Porter and Bailey: Using measurements of muscle cell nuclear RNA to assess larval condition of Gadus chalcogrammus 
339 
gies Corp. 2 ; Molecular Probes, Inc. 3 ). Syto RNASelect 
stain is known to also bind to DNA and emit an ex- 
tremely weak fluorescent signal; therefore, we exam- 
ined through the use of a staining study the effect that 
varying concentrations of Syto RNASelect stain had on 
the DAPI-DNA interaction to confirm that the stain 
concentration we used did not interfere with DAPI 
staining. Three concentrations of Syto RNASelect stain 
were mixed in DAPI and tested: 500 nM (concentra- 
tion recommended by Invitrogen), 1000 nM (maximum 
concentration recommended by Invitrogen), and 2000 
nM. The optimal preservative and stain concentration 
was defined as that which provided the highest nRNA 
fluorescence that did not affect DNA fluorescence as 
measured by flow cytometry. Each preservative was 
tested with the 3 concentrations of RNA stain previ- 
ously described and a DAPI-only negative control that 
was used to determine background RNA fluorescence. 
For preservative and stain testing, larvae were re- 
moved from a rearing tank, anesthetized in a 1% solu- 
tion of tricaine methanesulfonate (MS222), measured 
for their SL with an ocular micrometer, and placed in a 
1.5-mL microcentrifuge tube that was either filled with 
methanol or inserted into a gel-filled microcentrifuge 
tube holder that was frozen at -80°C. Tubes were kept 
in the holder until they were transferred to a -80°C 
freezer. The nuclei of 29 always-fed larvae were pooled 
to have enough material to simultaneously test 3 stain 
concentrations and the control group for each type of 
preservative. Six replicate aliquots were taken for each 
stain concentration and control. A specific volume of 
Syto RNASelect stain was then added to each aliquot 
to equal the desired final concentration. Larvae were 
processed and analyzed as described in the section 
Materials and methods, Flow cytometry. To determine 
optimal RNA stain concentration, analysis of variance 
(ANOVA), Tukey’s tests, and Dunnett’s tests were used 
to compare nRNA and DNA fluorescence associated 
with each RNA stain concentration and the control. 
SYSTAT, vers. 13 (Systat Software, Inc., Chicago), was 
used for all statistical testing. 
After the optimal preservation method and stain 
concentration were determined, nRNA staining was 
confirmed through comparison of untreated muscle 
cell nuclei to those nuclei treated with RNAse. Nuclei 
were treated in a mixture of DAPI and RNAse A (50 
pg mL _1 RNAse A, Sigma R6513, DNAse free) at room 
temperature for 25 min before adding the Syto RNAS- 
elect stain. The nuclei of 12 always-fed larvae were 
pooled to have enough nuclei to simultaneously test 
2 Life Technologies Corp. 2013. Life Technologies: Syto 
RNASelect green fluorescent cell stain — 5 mM solution in 
DMSO. Life Technologies Corp., Carlsbad CA. [Product 
information webpage; Available from http://products. invitro- 
gen. com/ivgn/product/S32703, accessed August 2013.] 
3 Molecular Probes, Inc. 2004. Syto RNASelect green fluo- 
rescent cell stain (S32703). Molecular Probes, Inc., Eugene, 
OR. [Product information document; Available from http:// 
tools, invitrogen.com/content/sfs/manuals/mp32703. pdf.] 
3 treatments: a positive control (DAPI+RNA stain), a 
negative control (DAPI only), and a treatment (RNAse 
A+DAPI+RNA stain). Four replicate aliquots were tak- 
en for each treatment, and ANOVA and Tukey’s tests 
were used to compare RNAse A-treated nuclei to both 
the positive and negative controls. 
After the staining protocol, an nRNA covariate to 
test in a larval condition model was determined with 
larvae from the always-fed and unfed treatments. Ten 
individuals were taken from each replicate tank of both 
treatments at first feeding (defined as the day when 
50% of the larvae had prey in their gut) and then at 
4, 8, 11, and 14 days after first feeding. Five potential 
nRNA covariates were investigated: geometric mean 
fluorescence of nuclei in the GO and G1 (G0/G1), S, and 
G2/M phases of the cell cycle pooled, mean fluorescence 
of each cell-cycle phase (G0/G1, S, and G2/M) sepa- 
rately, and the ratio of the number of S-phase nuclei 
to the number of Gl-phase nuclei with high RNA con- 
tent (hereafter, this ratio will be referred to as RSG1; 
see Materials and methods, Flow cytometry section). 
RSG1 is a measure of potential cell division based 
on progression of nuclei from the G1 to S phase. An 
unhealthy larva would not be expected to grow or it 
would grow more slowly than a healthy individual. 
Therefore, an unhealthy larva would have fewer di- 
viding cells and potentially less S-phase nuclei, and 
its ratio of S-phase nuclei to Gl-phase nuclei with 
high RNA content would be smaller than the ratio for 
healthy individuals. Nuclear RNA fluorescence between 
treatments was compared with the 2-sample /-test. The 
Mann-Whitney U test was used to compare RSG1 be- 
tween treatments. 
2010 experiment: nRNA, temperature, and condition 
Five larvae were sampled from each always-fed tank 
on 3 separate days after feeding began. A degree-day 
model (degree-day=temperature*fish age in days) was 
used so that larvae were sampled on the day of simi- 
lar developmental stage after first feeding, not on the 
same calendar day after first feeding (Table 1). Eight 
larvae from the unfed treatment were sampled from 
each tank daily, beginning at first feeding and ending 
at yolk exhaustion. All larvae were frozen at -80°C. 
Geometric mean nRNA fluorescence for nuclei in the 
G0/G1, S, and G2/M phases of the cell cycle pooled, 
and RSG1 for each larva was determined (see Materi- 
als and methods, Flow cytometry section). ANOVA and 
Tukey’s tests were used to examine differences in fluo- 
rescence and RSG1 between feeding treatments and 
among temperatures. 
2011 experiment: effect of freezing and storage on nRNA 
and DNA 
After 4 days of feeding, 20 control larvae (not frozen) 
were processed directly from the rearing tanks (10 
from each replicate tank) and analyzed by flow cytome- 
