2.3 Microstructure examination of O. oratoria compound eye
To study the microstructure of the compound eye, we dissected the bilateral compound eyes of one O. oratoria exposed to nature light after the habituation period and removed the cornea of the compound eyes under the stereomicroscope and immersed the compound eyes in filtered seawater. The outer shell of the compound eye was removed anatomically using corneal scissors and ophthalmic forceps along the base of the eyestalk, and the remaining compound eyes were fixed in 4% paraformaldehyde for 30 minutes at room temperature. The compound eyes were dehydrated according to the following procedures: 30% ethanol (60 minutes)→50% ethanol (60 minutes)→70% ethanol (60 minutes)→95% ethanol (60 minutes)→ethanol (30 minutes)→ethanol (30 minutes)→1:1 xylene: ethanol (5 minutes)→xylene (3 minutes)→xylene (3 minutes)→paraffin (120 minutes). The parrafin used was a mixture of equal proportions of high- and low- melting point paraffin to then use the mixture for sample embedding. The embedded compound eyes were cut into 4-micron sections using the microtome (Leica). The excised compound eyes were expanded in 37 ℃ water and then affixed to glass slides coated with poly-L-lysine (Sigma). The compound eye slices were then incubated at 37 ℃ for 12 hours before deparaffinization and rehydration according to the following procedures: xylene (10 minutes)→1:1 xylene: ethanol (3 minutes)→95% ethanol (3 minutes)→80% ethanol (3 minutes)→70% ethanol (3 minutes)→50% ethanol (3 minutes)→30% ethanol (3 minutes)→distilled water (3 minutes). After undergoing hematoxylin-eosin staining, compound eyes were sealed and stored using a sterile aqueous solution of 50% glycerol (Sangon Biotech). Finally, the transverse and longitudinal sections were examined and then photographed under digital microscope (Leica).
Additionally, to observe the surface ultrastructure of O. oratoria compound eye, the O. oratoria compound eyes were excised under the stereomicroscope (Leica) and immersed in warm water (~40 ℃) for 2 days to achieve softening. The dirt on the surface of the compound eyes was washed in ultrasonic cleaning instrument (Skymen) for 10 minutes. The compound eyes were dehydrated in multiple gradient concentrations of ethanol (50%, 70%, 80%, 90%, 95%, and 100%) for 10 minutes, and then dehydrated 3 times continuously with absolute ethanol. After the compound eyes were dried at room temperature, they were glued to the sample stage with the help of a stereoscopic microscope (Nikon) and conducting resin (Ausbond) by adjusting the sample orientation slightly with tweezers. The compound eyes were sprayed with gold in the sputter coater (Cressington) and photographed under the scanning electron microscopy (ZEiSS) to scan the surface ultrastructure. Meanwhile, the internal ultrastructure of theO. oratoria compound eye with the cornea removed was observed by referring to the surface ultrastructure observation process mentioned above.
Finally, to observe the refined structure of the rhabdom of O. oratoria compound eye, we immobilized the cornea-removed compound eyes with 5% glutaraldehyde for 2 hours and then applied phosphate buffer to the fixed compound eyes three times for 30 minutes each time. The treated compound eyes were re-fixed with 2% osmic anhydride for 90 minutes and rinsed with phosphate buffer. The compound eyes were then dehydrated using the subsequent processes: 30% ethanol (5 minutes)→50% ethanol (5 minutes)→70% ethanol (10 minutes)→80% ethanol (10 minutes)→85% ethanol (15 minutes)→90% ethanol (15 minutes)→95% ethanol (15 minutes)→100% ethanol (20 minutes)→100% ethanol (20 minutes)→acetone (15 minutes)→acetone (15 minutes). Subsequently, Epon 812 and epoxy-coated compound eyes were cut into ultrathin (50 nm) sections using the diamond knife and then stained with saturated uranyl acetate and citric acid (PH = 12). Finally, the sections were observed and photographed using transmission electron microscope (JEOL).
2.4 Light exposure experiments
After 30 days of temporary rearing in natural seawater, 90 healthyO. oratoria s were randomly transferred to 15 separated circulating tanks. 15 tanks were divided into five lighting scenarios (dark [DL], natural light [NL], linearly polarized light [LPL], left-rotation CPL [LCPL], and right-rotation CPL [RCPL]) by matching light sources and polarizers, and 3 tanks were present for each lighting scenario. We used light-tight boxes to envelop the 15 tanks to avoid external light affecting the set lighting scenarios. Meanwhile, 6 O. oratoria s were reared in each tank for 7 days in each scenario (Figure.1). During the experimental period, clams were fed to avoid cannibalism among the O. oratoria s per tank and the seawater was changed every day. Additionally, it is worth noting that because half of the O. oratoria s in the LPL scenario died, the culture experiment in the LPL scenario was repeated three times to ensure the number of samples required for transcriptome and proteome sequencing.