Bombykol is the first sex pheromone that was chemically identified from silkmoth Bombyx mori. To date, the biosynthetic mechanism of bombykol in the pheromone gland (PG) of B. mori has been intensively investigated. The most prominent characteristic of pheromone-producing cells in B. mori is the abundance of cytoplasmic lipid droplets (LDs). During sex pheromone biosynthesis, LDs in the PG have been implicated to play important roles as lipid storage organs for the pheromone precursors. However, the presence of LDs in other moth species except for B. mori remains poorly described. Thus, we hypothesized that LD formation in the PG of B. mori may be caused by corpulence under domestication resulting from the loss of flight through hybridization for generating commercial lines with enlarged body size. To verify our hypothesis, we explored LDs in the PG of a closely related species, B. mandarina. A bright-field microscopic observation of B. mandarina PG revealed the presence of candidate LDs with a diameter of approximately 10μm. Fluorescent microscopy revealed that these candidate LDs could be stained with a Nile red. Subsequent observation under a confocal laser-scanning microscopy confirmed that these candidate LDs were localized in the cytoplasm of the sex pheromone-producing cells in the PG of B. mandarina, indicating that these candidates were LDs. Therefore, we concluded that LD formation is a conserved trait between B. mori and B. mandarina, and the formation of LDs in B. mori may be not caused by domestication.

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The development of genome editing techniques has allowed the generation of tremendous genetic diversity, even in non-model insects. The major obstacle for maintenance of genetically diverse stocks in non-model insects is the need for constant rearing in the laboratory, which is labor-intensive and time-consuming. The maintenance of insect colonies in the laboratory is further complicated by risk factors such as disease contamination, human error, and genetic changes by natural mutations that can lead to the loss of desirable genotypes. To avoid these risk factors, cryopreservation is the most desirable option. Here, we present a method we developed for cryopreservation of ovary from the multicolored Asian ladybird beetle, Harmonia axyridis, adapted from techniques for ovary cyropresevation in the silkworm, Bombyx mori. We used last (4th) instar beetle larvae as both donors and recipients for the cryopreserved ovaries. The best and average success rates of cryopreservation were 40% and 26%, respectively, based on the percentage of beetles receiving cryopreserved ovary transplants that subsequently laid viable eggs. This success rate is much higher than that reported for B. mori using last instar larvae. The 26% average success rate is sufficient for the technique to be of practical use for maintaining genetically diverse lines of H. axyridis, and reduces the laborious tasks required for constant maintenance of numerous colonies. This is the first reported success in ovary cryopreservation for a small non-model insect. The techniques that we developed should also be useful for ovary cryopreservation in other small non-model insects.

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Plutella xylostella is the most destructive pest in cruciferous crops and Bacillus thuringiensis (Bt) has been widely used as a control measure. However, sequential application of Bt insecticides resulted in development of Cry1Ac-resistant P. xylostella. In order to use Bt effectively and continuously, it is urgent to address the mechanism of how P. xylostella has acquired resistance against Cry1Ac. A recent report suggested a mutation in the ATP-binding cassette (ABC) transporter gene (ABCC2) was related to Cry1Ac-resistance. However, there was no direct evidence of interaction between ABCC2 and resistance. In this study, we examined the receptor function of PxABCC2 against Cry1Ac toxin to understand the interaction resulting in resistance. We overexpressed PxABCC2 ectopically on Sf9 cells using a baculovirus (AcMNPVs) expression system, and assessed Cry1Ac susceptibility. 10nM of Cry1Ac had an effect on Sf9 cells that expressed PxABCC2 and swelled 53.84% of the effected cells. We also established S9 cells expressed PxABCC2 with a mutation and treated with Cry1Ac, resulted in no swelling cells, and no susceptibilities were observed. These results suggest PxABCC2 functions as a receptor and the mutation of PxABCC2 caused a loss of receptor function, which is responsible for Cry1Ac resistance. Furthermore, we visualized Cry1Ac and PxABCC2 using double immunostaining methods to observe the interaction between them. When Cry1Ac was treated with Sf9, cells expressed PxABCC2, Cry1Ac signals were scattered to surround PxABCC2 signals. Cry1Ac bound to PxABCC2 on the cell membrane. Conversely, no Cry1Ac signals were observed around mutated PxABCC2 signals, as no susceptibility against Cry1Ac was observed. These results indicate that PxABCC2 would lose its Cry1Ac binding ability when a mutation occurs, and this results in the loss of receptor function. This study demonstrates a mutation in PxABCC2 which involves Cry1Ac resistance and the loss of bindings between cry toxins and receptors might be mode of action to acquire resistance.

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Virus-like particles (VLPs) are multi-protein complex, which mimic viral particles without viral genomes. Recently various applications of VLPs for medical and pharmaceutical fields are developed. Furthermore, surface modification of VLPs is expected to extend their functional versatility. As an enzymatic strategy for the modification, microbial transglutaminase (MTG) that catalyzes the covalent bond between a glutamine residue and a lysine residue or a primary amine is suitable for efficient protein ligation. In the previous study, we demonstrated the efficient production of immunogenic VLPs of porcine circovirus type 2 (PCV2) using silkworm-baculovirus expression vector system (silkworm-BEVS). Herein, we established the display system of exotic molecules to VLPs by enzymatic covalent cross-linking using MTG. For the target modification, Q-tag (YPLQMRG) that is MTG reactive sequence were introduced into the N-terminus, C-terminus, or loop BC of capsid protein of PCV2 and successfully expressed as VLPs in silkworm pupae. Of these, PCV2 VLPs with Q-tag at the loop BC and C-terminus were efficiently conjugated with FITC-cadaverine and K-tagged (MRHKGS) EGFP by MTG. These results showed that flexible surface modification of VLPs mediated by MTG could be used for development of several therapeutic tools such as multivalent vaccines.

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We constructed the first trichopteran bacterial artificial chromosome (BAC) library in Stenopsyche marmorata. The BAC library consists of 32,256 clones with an average insert size of 65.38kb. We also made a cDNA library from an S. marmorata adult male and determined 598 unique sequences. Using the obtained sequences we designed putative single tagged site (STS) primers and selected 81 BACs containing orthologs of Bombyx mori genes by three-step PCR selection method. These BACs will be used for gene-based comparative mapping by fluorescence in situ hybridization (FISH) to identify regions of conserved synteny between the trichopteran and lepidopteran genomes.

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