J-STAGE Advance published date: May 12, 2021

AGE ESTIMATION FROM TEETH IN LONGMAN’S BEAKED WHALESAGE ESTIMATION FROM TEETH IN LONGMAN’SBEAKED WHALES (INDOPACETUS PACIFICUS)STRANDED IN NEW CALEDONIA (SOUTH PACIFIC)
Christina LOCKYER1* and Claire GARRIGUE2, 3
Age Dynamics, Huldbergs Alle 42, 2800 Kongens Lyngby, Denmark
Institut de Recherche pour le Développement UMR9220 ENTROPIE (IRD, Université de La Réunion,
CNRS, Université de la Nouvelle-Calédonie, IFREMER, Laboratoire d’Excellence–CORAIL,
BPA5, 98848 Noumea, New Caledonia)
3
Opération Cétacés, BP12827, 98802 Noumea, New Caledonia*Corresponding author: agedynamics@mail.dk
1

2

Abstract
Seven Longman’s beaked whales mass stranded in New Caledonia in November 2013, of
which 4 ultimately died, in a ﬁrst worldwide event reported for this poorly known Ziphioid
species. Teeth were extracted, collected and thoroughly cleaned of gum tissue from 3 females
ranging from juvenile to adult and one adult male. These were sectioned (crown-root) and prepared using two diﬀerent methods and examined under microscope magniﬁcation when Growth
Layer Groups (GLGs) in both dentine and cement were successfully identiﬁed. The methods
employed for aging included 1) sectioning centrally at approx. 150 µm through crown and root
on an Isomet circular diamond saw and examining under a microscope using both transmitted
polarised light and plain light; and, 2) thick sectioning (wafering) at approx. 2.5 mm and subsequent decalciﬁation in RDO™ (a proprietory brand, Illinois, USA) and then thin sectioning thewafer at 10–25 µm and staining with Ehrlich’s acid haematoxylin. GLGs were investigated inboth dentine (25 micron) and cementum (10–15 µm). Layering was evident in both tissues but
higher counts were more evident in thin stained sections of cementum. Although dentinal GLGs
in untreated tooth sections have been used successfully for aging in Ziphioid species Hyperoodon ampullatus (Christensen 1973, Feyrer et al., 2020), it is believed this is the ﬁrst time that
teeth have been used for estimating age from GLGs in this tropical species.
Key words:

Age estimation, Ziphioid whales, Indopacetus paciﬁcus, teeth.

Introduction
Longman’s beaked whale (Indopacetus paciﬁcus), ﬁrst described as a large species of the genus Mesoplodon (Longman, 1926) from a beachcast skull found in northern Queensland, Australia, was later
assigned to its own genus, Indopacetus, after comprehensive morphological evaluation (Moore, 1968)
and the discovery of a second skull in Somalia (Azzaroli, 1968). This species has long been considered one of the rarest and least known of all whales. Groups of Indopacetus are encountered withsome regularity in the tropical Indian and Paciﬁc Oceans (Anderson et al., 2006; Afsal et al., 2009;
Yamada et al., 2012), and there have been occasional reports of strandings (<20 stranded animals todate, Yamada et al., 2012) most of which have been singletons, with the exception of a cow-calf pairin Taiwan (Yao et al., 2012). More recently, a stranding was reported oﬀ the Phillipines (Acebes etal., 2019), and two separate strandings were reported oﬀ Okinawa, Japan, including a neonate (Kobayashi et al., 2021, 2021a).
This paper focuses on tooth material and relevant data from a mass stranding of Longman’s beaked
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whales in New Caledonia in the southwest Paciﬁc Ocean, and describes the details of the aging methods used, which may be helpful for researchers investigating more precise age in other beaked whales.
Among the samples collected from three females ranging from juvenile to adult and one adult male,
were the paired teeth, extracted and thoroughly cleaned of gum tissue. The details of the stranding
event are described by Garrigue et al. (2016) when seven animals came ashore together on the south
coast of the Grande Terre on 16 November 2013, four of which ultimately died. Only a brief accountof the age estimation was reported at this time. This is believed to be the ﬁrst worldwide mass stranding event for this poorly known species, and the ﬁrst time tooth layering has been investigated forLongman’s beaked whale.

Methods
Age was estimated using a single tooth from each of the four animals examined and was determined
without other detailed biological information apart from sex and body length. The remaining tooth
was retained untouched for other analyses. Two methods of aging were applied to investigate whichmight be more informative, and are detailed here. Although aging methods have been detailed for other beaked whales—Northern Bottlenose whale Hyperoodon ampullatus (Christensen, 1973; Feyrer etal., 2020)—Longman’s beaked whale is a tropical species, and in general little is known about aging
in beaked whales.
Fig. 1 shows the size and shape of a tooth. Each tooth was thoroughly cleaned of gum tissue and 2–3
sections, ca. 150 µm thick, were cut centrally through the crown and root of each tooth, using an Isomet

Fig. 1. The series of photos shows the size and dimensions of a tooth from specimen #4: a) and b) the lengthof the tooth in diﬀerent aspects; c) the greatest width of the tooth; d) the elliptical open pulp cavity at the root.
Only the top 10 mm of the tooth crown are exposed above the gum line which is clearly visible from the darker colour.

Fig. 2. Buehler Isomet low-speed diamond saw for sectioning teeth. The chuck and wood block mount for
the tooth are shown. The tooth is aﬃxed to the wood block using a standard hobby glue gun employing glue
sticks. The glue is easily removed after cutting and can be made easier by soaking the tooth sections and blockin water for some minutes.

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slow-speed circular diamond saw (see Fig. 2).
These thin sections were examined under a binocular dissecting microscope at magniﬁcationx7–x40 using alternately transmitted plain lightand polarized light, focusing on Growth LayerGroups (GLGs) in both dentine and cementum(see Perrin and Myrick Jr., 1980; Lockyer et al.,2016 for GLG deﬁnition). Each GLG observed
was assumed to have been deposited over a
1-year period as validated in other odontocetes
e.g. Physeter macrocephalus (Gambell, 1977),
Globicephala melas (Lockyer, 1993), Tursiops
truncatus (Hohn et al., 1999), Delphinapterus
leucas (Lockyer et al., 2016), and various mamFig. 3. A portable benchtop freezing microtome
mal species (Read et al., 2018). In addition,used for sectioning decalciﬁed teeth. Note the horia thicker central section (wafer) (2–2.5 mm)
zontal cutting plane, the ﬁxed freezing stage and thewas removed from each tooth, again throughmobile blade. The specimen is frozen by means of acrown-root axis adjacent to the ﬁrst thin section,
Peltier device with ﬂowing water in counter current.
and decalciﬁed in a rapid decalcifying solution
The blade is not cooled and the microtome is operRDO™ (a proprietory brand, Illinois, USA) for
ated at room temperature. The temperature of thefreezing stage is generally around −12°C and canup to 15 h. Thin sections of (10–25 µm) of thisbe adjusted by altering the water ﬂow to the device.
central wafer were then made using a freezing
microtome (see Fig. 3) and stained with Ehrlich’s
Acid Hæmatoxylin (EAH) as described for other species (Lockyer, 1993; Lockyer and Braulik 2014;
Read et al., 2018). The thinnest sections were destined for cemental examination and the thicker sections
for dentinal study. In this instance, the thin decalciﬁed sections were placed in large histo-cassettes in an
agitated ripe solution of EAH for up to 45 min until stain uptake was deemed optimal. The sections werethen transferred to water in the histo-cassettes and rinsed thoroughly of excess stain for several minutes.
Subsequently, the sections were transferred into a weak ammonia solution (just a few drops of ammonia
in the solution enough to turn the pH alkaline) and soaked here for about 3 min. until the sections hadturned uniformly from deep red to purple-blue. After a second rinsing in water, the sections were thenexamined wet under a microscope and the best sections were removed for mounting. Sections used forage estimation were ﬂoated onto 5% gelatin-coated microscope slides under water and arranged with amaximum of 2 sections per slide. The slides were then carefully blotted, section downwards, on absorbent paper (a type that does not shed ﬁbres) and then air-dried in a fume cupboard for about 30 min. andpermanently mounted under glass cover slips with DPX, a fast drying mixture of distyrine (a plasticizer)
and xylene. The slides were then allowed to completely dry in the fume cupboard for several days before
examination. Plain transmitted light was then used for examination under a microscope as for the unstained thick sections. Age data estimated from both preparation methods were recorded in both dentine
and cement.

Results
The ages estimated from the diﬀerent sections are presented in Table 1 where both stained anduntreated sections are recorded as well as GLGs in both the dentine and cementum. The ﬁrst point tonote is that for all specimens, the stained sections are generally providing higher GLG counts in both
dentine and cementum, with the exception of the dentine in specimen #6 where no identiﬁable GLGs
could be seen in the stained section. The other point is that cementum is generally providing higher
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counts and greater clarity than the dentine. The most consistent ages for both the adult female #6 andadult male #2 were estimated as 20–22 and >20–26 GLGs respectively, which by analogy with GLG
deposition rate in other cetaceans may be equivalent to years (see review in the Methods). Ages ofthe sub-adult females #4 and #5 were less clear, but #4 was estimated as 8–9 GLGs and #5 as 14–17
GLGs. Figs. 5–7 incl. show the untreated and stained sections of cementum for specimens #2, #5 and#6, respectively. In each case, more GLGs appear to be visible in the stained than in the untreatedsections. The GLGs identiﬁed are those that fall in the range shown for cementum in Table 1. At thisstage when the method of age estimation is experimental, it is unclear whether some of the identiﬁed
Table 1. Age estimates for four whales based on the examination of dentinal and cementum Growth LayerGroups (GLGs) under two diﬀerent treatments (After Garrigue et al., 2016).
SpecimennumberAge-sex classTreatment#2aAdult M,
length 590 cm#4b

No of GLGs2 observed

Overall Age1 Estimate(GLGs)2

DentineCementumunstained
stained

7+++3
20

20+
26

Sub adult F,
length 564 cm

unstained
stained

8
9–12

5++3
8+3

#5c

Sub adult F,
length 590 cm

unstained
stained

8++3
12+3

14
173

14–17 (range 8++ to 17)#6dAdult F,
length 618 cm

unstained
stained12++3
no GLGs
visible20
22

20–22 (range 12++ to 22)

20–26 (range 7++ to 26)
8–9 (range 5++ to 12)

Overall age was based on the modal age from several counts.
One GLG is assumed to be equal to one year but this requires future validation.
3
Addition symbol (+) indicates that more GLGs were present, but were too indistinct to be counted, and multiple+symbols reﬂect therelative amount of indistinct GLGs. In addition, the following comments relate to individual teeth.
aCrown worn down. Some GLGs may be missing. Dentinal resorption in root.
b
The presence of pulp stones interfered with the GLG pattern in the dentine such that it was diﬃcult to count at the base. Cement poorly
stained and ill-deﬁned. Dentinal resorption or decay in the root.
c
GLGs had poor deﬁnition, including double laminae. Dentinal resorption, or decay in root.
d
No dentinal resorption in root.
1
2

Fig. 4. Untreated section 150 microns thickness, of specimen #4: a) complete section examined under plaintransmitted light showing central dentine, surrounding cementum, and large areas of resorption at the dentine-cementum junction in the root; b) dentine examined under polarised ﬁlters, showing GLGs; c) close up of
the resorption area from a) examined under polarised ﬁlters showing pulp stones in the dentine root.

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Fig. 5.

Comparison of cementum in untreated and decalciﬁed stained sections of specimen #2.

Fig. 6.

Comparison of cementum in untreated and decalciﬁed stained sections of specimen #5.

GLGs might actually be accessory lines. Figs. 5–7 all show identiﬁed GLGs in the decalciﬁed andstained cementum, but it is clear that some are darkly stained and others paler; in addition, sometimes
the spacing between layers is not regular but very close appearing double (for example, see GLGs 19
and 21, Fig. 7) suggesting possible accessory lines. The untreated cementum in Fig. 7 shows identiﬁes
only 20 GLGs, indicating that staining may highlight other ultrastructures in addition to GLGs. Readet al. (2018) discuss the signiﬁcance and possible causes of accessory lines in marine mammalianteeth under the topic of tooth anomalies, including periodic changes in nutrition, hormonal levels andenvironmental factors. Presently it is diﬃcult to identify likely factors for this tropical species, notknowing much about the life history.
The specimens #2, #4 and #5 all had evidence of extensive mineral resorption at the dentine-cemenCetacean Popul. Stud. (CPOPS), Full paper
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Fig. 7.

Comparison of cementum in untreated and decalciﬁed stained sections of specimen #6.

Fig. 8.

Dentinal GLGs in an untreated section of a tooth of Hyperoodon ampullatus.

tum junction in the root region (Fig. 4a, c), while #6 had no such condition.

Discussion and conclusions
Both methods of preparation detailed here were adequate for identifying GLGs in cementum, withslightly higher counts in stained sections. Generally, GLGs were diﬃcult to identify in the dentine which
presented a lower age count than cementum. Thus cemental GLGs would appear to be the preferredtissue for age estimation in Longman’s beaked whale. Stained sections tended to produce higher GLG
counts, but at the moment it is unclear whether accessory lines may be highlighted and what time period

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AGE ESTIMATION FROM TEETH IN LONGMAN’S BEAKED WHALESa GLG represents. However, as suggested above, the norm in most mammals is an annual deposition of
GLGs (Gambell, 1977; Lockyer 1993; Hohn et al., 1999; Lockyer et al., 2016; Read et al., 2018). In thisstudy, the dentine did not provide clear diﬀerentiation of GLGs so that this tissue must be regarded as
unreliable for total age estimation in this species, based on this limited investigation.
Generally, dentinal GLGs are preferred for aging in cetacean teeth (Stewart and Stewart 2014;
Read et al., 2018). In Hyperoodon ampullatus, the dentinal GLGs in untreated tooth sections are thepreferred aging tool, as noted earlier (Christensen, 1972; Feyrer et al., 2020). This is demonstrated inFig. 8 where an untreated section (prepared in the same manner as Indopacetus teeth) shows up to 40
dentinal GLGs. There is no wear at the crown so that all GLGs are represented. There is limited information about aging in Ziphioid whales, and the authors note that teeth of Ziphius cavirostris (prepared

Fig. 9. Comparison of dentinal GLGs in a) untreated and b) decalciﬁed stained sections of teeth of Ziphiuscavirostris.
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in a similar manner as Indopacetus paciﬁcus teeth) presented good GLG resolution in both untreated
and decalciﬁed stained sections of dentine (Fig. 9). It should be noted however, that the cementum inboth Ziphius and Hyperoodon is thinner and less conspicuous than in Indopacetus (compare Fig. 4).
Additionally, unlike Ziphius and Hyperoodon, for which there have been past ﬁsheries (Japan, Norway) when some life history information was learned, there is almost nothing known about Indopacetus, including the extent of its distribution outside tropical waters, if at all. All that can be drawn fromthe methods tried presently is that cementum appears to be the best tissue to use for aging, and decalciﬁying and staining the tissue tends to reveal more detail and thus higher counts than the untreatedsections.
With respect to the presence of mineral resorption in the roots of the teeth of Indopacetus, the cause
is unknown (Fig. 4a, c). It is reported that high levels of cadmium (Cd) in the body can lead to interference in bone mineral structure, speciﬁcally calcium (Kazantsis, 2004; Sughis et al., 2011). Garrigue
et al. (2016) reported high concentrations of Cd in the kidneys and liver of #4, #5 and #6, but coincidentally noted that the lowest concentration was observed in #6, the only animal with no resorption.
In addition #4 had many pulp stones present in the dentine (Fig. 4b, c), the cause of which is again unknown. However, the presence of these structures made reading the dentinal GLGs very diﬃcult. Suchinclusions do not occur in cementum.
It is believed this is the ﬁrst time that teeth have been used for estimating age from GLGs in thisspecies, Indopacetus. These initial results are very promising and may be helpful to others who may
encounter similar specimens. The method may also be helpful in investigating some other beaked
whale species.

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Received: November 1, 2020
Accepted: March 5, 2021
Published online: May 12, 2021

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