De Novo Transcriptome Analysis of Differential Functional Gene Expression in Largemouth Bass (Micropterus salmoides) after Challenge with Nocardia seriolae

Abstract

Largemouth bass (Micropterus salmoides) are common hosts of an epizootic bacterial infection by Nocardia seriolae. We conducted transcriptome profiling of M. salmoides to understand the host immune response to N. seriolae infection, using the Illumina sequencing platform. De novo assembly of paired-end reads yielded 47,881 unigenes, the total length, average length, N50, and GC content of which were 49,734,288, 1038, 1983 bp, and 45.94%, respectively. Annotation was performed by comparison against non-redundant protein sequence (NR), non-redundant nucleotide (NT), Swiss-Prot, Clusters of Orthologous Groups (COG), Kyoto Encyclopaedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Interpro databases, yielding 28,964 (NR: 60.49%), 36,686 (NT: 76.62%), 24,830 (Swissprot: 51.86%), 8913 (COG: 18.61%), 20,329 (KEGG: 42.46%), 835 (GO: 1.74%), and 22,194 (Interpro: 46.35%) unigenes. Additionally, 8913 unigenes were classified into 25 Clusters of Orthologous Groups (KOGs) categories, and 20,329 unigenes were assigned to 244 specific signalling pathways. RNA-Seq by Expectation Maximization (RSEM) and PossionDis were used to determine significantly differentially expressed genes (False Discovery Rate (FDR) < 0.05) and we found that 1384 were upregulated genes and 1542 were downregulated genes, and further confirmed their regulations using reverse transcription quantitative PCR (RT-qPCR). Altogether, these results provide information on immune mechanisms induced during bacterial infection in largemouth bass, which may facilitate the prevention of nocardiosis.

Keywords: Illumina paired-end sequencing; Nocardia seriolae; immune response; largemouth bass (Micropterus salmoides); transcriptome.

Figure 1

The cluster of orthologous groups (COG) classification. 8913 (23.63% of the total annotated putative proteins) were grouped into 25 different categories.

Figure 2

Functional distribution of GO annotation.

Figure 3

KEGG classification of assembled unigenes from control and treated groups. (A) Cellular processes; (B) Environmental information processing; (C) Genetic information processing; (D) Human diseases; (E) Metabolism; and (F) Organismal systems.

Figure 4

Scatterplot of the top 20 enriched KEGG pathways. Rich Factor is the ratio of differentially expressed gene numbers annotated in this pathway terms to all gene numbers annotated in this pathway term. q ≤ 0.05 as significantly enriched.

Figure 5

Comparative gene expression analysis from qPCR and RNA-Seq in spleen from the infected largemouth bass with N. seriolae and compared with those in the control at the 24 h time point. Expression of target genes was normalized to β-actin as a reference gene. Statistically significant differences from control are presented, with * p < 0.05.