78

Abstract— A male whale shark(Rhincodon typus) has been kept in
captivity at the Okinawa Churaumi
Aquarium, Japan, for 23 years, providing a rare opportunity to observe
sexual maturation of a male. In
2012, when the individual was 8.5
m in total length (TL), the claspers
began to elongate, and the distal
ends of the claspers changed into
cauliflower-shaped forms. Thesemorphological changes in the claspers were completed in 11 months.
Plasma concentrations of sex hormones (testosterone and progesterone) increased in association withclasper elongation. After elongation,
additional movements, including
the adduction and crossing of claspers, were displayed, and a whitish
fluid, which may have been semen,
was discharged through the clasper
grooves, indicating that the male
was functionally mature after clasper elongation. These observations indicate that this captive whale shark
reached maturity when it was 8.5 m
TL and over 25 years in age. Thissize at maturity is consistent withobservations in the wild. However,maturity in this individual captive
animal may have occurred later
than has been previously reported
in nature.

Manuscript submitted 7 September 2018.
Manuscript accepted 11 April 2019.
Fish. Bull. 117:78–86 (2019).
Online publication date: 26 April 2019.
doi: 10.7755/FB.117.1-2.9
The views and opinions expressed or
implied in this article are those of theauthor (or authors) and do not necessarilyreflect the position of the National
Marine Fisheries Service, NOAA.

Sexual maturation in a male whale shark(Rhincodon typus) based on observations
made over 20 years of captivity
Rui Matsumoto (contact author)1,2
Yosuke Matsumoto1
Keiichi Ueda1,2
Miwa Suzuki3
Kiyoshi Asahina3
Keiichi Sato1,2
Email address for contact author: r-matsumoto@okichura.jp
1 Okinawa Churaumi Aquarium

Ishikawa 424
Motobu, Okinawa 905-0206, Japan
2 Okinawa Churashima Research Center

Ishikawa 424
Motobu, Okinawa 905-0206, Japan
3 College of Bioresource Sciences

Nihon University
Kameino 1866
Fujisawa, Kanagawa 252-0880, Japan

In the wild, tracking animals, especially migratory aquatic vertebrates,
throughout their entire life histories
is extremely difficult. Animals often
move over vast oceans and have longlife spans, sometimes longer thanthose of humans. Therefore, the life
history traits of such animals generally are based on fragmented information, obtained from dead specimens acquired opportunistically.
The process of sexual maturationin the whale shark (Rhincodon typus), the world’s largest fish, remains
poorly understood because of the difficulties associated with following individuals over long periods of time,
both in the wild and in captivity. The
Okinawa Churaumi Aquarium, located in Okinawa, Japan, has successfully maintained a whale shark forover 20 years in captivity, providing
a unique opportunity to better follow maturation history in this spe-

cies (e.g., Uchida et al., 2000; Martin, 2007). This male whale shark,
named Jinta, has been exhibitedsince 1995 at the Ocean Expo Aquarium (renamed the Okinawa Churaumi Aquarium in 2002), representing
the longest period of captivity forthis species in the world. From 1995
to 2018, the total length (TL) of theanimal increased from 4.6 to 8.7 m.
From our study of this shark, wereport the process of sexual maturation in the male whale shark, focusing on morphological changes andmovements of the claspers, possible
semen ejection, and changes in thecirculating levels of sex hormones.
For male elasmobranchs, the degreeof clasper elongation and calcification and increase in sex hormone
levels are generally used as indicators of sexual maturation (Gelsleichter and Evans, 2012); however, continuous changes in these indicators

Matsumoto et al.: Sexual maturation in a captive Rhincodon typus based on observations made over 20 years

during the maturation process have never before been
documented in whale sharks. This study was based ondata from only one individual, but it is still noteworthy
because this individual is the only whale shark thathas been successfully maintained in captivity for sucha long period. Our data provide important insights into
the maturation of male whale sharks.

Materials and methods
EthicsThis study was conducted as part of the health monitoring for captive animals at the Okinawa Churaumi
Aquarium. Animal welfare during rearing was maintained in strict accordance with the ethical guidelinesof 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 association. Maintenance, animal handling, and all procedures associated with this study also were conductedin accordance with the ethical guidelines of Okinawa
Churashima Foundation (OCF2), which specifies procedures for the care and welfare of higher vertebrates(reptiles, birds, and mammals); these guidelines did
not contradict the guidelines of the Japanese Association of Zoos and Aquariums.
Captivity conditions
The male whale shark is kept at the Okinawa Churaumi 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 withnatural seawater pumped directly from the East China
Sea. The temperature and chemistry of the water inthe tank are the same as those in the external environment. The roof of the tank is transparent; therefore,
light intensity in the tank reflects that of natural sunlight. The shark is fed 0.5–0.8% of its body weight ineuphausiids and sergestids every day.
GrowthThe TL of the male whale shark was estimated by measuring the length from the snout-tip to the origin of
the first dorsal fin (pre-first dorsal fin length [PD1]), toavoid measurement errors caused by lateral tail beats
when TL was measured directly. The following equation, from Matsumoto et al. (2017), was used to estimate 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 forcare and use of laboratory animals of Okinawa Churashima
Foundation, 11 p. [Available from Okinawa Churashima
Found., Ishikawa 888, Motobu, Okinawa 905-0206, Japan.]

79

LogTL = 0.964 × logPD1 + 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 growthstage by using segmented regression models (SRMs;
Muggeo, 2003; Segura et al., 2013). The growth of elasmobranchs is generally divided into 2 stages, immature(juvenile) and mature (adult) (Jensen et al., 2002), or
into 3 stages: immature, maturing, and mature (Saïdi
et al., 2009). Therefore, we applied 2 SRMs that estimated 1 or 2 break points (BKs) expected to be turning points of the growth stages, following the method
described by Muggeo (2003).
Clasper morphology
The morphological features of the claspers are used toindicate sexual maturation in elasmobranchs. We calculated the ratio of clasper outer length (CLO) to pelvic
inner margin length (P2I) (i.e., CLO:P2I), as describedby Compagno (1984). The CLO:P2I is a relative valuethat represents the degree of elongation from the posterior end of the pelvic fin. The CLO and P2I were analyzed with photogrammetry by using ImageJ, vers. 1.45
(Schneider et al., 2012) to take measurements fromphotographs of the ventral side of the pelvic region of
the shark. The photographs were taken underwater annually from October 2005 through August 2011, andthen they were taken monthly until October 2013.
Sex steroid hormones
The plasma concentrations of testosterone and progesterone (T and P4, respectively) were analyzed by using
blood samples. Veterinarians took blood underwaterfrom the radial vein of the pectoral fin, using a syringe with an 18-gauge needle, monthly from May 2008
through December 2013 (Ueda et al., 2017). Blood samples were then transferred to a blood-collection vacuum
tube treated with heparin (VP-H050K3, Terumo Corp.,
Tokyo, Japan). After centrifugation (3000 rpm for 15min), plasma was collected and immediately stored at−80°C for subsequent hormonal analysis. After thawing, the plasma was mixed with diethyl ether, and steroids were extracted by using a common procedure (Suzuki 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 concentrations of both steroid hormones were determinedthrough time-resolved fluorescent antibody analysis.
We compared the average blood levels of the hormones among the 3 observable developmental periodsof the clasper: short (clasper tips and posterior marginof pelvic fins are at the same horizontal position), elon3 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.

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Fishery Bulletin 117(1–2)

Table
Fitted parameters from 2 segmented regression models (SRMs) for estimating the growth of the captive male whale shark(Rhincodon typus) at the Okinawa Churaumi Aquarium in Okinawa, Japan. The SRMs used 1 or 2 break points (BKs) expected to be turning points between the 3 growth stages: immature, maturing, and mature. The average monthly changes
in growth of body length, during the immature stage (α1), during the mature stage in the model with 1 BK or the maturing
stage in the model with 2 BKs (α2), and during the mature stage in the model with 2 BKs (α3), are given with standard
errors of the means (SEs). The turning points from the immature to mature stage in the 1-BK model (BK1), from the immature to maturing stage in the 2-BK model (BK 1), and from the maturing to mature stage in the 2-BK model (BK 2) are
given with 95% confidence intervals (CI). r2=coefficient of determination.
SlopesModel with 1 BK
Model with 2 BKs

α1 (SE)

α2 (SE)

α3 (SE)

BK1 (95% CI)

BK2 (95% CI)

2.243 (0.072)
2.281 (0.086)

0.467 (0.320)
1.375 (0.428)–
0.050 (0.729)

170.5 (154.5–186.4)
149.7 (117.8–181.7)–
195.6 (162.8–228.3)

r2

449.9 (8.2) 0.994
447.8 (8.6) 0.993

gating (claspers extending behind the posterior
margin of pelvic fins), and completed (claspers
no longer extending). Statistical analyses wereconducted by using the nonparametric Kruskal–Wallis test followed by the nonparametric
Steel–Dwass post-hoc test. The relationshipbetween hormone levels and water temperature also was tested by using a regression
analysis. Measurements are reported as means
with standard errors (SEs), unless otherwise
noted.

Total length (cm)A

Captive period (month)

Results
GrowthTotal length (cm)

B

Intercept (SE)Captive period (month)Figure 1
Change in monthly body length of a male whale shark (Rhincodon typus) in captivity at the Okinawa Churaumi Aquariumin Okinawa, Japan, given in centimeters in total length and represented by segmented regression models (SRMs) with (A) 1 and(B) 2 break points. The dotted lines indicate the 95% confidential intervals. The data used in the SRMs were collected fromMarch 1995 through December 2013.

According to the SRM that used 1 BK, the average growth rate for body length (slope) fromMarch 1995 through May 2009 was 2.24 cm/
month (SE 0.07) (Fig. 1A, Table). During thisperiod, the TL increased from 460 to 832 cm.
The average growth rate between May 2009and December 2013 decreased to 0.47 cm/
month (SE 0.32), ºwith TL changing from 832
to 854 cm. Based on the estimates from theSRM that used 2 BKs (Fig. 1B), the averagemonthly growth from March 1995 throughAugust 2007 was 2.28 cm (SE 0.07), with TL
increasing from 460 to 789 cm (Table). From
August 2007 through June 2011, the growthrate was 1.38 cm/month (SE 0.43) with TL
changing from 789 to 852 cm. The growth ratefrom June 2011 through December 2013 was
0.05 cm/month (SE 0.73) with TL increasingfrom 852 to 854 cm. Both SRMs revealed thatthe individual grew in length rapidly until itreached approximately 800 cm TL. Considering annual growth in TL, the whale shark grewfrom 460 cm to >800 cm at a rate of 26.9 cm/

Matsumoto et al.: Sexual maturation in a captive Rhincodon typus based on observations made over 20 years

81

ril
Ap

ne
Ju

M

ar
ch

ay
M

ly
Ju

ril
Ap

ne
Ju

M

ar
ch

CLO:P2I

TL (cm)Figure 2
Relationship between the ratio of the clasper outer length to the pelvic inner length(CLO:P2I) and body length, given in centimeters in total length (TL), from October 2005
through October 2013 for a male whale shark (Rhincodon typus) in captivity at the Okinawa
Churaumi Aquarium in Japan.

year (1-BK model) or 27.4 cm/year (2BK model) from 1995 through 2008;
after that time period, the growth rate
decreased to 0.6–5.6 cm/year.

A

B

C

D

Clasper development and functionality
Clasper elongation was complete in
11 months (Fig. 2). Before August
2011, CLO:P2I was almost constant(1.00–1.09). After the claspers began
to elongate posteriorly beyond the tipof the pelvic fin, CLO:P2I increased to1.60 by July 2012; then this elongation ended, and the CLO:P2I remained
constant.
The shape of the claspers changed
as the claspers elongated (Fig. 3). Cauliflower-shaped tissues were formed at
the distal ends of the claspers, andcartilage associated with the rhipidion(small cartilaginous elements locatedat the distal end of the clasper) was
observed along clasper grooves.
The clasper became functional during a period of rapid elongation withskeletal development of the rhipidion.
The whale shark began to display a
new behavior in April 2012. The animal turned upside down, to an inverted position, while crossing bothclaspers simultaneously (Fig. 4, Suppl.
Video). The tips of both claspers werespread along the groove and inflected

Figure 3
Images with ventral views showing morphological changes in the claspersof the male whale shark (Rhincodon typus) in captivity at the Okinawa
Churaumi Aquarium, Japan, taken in (A) October 2005, (B) August 2011,
and (C) July 2012, as well as (D) an image of a posterior view taken in
February 2013 of the same whale shark.

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Fishery Bulletin 117(1–2)

Figure 4
Image with a ventral view of the rotational behavior of the clasper of the male whale shark (Rhincodon typus) in captivity at the
Okinawa Churaumi Aquarium, Japan, taken in June 2012.

dorsally while they were crossed. The clasper that wasplaced on the top of the other varied among events,
and both claspers had the top position interchangeably.
In addition, starting in August 2012, a white milky fluid was released from the claspers when this behavior
was displayed (Suppl. Video). However, it was not possible to confirm whether this fluid was semen because
it dispersed and became diffused in the tank immediately after release.
Changes in hormonal levels
Changes in plasma concentrations of T and P4 were recorded among the 3 observable developmental periodsof clasper development (Fig. 5). There were minimalchanges in the concentrations of T and P4 during ashort period (from May 2008 to July 2011), with mean
T concentrations of 6.23 ng/mL (SE 0.52) and mean P4
concentrations of 0.50 ng/mL (SE 0.08) (Suppl. Table).
However, T concentrations had large fluctuations during the elongating period (from August 2011 to July2012), ranging between 31.95 and 8.42 ng/mL with amean of 18.66 ng/mL (SE 3.06). In the elongating period, no change was notable in the levels of P4, witha mean of 0.79 ng/mL (SE 0.16) (Suppl. Table). In thecompleted period (from August 2012 to December 2013),
T and P4 levels were higher, with means of 36.74 ng/
mL (SE 9.46) and 1.21 ng/mL (SE 0.25), respectively,
than those in the other 2 periods.
Among the 3 periods of clasper development, there
was significant variation in levels of T (Kruskal–Wallis
test: 29.40, P<0.01) and P4 (Kruskal–Wallis test: 7.30,
P=0.03) (Fig. 6). Notably, T concentrations were significantly lower in the short period than in the elongat-

ing and completed periods (Steel–Dwass test:
P<0.01 for both periods). However, there wereno significant differences in T levels between
the elongating and completed periods (Steel–
Dwass test: P=0.95). Concentrations of P4
were also lower in the short period than in thecompleted period (Steel–Dwass test: P=0.03),
although there were no significant differencesin P4 levels between the short and elongating
periods (P=0.23) or between the elongating andcompleted periods (P=0.82).
There were significant relationships between water temperature and concentrationsof T (coefficient of determination [r2]=0.42,
P<0.01) and P4 (r2=0.26, P<0.01) during theshort period; whereas, these relationships
were not significant during the elongating (T:r2=0.06, P=0.59; P4: r2=0.14, P=0.40) and completed (T: r2=0.14, P=0.17; P4: r2=0.01, P=0.78)
periods (Fig. 7).

Discussion
Abrupt clasper elongation and increased levelsof sex hormones observed in the male whale
shark strongly indicate that the individual is sexuallymature. Increases in hormone levels (T or P4) associated with male sexual maturation have been reported invarious elasmobranchs (Rasmussen and Gruber, 1993),
although the mode of action of these hormones remains
unclear. Previous studies have hypothesized several
functions of sex hormones (such as T), including theinitiation of mating behavior (Rasmussen and Murru,
1992; Rasmussen and Gruber, 1993), spermiogenesis
and spermiation (Tricas et al., 2000; Henningsen et al.,2008), and clasper elongation (Heupel et al., 1999; Sulikowski et al., 2005; Sulikowski et al., 2006; Awruch etal., 2008). Hormone levels were slightly correlated withchanges in water temperature when the shark was immature; however, these correlations were not observed
during and after the clasper elongation period, andhormone levels were also significantly greater after the
shark started to mature sexually. Such patterns havenot been previously reported in other elasmobranch species. Further studies are needed to clarify the relationships between hormone levels and water temperatureduring each growth stage.
Our data allow comparisons between wild and captive whale sharks. The individual studied here wasconsidered mature at 8.5 m TL. This finding is consistent with the maturation size estimated for wild male
whale sharks off the northwestern coast of Australia
on the Ningaloo Reef, where 50% of 275 wild male
whale sharks had attained maturity at 8.1 m TL and
95% of those sharks had done so by 9.1 m TL (Norman
and Stevens, 2007). We were not able to determine theprecise age of maturation in our shark because theshark’s age was unknown when he was brought into
the aquarium.

Matsumoto et al.: Sexual maturation in a captive Rhincodon typus based on observations made over 20 years

ay

ay

M

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ay
M

ay
M

ay
M

ay

CLO:P2I

T concentration (ng/mL)
P4 concentration (ng/mL)

ay

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M

M

ay
M

ay

ay
M

M

ay
M

CLO:P2I

M

83

Figure 5
Relationships between the ratio of the clasper outer length to pelvic inner length (CLO:P2I)
and concentrations of (A) testosterone (T) and (B) progesterone (P4) from May 2008 throughNovember 2013 for a male whale shark (Rhincodon typus) at the Okinawa Churaumi Aquarium in Japan.

The age of live elasmobranchs can be estimatedonly by using growth models. Two studies have reported growth curves for the whale shark. Wintner (2000)
estimated the ages of 15 dead whale sharks by counting the number of growth bands in vertebrae, assuming that a single pair of growth bands corresponds to1 year, and generated a growth curve of this species.
By using that growth curve, the age of our shark at
capture (at a size of 460 cm TL) was estimated to be
10 or 14 years old. More recently, Hsu et al. (2014)provided new growth curves using a greater sample
size (n=92) of species collected from the populationin the northwestern Pacific Ocean. They estimated 2
growth curves on the basis of growth bands being deposited either once or twice a year, and they concluded that the band pairs were formed twice a year. The
growth curve provided by Hsu et al. (2014) is likely

applicable to estimate the age of our shark at capture
because the shark was taken from the same region.
Based on the growth curve of Hsu et al. (2014), theage of our shark at the time of capture was estimatedto be 7.6 years. Consequently, the estimated age at thetime that clasper elongation ceased was 24.9 years. This
result indicates that sexual maturation of our shark occurred later than the age at maturity estimated for wild
individuals in the Indo-Pacific region (17 years old; Hsu
et al., 2014). Meanwhile, Perry et al. (2018) estimatedthat males in the Maldives mature at 25 years, following
the methods described by Hsu et al. (2014). This estimation is comparable to our result. The timing of sexual
maturity of the whale shark may be associated moreclosely with size than with chronological age, a notionthat is consistent with previous findings (Gelsleichter etal., 2002); however, further investigations are needed.

Fishery Bulletin 117(1–2)

T concentration (ng/mL)

84

Elongating

Completed

Short

Elongating

Completed

P4 concentration (ng/mL)

Short

Figure 6
Comparisons of concentrations of the hormones (A) testosterone (T) and (B) progesterone (P4) from May 2008 to December
2013 in a male whale shark (Rhincodon typus) at the Okinawa
Churaumi Aquarium, Japan, during the 3 developmental periodsof the clasper: short, elongating, and completed. The box indicates the 25th and 75th percentiles, the whiskers indicate theminimum and maximum values, and the line within each box
indicates the median value. A circle indicates an outlier.

The results of this study indicate that morphological and functional changes in the claspers during maturation occurred over a relatively short time,less than 1 year, in our shark. The animal began todisplay new clasper movements during the period of
rapid clasper growth, behavior that has hitherto notbeen reported. These movements have been observed
during the mating season in other elasmobranchs, including the tawny nurse shark (Nebrius ferrugineus),zebra shark (Stegostoma fasciatum), bull shark (Carcharhinus leucas), sandbar shark (C. plumbeus), anddasyatid rays (senior author, unpubl. data), and theysometimes occur in the absence of females (Ritterand Compagno, 2013). Moreover, a white milky fluid, which we suspect to be semen, was released fromthe tip of the claspers as the claspers crossed. Basedon these observations, it is likely that the functional maturation of the claspers occurs in parallel withmorphological changes.
In summary, observations of a captive male whale
shark made over a period of 20 years have improved
our understanding of the process of sexual maturation in this species. Maturation was associated withincreased plasma concentrations of T and P4 and therapid morphogenesis and functioning of claspers. Additional studies involving both captive and wild individu-

85

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Water temperature (°C)

P4 concentration (ng/mL)M

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T concentration (ng/mL)

Matsumoto et al.: Sexual maturation in a captive Rhincodon typus based on observations made over 20 years

Figure 7
Relationship between water temperature and concentrations of (A) testosterone (T) and (B)progesterone (P4) from May 2008 through December 2013 in a male whale shark (Rhincodon
typus) at the Okinawa Churaumi Aquarium in Japan. The period covers the 3 developmental
periods of the clasper: short (T: r2=0.42, P<0.01; P4: r2=0.26, P<0.01), elongating (T: r2=0.06,
P=0.59; P4: r2=0.14, P=0.40), and completed (T: r 2=0.14, P=0.17; P4: r2=0.01, P=0.78). The
background colors of white, light gray, and dark gray indicate the short, elongating, andcompleted periods, respectively.

als are needed to further advance the biology of thewell-known but mysterious whale shark.

Acknowledgments
We express our gratitude to the Yomitan Fisheries Cooperative for collecting the whale shark used for theseobservations. We acknowledge H. Miyahara for providing us the opportunity to perform this research. We
thank both A. Uno and K. Aoki from the Nihon University, for assistance with laboratory analyses, and R.
Nozu, T. Tomita, and Y. Obayashi for their commentson this manuscript. We recognize S. Uchida, M. Toda,
M. Nakazato, A. Yamashiro, K. Murakumo, and othercolleagues for their valuable information and advice.

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