Fujinami et al.: Age determination and growth of Prionace glauca in the western North Pacific Ocean 
109 
Figure 2 
Images of vertebrae from blue sharks f Prionace glauca) captured between 
2010 and 2016 in the western North Pacific Ocean. Vertebra were treated 
to enhance growth bands by using (A) a burn method and (B and C) thin- 
sectioning after staining with alizarin red. Black arrows indicate observed 
growth bands for 2 sharks of different lengths, one less than and another 
greater than 200 cm in precaudal length (PCL): (A) 181.0 cm PCL and 
(B) 252.0 cm PCL. Panel C provides an enlarged image of the vertebra 
in panel B. 
using a research vessel. Only DL was 
measured for sharks caught by com¬ 
mercial vessels because the head and 
viscera were removed prior to mea¬ 
surement; DL was converted to PCL 
following Fujinami et al. (2017). 
Age determination 
Cervical vertebrae were excised from 
the region above the branchial cham¬ 
ber and stored frozen until process¬ 
ing. Vertebral centra were boiled for 
approximately 20 min to remove most 
connective tissue, then stored in 70% 
ethanol before being washed in run¬ 
ning water, soaked in sodium hydrox¬ 
ide solution (5000 mol/m 3 NaOH), and 
scrubbed with a polishing buff (20-cm 
microfiber cloth; Sankei Co. 1 * , Tokyo, 
Japan) to remove residual connective 
tissue from their surfaces. After clean¬ 
ing, centra were washed in running 
water, cut longitudinally into 2 sec¬ 
tions by using a diamond saw (MC- 
110; Maruto Instrument Co., Tokyo, 
Japan) with the focus slightly to one 
side to avoid cutting the focus (Fig. 
2A); each half-cut section was then 
air-dried for 24 h. The centrum radius 
(CR), from the focus to the edge of the 
centrum perpendicular to the direction 
of the cutting plane, was measured to 
the nearest 0.01 mm by using a digi¬ 
tal microscope (VH-8000; Keyence 
Corp., Osaka, Japan). The CR to PCL 
relationship was estimated by using 
linear regression and was compared 
by sex by using analysis of covariance 
(ANCOVA). 
For blue sharks, Fujinami et al. 
(2018a) recommended use of a burn method (for young¬ 
er individuals) simultaneously with other methods, 
such as thin sectioning, bomb radiocarbon dating, or 
tag-recapture dating (for older individuals). Accord¬ 
ingly, we used a burn method, which is highly efficient 
and is accurate for aging of small- and medium-sized 
blue sharks (<200 cm PCL) (Fujinami et al., 2018a), 
and a thin-sectioning method, which is useful for aging 
older sharks (e.g., Matta et al., 2017). 
Growth bands on vertebrae of specimens less than 
200 cm PCL were enhanced by burning the centrum in 
accordance with Fujinami et al. (2018a). Vertebral cen¬ 
tra were heated to 250°C in a drying oven (DO-300A; 
AS ONE Corp., Osaka, Japan) for 6-12 min (Fig. 2A). 
For larger specimens (>200 cm PCL), centra were sec¬ 
1 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. 
tioned (1.0 mm) by using a sliding microtome (Retora- 
tome REM-710; Yamato Kohki Industrial Co., Asaka, 
Japan) without embedding, after they were cleaned 
and cut in half as described previously. Sections were 
stained with alizarin red for 2 min in accordance with 
Berry et al. (1977) and rinsed in running tap water for 
approximately 10 min (Fig. 2B). Finally, stained sec¬ 
tions were dehydrated through a graded ethanol series 
(70%, 80%, 90%, and 100%) and mounted on micro¬ 
scope slides. 
Burned centra were observed by using the shad¬ 
owing method (Francis and Maolagain, 2000; Semba 
et al., 2009) with a digital microscope and fiber-optic 
light. Sections were observed by using an SZX7 ste¬ 
reo microscope (Olympus Corp., Tokyo, Japan) with 
reflected light. We defined a growth-band pair as one 
convex band (dark, narrow) and one concave band 
(light, broad) on the centrum surface when the burn 
method was used, and we defined a band pair as one 
