A fluorescent triple staining method was developed to stain the cytoplasm of neurons red, the nuclei of all kinds of cells, including neurons, blue and the nuclei of apoptotic neurons in cyan in the twelve ventral ganglia (VG) of the Bombyx mori ventral nerve cord. This differential staining method was used to distinguish between apoptotic and normal neurons in the suboesophageal ganglion (SOG), thoracic ganglia (TG)1 to TG3 and abdominal ganglia (AG)1 to AG8 and also determine the changes in the numbers of apoptotic neurons that occur during postembryonic development. In most of the VG tested, neuronal apoptosis was most marked during the period from the end of larval life to the mid pupal stage. The greatest number of apoptotic neurons was found in SOG of day-5 pupae, TG1 to TG3 and AG1 to AG4 of day-1 pupae, and AG5 to AG8 of day-4 pupae. In vivo injection of 20-hydroxyecdysone (20E) into day-8 5th instar larvae resulted in both a considerable increase in the number of apoptotic neurons and cleavage of procaspase-3 into caspase-3, which induced neuronal apoptosis in SOG and AG6 to AG8 in day-1 pupae, and a slight increase in the number of apoptotic neurons in TG1. In TG3 and AG4, however, it had little effect on the number of apoptotic neurons or cleavage of procaspase-3. Treatment of the VG of both day-8 5th instar larvae and day-2 pupae with protein synthesis inhibitors by in vivo injection triggered a significant inhibition of neuronal apoptosis and procaspase-3 cleavage in most of these ganglia in day-1 pupae and day-4 pupae, but not TG3 and AG4, in which there was little procaspase-3 and caspase-3. In vivo injection of caspase-8 and -3 inhibitors into day-8 5th instar larvae and day-2 pupae led to a substantial inhibition of neuronal apoptosis and of procaspase-3 cleavage in SOG, AG6 and TAG, but not in TG3 or AG4 of day-1 pupae and day-4 pupae. These findings suggest that neurons that die in SOG, TG1 and AG6 to AG8 in day-1 and -4 pupae may undergo apoptosis induced by the synthesis of a new protein and caspase-8- and -3-implicated signal transduction by the increase in titre of 20E in the haemolymph but not the neuronal aopotosis in TG3 and AG4. This study provides neurobiologists with valuable information and a means of studying neuronal apoptosis in the nervous system of insects.
Two glutathione S-transferase (GST) cDNAs, GSTD2 and GSTS2, were cloned from the silkworm Bombyx mori. The B. mori GSTD2 (BmGSTD2) gene spans 4371 bp and consists of four introns and five exons that encode 222 amino acid residues. The deduced amino acid sequence of BmGSTD2 showed 58% protein sequence identity to the Delta-class GST of Maduca sexta. The B. mori GSTS2 (BmGSTS2) gene spans 3470 bp and consists of three introns and four exons that encode 206 amino acid residues. The deduced amino acid sequence of BmGSTS2 revealed 67%, 63%, and 61% protein sequence identities to the Sigma-class GSTs from B. mori, Platynota idaeusalis, and M. sexta, respectively. The BmGSTD2 and BmGSTS2 cDNAs were expressed as 25 kDa and 23 kDa polypeptides, respectively, in baculovirus-infected insect Sf9 cells. Northern blot and Western blot analyses showed that BmGSTD2 and BmGSTS2 were specifically expressed in three gut regions, indicating that the gut is the prime site for BmGSTD2 and BmGSTS2 synthesis in B. mori larvae.
Hexokinase (HXK) is the first key enzyme in the glycolytic pathway and plays an extremely important role in energy metabolism. By searching the microsporidian database, we found a sequence (NBO_27g0008) of Nosema bombycis Nägali, 1857 with high similarity to hexokinase-2, and named it as NbHXK2. The NbHXK2 gene has 894 bp and encodes 297 amino acids with 34.241 kD molecular weight and 5.26 isoelectric point. NbHXK2 contains 31 phosphorylation sites and 4 potential N-glycosylation sites with signal peptides and no transmembrane domain. Multiple sequence alignment showed that NbHXK2 shares more than 40% amino acid identity with that of other microsporidia, and the homology with hexokinase-2 of Nosema tyriae Canning, Curry, Cheney, Lafranchi-Tristem, Kawakami, Hatakeyama, Iwano et Ishihara, 1999, Nosema pyrausta (Paillot, 1927) and Nosema ceranae Fries, Feng, da Silva, Slemenda et Pieniazek, 1996 was 89.17%, 87.82% and 69.86%, respectively. Phylogenetic analysis based on the amino acid sequence of hexokinase showed that all microsporidia cluster together in the same clade, and are far away from animals, plants and fungi, and that N. bombycis is closely related to N. tyriae; N. pyrausta; N. ceranae and Nosema apis Zander, 1909. Immunolocalisation with the prepared polyclonal antibody showed that NbHXK2 was mainly distributed in the cytoplasm and plasmalemma in proliferative, sporulation stage and mature spore of N. bombycis. qRT-PCR assay showed that the NbHXK2 expressed at higher level during spore germination and at early stage of proliferation. These results indicate that N. bombycis may use its own glycolytic pathways to supply energy for infection and development, especially germination and in the early stage of proliferation, and acquire energy from the host through certain ways as well.