Optimization of mosquito egg production under mass rearing setting: effects of cage volume, blood meal source and adult population density for the malaria vector, Anopheles arabiensis

Background: 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. Methods: 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. Results: 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. Conclusion: 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

Ovitrap Monitoring of Aedes aegypti and Aedes albopictus in Two Selected Sites in Quezon City, Philippines

In recent years, Aedes mosquitoes have become a serious health threat in the Philippines, causing a dramatic increase in dengue incidence. To design and implement adapted vector control measures, knowledge of vector composition and abundance is essential. Surveillance of mosquito populations using ovitraps was conducted for about two years (from April 2018–February 2020) in two selected sites (Sitio Payong and Villa Beatriz in Old Balara, Quezon City). These areas were identified as potential sites for small-scale pilot trials of the sterile insect technique. Aedes aegypti and Ae. albopictus were present in both sites, but Ae. aegypti was about 20 times more abundant than Ae. albopictus. There was a significant difference between the two sites in the number of eggs collected weekly but not in the OI. The number of Aedes spp. eggs collected were found to positively correlate with the maximum daily temperature. These data provide comprehensive, evidence-based information that will help in the design and implementation of Aedes control measures, as part of wider public health interventions for prevention and control

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

[EN] 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.The authors are grateful to Empresa de Transformación Agraria S.A., S.M.E, M.P. (TRAGSA), Spain, and to Wolbaki, China, for donating their strain for testing. They are grateful to the two reviewers for their useful comments/suggestions that improved our manuscript. This research was funded by the United States of America under the grant to the IAEA entitled ¿Surge expansion for the sterile insect technique to control mosquito populations that transmit the Zika virus.¿ The funders and the agency had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.Maïga, H.; Lu, D.; Mamai, W.; Somda, NSB.; Wallner, T.; Bakhoum, MT.; Masso, OB.... (2022). Standardization of the FAO/IAEA Flight Test for Quality Control of Sterile Mosquitoes. Frontiers in Bioengineering and Biotechnology. 10:1-14. https://doi.org/10.3389/fbioe.2022.8766751141

Sterile Insect Technique (SIT) field trial targeting the suppression of Aedes albopictus in Greece

The sterile insect technique (SIT) involves releasing large numbers of sterile males to outcompete wild males in mating with females, leading to a decline in pest populations. In the current study, we conducted a suppression trial in Greece against the invasive dengue vector mosquito Aedes albopictus (Skuse) through the weekly release of sterile males for 22 weeks from June to September 2019. Our approach included the long-distance transport of sterile mosquitoes, and their release at a density of 2,547 ± 159 sterile males per hectare per week as part of an area-wide integrated pest management strategy (AW-IPM). The repeated releases of sterile males resulted in a gradual reduction in egg density, reaching 78% from mid-June to early September. This reduction remained between 70% and 78% for four weeks after the end of the releases. Additionally, in the SIT intervention area, the ovitrap index, representing the percentage of traps containing eggs, remained lower throughout the trial than in the control area. This trial represents a significant advance in the field of mosquito control, as it explores the viability and efficacy of producing and transporting sterile males from a distant facility to the release area. Our results provide valuable insights for future SIT programmes targeting Ae. Albopictus, and the methodology we employed can serve as a starting point for developing more refined and effective release protocols, including the transportation of sterile males over long distances from production units to intervention areas

Longevity of mass-reared, irradiated and packed male Anopheles arabiensis and Aedes aegypti under simulated environmental field conditions

Abstract Background To ensure the success of a mosquito control programme that integrates the sterile insect technique (SIT), it is highly desirable to release sterile males with a maximal lifespan to increase release effectiveness. Understanding sterile male survival under field conditions is thus critical for determining the number of males to be released. Our study aimed to investigate the effect of mass rearing, irradiation, chilling, packing and release time on irradiated male mosquito longevity. Methods Anopheles arabiensis and Aedes aegypti immature stages were mass-reared using a rack and tray system. Batches of 50 males irradiated at the pupal stage were immobilised, packed into canisters and chilled for 6 hours at 6 °C. Mosquitoes were then transferred either in the early morning or early evening into climate chambers set to simulate the weather conditions, typical of the beginning of the rainy season in Khartoum, Sudan and Juazeiro, Brazil for An. arabiensis and Ae. aegypti, respectively. The longevity of experimental males was assessed and compared to mass-reared control males subjected either to simulated field or laboratory conditions. Results The combined irradiation, chilling and packing treatments significantly reduced the longevity of both An. arabiensis and Ae. aegypti under simulated field conditions (P < 0.001). However, packing alone did not significantly reduce longevity of Ae. aegypti (P = 0.38) but did in An. arabiensis (P < 0.001). Overall, the longevity of mass reared, irradiated and packed males was significantly reduced, with the median survival time (days) lower following an early morning introduction (4.62 ± 0.20) compared to an evening (7.34 ± 0.35) in An. arabiensis (P < 0.001). However, there was no significant difference in longevity between morning (9.07 ± 0.54) and evening (7.76 ± 0.50) in Ae. aegypti (P = 0.14). Conclusions Our study showed that sterile mass-reared males have a reduced lifespan in comparison to laboratory-maintained controls under simulated field conditions, and that An. arabiensis appeared to be more sensitive to the handling process and release time than Ae. aegypti. Longevity and release time are important parameters to be considered for a successful area-wide integrated vector control programme with a SIT component

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

A mosquito&rsquo;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 &micro;S/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

Développement larvaire et production de pupes d’Aedes aegypti dans le système d’élevage de masse de la FAO/AIEA et facteurs influençant l’efficacité de la séparation des sexes.

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 for Aedes aegypti and 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.La production d’un grand nombre de moustiques de haute qualité biologique et le tri des sexes avant les lâchers sont des défis clés lors de l’application de la technique des insectes stériles, dans le cadre d’une approche de lutte intégrée contre les ravageurs à l’échelle d’une zone. Il est nécessaire d’évaluer pleinement la capacité de production des équipements développés afin de planifier et de maintenir un niveau de production quotidien pour les activités de libération opérationnelle à grande échelle. Cette étude visait à évaluer l’utilisation potentielle de l’unité d’élevage larvaire FAO/AIEA pour Aedes aegypti et le taux de contamination par des femelles après le tri sexuel avec un séparateur en verre Fay–Morlan. Les plateaux de chaque rack ont été inclinés et leur contenu trié soit pour chaque plateau, soit après avoir mélangé le contenu de tous les plateaux du rack. La production de pupes et le taux de contamination par des femelles ont été estimés en fonction du jour de collecte, de la position du bac, du type de collecte des pupes et de l’opérateur du tri. Les résultats ont montré une variabilité quotidienne significative de la production de pupes et du taux de contamination par des femelles, avec un niveau élevé de production de pupes mâles atteint le deuxième jour de collecte et la contamination estimée des pupes mâles par des femelles a atteint environ 1 %. Ni la position du plateau ni le type de collecte des pupes n’ont affecté la production de pupes et le taux de contamination par des femelles. Cependant, l’opérateur avait un effet significatif sur le taux de contamination par les femelles. Ces résultats mettent en évidence la nécessité d’optimiser la production des pupes dès les premiers jours de la collecte et de développer une méthode de séparation des sexes plus efficace et automatisée

Radiation dose-fractionation in adult

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

Metabolomic and Ecdysteroid Variations in Anopheles gambiae s.l. Mosquitoes Exposed to the Stressful Conditions of the Dry Season in Burkina Faso, West Africa

International audienceThis study explored the metabolic adjustments prompted by a switch between the rainy and dry season conditions in the African malaria mosquitoes Anopheles gambiae (M and S molecular forms) and Anopheles arabiensis. Mosquitoes were reared in contrasted experimental conditions reflecting environmental variation in Burkina Faso. Thirty-five metabolites (including sugars, polyols, and amino acids) were monitored in newly emerged males and females, and their ecdysteroid titers were determined. Metabolomic signatures were remarkably similar across species, when specimens of same age and sex were reared under identical experimental conditions. In males and females, amino acids (including glycine, leucine, phenylanine, serine, threonine, and valine) were accumulated in 1-hold mosquitoes, then decreased 24 h after emergence, probably reflecting adult maturation and the amino acid-consuming process of cuticle sclerotisation. In turn, elevated amounts of alanine and proline in 24-h-old mosquitoes may assist the development of flight ability. Lower concentration of tricarboxylic acid cycle intermediates and isoleucine characterized older females reared under dry season conditions, suggesting metabolic and reproduction depression. In all cases, ecdysteroid concentration was much higher in males than in females, with significant seasonal variation in males. This might reflect a unique role of these hormones in shaping reproductive strategies and population demography in the An. gambiae s.l. species complex, further contributing to local adaptation in a highly fluctuating environment