Indication of the sex chromosome pair bearing Ag-NORs in a brackish water fish, Scatophagus argus showing male heterogamety

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1 Indian Journal of Experimental Biology Vol. 37, August 1999, pp Indication of the sex chromosome pair bearing Ag-NORs in a brackish water fish, Scatophagus argus showing male heterogamety A R Khuda Bukhsh & J Chakrabarti Department of Zoology, University of Kalyani, Kalyani , India Received 6 August 1998; revised 5 April 1999 Morphologically distinguishable sex elements were detected in a brackish water teleqstean fish, S. argus. While the female specimens had 24 homomorphic pairs of rod like chromosomes (2n = 48; NF = 48), in male specimens the karyotype differed having a heteromorphic pair, comprising one metacentric ("Y") and an acrocentric ("X") chromosome (2n = 48; NF = 49). The suspected XX and XY elements showed Ag-NOR locations at and around their centromeric regions, thus indicating some form of association existing between nucleolar organising regions and sex determining genes in this species. Although over 1600 species of fish have now been karyotypically studied, morphologically distinguishable or heteromorphic sex chromosome elements have only been detected in about I {)() species l - 5. During investigation on localization of Ag NORs in different species of Indian teleosts6-1o we have come across an interesting case in which the y chromosome could be morphologically distinguished from the other autosomes and X-chromosome and the sex chromosome pair appeared to bear the Ag-NORs which are reported here. Materials and Methods Altogether 40 specimens of both sexes of Scatophagus argus (Family. Scatophagidae) weighing between 20 and SO g were collected from the Hooghly river near Kakdwip (Estuarine belt of the Sunderbans), West Bengal, India and used after acclimatising them in brackish water cisterns of Central Institute of Brackish water Aquaculture, Kakdwip Research Centre. Since the usual doses of % and even 0.1 % of 1 mlil {)() g body weight, failed to arrest divisional stages at metaphase, a higher dose of 0.2% 1 mill {)() g body weight had to be used to get the satisfactory results. The somatic chromosomes were prepared from kidney and gill tissues of both sexes and germinal chromosomes from testis of male S. argus, sacrificing them after 3 hr of injection and by adopting the air drying-giemsa stain schedule described elsewhere II. One-step silver nitrate method of Howell and Blackl2 was followed for localization of Ag-NORs in the metaphase chromosomes. Results Out of 50 Giemsa-stained well spread metaphase plates studied in both male (Figs 1-3) and female (Fig. 7) S. argus, over 70% had 48 chromosomes, which could, be ascribed as the diploid number for this species. The male Giemsa-karyotype (Fig. 5) contained 23 homomorphic pairs and a heteromorphic pair comprising one metacentric and an acrocentric chromosome while the female Giemsa-karyotype (Fig. 9) contained all 24 homomorphic pairs. All the chromosomes were acrocentric and very gradually seriated measuring between 1.70 and 0.84 ~m in female and 1.68 and 0.92 ~m in male except for one metacentnc chromosome suspected as the "Y" element which measured 1.80 ~m in length. However, in the Giemsa-karyotype of either male or female the X-chromosome could not be distinguished from the other autosomes and was only arbitrarily shown in the karyotypes (Figs 5 and 9). In silver-stained preparations of metaphase plates in both male (Fig. 2) and female (Fig. 8), one pair of chromosomes showed positive staining for NORs. While in the female sex, a homomorphic pair had their NOR locations at the terminal centromeric regions (Figs 8 and 10), in male, the Ag-NOR locations were heterologous, one chromosome showing its tip NOR positive like that of female while the other was located in the median region

2 794 INDIAN 1. EXP. BIOL., AUGUST 1999 I t 1 2 ~.... '", ;'.~-- " <'""I' \ t ~ r., {~ ~" ;;.. (... ""..., l' v 3 4 5, : ". it< P, 6 "t ~l ~ Figs 1-6--Giemsa stai ned somatic metaphase complements of male S. argus. (Figs I and 3) and primary spermatocytic late diakinesis (Fig. 4): metacentri c "Y" chromosome has been indicated by arrows (Figs I, 3 and 4); karyotype (Fig. 5) prepared fro m Fig. 3; silver stained metaphase complement of male (Fig. 2) and its karyotype (Fig. ~ ); NOR-bearing chromosomes have been indicated by arrows and underlined in karyotypes. Bar = 10 flm.

3 KHUDA BUKHSH & CHAKRABARTI: SEX DETERMING ELEMENTS IN TELEOST I: Fig ~ 7-1 O-Giemsa stained metaphase complement of female S. argus (Fig. 7) and its karyotype (Fig. 9); silver stained metaphase complement of female (Fig. 8) and its karyotype (Fig. 10); NOR-bearing chromosomes have been indicated by arrows and underlined in karyolype. Bar = 10 jjm.

4 796 INDIAN J. EXP. BIOL., AUGUST 1999 (peri centric ) of the metacentric chromosome suspected as the "Y" element. Efforts to study the meiotic behaviour of the suspected XY did not meet with much success as no divisional activity was obtained in some 18 male specimens tried, most of which were sexually immature. However, in one male specimen a few divisional stages of late diakinesis only were obtained (Fig. 4) which showed 24 bivalents with single terminalised chiasma, in all the bivalents confirming the diploid number in the spec.ies to be 48 as reported earlier'3.'4 but not allowing the detailed study of meiotic behaviour of the suspected XY elements. Apparently one X shaped element (Fig. 4, arrow) was found in each of the plates studied which would possibly indicate that the heteromorphic X and the Y successfully paired atleast for some of their lengths. Discussion Khuda Bukhsh and Manna l3 reported diploid number of 48 chromosomes in S. argus which was later confirmed by Chowdhury et az 14. However, while Khuda-Bukhsh and Manna lj obtained 48 acrocentric chromosomes in females and 47 rods and one distinctly di sposed metacentric chromosome in male S. argus collected from Kakdwip, West Bengal, Chowdhury et al. 14 reported 2n=46A+2Sm chromosomes in marine specimens of both sexes of S. argus collected from Gopalpur-on-sea, Orissa state. Findings of the present investigation collaborate well with the observations of Khuda-Bukhsh and Manna l3. However, in the present study, the Ag-NORs also clearly demonstrate locations on two different sites in the male sex, indicating heteromorphic behaviour of one pair of chromosomes. Absence of heteromorphic sex pair in overwhelmjng majority of fish species studied so far has been attributed to their "primitive" state, the sex chromosomes being very little differentiated from autosomes 15. Further, out of nearly 100 species showing morphologically detectable sex. chromosomes, the most prevalent types of sex determination were XX female : XY male encountered in some 45 species, followed by some 30 species having yz male: ZW female while XO male: XX female and multiple sex chromosome mechanisms of XIX2XiXi female: XIX 2 Y male have also been reported in a few other species IS. However, the sex chromosome pair suspected in most of these cases were not verified by any banding studies. On the other band, Park and Grimm '9 could not find C-band and fluorescent staining technique to be of diagnostic use to differentiate ZW pair in either the European or the American Eels, Anguilla anguilla and A. rostrata, respectively. Interestingly, in the present study, the location of the NOR in the metacentric "Y" would imply that the other acrocentric NOR bearing chromosome should be "X" chromosome, particularly when two such acrocentric homologues showed the same pattern of NOR location in female sex. Although banding techniques are among the essential tools for the detailed analysis of chromosomes, methodological difficulties have limited the amount of information available on the banded chromosomes of fish lo,2()..24. In general 2n = 48 rod like chromosomes are most prevalent among the fish species cytologically studied so far and are believed by many to represent the pnmltlve... f' IS h k aryotype' 4 16, ccurrence 0 f a single metacentric chromosome in male sex of S. argus, therefore, could have been derived by a pericentric inversion of one of the X-chromosomes that would have more male detennining genes. Incidentally in the pnnutive vertebrates the chromosome morphology and behaviour of some sex linked genes indicate that the sex chromosome pair has remained largely homologous and sex determination is believed to be at the genic rather than the chromosomal levee 7. Differentiation of heteromorphic sex chromosomes probably resulted from inhibition of crossing over between the originally homologous pair, thus isolating the sex determining loci and stabilising differential accumulation of the two kinds of sex determining genes 2S. Morphological differentiation of Z and W or X and Y chromosomes could therefore imply that the initial homologue would either convert into heterologue through structural rearrangemene S. 29 or through initial heterochromatization prior to structural rearrangemeneo. Our efforts so far to induce C-bands in this species remain unsuccessful and therefore the heterochromatin distribution in the suspected sex chromosome pair remains unknown. Notwithstanding that, however, the present study would raise a possibility of some form of association of NORbearing chromosomes and sex determining genes in fish. Usually the Ag-NORs represent the chromosomal sites of l8s and 28s rr...1\la genes which were presumably transcribed in the proceeding interphase 31. The secondary constrictions of

5 KHUDA BUKHSH & CHAKRABARTI: SEX DETERMING ELEMENTS IN TELEOST 797 metaphase chromosomes are also associated closely with the nucleolar organiser regions. In over 250 species of fish studied so far for their NOR locations, the majority of species have been reported to have two NORs, which have been claimed to be the "primitive" or "fundamental" type in fish However, the number of NOR varies from 4 to 8 in some species and rarely one in a couple of species' , an d h eteromorp h" Ism 10 NOR I ocatlon. h as b een reporte d. 10 a f ew species ntis h' count S. argus shows NOR locations of fundamental nature but shows sex specific differences in location as also reported in the mosquito fish Aplocheilus panchax 10 in a different manner. Acknowledgement Grateful acknowledgements are made to Department of Environment and Forests, Government of India, New Delhi, for financial support. Authors are also thankful to the Central Institute of Brackishwater Aquaculture, Kakdwip Research Centre, Kakdwip, West Bengal, India, for help in collection of th e specimens. References I Vasi liev V P. lc/zthyol, 20 ( 1980) Sola L. Cataudella S &' Capanna E, GeneTica, 54 ( 198 I) Manna G K. Genetical Research in India: 15th Int. Congo Genet., (ICA R, New Delhi ), ( 1983) Manna G K, Nllcleus, 27 (1984) Ojima Y, Fish chromosome data retrieval list (Ojima Lab, K wansei Kak uin University, Nishinomiya, Japan) 1985, Kh uda-bukhsh A R & Tiwary S, Proc Zool Soc, 44 ( 1991 ) Khuda-Bukhsh A R & Tiwary S, J Illland Fish Soc Ind, 24 ( 1992) I I. 8 Khuda- Bukhsh A R & Tiwary S, Systematics and Evolution of III do Pacific Fish es: Proc 4th Indo Pacific Fish Conf, Bangkok, Khu da-blik hsh A R & Tiwary S, Nllclells, 39 ( 1996) Khllda-Bukhsh A R & Datta S, Indian J Exp Bioi, 35 ( 1997) 1 I I I. I I Khllda-Bu khsh A R, Ca rvoloria, 32 (1979) Howell W M & Black D A, Experientia, 36 ( 1980) Khuda-Bukhsh A R & Manna G K, CIS, Japan, 17 (1974) Chowdhury R C, Prasad R & Das C C, Caryologia, 32 (1979) White M J D, in Animal Cytology and Evolution, 3rd ed. (Cambridge University Press, Cambridge) 1973, Ojima Y, in Chromosomes in evolution of eukaryotic groups, edited by A K Sharma & A Sharma (CRC Press) 1983, Ill. 17 Khuda-Bukhsh A R, Chanda T & Barat A, Proc Sym on "Conservation and fish genetic resources of India ", in Fish genetics in India, edited by Das P & Jhingran A G (Today & Tomorrow's Printers and Publishers, New Delhi ) 1986, Khuda- Bukhsh A R & Baral A, Caryologia, 40 (1987) Park E H & Grimm H, Cytogenet Cell Genet, 3 1 (1981) Takai A & Ojima Y, Indo-Pacific Fish Biology: Proc 2nd Int Conf Indo-Pacific Fishes, edited by T Uyeno, R Arai, T Tanuichi & K Matsura (Jcthyo1 Soc Jpn, Tokyo) 1986, Gold J R, Li Y C, Shipley N S & Powers P K, J Fish Bioi, 37 (1990) Rab P & Crossman E J, Can J Zool, 72 (1994) Rab P, Reed K M, Leon A P de & Phillips R B, BioTechnique and Histochemistry, 71 ( 1996) Khuda-Bukhsh A R & Chakrabarti C, Indian J Exp Bioi, 34 ( 1996) Nogusa S, Mem Hyogo Univ Agric, 3 (1960) Ohno S, Wolf U & Atkin N B, Hereditas, 59 ( 1968) Ohno S, in Animal cytogenetics, vol 4. Chordata I: Protochordata, Cyclostomata and Pisces (Gebrtider Bomtraeger, Berlin/Stuttgart) Lyon M F, Nature, 250 ( 1974) Ohno S, in Th e evolution of reproduction edited by C R Austin & R V Short (Cambridge University Press, Cambridge) 1976, Singh L, Purdom 1 F & Jones K W, Chromosoma, 79 (1980) Howell W M, Chromosoma, 62 (1977) Amemiya C T & Gold J R, Genetica, 76 ( 1988) Amemiya C T & Gold J R, Copeia, 1 (1990a) Amemiya C T & Gold J R, Hereditas, I 12 ( I 990b) Amemiya C T, Powers P K & Gold J R, in Systematics, Historical Ecology & Fres})water Fishes edited by R L Mayden (S tanford University Press, Stanford, CAl 1992, John G, Barat A & Lakra W S, Nucleus, 35 (1992) Gold J R, Copeia, 1 ( 1984) Gold J R & Zoch P K, Th e South western Na tu ralist, 35 ( 1990) 211.