340 
Fishery Bulletin 1 1 1(4) 
Table 1 
Days after first feeding when laboratory-reared larvae 
of Walleye Pollock ( Gadus chalcogrammus ) were sam- 
pled to determine the effect of temperature on nuclear 
RNA. Rearing tanks were maintained at 3 different 
temperatures. Because temperature can affect the 
growth rate of fishes, a “degree-day” model (degree- 
day=temperature*fish age in days) was used to ensure 
that that fish were taken at similar developmental 
stages. 
Rearing 
temperature 
PC) 
Sampling days after 
first feeding 
(determined by 
degree-day model) 
Sampling days 
(calendar days) 
after first feeding 
2.9 
17, 35, 61 
6, 12, 21 
5.9 
18,35,65 
3,6, 11 
8.7 
17,33,58 
2,4,7 
try (see Materials and methods, Flow cytometry section). 
On the same day, 60 larvae were frozen at -80°C in in- 
dividual tubes (30 from each tank) for flow-cytometric 
cell-cycle analysis at later dates. At 4 weeks after they 
were frozen and at additional intervals of 3, 6, and 
10 months, 15 of these frozen larvae were randomly 
selected and analyzed with flow cytometry. Geometric 
mean nRNA fluorescence for nuclei in the G0/G1, S, 
and G2/M phases of the cell cycle pooled and RSG1 was 
determined for each larva (see Materials and methods, 
Flow cytometry section). The fraction of nuclei in the 
G0/G1, S, and G2/M phases of the cell cycle also was 
determined for each larva (see Materials and methods, 
Flow cytometry section). ANOVA, Dunnett’s tests, and 
Tukey’s tests were used to compare nRNA fluorescence, 
DNA fluorescence, and fractions of nuclei in the GO/ 
Gl, S, and G2/M phases between the control and frozen 
samples and among the frozen samples over time. 
Flow cytometry 
The tissue preparation protocol described in Porter 
and Bailey (2011), modified from Theilacker and Shen 
(2001), was followed. A frozen larva was placed on ice 
to thaw just before it was processed. A methanol-pre- 
served larva was processed directly from the preserva- 
tive. The larva was then placed on a glass depression 
slide into an approximately 100-pL mixture of DAPI 
and Syto RNASelect stains. The head and gut were dis- 
sected away from the trunk musculature, and the mus- 
cle tissue was sliced into 4 or 5 pieces with 2 scalpels. 
Only the pieces of muscle tissue were transferred 
into a microcentrifuge tube that contained a 230-pL 
mixture of DAPI and Syto RNASelect stains and the 
mixture was triturated 6 times with a 1-mL syringe 
with a 25-gauge needle to release the nuclei from the 
muscle cells. The solution was filtered through a 48- 
pm filter into another microcentrifuge tube to separate 
the stained nuclei from large cellular debris. Prepared 
samples were kept on ice until they were analyzed 
with a BD Biosciences Influx flow cytometer (BD Bio- 
sciences, San Jose, CA) typically within 4-5 h of prepa- 
ration. DAPI was excited with a 350-nm UV laser, and 
Syto RNASelect stain was excited with a 488-nm laser. 
The DAPI/DNA detector filter was 450/40, and the de- 
tector filter for Syto RNASelect stain/RNA was 525/30. 
Flourochrome compensation for overlapping emissions 
spectra of RNA and DNA stains is unnecessary when 
exciting DAPI and Syto RNASelect stain with the BD 
Biosciences Influx flow cytometer. The beams of the 
350-nm and 488-nm lasers intercept the stream at 
spatially separate points. Each beam excites only the 
stain for DNA (350 nm) or RNA (488 nm). The emis- 
sion light is focused on separate mirror pinholes and is 
detected in separate modular detection blocks. Chicken 
and trout erythrocyte nuclei (Biosure, Inc., Grass Val- 
ley, CA) stained with the same mixture of DAPI and 
Syto RNASelect stains used for the muscle cell nuclei 
were used as controls. 
At the beginning of each flow cytometry session, 
each control type was run and necessary adjustments 
were made to the flow cytometer to keep control fluo- 
rescence values similar to previous sessions. DNA and 
RNA fluorescence values for larvae within the same ex- 
periment but measured during different flow cytometry 
sessions were made comparable by standardizing to a 
common control value. Samples that had <5000 nuclei 
analyzed or a coefficient of variation >9.00 for the cell- 
cycle phase of G0/G1 were not used in further analy- 
ses, and the use of this criteria resulted in rejection of 
about a third of all samples. 
For each larva, the fraction of nuclei in the G0/G1, 
S, and G2/M phases was calculated with MultiCycle A V 
software, vers. 4.0 (Phoenix Flow Systems, San Diego, 
CA). FCS Express flow cytometry analysis software, 
vers. 3.0 (De Novo Software, Los Angeles, CA) was used 
to calculate RSG1 and geometric mean fluorescence 
values for nRNA and DNA. DAPI area (DNA content, 
linear scale) in relation to DAPI height (DNA content, 
linear scale) was plotted for each larva, and a gate 
(a boundary used to enclose specific data points) was 
made to exclude debris, doublets, triplets, and large ag- 
gregates from nuclei in the G0/G1, S, and G2/M phases 
(Fig. 1A). Nuclei in those phases were located within 
the gate (Fig. IB). 
To calculate geometric mean values of nRNA and 
DNA fluorescence for each larva, a scatter plot of nRNA 
fluorescence (log scale) values in relation to DNA fluo- 
rescence (linear scale) values was made from the data 
enclosed within the gate for nuclei in the G0/G1, S, 
and G2/M phases. Each phase was gated separately for 
mean fluorescence values (Fig. 2), and a single gate that 
enclosed nuclei of all phases was used for the pooled 
mean fluorescence value. RSG1 was calculated with the 
same nRNA and DNA fluorescence scatter plot. 
