Matsumoto et al.: Sexual maturation in a captive Rhincodon typus based on observations made over 20 years 
79 
during the maturation process have never before been 
documented in whale sharks. This study was based on 
data from only one individual, but it is still noteworthy 
because this individual is the only whale shark that 
has been successfully maintained in captivity for such 
a long period. Our data provide important insights into 
the maturation of male whale sharks. 
Materials and methods 
Ethics 
This study was conducted as part of the health moni¬ 
toring for captive animals at the Okinawa Churaumi 
Aquarium. Animal welfare during rearing was main¬ 
tained in strict accordance with the ethical guidelines 
of the Japanese Association of Zoos and Aquariums 
(JAZA 1 ), which does not stipulate the experimental 
procedures to be followed. Therefore, this study did 
not require separate, specific approval from this asso¬ 
ciation. Maintenance, animal handling, and all proce¬ 
dures associated with this study also were conducted 
in accordance with the ethical guidelines of Okinawa 
Churashima Foundation (OCF 2 ), which specifies proce¬ 
dures for the care and welfare of higher vertebrates 
(reptiles, birds, and mammals); these guidelines did 
not contradict the guidelines of the Japanese Associa¬ 
tion of Zoos and Aquariums. 
Captivity conditions 
The male whale shark is kept at the Okinawa Churau¬ 
mi Aquarium in an exhibition tank (Kuroshio Tank) 
with a length, width, and depth of 27, 35, and 10 m. 
The water in the tank is replaced continuously with 
natural seawater pumped directly from the East China 
Sea. The temperature and chemistry of the water in 
the tank are the same as those in the external environ¬ 
ment. The roof of the tank is transparent; therefore, 
light intensity in the tank reflects that of natural sun¬ 
light. The shark is fed 0.5-0.8% of its body weight in 
euphausiids and sergestids every day. 
Growth 
The TL of the male whale shark was estimated by mea¬ 
suring the length from the snout-tip to the origin of 
the first dorsal fin (pre-first dorsal fin length [PD 1]), to 
avoid measurement errors caused by lateral tail beats 
when TL was measured directly. The following equa¬ 
tion, from Matsumoto et al. (2017), was used to esti¬ 
mate TL in centimeters: 
1 JAZA (Japanese Association of Zoos and Aquariums). 1984. 
Japan Arboretum Aquarium Association ethics summary 
[Available from website.] 
2 OCF (Okinawa Churashima Foundation). 2016. Guide for 
care and use of laboratory animals of Okinawa Churashima 
Foundation, 11 p. [Available from Okinawa Churashima 
Found., Ishikawa 888, Motobu, Okinawa 905-0206, Japan.] 
LogTL = 0.964 x logPDl + 0.443. 
The PD1 has been measured underwater directly by 2 
scuba divers with a measuring tape, every year since 
the first year of captivity. 
The growth rate was calculated for each growth 
stage by using segmented regression models (SRMs; 
Muggeo, 2003; Segura et al., 2013). The growth of elas- 
mobranchs is generally divided into 2 stages, immature 
(juvenile) and mature (adult) (Jensen et al., 2002), or 
into 3 stages: immature, maturing, and mature (Saidi 
et al., 2009). Therefore, we applied 2 SRMs that esti¬ 
mated 1 or 2 break points (BKs) expected to be turn¬ 
ing points of the growth stages, following the method 
described by Muggeo (2003). 
Clasper morphology 
The morphological features of the claspers are used to 
indicate sexual maturation in elasmobranchs. We cal¬ 
culated the ratio of clasper outer length (CLO) to pelvic 
inner margin length (P2I) (i.e., CLO:P2I), as described 
by Compagno (1984). The CLO:P2I is a relative value 
that represents the degree of elongation from the pos¬ 
terior end of the pelvic fin. The CLO and P2I were ana¬ 
lyzed with photogrammetry by using ImageJ, vers. 1.45 
(Schneider et al., 2012) to take measurements from 
photographs of the ventral side of the pelvic region of 
the shark. The photographs were taken underwater an¬ 
nually from October 2005 through August 2011, and 
then they were taken monthly until October 2013. 
Sex steroid hormones 
The plasma concentrations of testosterone and proges¬ 
terone (T and P4, respectively) were analyzed by using 
blood samples. Veterinarians took blood underwater 
from the radial vein of the pectoral fin, using a sy¬ 
ringe with an 18-gauge needle, monthly from May 2008 
through December 2013 (Ueda et al., 2017). Blood sam¬ 
ples were then transferred to a blood-collection vacuum 
tube treated with heparin (VP-H050K 3 , Terumo Corp., 
Tokyo, Japan). After centrifugation (3000 rpm for 15 
min), plasma was collected and immediately stored at 
-80°C for subsequent hormonal analysis. After thaw¬ 
ing, the plasma was mixed with diethyl ether, and ste¬ 
roids were extracted by using a common procedure (Su¬ 
zuki et al., 1998). The extract was diluted 2.5 fold with 
0.05 M Tris-HCl (pH 7.75) buffer solutions, so that it 
could be used for measurements. The plasma concen¬ 
trations of both steroid hormones were determined 
through time-resolved fluorescent antibody analysis. 
We compared the average blood levels of the hor¬ 
mones among the 3 observable developmental periods 
of the clasper: short (clasper tips and posterior margin 
of pelvic fins are at the same horizontal position), elon- 
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. 
