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The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), is among the most destructive and cosmopolitan species of the family. It has been studied extensively and has become a model species for sterile insect technique (SIT) applications. More than 3 decades of coordinated research led to the development and continuous improvement of genetic sexing strains (GSSs) for this pest. The last generation of these strains (Vienna 7 and Vienna 8), developed at the FAO/IAEA Insect Pest Control Laboratory (IPCL) in Seibersdorf, Austria, presents a combination of characters that calls for specialized control and evaluation methods, especially in mass-rearing conditions. In this paper, we present the results of two small-scale laboratory tests that can evaluate the stability, productivity, and other properties of these strains and have been used in IPCL since their development. These tests were used for the analysis of the Vienna 7 and Vienna 8 GSSs after their re-introduction to the IPCL from the different rearing facilities. Our data show the robustness and stability of the ‘sexing’ character of these strains but also point to differences among them regarding egg hatch, pupation, adult emergence rates, and male tolerance to elevated temperatures. Well-defined, small-scale protocols allow the continuous monitoring of the properties of putative important strains; they can be used as standard operating protocols to enable unbiased comparison among strains available in different mass-rearing facilities.

Wolbachia are obligatory intracellular and maternally inherited bacteria that infect and spread through natural arthropod populations by inducing male-killing, feminization, parthenogenesis, and, most commonly, unidirectional and bidirectional cytoplasmic incompatibility (CI). Cytoplasmic incompatibility can be used to control natural populations of insect pests, in a way analogous to the Sterile Insect Technique (SIT), namely through the Incompatible Insect Technique (IIT). For the successful application of the IIT (based on a unidirectional CI approach) against a target species, it is essential that only males are released, as the release of females would lead to fertile matings between the released males and the released females and the establishment of a Wolbachia-carrying field population. In the present study, we describe a Wolbachia-infected line of the VIENNA 8 genetic sexing strain of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), that carries the selectable marker temperature sensitive lethal (tsl). We show that (1) transferred Wolbachia induce high levels of CI even after the temperature treatment required for the male-only production, and (2) the Wolbachia-infected genetic sexing C. capitata line can be used in cage population suppression experiments analogous to the SIT. We also discuss our results in a comparison between IIT and SIT, investigating whether irradiation and cytoplasmic factors can be combined toward the development of novel strategies for insect pest control.

A dominant mutation, Sergeant (Sr2), which affects expression of a third stripe on the abdomen of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), was recently isolated and the results of an extended genetic analysis are reported here. In addition, field cage data are presented on the mating competitiveness of strains carrying this mutation, including a genetic sexing strain (GSS). The incorporation of a phenotypic marker, such as Sr2, in a Mediterranean fruit fly GSS would complement, and could replace, the use of fluorescent dye for monitoring sterile insect technique programs and increase program efficiency by improving sterile and wild fly identification procedures. The mutation is homozygous lethal, and it is located on chromosome 5 at position 78B and 60D on the salivary and trichogen polytene maps, respectively. The development of the Sr2 GSS is described here, together with data on fitness and penetrance of the mutation. The field-cage results demonstrated 1) random mating bet...

The sterile insect technique (SIT) is an environmentally safe technology to control insect pests. To improve this technology, genetic sexing strains (GSS) have been developed for the Mediterranean fruit fly, Ceratitis capitata. Such strains are based on Y-autosome translocations linking a selectable marker to the male sex and their long-term stability, especially under large-scale mass rearing conditions, is threatened by genetic recombination in the heterozygous males. We have measured male recombination in order to be able to construct GSS that are more stable. Our results show that male recombination occurs at very low frequencies, that is, below 1% per generation. Furthermore, recombination in medfly males occurs premeiotically. By selecting strains where the Y-autosome translocation breakpoint and the selectable marker are closely linked, the deleterious effects of recombination on the stability of GSS can be minimized. In such strains recombination is reduced by ca. 80% as compared to previously studied GSS. Although recombinants still occur at very low frequencies they still pose a threat to the integrity of the sexing system if they possess a selective advantage. Under mass rearing condition such recombinants will accumulate according to their relative fitness and additional measures, such as improved mass rearing strategies, are required to preserve the accuracy of the sexing system. As a conclusion it is shown that current GSS are stable enough to allow mass rearing at levels exceeding 1000 million male medflies per week.

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.

Effects of gamma radiation on the egg and larval stages of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), were examined. Eggs and larvae were exposed at different ages to a series of gamma radiation doses ranging from 5 to 1,280 Gy. Eggs in the 1st half of their development (1 and 24 h) were very sensitive to irradiation treatment (20 Gy prevented egg hatch). However, mature eggs (48 h old) were much more tolerant. When mature eggs were irradiated, a dose of 640 Gy prevented egg hatch. Pupariation of eggs treated at 1 and 24 h was significantly affected at a dose of 20 Gy; adult emergence of eggs at 1, 14, and 48 h was prevented at this dose. The larval stage was significantly more resistant to radiation than the egg stage. Survival to the pupal stage increased with increasing age of larvae, and decreased with increasing dose. The minimum dose required to prevent pupariation ranged from over 160 Gy for the 1st instar to > 640 Gy for mature 3rd instars. In contrast, doses required to prevent adult emergence from irradiated larvae were relatively low and ranged from 10-20 to 20-40 Gy depending on the age of the insects when irradiated. Tests in which > 100,000 mature larvae were treated in air with a dose of 40 Gy resulted in no adult emergence. Similar results were obtained when 3rd instars were irradiated inside natural host fruits in a small scale laboratory experiment.

The effect of gamma radiation on the phenoloxidase activity in larvae of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), was examined. Phenoloxidase activity was determined by spectrophotometrically measuring the increase in optical density at 490 nm because of the conversion of L-Dopa to Dopachrome, or by observing the degree of melanization in larvae killed by freezing. Both methods produced comparable results. In unir-radiated larvae, phenoloxidase activity can be detected on day 4 (late 2nd instar), and the activity continues to accumulate through day 7 (late 3rd instar). This accumulation was not observed when larvae were irradiated with a minimum dose of 40 Gy before the 3rd instar. However, irradiating larvae in which enzyme activity was already high (days 5 and 6) did not remove activity but only reduced the rate of further accumulation. These results indicate that measuring the activity of this enzyme in C. capitata larvae is a good, sensitive, and reliable indicator of irradiation treatment provided that the limits mentioned above are observed.

The Mediterranean fruit fly Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is one of the most important pest of fruits and vegetables in tropical and subtropical countries. The sterile insect technique (SIT) as a component of area-wide integrated pest management (AW-IPM) approaches is being used for the successful management of this pest. VIENNA 8 is a genetic sexing strain (GSS) that has a white pupae (wp) and temperature sensitive lethal (tsl) mutation, the latter killing all female embryos when eggs are exposed to high temperatures (34°C). The use of this GSS permits production and the release of only males which has increased the cost effectiveness of the SIT several fold for this pest. An efficient method of identification of recaptured sterile males can further increase the cost effectiveness of the SIT for this pest. Therefore, VIENNA 8-Sergeant2 (Sr2) strain and the transgenic strain VIENNA 8-1260 having visible markers were constructed. All three strains were evaluated for egg production, egg hatch, and egg sterility parameters under semi mass-rearing conditions and mating competitiveness in field cages. VIENNA 8-1260 females produced significantly fewer eggs as compared with the two other strains, which produced similar numbers of eggs. However, egg hatch of all strains was similar. Egg hatch of eggs produced by untreated females that had mated with adult males that had been irradiated with 100 Gy as pupae 2 days before emergence, was different for the three strains, i.e., egg hatch of 0.63%, 0.77%, 0.89% for VIENNA 8, VIENNA 8-1260, and VIENNA 8-Sr2, respectively. Differences in male mating competitiveness of the three strains against wild-type males were gradually reduced with successive generations under semi mass-rearing conditions. However, VIENNA 8 males adapted faster to laboratory conditions as compared with VIENNA 8-Sr2 and VIENNA 8-1260 males with respect to mating competitiveness. VIENNA 8 males of the F10 generation were equally competitive with wild-type males, whereas the mating competitiveness of VIENNA 8-Sr2 and VIENNA 8-1260 males was similar but lower as compared with wild-type males. Males from all three strains copulated earlier than wild-type males. Results are discussed in relation with the potential benefits of incorporating novel strains for more effective SIT application.

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.

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.

In the medfly Ceratitis capitata, the Y chromosome is responsible for determining the male sex. We have mapped the region containing the relevant factor through the analysis of Y-autosome translocations using fluorescence in situ hybridization with two different probes. One probe, the clone pY114, contains repetitive, Y-specific DNA sequences from C. capitata, while the second clone, pDh2-H8, consists of ribosomal DNA sequences from Drosophila hydei. Clone pY114 labeled most of the long arm and pDh2-H8 hybridizes to the short arm and the centromeric region of the long arm. In 12 of the analyzed 19 Y-autosome translocation strains, adjacent-1 segregation products survive to the late pupal or even adult stage and can, therefore, be sexed. This was correlated with the length of the Y fragment still present in these aberrant individuals and allowed us to map the male-determining factor to a region of the long arm representing ~15% of the entire Y chromosome. No additional factors, affecting for example fertility, were detected outside the male-determining region.

The sex chromosomes of the tephritid fruit fly Ceratitis capitata (Wiedemann) are heteromorphic. The male-determining region was located on the Y chromosome by deletion mapping using unbalanced offspring from several translocation strains. In addition, we showed that only 15% of the Y chromosome is required for male determination and male fertility. Based on this result, we expected to find Y-chromosomal length polymorphism in natural populations. Using fluorescence in situ hybridization with two repetitive DNA probes that label the Y chromosome, no obvious size differences were detected in seven wild-type strains and three mutant strains. As the medfly is probably of East African origin, we also analyzed two wild-type strains established recently from pupae sampled in Kenya. The Y chromosomes show a polymorphism in the hybridization pattern of a repetitive Y-specific medfly clone. However, the overall size of the Y chromosome is similar to that of the other strains. Besides C. capitata, the tephritid fruit flies Ceratitis (Pterandrus) rosa Karsch and Trirhithrum coffeae Bezzi also emerged from pupae sampled in Kenya. Their karyotype was analyzed by C-banding. Furthermore, the ribosomal genes were mapped to the sex chromosomes in these two species. Key words : Ceratitis capitata, Tephritidae, C-Banding, FISH, rDNA.

A cytogenetic map of chromosome 5 from the Mediterranean fruit fly, Ceratitis capitata, was constructed. Six mutations were located by translocation, transposition or deletion mapping. This knowledge allowed alignment and orientation of the existing linkage map with the polytene chromosomes. In addition, mapping of mutations used as selectable markers in genetic sex-separation strains is an essential prerequisite for the improvement of genetic stability during mass-rearing.

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-α) 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.