Select Faculty Member Mark R. Brown Donald E. Champagne Daniel G. Colley Harry W. Dickerson, Jr. Roberto Docampo Stephen L. Hajduk Jessica Kissinger Patrick J. Lammie Kojo A. Mensa-Wilmot Julie M. Moore Silvia N.J. Moreno Ynes R. Ortega David S. Peterson Pejman Rohani Robert Sabatini Mike R. Strand Boris Striepen Rick L. Tarleton

Mark R. Brown
Professor, Department of Entomology
Ph.D., University of Georgia, 1985

413 Biological Sciences Building
Phone: (706) 542-2317
Email: mbrown@bugs.ent.uga.edu

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Overview
Reproduction in female insects encompasses a highly regulated sequence of behavioral, metabolic, and synthetic processes that result in the production of eggs. As in all other animals, peptide hormones bind to cell receptors that activate signal transduction pathways to provide precise and short-term regulation of physiological processes, including the synthesis of steroid or juvenile hormones that also modulate reproduction. Mosquitoes are exceptional model systems for this research because each successive cycle of egg maturation begins with a blood meal and ends with egg deposition two to three days later. Blood provides females with nutrients for egg maturation and metabolic storage, thus enabling survival to initiate another cycle. Understanding the regulation of reproduction in mosquitoes will give us insight into how pathogens, such as malaria and arboviruses that are ingested in a blood meal from an infected host, can multiply in the female's body during a reproductive cycle. Then, with another meal, the pathogens are transmitted to a different host, resulting in further dissemination.

The primary objective of my research program is to characterize the structure and function of peptide hormones and their receptors that activate key reproductive processes in two mosquito species: the yellow fever mosquito, Aedes aegypti, and the African malaria mosquito, Anopheles gambiae. The development of genome databases for both species has greatly aided the identification of genes and cDNAs encoding such proteins. Tissue and hemolymph expression profiles during development and oogenesis in females are being defined for the peptides and receptors of interest. As well, the receptors have been expressed for ligand binding studies. Conservation of peptide signaling and function in both species is being evaluated with bioassays.

Riehle, M. A., S. F. Garczynski, J. W. Crim, C. A. Hill, and M. R. Brown. 2002. Neuropeptides and Peptide Hormones in Anopheles gambiae. Science 298: 172-175.

Aedes aegypti Head Peptide I (Aea-HP-I)
This is the first neuropeptide to be isolated from mosquitoes, and it is a member of the extensive "RFamide" family of animal neuropeptides. In collaboration with Dr. Marc Klowden (University of Idaho), we showed that Aea-HP-I inhibits the host-seeking behavior of female Ae. aegypti. More recently, the Aea-HP-I gene and its expression during development and a reproductive cycle have been characterized.

Brown, M. R., M. J. Klowden, J. W. Crim, L. Young, L. Shrouder, and A. O. Lea. 1994. Endogenous regulation of mosquito host-seeking behavior by a neuropeptide. J. Insect Physiol. 40: 399-406.

Stracker, T. H., S. Thompson, G. L. Grossman, M. A. Riehle, and M. R. Brown. 2002. Characterization of the AeaHP gene and its expression in the mosquito, Aedes aegypti (Diptera: Culicidae). J. Medical Entomology 39(2): 331-342.

Ovary Ecdysteroidogenic Hormone (OEH)
As the only steroidogenic gonadotropin identified in invertebrates, to date, it is the functional equivalent of follicle-stimulating hormone and luteinizing hormone in vertebrates. Neurosecretory cells in the brain and abdominal ganglion secrete OEH into the hemolymph for several hours after a blood meal, and during this time, ovaries of female mosquitoes begin secreting ecdysteroid hormones, in response to OEH. The rising titer of ecdysteroids stimulates the fat body to synthesize yolk proteins, which are stored in mature eggs and used during embryonic development. Because this peptide is present in larvae and males, it likely has other functions.

Brown, M. R., R. Graf, K. M. Swiderek, D. Fendley, T. H. Stracker, D. E. Champagne, and A. O. Lea. 1998. Identification of a steroidogenic neurohormone in female mosquitoes. J. Biol. Chem. 273: 3967-3971.

Brown, M. R. and C. Cao. Distribution of ovary ecdysteroidogenic hormone I in the nervous system and gut of mosquitoes. 13 pp. J. Insect Science

Insulin-Related Peptides (ILP's)
In vertebrates, insulins are important growth factors and multifunctional regulators of metabolism. For a few insect species, including Anopheles gambiae, sequences for ILPs are known, but their function is uncertain. Earlier studies from mosquitoes indicated that head factors regulated carbohydrate and lipid stores in the same way as insulins and, interestingly, that the development of malaria in mosquitoes was stimulated by the ingestion of insulin. With immunocytochemistry, we have identified brain neurosecretory cells and midgut endocrine cells in larvae and adults that secrete ILPs. Now, we are trying to obtain nucleotide or amino acid sequences for ILPs in Aedes aegypti and to define regulatory roles for ILPs in mosquitoes. In collaboration with Dr. Rolf Graf (University of Zurich, Switzerland), we showed that vertebrate insulins stimulate steroidogenesis and protein synthesis in mosquito ovaries and identified an insulin receptor in ovaries. Activation of steroidogenesis in ovaries was shown to follow the same signal transduction pathway, as used by insulin in regulating carbohydrate metabolism, apoptosis, and immune response in mammalian cells. Recently, expression of the insulin receptor and a serine/threonine kinase, Aktan intracellular regulatory nexus, have been described during development and egg maturation in female Aedes aegypti.

Graf, R., S. Neuenschwander, M. R. Brown, and U. Ackermann. 1997. Insulin mediated secretion of ecdysteroids from mosquito ovaries and molecular cloning of the insulin receptor homologue (MIR) from ovaries of bloodfed Aedes aegypti. Insect Molec. Biol. 6: 151-163.

Riehle, M. A. and Brown, M. R. 1999. Insulin stimulates ecdysteroid production through a conserved signaling cascade in the mosquito Aedes aegypti. Insect Biochem. Molec. Biol. 29: 855-860.

Cao, C. and M. R. Brown. 2001. Localization of an insulin-like peptide in brains of two flies. Cell and Tissue Research 304: 317-321.

Riehle, M. A. and M. R. Brown. 2002. Insulin receptor expression during development and a reproductive cycle in the ovary of the mosquito Aedes aegypti. Cell and Tissue Research 308(3): 409-420.

Riehle, M. A. and Brown, M. R. 2003. Molecular analysis of the serine/threonine kinase Akt and its expression in the mosquito, Aedes aegypti. Insect Molecular Biology 12(3): 225-232.

Krieger, M. B. J., Jahan, N., Riehle, M. A., Cao, C., and Brown, M. R. 2004 Molecular characterization of insulin-like peptide genes and their expression in the African malaria mosquito, Anopheles gambiae. Insect Molecular Biology 13: 305-315.

Neuropeptide F
The midgut endocrine system of insects is an important source of peptide hormones, which are presumed to regulate appetite and digestion in the same ways as gut peptides from the same system in vertebrates. A second objective of my research program is to identify specific peptides originating from this system in insects and to define their regulatory roles.

In collaboration with Dr. Joe Crim (University of Georgia), the midgut endocrine system in the corn earworm, Helicoverpa zea, was described, and a short peptide was isolated from larval midguts that has tantalizing similarity to the neuropeptide Y (NPY) family in vertebrates. With an immunoassay specific for this H. zea peptide, we obtained a peptide from an extract of Drosophila melanogaster that is an authentic member of the aforementioned family, which also includes the neuropeptide F's (NPF) identified in other invertebrates. The Drosophila peptide is the first NPF to be identified in arthropods. Peptides in the NPY family are known to be potent stimulators of appetite and to regulate enzyme secretion in mammals, and in a nematode, mutations in the gene encoding the "NPY" receptor affect feeding behavior. With Dr. Ping Shen (University of Georgia), we characterized the gene encoding the Drosophila NPF and found that it is expressed in both the brain and gut of all stages. Molecular probes developed from our Drosophila studies also led to the identification of a related peptide in Aedes aegypti. The gene encoding the mosquito neuropeptide F and its expression have been described, and currently, our focus is to identify its receptor and function.

Brown, M. R. and A. O. Lea. 1990. Neuroendocrine and midgut endocrine systems in the adult mosquito. In Advances in Disease Vector Research, Vol. 6, Ed. K. F. Harris, p. 29-58.

Huang, Y-Q, M. R. Brown, T. D. Lee, and J. W. Crim. 1998. RF-amide peptides isolated from the midgut of the corn earworm, Helicoverpa zea, resemble pancreatic polypeptides. Insect Biochem. Molec. Biol. 28: 345-356.

Brown, M. R., Crim, J. W., Arata, R. C., Cai, H. N., Chun, C., and Shen, P. 1999. Identification of a Drosophila Brain-Gut Peptide Related to the Neuropeptide Y Family. Peptides 20, 1035-1042.

Garczynski, S. F., M. R. Brown, P. Shen, T. F. Murray, and J. W. Crim. 2002. Characterization of a functional neuropeptide F receptor from Drosophila melanogaster. Peptides 23: 773-780.

Stanek, D. M., J. Pohl, J. W. Crim, and M. R. Brown. 2002. Neuropeptide F and its expression in the yellow fever mosquito, Aedes aegypti. Peptides 23: 1367-1378.

Brown, M. R. 2003. Insect gut as an endocrine organ. In: Encyclopedia of Hormones (Henry, H. L. and Norman, A. W., Eds.), pp. 328-333. Academic Press.

Summary
Progress in this research contributes to two concepts shared by insect and vertebrate endocrinology. First, peptide hormones, as chemical messengers, are conserved to a high degree both in structure and function across the phyla of multicellular animals. Second, the nervous and digestive systems of animals use these messengers to coordinate metabolism and homeostasis, so that development and reproduction can occur. The elucidation of key regulatory pathways in mosquitoes can lead to stable and functional peptide mimics or to genetic transformation that may offer a new way to control their development or block pathogen transmission.