Sipe and Chittenden: A comparison of calcified structures for aging Paralichthys dentatus 
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age determination with whole otoliths and scales. Pre- 
vious studies have never evaluated sectioned otoliths in 
summer flounder, even though sectioned otoliths have of- 
ten proven a superior structure in other species, especially 
at older ages when scales and other structures can under- 
age fish (Beamish and McFarlane, 1983). Further study is 
especially needed because the location of the first mark on 
otoliths has recently been determined (Szedlmayer et al., 
1992). In addition, no work has been done to determine if 
right-left differences in the location of the focus result in 
differences in age determination. 
The main objective of our study was therefore to evalu- 
ate and compare whole otoliths, sectioned otoliths, scales, 
and opercular bones for aging summer flounder. We in- 
cluded opercular bones because many studies, on a variety 
of species, have found them to be superior to other struc- 
tures and to have very distinct and easy to read marks (for 
examples, see LeCren, 1947; Donald et al., 1992; Hostet- 
ter and Munroe, 1993). A second objective was to compare 
right and left otoliths for potential differences in age based 
on differences in the location of the focus. Calcified struc- 
tures were evaluated in terms of preparation and reading 
times, confidence in presumed annual mark clarity, agree- 
ment between repeated age readings, structure growth 
with fish growth, age agreement between different struc- 
tures of the same fish, and increases in the number of pre- 
sumed annual marks with structure size and fish size. Fi- 
nally, we discuss the formation of early, presumably false, 
marks on summer flounder otoliths and scales that result- 
ed in difficulties in age interpretation 
Methods 
Sample collection 
To minimize difficulties interpreting marks on the edge of 
the structures, collections of summer flounder were made 
far from the time of presumed annual mark formation, 
which occurs in May and June on the scales of Chesa- 
peake Bay summer flounder (Desfosse, 1995). Summer 
flounder were collected from commercial fisheries in the 
Chesapeake Bay region from September through Novem- 
ber of 1998 (n=165). Additional juvenile fish (n- 11) were 
collected by the Virginia Institute of Marine Science juve- 
nile bottom trawl survey in October of 1998 in the lower 
Chesapeake Bay and James River. 
Fish were processed for total length (TL), total weight 
(TW), and sex, and the calcified structures were removed 
as follows. Both saggital otoliths were removed, wiped 
clean, and stored dry in tissue culture cell wells. Scales 
were removed from just above the lateral line anterior 
to the caudal peduncle (Shepherd, 1980; Dery, 1988) and 
stored in coin envelopes. Both opercular bones were re- 
moved according to the methods of LeCren (1947), stored 
in coin envelopes, and frozen. 
The collection of summer flounder was stratified into six 
length-based categories of 100 mm each to include as many 
age groups as possible in the final study sample. A ran- 
dom sample of 15 fish was then chosen from the first five 
categories. The last category included the six largest fish, 
all of which were used in the comparison, for a total of 81 
fish. All calcified structures in the final study sample were 
assigned random numbers before preparation and aging. 
Summer flounder in the final study sample ranged in size 
from 209 to 758 mm TL and from 80.8 to 7304.6 g TW 
and in age from 0 to 10 years (determined from sectioned 
otoliths, as reported in this study). 
Preparation of calcified structures 
for age determination 
Whole otoliths were examined in water on a dark back- 
ground with reflected light at 120 to 240x magnification. 
Thin opaque bands, which appeared white under reflected 
light, were presumed to represent annual marks (Fig. 1A). 
Two counting paths were used for mark enumeration. The 
primary counting path was from the focus to the anterior 
margin of the otolith. The secondary counting path, used 
to verify the primary counting path reading, was from the 
focus to the posterior margin of the otolith. With calipers 
to 0.05 mm, whole otolith total length (WOTL) was mea- 
sured as the largest distance from the anterior to the pos- 
terior edge and whole otolith radial length (WORD was 
measured from the center of the focus to the tip of the 
anterior edge. A paired sample t-test was used to test for 
right-left differences in WORL. 
After all whole otolith readings were made, right and 
left otoliths were mounted sulcal groove down onto card- 
board with crystal bond adhesive and sectioned trans- 
versely through the focus with a variable speed Beuhler 
Isomet saw. The resulting sections, about 0.5 mm thick, 
were mounted on clear glass slides and immersed in crys- 
tal bond. Sections were viewed with transmitted light and 
bright field at 240x magnification. Thin opaque bands, 
which appeared dark with transmitted light, were pre- 
sumed to represent annual marks (Fig. IB) and were 
counted along the ventral side of the sulcal groove. Sec- 
tioned otolith radial length (SORL) was measured to 0.001 
mm along the ventral arm of the sulcal groove from the 
center of the focus to the otolith edge by using a compound 
video microscope with the Optimas image analysis sys- 
tem (Media Cybernetics, 1999). Broken otoliths were not 
measured if they were fractured along the focus. A paired 
sample t-test was used to test for right-left differences in 
SORL. 
Opercular bones were prepared according to the meth- 
ods of LeCren (1947). Briefly, they were soaked in cold tap 
water for several minutes to thaw and to partially loosen 
surrounding skin, then soaked for 1 minute in simmering 
water, after which the skin was easily removed with a 
toothbrush. The opercular bones were then rinsed with 
cold tap water and air-dried. Opercular bones were exam- 
ined dry with transmitted light and in water with reflected 
light on a dark background. Presumed annual marks (Fig. 
1C) were defined as sharp transitions from relatively nar- 
row translucent zones to relatively wide opaque zones that 
were continuous from the anterior to the posterior mar- 
gin of the bone (Bagenal and Tesch, 1978; Hostetter and 
Munroe, 1993). Translucent zones appeared white under 
