Immunoreactive intensity of FXPRL amide neuropeptides in response to environmental conditions in the silkworm, Bombyx mori

Abstract

In the silkworm Bombyx mori, the diapause hormone-pheromone biosynthesis activating neuropeptide gene, DH-PBAN, is a neuropeptide gene that encodes a polypeptide precursor consisting in five Phe-X-Pro-Arg-Leu-NH(2) (FXPRL) amide (FXPRLa) neuropeptides; DH (diapause hormone), PBAN (pheromone-biosynthesis-activating neuropeptide) and α-, β- and γ-SGNPs (subesophageal ganglion neuropeptides). These neuropeptides are synthesized in DH-PBAN-producing neurosecretory cells contained within three neuromeres, four mandibular cells, six maxillary cells, two labial cells (SLb) and four lateral cells of the subesophageal ganglion. DH is solely responsible, among the FXPRLa peptide family, for embryonic diapause. Functional differentiation has been previously suggested to occur at each neuromere, with the SLb cells releasing DH through brain innervation in order to induce embryonic diapause. We have investigated the immunoreactive intensity of DH in the SLb when thermal (25°C or 15°C) and light (continuous illumination or darkness) conditions are altered and following brain surgery that induces diapause or non-diapause eggs in the progeny. We have also examined the immunoreactivity of the other FXPRLa peptides by using anti-β-SGNP and anti-PBAN antibodies. Pupal SLb somata immunoreactivities seem to be affected by both thermal and light conditions during embryogenesis. Thus, we have been able to identify a close correlation between the immunoreactive intensity of neuropeptides and environmental conditions relating to the determination of embryonic diapause in B. mori.

Embryonic thermosensitive TRPA1 determines transgenerational diapause phenotype of the silkworm,Bombyx mori

“…3 A, B, and E). As described previously (16), this finding indicates that DH is actively released from these neurosecretory cells in 25DD pupae and that its activity is closely correlated with the determination of diapause type. (23) lane : 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 TRPA1 ActA3 lane: 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 lane: 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 lane: 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 If BmTRPA1 indeed affects diapause status in progeny through control of DH release, it is predicted that the immunoreactive intensity of SLb cells would be altered by RNAi.…”

“…Bivoltine (Kosetsu) and polyvoltine (N4) strains of B. mori were used in these experiments. Insects were reared as described previously (16). During the larval stages, animals were reared at 25-27°C under a 12-h light/ dark cycle and relative humidity of 50-60%.…”

“…Standard protocols were used for immunohistochemistry as previously described (16,29). For anti-BmTRPA1 immunohistochemistry, we used an anti-BmTRPA1 mouse antibody prepared from mice immunized with BSA-conjugated 14-aa coding sequences from R1134 to A1147 of the BmTRPA1.…”

“…These changes can result in active release of DH into the hemolymph during the middle pupal stages, during which the developing ovaries are sensitive to DH (12,16). Thus, environmental thermal information is stored for a long time period (approximately 1 mo) until the middle pupal stage, and is transduced to neuroendocrine signals (DH release) to induce progeny diapause.…”

“…DH acts on a G protein-coupled receptor in the developing ovaries of females during pupal-adult development (12,13), and is produced in DH-pheromone-biosynthesis-activating neuropeptide-producing neurosecretory cells (DHPCs) located within the subesophageal ganglion (SG) (14)(15)(16). DH levels in the SG and SG-brain complex are markedly lower in 25DD pupae than in 15DD pupae, although level of mRNA expression is higher in 25DD than in the 15DD pupae at the early to middle stages of pupal-adult development (11,17).…”