The sterile insect technique (SIT), a species-specific and environment-friendly method, is a promising technique for the area-wide integrated management of vector mosquitoes, which cause diseases such as dengue, yellow fever, and Zika (Dyck et al., 2021). Indeed, many field trials to demonstrate the effectiveness of the SIT against Aedes aegypti and Aedes albopictus have shown satisfactory results on a small scale (Bellini et al., 2013, 2021; Gato et al., 2021; Becker et al., 2022). Although significant advances have been made in its development (Culbert et al., 2018; Bimbilé Somda et al., 2019; Mamai et al., 2019; Mamai et al., 2020; Yamada et al., 2019; Bouyer et al., 2020; Maïga et al., 2020), there is considerable scope for further development, especially for large-scale implementation. The SIT package includes mosquito mass rearing, sex separation, male sterilization, transport, and release (Dyck et al., 2021). Sterilizing large numbers of males in a reliable manner while maintaining their quality remains one of the most challenging steps. Chemosterilization (Patterson et al., 1970), ionizing radiation (Patterson et al., 1975), and genetic manipulations (Catteruccia et al., 2009) are the main methods explored for mosquito sterilization. However, because of limited effectiveness or ethical, health, and environmental concerns, ionizing radiation, which does not release residues that could be harmful to human health or the environment (Helinski et al., 2009), is the most used method in current SIT programs. Gamma radiation from a ⁶⁰Co or ¹³⁷Cs source and X-ray radiation are commonly used because of their high energy and penetration (Helinski et al., 2009). However, the overall response of mosquitoes to radiation, as with all biological material, has been shown to be affected by many critical factors, including the radiation source, dose rate, dose amount, environment during irradiation, mosquito species, strain, life stage, and handling procedures (Helinski et al., 2009; Yamada et al., 2019; Yamada et al., 2020). For example, irradiation of eggs or larvae causes a high mortality rate even at low irradiation doses and cannot be considered for the SIT programs (Wakid et al., 1976). The pupal and adult stages were found to be more eligible for irradiation (Helinski et al., 2006; Helinski et al., 2009). However, optimal pupae irradiation has many requirements: synchronized pupal production is needed for collection in a small age range, and generally, older pupae (>36 h) should be used to avoid variability in induced sterility. Indeed, younger pupae are more radiosensitive than older pupae, and irradiating young pupae also results in increased somatic damage and mortality (Yamada et al., 2019). Larger numbers of pupae also need to either be submerged in water to allow compacting the pupae in a small container without crushing those at the bottom (note that the pupae would be subjected to hypoxia) or undergo relaxed compaction (single layers of pupae) to avoid crushing, but where pockets of hypoxia can form in which a subset of the pupae may be underdosed (Yamada et al., 2020). These conditions are not easy to achieve or reproduce, especially for mass irradiation. While efforts are still being devoted to overcome these difficulties, the focus is now shifting to irradiation during the adult stage. It has been reported that adult Aedes mosquitoes are similarly or slightly more radiosensitive than old pupae with a better quality after irradiation in some cases (Du et al., 2019; Ernawan et al., 2022). However, the main challenge is the immobilization and compaction of a large number of adult males for homogenous irradiation with minimal deleterious effects on their downstream quality. In general, cold temperatures are used to knock down the mosquitoes (Zhang et al., 2020; Ernawan et al., 2022). However, the quality of the sterile males can be compromised if the chilling temperatures and duration are not carefully controlled. Indeed, many studies on the effects of chilling on insect quality have reported negative effects (reviewed in Yamada et al., 2022). Aiming for a more reliable outcome regarding quality following adult mosquito irradiation, the role of nitrogen has been investigated (Helinski et al., 2009). Beneficial effects of the use of nitrogen in insect irradiation have been reported in fruit flies (Fisher, 1996) and tsetse flies (Vreysen, 1995; Mutika and Parker, 2006). However, its impact on mosquitoes is debated. Indeed, tests performed on Anopheles gambiae pupae and Culex quinquefasciatus pupae and adults showed no beneficial effect (Curtis, 1976; El-Gazzar et al., 1983). In contrast, the irradiation of adult male Ae. aegypti in nitrogen resulted in better competitiveness compared with irradiation in air, although higher doses were required to achieve full sterility (Hallinan and Rai, 1973). A recent study (Yamada et al., 2022) involving Ae. albopictus also showed a radioprotective effect of nitrogen at 45 Gy.

The present study aims to further investigate the possibility to use nitrogen in the mass irradiation of adult Ae. albopictus and Ae. aegypti mosquitoes in order to optimize the effectiveness of the SIT. Two nitrogen treatments were assessed in comparison to irradiation in air. The irradiation dose–response curves under these nitrogen treatments were determined considering gamma radiation doses of 0–125 Gy at low and high mosquito densities. The effects on male flight ability and longevity were further evaluated. A preliminary study was carried out to determine knock-down and wake-up times of adult male Aedes mosquitoes following their exposure to nitrogen.

Ionizing radiation is the method of choice for the sterilization of male mosquitoes in SIT programs (Helinski et al., 2009). Radiation-induced sterility is the result of dominant lethal mutations in the germ cells caused by radiation (LaChance, 1967). Although germ cells are more sensitive to radiation and are targets for mosquito sterilization, damage also occurs in somatic cells, especially in those undergoing mitotic division, leading to reduced quality traits such as longevity and competitiveness (Proverbs, 1969). For the SIT to be successful, it is important to minimize the negative effects of irradiation. Irradiation in low-oxygen environments (for example, in nitrogen) has been reported to have a radioprotective effect with a beneficial impact on the quality of sterile males in many insect species (Vreysen, 1995; Fisher, 1996; Mutika and Parker, 2006). However, its effect on mosquitoes was not clear (Hallinan and Rai, 1973; Curtis, 1976; El-Gazzar et al., 1983; Yamada et al., 2022) and needed to be investigated further.

The present study investigated the possibility to use nitrogen for adult immobilization in the mass irradiation of Ae. albopictus and Ae. aegypti without inducing adverse effects on their quality. Overall, significant differences were observed between the irradiation environments in the dose–response curves of the egg hatch rate within each species. The doses needed to achieve acceptable sterility were, by order of increase, higher in PreN₂ and N₂ compared with that for irradiation in air. These results are consistent with the results of previous studies that reported that higher radiation doses were required under nitrogen treatments to achieve adequate induced sterility in many insects, including fruit flies, tsetse flies, and mosquitoes (Hallinan and Rai, 1973; Curtis, 1976; El-Gazzar et al., 1983). Oxygen is known to be a radiosensitizer (Forshier, 2012). Therefore, the high sensitivity of insects to ionizing radiation in air is commonly attributed to the high level of oxygen. In contrast, the radioresistance under N₂ is likely because of the absence or low level of oxygen in the cells, as reported previously (Condon et al., 2017; Sassù et al., 2019). The radioresistance observed in males pretreated with nitrogen is also likely because of low oxygen saturation in the tissues during irradiation, which is also the main mechanism responsible for paralysis, which can last for ∼15 min after removing the N₂, and a lower metabolic rate. Immobilization of mosquitoes in nitrogen decreases the physiological and biochemical reactions that normally interact with radiation, resulting in radioresistance, since cells that are actively undergoing mitosis are known to be more sensitive to radiation (Proverbs, 1969). The hypothesis of anesthesia-induced radioresistance is supported by studies that assessed the effect of temperature during irradiation. Indeed, it has been reported that lower temperatures decreased the induced sterility of Ae. aegypti irradiated at the pupal or adult stages (Ernawan et al., 2022).

In our study, irradiation of high-density adult male Aedes either in air or in nitrogen treatments had similar extents of induced sterility as irradiation at low mosquito density. This result shows the potential of using nitrogen in adult mass irradiation. However, besides induced sterility, good quality of males is an important requirement to achieve the goal of the SIT.

Our results showed that the male flight ability was reduced by irradiation and that the effect was more pronounced under nitrogen treatments. These results corroborate those of previous studies that reported a negative effect of high ionizing radiation on sterile male quality, commonly attributed to the deleterious effect provoked in somatic cells (Forshier, 2012; Culbert et al., 2018). A recent study also found a reduction in the male flight ability of Ae. albopictus irradiated at 45 Gy in nitrogen compared with irradiation in normoxia as well as unirradiated males (Yamada et al., 2022). However, in our study, the relative differences observed in the male flight ability between the doses inducing nearly full sterility in air and nitrogen treatments were only approximately 10%. Therefore, it remains to be elucidated whether this extent of difference would affect the relative male competitiveness. Indeed, adult male Ae. aegypti irradiated at 100 Gy in nitrogen were found to be fully sterile and were as competitive as unirradiated males, while males irradiated in air at 35, 70, or 100 Gy were less competitive (Hallinan and Rai, 1973). A longer recovery time may enhance the flight ability of the males irradiated under nitrogen treatments and improve their competitiveness, as the flight tests were performed only 1 day after irradiation in the current study. This hypothesis is supported by the beneficial effect of irradiation in nitrogen treatments observed on male survival, as expected (Vreysen, 1995; Fisher, 1996). Overall, high survivorship (>90%) was recorded at day 10 postemergence (7–8 days after irradiation) regardless of the irradiation dose and environment. This suggests that the lifespan of males irradiated in our study conditions would be suitable for the SIT programs as male mosquitoes exhibit a higher mating ability within 10 days after emergence (Sawadogo et al., 2013; Damiens et al., 2016).

Furthermore, this study showed that PreN₂ treatment could be a reliable method for immobilizing adult mosquitoes for irradiation. Indeed, in contrast to the control, where mobility was observed during the irradiation period, mosquitoes stayed immobilized in both types of nitrogen treatment. However, long-term immobilization could be detrimental to mosquitoes. The best option would thus be the PreN₂ treatment as it reduces the immobilization duration, and it requires a lower dose than that required in N₂ environments to achieve full sterility but with similar effects on male quality.

Nitrogen treatment requires additional methods such as a cooling system to knock down the mosquitoes for compaction before the nitrogen treatment as well as for handling and transport purposes after irradiation. It, however, reduces the duration of chilling and allows conduction of the irradiation process without reducing the temperature in the irradiator chamber. For the optimal use of nitrogen, further studies are necessary to develop standardized procedures including the type of container, time and pressure for filling nitrogen, and immobilization duration, considering mosquito species, age, and density.