Porter and Bailey: Using measurements of muscle cell nuclear RNA to assess larval condition of Gadus chalcogrammus 
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DAPI DAP1+RNA stain RNAse 
Treatment 
13,400 
13,300 
c 13,200 - 
0) 
Q 
CO 
<D 
§ 13,100 
< 
§ 13,000 
12,900- 
12,800 
B 
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DAPI DAPI+RNA stain RNAse 
Treatment 
Figure 5 
Mean fluorescence of (A) nuclear RNA and (B) DNA as a function of 
3 treatments used to confirm nuclear RNA staining — 4',6-diamidino- 
2-phenylindole (DAPI) stain only (negative control), DAPI+nuclear 
RNA stain (positive control), and ribonuclease (RNAse) A — of 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 stan- 
dard error of the geometric mean. Fluorescence values are arbitrary 
units. 
Plots of nRNA and DNA fluorescence 
showed that always-fed larvae had a dis- 
tinct group of aggregated S-phase nuclei 
that joined the G0/G1 and G2/M phases, 
and S-phase nuclei were fewer and dis- 
persed for unfed larvae (Fig. 3, A and B). 
Overall RSG1 was significantly larger for 
always-fed larvae than for unfed larvae 
(0.23 [standard error of the mean 0.01] for 
always-fed and 0.16 [SE 0.01] for unfed 
larvae; Mann-Whitney U test, (7=2299.5, 
Ni=63, N2=50, P<0.001), clearly distin- 
guishing larvae between the 2 feeding 
treatments. For always-fed larvae, there 
was an initial increase in RSG1 and then 
a gradual decline to its initial value after 
2 weeks of feeding (Fig. 6A). For unfed in- 
dividuals, RSG1 declined throughout the 
time period to less than half its initial 
value after 2 weeks of feeding (Fig. 6A). 
Nuclear RNA fluorescence did not show a 
distinct difference between feeding treat- 
ments until after 2 weeks of feeding (Fig. 
6B). 
Nuclear RNA, temperature, and condition 
Growth in the always-fed treatment at the 
warmest temperature (8.7°C) was poor; 
therefore, larvae from this treatment were 
not used for further analyses. A growth 
rate of about 0.15 mm d -1 would be ex- 
pected (senior author, unpubl. data), but 
larvae grew 0.08 mm d -1 . Both mean per- 
centage of nuclei in the S-phase (8.90) and 
mean RSG1 (0.17) were small for a typical 
healthy, feeding larva, supporting the ob- 
servation that those larvae were not grow- 
ing well. Growth rates at the temperatures 
of 5.9°C (0.10 mm d -1 ) and 2.9°C (0.06 mm 
d -1 ) were typical for larvae reared at those 
temperatures. 
There was no significant difference in 
overall nRNA fluorescence between the 
always-fed and unfed treatments (ANO- 
VA, F(i i61) =1.25, P=0.27; Table 3). Rearing 
temperature significantly affected over- 
all nRNA fluorescence. Larvae reared at 
2.9°C had a higher fluorescence than lar- 
vae reared at 5.9°C (ANOVA, F(i,6i)=4-47, 
P=0.04; Table 3), indicating more RNA in 
muscle nuclei from larvae reared at the colder temper- 
ature. RSG1 was significantly higher for the always-fed 
treatment than for the unfed treatment at both tem- 
peratures (ANOVA, F(i 87)=59.65, P<0.01, Tukey’s test, 
P<0.01; Table 3), similar to the result for the experi- 
ment conducted in 2009. 
The effect of temperature on RSG1 was dependent 
on feeding treatment. RSG1 was smaller for always- 
fed larvae reared at 5.9°C compared with RSG1 of lar- 
vae reared at 2.9°C (ANOVA, F(i,87)=18.56, P<0.001, 
Tukey’s test, P<Q.Q1; Table 3), and this result may 
indicate shorter duration for the cell cycle at warmer 
temperatures. RSG1 was not significantly different be- 
tween unfed larvae reared at 8.7°C and at 2.9°C (ANO- 
VA, P(2,55)=3.61, P=0.03, Tukey’s test, P= 0.97; Table 
3), indicating that temperature affected RSG1 only of 
