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.