We previously reported the microarray-based selection of three ovulation-related genes in zebrafish. We used a different selection method in this study, RNA sequencing analysis. An additional eight up-regulated candidates were found as specifically up-regulated genes in ovulation-induced samples. Changes in gene expression were confirmed by qPCR analysis. Furthermore, up-regulation prior to ovulation during natural spawning was verified in samples from natural pairing. Gene knock-out zebrafish strains of one of the candidates, the starmaker gene (stm), were established by CRISPR genome editing techniques. Unexpectedly, homozygous mutants were fertile and could spawn eggs. However, a high percentage of unfertilized eggs and abnormal embryos were produced from these homozygous females. The results suggest that the stm gene is necessary for fertilization. In this study, we selected additional ovulation-inducing candidate genes, and a novel function of the stm gene was investigated.

The current study attempted to evaluate the antagonistic activity of compounds isolated and purified from the marine algae Padina arborescens during cultivation. The compounds were collected on a filter, concentrated on ODS columns and separated by HPLC. Two peaks that showed competitive progesterone binding activity with membrane progesterone receptor α (mPRα) were purified. Their physiological activity was further uncovered by in vitro and in vivo oocyte maturation and ovulation-inducing assays using zebrafish. The compounds inhibited the induction of oocyte maturation and ovulation. Moreover, the results showed that the compounds have antagonistic activity against mPRα. The purified compounds with antagonistic activity against mPRα would be considered as new pharmaceutical candidate.

Using an in vivo assay, we selected 11 genes that were highly upregulated during the induction of ovulation in zebrafish using microarray analysis and RNA sequencing. The Starmaker gene (stm) was one of these genes. Although stm has been previously reported to be involved in otolith formation during the early development of zebrafish, we detected its expression in eggs and showed that stm was related to fertilization by establishing an stm gene knockout strain using the CRISPR/Cas9 system. Further phenotypic analysis of stm knockout fish was conducted in this study. With a higher nonfertilization rate, the stm mutant strain showed an extremely low survival rate. Otoliths of stm homozygous mutant zebrafish showed abnormal morphology in embryos and adult fish. However, fish did not show any abnormalities in swimming behaviour in either embryos or adults. Stm proteins were detected on the chorion of ovulated eggs before spawning. Fibre-supported knob-like structures on the fertilization envelope (FE) also showed abnormal structures in stm mutants. The Stm protein is necessary for otolith formation, and a lack of Stm causes abnormal otolith formation. The partial defect of otolith formation does not cause defects in swimming behaviour. The Stm protein is expressed in the chorion and is responsible for the formation of fibre-supported knob-like structures on the FE. It was suggested that a lack of Stm caused a lower fertilization rate due to inadequate formation of the FE.

Highlights Different fishes were collected and were subjected to form an in vitro biofilm. Huge array (up to 10 7 cfu/ml or g) of pathogenic bacteria. Few of the isolates were sensitive and few were resistant against the antibiotics but after bio-film formation all the species acquired resistance.

Foods are the major source of energy and nutrition for all the living organisms. Measuring food safety and security is a vital concern nowadays. The present study attempted to evaluate a complete microbiological profile of some popular food items collected from different restaurants along with their drug-resistant profile. The main focus of this study was to evaluate the efficacy of 60 seconds microwave oven heat on the growth of microorganisms present in the collected food items. Presence of bacterial and fungal microbiological profiling of the food items and their drug-resistant pattern were determined through conventional cultural, biochemical and Kirby Bauer methods. After that, the food was heated in the microwave oven for 60 seconds. All the food items (chicken sandwich, French pizza, chicken pie, pasta, hot dog) were found to be highly contaminated with total viable bacteria and fungus up to 107CFU/g while the pathogenic bacteria such as Escherichia coli, Staphylococcus spp., Pseudomonas spp. was estimated up to 106CFU/g. After 60-second microwave oven heat treatment, the existence microbial load was significantly reduced. Meanwhile, fecal coliform was only detected in chicken sandwich before the heat treatment (102CFU/g) but that was completely eliminated by heat treatment. Staphylococcus spp. and Pseudomonas spp. were found in untreated chicken pie, pasta and French pizza samples although after heat treatment the growth was 100% eliminated. Only in chicken sandwich, 2-log reduction of Staphylococcus spp. was notified after 60-second heat. Moreover, the identified bacteria were found to be 100% resistant against commonly used antibiotics but only streptomycin (10 µg), azithromycin (15 µg) and gentamycin (10 µg) were found as effective drugs against all the isolates. The presence of resistant strain in food is very alarming which indicates the poor management set up and lack of proper legislation in food sector. However, the 60-second microwave oven heat treatment was so effective to reduce the substantial amount of bacteria.