Enhancement of growth performance, digestive enzyme activities and disease resistance in black tiger shrimp (Penaeus monodon) postlarvae by potential probiotics

Outbreaks of multidrug-resistant pathogens have been constraint on aquaculture production in Thailand, thereby controlling shrimp pathogens by preventive probiotics being importance to sustain the aquaculture system. In this study, the effect of potential probiotics, Bacillus subtilis and Enterococcus sp. was related to growth, digestive enzyme activities (trypsin and chymotrypsin) and pathogenic resistance by postlarval black tiger shrimp (Penaeus monodon). The experiment was divided into two treatments, in triplicate, with and without supplementation of probiotics as a food additive. Shrimp fed with probiotics for a culture period of 84 days showed a significant increase (P < 0.05) in weight (2.03 ± 0.29 g) and survival (71.91 ± 3.15 %) in comparison with non-treated shrimp (1.53 ± 0.28 g and 65.20 ± 5.68 %, respectively). Trypsin activity of treated shrimp was significantly higher (P < 0.05) than that of control shrimp whereas chymotrypsin activities of the two treatments were not significantly different (P > 0.05). After challenging P. monodon postlarvae with a shrimp pathogen, Vibrio harveyi for 10 days, percentage of mortality of P. monodon postlarvae fed with probiotics was 46.67 ± 1.44 %, significantly lower (P < 0.05) than that of non-treated shrimp (61.67 ± 6.29 %). This study showed that potential probiotics was appropriate for application in P. monodon postlarvae cultivation under laboratory condition due to improvement of shrimp weight and survival, enhancement of trypsin activity and reduced mortality causing by pathogenic V. harveyi. This is the first publication reported the effect of probiotics on trypsin and chymotrypsin activities of P. monodon postlarvae.     

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 salinity and pH on ion-transport enzyme activities, survival and growth of Litopenaeus vannamei postlarvae

Effects of the salinity and pH on ion-transport enzyme activities, survival and growth of Litopenaeus vannamei postlarvae were investigated. Shrimp were transferred from salinity 31‰ and pH 8.1 to different salinity levels of 22, 25, 28, 31 (control), and to different pH levels of 7.1, 7.6, 8.1 (control), 8.6 and 9.1. The results showed ion-transport enzyme activities and weight gains of postlarvae were significantly affected by salinity and pH variation, which had no obvious effect on survival rate. The changing salinity affected the activities of total ATPase and Na⁺-K⁺-ATPase notably (F > F_0.05), meanwhile, non-significantly to the activities of V-ATPase, HCO₃⁻-ATPase. Within 48 h of salinity changing, the activities of ATPase, Na⁺-K⁺-ATPase in each treatment group gradually increased with the sampling time and reached their climax at 48 h, and then stabilized, showing negative correlation with salinity. The changing of pH affected greatly the activities of ATPase, Na⁺-K⁺-ATPase, V-ATPase and HCO₃⁻-ATPase (F > F_0.05), the activities of ATPase, Na⁺-K⁺-ATPase in each treatment group (pH = 7.1, 7.6, 8.6, 9.1) showed peak change within 72 h and stabilized afterwards, and the Na⁺-K⁺-ATPase activities came back to the level of the control group; Meanwhile the changing extent of V-ATPase and HCO₃⁻-ATPase activity corresponded with the grads of pH, and these ATPase activities showed negative correlation with pH changing, the activities of V-ATPase, HCO₃⁻-ATPase in postlarvae of each treatment group came to stable level after 24 h. The experiment also indicated the strength order of these ion-transport enzyme activities were as follows: Na⁺-K⁺-ATPase > V-ATPase > HCO₃⁻-ATPase. Na⁺-K⁺-ATPase was the chief undertaker of osmoregulation under the salinity effects, while V-ATPase and HCO₃⁻-ATPase were the chief osmoregulation undertakers under pH changing. In different salinity environment, the contributions of Na⁺-K⁺-ATPase, V-ATPase and HCO₃⁻-ATPase of L. vannamei postlarvae approximately accounted for 62.0-78.0%, 15.9-29.0% and 2.03-4.12% of ATPase activities in total, respectively. Meanwhile, in different pH medium, the contributions of these ATPases approximately accounted for 50.7-67.4%, 21.3-31.8% and 2.15-7.90% of total ATPase activities, respectively. Weight gain of shrimp transferred to salinity 31 (control) and 28‰ was significantly higher than that of shrimp reared at 25 and 22‰, and weight gain of shrimp transferred to pH 8.1 (control) and 8.6 was significantly higher that that of shrimp transferred to pH 7.1, 7.6 and 9.1. It was suggested that during the process of desalting and culturing of postlarvae, the salinity changing should not exceed 3 and pH variety not more than 0.5.     

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.     

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.     

Delivery of HUFA, probionts and biomedicine through bioencapsulated Artemia as a means to enhance the growth and survival and reduce the pathogenesity in shrimp Penaeus monodon postlarvae

One of the major problems in the shrimp culture industry is the difficulty in producing high-quality shrimp larvae. In larviculture, quality feeds containing a high content of highly unsaturated fatty acids (HUFA) and ingredients that stimulate stress and disease resistance are essential to produce healthy shrimp larvae. In the present study, Penaeus monodon postlarvae (PL15) were fed for 25 days on an unenriched Artemia diet (control; A) or on a diet of Artemia enriched with either HUFA-rich liver oil of the trash fish Odonus niger (B), probionts [Lactobacillus acidophilus (C1) or yeast-Saccharomyces cerevisiae (C2)] or biomedicinal herbal products (D) that have anti-stress, growth-promoting and anti-microbial characteristics. P. monodon postlarvae fed unenriched Artemia exhibited the lowest weight gain (227.9 ± 8.30 mg) and specific growth rate (9.95 ± 0.05%), while those fed the HUFA-enriched Artemia (B) exhibited the highest weight gain and specific growth rate (362.34 ± 12.56 mg and 11.77 ± 0.08%, respectively). At the end of the 25-day rearing experiment, the shrimp postlarvae (PL40) were subjected to a salinity stress study. At both low and high (0 and 50‰) salinities, the group fed the control diet (A) experienced the highest cumulative mortality indices (CMI) 935.7 ± 2.1 and 1270.7 ± 3.1, respectively. Those fed diet D showed the lowest stress-induced mortality, and CMI were reduced by 31.1 and 32.3% under conditions of low and high salinity stress, respectively. A 10-day disease challenge test was conducted with the P. monodon postlarvae (PL40–PL50) by inoculating the shrimp with the pathogen Vibrio harveyi at the rate of 10⁵–10⁷ CFU/ml in all rearing tanks. P. monodon postlarvae fed probiont-encapsulated Artemia diets (C1 and C2) exhibited the highest survival (94.3 and 82.3%, respectively) and lowest pathogen load (V. harveyi) in hepatopancreas (5.2 × 10² ± 9.0 × 10 and 4.6 × 10² ± 9.0 × 10 CFU g⁻¹, respectively) and muscle (2.0 × 10² ± 6 × 10 and 1.7 × 10² ± 8.6 × 10 CFU g⁻¹, respectively) tissues. The shrimp that were fed the unenriched Artemia (Control; A) showed the lowest survival (26.33%) and highest bacterial load in the hepatopancreas (1.0 × 10⁵ ± 5 × 10³ CFU g⁻¹) and muscle (3.6 × 10⁴ ± 6 × 10² CFU g⁻¹). The shrimp fed the herbal product (D)-enriched Artemia also exhibited enhanced survival and reduced V. harveyi load in the tissues tested compared to the control diet (A) group. The results are discussed in terms of developing a quality larval feed to produce healthy shrimp larvae.     

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).     

Water quality dynamics and shrimp (Litopenaeus vannamei) production in intensive, mesohaline culture systems with two levels of biofloc management

A dense microbial community develops in the water column of intensive, minimal-exchange production systems and is responsible for nutrient cycling. A portion of the microbial community is associated with biofloc particles, and some control over the concentration of these particles has been shown to provide production benefits. To help refine the required degree of control, this study evaluated the effects of two levels of biofloc management on water quality and shrimp (Litopenaeus vannamei) production in commercial-scale culture systems. Eight, 50 m³ raceways were randomly assigned to one of two treatments: T-LS (treatment-low solids) and T-HS (treatment-high solids), each with four replicate raceways. Settling chambers adjacent to the T-LS raceways had a volume of 1700 L with a flow rate of 20 L min⁻¹. The T-HS raceways had 760 L settling chambers with a flow rate of 10 L min⁻¹. Raceways were stocked with 250 shrimp m⁻³, with a mean individual weight of 0.72 g, and shrimp were grown for thirteen weeks. Raceways in the T-LS treatment had significantly reduced total suspended solids, volatile suspended solids, and turbidity compared to the T-HS treatment (P ≤ 0.003). The T-LS raceways also had significantly lower nitrite and nitrate concentrations, and the T-HS raceways had significantly lower ammonia and phosphate concentrations (P ≤ 0.021). With the exception of nitrate, there were no significant differences between the change in concentration of water quality parameters entering and exiting the settling chambers in the T-LS versus the T-HS treatment. Nitrate never accumulated appreciably in the T-LS raceways, possibly due to denitrification in the settling chambers, bacterial substrate limitations in the raceways, or algal nitrate assimilation. However, in the T-HS raceways nitrate did accumulate. The T-HS settling chambers returned a significantly lower nitrate concentration and significantly greater alkalinity concentration than what entered them (P ≤ 0.005), indicating that denitrification may have occurred in those chambers. There were no significant differences in shrimp survival, feed conversion ratio, or final biomass between the two treatments. However, shrimp in the T-LS treatment grew at a significantly greater rate (1.7 g wk⁻¹ vs. 1.3 g wk⁻¹) and reached a significantly greater final weight (22.1 g vs. 17.8 g) than shrimp in the T-HS treatment (P ≤ 0.020). The results of this study demonstrate engineering and management decisions that can have important implications for both water quality and shrimp production in intensive, minimal-exchange culture systems.     

Improvement of growth performance,water quality and disease resistance against Vibrio harveyi of postlarval whiteleg shrimp (Litopenaeus vannamei) by administration of mixed microencapsulated Bacillus probiotics

The objective of this study was to investigate the effects of mixed Bacillus on growth, water quality and disease resistance against Vibrio harveyi in whiteleg shrimp (Litopenaeus vannamei). Postlarval shrimp (PL₃₀) were fed with (a) a basal diet (the control), (b) a diet containing mixed freeze‐dried Bacillus probiotics (FB) and (c) addition of mixed microencapsulated Bacillus probiotics (MB) in culture water. Addition of FB and MB probiotics improved (p < .05) growth, feed efficiency, survival and culture water quality (ammonia and nitrite) compared to the control group although there was no difference (p > .05) between the two treated groups. Bacillus numbers in gastrointestinal tracts and culture water of FB‐ and MB‐administrated shrimp were higher (p < .05) than in the control. After a 30‐day culture, shrimp were infected with V. harveyi and monitored for 10 days. A significant reduction (p < .05) in cumulative mortality was observed in FB‐ and MB‐supplemented shrimp (43.24% and 45.05%, respectively), compared to the control (63.06%). This finding demonstrated that administration of microencapsulated probiotics was as effective as freeze‐dried probiotics for improving growth, feed efficiency, survival, Bacillus in gastrointestinal tracts, water quality (ammonia and nitrite) and conferring disease resistance to V. harveyi.     

Intensive production of juvenile tiger shrimp Penaeus monodon: An evaluation of stocking density and artificial substrates

Tiger shrimp Penaeus monodon were intensively grown from PL₁₅ for 56 d in tank systems at stocking densities of 1000 and 2000 shrimp m^− 3, with and without the addition of artificial substrates (AquaMat® (buoyant and non-buoyant) and polyethylene mesh) at each density. Shrimp growth was significantly greater at the lower density and when substrates were added. Mean shrimp weight at harvest ranged from 0.64 ± 0.06 g (2000 shrimp m^− 3, no added substrate) to 1.17 ± 0.01 g (1000 shrimp m^− 3, added substrate). Survival was high and averaged 79.5 ± 2.7% across all treatments. The addition of substrates significantly increased survival at both stocking densities; however, survival was not significantly affected by stocking density. A maximum harvest density of 1645 shrimp m^− 3 and biomass of 1.27 kg m^− 3 were produced at a stocking density of 2000 m^− 3 with added substrates. Both harvest density and biomass significantly increased with stocking density and addition of substrates. The feed conversion ratio (FCR) of formulated feed was significantly lower when substrates were added. The results show that growth of P. monodon juveniles was inversely related to stocking density during intensive production. However, production output was significantly increased by addition of artificial substrates, which enhanced both growth and survival.     

Characterization by whole-cell hybridization of bacterial populations associated with shrimp hatchery biofilms

The impact of shrimp larvae development, as well as water and food inputs upon the increase of bacterial populations within the bacterial community of hatchery tank biofilms, was studied. For this study, a total of 68 biofilm samples were collected from concrete tanks at three larvae production times in a commercial shrimp hatchery. Seventeen samples were taken at each larval development stage (Zoea I, Mysis I, postlarvae 1 and postlarvae 16), as well as 37 samples from water, shrimp nauplii and food, introduced into the shrimp hatchery tanks. Culturable and direct bacterial counts were performed and 16S‐rRNA‐targeted oligonucleotide probes were used to quantify the presence of specific bacterial groups. An average of 27–70% of DAPI total cell counts were detected with the EUB338 probe, while the GAM42a probe signal ranged from 1% to 11%. Vibrio‐like bacteria (VLB) counts in TCBS agar ranged from <10 to 10¹ VLB/cm⁻², with a tendency to increase at the last postlarvae stage. The most significant external source of bacteria registered with GAM42a probe and TCBS agar were found in live Artemia nauplii, used as food; nevertheless, biofilms remain with low counts of these groups.     

A brackishwater isolate of Pseudomonas PS-102, a potential antagonistic bacterium against pathogenic vibrios in penaeid and non-penaeid rearing systems

A Pseudomonas sp PS-102 recovered from Muttukkadu brackish water lagoon, situated south of Chennai, showed significant activity against a number of shrimp pathogenic vibrios. Out of the 112 isolates of bacterial pathogens comprising Vibrio harveyi, V. vulnificus, V. parahaemolyticus, V. alginolyticus, V. fluvialis, and Aeromonas spp, 73% were inhibited in vitro by the cell-free culture supernatant of Pseudomonas sp PS-102 isolate. The organism produced yellowish fluorescent pigment on King's B medium, hydrolysed starch and protein, and produced 36.4% siderophore units by CAS assay and 32 μM of catechol siderophores as estimated by Arnow's assay. The PS-102 isolate showed wide ranging environmental tolerance with, temperatures from 25 to 40 °C, pH from 6 to 8, salinity from 0 to 36 ppt, while the antagonistic activity peaked in cultures grown at 30 °C, pH 8.0 and at 5 ppt saline conditions. The antagonistic activity of the culture supernatant was evident even at 30% v / v dilution against V. harveyi. The preliminary studies on the nature of the antibacterial action indicated that the antagonistic principle as heat stable and resistant to proteolytic, lipolytic and amylolytic enzymes. Pseudomonas sp PS 102 was found to be safe to shrimp when PL-9 stage were challenged at 10⁷ CFU ml^− 1 and by intramuscular injection into of ∼5 g sub-adults shrimp at 10⁵ to 10⁸ CFU. Further, its safety in a mammalian system, tested by its pathogenicity to mice, was also determined and its LD₅₀ to BALB/c mice was found to be 10⁹ CFU. The results of this study indicated that the organism Pseudomonas sp PS 102 could be employed as a potential probiont in shrimp and prawn aquaculture systems for management and control of bacterial infections.     

Effects of Vibrio harveyi on plasma clotting protein (coagulogen) of white shrimp Litopenaeus vannamei

Blood clotting exhibits various important functions, including the prevention of body fluid loss and invasion of pathogens in shrimp. The effects of pathogenic Vibrio harveyi on plasma of white shrimp (Litopenaeus vannamei) in vitro and in vivo were investigated in this study. The clotting protein (coagulogen) in plasma of white shrimp pre‐incubated with extracellular products (ECP) of V. harveyi was found apparently decreased and fast‐migrated in crossed immunoelectrophoresis (CIE) gels. In addition, the coagulogen had been degraded to many low molecular‐weight protein bands in plasma pre‐incubated with ECP on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gels. When pre‐challenged with bacterial cells and ECP of V. harveyi, the white shrimp began to die at about 30 and 16 h respectively. Moreover, plasma coagulogen was decreased more obvious in shrimp challenged with ECP than that with bacterial cells as visualized in CIE gels, and total plasma protein in both group of shrimp were all decreased. Haemolymph withdrawn from moribund shrimp pre‐challenged with V. harveyi or its ECP was observed unclottable. However, the addition of clotting factors (transglutaminase and/or Ca²⁺) to these unclottable plasma could apparently promote their re‐clotting ability as jelly‐like solid observed in microtubes. The recovery of clotting ability of plasma from moribund shrimp was due to the reformation of coagulogen (200 kDa) after adding the two clotting factors as shown on CIE and SDS‐PAGE gels. The present results suggest that the infection of V. harveyi in white shrimp may not only degrade coagulogen but also influence the presence of transglutaminase and Ca²⁺ ion.     

Serotonin stimulates ovarian maturation and spawning in the black tiger shrimp Penaeus monodon

Serotonin (5-hydroxytryptamine, 5HT) has been reported to induce ovarian maturation and spawning in the crayfish Procambarus clarkii and white Pacific shrimp Litopenaeus vannamei. The aim of this study was to explore the role of exogenous 5HT on the reproductive performance of the black tiger shrimp Penaeus monodon. 5HT solution was injected into domesticated P. monodon broodstock at 50 μg/g body weight and ovarian maturation and spawning were recorded. The presence of 5HT in the ovary and oviduct of P. monodon was also studied by immunohistochemistry and its levels in the ovary by enzyme link immunoabsorbance assay (ELISA). The 5HT-injected P. monodon developed ovarian maturation and spawning rate at the level comparable to that of unilateral eyestalk-ablated shrimp. Hatching rate and the amount of nauplii produced per spawner were also significantly higher in the 5HT-injected shrimp, compared to the eyestalk-ablated shrimp. 5HT-positive reactions were found in the follicular cells of pre-vitellogenic oocytes, in the cytoplasm of early vitellogenic oocytes and on the cell membrane and cytoplasm of late vitellogenic oocytes. 5HT in the ovary was present at 3.53 ± 0.26 ng/mg protein level in previtellogenic stage and increased to 17.03 ± 0.57 ng/mg protein level in the mature stage of the ovary. The results suggest a significant role of 5HT, possibly directly on the ovary and oviduct, on the reproductive function of female P. monodon.     

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.     

The tetrasporophyte of Asparagopsis armata as a novel seaweed biofilter

The red seaweed Asparagopsis armata (Harvey; Rhodophytae, Bonnemaisoniaceae) produces biologically active secondary metabolites that are valuable natural ingredients for cosmetics and medicine and its cultivation may therefore be a profitable venture. The tetrasporophyte of this species (“Falkenbergia rufolanosa”) was successfully tank-cultivated as a continuous biofilter for the effluent of a commercial fish farm in southern Portugal. Optimal stocking density for highest biomass yield and a low level of other algal species in winter and late spring was 5×g centrifuged fresh weight l^− 1. The effect of total ammonia nitrogen supply (TAN flux) on biofiltration and biomass yield was investigated in winter and spring. Results revealed that A. armata is currently the seaweed-biofilter with the highest TAN removal of up to 90 μmol l^− 1 h^− 1 at a TAN flux of about 500 μmol l^− 1 h^− 1. In the tanks used, this is equivalent to a removal of up to 14.5 g TAN m^− 2 day^− 1. At a lower TAN flux of about 40 μmol l^− 1 h^− 1, TAN removal by A. armata is more than double to what is reported at this flux for another successful seaweed biofilter, the genus Ulva. Monthly variation of A. armata biomass yield peaked in May and was lowest in January. At TAN fluxes between 300 and 400 μmol l^− 1 h^− 1, an average water temperature of 21.7 °C and a total daily photon flux density of 47 Mol m^− 2, seaweed yield was over 100 g DW m^− 2 day^− 1 with a recorded maximum of 119 g. During spring, autumn and early summer, the biomass of A. armata within the experimental tanks doubled every week. A model for the up scaling of this finfish integrated aquaculture of A. armata varies the investment in biofilter surface area and estimates the return in biofiltration and biomass yield. Highest TAN removal efficiencies will only be possible at low TAN fluxes and a very large biofilter area, resulting in a low production of biomass per unit area. To remove 50% of TAN from the effluent (1 mt Sparus aurata; 21 °C), 28 m² of biofilter, designed to support a water turnover rate of 0.8 Vol h^− 1 would be necessary. This system produces 6.1 kg FW (1.5 kg DW) of A. armata per day and has the potential to turn biofiltration into an economically sustained, beneficial side effect.     

Experimental integrated aquaculture of fish and red seaweeds in Northern Portugal

Three potentially valuable red seaweeds, Chondrus crispus Stackhouse, Gracilaria bursa pastoris (S.G. Gmelin) P.C. Silva and Palmaria palmata (L.) O. Kuntze, collected in northern Portugal, were cultivated using the nutrient-rich effluents from a local turbot (Scophthalmus maximus Linnaeus) and sea bass (Dicentrarchus labrax Linnaeus) farm. The algae were cultivated in a two level cascade system. Several arrangements of the cascade system, stocking densities (3, 5, 7 and 8 kg m^− 2) and water fluxes (140 and 325 l h^− 1) were tested to optimize biomass yield and nitrogen uptake rate and efficiency. The yield and the total ammonium nitrogen (TAN) uptake of the three species were highly seasonal. Palmaria could not survive culture conditions during the summer when water temperature was above 21 °C. In the spring, Palmaria had an average yield of 40.2 (± 12.80) g DW m^− 2 day^− 1 and a nitrogen uptake efficiency (NUE) of 41.0% (± 17.26%). NUE expresses, in percentage, the average reduction in TAN concentration between the inflows and the outflows of the tanks. Chondrus performed better in summer with an average yield of 37.0 (± 11.10) g DW m^− 2 day^− 1 and removing 41.3% (± 17.32%) of nitrogen. Gracilaria grew year round, but also performed better during spring/summer, producing an average of 29.1 (± 2.90) g DW m^− 2 day^− 1, and only 7.3 (± 5.08) g DW m^− 2 day^− 1 during autumn. Yield of C. crispus did not differ significantly when grown at two different stocking densities (5 kg m^− 2 and 8 kg m^− 2). On the other hand, Gracilaria had significantly higher yields at 5 than at 7 kg m^− 2. Better NUE, on average 76.7% (± 22.13%), was also obtained with 5 kg m^− 2 stocking density and only 63.8% (± 24.62%) with 7 kg m^− 2. The yield of Gracilaria increased significantly with the increase of water flux from 140 to 325 l h^− 1 and more nitrogen was removed from the water. However, NUE decreased from 48.4% to 33.4% at 140 and 325 l h^− 1, respectively. Biofiltration was highly improved by a cascade system with a NUE as high as 83.5%.     

A study on the influence of selected Indian herbal active principles on enhancing the immune system in Fenneropenaeus indicus against Vibrio harveyi infection

Five herbs such as Acalypha indica, Hygrophila spinosa, Picrorhiza kurooa, Tinospora cordifolia and Zingiber officinale were selected to screen for the in vitro immunostimulant activity against the shrimp pathogen Vibrio harveyi using different organic polar and non-polar solvents. After initial screening, the best extract of the five herbs was mixed thoroughly in equal proportion and made it an artificial immunostimulant diets (ISD) such as ISD-1(500), ISD-2 (1,000), ISD-3 (1,500) and ISD-4 (2,000) mg?kg^?1. The control diet was prepared without adding immunostimulant herbal extracts. The Indian white shrimp, Fenneropenaeus indicus having the weight of 8.0?±?1.0?g, were fed with the above diets for 60?days. After the completion of feeding trail, they were challenged with V. harveyi at the rate of 10⁷CFU/ml and found out the cumulative mortality, growth parameters, haematological and immunological parameters. The weight gain and specific growth rate were significantly (P?<?0.05) increased from the control group to experimental group. The immunostimulant active principles helped to decrease the coagulation time and improved the total haemocyte count (THC), phagocytosis, phenol oxidase (PO) activity, haemagglutinin activity and bacterial clearance.     

Identification of Vibrio harveyi,Vibrio ichthyoenteri,and Photobacterium damselae isolated from olive flounder Paralichthys olivaceus in Korea by multiplex PCR developed using the rpoB gene

Major fish bacterial diseases in Korea are edwardsiellosis, streptococcosis, and vibriosis. Among vibrionaceae, Listonella anguillarum, Vibrio harveyi, V. ichthyoenteri, and Photobacterium damselae were identified as causative organisms of vibriosis in flounder. In this study, we developed a multiplex PCR method using the RNA polymerase β subunit (rpoB) gene, known as a housekeeping gene for identification of Vibrio spp. causing vibriosis in flounder. Three pairs of PCR primers were designed based on the rpoB sequence of three species, V. harveyi, V. ichthyoenteri, and P. damselae. The PCR assay, using a mixture of six primers, yielded amplicons of 601, 434, and 533 bp in V. harveyi, V. ichthyoenteri, and P. damselae. None of the untargeted species yielded an amplicon. The detection limits for pure culture in kidney were 2.5 × 10⁴ cfu/g kidney for V. harveyi, 2.5 × 10⁵ cfu/g kidney for V. ichthyoenteri, and 2.5 × 10⁶ cfu/g kidney for P. damselae. From the colonies on TCBS agar plates of different samples, 632 Vibrio spp. isolated from aquacultured flounder between 2004 and 2010 were identified by the multiplex PCR method. As a result, 265 strains (41.9 %) were V. ichthyoenteri; 115 strains (18.2 %) were V. harveyi and 72 strains (11.4 %) were P. damselae.     

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.