Comprehensive transcriptome profiling and functional analysis of the meagre (Argyrosomus regius) immune system.

Meagre (Argyrosomus regius) belongs to the family Sciaenidae and is a promising candidate for Mediterranean aquaculture diversification. As a relatively recent species in aquaculture, the physiological consequences of the immune system activation in meagre are understudied. Spleen, as a primary lymphoid organ has an essential role in meagre immune and inflammatory responses. In this study, we have evaluated the in vivo effects of lipopolysaccharide (LPS) on the spleen transcriptome of meagre by RNA-seq analysis at 4 and 24 h after injection.

Physiological responses of reared sea bream (Sparus aurata Linnaeus, 1758) to an Amyloodinium ocellatum outbreak.

Amyloodiniosis represents a major bottleneck for semi-intensive aquaculture production in Southern Europe, causing extremely high mortalities. Amyloodinium ocellatum is a parasitic dinoflagellate that can infest almost all fish, crustacean and bivalves that live within its ecological range. Fish mortalities are usually attributed to anoxia, associated with serious gill hyperplasia, inflammation, haemorrhage and necrosis in heavy infestations; or with osmoregulatory impairment and secondary microbial infections due to severe epithelial damage in mild infestation. However, physiological information about the host responses to A. ocellatum infestation is scarce. In this work, we analysed the proteome of gilthead sea bream (Sparus aurata) plasma and relate it with haematological and immunological indicators, in order to enlighten the different physiological responses when exposed to an A. ocellatum outbreak. Using 2D-DIGE, immunological and haematological analysis and in response to the A. ocellatum contamination we have identified several proteins associated with acute-phase response, inflammation, lipid transport, homoeostasis, and osmoregulation, wound healing, neoplasia and iron transport. Overall, this preliminary study revealed that amyloodiniosis affects some fish functional pathways as revealed by the changes in the plasma proteome of S. aurata, and that the innate immunological system is not activated in the presence of the parasite.

The effect of live feeds bathed with the red seaweed Asparagopsis armata on the survival, growth and physiology status of Sparus aurata larvae.

Larval rearing is affected by a wide range of microorganisms that thrive in larviculture systems. Some seaweed species have metabolites capable of reducing the bacterial load. However, no studies have yet tested whether including seaweed metabolites on larval rearing systems has any effects on the larvae development. This work assessed the development of Sparus aurata larvae fed preys treated with an Asparagopsis armata product. Live prey, Brachionus spp. and Artemia sp., were immersed in a solution containing 0.5% of a commercial extract of A. armata (Ysaline 100, YSA) for 30 min, before being fed to seabream larvae (n = 4 each). In the control, the live feed was immersed in clear water. Larval parameters such as growth, survival, digestive capacity (structural-histology and functional-enzymatic activity), stress level (cortisol content), non-specific immune response (lysozyme activity), anti-bacterial activity (disc-diffusion assay) and microbiota quantification (fish larvae gut and rearing water) were monitored. Fish larvae digestive capacity, stress level and non-specific immune response were not affected by the use of YSA. The number of Vibrionaceae was significantly reduced both in water and larval gut when using YSA. Growth was enhanced for YSA treatment, but higher mortality was also observed, especially until 10 days after hatching (DAH). The mortality peak observed at 8 DAH for both treatments, but higher for YSA, indicates larval higher susceptibility at this development stage, suggesting that lower concentrations of YSA should be used until 10 DAH. The application of YSA after 10 DAH onwards promotes a safer rearing environment.

Micro-anatomical characterization of vertebral curvatures in Senegalese sole Solea senegalensis.

The micro-anatomical changes associated with lordotic and kyphotic vertebral curvatures (VC) in juvenile and adult Senegalese sole Solea senegalensis are described. In addition, it is demonstrated that the tissue and cellular structures of individual vertebrae can be severely affected. Two main conformations were found in deformed juvenile specimens: flattened vertebrae with dorso-ventral compression and trapezoidal vertebrae forming concave and convex sides under compressive and tensile stresses. Histological analyses revealed the occurrence of an ectopic cartilaginous tissue within the acellular bone, both in juveniles and adults, possibly to cope with altered mechanical stress in deformed vertebrae. The results suggest that the alteration in loading to which curved vertebral columns are subjected might trigger vertebral reshaping and differentiation of cells towards this ectopic tissue. In addition, mesenchymal cells appear to play an important role in its formation. It is here proposed that the acellular bone of S. senegalensis is capable of adaptively responding to altered loading regimes at the structural level by reshaping vertebrae and at the micro-anatomical level by recruiting chondrocyte-like cells to areas of altered mechanical stress.

Larval and juvenile development of dusky grouper Epinephelus marginatus reared in mesocosms.

The larval development of the dusky grouper Epinephelus marginatus up to the benthic juvenile stage is described in detail to establish a reference for their larval identification. Development is described in terms of ontogenetic changes in morphology, growth, pigmentation, fin structure and skeletal structure. Larvae were reared in mesocosms at a mean temperature of 24·3° C, salinity of 36·5, dissolved oxygen of 6·4 mg l(-1) and pH of 8·2. Newly hatched larvae had an estimated total length (LT ) of 2·3 mm. On the second day post hatching the yolk was almost fully absorbed with traces of the oil globule still present, the eyes were already pigmented and mouth and gut functional. At this stage the cranial skeletal elements for feeding and breathing (mouth and gills) and the pectoral-fin support were already present. About 50% of the observed larvae had food in their guts. Pigmentation was very characteristic, consisting of two large chromatophores visible on the edge of the primordial fin, close to the midpoint of the post-anal region of the body and over the midgut and hindgut and post-anal portion of the body. At 2·9 mm LT the emergence of the second dorsal-fin spine, characteristic of the Epinephilinae, was clearly visible. The pre-flexion stage started in larva of 3·2 mm LT . At 5·5 mm LT the larvae possessed posterior preopercular angle spines, and the dorsal and pelvic spines presented serrated edges and were pigmented. The water surface-tension-related death of the yolk sac and pre-flexion larvae described in the rearing of several other grouper species did not occur during E. marginatus culture. Notochord flexion, with initial ossification of the caudal-fin supporting elements, started at 6·6 mm LT . At this stage the major melanophores, preopercular, dorsal and pelvic spines and mandibular teeth were already present. Transformation of larvae into juveniles occurred when larvae averaged 13·8 mm LT . Juveniles with a mean LT of 20·1 mm started to settle and most of them were benthic with a mean LT of 26·8 mm.

Behavioural responses of sardines Sardina pilchardus to simulated purse-seine capture and slipping.

The behavioural effects of confinement of sardine Sardina pilchardus in a purse seine were evaluated through three laboratory experiments simulating the final stages of purse seining; the process of slipping (deliberately allowing fishes to escape) and subsequent exposure to potential predators. Effects of holding time (the time S. pilchardus were held or entangled in the simulation apparatus) and S. pilchardus density were investigated. Experiment 1 compared the effect of a mild fishing stressor (20 min in the net and low S. pilchardus density) with a control (fishing not simulated) while the second and third experiments compared the mild stressor with a severe stressor (40 min in the net and high S. pilchardus density). In all cases, sea bass Dicentrarchus labrax were used as potential predators. Results indicated a significant effect of crowding time and density on the survival and behaviour of slipped S. pilchardus. After simulated fishing, S. pilchardus showed significant behavioural changes including lower swimming speed, closer approaches to predators and higher nearest-neighbour distances (wider school area) than controls, regardless of stressor severity. These results suggest that, in addition to the delayed and unobserved mortality caused by factors related to fishing operations, slipped pelagic fishes can suffer behavioural impairments that may increase vulnerability to predation. Possible sub-lethal effects of behavioural impairment on fitness are discussed, with suggestions on how stock assessment might be modified to account for both unobserved mortality and sub-lethal effects, and possible approaches to provide better estimates of unobserved mortality in the field are provided.

Effect of guar gum on glucose and lipid metabolism in white sea bream Diplodus sargus.

The aim of this study was to assess the role of soluble non-starch polysaccharide (guar gum) on white sea bream Diplodus sargus, glucose and lipid metabolism. A control diet was formulated to contain 40 % crude protein, 14 % crude lipids and 35 % pregelatinized maize starch, and three other diets were formulated similar to the control diet except for guar gum, which was included at 4 % (diet GG4), 8 % (diet GG8) or 12 % (diet GG12). Diets were fed to the fish for 9 weeks on a pair-feeding scheme. Guar gum had no effect on growth performance, feed efficiency, glycaemia, cholesterolaemia and plasma triacylglyceride levels. Hepatic glucokinase and pyruvate kinase activities, liver glycogen content and liver insulin-like growth factor-I gene expression were not affected by dietary guar gum, while fructose-1,6-bisphosphatase activity was lower in fish fed guar gum-supplemented diets. Hepatic glucose-6-phosphate dehydrogenase activity was higher in fish fed diets GG4 and GG8 than in the control group. Overall, data suggest that in contrast to mammals guar gum had no effect on white sea bream glucose utilization and in lowering plasma cholesterol and triacylglyceride levels. However, it seems to contribute to lower endogenous glucose production.

Sarcoma in the thymus of juvenile meagre Argyrosomus regius reared in an intensive system.

Juvenile meagre Argyrosomus regius (Asso, 1809) maintained in experimental conditions developed lateral and/or bilateral circular-shaped sarcoma within the opercular cavity. The sarcoma was dense, reddish and its growth from the branchial arch exerted pressure on the operculum forcing it to open. Histologically, the neoplasm exhibited marked proliferation of mesenchymal connective tissue composed largely of fusiform cells, which developed in a solid pattern accompanied by abundant mononuclear cell types. Multifocal areas of discrete necrosis were also observed, compatible with a sarcomatous proliferation. The immunological parameters analysed suggested an inflammatory response. No bacteria were isolated from the hematopoietic organs. However, Vibrio species, components of the normal seawater flora, were isolated from the tumour, which may have had a role in eliciting the immune response. No evidence of viral pathogens was found by electron microscopy. In order to look for cytogenetic alterations often linked to sarcomas, the diploid number and karyotype of this species were determined for the first time. An increase in the aneuploidy level was observed in sarcoma cell metaphase stages compared to other tissues. The aetiology of this tumour remains unknown.

Glycemic and insulin responses in white sea bream Diplodus sargus, after intraperitoneal administration of glucose.

A glucose tolerance test was performed in white sea bream Diplodus sargus, juveniles to evaluate the effect of a glucose load on plasma glucose, insulin, triacylglyceride levels, and on liver glycogen storage in order to study the capability of glucose utilization by this species. After being fasted for 48 h, fish were intraperitoneally injected with either 1 g of glucose per kg body weight or a saline solution. Plasma glucose rose from a basal level of 4 to a peak of 18-19 mmol l(-1), 2-4 h after glucose injection and fish exhibited hyperglycemia for 9 h. An insulin peak (from 0.5 to 0.8 ng ml(-1)) was observed 2-6 h after glucose injection, and basal value was attained within 9 h. Liver glycogen peaked 6-12 h after the glucose load and thereafter decreased to the basal value which was attained 24 h after injection. Plasma triacylglycerides in glucose-injected fish were only significantly higher than the basal value 12 h after injection. Glucose-injected fish generally showed lower plasma triacylglyceride levels than control fish. Our results indicate that under these experimental conditions, glucose acts as an insulin secretagogue in white sea bream juveniles. Moreover, insulin may have contributed to restoring basal plasma glucose levels by enhancing glucose uptake in the liver. Further studies are needed to corroborate the lipolytic action of glucose. Clearance of glucose from the blood stream was fast, comparatively to other species, indicating that white sea bream has a good capability of glucose utilization.

Single and combined effects of ultraviolet radiation and triclosan during the metamorphosis of Solea senegalensis.

Ultraviolet radiation (UV) and triclosan (TCS) affect the early development of marine fish; however, the corresponding molecular mechanisms are still not fully understood. Therefore, this work aims to study the effects of the single and combined exposure to these stressors during the thyroid-regulated metamorphosis of the flatfish Solea senegalensis. Sub-lethal exposure (5.89 kJ m-2 UV and/or 0.546 and 1.090 mg L-1 TCS for 48 h) was performed at the beginning of metamorphosis (13 days after hatching, dah), followed by a period in clean media until complete metamorphosis (24 dah). Malformations, metamorphosis progression, length, behavior and the expression of thyroid axis-related genes were studied. TCS induced malformations, decreased swimming performance, and induced metamorphosis acceleration at 15 dah, followed by a significant metamorphosis delay. Such effects were more noticeable in the presence of UV. The down-regulation of five thyroid axis-related genes occurred after exposure to TCS (15 dah), and after 9 days in clean media two genes were still down-regulated. UV exposure increased the effect of TCS by further down-regulating gene expression immediately after the exposure. Since several effects persisted after the period in clean media, implications of these stressors (mainly TCS) on the ecological performance of the species are suggested.

Genetic differences between wild and hatchery populations of Diplodus sargus and D. vulgaris inferred from RAPD markers: implications for production and restocking programs design.

Restocking and stock enhancement programs are now recognized as an important tool for the management of fishery resources. It is important, however, to have an adequate knowledge on the genetic population structure of both the released stock and the wild population before carrying out such programs. In this study, random amplified polymorphic DNA (RAPD) markers were applied to assess genetic diversity and population structure of wild and hatchery populations of the white seabream Diplodus sargus and the common two-banded seabream D. vulgaris (Sparidae). The estimated values for intrapopulation genetic variation, measured using the percentage of polymorphic loci (%P), Shannon index (H'), and Nei's gene diversity (h), showed high values for all populations. The percentage of genetic variation within D. sargus and D. vulgaris populations, based on coefficient of gene differentiation, reached 82.5% and 90% of the total genetic variation, respectively. An undeniable decrease in genetic variation was found in both hatchery populations, particularly in D. sargus, compared to the wild ones. However, the high values of variation within all populations and the low levels of genetic variation among populations did not indicate inbreeding or depression effects, thus indicating a fairly proper hatchery management. Nevertheless, the results of this study highlight the importance of monitoring the genetic variation of hatchery populations, particularly those to be used in restocking programs. The creation of a genetic baseline database will contribute to a more efficient conservation management and to the design of genetically sustainable restocking programs.

Lunar and daily spawning rhythms of Senegal sole Solea senegalensis.

A periodicity of 29 days was observed in spawning rhythms in Senegal sole Solea senegalensis, with an acrophase around the last quarter and the new moon. In both spring and autumn, a very marked nocturnal spawning rhythm was registered, with spawning beginning after dusk and the acrophase occurring around 2300 hours. When the photoperiod was artificially extended (from 10L:14D to 14L:10D), S. senegalensis synchronized to the new photoperiod: spawning took place after the new 'dusk', the beginning gradually shifting from 2100 to 2300 hours and the acrophase from 2325 to 0032 hours. Under continuous light conditions, fish sustained rhythmicity for 2 days, with an acrophase at 2249 hours, which suggested the existence of an endogenous pacemaker controlling the daily spawning rhythm. These findings provided new insights for better understanding the reproductive physiology of this species and for optimizing the timing protocols of egg collection and larvae production in S. senegalensis aquaculture.

Methodology for assessing the individual role of fish, oyster, phytoplankton and macroalgae in the ecology of integrated production in earthen ponds.

Production costs in extensive and semi-intensive fish culture in earthen ponds are often too high to offer sustainable economic activity due to the low productivity of these systems. The right combination of commercial finfish species with inorganic (primary producers) and organic extractive (bivalves) species in Integrated MultiTrophic Aquaculture (IMTA) create a balanced system with higher profitability and risk reduction. To achieve this, it is crucial to understand the role of each functional groups within the system what we did by comparing three different IMTA production three different IMTA production treatments with distinct combinations of trophic levels: •fish, filter feeders, phytoplankton and macroalgae,•fish, filter feeders and phytoplankton•fish, phytoplankton and macroalgae Each treatment was carried out in two similar ponds under semi-intensive conditions and flow through system, in a total of 6 earthen ponds of 500 m2 surface and depth of 1.5 m. Results showed that the presence of oysters in the ponds enhanced water quality by decreasing turbidity and by controlling phytoplankton which led to regulation of dissolved oxygen levels. The enhanced water quality in these systems lead to improved fish performance and higher biomass production contributing to greater profitability. The combination of fish, oyster, phytoplankton and macroalgae was particularly good providing much more fish supply compared with the other two treatments. •Oysters enhanced water quality in the ponds by decreasing turbidity and controlling phytoplankton which regulated the dissolved oxygen levels.•The enhanced water quality in systems with oysters improve fish performance resulting in higher biomass production and greater profitability.•The combination of fish, oyster, phytoplankton and macroalgae was particularly good providing much more fish supply compared with the other two treatments.

Metabolism of tryptophan, methionine and arginine in Diplodus sargus larvae fed rotifers: effect of amino acid supplementation.

Dietary amino acids imbalances have been described when fish larvae are fed rotifers, what may lead to a reduction in growth rate. The tube-feeding technique can be used to assess the effect of free amino acid short term supplementation. In this study supplementation of tryptophan, methionine and arginine were tested in Diplodus sargus. Single crystalline (14)C amino acids as well as a mix of (14)C amino acids were used as tracers to compare results of individual amino acids metabolism with the average of all amino acids. The results show low absorption efficiencies for tryptophan (70%) and arginine (80%) and similar absorption for methionine (90%) when compared with the average of all amino acids. Supplementation of these amino acids seems to be viable but it did not result in higher retention compared to the amino acid mix. This means that tryptophan, methionine and arginine are probably not the limiting amino acid when Diplodus sargus larvae are fed rotifers. However, supplementation in these IAA may be required for their roles as precursors of important molecules other than proteins, in order to improve larval quality and/or performance.

Composition and bioaccessibility of elements in green seaweeds from fish pond aquaculture.

The elemental composition of five species of green seaweeds (Chaetomorpha linum, Rhizoclonium riparium, Ulva intestinalis, Ulva lactuca, Ulva prolifera) grown in fish pond aquaculture systems were studied. The elemental bioaccessibility in these species was also investigated through the application of an innovative in vitro digestive model of the human gastrointestinal tract. It was observed that R. riparium had the highest levels of Mn, Sr, Cd, Sn, and I and that U. lactuca had the highest Ni and Cu concentrations. The daily amounts of dried green seaweed required for achieving specific dietary intakes were calculated, namely: 7g of dried U. lactuca (for meeting Cu Recommended Daily Allowance, RDA); 173g of dried U. lactuca (Zn RDA); 78g of dried C. linum (Se RDA); 41g of dried C. linum (Mo RDA); and 0.5g of dried R. riparium (I Dietary Reference Intake, DRI). Concerning elemental bioaccessibility, Mn and Cu had the highest values, always above 50%, I values were in the lower range, between 14 and 31%. The elemental bioaccessibility range of R. riparium (31-100%) was higher than the ranges for other species, particularly C. linum (0-56%). The bioaccessibility results entailed higher quantities of dried seaweed for reaching dietary intakes: 10g of dried U. lactuca (Cu RDA); 290g of dried R. riparium (Zn RDA); and 2g of dried R. riparium (I DRI). Accordingly, R. riparium is a very rich I source. This study showed the importance of taking into account bioaccessibility results in estimating dietary intakes.