Life-history traits of a fluorescent Anopheles arabiensis genetic sexing strain introgressed into South African genomic background.

BACKGROUND: South Africa has set a mandate to eliminate local malaria transmission by 2023. In pursuit of this objective a Sterile Insect Technique programme targeting the main vector Anopheles arabiensis is currently under development. Significant progress has been made towards operationalizing the technology. However, one of the main limitations being faced is the absence of an efficient genetic sexing system. This study is an assessment of an An. arabiensis (AY-2) strain carrying the full Y chromosome from Anopheles gambiae, including a transgenic red fluorescent marker, being introgressed into a South African genetic background as a potential tool for a reliable sexing system. METHODS: Adult, virgin males from the An. arabiensis AY-2 strain were outcrossed to virgin females from the South African, Kwazulu-Natal An. arabiensis (KWAG strain) over three generations. Anopheles arabiensis AY-2 fluorescent males were sorted as first instar larvae (L1) using the Complex Object Parametric Analyzer and Sorter (COPAS) and later screened as pupae to verify the sex. Life history traits of the novel hybrid KWAG-AY2 strain were compared to the original fluorescent AY-2 strain, the South African wild-type KWAG strain and a standard laboratory An. arabiensis (Dongola reference strain). RESULTS: The genetic stability of the sex-linked fluorescent marker and the integrity and high level of sexing efficiency of the system were confirmed. No recombination events in respect to the fluorescent marker were detected over three rounds of introgression crosses. KWAG-AY2 had higher hatch rates and survival of L1 to pupae and L1 to adult than the founding strains. AY-2 showed faster development time of immature stages and larger adult body size, but lower larval survival rates. Adult KWAG males had significantly higher survival rates. There was no significant difference between the strains in fecundity and proportion of males. KWAG-AY2 males performed better than reference strains in flight ability tests. CONCLUSION: The life history traits of KWAG-AY2, its rearing efficiency under laboratory conditions, the preservation of the sex-linked fluorescence and perfect sexing efficiency after three rounds of introgression crosses, indicate that it has potential for mass rearing. The potential risks and benefits associated to the use of this strain within the Sterile Insect Technique programme in South Africa are discussed.

A rapid quality control test to foster the development of the sterile insect technique against Anopheles arabiensis.

BACKGROUND: With the fight against malaria reportedly stalling there is an urgent demand for alternative and sustainable control measures. As the sterile insect technique (SIT) edges closer to becoming a viable complementary tool in mosquito control, it will be necessary to find standardized techniques of assessing male quality throughout the production system and post-irradiation handling. Flight ability is known to be a direct marker of insect quality. A new version of the reference International Atomic Energy Agency/Food and Agricultural Organization (IAEA/FAO) flight test device (FTD), modified to measure the flight ability and in turn quality of male Anopheles arabiensis within a 2-h period via a series of verification experiments is presented. METHODS: Anopheles arabiensis juveniles were mass reared in a rack and tray system. 7500 male pupae were sexed under a stereomicroscope (2500 per treatment). Stress treatments included irradiation (with 50, 90, 120 or 160 Gy, using a Gammacell 220), chilling (at 0, 4, 8 and 10 °C) and compaction weight (5, 15, 25, and 50 g). Controls did not undergo any stress treatment. Three days post-emergence, adult males were subjected to either chilling or compaction (or were previously irradiated at pupal stage), after which two repeats (100 males) from each treatment and control group were placed in a FTD to measure flight ability. Additionally, one male was caged with 10 virgin females for 4 days to assess mating capacity (five repeats). Survival was monitored daily for a period of 15 days on remaining adults (two repeats). RESULTS: Flight ability results accurately predicted male quality following irradiation, with the first significant difference occurring at an irradiation dose of 90 Gy, a result which was reflected in both survival and insemination rates. A weight of 5 g or more significantly reduced flight ability and insemination rate, with survival appearing less sensitive and not significantly impacted until a weight of 15 g was imposed. Flight ability was significantly reduced after treatments at 4 °C with the insemination rate more sensitive to chilling with survival again less sensitive (8 and 0 °C, respectively). CONCLUSIONS: The reported results conclude that the output of a short flight ability test, adapted from the previously tested Aedes FTD, is an accurate indicator of male mosquito quality and could be a useful tool for the development of the SIT against An. arabiensis.

Does mosquito mass-rearing produce an inferior mosquito?

BACKGROUND: The success of the sterile insect technique depends, among other things, on continuous releases of sexually competitive sterile males within the target area. Several factors (including high rearing density and physical manipulation, such as larvae and pupae separation) can influence the quality of males produced in mass-rearing facilities. The different steps in mass production in the laboratory may modify the behaviour of mosquitoes, directly or through loss of natural characters as a result of adaptation to lab rearing, and lead to the competitiveness of sterile male being reduced. In the present study, the objective was to evaluate the effect of mass-rearing conditions on sterile male sexual competitiveness in semi-field cages compared to routine small scale laboratory rearing methods. METHODS: Anopheles arabiensis immature stages were reared both on a large scale using a rack and tray system developed by the FAO/IAEA (MRS), and on a small scale using standard laboratory rearing trays (SRS). Mosquito life history traits such as pupation rate, emergence rate, adult size as well as the effect of irradiation on adult longevity were evaluated. Moreover, 5-6 day old mosquitoes were released into field cages and left for two nights to mate and the mating competitiveness between sterile mass-reared males and fertile males reared on a small scale when competing for small scale reared virgin females was investigated. Resulting fertility in a treatment ratio of 1:1:1 (100 irradiated males: 100 non-irradiated males: 100 virgin females) was compared to control cages with 0:100:100 (non-irradiated control) and 100:0:100 (irradiated control). RESULTS: No significant differences in life history parameters were observed between rearing methods. The competitiveness index of mass reared males (0.58) was similar to males reared on a small scale (0.59). A residual fertility rate of 20% was observed in the irradiated control (100:0:100), measured as the percentage of eggs collected from the cages which developed to adulthood. No significant difference was observed (t = 0.2896, df = 4, P = 0.7865) between the rearing treatments (MRS and SRS) in the fertility rate, a measure of mating competitiveness. CONCLUSIONS: The results showed that the FAO/IAEA mass-rearing process did not affect mosquito life history parameters or the mating competitiveness of males.

The effects of genetic manipulation, dieldrin treatment and irradiation on the mating competitiveness of male Anopheles arabiensis in field cages.

BACKGROUND: To enable the release of only sterile male Anopheles arabiensis mosquitoes for the sterile insect technique, the genetic background of a wild-type strain was modified to create a genetic sexing strain ANO IPCL1 that was based on a dieldrin resistance mutation. Secondly, the eggs of ANO IPCL1 require treatment with dieldrin to allow complete elimination of female L1 larvae from the production line. Finally, male mosquito pupae need to be treated with an irradiation dose of 75 Gy for sterilization. The effects of these treatments on the competitiveness of male An. arabiensis were studied. METHODS: The competitiveness of ANO IPCL1 males that were treated either with irradiation or both dieldrin and irradiation, was compared with that of the wild-type strain (An. arabiensis Dongola) at a 1:1 ratio in 5.36 m3 semi-field cages located in a climate-controlled greenhouse. In addition, three irradiated: untreated male ratios were tested in semi-field cages (1:1, 5:1 and 10:1) and their competition for virgin wild-type females was assessed. RESULTS: The ANO IPCL1 males were equally competitive as the wild-type males in this semi-field setting. The ANO IPCL1 males irradiated at 75 Gy were approximately half as competitive as the unirradiated wild-type males. ANO IPCL1 males that had been treated with dieldrin as eggs, and irradiated with 75 Gy as pupae were slightly more competitive than males that were only irradiated. Ratios of 1:1, 5:1 and 10:1 irradiated ANO IPCL1 males: untreated wild-type males resulted in 31, 66 and 81% induced sterility in the female cage population, respectively. CONCLUSIONS: An irradiation dose of 75 Gy reduced the competitiveness of male ANO IPCL1 significantly and will need to be compensated by releasing higher numbers of sterile males in the field. However, the dieldrin treatment used to eliminate females appears to have an unexpected radioprotectant effect, however the mechanism is not understood. A sterile to wild-type ratio of 10:1 effectively reduced the population's fertility under the experimental field cage conditions, but further studies in the field will be needed to confirm the efficiency of sterile ANO IPCL1 males when competing against wild males for wild females.

Sperm Storage and Use Following Multiple Insemination in Aedes albopictus: Encouraging Insights for the Sterile Insect Technique.

The key to success in the application of the sterile insect technique (SIT) relies on the ability of released, sterile males to outcompete their fertile wild male counterparts to mate with wild females. However, many insect species exhibit multiple-mating behavior, which can be a way for females to select paternity for their progeny. This study aims to recognize the consequences of potential double-matings during an SIT program and to detect any evidence of sperm selection favoring sperm from fertile mates. This report provides a descriptive analysis of the storage and use of sperm by female Aedes albopictus. Stable isotopes were used to mark the sperm of fertile and sterile males. Mated females were allowed to oviposit before dissecting the spermathecae to link the presence of each type of sperm to the sterility of the eggs laid. It was found that sperm in females inseminated by both males was distributed in the three spermathecae with no obvious pattern, mostly mixed but also separately, and no evidence of any mechanism for sperm selection, sperm precedence, or sperm competition in Ae. albopictus females could be found. The fact that only a few double-mated females were double-inseminated and could also produce semi-sterile eggs, together with the finding that the sperm of sterile males appeared to be no less viable than that of fertile males, is an encouraging outcome for SIT approaches.

Upscaling irradiation protocols of Aedes albopictus pupae within an SIT program in Reunion Island.

The implementation of the sterile insect technique against Aedes albopictus relies on many parameters, in particular on the success of the sterilization of males to be released into the target area in overflooding numbers to mate with wild females. Achieving consistent sterility levels requires efficient and standardized irradiation protocols. Here, we assessed the effects of exposure environment, density of pupae, irradiation dose, quantity of water and location in the canister on the induced sterility of male pupae. We found that the irradiation of 2000 pupae in 130 ml of water and with a dose of 40 Gy was the best combination of factors to reliably sterilize male pupae with the specific irradiator used in our control program, allowing the sterilization of 14000 pupae per exposure cycle. The location in the canister had no effect on induced sterility. The results reported here allowed the standardization and optimization of irradiation protocols for a Sterile Insect Technique program to control Ae. albopictus on Reunion Island, which required the production of more than 300,000 sterile males per week.

Mating harassment may boost the effectiveness of the sterile insect technique for Aedes mosquitoes.

The sterile insect technique is based on the overflooding of a target population with released sterile males inducing sterility in the wild female population. It has proven to be effective against several insect pest species of agricultural and veterinary importance and is under development for Aedes mosquitoes. Here, we show that the release of sterile males at high sterile male to wild female ratios may also impact the target female population through mating harassment. Under laboratory conditions, male to female ratios above 50 to 1 reduce the longevity of female Aedes mosquitoes by reducing their feeding success. Under controlled conditions, blood uptake of females from an artificial host or from a mouse and biting rates on humans are also reduced. Finally, in a field trial conducted in a 1.17 ha area in China, the female biting rate is reduced by 80%, concurrent to a reduction of female mosquito density of 40% due to the swarming of males around humans attempting to mate with the female mosquitoes. This suggests that the sterile insect technique does not only suppress mosquito vector populations through the induction of sterility, but may also reduce disease transmission due to increased female mortality and lower host contact.

Radiation dose-fractionation in adult Aedes aegypti mosquitoes.

Balancing process efficiency and adult sterile male biological quality is one of the challenges in the success of the sterile insect technique (SIT) against insect pest populations. For the SIT against mosquitoes, many stress factors need to be taken into consideration when producing sterile males that require high biological quality to remain competitive once released in the field. Pressures of mass rearing, sex sorting, irradiation treatments, packing, transport and release including handling procedures for each step, add to the overall stress budget of the sterile male post-release. Optimizing the irradiation step to achieve maximum sterility while keeping off-target somatic damage to a minimum can significantly improve male mating competitiveness. It is therefore worth examining various protocols that have been found to be effective in other insect species, such as dose fractionation. A fully sterilizing dose of 70 Gy was administered to Aedes aegypti males as one acute dose or fractionated into either two equal doses of 35 Gy, or one low dose of 10 Gy followed by a second dose of 60 Gy. The two doses were separated by either 1- or 2-day intervals. Longevity, flight ability, and mating competitiveness tests were performed to identify beneficial effects of the various treatments. Positive effects of fractionating dose were seen in terms of male longevity and mating competitiveness. Although applying split doses generally improved male quality parameters, the benefits may not outweigh the added labor in SIT programmes for the management of mosquito vectors.

Title: Fractionnement de la dose d'irradiation chez les moustiques Aedes aegypti adultes. Abstract: Équilibrer l'efficacité du processus et la qualité biologique des mâles adultes stériles est l'un des défis du succès de la technique des insectes stériles (TIS) contre les populations d'insectes nuisibles. Pour la TIS contre les moustiques, de nombreux facteurs de stress sont à prendre en compte lors de la production de mâles stériles qui nécessitent une haute qualité biologique pour rester compétitifs une fois relâchés au champ. Les pressions de l'élevage en masse, du triage par sexe, des traitements d'irradiation, de l'emballage, du transport et de la libération, y compris les procédures de manipulation pour chaque étape, s'ajoutent au budget de stress global du mâle stérile après la libération. L'optimisation de l'étape d'irradiation pour atteindre une stérilité maximale tout en minimisant les dommages somatiques hors cible peut améliorer considérablement la compétitivité de l'accouplement des mâles et il est donc important d'examiner divers protocoles qui se sont révélés efficaces chez d'autres espèces d'insectes, comme le fractionnement de dose. Une dose entièrement stérilisante de 70 Gy a été administrée aux mâles Aedes aegypti en une dose unique ou fractionnée en deux doses égales de 35 Gy, ou une faible dose de 10 Gy suivie d'une seconde dose de 60 Gy. Les deux doses étaient séparées par des intervalles de 1 ou 2 jours. Des tests de longévité, d'aptitude au vol et de compétitivité à l'accouplement ont été réalisés pour identifier les effets bénéfiques des différents traitements. Des effets positifs de la dose de fractionnement ont été observés en termes de longévité des mâles et de compétitivité à l'accouplement. Bien que l'application de doses fractionnées améliore généralement les paramètres de qualité des mâles, les avantages peuvent ne pas compenser le travail supplémentaire dans les programmes TIS pour la gestion des moustiques vecteurs.

Suitability of Raycell MK2 Blood X-ray Irradiator for the Use in the Sterile Insect Technique: Dose Response in Fruit Flies, Tsetse Flies and Mosquitoes.

The sterile insect technique (SIT) is based on the inundatory field release of a target pest following their reproductive sterilization via exposure to radiation. Until recently, gamma irradiation from isotopic sources has been the most widely used in SIT programs. As isotopic sources are becoming increasingly expensive, especially for small programs, and regulations surrounding their procurement and shipment increasingly strict, irradiation capacity is one of the limiting factors in smaller or newly developing SIT projects. For this reason, the possibility of using X-ray irradiators has been evaluated in the recent decade. The availability of "off-the-shelf" blood X-ray irradiators that meet the technical requirements for insect irradiation can provide irradiation capacity for those SIT projects in which the acquisition of gamma ray irradiators is not feasible. Following the recent technical characterization of a Raycell MK2 X-ray blood irradiator, it was found in this study, that MK2 instruments were suitable for the sterilization of fruit flies, tsetse flies and mosquitoes, inducing comparable, even slightly higher, sterility levels compared to those achieved by gamma ray irradiation. This, together with its estimated processing efficiency, shows that MK2 irradiators are suitable for small- to mid-sized SIT programs.

From the Lab to the Field: Long-Distance Transport of Sterile Aedes Mosquitoes.

Pilot programs of the sterile insect technique (SIT) against Aedes aegypti may rely on importing significant and consistent numbers of high-quality sterile males from a distant mass rearing factory. As such, long-distance mass transport of sterile males may contribute to meet this requirement if their survival and quality are not compromised. This study therefore aimed to develop and assess a novel method for long-distance shipments of sterile male mosquitoes from the laboratory to the field. Different types of mosquito compaction boxes in addition to a simulation of the transport of marked and unmarked sterile males were assessed in terms of survival rates/recovery rates, flight ability and morphological damage to the mosquitoes. The novel mass transport protocol allowed long-distance shipments of sterile male mosquitoes for up to four days with a nonsignificant impact on survival (>90% for 48 h of transport and between 50 and 70% for 96 h depending on the type of mosquito compaction box), flight ability, and damage. In addition, a one-day recovery period for transported mosquitoes post-transport increased the escaping ability of sterile males by more than 20%. This novel system for the long-distance mass transport of mosquitoes may therefore be used to ship sterile males worldwide for journeys of two to four days. This study demonstrated that the protocol can be used for the standard mass transport of marked or unmarked chilled Aedes mosquitoes required for the SIT or other related genetic control programs.

Effects of radiation on the fitness, sterility and arbovirus susceptibility of a Wolbachia-free Aedes albopictus strain for use in the sterile insect technique.

BACKGROUND: The sterile insect technique (SIT) is a green and species-specific insect pest control technique that suppresses target populations by releasing factory-reared, radiosterilized males into the wild. Once released, it is important to be able to distinguish the released males from the wild males for monitoring purposes. Several methods to mark the sterile males exist. However, most have limitations due to monetary, process efficiency, or insect quality. Aedes albopictus is naturally infected with Wolbachia at a high prevalence, therefore the elimination of Wolbachia can serve as a biomarker to distinguish factory-reared male mosquitoes from wild conspecifics. RESULTS: In this study, a Wolbachia-free Ae. albopictus GT strain was developed and its fitness evaluated, which was found to be comparable to the wild GUA strain. In addition, GT male mosquitoes were irradiated at the adult stage and a dose of 20 Gy or more induced over 99% sterility. Moreover, a dose of 30 Gy (almost completely sterilizing male and female mosquitoes) had limited effects on the mating competitiveness of GT males and the vector competence of GT females, respectively. However, radiation reduced mosquito longevity, regardless of sex. CONCLUSION: Our results indicate that the Ae. albopictus GT strain can be distinguished from wild mosquitoes based on Wolbachia status and shows similar fitness, radio-sensitivity and arbovirus susceptibility to the GUA strain, indicating that it is feasible to use the GT strain to suppress Ae. albopictus populations for SIT programmes. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Genetic sex separation of the malaria vector, Anopheles arabiensis, by exposing eggs to dieldrin.

BACKGROUND: The sterile insect technique (SIT) has been used with success for suppressing or eliminating important insect pests of agricultural or veterinary importance. In order to develop SIT for mosquitoes, female elimination prior to release is essential as they are the disease-transmitting sex. A genetic sexing strain (GSS) of Anopheles arabiensis was created based on resistance to dieldrin, and methods of sex separation at the egg stage were developed. The use of this strain for SIT will require sexually sterile males: useful radiation doses for this purpose were determined for pupae and adults. METHODS: For the creation of the sexing strain, dieldrin-resistant males were irradiated with 40 Gy using a 60Co source and were subsequently crossed to homozygous susceptible virgin females. Individual families were screened for semi-sterility and for male resistance to dieldrin. For sex separation, eggs of a resulting GSS, ANO IPCL1, were exposed to varying concentrations of dieldrin for different durations. Percent hatch, larval survival, and male and female emergence were recorded. Radiation induced sterility was determined following adult and pupa exposure to gamma rays at 0-105 Gy. Mortality induced by dieldrin treatment, and levels of sterility post radiation were investigated. RESULTS: ANO IPCL1 contains a complex chromosome aberration that pseudo-links the male-determining Y chromosome and dieldrin resistance, conferring high natural semi-sterility. Exposure of eggs to 2, 3, and 4 ppm dieldrin solutions resulted in complete female elimination without a significant decrease of male emergence compared to the controls. A dose of 75 Gy reduced the fertility to 3.8 and 6.9% when males were irradiated as pupae or adults respectively, but the proportions of progeny of these males reaching adulthood were 0.6 and 1.5% respectively CONCLUSION: The GSS ANO IPCL1 was shown to be a suitable strain for further testing for SIT though high semi-sterility is a disadvantage for mass rearing.

Response of male adult Aedes mosquitoes to gamma radiation in different nitrogen environments.

The developmental stage of the mosquito is one of the main factors that affect its response to ionizing radiation. Irradiation of adults has been reported to have beneficial effects. However, the main challenge is to immobilize and compact a large number of adult male mosquitoes for homogenous irradiation with minimal deleterious effects on their quality. The present study investigates the use of nitrogen in the irradiation of adult Aedes albopictus and Ae. aegypti. Irradiation in nitrogen (N2) and in air after being treated with nitrogen (PreN2) were compared with irradiation in air at gamma radiation doses of 0, 55, 70, 90, 110, and 125 Gy. In both species, approximately 0% egg hatch rate was observed following doses above 55 Gy in air versus 70 Gy in PreN2 and 90 Gy in N2. Males irradiated at a high mosquito density showed similar egg hatch rates as those irradiated at a low density. Nitrogen treatments showed beneficial effects on the longevity of irradiated males for a given dose, revealing the radioprotective effect of anoxia. However, irradiation in N2 or PreN2 slightly reduced the male flight ability. Nitrogen treatment was found to be a reliable method for adult mosquito immobilization. Overall, our results demonstrated that nitrogen may be useful in adult Aedes mass irradiation. The best option seems to be PreN2 since it reduces the immobilization duration and requires a lower dose than that required in the N2 environment to achieve full sterility but with similar effects on male quality. However, further studies are necessary to develop standardized procedures including containers, time and pressure for flushing with nitrogen, immobilization duration considering mosquito species, age, and density.

Adult mosquito predation and potential impact on the sterile insect technique.

The sterile insect technique is a promising environmentally friendly method for mosquito control. This technique involves releasing laboratory-produced sterile males into a target field site, and its effectiveness may be affected by the extent of adult mosquito predation. Sterile males undergo several treatments. Therefore, it is vital to understand which treatments are essential in minimizing risks to predation once released. The present study investigates the predation propensity of four mantis species (Phyllocrania paradoxa, Hymenopus coronatus, Blepharopsis mendica, Deroplatys desiccata) and two gecko species (Phelsuma standingi, P. laticauda) on adult Aedes aegypti, Ae. albopictus and Anopheles arabiensis mosquitoes in a laboratory setting. First, any inherent predation preferences regarding mosquito species and sex were evaluated. Subsequently, the effects of chilling, marking, and irradiation, on predation rates were assessed. The selected predators effectively preyed on all mosquito species regardless of the treatment. Predation propensity varied over days for the same individuals and between predator individuals. Overall, there was no impact of laboratory treatments of sterile males on the relative risk of predation by the test predators, unless purposely exposed to double the required sterilizing irradiation dose. Further investigations on standardized predation trials may lead to additional quality control tools for irradiated mosquitoes.

Radiation dose-rate is a neglected critical parameter in dose-response of insects.

Reproductive sterility is the basis of the sterile insect technique (SIT) and essential for its success in the field. Numerous factors that influence dose-response in insects have been identified. However, historically the radiation dose administered has been considered a constant. Efforts aiming to standardize protocols for mosquito irradiation found that, despite carefully controlling many variable factors, there was still an unknown element responsible for differences in expected sterility levels of insects irradiated with the same dose and handling protocols. Thus, together with previous inconclusive investigations, the question arose whether dose really equals dose in terms of biological response, no matter the rate at which the dose is administered. Interestingly, the dose rate effects studied in human nuclear medicine indicated that dose rate could alter dose-response in mammalian cells. Here, we conducted experiments to better understand the interaction of dose and dose rate to assess the effects in irradiated mosquitoes. Our findings suggest that not only does dose rate alter irradiation-induced effects, but that the interaction is not linear and may change with dose. We speculate that the recombination of reactive oxygen species (ROS) in treatments with moderate to high dose rates might minimize indirect radiation-induced effects in mosquitoes and decrease sterility levels, unless dose along with its direct effects is increased. Together with further studies to identify an optimum match of dose and dose rate, these results could assist in the development of improved methods for the production of high-quality sterile mosquitoes to enhance the efficiency of SIT programs.

Standardization of the FAO/IAEA Flight Test for Quality Control of Sterile Mosquitoes.

Successful implementation of the sterile insect technique (SIT) against Aedes aegypti and Aedes albopictus relies on maintaining a consistent release of high-quality sterile males. Affordable, rapid, practical quality control tools based on the male's flight ability (ability to escape from a flight device) may contribute to meeting this requirement. Therefore, this study aims to standardize the use of the original FAO/IAEA rapid quality control flight test device (FTD) (version 1.0), while improving handling conditions and reducing the device's overall cost by assessing factors that could impact the subsequent flight ability of Aedes mosquitoes. The new FTD (version 1.1) is easier to use. The most important factors affecting escape rates were found to be tube color (or "shade"), the combined use of a lure and fan, mosquito species, and mosquito age and density (25; 50; 75; 100 males). Other factors measured but found to be less important were the duration of the test (30, 60, 90, 120 min), fan speed (normal 3000 rpm vs. high 6000 rpm), and mosquito strain origin. In addition, a cheaper version of the FTD (version 2.0) that holds eight individual tubes instead of 40 was designed and successfully validated against the new FTD (version 1.1). It was sensitive enough to distinguish between the effects of cold stress and high irradiation dose. Therefore, the eight-tube FTD may be used to assess Aedes' flight ability. This study demonstrated that the new designs (versions 1.1 and 2.0) of the FTD could be used for standard routine quality assessments of Aedes mosquitoes required for an SIT and other male release-based programs.

Characterization and dose-mapping of an X-ray blood irradiator to assess application potential for the sterile insect technique (SIT).

Self-contained gamma irradiators have been extensively used to reproductively sterilize insects for the sterile insect technique (SIT). More recently, the use of X-ray generators has gained attention due to the reduced investment, logistic, regulatory and safety requirements involved in the procurement, transport and operation of these machines compared with gamma irradiators. In this study, we evaluated a commercially available, "off-the-shelf" X-ray blood irradiator and found it suitable for insect irradiation in the frame of the SIT.

Does Tap Water Quality Compromise the Production of Aedes Mosquitoes in Genetic Control Projects?

A mosquito's life cycle includes an aquatic phase. Water quality is therefore an important determinant of whether or not the female mosquitoes will lay their eggs and the resulting immature stages will survive and successfully complete their development to the adult stage. In response to variations in laboratory rearing outputs, there is a need to investigate the effect of tap water (TW) (in relation to water hardness and electrical conductivity) on mosquito development, productivity and resulting adult quality. In this study, we compared the respective responses of Aedes aegypti and Ae. albopictus to different water hardness/electrical conductivity. First-instar larvae were reared in either 100% water purified through reverse osmosis (ROW) (low water hardness/electrical conductivity), 100% TW (high water hardness/electrical conductivity) or a 80:20, 50:50, 20:80 mix of ROW and TW. The immature development time, pupation rate, adult emergence, body size, and longevity were determined. Overall, TW (with higher hardness and electrical conductivity) was associated with increased time to pupation, decreased pupal production, female body size in both species and longevity in Ae. albopictus only. However, Ae. albopictus was more sensitive to high water hardness/EC than Ae. aegypti. Moreover, in all water hardness/electrical conductivity levels tested, Ae. aegypti developed faster than Ae. albopictus. Conversely, Ae. albopictus adults survived longer than Ae. aegypti. These results imply that water with hardness of more than 140 mg/l CaCO3 or electrical conductivity more than 368 µS/cm cannot be recommended for the optimal rearing of Aedes mosquitoes and highlight the need to consider the level of water hardness/electrical conductivity when rearing Aedes mosquitoes for release purposes.

The Insect Pest Control Laboratory of the Joint FAO/IAEA Programme: Ten Years (2010-2020) of Research and Development, Achievements and Challenges in Support of the Sterile Insect Technique.

The Joint FAO/IAEA Centre (formerly called Division) of Nuclear Techniques in Food and Agriculture was established in 1964 and its accompanying laboratories in 1961. One of its subprograms deals with insect pest control, and has the mandate to develop and implement the sterile insect technique (SIT) for selected key insect pests, with the goal of reducing the use of insecticides, reducing animal and crop losses, protecting the environment, facilitating international trade in agricultural commodities and improving human health. Since its inception, the Insect Pest Control Laboratory (IPCL) (formerly named Entomology Unit) has been implementing research in relation to the development of the SIT package for insect pests of crops, livestock and human health. This paper provides a review of research carried out between 2010 and 2020 at the IPCL. Research on plant pests has focused on the development of genetic sexing strains, characterizing and assessing the performance of these strains (e.g., Ceratitis capitata), elucidation of the taxonomic status of several members of the Bactrocera dorsalis and Anastrepha fraterculus complexes, the use of microbiota as probiotics, genomics, supplements to improve the performance of the reared insects, and the development of the SIT package for fruit fly species such as Bactrocera oleae and Drosophila suzukii. Research on livestock pests has focused on colony maintenance and establishment, tsetse symbionts and pathogens, sex separation, morphology, sterile male quality, radiation biology, mating behavior and transportation and release systems. Research with human disease vectors has focused on the development of genetic sexing strains (Anopheles arabiensis, Aedes aegypti and Aedes albopictus), the development of a more cost-effective larvae and adult rearing system, assessing various aspects of radiation biology, characterizing symbionts and pathogens, studying mating behavior and the development of quality control procedures, and handling and release methods. During the review period, 13 coordinated research projects (CRPs) were completed and six are still being implemented. At the end of each CRP, the results were published in a special issue of a peer-reviewed journal. The review concludes with an overview of future challenges, such as the need to adhere to a phased conditional approach for the implementation of operational SIT programs, the need to make the SIT more cost effective, to respond with demand driven research to solve the problems faced by the operational SIT programs and the use of the SIT to address a multitude of exotic species that are being introduced, due to globalization, and established in areas where they could not survive before, due to climate change.

Demonstration of resistance to satyrization behavior in Aedes aegypti from La Réunion island.

Aedes aegypti and Aedes albopictus are competent vectors of arboviruses such as dengue and chikungunya viruses which co-exist in some areas, including La Réunion island, Indian Ocean. A type of reproductive interference called satyrization has been described in sympatric species where dominant species mating fails to produce hybrids and thus reduces the fitness and tends to control the spread of the other species. Here, we investigated satyrization in laboratory experiments to provide insights on the potential impact on Ae. aegypti of a control campaign including a sterile insect technique component against Ae. albopictus. Different mating crosses were used to test sympatric, conspecific-interspecific and allopatric effects of irradiated and non-irradiated male Ae. albopictus on female Ae. aegypti, including in a situation of skewed male ratio. Our results suggest that there was only a low level of satyrization between sympatric populations of Ae. aegypti and Ae. albopictus colonized from La Réunion island. A male Ae. albopictus to female Ae. aegypti ratio of 3:1 did not increase the level of satyrization. Female Ae. aegypti previously mated to male Ae. albopictus were not prevented from being inseminated by conspecific males. A satyrization effect was not seen between allopatric Ae. albopictus and Ae. aegypti strains from La Réunion Island either. The tested Ae. aegypti strain from La Réunion island has therefore developed full resistance to satyrization and so releasing sterile male Ae. albopictus may not suppress Ae. aegypti populations if an overflooding of irradiated male Ae. albopictus leads to similar results. The management strategy of two competent species in a sympatric area is discussed.

TITLE: Démonstration de la résistance au comportement de satyrisation chez Aedes aegypti de l'île de La Réunion. ABSTRACT: Aedes aegypti et Aedes albopictus sont des vecteurs compétents d'arbovirus, tels que les virus de la dengue et du chikungunya, qui coexistent dans certaines régions, y compris à La Réunion, dans l'Océan Indien. Une interférence sur la reproduction appelée satyrisation a été décrite chez les espèces sympatriques où l'accouplement d'une espèce dominante ne produit pas d'hybrides et donc réduit le succès reproducteur et tend à contrôler la propagation des autres espèces. Ici, nous avons étudié la satyrisation dans des expériences de laboratoire pour fournir des informations sur l'impact potentiel sur Ae. aegypti d'une campagne de lutte contre Ae. albopictus basée sur la technique de l'insecte stérile. Différents croisements d'accouplements ont été utilisés pour tester les effets sympatriques, conspécifiques-interspécifiques et allopatriques de mâles Ae. albopictus irradiés et non irradiés sur les femelles Ae. aegypti, y compris dans une situation de ratio masculin asymétrique. Nos résultats suggèrent qu'il n'y avait qu'un faible niveau de satyrisation entre les populations sympatriques d'Ae. aegypti et Ae. albopictus colonisées à La Réunion. Un rapport mâles Ae. albopictus sur femelles Ae. aegypti de 3 contre 1 n'a pas augmenté le niveau de satyrisation. Les femelles Ae. aegypti déjà accouplées à un mâle Ae. albopictus n'ont pas été empêchées d'être inséminées par des mâles conspécifiques. Aucun effet de satyrisation n'a été observé entre les souches allopatriques d'Ae. albopictus et d'Ae. aegypti de La Réunion. La souche testée d'Ae. aegypti de La Réunion a donc développé une résistance totale à la satyrisation et en conséquence la libération de mâles stériles d'Ae. albopictus peut ne pas supprimer les populations d'Ae. aegypti si des lâchers massifs de mâles irradiés d'Ae. albopictus conduisent à des résultats similaires. La stratégie de gestion de deux espèces compétentes dans une zone sympatrique est discutée.