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

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.

A Novel Genetic Sexing Strain of Anastrepha ludens for Cost-Effective Sterile Insect Technique Applications: Improved Genetic Stability and Rearing Efficiency.

Anastrepha ludens (Loew) is one of the most destructive insect pests damaging several fruits of economic importance. The sterile insect technique (SIT) is used under an area-wide integrated pest management approach, to suppress these pest populations. Mass rearing facilities were initially established to produce sterile males of bi-sexual strains in support of SIT. The first genetic sexing strain (GSS) for A. ludens, Tapachula-7, based on pupal color dimorphism, was a key development since the release of males-only significantly increases the SIT efficiency. In this study, we document the development of a novel pupal color-based GSS. Twelve radiation-induced translocation lines were assessed as potential GSS in terms of recombination rates and rearing efficiency at a small scale. The best one, GUA10, was cytogenetically characterized: it was shown to carry a single translocation between the Y chromosome and chromosome 2, which is known to carry the black pupae marker. This GSS was further evaluated at medium and large scales regarding its genetic stability, productivity and quality versus Tapachula-7. GUA10 presented better genetic stability, fecundity, fertility, production efficiency, flying ability, and male mating, clear indicators that GUA10 GSS can significantly improve the efficacy and cost-effectiveness of SIT applications against this pest species.

Development and characterization of a pupal-colour based genetic sexing strain of Anastrepha fraterculus sp. 1 (Diptera: Tephritidae).

BACKGROUND: Area-wide integrated pest management programs (AW-IPM) incorporating sterile insect technique (SIT) have been successful in suppressing populations of different fruit fly species during the last six decades. In addition, the development of genetic sexing strains (GSS) for different fruit fly species has allowed for sterile male-only releases and has significantly improved the efficacy and cost effectiveness of the SIT applications. The South American Fruit Fly Anastrepha fraterculus (Diptera: Tephritidae) is a major agricultural pest attacking several fruit commodities. This impedes international trade and has a significant negative impact on the local economies. Given the importance of sterile male-only releases, the development of a GSS for A. fraterculus would facilitate the implementation of an efficient and cost-effective SIT operational program against this insect pest species. RESULTS: For potential use in a GSS, three new morphological markers (mutants) were isolated in a laboratory strain of A. fraterculus sp. 1, including the black pupae (bp) gene located on chromosome VI. The black pupa phenotype was used as a selectable marker to develop genetic sexing strains by linking the wild type allele (bp+) to the Y-chromosome -via irradiation to induce a reciprocal Y-autosome translocation. Four GSS were established and one of them, namely GSS-89, showed the best genetic stability and the highest fertility. This strain was selected for further characterization and cytogenetic analysis. CONCLUSIONS: We herein report the development of the first genetic sexing strain of a major agricultural pest, A. fraterculus sp. 1, using as a selectable marker the black pupae genetic locus.

Τhe Complete Mitochondrial Genome of Bactrocera carambolae (Diptera: Tephritidae): Genome Description and Phylogenetic Implications.

Bactrocera carambolae is one of the approximately 100 sibling species of the Bactrocera dorsalis complex and considered to be very closely related to B. dorsalis. Due to their high morphological similarity and overlapping distribution, as well as to their economic impact and quarantine status, the development of reliable markers for species delimitation between the two taxa is of great importance. Here we present the complete mitochondrial genome of B. carambolae sourced from its native range in Malaysia and its invaded territory in Suriname. The mitogenome of B. carambolae presents the typical organization of an insect mitochondrion. Comparisons of the analyzed B. carambolae sequences to all available complete mitochondrial sequences of B. dorsalis revealed several species-specific polymorphic sites. Phylogenetic analysis based on Bactrocera mitogenomes supports that B. carambolae is a differentiated taxon though closely related to B. dorsalis. The present complete mitochondrial sequences of B. carambolae could be used, in the frame of Integrative Taxonomy, for species discrimination and resolution of the phylogenetic relationships within this taxonomically challenging complex, which would facilitate the application of species-specific population suppression strategies, such as the sterile insect technique.

The chromosomes of Drosophila suzukii (Diptera: Drosophilidae): detailed photographic polytene chromosomal maps and in situ hybridization data.

The spotted wing drosophila, D. suzukii, is a serious agricultural pest attacking a variety of soft fruits and vegetables. Although originating from East Asia it has recently invaded America and Europe raising major concern about its expansion potential and the consequent economic losses. Since cytogenetic information on the species is scarce, we report here the mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of D. suzukii. The mitotic metaphase complement contains three pairs of autosomes, one of which is dot-like, and one pair of heteromorphic (XX/XY) sex chromosomes. The salivary gland polytene complement consists of five long polytene arms, representing the two metacentric autosomes and the acrocentric X chromosome, and one very short polytene element, which corresponds to the dot-like autosome. Banding pattern as well as the most characteristic features and prominent landmarks of each polytene chromosome arm are presented and discussed. Furthermore, twelve gene markers have been mapped on the polytene chromosomes of D. suzukii by in situ hybridization. Their distribution pattern was found quite similar to that of D. melanogaster revealing conservation of synteny although the relative position within each chromosome arm for most of the genes differed significantly between D. suzukii and D. melanogaster. The chromosome information presented here is suitable for comparative cytogenetic studies and phylogenetic exploration, while it could also facilitate the assembly of the genome sequence and support the development of genetic tools for species-specific and environment-friendly biological control applications such as the sterile insect technique.

The chromosomes and the mitogenome of Ceratitis fasciventris (Diptera: Tephritidae): two genetic approaches towards the Ceratitis FAR species complex resolution.

Ceratitis fasciventris is a serious agricultural pest of the Tephritidae family that belongs to the African Ceratitis FAR species complex. Species limits within the FAR complex are obscure and multidisciplinary approaches have attempted to resolve phylogenetic relationships among its members. These studies support the existence of at least three additional species in the complex, C. anonnae, C. rosa and C. quilicii, while they indicate the presence of two structured populations (F1 and F2) within the C. fasciventris species. In the present study we present the mitotic karyotype, polytene chromosome maps, in situ hybridization data and the complete mitochondrial genome sequence of an F2 population of C. fasciventris. This is the first polytene chromosome map and complete mitogenome of a member of the FAR complex and only the second reported for the Ceratitis genus. Both polytene chromosomes and mitochondrial sequence could provide valuable information and be used as reference for comparative analysis among the members of the complex towards the clarification of their phylogenetic relationships.

Cryopreservation of Embryos of the Mediterranean Fruit Fly Ceratitis capitata Vienna 8 Genetic Sexing Strain.

The Mediterranean fruit fly, Ceratitis capitata, is one of the most serious pests of fruit crops world-wide. During the last decades, area-wide pest management (AW-IPM) approaches with a sterile insect technique (SIT) component have been used to control populations of this pest in an effective and environment-friendly manner. The development of genetic sexing strains (GSS), such as the Vienna 8 strain, has been played a major role in increasing the efficacy and reducing the cost of SIT programs. However, mass rearing, extensive inbreeding, possible bottleneck phenomena and hitch-hiking effects might pose major risks for deterioration and loss of important genetic characteristics of domesticated insect. In the present study, we present a modified procedure to cryopreserve the embryos of the medfly Vienna 8 GSS based on vitrification and used this strain as insect model to assess the impact of the cryopreservation process on the genetic structure of the cryopreserved insects. Forty-eight hours old embryos, incubated at 24°C, were found to be the most suitable developmental stage for cryopreservation treatment for high production of acceptable hatch rate (38%). Our data suggest the absence of any negative impact of the cryopreservation process on egg hatch rate, pupation rates, adult emergence rates and stability of the temperature sensitive lethal (tsl) character on two established cryopreserved lines (flies emerged from cryopreserved embryos), named V8-118 and V8-228. Taken together, our study provides an optimized procedure to cryopreserve the medfly Vienna 8 GSS and documents the absence of any negative impact on the genetic structure and quality of the strain. Benefits and sceneries for utilization of this technology to support operational SIT projects are discussed in this paper.

Cytogenetic Analysis of the South American Fruit Fly Anastrepha fraterculus (Diptera:Tephritidae) Species Complex: Construction of Detailed Photographic Polytene Chromosome Maps of the Argentinian Af. sp.1 Member.

Genetic and cytogenetic studies constitute a significant basis for understanding the biology of insect pests and the design and the construction of genetic tools for biological control strategies. Anastrepha fraterculus is an important pest of the Tephritidae family. It is distributed from southern Texas through eastern Mexico, Central America and South America causing significant crop damage and economic losses. Currently it is considered as a species complex; until now seven members have been described based on multidisciplinary approaches. Here we report the cytogenetic analysis of an Argentinian population characterized as Af. sp.1 member of the Anastrepha fraterculus species complex. The mitotic karyotype and the first detailed photographic maps of the salivary gland polytene chromosomes are presented. The mitotic metaphase complement consists of six (6) pairs of chromosomes, including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the salivary gland polytene complement shows a total number of five long chromosomes that correspond to the five autosomes of the mitotic karyotype and a heterochromatic network corresponding to the sex chromosomes. Comparison of the polytene chromosome maps between this species and Anastrepha ludens shows significant similarity. The polytene maps presented here are suitable for cytogenetic studies that could shed light on the species limits within this species complex and support the development of genetic tools for sterile insect technique (SIT) applications.

Achilles, a New Family of Transcriptionally Active Retrotransposons from the Olive Fruit Fly, with Y Chromosome Preferential Distribution.

Sex chromosomes have many unusual features relative to autosomes. The in depth exploration of their structure will improve our understanding of their origin and divergence (degeneration) as well as the evolution of genetic sex determination pathways which, most often are attributed to them. In Tephritids, the structure of Y chromosome, where the male-determining factor M is localized, is largely unexplored and limited data concerning its sequence content and evolution are available. In order to get insight into the structure and organization of the Y chromosome of the major olive insect pest, the olive fly Bactrocera oleae, we characterized sequences from a Pulse Field Gel Electrophoresis (PFGE)-isolated Y chromosome. Here, we report the discovery of the first olive fly LTR retrotransposon with increased presence on the Y chromosome. The element belongs to the BEL-Pao superfamily, however, its sequence comparison with the other members of the superfamily suggests that it constitutes a new family that we termed Achilles. Its ~7.5 kb sequence consists of the 5'LTR, the 5'non-coding sequence and the open reading frame (ORF), which encodes the polyprotein Gag-Pol. In situ hybridization to the B. oleae polytene chromosomes showed that Achilles is distributed in discrete bands dispersed on all five autosomes, in all centromeric regions and in the granular heterochromatic network corresponding to the mitotic sex chromosomes. The between sexes comparison revealed a variation in Achilles copy number, with male flies possessing 5-10 copies more than female (CI range: 18-38 and 12-33 copies respectively per genome). The examination of its transcriptional activity demonstrated the presence of at least one intact active copy in the genome, showing a differential level of expression between sexes as well as during embryonic development. The higher expression was detected in male germline tissues (testes). Moreover, the presence of Achilles-like elements in different species of the Tephritidae family suggests an ancient origin of this element.

Cytogenetic and symbiont analysis of five members of the B. dorsalis complex (Diptera, Tephritidae): no evidence of chromosomal or symbiont-based speciation events.

The Bactrocera dorsalis species complex, currently comprising about 90 entities has received much attention. During the last decades, considerable effort has been devoted to delimiting the species of the complex. This information is of great importance for agriculture and world trade, since the complex harbours several pest species of major economic importance and other species that could evolve into global threats. Speciation in Diptera is usually accompanied by chromosomal rearrangements, particularly inversions that are assumed to reduce/eliminate gene flow. Other candidates currently receiving much attention regarding their possible involvement in speciation are reproductive symbionts, such as Wolbachia, Spiroplasma, Arsenophonus, Rickettsia and Cardinium. Such symbionts tend to spread quickly through natural populations and can cause a variety of phenotypes that promote pre-mating and/or post-mating isolation and, in addition, can affect the biology, physiology, ecology and evolution of their insect hosts in various ways. Considering all these aspects, we present: (a) a summary of the recently gained knowledge on the cytogenetics of five members of the Bactrocera dorsalis complex, namely Bactrocera dorsalis s.s., Bactrocera invadens, Bactrocera philippinensis, Bactrocera papayae and Bactrocera carambolae, supplemented by additional data from a Bactrocera dorsalis s.s. colony from China, as well as by a cytogenetic comparison between the dorsalis complex and the genetically close species, Bactrocera tryoni, and, (b) a reproductive symbiont screening of 18 different colonized populations of these five taxa. Our analysis did not reveal any chromosomal rearrangements that could differentiate among them. Moreover, screening for reproductive symbionts was negative for all colonies derived from different geographic origins and/or hosts. There are many different factors that can lead to speciation, and our data do not support chromosomal and/or symbiotic-based speciation phenomena in the taxa under study.

The Bactrocera dorsalis species complex: comparative cytogenetic analysis in support of Sterile Insect Technique applications.

BACKGROUND: The Bactrocera dorsalis species complex currently harbors approximately 90 different members. The species complex has undergone many revisions in the past decades, and there is still an ongoing debate about the species limits. The availability of a variety of tools and approaches, such as molecular-genomic and cytogenetic analyses, are expected to shed light on the rather complicated issues of species complexes and incipient speciation. The clarification of genetic relationships among the different members of this complex is a prerequisite for the rational application of sterile insect technique (SIT) approaches for population control. RESULTS: Colonies established in the Insect Pest Control Laboratory (IPCL) (Seibersdorf, Vienna), representing five of the main economic important members of the Bactrocera dorsalis complex were cytologically characterized. The taxa under study were B. dorsalis s.s., B. philippinensis, B. papayae, B. invadens and B. carambolae. Mitotic and polytene chromosome analyses did not reveal any chromosomal characteristics that could be used to distinguish between the investigated members of the B. dorsalis complex. Therefore, their polytene chromosomes can be regarded as homosequential with the reference maps of B. dorsalis s.s.. In situ hybridization of six genes further supported the proposed homosequentiallity of the chromosomes of these specific members of the complex. CONCLUSIONS: The present analysis supports that the polytene chromosomes of the five taxa under study are homosequential. Therefore, the use of the available polytene chromosome maps for B. dorsalis s.s. as reference maps for all these five biological entities is proposed. Present data provide important insight in the genetic relationships among the different members of the B. dorsalis complex, and, along with other studies in the field, can facilitate SIT applications targeting this complex. Moreover, the availability of 'universal' reference polytene chromosome maps for members of the complex, along with the documented application of in situ hybridization, can facilitate ongoing and future genome projects in this complex.

Development, genetic and cytogenetic analyses of genetic sexing strains of the Mexican fruit fly, Anastrepha ludens Loew (Diptera: Tephritidae).

BACKGROUND: Anastrepha ludens is among the pests that have a major impact on México's economy because it attacks fruits as citrus and mangoes. The Mexican Federal government uses integrated pest management to control A. ludens through the Programa Nacional Moscas de la Fruta [National Fruit Fly Program, SAGARPA-SENASICA]. One of the main components of this program is the sterile insect technique (SIT), which is used to control field populations of the pest by releasing sterile flies. RESULTS: To increase the efficiency of this technique, we have developed a genetic sexing strain (GSS) in which the sexing mechanism is based on a pupal colour dimorphism (brown-black) and is the result of a reciprocal translocation between the Y chromosome and the autosome bearing the black pupae (bp) locus. Ten strains producing wild-type (brown pupae) males and mutant (black pupae) females were isolated. Subsequent evaluations for several generations were performed in most of these strains. The translocation strain named Tapachula-7 showed minimal effect on survival and the best genetic stability of all ten strains. Genetic and cytogenetic analyses were performed using mitotic and polytene chromosomes and we succeeded to characterize the chromosomal structure of this reciprocal translocation and map the autosome breakpoint, despite the fact that the Y chromosome is not visible in polytene nuclei following standard staining. CONCLUSIONS: We show that mitotic and polytene chromosomes can be used in cytogenetic analyses towards the development of genetic control methods in this pest species. The present work is the first report of the construction of GSS of Anastrepha ludens, with potential use in a future Moscafrut operational program.

Mitotic and polytene chromosomes analysis of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae).

The Oriental fruit fly, Batrocera dorsalis s.s. (Hendel) is one of the most destructive agricultural pests, belonging to a large group of difficult to distinguish morphologically species, referred as the B. dorsalis complex. We report here a cytogenetic analysis of two laboratory strains of the species and provide a photographic polytene chromosome map from larval salivary glands. The mitotic complement consists of six chromosome pairs including a heteromorphic sex (XX/XY) chromosome pair. Analysis of the polytene complement has shown a total of five polytene chromosomes (10 polytene arms) that correspond to the five autosomes. The most important landmarks of each polytene chromosome and characteristic asynapsis at a specific chromosomal region are presented and discussed. Chromosomal homology between B. dorsalis and Ceratitis capitata has been determined by comparing chromosome banding patterns. The detection of chromosome inversions in both B. dorsalis strains is shown and discussed. Our results show that the polytene maps presented here are suitable for cytogenetic analysis of this species and can be used for comparative studies among species of the Tephritidae family. They also provide a diagnostic tool that could accelerate species identification within the B. dorsalis complex and could shed light on the ongoing speciation in this complex. Polytene chromosome maps can facilitate the development of biological control methods and support the genome mapping project of the species that is currently in progress.

Sex and the single embryo: early deveiopment in the Mediterranean fruit fly, Ceratitis capitata.

BACKGROUND: In embryos the maternal-to-zygotic transition (MTZ) integrates post-transcriptional regulation of maternal transcripts with transcriptional activation of the zygotic genome. Although the molecular mechanisms underlying this event are being clarified in Drosophila melanogaster, little is know about the embryogenic processes in other insect species. The recent publication of expressed sequence tags (ESTs) from embryos of the global pest species Ceratitis capitata (medfly) has enabled the investigation of embryogenesis in this species and has allowed a comparison of the embryogenic processes in these two related dipteran species, C. capitata and D. melanogaster, that shared a common ancestor 80-100 mya. RESULTS: Using a novel PCR-based sexing method, which takes advantage of a putative LTR retrotransposon MITE insertion on the medfly Y chromosome, the transcriptomes of individual early male and female embryos were analysed using RT-PCR. This study is focused on two crucial aspects of the onset of embryonic development: sex determination and cellular blastoderm formation. Together with the three known medfly genes (Cctransformer, Cctransformer2 and Ccdoublesex), the expression patterns of other medfly genes that are similar to the D. melanogaster sex-determination genes (sisterlessA, groucho, deadpan, Sex-lethal, female lethal d, sans fille and intersex) and four cellular blastoderm formation genes (Rho1, spaghetti squash, slow-as-molasses and serendipity-alpha) were analyzed, allowing us to sketch a preliminary outline of the embryonic process in the medfly. Furthermore, a putative homologue of the Zelda gene has been considered, which in D. melanogaster encodes a DNA-binding factor responsible for the maternal-to-zygotic transition. CONCLUSIONS: Our novel sexing method facilitates the study of i) when the MTZ transition occurs in males and females of C. capitata, ii) when and how the maternal information of "female-development" is reprogrammed in the embryos and iii) similarities and differences in the regulation of gene expression in C. capitata and D. melanogaster. We suggest a new model for the onset of the sex determination cascade in the medfly: the maternally inherited Cctra transcripts in the female embryos are insufficient to produce enough active protein to inhibit the male mode of Cctra splicing. The slow rate of development and the inefficiency of the splicing mechanism in the pre-cellular blastoderm facilitates the male-determining factor (M) activity, which probably acts by inhibiting CcTRA protein activity.

Isolation and characterization of microsatellite markers from the Mediterranean fruit fly, Ceratitis capitata: cross-species amplification in other Tephritidae species reveals a varying degree of transferability.

The Mediterranean fruit fly, Ceratitis capitata, is a pest of major economic importance and has become a model for the development of SIT control programs for insect pests. Significant information has been accumulated on classical and population genetics of this species during the past 2 decades. However, the availability of molecular markers is limited. Here, we present the isolation and characterization of 159 microsatellite clones and the development of 108 polymorphic microsatellite markers for this insect pest. Mapping by in situ hybridization to polytene chromosomes of 21 microsatellite clones enriched the cytogenetic map that was previously constructed by our group. The enriched map provides a large number of STSs for future genome mapping projects. Cross-species amplification of these microsatellite loci in 12 Tephritidae species and sequence analysis of several amplification products indicated a varying degree of transferability and their possible usefulness as molecular and genetic markers in these species where genetic and molecular tools are limited.

Conditional embryonic lethality to improve the sterile insect technique in Ceratitis capitata (Diptera: Tephritidae).

BACKGROUND: The sterile insect technique (SIT) is an environment-friendly method used in area-wide pest management of the Mediterranean fruit fly Ceratitis capitata (Wiedemann; Diptera: Tephritidae). Ionizing radiation used to generate reproductive sterility in the mass-reared populations before release leads to reduction of competitiveness. RESULTS: Here, we present a first alternative reproductive sterility system for medfly based on transgenic embryonic lethality. This system is dependent on newly isolated medfly promoter/enhancer elements of cellularization-specifically-expressed genes. These elements act differently in expression strength and their ability to drive lethal effector gene activation. Moreover, position effects strongly influence the efficiency of the system. Out of 60 combinations of driver and effector construct integrations, several lines resulted in larval and pupal lethality with one line showing complete embryonic lethality. This line was highly competitive to wildtype medfly in laboratory and field cage tests. CONCLUSION: The high competitiveness of the transgenic lines and the achieved 100% embryonic lethality causing reproductive sterility without the need of irradiation can improve the efficacy of operational medfly SIT programs.

Isolation and characterization of microsatellite markers from the olive fly, Bactrocera oleae, and their cross-species amplification in the Tephritidae family.

BACKGROUND: The Tephritidae family of insects includes the most important agricultural pests of fruits and vegetables, belonging mainly to four genera (Bactrocera, Ceratitis, Anastrepha and Rhagoletis). The olive fruit fly, Bactrocera oleae, is the major pest of the olive fruit. Currently, its control is based on chemical insecticides. Environmentally friendlier methods have been attempted in the past (Sterile Insect Technique), albeit with limited success. This was mainly attributed to the lack of knowledge on the insect's behaviour, ecology and genetic structure of natural populations. The development of molecular markers could facilitate the access in the genome and contribute to the solution of the aforementioned problems. We chose to focus on microsatellite markers due to their abundance in the genome, high degree of polymorphism and easiness of isolation. RESULTS: Fifty-eight microsatellite-containing clones were isolated from the olive fly, Bactrocera oleae, bearing a total of sixty-two discrete microsatellite motifs. Forty-two primer pairs were designed on the unique sequences flanking the microsatellite motif and thirty-one of them amplified a PCR product of the expected size. The level of polymorphism was evaluated against wild and laboratory flies and the majority of the markers (93.5%) proved highly polymorphic. Thirteen of them presented a unique position on the olive fly polytene chromosomes by in situ hybridization, which can serve as anchors to correlate future genetic and cytological maps of the species, as well as entry points to the genome. Cross-species amplification of these markers to eleven Tephritidae species and sequencing of thirty-one of the amplified products revealed a varying degree of conservation that declines outside the Bactrocera genus. CONCLUSION: Microsatellite markers are very powerful tools for genetic and population analyses, particularly in species deprived of any other means of genetic analysis. The presented set of microsatellite markers possesses all features that would render them useful in such analyses. This could also prove helpful for species where SIT is a desired outcome, since the development of effective SIT can be aided by detailed knowledge at the genetic and molecular level. Furthermore, their presented efficacy in several other species of the Tephritidae family not only makes them useful for their analysis but also provides tools for phylogenetic comparisons among them.

cDNA cloning, characterization, and developmental expression of the 20S proteasome alpha5 subunit in the Mediterranean fruit fly Ceratitis capitata.

In the present study, we report the cDNA cloning, characterization, and developmental expression of the 20S proteasome alpha5 subunit from the Mediterranean fruit fly Ceratitis capitata (medfly). Using an RT-PCR fragment that corresponds to the amino-terminal region of the Drosophila melanogaster 20S proteasome alpha5 subunit, we isolated a 987-bp cDNA that encodes the complete coding region of the medfly ortholog, which was named CcPSMA5. CcPSMA5 consists of 241 amino acids and has a predicted molecular weight of 26.4 kDa and pI 4.75. Comparison of the CcPSMA5 amino acid sequence with the sequences of all known 20S proteasome alpha5 subunits from different organisms indicated that the medfly 20S proteasome alpha5 subunit has the strongest homology to that of Drosophila. In situ hybridization showed that the CcPSMA5 gene is mapped in the region 44B of chromosome 4. Northern blot hybridization analysis showed that the CcPSMA5 mRNA has a size of approximately 1.2 kb. High levels of the CcPSMA5 mRNA were detected in freshly laid eggs, indicating that they were maternally deposited. The mRNA expression pattern during medfly development suggests that the CcPSMA5 gene is upregulated before mid-embryogenesis and at the onset of metamorphosis.

Structural characterization of the medfly hsp83 gene and functional analysis of its proximal promoter region in vivo by germ-line transformation.

In order to define the regulatory elements responsible for the expression of the medfly hsp83 (Cchsp83) gene, we determined the sequence of a genomic region of the gene that included 3,536 bp upstream of the transcription initiation site, the first untranslated exon of 144 bp, a 275-bp intron, and 516 bp of the second coding exon. Structural analysis of the 5' flanking region revealed the presence of a typical TATA box, 28 bp upstream of the transcription start site, and seven putative heat shock elements (HSEs) further upstream. The 5' untranslated region of the Cchsp83 mRNA was found to contain extensive secondary structure in the first 126 nucleotides. We carried out deletion functional analysis of the proximal promoter region (-380/+139) in vivo by germ line transformation using the lacZ as a reporter gene. We found that sequences in the -380/-86 region are essential for the constitutive expression of the Cchsp83 gene. Under normal conditions, the -380/+139 region was able to drive significant levels of transgene expression in all developmental stages of the medfly as well as in the ovaries and testis. In most stages, the temporal expression pattern of the reporter gene was similar to the respective pattern of the endogenous Cchsp83 gene. Although the -380/+139 promoter region contained two putative HSEs, it was found unable to confer any heat-induced expression in the reporter gene.

An integrated genetic and cytogenetic map for the Mediterranean fruit fly, Ceratitis capitata, based on microsatellite and morphological markers.

A genetic map based on microsatellite polymorphisms and visible mutations of the Mediterranean fruit fly (medfly), Ceratitis capitata is presented. Genotyping was performed on single flies from several backcross families. The map is composed of 67 microsatellites and 16 visible markers distributed over four linkage groups. Fluorescence in situ hybridization of selected microsatellite markers on salivary gland polytene chromosomes allowed the alignment of these groups to the second, fourth, fifth and sixth chromosome. None of the markers tested showed segregation either with the X or the third chromosome. However, this map constitutes a substantial starting point for a detailed genetic map of C. capitata. The construction of an integrated map covering the whole genome should greatly facilitate genetic studies and future genome sequence projects of the species.