Edwin Mauricio Ramírez‐Santos

The Sterile Insect Technique (SIT) is an effective strategy for controlling insect pests, such as the Mediterranean fruit fly ( Ceratitis capitata , Wiedemann). The effectiveness of the SIT depends on the ability of the sterile males to mate and their capacity to induce sterility in wild females. This study evaluated how the irradiation age affects their sexual performance, measured by the outcome of female remating events. Males of the GSS VIENNA 8 ^D53− were irradiated at eleven different ages, from 72 h before emergence (pupal stage) to 72 h after emergence (adult stage) and mated with wild females. These females were subsequently allowed to mate with fertile males from the fluorescent TSS VIENNA 8 1260. The presence of fluorescent offspring was used as indicator of second‐male paternity. Results showed that males irradiated at post‐emergence ages produced the lowest egg‐to‐pupae conversion rate, indicating a greater ability to prevent females from producing offspring after remating with a fertile male. In contrast, males irradiated at pre‐emergence ages were associated with higher numbers of fluorescent offspring. Although no significant differences were found in mating competitiveness (RSI), the outcome of the remating showed differences in the effectiveness of initial matings. These findings highlight the importance of considering the age of flies at time of irradiation in mass‐rearing protocols to enhance the efficacy of SIT programs targeting C. capitata and suggest that irradiating males later in their life cycle, such as adult stages or in pupae close to adult emergence, limit offspring if females remate with fertile males in the field.

Sterile Insect Technique (SIT) applications against major insect pests and disease vectors rely on the cost‐effective production of high‐quality sterile males. This largely depends on the optimal management of target pest colonies by maximizing the benefits provided by a genetically rich and pathogen‐free mother colony, the presence of symbiotic microorganisms, and efficient domestication, mass‐rearing, irradiation, and release processes. At the same time microbial (bacteria, fungi, microsporidia, and viruses) pathogen outbreaks should be minimized or eliminated, and the use of hazardous chemicals restricted. The optimization of the colony management strategies for different SIT target insects will ensure a standardized high‐quality mass‐rearing process and the cost‐effective production of sterile males with enhanced field performance and male mating competitiveness. The aims of the Coordinated Research Project (CRP) were to develop best practices for insect colony management for the cost‐effective production of high‐quality sterile males for SIT applications against major insect pests and disease vectors through a multidisciplinary approach involving entomologists, geneticists, ecologists, microbiologists, pathologists, virologists, and mass‐rearing experts.

The sterile insect technique (SIT) for controlling the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), requires the mass‐rearing of males in bio‐facilities. This study summarizes the implementation of a Pre‐Filter system in the mass‐rearing process of C. capitata at the Moscamed program's El Pino facility in Guatemala. The Pre‐Filter selection process as presented here was aimed to increase the longevity of the VIENNA 8 D53‐ strain males released for pest control. Over a 9‐year period (2015–2024), the longevity of these males was systemically monitored, and the impact of the Pre‐Filter system on mortality, half‐life, and survival rate were analyzed. Results indicated a significant reduction in mortality at 48 h, declining from 16.47% in 2017 to 2.56% in 2024. Concurrently, the half‐life of males increased from 79.55 h in 2018 to 85.97 h in 2024. The Pre‐Filter system shifted the frequency distribution of half‐lives toward longer durations, with a peak observed between 76.14 and 86.14 h, whereas populations without the Pre‐Filter exhibited shorter half‐lives, peaking between 56.14 and 66.14 h. Comparative analyses with historical data (2005–2024) demonstrated that the Pre‐Filter system significantly improved the male longevity, compared to pre‐implementation periods during which mortality rates were higher and more variable. Additionally, the results revealed a gradual reduction in longevity variability within the population, indicating a more homogeneous longevity among the mass‐reared males.

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.

The transgenic strain of the Mediterranean fruit fly (medfly), Ceratitis capitata (Wied.) VIENNA 8 1260, developed from the classical genetic sexing strain VIENNA 8, has two molecular markers that exhibit red fluorescence in the body and green fluorescence in testicles and sperm. These traits offer a precise tool to discriminate between mass-reared sterile males and wild fertile males, and they could potentially increase the effectiveness of control programs for this pest. To assess the risk of horizontal transfer of the fluorescence transgenes in natural ecosystems, we used the VIENNA 8 1260 strain and the medfly parasitoid Fopius ceratitivorus. The fluorescence signal and the inheritance of the fluorescence gene markers were monitored for over 16 generations (about two years) in both species using fluorescence microscopy and a PCR-based assay. The PCR analysis was performed in four independent laboratories. Both fluorescence microscopy and PCR analysis indicated that no horizontal gene transfer of the DsRed transgene occurred during 16 generations of medfly parasitoid rearing under experimental conditions.

The genetically modified strain of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) VIENNA 8 1260, was developed from the genetic sexing strain VIENNA 8. It has two molecular markers that exhibit red fluorescence in the body and green fluorescence in testis and sperm. These traits offer a precise tool to discriminate between mass-reared and wild males, increasing the effectiveness of sterile insect technique. The reproductive performance of the VIENNA 8 1260 and VIENNA 8 D53- (without the D53 inversion introduced to prevent recombination) was compared at different irradiation doses. The general effect of irradiation on VIENNA 8 1260 followed the same patterns documented in previous publications for VIENNA 8 D53-. Irradiation doses of 80 Gray or greater reduced fertility and induced high levels of sterility in wild females. Fecundity reduction was higher in VIENNA 8 1260 than in VIENNA 8 D53- females. Vertical transmission of the fluorescence gene was confirmed up to the F4 generation. Substerilization in the VIENNA 8 1260 could jeopardize the usefulness of the transgenic strain due to the possible vertical transfer of the fluorescence transgene from the sterile males to the wild flies. A biologically safe higher irradiation dose could result in reduced competitiveness of the VIENNA 8 1260 strain. Mating and remating experiments suggest that Mediterranean fruit fly females exhibit a relative precedence in the use of the sperm: though both sperms are mixed, sperm from the remating is spent first. Results suggest a lower fitness of VIENNA 8 1260 sperm, when compared with sperm from a nonfluorescent bisexual strain, which is consistent with the lower reproductive performance documented for the VIENNA 8 1260 strain.

The genetically modified strain of Ceratitis capitata (Wiedemann) VIENNA 8 1260 has two morphological markers that exhibit fluorescence in body and sperm. To assess the feasibility of its use in area-wide integrated pest management (AW-IPM) programs using the sterile insect technique, its rearing performance and quality control profile under small, medium, and large scales was evaluated, as well as in field cages. The VIENNA 8 1260 strain had a lower yield than the control strains, VIENNA 8 with D53 inversion (VIENNA 8) and without D53 inversion (VIENNA 8 D53-). At mass-rearing scale, yield gradually increased in three generations without reaching the control strain values. The VIENNA 8 1260 strain was stable in the genetic sexing mechanism (>99.9%) and expression of fluorescence (100%). In field cages, the VIENNA 8 1260 males reduced the mating potential of wild males in the same magnitude as the VIENNA 8, when evaluated in independent cage tests. However, the relative sterility index and the strain male relative performance index of VIENNA 8 1260 males were significantly lower than those of the VIENNA 8. There were no significant differences in longevity of these strains. The potential application of the VIENNA 8 1260 in AW-IPM programs is further discussed.