Next-generation sequencing was used to analyze the effects of toxic microcystin-LR (MC-LR) on silver carp (Hypophthalmichthys molitrix). Silver carps were intraperitoneally injected with MC-LR, and RNA-seq and miRNA-seq in the liver were analyzed at 0.25, 0.5, and 1 h. The expression of glutathione S-transferase (GST), which acts as a marker gene for MC-LR, was tested to determine the earliest time point at which GST transcription was initiated in the liver tissues of the MC-LR-treated silver carps. Hepatic RNA-seq/miRNA-seq analysis and data integration analysis were conducted with reference to the identified time point. Quantitative PCR (qPCR) was performed to detect the expression of the following genes at the three time points: heme oxygenase 1 (HO-1), interleukin-10 receptor 1 (IL-10R1), apolipoprotein A-I (apoA-I), and heme binding protein 2 (HBP2). Results showed that the liver GST expression was remarkably decreased at 0.25 h (P < 0.05). RNA-seq at this time point revealed that the liver tissue contained 97,505 unigenes, including 184 significantly different unigenes and 75 unknown genes. Gene Ontology (GO) term enrichment analysis suggested that 35 of the 145 enriched GO terms were significantly enriched and mainly related to the immune system regulation network. KEGG pathway enrichment analysis showed that 18 of the 189 pathways were significantly enriched, and the most significant was a ribosome pathway containing 77 differentially expressed genes. miRNA-seq analysis indicated that the longest miRNA had 22 nucleotides (nt), followed by 21 and 23 nt. A total of 286 known miRNAs, 332 known miRNA precursor sequences, and 438 new miRNAs were predicted. A total of 1,048,575 mRNA–miRNA interaction sites were obtained, and 21,252 and 21,241 target genes were respectively predicted in known and new miRNAs. qPCR revealed that HO-1, IL-10R1, apoA-I, and HBP2 were significantly differentially expressed and might play important roles in the toxicity and liver detoxification of MC-LR in fish. These results were consistent with those of high-throughput sequencing, thereby verifying the accuracy of our sequencing data. RNA-seq and miRNA-seq analyses of silver carp liver injected with MC-LR provided valuable and new insights into the toxic effects of MC-LR and the antitoxic mechanisms of MC-LR in fish.The RNA/miRNA data are available from the NCBI database Registration No. : SRP075165.

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We performed annealing control primer (ACP)-based differential-display reverse transcription-polymerase chain reaction (DDRT-PCR) to isolate differentially expressed genes (DEGs) from the stage IV ovary and ovotestis of the rice field eel, Monopterus albus. Using 20 arbitrary ACP primers, 14 DEG expressed-sequence tags were identified and sequenced. The transcriptional expression of one DEG, G2, was significantly greater in the ovotestis than the stage IV ovary. To understand the role of G2 in sex inversion, G2 cDNA was cloned and semi-RT-PCR, real time PCR were performed during gonad development. The full-length G2 cDNA was 650 base pairs (bp) and it comprised a 5′-untranslated region (UTR) of 82 bp, a 3′-UTR of 121 bp and an open reading frame of 444 bp that encoded a 148-amino acid protein. The expression of G2 was weak during early ovarian development until the stage IV ovary, but expression increased significantly with gonad development. We speculate that G2 may play an important function during sex inversion and testis development in the rice field eel, but the full details of the function of this gene requires further research.

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High-throughput sequencing was applied to analyze the effects of toxic Microcystis aeruginosa on the silver carp Hypophthalmichthys molitrix. Silver carps were exposed to two cyanobacteria species (toxic and non-toxic) for RNA-seq and miRNA-seq analysis. RNA-seq revealed that the liver tissue contained 105,379 unigenes. Of these genes, 143 were significantly differentiated, 82 were markedly up-regulated, and 61 were remarkably down-regulated. GO term enrichment analysis indicated that 35 of the 154 enriched GO terms were significantly enriched. KEGG pathway enrichment analysis demonstrated that 17 of the 118 enriched KEGG pathways were significantly enriched. A considerable number of disease/immune-associated GO terms and significantly enriched KEGG pathways were also observed. The sequence length determined by miRNA-seq was mainly distributed in 20–23 bp and composed of 882,620 unique small RNAs, and 53% of these RNAs were annotated to miRNAs. As confirmed, 272 known miRNAs were differentially expressed, 453 novel miRNAs were predicted, 112 miRNAs were well matched with 7,623 target genes, and 203 novel miRNAs were matched with 15,453 target genes. qPCR also indicated that Steap4, Cyp7a1, CABZ01088134.1, and PPP1R3G were significantly differentially expressed and might play major roles in the toxic, detoxifying, and antitoxic mechanisms of microcystin in fish.