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and flow-cytometric cell-cycle analysis is as responsive 
to starvation as the RNA:DNA ratio in that it is able 
to detect starvation within a few days of no feeding 
(Porter and Bailey, 2011). 
Flow cytometry has been used to measure the rela- 
tive amount of cellular RNA and DNA in the brain cells 
of fish larvae (Theilacker and Shen, 1993b, 2001). Brain 
cells in the GO and G1 phases were separated through 
the use of ribonuclease (RNAse): GO were quiescent 
cells with low RNA content, and G1 were growing cells 
with high RNA content (Theilacker and Shen, 1993b). 
Theilacker and Shen (1993b) showed that the propor- 
tion of high-RNA-content G1 cells differed between fed 
and starved larvae, and it was suggested that those 
cells gave an indication of short-term changes in past 
feeding history. Nuclear RNA (nRNA) may react faster 
to metabolic changes than does cellular RNA, making 
it more sensitive to environmental change (Piwnicka et 
al., 1983) and potentially useful for assessment of the 
condition of fish larvae. 
Most nRNA is contained in the nucleolus (Li et 
al., 2006), the site of ribosome biogenesis (Sirri et al., 
2008), and ribosome production correlates with cellu- 
lar growth (Caldarola et al., 2009). For many cell types 
grown in culture, nRNA content of G1 cells is highly 
variable, and a specific amount may be required for 
entry into the S phase (Darzynkiewicz et al., 1980; 
Piwnicka et al., 1983; Staiano-Coico et al., 1989). The 
threshold amount needed for G1 cells to progress into 
the S phase has been defined as the minimum nRNA 
content of S-phase nuclei determined from plots of 
nRNA and DNA fluorescence measured by flow cytom- 
etry (Darzynkiewicz et al., 1980). 
In a previous study with flow cytometry (Porter and 
Bailey, 2011), cell-cycle information (fraction of nuclei 
in the S and G2/M phases), larval standard length (SL), 
and temperature were used as covariates in a labora- 
tory-developed model for measurement of the condition 
of larvae of Walleye Pollock ( Gadus chalcogrammus). 
In the study that we describe here, we found that an 
additional covariate based on nRNA improved the clas- 
sification accuracy of a similar condition model by more 
clearly defining healthy (i.e., feeding, growing) and un- 
healthy (i.e., starving) larval conditions. We also exam- 
ined the effect of rearing temperature on nRNA mea- 
surements and the effect of storage on frozen tissue 
used for measurements with flow cytometry. 
Materials and methods 
Larval rearing 
Adult Walleye Pollock were collected by trawl in She- 
likof Strait, Gulf of Alaska, during the spawning sea- 
son in March 2009 and 2010 by the Alaska Fisheries 
Science Center (AFSC). For our experiments in 2009 
and 2010, eggs from a single fish pairing (one female 
and one male) were fertilized and maintained aboard 
ship in the dark at 3°C before they were transported to 
the AFSC in Seattle, Washington. Eggs used for 2011 
experiments came from a brood stock of adults kept 
at the AFSC laboratory at the Hatfield Marine Science 
Center in Newport, Oregon, and were also the result of 
a single fish pairing. 
In 2009 and 2010, larvae were reared in 2 feeding 
treatments: an always-fed treatment, in which larvae 
were considered healthy, and an unfed (starved) treat- 
ment in which larvae were considered unhealthy. Only 
the always-fed treatment was used in 2011. Rearing 
methods are described in Porter and Bailey (2011). Two 
replicate tanks were used for each feeding treatment. 
The size of the tanks varied with each experiment: 
120 L in 2009, 20 L in 2010, and 60 L in 2011. The 
diet of larvae in the always-fed treatment consisted of 
laboratory-cultured rotifers ( Brachionus plicatilis ) that 
were fed an algal diet (Isochrysis galbana and Pavlova 
lutheri ) and a commercial rotifer supplement. Rotifers 
were maintained in the rearing tanks at a concentra- 
tion of 10 mL -1 . Natural zooplankton, which included 
primarily copepod nauplii ( Acartia spp.) and gastro- 
pod veligers, were collected from a local lagoon and 
screened through 202-pm mesh; they also were main- 
tained in the always-fed rearing tanks, at a concentra- 
tion of 3 mL -1 . A 16-h daylight cycle with a light level 
of 2.5 pmol photon mr 2 s -1 from overhead, full-spectrum 
fluorescent lights was used, and larvae were sampled 
4-6 h after the lights turned on at 0600 h. To avoid 
sampling larvae that were not actively feeding and pos- 
sibly unhealthy, only larvae that had prey in their gut 
were sampled from the always-fed treatment. Rearing 
temperatures varied: 6.0°C in 2009, 2.9°C, 5.9°C, and 
8.7°C in 2010, and 6.5°C in 2011. 
2009 experiments: nRNA staining protocol and covariate 
Three methods for preservation of larvae were tested 
to determine which of them was optimal for simultane- 
ous staining of DNA and nRNA in muscle cell nuclei 
of larvae of Walleye Pollock: 1) storage at -80°C, 2) a 
methanol treatment, and 3) storage at -80°C followed 
by a methanol treatment for 15 min before tissue pro- 
cessing. To stain DNA for cell-cycle analysis with flow 
cytometry, 4',6-diamidino-2-phenyIindole (DAPI), a flu- 
orescent DNA stain, was used at a concentration of 10 
pg mL -1 , and nRNA was stained with Invitrogen Syto 
RNASelect 1 green fluorecent cell stain (S32703, Life 
Technologies Corp., Carlsbad, CA), hereafter referred 
to as Syto RNASelect stain. 
Preservation of tissue by freezing works well for 
DAPI-DNA staining (Theilacker and Shen, 2001), 
and methanol-preserved tissue has been stained suc- 
cessfully with Syto RNASelect stain (Life Technolo- 
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