Comparative Transcriptome Analysis Revealed Genes Involved in Sexual and Polyploid Growth Dimorphisms in Loach (Misgurnus anguillicaudatus)

Sexual and polyploidy size dimorphisms are widespread phenomena in fish, but the molecular mechanisms remain unclear. Loach (Misgurnus anguillicaudatus) displays both sexual and polyploid growth dimorphism phenomena, and are therefore ideal models to study these two phenomena. In this study, RNA-seq was used for the first time to explore the differentially expressed genes (DEGs) between both sexes of diploid and tetraploid loaches in four tissues (brain, gonad, liver, and muscle). Results showed that 21,003, 17, and 1 DEGs were identified in gonad, liver, and muscle tissues, respectively, between females and males in both diploids and tetraploids. Regarding the ploidy levels, 4956, 1496, 2187, and 1726 DEGs were identified in the brain, gonad, liver, and muscle tissues, respectively, between tetraploids and diploids of the same sex. When both sexual and polyploid size dimorphisms were considered simultaneously in the four tissues, only 424 DEGs were found in the gonads, indicating that these gonadal DEGs may play an important regulatory role in regulating sexual and polyploid size dimorphisms. Regardless of the sex or ploidy comparison, the significant DEGs involved in glycolysis/gluconeogenesis and oxidative phosphorylation pathways were upregulated in faster-growing individuals, while steroid hormone biosynthesis-related genes and fatty acid degradation and elongation-related genes were downregulated. This suggests that fast-growing loaches (tetraploids, females) have higher energy metabolism levels and lower steroid hormone synthesis and fatty acid degradation abilities than slow-growing loaches (diploids, males). Our findings provide an archive for future systematic research on fish sexual and polyploid dimorphisms.

Isolation, identification and molecular characterization of Aeromonas spp. from farmed loach (Misgurnus anguillicaudatus)

Gram-negative Aeromonas bacteria is known to contaminate aquaculture products and retains the ability to infect wide range of host including fishes, shrimps and humans. This study is designed to evaluate the presence of Aeromonas in healthy loaches (Misgurnus anguillicaudatus) collected from the markets in Hainan and Guangdong provinces of China. Based on the molecular sequencing and phylogenetic analysis with single 16S rRNA and concatenated genes (gyrA and ropD), 104 isolates were identified as Aeromonas species followed by further classification to A. veronii (83.65%), A. jandaei (9.62%), A. hydrophila (3.85%) and A. allosaccharophila(2.88%). More than half of the isolates displayed hemolytic activity of 87.5% and were able to form moderate biofilm (78.26%). Fourteen antibiotics from ten representative classes were screened that demonstrated complete resistance to ampicillin, 89.4% and 68.3% to lincomycin and nalidixic acid. Moreover, a notable detection and prevalence was found in screening of ten virulence-related and nine resistance genes. To our knowledge, it is the first report of its kind demonstrating potential threat of the commercial loaches carrying Aeromonas to human health. These findings will assist professionals working in clinical settings to optimize their prescription accordingly and researchers to access the impacts of Aeromonas resistance on human health.

Genome Survey of Misgurnus Anguillicaudatus to Identify Genomic Information, Simple Sequence Repeat (SSR) Markers and Mitochondrial Genome

The dojo loach Misgurnus anguillicaudatus is an important economic species in Asia because of its nutritional value and broad environmental adaptability. Despite its economic importance, genomic data from M. anguillicaudatus was unavailable. In the present study, we conducted a genome survey of M. anguillicaudatus using next-generation sequencing technology. Its genome size was estimated to be 1105.97 Mb by using K-mer analysis, and its heterozygosity ratio, repeat sequence content, GC content were 1.45%, 58.98%, and 38.03%, respectively. A total of 376,357 microsatellite motifs were identified and mononucleotides, with a frequency of 42.57%, were the most frequently repeated motifs, followed by 40.83% dinucleotide, 7.49% trinucleotide, 8.09% tetranucleotide, and 0.91% pentanucleotide motifs. The AC/GT, AAT/ATT, and ACAG/CTGT repeats were the most abundant motifs among dinucleotide, trinucleotide, and tetranucleotide motifs, respectively. Besides, a complete mitochondrial genome was sequenced. Based on the maximum likelihood and Bayesian inference analyses, M. anguillicaudatus in this study was the “introgressed” mitochondrial type. Furthermore, a total of 376,357 SSR motifs were detected from the genome survey assembly. Seventy microsatellite loci were randomly selected from these SSR loci to test polymorphic, of which, twenty microsatellite loci were assessed in 30 individuals from a wild population. The number of alleles (Na), observed heterozygosity (Ho), and expected heterozygosity (He) per locus ranged from 7 to 19, 0.400 to 0.933, and 0.752 to 0.938, respectively. All twenty loci were highly informative (PIC > 0.700). Eight loci deviated from Hardy–Weinberg equilibrium after Bonferroni correction (P < 0.05). This is the first report of a genome survey in M. anguillicaudatus, and genome information, mitochondrial genome, and microsatellite markers will be valuable for further studies on population genetic analysis, natural resource conservation, and molecular marker-assisted selective breeding.

Cloning and Expressions of Chop in Loach (Misgurnus Anguillicaudatus) and Its Response to Hydrogen Peroxide (H2O2) Stress

C/EBP [CCAAT/enhancer-binding protein]-homologous protein gene (chop) which plays an important role in endoplasmic reticulum stress-induced apoptosis, was investigated here by RACE and qPCR in an aquaculture animal for the first time. The full-length cDNA sequences of loach (Misgurnus anguillicaudatus) chop were 2533bp, encoding 266 amino acids. The expression levels of loach chop changed obviously during different early life stages, and the highest appeared at the 8-cell stage. Among different tissues, loach chop predominantly expressed in gill, spleen and gonad. Then, we performed a hydrogen peroxide (H2O2, a common-used disinfectant) stress trial to explore the role of loach chop, with three different concentrations (0 μM, 50 μM and 100 μM) of H2O2. Half of the loaches from the 100 μM group were dead, while all loaches from the other two treatment groups were alive. The activities of CAT, SOD and GPX in loach gill, liver and spleen decreased with extended stress time and increased H2O2 concentration. The expression levels of gill chop in loaches from the 100 μM group were significantly higher than those from the other two treatment groups between 12 and 24 hours of exposure. atf4 and bax, two proapoptotic genes, were significantly up-regulated in gills of loaches from the 100 μM group compared to the other two groups 18 hours and 24 hours after treatment, while bcl2, an antiapoptotic gene, presented an opposite trend. These results indicated a close relationship between H2O2 stress and fish apoptosis, and loach chop played an important role in H2O2 stress response.