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Bivoltine silkworms (Bombyx mori) are destined to respectively produce diapause or non-diapause eggs when they are reared under short or long days during the larval stage. The insulin signaling pathway is thought to play an essential role in regulating diapause in various insect species, but its involvement in silkworm diapause programming has not been investigated in detail. Therefore, we examined day-night expression of the insulin receptor (InR) gene in the silkworm larval brain under different photoperiods or in night interruption experiments in which larvae were exposed to light for 2 h during the nighttime of short days. Expression of the InR gene was photoperiod-dependent and InR mRNA levels decreased with increasing daylength. As the daylength increased, expression during the nighttime decreased to lower stable levels earlier than that during the daytime. During night interruptions that induced non-diapause, the nighttime expression of InR decreased to low levels like those during long days, although daytime expression was only moderately decreased. Nighttime InR expression was downregulated in silkworms reared under non-diapause-inducing conditions (long days and night interruptions). In contrast, abundant InR was expressed during the day and night in short days that induced diapause. Our findings suggested that InR expression in the larval brain is associated with programming the diapause status in the next generation of silkworms. Downregulated InR might suppress the insulin signaling pathway and cause non-diapause induction in the next generation. DOI: http://dx.doi.org/10.5281/zenodo.10413155

Rab, a low molecular weight GTP-binding protein, regulates the transmission of intracellular proteins. Insect neuropeptides that are directly involved in growth, development, reproduction, homeostasis, courtship, feeding, circadian rhythm and many other physiological processes are synthesized in neurons and ganglia in the brain and secreted by specific neurosecretory cells in tissues such as the corpus allatum and frontal ganglion. To clarify the relationship between Rabs, two neuropeptides, Allatotropin (AT) and Prothoracicotropic hormone (PTTH) and the circadian clock protein, Period (PER), were used to determine the locations of nine Rabs (Rab1, Rab3, Rab6, Rab7, Rab11, Rab14, Rab19, Rab21 and RabX4). Rab6-, Rab11-, Rab14- and Rab21-immunohistochemical reactivities (IRs) partially overlapped AT-IR in the brain in B. mori. Rab3-, Rab6-, Rab11-, Rab14-, and Rab21-IRs overlapped AT-IR in the frontal ganglion. Of the seven Rabs, only Rab11-IR overlapped PTTH-IR in the brain. Rab1-, Rab3-, Rab11-, Rab14-, Rab19-, Rab21-, RabX4- and NUF (nuclear fallout, an effector of Rab11)-IRs, overlapped PER-IR in the brain. Therefore, Rab may regulate the exocytosis of PTTH, AT and a protein associated with the circadian rhythm.

Maternal silkworms (Bombyx mori) of bivoltine strains are destined to produce either diapause or non-diapause eggs depending on environmental factors, such as, temperature and photoperiod experienced during the egg and larval stages. However, the molecular mechanisms that program diapause, which depend on information about the environment, remain unclear. We aimed to identify genes that are involved in programming diapause in the next generation in bivoltine silkworms. We therefore screened differentially expressed genes (DEGs) in the larval brains of diapause- and non-diapause-egg producers kept under three different diapause-inducing conditions using cap analysis of gene expression. Under each condition, only temperature, illumination or photoperiod was changed during the egg or larval stage as a diapause-controlling stimulus to induce the production of diapause or non-diapause eggs. We then verified the expression of DEGs that were common to all the three conditions using real-time quantitative PCR. We investigated the functional involvement of candidate genes in programming diapause using double-stranded RNA interference (RNAi) for gene knockdown. The results showed more abundant juvenile hormone acid methyltransferase (Jhamt) and proton-coupled folate transporter (Pcft) gene expression in the brains of fifth instar larvae of producers of diapause eggs than those of non-diapause eggs under the three conditions. Furthermore, RNAi against either of these genes significantly decreased the incidence of diapause in the next generation. These findings indicate that both Jhamt and Pcft are involved in the programming of diapause in the silkworm brain. These genes could function in retaining information that leads to diapause in the next generation.