Cytogenetic analysis of Malpighian tubulepolytene chromosomes of Culex pipiens(Diptera: Culicidae)

Anna Zambetaki, Nicole Pasteur, and Penelope Mavragani-Tsipidou

Abstract: A simple technique is described for obtaining well-spread and readable Malpighian tubule polytene nuclei ofCulex pipienson a routine basis. Detailed polytene chromosome maps are presented with a description of the mostprominent landmarks of each chromosome, the regions with asynapsis and the most frequent weak points identified inthe polytene arms. Usable Malpighian tubule polytene chromosomes should facilitate molecular cytogenetic, genetic,and potentially biosystematic studies on this medically important global vector of viral inducing encephalitis.

Key words: Culex pipiens, polytene chromosomes, Malpighian tubules, banding pattern, photomap.

Résumé: Une technique simple permettant d’obtenir de façon routinière des noyaux polytènes bien étalés et lisiblesest décrite pour tubules de Malpighi chez leCulex pipens. Des cartes détaillées de chromosomes polytènes sontprésentées ainsi qu’une description des repères les plus visibles pour chaque chromosome, des régions d’asynapsis etdes point faibles les plus fréquents chez les bras polytènes. De tels chromosomes polytènes devraient faciliter lesétudes en cytogénétique moléculaire, en génétique voire même en biosystématique chez cet insecte d’intérêt médical àtravers le monde en raison de son rôle comme vecteur de nombreux virus causant des encéphalites.

Mots clés: Culex pipiens, chromosomes polytènes, tubules de Malpighi, motifs de bandes, carte chromosomique.

[Traduit par la Rédaction] Zambetaki et al. 755

Cytogenetic analysis of Diptera has been greatly facili-tated by the existence of the polytene chromosomes whichhave proven to be excellent material for studying chromo-some structure and function, temporal gene activities, chro-mosomal rearrangements and genomic organization (forreviews see Sorsa 1988 and Ashburner 1989). Further, directmapping of known nucleotide sequences by in situ hybrid-ization is greatly facilitated by accurate photomaps of thepolytene chromosomes.

The Culicidae family includes species with great eco-nomic and medical importance, as vectors of some of themost serious human diseases. The prospect of their biologi-cal control has led to the accumulation of genetic andcytogenetic data for these Culicidae species. Previous re-ports have shown that their polytene complements consist ofthree long polytene chromosomes (six polytene arms) which

correspond to a mitotic chromosome number 2n = 6 (Sutton1942; Kitzmiller and Clark 1952; Kitzmiller and Keppler1961; Dennhofer 1968; Sharma et al. 1969; Kanda 1970;Tewfik and Barr 1974; Chaudhry 1981; Asman and Lock-wood 1986; Verma 1987; Patnaik et al. 1989; Nance et al.1990; Achary 1994).

Culex pipienswas the first object of cytological investiga-tion in the Culicidae family (Stevens 1910, 1911) and eventhough the study of the salivary gland polytene chromo-somes of this species started very early (Sutton 1942;Kitzmiller and Clark 1952; Kitzmiller and Keppler 1961),they were described in significant detail quite later(Dennhofer 1968). However, these descriptions, which arebased on drawings of polytene chromosomes, have provendifficult to use for cytological analysis. Thus, the construc-tion of photographic maps of the polytene chromosomes ofthis species was considered potentially very useful.

Although the analysis of salivary gland polytenes remainsrefractory, Malpighian tubule polytene nuclei proved to bethe best material for cytogenetic analysis ofC. pipiens. De-tailed photomaps of the Malpighian tubule polytene chromo-somes are presented, and a simple method is offered forproducing well-spread polytene nuclei of this species on aroutine basis. These maps will be the reference standard forongoing molecular cytogenetic investigation (Zambetakiunpublished data).

In this study the Cever strain ofC. pipienswas used. This strain,which was collected in southern France in 1994 and reared as lar-vae in the presence of 0.1% tetracycline, was derived from the

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751

Corresponding Editor: A.J. Hilliker.

Received February 16, 1998. Accepted June 23, 1998.

A. Zambetaki,1,2 N. Pasteur,2

and P. Mavragani-Tsipidou.1,3

1Department of Genetics, Development and MolecularBiology, Aristotle University of Thessaloniki, Faculty ofSciences, School of Biology, 54006 Thessaloniki, Greece.

2Institut des Sciences de l’Evolution (UMR CNRS 5554),Universite de Montpellier-2 (CC 065), 34095 Montpellier,France.

3Author to whom all correspondence should be addressed(e-mail: mavragan@bio.auth.gr).

Sphae strain described by Nielsen-LeRoux et al. (1997). The cyto-plasmic incompatibilities observed when crossing Sphae withmany laboratory and field populations (including those collectedwithin less than 10 km from the Sphae collection site) have disap-peared in the Cever strain. Tetracycline treatment of Cever is per-formed every odd generation.

Polytene chromosome preparations were made using several tis-sues that exhibit polytenized nuclei from fourth instar larvae (sali-vary glands and Malpighian tubules) and adults (ovaries andMalpighian tubules) ofC. pipiens. The Malpighian tubules fromfemales, that were recently blood fed, gave the best results. Forchromosome preparations, 50–100 females, which were reared aslarvae under standard conditions with rich food (baker’s yeast),were isolated in 20 × 20 × 20 cm cages. More than 50 one weekold females were used for the preparations. They were allowed toblood feed for the first time and their Malpighian tubules wereused for preparations 24 h later.

Polytene chromosome preparations were made following themethod described forBactrocera oleaeby Zambetaki et al. (1995)with minor modifications. The Malpighian tubules were dissectedin Ringer’s solution and the polytene tissue was transferred to 45%acetic acid for a few seconds. The tissue was then left in 1 M HClfor 1 min, dipped into lactoacetic acid, 80% lactic acid – 60% ace-tic acid (1:1), solution and finally stained in lactoacetic orcein for3 min (Gurr’s Orcein, BDH Product 34210). Excess stain was re-moved by washing the tissue in a drop of lactoacetic acid beforesquashing. Two polytene chromosome preparations were obtainedfrom a single female.

Photographs were taken on Agfapan 100 film using a Zeissphase–contrast microscope.

Although the polytene chromosomes ofC. pipienshavebeen difficult to work with, the protocol employed gavemany preparations with several well-spread nuclei, allowingthe identification of each polytene element. The best results,for cytogenetic and in situ hybridization protocols (unpub-lished data), were obtained when 1 week old females thathad recently received a blood meal were used (see Materialsand methods). However, workable preparations were alsomade with older females that were blood fed more than onceor even with females that had not taken a blood meal.

In the present study, the photographic maps of the threepolytene chromosomes derived from Malpighian tubules ofC. pipiensare given in Figs. 1, 2, and 3. The chromosomesare numbered according to the general convention forCulexspecies. Chromosome 1 is the shortest, while chromosome 3is the longest of the polytene chromosome complement. Thepolytene chromosome complement is divided into 70 sec-tions, following the standard polytene maps of culicine spe-cies (Sutton 1942; Kitzmiller and Clark 1952; Kitzmiller andKeppler 1961; Dennhofer 1968; Sharma et al. 1969; Kanda1970; Tewfik and Barr 1974; Chaudhry 1981; Asman and

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Fig. 1. Photographic map of Malpighian tubule polytene chromosome 1 ofCulex pipiens. R and L indicate the right and left arms ofthe chromosome. Arrowheads indicate the weak points.

Fig. 2. Photographic map of Malpighian tubule polytene chromosome 2 ofCulex pipiens. R and L indicate the right and left arms ofthe chromosome. Arrowheads indicate the weak points.

Lockwood 1986; Verma 1987; Patnaik et al. 1989; Achary1994). However, the labeling of our maps is not based onthe earlier study of Dennhofer (1968), due to difficulties inaligning the two maps, the photographic (present study) andthe diagrammatic one (Dennhofer 1968).

A typical chromocenter, as an accumulation ofheterochromatin does not occur in theC. pipienspolytenecomplement and the individual chromosomes are separatedfrom each other. Chromosome 1 includes sections 1–16(Fig. 1) and it is the shortest in the polytene complement.The right and left arms do not differ significantly in lengthsize. The most characteristic landmark of this chromosomeis its putative centromeric area (region 9) which is alwayseasily identified by its compact appearance and its roundshape (Figs.1, 4f, and 4g) even in nuclei without well-spreadchromosomes. Regions 7 and 9 always constitute prominentlandmarks in this element. Chromosome 2 includes sections17–41 (Fig. 2) and consists of two unequal arms. The 2Lchromosome arm, which is quite longer than the 2R, is eas-ily recognized by its very characteristic telomeric region,which is the most prominent landmark of this chromosome.In contrast, the 2R tip is the most difficult of the entire set tobe identified. This is mainly due to its poor banding pattern,entangled configuration, and the fact that this region is typi-cally found to be asynaptic. The putative centromeric regionof this chromosome (section 26), not as prominent as that ofchromosome 1, can be identified either as two sphericalpuffs or as a banded region (Fig. 2). A very prominent land-mark of 2R arm is region 25, which is easily recognized bya pair of two strongly stained bands while region 31 is themost characteristic landmark of 2L arm consisting of a hugepuff (Figs. 2, 4d, and 4e). This is the region where esteraseA and B genes (which are amplified in the Cever strain) are

located (Zambetaki unpublished data). Chromosome 3 is thelongest of theC. pipienspolytene complement and includessections 42–70 (Fig. 3). It is metacentric with the telomericregions (regions 42–43 and 70) easily identified. Regions55–58 are characterized by many successive strong bands al-ternating by extensive interbands. As there is no obviouscentromeric structure in this element, but based on mitotickaryotypes, regions 55–56 are considered as the putativecentromere. Verification of centromere region might be pos-sible through C-banding analysis. The three bands in region59 consist of the most prominent landmark in the 3L arm.Strong banding homology between the photographic maps ofpolytene chromosomes ofC. pipiens(present study) and thediagrammatic one of Dennhofer (1968) are difficult to dis-cern. However, regions 16, 66–67, and 70 in both studies aredisplaying obvious chromosomal homologies.

In the polytene chromosomes ofC. pipiens,a high num-ber of asynaptic regions are routinely observed (Table 1).Many of these regions were typically found asynaptic andthis is why they are incorporated in the photographic map(Figs. 2 and 3). A number of weak points, which may be theresult of local underreplication of the chromosomes duringpolytenization (Lifschytz 1983; Spierer and Spierer 1984;Lamp and Laird 1987) are also shown in the photographicmaps of the polytene chromosomes ofC. pipiens(Figs. 1, 2,and 3).

The technical difficulties in the preparation of thepolytene chromosomes of theCulexgenus are mentioned inmost of the papers dealing with this material. It has beensuggested that the existence of weak points, the great length

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Chromosome arms Asynaptic regions

1R 1 (part) 3 4-5 (part) 7-8 (part)1L 10-11 (part)2R 17-18 20 222L 29-30 31-32 (part)3R 43-44 (part) 54-55 (part)3L 57-58 (part) 61-62 (part) 63-64 (part)

Table 1. Typically asynaptic regions found in the polytene chromosome arms ofCulex pipiens.

Fig. 3. Photographic map of Malpighian tubule polytene chromosome 3 ofCulex pipiens. R and L indicate the right and left arms ofthe chromosome. Arrowheads indicate the weak points.

of chromosome arms, the inter- and intra-specific chromo-somal ectopic bonds, and asynapsis observed in the polytenecomplement are some of the factors responsible for the prob-lems encountered in this genus (Sutton 1942; Kitzmiller andClark 1952; Kitzmiller and Keppler 1961; Dennhofer 1968;Sharma et al. 1969; Kanda 1970; Tewfik and Barr 1974;Chaudhry 1981; Asman and Lockwood 1986; Verma 1987;Achary 1994). However, the present work shows that theprotocol employed for Malpighian tubule polytene chromo-somes of the adult female ofC. pipiensgave excellent re-sults and that the photographic maps constructed are suitablefor further cytological analysis. Thus, it seems that hereto-fore the lack of a suitable technique to deal with these chro-mosomes was the main reason for the poor cytogenetic dataavailable till now. As mentioned by other authors, the rear-ing of the insects is important (Sharma et al. 1978; Procunierand Post 1986). In theSimulium damnosumcomplex, the de-gree of chromosome polyteny was found to be correlatedwith the gonotrophic cycle and be dependent on food source(Procunier and Post 1986). In our study, special care hasbeen taken for the larval diet, the population density and theculture temperature. Moreover, the choice of the tissueseems to play an important role. After examining manypolytene tissues (see Material and methods), we found thatthe Malpighian tubule polytene chromosomes gave the bestresults. The quality of the preparations improved signifi-cantly by using females that had recently received a bloodmeal. The Malpighian tubules of adult females were also

found to be the best tissue for obtaining good polytenechromosomes inCulex quinquefasciatus(Achary 1994).However, the method proposed for the later species gaveonly moderately good preparations, when used inC. pipiens(data not shown). On the other hand the method proposedherein gave very good preparations on a routine basis. Thus,we believe that the procedure used in the present study forthe fixation and staining of the tissue is the most crucial fac-tor for obtaining well-spread chromosomes.

One of the most striking features observed inC. pipienspolytene chromosomes is the asynapsis detected in severalregions of all chromosome arms. High degree of asynapsis isalso observed in otherCulex species. In all these reportsasynapsis was presumed to occur randomly and the reasonsfor this phenomenon were thought to be the position of thechromosomes on the slide, the amount of pressure exertedby squashing, or some other mechanisms before squashing(Sharma et al. 1969; Kanda 1970; Tewfik and Barr 1974;Chaudhry 1981; Asman and Lockwood 1986; Verma 1987;Patnaik et al. 1989; Achary 1994). Contrary to these reports,our study ofC. pipienschromosomes shows that asynapsiswas very frequently found in particular regions of the chro-mosomes (Fig. 4; Table 1). Even when the homology of thematernal and paternal strand in the asynaptic regions is evi-dent, differences in the length of interbands or even in thedensity of bands are obvious (Figs. 2, 3, 4a, 4d, and 4e).However in other cases, the homology is not evident at all(Figs. 4b and 4c). We speculate that asynapsis inC. pipiens

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Fig. 4. Representative examples of asynapsis inCulex pipienschromosomes. (a, b, c) 1R chromosome arm, (d, e) 2L chromosomearm. (f,g) Configurations of the centromeric region of chromosome 1.

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polytene chromosomes may be at least partially due topolymorphisms for regional chromosomal structuralheterozygosities.

The present study of the polytene chromosomes of C.pipiens provides necessary cytogenetic support for futurestudies dealing with the physical mapping of specific DNAsequences in this species by in situ hybridization and the in-vestigation of possible structural mechanism(s) of gene am-plification associated with resistance to organophosphorusinsecticides inCulex mosquitoes (Guillemaud et al. 1996;Nielsen-Leroux et al. 1997).

We thank Mrs. D. Heyse and Dr. C. Chevillon for supply-ing the strain and their advice with its maintenance andMrs. J. Catalan for her help and encouragement during vari-ous parts of this work. We thank Drs. C.D. Kastritsis andZ.G. Scouras for their support and critical reading of themanuscript. We greatly appreciate the significant contribu-tion of Drs. A. Hilliker and W. Procunier in the appearanceof this study. This work was supported in part by grantsfrom the Ministère de l’Education Nationale (ACC-SV3–95.03.037), CEE (ERBCHRXCT930172), and theProgramme Environnement, Vie et Societes of CentreNational de la Recherche Scientifique (GRD-1105). This ispaper no. 98-000 of UMR 5554.

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