Transcriptomic features of Pecten maximus oocyte quality and maturation

The king scallop Pecten maximus is a high valuable species of great interest in Europe for both fishery and aquaculture. However, hatchery production is a relatively new industry and it is still underdeveloped. Major hurdles are spawning control and gamete quality. In the present study, a total of 14 scallops were sampled in the bay of Brest (Brittany, France) to compare transcriptomic profiles of mature oocytes collected by spawning induction or by stripping. To reach such a goal, a microarray analysis was performed by using a custom 8x60K oligonucleotide microarray Adult scallops (mean weight±SD: 174±32g, mean length: 111±7mm) were caught from the bay of Brest at the beginning of their natural spawning period and transferred to the experimental hatchery of Ifremer (Argenton, France) where they were conditioned for 1 month under suitable conditions for germ cells maturation. Briefly, scallops were placed in experimental raceways supplied with 1 µm-filtered running seawater at 17 ± 1.0°C and fed with a mixed diet of two microalgae (Chaetoceros gracilis and Tisochrysis lutea) at a daily ratio equal to 10 exp 9 cells of each algae species/scallop. Released oocytes were obtained by thermal stimulation to induce spawning of females, consisting on exposure to alternate cycles of 18°C (20 minutes) and 23°C (1 hour) [Gruffydd and Beaumont, 1970]. Once spawning was completed, the collected oocytes were filtered in a 20 µm sieve, to avoid self-fertilization. Oocytes from eight females were rinsed with iso-osmotic ammonium formate (3 % w/v) to remove salt. A total of 20,000 oocytes were homogenized in 1,5 ml of Extract-all (Eurobio) and stored at -80 °C for further transcriptomic analyses. Fertilization was then performed as described in Corporeau et al., 2014. Trochophores movement was estimate at 24h post fertilization using a CASA device, according to Suquet et al. [2012]. Then, the D-larval yield was assessed at 48h post fertilization (number of normal D-larvae/total number of oocytes) as described in de Sousa et al., 2015. In addition, gametes (20,000 oocytes per female) from six sexually mature females were dissected and oocytes were collected by “gamete stripping” as reported in Song et al., 2009. About 20,000 oocytes from each female were harvested and stored as described above. The remaining stripped oocytes from each female were fertilized (as described above) and D-larval rate was registered. RNA was isolated by following the Extract-all manufacturer instructions and combining the RNeasy Mini Kit (Qiagen) for the nucleic acid purification. A DNAse treatment was also carried out (Qiagen). Samples concentration was measured in a NanoDrop® ND-1000 spectrophotometer and the RNA quality was assessed through the Bioanalyzer 2010 instrument (Agilent). Gene expression profiling was performed using an Pecten maximus oligo-DNA microarray of 59,824 probes based on single-colour detection (Cyanine-3 only). Microarrays were scanned with Agilent scanner G2565BA at a resolution of 2 microns; all slides were scanned twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%); the scanner software created a unique ID for each pair of XDR scans and saved it to both scan image files. FeatureExtraction v10.7.3.1 used XDR ID to link the pairs of scans together automatically when extracting data. The signal left after all the FE processing steps have been completed is ProcessedSignal that contains the Multiplicatively Detrended, Background-Subtracted Signal.