Multiple Sex-Chromosomes in the Common Indian Krait, Bungarus caeruleus Schneider

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1 Chromosoma (Berl.) 31, (1970) 9 by Springer-Verlag 1970 Multiple Sex-Chromosomes in the Common Indian Krait, Bungarus caeruleus Schneider L. SI~G~, T. S~At~MA and S. P. P~A'f-CJ~AVD~U~I Cytogenetics Laboratory, Department of Zoology Banar~s Hindu University, Varanasi l~eceived June 23, 1970 / Accepted July 9, 1970 Abstract. The highly poisonous common Indian Krait, Bungarus caeruleus of the family Elapidae has 2n ~ = 44 and 2n ~ = 43. There is a sharp difference in size between macro and microchromosomes. The number of macrochromosomes is 24 in males and 23 in females whereas there are 20 microchromosomes in both the sexes. The difference in number of macro-chromosomes in the two sexes is explainable on the basis of translocation of a macro-autosome to the W chromosome resulting in a multiple sex-chromosome constitution of Z1Z~Z~Z2~/Z1ZeW % types. Autoradiographic studies using Ha-Tdl~ show that the W is late replicating except for the translocated part which finishes its replication along with the macroautosoraes. This is the first example of a multiple sex-chromosome complex in a vertebrate with female heterogamety. Introduction Snakes of the primitive family Boidae have homomorphie sexchromosomes in both sexes (Be~ak, 1965; Singh, Sharma and Ray- Chaudhuri, 1968a). The highly evolved families like Elapidae and Crotalidae have well differentiated W chromosome in females (Bepak, 1965; Singh, Sharma and Ray-Chaudhuri, in press) whereas the largest family Colubridae has species with both homomorphie as well as heteromorphic ZW pairs (Be~ak, 1965; Singh, Sharma and t~ay-chaudhuri, 1968b; Singh, Sharma and Ray-Chaudhuri, in press). Only ZZ/ZW types of sex-chromosome constitution has so far been reported in snakes. During the course of our study of chromosomes of Indian snakes, we discovered an interesting case of a multiple sex-chromosomes in the common Indian Krait, Bungarus caeruleus. Material and.methods Five males and four females of B. caeruleus are studied. The chromosome analysis is done on preparations made from spleen, intestine, marrow of ribs and leukocyte cultures. The procedure adopted for culturing the leukocytes is as follows.

2 Multiple Sex-Chromosomes in a Snake 387 Blood is taken out directly from the heart with the help of the heparinised syringe, transferred in a universal container and kept in a refrigerator for an hour to allow the erythrocytes to settle down. Of the supernatant fluid with suspended leukoeytes 0.3 ml is mixed with 5 ml of medium 199, 1 ml of denatured human AB serum and 0.1 ml of phytohaemagglutinin and then incubated at 30 ~ C for 96 hours. Colcemid mierogramme per ml is added four hours prior to harvesting the culture. Slides are prepared by the usual air-drying procedure and stained in carbol fuchsin. The microphotographs are taken with a Carl Zeiss Photomicroscope and the centromerie positions on the chromosomes are described according to the system proposed by Levan, Fredga and Sandberg (1964). The autoradiographic study is done by using H3-TdR l~c/ml of specific activity 20 C/raM (The Radiochemical Centre, Amersham, England). Kodak AR-10 stripping film is used and slides are developed after 15 days of exposure. Results Slides prepared from different tissues of all the five male and four female individuals yielded more than 600 good metaphase plates. The chromosome analysis has revealed a diploid number of 44 chromosomes in the males and of 43 in the females with a very high degree of consistency. There is sharp bimodality between macro- and microchromosomes (Figs. 1 and 2). Tile number of maerochromosomes is 24 in the males (Fig. 1) and 23 in the females (Fig. 2) whereas the mieroehromosomes are 20 in both the sexes. In the females one of the macroehromosomes is conspicuously large, -with its eentromere in the median region. It is the largest in the whole complement and occupies approximately 19.5% of the length of the haploid set, including the mierochromosomes and 23 percent excluding them. Such a prominent chromosome is absent in the males and therefore it is taken to be the W chromosome. Another chromosome of the heteromorphie pair in the female having a submedian centromere occupies about 10.3% of the length of the haploid set. This value agrees with the usual size of the Z chromosomes in snakes. This chromosome is similar to one of the homomorphic pairs in the males and it is termed the Z a. The Z 1 and the W, as the case may be, can be identified in every metaphase plate of both the sexes by their size and centromeric positions. There is an odd macrochromosome in the females having the eentromere in the terminal region. It is similar to one of the homomorphic pairs in the males and it is termed the Z~ chromosome. The relative size of this pair is about 5.1% and it is confused with the pairs no. 9 and 10. The rest of the macrochromosomes have their eentromere in the terminal region. Pair number 1 is easily recognisable because of the presence of the secondary constriction in the long arm of both the chromosomes. Occasionally the secondary constriction is seen only in one of the chromosomes of this pair. It is very difficult to distinguish between the chromosomes of

3 388 L. Singh, T. Sharma and S. 1 ~. l~ay-chaudhuri: Fig. i. Male karyotype of B. caeruleus from leukocyte culture showing multiple sex-chromosomes (Z1Z1ZBZ~) Fig. 2. Female karyotype of B. caeruleus from leukocyte culture showing multiple sex-chromosomes (Z1Z=W) pairs 2 and 3. Due to their graded size and similarity in their eentromeric positions, ~he rest of ~he macroehromosomes can not be identified individually. All the mieroehromosomes have terminal centromeres.

4 Multiple Sex-Chromosomes in a Snake 389 -)iscussion This species has earlier been studied by Bhatnagar (1960) who reported the spermatogonial number as 2n = 44. We confirm this diploid number of chromosomes in males. Our study of the somatic cells in females, on the other hand revealed not only an odd number of chromosomes (2n = 43) but also a multiple sex chromosome complex in this species. The longest member of the female complement has been identified as the W. Its relative size is 23 % of the haploid set excluding the micro-chromosomes. Begak (1965) has reported in the snake, Clelia occipitoleutia a W chromosome which is even larger than what is found in B. caeruleus, comprising 27 % of the haploid set. The multiple sexchromosome complex can be explained most simply by assuming a reciprocal translocation between the W and one of the medium sized autosomes (Fig. 3). This was virtually a whole arm transfer with the loss of a small centric fragment probably largely made up of heterochromatin. The neo-w is metacentric, the autosome being attached to the shorter arm of the original W. The original Z is termed Z 1 and the homologue of the autosome now attached to the short arm of W is Z 2. They are expected to pair during female meiosis with the two arms of the rico-w, the Z 1 pairing with the larger arm and Z 2 with the shorter arm, giving rise to an association of three and thus ensuring proper disjunction of W to one pole and the Z 1 and Z 2 to the other pole. This hypothesis requires confirmation from meiotic studies in females which is being planned. Autoradiographie study after treatment with HS-TdI~ shows (Fig. 4) that the long arm of the W along with a short proximal region of the short arm is late replicating. The rest of the short arm finishes its DNA synthesis along with the autosomes. We presume that this portion of the W has originated through transloeation with an autosome. We have already reported (Ray-Chaudhuri, Singh and Sharma, in press) that the giant W of this species forms a characteristic chromocentre in the interphase somatic nuclei of females. If our hypothesis of the origin of multiple sex-chromosome complex in this species is substantially correct, it must be assumed that the W even before receiving the transloeated autosome was bigger than the Z chromosome. Our measurements of the relative length of the different components of the female sex-chromosome complex given in Fig. 3 will corroborate the statement. The Z 1 has a total relative length of 12.2% (exluding the mieroehromosomes) which is about the same as of Z chromosomes in various species of snakes. It is not possible to accurately measure the part of the W which replicated synchronously with the autosomes. But a reasonably correct estimate of the relative length of this portion can be obtained from the measurement of the autosome

5 390 L. Singh, T. Sharma and S. P. t~ay-chaudhuri: Origina[ C~w Derivation Derived A W W A i Z =Z~ A=Zz A Z V L05,N J A )1 "~Z2 o* Z Fig. 3. Diagrammatic representation of the origin of multiple sex-chromosomes in B. caeruleus Fig. 4. Metaphase plate of B. caeruleus female from leukocyte culture after 5 hours treatment of H3-TdR and 1 hour treatment of colehicine showing long arm of the W chromosome labelled along with the small proximal region of the short arm. The translocated portion of the autosome as well as other macroehromosomes are almost unlabelled now termed Z~ which is 6.0%. The total relative length of the W is 22,7 % and if we deduct the relative length of Z~ from it we get a relative length of 16.7% which is defh~itely bigger than 12.2% which is the measurement of the Z r

6 Multiple Sex-Chromosomes in a Snake 391 The problem o5 explaining the mechanism of the derivation of the W which is longer than the Z chromosome can not be tackled before a more comprehensive study of the chromosomes of the females of various members of the family Elapidae about which we have no information to-date. Acknowledgements. The work has been supported by the University Grants Commission, India which is gratefully acknowledged. The authors are also thankful to the Director, Zoological Survery of India, for the identification of the materials. References Begak, W. : Constitui~o cromossomica e mechanismo de determina~o do sexo em ofidios sul-americanos. 1. Aspectos cariotipicos. Mem. Inst. Butantan 32, (1965). Bhatnagar, A.N.: Chromosomes of Bungarus caeruleus Schneider (Elapidae: Ophidia). Cytologia (Tokyo) 25, (19603). Levan, A., Fredga, K., Sandberg, A. A. : Nomenclature for centromeric position on chromosomes. Hereditas (Lund) 52, (1964). Ray-Chaudhuri, S.P., Singh, L., Sharma, T.: Sexual dimorphism in somatic interphase nuclei of snakes. Cytogenetics (in press). Singh, L., Sharma, T., Ray-Chandhuri, S. P. : Chromosomes and the classification of snakes of the family Boidae. Cytogenetics 7, (1968a) W chromosome in the Indian water snake (checkred keel back) Natrix piscator (Colubridae). Experientia (Basel) 24, (1968b) Chromosome numbers and sex chromosomes in a few Indian species of amphibia and reptiles. Mammal. Chrom. ~ewsl. (in press). L. Singh Cytogenetics Laboratory Department of Zoology Banaras Hindu University Varanasi-5 India