J-STAGE Advance published date: September 30, 2021

MAEDA AND KATOSEASONAL CHANGES IN THE EARPLUGGERMINAL LAYERS OF NORTH PACIFIC COMMONMINKE WHALES
Hikari MAEDA1* and Hidehiro KATO2, 3Fisheries Resources Institute, Japan Fisheries Research and Education Agency,
2–12–4 Fukuura, Kanazawa, Yokohama, Kanagawa 236–8648, Japan
2
Tokyo University of Marine Science and Technology, 4–5–7 Konan, Minato-ku, Tokyo 108–8477, Japan
3
Institute of Cetacean Research, 4–5 Toyomi-cho, Chuo-ku, Tokyo 104–0055, Japan*Corresponding author: hikarim@aﬀrc.go.jp
1Abstract
We investigated the annual accumulation rate of the most recently formed layer of earplugs,
the germinal layer (GL), of the North Paciﬁc common minke whales, which is important forage estimation. The pale GLs from Kushiro in September and October were found to be signiﬁcantly thicker than those from Ayukawa in April and May. The results suggest that the palelamina accumulated during an approximately six-month period from spring to autumn in theirfeeding season. This was consistent with the hypothesis that one pale and one dark lamina,
comprising one growth layer group, are formed annually in this species.
Key words:

earplug, common minke whale, age determination, germinal layer, accumulation
rate.

The earplugs of baleen whales were ﬁrst considered to show aging characteristics by Purves (1955).
The external auditory meatus lumen is ﬂattened dorso-ventrally, so that the dorsal and ventral epidermis walls are in apposition forming a “blind” section (Purves, 1955). Because of this, the earplug will
never fall out throughout a whale’s life. The earplug is composed of a “core” and an “outer covering”(Fig. 1). The core of the earplug (b in Fig. 2), where the growth layer group (dark lamina and palelamina) is formed, is composed of exfoliated and keratinized epidermis of a ﬁnger-sack-like tissue
called the glove ﬁnger (Fig. 1). It is surrounded by a layer of wax-like material from the external auditory canal membrane (outer covering, a in Fig. 2).
Currently, earplugs are used in age estimation for several species of baleen whales (Lockyer, 1984a;
Gabriele et al., 2010; Nielsen et al., 2012; Kitakado et al., 2013; Maeda et al., 2016). Many baleen
whales are known to migrate seasonally, breeding in low-latitude waters in winter and feeding in
high-latitude waters in summer. It is thought that this annual cycle is reﬂected in earplug growth layers, with a pale lamina forming during the summer feeding season and a dark lamina forming during
the winter breeding season (Fig. 2); therefore, one growth layer group represents one year (Roe, 1967;

Fig. 1. Diagram showing the anatomical position of the earplug in a typical baleen whale. After Purves (1955).

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Fig. 2. Bisected surface of an earplug of a common minke whale. a: (outside the dotted line), outer covering;b: (inside the dotted line), core with growth layers. Scale bar represents: 5 mm.

Lockyer, 1972, 1984b). Fat content tends to be lower in the dark lamina and higher in the pale lamina
according to histological observations of ﬁn whale (Balaenoptera physalus) earplugs (Roe, 1967).
Many other studies have examined whale earplugs; for example, Ohsumi (1964) investigated the annual accumulation rate of earplugs in southern-hemisphere ﬁn whales using the mark-recapture method, and Gabriele et al. (2010) conducted a comparative study of the earplug growth layer and longterm individual identiﬁcation in North Paciﬁc humpback whales (Megaptera novaeangliae).
Previously, age estimation from the earplugs of the common minke whale (B. acutorostrata) was
generally thought to be diﬃcult and impractical because of the softness of the earplugs and the poor
formation of growth layers (Sergeant, 1963; Mitchell and Kozicki, 1975; Christensen, 1981; Larsen
and Kapel, 1983; Christensen et al., 1990; Kato, 1992; Auðunsson et al., 2013). Under the Japanese
Research Program and a special permit in the Western North Paciﬁc (JARPNII), common minke
whale earplugs were carefully collected using the gelatinized extraction method (Maeda et al., 2013)
and attempts were made to read the growth layer groups. Maeda et al. (2016) reported age readability
and age estimation errors in common minke whales and concluded that the earplugs of this species canbe used as a valid age estimation tool.
However, the annual accumulation rate of growth layers has not been analyzed for North Paciﬁc
common minke whales, and age estimation was conducted on the assumption that one growth layergroup is formed per year as in other baleen species. In the case of the common minke whale, it is important to understand the periodicity of growth layer formation for age estimation. In this study, wefocused on the recently formed layer, the germinal layer (GL), of the earplug and aimed to determine
the periodicity of growth layer formation based on seasonal changes in the GL (Fig. 2).
Earplugs obtained from JARPNII Coastal Components from 2003 to 2016 were used. These surveys were conducted oﬀ Ayukawa (the Paciﬁc coast of northern Honshu; within a 50-mile radius of
Ayukawa Port 38°17′N, 141°30′E) in April and May and oﬀ Kushiro (the Paciﬁc coast of eastern
Hokkaido; within a 50-mile radius of Kushiro Port 42°59′N, 144°22′E) in September and October(Fig. 3).
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Fig. 3. Survey areas oﬀ Ayukawa (the Paciﬁc coast of northern Honshu; within a 50-mile radius of Ayukawa
Port 38°17′N, 141°30′E) in April and May, and oﬀ Kushiro (the Paciﬁc coast of eastern Hokkaido; within a50-mile radius of Kushiro Port 42°59′N, 144°22′E) in September and October.
Table 1. Pale and dark ratio of germinal layer (GL) of the earplug of common minke whales in Ayukawaand Kushiro.

The earplugs were preserved in 10% neutral buﬀered formalin solution. In the laboratory, after
cutting ﬂat along the central axis of the earplug by using a sharp blade, the earplug was ground on awet stone under running water to expose the neonatal line and growth layers (Fig. 2). Growth layers
were counted underwater using a stereoscopic microscope (magniﬁcation: 3.2–31.5). The age in years
was deﬁned as one growth layer group comprising a pair of pale and dark laminae in the core. The
earplugs of 360 individuals with clear growth layers from the neonatal line to the GL were selectedand observed to determine whether the GL was pale or dark (Ayukawa: 175 individuals; Kushiro: 185
individuals; age range: 1 to 44 years). For 204 of these individuals, the thickness of the pale GL wasmeasured using an image analysis software (ImageJ, a public domain, Java-based image processing
program, National Institute of Health, USA).
Pale GLs were observed in 63.4% of the earplugs from Ayukawa in April and May and in 100%from Kushiro in September and October (Table 1). For the pale GLs, the mean thickness from Ayukawa was 0.21 mm, while that from Kushiro was 0.40 mm (Fig. 4A). The pale GLs from Kushiro weresigniﬁcantly thicker than those from Ayukawa (t-test; p<0.05).
Since the thickness of the GL is expected to compress as the number of growth layers increases, thethicknesses of the pale GLs from Ayukawa and Kushiro were compared with the number of growthlayers. The GL thickness from both Ayukawa and Kushiro decreased as the number of growth layers
increased, but the Kushiro GLs were thicker than the Ayukawa GLs in all growth layer number classes(Fig. 4B). In growth-layer classes 1–5, 6–10 and 11–15, the pale GLs from Kushiro were signiﬁcantly
thicker than those from Ayukawa (t-test; p<0.05).

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Fig. 4. A: Comparison of mean thickness of the pale GL (mm) between Ayukawa in April to May and Kushiroin September to October. B: Comparison of mean pale GL thickness (mm) between Ayukawa in April to May
and Kushiro in September to October by type of growth layers.

Maeda et al. (2013) measured the length of earplugs in the North Paciﬁc common minke whale andexamined the growth of the earplug (the core and the outer covering) with age, and found that the outer covering that covers the core was not present in younger earplugs, but it became thicker with age.
In younger individuals (with fewer layers), the outer covering has not yet formed and there is more
space in the ear canal; the wax pushed out of the glove ﬁnger is not compressed and forms a thicker
layer (although younger individuals have a better metabolism). However, as the number of layers increases, it was considered that the outer covering developed and the core of the earplug becomes larger; there is less space in the ear canal and the thickness of the GL formed by extrusion from the glove
ﬁnger becomes thinner.
Lockyer (1972) examined ﬁn whales from the southern hemisphere and observed a distinctly
spaced growth layer that diminished suddenly, which was called the transition phase. The transition
phase in the earplug, where widely spaced growth layers abruptly become much closer together, indicates the age at sexual maturity. Maeda et al. (2017) reported the transition phase could be identiﬁedin the North Paciﬁc common minke whale and the mean age at transition phase was about 7 years oldfor both males and females. While some individuals showed the transition phase (rapid narrowing ofthe layer interval) as early as 4 years of age (Maeda et al., 2017), the large number of young individuals with relatively wide stratum widths suggests that the SD were greater in the 1–5 years old group.
Hatanaka and Miyashita (1997) reported that immature minke whale individuals are distributed
along the northern Paciﬁc coast in early summer; mature females are abundant in high-latitude waters(Sea of Okhotsk) in summer; and mature males are distributed south of mature females in summer,
mainly east of the Kuril Islands, and are abundant oﬀ eastern Hokkaido at the end of summer. To examine seasonal changes in the accumulation of the growth layers, the feeding season migration routeof minke whales was assumed to be as follows. In early summer, minke whales migrate northward
along the northern Paciﬁc coast to feed, then migrate to Sanriku oﬀ Ayukawa in April and May, whichis the early feeding season, and then further northward to their main feeding grounds. They then migrate southward to the waters oﬀ Kushiro in September and October, which is the latter half of thefeeding season.
While individuals with dark GLs were observed in the early feeding season in Ayukawa, no individuals with dark GLs were observed in the late feeding season in Kushiro. The pale GL was signiﬁcantCetacean Popul. Stud. (CPOPS), Short paper
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ly thicker in Kushiro than in Ayukawa, suggesting that a pale layer accumulated during the feedingseason from spring to autumn, which lasts approximately six months. This was consistent with thehypothesis that the growth layer (one pair of pale and dark laminae) is accumulated once a year incommon minke whales, as in ﬁn whales and other species.
In this study, earplugs with clear growth layers were used. However, Maeda et al. (2016) reported
that age readability of North Paciﬁc common minke whale was 45.2% for male, 41.2% for female, andmost of unreadable earplugs of mature animals had growth layers with unclear formation such as irregular lamination and partially-formed growth layers. Individuals with irregular growth layer groups
may not have a clear seasonal migration, and this point requires further investigation.
Acknowledgements

We would like to express our gratitude to all survey leaders, researchers, crew, and staﬀ of the research station that participated in JARPNII coastal surveys. We also would like to express our sincere appreciation to the anonymous reviewers who
reviewed this paper and provided useful suggestions and advice.

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Received: 31 January, 2021
Accepted: 18 May, 2021
Published online: September 30, 2021

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