Dataset Information

Molecular genetic analysis of midgut serine proteases in Aedes aegypti mosquitoes.

ABSTRACT: Digestion of blood meal proteins by midgut proteases provides anautogenous mosquitoes with the nutrients required to complete the gonotrophic cycle. Inhibition of protein digestion in the midgut of blood feeding mosquitoes could therefore provide a strategy for population control. Based on recent reports indicating that the mechanism and regulation of protein digestion in blood fed female Aedes aegypti mosquitoes is more complex than previously thought, we used a robust RNAi knockdown method to investigate the role of four highly expressed midgut serine proteases in blood meal metabolism. We show by Western blotting that the early phase trypsin protein (AaET) is maximally expressed at 3 h post-blood meal (PBM), and that AaET is not required for the protein expression of three late phase serine proteases, AaLT (late trypsin), AaSPVI (5G1), and AaSPVII. Using the trypsin substrate analog BApNA to analyze in vitro enzyme activity in midgut extracts from single mosquitoes, we found that knockdown of AaSPVI expression caused a 77.6% decrease in late phase trypsin-like activity, whereas, knockdown of AaLT and AaSPVII expression had no significant effect on BApNA activity. In contrast, injection of AaLT, AaSPVI, and AaSPVII dsRNA inhibited degradation of endogenous serum albumin protein using an in vivo protease assay, as well as, significantly decreased egg production in both the first and second gonotrophic cycles (P < 0.001). These results demonstrate that AaLT, AaSPVI, and AaSPVII all contribute to blood protein digestion and oocyte maturation, even though AaSPVI is the only abundant midgut late phase serine protease that appears to function as a classic trypsin enzyme.

SUBMITTER: Isoe J

PROVIDER: S-EPMC2818436 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Molecular genetic analysis of midgut serine proteases in Aedes aegypti mosquitoes.

Isoe Jun J Rascón Alberto A AA Kunz Susan S Miesfeld Roger L RL

Insect biochemistry and molecular biology 20091103 12

Digestion of blood meal proteins by midgut proteases provides anautogenous mosquitoes with the nutrients required to complete the gonotrophic cycle. Inhibition of protein digestion in the midgut of blood feeding mosquitoes could therefore provide a strategy for population control. Based on recent reports indicating that the mechanism and regulation of protein digestion in blood fed female Aedes aegypti mosquitoes is more complex than previously thought, we used a robust RNAi knockdown method to ...[more]

PMID: 19883761

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Project description:Microbiota studies of Aedes aegypti and other mosquitoes generally focus on the bacterial communities found in adult female midguts. However, other compartments of the digestive tract maintain communities of bacteria which remain almost entirely unstudied. For example, the Dipteran crop is a food storage organ, but few studies have looked at the microbiome of crops in mosquitoes, and only a single previous study has investigated the crop in Ae. aegypti. In this study, we used both culture-dependent and culture-independent methods to compare the bacterial communities in midguts and crops of laboratory reared Ae. aegypti. Both methods revealed a trend towards higher abundance, but also higher variability, of bacteria in the midgut than the crop. When present, bacteria from the genus Elizabethkingia (family Weeksellaceae) dominated midgut bacterial communities. In crops, we found a higher diversity of bacteria, and these communities were generally dominated by acetic acid bacteria (family Acetobacteriaceae) from the genera Tanticharoenia and Asaia. These three taxa drove significant community structure differences between the tissues. We used FAPROTAX to predict the metabolic functions of these communities and found that crop bacterial communities were significantly more likely to contain bacteria capable of methanol oxidation and methylotrophy. Both the presence of acetic acid bacteria (which commonly catabolize sugar to produce acetic acid) and the functional profile that includes methanol oxidation (which is correlated with bacteria found with natural sources like nectar) may relate to the presence of sugar, which is stored in the mosquito crop. A better understanding of what bacteria are present in the digestive tract of mosquitoes and how these communities assemble will inform how the microbiota impacts mosquito physiology and the full spectrum of functions provided by the microbiota. It may also facilitate better methods of engineering the mosquito microbiome for vector control or prevention of disease transmission.

Project description:We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted by this vector, our results highlight biochemical choke points that could be targeted to disrupt transmission of multiple pathogens by these mosquitoes.

Dataset Information

Molecular genetic analysis of midgut serine proteases in Aedes aegypti mosquitoes.

Publications

Molecular genetic analysis of midgut serine proteases in Aedes aegypti mosquitoes.

Similar Datasets

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