Complete repertoire of long‐chain polyunsaturated fatty acids biosynthesis enzymes in a cyprinid,silver barb (Barbonymus gonionotus): Cloning,functional characterization and dietary regulation of Elovl2 and Elovl4

The silver barb (Barbonymus gonionotus) is a valuable cyprinid with good deposition of long‐chain polyunsaturated fatty acids (LC‐PUFA) such as docosahexaenoic acid (DHA). We have previously isolated a fatty acyl desaturase (Fads2) and elongase (Elovl5), which fulfils the production of eicosapentaenoic acid (EPA) and arachidonic acid (ARA) from C18 polyunsaturated fatty acid (PUFA) substrates. The isolation, functional characterization and dietary regulation of two elongases, Elovl2 and Elovl4, are reported here. The Elovl2 displayed the capacity to elongate C20 and C22 PUFA substrates, while the showed low activities towards saturated fatty acids and C22 PUFA substrates. This discovery validates the existence of a complete set of enzymes of LC‐PUFA in silver barb. The elovl2 showed higher expression in liver, in limited dietary LC‐PUFA intake conditions. As for Elovl4, tissue expression showed prominence in eye tissue. We also showed that the expression of both genes was upregulated when fish was fed with diets devoid of LC‐PUFA. Fatty acid composition analysis indicates the utilization of C18 polyunsaturated fatty acids for LC‐PUFA biosynthesis.     

Family growth response to different laboratory culture environments shows genotype–environment interaction in the sea urchin Strongylocentrotus intermedius

This study investigated the genetic variation and genotype–environment interaction (GEI) in the early growth stage among seven full‐sib families of the sea urchin Strongylocentrotus intermedius. From each of the seven families, 180 or 270 sea urchins of the same size were exposed to three laboratory environments (E₁, E₂ and E₃). These environments were commonly used in breeding programs for S. intermedius. After a 102 days trial, test height, test diameter, body weight and the coefficient of variation of body weight were determined. The results revealed significant family effects on most growth traits in all the three environments (except for test diameter in environment E₂), and significant environmental effects on growth in several families (family 1, 5, 6, 7). The study also revealed that the coefficient of variation in body weight varied significantly among the families (P < 0.01) but not among the environments. Highly significant GEI effects were also recorded for all growth traits (P ≤ 0.001), except for the coefficient of variation in body weight (P > 0.05). Variances of GEI accounted for about 0–2.742% of phenotypic variances for the investigated traits. Significant GEI caused a certain degree family ranking inversions for the growth traits. The present study provides evidence for the existence of GEI in the family selection of sea urchin S. intermedius. More attention should be paid to the GEI to obtain satisfactory genetic gain in S. intermedius.     

Measuring density effects on growth and survival of two size classes of juvenile sea urchins (Strongylocentrotus droebachiensis) held in sea‐based holding systems and impacts on holding system design and management

Assessing density in intensive sea urchin culture systems by measuring the percentage coverage of available surface area is an effective means of calculating the available space within a holding system, the proximity of the animals in the system to each other and the probable effects of the stocking density. The results of this study show that density plays a critical role in regard to both somatic growth (increase in test diameter measured in millimetres) as well as mortality of juvenile sea urchins ranging in size from 5 mm to 26 mm test diameter (the size range tested in the current experiments). These effects appear to be greater for smaller urchins. The critical percentage coverage densities are in the order of 50%‐60% coverage for juvenile sea urchins, and the authors advise farmers growing Strongylocentrotus droebachiensis to maintain stocking densities below this point and to reduce the stocking densities as and when stocks reach these critical density points. Percentage cover must be constantly monitored as the results from this study indicate that the growth rates of juvenile urchins can alter percentage coverage rapidly and reductions in density may be required as frequently as every 3‐4 months at the early juvenile stage. Holding system design should take these factors into account and incorporate a method of rapidly reducing stocking densities with minimal stress and handling of sea urchin stocks.     

The effect of fresh seaweed and a formulated diet supplemented with seaweed on the growth and gonad quality of the collector sea urchin,Tripneustes gratilla,under farm conditions

This gonad enhancement study investigates the effect of different fresh and formulated feeds and feeding regimes on the growth and gonad quality of wild‐collected adult sea urchin, Tripneustes gratilla, under farm conditions for over 18 weeks. In the first 12 weeks (phase 1), urchins were fed fresh Ulva rigida (U); a 50:50 mixture of fresh U. rigida and Gracilaria gracilis (UG); fresh G. gracilis (G) and a formulated diet 20U (containing 20% U. rigida), and in the final 6 weeks (phase 2) of the study, diet was changed to a formulated feed (20U diet). By the end of phase 1, urchins fed the 20U diet produced gonads (50.72 ± 5.4 g) that were significantly heavier (p < .001) than the gonads of urchins fed the fresh seaweed diets (U, UG & G). By the end of phase 2, gonad weight of urchins in treatment groups UG‐20U and G‐20U were similar to those fed the 20U‐20U diet. Gonad colour of urchins in the G‐20U treatment became significantly lighter (ANOVA, p = .029) and poorer quality, compared with urchins in the U‐20U group. This gonad enhancement study, conducted on wild‐collected adult T. gratilla, has shown that a formulated feed (20U diet) can enhance gonad growth and produce commercially acceptable gonads. This farm‐based study supports previous findings from aquarium‐based studies by our group and indicates that short‐term sea urchin gonad enhancement can be carried out under farm conditions in South Africa.     

Recombinant expression and comparative bioactivity of tongue sole insulin‐like growth factor (IGF)‐1 and IGF‐2 in Pichia pastoris

Insulin‐like growth factor (IGF) plays a key role in the complex system that regulates bony fish growth, differentiation, and reproduction. In the current study, recombinant tongue sole IGF‐1 and IGF‐2 were obtained using the Pichia pastoris expression system and their comparative bioactivities were investigated. Tricine–SDS–PAGE and western blot analysis showed that the recombinant tongue sole IGFs were secreted into the culture medium and had a molecular weight of 8.7 kDa. The optimal incubation time and pH for recombinant expression of IGFs were 36 hr and 5.0 respectively. Functional analysis demonstrated that both recombinant tongue sole IGF‐1 and IGF‐2 significantly promoted cell proliferation of MFC‐7 in vitro. In addition, the recombinant tongue sole IGF‐1 and IGF‐2 proteins could suppress hepatic mRNA levels of igf‐1 and igf‐2 in vitro, which showed that they have similar physiological functions. Taken together, the biologically active recombinant tongue sole IGF‐I and IGF‐II proteins will allow us to further investigate their physiological roles in growth regulation of this species. Furthermore, the present results also hinted at the potential application of these two recombinant IGF‐I and IGF‐II proteins into the tongue sole farming industry.     

Effects of protein and carbohydrate levels on survival,consumption and gonad index in adult sea urchin Strongylocentrotus purpuratus (Stimpson 1857) from Baja California,Mexico

A greater understanding of dietary protein and carbohydrate levels with regard to gonad production in Strongylocentrotus purpuratus would increase our nutritional knowledge of this sea urchin and guide the development of formulated diets for such aquaculture target species. A total of 255 purple sea urchins were captured from Ensenada Bay, Mexico, and maintained in 200‐L tanks for 9 weeks. Formulated diets that contained 30%, 26%, 23%, 20% and 17% of protein and 42%, 46%, 50%, 54% and 58% carbohydrates were offered ad libitum. Survival was affected by diets; urchins that were fed high‐protein–low‐carbohydrate diet experienced decreased survival. No significant differences were found in gonad index, but gonad production efficiency was lower in urchins that were fed a medium‐low‐protein–medium‐high‐carbohydrate diet. Urchins that were fed high carbohydrate levels utilized protein more efficiently and showed better digestibility of the diet and protein. These data suggest that all of our diets support gonad growth, but in terms of consumption, a diet that contains protein levels of 17% and 23% with carbohydrate levels of 50% and 58% are beneficial for S. purpuratus.     

Effect of feed deprivation and refeeding on the MYP gene expression of the sea urchin (Strongylocentrotus intermedius)

To determine the main expression site of major yolk protein (MYP) gene and the mechanisms for adaptation to starvation and refeeding in Strongylocentrotus intermedius, MYP mRNA expression amounts were analysed using a real‐time RT‐PCR. The results showed that MYP could be transcribed in the intestine, stomach, gonad and coelomocytes, and that the intestine was the main expression site of MYP gene in non‐starved urchins. The MYP synthesis in the intestine decreased during 15 days of starvation (67.70%, 52.58% and 71.35% of the control at 5, 10 and 15 days of fasting respectively) and then increased dramatically by different amounts (the peaks were 2.71‐, 12.16‐ and 7.89‐fold that of the control respectively) during the refeeding stages. Nevertheless, the expression amounts in the gonads did not decline, but increased continuously during all periods of fasting (2.66‐, 3.72‐ and 13.19‐fold that of the control at 5, 10 and 15 days of starvation respectively) and during the refeeding stages. At the end of the recovery feeding experiment, the levels reached 9.58‐, 17.48‐ and 100.69‐fold that of the control. These data suggested that the ‘priority’ strategy for the sea urchin is to reduce MYP expression amounts in the intestine if food is limited and to increase MYP gene expression in the gonad to protect reproductive function.     

Nutritional stimuli of gilthead seabream (Sparus aurata) larvae by dietary fatty acids: effects on larval performance,gene expression and neurogenesis

The concept of nutritional programming raises the interesting possibility of directing specific metabolic pathways or functions in juvenile fish, for example, to improve the use of substitutes to fishmeal and oil, and hence to promote sustainability in aquaculture. The aim of the study was to determine effects of early nutritional stimuli of gilthead seabream larvae and check if nutritional programming of gilthead sea bream is possible between 16 days post hatching (dph) and 26 dph. A trial was conducted to determine the effects of early nutritional stimuli of gilthead seabream larvae. Five experimental microdiets (pellet size <250 μm) were formulated containing five different proportions of a marine lipid source rich in long‐chain polyunsaturated fatty acids (LC‐PUFA) and two vegetable lipid sources rich in linolenic and linoleic acids. The results of this study demonstrate that dietary n‐3 LC‐PUFA levels increased larval growth and survival affecting Δ6 desaturase gen (fads2) expression and retinal neurons density. However, the high mortalities obtained along on‐growing in fish fed low n‐3 LC‐PUFA at 16 dph constrained the feasibility of nutritional programming of gilthead seabream during this late developmental window and needs to be further investigated.     

Long‐chain polyunsaturated fatty acid metabolism in carnivorous marine teleosts: Insight into the profile of endogenous biosynthesis in golden pompano Trachinotus ovatus

Golden pompano Trachinotus ovatus is an important farmed carnivorous marine teleost. Although some enzymes for long‐chain polyunsaturated fatty acid (LC‐PUFA) biosynthesis have been identified, the ability of T. ovatus for endogenous biosynthesis is unknown. Here, we evaluated in vivo LC‐PUFA synthesis in a 56‐day culture experiment using six diets (D1–D6) formulated with linseed and soybean oils to produce dietary linolenic/linoleic acid (ALA/LA) ratios ranging from 0.14 to 2.20. The control diet (D0) used fish oil as lipid source. The results showed that, compared with the corresponding indices of fish fed D0, the weight gain rate and specific growth rate as well as the contents of eicosapentaenoic (EPA) and docosahexaenoic acids in tissues (liver, muscle, brain and eye) of D1–D6 groups were significantly lower (p < .05). These data suggested that T. ovatus could not synthesize LC‐PUFA from C₁₈ PUFA or such ability was very low. However, tissue levels of 20:4n‐3 in fish fed diets D1–D6 were higher than that of D0 fish (p < .05), and positively correlated with dietary ALA/LA ratio, while levels of EPA showed no difference among the D1–D6 groups. These results indicated that Δ5 desaturation, required for the conversion of 20:4n‐3 to EPA, may be lacking or very low, suggesting incomplete LC‐PUFA biosynthesis ability in T. ovatus.     

Carotenoid composition of a New Zealand (Evechinus chloroticus) and an Australian (Heliocidaris erythrogramma) sea urchin in relation to gonad colour

Sea urchins are commercially harvested for their edible gonads, also known as roe or uni. Most species, particularly those from the Northern hemisphere are reported to exhibit limited variation in gonad colour, which is proposed to be due to carotenoid pigments. The commercially harvested species from New Zealand, Evechinus chloroticus, and from Australia, Heliocidaris erythrogramma, however, exhibit considerable gonad colour variation, ranging from a culinary desirable light yellow/orange to an undesirable brown. Gonads from E. chloroticus and H. erythrogramma were characterized spectrophotometrically and the carotenoid content extracted and analysed by reversed‐phase high‐performance liquid chromatography. The main carotenoid found in the gonads of both species was 9′‐cis‐echinenone, with lower levels of all‐trans‐echinenone, lutein and isozeaxanthin. Carotenoid composition contributed to the yellow/orange colour components of the gonads, but was not correlated with the darker roe coloration observed in some E. chloroticus individuals. Our results highlight important considerations relating sea urchin gonad coloration to carotenoid content and as such will provide useful information for the future aquaculture and processing of these two species.     

Effects of dietary n‐3 PUFA supplements on composition of n‐3 PUFA and expression of fatty acid elongase 5 (AJELOVL5) in sea cucumber,Apostichopus japonicus

This is the first comprehensive study on the effect of dietary polyunsaturated fatty acid (PUFA) levels on the expression of fatty acid elongase 5 (AJELOVL5), PUFA composition, and growth in juvenile sea cucumbers. The specific growth rate (SGR_w) was improved in n‐3 PUFA‐rich diets compared to low n‐3 PUFA diets. AJELOVL5 expression was apparently upregulated in juveniles fed lower PUFA diets relative to higher PUFA diets, with higher expression in the body wall and respiratory tree of juveniles fed diets without ɑ‐linolenic acid (ALA, 18:3n‐3) compared to juveniles fed higher ALA level diets; similar results were also detected in juveniles fed diets with lower eicosapentaenoic acid (EPA, 20:5n‐3), docosahexaenoic acid (DHA, 22:6n‐3), and none of ALA, EPA, or DHA respectively. The concentrations of ALA, EPA, and DHA in tissues were positively related to the content of dietary corresponding PUFA, with higher ALA content in juveniles fed diet ALA_12.71 than in the ALA_7.46 and ALA₀ groups. Similar results were also obtained in sea cucumber fed diets enriched with either EPA or DHA. Interestingly, considerable levels of EPA and DHA were found in the tissues of juveniles fed diets of CK₀ and DHA₀, with no specific input of EPA or DHA, showing that the sea cucumber was capable of biosynthesizing EPA and DHA from their corresponding precursors as ALA and linoleic acid (LA, 18:2n‐6).     

Identification and functional characterization of Histone‐like DNA‐binding protein in Nocardia seriolae (NsHLP) involved in cell apoptosis

Nocardia seriolae, a facultative intracellular bacterium, is the main pathogen of fish nocardiosis. Bioinformatic analysis showed that the histone‐like DNA‐binding protein (HLP) gene of N. seriolae (nshlp) encoded a secreted protein and might target the mitochondria in the host cell. To further study the preliminary function of HLP in N. seriolae (NsHLP), the gene cloning, extracellular products identification, subcellular localization, overexpression and apoptosis detection assay were carried out in this study. Mass spectrometry analysis of the extracellular products from N. seriolae showed that NsHLP was a secreted protein. Subcellular localization of HLP‐GFP fusion proteins mainly assembled in the nucleus, which indicated that the NsHLP was co‐located with the nucleus rather than mitochondria in fathead minnow (FHM) cells. Notably, the expression of NsHLP had changed the distribution of mitochondria into lumps in the FHM cell. In addition, apoptotic features were found in the transfected FHM cells by overexpression of NsHLP. Quantitative assays of mitochondrial membrane potential value, caspase‐3 activity and pro‐apoptotic genes mRNA (Bad, Bid and Bax) expression level demonstrated that the cell apoptosis was induced in the transfected FHM cells. All the results presented in this study provided insight on the function of NsHLP, which suggested that it may participate in the cell apoptosis regulation and play an important role in the pathogenesis of N. seriolae.     

Effects of diet and temperature on post‐settlement growth and survival of the short‐spined sea urchin Strongylocentrotus intermedius

Effects of diet and temperature on post‐settlement growth and survival of a sea urchin Strongylocentrotus intermedius, which fed on one of six mono‐cultured benthic diatoms, gametophytes of a brown alga Undaria pinnatifida, a green alga Ulvella lens plus mixed benthic diatoms, or no food at 6, 9, 12 and 15°C, were examined. Digestion efficiency (DE), ingestion rate (IR), excretion rate (ER) and digested cell content index (DCCI) of each diatom species were measured. Growth rates largely differed among the dietary treatments from 7 days post‐settlement. Sea urchins fed on a diatom Cocconeis sublittoralis showed considerably higher growth (7.85‐34.67 μm day^?1) than those fed on the other diets and high survival rates (100%). DCCI was also highest in C. sublittoralis. These results suggest that the quantity of diatom cell contents digested is an important factor affecting growth and survival of the sea urchins. Growth rates of sea urchins were higher in higher water temperatures, and significant differences in growth rate were observed between 6‐9 and 12‐15°C. ERs of C. sublittoralis were generally higher at higher temperatures, but DEs did not differ among the temperature treatments. Variations in growth rate among different water temperature appear to be caused by different food intakes of the urchins.