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 ml
y1
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 10
4
CFU per larva at day 5 post hatch to approximately 10
5
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.,
. 1976 .
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
no. at day 11
F, A or S isolates
per tank I
1 30
A 10
1a, 1b, 2, 15, 31, 32, 41, 42, 51, 52
2 27
A 7
3a, 3b, 4, 5, 19, 54, 55 3
44 S
13 6, 7, 8, 21, 22, 33, 34,
35, 43, 44a, 44b, 45, 53 4
30 A
8 9, 10a, 10b, 23, 24,
25, 57, 58 5
17 A
5 11, 12, 39, 59, 60
6 36
S 12
13a, 13b, 14, 28, 30, 36, 37, 47, 48, 49, 61a, 61b
II 7
F 11
63, 65, 66, 67, 102, 103, 104, 105, 106, 107, 108
8 42
S 13
69, 70, 71, 72, 73, 74, 109, 110, 111, 112, 113, 114, 115
9 F
14 75, 76, 77, 79, 80, 81, 116,
117, 118, 119, 120, 122, 123, 124
10 F
12 82, 83, 84, 85, 86, 87, 88,
125, 126, 128, 129, 130 11
12 A
11 89, 90, 91, 92, 93, 94, 96,
131, 132, 133, 135 12
38 S
11 95, 97, 98, 99, 100, 101,
137, 139, 140, 141, 149
a
Sssuccessful culture, A saverage culture, F s failure.
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,
. G and K did not grow on TSA or BHI and remained unidentified. Cluster B was readily
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
Ža isolatesr .
cluster Ž .
A 8 13a, 14, 53, 86, 93,
no growth on TSA no growth on BHI
98, 112, 139 Ž
. B 11
1b, 10b, 11, 13b, 22, Pseudomonas sp.
no match found 24, 28, 61a, 61b,
69, 89 Ž .
C 5 2, 9, 10a, 12, 52
no growth on TSA no match found
Ž . D 2
85, 99 no growth on TSA
no growth on BHI Ž
. E 22
4, 6, 7, 32, 33, 34, 37, Pseudoalteromonas
no match found 39, 54, 57, 60, 63, 71,
haloplanktis Ž
. 75, 82, 91, 96, 120,
4, 7, 32, 34, 39, 54 125, 129, 131, 137
Ž . F 3
70, 77, 100 no growth on TSA
no growth on BHI Ž .
G 2 3a, 3b
no growth on TSA no growth on BHI
Ž . H 9
51, 59, 65, 80, 90, 97, no growth on TSA
no match found 102, 116, 128
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
Ž .
45, 104, 111, 126 Ž .
J 4 19, 35, 36, 47
Vibrio sp. V. nereis or V. costicola
Ž .
35, 36, 47 Ž .
K 2 79, 83
no growth on TSA no growth on BHI
Ž .
L 35 5, 8, 15, 23, 25, 30, 31,
no growth on TSA Vibrio campbellii
Ž 41, 43, 72, 73, 74, 76,
8, 25, 30, 43, 76, .
81, 84, 88, 94, 101, 94, 105, 110, 133
103, 105, 106, 107, 108, 110, 114, 115, 117, 119,
122, 123, 124, 130, 133, 135 , 141
recognisable as Pseudomonas with the FAME technique but identification down to strain level was not possible. Likewise, no match was found with the BIOLOG
technique as the databank only contains reference strains of Vibrio species. Clusters C and H, which did not grow on TSA probably due to the lack of sufficient salt, showed
with BIOLOG less than 40 similarity with the Vibrio genera. Cluster E was recognised as Pseudoalteromonas haloplanktis with FAME analysis while there was no match
found with BIOLOG. According to BIOLOG, four isolates of cluster I were identified as being V. mediterranei while four other isolates were recognised as Vibrio nereis or
Vibrio costicola. According to the FAME analysis, all the isolates of cluster J were assigned to the genus Vibrio but without species specification. The results of BIOLOG
indicated that cluster J was V. nereis or V. costicola. The FAME-method was unable to
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
a a
a a
Ž . Ž
. Ž .
Ž . Ž
. Ž .
Ž . Ž .
Ž .
Ž . 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
6 2
9 5
4 7
4 8
2 3
6 1
23 9
5 7
3 3
c Success rate of larÕal cultures F s failure
1 1
6 1
5 2
2 15
A saverage 1
5 5
9 2
3 2
1 9
Sssuccess 6
6 1
7 2
1 9
3 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 .
P s Sr F q Aq S .
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 14.298.54
60.837.39 50.003.00
75.0015.84 15.8410.55
a
V. med. Q40 55.2412.00
76.6712.17 73.4011.30
81.6712.22 70.6310.47
Cluster A ND
85.8313.44 60.6011.24
ND 79.3010.81
Cluster B ND
44.1714.24 57.4013.35
ND ND
Cluster I ND
ND 66.6010.36
ND ND
Cluster J ND
ND 66.8015.38
ND ND
NDs not 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
