Molecular characterization, cytoprotective, DNA protective, and immunological assessment of peroxiredoxin-1 (Prdx1) from yellowtail clownfish (Amphiprion clarkii).

Peroxiredoxin-1 (Prdx1) is a thiol-specific antioxidant enzyme that detoxifies reactive oxygen species (ROS) and regulates the redox status of cells. In this study, the Prdx1 cDNA sequence was isolated from the pre-established Amphiprion clarkii (A. clarkii) (AcPrdx1) transcriptome database and characterized structurally and functionally. The AcPrdx1 coding sequence comprises 597 bp and encodes 198 amino acids with a molecular weight of 22.1 kDa and a predicted theoretical isoelectric point of 6.3. AcPrdx1 is localized and functionally available in the cytoplasm and nucleus of cells. The TXN domain of AcPrdx1 comprises two peroxiredoxin signature VCP motifs, which contain catalytic peroxidatic (Cp-C52) and resolving cysteine (CR-C173) residues. The constructed phylogenetic tree and sequence alignment revealed that AcPrdx1 is evolutionarily conserved, and its most closely related counterpart is Amphiprion ocellaris. Under normal physiological conditions, AcPrdx1 was ubiquitously detected in all tissues examined, with the most robust expression in the spleen. Furthermore, AcPrdx1 transcripts were significantly upregulated in the spleen, head kidney, and blood after immune stimulation by polyinosinic:polycytidylic acid (poly (I:C)), lipopolysaccharide (LPS), and Vibrio harveyi injection. Recombinant AcPrdx1 (rAcPrdx1) demonstrated antioxidant and DNA protective properties in a concentration-dependent manner, as evidenced by insulin disulfide reduction, peroxidase activity, and metal-catalyzed oxidation (MCO) assays, whereas cells transfected with pcDNA3.1(+)/AcPrdx1 showed significant cytoprotective function under oxidative and nitrosative stress. Overexpression of AcPrdx1 in fathead minnow (FHM) cells led to a lower viral copy number following viral hemorrhagic septicemia virus (VHSV) infection, along with upregulation of several antiviral genes. Collectively, this study provides insights into the function of AcPrdx1 in defense against oxidative stressors and its role in the immune response against pathogenic infections in A. clarkii.

Genotypic assessment of a dichotomous key to identify Vibrio coralliilyticus, a coral pathogen.

Vibrio coralliilyticus is a known pathogen to corals and larvae of bivalves. Its identification is made based on phenotypic and genotypic characters of isolated strains. To evaluate the efficiency of the phenotypic identification, 21 strains identified as V. coralliilyticus using a widely used dichotomous key were analyzed by qualitative PCR and sequencing of the 16S rDNA region. The results obtained by the behavioral test, amino acids usage, allow us to distinguish 3 A/L/O profiles: (1) A+/L-/O+; (2) A+/L+/O+; and (3) A-/L+/O+. In the genotypic tests, all strains tested positive with primers specific for the Vibrio genus. However, when primers were used for species identification, the results did not match those obtained with the dichotomous key chosen. The phenotypic characteristics taken into account to set apart V. coralliilyticus and other species were not proven to be efficient. More information about the morphological diversity of colonies and enzymatic activities should be considered in the formulation of phenotypic keys for V. coralliilyticus and related species.

Comparative assessment of Vibrio virulence in marine fish larvae.

Vibrionaceae infections are a major obstacle for marine larviculture; however, little is known about virulence differences of Vibrio strains. The virulence of Vibrio strains, mostly isolated from vibriosis outbreaks in farmed fish, was tested in larval challenge trials with cod (Gadus morhua), turbot (Scophthalmus maximus) and halibut (Hippoglossus hippoglossus) using a multiwell dish assays with single-egg/larvae cultures. The strains differed significantly in virulence as some caused a high mortality of larva reaching 100% mortality after a few days, while others had no or only marginal effects on survival. Some Vibrio strains were pathogenic in all of the larva species, while some caused disease only in one of the species. Twenty-nine of the Vibrio anguillarum strains increased the mortality of larvae from at least one fish species; however, pathogenicity of the strains differed markedly. Other Vibrio species had no or less pronounced effects on larval mortalities. Iron uptake has been related to V. anguillarum virulence; however, the presence or absence of the plasmid pJM1 encoding anguibactin did not correlate with virulence. The genomes of V. anguillarum were compared (D. Castillo, P.W. D'Alvise, M. Middelboe & L. Gram, unpublished data) and most of the high-virulent strains had acquired virulence genes from other pathogenic Vibrio.

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.

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 × 10(7) or 3 × 10(5) total bacteria per shrimp resulted in mortality with higher levels in the case of V. alginolyticus (100% mortality 18 h postinjection of 3 × 10(7) 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.

Resource partitioning and sympatric differentiation among closely related bacterioplankton.

Identifying ecologically differentiated populations within complex microbial communities remains challenging, yet is critical for interpreting the evolution and ecology of microbes in the wild. Here we describe spatial and temporal resource partitioning among Vibrionaceae strains coexisting in coastal bacterioplankton. A quantitative model (AdaptML) establishes the evolutionary history of ecological differentiation, thus revealing populations specific for seasons and life-styles (combinations of free-living, particle, or zooplankton associations). These ecological population boundaries frequently occur at deep phylogenetic levels (consistent with named species); however, recent and perhaps ongoing adaptive radiation is evident in Vibrio splendidus, which comprises numerous ecologically distinct populations at different levels of phylogenetic differentiation. Thus, environmental specialization may be an important correlate or even trigger of speciation among sympatric microbes.

Assessment of fluorescent-labeled bacteria for evaluation of in vivo uptake of bacteria (Vibrio spp.) by crustacean larvae.

Available methods to study crustacean digestive tract colonization by bacteria are laborious, time-consuming, and do not permit in vivo assays and observation. This paper reports on a rapid and consistent technique to apply a fluorescent label to bacteria, which can then be presented to filter-feeding crustacea such as Artemia and penaeid larvae for later in situ bacterial distribution observation. Three luminescent Vibrio spp. were stained and observed inside Artemia nauplii, shrimp zoea and mysis stages, Vibrio harveyi type strain ATCC 14126, M(1) (pathogenic) and Ea (non-pathogenic). Factors such as dye (DTAF) concentration, exposure time/temperature and sonication time were evaluated. Viability of the dye and stained bacteria were tested at 4, -20 and -70 degrees C storage temperatures for up to 81 days. Results show that 4 and -20 degrees C storage temperatures are not recommended. At -70 degrees C, both bacteria and dye are optimally preserved. Monodispersed fluorescent-labeled bacterial cells can be observed inside the digestive tract of crustacean larvae at a density of inoculation as high as 5.2 x 10(6) CFU ml(-1). After 2 to 4 h, some leaching occurs, increasing difficulty in observation, although after 24 h, it is still possible to observe monodispersed FLB inside the digestive tract of crustacean larvae. Autofluorescence may complicate observation when filter-feeding crustacean larvae are co-fed with microalgae.

Pesticide toxicity assessment using an electrochemical biosensor with Pseudomonas putida and a bioluminescence inhibition assay with Vibrio fischeri.

Two different toxicity tests, an electrochemical biosensor Cellsense and a bioluminescence inhibition assay ToxAlert were performed in order to establish and compare the acute toxicity responses of different types of raw and spiked water for a selected group of pesticides. The selected compounds were endosulfan, chlorfenvinphos, dimethoate, fenamiphos, ametryn, deltamethrin and alpha-cypermethrin; all of them are used in large quantities for agricultural purposes. In the first step, the study of the toxicity responses for each individual pesticide with Milli-Q water was carried out. Next, the toxic responses of different mixtures of these pesticides in different water matrices, i.e., Milli-Q water, surface water, groundwater and wastewater were studied in order to evaluate (i) device advantages and limitations for the toxicity evaluation of real environmental samples, (ii) antagonistic or synergistic effects and (iii) the influence of the water matrices. The survey of pesticides in real samples was carried out using a combined method involving both chemical analysis and toxicity bioassays. Chemical analysis involved the use of solid-phase micro-extraction (SPME) followed by gas chromatography with electron capture detection (GC/ECD) or thermoionic specific detection (GC/TSD) with mass spectrometric confirmation (GC/MS).

Bioassays with Vibrio fischeri for the assessment of delayed toxicity.

The standardized bioluminescence assay with Vibrio fischeri underestimates the aquatic toxicity of chemicals which interfere with metabolic pathways supporting long term processes like growth and reproduction due to its short incubation time (30 min). Therefore this short term assay was compared with two alternative bioassays with prolonged incubation times using the same test organism: the growth inhibition assay (7 h) and the long term bioluminescence assay (24 h). Two sets of compounds were selected to reflect acute and delayed toxicity. The first group comprised pentachlorophenol, dodecylpyridiniumbromide and 3,4-dichloroaniline and the second nalidixic acid, chloramphenicol and streptomycinsulfate. The effects of compounds with acute toxicity are determined with similar sensitivity in all bioassays. Substances with delayed toxicity show only minor or no toxicities in the standardized short term bioassay but severe effects in both long term bioassays independent of the parameter used. It is concluded that the standardized short term bioluminescence assay exhibits serious limitations for the assessment of aquatic toxicity. The long term bioassays, however, may help to overcome these limitations.