• Faculty Directory
• Science Education/Courses
• Curriculum Vitae
• List of Publications
• Research Group Homepage

Roger Miesfeld

Honors

• UA Honors College Faculty Excellence Award, 2012
• Chair, American Cancer Society TBE Panel, 1999-2001
• Chair, Gordon Conference on Cancer, 1994
• Fellow & Scholar, Leukemia Society America, 1986-1994
• Fellow, Jane Coffin Childs Memorial Fund, 1983-1986

Education and Appointments

• BS, MS 1973-79, San Diego State
  
• PhD 1979-83, SUNY Stony Brook
  
• Fellow 1983-87, UC San Francisco
  

Research Interests

• Biochemistry
  
• "Metabolism, Signaling, and Regulation"
  

Research Summary

Blood Meal Metabolism 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 transmit human pathogens. 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 ~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 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.

Selected Publications

Isoe, J., Mack, D.J., Njardarson, J.T., and Miesfeld, R.L. Biochemical inhibitors of COPI vesicle transport have differential metabolic effects on Diptera insects, in preparation.

Isoe, J., Stover, W., Miesfeld, R.B. and Miesfeld, R.L. Efficient blood meal metabolism in vector mosquitoes is dependent on GBF1 activation of ADP ribosylation factor 1, in preparation.

Mack, D.J., Isoe, J., Miesfeld, R.L. and Njardarson, J.T. (2012) Distinct biological effects of Golgicide A derivatives on larval and adult mosquitoes, Bioorg. Med. Chem. Lett., 22:5177-5188.

Alabaster, A., Isoe, J., Zhou, G., Lee, A., Murphy, A. and Miesfeld, R. (2011) Deficiencies in acetyl-CoA carboxylase and fatty acid synthase 1 differentially affect eggshell development and blood meal digestion in Aedes aegypti. Insect. Bioc. Mol. Biol., 41:946-955.

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.

Zhou, G., Isoe, J., Day, W.A., and Miesfeld, R. (2011) Alpha-COPI transport function is required for rough endoplasmic reticulum whorl formation in mosquito midgut epithelial cells, PLoS ONE 6(3): e18150.

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.

Wei, J., Wysocki, V.H., Miesfeld, R. and Scaraffia, P.Y. (2010) Differentiation and quantification of C1 and C2 13C-labeled glucose by tandem mass spectrometry, Analytical. Bioc., 404:40-44.

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.

Isoe, J., Rascon, A., Kunz, S., and Miesfeld, R. (2009) Molecular genetic analysis of midgut serine proteases in Aedes aegypti mosquitoes, Insect Bioc. Mol. Biol., 39:903-912.

Zhou, G. and Miesfeld, R. (2009) Differential utilization of blood meal amino acids in Aedes aegypti mosquitoes, Open Access Insect Physiology, 1:1-12.

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.