Disinfection of rotifer cysts leading to bacteria-free populations
Introduction
Rotifers of the genus Brachionus have become important research tools for several reasons. First, they are cyclical parthenogens (Birky and Gilbert, 1971) and, as a result of asexual reproduction, clones of genetically identical individuals can be produced from single females. Second, sexual reproduction leads to the production of thick-walled cysts that can be stored for years and then hatched to build populations as needed. Third, they are easy to collect and culture. Fourth, their lifespan is short, and bioassays can be conducted relatively quickly (Snell and Persoone, 1989). Fifth, they are valuable and in many cases indispensable food organisms for the culture of a large variety of marine finfish and crustacean larvae (Watanabe et al., 1983, Lubzens, 1987). However, rotifers carry a large bacterial load in their digestive tract (Nicolas et al., 1989, Skjermo and Vadstein, 1993), and bacteria have been found to be a major cause of variation in rotifer culture systems (Maeda and Hino, 1991). The development of axenic rotifers for research purposes has long been attempted to eliminate the interaction with microbes (Dougherty, 1963).
The relevance of disinfecting rotifers for different purposes is demonstrated by the abundant literature on methods to treat embryos of amictic females (Gilbert, 1970, Droop, 1976), cysts (Nathan and Laderman, 1959, Dougherty et al., 1960, Pourriot, 1965, Hirayama et al., 1979, Hagiwara et al., 1994) or adults (Plasota et al., 1980, Gatesoupe, 1982, Koutsikopoulos, 1985, Rodriguez et al., 1991, Tanasomwang and Muroga, 1992). The disinfecting compounds used either independently or in combination are sodium hypochlorite and diverse antibiotics. The use of the latter compounds induces microbial resistance, making them unreliable (Kapetanaki et al., 1995). Sodium hypochlorite has been reported effective for the disinfection of rotifer cysts; however, the concentrations and exposure times used range widely (e.g., Nathan and Laderman (50–100 ppm for 1 min); Hagiwara et al. (0.5–0.25 ppm for 60 and 30 min, respectively)). An evaluation of the effects of concentration and time of exposure of cysts to sodium hypochlorite was undertaken to develop a reliable method to produce bacteria-free rotifer populations.
Section snippets
Disinfection protocol
Cysts from the rotifer Brachionus plicatilis Müller (formerly called L-type B. plicatilis) were purchased from Aquaculture Supply, Florida. Cysts of B. rotundiformis Tschugunoff (formerly called S-type B. plicatilis) were either purchased from Aquaculture Supply, or kindly provided by Dr. Terry W. Snell (Georgia Institute of Technology, Atlanta, GA). Approximately 2 mg of cysts were placed in each of 18 2-ml Nalgene cryovials. Commercial-grade sodium hypochlorite (5.25% NaOCl) was used for this
Results
Hatching rates were significantly affected by the different concentrations of sodium hypochlorite (multi-way ANOVA, P<0.001) and by the time of exposure to the disinfectant (multi-way ANOVA, P<0.001), but were not different between experimental runs (multi-way ANOVA, P=0.11). Data of the three experimental runs were combined, resulting in a data set of three staggered replicates per treatment (Fig. 1). There was a slight improvement in hatching rates, compared with untreated controls, by
Discussion
The method developed to obtain axenic populations proved reliable in successive experiments with two species of rotifers. No adverse long-term effects on rotifer growth rate resulted from the disinfection treatment in 20 independent experiments (Douillet, unpubl.). The concentrations of sodium hypochlorite determined in this study as effective for disinfection of rotifer cysts were much higher than those reported in the literature (Nathan and Laderman, 1959, Hagiwara et al., 1994). Similarly,
Acknowledgements
This work was supported by Grant # NA56RG0388 from the National Oceanic and Atmospheric Administration through the National Sea Grant College Program. The views expressed therein are those of the author and do not necessarily reflect the views of NOAA or any of its sub-agencies. I am indebted to Dr. R. Benner for the use his epifluorescence microscope. I am grateful to Dr. T.W. Snell at the Georgia Institute of Technology, for supplying rotifer cysts and commenting on the manuscript.
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Growth performance of the very small rotifer Proales similis is more dependent on proliferating bacterial community than the bigger rotifer Brachionus rotundiformis
2017, AquacultureCitation Excerpt :Some Pseudomonas species, for instance, synthesize vitamin B12 which can be a limiting factor for rotifers under culture conditions (Yu et al., 1988). The performance of a rotifer culture in bacteria-free conditions was worse than in a culture supplemented with a microbial community (MC) (Douillet, 1998; Rombaut et al., 1999; Tinh et al., 2006) indicating that bacteria are important to rotifers. Although bacteria can hence be considered as an alternative feed for rotifers, they are considered to be a nuisance for a successful culture of marine fish larvae, and several methods have been developed to limit the transfer of bacteria from rotifer cultures to larval fish tanks (Makridis et al., 2000; Muroga and Yasunobu, 1987; Munro et al., 1993, 1994; Rombaut et al., 2003, Qi et al., 2009; Skjermo et al., 1997, Skjermo and Vadstein, 1999; Tanasomwang and Muroga, 1989; Verschuere et al., 1997 and Vadstein et al., 1993).
Rotifers, Artemia and copepods as live feeds for fish larvae in aquaculture
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2011, AquacultureCitation Excerpt :Even if the large majority of bacteria present in rotifers are not pathogenic, Dhert (1996) showed that fish larvae could be affected by the accumulation of those non-pathogenic bacteria. Reducing bacterial load has some advantages, but several studies (Gatesoupe et al., 1989; Yu et al., 1989; Hagiwara et al., 1994; Harzevili et al., 1997; Douillet, 1998; Hirata et al., 1998; Gatesoupe, 1999; Munro et al., 1999; Rombaut et al., 1999; Douillet, 2000; Verschuere et al., 2000) have demonstrated that the microbial community present in cultures contributes to the success of live feed culture. Our data suggest that the enrichment phase could increase the bacterial load in both rotifers (Table 4) and Artemia (Table 5).
Rotifer (Brachionus "Cayman") culture performance improvement through l-carnitine addition is not related to fatty acid metabolism
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