Hereditas 109,245-251 (1988)

The karyotype of the long-finned pilot whale, Globicephala melaena LISELO'TTE WESLEY ANDERSEN' and URSULA FRIEDRICH'

Department of Ecology and Genetics' and Department of Human Genetics2, University of Aarhus, Denmark

ANIX-.K5E\. 1.. W and FKIl-.I)KICH. 1J. I O X X . 'The karyotype o f the long-lmned pilot whale, Glohicephula melaena. - Heredim 109: 245-2.51. Lund. Swedcn. ISSN oOlX-066l. Received December 28. 1987

The chromo\omes ohtained by culturing lymphocytes from blood samples of the long-finned pilot whale Glohiwphula meluena, were htudied by Q- and C-banding. The culture method was very difficult to handle Thc karyotype of the long-finned pilot whale has a iypical cetacean ?n = 44 chromosome numhcr. The U- hand revealed a characteristic handing pattern, which showed a heteromorphism between individuals but ndt within individuals. These observations were supported by the C-banding, which disclosed clear size var- iation o f heterochromatin in some chromosome pairs. A species specific C-band pattern was observed in coinpari~iin between Glohicephala melacnu and Ghhirephulu macrorhynchuy, the short-finned pilot whale. 0- and C-band markers can be used in family- and population studies i f a good technical quality can be nchicved. The finding of an inactivation of one of the X-chromosomes in females opens the possibility of using thc method of staining the sex chromatin tn determine the sex dsmal l foetuses ( < I 2 cm).

I.i.selotw Wesley Andersen, Depanment of Ecolo~y and f;eaeric.s. Aarhirs University, N y Munkegade, Build- ing 5.70. L)K-8000 Aurhus C, Denmark

The cetacean karyotype is well known and de- scribed by ARNASON (1974). The cetaceans are di- vided into two suborders, the Mysticeti and Odon- toceti, but there is no pronounced difference in the characteristic karyotype of the two suborders. The general diploid number for both suborders is 2n=44, although the number 2n=42 is observed in the two odontocete families Physeteridae (ARNASON 1970; ARNASON and BF,NIKSCHKE 1973) and Ziphiidae (ARNASON et al. 1977) and in the mysticete family Balaenidae (JARRFL 1979).

The karyotype of the long-finned pilot whale, Globicephala rnelaena, belonging to the Odon- toceti, has not previously been investigated. Only the karyotypes from one female and one male of the short-finned pilot whale, Glohicephala rnacrorhyn- chus (scammoni), have been studied previously by WALEN and MADIN (1965) using conventional aceto- orcein staining, and by ARNASON (1Y74) using con- ventional staining, C-banding and autoradiography in one female. The aim of the present study has been:

(1) to investigate the chromosomes of Globicephala melaena by use of a simple method that is applicable in the field;

(2) to compare the karyotype of Globicephala rnelaena with that of Glohicephala rnacrorhynchus;

(3) to evaluate the C-band heteromorphism as a marker in family- and population studies;

(4) to compare the Globicephala rnelaenu karyotype with the characteristic odontocete karyotype; and

( 5 ) to examine the sex-chromatin in small foetuses (<12 cm) of Globicephala melaena, where sex-determination on basis of secondary sex-charac- ters is impossible.

Materials

The short-term culture of peripheral blood was cho- sen toadapt, asgoodaspossible, t o the difficultcon- ditions in the field. The blood samples were col- lected during the professional catch of the long-fin- ned pilot whale Globicephala melaena in collabora- tion with the international pilot whale programme managed by the Faroese Government.

The fishermen killed the whales by opening the vena jugularis in the neck region. A sterile tube con- taining a penicillin-streptomycin and heparin solu- tion was immediately placed in the opening and 50 mi blood were collected.

The total number of samples collected were 337, distributed over 10 schools (Fig. 1 ) .

Methods

The chromosome preparations were obtained from whole blood. T o optimize the growth method for

246 L. WESLFY ANIIEKSEN A N D 11 I KII-I)KI( I I

I B I O I A L

the pregnant l'emwles, unavoidably caught, carried small foetuses (<12 cm), where the secondary sex characters are indifferent. we have tried to stain in- terphase cells For sex chromatin.

The cells wcre scraped from the soft palate of the small foetuses, stroked an slides, fixed in alcohol, and stained with Cresyl Fast Violet. 100 cells were screened for the existence o f sex chromatin in each foetus.

Results The results were very disappointing. All blood sam- ples from the first three schools (Gotc, Leynar and Bow) were heavily infected. The blood samples from the Sandur school wcrc brought to Denmark, because we thought that the infections were due to laboratory conditions. The improved results from this school gave metaphaser from 14 individuals out of 43. The blood samples from Funningsfjordur were sent to Denmark, and 12 out of 36 blood cul- tures contained metaphnses. I.- The quality of the- metaphases was bad and in

Fig. 1. Map o f the Faroe Islands. (Nuinher of blood sarn- pies marked nn) .

the peripheral whale blood cultures, growth tem- perature, culture medium, mitogenes, antimycotica (fungicide), sera. and colcemide exposure were var- ied. The present results are based upon the follow- ing method using 4 ml Eagle's medium, 2 ml foetal calf serum, 0.15 nil phytohemagglutinin, penicillin- streptomycin (80-100 PI). This was cultured at 37°C for 72 hours. Two hours before harvesting, 0.1 ml colcemide (10 pg/ml) was added. 10 mlO.56 YO KC1 was used as hypotonic treatment for 5 min, and the metaphases were fixed three times in 3: 1 solution of methanol and glacial acetic acid.

Slides were stained in Quinacrine Murtard as de- scribed by SFHESTED( 1975), and the C-band waspre- pared as described by H O L B E K ~ ~ al. 1974.

The classification used for arranging the karyo- type is described by ARNASON (1974), and the chromosome measurements were performed with small-arm Vernier calipers on 7 females and 8 males.

The long-finned pilot whales have a very strong school instinct (SERGEANT 1962), and normally a whole school of whales are caught. Because some of

total only 15 individuals, 7 females and 8 males, were analysed. The blood samples from the rest of the schools failed in providing metaphases, because they apparently were too old because of delays in the transportation from the Faroe Islands to Den- mark.

The karyotyping of the long-finned pilot whale revealed a diploid chromosome number of 2n=44. There are 21 pairs of autosomes and two sex chromosomes, XX in the female and XY in the male. The autosomes arc arranged in four groups as previously described for the short-finned pilot whale ( A R N ~ S O N 1974). with six pairs of metaccntric (m). eight pairs o f submctacentric (sm), three pairs of subtelocentric (st) with small arms or satellites on the short arms, and four pairs of telocentric (t) chromosonies (Fig. 2 and 3). The Xis a submetacen- tric chromosome niewuring 5.0 'YO of A + X (auto- somes + X-chrornosomc), while the Y chromosome is minute and telocentric.

The Q-banding showcd a characteristic banding pattern in the single chromosome pair. We ob- served pale stained regions of variable size in the short arms of nos. sml . s t I ~ and the long arms of t l . This heteromorphism was noticed interindividually, but was constant intraindividually, indicating a nor- mal Q-band variation. No brilliantly fluorescing heteromorphism in the Q-banding was observed. Frequently, associations between the smallest sub-

Hereditas 109 (19nn)

Fig. 2. The Q-banded karyotype o f Globicephala melaetia, male

KARYOTYPE OF LONG-FINNED PILOT WHALE 247

248 L WESLEY ANDERSEN A N D [I rRIEDRlCH

Pig. 3. The O-banded karyotype o f Glohicephula melaena, female.

Hereditas 109 (1988)

Hereditas 109 (1988) KARYOTYPE OF LONG-FINNED PILOT WHALE 249

Fig. 4. The C-banded karyotype of Giobicephala rnelaena, female.

metacentric, subtelocentric, and telocentric chro- mosomes were noticed.

Comparable heteromorphisms were seen in C- banding. Here, constitutive heterochromatic blocks of variable size were observed in the short arms of thesml , sm2, and sm3, s t l , st2 andst3, and terminal in the long arms of t l and t2. These regions corre- sponded to the pale stained heteromorphisms ob- served in Q-banding. The size of these blocks of constitutive heterochromatin varied between the in- dividuals, but were constant within the individual. (Fig. 3 and 4).

The chromosome measurements of G. rnelaena are shown in Table 1. The absolute average length of the female haploid set was 54.1 p m . The arm ratio was not calculated in the telocentric group, be- cause of the bad quality of the chromosomes.

Examination of the sex chromatin of interphase cells in the small foetuses revealed positive X- chromatin in females and negative X-chromatin in males.

Discussion and conclusions (1) The cultivation of chromosomes from blood cul- tures gave poor results. The attempt was based on the good experience of using peripheral human whole blood to grow human chromosomes. The un- derlying motive was, first, to reduce the possibility of infection by using a short-term culture and, sec- ond, the low demand of sophisticated equipment in the field by this method.

One reason for the bad quality and poor results were the infections. Two types of infections were observed, a bacterial and a fungal. The fungus was identified as an Aspergillus sp., while the bacterial infections were caused by monococci and diplo- cocci. The source of the infection of Aspergillus sp. wcre probably the surroundings, where the spores are known to occur. This is supported by the fact that most individuals in those schools with fungal in- fections had Aspergillus infections. The bacterial in- fections in the Gotc, Leynar, and Bour schools were

250 L WESLEY ANDERSFN A N D u FRIEDRICH Heredrtas 109 ( I 988)

Table I Chromosome measurement\ o f Glohir.i~phalu meluena hdsed on 7 females and 8 male3

Chromosome Relativelength Arm ratio

Mean SE Mean SE

ni I m2 m3 m4 m5 m6

tm 1 sm2 X sm3 sm4 sm.5 sm6 sm7 sm8

ct 1 st2 st3

t I t2 13 14

Y

44.') 5.w I .OM 0.08 39.5 3.57 1.14 0.09 35.0 2.47 1.13 0.24 33.4 2.23 1.10 0.11 30.9 2.04 1.12 n 10 28.2 2.60 1.17 0.11

68.6 6.97 1.86 0.21 55.5 4.41 1.79 0 41 49.6 4.53 1.54 0.26 48.Y 2.80 1.7X 0.29 45.4 2.79 1.72 0 24 41.X 2.43 1.55 0.22 38.1 3.16 1.62 0.30 31.8 3.31 1.51 0 25 22.9 3.73 1.61 0 28

75.9 6.20 2 86 0.s9 65.7 4.24 3.21 0.65 60.9 3.38 3.16 0.64

59.3 7.06 47.4 3.71 39.1 4.50 34.5 2.84

20.53 2.43 ~

Relative length = lengthof autosome, + X-chromosome n

n = number of chromosomes in the haplod set. based upon the longe\t chromosome in this set

not identical. The infections in these three schools seemed to be specific for the single school, which in- dicates that the source of the bacterial infections could have been the whales themselves. This possi- bility could be an explanation of the observed differ- ences in the facility of driving and catching the dif- ferent schools. The schools, easy to drive and catch, could be less refractory because the whales had in- fections and were ill. Meanwhile, this hypothesis could be tested by a bacteriological study of the existing slides.

Another reason for the poor results could be the occurrence of high concentrations of stress hor- mone in the blood, changing the normal physiologi- cal balance of the blood, and thereby inhibiting the growth of the lymphocytes.

(2) The karyotype 0 1 G. melurnu resembles very much that o f G. niucrmrhynchus (WA1,t.N and MADIN 1965; AIINASON 1974). 'The examination of 15 indi- viduals revealed a Q-band hetcromorphism. This hctcromorphism was observed a s ii s i x variation o f

heterochromatic blocks in the short arms of the sub- metacentric and subtelocentric chromosomes, and in the terminal region of the long arms of the telocentric chromosomes. This was confirmed by the C-band analysis.

In the C-banding, we can expect variation also in the position of heterochrornatin in the centromeric region, as revealed by the excellent C-banding of one G. macrorhynchus shown by ARNASON (1974). Unfortunately. we had only C-banding from 2 indi- viduals with a bad quality.

The chromosome measurements of G. melaerta (Table 1) revealed a rather broad variation in the length of the chromosomes both between single pairs as well as between single individuals. These differences were caused by the extensive heteromorphism observed in Q- and C-banding. The relative length of G. rnelaena and G. mac- rorhynchus chromosomes did not show any signifi- cant differences.

A clear difference in the karyotypes of the two species was noticed in the largest telocentric chromosome pair. Here an interstitial heterochro- matic block oivariable size was observed in C-band- ing proximally to the ccntromere in G. rnacrorhyn- chus, whereas a heterochromatic block of variable size was observed in the terminal region in G. melaena (Fig. 4). The easiest explanation of this var- iation in position of the heterochromatin is a paracentric inversion. by which the interstitial heterochromatic block observed in G. macrorhyn- chus is turned from the centromeric to the terminal region of the chromosome observed in G. melaena. This indicates that gene exchange between the two species should be very rare. Homologous pairing in the pachytene at meiosis I of an inverted and a non- inverted chromosome is only possible by forming a loop. If recombination occurs in this loop, chromo- somal imbalance will arise in the offspring, and this is not compatible with normal survival.

(3) We observed a clear size variation of the heterochromatin between the largest submetacen- tric pairs, the different subtelocentric, and the telocentric pairs. This variation was constant in the single individual, but differed among individuals. These so-called Q-band and C-band markers would be ideal for family- and population studies, if a good technical quality o f banding could be achieved.

Even though the results in this study are obtained from two different schools of G. rneluena, the sam- ple size and the quality o f the chromosomes d o not allow to make any distinction within and between the schools.

Herediras I09 (1988) KAKYOTYPE OF LONGFINNED PILOT WHALE 251

These C-band markers may have arisen as a result of unequal crossing-over and subsequent duplica- tion or deletion of heterochromatin. These hetero- chromatin variants do not have any phenotypic ef- fect since the heterochromatin does not contain structural genes.

(4) Furthermore, it can be concluded from this study, that the G. melaena karyotype is in very good accordance with the characteristic odontocete karyotype described by AKNASON (1974, 1980), AR- NASoNet al. (1977), J A R K F L and ARNASON (1981), and SrOCK (1981).

( 5 ) The finding of positive sexchromatin in the female interphase cells implies that one of the X- chromosomes in females must be inactivated. This opens the possibility of using this method to deter- mine the sex of the small foetuses (<12 cm), and thereby facilitate the cvaluation of the actual sex ratio in the stock/stocks of G. mefaena off the Faroe Islands.

Acknowledgmenn. - We want t o thank KolmPrden Dol- phinarium. Sweden, and Hagenbeck Titrpark, Germany, for pro- viding the blood for the initial phase of the experiment. Fortheir in- valuable help. we want to thank the Director of Fisheries, Kjartan Hoydal. Faroe Islands, and the international scientific team, Do- rete Bloch, R o p e Mouritsen. Susan Skaaning, Geneviive Des- portes. and the rest of the technical staff Foroya Natturugripasdvn, Faroe Islands; Christina Lockyer, Tony Martin, Tim Waters, Sea Mammal Research Unit. Cambridge. UK; Bill Amos, Department of Genetics, Cambridge University, U K ; and Florence Jean-Cour- ant, Centre d e Dosage des Element5 Traces, France. A special thank to A. J . Therkelsen. head of the Department of Human Ge- netics. Aarhus University. DK. for allowing us to use the labnra- tory facilities, and to the photographer Kirsten Millgaard for the good photos.

This study was supported by funds from the Carlsberg Founda- tion and the Dani5h Natural Science Research Council, grant no. 11-5999.

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