Sexing beluga whales (Delphinapterus leucas) by means of DNA markers

MOIRA W. BROWN Department of Zoology, University of Guelph, Guelph, Ont., Canada NlG 2 WI

REE HELBIG' AND PETER T. BOAG Department of Biology, Queen's University, Kingston, Ont., Canada K7L 3N6

DAVID E. GASKIN Department of Zoology, University of Guelph, Guelph, Ont., Canada NlG 2WI

AND

BRADLEY N. WHITE' Department of Biology, Queen's University, Kingston, Ont., Canada K7L 3N6

Received May 1, 1990

BROWN, M. W., HELBIG, R., BOAG, P. T., GASKIN, D. E., and WHITE, B. N. 1991. Sexing beluga whales (Delphinapterus leucas) by means of DNA markers. Can. J. Zool. 69: 1971-1976.

Few methods are available for determining the sex of free-ranging individual whales, dolphins, and porpoises of species that are not obviously sexually dimorphic. We have developed a technique for sexing beluga whales (Delphinapterus leucas) by using a Y-chromosome-specific DNA restriction fragment. Genomic DNA was extracted from liver samples of 18 beluga whales (9 males, 9 females) sexed at dissection. DNA from males and females was digested with five restriction enzymes, electrophoresed, and transferred to membranes by Southern blotting. When probed with the labelled human Y-chromosome zinc finger protein gene probe pDP1007, male-specific bands and bands common to both sexes, but more intense in females than in males, were observed. The DNA digested with EcoRI provided the clearest sex-discriminating banding pattern. Even when DNA of various qualities digested with EcoRI was used, all the males showed a 3.4-kilobase (kb) band, presumably from the Y-chromosome, as well as a 2.1-kb band. Females showed the 2.1-kb band, but all lacked the 3.4-kb band. This 3.4-kb EcoRI male-specific band unambiguous sex determination, which will facilitate examination of sex-related differences in population structure and habitat use of belugas, which have important implications for management decisions.

BROWN, M. W., HELBIG, R., BOAG, P. T., GASKIN, D. E., et WHITE, B. N. 1991. Sexing beluga whales (Delphinapterus leucas) by means of DNA markers. Can. J. Zool. 69 : 197 1-1976.

11 existe peu de mCthodes qui permettent de distinguer le sexe chez les espkces de baleines, dauphins ou marsouins en nature qui n'ont pas de dimorphisme sexuel Cvident. Nous avons mis au point une technique de dktennination du sexe chez des bklugas (Delphinapterus leucas) en utilisant un fragment d' ADN du chromosome Y obtenu par traitement aux endonuclCases de restriction. De I'ADN gCnomique a CtC extrait d'kchantillons de foie chez 18 bClugas (9 mdles, 9 femelles) reconnus par dissection du systkme gknital. L'ADN de mdles et de femelles a CtC digCrC par plusieurs enzymes de restriction, soumis i 1'Clectrophorkse et transfCrC i des membranes par la mCthode Southern. En prCsence de la sonde marguke spCcifique au gkne du chromosome Y humain codat pour la protCine digitCe stabilisCe par le zinc (ZFY), le pDP1007, des bandes spCcifiques aux mdles et des bandes communes aux deux sexes, mais plus intenses chez les femelles, sont appaiues. L'ADN digCrC i 1'EcoRI est celui qui a permis d'obtenir les bandes les plus claires et les plus discriminantes du sexe. Meme en utilisant des ADN de diverses qualitks digCrCs i l'EcoRI, tous les mdles ont produit une bande de 3,4 kb, que l'on suppose relike au chromosome Y, et une bande de 2,l kb. Toutes les femelles ont produit la bande de 2,l kb, mais aucune n'a produit la bande 3,4 kb. Cette bande de 3,4 kb EcoRI spkcifique aux mdles donne une dktermination sClre du sexe et permettra l'examen des diffkrences relikes au sexe dans la structure de la population et l'utilisation de l'habitat, facteurs qui auront des impacts importants sur les dCcisions relatives i I'amCnagement.

[Traduit par la rkdaction]

Introduction belugas in the Nastapoka estuary (eastern Hudson Bay,

studies of the social stmcture and population biology of ~uebec) . However, thedrawback of this technique is that only

beluga whales ( ~ ~ l ~ h i ~ ~ ~ ~ ~ ~ ~ ~ leucas) have depended on reproductively active females are sexed, and correct identifica-

inference of an individual9 s sex from field observations of size 'ion - of - the - calf - s mother may be confounded by the presence

differences, association patterns, and behaviours assumed to be Of Other

sex specific. Size differences are used during aerial surveys to Few direct methods for sexing cetaceans in the are

distinguish between male and female belugas (T. G. smith, available, and their success is often specific to the species or

personal communication). Although adult male belugas are to a particular situation. Cetacean species with sex-specific

larger than females (Sergeant and Brodie 1969; Finley et a/. differences in m o r ~ h o l o g ~ and (or) behaviour are rare and the

1982), the utility of size differences for determining sex is observational criteria are not necessarily reliable unless

limited by age-related variation in size. caron and smith substantiated by other means (Matthews et al. 1988; Lambert-

(1990) used persistent adult-calf associations to identify female Sen 1988). In some Vecies (such as and right whales, Eubalaena spp.), it is possible to determine an

'Present address: Department of Biology, McMaster University, individual's sex by observing the genital region during above- Hamilton, Ont., Canada L8S 4K1. water activities (Caron 1988; Kraus et al. 1986; Payne et al.

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1972 CAN. J . ZOOL. VOL. 69, 1991

1983). This technique is of limited application in most species that exhibit little above-water activity. Glockner's (1983) method of sexing humpbacks whales (Megaptera novae- angliae) visually underwater is reliable, but depends on observa- tion in clear water and on close proximity of the observer to the whale. Wells (1986) used temporary-capture techniques to determine the sex of individual bottlenosed dolphins (Tursiops truncatus), but this method is not practical with larger ceta- ceans.

As an alternative, some researchers have employed karyo- typing and sex chromatin (Barr body) analysis to try to distinguish between male and female cetaceans. In mammals, males have one X-chromosome and one Y-chromosome whereas females have two X-chromosomes, one of which is inactivated in the form of a Barr body (Barr 1966). Winn and his co-workers (1973) were able to confirm the sex of eight individuals from three species of odonocetes (two belugas, two sperm whales, Physeter catadon, and four True's porpoises, Phocoenoides dalli truei) of known sex by the presence (females) or absence (males) of sex chromatin bodies in skin cells. Subsequent attempts to use sex chromatin analysis to determine the sex of mysticetes have met with little success. Hoelzel et al. (1983) examined 17 skin samples from three species of baleen whales of known sex and found no relation- ship between the occurrence of sex chromatin bodies and the sex of the animal. Nor could Matthews (1986) distinguish between male and femak gray whales (Eschrichtius robustus) because "sex chromatin-like structures" were present in similar proportions in both sexes. Both Hoelzel et al. (1983) and Matthews (1986) found inconsistencies in counts of sex chromatin bodies from two tissue samples from the same individual.

Sex chromatin analysis was initially more attractive because it makes use of fixed, prepared tissues, but the lack of consis- tent results forced cetacean researchers to try other methods. Karyotyping permits examination of the general appearance and structure of the chromosomes in the nucleus, but this technique requires the establishment and maintenance of healthy living cell cultures from a sterile tissue sample which may not be a simple undertaking in remote land- or sea-based operations. Lambertsen et al. ( 1988) successfully karyotyped fibroblast cultures from 10 of 32 Alaskan humpback whales, using skin obtained by remote biopsy (Lambertsen 1987) from free-ranging whales. Chromosomal identification of sex was used to verify the sex of the 10 karyotyped individuals (Lambertsen et al. 1988). Using the fibroblast cultures, the success rate relative to the number of samples obtained was low (3 1 %) and the authors admitted that further refinement of culture techniques was needed.

Determining the sex of individual cetaceans is important for understanding social behaviour, mating systems, and population dynamics. The purpose of the present study was to develop a reliable method for determining the sex of cetaceans by means of DNA markers. Our objective was to establish a method that could be applicable to other species of cetaceans when sex determination was important for elucidating social structure and sex-specific roles in situations where external identification of sex is not possible. Belugas were selected for the initial study because tissue is readily obtained during an Arctic Inuit subsistence hunt, and the sex of each specimen was established at dissection. We chose the human Y-chromosome sexing system rather than an X-chromosome sexing method to avoid

the confounding effects that can arise from comparing relative band intensities and because of the consistent results obtained with a wide variety of mammalian species by Page et al. (1987). This paper represents work that was done as part of a larger study examining the genetic structure of belugas to compare the genetic relatedness of the six 'management stocks' designated by the Department of Fisheries and Oceans (St. Lawrence River, eastern and western Hudson Bay, Cumberland Sound, the High Arctic, and the Beaufort Sea) in Canadian waters (Helbig et al. 1989).

Materials and methods Restriction enzymes were obtained from Bethesda Research

Laboratories. Gene Screen Plus'" nylon membrane and ',P-labelled dCTP were obtained from New England Nuclear Corporation, Mississauga, Ont., and Immobilon-N'" membrane was obtained from Millipore, Mississauga, Ont. The zinc finger probe (ZFY), pDP1007, was generously donated by D. C. Page (Page et al. 1987). Probe pDP1007 is a 1.3-kilobase (kb) HindIII fragment in plasmid pDPl007 from the 1A2 interval of the human Y-chromosome and has been shown to recognize a single or low-copy sequence on the Y-chromo- some of some mammals (Page et al. 1987).

Tissue sample collection Samples of liver tissue were obtained from belugas killed in the

1987 Inuit hunts on the Nastapoka River (56"55'N, 76"33'W) in eastern Hudson Bay, at Eskimo Point (61°06'N, 93"59'W) in western Hudson Bay, and in Grise Fiord (76"35'N, 83"14'W) on South Ellesmere Island. Samples were obtained through the cooperation of the Arctic Biological Station, the Freshwater Institute, and the Fisheries Joint Management Committee. The tissue samples were collected on the hunting grounds and frozen as soon as possible (up to 7 h post mortem). Samples were shipped to Queen's University on frozen ice paeks, or on dry ice when available, and stored at -20°C upon arrival.

DNA extraction and restriction enzyme digests DNA was extracted from liver tissue samples by means of an

Applied Biosystems Nucleic Acid Extractor (ABI), Model 340A, using techniques similar to those described in Helbig et al. (1989). Samples of frozen liver (0.18-0.30 g) were ground to a fine powder with a mortar and pestle cooled by liquid nitrogen and suspended in 3.5 mL of ABI lysis buffer (4 M urea, 0.2 M NaCl, 0.1 M Tris-HC1, pH 8.0, 0.5% n-lauroylsarcosine, 10 mM EDTA) for 24 h. This was followed by a proteinanse-K digestion (72 units in 0.5 mL) at 37°C for at least 16 h. The samples were loaded onto the ABI automated extractor which performed two extractions with equal volumes of phenol and chloroform (5050) and one extraction with chloroform. The DNA was then precipitated with sodium acetate to a final concentration of 0.15 M and two volumes of 95% ethanol, collected on Teflon-coated filters, rinsed with 70% ethanol, and redissolved in 0.5 rnL TNE, (10 mM Tris-HC1, 10 mM NaCl, 2 mM EDTA, pH 8.0) overnight at 65°C.

Genomic DNA (5 kg) was cleaved with EcoRI, HindIII, PstI, Hinfl, and AluI restriction enzymes, under the conditions recom- mended by the manufacturer, Bethesda Research Laboratories, using 3 units of enzymelkg DNA.

Agarose gel electrophoresis and Southern blots DNA restriction fragments were separated electrophoretically

through a 0.8%, 20 cm long agarose gel run for 12 h at 35 V. The DNA fragments within the gel were depurinated by acid treatment and transferred to a positively charged membrane (Gene Screen plusTM or Immobilon-N) by the capillary blot procedure, following the techniques of Southern (1975) and the manufacturer's guidelines (New England Nuclear and Millipore, respectively). The membranes were dried at room temperature, then baked in a vacuum oven for 2 h (1 h for Immobilon-N) at 80°C.

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NOTES

FIG. 1. Identification of male-specific restriction fragments in belugas by means of the ZFY probe. DNA from one female (a) and one male (H) beluga (from eastern Hudson Bay) were digested with EcoRI, HindIII, PstI, HinfI, and AluI and blotted on a nylon membrane (Gene Screen PlusTM) and hybridized with the human Y zinc finger protein gene probe pDP1007. After a 4-day exposure, the autoradiograph shows male- specific bands and bands common to both sexes. The size marker in lane 1 is h-EcoRI-digested fragments.

Probe labelling and hybridization some zinc finger protein gene probe (ZFY) pDP1007 (Page et al. The baked membranes were washed in 0.1 X 0.15 M NaCl + 1987) was radioactively labelled by primer extension using 50 kCi

0.015 M sodium citrate (SSC) and 0.5% SDS for 1 h at 65OC and (1 Ci = 37 GBq) of 3 2 P - d C ~ ~ to specific activities of 7 X 10' - 1.5 prehybridized overnight in a solution of 10% dextran sulfate, 1% X lo9 dprnlkg (1 dpm = 0.0167 Bq) (Feinberg and Vogelstein 1984). denatured salmon sperm DNA, and 1% SDS. The human Y-chromo- Blots were hybridized with labelled pDP1007 overnight at 65°C. The

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CAN. J . ZOOL. VOL. 69, 1991

X................

FIG. 2. Confirmation that the 3.4-kb EcoRI fragment was male-specific. Probe pDP1007 was hybridized with EcoRI-digested DNA from eight females (a) and eight males (m) blotted on Immobilon-N. The DNAs in lanes 2-6 and 10-12 are from eastern Hudson Bay belugas; western Hudson Bay belugas are represented in lanes 7, 8, and 13, and belugas from the High Arctic in lanes 9 and 14-17. The size marker in the left lane is A-HindIII-digested fragments.

blots were washed twice in 2 X SSC and 0.1% SDS at room temperature, then transferred to 1 X SSC and 0.5% SDS for two washes of 15 min each, one at room temperature, the second at 65°C. Once washed and air-dried, the blots were autoradiographed for 4-5 days with Dupont Cronex film and Dupont Lightning PlusTM intensify- ing screen at -70°C.

Results To identify restriction fragments specific to the Y-chromo-

some, DNA (5 pg) from one male and one female beluga from the Nastapoka estuary (eastern Hudson Bay) was digested with EcoRI, HindIII, PstI, Hinfl, and AluI, and hybridized with the human ZFY probe pDP1007. The beluga DNA hybridized well with the human probe and provided clear signals on the autoradiographs. The probe detected male (presumably Y)

specific bands with all five enzymes (Fig 1). The probe also recognized fragments common to both sexes with a signal approximately twice as intense in the female as in the male. This presumably represents the homologous X-chromosome sequences found in humans and other mammals (Page et al. 1987). The pattern of a band specific to the male and a band common to both sexes was present in each of the enzymes tested (Table 1). The results from the EcoRI-digested DNA provided the clearest difference between male and female; the male showed a band at 3.4 kb that was absent in the female.

To confirm the 3.4-kb band as a male-specific marker rather than a polymorphism, DNA (5 pg) from eight male and eight female belugas from three different management stocks in eastern Hudson Bay (three males and five females), western

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NOTES 1975

TABLE 1. Genomic restriction fragments of one male and one female segregation which is important in conservation and manage- beluga whale produced by five enzymes hybridized with ZFY probe ment. For example, the belugas found in the Nastapoka estuary

pDP 1007 during summer continue to be so heavily exploited that present harvest levels may well jeopardize the survival of this popula-

Fragment size (kb) tion (Finley et al. 1982). These site-tenacious belugas, believed

EcoRI Hind111 PstI HinfI AluI

Hudson Bay (one male and two females), and Grise Fiord (four males and one female) were examined. All had been sexed at dissection. The 3.4-kb band was present in all males and absent in all females (Fig. 2). In addition, the 2.1-kb band common to both sexes was generally more intense in females than males. The quality of the DNA used was variable and significantly degraded in some cases. This correlated with the fact that the liver samples had thawed in transit. Even with these badly degraded DNAs, it is possible to determine unequivocally the beluga's sex by the presence or absence of a 3.4-kb band in EcoRI-digested DNA.

Discussion Using Y-specific DNA markers provides a reliable method

for determining the sex of individual belugas. Since differentia- tion of the sexes by means of Y-specific markers is based on the presence or absence of a particular band and not on the relative intensity of the signal, DNAs of variable quality still gave unambiguous results. One advantage of this approach over cytogenetics for the field biologist is that once tissue samples are obtained they need only be frozen or pickled (Helbig et aC 1989, Seutin et al. 1991). Amos and Hoelzel (1990) found that although freezing at -20°C or below was the best preservative, DNA of sufficient quality be obtained from whole-skin samples preserved in a pickling solution of 20% dimethylsulphoxide (DMSO) in water saturated with sodium chloride and varying amounts of ethylenediamine- tetraacetic acid (EDTA).

The liver tissue samples for the present study were obtained from dead belugas. Beluga skin is also a viable source of DNA for molecular studies. Helbig et al. (1989) report good DNA yields of 300-500 k g from 0.15-0.18 g of both dermis and epidermis of stranded belugas. High-molecular-weight DNA has been extracted successfully from both dermal and epider- mal skin tissue from humpback whales (Baker et al. 1987), right whales (Schaeff et al. 1991), and killer whales (Orcinus orca) (Amos and Hoelzel 1990) collected by remote biopsy sampling. It is also possible to obtain skin samples from belugas in this manner. In 1988, T. Smith (unpublished data) successfully obtained, using remote sampling techniques, 30 skin biopsy samples from free-ranging belugas, from which high-molecular-weight DNA has been extracted (unpublished data).

Molecular methods of sex determination are a powerful tool that can be used to confirm an individual beluga whale's sex, which could previously only be inferred by associations size and (or) assumed sex-specific behaviours. With further field research on animals of known sex, we expect this tool will help researchers to describe less obvious sex-related roles and behaviours and address theories on maternal behaviour patterns and sex ratio, as well as provide information on sexual

to be mostly females and their calves, may represent the reproductive core for this 'stock' (Finley et al. 1982); Smith and Hammill 1986; Caron and Smith 1990). Calf counts can be made in the field, and remote skin biopsy sampling and subsequent molecular determination of sex (of recognizable individuals where possible) would provide enough information to confirm the sexual composition of the adult population and estimate calf production without the need to kill animals to obtain such base-line data. In addition, the DNA, once ex- tracted, is available for a suite of other genetic analyses that can provide information on a population's taxonomic status, level of genetic variability, and family relationships.

Acknowledgements We thank T. G. Smith and D. W. Doidge from the Arctic

Biological Station in Sainte Anne-de-Bellevue, Quebec, and J. W. Clayton at the Freshwater Institute in Winnipeg, Mani- toba, for their generous cooperation in obtaining tissue samples. We are grateful to David Page (Whitehead Institute, Cambridge, Massachusetts) for providing us with the human Y- chromosome zinc finger protein gene probe, pDP1007. We thank Andrew Read, whose review comments improved the manuscript. The support for this work was provided by operating grants to B.N.W., P.T.B., and D.E.G. from the Natural Sciences and Engineering Research Council of Canada (NSERC), and funding to B.N.W. by the St. Lawrence Action Plan, Department of Fisheries and Oceans. M. Brown was supported by an NSERC scholarship, and R. Helbig received a Lerner-Gray award from the American Museum of Natural History.

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Erratum: Organization of the adluminal and retractor cells in the coelomic lining from the tube foot of a phanerozonian starfish, Luidia foliolatal

MICHAEL J. CAVEY Musculo-Skeletal Research Group, Department of Biological Sciences, University of Calgary, Alta., Canada T2N IN4

AND

RICHARD L. WOOD Department of Anatomy and Cell Biology, School of Medicine, University of Southern California,

Los Angeles, CA 90033, U. S. A. (Ref. Can. J. Zool. 69: 91 1-923. 1991.)

The title of this article as given above the English and French abstracts on p. 9 11 was incorrect, and should be as shown above.

'Received at NRC July 16, 1991. Rinted in Canada i Irnprirnt au Canada

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