Pantelis Katharios

An application of different bioindicators for assessing water quality: a case study in the rivers Alfeios and Pineios (Peloponnisos, Greece)

Abstract A number of bioindicators and biotic indices and scores based on benthic macroinvertebrates, diatoms, fishes, aquatic and riparian vegetation in relation to physicochemical parameters have been applied in assessing the water quality of the rivers Alfeios and Pineios (Peloponnisos, Greece). According to the findings, the water quality in both rivers varied from very poor to very good. Among the bioindicators used, the benthic macroinvertebrates seem to be the most reliable. The BBI and IBE were the most applicable indexes while the applicability of the IBMWP and IASPT in the Greek region can be enforced with the inclusion of the Diptreran family Rhagionidae and the Coleopteran family Elminthidae.

Pathological obstacles of newly-introduced fish species in Mediterranean mariculture: a review

Species diversification has been extensively employed in Mediterranean mariculture industry as a tool to relief the crisis resulting from market saturation and overproduction of the two main representatives, sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata). Numerous new promising candidates belonging to several fish families such as Sparidae, Polyprionidae, Serranidae, Sciaenidae, Soleidae, Carangidae and Scombridae have been introduced to provide alternative culture choices in Mediterranean. The farming potential of these species in production scale has received some or little success partly due to the fact that their entrance has not been accompanied without the presence of considerable pathological problems. Bacterial outbreaks causing serious problems have been mainly induced by Photobacterium damsella subsp. piscicida, Vibrio alginolyticus, V. harveyi, Tenacibaculum maritimum and Flavobacterium sp. Detrimental parasitic pathogens included the myxosporean Enteromyxum leei, the ciliates Cryptocaryon irritans and Uronema spp., the flagellate Amyloodinium occelatum, the monogenean Zeuxapta seriolae and the digeneans Paradeontacylix-like spp. Viral encephalopathy and retinopathy infections and the chlamydia-like bacteria causing Epitheliocystis disease are also of significance importance. Numerous other agents of lesser pathogenic significance as well as other occasionally recorded pathogens are also mentioned in this review. Additional research effort has to be devoted to the pathogenesis of important pathogens and the interactions with their hosts to assist the viability of new fish species farming in Mediterranean region.

Sperm ultrastructure of the gall bladder fluke Anisocoelium capitellatum (Digenea: Cryptogonimidae), a parasite of Uranoscopus scaber (Pisces: Uranoscopidae)

Ultrastructure of mature spermatozoon of Anisocoelium capitellatum is described with transmission electron microscopy. The description gave evidence of some characteristics of this digenean. The male gamete presents features allowing the distinction between A. capitellatum and other digenetic trematodes. It is characterized by original anterior and posterior extremities. The peripheral doublets appear prior to the central cores in the anterior extremity of axoneme. In the distal part of the spermatozoon, these ultrastructural features are also the first elements disappearing in the axoneme 1 and the last remaining in the axoneme 2. Another important peculiarity of the spermatozoon is the presence of three mitochondria.

Environmental marine pathogen isolation using mesocosm culture of sharpsnout seabream: striking genomic and morphological features of novel Endozoicomonas sp.

Aquaculture is a burgeoning industry, requiring diversification into new farmed species, which are often at risk from infectious disease. We used a mesocosm technique to investigate the susceptibility of sharpsnout seabream (Diplodus puntazzo) larvae to potential environmental pathogens in seawater compared to control borehole water. Fish exposed to seawater succumbed to epitheliocystis from 21 days post hatching, causing mortality in a quarter of the hosts. The pathogen responsible was not chlamydial, as is often found in epitheliocystis, but a novel species of the γ-proteobacterial genus Endozoicomonas. Detailed characterisation of this pathogen within the infectious lesions using high resolution fluorescent and electron microscopy showed densely packed rod shaped bacteria. A draft genome sequence of this uncultured bacterium was obtained from preserved material. Comparison with the genome of the Endozoicomonas elysicola type strain shows that the genome of Ca. Endozoicomonas cretensis is undergoing decay through loss of functional genes and insertion sequence expansion, often indicative of adaptation to a new niche or restriction to an alternative lifestyle. These results demonstrate the advantage of mesocosm studies for investigating the effect of environmental bacteria on susceptible hosts and provide an important insight into the genome dynamics of a novel fish pathogen.

Ontogenesis of the HPI axis and molecular regulation of the cortisol stress response during early development in Dicentrarchus labrax .

The cortisol stress response and the molecular programming of the corticoid axis were characterized for the first time during early ontogeny in a Mediterranean marine teleost, the European sea bass (Dicentrarchus labrax). Sea bass embryos, pre-larvae and larvae at specific points of development were exposed to acute stressors and the temporal patterns of cortisol whole body concentrations and the expression of genes involved in corticosteroid biosynthesis, degradation and signaling were determined. Expression of genes (gr1, gr2, mr, crf) involved into the corticoid response regulation combined with histological data indicated that, although a cortisol stress response is evident for the first time around first feeding, a pattern becomes established in larvae at flexion until the formation of all fins. Moreover, mRNA transcript levels of 11β-hydroxylase and 11β-hsd2 showed a strong correlation with the whole body cortisol concentrations. Concluding, our data reveal the presence of an adaptive mechanism in European sea bass at early ontogeny enabling to cope with external stressful stimuli and provide a better insight into the onset and regulation of the stress response in this species.

Severe mortality in mesocosm-reared sharpsnout sea bream Diplodus puntazzo larvae due to epitheliocystis infection.

This paper describes severe mortalities recorded in sharpsnout sea bream Diplodus puntazzo larvae reared in mesocosms. The mortalities were attributed to epitheliocystis infection. The pathology associated with the disease is described using histological techniques. Microscopical examination showed a massive infection of the skin, fins, and oral cavity, with impaired feeding, respiration, and osmoregulation being the most likely cause of death. This is the first report of epitheliocystis disease in sharpsnout sea bream and in fish at such an early developmental stage.

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.

Accumulation of ivermectin in the brain of sea bream, Sparus aurata after intraperitoneal administration.

Ivermectin, which is widely used in veterinary and human, has been considered safe due to its inability to penetrate into the central nervous system of higher vertebrates. This paper presents data on the ability of the drug to cross the blood-brain barrier of the marine teleost sea bream, Sparus aurata and accumulate in the brain. The concentration of the drug in the brain and the serum of the fish was assessed by the use of a direct competitive ELISA commercial kit. Our results showed a rapid uptake of the substance by the brain of the fish reaching a maximum concentration of 98.9ngg(-1) 8h post treatment. The trend of the absorption of the drug in brain followed that of the blood. Concentration of the drug in the brain remained high at each sampling point over the 24h duration of the experiment. In view of these findings, the need of study of the role of the blood-brain barrier and particularly the multidrug resistance mechanism in sea bream is outlined.

Emerging pathogens of gilthead seabream: characterisation and genomic analysis of novel intracellular β-proteobacteria

New and emerging environmental pathogens pose some of the greatest threats to modern aquaculture, a critical source of food protein globally. As with other intensive farming practices, increasing our understanding of the biology of infections is important to improve animal welfare and husbandry. The gill infection epitheliocystis is increasingly problematic in gilthead seabream (Sparus aurata), a major Mediterranean aquaculture species. Epitheliocystis is generally associated with chlamydial bacteria, yet we were not able to localise chlamydial targets within the major gilthead seabream lesions. Two previously unidentified species within a novel β-proteobacterial genus were instead identified. These co-infecting intracellular bacteria have been characterised using high-resolution imaging and genomics, presenting the most comprehensive study on epitheliocystis agents to date. Draft genomes of the two uncultured species, Ca. Ichthyocystis hellenicum and Ca. Ichthyocystis sparus, have been de novo sequenced and annotated from preserved material. Analysis of the genomes shows a compact core indicating a metabolic dependency on the host, and an accessory genome with an unprecedented number of tandemly arrayed gene families. This study represents a critical insight into novel, emerging fish pathogens and will be used to underpin future investigations into the bacterial origins, and to develop diagnostic and treatment strategies.

Host-associated genomic features of the novel uncultured intracellular pathogen Ca. Ichthyocystis revealed by direct sequencing of epitheliocysts

Advances in single-cell and mini-metagenome sequencing have enabled important investigations into uncultured bacteria. In this study, we applied the mini-metagenome sequencing method to assemble genome drafts of the uncultured causative agents of epitheliocystis, an emerging infectious disease in the Mediterranean aquaculture species gilthead seabream. We sequenced multiple cyst samples and constructed 11 genome drafts from a novel beta-proteobacterial lineage, Candidatus Ichthyocystis. The draft genomes demonstrate features typical of pathogenic bacteria with an obligate intracellular lifestyle: a reduced genome of up to 2.6 Mb, reduced G + C content, and reduced metabolic capacity. Reconstruction of metabolic pathways reveals that Ca. Ichthyocystis genomes lack all amino acid synthesis pathways, compelling them to scavenge from the fish host. All genomes encode type II, III, and IV secretion systems, a large repertoire of predicted effectors, and a type IV pilus. These are all considered to be virulence factors, required for adherence, invasion, and host manipulation. However, no evidence of lipopolysaccharide synthesis could be found. Beyond the core functions shared within the genus, alignments showed distinction into different species, characterized by alternative large gene families. These comprise up to a third of each genome, appear to have arisen through duplication and diversification, encode many effector proteins, and are seemingly critical for virulence. Thus, Ca. Ichthyocystis represents a novel obligatory intracellular pathogenic beta-proteobacterial lineage. The methods used: mini-metagenome analysis and manual annotation, have generated important insights into the lifestyle and evolution of the novel, uncultured pathogens, elucidating many putative virulence factors including an unprecedented array of novel gene families.