species of extant snakes currently are recognized, divided into 17 families (Pough et al., 2001), the phylogeny of which has not been conclusively determined (Lawson et al., 2005; Vidal et al., 2007). Four of these families (Atractaspididae, Colubridae, Elapidae, and Viperidae) compose the traditional taxon superfamily Colubroidea (Figure 1), which contains most (approximately 2430) species of snake (Cadle, 1988; Kraus and Brown, 1998; Pough et al., 2001; Slowinski and Lawson, 2002; Vidal, 2002; but see Lawson et al., 2005, Vidal et al., 2007, and Zaher et al., 2009 for recent revisions). All venomous snake species belong to the Colubroidea, and it is generally held that venom arose in the ancestor of th is clade followed by alterations in venom composition, glandu lar morphology, and tooth shape (Vidal, 2002; Jackson, 2003). However, recent research has shown an earlier reptilian origin of venom, with venom being found in multiple liza rd families, indicating that only the type of modified venom apparatus (but not venom itself) may be unique to the Colubroidea (Fry et al., 2006). Further confusing the issu e, however, is the absence of venom in all of the snakes basal to the Colubroidea, which may indicate multiple loss and gain events throughout snake evolution.

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15 The total number of venomous snake specie s is not currently known, as many species that have traditionally been cons idered harmless have not been analyzed for the presence of venom. The discovery of bi ologically-active oral secretions from “harmless” colubrid snakes has had tragic endings in the cases of Dr. Karl P. Schmidt of the Field Museum of Natural History, who was killed from the bite of a boomslang ( Dispholidus typus ; Pope, 1958), and Dr. Robert Mehrtens, who was killed by the bite of a twig-mimic snake ( Theoltornis capensis ; Greene, 1997). Histo logical studies have indicated the presence of venom glands in species considered to be without venom (Taub, 1967), and studies of gene expression hav e shown production of toxins from oral glands in snakes considered harmless (Fry et al., 2003b). The increase in popularity of herpetoculture will likely increase the number of snake species known to be venomous. Variation in venom, both qualitatively and quantitatively, is considerable and occurs at almost every taxonomic level fr om the individual (i.e. ontogenetic shift: Mackessy, 1988) to the family level (among fam ilies of the Colubroidea; Chippaux et al., 1991). In general, snake venoms consist mostly of soluble polypeptides in serous or mucus secretions, but may also include ca rbohydrates, lipids, metal ions, and other organic compounds, including amines (Mebs, 2001) and purines (Aird, 2002). Up to 90% of the dry weight of most venom is comp rised of polypeptides of three size classes: low molecular weight components (< 1.5 kDa) polypeptide toxins (5 to 10 kDa), and enzymes (10 to 150 kDa) (Hider et al., 1991) One individual snake may secrete venom that contains numerous chemical co mpounds from all three categories. Venom can affect a wide range of physiol ogical functions in the envenomated organism, and each individual venom component may have a unique function. Most

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16 snake venoms contain phospholipases A2 (PLA2), cell-destroying enzymes that can be edema-inducing, lipolytic, or myolytic, depending on their specific three-dimensional structure (Chijiwa et al., 2003; Lomonte et al., 2003a). They may also be chemically altered to become toxins that can block ac etylcholine release fr om the presynaptic motor end plate (Mebs, 2002). Other com pounds that degrade ubiquitous biological structures, such as proteas es that degrade peptides or hyaluronidases that destroy hyaluronic acid, are also often found in venoms. Some venoms contain components that affect specific physiological functions, such as enzymes that either potentiate or prevent part of the mammalian blood clo tting cascade (Mebs, 2002), while others contain functionally-diverse mixtures of components. Some snake venoms may cause localized swelling in humans, whereas other s cause rapid death through loss of neural function. Components of viperid venom include a wi de array of proteins with enzymatic activity and non-enzyme polypeptides. Am ong the enzymes, the major groups are phosphodiesterases, 5’nucleotidases, alka line phosphomonoesterases, hyaluronidases, L-amino acid oxidases, metalloproteinases, serine proteases, arginine esterases, and phospholipases A2 (PLA2). Among the non-enzymatic polypeptides are cysteine-rich secretory proteins (CRiSPs), nerve growth factors, PLA2-based neurotoxins, non-PLA2 myotoxins, C-type lectins, disintegrins, brad ykinin potentiators, and tripeptide inhibitors (Mackessy, 2009). This is not an exhaustive list of all venom component groupings, as some venom constituents have not as of yet been categorized. Indeed, new venom components are being described often (Pawlak et al., 2006; Nair et al., 2007), and the venom from many species has not been thoroughly studied.

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17 Most venom enzymes work by hydrolyzi ng biomolecules in the prey, thereby reducing function and altering normal homeos tasis of the envenomated organism. These enzymes are usually examined in one of two ways: either they are isolated and characterized for their specific activity (enzyme-based study) or they are examined for their effect on a specific substrate (substrat e-based study). This can lead to confusion as one enzyme may affect multiple subs trates and may also have more than one moniker, depending on the manner in which it has been studied. Further, there may be overlapping activities from many different enzyme groups There are, however, generalities in terms of the chemical func tion and biological activity of many enzyme groups. Phosphodiesterases, as their name im plies, hydrolyze phosphodiester bonds. This catalysis is mostly seen as a degradatio n of nucleic acids, including DNA and two ribonucleic acids (RNAs), ribosomal RNA and transfer RNA, but the enzymes can affect many other nucleotides and nucleic acids as well (Dhananjaya et al., 2009). Although the overall effect has not been completely determined, it appears that the depletion of such nucleotides results in hypotens ion and/or shock (Mackessy, 1998). 5’nucleotidases attack nucleic acid at the 5’ carbon position, degrading the sugar moieties of both DNA and RNA (Dhananjaya et al., 2009; Rael, 1998). The overall effect of 5’nucleotidases is to release nucleosides fr om nucleic acids (Macke ssy, 2009). A third enzyme group that affects nucleic acids is the alkaline phosphomonoesterases, which hydrolyze phosphomonoesterases at pH abov e neutral (Dhananjaya et al., 2009; Rael, 1998). The biological effect of the alka line phosphomonoesterases is uncertain, but

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18 these three groups of enzymes attack nucleoti des in different manners, and all three are fairly ubiquitous in viperid venoms (Dhananjaya et al., 2009). Hyaluronidases are also commonly call ed “spreading factors” because of their capacity to degraded hyaluronic acid. Hyaluroni c acid is a ubiquitous component of the extracellular matrix of tissues, and is at l east partially responsible for cementing cells together. Hyaluronid ases have been found in all elapid and viperid venom examined thus far (Girish et al., 2002) and although they appear to be at higher activity levels in the elapids, there is a broad r ange of activity throughout the vi perids. When hyaluronic acid is degraded by hyaluronidase, the remaining venom components are able to spread through the tissue, since their movement is delimited. This can lead to localized necrosis, as nearby cells are destroyed by other venom componen ts, or to systemic effects, as other venom components spread into blood vessels made “leaky” by the hyaluronidase. L-Amino acid oxidases (LAAOs) are the majo r exception to venom enzymes in that they catalyze the oxidation of L-amino acids through a twostep deamination process (Chippaux, 2006). The result of the deamination is a gener al degradation of amino acids that may result in cell dama ge or apoptosis (Tan and Fung, 2009). Proteolytic enzymes are those enzymes t hat lead to the degradat ion of structural proteins into component pepti des or amino acids. They have great digestive capability and can hydrolyze proteins in their nativ e (non-denatured) stat e through cleavage of peptide and ester bonds (Mebs, 2002). Some of these enzymes have been categorized as metalloproteinases (because of their reliance on metal ion co-factors) and serine proteases (because of their similarity to blood factors). Metalloproteinases cause

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19 hemorrhage and necrosis, but may also be respons ible for digestion of prey. Serine proteases disrupt hemostasis and may do so through multiple mechanisms that disrupt the blood clotting cascade (Marsh, 1994; Mackessy, 2009). Serine proteases are generally placed into three categories, depending on their mode of action. The thrombin-like serine proteases cleave fibrinogen at the same location as thrombin, leading to a rapid deplet ion of fibrinogen. The resulting fibrin, however, is not coagulable due to a lack of fibr in stabilizing factor leading to an overall anti-coagulation effect and possible circulatin g clots in the blood stream (Marsh, 1994; Markland, 1998; Swenson and Markland, 2005). Kallikrein-like serine proteases cause release of bradykinin from high molecula r weight kininogen and degradation of angiotensin. The result of both of these is a precipitous dr op in blood pressure (Nikai and Komori, 1998). Although they have enzymatic activities against peptide and ester substrates, the biological effects of arginine estera ses are not fully understood (Mackessy, 2009). PLA2s are a very interesting group of enzymes found in almost all venomous species, including viperids, elapids, and some colubrids. In general, PLA2s are active in destroying the phospholipid layer of cells. However, it is possible for very small changes in the structure of PLA2s to cause a change in its physiological activity (Chijiwa et al., 2003; Kini and Chan, 1999). As previously noted, PLA2s can be edema-inducing, lipolytic, myolytic, or acetylcholine releas e inhibiting (Mebs, 2002). The diversity of function and apparently wide stru ctural variation of PLA2s makes them interesting for studying molecular evolution (Chijiwa et al ., 2003; Creer et al., 2003; Li et al., 2005).

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20 The study of snake venom CRiSPs is fairly new, with most work being done in the last 15 years. CRiSPs have been found to have diverse functi ons in different species, ranging from disruption of potassium or ca lcium currents in neurons to induction of hypothermia (Yamazaki and Morita, 2004; Heyborne and Mackessy, 2009). The functions of many similar CRiSPs in other species, however, have not been elucidated. Nerve growth factors stimulat e growth of nerve cells and are found in both viperid and elapid venoms. The actual mechanism of function in prey is not fully understood, nor is the biological function (Kostiza and Meier, 1996), and the number of studies has been limited. PLA2-based presynaptic neurotoxins functi on by blocking acetylcholine release from axon terminals, thereby causing a fl accid paralysis. These have very strong neurotoxic functions, and they include the Mo jave toxin found in some rattlesnake species (John et al., 1994). Other viperid toxins, the non-PLA2-based myotoxins, disrupt voltage-sensitive sodium channels leading to prey immobilization and myonecrosis (Mackessy, 2009). C-type lectins and disintegrins both effect ively alter the blood clotting cascade, but in different manners. C-type le ctins bind to platelets, and th is can either cause initial clotting, or it can cause platelets to be removed from forming clots, depending on the specific form of the lectin (Komori et al ., 1999; Du and Clemetson, 2009). Disintegrins act by disrupting platelet aggregation, and they are often found grouped with a metalloproteinase (the comb ination being called an ADAM—A D isintegrin a nd M etalloproteinase). Some disintegrins halt initial aggregation, wh ile others work by disrupting formed aggregates (Okuda and Morita, 2001).

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21 Bradykinin potentiating peptides (BPPs) functi on by effectively being an inhibitor of angiotensin converting enzyme (ACE). ACE is responsible for converting angiotensin I into angiotensin II, and angiotensin II causes vasoconstriction. Besides blocking ACE activity, BPPs may have a direct effect on a ngiotensin II, but both actions lead to a reduction in vasoconstriction, allowing bradyki nin to cause vasodilation and hypotension (Ferreira et al., 1995, 1999). Besides substances that are effective biologically against prey, some snake venoms also contain factors that appear to help stabilize these other components. These include tripeptide inhibitors (Francis and Kaiser, 1993) and citrate (Freitas et al., 1992). These apparently work by inhibiting th e enzyme interactions in the venom, so that the biological functions do not occur until the venom has been injected into potential prey and dispersed. There are many other venom component s that have been characterized from species outside of Viperidae. These includ e many other toxins, some blood clot disruption factors, and some enzymes. I ndeed, colubrid venoms have just recently become the subjects of many studies (Ma ckessy, 2002), and the total diversity of components has probably only been superficially recognized. As the focus of this introduction is on a viperid snake, non-vipe rid venom component categories will only be described briefly. Toxins are a large and diverse group of non-enzymatic peptides that have various functions. They normally affect the ner vous system by alte ring presynaptic or postsynaptic functions at a neuromuscular juncti on. Toxins that affect these junctions presynaptically have two primary functions: they can interfere with the release of

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22 acetylcholine from the nerve endplate, or they can block specific ion channels, thereby blocking repolarization and increa sing the amount of acetylcholin e released. In the first case, nerve signals are not fully propagated to t he motor unit, so that a flaccid paralysis occurs; in the second case, signals may be propagated unintentionally or with uncontrolled rapidity leading to spasms and possible tetany. Other toxins affect neuromuscular junctions postsynaptically by bi nding to acetylcholine receptors, thereby blocking acetylcholine uptake and leading to paralysis. Wh ile presynaptic toxins are found in both viperids and elapids, they are much more prevalent in elapid venoms and cause a much greater response in prey. Postsynaptic neurotoxins are predominantly of elapid origin. Another group of toxins, the cardiotoxins, are found primarily in elapids, but may be in smaller concentrations in viperids. T hese toxins can cause hemolysis of red blood cells, lysis of other cells, depolarization of excitable membranes, and activation of phospholipases A2 into toxins. There are other venom components that ar e mostly limited to elapids. These include acetylcholinesterases and fasciculin s. It is logical to assume that acetylcholinesterases function to degrade acet ylcholine at the neuromuscular junction (as endogenous forms do). However, in venom these enzymes are so large that they do not seem to actually reach the site w here they could do the most harm (i.e. the neuromuscular junction; Mebs, 2002), so th eir actual effect is not known. Acetylcholinesterases have been found in col ubrid venom, as well (Mackessy et al., 2006). The fasciculins have the opposite effect on acetylcholine transmission, as they are potent inhibitors of acetylcholinesterase. By reducing acetylcholinesterase activity,

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23 more acetylcholine travels across t he neuromuscular junction and is detected postsynaptically. This can cause spasms and tetany. The diversity of venom components, bot h structurally and functionally, is enormous, and is only touched upon here. O ften venom components are categorized by function, whereas they are at other times categorized by structure. This leads to much confusion as components of similar st ructure can cause different effects, and components of different structure can cause similar effects. Al so, the number of distinct enzymes and toxins known is increasing, as is the number of known unique toxin families. All of this makes for an interesting and dynamic field of study. Venom Delivery Systems In snakes, effective use of venom is accomplished by the venom delivery system (VDS). The VDS includes the venom, an associated gland used for production and storage, and specialized teeth used for delivery. The evolution of components of VDSs, including a presumably co-opt ed salivary gland, allowed for the improved success of snakes because species with these adaptations were more readily able to capture and subdue prey, some of which may have been potentially harmful to the snake. Venomous snakes can either envenomate prey, without any further contact, and consume it after it has died, or they can envenomate the prey while grasping it. In the first case, snakes need not be in contact with prey item except fo r initial envenomation and final consumption. In the second case, t he venom reduces the time the snake is in contact with the prey item (Shine and Sc hwaner, 1985); this is important because struggling prey can injure or kill snakes. Some species even have the capacity to track envenomated prey through the unique chemical si gnature of the venom (Furry et al.,

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24 1991; Lavin-Murcio et al., 1993). Reduced pr ey handling time reduces energy spent struggling with prey and significa ntly decreases the potential of injury to the snake. Since the origin of a rudimentary venom gland, the basic pattern has been altered in different snake lineages. Four venom gland morphologie s typically are recognized, based on traditional phylogeny: 1) the atra ctaspidid type; 2) the colubrid type (historically referred to as Duvernoy’s gland) ; 3) the elapid type; and 4) the viperid type. There is, however, high variability among t he morphologies of different species within one family, with the most consistent mor phology found in the Viperidae and Elapidae, and the least consistent in the Atractas pididae and Colubridae (Kochva and Gans, 1970). In general, the atractaspidid venom gl and is large, tubular, and elongated posteriorly behind the eye (well beyond the head in some species, such as Atractaspis engaddensis (Kochva, 1987)). It comprises most ly unbranched tubules radiating from a central lumen. These glands are not asso ciated with an accessory gland, but mucosal cells line the major portion of the length of th e lumen. Also, the terminal duct of the gland empties venom into anterior, tubul ar fangs (Kochva et al., 1967). In atractaspidids, venom appears to be forcibly ejected through the compression action of an adductor externus medialis muscle that extends from the parietal bone around the posterior end of the gland to the cor ner of the mouth (Jackson, 2003). The venom gland of colubrid snakes, if pres ent, is relatively small in most species and also is located posteriorly and ventrally to the eye (Ovadia, 1984, 1985). This gland is somewhat oval in morphology, is latera lly compressed, and is considered to be the ancestral state of the Colubroidea (Vidal, 20 02; Jackson, 2003). In Boiga irregularis

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25 one of the few species in which it has been examined, the gland is multi-lobed, with secondary branching occurring within each lobe (Zalisko and Kardong, 1992). The gland is usually encased in a thin connective ti ssue layer, and consists mostly of serous cells. There is a small central cistern that is analogous to the lumen of other venom glands, but is greatly reduced in com parison (with exceptions such as Dispholidus typus and Thelotornis capensis— Greene, 1997). One main duct em pties the central cistern into the oral epithelium near the corner of the mouth. No accessory glands are associated with the colubrid venom gl and, and generally there are no associated compressor muscles. Taub (1967) noted that Duvernoy’s glands contain either purely serous cells (as in Boiga dendrophila Diadophis punctatus Erythrolamprus bizona and others), or a mixture of ser ous and mucosal cells (e.g., Nerodia sipedon and Thamnophis elegans ), with mucous cells being found in the duct (Kochva, 1987). Elapid snakes have an oval-shaped serous gland that is associated with a mucous accessory gland. The gland is located ventrally and posteriorly to t he eye, with the main part of the gland composed of tubules with simple or complex branching patterns. These tubules empty into a narrow lumen that is surrounded by the accessory gland. After passing through the accessory gland, venom is transported to the anterior fangs. A split adductor externus superficialis muscle that exerts force on the dorsal and ventral aspects of the gland forcibly ejects content s of the gland. It appear s that in elapids venom is stored mostly within cells, ra ther than in the lumen (Jackson, 2003). The viperid type of venom gland is the most widely studied, and appears to have the most conserved morpholog y of the families of the Co lubroidea (Kochva and Gans, 1970). It is a large, triangular (in profile) gland that is divided into several lobules by

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26 folds in the surrounding tissue (Kochva, 1987) The accessory gland is located just posterior to a secondary duct that empties into large, grooved fangs at the anterior end of the mouth. Between the mucosal access ory gland and the main serous venom gland is a primary duct. This duct is connected to a large lumen fed by multiple, complex branched tubules. The posterior end of the gland expands dorsa lly to the level of the eye. The large store of v enom in the viperid gland lum en is ejected by a complex pattern of musculature that includes the adductor externus profundus muscle (also often termed the compressor glandulae; Jackson, 2003). Along with these gland types, multiple form s of associated teeth are used to aid in venom delivery by rupture of the skin of the prey and/or c onduction of the venom along dental grooves. Tooth types include: 1) aglyphous—small, ungrooved teeth throughout the oral cavity; 2) opisthoglyph ous—small (but larger than other teeth in the oral cavity), grooved teeth located in the dorsa l, posterior aspect of the m outh in the proximity of the duct that empties the venom gland; 3) proteroglyphous—l arger, fixed fangs located anteriorly on the maxilla that have deep grooves (forming partially to fully enclosed tubes in some species); and 4) solenoglyphou s—very large, grooved, rotating fangs that are located anteriorly on the maxilla and fold against the roof of the mouth when it is closed. Examples of venom-producing agly phous snakes include many colubrids (such as Natrix tessellata (Ovadia, 1984), Spalerosophis cliffordi (Ovadia, 1985; Rosenberg et al., 1985) and Thamnophis sirtalis (Kochva, 1965)) as well as some atractaspidids and (under the phylogeny of Laws on et al., 2005) elapids. Opisthoglyphous fangs are seen in many colubrid snakes, including Boiga irregularis (Zalisko and Kardong, 1992) and Telescopus fallax (Kochva, 1965). The proteroglyphous condition is seen in the snakes

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27 of the Elapidae. All viperids (such as Causus rhombeatus —Shayer-Wollberg and Kochva, 1967) are solenoglyphous, as are some atractaspidids (such as Atractaspis engaddensis —Kochva et al., 1967), although the groups have different fang morphology. A recent examination of t he evolution of different f ang types indicates that the ancestral state in snakes is to have no di fferentiation of teeth along the entire dental lamina (i.e. no fangs). Next, a secondary dental lamina, located in the posterior region of the oral cavity, allowed fo r elongated and specialized teeth to form in proximity to the venom gland. More recently, in what appe ars to be two independ ent occurrences, the anterior dental lamina that orig inally gave rise to normal t eeth was lost in both elapids and viperids. This allowed fangs to be produc ed in the anterior part of the oral cavity— in neonates of these species the fang originates at the posterior aspect of the oral cavity and then migrates to the anterior positi on during ontogeny (Vonk et al., 2008) Venoms historically have been considered either hemotoxic (affecting the blood) or neurotoxic (affecting the nervous syst em), although those terms do not adequately describe venom complexity. Indeed, a si ngle venom sample may contain compounds with both general functions (e.g ., crotaline snakes of th e western United States—Glenn et al., 1983; Glenn and Straight, 1985, 1989, 1990; Snchez et al., 2005). Further, the use of these two terms oversimplifies the action of a mixture that may attack many aspects of physiology other than the circulatory and nervous systems. The physiological functions of snake venoms are di verse and vary by species. Variation within venoms can be found at many taxonomi c levels, and is a worthwhile topic for study not simply because of potentia l human health consequences (antivenom and

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28 pharmaceutical research), but also bec ause it can be used to examine interand intra-specific mechanisms of evolution. Variables That May Affect Snake Venom Evolution Many factors may potentially affect the evolution of snake venom. First among these is the ability to capture prey easier through the use of venom It is easy to imagine that an ancestral specie s of snake with a salivary toxi n affecting its prey would be more able to subdue prey than snakes lacking this toxin. This increased prey capture ability could translate into increas ed reproductive success by allowing for rapid growth and enhanced body condition. Rapid grow th would allow snakes to avoid some predators because the snakes would reach la rger sizes in less time. Enhanced body condition could lead to increased fecundity in females, increased mating success in males of species with male-male combat, and increased male mating success, in general, through differential selection by females. The ability to obtain prey more easily allows organisms to allocate more ener gy toward reproduction, and less toward foraging. The ability of the venom to affect different prey specie s must also be considered. To be successful for prey capture, venom must either contain toxins that are effective toward specific prey species or are general enough to subdue multiple prey species. It is possible that snake species with many di fferent types of toxins (and thus greater variation) would be more able to obtain prey simply by increasing their prey pool. On the other hand, it is also possible that s nakes could become more specialized (and thus have less variation) by utilizing an unexploit ed food source. If this were true, venom variation should be maintained in generalis t feeders and reduced in specialists.

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29 The relative danger of potential prey coul d also influence the evolution of venom composition. If a snake has a prey pool that includes species that can cause the snake physical harm, then more rapidly-acting v enom would decrease injury. Conversely, species that consume prey lacking dangerous defenses would likely evolve less toxic venom, assuming that there is a cost of production of strong venom (Li et al., 2005). As the venom would be less toxic, species consuming defenseless prey should have less venom variation than species consuming potent ially harmful prey. This lower variation would be due to loss of toxic venom components. The energetic cost of production and ma intenance of venom has not been studied thoroughly, but has been shown to affect metabolic rates in recently-milked snakes (McCue, 2006). If venom has a high energetic cost, th en it should be produced only when it confers an advantage to the snake s pecies possessing it. A high energetic cost should also reduce the variation of the venom, as wider variation in chemical production would lead to an unnecessary increase in metabo lic cost by co-opting multiple synthesis pathways. On the other hand, if venom is not energetically costly, no such constraint should exist, and individual venoms could easily contain a high diversity of components. Of course, different venom co mponents could have different me tabolic costs, confusing the issue even further. Studies of Venom Va riation—General Chippaux et al. (1991) revi ewed research on venom variation and the techniques used to analyze it. The review indicated vari ability in venom composition at multiple taxonomic levels, and gave many examples fr om each taxonomic level. Interest in venom variation has not diminished. Differe nces in venom enzymatic activities (Tan and Ponnudurai, 1991, Cavinato et al., 1998, M onteiro et al., 1998, Otero et al., 1998,

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30 Prasad et al., 1999, Lpez-Lozano et al., 2002, Shashidharamurthy et al., 2002, Saldarriaga et al., 2003, Ba lija et al., 2005) and protein co mposition (Straight et al., 1991, Daltry et al., 1996a, Forstner et al., 1997, Cavinato et al., 1998, Monteiro et al., 1998, Rodrigues et al., 1998, Prasad et al., 1999, Saravia et al., 2002, Shashidharamurthy et al., 2002, Creer et al., 2003) have been shown within a single species. These differences were ontogenetic (Monteiro et al., 1998, Andrade and Abe 1999, Lpez-Lozano et al., 2002, Saravia et al., 2002, Saldarriaga et al., 2003), intraspecific (Tan and Ponnudurai, 1991, Montei ro et al., 1998), and interpopulational (Straight et al., 1991, Daltry et al., 1996a, Forstner et al ., 1997, Cavinato et al., 1998, Otero et al., 1998, Prasad et al., 1999, Fr y et al., 2001, Saravia et al., 2002, Shashidharamurthy et al., 2002, Creer et al., 2003, Wickramar atna et al., 2003, Balija et al., 2005). Studies of Venom Va riation—Evolutionary Implications Previous work has examined the corre lation of venom components with other parameters. In an attempt to determine how venom evolves, Daltry et al. (1996b) studied the Malayan pitviper ( Calloselasma rhodostoma ) in the Malay Peninsula (Vietnam, Thailand, and Malaysi a) and Java (Indonesia). They visualized whole protein composition of venom from adult snakes us ing isoelectric focusing and compared it to phylogenetic relatedness, geogr aphic proximity, and local prey type. Relatedness was determined by using restriction-fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA). Prey type wa s determined by exam ination of fecal samples from wild snakes combined with gu t content analysis of museum specimens from known localities (Daltry et al., 1998).

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31 Daltry et al. (1996b) considered thr ee general hypotheses to explain venom variation over their area of study: 1) geographically cl ose populations have similar venom composition based on gene sharing th rough local mating or local environmental conditions, indicating that gene flow is a major factor; 2) similarities are based on phylogeny, so that populations with recent common ancestors would have more similar venom composition, indicating that genetic dri ft is in operation; or 3) variation in local diet explains venom variation because venoms more effective toward locally abundant prey are selected over less locally-effective v enom, indicating that natural selection is in action. Their analysis indicated that venom composition was more closely correlated with local prey type than wit h geographic proximity or phy logeny, and led them to suggest that natural selection was acting th rough local prey type as the prime factor influencing venom composition ov er gene flow and genetic drift. Sasa (1999a, 1999b) expressed skepticis m about this result, and postulated a fourth possibility—that venom composition variation has no adaptive value. Under this hypothesis, variation in venom composition does not causally affect fitness and should not show any correlation. In contrast to Daltry et al. (1996b) is a study of Notechis ater niger by Williams et al. (1988), where venom samples from populations of this Australian elapid snake were subjected to gel filtration chromatography and SDS polyacrylamide gel electrophoresis (SDS-PAGE). Twenty different SDS-PAGE bands were present in venoms from snakes throughout the region, but differ ed by population. In short, the 12 different populations logically grouped into 7 different elution patte rns. The populations of similar banding pattern were not correlated with available pr ey or with local environment, but rather

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32 geographic distance. T he populations studied all were in sular, and their venom profile was also correlated with time since separati on from nearby landmasses, as determined by water depth. The authors thus concluded t hat variation in venom composition in this species was more directly linked to vicari ance events followed by genetic drift than to any selective pressures. Creer et al. (2003) examined the viperid Trimeresurus stejnegeri in Taiwan to determine correlations among venom compos ition (as determined by mass-to-charge peak profiles using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)), genetic relatedness (as determined by mtDNA analysis), geographic location, and diet (based on dissections of field-collected snakes (Creer et al., 2002)). They noted that venom composition was not tightly correlated with phylogeny, and postulated that variation in venom composition may be caused by natural selection due to regional diets or by founder effects. The i dea that evolution of venom composition may be influenced by differ ent modes of evolution under different environmental conditions has not been directly examined. More recently, Barlow et al. (2009) found that venom toxicity was correlated with the relative amount of in vertebrate prey upon which sn akes of the viper genus Echis fed. They noted that ther e was great variation in v enom composition among the species examined, and that the same was tr ue for diet variation. However, those species that were known to feed to a great er amount on scorpions had a venom toxicity that was much greater against scorpions th an other species (analyzed via lethal dose comparisons). This study indicates natural selection of venom components that are useful against specific prey types.

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33 To fully determine how venom is evolving in snakes, it is necessary to separate out the relative importance of the different modes of evolution. These may be different under various ecological conditi ons, but they repr esent a first step towards predicting evolutionary trajectories. Recent work (Reza et al. 2006) has indicated that gene duplication with resulti ng neoor sub-functionalization ma y be responsible for adding to the variation found within venoms. This study, those examining novel venom components, and those analyzing differences in venom activity within and among populations indicate the vast diversity of venom components. The Florida Cottonmouth—A Model for Study of Venom Evolution The Florida cottonmouth snake (also often referred to as the water moccasin), is a viperid snake common throughout Florida. The species has been well studied (Gloyd and Conant, 1990) and its venom has been t he subject of numerous studies (see Chapter 2). It makes a good m odel species for examinations of venom evolution due to its abundance, well-described ecology, and rich venom lit erature. Although this species is considered conti nuous throughout its range, it is mostly associated with fresh water habitats, so it tends to have local abundance in favorable habitat. Furthermore, there is one populatio n on the island of Seahorse Key that appears geographically isolated a nd has a very unique ecology. Snakes on this island subsist mostly on fish that are dropped or regurgitated by colonially-nesting seabirds (Wharton, 1969; Lillywhite and McCleary, 2008). This seasonally abundant food source raises questions as to how diet may affect venom composition. As a comparison, snakes on the mainland are known to feed on a wide range of prey and must either actively forage or use sit-and-wait tactics to catch prey. They are also known to be scavengers.

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34 The dichotomy of mainland versus island snakes offers a chance to compare the relative influence of prey on venom compositi on in the cottonmouth. It also affords the opportunity to characterize natural variati on in phenotypic venom characteristics among natural populations. As natural variation is one factor necessary for evolution to occur, simply cataloging any occurring variation is ve ry important. By c haracterizing interand intra-population genetic relat edness, venom characterist ics, dietary breadth, and correlations among these, it should be po ssible to better understand the manner by which these populations, in general, and thei r venom, in particular, are evolving.

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35 Figure 1-1. Phylogenetic relationships among venomous snake families. This phylogeny, adapted from Pough et al. (2001), shows the traditionally accepted relationships of the advanc ed snakes, although there is much current dissent in the lit erature. Many basal fa milies have been excluded as they do not produce venom. Number s shown indicate the taxa: 1) Colubroidea, 2) Caenophidia, and 3) Macrostomata. Atractaspididae Elapidae Colubridae Viperidae Acrochordidae Tropidophiidae Xenophidiidae Bolyeriidae Boidae Loxocemidae Xenopeltidae 1 2 3

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36 CHAPTER 2 THE VENOM OF THE COTTONMOUTH Introduction The toxic secretions of venomous snakes have been studied due to their importance in diagnosis and treatment of snak ebite symptoms. Although not as toxic to humans as related rattlesnakes, the cottonmouth is responsible for both lethal and non-lethal bites throughout its ra nge. It is one of the most common snakes in the state of Florida (pers. obs.), and is likely to co me in contact with humans in any area where both species utilize resources, which includes most fresh water habitats. In general, cottonmouth envenomation in humans causes local tissue damage including direct hemolysis, hemorrhaging, and disruptions in the blood clotting cascade. Local tissue damage can be caused by the combined effects of many different enzymes, including phosphodiesteras es, 5’-nucleotidases, alkaline phosphomonoesterases, L-amino acid oxidases, metalloproteinases, arginine esterases, and phospholipases. The enzym ology of cottonmouth venom has been examined both in terms of the effects of sp ecific venom enzymes (enzyme-based study) and the overall effects of either crude or separated fractions of venom on potential substrates (substrate-based st udy). While enzyme-based study is difficult because it requires pure enzyme, it results in data specific to one compound, although this may obscure potential synergistic reactions with other venom or target components. Substrate-based studies can indicate over all effect on one substance, but without immediate determination of t he responsible enzyme or enzymes. In some cases, multiple enzymes, cofactors, and/or s ubstates combine to cause an effect.

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37 One enzyme that has been extensively studied in cottonmouth venom is phospholipase A2. This enzyme has been examined for its effects on target tissues, but the specific forms found in cottonmout h venom also have been used as models for studies of interfacial lipid chem istry. Further, cottonmouth PLA2s have been used as a tool in examining dynamics of nerves and cell clusters. Physiological research using cottonmouth venom has led to examinations of its multiple modes of action in different spec ies. Among these examinations are numerous studies of treatments for va rious human blood diseases. Besides human testing, other animal species have been utilized as models to examine local and systemic physiological effects of the venom. Further, cottonmouth venom has been util ized to explore ecological and evolutionary questions. Such explorations in clude examinations of the effects of venom on chemosensory ability in snakes, comparis ons of venom composition among related species, and ability of potential predators and prey to withstand envenomation. Recent research utilizing aspects of cottonmouth venom have focused on examinations of novel components, such as vascular endothelial growth factor. Still other studies are beginning to examine t he shear diversity of venom components expressed utilizing modern s ophisticated biochemical and molecular techniques. The study of cottonmouth venom has mirror ed that of the study of snake venoms in general, and has a rich literature reachi ng back over 150 years (see Ingalls, 1843). While this research has answered a lot of questions and garnered much data, there is still much to be studied. This review is specifically concerned with research on venom of the cottonmouth, Agkistrodon piscivorus and is not an attempt to explain everything

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38 that is known about snake veno m. In some cases, extr a information from the general literature is included for clarity. This is an attempt to be as complete as possible, but the shear volume of work done thus far makes it likely that some ancillary research may have been missed. Human Envenomations by Cottonmouths Demographics of Human Envenomation Cottonmouths are occasionally res ponsible for human envenomations, and although comprehensive information is lacking, many studies have examined cottonmouth bites in terms of frequency, lo cation, and other demographic categories. Interpretation of studies reporting snake bite s is difficult because of the potential for improper identification of species, so most information must be considered carefully. For many of these, snakebite treatment is not completely standardized, and each case may have different data collected. Sometimes snake bite data are r eported as a group, regardless of species or demographic informati on concerning the afflicted individuals. Often, bite location (anatomical and/or geographi c) is not reported or the time frame of the study is not given (Snyder and Knowles, 1963; Parrish et al., 1965). However, there are usable data in almost ever y reported study. One notable exception to this rule was the case of a human bitten by a “moccasin,” in which the patient recovered after the anuses of two live chickens were used to “draw out” the venom (both chickens died) and the patient was prescribed ammonia as medication (Ingalls, 1843). Table 2-1 shows a comparison of published studies and indicates (where possible), number and overall percentage of snake bites attributed to cottonmouths, as well as reports of mortalities for the specific ti meframe and geographic region covered.

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39 Photos of cottonmouth envenomation in humans have been published (Snyder et al., 1968, 1972). Also, detailed case studies of humans bitten by cottonmouths have been described (Parrish, 1967; Henderson and Dujon, 1973; Roberts et al., 1985). General Physiological Effects (Human) In cases of humans bitten by cottonm ouths, the symptoms range from nothing (likely due to lack of venom injection) to death. Case studies of such instances indicate a general sequence of events, but there may be variation depending on the site of the bite, amount of venom injected, and the treatment rendered fo llowing the bite. Prior to the development of effective antivenom tr eatment, symptoms and sequelae were much harsher than in modern bite s treated with advanced techni ques. Reports of these previous instances give an indication of t he general sequence of events in humans with untreated bites. After envenomation, there is severe pain at the site of the bite. This pain is followed by localized edema and erythema which continues to get worse without treatment (Parrish, 1967) and can lead to compar tment syndrome (Roberts et al., 1985). If the venom spreads throughout the circ ulatory system, hemolysis of blood and fibrinolysis of clots lead to a drop in bloo d pressure, which can render the victim very weak and eventually helpless (McCollough and Gennaro, 1966; Parrish, 1967). If the envenomation is not treated, the loss of bl ood pressure can lead to organ failure and eventually death; although death from cottonm outh bites is quite rare (Table 2-1). The sequence of symptoms leading up to death has been described for dogs (Brown, 1941; Vick et al., 1967) and cats (Ru ssell and Bohr, 1962) and is similar to that in humans.

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40 Specific Physiological Effects (Comparative) Epithelial tissue Agkistrodon piscivorus venom has been used in comparative physiological research to examine direct ef fects on skin. Direct application induces purpura in rat skin (Spaet, 1952), and petechial hemorrhaging in t he hamster cheek (Arendt et al., 1953; Fulton et al., 1956) and dog lung surfac e (Bonta et al., 1969, 1970a, 1970c). Subdermal tissue In studies where venom is injected intr adermally, there are local effects ranging from edema to necrosis. Likely due to t he presence of hyaluronidase, venom often spreads from the injection site to other areas, making it difficult to separate strictly local effects from potentially system ic effects. The induction of edema in the rat paw has been used to study potential anti-inflammatory substances (Rocha e Silva et al., 1969) and venom inhibitors (Marshall et al., 1989), and such untreated edema eventually leads to hemorrhage and myonecrosis (Mebs et al., 1983). Blood—hemolysis Very early in the study of cottonmouth venom, the ability to lyse red blood cells (RBCs) was either conjectured or shown. Mitchell and Reichert ( 1886) postulated that hemolysis may be occurring in the pigeons upon wh ich they did their studies. Flexner and Noguchi (1902) noted that a very dilute concentration of cottonmouth venom had great ability to hemolyse dog and rabbit blood in vitro, even when they heated venom to 96C prior to use. They noted that RBCs separated from serum were not lysed by venom, indicating a factor in serum was necessa ry for hemolysis to o ccur. Further work during this time noted that separated horse RBCs hemolysed within 40 min of exposure

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41 to venom (Noc, 1904), and confirmed t he low doses needed (Noguchi, 1909) and general temperature insensitiv ity (Madsen and Noguchi, 1904). Decades later, it was stated that the hemolytic activity of cottonmouth venom was very sensitive to heat, was most stable at pH 5. 0 to 7.0, quickly lost activity in solution, and was not affected by exposure to ch loroform (Peck and Ma rx, 1937). These researchers also indicated that the hemolyt ic activity was caused by two different substances, called hemolysins A and B, t hat could be separated electrophoretically (Marx and Peck, 1938). The hem olytic factor of the venom works rapidly to destroy lymphocytes, but polymorphonuclear leukocytes are more resistant to lysis (Schrek, 1943). In general, a dose-dependent effect of venom on hemolysis using separated human RBCs was noticed, and the addition of serum increased this action (Philpot, 1949). The hemolytic ability against RBCs was furt her examined in rabbits (Minton, 1956) and guinea pigs (Bhargava et al., 1970; Vincent et al., 1972), and one study showed hemolysis of RBCs from humans, capuchin monkeys, dogs, pigeons, rabbits, snakes ( Bothrops asper ) and toads ( Bufo marinus ) (Gomez-Leiva, 1976). A further examination showed 35 to 95% hemolysis for RBCs from t en different mammal species (Kelen et al., 1960-62). Addition of heparin (Vincent et al ., 1972), dextrane sulfate (Vincent et al., 1972), and albumin (Gul et al., 1974) in creased the hemolytic activity; but ethylenediaminetetraacetic ac id (EDTA) reduced hemolysis (Deutsch and Diniz, 1955). Interestingly, A. piscivorus RBCs appear to be resistant to hemolysis by venom (Minton, 1976).

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42 Using Sephadex G-50 to separate the venom no hemolysis activity was seen in any of the three resulting fractions (Bonta et al., 1970b). However, ac tivity was seen in fractions collected after ion exchange chro matography of whole venom (Clark and Higginbotham, 1971). There is some disagreement as to the rela tive level of hemolysis brought about by cottonmouth venom. Deutsch an d Diniz (1955) state that A. piscivorus venom has the highest hemolytic rate of the snake venoms t hey examined, but Rosenfeld et al., (196062) state that it has only weak hemolytic activity. Utilizing blood agar plates and fractionated venom, Gennaro and Ramsey (1959a) found only minor hemolytic activity. Blood—coagulation While the overall effect of A. piscivorus venom on blood is anti-coagulant, some aspects of the venom enhance coagulation, l eading to clot formation. Boiling venom prior to injection into an animal model caus es at least initial coagulation of blood (Mitchell and Reichert, 1886), and fairly small c oncentrations are necessary for initiation of coagulation (Noguchi, 1909). When veno m is combined with horse fibrinogen or prothrombin (precursors to bl ood clots) at low concentration there is a coagulant effect that disappears with time or higher concent rations (Eagle, 1937). Likewise, rabbit RBCs show coagulation at low venom conc entrations (Schrek, 1943; Minton, 1956), and human RBCs show great initia l agglutination in short ti me periods with concentrated venoms (Philpot, 1949). These quickly decline wi th time, and the same is true for dogs injected with venom (Houssay and Sordelli, 1919) Oshima et al. ( 1969) indicate that fibrin clots appear within 2 hours of venom mixing with RBCs. Agkistrodon piscivorus venom does contain lectins, which are known to aid in agglutination and may account for initial clotting activity (Ogilvie and Gartner, 1984).

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43 Blood—anti-coagulation Inhibition of blood coagulation has been noted for some time (Mitchell and Reichert, 1886; Noc, 1904) as has the incr ease in clotting time of blood mixed with cottonmouth venom (Houssay and Sordelli, 1919) Taylor and Mallick (1936) suggested that the prolongation of clo tting time indicated that A. piscivorus venom was anti-thrombic, as did Fleckenstein and Fetti g (1952), and Eagle (1937) indicated that the anti-coagulant effect was due to some form of proteolysis of blood clotting proteins. Others later suggested that t he venom was either fibrinogeno lytic (Didisheim and Lewis, 1956) or fibrinolytic (Rosenfeld et al., 1959) This has led to disagreements in the literature, with some indica ting low (Mebs, 1970) or high (Bhargava et al., 1972) kinin-releasing activity, no (B ajwa et al., 1982) or mild (C opley et al., 1973) thrombin activity, and no (Kornalk, 1966; Bajwa et al., 1982) or very low (Kornalk and Stblov, 1967) plasminogen activation. Although there have been many examinations of the way venom may disrupt different parts of the blood clotting cascade, re search in this area is ongoing. What is known is that cottonmouth venom contains a protein C activator (Exner et al., 1985), which can act as an anti-coagulant. This substance in venom is 39 to 42 kDa in molecular weight and is stable after heating to 70C at pH 3.0 (Stocker et al., 1986). Two fibrinogenolytic enzymes, piscivorase I and II, (Hahn et al., 1995; Markland, 1998) have been purified from cottonm outh venoms, as have two di sintegrins—applaggin and piscivostatin. All of these substances hav e resulting anti-coagulant activities, and they all may work in combination to exacerbat e overall disruption of the blood clotting cascade.

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44 Muscle Because of their proximity, it is someti mes difficult to sepa rate the effects of venom on skin and underlying tissue such as mu scle or small blood vessels. However, examination of muscle effects generally show sw elling at the site of venom injection with underlying hemorrhaging and eventual myonecro sis (Homma and Tu, 1971). Mebs et al. (1983) found venom injected intramuscula rly into rats caused creatine kinase production (an indicator of mu scle degradation) at four hours that was reduced at 16 and 48 hours. Also, direct injection in to pigeon breast muscle showed major hemorrhaging of surrounding tiss ue (Mitchell and Reichert, 1886). Envenomation in dogs makes skeletal muscle unresponsive to direct or nervous stimulation, but this is likely a secondary effect (Hadidian, 1956). Venom added directly to guinea pig muscle showed decreased invoked muscle contractions followed by complete block (Russell and Long, 1961). Yama zaki et al. (2003) indicate that the presence of cysteine-rich secretory prot eins in venom leads to reduced muscle contraction in rat tails, and the venom fracti ons responsible for myonecrosis have been separated using Sephadex G-75 and CM cellulose chromatography (Mebs and Samejima, 1986). The Lys49 (AppK49) form of A. piscivorus venom phospholipase A2 has been determined to cause direct myotoxicity by binding long-chain fatty acids (Pedersen et al., 1995). By creating a synthetic peptide containing the C-terminal sequence of AppK49, Nez et al. (2001) demonstrated its ability to lyse skeletal muscle, and the amino acid residues 115 through 129 have been shown to be the most important structural aspects for this function ( Lomonte et al., 2003b; Yamazaki et al., 2005a, 2005b). AppK49 is actually a homodimer, a nd dissociation of its tertiary structure

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45 greatly reduces myotoxic activity (Angulo et al., 2005). Lomonte et al. (2003a) have reviewed the myotoxic action of AppK49. Nervous tissue Rosenberg and colleagues have conducted a se ries of experiments using nerves, especially the giant squid axon, to examine venom effects. T hey found that, in general, small concentrations of venom make the nerve susceptible to other exogenous compounds, by increasing nerve membrane permeability (Rosenberg and Podleski, 1962; Rosenberg and Ng, 1963; Rosenber g and Podleski, 1963; Hoskin and Rosenberg, 1965; Rosenberg, 1965; Ros enberg and Hoskin, 1965; Rosenberg and Dettbarn, 1967; Condrea et al., 1967). A similar phenomenon was shown using frog nerves (Hadidian, 1956), and the factor responsible for this action was hypothesized to be PLA2 (Rosenberg and Podleski, 1962; Rosenber g and Hoskin, 1963; Rosenberg and Ng, 1963; Condrea, 1967). Russell and Long (1961) showed that venom added to guinea pig phrenic nerve preparations initially caused muscle contra ctions that decreas ed in amplitude until neuromuscular block at 21 min postapplication. Pr e-treatment with triO -cresylphosphate made hen sciatic nerve pr eparations more susceptible to A. piscivorus venom effects (Morazain and Ro senberg, 1970). Guinea pig brain preparations were used to examine the ability of A. piscivorus and three other venoms to inhibit brain enzymes that oxidize pyruvate, and boiled cottonmouth venom was found to have the lowest inhibitory effect of the venoms studied (Braganca and Quastel, 1953). Cottonmouth venom contains a nerve growth factor (Levi-Mont alcini and Cohen, 1956; Cohen, 1959), although it s mode of action in envenomated prey has not been

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46 examined. Although neurot oxic effects have been hypothesized (Micheel and Jung, 1936; Brown, 1941; Braganca, 19 55; Jimenez-Porras, 1968), it is unlikely that cottonmouth venom contains a strict neur otoxin, and that any symptoms resembling neurotoxic effects are secondarily caused by other venom components. Respiratory system Alterations in respiration have been not ed in animals injected with cottonmouth venom. Rabbits have shown immediate increas es in rate, followed by decrease and, in some cases, cessation (Mitchell and Reichert, 1886). Dogs injected directly into the femoral vein with higher venom concentrati ons died from respiratory arrest (Brown, 1941), but it was also noted that most dogs do not die from (or do not have) respiratory complications (Hadidian, 1956). In short, respiratory effects appear to be secondary symptoms of envenomation. Circulatory system In general, the venom of the cottonmouth affects the circ ulatory system by causing hemorrhaging of blood vessels, hemolysis of RB Cs, and disruption of the blood clotting cascade. This triple effect results in a precipitous drop in blood pressure that incapacitates or kills prey. Cottonmouth venom has been found to cause hemorrhagic activity in pigeons (Mitchell and Reichert, 1886; Taylor and Malli ck, 1936), rabbits (Mitchell and Reichert, 1886; Peck and Marx, 1937; Flow ers, 1963), chicken embryo s (Witebsky et al., 1935), mice (Ohsaka et al., 1966; Ownby et al ., 1994), and dog lung surface (Bonta et al., 1970a, 1970b, 1970c; Bhargava et al., 1970), but not on rat lung (Almeida et al., 1977). The mode of action is characterized as diapedesis (leaking through basement

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47 membranes) or loss of structural integrity of sma ll blood vessels (Malucelli and Mariano, 1980). The metal chelators EDTA (Goucher and Flowers, 1964; Bonta et al., 1970a) and -mercapto-(2-furyl)acrylic acid (MFA; Giroux and Lachemann, 1981) both reduce the hemorrhagic effect of cottonmouth venom, indi cating the presence of metal ions in the hemorrhagic factor. This fact led to the determination of metalloproteinases as the major factors responsible for blood ve ssel damage (Spiekerman et al., 1973). Hemorrhagic activity was also reduced or destroyed by X-ray (Flowers, 1963) or ultraviolet (Tejasen and Ottolenghi, 1970) ir radiation. Treating the venom with heparin causes a precipitate to form but does not appear to affect hemorrhagic activity (Bhargava et al., 1970; Bonta et al., 1970b, 1970c), nor does treatment with chloroform (Peck and Marx, 1937). Hemorrhagic activity is greatest when pH is between 6.0 and 8.0, but the factor appears to be denatured outside this range (P eck and Marx, 1937). Boiling of the venom does not seem to destr oy the hemorrhagic effect (Mitchell and Reichert, 1886). Hemorrhagic activities have been repor ted for all three subspecies of A. piscivorus (Tan and Ponnudurai, 1990). The hemorrhagic factor was initially descr ibed as a basic protein of approximately 10 kDa molecular weight (Bonta et al., 1970b, 1970c), but has more recently been characterized as an acidic protein of 69 kDa molecular weight (Dinh et al., 1985). This latter value is more in line with known me talloproteinases (Mackessy, 2009). The hemorrhagic factor can be separated from hem olytic factors via electrophoresis (Marx and Peck, 1938), and also can be separat ed from other venom components via

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48 Sephadex G-50 (Bhargava et al., 1970; Bont a et al., 1970b), Sephadex G-100 (Dinh et al., 1985) and ion exchange (Clark and Hig ginbotham, 1971; Dinh et al., 1985) chromatography. Cottonmouth venom appears to inhibit angiotensin converting enzyme (ACE) through the action of a bradyki nin potentiating peptide, AppF (Ferreira et al., 1995, 1999). The overall effect of such a substanc e is to reduce or stop vasoconstriction, thereby adding to an overall drop in blood pressure. Bhargava et al. (1970) also described a hypotensive substance call ed slow reacting substance C. Weak dilutions of venom affect the heart, although no cardiotoxin has been isolated. Magenta (1922) found that very weak dilutions of venom placed directly on the heart of L. ocellatus (genus not reported) caused cessa tion of beating within 44 min, while isolated frog hearts showed dosedependent declines in both heart rate and stroke volume (Brown, 1940). Further, isol ated rabbit hearts quickly ceased activity after venom was placed on them (Essex, 1932). By using radio-labeled A. p. piscivorus venom injected into mice, it was found that large amounts of radioactivity localized to the heart and lungs (Gennaro and Ramsey, 1959b). Other circulatory effects are based on direct effects on blood (hemolysis) and disr uptions in the blood clotting cascade (see Blood section above). Immune system Guinea pig serum mixed with A. piscivorus venom showed reduced activity of complement proteins C2, C3c, and C4 in on e study (Zarco et al., 1967) and of C2 through C9 in another (Birdsey et al., 1971). Very little ac tivity against C1 was seen (Birdsey et al., 1971).

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49 Mast cell research has been conducted utiliz ing cottonmouth venom, as mast cells release histamines at the site of envenom ation (Zahl and Nowak, 1951). Injection of venom intradermally into guinea pigs causes mast cell degranulati on (Raab and Kaiser, 1965). Clark and Higginbotham (1971) described A. piscivorus venom as having a mastocytolytic cationic protein that induc es local and systemic allergic reactions. Lymphatic system Cottonmouth venom injected into tissues c auses edema formation in rats (Rocha e Silva et al., 1969; Bhargava et al., 1972; Me bs, 1983) and mice (Marshall et al., 1989). Sephadex G-50 separation was used to isol ate the substance responsible for edema formation, and it was found to have a molecula r weight of about 8 kDa (Bhargava et al., 1972). Marshall et al. (1989) suggested that edema formation may be caused by phospholipase activity via generation of eico sanoids and platelet -activating factor. Excretory system Very little research has been conducted on t he effects of venom on the excretory system of envenomated prey. Huang et al. ( 1972) used tritiated venom injected into rats to examine excretory patterns. They found an increase in both urinary and fecal excretion of tritium up to 20 hours after envenomation followed by return to normal levels over the subsequent 60 hours. Fecal ex cretion was the major route of clearance of venom, and liver and intestinal tissue show ed the greatest radioactivity distribution. Direct effects of venom on kidneys have also been examined. When venom is injected into rats, alkaline phosphatase and l eucine aminopeptidase activities increase in the urine, indicating nephrotox icity (Raab and Kaiser, 1966a, 1966b).

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50 Treatment of Cottonmouth Bite Medical treatment Treatment for cottonmouth bite follows guidelines for typical pit viper bites in North America (Smith and Bush, 2009). General recommendations include keeping the envenomated patient calm and immediately seek ing professional medical attention. The use of traditional snake bite treatment s such as suction, cutting, pressure bandages, and tourniquets are not helpful and may actually cause secondary problems. Spread of tissue damage in rats injected wit h cottonmouth venom was not affected by local application of either co ld or heat (Cohen et al., 1992). Medical treatment generally includes observation of symptoms followed by antivenom use, if warranted (Smith and Bu sh, 2009). Although prev ious studies have found fasciotomy to be useful for compartm ent syndrome (Roberts et al., 1985), it is currently not advised in favor of vigor ous antivenom use to reduce compartment pressure (Smith and Bush, 2009). Antivenom research Agkistrodon piscivorus venom has been used for antivenom development, as cottonmouths are responsible for human envenom ation annually. Early on, it was found that antivenom had specificity fo r individual venoms, although some cross-reactivity was also noticed (Noguchi, 1906a, 1906b). These in itial studies showed that antimoccasin antivenom was more efficacious in reducing toxi city and hemolysis effects, and that this efficacy was both timeand dose-dependent. In one early clinical report, the instance of death in humans bitten by cottonmouths was lower when antivenom was used (4/167 or 2.4%) than when it was not (4 /27 or 14.8%), as is expec ted (Githens, 1935). Current antivenom is more efficacious, and mortality is highly unlikely in treated individuals.

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51 Although the normal mode of antivenom producti on is to inject small amounts of venom into a laboratory animal and then collect the resulting antibodies, some research has gone into finding alternatives to this met hod. In one series of tests, researchers used venom initially exposed to the metal c helator EDTA to reduce negative effects of the venom on antibod y-producing animals (Goucher and Flowers, 1964; Flowers and Goucher, 1965). They found that local tissue damage and necrosis was reduced in animals receiving venom mixed with EDTA, but that other venom effects were not significantly reduced (Flowers and Goucher 1965). X-ray irradiated venom was found to lose lethality but maintain antigenici ty in laboratory rabbits (Flowers, 1966), and thiabendazole treatment of v enom inhibited hemorrhagic acti vity in guinea pigs (Stone et al., 1966). In this last study, there was also a reduction in venom effects when thiabendazole was injected into animals following envenomat ion. Further, photooxidized venom injected into mice and r abbits over 3 to 4 weeks afforded some protection against native venom (Kocholaty and Ashley, 1966). Mice given serum from inoculated rabbits were also afforded protec tion from native venom, and the intravenous route of antivenom was found to be more efficacious than t he intraperitoneal (IP) route (Kocholaty and Ashley, 1966; Kocholaty et al., 1968). In North America, polyvalent antivenom s are created by combining antibodies against the venoms of locally common crotalid snakes (usually genera Crotalus and Agkistrodon ). Examination of polyvalent ant ivenoms have shown the ability to neutralize 40.8 murine median lethal doses (LD50s) per mg antivenom (Gingrich and Hohenadel, 1956), and the ability of polyvale nt antivenom to protect dogs against A. piscivorus venom also has been shown (Snyder et al., 1968). More recently, polyvalent

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52 antivenom was effective against 2 (but not 5) LD50s of cottonmouth venom in mice, in terms of hemorrhagic, myotoxic and defibrinating activities (A rce et al., 2003). Snchez et al. (2003) examined three commerci ally-produced antivenoms and found that fibrinolytic and hide powder azure activities were not inhibited by any of these, but hemorrhagic and gelatinase activity inhibition was case-specific. Venom-Associated Anatomy The venom apparatus of A. piscivorus is typical of that for viperid snakes, and is paired, with both left and right apparatuses. It contains a main gland with a lumen in which venom is stored, a prim ary duct through which venom l eaves the main gland, an accessory gland into which the primary duc t empties, a secondary duct through which venom travels, and a tubular fang through which venom leaves (Gennaro et al., 1960, 1963, 1968). The histology of the glandular tissue indicate s that the main gland is serous in nature and the accessory gland is mucous-secreting (Gennaro et al., 1963; Rhoades et al., 1967). Venom is produced in t he cells of the main gland, and stored in vesicles prior to being secreted into the lume n (Gennaro et al., 1968). Within the lumen, the venom is serous in natur e and does not appear to have a mucosal aspect to it until it passes through the accessory gland (Gennaro et al., 1963). The microstructure of both the venom and accessory glands has been exam ined with electron microscopy (Odor and Gennaro, 1960; Odor, 1965; Andrews et al., 1968; Gennar o et al., 1968), and basic histological staining techniques for t he glands have been described (Gennaro et al., 1960). It has been suggested that the accessory gland may be responsible for increasing the toxicity of the venom (Gennaro et al., 1963; Rhoades et al., 1967). Rhoades et al., (1967) noted that the hi stology of the accessory gland was typical of that seen in

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53 mucous-secreting glands and that it contained much more uronic acid than the main gland. More recently, a factor that causes lung collapse in frogs was isolated from the accessory gland, but was not found in the main venom gland (Gennaro et al., 2007). As part of a cDNA library construction, the gene for parvalbumin was isolated from venom gland tissue and cloned (Jia and Perez, 2009). It was localized to the gland, but not to the venom, indicating that it is not secreted by the gland into the lumen. The musculature surrounding the glandul ar region, including those muscles involved in venom ejection, have been descr ibed in detail by Kardong (1973). The effects of occlusion of the primary duct have been examined in A. p. piscivorus and A. p. leucostoma (Glenn et al., 1973), and no adverse hea lth effects were seen in the snakes, as it appears that the venom gland initially fills and then stops producing venom. Venom Characteristics Basic Description Volume Although the amount of venom stored within the lumen of the main venom gland is likely correlated with animal size, an average of 550 l per extraction was found for 315 animals (sizes not given) over a two y ear period (Wolff and Githens, 1939a), 320 l for “young” snakes, 420 l for adults, and 530 l fo r “old” specimens (Do Amaral, 1928). These researchers found that the volume ej ected by individuals did not decrease with multiple extractions over the two year dur ation of their study. Maximum amounts of venom have been reported as 3.5 and 4.0 ml of venom at two diffe rent times from one individual (Wolff and Githens, 1939b), and an av erage of 1.05 ml for “exceptional” specimens (Do Amaral, 1928).

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54 In a typical predatory strike, snakes do not empty their entire veno m store. To this effect, it has been stated that Agkistrodon (species not stated) eject only 25 to 75% of their venom store in any one bite thr ough a rubber membrane (Do Amaral, 1928). Using radioiodine as a marker, it was also noted that cottonmouths deliver less venom to smaller prey and less overall than Crotalus atrox (western diamondback rattlesnake; Gennaro et al., 1961). Specific gravity Mitchell and Reichert (1886) report A. piscivorus venom as having a specific gravity of 1.032 g/ml. This is the only known study to specifical ly state specific gravity of this venom. Protein content While some studies examine protein cont ent to report activities on a per mg protein basis, there is one report of protein comprising 68% of A. piscivorus venom (Kocholaty et al., 1971). This indicates that enzymes and peptides comprise the majority of the venom. Glycoprotein content Of carbohydrate associated with A. piscivorus venom (51.7 g/mg venom protein), neutral sugars account for 15.0 g/mg, amin o sugars account for 28.2 g/mg, and sialic acid accounts for 8.5 g/mg. The ratio of neutral sugars (D-galactose: D-mannose: Lfucose) was determined to be 1.0:0. 81:0.72 (Oshima and Iwanaga, 1969). Dry mass As venom is often dried for storage and then reconstituted in solution prior to use, many authors have given numbers for the dry mass of venom per individual. Unfortunately, these numbers ar e not accompanied by the mass of the animals. It has

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55 been reported that the dry ma ss of venom was 27.42% initial wet mass (Mitchell and Reichert, 1886); 28 to 28.6% initial we t mass, depending on animal size (Do Amaral, 1928); and 28 g/100 ml venom (28%; Ellio tt, 1978 quoting Wolff and Githens, 1939a, 1939b). The Mitchell and Reichert (1886) value is reported in their paper as the percent loss on drying, which would indicate a dry mass of 71.58% initial mass, but this was interpreted as 27.42% by Do Amaral (1928) and is much closer to other reported values. Dry mass per extraction has been repor ted in terms of average as 98 mg (for 264 specimens; Githens, 1935), 100 to 150 mg (Minton, 1974), and 158 mg (Wolff and Githens, 1939a). One study t hat grouped animals into size classes gave dry mass per extraction as 90 mg for adults 120 mg for older animals, and 300 mg for exceptional individuals (Do Amaral, 1928), and a maxi mum (of 264 specimens) for another study was reported as 190 mg (Githens, 1935). Average dry mass, without information on animal size, has been reported as 150 mg (V ick, 1973), 90 to 148 mg (Kochva, 1978) and 90 to 170 mg (Russell, 1980). Spectrophotometry Singer and Kearney (1950a) published the absorbance spectrum (250 to 560 nm) for A. piscivorus venom showing the whole enzyme. They also provide a spectrogram of the prosthetic (flavi n adenine dinucleotide) group. Stability Although storage methods for venom vary, reports specifically for A. piscivorus venom indicate that different methods of preservation yield di fferent results, indicating that preservation method is important (Schttler, 1951b). This study noted that opening vials of venom multiple times reduced acti vity. Jones (1976) reported that lyophilized

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56 venom gave the same activity one year after drying, and Gennaro and Hood (1961) reported that phospholipase was still ac tive 8 and 10 years after drying. Variation The many components and activities found within cottonmouth venom have been compared against those of many other relat ed and unrelated species. However, very little work has been done on natural variati on of venom components within the species, among subspecies, among populations, or within populations. Po tential variation is very important because it can influence the efficacy of antivenom. It is also interesting as a phenotypic character that could be used in studying evolutionary relationships among and within snake species. Early on, variat ion was noticed in its ability to abate the Shwartzman phenomenon (Peck and Rosenthal 1935) and stop nosebleeds (Goldman, 1936) in the same patient. The high level of venom variation found within populations of snakes has indicated that venom is not a good phenotypic character for determining taxonomic relationships among snake specie s and populations (Jones, 1976; Gibson, 1977). Cottonmouths appear to detect their i ndividual venom in prey and use it as a search cue, so venom variation may be import ant for prey tracking (Chiszar et al., 1992; Greenbaum et al., 2003). Perhaps the best example of venom variation within the cottonmouth is the wide values found using commercially-supplied venom from three sources to examine multiple physiologic al activities (Tan and Ponnudurai, 1990). Toxicity The median lethal dose (LD50) of cottonmouth venom has been determined in many different studies, usually utilizing mice, and by different routes of administration. These are shown in Table 2-2, as are other lethality values for animals either using a different toxicity statistic or animals with special physiological conditions.

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57 Many different measures of toxicity have been reported, and it is often difficult to compare them because of the sheer diversit y of techniques or lack of important information. One of the earliest examinations of A. piscivorus venom effects on pigeons showed that heating venom to 78C did not affe ct the toxicity of the venom, but boiling caused a reduction (Mitchell and Reichert, 1886). In the same study the following items were noted: filtering venom through “anima l charcoal” rendered venom non-toxic, co-injection of an iron chloride solution r educed venom effects, and addition of iron to the venom solution made no differ ence. Further, it was noted t hat IP injection of boiled, filtered venom was lethal to pigeons, but without the hemorrhaging normally seen. In another early study a rabbit injected with venom was dead within 11 min and did not show signs of intravascular blood c oagulation (Houssay and Sordelli, 1919). Gloyd (1933) reported a cottonmout h envenomating an d killing one Crotalus atrox and Conant (1934) reported one captive cottonmouth that envenomated and killed two rattlesnakes ( Crotalus confluentus oreganus ) and another cottonmouth. A study examining the effects of venom on other snake species indicated that A. contortrix mokeson may have some immunity toward cottonmouth venom, as do conspecifics, but both can die from the ev enomation (Swanson, 1946). Minimum and other (average, maximum) lethal doses for cottonmouths have also been reported in a number of studies (Table 2-3) It was calculated, in one study, that the cottonmouth had enough toxic venom to kill 1755 pigeons (Wolff and Githens, 1939), and a later study calculated average time to death in mice (98 min) and dogs (240 min; Vick et al., 1967). Another study showed less than 8 hour survival time in dogs bitten by cottonmouths in a l aboratory environment (Vick, 1973).

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58 Separation of venom by Sephadex G-75 column chroma tography showed primary toxicity in the first peak (Clark and Higginbotham, 1965a), and separation by P-10 biogel column chromatography showed higest lethality in fraction Ic (Moran and Geren, 1979). Further, using coarboxymethyl cellulo se ion-exchange chromatography, lethality was noted in all four fractions, but with fractions 1 and 2 having greater potency than fractions 3 and 4 (Powlick and Geren, 1981). Recently, Gennaro et al. (2007) noted that a dose of 0.5 mg/kg was lethal to rabbi ts but not cats, whereas a dose of 5.0 mg/kg was lethal to both. UV irradiation of venom reduced its toxicity (Tejas en and Ottolenghi, 1970) in mice, as did heating to 80C before use (Noc, 1904). Wolff and Githens (1939) found that multiple venom extraction events did not reduce toxicity toward pigeons. Heatwole et al. (1999) found that the LD50 of venom against adult bullfrogs was higher than the LD50 against tadpoles of the same species, indicating an ontogenetic shift whereby animals more likely to be envenomated are afforded more protection. Cottonmouth venom also has been found to be toxic again st tumor cells, but was not found to preferentially kill tumor cells over normal ones (Tu and Giltner, 1974). The concentration of venom that killed 50% of stomach cancer cells was found to be <3.0 g/ml (Ahn et al., 1997). Separation techniques Separation techniques are commonly used to isolate specific components of venoms, and A. piscivorus venom has been separated using many different techniques. In the earliest report, it was noted that so lids precipitate from liquid venom simply through the effects of gravity (Mitchell and Reichert, 1886). Through dialysis with water, these researchers separated “globulins” from the remainder which contained

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59 “peptones.” “Globulins” were defined as pr oteins insoluble in distilled water and coagulable by boiling. “Peptones” were consi dered to be proteins soluble in water and not coagulable by boiling. Furt her, “globulins” were broken into the categories of watervenom-, copper-venom-, and dialysis-v enom-globuins based on the separation technique that yielded them. Because separation procedures such as pr ecipitation, extrac tion, and absorption often led to destruction of venom componen ts, and because much of the venom was thought to be protein, electr ophoresis was attempted with A. piscivorus venom (Marx and Peck, 1938). It was found that two subs tances they called hemolysins A and B migrated to different ends of the electrophoresis chamber and that the hemorrhagic factor could be separated from the hemolys ins. It was also determined that the hemorrhagic factor’s isoelectric point was between pH 4 and 5. Later, the isoelectric point of L-amino acid oxidase was found to be between pH 5.5 and 5.6 (Singer and Kearney, 1950a). Among the forms of electrophoresis reported for separating A. piscivorus venom, paper electrophoresis isolated 8 bands (Habermann and Neumann, 1954), starch electrophoresis showed 13 bands (Bertke et al., 1966), disc electrophoresis yielded 4 to 8 major and 3 to 9 minor bands depending on pH (Basu et al., 1969, 1970; Mebs and Samejima, 1986), and gel electrophoresis f ound ten bands (Powlick and Geren, 1981). Moving boundary electrophoresis (Wagner et al., 1968) and cellulose acetate strips have also been used, the latter examining a ll three subspecies (an Illinois population indicated as A. p. conanti is well outside of its range, and could only be A. p. piscivorus based on locality; Jones, 1976).

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60 Chromatography methods used include paper (Franklin et al., 1951), Sephadex G50 (four fractions, Bhargava et al., 1970) Sephadex G-75 (three peaks, Clark and Higginbotham, 1965a; Mebs and Sameji ma, 1986), Sephadex G-100 (three peaks Bajwa et al., 1982), P-cellulose (12 fr actions, Clark and Higginbotham, 1965b), CM cellulose (Mebs and Samejima, 1986), anion exchange (Wagner et al., 1968), gel filtration (Wagner et al., 1968), ion exchan ge (16+ fractions, Clark and Higginbotham, 1971; Spiekerman et al., 1973; Pr escott and Wagner, 1976; six pe aks, Dinh et al., 1985; Ramrez et al., 1999), P-10 Bio-gel colu mn (Moran and Geren, 1979; Powlick and Geren, 1981), carboxymethyl ce llulose ion exchange (four peaks, Powlick and Geren, 1981; four peaks, Dinh et al., 1985), high-performance liquid (R amrez et al., 1999), and concanavalin A Sepharose (specifically for A. p. leucostoma Soper and Aird, 2007). Combinations of separation techniques have been used for secondary separation of primary fractions (e.g. Clark and Higginbotham, 1971; Powlick and Ge ren, 1981; Dinh et al., 1985). Sedimentation also has been us ed for separation of venom components (Wagner et al., 1968). Separation techniques have been utilized in an attempt to isolate fractions containing specific activities or com ponents of the venom. These have been done to examine allergenicity (Clark and Higginbotham, 1965b), L-amino acid oxidase (Powlick and Geren, 1981), aminopeptidas e (Wagner et al., 1968; Pr escott and Wagner, 1976), bradykinin-releasing activity (Powlick and Geren, 1981), esterase (Prescott and Wagner, 1976; Powlick and Geren, 1981), fibrinol ytic activity (Retzios and Markland, 1990; Hahn et al., 1995; Ramrez et al., 1999) hemolytic activity (Powlick and Geren, 1981), hemorrhagic activity (Powlick and Ger en, 1981; Dinh et al., 1985), hyaluronidase

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61 (Powlick and Geren, 1981), hypotensive activity (Habermann and Neumann, 1954), metalloproteinase (Spiekerman et al., 1973) myotoxicity (Mebs and Samejima, 1986), necrotizing activity (Clark and Higgi nbotham, 1965b), phosphodiesterase (Powlick and Geren, 1981), phospholipase (Habermann and Neumann, 1954; Powlick and Geren, 1981), proteolytic activity (Prescott and Wagner, 1976; Powlick and Geren, 1981), and toxicity (Clark and Higginbotham, 1965b; Mor an and Geren, 1979; Po wlick and Geren, 1981). Specific Components Enzymes Studies of venom enzyme activity are many and varied. Although they are a method for distinguishing specific activiti es (either enzyme-based or substrate-based), their real value comes in comparisons bas ed among individuals, groups, species, or higher levels. It is especially difficult to compare reported values against each other, as there are many different enzymatic technique s that utilize various assay conditions and report results using varied and (sometimes) conf using units. Luckily, the majority of results are reported on a per mg venom or per mg protein basis, which allows some conformity. Phosphodiesterase. Cottonmouth venom has been shown to have phosphodiesterase activity that has been c haracterized as low to moderate (Mebs, 1970), moderate (Gulland and Jackson, 1938a; Tan and Ponnudurai, 1990), and moderately high (Kocholaty et al., 1971). Repor ted values of phosphodiesterase activity include 7800 enzyme units/mg dry venom (R ichards et al., 1965), 47 units/mg protein (Kocholaty et al., 1971), 0.046 0.011 internat ional units/mg (Moran and Geren, 1979), and 10 to 27 nmol/mg/min (Tan and Ponnudura i, 1990). Carboxymethyl cellulose

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62 ion-exchange chromatography shows phosphodies terase elution in the second fraction (Powlick and Geren, 1981), and UV irr adiation of venom appears to destroy phosphodiesterase activity in a time-depen dent manner (Tejasen and Ottolenghi, 1970). Tan and Ponnudurai (1990) examined pool ed venom samples of all three A. piscivorus subspecies from commercial sources and found that A. p. conanti had low variation, while A. p. piscivorus and A. p. leucostoma both had high variation. 5’-nucleotidase. After initial indications that it does have 5’-nucleotidase activity (Gulland and Jackson, 1938b; Zeller, 1951), moderate (Mebs, 1970) and moderate to high (Tan and Ponnudurai, 1990), and high (Braganca, 1955) activity has been noted for A. piscivorus venom. Gulland and Jackson (1938b) indicated that venom could dephosphorylate both adenosineand inosine-5-phosphate. T he two specific values reported for this activity are 495000 enzyme uni ts/mg dry venom (Richards et al., 1965) and 3.2 to 7.3 nmol phosphate produced/mi n/mg venom (Tan and Ponnudurai, 1990). Alkaline phosphomonoesterase. Multiple reports of phosphomonoesterase in A. piscivorus venom indicate that there is little acti vity. Specifically, no activity (Gulland and Jackson, 1938a; Zeller, 1951; Braganca, 1955), 550 enzyme units/mg dry venom (Richards et al., 1965) and 2 to 11 nmol/mg/min (Tan and Ponnudurai 1990) have been reported. This enzyme also was called monophosphatidase by Richards et al. (1965). Hyaluronidase. Presence of hyaluronidase was in itially seen by mixing venom with India ink and noting a dosedependent increase in diameter of lesions produced in rabbit skin (Duran-Reynals, 1938 1939). At the time, it was considered a “spreading factor,” but Zeller (1948) equates this term with the enzyme hyaluronidase.

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63 A period of confusion in the literatur e is seen when Haas (1946a) indicated cottonmouth venom hyaluronidase should be called “invasin,” to which mammals had an inhibitor termed “antinvasin” in their serum. In the same series of papers, “proinvasin I” was described as a venom substance t hat destroyed “antinvasin I” (Haas, 1946b), and it was hypothesized that this allowed hy aluronidase to be active (Zeller, 1948). Further confusing was the description of “antin vasin II” which destroys “proinvasin I” as well as the hypothesized existence of “proinva sin II” and “antinvasi n III” (Haas, 1946c). Later work determined that “pro invasin I” was simply the pr oteolytic capability of the venom, which degrades the serum that normally inactivates hyaluronidase. By doing so, hyaluronidase is effectively activated (Hadidian, 1953, 1956). Boquet et al. (1958) determined the activity of A. piscivorus venom hyaluronidase to be 16.1 total reducing units/s/mg venom, wh ich was the lowest of the species they studied. Contrary to this, a more recent examination of the enzyme indicated high activity and variability at 66 to 200 National Formulary units/mg venom (Tan and Ponnudurai, 1990). Powlick and Geren ( 1981) used carboxymethyl cellulose ion-exchange chromatography to isolate hyaluronidase. L-amino acid oxidase. L-amino acid oxidase (LAAO) degrades L-amino acids via a two step process whereby the amino group is replaced by an oxygen molecule that is double covalently bonded to the central carbon, the overall mechanism being oxidation (Chippaux, 2006). Although the general activi ty of the enzyme has been examined in A. piscivorus venom, the majority of details co ncerning LAAO have been discovered in other species, so what work that has been done specifically on A. piscivorus LAAO is

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64 fairly sparse. Besides its direct activity, LAAO also has been implicated in activating proteases in the same venom (Zeller, 1951) The enzyme itself has been described as about 59 (Powlick and Geren, 1986) to 60 kDa (Singer and Kearney, 1950b), with a flavin adenine dinucleotide (FAD) prosthetic group (Singer and Kearney, 1950a). The enzyme’s cellulose electrophoresis mobility is reported as -1.78 to -1.81 X 10-5 cm2/s/V (Wellner and Meister, 1960), and its isoelectric point is reported as pH 5.5 to 5.6 (Singer and Kearney, 1950a). In general, the concentration of subs trate limits the r eaction, with high concentrations reducing activity. However, in the presence of pure oxygen, higher substrate concentrations will not reduce acti vity (Meister and Wellner, 1963). Under anaerobic conditions, activity is reduced but still occurs with L-leucine as substrate (Braganca and Quastel, 1952). The enzyme reaches optimal activity around pH of 7.4 (Wellner and Meister, 1960), wit h a steady decline with lower pH and a sharp decline at higher pH (Meister and Wellner, 1963). Phosphate, in the form of phosphate buffer, has a reversible (Kearney and Singer, 1951b; Meis ter and Wellner, 1963) inhibitory effect (Kearney and Singer, 1949, 1951c), as does pure water, but presence of chloride ions maintains activity (Kearney and Singer, 1951a). When the pH is lo wered, the enzyme appears to dissociate into smaller fragments, indicating the presence of one or more labile covalent bonds (Powlick and Geren, 1 986). The optimal reac tion temperature has been reported as 38C (Singer and Kearney, 1950b) Singer and Kearney (1950c) give informa tion on isolating LAAO from crude venom, but later attempts to crystallize the pure form were unsuccessful (Wellner and Meister, 1960). The carboxymethyl cellul ose ion-exchange chromatography profile for

PAGE 65

65 A. piscivorus venom has been published, and it indicates where LAAO separates (Powlick and Geren, 1981). This same study further separated the LAAO-containing fraction on a bio-gel column. As with other venom enzymes, reported activity values suffer due to lack of standardization of assay conditions, assay methods, and units reported. Relative descriptions of A. piscivorus LAAO activity have included 1) higher than most elapids, but medium to low for viperids (Zeller, 1948); 2) very high (Mebs, 1970); 3) in the midrange of those tested (Kochol aty et al., 1971), and 4) m oderate activity with marked variation (Tan and Ponnudurai, 1990). Activity of A. piscivorus venom LAAO has been reported as 380 l O2 consumed/h/mg venom (Zeller, 1948), 3100 molecules substrate oxidized/enzyme molecule/min (Singer and Kearney, 1950b), 277 units/mg protein (based on absorbance change X1000; Kocholaty et al., 1971), 0.40 0.03 international units/mg (Moran and Geren, 1979), 105 to 3 01 National Formulary units/mg venom (Tan and Ponnudurai, 1990), and 7.28 U/mg venom (Ahn et al., 1997). Metalloproteinase. Although the hemorrhagic capacity of A. piscivorus venom has been well studied, very few examinations of the specific form of metalloproteinase responsible for this action have been conducted. Leucostoma peptidase A was described a based on its size (MW = 22.5 kD a) and its ability to hydrolyze many L-amino acids (Wagner et al., 1968). This same paper reported its relative amino acid composition, determined its isoelectric point at pH 6.5, and considered it an endopeptidase protease (also called arylamylase). It was later considered a metalloproteinase due to its Ca2+ to Zn2+ ratio (2:1), and its failure to act when mixed with the metal chelator EDTA (Spiekerman et al., 1973). This study also confirmed the

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66 molecular weight of 22.5 kDa. It appears to be an endopeptidase because it only hydrolyzes peptides with basic or aromatic C-terminal residues. It is active over the entire pH range studies (7.0 to 10.0), but is optimal at pH 8.5. Leucostoma peptidase A can be purified via a published protocol (Wagner et al., 1968; Prescott and Wagner, 1976). More recently, Jia et al. (2009) utiliz ed a cDNA library to characaterize two different forms of me talloproteinase from A. p. leucostoma venom gland. Both forms showed activity against fibrinogen. Serine protease I (thrombin-like). Although much work has been done examining the effects of cott onmouth venom on the blood clotting cascade, very little of that work has focused on the presence of thro mbin-like activity. Copley et al. (1973) indicated that the venom shows mild thrombin -like activity; whereas Bajwa et al. (1982) state that it has no thrombin-like activity. Utilizing venom from all three subspecies, Tan and Ponnudurai (1990) found no thrombin-like activity. Hadidian (1956) states that A. piscivorus venom destroys prothrombin, indicating it has activity reducing any thrombin-like effe cts. Other studies have also indicated antithrombin activity (Kornalk, 1966, 1971), in cluding activity against antithrombin III, the major thromboprotective plasma pr otein (Kress and Catanese, 1980). These studies indicate a potentially complex interact ion of venom with multiple factors leading to (or reducing) thrombin activity of converting fibrinogen to fibrin. Serine protease II (kallikrein-like). Cottonmouth venom appears to have kallikrein-like activity (Bonta et al., 1970a), wh ich may be important for alterations in the blood clotting cascade. Venom mixed with hu man plasma releases bradykinin in a

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67 dose-dependent manner (Philpot et al., 1978), i ndicating kallikrein activity of cleaving kininogen (Webster and Pierce, 1963). Compared to other venomous snakes, cottonmouth venom is reported to have moderate kallikrein-like activity (Deutsch and Diniz, 1955; Oshima et al., 1969). Using carboxymethyl cellulo se ion-exchange chromatography, activity was isolated in the first and second fractions (Powlick and Geren, 1979). A kallikrein-like enzyme with a molecular weight of 29 kDa has been isolated and characterized as cleaving kininogen and the B chain of fibrinogen (Nikai et al., 1988a), and kinin-releasing activity in A. p. piscivorus venom has been reported as 5.1 0.6 g kinin released/min/mg venom (Bailey et al., 1991). Serine protease III (arginine esterase). Arginine esterase (also called arginine ester hydrolase) activity in A. piscivorus venom has been reported as 4.6 mol N-tosyl L-arginine methyl ester hydrolyzed/min/mg pr otein (Oshima et al., 1969) and 6.8 to 10.8 mol -benzoyl-L-arginine ethyl ester consumed/ min/mg venom (Tan and Ponnudurai, 1990). Moderate activity toward p -tosyl-L-arginine methylester also has been reported (Kocholaty et al., 1971). Phospholipase I (phospholipase A2). Of the research conducted on cottonmouth venom, by far the most studied component has been the enzyme phospholipase A2 (PLA2). A. piscivorus venom has very high PLA2 activity (Mebs, 1970) and has been reported as having higher activity than many other venoms (Kocholaty et al., 1971), including those of Crotalus adamanteus Daboia russelli Naja naja and Ophiophagus hannah (Rosenberg and Ng, 1963). The A2 forms of phospholipases work generally to hydrol yze phospholipids bound to membranes (e.g. those found in the plasma membrane of cells) at the sn -2 acyl bond, resulting in

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68 arachidonic acid and lysophospholipid produc ts (Augustyn and Elliott, 1970; see Ownby et al., 1999, for a review). Types of cells affected include RBCs, which results in hemolysis (Gul et al., 1974). Phospholipase also has been referred to in the older literature as phosphatidase (Chargaff and Cohen, 1939), and the forms found in A. piscivorus venom have been used for studies of venom activity, nerve conduction, and interfacial chemistry. Initially, PLA2 was examined for activity eit her from whole venom or from separated fractions. In these cases, A. piscivorus venom phospholipases have been found to hydrolyze many different lipid s ubstrates, including egg yolks (Chargaff and Cohen, 1939; Tan and Ponnudurai, 1990), le cithin (Chargaff and Cohen, 1939; Fairbairn, 1945), cephalins (Fairbairn, 1945), ovolecithin (Long and Penny, 1957) phosphatidylcholine (Rosenber g, 1976), phosphotidylethanolam ine (Rosenberg, 1976), phosphatidylserine (Rosenberg, 1976), 1-acyl-2 -acyl-glycero-3 phosphorylcholine (Waku and Nakazawa, 1972), 3-(acyloxy)-4 nitrobenzoic acids (Cho et al., 1988b), 1,2-dioctanoyl-3sn -glycerophosphorylcholine (Van den Bergh et al., 1988), and 2-arachidonoyl-1-stearoyl-L-3-phosphatidylcholine (Yamaguchi et al., 1997). Other substrates that are not hydrolyzed include cerebrosides (Fairbairn, 1945), sphingomyelins (Fairbairn, 1945), acet al phospholipids (Fairbairn, 1945), lysophospholipids (Fairbairn, 1945), cephalin (Chargaff and Cohen, 19 39, contrary to above), sphingomyelin (Rosenberg, 1976), 1O -alkenyl-2-acyl-glycero-3phosphorylcholine (Waku and Nakazawa, 1972), and 1O -alkyl-2-acyl-glycero-3phosphorylcholine (Waku and Nakazawa, 1972).

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69 Conditions for assaying PLA2 activity have been quite diverse, with many different additives and assay conditions (e.g. temperat ure, pH) being tested. Substances found to enhance activity include Ca2+ (Marinetti, 1965), Mg2+ (small—Marinetti, 1965), albumin (Gul et al., 1974), l ysolecithin (Bell and Biltonen, 1989a), fatty acids (Bell and Biltonen, 1989a), and glycerides (Bell et al., 1995). Indeed, it has been determined that Ca2+ presence is nearly essential for proper PLA2 function (Bell and Biltonen, 1989a; Van den Bergh, 1989; Lathrop and Biltonen, 1992; Scott et al., 1992) and that the overall (Long and Penny, 1957; Bell and Biltonen, 1989a) and initial (Bell and Biltonen, 1989a) rates of reaction are Ca2+ concentration-dependent. It also has been shown that Ca2+ reduces dimerization of PLA2. Cho et al. (1988b) found that activity was fairly similar when either Ba2+ or Sr2+ was substituted for Ca2+ (when using benzoic acid substrates). No observable effect was found with addition of Mn2+ (Marinetti, 1965), Fe3+ (Marinetti, 1965), sodium iodoac etate (Long and Penny, 1957), sodium p chloromercuribenzoate (Long and Penny, 1957) or sodium mercaptoacetate (Long and Penny, 1957). Inhibition was noted after the addition of Al3+ (Marinetti, 1965), Cu2+ (Long and Penny, 1957; Marinetti, 1965), Mg2+ (Cho et al., 1988b—for benzoic acid substrates), Zn2+ (Marinetti, 1965; Cho et al., 1988b— for benzoic acid substrates), EDTA (Long and Penny, 1957), heparin (Dua and Cho, 1994), and fucoidin (Angulo and Lomonte, 2003—specifically for the Lys49 PLA2). Activity patterns have been noted in mult iple studies, with higher temperatures generally yielding higher activities, up to a poi nt. Marinetti (1965) found higher activity at 41C than at 25C (Marinetti, 1965), and optimal temperature between 25 and 75C was found to occur at 65C (Nair et al., 1976). However, another study showed the

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70 maximal temperature (bet ween 38 and 48C) was 41 to 44C, depending on assay conditions (Bell et al., 1995). Increased dimerizaton has been seen with increased pH (over the range 4.2 to 7.0; My att et al., 1991), and optimal pH for lecithin hydrolysis is 6.5 to 7.0 (Magee and Thompson, 1960). General studies of traditional enzyme kinetics have also been conducted (Fairbairn, 1945; Magee and Thompson, 1960; Marinetti, 1965) Phospholipase activity was reduced when venom was exposed to X-ray (Flowers, 1963) or UV radiation (Tejasen and Ottolenghi 1970). In terms of stability, Gennaro and Hood (1961) found phospholipase to be active in dried A. piscivorus venom extracted 8 and 10 years earlier. Many techniques for separation of the PLA2-containing fraction of cottonmouth venom have been described, including those utilizing sephadex G-50 (Bhargava et al., 1970; Bonta et al., 1970b), gel filtrati on (Augustyn and Elliott, 1970), carboxymethyl cellulose ion-exchange chromatography (P owlick and Geren, 1981), diethylaminoethyl (DEAE) cellulose chromatography (P owlick and Geren, 1981), sephadex G-75 (Marinetti, 1965), and sedimentation (Augustyn and Elliott, 1970). Initial estimates of the molecular mass of purified PLA2 were 13.5 0.25 kDa by gel filtration and 13.3 to 15.4 kDa by sedimentation. Reported values for activity of PLA2 suffer in that they are not standardized by substrate type, assay conditions, or reporte d activity units. Using clearing of an egg yolk emulsion as indication of activity, Mari netti (1965) found three values of 25, 200, and 270 absorbance units/min. Other values include 54 equivalents of fatty acids titrated/mg protein (Kocholaty et al., 1971), 18.9 1.4 inte rnational units/mg (Moran and

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71 Geren, 1979), 10.80 turbidimetric units/g PLA2 (fractionated; Bhargava et al., 1970), and 306 to 825 mol product/min/mg (603 to 825, conanti ; 306 to 625, leucostoma ; 430 to 771, piscivorus ; Tan and Ponnudurai, 1990). Studies of A. piscivorus PLA2 published prior to 1984 have suffered in that what is reported is activity based on s ubstrate used or product produced. This is a non-specific assay, in which results are clouded by the presence of multiple forms of PLA2 and possible effects from other venom components. While these types of studies are useful, they do not give indications of the separ ate mechanisms of acti on of the two known PLA2 forms. The two forms di ffer in some small ways, but are named based on the amino acid residue located in the 49th position. The first group of described PLA2s is termed Asp-49 (or AppD49, for A. p. piscivorus aspartate 49) due to the presence of an aspartate, and this was thought to be the case for all PLA2s. However, a form in which the aspartate is replaced by a lysine residue (group name Lys-49; AppK49) was subsequently described (Maraganore et al., 1984) AppD49 is found in dimeric form with a specific confor mation that allows Ca2+ ions to activate i t, but AppK49 does not have this binding region (Mar aganore and Heinrikson, 1986). The mechanism of action of AppD49 against phospholipids has been extensively studied, and the general scheme follows. A monomer of AppD49 normally binds a Ca2+ ion, thereby giving a conformational change that allows the PLA2 to bind a lipid substrate (Bell and Biltonen, 1989a). Al though AppD49 may also bind without Ca2+, it has no enzymatic function without it (Scott et al., 1986). Follo wing binding of Ca2+, each PLA2 undergoes autocatalyic acylation and di merizes to become an active enzyme (Cho et al., 1988a). It appears that the mo nomer form of AppD49 has very little

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72 enzymatic activity, and that the time required fo r it to dimerize acc ounts for the lag time seen in enzymatic reactions (Bell and Bilt onen, 1989b, 1992; Bell et al., 1995). Lathrop et al. (2001) further exami ned lipid binding of AppD49 and conjectured that there is either a conformational change when the enzyme is activated, or there is a ternary complex formed by the combi nation of lipid, enzyme, and Ca2+. Further exhaustive studies of the kinetics of A. piscivorus PLA2 have examined the molecular dynamics, including electrostatic interactions (Stahelin and Cho, 2001; Tatulian, 2001; Leidy et al., 2004; Diraviyam and Murray, 2006). AppK49 was, for a time, consi dered an inactive form of PLA2 because of the inability to dimerize and lack of lipid-catal ylzing enzymatic activity (Scott et al., 1992; Baker et al., 1994). However, it was late r determined that AppK49 has physiological functions that do not mirror those of AppD49, and is just as active in other respects (Dhillon et al., 1987). Although it was initially thought to have lower lipid catalytic ability, AppD49 does not catalyze lipid substrates at all (Van den Bergh et al., 1988). It does, however, affect tissues in mouse heart and nerve diaphragm preparations (Dhillon et al., 1987; Condrea, 1989) and acts as a myotox in (Pedersen et al., 1995) by binding to long-chain fatty acids. By utilizing the te rminal amino acid sequence of AppK49 and making a synthetic peptide, it has been determined that the myotoxic e ffects are a direct result of the sequence of amino acids in the 115 to 129 residue range (Nez et al., 2001; Lomonte et al., 2003b). AppK49 wo rks as a homodimer and also has been determined to be a vascular endothelial growth factor (Yamazaki et al., 2005a, 2005b). The synthetic C-terminal end sequence has been utilized to examine effects of AppK49 on toad bladder cells, where it did not increas e permeability (Leite et al., 2004), and has

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73 been found to not discriminate between normal and tumor muscle cells (Araya and Lomonte, 2007). Further, the synthetic pepti de (which is only in monomer form) has reduced activities with lower pH (Angulo et al., 2005). The structures of both forms have been eluc idated as to their amino acid chain, and crystal and NMR structures They have also been examined for active sites via site-directed mutagenesis. The primary ami no acid structures of both forms have been reported (Maraganore and Hein rikson, 1986, 1993; Maraganore et al., 1987; Van den Bergh et al., 1989; Welches et al., 1993), and i ndicate many conserved regions, but with important differences. The failure to bind Ca2+ has been conjectured as the reason for lack of lipid catalytic activity in AppK49 (Van den Bergh et al., 1989), as the conformation of this form apparent ly causes the space where Ca2+ would normally bind to be filled, thereby blocking Ca2+ (Scott et al., 1992). T he 7 amino acid extension found on the AppD49 form is not considered the reason for their ability to dimerize (Welches et al., 1993). Angulo et al. (2005) examined AppK49 and indicated that although dimerization increases myotoxic and cytot oxic activities, it is not essential for some effect. AppD49 has been cloned into bacterial cells to produce the molecule for studies of its structure (Lathrop et al., 1992). The crystal struct ure has been examined for both AppD49 (Han et al., 1997) and AppK49 (Sco tt et al., 1986, 1992; Holland et al., 1990), and the secondary structure of AppD49 has been determined via NMR spectroscopy (Jerala et al., 1996). Detailed examinations of the dynamics of PLA2 have been conducted for elucidating the catalytic network (conserv ed, functional residues—Demaret and Brunie,

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74 1992) and examining effects of rippling bilayers (Leidy et al., 2004) on activity. Other studies have modulated assay conditions to examine interfacial kinetics between PLA2 and lipid substrates (Bell and Biltonen, 1989 a; Bell et al., 1992; Dua and Cho, 1994; Baker et al., 1994; Bell et al., 1995; Sheffi eld et al., 1995), and it has been proposed that the two PLA2 isoforms may act synergistically to affect a larger array of lipid structures than either form coul d individually (Shen and Cho, 1995b). There has been some slight disagreem ent in the classification of PLA2, with some considering it an acidic protein (Powlick and Geren, 1981; Lynch, 2007) or a basic one (Augustyn and Elliott, 1970). Possibly clarif yng the issue, Mar aganore et al. (1987) stated that there is an acidic dimer (the dimerized form of AppD49) and two basic monomers (the two components of the dimerized AppK49). In a series of experiments, A. piscivorus venom applied to squid nerve axons allowed better transport of neurotransmitte rs and other chemicals across the cell membrane (Rosenberg and Ng, 1963; Rose nberg and Podleski, 1963; Hoskin and Rosenberg, 1965; Rosenberg, 1965; Ros enberg and Hoskin, 1965; Condrea et al., 1967; Rosenberg and Dettbarn, 1967), indicating PLA2 activity. This occurred both with whole venom and an isolated fr action that contained PLA2 (Condrea et al., 1967). Treatment also reduced electrical activity normally elicited by acetylcholine (Rosenberg and Podleski, 1963). This effect was s een more with crudely-dissected axons, indicating the venom may be responsible for disrupting Schwann cells, but not the axolemma (Rosenberg and Hoskin, 1965). Furt her evidence of this was the Schwann cell structural changes (vacuol ation of cytoplasm) seen in some preparations (Martin

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75 and Rosenberg, 1968). Venom from Agkistrodon contortrix was not as effective at making squid cells competent to cu rare (Rosenberg and Podleski, 1963). Cottonmouth venom also has been examined for its effects on the electroplax of the electric eel, Electrophorus electricus It causes irreversible depolarization at higher concentrations and antagonizes depolarizatio n caused by carbamylcholine at lower concentrations (Bartels and Rosenberg, 1972), bot h of which indicate activity of PLA2. At the concentrations used in this series of studies, PLA2 was found to disrupt mitochondrial function without destroying t hem (Rosenberg, 1976). Previous studies have also noticed the ability of App PLA2 to cause increased mitochondrial respiration with small concentrations and severe inhi bition of electron transport at higher concentrations (Petrushka et al., 1959; Augu styn et al., 1970). Another study utilized A. piscivorus PLA2 to examine changes in the bacteria Escherichia coli after exposure to bactericidal/permeability-incr easing proteins from neutroph ils (Forst et al., 1987). Further animal studies with A. piscivorus venom have indicated that PLA2 is responsible for edema formation in the mouse paw model (Marshall et al., 1989). It is reported that response is dose-dependent, and the mode of action may be via a histamine/serotonin release from basophils or mast cells (Calhoun et al., 1989). When AppK49 was used alone, edema occurred, bu t skeletal muscle was also lysed (Nez et al., 2001). Cottonmouth PLA2 has been investigated fo r potential treatment of hypercholesterolemia in humans (Shen and Cho, 1995a), and also has been studied as a potential method of molecu lar packaging, due to its c onformation (Shen et al., 1994). Phospholipase II (phospholipase B). Two studies have indicated the presence of phospholipase B (PLB) activity in cottonmout h venom. This activity was found only at

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76 high pH, and increased from pH 8.5 to 10.5, with no activity being seen from pH 4.5 to 8.0 (Doery and Pearson, 1964). Using lysophosphatidylcholine as substrate, the activity of PLB was recorded as 0.51 0.10 equivalents of free fatty acids at pH 9.4 (Fletcher et al., 1979). Other enzymatic activities Aminopeptidase. A. piscivorus venom has been found to have leucine aminopeptidase activity because it strongly (Michl and Molzer, 1965) or moderately (Tu and Toom, 1967) degrades the substrate L-leucyl-naphthylamide. This activity has been seen in other studies, as well, by separating a fraction that contains the activity (Wagner et al., 1968; Prescott and Wagner, 1976). Although they later indicate aminopeptidase activity, Wagner and Prescott (1966a, 1966b) in itially indicated that hydrolysis of L-leucyl-naphthylamide and L-alanyl-naphthylamide were not done by a true aminopeptidase. ATPase. Activity toward ATP has been seen at both a low (Zeller, 1950, 1951) and moderate (Mebs, 1970) le vel. Addition of Mg+2 did not affect activity (Zeller, 1950). Cholinesterase. Despite multiple attempts, no c holinesterase activity has been found in A. piscivorus venom (Zeller, 1947, 1948, 1949; H adidian, 1956; Mebs, 1970). Because multiple studies yielded the same results, this seems to indicate either complete absence or below-detection-leve l activities of cholinesterase in A. piscivorus venom. Collagenase. Direct digestive effects of A. piscivorus venom on collagen have been examined, with basically no or low ac tivity reported. Although no collagen digestion was seen, mice injected wit h venom showed an increase in urine hydroxyproline, which is an indicator of collagen degradation (Kaiser and Rabb, 1971).

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77 Rat collagen shows a reduction in viscosity when exposed to A. p. leucostoma venom (Simpson et al., 1971). Weak collagenase activity has been seen (Moran and Geren, 1979), but has been attributed to the action of proteas es (Szabo and Gennaro, 1978). Elastase. One study has indicated el astinolytic activity in A. piscivorus venom, and indicated that it was among the highest of those species examined (Bernick and Simpson, 1976). However, no other examin ations of this enzyme in cottonmouth venom have been conducted. Fibrinogenase. Atlhough thrombin-like enzymes cause the conversion of fibrinogen into fibrin, thereby enhancing in itial blood clotting, fibrinogenases are enzymes responsible for cleaving fibrinogen in a manner that does not lead to clotting (Markland, 1991, 1998). The fact or responsible for fibrinogenase activity in cottonmouth venom has been characterized and is called -fibrinogenase (Nikai et al., 1988b). It is a 33.5 kDa molecular weight molecule with an isoelectric point at pH 4.5 and a venom concentration of 2.5 mg/g crude venom. It is stable at pH 2 to 10 and when mildly heated, and is inactivated by benzamidine, 2-mercaptoethanol, N-bromosuccinimide, and diisopropylfluorophosphate, wh ich indicates its serine structure is important for activity. It has both esterase and kinin-releasing activity. Hahn et al. (1995) have described two fibr inolytic enzymes (piscivorase I and II) that also have fibrinogenolytic activity. Pisc ivorase I has a molecular weight of 23.4 kDa and cleaves both the A and B chains of fibrinogen, while pi scivorase II is 29.0 kDa in size and primarily cleaves the A chain.

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78 Nucleosidase (NADase). Two studies have examined cottonmouth venom nicotinamide adenine dinucleosidase (NADase). In these studies, NADase had either weak (Moran and Geren, 1979) or moderat e (Tatsuki et al., 1975) activity. Non-specific esterase. Other than studies of specific activities of phosphodiesterase, alkaline phosphomonoeste rase, and arginine esterase, many researchers have examined the ability of sn ake venom to hydrolyze ester compounds in terms of overall venom activity. Cottonmout h venom is reported to have low activity toward t-BOC-L-alanine p -nitrophenyl esterase (Bernick and Simpson, 1976) and no activity toward -naphthyl acetate. Using Sephadex G-50 column separation, general esterase activity was found to be greatest in the first fraction (Bonta et al., 1970b; Bhargava et al., 1970, 1972). Venom esterase was not inhibited by snak e serum from an unreported species (Philpot et al., 1978). Non-specific protease. Multiple proteolytic enzymes occur in cottonmouth venom, and many studies have ex amined general protease acti vity toward a substrate rather than isolating spec ific enzymes. The terms protease and proteinase are interchangeable and can be found in both forms throughout the literature. Many studies reporting proteolytic activity have utilized ester-containing substrates for hydrolysis studies. These are not included here (see N on-specific esterase section above). Proteolytic activity (hydrolysis) ha s been found against prothrombin (Hadidian, 1956), fibrinogen (Hadidian, 1956), urea-de natured hemoglobin (Wagner and Prescott, 1966), casein (Kocholaty and Ashley, 1966; Wagner and Prescott, 1966; Kaiser and Rabb, 1967; Oshima et al ., 1969; Mebs, 1970; Kocholaty et al., 1971; Moran and

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79 Geren, 1979; Powlick and Geren, 1981), gel atin (Kaiser and Rabb, 1967; Tan and Ponnudurai, 1990; Ownby et al., 1994), az ocoll (Kaiser and Rabb, 1967; Szabo and Gennaro, 1978; Ownby et al., 1994), L-amino acids (Spiekerman and Prescott, 1968), N-benzoyl-DL-argininep -nitroanilide (Kocholaty et al ., 1971), trypsin (Kress and Paroski, 1978), chymotrypsin (Kress and Paroski, 1978), and hide powder azure (Powlick and Geren, 1981). Another study (Osaka et al., 1966) reported proteinase activity, but without indicating the substrate used. Proteolytic activity against hemoglobin was not affected by presence of Mg2+, Ca2+ or Zn2+ (Wagner and Prescott, 1966). General protease activity was inhibited by the use of cyanide (Hadidian, 1956); Co2+, Mn2+, Ni2+ or Cd2+ ions (Wagner and Prescott, 1966); EDTA (Philpot, 1959; Wagner and Prescott, 1966); p -chloromecuribenzoate (Wagner and Prescott, 1966), cysteine (Wagner and Prescott, 1966), penicillamine (Philpot, 1959), and MFA (Giroux and Lachemann, 1981). Protease activity was also found to be inhibited by the presence of se rum (Philpot et al., 1978), presence of human plasma 2 macroglobulin (Kress and Catanese, 1981) and exposure to ultraviolet light (Tejasen and Ottolenghi, 1970). In this last st udy, they noted that the loss of activity was not strictly a time-dependent function, indicating there may be more than one proteolytic factor. Bonnett and Guttman (1 971) found that protease activity was inhibited by the seru m of the kingsnake. The factor responsible for proteolytic activity in cottonmouth venom has been described as having a molecular weight of 22.5 kDa, with an isoelectric point at pH 6.5 (Wagner et al., 1968). In another study, it was determined to have a molecular weight of 26 kDa, contain 1 mo le of Zn and 2 of Ca2+ per mole of enzyme, and function

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80 optimally at pH 8.5 (Wagner and Prescott, 1966) Kornalk (1971) indicated that its function and structure ar e not identical to either plas min or trypsin and others have stated that is not the same as kallikre in or trypsin (Bhargava et al., 1972). Separation studies have utilized ion exch ange (Clark and Higginbotham, 1970) and Sephadex A-50 column chromatography (P rescott et al., 1976). This last study noted five different proteolytic components from their second fraction, indicating multiple factors with proteolytic capacity. Non-specific Peptidase. Outside of studies of aminopeptidase, the only substrate-based examination of the ability of cottonmouth venom to degrade small peptides was conducted by Tu and Toom (1968). They found most di-, tri-, and tetrapeptides examined were degraded by venom. Nonenzymatic proteins/peptides Cysteine-rich secretory proteins. One instance of cyst eine-rich secretory proteins (CRISPs) has been reported in A. piscivorus venom (Yamazaki et al., 2003). The authors isolated the protein, reported its amino acid sequenc e, and used it to show reduced muscle contractility of injected rat tails. Nerve growth factors. Although its function in enven omated prey has not been examined, a nerve growth factor (NGF ) has been isolated from cottonmouth venom (Cohen and Levi-Montalcini, 1956a). This factor was isolated (by DEAE cellulose electrophoresis) and used to increase growth in both sensory and sympathetic nerve ganglia (Cohen and Levi-Montalcini, 1956b). Both crude venom and the isolated fraction were found to increase nerve growth in a linear fashion, and venom had 1000x the potency of mouse tumor growth factor (Levi-Montalcini and Cohen, 1956). The

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81 factor is approximately 20 kDa in mole cular weight (Cohen, 1959) and is similar, immunologically, to other snake, but not mouse, NGFs (Bailey et al., 1976). C-type lectins. A. piscivorus venom contains lectins, wh ich aid in agglutination. Gartner and Ogilvie (1984) reported lectin concentrations of 9.8 mg/g venom and indicated that the venom had aggl utination capability of 4.7 X 108 units/mg lectin (Gartner and Ogilvie, 1984). They described the isolated lectin as dependent on Ca2+ and inhibited by chelators such as EDTA and di thiothretol. Their lectin had a molecular mass of 28 kDa (two 14 kDa units connect ed by disulfide bridges) and an isoelectric point at pH 7.2 to 7.3. A late r study described APL, the specific A. piscivorus lectin, as being composed of homodimers, each having a mo lecular weight of 16.2 kDa, linked by a disulfide bridge (K omori et al., 1999). Disintegrins. Two forms of disintegrins, whic h are inhibitors of platelet aggregation in the blood, have been described from A. piscivorus venom: applaggin and piscivostatin. Applaggin was initially described as a homodimer because of its 17.7 kDa size when unreduced by thiol and 9.8 kD a when reduced (Chao et al., 1989), but others have indicated it does not dimerize (Wencel-Drake et al., 1993). Regardless of dimerization capability, the monomeric form is 71 amino acids in length and binds to the Arg-Gly-Asp region of the integrin GPIIb /IIIa, which is necessary for normal blood clotting activity (Savage et al., 1990). By bi nding to this region, app laggin distrupts the clotting cascade. Attempts to determine the structur e of applaggin by X-ray crystallography have not been successful, due to low diffraction (Arni et al., 1999). The second A. piscivorus venom disintegrin, piscivost atin, was more recently described as having a heter odimeric structure with and chains of 65 and 68 amino

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82 acids, respectively (Okuda and Morita, 2001) It appears to work by inhibiting both platelet aggregation and (i f aggregation does occur) platelet disaggregation (Okuda and Morita, 2001). The crystal structure of piscivostatin has been examined (Fujii et al., 2002). Studies on both disintegrins have been reviewed in comparison with other known disintegrins from snake venom (McLane et al., 2007). Bradykinin potentiating protein. A. piscivorus venom appears to have activity similar to the bradykinin pot entiating peptide (BPP) found in Bothrops jararaca BPP is an inhibitor of both angiotensin converting enzyme (ACE) and angiotensin I, so it works to reduce vasoconstriction, leading to a drop in blood pressure Cottonmouth venom has been found to be a mild inhibitor of ACE in rabbits (Sander et al., 1972). Ferreira et al. (1995) found one separated fraction, which they termed AppF, to have BPP activity on isolated guinea pig ileum and a molecular weight of 1224.2 Da, determined by mass spectrometry. They later examined the isolated AppF using NMR spectroscopy and found it to have two conformati ons (Ferreira et al., 1999). Small organic compounds Citrate. Citrate has been found to be the major organic acid in A. piscivorus venom (Freitas et al., 1992). It is likely that presence of citrate stabilizes venom activity by inhibiting enzymes from catalysis within the venom gland. Neurotransmitters. Some work has been done in an attempt to find neurotransmitters in A. piscivorus venom, with somewhat equivocal results. Norepinephrine was present in venom gland extract, but neither it nor serotonin was detected in cottonmouth venom (Ant on and Gennaro, 1965). Indole-reacting compounds have been detected in cottonmouth venom using three different assay methods, although the author of this study cautioned that the nonspecificity of the

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83 assay makes it impossible to definitively stat e that serotonin is present (Welsh, 1966). In a similar study, an acet ylcholine-like substance was detected (Welsh, 1967). Toxins Neurotoxins. Although it has been suggested t hat cottonmouth venom has a neurotoxic effect (Brown, 1941), a proven neur otoxin has never been isolated. Indeed, venom effects on nerves seem to be limited to phospholipase A2 activity causing degradation of cell membranes Hadidian (1956) indicated some evidence for neurtoxicity, but concluded that loss of nerve impulses were secondary effects. One study (Braganca, 1955) has reported a neurotox in of molecular weight 4 kDa, but another (Micheel and Jung, 1936) reported an i nability to purify a neurotoxin. If a neurotoxin does exist in cottonmouth venom, it does not seem to have a major effect. Mojave toxin. A. piscivorus venom has not been found to contain Mojave toxin, unlike some species of crotaline rattlesnakes (W einstein et al., 1985). While it is likely that this particular component is not found in co ttonmouths, it is possible that it is simply present below detection limits. Vascular endothelial growth factor The Lys49 form of A. piscivorus phospholipase A2 has been characterized as a vascular endothelial growth factor because of it s ability to stimulate cell proliferation in vitro (Yamazaki et al., 2005a, 2005b). The snake-derived form is called KDR binding protein (KDR-bp), and it also i nduces hypotension in rats and binds with high affinity to kinase insert domain-containing receptor (KDR, also known as vascular endothelial growth factor receptor 2). KDR-bp is a weak vascular permeability enhancer (Yamazaki et al., 2007), and its functional binding site appears to be the C-terminal loop (Fujisawa et al., 2008).

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84 Potential Components Recently, Jia et al. (2008) constr ucted and analyzed a complementary DNA (cDNA) library for cottonmouth ( A. p. leucostoma ) venom gland tissue. This information can now be used to examine homology amo ng similar components in different species and to find and characterize novel toxins. From this work the first venom gland-specific, non-venom component—parvalbumin—has been characterized and expressed in E. coli (Jia and Perez, 2009). The cDNA library also has been used to characterize two different forms of metalloprot einase from the venom, both of which have activity against fibrinogen (Jia et al., 2009). Venom Studies Human Disease Clinical Research Cottonmouth venom has been examined as potential treatment for a number of human diseases, although it wa s noted that intradermal inje ction of venom in humans causes reddening of the skin (Essex, 1932). Most of these studies occurred in the 1930’s and were concerned with various he matological conditions. In general, intradermal injection of weak concentrations of venom causes a wheal with surrounding redness and ecchymoses, and these conditi ons usually disappear after multiple injections (Peck, 1933a; Peck and Rosenthal, 1935). However, in some cases (75% of patients considered “allergic” and 50% of patients considered “non-allergic), hypersensitivity occurs and is expressed as a hot, red, tender swollen area (Peck, 1933a). In such cases, desensitization by reducing venom concentration was usually successful (Peck, 1933a; Peck and Rosenthal, 1935). In a number of cases, patients had bad reactions and needed to be removed from venom therapy (Peck and

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85 Goldberger, 1933), and there appears to be a bal ance between the efficacy of treatment and hypersensitivity (Peck and Rosenthal, 1935). Venom was used as a treatment, wit h at least some success, for thrombocytopenic purpura (Peck, 1932; Greenw ald, 1935), Schonlein-Henoch’s purpura (Peck, 1932), hemophilia (Pe ck, 1932), uterine bleeding (Peck and Goldberger, 1933; Goldberger and Peck, 1937), postpartum bleedi ng (Davin et al., 1937), epistaxis (Goldman, 1936; Dack, 1935), urticaria (Coh en, 1951). Venom therapy does not seem to have an effect on congenital hemophilia (Peck and Rosenthal, 1935), scarlet fever (Schneierson et al., 1936), or uterine bl eeding caused by fibromyomas (Goldberger and Peck, 1937). For the study of the use of venom in reduc ing post-partum bleeding, it was found that reduced blood loss during birt h occurred when venom was given prior to birth. The offspring of such births we re apparently not affe cted by the pre-partum venom, as indicated by a lack of antivenom in the cord blood (Davin et al., 1937). In a study of the use of venom in treating th rombocytopenic purpura, active bleeding was stopped, general bleeding was much reduced, and platelet counts increased, indicating that venom was working to reduce blood lo ss from capillaries (Greenwald, 1935). The venom also has been used as a supplementary test for diagnosis of various blood diseases via examination of capillaries. A positive test (when capillaries rupture due to the venom) can indicate or rule out ce rtain diseases, and a change in this test result can indicate recovery (Peck et al., 1936, 1937a). Diseases in which this test has shown to be diagnostic or non-effective hav e been tabulated (Peck, 1937b). Brabec and Kornalk (1977) examined the effect of A. piscivorus venom on blood from normal and diseased patients. They found that, co mpared to RBCs from normal patients,

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86 RBCs from patients with autoi mmune hemolytic anemia were not differently lysed, those from patients with hereditary spherocytosis s howed slightly greater lysis, and those from patients with paroxysmal nocturnal haemoglobinur ia showed much greater lysis. These effects only occurred in the presence of plasma indicating complement is activated. In an additional study of the effects on A. piscivorus venom on blood from patients with 16 different hematological diseases, high hemol ysis was only seen in blood from patients with paroxysmal nocturnal hemat oglobinuria, and only if complement was also present (Žk et al., 1984; Brabec et al., 1985). Immunology Cottonmouth venom has been used for imm unological studies because of its ability to reduce the Shwartzman phenonmenon normally seen with multiple exposures to allergens. This reduction was seen both in humans (Peck and Rosenthal, 1935) and rabbits (Peck and Sobotka, 1931; Peck, 1933b), and this ability was reduced in the presence of either A. piscivorus antivenom or horse serum (Peck, 1934). Agkistrodon.piscivorus venom has been examined for immunological reactivity toward venoms or antivenoms produced using the venom of other species. It was found that neither Daboia nor Echis antivenom protected pigeons against cottonmouth venom, although Daboia antivenom did specific ally neutralize the hemo rrhagic effects (Taylor and Mallick, 1965). In agar plate immunodiffu sion studies, there was evidence of antigens that were similar to or shared with Agkistrodon contortrix and Crotalus atrox venom (Minton, 1957). Tu and Adams (1968) used immunodiffusion to show A. piscivorus venom to be more similar to A. contortrix than to A (= Calloselasma ) rhodostoma venom. Using other venom s, it was found that co ttonmouth venom did not immunoreact with Bungarus fasciatus or Naja naja venom or antivenom, although it did

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87 show precipitation with polyvalent crota lid antiserum (Munjal and Elliott, 1972). Polyvalent antivenom, conspecific antivenom, and heparin all visibly complexed with cottonmouth venom in vitro (Clark and Higginbotham, 1965a). Although both treatments caus e loss of toxic activities, venom exposed to X-ray (Flowers, 1963, 1966) or ultraviolet radiat ion (Tejasen and Ottolenghi, 1970) maintains its antigenicity. Both of these studies were attempts to ma ke inoculation of antivenom-producing animals less toxic wh ile still creating useful antivenom. The ability of serum from other snake species to neutralize cottonmouth venom has been examined for both venomous and non-v enomous species. Some neutralizing capacity was seen in serum from the venomous species Crotalus adamanteus (Philpot and Deutsch, 1956; Philpot et al., 1978), Agkistrodon contortrix (Philpot et al., 1978; Weinstein et al., 1991), and conspecifics (Philp ot et al., 1978). Non-venomous genera showing neutralizing capacity include Coluber Natrix Elaphe and Lampropeltis (Philpot et al., 1978). The genus Lampropeltis (king and milk snakes) has been examined in depth due to their habits of consuming veno mous snakes as part of their diets. Lampropeltis species known to show venom neutralizin g effects in their sera include the species L. getula (Ditmars, 1928; Bonnett and Guttman, 1971) and the subspecies L. g. floridana (Philpot and Smith, 1950a, 1950b; Philpot and Deutsch, 1956; Weinstein et al., 1992). Besides L. g. floridana Weinstein et al. (1992) al so found neutralizing capacity in L. alterna L. calligaster L. g. getula L. g. californiae L. g. holbrooki L. mexicana greeri L. triangulum hondurensis L. t. triangulum and L. ruthveni (but not for L. g. splendida ). Resistance to cottonmouth venom also has been seen in mammals including the opossum ( Didelphis viriginiana —Kilmon, 1976; Werner and Vick, 1977;

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88 Ramrez et al., 1999), the woodrat ( Neotoma micropus —Perez et al., 1978; Ramrez et al., 1999) the Mexican ground squirrel ( Spermophilus mexicanus —Ramrez et al., 1999), and the cotton rat ( Sigmodon hispidus —Ramrez et al., 1999). Monoclonal antibodies developed using Crotalus atrox venom had strong reactions against A. piscivorus venom (Perez et al., 1984), as did anti-protein C activator developed using A. contortrix venom (Stocker et al., 1987). A further cross-reaction was found between antifibrolase antibody from A. contortrix contortrix and fibrinolytic enzyme from A. p. conanti (Chen et al., 1991). Conclusion Studies of the venom of A. piscivorus are diverse and many. While they have examined overall effects of venom on humans and different animal models, they have also specifically scrutinized individual veno m components to the molecular level. This research has indicated that cottonmouth venom has many different components with various physiological effects, some of which have been characterized, and some of which remain to be studied. Most of the work has focused on physiological effects or efficaciousness of antivenom, and much has also been done using venom as a research tool. However, mu ch work remains to be done in the areas of ecology and evolution of the venom in order to truly understand it.

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89 Table 2-1 Published records of cottonmouth bites and bite-caused fatalities. Where possible, number of mortalities directly credited to cottonmouths and geogr aphic locations are given. Each study was done slightly differently and had different methods of analyzing data. Unreported values are indicated by “--.” Overall, the data show that cottonmouths are responsible for a high percentage of snake bites in the United States, but are not responsible for many deaths. Bites by Bites by Cottonmouth Mortalities Time span Geographic location source cottonmouths all snakes bites (%) 43 458 9.411928United Stat es Hutchison, 1929 194 1973 9.88--United States Githens, 1935 7 51 7.301948Florida Andrews and Pollard, 1953 46 179 25.7--1962Florida Sowder and Gehres, 1963 33 461 7.2--1958-1959Texas Parrish, 1964 133 1538 8.6--1958-195910 US statesa Parrish et al., 1965, 1966 9 64 14.101927-1965Florida and Georgiab Watt and Gennaro, 1965 208 2836 7.3--1958-1959United Statesc Parrish, 1966 755 6680 11.3--1959United Statesd Parrish, 1967; Parrish and Donnell, 1967 69 382 18.101963-1966Florida Andrews et al., 1968 6 204 2.9----Western United Statese Russell, 1969 46 177 26.0--1962Florida Sowder and Gehres, 1968 38 168 22.6--1963Florida Sowder and Gehres, 1968 38 185 20.5--1964Florida Sowder and Gehres, 1968 38 198 19.2--1965Florida Sowder and Gehres, 1968 49 190 25.8--1966Florida Sowder and Gehres, 1968 32 158 20.3--1967Florida Sowder and Gehres, 1968 7 45 15.6--1969-1976South Carolinaf Sabback et al., 1977 26 107 24.301927-1977Florida and Georgiaf Watt, 1978 1 32 3.101970-1977United Kingdomg Reid, 1978 aThe states in this study were Arkansas, Arizona, Georgia, Loui siana, Mississippi, North Carolina, Oklahoma, South Carolina, Tex as, and West Virginia. bTwo counties were analyzed from each state. cBased on data reported by hospitals. dSame data as Parrish (1966), except combined with physicians reports and estimat ed for non-participating hospitals. eIncludes non-native, captive snakes. fBoth studies included data from one hospital. gInstances of bites from non-native specimens.

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90 Table 2-2. Published toxicity values for cottonm outh venom. Median lethal dose (LD50) is the concentration of venom required to kill 50% of study animals. Units are mg ly ophilized venom/kg body weight, unless otherwise noted. The routes of administration are: IP—intraperitoneal, IV—intravenous SC—subcutaneous. For comparison, variations on these studies are listed at the bottom of the table. These studies included determinations of LD50 in physiologically altered animals and determi nations of alternate toxicity values (LD99 and LD100). LD50 Route Animal Source Notes model LD50 studies: 5.11 IP Russell, 1967b 4.844 IP mouse Kocholaty et al., 1971 5 to 6 IP mouse Hadidian, 1956 5.10 IP mouse Russell, 1980 5.1 IP mouse Weinstein et al., 1991 5.33 IP mouse Weinstein et al., 1992 6 IP mouse Powlick and Geren, 1981 6.85 IP mouse Minton, 1956 10 IP mouse Huang et al., 1972 4.00 IV Russell, 1967b 60.0a IV mouse Ohsaka et al., 1966 64a IV mouse Clark and Higginbotham, 1965a 75 5.5a IV mouse Clark and Higginbotham, 1971 2.044 IV mouse Kocholaty et al., 1971 3.35 IV mouse Flowers, 1963 3.9 IV mouse Gingrich and Hohenadel, 1956 4.17 IV mouse Russell, 1980 11.3 to 13.6 SC mouse Moran and Geren, 1979 25.10 SC mouse Russell, 1980 25.85 SC mouse Minton, 1956

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91 Table 2-2. Continued LD50 Route Animal Source Notes model Other toxicity studies: 3a IV mouse Clark and Higginbotham, 1965a Immunized, adrenalectomized 39a IV mouse Clark and Higginbotham, 1965a Immunized 44a IV mouse Clark and Higginbotham, 1965a Non-immunized, adrenalectomized 3 0.5a IV mouse Clark and Higginbotham, 1971 Adrenalectomized, sensitized 20 IV mouse Flowers, 1963 X-ray irradiation 5.26 IV mouse Vick et al., 1967 LD99 0.75 IV dog Vick et al., 1967 LD99 40 IP frog Hall and Gennaro, 1961 LD100 6.2 IP mouse Hall and Gennaro, 1961 LD100 aValues listed are in g/animal. bAnimal model not reported.

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92 Table 2-3. Reported minimum and mean lethal doses for cottonmouth venom. Minimum lethal doses are defined as the lowest dose used that killed the species studied. Mean lethal dose is t he average of doses that killed animals in the study. The routes of administrat ion are as follows: IP—intraperitoneal, IV—intravenous, SC—subcutaneous. Value Units Animal Route Source model Minimum lethal dose: 4 mg/kg mouse IP Gennaro and Ramsey, 1959a 7 mg/kg mouse IP Gennaro and Ramsey, 1959a 2.5 mg/kg rabbit IP Noguchi, 1909 0.06 mg/animal pigeon IV Githens, 1935 0.1 mg/animal pigeon IV Taylor and Mallick, 1936 0.15 mg/kg mouse IV Izard and Boquet, 1953 2 mg/kg rabbit IV Izard and Boquet, 1953 2.46 mg/kg dog IV Vick, 1973 0.015 mg/animal mouse SC Schttler, 1951a 1 mg/animal mouse SC Noc, 1904 30 to 50 mg/animal mouse SC Noc, 1904a 0.10 mg/kg mouse SC Izard and Boquet, 1953 1 mg/kg guinea pig SC Izard and Boquet, 1953 Mean lethal dose: 0.11 mg/animal pigeon IV Githens, 1935 32.4 mg/kg mouse SC Schttler, 1951a aVenom heated to 80C prior to use.

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93 CHAPTER 3 VENOM EXTRACTION FROM ANESTHE TIZED FLORIDA COTTONMOUTHS Introduction Researchers who work with venomous or ganisms must ensure the well-being of both their animals and themselves through good safety practices. These precautionary practices are complex, especially when studi es require venom extraction from live snakes. Collection techniques should fa cilitate the study’s goals and experimental design, proper treatment of the animals, and safety of th ose coming into contact with the animals. Safety precautions will, theref ore, vary depending on t he study goals. In commercial antivenin production the goal is to maximize venom yield. In long-term studies of recaptured wild snakes, however, c onsistency, repeatability, and portability in the field are equally important These goals require diffe rent extraction techniques. Many studies utilizing venom do not deta il collection methods, but previously described venom collection techniques for snak es include spontaneous ejection (where the snake bites through a membrane into a colle cting cup—di Tada et al., 1976; Tare et al., 1986), forced ejection by glandular mass age (di Tada et al., 1976; Mackessy, 1988) and evoked ejection using electrical stimul ation (Johnson, 1938; Gans and Elliott, 1968; Glenn et al., 1972; Marsh and Glatston, 1974; Johnson et al., 1987; and Vieira et al., 1988). For any method the animal can be conscious or anesthetized. The use of halothane for inhalant anesthesia was firs t described by Hackenbrock and Finster (1963), and other inhalants (i.e. methoxyflurane, isoflurane, and sevoflurane) have also been utilized. Some studies have used inject able anesthetics (i.e. ketamine, propofol),

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94 and others have combined anesthesia with saliva-inducing chemicals such as pilocarpine to increase venom yield (Hill and Mackessy, 1997). Grasping snakes behind the head and allowing them to bite through a membrane over a collecting vessel (spontaneous ejection) yields large amounts of venom. It can be, however, unsafe and difficult to standardize The snakes are made to bite multiple times, with decreased venom yields in consec utive bites, indicating incomplete emptying of stored venom. Further, there is contr adicting evidence concerning the ability of snakes to meter venom ejection (Hayes, 1993, 1995; Hayes et al., 1995; Young et al., 2002, Young and Zahn, 2001), a fu rther factor potentially addi ng to the difficulty of quantifying total venom volume for snakes t hat are conscious. Any physical restraint during extraction is likely stressful to the animals, possibly resulting in altered behavior and physiology. Most importantly, manual rest raint of venomous snakes increases the chance of human envenomation Anesthesia has previously been used to increase safety and potentially reduce stress in snakes during venom collection by reducing the contact time in which the animals are conscious. In most studies the venom glands were massaged to eject the venom into a collecting vessel or capi llary tube (Mackessy, 1988). Although this technique works well with large viperids, it does not ensure collection of the entire venom store due to variation in squeeze force and massage technique. Further, massage may alter venom composition, which in at least one case has been observed as differences in turbidity and color (Ma ckessy, 1988). Also, in the neotropical rattlesnake ( Crotalus durissus ), massage has been associated with higher incidence of glandular infection (di Tada et al., 1976).

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95 Electrical stimulation is common fo r venom collection from arthropods (Hemiptera—Sahayaraj et al., 2006; Hy menoptera—Davies et al., 2004; BrochettoBraga et al., 2006; Araneae—Escoubas et al., 2002; and Scorpiones—Candido and Lucas, 2002) and other invertebrates (Anthozoa— Malpezzi et al., 1993). While it can cause death of small invertebrates, it has been used in conscious snakes without apparent adverse effect (Johnson, 1938; G ans and Elliott, 1968; Glenn et al., 1972; Marsh and Glatston, 1974; Johns on et al., 1987; and Vieira et al., 1988). A few studies have compared collection techniques with ambiguous conclusions. The authors do, however, agree that manual mani pulation and electrical stim ulation yield more venom than does spontaneous ejection in the same s nakes (di Tada et al., 1976; Tare et al., 1986). Here we describe venom collection fr om anesthetized Florida cottonmouths ( Agkistrodon piscivorus conanti ) using electrical stimulation to enhance quantification, and to decrease handling time and envenomation hazard to researchers. Further, our technique uses a relatively inexpensive, por table human medical nerve stimulator that can be used in either the laboratory or the field. Materials and Methods Animals As part of a larger study, 70 wild cottonmouth snakes ( Agkistrodon piscivorus conanti ) were collected from three lo cations in Florida. While in captivity, snakes were maintained individually in commercial fiber glass or plastic caging with newspaper substrate and ad libitum access to water, but were not fed until after venom collection. Venom was collected mostly from adult snakes, but some juveniles were also used for

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96 comparison. Further, three clutches of neonate snakes (n = 17) born in captivity from wild-caught gravid females, were compared. Anesthesia Isoflurane (5% induction, 2.5% maintenance) in oxygen (2 L/min) administered with a precision vaporizer and flow-meter was used in itially for anesthesia (n = 2). This technique produced consistent, reversible a nesthesia but was rejected because of prolonged induction and recovery times, and difficu lty of use in remote field situations. A combination of medetomid ine (100 g/kg) and ketamine (10 mg/kg) administered intramuscularly (IM) in the paravertebral musculature pr oduced short-term immobility in a third (n = 1) snake (395 g), but a higher combination dosage (200 g/kg and 20 mg/kg IM, respectively) failed to produce reliable, safe immobility in a fourth (n = 1) snake (434 g). Glandular massage was attempted in th ese four and two other snakes (n = 2) anesthetized with propofol (10 mg/kg). We were unable to eject venom from any of these snakes using this technique, so altern ate methods of extraction were explored. These six individuals are not included in statistical analysis. Anesthesia in the rest of the snakes (total n = 81; 64 wild caught, 17 captive reared) was accomplished by an intrava scular injection of propofol (Rapinovet, Schering-Plough Animal Health Corporation; 10 mg/ml, 8 to 10 mg/kg body mass) into the ventral coccygeal vessels posterior to the cloaca (Heard, 2001). For physical restraint each snake was induced to enter a hard plastic, transparent snake tube (Lock, 2008) which allowed safe access to the vess els for injection and visualization of the snake during induction. The snake was assessed to be sufficiently relaxed when it could not right itself, could not raise its head wh en in dorsal recumbency, and did not respond

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97 to a strong pinch in the cloacal region. If induction did not occur within 10 to 15 min, a supplemental propofol injection (n = 33), isof lurane (5%) in oxygen and nitrous oxide (n = 4) insufflation into the snake tube, or a sequential combination (n = 2) was administered. These last two groups of animals are not included in subsequent data analysis so that only animals anesthetized sole ly with propofol (n = 75) are compared. Supplemental propofol injections were admin istered via caudal vein (n = 24) or by intracardiac injection (n = 9; Isaza et al., 2004). Venom Extraction Once the snake was determined to be suffi ciently relaxed, its head was extended beyond the end of the snake tube (Figure 3-1). If the animal should move in this position, it could rapidly be pulled back into the tube by grasping its tail and caudal body. Venom was extracted by electrical stimul ation using a portabl e constant current peripheral nerve stimulator (Fisher & Paykel Healthcare). Depending on size, the amperage was set at 10, 20, or 30 mA, with 20 mA being effective for most snakes. Initial attempts using a single twitch or a “train of four” failed to elic it venom expulsion. All successful venom collections used a tetanic pulse of 5 s duration. The alligator clips of the stimulator pr obes were attached across the venom gland, pinching the loose skin near the articulation of the jaw and directly behind or under the eye (Figure 3-2). To provide good contac t for current transmission, the clips were moistened with alcohol. The right and left ve nom glands were stimulated in succession, and venom from both glands was pooled into a clean beaker for quantification. Stimulation of one gland sometimes yielded v enom from both, indicating the pulse was sufficient to elicit muscle contraction over both glands. Crude venom from each

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98 individual was centrifuged at 5000 rpm for 2 min, and the supernatant was aliquoted in volumes of 25 to 100 l for quantification to the nearest 25 l. Demographic Data The sex of each animal was determined usi ng commercial stainless steel sexing probes, and the following measurements were made: mass, snout-to-vent length (SVL), and tail length. Relationships between propofol dose or induction time and animal mass were analyzed using linear regression analysis. Results Induction times after administration of pr opofol into the ventral coccygeal vessels were 12.2 8.5 min (mean 1 standard deviation) whether with (19.0 7.8 min; n = 33) or without (6.8 4.1 min; n = 42) supplemental doses of pr opofol only (no isoflurane). We initially used 10 mg/kg body mass propofol to induce unconsciousness, and although lower concentrations (9.8 1.7 mg/kg) were sufficient in some cases, at other times supplemental doses (total 18.8 5. 6 mg/kg) were needed (P << 0.001; Figure 33). Time to induction was mass-specific, with larger animals taking longer to be affected by anesthesia (P < 0.001; Figure 3-4). For the animals for which we have specific data (n = 15), initial recovery (time when anima ls started moving on their own) occurred in 74.1 +/38.2 min. Venom volume was directly correlated wit h snout-vent length (P << 0.001; Figure 3-5), and mass (P << 0.001; data not shown) with larger anima ls having greater volume. A few animals produced less venom than would be expected based on their mass, including one animal that was later found to have an abscessed liver and one that was

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99 likely hit by a car prior to collection. When repeatedly stimulated, however, no more venom was ejected, suggesting co mplete emptying of the glands. Discussion A commercially-available medical nerve stimulator produced reliable, safe collection of venom from anes thetized cottonmouth snakes. The short-acting anesthetic propofol induced anesthetic immobility in most snakes at dosages of 8 to 10 mg/kg IV. Induction times were variable when the drug was administered into the vessels of the ventral tail, and several snakes required su pplemental injections. Induction after intracardiac administration was very rapi d, although overall induction times were calculated after initial ventral tail injection. Most of the snakes continued breathi ng throughout the procedure. Further, the snakes recovered fairly rapidly, allowing them to quickly resume normal behavior. A major problem with the use of propofol in these and other animals is that it must be injected IV to be effective. This require s skill in venepuncture in these and smaller snakes, and even then, many of the animals require supplemental injections. The prolonged induction times in snakes adminis tered propofol in the ventral coccygeal vessels may be due either to failure to inje ct the full dose into the caudal vein or prolonged circulation from the caudal portion of the body. The rapid and consistent responses to intracardiac injection of the lo wer propofol dosages (5 mg/kg) support this. Although intracardiac injection is relatively easier to achi eve, care must be taken to prevent injury to the heart. The other conc ern is administering a cardiodepressant drug in high concentration to the myocardial tissue. Although the majority of this study was conducted using propofol injected IV, we hav e subsequently found IC injection to reduce

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100 handling time further (data not shown). Such injections should only be conducted, however, by those familiar with the techni que to prevent damage to the heart. A consistent and efficacious injection is import ant for this technique to work in a timely manner, and practice is needed under proper guidance. A major advantage of propofol for field work in reptiles is that it can be readily transported and requires no more equipment t han a syringe and needle to administer. A hand-held ventilator, such as an Ambu bag, and endotracheal tube are all that are necessary to ventilate an anesthetized animal, and these are not typically needed. The constant current peripheral ner ve stimulator simplifies venom extraction both in a field situation and in the laborator y. The model described is por table and uses AA batteries, making it very convenient. The combinatio n of anesthesia and electrical stimulation leads to greater safety, better repeatability, easier quantification, and lowered handling time. Since the animals are unconscious during t he procedure, they are unlikely to bite, thereby enhancing safety. They are also un able to resist muscle flexion around the gland, increasing repeatability and quantif ication, and are unaware of handling and sample collection, likely reducing stress. Care must still be taken to avoid accidental contact, and we do this by maintaining the ani mal’s heads within clear plastic restraining tubes, except during venom extraction. It is possible some venom remains in the gl and after electrical stimulation, creating errors in volume measuremen t. With repeated stimulation, however, we saw no greater venom yield. In comparison, spontaneous ej ection results in more venom with greater number of bites. A further pr oblem with quantification of yiel d from wild snakes is that

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101 conditions prior to capture can not be deter mined. It is always possible the animal recently fed or used venom in defense, t hereby reducing availabl e volume. Captive neonates fed two weeks after venom collecti on were able to subdue prey with venom, but many held prey after envenomation, suggesting a possible reduction in available venom (data not shown). It is also possibl e that removing all venom from the glands increases the amount of cellular material ejected with the venom. As most studies centrifuge venom and remove cellular debris a fter collection but prior to lyophilization, this may not present a problem. We recaptured three snakes within twelve m onths of initial capture and release. All of these animals were in good health, sim ilar body condition, and with equal or greater venom yield during the second co llection. While this is a small sample size, it does indicate that our methods do not irreversibly harm the animals. Future research into the effects of this method on snakes should include multiple captures or long term maintenance in the laboratory as well as ho rmonal assays for typica l stress indicators, such as cortisol. Likewise, a thorough co mparison of venom yiel d among this and other extraction techniques would be useful. In summary, the combination of inject able propofol anesthetic and electrical stimulation for venom extraction yield measur able, repeatable results. This technique allows for reduced handling time for t he animals and increased safety for the researchers, and is portable to most field situations. It should be considered as an alternative or addition to ot her extraction techniques.

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102 Figure 3-1. Anesthetiz ed Florida cottonmouth ( Agkistrodon piscivorus conanti ) in tube with only head protruding. The animal is lying in dorsal recumbency and easy to bring back into the tube by pulling t he anterior portion of the body, should it prematurely revive.

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103 Figure 3-2. Anesthetiz ed Florida cottonmouth ( Agkistrodon piscivorus conanti ) showing placement of electrodes on the skin surface across the venom gland. Figure 3-3. Anesthesia induc tion in Florida cottonmouths, Agkistrodon piscivorus conanti Snakes (n = 75) were initiall y injected with 10 mg/kg body mass or less, and some (n = 33) had supplemental injections (up to 10 mg/kg body mass). Induction occurred in 12.2 8.5 mi n of initial injection. There was a significant mass-associated positive trend between amount of propofol needed for induction and animal mass (regression analysis). -10 0 10 20 30 40 50 60 70 050010001500200025003000 Mass (g) Total Propofol Used for Induction (mg) y = 0.0144x – 0.9484 R2 = 0.8078 P << 0.001

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104 Figure 3-4. Mass-specific e ffects of propofol on induction time in Florida cottonmouths, Agkistrodon piscivorus conanti Snakes were initially injected with propofol (10 mg/kg body mass), and monitored for lo ss of righting reflex and failure to respond to a mild cloacal pinch. Induc tion time was positively correlated with snake mass (regression analysis). Figure 3-5. Mass-specific venom yield from Florida cottonmouths, Agkistrodon piscivorus conanti by electrical stimulation. All animals were anesthetized with propofol (10 mg/kg body mass) prio r to venom collection, and venom yield was significantly correlated wit h animal snout-vent length (regression analysis). 0 10 20 30 40 50 050010001500200025003000 Mass (g) Induction Time (min) y = 0.004x + 9.5399 R2 = 0.1046 P < 0.005 0 400 800 1200 1600 0200400600800100012001400 Snout-vent Length (mm) Total Venom Yield (l) y = 1.0197x – 271.91 R2 = 0.6523 P << 0.001

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105 CHAPTER 4 CROSS SUBFAMILY AMPLIFICATION: A WORKING SET OF MICROSATELLITE PRIMERS FOR USE IN THE FLORIDA COTTONMOUTH Introduction In the United States, there are three genera of viperid snakes, and all are members of the subfamily Crotalinae (rattlesnakes and their allies). These genera are: Crotalus (rattlesnakes), Sistrurus (pigmy rattlesnakes), and Agkistrodon (copperheads and cottonmouths). All are venomous and range in abundance, depend ing on habitat specificity. The Florida cottonmouth ( A. piscivorus conanti ) is associated with fresh water habitats throughout most of the st ate of Florida, where it f eeds on a wide range of mostly vertebrate prey. The cottonm outh in general has been the focus of many wide-ranging studies of ecology and evolution (Gloyd and Conant, 1990), and previous phylogenies have utilized mtDNA to examine phylog eography and relationship with the copperhead, A. contortrix (Guiher and Burbrink, 2008). However, previously no microsatellite loci have been specifically developed for use in this species. Many previous studies of snakes have developed microsatellites to analyze genetic relatedness among or within populations of a certain species (hereafter termed target species; Villarreal et al., 1996; Gibbs et al., 1998; Burns and Houlden, 1999; McCracken et al., 1999; Prosser et al., 1999; Jordan et al ., 2001; Matson et al., 2001; Garner et al., 2002; Holycross et al., 2002; Carlsson et al., 2003; Bond et al ., 2005; Manier and Arnold, 2005; Stapley et al., 2005; Lane et al ., 2008; Munguia-Vega et al., 2009). Some of these studies have examined the utility of previously-developed loci in species other than the target species (her eafter termed non-target species) and have yielded different

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106 results including: 1) no PCR am plification (Gibbs et al., 1998; Prosser et al., 1999; Holycross et al., 2002; Blouin-Demers and Gibbs 2003; Carlsson et al., 2003; Stapley et al., 2005; Meister et al., 2009), 2) amplification but without reporting allelic diversity (Gibbs et al., 1998; Prosser et al., 1999; Blouin-Demers and Gibbs, 2003; Bond et al., 2005; Clark et al., 2008), and 3) amplification with multiple alleles reported (Jordan et al., 2001; Matson et al., 2001; Hille et al., 2002; Holycross et al., 2002; Carlsson et al., 2003; Manier and Arnold, 2005; Stapley et al ., 2005; Anderson, 2006; Lane et al., 2008; Meister et al., 2009). This last category of studies indicates that some snake microsatellite loci may be broadly useful for g enetic studies of non-target snake species. Indeed, a few studies have utilized loci dev eloped in one species to answer population genetics questions in another (Scott et al., 2 001; Manier and Arnold, 2005; Clark et al., 2008). Materials and Methods As part of a larger study, Florida cottonmouth (N = 89) DNA samples were collected from two locations in Florida: Pa ynes Prairie Preserve State Park (PP) and Lower Suwannee National Wildlife Refuge (LS) both located in northern Florida, separated by approximately 90 km To preserve DNA, tissue samples were either mixed with lysis buffer (for blood from live anima ls; 0.1 M Tris-HCl with 100 mM EDTA and 10 mM NaCl, pH 8.0) or tissue buffe r (for body wall from road-kil led animals; saturated NaCl solution with 250 mM EDTA and 20% DMSO, pH 7.5). DNA extraction was done using the Qiagen DNEasy kit, and resulting DNA was examined quantitativel y and qualitatively using a NanoDrop 8000, with in itial concentrations rang ing from 8.6 to 90.0 ng/ l. Primers from previously published (Villarreal et al., 1996; Gibbs et al., 1998; Holycross et

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107 al., 2002; Carlsson et al., 2003; Goldberg et al., 2003; Oyler-McCance et al., 2005; Clark, 2008; Table 4-1) studies were purchased from MWG Biotech, and used initially to amplify 12 to 24 cottonmouth DNA samples. We utilized the published primers that recorded the greatest variability, and did not examine any locus with fewer than 3 alleles in the original target species. A gradient thermocycler was used to optimize temperature and other parameters (DNA, Mg2+, and primer concentrations) to give repeatable, clear results. Products were visualized on 2% agarose gels stained with ethidium bromide and examined for presence of two or more bands of different size. If optimization failed to yield a product after multiple attempts, or only one band was visible, the individual locus was dropped. Results Table 4-1 shows which loci ampl ified cottonmouth DNA and which gave variable-sized products. Overa ll, 71% (22/31) of tested loci amplified and 32% (10/31) yielded visual variable fragment sizes. Tw o other loci (6.5%) yielded what appeared to be visible differences; howev er, after fluorescent label ing, CH2D was found to be monotypic. Because of the low visible variation in Scu05 it was not utilized further. Forward primers for the ten va riable loci were fluorescently labeled on the 5’ end with either HEX or 6-FAM, and PCR conditions we re optimized for each locus (Table 4-2). Slight deviations in cycle number and/or temper ature were found to not adversely affect amplification of products. Genotypes were determined for all individ uals at all ten loci by analyzing PCR products on either a M egaBACE 1000 or Applied Bi osystems 3730 automated sequencer with internal standard ladders, and fragment sizes were determined using

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108 GENEMARKER software. MICRO-CHECKER version 2.2.0.3 (van Oosterhout et al., 2004) was used to examine the data fo r large allele dropout, null alle les, and stuttering signals, and only the LS population showed a potential null allele and stuttering, at locus Scu26 CERVUS version 3.0 (Marshall et al., 1998) was used to determine observed and expected heterozygosities, and GENEPOP version 4.0 (Raymond and Rousset, 1995) was used to test for linkage disequilibrium and si gnificant deviations from Hardy-Weinberg equilibrium. Following sequential Bonferroni tests, no linkage disequilibrium was found for any loci pair. One locus in the LS population (CH1A) and two loci in the PP population (CH2E and CH5 A) were found to deviate sign ificantly from Hardy-Weinberg equilibruim. Allele sizes fell within or over lapped those reported for the original target species. Discussion Development of species-specific microsate llite loci, while much easier than it once was, is a time-consuming and potentially cost ly endeavor. However, a relatively easy and inexpensive substitute is to attempt the use of marker s developed in closely related species. For snake species in particular some microsatellite loci have been found to amplify products in even distantly related spec ies (Gibbs et al., 1998; Prosser et al., 1999; Bond et al., 2005; Hille et al., 2002). Al though this wide-ranging cross-reactivity in snakes may be an exception, other studies have described the usefulness of non-target species loci for non-snake organisms (Donaldson and Vercoe, 2008). The cottonmouth is a well-studied viperid sn ake, yet no microsatellite markers have been developed for the species. The ten loci reported here have been shown to be polymorphic in the Florida cottonmouth, and may be useful for future studies of

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109 population genetics. Further, we have shown that there is potential for these and other loci, developed in closely (or even distantly) re lated species, to be useful for genetic studies of other crotaline snakes, although ea ch must be examined in a case-by-case fashion. While we tended to use primers with known pol ymorphism in their ta rget species, it is possible that polymorphism may be even great er in non-target species. While some loci from snakes of the genus Crotalus and Sistrurus were polymorphic in A. p. conanti two loci developed in C. viridis concolor (a North American rattlesnake) and two developed in Vipera berus (a European viperine snake) did not amplify regions in A. p. conanti DNA. This may be expected for V. berus as it is distantly related, but is slightly surprising for C. v. concolor It would be of interest to examine South American crotaline and/or Asian viperine snakes to see if microsatellite usefulness can be mapped phylogenetically. Additional recently published crotaline loci (Munguia-Vega et al., 2009) may also be useful for such studies. As previously shown, published primer s may be a useful starting point for population genetic studies in non-t arget species. By validating such primers for use in non-target species, researchers may save both time and money and may be able to expand the set of useful micros atellites for a given species. Crotaline snakes appear to be a good example of a taxon with many publish ed loci overall and with multiple studies indicating cross-reactivity. We have validated a set of 10 microsatellite loci developed in related species for use in the non-target cottonmouth snake.

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110 Table 4-1. Utility of 31 microsatellit e primers for use with cottonmouths ( Agkistrodon piscivorus ). Target species, accession number (where published), and number of alleles (NA) from original study are given. Results from the current study are abbrevia ted as follows: amp/no amp = relative ability to amplify PCR product; var/no var = size va riation in amplified PCR product. Locus Target GenBank Reference Target Result species accession species number NA 3-155 Crotalus horridus -Villareal et al., 1996 4no amp 5A Crotalus horridus -Villareal et al., 1996 8 (11*)amp, var 5-183 Crotalus horridus -Villareal et al., 1996 7no amp 7-87 Crotalus horridus -Villareal et al., 1996 3 (7*)amp, no var CH1A Crotalus horridus -Clark, 2006 8amp, var CH2D Crotalus horridus -Clark, 2006 undeterminedamp, no var CH2E Crotalus horridus -Clark, 2006 13amp, var CH3A Crotalus horridus -Clark, 2006 undeterminedamp, no var CH3B Crotalus horridus -Clark, 2006 undeterminedno amp CH4B Crotalus horridus -Clark, 2006 13amp, var CH4C Crotalus horridus -Clark, 2006 9amp, no var CH4D Crotalus horridus -Clark, 2006 7amp, no var Crti05 Crotalus tigris AY298757 Goldberg et al., 2003 22amp, no var Crti08 Crotalus tigris AY298762 Goldberg et al., 2003 26no amp Crti09 Crotalus tigris AY298759 Goldberg et al., 2003 41amp, var Crti10 Crotalus tigris AY298760 Goldberg et al., 2003 22amp, var MFRD5 Crotalus viridis concolor AY770999 Oyler-McCance et al., 2005 9no amp MFR9 Crotalus viridis concolor AY771002 Oyler-McCance et al., 2005 9no amp Cw A14 Crotalus willardi obscurus AY130982 Holycross et al., 2002 7amp, no var Cw B6 Crotalus willardi obscurus AY130985 Holycross et al., 2002 5amp, no var Cw B23 Crotalus willardi obscurus AY130984 Holycross et al., 2002 12amp, var Cw C24 Crotalus willardi obscurus AY130986 Holycross et al., 2002 24amp, var Cw D15 Crotalus willardi obscurus AY130987 Holycross et al., 2002 5amp, no var Scu01 Sistrurus catenatus -Gibbs et al., 1998 12amp, no var Scu05 Sistrurus catenatus -Gibbs et al., 1998 11amp, no var Scu07 Sistrurus catenatus -Gibbs et al., 1998 8amp, no var

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111 Table 4-1. Continued: Locus Target GenBank Reference Target Result Species accession species number NA Scu11 Sistrurus catenatus -Gibbs et al., 1998 7amp, var Scu16 Sistrurus catenatus -Gibbs et al., 1998 4no amp Scu26 Sistrurus catenatus -Gibbs et al., 1998 5amp, var Vb11 Vipera berus AJ496617 Carlsson et al., 2003 38no amp Vb37 Vipera berus AJ496619 Carlsson et al., 2003 12no amp *Clark, 2006 data for Crotalus horridus

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112 Table 4-2. Information for microsatellite loci used with Agkstrodon piscivorus conanti DNA. Optimal PCR conditions including ambient temperature (Ta), MgCl concentration and number of cycles are given. Also presented are overall alle le size ranges, and number of alleles (NA), observed heterozygosity (HO) and expected heterozygosity (HE) for two populations. Locus Label PCR MgCl CyclesSize Paynes Prairie Lower Suwannee Reference Ta (C) (mM) (bp) NA HO HE NA HO HE CH1A 6-FAM 59.51.536200-22570.608 0.64890.6580.768aClark, 2006 CH2E HEX 59.52.036167-203110.588 0.651a100.8950.876Clark, 2006 CH4B 6-FAM 57.52.036146-16660.745 0.74840.3950.391Clark, 2006 CH5A HEX 57.52.038135-14640.490 0.558a40.3160.355Clark, 2006 Crti09 HEX 52.32.040309-379120.824 0.84 870.7110.821Goldberg et al., 2003 Crti10 6-FAM 52.62.036200-316170.882 0.91 5140.8950.898Goldberg et al., 2003 Cw B23 6-FAM 52.62.036200-238110.725 0.68 470.7630.802Holycross et al., 2002 Cw C24 HEX 55.62.036202-286210.863 0.932160.8420.921Holycross et al., 2002 Scu11 6-FAM 52.92.033143-182140.902 0.86480.7890.855Gibbs et al., 1998 Scu26 HEX 57.52.038128-175110.725 0.83160.5260.723Gibbs et al., 1998 aSignificant deviation from Hardy-Weinberg e quilibrium after Bonferroni correction.

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113 CHAPTER 5 POPULATION GENETIC ANALYSIS OF ONE INSULAR AND THREE MAINLAND POPULATIONS OF THE FLORIDA COTTONMOUTH Introduction The North American snake genus Agkistrodon is comprised of four species: bilineatus (the cantil), contortrix (the copperhead), piscivorus (the cottonmouth), and taylori (Taylor’s cantil). It has been hypothesized that the common Agkistrodon ancestor developed sometime in the late Oligocene (28 to 24 mya), and that piscivorus separated from the remainder of the species during the mi ddle Miocene (~16 to 15 mya) (Van Devender and Conant, 1990). Contrary to this, Parkinson et al. (2000) utilized mitochondrial and transfer RNA sequences and indicated that contortrix separated earliest, with piscivorus second. Guiher and Burbrink (2008) used mitochondrial DNA sequences of the cytochrome b gene and f ound support for the same pattern of relationship as Parkinson et al. (2000). They also determined that the initial separation of contortrix occurred about 6.60 mya with piscivorus separating around 5.30 mya. The cottonmouth is generally held to contain three subspecies: conanti (Florida cottonmouth), leucostoma (western cottonmouth), and piscivorus (eastern cottonmouth) (Gloyd and Conant, 1990). Only conanti is located in peninsular Florida, although it may interbreed with leucostoma on the northwestern par t of its range and with piscivorus on the northeastern portion of its range. Recent molecular data also indicate that leucostoma and piscivorus should be grouped and consi dered a sister taxon to conanti (Guiher and Burbrink, 2008). In a recent study, Douglas et al. ( 2009) examined the genetic relationship among the species and subspecies of Agkistrodon They analyzed mitochondrial DNA sequences, and their data supported both the late separation of A. piscivorus

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114 (subsequent to A. contortrix ) and the grouping of A. p. leucostoma and A. p. piscivorus (with A. p. conanti being separate). They also r eported support for the origin of A. p. conanti from Mexico via rafting across the Gulf of Mexico. Interestingly, their data indicated that A. p. conanti was limited in it range expa nsion due to 1) separation of Peninsular Florida from the mainland by a midto late-P leistocene incurrence of sea water and 2) competition from established A. p. piscivorus after reunion of Peninsular Florida and the mainland. Among snake species, the cottonmouth has been extensively studied in terms of its natural history and ecology (Gloyd and Conan t, 1990). Within Florida, this snake is usually found in or near freshwater habitats, and it is one of the most common snakes. Possible reasons for the success of this species in Florida include the widespread occurrence of freshwater (much habitat), t he relative low altit udes (low barriers to dispersal), and the ubiquitous nature of its dietary prefer ences (wide trophic niche breadth; Lillywhite and McCl eary, 2008). The relative abundance of individuals makes this species suitable for population-level stud ies, especially baseline studies for later comparison. Although it is well known that insular populations of animals are under different ecological constraints t han mainland populations and may, therefore, evolve along different trajectories, predicting the manner in which these populations evolve is problematic. Among snakes, previous compar isons of insular populations with their mainland counterparts have examined phenotyp ic and ecological characters such as morphometrics (Shine, 1987), venom activity or composition (Mebs, 1970; Selistre and Giglio, 1987; Williams and White, 1987) prey base (Wharton, 1966, 196 9; Shine et al.,

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115 2002; Wster et al., 2005; Lillywhite and McCl eary, 2008) and interspecies interactions (Bonnet et al., 1999; Shine et al., 2002). There is one insular population of Flor ida cottonmouth—that on the island of Seahorse Key (SHK)—that is of special interest. This population was intensively studied by Wharton (1966, 1969), and was found to have a unique and interesting natural history. The SHK population feeds pr imarily on fish that are regurgitated or dropped by colonially-nesting birds, including Brown Pelicans ( Pelecanus occidentalis ), Double-crested Cormorants ( Phalcrocorax auritus ), and White Ibises ( Eudocimus albus ). This relatively easy source of hi gh-quality food has apparen tly allowed the SHK population to be maintained in high density, with many very large individuals in the population. SHK is a ~67 ha island located ~5 km off of the coast of mainland Florida and is separated from the mainland by salt water (Figure 5-1). Th is marine corridor may act as a dispersal barrier, reducing ge ne flow from the mainland to the island and vice versa. The island is thought to have been isol ated from the mainla nd for about 3300 to 3900 years, based on consistent rates of sea level rise since the late Holocene (Roark, 2003). However, because of occasional heavy ra ins and large freshwater runoff via the Suwannee River, it is likely t hat water between the mainland and SHK is highly variable in salinity. The relative isolation and abund ant food source leads to the question as to whether the SHK population of snakes is ge netically isolated from mainland populations and is possibly inbred. To examine possible genetic isolation of the SHK population, comparisons were made with snakes from three other geographica lly defined populations: Paynes Prairie

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116 Preserve State Park (PP), Lower Suwannee National Wildlife Refuge (LS), and Big Cypress National Preserve (BC). LS is a 21450 ha refuge located on the mainland northeast of SHK. It contains a mixture of floodplain cypress swamps and estuarine habitats, and is closest to SHK of the areas studied. PP is located in north central Florida and is 8500 ha in area. Year-to-year rainfall amounts for t he prairie range from times of relative dryness with small ponds to times in which much of the park is flooded, usually due to tropical storm action; it is surrounded by upland live oak forest and a series of interconnecting streams, swamps, and seasonally flooded fo rests. Finally, BC is located in south Florida, is ~291400 ha in area, and is part of the Everglades. It contains diverse habitats dominated by seas onally flooded sawgrass fields and includes cypress swamp and permanent ponds and ditches. In order to examine the e ffects of local ecology on population evolution, it must first be shown that populations have separate genet ic identities. In order to do this, and to examine whether the salt water corridor is an effective barrier for isolation of the insular population, the genetic relationshi ps among the described populations were examined. This comparative genetic analysi s was done utilizing microsatellite markers, which are useful for discerning fine populat ion structure. Exam ination of population structure within a common species is of interest in areas like Florida because of potential habitat fragmentat ion due to human development. Materials and Methods Animals Genetic samples from a total of 129 ani mals were used, spread amongst the four populations (N = 25, SHK; N = 51, PP; N = 38, LS; and N = 15, BC). Live animals were found by searching trails, stream banks, and ponds or by slow road cruising in an

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117 automobile; road-killed animals were visually spotted by road cruising. For each individual, spatial data were recorded as Gl obal Positioning System (GPS) coordinates. Live animals were transported to the laborat ory for sample collection. Blood from live animals or body wall from fresh road-killed an imals were preserved either in lysis or tissue buffer, respectively. DNA was extr acted using a Qiagen DNEasy DNA extraction kit, and initial concentrations of DNA were determined using a NanoDrop 8000. Analysis Previously published crotalid microsatelli te loci primers were purchased from MWG Biotech AG and the forward primers were fluorescently la beled either with HEX or 6-FAM (Table 5-1). These loci were developed using different crotalid species, so we initially validated their use in cottonmouths. The loci used were as follows: CH1A, CH2E, and CH4B (Clark, pers. comm.); CH5A (V illarreal et al., 1996); Crti09 and Crti10 (Goldberg et al., 2003); Cw B23 and Cw B24 (Holycross et al., 2002); and Scu11 and Scu26 (Gibbs et al., 1998). DNA fragments were amplified via PCR, with conditions dependent upon the specific locus in question, with an annea ling temperature of 72C and elongation temperatures between 52.3 and 59.5C (Table 5-1). Fluor escently-labeled amplified products were analyzed by capillary electr ophoresis on either an Applied Biosystems 3730 or MegaBACE 1000, and fragment sizes were determined using GENEMARKER software. MICRO-CHECKER version 2.2.0.3 (van Oosterhout et al., 2004) was used to examine the data for large allele dropout, null alleles, and stuttering signals. CERVUS version 3.0 (Marshall et al., 1998) was used to determi ne observed and expected heterozygosities, and GENEPOP version 4.0 (Raymond and Rousset, 1995) was used to test for linkage

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118 disequilibrium and significant deviations fr om Hardy-Weinberg equilibrium (HWE). GenAlEx 6.2 (Beck et al., 2008; Smouse et al., 2008) was also utilized to determine migration rates among the populations. To determine the population iden tity of each individual, STRUCTURE 2.2 (Pritchard et al., 2000; Falush et al., 2003) was used. This program uses a Bayesian model clustering system to determine population stru cture and assign every individual to a specific population. It is based on overall allele frequencies and uses a Markov chain Monte Carlo (MCMC) simulation. The progr am further analyzes the data to determine the most likely number of populations being r epresented by the data set overall. The data were analyzed using a burn-in period of 50000 followed by 200000 MCMC repeats, and the admixture model was used. Initial, smaller analyses of k = 1 to 10 populations indicated that the data did not support models for k = 1, 2, 7, 8, 9 or 10 population, so the final analysis was run 7 times for each of k = 3 through 6 independent populations. The program TESS, which is a similar Bayesian model that utilizes a Hidden Markov Random Field simulation and incorporation spatial data (GPS), was also used to determine population identity fo r each individual animal. Results The LS population showed a potential null allele and stuttering at locus Scu26 and the SHK population showed a potential null allele and stuttering at locus CH2E. Following sequential B onferroni tests, no linkage disequilibrium was found for any loci pair. However, because of high allelic divers ity, each of the individuals from BC had a unique allelic identity for locus Cw C24. Also, because loci CH4B and CH5A were monotypic within the SHK popul ation, meaningful disequilibr ium comparisons could not be performed. One locus in the LS populatio n (CH1A) and two loci in the PP population

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119 (CH2E and CH5A) were f ound to deviate significantly from HWE, but all BC loci were in HWE (Table 5-2). Three loci in the SHK population were monotypic (CH4B and CH5A) or nearly monotypic ( Cw B23), so tests for HWE could not be done. The results of the STRUCTURE 2.2 analysis indicated that the most likely scenario includes k = 4 populations (lnP(D) = -4154.8) The grouping of indi viduals into these populations can be seen in Figure 5-2, and it shows clearly defined delineations among the four populations. With minor deviations, individuals collected from an area identified genetically with other i ndividuals from that same populat ion, indicating little mixing among the populations. FST values, which indicated lik elihood of group segregation based on variation within each population and for all populat ions combined, indicated that all four populations are genetically separate. Using standard published numbers (Pritchard et al., 2000), the FST values were found to indicate moderate (0.051, BC; 0.085, LS), great (0.151, PP) and very gr eat (0.581, SHK) genet ic isolation. Results from the TESS run were similar to the STRUCTURE results. Once again, all individuals from one geogr aphic population were genetical ly grouped with the other individuals from that region (Figure 5-3). The same mi nor exceptions were seen using the TESS program. TESS also gives a spatial visual representation of the populations by incorporating GPS data (Figure 5-4). GenAlEx results indi cated low migration rates, with the greatest rate (4.44 individuals per generation) being found between PP and LS, and the lowest rate (0.96 individuals per generation) being between PP and SHK. Discussion Based on these results, the four studied populat ions are genetically different from one another, indicating at least partially di fferent evolutionary trajectories. More importantly, FST values indicate that the insular population (SHK) is more genetically

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120 isolated than the other three popul ations. This is of interest, because it indicates that the marine corridor separating SHK from the mainland is an effective barrier to migration of the animals and, therefore, gene flow between the populations. Despite the proximity of the LS population to the SHK popul ation, LS is more genetically close to both PP and BC than it is to SHK. Allelic diversity among the populations indi cates potential inbreeding in the SHK population, as it had the fewest number of al leles for all loci and the lowest average number of alleles per i ndividual for all but locus CH1A. The most extreme examples of this are loci CH4B and CH5A, which are monoal lelic in the SHK population. As reduced allelic diversity is an indication of an inbred population (either through genetic bottleneck or founder effects), this indicate s inbreeding in t he SHK population. In order for populations of organisms to ev olve independently, they must first be isolated by some means, and geographic barriers can be highly effective. In the case of the cottonmouth populations studied here, sea water appears to be involved in isolation of the SHK population. Beside this, howev er, the other three mainland populations showed levels of isolation, albeit less t han the SHK population. This is understandable in the case of BC, because it is s eparated from the north ern populations by approximately 400 km. Howeve r, the distance between SHK and LS (~30 km) is much shorter than between LS and PP (~80 km). The establishment of effect isolation is t he first step in examin ing the evolution of phenotypic characters in populati ons. With this knowledge, it is possible to examine differences among populations meaningfully. The Florida cottonmouth, and especially the SHK population, makes a great model for studyi ng evolution of phenotypic

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121 characters in a natural setting. Specifically, because of the rich history of research into its venom, replete with numerous conflicting reports in the lit erature, and the relative common occurrence of the species throughout its range, the Florida cottonmouth is a worthy study species. The results of this study are also import ant because they give baseline data into the population genetics of an anima l that is not currently c onsidered threatened. As interest in many species occurs once that species has reached a crit ical level, baseline data is usually lacking or severely limited. In the state of Florida, where land development occurs at a very rapid pace, these data can serve as standards for future examinations of habitat fragmentat ion due to human land development.

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122 Table 5-1. Specifics of microsatellite amplification. Elongation temperatures varied depending on the locus, as did the number of cycles. Locus Fluorescent Primer Sequences PCR MgCl Cycles Reference Label Ta (C) (mM) CH1A 6-FAM F-GCAGGCACTGTTGTCA CTGT 59.5 1.5 36 Clark, 2006 R-TCAACCAAATTTGCTCATGC CH2E HEX F-TCTGATTGCAAGCTCT GGTTT 59.5 2.0 36 Clark, 2006 R-TTCCAGGAAGTCACCAGGAG CH4B 6-FAM F-CAGGTGACTCAGAAGGCACA 57.5 2.0 36 Clark, 2006 R-ATCAGTTCTGGGTGGACGTT CH5A HEX F-CCAGAGCCATCAAGGCCCTT 57.5 2.0 38 Clark, 2006 R-TGCAGAGGCAGCACTTTGTTA Crti09 HEX F-TAGGAATAAGAAATGTCAGG 52.3 2.0 40 Goldberg et al., 2003 R-TAATGTAATGTGGTTCAGGA Crti10 6-FAM F-ATGACCTGGATACTGTGTT 52.6 2.0 36 Goldberg et al., 2003 R-ACTGCTATACTTAGAGTGAA Cw B23 6-FAM F-TGGTGTCATCTGGAGTTAAATC 52.6 2.0 36 Holycross et al., 2002 R-GCTTTTGTTTATATGGAGAGTCG Cw C24 HEX F-ATTGGATAGAAGTAGTTTTGGTA 55.6 2.0 36 Holycross et al., 2002 R-CCCCCCTTTTTTTATGGCAGC Scu11 6-FAM F-AATCAGCATGTGGCTTAAATC 52.9 2.0 33 Gibbs et al., 1998 R-GCTGCTTGGCTACATATGC Scu26 HEX F-GAAATTGGTGGAAGAGACCTG 57. 5 2.0 38 Gibbs et al., 1998 R-GTCCAGGATATGAGGGATCTG

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123 Table 5-2. Locusand population-specific data for microsate llite analysis of cottonmouths. A total of 129 individual snakes were used for the comparisons, and the number for each population is given. NA is the number of alleles per locus for each population and in total. HO is observed heterozygosity and HE is expected heterozygosity. Locus Total Total SHK (n = 25) PP (n = 51) LS (n = 38) BC (n = 15) Size NA NAHO HE NAHO HE NA HO HE NAHO HE (bp) CH1A 200-225 10 50.4400.37770.6080.648 9 0.6580.768a50.8000.733 CH2E 167-203 13 30.4000.598110.5880.651a 10 0.8950.87670.8000.777 CH4B 144-166 7 10.0000.00060.7450. 748 4 0.3950.39140.7330.690 CH5A 135-146 4 10.0000.00040.4900.558a 4 0.3160.35530.2000.191 Crti09 309-379 14 30.6400.496120.824 0.848 7 0.7110.82170.5330.690 Crti10 200-316 18 40.6000.500170.882 0.915 14 0.8950.898100.7330.885 Cw B23 200-238 13 20.0400.040110.7250.684 7 0.7630.80260.7330.837 Cw C24 202-286 23 50.4400.504210.8630.932 16 0.8420.921160.7330.938 Scu11 143-182 15 30.6000.571140.9020.864 8 0.7890.85540.2670.251 Scu26 128-175 11 20.2800.301110.7250.831 6 0.5260.72350.7330.701 aLoci with significant deviations from Hardy-Weinberg equilibrium

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124 Figure 5-1. Locations of cottonmouth popul ations studied. The image was created using Google Earth. Big Cypress Lower Suwannee Seahorse Key Paynes Prairie

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125 Figure 5-2. STRUCTURE population identity for individual cottonmouths in Florida. This image was generated using STRUCTURE 2.2. Each vertical bar represents one individual, and colors indicate like lihood that an indi vidual genetically identifies with that population. With few exceptions, individuals identify genetically with the popul ation in the region from wh ich they were sampled. Specimens with multiple colors (such as the large maroon bar in the Paynes Prairie region) indicate mixed identi ty, and the amount of one color indicates the percentage with which the individu al identifies with that population. Figure 5-3. TESS Population identity for individual cott onmouths in Florida. This image was generated using TESS 2.2. Each vertical bar represents one individual, and colors indicate likeliho od that an individual genetic ally identifies with that population. With few exceptions, i ndividuals identify genetically with the population in the region from which t hey were sampled. Specimens with multiple colors (such as the large ma roon bar in the Paynes Prairie region) indicate mixed identity, and the amount of one color indicates the percentage with which the individual ident ifies with that population.

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126 Figure 5-4. Spatial and genetic relationships of individual Florida cottonmouths. This image was created using Tess 2.2. Each black dot represents one individual, and each polygon indicates the population with which that individual identifies genetically. Colors, in general, indicate the following: green—Big Cypress; blue—Paynes Prairie; maroon—Low er Suwannee; and orange—Seahorse Key. Distances between dots are to scale with GPS data.

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127 CHAPTER 6 ENZYMATIC ACTIVITY IN THE VENOM OF THE FLORIDA COTTONMOUTH: A POPULATION-LEVEL COMPARISON Introduction The Florida cottonmouth snake, Agkistrodon piscivorus conanti is a common snake associated with fresh water habitats thr oughout the Florida peninsula. It is known to be a dietary generalist (Lillywhite and McCl eary, 2008), consuming various species of vertebrate prey. Its venom is complex, containing many di fferent biologically active enzymes and functional non-enzymatic peptides Although there may be accompanying local tissue damage, the major effect of cottonmouth envenomation is a disruption of hemostasis. This disruption includes hemol ysis of red blood cells, perturbation of the blood clotting cascade, and hemorrhaging from bl ood vessels, all of which result in a precipitous drop in blood pressure. This fa ll in blood pressure leads to incapacitation and possible death. The majority of active components of cottonmouth venom are enzymes. Among these, cottonmouths are known to have specific phosphodiesterase (Mebs, 1970), 5’-nucleotidase (Richards et al., 1965), alkaline phosphomonoesterase (Tan and Ponnudurai, 1990), hyaluronidas e (Powlick and Geren, 1981), L-amino acid oxidase (Ahn et al., 1997), metalloproteinase (Jia et al., 2009), serine prot eases (thrombin-like— Kress and Catanese, 1980; kallikrein-like—Ba iley et al., 1991; and arginine esterase— Kocholaty et al., 1971), and phospholipase A2 (Ownby et al., 1999). There are also general enzymatic activities, based on their effects on substrate, and these include aminopeptidase (Prescott and Wagner, 1976) ATPase (Mebs, 1970), fibrinogenase (Nikai et al., 1988b), non-specific esterase (Bernick and Simpson, 1976), and non-specific protease (Wagner and Prescott, 1966a).

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128 Previous studies using cottonmouth venom ha ve noted large variation in activity or effects when different individuals or comme rcially-produced lots were utilized (Tan and Ponnudurai, 1990). Venom variat ion is of interest because it has implications for snakebite treatment including antivenom t herapy, and cottonmouths are traditionally responsible for human envenomations (Watt, 1978). Further, as venom is likely important for prey capture and appears to evol ve quite rapidly (Lynch, 2007), it is a useful phenotypic trait for ex aminations of evolution. Several previous studies have exami ned potential correlations among venom characteristics and other factors such as geographic location, interand intra-populational phylogeny, and diet. Daltry et al. ( 1996b) found individual Calloselasma rhodostoma to have venom correlated with local prey species, indicating that natural selection for prey is very impor tant in influencing venom evolution. Williams et al. (1988) found that insular populations of Notechis ater niger had venom more tightly correlated with distanc e from the mainland, indica ting vicariance, and therefore genetic drift, was important for venom evolution. In a study of Trimeresurus stejneger Creer et al. (2003) postulated t hat natural selection was likel y the most important factor influencing temporal changes in venom compos ition. More recently, Barlow et al. (2009) found venom compositi on in four species of Echis to correlate with prey species, again indicating natural selection. There is some evidence that venom production in snakes is energetically costly (McCue, 2006), so it may be possible that optimizing venom for certain prey is necessary for overall fitness. Indeed, alt hough most hydrophiine sea snakes are highly

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129 venomous, one species that is a di etary specialist of fish eggs, Aipysurus edouxii has effectively lost its main neur otoxin (Li et al., 2005). To examine potential differences in venom composition within the Florida cottonmouth, three geographica lly isolated populations we re selected. The focal population was that found on the island of Seahorse Key (SHK; Figure 6-1). Snakes on this island have a unique ecology in that they feed primarily on marine fishes that are dropped or regurgitated by colonially nes ting water birds. The other population locations were Paynes Prairie Preserve St ate Park (PP), which is located in north central Florida and is a seasonally flooded prairie with associated fresh water ponds and streams, and Lower Suwannee National Wild life Refuge (LS), which is located on the Gulf coast of Florida (near SHK) and ha s fresh and brackish water habitats. The two mainland populations appear to have the di verse diets normally attributed to the species. Because of this, it was of intere st to determine whether the three populations showed any differences in venom enzymatic activities. Although Tan and Ponnudurai (1 990) examined the activiti es of many different venom enzymes, they utilized venom provided by commercial sources. These venoms were pools of a number of individu al snakes from the respective sources, so no levels of among-population variation coul d be determined. They did, however, find variation among the venoms from differ ent sources. Among the en zymes showing the greatest amount of variation were protease, hyaluronidase, L-amino acid oxidase (LAAO), and phospholipase A2 (PLA2). The protease assay utilizes casein as substrate and is an indicator of the overall effects of possi bly many different proteolytic enzymes. Proteases are generally responsible for hydr olysis of structural proteins, may cause

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130 local and systemic tissue damage, and ma y be useful for prey digestion. Hyaluronidase, causes hydrolysis of hyal uronic acid between cells, thereby allowing other venom components to trav el through cell barriers. Hyaluronidase is sometimes considered a “spreading factor” because of this activity. LAAO causes oxidation of Lamino acids via a two-step deamination process. This catalysis may lead to cell death via apoptosis, but the overa ll biological effects have not been determined. PLA2 is generally responsible for degradation of cell me mbranes, as it catalyzes break down of phospholipids. Materials and Methods Animals Snakes were collected from the three different regions by either active searching on foot along trails and waterways or by road cr uising. A total of 76 animals (SHK = 25; PP = 25; LS = 26) were captured and transported to the laboratory for venom extraction. Animals were anesthetized using intravascular or intracardiac injections of Propofol (Rapinovet, Schering-Plough Animal Health Co rporation; 10 mg/ml, 8 to 10 mg/kg body mass), and venom was extracted from anest hetized animals using a human constant current peripheral nerve stimul ator (10 to 30 mA; Fisher & Paykel Health Care) applied across each venom gland (McCleary and Heard, 2009). For each individual, venom from both glands was pooled. Morphometric and demographic data were also collected for all individuals, and these in cluded mass, snout-to-vent l ength (SVL), total length, and sex. Sex was determined using stainless steel sexing probes. Venom Preparation Venom was collected in polypropylene beak ers and aliquoted into microcentrifuge tubes for quantification and lyop hilization. All venoms were reconstituted in 0.85% NaCl

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131 to approximately 0.5 mg prot ein/ml venom prior to use in enzyme assays, and venom was utilized within 24 hours of reconstitution. Select individual venom samples (N = 64) were visualized by SDS-PAGE gel electrop horesis to examine them for obvious differences. For electrophoresis, samples of 25 l (pre-lyophilization) were reconstituted and run on 12% acrylamide gels with 2-( N -morpholino)ethanesulfonic acid running buffer. Enzyme Assays Enzyme assays were conducted as prev iously described (Tan and Ponnudurai 1988, 1990), with slight modifications, and al l assays were validated using pooled venom samples to verify linear kinetic reacti ons. For the protease assay, venom (20 l) was added to 150 l 2% casein (sodium sa lt, Sigma) solution in 0.25 M phosphate buffer, pH 7.75, and mixed in a shaking inc ubator at 37C and 125 rpm. After 120 min, all reactions were stopped by addition of 150 l 5% trichloroacetic acid. Samples were then centrifuged for 5 min at 10, 000 rpm to precipitate unreac ted casein. Aliquots (200 l) of supernatant were transferred to a UV microplate for spectrophotometric analysis at 280 nm. Tyrosine dilutions (0 to 1.25 m M) were used to create a standard curve by which tyrosine production could be determined, and results are calculated as nmol tyrosine/mg venom protein/h. Negative controls were done utilizing 0.85% saline without venom, and these blank values were subtracted from all experimental values. Hyaluronidase activity was examined utilizing hyaluronic acid as substrate. In a microplate, 20 l venom samples were adde d to 50 l of 0.75 mg/ml human umbilical cord hyaluronic acid (Sigma) in 0.2 M sodi um acetate buffer with 0.15 M NaCl. These samples were incubated at 37C and shaken ev ery 60 s. After 60 min, all reactions were halted using 100 l 2.5% cetyltrime thylammonium bromide in 2% NaOH. All

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132 samples were then read spectrophotometrica lly on the plate reader at 400 nm. A standard curve containing hyaluronic acid di lutions (0 to 0.75 mg/ml) was used to determine activity. Bovine testes hyaluronid ase (Sigma) was used as a positive control, and 0.85% NaCl without venom was used as a negative control. Saline blank values were subtracted from all ot hers, and the results are repo rted as g hyaluronic acid used/mg venom protein/h. LAAO activity was determined using L-leucine as substrate. Briefly, 10 l of 0.0075% horseradish peroxidase was mixed wit h 180 l of 0.2 M tr iethanolamine buffer with 0.007% o -dianisidine and 0.1% L-leucine in a microplate. These mixtures were pre-incubated at 37C in the plat e reader for 5 min. At that time, 10 l of venom sample were added to each well to initiate the reac tion. Absorbances were read at 436 nm every 15 s for 45 min, and samples were shaken prior to each reading. Reaction velocities were calculated over the linear portion of each reaction and compared to a standard curve of hydrogen per oxide concentrations. Crotalus atrox LAAO (Sigma) was used as a positive control, 0.85% saline wa s used as a negative control, and results are reported as nM o -dianisidine reduced/ng venom protein/min. PLA2 activity was determined using a commerc ial kit (Cayman Chemical) in which activity is detected spectrophotometrically. In general, 5 l of assay buffer, 10 l of diluted venom (reconstituted in assay buffer), 10 l of 5,5’-dithio-bis-2-nitrobenzoic acid (DTNB), and 200 l of diheptanoyl thio-phosphat idylcholine substrate were combined in a microplate well, and the rate of change was determined from 0.5 to 3 min at 414 nm. In the assay, PLA2 from the venom removes a fatty ac id chain from the substrate and half of the DTNB (now 5-thio-2-nitrobenzoic ac id) takes its place. The remaining 5-thio-

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133 2-nitrobenzoic acid is detected via spectrophotometer. The subs trate provided in the kit was further diluted 1:3 in assay buffer ( 25 mM Tris-HCl, pH 7.5, with 10 mM CaCl2, 100 mM KCl, and 0.3 mM Triton X-100), and venom was diluted to approximately 0.001 to 0.005 mg/ml prior to use in the assay. Posi tive controls were conducted using provided bee venom PLA2, and negative controls used assay buffe r instead of diluted venom. For each enzyme assay, every individual animal’s venom was run in quadruplicate (except the PLA2 assay, which was run in duplicate or triplicate), and t he average of the replicates was used for data analysis. Protein concentration of each venom sample was determined using the Pierce bicinchoni nic acid method with bovine serum albumin standards (Thermo Scientific). Differenc es in enzyme activities among populations were determined using analysis of variance (ANOVA) with Tukey’ s post-hoc test and between sexes using two-tailed, unpaired t-tests. Exami nations of correlation among different venom activities and demographic dat a were done utilizing regression analysis. To examine combined effects among the enzym e activities, a multivariate analysis of variance (MANOVA) was conducted with popul ation as the independent variable. Results Electrophoresis The samples run through sodium dode cyl sulfate polyacrylamide gel electrophoresis showed much similarity in pr otein banding pattern, at least in terms of the number of bands present (Fi gure 6-2). There is some obv ious individual variation, both in terms of numbers of bands and intensity of specific bands. However, there is no readily apparent populati on-level pattern among these differences.

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134 Protease There was a significant difference in m ean protease activity between the LS and PP populations, with the SHK population interm ediate and not different from either of the other two (Figure 6-3; P < 0.001; one-wa y ANOVA with Tukey post-hoc test). There was no significant difference in protease acti vity between male and female snakes (P = 0.57), and there was no correlation between prot ease activity and either SVL (P = 0.46), mass (P = 0.79), or venom volume (P = 0.57). Hyaluronidase There were no significant differences in mean hyaluronidase activity among the three populations (Figure 6-4; P = 0.43). There was no signifi cant difference in activity between the sexes (P = 0.06), although the dat a are approaching significance. Further, there was no correlation between hyaluronidas e activity and either SVL (P = 0.47), mass (P = 0.40), or venom volume (P = 0.27). L-Amino Acid Oxidase SHK snakes showed elevated mean LAAO ac tivity over both LS and PP snakes (Figure 6-5; P < 0.001; one-way ANOVA with Tukey post-hoc test). There was no significant difference in LAAO activity bet ween male and female snakes (P = 0.87), and there was no correlation between LAAO activi ty and either SVL (P = 0.87), mass (P = 0.79), or venom volume (P = 0.15). Phospholipase A2 LS snakes showed a significantly elevated PLA2 activity over PP snakes, with SHK snakes being intermediate (Figure 6-6; P < 0.001; one-way ANOVA with Tukey post-hoc test). There was no significant difference in PLA2 activity between males and females

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135 (P = 0.92), and no correlation between PLA2 activity and either SVL (P = 0.13), mass (P = 0.12), or venom vo lume (P = 0.30). Venom Correlations To examine any potential correlations among the enzymes examined, pairwise regression analyses were done. For all pairs (protease vs. hyaluronidase, protease vs. LAAO, protease vs. PLA2, hyaluronidase vs. LAAO, hyaluronidase vs. PLA2, and LAAO vs. PLA2), there were no significant correlations (P = 0.16, 0.96, 0.28, 0.67, 0.77, and 1.0, respectively). Preliminary assu mption testing for MANOVA analysis examined normality, linearity, outliers, homogeneity, and mult icolinearity, with no serious violations found. There was a statistically signifi cant difference among populations on the combined dependent variables (F(8,140) = 5. 44; P < 0.001; Wilks’ Lambda = 0.58; partial 2 = 0.237). When considered separat ely utilizing sequential Bonferroni corrections, the same patterns were found as with the ANOVA examinations described. Statistically significant differences am ong the populations were seen in protease (F(2,73) = 9.23; P < 0.001; partial 2 = 0.202), LAAO (F(2,73) = 7.84; P = 0.001; partial 2 = 0.177), and PLA2 (F(2,73) = 4.50; P = 0.014; partial 2 = 0.110). Discussion The results indicate that there are some differences in venom composition among the different populations studied. In particula r, the SHK population has a greater activity of LAAO than the other two, and intermedi ate activity for both protease and PLA2. This population is separated from the mainland by a salt water corridor that may effectively reduce migration to and from the island. Su ch separation may lead to differential rates of evolution when compared with snakes on t he mainland, and this could be manifested as a phenotypic change in venom compositi on or activity. However, the results

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136 presented here show an unclear pi cture of venom evolution and indicate that more than isolation may be involved. The SHK population seems to be a good c andidate for differences in venom not simply because of its insular habitat, but also because of its unique diet on the island. It would be logical to expect, if venom production is costly as indicated in previous studies (McCue, 2006), that snakes feeding on a ready supply of easy prey would eventually lose venom toxicity. Under such a scenario the cost of venom production would have to create a large enough drain on resources to make a difference in fitness. If, however, the abundance of prey offsets the energetic costs of venom production, venom would be less likely to take a directional evolutio nary trajectory and may instead evolve simply due to stochastic factors (i.e. genetic drift). One very interesting result of this study is the wide variation in activity found for all enzymes assayed. Not only is there a major ov erall variation, but there is also so much variation that any population-level differences may be obscured. This is interesting in that natural variation is important for natural selection to affect evolution. It is possible, however, that the wide diet seen normally in ma inland snakes is subject to selection so that cottonmouths may switch prey species under suboptimal conditions. This might make even the island snakes evolutionarily pr edisposed to maintaining venom variation. A further confounding factor for the SHK po pulation is in their capacity as carrion feeders. While many snake species are k nown to consume dead prey opportunistically, the majority of the diet of SHK snakes is made up of carrion. Although individual snakes on this island have been seen to bi te dead fish before consuming them, they also often simply swallow fish without env enomation. This is important because

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137 besides incapacitation of prey, venom is likel y used for aiding in diges tion, especially of large prey items. If venom is not being used for either in capacitation or digestion, then it is even less likely to be under the influence of natural selection. It is possible that venom toxicity could be maintained in SHK snakes due to longterm fluctuations in local climate. Duri ng periods of prolonged drought, when birds are less likely to nest successfully on the island, the snakes may have to rely on alternative prey such as introduced rats. Likewise, alter nate prey such as lizards may be important for young snakes on the island, making toxicity useful. This is the first known report examin ing individual variation within and among natural populations of the cottonm outh. Overall, it indicates that natural levels of venom variation are high. Although some diffe rences in population-level venom enzyme activity were seen, caution must be used when interpreting these results, as many other ecological factors may be involved in shaping venom composition. This is a first step in examining venom evolution at the level at which ev olution acts the most—the population.

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138 Figure 6-1. Map of Flori da with population locations indicated. The inset is an enlargement of the norther n part of the state, and the populations are as follows: LS, Lower Suwannee National Wi ldlife Refuge; PP, Paynes Prairie Preserve State Park; and SHK, Seahorse Key. Maps were generated using Google Earth. PP LS SHK

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139 Figure 6-2. Electrophoresis banding pattern s for individual cottonmouth venom samples. Individual samples were run on 12% acrylamide gels with 2-( N -morpholino)ethanesulfonic acid running buffer. Multiple individual samples from each population were us ed for comparison, including six animals from Big Cypress National Pres erve. Relative molecular masses are given on the left side of each gel and are calculated from molecular ladders of known sizes. Possible venom com ponents are bracketed and described on the right side of each gel. LS = Lo wer Suwannee National Wildlife Refuge and PP = Paynes Prairie Preserve State Park.

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140 Figure 6-3. Comparison of mean protease activities for three populations of Florida cottonmouth. Enzyme assays utilized venom samples from individual snakes from three different geographi c locations. SHK is Seahorse Key, LS is Lower Suwannee National Wildlife Refuge, and PP is Paynes Prairie Preserve State Park. Letters indicate significant groupings (P < 0.001) using a one-way ANOVA with Tukey post-hoc test. Activi ties are reported as nmol tyrosine produced per mg venom protein per hour, and error bars indicated 1 standard error.

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141 Figure 6-4. Comparison of mean hyaluronidase activiti es for three populations of Florida cottonmouth. Enzym e assays utilized venom samples from individual snakes from three different geographic lo cations. SHK is Seahorse Key, LS is Lower Suwannee National Wildlife Refuge, and PP is Paynes Prairie Preserve State Park. No significant differences were seen among the populations using a one-way AN OVA with Tukey post-hoc test. Activities are reported as g hyaluronic acid cons umed per mg venom protein per hour, and error bars indicated 1 standard error.

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142 Figure 6-5. Comparison of mean L-amino acid oxidase (LAAO ) activities for three populations of Florida cottonmouth. Enzyme assays utilized venom samples from individual snakes from three different geographic locations. SHK is Seahorse Key, LS is Lower Suwannee National Wildlife Refuge, and PP is Paynes Prairie Preserve State Park. Let ters indicate significant groupings (P < 0.001) using a one-way ANOVA with Tukey post-hoc test. Activities are reported as nM o -dianisidine reduced per mg venom protein per min, and error bars indicated 1 standard error.

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143 Figure 6-6. Comparison of mean phospholipase A2 activities for three populations of Florida cottonmouth. Enzym e assays utilized venom samples from individual snakes from three different geographic lo cations. SHK is Seahorse Key, LS is Lower Suwannee National Wildlife Refuge, and PP is Paynes Prairie Preserve State Park. Letters indicate significant groupings (P < 0.001) using a one-way ANOVA with Tukey post-hoc test. Activities are reported as nM o dianisidine reduced per mg venom protein per min, and error bars indicated 1 standard error.

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144 CHAPTER 7 A POPULATION-LEVEL COMPARISON OF THE DIET OF THE FLORIDA COTTONMOUTH, AGKISTRODON PISCIVORUS CONANTI Introduction The Florida cottonmouth ( Agkistrodon piscivorus conanti ) is known to consume various vertebrate prey species including fishes, amphibians, reptiles, birds, and mammals in the wild (Lillywhite and McCl eary, 2008). It is also known to be a scavenger of both natural prey and human re fuse (Wharton, 1969), and will consume many species in captivity that would not normally be experienced in the wild (Allen and Swindell, 1948). Many reports of ingested prey have focused on individual snakes, rather than on overall populat ion-level prey base, with only one published study detailing the specific diet of a population fr om one location (Wharton, 1966, 1969). This study focused on the snakes of the island of Seahorse Key (SHK), located ~5 km from the western Florida coast and found that this population greatly utilized dead fish scavenged under colonies of nesting birds. No other cottonmouth population has been reported to have such a carrion-based diet. Previous studies examining the relati onship between diet and venom composition have indicated that venom is more tightly co rrelated with local prey type than with other parameters (Daltry et al., 1996; Creer et al., 2003). One other study found a different pattern, in which venom composition in snak es on islands was more closely associated with the time the island had been isolated from the mainland, rather than with diet (Williams et al., 1988). The relationships of the first two studies indicate that venom composition may be evolving via natural sele ction, but the last study indicates that genetic drift may be mo re important.

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145 It is understandable that prey could be a major factor in the evol ution of animals, especially venomous snakes. The venom must be efficacious enough to facilitate prey capture, so alterations in its composition could greatly enhance or hinder the ability of the species to feed. This, in turn, coul d lead to alterations in reproduction and abundance of the species. In order to determine whether prey base may have an influence on phenotypic characters relating to venom composition, it is necessary to characterize diets of defined populations. As an attempt at th is, cottonmouths from three different geographic regions were examined for their dietary preference. These populations included SHK and two mainland sites, Paynes Prairie Preserve State Park (PP) and Lower Suwannee National Wildlife Refuge (LS). PP is located in north central Florida and includes a grassland area with multi-year flooding cycles, numerous fresh water ponds and streams, and patchy forest. Lower Suwannee is located on the western coast of Florida, separated from SHK by salt water, and contains both fresh and salt water habitats. The differences in habitats may lead to differences in prey base, although both PP and LS are very heter ogeneous in their habitat composition. Prey determination can be difficult for m any reasons. First, many animals are encountered because they are actively seek ing food (Daltry et al., 1998), whereas recently fed animals would be more likely to take shelter to digest. Because of this, many collected snakes are likely to lack stomac h and intestinal prey items. Second, in animals where food items are present, they are often found in a highly degraded state, making identification to any leve l difficult and to the specie s level impossible. Third, some animals are digested more quickly than others. This could lead to their reduced

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146 presence in the intestines and, especially, t heir feces. The only way to realistically determine all food items is through dissection of each individual. This practice is not tenable in endangered species and is often not practical for non-endangered species. It is, however, viable in dead animals. With these limitations in mind, useful data on diet can be collected from animals recently killed in the wild (by automobiles) or preserved in museum collections. Many vertebrate body parts either ar e not digestible by snakes (mammalian hair, etc.) or are resistant to digestion (large bones). In many cases, identifi cation to the class level is relatively easy, and for some well-preserv ed or unique features it may be possible to identify the dietary organism to the species level. In some cases it may even be possible to determine the number of prey i ngested by the snake (such as when two copies of the same bone are found). By utili zing metrics collected for museum research collections, it may even be possible to determine t he size of the prey in life. Overall, the less destructive use of road-killed animals can be very informative. Stable isotope analysis is often used to examine the flow of energy through ecosystems (Post 2002) or the specific di ets of certain species (Dalerum and Angerbjrn, 2005). In order to determine the importance of sp ecific prey species to a predator, however, the specific isotopic si gnature must be deter mined for all potential diet items. These can then be compared to the isotopic signat ures of the predator to look for similarities. Although not specific enough to completely determine diet, simple analysis of stable isotope si gnatures of predators from di fferent regions can give an indication of their similarities in diet.

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147 Materials and Methods Diet items were identified from snakes from each of the populations by a combination of three different methods. Firs t, fresh road-killed an imals from the areas of interest were collected when possible and di ssected (N = 33). Stomach and intestinal contents were collected and sorted in 70% ethanol. After isolating fragments of bone, scales, hair, feathers, or other identifiable an atomical parts (such as otoliths and lenses of fishes), each was ident ified to the lowest tax onomic level utilizing the zooarcheological research collect ion at the Florida Museum of Natural History (FLMNH) and through the resources of the Herpetology, Ichthy ology, Ornithology, and Mammalogy sections of FLMNH. Second, the same basic procedure was used to identify fragments located in t he gastrointestinal tract of s pecimens from the American Museum of Natural History (N = 1) and FLMNH (N = 13). Thir d, the feces of short-term captive live animals were analyzed in a sim ilar fashion (N = 35). Of these animals, many (N =17) did not contain diet item s and so were removed from analysis. Snakes were categorized in terms of type of prey from gut or fe cal contents. Prey items were all identif ied to order, as this was the deepest taxonomic level possible in many cases due to degradation of the sample. The number of animals containing each order of vertebrate prey was determined for each population. These tallies were then compared against each other. As an example, a snake contai ning three fish would be included in the fish category once, and a s nake containing a fish, a snake, and a lizard would be included in both the fish and squamat e categories (once each). To boost the overall sample size, vertebrate prey dat a from Wharton (1969) were added to the analysis. Although Wharton (1969) reports th e presence of invertebrates in the gut contents of cottonmouths, thes e were excluded from analysis. The reason for this is

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148 that there have been no direct reports of cottonmouths consuming invertebrate prey, and many of the vertebrate prey they do cons ume (frogs, fish, lizards, etc.) tend to have their own gut contents when consumed by th e snake. This means that undigested fragments of invertebrate exo skeletons may appear in the gut and fecal contents. In many cases, the invertebrat e fragments are accompanied by parts of a potential prey species. Further, some of the invertebrates are very small, and would not be expected to be ingested by a cottonmouth. To determine whether there were any significant differences in dietary composition, a 2 test was performed. Expected values were obtained by adding all dietary numbers from all three populat ions together and determining a common proportion. Observed values used were from tabulated diet numbers from each population. These tests were performed on mult iple data sets using different criteria including: 1) all new diet it ems from this study combined with data from Wharton (1969) (NTOTAL = 158; NSHK = 110; NPP = 27, NLS = 21); and 2) diet items only new to this study, from snakes over 500 mm snout to vent length (SVL) and 300 g (NTOTAL = 58, NSHK = 16, NPP = 23, NLS = 19). For all analyses, a posthoc Monte Carlo simulation using 10000 random draws of N sa mples (dependent on population size for each analysis) was conducted using specific sample sizes, and the resulting P-values are indicated as PMC. A further preliminary analysis was done as part of a more extensive study (Lillywhite, unpublished) to compare the stab le isotope signatures of animals from the three regions. This was done by using tissue fr om either scale clips or tail tips. These samples were prepared in a drying oven and analyzed by mass spectrometry. Samples

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149 loaded into tin capsules were placed in a Carlo Erba NA1500 CNS elemental analyzer. They were combusted under oxygen-rich air in a quartz column at 1000C, and the sample gas was transported in a helium carri er stream through a hot reduction column (650C) consisting of elemental copper for oxyg en removal. To re move water from the samples, the effluent stream was passed thr ough a chemical trap. It was then passed into a ConFlo II preparation system and finally into a Finnigan-MAT 252 mass spectrometer running in continuous flow m ode to measure sample gas relative to a laboratory reference gas. For this porti on of the study, animals from SHK (N = 127) were of all sizes, while snakes from PP (N = 18) and LS (N = 20) were at least 300 g and 500 mm SVL. The stable isotopes ex amined were nitrogen-15 and carbon-13. Results The three main categories of vertebrate pr ey found were fish, squamates (lizards and snakes), and mammals, with birds being represented only in t he SHK population. Because of this, analyses were run with the fi rst three categories a nd a fourth “other” category that combined remainin g items or with all four of these cat egories and a fifth “other” category. Besides these categorie s, amphibians (both urodele and anuran) and turtles were identified in gut contents. The relative pr oportions of the four-category totals can be seen in Figure 7-1, and of the fi ve-category totals in Figure 7-2. These are both for the combined data set. Using diet items determined from this study alone that met mass and SVL restrictions, there were no significant differ ences among the populations if the data were separated into 4 categories. However, w hen birds were considered as their own category, the SHK populatio n was significantly differ ent from expected dietary proportions ( 2 = 11.51, df = 4, P = 0.0214, PMC = 0.0234). When the data from

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150 Wharton (1969) and animals outside the strict cr iteria were used, th ere were significant differences among all three populations. Th is was true when the data was analyzed as four categories (SHK, 2 = 10.6, df = 3, P = 0.0141, PMC = 0.0147; PP, 2 = 15.78, df = 3, P = 0.0013, PMC = 0.0021; LS, 2 = 11.22, df = 3, P = 0.0106, PMC = 0.0115) and as five categories (SHK, 2 = 334.66, df = 4, P < 0.0001, PMC < 0.0001; PP, 2 = 30.67, df = 4, P < 0.0001, PMC = 0.0001; LS, 2 = 13.96, df = 4, P = 0.0074, PMC = 0.0087). The preliminary stable isot ope analysis can be seen in Figure 7-3. For these data, the two analyzed isotopes are plotted agains t each other. The figure indicates a difference in the SHK population isotopic sig nature compared to the other two sites. Discussion Although there was an apparent difference among the diets of the adult snakes analyzed specifically for this study, ther e were no statistical differences between observed and expected values (with the ex ception of SHK when utilizing 5 prey categories). It is likely that this is due to the small samp le size caused by strict limitations on included sample s, as inclusion of smalle r individuals and data from Wharton (1969) yielded significant differ ences in dietary structure among all populations, whether using 4 or 5 categories of data. The significant differences found when Wharton’s data was included must be viewed carefully, because it is possible t hat the large sample size for one population skews the overall expected values of the ot her populations. At the same time, however, the majority of prey items from SHK were fish, whereas they accounted for 38% of items from LS and only 10.3% of item s from PP. These values i ndicate a real difference in fish consumption among the populations. Other prey categorie s, specifically

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151 squamates, also are widely divergent among the populations, indicating a real difference in either availability or utiliz ation of prey in the different regions. The stable isotope analysis indicates that these two particular isotopes are being incorporated from different sources, with SHK being dist inct from the two mainland populations. This does not necessarily mean different prey species, however, as isotopes are transferred up trophic levels and may be altered by both lower level consumer and primary producer species (Post, 2002). The clear delineation between the SHK and mainland animals is interesting nonetheless. On SHK, snakes consume dead fish dr opped by seabirds, but scavenging was also seen in the mainland populations. Th is scavenging was indicated by both the presence of larvae of carrion-consuming in vertebrates and the gener al condition of some prey items. As an ex ample, two road-killed snakes from the PP population were found to contain whole frogs that appeared to have first also been killed on the road, as they were in a mangled condition. Further, a specimen from the LS population contained a lizard that was in multiple pi eces but only slightly digested. As cottonmouths do not tear apart their prey, th is indicated the lizard was dead prior to ingestion by the snake. All of this point s to the general opportunistic nature of the cottonmouth. Although it has previously been i ndicated that amphibians may be underrepresented in snake gut and fecal content s due to their ease of digestion (Daltry et al., 1998), it is notable that many am phibian fragments were identified in fecal samples from live snakes. It is possible that the stress from manipulat ion of live snakes decreases their gut passage time, allowing for less degraded samples in the feces.

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152 However, this does indicate that amphibi an structures can be f ound in feces of the snakes that consume them. Road-killed animals were useful in exam ining population-specif ic diets of the cottonmouth. Although they may be less likely to contain prey in their gastrointestinal tracts, they did yield useful information. It would be of interest to examine the relative digestibility of various prey species in the laboratory to determine whether different prey items are consistently detectable. Overall, this study adds to the understanding of the dietary hab its of the Florida cottonmouth and supports the rat her unique feeding style of the population on SHK. It supports the idea that cottonmouths are opportunistic feeders, as evidenced by scavenging and a wide array of prey specie s. It further indicates that these characteristics may allow the cottonmouth to adapt quickly to different ecosystems. This, in turn, may help explain the presence and relative abundance of the Florida cottonmouth throughout its range.

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153 Figure 7-1. Four-category diet composition comparison for three populations of Florida cottonmouth. The “other” category incl udes amphibians, turtles, and birds. Charts made only from new data from s nakes over 300 g are on the left, while previously published data and smaller ani mals are included on the right. SHK indicates the Seahorse Key population, PP is the Paynes Prairie population, and LS is the Lower Suwannee populati on. The populations in the “All” column were significantly differ ent from expected values using a 2 test (P < 0.02 for all). Mammal Squamate Fish Other New only All SHK PP LS

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154 Figure 7-2. Five-category diet composition comparison for three populations of Florida cottonmouth. The “other” category incl udes amphibians and turtles. Charts made only from new data from snakes over 300 g are on the left, while previously published data and smaller ani mals are included on the right. SHK indicates the Seahorse Key population, PP is the Paynes Prairie population, and LS is the Lower Suwannee population. SHK from the “New only” column (P < 0.03) and all populations in the “A ll” column (P < 0.01 for all) were significantly different from expected values using a 2 test. New only All SHK PP LS Mammal Squamate Fish Other Bird

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155 -30 -25 -20 -15 5101520 15N SHK PP LS 13C Figure 7-3. Stable isotope analysis of scale tissue from Florida cottonmouths. Samples were collected from three different populations (SHK = Seahorse Key, PP = Paynes Prairie, and LS = Lower Suwannee).

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156 CHAPTER 8 EVOLUTIONARY IMPLICATIONS AND SUMMARY Introduction Previous studies have concluded that the phenotypic character of venom composition is evolving under the influence of natural selection (D altry et al., 1996b; Creer et al., 2003) or genetic drift (Williams et al., 1988). In an attempt to test these hypotheses, I examined the genetic relationshi ps among four populati ons of the Florida cottonmouth, Agkistrodon piscivorus conanti and further compared venom enzyme activities in three of those populations. As a first step, it was necessary to sele ct venom characteristics that were known to have fairly high variation, as lack of vari ation would be more likely to simply lead to similar results among populations. Although mu ch work has been done on the effects of cottonmouth envenomation an d the composition and activi ty of cottonmouth venom, a review of the literature s howed some differing results. Among the studies examining enzymatic activities, Tan and Ponnudurai (1988, 1990) specifically studied the effects of cottonmouth venom, and they found great variat ion in a number of enzymes. However, they did not examine individual s, but rather pools of v enom. In order to get at differences among populations, it is necessary to look at phenotypic characters of individuals within those populations. It was also necessary to devise a techniq ue by which venom could be collected in a standard fashion. Previous published tech niques of venom extraction were developed either to produce as much venom as possible or a sample of an arbitrary size for quick studies, and many either did not work with cottonmouths specifically or were not practical. Using a combination of previous ly utilized techniques, but with more updated

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157 methods of anesthesia and electrical stimulati on, we were able to increase safety for humans involved in the study, reduce stress on the animals, and collect venom in a standard and repeatable fashion. After determining the manner of venom co llection and the specific components of venom to be studied, we needed to find a me thod by which the relationships among the snake populations could be determined. Previous work utilizing mitochondrial DNA (Parkinson et al., 2000; Guiher and Burb rink, 2008) or assorted fragment length polymorphisms either focused on subspecies-level relationships or were unsuccessful at verifying population-level identity (Roark, 2006). Although no microsatellite loci had been developed specifically for use in the co ttonmouth, they are powerful markers for discerning fine-scale population structure. We screened micr osatellite loci developed in closely related genera of snakes and although many either di d not amplify a product in cottonmouths or did not show any variation, we found ten useful markers that did both. After collecting 129 DNA samples from both live and road-killed snakes, we genotyped them using the ten mi crosatellite loci. Multiple analyses of these samples indicate that our four populati ons of interest are genetically isolated from each other to a significant extent. Our insular population, found on the island of Seahorse Key (SHK), was found to be the most isolat ed of the populations regardless of its proximity to the Lower Suwannee National Wildlife Refuge (LS) population. SHK was also found to have indications of inbreeding within its popu lation. Further, the LS population was less related to SHK than it was to the distant Paynes Prairie Preserve State Park (PP) population or the far distant Big Cypress National Preserve (BC) population.

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158 For three of these populations, we collect ed venom samples. Unfortunately, the BC population was much more scattered than the others, and several searching trips yielded too few live animals (N = 6) to make a statistically sound comparison. Using the other three populations, however, we did find significant differ ences in three of the four venom enzymes studied. Pr otease and phospholipases A2 (PLA2) activities were found to be significantly different between the LS (highest) and PP (lowest) populations, with SHK being intermediate; hyal uronidase activity was not found to be significantly different among any of the populations; and L-amino acid oxidase (LAAO) activity was found to be significantly elevated in the SHK population. There are significant differences in the activities of the venom of the three populations studied, but it is difficult to determine whether these are biologically meaningful Although it seems possible that the SHK snakes are fe eding upon a prey type that is more susceptible to a high L AAO activity, the dependence of these snakes upon carrion fish makes that seem unlikely. One previous study indicated that LAAO might help potentiate proteases, which could be useful with a high-pro tein fish diet. It may be just as possible that snakes on SHK are not under the same types of survival pressures as the mainland populations. SHK has a seasonal abundance of food and few potential snake predators outside of some diurnal birds (cannibalism notwithstanding). It is possible that LAAO is energetically expensive to make (but highly useful) so that the island snakes are not affected to the same extent that mainland snakes would be. The instance of higher protease activity in the venom of LS snakes could be related to the specifics of their environm ent. Unlike SHK, LS has very diverse

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159 substructure within its overall environment. This leads to a more diverse range of potential prey, so it may be more useful for snakes to have varied venom. Because protease activity is a general assay of multip le proteolytic enzymes, it is impossible to say exactly which proteases are increased in LS venom, or whether it may be all of them. Proteases are responsible for overa ll degradation of protein structures in the prey, so it is possible that LS snakes ar e consuming high-protein food items, such as fish, and that the elevated protease is si mply an evolutionary response to enhance digestion. The presence of brackish water in this system makes the prevalence of fish prey seem a little greater, al though there are many other potential prey species in this habitat. It is difficult to draw conclusions c oncerning the PP population and its lower protease and LAAO activities. However, this is the only population studied that is in a purely fresh water environment. It coul d be that none of the enzymes studied are necessary in high amounts for incapacitati on and digestion of prey in the prairie and fresh water swamp setting. The similarity in hyaluronidase activity among the populations indicates that it is likely not a major factor in t he biological activity of the venom, at least in terms of differential reproductive success. Hyaluroni dase acts by degrading hyaluronic acid between cells to decrease their function as barriers. This allows the other venom components to slip through tissue layers and po ssibly become systemic. It also allows enzymes to spread, potentiating digestive enzym e effects. It may be that this enzyme functions more in a digestive capacity after the death of a prey animal than in aiding in

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160 incapacitation. It could be that higher activiti es do not yield greater results, so elevation of this component costs ener gy without any added benefits. Differences in PLA2 activity among the populations also do not indicate a clear pattern. The same pattern f ound with protease wa s found with PLA2, in that LS animals had higher activity than PP animals, wit h SHK snakes intermediate. PLA2 is used, in general, to degrade cell membranes, but it does have very different functions with minor structural changes. Any changes in func tion would not be detected with the assay utilized in this study. It is possible that the prey being c onsumed by LS snakes is more susceptible to PLA2 activity. The difference in prey base is likely at the root of the differences in venom composition among the populations studied. Dietary anal ysis indicated that the prey base was different for each population, so venom composition may be evolving activity toward certain prey species. However, unlik e the Daltry et al. ( 1996b) study, it may not be the specific prey items, but rather the overall amount of food energy available that is important. If food is lim ited, venom is expected to evolve toward greater incapacitation of available prey. If, however, food is not the limiting factor, venom may simply evolve in a random fashion (as was indicated by W illiams et al., 1988). This leads to the question of whether insular populations ar e generally under different evolutionary constraints than mainland populations. Quantitative Trait Loci Although there is seemingly no clear-cut answer to the question of how venom is evolving in these populations, there is evidence that pr ey may be important. The populations of interest are genet ically distinct, have overall venom activity that is different, and have differences in diet composit ion. It can not be st ated definitively that

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161 prey base differences are causing the observ ed venom activity differences, but it is possible to compare venom differences with genetic differences. Quantitative traits ar e those phenotypic characters t hat are influenced by multiple genes and have a continuous distribution over t heir range of potential values, such as the venom activities examined in this study. They differ from discr ete traits that are controlled by one gene, and are instead influenced by a suite of genes called quantitative trait loci (QTLs). These loci may be strongly influenced by environment. Recently, quantitive traits have been used to determine quantitative genetic differentiation, and these data have been compared to neutral marker gene differentiation. Quantitative genetic di fferentiation can be calculated as a QST value that considers the both within and bet ween population variances in a phenotypic character. The QST is analogous to the FST statistic calculated for neutral genetic traits. In the case of the FST, heterozygosities are calculated to determine the relative importance of variance between populations compar ed to the species as a whole. Comparisons of QSTs and FSTs values have been popular rece ntly for inferring the effect of natural selection on quantitative traits (Leinonen et al., 2008). Lande (1992) considered that if QST and FST values were similar, then there was no selection on the quantitative trait in question, and Yang et al (1996) indicated that in these cases genetic drift can explain any differentiati on. Meril and Crnokrak (2001) published a model of QST/FST proportions that considered values near 1.0 to be indi cative of drift alone, values above 1.0 to be indicative of di versifying selection, and values below 1.0 to indicate stabilizing selection. Althoug h there is an assumption of heritability when using QSTs, it is often not possible to determine whether traits truly are heritable. The

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162 Florida cottonmouth is not a model organism fo r these types of studies because of their relatively late reproductive age and twoto th ree-year reproductive cycles. However, it is still worthwhile to compare QSTs and FSTs to get an indication of the potential importance of natural selection on enzyme characteristics. Pairwise FST values were determined for each pair of populations using Structure 2.2, and QST values were determined by calcul ating within and between population variances in enzyme activity for each veno m enzyme and each pair of populations. The QST/FST proportions indicate that most of th e enzyme activities are under diversifying selection among all population co mparisons (Figure 8-1). The exceptions to this trend are two comparisons (SHK to LS and SHK to PP) of hyaluronidase activity. These values indicate that selection has less of an impact on values than does genetic drift. Interpretation of these data must be c onsidered carefully, however. Whitlock (1999) stated that diversifying selecti on in newly formed populations may not be detectable simply because too little time may have passed to increase QST values, even when populations truly are diversifying. This indicates that these calculations are too conservative to detect new species. On t he other hand, Hendry (2002 ) indicated that it is more difficult to detect diversifying select ion in populations that have been isolated for long periods of time. As populations diverge, the FST value increases towards 1.0, which would obscure any large values of QST. In these cases, diversifying selection would not be detected. At that point, however, it could be considered that the populations belong to different, distinct species. Conclusion The three populations of Florida cottonmout h utilized in this study were found to be genetically distinct fr om one another using microsatellite loci. These same populations

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163 were found to have differences in activities of three of four ve nom enzymes. Further, the populations were found to have differences in prey type found in gut and fecal contents. Correlative comparisons of enzyme activity and neutral genetic markers indicate that the enzymatic activities ar e (for the most part) undergoing diversifying selection. Together, these data point to a divergence of venom activity and genetic relatedness that is at least correlated wit h prey differences, although diet can not definitively be considered to be driving this divergence. Since we now have the means to co llect cottonmouth venom in a standard fashion, and also can determine genetic relationships among these snakes using a powerful set of microsatellite loci, getting at the essence of the relative importance of natural selection in venom evolution shoul d be easier. Without fully understanding the components present in the venom and their spec ific physiological action on prey, it is more difficult, so further work is needed in th ese areas. Although it is also difficult to catalogue the relative importance of different prey species in such a generalist feeder, diet breadth will also be important for future work. We now also have an understanding of the natural variation in activity of f our enzymes, and this may lead to discovery of novel venom components that may be useful not only for studies of evolution, but also for practical use in antivenom ther apy or pharmaceutical development.

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164 Figure 8-1. QST/FST comparison of enzyme activities for each of three populations of Florida cottonmouths. Pairwise QST values were calculated as described in the text and pairwise FST values were determined using STRUCTURE 2.2. A 1:1 line is included for reference. Using the model of Meril and Crnokrak (2001), values above the line indicate characters undergoing diversifying selection, values on or near the line indicate characters not undergoing selection (but rather drift), and values below the line indicate characters undergoing uniform or stabilizing selection.

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165 LIST OF REFERENCES Ahn, M.Y., Lee, B.M., Kim, Y.S., 1997. Cytotoxicity and L-amino acid oxidase activity of animal venoms. Archives of Pharmacal Research 20(1), 13-16. Aird, S.D., 2002. Ophidian envenomation strategies and the role of purines. Toxicon 40, 335-393. Allen, E.R., Swindell, D ., 1948. Cottonmouth moccasin of Florida. Herpetologica 4(supplement 1), 1-16. Almeida, O.P., Bhm, G.M., Bonta, I.L., 1977. Morphologica l study of lesions induced by snake venoms ( Naja naja and Agkistrodon piscivorus ) in the lung and cremaster vessels of rats. J ournal of Pathology 121, 169-176. Anderson, C.D., 2006. Utility of a set of microsatelli te primers developed for the massasauga rattlesnake ( Sistrurus catenatus ) for population genetic studies of the timber rattlesnake ( Crotalus horridus ). Molecular Ecology Notes 6, 514-517. Andrade, D.V., Abe, A.S., 1999. Relations hip of venom ontogeny and diet in Bothrops Herpetologica 55(2), 200-204. Andrews, C.E., Dees, J.E., Edwards, R.O., Jackson, K.W., Snyder, C.C., Moseley, T., Gennaro, J.F., Jr, Gehres., G. W., 1968. Venomous snakebite in Florida. Journal of the Florida Medical Association 55, 308-316. Andrews, E.H., Pollard, C.B., 1953. Report of snake bites in Florida and treatment: venoms and antivenoms. Journal of the Flor ida Medical Association 40(6), 388397. Angulo, Y., Gutirrez, J.M., Soares, A.M. Cho, W., Lomonte, B., 2005. Myotoxic and cytolytic activities of dimeric Lys49 phospholipase A2 homologues are reduced, but not abolished, by a pH-induced di ssociation. Toxicon 46, 291-296. Angulo, Y., Lomonte, B., 2003. Inhibitory effect of fucoi dan on the activities of crotaline snake venom myotoxic phospholipases A2. Biochemical Pharmacology 66, 19932000. Anton, A.H., Gennaro, J.F., Jr., 1965. Nor epinephrine and serotonin in the tissues and venoms of two pit vipers. Nature 208(5016), 1174-1175. Araya, C., Lomonte, B., 2007. An titumor effects of cationic synthetic peptides derived from Lys49 phospholipase A2 homologues of snake venoms. Cell Biology International 31(3), 263-268.

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168 Bhargava, N., Zirinis, P., B onta, I.L., Vargafti g, B.B., 1970. Comparison of hemorrhagic factors of the venoms of Naja naja Agkistrodon piscivorus and Apis mellifera Biochemical Pharmacology 19, 2405-2412. Birdsey, V., Lindorfer, J., Gewurz, H., 1971. Interaction of toxic venoms with the complement system. Immunology 21:299-310. Blouin-Demers, G., Gibbs, H. L., 2003. Isolation and characterization of microsatellite loci in the black rat snake ( Elaphe obsoleta ). Molecular Ecology Notes 3, 98-99. Bond, J.M., Porteous, R., H ughes, S., Mogg, R.J., Gardner M.G., Reading, C.J., 2005. Polymorphic microsatellite markers, isolat ed using a simple enrichment procedure, in the threatened smooth snake ( Coronella austriaca ). Molecular Ecology Notes 5, 42-44. Bonnett, D.E., Guttman S.I., 1971. Inhibition of moccasin ( Agkistrodon piscivorus ) venom proteolytic activity by t he serum of the Florida kingsnake ( Lampropeltis getulus floridana ). Toxicon 9, 417-425. Bonnet, X., Bradshaw, D., Shi ne, R., Pearson, D., 1999. Why do snakes have eyes? The (non-)effect of blindness in island tiger snakes ( Notechis scutatus ). Behavioral Ecology and Sociobiology 46, 267-272. Bonta, I.L., Bhargava, N., Vargaftig, B.B., 1970a. Hemorrhagic snake venoms and kallikrein inhibitors as tools to study fa ctors determining the in tegrity of the vessel wall. Advances in Experimental Medicine and Bi ology 8, 191-199. Bonta, I.L., Bhargava, N., Vargaftig, B.B., 1970b. Dissociati on between hemorrhagic, enzymatic and lethal activity of some snake venoms and of bee venom as studied in a new model. In: de Vries, A., Kochva, E. (Eds.), Toxins of Animal and Plant Origin. Gordon and Breach Science Publishers, New York, pp. 707-719. Bonta, I.L., Vargaftig, B.B ., Bhargava, N., De Vos, C. J., 1970c. Method for study of snake venom induced hemorrhages. Toxicon 8, 3-10. Bonta, I.L., Vargaftig, B.B ., De Vos, C.J., Grijsen, H ., 1969. Haemorrhagic mechanisms of some snake venoms in relation to prot ection by estriol succinate of blood vessel damage. Life Sciences 8, 881-888. Boquet, P., Izard, Y., Detrait, J., 1958. Recherches sur le fa cteur de diffusion des venins de serpents. Comptes Rendus des Sances de la Socit de Biologie 152(10), 1363-1365. Brabec, V., Kornalk, F., 1977. Lysis of r ed blood cells in some haemolytic anaemias— the lytic effect of Agkistrodon piscivorus venom in vitro Folia Haematologica. Internationales Magazin fur Klinisc he und Morphologische Blutforschung 104(3), 428-435.

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169 Brabec, V., Kornalk, F., Zak, J., Dyr J.E. 1985. Use of some snake venoms in the diagnostics of paroxysmal nocturnal hemogl obinuria (PNH). Folia Haematologica. Internationales Magazin fur Klinisc he und Morphologische Blutforschung 112(2), 293-302. Braganca, B.M., 1955. Neurochemical effects of snake venoms. In: Elliott, K.A.C., Page, I.H., Quastel, J.H. (Eds.), Neurochemis try, the Chemical Dynamics of Brain and Nerve. Charles C Thomas, Springfield, pp. 612-630. Braganca, B.M., Quastel, J.H., 1952. Amino acid oxidations by snake venoms. Archives of Biochemistry and Biophysics 40, 130-134. Braganca, B.M., Quastel, J.H. 1953. Enzyme inhibitions by snake venoms. Biochemical Journal 53(1), 88-102. Brochetto-Braga, M.R., Palma, M.S., Carm ona, E.C., Chaud-Netto, J., Rodrigues, A., da Silva, G.P., 2006. Influence of t he collection methodology on the Apis mellifera venom composition: peptide analysis. Sociobiology 47 (3), 759-770. Brown, R.V., 1940. Action of water mo ccasin venom on the isolated frog heart. American Journal of Ph ysiology 130(4), 613-619. Brown, R.V., 1941. Effects of water mocca sin venom on dogs. American Journal of Physiology 134, 202-207. Burns, E.L., Houlden, B.A., 1999. Isolati on and characterization of microsatellite markers in the broad-headed snake Hoplocephalus bungaroides Molecular Ecology 8, 520-521. Cadle, J.E., 1988. Phylogenetic relations hips among advanced snakes: a molecular perspective. University of California Press, Berkeley. Calhoun, W., Yu, J., Sung, A ., Chau, T.T., Marshall, L.A ., Weichman, B.M., Carlson, R.P., 1989. Pharmacologic modul ation of D-49 phospholipase A2-induced paw edema in the mouse. Agents and Actions 27(3-4), 418-421. Candido, D.M., Lucas, S., 2002. Main tenance of scorpions of the genus Tityus Koch (Scorpiones, Buthidae) for venom obtenti on at Instituto But antan, So Paulo, Brazil. Journal of Venomous Animals and Toxins Including Tropical Diseases 10 (1), 86-97. Carlsson, M., Isaksson, M., Hoggren, M., T egelstrom, H., 2003. Characterization of polymorphic microsatellit e markers in the adder, Vipera berus Molecular Ecology Notes 3, 73-75. Cavinato, R.A., Remold, H ., Kipnis, T.L., 1998. Purification and variability in thrombin-like activity of Bothrops atrox venom from different geographic regions. Toxicon 36(2), 257-267.

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170 Chargaff, E., Cohen, S.S., 1939. On lysopho sphatides. The Journa l of Biological Chemistry 129, 619-628. Chijiwa, T., Yamaguchi, Y., Ogawa, T., Deshimaru, M., Nobuhisa, I., Nakashima, K., Oda-Ueda, N., Fukumaki, Y., Hattori, S., Ohno, M., 2003. Interisland evolution of Trimeresurus flavoviridis venom phospholipase A2 isozymes. Journal of Molecular Evolution 56, 286-293. Chippaux, J.-P., 2006. S nake Venoms and Envenomations. Krieger Publishing Company, Malabar. Chippaux, J.-P., Williams, V., White, J., 1991. Snake venom variability: methods of study, results and interpretation. Toxicon 29(11), 1279-1303. Chiszar, D., Lee, R.K.K., Radc liffe, C.W., Smith, H.M., 1992. Searching behaviors by rattlesnakes following predatory strikes. In: Campbell, J.A., Brodie, E.D., Jr. (Eds.), Biology of the Pitvipers. Selva, Tyler, pp. 369-382. Cho, W., Tomasselli, A.G., Heinrikson, R.L. Kzdy, F.C., 1988a. The chemical basis for interfacial activation of monomeric phospholipases A2. The Journal of Biological Chemistry 263(23), 11237-11241. Cho, W., Markowitz, M.A., Kzdy, F. J., 1988b. A new class of phospholipase A2 substrates: kinetics of the phospholipase A2 catalyzed hydrolysis of 3-(acyloxy)-4-nitrobenzoic acids. Journal of the Ameican Chemical Society 110, 5166-5171. Clark, A., 2006. Using microsat ellite loci to determine the fine scale genetic structure of a complex of timber rattlesnake (Crota lus horridus) dens in northeastern New York. Unpublished master’s thes is, University of Florida. Clark, J.M., Higginbotham, R.D., 1965a. Separation of toxic an d allergenic fractions of Agkistrodon piscivorus venom. Texas Reports on Biology and Medicine 23(1), 141. Clark, J.M., Higginbotham, R.D. 1965b. Separation of toxic and allergenic fractions of Agkistrodon p. piscivorus venom. Federation Proceedings 24(2P1), 251. Clark, J.M. Higginbotham, R. D., 1970. Influence of adrenalectomy and/or cortisol treatment of resistance to moccasin ( Agkistrodon p. piscivorus ) venom. Toxicon 8, 25-32. Clark, J.M. Higginbotham, R. D., 1971. Cottonmouth moccasi n venom: fractionation of toxic and allergenic components and intera ction with tissue mast cells. Texas Reports on Biology and Medicine 29, 181-192.

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206 Zalisko, E.J., Kardong, K.V., 1992. Histology and histochemistry of the Duvernoy’s gland of the brown tree snake Boiga irregularis (Colubridae). Copeia 1992(3), 791799. Zarco, R.M., Schultz, D., Vroon, D.H., 1967. Inactivation of guinea pig complement by Agkistrodon piscivorus (cottonmouth moccasin). Federation Proceedings 26, 362. Zeller, E.A., 1947. ber das vorkommen und die natur der cholinesterase der schlangengifte. Experi entia 3(9), 375-376. Zeller, E.A., 1948. Enzymes of snake v enoms and their biological significance. Advances in Enzymology 8, 459-495. Zeller, E.A., 1949. ber die cholinestera se der schlangengifte. 5. mitteilung ber biochemie der tierischen gifte. Helv etica Chimica Acta 32(1), 94-105. Zeller, E.A., 1950. ber phosphatesen II ber eine neue adenosintriphosphatase. Helvetica Chimica Acta 33(4), 821-833. Zeller, E.A., 1951. Enzymes as essential co mponents of bacterial and animal toxins. In: Sumner, J.B., Myrbck, K. (Eds.), The Enzymes, Chemistry and Mechanism of Action, vol. 1, part 2. Academic Press, New York, pp. 986-1013.

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207 BIOGRAPHICAL SKETCH Ryan J. R. McCleary began a fascination wi th reptiles and amphibians at an early age when he lived near herpetologist Dr. Jame s Gillingham. As a high school student, he was greatly influenced by his biology teacher, Mr. Steve Robbins, who encouraged him to pursue his reptilian interests. He graduated from Western Mi chigan University in 1993 with an undergraduate degree in biology and from Virginia Tech in 2001 with a master’s degree in the same field. At Virginia Tech, he worked with Dr. F. M. Anne McNabb examining the effects of pollutant c hemicals on endocrine function in birds. During his tenure as a student, he has completed a summer natural history internship at the National Museum of Natural History (Smi thsonian Institution) in Washington, DC, under the supervisor of Dr. Roy W. McDiarmid and a summer fellowship at the Lincoln Park Zoo in Chicago, IL. He has also conducted research in the Bahamas, Tobago, and Taiwan as well as parts of Florida. Recently, he completed the National Science Foundation East Asia Pacific Summer Institute program in Singapore.