Overall development of a bioprocess for the outdoor production of Nannochloropsis gaditana for aquaculture

In this paper, a systematic methodology is shown for the scaling‐up of Nannochloropsis gaditana production for aquaculture uses. First, an adequate culture medium was developed, prepared using fertilizers instead of pure chemicals. Subsequently, the performance of N. gaditana was modelled as a function of average irradiance; this model being validated in continuous culture experiments. The model was used to determine the optimal dilution rate as well as the expected biomass concentration and productivity at optimal conditions. Finally, outdoor experiments were performed to confirm the model's validity and to determine optimal conditions at real production step. Biomass productivity values of up to 0.08 g L^?1 day^?1 were obtained at an optimal dilution rate of 0.25 per day in 0.2 m wide flat‐panel reactors using culture medium containing 0.4 g/L NO^?₃ and 0.034 g/L·PO₄^?3. Fish trials with Atlantic Salmon demonstrated that the inclusion of produced biomass into fed increase the final weight up to 5%, thus confirming the adequacy of the biomass produced for aquaculture uses. The growth model and the scaling‐up strategy proposed here are necessary to develop real industrial‐scale processes capable of supplying microalgal biomass to the aquaculture markets, which in turn require a guaranteed supply and quality of the raw materials provided.     

Histological alterations in gills of shrimp Litopenaeus vannamei in low‐salinity waters under different stocking densities: Potential relationship with nitrogen compounds

Two experimental modules with different stocking densities (M1 = 70 and M2 = 120 shrimp /m²) were examined weekly over a culture cycle in tanks with low‐salinity water (1.9 g/L) and zero water exchange. Results showed survival rates of 87.7 and 11.9% in M1 and M2, respectively. Water temperature, pH, dissolved oxygen, electrical conductivity and chlorophyll a were not significantly (p > .05) different between modules. In contrast, the concentrations of nitrogen compounds were significantly (p < .05) different between modules, except nitrite‐N (M2 were 2.31 ± 1.38 mg/L N‐TAN, 0.18 ± 0.49 mg/L N‐NO₂^? and 6.83 ± 6.52 mg/L N‐NO₃^?; in M1: 0.97 ± 0.73 mg/L N‐TAN, 0.05 ± 0.21 mg/L N‐NO₂^? and 0.63 ± 0.70 mg/L N‐NO₃^?). When waters of both modules reached higher levels of ammonia and nitrite, histological alterations were observed in gills. The histological alterations index (HAI) was higher in M2 (5‐112) than in M1 (2‐22).     

Biomass production,proximate composition and fatty acid profile of the local marine thraustochytrid isolate,Schizochytrium sp. LEY7 using low‐cost substrates at optimum culture conditions

This study was conducted to investigate low‐cost substrate alternative and the optimum culture conditions in mass producing the local marine thraustochytrid, Schizochytrium sp. LEY7 isolated from the mangrove leaves of Baybay, Southern Leyte Philippines. Results showed that Schizochytrium sp. LEY7 is able to utilize commercial grade glucose and yeast extract from NaCl‐treated baker's yeast as source of carbon and micronutrients respectively. Cost of mass producing the thraustochytrid isolate using the alternative production substrates was substantially reduced. Incubation temperature and salinity levels were the two growth factors significantly affecting the biomass production of the isolate. The short duration of lag phase shown by the isolate suggests a growth advantage in that cells are readily able to adapt to their new environment. Total lipids averaged to 19.4%. Principal fatty acids were palmitic acid (C16:0) with 33.52% and docosahexanoic acid (DHA, C22:6n‐3) with 39.92% proportion to total fatty acid. Eicosapentaenoic (C20:5n‐3, 1.01%) and arachidonic (C20:4n‐3, 0.90%) are present but in relatively lower amount. Our findings showed the potential of low‐cost substrate in mass producing the local thraustochytrid isolate, Schizochytrium sp. LEY7 as lipid and polyunsaturated fatty acid source in aquaculture. Biomass production was enhanced by optimizing the culture conditions.     

Long‐term low‐salinity stress affects growth,survival and osmotic related gamma‐aminobutyric acid regulation in the swimming crab Portunus trituberculatus

A 30‐day experiment was conducted to investigate the effects of long‐term low‐salinity stress on the growth performance and osmotic related chlorine ion channel ligand regulation: gamma‐aminobutyric acid (GABA) content and GABA_A receptor‐associated protein (GABARAP) expression in Portunus trituberculatus. The salinity levels of both the control group and the experimental group were 30 and 12 psu respectively. After rearing for 30 days, the specific growth rate and survival rate were compared between the two groups, and salinity 6 psu was used to test the salinity tolerance. The results were as follows: (a) Both the specific growth rate and survival rate were significant lower in the experimental group (p < 0.05) after 30 days; (b) After challenge with salinity 6 psu for 72 hr, the crabs of experimental group had a 100% survival rate, whereas the crabs of the control group were all dead within 48 hr; (c) The content of GABA and the gene expression level of GABARAP in experimental group were significant different from control group (p < 0.05) after challenge via salinity 6 psu. In the control group, the GABA content increased rapidly from 9.96 ± 2.09 to 42.00 ± 5.94 µg/g; however, in the experimental group, it only increased to 27.82 ± 2.55 µg/g; the gene expression of GABARAP in the experimental group increased to the maximum at 24 hr, then decreased and stabilized at 48 hr, suggesting that GABA and GABARAP were trigged during the early stage of low‐salinity stress resistance.     

Influence of variation in water temperature on survival,growth and yield of Pacific white shrimp Litopenaeus vannamei in inland ponds for low‐salinity culture

There is considerable interest in the culture of whiteleg shrimp (Litopenaeus vannamei) in inland low‐salinity water in Alabama and other states in the Sunbelt region of the US. However, the growing season is truncated as compared with tropical or subtropical areas where this species is typically cultured, and temperature is thought to be a major factor influencing shrimp production in the US. This study, conducted at Greene Prairie Aquafarm located in west‐central Alabama, considered water temperature patterns on a shrimp farm in different ponds and different years; and sought possible effects of bottom water temperature in ponds on variation in shrimp survival, growth and production. Water temperature at 1.2 m depth in 22 ponds and air temperature were monitored at 1‐hr intervals during the 2012, 2013, 2014 and 2015 growing seasons. Records of stocking rates, survival rates and production were provided by the farm owner. Correlation analysis and linear mixed model analysis of variance were used. Results showed that hourly water temperatures differed among ponds. The range of water temperature in each pond explained 41% of the variance in average final weight of shrimp harvested from each pond. In conclusion, the results suggest that variation in water temperature patterns has considerable influence on shrimp growth and survival in ponds.     

Aeromonas salmonicida infection levels in pre‐ and post‐stocked cleaner fish assessed by culture and an amended qPCR assay

Due to increasing resistance to chemical therapeutants, the use of ‘cleaner fish’ (primarily wrasse, Labridae, species) has become popular in European salmon farming for biocontrol of the salmon louse, Lepeophtheirus salmonis (Krøyer). While being efficient de‐licers, cleaner fish mortality levels in salmon cages are commonly high, and systemic bacterial infections constitute a major problem. Atypical furunculosis, caused by Aeromonas salmonicida A‐layer types V and VI, is among the most common diagnoses reached in clinical investigations. A previously described real‐time PCR (qPCR), targeting the A. salmonicida A‐layer gene (vapA), was modified and validated for specific and sensitive detection of all presently recognized A‐layer types of this bacterium. Before stocking and during episodes of increased mortality in salmon cages, cleaner fish (primarily wild‐caught wrasse) were sampled and screened for A. salmonicida by qPCR and culture. Culture indicated that systemic bacterial infections are mainly contracted after salmon farm stocking, and qPCR revealed A. salmonicida prevalences of approximately 4% and 68% in pre‐ and post‐stocked cleaner fish, respectively. This underpins A. salmonicida's relevance as a contributing factor to cleaner fish mortality and emphasizes the need for implementation of preventive measures (e.g. vaccination) if current levels of cleaner fish use are to be continued or expanded.     

Effect of different dietary microalgae combinations on growth and survival of black‐lip pearl oyster (Pinctada margaritifera) larvae and the feasibility of replacing microalgae with a dietary lipid emulsion

A 10‐day experiment was performed to examine different mono, binary and ternary dietary combinations on survival and growth of D‐shaped and umbone black‐lip pearl oyster, Pinctada margaritifera, larvae. The three tropical microalgae species were the flagellate Isochrysis galbana clone T. Iso (CS‐177) and diatoms Chaetoceros calcitrans (CS‐178) and Chaetoceros muelleri (CS‐176) which were fed to D‐shaped and umbone larvae at a density of 7000 and 14 000 cells mL^?1, respectively. A second experiment was performed to investigate the feasibility of replacing T. Iso with a lipid emulsion for both D‐shaped and umbone larvae for 10 and 12 days, respectively. The treatments included only T. Iso, unfed and lipid emulsion to substitute T. Iso at levels of 10% (LIP10), 30% (LIP30) and 100% (LIP100). In the first experiment, results showed that a monospecific diet of T. Iso led to significantly higher (P < 0.05) survival and growth of D‐shaped larvae than all the other treatments. Meanwhile, D‐shaped larval survival was significantly lower when only fed C. calcitrans as well as growth for those fed C. calcitrans or in combination with C. muelleri. However, for umbone larvae, survival and growth were significantly higher when fed a binary combination of T. Iso and C. muelleri or the ternary combination of T. Iso, C. muelleri and C. calcitrans compared with all other treatments. For the second experiment, results showed that with increasing lipid emulsion replacement, survival of both D‐shaped and umbone larvae significantly decreased (P < 0.05); however, the LIP100 treatment was not significantly different (P > 0.05) from the unfed treatment. For D‐shaped larvae, no significant growth difference was detected (P > 0.05) between the T. Iso and LIP10 fed treatments while for umbone larvae, the T. Iso, LIP10 and LIP30 were not significantly different (P > 005). These results indicate that microalgae combinations appear more necessary for later staged P. margaritifera larvae. In addition, the use of a lipid emulsion appeared to provide some nutrition to the larvae, although more research should be conducted to improve the use of such replacements.     

A partial substitution of microalgae with single cell detritus produced from seaweed (Porphyra haitanensis) for the nursery culture of tropical oyster (Crassostrea belcheri)

Oyster seed production in hatchery and nursery systems has relied on the production of microalgae, which is cost effective, but often unpredictable. The development of a satisfactory substitute diet would greatly reduce production costs of hatchery operations. Single cell detritus (SCD) production from seaweed (Porphyra haitanensis) may have great potential as a partial substitute for microalgae. In this experiment, a technique for producing SCD was developed and the product was used as a partial substitute for microalgae for the nursery culture of juvenile tropical oysters (Crassostrea belcheri) in a hatchery setting. A reverse osmosis technique was used on ground seaweed (particle size <200 μm) immersed in freshwater and placed on a shaker at 100 rpm for 2 h before being returned to seawater. This technique was shown to be highly effective for SCD production with a density of 33.7 ± 7.0 × 10⁴ cell mL^?1 and the percentage of SCD particle diameter <20 μm was 89.2%. A partial replacement of microalgae with SCD was found to be unsuitable for nursing oyster larvae. For juvenile oysters (shell width 1.85 ± 0.03 mm and shell length 1.78 ± 0.06 mm) substituting 75% of microalgae with SCD showed lower absolute shell growth, and lower daily yields and survival rates when compared to rates substituting 50% or lower substitution with SCD, or 100% microalgae (P < 0.05). It was found that substituting 50% of the traditional microalgae with SCD produced from seaweed (P. haitanensis) can be used as a partial microalgae substitute for the nursery culture of the juvenile tropical oyster.     

Development of low‐lipid formulated feeds with different protein/energy ratios for Octopus vulgaris ongrowing

We studied the feeding and growth rates, feed efficiency and nutritional composition of subadult octopuses (689 ± 77 g) fed low‐lipid diets with two different protein/energy ratios (P/E): Medium P/E diet (30.5 g protein MJ^?1, 662 g kg^?1 protein dry weight (dw)) or High P/E diet (36.8 g protein MJ^?1, 843 g kg^?1 protein dw). Survival was 100% for the Medium P/E and 87.5% for the High P/E diet. The specific feeding rates were 1.91% and 1.67% body weight day^?1 for the Medium P/E and High P/E diet, respectively (P < 0.05). The specific growth rates (0.92% and 1.25% body weight day^?1), feed efficiency indices (47.3% and 72.7%), productive protein value (23.9% and 29.1%) and digestive gland index (5.6% and 6.4%) were significantly better in animals fed the High P/E than the Medium P/E diet (P < 0.05). The proximate composition of the digestive gland was similar for both diets, but the total lipid content of carcass was higher in animals fed the Medium P/E diet (P < 0.05). Our results suggest that the protein content of the feed could be a factor regulating feeding rates in octopus.     

Immunostimulatory effect of mangrove‐derived marine yeasts in Penaeus monodon

This study attempted to assess the efficacy of mangrove‐derived marine yeasts as a source of immuno‐stimulant in the tiger shrimp, Penaeus monodon. The shrimp was fed on the diets in supplementation with yeast biomass at 10% for a period of 15 days and then challenged with the pathogenic Vibrio cholerae. The animals were assessed for immune responses in terms of total haemocyte counts, phenol oxidase and endobiotics. Among the yeasts tested, Rhodotorula minuta was found to have high immunostimulatory effect in the shrimps especially after challenge with the pathogenic vibrio.     

Use of Nannochloropsis sp. isolated from the East China Sea in larval rearing of sea cucumber (Apostichopus japonicus)

The supply of microalgae to hatcheries is a limiting factor for the mass larval production of sea cucumber (Apostichopus japonicus) in Fujian Province, China. In this study, Nannochloropsis sp. isolated from the East China Sea was tested as food for A. japonicus larvae. The first trial compared the effect of mono‐, bi‐ and trialgal diets comprising three microalgae (Chaetoceros muelleri, Dunaliella tertiolecta and Nannochloropsis sp.) on A. japonicus larval growth, survival, settlement and juvenile growth. The results showed that there were no significant differences in survival and settlement between larvae fed with Nannochloropsis sp. and other diets. All diet treatments yielded similar juvenile sea cucumber output. In the second trial, A. japonicus larvae were fed equally four times daily at three different rations (5000, 20 000 and 40 000 cells mL^?1 day^?1). Larvae fed 20 000 cells mL^?1 day^?1 were significantly larger than larvae in other groups and experienced the highest survival rate. In the third trial, A. japonicus larvae were fed 20 000 cells mL^?1 day^?1 in three different frequency (2, 3 and 4 meals day^?1). The greatest body length was observed in larvae that received 3 meals day^?1. Survival and settlement of larvae fed 2 meals day^?1 were significantly lower than other two groups. These results suggest that Nannochloropsis sp. can be used as a diet for the large‐scale production of A. japonicus seed, and larvae fed three times daily at a ration of 20 000 cell mL^?1 day^?1 are recommended for hatchery production of A. japonicus.     

Acute toxicity of nitrite on white shrimp Litopenaeus vannamei (Boone) juveniles in low‐salinity water

The nitrite toxicity was estimated in juveniles of L. vannamei. The 24, 48, 72 and 96 h LC₅₀ of nitrite‐N on juveniles were 8.1, 7.9, 6.8 and 5.7 mg L^?1 at 0.6 g L^?1; 14.4, 9.6 8.3 and 7.0 mg L^?1 at 1.0 g L^?1; 19.4, 15.4, 13.4 and 12.4 mg L^?1 at 2.0 g L^?1 of salinity respectively. The tolerance of juveniles to nitrite decreased at 96 h of exposure by 18.6% and 54.0%, when salinity declined from 1.0 to 0.6 g L^?1 and from 2.0 to 0.6 g L^?1 respectively. The safe concentrations at salinities of 0.6, 1.0 and 2.0 g L^?1 were 0.28, 0.35 and 0.62 mg L^?1 nitrite‐N respectively. The relationship between LC₅₀ (mg L^?1), salinity (S) (g L^?1) and exposure time (T) (h) was LC₅₀ = 8.4688 + 5.6764S – 0.0762T for salinities from 0.6 to 2.0 g L^?1 and for exposure times from 24 to 96 h; the relationship between survival (%) and nitrite‐N concentration (C) for salinity of 0.6–2.0 g L^?1, nitrite‐N concentrations of 0–40 mg L^?1 and exposure times from 0 to 96 h was as follows: survival (%) = 0.8442 + 0.1909S – 0.0038T – 0.0277C + 0.0008ST + 0.0001CT–0.0029SC, and the tentative equation for predicting the 96‐h LC₅₀ to salinities from 0.6 to 35 g L^?1 in L. vannamei juveniles (3.9–4.4 g) was 96‐h LC₅₀ = 0.2127 S² + 1.558S + 5.9868. For nitrite toxicity, it is shown that a small change in salinity of waters from 2.0 to 0.6 g L^?1 is more critical for L. vannamei than when wider differences in salinity occur in brackish and marine waters (15–35 g L^?1).     

Quantitative dietary leucine requirement of juvenile Pacific white shrimp,Litopenaeus vannamei (Boone) reared in low‐salinity water

The experiment was conducted to determine the leucine requirement of juvenile Pacific white shrimp Litopenaeus vannamei (Boone) in low‐salinity water (0.50–1.20 g L^?1). Six diets were formulated to contain 410 g kg^?1 crude protein with fish meal, peanut meal and precoated crystalline amino acids with different concentration of l ‐leucine (16.72, 19.60, 22.06, 24.79, 27.28 and 30.16 g kg^?1 dry diet). Each diet was randomly assigned to triplicate groups of 30 shrimps (0.38 ± 0.002 g), and the feed trial lasted for 8 weeks. The results indicated that the maximum weight gain was observed at 24.95 g kg^?1 dietary leucine group, whereas the diets containing higher leucine concentration conversely reduced the growth performance (P < 0.05). Moreover, the highest body protein content and body protein deposition and the lowest haemolymph AST and ALT activities were also found at 24.95 g kg^?1 dietary leucine group. With the increase in leucine in diets, a dose‐dependent increase was found in body lipid content and haemolymph urea concentration. The polynomial regression calculated using weight gain, feed efficiency and body protein deposition indicated that the optimal dietary leucine requirement for L. vannamei reared in low‐salinity water was 23.73 g kg^?1 leucine of dry diet, correspondingly 57.88 g kg^?1 of dietary protein.     

Effect of using sodium bicarbonate to adjust the pH to different levels on water quality,the growth and the immune response of shrimp Litopenaeus vannamei reared in zero‐water exchange biofloc‐based culture tanks

The bioflocs technology proved to be a sustainable technique used in zero‐water exchange shrimp culture systems. However, the pH and alkalinity may decrease due to the biofloc formation process and Nitrification. A 48‐day experiment was performed to investigate the effects of different pH (7.1–7.6; 7.6–8.1) conditions on water quality, the growth and the health status of shrimp in biofloc technology (BFT) through using sodium bicarbonate to adjust pH respectively. Two pH treatments and one control were compared: T0 — control, T1 — pH 7.6 — NaHCO₃, T2 — pH 8.1 — NaHCO₃, each treatment consisted of three replicate tanks (90 L water volume) and each replicate stocked with 30 shrimp (equivalent to 333 shrimp m^?3). Significant physical, chemical and biological differences (P < 0.05) were detected among treatments. At the end of the experiment, water quality, the growth and the immune response of shrimp in control were significantly lower (P < 0.05) than the other treatments. Moreover, the T2 treatment had a better performance in these three aspects. The results indicated that it was necessary to adjust the pH and alkalinity in the BFT, and a higher pH as well as alkalinity for shrimp growth and the stability of the BFT were more favourable.     

Hatchery cultivation of the common cockle (Cerastoderma edule L.): from conditioning to grow‐out

This study describes for the first time the cultivation of Cerastoderma edule on a commercial scale. A protocol to grow F₂ generation cockles was developed, which led to fine‐tuning experiments for broodstock conditioning and spat growth. Broodstock animals were conditioned with diets of Isochrysis galbana (T‐Iso) or Tetraselmis suecica, whereas a third group was not fed. The best diet, T. suecica, induced 12 females out of 100 animals to spawn a total of 3 380 000 eggs. The non‐fed group did not spawn. Cockle spat (4.9 ± 1.0 mm) grew best when given a mixed diet of C. muelleri, T‐Iso and Sceletonema costatum, or a mixture of P. tricornutum and S. costatum at a concentration of 240 cells μl^?1 day^?1, resulting in a tripling of their wet weight after 14 days. The impact of density, burrowing substrate and food availability on cockle spat growth (41 days old, 5.6 ± 1.2 mm) was studied for 11 weeks. Best results were obtained by culturing spat at ad libitum food conditions at 500 ind m^?2, resulting in an average growth rate of 168 μm day^?1, an average final size of 19.0 ± 1.9 mm and a total final biomass of 1040 g m^?2.     

Stocking location and predation by marine fishes affect survival of hatchery‐reared Atlantic salmon smolts

Abstract Release strategies of hatchery‐reared Atlantic salmon, Salmo salar L., smolts were compared by studying survival and migration of smolts (n = 99) and their predators (Atlantic cod, Gadus morhua L., n = 8; and saithe, Pollachius virens (L.), n = 2) during the first 37 km of the marine migration using acoustic telemetry. Survivorship was higher in smolts released at the river mouth (30%) compared with smolts released in the river (12%). This was likely due to mortality or reduced migratory behaviour in fresh water. The marine mortality was 37% during the first 2 km after leaving the river (at least 25% mortality because of predation from marine fishes), and total marine mortality over 37 km was 68%. Detection‐depth data were useful for evaluating whether the tagged smolts were alive or predated; mortality during the first 2 km of outward migration would have been underestimated at 26% instead of 37% without the analysis of depth detection. Transmitters from consumed post‐smolts remained in predators for up to 47 days (average 29 days).     

Use of marine by‐product meals in diets for juvenile longfin yellowtail Seriola rivoliana

A 60‐day feeding trial to determine the nutritional value of marine by‐product meals in diets for longfin yellowtail Seriola rivoliana juveniles (48.1 ± 0.6 g initial weight) was conducted. Five diets were evaluated: a reference diet (RD; 500 g kg^?1 CP, 130 g kg^?1 L), containing 500 g kg^?1 of fish meal (FM); three experimental diets with 125 g kg^?1 of shrimp head (SM), Catarina scallop viscera (CM) or Pen shell viscera (PM) meals; and one diet (SCP) containing 125 g kg^?1 of each of the experimental meals, to partially replace FM. Survival was not significantly affected by any treatment. Individual weight gain per day was high for the PM (5.3 ± 0.51 g d^?1) and SM (4.7 ± 0.32 g d^?1) diets, being significantly higher than the RD (3.5 ± 0.23 g d^?1) and the other treatments (<1.2 g d^?1). Feed intake was high in PM and SM diets, and very low in SCP and CM diets. Biochemical and haematological parameters were similar among treatments RD, PM and SM, while fish fed CM and SCP exhibited lower levels of total protein, cholesterol, haematocrit and haemoglobin. The results indicate that SM or PM can be used to partially replace FM in diets for yellowtail juveniles.