Manila clam - Perkinsus olseni interaction based on gene expression analysis of clam hemocytes and parasite trophozoites [Manila clam]

The Manila clam (Ruditapes philippinarum) is the bivalve species with the highest world production from both fisheries and aquaculture, but its production is seriously threatened by perkinsosis, a disease caused by the protozoan parasite Perkinsus olseni. To understand the molecular mechanisms underlying R. philippinarum–P. olseni interaction, we analyzed the gene expression profiles of in vitro challenged clam hemocytes and P. olseni trophozoites, using two oligo-microarray platforms, one previously validated for R. philippinarum hemocytes and a new one developed and validated in this study for P. olseni. Manila clam hemocytes were in vitro challenged with trophozoites, zoospores, and extracellular products from P. olseni in vitro cultures, while P. olseni trophozoites were in vitro challenged with Manila clam plasma along the same time-series (1 h, 8 h, and 24 h). The hemocytes showed a fast activation of the innate immune response, particularly associated with hemocyte recruitment, in the three types of challenges. Nevertheless, different immune-related pathways were activated in response to the different parasite stages, suggesting specific recognition mechanisms. Furthermore, the analyses provided useful complementary data to previous in vivo challenges, and confirmed the potential of some proposed biomarkers. The combined analysis of gene expression in host and parasite identified several processes in both the clam and P. olseni, such as redox and glucose metabolism, protease activity, apoptosis and iron metabolism, whose modulation suggests cross-talk between parasite and host. This information might be critical to determine the outcome of the infection, thus highlighting potential therapeutic targets. Altogether, the results of this study aid to understand the response and interaction between R. philippinarum–P. olseni and will contribute for developing effective control strategies for this threatening parasitosis. R. philippinarum hemocytes (5x106) were challenged in vitro with P. olseni trophozoites (5x106), zoospores (5x106) and extracellular products (2.5 mL of culture media enriched with extracellular products) separately in IWAKI 6-well plates. Each challenge included three biological replicates for both treatment and control (only culture media) groups, and each biological replicate was a pool of hemocytes from 10 different clams, thus averaging individual biological variation. Samples for RNA extraction were collected at 1, 8 and 24 h after the start of the challenge. Thirty-two microarrays (four slides) were used. R. philippinarum control replicates at each time point were pooled, so a single control microarray was used for each sampling time (1-C, 8-C, 24-C); in addition, the low RNA amount in the controls of the zoospore challenge determined pooling all controls in a single microarray hybridization.