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									                       The Role of Odorant Binding Proteins (OBPs) in Host-seeking Behavior of Mosquitoes
                                        Dr. Marika Walter, Dr. Osvaldo Marinotti, M. Biessmann, A. Maranhao, B. Rusconi, T. Thompson
                                        Developmental Biology Center, University of California, Irvine
                                         The mosquito, Anopheles gambiae (Fig.1), is                                                                        The olfactory system of A. gambiae is located in
                                         the major sub-Saharan vector for the malaria                                                                       the antennae (Fig.3). Two olfactory neurons
                                         parasite, Plasmodium falciparum. Globally,                                                                         extend in the sensillum. The cuticle has pores to
                                         400 million people are infected with malaria                                                                       allow the odors to enter into the hemolymph
                                         resulting in 3 million deaths annually (most are                                                                   (Fig. 4). Odorant binding proteins (OBPs) are
                                         children under the age of 5). Currently there is                                                                   produced by the accessory cells and secreted
                                         no vaccine against malaria, though there are                                                                       into the hemolymph. In this model the odor in the
                                         several in clinical trials. The treatment of                                                                       hemolymph binds to an OBP, which then
    Fig.1 Pink Eye (top) and             malaria is becoming increasingly complicated
    Wildtype Mosquitoes Heads
                                                                                                                                                            presumably transports it to the receptor. It is not         Fig.5 Molecular Model of Odor Recognition
                                         due to a rise in parasite resistance. This                          Fig.3 Male and Female Antennae of A.gambiae
                                                                                                                                                            clear, if the OBP still binds the odor once it has
                                         situation is further exacerbated by some of the                                                                    reached the receptor, or if it detaches (Fig.5). The
                                         detrimental effects of past chemical control                                                                       binding to the receptor elicits a depolarization of
                                         agents such as DDT.                                                                                                the membrane. Every neuron carries only two
                                                                                                                                                            receptors on its surface, a conserved (OR7) and
                                                 Female A. gambiae utilize their                                                                            a divergent. They form a ligand gated Ca2+ ion
                                         olfactory system to seek out a host for blood                                                                      channel. Released odors are degraded by
                                         meals in order to acquire the nutrients                                                                            odorant degrading enzymes (ODE).                                                QuickTime™ an d a
                                         necessary to produce eggs. Olfaction also                                                                          There are over 60 predicted OBPs. Thirty were
                                                                                                                                                                                                                                      are need ed to see this picture .

                                         plays an important role in oviposition. Since                                                                      determined through RT-PCR to have divergent
                                         olfaction is linked to crucial behaviors,                                                                          levels in females compared to males or have a
                                         understanding olfactory processes in more                                                                          changed expression after a blood meal (Table 1).
                                         detail can lead to improved insect control                                                                         The Aim of our group is to find ligands for
                                         strategies. The malaria parasite is transferred                     Fig.4 Model of an Olfactory Sensillum          those OBPs.
                                         through the saliva of the mosquito during                                                                                                                                    Table 1: OBPs known to be expressed
                                         feeding (Fig. 3).                                                                                                                                                            differently in males to females. FH: female head,
                                                                                                                                                                                                                      MH: male head, BFH: blood fed female head
Fig.2 Model of the Malaria Life Cycle                                                                                    The binding pocket residues for Agam OBP1 were
                                                                                                                         identified using the crystal structure of native and
                                                                                                                         indole-bound protein. A series of Agam OBP1
Olfactory perception results from the molecular                                                                          proteins with binding pocket residues mutations were
recognition of odor ligands by specific proteins. The                                                                    generated to study the binding capabilities. These
ligand specificity of an odorant-binding protein (OBP) can                                                               mutated Agam OBP1 proteins will then be used in
be determined by using a high-throughput fluorescence-                                                                   several protein-ligand binding assays to determine
based assay developed by Inscent Inc. This fluorescence                                                                  the residues most critical to ligand binding affinity.
assay uses a dye, N-phenyl-1-naphthylamine (1-NPN),                                                                      Mass spectrometry will be used to quantify ligand               Fig.9 Electrodes on Mosquito Antennae (left), EAG with Chromatogram of the
which binds to the hydrophobic pockets of proteins. Odor                                                                 binding.                                                        Ligand Pulse
ligands specific to an OBP will compete for binding to the
subject protein, displacing 1-NPN resulting in decreased                  Fig.7 Binding Kinetics of Indole                                                                               To determine the role of OBPs in olfaction, RNA
fluorescence.                                                             Measured via Flashplate Assay                                                                                  interference can be used. dsRNA is injected into living
                                                                                                                                                                                         mosquitoes. The response to an odorant stimulus is
                                                                        Scintillation proximity assaying (SPA)
                                                                                                                                                                                         recorded with an electrode placed on the antennae. The
                                                                        provides another method for assessing
                                                                                                                                                                                         readout is an electroantennogramm (EAG) (Fig.9). A short
                                                                        protein-ligand interaction. Purified
                                                                                                                                                                                         pulse of the odorant of interest is released and the
                                                                        recombinant protein is bound to the well of Fig.8 Model of OBP1-Indole Interaction
                                                                                                                                                                                         response recorded. A knock down mosquito will not show
                                                                        a Perkin Elmer FlashPlate containing
                                                                                                                                                                                         an electric pulse, when exposed to a previously
                                                                        aqueous radiolabeled ligand. Though the
                                                                                                                                                                                         determined ligand.
                                                                        radioactivity of the radioligand is quenched
                                                                        in solution, protein-ligand interaction brings                    Inscent Inc, Irvine, US
 Fig.6 NPN-1 Competition Assay                                          the radioactive ligand into close proximity of                    Dr.. P. Guerin, University of Neuchâtel, Switzerland
                                                                        the scintillant resulting in a detectable                         Prof..E. Eliopoulos, Agricultural University of Athens; Greece,
                                                                        signal.                                                           Prof.. K. Iatrou, National Centre for Scientific Research” Demokritos”,

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