Effective Oral RNA Interference (RNAi) Administration to Adult <em>Anopheles gambiae</em> Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) ...

Centers for Disease Control and Prevention

Mosquito-borne diseases affect millions worldwide, with malaria alone killing over 400 thousand people per year and affecting hundreds of millions. To date, the best strategy to prevent the disease remains insecticide-based mosquito control. However, insecticide resistance as well as economic and social factors reduce the effectiveness of the current methodologies. Alternative control technologies are in development, including genetic control such as the sterile insect technique (SIT). The SIT is a pivotal tool in integrated agricultural pest management and could be used to improve malaria vector control. To apply the SIT and most other newer technologies against disease transmitting mosquitoes, it is essential that releases are composed of males with minimal female contamination. The removal of females is an essential requirement because released females can themselves contribute towards nuisance biting and disease transmission. Thus, females need to be eliminated from the cohorts prior to release. Manual separation of Anopheles gambiae pupae or adult mosquitoes based on morphology is time consuming, is not feasible on a large scale and has limited the implementation of the SIT technique. The doublesex (dsx) gene is one of the effector switches of sex determination in the process of sex differentiation in insects. Both males and females have specific splicing variants that are expressed across the different life stages. Using RNA interference (RNAi) to reduce expression of the female specific (dsxF) variant of this gene has proven to have detrimental effects to the females in other mosquito species, such as Aedes aegypti. We tested oral RNAi on dsx (AgdsxF) in An. gambiae.

Downregulation of female doublesex expression by oral-mediated RNA interference reduces number and fitness of Anopheles gambiae adult females.

Larval mosquitoes are aquatic omnivorous scavengers which scrape food from submerged surfaces and collect suspended food particles with their mouth brushes. The composition of diets that have been used in insectaries varies widely though necessarily provides sufficient nutrition to allow colonies to be maintained. Issues such as cost, availability and experience influence which diet is selected. One component of larval diets, essential fatty acids, appears to be necessary for normal flight though deficiencies may not be evident in laboratory cages and are likely more important when mosquitoes are reared for release into the field in e.g. mark-release-recapture and genetic control activities. In this study, four diets were compared for rearing Anopheles gambiae and Aedes aegypti, all of which provide these essential fatty acids. Two diets were custom formulations specifically designed for mosquitoes (Damiens) and two were commercially available fish foods: Doctors Foster and Smith Koi Staple Diet and TetraMin Plus Flakes. Development rate, survival, dry weight and adult longevity of mosquitoes reared with these four diets were measured. The method of presentation of one diet, Koi pellets, was additionally fed in two forms, pellets or a slurry, to determine any effect of food presentation on survival and development rate. While various criteria might be selected to choose 'the best' food, the readily-available Koi pellets resulted in development rates and adult longevity equal to the other diets, high survival to the adult stage and, additionally, this is available at low cost.

Pragmatic selection of larval mosquito diets for insectary rearing of Anopheles gambiae and Aedes aegypti.

Mosquito rearing containers contain organic-rich water that nourishes numerous bacteria, some of which are capable of forming biofilms. Biofilm is broadly an extracellular polymeric matrix (EPS) in which living bacteria occur, and the accumulation of biofilm is possible during routine stock-keeping as most of these containers are re-used. Whether biofilm has an effect on the mosquito rearing is not a question that has been investigated, nor have measures to reduce biofilm in this context been systematically studied.

Measuring and reducing biofilm in mosquito rearing containers.

As a means of obtaining reproducible and accurate numbers of larvae for laboratory rearing, we tested a large-particle flow-cytometer type device called the 'Automated Particle Counter' (APC). The APC is a gravity-fed, self-contained unit that detects changes in light intensity caused by larvae passing the detector in a water stream and controls dispensing by stopping the flow when the desired number has been reached. We determined the accuracy (number dispensed compared to the target value) and precision (distribution of number dispensed) of dispensing at a variety of counting sensitivity thresholds and larva throughput rates (larvae per second) using < 1-day old Anopheles gambiae and Aedes aegypti larvae. All measures were made using an APC algorithm called the 'Smoothed Z-Score' which allows the user to define how many standard deviations (Z scores) from the baseline light intensity a particle's absorbance must exceed to register a count. We dispensed a target number of 100 An. gambiae larvae using Z scores from 2.5-8 and observed no difference among them in the numbers dispensed for scores from 2.5-6, however, scores of 7 and 8 under-counted (over-dispensed) larvae. Using a Z score ≤ 6, we determined the effect of throughput rate on the accuracy of the device to dispense An. gambiae larvae. For rates ≤ 98 larvae per second, the accuracy of dispensing a target of 100 larvae was - 2.29% ± 0.72 (95% CI of the mean) with a mode of 99 (49 of 348 samples). When using a Z score of 3.5 and rates ≤ 100 larvae per second, the accuracy of dispensing a target of 100 Ae. aegypti was - 2.43% ± 1.26 (95% CI of the mean) with a mode of 100 (6 of 42 samples). No effect on survival was observed on the number of An. gambiae first stage larvae that reached adulthood as a function of dispensing.

Trials of the Automated Particle Counter for laboratory rearing of mosquito larvae.

Laboratory rearing of mosquitoes is commonly practiced by researchers studying mosquito-borne infectious diseases and vector control methods. In the absence of cryopreservation methods to stabilize unique or genetically modified strains, mosquito lines must be continuously maintained, a laborious process that risks selection effects, contamination, and genetic drift. Towards the development of a cryopreservation protocol, several commonly used cryoprotectants were systematically characterized here both individually and as cocktails. Among first instar, feeding-stage An. gambiae and An. stephensi larvae, cryoprotectant toxicity followed the order of dimethyl sulfoxide > ethylene glycol > methanol. The resulting LD values were used as the basis for the development of cryoprotectant cocktail solutions, where formulation optimization was streamlined using Taguchi methods of experimental design. Sensitivity to hypothermia was further evaluated to determine the feasibility of cryoprotectant loading at reduced temperatures and slow cooling approaches to cryopreservation. The information described here contributes to the knowledge base necessary to inform the development of a cryopreservation protocol for Anopheles larvae.

Cryoprotectant toxicity and hypothermic sensitivity among Anopheles larvae.

Shipments of living mosquitoes and other arthropods require temperatures that are within a range that is compatible with their health and survival. In addition to express shipping and insulated containers, shipments often include materials that either store heat (i.e., have thermal mass) or otherwise stabilize the temperature. In this paper, we present the results of comparisons of thermal mass and phase change materials to stabilize the temperature under various conditions. We compared a rigid foam refrigerant and a number of phase change materials to bubble wrap for their capacity to moderate temperature change by measuring the temperatures in standard uninsulated shipping containers during exposure to high (37°C), cold (4°C), and freezing (-20°C) temperatures. We make recommendations for shipments depending on the ambient conditions that are expected to be experienced en route.

Catherine Hunt

Division of Parasitic Diseases and Malaria,
Entomology Branch,
Division of Parasitic Diseases and Malaria, Entomology Branch

Centers for Disease Control and Prevention

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

Catherine Hunt

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Parasitic Diseases and Malaria,Entomology Branch,Division of Parasitic Diseases and Malaria, Entomology Branch

Centers for Disease Control and Prevention

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

Division of Parasitic Diseases and Malaria,
Entomology Branch,
Division of Parasitic Diseases and Malaria, Entomology Branch