Juvenile Grouper Research Articles

Gut microbiota dynamics in carnivorous European seabass (Dicentrarchus labrax) fed plant-based diets

A healthy gastrointestinal microbiota is essential for host fitness, and strongly modulated by host diet. In aquaculture, a current challenge is to feed carnivorous fish with plant-feedstuffs in substitution of fish meal, an unsustainable commodity. Plants have a limited nutritive value due to the presence of non-starch polysaccharides (NSP) which are not metabolized by fish. In this work we assessed the effects of NSP-enriched diets on European seabass gut microbiota and evaluate the selective pressure of plant feedstuffs towards gut microbes with NSP-hydrolytic potential, i.e. capable to convert indigestible dietary constituents in fish metabolites. Triplicate groups of European seabass juveniles were fed a fish meal-based diet (control) or three plant-based diets (SBM, soybean meal; RSM, rapeseed meal; SFM, sunflower meal) for 6 weeks, before recovering intestinal samples for microbiota analysis, using the Illumina’s MiSeq platform. Plant-based diets impacted differently digesta and mucosal microbiota. A decrease (p = 0.020) on species richness, accompanied by a decline on the relative abundance of specific phyla such as Acidobacteria (p = 0.030), was observed in digesta samples of SBM and RSM experimental fish, but no effects were seen in mucosa-associated microbiota. Plant-based diets favored the Firmicutes (p = 0.01), in particular the Bacillaceae (p = 0.017) and Clostridiaceae (p = 0.007), two bacterial families known to harbor carbohydrate active enzymes and thus putatively more prone to grow in high NSP environments. Overall, bacterial gut communities of European seabass respond to plant-feedstuffs with adjustments in the presence of transient microorganisms (allochthonous) with carbohydrolytic potential, while maintaining a balanced core (autochthonous) microbiota.

Importance of experienced thermal history: Effect of acclimation temperatures on the high-temperature tolerance and growth performance of juvenile marbled flounder.

Experienced thermal history often affects the temperature tolerance of fish; however, the effect of thermal history on growth performance is unclear. To contribute to effective stocking (release of hatchery-reared juveniles in the field), we conducted four laboratory experiments using juvenile marbled flounder (Pseudopleuronectes yokohamae, around 30mm standard length and 0.3g body wet weight) acclimated at 12°C and 24°C for approximately 2 weeks to investigate the effects of acclimation temperature on high-temperature tolerance, food consumption, and growth performance. The acclimation to 24°C increased tolerance to high temperatures, as shown in a 24-h exposure experiment and in a temperature elevation experiment. The 50% lethal temperature (upper incipient lethal temperature) was estimated to be 25.9°C and 29.0°C for the 12°C and 24°C acclimation groups, respectively. In subsequent experiments, we tested the effects of high and low temperature acclimation on the food consumption and growth performance of two size groups of juveniles (28.7±2.0 and 34.5±2.9mm, mean±SD), that were reared at temperatures ranging from 14°C to 23°C. The optimal temperature for growth was 20°C and did not differ between the acclimation temperatures or between the size groups. However, food consumption and growth performance were suppressed by acute temperature changes. Specifically, feeding and growth were lower in the 24°C-acclimated group than in the 12°C-acclimated group when exposed to 14°C, which is close to the natural water temperature at release in the field. These results suggest that experienced thermal history does not affect the optimal temperature but can affect the growth performance of juveniles. To maximize the post-release growth of hatchery-reared juveniles, the influence of thermal history should be taken into consideration and acute thermal changes before release should be avoided.

Key elements in the implementation of escape gaps recommendations for the protection of juvenile vulnerable squaretail coralgroupers (Plectropomus areolatus) in Karimunjawa Island

IUCN classified squaretail coralgroupers into vulnerable status. If this is ignored, there may be a risk of threatening its sustainability and changing its status to endangered. This condition needs proper regulation concerning juvenile protection. Escape gap recommendations are considered to be the proper solution for this particular issue because juveniles were able to release without reducing fishermen's revenues. When implementing an escape gap, various elements support failure and success. Therefore, it is necessary to know what elements will have an effect so that the program could implement successfully. This study aims to identify key elements in escape gap implementation. The key elements are identified through Interpretive Structural Modelling. This method is carried out systematically by ranking several elements that influence the implementation. The result showed that the implementation of the escape gap required prioritizing key elements. These keys are releasing juvenile grouper with high survivability (objectives), trial use of the escape gap on the trap (activities), worries of income reduction (obstacles), Marine and Fisheries Departments of Central Java (institutions involved), and fishermen (affected communities). These key sub-elements will drive other sub-elements to implement the escape gap in trap fisheries successfully.

Metamorphosis and skeletal development of hybrid Epinephelus awoara (♀) and Epinephelus tukula (♂) progenies

Here, we explored the metamorphosis and skeletal development of hybrids between Epinephelus awoara (♀) and Epinephelus tukula (♂) (EAT). Currently, many varieties with good characters have been produced by cross breeding, which plays an important role in the development of aquaculture. However, few basic studies have focused on the developmental biology of hybrid progeny. We studied the morphological characteristics and body structure of larvae and juveniles of the hybrid and measured their body length. The results showed that the metamorphosis process of the hybrid grouper was comparable to that of other groupers. Moreover, using whole-mount bone-staining technology, the ontogeny and ossification of the cranium, appendage skeleton and vertebral column were observed and described in the larvae and juveniles. Newly hatched larvae did not have a skeleton. Head cartilage and the pectoral fin developed at 3 days after hatching (DAH), and the total length (TL) was 2.74 ± 0.06 mm. Cartilage of the dorsal fin and pelvic fin developed at 15 DAH (3.90 ± 0.97 mm TL). The vertebral column and caudal fin cartilage appeared at 18 DAH (4.69 ± 0.58 mm TL). Cartilage was observed in the anal fin at 22 DAH (6.16 ± 1.76 mm TL). The second dorsal fin spine and pelvic fin spine were fully ossified between 24 and 26 DAH (8.01 ± 1.41–9.29 ± 1.64 mm TL). The first skeletal part that completed ossification was the pelvic fin, which occurred at the 44 DAH (24.64 ± 3.63 mm TL). The vertebral column was fully ossified at 47 DAH (29.70 ± 1.97 mm TL). The dorsal fin and anal fin were ossified at 51 DAH (34.64 ± 1.92 mm TL) and the cephalic skeleton, caudal fin and pectoral fin were fully ossified at 68 DAH (41.89 ± 2.16 mm TL). These results provide abundant biological data on the metamorphosis and skeletal development of larvae and juvenile groupers, as well as analysis of the growth and survival rate advantage of hybrid offspring.

Prevalence of Infectious Spleen and Kidney Necrosis Virus (ISKNV), Nervous Necrosis Virus (NNV) and Ectoparasites in Juvenile Epinephelus spp. Farmed in Aceh, Indonesia

A cross-sectional survey was used to estimate the prevalence of infections with the Infectious spleen and kidney necrosis virus (ISKNV, Megalocytivirus), nervous necrosis virus (NNV, Betanodavirus), and infestations with ectoparasites during the rainy season in juvenile grouper (Epinephelus spp.) farmed in Aceh, Indonesia. The survey was intended to detect aquatic pathogens present at 10% prevalence with 95% confidence, assuming 100% sensitivity and specificity using a sample size of 30 for each diagnostic test. Eight populations of grouper from seven farms were sampled. Additional targeted sampling was conducted for populations experiencing high mortality. Infection with NNV was detected at all farms with seven of the eight populations being positive. The apparent prevalence for NNV ranged from 0% (95% CI: 0–12) to 73% (95% CI: 54–88). All of the fish tested from the targeted samples (Populations 9 and 10) were positive for NNV and all had vacuolation of the brain and retina consistent with viral nervous necrosis (VNN). Coinfections with ISKNV were detected in five populations, with the highest apparent prevalence being 13% (95% CI: 4–31%). Trichodina sp., Cryptocaryon irritans and Gyrodactylus sp. were detected at three farms, with 66% to 100% of fish being infested. Hybrid grouper sourced from a hatchery were 5.4 and 24.9 times more likely to have a NNV infection and a higher parasite load compared to orange-spotted grouper collected from the wild (p < 0.001). This study found that VNN remains a high-impact disease in grouper nurseries in Aceh, Indonesia.

Effects of different dietary carbohydrate‐to‐lipid ratios on growth, plasma biochemical indexes, digestive and immune enzymes activities of juvenile orange‐spotted grouper Epinephelus coioides

An 8-week feeding trial was conducted to investigate the effects of dietary carbohydrate-to-lipid (CHO:L) ratios on growth, liver and muscle glycogen content, haematological indices, and liver and intestinal enzyme activity of juvenile grouper (Epinephelus coioides). Five isonitrogenous (496.0 g/kg protein) and isoenergetic (21.6 KJ/g gross energy) diets with varying CHO:L ratios of 0.65 (D1), 1.31 (D2), 2.33 (D3), 4.24 (D4) and 8.51 (D5), respectively, were fed to triplicate groups of 20 fish (average 10.02 ± 0.1 g) for 8 weeks. Results showed that the weight gain rate, specific growth rate and protein efficiency ratio (PER) of juvenile grouper increased first and then decreased with the increase in CHO:L ratio, reaching a maximum value in the D4 (CHO:L = 4.24) diet. The trend for the feed conversion ratio was opposite to the PER. Along with the diet CHO:L ratios, the apparent digestibility coefficients (ADC) of crude lipid and energy for the juvenile groupers decreased gradually, while the ADC of dry matter, liver and muscle glycogen level increased gradually. Total protein, triglycerides and cholesterol in serum were all maximized in the D4 diet and glucose in the D5 (CHO:L = 8.51) diet. Digestive enzyme activity in the intestine was significantly affected by dietary CHO:L ratio. Liver hexokinase, phosphofructokinase and glucose-6-phosphate dehydrogenase activity increased significantly as CHO:L ratio increased. Liver lysozyme and acid phosphatase activity in the groupers fed the D3 (CHO:L = 2.33) diet was significantly higher than that of other diets. Liver fructose-1,6-bisphosphatase and alkaline phosphatase activity reached a maximum value in the D4 diet and was significantly higher than that in the D1 diet. Taking the above results together, it can be concluded that an optimal dietary CHO:L ratio of 2.33 is suitable for grouper culture concerning growth performance and health.

Occurrence depth of juvenile freshwater pearl mussels (Margaritifera margaritifera) in a river bed tested by experimental mesh tubes

Sediment depth preference was tested on 1-year-old freshwater pearl mussel juveniles. Mesh tubes allowing vertical migration with a possibility to leave hyporheic space, filled with 1–2-mm granulometry size substrate and populated with groups of juveniles, were placed in the natural river bed of the Tepla Vltava River, Czech Republic. The depth position of the juveniles was recorded after 1- and 2-month exposure during the summer and after 8 months including wintering. The juveniles showed a tendency not to stay on the sediment surface, but to penetrate into a very shallow depth, and to accumulate and stay 2–3 cm deep in the substrate in the summer period. No juvenile was found below 8 cm sediment depth, neither in the summer nor after wintering. In the experimental tubes, various survival rates (0–100%) were recorded in close relation to substrate oxygenation development at the tested sites. Oxygen decrease was accompanied by juveniles trying to escape the hyporheal.

Restored river habitat provides a natural spawning area for a critically endangered landlocked Atlantic salmon population.

Supplementing endangered fish populations with captive bred individuals is a common practice in conservation management. The aim of supplementary releases from hatchery broodstocks is to maintain the viability of populations by maintaining their genetic diversity. Landlocked Lake Saimaa salmon (Salmo salar m. sebago) has been critically endangered for the past half-century. As a result of anthropogenic disturbance, especially construction of hydroelectric power plants, the Lake Saimaa salmon has become completely dependent on hatchery broodstock. Recently, habitat restoration has been done in one of the former spawning rivers with the aim of creating a new natural spawning ground for the critically endangered population. Hatchery fish releases have also been revised so that in addition to juveniles, adult fish from the hatchery and from the wild have been released into the restored river. We assessed here if a restored river stretch can be used as a natural spawning ground and juvenile production area with the aim of improving genetic diversity of the critically endangered Lake Saimaa salmon. By constructing a pedigree of the released adults, and juveniles sampled from the restored river, we found that the majority of the released adults had produced offspring in the river. We also found that wild-caught spawners that were released into the restored river had much higher reproductive success than hatchery-reared parents that were released into the restored river at the same time. We found no significant differences in genetic diversity between the parent and offspring generations. Meanwhile, relatedness among different groups of adults and juveniles varied a lot. For example, while the hatchery-reared females were on average half-siblings, wild-caught females showed no significant relatedness. This highlights the importance of using pedigree information in planning the conservation and management of endangered populations, especially when artificial propagation is involved.

Growth, body composition, and distal intestine histology of Florida pompano Trachinotus carolinus in response to dietary inclusion of hydrolyzed salmon meal and pH adjustment

ABSTRACT In this study, we aimed to investigate the inclusion effect of hydrolyzed salmon meal (HSM) in combination with enzyme-treated soybean meal (ESBM) with or without pH adjustment to completely replace dietary FM on the growth, body composition, and the distal intestine condition of the fish. For that purpose, four experimental diets were formulated to be iso-nitrogenous and iso-lipidic and to contain 400 g kg−1 crude protein and 80 g kg−1 lipid. A reference diet contained 150 g kg−1 FM, 450 g kg−1 soybean meal (SBM), and 39.5 g kg−1 ESBM, and the second diet was formulated replacing FM with 190.6 g kg−1 ESBM. The third diet was produced by supplementing 40 g kg−1 hydrolyzed salmon meal (4% HSM) into the diet formulation at the expense of ESBM, and the fourth diet was produced similar with the third diet, but the pH of the diet was adjusted to match the value in the reference diet (4% HSMA). All test diets were supplemented with taurine at the level of 5 g kg−1. Diets were fed to apparent satiation to triplicate groups of Florida pompano juveniles (mean weight 11.88 ± 0.18 g). After 8 weeks of growth, there were no significant differences in the final weight, percentage weight gain, thermal growth coefficient, feed intake, survival rate, and feed conversion ratio in fish offered the various diets. Nor were significant differences observed in the dry matter composition of the fish in all treatments and only minor changes in the histomorphological of distal intestine as the FM was replaced by plant ingredients. Overall, the adjustment of pH level of the diet containing 4% HSMA did not have any significant effect on the growth and physiological function of fish compared to the group of fish fed with diet without pH adjustment.

Within-Generation Polygenic Selection Shapes Fitness-Related Traits across Environments in Juvenile Sea Bream.

Understanding the genetic underpinnings of fitness trade-offs across spatially variable environments remains a major challenge in evolutionary biology. In Mediterranean gilthead sea bream, first-year juveniles use various marine and brackish lagoon nursery habitats characterized by a trade-off between food availability and environmental disturbance. Phenotypic differences among juveniles foraging in different habitats rapidly appear after larval settlement, but the relative role of local selection and plasticity in phenotypic variation remains unclear. Here, we combine phenotypic and genetic data to address this question. We first report correlations of opposite signs between growth and condition depending on juvenile habitat type. Then, we use single nucleotide polymorphism (SNP) data obtained by Restriction Associated DNA (RAD) sequencing to search for allele frequency changes caused by a single generation of spatially varying selection between habitats. We found evidence for moderate selection operating at multiple loci showing subtle allele frequency shifts between groups of marine and brackish juveniles. We identified subsets of candidate outlier SNPs that, in interaction with habitat type, additively explain up to 3.8% of the variance in juvenile growth and 8.7% in juvenile condition; these SNPs also explained significant fraction of growth rate in an independent larval sample. Our results indicate that selective mortality across environments during early-life stages involves complex trade-offs between alternative growth strategies.

Photodegradation Elevated the Toxicity of Polystyrene Microplastics to Grouper (Epinephelus moara) through Disrupting Hepatic Lipid Homeostasis

Microplastics (MPs) have caused increasing global concerns due to their detrimental effects on marine ecosystems. However, the role of photodegradation in altering toxicity of MPs to marine organisms is poorly understood. We therefore investigated the photolytic transformation of pristine polystyrene fragments (P-PS) by 60-day ultraviolet (UV) irradiation, and compared the toxicity of P-PS, photodegraded PS (PD-PS), and commercially available polystyrene microbeads (C-PS) to juvenile grouper (Epinephelus moara). Photodegradation reduced the size from ∼55.9 μm of P-PS to ∼38.6 μm of PD-PS, even produced nanoparticles (∼75 nm) with a yield of 7.03 ± 0.37% (w/w), and induced surface oxidation and formation of persistent free radicals (e.g., CO•, COO•). Also, endogenous pollutants (chemical additives and polymer fragments) were leached out. Thus, PD-PS had the highest growth inhibition and lipidosis-driven hepatic lesions of grouper, followed by P-PS and C-PS, which was mainly explained by increased hepatic bioaccumulation of MPs/NPs and released endogenous toxicants. Furthermore, oxidative stress-triggered mitochondrial depolarization, suppression of fatty acid oxidation and transport, and promotion of inflammation were identified as the key mechanisms for the enhanced hepatotoxicity after photodegradation. This work provides new insight into the potential hazard and harm of MPs in marine environments after photodegradation.

High-level expression and characterization of a lipase from Trichosporon fermentans Y3 and its application as an aquafeed additive for grouper (Epinephelus coioides)

A lipase gene was cloned from Trichosporon fermentans Y3, subcloned into pPICZαA, and then expressed in Pichia pastoris X33. The lipase (lipRT), which was obtained using a 50-L bioreactor, had maximum activity of 6000 U/mL. It had an optimum pH 8.0 and remained stable between pH 3.0 and 11.0, while its optimum temperature was 45 °C, maintaining more than 55% of optimum activity between 20 and 35 °C. To evaluate the potential application of lipRT as an aquafeed additive for juvenile grouper (Epinephelus coioides), fish with initial weight 13.31 ± 0.26 g were randomly divided into 12 sea cages and fed five formulated diets (labeled diets 1 to 4). Diets 1–4 were formulated to include 8% crude lipid and serial levels of lipRT (0 U/kg, 50 U/kg, 500 U/kg, and 1000 U/kg). The results showed that WG, FE, and PER of fish as well as ACH50, LSZ, and O2− production ratio in serum of fish on diets 3 and 4 were significantly (P < 0.05) higher than those on diets 1 and 2. The present data suggests that lipRT could improve the utilization of lipid and some nonspecific immunological parameters, and therefore could potentially be used as an aquafeed additive.

Functional correlations of axial muscle fiber type proportions in the waterfall-climbing Hawaiian stream fish Sicyopterus stimpsoni.

Assessing the factors that contribute to successful locomotor performance can provide critical insight into how animals survive in challenging habitats. Locomotion is powered by muscles, so that differences in the relative proportions of red (slow-oxidative) vs. white (fast-glycolytic) fibers can have significant implications for locomotor performance. We compared the relative proportions of axial red muscle fibers between groups of juveniles of the amphidromous gobiid fish, Sicyopterus stimpsoni, from the Hawaiian Islands. Juveniles of this species migrate from the ocean into freshwater streams, navigating through a gauntlet of predators that require rapid escape responses, before reaching waterfalls which must be climbed (using a slow, inching behavior) to reach adult breeding habitats. We found that fish from Kaua'i have a smaller proportion of red fibers in their tail muscles than fish from Hawai'i, matching expectations based on the longer pre-waterfall stream reaches of Kaua'i that could increase exposure to predators, making reduction of red muscle and increases in white muscle advantageous. However, no difference in red muscle proportions was identified between fish that were either successful or unsuccessful in scaling model waterfalls during laboratory climbing trials, suggesting that proportions of red muscle are near a localized fitness peak among Hawaiian individuals.

Fat powder can be a feasible lipid source in aquafeed for the carnivorous marine teleost golden pompano, Trachinotus ovatus

In the production of aquafeeds, exogenous liquid oils are usually added directly or by vacuum coating. In order to evaluate the practicality of fat powder in aquafeeds and aquaculture, four isoproteic (46%) and isolipidic (12%) diets named FOl, BOl, FOp, and BOp were prepared with fish oil (FO) or blend oil (BO, consisting of fish, soybean, rapeseed, and perilla oils) or their corresponding fat powder as dietary lipid, respectively. Each diet was fed to triplicate groups of golden pompano Trachinotus ovatus juveniles (initial body weight about 15.10 g) in floating sea cages (1.0 × 1.0 × 2.0 m, 30 fish per cage) for 10 weeks. Fish were fed to nearly satiation at two times each day. The results showed that the final weight, weight gain rate (WGR), specific growth rate (SGR), food conversion ratio (FCR), condition factor (CF), viscerosomatic index (VSI), and survival rate (SUR) displayed no significant difference among the four dietary groups (P > 0.05), while the hepatosomatic index (HSI) in the BOp group (1.01) was significantly lower than that in FO-base groups (1.19–1.23) (P < 0.05). The fatty acid (FA) composition in livers or dorsal muscle showed no difference between the fish fed the diets from liquid oil and corresponding fat powder. A significantly high serum alkaline phosphatase (ALP) activity and low hepatic malondialdehyde (MDA) level were detected in the BOp group (P < 0.05). Moreover, the mRNA levels of hepatic glutathione peroxidase (gsh-px), superoxide dismutase (sod), and catalase (cat), as well as intestinal FA binding protein 2 (fabp2) and FA transport protein 4 (fatp4) genes in one or both of the fat powder groups, were significantly higher than those in the corresponding liquid oil groups (P < 0.05). Additionally, the peroxide value (POV) and MDA level in FOp and BOp diets were less than 27% of those in FOl and BOl diets after stored at − 20 °C for 3 months. These results demonstrated that fat powder diets, especially BOp, displayed better effects than liquid oil diets in lipid oxidation stability, liver health, and FA transportation in T. ovatus, which indicated that fat powder can be a feasible lipid source in aquafeed. To our knowledge, this is the first report on the practical application of fat powder in aquaculture, which may provide a new form of lipid addition in the aquafeeds industry.

Valorisation and enhanced sustainability of Senegalese sole (Solea senegalensis) aquaculture by dietary use of ditch shrimp (Palaemonetes varians) meal

The European Blue Growth Strategy requires innovative proposals contributing to sustainable development of the aquaculture activity. Within this context, ditch shrimp (Palaemonetes varians) meal obtained from extensively managed aquaculture wetlands was assessed as a potential source to substitute dietary fishmeal (FM) in compound diets for Senegalese sole (Solea senegalensis) juveniles. Estuarine ditch shrimp biomass from an integrated aquaculture system was converted into a meal that contained 61.9% protein and 9.2% lipid on a dry matter (DM) basis. The n-3 highly unsaturated fatty acids (HUFA) constituted the main fraction of the polyunsaturated fatty acids (PUFA). Three isonitrogenous (55% DM), isolipidic (12% DM) and isoenergetic (17 kJ g−1 DM) diets containing three substitution levels of FM by ditch shrimp meal (SM) were elaborated: 100% FM + 0% SM (SM0); 50% FM + 50% SM (SM50) and 0% FM + 100% SM (SM100). The experimental diets were tested on duplicate groups of sole juveniles (~4 g body weight) in an experiment that lasted 63 days. The substitution of dietary FM with SM did not influence growth and feed intake of the reared fish. Juveniles fed SM100 significantly decreased their muscle and skin lipid depositions in comparison with soles fed SM0. Moreover, muscle lipid analyses showed arachidonic acid (ARA) enrichment caused by the SM dietary use and a major presence of the n-3 over n-6 PUFA. Overall results demonstrate that dietary incorporation of P. varians meal in compound diets for Senegalese sole would add nutritional value to the harvested product with no impact on growth performance of reared fish. The dietary use of a low trophic level marine resource is an innovative practice that may contribute to a further sustainable development of aquaculture of carnivorous fish species.

English

Recognized for its traditional roles, estrogen is ever-present in all vertebrates, regulates reproduction by binding and activating estrogen receptors (ERs), and also controls several functions of vertebrates, including reproductive immune, and central nervous systems. In order to access any other possible functions of the estrogen receptors in the development of the juvenile Hybrid grouper (Epinephelus fuscoguttatus â™€× Epinephelu polyphekadion ♂), full-length of ERs cDNA sequences were isolated and analyses were found to be 2391 bp for hgERα, 2626 bp for gERb1 and 2339 bp for hgERb2, respectively. The results of amino acid and phylogenetic analysis revealed that each hgER was grouped in consistent taxonomic groups of perciformes and demonstrated great evolutional conservation in functional domains. Real-time PCR examination discovered that the receptors expressed in all tissues examined, though, at a different level, the ERα mRNA level expressed higher than ERβ1, and ERβ2 in tissues examined. The ERα mRNA level of expression was found to be highest in the tissue of the heart, followed by muscle, and liver. The ERβ1 mRNA level was greatest in heart tissue, trailed by liver and muscle and ERβ2 was highest in the heart trailed by stomach and liver. The minimal expression was recorded in the kidney, the gill, and the brain for ERα, ERβ1, and ERβ2 respectively. These results put forward that steroid hormone estrogen receptors might be playing a significant part in the controlling of social behavior complexity, plasticity behavior, and the assessment of a gratifying inducement in Hybrid grouper. Key words: Estrogen receptors, Real-time POR, tissue expression, hybrid grouper.

Growth and metabolic responses of juvenile grouper (Epinephelus coioides) to dietary methionine/cystine ratio at constant sulfur amino acid levels

An eight-week feeding trial was conducted to investigate the effects of dietary methionine/cystine (Met/Cys) ratio on juvenile grouper growth performance, hepatic metabolic enzymes, serum immune-related parameters, liver antioxidant indices, and relative expression of genes in the mTOR pathway. Six isonitrogenous, isocaloric experimental diets with a fixed Met and Cys level (1.78%) and variable Met/Cys ratio (0.76–4.50) were formulated. Each diet was randomly assigned to triplicate groups of 30 juvenile fish with an average initial weight of 11.66 g. The fish were fed twice daily for 2 months. Weight gain and specific growth rates, feed efficiency and intake, and protein efficiency ratio had positive linear and/or quadratic responses to the dietary Met/Cys ratio. In contrast, the variations in dietary Met/Cys ratio had no apparent effect on the hepatosomatic index, condition factor, whole-body moisture, protein, lipid or ash content, or hepatic betaine homocysteine methyltransferase or S-adenosylhomocysteine hydrolase. Hepatic methionine synthase, cystathionine β-synthase, glutathione peroxidase, superoxide dismutase, and catalase activity, serum acid phosphatase, alkaline phosphatase, and lysozyme activity, and serum IgM and C3 content had positive linear and/or quadratic responses to the dietary Met/Cys ratio. In contrast, the malondialdehyde content had negative linear and/or quadratic responses to the dietary Met/Cys ratio. Hepatic RagA/B, RagC/D, Raptor, and eIF4B mRNA levels were unaffected by the dietary Met/Cys ratio. On the contrary, muscular upstream and downstream mTOR signal pathway gene mRNA levels had positive linear and/or quadratic responses to the dietary Met/Cys ratio. However, the 4E-BP mRNA levels had negative linear and quadratic responses to the dietary Met/Cys ratio. Cys could replace up to 41% of the Met in the fish diets. The use of appropriate Met/Cys ratios (range 1.93–2.77) in the feed may help improve grouper growth performance, immune function, and antioxidant ability and positively regulate genes in the mTOR signaling pathway.

Red grouper nervous necrosis virus (RGNNV) induces autophagy to promote viral replication

Autophagy is an evolutionarily conserved cellular degradation process that is essential for homeostasis. As a cell steward, autophagy is thought to be a process that may have evolved to combat intracellular pathogens. However, some virus can subvert or utilize autophagy-related membrane structures to increase viral replication. The red-spotted grouper nervous necrosis virus (RGNNV) is a fish pathogen which leads to disastrous viral nervous necrosis in larvae and juvenile groupers and other marine fishes. To better comprehend the pathogenesis and replication mechanism of RGNNV, we investigated the relationship between RGNNV and autophagy. Here, we demonstrated that RGNNV induced autophagy in grouper spleen (GS) cells, as the significant increase in ultrastructural autophagosome-like vesicles, fluorescent punctate pattern of microtubule-associated protein 1 light chain 3 (LC3), and the conversion of LC3-I to LC3-II. Additionally, ultraviolet-inactivated RGNNV and the capsid protein also triggered autophagy. Enhancement of autophagy contributed to RGNNV replication, whereas blocked autophagy decreased RGNNV replication. Moreover, impeded fusion of autophagosomes and lysosomes also reduced RGNNV replication, indicating that RGNNV utilized the different steps of autophagy pathway to facilitate viral replication. The further study showed that RGNNV induced autophagy through activating the phosphorylation of eIF2α and inhibiting the phosphorylation of mTOR. These results will assist the search for novel drugs targets and vaccine design against RGNNV from the perspective of downregulating autophagy.

Dietary supplementation of vitamin E and citric acid could significantly promote the relative expression of PPARα and aconitase genes, concentration of polyunsaturated fatty acids, antioxidant enzyme activities, and growth of juvenile cobia

The experiment was to study the effect of different dietary vitamin E (VE) and/or citric acid (CA) supplementation on the peroxisome proliferator-activated receptor α (PPARα) and aconitase (ACO) gene expression, fatty acids, antioxidant enzyme activities, and growth of juvenile cobia (Rachycentron canadum). Seven groups of cobia juveniles in triplicate were cultured in tanks using filtered and aerated seawater. The juveniles were fed twice a day using 7 specific diets: control diet (D0) without supplementation; Diets 1 to 6 (D1 to D6) were supplemented with CA 0, 12, 12, 6, 3, 1.5 g and VE 100, 0, 100, 75, 50, 25 IU per kg of dried feed, respectively. The juveniles were fed for 12 weeks and their liver, muscle, and serum were sampled randomly for analysis in weeks 0 and 12. The results showed that the supplementation of VE plus CA could significantly promote the relative mRNA expression of ACO and PPARα genes, concentration of polyunsaturated fatty acids (PUFAs), activity of antioxidant enzymes, and growth of juvenile cobia. The effect of both dietary VE plus CA supplementation on the above indexes was significantly greater (P < 0.05) than a single VE (D1) or CA (D2) supplementation. The optimum diet was D5, with a VE of 50 IU and a CA of 3 g supplementation per kg of dried feed in the tested diets.

Effect of dietary ARA/EPA/DHA ratios on growth performance and intermediary metabolism of gilthead sea bream (Sparus aurata) juveniles

Vegetable oils (VO) are generally accepted as valuable alternatives to fish oils but are devoid of n-3 LC-PUFA (Long-Chain-Polyunsaturated Fatty Acids) and rich in C18-PUFA. Marine fish have limited capacity to express Δ6/Δ5 desaturases (FADS1 and FADS2) and elongases of very-long-chain fatty acids (Elovl), which severely limits their capacity to produce LC-PUFA from C18-PUFA. This study aimed to evaluate the effects of dietary essential fatty acids(EFA) ratios, arachidonic (ARA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids on growth performance, feed utilization, whole-body composition, hepatic and intestinal activity of key-enzymes of lipid and carbohydrate metabolism, and expression of lipid metabolism-related genes. Diets were formulated to include ARA/EPA/DHA ratios of 2.0/0.2/0.1 (Diet A); 1.0/0.4/0.4 (Diet B); 0/0.6/0.6 (Diet C); and 0/0.3/1.5 (Diet D) and were fed to triplicate groups of gilthead sea bream juveniles for 56 days.Dietary EFA ratios did not affect growth performance and whole-body composition. Nevertheless, feed and protein utilization were higher with diet B than diets C or D. Except for ARA, muscle EFA profile followed that of the diets. Plasma triglycerides were higher with diet B and C than A, and plasma cholesterol decreased in fish fed the higher DHA diet. Hepatic ELOVL5 and phospholipase A2 were upregulated in fish fed diet C compared to diet A. In the intestine, ELOVL5 levels were down-regulated in fish fed diet A and D compared to fish fed diet B.Overall, results showed that besides EPA and DHA, adequate dietary ARA level needs to be provided to optimize feed utilization efficiency of gilthead sea bream juveniles.