Oxygen depletion and sulphide formation, resulting from the accumulation of organic waste, are common challenges in shrimp ponds that could result in complete harvest failure. The stage at which these circumstances occur during the shrimp growth period remains elusive, yet, knowledge of the timing of oxygen depletion and sulphide formation is essential to enable remediating actions. Here, we used an experimental shrimp pond model at different stages in the shrimp growth period to determine when oxygen depletion and sulphide production occur. Microscale depth measurements of oxygen and H2S were determined using microelectrodes to visualize their profiles at different depths of the water-sediment interface and the sediment. We evaluated the potential of different molybdate concentrations at different stages to determine the optimal conditions to suppress H2S formation. Oxygen depletion and sulphide production took place in the middle of the shrimp growth cycle in the simulated model of waste accumulation. The addition of molybdate was only effective in the early stages of the onset of oxygen depletion and H2S formation, and residual molybdate was required to ensure a continuous suppression of sulphide production. However, oxygen depletion could not be prevented and reintroduction of oxygen did not occur when molybdate was added. In conclusion, molybdate appeared to be an effective strategy to suppress H2S formation at the onset of its production in a shrimp pond bottom model.

The bioactivities of two commercially available probiotics and one chemical disinfectant were tested against strains of Vibrio parahaemolyticus (VPAHPND) and V. harveyi. This study aimed to determine shrimp pathogenic Vibrios' in vitro and in vivo sensitivities to commercial probiotics and a chemical disinfectant. The probiotics and disinfectant were tested first in vitro, followed by the in vivo trials. Results showed that upon administration of probiotics either through diet or adding into the tank water, the survivability of shrimp was increased during challenge with VPAHPND and V. harveyi. Also, the disinfectant was tested against the same pathogens and showed positive bactericidal effects at 2,500 ppm and 5,000 ppm. The present findings suggest that adding probiotics to the rearing water or the shrimp feeds effectively prevents infection by lowering the load of pathogenic bacteria. In comparison, the effectiveness of the disinfectant (PUR) depends on its appropriate concentration and timing of application. It is not only limited to rearing water but is also applicable for decontaminating pond liners, tanks, and other paraphernalia.

Shrimp are commonly cultured in earthen aquaculture ponds where organic-rich uneaten feed and faeces accumulate on and in the sediment to form anaerobic zones. Since the pond water is rich in sulphate, these anaerobic conditions eventually lead to the production of sulphide. Sulphides are toxic and even lethal to the shrimp that live on the pond sediment, but physicochemical and microbial reactions that occur during the accumulation of organic waste and the subsequent formation of sulphide in shrimp pond sediments remain unclear. Molybdate treatment is a promising strategy to inhibit sulphate reduction, thus, preventing sulphide accumulation. We used an experimental shrimp pond model to simulate the organic waste accumulation and sulphide formation during the final 61 days of a full shrimp growth cycle. Sodium molybdate (5 and 25 mg/L Na2MoO4.2H2O) was applied as a preventive strategy to control sulphide production before oxygen depletion. Molybdate addition partially mitigated H2S production in the sediment, and delayed its transfer to the bulk liquid by pushing the higher sulphide concentration zone towards deeper sediment layers. Molybdate treatment at 25 mg/L significantly impacted the overall microbial community composition and treated samples (5 and 25 mg/L molybdate) had about 50% higher relative abundance of sulphate reducing bacteria than the control (no molybdate) treatment. In conclusion, molybdate has the potential to work as mitigation strategy against sulphide accumulation in the sediment during shrimp growth by directly steering the microbial community in a shrimp pond system.

Shrimp are commonly cultured in earthen aquaculture ponds where organic-rich uneaten feed and faeces accumulate on and in the sediment to form anaerobic zones. Since the pond water is rich in sulphate, these anaerobic conditions eventually lead to the production of sulphide. Sulphides are toxic and even lethal to the shrimp that live on the pond sediment, but physicochemical and microbial reactions that occur during the accumulation of organic waste and the subsequent formation of sulphide in shrimp pond sediments remain unclear. Molybdate treatment is a promising strategy to inhibit sulphate reduction, thus, preventing sulphide accumulation. We used an experimental shrimp pond model to simulate the organic waste accumulation and sulphide formation in a long-term experiment (61 days). Sodium molybdate (5 and 25 mg/L Na2MoO4.2H2O) was applied as a preventive strategy to control sulphide production before oxygen depletion. Molybdate addition mitigated H2S production in the sediment, and delayed its transfer to the bulk liquid by pushing the higher sulphide concentration zone towards deeper sediment layers. Molybdate treatment at 25 mg/L significantly impacted the overall microbial community composition and treated samples (5 and 25 mg/L molybdate) had about 50% higher relative abundance of sulphate reducing bacteria than the control (no molybdate) treatment. In conclusion, molybdate worked as long-term mitigation strategy against sulphide accumulation by directly steering the microbial community in a shrimp pond system.

The microbial rearing quality influences the survival of marine larvae. Microbially matured water treatment systems (MMS) provide a more favourable rearing water microbiome than flow-through systems (FTS). It has previously been hypothesised, but not investigated, that initial rearing in MMS leaves a protective legacy effect in Atlantic cod larvae ( Gadus morhua ). We tested this hypothesis through a crossover 2 × 2 factorial experiment varying the rearing water treatment system (MMS vs FTS) and the microbial carrying capacity (+ /− added organic matter). At 9 days post-hatching, we switched the rearing water treatment system. By comparing switched and unswitched rearing tanks, we evaluated if legacy effects had been established in the larvae or their surrounding rearing water bacterial community. We analysed the bacterial communities with flow cytometry and 16S rRNA gene sequencing. We found no evidence that the initial rearing condition left a legacy effect in the communities by evaluating the bacterial community diversity and structure. Instead, the present rearing condition was the most important driver for differences in the rearing water microbiota. Furthermore, we found that MMS with high microbial carrying capacity appeared to seed a stable bacterial community to the rearing tanks. This finding highlights the importance of keeping a similar carrying capacity between the inlet and rearing water. Moreover, we reject the hypothesis that the initial rearing condition leaves a protective legacy effect in larvae, as the larval survival and robustness were linked to the present rearing condition. In conclusion, our results highlight the importance of maintaining a beneficial microbial rearing environment from hatching and throughout the larval rearing period.

The development of effective management strategies to reduce the occurrence of diseases in aquaculture is hampered by the limited knowledge on the microbial ecology of these systems. In this study, the dynamics and dominant community assembly processes in the rearing water of Litopenaeus vannamei larviculture tanks were determined. Additionally, the contribution of peripheral microbiomes, such as those of live and dry feeds, to the rearing water microbiome were quantified. The community assembly in the hatchery rearing water over time was dominated by stochasticity, which explains the observed heterogeneity between replicate cultivations. The community undergoes two shifts that match with the dynamics of the algal abundances in the rearing water. Source tracking analysis revealed that 37% of all bacteria in the hatchery rearing water were introduced either by the live or dry feeds, or during water exchanges. The contribution of the microbiome from the algae was the largest, followed by that of the Artemia, the exchange water and the dry feeds. Our findings provide fundamental knowledge on the assembly processes and dynamics of rearing water microbiomes and illustrate the crucial role of these peripheral microbiomes in maintaining health-promoting rearing water microbiomes.

The production of shrimp is often performed in earthen outdoor ponds in which the high input of feed and faeces on the bottom can result in deterioration of the water quality, which negatively impacts the animals and the environment. Here, we investigate the potential of sodium molybdate (Na₂MoO₄·2H₂O), sodium nitrate (NaNO3) and sodium percarbonate (Na2CO3·1.5H2O2) to control sulphide production in a simulated shrimp pond bottom system that included the sediment, overlaying artificial seawater and organic matter input in the form of shrimp feed and shrimp faeces. Sediment depth gradient measurements of oxygen, H2S and pH were obtained during 7 days of incubation using microelectrodes. The most significant impact in terms of H2S, was observed for 50 mg/L sodium molybdate. At the water-sediment interface, there was up to 73% less H2S detected for this treatment in comparison to a control treatment, while in the deeper layers of the sediment it was up to 47% less H2S. The residual sulphate concentrations in the molybdate treated samples were 16 ± 4% higher than the control, indicating an inhibition in sulphate reduction. Nitrate and sodium percarbonate treatments also showed a limited capacity to decrease H2S entering in the water column, yet no clear difference in H2S concentrations in the sediment compared to the control were observed. Molybdate treatment appears to work through the inhibition of sulphate reducing bacteria in situ for the control of H2S production better than the chemical oxygen boosters or nitrate treatment.

A preliminary study was conducted to demonstrate fate of the bacterial storage compound poly-beta-hydroxybutyrate (PHB) once ingested and degraded in vivo in crustaceans. The 2% supplementation of C-13-labeled Ralstonia eutropha DSM545 containing 75% PHB on dry weight in postlarval whiteleg shrimp, Litopenaeus vannamei resulted in consistent enrichment of two fatty acids (containing 14-24 carbons [C] in length) pentadecanoic acid (15:0) and palmitic acid (16:0) in all lipid fractions. This could indicate that C-13 signal may not only originate from the PHB but partly from the structural components of the bacterial cell. There was a trend of lower C-13 enrichment in various lipid fractions of the postlarvae as compared to the diet. These findings may have important implications as to potential of PHB as energy source rather than as building block. There was a rapid transfer of C-13 in the postlarvae as seen by the significant C-13 enrichment in the tissues 4 hr after feeding with labeled bacterial cells. Overall, the assimilation of PHB in crustacean tissues is demonstrated for the first time. Our results indicate that PHB once ingested is rapidly assimilated in the tissues and could probably act as an energy source rather than as a building block.

The production of shrimp is often performed in earthen outdoor ponds in which the input of feed and faeces on the bottom results in deterioration of the water quality that results in a negative impact on the aquatic animals and the environment. Nitrate application in shrimp ponds could improve pond water and sediment quality by oxidizing anaerobic zones of the sediment. This study was conducted to evaluate the possible benefits of nitrate treatment in combination with probiotics application to decrease sulphide formation in shrimp ponds, which is a crucial problem during the growth phase of shrimp. Three sets of experiments were conducted to describe the mechanism of nitrate treatment: a dual dose nitrate treatment at the start and after 14 days that investigated nitrate treatment with concentrations of 50, 100 and 200 mg/L NO3-, a multiple dose nitrate treatment where lower doses of nitrate (12.5, 25 and 50 mg/L NO3-) were tested with more frequent application (every 4 days) and a microbially assisted nitrate treatment in which 50 mg/L NO3- treatment was applied every 4 days with and without presence of a single probiotic application. A batch system including shrimp feed and faeces, representing the bottom waste produced during 90 days of shrimp culture, was used to test the effect of nitrate addition under anaerobic conditions that mimic the dead zones in shrimp pond bottoms where high amounts of organic matter accumulate. Nitrate addition acted as a curative solution for hydrogen sulphide (H2S) accumulation, rather than preventive. Nitrate amended treatments (12.5 mg/L.day NO3-) decreased the accumulated H2S in the headspace of incubation bottles by 93 +/- 3% compared to the controls. Nitrate addition increased pH and decreased organic acids accumulation to trace amounts. To justify the decrease in H2S concentration apart from the effect sulphur speciation through pH increase, a separate test was conducted to compare the effect of NaOH with nitrate treatment, in which nitrate treatment resulted in a 62.8% higher decrease in H2S concentrations than the NaOH treatment. The use of a denitrifying strain did not improve the beneficial effect of nitrate, indicating that intrinsic capacity for anoxic conversion of waste by the present community was sufficient. In conclusion, nitrate treatment showed a potential to control H2S accumulation in the shrimp ponds that is a critical problem during the growth of shrimp.

The effects of dietary poly-beta-hydroxybutyrate (PHB) on the lipid and fatty acids (FA) in crustaceans were investigated usingArtemiaas model species. Supplying PHB either in crystalline or amorphous form significantly increased the whole-body lipid contents of starvedArtemia. Co-supplying dietary PHB with bacterial PHB degraderComamonas testosteroniat 10(6) cells ml(-1) for 24 hr also significantly increased this parameter. These findings have important implications on the lipid-saving effects of PHB in crustacean tissues. Dietary PHB, however, did not contribute to the increase in docosahexaenoic acid (DHA) of the naturally DHA-deprivedArtemia. Alternative strategy of co-supplying dietary PHB with highly unsaturated fatty acid (HUFA)-rich emulsion for 24 hr effectively improved the nutritional contents ofArtemiaand at the same time assured efficient delivery of PHB to the larval predator. According to a C-13 stable isotope tracer study, the significantly higher delta C-13 levels measured inArtemia2 hr postfeeding with C-13-labelledR. eutropha DSM545 indicate that PHB offered in natural matrix is rapidly assimilated. Overall, this study demonstrated the lipid-saving effects of PHB inArtemia. PHB assimilation following gastrointestinal degradation could attribute to its reported beneficial effects in various aquatic farmed species.

Biofloc systems are microbial mature environments that are potentially less conducive disease outbreaks. We hypothesized that the way in which biofloc microbial communities are managed determines the level of disease protection. To investigate such hypothesis, Litopenaeus vannamei post-larvae were cultured for 21 days in biofloc environments created by different water management procedures. Five different types of bioflocs were created: autotrophic bioflocs without probiotics, autotrophic bioflocs with probiotics, heterotrophic bioflocs without probiotics, heterotrophic bioflocs with probiotics, and a flow-through system as a control. Heterotrophic bioflocs were obtained by daily addition of carbon (glucose) at an estimated C/N ratio of 18 throughout the experiment. For autotrophic bioflocs this input of carbon was applied only to start up the system and upon appearance of bioflocs (TSS > 100 mg L-1) and a drop in total ammonium nitrogen concentration below 0.05 mg L-1, carbon dosing was stopped. Bioflocs cultured with addition of probiotics received a 0.5 ppm dose every 48 hours. After 21-d culture period, a 96 h challenge test was performed with a Vibrio parahaemolyticus strain known to cause AHPND. For each biofloc type, this challenge was performed in three different approaches: 1- Shrimp were taken out of their biofloc tanks and challenged by applying new seawater; 2-Shrimp from biofloc tanks were challenged in their respective biofloc suspensions; and 3- Non-experimental shrimp, randomly selected from a recirculation (RAS) system were challenged in the types of biofloc suspensions. Mortality was high when shrimp were challenged in new seawater, independent of treatment. When challenged in their respective biofloc suspensions shrimp survival was the highest in heterotrophic bioflocs with and without probiotic supplementation and the autotrophic bioflocs with probiotics, whereas shrimp survival in autotrophic bioflocs without probiotics was 50%. These results were similar when non-experimental shrimp originating from a RAS system were challenged in these biofloc suspensions. Taken together, results suggest that bioflocs as such can decrease the impact of a Vibrio parahaemolyticus challenge and that this protection depends on the operational parameters of the biofloc system. Moreover, probiotics can be used to complement the protective effect of bioflocs. This information reinforces the importance of microbial community management as a tool to reduce the risk of disease and establish highly biosecure systems.

The use of artificial substratum consisting of poly-beta-hydroxybutyrate (PHB)-based biodegradable plastic for penaeid shrimp culture was investigated in the present study. The survival of postlarval tiger shrimp Penaeus monodon (30 +/- 5 mg) provided with PHB substratum made out of PHB type DP9002 (Metabolix GmbH, Koln, Germany) was 88.7 +/- 3.4% and this was significantly higher as compared to postlarvae provided conventional substratum consisting of polyvinylchloride (PVC) pipes (67.3 +/- 6.5%). However, no significant weight improvement was observed for the postlarval tiger shrimp indicating that PHB could not be used as growth promoter. Nevertheless, a trend of improved robustness against adverse environmental conditions (lethal ammonium chloride concentration) and increased resistance to pathogenic Vibrio was observed in postlarval tiger shrimp provided with PHB substratum as compared to postlarvae provided with PVC substratum. Results indicate higher preference by postlarvae on PHB substratum over PVC substratum. Overall, this study indicates the potential of artificial substratum consisting of PHB-based biodegradable plastic as replacement for conventional substratum consisting of PVC pipes in enhancing the survival of postlarval tiger shrimp and improving its performance against adverse environmental conditions and disease resistance.

Marine invertebrates rely mainly on innate immune mechanisms that include both humoral and cellular responses. Antimicrobial peptides (AMPs), lysozyme and phenoloxidase activity, are important components of the innate immune defense system in marine invertebrates. They provide an immediate and rapid response to invading microorganisms. The impact of amorphous poly-beta-hydroxybutyrate (PHB-A) (1 mg PHB-A L-1) on gene expression of the AMPs mytimycin, mytilinB, defensin and the hydrolytic enzyme lysozyme in infected blue mussel larvae was investigated during "in vivo" challenge tests with Vibrio coralliilyticus (10(5) CFU mL(-1)). RNAs were isolated from mussel larvae tissue, and AMPs were quantified by q-PCR using the 18srRNA gene as a housekeeping gene. Our data demonstrated that AMPs genes had a tendency to be upregulated in challenged mussel larvae, and the strongest expression was observed from 24 h post-exposure onwards. The presence of both PHB-A and the pathogen stimulated the APMs gene expression, however no significant differences were noticed between treatments or between exposure time to the pathogen V. coralliilyticus. Looking at the phenoloxidase activity in the infected mussels, it was observed that the addition of PHB-A significantly increased the activity.

The natural amorphous polymer poly-β-hydroxybutyrate (PHB-A: lyophilized Ralstonia eutropha containing 75% PHB) was used as a biological agent to control bacterial pathogens of blue mussel (Mytilus edulis) larvae. The larvae were supplied with PHB-A at a concentration of 1 or 10 mg/L for 6 or 24 hr, followed by exposure to either the rifampicin-resistant pathogen Vibrio splendidus or Vibrio coralliilyticus at a concentration of 105 CFU/ml. Larvae pretreated 6 hr with PHB-A (1 mg/L) survived a Vibrio challenge better relative to 24 hr pretreatment. After 96 hr of pathogen exposure, the survival of PHB-A-treated mussel larvae was 1.41- and 1.76-fold higher than the non-treated larvae when challenged with V. splendidus and V. coralliilyticus, respectively. Growth inhibition of the two pathogens at four concentrations of the monomer β-HB (1, 5, 25 and 125 mM) was tested in vitro in LB35 medium, buffered at two different pH values (pH 7 and pH 8). The highest concentration of 125 mM significantly inhibited the pathogen growth in comparison to the lower levels. The effect of β-HB on the production of virulence factors in the tested pathogenic Vibrios revealed a variable pattern of responses.

The bacterial storage compound poly-β-hydroxybutyrate, a polymer of the short-chain fatty acid 3-hydroxybutyrate, has been reported to protect various aquatic animals from bacterial disease. In order to obtain a better mechanistic insight, we aimed to (1) investigate whether 3-hydroxybutyrate is released from poly-β-hydroxybutyrate within sterile brine shrimp larvae, (2) determine the impact of 3-hydroxybutyrate on the virulence of Vibrio campbellii to brine shrimp larvae and on its cell density in the shrimp, and (3) determine the impact of this compound on virulence factor production in the pathogen. We detected 3-hydroxybutyrate in poly-β-hydroxybutyrate-fed brine shrimp, resulting in 24 mM 3-hydroxybutyrate in the intestinal tract of shrimp reared in the presence of 1000 mg l −1 poly-β-hydroxybutyrate. We further demonstrate that this concentration of 3-hydroxybutyrate does not affect the growth of V . campbellii , whereas it decreases the production of different virulence factors, including hemolysin, phospholipase and protease activities, and swimming motility. We hypothesize that by affecting all these virulence factors at once, 3-hydroxybutyrate (and thus also poly-β-hydroxybutyrate) can exert a significant impact on the virulence of V . campbellii . This hypothesis was confirmed in a challenge test showing that 3-hydroxybutyrate protected gnotobiotic brine shrimp from pathogenic V . campbellii , without affecting the number of host-associated vibrios.

The use of artificial substratum made out of poly-β-hydroxybutyrate (PHB) based biodegradable plastic for penaeid shrimp culture was investigated for the first time. Provision of PHB substratum consisting of PHB type P209 (Biomer, Krailling, Germany) to postlarval whiteleg shrimp Litopenaeus vannamei (106 ± 52 mg) resulted in significantly higher survival (P ≤ 0.05) of 83.6 ± 3.4% as compared to 73.9 ± 3.0% for postlarvae provided with conventional substratum consisting of polyvinylchloride (PVC) pipes. Results on final weight showed significantly higher weight (P ≤ 0.05) of 1008.2 ± 4.5 mg in postlarval whiteleg shrimp provided PHB substratum as compared to 893.4 ± 30.0 mg for postlarvae provided with PVC substratum. The trends of higher visit and preference by postlarval whiteleg shrimp on 10-day and 70-day aged PHB substrata over 10-day and 70-day aged PVC substrata suggest the importance of PHB substratum as grazing area rather than as shelter. Test conducted on water quality also showed a trend towards higher total ammonia-nitrogen (TAN) conversion from an initial concentration of 1.75 ± 0.0 mg L−1 to 0.35 mg ± 0.04 mg L−1 after 72 h with PHB substratum while using no substratum and PVC substratum lead to TAN concentrations of 1.28 ± 0.06 mg L−1 and 1.23 ± 0.10 mg L−1, respectively. Overall, this study indicates that artificial substratum consisting of PHB-based biodegradable plastic increases the quality of postlarval whiteleg shrimp and that it contributes to maintaining good water quality.

In this study, the Bacillus sp. JL47, a superior PHB-accumulating Bacillus strain identified from the previous work, was tested for its protective effects in gnotobiotic Artemia franciscana during a pathogenic Vibrio campbellii challenge. The utilization of gnotobiotic Artemia is important in this experiment because any possible microbial interference (which are naturally present in conventional culture system) are eliminated in this model culture system, hence the interpretation of the results in this mechanistic study can be more conclusive. The survival of the Artemia fed the Bacillus sp. JL47 at 1×107cellsmL−1 was significantly higher as compared with the challenged control and the survival was almost doubled when the dose was increased to 5×107cellsmL−1. However, feeding the Artemia at 106cellsmL−1 or lower showed no significant protective effects. Based on these densities, the estimated concentration of amorphous PHB showing a significant protection in Artemia was c. 2.44mgL−1 and the effects were even better when the amorphous PHB level was increased to 12.19mgL−1. Furthermore, feeding Bacillus sp. JL47 containing 55% amorphous PHB (on CDW) to Artemia showed a significantly higher survival in a Vibrio challenge relative to Bacillus containing 29% PHB. The data suggest that the amorphous PHB accumulated in the Bacillus sp. JL47 strain is an important determinant for the increased survival of challenged Artemia.