Hibernation with Rhythmicity in the Retina, Brain, and Plasma but Not in the Liver of Hibernating Giant Spiny Frogs (Quasipaa spinosa)

SIMPLE SUMMARY: Aquatic ectotherms experience hypoxia under water during hibernation, which enables them to move denoting some level of consciousness, unlike terrestrial hibernators. However, how aquatic ectotherms modulate their clocks and clock-controlled genes in different tissues and plasma melatonin and corticosterone in light-dark cycles under natural environments before and during hibernation, remains to be largely unexplored. To achieve these, in this study, we investigated circadian clock genes, circadian clock-controlled genes, antioxidant enzyme genes, and related hormones in giant spiny frog (Quasipaa spinosa). Our results demonstrated that, despite the hypometabolic state of hibernation, the retina and the brain displayed some circadian rhythms of clock and antioxidant genes, as well as melatonin, while the liver was inactive. These novel findings may contribute to an understanding of how aquatic ectotherms use their circadian system differentially to modulate their physiology in escaping hypoxia during hibernation and preparing for arousal. ABSTRACT: Hibernation in ectotherms is well known, however, it is unclear how the circadian clock regulates endocrine and antioxidative defense systems of aquatic hibernators. Using the giant spiny frog (Quasipaa spinosa), we studied mRNA expression levels of (1) circadian core clock (Bmal1, Clock, Cry1 and Per2), clock-controlled (Ror-α, Mel-1c and AANAT), and antioxidant enzyme (AOE) (SOD1, SOD2, CAT and GPx) genes in retina, brain, and liver; and (2) plasma melatonin (MT) and corticosterone (CORT) levels, over a 24-hour period at six intervals pre-hibernation and during hibernation. Our results showed that brain Bmal1, Cry1, Per2 and Mel-1c were rhythmic pre-hibernation and Clock and Ror-α during hibernation. However, the retina Bmal1, Clock and Mel-1c, and plasma MT became rhythmic during hibernation. All brain AOEs (SOD1, SOD2, CAT and GPx) were rhythmic pre-hibernation and became non-rhythmic but upregulated, except SOD1, during hibernation. However, plasma CORT and liver clocks and AOEs were non-rhythmic in both periods. The mRNA expression levels of AOEs closely resembled those of Ror-α but not plasma MT oscillations. In the hibernating aquatic frogs, these modulations of melatonin, as well as clock and clock-controlled genes and AOEs might be fundamental for them to remain relatively inactive, increase tolerance, and escape hypoxia, and to prepare for arousal.