Integrative study of physiological changes associated with bacterial infection in Pacific oyster larvae

Abstract

Background: Bacterial infections are common in bivalve larvae and can lead to significant mortality, notably in hatcheries. Numerous studies have identified the pathogenic bacteria involved in such mortalities, but physiological changes associated with pathogen exposure at larval stage are still poorly understood. In the present study, we used an integrative approach including physiological, enzymatic, biochemical, and molecular analyses to investigate changes in energy metabolism, lipid remodelling, cellular stress, and immune status of Crassostrea gigas larvae subjected to experimental infection with the pathogenic bacteria Vibrio coralliilyticus.

Findings: Our results showed that V. coralliilyticus exposure induced (1) limited but significant increase of larvae mortality compared with controls, (2) declined feeding activity, which resulted in energy status changes (i.e. reserve consumption, β-oxidation, decline of metabolic rate), (3) fatty acid remodeling of polar lipids (changes in phosphatidylinositol and lysophosphatidylcholine composition`, non-methylene-interrupted fatty acids accumulation, lower content of major C20 polyunsaturated fatty acids as well as activation of desaturases, phospholipase and lipoxygenase), (4) activation of antioxidant defenses (catalase, superoxide dismutase, peroxiredoxin) and cytoprotective processes (heat shock protein 70, pernin), and (5) activation of the immune response (non-self recognition, NF-κκ signaling pathway, haematopoiesis, eiconosoids and lysophosphatidyl acid synthesis, inhibitor of metalloproteinase and antimicrobial peptides).

Conclusion: Overall, our results allowed us to propose an integrative view of changes induced by a bacterial infection in Pacific oyster larvae, opening new perspectives on the response of marine bivalve larvae to infections.

Figure 1. Mortality and clearance rate for Pacific oyster Crassostrea gigas larvae as a function of experiment duration and bacterial challenge.

Larvae were challenged with Vibrio coralliilyticus for a period of 24 h or 48 h. Data from different treatments were pooled when this effect was not significant. Chal: challenged larvae; Unchal: unchallenged larvae. Data are means±SD of duplicate tanks. Different letters indicate significant differences.

Figure 2. DGGE gels performed with 16 s cDNA extracted from larvae (L) and surrounding water (W).

Profiles were compared with (Chal) or without (Unchal) bacterial challenge and after 24 h and 48 h of experiment. The cDNA from reference sample (Ref), diet (D) and bacterial control (B) was used as ladder. Due to their high similarity, cDNA from duplicate samples were pooled. B1 and 2 correspond to the retrieved bands used to Vibrio coralliilyticus detection by sequencing analysis.

Figure 3. RDA ordination biplot representing the significant (p<0.05) challenge effect after 24 (Chal24) and 48 h (Chal48) as explanatory variables (arrows) on selected response variables (lines) related to energy metabolism (red), lipids (blue), cellular stress (green), and immunity (pink).

Response variables related to larval performance and biochemical analysis were formatted in normal text, to enzymatic analysis in bold and to molecular analysis in italic. Black symbols: challenged larvae, white symbols: unchallenged larvae, squares: 24 h of exposure, circles: 48 h of exposure. Response variables abbreviations: -A, enzymatic activity; -G, gene expression; N-, neutral lipids; P-, polar lipids; Σ, sum of; cLEC, c-type lectin-1; AGL, a-agglutinin attachment subunit; LBP/BPI, LPS binding/bactericidal-permeability-increasing protein; MYD88, Myeloid differentiation primary response (88); TRAF, TNF receptor associated factor; REL, REL protein; DRAC3, drosophila rho GTPase 3; TAL, hematopoietic transcription factors; TIMP, tissue inhibitor metalloproteinase; Defh2, defensine 2; GR, glutathione reductase; SOD, superoxide dismutase; CAT, catalase; GPX5, glutathione peroxidase 5; PRDX4-5, peroxiredoxine 4 and 5; PRN, Pernin; HSP70, Heat shock protein 70; NADHOX, Mitochondrial nadh:ubiquinone oxidoreductase; CCOi, Cytochrome c oxidase subunit i; AS6, ATP synthase f0 subunit 6; PK, pyruvate kinase; CCO, cytochrome c oxidase; ADH, Acyl-CoA dehydrogenase; ECH, Enoyl-hydratase isomerase family protein; ACS, Acyl-CoA synthetase; Lipstor, Adipophilin; Delta5, Stearoyl-desaturase 5; Delta6, Fatty acid desaturase 2; PLA2, Phospholipase a2 receptor 1; PLD1, Phospholipase delta 1; AA15LX, Arachidonate 15-lipoxygenase; TOT, total lipids; PI, phosphatidylinositol; LPC, lysophosphatidylcholine; TAG, triacylglycerols; FFA, free fatty acids; AA, arachidonic acid; EPA, eicosapentaenoic acid; DPA, Docosapentaenoic acid; DHA, Docosahexaenoic acid; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; NMI, non-methylene–interrupted fatty acids; Pind, peroxidation index; Protein, total proteins content; Feeding, feeding activity; Mortality, mortality rate.

Figure 4. Schematic diagrams of physiological changes induced during bacterial infection in C. gigas larvae.

Infection impact on larval physiology was investigated through immunity (1), cellular stress (2), energetic metabolism (3) and lipid metabolism (4) using physiological, enzymatic, biochemical and molecular analysis. Results suggest that bacterial infection induce the activation of the immune response (non-self recognition (1a), NF-κB signaling pathway (1b), haematopoiesis (1d), synthesis of inhibitor of metalloproteinase, antimicrobial peptide (1c) and phagocytosis (1e) allowing the destruction of pathogenic bacteria. The production of reactive oxygen species (ROS) during the phagocytosis process was managed by antioxidant defenses (2a) and cytoprotective proteins (2b). Infection affects the feeding activity (3a) which change the energy status of larvae (decline of metabolic rate (3b), energy reserve consumption (3c), β-oxidation activation (3d) and lower lipids storage (3e)). Besides metabolic changes, fatty acid remodeling in polar lipids (4a) is associated to pathogen exposure, as suggested by changes in phosphatidylinositol and lysophosphatidylcholine composition, non-methylene–interrupted fatty acids accumulation, lower content of major C₂₀ polyunsaturated fatty acids and activation of desaturases. Finally, infection induces the activation of phospholipase and lipoxygenase (4b) probably through NF-κB regulation (4c) to initiate eicosanoïdes and lysophosphatidic acid (LPA) pathways. See discussion for details.