Sea Bream Larvae Research Articles

Dietary taurine improves vision in different age gilthead sea bream (Sparus aurata) larvae potentially contributing to increased prey hunting success and growth

The effect of different levels of dietary taurine on vision, prey hunting success and growth was tested in developing larval gilthead sea bream (GSB; Sparus aurata). Fifteen 17 L aquaria were stocked with 3000 fertilized eggs aquarium−1 from a local GSB broodstock, which allowed the testing of 5 prey taurine treatments in triplicate aquaria treatment−1. Nine aquaria tested the effect of the non-taurine enriched rotifer (Brachionus rotundiformis) control (1.7 mg g−1 DW) and two taurine enriched rotifer treatments (6.6 and 11.0 mg g−1 DW) in 2 to 13 dph larvae followed by feeding non-taurine enriched Artemia nauplii (36.6 mg g−1 DW) to all treatments from 14 to 31 dph. Larvae in another 6 aquaria were fed control rotifers (1.7 mg g−1 DW) from 2 to 13 dph followed by testing the effect of taurine enriched Artemia nauplii (51.5 and 61.9 mg g−1 DW) on 16 to 31 dph larvae. In parallel, GSB fertilized eggs from the same broodstock were stocked (100 eggs L−1) in fifteen 400 L conical, polypropylene tanks, which tested the 5 rotifer and Artemia taurine levels in triplicate tanks treatment−1. Only rotifer consumption (mastax number) in 7 and 13 dph larvae and dry weight of 31 dph larvae were determined in the aquaria, while total length, essential fatty acid profile, taurine level, opsin abundance and gene expression of selected opsin genes in different age larvae were determined from the much larger biomass of the 400 L tanks.The growth of 3 and 13 dph larvae were significantly (P < 0.05) correlated with rotifer and larval body taurine levels in a dose-dependent manner. Moreover, rotifer ingestion (mastax number) significantly (P < 0.05) improved in 13 dph larvae with rotifer taurine level. The growth promoting effect of rotifer taurine continued to significantly (P < 0.05) influence larval length in older 21and 26 dph larvae, although this effect diminished (P > 0.05) in 31dph larvae. The contribution of taurine to improved vision was supported in 31 dph larvae, which exhibited significantly (P < 0.05) increasing retinal opsin protein abundance with rotifer taurine level in a dose dependent manner. In fact, dietary taurine markedly (P < 0.05) improved the gene expression of 5 different opsins in 8 and 31 dph larvae. Nevertheless, the correlation between rotifer taurine and larval fatty acid content suggests that the taurine-modulated production of bile salts facilitated lipid digestion and absorption, which also might have promoted growth.

Effect of dietary microminerals in early weaning diets on growth, survival, mineral contents and gene expression in gilthead sea bream (Sparus aurata, L) larvae

In previous studies, combined inclusion of Zn, Mn and Se in early weaning diets improved larval growth, but suggested a potential toxicity by one of these elements. The present study aimed to determine the effect of the single inclusion of Zn, Mn, Se or Cu, their combination (Control+) or their absence (Control−) on larval diets. At the end of the trial, survival was significantly (p < .05) lowest in fish fed C+ diet (17.16 ± 7% mean ± SD), followed by that of larvae fed Mn diet (21.91 ± 7%). The highest survival was obtained by Cu diet (35.27 ± 15%), followed by C− diet (34.58 ± 9%). Cu and Se supplementation significantly improved total length and body weight, in comparison with the C− fish. On the contrary, fish fed Mn and C+ showed the lowest growth. Supplementation with Zn or Cu significantly increased CuZnsod, whereas gpx was significantly upregulated in fish fed Se and C+ diets. ARA/EPA level was significantly highest and DHA/EPA lowest in larvae fed the Cu diet in fish fed C+ diet. The results pointed out the importance of supplementation with Cu, as well as Se and Zn, on early weaning diets for gilthead sea bream, and the potential toxic effect of Mn.

Effects of tank shapes and aerations on survival, growth and swim bladder inflation of red seabream Pagrus major larvae

This study aimed to seek for the optimal condition for small-scale larviculture of red seabream Pagrus major larvae. We examined the effects of tank shapes and aerations, which were assumed to influence the larval survival, growth and swim bladder inflation of P. major larvae. Seawater (50-l) was filled into three cylindrical (CT: 1.7 × 103 cm2 water surface area, 30 cm deep) and six rectangular (RT: 1.8 × 103 cm2 water surface area, 28 cm deep) tanks. One air stone with 100 ml/min aeration rate was set at the bottom center of three CT and RT (RT1AS), and two air stones with 50 ml/min aeration rate were set at the half bottom center of three RT (RT2AS). Five hundred eggs were distributed into each experimental tank. Rotifers were fed to larvae at 10 individuals/mL on 3 days post hatching (dph) and their distribution in tanks were measured. Survival rate at 14 dph in CT (54.7 ± 11.0 %) and RT1AS (55.3 ± 6.0 %) were significantly higher than that in RT2AS (29.6 ± 9.3 %, p < 0.05). However, the growth of larvae was not significantly different between tank shapes and aerators. Swimbladder inflation rates were not different between tank shapes and aerations, however, CT (58.9±28.3 %) showed lower trend (RT1AS 94.4±6.9 %, RT2AS 92.2±10.7 %). Rotifer distribution in tanks was higher at tank bottom (p < 0.05). Low flow regions were observed along the side walls of the tanks and bottom areas in CT and RT1AS due to a single-pair vortex system and formed at the center (between air stones) and from the air stone to the tank walls in RT2AS due to two single-pair vortex systems. These low-flow areas were coincided with higher rotifer distribution areas at the tank bottom indicating that measuring rotifer density can estimate the flow in a tank. We recommend the rectangular tank with one air stone system for red seabream larvae.

Daily rhythms in endocrine factors of the somatotropic axis and their receptors in gilthead sea bream (Sparus aurata) larvae

Living organisms have adapted to environmental oscillations in light and temperature through evolving biological clocks. Biological rhythms are pervasive at all levels of the endocrine system, including the somatotropic (growth) axis. The objective of the present research was to study the existence of daily rhythms on the somatotropic axis of a marine teleost species, specifically, the gilthead sea bream (Sparus aurata). Larvae of S. aurata at 30 dph (days post hatching), kept under a 9 L:15D (light-dark) photoperiod, were collected every 3 h throughout a 36 h cycle. The expression of the following somatotropic axis genes was analyzed by quantitative PCR: pituitary adenylate cyclase-activating polypeptide 1 (adcyap1), prepro-somatostatin-1 (pss1), growth hormone (gh), growth hormone receptor types 1 and 2 (ghr1 and ghr2, respectively), insulin-like growth factor 1 (igf1) and igf1 receptor a (igf1ra). All genes displayed significant differences among time points and, with the exception of adcyap1, all showed statistically significant daily rhythms. The acrophases of gh, ghr1, ghr2, igf1 and igf1ra were located around the end of the dark phase, between ZT19:44 and ZT0:48 h, whereas the highest expression levels of adcyap1 occurred at ZT18 h. On the other hand, the acrophase of pss1, an inhibitor of Gh secretion, was located at ZT10:16 h, hence it was shifted by several hours with respect to the other genes. The present results provide the first thorough description of somatotropic axis rhythms in gilthead sea bream. Such knowledge provides insights into the role of rhythmic regulation of the Gh/Igf1 axis system in larval growth and metabolism, and it can also improve the implementation of more species-specific feeding regimes.

Viral nervous necrosis outbreaks caused by the RGNNV/SJNNV reassortant betanodavirus in gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax)

Mediterranean marine aquaculture has suffered significant economic losses due to viral nervous necrosis (VNN) outbreaks mainly caused by different RGNNV betanodavirus strains. In recent years, the marine aquaculture sector has experienced the emergence of the RGNNV/SJNNV reassortant betanodavirus, harbouring the RNA1 segment of RGNNV genotype and the RNA2 segment of SJNNV genotype. So far, the reassortant strains caused massive mortality outbreaks in gilthead sea bream (Sparus aurata) larvae sparing the European sea bass (Dicentrarchus labrax). In this study, multiple mortality outbreaks occurred in one Italian marine hatchery involving both European sea bass and gilthead sea bream at different life stages were investigated through a complete microbiological and molecular analysis. Gilthead sea bream larvae and juveniles have recorded the highest mortality rates, however, both European sea bass and gilthead sea bream incurred a RGNNV/SJNNV reassortant betanodavirus persistent infection, able to act as asymptomatic carriers and viral source for susceptible fish. These new epidemiological data on nervous necrosis virus (NNV) reassortant infection provide precious advice on how to manage fish to reduce VNN spread in Mediterranean aquaculture. Evidence of interspecies transmission of RGNNV/SJNNV reassortant strains and the persistent infection in both European sea bass and gilthead sea bream, point out the importance to enforce a wide surveillance and a strict biosecurity programme addressing both RGNNV and reassortant RGNNV/SJNNV betanodaviruses in Mediterranean European sea bass and gilthead sea bream farms. Furthermore, the presence assessment of betanoviruses in all newly-introduced fish batches in the farm, regardless of the species and a strict segregation between European sea bass and gilthead sea bream batches within farms can significantly reduce the risk of NNV transmission. Finally, surviving fish can act as carrier fish, and thereby must be segregated from other batches and protected from stress conditions that could trigger a new clinical phase.

Application of probiotic bacteria for the improvement of sea bream (Sparus aurata) larval production

The main object of this study was to evaluate the effect of two probiotic bacterial strains Bacillus sp. R2 and Planococcus sp. R11 and their mixture (1:1) on live food cultures (rotifers and Artemia) and study its effect on gilthead sea bream larvae until 40 day after hatching (DAH). Probiotics were supplemented in the larval rearing system in triplicates in different strategies, which starting the exogenous feeding, by introducing to live food through bio-encapsulation or in tank water or both. There was significance difference (P<0.05) in the survival and growth of S.aurata larvae that had probiotic achieved compared to control. Meanwhile total proteins, total lipids, fatty acid profiles were determined for live food during encapsulation and for the produced larvae after feeding. The results showed increasingly significant difference (P<0.05) towards R11 probiotic treatments especially in bioencapsulation among all other treatments against control. Protease activity showed significant increase (P<0.05) in sea bream enriched with Planococcus sp. R11 led to the highest value (8.95 ± 0.21 U/mg) of enzyme activity in comparison to control (1.3±0.03 U/mg). Otherwise, the digestive system histology of larvae supplemented with bacteria in the period from 3-20 DAH showed improving number of goblet cells (37± 7.5), as well as number and length of villi (22±2.5and 35±9µm, respectively). Similarly,in the second duration from 20-40 DAH the number of goblet cells (96±14.4)was also increased, as well as the number and length of villi (27±2.5 and 126 ±25 µm, respectively(P<0.05)

Modelling the ingress of a temperate fish larva into a nursery coastal lagoon

The development of sensorial acuity and swimming capabilities of coastal fish larvae and their responses to coastal nursery cues are essential for recruitment success. Most studies of the response of fish larvae to environmental cues and their recruitment into nursery areas have focused on coral reef fish and only a few studies focused on fish species from temperate regions. The Sense Acuity and Behavioural (SAAB) Hypothesis proposes that fish larvae in temperate regions can sense nursery cues and ingress into estuarine ecosystems by using several active swimming strategies. We tested the SAAB hypothesis by studying the ingress of a temperate fish larvae – white seabream Diplodus sargus (Linnaeus, 1758) – into a coastal nursery area, the Ria Formosa Lagoon, Portugal. We combined the results from studies of sensory acuity, swimming capabilities and personality traits of post-flexion larvae with a fine-scale biophysical model to quantify the ingress of the white seabream into the lagoon. Data showed that the location of spawning sites and the directional swimming capabilities are both important for the successful ingress of white seabream larvae into the lagoon. Recruitment was higher when spawning grounds were located in areas with depths between 15.1 and 16.9 m and when post-flexion larvae used their directional swimming capabilities. The larvae ingressed passively into the lagoon with the tidal currents at pre-flexion stage and actively by using their directional swimming capabilities at post-flexion stage. Directional swimming also prevented larval export into the coastal area. When spawning occurred away from the seagrass smell plume emanating from the lagoon, the fish larvae were advected away and lost at sea. This work demonstrated the relevance of combining fish larvae behaviour and oceanography processes in modelling the dispersion of fish larvae to estimate recruitment.

Seabream Larval Physiology under Ocean Warming and Acidification

The vulnerability of early fish stages represents a critical bottleneck for fish recruitment; therefore, it is essential to understand how climate change affects their physiology for more sustainable management of fisheries. Here, we investigated the effects of warming (OW; +4 °C) and acidification (OA; ΔpH = 0.5) on the heart and oxygen consumption rates, metabolic enzymatic machinery—namely citrate synthase (CS), lactate dehydrogenase (LDH), and ß-hydroxyacyl CoA dehydrogenase (HOAD), of seabream (Sparus aurata) larvae (fifteen days after hatch). Oxygen consumption and heart rates showed a significant increase with rising temperature, but decreased with pCO2. Results revealed a significant increase of LDH activity with OW and a significant decrease of the aerobic potential (CS and HOAD activity) of larvae with OA. In contrast, under OA, the activity levels of the enzyme LDH and the LDH:CS ratio indicated an enhancement of anaerobic pathways. Although such a short-term metabolic strategy may eventually sustain the basic costs of maintenance, it might not be adequate under the future chronic ocean conditions. Given that the potential for adaptation to new forthcoming conditions is yet experimentally unaccounted for this species, future research is essential to accurately predict the physiological performance of this commercially important species under future ocean conditions.

Swimming Abilities of Temperate Pelagic Fish Larvae Prove that They May Control Their Dispersion in Coastal Areas

The Sense Acuity and Behavioral (SAAB) Hypothesis proposes that the swimming capabilities and sensorial acuity of temperate fish larvae allows them to find and swim towards coastal nursery areas, which are crucial for their recruitment. To gather further evidence to support this theory, it is necessary to understand how horizontal swimming capability varies along fish larvae ontogeny. Therefore, we studied the swimming capability of white seabream Diplodus sargus (Linnaeus, 1758) larvae along ontogeny, and their relationship with physiological condition. Thus, critical swimming speed (Ucrit) and the distance swam (km) during endurance tests were determined for fish larvae from 15 to 55 days post-hatching (DPH), and their physiological condition (RNA, DNA and protein contents) was assessed. The critical swimming speed of white seabream larvae increased along ontogeny from 1.1 cm s−1 (15 DPH) to 23 cm s−1 (50 and 55 DPH), and the distance swam by larvae in the endurance experiments increased from 0.01 km (15 DPH) to 86.5 km (45 DPH). This finding supports one of the premises of the SAAB hypothesis, which proposes that fish larvae can influence their transport and distribution in coastal areas due to their swimming capabilities. The relationship between larvae’s physiological condition and swimming capabilities were not evident in this study. Overall, this study provides critical information for understanding the link between population dynamics and connectivity with the management and conservation of fish stocks.

Modeling larval dispersal for the gilthead seabream in the northwestern Mediterranean Sea

To investigate dispersal and connectivity between spawning and lagoon nursery habitats of the gilthead seabream, Sparus aurata, in the Gulf of Lions (northwestern Mediterranean Sea), we modeled the potential transport of the species’ larvae between its supposed main spawning site in the region (the Planier Island) and two of its main local nursery areas (the coastal lagoons of Thau and Salses-Leucate). Passive larval drift simulations using a dispersal biophysical model showed a large variability in the possible trajectories from spawning to nursery areas and in the predicted ages for larvae arrival on the two nursery sites. The most common ages at arrival obtained in the simulations (20–60 days) are broadly consistent with previous modeling studies but contrast with the actual ages of the S. aurata post-larvae collected in 2016 and 2017 at time of the lagoon entrances (60–90 days, from otolith readings). The period between 25 and 70 days being critical for gilthead seabream larvae to acquire sufficient swimming, osmoregulatory, and olfactory abilities to enter coastal lagoons, we argue that ontogenic development plays a crucial role in the transport and local retention of S. aurata larvae in the studied region, explaining the discrepancy between simulation results and observed data.

Early ontogenetic development, digestive enzymatic activity and gene expression in red sea bream (Pagrus major)

Red sea bream (Pagrus major) is an important aquaculture species in Japan. This study aimed to determine the ontogenetic development of the digestive system, enzyme activity and related gene expressions of red sea bream larvae from hatching to post-metamorphosis (40 days after hatching, DAH). The larvae were fed with the euryhaline rotifer Brachionus plicatilis species complex L-type enriched with Chlorella vulgaris containing DHA from 3 to 15 DAH followed by Artemia nauplii from 16 to 40 DAH. Post culture analysis included, histological analysis of the larval gut, enzyme assays (trypsin, chymotrypsin, lipase, pepsin) and real time PCR with larvae sampled from 1 to 40 DAH. At hatching, the larval digestive tract was undifferentiated and resembled a straight tube. There was low detection of enzyme activity and gene expression of trypsin, chymotrypsin, lipase, and amylase during yolk-sac stage, before mouth opened. At 3 DAH, the mouth opened and the larvae began to exogenous feeding followed by the differentiation of the digestive tract into buccopharyngeal capacity and intestine at 5 DAH. The pancreas, liver, and gallbladder were functional indicating the transition from endogenous to exogenous feeding. The enzyme activity and gene expression of trypsin, chymotrypsin, lipase, and amylase increased and fluctuated throughout the larval development, a pattern that is common among marine fish species. Pepsin activity and gene expression were not detected until 15 DAH and strongly increased from 20 DAH. Metamorphosis was complete by 40 DAH following the regular arranging of microvilli and the abundance of vacuoles in intestine.

First indication of deleterious impacts in white-seabream larvae (Diplodus sargus) survival and behaviour following acute venlafaxine exposure.

Wastewater effluents are teeming with organisms, nutrients and chemical substances which water treatment processes fail to remove. Among these substances, pharmaceuticals such as antidepressants are a frequent occurrence, and have been reported to lead to severe effects in the physiology and behaviour of non-target marine species across taxa. Venlafaxine (VFX) is one of the most consistently prescribed substances for the treatment of human depressive disorders, acting as a serotonin and norepinephrine reuptake inhibitor. In the present study, the potential effects of this antidepressant on the survival and key behaviours (i.e. movement, aggression and foraging) of white seabream (Diplodus sargus) larvae were addressed. Larvae were submitted to an acute exposure of two different VFX treatments (low concentration, 10 µg L-1; and high concentration, 100 µg L-1) for a total of 48 h. Sampling took place after 24 and 48 h of exposure. Overall, results showed a significant effect of a two-day exposure to VFX in larvae of D. sargus. Survival was significantly reduced by exposure to a high concentration, but behavioural effects of antidepressant exposure were subtle: i.e. increased attack frequency and temporary modulation of capture success. Further research efforts should be directed towards evaluating the potential chronic effects of antidepressants in marine species, if we are to anticipate possible pressures on natural populations, and effectively advice policymakers towards the investment in new and more efficient methods of wastewater treatments.

Effects of dietary orange peel on growth performance, antioxidant activity, intestinal microbiota and liver histology of Gilthead sea bream ( Sparus aurata ) larvae

Effects of dietary orange peel on growth performance, antioxidant activity, intestinal microbiota and liver histology of Gilthead sea bream ( <i>Sparus aurata</i> ) larvae

GROWTH PERFORMANCE AND SURVIVAL OF GILTHEAD SEABREAM SPARUS AURATA LARVAE FED ROTIFER AND ARTEMIA

The experiment was carried out in order to study the effect of a rotifer Brachionus plicatilis and encapsulated Artemia nauplii as a live food on survival rate and growth performance for gilthead sea bream Sparus aurata larvae. Gilthead sea bream larvae (20 days old) with body length and weight of (7.3 + 0.20 mm) (5.4 + 0.10 mg) respectively, were stocked in twelve white fiberglass tanks (each of 400 Liter volumes) at a density of 1200-larvae/tank. Treatments were tested as follows, 1) low rotifers and low artemia (LRLA); 2) high rotifers and low artemia (HRLA) 3) low rotifers and high artemia (LRHA); and 4) high rotifers and high artemia (HRHA) for 24 days (10, 30 rotifers and 100, 200 artemia /ml, during the periods 1st, 8th, 9th , 16th, 17th and 24th days of the feeding experiment). Feeding of higher levels of rotifers and artemia increased the survival rate of S. aurata to 48.96%, however, the lower levels of both resulted in only 12.17 % survival rate. Other intermediate treatments of HRLA or LRHA resulted in better improvements in the survival rates (19.08 and 32.21% respectively) of S. aurata larvae. Values of growth performance of S. aurata larvae (gain in length and weight; average daily (gain in length and weight; and specific growth rate) were significantly increased with increasing the levels of live food from both rotifers and artemia. The results clearly showed the superiority of the higher levels of live artemia in enhancing growth performance of S. aurata larvae than rotifers. Finally, a suitable live food program to improve the survival rates of S. aurata larvae by feeding high levels of rotifers and artemia could be recommended.

The impact of propranolol, 17α-ethinylestradiol, and gemfibrozil on early life stages of marine organisms: effects and risk assessment.

Pharmaceuticals are ubiquitously detected in the marine environment at the ng-μg/L range. Given their biological activity, these compounds are known to induce detrimental effects on biota at relatively low exposure levels; however, whether they affect early life stages of marine species is still unclear. In this study, a set of bioassays was performed to assess the effects of propranolol (PROP), 17-α ethinylestradiol (EE2), and gemfibrozil (GEM) on gamete fertilization and embryonic development of mussels (Mytilus galloprovincialis) and sea urchins (Paracentrotus lividus), and on the survival of seabream (Sparus aurata) larvae. Treatments of PROP (500, 5000, 50,000ng/L), EE2 (5, 50, 500ng/L), and GEM (50, 500, 5000ng/L) were selected to encompass levels comparable or superior to environmental concentrations. Obtained data were tested for dose-response curve fitting and the lowest EC10/LC10 used to calculate risk quotients (RQs) based on the MEC/PNEC. No alteration was induced by PROP on the mussel gamete fertilization, while inhibitory effects were observed at environmental levels of EE2 (500ng/L) and GEM (5000ng/L). Fertilization was significantly reduced in sea urchin at all PROP and EE2 dosages. The 48-h exposure to all pharmaceuticals induced the onset of morphological abnormalities in either mussel or sea urchin embryos. Alterations were generally observed at environmentally relevant dosages, except for PROP in mussels, in which alterations occurred only at 50,000ng/L. A decreased survival of seabream larvae was recorded after 96-h exposure to PROP (all treatments), EE2 (50-500ng/L), and GEM (500ng/L). A median RQ > 1 was obtained for all pharmaceuticals, assigning a high risk to their occurrence in marine environments. Overall, results showed that current levels of contamination by pharmaceuticals can impact early stages of marine species, which represent critical junctures in the resilience of coastal ecosystems.

EFFECTS OF REPLACEMENT OF LIVE FOOD WITH DRY DIET ON GROWTH AND SURVIVAL RATE FOR SEABREAM (SPARUS AURATA) LARVAE

The present study was carried out to study the possibility of early weaning of gilthead sea bream (Sparus aurata) larvae. Therefore seabream larvae divided into different groups and weaned at 35, 40, 45, 50, 55and 60 days post hatching (dph). At the end of the experiment the mean wet weight and length of gilthead seabream were recorded for larvae. Showed that, the late weaning (50, 55 or 60 dph) significantly increased the wet weight and length of larvae compared to larvae groups that early weaned (35 and 40 dph) and the same trend was also observed for larvae after 60 dph. During the entire experimental period the highest SGR values were recorded for groups 60 and 55 dph (3.75 and 3.19), while the lowest SGR recorded for group 35 dph (1.59) indicating that, the late weaning age released the best SGR was compared to the early weaning of gilthead seabream larvae . The obtained results indicated that the late weaning of larvae (60 dph) significantly increased each of protein and fat content of larvae when compared to the early weaning of larvae (35 dph). In conclusion, it can be concluded that, the early weaning could not only improve the growth performance, but also keep the survival rate within the same limits for the original late weaning age.

Temperature responsiveness of gilthead sea bream bone; an in vitro and in vivo approach

This study aimed to characterize the molecules involved in osteogenesis in seabream and establish using in vitro/in vivo approaches the responsiveness of selected key genes to temperature. The impact of a temperature drop from 23 to 13 °C was evaluated in juvenile fish thermally imprinted during embryogenesis. Both, in vitro/in vivo, Fib1a, appeared important in the first stages of bone formation, and Col1A1, ON and OP, in regulating matrix production and mineralization. OCN mRNA levels were up-regulated in the final larval stages when mineralization was more intense. Moreover, temperature-dependent differential gene expression was observed, with lower transcript levels in the larvae at 18 °C relative to those at 22 °C, suggesting bone formation was enhanced in the latter group. Results revealed that thermal imprinting affected the long-term regulation of osteogenesis. Specifically, juveniles under the low and low-to-high-temperature regimes had reduced levels of OCN when challenged, indicative of impaired bone development. In contrast, gene expression in fish from the high and high-to-low-temperature treatments was unchanged, suggesting imprinting may have a protective effect. Overall, the present study revealed that thermal imprinting modulates bone development in seabream larvae, and demonstrated the utility of the in vitro MSC culture as a reliable tool to investigate fish osteogenesis.

Interaction between taurine, vitamin E and vitamin C in microdiets for gilthead seabream (Sparus aurata) larvae

The negative effects of oxidative damage on larval development, growth and survival are among the factors markedly affecting marine fish larvae production success. Whereas α-tocopherol (vit E) and l-ascorbic acid (vit C) are strong antioxidants, the potential antioxidant role of taurine (Tau) in fish diets and its relation with other antioxidant nutrients needs further clarification. The present study aimed to determine the potential interactions between the dietary levels of vit E plus vit C and Tau on survival, growth, bone development and oxidative metabolism in gilthead seabream larvae. Six micro-bound diets containing two levels of Tau combined with three levels of vit E plus vit C were manually fed 24 times/day to 16 dph larvae for 15 days. Elevation of dietary vit E and vit C over 3000 mg/kg, led to unbalanced body vit C/vit E ratio and up-regulation of genes coding for antioxidant enzymes, the highest incidence of bone anomalies and the lowest larval survival. Increase in dietary vit C + vit E up to 1800 mg/kg, down-regulated GPX expression, up-regulated OC expression and reduced maxillary bone anomalies. Increase in dietary Tau up to 7.1 g/kg significantly improved larval growth, down-regulated antioxidant enzyme genes and reduced the incidence of bone anomalies. Moreover, the pro-oxidative effects of the high supplementation of vit C and E where mitigated by increase in dietary Tau levels. Finally, increase in dietary vit E, vit C and Tau up to 1783 mg/kg, 1921 mg/kg and 7 g/kg lead to the highest larval survival and growth, the highest OC gene expression and the lowest incidence of bone anomalies, including lordosis, kyphosis, branchiostegal and maxillary bones anomalies.

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.

Determining gut transit rates in gilthead seabream larvae fed microdiets

In this study we assessed an easy methodology for testing gut filling dynamics in early fish larvae fed on microdiets by means of image analysis. As model for this study we used gilthead seabream Sparus aurata, a species whose feeding physiology during the larval stage is well-known. Ingestion and food transit assays were carried out at different ages between 9 and 27 days after hatching (dph). Gut content was examined by image analysis in 10 larvae sampled respectively at 0, 20, 40, 60, and 120 min after the first morning meal. Gut fullness level was determined measuring the pigmented area within the digestive cavity. In each sampling point and larvae, relative percentage of shadowed area respect to the total area of intestinal cavity was considered as fullness percentage, assuming that intestinal area and volume are highly correlated. Data obtained were adjusted to an exponential model with maximum asymptotic: C = Cmax(1−e(−bt)), where C = % fullness; Cmax is the asymptote (theoretical percentage of maximum fullness), t = sampling time (min) and b is a regression parameter indicating the slope of the filling curve. Ingestion rate (I) was calculated as I = Cmax∗b. Ingestion rate was considered as Filling rate taken that the determined data are percentages instead of a number of prey items as usual. Filling rate was expressed as % of total gut volume per time unit. The food transit time (time required to fill completely the gut) was calculated as 100/I. Our results demonstrate that the methodology based in the image analysis of the shadowed area by food within the gut is a useful tool to build saturation curves as a function of time in small early larvae, using early seabream larvae as model. Our results also showed that although food intake increases with age the filling rate of digestive tract is practically constant in the range of age assessed. In summary, feeding rates and gut transit time can be estimated by image analysis in early fish larvae while the body still remains transparent. This methodology is particularly useful for advancing in the feeding behaviour of larvae fed on microdiets.