Novelty in three-finger toxin evolution

Daniel Dashevsky B.A.

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2019 School of Biological Sciences Abstract

Snakes are a striking taxon on many levels and have an indelible place in the collective culture of humanity. This is due to a number of unusual features including their limblessness, ability to ingest large prey, but most importantly their venom. From a biological perspective, snakes are equally fascinating, showing how a simple body plan can be used to great effect in an extremely broad variety of niches. Snakes are globally distributed and ecologically diverse; as a result, since recent research has shown that most snakes are venomous to some degree, their venom is equally diverse. This thesis focuses on one major toxin family: three-finger toxins (3FTx). These toxins can be found in virtually all venomous snake lineages and are often neurotoxic. Since their original recruitment there have been numerous instances of structural and functional innovation within this toxin family. The research in this thesis pertains to several such novel adaptations in snakes of the colubrid and elapid families, many of whose venom is composed primarily of these toxins.

Chapter 2 deals with the ancestral version 3FTx and how, in the colubrine genus Boiga, simple mutations in toxins can lead to structural changes, in this case additional cysteines enable the formation of dimeric toxins with increased potency. These novel toxins can, in turn, open up new niches to the snakes which possess those toxins. In B. irregularis they facilitated a destructive invasion event and may have similarly assisted in the spread of an ancestral Boiga since our research shows that several other species possess similar dimeric toxins. This echoes a process that occurred in the ancestor of the elapids where the loss of a pair of cysteines and their disulfide bond was followed by an incredible diversification of these toxins and the snakes that produce them. Chapter 3 explores venom composition and activity of the genus Calliophis, the most basal genus of the elapids. C. bivirgatus and C. intestinalis both possess extremely elongated venom glands and each produce 3FTx with novel sodium channel toxicity. However, the two venoms produce opposite effects from one another. These results suggests some intriguing possibilities about the evolutionary history of these novel traits in Calliophis and the general composition of their venoms inform our understanding of how the venoms of more derived elapids evolved.

Chapters 4 and 5 focus on the genus Micrurus. This genus from the Americas is by far the most speciose genus of elapids and belongs to the coral snake clade which is the next branch in the phylogeny of the elapids after Calliophis. The venoms of these snakes all contain a high number of unique 3FTx, though some venoms may be dominated by PLA2 toxins in terms of overall composition. Chapter 4 focuses on the sequence diversity of 3FTx from this genus and the selection regimes under which they operate. This diversity may lay the groundwork for rapid changes in the toxin composition of the venom and accompanying changes in venom activity. Chapter 5 directly assesses the neurotoxic activity of 3FTx by testing the affinity of toxins in Micrurus venoms to the binding site of nicotinic acetycholine receptors from a wide sampling of other vertebrates. This is the known site of action for many 3FTx and bears out some of the predictions made in Chapter 4. The venoms in this study represent a diverse selection of species in terms of geography, phylogeny, and ecology and the variation in venom efficacy across targets shows clear phylogenetic trends, but that changes in expression levels or toxin sequences can facilitate the shift from one niche to another. Because of the fairly direct link from genotype to phenotype, relatively small genetic changes can create biochemical novelties in certain toxins which potentially allow large ecological shifts and influence the evolution of large clades of snakes and those species that coevolve with them. Taken together, this research provides a look at several such novelties and their continuing impact on the relevant organisms. There have been many more such occurrences within the 3FTx alone, to say nothing of other toxin families. If the patterns we find in our research hold true, then many other major events in the history of snake evolution may have co-occurred with similar novel venom phenotypes. Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis. Publications included in this thesis

Incorporated as Chapter 2 Dashevsky D, Debono J, Rokyta D, Nouwens A, Josh P, and Fry BG. “Three-Finger Toxin Diversifi- cation in the Venoms of Cat-Eye Snakes (Colubridae: Boiga)”. Journal of Molecular Evolution 86.8: 531–545, (2018).

Author Contribution Conception and design (25%) Daniel Dashevsky Bioinformatics (80%) (candidate) Writing (70%) Editing(20%) Conception and design (25%) Jordan Debono Writing (30%) Editing(20%) Bioinformatics (20%) Darin Rokyta Editing (30%) Amanda Nouwens Generate MS/MS data (100%) & Peter Josh Conception and design (50%) Bryan Fry Editing (30%)

Incorporated as Chapter 4 Dashevsky D and Fry BG. “Ancient Diversification of Three-Finger Toxins in Micrurus Coral Snakes”. Journal of Molecular Evolution 86.1: 58–67, (2018).

Author Contribution Conception and design (60%) Collate data (100%) Daniel Dashevsky Analysis(100%) (candidate) Writing (100%) Editing (60%) Conception and design (40%) Bryan Fry Editing (40%)

Submitted manuscripts included in this thesis

No manuscripts submitted for publication

Other publications during candidature

Peer-Reviewed Papers: 1. Debono J, Dashevsky D, Nouwens A, Fry BG. “The sweet side of venom: Glycosylated prothrombin activating metalloproteases from Dispholidus typus (boomslang) and Thelotornis mossambicanus (twig snake)”. Comparative Biochemistry and Physiology, Part C, In Press, (2019).

2. Zdenek CN, op den Brouw B, Dashevsky D, Gloria A, Youngman N, Watson E, Green P, Hay C, Dunstan N, Allen L, Fry BG. “Clinical implications of convergent procoagulant toxicity and differential antivenom efficacy in Australian elapid snake venoms”. Toxicology Letters, In Press, (2019).

3. Dashevsky D, Debono J, Rokyta D, Nouwens A, Josh P, and Fry BG. “Three-Finger Toxin Diversification in the Venoms of Cat-Eye Snakes (Colubridae: Boiga)”. Journal of Molecular Evolution 86.8: 531–545, (2018).

4. Dashevsky D and Fry BG. “Ancient Diversification of Three-Finger Toxins in Micrurus Coral Snakes”. Journal of Molecular Evolution 86.1: 58–67, (2018).

5. Baumann K, Dashevsky D, Sunagar K, Fry BG. “Scratching the Surface of an Itch: Molecular Evolution of Aculeata Venom Allergens”. Journal of Molecular Evolution 86.7: 484–500 (2018).

6. Goldenberg J, Cipriani V, Jackson TNW, Arbuckle K, Debono J, Dashevsky D, Panagides N, Ikonomopoulou MP, Koludarov I, Li B, Castro Santa R, Nouwens A, Jones A, Hay C, Dunstan N, Allen L, Bush B, Miles JJ, Ge L, Kwok HF, Fry BG. “Proteomic and functional variation within black snake venoms (Elapidae: Pseudechis)”. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 205: 53–61 (2018).

7. Lister C, Arbuckle K, Jackson TNW, Debono J, Zdenek CN, Dashevsky D, Dunstan N, Allen L, Hay C, Bush B, Gillett A, Fry BG. “Catch a tiger snake by its tail: Differential toxicity, co-factor dependence and antivenom efficacy in a procoagulant clade of Australian venomous snakes.” Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 202: 39–54, (2017).

8. Rogalski A, Soerensen C, op den Brouw B, Lister C, Dashevsky D, Arbuckle K, Gloria A, Zdenek CN, Casewell NR, Gutierrez´ JM, Wuster¨ W, Ali SA, Masci P, Rowley P, Frank N, Fry BG. “Differential procoagulant effects of saw-scaled viper (Serpentes: Viperidae: Echis) snake venoms on human plasma and the narrow taxonomic ranges of antivenom efficacies”. Toxicology Letters 280: 159–170, (2017)

9. Koludarov I, Jackson TN, op den Brouw B, Dobson J, Dashevsky D, Arbuckle K, Clemente CJ, Stockdale EJ, Cochran C, Debono J, Stephens C, Panagides N, Li B, Manchadi M-LR, Violette A, Fourmy R, Hendrikx I, Nouwens A, Clements J, Martelli P, Kwok HF, Fry BG. “Enter the dragon: the dynamic and multifunctional evolution of Anguimorpha lizard venoms”. Toxins 9.8: 242, (2017). 10. Yang DC, Dobson J, Cochran C, Dashevsky D, Arbuckle K, Benard M, Boyer L, Alagon´ A, Hendrikx I, Hodgson WC, Fry BG. “The bold and the beautiful: a neurotoxicity comparison of new world coral snakes in the Micruroides and Micrurus genera and relative neutralization by antivenom”. Neurotoxicity Research 32: 487–495, (2017).

11. Jackson TNW, Koludarov I, Ali SA, Dobson J, Zdenek CN, Dashevsky D, op den Brouw B, Masci PP, Nouwens A, Josh P, Goldenberg J, Cipriani V, Hay C, Hendrikx I, Dunstan N, Allen L, Fry BG. “Rapid radiations and the race to redundancy: an investigation of the evolution of Australian elapid snake venoms”. Toxins 8.11: 309, (2016).

12. Yang DC, Deuis JR, Dashevsky D, Dobson J, Jackson TNW, Brust A, Xie B, Koludarov I, Debono J, Hendrikx I, Hodgson WC, Josh P, Nouwens A, Bailie GJ, Bruxner TJC, Alewood PF, Lim KKP, Frank N, Vetter I, Fry BG. “The snake with the scorpion’s sting: novel three-finger toxin sodium channel activators from the venom of the long-glanded blue coral snake (Calliophis bivirgatus)”. Toxins 8.10: 303, (2016).

Conference abstracts

1. Dashevsky D, Debono J, Rokyta D, Nouwens A, Josh P, and Fry BG. “Boiga: venom and invasion”. Instituto Butantan: Guest Seminar, (2019).

2. Dashevsky D, Rokyta D, Vetter I, and Fry BG. “Electric blue: venom composition and activity of the long-glanded blue coral snake (Calliophis bivirgatus)”. Australian Society of Herpetology: Oral Presentation, (2018).

3. Dashevsky D and Fry BG. “Using sequence-based bioinformatics to guide toxin biodiscovery”. Venoms to Drugs: Rapid Fire Talk, (2017).

4. Dashevsky D and Fry BG. “Ancient diversification of three-finger toxins in Micrurus coral snakes”. Biology of Snakes: Oral Presentation, (2017).

5. Streicher JW, Pang L, Dashevsky D, Schaack S, Fujita MK, Meik JM. “Patterns of mito-genomic evolution in rattlesnakes: do disparate phylogenetic signals indicate historical recombination?”. Society for Molecular Biology & Evolution: Poster, (2017).

Contributions by others to the thesis

The contributions by many people to sections of this thesis have already been noted above in the publications sections, but there are those helping on projects that are not yet publications and those whose contributions go beyond simply helping publications make it out the door. Bryan Fry and Irina Vetter have been valuable advisors, helping me push my research forwards while remaining rigorous. Pioneering members of the Venom Evolution Lab including Jordan Debono, Richard Harris, Bianca op den Brouw, and Christina Zdenek have developed many of the techniques that are used in my research. Additional training in the lab was provided by James Dobson, Ivan Koludarov, and Nicholas Youngman. Darin Rokyta provided key insights, guides, and friendly lab to visit and learn various bioinformatics, venom extraction, and other techniques. Venoms used in this study were provided by many people including Alejandra Alagon, Chip Cochran, Nathaniel Frank, Choo Tan Hock, and Ana Moura da Silva.

Statement of parts of the thesis submitted to qualify for the award of another degree

No works submitted towards another degree have been included in this thesis.

Research involving human or animal subjects

No animal or human subjects were involved in this research. Acknowledgments

The obvious starting point is to thank my family. They’ve been supportive the whole way and never tried to dissuade me from moving to the opposite side of the globe to specifically look for incredibly toxic snakes. On top of that, they’ve gone above and beyond to come visit me and travel to wonderful parts of this country. I literally wouldn’t exist without my parents, but it all goes way beyond that. From Sam coming with me to help set up life in Brisbane to Margi letting me drag her around crocodile infested lakes with snakes under every bush, I’ve felt the love and trust constantly. I do my best to return it. The entire crew of the Venom Evolution Lab has been a dream to work with. Bryan Fry is a friend and and interesting person on top of being a great advisor who has always made sure that the entire PhD experience has been working to my interest and benefit. James Dobson is a fantastic friend, sounding board for ideas, enabler for spending money on food and beer, and herping partner. My entire cohort of Jordan Debono, Bianca op den Brouw, and Christina Zdenek have been there with me every step of the way, learning and growing together. Rich Harris has good taste in books which has been sorely lacking since Ivan Koludarov left. Tim Jackson was always good to drink beer and talk philosophy with. Renan Castro Santana let me dig a bunch of holes in the desert for him. The Brisbane Ultimate scene has been instrumental in keeping me sane. Within a week of starting my degree, I’d already been conscripted onto a team called the Cobras, so that was a good start. Through injuries, rain, sunburn, beetles eaten, beards half-shaved, tough matches lost I’ve enjoyed every practice, game, tournament, and party. Zev Permack, Viv Yuen, and Sam McGuckin were fantastic friends and housemates, I’m glad we got a year to hang out. The entire School of Biological Sciences has been excellent too, even if they did try to forget about us in an annex. Rooftop beers is a glorious institution, tutoring field courses on K’gari is a ridiculously nice job, and all the students are fantastic friends to have. I also want to thank everyone who has made my various research trips so enjoyable: Christina invited me to look for death adders on Magnetic Island; Rachel Beasley pulled strings to get me a place to stay in Arizona and Gordon Schuett provided the bed; Darin Rokyta’s lab was particularly welcoming and accomodating; special shout out goes to Mike Hogan, someday we’ll hang out and get to spend some shop time together; Stephanie Sgroi, Flavio Somogyi, Scott, Tangles, and Tara Brown let me tag along on a trip to one of the holy sites of snakedom; Felipe Grazziotin and the rest of everyone at Instituto Butantan; special thanks to Nora, the other Felipe, Joao,˜ and Ana for making sure I had a roof over my head and showing me how to have a good time in Sao˜ Paulo. Financial support

This research was supported by a University of Queensland Centennial Scholarship along with an International Postgraduate Research Scholarship from the Australian Government Department of education. This funding scheme has since transitioned to the Graduate School Scholarship and Research Training Program Scholarship provided by the same organizations, respectively.

Keywords

Snake venom, three-finger toxin, novelty, elapid, molecular evolution, neurotoxicity

Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 060409 Molecular Evolution, 40% ANZSRC code: 060199 Biochemistry and Cell Biology not elsewhere classified, 30% ANZSRC code: 060399 Evolutionary Biology not elsewhere classified, 30%

Fields of Research (FoR) Classification

FoR code: 0604, Genetics, 40% FoR code: 0601, Biochemistry and Cell Biology, 30% FoR code: 0603, Evolutionary Biology, 30% Contents

Abstract ...... ii

Contents xi

List of figures xiv

List of tables xvi

List of abbreviations xvii

1 Introduction 1 1.1 Origin of Snakes ...... 1 1.2 Snake Venoms ...... 1 1.3 Three-Finger Toxins ...... 3

2 Three-finger toxin diversification in the venoms of cat-eye snakes (Colubridae: Boiga)7 2.1 Abstract ...... 7 2.2 Introduction ...... 8 2.3 Results ...... 9 2.3.1 Transcriptomics ...... 9 2.3.2 Phylogenetics and Protein Similarity Network ...... 10 2.3.3 Signals of Selection ...... 13 2.3.4 Proteomics ...... 15 2.4 Discussion ...... 15 2.5 Materials and Methods ...... 20 2.5.1 Venom and Tissue Supplies ...... 20 2.5.2 Transcriptomics ...... 20 2.5.3 Proteomics ...... 22 2.5.4 Analysis ...... 22

3 Electric blue: transcriptomics, venom composition, and toxin evolution in the long- glanded coral snake species Calliophis bivirgatus and C. intestinalis 25 3.1 Abstract ...... 25 xi xii CONTENTS

3.2 Introduction ...... 26 3.3 Results ...... 28 3.3.1 Transcriptomics ...... 28 3.3.2 Signals of Selection ...... 31 3.3.3 Proteomics ...... 32

3.3.4 NaV1.4 Assays ...... 32 3.4 Discussion ...... 34 3.5 Materials and Methods ...... 38 3.5.1 Venom and Tissue Acquisition ...... 38 3.5.2 RNA extraction ...... 38 3.5.3 Sequencing ...... 38 3.5.4 Assembly ...... 38 3.5.5 Annotation ...... 39 3.5.6 Phylogenetic Reconstruction ...... 40 3.5.7 Similarity Network ...... 40 3.5.8 Protein Clustering ...... 40 3.5.9 Tests for Selection ...... 40 3.5.10 Protein Modelling ...... 41 3.5.11 HPLC ...... 41 3.5.12 MS/MS Sample Preparation ...... 41 3.5.13 MS/MS ...... 42

3.5.14 NaV1.4 Assays ...... 42

4 Ancient diversification of three-finger toxins in Micrurus coral snakes 43 4.1 Abstract ...... 43 4.2 Introduction ...... 44 4.3 Results ...... 44 4.4 Discussion ...... 48 4.5 Materials and Methods ...... 51 4.5.1 Phylogeny ...... 51 4.5.2 Similarity Network ...... 52 4.5.3 Protein Clustering ...... 52 4.5.4 Functional Residues ...... 52 4.5.5 Tests for Selection ...... 52 4.5.6 Protein Modeling ...... 52

5 Red-on-yellow queen: the binding of Micrurus venoms to a range of nicotinic receptor mimics reveal patterns of prey specificity and toxin resistance 55 5.1 Abstract ...... 55 5.2 Introduction ...... 56 CONTENTS xiii

5.3 Results ...... 58 5.4 Discussion ...... 60 5.5 Materials and Methods ...... 62 5.5.1 Venom Samples ...... 62 5.5.2 Mimotope production and preparation ...... 62 5.5.3 Bio-layer Interferometry ...... 63 5.5.4 Data Processing and Analysis ...... 64

6 Conclusion 65

Bibliography 69

A R script to convert all-to-all BLAST results to network file 95

B Calliophis MSMS results 97 List of figures

1.1 Higher-level squamate phylogenies ...... 2 1.2 Representative 3FTx structures ...... 4

2.1 Schematic phylogeny and alignment of representative denmotoxin-like 3FTx sequences . 11 2.2 Phylogeny of ten-cysteine 3FTx ...... 12 2.3 Protein similarity network of ten-cysteine 3FTx ...... 13 2.4 Schematic phylogeny, structure predictions, and signal of selection results for various clades of denmotoxin-like 3FTx ...... 14 2.5 Protein gels indicating dimerization of toxins in Boiga dendrophila venom ...... 16

3.1 Calliophis appearance, venom glands, and ophiophagy ...... 27 3.2 Expression levels of toxin contigs in Calliophis bivirgatus transcriptome ...... 29 3.3 Phylogeny of representative eight-cysteine 3FTx and Calliophis bivirgatus 3FTx . . . . . 30 3.4 Protein similarity network of representative eight-cysteine 3FTx and Calliophis bivirgatus 3FTx...... 31 3.5 Signals of selection in Calliophis bivirgatus 3FTx ...... 32 3.6 HPLC trace of Calliophis bivirgatus venom with active fractions highlighted ...... 33 3.7 Calliophis bivirgatus venom produces an immediate, dose-dependent, FLIPR response . 33 3.8 TTx abolishes the initial response to Calliophis bivirgatus venom, but not the longer term cytotoxicity caused by high doses ...... 34

3.9 Calliophis intestinalis venom blocks NaV channels ...... 35

4.1 Phylogeny of representative eight-cysteine 3FTx and Micrurus 3FTx ...... 45 4.2 Protein similarity network of representative eight-cysteine 3FTx and Micrurus 3FTx . . . 46 4.3 Alignments of Micrurus clades with functional residues highlighted ...... 47 4.4 Schematic phylogeny of representative eight-cysteine 3FTx and Micrurus 3FTx, structure predictions, and signal of selection results ...... 49

5.1 Alignment of α1-nAChR orthosteric binding sites ...... 57 5.2 Micrurus neurotoxicity varies greatly depending on the individual venom as well as the target ...... 59 xiv LIST OF FIGURES xv

5.3 Snake nAChR are consistently less vulnerable to α-neurotoxins, partially due to a W!S mutation in the ligand binding pocket ...... 60 List of tables

2.1 Assembly statistics of Boiga transcriptomes ...... 10 2.2 Presence and absence of irditoxin-like cysteines ...... 10 2.3 Tests of selection on the various clades of denmotoxin-like 3FTx ...... 14 2.4 Sample collection locations and analyses performed ...... 20

4.1 Tests of selection on the various clades of Micrurus 3FTx ...... 48 4.2 Composition of the eight-cysteine 3FTx dataset ...... 51 4.3 Phyre2 results for the various clades of Micrurus 3FTx ...... 53

B.1 MSMS results for Calliophis bivirgatus fraction 32 ...... 98 B.2 MSMS results for Calliophis bivirgatus fraction 34 ...... 99 B.3 MSMS results for Calliophis bivirgatus fraction 36 ...... 100 B.4 MSMS results for Calliophis intestinalis whole venom, pt. 1 ...... 101 B.5 MSMS results for Calliophis intestinalis whole venom, pt. 2 ...... 102

xvi List of abbreviations

Abbreviations 3FTx Three-Finger Toxins SVMP Snake Venom Metalloprotease PLA2 Phospholipase A2 TPM Transcripts Per Million nAChR Nicotinic Acetylcholine Receptor

NaV Voltage-Gated Sodium Channel

xvii

Chapter 1

Introduction

1.1 Origin of Snakes

The higher-level phylogeny of squamate reptiles has been somewhat controversial due to discrepancies between morphological and molecular methods. Morphological analyses consistently indicated that Iguania was the most basal group of squamates and that snakes belonged within the broad clade Scincomorpha (Figure 1.1) [1, 2]. On the other hand, molecular phylogenies placed Iguania much nearer the crown and indicated a close relationship between Iguania, Anguimorpha, and Serpentes (Figure 1.1) [3, 4]. The proposed clade formed by these three groups was named Toxicofera due to apparent homology in oral gland morphology and the proteins secreted by them which form the basis for the venom system of some snakes and lizards [5, 6]. The topology suggested by the molecular data has largely been supported by more sophisticated analyses that include vast amounts of molecular data and those that combine molecular and morphological datasets [7–10]. Perhaps the most notable traits shared by all snakes are their limblessness and their highly mobile skulls [11, 12]. While many other tetrapods have reduced or absent limbs [13], the skulls of snakes are far more unique and allow them to ingest much larger prey than other limbless taxa; this skull kinesis can be observed in an intermediate state amongst the mosasaurs (Cretaceous marine reptiles which include the largest squamate ever [14]) which are thought to be a sister group to snakes (Figure 1.1) [9, 15]. This greater breadth of diet may help explain the widespread success of snakes since their origin in the Cretaceous and has allowed for the subsequent evolution of novel predatory strategies including constriction and the use of venom [11, 16].

1.2 Snake Venoms

Venoms are often defined as toxic substances that are transferred from one organism into another by a delivery mechanism and mechanical injury [17]. Snake venoms are primarily used to subdue prey [18–21] though they can be used during defensive bites as well. Some species of the genus Naja (cobras) have adapted their venom and venom delivery system to be able to spit their venom 1 2 CHAPTER 1. INTRODUCTION

Figure 1.1: Higher level squamate phylogenies demonstrating the conflict between morphological and molecular methods as well as the Toxicofera clade. Mophological and molecular phylogenies adapted from [1] and [9], respectively.

as a long-distance deterrent to would-be predators [22–24]—qualifying it as a toxungen under the definitions proposed by Nelsen et al. [17]—in addition to its function as a predatory venom. Venom is an adaptation that is particularly central to the ecology and evolution of the front-fanged snakes from the families Elapidae and Viperidae as well as the subfamily Atractaspidinae [25–28]. In non-front-fanged snakes, ducts leading from the venom glands open into the mouth near the back of the maxilla [29]. All three of the front-fanged lineages have independently evolved maxillae that are reduced in size and number of teeth, which brings these anterior teeth and associated venom ducts towards the front of the mouth and simplifies the mechanical process of envenomation [30, 31]. At the same time, these families’ teeth were further adapted to become large (especially in Viperidae and Atractaspidae), tubular fangs which can conduct venom directly from the venom duct into the snake’s target [32]. Along with these changes in dentition, front-fanged snakes developed compressor muscles to eject venom from the lumen of the venom gland, along the venom duct, and out the end of the fang [18]. The benefits of venom to these snakes can be clearly illustrated by the fact that those snake species which occupy the most extreme environments are virtually all elapids or vipers. Elapids have evolved marine lifestyles twice [33] including species that can dive deeper than 200 m [34] and Hydrophis platurus (yellow-bellied sea snake), a truly pelagic generalist that—prior to human intervention—was the most widely distributed reptile species in the world [35]. Meanwhile, species of vipers hold the records for highest latitude (above the arctic circle) [36] and highest altitude (almost 5,000 m) [37] inhabited by snakes. Highly venomous species are able to eke out a living in other inhospitable environments including harsh deserts [38] and isolated islands with very scarce access to food and water [39–41] Snakes lacking prominent fangs were thought, until recently, to be non-venomous as—with a few notable exceptions—they posed little medical threat to humans [6,42–44]. A combination of factors, 1.3. THREE-FINGER TOXINS 3 including the recognition that many of these non-front-fanged snakes possess enlarged, grooved, teeth in the back part of the mouth [27,45,46], studies showing the homology between the venom glands of colubroid snakes [18, 47], adoption of a more inclusive definition of venom among the research community [17, 19, 28, 48–50], and actual extraction and analysis of secretions from the oral glands of these snakes [43, 44, 51, 52] has led to the realization that many non-front-fanged snakes should be considered venomous. While an anthropocentric lens may obscure some of the evolutionary and ecological dynamics of snake venoms, the global impact of snakebite should not be understated. Current estimates suggest that as many as 2.7 million people are envenomed every year and that perhaps 140,000 of those cases result in death [53–55]. However, even non-fatal bites can have devastating consequences and the total disease burden of snakebite is thought to outweigh that of more traditional sorts of diseases like dengue or leishmaniasis in similar regions [56]. Because of this, the World Health Organization recently classified snakebite as neglected tropical disease and has started aggressively supporting efforts to combat the problem [57]. While the research contained in this thesis does not deal directly with snakebite, it may have some relevance to the problem because it concerns the toxins that are often found in the venoms of elapid snakes. Elapids are the most diverse of the three lineages of front-fanged snakes and, along with the family Viperidae, comprise the vast majority of medically significant snake species [58,59]. Elapids are a prominent component of human-snake conflict in much of Sub-Saharan Africa, across Asia, and through Papua New Guinea and Australia [60–62]. Of the ‘Big Four’ species of medically important snakes in India, two are elapids: Naja naja (the Indian cobra) and Bungarus caeruleus (the common krait) [63]. The latter poses a non-standard challenge to snakebite prevention because many of the bites from this species are a result of the snake entering human houses at night while the occupants are sleeping [64]. Similar behavior has also been reported in some African species of the genus Naja [65, 66].

1.3 Three-Finger Toxins

The other necessary component of any venom system, beyond the morphological adaptations to deliver the venom, are the actual toxins that comprise the venom itself. Because venoms are delivered to prey via mechanical injury, there is no need for the toxins to be able to travel through the air, be absorbed across membranes, or any other constraints that often restrict which chemicals might be appropriate for such a biological task [17]. Thus, snakes were able to take advantage of the complexity and pre-existing diversity of protein biochemistry as their venom systems evolved. Venom genes are found to be subject to positive selection more frequently than housekeeping genes or the physiological homologs of toxin proteins [20, 28, 67–71]. This positive selection drives toxins to evolve rapidly through mechanisms such as the rapid accumulation of variation at exposed residues or extensive duplication and neofunctionalization [69,72]. One of the most significant sources of these selective pressures are the highly varied niches that snakes occupy, especially the correspondingly broad range 4 CHAPTER 1. INTRODUCTION

Figure 1.2: Representative 3FTx structures including the plesiotypic ten-cysteine form, the eight- cysteine toxins that are unique to elapids, and the long-chain toxins which are further derived in some elapids. Cysteine residues and the disulfide bonds between them are colored yellow.

of prey species [69, 73, 74]. Coupled with the rapid rate of venom evolution, this leads to many snakes evolving toxins that are particularly potent against their specific prey items [20, 47, 75, 76]. Studies of prey specificity across a broad selection of snakes and prey taxa have led to the rule of thumb that small non-enzymatic neurotoxins are particularly useful against diapsid prey (birds and reptiles), while synapsids (mammals) tend to be more susceptible to enzymatic coagulotoxins [18, 47,52,76,77]. Most elapid venoms are neurotoxic and the most prevalent neurotoxins across the family belong to the three-finger toxin (3FTx) protein family [18,43]. These toxins are estimated to have first appeared over 100 million years ago, relatively early in the evolutionary history of snakes, and as a result have been reported in transcriptomes from most families of alethinophidian snakes [6, 78]. The toxin family derives its name from the characteristic tertiary structure of three β-stranded loops, or ‘fingers’, that emerge from a core that is stabilized by cysteine-cysteine bonds (Figure 1.2) [79]. Because the amino acids on these loops are far more exposed than those closer to the center, these domains almost always contain the sites that interact with the toxins’ targets [78, 80]. The ancestral forms of 3FTx contain ten cysteine residues which form five disulphide bonds between particular pairs (Figure 1.2) [43, 81]. 3FTx are particularly prevalent in the venoms from the families Colubridae and Elapidae [44]. While colubrid snakes retain the plesiotypic ten-cysteine 3FTx, a common ancestor of elapids evolved a form of 3FTx that did not include the second and third ancestral cysteines, leaving only four pairs (Figure 1.2); these apotypic 3FTx diversified greatly and are far more highly expressed in elapid venoms than the ten-cysteine forms [82, 83]. 1.3. THREE-FINGER TOXINS 5

All the ten-cysteine 3FTx that have been characterized show similar toxic activities: binding to and thereby blocking the activity of post-synaptic nicotinic acetylcholine receptors (nAChRs) on the skeletal muscles (antagonistic binding) [43, 52, 84]. In snake venoms, this is often referred to as α-neurotoxicity and these toxins usually display a greater affinity for the receptors of diapsids (birds and reptiles) than those of synapsids (mammals) [85, 86]. Even novel modifications to the structure of these toxins, such as the covalently bonded heterodimers from the colubrid species Boiga irregularis and its close relatives increase the toxicity rather than changing the activity of these toxins [87,88]. The derived eight-cysteine toxins found in elapids, on the other hand, have evolved a several further structural alterations that modify their neurotoxic activity such as the presynaptic neurotoxins which can block calcium channels or acid sensing ion channels, κ-neurotoxins which are non-covalently linked dimers, and the long-chain neurotoxins which possess a novel pair of cysteines in the Loop II region (Figure 1.2) [83,89–91]. Beyond this, a number of these toxins have been shown to cause non-neurotoxic symptoms such as cytotoxicity and platelet inhibition [69, 83, 92]. It has also been shown that directed evolution can lead to the production of 3FTx with totally novel activity such as serine protease inhibition [93]. Many of the eight-cysteine toxins that did retain the ancestral activity have evolved greatly increased toxicity to the extent that α-bungarotoxin has seen widespread use as an experimental tool because of its potency and specificity [79, 94]. These 3FTx are the primary functional toxins in many elapid venoms and are responsible for the danger posed by many including many members of the genera Naja (cobras), Dendroaspis (mambas), and Micrurus (coral snakes) as well as the subfamily Hydrophiinae (sea snakes and Australian elapids) [76, 95–98].

3FTx are interesting beyond their significant role in snakebites across the world because of their potential as a biodiscovery source for new laboratory tools or medications. For instance, α- bungarotoxin is used widely in neuroscience research because of its potent and specific blockage of nAChRs [94]. 3FTx are also promising for drug development because of their small size and disulphide bonded structural core which makes them easier to produce synthetically than larger enzymatic toxins and increases the likelihood that they can be delivered to patients without the need for injection [99,100]. Both of these issues have challenged the development of existing venom derived drugs [101]. In fact, several 3FTx from other elapid lineages have been studied for their therapeutic potential. Diochot et al. found that several 3FTx from Dendroaspis polylepis (black mamba) venom that block acid sensing ion channels—which they called mambalgins—were non-toxic and just as effective against pain as morphine when injected into mice [102]. What is more, these 3FTx did not cause respiratory distress or tolerance like opioids do. Similarly, Pu et al. investigated the anti-nociceptive properties of hannalgesin, a 3FTx found in Ophiophagus hannah (king cobra) venom [103]. Intriguingly, this toxin produced its pain-killing effects even when ingested orally, whereas most venom proteins are thought to be entirely ineffectual if they are not injected directly into the target organism [17]. A comparable activity has been described from the venom of Micrurus lemniscatus; mice that ingested crude M. leminscatus venom were found to react much less strongly compared to controls on various behavioral measures of pain [104]. The similarity to hannalgesin and the fact that the venom of M. lemniscatus is known to be dominated by 3FTx suggest that the venom component responsible for this effect might 6 CHAPTER 1. INTRODUCTION also be a 3FTx [105]. This thesis will focus on 3FTx to try to achieve a better understanding of their current diversity and evolutionary history as well as the toxic effects they cause and the selection pressures that have shaped them. Chapter 2

Three-finger toxin diversification in the venoms of cat-eye snakes (Colubridae: Boiga)

Dashevsky D, Debono J, Rokyta D, Nouwens A, Josh P, and Fry BG. “Three-Finger Toxin Diversifi- cation in the Venoms of Cat-Eye Snakes (Colubridae: Boiga)”. Journal of Molecular Evolution 86.8: 531–545, (2018). Author Contribution Conception and design (25%) Daniel Dashevsky Bioinformatics (80%) (candidate) Writing (70%) Editing(20%) Conception and design (25%) Jordan Debono Writing (30%) Editing(20%) Bioinformatics (20%) Darin Rokyta Editing (30%) Amanda Nouwens Generate MS/MS data (100%) & Peter Josh Conception and design (50%) Bryan Fry Editing (30%)

2.1 Abstract

The Asian genus Boiga (Colubridae) is among the better studied non-front-fanged snake lineages because their bites have minor, but noticeable, effects on humans. Further, B. irregularis has gained worldwide notoriety for successfully invading Guam and other nearby islands with drastic impacts on the local bird populations. One of the factors thought to allow B. irregularis to become such a noxious pest is irditoxin, a dimeric neurotoxin composed of two three-finger toxins (3FTx) joined by a covalent bond between two newly evolved cysteines. Irditoxin is highly toxic to diapsid (birds and reptiles) prey, but roughly 1000⇥ less potent to synapsids (mammals). Venom plays an important role 7 CHAPTER 2. THREE-FINGER TOXIN DIVERSIFICATION IN THE VENOMS OF CAT-EYE SNAKES 8 (COLUBRIDAE: BOIGA) in the ecology of all species of Boiga, but it remains unknown if any species besides B. irregularis produce irditoxin-like dimeric toxins. In this study, we use transcriptomic analyses of venom glands from five species (B. cynodon, B. dendrophila dendrophila, B. d. gemmicincta, B. irregularis (Brisbane population), B. irregularis (Sulawesi population), B. nigriceps, B. trigonata) and proteomic analyses of B. d. dendrophila and a representative of the sister genus Toxicodryas blandingii to investigate the evolutionary history of 3FTx within Boiga and its close relative. We found that 92.5% of Boiga 3FTx belong to a single clade which we refer to as denmotoxin-like because of the close relation between these toxins and the monomeric denmotoxin according to phylogenetic, sequence clustering, and protein similarity network analyses. We show for the first time that species beyond B. irregularis secrete 3FTx with additional cysteines in the same position as both the A and B subunits of irditoxin. Transcripts with the characteristic mutations are found in B. d. dendrophila, B. d. gemmicincta, B. irregularis (Brisbane population), B. irregularis (Sulawesi population), and B. nigriceps. These results are confirmed by proteomic analyses that show direct evidence of dimerization within the venom of B. d. dendrophila, but not T. blandingii. Our results also suggest the possibility of novel dimeric toxins in other genera such as Telescopus and Trimorphodon. All together this suggests that the origin of these peculiar 3FTx is far earlier than was appreciated and their evolutionary history has been complex.

2.2 Introduction

While present in the venoms of multiple snake families, 3FTx are at their highest relative abundance in the families Colubridae and Elapidae [18, 44]. This broad trend holds true in the genus Boiga, with various studies finding that 3FTx are the predominant toxins in their venoms [44, 106–108]. Plesiotypic 3FTx—the type found in colubrid venoms—are characterised by ten conserved cysteine residues (Figure 1.2), α-neurotoxicity, and display greater affinity towards the post-synaptic nicotinic acetylcholine receptors (nAChRs) of birds and reptiles than those of rodents [43, 52, 86, 87]. Because early studies of the activity of these plesiotypic toxins used synapsid animal models (rodents) rather than diapsid models—which would have been more similar to the avian and reptilian natural prey of the snakes being studied—the ten-cysteine 3FTx were initially mistakenly referred to as “weak neurotoxins” [84]. The prevalence of such anthropocentric experimental design in investigations of the toxicity of the venoms of non-front-fanged snakes led to some authors concluding Boiga species were non-venomous [109]. More recently however, their venom has been well characterized and boigatoxin-A, denmotoxin, and irditoxin, the earliest characterized Boiga toxins, were found to be particularly potent in diapsid (avian) tissue assays [51, 52,87]. Irditoxin, from the venom of B. irregularis, is particularly notable among 3FTx because the mature toxin is a covalently linked dimer of two distinct 3FTx which exhibits a ten-fold higher potency than the monomeric denmotoxin [52,87]. Due in part to its effective venom, B. irregularis has become a noxious pest that is responsible for the extirpation of bird populations on several Pacific islands that were previously snake-free [110,111]. Since bird populations have crashed after their introductions, lizards formed the majority of the diet for B. irregularis on Guam, especially smaller individuals [112]. 2.3. RESULTS 9

Irditoxin may have played a key role in the invasion, its diapsid-specific toxicity enabling the snakes to efficiently exploit the local bird populations and transition to eating lizards once birds became scarce, but it remains unknown if irditoxin is unique to B. irregularis or if closely related Asian or African species also produce irditoxin-like dimers [3,113]. Modahl and Mackessy [107] suggested, based on sequence similarity, that the presence of such dimers in B. cynodon venom is likely. However, this study did not explore whether the newly-evolved cysteines characteristic of irditoxin were present in the sequences of species outside of B. irregularis. To investigate these questions we sequenced the venom gland transcriptomes of seven individuals from five species of Boiga ranging from the semi-terrestrial B. trigonata which is found in the Middle East and South Asia [114], to highly arboreal Southeast Asian species including two subspecies of B. dendrophila (B. d. dendrophila and B. d. gemmicincta), populations of B. irregularis from Sulawesi in western Indonesia and Brisbane in eastern Australia, as well as B. cynodon and B. nigriceps [115]. On separate occasions, we collected venom samples from B. d. dendrophila and Toxicodryas blandingii for proteomic investigation. These species encompass almost all the genetic, geographical, ecological and morphological diversification within this clade of colubrine snakes. T. blandingii is a sister taxon to the genus Boiga and is prevalent within the tree canopies of sub-Saharan Africa, where this clade is suspected to have originated [3, 113]. T. blandingii is one of the largest of species in this niche, exceeding three meters in length, and predates largely upon amphibians, birds and lizards [116]. B. d. dendrophila is widespread in Southeast Asia and also feeds on amphibians, birds and lizards [52].

2.3 Results

2.3.1 Transcriptomics

The summary statistics describing the concatenated transcriptomes for the various species are very similar, yet they contained highly variable numbers of unique 3FTx isoforms (Table 2.1). The most unusual transcriptome in terms of these statistics is that of the B. irregularis from Sulawesi which had a much greater portion of the reads left unpaired, more contigs, and a greater N50 length; despite these differences, the number of 3FTx isoforms in this sample roughly similar to the number from our B. irregularis from Brisbane. Similarly, the B. cynodon assembly contained only one full-length 3FTx (and fragmentary contigs indicating the likely presence of a second isoform at low expression levels) despite containing the second highest number of contigs. Our transcriptomics also confirm the presence of sequences with one or both of the additional cysteines that characterize the irditoxin dimer complex in a range of taxa beyond B. irregularis, where it was originally discovered (Table 2.2). Interestingly, we did not recover toxins with either irditoxin-like cysteine from the B. trigonata transcriptome even though several taxa more distantly related to B. irregularis contain one or both. It remains unclear if this is a genuine result or an artefact of the assembly process, especially given that B. trigonata’s assembly had the lowest number of contigs and N50 lengths (Table 2.1). The presence of single toxins which contained the additional cysteines that characterize both the CHAPTER 2. THREE-FINGER TOXIN DIVERSIFICATION IN THE VENOMS OF CAT-EYE SNAKES 10 (COLUBRIDAE: BOIGA)

Table 2.1: Assembly statistics for concatenated Boiga transcriptomes

Sample Reads Reads Reads Contigs N50 length 3FTx (merged) (unmerged pairs) (total) Boiga cynodon 1301770 70802 1372572 258965 47418 1 B. dendrophila 821221 41517 862738 203518 42443 43 dendrophila B. d. gemmicincta 935846 50831 986677 250557 49671 12 B. irregularis 1003728 48410 1052138 210826 42996 19 Brisbane B. irregularis 8544 905435 913979 297697 55737 14 Sulawesi B. nigriceps 859952 39583 899535 221907 44572 7 B. trigonata 1023112 38033 1061145 175745 35975 7

Table 2.2: Presence and absence of irditoxin-like cysteines

Taxon Irditoxin-like B Irditoxin-like A Boiga cynodon • B. dendrophila dendrophila • • B. d. gemmicincta • • B. irregularis Brisbane • • B. irregularis Sulawesi • • B. nigriceps • • B. trigonata Telescopus dhara • Trimorphodon biscutatus • Trimorphodon lambda •

A and B subunits of irditoxin (Figure 2.1) was an unexpected finding. While it is possible that these sequences are chimeric or otherwise represent assembly error, when we examined the coverage of the contigs and the sequences of the individual reads from which they were assembled, there was no evidence of assembly error. The potential structural and functional impact of a novel cysteine pair within the same sequence on the mature toxins’ structures and activity are entirely unknown.

2.3.2 Phylogenetics and Protein Similarity Network

We found that 56.4% of published ten-cysteine 3FTx come from the genus Boiga. This is likely due in large part to publication bias: Boiga have long been recognized as venomous, and B. irregularis is one of the most notorious invasive species in the world [42, 117]. The other well-studied non- front-fanged snake venom is that of Dispholidus typus, the dangerously toxic boomslang, which is primarily composed of P-III snake venom metalloproteinases and so contributes relatively few sequences to our data [21, 118]. Among the front-fanged snakes, the stereotypical toxins for each lineage are something besides ten-cysteine 3FTx: viperid venoms are primarily enzymatic [119], 2.3. RESULTS 11

Figure 2.1: Mature peptide sequences of notable denmotoxin-like sequences and additional rep- resentatives of each subclade with the sites of the 10 canonical cysteines and irditoxin A and B cysteines highlighted in color. These sequences include denmotoxin (a previously characterized monomeric toxin), irditoxin A and B subunits (a previously characterized dimeric toxin), sequences with cysteines in the characteristic position of Irditoxin A or B from Trimorphodon and Telescopus, and the two sequences which with irditoxin-like cysteines at both positions. Phylogeny and sub- clades are derived from Figure 2.2. Corresponding IDs for the selected sequences: Clade A—Boiga irregularis Brisbane 3FTx 00; Clade B—Boiga dendrophila dendrophila 3FTx 14; Trimorphodon B—Trimorphodon lambda A0A193CHM1; Clade C—Coelognathus radiatus P83490; Trimorphodon A—Trimorphodon biscutatus A7X3S2; Irditoxin B—Boiga irregularis A0S865; Clade D—Boiga cynodon A0A193CHL2; Telescopus B—Telescopus dhara A7X3V0; Clade E—Boiga dendrophila gemmicincta 3FTx 09; B. d. gemmicincta A & B—Boiga dendrophila gemmicincta 3FTx 05; Denmotoxin—Boiga dendrophila Q06ZW0; Clade F—Boiga dendrophila dendrophila 3FTx 08; B. d. dendrophila A & B—Boiga dendrophila dendrophila 3FTx 41; Irditoxin A—Boiga irregularis A0S864; Clade G—Boiga nigriceps 3FTx 03.

elapid venoms often dominated by derived eight-cysteine 3FTx [120], and atractaspidine venoms by blood-pressure-affecting serotoxin peptides and procoagulant enzymes [121–123]. Together, these factors make it so that there are far more ten-cysteine 3FTx known from Boiga than any other snake lineage. However, the sheer number of sequences from Boiga does indicate that the 3FTx toxin family has undergone extensive duplication during their evolution.

Of these Boiga toxins, 92.5% belong to a clade that we refer to as denmotoxin-like (Figure 2.2). Independent protein clustering and protein similarity network analyses confirmed this subdivision within our dataset (Figure 2.3). Within the denmotoxin-like sequences we designated monophyletic clades for further analysis (Figures 2.1 and 2.2): Clade A is composed exclusively of Boiga sequences, they retain the ancestral state of only 2 amino acids before cysteine #1 (as opposed to 9 in all other denmotoxin-like clades), and none possess either of the derived irditoxin-like cysteines; Clade B contains Boiga and Telescopus sequences none of which possess either of the irditoxin-like cysteines; Clade C contains sequences from Coelognathus, Oxybelis, and Trimorphodon, the latter of which includes toxins with the irditoxin-like A and B cysteines separately (though the T. biscutatus sequence also lacks the canonical cysteines #2 and #3); Clade D and E both contain only Boiga sequences with the irditoxin-like B cysteine; Clades F and G are also composed entirely of Boiga sequences, but contain a mix of those with the irditoxin-like A cysteine, neither, and one sequence each with both the irditoxin-like A and B cysteines. Clades E, F, and G are part of a polytomy that is separated from Clade D by two monotypic branches including a Telescopus sequence with the irditoxin-like B cysteine. CHAPTER 2. THREE-FINGER TOXIN DIVERSIFICATION IN THE VENOMS OF CAT-EYE SNAKES 12 (COLUBRIDAE: BOIGA)

Python_regius_A0A098LWQ5 0.98 1 Aspidites_melanocephalus_M9T271 1 Aspidites_melanocephalus_M9T1L2 Cylindrophis_ruffus_M9SZR1 Leioheterodon_madagascariensis_A7X3R6 Ahaetulla_prasina_A0A193CHL5 Thrasops_jacksonii_A7X3U7 0.86 0.99 Thrasops_jacksonii_A7X3U4 Dispholidus_typus_A7X3U0 1 1 1 Thrasops_jacksonii_A7X3T6 Dispholidus_typus_A7X3T2 1 Boiga_trigonata_3FTx_00 0.99 Boiga_trigonata_3FTx_02 0.99 1 Boiga_dendrophila_dendrophila_3FTx_26 Boiga_dendrophila_gemmicincta_3FTx_08 Boiga_dendrophila_dendrophila_3FTx_23 Boiga_nigriceps_3FTx_01 Boiga_dendrophila_dendrophila_3FTx_24 0.97 0.81 Boiga_trigonata_3FTx_04 Boiga_trigonata_3FTx_01 1 0.88 0.7 0.97 Boiga_dendrophila_gemmicincta_3FTx_02 Boiga_dendrophila_gemmicincta_3FTx_04 1 Boiga_dendrophila_gemmicincta_3FTx_03 Boiga_dendrophila_gemmicincta_3FTx_01 0.99 0.58 0.5 0.96 Boiga_dendrophila_dendrophila_3FTx_01 0.57 Boiga_dendrophila_dendrophila_3FTx_03 Boiga_dendrophila_dendrophila_3FTx_02 0.98 Boiga_dendrophila_dendrophila_3FTx_19 0.68 Boiga_dendrophila_dendrophila_3FTx_20 Boiga_dendrophila_dendrophila_3FTx_21 0.73 Boiga_dendrophila_dendrophila_3FTx_18 1 Boiga_dendrophila_dendrophila_3FTx_22 1 Boiga_irregularis_Sulawesi_3FTx_01 1 Boiga_irregularis_A0A0B8RZX3 Boiga_irregularis_A0A0B8RS37 0.98 Boiga_irregularis_A0A0B8RSY6 Boiga_irregularis_A0A0B8RYX1 Boiga_irregularis_A0A193CHL6 0.65 Boiga_irregularis_Brisbane_3FTx_06 0.59 Boiga_irregularis_Brisbane_3FTx_11 Boiga_irregularis_Brisbane_3FTx_09 0.57 Boiga_irregularis_Brisbane_3FTx_10 0.73 Boiga_irregularis_Brisbane_3FTx_08 0.9 Boiga_irregularis_Brisbane_3FTx_07 Clade A 0.65 Boiga_irregularis_A0A0B8RSZ3 Boiga_irregularis_A0A0B8RS48 Boiga_irregularis_Sulawesi_3FTx_02 1 Boiga_irregularis_A0A0B8RZY0 Boiga_irregularis_A0A0B8RZX9 1 Boiga_irregularis_A0A0B8RVC5 Boiga_irregularis_A0A0B8RYY9 Boiga_irregularis_Sulawesi_3FTx_04 Boiga_dendrophila_dendrophila_3FTx_04 1 Boiga_dendrophila_dendrophila_3FTx_25 1 Boiga_dendrophila_dendrophila_3FTx_17 0.69 0.87 Boiga_dendrophila_gemmicincta_3FTx_00 Boiga_dendrophila_gemmicincta_3FTx_07 0.67 Boiga_nigriceps_3FTx_02 0.64 Boiga_irregularis_A0A193CHL9 0.56 0.98 Boiga_irregularis_Brisbane_3FTx_00 Boiga_irregularis_Brisbane_3FTx_01 1 Boiga_irregularis_Sulawesi_3FTx_03 Boiga_irregularis_A0A193CHM0 Boiga_irregularis_A0A0B8RV99 Boiga_irregularis_A0A0B8RYW2 0.57 Boiga_irregularis_A0A193CHL4 Boiga_irregularis_A0A0B8RVA2 Boiga_irregularis_A0A0B8RZW9 Boiga_irregularis_A0A0B8RYW4 Boiga_irregularis_Sulawesi_3FTx_05 1 Telescopus_dhara_A7X3S8 Telescopus_dhara_A7X3S5 0.99 Boiga_dendrophila_dendrophila_3FTx_15 1 Boiga_dendrophila_dendrophila_3FTx_31 0.95 Clade B Boiga_dendrophila_dendrophila_3FTx_14 Trimorphodon_lambda_A0A193CHM1 Oxybelis_fulgidus_A0A193CHK9 1 0.98 0.68 Coelognathus_radiatus_P83490 Oxybelis_fulgidus_C0HJD3 0.76 1 0.9 Trimorphodon_lambda_A0A193CHM4 Trimorphodon_biscutatus_A7X3S0 Clade C 1 0.99 Trimorphodon_lambda_A0A193CHM5 Trimorphodon_biscutatus_A7X3S2 Boiga_irregularis_A0S865 1 0.96 Boiga_irregularis_A0A193CHL7 Boiga_cynodon_A0A193CHL2 0.55 Boiga_nigriceps_3FTx_06 0.99 0.63 Boiga_irregularis_Brisbane_3FTx_03 Boiga_irregularis_A0A0B8RZX5 0.98 Boiga_irregularis_A0A0B8RVB7 1 Boiga_irregularis_A0A0B8RYX5 Clade D Boiga_irregularis_A0A0B8RS42 Boiga_irregularis_A0A0B8RVB2 0.96 Boiga_irregularis_A0A0B8RS39 Boiga_irregularis_Sulawesi_3FTx_13 Boiga_trigonata_3FTx_06 Irditoxin-like Telescopus_dhara_A7X3V0 0.87 Boiga_dendrophila_dendrophila_3FTx_11 Boiga_dendrophila_dendrophila_3FTx_39 A 0.51 0.62 Boiga_dendrophila_gemmicincta_3FTx_09 1 Boiga_dendrophila_dendrophila_3FTx_05 0.98 Clade E Boiga_dendrophila_dendrophila_3FTx_28 B 0.54 Boiga_dendrophila_gemmicincta_3FTx_05 0.6 0.58 Boiga_dendrophila_dendrophila_3FTx_29 Boiga_dendrophila_Q06ZW0 0.89 Boiga_dendrophila_dendrophila_3FTx_08 0.93 Boiga_dendrophila_dendrophila_3FTx_13 Both 0.55 0.96 Boiga_dendrophila_dendrophila_3FTx_30 0.87 Boiga_dendrophila_dendrophila_3FTx_07 Boiga_dendrophila_dendrophila_3FTx_12 Clade F 0.89 Neither 1 Boiga_dendrophila_dendrophila_3FTx_06 Boiga_dendrophila_dendrophila_3FTx_27 Boiga_dendrophila_dendrophila_3FTx_40 0.89 1 Boiga_dendrophila_dendrophila_3FTx_16 Boiga_dendrophila_dendrophila_3FTx_42 Boiga_dendrophila_dendrophila_3FTx_41 Boiga_dendrophila_gemmicincta_3FTx_11 0.7 Boiga_dendrophila_dendrophila_3FTx_33 0.96 Boiga_dendrophila_dendrophila_3FTx_32 Boiga_dendrophila_dendrophila_3FTx_09 1 Boiga_dendrophila_dendrophila_3FTx_37 1 0.98 0.99 Boiga_dendrophila_dendrophila_3FTx_38 1 Boiga_dendrophila_dendrophila_3FTx_10 Boiga_dendrophila_gemmicincta_3FTx_10 1 1 Boiga_dendrophila_dendrophila_3FTx_36 1 Boiga_dendrophila_dendrophila_3FTx_34 Boiga_dendrophila_dendrophila_3FTx_35 Boiga_nigriceps_A0A193CHL8 Boiga_irregularis_A0S864 0.74 Boiga_nigriceps_3FTx_03 0.87 Boiga_nigriceps_3FTx_04 0.51 Boiga_irregularis_A0A0B8RSZ1 Boiga_irregularis_A0A0B8RZX8 0.97 Boiga_irregularis_A0A0B8RYY5 1 0.54 Boiga_irregularis_Sulawesi_3FTx_12 Boiga_irregularis_Sulawesi_3FTx_11 Boiga_irregularis_A0A0B8RS46 0.55 Boiga_irregularis_A0A0B8RS45 Boiga_irregularis_A0A0B8RS43 0.89 Boiga_irregularis_Brisbane_3FTx_12 Boiga_irregularis_Brisbane_3FTx_02 0.65 Boiga_irregularis_A0A0B8RZX6 1 Boiga_irregularis_A0A0B8RYY3 0.63 Boiga_irregularis_A0A0B8RYY0 Clade G 0.99 Boiga_irregularis_A0A0B8RZX7 0.73 Boiga_irregularis_A0A0B8RVB9 Boiga_irregularis_A0A0B8RSZ0 Boiga_irregularis_Sulawesi_3FTx_08 Boiga_irregularis_A0A0B8RZX0 0.99 Boiga_irregularis_A0A0B8RSX8 0.92 Boiga_trigonata_3FTx_05 0.83 Boiga_cynodon_A0A193CHL1 Boiga_irregularis_A0A0B8RZX1 Boiga_irregularis_A0A0B8RVA3 Boiga_irregularis_Sulawesi_3FTx_10 0.78 Boiga_irregularis_Sulawesi_3FTx_09 Boiga_irregularis_A0A0B8RS19 Boiga_irregularis_Sulawesi_3FTx_07 Boiga_irregularis_A0A0B8RVA5 0.99 Boiga_irregularis_A0A0B8RS22 0.94 Boiga_irregularis_A0A0B8RYW8 1 Boiga_irregularis_Sulawesi_3FTx_06 Boiga_irregularis_A0A0B8RSY0 Boiga_irregularis_Brisbane_3FTx_16 Boiga_nigriceps_3FTx_05 1 Boiga_irregularis_Brisbane_3FTx_17 Boiga_irregularis_Brisbane_3FTx_13 1 Boiga_irregularis_Brisbane_3FTx_14 Boiga_irregularis_Brisbane_3FTx_15 Bungarus_multicinctus_Q8JFX7 1 Bungarus_multicinctus_Q9YGH0 Bungarus_multicinctus_Q9PW19 Bungarus_multicinctus_O12963 1 0.54 Bungarus_multicinctus_Q9YGH9 0.58 Bungarus_multicinctus_Q9YGJ0 Micrurus_surinamensis_P86099 1 Micrurus_frontalis_P86423 0.61 0.9 1 Micrurus_mipartitus_C0HJR2 Micrurus_mipartitus_C0HJR1 0.92 Micrurus_frontalis_P86424 0.64 1 Micrurus_surinamensis_P86096 Micrurus_surinamensis_P86098 Micrurus_corallinus_P58370 1 0.83 Bungarus_multicinctus_Q70WS8 Bungarus_candidus_P81783 0.94 Bungarus_multicinctus_P15818 Bungarus_flaviceps_D5J9P6 1 0.92 Bungarus_flaviceps_D5J9P8 Bungarus_flaviceps_D5J9P5 Naja_atra_E2ITZ2 Bungarus_fasciatus_A2CKF6 1 Bungarus_candidus_Q8AY49 1 1 0.94 Bungarus_candidus_Q8AY50 Bungarus_candidus_Q8AY51 1 Bungarus_fasciatus_A2CKF7 0.87 Bungarus_candidus_Q6IZ95 0.99 Bungarus_flaviceps_D5J9Q0 0.9 0.95 Bungarus_flaviceps_D5J9P9 0.87 Bungarus_flaviceps_D5J9Q1 Naja_melanoleuca_P01400 0.8 Ophiophagus_hannah_Q2VBN2 0.83 Walterinnesia_aegyptia_C1IC49 Naja_naja_P29179 Naja_haje_haje_P01401 0.62 Naja_oxiana_P85520 0.84 Naja_atra_E2ITZ6 0.59 0.99 Naja_naja_P29180 0.88 Naja_atra_Q9YGI1 0.71 Naja_atra_O93422 Naja_atra_Q9YGI2 Naja_annulifera_P01399 1 Naja_nivea_P25680 Naja_sputatrix_Q802B2 1 0.76 1 Naja_naja_P29182 Naja_naja_P29181 0.88 1 Naja_kaouthia_P25679 Naja_kaouthia_P82935 1 Naja_atra_Q9YGI4 0.62 Naja_atra_P60814 0.51 Naja_sputatrix_O42256 0.83 Naja_sputatrix_O42255 0.99 Naja_sputatrix_Q9W7I3 Naja_sputatrix_Q802B3 Psammophis_mossambicus_A7X3M3 Protobothrops_flavoviridis_U3T794 1 Crotalus_adamanteus_A0A1W7RBD9 Sistrurus_catenatus_edwardsii_A5X2W8 0.68 Dispholidus_typus_A7X3Q4 Pantherophis_guttatus_A0A098LYI5 0.92 Erythrolamprus_poecilogyrus_A7X3P2 1 0.96 Hypsiglena_sp_A0A098M253 0.6 Erythrolamprus_poecilogyrus_A7X3P8 1 Phalotris_mertensi_A0A182C5Y1 0.54 Phalotris_mertensi_A0A182C5S7 1 Opheodrys_aestivus_A0A098LYA2 Python_regius_A0A098LY61 0.94 Boiga_irregularis_A0A0B8RZX2 Boiga_irregularis_A0A0B8RSY1 1 Boiga_irregularis_A0A0B8RS24 0.68 0.96 Boiga_irregularis_Sulawesi_3FTx_00 Boiga_irregularis_A0A0B8RVA6 0.51 Boiga_nigriceps_3FTx_00 1 Boiga_irregularis_Brisbane_3FTx_04 0.82 Boiga_irregularis_Brisbane_3FTx_05 Telescopus_dhara_A7X3N6 0.95 Boiga_trigonata_3FTx_03 0.99 Boiga_cynodon_3FTx_00 0.84 0.99 Boiga_dendrophila_dendrophila_3FTx_00 Boiga_dendrophila_gemmicincta_3FTx_06 Ahaetulla_prasina_A0A193CHL3 0.98 Dispholidus_typus_A7X6J0 0.59 Thrasops_jacksonii_A7X6I4 0.99 Trimorphodon_biscutatus_A7X3Q9 0.81 1 Pantherophis_guttatus_A0A098LYG6 0.67 Pantherophis_guttatus_A0A098LYI1 Opheodrys_aestivus_A0A098LWU4 1 Erythrolamprus_poecilogyrus_A7X3M9 0.96 Atractaspis_aterrima_GAMF01050194 1 Atractaspis_aterrima_GAME01026808 1 Atractaspis_aterrima_GAMF01005539 1 1 Atractaspis_aterrima_GAMF01055489 Atractaspis_aterrima_GAMF01040875 Azemiops_feae_M9T2J4 1 1 1 Opheodrys_aestivus_A0A098LWV3 Echis_coloratus_A0A0A1WCI9 0.5 Crotalus_adamanteus_A0A1W7RBC9 1 0.84 Sistrurus_catenatus_edwardsii_B4Y143 Sistrurus_catenatus_edwardsii_A5X2W7 1 Sistrurus_catenatus_edwardsii_A5X2X0 1 Sistrurus_catenatus_edwardsii_B4Y145 1 1 Sistrurus_catenatus_edwardsii_A5X2W9 Sistrurus_catenatus_edwardsii_B4Y144 Opheodrys_aestivus_A0A098LYB1 1 0.8 Python_regius_A0A098LY55 Opheodrys_aestivus_A0A098LWU5 Pseudoferania_polylepis_A7X3L3 1 0.97 Micrurus_fulvius_U3FVH6 Micrurus_altirostris_F5CPD4 1 Dendroaspis_jamesoni_kaimosae_P25682 0.73 0.79 Walterinnesia_aegyptia_C0HKZ8 Bungarus_candidus_P81782 Pantherophis_guttatus_A0A098LYH6

0.3

Figure 2.2: Phylogenetic tree of all publicly available ten-cysteine 3FTx and those from our Boiga transcriptome assemblies. Denmotoxin-like sequences are divided into monophyletic clades for further analyses and colored according to their irditoxin-like cysteines. 2.3. RESULTS 13

Figure 2.3: Protein similarity network f all publicly available ten-cysteine 3FTx and those from our Boiga transcriptome assemblies based on BLAST e-values. Labels correspond to CD-HIT clusters. Both the network and the clusters confirm the distinction between the denmotoxin-like sequences and others. Denmotoxin-like sequences are colored according to their irditoxin-like cysteines.

2.3.3 Signals of Selection

We examined the signals of selections on the various clades of denmotoxin-like sequences using whole gene as well as site-specific algorithms (Table 4.1). We found Clade D to be subject to negative selection, Clade C neutral, Clades A, E, F, and G positive, and Clade B extreme positive selection. However, Clades B, C, and E included few sequences which increases the potential error and decreases the statistical power of these estimates. This is illustrated by the fact that, despite Clade B’s extreme ω value across the whole gene, our site-specific analyses found relatively few sites that met their statistical significance thresholds. A possible reason for the high ω value in Clade B is because the taxa within it span a broad range of the colubrines. Though many sites failed to reach statistical significance, the pattern of estimated selection across the proteins for each clade can be seen in Figure 2.4. CHAPTER 2. THREE-FINGER TOXIN DIVERSIFICATION IN THE VENOMS OF CAT-EYE SNAKES 14 (COLUBRIDAE: BOIGA)

Table 2.3: Tests of selection on the various clades of denmotoxin-like 3FTx

Clade Sequences ω FUBAR (-)a FUBAR (+)b MEMEc FUBAR&MEMEd A 54 1.25 5 12 18 11 B 5 3.02 0 4 1 0 C 6 0.97 4 1 5 1 D 12 0.67 2 0 1 0 E 5 1.42 0 2 0 0 F 13 1.4 4 6 6 2 G 54 1.45 4 16 14 11 a Number of codons under negative selection according to FUBAR b Number of codons under positive selection according to FUBAR c Number of codons under episodic diversifying selection according to MEME d Number of codons that fit both citeria b and c

Figure 2.4: Schematic phylogeny of the clades within the denmotoxin-like 3FTx. Branches for each clade are colored and tips are labeled according to ω values. Protein models show front and back views colored according to the estimated strength of selection (β − α) from FUBAR (left) and MEME (right). 2.4. DISCUSSION 15

2.3.4 Proteomics

The combined 1D and 2D gel approach revealed that both T. blandingii and B. d. dendrophila were dominated by 3FTx (Figure 2.5). Comparison of reduced and non-reduced B. d. dendrophila 1D gels revealed the presence of a band in the non-reduced gel between the 15 and 20 kDa markers (Figure 2.5A) while in the reduced gel it was apparent that the monomeric 3FTx band sitting just below the 10 kDa marker was correspondingly thicker and LC-MSMS identified the additional band as 3FTx (Figure 2.5A). This confirmed the presence of a dimeric complex made up of two 3FTx. Homologous bands were either absent entirely from the T. blandingii venom or was too faint to detect in both the reduced and non-reduced gels (Figure 2.5A). The 2D gels (Figure 2.5B and C) further demonstrate the diversity of 3FTx isoforms found in both venoms.

2.4 Discussion

Non-front-fanged snake venoms remain a neglected area of research. This is in part due to the comparative difficulty of obtaining venom samples [124], the fact that bites from most of these snakes have negligible impacts on human health [42], and because it has only been relatively recently that the research community has begun to regard many of these snakes as venomous at all [17, 28, 43, 48]. This study focuses on the evolutionary dynamics of one toxin family and one genus of colubrines; there remains much work to be done on other toxin types and other non-front-fanged snakes. Proteomic and transcriptomic studies of Boiga venoms revealed that diapsid-specific neurotoxic 3FTx are the predominant toxins secreted by this amphibian, bird, and reptile feeding genus. Previous investigation into this genus revealed a unique disulphide-bond linked 3FTx dimer called irditoxin isolated from B. irregularis, an invasive species responsible for devastating decreases in bird popu- lations on the island of Guam [87,110,112]. This was especially significant because it was the first known covalently linked 3FTx-3FTx dimer toxin. Irditoxin was shown to have a particularly strong diapsid-specific (bird and reptile) neurotoxicity, ten-fold that of monomeric toxins [87]. Despite this toxin being isolated almost a decade ago, the presence or absence of homologous toxins in other Boiga species has not been thoroughly investigated until now. Our transcriptomic analyses revealed for the first time that species besides B. irregularis produce transcripts with the necessary additional cysteines to form both the A and B chains of irditoxin homo- logues. Such pairs of transcripts were recovered from B. dendrophila dendrophila, B. d. gemmicincta and, B. nigriceps (Table 2.2). In both B. irregularis and B. dendrophila (the two species which included transcriptomes from multiple individuals), these presence/absence data were consistent across species. We also found, from the alignment of our data and previously published toxin sequences, that two papers had published toxins with cysteines at the characteristic irditoxin sites prior to this paper. Fry et al. [18] published a toxin sequence from Trimorphodon biscutatus (Trimorphodon biscutatus A7X3S2) with the A cysteine and one with the B cysteine from Telescopus dhara (Telescopus dhara A7X3V0) the year prior to the discovery of irditoxin [87]. Thus, the earlier study did not recognise CHAPTER 2. THREE-FINGER TOXIN DIVERSIFICATION IN THE VENOMS OF CAT-EYE SNAKES 16 (COLUBRIDAE: BOIGA)

Figure 2.5: A) 1D SDS-PAGE glycine produced under reducing and non-reducing conditions stained with colloidal coomassie brilliant blue G-250. Toxins of interest, were identified by LC/MS-MS of excised bands. From left; Toxicodryas blandingii (Tbl) reduced (R), non-reduced (NR), ladder (250-10 kDa), Boiga dendrophila (Bdd) reduced (R), non-reduced (NR). 5B) 2D SDS-PAGE reduced electrophoresis mini gel. B) Toxicodryas blandingii crude venom stained with colloidal coomassie brilliant blue G-250. First dimension: isoelectric focusing (pH 3–10 non-linear gradient); second dimension molecular weight 250-10 kDa: 12% SDS PAGE. The pH gradient and the molecular weight marker positions are shown. 3FTx were identified by weight and comparison to 1D results. 5C) 2D SDS-PAGE reduced electrophoresis mini gel. C) Boiga dendrophila crude venom stained with colloidal coomassie brilliant blue G-250. First dimension: isoelectric focusing (pH 3–10 non-linear gradient); second dimension molecular weight 250-10 kDa: 12% SDS PAGE. The pH gradient and the molecular weight marker positions are shown. 3FTx were identified by weight and comparison to 1D results. 2.4. DISCUSSION 17 the significance of these cysteines. Modahl and Mackessy [107] discussed the similarity of their two B. cynodon sequences to irditoxin A (Boiga cynodon A0A193CHL1, 83% identical) and B (Boiga cynodon A0A193CHL2, 93% identical), however only the sequence similar to the B subunit actually contained the characteristic cysteine and the presence of this cysteine was not commented upon. Modahl and Mackessy [107] also published—but did not discuss—a B. nigriceps sequence that contained the A cysteine and a Trimorphodon lambda sequence that contained the B cysteine; nor did they discuss the presence or absence of the cysteines necessary for forming covalent bonds between the subunits when they speculated on the possible occurrence of dimeric complexes homologous to irditoxin in species besides B. irregularis. Conspicuously, our phylogenetic, clustering, and network analyses (Figures 2.2 and 2.3) demon- strate that sequence similarity does not reliably distinguish between toxins with or without the A and B cysteines. Our transcriptomic analyses confirm the finding of toxins with the A cysteine from B. nigriceps, but they did not recover any sequence with the B cysteine from B. cynodon. Because their technique of sequencing mRNA from venom rather than gland tissue resulted in very low yields, Modahl and Mackessy [107] did not recover toxins with both the A and B cysteines from any species including B. irregularis. Thus, our analyses are the first evidence that species besides B. irregularis transcribe both the A and B subunits. Despite the fact that our data include Trimorphodon toxins with cysteines at the characteristic site of both the A and B chain of irditoxin, they are found in different species with the A chain in T. biscutatus and the B chain in T. lambda. The T. biscutatus toxin with the A cysteine is particularly unusual because it has additionally mutated the 3rd and 4th canonical cysteines to phenylalanine and threonine respectively. Whether either species actually secretes both chains and whether they form a dimeric toxin will require a thorough proteomic investigation of their venoms. The function of the putative toxin is also of interest; while it seems somewhat unlikely these terrestrial snakes would possess a bird-specific toxin these toxins are also potent against other diapsids such as lizards. However, B. trigonata, which is morphologically and ecologically convergent with Telescopus and Trimorphodon, did not yield contigs with either of the characteristic irditoxin cysteines. Specific biochemical investigation of B. trigonata venom would be necessary to conclusively confirm or refute this finding. The distribution of these toxins—with the Trimorphodon A subunit more closely related to the Trimorphodon B subunit than the Boiga A subunit and the Telescopus B subunit nested within the Boiga B subunits (Figure 2.2)—suggests that the B subunit may have arisen in a common ancestor before much of the colubrine radiation and that toxins similar to the A subunit has evolved independently in Boiga and Trimorphodon. However, given how closely related the A and B subunits are in our phylogeny, it must also be considered that this denmotoxin-like clade of 3FTx may have been exapted for forming dimeric complexes and that both subunits could have evolved independently on multiple occasions. While the latter scenario is less parsimonious, in order to settle the issue, a greater diversity of colubrine sequences must be isolated and characterised in order to elucidate the likely ancestral forms of these proteins, as has been done with dimeric phospholipase A2 toxins from crotaline venoms CHAPTER 2. THREE-FINGER TOXIN DIVERSIFICATION IN THE VENOMS OF CAT-EYE SNAKES 18 (COLUBRIDAE: BOIGA)

(Whittington et al., 2018). Somewhat surprisingly, we did not find evidence for widespread positive selection across the denmotoxin-like 3FTx. In snake venom proteins, extensive duplication is typically accompanied with high ω values [69, 71, 125]. The only extreme positive selection we measured was in Clade B (ω=3.02) and, as discussed earlier (section 4.4), this value may be an artefact from low sample size and wide taxonomic range that would decrease with further sampling. On the other hand, we found Clade D, which includes the canonical irditoxin B chain, to be under fairly strong negative selection (ω=0.67). Perhaps this indicates that the structural constraints of dimerization may strengthen the negative selection acting on a toxin, as has been shown for non-covalently linked dimeric 3FTx [69]. The overall low ω values may help explain how these denmotoxin-like 3FTx have remained relatively similar across much of the colubrine radiation and the extensive diversification within Boiga. We also expected to see a pattern among site-specific signals of selection similar to that found in the eight-cysteine 3FTx, where the loops of the toxins—the domains that interact with the toxins’ targets—tended to be enriched in sites experiencing positive selection and the cores which are essential to the overall structure of the protein were enriched for those experiencing negative selection [69,71]. However, no such pattern is apparent in the clades of denmotoxin-like 3FTx (Figure 2.4). This may be partly due to the overall strength of positive selection being lower. We also see little variation in the predicted protein structures of the various clades. This is certainly due in part to the fact that only three structures have ever been published of ten-cysteine 3FTx: irditoxin B chain (Clades A, B, C, and D), denmotoxin (Clades E and F), and irditoxin A chain (Clade G). However, the relatively high similarity between the clades and low ω values also play a role in this. For these reasons, we suspect that the main source of functional diversity within the denmotoxin-like 3FTx is the actual dimerization, i.e. all the monomeric toxins are likely to exhibit similar activity to other monomeric toxins such as denmotoxin while all the Boiga dimeric toxins are likely to be very similar to irditoxin. As discussed earlier, the function of the putative dimeric Trimorphodon toxin remains speculative, but diapsid-specific toxicity seems likely. Given the apparent conservation of function within the denmotoxin-like 3FTx, it seems likely that the extensive duplication serves the proximal evolutionary purpose of increasing venom yield through gene dosage effects, a pattern of venom evolution that has been demonstrated in rattlesnakes [72]. T. blandingii and B. d. dendrophila were used for our venom proteomics because they bracket much of the genetic, morphological, geographical and ecological diversity found within this clade of colubrine snakes: T. blandingii is restricted to Africa, where this radiation is thought to have started, while B. d. dendrophila is representative of the recently derived Southeast Asian lineage within Boiga [3, 113]. Proteomic investigations via 1D and 2D SDS PAGE confirmed that both venoms were dominated by 3FTx (Figure 2.5). It was hypothesised that B. d. dendrophila would produce 3FTx homologous to those of its close relative, B. irregularis, since they occupy similar ecological niches and we had assembled transcripts of such homologues. A series of analyses including 1D (reduced and non- reduced) SDS-PAGE, 2D (reduced) SDS-PAGE, LC-MS/MS peptide sequencing, and transcriptomics supported this hypothesis (Figure 2.5). The protein band between 15 and 20 kDa that can be seen in 2.4. DISCUSSION 19 the B. d. dendrophila—but not T. blandingii—venom under non-reducing conditions matches what we would expect from irditoxin: the combined mass of its A and B chains is approximately 17 kDa (Figure 2.5A and B) [87]. It is apparent that this band contains dimeric complexes because under reducing conditions (which break disulphide bonds) this heavier band disappears and a new monomeric 3FTx band appears at a lower molecular weight (Figure 2.5A). The double band pattern is also seen in the 2D reduced gel for B d. dendrophila, but not T. blandingii (Figure 2.5B and C). This suggests that B. d. dendrophila produces toxins of similar molecular mass and biochemical structure to irditoxin while T. blandingii does not. These toxins were confirmed to be 3FTx by LC-MS/MS sequencing of proteins isolated from gel bands. This indicates that the evolution of dimeric toxins with both subunits homologous to those of irditoxin occurred after the genus Boiga separated from its sister genera and may help explain the patterns of migration and speciation we see in the genus.

Our results suggest that the idea that the evolution of dimeric toxins was the key evolutionary innovation exclusive to B. irregularis which facilitated it being a unique and highly successful invasive species is likely too simplistic because similar toxins can be found in several other species. Perhaps all Boiga species, or at least those expressing irditoxin-like dimers, pose similar risks and it is merely chance that it was B. irregularis invaded Guam rather than another species. If this is the case, then irditoxin may have underpinned the rapid evolution and migration of the modern Boiga species. The havoc B. irregularis has wreaked on Guamanian bird populations demonstrates how effective these toxins and the snakes employing them might have been as they encountered na¨ıve prey populations during their invasion of Southeast Asia and Oceania. A positive feedback between their arboreal lifestyle and this potent, diapsid-specific, toxin could explain why there are so many species of Boiga occupying highly arboreal niches throughout the Asian region. However, this would raise the question of why none of the other species have invaded other locations: perhaps Guam’s ecology was unique in the opportunities Boiga was given to invade in the first place, the lack of native snake species, or some other factor. While it is possible that there is something else distinctive about B. irregularis that makes it more noxious than its close relatives, no aspect of their ecology is an obvious culprit. For instance, B. irregularis is known to be highly food-motivated and somewhat generalist in feeding habits (Chiszar et al., 1993) which are traits often found in invasive species, but many other Boiga species have similarly broad diets (Stuebing et al., 1999). Thus, the invasion of Guam and surrounding islands by B. irregularis may simply have been a chance event based on which species was first transported to these islands and that other large species such as B. cynodon or B. dendrophila may have been just as invasive had either been given the opportunity. Irditoxin itself could still be the answer if the B. irregularis toxins happen to be more potent than their homologs in closely related species. However, neurotoxicity testing has shown that B. cynodon, for example, is of comparable toxicity (Lumsden et al., 2004). Whatever the reason, the fact remains that B. irregularis is a proven threat as an invasive species while other Boiga species are not. The currently available evidence neither disproves nor supports the idea that this is due to differences in their biology rather than a contingent historical fact. CHAPTER 2. THREE-FINGER TOXIN DIVERSIFICATION IN THE VENOMS OF CAT-EYE SNAKES 20 (COLUBRIDAE: BOIGA)

Table 2.4: Sample collection locations and analyses performed

Species Collection location Analysis Toxicodryas blandingii Tanzania Proteomics Boiga cynodon West Java, Indonesia Transcriptomics Boiga dendrophila dendrophila West Java, Indonesia Proteomics & Transcriptomics Boiga dendrophila gemmicincta South Sulawesi, Indonesia Transcriptomics Boiga irregularis Sulawesi, Indonesia Transcriptomics Boiga irregularis Brisbane, Australia Transcriptomics Boiga nigriceps West Java, Indonesia Transcriptomics Boiga trigonata Pakistan Transcriptomics

2.5 Materials and Methods

2.5.1 Venom and Tissue Supplies

Tissue samples in the form of venom glands and venom samples were collected from localities outlined in Table 2.4.

2.5.2 Transcriptomics

A multi-step process was used to sequence and align Boiga 3FTx from the SE Asian clade.

Species examined

To determine the sequence variability of Boiga 3FTx, venom glands were obtained from Boiga cynodon, B. d. dendrophila, B. d. gemmicincta, B. irregularis (Sulawesi), B. irregularis (Brisbane), B. nigriceps, and B. trigonata. All glands were dissected from freshly euthanized specimens 4-5 days post venom extraction, as this is the time when toxin mRNA transcriptions are being transcribed at the highest rate [52, 87, 126]. Toxicodryas blandingii venom glands were not available and therefore were not included (Table 2.4).

RNA extraction and mRNA purification

Venom gland tissue (20 mg) was homogenized using a rotor homogenizer and total ribonucleic acid (RNA) extracted using the standard TRIzol Plus methodology (Invitrogen). RNA quality was assessed using a Nanodrop (Nanodrop 2000/2000c, v1.4.2 Spectrophotometer, Thermo Scientific, USA). mRNA was extracted and isolated using and following standard Dynabeads mRNA DIRECT Kit (Life Technologies Ambion, 1443431, Thermo Fisher Scientific, USA).

Sequencing

Total RNA was extracted from venom glands using the standard TRIzol Plus method (Invitrogen). Extracts were enriched for mRNA using standard RNeasy mRNA mini kit (Qiagen) protocol. mRNA 2.5. MATERIALS AND METHODS 21 was reverse transcribed, fragmented and ligated to a unique 10-base multiplex identifier (MID) tag prepared using standard protocols and sequenced on a MiSeq platform (Australian Genome Research Facility). MID reads informatically separated sequences from the other transcriptomes on the plates, which were then post-processed to remove low quality sequences before de novo assembly into contiguous sequences (contigs).

Assembly

Illumina reads that were likely to be cross contamination between multiplexed samples were removed from our read files by identifying 57 nucleotide kmers in our focal read set that were present in another read set from the same lane at a 1000-fold or higher level. Reads with 25% or more of their sequence represented by such kmers were filtered from the data set. This was accomplished using Jellyfish 2.2.6 [127] and Kmer Analysis Toolkit (KAT) 2.3.4 [128]. We then removed adaptors and low quality bases from the reads and removed any reads shorter than 75 base pairs using Trim Galore version 0.4.3 [129]. We then used PEAR 0.9.10 [130] to combine pairs of reads whose ends overlapped into one, longer, merged read. We then carried out several independent de novo assemblies of these reads using the programs Extender version 1.04 [126], Trinity version 2.4.0 [131], and SOAPdenovo version 2.04 [132]. SOAPdenovo was run repeatedly with k-mer sizes of 31, 75, 97, and 127. The raw reads may be found in the NCBI Sequence Read Archive under the accession number SRP155444.

Annotation

The de novo assemblies were concatenated and searched against reference toxin sequences obtained from UniProt using BLAST version 2.7.1 [133, 134]. We then removed all remaining contigs that did not contain complete coding sequences. Those that did were screened by visualizing the read coverage for all contigs whose highest and lowest coverage differed by a at least factor of 10 using the Burrows-Wheeler Aligner (BWA) 0.7.16a [135], the Genome Analysis ToolKit (GATK) 3.8 [136], and BEDTools 2.26.0 [137]. Those that showed sharp discontinuities indicative of chimerical assembly were removed from the dataset. In cases where two samples contained transcripts for identical amino acid sequences, we aligned the raw reads to all the 3FTx from each sample using BWA to check if either of them was expressed at unusually low levels which might indicate contamination. In every case, the number of reads aligned to these contigs was within an order of magnitude of the highest number aligned to any toxin from that sample. Finally, we used CD-HIT version 4.7 to cluster the remaining sequences and remove duplicates [138,139]. The sequences of these contigs are available at the NCBI Transcriptome Shotgun Assembly Sequence Database under the following accession numbers: GGUA00000001( B. cynodon), GGUB00000001–GGUB00000043 ( B. d. dendrophila), GGUC00000001–GGUC00000012 ( B. d. gemmicincta), GGUD00000001–GGUD00000019 ( B. irregularis, Brisbane), GGUE00000001–GGUE00000014 ( B. irregularis, Sulawesi), GGUF00000001– GGUF00000007 ( B. nigriceps), and GGUG00000001–GGUG00000007 ( B. trigonata). CHAPTER 2. THREE-FINGER TOXIN DIVERSIFICATION IN THE VENOMS OF CAT-EYE SNAKES 22 (COLUBRIDAE: BOIGA)

2.5.3 Proteomics

Our proteomic investigations included using a combined approach of 1D and 2D SDS-PAGE, excision of gel bands and spots, and LC-MS/MS identification of the proteins therein. Proteomic methods were performed as previously described by us [21, 140, 141]. Figure 2.5A shows bands picked for each species for MSMS processing.

2.5.4 Analysis

Phylogenetic reconstruction

Protein sequences for all ten-cysteine 3FTx that were available from the UniProt database were combined with the translations of our 3FTx transcripts [134]. The sequences were aligned using a combination of manual alignment of the conserved cysteine positions and alignment using the MUltiple Sequence Comparison by Log-Expectation (MUSCLE) algorithm implemented in AliView for the blocks of sequence in between these sites [142, 143]. This alignment contained 282 total sequences of which 159 sequences were from Boiga. We reconstructed the phylogeny of these sequences using MrBayes 3.2 for 15,000,000 generations and 1,000,000 generations of burnin with lset rates=invgamma (allows rate to vary with some sites invariant and other drawn from a γ distribution) and prset aamodelpr=mixed (allows MrBayes to generate an appropriate amino acid substitution model by sampling from 10 predefined models) [144]. The run was stopped when convergence values stabilized at approximate 0.013. Two replicate runs recovered virtually identical topologies.

Similarity Network

An all-vs-all Basic Local Alignment Search Tool (BLAST) search was conducted on the same dataset of protein sequences as was used for the phylogeny with -outfmt "10 qacc sacc qcovs evalue" [133]. The results of this search were filtered using a custom R script (see Appendix A) to remove self-to-self results, collapse bidirectional results into one entry, and create a similarity score −17 defined as −log10(e − value) for each entry. Edges with coverage < 70% or e − value > 1 ⇥ 10 were excluded from the analysis and the network was created in Cytoscape 3.5.1 using the Prefuse Force Directed OpenCL Layout on the similarity scores [145].

Protein Clustering

Clustering was carried out using the CD-HIT 4.7 algorithm with options -c 0.45 -n 2 -d 0 -sc 1-g1[138,139]. This sets the similarity threshold of the clusters to 45% and sorts the clusters by the number of sequences they contain. 2.5. MATERIALS AND METHODS 23

Tests for Selection

Coding DNA sequences for denmotoxin-like sequences were compiled from GenBank [146]. The sequences were trimmed to only include those codons which translate to the mature protein, translated, aligned, and reverse translated using AliView and the MUSCLE algorithm [142, 143]. Phylogenetic trees for each clade were generated from the resulting codon alignments using the same methods as described above. These tree topologies were used for all subsequent analyses. We used several of the tests for selection implemented in HyPhy version 2.220150316beta due to their different emphases [147]. The AnalyzeCodonData analysis generates overall ω values for an alignment while the Fast Unconstrained Bayesian AppRoximation (FUBAR) method gauges the strength of consistent positive or negative selection on individual amino acids [148]. In contrast, the Mixed Effects Model of Evolution (MEME) method identifies individual sites that were subject to episodes of positive selection in the past [149].

Protein Modelling

Custom models for each clade of 3FTx were generated by inputting representative sequences (Clade A—Boiga irregularis Brisbane 3FTx 00; Clade B—Boiga dendrophila dendrophila 3FTx 14; Clade C—Boiga irregularis A0A0B8RS39; Clade D—Trimorphodon biscutatus A7X3S0; Clade E—Boiga dendrophila dendrophila 3FTx 28; Clade F—Boiga dendrophila dendrophila 3FTx 38; Clade G— Boiga dendrophila dendrophila 3FTx 29) to the Phyre2 webserver using the Intensive option [150]. Alignments of each clade were trimmed to match these structures and attribute files were created from FUBAR and MEME results. Conservation scores were calculated using the default settings of AL2CO [151]. The structures were rendered and colored according to these attributes in UCSF Chimera version 1.10.2 [152].

Chapter 3

Electric blue: transcriptomics, venom composition, and toxin evolution in the long-glanded coral snake species Calliophis bivirgatus and C. intestinalis

3.1 Abstract

The genus Calliophis is the most basal branch of the family Elapidae and the species therein are mostly small bodied, semi-fossorial, and ophiophagous. A clade pf three species within the genus have evolved larger bodies and highly elongated venom glands. Recent research has shown that C. bivirgatus has evolved a similarly unique toxin (calliotoxin) that produces spastic paralysis in their prey by acting on the voltage-gated sodium (NaV) channels. We assembled a transcriptome from C. bivirgatus to investigate the molecular characteristics of these toxins and the venom as a whole. Toxins that resemble calliotoxin are responsible for a substantial minority of the venom composition and form a distinct clade within a larger, more diverse clade of C. bivirgatus three-finger toxins. We also performed in vitro assays that replicated the previously reported activity and showed that this unique neurotoxicity is independent of the cytotoxic activity which the venom also causes in high concentrations. Beyond that, we also demonstrated that the venom of C. intestinalis—the nearest relative of C. bivirgatus—also effects NaV channels. C. intestinalis venom however, acts as an antagonist and produces in vitro results and reported symptoms similar to tetrodotoxin. Proteomic results indicate that C. intestinalis venom contains toxins with very similar sequences to calliotoxin and we hypothesize that these may be responsible for the NaV activity in C. intestinalis as well. We also suggest that these toxins may have been selected for in response to widespread resistance to more traditional elapid neurotoxicity amongst the snakes that Calliophis often prey on. 25 CHAPTER 3. ELECTRIC BLUE: TRANSCRIPTOMICS, VENOM COMPOSITION, AND TOXIN EVOLUTION IN THE LONG-GLANDED CORAL SNAKE SPECIES CALLIOPHIS BIVIRGATUS AND C. 26 INTESTINALIS 3.2 Introduction

The origins of the elapids remain somewhat unclear. They likely arose in Africa or Asia 30 million years ago before quickly radiating out to southern Asia, Australia, the Americas, as well as the Pacific and Indian oceans [153–155]. What triggered this radiation remains uncertain. The most obvious candidate feature that may have contributed to their success is their venom system. Elapids are one of three taxa of front-fanged snakes along with viperids and atractaspidines [28] and their venom delivery system is distinct from the other front-fanged families; slightly different muscles were co-opted for use as a venom gland compressor muscle and, unlike the large mobile fangs of viperids and atractaspidines, elapid fangs are relatively short and fixed in place relative to the rest of the skull [156, 157]. Virtually all elapid venoms that have been studied to date contain eight-cysteine 3FTx (Figure 1.2). Given that these potent toxins are not found in any other snake venoms, it is clear that they first evolved very early in the evolutionary history of the family, but after they had diverged from other lineages. It remains unclear if either the origin of these unique toxins or the morphological adaptations to more effectively deliver them preceded the other or whether they evolved in concert. One way to address questions about the order in which these key innovations evolved and what role they might have played in the radiation of the elapids is to examine the venom system of the most basal members of the family. The ecology, anatomy, and physiology of these snakes may provide insight about the niche occupied by the common ancestor of elapids and the state of these characteristic traits in those species may inform us about their evolutionary trajectories.

The genus Calliophis (Asian coral snakes) is the most basal group in Elapidae [4, 113, 159]. These snakes can be found from India through much of Southeast Asia and are mostly small, fossorial, and ophiophagous (Figure 3.1) [158,160–162]. Virtually all species will display their brightly-colored tails when threatened and many species also have brightly-colored stripes (Figure 3.1A) [115, 163]. There is a clear distinction within the genus between the short-glanded and long-glanded species [153, 159]. This refers to the fact that 3 (C. bivirgatus, C. intestinalis, and C. salitan) of the 11 currently described species possess extraordinarily elongated venom glands that extend into the body cavity and range from 1 1 4 to more than 3 of the body length (Figure 3.1B) [59,161,164,165]. Bites from short-glanded species are generally regarded as medically insignificant [166] while long-glanded species can pose more of a threat [167–171]. However, these species are rarely encountered so the available information regarding the hazards of a bite is slim. C. salitan is known only from a single recently described specimen, and so has no known bite reports [159]. C. intestinalis is responsible for the two best documented bite reports from these species: one afflicting a Major in the British armed forces who suffered almost exclusively local symptoms [172] and the other a herpetologist who documented his own experience including serious symptoms of systemic neurotoxicity [173]. C. bivirgatus is the only member of the genus responsible for a published account of a lethal bite: a two year old child in Malaysia was bitten on the hand a died roughly 45 minutes later [174,175]. This species has also been implicated in the death of a Singaporean man who shouted about a snake in his shower and was found dead with fang marks upon his toe [176]. However, the identification of the species responsible as C. bivirgatus is 3.2. INTRODUCTION 27

Figure 3.1: A) The striking colors of Calliophis bivirgatus, image by Yu Ching Tam via iNaturalist under CC BY-NC; B) The dissected venom glands of C. bivirgatus; C) C. bivirgatus preying upon Oligodon signatus, image by Xu Weiting [158]; D) Attempted cannibalism by C. intestinalis, image by Gan Gim Chuah via iNaturalist under CC BY-NC.

highly suspect at best; the only person who saw the snake in question died without describing it to anyone and the veterinarian at the Singapore Zoological Guardians told the coroner that the fang marks could have been made by C. bivirgatus, but that other species could also be responsible [176]. Despite the dubious nature of these claims, they have been repeated in many subsequent publications such as field guides, natural history books, and even a manual published by the United States Navy on the topic of venomous snakes and snakebite [170, 177, 178]. Several lines of in vitro evidence also underscore the danger of bites from C. bivirgatus. There is no specific antivenom developed against Calliophis venom, and preliminary tests of local elapid antivenoms found that the most effective antivenom still required large doses to protect mice from challenges of 2.5 LD50 and was ineffective against challenges of 5 LD50 [179]. A subsequent study involving a wider range of regional antivenoms noted that “[a]gainst a lethal challenge dose (2.5 LD50) of C. bivirgat[us] venom, all tested antivenoms failed to protect the mice, with the animals noted to die in spasticity (convulsion-like feature with myoclonus followed by muscle spasm, curled front limbs and out-stretched hind limbs) within 1 min post-injection of the venom-antivenom mixtures” [180]. These symptoms could be explained by experiments that demonstrate that C. bivirgatus venom contains toxins which functionally act as agonists to voltage-gated sodium channels associated with CHAPTER 3. ELECTRIC BLUE: TRANSCRIPTOMICS, VENOM COMPOSITION, AND TOXIN EVOLUTION IN THE LONG-GLANDED CORAL SNAKE SPECIES CALLIOPHIS BIVIRGATUS AND C. 28 INTESTINALIS skeletal muscle (NaV1.4) by delaying the inactivation of these channels; when added to an organ bath, the venom elicited spontaneous contractions from nerve-muscle preparations [165]. These spasms develop rapidly and may help explain the relatively swift death of the one confirmed bite victim of C. bivirgatus [174,175]. The effects of these toxins are functionally equivalent to the spastic paralysis that electric eels produce in their prey by the application of high-voltage electric discharge [181]. This toxic activity is in stark contrast to the usual α-neurotoxicity of elapid snakes. The clade referred to as the true coral snakes (containing the genera Sinomicrurus, Micruroides, and Micrurus) are a good point of comparison to Calliophis evolutionarily and ecologically. This clade forms next most basal branch of the elapid phylogeny after Calliophis [3,4,113,155] and most of the species that belong to it are fossorial, brightly-colored, and specialize in eating elongate ectothermic prey [12,35, 182]. The symptoms that occur when these snakes bite humans are markedly different from those produced by bites from long-glanded Calliophis. For instance, the single published account of a Sinomicrurus macclellandi bite indicated that the victim felt nothing until motor issues started developing 6 hours after the bite and culminated in lethal respiratory paralysis after 8 hours [183]. Similarly, bites from snakes of the genus Micrurus often exhibit few symptoms besides paralysis [184–186]. Clearly these symptoms are very different from those produced by C. bivirgatus venom, but interestingly the symptoms from C. intestinalis differ from both. In the sole account of a systemic envenoming, the author described suffering from dizziness, headaches, repeated attacks of difficulty breathing, vomiting, and diarrhea [173]. While these symptoms are not unheard of in snakebites, they do not definitively point to a particular mechanism of action [187]. However, it is clear that the toxins in C. intestinalis venom produce symptoms more similar to stereotypical elapid α-neurotoxicity than the novel activity of its sister species, C. bivirgatus. As with the elapid family as a whole, the genus Calliophis seems to have innovated in both the biological activity of the venom and the anatomy of the venom delivery system. We further investigate the composition and activity of C. bivirgatus venom due to the unique results of previous studies and also examine the venom of C. intestinalis as a point of comparison and to search for other novel results. This study’s investigation into the venom of these snakes can hopefully lend insight into the evolutionary dynamics that have shaped the genus as well as the family as whole.

3.3 Results

3.3.1 Transcriptomics

Our Calliophis bivirgatus transcriptome contained 125 unique toxins after rigorous quality control and filtering. Only three toxin families had a combined expression level greater than 1% of the total. In descending order of both expression level and number of unique contigs these families were 3FTx, kunitz peptides, and phospholipase A2s (PLA2s; Figure 3.2). The remaining 21 toxin contigs included cysteine-rich secretory proteins (CRiSPs), cystatins, hyaluronidase, kallikrein, nerve growth factors, natriuretic peptides, phopsphodiesterase, phospholipase B, snake venom metalloproteases (SVMPs), 3.3. RESULTS 29

Figure 3.2: The venom gland transcriptome of Calliophis bivirgatus is dominated by 3FTx, kunitz peptides, and PLA2: A) TPM normalized expression levels for all 125 toxin contigs, black borders to indicate those which had matches in our proteomics, orange crosses indicate contigs with similar sequence to calliotoxin; B) Proportion of total TPM accounted for by the major toxin families; C) Total number of unique contigs recovered from the major toxin families.

vespryn, and waprin. While SVMPs are common in many other snake venoms (espcially viperid or colubrid venoms), the other families are more obscure. They have been reported from a variety of venom transcriptomes, but are rarely if ever recovered from in notable quantities from actual venom and their functions remain almost entirely unknown [188]. Many of these contigs matched to the spectra of ion fragments produced by MS/MS proteomics including 15 of the 20 highest expressed toxins as well as toxins from all three major toxin families, SVMPs, CRiSP, and vespryn. Our phylogenetic analysis placed the 3FTx recovered from C. bivirgatus into the context of the previously known diversity of the toxin family (Figure 3.3). The fact that C. bivirgatus toxins could be found across the tree suggests that the eight-cysteine 3FTx had already begun to diversify before the common ancestor of Calliophis and other elapids. However, the closest non-Calliophis relative of any given C. bivirgatus toxin is, for the most part, a Micrurus toxin, which is what one would expect given their relative position in the species tree of Elapidae. Additionally, the majority of C. bivirgatus 3FTx, including those with extreme sequence similarity to calliotoxin, were recovered within two CHAPTER 3. ELECTRIC BLUE: TRANSCRIPTOMICS, VENOM COMPOSITION, AND TOXIN EVOLUTION IN THE LONG-GLANDED CORAL SNAKE SPECIES CALLIOPHIS BIVIRGATUS AND C. 30 INTESTINALIS

Figure 3.3: Unrooted tree of representative eight-cysteine 3FTx from across major taxonomic and functional divisions (black) as well as those from Calliophs bivirgatus. Most C. bivirgatus toxins are colored blue, while those sequences which are highly similar to calliotoxin are colored orange. Scale bar represents an average of 0.5 substitutions per site. 3.3. RESULTS 31

Figure 3.4: Protein similarity network of representative eight-cysteine 3FTx from across major taxonomic and functional divisions (black) as well as those from Calliophs bivirgatus. Most C. bivirgatus toxins are colored blue, while those sequences which are highly similar to calliotoxin are colored orange. The two major clades of C. bivirgatus toxins are indicated by arrows.

monophyletic clades that do not include toxins from any other species. Clade A will refer to the one that contains the calliotoxin sequences (among others) and Clade B will refer to the other. These two toxin clades were part of a polytomy that included several smaller Calliophis clades, several Micrurus clades, one clade that was a mixture of both, and a branch that led to the rest of the phylogeny. A protein similarity network analysis independently confirmed these results (Figure 3.4). It also showed that C. bivirgatus toxins are widely distributed amongst those of other elapids and that those toxins which were highly similar to calliotoxin formed a distinct cluster with each other amongst the other C. bivirgatus toxins.

3.3.2 Signals of Selection

dN Signals of selections analyses based on ratios of non-synonymous and synonymous mutations ( dS ) dN suggested that the 3FTx in both Clade A and B were subject to positive selection. The overall dS estimated for the two clades was very similar (Clade A: 2.40, Clade B: 2.37), but protein structure dN prediction and site-specific dS analyses suggested real differences between the two (Figure 3.5). One of the major structural differences was that the sequences in Clade A tended to be longer than those of Clade B, due to insertions in the Loop III region. With some notable exceptions in the Loop II region CHAPTER 3. ELECTRIC BLUE: TRANSCRIPTOMICS, VENOM COMPOSITION, AND TOXIN EVOLUTION IN THE LONG-GLANDED CORAL SNAKE SPECIES CALLIOPHIS BIVIRGATUS AND C. 32 INTESTINALIS

Figure 3.5: Signals of selection and protein structure prediction highlights differences between clades of Calliophis bivirgatus 3FTx: A) FUBAR β − α estimation of selection pressure mapped over the predicted structure of Clade A 3FTx; B) MEME weighted β − α estimation of selection pressure mapped over the predicted structure of Clade A 3FTx; C) FUBAR β − α estimation of selection pressure mapped over the predicted structure of Clade B 3FTx; D) MEME weighted β − α estimation of selection pressure mapped over the predicted structure of Clade B 3FTx.

dN of Clade A, the FUBAR and MEME site-specific dS analyses indicated similar selection regimes for similar regions of the toxins. The loops of Clade A seemed to be more highly enriched for sites under positive selection than their counterparts in Clade B, which can be indicative of neofunctionalization.

3.3.3 Proteomics

We used cell-based fluorescence assays on a Fluorescent Imaging Plate Reader (FLIPR) platform to screen HPLC fractions of C. bivirgatus venom for calliotoxin activity. Fractions 32, 34, and 36 displayed this activity and corresponded to the final three peaks of the HPLC trace (Figure 3.6). These fractions, along with crude C. intestinalis venom were analyzed by shotgun proteomics. As is to be expected on an initial fractionation of venom, each fraction contained multiple toxins, but the only shared toxins between all three were 3FTx with strong sequence similarity to calliotoxin (Tables B.1, B.2, and B.3). Whole venom shotgun proteomics of C. intestinalis also contained peptide hits to similar toxins (Tables B.4 and B.5).

3.3.4 NaV1.4 Assays

When we added C. bivirgatus venom to SH-SY5Y human neuroblastoma cells, it elicited an immediate, mg dose-dependent, response with an EC50 of roughly 0.24 ml of venom (Figure 3.7). Higher concentra- tions of C. bivirgatus also had a cytotoxic effect because these concentrations produced a long slow increase in the FLIPR measurement. Because we used a calcium dye to measure the response of the 3.3. RESULTS 33

Figure 3.6: HPLC trace of Calliophis bivirgatus venom in blue with active fractions highlighted in orange. Black line indicates solvent gradient.

Figure 3.7: Calliophis bivirgatus venom produces an immediate, dose-dependent, FLIPR response: A) Representative FLIPR traces for an effective dose of C. bivirgatus venom (blue) and empty buffer (black) demonstrating the initial peak caused by the venom; B) Semi-log scale dose response curve of the height of the initial peak produced by different amounts of C. bivirgatus venom (blue) and the average value produced by negative controls (black). CHAPTER 3. ELECTRIC BLUE: TRANSCRIPTOMICS, VENOM COMPOSITION, AND TOXIN EVOLUTION IN THE LONG-GLANDED CORAL SNAKE SPECIES CALLIOPHIS BIVIRGATUS AND C. 34 INTESTINALIS

Figure 3.8: TTx abolishes the initial response to C. bivirgatus venom, but not the longer term cytotoxicity caused by high doses: A) Representative FLIPR traces for a cytotoxic dose of C. bivirgatus venom both with (tan) and without (blue) the addition of TTx; B) Maximum response of the initial mg peak and cytotoxic ramp caused by 2.67 ml of C. bivirgatus venom. The initial peak is significantly lower when treated with TTx while the cytotoxic effect remains the same.

cells, this likely indicates compromised membranes which would allow for a steadily increasing influx of extracellular calcium. Visual inspection of the cells after the assay revealed that high concentrations of C. bivirgatus venom led to unusual cell morphology consistent with this hypothesis. However, since the addition of the NaV channel antagonist tetrodotoxin (TTx) abolished the initial peak, but not the slower increase, we conclude that the immediate response was in fact caused by the previously described delay of NaV channel inactivation [165] and not due to cytotoxicity (Figure 3.8). When we added C. intestinalis venom to the same cell line, it did not produce an immediate response. However when we performed a second addition of the NaV channel agonist veratridine to elicit a response from the NaV1.4 channels, those cells that had been exposed to C. intestinalis venom did not respond (Figure 3.9).

3.4 Discussion

Given that Calliophis is the most basal extant genus that produces the eight-cysteine 3FTx characteristic to the elapids, we hoped that our transcriptome would help clarify the evolution of these toxins. The 3.4. DISCUSSION 35

Figure 3.9: Calliophis intestinalis venom prevents NaV channel from responding to veratridine: A) Representative FLIPR traces showing the response of cells to veratridine after being treated with C. intestinalis venom (brown), TTx (tan), or empty buffer (grey); B) Maximum response produced by 20 mg µM veratridine. Cells treated with 2.50 ml of C. intestinalis venom (brown) or 0.1µM TTx (tan) show significantly decreased response compared to negative controls.

small size and high rates of evolution of the 3FTx make investigations of their precise history difficult and many analyses of these toxins have resorted to creating unrooted phylogenies [71,78,88]. Because of this, it is currently unclear which clades of eight-cysteine 3FTx sit near the base of the radiation after they evolved from the 10-cysteine forms. Our analyses suffer from these same problems, but offer some suggestive results (Figure 3.3). Calliophis sequences appear across much of the tree indicating that the common ancestor of all extant elapids most likely possessed multiple isoforms of the derived eight-cysteine 3FTx already. Our phylogeny remains unrooted, but for visual purposes we have placed the root on the branch between the polytomy containing the two major C. bivirgatus clades and the rest of the tree. It seems that data beyond the transcribed sequence of eight-cysteine 3FTx will be necessary to discern the details of their evolution. What does stand out is that of the Calliophis 3FTx sequences in our transcriptome, 52 out of 67 fall into two clades which are so closely related that they belong to the same polytomy. We have designated these Clades A and B and the sequences that bear significant resemblance to calliotoxin form a smaller clade nested within Clade A. The similarity in terms of morphology and toxin sequence of the venom system of Calliophis and other more derived elapids indicates that these traits were well established in the common ancestor of all elapids. The fact CHAPTER 3. ELECTRIC BLUE: TRANSCRIPTOMICS, VENOM COMPOSITION, AND TOXIN EVOLUTION IN THE LONG-GLANDED CORAL SNAKE SPECIES CALLIOPHIS BIVIRGATUS AND C. 36 INTESTINALIS that no extant lineage possesses what we might consider a transitional form of the elapid venom system suggests that the toxins and morphology both evolved rapidly and in concert which then allowed the descendants with fully functional venom systems to rapidly diversify and spread across the globe. The analyses we ran looking for signals of natural selection in the coding sequence of C. bivirgatus 3FTx indicated that these toxins are largely subject to positive selection. This is often the case for 3FTx (see Chapters 2 and 4) [69,71,88,125] and is the likely cause of the rapid rates of evolution that the toxin family exhibits. Positive selection can be the result of biological arms races which are a relatively common scenario in toxin evolution. The comparison between the two clades of C. bivirgatus toxins suggests some differences in terms of the structure of the toxins and the distribution of selection across them (Figure 3.5). The stronger selection on the loops of Clade A as well as the longer Loop III domain suggest a possible evolutionary explanation for how this clade was able to evolve the novel activity seen in calliotoxin. The loops of 3FTx are known to be the usual site of interaction between the toxin and the target and structural changes in these regions have been associated with shifts in neurotoxic activity before, such as in the long-chain neurotoxins which have evolved an additional disulfide bridge in the Loop II domain (note that this is different from the extra disulfide bridge which was lost when the eight-cysteine toxins evolved) and bind to different classes of receptors than those without [83, 189]. Our FLIPR results with C. bivirgatus venom are consistent with previous research that showed that it can shift the voltage dependence and delay inactivation of NaV1.4 channels [165]. Our results also indicate that high dosages can be cytotoxic which are consistent with reports of minor local tissue damage in the only detailed bite report from this species [174, 175]. The exact origin of this activity remains unclear; while a number of 3FTx are known to cause cytoxicity [83], none of the 3FTx sequences recovered from our transcriptome bear much similarity to those toxins (represented by Q9W716, Q91996, and Q69CK0 in our phylogeny). What is clear is that the cytotoxic activity is entirely independent from that of calliotoxin since the effect of calliotoxin can be detected at much lower venom concentrations and the addition of TTx completely blocks the effect of calliotoxin while not impacting the cytotoxicity signals at venom concentrations where both are apparent (Figure 3.8). While neither is a perfect measure of abundance of a given toxin in the actual venom, our transcriptomics (Figure 3.2) and HPLC (Figure 3.6) indicate that calliotoxin-like sequences are a significant but not dominant part of the overall venom composition of C. bivirgatus. This suggests that calliotoxin and other similar toxins with the same activity are likely more potent than the EC50 of mg 0.24 ml of our dose response curve suggests since that figure indicates the quantity of whole venom including the many other toxins which do not act upon NaV channels.

Perhaps the most intriguing finding of our research is that C. intestinalis venom can block NaV channels (Figure 3.9), which would produce a flaccid paralysis instead of the spastic paralysis produced by C. bivirgatus. This is consistent a well-documented bite report caused by this species: the symptoms described therein match very closely to those of a mild TTx envenomation from a blue-ringed octopus which supports our in vitro evidence suggesting a similar mechanism of action [173,190]. Based on the fact that C. intestinalis venom contains toxins that are highly similar to calliotoxin and also possesses 3.4. DISCUSSION 37 a novel NaV activity, we suspect that these toxins are responsible for the sodium channel blockage caused by C. intestinalis. We further speculate that this may represent the ancestral state for these toxins, since it seems simpler biochemically to bind to a channel in an antagonistic fashion than an agonistic one. In this scenario, the flaccid paralysis caused by NaV antagonism could have provided an evolutionary stepping stone that allowed the development of the more sophisticated spastic paralysis caused by C. bivirgatus venom.

While the mutations that lead to this ancestral NaV activity would have been pure happenstance, we suspect that it would have been of immediate selective advantage because of resistance to α- neurotoxins. Variants of this trait have evolved repeatedly, including a tryptophan!serine mutation at the beginning of the acetylcholine binding pocket of the α1 nicotinic acetycholine receptor that confers partial resistance in all snakes (See Chapter 5) and a threonine!asparagine mutation with accompanying glycosylation two amino acids later in elapids which grants near total immunity to the standard α-neurotoxic 3FTx [191]. For any ophiophagous snake, and especially one that routinely preys upon elapids like the long-glanded Calliophis do (Figure 3.1), any way around this resistance in potential prey items would be of enormous evolutionary benefit as it would greatly increase the amount of prey the snake could reasonably feed on. The evolution of a 3FTx that targets an entirely different family of receptor would have neatly sidestepped the issue of α-neurotoxin resistance. This would then have laid the groundwork for the later evolution of calliotoxin which has been suggested to bring further benefits to C. bivirgatus by immobilizing prey much quicker and therefore decreasing the chances of retaliation or escape [165]. These results are also relevant to the question of why a lineage of Calliophis evolved such extraordinarily long venom glands. They are almost certainly a compromise between the need to produce larger venom yields while maintaining a slim profile. Calliophis are all semi-fossorial and their slender build allows them to move through leaf litter, soil, and undergrowth with relatively little resistance. This lifestyle would be hindered by the development of large venom glands like those found in some cobras or Australian elapids which increase in circumference and give those snakes very broad heads. In the African viperid genus Causus, one species has developed similarly elongated glands, and this is hypothesized to be in response to evolutionary and ecological forces that drove that species to take larger prey items [192]. One possible explanation for this adaptation in Calliophis is that, prior to the evolution of NaV toxins, the ancestral long-glanded lineage adapted to α-neurotoxin resistance in their prey with increased venom yield. However, that does not explain why the development of these elaborate structures has not been subsequently selected against now that these species possess toxins which circumvent that resistance. Additionally, there are several other lineages of elapids (e.g., Bungarus) that face similar selective pressures, but have not developed enlarged venom glands of any sort. It is likely, instead, that the co-occurrence of the long glands and NaV toxins in the same species is not a coincidence. While these toxins are very dangerous at high doses, the murine intravenous LD50 of C. bivirgatus venom is relatively high [179, 180]. This suggests that these snakes might need to deploy a similarly large amount of venom to effectively subdue their prey with NaV toxins. In this case, the evolution of NaV toxins and elongated venom glands would be another instance of venom/delivery CHAPTER 3. ELECTRIC BLUE: TRANSCRIPTOMICS, VENOM COMPOSITION, AND TOXIN EVOLUTION IN THE LONG-GLANDED CORAL SNAKE SPECIES CALLIOPHIS BIVIRGATUS AND C. 38 INTESTINALIS system coevolution much like the origin of elapids and their eight-cysteine 3FTx.

3.5 Materials and Methods

3.5.1 Venom and Tissue Acquisition

Pooled C. bivirgatus venom from two adult females and one subadult male was provided by Nathaniel Frank of MToxins Venom Lab. C. intestinalis venom was provided by Dr. Choo Tan Hock of the University of Malaysia, and venom glands were provided by Iwan Hendrikx from a captive specimen of Malaysian stock. All venoms and tissues were imported under Australian Quarantine and Inspection Service permit 0001804439.

3.5.2 RNA extraction

Venom gland tissue (20 mg) was homogenized using a rotor homogenizer and total ribonucleic acid (RNA) extracted using the standard TRIzol Plus methodology (Invitrogen). RNA quantity was assessed using a Nanodrop 2000, spectrophotometer (Thermo Scientific). mRNA is extracted and isolated using standard Dynabeads mRNA DIRECT Kit (Life Technologies Ambion). Dynabeads work by hybridising poly-thymine (polyT) sequences, covalently bound to the Dynabead surface, to the mRNA poly-aderine (polyA) tail. This selectively enriches the sample for mRNA during the centrifugation and washing stages, since other RNA forms (which do not have a polyA tail) are removed. This facilitates a higher concentration of mRNA yield for sequencing purposes.

3.5.3 Sequencing

High throughput sequencing was conducted by the IMB Sequencing Facility (Institute of Molecular Bioscience, UQ, Australia). Libraries were prepared using TruSeq Stranded mRNA Library Preparation Kit (Illumina), following the manufacturer’s instructions, with 1.0 µg RNA input, a fragmentation time of 2 min, and 15 cycles of amplification. Libraries were sequenced on an Illumina NextSeq 500, using a 2x150bp High Output V2 kit. The transcriptome described in this paper was one of several species that were multiplexed together as part of a single sequencing run. Throughout our workflow GNU parallel 20170822 was used to run similar tasks in simultaneously, CD-HIT 4.8.1 was used to remove duplicate sequences, and Seqtk 1.2 was used to obtain subsets of sequence files [138, 139, 193, 194]. Also as a result of multiplexing, we experienced the common problem of low, but meaningful amounts of cross contamination between our multiplexed samples [195]. We attempted to mitigate this issue at several points in our methodology.

3.5.4 Assembly

Illumina reads that were likely to be cross contamination between multiplexed samples were removed from our read files by identifying 57 nucleotide k-mers in our focal read set that were present in another 3.5. MATERIALS AND METHODS 39 read set from the same lane at a 1000-fold or higher level. Reads with 25% or more of their sequence represented by such k-mers were filtered from the data set. This was accomplished using Jellyfish 2.2.6 [127] and K-mer Analysis Toolkit (KAT) 2.3.4 [128]. We then removed adaptors and low-quality bases from the reads and removed any reads shorter than 75 base pairs using Trim Galore version 0.4.3 [129]. We then used PEAR 0.9.10 [130] to combine pairs of reads whose ends overlapped into one, longer, merged read. We then carried out several independent de novo assemblies of these reads using the programs Extender version 1.04 [126], Trinity version 2.4.0 [131], and SOAPdenovo version 2.04 [132]. SOAPdenovo was run repeatedly with k-mer sizes of 31, 75, 97, and 127.

3.5.5 Annotation

The de novo assemblies were concatenated and TransDecoder 5.2.0 was used to predict open reading frames within the contigs [196]. We removed any ORFs that did not include both a start and stop codon. The remaining ORFs were then compared against reference toxin sequences obtained from UniProt using standalone BLAST 2.6.0 [133,134,197]. The resulting ORFs with similarity to known toxins were further quality controlled by manual inspection and BLAST searches against the Reptilia subsection of the NCBI Nucleotide database [133,197,198]. The Burrows-Wheeler Aligner (BWA) 0.7.16a and SAMtools 1.5 were used to align the original reads from each species to the total list of annotated ORFs from every assembly [135,199]. These results were visualized using Tablet 1.15 to screen for signs of chimerical assembly [200]. Those that showed sharp discontinuities in coverage maps across all species were removed from further analysis. A combination of read coverage, assembly quality, and BLAST search results were used to confirm the species of origin for each ORF. Given the evolutionary distance between the species we sequenced and the rate at which toxin genes mutate, it is extremely unlikely that any two of these species would express identical toxins. Because of this, we interpreted reads from multiple species aligning to a single contig as instances of contamination and tentatively assign the contig to the species with significantly higher expression levels. This assignation can be further reinforced if our BLAST results indicate that the sequence is question is more similar to the high-expression taxon than the lower one. The distribution of reads mapped to the sequence can also be informative if the pattern matches the norm for that toxin family in one species but not the others. For most sequences, all three indicators were unambiguous and in agreement while in a small minority of cases two other indicators could be used to decide between somewhat equivocal results of a third. No sequences that had made it to this point of the quality control process could not be confidently assigned using these methods. Once each ORF was properly assigned to its species of origin, similar isoforms were clustered together using a 99% similarity threshold. Expression levels were measured for each contig by using BWA and SAMtools to map the reads from the species of origin to these final contigs. Read levels were then normalized according to length and overall number of reads using the Transcripts Per Million (TPM) formula in an attempt to correct for size bias and make the data more easily comparable to other transcriptomes [201]. CHAPTER 3. ELECTRIC BLUE: TRANSCRIPTOMICS, VENOM COMPOSITION, AND TOXIN EVOLUTION IN THE LONG-GLANDED CORAL SNAKE SPECIES CALLIOPHIS BIVIRGATUS AND C. 40 INTESTINALIS

3.5.6 Phylogenetic Reconstruction

Protein sequences for major toxin families (3FTx, Kunitz, and PLA2) were downloaded from the UniProt database [134]; protein clustering and phylogenetic analyses were used to select a sub- set of sequences that represent the diversity of each family. These were then combined with the translated sequences from our C. bivirgatus transcriptome. The sequences were aligned using a com- bination of manual alignment of the conserved cysteine positions and alignment using the MUltiple Sequence Comparison by Log-Expectation (MUSCLE) algorithm implemented in AliView 1.18 for the blocks of sequence in between these sites [142, 143]. We reconstructed the phylogeny of these sequences using MrBayes 3.2 for 15,000,000 generations and 1,000,000 generations of burnin with lset rates=invgamma (allows rate to vary with some sites invariant and other drawn from a γ distribution) and prset aamodelpr=mixed (allows MrBayes to generate an appropriate amino acid substitution model by sampling from 10 predefined models) [144]. The runs were stopped when convergence values reached 0.01.

3.5.7 Similarity Network

An all-vs-all Basic Local Alignment Search Tool (BLAST) search was conducted on the same dataset of protein sequences as was used for the phylogeny with -outfmt "10 qacc sacc qcovs evalue" [133]. The results of this search were filtered using a custom R script (see Appendix A) to remove self-to-self results, collapse bidirectional results into one entry, and create a similarity score −9 defined as −log10(e − value) for each entry. Edges with coverage < 70% or e − value > 1 ⇥ 10 were excluded from the analysis and the network was created in Cytoscape 3.5.1 using the Prefuse Force Directed Layout on the similarity scores [145].

3.5.8 Protein Clustering

Clustering was carried out using CD-HIT 4.8.1 with options -c 0.45 -n 2 -d 0 -g 1 -sc 1 -sf 1 [138, 139]. This sets the similarity threshold of the clusters to 45% and sorts the clusters by the number of sequences they contain.

3.5.9 Tests for Selection

Based on our phylogenetic analyses, we identified distinct clades of C. bivirgatus 3FTx. Coding DNA sequences for these toxins were trimmed to only include those codons which translate to the mature protein, translated, aligned, and reverse translated using AliView and the MUSCLE algorithm [142, 143]. Phylogenetic trees for each clade were generated from the resulting codon alignments using the same methods as described above. These tree topologies were used for all subsequent analyses. We used several of the tests for selection implemented in HyPhy version 2.220150316beta due to their different emphases [147]. The AnalyzeCodonData analysis generates overall ω values for 3.5. MATERIALS AND METHODS 41 an alignment while the Fast Unconstrained Bayesian AppRoximation (FUBAR) method gauges the strength of consistent positive or negative selection on individual amino acids [148]. In contrast, the Mixed Effects Model of Evolution (MEME) method identifies individual sites that were subject to episodes of positive selection in the past [149].

3.5.10 Protein Modelling

Custom models for each clade of 3FTx were generated by inputting representative sequences to the Phyre2 webserver using the Intensive option [150]. Alignments of each clade were trimmed to match these structures and attribute files were created from FUBAR and MEME results. Conservation scores were calculated using the default settings of AL2CO [151]. The structures were rendered and colored according to these attributes in UCSF Chimera version 1.10.2 [152].

3.5.11 HPLC

Reversed phase high pressure liquid chromatography was used to fractionate crude venoms by the relative hydrophobicity of the toxins present. 2.5 - 3 mg of each venom was lyophilised before being resuspended in 1.5 ml of 3% of buffer B (90% ACN/0.043% trifluoracetic acid). Venom samples were then run through a Shimadzu RP HPLC reader. An acetonitrile gradient from 3% to 80% over 60 mins was applied. Fractions were automatically collected in every minute at a rate of 5 ml/minute. The protein concentration of these fractions was measured using a Nanodrop 2000, spectrophotometer (Thermo Scientific).

3.5.12 MS/MS Sample Preparation

20 µg of protein from selected HPLC fractions were dried using a lyophilizer and resuspended in 20 µL 8 M urea. To this we added 10 µL of 15 mM dithiothreitol (DTT) before incubating for 30 min at 56 ◦C. Once the samples had cooled to room temperature we added 10 µL of 15 mM iodoacetamide (IAA) and incubated for 30 min in the dark before adding another 10 µL of 15 mM DTT. To begin enzymatic digestion, the samples had 50 µL of 50 mM ammonium bicarbonate (ABC) and 500 ng/ of trypsin in dilute hydrochloric acid while sitting chilled in ice. An overnight incubation allowed the trypsin to digest the proteins in the samples. The mixture was then dried in a lyophilizer. The dried samples were resuspended in 10 µL 2.5% acetonitrile (ACN)/1% formic acid (FA). Pep- tides from the samples were separated and eluted via ZipTip%R (MilliPore) procedure: tips moistened with 10 µL 100% ACN repeated 3 times, tips equilibrated with 10 µL 5% ACN/0.1% FA repeated 3 times, sample loaded into tip pipetting up and down 8 times, tip washed in 10 µL 5% ACN/0.1% FA repeated 3 times, and sample is then eluted with 10 µL of 80% ACN/0.1% TFA into a new tube. The final volume of each sample was made up to 20 µL with 5% ACN/0.1% FA. CHAPTER 3. ELECTRIC BLUE: TRANSCRIPTOMICS, VENOM COMPOSITION, AND TOXIN EVOLUTION IN THE LONG-GLANDED CORAL SNAKE SPECIES CALLIOPHIS BIVIRGATUS AND C. 42 INTESTINALIS

3.5.13 MS/MS

Samples were separated using reversed-phase chromatography on a Shimadzu Prominence nanoLC system. Using a flow rate of 30 µL/min, samples were desalted on an Agilent C18 trap (0.3 x 5 mm, 5 µm) for 3 min, followed by separation on a Vydac Everest C18 (300 A, 5 µm, 150 mm x 150 µm) column at a flow rate of 1 µL/min. A gradient of 10-60% buffer B over 30 min where buffer A = 1 % ACN / 0.1% FA and buffer B = 80% ACN / 0.1% FA was used to separate peptides. Eluted peptides were directly analysed on a TripleTof 5600 instrument (ABSciex) using a Nanospray III interface. Gas and voltage settings were adjusted as required. MS TOF scan across m/z 350-1800 was performed for 0.5 sec followed by information dependent acquisition of up to 20 peptides across m/z 40-1800 (0.05 sec per spectra). Data was searched in ProteinPilot 5.0.2 using a custom database containing all contig isoforms assigned to Calliophis bivirgatus from our transcriptomics protocol before the clustering step, all reviewed elapid sequences from UniProt, and a database of common proteomics contaminants [134, 202].

3.5.14 NaV1.4 Assays

A FLuorescent Imaging Plate Reader (FLIPR) Tetra High-Throughput Cellular Screening System (Molecular Devices, Sunnyvale, CA, USA) was used to measure the real-time fluorescence responses (excitation, 470–495 nm; emission, 515–575 nm) of SH-SY5Y human neuroblastoma cells loaded with Ca2+ sensitive dye. These cells form a strong Ca2+ gradient across the membrane that can be altered even though depolarization of the in these cells is largely a result of Na+ ion flow [203,204]. To screen for NaV1.4 agonists, we incubated the cells with 0.1 µM tetrodotoxin (TTx) to selectively block the activity of NaV1.4 channels [205]; to screen for NaV1.4 antagonists, we added 20 µM veratridine as the second addition in our FLIPR assays which stimulates NaV1.4 activity [206, 207]. Statistical analyses of the data and the creation of graphs were carried out in the RStudio 1.1.456 implementation of R 3.2.3 [208, 209]. Chapter 4

Ancient diversification of three-finger toxins in Micrurus coral snakes

Dashevsky D and Fry BG. “Ancient Diversification of Three-Finger Toxins in Micrurus Coral Snakes”. Journal of Molecular Evolution 86.1: 58–67, (2018).

Author Contribution Conception and design (60%) Collate data (100%) Daniel Dashevsky Analysis(100%) (candidate) Writing (100%) Editing (60%) Conception and design (40%) Bryan Fry Editing (40%)

4.1 Abstract

Coral snakes, most notably the genus Micrurus, are the only terrestrial elapid snakes in the Ameri- cas. Elapid venoms are generally known for their potent neurotoxicity which is usually caused by three-finger toxin (3FTx) proteins. These toxins can have a wide array of functions that have been characterized from the venom of other elapids. We examined publicly available sequences from Micrurus 3FTx to show that they belong to eight monophyletic clades that diverged as deep in the 3FTx phylogenetic tree as the other clades with characterized functions. Functional residues from previously characterized clades of 3FTx are not well conserved in most of the Micrurus toxin clades. We also analyzed the patterns of selection on these toxins and find that they have been diversifying at a different rates, with some having undergone extreme diversifying selection. This suggests that Micrurus 3FTx may contain a previously underappreciated functional diversity that has implications for the clinical outcomes of bite victims, the evolution and ecology of the genus, as well as the potential for biodiscovery efforts focusing on these toxins. 43 CHAPTER 4. ANCIENT DIVERSIFICATION OF THREE-FINGER TOXINS IN MICRURUS CORAL 44 SNAKES 4.2 Introduction

Coral snakes are a clade of elapid snakes that are known for their striking colors and patterns as well as their life-threatening bites. After the genus Calliophis, the coral snakes are the second-most basal branch of the elapid family [3, 155]. The clade originated in Asia and are currently represented there by the genus Sinomicrurus (five species), but migrated across the Bering land bridge to the Americas roughly 30 million years ago [59,154,155]. Once in the Americas coral snakes divided into the genera Micruroides (one species) and Micrurus (81 species, including the four species often placed in the genus Leptomicrurus) which rapidly diversified and can be found from the southern United States to central Argentina [12, 59,182]. Due to a combination of their small size, bright colors, and secretive habits, coral snakes are responsible for relatively few bites compared to other venomous snakes in the same area [184,210], but they nonetheless claim lives throughout their entire range [211,212]. Like those of many elapids, bites from coral snakes are often dangerously neurotoxic [184,185,211,213]. Analysis of various coral snake venoms has recently shown that many species produce more phospholipase A2 (PLA2) toxins than 3FTx [214]. The enzymatic activities of Micrurus venoms vary widely, but for most species, the role each toxin family and the specific proteins within them plays remains largely unknown [96]. In this study we examine the diversity and evolutionary history of known 3FTx from Micrurus.A better understanding of the molecular evolution of these toxins will be useful for those studying the ecology and medical significance of these snakes as well as providing guidance for any biodiscovery efforts focused on these venoms. Recent discoveries like that of calliotoxin, a 3FTx from Calliophis bivirgatus that is the first vertebrate toxin known to activate voltage-gated sodium ion channels illustrate the potential of this toxin family [165]. Examining the diversity of 3FTx in Micrurus may allow future studies to make similar discoveries and the location of the coral snakes near the base of the elapids may shed light on the impact that the evolution of eight-cysteine 3FTx had on the radiation of the elapid snakes.

4.3 Results

Our phylogenetic analysis of eight-cysteine 3FTx from across the elapids (Figure 4.1) show that those produced by Micrurus belong to eight distinct monophyletic clades (counting those with at least 5 sequences). These clades diverge from each other relatively early in the tree both in terms of branch length and overall topology. Many of the Micrurus clades are phylogenetically closer to sequences from other species than they are to other Micrurus clades. Those eight-cysteine Micrurus sequences that have been previously characterized are marked in Figure 4.1. The only reported activity for any of these sequences is α-neurotoxicity. Note that of the studies referenced, only Rey-Suarez´ et al. tested whether the toxin in question had any activity beyond nAChR antagonism [217]. Carbajal-Saucedo et al. and Olamendi-Portugal et al. only investigated nAChR antagonistic activity while Moreira et al. relied on suppression of electrically stimulated 4.3. RESULTS 45

5 9 Micrurus_tener_A0A194AT81 Micrurus_fulvius_U3FVG5 Micrurus_tener_A0A194APB1 9 4 Micrurus_fulvius_U3F5B5 Micrurus_fulvius_U3FVG7 Micrurus_fulvius_U3FAC0 8 6 Micrurus_fulvius_U3F5B1 Micrurus_diastema_A0A0H4BKJ0 9 8 Micrurus_browni_A0A0H4J516 9 3 Micrurus_tener_A0A194ARE1 6 6 Micrurus_fulvius_U3FYQ0 Micrurus_fulvius_U3EPI1 5 1 100 Micrurus_diastema_A0A0H4BLZ2 100 Micrurus_browni_A0A0H4BJD8 100 Micrurus_diastema_A0A0H4BFQ4 Micrurus_laticollaris_A0A0H4BJD1 Micrurus_laticollaris_A0A0H4BLY9 Clade A 100 Micrurus_diastema_A0A0H4BJD4 Micrurus_tener_A0A0H4J521 100 Micrurus_fulvius_U3EPL9 Micrurus_fulvius_U3FYQ7 100 Micrurus_fulvius_U3FVI0 Micrurus_fulvius_U3FYR0 5 9 9 7 6 4 Micrurus_tener_A0A194ARC5 9 4 Micrurus_fulvius_U3FYQ5 Micrurus_fulvius_U3FVI5 8 6 7 7 Micrurus_corallinus_C6JUP5 Micrurus_tener_A0A194ARJ0 6 2 Micrurus_tener_A0A194ASE3 6 7 Micrurus_tener_A0A194ARE8 100 Micrurus_browni_A0A0H4IS74 Micrurus_corallinus_C6JUP4 Micrurus_altirostris_F5CPE6 9 0 Micrurus_fulvius_A0A0F7YZP6 Micrurus_fulvius_U3FAE1 Micrurus_tener_A0A194ATC0 Micrurus_tener_A0A194ARJ5 9 3 Micrurus_tener_A0A194ASE5 Micrurus_tener_A0A194APG5 Micrurus_tener_A0A194APH0 Micrurus_tener_A0A194ATC3 6 6 Micrurus_corallinus_C6JUP1 9 0 Micrurus_tener_A0A194ARC1 Micrurus_tener_A0A194ATB5 9 3 Micrurus_tener_A0A194ARK4 Clade B 8 9 Micrurus_tener_A0A194APH5 100 Micrurus_frontalis_P86421 5 5 9 0 Micrurus_altirostris_F5CPE0 9 4 Micrurus_mipartitus_B3EWF8 9 2 Micrurus_altirostris_F5CPE2 9 8 Micrurus_laticollaris_A0A0H4BKI7 Micrurus_surinamensis_P86094 100 Micrurus_laticollaris_K9MCX0 Micrurus_laticollaris_A0A0H4BFP9 100 Naja_annulifera_P25677 9 2 Hemachatus_haemachatus_P01402 9 9 Dendroaspis_polylepis_P0DKR6 ASIC1 Blocker 100 Ophiophagus_hannah_Q2VBP1 5 2 Ophiophagus_hannah_P83302 Bungarus_fasciatus_P14534 Pseudonaja_textilis_Q9W7K0 Micrurus_fulvius_U3FYQ9 Type III α-ntx 100 Micrurus_tener_A0A0H4IS80 7 5 Micrurus_tener_A0A194ASF5 8 1 Clade C 9 9 Micrurus_browni_A0A0H4BLZ7 Micrurus_diastema_A0A0H4BEF7 100 Bungarus_candidus_P83346 Bungarus_multicinctus_Q9YGI8 100 Micrurus_altirostris_F5CPD1 100 Micrurus_corallinus_C6JUP0 7 0 Micrurus_tener_A0A0H4IUP1 6 2 8 0 Micrurus_fulvius_U3FVH8 6 6 Micrurus_tener_A0A194ATD7 6 1 Micrurus_browni_A0A0H4BEG5 Clade D 8 1 Micrurus_tener_A0A194API9 Micrurus_tener_A0A194ARL8 Micrurus_tener_A0A194ARF2 100 Micrurus_tener_A0A194ARB6 Naja_atra_Q9W717 7 5 10Dendroaspis_angusticeps_P176960 Dendroaspis_angusticeps_P01409 Synergistic Toxins 9 8 9 5 100 Dendroaspis_angusticeps_Q9PSN1 6 6 Dendroaspis_angusticeps_P86419 7 8 Dendroaspis_angusticeps_P85092 8 7 Dendroaspis_polylepis_P80495 Aminergic ntx Naja_siamensis_P82462 100 Naja_siamensis_P82463 Micrurus_altirostris_F5CPE5 9 2 100 Micrurus_fulvius_U3EPM9 Micrurus_tener_A0A194APF0 100 Hemachatus_haemachatus_P25676 Naja_haje_P01415 8 0 Micrurus_corallinus_C6JUP3 5 6 100 Bungarus_multicinctus_P79688 100 Bungarus_multicinctus_Q9YGI0 9 9 Naja_kaouthia_P82464 Bungarus_multicinctus_Q9W727 Micrurus_tener_A0A194ASE0 8 8 Micrurus_tener_A0A194ATB0 Micrurus_nigrocinctus_P80548 7 2 Micrurus_diastema_A0A0G3YJD1 Clade E 9 0 Micrurus_browni_A0A0H4J512 Micrurus_browni_A0A0H4IVP7 Micrurus_altirostris_F5CPE3 5 8 Micrurus_laticollaris_A0A0G3YLL2 9 9 Micrurus_fulvius_U3F5E9 7 6 Micrurus_tener_A0A194ASE2 Clade F 6 0 100 Micrurus_corallinus_Q9PUB7 Micrurus_nigrocinctus_JC7187 Micrurus_surinamensis_P86097 8 6 100 Micrurus_altirostris_F5CPD2 Micrurus_frontalis_P86422 9 8 Clade G 100 Micrurus_altirostris_F5CPD9 Micrurus_altirostris_F5CPD6 Micrurus_altirostris_F5CPD5 8 4 Micrurus_pyrrhocryptus_P0CAR1 7 5 Micrurus_frontalis_P86420 Micrurus_surinamensis_P86095 Dendroaspis_angusticeps_P18329 5 7 Dendroaspis_jamesoni_P01406 5 4 100 Dendroaspis_angusticeps_P0C1Y9 Dendroaspis_polylepis_P25681 AChE Inhibitors 100 Dendroaspis_polylepis_P22947 2+ Dendroaspis_polylepis_P01414 L-type Ca ntx 100 Dendroaspis_angusticeps_P81946 Dendroaspis_jamesoni_P28375 Platelet Inhibitors 100 Laticauda_laticaudata_P10459 Laticauda_semifasciata_P60775 Micrurus_altirostris_F5CPD8 Type I α-ntx Micrurus_laticollaris_K9MCH1 Dendroaspis_angusticeps_P60237 Naja_kaouthia_P14613 Type B Muscarinic 100 Micrurus_tener_A0A194APG0 Micrurus_fulvius_A0A0F7Z3S3 8 9 6 2 Micrurus_browni_A0A0H4IRA8 Micrurus_browni_A0A0H4IUN8 100 Micrurus_fulvius_A0A0F7YZ34 8 9 Micrurus_fulvius_U3EPK7 6 5 Micrurus_fulvius_A0A0F7YYX2 Micrurus_clarki_C0HK04 100 Micrurus_tener_A0A194ARK8 100 6 1 Micrurus_tener_A0A194ATD1 9 6 Micrurus_diastema_A0A0H4BKJ5 Micrurus_tener_A0A194ARL3 Micrurus_altirostris_F5CPD3 Micrurus_fulvius_U3FAF0 Micrurus_fulvius_U3F5D3 Clade H 8 7 Micrurus_fulvius_U3FAE7 Micrurus_fulvius_A0A0F7Z3S7 100 Micrurus_fulvius_U3EPM4 Micrurus_tener_A0A0H4IVQ6 Micrurus_diastema_A0A0H4BEG1 8 7 9 3 Micrurus_corallinus_C6JUP2 Micrurus_tener_A0A194APE1 Micrurus_tener_A0A194ARH7 7 8 Micrurus_tener_A0A0H4IRB3 8 7 Micrurus_tener_A0A194ATA3 Micrurus_tener_A0A194ATA6 6 3 Micrurus_tener_A0A194ASD1 Micrurus_tener_A0A194ARB2 Hemachatus_haemachatus_P24778 9 9 Naja_atra_Q9W716 7 1 Naja_atra_Q91996 Aspidelaps_scutatus_P19004 7 2 8 2 8 5 Ophiophagus_hannah_Q69CK0 Aspidelaps_scutatus_P19003 Cytotoxins 8 9 Naja_mossambica_P01452 9 4 Naja_mossambica_P01467 8 7 Hemachatus_haemachatus_P01471 Hemachatus_haemachatus_P24777 Bungarus_multicinctus_P43445

0.2

Figure 4.1: Phylogenetic tree of publicly available 3FTx sequences. Clades with previously character- ized activities and clades of Micrurus sequences are highlighted. Circle (•) indicates Micrurus toxins with previously observed α-neurotoxic activity [215–218]. Scale bar represents an average of 0.2 substitutions per site. CHAPTER 4. ANCIENT DIVERSIFICATION OF THREE-FINGER TOXINS IN MICRURUS CORAL 46 SNAKES

Taxon ASIC1 Blocker H H A H H H A H H Non-Micrurus H H A H A A HH H A A H H A Micrurus A A H A A A A A H A H B H H H H B HH H B B B H A B B H B AA Cluster B H A A B A A B Type III -ntx A A BB B Cytotoxin A B A A A 0 B A A A A Cytotoxin B B CytCotytoxiotoxinn B B CytotoxiCytnotoxin 1 Synergistic Toxin Cytotoxin Cytotoxin D Cytotoxin D D 2 B D DD Aminergic Toxin D D Cytotoxin D PlatelePlatelett inhibi Inhibitotor r F 3 Aminergic Toxin Aminergic ToxiAminergicn Toxin C Aminergic Toxin L-type Ca2+ ntx C C Aminergic Toxin L-type Ca2+ ntx 4 C FF F 5 C F E Syngergistic Toxin E 6 E F AChE Inhibitor E G AChE Inhibitor E 7 Type I -nG tx E Type B Muscarinic G Other G G

Figure 4.2: Protein similarity network of publicly available 3FTx sequences based on BLAST e-values. Colors correspond to CD-HIT clusters containing at least five sequences and labels correspond to those found in Figure 4.1.

neuromuscular response [215,216,218]. The distribution of α-neurotoxicity suggests that phylogenetic position alone is not enough to assign putative function to Micrurus 3FTx sequences. The clades from our phylogenetic analysis are largely corroborated by the independent similarity network and protein clustering analyses (see Figure 4.2). This gives greater confdence that these are meaningful subdivisions within the 3FTx. In all three analyses many of the groups of Micrurus sequences are more akin to sequences from other elapids, including those with diverse modes of action, than they are to other groups of Micrurus toxins. The sequences of the toxins also suggest that there may be biological activities beyond α- neurotoxicity. Figure 4.3 shows that only Clades E and G have greater than 50% identity at the residues homologous to the functional residues of erabutoxin-a, a well-characterized Type I α-neurotoxin [80]. It must be noted that two of the Micrurus toxins with published α-neurotoxic activity belong to Clade B and only display 19% conservation of these sites [216,217]. This could be due to Clade B’s close relationship to the Type III α-neurotoxins, a preservation of the putative ancestral activity despite these amino acid changes, or convergent evolution in function. There are also clear differences in the rates and patterns of molecular evolution among the four 4.3. RESULTS 47

Figure 4.3: Alignments of Micrurus 3FTx where the functional residues of α-neurotoxic 3FTx are highlighted. Amino acids are colored with AliView default colors: hydrophobic residues in blue, polar in teal and green, and charged residues in red and purple (chemically unique residues like C, H, G, and P are given unique colors). Percent identity at the functional sites: A–4%; B–19%; C–33%; D–48%; E–83%; F–17%; G–67%; H–7%. CHAPTER 4. ANCIENT DIVERSIFICATION OF THREE-FINGER TOXINS IN MICRURUS CORAL 48 SNAKES

Table 4.1: Tests of selection on the various clades of Micrurus 3FTx

Clade ω FUBAR (-)a FUBAR (+)b MEMEc FUBAR&MEMEd A 3.39 0 24 19 16 B 2.16 1 11 10 6 D 1.51 1 2 0 0 H 1.78 2 12 10 6 a Number of codons under negative selection according to FUBAR b Number of codons under positive selection according to FUBAR c Number of codons under episodic diversifying selection according to MEME d Number of codons that fit both citeria b and c of these clades for which sufficient nucleotide sequences were available for analysis (see Table 4.1). Clade A was the highest in terms of the overall ω value (a measure of the strength of selection based on rates of synonymous and non-synonymous mutations, also known as the dN ratio or Ka ratio), the dS Ks number of sites that FUBAR identified as being positively selected, the number of sites that MEME determined had been subject to episodes of diversifying selection, and the number of sites that were called by both FUBAR and MEME. On the other hand, Clade D had a much lower ω value and very few codons were indicated to be under positive selection by either site-by-site algorithm. Figure 4.4 combines these tests for selection with protein structures predicted by the Phyre2 server and provides additional phylogenetic context. It should be noted that each clade is predicted to have a different structure than the others which once again suggests the potential for different activities as well. This reinforced by the tendency for the diversifying selection to be more concentrated on the three loops, or fingers, of the proteins rather than the core. This is in spite of the fact that the eight structural cysteine residues, which are almost perfectly conserved across and within all eight clades, do not appear to be subject to strong negative selection. This somewhat paradoxical result comes from the fact that site-by-site selection algorithms will predict a very low rate of non-synonymous mutation for codons which have little to no synonymous mutations; when these codons also display very low rates of non-synonymous mutations, measures that compare the two—such as ω values or FUBAR’s β − α—will tend to show neutral or weak negative selection.

4.4 Discussion

The pattern of evolution we see in the Micrurus 3FTx, where the amino acids in the core of the protein are subject to purifying selection while positive selection acts strongly on the rest is not a novel finding. This pattern of selection was previously reported and thoroughly discussed for other clades of 3FTx by Sunagar et al. [69]. What is notable are the extremely high overall ω values exhibited by some clades of Micrurus toxins (see Table 4.1). Of all the functional divisions studied by Sunagar et al. [69] the highest reported ω value was 2.59 (belonging to the Type III α-neurotoxins, see Figure 4.4). Margres et al. analyzed all of the Micrurus 3FTx—for which there were nucleotide sequences available at 4.4. DISCUSSION 49

Figure 4.4: Schematic phylogeny of Micrurus 3FTx clades and previously known functional divisions. Branches are colored according to ω values. Protein models show front and back views colored according to FUBAR’s estimated strength of selection (dN-dS, left) and MEME’s significance levels (right).

the time—together and found significant positive selection for over half the sites in their alignment averaging an ω value at those sites of 3.79 [125]. When averaged with the neutral and negatively selected sites from that analysis, the overall ω value for the Micrurus 3FTx was 2.43. This is in strong agreement with our results: the weighted mean of our ω values returns a value of 2.42. That being said, our phylogenetic analysis suggests that a clade-by-clade approach is more appropriate for these toxins and Clade A’s ω value of 3.39 is considerably higher than any other that has been reported yet. For context, the highest reported ω value for a gene from the human male reproductive system (which “have evolved much more rapidly than other types of character”) was 2.89 for Protamine 1 [219, pg. 304]. Many elapids produce 3FTx with a number of different activities and in Figure 4.1 we can see that a given toxin is often more closely related to those from other species (including Micrurus) than they are to toxins with different activity from within the same species’ venom. This suggests that the seeds of this functional diversity were sown before the coral snakes had diverged from the more derived elapids. Our results show that the toxins produced by Micrurus alone show a similar level of evolution (in terms of sequence diversity and diversifying selection) amongst themselves as do the 3FTx produced by all other elapids. When this is considered along with the fact that only two of the eight clades have been shown to have greater than 50% conservation of the functional α-neurotoxic residues, it seems that snakes of the genus Micrurus have evolved an array of 3FTx that are likely to have activities beyond α-neurotoxicity. It should be noted that it is still possible that the ancestral eight-cysteine 3FTx was α-neurotoxic and all of the Micrurus toxins have retained this activity in the CHAPTER 4. ANCIENT DIVERSIFICATION OF THREE-FINGER TOXINS IN MICRURUS CORAL 50 SNAKES face of rapid evolution at the interacting sites. This may seem unlikely, but those Micrurus sequences which have been shown to exhibit α-neurotoxic activity are not particularly closely related to each other (see Figure 1). Most of these α-neurotoxins (P86095, P86422, K9MCH1, P86097) conserve all but one or two of the previously characterized functional residues, but two of the toxins (B3EWF8, P86421) do not conserve any of them (see Figure 3). The discovery of two plesiotypic ten-cysteine 3FTx from Micrurus mipartitus that modulate GABAA activity, however, shows that novel activities are certainly possible within this particular toxin family and genus [220]. Functional testing of specific toxins must be performed across the full diversity of Micrurus 3FTx to settle the matter conclusively.

To date most studies of Micrurus 3FTx have not pursued the activities of individual toxins and those that have have specifically screened for the standard α-neurotoxicity [221–223]. This makes a certain amount of sense given that the paralysis typical of α-neurotoxicity is the most significant clinical symptom of Micrurus bites [211, for example]. Unfortunately these methods will never discover any toxins which exhibit activities beyond α-neurotoxicity. Since Micrurus mostly eat non-mammalian prey, an anthropocentric approach to studying their venom may overlook much of the relevant evolutionary history. In addition, it is these potential novel activities that hold the greatest possibilities for developing new scientific tools or laboratory techniques.

These results may even be relevant from a clinical perspective. If coral snake venoms do in fact contain 3FTx with a diverse range of activities then simple regulatory changes could easily lead to major differences in the overall effects of the venoms on short time scales or between closely related populations. Even if this did not lead to a significant change in the function of the venom it could affect the cross-reactivity of antivenom against species that were not included in its production. Along with shifts between 3FTx and PLA2 dominated venoms, shifts within the 3FTx makeup could help explain the somewhat patchy cross-reactivity of Micrurus antivenoms [214, 224].

In conclusion, most of the 3FTx produced in Micrurus venoms can be grouped into eight separate clades, which exhibit differences in their relationships to characterized 3FTx from other taxa, predicted structure, and rate of evolution. It was well known that Micrurus venoms contained a diversity of 3FTx, but the existence of these distinct clades, their affinity to better characterized toxins, and the difference in predicted structure was not known before now because other studies of Micrurus 3FTx had not included representative sequences from other genera to compare against. This variation in the sequence and structure of these toxins, especially in conjunction with evidence of diversifying selection, suggests that some of these clades may well have undergone neofunctionalization. Because of this we recommend that researchers use a wider variety of in vitro assays when characterizing the function of Micrurus 3FTx. Hopefully this will allow for the discovery of toxins with previously unknown functions within these venoms which would benefit our understanding of the evolutionary history of the genus as well as well as increasing their potential for biodiscovery. 4.5. MATERIALS AND METHODS 51

Table 4.2: Composition of the eight-cysteine 3FTx dataset

Micrurus sequences 124 Species 13 M. altirostris 12 M. browni 9 M. clarki 1 M. corallinus 7 M. diastema 8 M. frontalis 3 M. fulvius 28 M. laticollaris 7 M. mipartitus 1 M. nigrocinctus 2 M. pyrrhocryptus 1 M. surinamensis 3 M. tener 42 Representative sequences 47 Genera 8 Aspidelaps 2 Bungarus 7 Dendroaspis 16 Hemachatus 5 Laticauda 2 Naja 11 Ophiophagus 3 Pseudonaja 1

4.5 Materials and Methods

4.5.1 Phylogeny

Protein sequences for Micrurus 3FTx and representative 3FTx with known functions were obtained from the UniProt database [134]. This study was limited to eight-cysteine 3FTx because they account for the vast majority of reported sequences and analyses that included the nine and ten-cysteine forms failed to converge properly [69]. Only the mature peptide sequences were aligned and used as input due to the fact that the signal peptide was not available for all sequences. Details of the dataset can be found in Table 4.2. Many diverse, but monophyletic clades of 3FTx (such as those produced by Australian elapids) are represented by a single sequence. We reconstructed the phylogeny of these sequences using MrBayes 3.2 for 15,000,000 generations and 1,000,000 generations of burnin with lset rates=invgamma (allows rate to vary with some sites invariant and other drawn from a γ distribution) and prset aamodelpr=mixed (allows MrBayes to generate an appropriate amino acid substitution model by sampling from 10 predefined models) [144]. The run was stopped when convergence values fell below 0.01. CHAPTER 4. ANCIENT DIVERSIFICATION OF THREE-FINGER TOXINS IN MICRURUS CORAL 52 SNAKES

4.5.2 Similarity Network

An all-vs-all BLAST search was conducted on the same dataset of protein sequences as was used for the phylogeny with -outfmt "10 qacc sacc qcovs evalue" [133]. The results of this search were filtered using a custom R script (see Appendix A) to remove self-to-self results, collapse bidirectional

results into one entry, and create a similarity score defined as −log10e-value for each entry. Edges with coverage < 70% or e-value> 1 ⇥ 10−10 were excluded from the analysis and the network was created in Cytoscape 3.5.1 using the Prefuse Force Directed OpenCL Layout on the similarity scores [145].

4.5.3 Protein Clustering

Clustering was carried out using the CD-HIT 4.7 algorithm with options -c 0.4 -n 2 -d 0 -sc 1 -g 1 [138,139]. This sets the similarity threshold of the clusters to 40% and sorts the clusters by the number of sequences they contain.

4.5.4 Functional Residues

The amino acid sequence of erabutoxin-a was aligned with each clade of Micrurus 3FTx in AliView 1.18 using the MUSCLE algorithm [142, 143]. All residues besides the structural cysteines and those identified by Antil et al. as functional residues were converted to gap characters for legibility. All resulting gap-only columns were then deleted [80].

4.5.5 Tests for Selection

Coding DNA sequences for all possible Micrurus 3FTx were compiled from GenBank [146]. The sequences were trimmed to only include those codons which translate to the mature protein, translated, aligned, and reverse translated using AliView and the MUSCLE algorithm [142, 143]. Phylogenetic trees for each clade were generated from the resulting codon alignments using similar methods as described above. This tree topology was used for all subsequent analyses. We used several of the tests for selection implemented in HyPhy version 2.220150316beta due to their different emphases [147]. The AnalyzeCodonData analysis generates overall ω values for an alignment while the FUBAR method gauges the strength of consistent positive or negative selection on individual amino acids [148]. In contrast, the MEME method identifies individual sites that were subject to episodes of diversifying selection in the past [149].

4.5.6 Protein Modeling

Custom models for each clade of Micrurus 3FTx were generated by inputing representative sequences (Clade A–U3EPI1; Clade B–A0A194APH0; CladeD–A0A0H4BEG5; CladeH–A0A194ATA3) to the Phyre2 webserver using the Intensive option [150]. The details of the queries and results can be found in Table 4.3. 4.5. MATERIALS AND METHODS 53

Table 4.3: Phyre2 results for the various clades of Micrurus 3FTx

Clade Query PDB ID %ID Organism Resolution A U3EPI1 1LSI 50 Laticauda semifasciata NMR B A0A194APH0 4RUD 97 Micrurus fulvius 1.95A˚ D A0A0H4BEG5 2VLW 42 Dendroaspis angusticeps 1.39A˚ H A0A194ATA3 3PLC 47 Ophiophagus hannah 2.41A˚

Alignments of each clade were trimmed to match these structures and attribute files were created from FUBAR and MEME results. Conservation scores were calculated using the default settings of AL2CO [151]. The structures were rendered and colored according to these attributes in UCSF Chimera version 1.10.2 [152].

Chapter 5

Red-on-yellow queen: the binding of Micrurus venoms to a range of nicotinic receptor mimics reveal patterns of prey specificity and toxin resistance

5.1 Abstract

Many elapid venoms rely primarily on three-finger toxins (3FTx) that block the α1 subunit of nicotinic acetylcholine receptors (nAChRs) (α-neurotoxins) to immobilize their prey. The best studied of these is α-bungarotoxin from Bungarus multicinctus, a long-chain neurotoxin which get their name from insertions in the Loop II region which increase the overall length of the toxin and introduce a new disulfide bridge. Short-chain neurotoxins without these insertions are perhaps even more common and have somewhat different pharmacology from the long-chain neurotoxins. We used a bio-layer interferometry technique to measure the interaction between Micrurus venoms—which are devoid of long-chain neurotoxins—and mimotopes designed to resemble the orthosteric binding region of the α1-nAChR subunit from a range of taxa. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than patterns of venom composition. Venoms from the long-tailed clade of Micrurus tends to have greater effect than their short-tailed relatives. This may be related to the results of previous research which indicate that long-tailed Micrurus 3FTx are particularly diverse and subject to very strong positive selection. We also observed variation in the potency of the venoms depending on the target they were binding to. Rather than a pattern of prey-specific toxicity, we actually found that mimotopes modelled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is due, in part, to a characteristic tryptophan!serine mutation at the beginning of the orthosteric binding region of the nAChR α1 subunit in snake sequences. This resistance may be part of an arms race between elapid snakes and their prey which contributes to the generally high toxicity of venoms from these snakes. 55 CHAPTER 5. RED-ON-YELLOW QUEEN: THE BINDING OF MICRURUS VENOMS TO A RANGE OF NICOTINIC RECEPTOR MIMICS REVEAL PATTERNS OF PREY SPECIFICITY AND TOXIN 56 RESISTANCE 5.2 Introduction

The genus Micrurus is part of the group of elapids known as the ‘true coral snakes’ along with Sinomicrurus and Micruroides. Micrurus is the most speciose genus of elapids and can be found from Argentina all the way to the southern United States of America [35]. Genetic and morphological evidence shows a clear distinction between two groups within the genus, short-tailed and long-tailed Micrurus [35]. In general the short-tailed species are found in South America and possess a triadal color pattern (e.g. red-black-white-black-white-black-red). The long-tailed species are somewhat more variable with some species found in South America, but most in Central and North America; they include species with bicolored, monadal (e.g. red-yellow-black-yellow-red), and triadal patterns [35]. The venom composition of the two groups also differs somewhat in that the venom from long-tailed species are usually (though not exclusively) dominated by 3FTx while short-tailed species produce venoms that range from being almost entirely composed of 3FTx to mostly PLA2 [214].

One of the most widespread functions amongst the 3FTx is referred to as α-neurotoxicity because it inhibits the activity of the α subunit of the nicotinic acetylcholine receptor (nAChR). The nAChR is a pentameric transmembrane protein that play an important role in synaptic transmission, especially at the neuromuscular junction [225]. A toxin from the venom of Bungarus multicinctus was found to inhibit the function of these channels and was named α-bungarotoxin (coincidentally due to the original order of discovery rather than its site of action) [226]. After this toxin was shown to function by competitively excluding acetylcholine from binding to the nAChR, it and others toxins like it were used by researchers to probe the structure and function of nAChR leading to our current understanding of the different subunits [227]—there are currently seventeen known subtypes—and their distribution throughout the nervous system [228]. α-bungarotoxin is an example of what is known as a ‘long-chain neurotoxin’ because it comes from the clade of the 3FTx that have evolved an additional pair of cysteines in the Loop II region that form a novel disulfide bond with each other (Figure 1.2) [91]. On the other hand, ‘short-chain neurotoxins’ only have the standard eight cysteines characteristic of derived elapid 3FTx (Figure 1.2). Long-chain and short-chain neurotoxins are known to compete with each other indicating that they bind to similar locations on the nAChR, yet they exhibit different specificities and kinetics (short-chain toxins bind to and dissociate from the nAChR much quicker) indicating slightly different biochemical mechanisms of action [189].

We use a recently described bio-layer interferometry technique to assess the the α-neurotoxicity of Micrurus venoms [229]. This technique uses mimotopes—small peptides designed to mimic the region of interest of a larger protein—attached to optical biosensors to measure the binding of ligands [230].

In this case, we used mimotopes designed after the orthosteric binding region of the nAChR α1 subunit from a range of different taxa. Past studies have shown that similar mimotopes interact strongly with α-bungarotoxin, but this has not been explicitly demonstrated for short-chain neurotoxins [231–234]. Another study has shown that mimotopes may have potential as a therapeutic treatment to inhibit the α-neurotoxic activity of snake venoms by acting as decoys that keep the toxins from binding to the victim’s actual receptors [235]. 5.2. INTRODUCTION 57

Figure 5.1: Alignment of α1-nAChR orthosteric binding sites used to create mimotopes for this study. Amino acids that differ from the majority consensus are highlighted. Publicly available sequences are annotated with UniProt accession codes (except for the gecko sequence which is only available one GenBank). Other sequences were generated as part of an ongoing collaboration.

The long-chain neurotoxins seem to have originally evolved shortly after the common ancestor of Micrurus and the more derived elapids diverged. 3FTx with the additional cysteines characteristic of long-chain neurotoxins have been published from all lineages of elapids besides Calliophis and the true coral snakes [134]. Therefore our results regarding the α-neurotoxic activity of Micrurus venoms will not be confounded by the difference between long-chain and short-chain neurotoxins. This makes these venoms an ideal test of whether short-chain neurotoxins bind to mimotopes and how they behave on this mimotope assay. No other research has examined the binding of short-chain neurotoxins to

α1 subunit mimotopes specifically and the study demonstrating this method only used venoms that contain both short and long-chain neurotoxins [229]. One of the interesting results of the previous study using this methodology was that venom from the two species studied with specialized diets (Aipysurus laevis and Ophiophagus hannah) showed much greater effects on the mimotopes designed after the receptors of their preferred prey type (fish and snakes, respectively) than those from other taxa [229]. Prey specificity is a widespread phenomenon in snake venoms and these results suggest that our assay should be able to detect if Micrurus venoms have undergone similar adaptation [20, 47,75,76]. However, it must also be considered that there is an opposing evolutionary pressure for the prey of Micrurus to become less susceptible to the toxins of their predators. Resistance and functional immunity to snake α-neurotoxins has been reported from a number of taxa [236]. Mongooses, pigs, and hedgehogs which are known to prey on venomous snakes are almost entirely immune to elapid

α-neurotoxins due to convergent point mutations in the orthosteric binding site of the α1-nAChR subunit and similar mutations can be found in elapids themselves [237–239]. Whether the immunity in elapids evolved in response to self envenomation, intra-guild predation, or both is unclear. Reptile taxa CHAPTER 5. RED-ON-YELLOW QUEEN: THE BINDING OF MICRURUS VENOMS TO A RANGE OF NICOTINIC RECEPTOR MIMICS REVEAL PATTERNS OF PREY SPECIFICITY AND TOXIN 58 RESISTANCE including some lizards and other snakes have also been shown to at least partially resist the toxic effects of both long-chain and short-chain neurotoxins [240]. There have even been some studies addressing the effects of M. nigrocinctus venom on different taxa including one demonstration that cows are less susceptible than horses and another showing a marked difference between two genera of dipsadine snakes that M. nigrocinctus is known to prey on, Geophis (susceptible) and Ninia (resistant) [241,242]. The latter study is particularly interesting because incubating M. nigrocinctus venom with N. maculata serum had a protective effective when injected into mice; this suggests that there is some venom inhibitor in the serum of N. maculata rather than a mutation to their α1-nAChR subunit [242]. Because of these findings we wanted to test the effects of Micrurus venoms on mimotopes designed to resemble the orthosteric binding site of the α1-nAChR from a range of taxa. Figure 5.1 shows an alignment of the amino acid sequences that the mimotopes in this study were based off of. The positions of this alignment will be used throughout this paper rather than a whole gene alignment; for instance we will refer to the characteristic substitution from tryptophan to serine at the first amino acid of this alignment in the snake sequences as a W1S mutation.

5.3 Results

The interaction between venom and mimotope varied depending on the species of origin for both (Figure 5.2). Almost all the venoms we tested elicited less of a response from mimotopes derived from snakes than those from other taxa. However, because of the variability between the different Micrurus venoms, we could not simply compare the means for each mimotope. Instead, we created a factor denoting whether a mimotope was derived from a non-snake taxon (n = 360) or from a snake taxon (n = 180) and used the non-parametric Aligned Rank Transform to simultaneously test the effects of this factor, the venom, and their interaction. Heterogeneity of variance between groups of different sample sizes necessitated the use of this test rather than a more traditional ANOVA. This confirmed that there was significant variation between the efficacy of the venom from different species −16 2 (F14,510 = 155, p < 2 ⇥ 10 ,η = 0.81), that non-snake mimotopes were more easily bound by −16 2 Micrurus venoms than those derived from snakes (F1,510 = 492, p < 2 ⇥ 10 ,η = 0.49), and these −16 2 variables interacted significantly as well (F14,510 = 15, p < 2 ⇥ 10 ,η = 0.29). To test the difference the W1S mutation may have made to the ancestral snake lineage in which it arose, we created a pseudo-ancestral mimotope by modifying the sequence of the most basal snake mimotope (blindsnake) to revert the derived snake serine back to the tryptophan that all other taxa pos- sess at that position and used Aligned Rank Transform to test the effect of pseudo-ancestral mimotope (n = 45) compared to the wild-type (n = 45). This test showed that the normal blindsnake mimotope −10 2 was more resistant to Micrurus venoms (F1,60 = 55, p = 5 ⇥ 10 ,η = 0.48), variation between ven- −16 2 oms from different species of Micrurus remained significant (F14,60 = 42, p < 2 ⇥ 10 ,η = 0.91), −3 2 and the interaction between the two variables was also significant (F14,60 = 3, p = 1⇥10 ,η = 0.41). While these results clearly indicated that the W1S mutation confers partial resistance to the binding of Micrurus toxins, Welch’s two-sample t-test showed that the ratio of the average responses to a given 5.3. RESULTS 59

Figure 5.2: Micrurus neurotoxicity varies greatly depending on the individual venom as well as the target: Phylogeny on the left displays relationships between the venoms tested (short-tailed and long- tailed clades indicated by arrows), while the one on top displays the relationship between organisms from which mimotopes were derived (clade of mimotopes based on snake sequences indicated with arrow). Phylogenies were compiled from several sources [3, 4, 113, 155, 214, 243–245].Cells show the area under the curve (Mean ± Standard Deviation, n = 3) for the binding of each venom to each mimotope. CHAPTER 5. RED-ON-YELLOW QUEEN: THE BINDING OF MICRURUS VENOMS TO A RANGE OF NICOTINIC RECEPTOR MIMICS REVEAL PATTERNS OF PREY SPECIFICITY AND TOXIN 60 RESISTANCE

Figure 5.3: Snake nAChR are consistently less vulnerable to α-neurotoxins, partially due to the W1S mutation: responses of non-snake mimotopes to each Micrurus venom are on average higher than the responses of snake mimotopes to that same venom (indicated by the ratios greater than 1), similarly the pseudo-ancestral blindsnake mimotope without the W1S mutation are more vulnerable than the normal blindsnake mimotope.

venom of non-snake mimotopes to that of snake mimotopes (2.02 ± 0.65) was significantly greater than that the ratio between the pseudo-ancestral blindsnake mimotope and the normal one (1.43±0.40, p = 6 ⇥ 10−3, Figure 5.3).

5.4 Discussion

As we expected, the bio-layer interferometry method worked quite well with our samples of Micrurus venoms and interesting patterns emerged both in terms of the differences within Micrurus and between the targets (Figure 5.2). Since Micrurus venoms do not contain long-chain neurotoxins, this confirms that the assay can be used for short-chain neurotoxins as well. A previous study that investigated the interaction of short-chain neurotoxins and nAChR mimotopes had reported very little binding, but those mimotopes were designed to resemble the orthosteric binding region of the α7 subunit [235]. Short- chain neurotoxins are known to bind almost exclusively to the α1 rather than the α7 subunit [189,246]; because our mimotopes were designed to resemble the α1 subunit, this easily explains why our assay was able to measure the effects of these toxins where previous research had not. This also suggests that decoy peptides that resemble the α1 subunit could have even greater potential as a potential therapeutic tool to help mitigate the neurotoxic symptoms of snakebite than the α7 derived ones that have been 5.4. DISCUSSION 61 studied. When interpreting these results it is important to keep in mind the limitations of these methods. Most importantly, the mimotopes used in this study were designed based on the orthosteric binding region of the α1 nAChR. Given the range of targets that are bound by various 3FTx it is entirely possible that some toxins may interfere with the normal function of the α1 nAChR without binding to this specific site. This has not been explicitly demonstrated, but there is some suggestive evidence—e.g. the different functional residues between short and long-chain neurotoxins [80]—that this may in fact be the case. This could help explain the seemingly paradoxical result of species with known neurotoxic venoms, such as M. surinamensis, which produce virtually no response in our assay. Mimotopes can also fail to properly capture the behavior of whole proteins when the secondary or tertiary structure of that protein leads to residues from different parts of the chain interacting with each other in a biochemically significant fashion through charge, steric interactions, or some other mechanism. However, previous studies have shown that α-neurotoxic 3FTx tend to interact with the orthosteric site in a fairly straightforward manner and that mimotopes can bind to these toxins quite well [231–234]. One of the striking trends in our data was the variability within and between species of Micrurus. The venom with the largest effect on average (and for every individual mimotope) was a sample of M. corallinus, while the other individual of this species that we tested had only the 6th highest average effect. There also seems to be a phylogenetic trend where the long-tailed Micrurus (with the exception of M. tener and M. fulvius, the two North American species) elicited a much greater response than their short-tailed relatives. This is despite the fact that the venom composition for a number of these species is dominated by PLA2s rather than 3FTx [214]. This shift in activity could be related to the large clades of 3FTx from long-tailed Micrurus that display strong signals of positive selection, as discussed in Chapter 4 [71]. Another noteworthy source of variation in our results is the fact that mimotopes derived from animals other than snakes tend to be more sensitive to the venoms we tested than snake-based mimotopes (Figure 5.3). This is precisely the opposite result that we would have expected under a prey-specificity scenario of venom evolution. Instead, we found that a W1S mutation in the ligand binding region of the nAChR α1 subunit confers partial resistance to short-chain neurotoxins The difference between a blindsnake mimotope without this mutation and one with it seems to be smaller than the difference between those from other animals and those from snakes (Figure 5.3). The source of this increased resistance is somewhat enigmatic since there are no other derived mutations found in all and only snake sequences. What is interesting about the resistance conferred by this mutation is that it seems to operate through different biochemical mechanisms than the increased potency of the toxins in the venom from long-tailed species. The long-tailed species have similar ratios of responses between non-snake and snake mimotopes as the short-tailed species do. While 3FTx are a relatively ancient family of snake toxins and have even been reported from henophidian snakes (see Figure 2.2), it seems unlikely that they evolved before the divergence of the scolecophidian snakes [88]. Given that α-neurotoxic 3FTx had not evolved before the most recent CHAPTER 5. RED-ON-YELLOW QUEEN: THE BINDING OF MICRURUS VENOMS TO A RANGE OF NICOTINIC RECEPTOR MIMICS REVEAL PATTERNS OF PREY SPECIFICITY AND TOXIN 62 RESISTANCE common ancestor of all extant snakes, selection to resist these toxins can not explain the widespread occurrence of this mutation. Since this residue is largely conserved in most other chordate taxa, it seems plausible that there is some negative selection acting on that site. A scenario like this has been observed in lineages that can resist toad toxins, but related taxa in toad-free environments tend to lose the adaptation [247]. It is unclear whether the W1S mutation arose as a deleterious trait and persisted through chance, if it was a neutral mutation because something about the biology of snakes negated the penalty that other taxa would face, or if the common ancestor of snakes was exposed to some selective pressure that made the mutation adaptive at the time. Since then however, snakes producing α-neurotoxic 3FTx, many of whom consume other snakes, have become widespread. Whatever its origins the W1S mutation certainly seems to have been exapted to help protect these snakes from the risk of self envenomation and, perhaps more importantly, to protect snakes broadly from predation by their relatives. Another factor that is not captured by our results is the possibility of prey animals producing venom inhibitors rather than altering the targets, as has been well characterized for other families of snake toxins [236]. Indeed there is preliminary evidence suggesting that some snakes have done just that [241]. Coupled with the W1S mutation, such venom inhibitors could have a synergistic effect and grant high levels of resistance to α-neurotoxicity. This multi-faceted resistance may also help explain why so many neurotoxic snake venoms seem to be toxic to the point of overkill [248]: if their prey (snakes) are resistant and the test organisms (usually mice) are not, then the snake would need to deliver higher doses of venom to survive than one would surmise from the test results. With this in mind, the widespread resistance we observed amongst snakes to α-neurotoxins may be just part of an ongoing arms race between predator and prey and all the other victims are simply collateral damage.

5.5 Materials and Methods

5.5.1 Venom Samples

Some lyophilized venoms were sourced from long-term cryogenic collections in the Venom Evolution Lab while others were provided by Nathaniel Frank of MToxins Venom Lab, Alejandro Alagon of Universidad Nacional Autonoma´ de Mexico,´ and Ana Moura da Silva of Instituto Butantan. These venoms were resuspended in water, centrifuged (4C, 5min at 14,000 RCF), and diluted into a solution mg of 1 mL of venom in a 1:1 mixture of water and glycerol. Protein concentrations were measured using a NanoDrop 2000 UV-Vis Spectrophotometer (Thermofisher, Sydney, NSW, Australia).

5.5.2 Mimotope production and preparation

The sequences for the α1-nAChR were downloaded from the following UniProt entries: Tetronarce californica (P02710), Xenopus tropicalis (F6RLA9), Sarcophilus harrisii (G3W0J0), Rattus norvegi- cus (P25108), Gallus gallus (E1BT92), Gekko japonics (GenBank: XM015426640), and Anolis carolinensis (H9GA55). Additionally, the region of the orthosteric binding site of the α1-nAChR was 5.5. MATERIALS AND METHODS 63

Sanger sequenced for the following species: Barisia imbricata, Anilios bituberculatus, Boa constrictor, Oxyrhopus rhombifer, and Pantherophis guttatus. These sequences were then aligned using AliView 1.18 [143] and were trimmed down to the 14 amino acids of the orthosteric binding site. Subsequently, following previous protocols [231–233,249] mimotopes of these sequences were developed by GenicBio Ltd (Shanghai, China). As per previous studies [231], the cysteine doublet in the orthosteric binding site sequence was replaced in peptide synthesis steps with serine doublet to avoid uncontrolled postsynthetic thiol oxidation. Research has shown this has no effect on the analyte-ligand complex formation [250–252]. The mimotope was further synthesised to a biotin linker bound to a two aminohexanoic acid (Ahx) spacers to form a 30 A˚ linker between biotin and the peptide. The purpose of adding the biotin-Ahx complex is to allow the biotin molecule to bind non-covalently to the avidin pocket of the streptavidin coated disposable biosensors used in the bio-layer interferometry assay. The Ahx-Ahx linker allows for crucial clearance between the biotin and mimotope for the biotin to effectively reach the avidin binding pocket and for the mimotope to maintain its natural conformational freedom when binding to the analyte in solution. Dried stocks of synthesised mimotopes were solubilised in 100% dimethyl sulfoxide (DMSO) and µg then diluted 1:10 in deionised water to make a final working stock concentration of 50 ml and stored at −80 ◦C until use.

5.5.3 Bio-layer Interferometry

Binding kinetics were analysed by bio-layer interferometry utilising the Octet Red 96 system (ForteBio). All assays were conducted in a standard Greiner black 96 microtiter well plate. Analyte (venom) samples were diluted 1:20 from the working stock to make a final experimental concentration of 50 µg ml in the well (10 µg per well). Mimotope aliquots were diluted 1:50 to have a final concentration µg of 1 ml per well (0.2 µg per well). Assay running buffer was 1X DPBS with 0.1% BSA and 0.05% Tween-20. This buffer inhibits non-specific binding to the surface of the sensor and other proteins. Prior to experimentation, streptavidin sensors were hydrated in running buffer for 30-60 min, whilst being agitated at 2.0 RPM on a shaker. To regenerate the sensor tips during experimentation, the dissociation of analytes occurs using a standard acidic solution (glycine buffer), made up of 10 mM glycine (pH 1.5-1.7) in deionised water. Octet RED 96 assay methodology in the ForteBio Data Acquisition 9.0 program was set as follows: 60 s baseline, 50 s loading, 120 s baseline, 120 s association, 120 s dissociation, and 80 s regeneration/neutralisation step. The regeneration/neutralisation step consists of four cycles, lasting 10 s each, alternating between dipping in glycine buffer (regeneration) and then in running buffer (neutralisation) per cycle. For each baseline step throughout the experiment the same running buffer was used a maximum of three times per well. Experiments were run at 30 ◦C with the orbital agitation of the microplate set to 1000 rpm. Experiments were limited to less than 1 h to limit the change in analyte concentration due to evaporation on the warmed plate [230]. Analytes were set up in rows A-H, with triplicates set up in columns 1-3 and 4-6. To account for CHAPTER 5. RED-ON-YELLOW QUEEN: THE BINDING OF MICRURUS VENOMS TO A RANGE OF NICOTINIC RECEPTOR MIMICS REVEAL PATTERNS OF PREY SPECIFICITY AND TOXIN 64 RESISTANCE any potential evaporation effect in the wells during experimentation, the column running order was set to 1, 4, 2, 5, 3, 6 (rather than 1, 2, 3, 4, 5, 6). The mimotopes were set in column 7, running buffer was set in columns 8-10 and regeneration step (glycine buffer) and neutralisation step (running buffer) were columns 11 and 12 respectively. Negative controls were performed by replacing the venom sample with a 1:1 mixture of water and glycerol. Bungarus multicinctus venom was used as a positive control, as it has been shown to bind nAChR mimotopes [231–233].

5.5.4 Data Processing and Analysis

Data were processed as follows: 1) raw output folders (one per plate run) containing multiple running files were opened in the ForteBio Data Analysis 9.0 program, and in this program: 2) the ‘sensor tray’ was aligned with the location of sensors on our experimental plate, 3) an inter-step correction was performed whereby the data were aligned to baseline according to the y-axis of the initial baseline step (0.1–59.9 s), 4) a Savitsky-Golay filter was applied to the data, 5) the data were processed according to the above parameters, and 6) exported to a .csv file. Area under the curve for the association step of each samples was estimated using trapezoidal approximation in Microsoft Excel 15.28. Further statistical analyses were carried out in the RStudio 1.1.456 implementation of R 3.2.3 [208, 209]. Chapter 6

Conclusion

It is clear from my research—and the bodies of previous work upon which it is based—that the 3FTx family is highly adaptable and undergoing dynamic evolution in a wide range of snake taxa. One of the primary lines of evidence to indicate that this is a result of adaption rather than other evolutionary dN mechanisms is the analysis of dS ratios (Chapters 2, 3, 4). If non-synonymous mutations (which have a biochemical effect on the gene product) are fixed in a population more frequently than synonymous ones (which are nearly neutral), this suggests that the changes produced by the non-synonymous mutations confer some sort of fitness benefit. Occasionally, population demographic history can dN produce false positives using this method [262], but observations of elevated dS ratios amongst snake toxins are too widespread and consistent for this to be a likely explanation [20, 28, 67–71, 88]. In this thesis alone we have seen that separate datasets of 3FTx from five species of Boiga (Chapter 2), a single species Calliophis (Chapter 3), or 13 species of Micrurus (Chapter 4) all show signs of positive selection despite the very different demographic histories of these lineages. Boiga speciated into 33 species while migrating from Africa through to Australasia beginning roughly 26 million years ago [4, 59], Calliophis diverged from other elapids between 30 and 39 million years ago but remains confined to South and Southeast Asia and includes 11 species (only three of which belong to the long-glanded clade) [4, 59, 154, 155, 245], while Micrurus passed through a population bottleneck while crossing the Bering Land Bridge before radiating explosively to lead to 81 currently described species [4,59,154,155]. Given that the pattern of rapid diversification, strong positive selection, and variable toxic activity is robust and widespread amongst 3FTx [20, 69, 78, 82, 83, 92, 125, 263] the salient questions are how and why this phenomenon occurs.

When discussing the mechanistic origins of genetic variation, it is important to consider the genomic context of the loci in question. A chromosome-level assembly of a Crotalus viridis genome indicated that the microchromosomes were enriched in toxin genes [264]. Bird microchromosomes are known to experience higher mutation and recombination rates than the macrochromosomes [265, 266] which could facilitate the rapid duplication and sequence divergence seen in other toxin families. Schield et al. [264] also found that C. viridis microchromosomes “exhibit fundamentally different patterns of chromatin contact, with proportionally higher inter-chromosomal contact” than the macrochromosomes, 65 66 CHAPTER 6. CONCLUSION which could also potentially make them subject to higher mutation rates. Unfortunately, no published colubroid snake genome has been able to conclusively resolve the chromosomal location of the 3FTx gene cluster [70,267–269]. BLAST searches between 3FTx containing scaffolds from other assemblies and the C. viridis genome reveal similarity between the 3FTx scaffolds and an unassigned scaffold in the C. viridis (as well as more minor matches to Chromosome 1). Because of this, it remains unclear whether the increased mutation and recombination rates of microchromosomes might play a role in the accelerated evolution of 3FTx. Analysis of the C. viridis genome revealed another interesting trait shared by venom genes: virtually all of them of them were associated with regions of high-frequency chromatin contact that were flanked by CTCF insulator elements [264] which control chromatin structure and help promote gene transcription [270]. These features are partially responsible for the expression of the venom genes in the venom gland alone, but genes that are located in looser loops of chromatin that interact with other pieces of chromatin more frequently can also be more error-prone. The exact impacts of these factors have yet to be thoroughly investigated, but it seems that venom genes have several common genomic characteristics that would bias them towards higher mutation rates upon which positive selection can act. While the mechanisms through which the rapid molecular evolution we observe occurs are impor- tant, the research in this thesis deals more directly with how these evolutionary changes lead to novel toxin phenotypes. The simplest possible explanation is just that non-synonymous changes in the gene sequence can change the biochemistry of the mature toxin without significantly altering higher-level structures of the protein. This certainly can and does happen with 3FTx: cytotoxic 3FTx have very similar structure to neurotoxic forms [271], Calliophis 3FTx can target NaV channels (Chapter 3), and α-neurotoxins can vary greatly due to synthetic mutations at key residues [80] or due to phylogeny within Micrurus (Chapter 5). However, the structure of 3FTx lend them to more complex shifts in secondary and tertiary structure as well. The most prominent examples are the various alterations to the pattern of cysteine bonds that have happened throughout the evolutionary history of the toxin family. The deletion of one pair of cysteines helped give rise to the elapid radiation (Chapters 1 and 3) [69,78,82] while the evolution of an entirely novel pair led to more sophisticated α-neurotoxins in advanced elapids (Figure 1.2) [91, 189]. 3FTx are also known to form protein complexes that alter their function [90]. In Chapter 2 the evolutionary origins of such a complex are explored and they are found to have likely facilitated the diversification of the genus Boiga in Southeast Asia as well as playing a crucial role in the invasion of Guam and subsequent extirpation of a number of local bird populations by B. irregularis. The formation of these complexes was made possible by novel cysteines in two different 3FTx which allowed them to form an intermolecular disulfide bond [87]. Because of the straightforward relationship between venom genotype and phenotype, it is clear that relatively simple changes on the level of toxin genes can easily have large impacts on the level of the organism itself and the rest of its ecosystem. Chapters 3 and 5 also touch on the evolutionary reasons that might select for rapid evolution of venom phenotypes. In particular, those chapters emphasize the importance of predator-prey arms races. Chapter 5 demonstrates that virtually all snakes possess unusual mutations in the sequence of 67 the orthosteric binding region of the α1 subunit of the nAChR that grants them partial resistance to α-neurotoxic 3FTx. This trait is almost certainly advantageous in any ecosystem that also includes ophiophagous elapids (which is to say most of the world). When predator and prey each have a trait that can counteract the other, we often observe arms races of rapid evolution [272]. In such arms races, the selection on prey is usually thought to be stronger than the selection on predators; this is sometimes referred to as the “life-dinner principle” in reference to the somewhat different stakes the two organisms face (in any one predation attempt the predator risks losing out on a meal while the prey risks dying) [273]. However, when we consider the matter from the perspective of potential mutations, the stakes are often reversed. Toxins usually target vital physiological systems in the target (how else would they disable the prey?) [49], so mutations to those targets could have potentially lethal consequences. On the other hand, the worst case scenario for a mutation in a venom gene is that the mature toxin is rendered useless, all the energy invested in producing that toxin is wasted, and predation success rate might be decreased; while this is not beneficial, this scenario is far less likely to be immediately fatal. These repercussions are further attenuated by the fact that many venom genes—especially those that account for major portions of the composition of a snake’s venom—are highly duplicated in the genome (Chapters 2 and 3) [70, 263, 264, 267–269, 274, 275]. This increased copy number is thought to arise from selection for increased yield of those particular toxins [72]; the simple regulatory control of venom genes means that increased copy number leads to increased dosage of the toxin in question. These arrays of venom genes under strong positive selection then lay the groundwork for those happiest of accidents for a predator locked in a evolutionary arms race: mutations that lead to novel routes of attack [276]. We suspect that a scenario very much along these lines is what led to the evolution of 3FTx that can target NaV channels in Calliophis (Chapter 3). The diversity of eight-cysteine 3FTx in their genome allowed evolution to tinker with the different copies until some happened to develop this novel activity, all without losing the potent α-neurotoxicity that they had relied on until that point. These NaV neurotoxins then sidestepped any resistance their prey might have evolved to α-neurotoxins and allowed the long-glanded Calliophis to become the consummate snake eaters we are familiar with. Venom is a valuable model system for researching a number of other evolutionary phenomena for many of the same reasons that it is such an effective strategy in predator-prey arms races. It is a trait with a very simple relationship between genotype and phenotype that evolves quickly and directly influences the ecology of the organism. Because of this it is excellent for studying the the history of gene duplication [274, 275, 277], local adaptation [278–280], and the evolution of novelty [281–284]. The sheer number of venomous lineages, duplication of venom genes, and relative lack of evolutionary constraints means that ample data can be brought to bear on these areas of research. This thesis adds to that body of data by demonstrating how novel venom phenotypes have arisen amongst the 3FTx in several different episodes of snake evolution. Our conclusions suggest that the evolution of novel venom traits often accompany morphological adaptations during key adaptive periods in the evolution of several different taxa such as the diversifi- cation of arboreal Boiga in Southeast Asia (Chapter 2), the origin of elapids as whole (Chapter 3), the 68 CHAPTER 6. CONCLUSION evolution of long-glanded Calliophis (Chapter 3), and the radiation through the Americas by Micrurus (Chapter 4 and 5). While this thesis focused on the 3FTx, many of the trends identified by this research are applicable to other toxin families and venom evolution as a whole. Many of the major toxin families in snake venom (and even more in invertebrates) are similar to 3FTx in that they are small, non-enzymatic proteins with largely conserved structures [49,285]. These sorts of toxins are likely to display similar evolutionary dynamics, especially because venom resistance is a trait that is roughly as widespread as venom itself. For most of the major toxic activities that are found in venoms, there are prey animals which have evolved defenses to that particular route of attack [236]. Indeed, many venoms, especially those from “younger lineages” (those where venom evolved relatively recently) are known to be broadly under positive selection [286], much like the 3FTx studied in this thesis. If indeed our results can be generalized to other toxin families, snake venoms as a whole, or even venoms in general, we should expect novel venom phenotypes to co-occur with morphological innovations and adaptive radiations in other taxa as well. Bibliography

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Appendix A

R script to convert all-to-all BLAST results to network file

# !/usr / bin / env Rscript

#interprets the first argument as the input file and the second as the desired output name #gives and error and stops if there are no arguments args <− commandArgs (TRUE) if ( length ( args ) <2){ stop (”Missing arguments\ nUsage : BLAST2Network .R [INPUT FILE ] [OUTPUT FILE]\ n”)} infile <− as . character ( args [1]) outfile <− as . character ( args [2])

origin <− read . csv (infile) origin$ query <− as . character (origin$ query ) origin$ subject <− as . character (origin$ subject )

#make new data frame with same columns new =origin[FALSE,]

#loop through original results for (i in seq ( nrow (origin))){

95 96 APPENDIX A. R SCRIPT TO CONVERT ALL-TO-ALL BLAST RESULTS TO NETWORK FILE

#print progress every 1000 iterations if (i%%1000==0){ prog <− cat ( round (i/nrow(origin),2)⇤ 100,”%\n” , sep= ””)}

#skip any entry that is queried against itself if (identical(origin[i,1],origin[i,2])){ next} else{

#check for inverses of current row already in output data for (j in seq (from = 0, to = nrow ( new ))){

#if you find a reciprocal , use the lower e−value and the higher coverage then go to the next entry in the original data if (identical(origin[i,1],new [j ,2]) && identical (origin[i ,2] ,new [j ,1])){ new$e.value[j] <− min ( new$e.value[j], origin$e.value[i]) new$ coverage [ j ] <− max ( new$ coverage [ j ] , origin$ coverage [ i ]) break }

#if there is no reciprocal and the e−value is less than 1e−5, add the current row to the output if (j==nrow ( new ) && origin$e.value[i]<1e−5){ new [ nrow ( new )+1,] <− origin [i ,] } } } }

#define a new column new$ similarity <−−log10 ( new$e.value)

#write the new data to an output file without row numbers write . csv ( new , file=outfile , row . names=FALSE) Appendix B

Calliophis MSMS results

97 98 APPENDIX B. CALLIOPHIS MSMS RESULTS 6 6 6 6 6 6 6 6 6 6 6 4 4 5 8 1 5 5 5 4 17 17 10 10 10 Peptides(95%) 0 052 038 011 010 009 003 095 120 103 090 092 007 004 113 008 026 023 022 030 112 111 110 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx Vespryn Name Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi fraction 32 Vespryn-21 OS=Drysdalia coronoides OX=66186 PE=1 SV=1 0 DRYCN 052 038 011 010 009 003 095 120 103 090 092 007 004 113 008 026 023 022 030 112 111 110 PIG Calliophis bivirgatus VESP | 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx Vespryn TRYP | Accession sp Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi F8RKW2 | sp %Cov(95) 57.6300025 57.6300025 57.6300025 33.32999945 33.32999945 42.86000133 38.02999854 45.75999975 45.75999975 34.47999954 35.08999944 35.08999944 35.08999944 34.47999954 33.79999995 33.79999995 16.89999998 16.89999998 28.97000015 11.58000007 42.37000048 63.63999844 63.63999844 63.63999844 7.791999727 Table B.1: MSMS results for %Cov(50) 57.6300025 57.6300025 57.6300025 33.32999945 33.32999945 65.07999897 57.74999857 69.48999763 45.75999975 34.47999954 35.08999944 35.08999944 35.08999944 34.47999954 33.79999995 33.79999995 16.89999998 16.89999998 28.97000015 11.58000007 42.37000048 63.63999844 63.63999844 63.63999844 7.791999727 %Cov 50.849998 80.9499979 55.00000119 55.00000119 71.82999849 86.44000292 62.70999908 81.02999926 82.45999813 82.45999813 82.45999813 81.02999926 33.79999995 33.79999995 16.89999998 16.89999998 47.65999913 18.95000041 66.10000134 66.10000134 66.10000134 83.63999724 83.63999724 83.63999724 7.791999727 4 4 4 2 4 4 4 8.02 8.02 5.68 5.68 5.68 5.68 5.33 3.79 3.79 3.79 3.78 4.55 4.55 3.68 3.68 4.21 2.02 2.05 Total 0 0 0 0 0 0 0 0 0 0 0 0 4 2 0 0 4 0 0 8.02 5.68 5.31 4.47 4.17 2.05 Unused 1 2 2 2 2 3 3 3 3 3 4 4 4 4 5 5 6 6 6 6 7 7 7 8 1 N 99 4 4 4 2 2 2 2 2 2 2 2 2 4 4 3 3 3 3 2 Peptides(95%) 2 1 3 117 034 021 035 033 029 016 116 032 031 030 112 111 110 118 CRiSP CRiSP CRiSP 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx Name Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi fraction 34 Delta-elapitoxin-Cb1a OS=Calliophis bivirgatus OX=8633 PE=1 SV=1 2 1 3 CALBG Calliophis bivirgatus 117 034 021 035 033 029 016 116 032 031 030 112 111 110 118 CRiSP CRiSP CRiSP 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3SXNA | Accession Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi P0DL82 | sp %Cov(95) 20.0000003 33.32999945 33.32999945 33.32999945 15.79000056 15.79000056 15.79000056 15.79000056 15.79000056 15.79000056 15.79000056 15.79000056 15.25000036 11.37000024 11.37000024 6.634999812 38.17999959 38.17999959 38.17999959 Table B.2: MSMS results for %Cov(50) 20.0000003 33.32999945 33.32999945 33.32999945 15.79000056 15.79000056 15.79000056 15.79000056 15.79000056 15.79000056 15.79000056 15.79000056 15.25000036 11.37000024 11.37000024 6.634999812 38.17999959 38.17999959 38.17999959 24.56 24.56 24.56 24.56 24.56 %Cov 20.0000003 42.10999906 42.10999906 42.10999906 15.79000056 15.79000056 15.79000056 15.25000036 11.37000024 11.37000024 6.634999812 38.17999959 38.17999959 38.17999959 4 4 4 2 2 2 2 2 2 2 2 2 4 4 2 2 2.05 2.05 2.05 Total 4 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 2.05 Unused 1 1 1 1 1 1 1 1 1 1 1 2 2 2 3 3 3 3 1 N 100 APPENDIX B. CALLIOPHIS MSMS RESULTS 3 9 9 8 8 8 8 8 8 8 8 8 8 4 2 2 28 41 18 28 28 26 26 11 Peptides(95%) 0 11 089 067 047 043 016 117 035 034 033 029 021 116 032 031 030 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx SVMP Vespryn Name Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi fraction 36 Ohanin OS=Ophiophagus hannah OX=8665 PE=1 SV=2 Vespryn-21 OS=Drysdalia coronoides OX=66186 PE=1 SV=1 Delta-elapitoxin-Cb1a OS=Calliophis bivirgatus OX=8633 PE=1 SV=1 0 CALBG DRYCN Calliophis bivirgatus 11 OPHHA 089 067 047 043 016 117 035 034 033 029 021 116 032 031 030 PIG HUMAN HUMAN HUMAN VESP | 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx SVMP Vespryn VESP 3SXNA | | TRYP | Accession K22E K2C1 sp K1C10 | | | Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi sp sp sp P83234 | F8RKW2 P0DL82 | | sp sp sp %Cov(95) 20.23999989 19.91000026 15.50000012 53.32999825 53.32999825 33.32999945 33.32999945 8.553999662 9.523999691 56.13999963 56.13999963 15.79000056 15.79000056 15.79000056 15.79000056 15.79000056 15.79000056 15.79000056 15.79000056 15.79000056 15.25000036 21.50000036 7.367999852 7.367999852 Table B.3: MSMS results for %Cov(50) 20.39999962 22.26999998 17.04999954 53.32999825 53.32999825 33.32999945 33.32999945 9.952999651 9.523999691 56.13999963 56.13999963 15.79000056 15.79000056 15.79000056 15.79000056 15.79000056 15.79000056 15.79000056 15.79000056 15.79000056 15.25000036 21.50000036 7.367999852 7.367999852 75 %Cov 13.8500005 12.1100001 23.78000021 40.27999938 23.26000035 60.00000238 61.66999936 61.66999936 16.79999977 64.91000056 64.91000056 26.32000148 26.32000148 26.32000148 26.32000148 26.32000148 26.32000148 15.79000056 15.79000056 15.79000056 15.25000036 29.91000116 7.367999852 4 2 2 9.6 9.6 2.2 8.03 8.03 8.96 2.59 2.59 2.06 2.06 2.06 2.06 2.06 2.06 2.06 2.06 2.06 2.06 Total 23.56 20.24 17.81 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 0 9.6 6.41 2.59 2.09 23.56 20.24 17.81 Unused 1 2 3 4 4 4 4 5 6 7 7 7 7 7 7 7 7 7 7 7 7 8 8 8 N 101 4 5 5 4 8 8 8 6 74 35 34 37 89 89 89 10 10 11 11 11 11 11 12 12 11 15 30 30 15 30 15 18 Peptides(95%) 001 003 004 005 006 007 008 009 010 011 013 014 020 022 022 023 023 024 025 026 027 030 031 032 038 043 047 052 067 068 082 087 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx Name Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi whole venom, pt. 1 001 003 004 005 006 007 008 009 010 011 013 014 020 022 022 023 023 024 025 026 027 030 031 032 038 043 047 052 067 068 082 087 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx Accession Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi 25 25 %Cov(95) 80.9499979 44.44000125 64.41000104 50.70000291 50.70000291 70.42000294 86.44000292 71.82999849 53.32999825 53.32999825 35.58999896 35.58999896 35.58999896 35.58999896 35.58999896 36.21000051 36.21000051 35.58999896 35.58999896 35.58999896 36.84000075 36.84000075 33.32999945 33.32999945 33.32999945 33.32999945 33.32999945 37.92999983 60.71000099 46.66999876 Calliophis intestinalis 25 25 %Cov(50) 80.9499979 44.44000125 64.41000104 50.70000291 50.70000291 70.42000294 86.44000292 71.82999849 53.32999825 53.32999825 35.58999896 35.58999896 35.58999896 35.58999896 35.58999896 77.59000063 77.59000063 35.58999896 35.58999896 35.58999896 36.84000075 36.84000075 33.32999945 33.32999945 33.32999945 33.32999945 33.32999945 37.92999983 60.71000099 46.66999876 %Cov 54.9300015 80.9499979 44.44000125 88.13999891 66.20000005 64.06000257 64.06000257 70.42000294 86.44000292 87.31999993 53.32999825 53.32999825 88.13999891 88.13999891 88.13999891 88.13999891 88.13999891 96.54999971 96.54999971 88.13999891 45.75999975 88.13999891 47.36999869 87.72000074 94.99999881 69.99999881 69.99999881 94.99999881 69.99999881 48.28000069 71.42999768 61.66999936 8 4 4 8 4 4 4 4 4 4 8 6 12 10 4.02 4.01 4.03 4.03 4.03 4.03 4.03 4.03 4.01 4.08 4.55 4.55 4.08 4.55 Total 12.64 14.74 14.74 14.74 Table B.4: MSMS results for 0 0 0 4 4 0 0 0 0 4 0 0 0 0 0 0 4 2 0 0 0 0 0 0 4 0 6 10 4.01 4.02 4.15 14.74 Unused 2 2 2 2 4 4 8 N 19 14 17 11 11 15 12 14 19 14 18 18 17 11 17 11 17 17 17 11 11 10 10 15 10 102 APPENDIX B. CALLIOPHIS MSMS RESULTS 8 4 5 5 5 5 2 3 6 6 7 6 8 7 6 6 6 8 6 4 4 30 25 25 31 25 36 36 36 38 11 25 11 36 25 Peptides(95%) 0 01 04 07 10 12 13 18 19 20 21 02 06 07 08 16 18 19 24 31 34 089 090 092 095 103 110 111 112 113 114 116 120 IB IB IB IB IB IB IB IB IB IB 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx Kunitz Kunitz Kunitz Kunitz Kunitz Kunitz Kunitz Kunitz SVMP SVMP Vespryn Name PLA2 PLA2 PLA2 PLA2 PLA2 PLA2 PLA2 PLA2 PLA2 PLA2 Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi whole venom, pt. 2 Ohanin OS=Ophiophagus hannah OX=8665 PE=1 SV=2 Vespryn-21 OS=Drysdalia coronoides OX=66186 PE=1 SV=1 0 01 04 07 10 12 13 18 19 20 21 DRYCN 02 06 07 08 16 18 19 24 31 34 OPHHA 089 090 092 095 103 110 111 112 113 114 116 120 IB IB IB IB IB IB IB IB IB IB VESP | 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx 3FTx Kunitz Kunitz Kunitz Kunitz Kunitz Kunitz Kunitz Kunitz SVMP SVMP Vespryn VESP | PLA2 PLA2 PLA2 PLA2 PLA2 PLA2 PLA2 PLA2 PLA2 PLA2 Accession Calliophis intestinalis Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi Cbivi P83234 | F8RKW2 | sp sp 100 100 100 %Cov(95) 55.1699996 33.32999945 35.08999944 34.47999954 35.08999944 70.42000294 36.84000075 36.84000075 35.08999944 40.68000019 57.88999796 22.81000018 22.81000018 22.81000018 22.40999937 19.05000061 36.50999963 32.80000091 32.80000091 28.79999876 32.80000091 43.20000112 28.79999876 32.53999949 32.53999949 32.53999949 43.20000112 26.30000114 14.18000013 21.50000036 7.367999852 7.367999852 100 100 100 %Cov(50) 33.32999945 36.84000075 36.21000051 56.90000057 36.84000075 70.42000294 36.84000075 36.84000075 36.84000075 40.68000019 57.88999796 43.86000037 43.86000037 43.86000037 43.09999943 19.05000061 36.50999963 32.80000091 32.80000091 28.79999876 32.80000091 43.20000112 28.79999876 32.53999949 32.53999949 32.53999949 43.20000112 30.09000123 14.41999972 28.97000015 11.58000007 7.367999852 Table B.5: MSMS results for 50 100 100 100 %Cov 44.8300004 82.4000001 61.1100018 66.67000055 47.36999869 46.54999971 67.23999977 47.36999869 81.69000149 47.36999869 47.36999869 47.36999869 40.68000019 71.92999721 52.63000131 52.63000131 52.63000131 36.50999963 44.44000125 85.60000062 56.00000024 74.40000176 62.40000129 47.99999893 58.73000026 52.38000154 81.59999847 30.25999963 70.09000182 37.36999929 30.00000119 8 4 4 2 6 6 8 6 8 8 2 2 8.1 4.55 4.64 4.64 4.64 9.14 9.14 9.14 4.64 4.01 3.05 2.01 2.01 2.01 2.01 3.82 6.07 6.07 6.07 8.91 4.01 Total 12.01 21.39 0 0 0 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 9.11 4.01 2.37 2.01 3.17 8.08 4.72 12.01 21.39 Unused 7 7 7 7 5 5 5 3 7 6 6 6 6 6 6 6 6 6 6 1 9 N 17 11 13 22 16 10 21 22 22 22 20 20 16 16