In the developing world, the identification of clean, potable water continues to pose a pervasive challenge, and waterborne diseases due to fecal contamination of water supplies significantly threaten public health. The ability to efficiently monitor local water supplies is key to water safety, yet no low-cost, reliable method exists to detect contamination quickly. We developed an in vitro assay utilizing an odorant-binding protein (OBP), AgamOBP1, from the mosquito, Anopheles gambiae, to test for the presence of a characteristic metabolite, indole, from harmful coliform bacteria. We demonstrated that recombinantly expressed AgamOBP1 binds indole with high sensitivity. Our proof-of-concept assay is fluorescence-based and demonstrates the usefulness of insect OBPs as detector elements in novel biosensors that rapidly detect the presence of bacterial metabolic markers, and thus of coliform bacteria. We further demonstrated that rAgamOBP1 is suitable for use in portable, inexpensive dipstick biosensors that improve upon lateral flow technology since insect OBPs are robust, easily obtainable via recombinant expression, and resist detector fouling. Moreover, due to their wide diversity and ligand selectivity, insect chemosensory proteins have other biosensor applications for various analytes. The techniques presented here therefore represent platform technologies applicable to various future devices.

There is interest in using ligands of chemosensory binding proteins (CBP) to augment an insect’s responsiveness to chemosensory cues. We showed previously that combining a synthetic ligand of a CBP with limonene, a common citrus volatile, enhanced the probing response of Asian citrus psyllid (Diaphorinacitri). Here, we determined whether synthetic compounds, which were ligands of D. citri olfactory binding protein (OBP) DCSAP4, influenced the settling and aggregation levels of psyllids on young citrus shoots. The test ligands and Cmac scent were dispensed from a droplet of an emulsified wax product (SPLAT) placed on the bottom of each vial. The shoots were presented: (1) alone (shoot + blank SPLAT), (2) with a mixture of citrus volatiles (“Cmac scent”) (shoot + SPLAT with Cmac scent), or (3) with different concentrations of test ligands (shoot + SPLAT with test ligand at concentration 1, shoot + SPLAT with test ligand at concentration 2, etc.). Depending on the availability of test ligands, sprigs, and psyllids, each test included from two to four replicates of each treatment (i.e., shoot only, shoot + Cmac scent, shoot + test ligand at concentration 1, shoot + test ligand at concentration 2, etc.); only a single test ligand was presented in each test. For each test, 200 D. citri were released in the test area and the numbers of psyllids on each sprig were counted 24 h later. Sprigs with ≥7 psyllids were considered to be an aggregation. A total of seven ligands were tested individually. Four of the ligands (654, 717, 784, and 861) modulated psyllid settling and aggregation response, causing greater settling and aggregation to sprigs presented with the Cmac scent than to those sprigs with blank SPLAT. Presentation of one of the ligands (019) resulted in an opposite effect in which psyllid settling and aggregation levels were lower on sprigs with Cmac scent than on those with blank SPLAT. There were no differences in settling levels in the different treatment vials in the Ligand 905 experiment. In the Ligand 937 experiment, settling levels did not vary significantly between treatment vials although settling levels were relatively high in all treatment vials and there was a significant treatment effect. Increased settling and aggregation levels were largely not observed with in the vials with only the test ligands, and there was little effect of ligand concentration on psyllid response levels. This suggests that the test ligands themselves did not attract the psyllids but rather modulated the psyllid’s response to the Cmac scent. The results suggest that synthetic ligands of D. citri CBPs can be used to increase the effectiveness of citrus scent lures used to attract psyllids to monitoring traps and attract and kill devices.

Author(s): Patt, J.; Woods, D.; Dimitratos, S.; Meikle, W.; Stockton, D.; Lapointe, S.; Mafra-Neto, A. | Abstract: In the absence of pheromone attractants, host-plant volatiles offer the most likely means of improving capture levels of ACP with sticky cards and other types of visual traps. However, developing scent lures that can compete with the attractiveness of actual host-plants, especially those in flush, is challenging. We are developing a new class of synthetic scent lures that may enhance the attractiveness of naturally-occurring host-plant volatiles. These compounds are synthetic ligands that bind to chemosensory proteins (CSPs) found in the olfactory sensilla of target insects. These ligands may mimic naturally-occurring odorants and function as super-stimuli because of their strong affinity to CSPs. In our study, CSPs from ACP antennae were identified based on their reactivity to petitgrain oil (an essential oil extracted from sour orange leaves), an ACP attractant. Two behavioral assays were used to assess the biological activity of several candidate ligands. One assay measured ACP probing frequency into a line of emulsified wax (SPLAT®, ISCA Technologies) containing a test ligand, the other assay measured the retention time of psyllids in an airstream carrying the ligand. One ligand, nicknamed ‘Titan’, was more stimulatory than limonene, a common citrus volatile, while a mixture of Titan and limonene was significantly more stimulatory than either alone. Subsequent assays showed that Titan was as stimulatory to ACP as the odor emitted by flushing sprigs of orange jasmine, a favored host-plant. These results indicate that CSP ligands may synergize the attractiveness of naturally-occurring citrus volatiles and boost their effectiveness as scent lures for ACP.

Huanglongbing, a devastating disease of citrus, is spread by the Asian citrus psyllid (ACP) (Diaphorina citri (Kuwayama)). Area wide management plans aimed at reducing the incidence and spread of Huanglongbing rely upon sampling ACP with yellow sticky cards, which are not always reliable. The development of highly effective scent attractants may improve trapping efficacy, which, in turn, is needed to increase the reliability of vector detection and monitoring and to reduce costs associated with these activities. The protein components of the insect olfactory system can control and alter insect behavior; thus, these proteins are suitable targets for the development of novel control products using rational design, as has been accomplished for drug discovery in the pharmaceutical industry. Odorant binding proteins (OBPs) from ACP were recombinantly expressed in vitro and screened for their ability to bind to petitgrain oil, an essential oil from sour orange foliage known to attract ACP, in order to identify OBP(s) responsible for attraction behavior. The OBP that bound petitgrain oil was then screened with a combinatorial chemical library comprising 30,000 small molecules in order to isolate synthetic ligands; these ligands were subsequently evaluated for their behavioral effects on ACP. A synthetic ligand named 'Titan' was found to be behaviorally active in preliminary tests; Titan was tested further with respect to its abilities to stimulate two foraging behaviors of ACP and to interact with limonene, a monoterpene emitted by many ACP host plant species. The addition of Titan to limonene increased the amount of probing by ACP into an artificial midrib composed of an emulsified wax relative to the probing level elicited by limonene alone. Probing level to Titan alone was influenced by the concentration of Titan in the wax midrib, with the lower concentration being more stimulatory than the higher concentration. In olfactometer tests, response to Titan was similar to the odor of young orange jasmine foliage. Olfactory response was also higher to a mixture of limonene and Titan than to limonene alone. These results demonstrated that Titan is highly stimulatory and modifies psyllid response to limonene. Further tests will determine whether Titan can be used in concert with naturally occurring terpenes to develop potent scent attractants for ACP.

The initial steps of odorant recognition in the insect olfactory system involve odorant binding proteins (OBPs) and odorant receptors (ORs). While large families of OBPs have been identified in the malaria vector A. gambiae, little is known about their expression pattern in the numerous sensory hairs of the female antenna. We applied whole mount fluorescence in Situ hybridization (WM-FISH) and fluorescence immunohistochemistry (WM-FIHC) to investigate the sensilla co-expression of eight A. gambiae OBPs (AgOBPs), most notably AgOBP1 and AgOBP4, which all have abundant transcripts in female antenna. WM-FISH analysis of female antennae using AgOBP-specific probes revealed marked differences in the number of cells expressing each various AgOBPs. Testing combinations of AgOBP probes in two-color WM-FISH resulted in distinct cellular labeling patterns, indicating a combinatorial expression of AgOBPs and revealing distinct AgOBP requirements for various functional sensilla types. WM-FIHC with antisera to AgOBP1 and AgOBP4 confirmed expression of the respective proteins by support cells and demonstrated a location of OBPs within sensilla trichodea. Based on the finding that AgOBP1 and AgOBP4 as well as the receptor type AgOR2 are involved in the recognition of indole, experiments were performed to explore if the AgOBP-types and AgOR2 are co-expressed in distinct olfactory sensilla. Applying two-color WM-FISH with AgOBP-specific probes and probes specific for AgOR2 revealed a close association of support cells bearing transcripts for AgOBP1 and AgOBP4 and neurons with a transcript for the receptor AgOR2. Moreover, combined WM-FISH/-FIHC approaches using an AgOR2-specific riboprobe and AgOBP-specific antisera revealed the expression of the "ligand-matched" AgOBP1, AgOBP4 and AgOR2 to single trichoid hairs. This result substantiates the notion that a specific response to indole is mediated by an interplay of the proteins.

The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues containing single point mutations of interest. Circular dichroism (CD) and ligand-binding assays provided data necessary to probe the effects of the point mutations on ligand binding and the overall structure of AgamOBP1. Far-UV CD spectra of mutated AgamOBP1 variants displayed both substantial decreases to ordered α-helix structure (up to22%) and increases to disordered α-helix structure(up to 15%) with only minimal changes in random coil (unordered) structure. In mutations Y54A, Y122A and W114Q, aromatic side chain removal from the binding site significantly reduced N-phenyl-1-naphthylamine binding. Several non-aromatic mutations (L15T, L19T, L58T, L58Y, M84Q, M84K, H111A, Y122A and L124T) elicited changes to protein conformation with subsequent effects on ligand binding. This study provides empirical evidence for the in silico predicted functions of specific amino acids in AgamOBP1 folding and ligand binding characteristics.

Haematophagous insects are frequently carriers of parasitic diseases, including malaria. The mosquito Anopheles gambiae is the major vector of malaria in sub-Saharan Africa and is thus responsible for thousands of deaths daily. Although the role of olfaction in A. gambiae host detection has been demonstrated, little is known about the combinations of ligands and odorant binding proteins (OBPs) that can produce specific odor-related responses in vivo. We identified a ligand, indole, for an A. gambiae odorant binding protein, AgamOBP1, modeled the interaction in silico and confirmed the interaction using biochemical assays. RNAi-mediated gene silencing coupled with electrophysiological analyses confirmed that AgamOBP1 binds indole in A. gambiae and that the antennal receptor cells do not respond to indole in the absence of AgamOBP1. This case represents the first documented instance of a specific A. gambiae OBP-ligand pairing combination, demonstrates the significance of OBPs in odor recognition, and can be expanded to the identification of other ligands for OBPs of Anopheles and other medically important insects.

In this report we present results from a comprehensive study undertaken toward the identification of proteins interacting with odourant-binding proteins (OBPs) of the African malaria vector Anopheles gambiae with a focus on the interactions among different OBPs. From an initial screen for proteins that interact with a member of the Plus-C group of OBPs, OBP48, which is primarily expressed in female antennae and downregulated after a blood meal, a number of interacting proteins were identified, which included five classic OBPs and OBP48 itself. The interacting OBPs as well as a number of other classic and Plus-C group OBPs that were not identified in the initial screen, were expressed in lepidopteran cells and subsequently examined for in vitro interactions in the absence of exogenously added ligands. Co-immunoprecipitation and chemical cross-linking studies suggest that OBP48 is capable of homodimerizing, heterodimerizing and forming higher order complexes with those examined examples of classical OBPs identified in the initial screen but not with other classical or Plus-C group OBPs that failed to appear in the screen. The latter OBPs are, however, also capable of forming homodimers in vitro and, at least in the case of two examined classic OBPs, heterodimers as well. These results suggest a previously unsuspected potential of nonrandom combinatorial complexity that may be crucial for odour discrimination by the mosquito.

To obtain a better understanding of the olfactory processes that allow mosquitoes to identify human hosts, a molecular study has been performed to identify and characterize molecules in the olfactory signalling pathway of the African malaria vector Anopheles gambiae. Using cDNA libraries from antennae of females and males, a collection of cDNAs encoding odorant binding proteins and other novel antennal proteins were isolated and characterized, which represent various families of putative carrier proteins with homologues in other insects. Using filter array hybridizations and quantitative RT PCR, regulation and gender specificity of expression of these genes was investigated. Significant differences in steady‐state levels of some of these putative carrier protein genes were detected between the sexes and after blood feeding in females.

In spite of advances in medicine and public health, malaria and other mosquito-borne diseases are on the rise worldwide. Although vaccines, genetically modified mosquitoes and safer insecticides are under development, herein we examine a promising new approach to malaria control through better repellents. Current repellents, usually based on DEET, inhibit host finding by impeding insect olfaction, but have significant drawbacks. We discuss how comparative genomics, using data from the Anopheles genome project, allows the rapid identification of members of three protein classes critical to insect olfaction: odorant-binding proteins, G-protein-coupled receptors, and odorant-degrading enzymes. A rational design approach similar to that used by the pharmaceutical industry for drug development can then be applied to the development of products that interfere with mosquito olfaction. Such products have the potential to provide more complete, safer and longer lasting protection than conventional repellents, preventing disease transmission by interrupting the parasite life cycle.

One way of controlling disease transmission by blood‐feeding mosquitoes is to reduce the frequency of insect–host interaction, thus reducing the probability of parasite transmission and re‐infection. A better understanding of the olfactory processes responsible for allowing mosquitoes to identify human hosts is required in order to develop methods that will interfere with host seeking. We have therefore initiated a molecular approach to isolate and characterize the genes and their products that are involved in the olfactory recognition pathway of the mosquito Anopheles gambiae, which is the main malaria vector in sub‐Saharan Africa. We report here the isolation and preliminary characterization of several cDNAs from male and female A. gambiae antennal libraries that encode putative odourant binding proteins. Their conceptual translation products show extensive sequence similarity to known insect odourant binding proteins (OBPs)/pheromone binding proteins (PBPs), especially to those of D. melanogaster. The A. gambiae OBPs described here are expressed in the antennae of both genders, and some of the A. gambiae OBP genes are well conserved in other disease‐transmitting mosquito species, such as Aedes aegypti and Culex quinquefasciatus.

Host plant resistance research to date indicates that Brassica plants expressing the glossy leaf wax characteristic show some resistance to diamondback moth, Plutella zylostella (L.). In the present study oviposition and first-instar feeding preferences were examined on ‘Glossy’ and ‘Waxy’ near isogenic lines of Brassica rapa (L.). Feeding preference, growth and survival of later instar diamondback moth larvae also were examined. Although females did not discriminate between ‘Waxy’ and ‘Glossy’ plants for oviposition, there was a strong preference among first-instar larvae for ‘Waxy’ plants in the choice experiment. There were no significant differences in larval survival or fourth-instar feeding preference on any of the lines tested. The present experiment indicates that B. rapa expressing the glossy leaf wax characteristic shows some resistance to diamondback moth, similar to that observed previously with glossy B. oleracea. The resistance appears to have a behavioural basis and is expressed against early instar larvae.

Frizzled (Fz) signaling regulates cell polarity in both vertebrates and invertebrates. In Drosophila, Fz orients the asymmetric division of the sensory organ precursor cell (pI) along the antero-posterior axis of the notum. Planar polarization involves a remodeling of the apical-basal polarity of the pI cell. The Discs-large (Dlg) and Partner of Inscuteable (Pins) proteins accumulate at the anterior cortex, while Bazooka (Baz) relocalizes to the posterior cortex. Dlg interacts directly with Pins and regulates the localization of Pins and Baz. Pins acts with Fz to localize Baz posteriorly, but Baz is not required to localize Pins anteriorly. Finally, Baz and the Dlg/Pins complex are required for the asymmetric localization of Numb. Thus, the Dlg/Pins complex responds to Fz signaling to establish planar asymmetry in the pI cell.

Mdm2 acts as a major regulator of the tumor suppressor p53 by targeting its destruction. Here, we show that the mdm2 gene is also regulated by the Ras-driven Raf/MEK/MAP kinase pathway, in a p53-independent manner. Mdm2 induced by activated Raf degrades p53 in the absence of the Mdm2 inhibitor p19ARF. This regulatory pathway accounts for the observation that cells transformed by oncogenic Ras are more resistant to p53-dependent apoptosis following exposure to DNA damage. Activation of the Ras-induced Raf/MEK/MAP kinase may therefore play a key role in suppressing p53 during tumor development and treatment. In primary cells, Raf also activates the Mdm2 inhibitor p19ARF. Levels of p53 are therefore determined by opposing effects of Raf-induced p19ARF and Mdm2.