Growth, survival and biochemical composition of spider crab Maja brachydactyla (Balss, 1922) (Decapoda: Majidae) larvae reared under different stocking densities, prey:larva ratios and diets

The spider crab Maja brachydactyla is overexploited on the NW coast of Spain. Aquaculture of this species can be the solution to the problem, and consequently, several attempts of intensive larval rearing have been conducted. However, most of the studies already published do not provide enough zoo technical data, especially in terms of larval and prey densities or the nutritional quality of diets used for rearing.Three experiments were carried out to evaluate the conditions for intensive larval rearing of M. brachydactyla. Larval stocking density (10, 50 and 100 larvae L^− 1), prey:larva ratio (15, 30 and 60 enriched Artemia larva^− 1) and diet (enriched Artemia, non-enriched Artemia and polychaete supplement) effects on growth and survival of this species were studied. For larval culture nine, 35 L, 150 μm mesh-bottomed PVC cylinders (triplicates for each treatment and larval stage) connected to a recirculation unit, were used. Temperature and salinity were kept constant at 18 °C and 36‰ respectively. A 12 to 18 day trial was conducted for each experiment and samples of larvae were collected at each larval stage (zoea I, zoea II, megalopa) in the inter-molt phase and at first juvenile. Survival, carapace length and width, dry weight (DW), and proximate biochemical content (protein, carbohydrates and total lipid) as well as lipid class composition were determined.Stocking densities of 100 larvae L^− 1 resulted in higher growth in DW and higher content in lipids and protein for zoea I (ZI) and zoea II (ZII) than 10 larvae L^− 1. However, survival decreased with increasing stocking density.The use of 60 preys larva^− 1 produced larvae with significantly higher DW and protein content, especially at ZII stage, than lower prey densities. Survival rate obtained feeding 60 preys larva^− 1 up to the megalopa stage was almost two-fold (42.2%) the rate obtained using 15 preys larva^− 1 (24.8%).Larvae fed on enriched Artemia (EA) showed an increase in weight up to megalopa (518.9 ± 26.5 μg) in contrast to larvae fed on non-enriched prey (A) (467.9 ± 6.9 μg). Variation in DW correlated with the total lipid content (L) of the larvae (L_EA = 70.1 ± 37.5 μg ind^− 1; L_A = 28.9 ± 3.2 μg ind^− 1) especially in terms of neutral lipids. The use of an initial density of at least 50 larvae L^− 1 and 60 enriched Artemia larva^− 1 can be considered the most adequate rearing parameters in order to obtain good results in growth and survival of M. brachydactyla.     

Development of lipid microbeads for delivery of lipid and water-soluble materials to Artemia

Lipid spray beads (LSB) containing high concentrations of phospholipids were produced in order to improve their dispersion in both fresh and saltwater. The beads were developed to deliver both fat-soluble and water-soluble micronutrients to Artemia and other suspension feeders. LSB were prepared by spraying molted lipid into a chamber that was cooled with liquid nitrogen in order to solidify the lipid beads. Addition of soy lecithin to LSB did not affect retention of glycine when the beads were suspended in distilled water. There was an initial loss of 80% incorporated glycine after LSB were suspended in water for 20min. Artemia readily ingested riboflavin-containing LSB and their full guts were evident within 30min of feeding. The riboflavin content of Artemia could be increased from 55 ±  0.6 mg kg^− 1 (dw) to 329 ± 62 mg kg^− 1 (dw) after 1h enrichment. LSB prepared with phospholipids are promising vehicles for enrichment of suspension-feeding organisms used as feed for larval marine fish and crustaceans as well as other suspension feeders.     

Bacterial flora associated with the giant freshwater prawn Macrobrachium rosenbergii, in the hatchery system

The objective of this study was to understand the microbial flora associated with the hatchery system of giant fresh water prawn, Macrobrachium rosenbergii during an entire rearing cycle. Bacteriological and physico-chemical analysis was done for different samples of water, larvae, and Artemia. The total bacterial load in well water, seawater and inlet water varied from 10¹ to 10⁵ cfu ml^− 1 with higher counts seen in larval rearing tank (LRT) water. The Vibrio count ranged between 10¹ to 10³ cfu ml^− 1. Larval samples harboured a bacterial load of 10⁶ to 10⁷ cfu/10 larvae. The bacterial load in Artemia hatching water ranged from 4.90 × 10⁴ to 5.63 × 10⁶ cfu ml^− 1 while Artemia had a load ranging from 1.08 × 10⁷ to 2.09 × 10⁹ cfu g^− 1. Vibrio count in the LRT water ranged from 10¹-10³ cfu ml^− 1 while the count in larvae ranged from 10² to 10⁴ cfu/10 larvae. The bacterial genera were predominantly Gram-negative and comprised of Aeromonas spp., Pseudomonas spp., Vibrio spp. and Bacillus spp. and non-spore formers (NSF) were the dominant Gram-positive bacteria. This study documents the bacterial flora associated with Macrobrachium hatchery system during a regular normal run. Knowledge of the qualitative and quantitative aspects of bacterial flora in the hatchery would help to understand disturbances, if any, brought about during disease outbreaks.     

Selective particle feeding by the Chilean oyster, Ostrea chilensis; implications for nursery culture and broodstock conditioning

Hatchery broodstock conditioning and nursery culture of the Chilean flat oyster Ostrea chilensis have been hampered by the poor performance of oysters fed typical microalgal hatchery diets. To determine the feeding capabilities of this species the selective removal and consumption of natural planktonic assemblages and artificial inert particles (polystyrene beads) by juvenile and adult oysters were examined experimentally. The arrangement of the eulaterofrontal cirri of the ctenidia was also examined to infer their potential efficiency of particle selection for feeding. Polystyrene beads of 45 and 15 μm in diameter had high rates of removal from suspensions by both juvenile (45 μm = 70%, 15 μm = 73%) and adult (45 μm = 88%, 15 μm = 87%) oysters. In contrast, beads of 6 μm diameter had lower rates of removal (adults = 68%, juveniles = 53%), while 1 μm beads were not removed at all. Both adult and juvenile oysters feeding upon natural plankton assemblages removed only microphytoplankton (20-75 μm in size) despite the presence of nanophytoplankton (2-20 μm), picophytoplankton (< 2 μm), cyanobacterium Synechoccocus spp. (< 2 μm), and bacterial cells (< 75 μm) in the experimental suspensions. Eulaterofrontal cirri of both juvenile (15.2 μm ± 0.9 SE) and adult oysters (18.9 μm ± 0.3 SE) are the shortest that have been reported for any ostreid species which helps to explain the inability of this species to retain small food particles. The clearance rates for oysters feeding on microphytoplankton only were 1.49 (± 0.05 SE) and 7.1 (± 1.2 SE) l h^− 1g^− 1 for juveniles and adults respectively. These values are much higher than previously reported for this species being fed smaller sized cultured microalgae. Our results strongly suggest that the difficulties in the nursery and broodstock hatchery culture of this oyster may be due to inappropriate phytoplankton diets. We recommend the provision of cultured microalgae of 20-75 μm in diameter for improving the performance of hatchery maintained juvenile and adult O. chilensis.     

Suitability of the copepod, Acartia clausi as a live feed for Seabass larvae (Lates calcarifer Bloch): Compared to traditional live-food organisms with special emphasis on the nutritional value

Though artificial propagation of Asian seabass Lates calcarifer (Bloch) in captivity through induced breeding techniques is standardized under Indian conditions, larval and nursery rearing techniques including suitable nursery feeds have to be standardized to obtain better survival and growth. Feeding experiments in triplicate were conducted to evaluate the suitability of the marine copepod Acartia clausi as live prey for fourteen day-old seabass larvae (6.53 ± 0.06 mm; 8.58 ± 0.33 mg) and compared with the traditional live prey, rotifers and Artemia nauplii. While A. clausi and rotifers were mass produced using algae Isochrysis galbana, Chaetoceros affinis and Chlorella marina, Artemia nauplii were produced using cysts. Nutritional quality of cultured copepods was evaluated based on the proximate composition, amino acid and fatty acid composition, and compared with that of rotifers and Artemia nauplii. Proximate composition varied significantly (P < 0.05) among the different live feeds. A. clausi showed higher protein (63.12%) and lipid (16.65%) content than Artemia nauplii and rotifers. Total essential amino acids content was 2% lower in A. clausi compared to that in Artemia nauplii. Fatty acid profiles of the live feed organisms showed that A. clausi is a rich source of n − 3 fatty acids. The total n − 3 fatty acid content of A. clausi was 33.94%. Length, weight overall weight gain and survivorship were significantly (P < 0.05) different among the dietary treatments, and weight gain was comparatively higher in A. clausi fed larvae. Survival of seabass larvae fed A. clausi was obtained highest as 58.13% against the lower values of 39.93% and 41.62% in larvae fed rotifer and Artemia nauplii respectively. Final carcass composition of the larvae of L. calcarifer fed different live-food organisms showed significant differences (P < 0.05) among the dietary treatments. The fatty acid composition of the dietary treatments was reflected to a certain extent in the fatty acid composition of the seabass larvae. The present investigation revealed the nutritional value of calanoid copepod and thus underlining its usefulness as a suitable live-food organism for rearing larvae of the commercially valuable Asian seabass.     

Successful culture of larvae of Litopenaeus vannamei fed a microbound formulated diet exclusively from either stage PZ2 or M1 to PL1

In three separate experiments, harpaticoid copepods Tisbe monozota (alive and dead) and a microparticulate microbound diet were evaluated as alternatives to live Artemia nauplii as food, beginning at either stage PZ2 or M1, in the larval culture of Litopenaeus vannamei. Larvae were cultured in 2 L round bottom flasks at a density of 150 L^− 1 (Experiment 1) and 100 L^− 1 ( 3.2 and 3.3) at 28 °C, 35‰ salinity and 12:12 LD photoperiod, and fed 4×/day^- 1. Larvae were initially fed a mixture of phytoplankton to stages PZ2 or M1 and then fed either live Artemia, live or dead copepods, or a microparticulate microbound diet. The experiments were terminated and all larvae were harvested when more than 80% of larvae had molted to postlarvae 1 (PL1) within any flask representing any of the treatments. The comparative value of the different diets and feeding regimes was determined by mean survival, mean dry weight and total length of individual larva, and percentage of surviving larvae that were PL1. Trypsin activity of samples of larvae from each treatment was also determined. The microparticulate microbound diet effectively served as a complete substitute for Artemia nauplii when fed beginning at stage M1. When fed at the beginning of the PZ2 stage, survival was comparable to that of larvae fed Artemia, but mean dry weight, mean total length, and percent of surviving larvae that were PL1 generally were significantly less. Responses to the feeding of copepods, whether fed dead or live, as a substitute were generally significantly less than those of larvae fed either the Artemia nauplii or the microparticulate diet. Values of trypsin activity (10^− 5 IU/μg^- 1 dry weight) corresponded to the relative proportions of the different larval stages within a treatment, with higher activity being characteristic of early stages. Previously demonstrated successful results with another species of crustacean suggest that the microparticulate microbound diet has characteristics that should be effective in the culture of the carnivorous stages of other crustacean and fish larvae that are currently fed live Artemia nauplii.     

Morphological, structural, and functional characterization of the haemocytes of the scallop, Argopecten irradians

Argopecten irradians is one of the most important commercial species of Pectinidae family in China. The internal defense system of mollusks consists of circulating haemocytes. In order to characterize the haemocytes of the scallop A. irradians, light and electron microscopical studies were carried out. Four types of haemocytes were recognized: type I small hyalinocytes (2.38 ± 0.08 μm, 30-35%), type II large hyalinocytes (4.41 ± 0.33 μm, 15-20%), type III small granulocytes (4.15 ± 0.26 μm, 20-25%), and type IV large granulocytes (8.26 ± 0.52 μm, 25-30%). Granulocyte types showed smaller N/C ratios than hyalinocytes. The mean haemocyte concentration was about (3.75 ± 0.65) × 10⁷ cells ml^− 1 of haemolymph. Among haemocytes, 44.7% are granular and 55.3% are agranular. These gave a relatively systematic classification scheme for haemocytes of A. irradians. Three types of granules were identified: type I, with high electron-density; type II, with low electron-density; and type III, with a middle level of electron-density, based on TEM studies. Different haemocyte types were not separated with DDGC of Percoll in this study. Both granulocytes and hyalinocytes showed a phagocytic response to the two strains of bacteria, Escherichia coli and RLOs. The phagocytic ability of granulocyte was significantly higher (41-48%) than that of hyalinocyte (9.2-11.2%).     

Prey ingestion and live food selectivity of marble goby (Oxyeleotris marmorata) using rice field prawn (Macrobrachium lanchesteri) as prey

Marble goby (Oxyeleotris marmorata), a carnivorous fish native to freshwater in Asia-Pacific region, is a high-valued species in many Asian countries. The present study consisting of three experiments was conducted to determine the appropriate density, size and ingestion time of marble goby fingerlings on rice field prawn (Macrobrachium lanchesteri) as prey. Results showed that the ingestion rate of marble goby fingerlings (0.26-1.43 g size) on prawns (9-12 mm in length) had an asymptotic pattern peaked at a predator to prey ratio of 1:20. Marble goby fingerlings did catch prawns during both daytime (07:00-17:00 h) and nighttime (17:00-07:00 h), but the ingestion peaked during nighttime period. The hourly ingestion rate during nighttime was significantly higher than that during daytime for the smaller-size marble goby fingerlings of 0.26-0.87 g, while there was no significant difference in the hourly ingestion between nighttime and daytime for the larger-size marble goby fingerlings of 1.43 g. The daily ingestion of marble goby fingerlings on prawns (mg consumed prawns/goby/day) increased significantly with increasing average weights (g) of marble goby fingerlings (Y = 27.6 + 47.8X, n = 30, r² = 0.86, p < 0.05). Results also showed that the preferred prawn size of marble goby fingerlings of average weights of 0.26-4.18 g was small (9-12 mm) with positive electivity index of 0.19-0.33, and the shift from small- to medium-size (12-14 mm) prawns was slow. The present study has demonstrated the feasibility of using rice field prawns as live foods to nurse marble goby fingerlings.     

The effects of supplemental dietary cholesterol on growth, development and survival of mud crab, Scylla serrata, megalopa fed semi-purified diets

The effects of varying levels of dietary cholesterol on growth, development time and survival of mud crab, Scylla serrata megalopa were investigated using semi-purified microbound diets (MBD). Five iso-energetic diets containing different level of cholesterol ranging from 0.14% to 1% of dry weight of the diet were tested. Fifteen megalopa were reared individually for each dietary treatment, and development time and survival were recorded on a daily basis. More than 25% of megalopa from all treatments were able to metamorphose into the first crab stage, suggesting that the endogenous level of cholesterol in the basal diet (0.14%) was sufficient to support development of the megalopa stage of this species. Widest mean carapace width (3.53 ± 0.08 mm) and highest mean dry weight (2.11 ± 0.22 mg) were recorded for juveniles that molted from megalopa fed live Artemia, whereas no megalopa in the unfed control treatment metamorphosed into crabs. The average development time from megalopa to the juvenile crab stage varied between the treatments, where megalopa fed live Artemia or MBD containing 0.2%, 0.4% or 0.8% total cholesterol showed the most synchronized molting (between 8.0 and 9.9 days). Longest development time was recorded for the megalopa fed diets containing 0.14% or 1% total cholesterol (both 11 days). Highest survival (74.3%) was recorded for the megalopa fed a diet containing 0.8% cholesterol. The results of this study are valuable in research to develop formulated diets for mud crab larvae as a replacement for live food in hatchery culture.     

Influence of dietary soybean meal levels on growth, feed utilization and gut histology of Egyptian sole (Solea aegyptiaca) juveniles

The present study was undertaken in order to determine the effect of a dietary incorporation of soybean meal (SBM) on growth performance, nutrient utilization and gut histology of Egyptian sole Solea aegyptiaca. This species, which is genetically and morphologically close to Solea solea, is currently present in the Mediterranean Sea and reared in some Italian farms, using the same techniques for S. solea production and it could represent a promising new species for Mediterranean aquaculture.Three isoproteic and isolipidic extruded diets (crude protein, 47%, crude fat, 20%) were formulated containing 0 (diet 1), 18 (diet 2) and 30% SBM (diet 3). 540 juveniles (initial body weight of 6.9 ± 0.2 g) were equally distributed into nine 150 l squared tanks (bottom surface: 520 cm²) connected with a recirculation system (temperature 20 ± 1 °C and dissolved oxygen above 6 ppm). Animals have been hand-fed twice a day (at 9.00 a.m. and 5.00 p.m.) at a daily ratio of 1.5% body weight^− 1. Diets were tested in triplicate for 87 days. The fish were weighed at days 28, 57 and 87, and they were sampled at days 0, 57 and 87 for nutrient retention determination. One-way ANOVA with Newman-Keuls' post-test were used to analyze data (P ≤ 0.05).No differences in terms of palatability were observed and any diet has been consumed completely all throughout the experiment. At day 87, fish fed diet 1, 2 and 3 reached weights of 25.8 ± 0.8 g, 27.5 ± 0.9 g and 26.1 ± 1.5 g, respectively and no differences in performances or nutrient retention were found.Histopathological gut examinations have revealed no noticeable differences in the appearances of the intestines between any of diet groups. Intestinal mucosal cells presented well vacuolated upper, with well defined microvillar brush borders. On the basis of the results of this trial, SBM seems to be a good protein source for Egyptian sole and can be added in the diet up to 30% without any reduction in growth rate and no adverse effect on gut histology.     

Distribution of a fish pathogen Listonella anguillarum in the Japanese flounder Paralichthys olivaceus hatchery

The present study was undertaken to investigate the distribution of Listonella anguillarum in a Japanese flounder (Paralichthys olivaceus) hatchery. A total of 2704 isolates were obtained from the developing fish, live diets and artificial feeds of Japanese flounder and their rearing water, 439 of which were identified as L. anguillarum by the combining incubation on thiosulfate-citrate-bile salt-sucrose (TCBS) agar at 35 °C overnight with polymerase chain reaction (PCR) detection for the VAH1 hemolysin gene. L. anguillarum was detected in all seven rotifer samples, with densities of 2.5 × 10³ to 4.6 × 10⁶ colony forming units (CFU) g^− 1. Both the analyzed samples of Nannochloropsis oculata contained this bacterium at densities of 1.6 × 10⁴ to 1.4 × 10⁵ CFU g^− 1. L. anguillarum was detected in only one of four samples of Artemia nauplii with a density of 4.8 × 10⁵ CFU g^− 1 (35%) and it was not detected in the two analyzed artificial feed samples. L. anguillarum was detected in 11 of 18 specimens of larval and juvenile Japanese flounder at densities of 5.0 × 10¹ to 7.4 × 10⁵ CFU g^− 1, while it was not detected in the two analyzed egg specimens of Japanese flounder. These results indicate that L. anguillarum associated with the developing Japanese flounder is likely derived from rearing water and live diets such as rotifers. Further, it is strongly suggested that L. anguillarum is a transient bacterium of the intestinal microflora for the Japanese flounder but is a permanently indigenous one for the Japanese flounder hatcheries.     

Oxytetracycline (OTC) accumulation and elimination in hemolymph, muscle and hepatopancreas of white shrimp Litopenaeus vannamei following an OTC-feed therapeutic treatment

The white shrimp, Litopenaeus vannamei, has become a very important species for the development of shrimp aquaculture in Northwest Mexico. However, viral and bacterial diseases are considered a major threat to the development of this industry. In the present study a trial was conducted to evaluate the tissue distribution, maximum concentration, and elimination of the widely used antibiotic oxytetracycline (OTC) in L. vannamei using indoor tanks under laboratory-controlled conditions. OTC was given to shrimp simulating a therapeutic treatment through medicated feed for 14 days followed by a period of feeding without antibiotic for another 14 days to evaluate the elimination pattern. Samples of hemolymph, muscle, and hepatopancreas were taken from medicated animals every two days for 28 days. All tissues were removed and frozen immediately in liquid nitrogen. OTC levels were analyzed by High Performance Liquid Chromatography (HPLC). Results showed an important OTC increase during consumption of medicated feed in all examined tissues. OTC maximum concentrations were 33.54 ± 11.19, 194.37 ± 16.11, and 18.79 ± 5.87 µg g^− 1 for muscle, hepatopancreas and hemolymph, respectively. Although the highest OTC level was found in the hepatopancreas, it required only two days after the start of dosing to reach this value, whereas the maximum OTC for muscle and hemolymph was detected after eight days of dosing. Ten days after the cessation of medicated feeding, the drug content in the shrimp tail muscle was under the detectable limit for the method (0.01 µg g^− 1 of OTC).     

The effects of fish hydrolysate (CPSP) level on Octopus maya (Voss and Solis) diet: Digestive enzyme activity, blood metabolites, and energy balance

As has been demonstrated in previous studies, Octopus maya can be fed on artificial diets. In the present study six different diets were assayed. Five diets were designed to test the effect of percentage of inclusion of fish protein concentrate (CPSP: 0, 5, 10, 15, and 20%) and were offered to octopuses as a specifically designed artificial diet. The sixth diet consisted of frozen crab (Callinectes spp) and was used as control diet. Blood metabolites and energy budget of octopuses were evaluated to determine how CPSP levels modulate the digestive capacity and allow retaining energy for growth. Wild animals (316.4 ± 9.8 g) were used in the study. Results showed that CPSP produced a positive specific growth rate (SGR, % day^− 1) with high value in octopuses fed 15% CPSP level. A maximum growth rate of 0.86% day^− 1 was recorded in these animals, a value that is extremely low when compared with the SGR obtained when animals were fed fresh crab (3.7% day^− 1). In general, blood metabolites were affected by diet composition, indicating that some metabolites could reflect the nutritional and/or physiological status of octopus. Preliminary reference values for O. maya fed crab were found for glucose (0.09 ±0.02 mg/ml), lactate (0.004 ± 0.002 mg/ml), cholesterol (0.16 ± 0.02 mg/ml), acylglycerol (0.14 ± 0.01 mg/ml), protein (0.37 ± 0.04 mg/ml), hemocyanin (1.85 ± 0.04 mmol/l), and digestive gland glycogen (1.86 ± 0.3 mg/g). Total energy content can be used as an indicator of tissue metabolic reserves. In the present study, higher energy content in the digestive gland and muscle was observed in octopuses fed crab, followed by animals fed 15% CPSP. Results from the digestive gland indicated that the retained energy derived from glycogen, suggesting that lipids and protein were the main sources of variation linked with energy content. In general, digestive gland proteases activity and trypsin were induced in octopuses fed 15% CPSP. The capacity of O. maya juveniles to adjust their digestive enzymes to different types of food was evidenced. Essential amino acid content (EAA) of the diet was not a limiting factor. When dietary EAA profiles were compared with O. maya EAA profiles, all dietary EAA resulted in a higher concentration than whole body octopus composition. In the present study, all experimental groups ingested between 3300 and 4106 kJ wk^− 1 kg^− 1 without statistical differences among treatments, indicating that experimental diets were as attractive as crab. Differences were recorded in the proportion of absorbed energy (Ab, %) between CPSP-based and crab meat diets, suggesting digestion limitations associated with artificial diets. The present results indicate that the 15% CPSP diet had characteristics that stimulate digestive enzymes and reduce energetic costs associated with its digestion (HiE or SDA), channeling more biomass production than the other experimental diets.     

Productivity improvement of Lysmata seticaudata (Risso, 1816) larval rearing protocol through modelling

The Monaco shrimp Lysmata seticaudata (Risso, 1816) is a marine ornamental species whose ecology and biology, as well as its larval culture has previously been addressed. The objective of the study was to predict and improve productivity of this species rearing protocol through modelling. The models developed intend to help aquaculturists to maximize survival to postlarva, decrease larval duration and increase synchronism of metamorphosis and newly metamorphosed postlarvae size by manipulating temperature, diet, first feeding period and stocking density.The models developed allow us to conclude that the L. seticaudata rearing protocol productivity can be improved by raising larvae at a density of 40 larvae L^− 1 and fed newly hatched Artemia nauplii since hatching to zoea V, and with Algamac 2000™ enriched Artemia metanauplii from zoea V to metamorphosis to postlarvae.By providing more productive protocols to aquaculturists, destructive practices and wild collection may be reduced.     

Effects of dietary vitamin C on survival, growth, and immunity of large yellow croaker, Pseudosciaena crocea

An 8 weeks growth experiment was conducted to determine the effects of dietary vitamin C on the survival, growth, tissue ascorbic acid contents and immunity of large yellow croaker (Pseudosciaena crocea) with initial weight of 17.82 ± 0.68 g. Seven practical diets were formulated to contain 0.1, 12.2, 23.8, 47.6, 89.7, 188.5 and 489.0 mg ascorbic acid equivalent kg^− 1 diet, supplied as l-ascorbyl-2-polyphosphate (LAPP). Each diet was fed to triplicate groups of fish in seawater floating cages (1.5 × 1.5 × 2.0 m), and each cage was stocked with 120 fish. Fish were fed twice daily (05:00 and 17:00) to apparent satiation for 8 weeks. The water temperature fluctuated from 19.5 to 25.5 °C, the salinity from 25 to 28‰ and dissolved oxygen content was approximately 7 mg l^− 1 during the experimental period. Results showed that the specific growth rate (SGR) (from 1.80 to 1.96% d^− 1) had an increasing trend with the increase of dietary vitamin C, but no significant difference was observed among dietary treatments. No gross deficiency signs were observed in any of the experimental fish. Survival rate, however, significantly increased with increasing dietary vitamin C (P < 0.05). The vitamin C contents in liver and muscle correlated positively with the vitamin C in diets. The vitamin C requirement was estimated to be 28.2 mg kg^− 1 based on survival, and 87.0 mg kg^− 1 on liver content of vitamin C. The activities of serum lysozyme and alternative complement pathway (ACP), phagocytosis percentage (PP) and respiratory burst activity of head kidney significantly increased with increasing dietary vitamin C. The challenge experiment with Vibrio harveyi showed that fish fed the diets with supplementation of vitamin C had significantly lower cumulative mortality compared to the control group (66.7%), and the cumulative mortality (16.7%) in fish with 489.0 mg kg^− 1 ascorbic acid was significantly lower than that (41.7%) in fish with 23.8 mg kg^− 1 ascorbic acid. These results suggested that vitamin C significantly influenced the immune response and disease resistance of large yellow croaker.     

Activity of digestive enzymes along the gut of juvenile red abalone, Haliotis rufescens, fed natural and balanced diets

Two carbohydrases (cellulase, lysozyme), three proteases (trypsin, aminopeptidase and non-specific protease), a non-specific lipase, and semiquantitative tests of 19 digestive enzymes were assayed in different gut sections of juvenile red abalone, Haliotis rufescens, in order to identify the regions where digestion takes place and investigate the extent to which diet composition can modify the digestive capacity of abalone. The abalone were fed either fresh kelp (K) or balanced diets containing 25 or 38% crude protein for 6 months. Enzyme assays were carried out on different sections of the abalone's gut at the end of this period. On a weight-specific basis, the digestive gland was the site containing most of the enzymes. On a protein-specific basis, two main digestion regions were identified: the digestive gland-stomach region that is characterized by high activities of cellulase and lysozyme, chymotrypsin and protease, and the mouth-intestine region with a typically high activity of lipase and amino peptidase. Significant dietary effects were observed on the activity of enzymes, especially in the digestive gland. Abalone fed with 25 and 38% crude protein diets exhibited higher cellulase (39.8 ± 4.6 and 14.2 ± 0.8 mU mg^− 1 protein, respectively) and lysozyme activities (88.0 ± 20.4 and 56.6 ± 15.7 U, respectively) than those fed with fresh kelp (5.5 ± 0.7 mU mg^− 1 protein and 17.1 ± 1.8 U). In contrast, higher protease activity was found in kelp-fed organisms (234.1 ± 20.4 μg product/mg protein) than those fed the 25 and 38% crude protein diets (109.5 ± 20.7 and 119.5 ± 20.5 μg product/mg protein, respectively). Semiquantitative API ZYM assays resulted in no clear food-specific effects on the activity of carbohydrases, proteases, ester hydrolases or phosphohydrolases, yet organ-specific differences were conspicuous in various cases, and generally agreed with quantitative results. It is suggested that the increased carbohydrase activity exhibited by organisms fed the balanced diets resulted from a combination of an increased number of resident bacteria in the abalone's gut and facilitated contact between dietary substrates and digestive cells. The present results indicate that H. rufescens can adjust their enzyme levels in order to maximize the acquisition of dietary protein and carbohydrates. This characteristic can be advantageously used to search for suitable diets in abalone aquaculture.     

The effects of algal diets on population growth and egg hatching success of the tropical calanoid copepod, Acartia sinjiensis

As natural diets of fish larvae, a number of calanoid copepod species are being investigated for use as live prey in aquaculture hatcheries. One of these, the tropical calanoid copepod, Acartia sinjiensis, has good potential as a live feed for tropical reef fish larvae. However, the rearing techniques for A. sinjiensis require further development to improve productivity. This study was carried out to investigate the population growth and egg hatching success of A. sinjiensis when fed a range of mono-and binary algal diets, including algae in the form of frozen paste.For the population growth experiment, the final A. sinjiensis population, including eggs, nauplii, copepodites and adults, was determined after feeding eight algal diets (two frozen algae, four live monoalgal and two live binary algal diets) for 8 days at temperature 28 ± 1 °C; salinity 34 ± 1 psu and photoperiod 12 L:12 D. Five replicates, with an initial 12 adult A. sinjiensis per replicate, were set up for each treatment. In a separate experiment, effects of diets on egg hatching success were examined after 48 h incubation of eggs produced by A. sinjiensis fed the same eight diets.The results showed that diet significantly affected both population growth and hatching success of A. sinjiensis. Of the diets tested, the binary algal diets were more successful than monoalgal diets, while the frozen algae had little dietary value. The highest population growth was recorded on A. sinjiensis fed a binary diet of Tetraselmis chuii and the Tahitian strain of Isochrysis sp. (T-ISO) (final population: 1091 ± 80), which was significantly higher (P < 0.001) than all other diets tested except for the other binary diet of Nannochloropsis sp. and T-ISO (final population: 897 ± 123). Diet also had a significant effect (P < 0.001) on egg hatching rate, though the highest hatch rate was recorded with eggs produced by A. sinjiensis fed binary diet Nannochloropsis sp. and T-ISO (88.1 ± 2.1%), this was not significantly different from that of eggs produced by A. sinjiensis fed either T-ISO alone (88.0 ± 1.7%) or the binary algal diet of T. chuii and T-ISO (76.4 ± 7.1%). The results of this study suggest that among the diets tested, a combination of live T. chuii and T-ISO was the best for the culture of A. sinjiensis.     

Ammonia toxicity and its effect on the growth of the South African abalone Haliotis midae Linnaeus

The current study investigated acute toxicity to ammonia of the South African abalone, Haliotis midae, from three size classes relevant to mariculture operations, and the chronic impact of sub-lethal ammonia levels on growth of juvenile abalone.Results showed that tolerance to ammonia (at pH 7.8 and T_a = 15 °C) increases with body size (i.e. age) as indicated by 36 h LC₅₀ values: juvenile abalone (1-2.5 cm shell length) had the lowest LC₅₀ of 9.8 μg l^− 1 FAN, whereas LC₅₀ was 12.9 μg l^− 1 FAN in “cocktail”-size abalone (5-8 cm shell length). The highest LC₅₀ of 16.4 μg l^− 1 FAN was observed in “brood stock”-size animals (10-15 cm). When “cocktail”-size abalone were allowed to acclimatize to sub-lethal ammonia levels for 48 h, their ammonia tolerance increased compared with non-acclimatized abalone of the same size: LC₅₀ was 2.0 μg l^− 1 FAN higher at 14.8 μg l^− 1 FAN.Growth of juvenile abalone (1-2.5 cm shell length) during chronic exposure to sub-lethal FAN levels is inhibited: specific growth rate (SGR) is significantly reduced by 58.7% to 0.10 ± 0.03% d^− 1 (weight) compared with 0.24 ± 0.06% d^− 1 of abalone of a control group (no ammonia).The results demonstrate the negative effects of ammonia not only on survival but also on growth of farmed abalone, both impair profitability of the farming operation. The information from the present study will be helpful in determining water quality requirements in South African abalone farms.     

The role of fatty acids enrichments in the larviculture of false percula clownfish Amphiprion ocellaris

At present, clownfishes are the best example of successfully captive bred ornamental specimens but little is known about the relationship between food enrichment and both larval growth and development. In fact, it is well known that a certain percentage of these fishes cultured in captive conditions show a miss-band pigmentation. In the present study, the effects of live prey enrichment on growth and pigmentation in false percula clownfish (Amphiprion ocellaris) larvae were tested.Newly hatched A. ocellaris larvae were divided in three different groups and fed as follows: group A fed on enriched (Algamac 2000) B. plicatilis (10 ind./mL) from day 1 to day 5 post-hatch (ph); group B fed on enriched (Algamac 2000) B. plicatilis (10 ind./mL) followed by Artemia nauplii (5 ind./mL;) and group C fed on Algamac 2000 enriched B. plicatilis and Algamac 2000 enriched Artemia nauplii.Samples of the larvae were collected on day 5 from group A and on day 11 ph in group B and C for morphometric and molecular analysis. On day 11 ph food enrichment resulted in a better growth of group C larvae respect to those of group B fed on not enriched Artemia nauplii (15.8 ± 0.2 mg and 8.78 ± 0.02 mm vs. 6.8 ± 0.2 mg and 6.93 ± 0.01 mm). Moreover, 36 ± 2% of the juveniles obtained from group B showed a miss-band pigmentation as compared to 29 ± 1% of the juveniles obtained from group C. At molecular level, the results obtained by Real-Time PCR are in agreement with the morphometric ones: a positive induction of the Insulin-like Growth Factor II (IGFII) and Peroxisome Proliferator Activated Receptor α and β (PPARα and PPARβ) gene expression and a reduction of Myostatin (MSTN) was observed in group C larvae fed on enriched live prey. IGFI gene expression was higher in group B.The present study provides clear evidences of the positive role of Algamac 2000 on growth and pigmentation of captive cultured false percula clownfish.     

In situ studies on the bioaccumulation of microcystins in the phytoplanktivorous silver carp (Hypophthalmichthys molitrix) stocked in Lake Taihu with dense toxic Microcystis blooms

The phytoplanktivorous silver carp is an important biomanipulation fish to control cyanobacterial blooms and is also a food fish with the greatest production in China. The accumulation of the hepatotoxic microcystins (MCs) determined by LC-MS in various organs of silver carp was studied monthly in Lake Taihu dominated by toxic Microcystis aeruginosa. Average recoveries of spiked fish samples were 78% for MC-RR and 81% for MC-LR. The highest content of MCs was found in the intestine (97.48 μg g^− 1 DW), followed by liver (6.84 μg g^− 1 DW), kidney (4.88 μg g^− 1 DW) and blood (1.54 μg g^− 1 DW), and the annual mean MC content was in the order of intestine > liver > kidney > blood > muscle > spleen > gallbladder > gill. Silver carp could effectively ingest toxic Microcystis cells (up to 84.4% of total phytoplankton in gut contents), but showed fast growth (from 141 g to 1759 g in 1 year in mean weight). Silver carp accumulated less microcystins in liver than other animals in the same site or other fish from different water bodies at similar level of toxin ingestion. There was possible inhibition of the transportation of the most toxic MC-LR across the gutwall. Muscle of silver carp in Lake Taihu should not be consumed during period of dense Microcystis blooms while viscera were risky for consumption in more months.