Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen of the domestic silkworm, Bombyx mori, whose infection drastically alters larval locomotory patterns and activity. As B. mori is the only insect that has been completely domesticated, its larvae are typically inactive except for the wandering stage. In contrast, BmNPV-infected larvae exhibit vigorous movement. Recently, we developed a method for locomotory analysis of BmNPV-infected B. mori larvae, which yielded high-resolution data on BmNPV-induced host locomotory alteration. However, this method had a disadvantage when analyzing the behavior of uninfected larvae, as they were typically inactive and difficult to distinguish from the background noises. In addition, the analysis processes involve many manual steps, which reduces analysis throughput. We have developed KaicoTracker, a new tool for automated locomotory analysis of BmNPV-infected B. mori larvae, using Python and OpenCV. This tool is an all-inclusive package that tracks and quantifies larval locomotion and verifies the accuracy of the tracking. Larval locomotion is tracked by motion tracking function based on k-Nearest Neighbor implemented in OpenCV. Based on the tracking results, the positions of the larvae are determined, and locomotory distance, speed, duration, and frequency are calculated. The larval positions are marked on the original video, allowing tracking precision to be examined. By analyzing previously obtained data, we confirmed that this tool provides a robust analysis of inactive larvae with fewer manual steps.

 The effect of corona discharge on the silkworm’s egg diapause, Bombyx mori, has been studied for more than 150 years ago and showed that it can effectively prevent embryonic diapause. In this study, we evaluated the applicability of the corona discharge to the transgenesis of the diapause silkworm strains. We developed our corona discharge equipment to optimize the treatment parameters for the transgenesis of the diapause strain. In the first experiment, we treated the diapause eggs with a corona discharge 2 h after oviposition, then microinjected the plasmid DNAs to produce transgenic silkworms and reared the hatched silkworms. In G1 embryos, when we screened the transgenic silkworms, we discovered that they were present in one-fourth of the broods. In the second experiment, we injected the genetic materials into the eggs <6 h after oviposition, incubated them for one night at 25°C in the second experiment, and then treated them with corona discharge. Consequently, the hatched larvae were reared, G1 eggs were obtained, and the G1 embryo was screened. In both experiments, we obtained numerous transgenic silkworms. Our results depict that both diapause eggs, when treated with corona discharge before and after the injection, could be convenient and effective in producing transgenic silkworms. The first experiment results concurred with the report by Zhang et al. (2022). Moreover, we included new findings in the second experiment depicting that the corona discharge after the microinjection benefits transgenesis.