Allatotropin, DH31, and proctolin reduce chill tolerance in the two-spotted cricket, Gryllus bimaculatus.

The ability of insects to tolerate low temperatures, known as chill tolerance, contributes to their global distribution. However, the mechanisms underlying insect chill tolerance remain poorly understood. At low temperatures, insects enter chill coma, a reversible state of paralysis, owing to disrupted ion and water homeostasis. Upon returning to normal temperatures, insects reestablish ion and water homeostasis and recover the ability to move. In this study, we used the two-spotted cricket, Gryllus bimaculatus, as an experimental model and unveiled the roles of neuropeptides in regulating chill tolerance, typically evaluated by the time taken to recover from chill coma. Screening of 37 neuropeptides revealed that Allatotropin, DH31, and Proctolin inhibited chill coma recovery and decreased the survival rate under cold stress. RT-qPCR analyses revealed that the receptors for Allatotropin and DH31 were predominantly expressed in the hindgut. Injection of the three neuropeptides decreased both hemolymph mass and gut water content at low temperatures, most likely by increasing water excretion from the hindgut due to their effects on the rectum contraction. Additionally, Allatotropin and DH31 were produced by the terminal abdominal ganglion (TAG) innervating the hindgut since they were partly co-localized in the TAG, and their mature peptides were detected in the TAG-hindgut nerves. Moreover, the transcriptional levels of the neuropeptides in the TAG and receptors in the hindgut changed with cold exposure and rewarming. Based on these findings, we propose that Allatotropin, DH31, and Proctolin affect the physiological activities of the gut, probably the hindgut, to disrupt water homeostasis at low temperatures, thereby reducing chill tolerance in crickets.