Poly-beta-hydroxybutyrate-accumulating bacteria protect gnotobiotic Artemia franciscana from pathogenic Vibrio campbellii

A poly-beta-hydroxybutyrate (PHB)-accumulating enrichment culture was obtained using activated sludge from a polyphosphate-accumulating reactor as inoculum. PHB accumulated by the enrichment culture significantly enhanced the survival of Artemia nauplii, infected with the virulent pathogen Vibrio campbellii LMG 21363. A strain was isolated from the enrichment culture, based on its ability to accumulate PHB, and 16S rRNA gene sequencing of the isolate revealed 99% sequence similarity to Brachymonas denitrificans AS-P1. The isolate, named PHB2, showed good PHB-accumulating activity (up to 32% of the cell dry weight). PHB accumulated by isolate PHB2 was able to protect Artemia completely from the V. campbellii strain. Our data indicate that PHB-accumulating bacteria, such as B. denitrificans PHB2, could be used as an an effective and economically interesting alternative strategy to control infections in aquaculture.     

Monitoring of Vibrio harveyi quorum sensing activity in real time during infection of brine shrimp larvae

Quorum sensing, bacterial cell-to-cell communication, has been linked to the virulence of pathogenic bacteria. Indeed, in vitro experiments have shown that many bacterial pathogens regulate the expression of virulence genes by this cell-to-cell communication process. Moreover, signal molecules have been detected in samples retrieved from infected hosts and quorum sensing disruption has been reported to result in reduced virulence in different host–pathogen systems. However, data on in vivo quorum sensing activity of pathogens during infection of a host are currently lacking. We previously reported that quorum sensing regulates the virulence of Vibrio harveyi in a standardised model system with gnotobiotic brine shrimp (Artemia franciscana) larvae. Here, we monitored quorum sensing activity in Vibrio harveyi during infection of the shrimp, using bioluminescence as a read-out. We found that wild-type Vibrio harveyi shows a strong increase in quorum sensing activity early during infection. In this respect, the bacteria behave remarkably similar in different larvae, despite the fact that only half of them survive the infection. Interestingly, when expressed per bacterial cell, Vibrio harveyi showed around 200-fold higher maximal quorum sensing-regulated bioluminescence when associated with larvae than in the culture water. Finally, the in vivo quorum sensing activity of mutants defective in the production of one of the three signal molecules is consistent with their virulence, with no detectable in vivo quorum sensing activity in AI-2- and CAI-1-deficient mutants. These results indicate that AI-2 and CAI-1 are the dominant signals during infection of brine shrimp.     

Quorum sensing-disrupting brominated furanones protect the gnotobiotic brine shrimp Artemia franciscana from pathogenic Vibrio harveyi, Vibrio campbellii, and Vibrio parahaemolyticus isolates

Autoinducer 2 (AI-2) quorum sensing was shown before to regulate the virulence of Vibrio harveyi towards the brine shrimp Artemia franciscana. In this study, several different pathogenic V. harveyi, Vibrio campbellii, and Vibrio parahaemolyticus isolates were shown to produce AI-2. Furthermore, disruption of AI-2 quorum sensing by a natural and a synthetic brominated furanone protected gnotobiotic Artemia from the pathogenic isolates in in vivo challenge tests.     

Luminescence, virulence and quorum sensing signal production by pathogenic Vibrio campbellii and Vibrio harveyi isolates

Aims:  To study the relationship between luminescence, autoinducer production and virulence of pathogenic vibrios. Methods and Results:  Luminescence, quorum sensing signal production and virulence towards brine shrimp nauplii of 13 Vibrio campbellii and Vibrio harveyi strains were studied. Although only two of the tested strains were brightly luminescent, all of them were shown to produce the three different types of quorum sensing signals known to be produced by Vibrio harveyi. Cell-free culture fluids of all strains significantly induced bioluminescence in the cholerae autoinducer 1, autoinducer 2 and harveyi autoinducer 1 reporter strains JAF375, JMH597 and JMH612, respectively. There was no relation between luminescence and signal production and virulence towards brine shrimp. Conclusions:  There is a large difference between different strains of Vibrio campbellii and Vibrio harveyi with respect to bioluminescence. However, this is not reflected in signal production and virulence towards gnotobiotic brine shrimp. Moreover, there seems to be no relation between quorum sensing signal production and virulence towards brine shrimp. Significance and Impact of the Study:  The results presented here indicate that strains that are most brightly luminescent are not necessarily the most virulent ones and that the lower virulence of some of the strains is not due to a lack of autoinducer production.     

The effect of copper concentration on the virulence of pathogenic Vibrio harveyi

AIM: To demonstrate the influence of copper on luminescence and toxin production in Vibrio harveyi. METHODS AND RESULTS: The effect of copper concentration on the expression of both luminescence and toxin of V. harveyi was investigated. Copper concentration of less than 40 ppm had no effect on the growth. While V. harveyi cultured with 40 ppm copper concentration showed decreased luminescence as measured by spectrofluorophotometer and as observed. LuxD gene, which is related to luminescence expression, was monitored using real-time RT-PCR. Result showed that the concentration of cDNA coding for luxD was lower in V. harveyi with copper. Toxic activity against both HeLa cells and shrimp haemocytes was also lower in the culture supernatant of V. harveyi grown with 40 ppm copper concentration. Moreover, V. harveyi extracellular proteins were analysed using SDS-PAGE. Results showed that culture supernatant from V. harveyi grown without copper had thicker band indicating a higher concentration of the putative cysteine protease, one of the major toxin of V. harveyi. CONCLUSIONS: This study proved that both luminescence and toxin were repressed by copper. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study demonstrated that copper inhibited expression of phenotype of V. harveyi. Furthermore, it may inhibit quorum sensing of V. harveyi.     

The bacterial storage compound poly-beta-hydroxybutyrate protects Artemia franciscana from pathogenic Vibrio campbellii

Infections caused by antibiotic-resistant luminescent Vibrios can cause dramatic losses in aquaculture. In this study, the short-chain fatty acid beta-hydroxybutyrate and its polymer poly-beta-hydroxybutyrate were investigated as possible new biocontrol agents. beta-Hydroxybutyrate was shown to completely inhibit the growth of pathogenic Vibrio campbelli at 100 mM. Moreover, the addition of 100 mM of this fatty acid to the culture water of Artemia nauplii infected with the V. campbelli strain significantly increased the survival of the nauplii. As Artemia is a non-selective and particle filter feeder, we also investigated whether poly-beta-hydroxybutyrate particles could be used to protect Artemia from the pathogenic V. campbellii. The addition of 100 mg l(-1) poly-beta-hydroxybutyrate or more to the Artemia culture water offered a preventive and curative protection from the pathogen as a significantly enhanced survival was noticed. If added as a preventive treatment, a complete protection of infected nauplii (no significant mortality compared with uninfected nauplii) was observed at 1000 mg l(-1) poly-beta-hydroxybutyrate. Our data indicate that the use of poly-beta-hydroxybutyrate might constitute an ecologically and economically sustainable alternative strategy to fight infections in aquaculture.     

Identification of a novel DNA methyltransferase 2 from the brine shrimp, Artemia franciscana

DNA methyltransferase 2 (Dnmt2) is a dual-specificity DNA methyltransferase, which contains a weak DNA methyltransferase and novel tRNA methyltransferase activity. However, its biological function is still enigmatic. To elucidate the expression profiles of Dnmt2 in Artemia franciscana, we isolated the gene encoding a Dnmt2 from A. franciscana and named it as AfDnmt2. The cDNA of AfDnmt2 contained a 1140-bp open reading frame that encoded a putative Dnmt2 protein of 379 amino acids exhibiting 32% approximately 39% identities with other known Dnmt2 homologs. This is the first report of a DNA methyltransferase gene in Crustacean. By using semi-quantitative RT-PCR, AfDnmt2 was found to be expressed through all developmental stages and its expression increased during resumption of diapause cysts development. Southern blot analysis indicated the presence of multiple copies of AfDnmt2 genes in A. franciscana.     

Isolation of a cDNA encoding a putative SPARC from the brine shrimp, Artemia franciscana

SPARC (Secreted protein, acidic, rich in cysteine) is an extracellular matrix-associated and anti-adhesive glycoprotein extensively studied in vertebrates. Its presence among invertebrates has been reported in nematodes and flies. We cloned a cDNA containing a complete open reading frame for SPARC from the brine shrimp, Artemia franciscana. The amino acid sequence identity between the Artemia and the fly SPARCs was 55%, whereas that of the Artemia and the nematode proteins was 45%. Artemia and vertebrates exhibited a sequence identity of 30% in the predicted aa sequences. The SPARC consisted of four domains commonly found among reported SPARCs. The protein comprised 291 amino acids, having a signal peptide, a follistatin-like domain, one N-glycosylation site and one calcium-binding EF-hand motif. Fourteen cysteine residues conserved among all the secreted forms of SPARCs were present in the Artemia SPARC, and four extra cysteine residues were also found in it. The extra residues were conserved among SPARCs of the arthropods and the nematode. Phylogenetic analyses showed that the sequences of SPARCs were grouped into those of vertebrates and invertebrates. Though the structural organization of SPARC was conserved among all the species studied, SPARC within a group was highly conserved within that group, but divergent between the two. Northern blots revealed the presence of a 1.1 kb mRNA, which was faintly expressed in embryos and considerably detected in prenauplii and nauplii. The isolation of a SPARC cDNA from Artemia franciscana provides intriguing features of the divergent protein, SPARC.     

Characterization of ATP-dependent proteolysis in embryos of the brine shrimp, Artemia franciscana

Under anoxia, embryos of Artemia franciscana enter a state of quiescence. During this time protein synthesis is depressed, and continued degradation of proteins could jeopardize the ability to recover from quiescence upon return to favorable conditions. In this study, we developed an assay for monitoring ATP/ubiquitin-dependent proteolysis in order to establish the presence of this degradation mechanism in A. franciscana embryos, and to describe some characteristics that may regulate its function during anoxia-induced quiescence. For lysates experimentally depleted of adenylates, supplementation with ATP and ubiquitin stimulated protein degradation rates by 92 ± 17% (mean ± SE) compared to control rates. The stimulation by ATP was maximal at concentrations ≥11 μmol · l⁻¹. In the presence of ATP and ubiquitin, ubiquitin-conjugated proteins were produced by lysates during the course of the 4-h assays, as detected by Western blotting. Acute acidification of lysates to values approximating the intracellular pH observed under anoxia completely inhibited ATP/ubiquitin-dependent proteolysis. Depressed degradation was also observed under conditions where net ATP hydrolysis occurred. These results suggest that ATP/ubiquitin-dependent proteolysis is markedly inhibited under cellular conditions promoted by anoxia. Inhibition of proteolysis during quiescence may be one critical factor that increases macromolecular stability, which may ultimately govern the duration of embryo survival under anoxia. Accepted: 2 November 1999     

Cytogenetic proof that the brine shrimp Artemia franciscana (Crustacea, Branchiopoda) is found in Argentina

Artemia brine shrimps are commercially important and they have been extensively studied. This branchiopod crustacean is extensively used in aquaculture and other commercial and applied practices. The genus also awakes an increasing interest worldwide as an experimental model in other basic areas of research such as evolution and cytogenetics. In the present work adult male meiosis and nauplii mitotic cells in two Artemia populations from Argentina (Mar Chiquita and Las Tunas, Córdoba Province) are analyzed and compared to the reference strains A. franciscana (Great Salt Lake, Utah, USA) and A. persimilis (Salinas Grandes de Hidalgo, La Pampa Province, Argentina). The Mar Chiquita population shows the diploid and haploid numbers characteristic of A. franciscana, and a regular male meiosis. In the Las Tunas population diverse diploid and haploid numbers are encountered, and an irregular meiosis is observed in some individuals. Fluorescent DAPI and CMA banding shows bright chromocenters of intermediate size and number in both populations. Cytogenetic and phenotypic data show that the population of Mar Chiquita could be assigned to A. franciscana; the variation found in chromocenter size and number reflects an heterochromatin polytypism already observed in this species. The cytogenetic and morphological traits in Las Tunas population suggest an occasional hybridization between A. franciscana and A. persimilis.     

Vinblastine-induced aggregation of brine shrimp (Artemia) tubulin

Tubulin from the brine shrimp Artemia readily assembles in vitro in the absence of microtubule-associated proteins under conditions which do not permit assembly of tubulin from brain. Heated microtubule-associated protein preparations from bovine brain do, however, interact with Artemia tubulin, resulting in stimulation of tubulin assembly and formation of morphologically normal cold-sensitive microtubules. Addition of vinblastine to mixtures containing microtubules assembled in the presence of neural microtubule-associated proteins caused a drop and then a rise in turbidity of the solution. The turbidity changes were accompanied by the appearance of coils, presumably derived from the microtubules which disappeared upon addition of vinblastine. Coils also resulted when microtubule-associated proteins and vinblastine were added to tubulin before polymerization was initiated. Vinblastine prevented normal assembly and caused disruption of Artemia microtubules polymerized in the absence of microtubule-associated proteins. Under these conditions clumped or compact coils, different in appearance from those formed in the presence of the microtubule-associated proteins, were observed. The data confirm that tubulin from Artemia, an organism that is phylogenetically far removed from mammals, has retained binding sites for vinblastine and microtubule-associated proteins and that the interrelationship of these sites has been at least partially preserved. The incomplete depolymerization of Artemia microtubules in response to vinblastine when microtubule-associated proteins are absent suggests that the longitudinal tubulin-tubulin interactions involved in microtubule formation are more stable for Artemia than for neural tubulin.     

Intestinal uptake of lipovitellin from brine shrimp (Artemia franciscana) by larval inland silversides (Menidia beryllina) and striped bass (Morone saxatilis)

Intestinal uptake of lipovitellin (LV) from brine shrimp (Artemia franciscana) in larval inland silversides (Menidia beryllina) and striped bass (Morone saxatilis) was described using immunocytochemistry. Polyclonal antisera were raised against two subunits of LV (LV68 and LV190). When tested by immunocytochemistry, anti-LV68 showed cross-reactivity with some of the pancreatic cells especially in inland silversides. Therefore anti-LV190 was used to localize immunoreactive LV. Inland silversides at 14 days after hatching were fed Artemia nauplii and then sampled 4, 8, 12 hr after feeding. Similar experiments were carried out by using striped bass at 5 days and 15 days of age. They were sampled at 2, 4, 8, and 12 hr after feeding. Anterior enterocytes showed no evidence of uptake; however, the brush border of the cells of inland silversides reacted with the antiserum. Posterior enterocytes took up the LV and/or, possibly, their immunoreactive breakdown products. The pattern of uptake included accumulation in supranuclear vacuoles and digestion in supranuclear vacuoles, as suggested by the decay of the immunoreactivity over time. Thus, the posterior intestine of these larval fishes is the site of uptake and digestion of LV, an important nutritive component in the food of many larval fishes; this supports earlier findings using non-nutritive marker proteins.     

beta-Hydroxybutyrate in developing nauplii of brine shrimp (Artemia franciscana K.) under feeding and non-feeding conditions

Body content of beta-hydroxybutyrate, and individual dry mass, carbon content, and survival rate, were studied in developing nauplii of the brine shrimp Artemia franciscana K. from hatching to 96-97 h post hatching at 27 +/- 1 degrees C. The effect of two diets was studied in the experiment: Super Selco (SS) with a high lipid content; and Protein Selco (PS) with a high protein content. A starving group (S) was used as reference. The level of beta-hydroxybutyrate at hatching was 0.6 nmol.ind-1; it increased to 1.0-1.5 nmol.ind-1 in the SS- and S-groups, while in the PS-group it remained stable between 0.6-0.8 nmol.ind-1. At 60-80 h post hatch in the SS- and S-groups, the levels of beta-hydroxybutyrate were similar to the initial levels. The survival rate remained higher than 95% until 24 h post hatching in all groups. At the end of the experiment, the survival rate was 63% in the PS-group, 13% in the S-group and 3% in the SS-group. The Artemia nauplii individual dry mass and carbon content remained relatively stable in the SS-group; both parameters showed a significant increase in the PS-group and a significant decrease in the S-group. The results suggest that Artemia nauplii utilise ketone bodies as a fuel during development and growth, but that ketone catabolism may be overloaded by excessive lipid feeding resulting in increased mortality and possibly a ketotic acidosis.     

Colonization and dispersal patterns of the invasive American brine shrimp Artemia franciscana (Branchiopoda: Anostraca) in the Mediterranean region

Cysts of the brine shrimp Artemia franciscana are harvested from the Great Salt Lake (GSL) and San Francisco Bay (SFB) saltworks in the USA, and marketed worldwide to provide live food for aquaculture. This species has become invasive across several countries. We investigated (1) if the introduced populations in the Mediterranean region could have originated from these USA populations, (2) how the genetic diversity of Mediterranean compares to that at GSL and SFB, and (3) if genetic patterns in the Mediterranean can shed light on colonization routes. We sequenced a fragment of the cytochrome c oxidase subunit I and screened microsatellites loci from Mediterranean populations and the two putative USA sources. Haplotypes from Mediterranean populations were identical or closely related to those from SFB and GSL, and not related to other available American populations. Microsatellite analyses showed a reduced population diversity for most Mediterranean populations suggesting bottleneck effects, but few populations were showing similar or higher genetic diversity than native ones, which are likely to be admixed from both GSL and SFB because of multiple introductions. Results suggest natural dispersal, potentially via flamingos, between two Spanish populations. Our analyses show that all invaded populations could have originated from those commercialized USA populations.     

The gnotobiotic brine shrimp (Artemia franciscana) model system reveals that the phenolic compound pyrogallol protects against infection through its prooxidant activity

The phenolic compound pyrogallol is the functional unit of many polyphenols and currently there has been a growing interest in using this compound in human and animal health owing to its health-promoting effects. The biological actions of pyrogallol moiety (and polyphenols) in inducing health benefitting effects have been studied; however, the mechanisms of action remain unclear yet. Here, we aimed at unravelling the underlying mechanism of action behind the protective effects of pyrogallol against bacterial infection by using the gnotobiotically-cultured brine shrimp Artemia franciscana and pathogenic bacteria Vibrio harveyi as host-pathogen model system. The gnotobiotic test system represents an exceptional system for carrying out such studies because it eliminates any possible interference of microbial communities (naturally present in the experimental system) in mechanistic studies and furthermore facilitates the interpretation of the results in terms of a cause effect relationship. We provided clear evidences suggesting that pyrogallol pretreament, at an optimum concentration, induced protective effects in the brine shrimp against V. harveyi infection. By pretreating brine shrimp with pyrogallol in the presence or absence of an antioxidant enzyme mixture (catalase and superoxide dismutase), we showed that the Vibrio-protective effect of the compound was caused by its prooxidant action (e.g. generation of hydrogen peroxide, H₂O₂). We showed further that generation of prooxidant is linked to the induction of heat shock protein Hsp70, which is involved in eliciting the prophenoloxidase and transglutaminase immune responses. The ability of pyrogallol to induce protective immunity makes it a potential natural protective agent that might be a potential preventive modality for different host-pathogen systems.