in paraffin wax. Five-micrometer sections were stained according to
Prenant-Gabe Trichrome protocol (Gabe, 1968). Slides were examined
under a Nikon Eclipse 80i microscope coupled to a Nikon
DXM1200-C camera allowing individual determination of sex, and
gametogenic and storage stages based on criteria described in
Berthelin et al. (2000a) for diploid animals and Jouaux et al. (2010)
for triploid animals. Briefly, for diploid gametogenesis stage 0 corresponds
to the sexual resting stage, stage I is defined as the early developmental
stage (gonial mitosis), stage II corresponds to the
germinal lineage development and stage III is characterized as the
ripe gonad. For triploid animals, the determination of reproductive
stages took into account the presence of locked or unlocked cellular
divisions (for α and β animals respectively) at stages I and II, and
the production of gametes at stage III (large production for α and
few mature gametes for β). The storage stages are the same in diploid
and triploid oysters: the storage tissue is growing at stage A, dense
and structured at stage B, in regression at stage C and almost
completely depleted at stage D (Berthelin et al., 2000b).
2.5. mRNA extraction and reverse transcription
Reagents were purchased from Thermofisher®. Trireagent was