Turbot Larvae Research Articles

Antimicrobial Activity, Genetic Relatedness, and Safety Assessment of Potential Probiotic Lactic Acid Bacteria Isolated from a Rearing Tank of Rotifers (Brachionus plicatilis) Used as Live Feed in Fish Larviculture.

Aquaculture is a rapidly expanding agri-food industry that faces substantial economic losses due to infectious disease outbreaks, such as bacterial infections. These outbreaks cause disruptions and high mortalities at various stages of the rearing process, especially in the larval stages. Probiotic bacteria are emerging as promising and sustainable alternative or complementary strategies to vaccination and the use of antibiotics in aquaculture. In this study, potential probiotic candidates for larviculture were isolated from a rotifer-rearing tank used as the first live feed for turbot larvae. Two Lacticaseibacillus paracasei and two Lactiplantibacillus plantarum isolates were selected for further characterization due to their wide and strong antimicrobial activity against several ichthyopathogens, both Gram-positive and Gram-negative. An extensive in vitro safety assessment of these four isolates revealed the absence of harmful traits, such as acquired antimicrobial resistance and other virulence factors (i.e., hemolytic and gelatinase activities, bile salt deconjugation, and mucin degradation, as well as PCR detection of biogenic amine production). Moreover, Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) analyses unveiled their genetic relatedness, revealing two divergent clusters within each species. To our knowledge, this work reports for the first time the isolation and characterization of Lactic Acid Bacteria (LAB) with potential use as probiotics in aquaculture from rotifer-rearing tanks, which have the potential to optimize turbot larviculture and to introduce novel microbial management approaches for a sustainable aquaculture.

Assessing the Effects of Rotifer Feed Enrichments on Turbot (Scophthalmus maximus) Larvae and Post-Larvae Gut-Associated Bacterial Communities

Live feed enrichments are often used in fish larvicultures as an optimized source of essential nutrients to improve larval growth and survival. In addition to this, they may also play an important role in structuring larval-associated microbial communities and may help improve their resistance to diseases. However, there is limited information available on how larval microbial communities and larviculture water are influenced by different live feed enrichments. In the present study, we investigated the effects of two commercial rotifer enrichments (ER) on turbot (Scophthalmus maximus) larval and post-larval gut-associated bacterial communities during larviculture production. We evaluated their effects on bacterial populations related to known pathogens and beneficial bacteria and their potential influence on the composition of bacterioplankton communities during larval rearing. High-throughput 16S rRNA gene sequencing was used to assess the effects of different rotifer enrichments (ER1 and ER2) on the structural diversity of bacterial communities of the whole turbot larvae 10 days after hatching (DAH), the post-larval gut 30 DAH, and the larviculture water. Our results showed that different rotifer feed enrichments were associated with significant differences in bacterial composition of turbot larvae 10 DAH, but not with the composition of larval gut communities 30 DAH or bacterioplankton communities 10 and 30 DAH. However, a more in-depth taxonomic analysis showed that there were significant differences in the abundance of Vibrionales in both 10 DAH larvae and in the 30 DAH post-larval gut fed different RE diets. Interestingly, the ER1 diet had a higher relative abundance of specific amplicon sequence variants (ASVs) related to potential Vibrio-antagonists belonging to the Roseobacter clade (e.g., Phaeobacter and Ruegeria at 10 DAH and Sulfitobacter at 30 DAH). In line with this, the diet was also associated with a lower relative abundance of Vibrio and a lower mortality. These results suggest that rotifer diets can affect colonization by Vibrio members in the guts of post-larval turbot. Overall, this study indicates that live feed enrichments can have modulatory effects on fish bacterial communities during the early stages of development, which includes the relative abundances of pathogenic and antagonist taxa in larviculture systems.

Effects of Dietary Supplement of Schizochytrium Meal on Growth, Fatty Acid Profile and Activities of Digestive Enzymes in Turbot (Scophthalmus maximus L.) Larvae

Effects of Dietary Supplement of Schizochytrium Meal on Growth, Fatty Acid Profile and Activities of Digestive Enzymes in Turbot (Scophthalmus maximus L.) Larvae

Feeding with poly(I:C) induced long-term immune responses against bacterial infection in turbot (Scophthalmus maximus)

Poly(I:C) is a kind of chemosynthetic double-stranded RNA (dsRNA) analogue which could act as TLR3 agonist and induce IFN production. It is widely applied in anti-virus treatment and immunoregulation, as well as vaccine adjuvant in farmed animals. However, whether poly(I:C) could activate innate immune response to defense against bacterial infection remains unclear. In this study, we established a feeding trial model with different dose of poly(I:C) in turbot larvae, then challenged with Edwardsiella piscicida after 3–7 weeks resting period. The results show that feeding turbot with poly(I:C) exhibited a stronger inflammatory response and antioxidant stress ability, and significantly elevated the survival rate within the decreased bacterial loads. Importantly, the bacterial infection-induced white feces in hindgut of turbot were significantly alleviated after poly(I:C) feeding, and this administration induced protection could last for about 7 weeks. Taken together, these findings indicate that feeding turbot with poly(I:C) could enhance a long-term intestinal mucosal immunity in response to bacterial infection, suggesting that poly(I:C) might be a promising immunostimulant in aquaculture.

A review of the cultivation potential of turbot (Scophthalmus maximus (L.)) in the Baltic Sea region: a promising candidate species for marine aquaculture in Russia

Turbot (Scophthalmus maximus (L.)) is regarded as a good quality product that enjoys widespread acceptance and is therefore a popular choice for marine aquaculture in Europe. This study examines the potential and feasibility of the artificial cultivation of turbot in the Baltic Sea region. It takes into consideration both commercial farming and the implementation of artificial reproduction for stock enhancement programs. With regard to the latter, it discusses observed short-term trends towards reducing species catches in ICES 22-32 subdivisions as well as trends in hydrological process dynamics during the spawning process (especially low water salinity, which decreases the natural reproduction efficiency). The article also considers the potential of the research performed in 2018-2019 at the AtlantNIRO research facility on the development of cultivation techniques for turbot larvae. The findings demonstrate the possibility of cultivating turbot larvae under artificial conditions and are important for the development of marine aquaculture in the Kaliningrad region of Russia, since they can lead to the introduction of new fish species for farming in the future.

Molecular identification of Uronema marinum (Protozoa, Ciliophora, Scuticociliatia) in cultured turbot (Psetta maxima) larvae.

Scuticociliates are dangerous parasitic pathogens causing systemic tissue destruction and high mortality in marine fish worldwide. In this study, the first identification of Uronema marinum (Ciliophora, Scuticociliatida) from cultured turbot (Psetta maxima) larvae using mitochondrial cytochrome c oxidase 1 (cox1) gene sequence as well as species-specific primers was reported. The mean prevalence values of infected fish were calculated, and partial sequencing obtained from the mitochondrial cox1 gene region was also compared with isolates registered in the Genbank database. The sequence comparison showed 93.00% identity to U. marinum, and the parasite has also been deposited in the GenBank database. This study is the first case of U. marinum infection in Turkish marine aquaculture, contributing to the systematics and molecular epidemiology of scuticociliate in Turkey.

The intestine of artificially bred larval turbot (Scophthalmus maximus) contains a stable core group of microbiota.

Generally speaking, fish intestinal microbiota is easily affected by food or water environment, and it may be dynamically changed along with body growth. However, it remains unclear whether fish gut microbiota can be affected under any conditions. In the present study, we focused on cultured larval turbot (Scophthalmus maximus) and tracked its artificial breeding process from eggs to larvae in two farms located in different regions of China. Through continuous sampling, we analyzed and compared characteristics of intestinal microbiota in turbot larvae and its correlation with the bacteria in water and food at different developmental stages. The results showed that there was a steady group of microbiota in larval gut, and the highest relative abundance of strain was same between the two farms. This microbiota was established soon after hatching of fertilized eggs. Particularly, the structure of this microbiota was nearly not changeable afterward 3-4months of development. The bacteria carried by fertilized eggs might play an important role during the formation of this microbiota. In conclusion, our findings suggested that there was a core microbiota represented by Lactococcus sp. in gut of artificially bred turbot larvae. The relative proportion of such strain in gut was higher than 30% at the initial stage of turbot life.

Changes in the Microbiome of Mariculture Feed Organisms after Treatment with a Potentially Probiotic Strain of Phaeobacter inhibens.

The Phaeobacter genus has been explored as probiotics in mariculture as a sustainable strategy for the prevention of bacterial infections. Its antagonistic effect against common fish pathogens is predominantly due to the production of the antibacterial compound tropodithietic acid (TDA), and TDA-producing strains have repeatedly been isolated from mariculture environments. Despite many in vitro trials targeting pathogens, little is known about its impact on host-associated microbiomes in mariculture. Hence, the purpose of this study was to investigate how the addition of a TDA-producing Phaeobacter inhibens strain affects the microbiomes of live feed organisms and fish larvae. We used 16S rRNA gene sequencing to characterize the bacterial diversity associated with live feed microalgae (Tetraselmis suecica), live feed copepod nauplii (Acartia tonsa), and turbot (Scophthalmus maximus) eggs/larvae. The microbial communities were unique to the three organisms investigated, and the addition of the probiotic bacterium had various effects on the diversity and richness of the microbiomes. The structure of the live feed microbiomes was significantly changed, while no effect was seen on the community structure associated with turbot larvae. The changes were seen primarily in particular taxa. The Rhodobacterales order was indigenous to all three microbiomes and decreased in relative abundance when P. inhibens was introduced in the copepod and turbot microbiomes, while it was unaffected in the microalgal microbiome. Altogether, the study demonstrates that the addition of P. inhibens in higher concentrations, as part of a probiotic regime, does not appear to cause major imbalances in the microbiome, but the effects were specific to closely related taxa.IMPORTANCE This work is an essential part of the risk assessment of the application of roseobacters as probiotics in mariculture. It provides insights into the impact of TDA-producing Phaeobacter inhibens on the commensal bacteria related to mariculture live feed and fish larvae. Also, the study provides a sequencing-based characterization of the microbiomes related to mariculture-relevant microalga, copepods, and turbot larvae.

The plasticity of vision and body development of turbotScophthalmus maximuslarvae Under different light spectra

This study was aimed to find out the effect of five light spectra (white, LDW; blue, LDB; green, LDG; red, LDR and yellow, LDY) on the visual development and growth performance of turbot (Scophthalmus maximus) larvae. The number (per 100 μm) of cone cells, outer nuclear cells, ganglion cells, the ratio of outer nuclear to cone and ganglion cells, lens diameter and the minimum separable angle were determined. Variations in growth performance in total length, body mass and body colour were also examined. The results showed that the LDB group underwent metamorphosis faster than did the other groups and attained the greatest total length, body mass and light sensitivity. The LDB group also completed the body colour variation prior to the other groups. The LDG group exhibited a high mortality rate since 25 dph (days post-hatching). We arrived at the conclusions that the spectra could affect the vision and body development of turbot larvae specifically and the blue light had the enhancing efficiency during this process, followed by the white and yellow light. The red light imposed an inhibiting effect on these variations. Our study highlighted the significance of light condition and provided the reference for the farming.

Growth, stress and non-specific immune responses of turbot (Scophthalmus maximus) larvae exposed to different light spectra

The welfare of farmed fish is an important issue in aquaculture. There is particular concern regarding ambient light in culture systems. In the current study, turbot (Scophthalmus maximus) larvae were exposed to four different light spectra: full spectrum (LDF), blue (LDB, peak at 450 nm), orange (LDO, peak at 595 nm) and red (LDR, peak at 629 nm) from the embryo stage to 60 days post hatching (dph). During this time, the growth and immune response of the turbot were evaluated. Whole fish tissue samples were collected and weighed. mRNA expression levels of insulin-like growth factor-I (IGF-I), heat shock protein 70 (HSP70), glutathione S-transferase (GST), catalase (CAT), metallothionein (MT), lysozyme (LZM), cathepsin D (CTSD), cathepsin F (CTSF) and the enzyme activity of superoxide dismutase (SOD), CAT, and peroxidase (POD) were measured at 10, 20, 30, 40, 50, and 60 dph. The results showed that LDB had a positive effect on the growth of turbot larvae at 20, 40, 50 and 60 dph, while this effect was not significant at 10 and 30 dph. Levels of IGF-I mRNA also showed a similar tendency, indicating the crucial interaction between IGF-I and somatic growth. The effect of light spectrum on the stress response of turbot larvae was stage specific. At 10 dph, prior to metamorphosis, long wavelengths could induce a stress response in turbot larvae. At 20 dph, metamorphosis began and no significant stress response was observed in larvae exposed to different light spectra; however, LDO significantly up-regulated SOD, CAT, POD and LZM activity. Metamorphosis continued until 56 dph. From 20 to 60 dph long wavelengths (LDR and LDO) resulted in a stress response characterized by high mRNA expression levels of HSP70, GST, CAT, MT, LZM, CTSD and CTSF. SOD, CAT, POD and LZM activities were inhibited, indicating increased enzyme consumption to maintain homeostasis. Overall, our results suggested that LDF should be used in turbot culture systems prior to metamorphosis and LDB should be used after metamorphosis. These findings are in keeping with the changes in light spectra experienced by turbot larvae undergoing metamorphosis in the wild.

Responses of microbial community structure in turbot (Scophthalmus maximus) larval intestine to the regulation of probiotic introduced through live feed.

Various bacteria that adhere to the gut are important for the health of fish. Regulating the microbial community in a desirable direction may be beneficial in aquaculture for preventing and controlling the diseases caused by pathogenic microbes. In this study, we investigated the changes in the microflora in the intestinal tracts of turbot (Scophthalmus maximus) larvae after introducing a probiotic (Bacillus amyloliquefaciens) after the first feed. B. amyloliquefaciens was added as part of a nutrient enrichment system in live feed (Branchionus plicatilis or Artemia sinica), so it passed into the intestinal tracts of the newly hatched turbot larvae. The turbot larvae were fed on live feed containing B. amyloliquefaciens in the experimental group, whereas live feed without the probiotic was provided to larvae in the control group. The total bacterial genomic DNA in the larval guts was extracted and sequenced with an Illumina HiSeq PE250 system. According to the sequencing results, the abundances of microbial species and the microflora diversity were lower in the intestines in the experimental group than the control. Throughout development, the microflora structure in the intestines was mainly constructed before the first feed and the composition of the dominant operational taxonomic units (OTUs) was stable, where the abundances of OTU8, OTU124, OTU150, OTU107, and OTU17 were always high. Compared with the control, the structures of the microflora in the intestines were similar on different days during the development and the growth of larvae in the experimental group. However, the similarity of the microflora structure between different treatments was low on the same day. Furthermore, the mean proportion of common OTUs was only 74.7% in different treatments on each day, which indicates that the introduction of B. amyloliquefaciens in the live feed changed the microflora structure in the intestine. During the early development stage (days 3–30), the average abundance of Pseudomonas was reduced by 0.8% whereas that of Lactococcus increased by 3.5% in the experimental group. Pseudomonas spp. are considered potentially pathogenic bacteria but there is no direct evidence for the pathogenicity of Lactococcus in turbot. Moreover, several Lactococcus species are regarded as probiotics in aquaculture. Therefore, the use of B. amyloliquefaciens could be beneficial for optimizing the microbial community structure in the intestines of turbot larvae, which may explain the probiotic effect of B. amyloliquefaciens. This study provides a theoretical basis for the biological regulation of the microflora structure in the intestinal tract during turbot breeding.

Effects of different light spectra on embryo development and the performance of newly hatched turbot (Scophthalmus maximus) larvae

Light is a key environmental factor that synchronizes various life stages from embryo development to sexual maturation in fish. For turbot, light spectra have the most influence at the larval and juvenile stages. In the current study, differences in the development of embryos and the performance of newly hatched turbot larvae exposed to five different spectra: full spectrum (LDF), blue (LDB, peak at 450 nm), green (LDG, peak at 533 nm), orange (LDO, peak at 595 nm) and red (LDR, peak at 629 nm), were examined. At 62.8 h post fertilization, a higher number of embryos exposed to short-wavelengths (LDG and LDB) had developed a heartbeat in comparison with embryos exposed to other wavelengths. Larvae exposed to the green spectrum had higher malformation rates than larvae exposed to the other spectra, indicating that larvae exposed to green light may have significantly reduced survival rates. The results of non-specific immunity parameters showed that the mRNA expression levels of cathepsin D (CTSD), cathepsin F (CTSF), catalase (CAT) and metallothionein (MT) in larvae exposed to LDB were significantly higher than those exposed to other spectra, but CAT activity in larvae exposed to LDB was significantly lower than larvae exposed to the other spectra. There was no significant difference in MT activity in larvae exposed to the five different spectra. The mRNA expression level of lysozyme (LZM) in larvae exposed to LDR was significantly higher than other spectra, while there was no significant difference in LZM activity observed in larvae exposed to LDR, LDG, LDB and LDF. The difference of the enzyme activity of total superoxide dismutase (T-SOD) was not significant among larvae exposed to the five spectra. mRNA expression of the heat shock protein 70 (HSP70) was significantly higher in newly hatched larvae exposed to LDB, LDR and LDG, indicating that larvae exposed to LDB, LDG and LDR exhibited a stress response. The mRNA expression level of the insulin-like growth factor-1 (IGF-1) and growth parameters in the newly hatched larvae exposed to the different spectra were not significantly different. The results of the present study indicate that LDO and LDF should be used for embryo incubation and newly hatched larvae when rearing turbot. This study provides a theoretical basis for optimizing the incubation light environment for fertilized turbot eggs, promoting immunity and reducing stress responses in newly hatched larvae.

Exploring the Effect of Phage Therapy in Preventing Vibrio anguillarum Infections in Cod and Turbot Larvae.

The aquaculture industry is suffering from losses associated with bacterial infections by opportunistic pathogens. Vibrio anguillarum is one of the most important pathogens, causing vibriosis in fish and shellfish cultures leading to high mortalities and economic losses. Bacterial resistance to antibiotics and inefficient vaccination at the larval stage of fish emphasizes the need for novel approaches, and phage therapy for controlling Vibrio pathogens has gained interest in the past few years. In this study, we examined the potential of the broad-host-range phage KVP40 to control four different V. anguillarum strains in Atlantic cod (Gadus morhua L.) and turbot (Scophthalmus maximus L.) larvae. We examined larval mortality and abundance of bacteria and phages. Phage KVP40 was able to reduce and/or delay the mortality of the cod and turbot larvae challenged with V. anguillarum. However, growth of other pathogenic bacteria naturally occurring on the fish eggs prior to our experiment caused mortality of the larvae in the unchallenged control groups. Interestingly, the broad-spectrum phage KVP40 was able to reduce mortality in these groups, compared to the nonchallenge control groups not treated with phage KVP40, demonstrating that the phage could also reduce mortality imposed by the background population of pathogens. Overall, phage-mediated reduction in mortality of cod and turbot larvae in experimental challenge assays with V. anguillarum pathogens suggested that application of broad-host-range phages can reduce Vibrio-induced mortality in turbot and cod larvae, emphasizing that phage therapy is a promising alternative to traditional treatment of vibriosis in marine aquaculture.

Effect of TDA-producing Phaeobacter inhibens on the fish pathogen Vibrio anguillarum in non-axenic algae and copepod systems.

SummaryThe expanding aquaculture industry plays an important role in feeding the growing human population and with the expansion, sustainable bacterial disease control, such as probiotics, becomes increasingly important. Tropodithietic acid (TDA)‐producing Phaeobacter spp. can protect live feed, for example rotifers and Artemia as well as larvae of turbot and cod against pathogenic vibrios. Here, we show that the emerging live feed, copepods, is unaffected by colonization of the fish pathogen Vibrio anguillarum, making them potential infection vectors. However, TDA‐producing Phaeobacter inhibens was able to significantly inhibit V. anguillarum in non‐axenic cultures of copepod Acartia tonsa and the copepod feed Rhodomonas salina. Vibrio grew to 106 CFU ml−1 and 107 CFU ml−1 in copepod and R. salina cultures, respectively. However, vibrio counts remained at the inoculum level (104 CFU ml−1) when P. inhibens was also added. We further developed a semi‐strain‐specific qPCR for V. anguillarum to detect and quantify the pathogen in non‐axenic systems. In conclusion, P. inhibens efficiently inhibits the fish larval pathogen V. anguillarum in the emerging live feed, copepods, supporting its use as a probiotic in aquaculture. Furthermore, qPCR provides an effective method for detecting vibrio pathogens in complex non‐axenic live feed systems.

Ontogenetic development of attack behaviour by turbot larvae when exposed to copepod prey

Identification of fish larval behavioural traits permitting capture of specific live prey sizes is an important part of optimizing production of marine larvae. We investigated the capture success of turbot larvae (Scophthalmus maximus) at two development stages, 8 and 10 days post‐hatch (DPH), when offered small nauplii (129–202 μm), large nauplii (222–278 μm) and copepodites (342–542 μm), of the calanoid copepod Acartia tonsa. At 8 DPH, turbot larvae had the highest capture success (67%) when offered small nauplii, with a lower capture success of large nauplii (27%) but totally lacked the capabilities to capture copepodites. At DPH 10, the larvae increased the capture success of large nauplii (47%) and achieved a few successful attacks on copepodites. Energetically, large nauplii were the most beneficial at both larval development stages. The swimming kinematics of the period prior to a strike by the larva on the copepod was examined, and the approach pattern of the larva was identified as a controlling mechanism for their strike distance, with the initial approach speed of larva at DPH 10 being significantly less than at DPH 8. In all successful attacks, the strike distance was less than 1.17 mm and was significantly lower than unsuccessful attacks. Since the approach pattern of the larva is linked to its capture success, it could be used as the basis for a feeding scheme based on the swimming performance of individual batches of turbot larvae.

Influence of swimming behavior of copepod nauplii on feeding of larval turbot (Scophthalmus maximus)

Feeding in larval fish is influenced by a range of factors and among these are the morphological and behavioral characteristics of their prey. We investigated the influence of the swimming behavior of two species of calanoid copepods, Acartia tonsa and Temora longicornis, on larval turbot feeding. The nauplii of these species represent two contrasting swimming behaviors: A. tonsa is a jump-sink type swimmer, while T. longicornis is a cruise swimming type. Three replicates of ten larvae aged 7 and 9 days post hatch (DPH) were observed feeding on one of the two copepod species using a 2-dimensional video setup. The results showed that the duration of aiming postures by turbot larvae was 2.3 times higher when turbot larvae approached T. longicornis as compared to A. tonsa nauplii, indicating that larvae can more easily position themselves, preparing for attack, when the prey is of the jump-sink type. The attack speed of turbot larvae feeding on A. tonsa nauplii decreased slightly from DPH 7 to DPH 9, whereas it increased when attacking T. longicornis nauplii. Capture success rate by turbot larvae feeding on A. tonsa was 58% and slightly higher, but not significantly different to capture success rate when feeding on T. longicornis (54%). We conclude that the differences between behavior and other characteristics of these prey species have only minor effect on larval fish feeding, suggesting that copepods species for live feed should be selected according to their ease to culture more than to their species-specific characteristics.

Patterns of catabolic capacities and energy metabolism in developing embryos and yolk-sac larvae of turbot (Scophthalmus maximus L.)

Patterns of catabolic capacities and energy metabolism in turbot eggs during embryogenesis and yolk-sac larvae were investigated. The catabolization of energy fuels were evaluated by specific enzymes including trypsin-like proteases (TRY) for protein and triglyceride lipase (LIP) for lipids. Meanwhile, citrate synthase (CS) and lactate dehydrogenase (LDH) were measured to assess energy production pathway. Regarding contribution to energy production, the enzymes of pyruvate kinase (PK), aspartate aminotransferase (AAT) and hydroxyacyl CoA dehydrogenase (HOAD) which was indicative of the mobilizations of carbohydrates, amino acids and fatty acids respectively and their ratios were detected.Results showed the metabolic enzymes and catabolic enzymes were present and involved in the embryogenesis of turbot. LIP activity exhibited a pattern of ‘low-high-low-high’. TRY activity dropped to the minimal value at blastula stage, and then increased significantly to the maximal value at 2days after hatching (DAH). The enzymatic activities of HOAD, AAT, LDH and CS all showed similar upward tendency and increased significantly as development went on. PK activity increased to the peak at cleavage stage, and then decreased to the lowest point at hatching day.Concerning enzymatic ratios, both AAT/HOAD and PK/HOAD presented similar “high-low” patterns, showing the maximal value 3.18 and 45.27 respectively at cleavage stage and the minimal value 0.46 and 0.84 respectively at 2 DAH. PK/AAT ratio declined from fertilized eggs (14.31) to 2 DAH (1.82). PK/CS ratio decreased significantly from cleavage stage (1.63) to 2 DAH (0.16). In contrast, a rising tendency of HOAD/CS ratio appeared as growth went on. AAT/CS ratio peaked at gastrula stage (0.13) and declined thereafter. LDH/CS ratio increased from fertilized eggs (1.42) to cleavage stage (1.85), and then decreased until 2 DAH (0.55). LDH/PK ratio reached the peak of 3.85 at hatching day.Combined with patterns of enzymatic ratios, it can be concluded that carbohydrates are predominantly mobilized before cleavage stage and are gradually substituted by amino acids from cleavage stage to gastrula stage and fatty acids during the following days, revealing their importance for morphogenesis in turbot. Regarding energy production pathway, the dominant mode was anaerobic pathway before cleavage stage and aerobic pathway during subsequent periods.

Ontogeny of the digestive enzymes, thyroid hormones and cortisol in developing embryos and yolk-sac larvae of turbot (Scophthalmus maximus L.)

Activities of the digestive enzymes and concentrations of thyroid hormones and cortisol in developing embryos and yolk-sac larvae of turbot (Scophthalmus maximus L.) were measured in this study. Results showed that the embryonic mass presented “decrease-increase-decrease-increase-decrease” trend, appearing the minimal value of 0.34mg at 7.5h after fertilization (HAF) before the onset of blastula stage. As for egg diameter, it increased on the whole from 1.027mm at 0.5 HAF to 1.079mm at 95.67 HAF.In general, digestive enzymes presented four patterns in the present work. The patterns of alkaline phosphatase (AKP), pepsin (PG), chymotrypsin (CP), trypsin (TRY), leucine aminopeptidase N (LAP), lactate dehydrogenase (LDH) and glutamic oxalo acetic transaminase (GOT) were similar, showing low level before segmentation stage followed by increases until hatching day. Besides AKP, the other enzymes reached the peaks at 2days after hatching (DAH). Enzyme activities of acid phosphatase (ACP), lipase (LIP) and pyruvate kinase (PK) presented similar “decrease-increase” changes on the whole. Leucine-alanine peptidase (LAPP) was detected from fertilized eggs onwards, increased till 86 HAF at segmentation stage and then decreased thereafter. Amylase (AMY) activity was undetectable before gastrula stage and presented upward tendency thereafter.T3 concentration declined to the lowest value of 61.82ng/g sample at 3.17 HAF and exhibited “increase-decrease-increase” patterns during cleavage stage, then it elevated markedly until 29.5 HAF, and then maintained high stable levels before the onset of yolk-sac larval days, reaching the highest value of 220.62ng/g sample at 72.5 HAF, followed by obvious decline until the end. T4 concentration showed opposite tendencies to that of T3 before segmentation stage with the maximal value of 160.95ng/g sample at 39.17 HAF and the minimal value of 85.94ng/g sample at 24 HAF, and remained high levels before 0 DAH, followed by a slight decrease thereafter.Cortisol concentration presented three obvious declines during 2.67–4.67 HAF, 5.67–9.67 HAF and 10.67–24 HAF. The maximal value was 147.86ng/g sample at 5.67 HAF and the minimal value was 73.60ng/g sample at 24 HAF, respectively.The above results are very valuable to illuminate the evolvement of enzymes and hormones to better understand their functions during endogenous nutritional periods and serve as a basis for future related studies in this fish.

A production season of turbot larvaeScophthalmus maximus(Linnaeus, 1758) reared on copepods in a Danish (56°N) semi-intensive outdoor system

Turbot were reared from yolk sack larvae to juvenile in an outdoor semi-intensive system. Three production cycles were monitored from May to September. A pelagic food chain was established with phytoplankton, copepods and turbot larvae. Abiotic and biotic parameters of lower trophic levels together with turbot larval survival, development, prey electivity and growth were monitored. A decreasing larval survival from 18.4% in May to 13.6% in July and just 7.0% in September was observed. The overall phytoplankton and copepod abundance decreased during the productive season. The turbot larval growth showed significant differences between larvae below (isometric) and above (allometric) 7 mm. Larval fish gut content showed no differences with available prey between production cycles. Therefore, it appears that the available prey concentration is governing their growth in this outdoor system. First-feeding turbot larvae exhibited active selection for nauplii whereas developed larvae switched to copepodites and adult copepods. Although developing turbot larva exhibited active selection towards copepod size classes, there was no evidence of selective feeding on either of the two dominant copepod species. The turbot larvae's prey ingestion was modelled together with the standing stock of copepod biomass. The model results indicated that the estimated need for daily ingestion exceeded the standing stock of copepods. Hence, the initially established food web was unable to sustain the added turbot larvae with starvation as a consequence. We therefore suggest several solutions to circumvent starvation in the semi-intensive system.

Isolation of TDA-producing Phaeobacter strains from sea bass larval rearing units and their probiotic effect against pathogenic Vibrio spp. in Artemia cultures

Fish-pathogenic Vibrio can cause large-scale crashes in marine larval rearing units and, since the use of antibiotics can result in bacterial antibiotic resistance, new strategies for disease prevention are needed. Roseobacter-clade bacteria from turbot larval rearing facilities can antagonize Vibrio anguillarum and reduce mortality in V. anguillarum-infected cod and turbot larvae. In this study, it was demonstrated that antagonistic Roseobacter-clade bacteria could be isolated from sea bass larval rearing units. In addition, it was shown that they not only antagonized V. anguillarum but also V. harveyi, which is the major bacterial pathogen in crustaceans and Mediterranean sea bass larvae cultures. Concomitantly, they significantly improved survival of V. harveyi-infected brine shrimp. 16S rRNA gene sequence homology identified the antagonists as Phaeobacter sp., and in silico DNA-DNA hybridization indicated that they could belong to a new species. The genomes contained genes involved in synthesis of the antibacterial compound tropodithietic acid (TDA), and its production was confirmed by UHPLC-TOFMS. The new Phaeobacter colonized live feed (Artemia) cultures and reduced Vibrio counts significantly, since they reached only 104CFUmL−1, as opposed to 108CFUmL−1 in non-Phaeobacter treated controls. Survival of V. anguillarum-challenged Artemia nauplii was enhanced by the presence of wild type Phaeobacter compared to challenged control cultures (89±1.0% vs 8±3.2%). In conclusion, TDA-producing Phaeobacter isolated from Mediterranean marine larviculture are promising probiotic bacteria against pathogenic Vibrio in crustacean live-feed cultures for marine fish larvae.