Differentiation in Asellus Aquaticus (Crustacea, Isopoda)

Heredity 75 (1995) 267—272 Received 6 January 1995

Simple DNA repeats and sex chromosome differentiation in Asellus aquaticus (Crustacea, Isopoda)

E. V. VOLPI, F. PELLICCIAI, V. LANZAI, R. BARZOTTIf & A. ROCCHI*t Istituto di Genetica Mo/eco/are del CNR, Porto Conte Research and Training Laboratories, Aighero, tOipartimento di Genetica e Biologia Moleco/are, Università 'La Sapienza Pie A/do Moro, 5, 00185 Roma, and Centro di Genetica Evo/uzionistica del CNR, Universitá 'La Sapienza Roma, Italy

Thecrustacean isopod species Asellus aquaticus does not usually have recognizable sex chromosomes. We previously identified a Y chromosome marked by two heterochromatic bands in some males of a population from the Sarno river near Naples. In this work we used oligonucleotide probes to test the presence and possible sex-specific distribution of five simple repeat motifs in the genome of male and female individuals from the Sarno population. The five oligonucleotide probes were hybridized to enzyme-restricted genomic DNAs and the chromosome location of two probes was tested using fluorescence in situ hybridization. Our results show that only the (TCC) repetitive simple sequence has a sex-specific hybridization pattern and presents a significant accumulation on the Y chromosome in the region included between the two heterochromatic areas. Moreover the GGAAT sequence is not present in the genome of A. aquaticus in any detectable quantity.

Keywords:Asellus,crustacean, isopod, sex chromosome, simple repeats.

nique. The heterochromatic areas fluoresce brightly Introduction when stained with the GC-specific fluorochrome Theexistence of morphologically differentiated sex chromomycin A3, remaining dark when stained with chromosomes in crustaceans has been revealed by DAPI(Rocchi eta!., 1980; Di Castro eta!., 1983). simple karyological analyses in some 40 species only. A heteromorphic chromosome pair was identified in Of the isopods, several species of marine asellotes of several males from a population collected from the the Jaera genus exhibit female heterogamety with Sarno river near Naples. One chromosome shows two multiple sex chromosomes ZW1W2 and one flabellifers intercalary heterochromatic areas, one on each arm, shows male heterogamety with an X0 chromosome which stain brightly with chromomycin A3; variant formula, whereas no cases of XY type male hetero- ribosomal sequences are associated with these areas gamety have ever been observed (Ginsburger-Vogel & (Volpi et a!., 1992). This heterochromosome is Charniaux-Cotton, 1982; Legrand et a!., 1987). How- inherited through the male line as a normal Y chromo- ever, it is worth pointing out that of the roughly 4000 some (Rocchi eta!., 1984). Although the chromosomes species belonging to this order only about 60 have in the heteromorphic pair often appear precociously been karyotyped. Furthermore, differential staining or separated in meiotic metaphase I, they do in fact retain molecular hybridization techniques have hardly ever the capacity to recombine during meiosis in the sub- been applied. terminal regions distal to the heterochromatic areas. The karyotype of the isopod crustacean Asellus Exchange also occurs very infrequently in the chromo- aquaticus consists of eight pairs of homomorphic some area situated between the two heterochromatic chromosomes in both sexes. The chromosomes of this areas, thus giving rise to chromosomes displaying only species cannot be differentiated by G- or R-banding one heterochromatic area. On the strength of these and techniques, whereas a variable number of terminal other observations (Volpi et a!., 1992), we may reason- heterochromatic areas associated with the nucleolar ably postulate an early stage of chromosome differen- organizers can be detected using the C-banding tech- tiation; that this differentiation does not involve chromosome rearrangements in the form of inversions *Correspondence but, probably, the transfer of heterochromatic and

1995 The Genetical Society of Great Britain. 267 268 E. V. VOLPI ETAL.

ribosomal sequences from telomeric regions to inter- with 2 x SSC, 0.1 SDS at 66°C followed, for the (CA) stitial sites; and that this transfer could be favoured by probe only, by 1 x SSC, 0.1 per cent SDS. the 'Rabi-orientation' that the chromosomes retain Chromosome preparations obtained from squashes during interphase (Schweizer & Loidl, 1987). of testes were hybridized in situ with two of the In recent years the existence of sex-specific distribu- synthetic probes (CA) and (TCC). The probes were tion patterns for various simple, tandemly repeated labelled with biotin-1 1-dUTP (Sigma) or biotin dATP DNA sequences has been demonstrated in a number of (Gibco, BRL) by nick translation. Chromosome organisms. Although usually scattered in the genome, preparations were denatured in 70 per cent forma- in these cases the simple sequences present accumula- mide/2 x SSC at 70°C for 3 miii and the hybridization tions on the heterochromosome thus contributing to was performed at 37°C in 4 x SSC, 20 per cent the evolution of a morphologically differentiated sex formamide, 10 per cent (w/v) dextran sulphate and 0.3 chromosome pair (for reviews see Epplen, 1988; u L of sonicated salmon sperm DNA. The washes after Nanda eta!.,1991,1992). hybridization were at room temperature in 20 per cent In a previous paper we demonstrated the presence formamide/4 x SSC (three times) followed by three of GATA repeats in the A. aquaticus genome and washes in 4 x SSC, 0.1 per cent Tween 20 at 42°C. established the absence of their sex-linked distribution Biotin-labelled DNA was detected using FITC- (Pelliccia eta!.,1991). conjugated avidin (Vector Laboratories). The speci- In this study we used five oligonucleotide probes mens were simultaneously stained with DAPI. Digital specific for simple repeat motifs such as CA, TCC, images were obtained using a computer-controlled CGA, CCTG and GGAAT. The five probes were Zeiss Axioscop epifluorescence microscope equipped hybridized to enzyme-restricted genomic DNA of male with a CCD camera. Digital imaging and computer and female individuals from the Sarno population and, software have been previously described (Pelliccia et for TCC and CA, the chromosome location was tested a!., 1994). using fluorescence in situ hybridization. Our results show that the repetitive simple sequence (TCC) has a sex-specific hybridization pattern and a significant accumulation on the heterochromosome in Results a region situated between the two heterochromatic Southernblots, obtained with DNA from A. aquaticus areas. Moreover the GGAAT sequence is not present males and females collected in the Sarno river and in the genome of A. aquaricus in any detectable digested with restriction enzymes, were hybridized quantity. with five oligonucleotide probes consisting of CA, TCC, CGA, CCTG and GGAAT sequences repeated in tandem. Sequences homologous to the first four Materialsand methods probes used were present in the genome of this organism. The (GGAAT ),sequencewas not detected Theresearch was carried out on a population of A. in our conditions of hybridization. The (CA) and aquaticus collected from the Sarno river near Naples. (TCC) probes detected heterogeneous DNA frag- The DNA was extracted separately from batches of 30 ments, most of which form a high molecular weight male or female individuals. Samples of DNA (2u g) smear, and some discrete bands (Figs 1 and 2). Only were digested with six restriction endonucleases: the simple (TCC) sequence displays a sex-specific BamHl, EcoRI,Hindill,PstI,Sad orSacli hybridization pattern, the male DNA differing from (Boehringer, Mannheim), then size-fractionated on a female DNA by exhibiting an extra band with all the 0.8 per cent agarose gel and blotted to nylon filters. restriction enzymes used. The supernumerary frag- Synthetic probes consisting of repeat arrays of the ments are 7 kb long for the Sacli enzyme, 6.3 kb for CA, TCC, CGA, CCTG and GGAAT simple the HindIII enzyme, 6.2 kb for the BamHI and EcoRI sequences were generated by PCR as described by Ijdo enzymes, 5.6 kb for the PstI enzyme and 2.3 kb for the et a!. (1991). This method produces heterogeneous Sad enzyme (Fig. 1). The (CGA) and (CCTG) probes populations of molecules of various lengths, up to 25 hybridized mainly to heterogeneous DNA fragments kb. The probes were labelled by the nick translation with all the enzymes used, forming smears between 2.5 technique using [a32P]dATP and {a32P]dCTP and 15kb. (Amersham, UK). Filters were prehybridized at 66°C Subsequent to in situ hybridization with probe for 2 h in 5 x Denhardt's, 6 X SSC, 0.5 per cent SDS. (CA) a scattered distribution of label is observed on Hybridization was carried out at the same temperature all chromosomes; moreover, some hybridization for 48 h in the same solution. The filters were washed signals are preferentially located in areas proximal to

The Genetical Society of Great Britain, Heredity, 75, 267—272. female individuals.LambdaHind!I! fragmentswereusedas sequence. Notethesamepattern ofbandsformaleand The probewasthe32P-labelled (CA)syntheticsimple with BamHI(lanes1),Sad 2)andHindu!(lanes3). (M) andfemale(F)individuals ofAsellusaquaticusdigested Fig. 2SouthernblothybridizationofgenomicDNAmale kb). markers (23.3,9.4,6.6,4.3,2.3and2.0 male-specific fragments.Lambda The probewasthe32P-labelled(TCC) size markers(23.3,6.6,4.3,2.3 and2.0kb). Hin dillfragmentswereusedassize sequence. Thearrowheadsindicatethe 4), Hind!!!(lane5)andPstI6). (lanes 2),Sac!3),EcoRI digested withSac!!(lanes1),BamHI (F) individualsofAsellusaquaticus genomic DNAofmale(M)andfemale Fig. 1Southernblothybridizationof The GeneticalSociety ofGreatBritain,Heredity,75, 267—272. M F MFM (lanes -3- S

I I II I p

* p SEX CHROMOSOMEDFFERENTATIONINACRUSTACEAN269 hybridization canbeseeninoneregionlocated also involved,examinationofthespecimensisbyno the centromereofcertainchromosomesandinatleast times morefrequentlythan equivalentrandommotifs in alleukaryoticgenomes examined(Nandaeta!., Simple relation tothissignal(Fig.3c). heterochromosomes exhibitaheteromorphictetrad in meiotic displays thistypeoflabel(Fig.3a—b).Moreover,the further fromthecentromere.Nootherchromosome heterochromatic bands,whichisalsothebandsituated centromere andthesmalleroftwointerstitial some showsaprominentsignalintheareabetween centromere ofafewchromosomes.TheYchromo- tended tolocalizeintheareasneartelomeresand hybridization signalswerescantandsomeofthem tatively poorerlabelthanthe(CA)probe.Scattered chromatic bands(Fig.3d). between thetelomereandoneoftwohetero- two heterochromaticbands,whereassomesignsof shows noparticularlabelinthearealyingbetween means simple.WiththisprobetheYchromosome of thechromosomesand,moreover,polymorphismis one interstitialarea.Asitisimpossibletoidentifymost 1991). Nearlyallpossible simplemotifsoccur5—10 Discussion

Hybridization withthe(TCC)probeyieldedquanti- I

tandemly repeatedDNAsequences arepresent III I tIuII; I $ metaphases " II I •I I of o individuals • I nfl I. I-fl with ii$ 270 E. V. VOLPI ETAL.

Fig.3 In situ hybridization of spermatogonial (a, b, d) and meiotic 1(c) metaphases of Asellus aquaticus. The probes were the biotin-labelled (TCC) sequence (a, b, c) and the biotin-labelled (CA),, sequence (d). The same spermatogonial metaphase is hybridized in situ (a) and stained with DAPI (a'). The arrow heads indicate theY chromosomes. Bar represents 10 m. in both coding and noncoding regions (Tautz et at., All these observations suggest that the sex chromo- 1986). These sequences have been implicated in a somes are favoured sites for the accumulation of a variety of functions, although they might arise by DNA number of simple repeats. replication slippage or unequal crossing-over and have It is widely held that the heteromorphism of sex no primary function. Some simple sequences are sex- chromosomes evolved independently in the various specifically organized and the evolution of morpho- taxa, starting from a pair of homomorphic chromo- logically differentiated sex chromosomes has been somes and using various molecular mechanisms (for a associated with their significant accumulation by some review see Bull, 1983). researchers (for reviews see Epplen, 1988; Nanda et One typical mechanism for this type of chromosome a!., 1992). diversification is the accumulation of repetitive Accumulations of (GATA),, and (GACA) elements. In fact, Y (or W) chromosomes often exhibit sequences have been observed in the heterochromo- large heterochromatic areas consisting mainly of highly somes of snakes (Jones & Singh, 1985), rats (Schafer et repeated DNA sequences (for a review see John, at., 1986) and a poeciliid fish (Nanda et at., 1990, 1988). These sequences can be hundreds of base pairs 1991, 1992). Other poeciliid species harbour the long or simple motifs of fewer than 10 base pairs (e.g. GGAT and CA sex-specifically organized simple Cooke, 1976; Nakahori, 1986). repeat motifs (Nanda et a!., 1992). Moreover, long The accumulation of these sequences resulting stretches of (TCC),, simple repeats characterize bird W fundamentally from transposition and amplification chromosomes (Nanda et at., 1991). processes is believed to take place at first on the Y (or

The Genetical Society of Great Britain, Heredity, 75, 267—272. SEX CHROMOSOME DIFFERENTIATION IN A CRUSTACEAN 271

W) chromosome in the vicinity of the sex determining region, favoured by the absence or low frequency of Acknowledgements recombination events in this area. Thiswork was supported by Ministero delI'Università By further inhibiting exchange, the heteromorphism e della Ricerca Scientifica (MURST), Italia and by the thus acquired by the sex chromosome pair would lead Consiglio Nazionale delle Ricerche (CNR), Italia. We to increasing isolation of the Y (or W) chromosome, thank the Cenci Bolognetti Foundation for supplying creating the possibility for further differentiation the 32P-labelled nucleotides. (Muller, 1964; Bull, 1983; Charlesworth, 1991). In previous papers we observed that certain males in an A. aquaticus population collected from the Sarno References river near Naples display a heteromorphic sex chromosome pair. The Y chromosome has two intercalary BULL,i. .1983.Evolution of Sex Determining Mechanisms, areas that harbour ribosomal variant sequences Benjamin/Cummings, Menlo Park, CA. CHARLESWORTII, B. 1991. The evolution of sex chromosomes. (Rocchi et a!., 1984; Volpi et al., 1992). Meiotic recombination within the heteromorphic pair often occurs in Science, 251, 1030—1033. COOKE, R .1976.Repeated sequence specific to human the subterminal regions distal to the heterochromatic males. Nature, 262, 182—186. bands, but is very rarely observed in the region situated DI CASTRO, M., PRANTERA, G,, CIPRIANI, L. AND ROCCHI, A. 1983. between them. Silver staining analysis of nucleolar organizer activity In this paper we demonstrate the presence of a during spermatogenesis of Asellus aquaticus (Crustacea, further heteromorphism involving the A. aquaticus sex Isopoda). Genetica, 60, 163—166. chromosome pair. Experiments by Southern and in situ EPPLEN, .'r.1988. On the simple repeated GATA sequences hybridization demonstrate that the Y chromosome is in animal genomes: a critical reappraisal. J. Hered., 79, enriched with (TCC) simple repeats precisely in an 409—417. area comprised between the centromere and one of the GISBURGER-VOGEL, T. AND CHARNIAUX-COTFON, H. 1982. Sex determination. In: Bliss, D. E. (ed.) Biology of Crustacea, two heterochromatic bands. Thus the (TCC) sequence pp. 257—28 1. Academic Press, New York, London. plays a part in the evolution of the heteromorphic GRADY, D. L., RATLIFF, R. L., ROBINSON, D. L., McCANLIE5, E. C., MEYNE, chromosome pair, its accumulation possibly being .j,ANDMoyzis, R. K. 1992. Highly conserved repetitive DNA favoured by the scarcity of recombination events in this sequences are present at human centromeres. Proc. Nat!. region which probably harbours the sex gene/genes Acad. Sci. U.S.A., 89, 1695—1699. and also contains the centromere. The area on the HAMADA, H., PETRINO, M. G. AND KAKUNAGA, T. 1982. A novel heterochromosome harbouring TCC simple repeat repeated element with Z-DNA-forming potential is widely accumulation shows no colorimetric differentiation found in evolutionarily diverse eukaryotic genomes. Proc. after C-banding or staining with specific base fluoro- Nat!. Acad. Sci. U.S.A., 79, 6465—6469. chromes, and is thus not to be considered a classical IJDO, J. W., WELLS, R. A., BALDINI, A. AND REEDERS, S. T. 1991. Improved telomere detection using a telomere repeat heterochromatic region. The (TCC) sequence is also probe (TTAGGG) generated by PCR. Nuc!. Acids Res., present in other genome locations, both as relatively 19, 4780. long stretches of TCC sequences and as relatively short JOHN, B. 1988. The biology of heterochromatin. In: Verma TCC repeat arrays interspersed with other sequences. R. S. (ed.) Heterochromatin: Molecular and Structural In the case of the other simple repeats tested on the Aspects, pp. 1—147. Cambridge University Press, A. aquaticus genome, Southern blot analysis shows no Cambridge. sex-specific hybridization of the (CGA), (CCTG) and JONES, K. W. AND SINGH, L. 1985. Snakes and the evolution of (CA) probes. The (CA) simple repeat is found in all sex chromosomes. Trends Genet., 1, 55—6 1. eukaryotic genomes examined, indicating extra- LEGRAND, J. J., LEGRAND-HAMELIN, E. AND JUCHAULT, P. 1987. Sex ordinary evolutionary conservation (e.g. Hamada et a!., determination in Crustacea. Biol. Rev. Camb. Phil. Soc., 1982). It has been speculated that it is involved in the 62,439—470. regulation of gene expression as it can adopt Z-DNA MULLER, H. J. 1964. The relation of recombination to muta- tional advance. Mutat. Res., 1, 2—9. conformation. NAKAORI, Y., MITANI, K., YAMADA, M. AND NAGAKOME, y1986.A The (GGAAT )pentanucleotidedoes not seem to human Y chromosome specific repeated DNA family be present in the A. aquaticus genome. This repeat (DYZ1) consists of a tandem array of pentanucleotides. shows exceptional evolutionary conservation; centro- Nucl. Acids Res., 14, 7569—7580. meric localization of the repeat has been demonstrated NANDA, I., FEICHTINGER, W.. SCHMID, M., SCHRODER, 1. H., ZISCHLER, in several organisms, and it has been suggested that the H. AND EPPLEN, J. T. 1990. Simple repetitive sequences are repeat also plays a role in the functional human centro- associated with differentiation of sex chromosomes in the mere (Grady eta!., 1992). guppy fish. J. Mo!. Evol., 30, 456—462.

The Genetical Society of Great Britain, Heredity, 75, 267—272. 272 E. V. VOLPI ETAL.

NANDA, 1., SCHARLT, M., FEICHTINGER, W., EPPLEN, J. T. AND SCI-IMID, ROCCHI, A., PRANTERA, 0., LANZA, V. AND Dl CASTRO, M. 1984. M. 1992. Early stages of sex chromosome differentiation in Incipient sex chromosome differentiation in an isopod fish as analysed by simple repetitive DNA sequences. crustacean species, Asellus aquaticus. Chromosoma, 89, Chro,nosoma, 101, 301-3 10. 193—196. NANDA, 1.. ZISCHLER, H., EPPLEN, C., GUTTENBACH, M. AND SCI-IMID, M. SCHAFER, R., ALl, S. AND EPPLEN, J. T. 1986. The organization of 1991. Chromosomal organization of simple repeat DNA the evolutionarily conserved GATAIGACA repeats in the sequences used for DNA fingerprinting. Electrophoresis, mouse genome. Chromosoma, 93,502-510. 12,193—203. SCHWEIZER, D. AND LOIDL, .i. 1987. A model for hetero- PELLICCIA, E, Dl CASTRO, M., LANZA, V., VOLPI, E. V. AND ROCCHI, A. chromatin dispersion and the evolution of C-band 1991. GATA repeats in the genome of Asellus aquaticus patterns. In: Stahl, A., Luciani, J. M. and Vagner- (Crustacea, Isopoda). Chromosoma, 100,152—155. Capodanno, A. M. (eds) Chromosomes Today, vol. 9, pp. PELLICCIA, F., VOLPI, E. V., LANZA, V., GADDINI, L, BALDINI, A. AND 6 1—74. Allen and Unwin, London. ROCCHI, A. 1994. Telomeric sequences of Asellus aquaticus TAUTZ, D., TRICK, M. AND DOVER, 0. A. 1986. Cryptic simplicity in (Crust. Isop.). Heredity, 72, 78—80. DNA is a major source of genetic variation. Nature, 322, ROCCI-il, A., PRANTERA, G. AND Dl CASTRO, M. 1980. A study of 65 2—6 56. heterochromatin of Asellus aquaticus (Crust. Isop.). VOLPI, F. V., PELLICCIA, F., LANZA, V., Dl CASTRO, M. AND ROCCHI, A. Caryologia, 33, 401—409. 1992. Morphological differentiation of a sex chromosome and ribosomal genes in Asellus aquaticus (Crust. Isop.). Heredity, 69,478—482.

The Genetical Society of Great Britain, Heredity, 75, 267—272.