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Nuclear RNA fluorescence 
Figure 2 
Scatter plot of nuclear RNA (log scale) and DNA fluorescence (linear scale) of muscle 
cell nuclei in the GO and G1 (G0/G1), S, and G2 and M (G2/M) phases of the cell cycle 
for a laboratory-reared larva of Walleye Pollock (Gadus chalcogrammus ) sampled 
in 2009. The rectangles outlined in black indicate the gates used to calculate geo- 
metric mean values of nuclear RNA and DNA fluorescence. Fluorescence values are 
arbitrary units. Phases of the cell cycle: Gl=gap 1 or cell growth before cell division, 
G0=resting state from Gl, S=DNA synthesis, G2=gap 2 or cell growth before mitosis, 
and M=mitosis. 
Model testing 
Data from all 3 years were pooled for model testing. 
One-third of the data were randomly removed from 
that data set and used for independent cross-valida- 
tion testing. The remaining data were used to formu- 
late discriminant analysis models similar to the model 
described in Porter and Bailey (2011) to classify larvae 
as healthy (feeding and growing) or unhealthy (starv- 
ing). Models had SL, temperature, fraction of cells in 
the S phase, and fraction of cells in the G2/M phases 
as covariates, and were tested with and without the 
nRNA covariate included. The arcsin 4 % transforma- 
tion was used to normalize the fraction of cells in the S 
phase, fraction of cells in the G2/M phases, and RSG1. 
Models were compared on the basis of the accuracy of 
their classification of the cross-validation data set and 
Akaike’s information criterion values (Burnham and 
Anderson, 2002). 
Results 
Nuclear RNA staining protocol 
Flow-cytometric cell-cycle analysis showed that the tis- 
sue from frozen samples had a small amount of debris 
and distinct peaks in G0/G1 and G2/M phases. Fixing 
larvae in methanol for either a short period of time or 
long-term storage did not work as well; samples con- 
tained a large amount of cellular debris and aggregates, 
and the nuclei did not disassociate from the tissue eas- 
ily. Samples from larvae preserved long term in metha- 
nol contained too much debris to be usable for further 
analysis. Debris was not as great in the samples that 
were frozen and then received a short-term methanol 
treatment as it was in the samples stored long term 
in methanol. The latter samples were usable, but they 
were not as clean as the samples that were frozen and 
not treated with methanol. Comparison of the RNA 
fluorescence between frozen tissues and tissues that 
were frozen and then treated with methanol indicated 
that short-term methanol preservation did not improve 
RNA staining; therefore, frozen tissue (stored at -80°C) 
worked best for preservation of muscle tissue from lar- 
vae of Walleye Pollock for nRNA and DNA staining and 
was used for all further tests and experiments. 
Syto RNASelect stain concentration affected both 
nRNA and DNA fluorescence. The fluorescence of 
nRNA for all stain concentrations was significantly 
higher than the values observed for the DAPI-only 
control (ANOVA, F( 3;19 )=165.94, P<0.001; Dunnett’s 
test, P<0.001 for each concentration; Fig. 4A). The 
nRNA fluorescence values of the DAPI+1000-nM Syto 
