International shipments of Wolbachia-infected mosquito eggs: towards the scaling-up of World Mosquito Program operations.

The Wolbachia insect control method, employed by the World Mosquito Program (WMP), relies on introgressing Wolbachia through target Aedes aegypti populations to reduce the incidence of dengue. Since 2010, the WMP has been producing Wolbachia-infected mosquitoes at numerous sites across the globe for release in 11 countries. As the technology has matured, greater focus has been placed on mosquito production at larger central facilities for transport to remote release sites, both domestically and internationally. Of particular note is the production of Wolbachia-infected mosquitoes at the WMP's Australian production facility for successful international deployments in Fiji, Vanuatu, Kiribati and Sri Lanka. This requires careful management of both production and supply-chain processes to ensure that the quality of the mosquito eggs, specifically the hatch rate and Wolbachia infection rate, is maintained. To ensure the cost-effectiveness and scalability of the Wolbachia method, these processes will be further refined to facilitate deployment from large centralised production facilities.

La méthode de contrôle des insectes mise en oeuvre par le World Mosquito Program (WMP) recourt à la bactérie Wolbachia et repose sur l'introgression de cette dernière par le biais des populations cibles d'Aedes aegypti dans le but de réduire l'incidence de la dengue. Depuis 2010, le WMP produit des moustiques infectés par Wolbachia à partir de nombreux sites répartis dans le monde entier en vue de leur lâcher dans 11 pays. Cette technologie s'étant bien développée, l'accent est désormais mis sur la production de moustiques dans des établissements centralisés de plus grande envergure afin de les expédier vers des sites de lâcher éloignés, tant sur le territoire national qu'à l'échelle internationale. Il importe de souligner la production de moustiques infectés par Wolbachia conduite par le WMP sur son site australien et leur déploiement international couronné de succès aux Fidji, au Vanuatu, à Kiribati et au Sri Lanka. Cela nécessite une gestion rigoureuse des procédures aussi bien lors de la production que tout au long de la chaîne d'approvisionnement afin de veiller au maintien de la qualité des oeufs de moustiques et plus spécifiquement du taux d'éclosion et du taux d'infection par Wolbachia. Afin d'assurer la rentabilité et l'évolutivité de la méthode basée sur Wolbachia, ces procédures seront encore affinées afin de faciliter le déploiement à partir de sites de production à grande échelle centralisés.

El método de control de insectos con Wolbachia que emplea el World Mosquito Program (WMP) reposa en la introgresión de Wolbachia a través de poblaciones de Aedes aegypti para reducir la incidencia del dengue. Desde 2010, el WMP viene generando mosquitos infectados por Wolbachia en numerosas instalaciones repartidas por todo el globo, mosquitos que después son liberados en 11 países. A medida que la tecnología ha ido madurando, la tendencia ha sido cada vez más la de concentrar la producción de mosquitos en grandes instalaciones centrales y desde allí enviarlos a los remotos lugares de suelta, ya estén en el mismo país o en otros países. Especialmente destacable es la producción de mosquitos infectados por Wolbachia en las instalaciones que el WMP tiene en Australia, utilizadas con éxito como centro de operaciones para aplicar el método en otros países como Fiji, Vanuatu, Kiribati y Sri Lanka. Ello exige una cuidadosa gestión de los procesos tanto de producción como de la cadena de suministro, que garantice un nivel constante de calidad de los huevos de mosquito, y más concretamente de las tasas de eclosión y de infección por Wolbachia. A fin de asegurar que el método ofrezca una buena relación costo-eficacia y sea reproducible a mayor escala, esos procesos serán perfeccionados para facilitar el despliegue de los insectos a partir de grandes instalaciones de producción centralizada.

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.

Development of Aedes albopictus (Diptera: Culicidae) Larvae Under Different Laboratory Conditions.

Critical to successful application of the sterile insect technique against Aedes albopictus (Skuse) is the development of an efficient and standardized rearing protocol to be employed in the mass production system. In this study, several life history traits of Ae. albopictus were analyzed to identify upper and lower thresholds of larval density and diet concentration. Survival to pupation, time to pupation, and sex ratio were evaluated under a range of larval densities (0.5-5 larvae/ml) and food levels (0.05-1.6 mg/larva/d) using two larval diets (one locally developed; one developed by the FAO/IAEA). The larvae reared at 28 °C, at a density of 2 larvae/ml and receiving a food dose equal to 0.6 mg/larva/d of a diet consisting of 50% tuna meal, 50% bovine liver powder (the FAO/IAEA diet), and, as an additive, 0.2 g of Vitamin Mix per 100 ml of diet solution, developed in 5 d and had 90% survival to the pupal stage. With this rearing regime male pupae production 24 h after the onset of pupation was the highest; these pupae were ∼94% male.

X-ray-induced sterility in Aedes albopictus (Diptera: Culicidae) and male longevity following irradiation.

The mosquito Aedes albopictus (Skuse, 1895) is a potent vector of several arboviral diseases, most notably chikungunya and dengue fever. In the context of the sterile insect technique (SIT), the sterilization of the male mosquitoes before their release can be achieved by gamma-ray irradiation. As gamma-ray irradiators are becoming increasingly problematic to purchase and transport, the suitability of an X-ray irradiator as an alternative for the sterilization of Ae. albopictus males was studied. The sterilization of up to 200,000 pupae at one time can be achieved with relative ease, and the sterility results obtained were comparable with those achieved by gamma irradiation, where 99% sterility is induced with a dose of 40 Gy. A significant reduction of longevity was observed in the latter stages of the males' life after irradiation treatments, especially at doses > 40 Gy, which is consistent with the negative effects on longevity induced by similar radiation doses using gamma rays. Females irradiated at 40 Gy were not only 100% sterile, but also failed to oviposit entirely, i.e., all of the females laid 0 eggs. Overall, it was found that the X-ray irradiator is generally suitable for the sterilization process for sterile insect technique programs, as it showed a high processing capacity, practicality, high effectiveness, and reproducibility.

Mass production cage for Aedes albopictus (Diptera:Culicidae).

Mass production is an important co mponent of any pest or vector control program that requires the release of large number of insects. As part of efforts to develop an area-wide program involving the sterile insect technique (SIT) for the control of mosquitoes, the Insect Pest Control Laboratory of the Food and Agriculture Organization-International Atomic Energy Agency (FAO-IAEA Joint Division) has developed a mass production cage (Aedes MPC) for brood stock colonies in a mass production system for Aedes albopictus (Skuse, 1895). A preliminary experiment using Plexiglas cages was carried out to estimate the impact of cage volume on egg productivity. Transparent Plexiglas cages of different dimensions but loaded with the same adult density were tested. Egg productivity (number of eggs laid per adult female) and adult survival were recorded and analyzed. According to the results, the optimal volume of 100 liters has been chosen to develop the Aedes MPC. The numbers of adults introduced into the Aedes MPC did not affect the egg production and adult survival in comparison with the Plexiglas cage experiment data, confirming the possible use of Aedes MPC for mass-rearing procedures. Finally, the modification of Aedes MPC and creation of a new prototype model of MPC (Anopheles MPC) to effectively contain Anopheles arabiensis (Patton, 1905) adults is discussed with major changes pioneered to oviposition devices and systems for automatic collection of the eggs.

Occurrence of natural Anopheles arabiensis swarms in an urban area of Bobo-Dioulasso City, Burkina Faso, West Africa.

The swarming behaviour of natural populations of Anopheles arabiensis was investigated by conducting transect surveys on 10 consecutive days, around dusk, from March to April and from September to October 2012 in Dioulassoba, a district of Bobo-Dioulasso city in Burkina Faso (West Africa). Swarms were observed outside, around identified larval breeding sites on the banks of the Houet River, as well as in the open-air courtyards found at the centre of many homes in the region. Swarms were found to occur in open sunlit spaces, mostly located above physical or visual cues somehow visually distinct from the surrounding area. Overall 67 and 78 swarms were observed, respectively, during the dry season (March-April) and the rainy season (September-October) of 2012, between 1.5m and 4.5m above the ground at their centre. 964 mosquitoes were collected and analysed from dry season swarms, of which most were male, and all were An. arabiensis, as were the few resting mosquitoes collected indoors. Larvae collected from breeding sites found on the banks of the Houet River mostly consisted of An. arabiensis and only a minority of Anopheles coluzzii (formerly identified as An. gambiae M form). Of 1694 mosquitoes analysed from 78 swarms in the rainy season collections, a few An. gambiae (formerly known as An. gambiae S form) males were identified, and the remainders were An. arabiensis. The majority of larvae collected during the wet season from the same breeding sites were identified as An. arabiensis followed by An. coluzzii and An. gambiae. The same pattern of species composition was observed in resting mosquitoes, though the proportion of An. arabiensis was less overwhelming. These data support the conclusion that An. arabiensis is the most prevalent species in this area, though the difference in species composition when using different population sampling techniques is noteworthy. Further studies are required for more detailed investigations of male dispersal, feeding behaviour and mating patterns in this urban setting.

Swarming behaviour in natural populations of Anopheles gambiae and An. coluzzii: review of 4 years survey in rural areas of sympatry, Burkina Faso (West Africa).

The swarming behaviour of natural populations of Anopheles gambiae and An. coluzzii (formerly known as An. gambiae S and M forms, respectively) were investigated through longitudinal surveys conducted between July 2006 and October 2009 in two rural areas of south-western Burkina Faso where these forms are sympatric. In both sites, the majority of swarms were recorded above visual markers localised among houses. In Soumousso, a wooded area of savannah, 108 pairs caught in copula from 205 swarms were sampled; in VK7, a rice growing area, 491 couples from 250 swarms were sampled. If segregated swarms were the norm in both sites, many visual markers were shared by the two forms of An. gambiae. Furthermore, mixed swarms were collected annually in frequencies varying from one site to another, though no mixed inseminations were recorded, corroborating the low hybrid rate previously reported in the field. The occurrence of inter-specific mate-recognition mechanisms, which allow individuals to avoid hybridisation, is discussed.

X-ray sterilization of the An. arabiensis genetic sexing strain 'ANO IPCL1' at pupal and adult stages.

With a view to area wide integrated pest management programs with a sterile insect technique (SIT) component against the malaria vector Anopheles arabiensis, the effects of X-ray exposure of males of the genetic sexing strain (GSS) of An. arabiensis, "ANO IPCL1" have been tested. The suitability of X-ray was assessed in comparison to gamma-ray for mosquito sterilization in operational settings where the acquisition of gamma ray irradiators may be problematic. Pupae and adult males were sterilized using X-ray irradiation at 5 increasing doses and effects were observed in terms of fecundity and fertility. Irradiated pupae and adults were further observed for effects on longevity. The adult stage appeared to be more sensitive to radiation exposure than the pupal stage, with a residual fertility of 1% in adults irradiated at 75Gy and 0% at 105Gy, compared to 8%at 75Gy and 4% at 105Gy in adults irradiated at the pupal stage. All treatment groups except those irradiated at 60Gy were statistically different from the control groups in terms of adult longevity post treatment, however overall survival was not highly compromised in the 75Gy treatment group which showed 96% of induced sterility. To ensure the fine balance between the induced sterility and the fitness of the insect produced is achieved, irradiation of adults at this dose may be a good compromise for the SIT as applied for this species, especially in specific cases such as sterilization after female elimination by spiking blood meals. Investigation into the combined effects of blood spiking and irradiation at the adult stage and additional competitiveness studies both in laboratory and field cages will provide more insight into the radiation dose and mosquito stage to be used in the frame of future SIT programs targeting this important malaria vector.

Efficiency of three diets for larval development in mass rearing Aedes albopictus (Diptera: Culicidae).

A fundamental step in establishing a mass production system is the development of a larval diet that promotes high adult performance at a reasonable cost. To identify a suitable larval diet for Aedes albopictus (Skuse), three diets were compared: a standard laboratory diet used at the Centro Agricoltura Ambiente, Italy (CAA) and two diets developed specifically for mosquito mass rearing at the FAO/IAEA Laboratory, Austria. The two IAEA diets, without affecting survival to the pupal stage, resulted in a shorter time to pupation and to emergence when compared with the CAA diet. At 24 h from pupation onset, 50 and 90% of the male pupae produced on the CAA and IAEA diets, respectively, had formed and could be collected. The diet received during the larval stage affected the longevity of adult males with access to water only, with best results observed when using the CAA larval diet. However, similar longevity among diet treatments was observed when males were supplied with sucrose solution. No differences were observed in the effects of larval diet on adult male size or female fecundity and fertility. Considering these results, along with the relative costs of the three diets, the IAEA 2 diet is found to be the preferred choice for mass rearing of Aedes albopictus, particularly if a sugar meal can be given to adult males before release, to ensure their teneral reserves are sufficient for survival, dispersal, and mating in the field.

Anopheles arabiensis sperm production after genetic manipulation, dieldrin treatment, and irradiation.

The use of the sterile insect technique relies on the release of sterilized mass-reared male insects which, before field releases, endure several unnatural treatments. In the case of Anopheles arabiensis (Patton) sterile insect technique program in Sudan, the genetic background of the original strain was first changed to create a genetic sexing strain that is based on a dieldrin-resistant mutation. Secondly, the eggs of the genetic sexing strain require treatment with dieldrin to allow complete elimination of female L1 larvae to enable the release of males only. Finally, male mosquitoes receive an irradiation dose of 70 Gy as pupae for sterilization. The effects of these treatments on sperm production were tested separately and in combination. Irradiation alone significantly decreased the initial sperm number and prevented new sperm production. However, the dieldrin treatment, aimed at eliminating females, appears to have an unexpected radioprotectant effect.

Different blood and sugar feeding regimes affect the productivity of Anopheles arabiensis colonies (Diptera: Culicidae).

The success of the sterile insect technique for the management of mosquito populations depends on the release of large numbers of competitive sterile male insects. Sustainable mosquito production can only be obtained when proper mass-rearing equipment and adequate methods are available, including those to feed blood to the female mosquitoes. The blood feeding apparatus Hemotek consists of a small aluminum plate to which a collagen membrane is fixed and filled with blood kept warm by an electric heating element. A larger aluminum plate was developed to feed a larger number of female mosquitoes with blood that is kept at a constant temperature. The effect of different blood feeding regimes (feeding frequency and time the blood is kept in the Hemotek) and sugar deprivation before blood feeding on egg production of female Anopheles arabiensis Patton was tested. Egg production was higher when blood was offered to the mosquitoes every day as compared with every 2 or 4 d. Sugar deprivation for 7 h before blood feeding enhanced egg production by 50% compared with female mosquitoes that had continuous access to sugar. Neither male nor female survival was impaired. Finally, we showed that the same blood could be kept warm and used over several hours to feed mosquitoes in multiple cages without any impact on egg production or hatch rate. Being able to use the same blood over extended periods would save considerable time, handling, and funds.

An inexpensive and effective larval diet for Anopheles arabiensis (Diptera: Culicidae): eat like a horse, a bird, or a fish?

A successful sterile insect technique program depends upon mass production of good-quality sterile insects for release into a target area. Specifically, to control Anopheles arabiensis Patton (Diptera: Culicidae) in a pilot area in northern Sudan, 1 million An. arabiensis sterile males per day are needed. To reach this production objective, mosquito mass rearing is indispensable and larval diet, a key parameter for the production of healthy male mosquitoes, needs to be cost-effective. The Koi Floating Blend fish food, previously used at the Food and Agriculture Organization/International Atomic Energy Agency Insect Pest Control Laboratory for the routine rearing of the An. arabiensis colony, is no longer available. The aim of this study was to find a cheap and effective substitute for the discontinued diet. Several candidate powdered diets that are commercially available were tested to determine the best diet. By using mixture experiment principles and response surface methodology, the combination of two components, bovine liver powder and tuna meal, showed the best results in terms of larval survival, developmental, rate and adult size. The addition of a vitamin mixture further improved the diet. These positive production results coupled with the relatively low cost of our blend demonstrated the possibility of its use for mass rearing purpose.

Comparisons of life-history characteristics of a genetic sexing strain with laboratory strains of Anopheles arabiensis (Diptera: Culicidae) from northern Sudan.

A genetic sex separation strain (GSS) has been created for Anopheles arabiensis (Patton) (Diptera: Culicidae), one of the major African malaria vectors, for use in controlling wild populations of this species via the sterile insect technique (SIT). This GSS strain, "ANO IPCL1," allows sex separation by a translocation linking a dieldrin resistance allele and the Y chromosome. Differences between ANO IPCL1 relative to wild strains might reflect its field performance and therefore are of concern. Of more immediate interest is how differences might affect production during mass rearing. Life-history parameters were measured for the ANO IPCL1 strain and the two wild strains from which it originated. Although developmental rate differences were found among them, none were large. However, a major observed variation was the very low intrinsic fertility of ANO IPCL1 because of the translocation itself. This resulted in a much lower rate of increase: ANO IPCL1 was able to double its population size, in 7.8 +/- 0.4 d, whereas Dongola and Sennar strains could do so in 4.9 +/- 0.5 and 5.6 +/- 0.4 d. The presence of the Y-autosome translocation mainly affected the natural fertility of the males, and this will require amplification steps during mass rearing.

A new larval tray and rack system for improved mosquito mass rearing.

The requirement for efficient mosquito mass rearing technology has been one of the major obstacles preventing the large scale application of the Sterile Insect Technique against mosquitoes. At the Food and Agriculture Organization/International Atomic Energy Agency (FAO/ IAEA) Insect Pest Control Laboratories we developed a larval rearing unit based on the use of a stainless steel rack that operates 50 thermoformed ABS plastic trays and is expected to be able to successfully rear 140,000-175,000 Anopheles arabiensis (Patton) adult mosquitoes per rack. The mechanized rearing unit is simple to handle, maintains minimal water temperature variation and negligible water evaporation and allows normal larval development. The mosquito mass-rearing tray was designed to provide a large surface area of shallow water that would closely mimic natural breeding sites. The trays stack into a dedicated rack structure and filling and draining were easily performed. The close stacking of the trays in the rack and the possibility to tightly line up several racks makes this rearing unit a valid solution for maximal use of the space thus reducing construction, heating, and cooling costs. The low amount of labor required to operate the system also reduces labor costs that represent one of the main expenditures in any mass rearing facility operation. Preliminary experiments performed on Aedes albopictus (Skuse) also confirm the possibility of successfully extending the use of this technology to other mosquito species. Our larval rearing unit could enhance any mosquito control strategy in which large-scale releases of mosquitoes are needed to suppress or replace natural populations.

Density-dependent effects in experimental larval populations of Anopheles arabiensis (Diptera: Culicidae) can be negative, neutral, or overcompensatory depending on density and diet levels.

Anopheles arabiensis Patton (Diptera: Culicidae) larvae were reared from hatching to the adult stage in the laboratory under a range of diet and larval concentrations using a factorial design. The range circumscribed most of the larval densities and diet concentrations that would allow larval growth and survival using the particular diet formulation and water volume we tested. We determined how these variables affected three outcomes, as follows: larval development rate, survival, and wing length. As has been reported previously, negative density dependence of survival as a function of increased larval density was the prevalent effect on all outcomes when diet was limiting. When diet was not limiting, density dependence was not observed, and three cases of overcompensatory survival were seen. We discuss these results in the context of diet and larval densities for mass rearing and the effect of larval competition on control strategies.