Bacterial Additives That Consistently Enhance Rotifer Growth Under Synxenic Culture Conditions 1

Aquaculture 182Ž. 2000 249±260 www.elsevier.nlrlocateraqua-online

Bacterial additives that consistently enhance rotifer growth under synxenic culture conditions 1. Evaluation of commercial products and pure isolates

P.A. Douillet ) The UniÕersity of Texas at Austin, Marine Science Institute, 1300 Port Street, Port Aransas, TX 78373, USA Accepted 20 July 1999

Abstract

Axenic rotifers Ž.Brachionus plicatilis MullerÈ were cultured under aseptic conditions; they were fed either a bacteria-free artificial dietŽ. AD , or axenic Isochrysis galbana, or a combination of axenic Chlorella minutissima and the bacteria-free AD. The medium was inoculated with commercial bacterial additives or cultured strains of marine bacteria. The highest improvements in growth rateŽ. GR of rotifer populations were obtained with laboratory grown bacteria. Addition of an Alteromonas strain and an unidentified Gram negative strainŽ. B3 consistently enhanced rotifer GR in all experiments, and under all feeding regimes in comparison with control cultures inoculated with microbial communities present in seawater, or maintained bacteria-free. None of the other isolates or commercial products were consistent in their enhancement of rotifer production. q 2000 Elsevier Science B.V. All rights reserved.

Keywords: Rotifer; Brachionus plicatilis; Isochrysis galbana

1. Introduction

The rotifer Brachionus plicatilis has become a valuable and, in many cases indis- pensable, food organism for first feeding of a large variety of cultured marine finfish and crustacean larvaeŽ. Watanabe et al., 1983; Lubzens et al., 1997 . However, suppressed

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0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S0044-8486Ž. 99 00271-9 250 P.A. DouilletrAquaculture 182() 2000 249±260 growth or unforeseen death of rotifers are frequently observed in mass cultures Ž.Hirayama, 1987; Ushiro et al., 1990; Maeda and Hino, 1991; Hino, 1993 . Rotifer cultures harbor very large bacterial populations, which have been estimated to be in the order of 107 cells mly1 Ž. Nicolas and Joubert, 1986; Nicolas et al., 1989 . Rapid successional processes in the microbiota have been observed during the culture of rotifersŽ. Maeda and Hino, 1991 , and changes in the microbial ecosystem have been postulated as the cause of the collapse of rotifer culturesŽ. Hino, 1993 . The effects of bacteria on rotifer cultures are strain specific, as demonstrated by the findings of Yasuda and TagaŽ. 1980 , Yu et al. Ž. 1988 , Gatesoupe et al. Ž. 1989 , Maeda and HinoŽ. 1991 and Hagiwara et al. Ž. 1994 . These authors reported strains, from diverse taxonomical groups, that were able to either decrease or increase the growth rate Ž.GR of B. plicatilis. However, bacterially mediated changes in rotifer GRs are caused by diverse mechanisms. A nutritional contribution of bacteria to rotifer diets has been demonstrated by supply of vitamin B12 Ž. Yu et al., 1988 or inorganic nutrients Ž Hessen and Andersen, 1990. . In contrast, production of bacterial toxins has been found to reduce rotifer survival ratesŽ. Yu et al., 1990 . Another possible effect of bacteria in rotifer cultures is the biochemical transformation of accumulated waste products. Nitrogen budgets carried out with rotifers fed Nanochloropsis sp. revealed that 82%± 84% of the ingested N was released into the water as metabolic excretion and feces Ž.Tanaka, 1991; Hino et al., 1997 . Accumulation of metabolic products and excess uneaten food cause deterioration of water qualityŽ. Lubzens, 1987 , which may affect rotifer growth and reproductionŽ. Tanaka, 1991 . In fact, rotifer densities have been reported to decrease with increases of either un-ionized ammoniaŽ Yu and Hirayama, 1986.Ž or nitrite Lubzens, 1987 . . Removal of waste products from rotifer cultures has been reported to extend the harvest periodŽ. Lubzens, 1987 . A bacterially mediated improvement in water quality might be a very plausible mechanism for increasing rotifer GRs. In this study, the effects of additions of laboratory-grown microbes and several commercial bacterial additives were evaluated on the GR of B. plicatilis cultured under synxenic conditions, i.e., rotifers were grown in the presence of a known number, one or more, of microbial species. Single strains and commercial products with diverse characteristics that might be beneficial for rotifers were selected so that the evaluation of microbes would cover different plausible bacterial mechanisms for rotifer culture enhancement. The screening of microbes was carried out under an artificial dietŽ. AD and different algae feeding regimes.

2. Materials and methods

2.1. Preparation of rotifers

Cysts of the rotifer B. plicatilis MullerÈ Ž. formerly called L-type B. plicatilis were purchased from Aquaculture Supply, Florida. Bacteria-free rotifers were obtained by disinfecting the external surface of the cysts with sodium hypochlorite and they were tested for microbial contamination according to the methods presented in Douillet P.A. DouilletrAquaculture 182() 2000 249±260 251

Ž.1998 . To confirm that the rotifers were axenic, incubation tests of samples of rotifers in broth and agar under aerobic and anaerobic conditions were continued for 30 days. Axenicity tests were also performed on axenic and starved cultures at the end of the experiments. The experiment was discarded if bacterial contamination was detected.

2.2. Preparation of diets

An AD was developed and tested in preliminary experiments. The diet was prepared by dissolving 8 g of microfine SpirulinaŽ 8±10 by 20 mm; Aurum Aquaculture, Washington.Ž and 8 g of Torula dried yeast Lake States Division of Rhinelander Paper, Wisconsin. in 1 l of seawater at 15 ppt salinity. The dissolved diet was autoclaved. After cooling, filter-sterilized cyanocobalamineŽ. vitamin B12 was added at a concentration of 120 mg ly1 to the flask of AD to be used for first feeding only. The adequacy of the diet was ascertained by observing no significant difference between rotifer production in cultures fed either this diet or the diet developed by Gatesoupe and LuquetŽ. 1981 . Axenic Isochrysis galbana Ž.clone C-ISO, CCMP463 and Chlorella minutissima Ž.clone 2341 used in Experiments 2 and 4 were obtained from the National Center for Culture of Marine PhytoplanktonŽ. Maine and The Culture Collection of Algae at The University of Texas at Austin, respectively. Algae were grown in fr2 mediaŽ Guillard and Ryther, 1962. at 20±25 ppt salinity. Algal cultures were maintained in an incubator at 258C under constant cool-white fluorescent light at an intensity of 2250±4600 lx. Axenicity of algae was determined as described above for rotifers. Both species of algae were grown in 200 ml Erlenmeyer flasks. The cells were concentrated by centrifugation and resuspended at high concentrations in FASW, so that their daily addition to rotifer culturesŽ. approx. 20 ml would have little impact on rotifer densities. Rotifer cultures fed AD only were amended daily with FASW to maintain similar volumes to algae-fed rotifer cultures.

2.3. Preparation of bacteria

Commercially available bacterial additives were added directly to rotifer cultures. Bacterial strains kindly provided by other scientists or isolated by the author were cultured on Difco marine agar for 2±3 days, resuspended in FASW, washed by centrifugationŽ. 10,000=g for 10 min and resuspended in FASW. Photosynthetic bacteriaŽ. PH were cultured on Rhodospirillum ATTC Medium 1308 Ž Atlas and Parks, 1993, p. 774. for 1 week at 258C, under constant cool-white fluorescent light at an intensity of 2250±4600 lx. All glassware was washed in 10% nitric acid and rinsed seven times with tap water. Heat sterilization was carried out for 15 min at 1218C and a pressure of 1.06 kg cmy2 . All manipulations were done under a laminar flow hood.

2.4. Experimental protocol

Axenic rotifers were counted and transferred to 50 ml screw cap test tubes containing 30 ml of FASW. Samples of rotifers were taken from identical test tubes not used in 252 P.A. DouilletrAquaculture 182() 2000 249±260 experiments to corroborate initial rotifer densities. Culture experiments were initiated by the addition of food and the different bacteria. Control cultures consisted of:Ž. 1 cultures fed the same diets but maintained bacteria- free,Ž. 2 cultures fed the same diets and inoculated with bacteria present in 100 ml samples of freshly collected seawater filtered through a 1-mm screenŽ.Ž. SW , 3 starved cultures in Experiment 3, andŽ. 4 rotifer cultures fed only axenic I. galbana in Experiment 1. Rotifers were fed AD andror algae daily. The first day of culture, the AD was added at a final concentration of 0.2 mg mly1 ; then, the ration was decreased to 0.14 mg mly1 dayy1. The final concentration of cyanocobalamine after the first feeding was 1.5 g mly1 , as recommended by Hirayama and FunamotoŽ. 1983 . This vitamin was added only with the first feeding. Rotifers were fed on AD in Experiments 1 and 3. In Experiment 2, rotifers were fed either AD or axenic I. galbana. Rotifers fed I. galbana received daily additions to maintain a final concentration of 2=106 cells mly1.In Experiment 4, rotifers were fed either AD or a combination of AD and axenic C. minutissima. Rotifers were fed the same concentrations of AD under both feeding treatments. Rotifers supplemented with C. minutissima received daily algal additions to maintain a final concentration of 1=107 cells mly1. Commercial bacterial additives and pure bacterial isolates were added only once to rotifer cultures, on day one of the experiments, at a final concentration of 2=107 cells mly1. Bacteria concentrations were derived from equations relating spectrophotometric absorbanceŽ. 600 nm and bacteria numbers; the latter value was determined by direct count using DAPI staining techniquesŽ. Porter and Feig, 1980 . Such equations were developed and used for each bacterial additive tested. Commercial bacterial products and cultured strains tested in this research are presented in Table 1. In order to determine consistency of beneficial effects, strains that improved rotifer GR over axenic controls were repeatedly tested and discarded if the beneficial effects were not maintained. Bacterial treatments referred by their code name in Table 1 and tested in Experiment 1 included nine commercial productsŽ Acc, A2, A5, A6, A1100, A1200, F9, Mplus and Sy.Ž as well as eight laboratory cultured strains Vibrio alginolyticus, Alteromonas sp., B1, B2, B3, B4, B5 and PH. . In Experiment 2, the strain Enterococcus faecium was tested along with nine inoculantsŽ A1200, A5, Alteromonas sp., B2, B3, B4, B5, PH and V.a. which were evaluated for a second time. In Experiment 3, the additives Acc and B1 were evaluated for a second time, and five strains Ž Alteromonas sp., B3, B4, B5 and PH. were evaluated for a third time. Finally, in Experiment 4, commercial additives ABA and AB1 were tested for the first time, inoculants A2 and A6 were tested for the second time and strains Alteromonas sp., B3, B4 and B5 were tested for the fourth time. Due to differences in concentrations of bacteria in stock suspensions, different volumes of the suspensions had to be added to obtain a similar bacterial density under all treatments. In order to maintain the same volumes in culture systems, rotifer cultures were amended with different quantities of FASW. Initial rotifer densities for the different experiments were between 2.3 and 6 mly1. Rotifers were cultured for 4 days on a reciprocating shaker with a displacement of 4 cm at 60 cycles miny1. The culture tubes were illuminated with incandescent light at an intensity of 1000±1300 lx, under a 12 h light±12 h dark photoperiod and at 258C. P.A. DouilletrAquaculture 182() 2000 249±260 253 Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. cultured at UTMSI Alken Murray, NY dry Alken Murray, NY dry Dr. Don Lewis, Texas A&MTX University, cultured at UTMSI Advanced Microbial Systems, MN liquid Medipharm, IA dry P. Bogaert, University of Gent, Belgium dry Ž. , Alken Murray, NY liquid , American Biosystems, VA dry sp., P. stutzeri , , Water Quality Science International, MO liquid , Alken Murray, NY dry Nitrosomonas B. licheniformis , , Aspergillus oryzae rods Seawater isolates cultured at UTMSI , B. licheniformis , B. plicatilis y sp. Fritz, TX liquid sp. Alken Murray, NY liquid B. polymixa E. faecium , P. putida B. licheniformis , Pseudomonas aeruginosa now B. megaterium , Nitrobacter Nitrobacter Escherichia hermanii two strains P. stutzeri P. stutzeri Ž. 2203 Sybron Chemicals, VA dry sp., sp., , , , Rumenococcus albus sp. Seawater isolates cultured at UTMSI sp. , B. licheniformis B. amyloliquefaciens B. cereus three strains , two strains , , , , Alteromonas P. aeruginosa B. subtilis Nitrosomonas Bacillus subtilis Nitrosococcus P. aeruginosa E. faecium B. subtilis B. subtilis Nitrosomonas P. fluorescens P. aeruginosa Nitrobacter B. subtilis B. polymyxa B. subtilis V. alginolyticus Streptococcus faecium Ž. Ž. Ž . Ž. Ž. Ž. Ž. Ž . Ž. Ž. 9F9 a Ž. Ž. A5 A6 B1, B2, B3, B4, B5 Five unidentified marine Gram V.a A.sp. PH Photosynthetic red non-sulfur bacteria Qingdao Oceanogr. University, China E.f Sy A2 Table 1 Bacterial additives or bacteria strains tested in four experiments with Commercial name code Bacterial composition Origin presentation AB-1 AB1 Fritz-Zyme Ž. Ž. Ž. Ž. Ž. Ž. Ž. Aqua-Bacta-Aid N2 ABA Accelobac Acc Alken Clear-Flo 1100 A1100 Ž. Microbials Plus Mplus Ž. Ž. Alken Clear-Flo 1200 A1200 Ž. Ž . Ž. 254 P.A. DouilletrAquaculture 182() 2000 249±260

2.5. Data collection and analysis

At the end of each experiment, starved and axenic controls were sampled and tested as described above for bacterial contamination. Cultures were then homogenized by shaking and five samplesŽ. 100±1000 ml were withdrawn for estimation of rotifer densities. Rotifer population GR for each culture tube was calculated as: GRs lnŽ.Ž. final density yln initial density rculture period in days Ž. 4 days Parametric assumptions were evaluated using Hartley's test for homogeneity of variances, Tukey's test for non-additivity and Wilk±Shapiro's test for normality. Bacterial treatments were tested under two different feeding regimes in Experiments 2 and 4. These data sets were initially analyzed by two-way ANOVA, with feeding regime and bacterial treatment as variables. GR in all experiments was then analyzed using one-way ANOVA, followed by Tukey's range testŽ. T-method, Sokal and Rohlf, 1981 to determine differences between bacterial treatments at the 0.05 level of probability. Coefficients of variation ŽV U .Žbetween replicates under four treatments axenic, SW, B3 and Alteromonas sp.. were calculated in each experiment as in Sokal and Rohlf Ž.1981 : V U s Ž.100 sd rmean Ž 1q1r4n . where sd is the standard deviation, mean is the average GR and n is the number of replicates Ž.ns4 . Independent V U were determined for the different treatments under each one of the diets in Experiments 2 and 4; therefore, six V U values were calculated for each treatment in the four experiments. V U values under the SW treatment were compared with the V U values determined under the axenic, B3 and Alteromonas sp. treatments using the t-test for paired comparisonsŽ. Sokal and Rohlf, 1981 . All tests were performed with the computer program Statistix 2Ž. NH Analytical Software .

3. Results

No evidence of bacterial contamination was found in either starved or axenic control cultures at the end of the culture period. Axenic rotifers populations grew with all diets tested. GRs in rotifer populations inoculated with SW bacteria did not differ from those obtained with axenic controlsŽ. Figs. 1 and 2 . Significant differences in GRs between treatments were found in all experiments. In Experiment 1, additions of eight cultured bacterial additivesŽ B5, Alteromonas sp., PH, B3, B2, B4, V. alginolyticus and B1.Ž and five commercial products A1200, A2, A6, Acc and A5. resulted in larger GRs than those determined in axenic control cultures Ž.Tukey's, p-0.05 . The second highest GR was obtained in control cultures fed only on axenic I. galbana Ž.Fig. 1a . In Experiment 2, significant differences in GR were determined between feeding regimes and bacterial treatmentsŽ. two-way ANOVA, p-0.05 . Therefore, data sets for each feeding regime were analyzed by one-way ANOVA. Under the AD feeding regime, GRs were significantly larger than in axenic controlsŽ. Tukey's, p)0.05 in cultures P.A. DouilletrAquaculture 182() 2000 249±260 255

Fig. 1. GRs of B. plicatilis cultured under synxenic conditions with different bacterial additives and fed axenic ADs in Experiment 1Ž. a , Experiment 2 Ž. b , Experiment 3 Ž. c and Experiment 4 Ž. d . Results of Tukey's range tests are displayed above the histogram. Circles that occur together on any one of the horizontal lines indicate mean values that are not different at the 0.05% level of significance. See Table 1 for details of diets.

inoculated with one commercial productŽ. A5 and five cultured bacterial additives Ž B5, Alteromonas sp., B3, B4 and PH.Ž Fig. 1b . . Under the I. galbana feeding regime, GRs were significantly improved over axenic controls with all bacterial treatmentsŽ Tukey's, p-0.05.Ž. except for A1200 and A5 Fig. 2a . In Experiment 3, a negative GR was observed in starved cultures due to mortality of initial rotifer populationsŽ. Fig. 1c . Addition of four strains ŽAlteromonas sp., B3, B5 and B4.Ž significantly enhanced GRs of rotifers over axenic control cultures Tukey's, p-0.05. . As in Experiment 2, significant differences in GRs were determined between feeding regimes and bacterial treatments in Experiment 4Ž. two-way ANOVA, p-0.05 . There- fore, data sets for each feeding regime were analyzed by one-way ANOVA. Under the AD feeding regime, addition of all commercial additives and strain B4 resulted in lower GRs than those determined in axenic controls; however, the differences were not significantŽ. Fig. 1d . Under the mixed feeding regime ŽC. minutissimaqAD . , addition of the commercial products AB1 and ABA resulted in lower GRs than in axenic controls, but the difference was significative only for AB1Ž. Tukey's, p-0.05 256 P.A. DouilletrAquaculture 182() 2000 249±260

Fig. 2. GRs of B. plicatilis cultured under synxenic conditions with different bacterial additives and fed either axenic I. galbana in Experiment 2Ž. a or a combination of axenic AD and C. minutissima in Experiment 4 Ž. b . Results of Tukey's range tests are displayed above the histogram. Circles that occur together on any one of the horizontal lines indicate mean values that are not different at the 0.05% level of significance. See Table 1 for details of diets.

Ž.Fig. 2b . Under both feeding regimes, addition of strains B3 and Alteromonas sp. did improve rotifer GR over axenic controlsŽ. Tukey's, p-0.05 , while addition of strain B5 resulted in GRs that did not differ from those of the axenic controls. V U values in cultures seeded with SW bacteria were found to be significantly larger than either those calculated in axenic cultures Ž.t-test, ps0.018 , cultures inoculated with B3 Ž.ps0.018 or cultures inoculated with Alteromonas sp. Ž.ps0.025 .

4. Discussion

Axenic rotifers were used to evaluate the effects of additions of several bacterial additives on the GRs of rotifer populations fed diverse diets. By using axenic rotifers, the effects determined under each treatment can be ascribed to the microbes added to the culture system without interference from bacterial contaminants that could carry out any of the different mechanisms that could affect rotifer GR. Rotifer populations were able to grow on all diets tested under axenic conditions. Lower GRs resulted in cultures of axenic rotifers fed bacteria-free ADŽ. 0.28"0.15, mean"sd, ns16 , than bacteria-free I. galbana Ž.0.81"0.07, ns8 or a mixed bacteria-free diet of AD and C. minutissima Ž.0.84"0.03, ns4 . The GRs of rotifers in cultures inoculated with several laboratory isolates were significantly higher than those recorded in cultures fed the same diet, but kept axenic. The largest GR improvement was determined with AD in Experiment 1, where the addition of strain B5 resulted in a GR 5.9 times larger than in axenic controls fed the same diet. The importance of a bacterial component in rotifer cultures was illustrated in this first experiment, whereas the addition of five bacteria strains to cultures of rotifers fed AD resulted in GR levels that did not differ from those observed in cultures of rotifers fed axenic algae. Rotifer populations inoculated with SW bacteria had GRs that did not differ from those of bacteria-free populations fed the same diet. However, addition of SW bacteria to rotifer cultures increased variability between replicates as demonstrated by signifi- P.A. DouilletrAquaculture 182() 2000 249±260 257 cantly larger V U values than in axenic cultures. Except for Experiment 4, lower GRs occurred in cultures seeded with SW bacteria compared with cultures seeded with any other bacterial amendment. Most marine fish and crustacean hatcheries use seawater for the culture of rotifers. However, filtration and chlorination, which are the most widely used methods of water disinfection in hatcheries, may not eliminate all seawater microbesŽ Douillet and Pickering, 1999. . So bacteria present in seawater may constitute one of the mayor sources of contamination in rotifer cultures. Most studies on the effects of bacteria on rotifer cultures have been carried out under xenic culture conditions; therefore, in the following discussion, the effects of bacterial additives were compared with control cultures inoculated with SW bacteria. Commercial bacterial products tested in this study were either beneficial or slightly adverse to rotifer production. The largest improvement caused by the addition of a commercial productŽ. Alken 1200, Experiment 1 was an increase in rotifer GR of 4.5 times the value determined in SW controls. Unfortunately, none of the commercial products that showed beneficial characteristics for rotifer culture was consistent in repeated trials. Furthermore, addition of isolate B5 in Experiment 1 resulted in rotifer GR 5.7 times larger than in SW controls. Therefore, the magnitudes of improvement of rotifer growth caused by commercial products were smaller than those obtained with laboratory grown microbes. Addition to rotifer cultures of the commercial products F9 and A1100, which contain nitrifying bacteria Ž.Nitrosomonas sp. and Nitrobacter sp. , did not result in any GR improvement. Several strains of Bacillus and Pseudomonas are well known for their ability to produce exoenzymes that break down organic matterŽ. Pollock, 1962 , and thus they are frequently included in commercial products for waste water treatment. The commercial product that most enhanced the GR of rotifersŽ. Alken 1200 is a blend of Bacillus strains and nitrifying bacteria. Unfortunately, no water quality analyses were carried out to determine a bacterially mediated water quality improvement mechanism. Photosynthetic bacteria are frequently used for wastewater treatment. The photosynthetic strains tested in this studyŽ. PH were isolated from the bottom of shrimp ponds, and are currently used as probiotics in Chinese hatcheries. PH bacteria were beneficial for rotifer growth in two out of three experiments. Bacteria strains that improve production of aquatic organisms can be either consistent or not in their enhancement propertiesŽ. Douillet and Langdon, 1993 . Alteromonas sp. and an unidentified Gram negative strainŽ. B3 were found in this study to be consistently beneficial to rotifers in all experiments and under all feeding regimes. Al- teromonas sp. tested in this study was evaluated in previous studies with larvae of the Pacific oyster Crassostrea gigas. Consistent enhancement of oyster larvae production was achieved with the addition of Alteromonas sp. under synxenicŽ Douillet and Langdon, 1993.Ž. and xenic culture conditions Douillet and Langdon, 1994 . The magnitude of the enhancement of rotifer GRs caused by the addition of Alteromonas sp. and B3 with respect to SW control cultures changed between experiments when using ADŽ 1.7±4.3 times increase in GR for Alteromonas sp. and 1.6±4.1 times for B3. . It was of less magnitude and more constant for cultures fed either algae or a combination of algae and ADŽ. 1.2±1.4 times for Alteromonas sp. and 1.3±1.4 times for B3 . Yu et 258 P.A. DouilletrAquaculture 182() 2000 249±260 al.Ž. 1988 found the magnitude of bacterial improvement in GRs of rotifers higher when using AD than when using algae. Using data from Figs. 4 and 5 in Yu's paper, the addition of a Pseudomonas producer of vitamin B12 resulted in a GR of rotifers 2.9 times higher than in controls when fed AD and 2.6 times higher than controls when fed algae. Nutritional supplementation leading to enhanced rotifer GR has been reported for algae with blue-green algaeŽ. Snell et al., 1983 , algae with yeast Ž Hirayama and Watanabe, 1973.Ž , dried algae with yeast Hirayama and Nakamura, 1976 . , and photosynthetic bacteria with either algae or yeastŽ. Sakamoto and Hirayama, 1983 . The bacterial enhancement of GR determined in rotifers fed AD, and to a lesser extend on algae, might be due to the fact that bacteria provide essential compounds such as vitaminsŽ. Yu et al., 1988 or inorganic nutrients Ž Hessen and Andersen, 1990 . deficient in the diets. Soluble compounds excreted by bacteria into the culture medium are strain specificŽ. Yu et al., 1989; Hagiwara et al., 1994 , which could explain variation in rotifer production under different bacterial treatments. A nutritional contribution of Al- teromonas sp. to oyster larvae was demonstrated by 14C-feeding techniques whereby this strain provided in some cases over 180% of the carbon metabolic requirements of the larvaeŽ. Douillet, 1993 . This study, carried out under synxenic conditions, showed a very significant variation in rotifer population growth resulting from changes in the species composition of the microbiota. Addition of a few commercial products significantly improved rotifer GRs; unfortunately, the beneficial effects were not consistent in repeated trials with any of these products. In contrast, selected bacterial strains were consistent in their enhancement of rotifer GRs under all feeding regimes. Furthermore, variations in GR between replicate cultures seeded with these selected strains were significantly lower than in cultures seeded with SW bacteria. GR is highly dependent on the previous life history, i.e., accumulated nutrients, reproductive condition, of the rotifers used to start popula- tionsŽ. Scott and Baynes, 1978; Snell et al., 1983 ; therefore, it is difficult to compare GRs from different studies. However, GRs determined in batch cultures of rotifers fed AD oscillate between 0.15 to 0.62Ž Hirayama and Nakamura, 1976; YuferaÂ and Pascual, 1980; Komis et al., 1991; Shiri Harzevili et al., 1995. , while those determined with rotifers fed algae fall between the wider range of 0.09±1.09Ž Theilacker and McMaster, 1971; Scott and Baynes, 1978; Sakamoto and Hirayama, 1983; Snell et al., 1983; Maeda and Hino, 1991; Lubzens et al., 1993.Ž. . Two findings from this research, a poor GRs occurred in cultures inoculated with SW bacteria, which are the microbes likely to colonize commercial rotifer production systems, andŽ. b consistent improvements in GR resulted by adding selected microbes, indicate the potential for microbial management to improve rotifer production in culture facilities.

Acknowledgements

This work was supported by Grant a NA56RG0388 from the National Oceanic and Atmospheric Administration through the National Sea Grant College Program. The views expressed herein are those of the author and do not necessarily reflect the views P.A. DouilletrAquaculture 182() 2000 249±260 259 of NOAA or any of its sub-agencies. I am indebted to Dr. Ron BennerŽ The University of Texas at Austin, Marine Science Institute. for the use of his epifluorescence microscope. I am grateful to P. BogaertŽ. University of Gent, Belgium and Dr. Don LewisŽ. Texas A&M University, USA for supplying bacteria samples. Photosynthetic microbes were kindly provided by Dr. Anja RobinsonŽ. Oregon State University , who obtained this product from researchers at Qingdao Oceanographic University, China. I am indebted to the following companies for supplying samples of their products: Advanced Microbial Systems, Alken Murray, American Biosystems, Medipharm, Sybron Chemicals and Water Quality Science International.

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