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The Manila clam (Ruditapes philippinarum) is the second most exploited bivalve in the world but remains threatened by diseases and global changes. Their associated microbiota play a key role in their fitness and acclimation capacities. This study aimed at better understanding the behavior of clam digestive glands and extrapallial fluids microbiota at small, but contrasting spatial and temporal scales. Results showed that environmental variations impacted clam microbiota differently according to the considered tissue. Each clam tissue presented its own microbiota, and showed different dynamics according to the intertidal position and sampling period. Extrapallial fluids microbiota was modified more rapidly than digestive glands microbiota, for clams placed on the upper and lower intertidal position, respectively. Clam tissues could be considered as different microhabitats for bacteria as they presented different responses to small-scale temporal and spatial variabilities in natural conditions. These differences underlined a more stringent environmental filter capacity of the digestive glands.

Environmental Vibrio strains represent a major threat in aquaculture, but the understanding of their virulence mechanisms heavily relies on the transposition of knowledge from human-pathogen vibrios. Here, the genetic bases of the virulence of Vibrio harveyi ORM4 toward the European abalone Haliotis tuberculata were characterized. We demonstrated that luxO, encoding a major regulator of the quorum sensing system, is crucial for the virulence of this strain, and that its deletion leads to a decrease in swimming motility, biofilm formation, and exopolysaccharide production. Furthermore, the biofilm formation by V. harveyi ORM4 was increased by abalone serum, which required LuxO. The absence of LuxO in V. harveyi ORM4 yielded opposite phenotypes compared with other Vibrio species including V. campbellii (still frequently named V. harveyi). In addition, we report a full Type III Secretion System (T3SS) gene cluster in the V. harveyi ORM4 genome. LuxO was shown to negatively regulate the promoter activity of exsA, encoding the major regulator of the T3SS genes, and the deletion of exsA abolished the virulence of V. harveyi ORM4. These results unveil virulence mechanisms set up by this environmentally important bacterial pathogen, and pave the way for a better molecular understanding of the regulation of its pathogenicity. This article is protected by copyright. All rights reserved.

The production of non-fish based docosahexaenoic acid (DHA) for feed and food has become a critical need in our global context of over-fishing. The industrial-scale production of DHA–rich Thraustochytrids could be an alternative, if costs turned out to be competitive. In order to reduce production costs, this study addresses the feasibility of the non-axenic (non-sterile) cultivation of Aurantiochytrium mangrovei on industrial substrates (as nitrogen and mineral sources and glucose syrup as carbon and energy sources), and its scale-up from laboratory (250 mL) to 500 L cultures. Pilot-scale reactors were airlift cylinders. Batch and fed-batch cultures were tested. Cultures over 38 to 62 h achieved a dry cell weight productivity of 3.3 to 5.5 g.L−1.day−1, and a substrate to biomass yield of up to 0.3. DHA productivity ranged from 10 to 0.18 mg.L−1.day−1. Biomass productivity appears linearly related to oxygen transfer rate. Bacterial contamination of cultures was low enough to avoid impacts on fatty acid composition of the biomass. A specific work on microbial risks assessment (in supplementary files) showed that the biomass can be securely used as feed. However, to date, there is a law void in EU legislation regarding the recycling of nitrogen from digestate from animal waste for microalgae biomass and its usage in animal feed. Overall, the proposed process appears similar to the industrial yeast production process (non-axenic heterotrophic process, dissolved oxygen supply limiting growth, similar cell size). Such similarity could help in further industrial developments.

The aetiological agent Perkinsus olseni is globally recognised as a major threat for shellfish production considering its wide geographical distribution across Asia, Europe, Australia and South America. Another species, Perkinsus chesapeaki, which has never been known to be associated with significant mortality events, was recently detected along French coasts infecting clam populations sporadically in association with P. olseni. Identifying potential cryptic infections affecting Ruditapes philippinarum is essential to develop appropriate host resource management strategies. Here, we developed a molecular method based on duplex real-time quantitative PCR for the simultaneous detection of these two parasites, P. olseni and P. chesapeaki, in the different clam tissues: gills, digestive gland, foot, mantle, adductor muscle and the rest of the soft body. We firstly checked the presence of possible PCR inhibitors in host tissue samples. The qPCR reactions were inhibited depending on the nature of the host organ. The mantle and the rest of the soft body have a high inhibitory effect from threshold of host gDNA concentration of 2 ng.µL−1, the adductor muscle and the foot have an intermediate inhibition of 5 ng.µL−1, and the gills and digestive gland do not show any inhibition of the qPCR reaction even at the highest host gDNA concentration of 20 ng.µL−1. Then, using the gills as a template, the suitability of the molecular technique was checked in comparison with the Ray’s Fluid Thioglycolate Medium methodology recommended by the World Organisation for Animal Health. The duplex qPCR method brought new insights and unveiled cryptic infections as the co-occurrence of P. olseni and P. chesapeaki from in situ tissue samples in contrast to the RFTM diagnosis. The development of this duplex qPCR method is a fundamental work to monitor in situ co-infections that will lead to optimised resource management and conservation strategies to deal with emerging diseases.

Environmental Vibrio strains represent a major threat in aquaculture, but the understanding of their virulence mechanisms heavily relies on the transposition of knowledge from human-pathogen vibrios. Here, the genetic bases of the virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata were characterized. We demonstrated that luxO, encoding a major regulator of the quorum sensing system, is crucial for the virulence of this strain, and that its deletion leads to a decrease in swimming motility, biofilm formation, and exopolysaccharide production. Furthermore, the biofilm formation by V. harveyi ORM4 was increased by abalone serum, which required LuxO. The absence of LuxO in V. harveyi ORM4 yielded opposite phenotypes compared with other Vibrio species including V. campbellii (still frequently named V. harveyi). In addition, we report a full type III secretion system (T3SS) gene cluster in the V. harveyi ORM4 genome. LuxO was shown to negatively regulate the promoter activity of exsA, encoding the major regulator of the T3SS genes, and the deletion of exsA abolished the virulence of V. harveyi ORM4. These results unveil virulence mechanisms set up by this environmentally important bacterial pathogen and pave the way for a better molecular understanding of the regulation of its pathogenicity.

International audience

Vibrio tapetis, the etiological agent of Brown Ring Disease, mainly affects the Manila clam Ruditapes philippinarum. Although this bacterium is mainly known as a clam pathogen, it has been isolated from several fish species. The main aim of the present study was to further explore the variability of 27 V. tapetis isolates from bivalves and fish, considering three different aspects; in vitro virulence based on the loss of clam hemocyte adhesion properties, detection of the gene virB4 encoding for an essential component of the Type IV Secretion System, and MALDI-TOF MS characterization based on whole cell extracts. Finally, these approaches were compared and evaluated for their ability to differentiate the potential pathogenicity of the 27 isolates against the Manila clams. Among the 11 V. tapetis isolates from the common dab isolated in 2018 in Belgium, only one (2BB) showed intermediate in vitro virulence against the Manila clam, and seven carried the virB4 gene while none of the V. tapetis previously isolated from fish in 2003 showed the presence of this particular gene. Finally, the peak protein profiles generated with MALDI-TOF MS analysis from all 27 V. tapetis strains showed a clear clustering of clam pathogenic and nonpathogenic isolates suggesting that a new isolate of V. tapetis that would cluster within the clam pathogenic isolates could be potentially pathogenic to the Manila clam. Thereupon, MALDI-TOF MS typing allows rapid and cost-efficient identification of V. tapetis isolates and can be defined as a complementary method of the traditional qPCR that opens new perspectives to study the virulence of V. tapetis isolates but also to perform environmental monitoring in order to prevent outbreaks.

The synthetic pathways responsible for the production of the polyunsaturated fatty acids 22:6n-3 and 20:5n-3 were studied in the Dinophyte Alexandrium minutum. The purpose of this work was to follow the progressive incorporation of an isotopic label (13CO2) into 11 fatty acids to better understand the fatty acid synthesis pathways in A. minutum. The Dinophyte growth was monitored for 54 hours using high-frequency sampling. A. minutum presented a growth in two phases. A lag phase was observed during the first 30 hours of development and had been associated with the probable temporary encystment of Dinophyte cells. An exponential growth phase was then observed after t30. A. minutum rapidly incorporated 13C into 22:6n-3, which ended up being the most 13C-enriched polyunsaturated fatty acid (PUFA) in this experiment, with a higher 13C atomic enrichment than 18:4n-3, 18:5n-3, 20:5n-3, and 22:5n-3. Overall, the 13C atomic enrichment (AE) was inversely proportional to number of carbons in n-3 PUFA. C18 PUFAs, 18:4n-3, and 18:5n-3, were indeed among the least 13C-enriched FAs during this experiment. They were assumed to be produced by the n-3 PUFA pathway. However, they could not be further elongated or desaturated to produce n-3 C20-C22 PUFA, because the AEs of the n-3 C18 PUFAs were lower than those of the n-3 C20-C22 PUFAs. Thus, the especially high atomic enrichment of 22:6n-3 (55.8% and 54.9% in neutral lipids (NLs) and polar lipids (PLs), respectively) led us to hypothesize that this major PUFA was synthesized by an O2-independent Polyketide Synthase (PKS) pathway. Another parallel PKS, independent of the one leading to 22:6n-3, was also supposed to produce 20:5n-3. The inverse order of the 13C atomic enrichment for n-3 PUFAs was also suspected to be related to the possible &beta, oxidation of long-chain n-3 PUFAs occurring during A. minutum encystment.

Infectious agents such as the bacteria Vibrio aestuarianus or Ostreid herpesvirus 1 have been repeatedly associated with dramatic disease outbreaks of Crassostrea gigas beds in Europe. Beside roles played by these pathogens, microbial infections in C. gigas may derive from the contribution of a larger number of microorganisms than previously thought, according to an emerging view supporting the polymicrobial nature of bivalve diseases. In this study, the microbial communities associated with a large number of C. gigas samples collected during recurrent mortality episodes at different European sites were investigated by real‐time PCR and 16SrRNA gene‐based microbial profiling. A new target enrichment next‐generation sequencing protocol for selective capturing of 884 phylogenetic and virulence markers of the potential microbial pathogenic community in oyster tissue was developed allowing high taxonomic resolution analysis of the bivalve pathobiota. Comparative analysis of contrasting C. gigas samples conducted using these methods revealed that oyster experiencing mortality outbreaks displayed signs of microbiota disruption associated with the presence of previously undetected potential pathogenic microbial species mostly belonging to genus Vibrio and Arcobacter. The role of these species and their consortia should be targeted by future studies aiming to shed light on mechanisms underlying polymicrobial infections in C. gigas. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the EU project H2020 VIVALDI Grant No. 678589. We thank all the producers and people helping with sampling collection at the farming sites. We also thank the Ifremer ECOSCOPA network for producing and maintaining the oysters in the Bay of Brest. SI

International audience; Vibrio campbellii BAA-1116 is renowned for its bioluminescence properties, and genetic tools are available to genetically track this strain. However, many other ecologically important V. harveyi strains exist, for which only few genetic tools are available. In this study, a rapid electroporation protocol was developed to transform replicative plasmids in various environmental V. harveyi and Pseudoalteromonas strains. Moreover, a mini-Tn7 delivery system was modified to site-specifically integrate mini-transposons in the genome of V. harveyi ORM4. As a proof-of-principle, replicative plasmids carrying bioreporters were introduced by electroporation in V. harveyi ORM4 cells, and gene expression was followed at the single cell level. We could demonstrate that a flagellar gene is subjected to bimodal gene expression in V. harveyi ORM4, being highly expressed in 10% of the population in stationary phase. This study extends the possibilities to study environmental Vibrio strains and uncovers the occurrence of phenotypic heterogeneity in flagellar expression in Vibrio.

The Brown Ring Disease (BRD) caused high mortality rates since 1986 in the Manila clam Venerupis philippinarum introduced and cultured in Western Europe from the 1970s. The causative agent of BRD is a Gram-Negative bacterium, Vibrio tapetis, which is also pathogenic to fish. Here we report the first assembly of the complete genome of V. tapetis CECT4600T, together with the genome sequences of 16 additional strains isolated across a broad host and geographic range. Our extensive genome dataset allowed us to describe the pathogen pan- and core genomes and to identify putative virulence factors. The V. tapetis core genome consists of 3,352 genes, including multiple potential virulence factors represented by haemolysins, transcriptional regulators, Type I restriction modification system, GGDEF domain proteins, several conjugative plasmids, and a Type IV secretion system. Future research on the coevolutionary arms race between V. tapetis virulence factors and host resistance mechanisms will improve our understanding of how pathogenicity develops in this emerging pathogen.

International audience; Paralytic shellfish toxins (PST) bind to voltage-gated sodium channels (Nav) and block conduction of action potential in excitable cells. This study aimed to (i) characterize Nav sequences in Crassostrea gigas and (ii) investigate a putative relation between Nav and PST-bioaccumulation in oysters. The phylogenetic analysis highlighted two types of Nav in C. gigas: a Nav1 (CgNav1) and a Nav2 (CgNav2) with sequence properties of sodium-selective and sodium/calcium-selective channels, respectively. Three alternative splice transcripts of CgNav1 named A, B and C, were characterized. The expression of CgNav1, analyzed by in situ hybridization, is specific to nervous cells and to structures corresponding to neuromuscular junctions. Real-time PCR analyses showed a strong expression of CgNav1A in the striated muscle while CgNav1B is mainly expressed in visceral ganglia. CgNav1C expression is ubiquitous. The PST binding site (domain II) of CgNav1 variants possess an amino acid Q that could potentially confer a partial saxitoxin (STX)-resistance to the channel. The CgNav1 genotype or alternative splicing would not be the key point determining PST bioaccumulation level in oysters.

Vibrio harveyi is a marine bacterial pathogen responsible for episodic epidemics generally associated with massive mortalities in many marine organisms, including the European abalone Haliotis tuberculata . The aim of this study was to identify the portal of entry and the dynamics of infection of V. harveyi in the European abalone. The results indicate that the duration of contact between V. harveyi and the European abalone influences the mortality rate and precocity. Immediately after contact, the epithelial and mucosal area situated between the gills and the hypobranchial gland was colonized by V. harveyi . Real-time PCR analyses and culture quantification of a green fluorescent protein-tagged strain of V. harveyi in abalone tissues revealed a high density of bacteria adhering to and then penetrating the whole gill-hypobranchial gland tissue after 1 h of contact. V. harveyi was also detected in the hemolymph of a significant number of European abalones after 3 h of contact. In conclusion, this article shows that a TaqMan real-time PCR assay is a powerful and useful technique for the detection of a marine pathogen such as V. harveyi in mollusk tissue and for the study of its infection dynamics. Thus, we have revealed that the adhesion and then the penetration of V. harveyi in European abalone organs begin in the first hours of contact. We also hypothesize that the portal of entry of V. harveyi in the European abalone is the area situated between the gills and the hypobranchial gland.

International audience; The marine bacterium Vibrio tapetis is the causative agent of the brown ring disease, which affects the clam Ruditapes philippinarum and causes heavy economic losses in North of Europe and in Eastern Asia. Further characterization of V. tapetis isolates showed that all the investigated strains harbored at least one large plasmid. We determined the sequence of the 82,266 bp plasmid pVT1 from the CECT4600(T) reference strain and analyzed its genetic content. pVT1 is a mosaic plasmid closely related to several conjugative plasmids isolated from Vibrio vulnificus strains and was shown to be itself conjugative in Vibrios. In addition, it contains DNA regions that have similarity with several other plasmids from marine bacteria (Vibrio sp., Shewanella sp., Listonella anguillarum and Photobacterium profundum). pVT1 contains a number of mobile elements, including twelve Insertion Sequences or inactivated IS genes and an RS1 phage element related to the CTXphi phage of V. cholerae. The genetic organization of pVT1 underscores an important role of horizontal gene transfer through conjugative plasmid shuffling and transposition events in the acquisition of new genetic resources and in generating the pVT1 modular organization. In addition, pVT1 presents a copy number of 9, relatively high for a conjugative plasmid, and appears to belong to a new type of replicon, which may be specific to Vibrionaceae and Shewanelleacae.

The Gram-negative bacteriumVibrio tapetisis known as the causative agent of Brown Ring Disease (BRD) in the Manila clamVenerupis(=Ruditapes)philippinarum. This bivalve is the second most important species produced in aquaculture and has a high commercial value. In spite of the development of several molecular methods, no survey has been yet achieved to rapidly quantify the bacterium in the clam. In this study, we developed a Taqman real-time PCR assay targeting virB4 gene for accurate and quantitative identification ofV. tapetisstrains pathogenic to clams. Sensitivity and reproducibility of the method were assessed using either filtered sea water or extrapallial fluids of clam injected with the CECT4600TV. tapetisstrain. Quantification curves ofV. tapetisstrain seeded in filtered seawater (FSW) or extrapallial fluids (EF) samples were equivalent showing reliable qPCR efficacies. With this protocol, we were able to specifically detectV. tapetisstrains down to 1.125 101bacteria per mL of EF or FSW, taking into account the dilution factor used for appropriate template DNA preparation. This qPCR assay allowed us to monitorV. tapetisload both experimentally or naturally infected Manila clams. This technique will be particularly useful for monitoring the kinetics of massive infections byV. tapetisand for designing appropriate control measures for aquaculture purposes.

A piezotolerant, mesophilic, marine lactic acid bacterium (strain LT20T) was isolated from a deep sub-seafloor sediment core collected at Nankai Trough, off the coast of Japan. Cells were Gram-positive, rod-shaped, non-sporulating and non-motile. The NaCl concentration range for growth was 0–120 g l−1, with the optimum at 10–20 g l−1. The temperature range for growth at pH 7·0 was 4–50 °C, with the optimum at 37–40 °C. The optimum pH for growth was 7·0–8·0. The optimum pressure for growth was 0·1 MPa with tolerance up to 30 MPa. The main cellular phospholipids were phosphatidylglycerols (25 %), diphosphatidylglycerols (34 %) and a group of compounds tentatively identified as ammonium-containing phosphatidylserines (32 %); phosphatidylethanolamines (9 %) were minor components. The fatty acid composition was dominated by side chains of 16 : 0, 14 : 0 and 16 : 1. The G+C content of the genomic DNA was 42 mol%. On the basis of 16S rRNA gene sequence analysis and the secondary structure of the V6 region, this organism was found to belong to the genus Marinilactibacillus and was closely related to Marinilactibacillus psychrotolerans M13-2T (99 %), Marinilactibacillus sp. strain MJYP.25.24 (99 %) and Alkalibacterium olivapovliticus strain ww2-SN4C (97 %). Despite the high similarity between their 16S rRNA gene sequences (99 %), the DNA–DNA hybridization levels were less than 20 %. On the basis of physiological and genetic characteristics, it is proposed that this organism be classified as a novel species, Marinilactibacillus piezotolerans sp. nov. The type strain is LT20T (=DSM 16108T=JCM 12337T).

A novel piezotolerant, mesophilic, facultatively anaerobic, organotrophic, polarly flagellated bacterium (strain LT13aT) was isolated from a deep sediment layer in the Nankai Trough (Leg 190, Ocean Drilling Program) off the coast of Japan. This organism used a wide range of organic substrates as sole carbon and energy sources: pyruvate, glutamate, succinate, fumarate, lactate, citrate, peptone and tryptone. Oxygen, nitrate, fumarate, ferric iron and cystine were used as electron acceptors. Maximal growth rates were observed at a hydrostatic pressure of 10 MPa. Hydrostatic pressure for growth was in the range 0·1–50 MPa. Predominant cellular fatty acids were 16 : 1ω7c, 15 : 0 iso, 16 : 0 and 13 : 0 iso. The G+C content of the DNA was 44·9 mol%. On the basis of 16S rRNA gene sequences, strain LT13aT was shown to belong to the γ-Proteobacteria, being closely related to Shewanella putrefaciens (98 %), Shewanella oneidensis (97 %) and Shewanella baltica (96 %). Levels of DNA homology between strain LT13aT and S. putrefaciens, S. oneidensis and S. baltica were T represents a novel species. Genetic evidence and phenotypic characteristics showed that isolate LT13aT constitutes a novel species of the genus Shewanella. Because of the deep origin of the strain, the name Shewanella profunda sp. nov. is proposed, with LT13aT (=DSM 15900T=JCM 12080T) as the type strain.