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. q 2001 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 Ž
. Scophthalmus 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
Ž .
Vanbelle 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
. Two turbot experiments Exp. I and Exp. II , in whom the bacterial flora of the larval
gut was sampled, were performed under the same larval rearing conditions but with a difference in time. Both experiments were composed of six replicates: Exp. I included
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 l
y1
and 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 10 mm 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
Ž .
TCBS; 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 added to 1 ml of 3.75 N NaOH in 50 aqueous methanol and heated for 30 min in a
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 Ž
. Ž
. Ž
. survival 35 , average survival from 10 to 35 or a failure survival - 10 .
For each cluster from the PCA analysis, a selectivity index P was calculated following Ž
. the equation P s Sr F q A q S
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
. was higher than 0.5 i.e., a lot of isolates belonging to a successful culture , the bacteria
from this cluster were catalogued as potential probionts.
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
