Resistance to white spot syndrome virus in the European shore crab is associated with suppressed virion trafficking and heightened immune responses

Abstract

Introduction: All decapod crustaceans are considered potentially susceptible to White Spot Syndrome Virus (WSSV) infection, but the degree of White Spot Disease (WSD) susceptibility varies widely between species. The European shore crab Carcinus maenas can be infected with the virus for long periods of time without signs of disease. Given the high mortality rate of susceptible species, the differential susceptibility of these resistant hosts offers an opportunity to investigate mechanisms of disease resistance.

Methods: Here, the temporal transcriptional responses (mRNA and miRNA) of C. maenas following WSSV injection were analysed and compared to a previously published dataset for the highly WSSV susceptible Penaeus vannamei to identify key genes, processes and pathways contributing to increased WSD resistance.

Results: We show that, in contrast to P. vannamei, the transcriptional response during the first 2 days following WSSV injection in C. maenas is limited. During the later time points (7 days onwards), two groups of crabs were identified, a recalcitrant group where no replication of the virus occurred, and a group where significant viral replication occurred, with the transcriptional profiles of the latter group resembling those of WSSV-susceptible species. We identify key differences in the molecular responses of these groups to WSSV injection.

Discussion: We propose that increased WSD resistance in C. maenas may result from impaired WSSV endocytosis due to the inhibition of internal vesicle budding by dynamin-1, and a delay in movement to the nucleus caused by the downregulation of cytoskeletal transcripts required for WSSV cytoskeleton docking, during early stages of the infection. This response allows resistant hosts greater time to fine-tune immune responses associated with miRNA expression, apoptosis and the melanisation cascade to defend against, and clear, invading WSSV. These findings suggest that the initial stages of infection are key to resistance to WSSV in the crab and highlight possible pathways that could be targeted in farmed crustacean to enhance resistance to WSD.

Keywords: RNA-Seq; apoptosis; differential susceptibility; endocytosis; miRNAs; transcriptional response; white spot disease (WSD); whiteleg shrimp.

Figure 1

Schematic of the WSSV exposure and sampling procedure for both resistant and susceptible crustaceans. The upper panel outlines the experimental WSSV injection trial performed to generate RNA-seq and miRNA-seq datasets for resistant crab (C. maenas). Crabs were randomly allocated into two groups and housed in individual compartments in tanks. These groups were injected with either saline solution or WSSV-infected shrimp homogenate for the disease trial and the gills of 8 crabs were sampled from saline and WSSV-injected treatment groups at 0, 6, 12, 24, 48, 168, 336 and 672 hours and snap frozen for transcriptome sequencing. The lower panel illustrates the equivalent time course experiment in the susceptible shrimp (P. vannamei) reported in a previously published WSSV-exposure dataset utilised for comparison (8).

Figure 2

Transcriptome analysis. (A) Boxplot of the percentage of processed sequencing reads aligning to the WSSV-CN genome [GenBank Accession AF332093.3 (12)] over time in control and WSSV treatments. (B) A stacked bar plot of differentially expressed transcripts over time following WSSV injection in both C. maenas and P. vannamei. (C) A stacked bar plot of differentially expressed miRNAs over time following injection in both C. maenas and P. vannamei. (D) Percentage of processed sequencing reads aligning to the WSSV-CN genome in crabs displaying evidence for WSSV replication. (E) Principal component analysis of differentially expressed C. maenas transcripts in response to WSSV injection demonstrating separation of crabs displaying evidence for WSSV replication according to principal component 1. (F) Bubble plot of KEGG pathways up- and down-regulated over time in C. maenas in response to WSSV injection where point size represented the number of transcripts identified in the KEGG pathway and colour represents the adjusted p-value for enrichment.

Figure 3

Comparison of susceptible and resistant host responses to WSSV infection. (A) Visual representation of overlapping significantly enriched GO IDs detected in WSSV-injected C. maenas (represented by large blue nodes with ‘c_’ labels) compared to susceptible P. vannamei (represented by large yellow nodes with ‘s_’ labels). Each GO ID is represented by a grey node connected to a large node representing the time point and species within which this term was significantly enriched. A table of the GO IDs associated with each time point is presented in Supplementary File 10 . The central cluster of virus-related GO terms is indicated with a red box. (B) Overlapping KEGG pathways in WSSV-injected P. vannamei (represented by large yellow nodes with ‘s_’ labels) and C. maenas (represented by large blue nodes with ‘c_’ labels). Each KEGG ID is represented by a small node connected to a large node representing the time point and species within which this pathway was significantly enriched. Down-regulated KEGGs are depicted by small blue nodes, down-regulated KEGGs are represented by small red nodes and shared KEGGs that were both up- and down-regulated are depicted by yellow nodes. A table of the KEGG pathway IDs associated with each time point is presented in Supplementary File 11 . These plots were created using DiVenn (41).

Figure 4

Proposed mechanisms responsible for differences in susceptibility to WSSV between resistant and susceptible hosts [adapted from Verbruggen et al. (61)] (A) highly susceptible Penaeus vannamei and (B) highly resistant Carcinus maenas. Host components are displayed in brown tones and viral components are displayed in purple tones. Arrows indicate the direction of change in transcription or activity of a proposed process. Differential responses of susceptible and resistant hosts are shown in red and blue, respectively.