Study 1: Is the cloaca a uniquely specialized region of the
gastrointestinal and reproductive tracts?
Sceloporus virgatus lizards are small insectivorous animals found
in the Madrean Sky Islands of the southwestern United States and south
into Mexico (Stebbins,
2003). Our study population is at the northern reach of their range, in
Cochise County, Arizona near the American Museum of Natural History’s
Southwestern Research Station (SWRS). To address the adaptive hypothesis
that the cloacal microbiome will be unique from that of the upper GI and
reproductive tracts, we collected gravid Sceloporus virgatusfemales (n = 8) between 28 June and 01 July 2019 using a loop of fishing
line tied to a retractable fishing pole. Female cloacae were swabbed
immediately following capture. Females were then kept in large outdoor
enclosures at SWRS with access to water bowls and naturally occurring
prey for 1-4 d until being shipped overnight to the University of Puget
Sound in individual plastic containers on ice packs. Upon arrival, we
euthanized females using buffered MS-222
(Conroy et al.,
2009). We collected transverse sections of tissue (~2-4
mm long) from the cloaca (expanded tissue immediately above the vent),
lower intestine (~10 mm above cloaca), upper intestine
(~5 mm below stomach), and oviduct (at the position of
the lowest egg on the left side) using heat sterilized instruments, and
did not attempt to separate or isolate mucosal vs. luminal contents.
Microbes were eluted from swabs into an Amies solution by manual shaking
and were stored at -80°C, as were tissue samples, until DNA extraction.