Inhibitory activity of probiotics <Emphasis Type="Italic">Streptococcus phocae</Emphasis> PI80 and <Emphasis Type="Italic">Enterococcus faecium</Emphasis> MC13 against Vibriosis in shrimp <Emphasis Type="Italic">Penaeus monodon</Emphasis>

Occurrence of widespread epizootics among larval and cultured shrimp has put on viable preventive approaches such as application of probiotics on a high priority in aquaculture. In the present study, four probiotics bacteria were isolated from marine fish and shrimp intestine based on the antagonistic activity and nonpathogenic to the host. The isolates of probiotics strains Streptococcus phocae PI80, Enterococcus faecium MC13, Lactococcus garvieae LC149, B49 and one commercial probiotics (ECOFORCE) were fed to post larvae of Penaeus monodon obtained from two different hatcheries to analyze the growth and protection against Vibrio harveyi and V. parahaemolyticus. Growth of P. monodon post larvae fed with probiotic strain S. phocae PI80 was significantly (P < 0.001) higher when compared with control and other three strains in both experiments. The treatment of post larvae with B49 reduced the growth as well as Specific growth rate. Among the three probiotic strains S. phocae PI80 and E. faecium MC13 have effectively inhibited the pathogens. In experiment I high survival (92%) were observed in S. phocae PI80 treated post larvae when challenged with Vibrio harveyi followed by E. faecium MC13 (84%), B49 (76%) and ECOFORCE (68%) but PI80 did not protect the post larvae in the same experiment when they were exposed to V. parahaemolyticus. The probiotic isolate of MC13 has protected the post larvae against V. parahaemolyticus when compared to other probiotics and control. Similarly in the second experiment feeding of S. phocae enhanced the survival of larvae when challenged with V. harveyi. The laboratory studies proved that bacterial probionts S. phocae and E. faecium isolated from shrimp and brackishwater fish has potential applications for controlling pathogenic vibriosis in shrimp culture.     

Induction of Ovarian Maturation and Spawning in <Emphasis Type="Italic">Penaeus monodon</Emphasis> Broodstock by Double-Stranded RNA

Ovarian maturation in crustacean is under the control of gonad-inhibiting hormone (GIH); a neuropeptide secreted from X-organ sinus gland complex in eyestalks. Unilateral eyestalk ablation that partially destroys GIH source is therefore a general practice in Penaeus monodon hatchery to induce ovarian maturation and spawning. Our previous report showed that silencing of GIH expression by GIH-specific double-stranded RNA (GIH-dsRNA) resulted in an increased expression level of vitellogenin in P. monodon, thus suggesting that GIH-dsRNA could be an alternative method to induce ovarian maturation in female P. monodon broodstock. In this study, we further demonstrated that a single injection of GIH-dsRNA into previtellogenic female P. monodon at the concentration of 3 μg GIH-dsRNA per gram body weight of shrimp was able to inhibit GIH expression for a minimum of 30 days. This dsRNA-mediated GIH silencing led to ovarian maturation and eventual spawning in both domesticated and wild female broodstock, particularly with a comparable effect to eyestalk ablation in wild shrimp. This is the first report that demonstrates a potential strategy to induce ovarian maturation in female P. monodon broodstock by GIH-dsRNA and thus provides a possible substitute for the cruel and detrimental eyestalk ablation practice.     

Chemical Constituent,Antimicrobial Activity,and Synergistic Effect of the Stem,Leaf, and Flower Essential Oil of the Artemisia fragrans Willd. from Khoy

Artemisia fragrans is commonly used as a folk medicine as antispasmodic, anti-pyretic, anti-inflammatory, and abortifacient agents. The villagers use its pungent odor to repel rodents, mites, and pests, as well as its essential oil and smoke after burning to treat lung infections after uprooting the plant. Herein, we extracted the essential oils (EOs) of different parts of the plant and analyzed their chemical compositions and antibacterial activities. The chemical analysis led to the identification of 73, 59, and 57 compounds in the EOs of the stem, leaf, and flower, respectively. All of the EOs exhibited antibacterial activities against both G+ and G− bacteria. The EOs of the leaf and flower were more effective against tested bacteria, except B. anthracis and P. aeruginosa, compared to that of the stem. The binary combination of the EOs (stem and flower) or (stem and leaf) showed a synergistic effect. Statistical analysis indicated EOs of leaf and flower are more potent than that of the stem. These findings suggest the application of leaf and flower of the plant, which not only can prevent its uprooting but also ensure better therapeutic function.     

Distribution of Putative Virulence Genes and Antimicrobial Drug Resistance in <Emphasis Type="Italic">Vibrio harveyi</Emphasis>

The marine-estuarine bacterium Vibrio harveyi is an important pathogen of invertebrates, most significantly, the larvae of commercially important shrimp Penaeus monodon. In this study, we analyzed V. harveyi isolated from shrimp hatchery environments for understanding the distribution of putative virulence genes and antimicrobial drug resistance. The putative genes targeted for PCR detection included four reversible toxin (Rtx)/hemolysin genes, a gene encoding homologue of Vibrio cholerae zonula occludens toxin (Zot) and a hemolysin-coregulated protein gene (hcp) by polymerase chain reaction (PCR). Of the four putative reversible toxin genes, vhh-1 was detected in 31% of the isolates, vhh-2 in 46%, vhh-3 in 23% and vhh-4 was detected in 27% of the isolates. A zot-like sequence of bacteriophage f237 was present in 15%, while hcp sequence was detected in 48% of the isolates. The antimicrobial susceptibility test revealed resistance to several groups of antibiotics including β-lactams, cephalosporins, macrolides, quinolones, nitrofurantoin and tetracycline.     

Isolation and characterization of 23 polymorphic microsatellite markers for diversity and stock analysis in tiger shrimp (Penaeus monodon)

The tiger shrimp (Penaeus monodon) is an important marine crustacean in terms of biological diversity and aquaculture resource. The shrimp is widespread across the Indo‐Pacific region and shows apparent genetic differentiation among geographical populations. It is common practice to transport female brooders between different countries to seed the shrimp farms, posing potential problems of unwanted population admixture. We developed 23 polymorphic microsatellites for P. monodon (average H_E = 0.936) and these microsatellites were applicable for studying population differentiation, identifying valid stocks and tagging nonindigenous farmed shrimps.     

The application of bioflocs technology to protect brine shrimp (Artemia franciscana) from pathogenic Vibrio harveyi

Aims: To study the potential biocontrol activity of bioflocs technology. Methods and Results: Glycerol‐grown bioflocs were investigated for their antimicrobial and antipathogenic properties against the opportunistic pathogen Vibrio harveyi. The bioflocs did not produce growth‐inhibitory substances. However, bioflocs and biofloc supernatants decreased quorum sensing‐regulated bioluminescence of V. harveyi. This suggested that the bioflocs had biocontrol activity against this pathogen because quorum sensing regulates virulence of vibrios towards different hosts. Interestingly, the addition of live bioflocs significantly increased the survival of gnotobiotic brine shrimp (Artemia franciscana) larvae challenged to V. harveyi. Conclusions: Bioflocs grown on glycerol as carbon source inhibit quorum sensing‐regulated bioluminescence in V. harveyi and protect brine shrimp larvae from vibriosis. Significance and Impact of the Study: The results presented in this study indicate that in addition to water quality control and in situ feed production, bioflocs technology could help in controlling bacterial infections within the aquaculture pond.     

The resistance to ammonia stress of Penaeus monodon Fabricius juvenile fed diets supplemented with astaxanthin

This study was aimed at determining if the increase of body astaxanthin content through dietary supplementation in tiger prawn Penaeus monodon juvenile could enhance its antioxidant defense capability and resistance to ammonia stress. Haemolymph total antioxidant status (TAS) and superoxide dismutase (SOD) were chosen as parameters of shrimp antioxidant capacity. Resistance to chemical stress was evaluated by shrimp survival rate, and haemolymph aspartate aminotransferase (AST) and alanine aminotransferase (ALT). P. monodon 5-day postlarvae were fed diets supplemented with 0 and 71.5 mg kg⁻¹ astaxanthin for 8 weeks. Shrimps were then subjected to 72-h exposure of ammonia at 0.02, 0.2, 2 and 20 mg l⁻¹. The survival rates of the astaxanthin-fed (AX) shrimp were higher than those of the control shrimp under all levels of ammonia except 20 mg l⁻¹, showing that the shrimp's resistance to ammonia stress had been improved by dietary astaxanthin. AX shrimp had higher TAS than control shrimp at ammonia levels higher than 0.02 mg l⁻¹ and lower SOD at all ammonia levels suggested that antioxidation capability had been greatly enhanced. AST in AX shrimp was lower than that in control shrimp under all levels of ammonia stress. ALT in AX shrimp was either lower than or equal to that in control shrimp under various levels of ammonia. Both AST and ALT reflected that shrimp hepatopancreatic function had been improved by dietary astaxanthin. Astaxanthin can become essential for P. monodon when the animal is under ammonia stress.     

Antimicrobial and anti‐inflammatory activity of five Taxandria fragrans oils in vitro

The antimicrobial activity of five samples of Taxandria fragrans essential oil was evaluated against a range of Gram‐positive (n= 26) and Gram‐negative bacteria (n= 39) and yeasts (n= 10). The majority of organisms were inhibited and/or killed at concentrations ranging from 0.06–4.0% v/v. Geometric means of MIC were lowest for oil Z (0.77% v/v), followed by oils X (0.86%), C (1.12%), A (1.23%) and B (1.24%). Despite differences in susceptibility data between oils, oils A and X did not differ when tested at 2% v/v in a time kill assay against Staphylococcus aureus. Cytotoxicity assays using peripheral blood mononuclear cells demonstrated that T. fragrans oil was cytotoxic at 0.004% v/v but not at 0.002%. Exposure to one or more of the oils at concentrations of ≤0.002% v/v resulted in a dose responsive reduction in the production of proinflammatory cytokines IL‐6 and TNF‐α, regulatory cytokine IL‐10, Th1 cytokine IFN‐γ and Th2 cytokines IL‐5 and IL‐13 by PHA stimulated mononuclear cells. Oil B inhibited the production of all cytokines except IL‐10, oil X inhibited TNF‐α, IL‐6 and IL‐10, oil A inhibited TNF‐α and IL‐6, oil C inhibited IL‐5 and IL‐6 and oil Z inhibited IL‐13 only. IL‐6 production was significantly inhibited by the most oils (A, B, C and X), followed by TNF‐α (oils A, B and X). In conclusion, T. fragrans oil showed both antimicrobial and anti‐inflammatory activity in vitro, however, the clinical relevance of this remains to be determined.     

RpoN gene,RAPD profile,antimicrobial resistance and plasmids of Vibrio anguillarum isolates from vibriosis infected Penaeus monodon

Aim: To evaluate the diversity of Vibrio anguillarum isolates from vibriosis‐infected Penaeus monodon collected from east coast of India. Methods and Results: Thirty‐six V. anguillarum were cultured from specific V. anguillarum medium, further identified using biochemical tests and confirmed by PCR detection of rpoN gene. Randomly amplified polymorphic DNA analysis revealed that in each location, the selected V. anguillarum isolates produced a unique band pattern, indicating that the members of this species are genetically heterogeneous. Antibiotic sensitivity results showed that 85%, 72%, 70%, 58%, 45% and 34% of the isolates were resistant to erythromycin, furazolidone, chloramphenicol, oxolinic acid, ciprofloxacin and nitrofurantoin, respectively. Plasmids were found in 70% of the isolates, and nine different plasmid profiles were observed. Conclusions: Wide ranges of diversity were noted in V. anguillarum isolates collected from P. monodon at different locations of east coast of India. Significance and Impact of the Study: Molecular typing, antibiotic resistance and plasmid profiles of V. anguillarum isolates from shrimp in India enables the prediction of possible risk for diseases in shrimp culture environment and the application of alternative management plans to prevent further spread of antibiotic resistance.     

Chemical Diversity and Antimicrobial Activity of Salvia multicaulis Vahl Essential Oils

The chemical compositions and antimicrobial activities of the essential oils (EOs) of aerial parts of Salvia multicaulis Vahl , collected during the same week from two different Lebanese regions, were investigated. The EOs were obtained by hydrodistillation using a Clevenger‐type apparatus and characterized by GC and GC/MS analyses. The minimum inhibitory concentrations of these EOs were determined against one Gram‐negative and two Gram‐positive bacteria, one yeast, and five dermatophytes using the broth microdilution technique. One EO was notably active against Staphylococcus aureus, methicillin‐resistant S. aureus, and all of the Trichophyton species tested. Nerolidol was found to be the major compound in the active oil; nerolidol was also absent from the inactive oil. This study demonstrated that nerolidol shows antimicrobial activity and therefore significantly contributes to the antimicrobial potential of the oil. The chemical diversity of worldwide S. multicaulis EOs was analyzed, revealing that the EOs of this study belong to two different chemotypes found in the literature. The nerolidol chemotype appears to be restricted to Lebanon, and it can be used as antimicrobial agent against external bacterial and fungal infections.     

Synergistic larvicidal and repellent effects of essential oils of three Origanum species on Rhipicephalus annulatus tick

Ticks are of great economic importance worldwide, both because they represent major obstacles to livestock productivity and because of their ability to transmit diseases to humans and animals. Although synthetic acaricides are the most common method for tick control, their overuse has led to the development of resistance as well as unacceptable residual levels in animal products and in the environment in general. There is therefore an urgent need to identify alternative treatments. Among such alternative approaches for tick control is plant essential oil (EO) therapy. In the present study, we investigated the synergistic effect of EOs of three oregano species—Origanum onites, O. majorana and O. minutiflorum—against Rhipicephalus annulatus larvae. Gas chromatography–mass spectrometry profiles of the three EOs revealed that carvacrol was their major component, with a concentration of 86.2% in O. majorana, 79.1% in O. minutiflorum and 77.4% in O. onites. The results of larvicidal assays revealed that the doses that lead to the death of 50% of the ticks (LC₅₀) were 22.99, 25.08 and 27.06 µL/mL for O. majorana, O. minutiflorum and O. onites EOs, respectively, whereas the doses that lead to the death of 99% (LC₉₉) were 41.26, 43.62 and 48.96 µL/mL. In addition, the LC₅₀ and LC₉₉ of the three oils combined was lower (viz., 4.01 and 6.97 µL/mL) than that of each oil alone. The tested EOs were also able to repel larvae of R. annulatus to varying degrees, with O. onites oil exhibiting the greatest repellent effect, as shown by the lowest RC₅₀ dose, followed by O. minutiflorum and O. majorana. Interestingly, this means that the oil that was least effective in killing the larvae was the most effective in repelling them. The calculated synergistic factor of any combination was higher than 1 which means that combinations have a synergistic effect. In conclusion, the combination of all three oils showed higher toxic and repellent activities than either oil separately or combinations of any two oils, suggesting synergistic effects with low doses. Further studies including field trials and the establishment of the mode of action and side effects are urgently needed to expand on these findings, and other tick stages such as adults should also be tested.

     

Impact of live food on survival and growth of hatchery‐reared sea trout (Salmo trutta trutta L.) parr in the wild

Survival rates and growth parameters of hatchery‐reared sea trout (Salmo trutta trutta L.) fry were determined after stocking in the wild. The larvae were hatchery‐reared for 12 weeks in two groups: fry were fed either on live zooplankton and live chironomidae larvae (LFG), or fed a pellet diet (PFG). The survival rate and specific growth rates were higher in the LFG than in the PFG group. Most effective for hatchery‐reared fish intended for stocking was the natural, live feed. The mean number of chironomid larvae found in the stomachs of fish that were initially captured in the wild was significantly higher in the LFG than in the PFG group. The live diet supplied in the rearing period had a positive impact on the foraging skills of the sea trout fry and their survival in the wild after their release on 24 April 2010.     

Effect of stocking density on growth and survival of Clarias batrachus (Linn.) larvae and fry during hatchery rearing

Experiments were conducted to determine optimum stocking density for Clarias batrachus larvae and fry during hatchery rearing. The increase in stocking density decreased the total weight, specific growth rate (SGR) and percent weight gain of Clarias larvae during a 13‐day experiment. Survival rate was highest at a stocking density of 1000 m^?2 and lowest at 5000 m^?2. Stocking density did not influence the total biomass production of larvae. Clarias batrachus fry performance was studied during a 28‐day hatchery rearing experiment whereby fry stocked at a density of 100 m^?2 attained the highest total body weight (P < 0.05). The survival rate greatly declined to 59–61% by a density increase to 300 m^?2 and above. Stocking density influenced growth and survival of C. batrachus larvae and fry during hatchery rearing. The best performance was obtained when larvae were stocked at 2000 m^?2; survival was highest with C. batrachus fry stocked at 200 m^?2.     

Presence and development of populations of the introduced brown alga Sargassum muticum in the southwest Netherlands

Of the three Brachionus species used in aquaculture, Brachionus rubens, B. calycilorus and B. plicatilis, the latter is most widely used in raising marine fish and shrimp larvae due to its tolerance to the marine environment. In freshwater aquaculture the use of B. rubens and B. calycilorus is limited, probably because inert food products are readily available as feed for freshwater larvae.The rotifer Brachionus plicatilis is used in large numbers as the first food organism in intensive cultures of marine fish and shrimp larvae. An adequate supply of these rotifers relies on mass cultures. The reproductive rate of rotifers in these cultures depends on food quality and quantity, salinity, temperature and pH of the medium. Removal of waste products from culture tanks leads to higher and more efficient production of rotifers over extended periods of time. Rotifers have to be enriched with polyunsaturated fatty acids, which are essential for proper development and survival of marine fish and shrimp larvae.The future use of preserved rotifers and their resting eggs may help to overcome unforeseen failures of live cultures and may lead to more efficient use of these organisms in raising freshwater and marine fish and shrimp larvae.     

Indole analogues decreasing the virulence of Vibrio campbellii towards brine shrimp larvae

Indole signalling has been proposed as a potential target for the development of novel virulence inhibitors to control bacterial infections. However, the major structural features of indole analogues that govern antivirulence activity remain unexplored. Therefore, we investigated the impact of 26 indole analogues on indole-regulated virulence phenotypes in Vibrio campbellii and on the virulence of the bacterium in a gnotobiotic brine shrimp model. The results demonstrated that 10 indole analogues significantly increased the fluorescence of indole reporter strain Vibrio cholerae S9149, 21 of them decreased the swimming motility of V. campbellii, and 13 of them significantly decreased the biofilm formation of V. campbellii. Further, we found that 1-methylindole, indene, 2,3-benzofuran, thianaphthene, indole-3-acetonitrile, methyl indole-3-carboxylate, 3-methylindole, and indole-2-carboxaldehyde exhibited a significant protective effect on brine shrimp larvae against V. campbellii infection, resulting in survival rates of challenged brine shrimp above 80%. The highest survival of shrimp larvae (98%) was obtained with indole-3-acetonitrile, even at a relatively low concentration of 20 μM. Importantly, the indole analogues did not affect bacterial growth, both in vitro and in vivo. These results indicate the potential of indole analogues in applications aiming at the protection of shrimp from vibriosis.     

Chemical Composition,Antimicrobial and Insecticidal Activities of Flowers Essential Oils of Alpinia zerumbet (Pers.) B.L.Burtt & R.M.Sm. from Martinique Island

Alpinia species, used as ornamental plants and flavoring agents, are renowned for their therapeutic properties and their subsequent use in traditional medicines throughout the world. Alpinia zerumbet (Pers .) B.L.Burtt & R.M.Sm . is the most common Alpinia species encountered in Martinique. Several essential oils (EOs) obtained by hydrodistillation of A. zerumbet flowers collected in various locations on the island at different seasons were analysed to evaluate the influence of the collection period and area on the EOs’ chemical compositions and to assess their bioactivity. A combination of GC‐FID and GC/MS techniques was used to examine the volatile constituents, leading to the identification of a total of 71 components accounting for 97.8 – 99.3% of the respective total GC‐FID areas: among them, nineteen compounds were characterized for the first time in A. zerumbet EOs. The antimicrobial activity of these EOs was assessed against eight bacterial, yeast and fungal strains and two multi‐resistant strains: some significant bacteriostatic and fungistatic activities of A. zerumbet flower oils were evidenced. Finally, an interesting insecticidal activity of the flower EO was highlighted for the first time against Aedes aegypti.     

Bdellovibrio and Like Organisms Enhanced Growth and Survival of Penaeus monodon and Altered Bacterial Community Structures in Its Rearing Water

In this study, a 96-h laboratory reduction test was conducted with strain BDHSH06 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF011103","term_id":"120561011","term_text":"EF011103"}}EF011103) as the test strain for Bdellovibrio and like organisms (BALOs) and 20 susceptible marine bacterial strains forming microcosms as the targets. The results showed that BDHSH06 reduced the levels of approximately 50% of prey bacterial strains within 96 h in the seawater microcosms. An 85-day black tiger shrimp (Penaeus monodon) rearing experiment was performed. The shrimp survival rate, body length, and weight in the test tanks were 48.1% ± 1.2%, 99.8 ± 10.0 mm, and 6.36 ± 1.50 g, respectively, which were values significantly (P < 0.05) higher than those for the control, viz., 31.0% ± 2.1%, 86.0 ± 11.1 mm, and 4.21 ± 1.56 g, respectively. With the addition of BDHSH06, total bacterial and Vibrio numbers were significantly reduced (P < 0.05) by 1.3 to 4.5 log CFU · ml⁻¹ and CFU · g⁻¹ in both water and shrimp intestines, respectively, compared to those in the control. The effect of BDHSH06 on bacterial community structures in the rearing water was also examined using PCR amplification of the 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE). The DGGE profiles of rearing water samples from the control and test tanks revealed that the amounts of 44% of the bacterial species were reduced when BDHSH06 was added to the rearing water over the 85-day rearing period, and among these, approximately 57.1% were nonculturable. The results of this study demonstrated that BDHSH06 can be used as a biocontrol/probiotic agent in P. monodon culture.     

Loop‐mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei in penaeid shrimp

Aims

Enterocytozoon hepatopenaei is an emerging microsporidian parasite that has been linked to recent losses caused by white faeces syndrome (WFS) in cultivated giant or black tiger shrimp Penaeus (Penaeus) monodon and whiteleg shrimp Penaeus (Litopenaeus) vannamei in Asia. To more accurately assess its impact on shrimp production and to determine reservoir carriers for control measures, our objective was to establish a loop‐mediated isothermal amplification (LAMP) assay combined with colorimetric nanogold (AuNP) for rapid, sensitive and inexpensive detection of this parasite.

Methods and Results

A set of six specific primers was designed to successfully detect the SSU rRNA gene of E. hepatopenaei by a LAMP reaction of 45 min at 65°C combined with visual detection of the amplification product via hybridization at 65°C for 5 min with a ssDNA‐labelled nanogold probe, followed by salt‐induced AuNP aggregation (total assay time, approximately 50 min). This method gave similar results to LAMP followed by electrophoresis or spectrophotometric detection, and it was more sensitive (0·02 fg total DNA) than a conventional nested PCR (0·2 fg total DNA). The new method gave negative results with shrimp DNA templates extracted from diseased shrimp containing other pathogens, indicating that the LAMP‐AuNP assay was specific for E. hepatopenaei.

Conclusions

Without sacrificing sensitivity or specificity, the new LAMP‐AuNP assay significantly reduced the time, ease and cost for molecular detection of E. hepatopenaei in shrimp.

Significance and Impact of the study

The new method employs simple, inexpensive equipment and involves simple steps making it applicable for small field laboratories. Wider application of the method to screen broodstock before use in a hatchery, to screen postlarvae before stocking shrimp ponds, to test for natural carriers and to monitor shrimp in rearing ponds would help to assess and reduce the negative impact of this parasite in shrimp farming.     

Stocking density‐dependent growth and survival of Asian sun catfish,Horabagrus brachysoma (Gunther 1861) larvae

The aim of the experiment was to evaluate the growth and survival of Horabagrus brachysoma larvae at different stocking densities (3, 7, 13, 20, 27 and 33 larvae L^?1) during their hatchery phase. Total length and wet weight of the larvae consistently decreased (P < 0.05) at the end of 14 and 28 days of rearing as the density increased. The specific growth rate was significantly (P < 0.05) highest at three larvae L^?1 compared to the other five densities. The percent weight gain and survival of larvae was also highest at lowest density. The observation corroborates that catfish larvae can be reared at low densities in stagnant water conditions. Considering the value of larval growth, survival and overall weight gain, the stocking density of seven larvae L^?1 has been identified as the maximum for larval rearing of H.  brachysoma under hatchery conditions.