Miesfeld Lab

A female Aedes aegypti mosquito
obtaining a human blood meal
(CDC, Atlanta)

Mosquitoes are human disease vectors that can transmit pathogens through blood feeding. These human pathogens include the malarial parasite, Dengue and yellow fever viruses, and West Nile virus. There are over 3,000 mosquito species, but only a small number are responsible for the majority of vector-borne human diseases. The three primary mosquito vectors belong to the Anopheles (malaria), Aedes (Dengue and yellow fever viruses), and Culex (West Nile virus) genera. 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 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 - wow! 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 300 mosquito progeny.

Control of Trypsin Synthesis in the Mosquito Midgut
Proteases are hydrolytic enzymes that are produced in midgut epithelial cells in response to feeding. Early phase proteases, such as early trypsin (ET), are translationally-regulated by feeding, whereas, the late phase protease, LPT-1, also known as late trypsin, is transcriptionally-regulated. Within 90 minutes of blood feeding, rough endoplasmic reticulum whorls in midgut epithelial cells unwind and translation of early trypsin transcripts is initiated. Whorl unwinding can be inhibited in in vitro midgut cultures by the juvenile hormone analogue methoprene. Experiments are underway to identify proteins associated with the whorls before and after feeding using high resolution 2D gel electrophoresis and mass spectrometry.

TOR (Target of Rapamycin) is a protein kinase that is activated by nutrient and hormone signaling in a variety of organisms. The TOR pathway has been shown to be stimulted by PI3K signaling, and is also linked to amino acid transporters through an unknown mechanism. TOR activates both transcription and translation, and one of these downstream effects involves the phosphorylation of 4E-BP, an inhibitor of the translation initiation factor eIF-4E. To determine if TOR signaling is required for ET translation in Ae. aegypti, we used both molecular genetic (RNAi) and pharmacological (rapamycin) approaches to inhibit TOR in the midgut of fed mosquitoes. TOR inhibition was found to block 4EBP phosphorylation and ET synthesis.

Early work from the Wells lab using crude preparations of soy bean trypsin inhibitor suggested that protein digestion by ET was required to release an unknown factor that regulates LPT-1 gene expression. In 2006, however, Stephen Lu and Mike Wells reported that inhibition of ET by a variety of selective trypsin inhibitors, or by ET RNAi knock-down, did not alter blood meal-induced expression of the LPT-1 gene. Therefore, one of the projects in the lab is to find the bona fide upstream regulators of late phase trypsin gene expression. 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 LPT-1 gene expression in response to feeding. Surprisingly, we found that decapitation before feeding, followed by a gamma globulin protein enema, was sufficient to induce the expression of four late phase protease genes up to 100-fold (LPT-1, 5G1, Cx-A1, Ser. Col.). 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 blood meal induced late phase protease gene expression.

Regulation of Nitrogen and Lipid Metabolism in Mosquitoes
About 85% of blood meal protein amino acids are deaminated to produce alpha-keto acids and ammonia. This amount of ammonia should lead to a lethal hemolymph concentration of 200 mM, however, 24 hours after a protein meal, we found that hemolymph ammonia was only ~2 mM. Where is all of the ammonia going? The surprising answer was that it is converted into glutamine and proline as a way to prevent ammonia toxicity. Using mass spectrometry to follow ¹⁵N-labeled isotopes, coupled with the use of specific enzyme inhibitors and RNAi, we also found that Ae. aegypti females produce urea using an amphibian-like uricolytic pathway. This was an exciting discovery!

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. Very little is known about the relative roles of lipid metabolizing enzymes in blood fed mosquitoes, nor if there are rate-limiting steps in this process that could serve as targets for vector control. In the past year, we have cloned and characterized numerous genes from Ae. aegypti that are likely involved in blood meal induced lipid metabolism. 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. We are now combining metabolic labeling studies using ¹⁴C-protein, with RNAi knockdowns, to investigate the role of select lipid metabolizing pathways in blood meal metabolism.

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. Since Cx. pipiens can harbor the West Nile virus over the winter, we are interested in finding ways to disrupt diapause using metabolic inhibitors. We recently began a collaboration with Dr. David Denlinger's lab at Ohio State University to investigate lipid metabolism during Cx. pipiens diapause and are now rearing these mosquitoes using chicken blood in an artificial feeding apparatus. We recently demonstrated that we could induce diapause using reduced temperatures and light cycle durations in a climate controlled growth chamber and have begun to collect metabolic labeling data by feeding newly emerged adults ¹⁴C-glucose. These studies will be used to establish baseline profiles for future metabolic studies involving RNAi knockdown of specific genes.

Cx. pipiens mosquito obtaining a human blood meal.