Anopheles odorant receptors that detect spatial mosquito repellents

Abstract

Methods for identifying odorant receptors reactive to mosquito repellent compounds and methods for screening repellent compounds as well as repellent compositions include newly identified repellent compounds and their applications for protecting a subject against malaria or repelling mosquitoes in an area.

Description

Atfy Ref: 002240.609807ANOPHELES ODORANT RECEPTORS THAT DETECT SPATIAL MOSQUITOREPELLENTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 637,220, filed April 22, 2024, which is incorporated by reference herein in its entirety for all purposes.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support grant W81XWH-18-1-0732 awarded by the Department of Defense and under grant R01AI137078 awarded by the National Institutes of Allergy and Infectious Diseases. The government has certain rights in the invention.BACKGROUND1. Technical Field

[0003] Currently claimed embodiments of the invention relate to methods for identifying odorant receptors reactive to mosquito repellent compounds, methods for screening repellent compounds as well as repellent compositions comprising newly identified repellent compounds and their applications for protecting a subject against malaria or repelling mosquitoes in an area.2. Discussion of Related Art

[0004] The deadliest of all anthropophilic (human preferring) mosquitoes are those of the genus Anopheles, capable of vectoring the malaria-causing Plasmodium parasite, which kills over 600 000 people each year. Blood-seeking female mosquitoes must detect and interpret information about their environment, and thus rely on different sensory modalities and a finely tuned sensory system. Olfaction is an important sensory modality for adult female mosquitoes as it is involved in foraging, host-searching, and oviposition site selection. Most importantly, it is primarily through olfaction that female mosquitoes locate and recognize a specific host for blood-feeding, and potentially transmit diseases. Therefore, targeting mosquito olfaction can reduce the number of infectious bites and presents opportunities for effective interventions.Atfy Ref: 002240.609807

[0005] Synthetic non-volatile compound N,N-diethyl-meta-toluamide (DEET) is the most widely used mosquito repellent in public use since 1957. However, DEET has some drawbacks, including high concentrations (>30%) are required to be effective, an unpleasant odor, and the ability to dissolve some plastics and synthetic rubber. Recently, it was discovered that DEET when mixed in a liquid phase (and other synthetic non-volatile repellents such as IR3535 and picaridin) is not sufficient to drive Anopheles coluzzii repulsion, rather suppressing the volatile odorants from the skin, making it more difficult for a mosquito to detect them. However, if DEET is released in the air at the same time as the odorants, the masking effect is lost, and olfactory receptor neurons respond to the odorants similarly to when DEET is absent. On the other hand, natural repellents such as PMD (p-menthane 3,8-diol, which derives from the oil of the Lemon Eucalyptus), and lemongrass oil may activate multiple types of olfactory receptor neurons. These studies underline the fact that repellents may have multiple modes of action: (i) may activate a select number of odorant receptors to trigger repellency (so-called ‘smell and repel’ model); (ii) may activate and inhibit many odorant receptors at the same time, thus leading to a confusing effect; (iii) may interfere with the detection of attractive odorants. Moreover, repellents may not affect all mosquito species in the same manner.

[0006] PMD is an Corymbia citriodora extract also known as lemon eucalyptus, is a potent natural repellent extracted from the leaves of lemon eucalyptus trees. It was discovered in the 1960s during mass screenings of plants used in Chinese traditional medicine. Lemon eucalyptus essential oil, comprising 85% citronellal, is used by cosmetic industries due to its fresh smell. However, it was discovered that the waste distillate remaining after hydro-distillation of the essential oil was far more effective at repelling mosquitoes than the essential oil itself. Many plant extracts and oils repel mosquitoes, with their effect lasting from several minutes to several hours. Their active ingredients tend to be highly volatile, so although they are effective repellents for a short period after application, they rapidly evaporate leaving the user unprotected. The exception to this is PMD, which has a lower vapour pressure than volatile monoterpines found in most plant oils and provides very high protection from a broad range of insect vectors over several hours, whereas the essential oil is repellent for around one hour. To date, PMD is the only plant-based repellent that has been advocated for use in disease endemic areas by the CDC (Centres for Disease Control), due to its proven clinical efficacy to prevent malaria

[0026] and isAtt’y Ref: 002240.609807 considered to pose no risk to human health. It should be noted that the essential oil of lemon eucalyptus does not have EPA (Environmental Protection Agency) registration for use as an insect repellent.

[0007] Clearly, a better understanding of how insect repellents affect mosquitoes’ olfactory system can be leveraged not only to improve or identify new repellents, but importantly to control malaria-vector mosquitoes.

[0008] The olfactory system of the Anopheles gambiae species of mosquitoes primarily consists of two organs: the antennae and maxillary palps. The labella is a third chemosensory organ on the head that might detect low volatile odorants. Each of these organs is covered with sensory hairs called sensilla, and each sensillum houses olfactory sensory neurons that may contain one of three types of chemoreceptors: odorant receptors (ORs); gustatory receptors (Grs); and / or ionotropic receptors (IRs). ORs are expressed in most olfactory neurons, and each OR is expressed along with the odorant receptor co-receptor (Oreo) to form a receptor complex that is either narrowly or broadly tuned to a variety of host-derived odors.

[0009] Electrophysiological experiments have been done on mosquito sensory appendages with little knowledge of the underlying molecular mechanisms of the neurons. To date, several olfactory receptors (especially in the OR family) have been functionally characterized.However, few have been connected to specific sensory appendages and identified as reactive to specific repellent molecules. Therefore, there is an unmet need to develop a method for identifying odorant receptors reactive to repellent compounds as well as reliable methods for screening effective repellent compounds that activate odorant receptors while remaining safe for human-use and the environment.SUMMARY

[0010] Provided herein is a method for screening a plurality of test compounds comprising: contacting an olfactory sensilla that expresses at least one odorant receptor (OR) gene product with at least one test compound, said at least one OR gene product being reactive to at least one odorant; recording an activity pattern characterized by a spike frequency and a spike amplitude in said sensilla by single sensillum electrophysiological recordings (SSR), thereby identifying whether said at least one test compound has a repellent capability, wherein said repellentAtfy Ref: 002240.609807 capability is characterized by a modification in said activity pattern compared to that of a nonrepellent control or said at least one odorant.

[0011] In some embodiments, the modification in said activity pattern occurs in at least one of said a spike frequency or a spike amplitude.

[0012] In some embodiments, the olfactory sensilla is of an Anopheles mosquito.

[0013] In some embodiments, the at least one OR gene product is at least one of AgOR20,AgOR48, AgOR9 or AgOR8 of Anopheles species.

[0014] In some embodiments, the odorant is at least one of lemongrass oil or PMD (p- menthane 3,8-diol).

[0015] In some embodiments, the olfactory sensilla that expresses the AgOR8 gene product is the capitate peg sensilla of the maxillary palp.

[0016] In some embodiments, the AgOR8 gene product is expressed on neuron B.

[0017] In some embodiments, the AgOR8 gene product responds to both lemongrass oil andPMD.

[0018] In some embodiments, the olfactory sensilla that expresses at least one of AgOR20 or AgOR8 gene products is the sensilla of the antennal.

[0019] In some embodiments, the olfactory sensilla comprises an empty neuron Drosophila melanogaster having a deleted endogenous odorant receptor 22a / b (OR22a / b) gene substituted by at least one OR of the Anopheles species.

[0020] In some embodiments, the olfactory sensilla is of a mutant Drosophila melanogaster fly.

[0021] In some embodiments, the method further comprising a close proximity assay.

[0022] Provided herein is also a method for identifying at least one odorant receptor (OR) reactive to a repellent compound comprising: contacting with said repellent compound anAtfy Ref: 002240.609807 olfactory organ comprising a plurality of olfactory neuron (ONs) expressing a plurality of ORs of a transgenic Anopheles mosquito, said transgenic Anopheles mosquito expressing a genetically encoded calcium indicator (GECI) capable of emitted a fluorescent signal, visualizing by microscopy said fluorescent signal emitted upon a repellent-induced response of at least one OR among said plurality of ORs expressed on said plurality of ONs, microdissecting said olfactory organ; and extracting and sequencing RNA to identify said at least one OR that is reactive to said repellent compound.

[0023] Provided herein is a repellent composition comprising at least one of the test compounds identified in the methods of the present disclosure.

[0024] Provided herein is a method of controlling an Anopheles population in an area of infestation, or area susceptible of infestation and / or combating an Anopheles population by spreading the repellent composition of the present disclosure.

[0025] Provided herein is a method of inhibiting, preventing, reducing, or controlling the incidence of Anopheles-borne diseases such as malaria in a subject, comprising contacting said subject with the repellent composition of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 present a schematic illustrating a method for identifying at least one OR reactive to a repellent is shown schematically.

[0027] FIG. 2 presents a schematic illustrating a method for identifying an odorant receptor reactive to a repellent.

[0028] FIG. 3 presents data showing that lemongrass oil activates AgOR20 and AgOR48, while PMD activates AgOR20.

[0029] FIGs. 4A-4B presents data showing OR20 response to lemongrass oil 1% (FIG. 4A) or PMD 1% (FIG. 4B).Att’y Ref.: 002240.609807

[0030] FIGs. 5A-5C presents data showing OR48 response to lemongrass oil 1% (FIG. 5A), PMD 1% (FIG. 5B), or PMD 10% (FIG. 5C).

[0031] FIGs. 6A-6B presents data showing OR8 response to lemongrass oil 1% (FIG. 6A) or PMD 1% (FIG. 6B).

[0032] FIGs. 7A-7B presents summary of the responses of OR20, OR48, and OR8 to lemongrass oil 1% (FIG. 7A) or responses of OR20 and OR8 to PMD1% and OR48 to PMD 10% (FIG. 7B).

[0033] FIG. 8 presents data showing Anopheles maxillary palp capitate peg sensilla response to lemongrass oil and PMD (likely the “B” neuron that expressed AgOR8).

[0034] FIG. 9 presents data showing that AgOR8 expressing olfactory neurons do responds to PMD and lemongrass oil.DETAILED DESCRIPTION

[0035] All publications and patent applications identified herein are incorporated by reference in their entirety.

[0036] Some embodiments of the current subject matter are discussed in detail below. In describing embodiments, specific terminology is employed for the sake of clarity. However, the embodiments are not intended to be limited to the specific terminology so selected. A person skilled in the relevant art will recognize that other equivalent components can be employed, and other methods developed, without departing from the broad concepts of the present subject matter. All references cited anywhere in this specification are incorporated by reference as if each had been individually incorporated.

[0037] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0038] As used throughout, the term “compound” and “molecule” are used interchangeably.Att’y Ref: 002240.609807

[0039] As used throughout, the term “odorant receptor (OR)” refers to a membrane protein that is activated upon binding of a chemical. For example, the chemical is an odorant.

[0040] As used throughout, an “odorant” refers to a known compound or molecule that binds to a specific OR triggering an olfactory response. The olfactory response may be characterized by an activity pattern defined by a spike frequency and a spike amplitude measured by single sensillum electrophysiological recordings (SSR) to analyze the electrophysiological properties of specific ORs. For example, the odorant is a at least one of lemongrass oil or PMD. An odorant can be a repellent.

[0041] As used throughout, the term “repellent” refers to a compound that is capable of modifying host-seeking behavior of an Anopheles by masking the host odorant, reducing the host odorant volatility, activating at least one OR and / or inhibiting at least one OR, and / or modifying the response of an OR towards other odorants. The repellent may bind to one or more ORs. The repellent may alternatively modify how an OR would normally respond to the odorant that normally activates that OR, either by inhibiting its response or make it respond more robustly. The repellent may be naturally derived or synthetic, volatile or non-volatile. The efficacy of a repellent may be evaluated using various conventional tests such as SSR, the close proximity assay or calcium imaging.

[0042] As used throughout, the term “natural repellent” refers to a molecule that is derived from a natural source, for example a plant, and drives Anopheles repulsion. Preferably, a natural repellent is capable of activating and / or inhibiting at least one OR localized onto at least one of the olfactory organs including the antennae and maxillary palps. For example, the activation of a OR can be recorded by means of SSR or calcium imaging. A repellent might also inhibit the activity of other neurons as a method of function — for example, neurons that normally signal attraction to host odors. By inhibiting those ‘attractive’ neurons, it could reduce attraction which would result in behaviors that appear as repulsion. Non-limiting examples are essential oils extracted from plants such as Citronelle, lemongrass, peppermint or eucalyptus. A natural repellent may be volatile or non-volatile. A natural repellent may potentially function as an odor masker. In general, a natural repellent is environmentally friendly and a safer alternative to synthetic molecules such as DEET, picaridin or IR3535 for use in human.

[0043] Methods for identifying at least one OR reactive to a repellentAtt’y Ref: 002240.609807

[0044] A certain embodiment of a method for identifying at least one OR reactive to a repellent is shown schematically in FIG. 1. An embodiment relates to a method for identifying at least one OR reactive to a repellent compound that includes: subjecting an olfactory organ comprising a plurality of olfactory neuron (ONs) expressing a plurality of ORs of a transgenic Anopheles mosquito to the repellent compound, the transgenic Anopheles mosquito expressing a genetically encoded calcium indicator (GECI) capable of emitted a fluorescent signal; visualizing by microscopy said fluorescent signal emitted upon a repellent-induced response of at least one OR among the plurality of ORs expressed on the plurality of ONs, microdissecting the olfactory organ; and extracting and sequencing the RNA to identify said at least one OR that is reactive to said repellent compound. A non-limiting example of GECI are GCaMP6s, GCaMP6f. (Chen TW, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, Schreiter ER, Kerr RA, Orger MB, Jayaraman V, Looger LL, Svoboda K, Kim DS. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature. 2013 Jul 18;499(7458):295-300. doi: 10.1038 / naturel2354. PMID: 23868258; PMCID: PMC3777791)

[0045] In one aspect of the embodiment, the repellent compound is a known or well identified odorant. For example, the odorant is at least one of lemongrass oil or PMD.

[0046] There are 79 ORs found in the Anopheles gambiae mosquito genome. They are named AgORl, AgOR2, AgOR3, AgOR4, AgOR5, AgOR6, AgOR7 (also called AgORCO), AgOR8, AgOR9, AgORlO, AgORl 1, AgOR12, AgOR13, AgOR14, AgOR15, AgOR16, AgOR17, AgOR18, AgOR19, AgOR20, AgOR21, AgOR22, AgOR23, AgOR24, AgOR25, AgOR26, AgOR27, AgOR28, AgOR29, AgOR30, AgOR31, AgOR32, AgOR33, AgOR34, AgOR35, AgOR36, AgOR37, AgOR38, AgOR39, AgOR40, AgOR41, AgOR42, AgOR43, AgOR44, AgOR45, AgOR46, AgOR47, AgOR48, AgOR49, AgOR50, AgOR51, AgOR52, AgOR53, AgOR54, AgOR55, AgOR56, AgOR57, AgOR58, AgOR59, AgOR60, AgOR61, AgOR62, AgOR63, AgOR64, AgOR65, AgOR66, AgOR67, AgOR68, AgOR69, AgOR70, AgOR71, AgOR72, AgOR73, AgOR74, AgOR75, AgOR76, AgOR77, AgOR78, AgOR79.

[0047] The Anopheles mosquito is genetically engineered to express a genetically encoded calcium indicator (GECI) in olfactory neurons of olfactory organs according to the method described herein. For example, such GECI may be GCaMP or variants thereof that reports changes in calcium based on a fusion between green fluorescent protein (GFP) and the calciumAtt’y Ref: 002240.609807 binding protein calmodulin. When calmodulin binds to Ca2+, a conformational change causes an increase in GFP fluorescence. A GECI allows the monitoring of neuronal activity in vivo by microscopy. Several variants of GCaMP have been engineered that have different molecular response properties. For example, GCaMP6f signals changes to intracellular calcium the most rapidly whereas GCaMP6s can be more sensitive as it responds more slowly but can fluoresce more brightly.

[0048] A transgenic Anopheles mosquito was generated to express the calcium indicator GCaMP6f in olfactory neurons under the control of the QF2 / QUAS binary system as described previously [citation: Afify A, Betz JF, Riabinina O, Lahondere C, Potter CJ. Commonly Used Insect Repellents Hide Human Odors from Anopheles Mosquitoes. Curr Biol. 2019 Nov 4;29(21):3669-3680.e5. doi: 10.1016 / j.cub.2019.09.007. Epub 2019 Oct 17. PMID: 31630950; PMCID: PMC6832857.]. Briefly, a transgenic Anopheles line containing the QF2 transcription factor under the control of the regulatory genomic region for orco (a neuron-specific enhancer) is crossed with a transgenic Anopheles line containing the reporter gene GCaMP6f which expression is under the control of QF2 that binds to the upstream sequence QUAS. When the mosquito lines are crossed, the QF2 is expressed, and it binds to the QUAS sequences, leading to the expression of GCaMP6f in the orco-expressing neurons. It is then possible to visualize a repellent-induced response in activated olfactory neurons (ONs) on a mosquito olfactory organ and examine how the repellent modulates or modify the responses of one or more ORs.Olfactory neuron activities before, during, and after odor stimulation are recorded as changes in GCaMP6f fluorescence. Neurons that are the most strongly activated by odors exhibit the largest increases in fluorescence.

[0049] Using microdissection, the olfactory organ containing a plurality of activated ONs is dissected, RNA is extracted and sequenced to identify at least one candidate OR expressed in the plurality of activated ONs. From the candidate list of ORs, the “empty” neuron system as described herein was used to express each candidate OR. SSR or odor-stimulation by means of close proximity assay to link the expression of the candidate OR and a responsiveness to at least one repellent.

[0050] A certain embodiment of a method for identifying an odorant receptor reactive to a repellent is shown schematically in FIG. 2. This embodiment includes the heterologousAtt’y Ref.: 002240.609807 expression of a transgenic AgOR in an empty olfactory neuron (ON) of a mutant D. melanogaster fly. For example, the ON is an olfactory neuron of the antennal basiconic sensilla type 3 (ab3). The empty ab3A ON lacks the native OR22a / b which is replaced by the Gal4-UAS transgene AgOR of interest. The transgenic AgOR is expressed in the ab3A ON which projects its dendrites into the large basiconic sensilla. AgOR reactivity may be evaluated by means of single sensillum electrophysiological recordings (SSR) when subjected to or stimulated with a repellent compound.

[0051] Such methodology may be applied to various AgOR expressed in sensilla of the antennae or the maxillary palp including but not limited to AgORl, AgOR2, AgOR3, AgOR4, AgOR5, AgOR6, AgOR7 (also called AgORCO), AgOR8, AgOR9, AgORlO, AgORl 1, AgOR12, AgOR13, AgOR14, AgORl 5, AgOR16, AgOR17, AgORl 8, AgOR19, AgOR20, AgOR21, AgOR22, AgOR23, AgOR24, AgOR25, AgOR26, AgOR27, AgOR28, AgOR29, AgOR30, AgOR31, AgOR32, AgOR33, AgOR34, AgOR35, AgOR36, AgOR37, AgOR38, AgOR39, AgOR40, AgOR41, AgOR42, AgOR43, AgOR44, AgOR45, AgOR46, AgOR47, AgOR48, AgOR49, AgOR50, AgOR51, AgOR52, AgOR53, AgOR54, AgOR55, AgOR56, AgOR57, AgOR58, AgOR59, AgOR60, AgOR61, AgOR62, AgOR63, AgOR64, AgOR65, AgOR66, AgOR67, AgOR68, AgOR69, AgOR70, AgOR71, AgOR72, AgOR73, AgOR74, AgOR75, AgOR76, AgOR77, AgOR78, AgOR79, preferably AgOR8, AgOr9. AgORl 0. AgORl 1, AgOR15, AgOR20, AgOR27, AgOR30, AgOR39, AgOR41, AgOR42, AgOR43, AgOR44, AgOR48, AgOR66 or AgOR73. Accordingly, the reactivity of each AgOR to a repellent can be evaluated. For example, while lemongrass oil activates AgOR20, AgOR48 and AgOR8, PMD activates AgOR20 and AgOR8. Both natural repellents are potent Anopheles repellents. Lemongrass oil and PMD do not activate AgRO44.

[0052] A method for screening repellent compounds

[0053] An embodiment is related a method for screening a plurality of test compounds that includes contacting an olfactory sensilla that expresses at least one OR gene product with at least one test compound, said at least one OR gene product being reactive to an odorant; recording an activity pattern characterized by at least one of a spike frequency or a spike amplitude in said sensilla by single sensillum electrophysiological recordings (SSR), thereby identifying whether said at least one test compound has a repellent capability. The repellent capability isAtfy Ref: 002240.609807 characterized by a modification in the activity pattern compared to that of a non-repellent control or the odorant. The modification in the activity pattern occurs in at least one of said a spike frequency or a spike amplitude. In one aspect of the embodiment, the modification in the activity pattern is defined by an increase in at least one of the spike frequency or the spike amplitude compared to that of a non-repellent control or the odorant. In another aspect of the embodiment, the modification in the activity pattern is defined by a decrease in at least one of the spike frequency or the spike amplitude compared to that of a non-repellent control or the odorant. For example, when the OR gene product is at least one of AgOR8, AgOR9, AgOR20 and AgOR48 the odorant is at least one of lemongrass oil or PMD. (Table 1)

[0054] An embodiment is related to a method for screening a plurality of test compounds that includes contacting an olfactory sensilla that expresses at least one OR gene product with at least one test compound among repellent compounds, the at least one OR being reactive to at least one of lemongrass oil or PMD; recording at least one of a spike frequency or a spike amplitude in said sensilla by single sensillum electrophysiological recordings (SSR), thereby identifying whether the at least one test compound has a repellent capability. The repellent capability is characterized by an increase in at least one of the spike frequency or the spike amplitude compared to that of a non-repellent control compound or by an activity pattern of at least one of the spike frequency or the spike amplitude modified compared to that of at least one of lemongrass oil or PMD. In one aspect of the embodiment, the at least one OR gene product is selected from the group consisting of AgOR8, AgOR9, AgOR20 and AgOR48. For example, the activity pattern of an OR induced by the test compound may be similar, increased or decrease compared to the activity pattern of the same OR induced by at least one of lemongrass oil or PMD.Att’y Ref.: 002240.609807Table 1

[0055] In certain embodiments, the activity pattern of at least one of a spike frequency or a spike amplitude recorded for the test compound is compared to the activity pattern of at least one of the spike frequency or the spike amplitude recorded for a composition comprising a lemongrass oil concentration of about 1 to 10 %, preferably 1 %. Preferably, the activity pattern of at least one of a spike frequency or a spike amplitude recorded for the test compound is compared to the activity pattern of at least one of a spike frequency or a spike amplitude recorded for a composition comprising a PMD concentration of about 1 to 10 %. In another aspect of the embodiment, at least one of AgOR20, AgOR48 or AgOR8 responds to a composition comprising a lemongrass oil concentration of about 1 %. In another aspect of the embodiment, at least one of AgOR20 or AgOR8 responds to a composition comprising a PMD concentration of about 1 %. In yet another aspect of the embodiment, AgOR40 responds to a composition comprising a PMD concentration of about 10 %.

[0056] If the activity pattern of at least one of a spike frequency or a spike amplitude recorded for the test compound is similar or increased compared to that of the reference compound (e.g., lemongrass oil or PMD), the test compound may be deemed as effective or may be subjected to additional evaluation(s). On the other hand, if the activity pattern of at least one of a spike frequency or a spike amplitude recorded for the test compound is decreased compared to that of the reference compound (e.g., lemongrass oil or PMD), the test compound may be deemed as ineffective. Such compound may also be evaluated in combination with other compound(s). A weak test compound may synergize with other compounds in activating a particular AgOR.

[0057] In an embodiment, the olfactory sensilla is of an Anopheles mosquito. In one aspect of the embodiment, the olfactory sensilla is of an antennal or a maxillary palp of an Anopheles mosquito. In one aspect of the embodiment, the olfactory sensilla expressed at least one Anopheles OR, for example at least one of AgOR8, AgOR9, AgOR20 or AgOR48. For example, AgOR8 is expressed on neuron B of the maxillary palp, and AgORZO and AgOR48 are expressed on the olfactory sensilla of the antennal.

[0058] In another embodiment, the olfactory sensilla is of a mutant Drosophila melanogaster fly. Preferably, the olfactory sensilla of a mutant Drosophila melanogaster fly comprises aAtfy Ref: 002240.609807 neuron having a deleted endogenous OR22a'b gene substituted by at least one of Anopheles ORs. In one aspect of the embodiment, the neuron expressed at least one of AgOR8, AgOR20 or AgOR48.

[0059] In an embodiment, the method further includes applying a close proximity assay to evaluate the repellent capability of the test compound.

[0060] Repellent composition

[0061] An embodiment provides a repellent composition comprising an effective amount of at least one test repellent compound identified by the method described herein. In one aspect of the embodiment, the repellent composition further comprises a surfactant, and / or a diluent. In another aspect of the embodiment, the repellent composition further comprises an adjuvant which improves the repellent effect.

[0062] In one aspect of the embodiment, the repellent composition is formulated for application on subject. In one aspect of the embodiment, the repelling composition is formulated as a liquid composition, for example a water-soluble liquid, an emulsion concentrate, an emulsion in water, a suspension concentrate.

[0063] In one aspect of the embodiment, the repellent composition is formulated as a lotion, cream, dust, cosmetic, perfume, spray, or incense.

[0064] In some embodiments, the repellent composition is dispersed with a vaporizer, evaporator, fan, heat, candle, or wicked apparatus.

[0065] In one aspect of the embodiment, the repellent composition comprising an effective amount of at least one the test repellent compound identified by the method described herein is formulated for application to target an Anopheles population or their situs.

[0066] In one aspect of the invention, the repellent composition is also useful in and can be employed along with other repellents to control an Anopheles population in their situs.

[0067] Applications

[0068] An embodiment relates to a method of controlling an Anopheles population in an area of infestation, or area susceptible of infestation and / or combating an Anopheles population by spreading the repellent composition described herein.Atfy Ref: 002240.609807

[0069] An embodiment relates to a method of inhibiting, preventing, reducing, or controlling the incidence of Anopheles-borne diseases such as malaria in a subject, comprising contacting said subject with the repellent composition described herein.

[0070] An embodiment relates to a method of inhibiting, preventing, reducing, or controlling the incidence of Anopheles-borne disease such as malaria in a subject, the method includes stimulating at least one OR in an Anopheles with at least one test repellent compound identified by the method described herein, wherein the response in said at least one OR is modified and attraction to the subject inhibited.EXAMPLES

[0071] The following describes some particular examples according to some embodiments of the current invention. The general concepts of this invention are not limited to these particular examples.

[0072] Material and methods

[0073] Imaging

[0074] Calcium imaging was performed as previously described. A 3-10 day old non-blood fed Anopheles female mosquito was inserted into a pipette tip and pushed to the end until the antennae were extended outside the pipette tip. Molding clay was then used to attach the pipette tip to a glass slide and the antennae were placed on a glass coverslip and immobilized using two pulled glass capillary tubes. The 11thantennal segment was imaged through 50x (LD EC Epipl an-Neofluar 50x / 0.55 DIC) objective on a Zeiss Axio Examiner DI microscope. A light source (Zeiss Illuminator HXP 200C), and eGFP fdter cube (FL Filter Set 38 HE GFP shift free) were used for imaging. An EMCCD camera (Andor iXon Ultra) and the Andor Solis software were used to record videos of the antennal neuron responses to odorants. For odorant stimulation, a Pasteur pipette containing 20pl of 30% lemongrass oil or p-Menthane-3,8-diol (PMD) was inserted into a hole in a serological pipette directed at the antenna. A stimulus controller (Syntech CS-55) was used to divert a 1 s pulse of charcoal-filtered air (5 ml / s) into the Pasteur pipette to deliver the odorant to the antenna. Imaging was performed using 24 female mosquitoes (12 mosquitoes were tested with each of the odorants).

[0075] DissectionAtfy Ref: 002240.609807

[0076] The recorded video was inspected and sensilla hairs on the mosquito antenna were used as landmarks to mark the position of responding neurons. After imaging, the glass slide containing the mosquito was transferred to a dissecting microscope (Zeiss Stemi 2000), and identified landmarks were used to cut out 1 / 3 of the antennal segment containing responding neurons using one of two sapphire blades (World Precision Instruments, blades #504076 and #504073). While the antenna was pinned down on the cover slip using two pulled glass capillary tubes, the sapphire blade was used first to make a cut on the distal side of the antenna, and then another cut was made at the proximal side, and the dissected tissue (1 / 3 of the antennal segment, now attached to the sapphire blade) was transferred to a PCR tube (kept on ice) containing 1 pl of lysis buffer mix (lysis buffer and Rnase inhibitor) and 9pl of Nuclease-Free water. The sapphire blade was dipped several times in the buffer mix to make sure the tissue is left in the mix. The sapphire blade was then visually inspected for any attached tissue under the dissecting microscope. The PCR tube containing the tissue was immediately stored at -20° C until it was sent to the Johns Hopkins Transcriptomics & Deep Sequencing Core for RNA purification and RNA-Seq. New pipette tips, slides, coverslips, and pulled glass capillary tubes were used for each mosquito while the sapphire blade was rinsed with ethanol (>99.5%, Fisher Scientific) after every use.

[0077] The “empty neuron” system in mutant J. melcmogaster fly

[0078] The empty neuron system (Dobritsa et al., Neuron, 2003) was used to study the response of AgOR20 and AgOR48 to repellents. In this system, Drosophila melanogastef s OR22a receptor (endogenously expressed in the A neuron of the ab3 sensillum) was replaced by either Anopheles gambiae AgOR20 or AgOR48 that were expressed in the ab3A neuron using the GNLA / UAS system (Lin and Potter, PLoS One, 2015; Carey et al., Nature, 2010). A fly was inserted into a pipette tip and pushed to the end until the antennae and half the head are extended outside the pipette tip. Molding clay was then used to plug the other end of the pipette tip. Molding clay was also used to attach the pipette tip to a glass slide, and the antennae were placed on a glass coverslip and immobilized using a pulled glass capillary tube. The slide was mounted on a Zeiss Axio Examiner DI microscope and visualized using the 50x objective (LD EC Epiplan-Neofluar 50x / 0.55 DIC). A light source (Zeiss Illuminator HXP 200C), and eGFP filter cube (FL Filter Set 38 HE GFP shift free) were used to identify GFP-expressing ab3 sensilla. AAtt’y Ref.: 002240.609807 glass recording electrode, fdled with Beadle-Ephrussi Ringer’s solution (7.5 g of NaCl + 0.35 g of KC1 + 0.279 g of CaC12-2H2O in 1 L H2O), was inserted into the shaft or base of an ab3 sensillum. A tungsten reference electrode was inserted into the fly eye. To deliver odorants, a Pasteur pipette containing 20pl of mineral oil, 1% lemongrass oil, or PMD was inserted into a hole in a serological pipette directed at the antenna. A stimulus controller (Syntech CS-55) was used to divert a 1 s pulse of charcoal -fdtered air (5 ml / s) into the Pasteur pipette to deliver the odorant to the antenna. Signals were amplified 100x using a USB-IDAC System (Syntech, Hilversum, the Netherlands), input into a computer via a 16-bit analog-digital converter and analyzed offline with AUTOSPIKE software (USB-IDAC System; Syntech).

[0079] RNA purification and RNA-Seq

[0080] To generate cDNA from antennal samples, the SMART-seq v4 kit (Takara Bio) was used. One antennal sample tested with lemongrass oil and 2 antennal samples tested with PMD did not produce cDNA. The Nextera kit (Illumina) was used to create cDNA library from all other samples. For sequencing, samples were run on NovaSeq, S Prime Flow Cell (Illumina) with 100 cycles. The kallisto program was then used to pseudoalign reads from each sample to a reference transcriptome (Anopheles gambiae, AgamP4) and generate “abundance” files that contain expression levels of all odorant receptors (ORs) genes in each sample. Abundance files were converted to .csv files and used to sort ORs based on their expression levels (transcriptome per million, TPM). An OR with >1 TPM was considered “present” in the sample and used to create a hitlist of ORs that are present in most samples. ORs present in most samples were heterologously expressed in the ab3 A neuron on the Drosophila melanogaster antenna (the empty neuron system) and tested using fluorescence-guided single sensillum recording to confirm their response to lemongrass oil and / or PMD.

[0081] Close Proximity Response Assay to monitor a mosquito direct response to odor (Slide 14)

[0082] A single mosquito was transferred to a cage (BugDorm, 30 X 30 X 30 cm). The mosquito was then allowed to land and rest on one of the cage mesh walls. The mosquito was then approached, from outside the cage, by a 1000 mL pipette tip containing a piece of filter paper soaked with an odorant. The pipette tip was rested on the outer side of the cage wall so that the mosquito was at a 0.5 cm distance from the filter paper soaked with odorant. TheAtt’y Ref.: 002240.609807 mosquito was observed for 30 s and the time it took to fly away was scored. The sequence of odorants tested with each mosquito was randomized, and the mosquito was given 2 minutes between odorants. If the mosquito flew off, it was allowed to land and rest for 2 minutes before the next odorant was tested. To analyze how long it takes for mosquitoes to fly in response to odorants, a Cox Proportional Hazard Model was used, which also accounted for the number of previous odorant exposures. Data was plotted using a Kaplan-Meier survival Estimates.

[0083] Example 1: PMD and Lemongrass oil are repellent to Anopheles mosquitoes, unlike the synthetic DEET, IR3535 and picaridin

[0084] Results from the close proximity response assay. Kaplan-Meier estimates shows the proportion of Anopheles coluzzii mosquitoes that remained on the cage wall over time in response to repellents and the paraffin oil control (n = 30 mosquitoes). Asterisks indicate significant differences from paraffin oil (Cox Proportional Hazard Model, ***P < 0.001).

[0085] Example 2: Lemongrass oil, PMD, and DEET are repellent to Aedes aegypti mosquitoes unlike the synthetic IR3535 and picaridin.

[0086] Results from the close proximity response assay. Kaplan-Meier estimates shows the proportion of Aedes aegypti mosquitoes that remained on the cage wall over time in response to repellents and the paraffin oil control (n = 30 mosquitoes). Asterisks indicate significant differences from paraffin oil (Cox Proportional Hazard Model, **P< 0.01, ***p < 0.001)

[0087] Example 3: Lemongrass oil leads to robust and specific olfactory neuron activation in transgenic mosquitoes

[0088] The QF2 binary expression system is used to genetically label olfactory neurons. The QF2 transcription factor is expressed from the Oreo promoter / enhancer in a majority of olfactory neurons. QF2 binds and activates the QUAS enhancer to drive expression of a gene of interest, such as GFP or GCaMP6. Anopheles mosquitoes that contain both QF2 and QUAS transgenes exhibit robust expression of the gene of interest in olfactory neurons in the antenna, maxillary palp, and labella. For calcium imaging, the QUAS-GCaMP6f reporter is used with Orco-QF2. A mosquito that contains Orco-QF2 transgene and QUAS-GCaMP6f transgene is placed in a cut pipette tip and the antenna held down onto a glass slide using glass electrodes. The olfactory neurons expressing GCaMP6f are visible in the antenna and can be directly imaged using aAtt’y Ref: 002240.609807 fluorescence microscope. A 1 -second puff of repellent odor (such as lemongrass oil) leads to increased fluorescence in 4 neurons in an antennal segment. These represent the 4 neurons in an antennal segment activated by lemongrass oil.

[0089] Example 4: Lemongrass oil and PMD activates AgORlO but not AgOR44, Lemongrass oil activates AgOR48

[0090] Representative results from the Drosophila empty neuron system used to characterize the response properties of AgORs. The “A” neuron (large spiking neuron) ectopically expresses the listed AgOR in the ab3 sensilla. Responses to odors are recorded by single sensillum recordings of the ab3 sensilla. Expression of AgOR44 in the empty neuron system does not result in responses to lemongrass oil or PMD. In contrast, expression of AgOR20 in the empty neuron system leads to robust responses to lemongrass oil and PMD indicating that AgOR20 is activated by these repellents. Expression of AgOR48 in the empty neuron system leads to even stronger responses to lemongrass oil but only mild responses to PMD. This indicates that AgOR48 directly responds to lemongrass oil. FIG. 3 presents data showing that lemongrass oil activates AgOR20 and AgOR48, while PMD activates AgOR20. FIGs. 4A-4B presents data showing OR20 response to lemongrass oil 1% (FIG. 4A) or PMD 1% (FIG. 4B). FIGs. 5A-5C presents data showing OR48 response to lemongrass oil 1% (FIG. 5A), PMD 1% (FIG. 5B), or PMD 10% (FIG. 5C). FIGs. 6A-6B presents data showing OR8 response to lemongrass oil 1% (FIG. 6A) or PMD 1% (FIG. 6B). FIGs. 7A-7B presents summary of the responses of OR20, OR48, and OR8 to lemongrass oil 1% (FIG. 7A) or responses of OR20 and OR8 to PMD1% and OR48 to PMD 10% (FIG. 7B).

[0091] Example 5: Identification of AgOR8 expressed in Neuron B of the maxillary palp is activated by the natural repellent lemongrass oil and PMD

[0092] The mosquito contains two main olfactory organs: the antenna and the maxillary palp. The antenna contains most olfactory neurons which expressed AgOR20 and AgOR48. Both ORs respond to PMD and lemongrass oil.

[0093] The maxillary palp contains capitate peg sensilla that house 3 olfactory neurons (called A, B, C). Single sensillum recordings was used to directly test the response of Anopheles mosquito maxillary palp olfactory neurons towards PMD and lemongrass oil. The “B” neuronAtt’y Ref: 002240.609807 expressing the AgOR8 receptor responds to PMD and lemongrass oil. Previously, using the empty neuron system, AgOR8 was found to respond to PMD and lemongrass oil. FIG. 8 presents data showing Anopheles maxillary palp capitate peg sensilla response to lemongrass oil and PMD (likely the “B” neuron that expressed AgOR8). FIG. 9 presents data showing that AgOR8 expressing olfactory neurons do responds to PMD and lemongrass oil.ReferencesAfify, A., Betz, J. F., Riabinina, 0., Lahondere, C. & Potter, C. J. Commonly Used Insect Repellents Hide Human Odors from Anopheles Mosquitoes. Current Biology 29 , 3669-3680 (2019).Afify, A. & Potter, C. J. Calcium Imaging of Anopheles coluzzii mosquito antennae expressing the calcium indicator GCaMP6f. Cold Spring Harb Protoc 2022, Pdb.protl07918 (2022).Bray, N. L., Pimentel, H., Melsted, P. & Pachter, L. Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol 34, 525-527 (2016).Carey, A. F., Wang, G., Su, C. Y ., Zwiebel, L. J. & Carlson, J. R. Odorant reception in the malaria mosquito Anopheles gambiae. Nature 464, 66-71 (2010).Lin, C.-C. & Potter, C. J. Re-Classification of Drosophila melanogaster Trichoid and Intermediate Sensilla Using Fluorescence-Guided Single Sensillum Recording. PLOS ONE 10, e0139675 (2015).

Claims

Atfy Ref: 002240.609807WE CLAIM:

1. A method for screening a plurality of test compounds comprising: contacting an olfactory sensilla that expresses at least one odorant receptor (OR) gene product with at least one test compound, said at least one OR gene product being reactive to at least one odorant; recording an activity pattern characterized by a spike frequency and a spike amplitude in said sensilla by single sensillum electrophysiological recordings (SSR), thereby identifying whether said at least one test compound has a repellent capability, wherein said repellent capability is characterized by a modification in said activity pattern compared to that of a non-repellent control or said at least one odorant.

2. The method of claim 1, wherein said modification in said activity pattern occurs in at least one of said a spike frequency or a spike amplitude.

3. The method of claim 1, wherein said olfactory sensilla is of an Anopheles mosquito.

4. The method of claim 3, wherein at least one OR gene product is at least one of AgORZO, AgOR48, AgOR9 or AgOR8 of Anopheles species.

5. The method of claim 4, wherein said odorant is at least one of lemongrass oil or PMD (p- menthane 3,8-diol).

6. The method of claim 1, wherein said olfactory sensilla that expresses the AgOR8 gene product is the capitate peg sensilla of the maxillary palp.

7. The method of claim 6, wherein said AgOR8 gene product is expressed on neuron B.

8. The method of claim 6, wherein said AgOR8 gene product responds to both lemongrass oil and PMD.Att’y Ref.: 002240.6098079. The method of claim 1, wherein said olfactory sensilla that expresses at least one of AgOR20 or AgOR8 gene products is the sensilla of the antennal.

10. The method of claim 1 wherein said olfactory sensilla comprises an empty neuron Drosophila melanogaster having a deleted endogenous odorant receptor 22a / b (OR22a / b) gene substituted by at least one OR of the Anopheles species.

11. The method of claim 10, said olfactory sensilla is of a mutant Drosophila melanogaster fly.

12. The method of claim 1, further comprising a close proximity assay.

13. A method for identifying at least one odorant receptor (OR) reactive to a repellent compound comprising: contacting with said repellent compound an olfactory organ comprising a plurality of olfactory neuron (ONs) expressing a plurality of ORs of a transgenic Anopheles mosquito, said transgenic Anopheles mosquito expressing a genetically encoded calcium indicator (GECI) capable of emitted a fluorescent signal, visualizing by microscopy said fluorescent signal emitted upon a repellent-induced response of at least one OR among said plurality of ORs expressed on said plurality of ONs, microdissecting said olfactory organ; and extracting and sequencing RNA to identify said at least one OR that is reactive to said repellent compound.

14. A repellent composition comprising at least one of the test compounds identified in the method of claim 1 .

15. A method of controlling an Anopheles population in an area of infestation, or area susceptible of infestation and / or combating an Anopheles population by spreading the repellent composition of claim 14.Att’y Ref.: 002240.60980716. A method of inhibiting, preventing, reducing, or controlling the incidence of Anopheles- borne diseases such as malaria in a subject, comprising contacting said subject with the repellent composition claim 14.

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