New insights into the Manila clam – Perkinsus olseni interaction based on gene expression analysis of clam hemocytes and parasite trophozoites through in vitro challenges

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 collected from a P. olseni-free area (Camariñas, NW Spain) were used to collect hemocytes for the in vitro challenge with P. olseni trophozoites, zoospores and extracellular products (proteins released into the culture medium by P. olseni trophozoites). Similarly, P. olseni trophozoites were challenged in vitro with R. philippinarum plasma collected from clams of the P. olseni-free area. The details for collection of hemocytes, trophozoites, zoospores and extracellular products have been described in Hasanuzzaman et al. (2017). The procedures to collect trophozoites from in vitro cultures (1-2 months old) and the isolation of Manila clam plasma have been described in Hasanuzzaman et al. (2016). Absence of P. olseni infection in every used clam was confirmed by PCR and incubation of gill pieces in Ray’s fluid thioglycollate medium. All experiments were carried out in the facilities of Centro de Investigacións Mariñas (CIMA; Spain). R. philippinarum hemocytes (5x10e6) were challenged in vitro with P. olseni trophozoites (5x10e6), zoospores (5x10e6) and extracellular products (2.5 mL of culture media enriched with extracellular products) separately in IWAKI 6-well plates (Fig.1A). 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. For the challenges, trophozoites and zoospores, obtained just before the challenge, were separately suspended in 2.5 mL filtered seawater (FSW) and added into a permeable insert (0.2 μm Anopore® membrane NUNC 25 mm) in each well. For hemocyte-extracellular products challenge, 2.5 mL of culture media enriched with hemocyte extracellular products were added into the inserts of the respective wells. The inserts allowed the flow of media but not the cells; hence, hemocytes and parasite cells were never in contact. Samples for RNA extraction were collected at 1, 8 and 24 h after the start of the challenge. Further details are available in Hasanuzzaman et al. (2017). P. olseni trophozoites (~ 5x10e6) resuspended in 2.5 mL FSW were placed in IWAKI 6-well plates, and 2.5 mL of plasma (treatment) or FSW (control) were added into a permeable insert (0.2 µm Anopore® membrane NUNC 25 mm) set in the plate-wells (Fig. 1B). Four pseudo-replicates (trophozoites from the same culture) for both control and treatment groups were collected at 1, 8 and 24 h since the onset of the challenge. Further details are available in Hasanuzzaman et al. (2016). The design of the R. philippinarum oligo-microarray has been described and validated in Hasanuzzaman et al. (2018). Briefly, this microarray was designed on an 8 × 15 k Agilent format and included 14,621 probes representing 11,052 transcripts of which 10,813 were annotated (97.8%). 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. To construct the P. olseni oligo-microarray, we selected 10,104 sequences from our previously published P. olseni trophozoite transcriptome (Hasanuzzaman et al., 2016). A total of 9,369 sequences were selected because they were annotated to transcripts in the NCBI nr protein database, while the remaining 735 non-annotated sequences were selected by their notable differential expression in our preliminary evaluation (Hasanuzzaman et al., 2016). One oligo-probe was designed for annotated sequences (known sense) and two probes (sense and antisense) were designed for the non-annotated sequences. We also included 4,158 technical replicates for microarray reproducibility evaluation. All processes for oligo-probe design and Agilent oligo-microarray procurement were similar to those followed for the Manila clam microarray (Hasanuzzaman et al., 2018). A total of 16 microarrays (two slides) were used for the experiment. A single pooled control was hybridized in two microarrays per slide as technical replicates. Twelve microarrays were used for the treatments across the time-series (1 h, 8 h and 24 h) including four replicates per time point. Hybridizations were performed at the Universidade de Santiago de Compostela (USC) Functional Genomics Platform using the Agilent Technology Gene Expression Unit following a one-color gene expression analysis protocol. All hybridizations were carried out by the same researcher in the same day. Hybridized slides were scanned using an Agilent scanner (G2565B, Agilent Technologies) and signals were captured and processed. The microrarray platforms (Agilent-072098 and Agilent-xxx) and data presented in this publication has been deposited in the NCBI’s Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are available under accession number xxxx. Microarray hybridization, data processing and quality filtering were carried out as previously described (Hasanuzzaman et al., 2018).