Genomic mining of Vibrio parahaemolyticus highlights prevalence of antimicrobial resistance genes and new genetic markers associated with AHPND and tdh + /trh + genotypes.

BACKGROUND: Acute Hepatopancreatic Necrosis Disease (AHPND) causes significant mortality in shrimp aquaculture. The infection is primarily instigated by Vibrio parahaemolyticus (Vp) strains carrying a plasmid encoding the binary toxin PirAB. Yet, comprehension of supplementary virulence factors associated with this relatively recent disease remains limited. Furthermore, the same holds for gastroenteritis in humans caused by other Vp genotypes. Additionally, given the prevalent use of antibiotics to combat bacterial infections, it becomes imperative to illuminate the presence of antimicrobial resistance genes within these bacteria. RESULTS: A subsampled number of 1,036 Vp genomes was screened for the presence of antimicrobial resistance genes, revealing an average prevalence of 5 ± 2 (SD) genes. Additional phenotypic antimicrobial susceptibility testing of three Vp strains (M0904, TW01, and PV1) sequenced in this study demonstrated resistance to ampicillin by all tested strains. Additionally, Vp M0904 showed multidrug resistance (against ampicillin, tetracycline, and trimethoprim-sulfamethoxazole). With a focus on AHPND, a screening of all Vibrio spp. for the presence of pirA and/or pirB indicates an estimated prevalence of 0.6%, including four V. campbellii, four V. owensii, and a Vibrio sp. next to Vp. Their pirAB-encoding plasmids exhibited a highly conserved backbone, with variations primarily in the region of the Tn3 family transposase. Furthermore, an assessment of the subsampled Vp genomes for the presence of known virulence factors showed a correlation between the presence of the Type 3 Secretion System 2 and tdh, while the presence of the Type 6 Secretion System 1 was clade dependent. Furthermore, a genome-wide association study (GWAS) unveiled (new) genes associated with pirA, pirB, tdh, and trh genotypes. Notable associations with the pirAB genotype included outer membrane proteins, immunoglobulin-like domain containing proteins, and toxin-antitoxin systems. For the tdh + /trh + genotypes (containing tdh, trh, or both genes), associations were found with T3SS2 genes, urease-related genes and nickel-transport system genes, and genes involved in a 'minimal' type I-F CRISPR mechanism. CONCLUSIONS: This study highlights the prevalence of antimicrobial resistance and virulence genes in Vp, identifying novel genetic markers associated with AHPND and tdh + /trh + genotypes. These findings contribute valuable insights into the genomic basis of these genotypes, with implications for shrimp aquaculture and food safety.

Biofloc technology and immune response of penaeid shrimp: A meta-analysis and meta-regression.

Biological flocs develop as heterogenious communities of detritus, heterotrophic and autotrophic bacteria, algae, fungi and a variety of other things through appropriate management of the carbon-nitrogen ratio in aquaculture farming systems. These microbial communities improve the water quality, reduce the concentrations of toxic inorganic species, and act as a source of food and immunostimulants. However, the information about their role as immunostimulants is widely variable. To obtain a clearer view of the situation, it is necessary to summarize the current literature on this topic through a quantitative review analysis: a meta-analysis. The present work aims to evaluate the effect of biofloc technology (BFT) implementation on culture water quality through meta-analysis, meta-regression, and correlation analysis. Pubmed, Science Direct, Scopus, and Google Scholar were searched from 2000 to 2022 on the influence of BFT on immunological parameters. Three hundred and thirty-four of the scientific articles used BFT for penaeid shrimp, but only 25 articles met the inclusion criterion of using BFT to evaluate its effect on the shrimp's immunity. The current analysis found that the use of BFT stimulates all the evaluated immunological parameters. Also, meta-regression with fixed data of study parameters showed that slow-release carbon sources, probiotics, and an estimated carbon/nitrogen ratio of 10 potentiated a larger effect on BFT immune stimulation. The meta-analysis demonstrated that the volumes of the settleable solids (SS) and the total suspended solids (TSS) are crucial for the effect of BFT on the shrimp immune system. When TSS was higher than 294 mg/L, the respiratory metabolism of the shrimp was compromised. The analysis found evidence of publication biases, indicating that the data had to be interpreted carefully. Nonetheless, this meta-analysis shows that the data quality was sound and the approach used was appropriate; moreover, it also showed that BFT can be an environmentally friendly alternative culture method that enhances the shrimp's immune system.

Prolonged ecological changes can affect morphometrics and gene expression profile? Focusing on Hsp-70 and NLHS-induced Hsp-70 of Artemia urmiana.

BACKGROUND: In recent years, many aquatic ecosystems, including Urmia Lake, have undergone severe ecological tensions. This lake, the largest natural habitat of the brine shrimp Artemia urmiana, has progressively desiccated and its salinity has dramatically increased over the last three decades. In the face of the long period environmental stresses, understanding the adaptation and ecological plasticity mechanisms is the most interesting challenges in genetic and applied ecology. These mechanisms may probably be driven by inducing expression of some genes involved in adaptation such as Hsp-70 and also adjusting morphological parameters. But they are yet to be understood. Hence, the present work aimed to study the mechanisms, along with testing the hypothesis that non-lethal heat shocked nauplii originating from drought period can evoke Hsp-70 expression more than those from rainy period. METHODS: This study measured and analyzed morphometrical characters of adult male and female Artemia urmiana over three decades. Then, the influence of three-decade ecological crisis on Hsp-70 and non-lethal heat shock (NLHS)-induced Hsp-70 expression levels of nauplii of Artemia urmiana habiting Urmia Lake using Real-time PCR technique, based on cyst collections in 1994 (rainy period) to 2020 (drought period), was evaluated. RESULTS: The morphometrics results showed that the morphological characters were significantly shrunk in 2020 compared to 1994 (CI 95%, p < 0.05). Furthermore, our results depicted that, Hsp-70 expression level was significantly upregulated in response to the prolonged ecological crisis, (CI 95%, P < 0.0001), and also interestingly, the nauplii exposed to longe-term ecological crisis (belong to 2020) were able to increase Hsp-70 expression more than other ones in response to environmental stressors including heat. CONCLUSIONS: The present results showed the involvement of Hsp-70 in the adaptation of Artemia urmiana to long term ecological alteration at the cost of shrinking morphometric parameters.

The shrimp nephrocomplex serves as a major portal of pathogen entry and is involved in the molting process.

Viruses, such as white spot syndrome virus, and bacteria, such as Vibrio species, wreak havoc in shrimp aquaculture [C. M. Escobedo-Bonilla et al., J. Fish. Dis. 31, 1-18 (2008)]. As the main portal of entry for shrimp-related pathogens remain unclear, infectious diseases are difficult to prevent and control. Because the cuticle is a strong pathogen barrier, regions lacking cuticular lining, such as the shrimp's excretory organ, "the antennal gland," are major candidate entry portals [M. Corteel et al., Vet. Microbiol. 137, 209-216 (2009)]. The antennal gland, up until now morphologically underexplored, is studied using several imaging techniques. Using histology-based three-dimensional technology, we demonstrate that the antennal gland resembles a kidney, connected to a urinary bladder with a nephropore (exit opening) and a complex of diverticula, spread throughout the cephalothorax. Micromagnetic resonance imaging of live shrimp not only confirms the histology-based model, but also indicates that the filling of the diverticula is linked to the molting cycle and possibly involved therein. Based on function and complexity, we propose to rename the antennal gland as the "nephrocomplex." By an intrabladder inoculation, we showed high susceptibility of this nephrocomplex to both white spot syndrome virus and Vibrio infection compared to peroral inoculation. An induced drop in salinity allowed the virus to enter the nephrocomplex in a natural way and caused a general infection followed by death; fluorescent beads were used to demonstrate that particles may indeed enter through the nephropore. These findings pave the way for oriented disease control in shrimp.

ß-1,3-Glucan/chitin unmasking in the Saccharomyces cerevisiae mutant, Δmnn9, promotes immune response and resistance of the Pacific oyster (Crassostrea gigas) to Vibrio coralliilyticus infection.

Yeast cells can play a crucial role in immune activation in fish and shellfish predominantly due to the cell wall component ß-1,3-glucan, providing protection against bacterial or viral infections. However, the immunostimulatory capacity of dietary yeast cells remains poorly studied in bivalves. To understand the role of yeast cell wall components (mannan, ß-glucan and chitin) as immune activators, this study characterized the surface carbohydrate exposure of the wild-type baker's yeast Saccharomyces cerevisiae (WT) and its Δmnn9 mutant, which presents a defective mannan structure, and compared these profiles with that of ß-glucan particles, using fluorescein isothiocyanate (FITC)-labeled lectin binding analysis. Then, a first trial evaluated the immunological response in Crassostrea gigas juveniles after being fed for 24 h with an algae-based diet (100A) and its 50% substituted version (based on dry weight) with WT (50A50WT) and Δmnn9 (50A50Y), and the posterior resistance of the juveniles against Vibrio coralliilyticus infection (trial 1). The mRNA expression was measured for ß-glucan-binding protein (CgßGBP), Toll-like receptor 4 (CgTLR4), C-type lectin receptor 3 (CgCLec-3), myeloid differentiation factor 88 (CgMyD88), nuclear factor-kappa B (CgNFκB), lysozyme (CgLys), interleukin 17-5 (CgIL17-5), and superoxide dismutase (CgSOD), in oysters, before and 24 h after the bacterial inoculation. A second trial tested the effect of incorporating Δmnn9 into the 100A diet for 24 h at different substitution levels: 0, 5, 10, 25, and 50% (100A, 95A5Y, 90A10Y, 75A25Y, and 50A50Y), followed by the bacterial challenge with V. coralliilyticus (trial 2). Our findings showed that the outer cell wall surface of WT is largely composed of mannan, while Δmnn9 presents high exposure of ß-glucan and chitin, exhibiting similar FITC-lectin binding profiles (fluorescence intensity) to ß-glucan particles. A significantly higher survival after the bacterial challenge was observed in oysters fed on 50A50Y compared to those fed 50A50WT and 100A in trial 1. This better performance of 50A50Y was supported by significantly higher gene expressions of CgLys, CgSOD, CgMyD88, and CgßGBP compared to 100A, and CgSOD and CgNFκB in relation to those fed on 50A50WT, prior to the bacterial inoculation. Furthermore, improved survival was observed in oysters fed 50A50Y compared to those offered lower Δmnn9 levels and 100A in trial 2. The superior performance of Δmnn9-fed oysters is mostly associated with the elevated presence of unmasked ß-glucans on Δmnn9 cell wall surface, facilitating their interactions with oyster hemocytes. Further studies are needed to evaluate administration dose and frequency of Δmnn9 to develop strategies for long-term feeding.

Selected essential oil components fail to induce an immunological response in Artemia but still protect against vibriosis.

In the present research, in order to screen out the best candidates from 12 different EOCs, we proposed three in vivo screening methods, namely the screening method of bioluminescence of V. campbellii associated with brine shrimp, regrowth performance of V. campbellii, and immune gene expression of brine shrimp without challenge. Our result showed that challenged with V. campbellii at 107 cells/mL, the survival of the brine shrimp at 48 h was significantly increased after treatment with the EOCs (at 0.0005%, v/v) of 4-allylanisole, R-(+)-limonene, S-(-)-limonene, (-)-terpinen-4-ol, (±)-citronellal, citral, trans-cinnamaldehyde and (+)-carvone, compared to the positive control group. Also, it was observed that the EOCs- of 4-allylanisloe, R-(+)-limonene, S-(-)-limonene, (-)-ß-pinene, geraniol, (±)-citronellal, citral, trans-cinnamaldehyde and (+)-carvone decreased significantly the in vivo bioluminescence of V. campbellii at 36 h after Vibrio exposure. The regrowth assay showed that independently from incubation time (1, 12 or 24 h), no difference was observed in the regrowth curve in all EOC treatment groups compared to the positive control group. The dscam gene expression in the (±)-citronellal group, and the sod gene in the citral group were observed to be significantly higher than in the negative control at 24 h, respectively. However, most of the immune genes were down-regulated in the EOC groups. Combining the survival data at 48 h with the bioluminescence result at 36 h, it was noted that the survival rate of brine shrimp was moderately correlated with in vivo bioluminescence of V. campbellii. The results indicate that the approach of determining in vivo bioluminescence of V. campbellii is a moderately reliable, fastest, and cheapest screening method for EOCs. As the regrowth performance assay of V. campbellii, and the immune genes expression assay of brine shrimp without challenge cannot predict Artemia survival properly, they cannot be used as screening methods for EOCs. Moreover, the immune genes expression assay is relatively slow, time-consuming and costly.

Supplementation of ex situ produced bioflocs improves immune response against AHPND in Pacific whiteleg shrimp (Litopenaeus vannamei) postlarvae.

The emergence of Vibrio diseases, including acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio spp., had resulted in heavy losses in global shrimp production. Biofloc technology is a closed aquaculture system developed as one of the sustainable solutions to increase system resilience in the shrimp industry. In this study, biofloc was formed externally (ex situ biofloc) with probiotics Bacillus sp. strain BME and Bacillus sp. strain BCE, diatom microalgae Chaetoceros calcitrans, and a consortium of nitrifying bacteria, in the ratio of 1:1:6:6 as a starter. The study showed that the ex situ biofloc supplementation in Pacific whiteleg shrimp (L. vannamei) postlarvae culture can increase the shrimp culture performance (shrimp survival and growth), reduce Vibrio counts in the water and shrimp body, and provide stimulation of the shrimp immune response through humoral immune responses, such as pattern recognition protein (C-type lectin) and melanization process (proPO). Overall, the results indicate that the supplementation of ex situ biofloc provided protection to shrimp under Vibrio infection, regardless of the timing of addition (before, simultaneously, or after addition of Vibrio sp. strain VPA). This suggests that the ex situ biofloc can be effective as a preventive and a supportive treatment against potential AHPND infection in L. vannamei postlarvae culture. Taken together, the ability of the ex situ biofloc to modulate immune-related gene expression and resistance of L. vannamei against potentially AHPND-causing Vibrio sp. strain makes it an effective aquaculture technology for infectious disease control in shrimp production with high-density and minimal water exchange culture. KEY POINTS: • Supplementation of ex situ produced biofloc in shrimp postlarvae culture. • Ex situ biofloc reduces Vibrio counts in the water and shrimp body. • Ex situ biofloc stimulates shrimp humoral immune responses and survival.

The genome of the extremophile Artemia provides insight into strategies to cope with extreme environments.

BACKGROUND: Brine shrimp Artemia have an unequalled ability to endure extreme salinity and complete anoxia. This study aims to elucidate its strategies to cope with these stressors. RESULTS AND DISCUSSION: Here, we present the genome of an inbred A. franciscana Kellogg, 1906. We identified 21,828 genes of which, under high salinity, 674 genes and under anoxia, 900 genes were differentially expressed (42%, respectively 30% were annotated). Under high salinity, relevant stress genes and pathways included several Heat Shock Protein and Leaf Embryogenesis Abundant genes, as well as the trehalose metabolism. In addition, based on differential gene expression analysis, it can be hypothesized that a high oxidative stress response and endocytosis/exocytosis are potential salt management strategies, in addition to the expression of major facilitator superfamily genes responsible for transmembrane ion transport. Under anoxia, genes involved in mitochondrial function, mTOR signalling and autophagy were differentially expressed. Both high salt and anoxia enhanced degradation of erroneous proteins and protein chaperoning. Compared with other branchiopod genomes, Artemia had 0.03% contracted and 6% expanded orthogroups, in which 14% of the genes were differentially expressed under high salinity or anoxia. One phospholipase D gene family, shown to be important in plant stress response, was uniquely present in both extremophiles Artemia and the tardigrade Hypsibius dujardini, yet not differentially expressed under the described experimental conditions. CONCLUSIONS: A relatively complete genome of Artemia was assembled, annotated and analysed, facilitating research on its extremophile features, and providing a reference sequence for crustacean research.

Can only one physiological trait determinate the adverse effect of green fluorescent protein (GFP) incorporation on Vibrio virulence?

Green fluorescent protein (GFP) has been used extensively for in situ animal studies that follow up bacterial infection under epifluorescence microscopy. It is assumed that GFP is acting as a "neutral" protein with no influence on the bacterial physiology. To verify this hypothesis, the virulence of Vibrio splendidus ME9, Vibrio anguillarum NB10, and their respective GFP-tagged strains ME9-GFP and NB10-GFP (transconjugants) was compared in vitro and tested in vivo towards blue mussel (Mytilus edulis) larvae. Results showed that the incorporation of GFP negatively impacted the growth and swimming motility of NB10 in vitro. Correspondingly, the mRNA levels of genes involved in bacterial swimming motility (flaA, flaE, and cheR) were significantly down-regulated in NB10-GFP. As for the strain ME9 on the other hand, GFP incorporation only had a negative effect on swimming motility. However, both the strains NB10-GFP and ME9-GFP showed almost the same virulence as their respective parental strain towards mussel larvae in vivo. Overall, the data presented here demonstrated that incorporation of GFP may cause modifications in cell physiology and highlight the importance of preliminary physiological tests to minimize the negative influence of GFP tagging when it is used to monitor the target localization. The study also supports the idea that the virulence of Vibrio species is determined by complex regulatory networks. Notwithstanding the change of a single physiological trait, especially growth or swimming motility, the GFP-tagged Vibrio strain can thus still be considered usable in studies mainly focusing on the virulence of the strain. KEY POINTS: • The effect of GFP incorporation on physiological trait of Vibrio strains. • The virulence in vibrios could be multifactorial. • The stable virulence of Vibrio strains after GFP incorporation.

The chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) controls cellular quiescence by hyperpolarizing the cell membrane during diapause in the crustacean Artemia.

Cellular quiescence, a reversible state in which growth, proliferation, and other cellular activities are arrested, is important for self-renewal, differentiation, development, regeneration, and stress resistance. However, the physiological mechanisms underlying cellular quiescence remain largely unknown. In the present study, we used embryos of the crustacean Artemia in the diapause stage, in which these embryos remain quiescent for prolonged periods, as a model to explore the relationship between cell-membrane potential (Vmem) and quiescence. We found that Vmem is hyperpolarized and that the intracellular chloride concentration is high in diapause embryos, whereas Vmem is depolarized and intracellular chloride concentration is reduced in postdiapause embryos and during further embryonic development. We identified and characterized the chloride ion channel protein cystic fibrosis transmembrane conductance regulator (CFTR) of Artemia (Ar-CFTR) and found that its expression is silenced in quiescent cells of Artemia diapause embryos but remains constant in all other embryonic stages. Ar-CFTR knockdown and GlyH-101-mediated chemical inhibition of Ar-CFTR produced diapause embryos having a high Vmem and intracellular chloride concentration, whereas control Artemia embryos released free-swimming nauplius larvae. Transcriptome analysis of embryos at different developmental stages revealed that proliferation, differentiation, and metabolism are suppressed in diapause embryos and restored in postdiapause embryos. Combined with RNA sequencing (RNA-Seq) of GlyH-101-treated MCF-7 breast cancer cells, these analyses revealed that CFTR inhibition down-regulates the Wnt and Aurora Kinase A (AURKA) signaling pathways and up-regulates the p53 signaling pathway. Our findings provide insight into CFTR-mediated regulation of cellular quiescence and Vmem in the Artemia model.

Environmental conditions steer phenotypic switching in acute hepatopancreatic necrosis disease-causing Vibrio parahaemolyticus, affecting PirAVP /PirBVP toxins production.

Bacteria in nature are widely exposed to differential fluid shears which are often a trigger for phenotypic switches. The latter mediates transcriptional and translation remodelling of cellular metabolism impacting among others virulence, antimicrobial resistance and stress resistance. In this study, we evaluated the role of fluid shear on phenotypic switch in an acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus M0904 strain under both in vitro and in vivo conditions. The results showed that V. parahaemolyticus M0904 grown at lower shaking speed (110 rpm constant agitation, M0904/110), causing low fluid shear, develop cellular aggregates or floccules. These cells increased levan production (as verified by concanavalin binding) and developed differentially stained colonies on Congo red agar plates and resistance to antibiotics. In addition, the phenotypic switch causes a major shift in the protein secretome. At 120 rpm (M0904/120), PirAVP /PirBVP toxins are mainly produced, while at 110 rpm PirAVP /PirBVP toxins production is stopped and an alkaline phosphatase (ALP) PhoX becomes the dominant protein in the protein secretome. These observations are matched with a very strong reduction in virulence of M0904/110 towards two crustacean larvae, namely, Artemia and Macrobrachium. Taken together, our study provides substantial evidence for the existence of two phenotypic forms in AHPND V. parahaemolyticus strain displaying differential phenotypes. Moreover, as aerators and pumping devices are frequently used in shrimp aquaculture facilities, they can inflict fluid shear to the standing microbial agents. Hence, our study could provide a basis to understand the behaviour of AHPND-causing V. parahaemolyticus in aquaculture settings and open the possibility to monitor and control AHPND by steering phenotypes.

Gut Microbiota of Migrating Wild Rabbit Fish (Siganus guttatus) Larvae Have Low Spatial and Temporal Variability.

We investigated the gut microbiota of rabbit fish larvae at three locations in Vietnam (ThuanAn-northern, QuangNam-intermediate, BinhDinh-southern sampling site) over a three-year period. In the wild, the first food for rabbit fish larvae remains unknown, while the juveniles and adults are herbivores, forming schools near the coasts, lagoons, and river mouths, and feeding mainly on filamentous algae. This is the first study on the gut microbiota of the wild fish larvae and with a large number of individuals analyzed spatially and temporally. The Clostridiales order was the most predominant in the gut, and location-by-location alpha diversity showed significant differences in Chao-1, Hill number 1, and evenness. Analysis of beta diversity indicated that the location, not year, had an effect on the composition of the microbiota. In 2014, the gut microbiota of fish from QuangNam was different from that in BinhDinh; in 2015, the gut microbiota was different for all locations; and, in 2016, the gut microbiota in ThuanAn was different from that in the other locations. There was a time-dependent trend in the north-south axis for the gut microbiota, which is considered to be tentative awaiting larger datasets. We found limited variation in the gut microbiota geographically and in time and strong indications for a core microbiome. Five and fifteen OTUs were found in 100 and 99% of the individuals, respectively. This suggests that at this life stage the gut microbiota is under strong selection due to a combination of fish-microbe and microbe-microbe interactions.

Beta-glucan's varying structure characteristics modulate survival and immune-related genes expression from Vibrio harveyi-infected Artemia franciscana in gnotobiotic conditions.

ß-Glucans have long been used as an immunostimulant in aquaculture. However, the relationship of its structure to its immunomodulatory properties are poorly understood. In this study, the particle size and chemical structure of ß-glucans extracted from wild-type strain of baker's yeast (Saccharomyces cerevisiae) and its null-mutant yeasts Gas1 were characterised. Using Sigma ß-glucan as a reference, the immunomodulatory properties of these polysaccharides in the germ-free Artemia franciscana model system in the presence of Vibrio harveyi bacterial challenge were investigated. The survival of the A. franciscana nauplii, upon challenge with V. harveyi, was significantly higher in all three glucan-treated groups compared to the control. The glucan Gas1 with a lower degree of branching and shorter side chain length had the most prominent V. harveyi-protective effects. The particle size did not affect the nauplii survival when challenged with V. harveyi. Results also showed that the salutary effect of the tested glucans was associated with the upregulation of innate immune genes such as lipopolysaccharide and ß-1,3-glucan-binding protein (lgbp), high mobility group box protein (hmgb), and prophenoloxidase (proPO). Interestingly, the up-regulation of superoxidase dismutase (sod) and glutathione-s-transferase (gst) was only observed in Gas1 treated group, indicating that Gas1 could function to induce higher reactive oxygen species and stronger immunomodulatory function in A. franciscana, and therefore higher survival rate. The expression of heat shock protein 70 (hsp70), peroxinectin (pxn), and down syndrome cell adhesion molecule (dscam) remain unaltered in response to glucan treatment. Taken together, this study provides insights into the structure-function relationship of ß-glucan and the results confirmed that ß-glucan can be an effective immunostimulant in aquaculture, especially the Gas1 glucan.

Labelling halophilic Archaea using 13C and 15N stable isotopes: a potential tool to investigate haloarchaea consumption by metazoans.

The use of stable isotope (SI) labelling and tracing of live diets is currently considered one of the most comprehensive tools to detect their uptake and assimilation by aquatic organisms. These techniques are indeed widely used in nutritional studies to follow the fate of specific microbial dietary components, unraveling trophic interactions. Nevertheless, to the current date our understanding of aquatic trophic relationships has yet to include a whole domain of life, the Archaea. The aim of the present research was, therefore, to describe a halophilic Archaea (haloarchaea) labelling procedure, using the SI 13C and 15N, to enable the application of SI tracing in future studies of haloarchaea consumption by aquatic metazoans. To this end, three 13C enriched carbon sources and two 15N enriched nitrogen sources were tested as potential labels to enrich cells of three haloarchaea strains when supplemented to the culture medium. Our overall results indicate 13C-glycerol as the most effective carbon source to achieve an efficient 13C enrichment in haloarchaea cells, with Δδ13C values above 5000‰ in all tested haloarchaea strains. As for 15N enriched nitrogen sources, both (15NH4)2SO4 and 15NH4Cl seem to be readily assimilated, also resulting in efficient 15N enrichment in haloarchaea cells, with Δδ15N values higher than 20,000‰. We believe that the proposed methodology will allow for the use of SI labelled haloarchaea biomass in feeding tests, potentially providing unambiguous confirmation of the assimilation of haloarchaea biomass by aquatic metazoans.

Ralstonia eutropha, containing high poly-ß-hydroxybutyrate levels, regulates the immune response in mussel larvae challenged with Vibrio coralliilyticus.

Marine invertebrates rely mainly on innate immune mechanisms that include both humoral and cellular responses. Antimicrobial peptides (AMPs), lysozyme and phenoloxidase activity, are important components of the innate immune defense system in marine invertebrates. They provide an immediate and rapid response to invading microorganisms. The impact of amorphous poly-ß-hydroxybutyrate (PHB-A) (1 mg PHB-A L-1) on gene expression of the AMPs mytimycin, mytilinB, defensin and the hydrolytic enzyme lysozyme in infected blue mussel larvae was investigated during "in vivo" challenge tests with Vibrio coralliilyticus (105 CFU mL-1). RNAs were isolated from mussel larvae tissue, and AMPs were quantified by q-PCR using the 18srRNA gene as a housekeeping gene. Our data demonstrated that AMPs genes had a tendency to be upregulated in challenged mussel larvae, and the strongest expression was observed from 24 h post-exposure onwards. The presence of both PHB-A and the pathogen stimulated the APMs gene expression, however no significant differences were noticed between treatments or between exposure time to the pathogen V. coralliilyticus. Looking at the phenoloxidase activity in the infected mussels, it was observed that the addition of PHB-A significantly increased the activity.

Effect of catecholamine stress hormones (dopamine and norepinephrine) on growth, swimming motility, biofilm formation and virulence factors of Yersinia ruckeri in vitro and an in vivo evaluation in rainbow trout.

In this study, we evaluated the impact of the catecholamines on growth, swimming motility, biofilm formation and some virulence factors activities of pathogenic Yersinia ruckeri. Norepinephrine and dopamine (at 100 µM) significantly increased the growth of Y. ruckeri in culture media containing serum. An increase in swimming motility of the pathogen was found following the exposure to the hormones; however, no effect was seen on caseinase, phospholipase and haemolysin productions. Further, antagonists for the catecholamine receptors were observed to block some of the influences of the catecholamines. Indeed, the effects of catecholamines were inhibited by chlorpromazine (the dopaminergic receptor antagonist) for dopamine, labetalol (α-and ß-adrenergic receptor antagonist) and phenoxybenzamine (the α-adrenergic receptor antagonist) for norepinephrine, but propranolol (the ß-adrenergic receptor antagonist) showed no effect. Pretreatment of Y. ruckeri with the catecholamines resulted in a significant enhancement of its virulence towards rainbow trout and the antagonists could neutralize the effect of the stress hormones in vivo. In summary, our results show that the catecholamines increase the virulence of Y. ruckeri which is pathogenic to trout through increasing the motility, biofilm formation and growth.

Does Ralstonia eutropha, rich in poly-ß hydroxybutyrate (PHB), protect blue mussel larvae against pathogenic vibrios?

The natural amorphous polymer poly-ß-hydroxybutyrate (PHB-A: lyophilized Ralstonia eutropha containing 75% PHB) was used as a biological agent to control bacterial pathogens of blue mussel (Mytilus edulis) larvae. The larvae were supplied with PHB-A at a concentration of 1 or 10 mg/L for 6 or 24 hr, followed by exposure to either the rifampicin-resistant pathogen Vibrio splendidus or Vibrio coralliilyticus at a concentration of 105 CFU/ml. Larvae pretreated 6 hr with PHB-A (1 mg/L) survived a Vibrio challenge better relative to 24 hr pretreatment. After 96 hr of pathogen exposure, the survival of PHB-A-treated mussel larvae was 1.41- and 1.76-fold higher than the non-treated larvae when challenged with V. splendidus and V. coralliilyticus, respectively. Growth inhibition of the two pathogens at four concentrations of the monomer ß-HB (1, 5, 25 and 125 mM) was tested in vitro in LB35 medium, buffered at two different pH values (pH 7 and pH 8). The highest concentration of 125 mM significantly inhibited the pathogen growth in comparison to the lower levels. The effect of ß-HB on the production of virulence factors in the tested pathogenic Vibrios revealed a variable pattern of responses.

Effect of quorum quenching bacteria on growth, virulence factors and biofilm formation of Yersinia ruckeri in vitro and an in vivo evaluation of their probiotic effect in rainbow trout.

Five N-acyl homoserine lactone-degrading bacteria (quorum quenching (QQ) strains) were selected to evaluate their impacts on growth, virulence factors and biofilm formation in Yersinia ruckeri in vitro. No difference was observed among the growth pattern of Y. ruckeri in monoculture and coculture with the QQ strains. To investigate the regulation of virulence factors by quorum sensing in Y. ruckeri, cultures were supplemented with 3oxo-C8-HSL. The results indicated that swimming motility and biofilm formation are positively regulated by QS (p < 0.05), whereas caseinase, phospholipase and haemolysin productions are not influenced by 3oxo-C8-HSL (p > 0.05). The QQs were able to decrease swimming motility and biofilm formation in Y. ruckeri. QQ bacteria were supplemented to trout feed at 108  CFU/g (for 40 days). Their probiotic effect was verified by Y. ruckeri challenge either by immersion or injection in trout. All strains could significantly increase fish survival with Bacillus thuringiensis and Citrobacter gillenii showing the highest and lowest relative percentage survival (RPS) values (respectively, 85% and 38%). Besides, there was no difference between the RPS values by either immersion or injection challenge expect for B. thuringiensis. The putative involvement of the QQ capacity in the protection against Yersinia is discussed.

Arcobacter haliotis sp. nov., isolated from abalone species Haliotis gigantea.

A Gram-negative, aerobic, polar-flagellated and rod-shaped, sometimes slightly curved bacterium, designated MA5T, was isolated from the gut of an abalone of the species Haliotis gigantea collected in Japan. Phylogenetic analyses based on 16S rRNA, gyrB, hsp60 and rpoB gene sequences placed strain MA5T in the genus Arcobacter in an independent phylogenetic line. Comparison of the 16S rRNA gene sequence of this strain with those of the type strains of the established Arcobacter species revealed A. nitrofigilis (95.1 %) as nearest neighbour. Strain MA5T grew optimally at 25 °C, pH 6.0 to 9.0 and in the presence of 2 to 5 % (w/v) NaCl under both aerobic and microaerobic conditions. The predominant fatty acids found were summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1 ω7c), C12 : 0 3-OH and C18 : 1 ω7c. Menaquinone-6 (MK-6) and menaquinone-7 (MK-7) were found as the major respiratory quinones. The major polar lipids detected were phosphatidylethanolamine and phosphatidylglycerol. Strain MA5T could be differentiated phenotypically from the phylogenetic closest Arcobacter species by its ability to grow on 0.05 % safranin and 0.01 % 2,3,5-triphenyl tetrazolium chloride (TTC), but not on 0.5 % NaCl. The obtained DNA G+C content of strain MA5T was 27.9 mol%. Based on the phylogenetic, chemotaxonomic and phenotypic distinctiveness of MA5T, this strain is considered to represent a novel species of the genus Arcobacter, for which the name Arcobacter haliotis sp. nov. is proposed. The type strain is MA5T (=LMG 28652T=JCM 31147T).