g analysis of mitotic and polytene chromosomes and … · 2013. 5. 7. · the melon ßy, bactrocera...

GENETICS

Analysis of Mitotic and Polytene Chromosomes and PhotographicPolytene Chromosome Maps in Bactrocera cucurbitae

(Diptera: Tephritidae)

A. ZACHAROPOULOU,1,2,3 W.A.A. SAYED,1,4 A. A. AUGUSTINOS,2 F. YESMIN,1,5

A. S. ROBINSON,1 AND G. FRANZ1

Ann. Entomol. Soc. Am. 104(2): 306Ð318 (2011); DOI: 10.1603/AN10113

ABSTRACT We report here a cytogenetic analysis of the melon ßy,Bactrocera cucurbitae, Coquillett(Diptera: Tephritidae), a species of signiÞcant agricultural importance. The mitotic karyotype anddetailed photographic maps of the larval salivary gland polytene chromosomes of the species arepresented. The mitotic karyotype consists of six pairs of chromosomes including one pair of heter-omorphic sex (XX/XY) chromosomes. The heterogametic sex is ascribed to the male. The analysis ofpolytene chromosomes has shown a total number of Þve long polytene elements (10 polytene arms)that correspond to the Þve autosomes. The characteristic features and the most prominent landmarksof each polytene chromosome are presented. The proposed chromosomal homology between B.cucurbitae and Mediterranean fruit ßy,Ceratitis capitata (Wiedemann), was determined by comparingchromosome banding patterns. The detection of heterozygous chromosome inversions in two strainsis shown and discussed. The current study provides workable polytene chromosome maps of thespecies and compares our results with previous reports. We show that these maps can be used forcytogenetic studies in the species and for comparative studies among the tephritid species. They alsocan support the development of control methods and clarify the taxonomic status of B. cucurbitae.

KEY WORDS Bactrocera cucurbitae, melon ßy, polytene chromosomes, chromosome inversions,Tephritidae

The melon ßy, Bactrocera cucurbitae Coquillett (Dip-tera: Tephritidae), is a species of signiÞcant agricul-tural importance. It is a polyphagous fruit ßy andattacks �120 plant species, being a major pest of theCucurbitaceae and Solanaceae species (Dhillon et al.2005, Piñero et al. 2006). The melon ßy is distributedover most of India, which is considered its nativehome, and throughout Pakistan, Nepal, China, NewGuinea, the Philippines, and the Mariana and the Ha-waiian Islands (Weems et al. 2001). Other populationswere reported in Africa (Egypt, Kenya, Tanzania, andEast Africa) where it is probably a recent invader(Weems et al. 2001). It invaded the south-westernislands of Japan from 1919 to 1974, but it was success-fully eradicated after an areawide integrated programinvolving the releaseof sterile insects lasting from1972to 1993 (Koyama et al. 2004).

Despite its wide distribution and importance as anagricultural pest, knowledge of the genetics of thespecies is still limited. Fifteen morphological markershave been described and assigned to Þve autosomallinkage groups (McCombs et al. 1996). Recently, agenetic sexing strain has been constructed based on apupal color marker McInnis et al. 2004). Genetic anal-yses of wild and natural populations have been re-ported using random ampliÞed polymorphic DNAmarkers (Haymer 1995) and mitochondrial DNA(Muraji and Nakahara 2001, Jamnongluk et al. 2003,Zhu et al. 2005, Hu et al. 2008). Cytogenetic studieshave been carried out, but the results are controversialconcerning both the description and the character-ization of mitotic and polytene chromosomes (Go-palan 1972, Gopalan and Dass 1972, Bhatnagar et al.1980, Singh and Gupta 1984, Shahjahan and Yesmin2002).

Dipteran polytene chromosomes are extremely use-ful for cytogenetic and genetic studies because of theircharacteristic species-speciÞc banding pattern. Poly-tene chromosomes have been used in studies relatedto chromosome structure and function, gene activity,genomic organization, and phylogenetic relationshipsamong species and in studies to distinguish membersof complex species (Coluzzi et al. 1979, Clayton andGuest 1986, Ashburner 1992, Krimbas and Powell 1992,

1 Joint FAO/IAEA Programme of Nuclear Techniques in Food andAgriculture, International Atomic Energy Agency, AgencyÕs Labora-tories, Seibersdorf A2444, Austria.

2 Corresponding author, e-mail: [email protected] Department of Biology, University of Patras, Patras 26500, Greece.4 Current address: Department of Biological Application, Nuclear

Research Centre, Atomic Energy Authority, Cairo, Egypt.5 Current address: Radiation Entomology Laboratory, Institute of

Food and Radiation Biology (IFRB), Atomic Energy Research Es-tablishment (AERE), Bangladesh Atomic Energy Commission(BAEC), Dhaka-1000, Bangladesh.

0013-8746/11/0306Ð0318$04.00/0 � 2011 Entomological Society of America

Zhimulev et al. 2004). They also provide a means toanalyze the structure of chromosome rearrangementsand to map the precise localization of genes throughin situ hybridization, thus contributing to the con-struction of detailed cytogenetic maps (Pardue andGall 1975).

For insects of economic importance such studiescan facilitate both the genetic analysis of natural pop-ulations and the development or improvement of ge-netic control methods. Polytene chromosome maps ofthe Mediterranean fruit ßy Ceratitis capitata (Wiede-mann) have helped to improve the sterile insect tech-nique by supporting the development of geneticsexing strains (reviewed in Robinson et al. 1999,Gariou-Papalexiou et al. 2002, Franz 2005).

Here, we present the mitotic karyotype and thesalivary gland polytene chromosome maps of B. cu-curbitae. Our primary interest was to provide work-able polytene chromosome maps of the species andcompare our results with previous reports. We showthat these maps could be used for cytogenetic studiesin the species and could also be used for comparativestudies among the tephritid species.

Materials and Methods

B. cucurbitae Strains. Two laboratory colonies of B.cucurbitae, a wild-type strain and a genetic sexingstrain (GSS) maintained in the Entomology UnitSeibersdorf laboratory (Austria), were used in thisstudy, to compare the chromosome structure in twostrains from different geographic origin. The wild-type strain was derived from pupae sent to Seibersdorfin 2007 from the Institute of Food and RadiationBiology, Atomic Energy Research Establishment,Dhaka, Bangladesh. The genetic sexing strain, createdwith a wild-type strain from Hawaii (McInnis et al.2004), was derived from pupae sent to Seibersdorf in2005. Adults were fed on a mixture (1:1:3) of yeast:wheat germ:sugar. Larvae of the wild-type strain werereared on sweet pumpkin (Cucurbita sp.). Larvae ofthe GSS were reared on an artiÞcial diet containing28% wheat bran, 7% brewerÕs yeast, 13% sugar, 0.28%sodium benzoate, and 1.7% HCl.Mitotic Chromosome Preparations. Chromosome

preparations were made from brain ganglia of latethird-instar larvae (Zacharopoulou 1987). Brain tissuewas dissected in saline solution (0.7% NaCl) and trans-ferred to hypotonic solution (1% sodium citrate) on adepression slide for at least 15 min and then Þxed for3 min in freshly prepared Þxative (3:1 methanol:aceticacid), with several changes to ensure the completeremoval of the water. By the end of Þxation, the Þx-ative was removed and a small drop of 60% acetic acidwas added. Working quickly under the dissecting mi-croscope, the tissue was dispersed by drawing up intoa micropipette several times. The cell suspension wasÞnally laid on a clean slide placed on a warm hotplate(40Ð45�C) for drying. Chromosomes were stainedwith 5% Giemsa in 10 mM phosphate buffer, pH 6.8. Cbanding was performed according to Selivon andPerondini (1997).

Polytene Chromosome Preparations. Polytenechromosome preparations were made from well-fedthird-instar larvae Zacharopoulou 1987, 1990). Larvaewere dissected quickly in 45% acetic acid, and salivaryglands were transferred to 3 N HCl on a depressedslide for 1 min. Glands were Þxed in glacial aceticacid:water:lactic acid (3:2:1) for �5 min (until beingtransparent) before staining in lacto-acetic-orcein for5Ð7 min. Excess stain was removed by washing theglands in lacto-acetic acid before squashing. Chromo-some slides were analyzed at 100� magniÞcation byusing a phase contrast microscope (Leitz, Wetzlar,Germany). Well spread nuclei or isolated chromo-somes were photographed using a CCD camera (Prog-Res CFcool, Jenoptik Jena Optical Systems, Jena,Germany).Construction of Photographic Polytene Maps. Pho-

tographs showing the best morphology for each chro-mosome region were selected. These regions wereassembled using the Photoshop CS2 software (AdobeSystems, Mountain View, CA) to construct the com-posite photographic map for each chromosome.

Results

Mitotic Chromosomes. Previous reports on the cy-tology ofB. cucurbitae (Gopalan 1972, Bhatnagar et al.1980, Singh and Gupta 1984, Shahjahan and Yesmin2002) have shown that the mitotic chromosome com-plement of the species consists of six pairs of somat-ically paired chromosomes, including an XX/XY sexchromosome pair; this was conÞrmed by the currentstudy. Figure 1 shows chromosome spreads from fe-

Fig. 1. B. cucurbitae mitotic karyotype. Giemsa stainingof mitotic metaphases from female larvae (a and b), a malelarva (c), and C-banding metaphase from a female larva (d);X, Y chromosomes (indicated by arrows) and the two sub-metacentric chromosomes, A1 and A2, are indicated. Aster-isks show the secondary constriction of X chromosome.

March 2011 ZACHAROPOULOU ET AL.: B. Cucurbitae POLYTENE CHROMOSOME MAPS 307

male and male larvae. Although the sex of the third-instar larvae could not be determined, the heteroga-metic sex (XY) is ascribed to the male, a situation thatholds for all tephritid species so far analyzed (seeDiscussion). Three of the autosomes are metacentric.Only the longest pair of these can be distinguishedbecause the other two pairs are very similar in size.The rest of the autosome pairs are submetacentric(named A1 and A2 in Fig. 1) and can be recognized bydifferences in their size and arm ratios. Due to thedifÞculties in distinguishing the Þve autosomes, theyhave not yet been numbered. It is preferable to cor-relate the mitotic and polytene chromosomes Þrst andthen use the same numbering system for both chro-mosome sets.

The sex chromosomes are the smallest of the com-plement and are more heavily stained than the auto-somes. This observation, combined with the absenceof chromatid separation, indicates that both sex chro-

mosomes are mainly heterochromatic. The X chro-mosome is the longer of the two and often shows twoconstrictions, the Þrst at about the middle and thesecond close to one end (Fig. 1a and d). As is evident(Fig. 1d), both constrictions are lightly stained afterthe C-banding, contrary to the heavy staining of therest of the chromosome. The Y chromosome is tinyand dot-like and is heavily stained.Polytene Chromosomes. Well-spread preparations

of B. cucurbitae polytene chromosomes are not easy toobtain mainly due to frequent ectopic pairing, the pres-ence of numerous weak points and the coiling of chro-mosomes. Polytene chromosomes are usually frag-mented, thus making it difÞcult to identify eachchromosome in its entirety. Because the quality of chro-mosomes is crucial for their analysis, efforts were con-centrated on improving the rearing of the larvae. Toobtain optimal chromosomes, we used a lower temper-ature for the rearing of the larvae (20�C instead of 25�C)

Fig. 2. B. cucurbitae polytene nucleus 1. The chromosome arms are shown. Thin arrows show centromeric regions (C)of the chromosomes and the thick arrow shows the breakpoints of an inversion on the chromosome arm 4L, In(4L)43-47.The dotted line shows the continuity of the 4L arm. The heterochromatic mass (H) is indicated. Characteristic landmarksare indicated by numbering the respective chromosome sections.

308 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 104, no. 2

and we avoided overcrowding in the larval cultures.Using this approach, a sufÞcient number of well spreadpreparations was obtained that enabled the identiÞca-tion of each chromosome and the construction of thecomposite polytene chromosome maps.

The polytene nuclei of B. cucurbitae have Þve long-banded chromosomes (Figs. 2 and 3), correspondingto the Þve autosomes. We never observed a sexualdimorphism in polytene nuclei, an indication support-ing the nonpolytenization of the sex chromosomes. Asis evident from Figs. 2 and 3, there is no typical chro-mocenter where all chromosomes of a nucleus areassociated through their heterochromatic centro-meric regions. However, in some well spread chro-mosome preparations, a limited heterochromaticstructure was observed, either connecting some of thechromosomes, or intertwined with the banded chro-mosomes (Figs. 2 and 3).

Absence of a typical chromocenter complicates thelocation of the centromere in each chromosome.Based on several criteria that have already been usedto localize the centromeres in other Tephritid species(Bedo 1987, Zacharopoulou 1990, Zhao et al. 1998,Mavragani-Tsipidou 2002, Garcia-Martinez et al.

2009), it was possible to determine the centromereposition for each chromosome element. Figure 4shows the centromeric positions of all polytene auto-somes, indicated by heterochromatic mass (Fig. 4aand b), bands with a diffused structure (Fig. 4c), or bya form of constriction (Fig. 4d and e), and/or byattenuated chromosome threads that connect thechromosomal arms (Fig. 4f and g).

Chromosome tips are sufÞciently characteristic toallow identiÞcation and differentiation of each chro-mosome (Figs. 2 and 3). An analogous situation holdsfor the proximal chromosome regions, as shown in Fig.4. In addition, many bands along each chromosomeexhibit their own distinctive appearance that providesimportant landmarks for chromosome identiÞcation.Several puffs were observed in each chromosome, inaccordance with the pufÞng phenomena observed indipteran polytene chromosomes.

One characteristic of the polytene chromosomesfound in several chromosome preparations is the ec-topic pairing between the telomeric regions of differ-ent chromosome arms. Figure 5 shows examples of thistype of pairing in which chromosome arm 3L is pairedeither with chromosome 4R or 6L. In most cases, this

Fig. 3. B. cucurbitae polytene nucleus 2. The centromeric regions are shown by thin arrows (C stands for centromere).Note the heterochromatic structure (partial chromocenter C2/3). The telomeres of the ten arms are indicated. The thickarrow shows the breakpoints of an inversion on the chromosome arm 2L, In(2L)6-9. The pericentromeric region ofchromosome Þve is indicated by numbering the respective chromosome sections.


type of pairing is so tight that it is difÞcult to identifythe free ends of the chromosomes involved.

We adopted the numbering system used for C.capitata chromosomes (Zacharopoulou 1990) to fa-cilitate comparison with other tephritid species.Following this system, the whole polytene genomewas divided into 100 sections, and each element wasallocated 20 sections irrespective of its size, usingthe most prominent or distinctive bands as sectionborders. Furthermore, the polytene chromosomeswere labeled from two to six to indicate homologywith the respective chromosomes of C. capitata,based on the similarity of the banding pattern ofspeciÞc chromosomal regions. B. cucurbitae poly-tene chromosome reference maps and their bandingpattern comparison with C. capitata chromosomemaps are presented in Figs. 6Ð10.Chromosome 2, Sections 1–20.The two free ends of

chromosome two are easily identiÞed and can be usedas diagnostic markers for this chromosome (Fig. 6).Moreover, the centromeric region of chromosometwo has a diffuse structure that always connects thetwo arms and is often connected to other centromericregions (Fig. 4a and b), thus forming a partial chro-mocenter. The left arm has a poor banding morphol-ogy, whereas the right arm has a distinctive bandingpattern that facilitates the identiÞcation of this arm.

Fig. 4. Centromeric regions of the Þve B. cucurbitae polytene chromosomes. Arrows indicate the heterochromatic massassociated with centromeres (a and b), diffused bands (c), constrictions (d and e), and attenuated threads (f and g). Thechromosome arms are indicated.

Fig. 5. Ectopic pairing of telomeres inB. cucurbitae chro-mosomes. (a and c) Contacts of the 4R and 3L telomeres. (b)Contacts of the 3L and 6L telomeres. Note the tight contactof the two tips (a and b) and the attenuated thread con-necting the two tips (c). Telomeres are indicated by arrowsmarked with an asterisk (*). Sections of the chromosomesalso are shown.


The most important landmarks of the chromosome areunderlined in Fig. 6.

Comparison of chromosome two of B. cucurbitaewith chromosome two of C. capitata showed a limitedsimilarity of banding patterns, restricted to telomeres,subtelomeric regions, and pericentromeric regions.One inversion was found on 2L, compared with C.capitata 2L arm (Fig. 6).Chromosome3, Sections 21–40.Chromosome three

of B. cucurbitae is the most “difÞcult” of the genome(Fig. 7). In addition to its poor banding morphology,it shows frequent breaks due to the numerous weakpoints. By analyzing and subsequently selecting thebest chromosome regions from a large number ofchromosome preparations, it was possible to constructthe composite map presented here. The centromerehas a diffuse structure and often shows an ectopiccontact with centromeric regions of other chromo-somes (Fig. 4a and b). The chromosomal regions thatcan be used as identiÞcation marks of this chromo-some are shown in Fig. 7.

Chromosome three of B. cucurbitae is homologousto chromosome three of C. capitata based mainly onthe characteristic bands of the 3L arm. Three trans-positions were found on B. cucurbitae 3L, in relationto C. capitata 3L, two of them with inverted orienta-tion. There is only limited similarity between the 3Rarms of the two species, restricted to the tips (Fig. 7).Chromosome 4, Sections 41–60. This chromosome

is easy to identify (Fig. 8). The centromeric region is

characterized by an attenuated chromatin thread, bydiffused heterochromatic bands that always connectthe two arms, or both, and it is usually ectopicallyconnected to other centromeres (Fig. 4). The mostimportant diagnostic features of the chromosome areshown in Fig. 8.

The homology of this chromosome with chromo-some four of C. capitata is supported by extendedbanding pattern similarities between the two ele-ments. One inversion was found in B. cucurbitae 4Larm relative to C. capitata 4L (Fig. 8).Chromosome5,Sections61–80.ChromosomeÞve is

the longest of the polytene complement and the leftarm is signiÞcantly longer than the right arm (Fig. 9).The left arm has regions with a distinctive bandingpattern along with regions of poor banding pattern,whereas the right arm has the best banding pattern ofthe set. The centromere position of the chromosomeis deÞned by a constriction at the boundaries of sec-tions 72 and 73, the attenuated thread that connectsthis constriction to other nonhomologous chromo-somes (Fig. 4d and f), or both. The most characteristicregions that constitute important landmarks for thischromosome are shown in Fig. 9.

The homology of chromosome Þve between B. cu-curbitae andC. capitata is readily apparent, despite thepresence of two transpositions on 5L, one paracentricinversion on 5R and one pericentric inversion (Fig. 9).Chromosome6, Sections 81–100.Chromosome six is

long with its left arm signiÞcantly longer than the right

Fig. 6. Reference map of B. cucurbitae (Bc) chromosome two (sections 1Ð20) and banding pattern comparison with theC. capitata (Cc) chromosome two (C, centromere). The most important landmarks of B. cucurbitae chromosome two areunderlined. Dotted lines connecting the chromosomes indicate sections with similar banding pattern and arrows show therelative orientation of these sections to each other. The tips of both arms can be correlated with the respective tips ofchromosome two of C. capitata. Sections 2 and 3 of B. cucurbitae 2L arm show similarity to sections 2Ð4 of C. capitata 2L arm,although in inverted orientation. Proximal 2L and 2R pericentromeric regions are similar in the two species.


arm, a situation that also is observed for chromosome5 (Fig. 10). The free end, section 81, is easily recog-nized by a bundle of thin dark bands covering almostthe Þrst half of this section. The free end of the rightarm, section 100, exhibited a characteristic morphol-ogy is easily identiÞed (Fig. 10). However, the wholearm is very fragile due to many weak points along itslength, thus making its identiÞcation difÞcult.

Banding pattern similarities between B. cucurbitaechromosome six and C. capitata chromosome six sup-port the proposed homology of these two chromo-somes. One inversion was observed on B. cucurbitae6L, compared with C. capitata 6L arm (Fig. 10).Chromosome Rearrangements.During the analysis

of numerous polytene chromosome preparations sev-eral paracentric inversions were observed. Examplesof such inversions are given in Fig. 11. Inversions aredistributed in four of the Þve chromosomes; no rear-rangements were observed in chromosome 5.

Discussion

Previous cytological reports for B. cucurbitaeshowed that the mitotic karyotype consists of six pairsof chromosomes, including a heteromorphic sex chro-mosome pair, that are consistent with the currentÞndings. However, there are differences concerning

the chromosomal arm ratios either among previousreports (Gopalan 1972, Bhatnagar et al. 1980, Singhand Gupta 1984, Shahjahan and Yesmin 2002), be-tween these reports and the current results, or both.According to the present results (Fig. 1), two of theautosome pairs (A1 and A2) are submetacentric,whereas three pairs are metacentric. The sex chro-mosomes, easily identiÞed by the heteromorphic pairof XX/XY chromosomes, are the smallest of the set.The Y chromosome is very small and dot-like. Both sexchromosomes are highly heterochromatic, as revealedby Giemsa staining and C-banding. In addition, theyshow a different degree of chromatid separation, asituation that is in agreement with their heterochro-matic nature. The heterogametic karyotype (XY) wasascribed to the male that is consistent with all tephritidspecies analyzed so far,C. capitata (Bedo 1986; Zacha-ropoulou, 1987, 1990),Bactroceraoleae(Gmelin)(Ma-vragani-Tsipidou et al. 1992), Bactrocera tryoni (Frog-gatt) (Zhao et al. 1998),B. cucurbitae (Bhatnagar et al.1980, Singh and Gupta 1984, Shahjahan and Yesmin2002), Bactrocera dorsalis (Hendel) (Baimai et al.1995, 1999, 2000),Rhagoletis cerasiL. (Kounatidis et al.2008) as well as several Anastrepha species (Selivonand Perondini 1997, Cevallos and Nation 2004, Selivonet al. 2005, Cáceres et al. 2009, Garcia-Martinez et al.2009). Male heterogamety in B. cucurbitae also is sup-

Fig. 7. Reference map of B. cucurbitae (Bc) chromosome three (sections 21Ð40) and banding pattern comparison withthe C. capitata (Cc) chromosome three (C, centromere). The underlined sections constitute identiÞcation markers for B.cucurbitae chromosome 3. Alternative appearance of 3R telomere is indicated by asterisk. Dotted lines connecting thechromosomes indicate sections with similar banding pattern and arrows show the relative orientation of these sections to eachother. Chromosome three ofB. cucurbitae is homologous to chromosome three ofC. capitatabased on the characteristic bandsof the 3L arm. Three transpositions are shown, two of them with inverted orientation in relation to C. capitata. The thirdtransposition (marked with asterisk) also is found to all other Bactrocera species analyzed so far, but in these cases showsan inverted orientation compared with C. capitata. The 3R arms show limited similarity; only the tips can be correlated.


ported by the construction of a genetic sexing strainbased on a Y-autosome translocation (McInnis et al.2004). The exact structure of this translocation wasalso determined genetically and cytogenetically (un-published data).

In B. cucurbitae polytene nuclei, Þve banded poly-tene elements were observed that correspond to theÞve autosomes. This is in agreement with the resultsfound in other tephritid species (Bedo 1986, 1987;Zacharopoulou 1987, 1990; Mavragani-Tsipidou et al.1992; Zambetaki et al. 1995; Zhao et al. 1998; Kouna-tidis et al. 2008; Garcia-Martinez et al. 2009). Contraryto these data, two early studies ofB. cucurbitae salivarygland polytene chromosomes (Gopalan 1972, Gopalanand Dass 1972, Singh and Gupta 1984) reported thepresence of six chromosomes, one of which was as-signed to the X chromosome. However, no evidenceof sex chromosome polytenization was found in thepresent work, although numerous chromosomespreads (�300 slides were analyzed) from both thewild-type and the genetic sexing strain were analyzed.Sexual dimorphism in polytene nuclei was never ob-served, an indication supporting the nonpolyteniza-tion of sex chromosomes as in all tephritid speciesanalyzed so far. An additional support to the hetero-chromatic nature of the X chromosome in Tephritidaeis that all C. capitata genes that are homologous toX-linked genes of Drosophila melanogaster (Meigen)were mapped by in situ hybridization to chromosomeÞve (Zacharopoulou et al. 1992; Gariou-Papalexiou et

al. 2002). This holds also for B. tryoni and B. oleae(Zhao et al. 1998, Mavragani-Tsipidou 2002). Further-more, a comparative analysis between the polytenemaps presented here and those reported previously(Gopalan 1972, Gopalan and Dass 1972, Singh andGupta 1984) was difÞcult because in the latter studieshand drawn maps were constructed. More recently,Shahjahan and Yesmin (2002) presented photo-graphic maps of Þve polytene chromosomes, but theirpoor chromosome quality prevented any search forhomology between these maps and the maps pre-sented here.

A typical chromocenter in which all chromosomearms are connected through their centromeres wasnot found, a situation that is in accordance with allpreviously analyzed tephritid species. However, akind of a loose chromocenter, which is easily dissoci-ated during slide preparation, can be seen. Evidencefor this is the partial chromocenter indicated by itsheterochromatic structure (Figs. 2Ð4) in which dif-ferent chromosomes are connected. An analogous sit-uation of a partial chromocenter in which two chro-mosomes are connected has been reported for B.tryoni and was attributed either to the large amount ofheterochromatin or to the high degree of polyteniza-tion at centromeric regions of these speciÞc chromo-somes (Zhao et al. 1998). Regardless, this structurepossibly represents pericentromeric chromosomal re-gions consisting of heterochromatic material. Possiblythe nonreplicated sex chromosomes may also be in-

Fig. 8. Reference map of B. cucurbitae (Bc) chromosome four (sections 41Ð60) and banding pattern comparison withthe C. capitata (Cc) chromosome four (C, centromere). Sections that represent identiÞcation landmarks for B. cucurbitaechromosome four are underlined. Dotted lines connecting the chromosomes indicate sections with similar banding patternand arrows show the relative orientation of these sections to each other. The homology of the two chromosomes is prominentand can be considered as mostly colinear. One inversion was found on B. cucurbitae 4L arm, in relation to C. capitata 4L arm.


volved in the formation of this structure. Further stud-ies including in situ hybridization with speciÞc probes,such as sex-speciÞc or highly repetitive DNA se-quences, would be required to verify the existence ofa loose chromocenter. Interestingly, both size anddensity of this structure are not comparable to thatfound in other Bactrocera species, such as B. tryoni(Zhao et al. 1998), B. oleae (Mavragani-Tsipidou et al.1992), and B. dorsalis (unpublished data). In the lastthree species, the heterochromatin is accumulated asa high-density mass, and in most cases at speciÞc cen-tromeric regions. This may reßect a different chro-mosome organization related to the amount and dis-tribution of the heterochromatin distinguishing thesespecies from B. cucurbitae.

The role of heterochromatin in chromosome evo-lution has been well documented in several groups ofspecies, including Diptera. Differences have been ob-served concerning both the distribution and the quan-tity of heterochromatin among chromosomes, char-acteristics that have provided useful tools fortaxonomy, especially among sibling species (reviewedin Baimai (1998).

The phenomenon of ectopic pairing between tips ofdifferent chromosomes also has been observed in B.oleae (Mavragani-Tsipidou et al. 1992), Anastrepha

ludens (Loew) (Garcia-Martinez et al. 2009), and B.dorsalis (unpublished data), and it is possibly relatedto the molecular structure and organization of thedistal terminal parts of chromosomes in these species.In D. melanogaster, the distal-most parts of chromo-somes are composed of arrays of speciÞc non-longterminal repeat retrotrasposons (Het-A and TART)that are arranged head to tail in tracts of variablelength among several strains. In speciÞc mutantstrains, these tracts are expanded, resulting in theextension of the chromosome ends and an increasedfrequency of ectopic contacts between telomeres (re-viewed in Zhimulev et al. (2004).

The detection of chromosomal inversions in a Bac-trocera species is interesting. They occur in both thewild strain from Bangladesh and the sexing strain fromHawaii. Naturally occurring inversions have neverbeen observed in the other two Bactrocera speciesanalyzed previously, i.e.,B. oleae(Mavragani-Tsipidouet al. 1992; Mavragani-Tsipidou, unpublished data)and B. tryoni (Zhao et al. 1998). The same holds truefor two other tephritid species, A. ludens and C. capi-tata. Especially in the latter species, many differentstrains or populations have been analyzed, and nonaturally occurring inversions were observed (A.Z.,unpublished data). Furthermore, chromosomal rear-

Fig. 9. Reference map of B. cucurbitae (Bc) chromosome Þve (sections 61Ð80) and banding pattern comparison with theC. capitata (Cc) chromosome Þve (C, centromere). The most characteristic sections that can be used as diagnostic landmarksfor B. cucurbitae chromosome Þve are underlined. Dotted lines connecting the chromosomes indicate sections with similarbanding pattern and arrows show the relative orientation of these sections to each other. The banding pattern similarity ofthis chromosome in the two species is apparent. The 5L telomere and the distal chromosomal region 61Ð63 are colinear withC. capitata 61Ð63C chromosome sections. Two transpositions on B. cucurbitae 5L arm relative to C. capitata 5L are shown.One paracentric inversion on 5R arm and a pericentric one also were found. Both inversions are common in all Bactrocerasanalyzed so far.


rangements have been recently reported for Anastre-pha fraterculus (Wiedemann). Cytogenetic analysis intwo laboratory strains from Peru and Argentina, aswell as in their hybrids, revealed many chromosomerearrangements, notably inversions, in Peru strain and

a high level of polytene chromosome asynapsis in thehybrids (Cáceres et al. 2009). However, in this par-ticular case it is possible that the two strains analyzedrepresent two different entities according to evi-dences that fully support suggestions forA. fraterculusbeing composed of several cryptic species (Cáceres etal. 2009).

Chromosomal rearrangements are common in a va-riety of taxa, including Diptera, and have been exten-sively studied inDrosophila.A considerable number ofstudies regarding the generation of inversions andtheir role in speciation have been performed. Currentevidence suggests an important association betweentransposable elements and the generation of chromo-some breaks and rearrangements in laboratory strainsas well as in natural populations (reviewed in Krimbasand Powell 1992), although alternative models havebeen proposed (Ranz et al. 2007). Recently, Handler(2003) reported the isolation of a newhAT transposon,the hopper element from B. dorsalis, which exhibitsfeatures consistent with transpositional activity. Inaddition, he showed that elements closely related tohopper also exist in B. cucurbitae. Further studies onthe correlation between the distribution of the trans-posable elements and the inversion breakpoints are

Fig. 10. Reference map of B. cucurbitae (Bc) chromosome six (sections 81Ð100) and banding pattern comparison withthe C. capitata (Cc) chromosome six (C, centromere). The identiÞcation landmarks of B. cucurbitae chromosome six areunderlined Alternative appearance of 6R telomere is indicated by asterisk. Dotted lines connecting the chromosomes indicatesections with similar banding pattern and arrows show the relative orientation of these sections to each other. Sections 81Ð85of B. cucurbitae show a great afÞnity to 81Ð85A of C. capitata. One inversion on B. cucurbitae 6L was found relative toC. capitata 6L.

Fig. 11. Heterozygous inversions observed in B. cucur-bitae polytene chromosomes. Arrows indicate the break-points. (a) In(2L)1Ð3. (b) In(2R)18Ð19. (c) In(4L)43-46.(d) In(6L)85-89.


necessary, including the analysis of natural popula-tions from different geographic origin.

The establishment of chromosomal homologies,based on banding patterns and in situ hybridization,among C. capitata, B. oleae, and B. tryoni (Zhao et al.1998, Zambetaki et al. 1999, Mavragani-Tsipidou 2002)encouraged us to search for homology between B.cucurbitae andC. capitata. Indeed, signiÞcant bandingpattern similarities were found that supported theproposed homology of the chromosomes in the twospecies. It is worthwhile mentioning thatB. cucurbitaechromosomes show extensive banding patterns simi-larity to both aforementionedBactrocera species (datanot shown). However, in situ hybridization and syn-teny analysis could verify the proposed homologiesand point to the chromosomal rearrangements thatdifferentiated these species, a work that is in our fu-ture plans including all tephritid species for whichpolytene maps are available. The numbering of thechromosomes of B. cucurbitae is in accordance withthose of C. capitata and will facilitate comparativestudies with other tephritid species in which chromo-some homology with C. capitata has been established.

Our preliminary comparative analysis of polytenechromosomes points to the presence of intrachromo-somal rearrangements, mainly inversions and trans-positions. In addition, most of the chromosome tipsand pericentromeric regions exhibit banding patternsimilarities between the two species, a Þnding thatcould be attributed to the nonrandom distribution ofnaturally occurring chromosome breakpoints. Thishas been found in many Drosophila species (Krimbasand Powell 1992), as well as in tsetse ßies (GlossinaWiedemann) (Gariou-Papalexiou et al. 2007).

One particular pericentric inversion that has beenobserved in the two previously analyzed Bactroceraspecies (Zhao et al. 1998) andB.dorsalis(unpublisheddata) relative to C. capitata, is also present in B. cu-curbitae, suggesting that the Þxation of this pericentricinversion occurred after the divergence of C. capitataand the Bactrocera species (Fig. 9). An additionalÞnding is the structure of the left arm of the B. cu-curbitae chromosome 3. This arm shows extensivebandingpattern similaritywith theC. capitata3Lchro-mosome arm (Fig. 7), despite the presence of threetranspositions, two of them with inverted orientation.Surprisingly, the structure of this arm is different fromits homolog in the otherBactrocera species analyzed sofar (Zhao et al. 1998) and also from B. dorsalis (man-uscript in preparation). This observation combinedwith the previously mentioned structure of the het-erochromatic material in polytene nuclei of B. cucur-bitae points to a degree of differentiation from theBactrocera species analyzed so far. This is in agreementwith previous phylogenetic studies in Tephritidae(White and Elson-Harris 1992, Malacrida et al. 1996,Segura et al. 2006, Han and Ro 2009). Behavioral,biochemical, and molecular evidences support theidea that B. cucurbitae has a closer relationship to thegenus Dacus than to the genus Bactrocera. Compara-tive cytogenetic studies among more species of thetwo genera could clarify the phylogeny of B. cucur-

bitae that is currently grouped within the Bactroceragenus.

In conclusion, the results of this study show clearlythat the B. cucurbitae salivary gland polytene chro-mosome maps presented here are suitable for cyto-genetic analysis of the species. This analysis can helpto clarify the phylogenetic status and support the de-velopment of biological control techniques for thisspecies.

Acknowledgments

This work forms part of the Joint Food and AgriculturalOrganization of the United Nations/International AtomicEnergy Agency research program for the development ofimproved control methodologies against fruit ßy pest species.

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Received 16 July 2010; accepted 18 October 2010.


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