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 ) 1692 Houghton Ct North, Dunwoody, GA 30338, USA. Tel.: q1-770-671-9393; E-mail: philippe± [email protected] 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.