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Odorant Receptor Expression Directs Behavior in Anopheles gambiae Mosquitoes

By: Carol A. Rouzer, VICB Communications
Published: May 14, 2013

Changes in odorant receptor expression following a blood meal facilitate the switch from blood meal-seeking to reproductive behavior.

Despite extensive ongoing research efforts to combat the disease, malaria remains a major public health threat. The 219 million cases of malaria in 2010 resulted in 660,000 deaths, 91% of which were in Africa. Malaria is caused by a mosquito-born parasite. Of the 41 genera of mosquitoes in the world, only the genus Anopheles (FIgure 1) can carry malaria, and of the 430 Anopheles species, only about 40 serve as vectors. The failure to develop an effective malaria vaccine and the increasing resistance to currently available therapies suggest that the best way to conquer malaria may be to control the mosquito vector. Mosquitoes transmit disease through blood feeding on hosts, such as humans, that are selected in a process primarily determined by sensing host-specific odorant molecules. This has led Vanderbilt Institute of Chemical Biology member Laurence Zwiebel and his laboratory to seek a detailed understanding of how mosquito odorant receptors work. In a recent report that brings together bioinformatics and chemical biology, they now show that taking a blood meal leads to a change in odorant receptor expression that has a direct effect on the mosquito’s life cycle and behavior [D. C. Rinker, et al. (2013) Proc. Natl. Acad. Sci. U.S.A., published online April 29, doi:10.1073.pnas.1302562110].

Figure 1. Anopheles freeborni mosquito taking a blood meal. Image reproduced from the Centers for Disease Control, CDC, public domain.

Mosquitoes of the species Anopheles gambiae are the most important malaria vectors. The subgroup An. gambiae sensu stricto, which limits its feeding behavior almost exclusively to humans, was the focus of the Zwiebel lab’s research. Both male and female An. gambiae use sugar from plant sources for energy, but the female requires a blood meal to complete her reproductive cycle. Once a full blood meal has been secured, she rests for a period of about two days and then lays her eggs. The female exhibits a diel pattern of behavior, with host-seeking and feeding occurring mostly at night, but the pattern is disrupted during the shift from feeding to reproductive behaviors (Figure 2). Prior studies of the antennae and odorant receptor neurons of female mosquitoes show that responsiveness varies throughout the day. These changes are linked to variations in the expression of genes for some olfactory proteins. Furthermore, female mosquitoes exhibit a reduced ability to respond to host odor stimulation following a blood meal. These findings led the Zwiebel lab to hypothesize that blood meal-related changes in the expression of odorant receptor genes is directly linked to the shift from host-seeking to egg-laying behavior of the mosquito.

Figure 2. Anopheles gambiae mosquitoes exhibit a diel cycle in host-seeking behavior, with greatest activity occurring at night. Once a blood meal has been obtained, host-seeking behavior is suppressed, and the female begins to search for a place to lay her eggs. The timeline shows the points at which samples of antenna tissue were obtained for blood meal-fed (Bf, red) and non-blood meal-fed (nBF, blue) mosquitoes, and the number of base pairs read by illumina sequencing at each time point. Image reproduced with permission from D. C. Rinker, et al. (2013) Proc. Natl. Acad. Sci. U.S.A., published online April 29, doi:10.1073.pnas.1302562110, copyright 2013, D. C. Rinker, et al.

There are five chemosensory gene families in An. gambiae mosquitoes, including odorant (AgOr), gustatory (AgGr), ionotropic glutamate (AgIr), and odorant binding proteins (AgObp). Recent identification of these genes, delineation of their expression patterns, and determination of the sensitivities of the expressed proteins has provided key information linking chemosensation to behavior. To test their hypothesis that chemosensory gene expression is linked to host-seeking versus reproductive behaviors, the Zwiebel lab provided a test group (Bf) of female mosquitoes with a blood meal, while maintaining a control group (nBf) on sucrose alone. The blood meal was provided in the middle of the dark phase of the diel cycle. Antennae tissue was then harvested from the mosquitoes at 1, 12, 24, 36, and 48 h after the blood feeding (Figure 2).

RNA sequencing of the antenna samples provided data for 8,995 genes, about 5000 of which showed abundance differences between the Bf and nBf groups at any one time point. The investigators selected 1,235 genes that showed at least a two-fold change at one or more time points for cluster analysis. The results identified fourteen clusters of genes that exhibited distinct patterns of change. For most of these, expression changed at only a single time point, although one cluster was observed to increase at all time points. The genes in a number of clusters coded for proteins that were unrelated to antenna functions. Their role in processes such as blood digestion, royal jelly production, and other aspects of ovarian function suggested that these genes were probably regulated by circulating factors that had been triggered by the blood meal. The cluster that included the largest group of olfactory-associated transcripts included nine AgObps genes. These genes were undetectable except at the 36 h time point, leading the investigators to hypothesize that they, too, were not primarily involved in antenna function.

One cluster comprising ten genes showed a strong diel pattern of expression that was phase-shifted as a result of the blood meal. This cluster included genes encoding five opsin G protein-coupled receptors and two arrestins. The abundance of the G protein Gαq exhibited a similar pattern of expression, leading the investigators to hypothesize that these genes may be involved in odorant reception and play a role in the strong diel pattern of blood-feeding behaviors.

The RNA sequencing data revealed high levels of transcripts for only 35 chemosensory genes, a result that was not surprising, since many of the odorant receptors are expressed in organs other than the antennae. The most abundant of the detected genes exhibited decreased expression in Bf mosquitoes except at the 24 h time point. The magnitude of the change was not the same for all genes, however, leading to a rank order shift in levels of abundance. As a result of having previously established the odorant response profiles for most of the Anopheline odorant receptors, the investigators were able to combine data on odorant response for each encoded protein together with the transcript abundance data to predict shifts in the insect’s odorant response spectrum. They were interested to find that the largest decreases in sensitivity were to linalool-oxide and 1-octen-3-ol, both of which are human skin odorants. Similarly interesting was the finding that the increased expression observed in many genes at 24 h would be predicted to enhance sensitivity to chemicals involved in oviposition behavior. The two compounds exhibiting the highest increase in receptivity at 24 h were 2-propylphenol and 4-methylcyclohexanol. The Zwiebel lab developed a dual-choice oviposition bioassay designed to determine if these chemicals served as signals for oviposition site selection (Figure 3). The results showed that, indeed, both compounds influenced egg-laying behavior, with 2-propylphenol serving as an oviposition attractant and 4-methylcyclohexanol serving as a repellant.

Figure 3. Design of the dual-choice oviposition assay. Gravid females in chamber (a) are released through opening (c) into the assay cage (b), which contains two cups of liquid (d). One cup contains water, while the other contains water plus the test odorant. Preference for each cup is scored on the basis of the egg-laying behavior of the mosquito. In these experiments, 2-propylphenol served as an attractant, and 4-methylcyclohexanol as a repellant. Expression of receptors sensitive to both of these compounds was increased in females following a blood meal. Image reproduced with permission from D. C. Rinker, et al. (2013) Proc. Natl. Acad. Sci. U.S.A., published online April 29, doi:10.1073.pnas.1302562110, copyright 2013, D. C. Rinker, et al.

It is important to note that each odorant receptor responds to multiple ligands, and each ligand stimulates multiple odorant receptors. Therefore, predicting the effects of changes in expression of a large number of receptors is quite complex. Nevertheless, the Zwiebel lab’s results support their hypothesis that changes in odorant receptor expression following a blood meal contribute to the subsequent change in insect behavior, guiding the mosquito away from host-seeking and toward reproduction. A clear understanding of how odorant receptor function directs mosquito behavior will be a valuable tool in the search for new ways to control the feeding and reproduction of this highly dangerous disease vector. The Zwiebel lab is currently working to develop new forms of insect repellants based on compounds that bind specifically to some of the odorant receptors. Other possibilities include discovering molecules that interact with the receptors and trick the mosquito into laying her eggs in an inappropriate place.





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