Effect of Light and Water Agitation on Hatching Processes in Clown Anemonefish Amphiprion ocellaris

Clown anemonefish (Amphiprion ocellaris) exhibit an environmentally cued hatching strategy, wherein parents generate water flow to encourage embryos to hatch after sunset on the eighth day post spawning. Although previous studies have shown that hatching requires complete darkness and water agitation, the regulatory mechanisms underlying these environmental cues remain elusive. This study investigated the expression patterns of hatching enzymes and how darkness and water agitation affect their secretion and the digestion of the egg envelope (chorion) in clown anemonefish. We revealed that hatching enzymes were expressed from the early developmental stages and decreased towards hatching day, similar to other fish species. The hatching gland cells were located on the tail, which may be associated with egg shape and chorion breakage. On the day of hatching, chorion digestion occurred exclusively in complete darkness, regardless of water agitation. In addition, ionomycin, a Ca2+ ionophore and an inducer of hatching enzyme secretion, triggered chorion digestion even under light-on conditions. Furthermore, water agitation expedited chorion digestion in the dark. In conclusion, our findings indicate that darkness triggers the secretion of hatching enzymes mediated by changes in Ca2+ levels, whereas water agitation assists chorion digestion activity and/or hatching enzyme secretion. These responses to environmental cues contribute to the rapid and synchronised hatching in clown anemonefish.