The expression of metabolic enzyme genes and heat-shock protein genes (Hsp) during early embryogenesis in diapause and non-diapause eggs of the silkworm Bombyx mori was quantified by semi-quantitative RT-PCR. The trehalase gene (Tre) was expressed in non-diapause eggs up-to nine days, while in diapause eggs was not up regulated. The glycogen phosphorylase gene (GPase) was expressed in non-diapause eggs, whereas in diapause eggs a high level was observed in early stage, but down regulated in later stage. The phosphofructokinase gene (PFK) and sorbitol dehyrogenase-2 gene (SDH-2) expression was fluctuated in non-diapause eggs, whereas in diapause eggs these were expressed only at early stage and not observed in later stage. The glucose-6-phosphate dehydrogenase gene (G6P-DH) in non-diapause eggs was highly expressed during the differentiation phase and decreased in the organogenesis phase. In contrast to this, expression in diapause eggs was of low level during differentiation phase and of high level observed in the organogenesis phase. In the tissues, PFK and SDH-2 were selectively expressed in cuticle and midgut, whereas Tre expression was high in midgut and ovary of larvae incubated at 15 (20.4, 20.8, 40, 70, and 90) were expressed in both diapause and non-diapause eggs. Their expression was, however, selective in tissues with Hsp20.4 in midgut and ovary, Hsp40 in head, Hsp70 in cuticle and Hsp90 in ovary and head in high amounts at 15• C. These results suggest that the metabolic enzyme genes studied except Hsp play a major role during embryogenesis of diapause and non-diapause silkworm.

Conservation of silkworm [Bombyx mori L. (Lepidoptera: Bombycidae)] germplasm aids in prevention of genetic erosion and race extinction. Multivoltine silkworm germplasm maintenance involves rearing five generations per year and this requires expenditure, manpower, and infrastructure, in addition to frequent exposure of the silkworms to diseases, pests, and predators. To minimize these problems, an alternative protocol was developed by way of induction of egg diapause in the multivoltine silkworms through rearing of the fourth and fifth instars at low temperature (18-20°C) under regulated photoperiod (L6:D18). This method allowed induction of egg diapause in the selected races at levels ranging from 26 to 94% and the diapause-induced eggs were maintained under cold preservation conditions. The stability of genotypic characters was confirmed in the diapause-induced silkworm batches after the seventh generation, by comparison with control batches of silkworm using inter simple sequence repeat-polymerase chain reaction (ISSR-PCR). Analysis of Nei's genetic distance for the different races indicated no significant variation between control and diapause-induced races. This reveals that all the selected races maintained genetic stability even after the seventh generation at the phenotypic and molecular level. Hence, it can be concluded that induction of egg diapause is an appropriate alternative method to preserve multivoltine races for longer periods of time.

Diapause hormone (DH) and pheromone biosynthesis-activating neuropeptide (PBAN) code for the diapause gene (DH-PBAN) of Bombyx mori Linnaeus. The DH-PBAN gene sequence from the BLAST database was searched against B. mori genomic gene sequences for similarity. Results showed maximum homology with a genomic contig (accession no. D16230). Primers were designed for the intron, exon and promoter regions of the DH-PBAN gene sequence. The corresponding regions were amplified using genomic DNA as template and products of diapause and non-diapause silkworm races were compared. There was no variation in the 425 bp intron and 254 bp exon regions. However, 1.3% nucleotide sequence variation was observed in selected silkworm races. The promoter region showed 4.2% single nucleotide variation in the diapause and nondiapause silkworm races. The promoter region of the diapause gene showed variations in the sequence of the POU (Pit 1, Oct 1 and Unc-86)-binding site of non-diapause and diapause races of B. mori. In phylogenetic analysis, B. mori formed a cluster with other silkworms of the Bombycidae family, whereas the Noctuidae family formed a separate cluster, indicating that the diapause gene is conserved across the insect taxa.