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Fishery Bulletin 111(4) 
Table 2 
Nuclear RNA fluorescence of muscle cell nuclei in various phases of the cell cycle from labora- 
tory-reared larvae of Walleye Pollock (Gadus chalcogrammus). Larvae were sampled from the 
always-fed and unfed treatments used in experiments in 2009. Fluorescence values are arbitrary 
units and were adjusted on the basis of controls to make samples comparable among sessions 
of flow cytometry. Standard errors of geometric means are reported in parentheses. Cell-cycle 
phases: Gl=gap 1 or cell growth before cell division, G0=resting state from G1 phase, S=DNA 
synthesis, G2=gap 2 or cell growth before mitosis, and M=mitosis. G0/Gl=fluorescence of GO and 
G1 phases combined, G2/M=fluorescence of G2 and M phases combined. 
Treatment 
RNA fluorescence 
Mean 
n 
Statistical test 
p 
Always-fed 
G0/G1, S, G2/M 
phases pooled 
54.9 (1.9) 
63 
2-sample f-test, 
#111=1.03 
0.31 
Unfed 
G0/G1, S, G2/M 
phases pooled 
52.1 (2.0) 
50 
- 
- 
Always-fed 
G0/G1 phases 
49.2 (1.6) 
63 
2-sample #-test, 
#111=1.01 
0.31 
Unfed 
G0/G1 phases 
46.9 (1.6) 
50 
- 
- 
Always-fed 
S phase 
87.2 (2.7) 
63 
2-sample *-test, 
*111=0.57 
0.57 
Unfed 
S phase 
90.5 (3.3) 
50 
- 
- 
Always-fed 
G2/M phases 
87.2 (2.7) 
63 
2-sample *-test, 
*111=0.24 
0.81 
Unfed 
G2/M phases 
86.3 (3.1) 
50 
- 
- 
growing larvae. The data for unfed larvae were re-an- 
alyzed with the 8.7°C treatment removed to determine 
if the exclusion of the data from the 8.7°C always-fed 
treatment biased the conclusions from this study. The 
results were unchanged; there was no significant differ- 
ence in RSG1 between unfed larvae at 2.9°C and 5.9°C 
(ANOVA, F (1>87 )=59.65, P<0.001, Tukey’s test, P=0.05). 
Model testing 
Data from experiments in 2009, in 2010 (temperatures: 
2.9°C and 5.9°C), and in 2011 were pooled for model 
testing (n= 237). The 8.7°C experiment in 2010 was ex- 
cluded because larvae grew poorly in the always-fed 
treatment. A control model used temperature, SL, arcsin 
\fx -transformed fraction of cells in the S phase, and 
arcsin \fx -transformed fraction of cells in the G2/M 
phases; and a test model added arcsin V* -trans- 
formed RSG1 to the covariates used in the control 
model (n=158). For independent cross-validation test- 
ing, 49 always-fed larvae ranging from 5.36 to 8.64 mm 
SL and 30 unfed larvae from 5.20 to 5.92 mm SL 
were used (n= 79). Both models significantly dis- 
criminated between the always-fed and unfed treat- 
ment groups (control model, Wilks’s lambda=0.46, 
F (4 i53)=44.19, P<0.001; test model, Wilks’s lambda=0.45, 
^(5A52)=37.13, P<0.001). 
The test model improved overall classification ac- 
curacy by 4%, and accuracy for both always-fed and 
unfed larvae increased (Table 4). The improvement in 
the always-fed treatment larvae was due to the correct 
classification of additional small, feeding larvae (<6.00 
mm SL). The classification accuracy in the test model 
for those larvae improved 14%, increasing from 53% 
(8/15) to 67% (10/15) when RSG1 was used. Classifica- 
tion accuracy of unfed treatment larvae improved 3%, 
and the test model correctly classified all the unfed 
larvae tested (Table 4). For larvae <6.00 mm SL from 
both the always-fed and unfed treatments, classifica- 
tion accuracy improved 7% (an increase from 37/45 to 
40/45). There was no difference in classification accu- 
racy between models for larvae >6.00 mm SL, and both 
models correctly classified all of those larvae. Akaike’s 
information criterion value for the test model was less 
than that value for the control model (-1130.53 and 
-1001.38, respectively), indicating that the addition of 
RSG1 improved model fit. 
Effect of freezing and storage on nRNA and DNA 
There was a loss of DNA when larvae were frozen and 
thawed, but that process did not affect nRNA mea- 
surements. The DNA fluorescence of fresh tissue was 
significantly greater than DNA fluorescence of frozen 
