Transcriptome and expression profiling analysis of the hemocytes reveals a large number of immune-related genes in mud crab Scylla paramamosain during Vibrio parahaemolyticus infection

Abstract

Background: Mud crab Scylla paramamosain is an economically important marine species in China. However, frequent outbreaks of infectious diseases caused by marine bacteria, such as Vibrio parahaemolyticus, result in great economic losses.

Methodology/principal findings: Comparative de novo transcriptome analysis of S. paramamosain infected with V. parahaemolyticus was carried out to investigate the molecular mechanisms underlying the immune response to pathogenic bacteria by using the Illumina paired-end sequencing platform. A total of 52,934,042 clean reads from the hemocytes of V. parahaemolyticus-infected mud crabs and controls were obtained and assembled into 186,193 contigs. 59,120 unigenes were identified from 81,709 consensus sequences of mud crabs and 48,934 unigenes were matched proteins in the Nr or Swissprot databases. Among these, 10,566 unigenes belong to 3 categories of Gene Ontology, 25,349 to 30 categories of KEGG, and 15,191 to 25 categories of COG database, covering almost all functional categories. By using the Solexa/Illumina's DGE platform, 1213 differentially expressed genes (P<0.05), including 538 significantly up-regulated and 675 down-regulated, were detected in V. parahaemolyticus-infected crabs as compared to that in the controls. Transcript levels of randomly-chosen genes were further measured by quantitative real-time PCR to confirm the expression profiles. Many differentially expressed genes are involved in various immune processes, including stimulation of the Toll pathway, Immune Deficiency (IMD) pathway, Ras-regulated endocytosis, and proPO-activating system.

Conclusions/significance: Analysis of the expression profile of crabs under infection provides invaluable new data for biological research in S. paramamosain, such as the identification of novel genes in the hemocytes during V. parahaemolyticus infection. These results will facilitate our comprehensive understanding of the mechanisms involved in the immune response to bacterial infection and will be helpful for diseases prevention in crab aquaculture.

Figure 1. Length statistic of all consensus sequences obtained from the mud crab S.paramamosain transcriptome library.

Figure 2. GO annotation of the unigenes in the V.parahaemolyticus infected crabs transcriptome.

Most unigenes can be divided into three major categories, including biological process, cellular component, and molecular function.

Figure 3. Histogram presentation of clusters of orthologous groups (COG) classification in S.paramamosain.

All putative proteins were aligned to the COG database and could be classified into at least 25 molecular families.

Figure 4. The differentially expressed genes upon V.parahaemolyticus infection.

By using Solexa/Illumina's DGE platform, 1213 differentially expressed genes were detected in comparative analysis of the expression profiles between V.parahaemolyticus-infected crabs and control crabs, including 538 up-regulated genes and 675 down-regulated genes (P<0.05).

Figure 5. Comparison of the expression profiles of randomly selected genes as determinated by Illuminia sequencing and qRT-PCR analysis.

Target gene abbreviations are as follows: Cd42, cell division cycle protein 42; PpC, phospholipid phospholipase C; Chy, chymotrypsin; Fam, farnesoic acid O-methyltransferase; ALF2, antilipopolysaccharide factor 2; Ras, Ras GTPase-activating protein; SS, son of sevenless; Pero, peroxinectin