Materials and methods Directory UMM :Data Elmu:jurnal:A:Aquaculture:Vol182.Issue3-4.Feb2000:

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 7 y1 Ž . order of 10 cells ml Nicolas and Joubert, 1986; Nicolas et al., 1989 . Rapid successional processes in the microbiota have been observed during the culture of Ž . rotifers Maeda and Hino, 1991 , and changes in the microbial ecosystem have been Ž . postulated as the cause of the collapse of rotifer cultures Hino, 1993 . The effects of bacteria on rotifer cultures are strain specific, as demonstrated by the Ž . Ž . Ž . findings of Yasuda and Taga 1980 , Yu et al. 1988 , Gatesoupe et al. 1989 , Maeda Ž . Ž . and Hino 1991 and Hagiwara et al. 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 Ž . Ž demonstrated by supply of vitamin B Yu et al., 1988 or inorganic nutrients Hessen 12 . and Andersen, 1990 . In contrast, production of bacterial toxins has been found to Ž . reduce rotifer survival rates 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 Ž . uneaten food cause deterioration of water quality Lubzens, 1987 , which may affect Ž . rotifer growth and reproduction Tanaka, 1991 . In fact, rotifer densities have been Ž reported to decrease with increases of either un-ionized ammonia Yu and Hirayama, . Ž . 1986 or nitrite Lubzens, 1987 . Removal of waste products from rotifer cultures has Ž . been reported to extend the harvest period 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 Ž . enhancement. The screening of microbes was carried out under an artificial diet AD and different algae feeding regimes.

2. Materials and methods

2.1. Preparation of rotifers Ž . Cysts of the rotifer B. plicatilis Muller 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 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 Ž by dissolving 8 g of microfine Spirulina 8–10 by 20 mm; Aurum Aquaculture, . Ž Washington 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 Ž . cooling, filter-sterilized cyanocobalamine vitamin B was added at a concentration of 12 120 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 Ž . cultures fed either this diet or the diet developed by Gatesoupe and Luquet 1981 . Ž . Axenic Isochrysis galbana clone C-ISO, CCMP463 and Chlorella minutissima Ž . clone 2341 used in Experiments 2 and 4 were obtained from the National Center for Ž . Culture of Marine Phytoplankton Maine and The Culture Collection of Algae at The Ž University of Texas at Austin, respectively. Algae were grown in fr2 media 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 Ž . cultures 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 Ž . centrifugation 10,000 = g for 10 min and resuspended in FASW. Photosynthetic Ž . Ž bacteria PH were cultured on Rhodospirillum ATTC Medium 1308 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 cm y2 . 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 experiments to corroborate initial rotifer densities. Culture experiments were initiated by the addition of food and the different bacteria. Ž . Control cultures consisted of: 1 cultures fed the same diets but maintained bacteria- Ž . free, 2 cultures fed the same diets and inoculated with bacteria present in 100 ml Ž . Ž . samples of freshly collected seawater filtered through a 1-mm screen SW , 3 starved Ž . cultures in Experiment 3, and 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 Ž . ml , as recommended by Hirayama and Funamoto 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 = 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 Ž . absorbance 600 nm and bacteria numbers; the latter value was determined by direct Ž . count using DAPI staining techniques 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 Ž included nine commercial products Acc, A2, A5, A6, A1100, A1200, F9, Mplus and . Ž Sy 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 Ž tested along with nine inoculants 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 Ž and B1 were evaluated for a second time, and five strains 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 1000–1300 lx, under a 12 h light–12 h dark photoperiod and at 258C. P.A. Douillet r Aquaculture 182 2000 249 – 260 253 Table 1 Bacterial additives or bacteria strains tested in four experiments with B. plicatilis Ž . Ž . Commercial name code Bacterial composition Origin presentation Ž . Ž . AB-1 AB1 Bacillus subtilis Advanced Microbial Systems, MN liquid Ž . Ž . Aqua-Bacta-Aid N2 ABA B. subtilis, B. amyloliquefaciens, B. licheniformis, Water Quality Science International, MO liquid Ž . Nitrosococcus two strains Ž . Ž . Accelobac Acc B. subtilis, B. cereus, B. megaterium, B. licheniformis, American Biosystems, VA dry B. polymyxa, Rumenococcus albus, Aspergillus oryzae Ž . Ž . Alken Clear-Flo 1100 A1100 Nitrosomonas sp., Nitrobacter sp. Alken Murray, NY liquid Ž . Ž . Ž . Alken Clear-Flo 1200 A1200 B. subtilis two strains , Pseudomonas aeruginosa, P. stutzeri, Alken Murray, NY liquid P. fluorescens, Escherichia hermanii, Nitrosomonas sp., Nitrobacter sp. Ž . Ž . Ž . A2 B. subtilis three strains , B. licheniformis, Alken Murray, NY dry P. aeruginosa, P. stutzeri Ž . Ž . A5 P. aeruginosa, P. stutzeri, P. putida Alken Murray, NY dry Ž . Ž . A6 B. subtilis, B. licheniformis, B. polymixa Alken Murray, NY dry Ž . Ž . Fritz-Zyme a9 F9 Nitrosomonas sp., Nitrobacter sp. Fritz, TX liquid Ž . Ž . Ž . Microbials Plus Mplus Streptococcus faecium now E. faecium Medipharm, IA dry Ž . Ž . Sy P. aeruginosa 2203 Sybron Chemicals, VA dry Ž . Ž . A.sp. Alteromonas sp. Seawater isolates cultured at UTMSI Ž . Ž . Ž . B1, B2, B3, B4, B5 Five unidentified marine Gram y rods Seawater isolates cultured at UTMSI Ž . Ž . E.f E. faecium P. Bogaert, University of Gent, Belgium 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 AM University, Ž . TX cultured at UTMSI 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 Ž . shaking and five samples 100–1000 ml were withdrawn for estimation of rotifer densities. Rotifer population GR for each culture tube was calculated as: GR s ln final density y ln initial density rculture period in days 4 days Ž . Ž . Ž . 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 Ž . one-way ANOVA, followed by Tukey’s range test T-method, Sokal and Rohlf, 1981 to determine differences between bacterial treatments at the 0.05 level of probability. Ž U . Ž Coefficients of variation V between replicates under four treatments axenic, SW, . B3 and Alteromonas sp. were calculated in each experiment as in Sokal and Rohlf Ž . 1981 : U V s 100 sd rmean 1 q 1r4 n Ž . Ž . where sd is the standard deviation, mean is the average GR and n is the number of Ž . U replicates 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. Ž . treatments using the t-test for paired comparisons Sokal and Rohlf, 1981 . All tests Ž . were performed with the computer program Statistix 2 NH Analytical Software .

3. Results