Fang et al. : Age, growth, and population structure of Ommastrephes bartrarrw in the North Pacific Ocean 
35 
relatively low success rate. Potential use of alterna- 
tive methods has been the topic of recent discussions 
(Moltschaniwskyj and Cappo, 2009). For example, the 
beak, which is the main feeding organ for squids, has a 
stable morphological structure because of its chitinous 
structure (Clarke, 1986) and could potentially be used 
for determination of a squid’s age. Removing a whole 
beak from the buccal mass is much easier than extract- 
ing a statolith from the statocyst. The beak, therefore, 
has been identified as an appropriate structure for 
studies of squid biology and ecology (Jackson et al., 
1997; Groger et al., 2000; Martinez et al., 2002; Cherel 
and Hobson, 2005; Guerra et al., 2010). 
Clarke (1965) first identified the incremental struc- 
ture in the beak of Moroteuthis mgens and demonstrat- 
ed its potential for estimating age. Raya and Hernan- 
dez-Gonzalez (1998) suggested the sagittal plane of a 
rostrum sagittal section (RSS) as the optimal plane for 
aging common octopus ( Octopus vulgaris), and longitu- 
dinal increments deposited in RSS were also observed 
to have periodic light and dark bands. The lateral wall 
surface (LWS) of the beak also can be used for reading 
growth increments, and a study over a specific period 
indicated that one ring represents 1 d of life in post- 
larvae (Hernandez-Lopez et al., 2001). Perales-Raya et 
al. (2010) improved beak aging methods and compared 
the precision of readable growth increments between 
RSS and LWS methods. They found that the RSS yield- 
ed more precise age estimates but that the LWS was 
quicker and simpler to prepare (Perales-Raya et al., 
2010). Canali et al. (2011) and Castanhari and Tomas 
(2012) also analyzed the upper beak microstructure 
and the relationship between increments and ML or 
body weight (BW). Examination of the upper beak has 
been recommended as a simple and effective approach 
for estimating cephalopod age (Canali et al., 2011; Cas- 
tanhari and Tomas, 2012). 
Previous studies on squid growth have focused pri- 
marily on seasonal and population variability in body 
size (Chen and Chiu, 2003; Ichii et al., 2004). Varia- 
tion of oceanographic conditions and feeding habits can 
greatly impact the growth of red flying squid (Wata- 
nabe et al., 2004; Ichii et al., 2009). Oceanic squid tend 
to migrate over long-distances between their spawning 
or nursery grounds and their feeding grounds (Sem- 
mens et al., 2007). Individuals are exposed to varied 
environmental conditions during ontogenesis. There- 
fore, age estimation is an important part of analyzing 
growth rates for the different life stages of squids. The 
age-based calculation of growth rates that is based on 
measurements of statoliths is a popular method and 
has been used in making growth estimations for oce- 
anic squid. The growth rates of ML and BW for some 
ommasterphid species have also been derived from 
statoliths (Markaida et al., 2004; Keyl, 2009; Chen et 
al., 2011, 2013). 
In this study, we used beak microstructure of red 
flying squid to identify its spawning season and to ex- 
amine age composition. The relationships between ML, 
BW, main beak morphometric variables, and age were 
defined in order to examine squid growth patterns. The 
relative differences between females and males were 
also investigated. This study is one of the first in which 
oceanic ommasterphid squid has been aged by using 
beak measurements. These methods can be applied to 
age and growth analyses of other oceanic cephalopods. 
Materials and methods 
Squid samples were randomly collected from Chinese 
commercial jigging vessels FV Jinhai 827 and FV 
Ningtai 21 in fishing grounds (between 154°E-174°W 
and 39°N-45°N) of the North Pacific Ocean from July 
through November in 2011 (Fig. 1). Samples were im- 
mediately frozen at -18°C for subsequent laboratory 
work. 
After samples were thawed in the laboratory, dorsal 
ML and BW were measured to the nearest 1 mm and 
1 g, respectively. The upper and lower beaks were then 
dissected from the buccal mass, washed with fresh wa- 
ter, and stored in a solution of 75% ethyl alcohol. The 
following 12 morphological variables were measured 
to the nearest 0.1 mm: upper hood length, upper crest 
length, upper rostrum length (URL), upper rostrum 
width, upper lateral wall length, upper wing length, 
lower hood length, lower crest length, lower rostrum 
length, lower rostrum width, lower lateral wall length, 
and lower wing length (Fig. 2A; Chen et al., 2012). 
For age estimation, the use of RSS has been reported 
to be more precise than the use of LWS (Perales-Raya 
et al., 2010). The lower beak is more likely to be sub- 
ject to erosion because it always covers the upper beak 
during biting and chewing. Moreover, the rostrum tip 
of the lower beak also has the function of paralyzing 
prey organisms (Nixon, 1973). The upper beak has a 
relatively complete rostrum tip, although erosion may 
still occur. Therefore, we measured the RSS of the up- 
per beak in this study. The upper beak was cut in half 
with scissors, producing 2 RSS pieces. To count incre- 
ments as accurately as possible, we cut the RSS into 2 
different sizes and used the larger one, which tended 
to have a preserved focal plane representing all growth 
rings in the central section. The larger half of the RSS 
was cleaned with water to remove mucus and was em- 
bedded in a small mold with epoxy resin. We ground 
the focal plane with 120-, 600-, and 1200-grit water- 
proof sandpaper until the growth increments became 
clearly visible. Finally, we polished the RSS slice with 
0.05-pm aluminium oxide powder to remove scratches 
on the surface. This preparation process is similar to 
that for statoliths (Dawe and Natsukari, 1991). 
The number of increments in each RSS were counted 
with a microscope (Olympus Corp., 1 Tokyo) at magnifi- 
cations of lOx, 40x, and 400x. Images of different sec- 
tions were taken with a charge coupled device (CCD) 
1 Mention of trade names or commercial companies is for iden- 
tification purposes only and does not imply endorsement by 
the authors or the National Marine Fisheries Service, NOAA. 
