Heredity 74 (1995) 524—530 Received l7August 1994 The Genetical Society of Great Britain

Localization and activity of rDNA genes in tiger (Coleoptera: Cicindelinae)

JOSÉ GALIAN*, JOSÉ SERRANO, PILAR DE LA RUA, EDUARD PETITP!ERREt & CARLOS JUANt Departamento de Biologia , Facu/tad de Veterinaria, Universidad de Murcia, 30071 Murcia, Spain, tLaboratori de Genètica, Departament de Biologia Ambiental, Universitat de les I/los Ba/ears, 07071 Pa/ma do Mal/orca, Spain and 4Schoo/ of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.

Silverstaining of male meiotic nuclei of six species of the tiger genus (tribe ), with multiple sex chromosomes, reveals the presence of active nucleolar organizing regions (NORs) in the sex vesicle. In one species, Cicindela melancholica, fluorescence in situ hybridization (FISH) with a ribosomal probe showed that rDNA genes are in one of the three X chromosomes and in the Y chromosome. Silver staining and FISH show that the related species Cicindela paludosa with a male XO system, has NORs located in one pair of autosomes. In euphratica (tribe Megacephalini) these techniques indicate that NORs are located in three autosomal pairs but not in the single X chromosome of males. In all these species the nucleolus can be seen from the onset of meiosis to the end of the diffuse stage; it disappears from diplotene to the end of meiosis and appears again during the spermatid stage. From these results it is concluded that: (i) the nucleolus does not seem to play a major role in the pairing and association of the multiple sex chromosomes during first meiotic prophase and metaphase; (ii) the occurrence of NORs in the heterosomes of species having multiple sex chromosomes is thought to be an ancestral condition for the genus Cicindela; and (iii) changes of location of NORs from the heterosomes to the autosomes have occurred within species of this genus, at least in species showing extensive karyotypic repatterning.

Keywords:Caraboidea,Cicindelinae, Coleoptera, FISH, NORs, rDNA.

Introduction sents the parachute proper and the y chromosome the load, which is connected to the X by two tenuous Littleattention has been paid to the localization of threads (Smith & Virkki, 1978). Since the early state- nucleolar organizers (NORs) in Adephagan beetles, in ments of John & Lewis (1960) that the nucleolus is contrast to the studies concerning species of the large necessary for the Xyp to achieve regular co-orientation suborder Polyphaga (Drets et a!., 1983; Postiglioni & during metaphase I, there have been other results Brum-Zorrilla, 1988; Virkki eta!., 1990, 1991; Juan et which show that the sex bivalent does not have a at., 1993). Weber (1971) reported the occurrence of typical nucleolus, although an argyrophilous substance NORs in the largest autosomal pair of Cam bus is developed between sex chromosomes that may carry auronitens and C. granulatus, using light and electron out the functions postulated by John & Lewis microscopy. Secondaryconstrictionsindicating (Postiglioni & Brum-Zorrilla, 1988; Virkki eta!., 1990, possible NORs have also been reported in a number of 1991). other carabid species (Serrano, 1986; Serrano et a!., Tiger beetles are carabids belonging to the subfamily 1986; Galián et a!., 1990), but no specific methodology Cicindelinae which are characterized by the presence has been worked out for analysing the nucleolus. of multiple sex chromosomes and form a nonchias- Despite this, there is much debate about the role of matic multivalent (Giers, 1977) so they are suitable for the nucleolus in the association of the typical Xyp sex studying the localization of NORs and the role of the chromosomes of the suborder Polyphaga (see for nucleolus during meiosis. The generalized karyotype example Juan eta!., 1993). The sex-bivalent Xyp has a consists of nine to 12 autosomes plus Xj, where n parachute-like shape where the X chromosome repre- varies between 2 and 4 (Serrano, 1980; Serrano et a!., 1986; Serrano & Collares-Pereira, 1989, 1992; Galián *Correspondence eta!., 1990). Only two species show an X0 sex chromo-

524 1995 The Genetical Society of Great Britain. rDNA GENES IN TIGER BEETLES 525

some mechanism, probably of unlike origin: Mega- Chromosome preparations cephala euphratica (tribe Megacephalini) and Cicindela paludosa (tribe Cicindelini; Serrano eta!., 1986). Malegonads from adult beetles were used to obtain The introduction of fluorescence in situ hybridiza- mitotic or meiotic chromosomes and nuclei. Gonads tion (FISH) in many animal and plant species including were dissected from beetles anaesthetized by ethyl beetles, has allowed the localization and mapping of acetate. A 0.04 M sodium acetate solution plus 0.05 per specific DNA sequences on chromosomes as for cent colchicine solution was injected 1 h prior to dis- JOSÉ GALIAN*, JOSÉ SERRANO, PILAR DE LA RUA, EDUARD PETITP!ERREt & example rDNA genes (Juan eta!., 1993, and additional section. Gonads were fixed in fresh ethanol-acetatic references therein). FISH differs from silver staining in acid solution (3:1) for 1 h. Routine staining was carried Departamento de Biologia Animal, Facu/tad de Veterinaria, Universidad de Murcia, 30071 Murcia, Spain, tLaboratori de that it reveals the actual number of chromosomes out with acetic orcein for 10 mm and squashing. For Genètica, Departament de Biologia Ambiental, Universitat de les I/los Ba/ears, 07071 Pa/ma do Mal/orca, Spain and carrying NORs whereas silver staining indicates which silver staining small sections of the gonads were 4Schoo/ of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK. of these are active in a particular stage of the cell cycle. squashed in 45 per cent acetic acid and the coverslip Moreover, silver staining may indicate the presence of removed after freezing in liquid nitrogen. Slides were staining of male meiotic nuclei of six species of the genus Cicindela (tribe a nucleolar-like substance within chromosomes lacking dried in a 60°C hot plate. For in situ hybridization Cicindelini), with multiple sex chromosomes, reveals the presence of active nucleolar organizing a NOR. Thus, the Polyphagan beetle Misolampus small sections of the gonads were placed in eppendorff regions (NORs) in the sex vesicle. In one species, Cicindela melancholica, fluorescence in situ goudoti (family Tenebrionidae) does not have NORs in tubes with 20 uL of 60 per cent acetic acid, to break up hybridization (FISH) with a ribosomal probe showed that rDNA genes are in one of the three X the Xyp chromosomes in contrast to the related species the tissue giving a cell suspension. Drops of this chromosomes and in the Y chromosome. Silver staining and FISH show that the related species Tenebrio molitor, but in both species the sexual biva- suspension (5 4uL) were placed on preheated slides and Cicindela paludosa with a male XO system, has NORs located in one pair of autosomes. In lent is heavily silver-stained up to anaphase I (Juan et dried on a 60°C hot plate. Megacephala euphratica (tribe Megacephalini) these techniques indicate that NORs are located in a!., 1993). In this paper we applied silver staining and fluore- three autosomal pairs but not in the single X chromosome of males. In all these species the Silverstaining nucleolus can be seen from the onset of meiosis to the end of the diffuse stage; it disappears from scence in situ hybridization with a ribosomal probe to diplotene to the end of meiosis and appears again during the spermatid stage. From these results it is tiger beetles' germ cells to show the chromosomal Asolution of 20 per cent AgNO3 in distilled water concluded that: (i) the nucleolus does not seem to play a major role in the pairing and association of localization of rDNA genes, their relationship to pair- previously adjusted to pH 3.0 with formic acid was the multiple sex chromosomes during first meiotic prophase and metaphase; (ii) the occurrence of ing of multiple sex chromosomes and their phylo- used. The solution was kept in the dark for 15 mm with NORs in the heterosomes of species having multiple sex chromosomes is thought to be an ancestral genetic significance. occasional shaking and was thereafter centrifuged at condition for the genus Cicindela; and (iii) changes of location of NORs from the heterosomes to 13000 g for 5 mm to separate the silver previously the autosomes have occurred within species of this genus, at least in species showing extensive precipitated. A 100 4uL aliquot of the supernatant was placed on the slides and incubated at 70°C during 5—15 Materials and methods mm in a humid chamber. The slides were rinsed thoroughly in distilled water, counterstained with 2 per Material cent Giemsa in phosphate buffer pH 6.8, washed, air- The species sampled in the study were collected in the dried and mounted in Eukitt. sents the parachute proper and the y chromosome the localities of southeastern Spain listed in Table 1. load, which is connected to the X by two tenuous attention has been paidthreads (Smithto the & Virkki, localization 1978). Since the early state-of nucleolar organizers (NORs)ments inof John Adephagan & Lewis (1960) that beetles, the nucleolus in is suborder Polyphaga (Dretsnecessary et a!., for the 1983; Xyp to achieve Postiglioni regular co-orientation & Brum-Zorrilla, 1988; Virkkiwhich eta!., show that 1990, the sex bivalent 1991; does Juan not have et a at., 1993). Weber (1971)typical reported nucleolus, althoughthe occurrence an argyrophilous substance of NORs in the largest autosomalis developed between pair sex chromosomes of Cam that may bus carry Table 1 Haploid chromosome numbers and collection sites of the eight species of auronitens and C. granulatus,out the usingfunctions lightpostulated and by Johnelectron & Lewis tiger beetles from southeastern Spain possible NORs have also been reported in a number of Species n Localities Tiger beetles are carabids belonging to the subfamily Tribe Megacephalini 1986; Galián et a!., 1990), Cicindelinaebut no whichspecific are characterized methodology by the presence Megacephala euphratica 15 + X La AzohIa (Murcia) Tribe Cicindelini Despite this, there is muchmatic multivalentdebate (Giers, about 1977) so the they are role suitable of for Cicindela campestris 9 + XXXY Balneario de Tus (Albacete) the nucleolus in the associationstudying the of localization the typical of NORs and Xyp the role sexof the C. maura 9 + XXXY Laguna del Hondo (Alicante) chromosomes of the subordernucleolus during Polyphaga meiosis. The generalized (see karyotype for C. deserticoloides 9+ X)(XY San Isidro de Albatera (Alicante) consists of nine to 12 autosomes plus Xj, where n C. litorea 9 + XXXY Salinas del Rassal (Murcia) example Juan eta!., 1993). The sex-bivalent Xyp has a C. me!ancho!ica 9+ XXXY El Algar (Murcia) C. f7exuosa 9+ XXXY Salinas de San Pedro (Murcia) 1986; Serrano & Collares-Pereira, 1989, 1992; Galián 7+ X HellIn (Albacete) eta!., 1990). Only two species show an X0 sex chromo- C. paludosa

The Genetical Society of Great Britain, Heredity, 74, 524—530. 526 J. GALIAN ETAL.

ualized chromosomes showing a bouquet disposition DNA probe (Fig. ic). Thereafter, from diplotene to metaphase I, Theribosomal probe used was pDm238 (Roiha et a!., the sex chromosomes show only terminal connections 1981) cloned in pBR322. It contains the 18S, 5.8S, which are probably of a nonchiasmatic nature (Fig. id). 28Sgenes, plus intergenic spacers of Drosophila The congeneric species Cicindela paludosa has a melanogaster. diploid chromosome number 2n =15(n =7+X) and an X0 sex chromosome system (Fig. le). Megacephala euphratica has the highest chromosome number so far In situ hybridization known in tiger beetles, 2 n =31, and also shows a single Methodsfor pretreatment of slide preparations, in situ X in males (Fig. if). hybridization and probe detection followed the proce- dure described by Juan et al. (1993) with minor modifications. Chromosome spreads were pretreated Silverstaining of NORs with DNase-free RNase in 2 x SSC for 1 h at 37°C, Silverstaining in the six species found to have multiple followed by treatment with 0.005 per cent pepsin in 10 sex chromosomes shows the same common pattern. mlvi HC1 for 10 mm, dehydration in a graded ethanol Two nucleolar precipitates are seen in interphase series, and air drying. The probe was labelled with nuclei. These come together from leptotene to late biotin-16-dUTP by nick translation. The hybridization pachytene and are associated to the sex vesicle (Fig. 2a, mixture contained 50 per cent formamide, 0.1 pg/1uL b,d). In the diffuse stage after pachytene the two denatured salmon sperm DNA, 0.1 1ug/uL yeast RNA precipitates are seen separated (Fig. 2c), and are joined and 4 ng/1uL of labelled probe. A 5 duL aliquot of this again at early diakinesis. As diakinesis progresses the mixture was placed on the slide and covered with a 20 x 20 mm coverslip. Denaturation of the probe and nucleolar precipitates disappear and reappear again in spermatid nuclei, where only one silver dot per nucleus chromosomes was performed at the same time on a is present (Fig. 2d inset). 80°C hot plate for 3 mm and they were then trans- Prophase I nuclei in C. paludosa show two silver ferred to a humid chamber at 37°C for hybridization dots from leptotene to zygotene (Fig. 2e right) that overnight. After hybridization coverslips were carefully become fused in pachytene (Fig. 2e left) and during the removed and the slides were then given a stringent diffuse stage (Fig. 2f). These dots disappear at diakine- wash three times for 5 mm each in 50 per cent forma- sis. No nucleolar precipitates are observed from meta- mide, 2 x SSC at 37°C. Sites of probe hybridization phase I to anaphase II, but thereafter every spermatid were detected with avidin-FITC (fluorescein). The nucleus shows one nucleolar precipitate. In ovariole signal was amplified twice using goat anti-avidin-biotin. cells of this species two precipitates are observed at Slides were counterstained with propidium iodide and interphase (not shown) as in male diffuse nuclei. mounted with antifade solution to reduce fade of Early prophase I nuclei in M. euphratica show from fluorescence. Slides were examined with a Zeiss one to four silver precipitates (Fig. 2g) that develop Axiophot photomicroscope and photographed with into four areas of silver dots during the diffuse stage Kodak Ektachrome. (Fig. 2h). In most spermatid nuclei only one nucleolus was formed since only one dot per cell was visible. Results Insitu hybridization with ribosomal probe Conventional staining FISHwith the ribosomal probe pDm238 was The male diploid chromosome number of Cicindela performed to test for the presence of ribosomal genes campestris, C. maura, C. deserticoloides, C. litorea, C. detected as active with silver staining. It was performed melancholica and C. flexuosa is 2n 22, with nine in one species with multiple sex chromosomes, pairsof autosomes and four heterosomes, Cicindela melancholica, and in two species with an X0 n 9 + XXXY(Table1, Fig. 1). Autosomal bivalents sex chromosome mechanism, C. paludosa and Mega- form chiasmata and show a regular meiosis. The cephala euphratica. Hybridization of spermatogonial multiple sex chromosomes form a heteropycnotic mitosis of C. melancholica showed the presence of condensed body or 'sex-vesicle' at early zygotene (Fig. ribosomal genes in two nonhomologous chromosomes, la), which is visible until late pachytene. Nuclear one in the distal part of the Y chromosome and the volume increases greatly during a diffuse stage that other in an interstitial region of one of the X chromo- follows pachytene at which stage the chromatin decon- somes (Fig. 3a). The intensity of fluorescence differs denses (Fig. ib). The sex vesicle evolves into individ- slightly between these two chromosomes. The two

The Genetical Society of Great Britain, Heredisy, 74, 524—530. euphratica, metaphaseI.Arrowsshowthemultiplesexchromosomesfrom(a)to(d)andunivalentin(e)(f).All Fig. All x2000. pachytene (left).(f)C.paludosa, diffusestage.(g)Megacephalaeuphratica,leptotene.(h) euphratica,diffusestage. melancholica, diffusestage.(d) C.melancholica,pachytene;inset:spermatidnucleus.(e) C. paludosa,zygotene(right)and Fig. 2Silverstainingoftigerbeetle meioticnuclei.(a)Cicindeladeserticoloides,pachytene. (b)C.flexuosa,latezygotene.(c) deserticoloides, diffusestage.(c)C.litorea,earlydiplotene.(d)diakinesis.(e)paludosa,(f)Megacephala x2000. The GeneticalSociety ofGreatBritain,Heredity,74, 524—530. 1 Meioticnucleioftigerbeetlespeciesafterconventionalorceinstaining.(a)Cicindelamelancholica,zygotene.(b)C.

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Fig.3 Fluorescence in situ hybridization of tiger beetle nuclei with the ribosomal probe pDm238. (a) Cicindela melancholica, mitotic metaphase showing two chromosomes with hybridization signal. (b) C. melancholica, diffuse stage showing two signals. (c) C. melancholica, two zygotene nuclei showing the sex vesicle with two signals. (d) C. melancholica, metaphase I with the hybridization signal in the sex quadrivalent; inset: two spermatid nuclei with a single signal. (e) C. paludosa, pachytene with signal in one bivalent (top) and spermatid nucleus with one signal (bottom). (1) Megacephala euphratica, metaphase I with signal in three autosomal pairs; inset: spermatid nucleus with three signals of different intensity. Bar represents 5 1um.Allx 2000.

signals are also seen at zygotene and the diffuse stage Discussion (Fig. 3b,c). Hybridization of first metaphase nuclei with the probe shows that the signal clearly appears in two Localization of NORs in cicindeild beetles chromosomes of the sex quadrivalent (Fig. 3d). This is further confirmed by studying the FISH pattern of Evidencefrom standard methods, silver staining and spermatids using the same probe. In this case, all FISH with a ribosomal probe indicates that cicindelid spermatids show only one hybridization signal, a find- beetles with multiple sex chromosomes have the NORs ing which was expected if the two hybridizing chromo- situated in two of these heterosomes, probably in one somes in the quadrivalent were one X and the Y (Fig. X (the largest one in C. melancholica, as inferred from 3d inset). the karyogram shown in Serrano & Collares-Pereira, FISH with the ribosomal probe in C. paludosa 1992) and in the Y chromosome. In particular, the shows hybridization in one autosomal pair, and no staining of the sex vesicle during prophase I with silver signal is present in the single X chromosome. This is and the hybridization of this vesicle in two sites with confirmed in spermatid nuclei, every one of which the rDNA probe are considered as conclusive evidence shows one signal (Fig. 3e). for such an inference. In Megacephala euphratica meiotic observations The two species with a male XO system show a show that the ribosomal cistrons are present in three different pattern. In C. paludosa the results indicate medium-sized autosomal pairs but not in the X that the NORs are present in one autosomal pair. chromosome (Fig. 3f). Accordingly, spermatids also Assuming that the karyotype of this species has show three fluorescent dots (Fig. 3f inset). The three evolved from an ancestral one with multiple sex chromosome pairs can be distinguished by a different chromosomes (Serrano et a!., 1986; Galián et a!., intensity of the signal even in spermatid nuclei. 1990), it must be concluded that the rDNA genes have

The Genetical Society of Great Britain, Heredity, 74, 524—530. rDNA GENES IN TIGER BEETLES 529 been translocated from the sex chromosomes to auto- species will be found among those of the subgenus somes during the evolution towards the more compact which are related to C. (Cylindera) paludosa. karyotype shown by this species. It not only has the heterosomes decreased in number from XXYto XO, and evolution of the pattern but also the autosomes from 18 to 14 (Serrano et a!., Origin 1986). Serranoet a!. (1986) have suggested that the occur- In Megacephala euphratica three autosomal pairs rence of multiple sex chromosomes is an ancestral have rDNA genes but the single X chromosome does condition for the subfamily Cicindelinae. As the not. The difference in intensity of the fluorescent signal species discussed above belong to different lineages between these chromosomes probably suggests that the within the large and cosmopolitan genus Cicindela, it number of copies of rDNA genes varies from one loca- may be concluded that the presence of NORs on one X tion to another. This structural difference might explain and the Y chromosome results from common ancestry. the different pattern of activity between the NORs of The relationship between this characteristic pattern this species, as up to four silver precipitates only can be and the occurrence of multiple sex chromosomes is clearly seen during meiotic prophase. uncertain and these might well be unrelated pheno- mena; that is, changes in the localization of NORs are independent of changes in the sex chromosome system. ActiveNORs, rDNA genes and mode of association of In any case, species showing a different pattern should the multiple sex chromosomes be considered as derived, as it happens with C. (Cylin- Asdiscussed above, the species of tiger beetles studied dera) paludosa. with silver staining and in situ hybridization with the Interestingly, rearrangement of NORs from hetero- ribosornal probe can be grouped into two categories: (i) somes to autosomes (or vice versa) has also been species with active NORs and rDNA genes located in reported in Polyphagan beetles, namely, some two of the sex chromosomes, and (ii) species with dermestid beetles (John & Shaw, 1967) and in the active NORs and rDNA genes in autosomal pairs but tenebrionids Tenebrio molitor (NORs present on Xyp) not in the single X chromosome. These results raise the vs. Misolampus goudoti (NORs in the autosomes; Juan question of the role of nucleolar material in the asso- et a!., 1993), without any change in the sex chromo- ciation and segregation of the multiple sex chromo- some system. somes in cicindelid beetles, as proposed for species of The results from Megacephala euphratica (tribe the suborder Polyphaga (John & Lewis, 1960). Megacephalini) are consistent with the phylogenetic As no silver precipitate is seen after the diffuse distance between this tribe and the Cicindelini based meiotic stage of tiger beetles, it might be concluded that on karyotypic and morphological grounds. As well as no nucleolar substance plays any role in the organiza- the differences in number of autosomes and type of sex tion and segregation of the sex quadrivalent from chromosomes (Serrano eta!., 1986), M euphratica also diakinesis to the onset of anaphase I. In agreement with differs in the position and number of NORs. Further this conclusion no staining of any kind is found studies on members of this tribe are needed to evaluate between the elements of the quadrivalent by classical properly the phylogenetic significance of these differ- staining methods; only telomeric connections are ences. observed. Moreover, Giers (1977) clearly showed in Cicindela hybrida that if protein synthesis is experi- Acknowledgements mentally inhibited with cycloheximid or puromycin, the telomeric connections between heterosomes are Weare grateful to A. Andüjar, J. L. Lencina, and A. S. not developed and the quadrivalent is not formed. Ortiz for their help in collecting material and Professor It is also doubtful if the nucleolar substance plays a G. M. Hewitt who kindly made suggestions that major role in the association of sex chromosomes improved the paper. This work has been supported by during the earlier stages of meiosis. It is known that no project DGICYT no. PB9O/0357 of the Spanish synaptonemal complex is formed between the sex Ministry of Education and Science. chromosomes (Giers, 1977), and only two of the heterosomes have NORs, and hence the ability to form their own nucleolus, thus making it possible to become References associated through this organelle. DRETS,M. E., CORBELLA, E., PANZERA, F. AND FOLLE, G. A, 1983. From these conclusions about the role of the nucleo- C-banding and non-homologous associations II. The lar substance, we may expect to find some species of 'parachute' Xyp sex bivalent and the behavior of hetero- Cicindela with multiple sex chromosomes and NORs chromatic segments in Epilachna paenulata. Chromo- located only in the autosomes. Perhaps some of these soma,88,249—255. The Genetical Society of Great Britain, Heredity, 74, 524—530. 530 J. GAUAN ETAL.

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The Genetical Society of Great Britain, Heredity, 74, 524—530.