University of Nevada, Reno Understanding Patterns Of

University of Nevada, Reno

Understanding Patterns of Resistance to Spider Venom in Lizards: Ecology and Phylogeny Matter

A thesis submitted in partial fulfilment of the requirements for the degree of Master of Science in Biology

Vicki L. Thill

Dr. Chris Feldman/Thesis Advisor

Dr. Mike Teglas/Thesis Co-advisor

December 2019

THE GRADUATE SCHOOL

We recommend that the thesis prepared under our supervision by

VICKI LEE THILL

entitled

Understanding Patterns of Resistance to Spider Venom in Lizards: Ecology and Phylogeny Matter

be accepted in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Christoffer R. Feldman, Ph.D., Advisor

Michael B. Teglas, Ph.D., Co-advisor

Jennifer L. Hoy, Ph.D., Graduate School Representative

David W. Zeh, Ph.D., Dean, Graduate School

December 2019 i

ABSTRACT Lizards and spiders often engage in predator-prey interactions, and many spiders can be dangerous as both predator and prey. However, we know little about how lizards tackle dangerous spider prey. And yet, some species are known to consume especially potent spider prey, like the western black widow

(Latrodectus hesperus). In particular, Elgaria multicarinata is known to preferentially consume venom-defended L. hesperus. I asked whether E. multicarinata possessed resistance to the venom of L. hesperus and evaluated resistance at two levels of biological organization: at the whole animal level, and at the muscle tissue level. I included one other sympatric species that will eat L. hesperus when offered (Sceloporus occidentalis), and one sympatric species that is known prey of L. hesperus (Uta stansburiana). To evaluate the whole animal resistance, I used sprint speed performance; to evaluate muscle tissue resistance, I used comparative histology. Lizards were tested against either a

control (sterile saline), “low” (1 mouse LD50), or “high” (5 mouse LD50) dose of black widow spider venom (BWSV). I found that E. multicarinata showed no response to any venom treatment, while U. stansburiana had tissue level responses for both low and high venom treatments and a whole animal response only for high venom treatment; S. occidentalis was somewhere in the middle, with no whole animal response and slight (but significant) muscle tissue response.

Given the variety of responses in these three lizards across response levels (whole animal, tissue) and treatments (low, high), and the lack of drastic ii susceptibility in U. stansburiana, I then asked whether resistance to BWSV could be rooted deep in the squamate lineage (i.e. an ancestral trait of all lizards). I investigated this by testing for whole animal and muscle tissue resistance to

BWSV using sprint speed performance and comparative histology on species representing a broader phylogenetic, ecological, and geographical scope of

Squamate taxa. I found that insectivorous species (Coleonyx variegatus and

Takydromus sexlineatus) were resistant to both low and high venom treatments at the whole animal, but not at the tissue level. I included a single herbivorous species, Iguana iguana, which showed drastic decreases in sprint performance and severe tissue responses for both low and high venom treatments. The variation in responses seen at different levels (whole animal, tissue) and at different treatments (low, high) combined with ecological traits, provides evidence that both ecology and phylogeny contribute to whether BWSV resistance is present, and at what level – whole animal and/or muscle tissue.

iii

ACKNOWLEDGEMENTS

This work would not have been possible without the dual guidance of my major advisors, Dr. Chris Feldman and Dr. Mike Teglas, who have both perfected the art of helpful criticism. Their support and responsiveness throughout this project was invaluable, and I will be forever grateful for their kindness and their positive and uplifting attitudes. Their enthusiasm for science was contagious and created a wonderful, encouraging space within which to question, to formulate ideas, to learn from mistakes, and to get things done. I also thank Dr. Jennifer Hoy for valuable contributions as a committee member, especially in regard to her advice about whole animal performance and histology data extractions. I sincerely thank

Dr. Matt Forister for thoughtful discussion regarding analyses.

I am thoroughly grateful to the late Dr. Nate Nieto for his early contributions, especially in regard to pilot trials that helped establish the viability of this project. I also thank Kris Wild for early project advice on racing lizards and video processing software and Jake Holland for track design and construction. I thank Dr. Bob Hansen for providing open use of his beautiful photographs of

Elgaria multicarinata, Sceloporus occidentalis, Uta stansburiana, and Latrodectus.

To all those who assisted with field collection in any way, thank you (in no particular order): Erica Ely, Dr. Bobby Espinoza, Kristin McCarty, Dr. Emily Taylor,

Jason Wurtz, Dr. Dean Leavitt, Jonathan DeBoer, Ally Xiong, Joshua Hallas,

Daniel Moore and finally, Jeff Wilcox at Sonoma Mountain Preserve for not only iv hosting during collection excursions but also being a science mentor and knowing exactly where to find Elgaria in the “off season”.

For assistance with live animal trials, video processing, and data extraction, I’d like to thank McKenzie Wasley, the most capable and cheerful lab tech and human being I’ve ever encountered; Leah Herbert and Molly McVicar, both of whom tackled the most tedious tasks without complaint; and live animal caregivers and lab techs Amber Durfee, Taylor Disbrow, Lizzy Sisson, Luke

Mauer, and Gabrielle Blaustein. I thank the Office of Animal Resources for accessibility and advice regarding best practices for live animal care, in particular

John Gray and Rebecca Evans, and the Institutional Animal Care and Use

Committee for approval of live animal protocols.

I thank Robert del Carlo and Jessica Reimche for their intelligent discourse, helpful feedback, and for providing a positive environment within which to work. For reassurance, informal advice and, sometimes, commiseration,

I thank my office colleagues, especially Dr. Anne Espeset. For assisting with almost every step of this project, from field collection to performance trials and manuscript review, I thank Haley Moniz, who is genuine, quick-witted, and impressively organized; she is a real human bean and my best friend.

Support for this research was provided by the UNR Graduate Student

Association Research Grant program, the Society for the Study of Amphibians and Reptiles (travel award), as well as funds to CRF, including generous donations by Ron Aryel. v

TABLE OF CONTENTS

Abstract ...... i Acknowledgements ...... iii Table of Contents ...... v List of Tables...... vii List of Figures ...... viii Thesis Overview ...... 1 Literature Cited ...... 6 Chapter 1. Preying dangerously: Black widow spider venom resistance in sympatric lizards ...... 9 Abstract ...... 9 Introduction ...... 10 Methods ...... 15 Animal collection and care ...... 15 Whole-animal performance ...... 15 Analyses ...... 17 Comparative histology ...... 18 Results ...... 19 Whole-animal performance ...... 19 Comparative histology ...... 21 Discussion ...... 24 Literature Cited ...... 29 Tables ...... 34 Figures ...... 40 Chapter 2. Spider venom resistance in lizards lingers across branches ...... 44 Abstract ...... 44 Introduction ...... 46 Methods ...... 49 Animal collection and care ...... 49 vi

Whole-animal performance ...... 50 Analyses ...... 52 Comparative histology ...... 52 Ancestral State Reconstruction ...... 54 Results ...... 55 Qualitative impacts of BWSV on lizards...... 55 Whole-animal performance ...... 56 Comparative histology ...... 58 Trait Mapping ...... 61 Discussion ...... 62 Literature Cited ...... 67 Tables ...... 70 Figures ...... 76 Concluding Remarks ...... 81 Supplementary Data ...... 82 Lack of resistance to Arizona bark scorpion venom in Elgaria multicarinata and Sceloporus occidentalis ...... 82 Introduction and Methods ...... 82 Results and Discussion ...... 84 Literature Cited ...... 86 Tables ...... 87 Figures ...... 88

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LIST OF TABLES

1.1 Linear Mixed Effect Model Ranks ...... 34 1.2 Species Level Linear Mixed Effect Model Ranks ...... 35 1.3 Differences between pre- and post-injection velocity across species and treatment ...... 36 1.4 Differences between untreated and treated tissue across species ...... 37 1.S1 List of individuals used, with collection data...... 38 2.1 Linear Mixed Effect Model Ranks ...... 70 2.2 Species Level Linear Mixed Effect Model Ranks ...... 71 2.3 Differences between pre- and post-injection velocity across species and treatment ...... 72 2.4 Differences between untreated and treated tissue across species ...... 73 2.S1 List of individuals used, with collection data...... 74 S.S1 List of individuals used, with collection data...... 87

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LIST OF FIGURES

1.1 Focal species images ...... 40 1.2 Percent performance (relative to baseline velocity) plotted over time and grouped by species and treatment ...... 41 1.3 Reference histology images and treatment images showing characteristic species responses by treatment ...... 42 1.4 Boxplots comparing untreated tissue response with treated tissue response for all muscle tissue variables ...... 43 2.1 Focal species images ...... 76 2.2 Ancestral state reconstructions for resistance levels and combined phylogenetic tree with ecological data ...... 77 2.3 Percent performance (relative to baseline velocity) plotted over time and grouped by species and treatment ...... 78 2.4 Reference histology images and treatment images showing characteristic species responses by treatment ...... 79 2.5 Boxplots comparing untreated tissue response with treated tissue response for all muscle tissue variables ...... 80 S.1 Focal species images ...... 88

THESIS OVERVIEW

Antagonistic relationships are ecological interactions that involve a benefit for one ecological partner at the cost of the other, often exerting selective pressure on the negatively affected side of the interaction (Dawkins and Krebs 1979, Brodie and Brodie 1999). These relationships can lead to varied ecological patterns, whether that be cyclic population cycles, like the classic (but not completely understood) relationship between lynx and snowshoe hares (Krebs et al. 2001) or switching from antagonistic to mutualistic interactions (Thompson 2014), or adaptations that sometimes lead to coevolutionary arms races (Benkman et al.

2003, Thompson 2005).

In predator-prey relationships, natural selection often promotes adaptations that allow prey species to “escape” from the negative effects of predation (Endler 1991, Lima and Dill 1990, Richardson and Anholt 2010). Such antipredator adaptations can take various forms, from pointy spines (Mikolajewski and Johansson 2004) to schooling behavior (Seghers 1974) to chemical deterrents that result in distastefulness or even poisoning (Williams 2008). Prey that are potentially dangerous can, in turn, lead to predator adaptations

(Mukherjee and Heithaus 2013). Predator adaptations, like those in prey, can take many forms, like changing prey handling behavior (Eisner et al. 1993, Mukherjee and Heithaus 2013) or physiological resistance to a toxin or venom (Rowe and

Rowe 2008, Feldman et al. 2012, Hopp et al. 2017). 2

Even though predator-prey interactions involving toxic or venomous traits are common in nature (Holding et al. 2016, McCabe and Mackessy 2016), only a handful have actually been explored. Mammals are best represented in the literature (e.g., Rowe and Rowe 2008, Jansa and Voss 2011), though still sparse.

Studying mammal systems has uncovered various mechanisms underlying venom resistance that might be broadly applicable across vertebrates (Holding et al. 2016). Nevertheless, there remains a lack of understanding in mechanisms of resistance at physiological and molecular levels (Holding et al. 2016, McCabe and Mackessy 2016). Thus, exploring patterns of venom resistance across a diverse group of vertebrate clades, like squamates, will add foundational data to further build our understanding of these complex ecological interactions and the adaptations they drive.

Within squamates, lizards are especially interesting because a large proportion of them are insectivorous (Espinoza et al. 2004) and prey on noxious arthropods of some kind (Pritchett 1903, Sherbrooke 2003, Stebbins 2003); of particular note are their relationships with spiders. Spiders are widely distributed

(Garb et al. 2004) and can be extremely abundant (Turnbull 1973), making them important prey for many taxa. Lizards can influence abundance and diversity of spiders both directly and indirectly (Spiller and Schoener 1988, Polis and Hurd

1995, Spiller and Schoener 1998, González-Suárez 2011), and can be the primary spider predator in some ecosystems (Polis and Hurd 1995). Many lizards across a variety of biomes consume some proportion of spiders in their diet 3

(Parker and Pianka 1974, Spiller and Schoener 1988, Gasnier et al. 1994,

Stebbins 2003), yet the vast majority of spiders are venomous (Sunagar and

Moran 2015), making them potentially dangerous prey.

In Chapter 1, I explored whether lizards that consume dangerous spider prey are resistant to their venom. I first selected an abundant and potently venomous spider, the western black widow (Latrodectus hesperus); then, I chose three insectivorous lizard species sympatric with L. hesperus that represent presumed resistant predators: Elgaria multicarinata (Cowles 1937, Cunningham

1956), Sceloporus occidentalis (pers. obs., in captivity) and presumed susceptible non-predator: Uta stansburiana.

I evaluated whether resistance was present at two broad scales: whole animal level (sprint performance) and muscle tissue level (comparative histology).

I exposed lizards to low (ecologically relevant) and high venom treatments and compared whole animal sprint performance and muscle tissue response to responses elicited by saline control. I expected to see no reduction in sprint speed and no muscle tissue response compared to saline controls in resistant lizards, and reduced sprint speed and evident muscle tissue response compared to saline controls in susceptible lizards.

Elgaria multicarinata exhibited no differences in whole animal sprint performance or muscle tissue response between control and venom treatments, suggesting that they are highly resistant to black widow spider venom (BWSV).

Sceloporus occidentalis showed no difference in whole animal sprint 4 performance compared to controls, but did show muscle tissue response to both low and high venom treatments. Like E. multicarinata, these results are indicative of resistance to BWSV; however, there is variation in the scale at which resistance is present. Uta stansburiana was the only species that had both reduced whole animal performance compared to control (at high venom treatment) and muscle tissue response (at low and high venom treatment).

Lizard predators of a dangerous but abundant spider seemed to have more effective defenses against the effects of BWSV compared to a lizard non- predator; however, even U. stansburiana did not exhibit complete susceptibility, tolerating ecologically relevant doses without reductions in sprint performance.

This led me to question whether whole animal resistance to BWSV is ancestral and present in many squamate taxa or if it is due to local predator-prey interactions.

In Chapter 2, I selected three additional lizard species to expand the phylogenetic scope of the work performed in Chapter 1; these species also represent additional ecological traits (herbivory) and broadened the geographic range to include species not sympatric with L. hesperus. I tested these species using the same venom treatments as in Chapter 1, at the whole animal and muscle tissue level.

Of the three additional species tested, two performed similarly to S. occidentalis in that there was no whole animal effect (sprint speed was not reduced compared to controls), but there was some muscle tissue effect. The 5 only herbivorous lizard, Iguana iguana, had drastic reductions in sprint speed for both low and high venom treatments as well as an adverse muscle tissue response indicative of total susceptibility to BWSV. Iguana iguana was also the only species that had issues with coordination of movement, including paralysis of the hind legs.

The gradient of resistance found across these species indicates three main takeaways. First, there is evidence that resistance to BWSV at the whole animal level is ancestral, and could be present in a wide variety of lizards.

Second, there is evidence that tissue level resistance could be due to predator- prey interactions, like that between E. multicarinata and its preferred prey. Finally, the ecological trait of herbivory seems to be associated with the loss of this putatively ancestral trait, although testing additional herbivorous species would lend support to that conclusion. Resistance to BWSV in this suite of lizards seems to be a result of both current ecological traits and phylogenetic history.

LITERATURE CITED

Benkman, C.W., Parchman, T.L., Favis, A. & Siepielski, A.M. Reciprocal Selection Causes a Coevolutionary Arms Race between Crossbills and Lodgepole Pine. The American Naturalist 162, 182–194 (2003). Brodie, E.D. III & Brodie, E.D., Jr. Predator-Prey Arms Races: Asymmetrical selection on predators and prey may be reduced when prey are dangerous. BioScience 49, 557-568 (1999). Cowles, R.B. The San Diegan Alligator Lizard and the Black Widow Spider. Science 85, 99–100 (1937). Cunningham, J.D. Food Habits of the San Diego Alligator Lizard. Herpetologica 12, 225-230 (1956). Dawkins, R. & Krebs, J.R. Arms races between and within species. Proceedings of the Royal Society B 205, 489-511 (1979). Eisner, T., Baldwin, I.T. & Conner, J. Circumvention of prey defense by a predator: ant lion vs. ant. Proceedings of the National Academy of Sciences 90, 6716–6720 (1993). Endler, J.A. Variation in the appearance of guppy color patterns to guppies and their predators under different visual conditions. Vision Research 31, 587-608 (1991). Espinoza, R.E., Wiens, J.J. & Tracy, C.R. Recurrent evolution of herbivory in small, cold-climate lizards: Breaking the ecophysiological rules of reptilian herbivory. Proceedings of the National Academy of Sciences 101, 16819–16824 (2004). Feldman, C.R., Brodie, E.D., Jr., Brodie, E.D. III & Pfrender, M.E. Constraint shapes convergence in tetrodotoxin-resistant sodium channels of snakes. Proceedings of the National Academy of Sciences 109, 4556–4561 (2012). Garb, J.E., González, A. & Gillespie, R.G. The black widow spider genus Latrodectus (Araneae: Theridiidae): phylogeny, biogeography, and invasion history. Molecular Phylogenetics and Evolution 31, 1127–1142 (2004). Gasnier, T.R., Magnusson, W.E. & Lima, A.P. Foraging Activity and Diet of Four Sympatric Lizard Species in a Tropical Rainforest. Journal of Herpetology 28, 187-192 (1994). González-Suárez, M., Mugabo, M., Decencière, B., Perret, S., Claessen, D. & Le Galliard, J-F. Disentangling the effects of predator body size and prey density on prey consumption in a lizard. Functional Ecology 25, 158–165 (2011). Holding, M.L., Drabeck, D.H., Jansa, S.A. & Gibbs, H.L. Venom Resistance as a Model for Understanding the Molecular Basis of Complex Coevolutionary Adaptations. Integrative and Comparative Biology 56, 1032–1043 (2016). Hopp, B.H., Arvidson, R.S., Adams, M.E. & Razak, K.A. Arizona bark scorpion venom resistance in the pallid bat, Antrozous pallidus. PLoS ONE 12, e0183215 (2017). 7

Jansa, S.A. & Voss, R.S. Adaptive Evolution of the Venom-Targeted vWF Protein in Opossums that Eat Pitvipers. PLoS ONE 6, e20997 (2011). Krebs, C.J., Boonstra, R., Boutin, S. & Sinclair, A.R.E. What Drives the 10-year Cycle of Snowshoe Hares? BioScience 51, 25 (2001). Lima, S.L. & Dill, L.M. Behavioral decisions made under the risk of predation: a review and prospectus. Canadian Journal of Zoology 68, 619–640 (1990). McCabe, T.M. & Mackessy, S.P. Evolution of Resistance to Toxins in Prey. in Evolution of Venomous Animals and Their Toxins (eds. Gopalakrishnakone, P. & Malhotra, A.), 1–19 (Springer Netherlands, 2016). Mikolajewski, D.J. & Johansson, F. Morphological and behavioral defenses in dragonfly larvae: trait compensation and cospecialization. Behavioral Ecology 15, 614–620 (2004). Mukherjee, S. & Heithaus, M.R. Dangerous prey and daring predators: a review: Daring predators. Biol Rev 88, 550–563 (2013). Polis, G.A. & Hurd, S.D. Extraordinarily high spider densities on islands: flow of energy from the marine to terrestrial food webs and the absence of predation. Proceedings of the National Academy of Sciences 92, 4382–4386 (1995). Pritchett, A. H. Some Experiments in Feeding Lizards with Protectively Colored Insects. The Biological Bulletin 5, 271–287 (1903). Richardson, J.M.L. & Anholt, B.R. Morphological defenses against enemies. In: Encyclopedia of Animal Behavior (eds. Breed, M. & Moore, J.), 493-499 (Elsevier 2010). Rowe, A.H. & Rowe, M.P. Physiological resistance of grasshopper mice (Onychomys spp.) to Arizona bark scorpion (Centruroides exilicauda) venom. Toxicon 52, 597–605 (2008). Seghers, B.H. Schooling Behavior in the Guppy (Poecilia reticulata): An Evolutionary Response to Predation. Evolution 28, 486–489 (1974). Sherbrooke, W.C. Introduction to horned lizards of North America. (University of California Press, 2003). Spiller, D.A. & Schoener, T.W. An Experimental Study of the Effect of Lizards on Web-Spider Communities. Ecological Monographs 58, 57–77 (1988). Spiller, D.A. & Schoener, T.W. Lizards Reduce Spider Species Richness by Excluding Rare Species. Ecology 79, 503–516 (1998). Stebbins, R.C. A field guide to western reptiles and amphibians. (Houghton Mifflin, 2003). Sunagar, K. & Moran, Y. The Rise and Fall of an Evolutionary Innovation: Contrasting Strategies of Venom Evolution in Ancient and Young Animals. PLoS Genet 11, e1005596 (2015). Thompson, J.N. The geographic mosaic of coevolution. (University of Chicago Press, 2005). Thompson, J.N. Interaction and coevolution. (London: The University of Chicago Press, 2014). 8

Turnbull, A. L. Ecology of the True Spiders (Araneomorphae). Annual Review of Entomology 18, 305–348 (1973). Williams, B.L. Distribution, ontogenetic profile, and anti-predator efficacy of tetrodotoxin in two species of blue-ringed octopuses (Hapalochlaena lunulata and H. fasciata). (University of California, Berkeley, 2008).

Preying dangerously: Black widow spider venom resistance in sympatric

lizards

ABSTRACT

In the Western U.S., at least two lizard species (Elgaria multicarinata and

Sceloporus occidentalis) are sympatric with, and prey on, venomous western black widow spiders (Latrodectus hesperus). The effects of black widow spider venom (BWSV) on some mammals are well understood, but the effects on reptiles have not been investigated even though lizards are major predators of spiders. We evaluated potential resistance to BWSV in these two spider predator species and a third potentially susceptible lizard species (Uta stansburiana) known to be prey of widow spiders. We used whole-animal performance trials and comparative tissue histology at two concentrations of BWSV, “low” (1 mouse

LD50) and “high” (5 mouse LD50). Maximum performance was not significantly affected at the whole animal level in either E. multicarinata or S. occidentalis for any treatment, in contrast to U. stansburiana which suffered significant performance reductions in high dose treatments. Furthermore, E. multicarinata showed minimal muscle tissue response compared to the muscle tissue response seen in U. stansburiana. Sceloporus occidentalis, while not significantly affected at the whole animal level, had similar levels of muscle tissue damage and immune system infiltration as U. stansburiana. These data suggest that predator-prey 10 relationships between lizards and spiders are complex, possibly leading to physiological and molecular adaptations that allow some lizards to tolerate the effects of potentially dangerous arachnid venoms.

INTRODUCTION

Antagonistic relationships, like those between predator and prey, can exert intense selective pressures as these interactions have life and death outcomes

(Dawkins and Krebs 1979, Brodie and Brodie 1999). The dynamic nature of these interactions can lead to diverse outcomes, from local extirpation of one ecological partner to patterns of coevolution (Benkman et al. 2003, Thompson 2005, Endara et al. 2017). Thus, understanding how natural opponents interact is an important aspect of community ecology and evolutionary biology. Despite the prevalence of predator-prey relationships in natural systems, only a handful of these interactions are well understood (Hörnfeldt 1978, Krebs and Boutin 1995, Brodie and Brodie 1999).

Lizards are important consumers, and most species prey largely on arthropods (Buckner 1966, Polis and Hurd 1995, Howard et al. 1999, Bouam et al. 2016). Many arthropods are well-protected against predation via possession of defensive and offensive chemicals (Pasteels et al. 1983, Laurent et al. 2005); nevertheless, many lizards appear to have overcome these chemical defenses, preying on poisonous insects or venomous arachnids with regularity. For example, species of Phrynosoma specialize on noxious native ants (Schmidt et al. 11

1989, Sherbrooke 2003), the western banded gecko (Coleonyx variegatus) is known to prey on various scorpions in the southwestern U.S. (Parker and Pianka

1974), and the Texas alligator lizard (Gerrhonotus infernalis) will readily eat southern black widow spiders (Latrodectus mactans) when offered (Pritchett

1903).

Predator-prey interactions between spiders and lizards are particularly interesting because spiders are major predators of other arthropods (Schoener and Toft 1983, Pekár et al. 2012), and even of small vertebrates (Nyffeler and

Vetter 2018), including lizards (Wilson 1991). Yet lizards appear to be important predators of spiders (Spiller and Schoener 1988, Kartzinel and Pringle 2015) and have the capacity to regulate spider abundance (and possibly diversity) in ecological communities (Polis and Hurd 1995, Spiller and Schoener 1998). What is less clear, however, is whether lizard predators have evolved specialized adaptations to overcome the dangerous venoms of their spider prey. Here, we examine whether two lizards that prey on a venom-defended arachnid have developed venom resistance.

Southern alligator lizards (Elgaria multicarinata) are known to consume western black widow spiders (L. hesperus) (Cowles 1937, Cunningham 1956) and seek out their toxic egg sacs (Cunningham 1956, Buffkin et al. 1971).

Similarly, diet studies on the western fence lizard (Sceloporus occidentalis) suggest that they regularly consume spiders (Johnson 1965, Clark 1973), and we have observed S. occidentalis readily take L. hesperus in captivity (CRF and VLT 12 pers. obs.). While the importance of this venomous spider as part of their respective diets remains unclear, L. hesperus tends to be locally abundant and occupies the same microsites as both lizard species (e.g. under bark, in the openings of small burrows) (Wilson 1991, Stebbins 2003, Lettink and Patrick

2006, Bradley 2019). Even if predation events involving L. hesperus are rare, the strong potency of black widow spider venom (BWSV) may still be an important selective pressure for some lizard species.

The venom of L. hesperus has an intraperitoneal (IP) mouse LD50 (lethal dose for 50% of individuals) of 0.64 mg/kg (Daly et al. 2001); to put this in perspective, the venom of the western diamondback rattlesnake (Crotalus atrox)

has an IP mouse LD50 of 0.72 mg/kg and the venom of sidewinders (C. cerastes)

has less than one third the potency of BWSV at 2.08 mg/kg IP mouse LD50

(Keegan and MacFarlane 1963). Latrodectus hesperus is capable of venom metering, so envenomation events can be completely “dry” or can inject up to

0.110 mg of venom in a single bite (Nelsen et al. 2014), which is enough to kill a

160 g vertebrate. The average envenomation injects 0.016 mg of venom (Nelsen et al. 2014).

Like many widow spiders, L. hesperus is euryphagous, eating a broad variety of prey (Pekár et al. 2012); they mainly consume insects within the

Coleoptera, Hymenoptera, Orthoptera, and Dermaptera, in addition to land crustaceans (e.g., isopods) (Salomon 2011). Black widow spider venom contains at least three taxon-specific sets of protein elements; two of them, 13 latrocrustatoxins and latroinsectotoxins, target main prey items - crustaceans and insects (Grishin 1998, Salomon 2011). For both of these groups, the venom acts as a presynaptic neurotoxin, causing muscle spasms and destroying nerve cells

(Griffiths and Smyth 1973, Rohou et al. 2007); in crustaceans, synaptic vesicles and mitochondria suffer heavy damage (Burmistrov et al. 1997).

Another important protein in BWSV, ⍺-Latrotoxin, targets vertebrates.

Unlike insects and crustaceans, vertebrates appear only occasionally in the diet of widow spiders (Hódar and Sánchez-Piñero 2002, Salomon 2011), though the list of partially consumed vertebrates in Latrodectus webs is impressive, ranging from mammals to snakes (Konig 1987, Orange 1990, Wilson 1991, Beaman and

Tucker 2014, Nyffeler and Vetter 2018). ⍺-Latrotoxin forms cation channels in presynaptic membranes of the neuromuscular junction, forcing massive neurotransmitter release and simultaneously blocking the action of neuro- mediators (Meldolesi et al. 1986, Grishin 1998). This neurotransmitter release translates to clinical effects characterized most often by severe muscle cramping

(Bond 1999), muscle fasciculation, local paralysis, and pain lasting for hours to days (Isbister and Fan 2011, Warrell 2012). Envenomation can even lead to death in humans, albeit rarely (Timms and Gibbons 1986, Isbister and Fan 2011).

For a small lizard, tackling this relatively large but chemically well- defended meal may be risky. At worst, envenomation can result in death, and even non-lethal bites have the potential to injure or temporarily immobilize a lizard, rendering it vulnerable to predation or harsh environmental conditions. If E. 14 multicarinata and S. occidentalis engage regularly with dangerous prey such as black widows, they may have evolved countermeasures to reduce or negate the effects of BWSV.

To examine whether lizard species possess resistance to BWSV, we exposed three insectivorous lizard species that are sympatric with L. hesperus to standardized doses of BWSV and used a simple performance assay comparing pre-injection performance ability to post-injection performance ability. In addition, we used comparative histology on relevant portions of muscle tissue as a secondary measurement of resistance. We hypothesize that: (1) lizards that regularly encounter and possibly predate on L. hesperus would possess resistance to its venom (E. multicarinata, S. occidentalis); and (2) a lizard species

(Uta stansburiana) that has been known prey of L. hesperus (Wilson 1991) would be susceptible to BWSV. If resistance is present, we expect no significant reduction in post-injection velocity and no significant difference in muscle tissue response compared to saline-injected controls. In a susceptible lizard, we expect to see significant reductions in post-injection velocity and significant muscle tissue response compared to saline-injected controls.

MATERIALS AND METHODS

Animal collection and care

We collected 47 lizards (16 Elgaria, 16 Sceloporus, 15 Uta) (Fig.1) from field sites in California and Nevada (Table S1) and transported animals to the University of

Nevada Reno (UNR). We housed lizards individually in 5- or 10-gallon glass tanks with mesh lids and provided heat (40 Watt heat bulbs) and UV light (Repti-sun,

10.0 UVA/UVB, ExoTerra (Hagen), Montreal, CAN). We maintained lizards on a

12l:12d light cycle, in a room with temperatures at 75 F (+/- 5 F) and humidity near 35% (+/- 5%). We fed lizards crickets or mealworms with occasional calcium supplementation (Rep-Cal Calcium with Vitamin D, Los Gatos, CA, USA). We recorded snout-vent length (SVL) to the nearest 0.1 cm monthly and body mass to the nearest 0.05 g every two weeks for each lizard during captivity. All live animal procedures were approved by the Institutional Animal Care and Use

Committee.

Whole-animal performance

We established baseline velocity performance for each individual lizard prior to injections, and then evaluated changes in velocity performance following injections. We adapted our whole animal performance assay from the well- developed bioassay used to evaluate tetrodotoxin resistance in garter snakes

(Thamnophis), under the assumption that highly resistant animals will be able to maintain their normal performance capability when exposed to a standardized 16 dose of toxin while susceptible animals will display dramatic reductions in performance given a dose of equal potency (Brodie et al. 2002, Ridenhour et al.

2004).

To measure baseline and post-injection velocity, we sprinted lizards on a

2.2 meter racetrack constructed of aluminum alloy, high-density polyethylene

(HDPE) plastic and removable polyester (PET) carpet lining. We recorded lizard body temperatures in situ using an infrared (IR) heat gun (Etekcity, Anaheim, CA,

USA) immediately prior to each performance assay. We video-recorded performance assessments using a HERO4 GoPro (1060 linear video, 60 frames per second), and analyzed each video for velocity using Physlets Tracker software v5.1.1 (Brown 2018). We measured distance travelled for every two frames, and the Physlets software calculated velocity using our calibrated distance measurements and known framerate of videos. Our baseline velocity is an average of the top ten velocity values (after outlier removal). Once baseline sprint performance was established, we divided lizards into treatment groups (1

mouse LD50, 5 mouse LD50, and a control group receiving sterile saline, hereafter referred to as “low”, “high”, and “control”, respectively). We obtained BWSV from SpiderPharm (Yarnell, AZ) as lyophilized 0.5 mg pellets and reconstituted them to a 0.1 mg/μL stock using sterile saline. We then serially diluted this stock to concentrations appropriate to our mass-adjusted doses (0.01 mg/μL, 0.001 mg/μL, 0.0001 mg/μL). 17

We injected lizards intramuscularly in the dorsal thigh of the right hind leg using a 3/10 cc disposable insulin syringe with a 31 gauge needle (UltiCare,

Excelsior, MN, USA). We attempted to keep injection volumes at or below 0.25% of body weight by volume (as per Diehl et al. 2001). Following injections, we performed three performance assessments: immediately after injection, 24 hours after injection, and 48 hours after injection. Upon completion of the final performance assessment, we monitored lizards for 4 days before humanely euthanizing each animal and harvesting the hind legs for histological examination.

Finally, we deposited each lizard in the Herpetological Collection of the University of Nevada, Reno, Museum of Natural History (UNR) (Table S1). Note that space constraints in our live animal space necessitated two separate trials (2017, 2018) and the results of each were pooled.

To ensure that our venom was acting as intended, we injected four mice

(Mus musculus) with a 1 mouse LD50 dose of BWSV. We provided mice with pain medication (Buprenorphin SR-Lab, 1 mg/kg) and monitored them for 24 hours before we humanely euthanized them. We used the grimace factor scale

(Langford et al. 2010) to quantify mouse discomfort and ensure that pain medication was working as intended.

Analyses

We analyzed all data in R v3.6.1 (R Core Team 2008). We first conducted linear mixed-effect regression models (LMM) with post-injection velocity ratio as the 18 response variable and a variety of fixed effects (treatment, species, time, trial, body condition, sex, temperature, volume of fluid injected as a percentage of body weight). We included interactions between variables, and individual as a random effect. To select the best model, we used Akaike’s Information Criterion

(AIC; Burnham and Anderson 2002), and then performed additional LMMs at the species level to evaluate finer scale effects.

Comparative histology

We harvested muscle tissue (dorsal segments of the femorotibialis externus and iliofemoralis) immediately following euthanasia from the injection site of the right leg; a mirror sample was obtained from the uninjected left leg for each individual as a negative control. Formalin preserved tissues were prepared and stained

(hematoxylin and eosin) by IDEXX Laboratories (Sacramento, CA). We compared treated muscle tissue from the right leg to untreated muscle tissue from the left leg of the same individual using ImageJ v1.52a (Schneider et al. 2012). We captured between three and five images per slide at 100x magnification per limb.

We analyzed each image using a randomized grid system, excluding grids from random selection if more than 10% edge white space was present. We quantified tissue damage using percent damaged area (PDA) (adapted from Woodell et al.

2010) and immune system response was quantified with nuclear counts (adapted from Giovannelli et al. 2018). Muscle was considered damaged if the muscle fiber was clearly undergoing necrosis or if there was evidence of recent regeneration 19

(Fig. 3). We performed nuclear counts (excluding red blood cell nuclei) and distinguished between “normal” nuclei (nuclei found where expected within muscle fibers) and “abnormal” (i.e. leukocytes, central nuclei); we also included a measure of the ratio of normal to abnormal nuclei (percent normal nuclei, or

PNN).

Percent damaged area and nuclear counts were performed using standard

ImageJ (Abramoff et al. 2004) and add-on Cell Counter. We averaged PDA and nuclear count variables (normal, abnormal, and PNN) across grids within images, with up to five replicates per limb. We used t-tests to evaluate significance of differences between left and right limbs grouped by species and treatment, followed by ANOVAs.

RESULTS

Whole animal performance

All lizards injected with saline or venom behaved normally in their enclosures and did not exhibit obvious ill-effects or discomfort during the monitoring period (i.e. no biting at treated limbs, no visible swelling of treated limbs, no observable difficulties with locomotion, and no appetite suppression); all individuals survived treatment.

The best linear mixed-effect (LMM) model to explain variation in our dependent variable, post-injection velocity, included species, treatment, and time as independent variables with individual as a random effect (Table 1). Additional 20 variables were either not significant in any model (trial, sex, body condition, volume injected) or were significant only in poorly performing models

(temperature). Given the highly significant differences in post-injection velocity between species (LMM, 휒2 = 54.369, df = 2, p < 0.0001), we conducted linear regressions for each species using variables from the top three LMM models in

Table 1 and ranked them using AIC (Table 2).

The LMM that best described the variation in post-injection velocity for E.

2 2 multicarinata (R c = 0.46, df = 8) included time (LMM, 휒 = 50.73, df = 2, p <

0.0003) and treatment (LMM, 휒2 = 3.71, df = 2, p > 0.15), though treatment did not have a significant effect. Elgaria multicarinata that were injected with a low or high BWSV treatment had similar post-injection velocities as those injected with the control (saline solution) (Fig. 2, Table 3).

2 The best LMM for S. occidentalis (R c = 0.11, df = 2) included only treatment, which did not have a significant effect (LMM, 휒2 = 4.20, df = 2, p >

0.12). Average reduction in post-injection velocity was similar across treatment groups (Table 3). Compared to E. multicarinata, the post-injection velocity in S. occidentalis was more variable (Fig. 2).

2 The best LMM for U. stansburiana (R c = 0.73, df = 8) included time and treatment, which were both significant (LMM, 휒2 = 107.36, df = 3, p < 0.0001; 휒2 =

11.091, df = 2, p < 0.003). Uta stansburiana had higher reductions in post- injection velocity than both E. multicarinata and S. occidentalis, and this pattern extended across all treatment groups (including control); however, only U. 21 stansburiana in the high venom group had significantly reduced post-injection velocities (Fig. 2, Table 3).

Temperature, though an important factor in the performance abilities of ectotherms (Angilletta et al. 2002), was not a descriptive factor for sprint performance in our trials, and was not retained in any top models (Table 2). All three focal species had body temperatures in the range of their preferred activity range during performance assessments (E. multicarinata, 푥̅ = 28.21° C, SE =

0.77, Kingsbury 1994; S. occidentalis, 푥̅ = 35.89° C, SE = 2.98, Brattstrom 1965;

U. stansburiana, 푥̅ = 35.84° C, SE = 2.935, Goller et al. 2014), and temperature was not retained in the best performing LMM model or in any species models

(Table 2,3).

Comparative histology

All negative control muscle tissues had similar statistics across species, with limited abnormal nuclei counts (<13) and PDA (<1.1%) as expected. Muscle tissues that received control injections were not significantly different from uninjected control tissues except for S. occidentalis, which had significantly higher abnormal nuclei counts, lower PNN, and higher PDA (Table 4). Normal nuclei when paired within species and treatment were not significantly different between control and injected muscle tissue (Table 4), except for the S. occidentalis low venom group. 22

ANOVAs for all dependent variables included only data for right legs and examined the effect of species, treatment, and the interaction between treatment and species. Additional variables were tested but were not near significance for any ANOVA (trial, sex, body condition, volume injected, temperature) and so were dropped. Across all species, normal nuclei were not significantly affected by

species (F2,229 = 2.95, p > 0.05) or treatment (F2,229 = 0.49, p > 0.5), but were

significantly affected by the interaction between species and treatment (F4,229 =

2.42, p < 0.05). Abnormal nuclei were significantly affected by species (F2,233 =

9.68, p < 0.0001), treatment (F2,233 = 4.87, p < 0.01), and the interaction between

species and treatment (F4,229 = 2.45, p < 0.05). Control and low venom treatment tissue had similar numbers, but high venom treatment tissue had significantly elevated abnormal nuclei counts (Tukey HSD, diff = 19.73, p < 0.05). Species level effects were driven mostly by E. multicarinata, which had reduced abnormal nuclei effects compared to both U. stansburiana and S. occidentalis (Tukey HSD, diff = 20.08, adj p < 0.05), especially when comparing high venom treatment groups between species (Tukey HSD, diff = 48.67, adj p < 0.01).

PNN was significantly affected by species (F2,229 = 10.29, p < 0.0001) and

treatment (F2,229 = 5.26, p < 0.01), but not the interaction between species and

treatment (F4,229 = 2.03, p > 0.05). High venom treated individuals had significantly lower PNN than low or control (Tukey HSD, diff = 10.58, p < 0.05). Sceloporus occidentalis had significantly lower PNN than both E. multicarinata and U. stansburiana regardless of treatment (Tukey HSD, diff = 12.34, p < 0.01). PDA 23

was significantly affected by species (F2,229 = 9.25, p <0.005), treatment (F2,229 =

8.29, p < 0.005), and the interaction between species and treatment (F4,229 = 3.20, p < 0.05). Elgaria multicarinata had significantly lower PDA compared to S. occidentalis (Tukey HSD, diff = 11.88, p < 0.0001), especially in high venom treatments (Tukey HSD, diff = 23.88, p < 0.0001). The high venom treatment group had significantly higher PDA across species compared to low and control groups (Tukey HSD, diff = 8.88, p < 0.01).

We found only slight evidence of muscle tissue response when comparing untreated and treated muscle tissue for E. multicarinata. Additionally, we found no significant difference in right leg muscle tissue between saline controls and venom treatments, with treated limbs averaging between 0.8% and 3.3% PDA across treatments (Fig. 4, Table 4). While these differences were not significant for any treatment (Table 4), Elgaria multicarinata did have slightly higher abnormal nucleus counts for low venom treated muscle tissue (t = -2.08, df =

39.4, p < 0.05; Fig. 4B).

Sceloporus occidentalis had significant differences in most muscle tissue response variables. Normal nuclei counts were significantly increased between untreated and low venom treated muscle tissue (t = -2.47, df = 37.28, p < 0.02;

Fig. 4A), but no significant differences were seen between other treatments

(Table 4). Abnormal nuclei counts were significantly elevated in all treated muscle tissue compared to untreated tissue (control: t = -2.86, df = 24.42, p < 0.01; low dose: t = -2.96, df = 30.33, p < 0.01; high dose: t = -5.58, df = 29.84, p < 0.0001; 24

Fig. 4B). PNN was similarly affected in all treated muscle tissue, showing significant reductions compared to untreated muscle tissue (control: t = 3.21, df =

32.13, p < 0.005; low dose: t = 2.27, df = 45.46, p < 0.05; high dose: t = 6.64, df =

31.78, p < 0.0001; Fig. 4C). This pattern continued for PDA, with all treated muscle tissue showing significant increases compared to untreated muscle tissue; this effect was especially strong in high treatment muscle tissue (control: t

= -2.44, df = 23.52, p < 0.05; low dose: t = -2.83, df = 29.11, p < 0.01; high dose: t

= -4.05, df = 29.01, p < 0.005; Fig. 4D).

Uta stansburiana had significant differences between untreated and venom treated muscle tissue for all muscle tissue response variables, and control tissue had no significant differences compared to untreated tissue (Fig. 4, Table

4). Abnormal nuclei increased significantly for venom treatments (low dose: t = -

2.50, df = 34.12, p < 0.05; high dose: t = -3.60, df = 28.32, p < 0.005; Fig. 4B).

PNN was significantly reduced for venom treatments (low dose: t = -2.50, df =

34.12, p < 0.05; high dose: t = -3.60, df = 28.32, p < 0.005; Fig. 4C). Finally, PDA was significantly increased for venom treatments (low dose: t = -2.78, df = 29.37, p < 0.01; high dose: t = -3.15, df = 28.06, p < 0.005; Fig. 4D).

DISCUSSION

For animals that consume venomous prey, the ability to avoid or withstand the effects of envenomation may be a critical adaptation (Zlotkin et al. 2003, Rowe and Rowe 2008, McCabe and Mackessy 2016). Thus, we expect that the high 25 costs of engaging with venomous prey will promote defensive adaptations, whether behavioral or physiological (McCabe and Mackessy 2016, Farrell et al.

2018). Here, we used whole animal performance assessments, coupled with comparative histology, to evaluate resistance to BWSV in three insectivorous lizard species sympatric with L. hesperus.

Our results suggest that two lizard species that are likely to encounter and consume L. hesperus possess resistance to BWSV. Elgaria multicarinata displays an impressive ability to tolerate BWSV at the whole animal level. They functioned at or near 100% of their baseline velocity, even when exposed to a dose five

times the mouse LD50 (Fig. 2). Given the evidence that E. multicarinata consumes

L. hesperus regularly, it is not surprising that these lizards show the strongest level of resistance, both at the whole animal level and at the tissue level, with no significant difference in muscle damage or immune cell infiltration between untreated and treated muscle tissue or control and treatment muscle tissue

(Table 4).

While S. occidentalis did not have a significant effect of treatment at the whole animal level, the significant effects at the tissue level suggest that their resistance to BWSV is not as sophisticated or extreme as that of E. multicarinata.

This disparity between whole animal and tissue level resistance could indicate that S. occidentalis prey on L. hesperus less often compared to E. multicarinata.

However, even five times the mouse LD50 only slowed S. occidentalis by approximately 11% on average (Table 2). Given their ability to run at speeds near 26 their baseline even though their muscle tissue was significantly damaged, it seems probable that their intermediate level of resistance is suitable to avoid severe ecological effects of envenomation in natural situations.

In contrast, we expected U. stansburiana to be affected by BWSV given their status as occasional prey to L. hesperus. This prediction was supported,

with U. stansburiana significantly affected by the highest treatment dose (5 LD50), showing reduced performance capabilities coupled with significantly higher muscle fiber damage and immune system infiltration for treated muscle tissue compared to untreated muscle tissue. The dramatic reduction in sprint speed in the high treatment group (~72%, Table 2) would almost certainly translate to significant ecological effects, impacting their ability to evade predation and to effectively capture prey or perhaps exposing them to unfavorable environmental conditions.

Given the apparent gradient of resistance, from very high in E. multicarinata, to intermediate in S. occidentalis, to low in U. stansburiana, resistance to BWSV clearly varies across species. However, it remains unclear whether this variation is due to the intensity of predator-prey interactions, lineage-specific differences (e.g. E. multicarinata is an Anguid, compared to the

Phrynosomatids S. occidentalis and U. stansburiana), or other physiological mechanisms. In addition, the similarities that we see in the post-injection velocity

between control (saline) and low (1LD50) treatment groups in all three species could be a signal that lizards in general possess a baseline or low-grade 27 resistance to presynaptic neurotoxic arachnid venoms. Both this variation in resistance and the possibility of ancestral resistance warrant further investigation.

Elgaria multicarinata are often found in suburban environments (Spear et al. 2017, pers. obs.), and L. hesperus is known to have high population densities in urban areas (Vetter et al. 2012). Therefore, the ability to consume this abundant but well-defended prey while suffering no ecologically relevant effect of envenomation is particularly useful. Further investigation is needed to understand whether this resistance can be overcome by higher concentrations of BWSV, whether there are population-level differences in BWSV resistance, and the underlying physiological processes of BWSV resistance in these species. From a mechanistic perspective, the lack of histological damage in E. multicarinata may provide a starting point for future investigations that seek to uncover the physiological method of resistance.

Predators that deal with chemically defended prey must either avoid or mitigate those defenses, whether through behavioral changes in prey recognition or handling techniques (Mukherjee and Heithaus 2013, Farrell et al. 2018) or through physiological changes that allow them to reduce or block the effects of toxins and venoms (Brodie and Brodie 1990, Rowe and Rowe 2008). We know that toxin and venom resistance has evolved in many systems, sometimes allowing predation on prey that possess neurotoxic venoms (Rowe and Rowe

2008) or neurotoxic secretions (Brodie and Brodie 1990) or allowing escape from predators that possess hemorrhagic venom (de Wit and Weström 1987). Clearly, 28 there are a variety of adaptations that can allow predators or prey to escape the effects of toxins and venoms. Occasionally, these predator-prey interactions lead to a coevolutionary arms race, where a cyclical escalation in offensive and defensive adaptations continues until some limit is reached or one party somehow “escapes” the cycle (Thompson 2005, Hanifin et al. 2008). Though there are many predator-prey systems involving defended prey or venomous predators, only rarely have these been shown to be coevolutionary. This work is a foundational step in understanding whether and how some lizards have entered a coevolutionary arms race with dangerous arachnid prey.

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Table 1. Mixed linear regression models, with post-injection velocity performance ratios as the dependent variable and allowing intercepts to vary at the individual level. The variables are defined as follows: “rx” refers to treatment

(control = saline, low = 1LD50, high = 5LD50); “species” refers to species of lizard (Elgaria multicarinata, Sceloporus occidentalis, Uta stansburiana); “time” refers to time post-injection (before, immediately after, 24 hours after, 48 hours after); “temp”, or temperature, is the body temperature of an individual at the time performance was assessed; “trial” refers to trial 1 or trial 2 as lizards were assessed in two distinct groups; “sex” is the sex of the lizard, male or female; “body con” is body condition, estimated by dividing mass by SVL (g/cm); “vol inj”, or volume injected, is the volume of fluid injected into the lizard as a percentage of the lizard’s body weight.

Linear Mixed Effect Models: Post-injection Velocity

Rank Fixed Effects Interactions df R2c AIC

1 rx, species, time None 10 0.506 -227.6

2 rx, species, time, temp None 11 0.519 -222.2

3 rx, species, time, temp rx:species 15 0.525 -202.3

4 rx, species None 7 0.243 -184.3

5 rx, species, time, temp, trial, sex rx:species, temp:species, temp:time 21 0.594 -161.5 rx, species, time, temp, trial, sex, rx:species, temp:time, temp:spec, 6 25 0.598 -157.9 body con, volume injected body con:vol injected

Table 2. Mixed linear regression models at the species level, with post-injection velocity performance ratios as the dependent variable and allowing intercepts to vary at the individual level. The fixed effects are defined as follows:

“rx” refers to treatment (control = saline, low = 1LD50, high = 5LD50); “time” refers to time post-injection (before, immediately after, 24 hours after, 48 hours after), and “temp”, or temperature, is the body temperature of an individual at the time performance was assessed. Only the top two models for each species are included.

Species Level Mixed Effect Linear Models

Rank Model Name Fixed Effects Interactions df R2c AIC

1 Elgaria 2 rx, time none 8 0.46 -111.63

2 Elgaria 1 rx, time, temp none 9 0.50 -106.63

1 Sceloporus 2 rx none 5 0.11 -57.8

2 Sceloporus 1 rx, time none 8 0.33 -50.18

1 Uta 1 rx, time none 8 0.73 -56.31

2 Uta 0 rx, time, temp none 9 0.72 -46.91 36

Table 3. Comparison of mean velocity values (cm/s) for each species, along with post-injection velocities by treatment group (Control: saline; Low: 1LD50; High: 5LD50), and the percent reduction in post-injection velocity. Note that Uta stansburiana has the highest percent reduction of velocity and is the only species with a significant effect of treatment on velocity (*).

Changes in mean velocity by species and treatment Species 풙̅ before (cm/s) SE Treatment 풙̅ after (cm/s) SE % Change p-value 141.08 4.41 control (n=4) 157.34 15.86 +11.53% 0.387

Elgaria multicarinata 120.9 6.68 1LD50 (n=6) 133.40 9.17 +10.34% 0.300

142.18 9.22 5LD50 (n=6) 130.20 7.50 -8.43% 0.338

158.66 17.86 control (n=4) 135.92 16.05 -14.33% 0.381

Sceloporus occidentalis 154.21 17.76 1LD50 (n=5) 159.38 10.9 +3.35% 0.812

163.47 13.38 5LD50 (n=5) 133.84 22.63 -18.13% 0.299 168.41 30.08 control (n=4) 113.91 23.06 -32.36% 0.204

Uta stansburiana 163.50 6.53 1LD50 (n=5) 121.70 5.95 -25.57% 0.002*

175.32 14.18 5LD50 (n=5) 101.99 8.11 -41.83% 0.004*

Table 4. Comparison of muscle tissue response by leg, treatment, and species. Variables include “norm”, which is a count of normal muscle fiber nuclei; “abnorm”, which is a count of abnormal nuclei (leukocytes or central nuclei); “PNN”, or percent normal nuclei, which is a ratio of normal nuclei to total nuclei, which gives us a measure of both how normal nuclei are responding and how immune system is responding via abnormal (white blood cell) increase; and “PDA”, or percent damaged area, which is a measurement of damaged muscle fiber. Significance denoted by * (p < 0.05), ** (p < 0.01), and *** (p < 0.001). Note that significant tissue effects were found in Sceloporus and Uta right legs across many variables, but only one in Elgaria.

Muscle Tissue Response Treatment L 풙̅ norm R 풙̅ norm L 풙̅ abnorm R 풙̅ abnorm L 풙̅ PNN R 풙̅ PNN L 풙̅ PDA R 풙̅ PDA Species (n) (SE) (SE) (SE) (SE) (SE) (SE) (SE) (SE)

Control 54.9 (3.8) 49.1 (2.6) 8.9 (1.2) 19.7 (5.6) 79.87 (2.7) 73.7 (5.1) 0.6 (0.2) 3.3 (1.7) (25)

Low (30) 62.8 (3.5) 8.0 (1.1) 7.6 (1.1) 13.5* (2.6) 88.59 (1.2) 81.0* (1.2) 0.3 (0.1) 2.1 (1.1) Elgaria 82.3 (3.4) 77.6 (3.2) 0 0.8 (0.6) multicarinata High (23) 43.9 (3.4) 44.7 (3.7) 10.0 (2.2) 13.0 (2.3)

Control 52.7 (5.0) 50.1 (4.5) 11.4 (2.4) 50.9* (13.6) 84.3 (2.7) 63.7** (5.8) 1.1 (0.5) 11.4* (4.2) (24)

Low (30) 34.1 (1.6) 44.8* (4.1) 9.2 (1.5) 38.0** (9.6) 80.1 (2.7) 67.5** (4.9) 0.2 (0.1) 5.4** (1.8) Sceloporus Sceloporus occidentalis High (30) 49.9 (3.9) 38.9 (4.0) 5.8 (1.2) 61.6*** (9.9) 90.9 (1.4) 48.6*** (6.2) 0.1 (0.1) 24.7** (6.1)

Control 53.2 (4.7) 45.9 (2.9) 13.0 (3.0) 9.5 (2.0) 83.5 (2.9) 84.1 (2.6) 0.2 (0.2) 0.3 (0.2) (17)

Low (30) 50.8 (2.5) 50.1 (3.6) 9.4 (2.2) 28.6* (7.3) 86.3 (2.3) 72.3** (1.3) 0.2 (0.1) 5.1** (1.8) Uta Uta 64.0*** (5.6) 0.4 (0.2) 16.1** (5.0) stansburiana High (28) 58.0 (3.8) 55.5 (4.7) 7.2 (1.1) 623** (15.2) 89.8 (1.35) 38

Table S1. Lizards used in black widow spider venom performance trials, with species, museum voucher number (UNR), collector number (Coll ID), general locality (county and state), sex, SVL (cm) and mass (g).

Species Museum ID Coll ID Location (Co. / State) Sex SVL (cm) Mass (g) Elgaria Unassigned CRF 2503 Yolo Co., CA M 13.5 39 multicarinata Unassigned CRF 3018 Yolo Co., CA M 13.5 47.5 Unassigned CRF 3142 Yolo Co., CA F 11.6 28 UNR 9818 CRF 3143 Yolo Co., CA M 11.5 31.35 UNR 9819 CRF 3144 Yolo Co., CA F 11 23.45 Unassigned CRF 3145 Yolo Co., CA M 11.4 30 UNR 9817 CRF 3241 Yolo Co., CA M 13 41 UNR 9823 CRF 3242 Yolo Co., CA M 11.3 28.4 UNR 9820 CRF 3263 Yolo Co., CA M 9 9.95 UNR 9822 CRF 3264 Yolo Co., CA F 10.6 22 Unassigned CRF 3096 Santa Cruz Co., CA F 12.2 38.5 Unassigned CRF 3148 Ventura Co., CA M 13.1 55.5 UNR 9816 CRF 3178 Ventura Co., CA M 12.1 46 UNR 9821 CRF UNK1 - M 12 29 Elgaria Unassigned CRF 3077 Fresno Co., CA M 12.5 32 coerulea Unassigned CRF 3113 Santa Cruz Co., CA M 11 25.5 Sceloporus Unassigned CRF UNK2 - F 6.5 11.7 occidentalis Unassigned KM 012 Washoe Co., NV F 5 6.5 UNR 9836 VLT 004 Washoe Co., NV M 6.7 14.5 Unassigned VLT 006 Washoe Co., NV M 6.8 18.5 Unassigned VLT 007 Washoe Co., NV M 7.5 21 Unassigned VLT 008 Washoe Co., NV M 7.8 19 Unassigned VLT 009 Washoe Co., NV M 8 18.5 Unassigned VLT 010 Washoe Co., NV F 8 1.5 UNR 9835 VLT 011 Washoe Co., NV M 7.1 15

SVL Species Museum ID Coll ID Location (Co./State) Sex Mass(g) (cm) Sceloporus UNR 9829 VLT 015 Washoe Co., NV M 7.3 11.7 occidentalis UNR 9834 VLT 026 Washoe Co., NV M 7.5 23.3 UNR 9830 VLT 027 San Luis Obispo Co., CA M 7.6 16.15 UNR 9832 VLT 028 San Luis Obispo Co., CA M 7.4 12.9 UNR 9833 VLT 029 San Luis Obispo Co., CA M 7.7 15.1 UNR 9837 VLT 030 San Luis Obispo Co., CA M 6.9 18.05 UNR 9831 VLT 031 San Luis Obispo Co., CA M 7.5 16 Uta Unassigned KM 001 Churchill Co., NV F 4.2 3.5 stansburiana Unassigned KM 002 Churchill Co., NV F 4.2 3 Unassigned KM 005 Churchill Co., NV M 4.5 2.5 Unassigned KM 007 Churchill Co., NV F 4.2 2.5 Unassigned KM 008 Churchill Co., NV M 4.9 3 Unassigned KM 010 Churchill Co., NV F 4.7 3 Unassigned VLT 002 Churchill Co., NV M 4.4 3 Unassigned VLT 003 Churchill Co., NV M 5.5 3 UNR 9838 VLT 017 Churchill Co., NV M 5.4 5.1 UNR 9839 VLT 018 Churchill Co., NV M 4.7 4.2 UNR 9840 VLT 019 Churchill Co., NV F 5.2 5.3 UNR 9841 VLT 020 Churchill Co., NV M 4.5 3.85 UNR 9842 VLT 023 Churchill Co., NV F 4.65 4.15 UNR 9843 VLT 032 Churchill Co., NV M 4.1 2.95 UNR 9844 VLT 033 Churchill Co., NV M 4.8 3.4

Figure 1. Focal species. Clockwise from top left: Elgaria multicarinata, Sceloporus occidentalis, Uta stansburiana, Latrodectus hesperus. Elgaria multicarinata (Anguidae) is a medium, elongate-bodied lizard native to the western U.S. (Stebbins 2003) that feeds on a variety of invertebrates, including western black widows and their egg sacs (Cowles 1937, Cunningham 1956), small vertebrates, and eggs (Stebbins 2003). Sceloporus occidentalis (Phrynosomatidae) is a common, relatively small, stout lizard native to the western U.S. that feeds on a variety of invertebrates, including spiders (Clark 1973, Stebbins 2003). Uta stansburiana (Phrynosomatidae ) is a small lizard native to the western U.S. and northern Mexico that feeds on a variety of invertebrates, including spiders (Stebbins 2003). Latrodectus hesperus (Theriididae) is a venomous spider known more commonly as the western black widow; it is native to the southwestern U.S. (Garb et al. 2004) and feeds mostly on invertebrates, crustaceans, and occasionally small vertebrates (Salomon 2011, Nyfeller and Vetter 2018).

Figure 2. Panels showing changes in post-injection velocity ratio over time. Percent performance is sprint performance scaled against baseline ability. (A) plots have been grouped by species (6 ≥ n ≥ 4) to visualize how percent performance changes in response to treatment (“Control” = saline,

“Low” = 1LD50, “High” = 5LD50) over time (immediately after injection, 24 hours after, 48 hours after). (B) plots have been grouped by treatment to show variation in response to treatment across species. Notice that in the bottom right panel, which is “high” treatment, U. stansburiana are the only species to have a statistically significant reduction in performance (*)

Figure 3. Histological images of lizard muscle tissue taken at 100x magnification. In A-C, images have been cropped to highlight specific morphological characteristics. Arrows point to a raft of nucleated red blood cells with associated white blood cells (A) and regenerating muscle fibers (C). In (B), the focal field is filled with necrotic fibers. Panel (D) highlights differences in muscle tissue response by species (columns) and treatment (rows). Notice that E. multicarinata has tissue with similar appearance for all treatments, while both S. occidentalis and U. stansburiana show muscle tissue response in the form of necrosis and white blood cell increases in venom treatments.

* *

Figure 4. Boxplots of various muscle tissue response variables. “L” refers to values from untreated tissue harvested from the left leg, used to establish baseline values for variables in the absence of treatment. “R” refers to values from treated tissue harvested from treated muscle tissue of the right leg (“Control” = saline, “Low” = 1LD50, “High” = 5LD50), used to quantify response of treatments. (A) shows normal nuclei counts, (B) shows abnormal nuclei counts, (C) shows percent normal nuclei (PNN) and (D) shows percent damaged area (PDA). Significant differences between saline control and treated tissue are denoted by * (p < 0.05), ** (p < 0.001), or *** (p <0.0001).

Spider venom resistance in lizards lingers across branches

ABSTRACT Lizards are major predators of arthropods, many of which are chemically defended. Spiders, in particular, represent potentially dangerous but important prey for lizards. We have previously shown that at least two lizard species, the southern alligator lizard (Elgaria multicarinata: Anguidae) and western fence lizard (Sceloporus occidentalis: Phrynosomatidae), possess some degree of resistance to the potent venom of sympatric western black widow spiders

(Latrodectus hesperus). These two lizard species are distantly related (occurring on different parts of the lizard evolutionary tree), begging the question as to whether resistance to black widow venom represents local adaptation or a deeply rooted trait found throughout the squamate lineage. Thus, we performed whole animal assays of resistance to black widow spider venom (BWSV) and comparative histology on species representing a wider phylogenetic and geographic array of squamate taxa. We tested two species that do not co-occur with L. hesperus, one insectivorous (Takydromus sexlineatus: Lacertidae) and one herbivorous (Iguana iguana: Iguanidae) and an insectivorous species

(Coleonyx variegatus: Eublepharidae) that is sympatric with L. hesperus. We found that post-injection velocity was not significantly affected at the whole animal level in either insectivorous species (T. sexlineatus or C. variegatus),

45 despite the fact that these species represent divergent parts of the lizard phylogeny, live in different parts of the world, and should represent different histories of association with widow spiders. In stark contrast, the herbivorous species (I. iguana) showed drastically reduced sprint performance when exposed to BWSV. Comparative histology results showed some muscle tissue damage in

T. sexlineatus and C. variegatus, but this was moderate compared to the muscle tissue damage shown in I. iguana. Combining these data with our previous work and mapping these traits onto the lizard phylogeny suggests that all lizards possess some baseline levels of resistance to BWSV compared to mammals. In addition, placing these data into a phylogenetic context indicates that resistance to BWSV has been elevated in E. multicarinata, the only known lizard predator of western black widows, and conversely, resistance has been lost in I. iguana, the only herbivore in our study. These results provide evidence that ecological interactions with widow spiders may drive patterns of venom resistance across the lizard phylogeny.

INTRODUCTION

Over 400 million years ago, predatory arachnids like scorpions and spiders developed venom to aid in subduing prey (Sunagar and Moran 2015). To this day, the vast majority of spiders still employ venom as a principle tool in predation (Sunagar and Moran 2015). Predator-prey interactions like those between these spiders and their prey can drive coevolutionary relationships

(Dawkins and Krebs 1979). When the potential cost of these antagonistic interactions is death, selective pressure can be intense (Brodie and Brodie 1999,

McCabe and Mackessy 2016).

In systems where one ecological partner possesses venom, these predator-prey interactions can lead to the development of venom or toxin resistance (McCabe and Mackessy 2016). If these interactions take place early in evolutionary history, then the resistant trait could potentially be maintained in subsequent descendants at a baseline level. For example, most garter snakes

(Thamnophis), and even their relatives, evolved a baseline level of resistance to tetrodotoxin (TTX) in prey (Motychak et al. 1999), probably through chance mutations in a handful of important genes (McGlothlin et al. 2016). Previous work

(Chapter 1) demonstrates that some lizards possess resistance to 훼-latrotoxin, which is the main vertebrate-specific neurotoxin present in black widow spider venom (BWSV). In addition to evidence of resistance to 훼-latrotoxin, we found that resistance varied across species.

Latrotoxins are a medically important neurotoxic component of venoms possessed by many spiders in the family Theridiidae (Garb and Hayashi 2013).

They have been used heavily in research over the last 40 years to understand the actions of nerve cells (Silva et al. 2009). Latrotoxins (or even the genes encoding latrotoxins) have been found in several genera within Theridiidae (Garb and

Hayashi 2013), but only a handful of spiders possess medically relevant latrotoxins: Parasteatoda, Steatoda and Latrodectus (Garb et al. 2004).

These genera are part of the Latrodectinae, which evolved in the early

Cretaceous (Liu et al. 2016). Thus, it is likely that latrotoxins have been around for

30 to 50 million years. While there is some debate around the squamate phylogeny, it is generally agreed that lizards were common by ~150 mya, in the

Middle Jurassic (Hutchinson et al. 2012), long before the Latrodectinae. Given that less than 2% of extant squamates are herbivorous (Espinoza et al. 2004) and that insectivory existed in ancient lizards (Modesto et al. 2009), it is likely that some of these early lizards were insectivorous and encountered, and possibly even preyed on, the ancestors of Latrodectus.

To determine whether resistance to BWSV is an ancestral trait and has been broadly retained within Squamata, we selected a small set of disparately related lizard taxa and exposed them to BWSV using techniques from Chapter 1.

We chose a lacertid insectivore (Takydromus sexlineatus) native to southeast

Asia (Arnold 1997), an iguanid herbivore (Iguana iguana) with an extensive range from Mexico through South America and the Caribbean (Rand et al. 1990), and

48 an eublepharid insectivore native to the Southwestern U.S. (Coleonyx variegatus)

(Fig. 1). We then added data from Chapter 1 which represent two additional families (Phrynosomatidae, Anguidae). Our species thus represent four major squamate groups (Anguimorpha, Gekkota, Iguania, and Lacertoidea; after Pyron et al. 2013 and Zheng and Wiens 2016; Fig. 2).

Given the evidence from Chapter 1 that resistance to ecologically relevant doses of BWSV is present even in a lizard that was presumed susceptible (Uta stansburiana), combined with the cosmopolitan distribution of 훼-latrotoxin possessing spiders (Garb et al. 2004), we predict that baseline resistance will have been retained in many squamates. We can further predict that baseline resistance manifests at the whole animal level, but not necessarily the tissue level and that baseline resistance should defend against ecologically relevant doses

(i.e., 1LD50) but not necessarily higher doses (i.e., 5LD50). If baseline resistance is present, we predict no reduction in sprint speed for low dose treatments, but significant reductions in sprint speed for high dose treatments (e.g., S. occidentalis response from Chapter 1).

Second, we hypothesize that lizards which preferentially consume L. hesperus or other venomous widow species will have elevated resistance levels in response to their interactions with dangerous prey. We would expect to see resistance at both the tissue and whole animal level, even at high doses (i.e.

5LD50). If resistance is elevated, then we predict that there will be no reduction in

49 sprint speed and no muscle tissue response (e.g., E. multicarinata response from

Chapter 1).

If there are lizards without baseline or elevated resistance, then we predict that sprint performance would be reduced for both low and high venom treatments, as well as muscle tissue response in both treatments. So there are three potential outcomes: resistance absent, baseline resistance, and elevated resistance, and the criteria we use to determine this is based on (1) where resistance is present: whole animal or tissue level and (2) at what dose resistance

is present: 1LD50 or 5LD50.

MATERIALS AND METHODS

Animal collection and care

We obtained 35 lizards (14 juvenile Iguana, 12 adult or sub-adult Coleonyx, 9 adult Takydromus) from licensed commercial vendors or field sites in Nevada and

Arizona under permits to CRF and VLT (Table S1) and maintained all animals at the University of Nevada Reno (UNR). We housed C. variegatus and T. sexlineatus singly in 5- or 10-gallon glass tanks with mesh lids or in pairs (male – female) in 10-gallon glass tanks with mesh lids, and were provided with heat (40

Watt bulbs) and UV light (Repti-sun, 10.0 UVA/UVB, ExoTerra (Hagen), Montreal,

CAN). We housed I. iguana in pairs in 30-gallon glass tanks with mesh lids, a large spot heat lamp, UV light, and 10 x 10 x 4 cm water dish.

We maintained lizards on a 12l:12d light cycle, in a room with temperatures at 75 F (+/- 5 F) and humidity near 35% (+/- 5%). We misted I. iguana daily and fed shredded butternut squash and leafy greens every other day. We fed Takydromus and Coleonyx crickets or mealworms with occasional calcium supplementation (Rep-Cal Calcium with Vitamin D, Los Gatos, CA, USA).

We recorded snout-vent length (SVL) (cm) monthly and body mass (g) every two weeks for each lizard during captivity. All live animal procedures were approved by the Institutional Animal Care and Use Committee.

Whole-animal performance

We established baseline sprint performance (velocity) for each individual lizard prior to injections, and then evaluated changes in velocity performance following injections. We adapted our whole animal performance assay from the well- developed bioassay used to evaluate tetrodotoxin resistance in garter snakes

(Thamnophis), under the assumption that highly resistant animals will be able to maintain their normal performance capability when exposed to a standardized dose of toxin, while susceptible animals will display dramatic reductions in performance given a dose of equal potency (Brodie et al. 2002, Ridenhour et al.

2004).

To measure baseline and post treatment velocity, we sprinted lizards on a

2.2 m racetrack constructed of aluminum alloy, high-density polyethylene (HDPE) plastic and removable polyester (PET) carpet lining. We recorded lizard body

51 temperatures using an infrared (IR) heat gun (Etekcity, Anaheim, CA, USA) immediately prior to each performance assay. We video-recorded performance assessments using a HERO4 GoPro (1060 linear video, 60 frames per second), and analyzed each video for velocity using Physlets Tracker software v5.1.1

(Brown 2018). We measured distance travelled for every two frames, and calculated velocity with our distance measurements and known framerate of videos using Physlets software. We then calculated baseline sprint speed as the average of the top ten velocity values (after outlier removal).

Once we established baseline sprint performance, we divided lizards into treatment groups (a control group receiving sterile saline, low BWSV treatment

group, receiving 1 mouse LD50, and a high BWSV treatment group, receiving 5

mouse LD50). We obtained BWSV from SpiderPharm (Yarnell, AZ) as lyophilized

0.5 mg pellets and reconstituted it to a 0.1 mg/μL stock using sterile saline. We then serially diluted this stock to concentrations appropriate to our mass-adjusted doses (0.01 mg/μL, 0.001 mg/μL, 0.0001 mg/μL). We administered intramuscular

(IM) injections to lizards, always in the dorsal thigh of the right hind leg, using a

300 cc disposable insulin syringe with a 31 gauge needle (UltiCare, Excelsior,

MN, USA). We attempted to keep injection volumes at or below 0.25% of body weight by volume (as per Diehl et al. 2001). Following injections, we performed three performance assessments: immediately after injection; 24 hours after injection; 48 hours after injection. Upon completion of the final performance assessment, we monitored lizards for 4 days before humanely euthanizing each

52 animal and harvesting the hind legs for histological examination. Finally, we deposited each lizard in the Herpetological Collection of the University of Nevada,

Reno, Museum of Natural History (UNR) (Table S1).

Analyses

We first conducted linear mixed regression models (LMM) with post-injection velocity as the response variable, a variety of fixed effects (treatment, species, time, body condition, sex, temperature, volume of fluid injected as a percentage of body weight) and their interactions, and included a random effect of individual.

We used Akaike’s Information Criterion (AIC) to select the best model (Burnham and Anderson 2002), and then performed additional linear mixed models at the species level to evaluate finer scale effects. We analyzed all data in R v3.6.1 (R

Core Team 2019).

Comparative histology

(hematoxylin and eosin) by IDEXX Laboratories (Sacramento, CA). We compared treated muscle tissue to untreated muscle tissue of the same individual using

ImageJ v1.52a (Schneider et al. 2012). We captured between three and five

53 images per slide at 100x magnification per limb. We analyzed each image using a randomized grid system, excluding grids from random selection if more than 10% edge white space was present.

We quantified tissue damage using percent damaged area (PDA) (adapted from Wooddell et al. 2011) and immune system response was quantified with nuclear counts (adapted from Giovannelli et al. 2018). Muscle was considered damaged if the muscle fiber was clearly undergoing necrosis or if there was evidence of recent regeneration (Fig. 3A-C). We performed nuclear counts

(excluding red blood cell nuclei) and distinguished between “normal” nuclei

(nuclei found where expected within muscle fibers) and “abnormal” (i.e. leukocytes, central nuclei); we also included a measure of the ratio of normal to abnormal nuclei (percent normal nuclei, or PNN). Nuclei are important for the normal function of muscle fibers (cite) and increased white blood cells are indicative of immune system response (cite), so these measures help characterize effects of venom on general muscle function as well as immune system response.

We scored percent damaged area and made nuclear counts using ImageJ

(Abramoff et al. 2004) with the add-on Cell Counter. We averaged PDA and nuclear count variables (normal, abnormal, and PNN) across grids within images, with up to five replicates per limb. We used t-tests to evaluate significance of differences between untreated muscle tissue and treated muscle tissue within

54 species, followed by ANOVAs of treated muscle tissue to evaluate significant differences across species and treatment.

Ancestral state reconstruction

To understand the evolution of BWSV resistance in lizards, as well as potential ecological correlates of BWSV evolution, we reconstructed the pattern of trait change on the lizard phylogeny. We conducted ancestral state reconstructions

(ASR) by tracing characters over the lizard tree in Mesquite 3.6 (Maddison and

Maddison 2018). We evaluated three characters as follows: 1. Spider prey: (0) spiders not in diet, (1) spiders in diet, not including widow spiders, (2) spiders in diet, including widow spiders; 2. Whole animal BWSV resistance: (0) absent—at

low dose (1LD50) species show significant reduction in sprint performance

compared to control treatment, (1) partially present—at low BWSV dose (1LD50) species show whole animal sprint performance on par with baseline speed, (2)

present—at high BWSV dose (5LD50) species show whole animal sprint performance on par with baseline speed; 3. Tissue level BWSV resistance: (0)

absent—at low BWSV dose (1LD50) species show significant muscle tissue response to compared to control dose treatments, (1) partially present— at low

BWSV dose (1LD50) species show muscle tissue response on par with saline

control treated tissue, (2) present – at high dose (5LD50) species show minimal muscle tissue response compared to saline control treatments. To score taxa, we used descriptions from the literature on the diet of lizards (Cowles 1937,

Cunningham 1956, Parker and Pianka 1974, Wilson 1991, Stebbins 2003,

Fukudome and Yamawaki 2016), and combined BWSV resistance data from

Chapter 1 and 2.

We mapped traits onto the currently accepted phylogeny for squamates

(Pyron et al 2013; Zheng and Wiens 2016). We then used the maximum parsimony (MP) method of ASR, which minimizes the amount of character change given a tree topology and character state distribution (Maddison 1994); one character state or another was assigned to a node if it created fewer steps, otherwise the node was considered equivocal. We also considered character transitions to be unordered (Fitch parsimony).

RESULTS

Qualitative impacts of BWSV on lizards

We made standard observations between performance assays (health status recorded every eight hours for seven days post-injection). These yielded no visible outward behavioral effects of BWSV on C. variegatus and T. sexlineatus.

However, we noted that T. sexlineatus appeared to switch more frequently from escape behavior (sprinting to flee pursuer) to more defensive postures and behaviors in post-injection performance assays (brief full body rigidity and adpression of limbs), a behavior not seen during baseline performance trials. On the other hand, I. iguana showed obvious visible signs of the effects of BWSV. In fact, one I. iguana treated with the low dose and two that treated with the high

56 dose venom died between observation periods. Two I. iguana individuals treated with low dose and one that was treated with high dose venom reached previously established humane endpoints and were euthanized prior to the end of the four- day observation period. All but one remaining I. iguana individual from the venom treatment groups showed severe signs of envenomation during observations, including partial limb paralysis and reduced appetite and movement.

Whole animal performance

The best linear mixed-effect (LMM) model to explain variation in our dependent variable, post-injection velocity, included time, treatment and the interaction of time and species as independent variables with individual as a random effect

(Table 1). Additional variables and their interactions were not significant in any model (temperature, sex, body condition, volume injected). Given the highly significant differences in post-injection velocity between species (LMM, 휒2 = 9.71, df = 2, p < 0.008), we conducted linear regressions for each species using variables from the top two LMM models and ranked them (Table 2).

The LMM that best described the variation in post-injection velocity for C.

2 variegatus (R c = 0.65, df = 8) included treatment, which was not significant

(LMM, 휒2 = 0.50 df = 2, p > 0.77) and time, which was highly significant (LMM, 휒2

= 60.62, df = 3, p < 0.0001). Coleonyx variegatus showed reduced velocity immediately following injection for all treatments and recovered nearly to baseline velocity by 48 hours post-injection for all treatments (Fig. 2, Table 3).

2 The best LMM for T. sexlineatus (R c = 0.08, df = 5) included treatment, which was not significant (LMM, 휒2 = 5.07, df = 2, p > 0.07); a second model that included time performed similarly well (Table 2), which was highly significant

(LMM, 휒2 = 22.04, df = 3, p < 0.0001). Average reduction in post-injection velocity was similar across venom treatment groups, and the control group experienced a significant reduction in speed immediately post-injection (Fig. 3, Table 3).

Individual variation in post-injection velocity was low compared to C. variegatus and I. iguana.

2 The best LMM for I. iguana (R c = 0.57, df = 6) included treatment and time, both of which were significant (LMM, 휒2 = 7.04, df = 2, p < 0.03; 휒2 = 42.19, df = 3, p < 0.0001). Iguana iguana had severely reduced post-injection velocity speeds for both venom treatments, with two individuals that were unable to complete a performance assessment at 48 hours post-injection. All but two individuals (one of these was excluded from all analyses) in venom treatment groups were reduced to less than 50% performance ability (Fig 2). By contrast, I. iguana that received control injections had post-injection performance that remained near 100% over time (Table 3).

Because temperature is an important physiological factor for ectotherms

(Angilletta et al. 2002), we ensured that lizards were within the range of their preferred body temperatures prior to performance assays (C. variegatus, 푥̅ =

28.04° C, SE = 0.97, Vance 1973; T. sexlineatus, 푥̅ = 29.3° C, SE = 2.13, Zhang and Ji 2004; I. iguana, 푥̅ = 30.26° C, SE = 2.13, McGinnis and Brown 1966).

Temperature was not an important factor in a lizard’s performance during our trials, and was not retained as a descriptive factor in any top models (Table 1).

Comparative histology

All untreated (left leg) muscle tissue had limited PDA as expected (< 3.0%), but some species (T. sexlineatus) started with elevated abnormal nuclear counts

(Table 4). There were no significant differences between untreated muscle tissue and saline control muscle tissue for any response variable except for C. variegatus, which had increased abnormal nuclei counts (t = -2.23, df = 27.72, p <

0.05; Fig. 5, Table 4). There were slightly increased levels of PDA across species within right leg control groups, but these were not significant.

We used ANOVAs to examine the influence of various predictors on tissue response in right (injected) legs, including species, treatment (control = saline,

low = 1LD50, high = 5LD50), and the interaction between treatment and species.

The additional variables we tested were not significant (sex, body condition, volume injected, and temperature) and so we removed them from subsequent analyses. Normal nuclei were significantly different in treated muscle tissue

between species (F2,159 = 21.14, p < 0.0001), treatment (F2,159 = 5.19, p < 0.01),

and the interaction between species and treatment (F4,159 = 5.65, p < 0.001).

Control tissue had significantly higher normal nuclei compared to venom treated tissue across species (Tukey HSD = 14.57, p < 0.05), and C. variegatus had a

59 lower normal nuclei count compared to T. sexlineatus and I. iguana across treatments (Tukey HSD = 29.29, p < 0.0001).

Abnormal nuclei were significantly affected by species (F2,159 = 8.05, p <

0.005), treatment (F2,159 = 10.05, p < 0.0001), and the interaction between species

and treatment (F4,159 = 4.47, p < 0.005). Both low and high venom treated tissue had significantly elevated abnormal nuclei compared to control (Tukey HSD =

34.11, p < 0.005). Species level effects across treatment were driven mostly by T. sexlineatus, which had reduced abnormal nuclei effects compared to both C. variegatus and I. iguana (Tukey HSD = 30.11, adj p < 0.001), especially in I. iguana high venom treatment (Tukey HSD = 72.75, adj p < 0.001).

PNN was significantly affected by species (F2,159 = 12.13, p < 0.0001),

treatment (F2,159 = 18.14, p < 0.0001), and the interaction between species and

treatment (F4,159 = 7.38, p > 0.0001). Venom treated muscle tissue had significantly lower PNN than control tissue (Tukey HSD = 22.49, p < 0.0001).

Takydromus sexlineatus had significantly higher PNN compared to C. variegatus and I. iguana across treatments (Tukey HSD = 17.5, p < 0.005). This was especially evident when comparing high treatment tissue of T. sexlineatus with high treatment tissue of I. iguana (Tukey HSD = 40.9, p < 0.0001).

PDA was significantly affected by species (F2,159 = 49.31, p <0.0001),

treatment (F2,159 = 25.72, p < 0.0001), and the interaction between species and

treatment (F4,159 = 10.58, p < 0.0001). There were significant differences between all three species (Fig. 5), with I. iguana showing increased PDA compared to C.

60 variegatus and T. sexlineatus (Tukey HSD = 31.22, p < 0.0001; Fig. 5D) and C. variegatus showing increased PDA compared to T. sexlineatus (Tukey HSD =

11.97, p < 0.05). Control tissue had significantly lower PDA across species

(Tukey HSD = 21.08, p < 0.0001). Iguana iguana venom treated muscle had significantly higher PDA compared to all other species and treatments (Fig 5), with high venom treated C. variegatus having the next highest PDA (Tukey HSD =

28.48, p < 0.005).

We found no evidence of muscle tissue response when comparing control treated and venom treated muscle tissue for C. variegatus. Venom treated limbs had significantly higher PDA (low dose: t = -2.80, df = 18.37, p < 0.05; high dose: t

= -3.74, df = 19.01, p < 0.005; Fig. 5D). Abnormal nuclei were significantly elevated for all treatments compared to untreated muscle tissue (t-test; control: t

= -2.13, df = 27.72, p < 0.05; low dose: t = -4.46, df = 20.15, p < 0.005; high dose: t = -4.61, df = 20.11, p < 0.005; Fig. 5B).

Takydromus sexlineatus showed significant differences between untreated tissue and high venom treated tissue, but not control or low venom treated tissue

(Table 4, Fig. 5). Normal nuclei counts were not significantly reduced for any treatment; actually, both low and high venom treated tissue showed non- significant increases in normal nuclei (Table 4, Fig. 5A). Abnormal nuclei counts were significantly elevated in high venom treated muscle tissue compared to untreated tissue (t = -4.61, df = 25.17, p < 0.005; Fig. 5B). PNN was significantly reduced only in high venom treated muscle tissue (t = 4.33, df = 34.18, p < 0.005;

Fig. 5C). This pattern continued for PDA, with only high venom treated muscle tissue showing a significant increase compared to untreated muscle tissue treatment (t = -2.89, df = 22.9, p < 0.01; Fig. 5D).

Iguana iguana showed significant differences between untreated and venom treated muscle tissue for all muscle tissue response variables (Table 4,

Fig. 5). Conversely, we found no significant differences between untreated tissue and control treated tissue for any variable (Table 4, Fig. 5). Normal nuclei increased significantly after venom treatment compared to untreated muscle tissue (low dose: t = 3.24, df = 37.30, p < 0.005; high dose: t = 5.49, df = 22.79, p

< 0.0001; Fig. 5A). Abnormal nuclei increased significantly for venom treated tissue compared to untreated tissue (low dose: t = -5.25, df = 24.50, p < 0.0001; high dose: t = 4.59, df = 19.30, p < 0.005; Fig. 5B). Similarly, PNN was significantly reduced for venom treatments compared to untreated tissue (low dose: t = 7.40, df = 25.69, p < 0.0001; high dose: t = 7.46, df = 20.28, p < 0.0001;

Fig. 5C). Finally, PDA significantly increased in venom treated muscle tissue compared to untreated muscle tissue (low dose: t = -6.13, df = 24.01, p < 0.0001; high dose: t = -11.18, df = 19.03, p < 0.0001; Fig. 5D).

Trait mapping

We mapped an ecological trait (diet) and BWSV resistance abilities onto the current lizard phylogeny to examine the evolution of these characters. Given the small number of taxa, and small number of state changes, we can easily

62 reconstruct the evolution of these traits. Insectivory is assigned as an ancestral trait, which is supported by the literature (Modesto et al. 2009), and in our case represents predation on spiders particularly. This changed to herbivory once among our representative species (in I. iguana). We also have a “switch” to specializing on black widows in E. multicarinata, again assigned a single change within our study species.

When we map our whole animal and tissue level venom resistance traits on the tree (Fig. 2), it appears that ancestral resistance involves both whole animal and tissue level resistance (e.g. S. occidentalis, C. variegatus, and T. sexlineatus; Fig. 2). We see that two species have partially or completely lost presumed baseline resistance (U. stansburiana and I. iguana, respectively). The only species that meets criteria for elevated resistance is E. multicarinata, which is also the only confirmed lizard predator of L. hesperus (Cowles 1937,

Cunningham 1956), and is characterized by a lack of response at both whole animal and tissue levels for low and high venom treatments.

DISCUSSION

Lizards and venomous spiders have likely been interacting for well over 100 million years (Simões et al. 2018). The continual predation of lizards on spiders over evolutionary history may have contributed to evolution of venom resistance in many squamate taxa. Here we used the neurotoxic 훼-latrotoxin because the

63 genera that possess this venom are cosmopolitan and relatively ancient (ca. 50 million years old; Liu et al. 2016), increasing the likelihood that a large percentage of squamate taxa are likely to have some interactions with these spiders.

Of the squamate species tested in this study, only one showed susceptibility to BWSV, I. iguana. Coincidentally, I. iguana, was also the only herbivorous species tested. The response was so strong, in fact, that several individuals perished before the completion of the observation period, and several others suffered systemic effects (e.g. full body paralysis) and had to be humanely euthanized.

If we combine species data from Chapter 1 with the results from this study, we can roughly categorize resistance level based on response to low and high venom treatments, and whether those responses occurred at the whole animal level, muscle tissue level, or both. Elgaria multicarinata, which had no effect of low or high venom treatment at the whole animal level, and very minimal muscle tissue response, showed the highest level of resistance. Because E. multicarinata is the only species known to preferentially consume Latrodectus, this may be due to ongoing selective pressure (Dawkins and Krebs 1979).

Species with baseline resistance showed no effect at the whole animal level, but had significant muscle tissue response in venom treated muscle tissue.

The species that meet these criteria include S. occidentalis, T. sexlineatus and C. variegatus. While we were not surprised that C. variegatus fit into this group because they are sympatric with L. hesperus and we often captured them in very

64 close proximity to these spiders, we were surprised to find that T. sexlineatus fit solidly into this group as well. Takydromus sexlineatus had no whole animal effect for any treatment by 48 hours post-injection, and showed tissue level response only at the high venom treatment. This is our smallest lizard species by weight (<

3 g on average), making it unlikely that they would be capable of predation on any female adult Latrodectus that might overlap their range. Especially noteworthy is that while T. sexlineatus had significantly higher PDA in high venom treated limbs, this was comparable to the non-significant PDA response in the

most resistant species from Chapter 1 (2.21% in 5LD50 treated T. sexlineatus vs.

2.1% in 1LD50 treated E. multicarinata). Given these results, the species that best fit our expectations for ancestral resistance are S. occidentalis, C. variegatus and

T. sexlineatus; these species possessed whole animal resistance for both low and high venom treatments, but had statistically significant muscle tissue response to high venom treatments.

Iguana iguana is extremely susceptible to BWSV and appears to have lost the presumed ancestral resistance seen across the lizard phylogeny. Post-

injection velocity was reduced (> 50%) at ecologically relevant (1LD50) doses and drastically reduced (>80%) in the high venom treatment group. This was also the only species that showed easily observable outward effects in venom treated groups at the whole animal level (hind limb and total body paralysis, uncoordinated movements, inability to eat or absence of appetite). Muscle tissue

65 response in I. iguana was significant for low and high venom doses, and far exceeded the response seen in any other species.

The variation in resistance for these species is consistent with phylogenetic relationships (Fig. 2), with notable exceptions in I. iguana and E. multicarinata indicating that ancestry is not the only explanation for resistance level. Perhaps unsurprisingly, slightly elevated species (S. occidentalis, C. variegatus, T. sexlineatus) are all sympatric with and either known or possible predators of some species of Latrodectus. Takydromus sexlineatus, which is native to southeast Asia, is the only exception; this species probably encounters

Latrodectus, and it likely eats spiders like others in this genus (Jackson and

Telford 1975); however, whether any Latrodectus are included in their diet is unknown as dietary research on this species and indeed, the entire genus, is sparse. Takydromus sexlineatus is sympatric with several species of widow spider (L. elegans, L. hasselti; Garb et al. 2004) and therefore may retain resistance due to predation, though this is speculative.

It’s apparent that resistance to at least one neurotoxic venom, 훼-latrotoxin, is present across disparate branches of the squamate phylogeny, and within these species we see considerable variation in the level of resistance (Fig. 2). Of our six lizard species, the three that are most closely related (Phrynosomatidae:

S. occidentalis, U. stansburiana and Iguanidae: I. iguana) show three levels of resistance, respectively: baseline, partial baseline, and absent. The next closely related species, Anguidae: E. multicarinata, shows resistance at both whole

66 animal and tissue level, unique among all species tested. The last two,

Lacertidae: T. sexlineatus and Eublepharidae: C. variegatus, both show baseline resistance, similar to S. occidentalis. What seems to best predict the retention of baseline resistance is the basic ecological trait of insectivory (Fig. 2).

While potency of BWSV varies across Latrodectus species (Daly et al.

2001, de Roodt et al. 2017), the components are similar enough that antivenoms from one species will be effective even for distantly related latrotoxin possessing species (e.g., antivenom from L. hasseltii is effective in neutralizing L. mactans, L. hesperus, and even Steatoda sp. venom; Daly et al. 2001, Graudins et al. 2001).

Venoms in ancient lineages like arachnids have been highly conserved over evolutionary history (Liu et al. 2015). Therefore, perhaps predator-prey interaction on either side of that equation, with most species of Latrodectus, is enough to maintain elevated or baseline resistance; highly elevated resistance only appears to be present in a preferential L. hesperus predator, E. multicarinata

(Cowles 1937, Cunningham 1956).

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(control = saline, low = 1LD50, high = 5LD50); “species” refers to species of lizard (Coleonyx variegatus, Takydromus sexlineatus, Iguana iguana); “time” refers to time post-injection (immediately after, 24 hours after, 48 hours after); “temp” is the body temperature of an individual at the time performance was assessed; “sex” is the sex of the lizard, male or female; “body con” is body condition, estimated by dividing mass by SVL (g/cm); “vol inj” is the volume of fluid injected into the lizard as a percentage of the lizard’s body weight. For all models, individual was included as a random effect.

Linear Mixed Effect Models: Post-injection Velocity

Rank Fixed effects Interactions df R2c AIC

1 time time:species 14 0.61 -23.58

2 time none 6 0.39 -12.04

3 rx, time, species rx:species, time:species 20 0.62 -5.74

4 rx, time, body condition, species rx:species, time:species 7 0.62 -1.71 rx, species, time, temp, sex, body 5 all possible 36 0.67 72.36 con, vol inj

“rx” refers to treatment, three levels (control = saline, low = 1LD50, high = 5LD50) and “time” refers to time, four levels (before injection, immediately after, 24 hours after, 48 hours after). Only the top two models for each species are included.

Species Level Linear Mixed Effect Models: Post-injection Velocity

Rank Species Fixed effects Interactions df R2c AIC 1 C. variegatus rx, time none 8 0.65 -38.1

2 C. variegatus rx none 5 0.05 -21.36

1 T. sexlineatus rx none 5 0.08 -39.27

2 T. sexlineatus rx, time none 8 0.44 -36.57

1 I. iguana rx, time none 8 0.59 29.23

2 I. iguana rx, time rx:time 14 0.69 37.43

Table 3. Comparison of mean velocity values (cm/s) for each species, along with post-injection velocities by treatment group (control = saline, low = 1LD50, high = 5LD50), and the percent reduction in post-injection velocity (% change). Note that Iguana iguana has the highest percent reduction of velocity and is the only species with a significant effect of treatment on velocity (*).

Changes in Mean Velocity by Species and Treatment Species Treatment 풙̅ before (cm/s) SE 풙̅ after (cm/s) SE % Change p-value

control (n=4) 93.03 16.05 86.65 17.75 -6.86% 0.6 Coleonyx 1LD (n=4) 81.38 3.52 73.92 16.63 -9.17% 0.53 variegatus 50

5LD50 (n=4) 99.00 8.84 83.34 12.44 -15.82% 0.14

control (n=3) 102.54 14.91 89.29 21.36 -12.92% 0.42 Takydromus 1LD (n=3) 98.13 8.81 85.49 8.32 -12.88% 0.11 sexlineatus 50

5LD50 (n=3) 105.24 9.46 98.07 1.83 -6.81% 0.08

control (n=4) 190.50 59.03 163.13 25.00 -14.37% 0.23

Iguana iguana 1LD50 (n=5) 172.57 57.42 79.38 46.58 -54.00% 0.01*

5LD50 (n=4) 152.21 27.79 28.39 45.66 -81.35% 0.006**

Table 4. Comparison of muscle tissue response by leg, treatment, and species. Variables include “norm”, count of normal muscle fiber nuclei; “abnorm”, count of abnormal nuclei (leukocytes or central nuclei); “PNN”, is the percent of normal nuclei, a combined measure of changes in normal and abnormal nuclei; and “PDA”, is the percent damaged area, a measurement of damaged muscle fiber. Note the especially dramatic differences between left (uninjected) and right leg (injected) for venom treatments in Iguana iguana.

Muscle Tissue Response

L x̅ Treatment L x̅ norm R x̅ norm R x̅ abnorm L x̅ PNN R x̅ PNN L x̅ PDA R x̅ PDA Species abnorm (n) (SE) (SE) (SE) (SE) (SE) (SE) (SE) (SE)

control (20) 46.6 (6.1) 39.9 (3.5) 17.0 (4.2) 31.2* (8.5) 75.9 (5.0) 60.71 (6.6) 2.8 (2.0) 8.0 (3.1)

low (18) 30.0 (1.4) 28.9 (3.5) 18.5 (3.8) 76.9** (12.5) 67.9 (4.2) 34.97*** (5.4) 0.8 (0.6) 9.1* (2.9) Coleonyx variegatus high (20) 31.7 (1.5) 22.3* (3.2) 8.3 (1.9) 59.7** (11.0) 81.7 (2.9) 38.17*** (7.3) 0.1 (0.1) 25.8* (6.9)

control (10) 51.3 (1.5) 49.1 (4.3) 25.3 (4.0) 36.3 (6.4) 68.8 (3.3) 59.32 (4.8) 0.4 (0.2) 0.8 (0.7)

low (9) 47.4 (2.8) 54.1 (6.6) 39.5 (6.4) 34.3 (6.6) 56.6 (3.3) 62.14 (4.8) 0.4 (0.2) 4.0 (2.2) sexlineatus

Takydromus high (19) 63.6 (5.3) 73.6 (4.6) 9.8 (1.5) 26.2** (3.2) 86.1 (1.6) 74.4** (2.1) 0.5 (0.2) 2.2** (0.6)

control (19) 80.5 (8.6) 86.2 (6.4) 11.2 (2.0) 15.6 (2.3) 87.0 (2.0) 85.8 (1.5) 2.3 (1.9) 7.5 (2.4)

low (24) 83.5 (5.2) 48.6** (9.4) 9.0 (1.3) 75.7*** (12.7) 90.0 (1.3) 36.1*** (7.2) 0.3 (0.1) 54.3*** (8.8)

high (20) 100.3 (3.1) 44.0*** (9.8) 15.6 (1.6) 99.0** (18.1) 86.8 (1.3) 33.5*** (7.0) 0.6 (0.2) 75.1*** (6.7) Iguana iguana

Species Museum ID Coll ID Location (Co./State) Sex SVL (cm) Mass(g) Coleonyx Unassigned VLT035 Pima Co., AZ M 5.2 3.75 variegatus

Unassigned VLT036 Pima Co., AZ F 4.7 2.3

Unassigned VLT037 Pima Co., AZ M 7 6.5 Unassigned VLT038 Pima Co., AZ F 4.5 2.35 Unassigned VLT039 Pima Co., AZ M 4.6 2.4 Unassigned VLT040 Clark Co., NV F 6.7 4.65 Unassigned VLT041 Clark Co., NV M 6.7 4.5 Unassigned VLT042 Clark Co., NV F 7.2 5.95 Unassigned VLT043 Clark Co., NV F 6.6 4.5 Unassigned VLT044 Clark Co., NV F 7 5.35 Unassigned VLT093 Southern CA M 6.7 7 Unassigned VLT094 Southern CA M 7 8 Takydromus Unassigned VLT046 Triple L Reptile M 5.8 3.1 sexlineatus Unassigned VLT047 Triple L Reptile F 5.4 2.9 Unassigned VLT048 Triple L Reptile F 5.2 2.8 Unassigned VLT050 Triple L Reptile F 5.3 2.8 Unassigned VLT052 Triple L Reptile M 5.8 3.3 Unassigned VLT053 Triple L Reptile F 5.3 2.8 Unassigned VLT054 Triple L Reptile F 6 2.9 Unassigned VLT056 Triple L Reptile M 5 2.2 Unassigned VLT057 Triple L Reptile F 5.1 3.15 Iguana iguana Unassigned VLT077 Reptile City M 12.2 51 Unassigned VLT078 Reptile City M 12 43 Unassigned VLT079 Reptile City F 13.2 57

Unassigned VLT081 Reptile City F 12.6 56

Species Museum ID Coll ID Location (Co./State) Sex SVL (cm) Mass(g) Iguana iguana Unassigned VLT082 Reptile City F 12.7 48 Unassigned VLT083 Reptile City M 11.9 49.5 Unassigned VLT085 Reptile City F 13.1 53 Unassigned VLT087 Reptile City M 9.4 26 Unassigned VLT088 Reptile City F 12.1 56 Unassigned VLT089 Reptile City M 9.1 27.5 Unassigned VLT090 Reptile City F 11.2 39 Unassigned VLT091 Reptile City F 10.7 36

Figure 1. Focal species. Clockwise from top left: Coleonyx variegatus, Takydromus sexlineatus, Iguana iguana, Latrodectus hesperus. Coleonyx variegatus (Eublepharidae) is a small lizard native to the southwest U.S. that feeds on invertebrates, including spiders and scorpions (Parker and Pianka 1974, Stebbins 2003). Takydromus sexlineatus (Lacertidae) is a small, slender lizard with an elongated tail native to southeast Asia (Arnold 1997) that feeds on invertebrates, including spiders (Jackson and Telford 1975). Iguana iguana (Iguanidae) is a medium to large lizard with a wide range that includes Mexico, Central and South America which feeds on plant matter, including foliage and fruits (Rand et al. 1990). Latrodectus hesperus (Theriididae) is a venomous spider known more commonly as the western black widow; it is native to the southwestern U.S. (Garb et al. 2004) and feeds mostly on invertebrates, crustaceans, and occasionally small vertebrates (Salomon 2011, Nyfeller and Vetter 2018).

Figure 2. Ancestral state reconstructions (top left) showing parsimonious character state transitions for whole animal and tissue level resistance in six lizard species (Sceloporus occidentalis, Uta stansburiana, Iguana iguana, Elgaria multicarinata, Takydromus sexlineatus, and Coleonyx variegatus. For both whole animal and tissue level resistance, there is phylogenetic signal, although there are some nodes unresolved for tissue level resistance. Main figure combines ecological traits (insectivory, herbivory) with phylogenetic relationships (Pyron et al. 2013, Zheng and Wiens 2016), mapped with the resistance data. Notice that unique character states of I. iguana and E. multicarinata (respectively, herbivory and black widow predation) are associated with unique tissue level resistance states (respectively, no resistance and resistance at 5LD50).

* *

Figure 3. Panels showing changes in post-injection velocity ratio over time. Percent performance is sprint performance scaled against baseline ability. (A) plots have been grouped by species (n between 4 and 6) to visualize how percent performance changes in response to treatment (“Control” = saline, “Low”

= 1LD50, “High” = 5LD50) over time (immediately after injection, 24 hours after, 48 hours after). (B) plots have been grouped by treatment to show variation in response to treatment across species. Notice that the only species with statistically significant reductions in performance (*) is I. iguana.

Figure 4. Histological images of lizard muscle tissue taken at 100x magnification. In A-C, images have been cropped to highlight specific morphological characteristics. Arrows point to a raft of nucleated red blood cells with associated white blood cells (A) and regenerating muscle fibers (C). In (B), the focal field is filled with necrotic fibers. Panel (D) highlights differences in muscle tissue response by species (columns) and treatment (rows). Notice that there is evidence for muscle tissue response in both C. variegatus and T. sexlineatus, but that it is subdued compared to the response shown in I. iguana venom treated muscle tissue.

Figure 5. Boxplots of various muscle tissue response variables. “L” refers to values from untreated tissue harvested from the left leg, used to establish baseline values for variables in the absence of treatment. “R” refers to values from treated tissue harvested from treated muscle tissue of the right leg (“Control” = saline, “Low” = 1LD50, “High” = 5LD50), used to quantify response of treatments. (A) shows normal nuclei counts, (B) shows abnormal nuclei counts, (C) shows percent normal nuclei (PNN) and (D) shows percent damaged area (PDA). Significant differences (p < 0.05) between saline control and treated tissue are denoted by *.

CONCLUDING REMARKS

This work helps to further establish foundational data for a network of lizard predator and venomous spider prey interactions which can contribute to our understanding of predator adaptations across vertebrate taxa, and further characterizing how venom resistance adaptations occur over time and space.

The unmatched resistance possessed by E. multicarinata encourages further work on this predator-prey system to discover the mechanism of resistance, while the absence of resistance in I. iguana encourages exploration into potential costs associated with resistance. We find that both phylogeny and ecology matter, and that BWSV resistance in lizards may be present even without direct predator-prey interaction, although natural history data is necessary to inform ecological traits to strengthen this conclusion.

Another important question to answer in this system is whether these resistance mechanisms are specific to 훼-Latrotoxin or broadly effective against many arachnid venoms. The complex interaction between phylogeny and ecology on potentially adaptive traits can be difficult to untangle, but our research shows that there are definite patterns to be found even at small scales; further work would expand and contextualize our results.

S1: Testing Resistance to Arizona Bark Scorpion Venom in Lizards that are

Resistant to Black Widow Spider Venom

INTRODUCTION AND METHODS

In chapter 1, I established that some lizards possess resistance to black widow spider venom; in chapter 2, I further established that many lizards possess resistance to black widow spider venom, and this could have possibly originated with predator-prey interactions in the distant past, leading to ancestral BWSV resistance across lizard taxa. Given that many venomous arachnids were present during the Cretaceous (Sunagar and Moran 2015), I hypothesized that ancestral venom resistance in lizards might be a generalized or broad defense against a variety of arachnid neurotoxins, similar to the resistance in didelphid marsupials to various components of venom across several snake species (Voss and Jansa

2012). To test this hypothesis, I chose a potently venomous arachnid

(Centruroides sculpturatus) with a neurotoxic venom that acts specifically on sodium channels in vertebrate membranes (Rowe and Rowe 2008, Hopp et al.

2017, Carcamo-Noriega et al. 2018), while specific membrane channel targets are still unknown in BWSV (Grishin et al. 1998). I then selected two lizard species from Chapter 1 that are known to be resistant to BWSV and not sympatric with this novel arachnid (except at the extreme southern edge of their respective ranges; Stebbins 2003, Carcamo-Noriega et al. 2018).

The Arizona bark scorpion is a small scorpion native to the desert southwest of the U.S. (Carcamo-Noriega et al. 2018) and is the most venomous scorpion in that region (Hopp et al. 2017). I chose to test resistance to Arizona bark scorpion venom (ABSV) in Elgaria multicarinata which exhibited the most elevated resistance level and was unaffected by injection with BWSV. I also included Sceloporus occidentalis which was shown to have baseline resistance, with response to venom at the tissue level but not the whole animal level.

We assume that whole animal resistance will be present even in the absence of tissue level resistance, so for this supplemental data we chose to focus on whole animal performance. If the resistance to BWSV that was observed in Chapter 1 is due to a generalized ancestral trait that grants resistance to a variety of arachnid neurotoxins, then exposure to venom from a non-sympatric arachnid (C. sculpturatus) should result in no reduction in sprint performance and no observable effects.

Methods for field collection of lizards, maintenance in the lab, sprint performance and injections followed those outlined in Chapter 1. Venom doses

were the same as in Chapter 1, but using the mouse LD50 value for C. sculpturatus venom which is 1.12 mg/kg (Valdez-Cruz et al. 2004). All live animal work was approved by the Institutional Animal Care and Use Committee.

RESULTS AND DISCUSSION

Both E. multicarinata and S. occidentalis had strong, adverse effects after injection to ABSV that were evident within two hours of injection. I observed lizards post-injection and noted behavioral changes including gaping, writhing, biting at the air, full immobility, uncoordinated movement, muscle spasms leading to full body paroxysm, and loss of righting reflex. In several cases, lizards met predetermined endpoints and I humanely euthanized them before harvesting muscle tissue for future histological review.

Of the 21 lizards treated with ABSV (E. multicarinata = 11, S. occidentalis

= 10), 11 were euthanized prior to the end of the four-day observation period (E. multicarinata = 1, S. occidentalis = 10) (Table S1). All lizards completed performance assessments but were clearly affected. Analysis of performance videos has not been completed, but many lizards had post-injection sprint performances that were essentially 100% reduced (to 0 ability), so we can safely draw tentative conclusions.

The lack of observable whole animal response to BWSV in most of our species from Chapters 1 and 2 of this research led us to hypothesize that lizards might be powerhouses of venom resistance, able to withstand a broad variety of arachnid venoms since they have likely been interacting as predators or prey for millions of years. However, the easily observable, strong, and adverse response of our two species (E. multicarinata, S. occidentalis) to ABSV has served to highlight that the resistance of lizards to BWSV seems tightly tied to specific

85 components within BWSV, perhaps 훼-latrotoxin. This supplemental data helps to guide future research in this system, especially in regard to uncovering starting points to look for underlying molecular mechanisms that may be responsible for resistance to BWSV. In addition, it may be worthwhile to test the resistance of C. variegatus to ABSV since they do engage in predator-prey interactions with C. sculpturatus, unlike the two species that we tested in this trial.

LITERATURE CITED

Carcamo-Noriega, E.N., Olamendi-Portugal, T., Restano-Cassulini, R., Rowe, A., Uribe-Romero, S.J., Becerril & B., Possani, L.D. Intraspecific variation of Centruroides sculpturatus scorpion venom from two regions of Arizona. Archives of Biochemistry and Biophysics 638, 52–57 (2018). Grishin, E.V. Black widow spider toxins: the present and the future. Toxicon 36, 1693–1701 (1998). Hopp, B.H., Arvidson, R.S., Adams, M.E. & Razak, K.A. Arizona bark scorpion venom resistance in the pallid bat, Antrozous pallidus. PLoS ONE 12, e0183215 (2017). Rowe, A.H. & Rowe, M.P. Physiological resistance of grasshopper mice (Onychomys spp.) to Arizona bark scorpion (Centruroides exilicauda) venom. Toxicon 52, 597–605 (2008). Stebbins, R.C. A field guide to western reptiles and amphibians. (Houghton Mifflin, 2003). Sunagar, K. & Moran, Y. The Rise and Fall of an Evolutionary Innovation: Contrasting Strategies of Venom Evolution in Ancient and Young Animals. PLoS Genet 11, e1005596 (2015). Voss, R. S. & Jansa, S.A. Snake-venom resistance as a mammalian trophic adaptation: lessons from didelphid marsupials. Biological Reviews 87, 822– 837 (2012).

Location Species Museum ID Coll ID Sex SVL (cm) Mass (g) (Co./State) Sceloporus 9920 CRF 3365 Washoe Co., NV M 7 10.25 occidentalis 9921 CRF 3367 Washoe Co., NV M 6.6 14.5 9922 CRF 3368 Washoe Co., NV F 6.2 9 9899 VLT 109 Washoe Co., NV F 7.8 17.25 9904 VLT 133 Washoe Co., NV M 7.5 15.25 9905 VLT 134 Washoe Co., NV M 7.7 21.75 9906 VLT 135 Washoe Co., NV M 7.7 17 9908 VLT 137 Washoe Co., NV M 6.6 16.75 9909 VLT 138 Washoe Co., NV M 6.9 12.5 9910 VLT 139 Washoe Co., NV F 7.6 17.75 9911 VLT 140 Washoe Co., NV F 7.8 17 9912 VLT 141 Washoe Co., NV M 8.1 20.75 9913 VLT 142 Washoe Co., NV F 7.9 21.75 9914 VLT 143 Washoe Co., NV M 7.1 16.75 Elgaria 9923 CRF 3369 Yolo Co., CA M 12.8 39.25 multicarinata 9924 CRF 3370 Yolo Co., CA M 12.4 31.75 9925 CRF 3371 Yolo Co., CA M 13.4 41 9926 CRF 3372 Yolo Co., CA F 11.3 33 9927 CRF 3373 Yolo Co., CA U 11.5 24.25 9928 CRF 3383 Contra Costa Co., CA F 10.1 19.75 9929 CRF 3409 Ventura Co., CA M 11.9 39.25 9919 VLT 146 Contra Costa Co., CA M 12.3 31.75 9918 VLT 145 Contra Costa Co., CA U 11.3 26.75 9897 HAM 002 Washoe Co., NV M 7 6 9898 VLT 100 Sonoma Co., CA F 13 48.25

9900 VLT 129 Sonoma Co., CA F 7.7 7.75 9901 VLT 130 Sonoma Co., CA M 12.6 23.75 9902 VLT 131 Sonoma Co., CA F 13.8 42.25 9903 VLT 132 Mendocino Co., CA U 12.1 23 9917 VLT 144 Los Angeles Co., CA M 12 32.25

Figure 1. Centruroides sculpturatus (Buthidae), a small scorpion native to the southwestern U.S. (Carcamo-Noriega et al. 2018) that is arguably the most potently venomous scorpion in North America (Valdez-Cruz et al. 2004).