Circadian Clock Gene Period Contributes to Diapause via GABAeric-Diapause Hormone Pathway in Bombyx mori

SIMPLE SUMMARY: Diapause in insects is a classic and long-term concern subject regulated by both circadian clock and endocrine system. Studies in many insects have shown that disturbance of circadian clock system can affect diapause occurrence. However, the specific molecular regulation mechanism and key nodes between circadian clock and endocrine hormones regulating diapause occurrence are still lack of insightful reports. Our work identified the molecular nodes and pathways through which the transcription-translation feedback loop of the silkworm circadian clock regulated the level and action of diapause hormones, based on the diapause change in a silkworm mutant line of Period gene knockout. This work confirmed that Period knocked out in silkworms changed the classic temperature- and photoperiodic-dependent diapause-destiny and changed the diapause through the GABA-DH neurotransmitter-endocrine hormone pathway, and showed that the GABA receptor gene, GRD, was controlled by both the circadian clock and endocrine system in silkworms. The results provided an example to explain the regulatory mechanism of the circadian clock on endocrine hormones in the silkworm. ABSTRACT: Diapause is a developmental transition in insects based on seasonal adaptation to adversity; it is regulated by a circadian clock system and the endocrine system. However, the molecular node and its mechanism underlying the effects of these systems are still unclear. Here, a mutant of Bombyx mori with the circadian clock gene Period (Per) knocked out was constructed, which dramatically changed the classic diapause-destined pathway. Per-knockout silkworms powerfully attenuated, but could not completely block, the predetermined effects of temperature and photoperiod on diapause determination, and this effect depended on the diapause hormone (DH) pathway. The impaired transcription-translation feedback loop of the circadian clock system lacking the Per gene caused direct up-regulation of the expression of GRD, a receptor of γ-aminobutyric acid (GABA), by changing expression level of Cycle. The synthesis of GABA in the tissue complex of brain-suboesophageal ganglion then increased and restricted the decomposition, which continuously promoted the GABAergic signal to play a role, and finally inhibiting (delaying) the release of DH to the hemolymph, and reducing the diapause-inducing effect of DH. The results provided an example to explain the regulatory mechanism of the circadian clock on endocrine hormones in the silkworm.