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The continuous feeding of turbot larvae, Scophthalmus maximus, and control of the bacterial environment of rotifers
Aquacultlcre, 89 (1990) 139-148 Elsevier Science Publishers B.V., Amsterdam
139
The continuous feeding of turbot larvae, Scophthalrnus maximus, and control of the bacterial environment of rotifers Franqois-Jog1 Gatesoupe INRAMFREMER, Centre de Brest, BP 70, 29280 Plouzank (France) (Accepted 2 1 November
1989)
ABSTRACT Gatesoupe, F.-J., 1990. The continuous feeding of turbot larvae, Scophthalmus maximus, and control of the balzterial environment of rotifers. Aquaculture, 89: 139-148. Turbot la.rvae were fed continuously with rotifers pumped from the culture tanks and rinsed before distribution. This technique improved the growth and survival rates of turbot, in comparison with the results obtained with a single daily distribution of rotifers enriched for 6 h. Special attention was paid to associated bacteria. Most bacteria detected in healthy rotifers and turbot were identified as Vibrio algirtolyticus.An opportunistic strain of Aeromonas sp. was observed in large numbers, but only in tanks where the larvae were on the verge of high mortality. A food additive containing live lactic bacteria improved the growth rate of turbot when it was put into the rotifer medium. A similar improvement was observed when the pH of this medium was lowered to 5.25-6, while the temperature was increased from 19 to 26°C. The combination of the bacterial additive and the pH-temperature changes was not efficient and might have led to the proliferation ofderomonas.
INTRODUCTION
Bacteria associated with rotifers have a detrimental effect on the growth and survival rates of turbot larvae (Gatesoupe, 1989). The main cause of the bacterial growth in rotifers is overnight enrichment with oil emulsion (Gatesoupe et al., 1989). The continuous distribution of rotifers needed to be reexamined in the light of this latter observation. In a previous experiment (Perez and Gatesoupe, 1988), rotifers were enriched with fish oil emulsion for 2 days. During the first day, they were disinfected with antibiotics. During the second day, they were semi-continuously distributed to turbot larvae. Continuation of the enrichment after the disinfection was not correct, since bacteria were able to grow again. This might be a reason why the growth and survival rates of turbot were not improved by this treatment. However, 22:6 n-3 fatty 0044-8486;90/$03.50
0 1990 -
Elsevier Science Publishers
B.V.
140
F.-J. GATESOUPE
acid reserves were increased in the larvae. Thus continuous distribution of rotifers can maintain their essential fatty acid content. The experiments presented here were therefore carried out with a view to testing the continuous distribution of rotifers without disinfection and further enrichment. Nevertheless, high numbers of bacteria were also observed in rotifers before enrichment, while these rotifers were not ingested by turbot (Nicolas et al., 1989). It was therefore important to control the bacterial environment of the rotifers. A food additive containing live lactic bacteria was found to limit the bacterial growth in rotifers (Gatesoupe et al., 1989). This additive had to be tested with the continuous distribution of rotifers. Moreover, the rotifers could endure a wide range of pH and temperature conditions (Snell et al., 1987). The effect of a change in these conditions was studied, with a view to enhancing a possible action of lactic bacteria. MATERIAL AND METHODS
Experimentaldesign Four grades of rotifers were tested: ( 1) the control rotifers C were reared at normal pH and temperature; (2) the pH was lowered and the temperature was increased for rotifers pT; (3) a food additive containing lactic bacteria ( Acosil ) was introduced into the medium of rotifers A, reared at normal pH and temperature; and (4) this additive was tested on rotifers PTA, reared at low pH and high temperature. These four kinds of rotifers were distributed continuously to turbot larvae (turbot C, A, pT and PTA). The effect of the continuous distribution was tested in comparison with rotifers pT and PTA distributed once a day after 6 h of enrichment (turbot DpT and DpTA). Four experiments were carried out, with triplicates per treatment in each experiment (Table 1). The effect of the continuous distribution was tested in experiments 1 and 2 with rotifers PTA and pT, respectively (treatments PTA, DpTA, pT and DpT ). The dietary values of the four kinds of rotifers distributed continuously were compared in the last three experiments (treatments pT and A in experiment 2, C, pT and pTA in experiment 3, and pT and PTA in experiment 4). Rotifer culture The rotifers were reared in 150-l tanks (rearing unit shown schematically in Fig. 1). A quarter of the volume was renewed daily with 18%0 salt water. The rotifers were fed with baker’s yeast (60.8 mg- ’ day- ’ ), cod liver oil (7.4 mg 1-l day- ’ ), soybean lecithin (0.6 mg 1--I day- * ), and vitamin premix (0.7 mg 1-l day-‘, Spyridakis et al., 1988). When the pH of the rotifer culture was lowered (rotifers pT and PTA), this food suspension was introduced into the renewing water of the rotifer tanks. This water was adjusted to pH 4 with normal HCl and distributed continuously with a peristaltic pump into
141
QUALITY OIF ROTIFER CULTURES FOR FEEDING LARVAL TURBOT
TABLE 1 Summary of the experimental design and rotifer culture conditions (D: rotifers distributed discontinuously, pT rotifers reared at low pH and high temperature, A: Acosil added to the diet, C: control) Treatment
Experiment 1 Experiment 2 Experiment 3 Experiment 4
DPT
DpTA
-
+
+
Rotifer culture: pH range Temperature (“C) Acosil (me. I-’ day-‘) Continuous distribution of rotifers
5.25-6 26 0
PT
PTA
C
A
+
-
+ + +
+ +
+ -
+ -
5.25-6 26 8
5.25-6 26 0
5.25-6 26 8
7-8 19 0
7-8 19 8
+
+
+
+
-
IOcm ROTI FER TREATME:N c ,A
PT , PTA
YEAST
ROTI FERS
RINSE
TURBOT
Fig. 1. Re#aring unit for the continuous feeding of turbot C, A, pT and PTA. Rotifers C and A were fed with the l-l bottle of yeast suspension. Rotifers pT and PTA were fed with yeast at pH 4 in the renewing water. Then the rotifers were pumped and rinsed continuously in this unit.
the rotifer tanks. In these tanks, the pH was checked daily and adjusted to 5.25. The value before adjustment was near to pH 6. The rearing temperature was kept at 26°C. When the pH was not modified (rotifers C and A), the
F.-J. GATESOUPE
142
food suspension was stirred constantly in a l-1 bottle and distributed semicontinuously to rotifers. The temperature was kept at 19 “C and a quarter of the culture volume was renewed daily at one go. Snell et al. ( 1987) found that the swimming activity of rotifers reared at 20°C and pH 7.5 was nearly the same as the activity of rotifers reared at 25°C and pH 5.2, while the maximum value was observed at 30” C and pH 7. Rotifers tolerated acid pH values to a greater extent than alkaline values, likely due to the toxicity of unionized ammonia (Yu and Hirayama, 1986). When Acosil, a dry extract of sprouting cereal grains fermented with lactic bacteria, was tested (rotifers A and PTA), it was delivered at the rate of 8 mg l- ’ day- ’ into a 40-pm net bag once a day, so that only small particles and soluble substances were introduced into the rotifer culture medium. Turbot rearing Newly hatched turbot were put in 150-l tanks at a density of 30 larvae l- ‘. When turbot were fed once a day (turbot DpT and DpTA), the amounts of rotifers were determined visually so as to obtain a slight excess overnight (Fig. 2). The same amount was also given discontinuously in the other tanks, The continuous distribution supplied surplus rotifers. These rotifers were continuously pumped and rinsed on a 80-pm net with seawater (35%0). They were again pumped continuously up to the larval tanks. One rotifer tank could feed
DAY
DIET
Fig. 2. Mean amounts of rotifers given daily per hatched larva in the four experiments. The same amount (Ro) as distributed discontinuously to tanks D (treatments DpT, DpTA) was also distributed discontinuously to the other tanks. The amounts of rotifers distributed continuously (R + ) were slightly different between treatments C, A pT and PTA.
QUALITY OF ROTIFER CULTURES FOR FEEDING LARVAL TURBOT
143
four larval tanks of the same volume. In these larval tanks, the water outlet passed through a 180-pm net, so as to draw away excess rotifers. The tanks with discontinuous feeding were fitted with 80-pm nets. The rotifers were enriched only for these latter tanks. The enrichment consisted of 6 h immersion in an emulsion (cod liver oil 46 mg l- ’ and soybean lecithin 4 mg l- ’ ) . The experiments ended at day 10 after hatching. The final growth and survival rates were compared by an a priori test and that of Student-NewmanKeuls, respectively (Sokal and Rohlf, 1969). Bacteria were counted in rotifers on Petrifilm SM plates, which gave bacterial numbers in seafood very close to those obtained with the standard agar method (Fung et al., 1986). About 5 ml of rotifer culture were sampled per treatment. Under sterile conditions, the rotifers were rinsed over a 80-pm net and resuspended in 4.5 ml of seawater. They were counted and crushed in a glass homogenizer. The samples were diluted 1000 times and inoculated on SM plates. Colonies were isolated from these plates and determined with the Api 20 E system (23, 12, 16 and 14 colonies were determined in rotifers C, A ‘pT, and PTA, respectively). At day 9 after hatching, live larvae from each tank were sampled for counting bacteria. Under sterile conditions, they were rinsed over a 180-pm net and treated in the same way as the rotifers for the inoculation of SM plates and the Api 20 E system ( 16,5 and 15 colonies were determined in the samples from 3, 4 and 3 tanks in experiments 2, 3 and 4, respectively). The proportions of the phenons were compared by a chi-square test whenever possible. RESULTS
In experiment 1, the mean weight obtained with continuous feeding (treatment PTA) was significantly higher than that obtained with discontinuous feeding (treatment DpTA, Table 2 ). The survival rates were not significantly different. In experiment 2, the treatment DpT with discontinuous distribution of rotifers resulted in mean weight and survival rates of turbot significantly lower than those observed with the two other treatments. Moreover, the mean weight obtained with treatment A was significantly higher than that with treatment pT. In experiments 3 and 4, the mean survival rates were not significantly different, due to a great variability of the survival rate obtained with treatment PTA (standard errors shown in Fig. 5 ) . This variability was due to a high mortality rate observed between day 9 and day 10 in one tank corresponding to treatment pTA in experiments 3 and 4. The mean weights obtained with treatment pT were significantly higher than those observed with treatments PTA and C. Two thousand bacteria were counted per rotifer in treatments C and PTA, whereas 7000 and 8000 bacteria were counted per rotifer in treatments pT and A, respectively. The dominant species, Vibrio alginolyticus, was respon-
144
F.-J. GATESOUPE
TABLE 2 Mean weight and survival rate of turbot at day 10. Superscripts a, b and c indicate significant differences Treatment DPT Mean weight (mg) Experiment 1 Experiment 2 Experiment 3 Experiment 4 Survival rate (Oh) Experiment 1 Experiment 2 Experiment 3 Experiment 4
0.38” -
DpTA 0.25b -
PT 0.55b 0.63” 0.63”
27 2gb -
69” 52 62
PTA 0.29” 0.52b 0.55b
61 22 66
C 0.50b -
46 -
A
0.66” -
82” -
1000 BACTERIA/ROTIFER
PHENONS
Fig. 3. Number of bacteria (thousands per rotifer). V. alginolyticus (VA) dominated and the other phenons (R) were not identified.
sible for most of the difference between the numbers of bacteria (Fig. 3). The other phenons, gathered as phenons R, represented about 1000 bacteria per rotifer in each samples. They could not be identified by the Api 20 E system. The differences observed between groups for the bacterial counts in larvae at day 9 were not significant. However, the numbers of bacteria within treatment PTA were especially variable. This variability might explain that of the survival rates, since the lots with a high number of bacteria at day 9 presented
QUALITY OF ROTIFER CULTURES FOR FEEDING LARVAL TURBOT
MILLION
Fig. 4. Number of bacteria (millions per larva) at day 9. Profile pTA1, with Aeromonas sp. ( Ae) dominant, corresponds to the tanks with a high mortality rate between day 9 and day 10. Profile pTA2 corresponds to tanks with normal survival rates. Like the profiles pT, DpT, A and C, it contains phenons observed in rotifers (VA, R) or not (phenons T).
survival rate (“/I
8
DPT
20 %
0
c 0
pTAl I
I
I
20
40
60
I
I
80
locl
Vibrio alginolyticus (‘/o) Fig. 5. Relation between the proportion of K afginolyticus in turbot flora and the larval survival rate. The standard errors of this rate are represented by vertical segments.
146
F.-J. GATESOUPE
a high mortality at day 10. This is the reason why two bacterial profiles were distinguished for treatment PTA (Fig. 4). Profile PTA 1 corresponded to the two tanks with a high mortality rate between day 9 and 10, whereas profile pTA2 corresponded to the other tanks. In experiment 3, one of the live colonies of profile pTA 1 was I/ alginolyticus, whereas the four others were Aeromonas sp. In experiment 4, the 15 colonies from pTA1 belonged to the same Aeromonas sp. as in experiment 3. This species would be A. hydrophila according to the Api 20 E system, but it could not grow in freshwater. Profile pTA2 was close to the profiles of the other treatments. V alginolyticus dominated and its proportion was significantly different between treatments (3 1, 42, 60 and 90% of the total phenons in profiles DpT, pTA2, pT and A, respectively). A positive relation was observed between the proportion of this Vibrio and the survival rate of turbot (Fig. 5). This species was associated with phenons R, already observed in rotifers, and others, gathered as phenons T. Among these latter phenons, Pseudomonas spp. and Vibrio spp. were identified. DISCUSSION
The continuous distribution of rotifers allows a high survival rate without any antibiotic addition. This confirms that the dietary value of rotifers can be improved without the enrichment phase causing an increase of bacteria. However, the importance of associated bacteria should not be underestimated. Two bacterial strains seemed to be specially important in these experiments. V. alginolyticus was the dominant species in SM plate counts of rotifers and turbot cultured under normal conditions. This species was reported to be pathogenic for black sea bream fry (Kusuda et al., 1986). The strain observed in the present experiments does not appear to be pathogenic. On the contrary, there was a positive relation between its proportion in the flora and the subsequent survival rate of turbot. The strain of Aeromonas seems opportunistic, since its proliferation was observed only in tanks of larvae on the verge of high mortality. A hypothetic competition between I! alginolyticus and opportunistic bacteria may give an explanation for the positive relation between the proportion of this species and the survival rate of turbot. The infected larvae seemed to be weakened by the combination of the bacterial additive and the pH or temperature change in rotifer culture. However, an improvement of the growth rate was observed with the separate treatments, i.e. pH and temperature changes, or the introduction of the bacterial additive at normal pH and low temperature. Further experiments are required to investigate the effects of pH and temperature on the bacterial additive. Acidophilic strains of lactic bacteria may be specially interesting for the combined treatment.
QUALITY OF ROTIFER CULTURES FOR FEEDING LARVAL TURBOT
147
CONCLUSION
If some improvements are still to be expected, these experiments bring a practical solution for the feeding of turbot with rotifers of good dietary and bacterial value. Considering that the proliferation of opportunistic bacteria was observed, hygienic rules should be strictly applied. The continuous distribution of rotifers fed on a fatty diet should be used, but the continuous culture of rotifers in the larval rearing room seems dangerous. Rather, it is advisable to introduce new rotifers into this room each day for the continuous feeding, while culturing the rotifers in another room. Feeding with Artemia will be e.xamined in a future communication. ACKNOWLEDGMENTS
Acosil was freely provided by Delporte S.A. (59283 Moncheaux, France). Valuable advice on the use of the Api 20 E system was given by Drs. J.L. Nicolas and M. Vigneulle, Mrs. D. Ansquer and Mr. A. Abiven. Thanks are due to Drs. J. Guillaume, Y. Harache and S.J. Kaushik for critical reading of the manuscript.
REFERENCES Fung, D.Y.C., Hart, R.A. and Chain, V., 1986. Rapid methods and automated procedures for microbiological evaluation of seafood. In: D.E. Kramer and J. Liston (Editors), Seafood Quality Determination, Proc. Int. Symp., Univ. Alaska, Sea Grant College Program, Anchorage, lo- I4 Nov. 1986. Elsevier, Amsterdam, pp. 247-253. Gatesoupe, F.J., 1980. Further advances in the nutritional and antibacterial treatments of rotifers as food for turbot larvae, .Scophrhafn?us maximus L. In: N. de Pauw, E. Jaspers, H. Ackefors and N. Wilkins (Editors), Aquaculture, a Biotechnology in Progress, Vol. 2. European Aquaculture Society, Bredene, Belgium, pp. 721-730. Gatesoupe. F.-J., Arakawa, T. and Watanabe, T., 1989. The effect of bacterial additives on the production rate and dietary value of rotifers as food for Japanese flounder, Paralichthys olivaceus. Aquaculture, 83: 39-44. Kusuda, R., Yokoyama, J. and Kawai, K., 1986. Bacteriological study on cause of mass mortalities in cultured black sea bream fry. Bull. Jpn. Sot. Sci. Fish., 52: 1745-I 75 1. Nicolas, J.L., Robic, E. and Ansquer, D., 1989. Bacterial flora associated with a trophic chain consisting of microalgae, rotifers and turbot larvae: influence of bacteria on larval survival. Aquaculture, 83: 237-248. PCrez Bena.vente, G. and Gatesoupe, F.-J., 1988. The continuous distribution of rotifers increases ,the essential fatty acid reserve of turbot larvae, Scophthalmus maximus. Aquaculture, 72: 109-I 14. Snell, T.W., Childress, M.J. and Boyer, E.M., 1987. Assessing the status ofrotifer mass cultures. J. World. Aquacult. Sot., 18: 270-277. Sokal, R.R. and Rohlf, F.J., 1969. Biometry. Freeman, San Francisco, CA, pp. 226-246. Spyridakis, P., Gabaudan, J., MCtailler, R. and Guillaume, J., 1988. DigestibilitC des protCines
148
F.-J. GATESOUPE
et disponibilitk des acides aminb de quelques matitres chez le bar (Dicentrarchus labrax). Reprod. Nutr. DCv., 28: 1509-I 5 17. Yu, J.P. and Hirayama, K., 1986. The effect of un-ionized ammonia on the population growth of the rotifer in mass culture. Bull. Jpn. Sot. Sci. Fish., 52: 1509- 15 13.
139
The continuous feeding of turbot larvae, Scophthalrnus maximus, and control of the bacterial environment of rotifers Franqois-Jog1 Gatesoupe INRAMFREMER, Centre de Brest, BP 70, 29280 Plouzank (France) (Accepted 2 1 November
1989)
ABSTRACT Gatesoupe, F.-J., 1990. The continuous feeding of turbot larvae, Scophthalmus maximus, and control of the balzterial environment of rotifers. Aquaculture, 89: 139-148. Turbot la.rvae were fed continuously with rotifers pumped from the culture tanks and rinsed before distribution. This technique improved the growth and survival rates of turbot, in comparison with the results obtained with a single daily distribution of rotifers enriched for 6 h. Special attention was paid to associated bacteria. Most bacteria detected in healthy rotifers and turbot were identified as Vibrio algirtolyticus.An opportunistic strain of Aeromonas sp. was observed in large numbers, but only in tanks where the larvae were on the verge of high mortality. A food additive containing live lactic bacteria improved the growth rate of turbot when it was put into the rotifer medium. A similar improvement was observed when the pH of this medium was lowered to 5.25-6, while the temperature was increased from 19 to 26°C. The combination of the bacterial additive and the pH-temperature changes was not efficient and might have led to the proliferation ofderomonas.
INTRODUCTION
Bacteria associated with rotifers have a detrimental effect on the growth and survival rates of turbot larvae (Gatesoupe, 1989). The main cause of the bacterial growth in rotifers is overnight enrichment with oil emulsion (Gatesoupe et al., 1989). The continuous distribution of rotifers needed to be reexamined in the light of this latter observation. In a previous experiment (Perez and Gatesoupe, 1988), rotifers were enriched with fish oil emulsion for 2 days. During the first day, they were disinfected with antibiotics. During the second day, they were semi-continuously distributed to turbot larvae. Continuation of the enrichment after the disinfection was not correct, since bacteria were able to grow again. This might be a reason why the growth and survival rates of turbot were not improved by this treatment. However, 22:6 n-3 fatty 0044-8486;90/$03.50
0 1990 -
Elsevier Science Publishers
B.V.
140
F.-J. GATESOUPE
acid reserves were increased in the larvae. Thus continuous distribution of rotifers can maintain their essential fatty acid content. The experiments presented here were therefore carried out with a view to testing the continuous distribution of rotifers without disinfection and further enrichment. Nevertheless, high numbers of bacteria were also observed in rotifers before enrichment, while these rotifers were not ingested by turbot (Nicolas et al., 1989). It was therefore important to control the bacterial environment of the rotifers. A food additive containing live lactic bacteria was found to limit the bacterial growth in rotifers (Gatesoupe et al., 1989). This additive had to be tested with the continuous distribution of rotifers. Moreover, the rotifers could endure a wide range of pH and temperature conditions (Snell et al., 1987). The effect of a change in these conditions was studied, with a view to enhancing a possible action of lactic bacteria. MATERIAL AND METHODS
Experimentaldesign Four grades of rotifers were tested: ( 1) the control rotifers C were reared at normal pH and temperature; (2) the pH was lowered and the temperature was increased for rotifers pT; (3) a food additive containing lactic bacteria ( Acosil ) was introduced into the medium of rotifers A, reared at normal pH and temperature; and (4) this additive was tested on rotifers PTA, reared at low pH and high temperature. These four kinds of rotifers were distributed continuously to turbot larvae (turbot C, A, pT and PTA). The effect of the continuous distribution was tested in comparison with rotifers pT and PTA distributed once a day after 6 h of enrichment (turbot DpT and DpTA). Four experiments were carried out, with triplicates per treatment in each experiment (Table 1). The effect of the continuous distribution was tested in experiments 1 and 2 with rotifers PTA and pT, respectively (treatments PTA, DpTA, pT and DpT ). The dietary values of the four kinds of rotifers distributed continuously were compared in the last three experiments (treatments pT and A in experiment 2, C, pT and pTA in experiment 3, and pT and PTA in experiment 4). Rotifer culture The rotifers were reared in 150-l tanks (rearing unit shown schematically in Fig. 1). A quarter of the volume was renewed daily with 18%0 salt water. The rotifers were fed with baker’s yeast (60.8 mg- ’ day- ’ ), cod liver oil (7.4 mg 1-l day- ’ ), soybean lecithin (0.6 mg 1--I day- * ), and vitamin premix (0.7 mg 1-l day-‘, Spyridakis et al., 1988). When the pH of the rotifer culture was lowered (rotifers pT and PTA), this food suspension was introduced into the renewing water of the rotifer tanks. This water was adjusted to pH 4 with normal HCl and distributed continuously with a peristaltic pump into
141
QUALITY OIF ROTIFER CULTURES FOR FEEDING LARVAL TURBOT
TABLE 1 Summary of the experimental design and rotifer culture conditions (D: rotifers distributed discontinuously, pT rotifers reared at low pH and high temperature, A: Acosil added to the diet, C: control) Treatment
Experiment 1 Experiment 2 Experiment 3 Experiment 4
DPT
DpTA
-
+
+
Rotifer culture: pH range Temperature (“C) Acosil (me. I-’ day-‘) Continuous distribution of rotifers
5.25-6 26 0
PT
PTA
C
A
+
-
+ + +
+ +
+ -
+ -
5.25-6 26 8
5.25-6 26 0
5.25-6 26 8
7-8 19 0
7-8 19 8
+
+
+
+
-
IOcm ROTI FER TREATME:N c ,A
PT , PTA
YEAST
ROTI FERS
RINSE
TURBOT
Fig. 1. Re#aring unit for the continuous feeding of turbot C, A, pT and PTA. Rotifers C and A were fed with the l-l bottle of yeast suspension. Rotifers pT and PTA were fed with yeast at pH 4 in the renewing water. Then the rotifers were pumped and rinsed continuously in this unit.
the rotifer tanks. In these tanks, the pH was checked daily and adjusted to 5.25. The value before adjustment was near to pH 6. The rearing temperature was kept at 26°C. When the pH was not modified (rotifers C and A), the
F.-J. GATESOUPE
142
food suspension was stirred constantly in a l-1 bottle and distributed semicontinuously to rotifers. The temperature was kept at 19 “C and a quarter of the culture volume was renewed daily at one go. Snell et al. ( 1987) found that the swimming activity of rotifers reared at 20°C and pH 7.5 was nearly the same as the activity of rotifers reared at 25°C and pH 5.2, while the maximum value was observed at 30” C and pH 7. Rotifers tolerated acid pH values to a greater extent than alkaline values, likely due to the toxicity of unionized ammonia (Yu and Hirayama, 1986). When Acosil, a dry extract of sprouting cereal grains fermented with lactic bacteria, was tested (rotifers A and PTA), it was delivered at the rate of 8 mg l- ’ day- ’ into a 40-pm net bag once a day, so that only small particles and soluble substances were introduced into the rotifer culture medium. Turbot rearing Newly hatched turbot were put in 150-l tanks at a density of 30 larvae l- ‘. When turbot were fed once a day (turbot DpT and DpTA), the amounts of rotifers were determined visually so as to obtain a slight excess overnight (Fig. 2). The same amount was also given discontinuously in the other tanks, The continuous distribution supplied surplus rotifers. These rotifers were continuously pumped and rinsed on a 80-pm net with seawater (35%0). They were again pumped continuously up to the larval tanks. One rotifer tank could feed
DAY
DIET
Fig. 2. Mean amounts of rotifers given daily per hatched larva in the four experiments. The same amount (Ro) as distributed discontinuously to tanks D (treatments DpT, DpTA) was also distributed discontinuously to the other tanks. The amounts of rotifers distributed continuously (R + ) were slightly different between treatments C, A pT and PTA.
QUALITY OF ROTIFER CULTURES FOR FEEDING LARVAL TURBOT
143
four larval tanks of the same volume. In these larval tanks, the water outlet passed through a 180-pm net, so as to draw away excess rotifers. The tanks with discontinuous feeding were fitted with 80-pm nets. The rotifers were enriched only for these latter tanks. The enrichment consisted of 6 h immersion in an emulsion (cod liver oil 46 mg l- ’ and soybean lecithin 4 mg l- ’ ) . The experiments ended at day 10 after hatching. The final growth and survival rates were compared by an a priori test and that of Student-NewmanKeuls, respectively (Sokal and Rohlf, 1969). Bacteria were counted in rotifers on Petrifilm SM plates, which gave bacterial numbers in seafood very close to those obtained with the standard agar method (Fung et al., 1986). About 5 ml of rotifer culture were sampled per treatment. Under sterile conditions, the rotifers were rinsed over a 80-pm net and resuspended in 4.5 ml of seawater. They were counted and crushed in a glass homogenizer. The samples were diluted 1000 times and inoculated on SM plates. Colonies were isolated from these plates and determined with the Api 20 E system (23, 12, 16 and 14 colonies were determined in rotifers C, A ‘pT, and PTA, respectively). At day 9 after hatching, live larvae from each tank were sampled for counting bacteria. Under sterile conditions, they were rinsed over a 180-pm net and treated in the same way as the rotifers for the inoculation of SM plates and the Api 20 E system ( 16,5 and 15 colonies were determined in the samples from 3, 4 and 3 tanks in experiments 2, 3 and 4, respectively). The proportions of the phenons were compared by a chi-square test whenever possible. RESULTS
In experiment 1, the mean weight obtained with continuous feeding (treatment PTA) was significantly higher than that obtained with discontinuous feeding (treatment DpTA, Table 2 ). The survival rates were not significantly different. In experiment 2, the treatment DpT with discontinuous distribution of rotifers resulted in mean weight and survival rates of turbot significantly lower than those observed with the two other treatments. Moreover, the mean weight obtained with treatment A was significantly higher than that with treatment pT. In experiments 3 and 4, the mean survival rates were not significantly different, due to a great variability of the survival rate obtained with treatment PTA (standard errors shown in Fig. 5 ) . This variability was due to a high mortality rate observed between day 9 and day 10 in one tank corresponding to treatment pTA in experiments 3 and 4. The mean weights obtained with treatment pT were significantly higher than those observed with treatments PTA and C. Two thousand bacteria were counted per rotifer in treatments C and PTA, whereas 7000 and 8000 bacteria were counted per rotifer in treatments pT and A, respectively. The dominant species, Vibrio alginolyticus, was respon-
144
F.-J. GATESOUPE
TABLE 2 Mean weight and survival rate of turbot at day 10. Superscripts a, b and c indicate significant differences Treatment DPT Mean weight (mg) Experiment 1 Experiment 2 Experiment 3 Experiment 4 Survival rate (Oh) Experiment 1 Experiment 2 Experiment 3 Experiment 4
0.38” -
DpTA 0.25b -
PT 0.55b 0.63” 0.63”
27 2gb -
69” 52 62
PTA 0.29” 0.52b 0.55b
61 22 66
C 0.50b -
46 -
A
0.66” -
82” -
1000 BACTERIA/ROTIFER
PHENONS
Fig. 3. Number of bacteria (thousands per rotifer). V. alginolyticus (VA) dominated and the other phenons (R) were not identified.
sible for most of the difference between the numbers of bacteria (Fig. 3). The other phenons, gathered as phenons R, represented about 1000 bacteria per rotifer in each samples. They could not be identified by the Api 20 E system. The differences observed between groups for the bacterial counts in larvae at day 9 were not significant. However, the numbers of bacteria within treatment PTA were especially variable. This variability might explain that of the survival rates, since the lots with a high number of bacteria at day 9 presented
QUALITY OF ROTIFER CULTURES FOR FEEDING LARVAL TURBOT
MILLION
Fig. 4. Number of bacteria (millions per larva) at day 9. Profile pTA1, with Aeromonas sp. ( Ae) dominant, corresponds to the tanks with a high mortality rate between day 9 and day 10. Profile pTA2 corresponds to tanks with normal survival rates. Like the profiles pT, DpT, A and C, it contains phenons observed in rotifers (VA, R) or not (phenons T).
survival rate (“/I
8
DPT
20 %
0
c 0
pTAl I
I
I
20
40
60
I
I
80
locl
Vibrio alginolyticus (‘/o) Fig. 5. Relation between the proportion of K afginolyticus in turbot flora and the larval survival rate. The standard errors of this rate are represented by vertical segments.
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a high mortality at day 10. This is the reason why two bacterial profiles were distinguished for treatment PTA (Fig. 4). Profile PTA 1 corresponded to the two tanks with a high mortality rate between day 9 and 10, whereas profile pTA2 corresponded to the other tanks. In experiment 3, one of the live colonies of profile pTA 1 was I/ alginolyticus, whereas the four others were Aeromonas sp. In experiment 4, the 15 colonies from pTA1 belonged to the same Aeromonas sp. as in experiment 3. This species would be A. hydrophila according to the Api 20 E system, but it could not grow in freshwater. Profile pTA2 was close to the profiles of the other treatments. V alginolyticus dominated and its proportion was significantly different between treatments (3 1, 42, 60 and 90% of the total phenons in profiles DpT, pTA2, pT and A, respectively). A positive relation was observed between the proportion of this Vibrio and the survival rate of turbot (Fig. 5). This species was associated with phenons R, already observed in rotifers, and others, gathered as phenons T. Among these latter phenons, Pseudomonas spp. and Vibrio spp. were identified. DISCUSSION
The continuous distribution of rotifers allows a high survival rate without any antibiotic addition. This confirms that the dietary value of rotifers can be improved without the enrichment phase causing an increase of bacteria. However, the importance of associated bacteria should not be underestimated. Two bacterial strains seemed to be specially important in these experiments. V. alginolyticus was the dominant species in SM plate counts of rotifers and turbot cultured under normal conditions. This species was reported to be pathogenic for black sea bream fry (Kusuda et al., 1986). The strain observed in the present experiments does not appear to be pathogenic. On the contrary, there was a positive relation between its proportion in the flora and the subsequent survival rate of turbot. The strain of Aeromonas seems opportunistic, since its proliferation was observed only in tanks of larvae on the verge of high mortality. A hypothetic competition between I! alginolyticus and opportunistic bacteria may give an explanation for the positive relation between the proportion of this species and the survival rate of turbot. The infected larvae seemed to be weakened by the combination of the bacterial additive and the pH or temperature change in rotifer culture. However, an improvement of the growth rate was observed with the separate treatments, i.e. pH and temperature changes, or the introduction of the bacterial additive at normal pH and low temperature. Further experiments are required to investigate the effects of pH and temperature on the bacterial additive. Acidophilic strains of lactic bacteria may be specially interesting for the combined treatment.
QUALITY OF ROTIFER CULTURES FOR FEEDING LARVAL TURBOT
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CONCLUSION
If some improvements are still to be expected, these experiments bring a practical solution for the feeding of turbot with rotifers of good dietary and bacterial value. Considering that the proliferation of opportunistic bacteria was observed, hygienic rules should be strictly applied. The continuous distribution of rotifers fed on a fatty diet should be used, but the continuous culture of rotifers in the larval rearing room seems dangerous. Rather, it is advisable to introduce new rotifers into this room each day for the continuous feeding, while culturing the rotifers in another room. Feeding with Artemia will be e.xamined in a future communication. ACKNOWLEDGMENTS
Acosil was freely provided by Delporte S.A. (59283 Moncheaux, France). Valuable advice on the use of the Api 20 E system was given by Drs. J.L. Nicolas and M. Vigneulle, Mrs. D. Ansquer and Mr. A. Abiven. Thanks are due to Drs. J. Guillaume, Y. Harache and S.J. Kaushik for critical reading of the manuscript.
REFERENCES Fung, D.Y.C., Hart, R.A. and Chain, V., 1986. Rapid methods and automated procedures for microbiological evaluation of seafood. In: D.E. Kramer and J. Liston (Editors), Seafood Quality Determination, Proc. Int. Symp., Univ. Alaska, Sea Grant College Program, Anchorage, lo- I4 Nov. 1986. Elsevier, Amsterdam, pp. 247-253. Gatesoupe, F.J., 1980. Further advances in the nutritional and antibacterial treatments of rotifers as food for turbot larvae, .Scophrhafn?us maximus L. In: N. de Pauw, E. Jaspers, H. Ackefors and N. Wilkins (Editors), Aquaculture, a Biotechnology in Progress, Vol. 2. European Aquaculture Society, Bredene, Belgium, pp. 721-730. Gatesoupe. F.-J., Arakawa, T. and Watanabe, T., 1989. The effect of bacterial additives on the production rate and dietary value of rotifers as food for Japanese flounder, Paralichthys olivaceus. Aquaculture, 83: 39-44. Kusuda, R., Yokoyama, J. and Kawai, K., 1986. Bacteriological study on cause of mass mortalities in cultured black sea bream fry. Bull. Jpn. Sot. Sci. Fish., 52: 1745-I 75 1. Nicolas, J.L., Robic, E. and Ansquer, D., 1989. Bacterial flora associated with a trophic chain consisting of microalgae, rotifers and turbot larvae: influence of bacteria on larval survival. Aquaculture, 83: 237-248. PCrez Bena.vente, G. and Gatesoupe, F.-J., 1988. The continuous distribution of rotifers increases ,the essential fatty acid reserve of turbot larvae, Scophthalmus maximus. Aquaculture, 72: 109-I 14. Snell, T.W., Childress, M.J. and Boyer, E.M., 1987. Assessing the status ofrotifer mass cultures. J. World. Aquacult. Sot., 18: 270-277. Sokal, R.R. and Rohlf, F.J., 1969. Biometry. Freeman, San Francisco, CA, pp. 226-246. Spyridakis, P., Gabaudan, J., MCtailler, R. and Guillaume, J., 1988. DigestibilitC des protCines
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et disponibilitk des acides aminb de quelques matitres chez le bar (Dicentrarchus labrax). Reprod. Nutr. DCv., 28: 1509-I 5 17. Yu, J.P. and Hirayama, K., 1986. The effect of un-ionized ammonia on the population growth of the rotifer in mass culture. Bull. Jpn. Sot. Sci. Fish., 52: 1509- 15 13.