The Psychological Record, 2010, 60, 217–226

Response of WesteRn DiamonDback Rattlesnakes (Crotalus atrox) to chemical cues of mice (Mus MusCulus) of DiffeRent GenDeRs anD

RepRoDuctive status

Anthony J. Saviola and David Chiszar

University of Colorado at Boulder

Matthew T. Bealor

University of Colorado at Denver

Hobart M. Smith

University of Colorado at Boulder

Eight western diamondback rattlesnakes (Crotalus atrox) were exposed to 6 stimuli: (1) clean, unused bedding; (2) an adult male mouse; (3) an adult lactat-ing female mouse; (4) an adult lactating female mouse with a litter; (5) 2 adult nonlactating female mice, to control for the extra surface area in Condition 4; and (6) a litter of newborn mice. All stimuli were presented in opaque plastic boxes with 18 perforations in each side; hence, chemical but not visual cues were available to the snakes. Our goal was to examine if chemical cues from ro-dents of different genders, lactating female rodents, and rodent litters influence predatory behavior in rattlesnakes. Results indicated that C. atrox responded to odors arising from adult mice (Mus musculus) with higher rates of tongue flicking than were seen in the control condition, but the 4 conditions involving adult mice did not differ significantly from each other. Neonatal mice did not pro-duce consistently higher rates of tongue flicking than did the control condition. Key words: western diamondback rattlesnake, Crotalus atrox, chemical cues, predatory behavior

Rattlesnakes recognize potential prey either by chemical cues associated with integument (Chiszar, Melcer, Lee, Radcliffe, & Duvall, 1990; Duvall, Chiszar, Hayes, Leonhardt, & Goode, 1990) or by visual and thermal cues (Hennessy & Owings, 1988; Kardong & Berkhoudt, 1999). Feeding experience among timber rattlesnakes (Crotalus horridus) increased the responsiveness of the snakes to chemical cues of the prey, facilitating location of potential ambush sites (Clark, 2004). Northern Pacific rattlesnakes (Crotalus oreganus) may use the behavior of adult female California ground squirrels

Correspondence concerning this article should be addressed to A. J. Saviola, Department of

Ecology and Evolutionary Biology, University of Colorado, Boulder 80309-0334. E-mail: anthony.

saviola@colorado.edu

218 SAVIOLA ET AL.

(Spermophilus beecheyi ) as a visual cue for locating litters (Hennessy & Owings, 1988). Although C. oreganus is the primary predator of ground squirrel pups (Coss, 1991; Swaisgood, Owings, & Rowe, 1999), rattlesnakes typically do not display predatory behaviors toward adult California ground squirrels (Hennessy & Owings, 1988), which have developed physiological resistance to rattlesnake venoms (Poran, Coss, & Benjamini, 1987; Coss, Guse, Poran, & Smith, 1993) and also demonstrate behavioral tactics of harassing and even attacking rattlesnakes (Owings & Coss, 1977).

Maternal female ground squirrels respond with increased time and effort directed toward snakes as compared with nonmaternal or male ground squirrels (Swaisgood et al., 1999), a behavior Hennessy and Owings (1988) suggested rattlesnakes recognize, thereby eliciting increased searching for the litter. Although this behavior of maternal females is suggested to be the reason why rattlesnakes will investigate the nearby region, an alternative hypothesis might be that rattlesnakes respond to chemical cues specifically associated with maternal rodents. The purpose of our study was, therefore, to examine if chemical cues from rodents of different genders, lactating female rodents, and rodent litters influence predatory behavior in rattlesnakes.

Materials and Methods

Eight long-term captive western diamondback rattlesnakes, Crotalus atrox, were observed in this study. They were housed individually in glass terraria (61 × 41 × 44.5 cm) containing a water bowl, newspaper bedding, and a rock to assist with shedding. The rattlesnakes were presented with an opaque plastic box (10 × 10 × 10 cm) with 18 perforations in each side (Chiszar, Krauss, Shipley, Trout, & Smith, 2009; Chiszar, Taylor, Radcliffe, Smith, & O’Connell, 1981). All perforations were circular drill holes, 2 mm in diameter, certainly large enough to permit passage of volatile chemicals but not large enough to permit visual examination of the interior of the box. The box contained one of six stimuli: (1) clean unused bedding; (2) an adult male mouse; (3) an adult lactating female mouse; (4) an adult lactating female mouse with a litter; (5) two adult nonlactating female mice, to control for the extra surface area in Condition 4; and (6) a litter of newborn mice. Litters in Conditions 4 and 6 were always less than 5 days old; hence, the pups were small and hairless and remained clustered. Thermal cues arising from prey should be equal in Conditions 2 and 3; aside from this equality, no other attempt was made to control thermal (infrared) properties of the stimulus boxes. Each rattlesnake was tested six times, once with each of the six stimuli. Trials were presented in random order (a different random order was selected for each snake), and each trial lasted 30 min. The plastic box with one of the six stimuli was placed into the snake’s terrarium, and total tongue flicks, tongue flicks directed at the box, and seconds spent investigating the box were recorded every minute for the duration of the trial. Rate of tongue flicking is a measure of the extent to which snakes are using their vomeronasal system to analyze nonvolatile chemical cues. Further, detection of volatile cues by the nasal system is known to trigger activation of the vomeronasal system (Cowles & Phelan, 1958). Therefore, rate of tongue flicking is a useful measure of nasal (volatile) as well as vomeronasal (nonvolatile) chemoreception. (See Burghardt, 1970, and Halpern, 1987, for reviews of chemoreception in reptiles.) The laboratory was maintained at

219RATTLESNAKE RESPONSE TO CHEMICAL CUES

26 °C and the photoperiod was automatically controlled on a 12:12 cycle. The snakes were fed one mouse weekly during the study, but never during tests or during the morning hours preceding tests, and successive tests were always separated by at least 1 week.

Results

Distributional properties of the dependent variables were assessed by comparing observed and expected frequencies of scores in four intervals (i.e., from 2 SD below the mean to 1 SD below, from 1 SD below the mean to the mean, from the mean to 1 SD above, and from 1 SD above the mean to 2 SD above). Expected frequencies were derived by multiplying N = 48 by the relevant proportions from the normal distribution. For total tongue flicks, observed and expected frequencies did not differ significantly, χ2 = 2.94, df = 3, p > 0.05, indicating that this variable was normally distributed. Tongue flicks directed at stimulus boxes and seconds spent investigating the boxes departed from normality, both χ2 = 16.91, df = 3, p < 0.05. Because analysis of variance (ANOVA) is robust against departures from normality (Winer, 1971), we used this test to assess effects of stimulus conditions, but we also developed a normally distributed composite variable that combined the three separate dependent variables (see below). That is, observed and expected frequencies for the composite variable did not differ significantly, χ2 = 1.51, df = 3, p > 0.05. Further, nonparametric tests were applied to supplement the parametric ones.

0

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Figure 1. Mean number of tongue flicks per 30-min trial. Note. 1 = box with clean, unused bedding; 2 = an adult male mouse; 3 = an adult lactating female mouse; 4 = an adult lactating female mouse with a litter; 5 = two adult nonlactating female mice; and 6 = a litter of newborn mice.

220 SAVIOLA ET AL.

Because each snake was tested si x t imes, it is possible that habituation or sensitization may have occurred, causing later trials to have systematically lower or higher scores than earlier ones, regardless of the stimulus conditions presented on the later trials. Even though the order of stimuli was randomized over the six trials, habituation or sensitization could complicate the eventual interpretation of a stimulus effect. Therefore, we assessed systematic change in behavior over successive trials by ANOVAs, finding no evidence for an effect of trials on any of the dependent variables (total tongue flicks: F = 2.17; tongue flicks at boxes: F = 0.89; seconds spent investigating boxes: F = 1.10; all dfs = 5, 35, all ps > 0.05).

Mean total numbers of tongue flicks emitted in each of the conditions are shown in Figure 1, where it is clear that the conditions had different stimulus value to the C. atrox, F = 2.47, df = 5, 35, p < 0.05.

The four conditions involving adult mice had significantly higher means than did the control and the litter-only conditions, F = 11.36, df = 1, 35, p < 0.05. However the four conditions involving adult mice did not differ significantly (all ps > 0.05 by Duncan’s new multiple range test). Thus, lactating females did not inspire greater searching by C. atrox than did either a male or nonlactating females.

Figures 2 and 3 present the mean number of tongue flicks aimed at stimulus boxes and the mean number of seconds spent investigating the boxes.

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Figure 2. Mean number of tongue flicks directed at the box. Note. 1 = box with clean, unused bedding; 2 = an adult male mouse; 3 = an adult lactating female mouse; 4 = an adult lactating female mouse with a litter; 5 = two adult nonlactating female mice; and 6 = a litter of newborn mice.

221RATTLESNAKE RESPONSE TO CHEMICAL CUES

0

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Figure 3. Mean number of seconds snakes spent investigating the box. Note. 1 = box with clean, unused bedding; 2 = an adult male mouse; 3 = an adult lactating female mouse; 4 = an adult lactating female mouse with a litter; 5 = two adult nonlactating female mice; and 6 = a litter of newborn mice.

In both cases, three of the four conditions involving adult mice had higher scores than either the control or the litter-only condition, but ANOVAs failed to reveal a significant main effect of conditions (tongue flicks directed at boxes: F = 1.54, df = 5, 35, p > 0.05; number of seconds spent investigating box: F = 1.52, df = 5, 35, p > 0.05). A serious problem with these data was the fact that on 31 of 48 trials there were zero tongue flicks aimed directly at the box and zero seconds spent investigating the box. On these trials the snakes may have been actively flicking their tongues and exploring their cages, but they were not focused on the stimulus boxes. Such behavior occurs for two reasons: (1) Volatile chemicals trigger tongue flicking, but in the absence of readily accessible nonvolatile or visual cues, snakes sometimes require significant time to discover and focus on the source of the volatiles, and (2) rattlesnakes, especially C. atrox, are prone to be somewhat defensive and to move about their cages for a while after being disturbed, even though volatile chemicals have triggered tongue flicking. The zero scores, of course, gave rise to skewed distributions as well as to large error variances (compare error bars in Figures 2 and 3 with those in Figure 1). Consequently, we formed composite scores in an effort to normalize the data. First, if the snake aimed tongue flicks at the box, we expressed this as a percentage of the snake’s total tongue flicks on that trial. Then the composite score was formed as follows: C = total number of tongue flicks emitted on the trial + % aimed at the box + number of seconds spent investigating the box. This composite has the effect of giving extra weight to investigation directed at the stimulus boxes, a procedure that seems reasonable since such

222 SAVIOLA ET AL.

investigation implies more direct sensation of stimuli than is the case for tongue flicks aimed elsewhere in the test environment (see Burghardt, 1970, and Arnold, 1978, for conceptually similar composite scores; see Cooper and Burghardt, 1990, for an analysis of statistical properties of various composite scores). Furthermore, the distribution of composite scores was not significantly different from normal by a chi-square goodness-of-fit test, as reported above. Mean composite scores for each stimulus condition are shown in Figure 4, revealing a pattern rather like that seen in Figure 1.

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1 2 3 4 5 6

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Figure 4. Mean composite scores for each stimulus condition. Note. 1 = box with clean, unused bedding; 2 = an adult male mouse; 3 = an adult lactating female mouse; 4 = an adult lactating female mouse with a litter; 5 = two adult nonlactating female mice; and 6 = a litter of newborn mice.

In fact, the statistical analysis confirmed this point, F = 2.49, df = 5, 35, p < 0.05, with all conditions involving adult mice having larger means than the remaining two conditions, F = 10.97, df = 1, 35, p < 0.05. As was the case for total tongue flicks, the four conditions including adult mice did not differ from each other (all ps > 0.05 by Duncan’s new multiple range test). Hence, the composite score did not provide greater resolution than the total tongue flicks.

A nonparametric test was also applied to all four dependent variables. We subtracted control scores for each snake from that animal’s scores in each of the other conditions, and we recorded the differences as either positive or negative. All differences were pooled, giving rise to a maximum of 40 signs; zeros were ignored, as is typically the case with sign tests (Siegel, 1956). For each dependent variable, the overall chi-square executed on signs was significant: χ2 = 8.10 for total tongue flicks, 10.28 for tongue flicks directed at stimuli, 12.25 for number of seconds spent investigating stimuli, and 8.10 for composite scores; all dfs = 1, all ps < 0.05. Hence, the control condition

223RATTLESNAKE RESPONSE TO CHEMICAL CUES

produced lower scores than all the other conditions combined. However, all sign tests comparing the remaining conditions with each other were not significant. Thus the litter-only condition differed from the control and not from the other four conditions. As was true for the ANOVAs, odors arising from the lactating females did not elicit greater chemosensory searching than did odors arising from other mice.

Discussion

The C. atrox responded to odors arising from adult M. musculus with a higher rate of tongue flicking than was seen in the control condition, a fact that agrees with numerous studies of chemical cue utilization by rattlesnakes (Chiszar, Radcliffe, Scudder, & Duvall, 1983; Cowles & Phelan, 1958; Duvall et al., 1990; Kardong, 1986). The ANOVAs applied to the data shown in Figures 1 through 4 implied that neonatal mice did not inspire greater chemosensory examination than did the control condition. This outcome might be taken to mean that neonates are less odoriferous than adults. Although this is probably true (Finlayson & Baumann, 1958; Hoffsten, Hill, & Klahr, 1975), the nonparametric analyses suggest that caution is necessary. Perhaps neonatal mice unaccompanied by an adult female present a weak or variable odor, especially when the neonates are relatively cool, such that less volatilization occurs. Hence, the rattlesnakes might be presented with an ambiguous cue of low concentration, giving rise to variable intensities of response by the predators. This could account for the differences between the parametric and nonparametric analyses. It must also be kept in mind that our neonates were always less than 5 days old; perhaps older, furred neonates would present stronger chemical stimuli to the snakes. Most important, the lactating females did not elicit greater chemosensory investigation than did males or nonlactating females. Thus, we have no evidence that the putative rattlesnake response to maternal females (Hennessy & Owings, 1988) is mediated by chemical cues unique to these animals.

It must be kept in mind that Hennessy and Owings (1988) studied Northern Pacific rattlesnake (C. oreganus) predation upon California ground squirrels (S. beecheyi ), whereas we studied C. atrox response to house mice (M. musculus). Generalization of our conclusions to other species of rattlesnakes and to other species of prey depends upon the extent to which our paradigm constitutes an appropriate model. This point cannot be confirmed or disconfirmed until replications are executed with other species of predator and prey. Nevertheless, because the original hypothesis by Hennessy and Owings (1988) emphasized visual cues arising from maternal ground squirrels rather than chemicals, our results can be tentatively regarded as consistent with that thinking.

An apparatus that could provide definitive results would involve a three-compartment terrarium, the center one occupied by the snake, one occupied by a maternal female rodent and her litter, and one occupied by a nonmaternal adult. The rodent compartments would need to be separated from the snake’s compartment by four types of plastic barriers on successive trials (opaque; opaque but perforated, allowing chemical cues but not visual cues to pass; transparent, allowing only visual cues to be detected by the snake; transparent and perforated, allowing both chemical and visual cues

224 SAVIOLA ET AL.

to pass). Measuring the snake’s investigation of the respective compartments under each barrier condition would reveal the relative importance of visual and chemical cues. One added advantage of this setting would be that the behavior of the maternal and nonmaternal rodents could be observed to determine if the presence of the predator affects them differently. Furthermore, this apparatus could easily be used with multiple species of predator and prey to explore generality of results.

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