Porter and Bailey: Using measurements of muscle cell nuclear RNA to assess larval condition of Gadus chalcogrammus 
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Figure 6 
For always-fed and unfed treatments of larvae of Walleye Pollock 
( Gadus chalcogrammus) reared in 2009, (A) mean ratios of the 
number of S-phase nuclei to the number of Gl-phase nuclei with 
high nuclear RNA content (RSG1) and (B) geometric mean fluo- 
rescence of nuclear RNA of all cell-cycle phases pooled. The G1 
phase of the cell cycle is when cell growth occurs before cell divi- 
sion, and the S phase is when DNA replicates. Error bars indicate 
±1 standard error of the mean. Fluorescence values are arbitrary 
units. 
tissue (ANOVA, F 4 48 =36. 67, P<0.001, Dunnett’s tests, 
P<0.05 for all frozen groups; Table 5), indicating that 
DNA was lost when tissue was frozen and thawed. The 
percentage of nuclei in the G2/M phases was not sig- 
nificantly different between the fresh and frozen tissue 
treatments (ANOVA, 2*4 48 = 2.00, P=0.11; Table 5), but 
freezing affected the percentage of nuclei in the GO/ 
G 1 and S phases. There was a significant increase in 
the percentage of nuclei in the G0/G1 phases between 
fresh and frozen tissue (ANOVA, P 4 48=19.90, 
P<0.001, Dunnett’s tests, P<0.01; Table 5), and 
freezing had the opposite effect on S-phase 
nuclei, namely a decrease in the percentage 
of nuclei in that phase (ANOVA, P 4 4g=19.14, 
P<0.001, Dunnett’s tests, P<0.01; Table 5). 
The increase in the percentage of nuclei in 
the G0/G1 phases in the freezing treatment 
may be due to a loss of DNA from S-phase 
nuclei that caused them to be identified as 
nuclei in the G0/G1 phases on the basis of 
their fluorescence signal. There was no signif- 
icant change in the percentage of G0/G1- or 
S-phase nuclei among the 4 frozen groups of 
larvae tested (Tukey’s tests, P >0.35; Table 5), 
indicating that DNA was stable during stor- 
age of frozen tissue. This result indicates that 
DNA was lost by S-phase nuclei either during 
the freezing process or subsequent thawing. 
There was no significant difference in nRNA 
fluorescence between fresh and frozen tissues 
(ANOVA, P 4 48=2.38, P=0.06; Table 5), indi- 
cating no loss of nRNA. RSG1 showed results 
similar to those as DNA in that RSG1 of fro- 
zen tissue was significantly less than RSG1 
of fresh tissue, and it was stable during the 
10 months of storage of frozen tissue (ANO- 
VA, F 4 16.70, P<0.001, Dunnett’s tests, 
P<0.01; Tukey’s tests >0.90; Table 5). 
Discussion 
We developed a protocol for staining nRNA in 
muscle cell nuclei of larvae of Walleye Pollock 
for use in flow cytometry, and we showed that 
the inclusion of an nRNA covariate in a mod- 
el resulted in more accurate measurement 
of physiological condition than did cell-cycle 
analysis alone. Accurate assessment of condi- 
tion of fish larvae is essential because small 
changes in mortality rate over a long period 
of time can strongly influence future recruit- 
ment (Houde, 1987). RSG1, based on nRNA 
fluorescence, proved to be an indicator of po- 
tential growth that was responsive to feeding 
conditions, and it contributed meaningful im- 
provement to the discriminant analysis model 
for assessment of larval condition, as shown 
by an increase in classification accuracy and 
a decrease in Akaike’s information criterion value. 
Data from 3 years were used for model testing; there- 
fore, results are not unique to a single group of larvae. 
The classification accuracy of small larvae (<6.00 mm 
SL) was most improved (7%). This result is important 
because that size class includes larvae that have just 
started to feed, and the S- and G2/M-phase fractions 
can be highly variable in first-feeding larvae and may 
not always distinctly indicate condition. Additionally, 
