Research Interests

 

The Molecular Genetics of Olfaction in Insect Disease Vectors

The major focus of the laboratory is the characterization of specific genes and their products that control important behavioral processes in the life cycle of insects which act as disease vectors. In this light we concentrate on host (i.e. blood-meal source) seeking/selection in the mosquito Anopheles gambiae which is the principal vector for malaria in Africa. Malaria is caused by a protozoan parasite of the genus Plasmodium that is transmitted to humans through blood feeding by female Anopheline mosquitoes. In this context, we are examining the molecular events of olfaction as this sense predominates the overall host preference behaviors in mosquitoes and other insects. This aspect of the mosquito's behavior is especially important as it makes a significant contribution to the vectorial capacity of this arthropod vector, as well as playing a similar role in the overall impact of many other insects of economic importance.

 

The Molecular Components of Olfactory Signal Transduction in Insects

The proteins that are graphically represented here are present on the inside surface of the dendritic membrane on olfactory receptor neurons. Signal transduction is initiated when odorants (either alone or in complexes with Odorant Binding Proteins) bind to members of seven transmembrane containing G-protein coupled odorant receptors (ORs). This binding causes a conformational change to the OR that allows it to interact with heterotrimeric G-proteins (abc) and thereby releasing the Ga subunit that in turn, will activate downstream effector enzymes that include adenyl cyclase (AC) or phospholipaseC (PLC). AC converts ATP to the second messenger cyclicAMP (cAMP) while PLC will convert PIP2 to the second messengers IP3 + DAG. Both cAMP and IP3 are capable of opening Na or Ca channels that allow influx of these ions leading to membrane depolarization and the induction of action potentials that effect neuronal signaling.

We use molecular and informatics based approaches to identify genes that are active in olfactory signal transduction in A. gambiae. The molecular characterization of genes which mediate olfaction in this Anopheline mosquito has started with the generation of cDNA libraries specifically derived from olfactory (i.e. the antennal and maxillary palps) and neural (heads that have been stripped of antennal and maxillary palps) structures of female adult mosquitoes. These hand-dissected structures have been used as substrate for the synthesis of subtracted cDNA libraries using novel PCR based methods that are specifically designed to facilitate the use of picogram amounts of mRNA starting material. Initial progress from screening of these libraries as well as a genomics based approaches resulted in the isolation of several olfactory genes from An. gambiae that are currently being characterized at the molecular and cellular levels.

These include AgArr1, a member of the arrestin family of proteins that play essential roles in regulating olfactory signal transduction cascade (Merrill et al, Insect Molecular Biology 2002). Arrestins are involved in the termination of signaling pathways and play essential roles in desensitization and adaptive responses. We have shown that AgArr1 is expressed at high levels in both the olfactory and visual systems of An. gambiae and have used the model insect system, Drosophila melanogaster to demonstrate the dual roles of arrestins in both of these sensory pathways (Merrill et. al. J. Neurobiology, in press). Current efforts of "Team Arrestin" (aka Will Walker, M. Rutzler, RJ PItts) include establishing robust behavioral, biochemical and electrophysiological assays for the role of arrestins in olfactory processes in both Drosophila and Anopheles.

In addition to the studies of arrestin function, we are focused on the molecular, biochemical and functional characterization of odorant receptor (OR) proteins in An. gambiae. "Team OR" (aka RJ Pitts, M. Rutzler, T. Lu, "C" Xia, W. Walker) uncovered several candidate OR genes in Anopheles (AgORs) marking the first occasion that such genes have been cloned in an insect other than D. melanogaster (Fox et al., PNAS 2001). Interestingly, AgOr1, one of the putative AgORs described in this study was shown to be expressed solely in female mosquitoes which is important because blood feeding and disease transmission is carried out only by females. We have also demonstrated that AgOr1's expression is down regulated in response to blood feeding in a fashion that mirrors behavioral and electrophysiological studies from the laboratory of Willem Takken (University of Wagenigen, The Netherlands) that showed immediately after a blood meal, female An. gambiae adults no longer respond to human olfactory cues. More recently, our laboratory has used bioinformatics and molecular approaches to catalog the complete set of 79 AgOR genes in this important vector mosquito (Hill et al. Science 2002) and we are currently focused on a detailed biochemical and molecular characterization of this important gene family. More recently we have extended this study to include the use of electrophysiology, cell culture and transgenic insect systems (e.g.Drosophila) in order to study the functional characteristics of AgORs. In another fruitful collaboration with the Carlson laboratory at Yale, we have successfully expressed two AgOR proteins in a defined olfactory neuron in Drosophila where it one one them (AgOr1) has been shown to be specifically "tuned" to respond to 4-methylphenol which is a component of human sweat known to excite An. gambiae females (Hallem et. al. Nature 2004)

A long-term objective of our work is the molecular characterization of the olfactory genes in general as well as the mechanisms that which are central in the marked preference for human blood meals (anthropophily) characteristic of An. gambiae s.s. In fact it is this component of the mosquito's behavior which makes it such an important disease vector. In contrast, a preference for bovine blood meals (zoophily) has been observed in the non-vector sibling species An. quadriannulatus . By using subtractive hybridization it may be possible to prepare anthropophilic and zoophilic enriched cDNA pools from which a more defined pool of olfactory and other behavioral genes may be isolated. For the present, we are engaged in a suite of studies to isolate and characterize orthologous ORs from species of anthropophilic and zoophilic mosquitoes as well as from other mosquito vectors for other human pathogens. These include Aedes aegypti, the vector for dengue and yellow fever that is widespread in Central and South America (A.A. Melo et. al. Chemical Senses 2004) and Culex pipens, the North American mosquito responsible for transmission of West Nile Virus.

We collaborate in these efforts with several other laboratories at Yale University (John Carlson), University of Illinois (Hugh Roberston), the Swedish Agricultural University (Bill Hansson) and The University of Wageningen (Willem Takken).