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Fishery Bulletin 111 (4) 
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Figure 4 
Mean fluorescence of (A) nuclear RNA and (B) DNA as a function of 
nuclear RNA stain concentration observed in muscle cell nuclei from 
larvae of Walleye Pollock ( Gadus chalcogrammus ) reared in 2009. The 
horizontal bar indicates fluorescence values that are not significantly 
different (P>0.05). Error bars indicate ±1 standard error of the geometric 
mean. Fluorescence values are arbitrary units. 
0.44 respectively; Fig. 4B). The 2000-nM concentration 
caused a significant reduction of DNA fluorescence com- 
pared with the DNA fluorescence of the control (ANO- 
VA, F (3 i 9 )= 10.90, P<0.001; Dunnett’s test, P-0.002; Fig. 
4B), indicating that a Syto RNASelect stain concentra- 
tion >1000 nM negatively affected DNA staining. Addi- 
tionally, no significant difference was observed in DNA 
fluorescence between the 500-nM and 1000-nM concen- 
trations (ANOVA, F (3 i9)=10.90, P<0.001; Tukey’s test, 
P=0.10; Fig. 4B). Therefore, the 1000-nM concentration 
was optimal for nRNA staining because 
it produced the highest nRNA fluores- 
cence and had no effect on DNA staining. 
The nRNA fluorescence of the RNAse- 
treated nuclei was significantly less 
than the values seen for the positive 
control (DAPI+ 1000-nM Syto RNASelect 
stain), indicating that nRNA was being 
stained (ANOVA, F (2 ,9)=386.26, P<0.0001; 
Tukey’s test, P<0.0001; Fig. 5A); however, 
the treated nuclei had a higher fluores- 
cence than the negative control (DAPI 
only; ANOVA, F (29 )=386.26, P<0.0001; 
Tukey’s test, P<0.0001; Fig. 5A), indicat- 
ing fluorescence signal from stained DNA 
or the incomplete removal of nRNA from 
the samples. There was no significant dif- 
ference in DNA fluorescence among the 
3 treatments (ANOVA, P ( 2 g)=0.009, 
P=0.99; Fig. 5B); therefore, it is most 
likely that the treatment with RNAse 
A did not completely remove all of the 
nRNA. Additionally, this experiment in- 
dependently confirmed that the 1000 - 
nM concentration of the Syto RNASelect 
stain does not affect DNA staining be- 
cause there was no significant difference 
in DNA fluorescence between the group 
stained with DAPI only and the group 
stained with DAPI+ 1000-nM Syto RNAS- 
elect stain. 
Nuclear RNA covariate 
For each treatment, larval SL was not 
significantly different between replicate 
tanks (always-fed treatment, 2 -sample 
f-test, £gi=0.196, P= 0.85; unfed treat- 
ment, 2-sample Utest, £ 43 = 0 . 45, P=0.65), 
indicating that larvae in those tanks 
responded similarly to the same treat- 
ment. Therefore, replicate measurements 
of nRNA fluorescence from each tank for 
each treatment were pooled. The growth 
rate of larvae in the always-fed treat- 
ment from hatching to 19 days after 
hatching was 0.11 mm d _1 , a rate that 
is typical for larvae of Walleye Pollock 
reared in a 6 °C laboratory (Porter and 
Theilacker, 1999). To formulate the nRNA covariate, we 
used 113 larvae, 63 always-fed (healthy) and 50 un- 
fed (unhealthy). There was no significant difference in 
nRNA fluorescence between feeding treatments when 
all phases of the cell cycle were pooled or when phases 
were examined separately (Table 2), indicating that 
nRNA fluorescence was not a useful indicator of condi- 
tion. Unlike nRNA fluorescence, RSG1 was responsive 
to feeding conditions; therefore, it was chosen as the 
nRNA covariate for model testing. 
