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.

Background: The sterile insect technique (SIT) for use against mosquitoes consists of several steps including the production of the target species in large numbers, the separation of males and females, the sterilization of the males, and the packing, transport and release of the sterile males at the target site. The sterility of the males is the basis of the technique; for this, efficient and standardized irradiation methods are needed to ensure that the required level of sterility is reliably and reproducibly achieved. While several reports have found that certain biological factors, handling methods and varying irradiation procedures can alter the level of induced sterility in insects, few studies exist in which the methodologies are adequately described and discussed for the reproductive sterilization of mosquitoes. Numerous irradiation studies on mosquito pupae have resulted in varying levels of sterility. Therefore, we initiated a series of small-scale experiments to first investigate variable parameters that may influence dose-response in mosquito pupae, and secondly, identify those factors that potentially have a significantly large effect and need further attention.Methods: In this study, we compiled the results of a series of experiments investigating variable parameters such as pupal age (Aedes aegypti), pupal size (Ae. aegypti), geographical origin of mosquito strains (Ae. aegyptiandAe. albopictus), exposure methods (in wetversusdry conditions,Ae. albopictus) and subsequently in lowversushigh oxygen environments [submerged in water (low O₂(< 5 %)] and in air [high O₂(~ 21 %)] on the radiosensitivity of male pupae (Ae. aegypti,Ae. albopictusandAnopheles arabiensis).Results: Results indicate that radiosensitvity ofAe. aegyptidecreases with increasing pupal age (99% induced sterility in youngest pupae, compared to 93% in oldest pupae), but does not change with differences in pupal size (P = 0.94). Differing geographical origin of the same mosquito species did not result in variations in radiosensitivity inAe. aegyptipupae [Brazil, Indonesia, France (La Reunion), Thailand] orAe. albopictus[Italy, France (La Reunion)]. Differences in induced sterility were seen following irradiation of pupae that were in wetversusdry conditions, which led to further tests showing significant radioprotective effects of oxygen depletion during irradiation procedures in three tested mosquito species, as seen in other insects.Conclusions: These findings infer the necessity to further evaluate significant factors and reassess dose-response for mosquitoes with controlled variables to be able to formulate protocols to achieve reliable and reproducible levels of sterility for application in the frame of the SIT.

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. 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). 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. The results showed that the FAO/IAEA mass-rearing process did not affect mosquito life history parameters or the mating competitiveness of males.

The production of a large number of mosquitoes of high biological qualities and reliable sex sorting before release are key challenges when applying the sterile insect technique as part of an area-wide integrated pest management approach. There is a need to fully evaluate the production capacity of the equipment developed in order to plan and maintain a daily production level for large-scale operational release activities. This study aimed to evaluate the potential use of the FAO/IAEA larval rearing unit forAedes aegyptiand the subsequent female contamination rate after sex sorting with a Fay–Morlan glass separator. Trays from each rack were tilted and their contents sorted either for each individual tray or after mixing the content of all trays from the rack. The pupal production and the female contamination rate were estimated with respect to day of collection, position of the tray, type of pupae collection, and sorting operator. Results showed significant daily variability of pupal production and female contamination rate, with a high male pupal production level achieved on the second day of collection and estimated female contamination of male pupae reached around 1%. Neither tray position nor type of pupae collection affected the pupal production and female contamination rate. However, the operator had a significant effect on the female contamination rate. These results highlight the need to optimize pupal production at early days of collection and to develop a more effective and automated method of sex separation.

Anopheles arabiensis is one of the major malaria vectors that put millions of people in endemic countries at risk. Mass-rearing of this mosquito is crucial for strategies that use sterile insect technique to suppress vector populations. The sterile insect technique (SIT) package for this mosquito species is being developed by the Insect Pest Control Subprogramme of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. To improve mass-rearing outcomes for An. arabiensis, the question of whether the egg production by females would be affected by the size of the adult holding cages, the source of the blood meal and the total number of pupae that could be loaded into the cages was addressed and finally the impact of adding additional pupae to the cage daily to maintain adult numbers on egg productivity assessed. Mass production cages of two different volumes, two different sources of blood meal (bovine and porcine) and two different population densities (cages originally loaded with either 15,000 or 20,000 pupae) were tested and evaluated on the basis of eggs produced/cage or per female. Males and females pupae with a ratio of 1:1 were added to the cages at day 1 and 2 of pupation. The emerging adults had constant access to 5% sugar solution and blood fed via the Hemotek membrane feeding system. Eggs were collected either twice a week or daily. A generalized linear model was used to identify factors which gave significantly higher egg production. Neither cage volume nor blood meal source affected egg production per cage or per female. However, increasing population density to 20,000 pupae had a negative effect on eggs produced per cage and per female. Although high density negatively impacted egg production, adding 1000 daily additional pupae compensating for daily mortality resulted in a substantial increase in egg production. Moreover, in all tests the first and the third egg batches collected were significantly higher than others eggs batches. With the equipment and protocols described here and routinely used at the Insect Pest Control Laboratory (IPCL), it was possible to produce up to 120,000 eggs/cage/day. These results demonstrated that 15,000 is the optimal number of pupae to be loaded into the Anopheles Mass production cages. Under this condition, an average of 40 eggs per female was obtained for five gonotrophic cycles. However, an improvement in egg production can be achieved by daily addition, to the original 15,000 pupae, of one thousand pupae a day. Interestingly, feeding females with bovine or porcine blood using both large and small versions of the mass production cage did not affect egg productivity.

Radiation induced sterility is the basis of the Sterile Insect Technique, by which a target insect pest population is suppressed by releasing artificially reared sterile males of the pest species in overflooding numbers over a target site. In order for the sterile males to be of high biological quality, effective standard irradiation protocols are required. Following studies investigating the effects of mosquito pupae irradiation in water versus in air, there is a need to investigate the oxy-regulatory behavior of mosquito pupae in water to better understand the consequences of irradiation in hypoxic versus normoxic conditions. Pupae of Aedes aegypti, Ae. albopictus, and Anopheles arabiensis were submerged in water inside air-tight 2 ml glass vials at a density of 100 pupae/ml and the dissolved oxygen (DO) levels in the water were measured and plotted over time. In addition, male pupae of Ae. aegypti (aged 40–44 h), Ae. albopictus (aged 40–44 h) and An. arabiensis (aged 20–24 h) were irradiated in a gammacell220 at increasing doses in either hypoxic (water with < 0.5% O2 content) or normoxic (in air) conditions. The males were then mated to virgin females and resulting eggs were checked for induced sterility. All three species depleted the water of DO to levels under 0.5% within 30 minutes, with An. arabiensis consuming oxygen the fastest at under 10 minutes. Following irradiation, the protective effect of hypoxia was observed across species and doses (P < 0.0001), increasing at higher doses. This effect was most pronounced in An. arabiensis. The consumption of dissolved oxygen by pupae submerged in water was significantly different between species, indicating that their oxy-regulatory capacity seems to have possibly evolved according to their preferred breeding site characteristics. This needs to be considered when sterilizing male mosquitoes at pupal stage in water. Depending on species, their DO consumption rates and their density, irradiation doses needed to achieve full sterility may vary significantly. Further assessments are required to ascertain optimal conditions in terms of ambient atmosphere during pupal irradiation to produce competitive sterile males, and temperature and density dependent effects are expected.