Roger L. Miesfeld
Professor of Biochemistry, and Molecular and Cellular Biology
Ph.D. 1983, State University of New York at Stony Brook
Metabolic Regulation in Mosquitoes
Research Interests
Mosquitoes are human disease vectors that transmit pathogens through blood feeding. These human pathogens include the malarial parasite, Dengue and yellow fever viruses, and West Nile virus. Out of over 3,000 mosquito species known worldwide, only a handful require a bloodmeal to complete the reproductive cycle. The major mosquito disease vectors belong to three different genera; Anopheles (malaria), Aedes (Dengue and yellow fever viruses), and Culex (West Nile virus). An. gambiae and Ae aegypti feed almost exclusively on humans (anthropophilic), whereas, Cx. pipiens feeds on various animals including humans (zoophilic). Since blood meal feeding creates a unique metabolic challenge as a result of the extremely high protein and iron content of blood, it is possible that interfering with blood meal metabolism could provide a novel control strategy for mosquito born diseases in infected areas.
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 ~40 fertilized eggs within 72 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 three times during her short two week lifetime, resulting in the production of as many as 150 mosquito progeny.
We have three projects in the lab that are currently funded by the National Institutes of Health.
Regulation of protease gene expression in the midgut of Ae. aegypti mosquitoes
It has been known for some time that blood meal feeding induces the expression of a numerous protease genes in midgut epithelial cells. One strategy has been to take a physiological approach to identify endocrine (midgut or fat body) or neuroendocrine (brain) molecules that may be involved in regulating LT gene expression in response to feeding. Surprisingly, we found that abdomen ligation 30 min post-feeding, or decapitation before feeding followed by a protein enema, was sufficient to induce LT gene expression. These results led us to focus on identifying signaling pathways within the midgut that are activated by a protein meal in the lumen. We are now using RNAi approaches to test if known nutrient-sensing transcription factors are responsible for late phase protease gene expression. This includes examining the role of TOR (Target of Rapamycin) in the early and late phases of blood meal digestion.
Regulation of energy metabolism in mosquitoes
Recent studies performed in the laboratory represent the first dynamic approach to determining the fate of amino acids derived from the blood meal of mosquitoes. The rates of oxidation of
14C substrates and the metabolism of 15N-ammonia in Ae. aegypti females revealed unexpected pathway fluxes as compared to what is known about similar pathways in vertebrates. For example, we discovered that Aa. aegypti contain a functional uricolytic pathway that converts uric acid to urea. Using traditional biochemical methods and modern mass spectrometry techniques, we are now in a position to probe the metabolic control of amino acid and fatty acid metabolism in more detail, and to develop quantitative models describing metabolic flux at different stages of mosquito development. These studies are being carried out in Aa. aegypti, as well as, Cx. pipiens, a vector of West Nile Virus. Cx. pipiens mosquitoes are somewhat unusual compared to Ae. aegypti and An. gambiae in that adult Cx. pipiens mosquitoes undergo diapause (a type of hibernation) over the winter by storing up lipid reserves as a source of energy.
Molecular analysis of lipid synthesis, transport and storage in Ae. aegypti mosquitoes
Mosquitoes need to convert carbon derived from amino acid deamination of blood meal proteins into metabolic energy that can be used to complete the gonotrophic cycle. In the past year, we have cloned and characterized numerous genes from Ae. aegypti that that are likely involved in blood meal induced lipid metabolism. These include acetyl-CoA carboxylase (ACC), two different fatty acid synthase (FAS) genes, cytosolic fatty acid binding protein (FABP), the fatty acid translocase (the mammalian CD36 ortholog), the plasmalemmal fatty acid binding protein (FABPpm), and the diacylglycerol acyl transferases (DGAT1, DGAT2a, DGAT2b). QRT-PCR analysis of these genes in blood fed mosquitoes, showed that with the exception of the two FAS genes, all of the others were differentially expressed in response to feeding. Moreover, RNAi knockdown revealed that decreased expression had clear effects on lipid metabolism and reproduction, with phenotypes ranging from loss of egg production, delayed oviposition, and decreased egg size.
Selected Publications
Zhou, G. and Miesfeld, R. (2009) Energy metabolism during diapause in Culex pipiens mosquitoes. J. Insect. Physiol., 55:40-46.
Isoe, J., Zamora, J. and Miesfeld, R. (2009) Molecular analysis of the Aedes aegypti carboxypeptidase gene family. Insect. Bioc. Mol. Biol., 39:68-73.
Scaraffia, P.Y., Tang, G., Isoe, J., Wysocki, V.H., Wells, M.A. and Miesfeld, R. (2008) Discovery of an alternate metabolic pathway for urea synthesis in adult Aedes aegypti mosquitoes, Proc. Nat. Acad. Sci., 105:518-523.
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.
Isoe, J., Kunz, S., Manhart, C., Wells, M.A. and Miesfeld, R. (2007) Regulated expression of microinjected DNA in adult Aedes aegypti mosquitoes. Insect Mol. Biol., 16:83–92.
Masakazu Yamamoto, M., Watt, C., Schmidt, R., Kuscuoglu, U., Miesfeld, R. and Goldhamer, D. (2007) Cloning and characterization of a novel MyoD enhancer-binding factor. Mech. Develop., 124:715-28.