Investigation of Aposematism in Amphibians of Southern Appalachian

Winebarger, Monica

General Survey Methods We will survey for amphibians using a variety of techniques (i.e., dip netting, log turning, rock lifting, night road cruising). Amphibians will be captured and held in appropriately sized containers with a moist paper towel and kept in coolers to mimic their preferred habitats (cool, dark, and moist). They will be transported to appropriate lab space in Rankin Science West at Appalachian State University where we will obtain the mass and length of each animal. Skin Secretion Collection We will also collect skin secretions by rinsing individuals with deionized water, placing them in plastic baggies, and gently harassing them for up to five minutes (see Clark, 2010). Animals will be removed from baggies, rinsed with filtered water, and kept at 5 o C for up to two hours in preparation for spectrometry. Skin secretion samples will be transferred to micro centrifuge tubes and stored in methanol at -20 o C until analysis. We will be using gas chromatography-mass spectrometry to characterize skin secretions. Spectral Measurements To measure dorsal coloration of each animal we will use an Ocean Optics reflectance spectrometer (S2000: Range 250-880 nm: Dunedin, FL, USA), placing the probe at five sites along the dorsal region. We may also take measurements of ventral coloration in certain species if it appears to be conspicuous, or if the species is known to display the ventral side when threatened or during courtship. Fungal Pathogen Monitoring As part of a larger amphibian monitoring program at Appalachian State University, we will test some amphibians for Batrachochytrium pathogens (i.e., Bd and Bsal). We will collect samples by rubbing a sterile swab along all surfaces of the body and then place the swabs in a -20 C freezer until analysis. We will determine the presence or absence of these pathogens using a standardized PCR Assay using both positive and negative controls. For more detailed protocol see Moffit et al. (2015). Vouchered Specimens While many amphibian skin secretion studies have vouchered specimens and used whole skins for their analyses, we will not be vouchering every animal. However, we will occasionally voucher amphibians in order to take small skin samples for pigment analysis. We will take a small sample of pigmented skin, then deposit the whole animal in the Appalachian State University Vertebrate Collection. Animals will be euthanized in MS-222 (Tricaine) for a minimum of 60 minutes in accordance with the 2013 Report from the AVMA Panel, and then transferred to ethanol (70% or higher). We will create a list of all vouchered specimen and their associated data and disseminate this information to the NPS. We will not voucher any species listed under state protection (Special Concern, Threatened, or Endangered), or voucher any specimens in areas with low population densities. All animals that are not vouchered will be returned to their site of capture upon completion of data collection. Occasionally data outlined above may be collected in the field or at another facility, only in the event that an animal is captured >50 miles from Appalachian State University.