In this paper, we report on the specific spore germination conditions of the microsporidia Trachipleistophora haruka (Old name Trachipleistophora sp. OSL-2012-10 was reported by Shigano et al. (2015)) isolated from the common cutworm, Spodoptera litura from the Ogasawara Islands, Japan. We analyzed germination rates in varying salt conditions and found that alkaline stress was required for spore germination. Nosema bombycis NIS-001 of the experiment control germinated regardless of temperature only by treatment of 0.1 M KOH or 0.1 M KCl+3% (w/v) H2O2. However, Trachipleistophora haruka treatment by 0.85% NaCl solution at 25°C showed germination behavior similar to treatment of 0.1 M KOH and 0.1 M KCl+3% (w/v) H2O2. The results indicated that this microsporidia requires sodium or potassium ions to germinate.
The Z chromosome-linked sex-linked translucent (os) mutation in the silkworm Bombyx mori causes a slightly translucent larval integument that results from the reduction of urate granules in epidermal cells. We previously concluded that mutations in an amino acid transporter gene belonging to the solute carrier family are responsible for the os mutant phenotype by positional cloning and sequence analysis of independent os mutant strains. However, direct evidence using functional analysis was missing. Therefore, we performed somatic mosaic analysis of the os candidate gene using the CRISPR/Cas9 system. An in vitro synthesized single guide RNA (sgRNA) and Cas9 protein were mixed and injected into eggs of the p50T strain. We successfully obtained mosaic larvae in which the epidermis comprised both white and slightly translucent areas. Various mutations had been induced at the target sites of the os gene in the genomic DNA extracted from G0 adult legs, indicating that the amino acid transporter gene is required for the sufficient accumulation of uric acid in epidermal cells granules. In addition, we searched OS homologous proteins in 24 genome-sequenced lepidopteran and 12 other insects. Phylogenetic analysis showed that the OS protein is conserved in all lepidopteran insects. On the other hand, OS proteins were not found in some insect species, such as Apis mellifera, Drosophila melanogaster, and Tribolium castaneum. It is considered that os genes play an important role in lepidopteran insects for their survival in nature.
Long-term exposure of human sperm cells to reactive oxygen species (ROS) can cause decreased motility and viability, as well as DNA fragmentation. An antioxidant defense system, called a preventive antioxidant system, is therefore needed to maintain low ROS concentrations in sperm and seminal plasma. Superoxide dismutase (SOD, EC 1.15.1. 1), one of the most important antioxidant enzymes, catalyzes the conversion of superoxide radical (O2•−) to hydrogen peroxide (H2O2) and molecular oxygen (O2). In this report, we documented a high SOD activity level in the reproductive organs of the male silkmoth (Bombyx mori, L.), particularly in the glandula lacteola, and found that about 90% of the SOD activity was transferred to females by ejaculation and maintained. We characterized three cDNAs from the adult male reproductive system: a soluble cytoplasmic copper/zinc SOD (Cu/Zn SOD, SOD1) and two extracellular forms of copper/zinc SOD (EC-SOD, SOD3). The levels of transcription and protein accumulation of Bombyx SOD1 indicated that it is abundantly present in the extracellular fluid of the male glandula lacteola, which transfers to the female during ejaculation. Furthermore, it was observed that some of the transferred SOD1 exists in the sperm fraction stored in the mating female’s bursa copulatrix. Our present results demonstrate the origins of seminal SOD activities and suggest that SOD might be a potential source and function of antioxidants in semen and also may control the amount of extracellular ROS involved in sperm quality maintenance.