Body condition indices and morphometric measurements (length and weight) are relatively insensitive to and unreliable for describing the physiological or nutritional status of an or-ganism (Fathallah et al., 2010). One can assess the physiologi-cal and nutritional status of bivalves by examining a variety of biochemical parameters or indices of instantaneous growth, usually expressed as ratios of nucleic acids (RNA/DNA). Nucleic acids play a major role in growth and development. While DNA content is stable under changing environmental conditions, and is thus used as an indicator of biomass and cell number (Dortch et al., 1983), RNA is directly involved in protein biosynthesis and growth is dependent on the amount of available RNA (Clemmesen, 1987). Food availability tends to be the main factor affecting growth and thus the RNA/DNA ratio in bivalves. Several studies have reported that the ratio of RNA/DNA is a reliable and useful index of nutritional con-dition and environmental stress in mollusks (Joyner-Matos et al., 2007; Heilmayer et al., 2008), crustaceans (Anger and Hirche, 1990; Chicharo et al., 2007), and fish. Furthermore, the RNA/DNA ratio is correlated with food quality (Naimo et al., 2000; Vrede et al., 2002) and nutrition level (Whyte et al., 1990), and it decreases rapidly during periods of starvation (Bracho et al., 2000; Chicharo et al., 2001).
Freshwater snails of the genera
The goals of this study were to gain information regarding suitable rearing conditions for a valuable aquaculture species,
Short-term rearing experiments for the melania snail,
A starvation experiment was performed in parallel with the rearing experiment to investigate the effect of fed vs. starved snails. In total, 200 snails were randomly transferred into each plastic tank (100 L) with filtered freshwater (10 ㎛) from each rearing tank (maintained with artificial food or with the natural food organism assemblages from a pond). Each treat-ment consisted of two replicate tanks. All experimental condi-tions were maintained equally. Sampling from the starvation treatments was performed daily each morning (10:00 h) for 25 days. Five snails were taken from each aquarium and their wet weight determined. The snail samples were stored at -80℃ for biochemical analysis.
For biochemical analysis, muscle tissue was dissected from the thawed snails, and the total nucleic acids were extracted from each sample individually by homogenization with a tis-sue grinder. During homogenization, the glass tube was kept cold by immersion in a plastic beaker with wet-ice. The SPRI-TE Nucleic Acid Extractor (Beckman Coulter, Fullerton, CA, USA) system was used for total nucleic acid extraction following the manufacturer’s instructions ( http://www.beck-mangenomics.com/documents/products/SPRI_TE_OpMan.pdf ). This is a fully automated system capable of extracting nucleic acids from a variety of sample types including plas-ma, serum, viral transport media, formalin-fixed paraffin-embedded tissue, and entire blood samples. The instrument is equipped with magnetic filtration technology. Reagent car-tridges contain a lysis solution, a binding reagent, a washing buffer, and an eluation buffer.
Fluorometric assays for the quantification of nucleic acids were based on the method described by Schmidt and Ernst (1995). Two aliquots (RNA and DNA) of the nucleic acid so-lution were processed in parallel. To the RNA aliquot (5 μL), 5 μL DNase solution (DNase I, RNase-free, 10 μg/μL; Roche, Darmstadt, Germany) and 40 μL DNase buffer (40 mM Tris-HCl, pH 7.9, 19 mM NaCl, 6 mM MgCl2, 10 mM CaCl2) were added. To the DNA aliquot (5 μL), 2 μL RNase buffer (Roche), 5 μL RNase solution (RNase, DNase -free, 0.5 μg/μL; Roche) and 33 μL nuclease-free water were added. After incubation for 1 h at 37℃, the reactions were terminated by cooling the solutions on ice. The concentrations of both RNA and DNA were estimated using a NanoVue spectrophotometer (GE Healthcare, Piscataway, NJ, USA).
Sample means were compared using an unpaired indepen-dent sample t-test between the two experimental groups. Dif-ferences were considered significant at
[Fig. 1.] Change of the RNA/DNA content of melania snail during a 2 day rearing experiment. Values are given as means ± SD of 5 snails. Significant differences between the experimental and natural group are indicated with * for DNA or ** for RNA (P < 0.05).
[Fig. 2.] Change of the RNA/DNA ratios of melania snail during a 3 day rearing experiment. Values are given as means ± SD of 5 snails. Significant differences between the experimental and natural group are indicated with * (P < 0.05).
The average weight of the snails in the artificial group was 509 ± 44 mg. The DNA contents of both groups were lower than the RNA contents and showed small variations between 75 μg and 150 μg (Fig. 1). The DNA contents of the natur-al group were significantly higher than those of the artificial group at most time points except 22:00 h (
The RNA contents of both groups ranged between 200 μg
[Fig. 3.] Change of mean body weight of melania snail during a starvation experiment. Values are given as means ± SD of 5 snails. Significant differences between the experimental and natural group are indicated with * (P < 0.05).
and 300 μg (Fig. 1). Total RNA content differed significantly among most time points, with the natural group exhibiting a higher total RNA content than the artificial group (
The RNA and DNA levels of the freshwater snails calculat-ed over the 3-day trial were 1.94 and 3.5, respectively (Fig. 2). In contrast to the total nucleic acid contents, the RNA/DNA ratio of the artificial group was significantly higher than that of the natural group at all time points except 22:00 h (
The body weights of freshwater snails showed minor variations in both of the starved groups (Fig. 3). Overall, snails in the natural group were heavier than those in the artificial group (
The RNA/DNA ratios of the freshwater snails in both groups dramatically decreased from days 3 to 5, after which that of the starved group remained constant at the end of the experiment (Fig. 5). Significant differences in the RNA/DNA ratios between the artificial and natural group were detected from the 3rd day to the 6th day of starvation.
[Fig. 4.] Change of the RNA and DNA content of melania snail during a starvation experiment for 25 days. Values are given as means ± SD of 5 snails. Significant differences between the experimental and natural group are indicated with * for DNA or ** for RNA (P < 0.05).
Growth measurements based on shell size and body weight are technically simple and inexpensive, but require costly long-term growth experiments to detect change (Wo et al., 1999). Growth is directly linked to protein synthesis, which is related to the amount of RNA in cells. Given that the amount of DNA in a cell remains relatively constant, and can thus serve as an index of cell number or biomass (Clemmesen, 1994), the RNA/DNA ratio should provide a good indication of the rate of protein synthesis and therefore the growth of organisms of different sizes.
In our short-term rearing experiments, the DNA and RNA contents in the natural group were higher than those in the artificial group, probably because the snails in the natural group were larger (heavier) than those in the artificial group. We also found that the RNA/DNA ratio of the artificial group was significantly higher than that of the natural group at most time points. Bulow (1987) suggested that for the golden shiner (
For the artificially fed group, the RNA/DNA ratio was high-est during the day, which may have been a result of the as-signed feeding schedule whereby food was provided at 09:00 h. The subsequent increase in the RNA/DNA ratio was likely due to a dramatic increase in the RNA content while the DNA content remained constant, indicating that the culture condi-tions were ideal and continuous growth was occurring. These results highlight the importance of suitable feeding conditions as well as of including an additional feeding at night.
In contrast to the artificially fed group, the RNA/DNA ratio of the naturally fed group was highest at night. Chicharo et al. (2001) reported similar results in two mollusks (
The goal of this study was to gain an understanding of the effect of starvation and dietary source on the growth of the me-lania snail by measuring the quantitative change in the ratio of RNA to DNA. Starvation is one of the most important factors affecting the recruitment of larvae (Tripathi and Verma, 2003). Negative growth during starvation may be the result of snails utilizing their body reserve to fuel vital processes at times of energy shortage. We observed decreases in the RNA content and no change in the DNA content throughout our study peri-od. Given that cellular RNA is essential for the biosynthesis of proteins, the amount of bulk RNA increases rapidly in grow-ing tissues while the amount of cellular DNA remains fairly constant. Clemmesen (1987) and Raae et al. (1988) reported similar results in fish larvae. They explained the higher DNA content in starved larvae as residual cellular energy being used for rapid unscheduled DNA synthesis due to the lack of suf-ficient nutrition. Herein, the RNA/DNA ratios ranged from 0.7 to 1.0 under starved conditions. Although the validity of direct comparisons among results of different experimental setups has been argued (Caldarone and Buckley 1991), several stud-ies have reported that RNA/DNA ratios less than 2.0 indicate starvation conditions in fish and mollusks (Sika and Layman, 1995; Chicharo et al., 2001; Vidal et al., 2006).
The RNA/DNA ratio of the artificial group decreased sub-stantially 3 days after starvation. This result is consistent with those of a previous report that demonstrated the validity of using the RNA/DNA ratio as an indicator of nutritional condi-tion in the Japanese turban shell (
In conclusion, we demonstrated that the RNA/DNA ratio can serve as a useful, fast, and sensitive indicator of starvation stress. Specifically, we showed that the RNA/DNA ratio can be used as an index of the growth and the starvation state of freshwater snails. Further studies are needed to apply these laboratory results to natural populations. Such studies could assess the growth and the nutritional status of the most valu-able snail species in Korean freshwater aquaculture.