48 
Fishery Bulletin 1 13(1) 
For many species, the otolith is the preferred struc- 
ture. Otoliths were found to be more suitable for aging 
than were scales for a large number of species, such 
as lake whitefish (Coregonus clupeaformis [Barnes and 
Power, 1984]), striped bass ( Morone saxatilis [Welch et 
ah, 1993; Secor et ah, 1995]), white crappie ( Pomoxis 
annularis [Boxrucker, 1986]), bluefish ( Pomatomus sal- 
tatrix [Robillard et ah, 2009]), bull trout (Salvelinus 
confluentus [Zymonas and McMahon, 2009]), Dolly Var- 
den ( Salvelinus malma [Stolarski and Sutton, 2013]), 
and summer flounder (. Paralichthys dentatus [Sipe and 
Chittenden, 2001]). Although comparisons between 
scales and otoliths have been relatively common, more 
comprehensive studies that involve multiple struc- 
tures are rarer. Scales, otoliths, vertebrae, opercula, 
and subopercula were used to age pontic shad (Alosa 
pontica ) in two recent studies that included the fam- 
ily Clupeidae [Yilmaz and Polat, 2002; Visnjic-Jeftic et 
al., 2009]), yet no such comprehensive study has been 
completed for the American shad. 
The Interstate Fishery Management Plan for Shad 
and River Herring requires that biological data, includ- 
ing ages, be collected for American shad annually by 
states from Maine to Florida (ASMFC 1 ). These data are 
used to make informed decisions regarding the man- 
agement of this species (ASMFC 2 ). Specifically, age 
data are used to estimate mortality, determine the age 
of recruitment into spawning populations, and char- 
acterize the age structure of populations by sex. Cur- 
rent data used for these purposes are based on scales, 
which have been shown to produce unreliable ages 
(McBride et ah, 2005; Duffy et al., 2011, 2012; Upton 
et al., 2012). Given the importance of age data for the 
assessment and management of American shad, it is 
imperative that an unbiased and precise aging method 
be used. The objective of this study was to compare 
the precision of age estimates obtained from the scales, 
otoliths, opercula, and vertebrae of American shad. 
Materials and methods 
American shad were collected by dip net from the fish 
lift at the Essex dam in Lawrence, Massachusetts, on 
the Merrimack River during May and June of 2008- 
2010. American shad were distinguished from other 
species by morphological characteristics described by 
Collette and Klein-MacPhee (2002). Samples were fro- 
1 ASMFC (Atlantic States Marine Fisheries Commission). 
1999. Amendment 1 to the Interstate Fishery Management 
Plan for Shad & River Herring. Fishery Management Report 
No. 35 of the Atlantic States Marine Fisheries Commission, 
77 p. [Available from http://www.asmfc.org/uploads/file/sha- 
daml.pdf] 
2 ASMFC (Atlantic States Marine Fisheries Commission). 
2012. River Herring Benchmark Stock Assessment, vol. 1. 
Stock Assessment Report No. 12-02 of the Atlantic States 
Marine Fisheries Commission, 392 p. [Available from http:// 
www.asmfc.org/uploads/file/riverHerringBenchmarkStockAs- 
sessmentVolumeIR_May2012.pdf.] 
zen and transported to the Annisquam River Marine 
Fisheries station in Gloucester, Massachusetts. After 
thawing overnight, TL in millimeters, weight in grams, 
and sex were recorded for each fish. Sagittal otoliths, 
scales, opercula, and vertebrae were removed and 
stored for processing. 
Otoliths were rinsed in water and stored dry in 
microcentrifuge vials. Whole otoliths were placed in 
a black dish filled with mineral oil and viewed with 
reflected light through a dissecting microscope at 
30-40x magnification for age determination. Left and 
right otoliths were examined side by side to aid in 
discerning between annuli and checks. The distal sur- 
face of the otoliths provided the clearest view of the 
annuli. 
Scales were stored dry in envelopes before being 
cleaned in an ultrasonic bath with a 5% pancreatin so- 
lution (Whaley, 1991). From 3 to 5 clean, nonregener- 
ated scales were dried with a paper towel and placed 
between 2 glass slides. Scales were viewed with trans- 
mitted light at 5-10 x on a digital computer imaging 
system that included Image-Pro Plus 3 image analysis 
software, vers. 6.2 (Media Cybernetics, Inc., Rockville, 
MD). All scales on a slide were examined to discern 
annuli from checks, but the scale with clearest annuli 
was used for age determination. 
Opercula were stored frozen before they were boiled 
for 2-3 min. A small brush was used to clean excess 
flesh from opercula after they were boiled. The opercu- 
la were then rinsed in clean water and air dried for at 
least 24 hours. Dry opercula were held up to a fluores- 
cent light source, and annuli were enumerated without 
magnification. Opercula were viewed as pairs to help 
discern between annuli and checks. 
Vertebrae numbers 4-10 were separated with a scal- 
pel. Excess flesh was removed with a scalpel before the 
vertebrae were soaked for 24-48 hours in a solution of 
3% hydrogen peroxide. After the soaking of the verte- 
brae, a small brush was used to remove any remain- 
ing flesh, and the vertebrae were allowed to air dry for 
at least 24 hours. Whole vertebrae were viewed under 
reflected light at 20-30x with a digital computer imag- 
ing system with Image-Pro Plus software. All vertebrae 
from each fish were examined to discern between an- 
nuli and checks. The vertebra with clearest annuli was 
selected for age determination. 
Annuli in the otoliths, opercula, and vertebrae were 
defined as the distal edge of each hyaline zone. We 
used Cating’s (1953) definition of annuli on scales, al- 
though we did so with disregard for their position in 
relation to the location of the transverse grooves be- 
cause the number of transverse grooves within each 
annulus as outlined by Cating (1953) was discredited 
by Duffy et al. (2011). Because all fish in this study 
3 Mention of trade names or commercial companies is for iden- 
tification purposes only and does not imply endorsement by 
the National Marine Fisheries Service, NOAA. 
