Establishment of a Sandwich ELISA System to Detect Diapause Hormone, and Developmental Profile of Hormone Levels in Egg and Subesophageal Ganglion of the Silkworm, Bombyx mori

Abstract

In the silkworm Bombyx mori, diapause hormone (DH) is produced in the female subesophageal ganglion (SG) and induces embryonic diapause by targeting developing ovaries. DH is processed from a precursor protein consisting of DH, pheromone biosynthesis activating neuropeptide (PBAN) and three other neuropeptides (SGNPs). Because these five neuropeptides share a common sequence, FXPRLamide, at the C-terminus, a direct and specific assay for DH itself is required in order to understand the profile of concentration changes. In this study, we produced a mouse monoclonal antibody (anti-DH[N] mAb) against the N-terminal region of DH and developed a sandwich enzyme-linked immunosorbent assay using the anti-DH[N] mAb and a rabbit polyclonal antibody against the C-terminus of DH. This procedure enabled us to specifically quantify the DH molecule at femtomolar levels (equivalent to 1/10 of SG). We then plotted DH levels in eggs and SGs during embryonic and post-embryonic development. DH was present in late-stage embryos that had been destined for the production of both diapause and nondiapause eggs. DH levels in SG gradually increased in both types during larval development and peaked at the early pupal stage. At the middle pupal stage, DH levels in SG and SG-brain complex decreased markedly in the diapause-egg producing type, thus indicating active release of DH into the hemolymph. From 5th instar larva to adult, no sexual differences in DH levels were observed in SGs or SG-brain complexes from diapause and nondiapause egg-producing types.

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

“…The DH content in the brain-SG complex has previously been shown to decrease from the middle pupal stage in both diapause types (Kitagawa et al 2005). Therefore, these results suggest that FXPRLa peptides are actively transported from the SG, although the precise developmental rate of FXPRLa biosynthesis remains unknown.…”

“…At the middle pupal stage, DH levels in the SG and SG-brain complex are markedly decreased in the DEP as compared with those of the NDEP (Kitagawa et al 2005). Furthermore, significant morphological and functional differences in the three neuromeres of the DHPCs have been detected following surgical ablation experiments (Ichikawa et al 1996) and electrophysiology experiments involving changes in firing activity patterns between DEP and NDEP (Ichikawa and Kamimoto 2003;Ichikawa 2003).…”

“…For the measurement of DH content in the brain-SG complex, we used sandwich enzyme-linked immunosorbent assay performed according to the method described by Kitagawa et al (2005) with some modifications. Briefly, the brain-SG complexes of five animals were dissected and suspended together in 250 μl ice-cold PBS.…”

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

“…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). The labial neuromere cell of the SG (SLb) somata are DHPCs, and transport of DH from the Significance Diapause has evolved as a specific subtype of dormancy in most insect species and as a seasonal polyphenism that ensures survival under unfavorable environmental conditions and synchronizes populations.…”

Molecular cloning and characterization of Bombyx mori CREB gene

“…Interestingly, the tenidium formation and late stages of embryogensis are sensitive periods for receiving environmental signals to determine whether its offsprings are diapause or non-diapause (Hasegawa, 1957;Yamashita and Hasegawa, 1985). Meanwhile, pupal-adult development is a key stage during which DH is synthesized and secreted that leads to the egg diapause (Yamashita and Hasegawa, 1985;Kitagawa et al, 2005). The sustained high expression of the BmCREB in the diapause type silkworm, especially at the two critical stages suggested that the activation of BmCREB in transcriptional level is related to the diapause signal reception in embryogenesis and retrieval in pupal stage.…”