Panagiotis Berillis

The presence of microcystins in fish Cyprinus carpio tissues: a histopathological study

The occurrence of heavy cyanobacterial blooms has become a worldwide problem, as a consequence of eutrophication of the aquatic ecosystems; furthermore, 60% to 75% of these blooms have been found to be toxic. Microcystins (MCYSTs), the predominant toxins of cyanobacterial blooms, are associated with mortality and illness in both animals and humans. Laboratory-controlled experiments studying the effects of different microcystins on the common carp (Cyprinus carpio) have revealed various histopathological alterations. The aim of the present study is to investigate the effect of chronic or subchronic exposure of fish to microcystins under natural environmental conditions by examining the possible histopathological changes associated with a dense cyanobacterial bloom and determining the microcystin contents of fish tissues. Common carps (C. carpio) were caught from Lake Karla (Greece), during a dense cyanobacterial bloom. The concentration of MCYSTs in the fish liver, kidney and muscle tissues was measured by enzyme-linked immunosorbent assay. The pseudogaster contents were analysed, and a histopathological examination was performed using light and electron microscopy. Severe alterations were detected in the liver and the kidney, suggesting that the toxic effects were caused by various pollutants that were particularly associated with microcystins. The histopathological findings are also discussed, taking into consideration the health conditions of the common carp as a commercial fish species. The mechanisms of expansion of the microcystins and the poisoning of aquatic organisms (e.g. fish) are not yet known in the Lake Karla ecosystem. Future research may focus on identifying the changes caused by microcystins and other factors that exert similar effects on fish tissues, as well as on establishing the overall combined effect of all these factors on fish health.

A novel image processing method to determine the nutritional condition of lobsters

The digestive gland of crustacean is involved in various metabolic activities, including the synthesis and secretion of digestive enzymes that begin the process of food digestion, intracellular digestion and absorption of nutrients, storage of reserves, and disposal of waste products. It consists of two glandular lobes which extensively subdivide to form a complex of blind-ending tubules, whose size, surface area, and digestive cells are associated with intracellular digestion and the nutritional status of the organism. The aim of this paper was to study the morphology of the digestive gland in various lobster species and calculate the surface area of tubules, lumen and digestive cells (R-, F-, and B-cells) and their ratios to total tubule surface area. The similarity in ratios obtained in this study between individual lobsters suggests that the method developed in this study can be successfully applied to a range of species. This study describes a novel image processing algorithm for the automatic measurement of the hepatopancreas structure using stained cross sections of digestive gland tubules. The proposed new methodology could be used for studying the physiology and nutrient metabolism of lobsters and other crustaceans. The computer-aided analysis described in this paper is accurate for the quantitative assessment of the lobsters digestive gland structure.

Harmful and parasitic unicellular eukaryotes persist in a shallow lake under reconstruction (L. Karla, Greece)

The reconstructed Lake Karla, Greece, has been undergoing its water-filling period since November 2009. In this paper, we aimed at investigating whether the unicellular eukaryotes, including the toxic/parasitic ones, that have been found during mass fish kills in the lake (March–April 2010), persist during the first warm period of the lake (May, August, November 2010). Given that microscopic characterization of some of these eukaryotes is not adequate for their identification, we analysed the 18S rRNA gene diversity of plankton samples. All the found phylotypes belonged to the phyla of Mesomycetazoa, Chlorophyta, Fungi, Alveolata, Cercozoa, Cryptophyta and Stramenopiles. Some members of these groups seem to persist in Lake Karla as they have been found in early spring as well. These microscopic eukaryotes are either ichthyotoxic/parasitic (e.g. Pfiesteria sp./Pseudopfiesteria shumwayae, some Fungi, Mesomycetazoa, Lagenidium sp., Cercozoa) or indicative of hyper-eutrophic conditions (e.g. Oocystis sp., Scenedesmus spp.) and were rather abundant during the first spring–autumn period of the lake’s refilling process. These complex microscopic communities are expected to shape highly dynamic and variable food webs with the risk of repeated fish kills.

Dynamics of biofilm formation by Listeria monocytogenes on stainless steel under mono-species and mixed-culture simulated fish processing conditions and chemical disinfection challenges

The progressive ability of a six-strains L. monocytogenes cocktail to form biofilm on stainless steel (SS), under fish-processing simulated conditions, was investigated, together with the biocide tolerance of the developed sessile communities. To do this, the pathogenic bacteria were left to form biofilms on SS coupons incubated at 15°C, for up to 240h, in periodically renewable model fish juice substrate, prepared by aquatic extraction of sea bream flesh, under both mono-species and mixed-culture conditions. In the latter case, L. monocytogenes cells were left to produce biofilms together with either a five-strains cocktail of four Pseudomonas species (fragi, savastanoi, putida and fluorescens), or whole fish indigenous microflora. The biofilm populations of L. monocytogenes, Pseudomonas spp., Enterobacteriaceae, H2S producing and aerobic plate count (APC) bacteria, both before and after disinfection, were enumerated by selective agar plating, following their removal from surfaces through bead vortexing. Scanning electron microscopy was also applied to monitor biofilm formation dynamics and anti-biofilm biocidal actions. Results revealed the clear dominance of Pseudomonas spp. bacteria in all the mixed-culture sessile communities throughout the whole incubation period, with the in parallel sole presence of L. monocytogenes cells to further increase (ca. 10-fold) their sessile growth. With respect to L. monocytogenes and under mono-species conditions, its maximum biofilm population (ca. 6logCFU/cm2) was reached at 192h of incubation, whereas when solely Pseudomonas spp. cells were also present, its biofilm formation was either slightly hindered or favored, depending on the incubation day. However, when all the fish indigenous microflora was present, biofilm formation by the pathogen was greatly hampered and never exceeded 3logCFU/cm2, while under the same conditions, APC biofilm counts had already surpassed 7logCFU/cm2 by the end of the first 96h of incubation. All here tested disinfection treatments, composed of two common food industry biocides gradually applied for 15 to 30min, were insufficient against L. monocytogenes mono-species biofilm communities, with the resistance of the latter to significantly increase from the 3rd to 7th day of incubation. However, all these treatments resulted in no detectable L. monocytogenes cells upon their application against the mixed-culture sessile communities also containing the fish indigenous microflora, something probably associated with the low attached population level of these pathogenic cells before disinfection (<102CFU/cm2) under such mixed-culture conditions. Taken together, all these results expand our knowledge on both the population dynamics and resistance of L. monocytogenes biofilm cells under conditions resembling those encountered within the seafood industry and should be considered upon designing and applying effective anti-biofilm strategies.

Collagen Fibrils in Cultured and Wild Sea Bream (Sparus aurata) Liver. An Electron Microscopy and Image Analysis Study

This study aims to measure liver collagen fibril diameter in cultured and wild sea breams (Sparus aurata). Cultured sea breams were fed three isonitrogenous diets. The organically produced feed contained sustainable certified fish meal (45%), fish oil (14%), and organic certified wheat; the laboratory feed contained fish meal (45%), fish oil (14%), wheat meal, and soya meal; and the commercial feed included fish meal (46%), fish oil (17%), soya meal, wheat meal, and corn gluten meal. The organic diet had higher amounts of vitamins A, C, and E; specific amino acids; and minerals that enhanced the biosynthesis of collagen. This study shows that fish fed the organic feed had significantly bigger collagen fibril diameters than the fish fed the conventional feed. Furthermore, the organically fed fish had similarly sized collagen fibril diameters as wild fish. More research is needed to understand the long-term effects and the mechanism and function of fish collagen peptide intake on lipid absorption and metabolism; and to identify dietary regimes that are able to improve whole body lipid profiles and suppress the transient increase of plasma triglycerides.

Improving aeration for efficient oxygenation in sea bass sea cages. Blood, brain and gill histology

Abstract An air diffusion based system (Airx) was developed to control the dissolved oxygen levels in aquaculture sea cages. The system was introduced and then tested for 37 days in a sea bass sea cage (aerated cage). A second sea bass sea cage, without the AirX, was used as a control. Oxygen levels were measured in both cages at the start of the trial, before the AirX system was introduced, and during the working period of the AirX system. Fish samples were collected 15 days after the AirX system was introduced and at the end of the experiment. Blood smears were prepared and examined microscopically. Erythrocyte major axis, minor axis and area of fish erythrocytes were measured. Leucocyte differentiation was also examined. In the control cage, the fish had significantly larger red blood cells when compared with the red blood cells of the fish in the aerated cage. Histological examination of the gills and brain revealed no morphological differences or alterations between the two groups of fish. This study demonstrated that an air diffuser system could improve the water quality of fish farmed in sea cages and enhance sea bass physiological performance, especially if DO levels fall below 60% oxygen saturation.

Measurement of the feed consumption of Nephrops norvegicus feeding on different diets and its effect on body nutrient composition and digestive gland histology

The feed consumption of Nephrops norvegicus (Linnaeus, 1758) reared in captivity and fed either mussel or a pelleted diet was measured for ten weeks. Both groups of lobsters showed a similar feed consumption, which averaged 54.7 and 49.9 mg of dry weight day−1 lobster−1, respectively. Tail muscle and digestive gland nutrient composition was unaffected by the different type of diet, except muscle lipid contents. The pelleted diet resulted in increased moults, but the mussel diet was more effective with reference to survival. A similar digestive condition was also evidenced between the groups despite the bigger B-cells area in the digestive gland tubules of the pellet-fed lobsters. These findings contribute to a better understanding of the nutrition and feeding capabilities of N. norvegicus reared in captivity, which is critical for the development of suitable diets for its potential in aquaculture.

Bacterial biofilm development during experimental degradation of Melicertus kerathurus exoskeleton in seawater

Chitinolytic bacteria are widespread in marine and terrestrial environment, and this is rather a reflection of their principle growth substrates ubiquity, chitin, in our planet. In this paper, we investigated the development of naturally occurring bacterial biofilms on the exoskeleton of the shrimp Melicertus kerathurus during its degradation in sea water. During a 12-day experiment with exoskeleton fragments in batch cultures containing only sea water as the growth medium at 18 °C in darkness, we analysed the formation and succession of biofilms by scanning electron microscopy and 16S rRNA gene diversity by next generation sequencing. Bacteria belonging to the γ- and α-Proteobacteria and Bacteroidetes showed marked (less or more than 10%) changes in their relative abundance from the beginning of the experiment. These bacterial taxa related to known chitinolytic bacteria were the Pseudolateromonas porphyrae, Halomonas aquamarina, Reinekea aestuarii, Colwellia asteriadis and Vibrio crassostreae. These bacteria could be considered as appropriate candidates for the degradation of chitinous crustacean waste from the seafood industry as they dominated in the biofilms developed on the shrimps exoskeleton in natural sea water with no added substrates and the degradation of the shrimp exoskeleton was also evidenced.

Aquaponics Software in Greece

A valuable and sustainable approach for food security could be the further expansion of aquaponics production. Aquaponics is the combination of fish farming in recirculating aquaculture systems (RAS) and cultivation of plants in water (hydroponic). Research on the sustainability of aquaponics is focused on system water quality parameters and availability of nutrients for the three components: the plants, the fish, and the bacteria (Tyson et al., 2011; König, et al., 2016). The technology works as a closed loop system that reduces the consumption of fresh water compared to conventional monocultures and uses the nutrients excreted by the fish as fertilizer for the plants. Nitrogen uptake depends on the plants species that play an important role in avoiding the accumulation of NO3-in aquaponics. In addition, carbohydrate addition to aquaculture systems stimulates heterotrophic bacteria growth that results in the removal of inorganic nitrogen through assimilation. Thus, all fish waste and feed nutrients can be utilized and recycled (Graber and Junge-Berberovic, 2009). The need for technological advancements is also addressed (König et al., 2016).

Skeletal Deformities in Seabreams. Understanding the Genetic Origin Can Improve Production

*Correspondence to: Panagiotis Berillis, Department of Ichthyology and Aquatic Environment, School of Agricultural Sciences, University of Thessaly, Fytoko Street, Nea Ionia Magnesia, Greece, Tel: (+30)2421093248; E-mail: pveril@apae.uth.gr Europe represents the largest market for fish in the world. In Europe, aquaculture accounts for about 20% of fish production and directly employs about 85,000 people (European Commission, 2017). EU aquaculture is renowned for its high quality, sustainability and consumer protection standards. In 2015, the European aquaculture production reached 2,350,278 tons. The 94% of that amount represented by salmon, trout, seabream, seabass and carp. In the Mediterranean region seabreams represented the 45% of the marine production (FEAP, 2016) with the juvenile production, for stocking on growing farms, to be stable at 1,100 million (FEAP, 2016). According the 2016 Annual Economic Report (2016) the total value of sales from the EU aquaculture sector is reported at 4,517 million € in 2014. This represents a 3% increase to the 4,365 million € reported in 2012, although there was a 4% decrease in the production.