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Search for beneficial bacterial strains for turbot
ž
Scophthalmus maximus L. larviculture
/
L. Huys
a,b,), P. Dhert
a, R. Robles
a, F. Ollevier
c, P. Sorgeloos
a,
J. Swings
c,da
Laboratory of Aquaculture and Artemia Reference Center, Ghent UniÕersity, Rozier 44, 9000 Gent, Belgium
b
Laboratory of Microbiology, Ghent UniÕersity, K.L. — Ledeganckstraat 35, 9000 Gent, Belgium
c
Laboratory of Aquatic Ecology, Zoological Institute, Catholic UniÕersity of LouÕain, De Beriotstraat 32,
3000 LouÕain, Belgium
d
BCCMrLMG Culture Collection, Ghent UniÕersity, K.L. — Ledeganckstraat 35, 9000 Gent, Belgium
Received 5 January 2000; received in revised form 10 July 2000; accepted 21 July 2000
Abstract
The aerobic bacterial flora in the gut of turbot larvae and their influence on larval survival was examined. Two turbot experiments were run with six replicates each time. Large variation, from 0% up to 44%, was observed in the survival percentage of turbot larvae. There was no correlation between the number of bacteria present in the gut of turbot larvae and the larval survival rate. During both experiments, all replicates followed nearly the same rate of bacterial development in the gut of turbot larvae going from circa 102 CFU larvay1 just before first feeding at day 3 post hatch to 105CFU larvay1at day 9 post hatch.
In total, 127 bacterial isolates from 12 rearing tanks were sampled for further investigation. Based on their fatty acid profile obtained by FAME-analysis, and using principal component
Ž
analysis, the isolates were subdivided in 12 major gaschromatographic-groups or clusters
GC-. Ž .
groups , 11 isolates remained unclustered. Four specific GC-groups namely cluster A, B, I and J were selected as potential beneficial bacteria for turbot larviculture as the majority of the isolates of these clusters derived from rearing tanks with a survival percentage higher than 35%. Representative isolates of these clusters were screened on their ability to enhance the survival rate as well as the poor reproducibility in larval survival in a small-scale turbot confrontation test. Also, a Vibrio mediterranei Q40 strain, isolated from sea bream larvae, was included in these small-scale confrontation tests. Only cluster A and the V. mediterranei Q40 strain had a distinct positive and reproducible effect on larval survival. In conclusion, cluster A and V. mediterranei
)Corresponding author. Tel.:q32-9-264-3754; fax:q32-9-264-4193.
Ž .
E-mail address: [email protected] L. Huys .
0044-8486r01r$ - see front matterq2001 Elsevier Science B.V. All rights reserved.
Ž .
Q40 seemed to play a role as first coloniser of the gut of turbot larvae and could prevent the colonisation of the gut by opportunistic bacteria. q2001 Elsevier Science B.V. All rights reserved.
Keywords: Turbot larvae; Bacteria; Microbial management; Probiotic
1. Introduction
Considerable progress has been accomplished in the commercial culture of turbot
ŽScophthalmus maximus L. but larval survival remains unpredictable, especially, in the. Ž
second week post hatch when mass mortalities are observed Planas, 1994; Ringø and
.
Vadstein, 1998; Ringø and Birkbeck, 1999 . Experiments have suggested that these
Ž
mortalities may be due to inadequate microbial conditions Minkoff and Broadhurst,
.
1994 , however, most of these mortalities could not be associated with primary or specific pathogens but rather with opportunistic bacteria that attack the host larvae under
Ž
stress conditions Olafsen, 1993; Vadstein et al., 1993; Munro et al., 1995; Verdonck
.
and Swings, 1995 .
Ž
Since fish larvae establish their bacterial flora partly in a non-selective way Hansen
.
and Olafsen, 1990; Cahill, 1990 , the initial bacterial environment is of utmost impor-tance. In this respect, the early colonisation of the gut by non-opportunistic bacteria may initiate a resident microflora which could prevent the proliferation and colonisation of
Ž
the gut of larvae by opportunistic andror pathogenic bacteria Westerdahl et al., 1994;
.
Bergh, 1995; Skjermo et al., 1997 . Colonisation of the digestive tract with beneficial or
AprobioticB micro-organisms is a well-recognised practice in veterinary medicine
ŽVanbelle et al., 1990 and this concept may be used in larval rearing. Ringø and.
Ž .
Vadstein 1998 were able to colonise the gut of turbot larvae with Vibrio pelagius when the bacterial species was added 2 days after hatching but lower densities were observed when the larvae were exposed to V. pelagius at day 5 or 8 post hatch. Therefore, it was concluded that V. pelagius has to compete with the microbiota already present in the larval gut for suitable attachment sites.
In the present research, the relationship between the intestinal bacterial flora of turbot larvae and larval survival rates was studied. Possible beneficial bacterial strains were isolated to investigate their effect on larval survival. The overall goal was to identify beneficial bacterial strains that may improve the hatchery output in terms of repro-ducibility and larval survival rates.
2. Materials and methods
2.1. LarÕal rearing conditions
Ž .
tanks 1 to 6 and the following executed Exp. II included tanks 7 to 12. The two experiments were run until 11 days post hatch during which the water was not renewed. The use of a stagnant system during the early rotifer-feeding stage of larval turbot is a usual practice in most hatchery facilities as newly hatched turbot larvae are very small
Ž .
and fragile personal communication, Ringø et al., 1996; Skjermo and Vadstein, 1999 . For each experiment, 1-day-old turbot larvae, deriving from a same spawn, were
Ž .
obtained from a commercial hatchery France Turbot, France and acclimated to
Ž .
seawater 35 ppt, 188C during a few hours. After acclimatisation, the larvae were
stocked in 60-l black conical tanks at a density of 60 larvae ly1and reared following the
Ž .
procedure of Dhert et al. 1993 . Tanks were filled with seawater that was filtered
Ž
through a membrane filter with a pore size of 0.2 mm, disinfected with NaOCl 10
.
ppm , aerated overnight and then neutralised by addition of sodium-thiosulphate.
Ž .
Rotifers Brachionus plicatilis were used as a live food source for larval turbot
Ž .
following the method described by Dhert 1996 . The survival of each tank was
evaluated at the end of the experiment on day 11.
2.2. Sampling and isolation of the gut microflora of larÕal turbot
Samples of the aerobic flora of the intestine of 50 turbot larvae were taken at days 3, 5, 7 and 9 post hatch. As the small size of turbot larvae renders dissection impractical, the aerobic bacterial flora of the gut was sampled according to the method of Muroga et
Ž .
al. 1987 . At each sampling, 50 larvae from each tank were captured with a sterile pipette and placed into a sterile beaker.
Ž .
To remove the surface bacteria, the larvae were caught on a sterile mesh 10mm and
Ž .
successively anaesthetised by immersion in a 0.1% wrv benzocaine solution for 10 s,
Ž .
disinfected in a 0.1% wrv benzalkoniumchloride solution for 10 s and then rinsed
Ž .
three times in a sterile Nine Salt Solution NSS; Olssen et al., 1992 , each rinse for 5 s. The larvae were aseptically transferred to a sterile plastic bag containing 25 ml of NSS
Ž .
and homogenised in a stomacher blender 400SN, Seward Medical, London, UK .
Ž y1 y2.
Dilutions of the homogenised sample solutions 10 and 10 were prepared using
Ž
sterile NSS and 0.1 ml volumes were plated out on Marine Agar 2216 MA; Difco
.
Laboratories, Detroit, MI, USA and on Thiosulphate–Citrate–Bile salt–Sucrose Agar
ŽTCBS; Oxoid, Basingstoke, Hampshire, England . TCBS-agar was used as a selective.
medium for isolation of Vibrios rather than for quantification. All the plates were incubated at 188C for 5 days. After incubation, all different colony types obtained from the first dilution, both on MA and TCBS, were isolated and further purified on MA. Pure strains were used for further characterisation.
2.3. Microbiological characterisation techniques
( )
2.3.1. Gas chromatographic analysis of cellular fatty acid methyl esters FAME Quantitative analysis of cellular fatty acid compositions was performed using the
Ž .
gas–liquid chromatographic procedure as described by De Boer and Sasser 1986 .
Ž .
Strains were grown for 24 h at 288C on MA Difco . Approximately 70 mg of cells were
boiling water bath for saponification. Methylation was achieved by adding 2 ml of 6 N hydrochloric acid in aqueous methanol and heating for 10 min at 808C. After cooling to room temperature, fatty acid methyl esters were extracted with a mixture of hexane and methyl-iso-butylether. Fatty acid methyl esters were analysed with a Hewlett-Packard
Ž
model 5898A gaschromatograph and identified using MIS software Microbial ID,
.
Newark, DE, USA . The isolates were compared and grouped into gaschromatographic
Ž .
groups GC-groups or clusters on the basis of the fatty acid fingerprints by principal
Ž .
component analysis PCA using the same software package. PCA represents each
Ž .
operational unit OUT as a point in a multidimensional space and the relationships between the OUT’s are represented by the Euclidean distances between the
representa-Ž .
tive points Dunn and Everitt, 1982 . Clusters were delineated at 90% similarity and isolates being part of the same cluster, were considered belonging to the same species. After grouping into clusters, two or more representatives of each cluster were selected
Ž .
for FAME analysis with Tryptic Soy Agar TSA, Difco Laboratories, Detroit, MI, USA as culture medium instead of MA. In that way it was possible to compare the FAME fingerprints of the unknown isolates with the FAME fingerprints of the reference strains
Ž .
in the commercial databank TSBA version no. 3.9, Microbial ID, Newark, DE, USA which were also cultured on TSA. It should be noted that not all the investigated strains grew on TSA probably due to the lack of sufficient salt in the culture medium.
2.3.2. BIOLOG fingerprints
The strains that did not grow on TSA and the strains belonging to a Vibrio cluster were further characterised by BIOLOG metabolic profiles. Strains were grown for 24 h
Ž .
at 258C on Brain Heart Infusion BHI, Difco Laboratories, Detroit, MI, USA
supple-Ž . Ž .
mented with 1.5% wrv sodium chloride. Inocula were prepared in 1.5% wrv
sodium chloride and the cell density was standardised between 0.26 and 0.30 O.D. using
Ž
a spectrophotometer at 590 nm. Each well in the BIOLOG GN microplate BIOLOG,
.
Hayward, CA, USA was inoculated with 150 ml of the cell suspension and these
microplates were incubated at 258C for 24 h. Changes in colour were measured using a
Ž .
Multiscan Multisoft filter photometer Labsystems, Helsinki, Finland at 590 nm. The BIOLOG profiles were compared to a database containing BIOLOG fingerprints of 850 Vibrio type- and reference strains, by numerical analysis using the Pearson product moment correlation coefficient. The strains were grouped by unweighted pair-group
Ž .
method of averages UPGMA .
2.4. Selection of potential probiotic strains for turbot larÕiculture
The results of the larval rearing cultures were arbitrarily evaluated as successful
Žsurvival )35% , average survival from 10% to 35% or a failure survival. Ž . Ž -10% ..
For each cluster from the PCA analysis, a selectivity index P was calculated following
Ž .
the equation PsSr FqAqS where S stands for the total number of isolates
deriving from a successful tank, A for the total number of isolates deriving from an average culture and F for the total number of isolates from a failure. When the index P
Ž .
2.5. Small-scale confrontation tests
For each selected potential probiotic gaschromatographic cluster, one representative isolate was pointed out for further screening on the effect of larval survival. The selected potential probiotic strains were tested on first feeding turbot larvae in a 1-week confrontation test. Therefore, 1 day after hatching, 30 larvae were stocked in a 1-l glass beaker filled with 500 ml UV-sterilised seawater. Each tested bacterial strain as well as the control group consisted of eight replicates. No aeration and no feeding were
supplied, the temperature was constantly 188C and the beakers were continuously
illuminated. Bacterial suspensions were prepared of each selected potential probiotic
Ž . 5
strain in a Nine Salt Solution NSS and added to the beakers at a concentration of 10 bacteria mly1 water. The effect on larval survival was evaluated daily and compared to
untreated control groups. In total, five small-scale confrontation tests were carried out following this procedure. Additionally, a Vibrio mediterranei Q40 strain isolated from
Ž .
sea bream larvae Grisez et al., 1997 was included during each trial.
3. Results
3.1. SurÕiÕal at day 11
Large variation, from 0% up to 44%, was observed in the survival percentage of
Ž .
larval turbot during both experiments Table 1 . According to the evaluation criteria, tanks 3 and 6 from Exp. I as well as tanks 8 and 12 from Exp. II were pointed out as successful culture tanks since they had a survival percentage above 35%. Tanks 7, 9 and 10, all used in Exp. II, collapsed completely and were therefore evaluated as failures.
Ž . Ž .
The remaining tanks 1, 2, 4 and 5 Exp. I , just as tank 11 Exp. II , were considered as average cultures.
3.2. Bacterial loading of the gut of turbot larÕae
The development of gut-associated bacteria in larval turbot was assessed as changes
Ž .
in the number of colony forming units CFU on MA. During both experiments, no significant differences between the tanks were obtained in the development of the bacterial flora in the gut of intensively reared turbot larvae. Just before first feeding at
Ž 2 .
day 3 post hatch, the turbot larvae had a few bacteria circa 10 CFUrlarva associated
with the gut. This level increased rapidly after feeding commenced until more than 104
CFU per larva at day 5 post hatch to approximately 105 CFU per larva at day 9 post
Ž .
hatch results not shown .
During both experiments, no Vibrio-counts were observed before addition of the first food at day 3 post hatch. Although this number increased rapidly after feeding had started, it rarely exceeded 10% of the value perceived on MA. As the Vibrio-count on
Ž .
TCBS is often found to be of limited value Bolinches and Egidius, 1987 , TCBS-agar is
Ž
used as a selective medium for Vibrios rather than for quantification Nicholls et al.,
.
Table 1
Survival percentages at day 11 of experiment I and II with notification of the isolate numbers per tank Experiment Tank Survival % Evaluation: Number of Isolate no.
a
110, 111, 112, 113, 114, 115
9 0 F 14 75, 76, 77, 79, 80, 81, 116,
Sssuccessful culture, Asaverage culture, Fsfailure.
3.3. Characterisation of the isolates by FAME and BIOLOG fingerprints
During these two turbot experiments, 149 isolates were obtained but when culturing them on MA, 15% did not grow and therefore only 127 isolates were considered for the
Ž .
fatty acid analysis FAME . Table 1 includes a schematic overview of the number of isolates originating in each tank.
The isolates were compared and grouped on the basis of the fatty acid fingerprints
Ž .
using principal component analysis PCA . This operation resulted in the definition of 12 major gaschromatographic FAME-groups or clusters while 11 isolates remained
Ž .
unclustered namely isolate no. 1a, 55, 58, 66, 67, 87, 92, 95, 109, 113 and 118 . The delineation of these clusters and single strains was also found in a numerical analysis of
Ž .
the fatty acid of the isolates dendrogram not shown , using the Euclidean distance
Ž
coefficient and clustering by the unweighted pairgroup average method Sneath and
.
Sokal, 1973 . For further identification of the clusters, 3 or more representatives of each cluster were selected for the FAME analysis with TSA as a culture medium and they
Ž
were also subjected to the BIOLOG technique. Table 2 shows that five clusters A, D, F,
.
Table 2
Survey of the 12 FAME-clusters obtained by FAME analysis with MA as growth medium. Representative
.
strains of each FAME-cluster were identified by comparison with databases of reference strains using i
.
FAME analysis with TSA as growth medium and ii BIOLOG metabolic fingerprinting
FAME cluster Isolate no. FAME identification BIOLOG Identification
Žaisolatesr
B 11 1b, 10b, 11, 13b, 22, Pseudomonas sp. no match found
24, 28, 61a, 61b,
E 22 4, 6, 7, 32, 33, 34, 37, Pseudoalteromonas no match found 39, 54, 57, 60, 63, 71, haloplanktis
Ž .
I 13 21, 42, 44a, 44b, 45, Vibrio sp. Vibrio mediterranei
Ž .
48, 49, 104, 111, 126, 21, 48, 49, 132
132, 140, 149 V. nereis or V. costicola
Ž45, 104, 111, 126.
identify cluster L as no growth was detected on TSA, while BIOLOG identified cluster L as Vibrio campbellii with a similarity level of 75%.
3.4. Distribution of the FAME clusters
There was a great variation between the twelve different tanks with regard to the presence of the FAME-clusters among the bacterial isolates from the intestine of turbot
Ž .
larvae of each tank Table 3 . Isolates of clusters C, G and J were detected in at most three tanks of the first experiment while isolates of clusters D, F and K appeared in maximally three tanks of the second experiment. On the other hand, isolates from cluster E and L were rather ubiquitously present in all tanks, i.e., that, with regard to the experimental design used in this work, P. haloplanktis and V. campbellii were both predominant bacteria in the intestinal tract of larval turbot. The remainder clusters A, B, H and I occurred in at least six tanks and appeared in both experiments.
Table 3
Ž . Ž .
Distribution of the number of isolates per FAME cluster according to a the tank number, b the sampling
Ž . Ž . Ž .
day 3, 5 and 9 and c the success rate of the larval culture S, A or F
Ž .
According to Number of isolates per FAME cluster total number of isolates per cluster
aŽ . aŽ . Ž . Ž . Ž . Ž . Ž . Ž . aŽ . aŽ . Ž . Ž .
A 8 B 11 C 5 D 2 E 22 F 3 G 2 H 9 I 13 J 4 K 2 L 35
( )a Tank no.
1 1 2 1 1 1 3
2 2 2 1 1
3 1 1 4 4 1 2
4 2 2 1 2
5 1 1 2 1
6 2 4 1 2 2 1
7 1 2 1 5
8 1 1 1 1 1 6
9 2 1 2 1 7
10 1 1 3 1 1 1 3
11 1 1 3 1 1 3
12 2 1 1 1 1 2 2
( )b Sampling day
3 4 4 1 2 9 3 0 6 0 0 2 9
5 4 7 4 0 8 0 2 3 6 1 0 23
9 0 0 0 0 5 0 0 0 7 3 0 3
( )c Success rate of larÕal cultures
Fsfailure 1 0 0 1 6 1 0 5 2 0 2 15
Asaverage 1 5 5 0 9 0 2 3 2 1 0 9
Sssuccess 6 6 0 1 7 2 0 1 9 3 0 11
b
Index P 0.75 0.55 0.00 0.50 0.32 0.67 0.00 0.11 0.69 0.75 0.00 0.31
a
Selected potential probionts.
b Ž .
Table 4
Ž
Small-scale confrontation tests: survival percentages at day 5 post hatch 48 h after administration of the
.
bacterial suspension
Ž .
Bacterial strain Survival %"SD day 5 post hatch
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
Control 14.29"8.54 60.83"7.39 50.00"3.00 75.00"15.84 15.84"10.55
a
V. med. Q40 55.24"12.00 76.67"12.17 73.40"11.30 81.67"12.22 70.63"10.47 Cluster A ND 85.83"13.44 60.60"11.24 ND 79.30"10.81
Cluster B ND 44.17"14.24 57.40"13.35 ND ND
Cluster I ND ND 66.60"10.36 ND ND
Cluster J ND ND 66.80"15.38 ND ND
NDsnot determined.
a Ž .
Vibrio mediterranei Q40: strain isolated from sea bream larvae Grisez et al., 1997 .
Table 3 shows that the clusters D, F and K only appeared in the first sampling at day 3. Later in the experiment, no more isolates of these clusters were detected, suggesting that the respective clusters could not be established in the intestinal tract of the larvae. The same conclusion can be made for cluster G that only appeared at sampling day 5. Clusters A, B, C and H were only isolated at sampling day 3 and 5 while clusters I and J were detected from day 5 onwards.
3.5. Small-scale confrontation tests
For the identification of a potential probiont, clusters with a high P-index were selected, meaning that the majority of the isolates of this cluster originated from a
Ž .
successful turbot culture, which was the case for clusters A, B, F, I and J Table 3 . Apart from cluster F that only contained three isolates and one of them belonging to a failure, representative isolates of these clusters were tested in vivo for their effect on the
Ž .
survival of turbot larvae in a small-scale confrontation test Table 4 . The influence on larval survival was most visible at day 5 post hatch, i.e., 48 h after addition of the bacterial strain, while around day 7 or 8 post hatch, the majority of the larvae were dead
Ž .
through lack of feed personal observation .
The strains representing the unidentified cluster A and V. mediterranei Q40 both had a distinctive positive and reproducible effect on the survival of turbot larvae compared to the untreated control groups. The isolates representing cluster I and J were only evaluated in the third trial and showed both a positive effect on larval survival compared to the control although they seemed to be less effective than cluster A and V. mediterannei Q40. The reproducibility of these results needs to be verified. No reproducible results were obtained with the strain representative of the Pseudomonas cluster B.
4. Discussion
than egg quality, nutrition and culture techniques. Many studies have revealed micro-organisms to be involved in the problems during the early larval stages of marine fish
Ž
larvae Verdonck and Swings, 1995; Skjermo and Vadstein, 1999; Hansen and Olafsen,
.
1999 .
Ž .
The results of this study, in line with Munro et al. 1994 , clearly demonstrated that there was no correlation between the number of bacteria present in the larval gut and larval survival rates as all replicates followed nearly the same bacterial development in
Ž
the larval gut throughout the whole experiment. Just before first feeding day 3 post
. Ž 2 .
hatch , turbot larvae contained few bacteria circa 10 CFUrlarva associated with the
gut. After feeding had started, this level increased to more than 104CFUrlarva at day 4
post hatch and grew further up to 105CFUrlarva at day 9 post hatch. These results are
Ž . Ž .
in accordance with the findings of Munro et al. 1993 , Ringø et al. 1996 and Ringø
Ž .
and Vadstein 1998 .
The observation that bacteria can enter the tract before first feeding has initiated, can be explained by the osmoregulation during which the marine fish larvae start drinking
Ž .
before the yolk is completely absorbed Reitan et al., 1998 . Moreover, Hansen and
Ž .
Olafsen 1999 suggest that ingestion of bacteria at the yolk sac stage might result in the establishment of a primary intestinal microflora, which seemed to persist beyond first feeding.
Ž .
Munro et al. 1995 postulated that control on the bacterial diversity present, rather than the total bacterial density in the gut might be important in ensuring high larval survival rates. If the gut bacteria influence the survival rate, there should be a difference in the composition of the gut flora between turbot larvae derived from a failure culture, an average culture or a successful culture during the two turbot experiments presented in this study. Although there was a great variation in the presence of the 12 FAME-clusters over the 12 rearing tanks, isolates from cluster E and L were ubiquitously present in all tanks at each sampling day. This implies that, with respect to the experimental design used in this research, P. haloplanktis and V. campbellii are predominant species in the gut of larval turbot. The dissimilarity in bacterial composition between the different tanks, observed in both Exp. I and Exp. II, highlight the inter-tank variability that was
Ž .
also reported by Grisez et al. 1997 . Yet, no explanation could be found for this tank-effect although it is remarkable that the presence of both the non-Vibrio cluster A
Ž
and the Vibrio cluster I was peculiar to all successful rearing cultures tanks 3, 6, 8 and
.
12 , while only one isolate belonging to cluster A was detected in solely one failure
Ž .
culture tank 10 . In conclusion, these observations indicate that the composition of the gut microflora of larval turbot may differ from one tank to another, even when the same rearing conditions are applied.
Importantly, a series of studies investigating the taxonomic composition of the larval
Ž .
gut of various marine fish species review Ringø and Birkbeck, 1999 demonstrated that the intestinal microflora is very diverse as the composition may change with age,
Ž .
nutritional status and environmental conditions Hansen and Olafsen, 1999 . Besides, it should be noted that differences in methodology make interlaboratory comparisons difficult, explaining conflicting statements in literature.
whom were attendant at all sampling days, most clusters were only temporarily present. It is interesting to note that the unidentified non-Vibrio cluster A was isolated before first feeding had commenced and disappeared later on in the experiment. The outcome that some bacterial strains may become established while others may be digested or expelled, can be attributed to the complex interactions between the host and the
Ž .
intestinal microbiota Hansen and Olafsen, 1999 , which reinforces the notion that further research on this matter is warranted.
Ž
As fish larvae establish their gut microflora partly in a non-selective way Hansen
.
and Olafsen, 1989; Cahill, 1990 and as there is proof for the existence of an indigenous
Ž
microflora in marine and fresh water fish Horsley, 1977; Sakata, 1990; Ringø and
.
Gatesoupe, 1998 , the initial transient bacteria are of utmost importance as they may become established and evolve into a more persistent microflora. Moreover, the ability of specific bacteria to occupy attachment sites in the larval gut preventing opportunistic bacteria proliferating and colonising the larval gut, is assumed to be an important defence mechanism, especially during very early larval stages when the immune system
Ž .
is not fully developed Vanbelle et al., 1990 .
Based on this hypothesis, representative isolates of cluster A, B, I and J were selected as potential beneficial strains and they were screened for their effect on larval survival in a small-scale confrontation test, in which also a V. mediterranei strain Q40 was included. Only the non-Vibrio cluster A and V. mediterranei Q40, administered at the moment of mouth opening, showed a distinct positive and reproducible effect on larval survival. It was concluded that both these strains could play a role as first colonisers of the gut of turbot larvae and as a consequence protect the gut from colonisation by possible harmful bacteria.
These observations indicate that the concept of introducing bacterial species to the rearing water at very early larval stages, may favour the growth of a protective normal flora and display a continued effect on the further bacterial development in the larval intestinal tract. This finding is supported by the earlier observations of Strøm and Ringø
Ž1993 , Ringø et al. 1996 and Ringø and Vadstein 1998 .. Ž . Ž .
Acknowledgements
LH acknowledges a grant of the Flemish Institute for the Promotion of Scientific
Ž .
Technological Research in the Industry IWT . This study was also supported by the
Ž .
Fund for Scientific Research — Flanders Belgium, FWO grant no. G0063.96 .
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