Introduction

The Tephritidae family consists of more than 500 genera and 4600 species being one of the most speciose groups of Diptera1. About 40% of the species of this family are characterized as true fruit flies since they infect a great variety of fruit producing plants and a significant number of them are considered serious agricultural pests. The Anastrepha, Bactrocera, Ceratitis, Dacus and Rhagoletis genera include some of the world’s most destructive insect pests, causing extensive damages in a wide variety of crops with enormous economic impact2.

Ceratitis fasciventris is an African fruit fly, which, together with C. anonae, C. rosa and the recently described C. quilicii comprise the“Ceratitis FAR complex”3, 4. All members of the complex are agricultural pests with C. rosa being the most aggressive one. They attack a great number of wild and cultivated plants belonging to more than 25 different families, and present greatly overlapping host range while each also has unique hosts5, 6. Ceratitis fasciventris is found mainly in eastern and western Africa sympatrically with C. anonae, while C. rosa and C. quilicii are distributed in eastern and southern Africa overlapping with each other and partially with C. fasciventris in Kenya and Tanzania4, 6,7,8. There is increasing concern about the possibility that these species could expand outside their native range similarly to the spread of C. capitata almost worldwide in the past century2, 3, 6, 9, 10. Populations of C. rosa and C. quilicii have been already introduced in the Indian ocean islands Mauritius and La Réunion4, 11 and their invasive potential is a major consideration taking into account their potential adaptation to a wider temperature range than C. capitata 6, 12,13,14 and their capability to attack fruits grown also in temperate climates (e.g. peaches and apples)4, 15.

Because of the high risk of their expansion, the delimitation and accurate identification of species within the FAR as well as other complexes of economic relevant tephritid pests is of crucial importance for regulatory agencies and agricultural countries. It affects the international trade and quarantine policies implemented on fruit and vegetable hosts with significant economic impact16. Besides, the clarification of genetic relationships among the different entities of species complexes is critical for the effective development and application of environmental friendly methods for their control, such as the Sterile Insect Technique (SIT)17, 18. Within the FAR complex the species boundaries are obscure and have been the object of research and discussion for some time.The members of the complex are almost identical morphologically. Their identification has been based on differences in the setal pattern of the adult male mid legs, while females are almost indistinguishable4, 19, 20. Due to the absence of clear diagnostic morphological features a number of molecular approaches, including mitochondrial markers and the ITS1 sequence, have been used unsuccessfully to clearly resolve the limits among the morphospecies of the complex3, 21,22,23. A more recent microsatellite analysis revealed the presence of five genotypic clusters within the complex: two (R1, R2) representing populations that were at the time considered as C. rosa, two (F1, F2) for C. fasciventris and one for C. anonae 8, thus complicating further the phylogenetic relationships and species discrimination in the FAR complex.

Recent studies have shown that efforts to resolve complex species status require multidisciplinary approaches24, 25, well-characterized material and extended sampling26,27,28. An example is the study of the members of the Bactrocera dorsalis complex, B. dorsalis s.s., B. papayae, B. philippinensis, B. invadens and B. carambolae, for which morphological/morphometric, behavioral/sexual compatibility, chemoecological, molecular/genetic and cytogenetic data lead to the synonymization of the four members, maintaining B. carambolae as a discrete entity29. Pluralistic approaches have been also followed for species delimitation within species complexes of other Tephritidae genera, such as the Anastrepha fraterculus complex18, 30,31,32,33,34,35. In this context, the issue of the number and the limits of species within the FAR complex has been recently addressed by an integrative approach on the basis of: (i) adult and larvae morphology36, 37, (ii) wing morphometrics38, (iii) microsatellite analysis8, (iv) cuticular hydrocarbons39, (v) pheromones40, (vi) developmental physiology41 and (vii) geographical and altitudinal distribution42. The above analysis supported the species identity of C. anonae 36. For C. rosa, it was proposed that it consists of two separate entities (R1 and R2) that should be considered as different species: C. rosa representing group R1 and the newly described species C. quilicii representing group R24, 36. Although two entities (F1 and F2) were identified for C. fasciventris as well, the available data are insufficient to support their separation in two different species36.

The above analysis, although incorporating a volume of data from several disciplines, lacks chromosome/cytogenetic and mitochondrial evidence. Cytogenetic analyses can be proven valuable in efforts to resolve phylogenetic relationships and species boundaries among closely related species. Many years of cytogenetic studies in Drosophila and mosquitoes have shown that chromosomal rearrangements (CRs), especially inversions, play a causative role in speciation and they can be used as interspecific phylogenetic markers43,44,45,46,47,48. Modern genomic support the above observations and propose that CRs enhance speciation through the restriction of recombination within and near inverted regions resulting in restriction of gene flow49,50,51,52,53,54,55,56,57,58.

In Tephritidae flies, mitotic karyotypes have been used to distinguish between different members of species complexes based on differences of the sex chromosomes33, 35, 59,60,61,62,63,64. Polytene chromosome maps are available for 11 tephritid species belonging to five genera65,66,67,68,69,70,71,72,73,74,75,76,77,78,79. Comparative analyses among most of them revealed specific CRs that are diagnostic at the genus, subgenus and species level70, 75,76,77,78, 80, supporting the possible involvement of CRs in speciation.

The mitochondrial DNA (mt DNA) is considered a very useful molecular marker for phylogenetic analyses as it can be informative at a variety of taxonomic levels81, 82. Partial mitochondrial gene sequences have been extensively used for inferring phylogeny among species of the Tephritidae family83,84,85,86,87,88,89,90,91,92,93,94, and within the Ceratitis genus, in particular3, 22, 23. However, the use of complete mitochondrial genome sequences, facilitated by the continuously increasing number of them in the databanks, has become a preferable approach for phylogenetic and molecular systematic studies in several insect groups95,96,97,98,99,100,101,102,103 including Tephritidae104,105,106,107,108,109,110. When the issue is the discrimination of closely related species for quarantine and management applications the availability of complete mitogenome data is particularly valuable, as it can allow identification of the most informative diagnostic markers/sequences through intra and inter-specific comparative analysis106.

In the current study, we present the mitotic karyotype, a detailed salivary gland polytene chromosome analysis, as well as the complete mitochondrial genome sequence of C. fasciventris. This is the first description of the mitotic and polytene chromosomes, as well as the first complete mitogenome sequence from a member of the Ceratitis FAR complex and only the second available for the whole genus, after the model species, C. capitata. Both cytogenetic and mitochondrial information provided can be used as reference for comparative studies towards species delimitation and resolution of phylogenetic relationships within this species complex, but also among other species of the Ceratitis genus.

Results and Discussion

Mitotic chromosomes

The mitotic karyotype of Ceratitis fasciventris consists of six pairs of chromosomes: five pairs of meta- or sub-metacentric autosomes and one pair of sex chromosomes (Fig. 1). According to the labeling system used for the Mediterranean fruit fly (medfly), C. capitata (Wiedemann)67, the sex chromosomes are designated as the first pair of the mitotic karyotype, while the five autosomes are labeled from 2 to 6, in order of descending size. The first autosome pair (2) of C. fasciventris is easily distinguished due to its larger size. The remaining four autosomes are almost equal in size. One of them is clearly submetacentric and has been designated as autosome pair 3 in accordance to C. capitata, while the other three cannot be reliably distinguished (Fig. 1b). The sex chromosomes are identified as the heteromorphic pair (XY) of heavily stained chromosomes in the mitotic complement (Fig. 1b). This karyotype is consistent with the karyotype of most Tephritidae species analyzed so far59,60,61, 65, 67, 68, 70, 71, 73,74,75,76,77, 79, 111.

Figure 1
figure 1

Mitotic karyotype of Ceratitis fasciventris. (a) Female karyotype. The X chromosomes are indicated. (b) Male karyotype. The autosomes 2 and 3 and the X and Y chromosomes are indicated. Chromosomes were stained with Giemsa.

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The above described mitotic karyotype of C. fasciventris is quite similar to the karyotype of C. capitata 66, 67. The most apparent difference between the two species is the size of the sex chromosomes. In C. capitata, the X chromosome is about the same size with the largest autosome, while the Y is the shorter chromosome of the mitotic complement. On the contrary, in C. fasciventris the X chromosome is shorter than any autosome, while the Y chromosome is significantly smaller with its length reaching about ¼ of the length of the X. Size and shape variation of the sex chromosomes is common among tephritids and, based on their highly heterochromatic nature, it could be attributed to the accumulation or loss of heterochromatin30, 31, 33, 35, 59,60,61, 63,64,65, 67, 68, 70,71,72,73,74,75,76,77,78,79, 111,112,113,114,115,116.

Polytene chromosomes

The analysis of the salivary gland polytene chromosomes of C. fasciventris showed that the polytene complement consists of five long, well banded chromosomes (10 polytene arms) corresponding to the five autosomes. The sex chromosomes are under-replicated in polytene tissues, do not form discrete polytene elements and are apparent as a heterochromatic network (Fig. 2). No typical chromocenter exists; the two arms of each individual chromosome are loosely connected or can be found separated from each other. The polytene chromosomes of C. fasciventris were numbered from 2 to 6 and divided into sections from 1 to 100 based on their banding pattern similarities to C. capitata. For each polytene chromosome, the longer arm is designated as left (L) and the shorter one as right (R) (Fig. 3).

Figure 2
figure 2

A polytene nucleus of Ceratitis fasciventris. The telomeres of the polytene elements are indicated. The heterochromatic network (hn) corresponding to the sex chromosomes is circled.

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Figure 3
figure 3

Photographic polytene chromosome map of Ceratitis fasciventris. C indicates the centromere.

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Detailed comparison of the polytene chromosomes of C. fasciventris to the reference map of C. capitata 67, 117 revealed significant similarities. Characteristic structures such as the distal ends (telomeres) and the centromeric region of each chromosome seem identical between the two species. Furthermore, extensive similarity in the banding pattern of eight out of the ten polytene arms, namely 2L, 2R, 3R, 4L, 4R, 5R, 6L and 6R, can be identified (Supplementary Figs S1–S8). However, some differences in the puffing pattern were observed, which could reflect species-specific differences, different developmental stages118 and/or differences at rearing conditions. Regarding chromosome arms 3L and 5L, despite the banding pattern similarity in both ends of each arm, differences can be observed in the inner parts. After thorough analysis of the banding pattern, specific chromosome segments that seem to be rearranged in comparison to C. capitata have been identified (Figs 4 and 5). These changes could have been derived from consecutive overlapping inversions resulting in both inverted and transposed chromosome fragments119. Comparative analyses of polytene chromosomes among several tephritids showed that CRs are restricted to specific chromosome arms, mainly to the 3L and 5L arms70, 76,77,78, 80.

Figure 4
figure 4

Comparison of the 3L polytene chromosome arms of Ceratitis fashiventris and Ceratitis capitata. (a) The 3L polytene chromosome arm of C. fasciventris; (b) The 3L polytene chromosome arm of C. capitata. Horizontal arrows of the same color show the position and relative orientation of the putative corresponding chromosome segments between the two species. Vertical black lines underneath the chromosomes show the proposed breakpoints. Black arrows indicate the hybridization site of the hsp70 probe in each species. C indicates the centromere.

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Figure 5
figure 5

Comparison of the 5L polytene chromosome arms of Ceratitis fashiventris and Ceratitis capitata. (a) The 5L polytene chromosome arm of C. fasciventris; (b) The 5L polytene chromosome arm of C. capitata. Horizontal arrows of the same color show the position and relative orientation of the putative corresponding chromosome segments between the two species. Vertical black lines underneath the chromosomes show the proposed breakpoints. Black arrows indicate the hybridization site of the w probe in each species. C indicates the centromere.

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Chromosome localization of molecular markers

Four gene markers, namely hsp70, w, sxl and st (Table 1), were localized on the polytene chromosomes of C. fasciventris by in situ hybridization. Each heterologous probe gave a unique hybridization signal (Table 1 and Fig. 6): (i) the hsp 70 specific probe on region 27 of the 3L arm (Fig. 6a), (ii) the w probe on region 67 of the 5L polytene arm (Fig. 6b), (iii) the sxl probe on region 79 of the 5R arm (Fig. 6c) and (iv) the st probe on region 83 of the 6L polytene arm (Fig. 6d).

Table 1 The hybridization probes and their localization sites on the polytene chromosomes of Ceratitis fasciventris and Ceratitis capitata.
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Figure 6
figure 6

In situ hybridization on the salivary gland polytene chromosomes of Ceratitis fasciventris. (a) hybridization site of the hsp70 probe; (b) hybridization site of the w probe; (c) hybridization site of the sxl probe and (d) hybridization site of the st probe. Arrows indicate the hybridization signals.

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The four gene markers studied here have been previously mapped in C. capitata at putative corresponding polytene chromosome regions70, 117, 120, 121. The localization of the sxl and st genes supports the conservation of the 5R and 6L polytene chromosome arms between the two species, as it was deduced by banding pattern similarities (Supplementary Figs S6 and S7). On the other hand, the hybridization of the hsp70 gene at region 27 of C. fasciventrisis is in discordance with the localization of the above gene at region 24 of C. capitata (Table 1 and Fig. 4), supporting the proposed rearrangements of the 3L polytene arm between the two species (Fig. 4). Similarly, the different chromosome region of the w locus (67 on 5L for C. fasciventris, 65 on 5L for C. capitata; Table 1 and Fig. 5) is further support for the rearrangements of the 5L chromosome arm between the two species suggested by banding pattern analysis (Fig. 5).

The banding pattern comparison and gene localization data provide evidence for the conservation of chromosome synteny between the closely related species C. capitata and C. fasciventris. However, a more thorough chromosome mapping analysis would be needed in order to confirm the above observation. The idea that the chromosome elements have maintained their basic content and identity was initially proposed for Drosophila species122, 123. Later comparative studies using biochemical and morphological markers showed a remarkable conservation of linked gene clusters among a wider phylogenetic range of higher Diptera124,125,126. In situ hybridization of well characterized genes on polytene chromosomes further supported the extensive conservation of linkage groups in Tephitidae70, 117, 127,128,129,130,131. On the other hand, the differences identified in the structure of the 3L and 5L polytene arms of C. fasciventris and C. capitata, are congruent with the concept that chromosome rearrangements, mainly paracentric inversions, are implicated in species differentiation in Diptera which is substantiated by comparative cytogenetic and genomic analyses in Drosophila and mosquitoes46,47,48,49,50,51,52,53,54, 56,57,58. In accordance with the above notion, comparisons among tephritids, reveal that inversions and/or transpositions on specific chromosomes, including chromosomes 3 and 5, differentiate species and support the potential value of chromosome rearrangements as phylogenetic and diagnostic markers among closely related species70, 75,76,77,78, 80, 111.

Mitochondrial genome

The mitochondrial DNA of C. fasciventris was amplified and sequenced in 27 overlapping fragments. The above analysis resulted in the nucleotide sequence of the complete C. fasciventris mitogenome consisting of 16,017 bp with very high A + T (77.17%) and very low G + C (22.83%) contents. The organization of the C. fasciventris mt genome is typical to that of other tephritids studied104, 106,107,108,109,110, 132,133,134,135,136. It comprises 13 protein-coding, two rRNA (12S and 16S rRNA) and 22 tRNA genes, and one major non-coding sequence, the control region (Table 2), all of which are similar in size to their counterparts in other insects.

Table 2 Organization of the Ceratitis fasciventris mitochondrial genome.
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Protein coding genes

The location of the 13 protein coding genes (PCGs) was determined by the identification of initiation and termination signals as well as by sequence comparisons to the respective genes of C. capitata. Nine of them are encoded by the H strand, while ND1, ND4, ND4L and ND5 are encoded by the L strand (Table 2). Only six protein coding genes (COII, ATP6, COIII, ND4, ND4L, CYTB) begin with the canonical initiation codon ATG, five of them (ND2, ATP8, ND5, ND6 and ND1) initiate with ATT, while for ND3 the first codon is ATA and for COI it is TCG (Table 2), similarly to other Tephritidae species104, 106,107,108, 110, 132, 133. In C. fasciventris, two of the protein coding genes (ND5 and ND1) possess an incomplete termination codon (T) (Table 2). This is common in animal mitochondrial DNA; the stop codon is likely completed by post-transcriptional polyadenylation137.

The overlapping of seven nucleotides between ATP8 and ATP6 genes is the longest observed between protein coding genes of C. fasciventris and is also common (presenting variable size) among tephritids104, 106, 132, 133. Overlapping restricted to one or two nucleotides can also be observed between ATP6 and COIII, ND4 and ND4L, ND6 and CYTB and CYTB and tRNA Ser (Table 2). However, in these cases one could assume an incomplete stop codon for the preceding gene, which could mean lack of overlap with the following one.

RNA genes

The C. fasciventris 16S rRNA and 12S rRNA genes consist of 1,341 (positions: 12,793–14,133) and 788 (positions: 14,206–14,993) nucleotides, respectively (Table 2). As in other insects, these genes are located in the L strand at the end of the mtDNA molecule between the gene for tRNA Leu (CUA) and the control region, and are separated by the tRNA Val gene. Dispersed among the protein-coding and the rRNA genes there are 22 tRNA genes; 14 on the H and 8 on the L strand. Their sizes range from 64–72 nucleotides (Table 2) and are predicted to fold into the expected cloverleaf secondary structures.

Non-coding regions

Following the rule for animal mitochondrial DNA, the mt genome of C. fasciventris contains only one long non-coding region with very high A + T content (90.24%) that regulates replication and transcription, the control region. It is located between the 12SrRNA and the tRNA Ile genes and its length is 1024 bp (positions 14,994–16,017; Table 2), within the range of the respective region in other Tephritidae species. At the 5′ end of the control region (positions 15,947–15,973) a poly(T) followed by a [TA(A)n]-like stretch can be observed. This feature is conserved among insect species and has been proposed to play a role in the control of transcription and/or replication107, 110, 133, 138. Two motifs, TTAAATTAATAATTAT and TATTTTTATTTTTAAATT, were found to be tandemly repeated three (positions 15,263–15,315) and two times (positions 15,871–15,909), respectively. Tandem repeats have been identified in the control regions of other Tephritidae species, as well107, 133.

The C. fasciventris mitogenome contains 20 intergenic spacers (IGS) of 232 bp total length (Table 2). The longest one (62 bp) is located between tRNA Ile and tRNA Gln, as is the case in B. zonata 107. In C. capitata, an IGS is present at the respective position but it is not the longer one. In the latter species, the longer IGS (46 bp) is found between tRNA Gln and tRNA Met  132 similarly to several Bactrocera species106, 108, 109. Quite long IGS in species of the Tephritidae family are also present between tRNA Cys and tRNA Tyr, which is able to form a secondary structure and between tRNA Arg and tRNA Asn  104, 106, 108,109,110, 132,133,134. The second longest IGS in C. fasciventris lies between tRNA Cys and tRNA Tyr; however it is only 20 bp long and cannot form a stem loop. On the other hand, IGS separating tRNA Arg and tRNA Asn is only 3 bp long. It has been observed thatspecific IGS (the ones between tRNA Glu and tRNA Phe, ND5 and tRNA His, tRNA Ser and ND1 and ND1 and tRNA Leu) are conserved in size and sequence among several tephritids and have their counterparts in the control region107, 133. This is also valid for C. fasciventris.

Sequence comparisons with C. capitata and other Tephritidae species

The C. fasciventris mitogenome is the second complete mt DNA sequence of the Ceratitis genus analyzed, with the first one being that of C. capitata 132. The mitochondrial genomes of these two species are highly similar both in terms of organization and structure as well as of sequence similarity. The overall sequence identity is 92.11%, while identity of the PCGs is 92.25% (Table 3). In comparison to other tephritid mitogenomes, the C. fasciventris mtDNA presents the lowest sequence identity with B. minax (79.18% for complete mitogenome; 79.31% for PCGs). The latter species presents the lowest identity percentages in comparison with any other tephritid, even the ones of the same genus, both in the complete mitosequence, as well as in the PCGs sequence (Table 3).

Table 3 Identity percentage matrix among 19 Tephritidae mitochondrial genome sequences.
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The phylogenetic analysis based on the concatenated sequences of the 13 PCGs of 19 Tephritidae species places the currently analyzed C. fasciventris sequence in the same clade with the sequence of C. capitata (Fig. 7) confirming the closer relationship of the two species22, 139. Furthermore, species of the Zeugodacus subgenus are grouped separately from all other Bactrocera subgenera in a sister clade to Dacus (Fig. 7), supporting the recent suggestion of raising Zeugodacus to the genus level, as well its closer phylogenetic relationship to Dacus in comparison to Bactrocera 88, 92, 94, 110, 140.

Figure 7
figure 7

Molecular phylogenetic analysis by Maximum Likelihood method. Tree based on 13 protein coding genes from complete mitochondrial genome sequences of 19 Tephritidae species. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Asterisk indicates the Ceratitis fasciventris sequence analyzed in the present study. Clustering based upon subgenera for the Bactrocera genus is indicated by bars along the right hand side of the phylogenetic tree. Drosophila melanogaster was used as out group to root the tree. Numbers at nodes are for bootstrap percentages from 1000 replicates; only the ones higher than 50 are presented. The GenBank accession numbers of the mtDNA sequences used are given in Supplementary Table S1.

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Conclusions

In the crucial issue of species boundaries within complexes of destructive insect pests, a multidisciplinary approach is considered the best way for reaching solid results and reliable species discrimination24, 25, 29. To this direction, cytogenetic and complete mitogenome analysis can provide important information and potentially reveal species diagnostic characters. The mitotic karyotype and polytene chromosome maps of C. fasciventris are the first presented for the FAR complex and the second for the genus Ceratitis. The comparison and the differences identified with the mitotic and polytene chromosomes of C. capitata proves the cytogenetic information on C. fasciventris is valuable for comparative analysis possibly providing important insight in the genetic relationships of the species of the FAR complex, as well as among other species of the Ceratitis genus. Likewise, the first complete mitochondrial sequence of a member of the FAR complex can be used as reference for future sequence comparisons among FAR species, aiming to reveal polymorphic mitochondrial regions suitable for the development of molecular diagnostic markers. The fact that the insects used originate from a well-characterized established colony eliminates possible problems resulting from sampling and species misidentification80 and supports the use of both cytogenetic and mitochondrial present results as reference material.

Methods

Specimens and rearing conditions

Insects used in the present study came from a C. fasciventris colony maintained at the FAO-IAEA Insect Pest Control Laboratory (IPCL), Seibersdorf Austria. The colony was established in the summer of 2013 from pupae found in field infested Citrus sp. fruits from Kenya. Male adults were identified as belonging to the F2 group of C. fasciventris, based on the coloration and setal ornamentation of the mid tibia36. Identified adult voucher specimens of the colony are preserved at the entomology collections of the Royal Museum for Central Africa (Tervuren, Belgium).

Rearing was accomplished by keeping adults in groups of 300–500 individuals, in 2-side fine-mesh covered, rectangular cages (50 × 30 × 20 cm). Adults had ad libitum access to water and adult diet consisting of yeast hydrolysate and sugar at 1:3 ratio. For oviposition, females were provided with bananas, which were pin punctured with 50–70 holes to serve egg deposit. The oviposited bananas were placed over a thin sawdust layer, in plastic trays. The fully developed 3rd instar larvae that exited the bananas and pupated in the sawdust were collected by sieving. The colony was reared under controlled temperature, humidity and light conditions (22 °C, 65 ± 2% RH, 14 h L: 10 h D).

Mitotic chromosome preparations

Spread chromosome preparations were made from nerve ganglia of third instar larvae67, 111. Brain tissue was dissected in 0.7% NaCl solution. The material was transferred to 1% sodium citrate on a well slide for at least 15 min and in fresh fixation solution (methanol/acetic acid 3:1) for 3 min. Samples were transferred to a small drop of 60% acetic acid and dispersed using a micropipette. The cell suspension was dried by laying it on a clean slide placed on a hotplate (40–45 °C). Chromosomes were stained with Giemsa solution (5% Giemsa in 10 mM phosphate buffer, pH 6.8). Chromosome slides were analyzed at 100× magnification, using a phase contrast microscope (Leica DMR), and photographs were taken using a CD camera (ProgResCFcool; Jenoptik Jena Optical Systems, Jena, Germany). More than 20 chromosome preparations representing 20 individual larvae and at least 10 well spread nuclei per preparation were analyzed.

Polytene chromosome preparations

Polytene chromosome preparations were made from well fed third-instar larvae or 1–2 days old pupae. Larvae were dissected in 45% acetic acid and salivary glands were carefully transferred to 3 N HCL on a depression slide for 1 min. Glands were fixed in glacial acetic acid:water:lactic acid (3:2:1) for about 5 min before stained in lacto-acetic-orcein for 5–7 min67, 111. Early pupae were dissected in Ringer’s solution and the salivary glands were transferred to 45% acetic acid for 2–3 min and post-fixed in 1 N HCL for 2 min. The material was passed through lacto- acetic acid (80% lactic acid:60% acetic acid, 1:1) and stained in lacto-acetic-orcein for 10–20 min. Excess stain was removed by washing the glands in lacto-acetic acid69, 111. For best chromosome preparations, each salivary gland was cut in two pieces, and each piece was squashed for one preparation. More than 200 chromosome slides (representing at least 150 single larvae or pupae) were prepared and observed at 63× and 100× objectives in a phase contrast microscope (Leica DMR). At least 200 well spread nuclei and/or isolated chromosomes were selected for analysis and photographed using a digital camera (see above). Selected photographs for each chromosome arm showing the best morphology were used for the construction of the C. fasciventris polytene chromosome maps, as well as for the comparison with the available maps of C. capitata 117.

In situ hybridization

Polytene chromosome preparations for in situ hybridization were made from salivary glands of late third instar larvae or young pupae (1–2 day old)128. Four heterologous gene sequences, namely hsp70, w, sxl and st were used as probes (Table 1). Labeling was performed using the DIG-High Prime and detection using the Anti-Digoxigenin-AP, Fab fragments and the NBT/BCIP Stock Solution, all purchased by ROCHE, Mannheim, Germany. Hybridization was performed at 60 °C for the hsp70, sxl and st probes and at 52 °C for the w probe. Hybridization and detection procedures were performed as previously described111, 128. Four to five preparations were hybridized with each probe, and at least ten well spread nuclei per preparation were observed at 63x or 100x magnification with a Nikon Eclipse 80i or a Leica DMR phase contrast microscope, respectively. Photographs were captured using a Nikon DS-5 M-U1 (63x) or a Jenoptik ProgRes (100x) CCD camera.

DNA isolation and mitochondrial genome amplification and sequencing

Total genomic DNA was extracted from single flies, using the CTAB protocol141.

The complete mtDNA sequence was obtained from a single specimen by standard PCR amplifications using: (i) heterologous primers that were designed based on the mitochondrial sequences of Ceratitis capitata and Bactrocera dorsalis (Supplementary Table S1) and (ii) homologous primers that were designed based on the sequences determined by the analysis in this study. In total 27 pairs of primers amplifying overlapping fragments were designed by the Oligoexplorer and Oligoanalyzer programs (Supplementary Table S2). The reaction mixture (25 μl) contained 1X PCR buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.5 μM of the appropriate primers and 1 U Taq polymerase (BIOTAQ, BIOLINE). Approximately 30 ng of template DNA was used in each reaction. PCR reactions comprised an initial denaturation at 94 °C for 3 min, followed by 40 cycles of 45 min denaturation at 94 °C, 30 sec primer annealing at 49–56 °C and 1 min DNA chain extension at 72 °C, for standard PCR. This was followed by a final extension at 72 °C for 7 min. The resulting PCR products were analyzed in a 1.0% agarose gel stained with ethidium bromide. PCR products were purified by the Nucleospin Gel and PCR Clean up kit (Macherey Nagel, Germany).

Sequencing reactions were performed by Macrogen Europe (Amsterdam, The Nederlands). The full-length mtDNA sequence was assembled using EMBOSS Merger142.

Sequence analysis

Nucleotide sequence analysis was performed using the programs BLASTn (www.ncbi.nlm.nih.gov) and ClustalOmega (www.ebi.ac.uk). The regions of the protein-coding, rRNA, and tRNA genes were initially identified by comparison with the corresponding known sequences of C. capitatα (Supplementary Table S1). Furthermore, the 13 mitochondrial protein-coding genes were defined by the presence of initiation and stop codons whereas the 22 tRNAs were checked for their capability to fold into cloverleaf secondary structures and the presence of specific anticodons by tRNAscan-SE (http://lowelab.ucsc.edu/tRNAscan-SE/)143 and MITOS (http://mitos.bioinf.uni-leipzig.de/index.py)144. Repeats in the control region were found by the “Tandem Repeat Finder” program (http://tandem.bu.edu/trf/trf.html)145.

Phylogenetic analysis based on the concatenated protein coding gene sequences from available Tephritidae mitogenomes (one for each species) (Supplementary Table S1) was performed using MEGA 7.0146. Multiple sequence alignment (11,242 positions total length) was constructed by ClustalW using default parameters. Phylogenetic trees were inferred by the Maximum Likelihood (ML) method based on the General Time Reversible (GTR) model with 1000 bootstrap replicates.

Data Availability

All data generated or analysed during this study are included in this published article (and its Supplementary Information files).