Hostname: page-component-76fb5796d-zzh7m Total loading time: 0 Render date: 2024-04-27T19:44:46.781Z Has data issue: false hasContentIssue false
  • English
  • Français

A comparative analysis of the chromosomes of three FARQ species complex members, Ceratitis rosa, C. quilicii, and C. fasciventris F2 (Diptera: Tephritidae)

Published online by Cambridge University Press:  16 June 2023

Elena Drosopoulou Open the ORCID record for Elena Drosopoulou [Opens in a new window] ,
Angeliki Gariou-Papalexiou ,
Georgia Gouvi ,
Antonios A. Augustinos ,
Kostas Bourtzis  and
Antigone Zacharopoulou
Elena Drosopoulou*
Affiliation:
Department of Genetics, Development and Molecular Biology, School of Biology, Faculty of Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece
Angeliki Gariou-Papalexiou
Affiliation:
Department of Biology, University of Patras, Greece
Georgia Gouvi
Affiliation:
Laboratory of Systems Microbiology and Applied Genomics, Department of Environmental Engineering, University of Patras, Greece Insect Pest Control Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Seibersdorf, Austria
Antonios A. Augustinos
Affiliation:
Department of Plant Protection Patras, Institute of Industrial and Forage Crops, Hellenic Agricultural Organization ‘DIMITRA’, Patras, Greece
Kostas Bourtzis*
Affiliation:
Insect Pest Control Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Seibersdorf, Austria
Antigone Zacharopoulou
Affiliation:
Department of Biology, University of Patras, Greece
*
Corresponding authors: Elena Drosopoulou; Email: edrosopo@bio.auth.gr; Kostas Bourtzis; Email: K.Bourtzis@iaea.org
Corresponding authors: Elena Drosopoulou; Email: edrosopo@bio.auth.gr; Kostas Bourtzis; Email: K.Bourtzis@iaea.org
Rights & Permissions [Opens in a new window]

Abstract

The Ceratitis FARQ species complex consists of four highly destructive agricultural pests of Africa, namely C. fasciventris, C. anonae, C. rosa, and C. quilicii. The members of the complex are considered very closely related and the species limits among them are rather obscure. Their economic significance and the need for developing biological methods for their control makes species identification within the complex an important issue, which has become clear that can only be addressed by multidisciplinary approaches. Chromosomes, both mitotic and polytene, can provide a useful tool for species characterization and phylogenetic inference among closely related dipteran species. In the current study, we present the mitotic karyotype and the polytene chromosomes of C. rosa and C. quilicii together with in situ hybridization data. We performed a comparative cytogenetic analysis among the above two species and C. fasciventris, the only other cytogenetically studied member of the FARQ complex, by comparing the mitotic complement and the banding pattern of the polytene chromosomes of each species to the others, as well as by studying the polytene chromosomes of hybrids between them. Our analysis revealed no detectable chromosomal rearrangements discriminating the three FARQ members studied, confirming their close phylogenetic relationships.

Keywords

in situ hybridizationinsect pestsmitotic chromosomespolytene chromosomessibling speciesTephritidae
Type
Research Paper
Information
Bulletin of Entomological Research , Volume 113 , Issue 4 , August 2023 , pp. 537 - 545
Check for updates
Creative Commons
Creative Common License - CCCreative Common License - BY
This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited.
Copyright
Copyright © The Author(s), 2023. Published by Cambridge University Press

Introduction

Tephritidae is a speciose family of Diptera, with a great number of species characterized as serious agricultural pests (Bickel et al., Reference Bickel, Pape and Meier2009). In particular, the genera of Anastrepha, Bactrocera, Ceratitis, Dacus, Rhagoletis, and Zeugodacus include some of the most destructive fruit flies that cause severe economic losses due to crop damaging of commercial fruit and vegetable and restrictions to global trade (White and Elson-Harris, Reference White and Elson-Harris1992; De Meyer et al., Reference De Meyer, Delatte, Mwatawala, Quilici, Vayssieres and Virgilio2015b). From the genus Ceratitis, the Mediterranean fruit fly, Ceratitis capitata, is the best studied species used as a model pest organism, because of its almost global distribution and its enormous economic impact (Malacrida et al., Reference Malacrida, Gomulski, Bonizzoni, Bertin, Gasperi and Guglielmino2007). In the recent years, attention has been also drawn to the Ceratitis species of the African FARQ complex. Until 2016, the complex was known as FAR species complex and was considered to consist of three closely related species, C. fasciventris, C. anonae, and C. rosa (Virgilio et al., Reference Virgilio, Backeljau, Barr and De Meyer2008). However, accumulating evidence from studies on molecular genetics (Virgilio et al., Reference Virgilio, Delatte, Quilici, Backeljau and De Meyer2013), morphometrics (Van Cann et al., Reference Van Cann, Virgilio, Jordaens and De Meyer2015), developmental physiology (Tanga et al., Reference Tanga, Manrakhan, Daneel, Mohamed, Fathiya and Ekesi2015), behavior and sexual compatibility (De Meyer et al., Reference De Meyer, Delatte, Ekesi, Jordaens, Kalinová, Manrakhan, Mwatawala, Steck, Van Cann, Vaničhová, Břizová and Virgilio2015a), chemoecology (Vaníčková et al., Reference Vaníčková, Břízová, Mendonça, Pompeiano and Do Nascimento2015), and environmental preferences (Mwatawala et al., Reference Mwatawala, Virgilio, Joseph and De Meyer2015) lead to the conclusion that C. rosa was, in fact, consisting of two entities, one of which has been described as a new species within the complex, C. quilicii (De Meyer et al., Reference De Meyer, Mwatawala, Copeland and Virgilio2016). Microsatellite analysis indicated the existence of two genotypic groups in C. fasciventris, as well, referred to as types F1 and F2 (Virgilio et al., Reference Virgilio, Delatte, Quilici, Backeljau and De Meyer2013). This was confirmed also by morphological data, however, in the absence of integrative evidence, C. fasciventris is still considered as one species (De Meyer et al., Reference De Meyer, Delatte, Ekesi, Jordaens, Kalinová, Manrakhan, Mwatawala, Steck, Van Cann, Vaničhová, Břizová and Virgilio2015a).

The four members of the FARQ complex are highly polyphagous attacking plants from more than 25 different families and are considered a major threat to the agricultural production and economy of many countries of the African continent, as well as species of quarantine significance (White and Elson-Harris, Reference White and Elson-Harris1992; Smith et al., Reference Smith, McNamara, Scott and Holderness1997; De Meyer et al., Reference De Meyer, Copeland, Lux, Mansell, Wharton, White and Zenz2002). C. fasciventris and C. anonae are distributed mainly through Western and Central Africa, found sympatrically in several regions, while C. rosa and C. quilicii present overlapping distribution in Eastern and Southern Africa (De Meyer et al., Reference De Meyer, Copeland, Lux, Mansell, Wharton, White and Zenz2002, Reference De Meyer, Mwatawala, Copeland and Virgilio2016; Copeland et al., Reference Copeland, Wharton, Luke, De Meyer, Lux, Zenz, Machera and Okumu2006). It has been reported that C. rosa occupies mainly lower altitude areas, while C. quilicii predominates in cooler highland regions (Mwatawala et al., Reference Mwatawala, Virgilio, Joseph and De Meyer2015), probably reflecting differences in the developmental and survival rates of the two species in respect to climate variation (Tanga et al., Reference Tanga, Manrakhan, Daneel, Mohamed, Fathiya and Ekesi2015). It should be noted that C. quilicii is the only one of the two sibling species found in the southernmost parts of Africa where the climate is more temperate (De Meyer et al., Reference De Meyer, Delatte, Ekesi, Jordaens, Kalinová, Manrakhan, Mwatawala, Steck, Van Cann, Vaničhová, Břizová and Virgilio2015a). However, because of the recent separation into different species, the specific distribution patterns for C. rosa and C. quilicii may need reevaluation. Among the FARQ pests, C. rosa and C. quilicii are the most aggressive ones causing significant destruction to a large variety of crops and presenting high expansion potential. Already, C. quilicii has been introduced in the Islands of Mauritius and La Réunion (White et al., Reference White, De Meyer and Stonehouse2000; De Meyer et al., Reference De Meyer, Mwatawala, Copeland and Virgilio2016) and a great concern has arisen about their possible expansion to more temperate climates outside Africa, since they can survive in a wide temperature and altitude range (Duyck and Quilici, Reference Duyck and Quilici2002; Copeland et al., Reference Copeland, Wharton, Luke, De Meyer, Lux, Zenz, Machera and Okumu2006; Geurts et al., Reference Geurts, Mwatawala and De Meyer2012; de Villiers et al., Reference de Villiers, Hattingh and Kriticos2013; Tanga et al., Reference Tanga, Khamis, Tonnang, Rwomushana, Mosomtai, Mohamed and Ekesi2018).

The species of the FARQ complex are extremely similar in morphology; males are hardly identified by subtle differences in the setal ornamentation and pigmentation of mid femur and tibia, while females are practically indistinguishable (De Meyer et al., Reference De Meyer, Delatte, Ekesi, Jordaens, Kalinová, Manrakhan, Mwatawala, Steck, Van Cann, Vaničhová, Břizová and Virgilio2015a, Reference De Meyer, Mwatawala, Copeland and Virgilio2016). Species delimitation and phylogenetic relationships among the four taxa are not fully resolved. Several approaches have been undertaken toward this direction including morphometrics (Van Cann et al., Reference Van Cann, Virgilio, Jordaens and De Meyer2015), interspecies hybridization and estimation of developmental stability (Erbout et al., Reference Erbout, De Meyer and Lens2008), biochemical characterization of pheromones and cuticular hydrocarbons (Vaníčková et al., Reference Vaníčková, Virgilio, Tomčala, Břízová, Ekesi, Hoskovec, Kalinová, Do Nascimento and De Meyer2014, Reference Vaníčková, Břízová, Mendonça, Pompeiano and Do Nascimento2015; Břízová et al., Reference Břízová, Vaníčková, Faťarová, Ekesi, Hoskovec and Kalinová2015), and molecular/genetic data of nuclear and mitochondrial sequences (Douglas and Haymer, Reference Douglas and Haymer2001; Barr and McPheron, Reference Barr and McPheron2006; Barr et al., Reference Barr, Copeland, De Meyer, Masiga, Kibogo, Billah, Osir, Wharton and McPheron2006, Reference Barr, Islam, De Meyer and McPheron2012; Virgilio et al., Reference Virgilio, Backeljau, Barr and De Meyer2008, Reference Virgilio, Jordaens, Breman, Backeljau and De Meyer2012), with the analysis of a specific microsatellite set conferring better resolution among populations of the complex (Delatte et al., Reference Delatte, Virgilio, Simiand, Quilici and De Meyer2013; Virgilio et al., Reference Virgilio, Delatte, Quilici, Backeljau and De Meyer2013, Reference Virgilio, Daneel, Manrakhan, Delatte, Meganck and De Meyer2019). Recently, a phylogenomic study based on genome-wide SNP analysis provided consistent resolution and better insights into the phylogenetic relationships of the FARQ members (Zhang et al., Reference Zhang, De Meyer, Virgilio, Feng, Badji and Li2021). A good understanding of the evolutionary relationships and the development of accurate, simple, and fast diagnostic tools for the sibling species of the FARQ complex is of great importance for the implementation of quarantine measures, as well as for biological control applications, including the sterile insect technique (SIT), against these pests.

The number and structure of chromosomes are fundamental genetic characteristics of species, while chromosome rearrangements are considered to play a major role in speciation. In Diptera, the occurrence of polytene nuclei in several juvenile tissues has greatly facilitated the study of chromosomes due to their enormous size and consistent banding pattern (Zhimulev and Koryakov, Reference Zhimulev and Koryakov2009). Numerous cytogenetic studies in Drosophila but also in mosquitoes have explored the evolutionary changes of chromosome structure among related species and, together with modern genomic data, substantiated that chromosome rearrangements and especially paracentric inversions promote speciation, mainly through suppressing recombination and, thus, preserving sets of co-adapted alleles, and suggested that they could be used as phylogenetic markers (Sturtevant and Dobzhansky, Reference Sturtevant and Dobzhansky1936; Coluzzi et al., Reference Coluzzi, Sabatini, Petrarca and Di Deco1979; Krimbas and Powell, Reference Krimbas and Powell1992; Noor et al., Reference Noor, Grams, Bertucci and Reiland2001; Rieseberg, Reference Rieseberg2001; Kirkpatrick and Barton, Reference Kirkpatrick and Barton2006; Kulathinal et al., Reference Kulathinal, Stevison and Noor2009; Faria and Navarro, Reference Faria and Navarro2010; McGaugh and Noor, Reference McGaugh and Noor2012; Lee et al., Reference Lee, Collier, Sanford, Marsden, Fofana, Cornel and Lanzaro2013). In Tephritidae, as well, differences in the size and structure of mitotic sex chromosomes have been descripted as diagnostic characters among closely related species (Hunwattanakul and Baimai, Reference Hunwattanakul and Baimai1994; Baimai et al., Reference Baimai, Trinachartvanit, Tigvattananont, Grote, Poramarcom and Kijchalao1995, Reference Baimai, Phinchonsgsakuldit, Sumrandee and Tigvattananont2000; Baimai, Reference Baimai1998; Goday et al., Reference Goday, Selivon, Perondini, Greciano and Ruiz2006; Cáceres et al., Reference Cáceres, Segura, Vera, Wornoayporn, Cladera, Teal, Sapountzis, Bourtzis, Zacharopoulou and Robinson2009; Hernández-Ortiz et al., Reference Hernández-Ortiz, Bartolucci, Morales-Valles, Frías and Selivon2012; Giardini et al., Reference Giardini, Milla, Lanzavecchia, Nieves and Cladera2015). Furthermore, comparative analyses of polytene chromosomes have identified specific rearrangements that could distinguish between genera, subgenera, or species (Augustinos et al., Reference Augustinos, Drosopoulou, Gariou-Papalexiou, Asimakis, Cáceres, Tsiamis, Bourtzis, Mavragani-Tsipidou and Zacharopoulou2015; Zacharopoulou et al., Reference Zacharopoulou, Augustinos, Drosopoulou, Tsoumani, Gariou-Papalexiou, Franz, Mathiopoulos, Bourtzis and Mavragani-Tsipidou2017; Gouvi et al., Reference Gouvi, Gariou-Papalexiou, Augustinos, Drosopoulou, Tsiamis, Bourtzis and Zacharopoulou2022). Cytogenetic information on tephritid pests has also been proved valuable for the development and characterization of genetic sexing strains essential for the implementation of certain control methods, such as SIT (Augustinos et al., Reference Augustinos, Drosopoulou, Gariou-Papalexiou, Asimakis, Cáceres, Tsiamis, Bourtzis, Mavragani-Tsipidou and Zacharopoulou2015; Zacharopoulou et al., Reference Zacharopoulou, Augustinos, Drosopoulou, Tsoumani, Gariou-Papalexiou, Franz, Mathiopoulos, Bourtzis and Mavragani-Tsipidou2017; Gouvi et al., Reference Gouvi, Gariou-Papalexiou, Augustinos, Drosopoulou, Tsiamis, Bourtzis and Zacharopoulou2022). Even so, taking into consideration that speciation is a complex procedure driven by variable factors one can understand that chromosome structure and cytogenetics could only be one of multiple tools for species delimitation. Especially in cases of recent or ongoing speciation, pools of independent data in the context of ‘integrative taxonomy’ (Schutze et al., Reference Schutze, Virgilio, Norrbom and Clarke2017a, Reference Schutze, Bourtzis, Cameron, Clarke, de Meyer, Hee, Hendrichs, Krosch and Mwatawala2017b) and modern genome-wide analyses (Zhang et al., Reference Zhang, De Meyer, Virgilio, Feng, Badji and Li2021) are necessary for clearer perception.

In this study, we describe the mitotic and polytene chromosome of C. rosa and C. quilicii and we conduct a comparative polytene chromosome analysis among the above species and C. fasciventris F2 by observation of polytene nuclei of each species as well as of F1 hybrids between them. Furthermore, we localized the hsp70 gene on the polytene chromosomes of the above species, since rearrangements which include the chromosome region where the hsp70 locus resides on the 3L polytene arm seem to be common among several tephritid species (Drosopoulou et al., Reference Drosopoulou, Pantelidou, Gariou-Papalexiou, Augustinos, Chartomatsidou, Kyritsis, Bourtzis, Mavragani-Tsipidou and Zacharopoulou2017; Zacharopoulou et al., Reference Zacharopoulou, Augustinos, Drosopoulou, Tsoumani, Gariou-Papalexiou, Franz, Mathiopoulos, Bourtzis and Mavragani-Tsipidou2017), some of them closely related (Gouvi et al., Reference Gouvi, Gariou-Papalexiou, Augustinos, Drosopoulou, Tsiamis, Bourtzis and Zacharopoulou2022). Our aim is to reveal possibly existing chromosome rearrangements that could be informative toward the better understanding of the phylogenetic relationships of the species and could be used as discriminating characters for species identification within the FARQ complex.

Materials and methods

Insects from five colonies maintained at the Insect Pest Control Laboratory (IPCL), Seibersdorf, Austria were used in the present study. The above colonies were established from insects originating from confirmed colonies of Ceratitis fasciventris F2 (hereafter C. fasciventris), C. rosa and C. quilicii maintained at ICIPE, Kenya and of C. rosa and C. quilicii maintained at CRI, South Africa. The colonies were reared under controlled temperature, humidity, and light conditions, as previously described (Drosopoulou et al., Reference Drosopoulou, Pantelidou, Gariou-Papalexiou, Augustinos, Chartomatsidou, Kyritsis, Bourtzis, Mavragani-Tsipidou and Zacharopoulou2017).

Mitotic chromosome preparations were spread from nerve ganglia of third instar larvae following the air-drying technique described in Mavragani-Tsipidou et al. (Reference Mavragani-Tsipidou, Zacharopoulou, Drosopoulou, Augustinos, Bourtzis, Marec and Sharakhov2014). Brain tissue was dissected in physiological solution, treated with hypotonic solution (1% sodium citrate) for about 15 min and fixed in fresh fixation solution (methanol/acetic acid 3:1) for 3 min. Samples were macerated in a small drop of 60% acetic acid, dripped onto a clean slide and placed on a hotplate (40–45 °C). After air-drying, preparations were stained in Giemsa solution (5% Giemsa in 10 mM phosphate buffer, pH 6.8) and observed with 100× magnification objective lens, using a phase contrast microscope (Leica DMR). Well spread nuclei were photographed using a CCD camera (ProgResCFcool; JENOPTIK Jena Optical Systems, Jena, Germany). About ten chromosome preparations from individual larvae from each strain and at least ten well spread nuclei per preparation were analyzed.

Polytene chromosome preparations for banding pattern analysis were made from salivary glands of third-instar larvae as described in Mavragani-Tsipidou et al. (Reference Mavragani-Tsipidou, Zacharopoulou, Drosopoulou, Augustinos, Bourtzis, Marec and Sharakhov2014). Salivary glands were dissected in 45% acetic acid, transferred to 3N HCL for 1 min, and fixed in fixation solution (3 parts glacial acetic acid: 2 parts water: 1 part lactic acid) for about 5 min. Staining was performed in lacto-acetic-orcein for 5–7 min. After excess stain was removed, the glands were squashed in lacto-acetic acid. About 50 chromosome slides from each strain were prepared and well spread nuclei and/or isolated chromosomes were observed at 63× and 100× objectives in a phase contrast microscope (Leica DMR) and photographed using a CCD camera (ProgResCFcool; JENOPTIK Jena Optical Systems).

Polytene chromosome preparations for in situ hybridization were made following the procedure described by Mavragani-Tsipidou et al. (Reference Mavragani-Tsipidou, Zacharopoulou, Drosopoulou, Augustinos, Bourtzis, Marec and Sharakhov2014). A genomic fragment of the hsp70 gene of Ceratitis capitata (Papadimitriou et al., Reference Papadimitriou, Kritikou, Mavroidis, Zacharopoulou and Mintzas1998) was used as probe. Labeling of the probe and detection of the signal was performed using the ‘DIG-DNA Labeling and Detection kit’ purchased by ROCHE, Mannheim, Germany and following the protocol described in Mavragani-Tsipidou et al. (Reference Mavragani-Tsipidou, Zacharopoulou, Drosopoulou, Augustinos, Bourtzis, Marec and Sharakhov2014). Hybridization was performed at 65 °C. Five preparations and at least ten well spread nuclei per preparation were observed at 100× magnification with a Leica DMR phase contrast microscope equipped with a CCD camera (ProgResCFcool, JENOPTIK Jena Optical Systems).

Results and discussion

Mitotic chromosomes

The karyotypes of C. rosa and C. quilicii (2n = 12) appear identical to each other consisting of five pairs of autosomes and one pair of heteromorphic sex chromosomes (XX/XY) (fig. 1). The largest metacentric (chromosome 2), as well as the only submetacentric (chromosome 3) autosome pair can be easily identified (fig. 1). The remaining three autosomes (namely 4, 5, and 6) being all metacentric of similar size cannot be easily distinguished by our analysis. The two sex chromosomes differ significantly in size: the X chromosome is submetacentric of medium size, while Y is a small metacentric chromosome (fig. 1b, d). The karyotypes of C. rosa and C. quilicii, presented after Giemsa staining, are in agreement with the C. rosa karyotype described by Willhoeft and Franz (Reference Willhoeft and Franz1996). They also appear very similar to the mitotic karyotype of the closely related member of the FARQ complex, C. fasciventris (Drosopoulou et al., Reference Drosopoulou, Pantelidou, Gariou-Papalexiou, Augustinos, Chartomatsidou, Kyritsis, Bourtzis, Mavragani-Tsipidou and Zacharopoulou2017), in which the X chromosome seems to be of slightly smaller size (relatively to the autosomes). Similarly, the main difference of all FARQ karyotypes to C. capitata is the considerably shorter X and Y chromosomes. Such variation in the size of the sex chromosomes, reflecting differences of the amount of heterochromatin, can be commonly observed among very closely related, e.g. within a complex (Baimai et al., Reference Baimai, Trinachartvanit, Tigvattananont, Grote, Poramarcom and Kijchalao1995, Reference Baimai, Phinchonsgsakuldit, Sumrandee and Tigvattananont2000; Selivon et al., Reference Selivon, Perondini and Morgante2005a; Cáceres et al., Reference Cáceres, Segura, Vera, Wornoayporn, Cladera, Teal, Sapountzis, Bourtzis, Zacharopoulou and Robinson2009) or a bit more distantly related, e.g. within a genus, (Hunwattanakul and Baimai, Reference Hunwattanakul and Baimai1994; Frias, Reference Frias2004; Selivon et al., Reference Selivon, Perondini and Rocha2005b; Zacharopoulou et al., Reference Zacharopoulou, Augustinos, Drosopoulou, Tsoumani, Gariou-Papalexiou, Franz, Mathiopoulos, Bourtzis and Mavragani-Tsipidou2017) species of tephritids.

Figure 1. Mitotic karyotypes of C. rosa (a and b) and C. quilicii (c and d). (a, c) Female; (b, d) male. The sex chromosomes, X and Y, as well as the autosomes 2 and 3 are shown.

Polytene chromosomes

The salivary gland polytene nuclei of two C. rosa colonies and two C. quilicii colonies have been studied. Analysis showed that the polytene complement of the above species consists of ten long polytene arms with distinct banding pattern, corresponding to the five autosomes, while a dispersed heterochromatic network represents the under-replicated sex chromosomes (Supplementary figs 1 and 2), similarly to other Tephritidae species (Zacharopoulou et al., Reference Zacharopoulou, Augustinos, Drosopoulou, Tsoumani, Gariou-Papalexiou, Franz, Mathiopoulos, Bourtzis and Mavragani-Tsipidou2017; Gouvi et al., Reference Gouvi, Gariou-Papalexiou, Augustinos, Drosopoulou, Tsiamis, Bourtzis and Zacharopoulou2022). Although no typical chromocenter was observed, the centromeric region of different chromosomes could be found loosely connected (Supplementary fig. 1a). Chromosomes were numbered from 2 to 6, chromosome arms were designated as L or R (Supplementary figs 1 and 2) based on the similarities to C. fasciventris polytene chromosome maps (Drosopoulou et al., Reference Drosopoulou, Pantelidou, Gariou-Papalexiou, Augustinos, Chartomatsidou, Kyritsis, Bourtzis, Mavragani-Tsipidou and Zacharopoulou2017) and following the numbering system proposed for the polytene chromosomes of the medfly, the first tephritid species analyzed cytogenetically (Zacharopoulou et al., Reference Zacharopoulou, Augustinos, Drosopoulou, Tsoumani, Gariou-Papalexiou, Franz, Mathiopoulos, Bourtzis and Mavragani-Tsipidou2017).

Detailed comparison of the polytene chromosome banding pattern failed to reveal differences either among the analyzed strains of C. rosa and C. quilicii nor between each analyzed strain and C. fasciventris (Supplementary figs 3 and 4). Aiming to confirm the identical banding pattern of the analyzed species, the polytene chromosomes of F1 hybrids between C. rosa and C. quilicii, as well as between C. rosa and C. fasciventris and C. quilicii and C. fasciventris were also examined. The analysis of the polytene nuclei of the hybrids did not reveal any chromosome rearrangements between the parental strains. Synapsis of the homologous chromosomes was almost perfect in the hybrids between C. rosa and C. quilicii (fig. 2), while in the hybrids with C. fasciventris minor polymorphic asynapses were observed (Supplementary figs 5 and 6). Asynapses were mainly located at or close to the telomeric and the centromeric regions of the polytene arms and their extent was limited although it could vary among different nuclei (figs 3 and 4). The number and frequency of minor asynaptic sites were higher in the hybrids between C. rosa and C. fasciventris compared to the ones between C. quilicii and C. fasciventris. The most evident asynapses were the ones at the tips of chromosome arms 2L, 2R, 3L, 4R, and 6R (figs 3 and 4).

Figure 2. Polytene nuclei of F1 hybrids between C. rosa and C. quilicii. The telomeres of the polytene elements are indicated. 5LC indicates the 5L centromere. No asynapses are observed.

Figure 3. Asynapses frequently observed in the nuclei of F1 hybrids between C. rosa and C. fasciventris. The asynaptic telomeres of the polytene elements are indicated. Variable extent of asynapsis observed for 4R and 6R telomeres is presented in (f) and (i), respectively. Arrows indicate asynapses in the inner parts of the polytene elements. 5LC indicates the 5L centromere.

Figure 4. Asynapses frequently observed in the nuclei of F1 hybrids between C. quilicii and C. fasciventris. The asynaptic telomeres of the polytene elements are indicated. Variable extent of asynapsis observed for 3L telomere is presented in (d–f). Arrows indicate asynapses in the inner parts of the polytene elements.

The above observations indicate that the chromosomes of C. rosa and C. quilicii, at least at the banding pattern level, can be considered as homosequential to each other and to C. fasciventris and the available polytene chromosome maps of C. fasciventris (Drosopoulou et al., Reference Drosopoulou, Pantelidou, Gariou-Papalexiou, Augustinos, Chartomatsidou, Kyritsis, Bourtzis, Mavragani-Tsipidou and Zacharopoulou2017) is suggested to be used as reference map for the three FARQ species.

The lack of detectable differences in the mitotic and polytene chromosomes of the three FARQ species indicates that they are very close genetically. This is also supported by previous molecular genetic studies, including analysis of nuclear and mitochondrial fragments, DNA barcoding and analysis of complete mitogenomes, that couldn't resolve phylogeny or provide robust discriminating tools for the members of the complex (Virgilio et al., Reference Virgilio, Backeljau, Barr and De Meyer2008, Reference Virgilio, Jordaens, Breman, Backeljau and De Meyer2012; Barr et al., Reference Barr, Islam, De Meyer and McPheron2012; Drosopoulou et al., Reference Drosopoulou, Pantelidou, Gariou-Papalexiou, Augustinos, Chartomatsidou, Kyritsis, Bourtzis, Mavragani-Tsipidou and Zacharopoulou2017, Reference Drosopoulou, Damaskou, Markou, Ekesi, Khamis, Manrakhan, Augustinos, Tsiamis and Bourtzis2021). The limitations of the above approaches seem to be overcome only by genome-wide sequencing data succeeding to provide a robust phylogenetic inference within the complex (Zhang et al., Reference Zhang, De Meyer, Virgilio, Feng, Badji and Li2021). Absence of chromosomal rearrangements has also been observed between the two members of the B. dorsalis complex, namely B. dorsalis and B. carambolae (Augustinos et al., Reference Augustinos, Drosopoulou, Gariou-Papalexiou, Asimakis, Cáceres, Tsiamis, Bourtzis, Mavragani-Tsipidou and Zacharopoulou2015), however, within other species complexes of Tephritidae chromosome differences have been used as differentiating characters and revealed incipient speciation (Selivon et al., Reference Selivon, Perondini and Rocha2005b, Reference Selivon, Perondini and Morgante2005a; Goday et al., Reference Goday, Selivon, Perondini, Greciano and Ruiz2006; Cáceres et al., Reference Cáceres, Segura, Vera, Wornoayporn, Cladera, Teal, Sapountzis, Bourtzis, Zacharopoulou and Robinson2009; Hernández-Ortiz et al., Reference Hernández-Ortiz, Bartolucci, Morales-Valles, Frías and Selivon2012).

Chromosome localization of the hsp70 gene

The hsp70 gene has been localized on the polytene chromosome of C. rosa and C. quilicii. A unique hybridization signal has been identified on the same chromosomal position (3L polytene chromosome arm, region 27) in all strains tested (fig. 5). The localization site of the hsp70 gene on C. rosa and C. quilicii is identical to the one observed in C. fasciventris (Drosopoulou et al., Reference Drosopoulou, Pantelidou, Gariou-Papalexiou, Augustinos, Chartomatsidou, Kyritsis, Bourtzis, Mavragani-Tsipidou and Zacharopoulou2017) (fig. 5), supporting the homosequentiality of the chromosomes of the three members of the FARQ complex. Nevertheless, it is acknowledged that the localization of a much greater number of probes is required to draw conclusions about genomic synteny among the studied species.

Figure 5. In situ hybridization of the hsp70 gene probe on the salivary gland polytene chromosomes of C. rosa and C. quilicii. Arrows indicate the hybridization signals. The telomere of the 3L polytene arm is indicated. Numbered divisions are shown, separated by lines. The reference map of the 3L arm and the hybridization locus of the hsp70 gene of C. fasciventris (Drosopoulou et al., Reference Drosopoulou, Pantelidou, Gariou-Papalexiou, Augustinos, Chartomatsidou, Kyritsis, Bourtzis, Mavragani-Tsipidou and Zacharopoulou2017) are presented on the top.

In comparison to C. capitata, the site of the hsp 70 gene is different in the FARQ complex species indicating intrachromosomal rearrangements (Drosopoulou et al., Reference Drosopoulou, Pantelidou, Gariou-Papalexiou, Augustinos, Chartomatsidou, Kyritsis, Bourtzis, Mavragani-Tsipidou and Zacharopoulou2017) that have differentiated the structure of the 3L chromosome arm of the above species. The presence of rearrangements, such as translocations and inversions, in the 3L polytene arm has also been revealed by previous comparative analyses among species of several Tephritidae genera (Zacharopoulou et al., Reference Zacharopoulou, Augustinos, Drosopoulou, Tsoumani, Gariou-Papalexiou, Franz, Mathiopoulos, Bourtzis and Mavragani-Tsipidou2017; Gouvi et al., Reference Gouvi, Gariou-Papalexiou, Augustinos, Drosopoulou, Tsiamis, Bourtzis and Zacharopoulou2022), supporting the role of chromosome rearrangements in speciation (Noor et al., Reference Noor, Grams, Bertucci and Reiland2001; Rieseberg, Reference Rieseberg2001; Kirkpatrick and Barton, Reference Kirkpatrick and Barton2006; Faria and Navarro, Reference Faria and Navarro2010; McGaugh and Noor, Reference McGaugh and Noor2012; Lee et al., Reference Lee, Collier, Sanford, Marsden, Fofana, Cornel and Lanzaro2013) and their potential informativeness for phylogenetic inference among related tephritid species (Mavragani-Tsipidou et al., Reference Mavragani-Tsipidou, Zacharopoulou, Drosopoulou, Augustinos, Bourtzis, Marec and Sharakhov2014; Zacharopoulou et al., Reference Zacharopoulou, Augustinos, Drosopoulou, Tsoumani, Gariou-Papalexiou, Franz, Mathiopoulos, Bourtzis and Mavragani-Tsipidou2017; Gouvi et al., Reference Gouvi, Gariou-Papalexiou, Augustinos, Drosopoulou, Tsiamis, Bourtzis and Zacharopoulou2022).

Conclusions

Our comparative mitotic and polytene chromosome analysis of the colonized material of C. rosa and C. quilicii from two different African locations (Kenya and South Africa) and of C. fasciventris from Kenya did not unravel any detectable fixed chromosome rearrangements among the three members of the FARQ complex. The above emphasizes the need for multidisciplinary modern approaches when addressing sensitive issues of species designation within complexes of important insect pests, as only by the accumulation and evaluation of data coming from different aspects of the insect biology we can be led toward a more solid phylogenetic resolution and reliable species identification.

Supplementary material

The supplementary material for this article can be found at https://doi.org/10.1017/S0007485323000214.

Acknowledgements

We thank Drs Sunday Ekesi, Fathiya Khamis, and Aruna Manrakhan for providing the biological material from Kenya and South Africa for the establishment of the insect colonies used in the present study.

Author contributions

E. D., A. A., K. B., and A. Z. designed the study. G. G., E. D., A. G.-P., and A. Z. performed the experiments. G. G., A. G.-P., and A. Z. took the pictures. G. G., E.D., A. G.-P., A. A., and A. Z. interpreted and analyzed the data. A. G.-P. prepared the figures. E. D. wrote the original draft manuscript. A. A. and K. B. critically revised the manuscript. All authors reviewed and approved the final version of the manuscript.

Financial support

The present study has been funded by the Insect Pest Control Subprogram of the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture through a research contract entitled ‘Cytogenetic analysis of members of Tephritidae complex species and cytogenetic characterization of Genetic Sexing Strains’ (contract number: 20674).

Competing interest

None.

Footnotes

*

Coauthor deceased in 2022.

References

Augustinos, AA, Drosopoulou, E, Gariou-Papalexiou, A, Asimakis, ED, Cáceres, C, Tsiamis, G, Bourtzis, K, Mavragani-Tsipidou, P and Zacharopoulou, A (2015) Cytogenetic and symbiont analysis of five members of the B. dorsalis complex (Diptera, tephritidae): no evidence of chromosomal or symbiont-based speciation events. ZooKeys 540, 273–298.Google Scholar
Baimai, V (1998) Heterochromatin accumulation and karyotypic evolution in some dipteran insects. Zoological Studies 37, 75–88.Google Scholar
Baimai, V, Trinachartvanit, W, Tigvattananont, S, Grote, PJ, Poramarcom, R and Kijchalao, U (1995) Metaphase karyotypes of fruit flies of Thailand. I. Five sibling species of the Bactrocera dorsalis complex. Genome 38, 1015–1022.CrossRefGoogle ScholarPubMed
Baimai, V, Phinchonsgsakuldit, J, Sumrandee, C and Tigvattananont, S (2000) Cytological evidence for a complex of species within the taxon Bactrocera tau (Diptera: Tephritidae) in Thailand. Biological Journal of the Linnean Society 69, 399–409.CrossRefGoogle Scholar
Barr, NB and McPheron, BA (2006) Molecular phylogenetics of the genus Ceratitis (Diptera: Tephritidae). Molecular Phylogenetics and Evolution 38, 216–230.CrossRefGoogle Scholar
Barr, NB, Islam, MS, De Meyer, M and McPheron, BA (2012) Molecular identification of Ceratitis capitata (Diptera: Tephritidae) using DNA sequences of the COI barcode region. Annals of the Entomological Society of America 105, 339–350.CrossRefGoogle Scholar
Barr, NB, Copeland, RS, De Meyer, M, Masiga, D, Kibogo, HG, Billah, MK, Osir, E, Wharton, RA and McPheron, BA (2006) Molecular diagnostics of economically important Ceratitis fruit fly species (Diptera: Tephritidae) in Africa using PCR and RFLP analyses. Bulletin of Entomological Research 96, 505–521.Google ScholarPubMed
Bickel, D, Pape, T and Meier, R (eds) (2009). Diptera Diversity: Status, Challenges and Tools. Leiden, The Netherlands: Brill. Available at https://brill.com/view/title/12518 (Accessed March 10, 2022).CrossRefGoogle Scholar
Břízová, R, Vaníčková, L, Faťarová, M, Ekesi, S, Hoskovec, M and Kalinová, B (2015) Analyses of volatiles produced by the African fruit fly species complex (Diptera, Tephritidae). Zookeys 540, 385–404. doi: 10.3897/zookeys.540.9630Google Scholar
Cáceres, C, Segura, DF, Vera, MT, Wornoayporn, V, Cladera, JL, Teal, P, Sapountzis, P, Bourtzis, K, Zacharopoulou, A and Robinson, AS (2009) Incipient speciation revealed in Anastrepha fraterculus (Diptera; Tephritidae) by studies on mating compatibility, sex pheromones, hybridization, and cytology. Biological Journal of the Linnean Society 97, 152–165.CrossRefGoogle Scholar
Coluzzi, M, Sabatini, A, Petrarca, V and Di Deco, MA (1979) Chromosomal differentiation and adaptation to human environments in the Anopheles gambiae complex. Transactions of the Royal Society of Tropical Medicine and Hygiene 73, 483–497.CrossRefGoogle ScholarPubMed
Copeland, RS, Wharton, RA, Luke, Q, De Meyer, M, Lux, S, Zenz, N, Machera, P and Okumu, M (2006) Geographic distribution, host fruit, and parasitoids of African fruit fly pests Ceratitis anonae, Ceratitis cosyra, Ceratitis fasciventris, and Ceratitis rosa (Diptera: Tephritidae) in Kenya. Annals of the Entomological Society of America 99, 261–278.CrossRefGoogle Scholar
Delatte, H, Virgilio, M, Simiand, C, Quilici, S and De Meyer, M (2013) Isolation and characterisation of sixteen microsatellite markers cross-amplifying in a complex of three African agricultural pests (Ceratitis rosa, C. anonae and C. fasciventris, Diptera: Tephritidae). Conservation Genetics Resources 5, 31–34.CrossRefGoogle Scholar
De Meyer, M, Copeland, RS, Lux, S, Mansell, M, Wharton, R, White, IM and Zenz, NJ (2002) Annotated check list of host plants for Afrotropical fruit flies (Diptera: Tephritidae) of the genus Ceratitis. Zoologische Documentatie Koninklijk Museum voor Midden Afrika 27, 1–92.Google Scholar
De Meyer, M, Delatte, H, Ekesi, S, Jordaens, K, Kalinová, B, Manrakhan, A, Mwatawala, M, Steck, G, Van Cann, J, Vaničhová, L, Břizová, R and Virgilio, M (2015 a) An integrative approach to unravel the Ceratitis FAR (Diptera, Tephritidae) cryptic species complex: a review. ZooKeys 540, 405–427.CrossRefGoogle Scholar
De Meyer, M, Delatte, H, Mwatawala, M, Quilici, S, Vayssieres, J-F and Virgilio, M (2015 b) A review of the current knowledge on Zeugodacus cucurbitae (Coquillett) (Diptera, Tephritidae) in Africa, with a list of species included in Zeugodacus. ZooKeys 540, 539–557.CrossRefGoogle Scholar
De Meyer, M, Mwatawala, M, Copeland, RS and Virgilio, M (2016) Description of new Ceratitis species (Diptera: Tephritidae) from Africa, or how morphological and DNA data are complementary in discovering unknown species and matching sexes. European Journal of Taxonomy 233, 1–23. doi: 10.5852/ejt.2016.233Google Scholar
de Villiers, M, Hattingh, V and Kriticos, DJ (2013) Combining field phenological observations with distribution data to model the potential distribution of the fruit fly Ceratitis rosa Karsch (Diptera: Tephritidae). Bulletin of Entomological Research 103, 60–73.CrossRefGoogle Scholar
Douglas, LJ and Haymer, DS (2001) Ribosomal ITS1 polymorphisms in Ceratitis capitata and Ceratitis rosa (Diptera: Tephritidae). Annals of the Entomological Society of America 94, 726–731.CrossRefGoogle Scholar
Drosopoulou, E, Pantelidou, C, Gariou-Papalexiou, A, Augustinos, AA, Chartomatsidou, T, Kyritsis, GA, Bourtzis, K, Mavragani-Tsipidou, P and Zacharopoulou, A (2017) The chromosomes and the mitogenome of Ceratitis fasciventris (Diptera: Tephritidae): two genetic approaches towards the Ceratitis FAR species complex resolution. Scientific Reports 7, 4877. doi: 10.1038/s41598-017-05132-3CrossRefGoogle ScholarPubMed
Drosopoulou, E, Damaskou, A, Markou, A, Ekesi, S, Khamis, F, Manrakhan, A, Augustinos, AA, Tsiamis, G and Bourtzis, K (2021) Τhe complete mitochondrial genomes of Ceratitis rosa and Ceratitis quilicii, members of the Ceratitis FAR species complex (Diptera: Tephritidae). Mitochondrial DNA Part B 6, 1039–1041.CrossRefGoogle Scholar
Duyck, PF and Quilici, S (2002) Survival and development of different life stages of three Ceratitis spp. (Diptera: Tephritidae) reared at five constant temperatures. Bulletin of Entomological Research 92, 461–469.CrossRefGoogle ScholarPubMed
Erbout, N, De Meyer, M and Lens, L (2008) Hybridization between two polyphagous fruit-fly species (Diptera: Tephritidae) causes sex-biased reduction in developmental stability. Biological Journal of the Linnean Society 93, 579–588.CrossRefGoogle Scholar
Faria, R and Navarro, A (2010) Chromosomal speciation revisited: rearranging theory with pieces of evidence. Trends in Ecology & Evolution 25, 660–669.CrossRefGoogle ScholarPubMed
Frias, D (2004) Importance of larval morphology and heterochromatic variation in the identification and evolution of sibling species in the genus Rhagoletis (Diptera: Tephritidae) in Chile. In Proceedings of the 6th International Symposium on fruit flies of economic importance (Stellenbosch, South Africa: Barnes, B.N.), 267–276.Google Scholar
Geurts, K, Mwatawala, M and De Meyer, M (2012) Indigenous and invasive fruit fly diversity along an altitudinal transect in Eastern Central Tanzania. Journal of Insect Science 12, 12.CrossRefGoogle ScholarPubMed
Giardini, MC, Milla, FH, Lanzavecchia, S, Nieves, M and Cladera, JL (2015) Sex chromosomes in mitotic and polytene tissues of Anastrepha fraterculus (Diptera, Tephritidae) from Argentina: a review. Zookeys 540, 83–94.CrossRefGoogle Scholar
Goday, C, Selivon, D, Perondini, ALP, Greciano, PG and Ruiz, MF (2006) Cytological characterization of sex chromosomes and ribosomal DNA location in Anastrepha species (Diptera, Tephritidae). Cytogenetic and Genome Research 114, 70–76.CrossRefGoogle Scholar
Gouvi, G, Gariou-Papalexiou, A, Augustinos, AA, Drosopoulou, E, Tsiamis, G, Bourtzis, K and Zacharopoulou, A (2022). The chromosomes of Zeugodacus tau and Zeugodacus cucurbitae: a comparative analysis. Frontiers in Ecology and Evolution 10, 854723. doi: https://www.frontiersin.org/articles/10.3389/fevo.2022.854723 (Accessed August 3, 2022).CrossRefGoogle Scholar
Hernández-Ortiz, V, Bartolucci, AF, Morales-Valles, P, Frías, D and Selivon, D (2012) Cryptic species of the Anastrepha fraterculus complex (Diptera: Tephritidae): a multivariate approach for the recognition of South American morphotypes. Annals of the Entomological Society of America 105, 305–318.CrossRefGoogle Scholar
Hunwattanakul, N and Baimai, V (1994) Mitotic karyotypes of four species of fruit flies (Bactrocera) in Thailand. Agriculture and Natural Resources 28, 142–148.Google Scholar
Kirkpatrick, M and Barton, N (2006) Chromosome inversions, local adaptation and speciation. Genetics 173, 419–434.CrossRefGoogle ScholarPubMed
Krimbas, CB and Powell, JR (1992) Drosophila Inversion Polymorphism. Boca Raton, FL: CRC Press.Google Scholar
Kulathinal, RJ, Stevison, LS and Noor, MAF (2009) The genomics of speciation in drosophila: diversity, divergence, and introgression estimated using low-coverage genome sequencing. PLoS Genetics 5, e1000550.CrossRefGoogle ScholarPubMed
Lee, Y, Collier, TC, Sanford, MR, Marsden, CD, Fofana, A, Cornel, AJ and Lanzaro, GC (2013) Chromosome inversions, genomic differentiation and speciation in the African malaria mosquito Anopheles gambiae. PLoS ONE 8, e57887.CrossRefGoogle ScholarPubMed
Malacrida, AR, Gomulski, LM, Bonizzoni, M, Bertin, S, Gasperi, G and Guglielmino, CR (2007) Globalization and fruitfly invasion and expansion: the medfly paradigm. Genetica 131, 1–9.CrossRefGoogle ScholarPubMed
Mavragani-Tsipidou, P, Zacharopoulou, A, Drosopoulou, E, Augustinos, AA, Bourtzis, K and Marec, F (2014) Tephritid fruit flies (Diptera). In Sharakhov, IV (ed.), Protocols for Cytogenetic Mapping of Arthropod Genomes. Boca Raton, Florida: CRC Press, pp. 1–62. doi: 10.1201/b17450.Google Scholar
McGaugh, SE and Noor, MAF (2012) Genomic impacts of chromosomal inversions in parapatric Drosophila species. Philosophical Transactions of the Royal Society of London B Biological Sciences 367, 422–429.CrossRefGoogle ScholarPubMed
Mwatawala, M, Virgilio, M, Joseph, J and De Meyer, M (2015) Niche partitioning among two Ceratitis rosa morphotypes and other Ceratitis pest species (Diptera, Tephritidae) along an altitudinal transect in Central Tanzania. Zookeys 540, 429–442.CrossRefGoogle Scholar
Noor, MAF, Grams, KL, Bertucci, LA and Reiland, J (2001) Chromosomal inversions and the reproductive isolation of species. PNAS 98, 12084–12088.CrossRefGoogle ScholarPubMed
Papadimitriou, E, Kritikou, D, Mavroidis, M, Zacharopoulou, A and Mintzas, AC (1998) The heat shock 70 gene family in the Mediterranean fruit fly Ceratitis capitata. Insect Molecular Biology 7, 279–290.CrossRefGoogle ScholarPubMed
Rieseberg, LH (2001) Chromosomal rearrangements and speciation. Trends in Ecology & Evolution 16, 351–358.CrossRefGoogle ScholarPubMed
Schutze, MK, Virgilio, M, Norrbom, A and Clarke, AR (2017 a) Tephritid integrative taxonomy: where we are now, with a focus on the resolution of three tropical fruit fly species complexes. Annual Review of Entomology 62, 147–164.CrossRefGoogle ScholarPubMed
Schutze, MK, Bourtzis, K, Cameron, S, Clarke, AR, de Meyer, M, Hee, AKW, Hendrichs, J, Krosch, M and Mwatawala, M (2017 b) Integrative taxonomy versus taxonomic authority without peer review: the case of the oriental fruit fly, Bactrocera dorsalis (Tephritidae). Systematic Entomology 42, 609–620.CrossRefGoogle Scholar
Selivon, D, Perondini, ALP and Morgante, JS (2005 a) A genetic–morphological characterization of two cryptic species of the Anastrepha fraterculus complex (Diptera: Tephritidae). Annals of the Entomological Society of America 98, 367–381.CrossRefGoogle Scholar
Selivon, D, Perondini, ALP and Rocha, LS (2005 b) Karyotype characterization of Anastrepha fruit flies (Diptera: Tephritidae). Neotropical Entomology 34, 273–279.CrossRefGoogle Scholar
Smith, IM, McNamara, DG, Scott, PR and Holderness, M (eds) (1997). Quarantine pests for Europe. Data sheets on quarantine pests for the European Union and for the European and Mediterranean Plant Protection Organization, 2nd Edn. Wallingford, UK: CAB International.Google Scholar
Sturtevant, AH and Dobzhansky, T (1936) Inversions in the third chromosome of wild races of drosophila Pseudoobscura, and their use in the study of the history of the species. Proceedings of the National Academy of Sciences 22, 448–450.CrossRefGoogle Scholar
Tanga, CM, Manrakhan, A, Daneel, J-H, Mohamed, SA, Fathiya, K and Ekesi, S (2015) Comparative analysis of development and survival of two natal fruit fly Ceratitis rosa Karsch (Diptera, Tephritidae) populations from Kenya and South Africa. Zookeys 540, 467–487.Google Scholar
Tanga, CM, Khamis, FM, Tonnang, HEZ, Rwomushana, I, Mosomtai, G, Mohamed, SA and Ekesi, S (2018) Risk assessment and spread of the potentially invasive Ceratitis rosa Karsch and Ceratitis quilicii De Meyer, Mwatawala & Virgilio sp. Nov. using life-cycle simulation models: implications for phytosanitary measures and management. PLoS ONE 13, e0189138.CrossRefGoogle Scholar
Van Cann, J, Virgilio, M, Jordaens, K and De Meyer, M (2015) Wing morphometrics as a possible tool for the diagnosis of the Ceratitis fasciventris, C. anonae, C. rosa complex (Diptera, Tephritidae). ZooKeys 540, 489–506.Google Scholar
Vaníčková, L, Virgilio, M, Tomčala, A, Břízová, R, Ekesi, S, Hoskovec, M, Kalinová, B, Do Nascimento, RR and De Meyer, M (2014) Resolution of three cryptic agricultural pests (Ceratitis fasciventris, C. anonae, C. rosa, Diptera: Tephritidae) using cuticular hydrocarbon profiling. Bulletin of Entomological Research 104, 631–638.CrossRefGoogle ScholarPubMed
Vaníčková, L, Břízová, R, Mendonça, AL, Pompeiano, A and Do Nascimento, RR (2015) Intraspecific variation of cuticular hydrocarbon profiles in the Anastrepha fraterculus (Diptera: Tephritidae) species complex. Journal of Applied Entomology 139, 679–689.CrossRefGoogle Scholar
Virgilio, M, Backeljau, T, Barr, N and De Meyer, M (2008) Molecular evaluation of nominal species in the Ceratitis fasciventris, C. anonae, C. rosa complex (Diptera: Tephritidae). Molecular Phylogenetics and Evolution 48, 270–280.CrossRefGoogle Scholar
Virgilio, M, Jordaens, K, Breman, FC, Backeljau, T and De Meyer, M (2012) Identifying insects with incomplete DNA barcode libraries, African fruit flies (Diptera: Tephritidae) as a test case. PLoS ONE 7, e31581. doi: 10.1371/journal.pone.0031581CrossRefGoogle ScholarPubMed
Virgilio, M, Delatte, H, Quilici, S, Backeljau, T and De Meyer, M (2013) Cryptic diversity and gene flow among three African agricultural pests: Ceratitis rosa, Ceratitis fasciventris and Ceratitis anonae (Diptera, Tephritidae). Molecular Ecology 22, 2526–2539.CrossRefGoogle Scholar
Virgilio, M, Daneel, J-H, Manrakhan, A, Delatte, H, Meganck, K and De Meyer, M (2019) An integrated diagnostic setup for the morphological and molecular identification of the Ceratitis FAR complex (C. anonae, C. fasciventris, C. rosa, C. quilicii, Diptera, Tephritidae). Bulletin of Entomological Research 109, 376–382.CrossRefGoogle Scholar
White, IM and Elson-Harris, MM (1992). Fruit Flies of Economic Significance: Their Identification and Bionomics. Wallingford, UK: CAB International.CrossRefGoogle Scholar
White, IM, De Meyer, M and Stonehouse, J (2000). A review of native and introduced fruit flies (Diptera, Tephritidae) in the Indian Ocean islands of Mauritius, R{é}union, Rodrigues and Seychelles. In Proceedings of the Indian Ocean Commission, Regional Fruit Fly Symposium (Flic en Flac, Mauritius), 15–21.Google Scholar
Willhoeft, U and Franz, G (1996) Comparison of the mitotic karyotypes of Ceratitis capitata, Ceratitis rosa, and Trirhithrum coffeae (Diptera: Tephritidae) by C-banding and FISH. Genome 39, 884–889.CrossRefGoogle ScholarPubMed
Zacharopoulou, A, Augustinos, AA, Drosopoulou, E, Tsoumani, KT, Gariou-Papalexiou, A, Franz, G, Mathiopoulos, KD, Bourtzis, K and Mavragani-Tsipidou, P (2017) A review of more than 30 years of cytogenetic studies of Tephritidae in support of sterile insect technique and global trade. Entomologia Experimentalis et Applicata 164, 204–225.CrossRefGoogle Scholar
Zhang, Y, De Meyer, M, Virgilio, M, Feng, S, Badji, K and Li, Z (2021) Phylogenomic resolution of the Ceratitis FARQ complex (Diptera: Tephritidae). Molecular Phylogenetics and Evolution 161, 107160.CrossRefGoogle Scholar
Zhimulev, IF and Koryakov, DE (2009) Polytene chromosomes. In Encyclopedia of Life Sciences (ELS). Chichester: John Wiley & Sons, Ltd. Available at 10.1002/9780470015902.a0001183.pub2Google Scholar
Figure 0

Figure 1. Mitotic karyotypes of C. rosa (a and b) and C. quilicii (c and d). (a, c) Female; (b, d) male. The sex chromosomes, X and Y, as well as the autosomes 2 and 3 are shown.

Figure 1

Figure 2. Polytene nuclei of F1 hybrids between C. rosa and C. quilicii. The telomeres of the polytene elements are indicated. 5LC indicates the 5L centromere. No asynapses are observed.

Figure 2

Figure 3. Asynapses frequently observed in the nuclei of F1 hybrids between C. rosa and C. fasciventris. The asynaptic telomeres of the polytene elements are indicated. Variable extent of asynapsis observed for 4R and 6R telomeres is presented in (f) and (i), respectively. Arrows indicate asynapses in the inner parts of the polytene elements. 5LC indicates the 5L centromere.

Figure 3

Figure 4. Asynapses frequently observed in the nuclei of F1 hybrids between C. quilicii and C. fasciventris. The asynaptic telomeres of the polytene elements are indicated. Variable extent of asynapsis observed for 3L telomere is presented in (d–f). Arrows indicate asynapses in the inner parts of the polytene elements.

Figure 4

Figure 5. In situ hybridization of the hsp70 gene probe on the salivary gland polytene chromosomes of C. rosa and C. quilicii. Arrows indicate the hybridization signals. The telomere of the 3L polytene arm is indicated. Numbered divisions are shown, separated by lines. The reference map of the 3L arm and the hybridization locus of the hsp70 gene of C. fasciventris (Drosopoulou et al., 2017) are presented on the top.

Supplementary material: File

Drosopoulou et al. supplementary material

Drosopoulou et al. supplementary material

Download Drosopoulou et al. supplementary material(File)
File 6.7 MB