Genetic and cytogenetic characterization of genetic sexing strains of Bactrocera dorsalis and Bactrocera cucurbitae (Diptera: Tephritidae).

In the current study, we performed genetic and cytogenetic analyses of two genetic sexing strains (GSSs), one for Bactrocera dorsalis s.s. and one for melon fly, Bactrocera cucurbitae Coquillett, the first such strains ever constructed in these species. In both strains, the genetic sexing mechanism is based on a pupal color dimorphism (white or brown) and is the result of a reciprocal translocation between the Y chromosome and the autosome bearing the white pupae (wp) locus. Based on genetic analysis and cytological data on mitotic metaphases and larval salivary gland polytene chromosomes, we succeeded in mapping the autosome breakpoints in the two Y-autosome translocations even though the Y chromosome is not visible in polytene nuclei. We show that polytene chromosomes can be used in cytogenetic analyses toward the development of genetic control methods in these pest species. The results of the genetic analysis are in full agreement with the cytological description of the strains.

Mitotic and polytene chromosome analysis in the Mexican fruit fly, Anastrepha ludens (Loew) (Diptera: Tephritidae).

The present study constitutes the first attempt to construct a polytene chromosome map of an Anastrepha species, Anastrepha ludens (Loew), a major agricultural pest. The mitotic karyotype has a diploid complement of 12 acrocentric chromosomes, including five pairs of autosomes and an XX/XY sex chromosome pair. The analysis of salivary gland polytene chromosomes has shown a total number of five polytene elements that correspond to the five autosomes. The characteristic features and the most prominent landmarks of each chromosome are described. By comparing chromosome banding patterns, the possible chromosomal homology between A. ludens and Ceratitis capitata (Wiedemann) is presented. This work shows that polytene maps of A. ludens are suitable for cytogenetic studies in this species and may be used as reference for other Anastrepha species, most of which are also serious agricultural pests.

The hsp27 gene of the Mediterranean fruit fly, Ceratitis capitata: structural characterization, regulation and developmental expression.

In the present study, a genomic DNA clone encoding the medfly homolog of Drosophila melanogaster hsp27 gene, named Cchsp27, was isolated. We sequenced a part of the clone containing the coding region, the 5' untranslated region and approximately 2.8 Kb of the 5' flanking region of the gene. Phylogenetic analysis of several insect small heat shock proteins, suggested that CcHsp27 is orthologous to Drosophila Hsp27 and Sarcophaga crassipalpis Hsp25. The Cchsp27 gene was mapped at the 81A division of the sixth chromosome which coincides with one of the major heat shock puffs of medfly. Structural analysis of the 5' flanking region of the Cchsp27 gene revealed the presence of five putative heat shock elements and one putative ecdysone response element. In addition to heat induction, the Cchsp27 gene was expressed at several stages of normal medfly development. In general, the developmental expression pattern of the Cchsp27 gene was similar to the respective pattern of Drosophila hsp27 gene. However, there were some important differences in certain developmental stages suggesting differential regulation of the hsp27 gene in the two dipterans species. Salivary gland culture experiments showed that the Cchsp27 gene is regulated by 20-hydroxyecdysone.

Polytene chromosome maps in four species of tsetse flies Glossina austeni, G. pallidipes, G. morsitans morsitans and G. m. submorsitans (Diptera: Glossinidae): a comparative analysis.

Photographic polytene chromosome maps from pupal trichogen cells of four tsetse species, Glossina austeni, G. pallidipes, G. morsitans morsitans and G. m. submorsitans were constructed and compared. The homology of chromosomal elements between the species was achieved by comparing banding patterns. The telomeric and subtelomeric chromosome regions were found to be identical in all species. The pericentromeric regions were found to be similar in the X chromosome and the left arm of L1 chromosome (L1L) but different in L2 chromosome and the right arm of L1 chromosome (L1R). The L2 chromosome differs by a pericentric inversion that is fixed in the three species, G. pallidipes, G. morsitans morsitans and G. m. submorsitans. Moreover, the two morsitans subspecies appeared to be homosequential and differ only by two paracentric inversions on XL and L2L arm. Although a degree of similarity was observed across the homologous chromosomes in the four species, the relative position of specific chromosome regions was different due to chromosome inversions established during their phylogeny. However, there are regions that show no apparent homology between the species, an observation that may be attributed to the considerable intra--chromosomal rearrangements that have occurred following the species divergence. The results of this comparative analysis support the current phylogenetic relationships of the genus Glossina.

Evaluation of the activities of the medfly and Drosophila hsp70 promoters in vivo in germ-line transformed medflies.

The promoter of the hsp70 gene of Drosophila melanogaster has been widely used for the expression of foreign genes in other insects. It has been generally assumed that because this gene is highly conserved, its promoter will function efficiently in other species. We report the results of a quantitative comparison of the activities of the medfly and D. melanogaster hsp70 promoters in vivo in transformed medflies. We constructed transformed lines containing the lacZ reporter gene under the control of the two promoters by using Minos-mediated germ-line transformation. The activity of each promoter was evaluated in 15 transformed lines by beta-galactosidase quantitative assays. The heat-inducible activity of the medfly promoter was found several times higher than the respective activity of the heterologous D. melanogaster promoter. These results were confirmed by northern blot analysis and indicate that the D. melanogaster promoter does not work efficiently in medfly. The -263/+105 medfly promoter region that was used in this study was found able to drive heat shock expression of the lacZ reporter gene in all stages of medfly, except early embryonic stages, in a similar fashion to the endogenous hsp70 genes. However the heat inducible RNA levels driven from this promoter region were significantly lower than the endogenous hsp70 RNA levels, suggesting that additional upstream and/or downstream sequences to the -263/+105 region may be necessary for optimum function of the medfly hsp70 promoter in vivo.

Microsatellite analysis of olive fly populations in the Mediterranean indicates a westward expansion of the species.

Bactrocera oleae is the major insect pest of the olive fruit. Twelve microsatellite loci isolated from the genome of this insect were used in a Mediterranean-wide population analysis. These loci were highly polymorphic with a mean number of alleles per locus of 10.42 and a mean effective number of alleles of 2.76. The analysis was performed on a sample of 671 flies collected from nineteen locations around the European part of the Mediterranean basin. Despite the high level of gene flow across the Mediterranean, results support the notion of a differentiation of three subpopulations: one of the Iberian Peninsula, one of Greece and Italy and one of Cyprus. In addition, the gradual decrease of heterozygosity from the Eastern to the Western part of the Mediterranean indicates a westward expansion of the species.

Developmental profiles and ecdysone regulation of the mRNAs for two ecdysone receptor isoforms in the Mediterranean fruit fly Ceratitis capitata.

Using 5' RACE with specific primers for the ecdysone receptor B1 isoform of the Mediterranean fruit fly (medfly), Ceratitis capitata, we isolated a cDNA clone encoding the specific region of the medfly ecdysone receptor A isoform (CcEcR-A). The CcEcR-A-specific region was very similar to the EcR-A-specific region of Drosophila melanogaster and less similar to the EcR-A-specific regions of Lepidoptera. The developmental expression of both CcEcR-A and CcEcR-B1 mRNAs was studied in whole animals, salivary glands and ovaries by RT-PCR, using isoform-specific primers. Both CcEcR mRNAs are present in very early embryos, decrease to very low levels during the first hours of embryogenesis and are highly expressed in all consequent embryonic stages. During metamorphosis both isoforms are present showing two peaks; the first at the larval-prepupal transition and the second during the second half of prepupal development. These peaks are correlated with the two puffing cycles and the two major 20-hydroxyecdysone (20E) increases that occur during medfly metamorphosis. CcEcR-B1 mRNA was directly induced in larval salivary glands in vitro by 20E, even at very low concentrations of the hormone, while CcEcR-A mRNA was slightly induced only by high 20E concentrations and in the absence of a protein synthesis inhibitor. During oogenesis, the CcEcR mRNAs were expressed synchronously, peaking at the beginning of both previtellogenic and vitellogenic phases.

Photographic polytene chromosome maps for Glossina morsitans submorsitans (Diptera: Glossinidae): cytogenetic analysis of a colony with sex-ratio distortion.

Photographic polytene chromosome maps from trichogen cells of pharate adult Glossina morsitans submorsitans were constructed. Using the standard system employed to map polytene chromosomes of Drosophila, the characteristic landmarks were described for the X chromosome and the two autosomes (L1 and L2). Sex-ratio distortion, which is expressed in male G. m. submorsitans, was found to be associated with an X chromosome (X8) that contains three inversions in each arm. Preliminary data indicate no differences in the fecundity of X(A)X(A) and X(A)X(B) females, but there are indications that G. m. submorsitans in colonies originating from Burkina Faso and Nigeria have genes on the autosomes and (or) the Y chromosome that suppress expression of sex-ratio distortion.

Identification and partial characterization of a new Ceratitis capitata-specific 44-bp pericentromeric repeat.

Tandem satellite DNA repeats are often associated with centromeres. In spite of their importance in the organization of the centromere, they do not seem to be broadly conserved among species and their role is still unclear. Here we report the identification of a new 44-bp tandem pericentromeric repeat from the medfly, Ceratitis capitata. The repeat is specific to this insect and is not found in any of the other closely related species tested. It localizes in four out of its five autosomes and in the X chromosome. It is organized in long arrays, interspersed by transposable elements and other less well-defined sequence motifs.

Ecdysone-regulated chromosome puffing in the salivary glands of the Mediterranean fruit fly, Ceratitis capitata.

The effect of ecdysone on the puffing activity of the polytene chromosomes of Ceratitis capitata has been studied in organ cultures of late-larval salivary glands. Culture of glands from 120-h-old larvae (puff stage 1) in the presence of ecdysone resulted in the initiation of the late-larval puffing cycle that is normally observed in 145-h-old larvae (puff stage 4). During a 7-h period in the presence of ecdysone, the puffing patterns of most loci resembled the in vivo patterns observed in the period between puff stages 4 and 10, indicating that the first puffing cycle can be initiated by the hormone and proceed almost to completion, in vitro. Culture of salivary glands in the presence of ecdysone and a protein-synthesis inhibitor, as well as ecdysone withdrawal and readdition experiments, indicated that most of the ecdysone-regulated puffs could be categorized into three classes: (i) the puffs that were suppressed immediately by ecdysone, even in the absence of protein synthesis; (ii) the puffs that were induced directly by ecdysone; and (iii) the puffs that were induced indirectly by ecdysone, that is, they were induced after a lag period of a few hours and required protein synthesis for their induction.

Hermes-mediated germ-line transformation of the Mediterranean fruit fly Ceratitis capitata.

We report the use of the Hermes transposable element for germ-line transformation of the Mediterranean fruit fly, Ceratitis capitata. Hermes was able to genetically transform this insect at an estimated frequency between 0.6 and 1.1%, which is comparable to the transformation frequencies obtained for this species when using other transposable elements. Hermes integrates into the medfly genome by a cut-and-paste mechanism and the sequences integrated into the genome are delimited by the terminal nucleotides of the Hermes inverted terminal repeats. Integration resulted in the generation of 8 bp target site duplications, the sequences of which conformed to the target site duplications generated by hAT element transposition in insects. The Hermes element is one additional genetic tool that can be deployed in manipulating and characterizing the medfly genome.

The construction of the first balancer chromosome for the Mediterranean fruit fly, Ceratitis capitata.

The construction of the first balancer chromosome, FiM1, for the medfly Ceratitis capitata is described. This chromosome has three overlapping pericentric inversions and is marked with dominant and recessive mutations. The inversion breakpoints of FiM1 suppress recombination throughout the length of the fifth chromosome, allowing lethal mutations to be recovered and maintained. This chromosome will provide a powerful tool for the manipulation of laboratory stocks, in particular, the recovery of new mutant and transgenic strains. We demonstrate the use of FiM1 for the recovery and maintenance of chromosomes carrying lethal mutations.

Ceratotoxins: female-specific X-linked genes from the medfly, Ceratitis capitata.

In this paper, we report the chromosomal localization of ceratotoxins, a gene family encoding antibacterial female-specific peptides from the mediterranean fruit fly Ceratitis capitata. The analysis of both polytene and mitotic chromosomes by in situ hybridization shows that ceratotoxins are the first case of female-specific X-linked genes from the medfly C. capitata. Southern blot analysis reveals that the ceratotoxin gene family is not specifically amplified in the female reproductive accessory glands of C. capitata.

Cloning and characterization of CcEcR. An ecdysone receptor homolog from the mediterranean fruit fly ceratitis capitata.

In order to understand the role that 20-hydroxyecdysone plays during development of the Mediterranean fruit fly Ceratitis capitata (medfly), a major agricultural pest, we have cloned a Ceratitis ecdysone receptor (CcEcR) and studied its expression and its binding properties to an ecdysone response element. Using the conserved DNA binding region of the Drosophila melanogaster ecdysone receptor (DmEcR) B1 cDNA as a probe, we isolated a medfly cDNA clone containing the coding region, a part of the 5'-untranslated region and the complete 3'-untranslated region of a CcEcR. The deduced CcEcR polypeptide contained all five domains typical of a nuclear receptor. Alignment comparisons and phylogenetic analyses indicated that CcEcR most closely resembled the B1 isoform of DmEcR and Lucilia cuprina EcR homolog (LcEcR) relative to all other known ecdysone receptors. In situ hybridization analysis showed that the CcEcR gene is mapped in the region 53B of the 4R chromosome arm, while Northern hybridization analysis showed that CcEcR transcripts have a size of approximately 8 kb. Significant levels of CcEcR transcripts were detected in eggs, middle and late embryos, late third instar larvae and middle prepupae. The levels of the CcEcR transcripts during the other larval stages as well as during pupal and adult stages were much lower, while during the early stages of embryogenesis were very low. Electrophoretic mobility shift assays indicated that CcEcR binds specifically to the Drosophila hsp27 ecdysone response element as a heterodimer with Drosophila USP, the product of the ultraspiracle gene. Our structural and biochemical data suggest that CcEcR is the functional homolog of the B1 isoform of DmEcR.

Mitotic and polytene chromosome analyses in the Queensland fruit fly, Bactrocera tryoni (Diptera: Tephritidae).

The Queensland fruit fly, Bactrocera tryoni, like the Mediterranean fruit fly, Ceratitis capitata, has a diploid complement of 12 chromosomes, including five pairs of autosomes and a XX/XY sex chromosome pair. Characteristic features of each chromosome are described. Chromosomal homology between B. tryoni and C. capitata has been determined by comparing chromosome banding pattern and in situ hybridisation of cloned genes to polytene chromosomes. Although the evidence indicates that a number of chromosomal inversions have occurred since the separation of the two species, synteny of the chromosomes appears to have been maintained.

Cloning and characterization of a cDNA encoding a male-specific serum protein of the Mediterranean fruit fly, Ceratitis capitata, with sequence similarity to odourant-binding proteins.

Male-specific serum proteins (MSSPs) are low molecular weight proteins which accumulate in high amounts in the haemolymph of adult males of the medfly Ceratitis capitata. By screening an expression library with anti-MSSP antibodies, we have isolated and determined the nucleotide sequence of a cDNA clone coding for one of the male-specific polypeptides (MSSP-alpha). The MSSP-alpha mRNA encodes a polypeptide of 144 amino acids with a secretory signal sequence of sixteen amino acids. Southern analysis indicated that there are multiple copies of MSSP genes in the medfly genome. Northern analysis showed that the MSSP mRNAs are synthesized only in adult males. The accumulation pattern of these mRNAs during development suggests that the expression of the MSSP genes is developmentally regulated at both transcriptional and translational levels. The predicted peptide sequence of MSSP-alpha shows significant similarity to a group of pheromone- and general odourant-binding proteins of insects.

The heat shock 70 gene family in the Mediterranean fruit fly Ceratitis capitata.

The cloning and the characterization of the heat shock 70 (hsp70) genes of the medfly C. capitata, a major agricultural pest, are presented. Six genomic clones were isolated by screening a medfly genomic library with an hsp70 genomic fragment of Drosophila melanogaster. They form two 30 kb contigs, both of which map cytogenetically in a single major heat shock puff (3L:24C) of the salivary gland polytene chromosomes. Restriction mapping and blot hybridization indicated the presence of six putative hsp70 genes in these two closely linked regions. The sequence of one of these genes suggests that it is a heat-inducible hsp70 gene. The 638-codon open reading frame shows 84% identity at the amino acid level (73.5% at the nucleotide level), relative to corresponding D. melanogaster sequences. The 5' untranslated leader sequence, approximately 200 bp long, is not interrupted by introns and is very rich (48%) in adenine residues, resembling Drosophila heat-inducible hsp70 genes. Furthermore, the promoter of this gene contains two characteristic heat shock elements close upstream from the TATA box. The levels of the hsp70 transcripts are very low at 25-30 degrees C, increase significantly at 33 degrees C and reach maximum at 39 degrees C.

Recovery of a marked translocation strain that will facilitate the isolation of balancer chromosomes in the Mediterranean fruit fly, Ceratitis capitata.

The results of two screens for mutations and chromosomal aberrations in Ceratitis capitata are presented. Three dominant mutations were recovered, including Sb, which is associated with a homozygous lethal translocation between the third and fifth chromosomes, T(3;5)Sb, with the fifth chromosome breakpoint adjacent to y. The T(3;5)Sb chromosome is maintained by selecting for Sb in a T(3;5)Sb, w2 Sb y2 wp/w2 y2 wp stock and can be used to distinguish between other chromosomes carrying differential combinations of the recessive markers w2 y2 wp. The ability to isolate particular marked chromosomes is essential in order to recover an inversion-based balancer chromosome. In addition to the recovery of dominant mutations, gamma-ray induced somatic mosaics of w2 and y2 and zygotic w mosaics were found. The generation of zygotic mosaics following mutagenesis can give mutants with a mosaic germ line that fail to breed true in the first generation. A screen of 22,830 irradiated chromosomes failed to recover variegating alleles of w, although such alleles might be recovered in a larger screen. The high frequency of dominant mutations and the instability at the w locus in our stocks implies a background level of dysgenic activity. These results have implications for the construction and long-term maintenance of genetically modified strains.

The white gene of Ceratitis capitata: a phenotypic marker for germline transformation.

Reliable germline transformation is required for molecular studies and ultimately for genetic control of economically important insects, such as the Mediterranean fruit fly (medfly) Ceratitis capitata. A prerequisite for the establishment and maintenance of transformant lines is selectable or phenotypically dominant markers. To this end, a complementary DNA clone derived from the medfly white gene was isolated, which showed substantial similarity to white genes in Drosophila melanogaster and other Diptera. It is correlated with a spontaneous mutation causing white eyes in the medfly and can be used to restore partial eye color in transgenic Drosophila carrying a null mutation in the endogenous white gene.