Aquaculture 305 (2010) 32–41

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Aquaculture

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

Surajit Das ⁎, Louise R. Ward, Chris BurkeNational Centre for Marine Conservation and Resource Sustainability, Australian Maritime College, University of Tasmania, Locked Bag 1370, Launceston, Tasmania 7250, Australia

⁎ Corresponding author. Present address: DepartmInstitute of Technology, Rourkela-769 008, Orissa, Infax: +91 661 2462022.

E-mail addresses: surajit@myself.com, surajit@nitrkl

0044-8486/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.aquaculture.2010.04.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 7 January 2010Received in revised form 30 March 2010Accepted 1 April 2010

Keywords:ArtemiaPenaeus monodonProbioticsStreptomycesVibrioSurvivalProtection

Marine Streptomyces strains (CLS-28, CLS-39 and CLS-45) were used to colonise Artemia nauplii (Instar I) and15 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 at106 CFU ml−1. A significant reduction in mortality (Pb0.001) was found by addition of 1% wet cell mass ofStreptomyces strains in nauplii and adult Artemia against both the pathogens. The best protective responseswere shown by CLS-39 in both nauplii and adults against V. harveyi and by CLS-39 in nauplii and CLS-28 inadults against V. proteolyticus. Shrimp feeds were supplemented with Streptomyces cell mass at 1% dosageand fed to black tiger shrimp Penaeus monodon postlarvae for 15 d in three treatments with two treatmentsof 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 inthe range of 1 to 2 ppm in all the treatments with significant differences between treatments (Pb0.05).Significant differences (Pb0.05) were also found in survival, total length and wet weight of the shrimppostlarvae 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 thanthe 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 (Pb0.05) andthe total bacterial counts showed no significant differences in the treatments (PN0.05). After challenge withV. harveyi at 107 CFU ml−1, highest survival was found in T1 and T5. Among the Streptomyces treatments, T1showed significantly higher survival compared to the control, followed by T2 and T3. Thus Streptomycesstrains show promise as probiotic agents in mariculture.

ent of Life Science, Nationaldia. Tel.: +91 661 2462684;

.ac.in (S. Das).

ll rights reserved.

© 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 theprophylactic application of antibiotics and biocides. Fuller (1989) defineda probiotic as ‘a live microbial feed supplement which beneficially affectsthe host animal by improving its intestinal microbial balance’. A modifiedand more appropriate definition was proposed by Verschuere et al.(2000a) — ‘a live microbial adjunct which has a beneficial effect on thehost by modifying the host-associated or ambient microbial community,by ensuring improveduseof the feedor enhancing its nutritional value, byenhancing the host response towards disease, or by improving the qualityof its ambient environment’.

Actinobacteria is a class with five subclasses that was proposed byStackebrandt et al. (1997) to group the highly diverse so calledactinomycetes based on chemical composition, DNA–DNA reassocia-tion and 16S rRNA gene sequence similarities. Members of the

Actinobacteria are prolific sources of secondary metabolites and thevast majority of these compounds are derived from the single genusStreptomyces. Streptomyces is a Gram-positive aerobic genus in theorder Actinomycetales, suborder Streptomycineae and family Strepto-mycetaceae (Stackebrandt et al., 1997) and has a DNA G+C content of69–78 mol%. Marine-derived Streptomyces have been studied forisolation of several novel secondary metabolites (Fenical and Jensen,2006; Das et al., 2006a). However, to date there have only been a fewstudies that have considered Actinobacteria for their application asprobiotics in aquaculture.

We have reported the prospects of using marine Actinobacteria asprobiotics in aquaculture (Das et al., 2008a) and began screening marineActinobacteria for use as new biocontrol agents for aquatic animals. Wereport here the effect of threemarine Streptomyces strains on Artemia andPenaeus monodon. Artemia has long been considered as a model/ testorganism to study the mode of action of probiotic bacteria due to itsadaptability 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 cystsproduction), small body size, and adaptability to variednutrient resources(Nunes et al., 2006). There have been several experiments carried out onArtemia in the search for new biocontrol agents for aquaculture

33S. Das et al. / Aquaculture 305 (2010) 32–41

(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 asprobiotics and practically none of the studies reported the toxicity ofthe Actinobacteria and protection from pathogens. In the currentstudy, marine Streptomyces were isolated and screened for potentialprobiotic activities. The LC50 of virulent Vibrio harveyi and V.proteolyticus were examined in Artemia to determine LC50 value, andthe efficacy of the marine Streptomyces strains to protect Artemia fromVibrio spp. challenge infection was assessed. P. monodon postlarvaewere fed with Streptomyces-supplemented feed and the protectionfrom the challenge infection by V. harveyi was evaluated.

2. Materials and methods

2.1. Isolation of marine Streptomyces

Marine sediment samples were collected from shrimp farms locatedat Queensland, Australia (lat 21°43′09″S and long 149°25′54″E). Toisolate the Streptomyces, aliquots (0.5 ml) of serially diluted (10−1 and10−2) sampleswere 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 ExtractMalt 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 withnystatin and cyclohexamide at 25 µg ml−1 and 10 µg ml−1 respectivelyto minimize contamination with fungi and 10 µg ml−1 nalidixic acid tominimize contaminantgrowth(Takizawaet al., 1993;Ravel et al., 1998).Plates were incubated for 7 to 15 d at 30 °C temperature and then thecolonieswith a tough or powdery texture, dry or folded appearance andbranching filamentswith orwithout aerialmycelia (Mincer et al., 2002)were sub-cultured and transferred on SCA and ISP 2 slants. Until furtheruse, 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 toget 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 wereincubated for 7 d at 30 °C temperature. The bulk of the colonies werescraped away with a sterile slide and the remaining growth was killedby exposure to chloroform (70%) for 15 min. The plates were then airdried for 10 min to remove any residual chloroform vapour.

Five fish-pathogenic Vibrio strains (V. harveyi V890, V. parahae-molyticus, V. proteolyticus V760, V. anguillirum V572 and V. alginoly-ticus 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 SoyaAgar (TSA, OXOID®) and TCBS medium (OXOID®). Cultures of Vibriospp. were grown for 18 h and streaked on each plate perpendicular tothe chloroform-killed actinomycetes strain. The plateswere incubatedfor 24 h at 30 °C temperature. The width of inhibition zones of eachVibrio species was measured in mm. Test strains that showed growthnear or around the actinomycetes strains with no inhibition wereconsidered resistant.

2.2.2. Exoenzymatic assayCultured actinomycetes were screened for hydrolytic exoenzy-

matic activities (amylase, protease and lipase). These tests wereconducted on Yeast Extract Malt Extract Agar (ISP 2) mediumcontaining starch for amylolytic activity, skimmedmilk for proteolyticactivity and Tween 80 for lipolytic activity at 1% concentration.Amylolytic activity was detected by flooding the plates with 1% iodinesolution. Presence of amylase was visualized by decolorized haloaround the culture due to starch digestion. Proteolytic activity wasobserved by clearing of the milk and lipolytic activities were observedby the formation of a halo of precipitated fatty acids around thecolony.

2.3. Mass culture of selected Streptomyces strains

Based on the results from cross streak and enzymatic assays, 3strains of Streptomyces (CLS-28, CLS-39 and CLS-45) were selected formass culture. They were each mass cultured in ISP 2 liquid medium in100 ml Schott flasks incubated at 30 °C for 7 d on a shaker. Thecultured cells were harvested by centrifugation (3000 rpm for10 min) and washed with sterile, normal saline solution (0.85%NaCl). The wet cell mass was kept at 4 °C until used for feedpreparation. Several batches of mass culture were run to get adequateamounts of cell mass.

2.4. 16S rRNA gene sequencing and identification of the selectedactinomycetes strains

Molecular identification of three selected actinomycetes strains(CLS-28, CLS-39 and CLS-45) as potential probionts were carried outby 16S rRNA gene (16S rDNA) amplification and sequencing.

2.4.1. DNA extractionThe biomass of three actinomycetes isolates was harvested by

centrifugation. Pellet was washed twice in sterile Tris–EDTA bufferand approximately 100 mg (wet weight) biomass was used for DNAextraction based on cetyltrimethylammonium bromide (CTAB)purification following Ausubel et al. (1999).

2.4.2. DNA amplification and sequencingColony 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 wasperformed in 50 µl volumes containing 2 mM MgCl2, 2.5 U Taqpolymerase (Bioline, Australia), 100 µM of each dNTP, 0.2 µM ofeach primer and 3 µl template DNA. The PCR programme used was aninitial denaturation at 96 °C for 5 min followed by 30 cycles of 95 °Cfor 15 s, 49 °C for 30 s and 72 °C for 1 min and a final extension at72 °C for 1 min. Amplified DNAs were purified by the Montage™ PCRcentrifugal filter device (Millipore Corp., USA) following manufac-turer's instructions and finally quantified by Turner TBS380 DNAfluorometer. The sequencing reactions were carried out by AustralianGenome Research Facility Ltd., Australia with 27F, 529F, 518R, 1073Rand 1492R primers to compare the chromatograms and get a clearconsensus sequence for each strain.

2.4.3. Phylogenetic analysisSequence data were compiled and consensus sequence was

obtained by using Geneious 3.8.5 programme and examined forsequence homology with the archived 16S rDNA sequences fromGenBank at www.ncbi.nlm.nih.gov/nucleotide, employing the BLAST.

Multiple alignments of sequences were performed with theClustalX (1.83) program (Thompson et al., 1997). A phylogenetictree was constructed using the neighbour-joining DNA distance

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

algorithm (Saitou and Nei, 1987) using DAMBE 5.0.25 (Data Analysisin Molecular Biology and Evolution, http://dambe.bio.uottawa.ca/dambe.asp). The resultant tree topologies were evaluated bybootstrap analysis (Felsenstein, 1985) of neighbour-joining data setsbased on 1000 resamplings.

2.4.4. GenBank submissionThe 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 strainsHarvested wet cell mass from three strains was examined for

toxicity in Artemia. Experiments were conducted in sterile polysty-rene 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 theexperiment. Untreated control animals were kept in sterile sea waterand the experiments was performed in quadruplicate. Instar I naupliiand adult Artemia were kept in the wells containing 5 ml of air-saturated sea water with different concentrations of the cell masssuspensions and the numbers of Artemia were counted. Plates wereclosed and incubated at 28 °C for 72 h. Mortality was determined after24, 48 and 72 h and the dead animals were removed. At the end of theexperiment, 0.5 ml of 16% formaldehydewas added to eachwell to killall remaining animals following Caldwell et al. (2003) and the exactinitial numbers of animals used in the experiments were crosschecked by summing up the number of individuals.

2.5.2. Virulence of V. harveyi and V. proteolyticusV. 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 incubatedfor 24 h at 28 °C. Viable cells were counted by haemocytometer andharvested by centrifuging at 3000 rpm for 10 min. The harvested cellswere suspended into an equal volume of sterilised sea water. This wasserially diluted to obtain 108 to 105 CFU ml−1 and used in theexperiments to determine virulence. Aliquots of each dilution wereplated on TSA to confirm the CFU ml−1. Virulence testing was carriedout on Artemia in 12-well cell culture plates. Nauplii and adults wereimmersed 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 percentagemortality (including moribund individuals) was calculated after24 h. The virulence of V. harveyi and V. proteolyticus was determinedfrom the LC50 (Azad et al., 2005) following the method of Reed andMuench (1938).

2.5.3. Artemia challenge experiment and protection by marineStreptomyces

The capacity of three Streptomyces strains (CLS-28, CLS-39 andCLS-45) to reduce mortality in Artemia during challenge by V. harveyior V. proteolyticuswas observed for nauplii and 15-day old Artemia. In12-well cell culture plates nauplii and adult Artemiawere grown withthree strains of Streptomyces at 1% concentration (v/v) in air saturatedsterile sea water together with 24 h cultures of V. harveyi or V.proteolyticus at the final concentration of 106 CFU ml−1. Controlanimals were kept in sea water. Plates were closed and incubated at28 °C. Survival was assessed by scoring the number of dead animals onthe bottom of each plate using an inverted microscope. Dead animalswere removed after counting and the percentage of surviving Artemiawas calculated after 24, 48 and 72 h by recording the number of deadanimals on the bottom of each plate. After 72 h, 0.5 ml of 16%formaldehyde was added to each well to kill all remaining animalsand the exact initial numbers of animals used in the experimentswere

cross checked by summing up the number of dead and survivingindividuals in each well.

2.6. Experiment with black tiger shrimp (P. monodon)

2.6.1. AcclimatizationHealthy black tiger shrimp (P. monodon) postlarvae (PL16) from

Rocky Point Hatchery, Queensland were acclimatised in a large glasstank with gravel bed prior to experimental trial. Unhealthy or injuredanimals were removed during this period. PL were fed to satiationwith a shrimp feed from Ridley Aqua-Feed, Queensland, Australia viaan automatic feeder. The salinity and temperature of the water rangedfrom 34 to 35 ppt and 25 to 28 °C respectively.

2.6.2. Experimental set-upPolyvinyl jugs of 2 l capacity were used in the experiment. Jugs

were filled with 1.5 l of water and each jug stockedwith 25 shrimps atPL33 stage. The average length and weight of the animals weredetermined by digital calliper and electronic balance (3 decimalpoints) respectively. Air was supplied to each jug via Millipore(0.22 µ) filters to prevent contamination during aeration and the topwas covered with polythene wrap to prevent spillage and escape ofshrimps. All experiments were conducted in triplicate. Controlanimals (without any treatment and fed with the reference feed)were also kept in triplicate jugs. A photoperiod of 12 h dark and 12 hlight was maintained throughout the experimental period.

2.6.3. Preparation of feedCommercial shrimp feed (Enhance starter feed, Ridley Aquafeeds,

Queensland) was used as the reference feed and 4 experimental feedswere prepared from it: i) Feed 1 — supplemented with 1% CLS-28 cellmass; ii) Feed 2 — supplemented with 1% CLS-39 cell mass; iii) Feed3 — supplemented with 1% CLS-45 cell mass; iv) Feed 4 —

supplemented with 1% Sanolife® MIC (as the treatments were alsoin the same concentration).

All the feeds were prepared by adding 30% water to the dryingredient mixture, extruding the dough through a 1 mm die(Italpast) and drying for 2–4 h to keep the final moisture level at10%. The reference feed was prepared by mixing with distilled water.Streptomyces cell mass were supplemented while preparing the feedat 1% concentration (w/v). For 200 g feed mixture the required cellmass was calculated as 2 ml. This cell mass and ∼60 ml water wasmixed with the feed mixture to make dough and small crumble feedwas prepared by extruding. Then the crumble feedwas air dried for 2–4 h to get ∼10% water. This dried feed was ground for theexperimental trial and kept in the freezer.

2.6.4. Virulence check of test pathogensA virulence test of V. harveyiwas carried out on PL40 (as during the

challenge experiments the treated animals were also about the sameage). V. harveyi V890 incubated in TSB for 24 h, at 28 °C centrifuged at3000 rpm for 10 min and the harvested cells serially diluted in sterilesea water to obtain 107 to 103 CFU ml−1. Aliquots of each dilutionwere plated on TSA to confirm the CFU ml−1. Shrimp postlarvae wereimmersed for 12 h in duplicate vessels containing either 103, 104, 105,106 or 107 CFU ml−1 of V. harveyi. Control postlarvae were immersedin sterile sea water for 12 h. After the immersion, postlarvae werereleased into the jugs and mortality was observed for 5 d. Thevirulence of V. harveyi on PL40 P. monodon was determined from theLC50 as described above for Artemia.

2.6.5. Protection of PL from V. harveyiP. 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 — referencefeed; (B) T1— Feed 1; (C) T2— Feed 2; (D) T3— Feed 3; (E) T4— Feed4; (F) T5 — reference feed+Sanolife® MIC in water (1 g/tonne after

35S. Das et al. / Aquaculture 305 (2010) 32–41

germination following manufacturer details). All treatments and thecontrol were replicated five times. All the shrimps received preparedfeed twice daily (at dawn and dusk) at approximately 5–8% bodyweight day−1. The amount of feed was determined by feeding adlibitum and altered based on previous consumption. Uneaten feed andfaces were removed by siphoning daily in the morning.

2.6.6. In-trial managementFeeds 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 werecollected daily from each jug and analysed for total heterotrophicbacteria (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 formicrobial 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. Everyweek, shrimp faeces and a few live shrimps from each jug (from T1, T2and T3) were assessed for the presence of Streptomyces by culture onSCA and ISP 2 media.

2.6.7. Pathogen challenge and survival/protection testAfter feeding experimental feeds to shrimps for 15 d, challenge

tests were performed with V. harveyi at 107 CFU ml−1 (Rengpipatet al., 1998). Shrimps were removed from their jugs and exposed to V.harveyi for 12 h, and then replaced into their original jugs and survivalmonitored for five days.

2.7. Statistical analyses

Data are presented as mean±standard error. Significance ofdifference between different treatment groups was tested usingone-way analysis of variance (ANOVA) and significant results werecompared with Tukey's HSD post-hoc test. Homogeneity of variancewas assessed by residual plots. For all the tests the significance wasdetermined at the level of Pb0.05.

3. Results

3.1. Isolation of marine Streptomyces and activities of selected strains

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

Table 1Growth characteristics of marine actinomycetes on culture media, enzymatic activities and

Strainnos.

Colour of the strainsa Growth onmediab

Enzymatic activitie

Obverse Reverse Amylolytic Pr

CLS-28 White White/Yellow ISP 2, MA +++ +CLS-29 White White/Yellow ISP 2, MA − +CLS-30 White White/Yellow ISP 2, SCA − ±CLS-31 White White/Yellow ISP 2, SCA − +CLS-32 White White/Pink ISP 2, SCA, MA ++ +CLS-33 White/Grey Yellow/Green ISP 2, SCA +++ +CLS-39 White/Grey Black ISP 2, SCA, MA +++ +CLS-42 White/Grey White ISP 2 +++ +CLS-43 White/Grey White ISP 2 +++ +CLS-45 White White/Grey ISP 2, MA − +CLS-46 White White/Grey ISP 2, SCA ++ +CLS-48 White White SCA, MA − +

a On Yeast extract malt extract agar (ISP 2) medium.b Different media: ISP 2 — Yeast extract malt extract agar; SCA — Starch casein agar; MAc Presence or absence of the activity determined by the magnitude of zones of activity: +d Against the listed pathogens — V.h — Vibrio harveyi, V.pa— Vibrio parahaemolyticus, V.pr—

good; ++medium; + slight; ± doubtful; − nil.

successful on ISP 2 medium. Out of 43 strains isolated, 12 strains werefound to have antimicrobial activity as demonstrated in the crossstreak assay. Of the 12 active strains, 2 were active against all 5pathogens, 8 against 4 pathogens, 1 against 3 and 1 was active against2 pathogens. All but one isolate was active against V. alginolyticus, butonly 8 isolates were inhibitory to V. proteolyticus or V. harveyi. All thestrains showed proteolytic activity, but variable amylolytic andlipolytic activities. The characteristics of the colony morphology andthe antagonism and enzymatic assays of the selected strains aredescribed in Table 1.

3.2. Identification of the selected Streptomyces strains

Evaluating the results from cross streak and enzymatic assaysthree strains, viz. CLS-28, CLS-39 and CLS-45 were chosen for furtherstudy. Based on the sequence of the 16S rRNA gene, the selectedisolates were identified as Streptomyces. The phylogenetic tree for thethree strains with other Streptomyces is shown in Fig. 1.

3.3. Experiments on Artemia

3.3.1. Biotoxicity test of marine Streptomyces on ArtemiaThe percentage of mortality of Artemia nauplii (Instar I) and adult

Artemia with different concentrations of the three Streptomycesstrains (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 andadults were 7.0 and 8.0% cell mass per unit volume respectively (after72 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 and7.0% respectively. Significantly higher (F=69.71, Pb0.01) mortalitywas observed by CLS-45 strain for both nauplii (67.7%) and adult(64.3%) with the increase of cell mass concentration.

3.3.2. Virulence of V. harveyi and V. proteolyticusV. proteolyticus was found to be more virulent than V. harveyi on

both nauplii and adult (Table 3). LC50 values for V. harveyi on Artemianauplii and adults were found to be 105.2 and 105.3 CFU ml−1

respectively and for V. proteolyticus those were 105 and105.1 CFU ml−1 respectively (Table 3). As the LC50 was higherthan 105 CFU ml−1 for both the stages of Artemia, the challengeand survival experiment was conducted with 106 CFU ml−1.

3.3.3. Protection of Artemia by Streptomyces strainsThe three Streptomyces strains (CLS-28, CLS-39 and CLS-45) were

able to protect the Artemia nauplii and adults from the pathogenic

results of cross streak assay against Vibrio pathogens.

sc Cross streak assayd

oteolytic Lipolytic V.h V.pa V.pr V.an V.al

+ − +++ +++ ++ + +++± + + − ++ ++++++ + + − ++ ++

+ + − ++ + + ++++ + − + ++ +++ −

± + + − + ++++ ++ +++ ++ +++ − +++ − + ++ − + ++++ − + ++ + − +++ + + +++ ++ + +

± − − ++ − +++++ + +++ − + ++ ++

— Milk agar.++, good; ++, medium; ±, doubtful; −, nil.

Vibrio proteolyticus, V.an— Vibrio anguillarum, V.al— Vibrio alginolyticus Inhibition: +++

Fig. 1. Phylogenetic tree showing the positions of three strains: CLS-28, CLS-39 and CLS-45 in the Streptomyces tree based on 16S rRNA partial gene sequence analysis. Numbersat nodes are bootstrap values (%) based on neighbour-joining analysis of 1000resampled datasets. The scale bar indicates the number of nucleotide substitutions persite.

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

action of V. harveyi and V. proteolyticus, as the survival rate of Artemiain the presence of each of these strains was higher than that whenonly V. harveyi or V. proteolyticus were administered in the experi-

Table 2Mortality (%) of Artemia after 24, 48 and 72 h immersion in different concentrations ofcell mass of three Streptomyces strains (CLS-28, CLS-39 and CLS-45).

Cellconc.

StagesofArtemia

CLS-28 CLS-39 CLS-45

24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h

Control Instar I 0 0 0 0 0 0 0 0 0Adult 0 0 0 0 0 0 0 0 0

0.1% Instar I 0 12.5 18.8 0 13.3 16.7 0 16.7 19.4Adult 0 6.7 13.3 0 12.5 15.6 0 14.7 17.6

0.5% Instar I 9.4 15.6 18.8 8.8 14.7 17.7 10.0 16.7 20.0Adult 7.7 11.5 15.4 6.7 13.3 16.7 8.8 14.7 17.7

1% Instar I 8.6 14.3 17.1 8.3 13.9 16.7 9.4 15.6 18.8Adult 6.9 13.8 17.2 6.1 12.1 15.2 7.7 15.4 15.4

5% Instar I 34.3 40.0 54.3 23.5 29.4 38.2 36.7 46.7 60.0Adult 31.4 37.1 48.6 22.2 25.0 33.3 36.1 41.7 52.8

10% Instar I 41.2 55.9 61.8 40.0 53.3 60.0 44.1 52.9 67.7Adult 37.5 46.9 59.4 35.7 46.4 53.6 42.9 53.6 64.3

ments (Figs. 2 and 3). V. harveyi at 106 CFU ml−1 killed all Artemianauplii in 72 h, but adults were more resistant and 27% survived after72 h. However, the survival of Artemia nauplii as well as of adults wassignificantly increased by the addition of Streptomyces strains(nauplii: F=111.176, Pb0.001, df=7; adults: F=29.25, Pb0.001,df=7). Significantly higher survival of Artemia nauplii (67%) andadults (61%) exposed to V. harveyi occurred with Streptomyces CLS-39than with Streptomyces CLS-45 after 72 h. Strain CLS-28 induced anintermediate survival rate compared to CLS-39 and 45.

All the nauplii and adults were killed by V. proteolyticus at106 CFU ml−1 after 72 h. However, addition of Streptomyces strainsenabled significantly higher survival (nauplii: F=138.266, Pb0.001,df=7; adults: F=56.58, Pb0.001, df=7). Greatest protection innauplii was shown with CLS-39 (survival = 52%) and in adults withCLS-28 (survival = 61%) after72 h, but the differences between thesetwo 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 asignificant reduction in Artemia survival compared to the control withboth of these strains. Strain CLS-45 also induced significantly highersurvival of Artemia under challenge with V. harveyi or V. proteolyticusexcept in adults challenged with V. harveyi. However, the survival withCLS-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 feedsStreptomyces were recorded from Feed 1, Feed 2 and Feed 3, but

not from the reference feed. It was assumed that the Streptomycespresent in the Feed 1, Feed 2 and Feed 3 were CLS-28, CLS-39 and CLS-45 respectively as these were the added strains. The highest THBcount was obtained in Feed 4 (≈106 CFU g−1), whereas the referencefeed, 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.

3.4.2. Stocking of the PLs for trialThe 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 respec-tively (mean±SE) (Table 4). At the time of stocking, the salinity of thewater was 35 ppt, temperature was 25 °C, pH was 7.0 and noammonia, nitrate or nitrite was observed.

3.4.3. In-trial physico-chemical parameters of water and survival ofanimals

The water quality parameters during the experiment are pre-sented in Table 4. Ammonia started developing in the control as wellas in all the treatments from day 1. Ammonia was in the range of 1 to2 ppm in all the treatments throughout and the differences were alsosignificant (Pb0.05). However, NO2

− and NO3− were not detected

throughout the trial. pH was almost consistent in all the treatmentswith the value of 7.0. The cumulative mortality in the each treatmentwas recorded and % survival calculated. The survival of animals in T3was significantly higher (Pb0.05) than in both the control and in T4which were not significantly different from each other. Survival in T1,T2, T4 and T5 treatments was intermediate to T3 and the control andnot significantly different to either. Animals in T1, T2 and T5 grewsignificantly better in terms of both length and weight than didcontrol animals.

3.4.4. In-trial microbial analysis and colonisation of StreptomycesTotal 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 allthe treatments (Fig. 4). This study showed that the Vibrio populationin T1 was significantly lower than in the control and treatments T2, T3

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

Cellconc.(CFU/ml)

Challenged (n±SD) Survived (n±SD) % mortality LC50 (CFU/ml)

Instar I Adult Instar I Adult Instar I Adult Instar I Adult

V. ha V. pb 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 29±5 32±2 21±3 35±2 6±2 2±1 5±1 3±1 79 94 80 91 105.2 105.04 105.3 105.14

×107 22±4 33±3 25±7 33±3 6±1 4±1 6±4 8±2 73 88 76 76×106 26±7 35±3 24±6 34±2 10±4 10±3 8±3 13±3 62 71 67 62×105 17±4 35±3 25±5 33±3 9±3 18±3 14±6 17±1 47 49 44 48

a Vibrio harveyi.b Vibrio proteolyticus.

37S. Das et al. / Aquaculture 305 (2010) 32–41

and T4 (Pb0.05), but not T5. The total bacterial counts showed nosignificant differences in the treatments (PN0.05).

Streptomyces spp. were isolated after weeks 1 and 2 from theanimals and from faeces from T1, T2 and T3 treatments, indicatingthat the Streptomyces strains reached the digestive system of shrimps.

3.4.5. LC50 of V. harveyi on P. monodonThe 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

Fig. 2. Protection (survival % mean±SE, n=4) of Artemia (A) nauplii and (B) adult from VibrTreatments with different letters differed significantly (Tukey's b; Pb0.05) within the mean

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 survivalAdministration of the Streptomyces in T1 and the commercial

probiotic in T5 significantly increased survival of P. monodon over thecontrol when challenged with V. harveyi (Table 5). Although theStreptomyces treatments T2 and T3 had lower survival than with T1only the T4 treatment with the commercial probiotic administered infood showed a significantly lower survival than T1 (Table 5).

io harveyi by addition of 1% (v/v) cell mass of Streptomyces (CLS-28, CLS-39 and CLS-45).survivals at 72 h observation.

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 CLS-45). Treatments with different letters differed significantly (Tukey's b; Pb0.05) within the mean survivals at 72 h observation.

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

4. Discussion

4.1. Biotoxicity of Streptomyces strains on Artemia

Despite some criticism (e.g. the absence of Artemia in most of themarine ecosystems, lack of sensitivity to chemical exposure due to theintrinsic resistance to extreme salinity conditions, see review byPersoone and Wells, 1987) against the use of Artemia in biotoxicitystudies, they have been reported to be able to detect lowerconcentrations of toxic compounds than other crustacean test

Table 4Water quality parameters (means across 15 d), growth and survival (initial number=25) osuperscripts in a column differed significantly (Tukey's b; Pb0.05).

Treatments Parameters

NH4 (ppm) % ofsurvivals

Total leng

Initial

Control 1.76±0.05b 74.67a 19.16±0.T1 (Feed+CLS-28) 1.48±0.06a 82.67a,b 19.55±0.T2 (Feed+CLS-39) 1.57±0.06a,b 85.33a,b 19.93±0.T3 (Feed+CLS-45) 1.62±0.06a,b 92.00b 19.74±0.T4 (Feed+Sanolife®) 1.57±0.06a,b 77.33a 19.53±0.T5 (Sanolife® in water) 1.46±0.06a 84.00a,b 19.49±0.

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

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

f P. monodon larvae during the 15 d trial in different treatments. Means with different

th (mm) Wet weight (g)

Final Initial Final

23 19.59±0.19a 0.0512±0.0018 0.0621±0.002a

55 20.62±0.21c,d 0.0566±0.0015 0.0832±0.004b,c

28 21.07±0.23d 0.0582±0.0022 0.0819±0.0033b

17 20.12±0.24a,b,c 0.0578±0.0018 0.0627±0.0036a

15 19.75±0.19a,b 0.0555±0.0016 0.0612±0.0024a

15 20.56±0.22b,c,d 0.0549±0.0015 0.0952±0.0048c

d wet weight.

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; Pb0.05).

39S. Das et al. / Aquaculture 305 (2010) 32–41

was anticipated that if Artemia could tolerate Streptomyces, and aprotective response was shown against pathogens, then the candidatespecies in aquaculture would also be able to get the beneficialprobiotic effect from Streptomyces.

To act as probionts, the isolated strains must be non-toxic andtolerable by the target animals (Gomez-Gil et al., 2000; Kesarcodi-Watson et al., 2008; Tinh et al., 2008). Lactic acid bacteria (LAB) andBacillus probiotic strains were reported to reach the digestive systemand 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 studywere also tolerated and found safe for both nauplii and adult Artemia.However, the assimilation into the digestive system was notspecifically checked, but rather assumed, as the Streptomyces weredetected in the shrimp postlarvae to which the Artemia were fed.

Although it was found that addition of Streptomyces led tomortality compared to negative control raising the question of safetyto target animals, this was mainly due to the aggregation offilamentous, viscous, live cell mass of Streptomyces which chockedthe respiration of Artemia. In the same well where there was noaggregation, Artemia were alive. Thus, it was assumed that themortality was not due to the toxicity of Streptomyces, it was due to theblocking of respiration. While the Vibrio and Streptomyceswere addedtogether to find out the protection, the pathogenicity of Vibrio wasfound to be reduced by the action of Streptomyces.

Nunes et al. (2006) mentioned that the age of the animals was amajor factor influencing results in toxicity testing. Barahona andSanchez-Fortun (1996) reported themost suitable age class of Artemiafor toxicological testing was 48 h old animals. The present study wasquite thus aptly conducted on 24, 48 and 72 h grown Artemia to get acomprehensive picture of biotoxicity and protective response throughthe addition of marine Streptomyces. Nauplii were found to be more

Table 5Survival and mortality of P. monodon in different treatments after exposure to virulentV. harveyi at 107 CFU ml−1 (n=3). Means with different superscripts in a columndiffered significantly (Tukey's b; Pb0.05).

Treatments Challenged (n±SE) Survived (n±SE) Mortality %

Control 15±0 6.33±0.33a 58.00T1 15±0 10.33±0.67b 31.33T2 15±0 9.33±0.67a,b 38.00T3 15±0 8.67±0.88a,b 42.00T4 15±0 6.67±0.88a 55.33T5 15±0 10.67±0.88b 28.67

susceptible and showed lower survival when exposed to Streptomycesin all the experiments indicating that adult Artemiaweremore robust.

4.2. Protection of Artemia from Vibrio

The experimental animals which received a combination ofpathogen and Streptomyces showed significantly higher survivalrates than the untreated control group (pathogen only). Higherprotection (survival 68%) was provided by prior addition of CLS-39and lower survival (39%)was shown by CLS-45 against V. harveyi. CLS-28 provided maximum protection (survival 61%) and CLS-45 showedminimum protection (survival 38%) protection against V. proteolyti-cus. It could be considered that the Streptomyces strains showedprobiotic activity and either improved the disease resistance ability ofnauplii and adult Artemia or inactivated the virulent Vibrio spp. tosome extent, or both. Other studies have reported improved survivalof Artemia challenged with pathogenic vibrios: Verschuere et al.(2000b) reported a higher survival rate of Artemia (∼80%) whenchallenged with V. proteolyticus and protected by preemptivecolonization of selected bacterial strains (Aeromonas spp. and Vibrioalginolyticus). Marques et al. (2006) reported 95% survival of Artemiaprotected with Bacillus sp. after 72 h challenge with V. proteolyticus.Patra and Mohamed (2003) also found 91% survival when Artemianauplii were protected by yeast from V. harveyi infection. Althoughthese studies reported higher survival than those in the present study,it was worthy to ascertain the potential of Streptomyces spp. whichcan protect Artemia from the action of Vibrio spp., because the spore-forming capacity of Streptomyces may make them a more practicalalternative than non-spore-forming microbes. Gatesoupe (1991)reported that the efficacy of Artemia nauplii in bioencapsulatingbacteria strongly depends on the type of bacteria used, time ofexposure, and status of the bacteria.We found that Artemia tolerated 3strains of Streptomyces (CLS-28, 39 and 45) very well and thus shouldbe able to transfer the Streptomyces to animals to which they are fed.

Streptomyces species are distributed widely in aquatic andterrestrial habitats (Pathom-aree et al., 2006) and are of commercialinterest due to their strong capacity to produce novel bioactivecompounds. It was also expected that Streptomyces species will have acosmopolitan distribution as they produce abundant spores which arereadily dispersed (Antony-Babu et al., 2008). There are several reportsof inhibition of Vibrio spp. by marine Streptomyces (Das et al., 2004;You et al., 2007). Marine Streptomyces are a major source ofantibacterial compounds (Fenical and Jensen, 2006). Therefore, theprotective effect of Streptomyces strains may be due to the productionof antibacterial compounds active against V. harveyi and V.proteolyticus.

40 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 ofshrimp P. monodon revealed that Streptomyces CLS-28 can beeffectively used probiotically in aquaculture. Results indicated thatthe Streptomyces-treated P. monodon culture treatment showed lowerVibrio counts in the T1 treatment than in the control, although totalheterotrophic bacterial counts were not significantly different. Theseresults 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 beassessed for safety to the host. Two strains of Streptomyces (CLS-28and 39) significantly increased the growth of PLs compared to thecontrol and the significant survival (Pb0.05) was found during 15 dtrial in all the treatments fed with Streptomyces fortified feed. Instead,after feeding for 15 d significant growth differences (Pb0.05) wasfound among the treatments. In addition, the concentration ofammonia in the treatments was the same as in the control and norwas nitrate or nitrite found indicating no adverse effect on the tankenvironment. Probiotics may improve digestive activity by synthesisof vitamins, cofactors or by improving enzymatic activity (Fuller,1989; Gatesoupe, 1999). These properties could have contributed tothe weight increase seen in T1 and T2 compared to the control(Table 4), by improving amylolytic and proteolytic activity in theshrimp digestive tract.

In this study, the presence of Streptomyces from the faeces and inthe whole animals indicated that it reached to the digestive system ofshrimps, but this does not confirm that Streptomyces colonised the gutas the shrimps were fed Streptomyces-fortified feed daily.

In most studies, the explanation for the mechanisms of action ofprobiotics is largely based on in vitro observations, neglecting that invivo physiology might be different from the metabolic process in vitro.Selective ingestion by the host (Riquelme et al., 2001), death in thedigestive tract (Vine et al., 2006), or a failure of a probiotic to maintainits in vitro physiology under circumstances of more complexmicrobialinteractions and/or nutritional environment are some of the chal-lenges that a probiotic might face inside a host. Moreover, theinteractions between the introduced probiotics and the indigenousgastrointestinal (GI) microbiota are still poorly understood (Tinhet al., 2008). Considering these views, the probiotic mode(s) of actionof the Streptomyces are yet to be determined.

The most important limitation to the use of probiotics is that inmany cases they are not able to maintain themselves, and so need tobe added regularly and at high concentrations (Vine et al., 2006),which makes this technique less cost-effective. Moreover, probioticsthat were selected in vitro based on the production of inhibitorycompounds 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 sys-tem(s), which will facilitate their growth and establishment in thehost. It was for this reason, that in the present study, Streptomyceswere isolated from the shrimp pond sediment samples.

The protection afforded the postlarvae by Streptomyces treatmentgroups (T1, T2 and T3) were not significantly different than from thecommercial probiotic (T5). The commercial probiotic used in thisexperiment was labelled as a mixture of Bacillus subtilis, B.licheniformis and B. pumilus. The efficiency of the Streptomyces couldbe improved by optimising the dose and the mode of application. Thestudy was conducted in the small jugs where water was exchangeddaily, which might not allow Streptomyces to colonise completely(even though Streptomyceswere recovered from the animals and theirfaeces). Microorganisms only produce metabolites during the sta-tionary growth phase and if complete colonisation does not occur inthe gut due to constant flushing, total protectionmay not be observed.In addition, as suggested by Vine et al. (2004) for other bacterialprobiotics, the growth of Streptomyces may be less than the rate of

flushing from the intestine, and Streptomycesmay be unable to attachto the intestine so that they will be flushed before reaching a viablepopulation level. Another point is that this trial was conducted onlyfor 15 d and the slowly growing Streptomyces perhaps needed a longertime to colonise the animals. However, it is to keep in mind thepotential setbacks of using Streptomyces in culture system whichinclude the production of clinical antibiotics and lateral gene transferof antibiotic-resistance gene (reviewed in Das et al., 2008a). But, asthe several clinical antibiotics are also produced by bacterial probionts(e.g. Bacillus) and lateral gene transfer of antibiotic-resistance gene isan ecological phenomenon, Streptomyces may be potentially used asprobiotics in aquaculture.

5. Conclusion

From this study it can be concluded that marine Streptomycesstrains can protect Artemia nauplii and adults from the infection ofVibrio spp. and are non-toxic to shrimp and are able to protect themfrom Vibrio spp. in culture systems. Thus, Streptomyces can beincluded among the potential biological control agents in aquaculture.However, to establish a probiotic nature of Streptomyces in diseaseprevention and/ or growth stimulator of aquaculture animals, furtherextensive trials with different target animals and a commercial cost–benefit analysis are necessary. It would be advantageous if a pond trialcan be done in future. Streptomyces are saprophytic and grow well inthe sediment, which will help them to colonise host animals. It isimportant to determine if a combination of Streptomyces strains canbe effectively used in aquaculture either as water or feed probiotics. Itis also essential to determine the optimum dose and the best mode ofapplication.

Acknowledgement

An Endeavour Research Fellowship to S.D by Department ofEducation, Employment and Workplace Relations, Australian Gov-ernment to carry out Postdoctoral research at University of Tasmaniais gratefully acknowledged. Funding to carry out the project wasprovided by the National Centre for Marine Conservation andResource Sustainability and is gratefully acknowledged. Specialthanks are due to Dr. Chris Bolch for his help in 16S rRNA genesequencing study. We also thank Mr. Detlef Planko, Dr. Mark Adams,Mr. Daniel Pountney and Mr. Jon Schrepfer for rendering theirtechnical help in the setting up of the tanks.

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