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Background The World Health Organization (WHO) has emphasized the urgent need for alternative strategies to chemical insecticides for controlling mosquito populations, particularly the invasive Aedes species, which are known vectors of arboviruses. Among these alternative approaches, the sterile insect technique (SIT) is experiencing rapid development, with numerous pilot trials being conducted worldwide. Main text This review aims to elucidate the principles of SIT and highlight the significant recent advancements that have facilitated its scalability. I also employ a phased conditional approach to categorize the progression of 39 projects, drawing on peer reviewed studies, press releases and direct communication with project managers. This review indicates that a substantial number of projects illustrate the efficacy of SIT in suppressing Aedes populations, with one project even demonstrating a reduction in dengue incidence. I offer several recommendations to mitigate potential failures and address the challenges of compensation and overcompensation when implementing SIT field trials. Furthermore, I examine the potential implications of male mating harassment on the effectiveness of SIT in reducing disease transmission. Conclusions This comprehensive assessment underscores the promise of SIT as a viable strategy for mosquito control. The insights gained from these trials not only contribute to the understanding of SIT’s effectiveness but also highlight the importance of careful project management and ecological considerations in the pursuit of public health objectives. Graphical Abstract

Background Aedes aegypti , the primary vector of dengue, chikungunya, and Zika viruses, poses a significant public health threat worldwide. Traditional control methods using insecticides are increasingly challenged by resistance and environmental concerns. The sterile insect technique (SIT) offers an eco-friendly alternative that has been successfully applied to other insect pests. This article aims to briefly review Ae. aegypti management in Cuba, highlighting the accomplishments, challenges, and future directions of the SIT. Main body Here we provide a brief summary of the extensive history of Ae. aegypti control efforts in Cuba. After a successful eradication campaign in the 1980s, a resurgence of dengue cases has been observed in recent years, suggesting that traditional control methods may have limited effectiveness under current conditions. In response, Cuba initiated a phased approach to develop and evaluate the feasibility of SIT for Ae. aegypti control, starting in 2008. Initial research focused on Ae. aegypti mating behavior and sterilization methods, followed by successful laboratory and semi-field trials that demonstrated population suppression. The first open-field trial in 2020 confirmed the efficacy of the SIT in reducing Ae. aegypti populations under real-world conditions. Currently, the research is in a phase involving a cluster-randomized superiority-controlled trial. This planned trial will compare the standard vector control program with the same program augmented by the SIT, aiming to assess the impact of the SIT on dengue incidence as the primary outcome. Implementing robust epidemiological trials to evaluate the effectiveness of the SIT is complex due to potential spillover effects from mosquito and human movement across study areas. Additionally, conducting the SIT requires significant development and operational investments. Despite these challenges, the ongoing Cuban trial holds promise for establishing the SIT as an effective and sustainable tool for Ae. aegypti control and for reducing the burden of mosquito-borne diseases. Conclusions The phased evaluation conducted in Cuba confirms the efficacy of the SIT against Ae. aegypti , highlighting its potential for sustainable mosquito-borne disease management. The effective implementation of multi-site trials will be crucial in providing evidence of the potential of the sterile insect technique as part of a strategy to reduce the incidence of arboviral diseases. Graphical Abstract

Background Lee County Mosquito Control District (LCMCD) is an independent taxing district that works to protect human health and improve quality of life in Lee County, Florida, USA. With local dengue transmission in southern Florida, LCMCD prioritized the control of Aedes aegypti. Due to the cryptic larval habitats of Ae. aegypti and insecticide resistance, effective control using conventional methods is difficult. Thus, the sterile insect technique (SIT) program, using X-ray irradiated male mosquitoes, was created to target Ae. aegypti. The goal of this program was to suppress Ae. aegypti through establishing a robust SIT program and performing a pilot study in the field to assess the impacts of SIT releases. Main text The SIT program at LCMCD released sterile male Ae. aegypti from 2020 to 2022 in Captiva Island, Florida. The SIT program works within a larger Integrated Mosquito Management (IMM) framework and is not a standalone tool. The SIT program consists of nine employees, one of which is dedicated to quality assurance. Quality assurance assessments are performed routinely and periodically. Due to widespread destruction throughout Captiva and Sanibel Islands from Hurricane Ian in September 2022, the SIT pilot in Captiva Island was concluded and moved to Fort Myers, Florida. During the pilot study on Captiva Island, various lessons were learned and this knowledge has been applied to efforts in Fort Myers. Conclusions LCMCD has established a successful SIT program to suppress populations of Ae. aegypti . Through connections with the International Atomic Energy Agency (IAEA) and the University of Florida, LCMCD received guidance from experts in the field to help ensure the program’s success. Stable funding through taxes levied specifically for mosquito control provided essential consistency, allowing the program to grow and evolve. Consistent trapping routines provided immense amounts of entomological data. Thoughtful and intentional community engagement was essential in ensuring acceptance of the SIT program in Lee County. Following the phased conditional approach suggested by IAEA, LCMCD has built an effective and resilient SIT program. The integration of the SIT as a tool of an area-wide mosquito control program is a feature that distinguishes LCMCD’s SIT program from others.

Background The invasive Asian tiger mosquito ( Aedes albopictus ) poses growing health risks across Europe. In Switzerland, a preliminary field trial was conducted to assess the feasibility of integrating the sterile insect technique (SIT) into existing integrated vector management (IVM), which includes breeding site removal and application of biological larvicides. SIT involves repeated releases of irradiated sterile males, which mate with wild females, producing non-viable eggs and leading to population decline. Methods Following a preliminary release test in 2022, a small-scale SIT trial took place in 2023 in Morcote, Switzerland. Approximately 150,000 sterile males were released weekly over a 45-hectare area throughout the entire mosquito activity season, from May to September. This SIT area also received routine IVM. Population dynamics were compared with a control area where only IVM was applied. Monitoring included egg counts, hatch rates, and adult female densities. Generalized additive mixed-effects models (GAMM) and generalized additive models (GAM) accounted for spatial, temporal, and random effects. Model selection used AIC, BIC, and Chi-square tests (significance at 5%). Results The SIT-treated area showed a significant mosquito population reduction. Egg counts dropped by 57% (GAMM regression coefficient: − 0.8513, P  < 0.001), with temporal patterns differing between SIT-treated and control areas ( P  < 0.001). Egg hatch rates were also lower in the SIT area, with odds of hatching reduced by 1.24 log-odds units ( P  < 0.001). Adult female densities declined by 66% (regression coefficient: − 1.0818, P  < 0.001). Spatial GAMs revealed heterogeneous effects: up to 90% egg reduction in the western release area, while the eastern edge, bordering untreated zones, showed up to 300% higher egg counts. Similar spatial trends were observed for hatch rates and adult females ( P  < 0.01). These findings highlight both the overall effectiveness of SIT and the influence of mosquito immigration on spatial patterns. Conclusions This trial demonstrated the potential of SIT as a complementary tool in Swiss vector control. Public interest and acceptance were high. To improve cost-effectiveness, further optimization of male production, sterilization, transport, and release processes is needed. Continued implementation over multiple seasons is recommended to enhance long-term effectiveness. Graphical Abstract

Background The sterile insect technique (SIT) requires distinguishing sterile from wild male mosquitoes to evaluate male qualities and maintain an appropriate release ratio for efficient population suppression. Current dye/powder marking methods have limitations and may affect SIT effectiveness, necessitating alternative discrimination strategies. Aedes albopictus naturally harbors two Wolbachia infections ( w AlbA/ w AlbB), which can be eliminated via tetracycline. Although Wolbachia removal minimally affect host fitness, its impact on microbiota remains unclear. Characterizing post-elimination microbial communities is the first step to identify novel endogenous biomarkers for SIT monitoring. Methods We analyzed the bacterial diversity and composition of two strains of wild-type GUA ( Wolbachia -infected) and GT ( Wolbachia -free) mosquitoes using the 16S r RNA V3-V4 region sequencing. qPCR was employed to confirm the relative abundance of four major bacterial genera, while PCR was used to validate selected biomarkers for distinguishing factory-reared sterile males from wild males. Kruskal-Wallis or Mann-Whitney test was used to analyze the comparable parameters between GUA and GT strains. Results Five-day-old GUA and GT females showed similar microbial diversity/composition, while young males shared diversity but differed in composition. The core microbiota in both strains consisted of Proteobacteria (64.27%), Firmicutes (16.09%), Actinobacteriota (11.22%), and Bacteroidota (4.96%). Asaia was dominant in both strains (GUA: 47.33%; GT: 32.69%), whereas Enterococcus increased in GT males with aging. Wolbachia was absent in GT mosquitoes, and Elizabethkingia was undetected in GUA males. qPCR further confirmed these trends. PCR analysis revealed that w AlbB exhibited higher stability in differentiating factory-reared GT males from their wild counterparts (96.7% infection in field males, n  = 60) compared to w AlbA (61.7%, n  = 60) or Enterococcus (65.8%, n  = 120). The mark-release-recapture experiment further confirmed the detectability using w AlbB biomarker. Conclusions Without obvious fitness costs observed previously in the Ae. albopictus GT strain compared to GUA strain, the removal of Wolbachia significantly changes the microbial composition in male mosquitoes in this study . Wolbachia w AlbB is recommended as a reliable biomarker for distinguishing sterile males from wild males when using GT strain in SIT programs targeting Ae. albopictus . Graphical Abstract

Background Genetic biocontrol interventions targeting mosquito-borne diseases require the release of male mosquitoes exclusively, as only females consume blood and transmit pathogens. Releasing only males eliminates the risk of increasing mosquito bites and spreading pathogens while enabling effective population control. The aim of this study is to develop robust sex-sorting methods for early larval stages in mosquitoes, enabling scalable male-only releases for genetic biocontrol interventions. Methods To address the challenge of sex-sorting in the Asian malaria vector Anopheles stephensi , we engineer Sexing Element Produced by Alternative RNA-splicing of a Transgenic Observable Reporter (SEPARATOR). This dominant fluorescent-based method, previously proven effective in Aedes aegypti , exploits sex-specific alternative splicing of a reporter to ensure exclusive male-specific expression early in development. The sex-specific alternative RNA splicing of the doublesex gene was selected as a target for engineering SEPARATOR due to its evolutionary conservation in insects. To expand SEPARATOR’s applicability for genetic sexing, we assessed the cross-species sex-specific RNA splicing activity of the An. gambiae doublesex ( AngDsx ) splicing module in An. stephensi . Male-specific enhanced green fluorescent protein (EGFP) expression was verified throughout the mosquito life cycle using a fluorescent stereomicroscope. Results Our results confirm that SEPARATOR regulates male-specific EGFP expression in An. stephensi and enables reliable positive male selection from the first instar larval stages. Molecular analysis demonstrates that male-specific EGFP expression is dependent on doublesex sex-specific splicing events. Additionally, the splicing module from An. gambiae operates effectively in An. stephensi , demonstrating evolutionary conservation in sex-specific splicing events between these species. Conclusions SEPARATOR’s independence from sex-chromosome linkage provides resistance to breakage that could be mediated by meiotic recombination and chromosomal rearrangements, making it highly suitable for mass male releases. By enabling precise male selection from the first instar larval stages, SEPARATOR represents a significant advancement that will aid in the genetic biocontrol for Anopheles mosquitoes. Graphical Abstract

Background Vector-borne diseases cause morbidity and mortality globally. However, some areas are more impacted than others, especially with climate change. Controlling vectors remains the primary means to prevent these diseases, but new, more effective tools are needed. The World Health Organization (WHO) prioritized evaluating novel control methods, such as sterile insect technique (SIT) for control of Aedes -borne diseases. In response, a multiagency partnership between the U.S. Centers for Disease Control and Prevention (CDC), the Special Programme for Research and Training in Tropical Diseases (TDR), WHO, and the International Atomic Energy Agency (IAEA) supported the operational implementation and evaluation of SIT against Aedes aegypti and arboviral diseases in the Pacific through a consortium of regional partners (PAC-SIT Consortium). Main text A workshop was held from 2 to 6 May 2023, during which PAC-SIT country participants, researchers, and stakeholders in SIT, scientific advisory committee members, and organizational partners came together to review the principles and components of SIT, share experiences, visit field sites and the SIT facility, and officially launch the PAC-SIT project. Working in groups focused on entomology, epidemiology, and community engagement, participants addressed challenges, priorities, and needs for SIT implementation. Conclusions The PAC-SIT workshop brought together researchers and stakeholders engaged in evaluating SIT for arboviral diseases in the Pacific region and globally. This training workshop highlighted that many countries are actively engaged in building operational capacities and phased testing of SIT. The workshop identified a key need for robust larger-scale studies tied with epidemiological endpoints to provide evidence for the scalability and impact on mosquito-borne diseases.

Background Aedes mosquitoes pose a significant global threat as vectors for several debilitating arboviruses, including dengue, Zika, yellow fever, and chikungunya. Their unique breeding habits, behavior, and daytime activity complicate control efforts, prompting the search for innovative solutions. The sterile insect technique (SIT) and incompatible insect technique (IIT) are promising new techniques under investigation. This review synthesizes findings from field trials on SIT and/or IIT for Aedes mosquito control. Methods A scoping review was conducted through comprehensive searches on Scopus, Web of Science, MEDLINE, PubMed, and preprint repositories up to April 25, 2024. Studies were initially screened for relevance based on their titles and abstracts, followed by a full-text review conducted by two independent extractors. Only field trials with control groups were included, with the final assessment focusing on trials reporting epidemiological outcomes. Data were abstracted into templates, emphasizing study design, intervention details, and outcomes. The review adhered to the PRISMA-ScR guidelines. Results The search identified 21 field trials in various countries against Aedes mosquitoes. These trials employed diverse methodologies and mosquito release strategies, achieving varying levels of mosquito population suppression. Notably, two SIT and two Wolbachia -based IIT trials reported epidemiological outcomes, including reductions in dengue incidence and associated risk ratios. However, the reliance on national surveillance data for assessing dengue incidence suggests caution due to the potential underreporting of subclinical cases. Conclusions The review underscores the promise of SIT and IIT for controlling Aedes mosquito populations, citing successful reductions in mosquito densities and dengue transmission. However, it calls for more rigorous study designs and standardized methodologies, as well as the adoption of comprehensive frameworks to accurately assess the effectiveness of these interventions. Future research should focus on bridging gaps in real-world effectiveness by addressing factors such as feasibility, acceptability, scalability, and cost, which are crucial for guiding their successful large-scale deployment in any country.

Background Understanding of mosquito spatiotemporal dynamics is central to characterize candidate field sites for the sterile insect technique (SIT) testing, and is critical to the effective implementation and evaluation of pilot sterile male release programs. Here, we present a detailed description of Aedes albopictus (Skuse) egg-laying activity over a 6-year period in urban areas identified as potential SIT testing sites on Reunion Island. Method Weekly entomological collections using ovitraps were carried out in residential and adjacent uninhabited habitats in two urban areas, Duparc and Bois Rouge, in the municipality of Sainte Marie, Reunion Island. Time-series data incorporating the frequency of positive ovitraps and the total number of eggs/ovitrap recorded each time at each locality during the study period from May 2013 to December 2018 were analyzed with multifaceted statistical approaches including descriptive statistics and spatiotemporal analyses incorporating the role of climatic factors on overall ovitrap productivity. Results During the ovitrap survey, the proportion of egg-positive ovitraps differed among study sites ( χ 2  = 50.21, df = 2, P  < 0.001), being relatively lower in Duparc (89.5%) than in Bois-Rouges (95.3%) and the adjacent buffer zone (91.2%). Within each neighborhood, Ae. albopictus egg abundance varied by month in a roughly seasonal pattern marked by a single peak occurring more regularly February each year, a decline at the onset of the austral winter in July, followed by a period of lower ovitrap productivity in August and September. Fluctuation in both positivity rate and eggs densities per ovitraps were related to annual and seasonal variations in local temperature and rainfall ( P  < 0.001 in all cases). The spatial analysis also captured substantial between- and within-habitats heterogeneity, whereby the overall ovitrap productivity was higher in residential areas than in the buffer zone. Conclusions Collectively, these results reveal that the distribution of Ae. albopictus oviposition activity is shaped by local habitat heterogeneity and seasonal climatic factors. Overall, this study provides baseline insights into the reproductive dynamics of Ae. albopictus , which would assist in planning locally tailored SIT interventions, while addressing concerns related to focal areas of high egg-laying intensity and potential immigration of females from natural areas.

Background The Sterile insect technique (SIT) has been successfully used in agricultural pest control, leading to interest in its application for public health, particularly in controlling Aedes mosquitoes in the USA, Italy, Cuba, and Greece. Malaysia has conducted a small-scale SIT pilot trial since 2019 for dengue control. This study evaluates mosquito populations in treated and untreated sites through three objectives: (1) comparing mean larvae per trap (MLT) and dengue cases for Ae. aegypti and Aedes albopictus ; (2) estimating survival rates and wild populations using mark-release-recapture (MRR); and (3) analysing spatial distribution in treated and untreated sites. Methods Ae. aegypti males, irradiated at 55 Gray, were released in three locations: Pangsapuri Kota Laksamana (KT), Malacca (19 months), Pangsapuri Taman Tasik Utama (TTU), Malacca (8 months), and the Customs, Immigration, and Quarantine Complex (CIQ), Johor (7 months). Statistical analyses assessed SIT effectiveness, including T-tests for larval density and ovitrap indices, Mulla’s formula and relative variance (RV) for population reduction, and the Lincoln Index for estimating wild male populations and probability of daily survival. Results Weekly releases of sterile Ae. aegypti males at doses of 1278–7942 males/ha achieved a sterile-to-wild male ratio of 5.85 and a mean daily survival rate of 0.61, leading to significant reductions in larval densities: 76.25% in Kota Laksamana (KT), 96.74% in Taman Tasik Utama (TTU), and 89.00% in CIQ Gelang Patah, thereby supporting dengue control efforts. In KT, the MLT was initially low but increased, although with suppression < 90%, there was a reduce of dengue cases throughout the release period. The MRR’s mean survival rate (± standard deviation) in KT was 0.61 (± 0.08). The spatial clustering of Ae. aegypti was observed in central blocks during the high MLT period. However, larval densities rebounded after releases ceased. Spatial clustering revealed no initial clustering, though clustering patterns emerged over time in KT. Conclusions SIT effectively suppressed Ae . aegypti populations and supported dengue control. Optimizing sterile-to-wild male ratios, spatial distribution, and monitoring strategies is essential for sustainable vector control. These findings provide insights for scaling up SIT field trials, with future efforts focusing on refining release and monitoring strategies to enhance SIT as an effective dengue control tool. Trial registration NMRR-17–2652-39,099 “Field evaluation of Sterile Insect for Aedes aegypti Suppression.”