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Indian Journal of Experimental Biology Vol. 38, May 2000, pp. 467-470 Chromosome banding studies in an Indian mullet: Evidence of structural rearrangements from NOR locations J Chakrabarti & A R Khuda-Bukhsh* Department of Zoology, University of Kalyani, Kalyani, Nadia-741 235, W.B. Received 22 November 1999; revised 24 January 2000 C- , G- and NOR bands have been studied in the female sex of Rhinomugil corsula. (Mugilidae, Pisces) by deploying the conventional methodologies with suitable modifications of minor nature. The diploid metaphase complements contained 48 acrocentric chromosomes. The localization of C-band heterochromatin was found to be mostly at or near the centromeric regions of the acrocentric chromosomes. The G-type bands were not so well defined, but some of the G-banded chromosomes also contained C-bands. Interestingly, silver-positive NORs were found at the telomeric ends of five acrocentric chromosomes, including one homologous pair having NORs in both chromatids, while one chromosome showed NORs in both of its chromatids and the other two had only one NOR localized at one of its chromatids. This would suggest th at one homologue of the second pair of NOR-bearing chromosomes possibly underwent a chromatid exchange with a nonNOR bearing chromosome. This is quite a unique situation not reported earlier in any species of fish. , though some other form of NOR-polymorphism /heteromorphism has rarely been reported. Therefore, further exploration in natural populations of this species to examine the other sex and to verify if there also exists other chromosomally polymorphi c races (in respect of NOR-polymorphism) of this species, would be rewarding. Chromosome banding studies in fish, particularly the C- and G- bands, have proved to be relatively difficult in fish because of the small size and large number of chromosomes in most of them. In course of our chromosome banding studies on in vivo metaphase chromosomes of fish I-S an interesting situation was encountered in an Indian mullet, Rhinomugil corsula (Fam: Mugilidae) where structural rearrangements between chromosome pairs could be inferred from their NOR locations . Materials and Methods In an effort to obtain adequate number of good metaphase spreads and to deterrrline if a particular concentration and dose of colchicine treatment could be ascribed as optimal, different specimens were injected intramuscularly with 0.03, 0.05, 0. 1, 0.2, 0.3 and 0.4% colc hi c ine solution @ I mill 00 g body weight separately and specimens injected with each of these doses were sacrificed at 3, 4, 5, 6, 8 and 12 hr, respectively, after injection. Although over 50 living specimens of both sexes of R.. corsula of varying weights and sizes collected from brackish water cisterns of Central Institute of Brackishwater Aquaculture, Kakdwip, were used for the present study, suitable metaphase spreads could be obtained *Correspondent auth or only in one female specimen injected with 0.3 % colchicine and sacrificed at 3 hr, permitting us to induce C-, G- and NOR bands. However, some other specimens treated with the same or other dose and concentration did occasionally yield some fairly good plates but those were not found suitable for banding studies, because only very good plates give reproducible results. But since the unique location of NORs in this specimen apparently showing no morphological distinction from the other specimens used, clearly demonstrated structural rearrangement between two chromosomes of sirrlilar size, one NOR bearing and one non NOR bearing, it was felt that thi s information might be cytologically interesting and useful to other researchers. For C- and G- band studies the method of Sumner · · . 5, 6, 10, II was use d . mo d 1'f'tcattons wtt h mmor 9 For NOR location the one-step silver nitrate method of Howell and Black 12 was followed. Results The typical diploid Giemsa stained metaphase spreads (Fig 1) contained 48 rod like chromosomes alignable into 24 homomorphic pairs (Fig 2). The Cbanded metaphase spreads (Fig 3) showed the distribution of constitutive heterochromatin mostl y at or near the centromeres excepting a few pairs which INDIAN 1 EXP BIOL, MAY 2000 468 ii s 'i: . J .~'ii' ..~ , ._..,...... ... . . r..· tli. 2 .- .'f '. . ~ ,, , . -.·~ '' I• ,.i ' .t / .. 4 5 6 Figs 1-6 - Showing Giemsa stai ned so matic metaphase co mpl ement (Fig. I) of female Rhinomugil corsula; karyotype (Fig. 2) prepared from Fig. I ; C-banded metaphase complement (Fig. 3); karyotype (Fig. 4) prepared from Fig. 3; G-banded metaphase co mplement (Fig. 5); karyotype (Fig. 6) prepared from Fig. 5. Arrows indicate lateral asymmetry in stri ations. Bar= I 0 J.lm. CHAKRABARTI & KHUDA-BUKHSH : CHROMOSOME BANDING STUDIES IN AN INDI AN MULLET 8 & I 0), however, it was revealed th at one pair was perfectly homologous hav ing the ir NOR located at both chromatids in the ir telomeric ends (chromosome no. 2). Interestingly enough while both chromatid ends of chromosome pair no. 3 contained NORs in one homologue, the other homologue (chosen by its similarity in size) did not conta in any NOR at either chromatid. On the other hand , the chromosome pair arranged at no. 4 in the karyotype comprised homologues, both of which had only one NOR located at one chromatid only at its terminal end . showed blocks of heterochromatin either at one end (chromosome no . 3, chromosome no . 5, chromosome no. 6, chromosome no. 9, chromosome no . 17, chromosome no. 23 and chromosome no. 24) or both ends of chromatids (chromosome no . 4) or whole length of chromosome (chromosome no. 2) (Fig 4). Interestingly, a rather unequal di sposition of heterochromatin was noted between homologues of pair no . 3 and 4 (Fig 4). The G-banded metaph ase spreads (Fig 5) showed lateral stri ation s of dark and light bands which when karyotyped (Fig 6) also showed a little asymmetry in banding pattern in respect of a few individual pairs (chromosome no. 3, chromosome no. 15, chromosome no. 19, chromosome no. 20), some of which might be due to unfavourable deposition , if not inherent in the material. A few chromosome pairs also showed C-bands (chromosome no. I , chromosome no. 6, chromosome no. 13 to 16, chromosome no. 22). The sil ver stained metaphase preparations (Figs 7 & 9) showed five chromosomes showing NOR positi ve regions. From the respective karyotypes (Figs Discussion The diploid karyotype of 2n=48 rods and n=24 had 13 earlier been reported by Khuda-Bukhsh and Manna in Rhinomugil corsula . The present study would confirm the diploid number of 48 chromosomes in thi s species. A careful analysis of the C-banded chromosomes revealed at least one pair of chromosomes which were C-heteroc hromatin positive for the whole length (c hromoso me no. 2, Fig-4) and another pair which had the greater part (chromosome ·.;?'- ~,J\ A ; ~-- ,..:.{! , _ "" ~· ~ ( f'J r ,., " ~ 469 -~ . ;i - .,w:#···.... .·- • .. . . . . ~ "[~. "i'A ;f¥:F.. "';~' 1\i\ -~·\ ll'f'l ~ct ~~r -,;< \.,; f\:<'"f""' 1. /l • "" • "1'4" 8 -' f'fl\ l\t\ " tl' ~ ---~ ""' ~- ~ _,.,, ~~ 11 11~" -~ - \1\ ,,., ~~ il-8 J':J\' ~ tlh rtf\ AI\ --1\ 'f'A -kft .Ai '~" ItA 10 Figs 7-10- Showing silver stai ned somatic metaph ase complements (Figs. 7 and 9) of fema le Rhinomugil corsula and karyotypes (Figs. 8 and I 0), prepared fro m Fig. 7 and Fig. 9, respectively. NOR-bearing chromosomes have been indicated by "arrows" and und erlined in karyotypes. (==?=NOR in both chro m atid s,~ = NOR in single chromatid), Bar= I 0 )lm. INDIAN J EXP BIOL, MAY 2000 470 no . 4, Fig-4) of the chromosome localizing Cheterochromatin . Interestingly, two other pairs tended to show unequal distribution of heterochromatin (chromosome nos . 3 and 5, Fig-4) . Therefore, it appeared that the C-heterochromatin possibly incftided or was confluent with the NOR bearing zones. However, in our earlier study on the localization of C-band heterochromatin in the male sex of this species collected from Haldia, West Bengal' the C-bands were apparently all localized at or near the centromeres. But since in our present study male specimens did not yield suitable plates for banding, the C-band distribution could not be compared between the sexes of this population of R. corsula. Lateral striations of the G-banded chromosomes could not throw any additional light possibly because of the smaller size and rather unsuitable resolution of the banded structure, characteristic of the fish in genera1 5 14 ' or in the absence of well defined "isocore" . f ts ' h 15 . structure m However, what makes the present study interesting is the location of NORs which clearly suggests that there had been a chromatid exchange occurring between one homologue of an NOR bearing chromosome and a nonNOR bearing chromosome. Incidentally, Khuda-Bukhsh and Chakrabarti 16 recently reported the association of Ag-NORs with sex chromosomes in another brackish water fish, Scatophagus argus showing morphologically distinguishable (heteromorphic) sex elements. Therefore, it was not conclusive if this unique NOR location in R. corsula has anything to do with the sex chromosome mechani sm, particularly because difference in NOR numbers and locations between sexes has also been reported in case of a mosquito fish, Aplocheilus panchax7. Further, because R. corsula can thrive well in both brackish and fresh waters, the unorthodox NOR location in thi s species may have some added significance. Acknowledgement Grateful acknowledgements are made to Department of Environment and Forests, Government of India, New Delhi, for their financial support. Authors are also thankful to the staff of Central Institute of Brackish-water Aquaculture, Kakdwip Research Centre, Kakdwip, West Bengal, India, for their help in collection of the specimens. References I Nayak K & Khuda-Bukhsh A R, Chromosome co mpl ements in Mugil corsula (Mugilidae, Pisces) with reference to somatic C-band localization. Proc Na~ Acad Sci Letters, 14 (1991) 195. 2 Khuda- Bukhsh A R & Tiwary S, Locali zation of Nucl eolar Organizer Regions (N ORs) in the met a_ hase chromosomes of the Indian climbing perch, Anabas testudineus. Proc Zoo/ Soc, 44 (1991 ) I 0 I. 3 Khuda-B ukhsh A R & Tiwary S, Localization of Nucleolar Organizer Regions (NORs) in the chromosomes of nine species of teleosts (Pisces) from India. Systematics and Evolution of lndoPacijic Fishes: Proc 4th lndoPacijic Fish Conf. Bangkok, 1994,22. 4 Khuda- Bukhsh A R & Tiwary S , NOR locations in metaphase chromosomes of six species of teleosts. Nucleus, 39 ( 1996) 128. 5 Khud a-Bukhsh A R & Chakrabarti ", Indu ction of serial bands akin to G-type on metaphase chromosomes of two species of fish , Rita rita (Bagridae) a.nd Hypophthalmicthys molitrix (Cyprin id ae). Indian J Exp Bioi, 34 ( 1996) 1271. 6 Khuda-Bukhsh A R & Chakrabarti C, D istribution of C-band heterochromatin in metaphase c hromoso mes of the sil ver carp , Hypophthalmichthys molitrix (C yprinidae, Pisces). Proc Zoo/ Soc, 52 ( 1999) I . 7 Khuda-Bukhsh A R & Datt a S , Sex-specific differences in NOR-location on metaphase chromoso mes of the mosquito tish, Aplocheilus panchax (Cyprinod•)n tidae). Indian J Exp Bioi, 35 (1997) II II. 8 Mallick P, Chakrabarti J & Khuda-Bukhsh A R , Ag- NOR locations on metaphase chromosomes of two species ol estuarine fi shes , Lates calcarifer (Fam: Centropomidae) and Etroplus suratensis (Fam: Cichlidae). Nucleus, 41 , ( 1998) 43 . 9 Sumner A T, A simpl e tec hn iqu e for d e mon s tra tin ~ centromeric heterochromatin . Exp Cel! Res, 75 ( 1972) 304. I 0 Datta S & Khud a-Buk hsh A R , Unorthodox distribution of C. band heterochromatin in mosquito fi sh, Aplocheilus pancha; with female heterogamety (Fam: Cyp rinodontidae). Uttw Pradesh J Zoo/, 18 ( 1998) 165. II Khuda-Bukhsh A R & Datta S, Induc tion of C- and G-typ( bands on in vivo metaphase chromosomes of three species o larv i vorou s fi shes. Uttarpradesh J Zoo/, 19 ( 1999) 143. 12 Howell W M & Blac k D A, Controlled silver staining o nucleolus organ izer regions with a protective colloida developer: a one-step method. Experientia, 36 ( 1980) I 014. 13 Khuda- Bukhsh A R & Manna G K, Chromosome analysis ir two species of Indi an mullets (Pisces). Indian Biologist, l ( 1976) 23 . 14 Gold J R , Li Y C , Shipley N S ·& Powers P K, lmprovec methods for working with fish chromoso mes with a review o metaphase chromosome banding. J Fish Bioi, 37 ( 1990) 563 . IS Sumner A T , in Chromosome Banding, (Unwin Hyman Ltd London , UK) 1990 I . 16 Khud a-Bukhsh A R & Chakraban i 1, Indi cation of se: chromosome pair bearing Ag-NORs in a brackish water fi sh Scatophagus argus (Fam: Scatop hagi dae) showing mall heterogamety. Indian J Exp Bioi, 37 ( 1999) 793 .