Improvements in the fluorimetric determination of the RNA and DNA content of individual marine fish larvae

Abstract

The RNA/DNA ratio is a useful indicator of the nutritional condition of fish larvae. The presented analytical procedure is an improvement of Clemmesen's (Meeresforschung 32: 134-143, 1988) methodology which involves purification of fish larvae tissue homogenates and subsequent fluorescence-photometric measurements using specific nucleic acid dyes. The modifications concern the homogenization and nucleic acid extraction procedures. A 'shaking mill' was compared to a potter Elvehjem microhomogenizer and a reduction in the washing and purification steps was achieved. Treatment of samples with ribonuclease A and subsequent fluorescence measurement using ethidium bromide was given preference compared to the DNA-bisbenzimidazole determinations due to problems arising from high self-fluorescence of the samples and the influence of 'quenching' substances disturbing the DNA-bisbenzmidazole determinations. Different RNase concentrations and their influences on RNA and DNA were checked. Recovery rates of standard RNA and DNA 'spikes' were determined. Fish larvae samples were analysed with the previous and the improved modified procedure and a correction factor to compare results measured with the 2 procedures was calculated. With the presented method the physiological condition of individual larvae and the amount of variabhty can be determined.

Larval condition and growth of <i>Sardinella brasiliensis </i> (Steindachner, 1879): preliminary results from laboratory studies

“…RNA/DNA ratios and tryptic enzyme activity were determined according to the analytical technique described by Clemmesen (1993) and Ueberschär (1995), on entire individual fish larvae, after measurement of total length and checking for possible damage. Damaged larvae were excluded from all analyses.…”

Impacts of copepods on marine seston, and resulting effects on Calanus finmarchicus RNA:DNA ratios in mesocosm experiments

“…The nutritional status of C. finmarchicus was determined with RNA:DNA measurements, and quantified fluorimetrically with ethidium bromide and ribonuclease A using a modification of the method of Clemmesen (1993). The response of the RNA:DNA ratios to changes in food availability and quality are dependent on the developmental stages of the copepods (Wagner et al 1998(Wagner et al , 2001.…”

“…A variability test was performed in order to define the variability between aliquots of one homogenate. This test should be used as an indicator of whether the method by Clemmesen (1993) was applicable to copepodite stages. In this test we measured 15 parallel aliquots from the same homogenate using a pooled sample of mixed stages of C. finmarchicus copepodites.…”

“…The ratio of the two (RNA:DNA) and the RNA content alone have proven to be a useful tool in determination of nutritional status of various zooplankton organisms (e.g. Ba˚mstedt and Skjoldal 1980;Ba˚mstedt 1983;Saiz et al 1998;Wagner et al 1998;Vrede et al 2002) and fish larvae and juveniles (Clemmesen 1993(Clemmesen , 1994(Clemmesen , 1996Clemmesen et al 2003). Typically, an increased RNA:DNA ratio indicates a higher growth potential.…”

Quantifying RNA and DNA in planktonic organisms with SYBR Green II and nucleases. Part B. Quantification in natural samples

“…However, we investigated other ways to improve the nucleic acid extraction. First, we tested the procedure described by Clemmesen (1993) that had been adapted from Molecular Biology protocols for obtaining purified nucleic acids extracts from fish larvae. The methodology used ethidium bromide as the fluorochrome, and SDS, EDTA, chloroform, phenol and isoamylalcohol that were subsequently removed by washing.…”