Dataset: Bugula neritina mesocosm symbiont titers

Adult B. neritina colonies were collected from Oyster, VA, and Beaufort, NC. Larvae were collected from individual colonies and settled into 6-well plates. Samples of the adult colonies were preserved in DNA/RNA stabilization solution. After settlement, the plates were transferred to mesocoms maintained at different temperatures (16°C and 22°C). In addition half of the mesocosms had 1-2 filter-feeding invertebrates, the solitary tunicate, Styela sp., that could potentially on larvae. The colonies were fed cultured Rhodomonas sp. phytoplankton. Individual colonies were collected at different time points (18 w, 23 w), and preserved in DNA/RNA stabilization solution. Metagenomic DNA was extracted from preserved colonies using the Qiagen QIAamp DNA mini kit following the manufacturer’s directions. The final eluate was tested through endpoint PCR, using BnCOI primers (see "Table 1" Supplemental File). The PCR conditions were: initial denaturation at 95°C for 15 min, 30 cycles of 95°C denaturation for 30 s, 50°C annealing for 30s, 72°C extension for 1 min, and a final extension at 72°C for 5 min. The reaction setup per 25 uL was 12.5 uL Apex Mastermix, 2.5 uL each 10 uM forward, reverse primers, 4.5 uL DI water, and 3 uL template. Visualization of amplicons after agarose gel electrophoresis confirmed successful metagenomic DNA extraction and amplification.

The metagenomic DNA from B. neritina colonies underwent multiplex (host and symbiont targets) quantitative PCR using the Applied Biosystems StepOne Plus Real-Time PCR System to determine the absolute quantity of the symbiont in the DNA using a symbiont target, bryA, and a host target, VDAC. The reaction mix was as follows (20 uL reaction): 10 uL Tonbo BioSciences Fastprobe/Cyberfast qPCR Mastermix, 0.8 uL 10 uM each host forward, reverse primer, 0.8 uL 10 uM each symbiont forward, reverse primer, 0.4 uL each 10 uM host, symbiont probe, 4 uL DI water. The reaction conditions were: initial denaturation for 20 s at 95°C, 40 cycles of 95°C denaturation for 5 s, followed by annealing and extension at 60°C for 20 s.

The same symbiont primers and probe were used for both Type N and S colonies, but the host VDAC primers and probe differed between the two host genotypes. Each sample was run in triplicate, and a standard curve was generated for each run using copy number standards (see below). The efficiency for each run and for each target ranged between 91% to 98%. The R-squared values for the standard curve ranged between 97% and 99%. The mean number of copies were calculated for each sample by fitting the CT value to the standard curve.

Quantitative PCR Copy Number Standards: Using DNA from parental samples, host VDAC (Type N and Type S) and symbiont bryA primers (see "Table 1" Supplemental File), and Apex Mastermix, PCR was performed with specific cycling parameters mentioned previously. Agarose gel electrophoresis followed by visualization of the amplicons was performed to confirm amplification. The PCR amplicons were purified using the Qiagen MinElute PCR Purification kit following the manufacturer’s directions. The samples were concentrated to create a stock solution for both host VDAC and symbiont bryA amplicons. A Nanodrop spectrophotometer was used to quantify the concentration of amplicons (average of three readings). Serial dilutions using nuclease-free water as a diluent were performed to produce standards ranging from 32 to 4,000 copies of DNA per mL for both VDAC and bryA stocks. Dilutions were aliquoted, stored at -80oC until use, and were never refrozen.