Co-Culturing Microalgae with Roseobacter Clade Bacteria as a Strategy for Vibrionaceae Control in Microalgae-Enriched Artemia.

Bacterial communities associated with fish larvae are highly influenced by the microbiota of live prey used as feed (rotifers or Artemia), generally dominated by bacterial strains with a low degree of specialization and high growth rates, (e.g., Vibrionaceae), which can be detrimental to larvae. Co-cultivation of microalgae used in the enrichment of Artemia (e.g., Phaeodactylum tricornutum, or Chlorella minutissima) with Vibrio-antagonistic probiotics belonging to the Roseobacter clade bacteria (e.g., Phaeobacter spp. or Ruegeria spp.) was studied. The introduction of the probiotics did not affect microalgae growth or significantly modify the composition of bacterial communities associated with both microalgae, as revealed by DGGE analysis. The inoculation of P. tricornutum with Ruegeria ALR6 allowed the maintenance of the probiotic in the scale-up of the microalgae cultures, both in axenic and non-axenic conditions. Using Ruegeria-inoculated P. tricornutum cultures in the enrichment of Artemia reduced the total Vibrionaceae count in Artemia by 2 Log units, therefore preventing the introduction of opportunistic or pathogenic bacteria to fish larvae fed with them.

Characterization of Host-Associated Microbiota and Isolation of Antagonistic Bacteria from Greater Amberjack (Seriola dumerili, Risso, 1810) Larvae.

Greater amberjack (Seriola dumerili) is a new species in marine aquaculture with high mortalities at the larval stages. The microbiota of amberjack larvae was analyzed using 16S rDNA sequencing in two groups, one added copepod nauplii (Acartia tonsa) in the diet, and one without copepods (control). In addition, antagonistic bacteria were isolated from amberjack larvae and live food cultures. Proteobacteria was the most abundant phylum followed by Bacteroidota in amberjack larvae. The composition and diversity of the microbiota were influenced by age, but not by diet. Microbial community richness and diversity significantly increased over time. Rhodobacteraceae was the most dominant family followed by Vibrionaceae, which showed the highest relative abundance in larvae from the control group 31 days after hatching. Alcaligenes and Thalassobius genera exhibited a significantly higher relative abundance in the copepod group. Sixty-two antagonistic bacterial strains were isolated and screened for their ability to inhibit four fish pathogens (Aeromonas veronii, Vibrio harveyi, V. anguillarum, V. alginolyticus) using a double-layer test. Phaeobacter gallaeciensis, Phaeobacter sp., Ruegeria sp., and Rhodobacter sp. isolated from larvae and Artemia sp. inhibited the fish pathogens. These antagonistic bacteria could be used as host-derived probiotics to improve the growth and survival of the greater amberjack larvae.

The Probiotic Phaeobacter inhibens Provokes Hypertrophic Growth via Activation of the IGF-1/Akt Pathway during the Process of Metamorphosis of Greater Amberjack (Seriola dumerili, Risso 1810).

Metamorphosis entails hormonally regulated morphological and physiological changes requiring high energy levels. Probiotics as feed supplements generate ameliorative effects on host nutrient digestion and absorption. Thereby, the aim of the present research was to investigate the impact of the probiotic Phaeobacter inhibens as a water additive on cellular signaling pathways in the metamorphosis of greater amberjack (Seriola dumerili). Activation of insulin-like growth factor type 1 receptor (IGF-1R), protein kinase B (Akt), mitogen-activated protein kinases (MAPKs) and AMP-activated protein kinase (AMPK), induction of heat shock proteins (Hsps), and programmed cell death were assessed through SDS-Page/immunoblot analysis, while energy metabolism was determined through enzymatic activities. According to the results, greater amberjack reared in P. inhibens-enriched water entered the metamorphic phase with greater body length, while protein synthesis was triggered to facilitate the hypertrophic growth as indicated by IGF-1/Akt activation and AMPK inhibition. Contrarily, MAPKs levels were reduced, whereas variations in Hsps response were evident in the probiotic treatment. Apoptosis and autophagy were mobilized potentially for the structural remodeling processes. Furthermore, the elevated enzymatic activities of intermediary metabolism highlighted the excess energy demands of metamorphosis. Collectively, the present findings demonstrate that P. inhibens may reinforce nutrient utilization, thus leading greater amberjack to an advanced growth and developmental state.

Antibacterial Activity against Four Fish Pathogenic Bacteria of Twelve Microalgae Species Isolated from Lagoons in Western Greece.

Microalgae may produce a range of high-value bioactive substances, making them a promising resource for various applications. In this study, the antibacterial activity of twelve microalgae species isolated from lagoons in western Greece was examined against four fish pathogenic bacteria (Vibrio anguillarum, Aeromonas veronii, Vibrio alginolyticus, and Vibrio harveyi). Two experimental approaches were used to evaluate the inhibitory effect of microalgae on pathogenic bacteria. The first approach used bacteria-free microalgae cultures, whereas the second approach used filter-sterilized supernatant from centrifuged microalgae cultures. The results demonstrated that all microalgae had inhibitory effects against pathogenic bacteria in the first approach, particularly 4 days after inoculation, where Asteromonas gracilis and Tetraselmis sp. (red var., Pappas) exhibited the highest inhibitory activity, reducing bacterial growth by 1 to 3 log units. In the second approach, Tetraselmis sp. (red var., Pappas) showed significant inhibition against V. alginolyticus between 4 and 25 h after inoculation. Moreover, all tested cyanobacteria exhibited inhibitory activity against V. alginolyticus between 21 and 48 h after inoculation. Statistical analysis was performed using the independent samples t-test. These findings suggested that microalgae produce compounds with antibacterial activity, which could be useful in aquaculture.

Oxygenation of European seabass (Dicentrarchus labrax L.) in cages through aeration and effect on lipid metabolism.

Two groups of the same batch of European seabass were used in an industrial-scale trial in sea cages in Vorios Evoikos, Greece. For about one month, one of the two cages was oxygenated by use of compressed air injected in seawater through an AirX frame (Oxyvision A/S, Norway) at 3.5 m depth, while oxygen concentration and temperature were monitored every 30 min. The liver, gut, and pyloric ceca samples were taken from fish of both groups for measurement of gene expression of phospholipase A2 (PLA2) and hormone-sensitive lipase (HSL), and for histological analysis at the middle and end of the experiment. Real-time qPCR was performed with housekeeping genes ACTb, L17, and EF1a. The expression of PLA2 increased in pyloric caeca samples from the oxygenated cage, suggesting that aeration improved the absorption rate of dietary phospholipids (p < 0.05). Expression of HSL increased significantly in liver samples from the control cage, in comparison with the aerated cage (p < 0.05). Histological examination of sea bass samples revealed an increased fat accumulation in the hepatocytes of fish in the oxygenated cage. The results of the present study showed increased lipolysis induced by low DO levels in farmed sea bass in cages.

Sublethal exposure to Microcystis aeruginosa extracts during embryonic development reduces aerobic swimming capacity in juvenile zebrafish.

In the last decades, cyanobacterial harmful algal blooms (CyanoHABs) pose an intensifying ecological threat. Microcystis aeruginosa is a common CyanoHAB species in freshwater ecosystems, with severe toxic effects in a wide range of organisms. In the present paper we examined whether transient and short (48 h) exposure of fish embryos to sublethal levels of M. aeruginosa crude extract (200 mg biomass dw L-1) affects swimming performance at later life stages (end of metamorphosis, ca 12 mm TL, 22,23 days post-fertilization). Pre-exposed metamorphosing larvae presented a significant decrease in swimming performance (9.7 ± 1.6 vs 11.4 ± 1.7 TL s-1 in the control group, p < 0.01), and a significant decrease in the ventricle length-to-depth ratio (1.23 ± 0.15 vs 1.42 ± 0.15 in control fish, p < 0.05). In addition, extract-exposed fish presented significantly elevated rates of vertebral abnormalities (82 ± 13% vs 7 ± 4% in the control group), mainly consisting of the presence of extra neural and haemal processes. No significant differences between groups were detected in survival and growth rates. Results are discussed in respect to the mechanisms that might mediate the detected cyanobacterial effects. This is the first evidence of a direct link between sublethal exposure to M. aeruginosa during the embryonic period and swimming performance at later life-stages. Decreased swimming performance, altered cardiac shape, and elevated vertebral abnormalities in response to early exposure to M. aeruginosa could have significant effects on fish populations in the wild.

Isolation of Phaeobacter sp. from Larvae of Atlantic Bonito (Sarda sarda) in a Mesocosmos Unit, and Its Use for the Rearing of European Seabass Larvae (Dicentrarchus labrax L.).

The target of this study was to use indigenous probiotic bacteria in the rearing of seabass larvae. A Phaeobacter sp. strain isolated from bonito yolk-sac larvae (Sarda sarda) and identified by amplification of 16S rDNA showed in vitro inhibition against Vibrio anguillarum. This Phaeobacter sp. strain was used in the rearing of seabass larvae (Dicentrarchus labrax L.) in a large-scale trial. The survival of seabass after 60 days of rearing and the specific growth rate at the late exponential growth phase were significantly higher in the treatment receiving probiotics (p < 0.05). Microbial community richness as determined by denaturing gradient gel electrophoresis (DGGE) showed an increase in bacterial diversity with fish development. Changes associated with the administration of probiotics were observed 11 and 18 days after hatching but were not apparent after probiotic administration stopped. In a small challenge experiment, seabass larvae from probiotic treatment showed increased survival (p < 0.05) after experimental infection with a mild pathogen (Vibrio harveyi). Overall, our results showed that the use of an indigenous probiotic strain had a beneficial impact on larval rearing in industry-like conditions.

Evaluation of a battery of marine species-based bioassays against raw and treated municipal wastewaters.

The present study evaluates a battery of marine species-based bioassays against chemically characterized municipal wastewater samples (raw and WWTP treated). We estimated Dunaliella tertiolecta growth rate inhibition (24-96h IC50 values), Artemia franciscana immobilization (24h LC50 values), mussel hemocytes viability and lipid peroxidation enhancement (in terms of neutral red retention assay/NRRT and malondialdehyde/MDA content, respectively) in influent- and WWTP effluent-treated species. We found algal growth arrest and stimulation respectively, almost similar 24hLC50 values in Artemia sp., and significantly higher adverse effects (in terms of NRRT and MDA levels) in influent-treated mussel hemocytes. Furthermore, the estimation of hatchability, yolk-sac larvae mortality (24-120hLC50) and spinal deformities (SD) in sea bream Sparus aurata showed slight variations over time, with the lowest LC50 and SD50 (representing spinal deformities at 50% of yolk-sac larvae) values to be observed in influent-treated larvae at 120h. Data interpretation (both chemical and biological) revealed that toxic endpoints, such as NRRT50, 96hIC50Dun, 120hLC50Sparus and 120hSD50Sparus, significantly related to WWTP removal efficiency and further mediated by the presence of dominant compounds, such as As and Cr, could be used for identifying main components of toxicity in wastewaters.

The Atlantic Bonito (Sarda sarda, Bloch 1793) transcriptome and detection of differential expression during larvae development.

The Atlantic bonito (Sarda sarda, Bloch 1793) belongs to the important marine fish species with a wide geographical distribution covering the Atlantic Ocean, the Mediterranean and its bordering seas. Aquaculture practices for this species are still in their infancies and scientific studies are seldom undertaken, mainly because of difficulties in sampling. Thus for small tuna species like the Atlantic bonito only little is known about its biology and regarding the molecular background even less information is available. In the production of marine fish it is known that the most critical period is the larval stages, as high growth rates as well as significant developmental changes take place. In this study we have investigated the transcriptome of the Atlantic bonito of five larvae stages applying Illumina sequencing technology. For non-model species like aquaculture species, transcriptome analysis of RNA samples from individuals using Illumina sequencing technology is technically efficient and cost effective. In the present study a total number of 169,326,711 paired-end reads with a read length of 100 base pairs were generated resulting in a reference transcriptome of 68,220 contigs with an average length of 2054 base pairs. For differential expression analyses single end reads were obtained from different developmental stages and mapped to the constructed reference transcriptome. Differential expression analyses revealed in total 18,657 differentially expressed transcripts and were assigned to five distinguished groups. Each of the five clusters shows stage specific gene expression. We present for the first time in the Atlantic bonito an extensive RNA-Seq based characterization of its transcriptome as well as significant information on differential expression among five developmental larvae stages. The generated transcripts, including SNP and microsatellite information for candidate molecular markers and gene expression information will be a valuable resource for future genetic and molecular studies.

Environmental conditioning of skeletal anomalies typology and frequency in gilthead seabream (Sparus aurata L., 1758) juveniles.

In this paper, 981 reared juveniles of gilthead seabream (Sparus aurata) were analysed, 721 of which were from a commercial hatchery located in Northern Italy (Venice, Italy) and 260 from the Hellenic Center for Marine Research (Crete, Greece). These individuals were from 4 different egg batches, for a total of 10 different lots. Each egg batch was split into two lots after hatching, and reared with two different methodologies: intensive and semi-intensive. All fish were subjected to processing for skeletal anomaly and meristic count analysis. The aims involved: (1) quantitatively and qualitatively analyzing whether differences in skeletal elements arise between siblings and, if so, what they are; (2) investigating if any skeletal bone tissue/ossification is specifically affected by changing environmental rearing conditions; and (3) contributing to the identification of the best practices for gilthead seabream larval rearing in order to lower the deformity rates, without selections. The results obtained in this study highlighted that: i) in all the semi-intensive lots, the bones having intramembranous ossification showed a consistently lower incidence of anomalies; ii) the same clear pattern was not observed in the skeletal elements whose ossification process requires a cartilaginous precursor. It is thus possible to ameliorate the morphological quality (by reducing the incidence of severe skeletal anomalies and the variability in meristic counts of dermal bones) of reared seabream juveniles by lowering the stocking densities (maximum 16 larvae/L) and increasing the volume of the hatchery rearing tanks (minimum 40 m(3)). Feeding larvae with a wide variety of live (wild) preys seems further to improve juvenile skeletal quality. Additionally, analysis of the morphological quality of juveniles reared under two different semi-intensive conditions, Mesocosm and Large Volumes, highlighted a somewhat greater capacity of Large Volumes to significantly augment the gap with siblings reared in intensive (conventional) modality.

Bioenergetic changes in the microalgal photosynthetic apparatus by extremely high CO2 concentrations induce an intense biomass production.

Unicellular green alga Chlorella minutissima, grown under extreme carbon dioxide concentrations (0.036-100%), natural temperature and light intensities (Mediterranean conditions), strongly increase the microalgal biomass through photochemical and non-photochemical changes in the photosynthetic apparatus. Especially, CO(2) concentrations up to 10% enhance the density of active reaction centers (RC/CS(o)), decrease the antenna size per active reaction center (ABS/RC), decrease the dissipation energy (DI(o)/RC) and enhance the quantum yield of primary photochemistry (F(v)/F(m)). Higher CO(2) concentrations (20-25%) combine the above-mentioned photochemical changes with enhanced non-photochemical quenching of surplus energy, which leads to an enhanced steady-state fraction of 'open' (oxidized) PSII reaction centers (q(p)), and minimize the excitation pressure of PSII (1 - q(p)) under very high light intensities (approximately 1700 micromol m(-2) s(-1) maximal value), avoiding the photoinhibition and leading to an enormous biomass production (approximately 2500%). In conclusion, these extreme CO(2) concentrations - about 1000 times higher than the ambient one - can be easily metabolized from the unicellular green alga to biomass and can be used, on a local scale at least, for the future development of microalgal photobioreactors for the mitigation of the factory-produced carbon dioxide.