title: negative extration 4
design: Coral fragments from P. daedalea colonies with mature (pigmented) gametes were collected at 3.5 m depth at Saadiyat Reef in Abu Dhabi (24 3548.79N, 54 2534.88E) during the predicted month of peak spawning in May 2017. P. daedalea is a hermaphrodite with mature oocytes and spermaries observed together in polyps prior to gamete release. Coral fragments were maintained in a recirculating aquarium system (with 1 m filtered seawater) at Zayed University in Abu Dhabi and separated into buckets at sunset each night to individually monitor spawning activity. Gamete bundles of sperm and eggs released on 10 May 2017 (1 night before full moon) from 7 colonies were collected, mixed, and left for 2 hours for fertilization to occur. Resultant embryos were then gently washed in filtered seawater. Coral embryos were subsequently cultured in 15 L larval rearing tanks throughout development into planula larvae (~3 days post-spawning) in 1 m filtered, autoclaved, and UV-sterilized seawater under temperature and salinity conditions matching the reef of origin (temperature: 28C; salinity: 44 PSU). Light reaching these tanks came from the ceiling lights in the laboratory plus natural light coming through a large window approximately 3 meters from the tanks (light levels never reached > 50 PAR). Triplicate samples of pools of 10-15 larvae were collected 15 days (early larval stage) and 28 days (late larval stage) post-spawning. In addition, gamete bundles were collected from 3 colonies that spawned on the second night on 11 May 2017 and carefully separated into eggs and sperm using plankton mesh (60 m). Further, triplicate samples of 1 m filtered seawater (with no larvae added) were collected at 28 days by passing 6 mL of seawater through a 0.2 m filter. All coral samples and filters were fixed in RNAlater (Thermo Fisher Scientific, UAE) in microtubes and incubated for several days at 4C prior to storage at -20 C until further processing. Overall, samples were collected from (i) parental eggs, (ii) parental sperm, (iii) mature 15-day-old planula larvae, (iv) mature 28-day planula larvae, and seawater in addition to 6 negative controls (i.e., 5 negative extractions and 1 PCR negative) for a total of 23 samples.