Open Access Research article

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

Rebeca Moreira1, Massimo Milan2, Pablo Balseiro1, Alejandro Romero1, Massimiliano Babbucci2, Antonio Figueras1, Luca Bargelloni2 and Beatriz Novoa1*

Author Affiliations

1 Instituto de Investigaciones Marinas (IIM), Consejo Superior de Investigaciones Científicas (CSIC), Eduardo Cabello 6, 36208 Vigo, Spain

2 Department of Comparative Biomedicine and Food Science (BCA), University of Padova, Viale dell’Università 16, 35020 Legnaro, Italy

For all author emails, please log on.

BMC Genomics 2014, 15:267  doi:10.1186/1471-2164-15-267

Published: 7 April 2014

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.

Keywords:
Ruditapes philippinarum; Vibrio alginolyticus; Hemocytes; Oligo-microarray; Gene ontology; Blast2GO; Immune response