Insect cell-baculovirus expression systems are widely used in the production of various proteins, and they have been utilized in the development of biopharmaceuticals in the recent years. Unlike Escherichia coli (E. coli) expression system, proteins expressed in this system undergo posttranslational modifications such as glycosylation. However, research has shown that glycosylation patterns in insect cell system are different from those in mammalian cell expression systems. In addition, the glycosylation mechanisms in insect cells have not been well understood. Here, we report on the N-linked glycan profiling derived from recombinant proteins expressed in the insect cell lines, Spodoptera frugiperda, IPLB-SF-21AE (Sf21) and Bombyx mori (BmN). Recombinant virus vectors were prepared using the genes from the Newcastle disease virus fusion protein (F) and hemagglutinin neuraminidase (HN) as model glycoproteins using host expanded hybrid baculoviruses, HyNPVs, which replicate and express the recombinant proteins in both Sf21 and BmN cells. The recombinant glycoproteins produced were identified using SDS-PAGE followed by Western blotting and were processed by peptide-N-glycosidase F, which cleaved N-linked glycans from proteins within the gel. Mass spectrometric analysis showed that the glycan pools from recombinant HN and F in our study included increased levels of high-mannose-type glycans compared with those previously reported. We also found differences between the collected N-linked glycans of expressed proteins in Sf21 and BmN cells. The high-mannose-type glycans in Sf21-derived N-linked glycan pools mainly contained seven to nine mannose residues, while those in BmN-derived N-linked glycan pools contained five to nine mannose residues. These results demonstrate the different glycan modifications of recombinant proteins expressed in Sf21 or BmN, which lead to the possible functional differences in these proteins.

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In the silkworm Bombyx mori, more than ten mutations exhibit an embryonic lethal phenotype. The lethal 19 (l-19) mutation is one of these, and embryos exhibiting this mutation die at blastokinesis. The l-19 mutation is controlled by a single recessive gene mapped to chromosome 19. However, the responsible gene has not yet been identified. In this study, we performed morphological observations of the eggs and embryos using microscopy and mapped l-19. Observations revealed that eggs with the l-19 mutation die before or after the body pigmentation stage, but only embryos in eggs before the body pigmentation stage were dead at blastokinesis. This result suggests that the phenotype of the l-19 mutation can be identified by sampling dead eggs at this stage. We next performed mapping of l-19 using primer sets designed for chromosome 19. However, we could not identify the l-19-linked region, because there were no regions where the genotypes of all F2 individuals were homozygous for the l-19 mutant, suggesting that l-19 does not map to chromosome 19. Therefore, we performed a linkage analysis using primer sets designed for each chromosome. Our analysis showed that l-19 does not map to chromosome 19, but instead maps to chromosome 12. Further mapping indicated that the l-19-linked region spanned approximately 10 Mb on chromosome 12.

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In lepidopteran species, the apical cell is located in the testicular follicle, which is similar to that of locusts and the hub in Drosophila. The origin and functions of the apical cell remain unknown in lepidopteran species. In this study, the behavior of the apical cell in Bombyx mori from the embryonic to adult stage was studied by immunofluorescence staining of microtubules (MTs). In the embryonic stage, apical cells in contact with the testicular membrane were distinguished from primordial germ cells by MT-rich cytoplasm. Four cell masses, including a single apical cell and primordial germ cells were distinguished before the testis was separated into four follicles. In the second instar, the apical cell grew larger, and the cytoplasm with fewer MTs appeared in the peripheral region. The apical cell was surrounded by the germinal stem cells, spermatogonia appeared outside the stem cells, and cysts occurred in multiplying spermatogonia. Thus, the germinal proliferation centers (GPCs) formed. During the stage from the third to the fourth instar, the apical cell moved inwards, and separated from the testicular epithelium. In the last instar, the apical cell, the GPCs as well, enlarged further. After the spinning stage, the MT-poor region of the apical cell diminished. Characteristics of GPCs remained unchanged during pupal stages. The apical cell persisted until adult emergence, but shrinkage of the nucleus appeared in surrounding germ cells. These findings indicate that the apical cell continues to function as niche for the germinal stem cells from the larval to the pupal stages.

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Wipfelkrankheit is a baculovirus-induced disease that causes caterpillars to migrate to the upper foliage of a plant, where they subsequently die. However, this enhanced locomotory activity (ELA) is not induced in Bombyx mori larvae infected with a mutant Bombyx mori nucleopolyhedrovirus (BmNPV) lacking a functional protein tyrosine phosphatase (ptp) gene. Previous studies have shown that BmNPV utilizes the PTP protein to establish adequate infection for ELA as a budded virus (BV)-associated structural protein rather than as an enzyme phosphatase. In this study, I investigated the importance of the stage when ptp is transcribed during virus-induced ELA. I generated a BmNPV mutant with a polyhedrin (polh) promoter-driven ptp gene in the polh locus, instead of with the endogenous ptp gene. This mutant induced typical ELA, which is similar to that observed upon infection with a control virus, indicating that the very late stage expression of the PTP protein is sufficient for ELA induction in BmNPV-infected larvae. The observation that this mutant recruited PTP protein to BVs strongly suggested PTP localization in BV to be crucial for ELA induction by BmNPV.

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