title: Standard Tank3 Adult Rep3
design: Xenopus laevis frogs were kept in recirculating water in Tecniplast/Marine Biotech systems in which the water was treated with a UV lamp and particulate, mechanical and carbon filters. The water temperature was kept at 18.5C (range 18-19 C), the conductivity at 1550 uS (range 1500-1600 uS, maintained by Tropic Marin/Instant Ocean sea salt dosing), the pH at 7.9 (range7.5-9.0, maintained using sodium bicarbonate dosing). The water was charcoal filtered Portsmouth mains water. Frogs were housed in 23 litres tanks at a stocking density of 7 adult females per tank. The flow rate of the recirculating water was 1.33 litres per minute in adult tanks and minimal flow in tadpole tanks. The light cycles were 13 hours of fluorescent light and 11 hours dark with night light. Tanks were supplemented with environmental enrichment of black plastic guttering and downpipe tubes cut in half in 20 cm lengths. Adult frogs were fed Horizon 23 trout pellets 2-3 times per day, a few pellets each. Tadpoles were fed Sera-micron algae daily with water flow turned to a slow drip. All animals were fed 5 days per week. Six tanks within a 9-tank rack were designated randomly for the experiment in a clean room (where embryos are surface sterilized before entering the housing system) and a standard room (where no sterilisation is performed). The remaining tanks were undisturbed. Three tanks per clean and standard rack were populated with adult female Xenopus laevis, and three tanks with 3-4 weeks old tadpoles.  The adults used originated from the same clutch of eggs as did the tadpoles and so each group was siblings of the same age. Adult Xenopus laevis, pre-metamorphic tadpoles and tank water were analysed. We focused on females since many laboratories do not keep male frogs due to the successful introduction of frozen sperm and supported by the fact that no statistical difference between male and female skin microbiomes was found in the Red-eye tree frog. Water (50 mL) was collected in sterile Falcon tubes from each of the designated tanks. Adult Xenopus laevis were taken from their tanks, rinsed by submersion into sterile distilled water to remove transient bacteria and swabbed with a sterile cotton swab (Medical Wire Equipment Co, MW-100) before being placed back in the system. To swab thoroughly and consistently, the same swabbing technique was followed for each sample and new nitrile gloves were used between samples. The pattern consisted of ten strokes on the ventral side, ten strokes on the dorsal side, five strokes under each front leg and five strokes between the toes. Tadpoles were trapped with a net, rinsed in distilled water and swabbed ten times on the dorsal side and ten times on the ventral side. Swabs were kept at 4C for up to one week before DNA preparation. Water samples were filtered through a 0.45 M Cellulose Nitrate Filter (Sartorius). These filters and the cotton swabs were suspended in 500 L of phosphate buffer saline (PBS) for 5 minutes with gentle agitation, 200 L of this solution was removed and mixed with 20 L of proteinase K. Each swab was processed following the DNeasy Blood and Tissue Kit (QIAGEN) manufacturers instructions. The extracted DNA was quantified by spectrophotometry and shipped to LGC Genomics GmbH. The 16S rRNA gene was targeted by PCR with primers 341F/785R used by Klindworth et al, 2013 (341F5-CCTACGGGNGGCWGCAG-3 and 785R 5-GACTACHVGGGTATCTAATCC-3), samples pooled, library prepared (incl. indexing*2) and quality controlled by gel electrophoresis before cluster generation and sequencing using Illumina MiSeq V3.
library_name: 341F-785R-30