Gene expression profile analysis of Manila clam (Ruditapes philippinarum) hemocytes after a Vibrio alginolyticus challenge using an immune-enriched oligo-microarray

Abstract

Background: The Manila clam (Ruditapes philippinarum) is a cultured bivalve with worldwide commercial importance, and diseases cause high economic losses. For this reason, interest in the immune genes in this species has recently increased. The present work describes the construction of the first R. philippinarum microarray containing immune-related hemocyte sequences and its application to study the gene transcription profiles of hemocytes from clams infected with V. alginolyticus through a time course.

Results: The complete set of sequences from R. philippinarum available in the public databases and the hemocyte sequences enriched in immune transcripts were assembled successfully. A total of 12,156 annotated sequences were used to construct the 8 × 15 k oligo-microarray. The microarray experiments yielded a total of 579 differentially expressed transcripts. Using the gene expression results, the associated Gene Ontology terms and the enrichment analysis, we found different response mechanisms throughout the experiment. Genes related to signaling, transcription and apoptosis, such as IL-17D, NF-κB or calmodulin, were typically expressed as early as 3 hours post-challenge (hpc), while characteristic immune genes, such as PGRPs, FREPs and defense proteins appeared later at 8 hpc. This immune-triggering response could have affected a high number of processes that seemed to be activated 24 hpc to overcome the Vibrio challenge, including the expression of many cytoskeleton molecules, which is indicative of the active movement of hemocytes. In fact functional studies showed an increment in apoptosis, necrosis or cell migration after the infection. Finally, 72 hpc, activity returned to normal levels, and more than 50% of the genes were downregulated in a negative feedback of all of the previously active processes.

Conclusions: Using a new version of the R. philippinarum oligo-microarray, a putative timing for the response against a Vibrio infection was established. The key point to overcome the challenge seemed to be 8 hours after the challenge, when we detected immune functions that could lead to the destruction of the pathogen and the activation of a wide variety of processes related to homeostasis and defense. These results highlight the importance of a fast response in bivalves and the effectiveness of their innate immune system.

Figure 1

Validation of the microarray. qPCR expression of selected genes: C3 (Complement component C3), IL-17D (Interleukin 17 D), LTBP-4 (Latent Transforming Growth Factor-β Binding Protein 4) and TRAF6 (tumor necrosis factor receptor-associated factor 6). Data are log2 transformed to facilitate the illustration of downregulated genes. All qPCR reactions were performed as technical triplicates and the expression level of analyzed genes was normalized using the 18S rRNA. Fold change units were then calculated by dividing the normalized expression values of hemocytes from infected clams by the normalized expression values of the controls. Each bar represents the mean and standard error of the biological replicates. Asterisks highlight the significant fold changes (t-test, p < 0.05). Microarray results (t-test, p < 0.01) are indicated in the boxes below the graph.

Figure 2

Distribution of regulated genes through time. Statistically significant gene modulation is subdivided according to intensity (fold change) and sense (up and down-regulation).

Figure 3

Venn diagram showing the common regulated genes among the four sampling times.

Figure 4

Biological process GO terms change in regulated genes through time. Numbers in abscissa refer to the percentage of GO term hits of the total of regulated sequences. GO terms with maximum representation in each sampling point are separately represented. A: 3 h, B: 8 h and C: 24 h. No GO processes exhibited a maximum representation 72 hours post-challenge.

Figure 5

Enrichment analysis results. Differential GO term distribution between the test set (significantly expressed genes for each sampling point and regulation, up/down) and the reference set (all of the sequences present in the microarray). Only significant analyses were represented. hpc, hours post-challenge.

Figure 6

Evaluation of the immune response after bacterial infection. (A) The cellular components of the hemolymph are granulocytes and hyalinocytes. Photographs were taken using Nomarski DIC objective. (B) Apoptotic and necrotic levels induced by V. alginolyticus in primary cultures of hemocytes (n = 6). (C)In vivo modulation of the cellular concentration in hemolymph after bacterial infection. A significant increment in the number of cells was observed 6 h after infection. Graph represents the measurement of 6 independent animals as well as the mean + SEM. (D) Apoptotic and necrotic levels in vivo induced by V. alginolyticus at different times post infection. In all graphs, bars represent the mean + SEM of 6 independent measurements. Data were analyzed using the Student’s t-test. (*) significant differences at p < 0.05.