Characterization of β-haemolytic Group B Streptococcus agalactiae in cultured seabream, Sparus auratus L., and wild mullet, Liza klunzingeri (Day), in Kuwait

Streptococcus agalactiae was isolated from cultured gilthead seabream, Sparus auratus L., and diseased wild Klunzinger's mullet, Liza klunzingeri (Day), in Kuwait Bay, Arabian Gulf. Isolates were catalase negative, β‐haemolytic, Gram‐positive cocci and serogroup B. Experimental infectivity trials with mullet and seabream brain isolates in Nile tilapia, Oreochromis niloticus L., caused 100 and 90% mortality, respectively, within 7 days post‐inoculation indicating virulent S. agalactiae as the bacterial pathogen responsible for the epizootic in Kuwait Bay.     

Seasonal expression of arginine vasotocin mRNA and its correlations to gonadal steroidogenic enzymes and sexually dimorphic coloration during sex reversal in the gilthead seabream (<Emphasis Type="Italic">Sparus aurata</Emphasis>)

Arginine vasotocin is a hormone produced in the hypothalamus of teleost fish that has been shown to regulate gonad development and sexual behavior. To study the role of arginine vasotocin in the gonadal cycle of the hermaphrodite gilthead seabream, Sparus aurata, we cloned the seabream arginine vasotocin (avt) complementary DNA (cDNA). We investigated the expression of brain avt throughout the gonad cycle using real-time quantitative PCR and compared its expression levels to the expression levels of two key gonadal steroidogenic enzymes, cyp19a1a and cyp11b2. In July, when the process of sex reversal is thought to begin, avt expression was elevated over the previous 2 months. Avt in the brain remained at or above the level of July until November then peaked again in December. There was no difference between males and females in the expression levels of brain avt throughout the year. However, only in ambisexual fish was the expression of the cyp19a1a gonadal aromatase correlated to the expression of avt in the brain. Cyp11b2 did not show any correlation to brain avt expression. We also found that females had more intense body coloration than males and that this intensity peaked prior to spawning. Avt expression and female coloration were positively correlated. The fact that brain avt expression was lowest during gonad quiescence, together with the observation of a correlation between brain avt with gonadal cyp19a1a and body coloration during that time suggests that avt may play a role during the process of sex reversal and spawning of the gilthead seabream.     

Influence of the dietary inclusion of <Emphasis Type="Italic">Gracilaria cornea</Emphasis> and <Emphasis Type="Italic">Ulva rigida</Emphasis> on the biodiversity of the intestinal microbiota of <Emphasis Type="Italic">Sparus aurata</Emphasis> juveniles

Partial substitutions of fish meal by 5, 15, or 25 % of Gracilaria cornea or Ulva rigida in experimental diets were evaluated to study their effects on biodiversity of intestinal microbiota composition in gilthead seabream (Sparus aurata) juveniles. The diets were offered to duplicate groups of 15 juvenile fish (14.0 ± 0.5 g) for 70 days, and at the end of the experiment the intestinal microbiota from four specimens of each treatment was analysed by denaturing gel gradient electrophoresis. Results showed that the substitution of fish meal by algae meal induced important modifications in the intestinal microbiota community, as a big reduction of the biodiversity when the highest percentage (25 %) of U. rigida was included. On the contrary, an increase on the number of species was detected when a 15 % of algae was included. Various Lactobacillus delbrueckii subspecies were selectively stimulated when G. cornea was included in the feed, and other bacterial species, such as those included in the Vibrio genus, were reduced.     

Nano-delivery of trace minerals for marine fish larvae: influence on skeletal ossification,and the expression of genes involved in intestinal transport of minerals,osteoblast differentiation,and oxidative stress response

Currently, the larviculture of many marine fish species with small-sized larvae depends for a short time after hatching, on the supply of high-quality live zooplankton to ensure high survival and growth rates. During the last few decades, the research community has made great efforts to develop artificial diets, which can completely substitute live prey. However, studies aimed at determining optimal levels of minerals in marine larvae compound feeds and the potential of novel delivery vectors for mineral acquisition has only very recently begun. Recently, the agro-food industry has developed several nano-delivery systems, which could be used for animal feed, too. Delivery through nano-encapsulation of minerals and feed additives would protect the bioactive molecules during feed manufacturing and fish feeding and allow an efficient acquisition of active substances into biological system. The idea is that dietary minerals in the form of nanoparticles may enter cells more easily than their larger counterparts enter and thus speed up their assimilation in fish. Accordingly, we evaluated the efficacy of early weaning diets fortified with organic, inorganic, or nanoparticle forms of trace minerals (Se, Zn, and Mn) in gilthead seabream (Sparus aurata) larvae. We tested four experimental diets: a trace mineral-deficient control diet, and three diets supplemented with different forms of trace minerals. At the end of the feeding trial, larvae growth performance and ossification, and the level of expression of six target genes (SLC11A2β, dmt1, BMP2, OC, SOD, GPX), were evaluated. Our data demonstrated that weaning diets supplemented with Mn, Se, and Zn in amino acid-chelated (organic) or nanoparticle form were more effective than diets supplemented with inorganic form of minerals to promote bone mineralization, and prevent skeletal anomalies in seabream larvae. Furthermore, nanometals markedly improved larval stress resistance in comparison to inorganic minerals and upregulated mRNA copy number of OC gene. The expression of this gene was strongly correlated with mineralization degree, thus confirming its potency as a good marker of bone mineralization in gilthead seabream larvae.     

Schizothorax prenanti corticotropin-releasing hormone (CRH): molecular cloning,tissue expression,and the function of feeding regulation

Corticotropin-releasing hormone (CRH) is a potent mediator of endocrine, autonomic, behavioral, and immune responses to stress. For a better understanding of the structure and function of the CRH gene and to study its effect on feeding regulation in cyprinid fish, the cDNA of the CRH gene from the brain of Schizothorax prenanti was cloned and sequenced. The full-length CRH cDNA consisted of 1,046 bp with an open reading frame of 489 bp encoding a protein of 162 amino acids. Real-time quantitative PCR analyses revealed that CRH was widely expressed in central and peripheral tissues. In particular, high expression level of CRH was detected in brain. Furthermore, CRH mRNA expression was examined in different brain regions, especially high in hypothalamus. In addition, there was no significant change in CRH mRNA expression in fed group compared with the fasted group in the S. prenanti hypothalamus during short-term fasting. However, CRH gene expression presented significant decrease in the hypothalamus in fasted group compared with the fed group (P < 0.05) on day 7; thereafter, re-feeding could lead to a significant increase in CRH mRNA expression in fasted group on day 9. The results suggest that the CRH may play a critical role in feeding regulation in S. prenanti.     

A bioenergetic and protein flux model to simulate fish growth in commercial farms: Application to the gilthead seabream

A model for fish growth simulation based on the bioenergetic factorial approach is presented. This work presents a novel approach that extends the traditional bioenergetic model by explicitly including the Energy and Protein fluxes (EP model). This is a valuable feature that allows the dynamic simulation of fish proximate composition. For the aquaculture industry it represents a trade-off between detailed process simulation and feasibility of model implementation, namely regarding data gathering on an operational setting. The EP model is targeted to simulate fish production in commercial farms. Farm data for feed intake, feed composition (energy and protein content), temperature over time and the initial fish body weight are the only required data to run the model. Furthermore, apparent digestibility coefficient (ADC) values of the feed used in the farm must be known or else ADC values of feeds with similar composition can be used. The EP model implementation is illustrated for the gilthead seabream (Sparus aurata) based on published experimental data. The model was validated (r = 0.997, p < 0.05, n = 12) using a published experimental data set for gilthead seabream reared in a range of temperatures that reproduce the conditions in most countries producing this species. For the entire growth period (488 days) the estimated mean absolute error (MAE) is 8.8 g.fish⁻¹ and the mean absolute percentage error (MAPE) is 8.3%. Simulation of fish growth in real operational conditions is evaluated with three datasets from a commercial farm that operates in earthen ponds with temperatures ranging between 12.6 °C and 24.8 °C. Overall the model outputs match well with the production data in the 3 batches. Initial weight ranged between 2.8 g and 3.7 g. The deviation between the data and simulated final weights is below 15 g, for a final weight around 435 g. The maximum absolute error is 21.1 g per fish (MAE) and in percentage 8.3% (MAPE).     

Enrichment of gilthead seabream (Sparus aurata L.) diet with microalgae: effects on the immune system

The present study assessed the effects of three orally administered microalgae (Nannochloropsis gaditana, Tetraselmis chuii and Phaeodactylum tricornutum) on different immune parameters and immune-related gene expression of gilthead seabream (Sparus aurata L.). Fish were fed a control or one of six experimental diets and sampled at 2 and 4?weeks of treatment. At the end of the trial, growth performance and different systemic and local immune activities were measured (natural haemolytic complement activity, IgM levels, phagocytosis and respiratory burst). The expression levels of different immune-associated genes (EF-1α, IgM_H, TCR-β, MHCIα, MHCIIα, CSF-1R and β-defensin) were analysed in head-kidney and gut. Administration of N. gaditana and T. chuii resulted in a significant increase in haemolytic complement activity, phagocytic capacity, as well expression level of β-defensin, as well as MHCIIα and CSF-1R, respectively. The P. tricornutum-supplemented diet provoked immunostimulation, and very little effect on gene expression was observed. These results suggest that dietary microalgae enhance gilthead seabream defence activity, which could be very important in order to consider such microalgae as a possible additive in fish diets.     

Cloning of a second variant of somatolactin-encoding cDNA from the gilthead seabream Sparus aurata

cDNA clones which include coding sequences of the gilthead seabream (Sparus aurata) somatolactin (SL) have been isolated from a cDNA library prepared from seabream pituitary gland poly (A)⁺ RNA. Flounder – SL cDNA was used as a hybridization probe. The complete nucleotide (nt) sequence of the seabream somatolactin has been determined. The clone encodes a polypeptide of 231 amino acid (aa) residues including 24 amino acid residues of signal peptide. Northern blot hybridization detected one band of approximately 1.8 kb mRNA. By comparing the sequences of this SL cDNA to the one recently published, it is suggested that two variants of the SL exist in seabream. By comparing the sequences of the aa of SL to the deduced aa sequences, it is possible that even a third variant of SL exists in this species.     

Use of the probiotic Shewanella putrefaciens Pdp11 on the culture of Senegalese sole (Solea senegalensis, Kaup 1858) and gilthead seabream (Sparus aurata L.)

The application of probiotics on aquatic animals is increasing for a better fish welfare status as well as an environment-friendly activity which are actual demands of modern aquaculture industry. A bacterium from skin mucus of healthy gilthead seabream (Sparus aurata L.) has been isolated and identified as Shewanella putrefaciens Pdp11. Different studies have been done to know its application as probiotic in the Senegalese sole and gilthead seabream farming. This article reviews the studies carried out with this probiotic microorganism focusing on the current knowledge of its in vitro and in vivo mechanisms of action. The results suggested that the probiotic S. putrefaciens Pdp11, due to its beneficial effects, could be used in the aquaculture activity of both species.     

Heat-shock protein 70 modulates apoptosis signal-regulating kinase 1 in stressed hepatocytes of Mugil cephalus

Oxidative stress causes damage at the cellular level and activates a number of signaling pathways. Heat-shock proteins (HSPs) play an important role in repair and protective mechanisms under cell response to stress conditions. HSP70 has been shown to act as an inhibitor of apoptosis. Apoptosis signal-regulating kinase-1 (ASK1) activity is regulated at multiple levels, one of which is through inhibition by cytosolic chaperons HSP70. The current study was aimed to investigate the alteration in signaling molecules that allow the fish to survive under stressed natural field conditions. The study also investigates the variation in biomolecular composition of hepatocytes by using Fourier transform infrared spectroscopy. The impact of stress on hepatocytes was assessed by measuring the level of lipid peroxides (LPO), catalase activity (CAT) and assessing the changes in hepatocytes of Mugil cephalus inhabiting Kovalam and Ennore estuaries. The expression of HSP70 and ASK1 were analyzed by immunoblot analysis and ELISA, respectively. The spectral analysis showed variations in biomolecular composition of hepatocytes at a wave number region of 4,000–400 cm^?1. There was significant decrease of CAT activity (p < 0.01) (25 %) with significant increase of LPO (p < 0.001) (35 %) and HSP70 (p < 0.001) and insignificant increase of ASK1 (p < 0.05) (16 %) in fish hepatocytes inhabiting Ennore estuary than Kovalam estuary. In conclusion, the present study suggests that the survival of fish in the Ennore estuary under stressed condition may be due to the upregulation of HSP70 that mediates the altered signal pathway which promotes cellular resistance against apoptosis.     

The bioactivity of gonadotropin releasing hormones and its regulation in the gilthead seabream,Sparus aurata: in vivo andin vitro studies

Thein vivo andin vitro potency of native and modified forms of gonadotropin releasing hormone (GnRH) to release gonadotropin (GtH) was studied inSparus aurata and correlated with their relative susceptibility to degradation by cytosolic-bound enzymes of the pituitary, kidney, and liver. Salmon (s) GnRH and luteinizing hormone-releasing hormone (LHRH) are equipotent whereas analogs of these peptides ((D-Arg⁶-Pro⁹NET)-sGnRH, (D-Ala⁶-Pro⁹NET)-LHRH, (D-Trp⁶)-LHRH) are superactive in inducingin vivo GtH release (at 10 μg/kg body weight). In anin vitro superfusion system of pituitary fragments all analogs are equipotent to the native peptides (at 10⁻¹⁰ to 2.5 × 10⁻⁷M). sGnRH and LHRH are rapidly degraded by cytosolic peptidases of the pituitary, liver, and kidney. The preferred site of cleavage is the Tyr⁵-Gly⁶ bond. Substitution of the position 6 glycine by D-amino acids renders the 5–6 bond resistant to degradation and shifts the main site of cleavage to the Pro⁹-Gly¹⁰NH₂ bond. Substitution at position 6 (as above) and at position 10 with Pro⁹NET results in analogs that are resistant to degradation. We propose that enzymatic cleavage terminates GnRH bioactivityin vivo and thus increased resistance to degradation is a major determinant of GnRH analog superactivity.     

The Competitive Advantages Arising from Different Environmental Conditions in Seabream,Sparus aurata,Production in the Mediterranean Sea

The economic performance of gilthead seabream, Sparus aurata, aquaculture in Mediterranean countries is not homogeneous despite the availability of a universalized technology and the absence of trade barriers. Differences in competitiveness among aquaculture facilities result, in part, from economic and environmental factors, which are conditioned by the geographical location of the facility. This article aims to identify and analyze the competitive advantages derived from the different environmental conditions in which seabream production has been developed along the Mediterranean Sea. A particle swarm optimization algorithm based on a seabream bioeconomic model was applied to the economic optimization of multibatch aquaculture production. The analysis involved hypothetical fattening farms located in the major areas in which this species is cultured. The water temperature was the only parameter that varied among locations. The average water temperatures in these locations were below the optimum growth temperature, around which growth progressively diminishes. The results of the study showed that the locations with higher average water temperatures and less variable water temperatures allowed the cultivation process to be more economically efficient. The seabream farms located in Greece and Turkey had a competitive advantage in terms of productivity and operating profitability because of more favorable environmental conditions.     

Sensory evaluation of farmed sharpsnout seabream (Diplodus puntazzo)

The first attempts at applying aquacultural techniques to sharpsnoutseabream (Diplodus puntazzo) have proved very promising.One of the factors to keep in mind when analysing the viability of a speciesforindustrial farming is that of consumer acceptance of the product, a variablethat can be measured by sensory evaluation tests. To that end, 107 participantsfrom a number of work centres were assessed with hedonic sensory tests usingsharpsnout seabream samples. For reference purposes, the tasting also includedgilthead seabream samples. Participants were asked to fill out a questionnairethat included questions concerning their preferences for any one fish. Weanalysed the sensory attributes of both fish species using a hedonic scale, andalso their acceptability (disposition to purchase) to consumers. The resultsproved fairly encouraging with a view to sharpsnout seabream large-scaleproduction. All attributes tested in the sensory profile analysis werepositively rated. About 60% of the surveyed population would consider buyingsharpsnout seabream at a price similar to that of gilthead seabream (10),and 85% found it to be pleasing to the eye. Aside from flavour, its texture,juiciness and fat level are the most important appraisal attributes. Efforts toimprove the organoleptic characteristics of the species should focus mainly onthese attributes.     

Differential expression of proteins in the gills of Litopenaeus vannamei infected with white spot syndrome virus

Determination of differentially expressed protein profile is necessary to understand the host response to viral infection. Proteomics can be applied as a tool to examine white shrimp Litopenaeus vannamei molecular responses against white spot syndrome virus (WSSV) infection, thus enabling development of effective strategies to reduce their impact on farms. In the present study, specific pathogen-free shrimp was tested against WSSV infection under several time intervals. Shrimps were submitted to a viral load of with 5.5 × 10⁶ viral copies in 100 μL/shrimp. The monitoring of infection was performed in intervals of 6, 12, 24, 48 and 72 h after infection. The analysis was realized using 2-DE, and differentially expressed proteins were identified by MALDI-TOF mass spectrometry (MS) peptide mass fingerprint (PMF). Between the differentially expressed proteins found in the infected animals, the most important were identified as caspase-2, ubiquitin and F1-ATP synthase. They are interesting candidates for biomarkers because could be related to the beginning of apoptosis process. The differentially expressed protein profile creates a new paradigm in the analysis of L. vannamei shrimp molecular response to WSSV infection and in virus–host relationship. Furthermore, it proposes potential biomarkers that allow strategies both selecting less susceptible individuals and reducing the impact of viruses on farms.     

Fish oil replacement by different microalgal products in microdiets for early weaning of gilthead sea bream (Sparus aurata,L.)

The aim of this study was to determine if algal products rich in DHA or ARA are able to completely replace fish oil in microdiets for marine fish larvae, gilthead seabream and if extra supplementation with EPA may further enhance larval performance. For that purpose, 20 day‐old gilthead seabream larvae of 5.97 ± 0.4 mm mean total length and 0.12 ± 0.001 mg mean dry body weight were fed with five microdiets tested by triplicate: a control diet based on sardine oil; a diet containing AquaGrow^® DHA (diet DHA) to completely substitute the sardine oil; a diet containing AquaGrow^® ARA (diet ARA); a diet containing both products, AquaGrow^® DHA and AquaGrow^® ARA to completely substitute the fish oil; and, a diet containing both products, AquaGrow^® DHA and AquaGrow^® ARA, together with an EPA source. Temperature, air and salinity activity tests were also performed to detect larval resistance to stress. At the end of the experiment, final survivals did not differ among groups. The microorganism produced DHA was able to completely replace fish oil in weaning diets for gilthead seabream without affecting survival, growth or stress resistance, whereas the inclusion of microorganism produced ARA did not improve larval performance. Moreover, addition of EPA to diets with total replacement of fish oil by microorganism produced DHA and ARA, significantly improved growth in terms of body weight and total length. The results of this study denoted the good nutritional value of microorganisms produced DHA as a replacement of fish oil in weaning diets for gilthead seabream, without a complementary addition of ARA. However, dietary supplementation of EPA seems to be necessary to further promote larval performance.     

Tissue distribution and depletion of sarafloxacin hydrochloride after in feed administration in gilthead seabream (Sparus aurata L.)

Tissue distribution and depletion of sarafloxacin was studied in gilthead seabream, under experimental field conditions at 25 and 18 °C, after in-feed administration of sarafloxacin hydrochloride for 5 days (10 mg/kg body weight/day). Ten fish per sampling point were examined during and after treatment. Muscle plus skin (n=10), liver, kidney and vertebra (pooled) were collected and analyzed by HPLC. Sarafloxacin concentrations in these tissues increased during the medication period, and then decreased rapidly. The highest sarafloxacin concentrations were recorded in liver (335.2 and 49.8 μg/g at 25 and 18 °C, respectively). In muscle plus skin, sarafloxacin concentrations were 193.0 and 42.0 μg/kg at 25 and 18 °C, respectively. Sarafloxacin residues were eliminated rapidly; at 36 h post treatment, the levels in muscle plus skin were 10.2 and 8.5 μg/kg at 25 and 18 °C, respectively. Elimination half-lives (t_1/2) were 17.8 and 32.5 h at 25 and 18 °C, respectively. Withdrawal period for the Maximum Residue Limit (MRL) of 30 μg/kg sarafloxacin in muscle plus skin (95% tolerance limit) at 25 °C was 42.2 h. The slow elimination from vertebra, relative to other tissues, suggests that vertebra behaves as a reservoir in gilthead seabream.     

Characterization and in vitro sensitivity of cholinesterases of gilthead seabream (<Emphasis Type="Italic">Sparus aurata</Emphasis>) to organophosphate pesticides

The characterization of cholinesterase activity in brain and muscle of gilthead seabream was carried out using four specific substrates and three selective inhibitors. In addition, K _m and V _max were calculated from the Michaelis–Menten equation for ASCh and BSCh substrates. Finally, the in vitro sensitivity of brain and muscle cholinesterases to three organophosphates (OPs) was also investigated by estimating inhibition kinetics. The results indicate that AChE is the enzyme present in the brain, whereas in muscle, a typical AChE form is present along with an atypical form of BChE. Very low ChE activity was found in plasma with all substrates used. The inhibitory potency of the studied OPs on brain and muscle AChEs based on bimolecular inhibition constants (k _i ) was: omethoate < dichlorvos < azinphosmethyl-oxon. Furthermore, muscle BChE was found to be several orders of magnitude (from 2 to 4) more sensitive than brain and muscle AChE inhibition by dichlorvos and omethoate.     

Dynamic expression pattern of corticotropin-releasing hormone,urotensin I and II genes under acute salinity and temperature challenge during early development of zebrafish

Corticotropin-releasing hormone (CRH), urotensin I (UI) and urotensin II (UII) are found throughout vertebrate species from fish to human. To further understand the role of crh, uI and uII in teleosts during development, we investigated the expression pattern of crh, uI, uIIα and uIIβ genes, and their response to acute salinity and temperature challenge during early development of zebrafish, Danio rerio. The results reveal that crh, uI, uIIα and uIIβ mRNA are detected from 0hpf, and the expression levels increase to a maximum at 6 days post fertilization (dpf), with the exception of uIIα that peak at 5dpf. Exposure of zebrafish embryos and larvae to acute osmotic (30ppt) stress for 15 min failed to modify expression levels of crh, uI, uIIα and uIIβ mRNA from levels in control fish except at 6dpf when uIIα and uIIβ were significantly (P < 0.05) modified. Exposure of embryos and larvae to a cold (18 °C) or hot stress (38 °C) generally down-regulated mRNA levels of crh, uI, uIIα and uIIβ apart from at 3dpf. The results indicate that the contribution of crh, uI, uIIα and uIIβ genes to the stress response in zebrafish may be stressor-specific during early development. Overall, the results from this study provide a basis for further research into the developmental and stressor-specific function of crh, uI, uIIα and uIIβ in zebrafish.