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.

Development of a novel genetic sexing strain of Ceratitis capitata based on an X-autosome translocation.

Genetic sexing strains (GSS), such as the Ceratitis capitata (medfly) VIENNA 8 strain, facilitate male-only releases and improve the efficiency and cost-effectiveness of sterile insect technique (SIT) applications. Laboratory domestication may reduce their genetic diversity and mating behaviour and hence, refreshment with wild genetic material is frequently needed. As wild males do not carry the T(Y;A) translocation, and wild females do not easily conform to artificial oviposition, the genetic refreshment of this GSS is a challenging and time-consuming process. In the present study, we report the development of a novel medfly GSS, which is based on a viable homozygous T(XX;AA) translocation using the same selectable markers, the white pupae and temperature-sensitive lethal genes. This allows the en masse cross of T(XX;AA) females with wild males, and the backcrossing of F1 males with the T(XX;AA) females thus facilitating the re-establishment of the GSS as well as its genetic refreshment. The rearing efficiency and mating competitiveness of the novel GSS are similar to those of the T(Y;A)-based VIENNA 8 GSS. However, its advantage to easily allow the genetic refreshment is of great importance as it can ensure the mass production of high-quality males and enhanced efficacy of operational SIT programs.

Does the Effect of Irradiation Dose Vary Between Flies Selected and Non-selected to Resist Desiccation? The Case of Anastrepha ludens (Diptera: Tephritidae).

The sterile insect technique (SIT), used to control different species of tephritid fruit flies (Diptera: Tephritidae), is an important element in sustainable agriculture because of its low negative impact on the environment. In SIT, flies are mass produced and sterilized in the laboratory and then released in a target area. However, once released, laboratory flies may confront harass environments that would reduce their performance and consequently SIT efficiency. Selecting flies that resist stressful conditions may help to improve the efficiency of the SIT by releasing males that resist desiccation, for example, ensuring, thus, their survival in environments with low relative humidity. However, the selection process may affect the resistance of flies to the stress of sterilization, since some life history traits are affected. Here, we studied the effect of irradiation on Anastrepha ludens (Loew) (Diptera: Tephritidae) desiccation resistant flies (DR) compared with nonselected flies (NS). We measured the effect of gamma irradiation dose (0, 20, 40, 60, and 80 Gy) on sterility (males and females) and quality parameters (emergence, flight ability, survival, and male sexual performance) in A. ludens adults of the DR and NS (control) strains. Our results indicate that irradiation affected equally the sterility of adults of both strains. None of the quality parameters differed between strains. The only difference was that DR flies survived longer than control flies. Thus, flies that are resistant to desiccation can be used in the SIT without altering the current process of irradiation and packing.

Improving the Phenotypic Properties of the Ceratitis capitata (Diptera: Tephritidae) Temperature-Sensitive Lethal Genetic Sexing Strain in Support of Sterile Insect Technique Applications.

The genetic sexing strain (GSS) of the Mediterranean fruit fly (Ceratitis capitata (Wiedemann)) Vienna 8D53- is based on a male-linked translocation system and uses two selectable markers for male-only production, the white pupae (wp) and the temperature sensitivity lethal (tsl) genes. In this GSS, males emerge from brown pupae and are resistant to high temperatures while females emerge from white pupae, are sensitive to high temperatures. However, double homozygous females (wp tsl/wp tsl) exhibit a slower development rate compared to heterozygous males (wp+tsl+/wp tsl) during the larval stage, which was attributed to the pleiotropic effects of the tsl gene. We present the first evidence that this slower development is due to a different gene, here namely slow development (sd), which is closely linked to the tsl gene. Taking advantage of recombination phenomena between the two loci, we report the isolation of a novel temperature sensitivity lethal strain using the wp mutation as a morphological marker, which showed faster development (wp tsl FD) during the larval stage and increased in its temperature sensitivity compared with the normal tsl strain. Moreover, the introgression of this novel wp tsl FD combined trait into the Vienna 8D53- GSS, resulted in a novel Vienna 8D53- FD GSS, where females showed differences in the thermal sensibility, larval development speed, and productivity profiles. The modification of these traits and their impact on the mass rearing of the GSS for sterile insect technique applications are discussed.

Slow Larvae Mutant and Its Potential to Improve the Pupal Color-Based Genetic Sexing System in Mexican Fruit Fly, (Diptera: Tephritidae).

For many years, an area-wide fruit fly control campaign against the Mexican fruit fly, Anastrepha ludens (Loew) has been implemented in some regions of Mexico and Texas, using the sterile insect technique (SIT) as its principal component. To improve the efficiency of the SIT, a genetic sexing strain based on black pupae mutation (bp) was developed for A. ludens, namely, 'Tapachula-7' (Tap-7 genetic sexing strains [GSSs]). This strain was introduced into the AW-IPM program recently and allows male-only releases for SIT applications. Here, we report the genetic and biological characterization of a new mutation, slow larvae (sl), which was introduced to the original translocation of the Tap-7 GSS resulting in two new GSS (slow-7 and Tap/slow-7). In both GSSs, the translocated wild-type males emerge from brown pupae that develop faster than females. The females are homozygous for sl mutation in the slow-7 GSS and homozygous for sl and bp mutations in the Tap/slow-7 GSS, reaching larval maturity 2 d after most of the wild-type males, allowing the separation of most males during pupariation. The potential use of the slow-7 and Tap/slow-7 GSSs in mass rearing and large-scale population suppression programs is discussed.

Comparison of classical and transgenic genetic sexing strains of Mediterranean fruit fly (Diptera: Tephritidae) for application of the sterile insect technique.

The development of genetic sexing strains (GSSs) based on classical genetic approaches has revolutionized the application of the sterile insect technique (SIT) against the Mediterranean fruit fly Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). The global use of Mediterranean fruit fly GSS for SIT applications as part of area-wide integrated pest management (AW-IPM) programmes is testimony to their effectiveness. During recent years, transgenic sexing strains (TSSs) have been developed through genetic engineering techniques offering the possibility to produce male-only progeny by introducing female embryonic lethal genes and to increase the efficacy to identify released sterile males by means of the expression of fluorescent transgene markers. Here, we present a comparative analysis of two Mediterranean fruit fly strains: the classical GSS VIENNA 8D53-/Toliman and the transgenic FSEL#32. The strains were compared for production efficiency and quality control indices under semi mass-rearing conditions, response to sterilizing irradiation doses, male mating performance in walk-in field cages, and production cost of male-only pupae. The results showed that, the FSEL #32 TSS had a similar fecundity but a higher production of male-only pupae than the VIENNA 8D53-/Toliman GSS. For some of the quality control parameters tested, such as pupal weight and survival under starvation conditions, the FSEL #32 TSS was inferior to the VIENNA 8D53-/Toliman GSS. Both the transgenic and the classical genetic sexing strains have shown acceptable and similar mating competitiveness when compared with wild males for mating with wild females. The cost production for both strains is similar but the FSEL#32 TSS may potentially be more cost effective at higher production levels. The results are discussed in the context of incorporating the transgenic strain for SIT application.

Different methods of methyl eugenol application enhance the mating success of male Oriental fruit fly (Dipera: Tephritidae).

Males of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) are strongly attracted to methyl eugenol (ME) (1,2-dimethoxy-4-(2-propenyl)benzene), a phenylpropanoid compound occurring in many plant species. Feeding on ME is known to enhance male B. dorsalis mating competitiveness, which can increase the effectiveness of the sterile insect technique (SIT) manifold. However, currently used systems for holding the mass-reared males in fly emergence and release facilities before release, do not allow for application of ME through feeding. Therefore, the current study was designed to evaluate different delivery systems of ME that would be applicable for large-scale application to sterile males held in such facilities. Males of a genetic sexing strain (GSS) of B. dorsalis treated by ME-aromatherapy or ME-airblown-aromatherapy that were competing with ME-fed males achieved a similar level of mating success in walk-in field cages, but the mating success was significantly higher when compared to untreated males. The results confirm the feasibility of developing ME-airblown-aromatherapy as a practical way of large scale ME delivery to enhance the mating competitiveness of sterile B. dorsalis males.

Pupariation time as a source of variability in mating performance in mass-reared Anastrepha ludens (Diptera: Tephritidae).

The effect of time of pupariation on pupal weight and adult sexual competitiveness under field cage conditions was studied in mass-reared Anastrepha ludens (Loew) males. Larvae that took 72 h to pupariate after separation from diet resulted in lighter pupae than those that took 24 and 48 h. Wild pupae were heavier than the 48- and the 72-h pupae but not the 24-h pupae. Interestingly, no differences in mating performance were found between males of the 24- and 48-h pupae despite differences in pupal weight. In general, lower-than expected levels of mating compatibility between sterile and wild A. ludens resulted from the interaction of both strains as more homotypic pairs were observed. Discussion focuses on the effect of the mass-rearing process on male fruit fly mating performance.

Fitness cost implications of PhiC31-mediated site-specific integrations in target-site strains of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae).

Site-specific recombination technologies are powerful new tools for the manipulation of genomic DNA in insects that can improve transgenesis strategies such as targeting transgene insertions, allowing transgene cassette exchange and DNA mobilization for transgene stabilization. However, understanding the fitness cost implications of these manipulations for transgenic strain applications is critical. In this study independent piggyBac-mediated attP target-sites marked with DsRed were created in several genomic positions in the Mexican fruit fly, Anastrepha ludens. Two of these strains, one having an autosomal (attP_F7) and the other a Y-linked (attP_2-M6y) integration, exhibited fitness parameters (dynamic demography and sexual competitiveness) similar to wild type flies. These strains were thus selected for targeted insertion using, for the first time in mexfly, the phiC31-integrase recombination system to insert an additional EGFP-marked transgene to determine its effect on host strain fitness. Fitness tests showed that the integration event in the int_2-M6y recombinant strain had no significant effect, while the int_F7 recombinant strain exhibited significantly lower fitness relative to the original attP_F7 target-site host strain. These results indicate that while targeted transgene integrations can be achieved without an additional fitness cost, at some genomic positions insertion of additional DNA into a previously integrated transgene can have a significant negative effect. Thus, for targeted transgene insertions fitness costs must be evaluated both previous to and subsequent to new site-specific insertions in the target-site strain.