James et al.: Validation of the use of vertebrae and dorsal-fin spines for age determination of Squalus acanthias 43 
times at liberty (TAL) greater than 1 year were used 
for validation. From each specimen, 1 centrum was pro- 
cessed for band-pair count (hereafter referred to as the 
histology section) and 1 centrum was processed for OTC 
mark analysis (hereafter referred to as the OTC section). 
The histology section was processed by using thin sec- 
tioning methods to enhance the band pairs following 
Natanson et al. (2007). The OTC section was embed- 
ded in TAP Clear-Lite casting resin (TAP Plastics, San 
Leandro, CA) and sectioned through the frontal plane 
with an IsoMet Low Speed Saw (Buehler, Lake Bluff, IL) 
by using paired diamond-edged blades separated by a 
0.6-mm spacer. Sections were stored individually in cap- 
sules in 70% ethanol and kept in the dark to preserve 
the OTC mark. Two photographs of the OTC section were 
taken under reflected light by using 1) white light and 2) 
long-wave ultraviolet light (366 nm) with a Nikon DSRi2 
digital camera (Nikon Corp., Tokyo, Japan) attached to 
a Nikon SMZ1500 stereomicroscope (Nikon Instruments 
Inc., Melville, NY). The number of band pairs (consisting 
of 1 opaque and 1 translucent band) distal to the OTC 
mark was counted. 
The distance from OTC mark to the centrum edge was 
measured in Adobe Photoshop CC 2017 (Adobe Inc., San 
Jose, CA) on the OTC section and transferred to the his- 
tology section to determine the location of the OTC mark 
relative to the band pairs. Because of the slight differences 
in sizes of adjacent vertebra, a ratio was used to calcu- 
late the position of the OTC mark on the histology section 
(Natanson, 1993). Band pairs past the OTC mark were 
then counted on the histology section; a half of a band pair 
indicated that the OTC mark was in the ultimate band 
(either translucent or opaque). 
Dorsal-fin spines were removed, scraped clean of excess 
tissue, and thoroughly dried. Digital images of the dried 
second dorsal-fin spines with OTC marks regardless of 
TAL were photographed by using an MTI CCD-72 video 
camera (Dage-MTI Inc., Michigan City, IN) attached to 
an SZX9 Olympus stereomicroscope (Olympus Corp., 
Tokyo, Japan) with long-wave ultraviolet light (366 nm). 
The OTC image was then superimposed over an identical 
image taken under reflected white light by using Adobe 
Photoshop CC 2017. Band pairs past the OTC mark were 
then counted. 
Linear regressions were performed on the data from 
each structure to determine if band-pair deposition was 
annual. Annual deposition is supported if the slope of 
the regression of the number of band pairs past the OTC 
mark against the TAL is not significantly different from 1 
(i.e., the 95% confidence interval [CI] overlaps 1). Linear 
regressions were calculated for data from the vertebral 
centra and the dorsal-fin spines in R, vers. 3.4.4 (R Core 
Team, 2018). 
Vertebral column analysis 
For preliminary analysis of the whole vertebral column, 
one adult female spiny dogfish (87.0 cm FL) was collected 
separately in 2019 to compare the number of band pairs 
among centra along the vertebral column. This individ- 
ual was not one of the recaptured individuals because a 
fresh, large individual was desired, and it was needed to 
avoid any potential shrinkage of the vertebrae during 
prolonged frozen storage. Fork length, TL, STL, weight, 
sex, and maturity status were recorded. Vertebra num- 
bers 1-80 from the head to the tail were measured to 
the nearest 0.1 mm in 3 dimensions: dorsoventral diam- 
eter (hereafter referred to as dorsal), lateral diame- 
ter (hereafter referred to as lateral), and rostrocaudal 
length (hereafter referred to as length). Each measure- 
ment was plotted against centrum number to observe 
changes in vertebral shape along the vertebral column. 
To determine if the number of band pairs varied along 
the vertebral column, every fifth vertebra was processed 
histologically and band pairs were counted as outlined 
above. No assumptions were made about the birth date 
of spiny dogfish; therefore, only whole band pairs are 
reported for this analysis. The band-pair count also 
was plotted by centrum number. The mean band-pair 
count and 95% CI of the mean were calculated among 
centra for the vertebral column. If the band-pair counts 
of more than 5% of the centra examined were outside 
of the calculated 95% CI, the band-pair counts differed 
significantly along the vertebral column (James and 
Natanson, 2020). 
Results 
Tagging and sample collection 
A total of 4306 spiny dogfish were injected with OTC, 
tagged, and released. Between July 2011 and September 
2016, 148 spiny dogfish were recaptured and returned 
whole to the Narragansett Laboratory. No OTC-injected 
spiny dogfish were returned after 2016. Time at liberty 
ranged from 1 d to 4.8 years. Eighty-seven individuals had 
TALs greater than 1 year; an OTC mark was observed in 
the vertebrae of 41 of these individuals (47.1%; 33 females 
and 9 males). Out of the 148 dorsal-fin spines examined, 
45 spines had an OTC mark (30.4%; 40 females and 
5 males). All 45 dorsal-fin spines with an OTC mark were 
included in analysis regardless of TAL to increase the 
sample size. Twenty-one individuals had an OTC mark in 
both the vertebrae and the dorsal-fin spine (Fig. 1). The 
size range of all examined individuals was 51.5-87.5 cm 
FL at tagging and 52.8-87.3 cm FL at recapture (Tables 1 
and 2). Some individuals shrank in FL over time, and this 
reduction in size is an artifact of the combination of small 
inaccuracies from measuring live spiny dogfish at capture 
and from measuring the recaptured spiny dogfish after 
being frozen. 
Validation of ages from vertebrae and dorsal-fin spines 
The number of band pairs past the OTC mark in vertebral 
centra does not support annual periodicity of band-pair 
formation. Vertebral centra had 0.5-2.5 band pairs past 
