Isolation and characterization of a novel Tenacibaculum species and a corresponding bacteriophage from a Mediterranean fish hatchery: Description of Tenacibaculum larymnensis sp. nov. and Tenacibaculum phage Larrie.

Tenacibaculum larymnensis sp. nov., a novel species of the Tenacibaculum genus was isolated from a commercial fish hatchery in Greece. The novel species is phylogenetically close to T. discolor and was biochemically and genetically characterized. The genome of T. larymnensis has 3.66 Mbps length, 31.83% GC content and the genomic analysis demonstrated that it harbors a wide enzymatic repertoire suggestive of increased degrading capacity but also several virulence factors including hemolysins, secretion systems, transporters, siderophores, pili and extracellular proteins. Using the novel strain, a virulent bacteriophage designated as Tenacibaculum phage Larrie was isolated and characterized. Larrie is a novel Siphovirus with relatively large genome, 77.5 kbps with 111 ORFs, a GC content of 33.7% and an exclusively lytic lifestyle. The new phage-host system can serve as an efficient model to study microbial interactions in the aquatic environment which contribute to the nutrient cycling.

A fish kill at the Aposelemis dam (Crete, Greece) caused by heavy parasitism by Ichthyobodo sp.

A fish kill was recorded at the Aposelemis Dam, which is the main reservoir of drinking water for the island of Crete in Greece. Hundreds of goldfish were found dead at a side stream which provides water to the reservoir. The affected fish had been entrapped in a small pond at the side of the stream with practically zero water renewal as the event occurred in August which is a dry season for the island of Crete. The event was alarming for the local community since anthropogenic pollution was initially suspected which could pose a significant human health threat. Following examination of the fish, the mortality was attributed to heavy infection by the parasitic flagellate, Ichthyobodo sp., whilst no pollutants were detected. The parasite was studied through light and scanning electron microscopy and was identified molecularly.

Vibrio Phage Artemius, a Novel Phage Infecting Vibrio alginolyticus.

Vibrio alginolyticus is an important pathogen of marine animals and has been the target of phage therapy applications in marine aquaculture for many years. Here, we report the isolation and partial characterization of a novel species of the Siphoviridae family, the Vibrio phage Artemius. The novel phage was species-specific and could only infect strains of V. alginolyticus. It could efficiently reduce the growth of the host bacterium at various multiplicities of infection as assessed by an in vitro lysis assay. It had a genome length of 43,349 base pairs. The complete genome has double-stranded DNA with a G + C content of 43.61%. In total, 57 ORFs were identified, of which 19 were assigned a predicted function. A genomic analysis indicated that Vibrio phage Artemius is lytic and does not harbor genes encoding toxins and antibiotic resistance determinants.

The Development of Bacteriophage Resistance in Vibrio alginolyticus Depends on a Complex Metabolic Adaptation Strategy.

Lytic bacteriophages have been well documented to play a pivotal role in microbial ecology due to their complex interactions with bacterial species, especially in aquatic habitats. Although the use of phages as antimicrobial agents, known as phage therapy, in the aquatic environment has been increasing, recent research has revealed drawbacks due to the development of phage-resistant strains among Gram-negative species. Acquired phage resistance in marine Vibrios has been proven to be a very complicated process utilizing biochemical, metabolic, and molecular adaptation strategies. The results of our multi-omics approach, incorporating transcriptome and metabolome analyses of Vibrio alginolyticus phage-resistant strains, corroborate this prospect. Our results provide insights into phage-tolerant strains diminishing the expression of phage receptors ompF, lamB, and btuB. The same pattern was observed for genes encoding natural nutrient channels, such as rbsA, ptsG, tryP, livH, lysE, and hisp, meaning that the cell needs to readjust its biochemistry to achieve phage resistance. The results showed reprogramming of bacterial metabolism by transcript regulations in key-metabolic pathways, such as the tricarboxylic acid cycle (TCA) and lysine biosynthesis, as well as the content of intracellular metabolites belonging to processes that could also significantly affect the cell physiology. Finally, SNP analysis in resistant strains revealed no evidence of amino acid alterations in the studied putative bacterial phage receptors, but several SNPs were detected in genes involved in transcriptional regulation. This phenomenon appears to be a phage-specific, fine-tuned metabolic engineering, imposed by the different phage genera the bacteria have interacted with, updating the role of lytic phages in microbial marine ecology.

Biological and Genomic Characterization of a Novel Jumbo Bacteriophage, vB_VhaM_pir03 with Broad Host Lytic Activity against Vibrio harveyi.

Vibrio harveyi is a Gram-negative marine bacterium that causes major disease outbreaks and economic losses in aquaculture. Phage therapy has been considered as a potential alternative to antibiotics however, candidate bacteriophages require comprehensive characterization for a safe and practical phage therapy. In this work, a lytic novel jumbo bacteriophage, vB_VhaM_pir03 belonging to the Myoviridae family was isolated and characterized against V. harveyi type strain DSM19623. It had broad host lytic activity against 31 antibiotic-resistant strains of V. harveyi, V. alginolyticus, V. campbellii and V. owensii. Adsorption time of vB_VhaM_pir03 was determined at 6 min while the latent-phase was at 40 min and burst-size at 75 pfu/mL. vB_VhaM_pir03 was able to lyse several host strains at multiplicity-of-infections (MOI) 0.1 to 10. The genome of vB_VhaM_pir03 consists of 286,284 base pairs with 334 predicted open reading frames (ORFs). No virulence, antibiotic resistance, integrase encoding genes and transducing potential were detected. Phylogenetic and phylogenomic analysis showed that vB_VhaM_pir03 is a novel bacteriophage displaying the highest similarity to another jumbo phage, vB_BONAISHI infecting Vibrio coralliilyticus. Experimental phage therapy trial using brine shrimp, Artemia salina infected with V. harveyi demonstrated that vB_VhaM_pir03 was able to significantly reduce mortality 24 h post infection when administered at MOI 0.1 which suggests that it can be an excellent candidate for phage therapy.

Native parasite affecting an introduced host in aquaculture: cardiac henneguyosis in the red seabream Pagrus major Temminck & Schlegel (Perciformes: Sparidae) caused by Henneguya aegea n. sp. (Myxosporea: Myxobolidae).

BACKGROUND: Henneguya Thélohan, 1892 (Myxobolidae) is one of the most species-rich genera of myxosporean parasites infecting fish. Although common in nature, there are few reports of these parasites causing important disease in aquaculture. In this paper, we describe a new species of Henneguya infecting Pagrus major (Temminck & Schlegel), a fish host introduced to the Mediterranean Sea from Japan in the late 1980s. RESULTS: Large plasmodia of the parasite were found in the bulbus arteriosus and in the ventricle of the infected fish. Spores were found mainly in the kidney and heart and were accompanied by melanized macrophages or vascular intimal proliferation mixed with a mild non-suppurative response, respectively. Comparisons of morphometric data for spore and polar capsule length and width, suggest a unique combination of features in the newly described species. Molecular analysis, based on 18S rDNA sequence of the parasite, followed by phylogenetic analysis, indicated that the parasite described here is a novel species of Henneguya, clustered with the marine congeneric species. CONCLUSIONS: Henneguya aegea n. sp. infects in aquaculture P. major, a host introduced as eggs to the Mediterranean from Japan. Despite the high host specificity of the myxobolid parasites, H. aegea n. sp. seems to be able to use P. major as a host and propagate successfully, causing morbidity and mortality. This could result in spillback of the new species from high density cultured non-native P. major to native fish hosts.

Diversification of Vibrio anguillarum Driven by the Bacteriophage CHOED.

Bacteriophages are an important factor in bacterial evolution. Some reports suggest that lytic bacteriophages can select for resistant mutant strains with reduced virulence. The present study explores the role of the CHOED bacteriophage in the diversification and virulence of its host Vibrio anguillarum. Nine phage-resistant strains were analyzed for their phenotype and different virulence factors, showing alterations in their fitness, motility, biofilm formation, lipopolysaccharide profiles and/or protease activity. Seven of the nine phage-resistant strains showed virulence reduction in a Sparus aurata larvae model. However, this is not generalized since two of the resistant strains show equal virulence compared with the parental strain. The genomic analysis of representative resistant strains displayed that the majority of the mutations are specific for each isolate, affecting genes related to lipopolysaccharide biosynthesis, quorum sensing, motility, toxin and membrane transport. The observed mutations were coherent with the phenotypic and virulence differences observed. These results suggest that the CHOED phage acts as a selective pressure on V. anguillarum, allowing proliferation of resistant strains with different genotypes, phenotypes and degrees of virulence, contributing to bacterial diversification.

Characterization of a Highly Virulent Edwardsiella anguillarum Strain Isolated From Greek Aquaculture, and a Spontaneously Induced Prophage Therein.

Edwardsiella-associated outbreaks are increasingly reported on both marine and freshwater aquaculture setups, accounting for severe financial and biomass losses. E. tarda, E. ictaluri, and E. hoshinae have been the traditional causative agents of edwardsiellosis in aquaculture, however, intensive studies due to the significance of the disease have just recently revealed two more species, E. piscicida and E. anguillarum. Whole genome sequencing that was conducted on the strain EA011113, isolated from farmed Diplodus puntazzo after an edwardsiellosis outbreak in Greece, confirmed it as a new clinical strain of E. anguillarum. Extensive phylogenetic analysis showed that this Greek strain is closely related to an Israeli E. piscicida-like clinical strain, isolated from diseased groupers, Epinephelus aeneus and E. marginatus in Red Sea. Bioinformatic analyses of E. anguillarum strain EA011113 unveiled a wide repertoire of potential virulence factors, the effect of which was corroborated by the mortalities that the strain induced in adult zebrafish, Danio rerio, under different levels of infection intensity (LD50 after 48 h: 1.85 × 104 cfu/fish). This strain was non-motile and according to electron microscopy lacked flagella, a fact that is not typical for E. anguillarum. Comparative genomic analysis revealed a deletion of 36 nt found in the flagellar biosynthetic gene (FlhB) that could explain that trait. Further in silico analysis revealed an intact prophage that was integrated in the bacterial genome. Following spontaneous induction, the phage was isolated, purified, characterized and independently sequenced, confirming its viability as a free, inducible virion as well. Separate genomic analysis of the prophage implies a plausible case of lysogenic conversion. Focusing on edwardsiellosis as a rapidly emerging aquaculture disease on a global scale, this work offers some insight into the virulence, fitness, and potential lysogenic conversion of a of a newly described, yet highly pathogenic, strain of E. anguillarum.

Isolation and characterization of a novel bacteriophage infecting Vibrio alginolyticus.

Vibrio alginolyticus is a common marine bacterium implicated in disease outbreaks in marine farmed fish and invertebrates. Due to the inappropriate use of antibiotics in aquaculture, alternative therapies have been proposed. One of the most promising options is the use of lytic bacteriophages to control pathogenic bacteria. This work describes the isolation and characterization of a lytic phage (VEN) against a V. alginolyticus strain (V2) isolated from a disease outbreak in common dentex (Dentex dentex) cultured at the Hellenic Centre for Marine Research (HCMR) in Crete, Greece. The bacteriophage is morphologically similar to phages from Podoviridae family and remained stable for 1 year at 4 °C and over 1 h when kept at 50 °C. VEN was able to lyse the host bacteria at several multiplicity of infection (MOI) (0.1-100) in liquid cultures. However, it was unable to infect other V. alginolyticus strains. Its genome consists of 44,603 bp with a GC content of 43.5%, while sequence analysis revealed the presence of 54 potential ORFs with a T7-like genomic organization. Almost 65% of the predicted ORFs presented homology with proteins of the vibriophages Vc1 and phi-A318 infecting Vibrio cyclitrophicus and Vibrio alginolyticus, respectively. Phylogenetic analysis applying the amino acid sequence of the large terminase subunit confirmed the close relationship of these phages. Furthermore, the comparison of the RNA polymerase of these phages revealed that the motifs A, B and C related to the catalytic activity and the recognition loop related to promotor identification were also conserved. VEN has an obligate lytic life cycle demonstrated by experimental data and genomic analysis. These results suggest that VEN may provide a good candidate to control recurrent diseases caused by V. alginolyticus at HCMR.

Isolation and characterization of a N4-like lytic bacteriophage infecting Vibrio splendidus, a pathogen of fish and bivalves.

A novel virulent bacteriophage, vB_VspP_pVa5, infecting a strain of Vibrio splendidus was isolated from a sea-cage aquaculture farm in Greece, and characterized using microbiological methods and genomic analysis. Bacteriophage vB_VspP_pVa5 is a N4-like podovirus with an icosahedral head measuring 85 nm in length and a short non-contractile tail. The phage had a narrow host range infecting only the bacterial host, a latent period of 30 min and a burst size of 24 virions per infected bacterium. Its genome size was 78,145 bp and genomic analysis identified 107 densely-packed genes, 40 of which could be annotated. In addition to the very large virion encapsulated DNA-dependent RNA polymerase which is the signature of the N4-like genus, an interesting feature of the novel phage is the presence of a self-splicing group I intron in the thymidylate synthase gene. A tRNAStop interrupted by a ~2.5kb open reading frame-containing area was also identified. The absence of genes related to lysogeny along with the high efficacy observed during in vitro cell lysis trials, indicate that the vB_VspP_pVa5 is a potential candidate component in a bacteriophage cocktail suitable for the biological control of V. splendidus in aquaculture.

Stumbling across the Same Phage: Comparative Genomics of Widespread Temperate Phages Infecting the Fish Pathogen Vibrio anguillarum.

Nineteen Vibrio anguillarum-specific temperate bacteriophages isolated across Europe and Chile from aquaculture and environmental sites were genome sequenced and analyzed for host range, morphology and life cycle characteristics. The phages were classified as Siphoviridae with genome sizes between 46,006 and 54,201 bp. All 19 phages showed high genetic similarity, and 13 phages were genetically identical. Apart from sporadically distributed single nucleotide polymorphisms (SNPs), genetic diversifications were located in three variable regions (VR1, VR2 and VR3) in six of the phage genomes. Identification of specific genes, such as N6-adenine methyltransferase and lambda like repressor, as well as the presence of a tRNAArg, suggested a both mutualistic and parasitic interaction between phages and hosts. During short term phage exposure experiments, 28% of a V. anguillarum host population was lysogenized by the temperate phages and a genomic analysis of a collection of 31 virulent V. anguillarum showed that the isolated phages were present as prophages in >50% of the strains covering large geographical distances. Further, phage sequences were widely distributed among CRISPR-Cas arrays of publicly available sequenced Vibrios. The observed distribution of these specific temperate Vibriophages across large geographical scales may be explained by efficient dispersal of phages and bacteria in the marine environment combined with a mutualistic interaction between temperate phages and their hosts which selects for co-existence rather than arms race dynamics.

Isolation and Characterization of Two Lytic Bacteriophages, φSt2 and φGrn1; Phage Therapy Application for Biological Control of Vibrio alginolyticus in Aquaculture Live Feeds.

Bacterial infections are a serious problem in aquaculture since they can result in massive mortalities in farmed fish and invertebrates. Vibriosis is one of the most common diseases in marine aquaculture hatcheries and its causative agents are bacteria of the genus Vibrio mostly entering larval rearing water through live feeds, such as Artemia and rotifers. The pathogenic Vibrio alginolyticus strain V1, isolated during a vibriosis outbreak in cultured seabream, Sparus aurata, was used as host to isolate and characterize the two novel bacteriophages φSt2 and φGrn1 for phage therapy application. In vitro cell lysis experiments were performed against the bacterial host V. alginolyticus strain V1 but also against 12 presumptive Vibrio strains originating from live prey Artemia salina cultures indicating the strong lytic efficacy of the 2 phages. In vivo administration of the phage cocktail, φSt2 and φGrn1, at MOI = 100 directly on live prey A. salina cultures, led to a 93% decrease of presumptive Vibrio population after 4 h of treatment. Current study suggests that administration of φSt2 and φGrn1 to live preys could selectively reduce Vibrio load in fish hatcheries. Innovative and environmental friendly solutions against bacterial diseases are more than necessary and phage therapy is one of them.

Morphological characterisation and phylogenetic relationships of Zschokkella candia n. sp. from the gall-bladder of Sparisoma cretense (L.) (Perciformes: Scaridae) in the Sea of Crete off Greece.

A new myxosporean parasite, Zschokkella candia n. sp., from the gall-bladder of the wild parrotfish Sparisoma cretense (L.) (Perciformes: Scaridae) is described based on light and scanning electron microscopy. Mature spores are elliptical, with mean dimensions 11.2 ± 0.5 × 7.8 ± 0.1 µm and possess spherical polar capsules with mean diameter of 2.3 ± 0.3 µm. The new species is differentiated from other similar species of the genus based on spore morphology, its coelozoic life-style and molecular data. The phylogenetic tree constructed using maximum likelihood analysis of small subunit (SSU) rDNA sequence data complements the characterisation of Zschokkella candia n. sp. by defining its phylogenetic position among the species of Zschokkella Auerbach, 1909 sequenced to date. The phylogenetic analysis supports the existing knowledge on the complicated polyphyletic relationships among the members of the genus Zschokkella.

Draft Genome Sequences of the Fish Pathogen Vibrio harveyi Strains VH2 and VH5.

Vibrio harveyi is an important marine pathogen that is responsible for vibriosis outbreaks in cultured fish and invertebrates worldwide. Here, we announce the draft genome sequences of V. harveyi strains VH2 and VH5, isolated from farmed juvenile Seriola dumerili during outbreaks of vibriosis in Crete, Greece.

First report of Edwardsiellosis in cage-cultured sharpsnout sea bream, Diplodus puntazzo from the Mediterranean.

BACKGROUND: Edwardsiella tarda, is a serious bacterial pathogen affecting a broad range of aquaculture fish species. The bacterium has also been reported as a human pathogen, however recent studies have dissociated the fish pathogenic Edwardsiella from those isolated from humans by placing them in a new species, E. piscicida. Here we report the first case of Edwardsiellosis in cultured sharpsnout sea breams, Diplodus puntazzo in Greece. CASE PRESENTATION: The disease has affected cultured sharpsnout sea breams of a commercial fish farm in a single location in East Greece. Two populations of sharpsnout sea breams stocked in two consecutive years in floating cages presented signs of disease which included nodules and abscesses in spleen and kidney, morbidity and cumulative mortality reaching 5.3 %. Using microbiological, biochemical and molecular tools we have identified Edwardsiella sp. as the main aetiological factor of the disease. Following phylogenetic analysis the bacterial isolates are grouped with the newly described Edwardsiella piscicida species. CONCLUSIONS: This is the first report of Edwardsiellosis in this species but most importantly in sea cage-cultured fish in the Mediterranean which may pose a serious threat for aquaculture fish species in this region.

Draft Genome Sequences of Vibrio alginolyticus Strains V1 and V2, Opportunistic Marine Pathogens.

We announce the draft genome sequences of Vibrio alginolyticus strains V1 and V2, isolated from juvenile Sparus aurata and Dentex dentex, respectively, during outbreaks of vibriosis. The genome sequences are 5,257,950 bp with a G+C content of 44.5% for V. alginolyticus V1 and 5,068,299 bp with a G+C content of 44.8% for strain V2. These genomes provide further insights into the putative virulence factors, prophage carriage, and evolution of this opportunistic marine pathogen.

Enteromyxum leei infection in parrotfish, Sparisoma cretense: histopathological, morphological and molecular study.

Enteromyxum leei (Myxozoa) has been identified as the aetiological agent of morbidity and mortality in captive feral parrotfish, Sparisoma cretense held at the facilities of Cretaquarium, the public aquarium of the Hellenic Centre for Marine Research in Crete. The parasite caused substantial mortality to the parrotfish exceeding 60% over a period of 3.5 months. Here we describe the course of disease, the effects of the parasite on the host based on histopathological observations and we provide morphological and molecular data on the parasite.

Description and relationships of two novel species of Ceratomyxa Thelohan, 1892 infecting the gallbladders of Aulopiformes: Atlantic lizardfish Synodus saurus Linnaeus, 1758 and royal flagfin Aulopus filamentosus Bloch, 1792 from Cretan Sea, Greece.

Two members of Aulopiformes (grinners) were examined for myxosporean parasites in their gallbladders. Synodus saurus and Aulopus filamentosus from Cretan Sea, Greece were infected by Ceratomyxa parasites which are described in this paper. Both species of Ceratomyxa are new and referred for the first time as Ceratomyxa cretensis n.sp. and Ceratomyxa filamentosi n.sp. The new species are described morphologically by light and scanning electron microscopy analysis, and characterized genetically by sequencing the small subunit (SSU) rDNA. The SSU rDNA sequences obtained from the two novel Ceratomyxa species were used for the construction of a phylogenetic tree with all the available Ceratomyxa SSU rDNA sequences.

Temperature sex determination in the European sea bass, Dicentrarchus labrax (L., 1758) (Teleostei, Perciformes, Moronidae): critical sensitive ontogenetic phase.

The temperature sex determination (TSD) mechanism in the European sea bass (Dicentrarchus labrax L.) was studied in respect to: a) the TSD sensitivity during the different developmental stages; and b) the intrapopulation correlation of sex determination with the growth rate up to the end of the TSD-sensitive period. At the stage of half-epiboly, eggs from the same batch were divided into four groups and subjected to different thermal treatments: a) 15 degrees C (G15 group) and b) 20 degrees C (G20 group) up to the middle of metamorphosis stage; c) 15 degrees C up to the end of yolk-sac larval stage and subsequently to 20 degrees C (G15-5 group); and d) 15 degrees C up to the end of the preflexion stage and then to 20 degrees C (G15-10 group). At the end of the treatments, size grading was applied and four additional populations were established from the upper (L) and lower (S) size portions of the G15 and G20 populations: G15L, G15S, G20L, and G20S. During the following growing phase, all populations were subjected to common rearing conditions. The sex ratios of each population were macroscopically determined at 190-210 mm mean total length. Female incidence was significantly affected (P < 0.05) by the different thermal treatments: 66.1% in the G15, 47.1% in the G15-10, 37.6% in the G15-5, and 18.1% in the G20 group. In addition, sex ratio was correlated with the growth rate of the fish up to the end of the TSD-sensitive period, with the larger fish presenting a significantly higher (P < 0.01) female incidence than the smaller fish in both thermal regimes tested: 73.1% in G15L vs. 57% in G15S, and 36.6% in G20L vs. 22.5% in G20S group. Results provide, for the first time, clear evidence that the sea bass is sensitive to TSD during all different ontogenetic stages up to metamorphosis, and that sex ratio is correlated with the growth rate of the fish well before the differentiation and maturation of the gonads.