Current Herpetology 31(1): 47–53, June 2012 © 2012 by The Herpetological Society of Japan
doi 10.5358/hsj.31.47
Antipredator Displays and Prey Chemical Preference of an Asian Natricine Snake, Macropisthodon rudis (Squamata: Colubridae) HIROHIKO TAKEUCHI* AND AKIRA MORI Department of Zoology, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606–8502, JAPAN
Abstract: Several Asian natricine snakes are known to possess special organs called nuchal glands. Extensive studies on the nuchal glands of Rhabdophis tigrinus have shown that the glands contain cardiac steroidal toxins known as bufadienolides, which are sequestered from its toad prey and are stored in the nuchal glands as defensive substances. In addition, several species of snakes with nuchal glands exhibit unique behaviors to enhance the effects of the glands (nuchal gland-related behaviors). Macropisthodon rudis is the only species that does not have the nuchal glands in the genus. We investigated its antipredator displays and chemical preference for toads to gain insight into the evolution of the nuchal glands. The results showed that M. rudis does not exhibit the nuchal gland-related behaviors such as neck arching, neck butting, and dorsal-facing posture. Additionally, this species showed high preference for toad chemicals. These results support a previous hypothesis that preference for toads predates the evolution of the nuchal glands and that the unique antipredator behaviors have evolved to enhance the defensive efficiency of the glands. Key words: Nuchal glands; Antipredator display; Chemical preference; Macropisthodon rudis; Toads
INTRODUCTION Snakes comprise a huge reptile group with over 2900 species and present a diversity of morphological, ecological, and behavioral characteristics (Vitt and Caldwell, 2009). Among them, one unique evolutionary innovation is a group of organs, called the nuchal glands, which are known only in several Asian natricine snakes (Nakamura, 1935; Smith, 1938). * Corresponding author. Tel: +81–75–753–4074; Fax: +81–75–753–4114; E-mail address:
[email protected]
Nuchal glands have been most well studied in Rhabdophis tigrinus from Japan: in this species, the glands contain cardiac steroidal toxins known as bufadienolides, which are sequestered from its toad prey and stored therein as a defensive substance (Hutchinson et al., 2007; Mori et al., in press). Rhabdophis tigrinus exhibits peculiar antipredator behaviors, such as neck arching, neck butting, and dorsal-facing posture. These behaviors have been assumed to be associated with the nuchal glands because the behaviors seem to enhance the defensive function of the chemical compounds in the glands (Fukada, 1961; Mutoh,
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1983; Mori et al., 1996; Mori and Burghardt, 2000, 2001). Mori and Burghardt (2008) investigated the ubiquitousness of these behaviors using 27 taxa of natricine snakes including both species that have the nuchal glands and species that lack them. As a result, neck arching and neck butting are exhibited only by snakes with the nuchal glands, suggesting that these peculiar behaviors and nuchal glands have co-evolved. Mori and Burghardt (2008) also postulated that neck-flattening, another antipredator display more common in natricines and other colubroid snakes, had predated the evolution of the nuchal glands and that these features were functionally subsequently associated. Currently, the nuchal glands have been described in three natricine genera, Rhabdophis, Macropisthodon, and Balanophis (Mori et al., in press). Several species belonging to Rhabdophis and one species of Macropisthodon, however, do not have the glands (Smith, 1938; Zhao, 1997; Mao and Chang, 1999). In order to reveal the co-evolutionary association between the nuchal glands and the defensive behaviors, investigation of antipredator displays of these snakes will provide an important clue. Nonetheless, no antipredator displays of either Macropisthodon species or Rhabdophis species that do not have the nuchal glands have ever been investigated. Exploitation of toads would be an essential prerequisite for the evolution of the nuchal glands if sequestration of toad toxins is essential to the evolutionary innovation of the glands. Thus, it is presumed that the common ancestor of Rhabdophis and Macropisthodon possessed the habit of eating toads. Therefore, it is possible that extant snakes without the nuchal glands but closely related to the species with the nuchal glands may still retain a dietary preference for toads. Macropisthodon rudis is the only species of the genus that does not have the nuchal glands (Smith, 1938). Although several anecdotal reports state that M. rudis preys upon toads (Zhao et al., 1998; Shang et al., 2009), its dependency on or preference for toads remains
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to be confirmed. In this study, we first tested the innate chemical preference for toads of M. rudis using a standardized experimental method. Then we investigate antipredator responses of newborn M. rudis to clarify whether the snake exhibits any “nuchal gland-related behaviors” (Mori and Burghardt, 2008).
MATERIALS AND METHODS Subjects A female Macropisthodon rudis (snout-vent length [SVL]=790 mm, body mass=188 g, measured after parturition) obtained from a pet dealer on 8 August 2010 gave birth to 18 baby snakes (3 males and 15 females) on 29 August 2010. Their mean SVL, tail length, and body mass measured immediately after birth were 173.7 mm, 42.0 mm, and 4.0 g for males, and 177.4 mm, 34.0 mm, and 4.2 g for females, respectively. The newborns were individually housed in transparent plastic cages (ca. 300×450×160 mm) with a paper floor covering and water dish at an ambient temperature of 25C. Chemical test The chemical response test was conducted on 7 September 2010. Preparation and presentation of chemical stimuli followed a wellestablished procedure employed for testing chemical discrimination by the vomeronasal organ in Squamata (Burghardt, 1970; Cooper, 1998). Cotton swabs bearing the chemical stimuli were presented to the snakes. The animals used for chemical stimuli included frogs (Ranidae, Fejervarya kawamurai and Rana nigromaculata), toads (Bufonidae, Bufo japonicus), lizards (Scincidae, Plestiodon japonicus), birds (Phasianidae, Coturnix japonica), and mammals (Muridae, Mus musculus). These amphibians and lizards were selected because these species or their close relatives are at least partially sympatric with Macropisthodon rudis in the wild. Distilled water and cologne (Shower Fresh, Ocean Citrus, Mandom; diluted to 10% with distilled water) were used as controls for the experi-
TAKEUCHI & MORI—NUCHAL GLAND-RELATED BEHAVIORS IN A SNAKE mental procedure and as detectable but biologically irrelevant odors, respectively. Approximately 12 h prior to the experiment, the cages were moved onto a testing table, and water dishes and paper floor coverings were removed. Each cage was separated by white paperboard to eliminate the visual effects of other snakes in adjacent cages. Approximately 10 s before each trial the cover of a cage was gently removed, and the tip of a cotton swab, which was either rolled over the external surface of the animals or dipped into the control fluids just before each trial, was presented 1–2 cm in front of the snout of the snake. The number of tongue flicks directed to the swab was counted for 60 s after the first tongue flick was observed. If no tongue flicks were made within 30 s after the presentation of the swab, the tip of the swab was gently touched to the snout of the snakes three times. If the snake did not flick its tongue for 60 s, the trial was terminated. A new stimulus was used for each snake. The order of the presentation of the stimuli was randomized and counterbalanced. If a snake did not show any tongue flicks for 60 s to a given stimulus, a trial with the same stimulus for the snake was conducted again after all seven stimuli were tested with the snake. At least a 15-min interval was set between trails using the same individual. Antipredator response test To elicit antipredator responses of the snakes, we used a standardized method developed for assessing levels and repertoires of antipredator reactions, in particular, those associated with the nuchal glands (Mori and Burghardt, 2000, 2008). A snake was gently removed from its home cage and introduced into an arena (280×450×300 mm) at an ambient temperature of 25C. After leaving the snake undisturbed for three minutes, the antipredator test began. Each trial lasted 1 min, during which the anterior and posterior parts of the snake`s body (excluding head and tail) were alternately pinned every 3 s for a total of 20 stimuli. Snakes were gently pinned
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with a long metal snake hook. This treatment simulates initial contact by a mammalian predator that tries to subdue the snake with a foreleg. All trails were conducted on 9 September 2010 and were videotaped (30 frames/ s) using a digital video camera (Sony, Handycam, DCR-HC62). To compare behavioral responses with those of the previous studies of natricine snakes (Mori et al., 1996; Mori and Burghardt, 2008), we focused on 18 behavioral responses (see Table 2 in Mori and Burghardt, 2008 for detailed description). Videotape analysis was done to record the occurrence of these 18 responses. If a given type of behavior was observed immediately after stimulus contact, we scored 1, otherwise 0. Thus, maximum and minimum scores for each behavior for each trial would be 20 and 0, respectively. Statistical analysis The effects of chemical stimulus on the number of tongue flicks were examined using the Friedman test, followed by pair-wise multiple comparisons using a Wilcoxon signed rank test. In multiple comparisons, we did not use Bonferroni correction for the adjustment of statistical significance level because of the conservative nature of this correction and the recent debates concerning the use of Bonferroni adjustments (e.g., Perneger, 1998; Moran, 2003; Nakagawa, 2004). Instead, we showed the results with the levels of P
