Observations on the chemosensory responses of the midget faded rattlesnake (Crotalus oreganus concolor): discrimination of envenomated prey in a type II venom species Anthony J. Saviola & Stephen P. Mackessy Journal of Ethology ISSN 0289-0771 Volume 35 Number 2 J Ethol (2017) 35:245-250 DOI 10.1007/s10164-017-0511-2 1 23 Your article is protected by copyright and all rights are held exclusively by Japan Ethological Society and Springer Japan. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”. 1 23 Author's personal copy J Ethol (2017) 35:245–250 DOI 10.1007/s10164-017-0511-2 SHORT COMMUNICATION Observations on the chemosensory responses of the midget faded rattlesnake (Crotalus oreganus concolor): discrimination of envenomated prey in a type II venom species 1,2 1 Anthony J. Saviola • Stephen P. Mackessy Received: 26 October 2016 / Accepted: 25 February 2017 / Published online: 3 March 2017 Ó Japan Ethological Society and Springer Japan 2017 Abstract Rattlesnakes use prey chemical cues for ambush type II venomous species can also discriminate between E site selection and for relocating envenomated (E) prey and NE chemical cues. following a predatory strike. The ability to discriminate between E and non-envenomated (NE) prey cues has been Keywords Behavior Á Chemical cues Á Chemosensory Á widely studied in rattlesnake species that produce type I Disintegrins Á Predation Á Tongue flicking Á Viper venoms, which show high levels of snake venom metal- loproteinase (SVMP) activity and low lethal toxicity [lethal dose which kills 50% of test animals (LD50) [1.0 lg/g]. Introduction However, E vs. NE prey discrimination studies have not been conducted on rattlesnake species that produce a type Research on squamate feeding strategies and prey recog- II venom that consists of low SVMP activity and high nition strongly indicates that many snakes and lizards have lethal toxicity (LD50\1.0 lg/g). In the current study, long- the ability to discriminate between chemical cues of dif- term captive Crotalus oreganus concolor, which produce a ferent prey (Burghardt 1970; Cooper et al. 1990; Clark type II venom, were tested for their ability to discriminate 2004; Saviola et al. 2012a). This chemosensory response between chemical cues of natural (Sceloporus undulatus may be innate (Mushinsky and Lotz 1980; Holding et al. and Peromyscus maniculatus) and non-natural (Hemi- 2016) or based on feeding experience (Burghardt et al. dactylus frenatus and Mus musculus) prey cues, as well as 2000), and it often results in fluctuating tongue flick rates for their ability to discriminate between E and NE mouse towards select prey cues. In squamates, tongue flicking is a carcasses, when prey envenomation occurred by a con- stimulus-seeking behavior and the main process for deliv- specific. Snakes showed significant levels of tongue flick- ering volatile and non-volatile cues to the vomeronasal ing towards the chemical extracts of P. maniculatus and M. organ (Halpern 1992; Schwenk 1995). Tongue flicking can musculus, suggesting that C. oreganus concolor exhibit be highly variable, and elevated in response to visual- both innate and experience-based plasticity in response to thermal stimuli (Chiszar et al. 1981; Saviola et al. 2011), prey chemical cues. In addition, C. oreganus concolor were vibratory cues (Saviola et al. 2012b), or chemical cues able to discriminate between E and NE prey sources, when (Burghardt 1970; Clark 2004; Saviola et al. 2010; Smith envenomation occurred by a conspecific, indicating that a et al. 2015), and it is often correlated with squamate for- aging ecology and prey preference (Cooper 1994, 1995, 2008). & Anthony J. Saviola Rattlesnakes exhibit multiple sensory modalities during [email protected]; predation, utilizing chemical stimuli to select ambush sites [email protected] (Roth et al. 1999; Clark 2004) and visual-thermal stimuli to 1 School of Biological Sciences, University of Northern deliver the envenomating strike (Hayes and Duvall 1991). Colorado, Greeley, CO, USA Following the release of prey, rattlesnakes then return to a 2 Present Address: Medical Laboratory and Radiation Sciences, reliance on chemical cues to relocate the envenomated University of Vermont, Burlington, VT 05405, USA (E) carcass (Chiszar et al. 1977; Parker and Kardong 2005). 123 Author's personal copy 246 J Ethol (2017) 35:245–250 This ability to relocate E prey and discriminate between E discrimination (Saviola et al. 2013), appear to be expressed and non-envenomated (NE) prey cues has been thoroughly at significantly higher concentrations in venoms of species studied (Dullemeijer 1961; Duvall et al. 1978; Chiszar with type I venoms (see Calvete et al. 2009, 2012). et al. 1992, 1999, 2008; Greenbaum et al. 2003; Parker and Therefore, the aim of this study was to examine the Kardong 2005). Rattlesnakes and other pit vipers (Chiszar chemosensory responses of C. oreganus concolor, which et al. 1992; Greenbaum et al. 2003) respond with increased produces a type II venom, and test whether this species can tongue flicking towards E prey when envenomation occurs discriminate between E and NE prey when envenomation by a conspecific, a closely related heterospecific (Duvall occurs by a conspecific. et al. 1978; Chiszar et al. 2008), or when lyophilized venom is manually injected into a previously euthanized mouse (Chiszar et al. 1999). Interestingly, rattlesnakes are Materials and methods unable to discriminate between E or NE carcasses when venom is painted on the integument of prey, indicating that Animals venom must be injected into tissue for chemical cue dis- crimination to occur (Chiszar et al. 1992). Further, when The subjects were five long-term captive adult C. oreganus testing responses of the western diamondback rattlesnake concolor, and all were feeding every 10–14 days on pre- (Crotalus atrox) to mice injected with fractionated venom, killed NSA mice (M. musculus). Mice were culls from the snakes were only able to discriminate between E and NE University of Northern Colorado Animal Research Facility cues when prey was injected with the venom peak con- and were euthanized by CO2 asphyxiation and frozen at taining the crotatroxin disintegrins (Saviola et al. 2013). -20 °C until feeding. Snakes were housed individually in This work identified these proteins as the ‘‘relocator’’ glass aquaria (61.0 9 41.0 9 44.5 cm) containing a paper molecule permitting successful recognition of E prey. floor, water bowls, and hide boxes, and maintained on a The midget-faded rattlesnake (Crotalus oreganus con- 12-h:12-h light:dark cycle and provided a thermal range of color) is a small (adult \700 mm snout-vent length) ter- 26 ± 1 °C. restrial rattlesnake native to extreme southcentral Wyoming, northeastern Utah, and western Colorado. This Chemical cue discrimination species feeds primarily on lizards, although small mam- mals have also been documented in their diet (Parker and To test the ability of C. o concolor to discriminate between Anderson 2007). Unlike many rattlesnake species that chemical cues of different prey, each snake was tested to undergo an ontogenetic shift in venom composition from five scent sources: distilled water (control), and extracts of higher toxicity and low snake venom metalloproteinase house gecko (Hemidactylus frenatus) and lab mouse (M. (SVMP) activity in neonates, to decreased toxicity and high musculus), non-natural prey for C. oreganus concolor, and SVMP activity in adults [type I venom (Mackessy extracts of prairie lizard (Sceloporus undulatus) and deer 2008, 2010)], the venom of C. oreganus concolor appears mouse (Peromyscus maniculatus), which represent prey to be paedomorphic (Mackessy et al. 2003), with high that are naturally encountered by this species (Parker and toxicity/low SVMP activity carried into adulthood. Adult Anderson 2007). Extracts were prepared by soaking live C. oreganus concolor, therefore, produce a type II venom prey in distilled water at a ratio of 1 mL of distilled water that exhibits low SVMP activity but high toxicity [lethal per 1 g of prey source for 10 min (Clark 2004; Saviola dose which kills 50% of test animals (LD50) \1.0 lg/g] et al. 2012a) and were always used within 1 h of prepa- toward rodents (Mus musculus), whereas adults of many ration. Snakes were fasted for 7–10 days before trials. rattlesnake species produce type I venoms containing During trials, extracts were presented to the snakes by higher SVMP activity with lower toxicities [LD50[1.0 lg/ soaking a cotton swab in the extract and placing the swab g (Mackessy 2008, 2010)]. To date, the studies that have approximately 2.5 cm in front of the snake’s nose (Cooper examined rattlesnake preferences towards E and NE prey and Burghardt 1990), and the rate of tongue flicking was cues have utilized only species with type I venoms (Duvall counted for 60 s. If the snake struck the cotton swab, the et al. 1978; Chiszar et al. 1992, 1999, 2008; Saviola et al. trial was terminated, and data were analyzed using the 2013). However, proteomic analyses of snake venoms tongue-flick attack score (TFAS) as reviewed in Cooper [termed ‘‘venomics’’ (Calvete et al. 2007)] continues to and Burghardt (1990). Tongue flicking for the 60-s trials identify significant venom variation with regards to general was recorded; however, if a snake struck the cotton swab, venom composition and overall abundance of specific this indicated maximal response, and TFAS was calculated protein families between and within species.