Efficiency assessment of a novel automatic mosquito pupae sex separation system in support of area-wide male-based release strategies.

This study provides a comparative analysis of two state-of-the-art automatic mosquito pupae sex sorters currently available: the ORINNO and the WOLBAKI Biotech pupae sex separation systems, which both exploit the sexual size dimorphism of pupae. In Aedes aegypti, the WOLBAKI sex sorter and the ORINNO with a sieve mesh size of 1.050 mm achieved sex separation with female contamination rates below 1%, low pupae mortality rates and high male flight capacity. However, in Ae. albopictus, there was more variability, with female contamination rates above the 1% threshold and pupae mortality reaching 27% when using the ORINNO sorter. On the other hand, the WOLBAKI sorter achieved a male pupae recovery of 47.99 ± 8.81% and 50.91 ± 11.77% in Ae. aegypti and Ae. albopictus, respectively, while the ORINNO sorter with a smaller sieve size achieved male pupae recoveries of 38.08 ± 9.69% and 40.16 ± 2.73% in Ae. aegypti and Ae. albopictus, respectively. This study provides valuable information for researchers and practitioners in the field, assisting in the selection of the most suitable system for mosquito control, management and research programs depending on their specific requirements.

Mass irradiation of adult Aedes mosquitoes using a coolable 3D printed canister.

In the last decade, the use of the sterile insect technique (SIT) to suppress mosquito vectors have rapidly expanded in many countries facing the complexities of scaling up production and procedures to sustain large-scale operational programs. While many solutions have been proposed to improve mass production, sex separation and field release procedures, relatively little attention has been devoted to effective mass sterilization of mosquitoes. Since irradiation of pupae en masse has proven difficult to standardise with several variables affecting dose response uniformity, the manipulation of adult mosquitoes appears to be the most promising method to achieve effective and reliable sterilization of large quantities of mosquitoes. A 3D-printed phase change material based coolable canister was developed which can compact, immobilize and hold around 100,000 adult mosquitoes during mass radio sterilization procedures. The mass irradiation and compaction treatments affected the survival and the flight ability of Aedes albopictus and Aedes aegypti adult males but the use of the proposed irradiation canister under chilled conditions (6.7-11.3 °C) significantly improved their quality and performance. The use of this cooled canister will facilitate adult mass irradiation procedures in self-contained irradiators in operational mosquito SIT programmes.

Effects of Chilling and Anoxia on the Irradiation Dose-Response in Adult Aedes Mosquitoes.

The success of the sterile insect technique (SIT) relies on the achievement of high levels of sterility and mating success of the factory-reared sterile males and thus their biological quality, which can be enhanced by the reduction of stress factors encountered during rearing, handling, and irradiation procedures. The achievement of consistent sterility levels requires reliable and standard irradiation protocols. Additionally, mosquito adults require immobilization prior to, and during irradiation to increase processing efficiency and to avoid physical damage caused by movement in restricted space. Common methods for immobilization include chilling and anesthetics such as nitrogen. Here we assessed the effects of chilling and exposure to nitrogen on the irradiation dose-response of Aedes mosquitoes, and their downstream effects on some male quality parameters including longevity and flight ability. We found that chilling does not incur damage in the insects in terms of longevity and flight ability when chilling duration and temperature are carefully controlled, and a recovery phase is provided. Irradiation in nitrogen shows high radioprotective effects during irradiation, resulting in reduced induction of sterility. Overall, longevity of males can be improved by irradiating in anoxia, however the exposure to nitrogen itself comes with negative impacts on flight ability. The results reported here will assist in the standardization and optimization of irradiation protocols for the SIT to control mosquito populations of medical relevance.

The efficiency of a new automated mosquito larval counter and its impact on larval survival.

To achieve consistent and standardized rearing for mosquito immature stages, it is crucial to control the initial number of larvae present in each larval tray. In addition, maintaining an optimal and synchronized development rate of larvae is essential to maximize the pupal production and optimize male sorting in a mass-rearing setting. Manual counting is labor intensive, time consuming and error prone. Therefore, this study aimed to investigate the use of a customized automated counter for the quantification of mosquito larvae. The present prototype of the mosquito larval counter uses a single counting channel consisting of three parts: a larvae dispenser, an electronic counting unit and computer control software. After the separation of the larvae from eggs and debris, batches of different numbers of Aedes aegypti first instar larvae were manually counted and introduced into the counter through the upper loading funnel and channeled out from the bottom of the counter by gravitational flow. The accuracy and repeatability of the mosquito larval counter were determined in relation to larval density and water quality. We also investigated its impact on larval survival. Results showed an impact of larval density and water quality on the accuracy of the device. A -6% error and a repeatability of +/- 2.56% average value were achieved with larval densities up to 10 larvae/mL of clean water. Moreover, the use of the mosquito larval counter did not have any effect on larval survival or development. Under recommended conditions, the mosquito larval counter can be used to enumerate the number of mosquito larvae at a given density. However, future developments involving the use of multiple channels or larger input larvae container would help to expand its use in large-scale facilities.

Monitoring Dry Season Persistence of Anopheles gambiae s.l. Populations in a Contained Semi-Field System in Southwestern Burkina Faso, West Africa.

To gain insight into the dry season survival strategies of Anopheles gambiae s.l., a new contained semi-field system was developed and used for the first time in Burkina Faso, West Africa. The system consisted of a screened greenhouse within which the local environment was reproduced, including all ecological requirements for mosquito development cycle completion. The system was seeded with the progenies of female Anopheles gambiae, Anopheles coluzzii, and Anopheles arabiensis collected in the vicinity of the greenhouse during the rainy season. After successful establishment in the semi-field system, mosquito populations were monitored over a 1-yr period by regular surveys of larval and adult specimens. We provided evidence for the persistence of adult mosquitoes throughout the dry season, in the absence of any suitable larval development site. During the hot and dry periods, adult insects were observed in artificial shelters (clay pots, building blocks, and dark corners). The mosquito population rapidly built up with the return of the rainy season in the area, when artificial breeding sites were refilled in the enclosure. However, only An. coluzzii and, later, An. arabiensis were detected in the subsequent rainy season, whereas no An. gambiae specimen was found. Our findings suggest that An. coluzzii and An. arabiensis may be able to aestivate throughout the dry season in Southwestern Burkina Faso, whereas An. gambiae might adopt a different dry-season survival strategy, such as long-distance re-colonization from distant locations. These results may have important implications for malaria control through targeted vector control interventions.

Combining two-dimensional gel electrophoresis and metabolomic data in support of dry-season survival in the two main species of the malarial mosquito Anopheles gambiae.

In dry savannahs of West-Africa, the malarial mosquitoes of the Anopheles gambiae sensu stricto complex annually survive the harsh desiccating conditions of the dry season. However, the physiological and biochemical mechanisms underlying how these mosquitoes survive such desiccating conditions are still undefined, and controversial. In this context, we provide the first work examining both proteomic and metabolomic changes in the two molecular forms of A. gambiae s.s (M and S forms) experimentally exposed to the rainy and dry season conditions as they experience in the field. Protein abundances of the mosquitoes were measured using a two-dimensional fluorescence difference gel electrophoresis (2D DIGE) coupled with a matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) and tandem mass spectrometry (MS) for protein identification. These assays were conducted by Applied Biomics (http://www.appliedbiomics.com, Applied Biomics, Inc. Hayward, CA, USA), and the mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000294. The metabolomic analysis was conducted using both Acquity UPLC(®) system (for amino acid identification), and a gas-chromatography-mass spectrometry platform (for sugars identification). Metabolomic fingerprintings were assessed in the University of Rennes 1, UMR CNRS 6553 EcoBio (France). A detailed interpretation of the obtained data can be found in Hidalgo et al. (2014) [1] (Journal of Insect Physiology (2014)).

Novel insights into the metabolic and biochemical underpinnings assisting dry-season survival in female malaria mosquitoes of the Anopheles gambiae complex.

The mechanisms by which Anopheles gambiae mosquitoes survive the desiccating conditions of the dry season in Africa and are able to readily transmit malaria soon after the rains start remain largely unknown. The desiccation tolerance and resistance of female An. gambiae M and S reared in contrasting environmental conditions reflecting the onset of dry season ("ods") and the rainy season ("rs") was determined by monitoring their survival and body water loss in response to low relative humidity. Furthermore, we investigated the degree to which the physiology of 1-h and 24-h-old females is altered at "ods" by examining and comparing their quantitative metabotypes and proteotypes with conspecifics exposed to "rs" conditions. Results showed that distinct biochemical rearrangements occurred soon after emergence in female mosquitoes that enhance survival and limit water loss under dry conditions. In particular, three amino acids (phenylalanine, tyrosine, and valine) playing a pivotal role in cuticle permeability decreased significantly from the 1-h to 24-h-old females, regardless of the experimental conditions. However, these amino acids were present in higher amounts in 1-h-old female An. gambiae M reared under "ods" whereas no such seasonal difference was reported in S ones. Together with the 1.28- to 2.84-fold increased expression of cuticular proteins 70 and 117, our data suggests that cuticle composition, rigidity and permeability were adjusted at "ods". Increased expression of enzymes involved in glycogenolytic and proteolytic processes were found in both forms at "ods". Moreover, 1-h-old S forms were characterised by elevated amounts of glycogen phosphorylase, isocitrate dehydrogenase, and citrate synthase, suggesting an increase of energetic demand in these females at "ods".

Metabolomic and ecdysteroid variations in Anopheles gambiae s.l. mosquitoes exposed to the stressful conditions of the dry season in Burkina Faso, West Africa.

This 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-h-old 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.

Physiology and development of the M and S molecular forms of Anopheles gambiae in Burkina Faso (West Africa).

In West Africa, M and S molecular forms of Anopheles gambiae sensu stricto (Diptera: Culicidae) Giles, frequently occur together, although with different population bionomics. The S form typically breeds in rain-dependant water collections and is present during the rainy season only whereas the M form can thrive all year long in areas with permanent breeding opportunities. In the present study, we explored physiological and developmental trade-offs at play in laboratory colonies and field populations of the M and S forms that originated from an area of sympatry in Burkina Faso, where M and S larvae exhibit such habitat segregation. In the laboratory, larvae of the M form developed slower than the S form (mean values 9.51 and 8.85 days, respectively, Wilcoxon's test, P < 0.001). Although wing length and dry weight at emergence showed large variations, M females were on average 8% heavier than S females of similar wing length. Higher nutritional reserves (proteins and lipids) in teneral adults explained part of this weight difference, reflecting a better ability of the M form to garner resources at the larval stage. Furthermore, a higher rate of ovarian maturation was observed in the M form after a single bloodmeal. The relevance of these findings for parasite transmission is discussed.