Ge (giant egg) is a sex-linked mutation in Bombyx mori which leads to the production of large-sized eggs caused by an increase in the number of follicular epithelial cells, each with decreased cell size. We previously determined that a gene orthologous to human Phytanoyl-CoA dioxygenase domain-containing 1 (PHYHD1) is specifically disrupted in Ge and Ge² mutants through positional cloning of the Ge locus. However, little is known about the function of the BmPHYHD1 gene in determination of the egg size. In this study, we knocked out BmPHYHD1 using CRISPR/Cas9. We employed a novel knockout protocol developed in other lepidopteran species in which a commercialized Cas9 mixed with two sgRNAs designed for a target gene is injected into newly-laid eggs. First, we confirmed that the protocol was applicable to B. mori with high efficiency. Second, we targeted the BmPHYHD1 gene and obtained G0 moths laid large size eggs. CRISPR/Cas9-mediated mutations were confirmed in G1 moths that laid large-sized eggs, demonstrating that the BmPHYHD1 gene controls egg size in B. mori. Finally, the dominant/recessive relationship between Ge and +^Ge is discussed and we propose the use of gi (giant egg) and gi² (giant egg 2) to represent giant egg mutations, Ge and Ge², respectively. We believe that Ge mutants are an appropriate model for the study of genetic mechanisms mediating the size and number of cells in animals.

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Bacillus thuringiensis has been available and utilized as a microbial pesticide. Currently, B. thuringiensis, which is used in microbial pesticide material, were often isolated strains in the natural environment. However, the rate of B. thuringiensis (BT index) was very low. If there is a more efficient isolation method than the conventional methods, it is possible to dramatically improve the possibility of discovering new B. thuringiensis strains. In this study, we were utilized two methods. One comparison of Small Sub-Unit rRNA (SSU rRNA) gene sequence, and the other is the Genome Profiling (GP) method that used the B. thuringiensis isolates strains by Yamamoto et al. (2014). A, comparison of each result was conducted. As a result, a SSU rRNA gene analysis was not found that detailed any differences. In addition, using the GP method confirmed that there was a difference in the genome and it revealed that the same strain is not present. For this reason, our results suggest that all isolates were different strains.

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The effectiveness of cryopreservation by vitrification has been reported in many organisms. However, successful silkworm preservation by this method has not been reported. To achieve this, we applied the V-cryoplate method for the cryopreservation of Bombyx mori embryos, a method originally developed in the field of plant cryopreservation. We performed surgery to remove the egg shells and attached the embryos that were enclosed within the serosal membranes to an aluminum cryoplate. Solution exchanges were applied by transferring the cryoplates. Unfortunately, many eggs were crushed and no embryos hatched after cryopreservation. Therefore, we modified the V-cryoplate protocol by mounting paper to the cryoplates with dechorionated eggs, and then removed the mounting paper from the cryoplate before immersion in liquid nitrogen. This modification avoided damage to the serosal membranes and made hatching possible. However, the larvae that hatched from this vitrification cryopreservation died without eating mulberry leaves (the diet of silkworm larvae). These results show that even though embryogenesis mostly progresses normally, some damage occurs. We could improve the method for vitrification for silkworm. However, there are still problems in achieving normal larval development.

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Recent technical advances on assembling DNA fragments has enabled generation of large, circular DNA of which length reached several hundred-kilobases. Adaptation of such technologies to baculoviral genomics would allow for flexible manipulation of baculoviral genomes and thus generation of designed viral genomes for both basic and applied biology. Here we report that a BmNPV genome-size DNA could be assembled from a set of PCR-amplified DNA fragments and was infectious to Bombyx mori ovary-derived BmN cell line. The DNA fragments were designed (1) to be excised mainly at intergenic regions and (2) with no homologous sequences on the terminals among the fragments for assembly purpose, and (3) with their lengths and Tm values of priming sequences adjusted for simultaneous amplification. Although these fragments were initially designed for assembling with Gibson assembly and in yeast in the first and subsequent steps, respectively, the bacmid propagated in yeast during a feasibility test showed a deletion during in-yeast assembly and/or propagation. Therefore, we assembled the fragments using only Gibson assembly throughout the assembling steps. An assembled clone was chosen and confirmed to carry the inverted vector sequence, a watermark to distinguish the assembled clone from the parental clone, and to be infectious to BmN upon transfection. The design and assembly strategy of DNA fragments and both amplified and assembled fragments become the foundation for further development of BmNPV-based, designed genomes.

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