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

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1 Ž. Aquaculture 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 Muller. were cultured under aseptic conditions; they were fed either a bacteria-free artificial diet Ž 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 rate Ž GR. of rotifer populations were obtained with laboratory grown bacteria. Addition of an Alteromonas strain and an unidentified Gram negative strain Ž 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 indispensable, food organism for first feeding of a large variety of cultured marine finfish and crustacean larvae Ž Watanabe et al., 1983; Lubzens et al., However, suppressed ) 1692 Houghton Ct North, Dunwoody, GA 30338, USA. Tel.: q ; philippe douillet@yahoo.com r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. Ž. PII: S

2 250 ( ) P.A. DouilletrAquaculture growth or unforeseen death of rotifers are frequently observed in mass cultures Ž Hirayama, 1987; Ushiro et al., 1990; Maeda and Hino, 1991; Hino, Rotifer cultures harbor very large bacterial populations, which have been estimated to be in the 7 y1 order of 10 cells ml Ž Nicolas and Joubert, 1986; Nicolas et al., Rapid successional processes in the microbiota have been observed during the culture of rotifers Ž Maeda and Hino, 1991., and changes in the microbial ecosystem have been postulated as the cause of the collapse of rotifer cultures Ž Hino, The effects of bacteria on rotifer cultures are strain specific, as demonstrated by the findings of Yasuda and Taga Ž 1980., Yu et al. Ž 1988., Gatesoupe et al. Ž 1989., Maeda and Hino Ž and Hagiwara et al. Ž 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 B Ž Yu et al., or inorganic nutrients Ž 12 Hessen and Andersen, In contrast, production of bacterial toxins has been found to reduce rotifer survival rates Ž Yu et al., 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., Accumulation of metabolic products and excess uneaten food cause deterioration of water quality Ž Lubzens, 1987., which may affect rotifer growth and reproduction Ž Tanaka, In fact, rotifer densities have been reported to decrease with increases of either un-ionized ammonia ŽYu and Hirayama, or nitrite Ž Lubzens, Removal of waste products from rotifer cultures has been reported to extend the harvest period Ž Lubzens, 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 diet Ž AD. and different algae feeding regimes. 2. Materials and methods 2.1. Preparation of rotifers Cysts of the rotifer B. plicatilis Muller 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

3 ( ) P.A. DouilletrAquaculture Ž 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 Preparation of diets An AD was developed and tested in preliminary experiments. The diet was prepared by dissolving 8 g of microfine Spirulina Ž8 10 by 20 mm; Aurum Aquaculture, Washington. and 8 g of Torula dried yeast ŽLake States Division of Rhinelander Paper, Wisconsin. in 1 l of seawater at 15 ppt salinity. The dissolved diet was autoclaved. After cooling, filter-sterilized cyanocobalamine Ž vitamin B. 12 was added at a concentration of 120 mg l y1 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 Luquet Ž Axenic Isochrysis galbana Ž clone C-ISO, CCMP463. and Chlorella minutissima Ž clone used in Experiments 2 and 4 were obtained from the National Center for Culture of Marine Phytoplankton Ž Maine. and The Culture Collection of Algae at The University of Texas at Austin, respectively. Algae were grown in fr2 media ŽGuillard and Ryther, at ppt salinity. Algal cultures were maintained in an incubator at 258C under constant cool-white fluorescent light at an intensity of 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 cultures Ž 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 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 centrifugation Ž 10,000 = g for 10 min. and resuspended in FASW. Photosynthetic bacteria Ž PH. were cultured on Rhodospirillum ATTC Medium 1308 ŽAtlas and Parks, 1993, p for 1 week at 258C, under constant cool-white fluorescent light at an intensity of 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 cm y2. All manipulations were done under a laminar flow hood 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

4 252 ( ) P.A. DouilletrAquaculture 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 bacteriafree, 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 screen Ž SW., Ž 3. starved cultures in Experiment 3, and Ž. 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 ml y1 ; then, the ration was decreased to 0.14 mg ml y1 day y1. The final concentration of cyanocobalamine after the first feeding was 1.5 g y1 ml, as recommended by Hirayama and Funamoto Ž 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=10 6 cells ml y1.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=10 7 cells ml y1. 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=10 7 cells ml y1. Bacteria concentrations were derived from equations relating spectrophotometric absorbance Ž 600 nm. and bacteria numbers; the latter value was determined by direct count using DAPI staining techniques Ž Porter and Feig, 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 products ŽAcc, A2, A5, A6, A1100, A1200, F9, Mplus and Sy. as well as eight laboratory cultured strains ŽVibrio alginolyticus, Alteromonas sp., B1, B2, B3, B4, B5 and PH.. In Experiment 2, the strain Enterococcus faecium was tested along with nine inoculants ŽA1200, 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 ml y1. Rotifers were cultured for 4 days on a reciprocating shaker with a displacement of 4 cm at 60 cycles min y1. The culture tubes were illuminated with incandescent light at an intensity of lx, under a 12 h light 12 h dark photoperiod and at 258C.

5 Table 1 Bacterial additives or bacteria strains tested in four experiments with B. plicatilis Ž. Ž. Commercial name code Bacterial composition Origin presentation AB-1 Ž AB1. Bacillus subtilis Advanced Microbial Systems, MN Ž liquid. Aqua-Bacta-Aid N2 Ž ABA. B. subtilis, B. amyloliquefaciens, B. licheniformis, Water Quality Science International, MO Ž liquid. Nitrosococcus Ž two strains. Accelobac Ž Acc. B. subtilis, B. cereus, B. megaterium, B. licheniformis, American Biosystems, VA Ž dry. B. polymyxa, Rumenococcus albus, Aspergillus oryzae Alken Clear-Flo 1100 Ž A1100. Nitrosomonas sp., Nitrobacter sp. Alken Murray, NY Ž liquid. Alken Clear-Flo 1200 Ž A1200. B. subtilis Ž two strains., Pseudomonas aeruginosa, P. stutzeri, Alken Murray, NY Ž liquid. P. fluorescens, Escherichia hermanii, Nitrosomonas sp., Nitrobacter sp. Ž A2. B. subtilis Ž three strains., B. licheniformis, Alken Murray, NY Ž dry. P. aeruginosa, P. stutzeri Ž A5. P. aeruginosa, P. stutzeri, P. putida Alken Murray, NY Ž dry. Ž A6. B. subtilis, B. licheniformis, B. polymixa Alken Murray, NY Ž dry. Fritz-Zyme a9 Ž F9. Nitrosomonas sp., Nitrobacter sp. Fritz, TX Ž liquid. Microbials Plus Ž Mplus. Streptococcus faecium Ž now E. faecium. Medipharm, IA Ž dry. Ž Sy. P. aeruginosa 2203 Sybron Chemicals, VA Ž dry. Ž A.sp.. Alteromonas sp. Seawater isolates Ž cultured at UTMSI. Ž B1, B2, B3, B4, B5. Five unidentified marine Gram Ž y. rods Seawater isolates Ž cultured at UTMSI. Ž E.f. E. faecium P. Bogaert, University of Gent, Belgium Ž dry. Ž PH. Photosynthetic red non-sulfur bacteria Qingdao Oceanogr. University, China Ž cultured at UTMSI. Ž V.a. V. alginolyticus Dr. Don Lewis, Texas A&M University, TX Ž cultured at UTMSI. P.A. DouilletrAquaculture 182 ( 2000 )

6 254 ( ) P.A. DouilletrAquaculture 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 samples Ž ml. were withdrawn for estimation of rotifer densities. Rotifer population GR for each culture tube was calculated as: GR s lnž final density. y lnž 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 test Ž T-method, Sokal and Rohlf, to determine differences between bacterial treatments at the 0.05 level of probability. Coefficients of variation ŽV U. between replicates under four treatments Žaxenic, SW, B3 and Alteromonas sp.. were calculated in each experiment as in Sokal and Rohlf Ž 1981.: U V s Ž 100 sd. rmean Ž 1q1r4n. where sd is the standard deviation, mean is the average GR and n is the number of U replicates Ž n s 4.. Independent V 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 comparisons Ž Sokal and Rohlf, All tests were performed with the computer program Statistix 2 Ž 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 controls Ž Figs. 1 and 2.. Significant differences in GRs between treatments were found in all experiments. In Experiment 1, additions of eight cultured bacterial additives ŽB5, Alteromonas sp., PH, B3, B2, B4, V. alginolyticus and B1. and five commercial products ŽA1200, A2, A6, Acc and A5. resulted in larger GRs than those determined in axenic control cultures Ž Tukey s, p 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 treatments Ž two-way ANOVA, p 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 controls Ž Tukey s, p) in cultures

7 ( ) P.A. DouilletrAquaculture Fig. 1. GRs of B. plicatilis cultured under synxenic conditions with different bacterial additives and fed axenic ADs in Experiment 1 Ž. 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 product Ž A5. and five cultured bacterial additives ŽB5, 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 treatments ŽTukey s, p except for A1200 and A5 Ž Fig. 2a.. In Experiment 3, a negative GR was observed in starved cultures due to mortality of initial rotifer populations Ž Fig. 1c.. Addition of four strains ŽAlteromonas sp., B3, B5 and B4. significantly enhanced GRs of rotifers over axenic control cultures ŽTukey s, p As in Experiment 2, significant differences in GRs were determined between feeding regimes and bacterial treatments in Experiment 4 Ž two-way ANOVA, p Therefore, 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 significant Ž Fig. 1d.. Under the mixed feeding regime Ž C. minutissimaq AD., addition of the commercial products AB1 and ABA resulted in lower GRs than in axenic controls, but the difference was significative only for AB1 Ž Tukey s, p

8 256 ( ) P.A. DouilletrAquaculture Fig. 2. GRs of B. plicatilis cultured under synxenic conditions with different bacterial additives and fed either axenic I. galbana in Experiment 2 Ž. 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 controls Ž Tukey s, p , 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, p s , cultures inoculated with B3 Ž p s or cultures inoculated with Alteromonas sp. Ž p s 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 AD Ž 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-

9 ( ) P.A. DouilletrAquaculture 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 microbes ŽDouillet and Pickering, 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 product Ž 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 matter Ž Pollock, 1962., and thus they are frequently included in commercial products for waste water treatment. The commercial product that most enhanced the GR of rotifers Ž Alken 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 study Ž 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 properties Ž Douillet and Langdon, Alteromonas sp. and an unidentified Gram negative strain Ž B3. were found in this study to be consistently beneficial to rotifers in all experiments and under all feeding regimes. Alteromonas 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 synxenic ŽDouillet and Langdon, and xenic culture conditions Ž Douillet and Langdon, 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 AD Ž times increase in GR for Alteromonas sp. and times for B3.. It was of less magnitude and more constant for cultures fed either algae or a combination of algae and AD Ž times for Alteromonas sp. and times for B3.. Yu et

10 258 ( ) P.A. DouilletrAquaculture al. Ž 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 algae Ž Snell et al., 1983., algae with yeast ŽHirayama and Watanabe, 1973., dried algae with yeast Ž Hirayama and Nakamura, 1976., and photosynthetic bacteria with either algae or yeast Ž Sakamoto and Hirayama, 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 vitamins Ž Yu et al., or inorganic nutrients Ž Hessen and Andersen, deficient in the diets. Soluble compounds excreted by bacteria into the culture medium are strain specific Ž Yu et al., 1989; Hagiwara et al., 1994., which could explain variation in rotifer production under different bacterial treatments. A nutritional contribution of Alteromonas sp. to oyster larvae was demonstrated by 14 C-feeding techniques whereby this strain provided in some cases over 180% of the carbon metabolic requirements of the larvae Ž Douillet, 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 populations Ž 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 0.62 ŽHirayama and Nakamura, 1976; Yufera 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 ŽTheilacker and McMaster, 1971; Scott and Baynes, 1978; Sakamoto and Hirayama, 1983; Snell et al., 1983; Maeda and Hino, 1991; Lubzens et al., Two findings from this research, Ž a. poor GRs occurred in cultures inoculated with SW bacteria, which are the microbes likely to colonize commercial rotifer production systems, and Ž. 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

11 ( ) P.A. DouilletrAquaculture of NOAA or any of its sub-agencies. I am indebted to Dr. Ron Benner ŽThe University of Texas at Austin, Marine Science Institute. for the use of his epifluorescence microscope. I am grateful to P. Bogaert Ž University of Gent, Belgium. and Dr. Don Lewis Ž Texas A& M University, USA. for supplying bacteria samples. Photosynthetic microbes were kindly provided by Dr. Anja Robinson Ž 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. References Atlas, R.M., Parks, L.C., Handbook of Microbiological Media. CRC, Boca Raton, 1079 pp. Douillet, P., Carbon contribution through bacterivory in larvae of the Pacific oyster Crassostrea gigas. Mar. Ecol. Prog. Ser. 102, Douillet, P., Disinfection of rotifer cysts leading to bacteria-free populations. J. Exp. Mar. Biol. Ecol. 224, Douillet, P., Langdon, C.J., Effects of marine bacteria on the culture of axenic oyster Crassostrea gigas Ž Thunberg. larvae. Biol. Bull. 184, Douillet, P., Langdon, C.J., Use of a probiotic for the culture of larvae of the Pacific oyster ŽCrassostrea gigas Thunberg.. Aquaculture 119, Douillet, P., Pickering, P., Seawater treatment for larval culture of the fish Sciaenops ocellatus Linnaeus Ž red drum.. Aquaculture 170, Gatesoupe, F.J., Luquet, P., Practical diet for mass culture of the rotifer Brachionus plicatilis: application to larval rearing of sea bass, Dicentrarchus labrax. Aquaculture 22, Gatesoupe, F.J., Arakawa, T., Watanabe, T., The effect of bacterial additives on the production rate and dietary value of rotifers as food for Japanese flounder, Paralichthys oliõaceus. Aquaculture 83, Guillard, R.R.L., Ryther, J.H., Studies on marine planktonic diatoms: I. Cyclotela nana Hustedt and Detonula conferõacea Cleve. Can. J. Microbiol. 8, Hagiwara, A., Hamada, K., Hori, S., Hirayama, K., Increased sexual reproduction in Brachionus plicatilis Ž Rotifera. with the addition of bacteria and rotifer extracts. J. Exp. Mar. Biol. Ecol. 181, 1 8. Hessen, D.O., Andersen, T., Bacteria as a source of phosphorus for zooplankton. Hydrobiologia 206, Hino, A., Present culture systems of the rotifer Ž Brachionus plicatilis. and the function of microorganisms. In: Lee, C.S., Su, M.S., Liao, I.L. Ž Eds.., Proceedings of Finfish Hatchery in Asia 91, December 1991, Tungkang, Taiwan. Tungkang Marine Laboratory Conference Proceedings 3, pp Hino, A., Aoki, S., Ushiro, M., Nitrogen-flow in the rotifer Brachionus rotundiformis and its significance in mass cultures. Hydrobiologia 358, Hirayama, K., A consideration of why mass culture of the rotifer Brachionus plicatilis with baker s yeast is unstable. Hydrobiologia 147, Hirayama, K., Funamoto, H., Supplementary effect of several nutrients on nutritive deficiency of baker s yeast for population growth of the rotifer Brachionus plicatilis. Bull. Jpn. Soc. Sci. Fish. 49, Hirayama, K., Nakamura, K., Fundamental studies on the physiology of rotifers in mass culture: V. Dry Chlorella powder as a food for rotifers. Aquaculture 8, Hirayama, K., Watanabe, K., Fundamental studies on physiology of rotifer for its mass culture: IV. Nutritional effect of yeast on population growth of rotifer. Bull. Jpn. Soc. Sci. Fish. 39, Komis, A., Candreva, P., Franicevic, V., Moreau, V., Van Ballaer, E., Leger, P., Sorgeloos, P., Successful application of a new combined culture and enrichment diet for the mass cultivation of the rotifer Brachionus plicatilis at a commercial hatchery scale in Monaco, Yugoslavia, France and Thailand. In: Lavens, P., Sorgeloos, P., Jaspers, E., Ollivier, F. Ž Eds.., Larvi 91 Fish and Crustacean Larviculture

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