16S rRNA gene sequencing of Lithobates clamitans tadpole gut and skin, as well as tank and pond water environmental samples. Raw sequence reads
16S rRNA gene sequencing of the V4 regions from Lithobates clamitans (Green Frog) tadpole guts and skin swabs, as well as tank water samples and pond water inoculants. In this study, we experimentally manipulated microbial exposure of captive Green frog, Lithobates clamitans (Ranidae, Aquarana), tadpoles. Specifically, we tested whether inoculating tadpoles with distinct sources of native pond water would yield distinct gut and skin microbiomes with effects on animal growth and survival. Further, we tested whether various concentrations of native pond water can increase microbiota richness in captive tadpoles and affect host fitness. Finally, we tested whether dietary tannic acid supplementation can alter amphibian microbiota and host fitness. Collectively, these results offer conceptual understanding towards community coalescence and inoculum dynamics, but also applied understanding towards developing practices for inoculating captive amphibians with natural microbial sources. Pond water was collected from two separate locations in Western Pennsylvania (PLE, and PM). Adult green frogs were caught in Western Pennsylvania and bred in a laboratory animal room. Resulting tadpoles were reared in reverse-osmosis mineral-supplemented water (laboratory water) until Gosner stage 21 before placement in treatment tanks. Study 1, Microbial Density Exposure: Tadpoles were randomly assigned to tanks (10 per tank, 3 tanks per treatment). We utilized six concentrations of each pond water source (PLE or PM): 0:100 (hereafter called laboratory water or "LW"), 1:99, 25:75, 50:50, 75:25, or 100:0 (pond water : laboratory water), plus two depletion treatments (autoclaved pond water and sterile filtered pond water) composed of 25% of the respective depleted pond water, and 75% laboratory water. Tadpoles were fed, ad libitum, a diet of 250 g commercial, alfalfa-based ground rabbit chow suspended in autoclaved agar (25 g agar, 1 L water). Study 2, Tannic Acids: Tadpoles were randomly assigned to tanks (10 per tank, 3 tanks per treatment). We utilized 2 water treatments of the PLE pond water: (1) 25% untreated PLE pond water : 75% untreated laboratory water, and (2) 25% autoclaved pond water : 75% untreated laboratory water. Tadpoles were fed, ad libitum, a diet of 250 g commercial, alfalfa-based ground rabbit chow suspended in autoclaved agar (25 g agar, 1 L water), supplemented with 2% tannic acid based on dry mass. Tadpoles were housed in a climate-controlled room maintained at 24C and 65% relative humidity, with a 12 hour on : 12 hour off light:dark cycle. Tadpoles were transferred weekly to new containers filled with 3-liters of their respective water. Mortality was monitored and recorded daily. At the end of week 4, tadpoles were euthanized (immersion in buffered MS-222 (10 g/L)), weighed, swabbed for skin microbiota (sterile swab was rolled gently across the tadpole for 10 seconds), and dissected for whole gut microbiota samples. Swabs and tissue samples stored at -80C until DNA extraction. For water samples, 45 mL of water was collected using sterile 25 mL serological pipettes and dispensed into sterile 50 mL conical tubes. The samples were spun at 1,000 x g for 10 minutes, the liquid eluted, pellets resuspended in 1 mL sterile PBS, and stored in microcentrifuge tubes at -20C until DNA extraction.