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Background: Mosquitoes are the deadliest animals in the world. Their ability to carry and spread diseases to humans causes millions of deaths every year. Due to the lack of efficient vaccines, the control of mosquito-borne diseases primarily relies on the management of the vector. Traditional control methods are insufficient to control mosquito populations. The sterile insect technique (SIT) is an additional control method that can be combined with other control tactics to suppress specific mosquito populations. The SIT requires the mass-rearing and release of sterile males with the aim to induce sterility in the wild female population. Samples collected from the environment for laboratory colonization, as well as the released males, should be free from mosquito-borne viruses (MBV). Therefore, efficient detection methods with defined detection limits for MBV are required. Although a one-step reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) method was developed to detect arboviruses in human and mosquito samples, its detection limit in mosquito samples has yet to be defined., Methods: We evaluated the detection sensitivity of one step RT-qPCR for targeted arboviruses in large mosquito pools, using pools of non-infected mosquitoes of various sizes (165, 320 and 1600 mosquitoes) containing one infected mosquito body with defined virus titers of chikungunya virus (CHIKV), usutu virus (USUV), West Nile virus (WNV) and Zika virus (ZIKV)., Results: CHIK, USUV, ZIKV, and WNV virus were detected in all tested pools using the RT-qPCR assay. Moreover, in the largest mosquito pools (1600 mosquitoes), RT-qPCR was able to detect the targeted viruses using different total RNA quantities (10, 1 and 0.1 ng per reaction) as a template. Correlating the virus titer with the total RNA quantity allowed the prediction of the maximum number of mosquitoes per pool in which the RT-qPCR can theoretically detect the virus infection., Conclusions: Mosquito-borne viruses can be reliably detected by RT-qPCR assay in pools of mosquitoes exceeding 1000 specimens. This will represent an important step to expand pathogen-free colonies for mass-rearing sterile males for programmes that have a SIT component by reducing the time and the manpower needed to conduct this quality control process.

Background: 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., Methods: 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% O 2 content) or normoxic (in air) conditions. The males were then mated to virgin females and resulting eggs were checked for induced sterility., Results: 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., Conclusions: 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.

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. aegypti and Ae. albopictus), exposure methods (in wet versus dry conditions, Ae. albopictus) and subsequently in low versus high oxygen environments [submerged in water (low O 2 (< 5 %)] and in air [high O 2 (~ 21 %)] on the radiosensitivity of male pupae (Ae. aegypti, Ae. albopictus and Anopheles arabiensis)., Results: Results indicate that radiosensitvity of Ae. aegypti decreases 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 in Ae. aegypti pupae [Brazil, Indonesia, France (La Reunion), Thailand] or Ae. albopictus [Italy, France (La Reunion)]. Differences in induced sterility were seen following irradiation of pupae that were in wet versus dry 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.

Background: Mass egg production is an important component of Aedes albopictus mosquito control programs, such as the sterile insect technique and incompatible insect technique, which requires the releases of large number of sterile males. Developing standard operating procedures and optimized cages for adult maintenance of Ae. albopictus can improve the mass rearing efficiency., Methods: Three different sex ratios of females to males with a total number of 4,000 mosquitoes were tested by evaluating the insemination rate, egg production (total number of eggs per cage), female fecundity and egg hatch rate in small cage (30 × 30 × 30 cm). Blood meals with adenosine triphosphate (ATP, 0.05 g/ml), cage structures (Big cage A: 90 × 30 × 30 cm; Big cage B: 90 × 30 × 50 cm or 90 × 50 × 30 cm) and rearing densities (12,000, 16,000 and 20,000 mosquitoes, corresponding to 0.9 cm 2 /mosquito, 0.675 cm 2 /mosquito and 0.54 cm 2 /mosquito, respectively) were also tested and evaluated on the basis of egg production, female fecundity and egg hatch rate. An adult rearing unit holding 15 of Big cage A with optimal egg production was designed to produce 10 million eggs per rearing cycle in a 1.8 m 2 space., Results: Female to male ratios at 3:1 in small cages resulted in higher egg production but did not affect insemination rate, female fecundity and egg hatch rate. A concentration of 0.05 g/ml of ATP added to blood meals improved the blood-feeding frequency and thus increased the overall egg production per cage. Cage structures affected the egg production per cage, but not egg hatch rate. A medium rearing density at 0.675 cm 2 /mosquito (16,000 mosquitoes) resulted in higher egg production compared to both low and high densities. An adult rearing unit for Ae. albopictus on the basis of Big cage A has been developed with the capacity of producing 10 million eggs within 15 days., Conclusions: Our results have indicated that the adult rearing methods and adult maintenance unit are recommended for Ae. albopictus mass rearing in support of the establishment of a medium-sized mosquito factory.

Background: Larval nutrition, particularly diet quality, is a key driver in providing sufficient numbers of high quality mosquitoes for biological control strategies such as the sterile insect technique. The diet currently available to mass rear Anopheles arabiensis, referred here to as the "IAEA diet", is facing high costs and difficulties concerning the availability of the bovine liver powder component. To promote more affordable and sustainable mosquito production, the present study aimed to find alternative diet mixtures. Eight cheaper diet mixtures comprised of varying proportions of tuna meal (TM), bovine liver powder (BLP), brewer's yeast (BY), and chickpea (CP) were developed and evaluated through a step by step assessment on An. arabiensis larvae and adult life history traits, in comparison to the IAEA diet which served as a basis and standard., Results: Four mixtures were found to be effective regarding larval survival to pupation and to emergence, egg productivity, adult body size and longevity. These results suggest that these different diet mixtures have a similar nutritional value that support the optimal development of An. arabiensis larvae and enhance adult biological quality and production efficiency, and thus could be used for mass rearing., Conclusions: Our study demonstrated that four different diet mixtures, 40 to 92% cheaper than the IAEA diet, can result in a positive assessment of the mosquitoes' life history traits, indicating that this mosquito species can be effectively mass reared with a significant reduction in costs. The mixture comprised of TM + BY + CP is the preferred choice as it does not include BLP and thus reduces the cost by 92% compared to the IAEA diet.

Background: Standardized larval rearing units for mosquito production are essential for the establishment of a mass-rearing facility. Two larval rearing units, developed respectively by the Guangzhou Wolbaki Biotech Co. Ltd. (Wolbaki) and Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture (FAO/IAEA-IPCL), are tested to assess their potential uses to mass-rear the larval stages of Aedes albopictus in support of the establishment of a medium-scale mosquito facility for the application of mosquito genetic control strategies., Methods: The triple Wolbachia-infected Ae. albopictus strain (HC strain) was used in this study. The effects of larval densities of two larval rearing trays (corresponding to 2.4, 3.0 and 3.6 larvae/cm 2 ) and tray size/position (top, middle and bottom layers) on the pupae production and larval survival were assessed when trays were stacked within the larval rearing units. The male pupae production, female pupae contamination after sex separation, and male mating competitiveness were also studied by using both larval rearing units in their entirety., Results: The optimal larval rearing density for Wolbaki-tray (Wol-tray) was 6,600 larvae (equal to 3.0 larvae/cm 2 ) and 18,000 larvae (3.6 larvae/cm 2 ) for the FAO/IAEA-IPCL tray (IAEA-tray). No significant difference in pupae production was observed when trays were stacked within top, middle or bottom layers for both units. At thirty-four hours after the first pupation, the average male pupae production was (0.89 × 10 5 ) for the Wol-unit and (3.16 × 10 5 ) for the IAEA-unit. No significant difference was observed in female pupae contamination between these two units. The HC males showed equal male mating competitiveness to wild type males for mating with wild type females in large cages, regardless of whether they were reared in the Wol-unit or IAEA-unit., Conclusions: The current study has indicated that both the Wol-unit and IAEA-unit are suitable for larvae mass-rearing for Ae. albopictus. However, the IAEA-unit, with higher male production and less space required compared to the Wol-unit, is recommended to be used in support of the establishment of a medium-sized mosquito facility.

Background: Quantification of eggs prior to rearing the immature stages of mosquitoes is an essential step in establishing a standardized mass rearing system. To develop a simple and accurate method of egg quantification for Aedes aegypti and Aedes albopictus, the relationship between egg number and weight, as well as egg number and volume, were studied., Methods: Known quantities of eggs (1,000, 3,000, 6,000, 12,000, 15,000, 18,000, 21,000 and 27,000) were counted and subsequently their weight and volume were measured. Best-fit curves and regression equations were used to describe relationships between Aedes egg number and both weight and volume., Results: Eighteen thousand Ae. aegypti eggs weighed 159.8 mg and had a volume of 277.4 μl, compared to measurements of 131.5 mg and 230.3 μl for Ae. albopictus. The eggs of Ae. aegypti were thus larger and heavier than those of Ae. albopictus. The use of weight and volume to quantify egg number was validated by counting volumes and weights of eggs expected to correspond to 3,000 and 18,000 eggs of each species; significant correlations were found in all cases except in the case of 3,000 Ae. albopictus eggs measured by volume., Conclusion: Methods for egg quantification were validated and shown to be a consistent and practical means to achieve uniform distribution of Aedes larvae between rearing trays, important for optimal mass rearing of the immature stages of Aedes mosquitoes.

Background: Ivermectin has longevity reducing effects in several insect species, including disease transmitting mosquitoes after feeding on hosts that have received ivermectin treatment. This has important implications in mosquito population control and thus the reduction of disease transmission. In addition, ivermectin could play an enormous role in mosquito control operations by its use in the female elimination process during mass-rearing, enabling the release of only sterile males in the context of the sterile insect technique (SIT)., Methods: Blood meals were spiked with various toxicants and were then offered to adult Anopheles arabiensis and killing effects were observed. Varying concentrations of the most effective substance were then tested in subsequent trials to obtain an optimal dose for quick and total female elimination. The remaining males were mated with untreated virgin females to assess whether their mating efficiency had been compromised. The most promising substance at the optimal concentration was further tested on a larger number of adults, after they had been irradiated and partially sterilised as pupae with 70 Gy to evaluate the feasibility of the method in a mass-rearing, and SIT context. The males resulting from the latter trial were also checked for mating efficiency post treatments., Results: Ivermectin (Virbamec®) at a concentration of 7.5 ppm was chosen from the toxicants tested as sufficiently effective in eliminating all female An. arabiensis in 4 days, the shortest time required for female elimination of all chemicals tested. Mating efficiency of the non-blood feeding male mosquitoes was not compromised significantly compared to controls even when they were kept for a total of 4 days (from emergence) before theoretical release. The irradiation treatment did not affect overall female feeding behaviour in this setting, nor were the sterile males less competitive for mating with virgin females after the treatments than virgin sterile males that had not been in the ivermectin treatment environment., Conclusions: Spiking bloodmeals with ivermectin has shown potential as a viable treatment to eliminate female An. arabiensis from laboratory colonies although its practical use in a mass-rearing facility still needs to be tested.