337 
Using measurements of muscle cell nuclear 
RNA with flow cytometry to improve 
assessment of larval condition of Walleye 
Pollock IGadus chalcogrammus ) 
Email address for contact author: steve.porter@noaa.gov 
Resource Assessment and Conservation Engineering Division 
Alaska Fisheries Science Center 
National Marine Fisheries Service, NOAA 
7600 Sand Point Way, NE 
Seattle, Washington 98115-6349 
Abstract— Nuclear RNA and DNA 
in muscle cell nuclei of laboratory- 
reared larvae of Walleye Pollock 
(Gadus chalcogrammus) were si- 
multaneously measured through the 
use of flow cytometry for cell-cycle 
analysis during 2009-11. The addi- 
tion of nuclear RNA as a covariate 
increased by 4% the classification 
accuracy of a discriminant analysis 
model that used cell-cycle, tempera- 
ture, and standard length to mea- 
sure larval condition, compared with 
a model without it. The greatest 
improvement, a 7% increase in accu- 
racy, was observed for small larvae 
(<6.00 mm). Nuclear RNA content 
varied with rearing temperature, in- 
creasing as temperature decreased. 
There was a loss of DNA when lar- 
vae were frozen and thawed because 
the percentage of cells in the DNA 
synthesis cell-cycle phase decreased, 
but DNA content was stable during 
storage of frozen tissue. 
Manuscript submitted 30 November 2012. 
Manuscript accepted 12 August 2013. 
Fish. Bull. 111:337-351. 
doi: 10.7755/FB.111.4.4 
The views and opinions expressed or 
implied in this article are those of the 
author (or authors) and do not necesarily 
reflect the position of the National 
Marine Fisheries Service, NOAA. 
Steven M. Porter (contact author) 
Kevin M. Bailey 
Marine fish larvae are most vulner- 
able to starvation during the first 
few weeks after they begin to feed 
(O’Connell, 1980; Theilacker, 1986; 
Theilacker and Porter, 1995). Star- 
vation may contribute directly to 
mortality, or it may do so indirect- 
ly by making larvae more vulner- 
able to predation (Bailey and Yen, 
1983; Folkvord and Hunter, 1986). 
Many methods, such as the RNA- 
DNA ratio (Buckley et al., 1999), 
lipid composition (Lochmann et al., 
1995), histological condition of tis- 
sues (Theilacker, 1978), morphologi- 
cal measurements (Theilacker, 1978), 
and flow cytometry (Theilacker and 
Shen, 1993a), have been developed 
to measure the physiological condi- 
tion of fish larvae. Mortality rate has 
been shown to correlate with nutri- 
tional condition of fish larvae (Thei- 
lacker et al., 1996). Hence, accurate 
assessment of condition can improve 
understanding of environmental pro- 
cesses that affect early life survival 
and recruitment. 
Previous studies have assessed 
the condition and growth of fish lar- 
vae with flow-cytometric cell-cycle 
analysis, which is a technique that 
measures the DNA content of indi- 
vidual cells in a population to deter- 
mine the proportion of cells in differ- 
ent phases of the cell cycle (Theilack- 
er and Shen, 1993b, 2001; Bromhead 
et al., 2000; Gonzalez-Quiros et al., 
2007; Porter and Bailey, 2011; Do- 
mingos et al., 2012). Cells that are in 
the process of dividing can have up 
to twice the amount of DNA as cells 
that are not dividing. The cell cycle 
consists of discrete phases: gap 1 
(Gl), DNA synthesis (S), gap 2 (G2), 
and mitosis (M) (Murray and Hunt, 
1993). Cell growth occurs during the 
Gl phase before cell division begins, 
and cells may enter a GO resting 
state from this phase in response 
to starvation or other unfavorable 
environmental conditions (Murray 
and Hunt, 1993). For cells to divide, 
they must first replicate their DNA 
(S phase) and then grow and produce 
the structures (G2 phase) necessary 
for mitosis. Cell division occurs dur- 
ing mitosis. 
The proportion of cells in the S, 
and G2 and M (G2/M) phases are in- 
dicative of cells that may divide, and 
the use of flow-cytometric cell-cycle 
analysis to assess physiological con- 
dition is founded on the premise that 
cell proliferation is related to condi- 
tion. Specific tissue types (e.g., Thei- 
lacker and Shen, 1993b; Gonzalez- 
Quiros et al., 2007; Porter and Bai- 
ley, 2011) or individual whole larval 
homogenates (Domingos et al., 2012) 
have been used with this method, 
