The effects of hybridization on divergent venom phenotypes: Characterization of venom from Crotalus scutulatus scutulatus × Crotalus oreganus helleri hybrids

Accepted Manuscript The effects of hybridization on divergent venom phenotypes: Characterization of venom from Crotalus scutulatus scutulatus × C. oreganus helleri hybrids Cara Francesca Smith, Stephen P. Mackessy PII:

S0041-0101(16)30234-3

DOI:

10.1016/j.toxicon.2016.08.001

Reference:

TOXCON 5429

To appear in:

Toxicon

Received Date: 3 June 2016 Revised Date:

27 July 2016

Accepted Date: 1 August 2016

Please cite this article as: Smith, C.F., Mackessy, S.P., The effects of hybridization on divergent venom phenotypes: Characterization of venom from Crotalus scutulatus scutulatus × C. oreganus helleri hybrids, Toxicon (2016), doi: 10.1016/j.toxicon.2016.08.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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THE EFFECTS OF HYBRIDIZATION ON DIVERGENT VENOM PHENOTYPES:

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CHARACTERIZATION OF VENOM FROM CROTALUS SCUTULATUS SCUTULATUS × C.

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OREGANUS HELLERI HYBRIDS

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Cara Francesca Smith and Stephen P. Mackessy*

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School of Biological Sciences, University of Northern Colorado, 501 20th Street, Greeley, CO

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80639-0017, USA

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*Corresponding Author: [email protected]

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Abstract Hybridization between divergent species can be analyzed to elucidate expression patterns of distinct parental characteristics, as well as to provide information about the extent of

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reproductive isolation between species. A known hybrid cross between two rattlesnakes with

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highly divergent venom phenotypes provided the opportunity to examine occurrence of parental

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venom characteristics in the F1 hybrids as well as ontogenetic shifts in the expression of these

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characters as the hybrids aged. Although venom phenotypes of adult rattlesnake venoms are

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known for many species, the effect of hybridization on phenotype inheritance is not well

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understood, and effects of hybridization on venom ontogeny have not yet been investigated. The

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current study investigates both phenomena resulting from the hybridization of a male snake with

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type I degradative venom, Crotalus oreganus helleri (Southern Pacific Rattlesnake), and a

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female snake with type II highly toxic venom, C. scutulatus scutulatus (Mojave

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Rattlesnake). SDS-PAGE, enzymology, Western blot and reversed phase HPLC (RP-HPLC)

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were used to characterize the venom of the C. o. helleri male, the C. s. scutulatus female and

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their two hybrid offspring as they aged. In general, Crotalus o. helleri  × C. s. scutulatus hybrid

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venoms appeared to exhibit overlapping parental venom profiles, and several different enzyme

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activity patterns. Both hybrids expressed C. o. helleri father-specific myotoxins as well as C. s.

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scutulatus mother-specific Mojave toxin. Snake venom metalloprotease activity displayed

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apparent sex-influenced expression patterns, while hybrid serine protease activities were

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intermediate to parental activities. The C. s. scutulatus  × C. o. helleri hybrid male's venom

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profile provided the strongest evidence that type I and type II venom characteristics are

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expressed simultaneously in hybrid venoms, as this snake contained distinctive characteristics of

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both parental species. However, the possibility of sex-influenced development of

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metalloprotease activity, as seen in the ontogenetic shifts of the hybrid female, may influence the

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levels of expression of both type I and type II characteristics in hybrid venoms. Ultimately, the

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chronological analysis of this known hybrid system reveals the most distinct characteristics that

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can be used in determining successful hybridization between snakes that follow the type I-type II

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trend in rattlesnake venom composition, namely the presence of metalloprotease activity and

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Mojave toxin.

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Keywords: Enzymes, HPLC, MALDI-TOF MS, metalloprotease, Mojave toxin, serine protease,

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phosphodiesterase, Western blot

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1.

Introduction The effectiveness of venom plays a critical role in the success of prey capture throughout

a rattlesnake’s lifetime. Ontogenetic shifts in venom composition are rooted in changes in diet

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preference as a snake ages (Andrade and Abe, 1999; Daltry et al., 1996; Gutiérrez et al., 1991;

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Mackessy, 1988; Mackessy et al., 2003), and venom composition has even been shown to

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undergo subtle changes as early as following the first natal shed, likely before a snake takes its

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first meal (Wray et al., 2015). Younger rattlesnakes are gape-limited to taking smaller prey

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items, primarily ectotherms including invertebrates, amphibians, and lizards, while adults tend to

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feed on larger endothermic prey (Mackessy, 1988). Prey availability also shifts as snakes get

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larger and are able to consume larger prey items. These dietary shifts often correlate with a shift

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in venom composition from a highly toxic venom with low metalloprotease activity (type II) to a

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largely proteolytic, lower toxicity venom (type I) (Durban, 2013; Mackessy, 1988, 2008,

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2010a,b;).

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Type I venoms typically have lower toxicity and are thought to aid in predigestion of larger mammalian prey, while type II venoms are likely optimized for rapid prey incapacitation,

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facilitating capture and handling (Mackessy, 1988, 2010a; Mackessy et al., 2003). Although the

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type I and type II venom compositional trend is widespread among adults of many rattlesnake

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species (Mackessy, 2008), and a type II to type I ontogenetic shift has been observed most

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frequently, a modified type I to type II ontogenetic shift in venom composition has been noted in

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C. v. viridis (Prairie Rattlesnake). This opposite trend involved a decrease in metalloprotease

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activity and an increase in myotoxin concentration, likely correlating with an ontogenetic shift in

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diet (Saviola et al., 2015). Because of the clear differentiation in venom components and

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activities observed in venoms of adult rattlesnakes, type I and type II venom characteristics are

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thought to be largely mutually exclusive (Mackessy, 2010b).

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Some snakes retain a highly toxic juvenile venom phenotype through adulthood, likely as a result of paedomorphosis (Calvete et al., 2009, 2012; Gutiérrez et al., 1991; Mackessy, 2010b;

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Mackessy et al., 2003). In rattlesnakes, this paedomorphic venom profile is typically

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characterized by the presence of multi-subunit PLA2-based neurotoxins, all homologs of

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crotoxin, which are largely responsible for the high toxicity of these venoms (Calvete et al.,

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2012; Mackessy et al., 2003; Saravia et al., 2002). Interestingly, this highly toxic phenotype is

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maintained through adulthood despite an age-related shift in diet (e.g., Mackessy et al., 2003).

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Crotalus scutulatus (Mojave Rattlesnake) is a snake typically found in deserts of the

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southwestern United States through central Mexico. Its venom contains a potently toxic PLA2-

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based neurotoxin (Mojave toxin), and adult C. s. scutulatus venom has been shown to contain

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very low protease activity (Glenn and Straight, 1978; Glenn et al., 1983; Mackessy, 1988).

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However, geographic variation in C. scutulatus venom has also been observed previously in a

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population in Arizona, USA (Glenn & Straight, 1983, 1990), and more recently in Mexico (Borja

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et al., 2014). Individuals that contained Mojave toxin and low metalloprotease activity were

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designated A venoms, and those that lacked Mojave toxin but had hemorrhagic venom were

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designated B venoms. Despite this geographic variation in venom composition, the type II

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venom characteristics of high neurotoxicity coupled with low metalloprotease activity is the most

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common and widespread venom phenotype observed in C. scutulatus.

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Crotalus o. helleri (Southern Pacific Rattlesnake) is found from southwestern California

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to Baja California, Mexico. Its venom has previously been reported to disrupt clotting

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mechanisms and affect smooth and striated muscle, among other activities (Metsch and Russell,

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1983; Ruiz et al., 1980). Crotalus o. helleri venom shifts from a more toxic type II phenotype to

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a more proteolytic type I phenotype as snakes age, and diet changes from primarily lizards to a

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diet that consists of mammalian prey (Mackessy, 1988). Proteolytic activity continues to

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increase as its capacity for larger mammalian prey also increases, suggesting a link between

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venom phenotype and prey taken. Though in most cases it fits a classical type I phenotype, C. o.

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helleri has been shown to have a moderate level of intraspecific diversity (Sunagar et al., 2014).

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Moreover, a highly localized population of C. o. helleri in southern California was determined to

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contain Mojave toxin and have reduced proteolytic activities. However, because these

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populations of Mojave toxin-positive C. o. helleri were geographically isolated from C. s.

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scutulatus, it was concluded that this novel venom phenotype was most likely not the result of

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recent introgression between C. o. helleri and C. s. scutulatus (French et al., 2004).

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Hybridization in rattlesnakes has been documented in scientific literature as early as 1942, when Bailey identified a hybrid between C. horridus horridus (Timber Rattlesnake) and

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Sistrurus catenatus catenatus (Eastern Massasauga) by conducting a thorough examination of

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scalation, body proportions and color patterns (Bailey, 1942). Instances of both intergeneric and

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interspecific hybridization between various species of rattlesnakes have been investigated via

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morphology, venom characteristics and, in one case, genetics. Morphologically, laboratory bred

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C. atrox (Western Diamondback Rattlesnake) x C. s. scutulatus (Mojave Rattlesnake) F₁ and F₂

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hybrids appeared to display overlapping parental characteristics (Aird et al., 1989). Characters

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such as the presence of dark flecks on dorsal scales, the number of scales between supraocular

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scales, position of the postocular light stripe, dorsal blotch patterning and light and dark tail rings

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were used to investigate inheritance patterns of morphological characteristics in both F1 and F2

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generations. Crotalus atrox × C. horridus hybridization was investigated by Meik et al. using

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scalation, color pattern and scanning electron microscopy of scale features (2008). A hybrid

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between C. s. scutulatus and C. atrox was genetically confirmed using allozyme data (Murphy

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and Crabtree, 1988). The hybrid individual presented with morphological characteristics

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intermediate to both parents, and allozyme data revealed that the hybrid contained species-

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specific markers of both C. s. scutulatus and C. atrox.

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Hybridization between rattlesnake species with highly divergent adult venoms provides the

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opportunity to characterize the venom variation that results from hybridization, to contextualize

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this variation within the type I-type II dichotomy of venom composition, and to study the venom

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gene inheritance patterns that result in a hybridized venom phenotype. Previously, only four

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studies have investigated rattlesnake hybridization using venom profiles. Glenn and Straight

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determined that hybridization was occurring between two species based on the presence of a type

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II neurotoxin in a type I species (1990). Snakes captured from an intergrade zone in southwestern

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New Mexico involving C. viridis viridis (Prairie Rattlesnake) and C. s. scutulatus showed a

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typical C. v. viridis morphology, but venom contained Mojave toxin, a distinct component of C.

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s. scutulatus that is not found in the venom of C. v. viridis. The authors concluded this was the

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result of historical hybridization and backcrossing of hybrids into C. v. viridis populations.

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However, this study assumed hybridization based on the presence of Mojave toxin in C. v. viridis

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populations without investigating metalloprotease activities as well. As such, it was not

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confirmed if hybrids displayed both type I and type II characteristics. Further characterizing

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hybridization between C. v. viridis and C. s. scutulatus in southwestern New Mexico, Zancolli et

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al. used both venomic and genomic data to determine that in this case, hybridization between

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snakes with type I and type II venoms did not result in the adaptive radiation of Mojave toxin

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into C. v. viridis populations, and ultimately these hybridized venoms remained confined to the

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hybrid zone (2016).

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Another study investigated hybrid morphology and venom profiles in a known hybrid system between one C. atrox and one C. s. scutulatus (Aird et al., 1989). Hybrids retained

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characteristics of both parents, but the C. s. scutulatus parent was described as having a type B

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venom (corresponding to type I venom), and once again, the presence of type I and type II

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characteristics resulting from hybridization was not confirmed. Aird et al. (2015) used venom

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gland transcriptomics and venomics to determine that Protobothrops flavoviridis × P. elegans

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hybrids, the result of species invasion (by P. elegans) due to human action on the Ryukya Islands

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of Japan, express overlapping parental venom profiles; however, P. flavoviridis and P. elegans

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have similar venom components and do not follow the type I/type II trend in venom composition

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seen in American rattlesnakes.

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the effects of hybridization on venom ontogeny have not been investigated. Because type I and

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type II venom characteristics are typically mutually exclusive, the venom phenotypes of hybrids

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between species that express these divergent venom phenotypes could reveal the mechanisms

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behind these potential incompatibilities. The current study outlines the venom phenotypes and

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ontogenetic shifts in venom composition resulting from the hybridization of a type I snake, C. o.

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helleri, and a type II snake, C. s. scutulatus. SDS-PAGE, venom enzymology, immunoblotting,

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MALDI-TOF MS and reversed phase HPLC were used to characterize the venom of a C. o.

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helleri male parent, a C. s. scutulatus female parent (both from southern California) and two of

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their offspring over a period of eight years (2007–2015). In addition, venoms from six adult C.

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o. helleri and six adult C. s. scutulatus individuals from southern California were used as

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parental reference samples to confirm that the venom activities and characteristics of the mother

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and father of the hybrids were distinctive and characteristic for each species. Based on the few

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published reports on hybridization and venom composition, we hypothesized that C. o. helleri ×

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C. s. scutulatus hybrids would display both the type I venom characteristic of high

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metalloprotease activity and the type II venom characteristic of expression of neurotoxic PLA2.

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2.

Materials and Methods

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2.1

Supplies and Reagents

Protein concentration reagents were purchased from BioRad, Inc. (Hercules, CA, USA).

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NuPage gels and Western blot materials were obtained from Life Technologies, Inc. (Grand

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Island, NY, USA). High performance liquid chromatography equipment and materials were

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obtained from Waters Corporation (Milford, MA, USA), and reversed phase columns were

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purchased from Phenomenex, Inc (Torrance, CA, USA). All other reagents (analytical grade or

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higher) were purchased from Sigma Biochemical Corp. (St. Louis, MO, USA).

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Venom Collection and Storage

Crotalus s. scutulatus × C. o. helleri hybrids and parents were obtained from Dan Grubb

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in 2007 when the two hybrid offspring (one male, one female) were approximately one year old.

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The male adult C. o. helleri and the female adult C. s. scutulatus originated from Los Angeles

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Co., California. All snakes were housed individually in the University of Northern Colorado

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(Greeley, CO) Animal Resource Facility. Venom was extracted from all snakes as previously

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described (Mackessy, 1988) at least once per year beginning in 2007, with the exception of 2012;

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the C. s. scutulatus mother died in 2010 and only three yearly samples were collected. All

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venoms were centrifuged at 9,500 x g for 5 minutes, lyophilized and stored at -20 °C.

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2.3.

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Protein Concentration Determination Dried venom samples were reconstituted at an apparent concentration of 4.0 mg/ml in

Millipore-filtered water, vortexed, centrifuged for 5 minutes at 9,500 x g, and protein

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concentration of the supernatant was determined using the Bradford protocol (1976) as modified

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by BioRad, Inc. and using bovine globulin as standard. Amount of material used in all

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subsequent assays was based on these determinations. Reconstituted samples were frozen at -20

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°C until used and then thawed and centrifuged at 9,500 x g for 5 minutes to pellet cellular debris.

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2.4.

Protein Gel Electrophoresis

Crude venom (20 µg) or lyophilized protein (approximately 5 µg - RP-HPLC) was

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loaded into wells of a NuPAGE Novex bis-tris 12% acrylamide mini gel and electrophoresed in

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MES buffer for 45 min at 175 V; 7 µl of Mark 12 standards were loaded for molecular weight

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estimates. Gels were stained with 0.1% Coomassie brilliant blue R-250 in 50% methanol and

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20% acetic acid (v/v) overnight with gentle shaking and destained in 30% methanol, 7% glacial

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acetic acid (v/v) in water until background was sufficiently destained (approximately 2 hours).

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Gels were then placed in storage solution (7% acetic acid, v/v) overnight with gentle shaking at

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room temperature and imaged on an HP Scanjet 4570c scanner.

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2.5.1

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Azocasein Metalloprotease Assay The azocasein metalloprotease assay procedure was performed as outlined in Aird and da

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Silva (1991), and all samples and controls were run in triplicate. Briefly, 20 µg of venom was

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incubated with 1 mg of azocasein substrate in buffer (50 mM HEPES, 100 mM NaCl, pH 8.0) for

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30 minutes at 37 ºC. The reaction was stopped with 250 µl of 0.5 M trichloroacetic acid,

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vortexed, brought to room temperature, and centrifuged at 2,000 rpm for 10 minutes.

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Absorbance of the supernatant was read at 342 nm, and all values were expressed as

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∆342nm/min/mg venom protein.

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2.5.2

L-amino acid oxidase assays were performed as outlined in Weissbach et al. (1960); all

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L-Amino Acid Oxidase

samples and controls were run in triplicate. L-kynurenine substrate was solubilized at 1.04

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mg/mL buffer (50 mM HEPES, 100 mM NaCl, pH 8.0), and 75 µl was added to 20 µg of venom

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in 645 µl buffer. The reaction was incubated at 37 ºC for 30 minutes and then terminated with

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750 µl of 10% trichloroacetic acid. Samples were brought to room temperature, and absorbances

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were read at 331 nm. Specific activity was calculated as nanomoles product formed/min/mg

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venom protein from a standard curve of the reaction product, kynurenic acid.

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2.5.3

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Phosphodiesterase Assay

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Phosphodiesterase assays were performed based on Laskowski’s (1980) modification of Björk (1963). 20 µg of crude venom was added to 200 µl of buffer (100 mM tris-HCl, 10 mM

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MgCl2, pH 9.0). 150 µl of 1.0 mM bis-p-nitrophenylphosphate substrate was added, and the

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reaction was incubated at 37 ºC for 30 minutes. The reaction was terminated with 375 µl of 100

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mM NaOH containing 20 mM disodium-EDTA, vortexed, brought to room temperature and

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absorbance read at 400 nm. Specific activity was expressed as ∆400nm/min/mg venom protein.

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Thrombin-like Serine Protease Assay

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Thrombin-like serine protease assays were performed as outlined in Mackessy (1993).

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Eight µg of crude venom was added to 373 µl of buffer (50 mM HEPES, 100 mM NaCl, pH

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8.0). Tubes were incubated at 37 ºC for approximately 3 minutes. 50 µl of substrate

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(BzPheValArg-pNA; Sigma) was added and the tube was vortexed and placed back at 37 ºC.

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Reactions were stopped after five minutes with 50% acetic acid. Tubes were read at 405 nm, and

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specific activity was calculated from a standard curve of p-nitroaniline and expressed as

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nanomoles product produced/min/mg of venom.

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2.5.5

Kallikrein-like serine protease assays were performed based on modifications of

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Kallikrein-like Serine Protease Assay

Mackessy (1993). Briefly, 0.8 µg of crude venom was added to 373 µl of buffer (50 mM

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HEPES, 100 mM NaCl, pH 8.0). Tubes were incubated at 37 ºC for approximately 3 minutes.

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50 µl of substrate (Bz-ProPheArg-pNA; Bachem) was added, and the tube was vortexed and

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placed back at 37 ºC. Reactions were stopped after three minutes with 50% acetic acid. Tubes

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were read at 405 nm, and specific activity was calculated from a standard curve of p-nitroaniline

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and expressed as nanomoles product produced/min/mg venom.

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2.6.

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Purification of Mojave Toxin and Concolor Toxin

Purification of the type II neurotoxins Mojave toxin and concolor toxin was achieved as outlined in Aird et al. (1986). 75 mg of crude C. s. scutulatus or C. o. concolor venom was

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dissolved in HEPES buffer and fractionated on a BioGel P-100 size exclusion column (2.6 x 89

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cm) using buffer (25 mM HEPES, 100 mM NaCl, 5 mM CaCl2, pH 6.8) at a flow rate of 6 mL/hr

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at 4 ºC. The toxin-containing peak was concentrated using a Millipore 3.0 kDa molecular weight

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cutoff centrifuge concentrator and further purified using a DEAE FF HiTrap 5 mL column in 50

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mM tris buffer pH 8.3 (0-1.0 M NaCl gradient) on a Life Technologies FPLC using Unicorn

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software. Concolor toxin was used as a positive control in Western blots and as a standard to

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identify the elution time of closely-related Mojave toxin on RP-HPLC (see below). The A

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subunit of Mojave toxin was purified and used to generate polyclonal antibodies in rabbits

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(GenScript, Piscataway, NJ, USA). Purified material was analyzed using a Bruker Microflex

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LRF MALDI-TOF mass spectrometer at the Proteomics and Metabolomics Facility at Colorado

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State University (Fort Collins, CO) to confirm expected mass. Concolor toxin identity was

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further confirmed by tryptic fragment analysis on an Orbitrap LC-MS/MS at the College of

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Medicine and Translational Science Core facility at Florida State University (Tallahassee, FL) as

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described in Modahl et al (2016).

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2.7.

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Western Blots

To test for the presence of Mojave toxin subunit A, 2 µg of purified concolor toxin or 20 µg of crude venom were loaded onto a NuPAGE Novex bis-tris 12% acrylamide gel and run in

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MES buffer at 175 V under reducing conditions for approximately 45 minutes as described

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above (mass standards - 7 µl of Novex Sharp pre-stained protein standard). Gels were blotted

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onto Novex 0.2 µm pore nitrocellulose membranes in a Novex Xcell II Blot Module in transfer

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buffer (25 mM bicine, 25 mM bis/tris, 1.0 mM EDTA and 0.05 mM chlorobutanol, pH 7.2 in

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20% methanol) for approximately 1 hour at ~175 mA in an ice bath. After blocking with 3%

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BSA in PBS and washing with PBS (3x), membranes were incubated with a 1:1500 dilution of

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rabbit anti-Mojave toxin subunit A polyclonal antibodies in 15 mL of 3% BSA in PBS with

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gentle shaking at room temperature overnight. Membranes were washed 3x in tris-buffered

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saline (TBS) and then incubated for 1 hour with alkaline phosphatase-conjugated goat anti-rabbit

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IgG secondary antibody (1:3000 dilution) in TBS. Membranes were washed again in TBS (3x),

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and antibody binding was detected with Sigma FAST BCIP/NBT (5-bromo-4-chloro-3-indolyl

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phosphate/nitro blue tetrazolium) tabs dissolved in 10 mL of Millipore-filtered water. The

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detection reaction was stopped with 20 mL of 20 mM disodium EDTA in PBS with gentle

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shaking for 10 minutes, rinsed in Millipore-filtered water and dried, and membranes were

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imaged on an Epson Perfection 4870 photo scanner.

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2.8.

High Performance Liquid Chromatography

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One mg of crude venom was resolubilized in 100 µl of Millipore-filtered water and centrifuged at 9,500 x g for 5 minutes to pellet cellular debris; supernatant was filtered with a

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0.45 µm syringe tip filter. Venom was subjected to reverse phase HPLC using a Phenomenex

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Jupiter C18 (250 x 4.6 mm, 5 µm, 300 Å pore size) column, and one minute fractions were

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collected at a flow rate of 1 mL/minute for a total run time of 100 minutes. To elute proteins, a

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gradient of 95% solution A (0.1% trifluoroacetic acid in Millipore-filtered water) to 95%

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solution B (0.1% TFA in 100% acetonitrile) was used (5% solution B for 10 minutes; 10–25% B

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over 10 minutes; 25–45% B over 60 minutes; 45–70% over 10 minutes; 70-90% over 2 minutes;

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90-95% over 3 minutes; and returning to 5% B over 5 minutes). Protein/peptide was detected at

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220 nm and 280 nm with a Waters 2487 Dual λ Absorbance Detector. Fractions corresponding

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to each peak were then frozen overnight at -80 ºC and lyophilized. These fractions were then

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analyzed along with 20 µg crude venom using SDS-PAGE as described above in order to

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determine mass and designate probable toxin families. Myotoxin, Mojave toxin and PLA2 peaks

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were also analyzed using a Bruker Microflex LRF MALDI-TOF mass spectrometer at the

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Proteomics and Metabolomics Facility at Colorado State University (Fort Collins, CO) to

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confirm protein identity. Approximately 1.0 µg protein was dissolved in 1.0 µL 50% acetonitrile

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containing 0.1% TFA, mixed with 1.0 µL sinapinic acid matrix (10 mg/mL, dissolved in the

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same solvent), and spotted onto target plates.

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2.9.

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Principal Coordinates Analysis HPLC peaks for 3 C. o helleri, 3 C. s. scutulatus and both hybrids were scored as present

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(1) or absent (0) and analyzed with GenAlEx (version 6.5 (http://www. http://biology-

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assets.anu.edu.au/GenAlEx); Peakall and Smouse 2006, 2012). Genetic distance was calculated

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for all 51 variable loci as a haploid binary system. Principal coordinate analysis was computed

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from genetic distance as a tri distance matrix using the standardized covariance method.

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2.10.

Crotalus s. scutulatus mother, C. o. helleri father and hybrid venoms were tested for

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Lethal Toxicity (LD50) Assays

lethal toxicity towards NSA mice (20-24 g). Mice were given intraperitoneal injections on the

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right side of the body. Doses of 0, 0.1, 0.25, 0.5, 1.0, 1.2 and 1.5 µg/g were adjusted for body

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mass and administered in a 100 µl bolus of 0.9% saline, and 24 hour survivorship was scored.

301

2.11.

Statistics

To test for significance between samples for enzymatic activities and reverse phase

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HPLC chromatogram peak areas, a two sample, two-tailed t-test assuming unequal variance was

304

run. Significance level was set at p<0.05.

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3.

Results

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3.1.

Gel Electrophoresis

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SDS-PAGE revealed a high level of complexity in both parents and adults, and each individual had a consistent electrophoretic profile from year to year, with the exception of one

310

band at approximately 10 kD that appeared between 2008 and 2009 in both hybrids and was

311

retained through adulthood (Fig. 1). Both hybrids venoms contained components specific to

312

each parent, and they showed overlapping profiles compared to parents. Notably, both hybrids

313

contained father-specific myotoxin bands at approximately 4 kD, though in lower abundance.

314

The dominant protein in C. s. scutulatus venom was a protein at approximately 14 kD (Mojave

315

toxin subunit B), which was also present at lower abundances in both hybrid venoms, but absent

316

in the male C. o. helleri.

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3.2.1 Azocasein Metalloprotease Activity Crude venoms were assayed for five enzymes commonly found in rattlesnake venoms. Metalloprotease activity was significantly different (p=0.004) between adult C. s. scutulatus

320

venom and adult C. o. helleri venom (Fig. 2A). The type I-type II dichotomy that encompasses

321

the C. viridis/oreganus complex (Mackessy, 2010) is also mirrored in the venom phenotypes of

322

C. o. helleri and C. s. scutulatus in the present study. Crotalus o. helleri venom metalloprotease

323

activity was typically orders of magnitude higher than the nearly undetectable metalloprotease

324

activity of C. s. scutulatus venom (see also Mackessy, 1988). In addition, although Crotalus o.

325

helleri adults consistently showed higher metalloprotease activity, they also displayed overall

326

higher variation in activity.

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The C. s. scutulatus female parent had consistently low azocasein metalloprotease activity that did not change over time, while the Crotalus oreganus helleri male parent had

329

metalloprotease activity that steadily increased from 2007 to 2015 (Fig. 3A). Azocasein

330

metalloprotease activity of C. s. scutulatus × C. o. helleri female hybrid venom dropped sharply

331

from 2007 to 2008, and remained low through adulthood, the typical trend seen in type II snake

332

venoms. The C. s. scutulatus × C. o. helleri hybrid male's venom had higher activity as a

333

neonate than the hybrid female, and it followed the typical type I venom trend of increasing

334

metalloprotease activity over time.

335

3.2.2 Thrombin-like Serine Protease Activity

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Average thrombin-like serine protease activities of all C. s. scutulatus and C. o. helleri

337

adult venoms did not differ significantly (p=0.168; Fig. 2B). Venom of the C. s. scutulatus

338

female parent had consistently high thrombin-like serine protease activity from 2007 through

339

2009 that was slightly variable from year to year (Fig. 3B). These values were concordant with

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the activities determined from the six other C. s. scutulatus adults. Venom from the male parent

341

Crotalus o. helleri had thrombin-like serine protease activity that was lower than the C. s.

342

scutulatus female parent, but it gradually increased between 2007 and 2015. Thrombin-like

343

serine protease activities of both hybrids fell below the male parent’s activity, but gradually

344

increased over time to fall intermediate to both parents by the time they reached adulthood.

345

3.2.3 Kallikrein-like Serine Protease Activity

Kallikrein-like activities of C. s. scutulatus and C. o. helleri reference venoms varied

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significantly (p=0.0002; Fig. 2C). Adult C. s. scutulatus venoms had a higher average activity

348

than all adult C. o. helleri, but C. o. helleri venoms had a wider range of activities. The C. s.

349

scutulatus female parent had consistently high kallikrein-like serine protease activity (Fig. 3C),

350

consistent with the activities determined from the six other C. s. scutulatus adult venoms.

351

Crotalus oreganus helleri venom had lower kallikrein-like serine protease activity than the C. s.

352

scutulatus female parent that increased slightly overtime. Activities of both hybrid venoms fell

353

intermediate between parent activities: the hybrid female decreased slightly and the hybrid male

354

increased slightly between 2007 and 2015.

355

3.2.4. L-Amino Acid Oxidase Activity

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Average activities of C. s. scutulatus and C. o. helleri reference venoms differed

357

significantly (p=0.008; Fig. 2D). Activities between both parents and hybrids remained

358

relatively consistent across all years, with all individuals increasing slightly overtime (Fig. 3D).

359

All individuals had comparable activities and no clear intermediate trend in activity was

360

apparent.

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3.2.5. Phosphodiesterase Activity Average activities of C. s. scutulatus and C. o. helleri reference venoms did not vary significantly (p=0.45; Fig. 2E). All individuals had phosphodiesterase activity that increased

366

over time; however, venom from the C. s. scutulatus female parent had consistently higher

367

activity than the C. o. helleri male parent (Fig. 3E).

368

3.3. Mojave Toxin and Concolor Toxin Purification

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Concolor toxin (C. o. concolor venom) and Mojave toxin (C. s. scutulatus venom) were

370

purified on a low pressure size exclusion BioGel P-100 column and an FPLC DEAE FF HiTrap

371

anion exchange column (data not shown). Identities of Mojave toxin and concolor toxin and

372

subunits were confirmed using MALDI-TOF MS and Orbitrap LC-MS/MS, and this material

373

was used in subsequent assays as a positive control (concolor toxin) and in antibody generation

374

(Mojave toxin subunit A: see below).

375

3.4. Western Blots

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Rabbit antibodies raised against the A subunit of Mojave toxin (produced by GenScript, Inc.)

377

were not monospecific and bound to the A subunit (a modified PLA2; 9.4 kDa actual mass), with

378

a diffuse band at approximately 9kD, the B subunit (a PLA2; actual mass ~14 kDa), with a band

379

at approximately 14 kDa, and to non-neurotoxic PLA2s, at approximately 14-15 kDa (Figure 4).

380

Crotalus s. scutulatus venom had a B subunit band at approximately 14 kDa and a more

381

prominent A subunit band at approximately 9 kD. Crotalus o. helleri venom had a prominent

382

PLA2 band at approximately 15 kDa, slightly higher than the C. s. scutulatus B subunit band and

383

lacked an A subunit band. Both hybrid venoms contained overlapping profiles relative to parents

384

with bands at approximately 15 kDa, 14kDa and 9 kDa. Neither negative control venoms (C. v.

385

viridis, C. o. cerberus) showed a band at 9 kDa.

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3.5.

High Performance Liquid Chromatography Reversed phase HPLC revealed a high level of complexity for all C. s. scutulatus, C. o.

helleri and hybrid venoms. However, SDS-PAGE demonstrated that similar protein components

389

eluted at comparable times regardless of species. The low abundance of smaller peaks from RP-

390

HPLC product did not adversely affect protein detection with SDS-PAGE, and amounts as low

391

as 2 µg were visible. The RP-HPLC chromatograms of three individual C. s. scutulatus

392

reference venoms revealed that 1) the dominant peak of C. s. scutulatus venom (Mojave toxin

393

subunit B) eluted at approximately 41 minutes, 2) no myotoxins were present in these C. s.

394

scutulatus venoms, and 3) metalloprotease peaks were minimal in all C. s. scutulatus

395

chromatograms (Fig. 5A, Fig. 7A, and Supplemental Figs. 1 and 2).

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Reversed phase HPLC fractionation of C. o. helleri venom revealed that myotoxins eluting at approximately 23 minutes were the dominant component (Supplemental Fig. 14). In

398

addition, all three C. o. helleri individuals lacked a 41 minute peak (Mojave toxin) and had

399

sizeable clusters of metalloprotease peaks eluting from approximately 84 to 90 minutes (Fig. 5B,

400

Fig. 7B, and Supplemental Figs. 3, 4, and 5).

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The C. o. helleri male parent’s metalloprotease peaks accounted for approximately 5% of

402

the total venom composition, while the C. scutulatus female parent’s metalloprotease peak made

403

up less than 1% of the venom (Figs. 5A and 5B). Both hybrid venoms also had metalloprotease

404

peaks in the two years that were analyzed; however, the female hybrid's peaks were a smaller

405

percentage of the total venom as a juvenile (in 2007; Table 1; Fig. 6A) and an adult (in 2015;

406

Fig. 5C) than the hybrid male (Figs. 6B and 6D, respectively).

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The female hybrid’s percentage of metalloproteases dropped slightly from 1.5% to 1.3% between 2008 and 2015 (Table 1; Figure 6A and 6C). This is consistent with the ontogenetic

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409

decrease seen in the female hybrid’s azocasein metalloprotease activity. Conversely, the male

410

hybrid’s percentage of metalloproteases increased from 5.1% to 7.9%, which also mirrors the

411

increase seen in the azocasein metalloprotease assay (Table 1; Figure 6B and 6D). The Mojave toxin B peak (41 min) comprised approximately 18% of the C. s. scutulatus

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female parent’s total venom (Fig. 5A). However, Mojave toxin comprised approximately 25%

414

of the total venom of two other C. s. scutulatus venoms (Supplemental Figs. 1 and 2). Both

415

hybrid chromatograms showed a 41 minute Mojave toxin subunit B peak; however, this was not

416

the dominant component as observed in the C. s. scutulatus individuals (Fig. 6, Fig. 7C and 7D).

417

Mass spectrometry revealed that both hybrid Mojave toxin peak components had masses

418

consistent with the subunit B of Mojave toxin present in the C. s. scutulatus female parent

419

(14,186 Da; Figure 7A, 7C, and 7D). The percentage of Mojave toxin increased slightly for both

420

the female and male hybrids as they aged, from 6.8% to 9.8% and 8.3% to 10.3%, respectively

421

(Table 1). This peak was absent from the male parent’s venom; the only PLA2 observed (RP-

422

HPLC peak 62 min) had a mass of 13, 665 Da (Figure 7B).

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3.6.

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Principal Coordinate Analysis (PCoA)

Principal coordinate analysis of reverse phase HPLC chromatograms of three C. s.

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scutulatus individuals, three C. o. helleri individuals and both hybrid venoms revealed three

426

distinct clusters (Fig. 8). All C. s. scutulatus and C. o. helleri individuals clustered tightly

427

together by species, and hybrids from all years analyzed clustered together, in between C. o.

428

helleri and C. s. scutulatus with regards to Coordinate 1.

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3.7.

Lethal Toxicity The male parent C. o. helleri venom had a substantially higher LD50 value than C. s.

431

scutulatus and both hybrid venoms (Fig. 9). The female parent Crotalus s. scutulatus and the

433

female and male hybrid venoms had LD50 values of 0.14, 0.14 and 0.18 µg/g, respectively.

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434 4.

Discussion

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The type I-type II dichotomy of venom composition seen in the majority of rattlesnakes

438

represents a tradeoff between highly toxic venom, resulting from the presence of PLA2-derived

439

neurotoxins, and degradative venom, characterized primarily by high metalloprotease activity

440

(Mackessy, 2010a). This dichotomy in venom composition is based on the observation that high

441

metalloprotease activity and neurotoxicity appear to be mutually exclusive characteristics of

442

many species, and these typically are consistent features of an entire species (Mackessy, 2010a).

443

However, in a small number of instances, both neurotoxic and proteolytic activities have been

444

shown to be expressed concurrently. For example, some populations of C. s. scutulatus were

445

reported to express both the hemorrhagic components of B venom (Type I venom) and the

446

neurotoxic Mojave toxin of A venom (Type II venom). These A + B venom profiles were

447

determined to be the result of an intergrade zone between A and B venom type populations in

448

southeastern Arizona (Glenn & Straight, 1989). This concept was further elaborated on by

449

Massey et al. (2012), who identified six (A-F) C. s. scutulatus venom phenotypes, in the same

450

southeastern region of Arizona, which formed a gradient of venom profiles from predominantly

451

degradative to predominantly neurotoxic. A similar trend was observed in the distribution of

452

Canebrake toxin among populations of C. horridus (Glenn et al., 1994). While the majority of

453

venoms clustered into type A and B designations, with type B venom being the most common

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phenotype observed throughout the entire range of this species, a few individuals from eastern

455

South Carolina, southwestern Arkansas, and northern Louisiana contained A + B venoms,

456

indicating the presence of an intergrade zone between snakes with A and B venoms in these

457

regions.

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The expression of Mojave toxin and high metalloprotease activity seen in the venoms of C. s. scutulatus × C. o. helleri hybrids is another exception to the rule that type I and type II

460

venom characteristics are usually mutually exclusive (Mackessy, 2010a). Moreover, the

461

concurrent expression of Mojave toxin and high metalloprotease activity most clearly

462

demonstrated in the venom profile of the hybrid male refutes French et al.'s (2004) assertion that

463

expression of metalloprotease genes may be suppressed by the expression of Mojave toxin genes.

464

However, the hybrids bred in captivity for this study are free from the ecological, trophic and

465

dietary selective pressures that likely contribute to the reinforcement of a type I-type II trade-off.

466

In an ecological context, it is unclear how selection would act on venom phenotypes that

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overcome the type I-type II dichotomy. Depending upon the success of snakes with hybridized

468

venoms in these areas, selection could play a critical role in the maintenance of a hybrid zone, in

469

the reinforcement of reproductive isolation and ultimately species divergence, or in the exchange

470

of specific toxin genes between species. Despite the assumption that type II neurotoxins are

471

inherently more effective at prey capture and that hybridization will result in the radiation of

472

these toxins into parental populations, Zancolli et al. (2016) reported that Mojave toxin found in

473

the venom of C. s. scutulatus × viridis hybrids in southwestern New Mexico did not permeate

474

into C. v. viridis populations and thus did not confer a selective advantage for snakes in these

475

areas. However, in another context, the expression of type I and type II venom characteristics in

476

conjunction with one another could broaden the range of prey items available, helping hybrids to

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477

capitalize on a more diverse array of prey items. On the other hand, hybridization could interfere

478

with a venom phenotype specifically adapted to a particular localized prey type, and ultimately

479

undermine the molecular mechanisms used for prey capture by snakes in these areas. The venom phenotypes and ontogenetic shifts that occur in venom composition resulting

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from the hybridization of a type I snake, C. o. helleri, and a type II snake, C. s. scutulatus,

482

suggest a complex pattern of inheritance of venom characteristics. Both hybrids expressed C. o.

483

helleri (father-specific) myotoxins as well as C. s. scutulatus (mother-specific) Mojave toxin.

484

This could indicate a dominant pattern of inheritance; however, SDS-PAGE band size and

485

intensity were reduced relative to parents, and percent area of RP-HPLC Mojave toxin peaks in

486

both adult hybrids were slightly higher than half (~10% of total venom protein) of the C. s.

487

scutulatus female parent’s percentage of Mojave toxin (~18% of total venom). Adult hybrid

488

myotoxin peaks made up 38-45% of the total venom profile of hybrids, while the C. o. helleri

489

male parent’s myotoxin peaks accounted for 62% of the total venom. This suggests a pattern of

490

codominant expression, as each hybrid would inherit myotoxin and Mojave toxin A subunit and

491

B subunit genes from only one parent, and express less of each toxin than a parent with two

492

alleles. Interestingly, hybrids had an LD50 value not significantly different from the C. s.

493

scutulatus mother, indicating that activity of Mojave toxin plus myotoxin is so potent that even a

494

reduced expression of the proteins is sufficient to induce a lethal dose.

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Both thrombin-like and kallikrein-like serine protease activities were intermediate to

496

parent activities, indicating possible codominant or additive expression of these compounds.

497

Thrombin-like activity also increased over time in both hybrids, while kallikrein-like serine

498

protease activity slightly decreased over time. Both of these trends in serine protease activity

499

have been previously noted in type II ontogenetic shifts in venom composition (Mackessy et al.,

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2003). However, thrombin-like serine protease activity was not significantly different between

501

the six C. o. helleri and six C. s. scutulatus adults used as representative samples and showed

502

wide variation in activity, particularly between C. o. helleri individuals. As such, thrombin-like

503

activity is likely not a good candidate for making inferences about hybrid status based on venom

504

profiles.

505

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Kallikrein-like serine protease activity was significantly higher in C. s. scutulatus than in C. o. helleri, with low variation in activity levels between individuals of both species.

507

Kallikrein-like serine protease activity may make a better marker for hybridization, as parent

508

activities are drastically different and expression appears to be codominant, so hybrids could fall

509

intermediate between parent activities. However, serine protease activities have been shown to

510

vary widely among Crotalus species (Mackessy, 2008, 2010a), and they are not associated with

511

the type I-type II dichotomy as strongly as metalloprotease activity. Use of kallikrein-like

512

activity to indicate hybridization may therefore be dependent on the species pair involved.

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L-amino acid oxidase activity, while significantly different between the two species, did

514

not show any significant differences in hybrid parents or definitive patterns in ontogenetic shifts

515

in either hybrid, though activity in all individual venoms increased gradually over time.

516

Phosphodiesterase activity was not significantly different between representative parent species,

517

but it also gradually increased over time for both parents and hybrids. Because both L-amino

518

acid oxidase and phosphodiesterase activities are not key components that consistently

519

characterize the differences between type I or type II venoms and are not toxic compounds that

520

contribute substantially to the lethality of a venom, they are not particularly useful in

521

characterizing the venom of C. s. scutulatus × C. o. helleri hybrids in the current study.

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Inheritance patterns of venom genes could be convoluted due to the large number of gene duplication events of functionally important toxins and the effects of random assortment and

524

genetic recombination (Casewell et al., 2011; Kondraschov, 2012; Vonk et al., 2013). The

525

extensive levels of gene duplication events and neofunctionalization result in large multigene

526

families with high numbers of similar toxin sequences that have the potential for highly variable

527

activities (Chang and Duda, 2012; Vonk et al., 2013). Thus, because of the randomizing effects

528

of assortment and genetic recombination on the multitude of repeatedly duplicated toxin genes,

529

hybrids will likely display varying levels of activities, regardless of further alterations to

530

expression via transcriptional and translational regulation, because they may inherit differing

531

quantities of various toxin genes.

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Venom profiles of hybrids may also be further complicated by epigenetic, transcriptional, or translational regulation that could alter the expression levels of inherited toxin genes

534

(Casewell et al., 2014; Gibbs et al., 2009; Margres et al., 2014). For example, transcripts

535

encoding three-finger toxins were sequenced from viperids, but were not expressed in the

536

venom, though the particular mechanism of translational repression was not elucidated

537

(Junqueira-de-Azevedo et al., 2006; Pahari et al., 2007). More recently, miRNAs have been

538

shown to play a role in the posttranscriptional regulation of crotoxin B-subunit and PIII

539

metalloprotease expression during the ontogenetic shift from a type II to a type I venom in C. s.

540

simus (Durban et al., 2013). As such, it is possible that some inherited toxin genes are

541

transcribed in hybrids, but are ultimately not expressed in the venom due to translational

542

regulation. Thus, the unique ‘hybridized’ venom profiles that result from hybridization between

543

snakes with divergent venom phenotypes are likely the result of complex patterns in gene

544

inheritance and the additive effects of transcriptional and translational regulation. However,

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despite these possible complications with inferring genotype from venom phenotype, there

546

appears to be a genotype-phenotype linkage in the concurrent expression of Mojave toxin

547

subunits A and B, simplifying hybrid status delineations of type I-type II hybrids based on

548

venom profiles (Zancolli et al., 2016).

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The ontogenetic shifts in azocasein metalloprotease activity of the hybrid male mirrored

550

the increase in the C. o. helleri male parent's activity and the typical increase in metalloprotease

551

activity over time seen in type I snakes. Following the trend seen in type II snakes, the hybrid

552

female's activity decreased from moderate to low activity over time. However, metalloprotease

553

activity seen in the female hybrid was not as low as the C. s. scutulatus mother (or other

554

reference C. s. scutulatus), which had nearly non-detectable activity. Because ontogenetic shifts

555

in metalloprotease activity of the female and male hybrid mirrored the trend of the C. s.

556

scutulatus mother and C. o. helleri father, respectively, this could indicate sex-influenced

557

inheritance or expression of metalloprotease genes. Therefore, in the event that hybridization is

558

unknown but suspected, sex may play a role in the ontogenetic shifts in venom composition that

559

occur and ultimately in the levels of metalloprotease activity observed in hybrids. We

560

acknowledge that the apparent sex-linked ontogenetic changes are based on a highly limited

561

sample size and therefore are speculative at this point. However, crosses of the F1 hybrids and

562

studies of the offspring produced by a C. o. helleri female and C. s. scutulatus male could further

563

elucidate the sex-influenced ontogenetic trends suggested in this study. Because sex

564

determination is genetic in snakes, further hybrid crosses could reveal a link between inheritance

565

of sex chromosomes and the expression of metalloprotease genes.

566

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5.

Conclusions Crotalus o. helleri × C. s. scutulatus hybrids appeared to exhibit a number of different

venom component expression patterns. This study presents an analysis of the ontogenetic shifts

570

of type I-type II hybrid venoms and confirms that the venom components that are most helpful in

571

determining hybrid status using venom profiles are the compounds that most clearly illustrate the

572

type I-type II dichotomy in venom composition. Thus, this known hybrid system can be used as

573

a model to generate venom profiles of potential type I-type II hybrids and allow for hybrid status

574

delineations based upon only two major venom components, despite the (likely) complexity of

575

parent and potential hybrid venoms. The C. s. scutulatus × C. o. helleri hybrid male's venom

576

profile provided the strongest evidence that venom profiles can be used to identify rattlesnake

577

hybrids, as this snake contains identifying characteristics of both parental species. However, the

578

possible influence of sex on metalloprotease activity development (as seen in the ontogenetic

579

shifts of the hybrid female) may lead to a wide range in activities of hybrid venoms. This should

580

be taken into account in the characterization and delineation of hybrid venoms due to the

581

possibility that sex influence may obfuscate the confirmation of hybridization between

582

rattlesnakes based on venom phenotypes alone.

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Understanding the inheritance and expression patterns of venom toxin genes as well as the regulatory mechanisms responsible for the venom profiles observed in known hybrid systems

585

(particularly those that span the type I-type II dichotomy) can help to clarify the diversity of

586

venom phenotypes that occur in an ecological context. Similar evolutionary mechanisms may

587

underpin the venom variation observed in the intergrade zones between rattlesnakes with

588

divergent venoms, allowing for the application of these venom profiling methods to new or

589

cryptic hybrid systems. Moreover, knowledge of prey availability and abundance at sites of high

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venom variation may help elucidate the adaptive roles of highly divergent venom types and the

591

evolutionary forces at work between snakes and available prey species. This often elusive

592

component of snake natural history can expand our understanding of not only the phenotypic

593

consequences of hybridization but also the effects of hybridization on venom markers under

594

selection. Hybridization events between species with divergent venom profiles may be a source

595

of novel venom phenotypes and could also further clarify the evolutionary and functional

596

consequences of intergrade zones between rattlesnake species.

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600

The authors thank Dan Grubb for donating the C. o. helleri male parent, the C. s. scutulatus

601

female parent and the hybrid offspring used in this study. Support for this project was provided

602

in part by a Provost Fund grant from the UNC Faculty Research and Publication Board (SPM)

603

and by grants from the UNC Graduate Student Association (CFS).

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References

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Aird, S.D., Kaiser, I.I., Lewis, R.V., Kruggel, W.G., 1986. A complete amino acid sequence for

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the basic subunit of crotoxin. Arch. Biochem. Biophy. 249, 296-300. Aird, S.D., Thirkhill L.J., Seebart C.S., Kaiser, I.I., 1989. Venoms and morphology of Western diamondback/Mojave rattlesnake hybrids. J. Herpetol. 23, 131-141.

Aird, S.D., da Silva, N.J., 1991. Comparative enzymatic composition of Brazilian coral snake (Micrurus) venoms. Comp. Biochem. Physiol. 99B, 287-294.

SC

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Aird, S.D., Aggarwal, S., Villar-Briones, A., Tin, M.M., Terada, K., Mikheyev, A.S., 2015.

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Snake venoms are integrated systems, but abundant venom proteins evolve more rapidly.

620

BMC Genomics 16, 647.

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618

Andrade, D. V., and Abe, A.S. 1999. Relationship of venom ontogeny and diet in Bothrops. Herpetologica 55, 200–204.

Bailey, R.M., 1942. An intergeneric hybrid rattlesnake. Am. Nat. 76, 376-385.

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Björk, W., 1963. Purification of phosphodiesterase from Bothrops atrox venom, with special

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consideration of the elimination of monophosphatases. J. Biol. Chem. 238, 2487-2490. Borja, M., Castañeda, G., Espinosa J., Neri, E., Carbajal, A., Clement, H., García, O., Alagon,

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A., 2014. Mojave Rattlesnake (Crotalus scutulatus scutulatus) with type B venom from

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Mexico. Copeia 2014, 7-13.

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

631

Calvete, J.J., Sanz, L., Cid, P., de la Torre, P., Flores-Díaz, M., Dos Santos, M.C., Borges, A.,

632

Bremo, A., Angulo, Y., Lomonte, B., 2009. Snake venomics of the Central American

633

rattlesnake Crotalus simus and the South American Crotalus durissus complex points to

ACCEPTED MANUSCRIPT

634

neurotoxicity as an adaptive paedomorphic trend along Crotalus dispersal in South

635

America. J. Proteome Res. 9, 528-544. Calvete, J.J., Pérez, A., Lomonte, B., Sánchez, E.E., Sanz, L., 2012. Snake venomics of Crotalus

637

tigris: the minimalist toxin arsenal of the deadliest neartic rattlesnake venom.

638

Evolutionary clues for generating a pan-specific antivenom against crotalid type II

639

venoms. J. Proteome Res. 11, 1382-1390.

RI PT

636

Casewell, N.R., Wagstaff, S.C., Harrison, R.A., Renjifo, C., Wüster, W., 2011. Domain loss

641

facilitates accelerated evolution and neofunctionalization of duplicate snake venom

642

metalloproteinase toxin genes. Mol. Biol. and Evol. 28, 2637-2649.

M AN U

SC

640

Casewell, N.R., Wagstaff, S.C., Wüster, W., Cook, D.A., Bolton, F.M., King, S.I., Pla, D., Sanz,

644

L., Calvete, J.J., Harrison, R.A., 2014. Medically important differences in snake venom

645

composition are dictated by distinct postgenomic mechanisms. Proc. Nat. Acad. Sci. 111,

646

9205-9210.

648 649

Cate, R.L., Bieber, A.L., 1978. Purification and characterization of Mojave (Crotalus scutulatus scutulatus) toxin and its subunits. Arch. Biochem. Biophys. 189, 397-408. Chang, D., Duda, T.F., 2012. Extensive and continuous duplication facilitates rapid evolution

EP

647

TE D

643

and diversification of gene families. Mol. Biol. Evol. doi: 10.1093/molbev/mss068.

651

Daltry, J.C., Wüster, W., Thorpe, R.S., 1996. Diet and snake venom evolution. Nature 379, 537-

652

AC C

650

540.

653

Durban, J., Pérez, A., Sanz, L., Gómez, A., Bonilla, F., Chacón, D., Sasa, M., Angulo, Y.,

654

Gutiérrez, J.M., Calvete, J.J., 2013. Integrated “omics” profiling indicates that miRNAs

655

are modulators of the ontogenetic venom composition shift in the Central American

656

rattlesnake, Crotalus simus simus. BMC Genomics 14, 234.

ACCEPTED MANUSCRIPT

657

French, W.J., Hayes, W.K., Bush, S.P., Cardwell, M.D., Bader, J.O., Rael, E.D., 2004. Mojave

658

toxin in venom of Crotalus helleri (Southern Pacific Rattlesnake): molecular and

659

geographic characterization. Toxicon 44, 781-791. Gibbs, H.L., Sanz, L., Calvete, J.J., 2009. Snake population venomics: proteomics-based

661

analyses of individual variation reveals significant gene regulation effects on venom

662

protein expression in Sistrurus rattlesnakes. J. Mol. Evol. 68, 113-125.

664

Glenn, J.L., Straight, R.C., 1978. Mojave rattlesnake Crotalus scutulatus scutulatus venom:

SC

663

RI PT

660

variation in toxicity with geographical origin. Toxicon 16, 81-84.

Glenn, J.L., Straight, R.C., Wolfe, M.C., Hardy, D.L., 1983. Geographical variation in Crotalus

666

scutulatus scutulatus (Mojave rattlesnake) venom properties. Toxicon 21, 119-130.

667

Glenn, J.L., Straight, R.C., 1989. Intergradation of two different venom populations of the

668

M AN U

665

Mojave rattlesnake (Crotalus scutulatus scutulatus) in Arizona. Toxicon 27, 411-418. Glenn, J.L., Straight, R.C., 1990. Venom characteristics as an indicator of hybridization between

670

Crotalus viridis viridis and Crotalus scutulatus scutulatus in New Mexico. Toxicon 28,

671

857-862.

TE D

669

Glenn, J.L., Straight, R.C., Wolt, T., 1994. Regional variation in the presence of canebrake toxin

673

in Crotalus horridus venom. Comparative Biochemistry and Physiology Part C. Toxicol.

674

Pharmacol. 107, 337-346.

AC C

EP

672

675

Gutiérrez, J.M., Dos Santos, M.C., de Fatima Furtado, M., Rojas, G., 1991. Biochemical and

676

pharmacological similarities between the venoms of newborn Crotalus durissus durissus

677 678 679

and adult Crotalus durissus terrificus rattlesnakes. Toxicon 29, 1273-1277.

Ho, C.L., Lee, C.Y., 1981. Presynaptic actions of Mojave toxin isolated from Mojave rattlesnake (Crotalus scutulatus) venom. Toxicon 19, 889-892.

ACCEPTED MANUSCRIPT

Junqueira-de-Azevedo, I.L., Ching, A.T., Carvalho, E., Faria, E., Nishiyama, M.Y., Ho, P.L.,

681

Diniz, M.R., 2006. Lachesis muta (Viperidae) cDNAs reveal diverging pit viper

682

molecules and scaffolds typical of cobra (Elapidae) venoms: implications for snake toxin

683

repertoire evolution. Genetics 173, 877-889.

686 687 688 689

environment. Proc. R. Soc. B. 279, 5048-5057.

Laskowski Sr, M., 1980. Purification and properties of venom phosphodiesterase. Methods

SC

685

Kondrashov, F.A., 2012. Gene duplication as a mechanism of genomic adaptation to a changing

Enzymol. 65, 276-284.

Mackessy, S.P., 1988. Venom ontogeny in the Pacific rattlesnakes Crotalus viridis helleri and C.

M AN U

684

RI PT

680

v. oreganus. Copeia 1988, 92-101.

Mackessy, S.P., 1993. Kallikrein-like and thrombin-like proteases from the venom of juvenile

691

northern Pacific rattlesnakes (Crotalus viridis oreganus). J. Nat. Toxins 2, 223-239.

692

Mackessy, S.P., Williams, K., Ashton, K.G., 2003. Ontogenetic variation in venom composition

693

and diet of Crotalus oreganus concolor: a case of venom paedomorphosis? Copeia 2003,

694

769-782.

Mackessy, S.P., 2008. Venom composition in rattlesnakes: trends and biological significance. In

EP

695

TE D

690

W.K. Hayes, K.R. Beaman, M.D. Cardwell, and S.P. Bush, eds., The Biology of

697

Rattlesnakes, p. 495-510. Loma Linda University Press, Loma Linda, CA.

698 699

AC C

696

Mackessy, S.P., 2010a. Evolutionary trends in venom composition in the western rattlesnakes (Crotalus viridis sensu lato): toxicity vs. tenderizers. Toxicon 55, 1463-1474.

700

Mackessy, S.P., 2010b. The field of reptile toxinology: snakes, lizards and their venoms. In

701

Mackessy, S.P. (ed.), Handbook of Venoms and Toxins of Reptiles, p. 3-23. CRC

702

Press/Taylor & Francis Group, Boca Raton, FL.

ACCEPTED MANUSCRIPT

703

Margres, M.J., McGivern, J.J., Wray, K.P., Seavy, M., Calvin, K., Rokyta, D.R., 2014. Linking

704

the transcriptome and proteome to characterize the venom of the eastern diamondback

705

rattlesnake (Crotalus adamanteus). J. Proteomics 96, 145-158. Massey, D.J., Calvete, J.J., Sánchez, E.E., Sanz, L., Richards, K., Curtis, R., Boesen, K., 2012.

707

Venom variability and envenoming severity outcomes of the Crotalus scutulatus

708

scutulatus (Mojave rattlesnake) from Southern Arizona. J. Proteomics 75, 2576-2587.

711 712

SC

710

Meik, J.M., Fontenot, B.E., Franklin, C.J., King, C., 2008. Apparent natural hybridization

between the rattlesnakes Crotalus atrox and C. horridus. Southwestern Nat. 53, 196-200. Ménez, A., ed. 2002. Perspectives in molecular toxinology. John Wiley & Sons. Chichester, England.

M AN U

709

RI PT

706

Metsch, R., Dray, A., Russell, F., 1983. Effects of the venom of the Southern Pacific rattlesnake,

714

Crotalus viridis helleri, and its fractions on striated and smooth muscle. Proc. West

715

Parmacol. Soc. 27, 395-398.

TE D

713

716

Modahl, C.M., Mukherjee, A.K., Mackessy, S.P., 2016. An analysis of venom ontogeny and

717

prey-specific toxicity in the Monocled Cobra (Naja kaouthia). Toxicon 119, 8-20. Murphy, R.W., Crabtree, C.B., 1988. Genetic identification of a natural hybrid rattlesnake:

719

Crotalus scutulatus scutulatus × C. viridis viridis. Herpetologica, 119-123.

EP

718

Pahari, S., Mackessy, S.P., Kini, R.M., 2007. The venom gland transcriptome of the Desert

721

Massasauga Rattlesnake (Sistrurus catenatus edwardsii): towards an understanding of

722 723 724 725

AC C

720

venom composition among advanced snakes (Superfamily Colubroidea). BMC Mol. Biol.

8, 115.

Parker, J.M., Anderson, S.H., 2007. Ecology and behavior of the Midget Faded Rattlesnake (Crotalus oreganus concolor) in Wyoming. J. Herpetol. 41, 41-51.

ACCEPTED MANUSCRIPT

727 728 729

Peakall, R., Smouse, P.E., 2006. GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol. Ecol. Notes 6, 288-295. Peakall, R., Smouse, P.E., 2012. GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research—an update. Bioinformatics 28, 2537-2539.

RI PT

726

Ruiz, C., Schaeffer, R., Weil, M., Carlson, R., 1980. Hemostatic changes following rattlesnake

731

(Crotalus viridis helleri) venom in the dog. J. Pharmacol. Exp. Ther. 213, 414-417.

732

Saravia, P., Rojas, E., Arce, V., Guevara, C., López, J.C., Chaves, E., Velásquez, R., Rojas, G.,

733

Gutiérrez, J.M., 2002. Geographic and ontogenic variability in the venom of the

734

neotropical

735

implications. Rev. Biol. Trop. 50, 337-346.

Crotalus

durissus:

pathophysiological

M AN U

rattlesnake

SC

730

and

therapeutic

Saviola, A.J., Pla, D., Sanz, L., Castoe, T.A., Calvete, J.J., Mackessy, S.P., 2015. Comparative

737

venomics of the Prairie Rattlesnake (Crotalus viridis viridis) from Colorado:

738

Identification of a novel pattern of ontogenetic changes in venom composition and

739

assessment of the immunoreactivity of the commercial antivenom CroFab®. J.

740

Proteomics 121, 28-43.

TE D

736

Sunagar, K., Undheim, E.A., Scheib, H., Gren, E.C., Cochran, C., Person, C.E., Koludarov, I.,

742

Kelln, W., Hayes, W.K., King, G.F., Antunes, A., Fry, B.G., 2014. Intraspecific venom

743

variation in the medically significant Southern Pacific Rattlesnake (Crotalus oreganus

745

AC C

744

EP

741

helleri): biodiscovery, clinical and evolutionary implications. J. Proteomics 99, 68-83.

ACCEPTED MANUSCRIPT

Vonk, F.J., Casewell, N.R., Henkel, C.V., Heimberg, A.M., Jansen, H.J., McCleary, R.J.,

747

Kerkkamp, H.M., Vos, R.A., Guerreiro, I., Calvete, J.J., Wüster, W., Woods, A.E.,

748

Logan, J.M., Harrison, R.A., Castoe, T.A., De Koning, A.P.J., Pollock, D.D., Yandell,

749

M., Calderon, D., Renjifo, C., Currier, R.B., Salgado, D., Pla, D., Sanz, L., Hyder, A.S.,

750

Ribeiro, J.M.C., Arntzen, J.W., Van Den Thillart, G.E.E.J.M., Boetzer, M., Pirovano, W.,

751

Dirks, R.P., Spaink, H.P., Duboule, D., McGlinn, E., Kini, M., Richardson, M.K., 2013.

752

The king cobra genome reveals dynamic gene evolution and adaptation in the snake

753

venom system. Proc. Nat. Acad. Sci. 110, 20651-20656.

SC

RI PT

746

Weissbach, H., Robertson, A., Witkop, B., Udenfriend, S., 1960. Rapid spectrophotometric

755

assays for snake venom l-amino acid oxidase based on the oxidation of l-kynurenine or

756

3,4-dehydro-l-proline. Anal. Biochem. 1, 286-290.

M AN U

754

Wooldridge, B., Pineda, G., Banuelas-Ornelas, J., Dagda, R., Gasanov, S., Rael, E., Lieb, C.,

758

2001. Mojave rattlesnakes (Crotalus scutulatus scutulatus) lacking the acidic subunit

759

DNA sequence lack Mojave toxin in their venom. Comp. Biochem. Physiol. B Biochem.

760

Mol. Biol. 130, 169-179.

762

Wray, K.P., Margres, M.J., Seavy, M., Rokyta, D.R., 2015. Early significant ontogenetic changes

EP

761

TE D

757

in snake venoms. Toxicon 96, 74-81. Zancolli, G., Baker, T.G., Barlow, A., Bradley, R.K, Calvete, J.J., Carter, K.C., de Jager, K.,

764

Owens, J.B., Price, J.F., Sanz, L., Scholes-Higham, A., Shier, L., Wood, L., Wüster, C.E.,

765 766 767 768

AC C

763

Wüster, W., 2016. Is hybridization a source of adaptive venom variation in rattlesnakes?

A test, Using a Crotalus scutulatus × viridis hybrid zone in Southwestern New Mexico. Toxins. doi: 10.3390/toxins8060188.

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Figure Legends

770 771 772 773 774 775

Fig. 1. Reducing SDS-PAGE of C. s. scutulatus female parent (S), C. o. helleri male parent (H), female hybrid (♀) and male hybrid (♂) venoms from 2007–2015 (except 2012). Protein families typically found at specific molecular masses are indicated by ovals. Note that the C. s. scutulatus female parent samples were only available through 2009. MW = Molecular weight standards (in kDa).

776 777 778 779

Fig. 2. Average azocasein metalloprotease (A), thrombin-like serine protease (B), kallikrein-like serine protease (C), L-amino acid oxidase (D), and phosphodiesterase activities of reference C. s. scutulatus (n=6) and C. o. helleri (n=6) venom. *, statistically different; p<0.01.

780 781 782 783 784

Fig. 3. Enzyme activities of parental and hybrid venoms. A) Azocasein metalloprotease, B) thrombin-like serine protease, C) kallikrein-like serine protease,D) L-amino acid oxidase, and E) phosphodiesterase activities of C. s. scutulatus (♀ parent), C. o. helleri (♂ parent) and male and female hybrids from 2007 through 2015.

785 786 787 788 789

Fig. 4. Western blot of C. s. scutulatus (S), C. o. helleri (H), hybrids (♀, ♂), C. v. viridis and C. o. cerberus venoms (negative controls), and purified concolor toxin (positive control), developed with anti-Mojave toxin subunit A antibodies, showing binding to the A subunit (~9 kD; red arrows) in C. s. scutulatus and hybrid venoms and purified concolor toxin.

790 791 792 793 794 795

Fig. 5. Reverse phase HPLC chromatograms, peak elution time tables, and gel of eluted peaks of C. s. scutulatus female parent (A), C. o. helleri male parent (B) and overlay of the two chromatograms (C); C. s. scutulatus (blue) and C. o. helleri (black). Note the high abundance of myotoxins in C. o. helleri, the presence of Mojave toxin in C. s. scutulatus and the lack of metalloproteases (SVMP) in C. s. scutulatus.

796 797 798 799

Fig. 6. Reverse phase HPLC chromatogram and peak elution time table of 1.0 mg venom protein from (A) hybrid female venom extracted in 2008, (B) hybrid male venom extracted in 2007, (C) hybrid female venom extracted in 2015, and (D) hybrid male venom extracted in 2015.

800 801 802 803

Figure 7. MALDI-TOF mass spectra of reverse phase HPLC fractions of (A) C. s. scutulatus female parent Mojave toxin subunit B, (B) C. o. helleri male parent PLA2, (C) hybrid female Mojave toxin subunit B, and (D) hybrid male Mojave toxin subunit B. Spectra are derived from HPLC fraction 41 for all samples except the male parent (fraction 62).

AC C

EP

TE D

M AN U

SC

RI PT

769

804 805 806 807

Fig. 83. Principal coordinate analysis of RP-HPLC results for three C. s. scutulatus, three C. o. helleri, and hybrids. Note the distinct clusters based on species, and that hybrids fall intermediate to C. o. helleri and C. s. scutulatus along coordinate 1.

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EP

TE D

M AN U

SC

RI PT

Fig. 9. Lethal toxicity (µg/g) of C. o. helleri male parent, C. s. scutulatus female parent and female and male hybrid neonate venoms to NSA mice.

AC C

808 809 810

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Table 1. Reverse phase HPLC chromatogram percent peak areas of myotoxins, Mojave toxin and SVMPs for C. s. scutulatus female parent and two reference C. s. scutulatus, C. o. helleri

2008 (♀) and 2015 (both).

RI PT

male parent and two reference C. o. helleri, and the male and female hybrids from 2007 (♂),

Sample

Myotoxins

Mojave toxin

C. s. scutulatus (n=3)

0

18-25

C. o. helleri (n=3)

49-62

0

Hybrid ♀ (2008)

39.9

6.8

1.5

Hybrid ♂ (2007)

42.3

8.3

5.1

Hybrid ♀ (2015)

44.7

9.8

1.3

Hybrid ♂ (2015)

37.6

10.3

7.9

0.5-2 6-20

SC

M AN U

TE D EP AC C

SVMPs

200.0 – 166.3 97.4 66.3 55.4

– – – –

S

HS

2009

HS

6.0 – 3.5 – 2.5 –

H

2013

H

2014

H

2015

H

Protein Family Nucleases, LAAO SVMP – PIII SVSPs CRiSP

PLA2

Myotoxins

EP

14.4 –

H

2011

AC C

21.5 –

H

TE D

36.5 – 31.0 –

2010

MW

2008

2007

M AN U

MW

SC

RI PT

ACCEPTED MANUSCRIPT

Smith & Mackessy Fig 1

Kallikrein-like (nmol product/min/mg)

PDE (A400 nm/min/mg)

0.7

0.6

0.5

0.4

0.3

0.2

0.1

A

C

E

L-AAO (nmol product/min/mg)

B

D

EP

800

600

400

200

60

50

40

30

20

10

0

*

C. s. scutulatus C. o. helleri

Smith & Mackessy Fig 2

AC C

Thrombin-like (nmol product/min/mg) 0

TE D

*

M AN U

*

SC

Metalloprotease (A342 nm/min/mg) 0.0

1000

800

600

400

200

0

0.5

0.4

0.3

0.2

0.1

0.0

RI PT

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0.2

0.1

0.0 2008

2010

2012

2014

1000

600 400 200 0 2008

2010

2012

2014

TE D

Year

1.2 1.0

E

EP

0.8 0.6 0.4

400 200 0

2008

2010

2012

2014

Year

70 60

D

M AN U

800

C

AC C

PDE (A400 nm/min/mg)

Kall SVSP (nmol product/min/mg)

Year

B 600

RI PT

A

800

SC

Thr SVSP (nmol product/min/mg)

0.3

L-AAO (nmol product/min/mg)

SVMP (A342 nm/min/mg)

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50 40 30 20 10 0

2008

2010

2012

2014

Year

C. s. scutulatus Female Hybrid C. o. helleri Male Hybrid

0.2 0.0

2008

2010

2012

2014

Year

Smith & Mackessy Fig 3

RI PT SC

Concolor Toxin (+) MW

C. o. cerberus (-)

M AN U

S

C. v. viridis (-)

ACCEPTED MANUSCRIPT

H

- 260

- 160 - 110 - 80 - 60 - 50 - 40

- 20 - 15

- 10

- 3.5

AC C

EP

TE D

- 30

Smith & Mackessy Fig 4

ACCEPTED MANUSCRIPT 0.245

41.722

A

100.00

30.00

40.00

50.00

77.875

66.068

42.717

60.00

85.269

60.807

20.00

70.00

80.00

Time (min)

SC

0.390

19.409

0.325

0.260

50.00

25.00

0.000 0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

88.442

91.306

84.770

86.998

78.016

73.258 74.553

27.321

11.626

0.065

64.679

50.678

0.130

55.537

60.887

0.195

M AN U

75.00

19.123

Absorbance 280 nm

100.00

100.00

0.455

67.232 68.799

B

23.558

0.520

0.00

90.00

80.00

%B (ACN, 0.1% TFA)

10.00

25.00

63.990

26.105

29.163

12.129

0.000 0.00

RI PT

48.657

31.691

0.070

0.035

50.00

57.14757.683

23.256

0.105

58.551

54.402

67.484

19.629

0.140

%B (ACN, 0.1% TFA)

75.00

0.175

21.426

Absorbance 280 nm

0.210

0.00 90.00

100.00

C

0.48

TE D

Time (min)

A b so rb an ce 280 n m

Myotoxins

Mojave toxin

EP

0.36

0.24

PLA2 SVSP

AC C

0.12

0.00 0.00

10.00

20.00

SVMP

SVSP

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

Time (min)

Smith & Mackessy Fig 5

ACCEPTED MANUSCRIPT Absorbance 280 nm

RI PT

SC

48.607 50.386

54.322 55.989 57.164 58.383

60.00

60.00

60.902

60.546 64.407

64.619 66.083

65.971 66.452

70.00

70.00

67.591

70.327

80.00

M AN U

80.00

78.022

77.771

84.665 87.870 91.235

100.00

%B (ACN, 0.1% TFA)

B

D

Absorbance 280 nm

75.00

%B (ACN, 0.1% TFA)

50.00

25.00

0.00

100.00

100.00

75.00

50.00

25.00

0.00

100.00

Absorbance 280 nm

11.657

20.00

20.00

19.156

0.28

0.24

0.20

0.16

0.12

0.08

10.00

10.00

11.582

0.04

0.00 0.00

0.245

0.210

0.175

0.140

0.105

0.070

0.035

0.000 0.00

TE D

90.00

90.00

91.311

23.326

23.730

24.367 25.690

22.632

25.708

30.00

30.00

28.651 30.919

54.724

60.971

41.604 42.683

48.659 50.392

54.320

60.00

58.689

58.423 60.629

64.576 66.098 67.532

64.822

70.00

70.00

67.776

80.00

80.00

78.041

77.791

84.669

84.792

90.00

90.00

88.429 91.323

87.819 91.272

%B (ACN, 0.1% TFA)

100.00

75.00

50.00

25.00

0.00

100.00

%B (ACN, 0.1% TFA)

100.00

75.00

50.00

25.00

0.00

100.00

Smith & Mackessy Fig 6

60.00

57.362

30.846

50.00

Time (min)

48.949 50.697

28.719

40.00

41.826 42.983

50.00

Time (min)

40.00

EP

67.392

70.368

84.760

AC C

0.245

54.683

41.498 42.600

50.00

Time (min)

48.935

56.289 57.403 58.587

40.00

50.00

Time (min)

40.00

41.745

42.950

0.210

22.693

30.00

30.00

30.955

0.175 19.297

21.120

25.698 26.987 28.633

25.716 28.710

0.140

12.222

20.00

20.00

22.743

0.105

10.00

10.00

19.248

0.070

0.035

0.000 0.00

0.245

0.210

0.175

0.140

0.105

0.070

0.035

0.000 0.00

12.064

23.754

A

C

Absorbance 280 nm

x10 4

14186.429

A

7096.511

In te n s . [a .u .]

In te n s . [a .u .]

ACCEPTED MANUSCRIPT

14,186.4

x10 4

13665.266

B

2.5

13,665.3

8

2.0

RI PT

6

1.5

4

6837.363

SC

1.0

2 0.5

M AN U

4729.079

0.0

0 0

2000

4000

6000

8000

10000

12000

14000

16000

18000

0

20000

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000 m/z

14182.200

C

5

In te n s . [a .u .]

x10 4

14,182.2

x10 4

14181.248

D

14,181.2

4

TE D

4

3

7095.275

EP

2

1

0 0

2000

AC C

In te n s . [a .u .]

m/z

4000

6000

8000

10000

12000

7094.481 3

2

1

0 14000

16000

18000

0

20000 m/z

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000 m/z

Smith & Mackessy Fig 7

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Smith & Mackessy Fig 8

100

RI PT

ACCEPTED MANUSCRIPT

Female C. s. scutulatus LD50 = 0.14 µg/g Male C. o. helleri LD50 = 1.07 µg/g

80

SC

Male hybrid LD50 = 0.18 µg/g

M AN U

60

40

0

TE D

20

EP

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5

Dose (µg/g)

AC C

% Mortality (24 hour)

Female hybrid LD50 = 0.14 µg/g

Smith & Mackessy Fig 9

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Highlights: •

C. o. helleri × C. s.scutulatus hybrid venoms display overlapping parental venom

RI PT

profiles. Hybrid venoms retain both the type I and type II venom characteristics.

•

Ontogenetic shifts in SVMP activity of hybrid venoms follow a sex-influenced trend.

•

Hybrids express intermediate kallikrein-like and thrombin-like serine protease activities.

AC C

EP

TE D

M AN U

SC

•

ACCEPTED MANUSCRIPT

Ethical statement

AC C

EP

TE D

M AN U

SC

RI PT

The authors hereby state that all procedures involving animals were conducted in a humane and ethical manner. All protocols were evaluated and approved (prior to initiating research) by the University of Northern Colorado Institutional Animal Care and Use Committee (UNC-IACUC).