An inhibitory compound produced by Pseudomonas with effectiveness on Vibrio harveyi

Persistence of the antivibrio property of the potential antagonistic probiotics, Pseudomonas MCCB 102 and 103, at different temperatures, pH and in organic solvents was studied. The antivibrio compound was extracted, purified and characterized using thin‐layer chromatography, high‐pressure liquid chromatography, liquid chromatography‐mass spectroscopy, UV–Vis and nuclear magnetic resonance spectroscopy and identified as N‐methyl‐1‐hydroxyphenazine, a phenazine antibiotic. The toxicity of the compound was tested in Penaeus monodon haemocyte culture and the IC₅₀ value was found to be 1.4 ± 0.31 mg L^?1. The compound was found to be bacteriostatic at 0.5 mg L^?1. Its stability to varying temperature, pH, organic solvents, prolonged shelf‐life and vibriostatic nature point to its suitability for prophylatic aquaculture application.     

Exclusion of Vibrio spp. by an antagonistic marine actinomycete Streptomyces rubrolavendulae M56

A marine actinomycete Streptomyces rubrolavendulae M56 isolated from the sediments of Bay of Bengal and displaying biogranulation property was used for the study. The strain showed antagonistic property against vibrios, the opportunistic pathogens in aquaculture. The efficacy of the biogranules of actinomycete M56 in competitive exclusion of Vibrio spp. was tested both in vitro and in vivo. Streptomyces rubrolavendulae M56 biogranules could significantly exclude the pathogenic Vibrio spp. in co‐culture experiments (in vitro). In vivo exclusion of Vibrio spp. in a Penaeus monodon postlarval rearing system was evaluated by treatment of the rearing water with biogranules of S. rubrolavendulae M56. The experiments proved that S. rubrolavendulae M56 biogranules could reduce the pathogenic Vibrio spp., while maintaining total heterotrophic bacterial count. Therefore, the actinomycete biogranules (M56) can be used as a promising alternative to antibiotics in the shrimp larval production system which is often affected by vibriosis.     

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.     

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.     

Application of prospective probionts at early stages of Atlantic cod (Gadus morhua L.) rearing

This work aimed at validating the use of two prospective probionts (Arthrobacter sp. and Enterococcus sp.) at early stages of cod (Gadus morhua L.) rearing. Ova at late post‐fertilized stage and larvae during their first 4 weeks of life were bathed with both probionts, isolated previously from the cod‐rearing environment. This treatment was compared with groups fed rotifers supplemented with a commercial probiotic (Remus^®) and those untreated. Microbiological analyses (total viable counts, presumptive Vibrio and lactic acid bacteria) were performed in rearing systems and larval survival, growth and development were assessed. Larval development was evaluated by proteolytic activity of larval lysates and immunological analysis of important proteins: apolipoprotein A‐I, haemoglobin, C‐reactive protein, C3 and cod serum proteins. Bacterial bathing led to a significantly higher larval weight, length and culturable microbial load in larval gastrointestinal (GI) tract when compared with the control and Remus groups. Development occurred earlier in bathed larvae. However, their survival was negatively affected compared with the control group, but was significantly higher than for the Remus group. The non‐pathogenicity of both probionts was demonstrated by intraperitoneal injection of 13 g cod juveniles. The results suggest that Arthrobacter and Enterococcus probionts affected the larval GI microbiota and contributed to growth, development and digestion, either directly or indirectly.     

Poly‐beta‐hydroxybutyrate‐enriched Artemia sp. for giant tiger prawn Penaeus monodon larviculture

The beneficial effects of PHB as supplement for giant tiger prawn Penaeus monodon postlarvae using a short‐term enrichment strategy via Artemia were examined. The effects of co‐supplementing with a lipid emulsion were also evaluated to determine whether it yielded an additional benefit. Results on the average weight and larval development were not significantly different among postlarvae fed the different dietary treatments, indicating that PHB supplementation could not be used to stimulate growth in P. monodon postlarvae while such positive results have been reported in other aquaculture species. Nonetheless, significantly higher survival was obtained in postlarvae fed PHB‐enriched Artemia irrespective of lipid enrichment. In addition, PHB increased the survival of the postlarvae after exposure to a lethal dose of ammonia. Lipid supplementation nullified this effect. The cumulative mortality of postlarvae subjected to a sublethal concentration of ammonia for 24 h and subsequent exposure to pathogenic Vibrio campbelli showed that PHB but not lipids could effectively enhance the resistance of the postlarvae. Co‐supplementing lipids even significantly decreased this outcome. Our study indicates that PHB supplementation increases the quality of larval P. monodon and their chance of surviving under adverse environmental conditions. The short‐term co‐supplementation with lipid emulsion did not add to these effects.     

Effect of probiotics on digestive enzyme activities and growth of cichlids,Etroplus suratensis (Pearl spot) and Oreochromis mossambicus (Tilapia)

In aquaculture, exposure to stressful conditions and problems related to diseases often occur resulting in serious economic loss. The use of probiotics that control pathogens through different mechanisms, aiding digestion by exoenzyme supply and establishment of beneficial microflora in the digestive tract has got increased attention recently. Two different bacterial species Micrococcus MCCB 104 and Bacillus MCCB 101 were tested as probiotics for two cichlid species Etroplus suratensis and Oreochromis mossambicus. The strains were administered separately and together, at a rate of 10³ CFU animal^?1 day^?1 for 28 days. The effects on digestive enzyme activity and growth were compared with those fed on control diet. FCR of E. suratensis was high in case of Bacillus alone as well as the mixed culture (Micrococcus and Bacillus together) than that of control. However, the SGR of E. suratensis was slightly lower than that of control during 14 days trial. SGR of both the treatments (Bacillus alone and mixed culture) was higher than that of the control on 28th day, and this might be due to the reason that specific duration is needed for the assimilation and conversion of nutrients into proteins. The intestinal digestive hydrolases of E. suratensis were profoundly enhanced mainly by Bacillus and mixed cultures. The hepatopancreatic versions of major digestive enzymes in E. suratensis were influenced variously by different probiotic treatments. The alpha‐amylase activity of hepatopancreas has been elevated significantly by mixed culture during 14 days experiments while on 28 days trials both Bacillus and mixed culture showed an enhancing effect. During 14 days feeding trials, the intestinal lipase activity has been prominently enhanced by all probiotic treatments. It is found that, in O. mossambicus, both 14 days and 28 days feeding experiments, the pepsin as well as intestinal digestive enzymes such as amylase, total alkaline protease, lipase and their hepatopancreatic counter parts showed a significant elevation in their specific activity. Intestinal alpha‐amylase and total alkaline proteases of O. mossambicus showed an increase in specific activities on both 14 days and 28 days feeding experiments with the Bacillus and Micrococcus combination. The hepatopancreatic enzymes were highly influenced by the Bacillus species in O. mossambicus. The results of this study suggest that live probiotic microorganisms such as Micrococcus MCCB 104 and Bacillus MCCB 101 may be incorporated while formulating the cost‐effective, nutritionally balanced diet of E. suratensis and O. mossambicus for better growth performance and nutrient utilization.     

Structural characteristics and succession of intestinal microbiota for Paralichthys olivaceus during the early life stage

The research of intestinal microflora structures for Paralichthys olivaceus larvae and juveniles will help us to master the ontogeny and developmental colonization of microflora during the larval rearing stage. In this study, we sequenced the total bacterial genomic DNA in larval and juvenile guts with an Illumina MiSeq PE300 system, and analysed the structural characteristics of these microbiota, feed and rearing water in live and formulated feeding periods. The structure of gut microbiota was gradually similar to those in wild P. olivaceus at the phylum level and the newly hatched ones with the growth, according to the distribution and abundance of intestinal microbiota. And the colonized rule of main microbial species in guts was decreased initially and then increased during the larvae and juveniles stage. Meantime, the core microflora of this study were obtained through the analysis of shared and dominant species, which included Bacteroides, Bacillus, Enterococcus, Lactobacillus, Lactococcus, Escherichia_Shigella, Acinetobacter, Pseudomonas, Vibrio, Nitrosomonas, and Glaciecola. The correlation analysis of microbiota between intestines and environmental factors suggested that microflora in feed and water could affect the distribution of larval and juvenile gut microbiota. Moreover, many species of Acinetobacter, Pseudomonas and Vibrio are treated as important potential pathogens in aquaculture. These all pointed out the microbial quality of feed and rearing water should be strictly controlled in fish breeding and farming, and supply theoretical bases for screening the native probiotics to artificially regulate the gut microbiota.     

Effects of dietary administration of two Patagonian probiotics on the zoeae I from the swimming crab Ovalipes trimaculatus

In this study, we analyzed the effects of supplying two alternative Patagonian-autochthonous probiotic strains of Bacillus sp. and Lactococcus sp. or no probiotic at all (control treatment) on the survival, stage duration, molting success to the next larval instar, and speed of vertical displacement of the zoeae I of the southern surf crab Ovalipes trimaculatus, also considering two alternative methods for their administration: (i) direct inoculation into the rearing seawater or (ii) bio-encapsulation in Artemia persimilis nauplii. Both probiotic strains were effectively bio-encapsulated by the nauplii at high CFU x naupli^?1. Probiotic inoculation in the rearing seawater improved the performance of the zoeae over the control treatment and also outperformed bio-encapsulation. Bio-encapsulation in Artemia nauplii resulted in lower survival and molting success to zoeae II compared to the control treatment. No significant differences were observed between the mean effects of either one of the two probiotics. Overall, our results show that the direct inoculation of any of both probiotics into rearing seawater improves the larval culture of the portunid crab.

     

Bacterial Change in the Gut and Hepatopancreas of Tiger Shrimp Reared in Bioaugmented System

The study was conducted in nine 150‐L capacity glass aquaria for 192 h to determine the changes in the microflora in the gut and hepatopancreas of tiger shrimp, Penaeus monodon, after bioaugmenting (water treatment and probiotics). Results showed that luminous bacterial counts were significantly lower in bioaugmented than in nonbioaugmented systems (range 0–5.9 × 10³ cfu/g and 0–3.2 × 10³ cfu/g in gut and hepatopancreas, respectively). Biochemical tests of isolates showed that the bioaugmented systems were dominated by Gram‐positive Bacillus and Streptococcus and the Gram‐negative Vibrio dominated the control. In water‐treated and probiotics‐fed bioaugmented system, the change in bacterial dominance to Bacillus became evident 2 h in the gut and 24 h in the hepatopancreas. Bacterial dominance shifted to Vibrio species after 120 h. Shrimp in the control were observed to become lethargic after 72 h as Vibrio species significantly increased in number. Dissolved oxygen and unionized ammonia levels were significantly lower in bioaugmented system compared with the control.     

Assessment of temperature or salinity effects on larval development by catecholamine‐induced metamorphosis of hatchery‐reared flat oyster,Ostrea angasi (Sowerby 1871) larvae

A need to improve larval rearing techniques led to the development of protocols for catecholamine‐induced settlement of flat oyster, Ostrea angasi, larvae. To further refine these techniques and optimize settlement percentages, the influence of salinity or temperature on development of O. angasi larvae was assessed using epinephrine‐induced metamorphosis. Larvae were reared between salinities of 15–35 and temperatures between 14.5 and 31°C. The greatest percentage survival, growth, development occurred when larvae were reared between 26 and 29°C and between salinities of 30 and 35. Larvae reared outside this salinity and temperature range exhibited reduced growth, survival and/or delayed development. Short‐term (1 h) reduction in larval rearing temperature from 26°C to 23.5°C significantly increased larval metamorphosis without affecting larval survival. Short‐term (1 h) increase in larval rearing temperature from 26°C to 29 and 31°C decreased larval survival and metamorphosis. To ensure repeatability in outcomes, tests showed that larvae sourced from different estuaries did not vary significantly in their metamorphic response to short‐term temperature manipulation and epinephrine‐induced metamorphosis.     

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.     

Pathogenicity of halophilic Vibrio harveyi in giant freshwater prawns (Macrobrachium rosenbergii De Man)

In this study, the phenotypic and pathogenic properties in freshwater host were characterized in 14 strains of halophilic Vibrio harveyi isolated from infected marine black tiger shrimp, Penaeus monodon. The lysogenic phenotype was assayed via prophage excision and mitomycin C induction. The bacteria were grouped into two types, corresponding to lysogenic and non‐lysogenic strains. The pathogenicity was determined via direct injection of bacterial cultures into post‐larval juvenile giant freshwater prawns, Macrobrachium rosenbergii De Man. All of the infected prawns showed similar symptoms and inflamed hepatopancreas. The V. harveyi isolates derived from the first‐injected infected prawns were re‐isolated and re‐injected into healthy giant freshwater prawns, in which they retained similar infectivity. Both lysogenic and non‐lysogenic Vibrio spp. showed identical virulence associated with 100% mortality within one day post‐injection. TEM micrographs showed hepatopancreatic nuclear deformation and lipid breakdown caused by lysogenic γ‐hemolytic VL19 and non‐lysogenic β‐hemolytic V33. However, the V33 strain was associated with severely disrupted mitochondria. None of the V. harveyi strains was able to produce a biofilm. Together, our findings indicate that the lysogenic and non‐lysogenic halophilic V. harveyi isolated from marine shrimps may use different virulence factors that are responsible for their pathogenicity in freshwater prawns.     

A Review of Probiotics in Shrimp Aquaculture

Development of shrimp aquaculture has been associated with increases in infectious diseases and environmental degradation. An effective alternative to chemicals and antibiotics used for the prevention of these problems is to administer probiotics into the rearing system. Three bacterial genera, Bacillus, Vibrio, and Pseudomonas, are commonly administered as probiotics in shrimp aquaculture. Candidate probiotics are species specific and need to be tested for their effectiveness for certain species in in vitro and in vivo. Supplementation into feed is more effective in conveying probiotics into animals compared to direct application into rearing systems. Overdosage or prolonged administration of probiotics can induce immunosuppression. A cell-density of 10⁵ colony-forming units (CFU) per ml is widely recommended. A combination of probiotics results in better outcomes for the host than individual probiotics. Probiotics improve water quality while reducing pathogenic bacteria. Probiotics show positive effects through an improvement in the physiological and immune responses of shrimps. Probiotics are increasingly becoming important and more common in any organic shrimp farming.     

Characterization of bacterial community in intestinal and rearing water of Penaeus monodon differing growth performances in outdoor and indoor ponds

Penaeus monodon provides a high‐quality protein source for humanity, and pond cultured shrimp often presents asynchronous growth. Microbial communities are important for the digestion and immunity of shrimp. Therefore, in this study, we investigated the bacterial characteristics of the intestine and rearing water of asynchronously growing P. monodon that were cultured in outdoor and indoor pond respectively. The results showed that the bacterial community of the rearing water was more complex than that of the intestine; the fast‐growing shrimp in the indoor pond had higher intestinal bacterial diversity. Besides, the dominant bacterial composition of the water was more complex than that of the intestinal. Specifically, the abundance of Proteobacteria in the intestine was consistent with the growth performance of shrimp in the outdoor pond, which was exactly the opposite in the indoor pond. At the genus level, two cyanobacteria, Limnothrix and Cyanobium PCC‐6307, were dominant in the indoor and outdoor water respectively. In the outdoor pond, Bacillus was dominant in the slow‐growing shrimp intestines, while Vibrio was dominant in the fast‐growing shrimp. The intestinal microbes of the fast‐growing shrimp had higher proteasome metabolic capacity. These results can provide new insights into microbiome characteristics involved in the asynchronous growth of shrimp.     

Improvements in larviculture of Crangon crangon as a step towards its commercial aquaculture

The European brown shrimp, Crangon crangon, is a highly valued commercial species in Europe. These shrimp fisheries are characterized by strong seasonal variations in average landings. Attempts to ‘catch and hold’ wild adults in land‐based rearing systems have been proven to be very difficult, due to inadequate feed, slow growth and high mortality. In this study, we have optimized design and operation procedures of a small‐scale static larval rearing system for the culture of C. crangon larvae. Focus was on optimizing larval survival via water temperature, feed selection, feeding regime and density. This is the first report that shows that C. crangon larvae can be reared at high densities of 300 larvae L^‐1 with high survival of 73.5 (± 5.4)% under laboratory conditions. In these systems, larvae can be fed exclusively Artemia nauplii according to a feeding regime which is adjusted based on major moulting events. Addition of microalgae may further increase survival by 10%. The information gathered during this research can be applied to further optimize larval development in either flow‐through or recirculation systems.     

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.     

In vivo evaluation of the effects of commercial Bacillus probiotics on survival and development of Litopenaeus vannamei larvae during the early hatchery period

Three in vivo experiments were conducted to measure the effectiveness of commercial Bacillus probiotics on survival and development of Litopenaeus vannamei larvae from nauplii 3–4 (N_3–4) to zoea 3 (Z₃) stages. Experiment I: Nine commercial Bacillus probiotics were individually added to larvae twice at N₆ and Z₁ at levels of 2, 20, and 100 mg L^?1. Only six of nine products at 20 mg L^?1 exhibited higher or significantly higher larval survival (P < 0.05) than the control. Experiment II: Two superior products from the first experiment were administered to larvae with six dose frequencies at 20 mg L^?1. For both products, three doses, once at each of N_3–4, N₆ and Z₁ stages, yielded the best larval survival and development rates (P < 0.05), and these were confirmed by enhanced activities of tryptase and amylase. Experiment III: The isolates of these two products, identified as Bacillus subtilis and Bacillus licheniformis, were delivered to larvae singly at concentrations of 1.0 × 10⁸ and 1.0 × 10⁹ CFU L^?1, or at the same concentrations by mixing the two equally. At 1.0 × 10⁹ CFU L^?1, B. subtilis exerted more beneficial effects on larvae than B. licheniformis or the mixture. Therefore, the optimal dosage and dose frequency of commercial Bacillus products should be evaluated prior to large‐scale application in shrimp hatcheries to avoid futility or even adverse effects, as spore counts and non‐bacterial ingredients are key factors influencing the efficacy of Bacillus probiotics.     

Effects of dietary probiotic on the growth performance,non‐specific immunity and disease resistance of cobia,Rachycentron canadum

The present study was performed to investigate the effects of a commercially available probiotic product (compound probiotic) containing Bacillus subtilis 7.0 × 10⁹ CFU g^?1, Bacillus licheniformis 3.0 × 10⁹ CFU g^?1, Lactobacillus spp. 5.0 × 10⁸ CFU g^?1 and Arthrobacter spp. 1.0 × 10⁸ CFU g^?1 on the growth performance, non‐specific immunity and protection against Vibrio harveyi infection in cobia (Rachycentron canadum). Fish were fed diets containing six graded levels of compound probiotic (0.0, 1.0, 2.0, 3.0, 4.0 and 5.0 g kg^?1) for 8 weeks. The results showed that the survival rate ranged from 81.1% to 84.4% with no significant difference among dietary treatments (P > 0.05) after feeding experiment. Dietary compound probiotic significantly increased the specific growth rate (SGR), serum lysozyme, alternative complement pathway (ACP) activity, phagocytosis percentage (PP) and respiratory burst activity of head‐kidney macrophages of cobia. Moreover, feeding of supplemented diets containing compound probiotic resulted in significantly lower mortality against the pathogens Vibrio harveyi compared with the control group. To elevate the growth and immune resistance ability of cobia, an optimal dose of dietary compound probiotic administration determined by second‐order polynomial regression analysis was 3.3 g kg^?1, on the basis of the SGR and mortality after challenge with V. harveyi.     

Probiotic bacteria effects on growth and bacterial composition of black tiger shrimp (Penaeus monodon)

The effects of Bacillus probiotic forms on growth performance and microbiota were examined in black tiger shrimp (Penaeus monodon). Shrimp were fed for 120 days with one of three diets; a control diet alone or supplemented with either probiotics consisting of live‐sprayed Bacillus (LS) or freeze‐dried Bacillus (FD). Culturable bacteria counts in the hepatopancreas, intestine and culture water of the three treatments were relatively constant during the experiment. Bacillus numbers in the hepatopancreas and intestines of probiotic‐treated groups increased significantly (P < 0.05) after 30 treatment days and remained relatively constant afterwards while no appreciable changes occurred in the control. Shrimp supplemented with LS and FD contained significantly lower concentrations of Vibrio (P < 0.05) in the hepatopancreas, intestine and culture water, compared to those in the control group. Shrimp treated with LS and FD probiotic bacteria showed a significant improvement (P < 0.05) in specific growth rate (SGR) and feed efficiency (FE), compared to those of control group. Probiotic bacteria have the potential to enhance shrimp culture by reducing Vibrio and improving growth performance.