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 b s t r a c t 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
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 LC
50
of V. harveyi and V.
proteolyticus was found to be 10
6
CFU ml
1
. V. proteolyticus was more pathogenic than V. harveyi at
10
6
CFU ml
1
. A signicant reduction in mortality (Pb0.001) 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 signicant differences between treatments (Pb0.05).
Signicant differences (Pb0.05) 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 10
8
and 10
6
CFU ml
1
respectively in all the treatments. The Vibrio population differed signicantly in the treatments (Pb0.05) and
the total bacterial counts showed no signicant differences in the treatments (PN0.05). After challenge with
V. harveyi at 10
7
CFU ml
1
, highest survival was found in T1 and T5. Among the Streptomyces treatments, T1
showed signicantly 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) dened
a probiotic as a live microbial feed supplement which benecially affects
the host animal by improving its intestinal microbial balance. A modied
and more appropriate denition was proposed by Verschuere et al.
(2000a) a live microbial adjunct which has a benecial effect on the
host by modifying the host-associated or ambient microbial community,
byensuring improveduseof thefeedor enhancingits nutritional value, by
enhancing the host response towards disease, or by improving the quality
of its ambient environment.
Actinobacteria is a class with ve subclasses that was proposed by
Stackebrandt et al. (1997) to group the highly diverse so called
actinomycetes based on chemical composition, DNADNA reassocia-
tion and 16S rRNA gene sequence similarities. Members of the
Actinobacteria are prolic 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 Strepto-
mycetaceae (Stackebrandt et al., 1997) and has a DNA G+C content of
6978 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, andadaptability to variednutrient resources
(Nunes et al., 2006). There have been several experiments carried out on
Artemia in the search for new biocontrol agents for aquaculture
Aquaculture 305 (2010) 3241
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: surajit@myself.com, surajit@nitrkl.ac.in (S. Das).
0044-8486/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2010.04.001
Contents lists available at ScienceDirect
Aquaculture
j our nal homepage: www. el sevi er. com/ l ocat e/ aqua- onl i ne
(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 LC
50
of virulent Vibrio harveyi and V.
proteolyticus were examined in Artemia to determine LC
50
value, and
the efcacy 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.
2. Materials and methods
2.1. Isolation of marine Streptomyces
Marine sediment samples were collected fromshrimp farms located
at Queensland, Australia (lat 214309S and long 1492554E). 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, K
2
HPO
4
: 2 g, KNO
3
: 2 g,
Casein: 0.3 g, MgSO
4
7H
2
O: 0.05 g, CaCO
3
: 0.02 g, FeSO
4
7H
2
O: 0.01 g,
Agar: 15 g, Filtered sea water: 1000 ml and pH: 7.00.1), 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, KNO
3
: 2 g,
K
2
HPO
4
: 2 g, Soluble starch: 0.5 g, Asparagine: 0.1 g, FeSO
4
.7H
2
O: 0.01 g,
CaCO
3
: 0.02 g, MgSO
4
.7H
2
O: 0.05 g, Agar: 15 g, Filtered sea water:
1000 ml and pH: 7.00.1). Each medium was supplemented with
nystatinand cyclohexamide at 25 g ml
1
and 10 g ml
1
respectively
to minimize contamination with fungi and 10 g ml
1
nalidixic acid to
minimizecontaminant 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 laments with or without aerial mycelia (Mincer et al., 2002)
were sub-cultured and transferred on SCAand 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 Modied 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: 7.00.1) 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 sh-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 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.
2.2.2. Exoenzymatic assay
Cultured actinomycetes were screened for hydrolytic exoenzy-
matic 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 ooding 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
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 mediumin
100 ml Schott asks 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 identication of the selected
actinomycetes strains
Molecular identication of three selected actinomycetes strains
(CLS-28, CLS-39 and CLS-45) as potential probionts were carried out
by 16S rRNA gene (16S rDNA) amplication and sequencing.
2.4.1. DNA extraction
The biomass of three actinomycetes isolates was harvested by
centrifugation. Pellet was washed twice in sterile TrisEDTA buffer
and approximately 100 mg (wet weight) biomass was used for DNA
extraction based on cetyltrimethylammonium bromide (CTAB)
purication following Ausubel et al. (1999).
2.4.2. DNA amplication 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 l volumes containing 2 mM MgCl
2
, 2.5 U Taq
polymerase (Bioline, Australia), 100 M of each dNTP, 0.2 M of
each primer and 3 l 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 nal extension at
72 C for 1 min. Amplied DNAs were puried by the MontagePCR
centrifugal lter device (Millipore Corp., USA) following manufac-
turer's instructions and nally quantied by Turner TBS380 DNA
uorometer. 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
33 S. Das et al. / Aquaculture 305 (2010) 3241
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 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 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 air-
saturated 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 10
8
to 10
5
CFU ml
1
and used in the
experiments to determine virulence. Aliquots of each dilution were
plated on TSA to conrm 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 10
5
to 10
8
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 LC
50
(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 nal concentration of 10
6
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 (PL
16
) 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 lled with 1.5 l of water and each jug stocked with 25 shrimps at
PL
33
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 ) lters 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 24 h to keep the nal 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 PL
40
(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 10
7
to 10
3
CFU ml
1
. Aliquots of each dilution
were plated on TSA to conrm the CFU ml
1
. Shrimp postlarvae were
immersed for 12 h in duplicate vessels containing either 10
3
, 10
4
, 10
5
,
10
6
or 10
7
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 PL
40
P. monodon was determined from the
LC
50
as described above for Artemia.
2.6.5. Protection of PL from V. harveyi
P. monodon PL
33
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
34 S. Das et al. / Aquaculture 305 (2010) 3241
germination following manufacturer details). All treatments and the
control were replicated ve times. All the shrimps received prepared
feed twice daily (at dawn and dusk) at approximately 58% body
weight day
1
. The amount of feed was determined by feeding ad
libitumand 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
(meanSD) 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. pHof water
(by Whatman pH paper), temperature (by centigrade thermometer)
and salinity (by refractometer) were also monitored daily. Every
week, shrimp faeces and a fewlive shrimps fromeach jug (fromT1, 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 10
7
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 ve days.
2.7. Statistical analyses
Data are presented as meanstandard error. Signicance of
difference between different treatment groups was tested using
one-way analysis of variance (ANOVA) and signicant results were
compared with Tukey's HSD post-hoc test. Homogeneity of variance
was assessed by residual plots. For all the tests the signicance was
determined 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 very
poor or nil. However, further sub-culture for purication was only
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. Identication 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 identied 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 LC
50
(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 LC
50
of CLS-39 on nauplii and adult was 8.0 and 9.3%
respectively and the LC
50
of CLS-45 on nauplii and adult was 6.5 and
7.0% respectively. Signicantly higher (F=69.71, Pb0.01) mortality
was observed by CLS-45 strain for both nauplii (67.7%) and adult
(64.3%) with the increase of cell mass concentration.
3.3.2. 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). LC
50
values for V. harveyi on Artemia
nauplii and adults were found to be 10
5.2
and 10
5.3
CFU ml
1
respectively and for V. proteolyticus those were 10
5
and
10
5.1
CFU ml
1
respectively (Table 3). As the LC
50
was higher
than 10
5
CFU ml
1
for both the stages of Artemia, the challenge
and survival experiment was conducted with 10
6
CFU ml
1
.
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
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 strains
a
Growth on
media
b
Enzymatic activities
c
Cross streak assay
d
Obverse Reverse Amylolytic Proteolytic Lipolytic V.h V.pa V.pr V.an V.al
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; MA Milk agar.
c
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.
35 S. Das et al. / Aquaculture 305 (2010) 3241
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 experi-
ments (Figs. 2 and 3). V. harveyi at 10
6
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
signicantly increased by the addition of Streptomyces strains
(nauplii: F=111.176, Pb0.001, df =7; adults: F=29.25, Pb0.001,
df =7). Signicantly 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
10
6
CFU ml
1
after 72 h. However, addition of Streptomyces strains
enabled signicantly higher survival (nauplii: F=138.266, Pb0.001,
df =7; adults: F=56.58, Pb0.001, 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 signicant 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
signicant reduction in Artemia survival compared to the control with
both of these strains. Strain CLS-45 also induced signicantly 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 CLS-
45 respectively as these were the added strains. The highest THB
count was obtained in Feed 4 (10
6
CFU g
1
), whereas the reference
feed, Feed 1, Feed 2 and Feed 3 had a THB of 5.5 to 8.510
4
CFU g
1
.
No Vibrio was recorded from any of the feed.
3.4.2. Stocking of the PLs for trial
The age of the stocked P. monodon was PL
33
and the mean length
and wet weight were 19.570.08 mm and 0.0550.0008 g respec-
tively (meanSE) (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 pre-
sented 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
signicant (Pb0.05). However, NO
2

and NO
3

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 signicantly higher (Pb0.05) than in both the control and in T4
which were not signicantly different from each other. Survival in T1,
T2, T4 and T5 treatments was intermediate to T3 and the control and
not signicantly different to either. Animals in T1, T2 and T5 grew
signicantly 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
2.510
8
CFU ml
1
and 3.5 to 1410
8
CFU ml
1
respectively in all
the treatments (Fig. 4). This study showed that the Vibrio population
in T1 was signicantly lower than in the control and treatments T2, T3
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. 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.
Table 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 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 0
Adult 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 19.4
Adult 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 20.0
Adult 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 18.8
Adult 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 60.0
Adult 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 67.7
Adult 37.5 46.9 59.4 35.7 46.4 53.6 42.9 53.6 64.3
36 S. Das et al. / Aquaculture 305 (2010) 3241
and T4 (Pb0.05), but not T5. The total bacterial counts showed no
signicant differences in the treatments (PN0.05).
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 systemof shrimps.
3.4.5. LC
50
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 10
7
CFU ml
1
dosages and the calculated LC
50
was 10
6.5
CFU ml
1
.
3.4.6. Challenge by V. harveyi and survival
Administration of the Streptomyces in T1 and the commercial
probiotic in T5 signicantly 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 signicantly lower survival than T1 (Table 5).
Table 3
LC
50
determination of virulent V. harveyi and V. proteolyticus on Artemia. Number of Artemia challenged and number surviving together with percentage mortality.
Cell
conc.
(CFU/
ml)
Challenged (nSD) Survived (nSD) % mortality LC
50
(CFU/ml)
Instar I Adult Instar I Adult Instar I Adult Instar I Adult
V. h
a
V. p
b
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
10
8
295 322 213 352 62 21 51 31 79 94 80 91 10
5.2
10
5.04
10
5.3
10
5.14
10
7
224 333 257 333 61 41 64 82 73 88 76 76
10
6
267 353 246 342 104 103 83 133 62 71 67 62
10
5
174 353 255 333 93 183 146 171 47 49 44 48
a
Vibrio harveyi.
b
Vibrio proteolyticus.
Fig. 2. Protection (survival % meanSE, n=4) of Artemia (A) nauplii and (B) adult fromVibrio harveyi by addition of 1% (v/v) cell mass of Streptomyces (CLS-28, CLS-39 and CLS-45).
Treatments with different letters differed signicantly (Tukey's b; Pb0.05) within the mean survivals at 72 h observation.
37 S. Das et al. / Aquaculture 305 (2010) 3241
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 specic benecial
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
Fig. 3. Protection (survival % meanSE, n=4) of Artemia (A) nauplii and (B) adult fromVibrio proteolyticus by addition of 1% (v/v) cell mass of Streptomyces (CLS-28, CLS-39 and CLS-
45). Treatments with different letters differed signicantly (Tukey's b; Pb0.05) within the mean survivals at 72 h observation.
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 signicantly (Tukey's b; Pb0.05).
Treatments Parameters
NH
4
(ppm) % of
survivals
Total length (mm) Wet weight (g)
Initial Final Initial Final
Control 1.760.05
b
74.67
a
19.160.23 19.590.19
a
0.05120.0018 0.06210.002
a
T1 (Feed+CLS-28) 1.480.06
a
82.67
a,b
19.550.55 20.620.21
c,d
0.05660.0015 0.08320.004
b,c
T2 (Feed+CLS-39) 1.570.06
a,b
85.33
a,b
19.930.28 21.070.23
d
0.05820.0022 0.08190.0033
b
T3 (Feed+CLS-45) 1.620.06
a,b
92.00
b
19.740.17 20.120.24
a,b,c
0.05780.0018 0.06270.0036
a
T4 (Feed+Sanolife) 1.570.06
a,b
77.33
a
19.530.15 19.750.19
a,b
0.05550.0016 0.06120.0024
a
T5 (Sanolife in water) 1.460.06
a
84.00
a,b
19.490.15 20.560.22
b,c,d
0.05490.0015 0.09520.0048
c
Values are in MeanSE. n=45 for NH
3
; n=3 for % survival and n=15 for total length and wet weight.
38 S. Das et al. / Aquaculture 305 (2010) 3241
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 benecial
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; Kesarcodi-
Watson 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 ora 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
specically 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
lamentous, 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 nd 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 inuencing 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
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 signicantly 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. CLS-
28 provided maximum protection (survival 61%) and CLS-45 showed
minimum protection (survival 38%) protection against V. proteolyti-
cus. 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 spore-
forming capacity of Streptomyces may make them a more practical
alternative than non-spore-forming microbes. Gatesoupe (1991)
reported that the efcacy 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.
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 signicantly
(Tukey's b; Pb0.05).
Table 5
Survival and mortality of P. monodon in different treatments after exposure to virulent
V. harveyi at 10
7
CFU ml
1
(n=3). Means with different superscripts in a column
differed signicantly (Tukey's b; Pb0.05).
Treatments Challenged (nSE) Survived (nSE) Mortality %
Control 150 6.330.33
a
58.00
T1 150 10.330.67
b
31.33
T2 150 9.330.67
a,b
38.00
T3 150 8.670.88
a,b
42.00
T4 150 6.670.88
a
55.33
T5 150 10.670.88
b
28.67
39 S. Das et al. / Aquaculture 305 (2010) 3241
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 signicantly 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) signicantly increased the growth of PLs compared to the
control and the signicant survival (Pb0.05) was found during 15 d
trial in all the treatments fed with Streptomyces fortied feed. Instead,
after feeding for 15 d signicant growth differences (Pb0.05) 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 conrmthat Streptomyces colonised the gut
as the shrimps were fed Streptomyces-fortied 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 fromthe 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 chal-
lenges 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 sys-
tem(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 signicantly 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 efciency 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 fromthe animals and their
faeces). Microorganisms only produce metabolites during the sta-
tionary growth phase and if complete colonisation does not occur in
the gut due to constant ushing, 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
ushing from the intestine, and Streptomyces may be unable to attach
to the intestine so that they will be ushed 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
benet 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 Gov-
ernment 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, 6374.
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--vis survival and growth of Penaeus monodon
postlarvae. Aquaculture 248, 227234.
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, 271278.
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, 6270.
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, 301306.
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 sh fry.
Fish Shellsh Immunol. 3, 451459.
Chythanya, R., Karunasagar, I., Karunasagar, I., 2002. Inhibition of shrimp pathogenic
vibrios by a marine Pseudomonas I-2 strain. Aquaculture 208, 110.
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, 13251335.
40 S. Das et al. / Aquaculture 305 (2010) 3241
Das, S., Lyla, P.S., Ajmal Khan, S., 2006b. Application of Streptomyces as a probiotic in the
laboratory culture of Penaeus monodon (Fabricius). Isr. J. Aquac.- Bamidgeh 58, 198204.
Das, S., Lyla, P.S., Ajmal Khan, S., 2008a. Distribution and generic composition of
culturable marine actinomycetes from the sediments of Indian continental slope of
Bay of Bengal. Chinese J. Oceanol. Limnol. 26, 166177.
Das, S., Ward, L.R., Burke, C., 2008b. Prospects of using marine actinobacteria as
probiotics in aquaculture. Appl. Microbiol. Biotechol. 81, 419429.
Defoirdt, T., Halet, D., Sorgeloos, P., Bossier, P., Verstraete, W., 2006. Short-chain fatty
acids protect gnotobiotic Artemia franciscana from pathogenic Vibrio campbelii.
Aquaculture 261, 804808.
Defoirdt, T., Boon, N., Sorgeloos, P., Verstraete, W., Bossier, P., 2007. Alternatives to
antibiotics to control bacterial infections: luminescent vibriosis in aquaculture as
an example. Trends Biotechnol. 25, 472479.
Dixon, B.A., Van Pucke, S.O., Chair, M., Demasque, M., Nelis, H.J., Sorgeloos, P., De
Leenheer, A.P., 1995. Bioencapsulation of the antibacterial drug safraoxacin in
nauplii of the brine shrimp Artemia franciscana. J. Aquat. Anim. Health 7, 4245.
Felsenstein, J., 1985. Condence limits on phylogenies: an approach using the
bootstrap. Evolution 39, 783791.
Fenical, W., Jensen, P.R., 2006. Developing a new resource for drug discovery: marine
actinomycete bacteria. Nat. Chem. Biol. 2, 666673.
Fuller, R., 1989. Probiotics in man and animals, a review. J. Appl. Bacteriol. 66, 365378.
Gatesoupe, F.J., 1991. Managing the dietary value of Artemia in larval turbot,
Scophthalmus maximus: the effect of enrichment and distribution techniques.
Aquacult. Eng. 10, 111119.
Gatesoupe, F.J., 1999. The use of probiotics in aquaculture. Aquaculture 180, 147165.
Gatesoupe, F.J., 2002. Probiotic and formaldehyde treatments of Artemia nauplii as food
for larval pollack, Pollachius pollachius. Aquaculture 212, 347360.
Gomez-Gil, B., Herrera-Vega, M.A., Abreu-Grobois, F.A., Roque, A., 1998. Bioencapsula-
tion of two different Vibrio species in nauplii of the brine shrimp (Artemia
franciscana). Appl. Environ. Microbiol. 64, 23182322.
Gomez-Gil, B., Roque, A., Turnbull, J.F., 2000. The use and selection of probiotic bacteria
for use in the culture of larval aquatic organisms. Aquaculture 191, 259270.
Hai, N.V., Fotedar, R., Buller, N., 2007. Selection of probiotics by various inhibition test
methods for use in the culture of western king prawns, Penaeus latisulcatus
(Kishinouye). Aquaculture 272, 231239.
Kesarcodi-Watson, A., Kaspar, H., Lategan, M.J., Gibson, L., 2008. Probiotics in
aquaculture: the need, principles and mechanisms of action and screening
processes. Aquaculture 274, 114.
Kumar, S.S., Philip, R., Achuthankutty, C.T., 2006. Antiviral property of marine actinomycetes
against white spot syndrome virus in penaeid shrimps. Curr. Sci. 91, 807811.
Marques, A., Francois, J.-M., Dhont, J., Bossier, P., Sorgeloos, P., 2004. Inuence of yeast quality
on performance of gnotobiotically grown Artemia. J. Exp. Mar. Biol. Ecol. 310, 247264.
Marques, A., Thanh, T.H., Sorgeloos, P., Bossier, P., 2006. Use of microalgae and bacteria
to enhance protection of gnotobiotic Artemia against different pathogens.
Aquaculture 258, 116126.
Mincer, T.J., Jensen, P.R., Kauffman, C.A., Fenical, W., 2002. Widespread and persistent
populations of a major new marine actinomycete taxon in ocean sediments. Appl.
Environ. Microbiol. 68, 50055011.
Nunes, B.S., Carvalho, F.D., Guilhermino, L.M., Stappen, G.V., 2006. Use of the genus
Artemia in ecotoxicity testing. Environ. Pollut. 144, 453462.
Pathom-aree, W., Stach, J.E.M., Ward, A.C., Horikoshi, K., Bull, A.T., Goodfellow, M., 2006.
Diversity of actinomycetes isolated from Challenger Deep sediment (10, 898 m)
from the Mariana Trench. Extremophiles 10, 181189.
Patra, S.K., Mohamed, K.S., 2003. Enrichment of Artemia nauplii with the probiotic yeast
Saccharomyces boulardii and its resistance against a pathogenic Vibrio. Aquacult. Int.
11, 505514.
Persoone, G., Wells, P.G., 1987. Artemia in aquatic toxicology: a review. In: Sorgellos, P.,
Bengtson, D.A., Decleir, W., Jaspers, E. (Eds.), Artemia Research and Its Applications.
: Morphology, Genetics, Strain Characterization, Toxicology, vol. I. Universa Press,
Wetteren, Belgium.
Ravel, J., Amoroso, M.J., Colwell, R.R., Hill, R.T., 1998. Mercury-resistant actinomycetes
from the Chesapeake Bay. FEMS Microbiol. Lett. 162, 177184.
Reed, L.J., Muench, H., 1938. A simple method of estimating fty percent endpoints. Am.
J. Epidemiol. 27, 493497.
Rengpipat, S., Phianphak, W., Piyatiratitivorakul, S., Menasveta, P., 1998. Effects of a
probiotic bacterium on black tiger shrimp Penaeus monodon survival and growth.
Aquaculture 167, 301313.
Rengpipat, S., Rukpratanporn, S., Piyatiratitivorakul, S., Menasaveta, P., 2000. Immunity
enhancement in black tiger shrimp (Penaeus monodon) by a probiont bacterium
(Bacillus S11). Aquaculture 191, 271288.
Rengpipat, S., Tunyanun, A., Fast, A.W., Piyatiratitivorakul, S., Menasveta, P., 2003.
Enhanced growth and resistance to Vibrio challenge in pond-reared black tiger
shrimp Penaeus monodon fed a Bacillus probiotic. Dis. Aquat. Org. 55, 169173.
Riquelme, C.E., Jorquera, M.A., Rojas, A.I., Avendano, R.E., Reyes, N., 2001. Addition of
inhibitor-producing bacteria to mass cultures of Argopecten purpuratus larvae
(Lamarck, 1819). Aquaculture 192, 111119.
Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstruct-
ing phylogenetic trees. Mol. Biol. Evol. 4, 406425.
Sorgeloos, P., Coutteau, P., Dhert, P., Merchie, G., Lavens, P., 1998. Use of brine shrimp,
Artemia spp., in larval crustacean nutrition: a review. Rev. Fish. Sci. 6, 5568.
Stackebrandt, E., Rainey, F.A., Ward-Raine, N.L., 1997. Proposal for a new hierarchic
classication system, Actinobacteria classis nov. Int. J. Syst. Bacteriol. 47, 479491.
Takizawa, M., Hill, R.T., Colwell, R.R., 1993. Isolation and diversity of actinomycetes in
the Chesapeake Bay. Appl. Environ. Microbiol. 59, 9971002.
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. CLUSTAL X
windows interface: exible strategies for multiple sequence alignment aided by
quality analysis tools. Nucleic Acids Res. 25, 48764882.
Tinh, N.T.N., Dierckens, K., Sorgeloos, P., Bossier, P., 2008. A review of the functionality
of probiotics in the larviculture food chain. Mar. Biotechnol. 10, 112.
Vaseeharan, B., Ramasamy, P., 2003. Control of pathogenic Vibrio spp. by Bacillus subtilis
BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon. Lett.
Appl. Microbiol. 36, 8387.
Verschuere, L., Rombaut, G., Huys, G., Dhont, J., Sorgeloos, P., Verstraete, W., 1999.
Microbial control of the culture of Artemia juveniles through preemptive
colonization by selected bacterial strains. Appl. Environ. Microbiol. 65, 25272533.
Verschuere, L., Heang, H., Criel, G., Sorgeloos, P., Verstraete, W., 2000a. Selected
bacterial strains protect Artemia spp. from the pathogenic effects of Vibrio
proteolyticus CW8T2. Appl. Environ. Microbiol. 66, 11391146.
Verschuere, L., Rombaut, G., Sorgeloos, P., Verstraete, W., 2000b. Probiotic bacteria as
biological control agents in aquaculture. Microbiol. Mol. Biol. Rev. 64, 655671.
Villamil, L., Figueras, A., Planas, M., Novoa, B., 2003. Control of Vibrio alginolyticus in
Artemia culture by treatment with bacterial probiotics. Aquaculture 219, 4356.
Vine, N.G., Leukes, W.D., Kaiser, H., 2004. In vitro growth characteristics of ve
candidate aquaculture probiotics and two sh pathogens grown in sh intestinal
mucus. FEMS Microbiol. Lett. 231, 145152.
Vine, N.G., Leukes, W.D., Kaiser, H., 2006. Probiotics in marine larviculture. FEMS
Microbiol. Rev. 30, 404427.
You, J., Xue, X., Cao, L., Lu, X., Wang, J., Zhang, L., Zhou, S., 2007. Inhibition of Vibrio
biolm formation by a marine actinomycete strain A66. Appl. Microbiol.
Biotechnol. 76, 11371144.
41 S. Das et al. / Aquaculture 305 (2010) 3241