Modification and comparison of 

 two fluorometric techniques for 

 determining nucleic acid contents 

 of fish larvae 



Michael F. Canino 



Alaska Fisheries Science Center 



National Marine Fisheries Service, NOAA 



7600 Sand Point Way NE. Seattle, Washington 981 1 5-0070 



Elaine M. Caldarone 



Northeast Fisheries Science Center 

 National Marine Fisheries Service, NOAA 

 Narragansett, Rhode Island 02882-1 199 



The ribonucleic acid (RNA) content 

 and the ratio of RNA to deoxyribo- 

 nucleic acid (DNA) have proven to 

 be reliable indices of the nutritional 

 condition of larval fish (Buckley, 

 1979, 1980, 1981, 1984; Wright and 

 Martin, 1985; Buckley and Lough, 

 1987; Clemmesen 1987, 1988; Rob- 

 inson and Ware, 1988; Canino et al., 

 1991; Richard et al., 1991; Canino, 

 1994). Cellular RNA content is cor- 

 related with the rate of protein syn- 

 thesis. DNA content, which re- 

 mains relatively constant in so- 

 matic tissues, may be used as an 

 index of cell number (Bulow, 1987). 

 The RNA/DNA ratio, therefore, re- 

 flects the protein synthesizing ca- 

 pability of larval fish and has been 

 used for estimating recent in situ 

 protein growth (see review by 

 Bulow, 1987; Robinson and Ware, 

 1988; Hovenkamp, 1990; Hoven- 

 kamp and Witte, 1991). 



Initial methods for determining 

 RNA and DNA concentrations in 

 tissue homogenates, based upon 

 ultraviolet light absorption (Munro 

 and Fleck, 1966; Buckley, 1979), 

 were limited by sample size, requir- 

 ing about 800 /ig dry weight of tis- 

 sue for a single analysis. The re- 

 quirement of pooled samples offish 

 larvae precluded quantitation of 



variability among individuals. Re- 

 cent development of highly sensi- 

 tive fluorometric techniques for di- 

 rect measurement of nucleic acid 

 contents of marine phytoplankton 

 (Berdalet and Dortch, 1991; Mordy 

 and Carlson, 1991), bacteria (Mordy 

 and Carlson, 1991), and individual 

 fish larvae (Clemmesen, 1988, 

 1993; Caldarone and Buckley, 1991; 

 Theilacker and Shen, 1993) now 

 provides a greater choice of meth- 

 ods. Several protocols are based upon 

 the fluorescence of the dye ethidium 

 bromide (EB), when bound to 

 nucleic acids. Fluorescence of the 

 nucleic acid-fluorochrome complex 

 is measured before and after diges- 

 tion of RNA by RNase (Karsten and 

 Wollenberger, 1972, 1977; Robinson 

 and Ware, 1988; Clemmesen, 1993), 

 or after sequential additions of 

 RNase and DNase (Bentle et al., 

 1981). Total fluorescence is then 

 partitioned into DNA and RNA com- 

 ponents and nucleic acid concentra- 

 tions are calculated indirectly by dif- 

 ference after enzymatic degradation. 

 A two-dye fluorometric method 

 for nucleic acid analysis of indi- 

 vidual fish larvae (Clemmesen, 

 1988) utilized EB to measure total 

 sample fluorescence and the DNA- 

 specific dye, Hoechst dye H33258 



(Hoechst), to measure DNA content 

 directly. A recent modification of 

 this method to a single-dye proce- 

 dure yields higher DNA estimates 

 and RNA estimates comparable to 

 the previous two-dye method 

 (Clemmesen, 1993). Both protocols 

 require extraction and purification 

 of crude homogenates before analy- 

 sis. While substances that interfere 

 with sample fluorescence may be 

 reduced or eliminated, the washing 

 and extraction steps of both meth- 

 ods restrict the number of samples 

 that can be processed in a day. 



Caldarone and Buckley ( 1991) de- 

 veloped a two-dye method for deter- 

 mining nucleic acid contents that 

 was coupled with an automated flow- 

 injection analysis (FLA) system in 

 which EB is used to estimate total 

 nucleic acid content and Hoechst is 

 used to measure DNA. This method 

 has the advantage of combining high 

 sensitivity and sample throughput 

 rate with a simple extraction proce- 

 dure. Unfortunately, an FIA system 

 may be too costly for many laborato- 

 ries. In this paper, we present an 

 adaptation of the FIA method for con- 

 ventional static fluorometric analy- 

 sis (CFA) and compare results using 

 the two procedures. 



Methods 



Fish larvae and juveniles from labo- 

 ratory culture and field samples from 

 six species — Atlantic cod, Gadus 

 morhua; inland silverside, Menidia 

 beryllina; haddock, Melanogrammus 

 aeglefinus; tautog, Tautoga onitis; 

 winter flounder, Pleuronectes 

 americanus; and walleye pollock, 

 Theragra chalcogramma — were 

 chosen to provide different species, 

 ages, and nutritional histories for 

 comparison (Table 1). Fish homo- 

 genates, except those of walleye 

 pollock, were prepared by homog- 

 enizing between 1 and 12 previ- 

 ously frozen individuals with deion- 



Manuscript accepted 23 May 1994. 

 Fishery Bulletin 93;158-165 (1995). 



158 



