Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology
GABA, 5-HT and amino acids in the rotifers Brachionus plicatilis and Brachionus rotundiformis
Introduction
Rotifers are microscopic metazoans with a simple nervous system consisting of a single large cerebral ganglion called the ‘brain’ and less than 300 nerve cells (Hyman, 1951, Clement et al., 1983). Nogrady and his coworkers reported the presence of acetylcholinerase and choline acetyltransferase (Nogrady and Alai, 1983, Nogrady and Keshmirian, 1986) and catecholamines and adrenergic receptors in rotifers (Keshmirian and Nogrady, 1988) but much more work is necessary before general conclusions can be made about neurotransmission in rotifers. The presence of γ-aminobutyric acid (GABA) and 5-hydroxytryptamine (5-HT, serotonin), which are important neurotransmitters and hormones in vertebrate and invertebrates, has not yet been confirmed in rotifers. In vertebrates, GABA and 5-HT are also present in non-nervous structures (Okada, 1986, Peroutka, 1988). They likewise have been reported in the nervous system of planarians and parasitic flatworms (Gustaffson et al., 1985, Fairweather et al., 1987, Eriksson and Panula, 1994, Eriksson et al., 1995). Since these organisms are phylogenetically close to rotifers, it seems plausible that GABA and 5-HT are also present in rotifers.
In our previous study, GABA and 5-HT were shown to increase reproduction of the rotifer Brachionus plicatilis (Gallardo et al., 1997). Further experiments showed that GABA (50 mg/l) increased rotifer asexual reproduction in stressful environmental conditions such as low food and high free ammonia levels (Gallardo et al., 1999) as in mass cultures (Gallardo et al., 2000b). In contrast, 5-HT enhanced sexual (mictic) reproduction (Gallardo et al., 2000a). To understand the mechanism of action of these chemicals, we wanted to know if they are present in rotifers. In this study, due to the difficulty of extracting protein from the nerve cells of this microscopic organism, we extracted and analyzed the free amino acids in the whole-body rotifer samples. Dot blot immunoasssay and high-performance liquid chromatography (HPLC) were employed to detect the presence and determine the concentration of GABA and 5-HT in rotifers.
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Rotifer samples
The rotifers B. plicatilis (NH3L and Kamiura strains) and Brachionus rotundiformis (Langkawi strain) were mass cultured separately on the microalga Nannochloropsis oculata. Prior to harvest, rotifers were starved for at least 24 h to remove the ingested algal food that might interfere with the results. Rotifers were harvested using a plankton net (45-μm mesh size) and washed with a physiological salt solution (1.35% NaCl) according to the protocol of Hara et al. (1984).
Preparation of extract
Rotifer sample (5 g) were
Dot blot immunoassay
Antibodies reacted with all three rotifer samples and standards for GABA and 5-HT immunoassays (Fig. 1). The reaction with GABA conjugated to BSA was clearer than GABA only. In the amino acid standard solution without GABA or 5-HT, and in TBS controls, no reaction appeared.
HPLC
Retention times for GABA and 5-HT were 20 and 70 min, respectively, in the amino acid standard and in all three rotifer samples. The concentrations of GABA and 5-HT were 71–188 and 12–64 pmol/mg, respectively (Table 1). GABA
Discussion
The presence of GABA and 5-HT in rotifers was confirmed by dot blot immunoassay and HPLC results. Antibody reactions with GABA in rotifer samples were clearly visible indicating their presence in rotifers. The possibility of obtaining dot blots that may be due to the reaction of rotifer endogenous peroxidase with the secondary antibody (anti-IgG conjugated to horseradish peroxidase) was eliminated by boiling the samples to inactivate the endogenous peroxidase. Dot blots of GABA conjugated to
Acknowledgements
Amino acid analysis by HPLC was done at Waters’ laboratory facilities in Osaka, Japan. This study was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 10660187) and Integrated Research Program for Effects of Endocrine Disrupters on Agriculture, Forestry, and Fisheries and their Action Mechanisms on Domestic Animals and Fishes (ED-99-II-3-1).
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