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.

Host-induced increase in larval sea bass mortality in a gnotobiotic challenge test with Vibrio anguillarum.

Vibrio anguillarum is the major cause of haemorrhagic septicaemia, vibriosis, which is a severe disease affecting marine fish. In this work, it was found that the mortality of gnotobiotic sea bass larvae challenged with V. anguillarum was dependent on the number of dead fish in the vials at the moment of challenge. Based on this finding, the effect of dead hosts (homogenised sea bass larvae or brine shrimp) on the virulence of V. anguillarum towards sea bass larvae was further investigated. Addition of homogenised hosts led to significantly increased larval mortality of challenged larvae, and this was observed for 3 different V. anguillarum strains, i.e. 43, NB 10 and HI 610. In contrast, the addition of similar levels of tryptone had no effect on mortality. In line with this, the motility of all 3 V. anguillarum strains was significantly increased by the addition of homogenised hosts but not by tryptone. These results suggest that dead hosts increase infectivity of V. anguillarum, not merely by offering nutrients to the bacteria, but also by increasing virulence-associated activities such as motility.

Development of a bacterial challenge test for gnotobiotic Nile tilapia Oreochromis niloticus larvae.

Gastrointestinal microbiota have an important impact on fish health and disease, stimulating interest in a better understanding of how these gastrointestinal microbial communities are composed and consequently affect host fitness. In this respect, probiotic microorganisms have been extensively used in recent aquaculture production. To study the use of probiotics in the treatment of infectious diseases, the establishment of a method of experimental infection to obtain consistent results for mortality and infection in challenge tests is important. In pathogen-screening tests, 4 candidate pathogenic bacteria strains (Edwardsiella ictaluri gly09, E. ictaluri gly10, E. tarda LMG2793 and Streptococcus agalactiae LMG15977) were individually tested on xenic Nile tilapia larvae. Only Edwardsiella strains delivered via Artemia nauplii, with or without additional pathogen delivery via the culture water, led to increased mortality in fish larvae. A gnotobiotic Nile tilapia larvae model system was developed to provide a research tool to investigate the effects and modes-of-action of probiotics under controlled conditions. A double disinfection procedure using hydrogen peroxide and sodium hypochlorite solution was applied to the fish eggs, which were subsequently incubated in a cocktail of antibiotic and antifungal agents. In the gnotobiotic challenge test, E. ictaluri gly09R was added to the model system via Artemia nauplii and culture water, resulting in a significant mortality of the gnotobiotic fish larvae. The developed gnotobiotic Nile tilapia model can be used as a tool to extend understanding of the mechanisms involved in host-microbe interactions and to evaluate new methods of disease control.

Convergent dynamics of the juvenile European sea bass gut microbiota induced by poly-ß-hydroxybutyrate.

Poly-ß-hydroxybutyrate (PHB) is a bacterial energy and carbon storage compound which exhibits a controlling effect on the gastrointestinal microbiota. Its beneficial activities for aquaculture have already been shown in terms of increased disease resistance and growth performance in a number of studies. However, the action of PHB on the intestinal microbial community in the treated animals has not yet been studied in depth. In this research, the effects of PHB on the microbiota composition in the intestinal tract of juvenile sea bass were examined. It was found that fish cohabiting in the same tank were on average 87% similar regarding the intestinal microbiota. When subjected to the same treatment and environmental conditions but reared in different tanks, the compositions of the enteric communities diverged. The provision of PHB overruled this tank effect by sustaining a microbial core community in the gut that represented 60% of the total bacterial diversity at the highest PHB level of 10%. The microbial community compositions converged between replicate tanks upon supplementation of PHB and were characterized by high dynamics and increased evenness. The results are discussed in the framework of hypotheses that try to relate the intestinal microbial community composition to the health status of the host organisms.

Recombinant DnaK Orally Administered Protects Axenic European Sea Bass Against Vibriosis.

Vibrio anguillarum causes high mortality in European sea bass (Dicentrarchus labrax) larviculture and is a hindering factor for successful sustainable aquaculture of this commercially valuable species. Priming of the innate immune system through administration of immunostimulants has become an important approach to control disease outbreaks in marine fish larviculture. This study was conducted to evaluate immunostimulation by Escherichia coli HSP70 (DnaK) in axenic European sea bass larvae in order to protect the larvae against vibriosis. DnaK stimulates the immune response in crustaceans and juvenile fish against bacterial infections. The use of axenic fish larvae allows to study immunostimulation in the absence of an interfering microbial community. At 7 days post-hatching, larvae received a single dose of alginate encapsulated recombinant DnaK. Two non-treated control groups in which animals either received empty alginate microparticles (C1) or no alginante microparticles (C2 and C3) were included in the study. Eighteen hours later, all larvae, except the ones from group C3 (non-infected control) were challenged with V. anguillarum (105 CFU, bath infection). Mortality was daily recorded until 120 h post infection and at 18, 24, and 36 h post infection, larvae were sampled for expression of immune related genes. Results showed that V. anguillarum induced an immune response in axenic sea bass larvae but that the innate immune response was incapable to protect the larvae against deadly septicaemic disease. In addition, we showed that administration of alginate encapsulated DnaK to axenic European sea bass larvae at DAH7 resulted in a significant, DnaK dose dependent, upreglation of immune sensor, regulatory and effector genes. Significant upregulation of cxcr4, cas1 and especially of hep and dic was correlated with significant higher survival rates in V. anguillarum infected larvae. In the future recombinant DnaK might perhaps be used as a novel immunostimulant in sea bass larviculture.

A review of the functionality of probiotics in the larviculture food chain.

During the past two decades, the use of probiotics as an alternative to the use of antibiotics has shown to be promising in aquaculture, particularly in fish and shellfish larviculture. This article reviews the studies on probiotics in larviculture, focusing on the current knowledge of their in vivo mechanisms of action. The article highlights that the in vivo mechanisms of action largely remain to be unravelled. Several methodologies are suggested for further in vivo research, including studies on gut microbiota composition, the use of gnotobiotic animals as test models, and the application of molecular techniques to study host-microbe and microbe-microbe interactions.

Recombinant ferritin-H induces immunosuppression in European sea bass larvae (Dicentrarchus labrax) rather than immunostimulation and protection against a Vibrio anguillarum infection.

Vibrio anguillarum causes high mortality in European sea bass (Dicentrarchus labrax) larviculture. In this study, we evaluated if the recombinant sea bass ferritin-H could stimulate the innate immune system of gnotobiotic European sea bass larvae resulting in protection against a V. anguillarum challenge. We also evaluated the effect of a V. anguillarum infection on the transcription of immune-related genes in gnotobiotic European sea bass larvae. Recombinant sea bass ferritin-H was produced, encapsulated in calcium alginate microparticles and orally delivered to sea bass larvae at seven days after hatching. Our results showed V. anguillarum caused an acute infection, resulting in high mortality. The infection significantly upregulated the expression of tlr3, tlr5, cas1, il1ß, tnfα, mif, il10, cc1, cxcl8 at 18, 24 and 36 h post infection, but not of the chemokine receptor genes cxcr4 and ccr9. There was no protective effect of ferritin-H. Remarkably, ferritin-H caused significantly higher transcript levels for cxcr4 and ccr9. Sea bass ferritin-H was more likely involved in immune-suppression and results point in the direction of a negative regulation of CXCR4 resulting in inhibition of cell proliferation, differentiation and migration which is detrimental to innate immunity and might explain the non-protective effect of ferritin-H in fish larvae.

Modulation of Innate Immune-Related Genes and Glucocorticoid Synthesis in Gnotobiotic Full-Sibling European Sea Bass (Dicentrarchus labrax) Larvae Challenged With Vibrio anguillarum.

Although several efforts have been made to describe the immunoendocrine interaction in fish, there are no studies to date focusing on the characterization of the immune response and glucocorticoid synthesis using the host-pathogen interaction on larval stage as an early developmental stage model of study. Therefore, the aim of this study was to evaluate the glucocorticoid synthesis and the modulation of stress- and innate immune-related genes in European sea bass (Dicentrarchus labrax) larvae challenged with Vibrio anguillarum. For this purpose, we challenged by bath full-sibling gnotobiotic sea bass larvae with 107 CFU mL-1 of V. anguillarum strain HI 610 on day 5 post-hatching (dph). The mortality was monitored up to the end of the experiment [120 hours post-challenge (hpc)]. While no variations were registered in non-challenged larvae maintained under gnotobiotic conditions (93.20% survival at 120 hpc), in the challenged group a constant and sustained mortality was observed from 36 hpc onward, dropping to 18.31% survival at 120 hpc. Glucocorticoid quantification and expression analysis of stress- and innate immunity-related genes were carried out in single larvae. The increase of cortisol, cortisone and 20ß-dihydrocortisone was observed at 120 hpc, although did not influence upon the modulation of stress-related genes (glucocorticoid receptor 1 [gr1], gr2, and heat shock protein 70 [hsp70]). On the other hand, the expression of lysozyme, transferrin, and il-10 differentially increased at 120 hpc together with a marked upregulation of the pro-inflammatory cytokines (il-1ß and il-8) and hepcidin, suggesting a late activation of defense mechanisms against V. anguillarum. Importantly, this response coincided with the lowest survival observed in challenged groups. Therefore, the increase in markers associated with glucocorticoid synthesis together with the upregulation of genes associated with the anti-inflammatory response suggests that in larvae infected with V. anguillarum a pro-inflammatory response at systemic level takes place, which then leads to the participation of other physiological mechanisms at systemic level to counteract the effect and the consequences of such response. However, this late systemic response could be related to the previous high mortality observed in sea bass larvae challenged with V. anguillarum.

Managing the Microbial Community of Marine Fish Larvae: A Holistic Perspective for Larviculture.

The availability of high-quality juveniles is a bottleneck in the farming of many marine fish species. Detrimental larvae-microbe interactions are a main reason for poor viability and quality in larval rearing. In this review, we explore the microbial community of fish larvae from an ecological and eco-physiological perspective, with the aim to develop the knowledge basis for microbial management. The larvae are exposed to a huge number of microbes from external and internal sources in intensive aquaculture, but their relative importance depend on the rearing technology used (especially flow-through vs. recirculating systems) and the retention time of the water in the fish tanks. Generally, focus has been on microbes entering the system, but microbes from growth within the system is normally a substantial part of the microbes encountered by larvae. Culture independent methods have revealed an unexpected high richness of bacterial species associated with larvae, with 100-250 operational taxonomic units associated with one individual. The microbiota of larvae changes rapidly until metamorphosis, most likely due to changes in the selection pressure in the digestive tract caused by changes in host-microbe and microbe-microbe interactions. Even though the microbiota of larvae is distinctly different from the microbiota of the water and the live food, the microbiota of the water strongly affects the microbiota of the larvae. We are in the early phase of understanding larvae-microbe interactions in vivo, but some studies with other animals than fish emphasize that we so far have underestimated the complexity of these interactions. We present examples demonstrating the diversity of these interactions. A large variety of microbial management methods exist, focusing on non-selective reduction of microbes, selective enhancement of microbes, and on improvement of the resistance of larvae against microbes. However, relatively few methods have been studied extensively. We believe that there is a lot to gain by increasing the diversity of approaches for microbial management. As many microbial management methods are perturbations of the microbial community, we argue that ecological theory is needed to foresee and test for longer term consequences in microbe-microbe and microbe-larvae interactions. We finally make some recommendations for future research and development.

N-acyl homoserine lactone-degrading microbial enrichment cultures isolated from Penaeus vannamei shrimp gut and their probiotic properties in Brachionus plicatilis cultures.

Three bacterial enrichment cultures (ECs) were isolated from the digestive tract of Pacific white shrimp Penaeus vannamei, by growing the shrimp microbial communities in a mixture of N-acyl homoserine lactone (AHL) molecules. The ECs, characterized by denaturing gradient gel electrophoresis analysis and subsequent rRNA sequencing, degraded AHL molecules in the degradation assays. Apparently, the resting cells of the ECs also degraded one of the three types of quorum-sensing signal molecules produced by Vibrio harveyi in vitro [i.e. harveyi autoinducer 1 (HAI-1)]. The most efficient AHL-degrading ECs, EC5, was tested in Brachionus experiments. EC5 degraded the V. harveyi HAI-1 autoinducer in vivo, neutralizing the negative effect of V. harveyi autoinducer 2 (AI-2) mutant, in which only the HAI-1- and CAI-1-mediated components of the quorum-sensing system are functional on the growth of Brachionus. This suggests that EC5 interferes with HAI-1-regulated metabolism in V. harveyi. These AHL-degrading ECs need to be tested in other aquatic systems for their probiotic properties, preferably in combination with specific AI-2-degrading bacteria.

Preparation and Characterization of Alginate Microparticles Containing a Model Protein for Oral Administration in Gnotobiotic European Sea Bass (Dicentrarchus labrax) Larvae.

Aquaculture is the fastest growing animal production sector. However, the production of marine fish is still hampered by the high mortality rate in the first few weeks after hatching. Mortality in larvae is often caused by microbial infections. Today, the incorporation of immunostimulants into microparticles provides us new tools to enhance disease resistance in marine larviculture. In this study, we prepared alginate microparticles loaded with the model antigen fluorescein isothiocyanate conjugated-bovine serum albumin. Optimum concentrations of alginate and CaCl2, the correct alginate viscosity and the appropriate preparatory conditions led to the creation of desirable microparticles with the correct size for oral feeding in gnotobiotic European sea bass larvae. The prepared alginate microparticles were stable in sea water and were successfully ingested by gnotobiotic sea bass larvae at day after hatching 7 without causing any negative effects. Results suggest the suitability of this drug delivery system for targeting the innate immune system of fish larvae in order to enhance disease resistance and thus reduce mortality in larviculture.

Identification of cultured Brachionus rotifers based on RFLP and SSCP screening.

The marine finfish industry worldwide depends greatly on the mass culture of Brachionus rotifers. Recently, molecular data have revealed a more complicated view about the species status of Brachionus rotifers than previous mainly morphological assessments. Under this view, Brachionus rotifers are comprised of many morphologically similar, albeit genetically differentiated, cryptic members of larger groups. A redefinition of the cultured rotifer species/biotypes is therefore needed if aquaculture is to reach higher levels of standardization and predictability. In this work, restriction fragment length polymorphism (RFLP) and single-strand conformational polymorphism (SSCP) methods are applied to the COI and 16S rRNA mitochondrial genes. A detailed COI restriction map was constructed, using sequence data from all known representatives of Brachionus phylogroups. Therefore, it is the first time that such an extended restriction database has been produced. Several restriction endonucleases are proposed for the discrimination of the different Brachionus species/biotypes. Furthermore, eight different SSCP gel alleles are described for the 16S region. Using these data, five Brachionus species/biotypes were identified in 78 samples collected from laboratories and hatcheries around the world.

Effect of poly-ß-hydroxybutyrate on growth and disease resistance of Nile tilapia Oreochromis niloticus juveniles.

The growth promoting effect of the bacterial storage compound poly-ß-hydroxybutyrate (PHB) has been studied for young fish of high trophic level (European sea bass) and intermediate trophic level (Siberian sturgeon). Here, the effect of PHB on growth, digestive enzyme activities, body composition and diseases resistance of juvenile Nile tilapia (Oreochromis niloticus) of low trophic level was investigated. Although dietary PHB supplementation (5, 25 and 50 g PHB kg(-1) formulated semi-purified diet) during 28 days resulted in a trend of increased weight gain, there was no significant difference in the mean final body weight (258-284 mg) when compared to the fish from the control group (on average 218 mg). Lipase activity increased significantly with about 20-40% by the supplementation of PHB in the diet, which may have led to the significant increase in total lipid content with about 10% in the PHB treatment groups. However, the profile of total (n-6) fatty acids (FAs), total monounsaturated FAs and total saturated FAs relative to the total lipid was similar among various PHB treatments. An additional challenge test on gnotobiotic Nile tilapia larvae using the pathogen Edwardsiella ictaluri gly09R showed that feeding challenged larvae with PHB-enriched Artemia nauplii resulted in a 20% higher survival as compared to the challenged control larvae. Overall, it is suggested that the trend of increased body weight gain resulted from intestinal lipid digestion, absorption and deposition and that PHB is effective as an antimicrobial agent for application in Nile tilapia larviculture.

Impact of mucin, bile salts and cholesterol on the virulence of Vibrio anguillarum towards gnotobiotic sea bass (Dicentrarchus labrax) larvae.

In this study, we investigated the impact of the host factors mucin, bile salts and cholesterol on the virulence of the economically important aquatic pathogen Vibrio anguillarum towards sea bass larvae. Pretreatment of V. anguillarum with either one of the host factors (at 10 mg l(-1)) prior to inoculation into the sea bass rearing water increased virulence of the bacterium, although the effect of cholesterol was not significant. Each of the three host factors significantly increased several virulence-related phenotypes in V. anguillarum, i.e. protease activity, flagellar motility, biofilm formation and exopolysaccharide production, whereas there was no effect on growth of the bacterium under these conditions. Furthermore, the host factors increased the expression of genes involved in these phenotypes, i.e. the metalloprotease empA, the flagellar transcriptional regulator fleQ, the flagellin gene flaA, the chemotaxis methyltransferase gene cheR, the exopolysaccharide biosynthesis gene wbfD and the exopolysaccharide export gene wza. Our results indicate that V. anguillarum uses host mucin, bile salts, and cholesterol as cues to promote the expression of several important virulence traits that enhance the success of transmission from one host to another.

RpoS and indole signaling control the virulence of Vibrio anguillarum towards gnotobiotic sea bass (Dicentrarchus labrax) larvae.

Quorum sensing, bacterial cell-to-cell communication with small signal molecules, controls the virulence of many pathogens. In contrast to other vibrios, neither the VanI/VanR acylhomoserine lactone quorum sensing system, nor the three-channel quorum sensing system affects virulence of the economically important aquatic pathogen Vibrio anguillarum. Indole is another molecule that recently gained attention as a putative signal molecule. The data presented in this study indicate that indole signaling and the alternative sigma factor RpoS have a significant impact on the virulence of V. anguillarum. Deletion of rpoS resulted in increased expression of the indole biosynthesis gene tnaA and in increased production of indole. Both rpoS deletion and the addition of exogenous indole (50-100 µM) resulted in decreased biofilm formation, exopolysaccharide production (a phenotype that is required for pathogenicity) and expression of the exopolysaccharide synthesis gene wbfD. Further, indole inhibitors increased the virulence of the rpoS deletion mutant, suggesting that indole acts downstream of RpoS. Finally, in addition to the phenotypes found to be affected by indole, the rpoS deletion mutant also showed increased motility and decreased sensitivity to oxidative stress.

Does virulence assessment of Vibrio anguillarum using sea bass (Dicentrarchus labrax) larvae correspond with genotypic and phenotypic characterization?

BACKGROUND: Vibriosis is one of the most ubiquitous fish diseases caused by bacteria belonging to the genus Vibrio such as Vibrio (Listonella) anguillarum. Despite a lot of research efforts, the virulence factors and mechanism of V. anguillarum are still insufficiently known, in part because of the lack of standardized virulence assays. METHODOLOGY/PRINCIPAL FINDINGS: We investigated and compared the virulence of 15 V. anguillarum strains obtained from different hosts or non-host niches using a standardized gnotobiotic bioassay with European sea bass (Dicentrarchus labrax L.) larvae as model hosts. In addition, to assess potential relationships between virulence and genotypic and phenotypic characteristics, the strains were characterized by random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (rep-PCR) analyses, as well as by phenotypic analyses using Biolog's Phenotype MicroArray™ technology and some virulence factor assays. CONCLUSIONS/SIGNIFICANCE: Virulence testing revealed ten virulent and five avirulent strains. While some relation could be established between serotype, genotype and phenotype, no relation was found between virulence and genotypic or phenotypic characteristics, illustrating the complexity of V. anguillarum virulence. Moreover, the standardized gnotobiotic system used in this study has proven its strength as a model to assess and compare the virulence of different V. anguillarum strains in vivo. In this way, the bioassay contributes to the study of mechanisms underlying virulence in V. anguillarum.