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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.

Rab proteins are small GTP-binding proteins and are the largest family in the Ras GTPase superfamily and mediate vesicular transport in cells. Diverse insulin-like peptides, such as bombyxin, are synthesized in the brain and secreted into the haemolymph by the corpus allatum (CA). In the brain of Bombyx mori, Rabs are expressed in a specific area; however, which Rabs actually link the secretion of bombyxin remains unknown. A double-staining analysis of nine Rabs (Rab1, 3, 6, 7, 14, 21, 26, 39 and X4) and bombyxin indicated that Rab3-, Rab7-, Rab39- and RabX4-immunohistochemical reactivity (ir) areas overlapped with bombyxin-ir in the brain and CA in B. mori, while Rab6-, Rab14- and Rab21-irs partially overlapped in the CA. Rab1-ir occurred in the other immunopositive areas in CA. Rab26-ir did not occur in the brain. Rab39-ir occurred in UNC104, Rab39- effector, -immunopositive neurons in the brain and CA. Thus, Rab3, 7, 39 and X4 may regulate the exocytosis of bombyxin.