Metabolic modulation of redox state confounds fish survival against Vibrio alginolyticus infection

Vibrio alginolyticus threatens both humans and marine animals, but hosts respond to V. alginolyticus infection is not fully understood. Here, functional metabolomics was adopted to investigate the metabolic differences between the dying and surviving zebrafish upon V. alginolyticus infection. Tryptophan was identified as the most crucial metabolite, whose abundance was decreased in the dying group but increased in the survival group as compared to control group without infection. Concurrently, the dying zebrafish displayed excessive immune response and produced higher level of reactive oxygen species (ROS). Interestingly, exogenous tryptophan reverted dying rate through metabolome re-programming, thereby enhancing the survival from V. alginolyticus infection. It is preceded by the following mechanism: tryptophan fluxed into the glycolysis and tricarboxylic acid cycle (TCA cycle), promoted adenosine triphosphate (ATP) production and further increased the generation of NADPH. Meanwhile, tryptophan decreased NADPH oxidation. These together ameliorate ROS, key molecules in excessive immune response. This is further supported by the event that the inhibition of pyruvate metabolism and TCA cycle by inhibitors decreased D. reiro survival. Thus, our data indicate that tryptophan is a key metabolite for the host to fight against V. alginolyticus infection, representing an alternative strategy to treat bacterial infection in an antibiotic-independent way.     

Loop‐mediated isothermal amplification method for rapid detection of Vibrio alginolyticus,the causative agent of vibriosis in mariculture fish

Aims: The purpose of this study was to develop a loop‐mediated isothermal amplification (LAMP) method for the rapid, sensitive and simple detection of Vibrio alginolyticus in mariculture fish. Methods and Results: LAMP primers were designed by targeting the gyrB gene. With Bst DNA polymerase, the target DNA can be clearly amplified for 60 min at 64°C in a simple water bath. The detection sensitivity of the LAMP assay for the detection of V. alginolyticus is about 3·7 × 10² CFU ml^?1 (3·7 CFU per reaction). LAMP products could be judged with agar gel or naked eye after the addition of SYBR Green I. There were no cross‐reactions with other bacterial strains indicating a high specificity of the LAMP. The LAMP method was applied to detect V. alginolyticus‐infected fish tissues effectively. Conclusions: The LAMP established in this study is a simple, sensitive, specific, inexpensive and rapid protocol for the detection of V. alginolyticus. Significance and Impact of the Study: This LAMP method provides an important diagnostic tool for the detection of V. alginolyticus infection both in the laboratory and field.     

Reduced redox-dependent mechanism and glucose-mediated reversal in gentamicin-resistant Vibrio alginolyticus

Strategy of managing antibiotic-resistant Vibrio alginolyticus, a bacterial pathogen that threatens human health and animal farming, is not available due to the lack of knowledge about the underlying mechanism of antibiotic resistance. Here, we showed that gentamicin-resistant V. alginolyticus (VA-R_GEN) has four mutations on metabolism and one mutation on a two-component system by whole-genome and PCR-based sequencing, indicating the metabolic shift in VA-R_GEN. Thus, metabolic profile was investigated by GC–MS based metabolomics. Glucose was identified as a crucial biomarker, whose abundance was decreased in VA-R_GEN. Further analysis with iPath, and gene expression and enzyme activity of the pyruvate cycle (the P cycle) demonstrated a global depressed metabolic pathway network in VA-R_GEN. Consistently, NADH, sodium-pumping NADH:ubiquinone oxidoreductase (Na(+)-NQR) system, membrane potential and intracellular gentamicin were decreased in VA-R_GEN. These findings indicate that the reduced redox state contributes to antibiotic resistance. Interestingly, exogenous glucose potentiated gentamicin to efficiently kill VA-R_GEN through the promotion of the P cycle, NADH, membrane potential and intracellular gentamicin. The potentiation was further confirmed in a zebrafish model. These results indicate that the gentamicin resistance reduces the P cycle and Na(+)-NQR system and thereby decreases redox state, membrane potential and gentamicin uptake, which can be reversed by exogenous glucose.     

Nutrient cycling in early coral life stages: Pocillopora damicornis larvae provide their algal symbiont (Symbiodinium) with nitrogen acquired from bacterial associates

The waters surrounding coral reef ecosystems are generally poor in nutrients, yet their levels of primary production are comparable with those reported from tropical rain forests. One explanation of this paradox is the efficient cycling of nutrients between the coral host, its endosymbiotic alga Symbiodinium and a wide array of microorganisms. Despite their importance for the animals' fitness, the cycling of nutrients in early coral life stages and the initial establishment of partnerships with the microbes involved in these processes has received little scrutiny to date. Nitrogen is an essential but limited nutrient in coral reef ecosystems. In order to assess the early nutrient exchange between bacteria and corals, coral larvae of the species Pocillopora damicornis were incubated with two coral‐associated bacteria (Alteromonas sp., or Vibrio alginolyticus), prelabeled with the stable nitrogen isotope ¹⁵N. The incorporation and translocation of nitrogen from Vibrio‐ and Alteromonas bacteria into P. damicornis coral larvae and specifically into the coral‐symbiotic Symbiodinium were detected by nanoscale secondary ion mass spectrometry (NanoSIMS). A significant increase in the amount of enriched ¹⁵N (two to threefold compared to natural abundance) was observed in P. damicornis larvae within 8 h of incubation for both bacterial treatments (one‐way ANOVA, F_5,53 = 18.03, P = 0.004 for Alteromonas sp. and F_5,53 = 18.03, P = 0.0001 for V. alginolyticus). These findings reveal that coral larvae acquire nutrients previously taken up from the environment by bacteria. The additional nitrogen may increase the survival rate and fitness of the developing coral and therefore contribute to the successful maintenance of coral reefs.     

Genotype and diet affect resistance,survival, and fecundity but not fecundity tolerance

Insects are exposed to a variety of potential pathogens in their environment, many of which can severely impact fitness and health. Consequently, hosts have evolved resistance and tolerance strategies to suppress or cope with infections. Hosts utilizing resistance improve fitness by clearing or reducing pathogen loads, and hosts utilizing tolerance reduce harmful fitness effects per pathogen load. To understand variation in, and selective pressures on, resistance and tolerance, we asked to what degree they are shaped by host genetic background, whether plasticity in these responses depends upon dietary environment, and whether there are interactions between these two factors. Females from ten wild‐type Drosophila melanogaster genotypes were kept on high‐ or low‐protein (yeast) diets and infected with one of two opportunistic bacterial pathogens, Lactococcus lactis or Pseudomonas entomophila. We measured host resistance as the inverse of bacterial load in the early infection phase. The relationship (slope) between fly fecundity and individual‐level bacteria load provided our fecundity tolerance measure. Genotype and dietary yeast determined host fecundity and strongly affected survival after infection with pathogenic P. entomophila. There was considerable genetic variation in host resistance, a commonly found phenomenon resulting from for example varying resistance costs or frequency‐dependent selection. Despite this variation and the reproductive cost of higher P. entomophila loads, fecundity tolerance did not vary across genotypes. The absence of genetic variation in tolerance may suggest that at this early infection stage, fecundity tolerance is fixed or that any evolved tolerance mechanisms are not expressed under these infection conditions.     

An in vitro and in vivo assessment of the potential of Vibrio spp. as probiotics for the Pacific White shrimp,Litopenaeus vannamei

Aims: The objective of the work was to determine whether known strains of nonpathogenic vibrios can act as probiotics for the control of Vibrio infections in the Pacific white shrimp, Litopenaeus vannamei. Methods and Results: Of the ten species tested, only Vibrio alginolyticus (NCIMB 1339) and Vibrio gazogenes (NCIMB 2250) showed antagonistic activity towards a panel of shrimp pathogenic vibrios. In the case of V. alginolyticus, this activity depended on the presence of live bacteria while in V. gazogenes both live and dead bacteria showed anti‐Vibrio activity. Injection of shrimp with either V. alginolyticus or V. gazogenes at 3 × 10⁷ or 3 × 10⁵ total bacteria per shrimp resulted in mortality with higher levels in the case of V. alginolyticus (100% mortality 18 h postinjection of 3 × 10⁷ bacteria). Juvenile shrimp were fed commercial diets top‐coated with either chitin (an immune stimulant) or chitin + V. gazogenes. Both chitin and V. gazogenes caused a significant decline in the number of Vibrio‐like bacteria in the fore and hind gut, and changes were also seen in the hepatosomatic index (a measure of digestive health) and the total number of blood cells in circulation. Analysis of mid/hindgut and faecal samples obtained using terminal restriction fragment length polymorphism showed that the gut microbiota of shrimp has limited bacterial diversity and that after 8 weeks exposure to the experimental diets there were significant changes in the microbial flora of the GI tract of shrimp as a result of the presence of V. gazogenes. Conclusions: Of the vibrios tested, V. gazogenes has potential as a probiotic for the control of bacterial diseases in shrimp. Significance and Impact of the Study: Overall, this study shows the promise of V. gazogenes together with chitin to improve the health and welfare of shrimp under aquaculture conditions.     

Shrimp that have received carrageenan via immersion and diet exhibit immunocompetence in phagocytosis despite a post-plateau in immune parameters

The effect of carrageenan on the immune response of white shrimp Litopenaeus vannamei, was studied in vitro and in vivo. Shrimp haemocytes receiving carrageenan at 1 mg ml⁻¹ experienced change in cell size, reduction in cell viability, increase in PO activity, serine proteinase activity, and RB in vitro. Shrimp received carrageenan via immersion at 200, 400 and 600 mg L⁻¹ after 3 h and orally at 0.5, 1.0 and 2.0 g kg⁻¹ after 3 weeks showed higher proliferation of haematopoietic tissues (HPTs) together with increases in haemocyte count and other immune parameters. Shrimp that fed a diet containing carrageenan at 0.5 g kg⁻¹ after 3 weeks significantly up-regulated gene expressions of several immune-related proteins. The immune parameters of shrimp that received carrageenan via immersion and orally increased to a plateau after 3 h and after 3 weeks, but decreased after 5 h and 6 weeks, respectively. Phagocytosis and clearance of Vibrio alginolyticus remained high in shrimp that had received carrageenan via immersion after 5 h and orally after 6 weeks, respectively. Resistances of shrimp against V. alginolyticus and white spot syndrome virus were higher over 24–144 h and 72–144 h, respectively in shrimp that received carrageenan at 600 mg L⁻¹ via immersion after 3 and 5 h. It was concluded that carrageenan effectively triggers an innate immunity in vitro, and increases mitotic index of HPT, immune parameters, gene expressions and resistance against pathogens in vivo. Shrimp received carrageenan via immersion and orally exhibited immunocompetence in phagocytosis and clearance of V. alginolyticus, and resistance to pathogen despite the trend in immune parameters to recover to background values.     

Melanic mutation causes a fitness decline in bean beetles infected by Wolbachia

Wolbachia cannot live outside a host, which is thought to be the reason for host‐Wolbachia coevolution toward benign parasitism, especially because the fitness of Wolbachia is traded against its host's fitness. Insect melanism has been reported to have a positive effect on pathogen resistance, but melanic mutants of Callosobruchus analis (Fabricius) and Callosobruchus chinensis (L.) (Coleoptera: Chrysomelidae) are infected with Wolbachia. Callosobruchus chinensis is infected with CI‐inducing Wolbachia, and melanic mutants exhibit fitness decline. Interestingly, this decline is not observed in C. analis melanic mutants that are infected with CI‐free Wolbachia. Our research question is whether the infection of CI‐inducing Wolbachia causes fitness decline of melanic hosts in C. analis. We examined fecundity, fertility, and longevity of C. analis melanic mutants and compared them between uninfected and infected hosts with CI‐inducing Wolbachia. Infected melanic mutants of C. analis exhibited fitness decline leading to reduced hatch rates even when parental combinations were compatible. Wolbachia can invade a host population by causing CI to decrease the fraction of uninfected hosts, but melanic mutant hosts decrease the number of infected hosts through fitness decline. Nevertheless, the melanism in hosts is not able to stop Wolbachia invasion in C. analis.     

The potential for arms race and Red Queen coevolution in a protist host–parasite system

The dynamics and consequences of host–parasite coevolution depend on the nature of host genotype‐by‐parasite genotype interactions (G × G) for host and parasite fitness. G × G with crossing reaction norms can yield cyclic dynamics of allele frequencies (“Red Queen” dynamics) while G × G where the variance among host genotypes differs between parasite genotypes results in selective sweeps (“arms race” dynamics). Here, we investigate the relative potential for arms race and Red Queen coevolution in a protist host–parasite system, the dinoflagellate Alexandrium minutum and its parasite Parvilucifera sinerae. We challenged nine different clones of A. minutum with 10 clones of P. sinerae in a fully factorial design and measured infection success and host and parasite fitness. Each host genotype was successfully infected by four to ten of the parasite genotypes. There were strong G × Gs for infection success, as well as both host and parasite fitness. About three quarters of the G × G variance components for host and parasite fitness were due to crossing reaction norms. There were no general costs of resistance or infectivity. We conclude that there is high potential for Red Queen dynamics in this host–parasite system.     

A novel approach to parasite population genetics: Experimental infection reveals geographic differentiation,recombination and host‐mediated population structure in Pasteuria ramosa,a bacterial parasite of Daphnia

The population structure of parasites is central to the ecology and evolution of host‐parasite systems. Here, we investigate the population genetics of Pasteuria ramosa, a bacterial parasite of Daphnia. We used natural P. ramosa spore banks from the sediments of two geographically well‐separated ponds to experimentally infect a panel of Daphnia magna host clones whose resistance phenotypes were previously known. In this way, we were able to assess the population structure of P. ramosa based on geography, host resistance phenotype and host genotype. Overall, genetic diversity of P. ramosa was high, and nearly all infected D. magna hosted more than one parasite haplotype. On the basis of the observation of recombinant haplotypes and relatively low levels of linkage disequilibrium, we conclude that P. ramosa engages in substantial recombination. Isolates were strongly differentiated by pond, indicating that gene flow is spatially restricted. Pasteuria ramosa isolates within one pond were segregated completely based on the resistance phenotype of the host—a result that, to our knowledge, has not been previously reported for a nonhuman parasite. To assess the comparability of experimental infections with natural P. ramosa isolates, we examined the population structure of naturally infected D. magna native to one of the two source ponds. We found that experimental and natural infections of the same host resistance phenotype from the same source pond were indistinguishable, indicating that experimental infections provide a means to representatively sample the diversity of P. ramosa while reducing the sampling bias often associated with studies of parasite epidemics. These results expand our knowledge of this model parasite, provide important context for the large existing body of research on this system and will guide the design of future studies of this host‐parasite system.     

Phenological asynchrony between host plant and gypsy moth reduces insect gut microbiota and susceptibility to Bacillus thuringiensis

The phenological synchrony between the emergence of overwintering herbivorous insects and the budding of host plants is considered a crucial factor in the population dynamics of herbivores. However, the mechanisms driving the interactions between the host plant, herbivores, and their pathogens are often obscure. In the current study, an artificially induced phenological asynchrony was used to investigate how the asynchrony between silver birch Betula pendula and gypsy moth Lymantria dispar affects the immunity of the insect to bacteria, its susceptibility to the entomopathogenic bacteria Bacillus thuringiensis, and the diversity in its midgut microbiota. The lysozyme‐like activity in both the midgut and hemolymph plasma and the nonspecific esterase activity and antimicrobial peptide gene expression in the midgut were studied in both noninfected and B. thuringiensis‐infected larvae. Our results provide the first evidence that phenologically asynchronous larvae are less susceptible to B. thuringiensis infection than phenologically synchronous larvae, and our results show that these effects are related to the high basic levels and B. thuringiensis‐induced levels of lysozyme‐like activities. Moreover, a 16S rRNA analysis revealed that dramatic decreases in the diversity of the larval gut bacterial consortia occurred under the effect of asynchrony. Larvae infected with B. thuringiensis presented decreased microbiota diversity if the larvae were reared synchronously with the host plant but not if they were reared asynchronously. Our study demonstrates the significant effect of phenological asynchrony on innate immunity‐mediated interactions between herbivores and entomopathogenic bacteria and highlights the role of nonpathogenic gut bacteria in these interactions.     

Distribution of some virulence related-properties of Vibrioalginolyticus strains isolated from Mediterranean seawater (Bay of Khenis, Tunisia): investigation of eight Vibriocholerae virulence genes

This study characterizes 28 Vibrio alginolyticus strains isolated from seawater from the Seacoast of Monastir (Khenis; Tunisia). V. alginolyticus were isolated using the TCBS modified agar plates and the biochemical activities were tested using RapID NF plus Strips. Proteases activities, hemolysis, antibiotics susceptibility, and adhesion to fish mucus and epithelial cell lines (Hep-2 and Caco-2) were also investigated. Eight Vibrio cholerae virulence genes (toxR, toxS, toxRS, toxT, ctxA, vpi, ace, zot) were investigated by PCR in genomes of V. alginolyticus strains. Most of the studied strains were β-haemolytic and produce many proteolytic enzymes. All isolates described here were resistant to several antibiotics tested. Six strains were able to adhere strongly to both Hep-2 and Caco-2 cell lines. The PCR investigation of V. cholerae genes showed a large distribution among the genomes of all V. alginolyticus strains. The toxR operon was found in 9 V. alginolyticus strains out of 28 studied. Only one strain was positive for the toxS and toxRS respectively. Five strains showed a positive amplification for the virulence pathogenic island (vpi), seven for the toxT, 3 for the ctxA and 9 for the Zonula occludens toxin (zot). The bay of Khenis harbors different genotypes of V. alginolyticus strains who inheritated several virulence genes from autochthones bacteria such as V. cholerae. These strains were able to produce several virulence enzymes and exhibit a high power to adhere to human epithelial cells and fish mucus.     

Temperature and oxygen regulated expression of a glutamine synthetase gene fromVibrio alginolyticus cloned inEscherichia coli

Glutamine synthetase (GS) synthesis inVibrio alginolyticus was regulated by temperature, oxygen and nitrogen levels. A GS gene,glnA fromV. alginolyticus was cloned on a 5.67 kb insert in the recombinant plasmid pRM210, which enabledEscherichia coli glnA, ntrB, ntrC deletion mutants to utilize (NH₄)₂SO₄ as a sole source of nitrogen. TheV. alginolyticus glnA gene was expressed from a regulatory region contained within the cloned fragment.V. alginolyticus glnA expression from pRM210 was subject to regulation by temperature, oxygen and nitrogen levels. GS specific activity in anE. coli wild-type strain was not affected by temperature or oxygen. pRM211 was a deletion derivative of pRM210 and GS production by pRM211 was not regulated by temperature, oxygen or nitrogen levels inE. coli.Abbreviation GS glutamine synthetase     

Characterization of role of the toxR gene in the physiology and pathogenicity of Vibrio alginolyticus

toxR, a conserved virulence-associated gene in vibrios, is identified in Vibrio alginolyticus ZJ51-O, a pathogenic strain isolated from diseased fish. To reveal the role of ToxR in the pathogenicity of V. alginolyticus, a deletion mutant was constructed by allelic exchange. The mutant showed the same level of growth in trypticase soy broth (TSB) and iron-limiting condition, as the wild type strain. However, deletion of toxR severely reduced resistance against bile salts and the capability of biofilm formation. Outer-membrane protein (OMP) analysis showed that a 37-kD protein was absent and a 43-kD protein was decreased in the mutant. By MS/MS, the two proteins are identified as the homologues of OmpT and OmpN, respectively. These data suggest that ToxR might have enhanced the bile resistance and biofilm formation through modulating the production of OMP without affecting the ability of iron acquisition and the virulence to the fish via injection. These results indicate that ToxR may assist V. alginolyticus to colonize on the surface of the fish intestine which is crucial for the initiation of the infection, though it may not be involved in the proliferation of the bacteria in the host tissue.     

Physiological and Biochemical Characteristics of the Halophilic Bacteria Vibrio parahaemolyticus and V. alginolyticus Isolated from Marine Invertebrates of Peter the Great Bay, Sea of Japan

One hundred strains of halophilic vibrios were isolated from 16 species of marine invertebrates of Peter the Great Bay. Based on their morphological and biochemical characteristics, the bacteria were identified as Vibrio parahaemolyticus and Vibrio alginolyticus. Bacterial isolates possessed virulence enzymes (DNAase, lecithinase, catalase) and were characterized by a high enterotoxigenicity. It was determined that 76% of the V. parahaemolyticus strains and 43% of the V. alginolyticus strains were Kanagawa-positive. The isolates showed a high adhesive capability, the average adhesion index was 18.06 cells per erythrocyte for V. parahaemolyticus and 12.55 for V. alginolyticus. The results of this study suggest a high pathogenic potential of the isolated halophilic vibrios, which are an epidemic hazard to marine invertebrates and to humans.     

Innate immunity of the white shrimp Litopenaeus vannamei weakened by the combination of a Vibrio alginolyticus injection and low-salinity stress

White shrimp Litopenaeus vannamei were reared at a salinity of 35‰ without a Vibrio alginolyticus injection (unchallenged group), and other shrimp were reared at 35‰, injected with tryptic-soy broth (TSB)-grown V. alginolyticus at 1.8 × 10⁵ colony-forming units (cfu) shrimp^?1 (challenged group), and then examined for the hyaline cell (HC) count, granular cell (GC, including semi-granular cell) count, total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB) and superoxide dismutase (SOD) activity after transfer to 35‰ (control), 25‰, 20‰, and 15‰ for 1, 6, 12, 24, 72, and 120 h. Results indicated that the haemocyte count, PO activity, RB, and SOD activity of unchallenged shrimp and challenged shrimp that were transferred to low-salinity levels all began to significantly decrease at 6, 6, 6, and 1 h, respectively, and reached the lowest levels at 12 h. HC, GC, the THC, PO activity, RB, and SOD activity of unchallenged shrimp that were transferred to 15‰ decreased by 53%, 41%, 49%, 68%, 39%, and 62%, whereas those parameters of challenged shrimp that were transferred to 15‰ decreased by 79%, 78%, 79%, 82%, 54%, and 72%, respectively after 12 h compared to control shrimp. These immune parameters began to recover after 24–72 h for both unchallenged shrimp and challenged shrimp. We concluded that the innate immunity was weakened in white shrimp L. vannamei that received combined stresses of a V. alginolyticus injection, and low-salinity transfer. It was also concluded that shrimp with respectively 21%, 18%, 46%, and 28% lower THC, PO activity, RB, and SOD activity of the original values would be killed due to decreases in their immunity, and resistance to V. alginolyticus infection. Shrimp farming should be maintained at a constant high salinity level to prevent exacerbated decreases in innate immune parameters of shrimp when infected by a pathogen coupled with low-salinity stress leading to mortality.     

Expression,purification and antibody preparation of flagellin FlaA from Vibrio alginolyticus strain HY9901

Aims: The main aims of this study were to clone and express flagellin flaA gene from Vibrio alginolyticus strain HY9901, also to prepare mouse anti‐FlaA polyclonal antibody for future pathogen or vaccine study. Methods and Results: The full‐length flaA gene was amplified by PCR with designed primers. The open reading frame of flaA gene contains 1131 bp, and its putative protein consists of 376 amino acid residues. Alignment analysis indicated that the FlaA protein was highly conserved. SDS–PAGE indicated that the FlaA protein was successfully expressed in Escherichia coli BL21 (DE3). Then, the recombinant FlaA protein was purified by affinity chromatography, and the mouse anti‐FlaA serum was produced. The expression of flaA gene was verified by various immunological methods, including western blotting, enzyme‐linked immunosorbent assay (ELISA) and immunogold electron microscopy (IEM). Conclusions: Flagellin flaA gene was cloned and identified from V. alginolyticus HY9901, the recombinant FlaA protein was expressed and purified, and high‐titre FlaA protein‐specific antibody was produced. Western blot analysis revealed that the prepared antiserum not only specifically react to FlaA fusion protein, but also to natural FlaA protein of V. alginolyticus. The expressed FlaA protein was demonstrated, for the first time, as the component of flagella from V. alginolyticus by IEM. Significance and Impact of the Study: This study may offer important insights into the pathogenesis of V. alginolyticus, provide a base for further studies on the diagnosis and evaluation that whether the FlaA protein could be used as an effective vaccine candidate against infection by V. alginolyticus and other Vibrio species. Additionally, the purified FlaA protein and polyclonal antibody can be used for further functional and structural studies.     

Experimental Vibriosis Induction with Vibrio alginolyticus of Larvae of the Catarina Scallop (Argopecten ventricosus =circularis) (Sowerby II, 1842)

Abstract Six-day-old larvae of the catarina scallop, Argopecten ventricosus (=circularis), were infected with different concentrations of Vibrio alginolyticus to determine virulence and to describe vibriosis in this species. The development of vibriosis was compared to the effect of the supernatant of a 24-h V. alginolyticus culture. An experimental larvae culture system (ELCS) yielded a maximum survival of 80% from the 6th to the 19th day (control and low concentrations of V. alginolyticus). No effect was shown with concentrations of V. alginolyticus below 0.5 × 10⁵ CFU ml⁻¹. At concentrations higher than 5.0 × 10⁵ CFU ml⁻¹, swimming depletion, empty stomachs, lipidic granules in the digestive system, velum degradation, and massive mortality were observed. The supernatant of V. alginolyticus culture showed similar effects to the highest concentrations of V. alginolyticus cells. Received: 13 November 1996; Accepted 28 March 1997