Herpetological Review 43(4), 2012 TECHNIQUES 579 Herpetological Review, 2012, 43(4), xxx–xxx. © 2012 by Society for the Study of Amphibians and Reptiles An Inexpensive, Minimally Invasive Method for Sampling Gastrointestinal Tract Bacteria in Snakes Gastrointestinal tract (GIT) microbiota perform important functions and provide multiple benefits to humans and agricul- turally important vertebrates and are an essential component of mammalian physiology (Bäckhed et al. 2004, Turnbaugh et al. 2006, Dethlefson et al. 2007). In humans and agricultural animals GIT microbiota protect hosts from pathogens, synthesize vita- mins, break down indigestible substances, and influence nutrient uptake and body fat accumulation (Xu and Gordon 2003, Turn- baugh et al. 2006). Our understanding of the diversity and func- tion of GIT microbiota is based primarily on studies of humans, laboratory mice, and agricultural animals but GIT microbiota may be of similar importance to wild vertebrates (Hill et al. 2008). If GIT microbiota of wild vertebrates perform functions similar those observed in humans and domesticated animals (immunity, digestion, vitamin synthesis) they may in turn influ- ence their survivorship, growth, and reproduction – important life history parameters that affect the population dynamics of all organisms (Stearns 1992). All wild vertebrates are subject to the possibility of periods of fasting due to variation in the availability of food in their environments (McCue 2006). The vertebrates for which the diversity and function of GIT microbiota have been well studied (humans, laboratory mice, and agricultural animals) feed at relatively constant intervals and have diets more uniform in composition than snakes. From a biodiversity standpoint the condition of experiencing periods without food is probably the more general and animals with a steady diet are the exceptions. If we are to understand the role of microbiota in vertebrates in general then their function should be studied in wild animals with variable feeding frequency. Relatively few studies have examined the diversity of GIT bacteria in wild vertebrates. Several studies have examined the microbial communities present in wild fish GITs (Rawls et al. 2006, Roeselers et al. 2011, Sullam et al. 2012). The majority of studies of GIT bacteria in wild vertebrates have been done on mammals including 4 wild African ungulate species (Nelson et al. 2003), the ceca of wild capybaras (Garcia-Amado et al. 2011), wild parrots (Xenoulis et al. 2010), and developing tammar wal- laby’s and brushtail possums (Lentle et al. 2006). Gastrointesti- nal tract bacteria of one wild and one captive snake species have been characterized (Hill et al. 2008, Costello et al. 2010). None of these studies have examined the function of GIT bacteria. The digestive physiology of snakes is quite different from mammals and birds (Secor and Diamond 2000) which facilitates their use as a model system to expand knowledge of the diversity and function of GIT microbiota. Snakes are capable of surviving much longer periods of starvation than most vertebrates (Wang et al. 2006). How do the snake GIT microbiota respond to periods when the GIT is devoid of food? Does the snake’s microbiota con- tribute to its ability to rapidly digest large prey items? The answers to these and other questions will broaden our understanding of host-microbe interactions and will shed light on the dynamics and functions of GIT microbiota in wild vertebrates in general. Few studies have used modern molecular methods to characterize the diversity of gastrointestinal tract bacteria in snakes. Preliminary studies have shown that snake GITs contain microbial communities similar in diversity and species compo- sition to mammals, including humans (Hill et al. 2008, Costello et al. 2010). Hill et al. (2008) quantified the GIT bacterial com- munities in individual specimens of Agkistrodon piscivorus and Crotalus horridus. They found that bacterial species diversity and composition was similar to that of mammals and birds and included bacteria from the phyla Firmicutes and Bacteroidetes. Costello et al. (2010) compared the GIT microbial communi- ties of fasted and digesting captive Burmese Pythons (Python molorus). They too found that Bacteroidetes and Firmicutes dominated the GIT flora but the structure of the community changed depending upon whether the snakes were fasting or digesting. Bacteroidetes dominated the GIT of fasting snakes but Firmicutes dominated the GIT of digesting snakes. Digest- ing snakes also contained more diverse microbial communities. Costello et al. (2010) pointed out the potential of snakes as model organisms for studying the function of GIT bacteria and the need for the development of a non-lethal procedure for sampling mi- crobiota in the snake GIT. Because snake GITs are a relatively straight tube it should be possible to sample the GIT microbiota without injuring the snake by inserting some type of device into the cloaca and an- teriorly to the intestine. The ability to sample GIT microbiota without injuring the snake would further enhance the utility of snakes as a model organism for studies of diversity and func- tion of GIT bacteria. In humans GIT bacteria have been sampled through the use of an endoscope (Knutson et al. 1982) but such a device is too large to be used in most snakes. In the present study we test the effectiveness of two versions of a minimally invasive sampling device for characterizing the bacterial communities in the gastrointestinal tracts of juvenile Prairie Rattlesnakes. Materials and Methods.—The five, 2-year old juvenile Prairie Rattlesnakes (Crotalus viridis) we used in this study were cap- tive raised offspring of snakes collected from South Dakota and North Dakota in 2008 and 2009. They had been used for labo- ratory experiments on starvation and metabolic rates but had been on a normal (non-starvation) diet for six months prior to JACQUES G. HILL III* Division of Amphibians and Reptiles, Department of Zoology, Field Museum of Natural History 1400 South Lakeshore Drive, Chicago, Illinois 60605-2496, USA STEVE BEAUPRE Department of Biological Sciences, University of Arkansas, Fayetteville, Arkansas 72701, USA STEVE RICKE Center for Food Safety IFSE and Department of Food Science, University of Arkansas, Fayetteville, Arkansas 72704, USA SIHONG PARK Cell and Molecular Biology Program, University of Arkansas, Fayetteville, Arkansas 72701, USA IRENE HANNING Department of Food Science and Technology, University of Tennessee, Knoxville, Tennessee 37996, USA *Corresponding author; e-mail: ngookhiew@yahoo.com