Interactions between the Parasite Philasterides dicentrarchi and the Immune System of the Turbot Scophthalmus maximus. A Transcriptomic Analysis

Abstract

The present study analyses the interactions between Philasterides dicentrarchi (a ciliate parasite that causes high mortalities in cultured flatfish) and the peritoneal cells of the turbot Scophthalmus maximus during an experimental infection. The transcriptomic response was evaluated in the parasites and in the fish peritoneal cells, at 1, 2 and 4 h post-infection (hpi) in turbot injected intraperitoneally (ip) with 107 ciliates and at 12 and 48 hpi in turbot injected ip with 105 ciliates. Numerous genes were differentially expressed (DE) in P. dicentrarchi, relative to their expression in control ciliates (0 hpi): 407 (369 were up-regulated) at 1 hpi, 769 (415 were up-regulated) at 2 hpi and 507 (119 were up-regulated) at 4 hpi. Gene ontology (GO) analysis of the DE genes showed that the most representative categories of biological processes affected at 1, 2 and 4 hpi were biosynthetic processes, catabolic processes, biogenesis, proteolysis and transmembrane transport. Twelve genes of the ABC transporter family and eight genes of the leishmanolysin family were DE at 1, 2 and 4 hpi. Most of these genes were strongly up-regulated (UR), suggesting that they are involved in P. dicentrarchi infection. A third group of UR genes included several genes related to ribosome biogenesis, DNA transcription and RNA translation. However, expression of tubulins and tubulin associated proteins, such as kinesins or dyneins, which play key roles in ciliate division and movement, was down-regulated (DR). Similarly, genes that coded for lysosomal proteins or that participate in the cell cycle mitotic control, glycolysis, the Krebs cycle and/or in the electron transport chain were also DR. The transcriptomic analysis also revealed that in contrast to many parasites, which passively evade the host immune system, P. dicentrarchi strongly stimulated turbot peritoneal cells. Many genes related to inflammation were DE in peritoneal cells at 1, 2 and 4 hpi. However, the response was much lower at 12 hpi and almost disappeared completely at 48 hpi in fish that were able to kill P. dicentrarchi during the first few hpi. The genes that were DE at 1, 2 and 4 hpi were mainly related to the apoptotic process, the immune response, the Fc-epsilon receptor signalling pathway, the innate immune response, cell adhesion, cell surface receptors, the NF-kappaB signalling pathway and the MAPK cascade. Expression of toll-like receptors 2, 5 and 13 and of several components of NF-κB, MAPK and JAK/STAT signalling pathways was UR in the turbot peritoneal cells. Genes expressing chemokines and chemokine receptors, genes involved in prostaglandin and leukotriene synthesis, prostaglandins, leukotriene receptors, proinflammatory cytokines and genes involved in apoptosis were strongly UR during the first four hours of infection. However, expression of anti-inflammatory cytokines such as Il-10 and lipoxygenases with anti-inflammatory activity (i.e., arachidonate 15-lipoxygenase) were only UR at 12 and/or 48 hpi, indicating an anti-inflammatory state in these groups of fish. In conclusion, the present study shows the regulation of several genes in P. dicentrarchi during the early stages of infection, some of which probably play important roles in this process. The infection induced a potent acute inflammatory response, and many inflammatory genes were regulated in peritoneal cells, showing that the turbot uses all the protective mechanisms it has available to prevent the entry of the parasite.

Severe Natural Outbreak of Cryptocaryon irritans in Gilthead Seabream Produces Leukocyte Mobilization and Innate Immunity at the Gill Tissue

“…Regarding the expression of NCC and CTL effectors, our data only documented an up-regulation of fasl in the gills but not of gzma or gzmb. Although the up-regulation of the granzymes as well as other cytotoxic effectors is documented in turbot infected with P. dicentrarchi [54], seabream infected with E. leei [24], or in rainbow trout infected with the myxosporean Ceratonova shasta [55], we failed to find this pattern. However, we found large-scale up-regulation of the fasl gene in the gills of infected seabream, which was also found in turbot infected with P. dicentrarchi [51], supporting the relevance of the granule-independent mechanism against parasites in fish [25].…”

Characterization of the turbot Scophthalmus maximus (L.) myeloperoxidase. An insight into the evolution of vertebrate peroxidases

“…dicentrarchi causes a potent inflammatory response in turbot (Valle et al, 2020), and leucocytes become highly stimulated after being in contact with the parasite (Piazzon et al, 2011). If turbot mpx behaves like human MPO, the decrease in expression could be a consequence of myeloid cell differentiation induced indirectly by the parasites.…”

“…For the in vitro experiments, parasites (Philasterides dicentrarchi, isolate I1) were cultured at 18 °C in L-15 Leibovitz medium containing 10% heat-inactivated foetal bovine serum (FBS), lipids (lecithin and Tween 80), nucleosides and glucose (Sigma-Aldrich, Madrid), as described by Valle et al (2020). Once the appropriate density was reached, the parasites were collected, centrifuged at 700 × g for 5 min, washed twice in L-15 medium with 2% FBS and resuspended in the same medium.…”

To React or Not to React: The Dilemma of Fish Immune Systems Facing Myxozoan Infections

“…Teleost fish have only three Ig isotypes: IgM, IgT/Z, and IgD (39), there is no switch of Igs in the secondary response, and the production of specific antibodies is temperature dependent (40, 41) (see section 3 for more details about the different functions of Igs). In the last decade our knowledge of the immune system of fish has notably increased (42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53), and in parallel, that of the immune responses against fish parasitic flagellates (54, 55), microsporidians (56), ciliates (57)(58)(59)(60), helminths (61)(62)(63)(64), amoeba (65,66) and crustaceans (67)(68)(69). However, there are methodological difficulties that limit our understanding of fish immune responses in comparison to other vertebrates.…”