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Learn more: PMC Disclaimer | PMC Copyright Notice J Econ Entomol . 2025 Nov 2;119(2):811–822. doi: 10.1093/jee/toaf268 Search in PMC Search in PubMed View in NLM Catalog Add to search Suitability of a chilled environmental box of the Precision X-RAD 320 cabinet style irradiator for the irradiation of mosquitoes and tsetse in the context of the sterile insect technique Hanano Yamada Hanano Yamada 1 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria Conceptualization, Data curation, Investigation, Methodology, Writing - original draft, Writing - review & editing Find articles by Hanano Yamada 1, ✉, # , Bénéwendé Aristide Kaboré Bénéwendé Aristide Kaboré 2 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing Find articles by Bénéwendé Aristide Kaboré 2, # , Samar Eisa Samar Eisa 3 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria 4 Department of Epidemiology, Tropical Medicine Research Institute, Khartoum, Sudan Investigation, Methodology Find articles by Samar Eisa 3, 4 , Didier Alexandre Kaboré Didier Alexandre Kaboré 5 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria 6 Institut de Recherche en Sciences de la Santé (IRSS), Bobo-Dioulasso, Burkina Faso Formal analysis, Investigation, Writing - review & editing Find articles by Didier Alexandre Kaboré 5, 6 , Andrew Gordon Parker Andrew Gordon Parker 7 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria Investigation, Validation, Writing - review & editing Find articles by Andrew Gordon Parker 7, 2 , Odet Bueno Odet Bueno 8 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria Investigation, Resources Find articles by Odet Bueno 8 , Wadaka Mamai Wadaka Mamai 9 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria Formal analysis, Resources, Validation, Writing - review & editing Find articles by Wadaka Mamai 9 , Thomas Wallner Thomas Wallner 10 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria Data curation, Investigation, Resources Find articles by Thomas Wallner 10 , Chantel Janet de Beer Chantel Janet de Beer 11 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria Formal analysis, Funding acquisition, Project administration, Supervision, Writing - review & editing Find articles by Chantel Janet de Beer 11 Editor: Daniel Hahn Author information Article notes Copyright and License information 1 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria 2 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria 3 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria 4 Department of Epidemiology, Tropical Medicine Research Institute, Khartoum, Sudan 5 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria 6 Institut de Recherche en Sciences de la Santé (IRSS), Bobo-Dioulasso, Burkina Faso 7 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria 8 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria 9 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria 10 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria 11 Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria ✉ Corresponding author. Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, P.O. Box 100, 1400 Vienna, Austria (Email: [email protected] ). 2 Present address for Andrew Gordon Parker: Roppersbergweg 15, 2381 Laab im Walde, Austria # Hanano Yamada and Bénéwendé Aristide Kaboré contributed equally to the manuscript. Roles Hanano Yamada : Conceptualization, Data curation, Investigation, Methodology, Writing - original draft, Writing - review & editing Bénéwendé Aristide Kaboré : Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing Samar Eisa : Investigation, Methodology Didier Alexandre Kaboré : Formal analysis, Investigation, Writing - review & editing Andrew Gordon Parker : Investigation, Validation, Writing - review & editing Odet Bueno : Investigation, Resources Wadaka Mamai : Formal analysis, Resources, Validation, Writing - review & editing Thomas Wallner : Data curation, Investigation, Resources Chantel Janet de Beer : Formal analysis, Funding acquisition, Project administration, Supervision, Writing - review & editing Daniel Hahn : Subject Editor Received 2025 Mar 4; Revised 2025 Aug 15; Accepted 2025 Aug 15; Collection date 2026 Apr. © The Author(s) 2025. Published by Oxford University Press on behalf of Entomological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License ( https://creativecommons.org/licenses/by-nc/4.0/ ), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact [email protected]. PMC Copyright notice PMCID: PMC13075812 PMID: 41176805 Abstract A cabinet-style small animal X-irradiator outfitted with an environmental chamber which can provide a consistent, chilled environment during irradiation was tested to sterilize the human and animal disease vectors Aedes aegypti Linnaeus (Diptera: Culicidae), Anopheles arabiensis Patton (Diptera: Culicidae), Glossina palpalis gambiensis Vanderplank (Diptera: Glossinidae) in the frame of the sterile insect technique (SIT). The environmental chamber enables the irradiation of immobilized, compacted adult insects avoiding mechanical damage incurred by movement and, thereby, maintaining better insect quality. For the species tested, there was no significant difference in dose response when irradiating late-stage pupae or adults, and chilling at 7 °C did not affect irradiation outcome in terms of sterility induced. The X-irradiator was shown to be effective and suitable for the sterilization of these important target species of the SIT and offers a practical means to sterilize insects at the adult stage which require chilling for immobilization. Keywords: X-ray, sterility, Aedes aegypti , Anopheles arabiensis , Glossina palpalis gambiensis Introduction Radiation exposure can be used for the reproductive sterilization of insects. This allows for the opportunity to manage pest populations as a type of “insect birth control” ( Dyck et al. 2021 ). This sterile insect technique (SIT) consists of the artificial mass-rearing of a target pest, the separation of sexes in cases where females have a negative impact (plant infestation or disease transmission), the reproductive sterilization of males which are then packed, transported, and released over a treatment area, and quality control (QC) tests to assess the biological quality and field performance of the released males. The technique is not suitable for all insect species, due to biological and ecological constraints such as complex mating behaviors, low dispersal capabilities, enhancing damage to crops or challenges in artificial mass-rearing, etc. However, many plant pests and disease vector species are indeed good candidates and have been successfully suppressed, contained, eradicated or their invasion and establishment prevented using the SIT ( Dyck et al. 2021 , Hendrichs et al. 2021 ). The Insect Pest Control Laboratory (IPCL) of Joint Food and Agriculture Organization (FAO)/International Atomic Energy Agency (IAEA) Centre of Nuclear Techniques in Food and Agriculture is continuing to develop and optimize the various components of the SIT package for several vectors of medical and veterinary importance: (i) tsetse flies ( Glossina spp) which feed on blood and can transmit human and animal trypanosomiasis, causing considerable morbidity and mortality in humans and preventing sustainable and profitable agriculture in a large part of sub-Saharan Africa; (ii) Aedes mosquito species [ Aedes aegypti Linnaeus { Diptera: Culicidae }, and Ae. albopictus Skuse { Diptera: Culicidae }] which are efficient vectors of numerous arboviral diseases and parasites in humans and animals, such as yellow fever, dengue fever, chikungunya, Zika, Mayaro, and dirofilaria in canines. Ae. aegypti alone has been implicated in the transmission of 52 different viruses according to the Walter Reed Biosystematic Unit [Walter Reed Biosystematics Unit {WRBU}]; and (iii) Anopheles mosquitoes [ Anopheles arabiensis Patton { Diptera: Culicidae } and An. stephensi Liston { Diptera: Culicidae }], efficient vectors of malaria parasites, which cause over 250 million cases and more than 600,000 deaths annually ( World Health Organization 2023 ). Significant progress has been made in terms of research and development for the various components of the SIT package for these target insects, reviewed in Vreysen et al. (2021) . One of the main areas of research is the sterilization process of the insects using ionizing radiation, either with gamma rays or X-rays. Historically, gamma irradiators were more common in SIT programmes, especially those programmes sterilizing more than a billion insects per week, where a large processing capacity of industrial, panoramic irradiators is required ( Yamada et al. 2023b ). Self-shielded Co 60 or Cs 137 gamma irradiators have also been commonly used in smaller programmes and have proven to be a reliable method for achieving the desired sterility levels in various insect species. Isotopic sources, however, present particular problems with safety, and security as they can potentially be misused, resulting in increasingly strict regulation of these sources. In addition, the transportation of the sources is problematic with frequent delays and refusals in shipment ( Gray 2011 , de Wright et al. 2016 ). More recently, the development of a new generation of X-ray irradiators with suitable dose rates, dose uniformity ratios and processing capacity have provided an adequate alternative to gamma irradiators for several SIT programmes ( Mastrangelo et al. 2010 ). As X-ray sources contain no radioactive material and produce no radiation when switched off, they avoid almost all of the regulatory and security issues. This has spurred research in the application potential of X-irradiators for the SIT, and the comparison of X- and gamma irradiators in terms of processing capacity and relative biological effectiveness (RBE) in insects ( Mastrangelo et al. 2010 , Ndo et al. 2014 , Kaboré et al. 2023 , Yamada et al. 2023a , 2023b ). Recently, an off-the-shelf blood X-irradiator (Raycell MK2) was evaluated for its potential use in insect sterilization and was found to achieve the target sterility levels in various insect species with similar doses as required for gamma irradiation ( Yamada et al. 2023a ). This X-irradiator had 2 X-ray tubes between which the 2 liter of insects can be irradiated at a dose rate of around 7 Gy/min, which would in theory accommodate the irradiation of around 30 million pupae or compacted Aedes mosquito adults, or 9 million tsetse pupae per week (5-d wk, with 6 h shifts per day) ( Yamada et al. 2023a ). However, it is important to note that insects irradiated at the adult stage need to be immobilized by chilling (or in anoxia) to be able to compact them and prevent movement resulting in physical damage to the fragile legs, wings, and antennae. Therefore, a container surrounded by phase change material, for example, could be used, which would decrease the usable volume for the sample ( Balestrino et al. 2024 ) and potentially attenuate the X-ray beam. Immobilization of adult mosquitoes by exposing them to Nitrogen during irradiation was also investigated Yamada et al ( Bimbilé Somda et al. 2022 , Yamada et al. 2022b ) and the results suggested that Anopheles mosquitoes seem to be more sensitive to low oxygen environments and thus immobilization in N 2 was not suitable, whereas Aedes mosquitoes were more tolerant to hypoxic conditions with N 2 providing highly radioprotective effects, but did seem to negatively affect flight ability in sterile males. The current study aims to assess a cabinet style, small animal X-irradiator “X-RAD 320” which has been outfitted with an environmental chamber (53 × 54 × 50 cm) that provides a chilled volume and is capable of holding and rotating 1 liter to 4.8 liter sample boxes. The advantage of the environmental chamber is that it can deliver chilled air over a range of temperatures (or a hypoxic/anoxic environment), which allows for the total volume to be used for exposing immobilized, compacted adult insects. Recently, a thorough dosimetric assessment was performed on the X-RAD 320 unit and the potential throughput of the unit in a mosquito SIT programme was estimated ( Parker et al. 2025 ). Assuming that around 150 (±50, depending on species and packing protocols) chilled, compacted mosquito males could be packed into 1 cm 3 , and the dose rates when using the standard settings for the 4.8 liter volume is around 4 Gy/min, around 40 to 48 million adult Aedes mosquitoes and approximately 25 to 30 million Anopheles males can be processed in a 5-d wk (with 6 h shifts per day) due to the larger usable chilled volume. Many parameters can be changed and adjusted in the X-RAD 320 unit which makes it a versatile irradiator. Smaller sample containers can be used to increase dose uniformity, and the source to sample distance can be decreased by moving the environmental chamber up and thereby increasing dose rates. Voltage and current can also be changed to study the potential effects of energy on dose response. For the series of experiments reported here, the standard settings for the maximum processing volume were selected (320 kV, 12.5 mA, source to sample distance of 43.5 cm) ( Parker et al. 2025 ), although the smaller 1 liter insect box was used for holding the small samples of insects. The dose response curves for pupae and adults of Aedes aegypti , adults only of Anopheles arabiensis , as well as pupae and adults of the tsetse species Glossina palpalis gambiensis were determined and compared to previous studies using different irradiators. The consistency of the chilled temperatures in the environmental chamber and the effects of chilling on the adult dose response were also determined. Materials and Methods Irradiation The X-irradiator, X-RAD 320 (Precision X-ray Inc., Madison, Connecticut, USA), outfitted with an environmental chamber (53 × 54 × 50 cm) was used for all irradiation exposures. All samples of insects were irradiated at a pre-defined standard reference point, which was in a small petri dish (Ø 5.5 cm), placed in the middle of a stack of 5 standard petri dishes (8.5 cm Ø) ( Fig. 1B ), or in smaller containers (with mesh on either side ( Fig. 1A top )), which were placed in a standard petri dish in the same location ( Fig. 1A bottom ), inside the small, 1 liter “insect box” provided with environmental chamber ( Fig. 1C ) of the X-RAD 320. Fig. 1. Open in a new tab Irradiation set-up: samples of insects were irradiated at a pre-defined standard reference point (source to sample distance), which was either a small petri dish (Ø 5.5 cm), placed in the middle of a stack of 5 standard petri dishes (8.5 cm Ø) (B), or in smaller containers with mesh on either side (A top), which were placed in a standard petri dish in the same location (A bottom), inside the small, 1 liter “insect box” provided with environmental chamber (C) of the X-RAD 320. The insect box holding the insects was programmed to rotate and flip automatically for each irradiation exposure. The dose uniformity ratio in the petri dish was 1.01. The chiller for the insect box was set to cool the samples to 7 °C for the chilled exposures. The X-RAD 320 was set at 320 kV and 12.5 mA for all exposures. Calculation of Dose Rates and Dosimetry Dose rates for each sample irradiation configuration were assessed and verified using a Farmer type 0.18-cm 3 free air ionization chamber (10 × 6 to 0.18, RadCal Corporation, Monrovia, California, USA) in conjunction with a digitizer and electrometer (AccuDose Model 9660A) as a reference dosimetry system to measure the dose rate and accumulated dose at a designated reference position. The ion chamber system was calibrated by the John Perry Laboratory (St George’s University Hospital Trust, London) with traceability to the National Physical Laboratory with a calibration factor of 1.0 and uncertainty of 3.3% (k = 2) in the energy range 40 to 1250 keV. Gafchromic HD-V2 (Lot # 02042102) or Maryland-V3 (Lot # 01222001) dosimetry films (International Specialty Products, New Jersey, USA) were packed in small (2 × 2 cm) paper envelopes which were placed near each insect sample to ensure the accuracy of the irradiation dose given in each irradiation event. A DoseReader 4 (Radiation General Ltd, Hungary) was used to read the films 24 h post-irradiation. The standard operating procedure for Gafchromic film dosimetry (FAO/IAEA 2022) was followed to determine the absorbed dose for each radiation event. The calibration used had an overall expanded uncertainty of 4.84% (k = 2, including the uncertainty value of the ion chamber). Rearing and Irradiation of Insects Mosquitoes Strains and Rearing The Ae. aegypti and An. arabiensis reference strains have been maintained at the IPCL following the “Guidelines for Routine Colony Maintenance of Aedes mosquitoes” ( FAO/IAEA 2017a ) and anopheline mass-rearing guidelines ( FAO/IAEA 2017b ), respectively. Aedes aegypti Dose Response in Pupae and Adults in Chilled vs. Non-Chilled Conditions Aedes aegypti eggs from 1 egg batch were collected and split in half to be hatched in 2 hatch events, 2 d apart (one for collecting adults, and one for collecting pupae for irradiation simultaneously). Pupae were collected in 4-h windows to ensure uniform pupal age. Pupae were sexed using a glass pupal sorter ( Focks 1980 ). Males were kept for irradiation and females were placed in individual tubes for emergence to ensure virginity for later mating. Adult males that emerged within an 8 h window were collected, batched in groups of 30, and kept in 15 × 15 × 15 cm Bugdorm cages (MegaView Science Co. Ltd, Taichung 40762, Taiwan). A sugar feeder was provided in each cage with a 5% sucrose solution. At the time of irradiation, pupae were >40 h old and adults were 24 to 32 h old. The doses for the dose-response curves for Ae. aegypti were selected according to the expected doses required to induce 50 to 100% sterility (in X-ray): 20, 55, and 70 Gy. Both the pupae and adults in each technical repetition were placed in 12 ml plastic cups with netted floors and lids, arranged around the center of a petri dish (8.5 cm Ø) ( Fig. 1A ). They were then irradiated in the X-RAD 320 at the same time at room temperature (22 to 24 °C). Another subset of adults from the same cohort was knocked down in the cold room (4 °C) for about 5 min, transferred into the small cups and irradiated with the same procedures and doses but with the environmental chamber of the X-RAD 320 chilled to 7 °C. The dose rate in the samples with the described settings was 3.9 Gy/min. Two biological repetitions each with 3 technical repetitions were performed for each treatment (pupae, adults, chilled vs non-chilled), dose and controls. Controls received the same handling (chilled and non-chilled) but were not irradiated. Anopheles arabiensis Dose Response in Chilled vs. Non-Chilled Adults Anopheles arabiensis pupae were collected and sexed visually using a stereomicroscope. Females were placed in individual tubes for emergence to ensure virginity and were kept for later mating. Batches of 30 male adults aged 24 to 36 h were knocked down in a cold room (4 °C) for approximately 5 min and transferred into the netted 12 ml plastic vials for irradiation ( Fig. 1A ). Those irradiated at room temperature were allowed to recover and warm up before irradiation, while those irradiated at chilled conditions were taken directly to the irradiator in a cooler box and placed in the chilled chamber of the X-RAD 320 for irradiation with 55, 70, and 90 Gy. The dose rate in the samples was 3.9 Gy/min. Two biological repetitions each with 3 technical repetitions were performed for each treatment (adults chilled vs non-chilled), dose and controls. Controls received the same handling (chilled and non-chilled) but were not irradiated. Assessment of Sterility For both species, following irradiation, the batches of male adults were placed in 15 × 15 × 15 cm Bugdorm cages and were supplied with a 5% sucrose solution. Aedes pupae were placed in cups with water in separate cages for emergence. Thirty virgin females were added to each cage when the adults reached 2 d of age (post-emergence) and were allowed to mate for 3 d before they were provided with 2 bloodmeals on consecutive days (days 6 & 7 post-emergence). Oviposition cups were added to each cage on day 8 for en masse egg collection (on days 9 & 10 post-emergence). Anopheles eggs were transferred to small cups with water for hatching directly after collection, and Aedes egg papers were slowly dried and stored for 10 d for egg maturation before hatching in cups with water and a drop of larval diet, closed with a lid ( FAO/IAEA 2017a , 2017b ). The total number of hatched and un-hatched eggs were counted using a stereomicroscope. Any non-hatched eggs were either opened with a dissection needle, or if many, were bleached to determine the fertility status ( FAO/IAEA 2019 ). Tsetse Strain and Rearing The Glossina palpalis gambiensis strain was originally established at Maisons-Alfort using wild material collected in Burkina Faso ( Mutika et al. 2013 ), the colony was later transferred to the Centre International de Recherche-Développement sur l’Elevage en zone Subhumide (CIRDES), in Burkina Faso. In 2009, the IPCL colony was established using pupae received from CIRDES. Maintained under laboratory conditions, the colony experiences a constant temperature and relative humidity (rH) of 24 ± 0.5 °C and 75 to 80%, respectively. It is also kept under subdued/indirect illumination, following a 12-h light/12-h dark photoperiod ( Feldmann 1992 ). Both the colony and experimental flies are fed 3 times per week using a defibrinated bovine blood diet with an artificial silicon membrane feeding system. G. p. gambiensis Dose Response of Pupae and Adult in Chilled vs. Non-Chilled Conditions Pupae were collected daily and stored in an incubator set at a constant temperature of 24 ± 0.5 °C and a relative humidity (rH) range of 75 to 80%. These pupae were subjected to sex sorting using the Near Infrared Pupae Sex Sorter (NIRPSS) approximately 23 to 24 d post larviposition ( Argilés-Herrero et al. 2023 ). Specifically, male pupae were selected from the cohort of pupae categorized as unmelanized. Male pupae (24 to 25-d-old) and newly emerged teneral male flies were immobilized under chilling (<3 min at 4 to 5 °C) and placed together in a small petri dish (Ø 5.5 cm), at the pre-defined standard reference point as described in the above section (“Irradiation set-up”) ( Fig. 1B ). The pupae and teneral males, co-located in the petri dishes, and were positioned immediately at the center of the radiation chamber. They were then irradiated with doses of 70, 90, 110, and 130 Gy either at room temperature or in chilled conditions at 7 °C. Overall, all samples were exposed to a total of 30 to 45 min of chilling treatment. The control group, which was not irradiated, underwent the same handling procedures. Regarding the cold treatment, the temperature was maintained at 7 °C throughout the irradiation period. Each treatment was replicated 3 times. Assessment of Sterility The control group consisted of non-irradiated pupae, and both irradiated and control pupae were handled and maintained under identical conditions. Incubation took place at 24 ± 0.5 °C and 75 to 80% relative humidity until emergence. Teneral males were placed in small cages [110 mm (Ø); 45 mm (H)] and fed following the previously described method until reaching sexual maturity. The flies irradiated as adults were also kept in cages and fed until their sexual maturity. In all treatment groups, 6 to 7-d-old male individuals were paired in standard colony cages (Ø 20 cm) with virgin females aged 3 to 4 d, maintaining a 1:2 male to female ratio for 4 d. Daily mortality monitoring was conducted. Subsequently, males and females were separated under chilling at 4 °C. The females were then transferred to 20 cm diameter cages, and their daily production and mortality were recorded over 60 d. The survival of males under the feeding regime in the small cages [110 mm (Ø); 45 mm (H)] was monitored for 90 d. Statistical Analysis Data were statistically analysed using R version 4.4.2 ( R Core Team 2024 ) under R studio version 2024.12.0 + 467 ( RStudio Team 2022 ). The tsetse pupae emergence rate was analysed using a generalized linear mixed model where the dose was considered as a fixed effect and the replicates as a random effect. The means comparison with the Tukey method was used to assess the difference between the irradiation dose treatments. Overdispersion was tested using the Pearson residual method (Bolker’s function), and no evidence of overdispersion was found. The random effect for replicate was included to account for between-batch variation, rather than to correct for overdispersion. An alternative model without the random effect was also fitted, and the inference regarding the fixed effect remained unchanged. Therefore, the random effect was retained in the final model to ensure design-based consistency and transparency. The induced sterility for the tsetse flies was calculated by subtracting from 100% (pupae production in the control group), the treatment production relative to control, which is obtained by dividing the pupae produced in each irradiation dose treatment by the pupae produced in the control group. All dose response data has been analysed using the dose response model under the drc package ( Ritz et al. 2015 ) with the drm function. The best model has been selected using the mselect function. Subsequently, the compParm function was employed to compare the curve parameters, and the ED function was utilized to determine the effective radiation doses that induce 50, 95, and 99% of sterility. The One Inflated Beta regression model has been utilized in conjunction with the dose-response model to compare the effectiveness of different treatments applied on tsetse. Furthermore, the residual fertility (RF) for the mosquitoes was calculated as a proportion of the control fertility of each treatment group (RF = HRtreatment/HRcontrol), where HRtreatment is the hatch rate of the treatment group, and HRcontrol is the hatch rate of the control group. Induced sterility (IS) was calculated by subtracting the RF from 1. The GLMM test was used to compare hatch rates between treatment groups. Ggplot2 ( Wickham 2016 ) was used to construct the dose-response curves for both tsetse and mosquitoes. Dose-Response Curve data used for Figs 6 and 7 were re-analyzed to support the relevant statements in the discussion section: Dose-response data for Aedes and Anopheles mosquitoes exposed to various irradiation dose rates were compiled from both original experiments and published sources. Data were analyzed to assess the relationship between dose rate and induced sterility. For each dose rate group, dose-response curves were fitted using Weibull functions and, where appropriate, logit-transformed logistic models. Parameters such as ED50 (dose for 50% sterility) and ED90 (dose for 90% sterility) were estimated from the fitted models. To evaluate differences between dose rates, visual comparisons of the fitted curves were conducted alongside numerical comparisons of ED50 and ED90 values. Where possible, bootstrapped confidence intervals were computed, and trend analyses (linear regression, quadratic regression, and nonparametric Spearman correlation) were used to detect systematic shifts in dose-response with increasing dose rate. Analyses were performed separately for Aedes and Anopheles . Fig. 6. Open in a new tab Dose–response curves for the induced sterility in Glossina palpalis gambiensis irradiated at room temperature versus chilled temperature (7 °C), fitted with the Weibull three-parameter model (W1.3). No significant difference was observed. Fig. 7. Open in a new tab Dose response curves of Aedes aegypti irradiated with X-ray and gamma-rays in various irradiators with increasing dose-rates (Gamma Beam GB127: 1.8 Gy/min; Theratron 780-c: 2.5 Gy/min; Raycell MK2: 7.7 Gy/min; RadSource RS2400: 9.11 Gy/min; GC220: 65 Gy/min; GC220: 84 Gy/min). The green line represents the results obtained with the X-RAD 320 (4 Gy/min) [The figure was modified from the original figure reported in ( Yamada et al. 2023b )]. Results The dosimetry confirmed that all doses received lay within the 4.84% uncertainty of the dosimetry system. Target dose values were used for the figures. The chilling in the environmental chamber was monitored throughout the duration of the exposures with both the built-in temperature probe of the X-RAD 320, as well as an added K-type thermocouple thermometer (Hawaii 93532R, HANNA Instruments, Woonsocket, Rhode Island), and was found to be very consistent for each exposure (7 °C ± 0.2), with temperatures only rising briefly when opening the environmental chamber to exchange samples. Mosquitoes Aedes aegypti Dose Response in Pupae, and Adults in Chilled vs. Non-Chilled Conditions For Ae. aegypti , >99% induced sterility was achieved with 55 Gy, regardless of life stage or exposure temperature. The best GLM model showed that the male sterility levels varied significantly according to the radiation dose (χ 2 = 3496.4; df = 2; P < 0.0001) as expected. There were also differences observed when comparing mosquito life stage (χ 2 = 66.0; df = 1; P < 0.0001). Concerning the life stage, at the lowest dose (20 Gy) the males irradiated as adults resulted in higher hatch rates in their offspring, meaning that the adult stage is more radioresistant as compared to that of the pupae (χ 2 = 62.098; df = 1; P < 0.0001) at this particular dose. At doses targeting higher sterility levels (50 to 70 Gy), and overall dose response curves of irradiated pupae and irradiated adults, there was no difference in radiosensitivity ( Fig. 2 ). Fig. 2. Open in a new tab Dose-response curve of the induced sterility of Aedes aegypti males irradiated at pupal or adult stage, fitted with the Weibull three-parameter (W1.3) model shows no significant difference in curve parameters. When considering the temperature effect, chilled males were slightly less sterile at the lowest tested dose of 20 Gy, but the difference was not significant (F = 2.084, P = 0.179), ( Fig. 3 ). Fig. 3. Open in a new tab Dose-response curve of the induced sterility of Aedes aegypti male adults following irradiation at room temperature versus chilled temperatures (7 °C), fitted with the log-logistic three-parameter (LL.3) model shows no significant difference in curve parameters. Anopheles arabiensis Dose Response in Chilled vs. Non-Chilled Adults For An. arabiensis , >99% induced sterility was reached with the tested dose of 90 Gy. Using the dose response model with the fitted model of Log-logistic 3-parameter (LL.3), the estimated effective doses that induce 50%, 95% and 99% of sterility are respectively 50.8 Gy, 72.4 Gy, 80.4 Gy ( Fig. 3 ). The induced sterility again varied significantly according to the radiation dose (χ 2 = 133.656; df = 2; P < 0.0001) as expected, but there was no significant difference between chilled and non-chilled treatment groups (F = 0.948, P = 0.353), ( Fig. 4 ). Fig. 4. Open in a new tab Dose-response curve of the induced sterility of Anopheles arabiensis adult males, following irradiation at room temperature versus chilled temperatures (7 °C), fitted with the log-logistic three-parameter (LL.3), shows no significant difference in curve parameters. However, based on model predictions (dashed lines), the non-chilled adult males are more radiosensitive at low doses (20 to 50 Gy). Tsetse Glossina palpalis gambiensis Dose Response to Irradiation as Pupae Under Chilled vs. Non-Chilled Conditions Adult emergence rate. When comparing the adult emergence rate while considering the cold treatment using the X-RAD 320 chilled environmental chamber, neither the temperature (χ 2 = 0.0109; df = 1; P = 0.917) nor the radiation dose (χ 2 = 0.6707; df = 4; P = 0.955) had a significant impact on the adult emergence rates ( Fig. 5 ). Fig. 5. Open in a new tab Adult emergence rate of male Glossina palpalis gambiensis irradiated as pupae in chilled vs. non-chilled conditions was analyzed using a generalized linear mixed model where the dose was considered as a fixed effect and the replicates as a random effect. The boxplot shows the median and upper and lower quartiles. Dots represent experimental data. Induced Sterility Modelling the induced sterility data using the One Inflated Beta regression showed that there was no significant difference in the sterility according to the temperature treatment ( P = 0.291) or the insect life stage ( P = 0.063) ( Table 1 ). Using the ED function allowed the determination of the estimated doses that induce 50, 95, and 99% of sterility ( Table 2 ). The doses responses curves presented in Fig. 6 shows that in chilled and non-chilled conditions, the pupae were slightly sensitive as compared to the adults at the lower radiation dose. Table 1. Glossina palpalis gambiensis dose response to irradiation as pupae under chilled vs. non-chilled conditions Estimate Std. Error t value P -value (Intercept) 2.6375 0.2709 9.735 9.5e-12 Temperature-Non-chilled −0.3573 0.3334 −1.072 0.2908 Stage-Pupae 0.6460 0.3366 1.919 0.0627 Open in a new tab Statistics of the regression of induced sterility in relation to temperature treatment and life stage using the 1 inflated beta model. Table 2. Estimated radiation doses inducing 50%, 95%, and 99% sterility in females Glossina palpalis gambiensis mated with males irradiated as pupae or adults under chilled and non-chilled conditions using the ED function Stage Treatments Estimated effective doses (Gy) 50% 95% 99% Pupae Chilled 45.1 72.3 97.3 Non-chilled 41.9 85.3 133.1 Adult Chilled 57.5 88.2 115.2 Non-chilled 50.3 86.3 121.0 Open in a new tab Discussion Using the Precision X-ray X-RAD 320 irradiator, pupae and adults of Ae. aegypti and G. p. gambiensis showed similar levels of radiosensitivity regardless of whether they were chilled or not chilled. Ae. aegypti males irradiated at adult stage exhibited similar levels of sterility compared to late-stage pupae irradiation, except at the lowest tested dose of 20 Gy, where males irradiated as adults were less sterile. Previous studies on the effects of development stage on dose response in this species reported a small, albeit significant difference in dose response ( Yamada et al. 2022b ), however, in this case, irradiated adults were the ones more radiosensitive, similar to the findings of Helinski et al for An. arabiensis ( Helinski et al. 2006 ). The strain and methodology were almost identical to the current pupae-adult comparative study, however, the previous study was performed using a Gammacell 220 (Nordion Inc., Canada) with significantly higher dose rates ( Yamada et al. 2022a ). Whether this difference in dose rates is the reason for the less pronounced difference observed here is not known. Similarly, the effects of chilling on the dose response of An. arabiensis and Ae. aegypti male adults were insignificant in this study, whereas the difference was small but significant in the study discussed above ( Yamada et al. 2022b ). This discrepancy could also be due to differences in the irradiator used (Gammacell 220), duration of the irradiation exposure, and thus also the chilling treatment, as well as the tested dose(s). For the previous study, a diagnostic dose of 45 Gy was used (in order to achieve sterility levels well under 99% for better comparison). At the time of the experiment, the Gammacell 220 had a dose rate of 65 Gy/min, providing an exposure time of around 42 s. However, the samples were kept at 7 °C for 1 h prior to irradiation ( Yamada et al. 2022b ). In the experiment reported here, the adult males were knocked down in the cold room and taken directly for exposure in the chilled chamber of the X-Rad320, which lasted 5 to 18 min (depending on target dose). This duration of chilling may not have been sufficient for observing any radioprotectant effects. This may also imply that the shorter overall chilling treatment may also reduce any negative effects on other quality parameters, such as flight ability. Although prolonged chilling can reduce overall flight ability when tested directly afterwards, a 2-d recovery period after chilling has been shown to fully reinstate flight capacity in Ae. aegypti and Ae. albopictus ( Maïga et al. 2022 , Yamada et al. 2022a ), for which Zhang et al. (2020) found the optimal treatment to be 5 to 10 °C, for a maximum of 3 h. Chilling was also shown not to reduce longevity of males exposed to 7 °C for 1 h in Ae aegypti ( Yamada et al. 2022b ), corroborating the results for this species tested at 4 to 10 °C for 1 to 24 h, and 2 to 10 °C for Ae. albopictus , which were found to be more sensitive to cold temperatures ( Culbert et al. 2019 ). Culbert et al. (2019) also reported no negative effects on longevity in An. arabiensis following cold treatments (4 to 10 °C) for up to 24 h. This level of tolerance provides ample time for handling, marking, packing, irradiation, and transport procedures while the sterile males are kept immobile, protecting them from excessive mechanical damage and potential added stress from, for example, being subjected to multiple knock-down events. It is important, however, to identify the threshold for the temperature and duration of the cold treatment beyond which the mosquitoes begin to show negative effects on downstream quality parameters. Regarding the dose response in the tsetse in this study, no significant differences were observed when G. p. gambiensis pupae or adults were irradiated under chilled and non-chilled conditions. Our results align with other studies on G. p. gambiensis , which showed that the cold treatment exhibited no discernible effect on the emergence rate ( Mutika et al. 2014 , 2019 ). The emergence rate ranged from 89.1% to 96.9% and was higher than the 83 to 91% and the 64% obtained for G. p. gambiensis and G. m. morsitans , respectively, stored at 9.2 ± 0.4 °C and 10 ± 1 °C ( Williamson et al. 1983 , Mutika et al. 2019 ). These observations imply that the potential impact of cold, whether protective or not, appeared to be exposure-time-dependent ( Yamada et al. 2022b ). However, additional stress factors, such as transportation and handling, may have contributed to the previous observed outcomes, such as reduced emergence rates and flight ability ( Diallo et al. 2019 ). Our research revealed that in both chilled and non-chilled conditions, the adults survived longer when irradiated as adults as compared to those irradiated at the pupal stage ( Kaboré 2024 ). This observation aligns with the existing body of knowledge in this domain, which indicates that storage of the pupae in low temperature followed by irradiation may result in a decrease of the survival time in G. p. gambiensis and G. m. morsitans ( Curtis and Langley 1971 , Mutika et al. 2019 ). As seen in the mosquitoes in this case, the cold treatment for both pupae and adult tsetse did not demonstrate a radioprotective effect. The pupae displayed a slightly higher sensitivity in both chilled and non-chilled conditions and corroborate the results on G.m. morsitans when pupae were exposed to 2 °C prior to irradiation ( Curtis and Langley 1971 ). This is consistent with the role of the life stage in insect radiosensitivity. In this regard, 25-d-old pupae are biologically very close to the fully developed teneral flies, which emerge around 33 d post-larviposition ( Kaboré et al. 2023 ), explaining the absence of a significant difference, as spermatogenesis occurs during the larval/pupal stage of tsetse ( Itard 1971 , Helinski et al. 2009 ). A level of 99% or higher sterility was achieved at slightly lower doses in the X-RAD 320 than when exposing the same strains of mosquitoes with the same handling and exposure protocols ( FAO/IAEA 2019 , Yamada et al. 2019 , Yamada et al. 2022a ) in the other irradiators available at the IPCL [Foss Model 812 gamma irradiator {Foss Therapy Services Inc., North Hollywood, California, USA}, Gammacell 220 irradiator {Nordion Ltd, Kanata, ON, Canada}, Raycell MK2 X-ray blood irradiator {Best Theratronics Ltd, Kanata, ON, Canada}, and RadSource RS 2400 X-ray irradiator {Rad Source Technologies Inc., Suwanee, Georgia}], and were comparable to historical data in available literature. As has been studied extensively and reported by Yamada et al. (2022a ) and Yamada et al. (2023a ), irradiation dose-rate again seems to be an important factor that affects dose response in these comparative studies. When plotting the results obtained for Ae. aegypti and An. arabiensis in this study together with the dose response curves from previous experiments and available literature [reported in Yamada et al. (2023b )], the new sterility curves fit in with the other curves according to dose rate ( Figs 7 and 8 , respectively). Thus, we re-evaluated the available dose-response curves to highlight a decreasing response trend with increasing dose rates for Aedes and Anopheles mosquitoes using both Weibull and logit-transformed logistic models. Dose rates examined ranged from approximately 1.8 to 93 Gy/min, across datasets compiled from both experimental results and values extracted from the published literature. In Aedes , Weibull model fitting revealed a consistent rightward shift in dose-response with increasing dose rate. ED50 values increased from ∼9 Gy at 2.5 to 4 Gy/min to ∼17 to 18 Gy at 7.7 to 84 Gy/min, indicating reduced sterilization efficiency at higher dose rates. Although linear regression and nonparametric trend tests yielded borderline statistical significance (Spearman ρ = 0.77, P = 0.072), bootstrapped ED50 confidence intervals reinforced this trend, especially for 7.7 Gy/min. These results align with radiobiological principles predicting that higher dose rates may reduce the biological effectiveness of ionizing radiation by limiting damage accumulation time ( Yamada et al. 2023a , and references therein). Fig. 8. Open in a new tab Dose response curves of Anopheles arabiensis irradiated with X-ray and gamma-rays in various irradiators with increasing dose-rates (Raycell MK2: 7.7 Gy/min; GC220: 16 Gy/min; GC220: 74 Gy/min; GC220: 84 Gy/min; GC220: 93 Gy/min). The green × points represent the results obtained with the X-RAD 320 (4 Gy/min) [The figure was modified from the original figure reported in ( Yamada et al. 2023b )]. In Anopheles , initial Weibull models suggested minimal dose rate effects, with most groups displaying saturated responses and identical ED50s (∼0.69 Gy), except for 16 Gy/min (ED50 ≈ 34.6 Gy). To better resolve curve differences, we applied logit transformations and fitted logistic models. This approach revealed clearer distinctions: while dose rates of 7.7, 74, and 84 Gy/min showed similar ED50s (25 to 27 Gy), higher values were observed for 16 and 93 Gy/min (36.5 and 54.1 Gy, respectively), suggesting decreased sterilization efficiency at both intermediate and high dose rates. Importantly, we acknowledge that some dose-response data—particularly for Anopheles —were extracted from previously published sources, which limited access to full replicate-level datasets. As a result, the precision and robustness of some fits may be constrained, and statistical power may be limited in certain comparisons. Nonetheless, the models provide consistent visual and quantitative evidence of a biologically relevant trend: higher dose rates may require higher total doses to achieve equivalent levels of sterility. To further assess the impact of dose rate on the sterilization response, we estimated ED90 values—the radiation dose required to achieve 90% sterility—for Aedes and Anopheles mosquitoes. In Aedes , Weibull model fits showed a clear dose rate-dependent increase in ED90: from 11.96 Gy at 4 Gy/min to 21.87 Gy at 65 Gy/min, and 23.17 Gy at 84 Gy/min. This represents nearly a 2-fold increase in the required dose to achieve target sterility at higher dose rates, consistent with the rightward shift observed in the corresponding dose-response curves and ED50 estimates. In contrast, Anopheles exhibited a more modest ED90 increase across similar dose rates. Logit-transformed logistic models yielded LD90 estimates of 39.31 Gy at 74 Gy/min and 41.36 Gy at 84 Gy/min, suggesting that while a slight rightward trend may exist, the dose rate effect on sterility is less pronounced in Anopheles. These findings may suggest a species-specific sensitivity to dose rate, however, it is more likely that the limited accessible datasets for dose response in referenced literature (for Anopheles ) is the reason for the less pronounced differences that we could identify here. Overall, these findings point to the potential for reduced efficiency at high or intermediate dose rates, and we anticipate that more dose response studies in the future will support these findings. A similar series of studies to assess dose response in tsetse report the same phenomenon, where X-irradiation achieved the target sterility at lower doses ( Mastrangelo et al. 2010 , Yamada et al. 2023b ). The effectiveness of 90 Gy aligns with earlier findings on this species ( Yamada et al. 2023a ) and emphasizes the suitability of the (chilled) environmental chamber and X-ray source for G. p. gambiensis sterilization. It is important to note that for this series of irradiation exposures, optimum conditions and set-up were aimed for. The container and volume used to hold the insect samples were kept very small to ensure a very high dose uniformity within the sample (DUR ∼0.01, as opposed to DUR 1.32 within the large sample box ( Parker et al. 2025 )) and all samples were accompanied with dosimetric film to ensure that any differences observed in dose response were not dose effects. Throughout the experiment, it was also ensured that all insects were the same age to avoid any age effects on dose response. In this and former studies by the authors, late-stage pupae were used in pupal dose response studies, as these are the most radioresistant and biologically very close to adults. In operational settings, such control and optimization are difficult to achieve, and biological and external effects are inevitable. Therefore, quality assurance and quality control protocols should be in place for the standardization of processes and detection of any deviations from expected outcomes. Overall, the X-RAD 320 together with its specialized environmental chamber provides a very versatile irradiator as numerous factors that are important in insect dose response can be tested, adjusted, and optimized. The highly stable climatic environment provides a suitable radiation exposure environment for insects at both pupal and adults stages, especially for those species that require high sterilizing doses and thus prolonged exposures in chilled conditions, such as moths, and potentially beetles. Ice packs or phase change material can only maintain stable temperatures for a limited period. Fluctuations in temperature are also likely to contribute to the overall stress budget that would cause a decline in sterile male quality. The possibility to irradiate in hypoxic or anoxic conditions also provides added benefit and opportunities to optimize irradiation protocols to potentially improve insect quality significantly, as has been shown in previous studies ( Baldwin and Salthouse 1959 , Curtis and Langley 1972 , Economopoulos 1977 , Ohinata et al. 1977 , Rananavare et al. 1991 ). Further investigations into energy effects are planned with this particular unit which can be performed by changing the settings of the X-RAD 320 or by adjusting filtration. Acknowledgements We would like to thank Mr. William McLaughlin, Director, Strategic Innovation, Precision X-ray Inc., for the training and guidance on the use of the X-RAD 320 and the SIT module, and Precision X-ray Inc. for the loan of the X-RAD 320 for the duration of this study. Contributor Information Hanano Yamada, Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria. Bénéwendé Aristide Kaboré, Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria. Samar Eisa, Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria; Department of Epidemiology, Tropical Medicine Research Institute, Khartoum, Sudan. Didier Alexandre Kaboré, Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria; Institut de Recherche en Sciences de la Santé (IRSS), Bobo-Dioulasso, Burkina Faso. Andrew Gordon Parker, Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria. Odet Bueno, Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria. Wadaka Mamai, Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria. Thomas Wallner, Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria. Chantel Janet de Beer, Insect Pest Control Section, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria. Author Contributions Hanano Yamada (Conceptualization [lead], Data curation [equal], Investigation [equal], Methodology [equal], Writing—original draft [lead], Writing—review & editing [equal]), Bénéwendé Aristide Kaboré (Data curation [equal], Formal analysis [lead], Investigation [equal], Methodology [equal], Writing—original draft [equal], Writing—review & editing [equal]), Samar Yousif Mohamed Eisa (Investigation [supporting], Methodology [supporting]), Didier Alexandre Kaboré (Formal analysis [supporting], Investigation [equal], Writing—review & editing [supporting]), Andrew Gordon Parker (Investigation [supporting], Validation [supporting], Writing—review & editing [supporting]), Odet Bueno (Investigation [supporting], Resources [supporting]), Wadaka Mamai (Formal analysis [supporting], Resources [supporting], Validation [supporting], Writing—review & editing [supporting]), Thomas Wallner (Data curation [supporting], Investigation [supporting], Resources [supporting]), and Chantel Janet de Beer (Formal analysis [supporting], Funding acquisition [equal], Project administration [equal], Supervision [equal], Writing—review & editing [equal]) Funding The research presented in this paper was funded by the United States of America under the grant to the IAEA entitled: Enhance Agency’s Capacity to Provide Support to Member States to Control Aedes Mosquitoes as Vectors of Human Pathogens, Particularly Zika Virus, Using Integrated Vector Management Approaches with a Sterile Insect Technique Component. 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