Mosquitoes are human disease vectors that transmit pathogens through blood feeding. These human pathogens include the malarial parasite, Dengue, Zika, and yellow fever viruses, and West Nile virus. Dengue fever and malaria continue to have a significant health and economic impact worldwide, and therefore new approaches are needed for controlling the spread of these diseases. Mosquitoes are highly evolved blood sucking insects that have been shown to efficiently ingest, metabolize, and transport blood meal nutrients from the midgut lumen to the ovaries to produce eggs. Although we have a basic knowledge of these processes at the physiological level, it requires a molecular understanding of metabolic regulation in mosquitoes in order to target blood meal metabolism as transmission blocking or vector control strategy. Since highly coordinated endocytotic and exocytotic processes in midgut, fat body, and ovary tissues likely play a central role in metabolic flux following the blood meal, we are investigating vesicle transport in Aedes aegypti and Anopheles stephensi mosquitoes.
Increase in reported cases of Dengue hemorrhagic fever (WHO)
Overview of Blood Meal Metabolism in Aedes aegypti Mosquitoes
Newly emerged female mosquitoes feed on nectar for several days until they are able to take their first blood meal (males do not blood feed). The blood meal is required for Ae. aegypti egg development and results in the deposition of ~100 fertilized eggs within ~60 hours of feeding. In order to produce this many eggs, blood meal metabolism requires efficient retrieval of nutrients and rapid excretion of toxic ammonia. This is an amazing accomplishment considering the mosquito's size. A typical female Ae. aegypti female mosquito weighs ~2.5 mg and can consume a blood meal of 2 ul in ~60 seconds. This 2.5 mg meal (including the water, protein, and lipid) is therefore equal in mass to her own body. This would be equivalent to a 125 lb. women drinking a 12 gallon smoothie that contains 25 lbs. of hamburger meat, 0.5 lb. of butter, and 2 tbls of sugar. Can you imagine not only drinking this mega smoothie in less than a minute, but completely digesting it, and then excreting all of the toxic waste products in just 24 hours? The female Ae. aegypti mosquito does this up to five times in her lifetime, resulting in the production of 500+ mosquito eggs over a two week period.
Role of COPI vesicle transport in blood fed mosquitoes
Blood feeding by vector mosquitoes provides the entry point for disease pathogens and presents an acute metabolic challenge that must be overcome to complete the gonotrophic cycle. Based on recent data showing that COPI vesicle transport is involved in cellular processes beyond Golgi-ER retrograde protein trafficking, we disrupted COPI functions in the Dengue mosquito Aedes aegypti to interfere with blood meal digestion. Surprisingly, we found that decreased expression of COPI coatomer proteins led to 89% mortality in blood fed mosquitoes by 72 hr post-feeding, compared to 0% mortality in control dsRNA injected blood fed mosquitoes and 3% mortality in COPI dsRNA injected sugar fed mosquitoes. We also examined midgut tissues by electron microscopy, quantitated heme in fecal samples, and characterized feeding-induced protein expression in midgut, fat body, and ovary tissues of COPI deficient mosquitoes. We found that COPI defects disrupt epithelial cell membrane integrity, stimulate premature blood meal excretion, and block induced expression of several midgut protease genes. In order to study the role of COPI transport in ovarian development, we injected COPI coatomer protein dsRNA after blood feeding, and found that while blood digestion was normal, follicles in these mosquitoes were significantly smaller by 48 hr post-injection and lacked eggshell proteins. Together, these data show that COPI functions are critical to mosquito blood digestion and egg maturation, a finding that could also apply to other blood feeding arthropod vectors.
Molecular analysis of lipid synthesis and storage in Ae. aegypti mosquitoes
To better understand the mechanism of de novo lipid biosynthesis in blood fed Ae. aegypti mosquitoes, we quantitated acetyl-CoA carboxylase (ACC) and fatty acid synthase 1 (FAS1) transcript levels in blood fed mosquitoes, and used RNAi methods to generate ACC and FAS1 deficient mosquitoes. Using the ketogenic amino acid 14C-leucine as a metabolic precursor of 14C-acetyl-CoA, we found that 14C-triacylglycerol and 14C-phospholipid levels were significantly reduced in both ACC and FAS1 deficient mosquitoes, confirming that ACC and FAS1 are required for de novo lipid biosynthesis after blood feeding. Surprisingly however, we also found that ACC deficient mosquitoes, but not FAS1 deficient mosquitoes, produced defective oocytes, which lacked an intact eggshell and gave rise to inviable eggs. This severe phenotype was restricted to the 1st gonotrophic cycle, suggesting that the eggshell defect was due to ACC deficiencies in the follicular epithelial cells, which are replaced after each gonotrophic cycle. Consistent with lower amounts of de novo lipid biosynthesis, both ACC and FAS1 deficient mosquitoes produced significantly fewer eggs than control mosquitoes in both the 1st and 2nd gonotrophic cycles. Lastly, FAS1 deficient mosquitoes, but not ACC deficient mosquitoes, showed delayed blood meal digestion, suggesting that a feedback control mechanism may coordinate rates of fat body lipid biosynthesis and midgut digestion during feeding. We propose that decreased ACC and FAS1 enzyme levels lead to reduced 14C-lipid biosynthesis and lower fecundity, whereas altered levels of the regulatory metabolites acetyl-CoA and malonyl-CoA account for the observed defects in eggshell formation and blood meal digestion, respectively.
Isoe, J., Goodwin, C., Isoe, Y.E., Lazarus, L.B., Day, W.A., McLean, J.A., and Miesfeld, R.L. Regulation of vesicle transport and eggshell synthesis in the ovaries of Aedes aegypti mosquitoes, submitted.
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Rascon, A., Gearin, J., Isoe, and Miesfeld, R. (2011) In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases in the Dengue vector mosquito Aedes aegypti. BMC Bioc. 12:43.
Isoe, J., Collins, J., Badgandi, H., Day, W.A., and Miesfeld, R. (2011) Defects in COPI transport cause blood feeding-induced mortality in Yellow Fever mosquitoes, Proc. Nat. Acad. Sci., 108:E211-E217.
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Scaraffia, P.Y., Zhang, Q., Thorson, K., Wysocki, V.H. and Miesfeld, R. (2010) Differential ammonia metabolism Aedes aegypti fat body and midgut tissues, J. Insect. Physiol., 56:1040-1049.
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Brackney, D.E., Isoe, J., Zamora, J., Black, W.C., Foy, B.D., Miesfeld, R. and Olson, K.E. (2010) Expression profiling and comparative analysis of seven novel midgut serine proteases from the Yellow Fever mosquito, J. Insect. Physiol. 56:736-744.
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Brandon, M.C., Pennington, J.E., Zamora, J., Isoe, J., Schillinger, A-S. and Miesfeld, R. (2008) TOR signaling is required for amino acid stimulation of early trypsin protein synthesis in the midgut of Aedes aegypti mosquitoes. Insect. Bioc. Mol. Biol., 38:916-922.
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