The transinfection of the endosymbiotic bacterium Wolbachia provides a method to produce functionally sterile males to be used to suppress mosquito vectors. ARwP is a wPip Wolbachia infected Aedes albopictus which exhibits a bidirectional incompatibility pattern with wild-types. We coupled a modelistic approach with laboratory experiments to test ARwP as a control tool and evaluate the possible occurrence of population replacement following the release of ARwP females in a wild-type (SANG) population. Repeated male-only releases were simulated and tested in the laboratory in comparison with releases contaminated with 1% ARwP females. Model simulations also investigated how migration affects the outcome of contaminated releases. Finally, the mean level of egg fertility and the long-term evolution of populations constituted by two Wolbachia infection types were studied by testing SANG and ARwP Ae. albopictus and performing more general model simulations. The model was parametrized with laboratory data and simulations were compared with results of biological trials. Small populations of ARwP males and females were theoretically and experimentally demonstrated to rapidly become extinct when released in larger SANG populations. Male-only releases at a 5:1 ratio with respect to the wild-type males led to a complete suppression of the SANG population in a few generations. Contaminated releases were efficient as well but led to population replacement in the long term, when the wild-type population approached eradication. Migration significantly contrasted this trend as a 5% population turnover was sufficient to avoid any risk of population replacement. At equal frequencies between ARwP and SANG individuals, the mean egg fertility of the overall population was more than halved and suppression was self-sustaining until one of the two infection types extinguished. In the case of bidirectional incompatibility patterns, the repeated release of incompatible males with small percentages of contaminant females could lead to population replacement in confined environments while it could be managed to target high efficiency and sustainability in wild-type suppression when systems are open to migration. This possibility is discussed based on various contexts and taking into consideration the possibility of integration with other control methods such as SIT and the use of larvicides.

Background: Releasing considerable numbers of radiation-sterilized males is a promising strategy to suppress mosquito vectors. However, releases may also include small percentages of biting females, which translate to non-negligible numbers when releases are large. Currently, the effects of irradiation on host-seeking and host-biting behaviors have not been exhaustively investigated. Information is also lacking regarding the effects of sterilizing treatment on the endosymbiotic bacteriumWolbachia, which is known to affect the vector competence of infected mosquitos.Methods: To ascertain the effects of irradiation on females, the pupae of twoAedes albopictusstrains, differing in their natural or artificialWolbachiainfection type, andAedes aegypti—which is not infected byWolbachia—were treated with various doses of X-rays and monitored for key fitness parameters and biting behavior over a period of 2 weeks. The effect of radiation onWolbachiawas investigated by quantitative polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) analysis.Results: PartialAedes albopictusfemale sterility was achieved at 28 Gy, but the number of weekly bites more than doubled compared to that of the controls. Radiation doses of 35 and 45 Gy completely inhibited progeny production but did not significantly affect the survival or flight ability ofAe. albopictusfemales and caused a tripling of the number of bites per female per week (compared to untreated controls). These results were also confirmed inAe. aegyptiafter treatment at 50 Gy.Wolbachiadensity decreased significantly in 45-Gy-irradiated females, with the greatest decreases in the early irradiation group (26 ± 2-h-old pupae).Wolbachiadensity also decreased as adults aged. This trend was confirmed in ovaries but not in extra-ovarian tissues. FISH analysis showed a strongly reducedWolbachia-specific fluorescence in the ovaries of 13 ± 1-day-old females.Conclusions: These results suggest that, under sterile insect technique (SIT) programs, the vector capacity of a target population could increase with the frequency of the irradiated females co-released with the sterile males due to an increased biting rate. In the context of successful suppression, the related safety issues are expected to be generally negligible, but they should be conservatively evaluated when large-scale programs relying on imperfect sexing and high overflooding release ratios are run for long periods in areas endemic for arboviral diseases. Also, the effects of irradiation on the vector competence deserve further investigation.Graphical

The Incompatible Insect Technique (IIT) is a species-specific, eco-friendly mosquito control method that relies on releasing Wolbachia-infected males, which induce cytoplasmic incompatibility (CI), rendering eggs inviable when mating with wild females. Aiming at optimizing IIT protocols in terms of cost-effectiveness, data on incompatible male dispersal and survival and the distance- and time-related impact of induced sterility are fundamental. This study plans to fill this gap and reports findings from a two-year field trial (2022–2023) at the ENEA-Casaccia Research Center, based on single-spot releases of incompatible Aedes albopictus males (ARwP strain). Male releases were carried out in late September 2022 (~15,000 released males) and the early Ae. albopictus season (at the end of June 2023; ~24,000 released males). Fifty-eight ovitraps were located at a 20–900 m distance from the ARwP release spot and were monitored weekly from May to November to assess egg hatching rates and measure CI effects in relation to both distance and time. Following the 2023 release, samples of adults were collected at increasing distances from the release site and at multiple post-release time points to assess, individually, wild female fertility and ARwP male dispersal and survival using Wolbachia as a genetic marker. Statistical analyses revealed that: (a) the highest reduction in the egg hatching was found within 100 m from the release spot (46.5% and 19.9%, respectively, in 2022 and 2023) but remained significant even at greater distances (29.9% and 7.7% at 300 m, respectively, in 2022 and 2023); (b) accordingly, the highest reduction in the wild female fertility occurred within 100 m from the release spot (47.3%), but similar effects were recognizable up to 600 m; (c) the overflooding ratio of the ARwP males did not significantly differ between 3 and 11 days after the release, with ARwP males remaining active up to 18 days and dispersing as far as 400 m. These results demonstrate the potential of localized, non-inundative IIT trials to furnish clues for the setup of spatially optimized release strategies, especially in scaled-up applications. The study also emphasizes the need for standardized assessment tools and further research regarding environmental and behavioral factors influencing long-term suppression outcomes.