RNA-seq analysis reveals significant transcriptome changes in turbot (Scophthalmus maximus) suffering severe enteromyxosis

Abstract

Background: Enteromyxosis caused by the intestinal myxozoan parasite Enteromyxum scophthalmi is a serious threat for turbot (Scophthalmus maximus, L.) aquaculture, causing severe catarrhal enteritis leading to a cachectic syndrome, with no therapeutic options available. There are still many aspects of host-parasite interaction and disease pathogenesis that are yet to be elucidated, and to date, no analysis of the transcriptomic changes induced by E. scophthalmi in turbot organs has been conducted. In this study, RNA-seq technology was applied to head kidney, spleen and pyloric caeca of severely infected turbot with the aim of furthering our understanding of the pathogenetic mechanisms and turbot immune response against enteromyxosis.

Results: A huge amount of information was generated with more than 23,000 identified genes in the three organs, amongst which 4,762 were differently expressed (DE) between infected and control fish. Associate gene functions were studied based on gene ontology terms and available literature, and the most interesting DE genes were classified into five categories: 1) immune and defence response; 2) apoptosis and cell proliferation; 3) iron metabolism and erythropoiesis; 4) cytoskeleton and extracellular matrix and 5) metabolism and digestive function. The analysis of down-regulated genes of the first category revealed evidences of a connexion failure between innate and adaptive immune response, especially represented by a high number of DE interferon-related genes in the three organs. Furthermore, we found an intense activation of local immune response at intestinal level that appeared exacerbated, whereas in kidney and spleen genes involved in adaptive immune response were mainly down-regulated. The apoptotic machinery was only clearly activated in pyloric caeca, while kidney and spleen showed a marked depression of genes related to erythropoiesis, probably related to disorders in iron homeostasis. The genetic signature of the causes and consequences of cachexia was also demonstrated by the down-regulation of the genes encoding structural proteins and those involved in the digestive metabolism.

Conclusions: This transcriptomic study has enabled us to gain a better understanding of the pathogenesis of enteromyxosis and identify a large number of DE target genes that bring us closer to the development of strategies designed to effectively combat this pathogen.

Figure 1

Samples hierarchical clustering by organ. Hierarchical clustering of all diseased and control samples for A Spleen, B Head kidney and C Pyloric caeca. Approximately unbiased P-values, computed by multi-scale bootstrap resampling, are displayed on branch nodes and clusters of samples with an approximately unbiased P-value > 0.95 are indicated with a dashed red box, indicating strong support.

Figure 2

DE genes Venn diagrams. Venn diagrams of A) all DE genes, B) up-regulated DE genes, C) down-regulated DE genes in the three organs (head kidney, spleen and pyloric caeca) are shown. The total number of DE expressed genes in each tissue and the number of unique and common genes between them is displayed.

Figure 3

Heatmap of 50 selected genes. Heatmap of the fifty selected genes presented in Table 2. Displayed are EdgeR [101] normalized counts for each sample and gene. Sample names are displayed at the bottom of the figure whilst gene symbols are shown to the right and have been hierarchically clustered according to their pearson correlation. The category assigned to each gene is also shown with a color code.

Figure 4

GO terms enrichment. GO enrichment (p < 0.01 FDR corrected) for DE genes in A) head kidney, B) spleen and C) pyloric caeca. The percentage of sequences with the associated GO term present in the full organ transcriptome is shown in blue, while the percentage of sequences with the GO term in the DE gene group is shown in red.

Figure 5

Enteromyxosis flowchart. Flowchart showing the main processes involved in severe turbot enteromyxosis. The flowchart has been divided in five sections according to the most representative processes occurring during Enteromyxum infection: I, blue, immune and defence response; II, purple, apoptosis and cell proliferation; III, red, iron metabolism and erythropoiesis; IV, green, metabolism and digestive function; V, orange, cytoskeleton and extracellular matrix.