Screening of marine Streptomyces spp. for potential use as probiotics in aquaculture

Aquaculture 305 (2010) 32–41

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Screening of marine Streptomyces spp. for potential use as probiotics in aquaculture Surajit Das ⁎, Louise R. Ward, Chris Burke National Centre for Marine Conservation and Resource Sustainability, Australian Maritime College, University of Tasmania, Locked Bag 1370, Launceston, Tasmania 7250, Australia

a r t i c l e

i n f o

Article history: Received 7 January 2010 Received in revised form 30 March 2010 Accepted 1 April 2010 Keywords: Artemia Penaeus monodon Probiotics Streptomyces Vibrio Survival Protection

a b s t r a c t Marine Streptomyces strains (CLS-28, CLS-39 and CLS-45) were used to colonise Artemia nauplii (Instar I) and 15 d old adult Artemia prior to challenge with Vibrio harveyi and V. proteolyticus. The LC50 of V. harveyi and V. proteolyticus was found to be ∼ 106 CFU ml− 1. V. proteolyticus was more pathogenic than V. harveyi at 106 CFU ml− 1. A significant reduction in mortality (P b 0.001) was found by addition of 1% wet cell mass of Streptomyces strains in nauplii and adult Artemia against both the pathogens. The best protective responses were shown by CLS-39 in both nauplii and adults against V. harveyi and by CLS-39 in nauplii and CLS-28 in adults against V. proteolyticus. Shrimp feeds were supplemented with Streptomyces cell mass at 1% dosage and fed to black tiger shrimp Penaeus monodon postlarvae for 15 d in three treatments with two treatments of commercial probiotic (T1: feed + CLS-28; T2: feed + CLS-39; T3: feed + CLS-45; T4: feed + Sanolife® commercial probiotic and T5: Sanolife® commercial probiotic in water). During this time, ammonia was in the range of 1 to 2 ppm in all the treatments with significant differences between treatments (P b 0.05). Significant differences (P b 0.05) were also found in survival, total length and wet weight of the shrimp postlarvae during the 15 d trial. T5 showed the best gains in terms of length and weight followed by T1, T2, T3 and T4. Streptomyces treatments T1, T2 and T3 showed better survival and higher length and weight than the control and T4. Total heterotrophic bacteria and Vibrio counts were in the range of 108 and 106 CFU ml− 1 respectively in all the treatments. The Vibrio population differed significantly in the treatments (P b 0.05) and the total bacterial counts showed no significant differences in the treatments (P N 0.05). After challenge with V. harveyi at 107 CFU ml− 1, highest survival was found in T1 and T5. Among the Streptomyces treatments, T1 showed significantly higher survival compared to the control, followed by T2 and T3. Thus Streptomyces strains show promise as probiotic agents in mariculture. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Probiotics which compete with bacterial pathogens for nutrients and/ or inhibit the growth of pathogens can be a valid alternative to the prophylactic application of antibiotics and biocides. Fuller (1989) defined a probiotic as ‘a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance’. A modified and more appropriate definition was proposed by Verschuere et al. (2000a) — ‘a live microbial adjunct which has a beneficial effect on the host by modifying the host-associated or ambient microbial community, by ensuring improved use of the feed or enhancing its nutritional value, by enhancing the host response towards disease, or by improving the quality of its ambient environment’. Actinobacteria is a class with five subclasses that was proposed by Stackebrandt et al. (1997) to group the highly diverse so called actinomycetes based on chemical composition, DNA–DNA reassociation and 16S rRNA gene sequence similarities. Members of the ⁎ Corresponding author. Present address: Department of Life Science, National Institute of Technology, Rourkela-769 008, Orissa, India. Tel.: +91 661 2462684; fax: +91 661 2462022. E-mail addresses: [email protected], [email protected] (S. Das). 0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.04.001

Actinobacteria are prolific sources of secondary metabolites and the vast majority of these compounds are derived from the single genus Streptomyces. Streptomyces is a Gram-positive aerobic genus in the order Actinomycetales, suborder Streptomycineae and family Streptomycetaceae (Stackebrandt et al., 1997) and has a DNA G + C content of 69–78 mol%. Marine-derived Streptomyces have been studied for isolation of several novel secondary metabolites (Fenical and Jensen, 2006; Das et al., 2006a). However, to date there have only been a few studies that have considered Actinobacteria for their application as probiotics in aquaculture. We have reported the prospects of using marine Actinobacteria as probiotics in aquaculture (Das et al., 2008a) and began screening marine Actinobacteria for use as new biocontrol agents for aquatic animals. We report here the effect of three marine Streptomyces strains on Artemia and Penaeus monodon. Artemia has long been considered as a model/ test organism to study the mode of action of probiotic bacteria due to its adaptability to wide ranges of salinity and temperature, short life cycle, high adaptability to adverse environmental conditions, high fecundity, parthenogenetic and sexual reproduction strategy (with nauplii or cysts production), small body size, and adaptability to varied nutrient resources (Nunes et al., 2006). There have been several experiments carried out on Artemia in the search for new biocontrol agents for aquaculture

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(Verschuere et al., 1999, 2000b; Villamil et al., 2003; Defoirdt et al., 2006; Marques et al., 2004, 2006). However, there have been only a few efforts (Das et al., 2006b; Kumar et al., 2006) to utilise marine actinobacterial resources as probiotics and practically none of the studies reported the toxicity of the Actinobacteria and protection from pathogens. In the current study, marine Streptomyces were isolated and screened for potential probiotic activities. The LC50 of virulent Vibrio harveyi and V. proteolyticus were examined in Artemia to determine LC50 value, and the efficacy of the marine Streptomyces strains to protect Artemia from Vibrio spp. challenge infection was assessed. P. monodon postlarvae were fed with Streptomyces-supplemented feed and the protection from the challenge infection by V. harveyi was evaluated.

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2.2.2. Exoenzymatic assay Cultured actinomycetes were screened for hydrolytic exoenzymatic activities (amylase, protease and lipase). These tests were conducted on Yeast Extract Malt Extract Agar (ISP 2) medium containing starch for amylolytic activity, skimmed milk for proteolytic activity and Tween 80 for lipolytic activity at 1% concentration. Amylolytic activity was detected by flooding the plates with 1% iodine solution. Presence of amylase was visualized by decolorized halo around the culture due to starch digestion. Proteolytic activity was observed by clearing of the milk and lipolytic activities were observed by the formation of a halo of precipitated fatty acids around the colony. 2.3. Mass culture of selected Streptomyces strains

2. Materials and methods 2.1. Isolation of marine Streptomyces Marine sediment samples were collected from shrimp farms located at Queensland, Australia (lat 21°43′09″S and long 149°25′54″E). To isolate the Streptomyces, aliquots (0.5 ml) of serially diluted (10− 1 and 10− 2) samples were inoculated by spread plate onto Starch Casein Agar (SCA) (Composition: Soluble starch: 10 g, K2HPO4: 2 g, KNO3: 2 g, Casein: 0.3 g, MgSO4·7H2O: 0.05 g, CaCO3: 0.02 g, FeSO4·7H2O: 0.01 g, Agar: 15 g, Filtered sea water: 1000 ml and pH: 7.0 ± 0.1), Yeast Extract Malt Extract Agar (ISP 2) (Composition: Yeast extract: 4 g, Malt extract: 10 g, Dextrose: 4 g, Agar: 15 g, Filtered sea water: 1000 ml and pH: 7.3) and Kuster's Agar (Composition: Glycerol: 10 g, Casein: 0.3 g, KNO3: 2 g, K2HPO4: 2 g, Soluble starch: 0.5 g, Asparagine: 0.1 g, FeSO4.7H2O: 0.01 g, CaCO3: 0.02 g, MgSO4.7H2O: 0.05 g, Agar: 15 g, Filtered sea water: 1000 ml and pH: 7.0 ± 0.1). Each medium was supplemented with nystatin and cyclohexamide at 25 µg ml− 1 and 10 µg ml− 1 respectively to minimize contamination with fungi and 10 µg ml− 1 nalidixic acid to minimize contaminant growth (Takizawa et al., 1993; Ravel et al., 1998). Plates were incubated for 7 to 15 d at 30 °C temperature and then the colonies with a tough or powdery texture, dry or folded appearance and branching filaments with or without aerial mycelia (Mincer et al., 2002) were sub-cultured and transferred on SCA and ISP 2 slants. Until further use, the slants were kept in cold room at 4 °C as described in Das et al. (2008b). 2.2. Screening of potential putative strains 2.2.1. Cross streak assay (Chythanya et al., 2002; Hai et al., 2007): As many as forty three actinomycetes strains were streaked about 0.5 cm width (in order to get 1 cm width culture after incubation) on Tryptone Soya Agar (TSA, OXOID®) and Modified Nutrient Agar (Composition: Glucose: 5 g, Peptone: 5 g, Malt extract: 3 g, Sodium chloride: 10 g, Agar: 15 g, Distilled water: 1000 ml and pH: 7.0 ± 0.1) and the plates were incubated for 7 d at 30 °C temperature. The bulk of the colonies were scraped away with a sterile slide and the remaining growth was killed by exposure to chloroform (70%) for 15 min. The plates were then air dried for 10 min to remove any residual chloroform vapour. Five fish-pathogenic Vibrio strains (V. harveyi V890, V. parahaemolyticus, V. proteolyticus V760, V. anguillirum V572 and V. alginolyticus V34) obtained from the culture collection of Dr. J. Carson (Department of Primary Industries, Parks, Water and Environment, Launceston, Tasmania, Australia) were cultured on Tryptone Soya Agar (TSA, OXOID®) and TCBS medium (OXOID®). Cultures of Vibrio spp. were grown for 18 h and streaked on each plate perpendicular to the chloroform-killed actinomycetes strain. The plates were incubated for 24 h at 30 °C temperature. The width of inhibition zones of each Vibrio species was measured in mm. Test strains that showed growth near or around the actinomycetes strains with no inhibition were considered resistant.

Based on the results from cross streak and enzymatic assays, 3 strains of Streptomyces (CLS-28, CLS-39 and CLS-45) were selected for mass culture. They were each mass cultured in ISP 2 liquid medium in 100 ml Schott flasks incubated at 30 °C for 7 d on a shaker. The cultured cells were harvested by centrifugation (3000 rpm for 10 min) and washed with sterile, normal saline solution (0.85% NaCl). The wet cell mass was kept at 4 °C until used for feed preparation. Several batches of mass culture were run to get adequate amounts of cell mass. 2.4. 16S rRNA gene sequencing and identification of the selected actinomycetes strains Molecular identification of three selected actinomycetes strains (CLS-28, CLS-39 and CLS-45) as potential probionts were carried out by 16S rRNA gene (16S rDNA) amplification and sequencing. 2.4.1. DNA extraction The biomass of three actinomycetes isolates was harvested by centrifugation. Pellet was washed twice in sterile Tris–EDTA buffer and approximately 100 mg (wet weight) biomass was used for DNA extraction based on cetyltrimethylammonium bromide (CTAB) purification following Ausubel et al. (1999). 2.4.2. DNA amplification and sequencing Colony PCR and PCR with extracted chromosomal DNA were conducted using 16 S universal primer — 16S-27F (5′ to 3′ AGAGTTTGATCMTGGCTCAG, M = A or C) and 16S-1492R (5′ to 3′ ACGGCTACCTTGTTACGA) (Geneworks, Australia) in a thermal cycler (Eppendorf Mastercycler Gradient). Polymerase Chain Reaction was performed in 50 µl volumes containing 2 mM MgCl2, 2.5 U Taq polymerase (Bioline, Australia), 100 µM of each dNTP, 0.2 µM of each primer and 3 µl template DNA. The PCR programme used was an initial denaturation at 96 °C for 5 min followed by 30 cycles of 95 °C for 15 s, 49 °C for 30 s and 72 °C for 1 min and a final extension at 72 °C for 1 min. Amplified DNAs were purified by the Montage™ PCR centrifugal filter device (Millipore Corp., USA) following manufacturer's instructions and finally quantified by Turner TBS380 DNA fluorometer. The sequencing reactions were carried out by Australian Genome Research Facility Ltd., Australia with 27F, 529F, 518R, 1073R and 1492R primers to compare the chromatograms and get a clear consensus sequence for each strain. 2.4.3. Phylogenetic analysis Sequence data were compiled and consensus sequence was obtained by using Geneious 3.8.5 programme and examined for sequence homology with the archived 16S rDNA sequences from GenBank at www.ncbi.nlm.nih.gov/nucleotide, employing the BLAST. Multiple alignments of sequences were performed with the ClustalX (1.83) program (Thompson et al., 1997). A phylogenetic tree was constructed using the neighbour-joining DNA distance

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S. Das et al. / Aquaculture 305 (2010) 32–41

algorithm (Saitou and Nei, 1987) using DAMBE 5.0.25 (Data Analysis in Molecular Biology and Evolution, http://dambe.bio.uottawa.ca/ dambe.asp). The resultant tree topologies were evaluated by bootstrap analysis (Felsenstein, 1985) of neighbour-joining data sets based on 1000 resamplings. 2.4.4. GenBank submission The partial sequences of the 16S rRNA gene of three isolates were submitted to NCBI GenBank and the assigned accession numbers are: CLS-28 (FJ200295), CLS-39 (FJ200296) and CLS-45 (FJ200297). 2.5. Artemia experimental design 2.5.1. Biotoxicity test of marine Streptomyces strains Harvested wet cell mass from three strains was examined for toxicity in Artemia. Experiments were conducted in sterile polystyrene 12-well cell culture plates. Five concentrations (0.1%, 0.5%, 1%, 5% and 10%) of cell mass in sterile seawater (v/v) were used in the experiment. Untreated control animals were kept in sterile sea water and the experiments was performed in quadruplicate. Instar I nauplii and adult Artemia were kept in the wells containing 5 ml of airsaturated sea water with different concentrations of the cell mass suspensions and the numbers of Artemia were counted. Plates were closed and incubated at 28 °C for 72 h. Mortality was determined after 24, 48 and 72 h and the dead animals were removed. At the end of the experiment, 0.5 ml of 16% formaldehyde was added to each well to kill all remaining animals following Caldwell et al. (2003) and the exact initial numbers of animals used in the experiments were cross checked by summing up the number of individuals. 2.5.2. Virulence of V. harveyi and V. proteolyticus V. harveyi V890 and V. proteolyticus V760 were grown at 28 °C on Tryptone Soya agar (TSA, OXOID®). Tryptone Soya broth (TSB, OXOID®) was then inoculated with single colonies and incubated for 24 h at 28 °C. Viable cells were counted by haemocytometer and harvested by centrifuging at 3000 rpm for 10 min. The harvested cells were suspended into an equal volume of sterilised sea water. This was serially diluted to obtain 108 to 105 CFU ml− 1 and used in the experiments to determine virulence. Aliquots of each dilution were plated on TSA to confirm the CFU ml− 1. Virulence testing was carried out on Artemia in 12-well cell culture plates. Nauplii and adults were immersed in 24 h cultures of either V. proteolyticus or V. harveyi at 105 to 108 CFU ml− 1. Control animals were kept in 4 fold diluted TSB. Plates were closed and incubated at 28 °C and the percentage mortality (including moribund individuals) was calculated after 24 h. The virulence of V. harveyi and V. proteolyticus was determined from the LC50 (Azad et al., 2005) following the method of Reed and Muench (1938). 2.5.3. Artemia challenge experiment and protection by marine Streptomyces The capacity of three Streptomyces strains (CLS-28, CLS-39 and CLS-45) to reduce mortality in Artemia during challenge by V. harveyi or V. proteolyticus was observed for nauplii and 15-day old Artemia. In 12-well cell culture plates nauplii and adult Artemia were grown with three strains of Streptomyces at 1% concentration (v/v) in air saturated sterile sea water together with 24 h cultures of V. harveyi or V. proteolyticus at the final concentration of 106 CFU ml− 1. Control animals were kept in sea water. Plates were closed and incubated at 28 °C. Survival was assessed by scoring the number of dead animals on the bottom of each plate using an inverted microscope. Dead animals were removed after counting and the percentage of surviving Artemia was calculated after 24, 48 and 72 h by recording the number of dead animals on the bottom of each plate. After 72 h, 0.5 ml of 16% formaldehyde was added to each well to kill all remaining animals and the exact initial numbers of animals used in the experiments were

cross checked by summing up the number of dead and surviving individuals in each well. 2.6. Experiment with black tiger shrimp (P. monodon) 2.6.1. Acclimatization Healthy black tiger shrimp (P. monodon) postlarvae (PL16) from Rocky Point Hatchery, Queensland were acclimatised in a large glass tank with gravel bed prior to experimental trial. Unhealthy or injured animals were removed during this period. PL were fed to satiation with a shrimp feed from Ridley Aqua-Feed, Queensland, Australia via an automatic feeder. The salinity and temperature of the water ranged from 34 to 35 ppt and 25 to 28 °C respectively. 2.6.2. Experimental set-up Polyvinyl jugs of 2 l capacity were used in the experiment. Jugs were filled with 1.5 l of water and each jug stocked with 25 shrimps at PL33 stage. The average length and weight of the animals were determined by digital calliper and electronic balance (3 decimal points) respectively. Air was supplied to each jug via Millipore (0.22 µ) filters to prevent contamination during aeration and the top was covered with polythene wrap to prevent spillage and escape of shrimps. All experiments were conducted in triplicate. Control animals (without any treatment and fed with the reference feed) were also kept in triplicate jugs. A photoperiod of 12 h dark and 12 h light was maintained throughout the experimental period. 2.6.3. Preparation of feed Commercial shrimp feed (Enhance starter feed, Ridley Aquafeeds, Queensland) was used as the reference feed and 4 experimental feeds were prepared from it: i) Feed 1 — supplemented with 1% CLS-28 cell mass; ii) Feed 2 — supplemented with 1% CLS-39 cell mass; iii) Feed 3 — supplemented with 1% CLS-45 cell mass; iv) Feed 4 — supplemented with 1% Sanolife® MIC (as the treatments were also in the same concentration). All the feeds were prepared by adding 30% water to the dry ingredient mixture, extruding the dough through a 1 mm die (Italpast) and drying for 2–4 h to keep the final moisture level at 10%. The reference feed was prepared by mixing with distilled water. Streptomyces cell mass were supplemented while preparing the feed at 1% concentration (w/v). For 200 g feed mixture the required cell mass was calculated as 2 ml. This cell mass and ∼ 60 ml water was mixed with the feed mixture to make dough and small crumble feed was prepared by extruding. Then the crumble feed was air dried for 2– 4 h to get ∼ 10% water. This dried feed was ground for the experimental trial and kept in the freezer. 2.6.4. Virulence check of test pathogens A virulence test of V. harveyi was carried out on PL40 (as during the challenge experiments the treated animals were also about the same age). V. harveyi V890 incubated in TSB for 24 h, at 28 °C centrifuged at 3000 rpm for 10 min and the harvested cells serially diluted in sterile sea water to obtain 107 to 103 CFU ml− 1. Aliquots of each dilution were plated on TSA to confirm the CFU ml− 1. Shrimp postlarvae were immersed for 12 h in duplicate vessels containing either 103, 104, 105, 106 or 107 CFU ml− 1 of V. harveyi. Control postlarvae were immersed in sterile sea water for 12 h. After the immersion, postlarvae were released into the jugs and mortality was observed for 5 d. The virulence of V. harveyi on PL40 P. monodon was determined from the LC50 as described above for Artemia. 2.6.5. Protection of PL from V. harveyi P. monodon PL33 were dispersed into 1.5 l volume jugs (25 animals per jug) and fed one of 6 diets for 15 days: (A) Control — reference feed; (B) T1 — Feed 1; (C) T2 — Feed 2; (D) T3 — Feed 3; (E) T4 — Feed 4; (F) T5 — reference feed + Sanolife® MIC in water (1 g/tonne after

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germination following manufacturer details). All treatments and the control were replicated five times. All the shrimps received prepared feed twice daily (at dawn and dusk) at approximately 5–8% body weight day− 1. The amount of feed was determined by feeding ad libitum and altered based on previous consumption. Uneaten feed and faces were removed by siphoning daily in the morning. 2.6.6. In-trial management Feeds were analysed for total heterotrophic bacteria (THB), Vibrio count and Streptomyces recovery before feeding. Shrimp survival (mean ± SD) was determined daily in each jug. Water samples were collected daily from each jug and analysed for total heterotrophic bacteria (on Johnson's marine agar), Vibrio population (on TCBS agar, OXOID®) and nutrients: ammonium, nitrate, nitrite (by API™ Saltwater Master Test Kit, USA) and expressed in CFU ml− 1 for microbial counts and ammonia, nitrate and nitrite in ppm. pH of water (by Whatman pH paper), temperature (by centigrade thermometer) and salinity (by refractometer) were also monitored daily. Every week, shrimp faeces and a few live shrimps from each jug (from T1, T2 and T3) were assessed for the presence of Streptomyces by culture on SCA and ISP 2 media. 2.6.7. Pathogen challenge and survival/protection test After feeding experimental feeds to shrimps for 15 d, challenge tests were performed with V. harveyi at 107 CFU ml− 1 (Rengpipat et al., 1998). Shrimps were removed from their jugs and exposed to V. harveyi for 12 h, and then replaced into their original jugs and survival monitored for five days. 2.7. Statistical analyses Data are presented as mean ± standard error. Significance of difference between different treatment groups was tested using one-way analysis of variance (ANOVA) and significant results were compared with Tukey's HSD post-hoc test. Homogeneity of variance was assessed by residual plots. For all the tests the significance was determined at the level of P b 0.05.

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successful on ISP 2 medium. Out of 43 strains isolated, 12 strains were found to have antimicrobial activity as demonstrated in the cross streak assay. Of the 12 active strains, 2 were active against all 5 pathogens, 8 against 4 pathogens, 1 against 3 and 1 was active against 2 pathogens. All but one isolate was active against V. alginolyticus, but only 8 isolates were inhibitory to V. proteolyticus or V. harveyi. All the strains showed proteolytic activity, but variable amylolytic and lipolytic activities. The characteristics of the colony morphology and the antagonism and enzymatic assays of the selected strains are described in Table 1. 3.2. Identification of the selected Streptomyces strains Evaluating the results from cross streak and enzymatic assays three strains, viz. CLS-28, CLS-39 and CLS-45 were chosen for further study. Based on the sequence of the 16S rRNA gene, the selected isolates were identified as Streptomyces. The phylogenetic tree for the three strains with other Streptomyces is shown in Fig. 1. 3.3. Experiments on Artemia 3.3.1. Biotoxicity test of marine Streptomyces on Artemia The percentage of mortality of Artemia nauplii (Instar I) and adult Artemia with different concentrations of the three Streptomyces strains (CLS-28, CLS-39 and CLS-45) is depicted in Table 2. The LC50 (lethal dose for 50% of a sample population) of CLS-28 on nauplii and adults were 7.0 and 8.0% cell mass per unit volume respectively (after 72 h). The LC50 of CLS-39 on nauplii and adult was 8.0 and 9.3% respectively and the LC50 of CLS-45 on nauplii and adult was 6.5 and 7.0% respectively. Significantly higher (F = 69.71, P b 0.01) mortality was observed by CLS-45 strain for both nauplii (67.7%) and adult (64.3%) with the increase of cell mass concentration.

3.1. Isolation of marine Streptomyces and activities of selected strains

3.3.2. Virulence of V. harveyi and V. proteolyticus V. proteolyticus was found to be more virulent than V. harveyi on both nauplii and adult (Table 3). LC50 values for V. harveyi on Artemia nauplii and adults were found to be 105.2 and 105.3 CFU ml− 1 respectively and for V. proteolyticus those were 10 5 and 105.1 CFU ml− 1 respectively (Table 3). As the LC50 was higher than 105 CFU ml− 1 for both the stages of Artemia, the challenge and survival experiment was conducted with 106 CFU ml− 1.

Actinomycete colonies were readily isolated from marine sediments on SCA and ISP 2, but on Kuster's agar growth was either very poor or nil. However, further sub-culture for purification was only

3.3.3. Protection of Artemia by Streptomyces strains The three Streptomyces strains (CLS-28, CLS-39 and CLS-45) were able to protect the Artemia nauplii and adults from the pathogenic

3. Results

Table 1 Growth characteristics of marine actinomycetes on culture media, enzymatic activities and results of cross streak assay against Vibrio pathogens. Strain nos.

Colour of the strainsa Obverse

Reverse

CLS-28 CLS-29 CLS-30 CLS-31 CLS-32 CLS-33 CLS-39 CLS-42 CLS-43 CLS-45 CLS-46 CLS-48

White White White White White White/Grey White/Grey White/Grey White/Grey White White White

White/Yellow White/Yellow White/Yellow White/Yellow White/Pink Yellow/Green Black White White White/Grey White/Grey White

a

Growth on mediab ISP 2, MA ISP 2, MA ISP 2, SCA ISP 2, SCA ISP 2, SCA, MA ISP 2, SCA ISP 2, SCA, MA ISP 2 ISP 2 ISP 2, MA ISP 2, SCA SCA, MA

Enzymatic activitiesc

Cross streak assayd

Amylolytic

Proteolytic

Lipolytic

V.h

V.pa

V.pr

V.an

V.al

+++ − − − ++ +++ +++ +++ +++ − ++ −

++ + ± ++ +++ + +++ +++ +++ +++ + +++

− ± +++ + + ± ++ − − + ± +

+++ + + − − + +++ + + + − +++

+++ + + ++ + + ++ ++ ++ +++ − −

++ − − + ++ − +++ − + ++ ++ +

+ ++ ++ + +++ + − + − + − ++

+++ +++ ++ ++ − ++ + ++ + + +++ ++

On Yeast extract malt extract agar (ISP 2) medium. Different media: ISP 2 — Yeast extract malt extract agar; SCA — Starch casein agar; MA — Milk agar. Presence or absence of the activity determined by the magnitude of zones of activity: +++, good; ++, medium; ±, doubtful; −, nil. d Against the listed pathogens — V.h — Vibrio harveyi, V.pa — Vibrio parahaemolyticus, V.pr— Vibrio proteolyticus, V.an — Vibrio anguillarum, V.al — Vibrio alginolyticus Inhibition: +++ good; ++ medium; + slight; ± doubtful; − nil. b c

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ments (Figs. 2 and 3). V. harveyi at 106 CFU ml− 1 killed all Artemia nauplii in 72 h, but adults were more resistant and 27% survived after 72 h. However, the survival of Artemia nauplii as well as of adults was significantly increased by the addition of Streptomyces strains (nauplii: F = 111.176, P b 0.001, df = 7; adults: F = 29.25, P b 0.001, df = 7). Significantly higher survival of Artemia nauplii (67%) and adults (61%) exposed to V. harveyi occurred with Streptomyces CLS-39 than with Streptomyces CLS-45 after 72 h. Strain CLS-28 induced an intermediate survival rate compared to CLS-39 and 45. All the nauplii and adults were killed by V. proteolyticus at 106 CFU ml− 1 after 72 h. However, addition of Streptomyces strains enabled significantly higher survival (nauplii: F = 138.266, P b 0.001, df = 7; adults: F = 56.58, P b 0.001, df = 7). Greatest protection in nauplii was shown with CLS-39 (survival = 52%) and in adults with CLS-28 (survival = 61%) after72 h, but the differences between these two treatments were not significant at post-hoc test. Therefore, two Streptomyces strains i.e. CLS-28 and CLS-39 at 1% (v/v) were found to protect Artemia nauplii and adults from the infection by V. harveyi and V. proteolyticus. However, in 3 out of 4 cases there was a significant reduction in Artemia survival compared to the control with both of these strains. Strain CLS-45 also induced significantly higher survival of Artemia under challenge with V. harveyi or V. proteolyticus except in adults challenged with V. harveyi. However, the survival with CLS-45 was noticeably less than with the other two strains. 3.4. Experimental trial on P. monodon 3.4.1. Microbial quality of the prepared feeds Streptomyces were recorded from Feed 1, Feed 2 and Feed 3, but not from the reference feed. It was assumed that the Streptomyces present in the Feed 1, Feed 2 and Feed 3 were CLS-28, CLS-39 and CLS45 respectively as these were the added strains. The highest THB count was obtained in Feed 4 (≈106 CFU g− 1), whereas the reference feed, Feed 1, Feed 2 and Feed 3 had a THB of 5.5 to 8.5 × 104 CFU g− 1. No Vibrio was recorded from any of the feed.

Fig. 1. Phylogenetic tree showing the positions of three strains: CLS-28, CLS-39 and CLS45 in the Streptomyces tree based on 16S rRNA partial gene sequence analysis. Numbers at nodes are bootstrap values (%) based on neighbour-joining analysis of 1000 resampled datasets. The scale bar indicates the number of nucleotide substitutions per site.

action of V. harveyi and V. proteolyticus, as the survival rate of Artemia in the presence of each of these strains was higher than that when only V. harveyi or V. proteolyticus were administered in the experiTable 2 Mortality (%) of Artemia after 24, 48 and 72 h immersion in different concentrations of cell mass of three Streptomyces strains (CLS-28, CLS-39 and CLS-45). Cell conc.

Stages of Artemia

CLS-28 24 h

48 h

72 h

24 h

48 h

72 h

24 h

48 h

72 h

Control

Instar Adult Instar Adult Instar Adult Instar Adult Instar Adult Instar Adult

0 0 0 0 9.4 7.7 8.6 6.9 34.3 31.4 41.2 37.5

0 0 12.5 6.7 15.6 11.5 14.3 13.8 40.0 37.1 55.9 46.9

0 0 18.8 13.3 18.8 15.4 17.1 17.2 54.3 48.6 61.8 59.4

0 0 0 0 8.8 6.7 8.3 6.1 23.5 22.2 40.0 35.7

0 0 13.3 12.5 14.7 13.3 13.9 12.1 29.4 25.0 53.3 46.4

0 0 16.7 15.6 17.7 16.7 16.7 15.2 38.2 33.3 60.0 53.6

0 0 0 0 10.0 8.8 9.4 7.7 36.7 36.1 44.1 42.9

0 0 16.7 14.7 16.7 14.7 15.6 15.4 46.7 41.7 52.9 53.6

0 0 19.4 17.6 20.0 17.7 18.8 15.4 60.0 52.8 67.7 64.3

0.1% 0.5% 1% 5% 10%

I I I I I I

CLS-39

CLS-45

3.4.2. Stocking of the PLs for trial The age of the stocked P. monodon was PL33 and the mean length and wet weight were 19.57 ± 0.08 mm and 0.055 ± 0.0008 g respectively (mean ± SE) (Table 4). At the time of stocking, the salinity of the water was 35 ppt, temperature was 25 °C, pH was 7.0 and no ammonia, nitrate or nitrite was observed. 3.4.3. In-trial physico-chemical parameters of water and survival of animals The water quality parameters during the experiment are presented in Table 4. Ammonia started developing in the control as well as in all the treatments from day 1. Ammonia was in the range of 1 to 2 ppm in all the treatments throughout and the differences were also − significant (P b 0.05). However, NO− 2 and NO3 were not detected throughout the trial. pH was almost consistent in all the treatments with the value of 7.0. The cumulative mortality in the each treatment was recorded and % survival calculated. The survival of animals in T3 was significantly higher (P b 0.05) than in both the control and in T4 which were not significantly different from each other. Survival in T1, T2, T4 and T5 treatments was intermediate to T3 and the control and not significantly different to either. Animals in T1, T2 and T5 grew significantly better in terms of both length and weight than did control animals. 3.4.4. In-trial microbial analysis and colonisation of Streptomyces Total heterotrophic bacteria and Vibrio were approximately 1.3 to 2.5 × 108 CFU ml− 1 and 3.5 to 14 × 108 CFU ml− 1 respectively in all the treatments (Fig. 4). This study showed that the Vibrio population in T1 was significantly lower than in the control and treatments T2, T3

S. Das et al. / Aquaculture 305 (2010) 32–41

37

Table 3 LC50 determination of virulent V. harveyi and V. proteolyticus on Artemia. Number of Artemia challenged and number surviving together with percentage mortality. LC50 (CFU/ml)

Cell conc. (CFU/ ml)

Instar I V. h

V. p

V. h

V. p

V. h

V. p

V. h

V. p

V. h

V. p

V. h

V. p

V. h

V. p

V. h

V. p

×108 ×107 ×106 ×105

29 ± 5 22 ± 4 26 ± 7 17 ± 4

32 ± 2 33 ± 3 35 ± 3 35 ± 3

21 ± 3 25 ± 7 24 ± 6 25 ± 5

35 ± 2 33 ± 3 34 ± 2 33 ± 3

6±2 6±1 10 ± 4 9±3

2±1 4±1 10 ± 3 18 ± 3

5±1 6±4 8±3 14 ± 6

3±1 8±2 13 ± 3 17 ± 1

79 73 62 47

94 88 71 49

80 76 67 44

91 76 62 48

105.2

105.04

105.3

105.14

a b

Challenged (n ± SD)

a

Survived (n ± SD) Adult

b

Instar I

% mortality Adult

Instar I

Adult

Instar I

Adult

Vibrio harveyi. Vibrio proteolyticus.

and T4 (P b 0.05), but not T5. The total bacterial counts showed no significant differences in the treatments (P N 0.05). Streptomyces spp. were isolated after weeks 1 and 2 from the animals and from faeces from T1, T2 and T3 treatments, indicating that the Streptomyces strains reached the digestive system of shrimps.

3.4.5. LC50 of V. harveyi on P. monodon The V. harveyi strain used in this study was found to be only lowly virulent to shrimp. It could not kill all individuals even after 5 days

exposure. The highest mortality observed was 55% at 107 CFU ml− 1 dosages and the calculated LC50 was 106.5 CFU ml− 1. 3.4.6. Challenge by V. harveyi and survival Administration of the Streptomyces in T1 and the commercial probiotic in T5 significantly increased survival of P. monodon over the control when challenged with V. harveyi (Table 5). Although the Streptomyces treatments T2 and T3 had lower survival than with T1 only the T4 treatment with the commercial probiotic administered in food showed a significantly lower survival than T1 (Table 5).

Fig. 2. Protection (survival % mean ± SE, n = 4) of Artemia (A) nauplii and (B) adult from Vibrio harveyi by addition of 1% (v/v) cell mass of Streptomyces (CLS-28, CLS-39 and CLS-45). Treatments with different letters differed significantly (Tukey's b; P b 0.05) within the mean survivals at 72 h observation.

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S. Das et al. / Aquaculture 305 (2010) 32–41

Fig. 3. Protection (survival % mean ± SE, n = 4) of Artemia (A) nauplii and (B) adult from Vibrio proteolyticus by addition of 1% (v/v) cell mass of Streptomyces (CLS-28, CLS-39 and CLS45). Treatments with different letters differed significantly (Tukey's b; P b 0.05) within the mean survivals at 72 h observation.

4. Discussion 4.1. Biotoxicity of Streptomyces strains on Artemia Despite some criticism (e.g. the absence of Artemia in most of the marine ecosystems, lack of sensitivity to chemical exposure due to the intrinsic resistance to extreme salinity conditions, see review by Persoone and Wells, 1987) against the use of Artemia in biotoxicity studies, they have been reported to be able to detect lower concentrations of toxic compounds than other crustacean test

organisms (Nunes et al., 2006). Barahona and Sanchez-Fortun (1996) also evaluated several age classes of Artemia and reported a greater relative sensitivity amongst 48-h old specimens. Moreover, juvenile and adult Artemia are used increasingly as suitable live feeds for diverse aquaculture species (Sorgeloos et al., 1998; Baylon et al., 2004) as well as being a vector for supplementing specific beneficial microbial cultures, vaccines and immunostimulants to the target species in aquaculture (Campbell et al., 1993; Dixon et al., 1995; Gatesoupe, 2002; Patra and Mohamed, 2003). This was described as bioencapsulation (Gomez-Gil et al., 1998) and in the present study it

Table 4 Water quality parameters (means across 15 d), growth and survival (initial number = 25) of P. monodon larvae during the 15 d trial in different treatments. Means with different superscripts in a column differed significantly (Tukey's b; P b 0.05). Treatments

Parameters NH4 (ppm)

Control T1 (Feed + CLS-28) T2 (Feed + CLS-39) T3 (Feed + CLS-45) T4 (Feed + Sanolife®) T5 (Sanolife® in water)

1.76 ± 0.05b 1.48 ± 0.06a 1.57 ± 0.06a,b 1.62 ± 0.06a,b 1.57 ± 0.06a,b 1.46 ± 0.06a

% of survivals

Total length (mm) Initial

Final

Wet weight (g) Initial

Final

74.67a 82.67a,b 85.33a,b 92.00b 77.33a 84.00a,b

19.16 ± 0.23 19.55 ± 0.55 19.93 ± 0.28 19.74 ± 0.17 19.53 ± 0.15 19.49 ± 0.15

19.59 ± 0.19a 20.62 ± 0.21c,d 21.07 ± 0.23d 20.12 ± 0.24a,b,c 19.75 ± 0.19a,b 20.56 ± 0.22b,c,d

0.0512 ± 0.0018 0.0566 ± 0.0015 0.0582 ± 0.0022 0.0578 ± 0.0018 0.0555 ± 0.0016 0.0549 ± 0.0015

0.0621 ± 0.002a 0.0832 ± 0.004b,c 0.0819 ± 0.0033b 0.0627 ± 0.0036a 0.0612±0.0024a 0.0952 ± 0.0048c

Values are in Mean ± SE. n = 45 for NH3; n = 3 for % survival and n = 15 for total length and wet weight.

S. Das et al. / Aquaculture 305 (2010) 32–41

39

Fig. 4. Total heterotrophic bacterial and Vibrio populations in P. monodon postlarvae after 15 d (n = 15). Means in Vibrio population with different superscripts differed significantly (Tukey's b; P b 0.05).

was anticipated that if Artemia could tolerate Streptomyces, and a protective response was shown against pathogens, then the candidate species in aquaculture would also be able to get the beneficial probiotic effect from Streptomyces. To act as probionts, the isolated strains must be non-toxic and tolerable by the target animals (Gomez-Gil et al., 2000; KesarcodiWatson et al., 2008; Tinh et al., 2008). Lactic acid bacteria (LAB) and Bacillus probiotic strains were reported to reach the digestive system and transform the gut micro flora to exhibit a probiotic effect (Rengpipat et al., 2000, 2003; Vaseeharan and Ramasamy, 2003; Villamil et al., 2003). Some Streptomyces strains in the present study were also tolerated and found safe for both nauplii and adult Artemia. However, the assimilation into the digestive system was not specifically checked, but rather assumed, as the Streptomyces were detected in the shrimp postlarvae to which the Artemia were fed. Although it was found that addition of Streptomyces led to mortality compared to negative control raising the question of safety to target animals, this was mainly due to the aggregation of filamentous, viscous, live cell mass of Streptomyces which chocked the respiration of Artemia. In the same well where there was no aggregation, Artemia were alive. Thus, it was assumed that the mortality was not due to the toxicity of Streptomyces, it was due to the blocking of respiration. While the Vibrio and Streptomyces were added together to find out the protection, the pathogenicity of Vibrio was found to be reduced by the action of Streptomyces. Nunes et al. (2006) mentioned that the age of the animals was a major factor influencing results in toxicity testing. Barahona and Sanchez-Fortun (1996) reported the most suitable age class of Artemia for toxicological testing was 48 h old animals. The present study was quite thus aptly conducted on 24, 48 and 72 h grown Artemia to get a comprehensive picture of biotoxicity and protective response through the addition of marine Streptomyces. Nauplii were found to be more

Table 5 Survival and mortality of P. monodon in different treatments after exposure to virulent V. harveyi at 107 CFU ml− 1 (n = 3). Means with different superscripts in a column differed significantly (Tukey's b; P b 0.05). Treatments

Challenged (n ± SE)

Survived (n ± SE)

Mortality %

Control T1 T2 T3 T4 T5

15 ± 0 15 ± 0 15 ± 0 15 ± 0 15 ± 0 15 ± 0

6.33 ± 0.33a 10.33 ± 0.67b 9.33 ± 0.67a,b 8.67 ± 0.88a,b 6.67 ± 0.88a 10.67 ± 0.88b

58.00 31.33 38.00 42.00 55.33 28.67

susceptible and showed lower survival when exposed to Streptomyces in all the experiments indicating that adult Artemia were more robust.

4.2. Protection of Artemia from Vibrio The experimental animals which received a combination of pathogen and Streptomyces showed significantly higher survival rates than the untreated control group (pathogen only). Higher protection (survival 68%) was provided by prior addition of CLS-39 and lower survival (39%) was shown by CLS-45 against V. harveyi. CLS28 provided maximum protection (survival 61%) and CLS-45 showed minimum protection (survival 38%) protection against V. proteolyticus. It could be considered that the Streptomyces strains showed probiotic activity and either improved the disease resistance ability of nauplii and adult Artemia or inactivated the virulent Vibrio spp. to some extent, or both. Other studies have reported improved survival of Artemia challenged with pathogenic vibrios: Verschuere et al. (2000b) reported a higher survival rate of Artemia (∼80%) when challenged with V. proteolyticus and protected by preemptive colonization of selected bacterial strains (Aeromonas spp. and Vibrio alginolyticus). Marques et al. (2006) reported 95% survival of Artemia protected with Bacillus sp. after 72 h challenge with V. proteolyticus. Patra and Mohamed (2003) also found 91% survival when Artemia nauplii were protected by yeast from V. harveyi infection. Although these studies reported higher survival than those in the present study, it was worthy to ascertain the potential of Streptomyces spp. which can protect Artemia from the action of Vibrio spp., because the sporeforming capacity of Streptomyces may make them a more practical alternative than non-spore-forming microbes. Gatesoupe (1991) reported that the efficacy of Artemia nauplii in bioencapsulating bacteria strongly depends on the type of bacteria used, time of exposure, and status of the bacteria. We found that Artemia tolerated 3 strains of Streptomyces (CLS-28, 39 and 45) very well and thus should be able to transfer the Streptomyces to animals to which they are fed. Streptomyces species are distributed widely in aquatic and terrestrial habitats (Pathom-aree et al., 2006) and are of commercial interest due to their strong capacity to produce novel bioactive compounds. It was also expected that Streptomyces species will have a cosmopolitan distribution as they produce abundant spores which are readily dispersed (Antony-Babu et al., 2008). There are several reports of inhibition of Vibrio spp. by marine Streptomyces (Das et al., 2004; You et al., 2007). Marine Streptomyces are a major source of antibacterial compounds (Fenical and Jensen, 2006). Therefore, the protective effect of Streptomyces strains may be due to the production of antibacterial compounds active against V. harveyi and V. proteolyticus.

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S. Das et al. / Aquaculture 305 (2010) 32–41

4.3. Probiotic effect on P. monodon The trial on growth, survival and protection from pathogens of shrimp P. monodon revealed that Streptomyces CLS-28 can be effectively used probiotically in aquaculture. Results indicated that the Streptomyces-treated P. monodon culture treatment showed lower Vibrio counts in the T1 treatment than in the control, although total heterotrophic bacterial counts were not significantly different. These results suggest that Streptomyces CLS-28 is antagonistic to Vibrio spp. in P. monodon culture, rather than to bacteria in general. In order to be considered as a probiotic, the strain has to be assessed for safety to the host. Two strains of Streptomyces (CLS-28 and 39) significantly increased the growth of PLs compared to the control and the significant survival (P b 0.05) was found during 15 d trial in all the treatments fed with Streptomyces fortified feed. Instead, after feeding for 15 d significant growth differences (P b 0.05) was found among the treatments. In addition, the concentration of ammonia in the treatments was the same as in the control and nor was nitrate or nitrite found indicating no adverse effect on the tank environment. Probiotics may improve digestive activity by synthesis of vitamins, cofactors or by improving enzymatic activity (Fuller, 1989; Gatesoupe, 1999). These properties could have contributed to the weight increase seen in T1 and T2 compared to the control (Table 4), by improving amylolytic and proteolytic activity in the shrimp digestive tract. In this study, the presence of Streptomyces from the faeces and in the whole animals indicated that it reached to the digestive system of shrimps, but this does not confirm that Streptomyces colonised the gut as the shrimps were fed Streptomyces-fortified feed daily. In most studies, the explanation for the mechanisms of action of probiotics is largely based on in vitro observations, neglecting that in vivo physiology might be different from the metabolic process in vitro. Selective ingestion by the host (Riquelme et al., 2001), death in the digestive tract (Vine et al., 2006), or a failure of a probiotic to maintain its in vitro physiology under circumstances of more complex microbial interactions and/or nutritional environment are some of the challenges that a probiotic might face inside a host. Moreover, the interactions between the introduced probiotics and the indigenous gastrointestinal (GI) microbiota are still poorly understood (Tinh et al., 2008). Considering these views, the probiotic mode(s) of action of the Streptomyces are yet to be determined. The most important limitation to the use of probiotics is that in many cases they are not able to maintain themselves, and so need to be added regularly and at high concentrations (Vine et al., 2006), which makes this technique less cost-effective. Moreover, probiotics that were selected in vitro based on the production of inhibitory compounds might fail to produce these compounds in vivo (Verschuere et al., 2000a). In addition, Defoirdt et al. (2007) recommended isolating candidate probiotics from the culture system(s), which will facilitate their growth and establishment in the host. It was for this reason, that in the present study, Streptomyces were isolated from the shrimp pond sediment samples. The protection afforded the postlarvae by Streptomyces treatment groups (T1, T2 and T3) were not significantly different than from the commercial probiotic (T5). The commercial probiotic used in this experiment was labelled as a mixture of Bacillus subtilis, B. licheniformis and B. pumilus. The efficiency of the Streptomyces could be improved by optimising the dose and the mode of application. The study was conducted in the small jugs where water was exchanged daily, which might not allow Streptomyces to colonise completely (even though Streptomyces were recovered from the animals and their faeces). Microorganisms only produce metabolites during the stationary growth phase and if complete colonisation does not occur in the gut due to constant flushing, total protection may not be observed. In addition, as suggested by Vine et al. (2004) for other bacterial probiotics, the growth of Streptomyces may be less than the rate of

flushing from the intestine, and Streptomyces may be unable to attach to the intestine so that they will be flushed before reaching a viable population level. Another point is that this trial was conducted only for 15 d and the slowly growing Streptomyces perhaps needed a longer time to colonise the animals. However, it is to keep in mind the potential setbacks of using Streptomyces in culture system which include the production of clinical antibiotics and lateral gene transfer of antibiotic-resistance gene (reviewed in Das et al., 2008a). But, as the several clinical antibiotics are also produced by bacterial probionts (e.g. Bacillus) and lateral gene transfer of antibiotic-resistance gene is an ecological phenomenon, Streptomyces may be potentially used as probiotics in aquaculture. 5. Conclusion From this study it can be concluded that marine Streptomyces strains can protect Artemia nauplii and adults from the infection of Vibrio spp. and are non-toxic to shrimp and are able to protect them from Vibrio spp. in culture systems. Thus, Streptomyces can be included among the potential biological control agents in aquaculture. However, to establish a probiotic nature of Streptomyces in disease prevention and/ or growth stimulator of aquaculture animals, further extensive trials with different target animals and a commercial cost– benefit analysis are necessary. It would be advantageous if a pond trial can be done in future. Streptomyces are saprophytic and grow well in the sediment, which will help them to colonise host animals. It is important to determine if a combination of Streptomyces strains can be effectively used in aquaculture either as water or feed probiotics. It is also essential to determine the optimum dose and the best mode of application. Acknowledgement An Endeavour Research Fellowship to S.D by Department of Education, Employment and Workplace Relations, Australian Government to carry out Postdoctoral research at University of Tasmania is gratefully acknowledged. Funding to carry out the project was provided by the National Centre for Marine Conservation and Resource Sustainability and is gratefully acknowledged. Special thanks are due to Dr. Chris Bolch for his help in 16S rRNA gene sequencing study. We also thank Mr. Detlef Planko, Dr. Mark Adams, Mr. Daniel Pountney and Mr. Jon Schrepfer for rendering their technical help in the setting up of the tanks. References Antony-Babu, S., Stach, J.E.M., Goodfellow, M., 2008. Genetic and phenotypic evidence for Streptomyces griseus ecovars isolated from a beach and dune sand system. Antonie Van Leeuwenhoek 94, 63–74. Ausubel, F.M., Brent, R., Kingstone, R.E., Seidman, J.G., Smith, J.A., Struhl, K., 1999. Short Protocols in Molecular Biology. Wiley, New York. Azad, I.S., Panigrahi, A., Gopal, C., Paulpandi, S., Mahima, C., Ravichandran, P., 2005. Routes of immunostimulation vis-à-vis survival and growth of Penaeus monodon postlarvae. Aquaculture 248, 227–234. Barahona, M.V., Sanchez-Fortun, S., 1996. Comparative sensitivity of three age classes of Artemia salina larvae to several phenolic compounds. Bull. Environ. Contam. Toxicol. 56, 271–278. Baylon, J.C., Bravo, M.E.A., Maningo, N.C., 2004. Ingestion of Brachionus plicatilis and Artemia salina nauplii by mud crab Scylla serrata larvae. Aquac. Res. 35, 62–70. Caldwell, G.S., Bentley, M.G., Olive, P.J.W., 2003. The use of a brine shrimp (Artemia salina) bioassay to assess the toxicity of diatom extracts and short chain aldehydes. Toxicon 42, 301–306. Campbell, R., Adams, A., Tatner, M.F., Chair, M., Sorgeloos, P., 1993. Uptake of Vibrio anguillarum vaccine by Artemia salina as a potential oral delivery system to fish fry. Fish Shellfish Immunol. 3, 451–459. Chythanya, R., Karunasagar, I., Karunasagar, I., 2002. Inhibition of shrimp pathogenic vibrios by a marine Pseudomonas I-2 strain. Aquaculture 208, 1–10. Das, S., Lyla, P.S., Rajagopal, S., Ajmal Khan, S., 2004. Antagonistic properties of deep sea actinomycetes isolated from Bay of Bengal. Proceedings of the Conference on Microbiology of the Tropical Seas. National Institute of Oceanography, Goa, India. Das, S., Lyla, P.S., Ajmal Khan, S., 2006a. Marine microbial diversity and ecology: importance and future perspectives. Curr. Sci. 25, 1325–1335.

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