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Villas-Bôas, S., Differential expression of novel metabolic and immunological biomarkers in oysters challenged with a virulent strain of OsHV-1, Developmental and Comparative Immunology (2017), doi: 10.1016/j.dci.2017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. and immunological responses in oyster larvae exposed to OsHV-1 µVar; some of which have 34 not previously been reported in molluscs. Multivariate analyses of entire metabolite profiles 35 were able to separate infected from non-infected larvae. Correlation analysis revealed the 36 presence of major perturbations in the underlying biochemical networks and secondary 37 pathway analysis of functionally-related metabolites identified a number of prospective 38 pathways differentially regulated in virus-exposed larvae. These results provide new insights 39 into the pathogenic mechanisms of OsHV-1 infection in oyster larvae, which may be applied 40 to develop disease mitigation strategies and/or as new phenotypic information for selective 41 breeding programmes aiming to enhance viral resistance. 42

From the first detection in the Eastern oysters (Crassostrea virginica) in 1972, herpes-like viruses/herpesviruses have been identified in at least 20 bivalve species and have caused massive mortalities in six cultured species of bivalves. Among them, the Pacific oyster (Crassostrea gigas) is most threatened by massive mortalities associated with detection of Ostreid herpesvirus 1 (OsHV-1) and its variants. These mortality events have been reported in more than 12 countries, and OsHV-1 itself has been reported in 15 countries worldwide. Traditionally, the genotype OsHV-1 (or OsHV-1 Var) is the main contributor to bivalve mortalities, but the emergence of variants (e.g. OsHV-1 lVar and related ones, acute viral necrosis virus-AVNV, OsHV-1-SB) is now responsible for new mortality outbreaks in Pacific oysters and other bivalve species. In addition to OsHV-1, Vibrio bacteria and other abiotic and host factors also significantly contribute to mortality outbreaks. To this end, the current contribution aims to synthesize the body of literature on our current knowledge of these mass mortality events to provide insights into the role of each risk factor and complex interactions in the disease process. Specifically, this review encompasses our current knowledge and critical gaps. Important suggestions for future investigation are also presented. Figure 1 Classification of main Pacific oyster (Crassostrea gigas) producing countries in 2016 in tonnes (data taken from FAO FishStatJ, version 3.01.0). The map was generated using http://mapchart.net. Asterisks (*) indicate countries with mass mortality events. ( ) >300 000 tonnes; ( ) >100 000 tonnes; ( ) >10 000 tonnes; ( ) >1000 tonnes; ( ) <1000 tonnes.

Vibrio coralliilyticus is a bacterial pathogen which can affect a range of marine organisms, such as corals, fish and shellfish, with sometimes devastating consequences. However, little is known about the mechanisms involved in the host-pathogen interaction, especially within molluscan models. We applied gas chromatography-mass spectrometry (GC-MS)-based metabolomics to characterize the physiological responses in haemolymph of New Zealand Greenshell™ mussels (Perna canaliculus) injected with Vibrio sp. DO1 (V. coralliilyticus/neptunius-like isolate). Univariate data analyses of metabolite profiles in Vibrio-exposed mussels revealed significant changes in 22 metabolites at 6 h post-infection, compared to non-exposed mussels. Among them, 10 metabolites were up-regulated, while 12 metabolites were down-regulated in infected mussels. Multivariate analyses showed a clear distinction between infected and non-infected mussels. In addition, secondary pathway analyses indicated perturbations of the host innate immune system following infection, including oxidative stress, inflammation and disruption of the TCA cycle, change in amino acid metabolism and protein synthesis. These findings provide new insights into the pathogenic mechanisms of Vibrio infection of mussels and demonstrate our ability to detect detailed and rapid host responses from haemolymph samples using a metabolomics approach.

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The antimicrobial role of itaconic acid (ITA) has been recently discovered in mammalian cells. In our previous studies, we discovered that marine molluscs biosynthesise substantial quantities of ITA when exposed to marine pathogens, but its antimicrobial function to Vibrio bacteria is currently unknown. Thus, in this study, we used an untargeted gas chromatography–mass spectrometry (GC-MS) platform to identify metabolic changes of Vibrio sp. DO1 ( V . corallyliticus / neptunius -like isolate) caused by ITA exposure. Vibrio sp. DO1 was cultured in Luria-Bertani broth supplemented with 3 mM sodium acetate and with different concentrations of ITA (0, 3 and 6 mM) for 24 h. The results showed that ITA completely inhibited Vibrio sp. growth at 6 mM and partially inhibited the bacterial growth at 3 mM. A principal component analysis (PCA) revealed a clear separation between metabolite profiles of Vibrio sp. DO1 in the 3 mM ITA treatment and the control, which were different in 25 metabolites. Among the altered metabolites, the accumulation of glyoxylic acid and other metabolites in glyoxylate cycle (cis-aconitic acid, isocitric acid and fumaric acid) together with the increase of isocitrate lyase (ICL) activity in the 3 mM ITA treatment compared to the control suggest that ITA inhibited Vibrio sp. growth via disruption of central carbon metabolism.

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