Molecular Identification of Nocardia seriolae and Comparative Analysis of Spleen Transcriptomes of Hybrid Snakehead (Channa maculata Female × Channa argus Male) With Nocardiosis Disease

Abstract

Hybrid snakehead (Channa maculata female × Channa argus male) is a new freshwater aquaculture fish species in southern China. During intensive aquaculture, hybrid snakeheads are often infected by Nocardia seriolae. In this study, hybrid snakehead infected suspiciously by N. seriolae in an artificial breeding pond were examined. Diseased hybrid snakeheads swam slowly without food intake, and the clinical symptoms included skin wound, anal swelling and ascites, and white granulomatous in liver, spleen, and kidney of fish. Through bacterial isolation, 16S rDNA sequencing, fluorescence in situ hybridization (FISH) and artificial infection experiment, the pathogen was identified as N. seriolae. Furthermore, the spleen samples from diseased and healthy male hybrid snakeheads in the same pond were used for RNA-Seq analysis. A total of 3,512 unique transcripts (unigenes) were identified as differentially expressed genes (DEGs), and 1,886 of them were up-regulated in diseased fish. The expression patterns of 20 DEGs were verified by quantitative polymerase chain reaction (qPCR). Several immune-related pathways and many immune-related genes were identified. qPCR results showed that the expression patterns of immune-related genes in the liver and kidney of diseased fish were comparable to that in the spleen. This study provides deep-sequencing data of hybrid snakehead spleen and will help understand the immune response of hybrid snakehead to N. seriolae. It is also helpful for the biomarker screening of fish-borne Nocardia spp. and the breeding of nocardiosis-resistant fish species.

Keywords: Nocardia seriolae; RNA-seq; fish; hybrid snakehead; immune response.

Figure 1

(A) Clinical signs of diseased hybrid snakehead body surface. Appearance of abdominal cavity (B), liver (C), spleen (D) and kidney (E). Histopathological sections of hybrid snakehead were shown. (F–H) correspond to the liver, spleen, and kidneys of the diseased fish; (I–K) correspond to the liver, spleen, and kidneys of the healthy fish. The white arrows indicate superficial lesions, black arrows indicate granules of N. seriolae, and yellow arrows indicate the necrosis and granuloma.

Figure 2

Phylogenetic analysis of 16S rDNA from the isolated bacteria, N. seriolae and other Nocardia sp. The phylogenetic tree was constructed using MEGA 6.0 with the Mmaximum likelihood method. Numbers of each node indicate the percentage of bootstrapping in 100 replications.

Figure 3

FISH of hybrid snakehead. The probe had strong hybridization signals in the liver (A), spleen (B) and kidney (C) of diseased fish, but weak hybridization signals in the liver (D), spleen (E) and kidney (F) of healthy fish. Scale bar = 50 mm.

Figure 4

Function annotation of unigenes based on BLAST against seven databases. (A) Numbers of unigenes annotated in seven databases. The X-axis represents different databases. The Y-axis corresponds to the number of unigenes.; (B) Venn diagram of unigenes annotated in seven databases; (C) Venn diagram of DEGs annotated in seven databases; (D) Volcano plot of DEGs comparing the diseased fish and healthy fish. The X-axis represents fold change between diseased and healthy groups. The Y-axis indicates the statistical significance of the difference of gene expression. Each dot in the diagram represents a gene, and the black dots represent no significant changes in expressed genes, while the red dots and the green dots represent up- and down- regulated in diseased fish, respectively. (E) Heat map of DEGs comparing the diseased fish and healthy fish. H1, H2 and H3 represent three samples of healthy individuals, and the D1, D2 and D3 represent three samples of diseased individuals. Each row represents a gene. Gene expression levels are shown with different colors. Negative and positive numbers indicate down- and up-regulation, respectively.

Figure 5

Bar graph description of Gene Ontology (GO) enrichment of DEGs. The X-axis corresponds to the number of DEGs. The Y-axis represents various GO terms. The healthy group was used as the control group, and the green bar and red bar represent down- and up-regulated DEGs, respectively.

Figure 6

Scatter diagram of the top 20 enriched KEGG pathways for up-regulated (A) and down-regulated (B) DEGs in diseased spleen sample. The X-axis corresponds to the rich factor of a pathway, and the rich factor is the ratio of DEGs to all the genes in a specific pathway. The Y-axis represents different pathways. The magnitude of the dots displays gene number from 10 to 40 (A) or 10 to 20 (B), and the q-value is described by the color classification.

Figure 7

Comparison of gene expression data between RNA-Seq and qPCR. (A) The relative expression levels of twenty DEGs detected by qPCR were compared with that of RNA-Seq. The X-axis represents the value of RNA‐Seq data and qPCR data, and the Y-axis represents the gene name. RNA-Seq data used log₂fpkm values of diseased and healthy fish samples, and qPCR data are presented as mean ± standard deviation (n=3). The p value of all genes in qPCR was less than 0.01. (B) Correlation of qPCR and RNA-Seq data. The X-axis represents the RNA-Seq data, and the Y-axis represents the qPCR data. The Pearson product-moment correlation coefficient (R²) of each plot was shown as an indication of positive linear correlation between the two methods. (C) The expression patterns of fifteen selected DEGs in the spleen, liver, and kidney. qPCR data are presented as mean ± SD (n=3). The X-axis represents the gene name, and the Y-axis represents the value of qPCR data. Statistically significant differences in gene expression levels between healthy and diseased fish in each tissue type are marked with asterisks (* means p < 0.05, ** means p < 0.01, *** means p < 0.001). The raw data (Ct values) of qPCR is in Supplementary Table 6 .