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Genetica 116: 45–57, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands. 45 Genetic and cytogenetic analysis of the olive fruit fly Bactrocera oleae (Diptera: Tephritidae) P. Mavragani-Tsipidou Department of Genetics, Development and Molecular Biology, School of Biology, Faculty of Science, Aristotle University of Thessaloniki (AUTH), GR-54124, Thessaloniki, Greece (Phone: +30-310-998393; Fax: +30-310- 998298; E-mail: [email protected]) Key words: Bactrocera oleae, Dacus oleae, fat body, in situ hybridization, Malpighian tubules, pest, polytene chromosomes, salivary glands, toroids Abstract The genetic and cytogenetic characteristics of one of the major agricultural pests, the olive fruit fly Bactrocera oleae, are presented here. The mitotic metaphase complement of this insect consists of six pairs of chromosomes including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the polytene complements of three larval tissues, the fat body, the salivary glands and the Malpighian tubules of this pest has shown (a) a total number of five long chromosomes (10 polytene arms) that correspond to the five autosomes of the mitotic nuclei and a heterochromatic mass corresponding to the sex chromosomes, (b) the constancy of the banding pattern of the three somatic tissues, (c) the absence of a typical chromocenter as an accumulation of heterochromatin, (d) the existence of reverse tandem duplications, and (e) the presence of toroid tips of the chromosome arms. The in situ hybridization of genes or DNA sequences to the salivary gland polytene chromosomes of B. oleae provided molecular markers for all five autosomes and permitted the estab- lishment of chromosomal homologies among B. olea, B. tryoni and Ceratitis capitata. The heat shock response of B. oleae, as revealed by heat-inducible puffing and protein pattern, shows a higher thermotolerance than Drosophila melanogaster. Introduction Bactrocera (Dacus) oleae (Gmelin) (Drew, 1989; White & Wang, 1992), a species belonging to the Tephritidae family of Diptera, is one of the major agricultural pests in olive producing countries. It is essentially monophagous, ovipositing and breeding on wild and cultivated Olea. Thus, its distribution coin- cides with the distribution of its host (Mediterranean countries, northern, eastern and southern Africa, Canary Islands, India, western Asia). Recently, it has also become established in California. In nature, the female oviposits a single egg into the olive fruit, up to 12 times per day and produces about 200–250 eggs in a lifetime. The larvae feed exclusively on the meso- carp of the olive fruits, while the adults feed on nectar, honey dew, and other sources of liquid or semi-liquid food. Damage is caused by tunneling of larvae which destroys the pulp, causes the fruit to drop, increases infections in the fruit and reduces the quality (acidity of the oil is increased) and quantity of the oil. The annual economic losses are very severe, especially in Mediterranean countries, where the favorable climatic conditions permit several generations per year (two to five generations, depending upon the latitude and temperature). During the last decades, there has been consider- able interest in developing procedures and methods for the biological control of this pest such as the use of natural enemies, mass trapping systems, and the sterile insect technique (SIT) (Kapatos, 1989). Cur- rently, the management and control of this pest is carried out using either cover sprays (with organo- phosphorous insecticides that kill adults and larvae

Genetic and Cytogenetic Analysis of the Olive Fruit Fly Bactrocera Oleae(Diptera: Tephritidae)

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Genetica 116: 45–57, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

45

Genetic and cytogenetic analysis of the olive fruit flyBactrocera oleae (Diptera: Tephritidae)

P. Mavragani-TsipidouDepartment of Genetics, Development and Molecular Biology, School of Biology, Faculty of Science, AristotleUniversity of Thessaloniki (AUTH), GR-54124, Thessaloniki, Greece (Phone: +30-310-998393; Fax: +30-310-998298; E-mail: [email protected])

Key words: Bactrocera oleae, Dacus oleae, fat body, in situ hybridization, Malpighian tubules, pest, polytenechromosomes, salivary glands, toroids

Abstract

The genetic and cytogenetic characteristics of one of the major agricultural pests, the olive fruit fly Bactroceraoleae, are presented here. The mitotic metaphase complement of this insect consists of six pairs of chromosomesincluding one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysisof the polytene complements of three larval tissues, the fat body, the salivary glands and the Malpighian tubulesof this pest has shown (a) a total number of five long chromosomes (10 polytene arms) that correspond to thefive autosomes of the mitotic nuclei and a heterochromatic mass corresponding to the sex chromosomes, (b)the constancy of the banding pattern of the three somatic tissues, (c) the absence of a typical chromocenter asan accumulation of heterochromatin, (d) the existence of reverse tandem duplications, and (e) the presence oftoroid tips of the chromosome arms. The in situ hybridization of genes or DNA sequences to the salivary glandpolytene chromosomes of B. oleae provided molecular markers for all five autosomes and permitted the estab-lishment of chromosomal homologies among B. olea, B. tryoni and Ceratitis capitata. The heat shock response ofB. oleae, as revealed by heat-inducible puffing and protein pattern, shows a higher thermotolerance than Drosophilamelanogaster.

Introduction

Bactrocera (Dacus) oleae (Gmelin) (Drew, 1989;White & Wang, 1992), a species belonging to theTephritidae family of Diptera, is one of the majoragricultural pests in olive producing countries. It isessentially monophagous, ovipositing and breeding onwild and cultivated Olea. Thus, its distribution coin-cides with the distribution of its host (Mediterraneancountries, northern, eastern and southern Africa,Canary Islands, India, western Asia). Recently, it hasalso become established in California. In nature, thefemale oviposits a single egg into the olive fruit, up to12 times per day and produces about 200–250 eggs ina lifetime. The larvae feed exclusively on the meso-carp of the olive fruits, while the adults feed on nectar,honey dew, and other sources of liquid or semi-liquid

food. Damage is caused by tunneling of larvae whichdestroys the pulp, causes the fruit to drop, increasesinfections in the fruit and reduces the quality (acidityof the oil is increased) and quantity of the oil. Theannual economic losses are very severe, especially inMediterranean countries, where the favorable climaticconditions permit several generations per year (twoto five generations, depending upon the latitude andtemperature).

During the last decades, there has been consider-able interest in developing procedures and methodsfor the biological control of this pest such as the useof natural enemies, mass trapping systems, and thesterile insect technique (SIT) (Kapatos, 1989). Cur-rently, the management and control of this pest iscarried out using either cover sprays (with organo-phosphorous insecticides that kill adults and larvae

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in the fruit by a semi-systemic effect) or bait sprays(with protein hydrolysate and insecticide which attractand kill the adults) (Economopoulos, 1989; Kapatos,1989; Roessler, 1989). However, the heavy relianceon chemical insecticides with the widespread environ-mental pollution, the presence of pesticide residues infood and water and their possible implications for hu-man welfare and health (Denholm & Rowland, 1992),together with the absence of species-specific mode ofaction call for more environment-friendly techniquesand strategies. The development of environment-friendly genetic methods of control, which have themajor advantage of species specificity (Gilmore, 1989;Robinson, 1989), can only be realized when there is athorough understanding and knowledge of the biology,ecology and the genetics of the target pest.

Many studies concerning the biology of B. oleaehave focused on biochemical and colonization gene-tics (Zouros & Loukas, 1989; Konstantopoulou,Economopoulos & Manoukas, 1996; Konstantopoulou,Economopoulos & Raptopoulos, 1999), mating beha-vior (Mazomenos, 1989), artificial diets for larvae andadults, oviposition substrates, and oviposition and lifehistory parameters (Fletcher, 1989; Tsitsipis, 1989;Tzanakakis, 1989). During the last decade, studieshave been carried out on the genetic and cytogeneticcharacteristics of B. oleae and this is the subject ofthis review.

Mitotic chromosomes

The first cytological data of the mitotic karyotype ofB. oleae were given by Frizzi and Springetti (1953),who reported the existence of five telocentric and oneacrocentric chromosome pair and subsequently themitotic metaphase chromosomes from brain gangliaof third instar larvae were described (Krimbas, 1963;Mavragani-Tsipidou et al., 1992). In these studies,there are only slight differences concerning the chro-mosomal arm ratio and both reports suggested theexistence of six pairs of chromosomes including onepair of sex chromosomes, with the male being theheterogametic sex (Figure 1). The sex chromosomes,readily identified by the heteromorphic pair of XX/XYchromosomes, are the smallest chromosomes of themitotic complement. The Y chromosome is a verysmall, dot-like chromosome, and X chromosome issmaller than the autosomes (being about half the sizeof the smallest autosome). The overall length ratio,Y/X, is 0.20. Following the labeling system used by

Figure 1. Mitotic metaphase chromosomes from nerve ganglia ofB. oleae: (a) female larva; (b) male larva (from Mavragani-Tsipidouet al., 1992).

Radu, Rossler and Koltin (1975) for the medfly Cer-atitis capitata, the sex chromosomes are labeled asthe first pair of the mitotic karyotype, while the fiveautosomes from 2 to 6 in order of descending size(Mavragani-Tsipidou et al., 1992). Using C-bandingtechniques it was shown that the Y chromosome, aswell as the short arm and the centromere-proximalportion of the long arm of the X chromosome, arehighly heterochromatic. The rest of the long arm of theX chromosome, although staining more lightly, doesnot reach the extent of chromatid separation seen in theautosomes, suggesting that this area has characteristicsof both euchromatin and heterochromatin (Mavragani-Tsipidou et al., 1992). The six chromosome pairsof the diploid complement in the olive fruit fly isconsistent with the modal number of chromosomepairs found in most of the Calyptrate Diptera (Boyes& Brink, 1965). Indeed, C. capitata (Bedo, 1986;Zacharopoulou, 1987), Dacus curcubitae (Singh &Gupta, 1984), Lucilia cuprina (Foster et al., 1980),and Bactrocera tryoni (Zhao et al., 1998) have thesame number of mitotic chromosomes as B. oleae.However, the relative length and the arm ratio ofboth autosomes and sex chromosomes are quite dif-ferent in these species, even in those species that arephylogenetically very close.

Polytene chromosomes

The first attempt to describe the polytene chromo-somes of B. oleae was by Krimbas (1963), who

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Figure 2. The IL and IR polytene chromosome arms of fat body (FB), salivary glands (SG) and Malpighian tubules (MT) of B. oleae (fromMavragani-Tsipidou et al., 1992; Zambetaki, Kleanthous & Mavragani-Tsipidou, 1995).

provided drawings of the salivary gland polytenechromosomes. He recognized 10 long polytenearms and two much shorter ones. Three decadeslater, detailed photographic maps of the polytenechromosomes of the fat body (Mavragani-Tsipidouet al., 1992), salivary glands and Malpighian tubules(Zambetaki, Kleanthous & Mavragani-Tsipidou, 1995)of this pest are available. The analysis of the polytenechromosomes of the three tissues showed that theB. oleae polytene complement consists of a total offive long chromosomes (10 polytene arms) that cor-respond to the five autosomes of the mitotic nuclei.The photographic maps of the five polytene chromo-somes found in the three somatic tissues as well astheir banding correlations are given in Figures 2–6.The data are derived from Mavragani-Tsipidou et al.(1992) and Zambetaki, Kleanthous and Mavragani-Tsipidou (1995), with slight corrections on the band–interband sequence. In all three tissues, no typical

chromocenter (as accumulation of heterochromatin)exists and this results in separation of the indi-vidual chromosomes. Five well-banded polytene chro-mosomes have also been reported for C. capitata(Bedo, 1986, 1987; Zacharopoulou, 1987, 1990;Bedo & Zacharopoulou, 1988), L. cuprina (Childress,1969; Foster et al., 1980; Bedo, 1982), Chrysomyabezziana (Bedo, 1992) and B. tryoni (Zhao et al.,1998).

The simultaneous analysis of the mitotic andpolytene nuclei of male and female B. oleae showedthat there was no difference in the number andquality of the polytene elements in the differ-ent sexes, indicating that no banded sex chromo-somes exist. However, a heterochromatic structure,which differed in size and density, was found andwas larger and more compact in the case of fe-males. It was proposed (Zambetaki, Kleanthous &Mavragani-Tsipidou, 1995) that the XX female

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Figure 3. The IIL and IIR polytene chromosome arms of FB, SG and MT of B. oleae (from Mavragani-Tsipidou et al., 1992; Zambetaki,Kleanthous & Mavragani-Tsipidou, 1995).

mitotic chromosomes corresponded to the large andmore compact heterochromatic network, while theXY male mitotic chromosomes were representedby the smaller and looser heterochromatic mass(Figure 7). Further analysis of X-linked sequencesand/or induced Y-autosome translocations has to becarried out to verify this. A heterochromatic struc-ture similar to that of B. oleae, corresponding tounder-replicating sex chromosomes, has also beenreported in C. capitata and L. cuprina (Childress,1969; Bedo, 1982, 1987; Bedo & Zacharopoulou,1988).

The analysis of the band–interband sequence ofthe polytene chromosomes of the three somatic tis-sues (fat body, Malpighian tubules and salivaryglands) of B. oleae (Mavragani-Tsipidou et al., 1992;Zambetaki, Kleanthous & Mavragani-Tsipidou, 1995)showed a good banding correspondence, indicatingconstancy of the banding pattern of different tis-sues in the same animal (Figures 2–6). Irregularitiesin the banding homology of some regions or even

chromosomal elements (due to differences in the spa-cing, staining, or even the number of bands) wereexplained by differential gene expression (Zambetaki,Kleanthous & Mavragani-Tsipidou, 1995). This no-tion was reinforced by the reverse tandem duplica-tions found in common in all three somatic tissuesof B. oleae (Figures 4 and 6) and by the identicalheterochromatic mass of the centromeric region ofeach chromosome element. Indeed, this heterochro-matic mass is identical in quality and quantity inthe three tissues, providing a remarkable landmarkfor the identification of each polytene chromosome(Figure 8). The constancy of the banding pattern ofthe polytene chromosomes in different tissues foundin B. oleae is in agreement with that observed inother species (Beermann, 1952; Pavan & Breuer,1952; Berendes, 1966; Richards, 1980; Redfern,1981; Roberts, 1988; Mavragani-Tsipidou, Scouras& Kastritsis, 1990). However, published data fromCalliphora erythrocephala (Ribbert, 1979) and C.capitata (Bedo & Zacharopoulou, 1988; Semeshin

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Figure 4. The IIIL and IIIR polytene chromosome arms of FB, SG and MT of B. oleae. Arrows indicate the outer limits of each pairedchromosomal duplication (from Mavragani-Tsipidou et al., 1992; Zambetaki, Kleanthous & Mavragani-Tsipidou, 1995).

et al., 1995) deviate significantly from the aboveconclusion.

Another characteristic of the polytene chromo-somes found in all three somatic tissues of B. oleaeis the extensive ectopic pairing of the telomeric re-gions of the different chromosome arms (Mavragani-Tsipidou et al., 1992; Zambetaki, Kleanthous &Mavragani-Tsipidou, 1995). This end to end pair-ing is so complete and tight that in many casesit is difficult to identify the free ends of the ele-ments involved (Figure 9). The chromosome armsmost frequently involved in ectopic pairing appear tohave one or more toroids at their tips. The toroidtips of the chromosome arms (Scouras & Kastritsis,1985) formed by the last dark band(s) (Figure 10)are usual in B. oleae (Mavragani-Tsipidou et al.,1992; Zambetaki, Kleanthous & Mavragani-Tsipidou,1995). Taking into account the structure, the function

and the complexity of the DNA sequences of thetelomeric regions, the preferential pairing of somechromosome arms observed in B. oleae could be ex-plained by the different degrees of homology of thechromosome tips (Renkawitz-Pohl & Bialojan, 1984).

No chromosomal rearrangements were found inthe laboratory stock of B. oleae with the exception ofa number of paired repeats (Figures 4 and 6), whichsuggest the existence of reverse tandem chromo-somal duplications (Mavragani-Tsipidou et al., 1992;Zambetaki, Kleanthous & Mavragani-Tsipidou, 1995).Chromosomal rearrangements were also not observedin natural populations of B. oleae. Indeed, studieson inversion polymorphism of B. oleae larvae, ob-tained from infested olive fruits collected from fivedifferent regions of Greece, showed that no inversionsor any other rearrangements were evident. Moreover,the examination of F1 hybrids from mass crosses of

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Figure 5. The IVL and IVR polytene chromosome arms of FB, SG and MT of B. oleae (from Mavragani-Tsipidou et al., 1992; Zambetaki,Kleanthous & Mavragani-Tsipidou, 1995).

females from the laboratory stock with wild malesfrom the above wild populations showed no differ-ence in the banding pattern (P. Mavragani-Tsipidou,unpublished data). Given that the insects in natureare exclusively dependent on the olive fruit, domes-tication and artificial rearing of B. oleae is difficult,the major problem being adaptation to a new oviposi-tion substrate. Thus, the only successful crosses of B.oleae from the field involve field males with laboratoryfemales. The fidelity of the banding pattern of chromo-somes in the F1 B. oleae hybrids together with theirsynapsis (P. Mavragani-Tsipidou, unpublished data)indicate that there are no homozygous inversions inthe B. oleae natural populations sampled. The factthat rearrangements were also not observed in naturalpopulations of C. capitata (Zacharopoulou, personalcommunication) may suggest chromosome fixation inthese Tephritid species. This could mean that the fre-quency of generation of rearrangements is rare or thatthe spontaneously occurring rearrangements are notmaintained in natural populations. The implication of

transposable elements in the generation of inversions(see Krimbas & Powell, 1992) provide a new perspec-tive to this problem. In B. oleae, much work still needsto be done to analyze large numbers of insects fromgeographically distant populations.

Molecular markers

The present status of the genome of an organism re-flects its evolutionary history. The in situ hybridizationof genes or DNA sequences to polytene chromosomeshas proved to be a powerful method to link the ge-netic and molecular information of an organism, and topermit the establishment of phylogenetic relationshipsamong different species.

Ten DNA-specific sequences (nine derived fromC. capitata and one from D. melanogaster) werehybridized, in situ, to the polytene chromosomesof B. oleae (Table 1) (Zambetaki et al., 1999;

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Figure 6. The VL and VR polytene chromosome arms of FB, SG and MT of B. oleae. Arrows indicate the outer limits of each pairedchromosomal duplication (from Mavragani-Tsipidou et al., 1992; Zambetaki, Kleanthous & Mavragani-Tsipidou, 1995).

Zambetaki, Mavragani-Tsipidou & Scouras, 2000).All the mapped sequences gave unique hybridizationsignals with the exception of sod1 and the major heatshock gene hsp70, which were found to hybridize totwo distinct bands, suggesting duplication events. InC. capitata there is molecular evidence for two sodgenes (Banks et al., 1995). The 10 mapped DNA se-quences provided molecular markers for all five auto-some polytene chromosomes of B. oleae (Table 1) andenabled chromosomal homologies among B. oleae, C.capitata and B. tryoni to be established.

Initial chromosomal homologies between B. tryoniand C. capitata with B. oleae have been proposedby Zhao et al. (1998), based exclusively on band-ing pattern similarities. These proposed homologiesfor chromosomes I, III, and V of B. oleae with 5,4, and 3 of B. tryoni and C. capitata, respectively,were confirmed based on in situ hybridization (Table1) (Zambetaki et al., 1999; Zambetaki, Mavragani-Tsipidou & Scouras, 2000). In addition, new homol-ogies were proposed for chromosomes II and IV of

B. oleae with 2 and 6, respectively, of B. tryoni and C.capitata, an assignment based on both banding patternsimilarities and in situ hybridization data (Zambetakiet al., 1999; Zambetaki, Mavragani-Tsipidou &Scouras, 2000). These homologies were further con-firmed by the location of two additional probes, thescarlet and the white DNA sequences derived fromB. tryoni (kindly provided by Dr. Zhao). The scar-let probe hybridized to chromosome IV and the whiteprobe to chromosome I of B. oleae (P. Mavragani-Tsipidou, unpublished data). In C. capitata and B.tryoni, these probes were mapped on chromosomes6 and 5, respectively (Zwiebel et al., 1995; Zhaoet al., 1998). The proposed chromosomal homologiesamong the three species of Tephritidae family as wellas the relative hybridization sites (Figure 11) show anextensive conservation of the chromosomal comple-ments of the three taxa. However, intra-chromosomalrearrangements such as paracentric or pericentric in-versions (Figure 11) may have occurred during theevolution of the chromosomal elements of the three

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Figure 7. Heterochromatic structure observed in the polytene nucleiof B. oleae: (a, b) female larva; (c) male larva. Arrows indicate thecompact heterochromatic region (from Zambetaki, Kleanthous &Mavragani-Tsipidou, 1995).

species. The conservation of the linkage groups foundin the Tephritidae family, seems to be extendedto distantly related Diptera (A. Zacharopoulou, thisissue).

Heat shock response of B. oleae

It is well known that the response of living organismsto a sudden increase of temperature or to a varietyof other stress conditions is the induction of the syn-thesis of a set of heat shock proteins (hsps) (Tissieres,Mitchell & Tracy, 1974; Ashburner & Bonner, 1979).In polytene chromosomes, a number of puffs can beseen after heat shock {first observed by Ritossa (1962)in D. bucksii} and these heat shock puffs are the lociof the major heat-inducible genes. These genes are

highly conserved through evolution and can be orga-nized in families based on their structural homologies(Morimoto, 1998).

The heat shock response of B. oleae as revealedby the heat-inducible puffing and protein pattern andthe localization of the major heat shock genes onthe salivary gland polytene chromosomes was re-cently investigated (Zambetaki, Mavragani-Tsipidou& Scouras, 2000) After temperature elevation, a set ofeight prominent puffs is induced in different regionsof the polytene chromosomes of B. oleae (Table 2).Two of these puffs are found to be the loci of themajor heat shock genes hsp70 and hsp83 (Zambetaki,Mavragani-Tsipidou & Scouras, 2000). Indeed, thehsp70-specific probe, derived from C. capitata, hy-bridized strongly to polytene chromosomes of B. oleae(at region 96 of VR chromosome arm), where avery prominent heat shock puff is observed (Table 2).The hybridization region consists of two adjacent dis-crete bands suggesting a duplication event. In D.melanogaster and Anopheles albimanus the hsp70genes are located at two discrete loci (Ish-Horowicz& Pinchin, 1980; Benedict, Cockburn & Seawright,1993), whilst in C. capitata and in many Drosophilaspecies they are found at a single locus (Drosopoulou,Konstantopoulou & Scouras, 1996; Konstantopoulou,Nikolaidis & Scouras, 1998; Papadimitriou et al.,1998).

The hsp83 specific DNA sequence, derived fromD. melanogaster, hybridized to a single band of IVLpolytene arm of B. oleae, where a moderately activatedpuff is observed after heat shock. The fact that thisprobe strongly hybridized to the polytene complementof B. oleae indicates the high level of homology ofthis gene between B. oleae and Drosophila. As theseparation time of the two genera is considered to be120 MYA (Beverly & Wilson, 1984), this gene mustbe under strong evolutionary constraints.

The newly synthesized polypeptides from thesalivary glands of B. oleae were analyzed in de-naturing acrylamide gels after gradual temperatureelevation from 25 to 42◦C (Zambetaki, Mavragani-Tsipidou & Scouras, 2000). The most heat-induciblepolypeptides of B. oleae had electrophoretic mobil-ities of 83, 70, 69, 27, 26 and 23 kDa. Whether ornot the induced 83 and 70 kDa polypeptides are ho-mologous to those of D. melanogaster is a matter offurther investigation. The “small” hsps were repre-sented in B. oleae by three polypeptides (27, 26 and23 kDa) (Zambetaki, Mavragani-Tsipidou & Scouras,2000).

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Figure 8. Centromeric regions of the five polytene elements of B. oleae: (a–e) MT; (f–j) SG; (k–o) FB (from Zambetaki, Kleanthous &Mavragani-Tsipidou, 1995).

Figure 9. Tight pairing observed among the tips of chromosomearms in B. oleae: (a–c) FB; (d–e) MT; (f–g) SG. Arrows indicate thetips of the different chromosome arms. Small arrows indicate thestretching of some areas owing to pairing. (f) Details of the prom-inent toroids of the VR telomeric region (from Mavragani-Tsipidouet al., 1992; Zambetaki, Kleanthous & Mavragani-Tsipidou, 1995).

As (a) B. oleae heat-inducible protein patterncan still be seen at 42◦C, (b) the maximum in-duction of hsp83 polypeptide in B. oleae is at

Figure 10. Details of the toroid tips of the chromosome arms of B.oleae: (a, b) 1R; (c) IIR; (d) IIIL; (e) IIIR; (f) IVL; (g) VR.

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Table 1. Distribution of molecular markers on the salivary gland polytene chromosome arms of B. oleae, C. capitataand B. tryoni (data derived from Zacharopoulou et al., 1992; Banks et al., 1995; Kritikou, 1997; Chrysanthakopoulou,Mintas & Zacharopoulou, 1998; Papadimitriou et al., 1998; Zhao et al., 1998; Zambetaki et al., 1999; Zambetaki,Mavragani-Tsipidou & Scouras, 2000)

Marker Chromosome position Marker reference

B. oleae C. capitata B. tryoni

s36 IL-10 5L-70B Konsolaki et al., 1990; Tolias et al., 1990

PS2a IL-12 5L-64B 5L-68C Brown et al., 1989

Sxl IR-20 5R-79B Saccone et al., 1998

β-tubulin IIR-38 2R-18A Brown & Savakis, unpublisheda

e2 IIIR-61 4R-57/58 Zacharopoulou et al., 1992

e3 IIIR-65 4R-59 Zacharopoulou et al., 1992

paramyosin IVL-74 6L-88B Vlachou et al., 1997

hsp83 IVL-68 6R-94A Holmgren et al., 1981

sod1 IVR-86 6R-99A 6R-97B Banks et al., 1995

hsp70 VR-96 3L-24C 3L-28B Papadimitriou et al., 1998

a A PCR fragment 1 kb in length derived from C. capitata.

37◦C, while in D. melanogaster it is at 33◦C(Lindquist, 1980) and (c) all the remaining hspsof B. oleae retain high activity at 40◦C, it wasproposed that B. oleae shows higher thermotoler-ance than D. melanogaster (Zambetaki, Mavragani-Tsipidou & Scouras, 2000). This is supported byevidence that eggs, larvae and adults of B. oleaeexhibit a higher heat tolerance in comparison withD. melanogaster (P. Mavragani-Tsipidou, unpublisheddata).

Conclusions and prospects

There is no doubt that many basic genetic andcytogenetic data on the olive fruit fly B. oleaeare now available. Moreover, efforts in the last 2years have resulted in the construction of a ge-nomic and two cDNA libraries (from adult maleand female flies), the cloning of the white andthe sex determining genes, sex lethal, doublesex and tra-2, and the isolation and characteriza-tion of microsatellite markers (A. Zacharopoulou;K. Komitopoulou; P. Mavragani-Tsipidou, unpub-lished data). However, many aspects on the bio-logy and genetics of this key insect pest of theolive crop remain unknown and, in spite of itseconomic impact, it remains one of the leaststudied pest species. Future work on this pest

must be focused on both classical and moleculargenetics.

Despite the importance of mutants as markers forthe development of ‘genetic control’ methods, no mor-phological mutants of B. oleae have been reported, asyet. Thus, the establishment of laboratory strains withmorphological and biochemical markers and muta-tions is of great importance. Moreover, these develop-ments will be greatly facilitated by further cytogeneticanalysis of the B. oleae genome, including chro-mosome mapping using multiple molecular markers.The isolation and cloning of B. oleae genes or DNAsequences and their mapping on the polytene chromo-somes will provide linkage group information and willpermit phylogenetic relationships to be established.Moreover, the localization by in situ hybridizationof these genes on both mitotic and polytene chromo-somes of B. oleae will enable correlations to be madebetween these two sets of chromosomal elements. Itwill also provide information on the fate of the sexchromosomes of the mitotic complement. The studyof genes involved in the sex determination pathwayis of great importance for the development of geneticsexing strains. The generation of genetic tools, suchas balancer chromosomes, the genetic and cytogen-etic studies on aneuploids or induced translocationsand the cytological survey of natural populations of B.oleae will elucidate many aspects of the genome andwill contribute to the development of new biologicalcontrol strategies.

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Figure 11. Schematic comparison of homologous chromosomes of B. oleae, C. capitata and B. tryoni.

Table 2. Behavior of the heat shock puffs of B. oleae at various temperatures and localization of the heat shock genes.(−) no induction, (+) relative induction of the puffs (from Zambetaki, Mavragani-Tsipidou & Scouras, 2000)

Puff Temperature (◦C) Chromosome arm hsp gene localization

25 37 40 42

6A ± ++ ++ ++ IL ?

18 ± ++ ++ + + + IR ?

61 ± ++ ++ ++ IIIR ?

63 ± ++ ± ± IIIR ?

65 ± ± ++ ++ IIIR ?

68 − +± +± + IVL hsp83

96 − ++ + + + + + + VR hsp70

99 + ++ − − VR ?

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Acknowledgements

I thank M. Hadzopoulou-Cladaras and Z. Scouras forcritical reading of the manuscript. This work was sup-ported by a grant from the Greek Agency of Researchand Technology.

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