Preparation and Use of a Yeast shRNA Delivery System for Gene Silencing in Mosquito Larvae

The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the dengue and Zika virus vector Aedes aegypti and the primary African malaria vector Anopheles gambiae. RNA interference (RNAi) has facilitated gene silencing experiments in both of these disease vector mosquito species and could one day be applied as a new method of vector control. Here, we describe a procedure for the genetic engineering of Saccharomyces cerevisiae (baker’s yeast) that express short hairpin RNA (shRNA) corresponding to mosquito target genes of interest. Following cultivation, which facilitates inexpensive propagation of shRNA, the yeast is inactivated and prepared in a ready-to-use dry tablet formulation that is fed to mosquito larvae. Ingestion of the yeast tablets results in effective larval target gene silencing. This technically straightforward and affordable technique may be applicable to a wide variety of mosquito species and potentially to other arthropods that feed on yeast

Lymphatic filariasis in Oceania

Lymphatic filariasis caused by the mosquito-transmitted helminth parasite Wuchereria bancrofti is an important problem in Oceania. Of the 33 countries and territories included in this review, 24 have been found to be endemic for this disease at some time in the past, and 18 of these were classified as endemic at the start of the Global Programme to Eliminate Lymphatic Filariasis in 2000. After the implementation of large mass drug administration campaigns and (to a lesser extent) vector control over the last 15 years, only ten Oceania countries and territories were still considered to have ongoing transmission of lymphatic filariasis in 2015. Through a systematic literature search and review, we identified 79 individual studies of filariasis in Oceania that were published in 70 papers between 1995 and 2015. Data on mosquito (by species) and human infection prevalence using all currently available diagnostic tests, as well as estimates of acute and chronic filariasis morbidity, were extracted from these publications and tabulated in chronological order by country and outcome measure, noting sampling method and sample size in order to evaluate study quality and precision. No studies were identified from Micronesia; most studies in Melanesia and Polynesia were found from Papua New Guinea (PNG) (30) and French Polynesia (16), respectively. All other countries in Melanesia and Polynesia were represented by 1–7 studies except Wallis and Futuna. The systematic review identified 19 published studies of mosquito infections and 62 of human infections but only 3 on acute morbidity (all from PNG in the 1990s) and 11 on chronic morbidity. Since Oceania has a diverse set of mosquito vectors, published reviews of relative efficiencies of different mosquito genera were examined to shed light on their transmission dynamics and hence the potential for elimination of filariasis in Oceania. The review indicates the need for collation of unpublished reports and studies in addition to more geographically representative studies of remaining filariasis infection distribution, as well as quantification of the disability (acute attacks, lymphoedema, elephantiasis and hydrocoele) that will remain once transmission is interrupted, in order to plan for services to alleviate these lifelong effects

Exploring the potential of computer vision analysis of pupae size dimorphism for adaptive sex sorting systems of various vector mosquito species

International audienceBackgroundSeveral mosquito population suppression strategies based on the rearing and release of sterile males have provided promising results. However, the lack of an efficient male selection method has hampered the expansion of these approaches into large-scale operational programmes. Currently, most of these programmes targeting Aedes mosquitoes rely on sorting methods based on the sexual size dimorphism (SSD) at the pupal stage. The currently available sorting methods have not been developed based on biometric analysis, and there is therefore potential for improvement. We applied an automated pupal size estimator developed by Grupo Tragsa with laboratory samples of Anopheles arabiensis, Aedes albopictus, Ae. polynesiensis, and three strains of Ae. aegypti. The frequency distribution of the pupal size was analyzed. We propose a general model for the analysis of the frequency distribution of mosquito pupae in the context of SSD-sorting methods, which is based on a Gaussian mixture distribution functions, thus making possible the analysis of performance (% males recovery) and purity (% males on the sorted sample).ResultsFor the three Aedes species, the distribution of the pupae size can be modeled by a mixture of two Gaussian distribution functions and the proposed model fitted the experimental data. For a given population, each size threshold is linked to a specific outcome of male recovery. Two dimensionless parameters that measure the suitability for SSD-based sorting of a specific batch of pupae are provided. The optimal sorting results are predicted for the highest values of SSD and lowest values of intra-batch variance. Rearing conditions have a strong influence in the performance of the SSD-sorting methods and non-standard rearing can lead to increase pupae size heterogeneity.ConclusionsSex sorting of pupae based on size dimorphism can be achieved with a high performance (% males recovery) and a reasonably high purity (% males on the sorted sample) for the different Aedes species and strains. The purity and performance of a sex sorting operation in the tested Aedes species are linked parameters whose relation can be modeled. The conclusions of this analysis are applicable to all the existing SSD-sorting methods. The efficiency of the SSD-sorting methods can be improved by reducing the heterogeneity of pupae size within rearing containers. The heterogeneity between batches does not strongly affect the quality of the sex sorting, as long as a specific separation threshold is not pre-set before the sorting process. For new developments, we recommend using adaptive and precise threshold selection methods applied individually to each batch or to a mix of batches. Adaptive and precise thresholds will allow the sex-sorting of mixed batches in operational conditions maintaining the target purity at the cost of a reduction in performance. We also recommend a strategy whereby an acceptable level of purity is pre-selected and remains constant across the different batches of pupae while the performance varies from batch to batch to fit with the desired purity