Seasonal epidermal molt in beluga whales, Delphinapterus leucas

D. J. ST. AUBIN Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ont., Canada NI G 2 WI

T. G. SMITH Arctic Biological Station, Department of Fisheries and Oceans, 555 St. Pierre Blvd., Sainte Anne-de-Bellevue, Que.,

Canada H9X 3R4

AND

J. R. GERACI Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ont.; Canada NIG 2WI

Received March 30. 1989

ST. AUBIN, D. J., SMITH, T. G., and GERACI, J. R. 1990. Seasonal epidermal molt in beluga whales, Delphinapterus leucas. Can. J. Zool. 68: 359-367.

Epidermal morphology and proliferation were examined in beluga whales during three phases of their annual cycle: spring migration from oceanic wintering grounds, summer occupation of estuaries in Hudson Bay, and return migration in fall. Incursion into relatively warm brackish water was associated with decreased thickness of the stratum externum and sloughing of a superficial layer of degenerative epidermal cells, changes that resulted in the loss of a distinctive yellow hue apparent over the dorsal body surface of whales examined during spring migration. Proliferation rate, determined by incorporation of tritiated thymidine in germinal cells, averaged 13.8- 16.6% in all three seasons, but exceeded 20% in 7 of 16 whales examined in the estuaries; similarly high values were not observed during spring migration, and in only one of nine animals sampled in the fall. Average proliferation rate in 13 captive belugas was 14.2-16.6%, two to three times higher than any reported value for other cetaceans or terrestrial mammals. Epidermal turnover time in a single whale studied over a 6-week period was estimated to be 70-75 days, comparable to that in bottlenose dolphins, but indicating a much higher rate of cell migration. In estuaries, elevated temperature and low salinity are presumably responsible for accelerating turnover of superficial cells, and may contribute to elevated proliferation rates by stimulating blood flow to the germinal layer.

ST. AUBIN, D. J., SMITH, T. G., et GERACI, J. R. 1990. Seasonal epidermal molt in beluga whales, Delphinapterus leucas. Can. J. Zool. 68: 359-367.

La morphologie et la prolifkration de I'kpiderme ont fait l'objet d'une ktude chez des Bklugas durant trois phases de leur cycle annuel : la migration du printemps au retour des refuges ockaniques d'hiver, l'occupation des estuaires de la Baie d'Hudson en kt6 et la migration de retour i?i l'automne. Leurs incursions dans les eaux saumltres relativement chaudes coincidaient avec une diminution de l'kpaisseur de la couche externe et le rejet d'une couche superficielle de cellules kpidermiques en dkgknkrescence, changements qui ont entrain6 la perte de la teinte jaune distinctive qui ornait la surface dorsale des baleines au cours de la migration de printemps. La vitesse de prolifkration cellulaire, kvaluke par incorporation de thymidine tritike dans les cellules gerrninatives, Ctait de 13,8-16,6% en moyenne au cours des trois phases, mais elle dkpassait 20% chez 7 de 16 baleines examinkes dans les estuaires; des valeurs aussi klevkes n'ont jamais kt6 observkes au cours de la migration de printemps et observkes chez une seule des neuf baleines examinees i?i I'automne. La vitesse de prolifkration moyenne a kt6 estimke i?i 14,2- 16,6% chez 13 Bklugas en captivitk, ce qui correspond i?i deux ou trois fois la valeur ordinairement e~egistrke chez d'autres cktacks ou chez des marnrnifkres terrestres. Le remplacement complet de l'kpiderme chez une Bkluga ktudik au cours d'une @node de 6 semaines a durk 70-75 jours, durke comparable i?i celle qui a kt6 e~egistrke chez des Dauphins i?i gros nez, mais durke indicatrice d'un taux beaucoup plus klevk de migration des cellules. En estuaire, la temp6rature klevke et la salinitC faible sont probablement responsables du taux de remplacement accklkrk des cellules superficielles et contribuent probablement aussi aux taux klevks de prolifkration par stimulation du flux sanguin vers la couche germinative.

[Traduit par la revue]

Introduction discolored epidermis. These seasonal events are reminiscent of

T~~ distinct ans sf om at ions occur in the color of the epi- a" epidermal molt, an event without parallel in other Cetaceans.

dermis of beluga whales, Delphinapterus [eucas. Born slate investigate this process 9 and pro-

grey, belugas of both sexes gradually become white over a liferation rate were determined in belugas during different stages

period of 10- 12 years (Benin and vladykov 1940; Be19kovich of their annual cycle in the eastern Canadian Arctic and, for

1964; Brodie 197 1 ; Sergeant 1973). The process is independent comparison, in in three research and display

of the onset of reproductive maturity ( ~ ~ ~ d i ~ 1971; sergeant institutions. Accelerated turnover of superficial layers and

1973) and is mediated by a reduction in melanin pigment enhanced proliferation of germinal cells in whales sampled

granules (Geraci et al. 1986). within the estuaries provide novel experimental evidence for An unrelated change occurs annually in the appearance of the in

epidermal surface. During winter, whales of all age classes Materials and methods develop a yellow coating, particularly over the dorsal surfaces

The principal study areas are shown in Fig. 1. Spring (June - early Of the 9 and flukes (K1einenberg 964). The July, 1985 and 1986) and fall (October-November, 1983 and 1985)

is most apparent during and is lost as the samples were obtained from whales hunted by Inuit along the coast of animals move into river estuaries in summer. There, whales rub Hudson strait (Table 1). During ~ ~ l ~ - ~ ~ ~ ~ ~ t , 1983-1985, whales themselves on the gravel river bottom (Finley 1982; Finley el al. were sampled in the estuary of the Nastapoka River in eastern Hudson 1987), a behavior which appears to result in shedding of Bay.

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360 CAN. J . ZOOL. VOL. 68, 1990

FIG. 1. Epidermal samples were collected from belugas taken by Inuit hunting in Hudson Strait (arrowheads) during spring and fall and in the estuary of the Nastapoka River (arrow) during summer. Whales captured in the estuary of the Seal River (star) were held at a nearby field camp for a 10-week study.

Tissues were collected from the whales within 20-30 min after death. Proliferation rate was measured by incorporating [6 -3~] thy- midine into the nuclei of cells synthesizing DNA (Marks et al. 197 1). Up to four specimens from the lateral body wall of each whale were excised using a 6-mm diameter biopsy punch. Each specimen consisted of the full thickness of epidermis and 2-3 mm of underlying dermis. A 1-cm, 25-gauge needle directed to the base of the epidermal pegs was used to inject each specimen with 1 mL of saline solution containing 20 kCi of [6-3~]thymidine (1 Ci = 37 GBq) having a specific activity of 5 Ci/mmol (Amersham Corporation, Arlington Heights, IL) . Each sample was then placed in a tube containing 1-2 mL of the same solution and incubated for 3 h at 10- 15OC. Specimens were then transferred to 10% neutrally buffered formalin. Routinely prepared paraffin sections, 5 k m thick, were stained with Ehrlich's hematoxylin and processed for autoradiography (Brown et al. 1983). Proliferation rate, expressed as labelling index (LI), was determined as the percentage of labelled germinal cells, based on counts averaging 3000 germinal cells per animal.

Incorporation of [3~] thymidine was also determined in vivo in seven belugas (four males, three females) captured in the estuary of the Seal River in western Hudson Bay (Fig. 1) in July, 1985. One male was tested within 12 h after capture, and released. The others were maintained at a field camp for 10 weeks in pools containing seawater drawn daily from Hudson Bay. After a 3-week acclimation period, and at irregular intervals thereafter, the belugas were injected subepider- mally in up to three sites on the dorsolateral aspect of the body, with 0.1 rnL of a solution containing 25 kCi of [3~]thymidine. After 1 h, a 6-mm diameter biopsy punch was used to excise the labelled tissues, which were then placed in 10% neutrally buffered formalin. For comparison with findings from samples tested in vitro, both techniques were used concurrently in five of the captive belugas. Biopsies obtained for in vitro injection and incubation were treated as for the hunted whales; then, all tissues were sectioned, stained, and processed for autoradiography.

To quantify epidermal cell turnover rate (Hicks et al. 1985), one of the whales held at the field camp was injected subepidermally at each of six sites with the same [3~]thymidine solution described above. Biopsies were obtained after 7, 15, 2 1, 24, 32, and 42 days, placed in 10% neutrally buffered formalin, and processed for autoradiography. Labelling index was determined in vivo on each day that a biopsy was taken for the turnover study.

Proliferation rates were also determined in vitro in five belugas held at the New York Aquarium (NYA), New York, NY, one at Mystic Marinelife Aquarium (MMA), Mystic, CT, and one at the Naval Ocean

TABLE 1. Epidermal samples examined for morphologic studies and assessment of proliferation rate (M, mature; I, immature; nd, not

determined)

Males Females

Source Season M I nd M I nd

Hudson Strait Spring 1 5 - 6 0 Fall 4 5 - 4 5 -

Nastapoka River Summer 6 8 4 9 3 2 Seal River Summer 0 4 - 1 2 - NYA Fall 1 1 - 1 2 - MMA Winter l o - - - - NOSC Summer 1 0 - - - -

NOTE: Maturity judged by body length and color, and by examination of reproductive organs and mammary glands.

Systems Center (NOSC), Kaneohe Bay, HI. At NYA, three of the whales were sampled in November, 1984,3 months after capture in the Churchill River. Two of these were tested again 1 year later, at which time two belugas that had been in captivity for 6-8 years were also studied. The beluga at MMA was sampled in January, 1984, and had been in captivity at various institutions for 23 years; the NOSC whale was captured in the Churchill River in 1977 and was sampled in June, 1982.

For seasonal comparison of epidermal morphology, samples of normal dorsolateral skin were selected from 49 whales. Tissues were fixed in 10% neutrally buffered formalin, processed according to standard histological techniques, sectioned at 5 km, and stained with hematoxylin and eosin. A microscope equipped with a calibrated ocular lens was used to measure the thickness of the entire epidermis and stratum externum. Statistical analyses were performed using a Newman-Keuls ANOVA.

Results The appearance of the epidermal surface was visibly different

in whales sampled within each seasonal habitat. In spring, the dorsal body wall and dorsal aspects of the flippers and flukes were distinctly yellow in color. The discoloration was particu- larly evident on white animals, but could also be observed on close inspection of grey juveniles. In addition, the skin of the head, neck, and mid-back was variably covered by a tenacious, fm layer up to 3-4 mm thick (Fig. 2).

Histologically, three strata are recognized in the epidermis of cetaceans: stratum externum, which consists of squamous parakeratotic cells and corresponds to the stratum corneum of terrestrial mammals, stratum intermedium, which is compara- ble to the stratum spinosum, and stratum germinativum (Harri- son and Thurley 1974; Geraci et al. 1986). In the dorsal epidermis of belugas sampled during spring migration, there was also a layer of irregular polyhedral cells overlying a relatively normal layer of flattened stratum externum cells; no superficial layer of squamous parakeratotic cells covered this zone of degenerated epidermal cells (Fig. 3). Foci of bacteria and unicellular organisms were observed within the zone, with no evidence of inflammatory reaction; underlying epidermal layers were morphologically normal. The layer of degenerated epidermal cells was also observed on some of the animals sampled within the estuary. However, in this setting, the tissue was hydrated and friable, and easily rubbed off to reveal an intact stratum externum below. Superficial cellular debris was not noted on any whale sampled in October or November, nor was the yellow hue on the dorsal aspect of flippers, flukes, and body wall.

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ST. AUBIN ET AL.

FIG. 2. Left view of the head region of a beluga taken during spring migration. The discolored area (outlined by dots) on the melon and dorsal thoracic region is raised above the level of the surrounding normal epidermis.

Measurement of stratum externum thickness revealed signifi- TABLE 2. Epidermal measurements (mm, 5 SD) in free-ranging cant seasonal differences (Table 2, Fig. 4). The layer was belugas thickest in spring samples and thinnest in fall; summer speci- mens were intermediate, with a much wider range than the other Sampling No. of Total Stratum two groups. All determinations were made on histologically whales thickness externum

- -

normal epidermis and thus, did not include specimens with superficial cellular debris.

In the germinal layer of epidermis collected during the summer, numerous mitotic figures were evident (Fig. 5). Cells in metaphase were obvious. Those in earlier and later stages of cell division were more difficult to identify; therefore, the technique of isotope labelling was employed to provide a more objective index of germinal cell proliferation.

The in vitro technique for labelling cells with [3~]thymidine was effective and reliable and, as in humans (Lachapelle and Gillman 1969), compared favorably with results obtained in vivo . Duplicate samples, injected and processed independently, were within 10% of the average value. Labelling indices determined in vivo in four captive whales were, on average, 6.4% higher than when determined by the in vitro technique (1 3.6% vs. 12.4%). Reproducibility of counts of labelled cells was assessed in five serial sections of a single biopsy from a captive beluga injected in vivo. In 19 independent counts by two observers, mean LI was 13.4 2 2.1% (CV, 15.7%); overall mean LI for 20 590 cells was 13.3%.

There was no significant difference in mean LI among the three groups, representing spring, summer, and fall (Table 3). However, the distribution of data within each group showed important differences (Fig. 6). The highest LI observed in samples collected in spring was 18.4%. By contrast, nearly half the whales examined in summer from the Nastapoka and Seal River estuaries had LIs greater than 20%, or more than 2 S D above the mean value for the spring sample; maximum LI in summer was 26.4%. In fall, only one of nine had an LI above

Spring 12 7.98k0.49 0.54k0. Summer 23 8.13k 1.34 0.35k0.32" Fall 14 8.81+1.11 0.21 k 0 . 0 7 ~

" p < 0.05 between the mean values indicated. bp < 0.01 between the mean values indicated.

20%. In six captive belugas sampled in November and January, LI averaged 14.7%; the captive whale tested in June had an LI of 13.3%.

Epidermal turnover was determined by charting the migration of labelled cells released from the germinal layer to their eventual disappearance from the stratum externum (Hicks et al. 1985). This was attempted in a brief study on one of the belugas held temporarily at an Arctic field camp. In samples collected 1 week after exposure to the isotope, most of the labelled cells were still within the germinal layer (Fig. 7). After 2 weeks, 46% of the cells had migrated away from the papillae, and were 2-4 rows from the stratum germinativum. By 21 -24 days, virtually all of the labelled cells were within the epidermal pegs, and some had reached the middle of the stratum intermedium. In the biopsy collected on day 42, the majority was in the mid-stratum intermedium; a few were at the base of the stratum externum. The study was concluded at that time because of the scheduled release of the whale. Assuming a constant rate of migration once the cells are released into the stratum intermedium, an average turnover time of 70-75 days was estimated.

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CAN. J. ZOOL. VOL. 68, 1990

FIG. 3. (A) Vertical section of the epidermis from the dorsum of a beluga sampled during spring migration. A layer of degenerative epidermal cells (dec) lies outside of the stratum externum (se); si, stratum intermedium; d, dermis. Scale bar, 2 mrn. (B) At higher magnification, an intact stratum externum can be demonstrated under the superficial cellular debris. The underlying stratum intermedium is normal. Scale bar, 500 Fm. (C) Colonies of bacteria and unicellular organisms are evident within a vertical section of the degenerative layer of epidermal cells. Scale bar, 100 Fm.

Discussion Growth and replacement of cetacean epidermis is generally

viewed as a continuous process (Ling 1972, 1984). This perception probably applies to most species occupying environ- ments that show little seasonal variation. In this respect, belugas are an exception. Their unique pattern of habitat utilization sets the stage for cyclical events in the epidermis, changes which can best be described as a molt. Before examining the evidence for such a process, it is useful to review the general characteristics of epidermal growth in this species.

In virtually all captive and free-ranging beluga whales examined, average proliferation rate determined by isotope incorporation was two to three times higher than has been reported for any other mammal (Stem et al. 197 1 ; Kreuger and Shelby 198 1 ; Camplejohn et al. 1984), including the bottlenose dolphin, Tursiops truncatus, the only other cetacean studied using this technique (Brown et al. 1983). This finding supports observations by Bel'kovich (1964) of numerous dividing cells in beluga epidermis. The most remarkable example of enhanced proliferation came from a whale tested 3 weeks after capture in

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ST. AUBIN ET AL. 363

FIG. 4. The stratum externum (se) is thickest in samples collected during spring migration (A), intermediate in samples from estuarine whales in summer (B), and thinnest during fall migration (C). Scale bar, 200 p,m.

TABLE 3. Epidermal proliferation rate determined by the incorporation of tritiated thy- midine (labelling index) in free-ranging and captive beluga whales (tests were performed

in vitro, except as noted)

Labelling index No. of Total no.

Sample whales of cells .i k SD Range Median

Free-ranging Spring 11 15 372 13.8k3.0 9.1-16.9 13.7 Summer 16 61 033 16.6k6.3 5.9-24.8 18.2 Fall 9 29 694 15.9k6.5 7.1-31.2 17.3

Captive Aquaria 7" 69 539 14.2k4.6 7.1-21.7 14.4 Field camp

In vitro 5 10 695 16.5k9.0 8.7-32.7 12.1 In vivo 7 19 861 16.6k 14.4 4.1-57.6 12.5

"Two whales were sampled on two occasions, a year apart, for a total of nine samples. whale was sampled on two occasions, and another on three, for a total of ten samples

the Seal River estuary. Over 50% of the germinal cells incorporated labelled thymidine following a single injection in vitro. Subsequent tests on the same whale during the next 7 weeks yielded values in the range of 10-15%, consistent with findings in the other captive whales. Such marked, but brief stimulation might be an exaggeration of a natural cycle, perhaps an artefact of captivity, but is nonetheless noteworthy in reveal- ing the remarkable capacity of epidermal cells to proliferate.

Turnover rate was examined over a 6-week period in the same

captive beluga. The pattern of cell migration was similar to that observed in Tursiops, but ,the rate was much greater (Hicks et al. 1985). After a delay of 1-2 weeks, labelled cells moved rapidly through the stratum intermedium, reaching the surface 4-5 weeks later. Assuming these cells originated from even the tips of the dermal papillae, they would have moved at least 4 mrn in approximately 30 days (0.13 mmlday), a rate roughly six times faster than for Tursiops. It would be imprudent to suggest that these observations on a single whale, tested at a time when

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364 CAN. I. ZOO L. VOL. 68, 1990

SPRING

FIG. 5 . Mitotic figures in epidermis from a beluga sampled during summer in the Nastapoka estuary. The stratum germinativum is 5-6 cell layers thick along a dermal papilla, sectioned in a plane perpendicular to the stratum externum. Scale bar, 100 Fm.

epidermal proliferation was perhaps maximally stimulated, are representative for the species. Nevertheless, the results demon- strate that epidermal turnover in belugas might, at times, be faster than expected (Geraci et al. 1986).

The suggestion that beluga epidermis is "renewed annually stems more from the grossly observable loss of discolored epidermis than from quantitative assessment of mitotic activity in the germinal layer. In spring, ,the epidermis is covered by a relatively thick stratum externum, suggesting that sloughing rate is relatively slow during the 8-10 months that belugas spend in areas where water temperature rarely exceeds 5°C. Intercellular lipids, which contribute to cellular cohesion, would be broken down more slowly at such temperatures (Menon et al. 1986), thereby prolonging the attachment of superficial cells. Incursion into warm water during summer months accelerates the rate of cellular dissociation, and the externum becomes significantly thinner by the time the whales leave on their fall migration.

In estuaries, belugas also shed a layer of degenerating cellular debris that is often present along the middorsal ridge. The nature and distribution of this material gives a clue as to its origin. During winter, belugas seek open water habitats, but neverthe- less must deal with newly forming ice in the leads, often by using their heads and backs to break through young ice up to 8 cm thick (Bel'kovich and Tarasevich 1964; Sergeant 1973). Abrasion against the undersurface of the ice might damage the stratum externum, exposing underlying epidermal cells to the

SUMMER

FALL

LABELLING INDEX (%) FIG. 6 . Proliferation rate, expressed as the labelling index (LI), was

determined on the basis of incorporation of tritiated thyrnidine. Seven of 16 whales sampled during the summer had an LI greater than 20%, or more than 2 SD above the mean LI for the spring samples; only one whale from the fall had a comparable LI.

osmotic forces of the sea. Left unprotected, cells of the stratum intermedium degenerate and are eventually isolated by rapidly maturing keratinocytes which form a new parakeratotic layer below. Similar changes have been observed following con- trolled incisions through the stratum externum in Tursiops (Bruce-Allen and Geraci 1985) and belugas (Geraci and Bruce-Allen 1987) and during the course of generalized epidermal sloughing in captive belugas (St. Aubin 1988).

Once externalized, the zone of degenerated epidermal cells should be readily sloughed, but appears to remain fixed to the surface for some time, judging from the abundance of microor- ganisms colonizing the devitalized tissue. Its persistence may

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~ I G . 7. Skin biopsies were collected 7 (A), 2 1 (B), 32 (C), and 42 (D) days after subepidermal injections of tritiated thymidine in a captive be1 ale. Intensely labelled cells are confined to the stratum germinativum for the 1 st week (A), move away from the papillae after 3 weeks (B), tributed throughout the stratum intermedium by 4-5 weeks (C), and begin to reach the surface after 6 weeks (D). Scale bar, 1 rnrn for A-C I mrn for D.

luga , are and

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also reflect the slow breakdown of intercellular macromolecules Sciences and Engineering Research Council of Canada (grant at low ambient temperatures. When whales reach the estuary, A6130 to J.R.G.), the World Wildlife Fund (grants to T.G.S. the unprotected cellular debris becomes hydrated, loses its and J.R.G.), and the Department of Fisheries and Oceans cohesiveness, detaches from the underlying stratum externurn, (through R. Moshenko, Section Head). and is sloughed.

A warm estuarine environment not only modifies the general appearance of the epidermis, but it seems to have a stimulatory effect on proliferation of germinal cells. The change might simply reflect passive elevation of skin temperature, which in turn would enhance the rate of all cutaneous metabolic processes. The suggestion parallels that of Feltz and Fay (1966), who proposed that pinnipeds haul out during molting periods to allow the epidermis to reach a temperature high enough to support epidermal growth and hair replacement. The critical temperature for pinniped epidermis was 15"C, within the range found in most estuaries occupied by belugas.

Increased ambient temperature would also promote cutane- ous blood flow, bringing nutrients and hormones capable of stimulating epidermal proliferation. Epidermal growth factor (EGF) (Cohen 1972), growth hormone (GH)(Holt and Marks 1976), and thyroid hormones (Holt et al. 1976; Holt and Marks 1977; Holt 1978) are among the substances that can signifi- cantly influence the mitotic activity of epidermal cells. There are as yet no data on EGF or GH in belugas. However, relatively high circulating levels of thyroxine and triiodothyronine have been observed in belugas within the estuarine environment (St. Aubin and Geraci 1988). The coincidental occurrence of enhanced epidermal growth and thyroid secretion is intriguing in view of the analogy to hormonally regulated molts in pinnipeds (Riviere et al. 1977; Ashwell-Erickson et al. 1986) and terrestrial mammals (Mohn 1958; Reineke et al. 1962; Boissin-Agasse et al. 198 1; Johnson 1984).

The proposed annual cycle in epidermal growth in belugas has no documented counterpart among cetaceans, though similar patterns might exist in species that migrate annually between polar feeding areas and tropical calving grounds. Such marked changes in environmental conditions can influence epidermal growth and turnover directly, or indirectly through the action of mediators such as thyroid hormones. For belugas, the process appears to be intimately associated with occupation of estuaries during summer months. Yet, it is likely that not all whales reach estuaries each year; some are observed in offshore waters throughout the summer (Smith and Hammill 1986). Is epidermal turnover retarded in such individuals? How critical is the loss of superficial accumulations of old epidermis? In answering these questions, we may gain some understanding of the importance of the beluga's dependence on the estuarine habitat.

Acknowledgements We thank D. W. Doidge, D. Hope, G. Horonowich, J. Orr,

and S. Waters (Department of Fisheries and Oceans), and T. Friesen, H. Mitchell, and C. Thomson (University of Guelph) for their support in the field studies, and W. Brown, J. Hunter, and L. Latta (University of Guelph) for laboratory assistance. L. Garibaldi (New York Aquarium), S. Spotte (Mystic Marinelife Aquarium), and Dr. P. Schroeder (Naval Ocean Systems Center) kindly provided access to captive whales. We are also grateful to the Inuit hunters of northern Quebec for their cooperation during the sample collection, John and George Hickes and their crew for live capture of whales, V. Lounsbury for cartography, and Dr. M. Fallding for reviewing the manuscript. Financial support was obtained from the Natural

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