RNAi-mediated gene knockdown and in vivo diuresis assay in adult female Aedes aegypti mosquitoes.
ABSTRACT This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis. The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.
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ABSTRACT: FlyNap (triethylamine) is commonly used to anesthetize Drosophila melanogaster fruit flies. The purpose of this study was to determine whether triethylamine is a suitable anesthetic agent for research into circulatory physiology and immune competence in the mosquito, Anopheles gambiae (Diptera: Culicidae). Recovery experiments showed that mosquitoes awaken from traditional cold anesthesia in less than 7 minutes, but that recovery from FlyNap anesthesia does not begin for several hours. Relative to cold anesthesia, moderate exposures to FlyNap induce an increase in the heart rate, a decrease in the percentage of the time the heart contracts in the anterograde direction, and a decrease in the frequency of heartbeat directional reversals. Experiments employing various combinations of cold and FlyNap anesthesia then showed that cold exposure does not affect basal heart physiology, and that the differences seen between the cold and the FlyNap groups are due to a FlyNap-induced alteration of heart physiology. Furthermore, exposure to FlyNap eliminated the cardioacceleratory effect of crustacean cardioactive peptide (CCAP), and reduced a mosquito's ability to survive a bacterial infection. Together, these data show that FlyNap is not a suitable substitute to cold anesthesia in experiments assessing mosquito heart function or immune competence. Moreover, these data also illustrate the intricate biology of the insect heart. Specifically, they confirm that the neurohormone CCAP modulates heart rhythms and that it serves as an anterograde pacemaker.PLoS ONE 07/2013; 8(7):e70414. DOI:10.1371/journal.pone.0070414 · 3.53 Impact Factor
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ABSTRACT: After taking vertebrate blood, female mosquitoes quickly shed excess water and ions while retaining and concentrating the mostly proteinaceous nutrients. Aquaporins (AQPs) are an evolutionary conserved family of membrane transporter proteins that regulate the flow of water and in some cases glycerol and other small molecules across cellular membranes. In a previous study, we found six putative AQP genes in the genome of the yellow fever mosquito, Ae. aegypti, and demonstrated the involvement of three of them in the blood meal-induced diuresis. Here we characterized AQP expression in different tissues before and after a blood meal, explored the substrate specificity of AQPs expressed in the Malpighian tubules and performed RNAi-mediated knockdown and tested for changes in mosquito desiccation resistance. We found that AQPs are generally down-regulated 24 hrs after a blood meal. Ae. aegypti AQP 1 strictly transports water, AQP 2 and 5 demonstrate limited solute transport, but primarily function as water transporters. AQP 4 is an aquaglyceroporin with multiple substrates. Knockdown of AQPs expressed in the MTs increased survival of Ae. aegypti under dry conditions. We conclude that Malpighian tubules of adult female yellow fever mosquitoes utilize three distinct AQPs and one aquaglyceroporin in their osmoregulatory functions.Scientific Reports 01/2015; 5:7795. DOI:10.1038/srep07795 · 5.58 Impact Factor
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ABSTRACT: Aedes aegypti is the major vector of dengue, yellow fever and chikungunya viruses that put millions of people in endemic countries at risk. Mass rearing of this mosquito is crucial for strategies that use modified insects to reduce vector populations and transmission of pathogens, such as sterile insect technique or population replacement. A major problem for vector mosquito mass rearing is the requirement of vertebrate blood for egg production since it poses significant costs as well as potential health hazards. Also, regulations for human and animal use as blood source can pose a significant obstacle. A completely artificial diet that supports egg production in vector mosquitoes can solve this problem. In this study, we compared different blood fractions, serum and red blood cells, as dietary protein sources for mosquito egg production. We also tested artificial diets made from commercially available blood proteins (bovine serum albumin (BSA) and hemoglobin). We found that Ae. aegypti performed vitellogenesis and produced eggs when given whole bovine blood, serum, or an artificial diet containing BSA. Conversely, egg production was impaired after feeding of the red blood cell fraction or an artificial diet containing only hemoglobin. We also found that egg viability of serum-fed mosquitoes were comparable to that of whole blood and an iron supplemented BSA meal produced more viable eggs than a meal containing BSA alone. Our results indicate that serum proteins, not hemoglobin, may replace vertebrate blood in artificial diets for mass mosquito rearing.PeerJ 05/2015; 3. DOI:10.7717/peerj.938 · 2.10 Impact Factor