AU2013327395A1 - Fruit fly olfactory attractant composition and use thereof - Google Patents

Abstract

The present invention relates to an olfactory attractant composition for fruit flies and its use in methods of attracting fruit flies, controlling fruit flies and monitoring for the presence of fruit flies. In particular, the olfactory attractant composition comprises at least two lower alkyl esters of the formula C

Description

WO 2014/053016 PCT/AU2013/001131 FRUIT FLY OLFACTORY ATTRACTANT COMPOSITION AND USE THEREOF Field of the Invention 5 The present invention relates to an olfactory attractant composition for fruit flies and its use in methods of attracting fruit flies, controlling fruit flies and monitoring for the presence of fruit flies. Dispensing devices for dispensing the composition and an apparatus for trapping fruit flies are also described. 10 Background of the Invention Fruit fly species in the family Tephritidae are recognised world wide as the most destructive and economically damaging insect pests of fruit and many above-ground vegetable crops. The subfamily Dacinae which is distributed across Africa, South East Asia and the Pacific regions contains some 48 major pest species and another 20 15 species of lesser importance. Annual costs of plant protection, quarantine surveillance programmes, losses of International trade and, in some countries like Australia, losses of interstate trade, run into billions of AU dollars. The average annual value of fruit fly susceptible 20 Australian horticulture is AU$4.8 billion, of which 25% is interstate trade. From 2003-2008, according to the Australian government and industry groups, the costs of fruit fly management was about AU$128 million. It is estimated by the International Centre for the Management of Pest Fruit Flies that Australian producers have annual expenses of pre-harvest and post-harvest fruit fly management to be about AU$200 25 million dollars and fruit fly induced crop losses and trade restrictions across South East Asia and the South Pacific regions is about AU$1 billion. Current management strategies for controlling and monitoring fruit flies include: WO 2014/053016 PCT/AU2013/001131 2 1. Field Pest Management * Insecticide cover sprays using organophosphates such as dimethoate and lebaycid. These insecticides are systemic and thus kill eggs and larvae within fruit. They are currently being banned for use. 5 * Protein bait sprays where substances such as yeast proteins are combined with an organophosphate insecticide such as malathion and are "spot" sprayed at the rate of 10 L/hectare. This method is successful in most crops but difficult to apply to some field vegetable crops and also breaks down under heavy rainfall conditions. It cannot be used in some countries where fruit crops are 10 susceptible to attack during the Summer monsoon period, e.g. Bhutan citrus. * A combination treatment of protein bait sprays and male lure "blocking". The protein baits are applied to crops as described above while the male lure (Cue Lure@ or methyl eugenol) is soaked in an absorbent material with an insecticide added, and these small "blocks" are attached to trees (host or non 15 host) at approximately 300-400 per km 2 . This approach is often expanded over large areas under the concept of Pest Free Areas or Areas of Low Pest Prevalence. These particular strategies are governed by policies set up under the World Trade Organisation. 2. Surveillance programmes 20 e Many countries such as Australia, New Zealand, USA, Japan and Pacific Island nations conduct permanent fruit fly surveillance using male lures in plastic traps. The lures, Cue-Lure@ and methyl eugenol, have an insecticide added to kill the flies once they enter the trap. One example is the North Australian Quarantine Survey (NAQS) first set up as a northern fruit fly survey 25 to detect exotic pest species entering Australia in the late 1970s. e Permanent fruit fly surveillance programmes are a compulsory strategy for International and interstate trade in fresh horticultural commodities. These surveys are conducted in production areas from which crops are harvested and exported. The surveys are based on male lure trapping using Cue-Lure® and 30 methyl eugenol, conducted under government guidance and regulations laid down by the World Trade Organisation.

WO 2014/053016 PCT/AU2013/001131 3 Studies into fruit fly biology and ecology show that: I. The host plant is the "centre of activity" for a localised fruit fly population. 2. The host plant with ripening fruit attracts sexually mature male flies and sexually immature female fruit flies. 5 3. After the female fruit flies reach sexual maturity, the males and females mate within the host plant. 4. After mating (i.e. fertilization of eggs), the female oviposits fertile eggs into ripening fruit. 10 While there are chemical lures available for attracting male fruit flies and protein baits can be attractants for female fruit flies in the subfamily Dacinae, these baits are not effective for attracting female flies into traps. Therefore current traps that include lures attract only male fruit flies. This leaves the females able to mate with untrapped males and therefore they are still able to oviposit in ripening fruit and vegetables. 15 Damage to crops still occurs. Furthermore, while there have been combinations of visual lures and chemical lures, these have not been used in traps. For example, visual lures impregnated with ammonia-based odours and coated with adhesive to which attracted fruit flies adhere when they alight on the visual lure are known. 20 A lure or olfactory attractant that is able to attract females as well as males into traps would advantageously reduce infestation in crops by luring and killing females before they can oviposit. There is a need for a fruit fly olfactory attractant and traps that attract or lure both 25 female and male fruit flies and thereby improve the control of fruit fly infestations. Summary of the Invention The present invention is predicated at least in part by the discovery that a combination of volatile lower alkyl esters is a suitable olfactory attractant for both female and male 30 fruit flies and it is effective while in use in a trap.

WO 2014/053016 PCT/AU2013/001131 4 In a first aspect of the invention, there is provided a fruit fly attractant composition comprising at least two lower alkyl esters of the formula (I):

C

1 AalkylC(O)OCI-4alkyl. 5 In another aspect of the invention, there is provided a dispensing device comprising the composition of the invention. In a further aspect of the invention, there is provided a fruit fly trap apparatus comprising the composition of the invention or a dispensing device of the invention. 10 In another aspect of the invention, there is provided a fruit fly trap apparatus comprising: (i) an olfactory attractant composition of the invention; and (ii) a visual attractant. 15 In yet a further aspect of the invention, there is provided a method of attracting fruit fly pests comprising exposing at least one fruit fly to a composition of the invention. In another aspect of the invention, there is provided a method of trapping fruit fly 20 pests comprising exposing at least one fruit fly to an apparatus of the invention. In yet another aspect of the invention, there is provided a method of monitoring for the presence of at least one fruit fly comprising positioning an apparatus of the invention in an environment which requires monitoring for fruit flies. 25 Brief Description of the Figures Figure 1A is a graphical representation of the percent of mature and-mated females attracted to a trap containing an attractant composition comprising methyl acetate, ethyl acetate, ethyl propionate and ethanol, during a 4 hour trial. 30 Figure lB is a graphical representation of the percent of males attracted to a trap containing an attractant composition comprising methyl acetate, ethyl acetate, ethyl WO 2014/053016 PCT/AU2013/001131 5 propionate and ethanol, during a 4 hour trial. Figure 1 C is a graphical representation of the percent of immature females attracted to. a trap containing an attractant composition comprising methyl acetate, ethyl acetate, 5 ethyl propionate and ethanol, during a 4 hour trial. Figure 2A is a graphical representation of the number of male (o) and female (e) Bactrocera tryoni fruit flies trapped during a first 7 day field cage trial set up within a laboratory. 10 Figure 2B is a graphical representation of the number of male (o) and female (9) Bactrocera tryoni fruit flies trapped during a second 7 day field cage trial set up within a laboratory. 15 Figure 3A is a graphical representation of the number of male (o) and female (e) Bactrocera cucumis fruit flies trapped during a first 7 day field cage trial set up within a laboratory. Figure 3B is a graphical representation of the number of male (o) and female (e) 20 Bactrocera cucumis fruit flies trapped during a second 7 day field cage trial set up within a laboratory. Figure 4 is a graphical representation of the number of male (o) and female (.) Bactrocera cacuminata fruit flies trapped during a 7 day field cage trial set up within 25 a laboratory. Figure 5A is a schematic front view representation of an exemplary aerosol dispenser, for the composition of the invention. 30 Figure 5B is a schematic side view representation of the aerosol dispenser of Figure 5A.

WO 2014/053016 PCT/AU2013/001131 6 Figure 6 is a schematic representation of an exemplary fruit fly trap apparatus. Figure 7 is a schematic representation of an exemplary fruit fly trap apparatus incorporating a visual lure. 5 Figure 8 is a schematic representation of a fruit fly trap incorporating a visual lure and an aerosol dispenser. Figure 9A is a graphical representation of the relative attractiveness of eight 10 headspace volatile compounds: ethanol, ethyl acetate, methyl acetate, ethyl propionate, ethyl butanoate, isoamyl acetate, isobutyl acetate and methyl butanoate to female B. tryoni flies. Figure 9B is a graphical representation of the relative attractiveness of eight headspace 15 volatile compounds: ethanol, ethyl acetate, methyl acetate, ethyl propionate, ethyl butanoate, isoamyl acetate, isobutyl acetate and methyl butanoate to male B. tryoni flies. Figure 9C is a graphical representation of the relative attractiveness of eight headspace 20 volatile compounds: ethanol, ethyl acetate, methyl acetate, ethyl propionate, ethyl butanoate, isoamyl acetate, isobutyl acetate and methyl butanoate to male and female B. tryoni flies. Figure 10 A is a graphical representation showing the percent of female fruit flies (B. 25 tryoni) trapped by Attractant formulations 1 and 2. Figure lOB is a graphical representation showing the percent of male fruit flies (B. tryoni) trapped by Attractant formulations 1 and 2. 30 Figure 1OC is a graphical representation showing the percent of male and female fruit flies (B. tryoni) trapped by Attractant formulations I and 2.

WO 2014/053016 PCT/AU2013/001131 7 Figure 11 A, is a graphical representation showing the mean percentage of female flies trapped in traps of different configurations. Figure II B is a graphical representation showing the mean percentage of male flies 5 trapped in traps of different configurations. Figure 11 C is a graphical representation showing the mean percentage of female and male flies trapped in traps of different configurations. 10 Detailed Description of the Invention 1. Definitions The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. 15 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers 20 or steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or 25 information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. As used herein, the term "about" refers to a quantity, level, value, dimension, size, or amount that varies by as much as 30%, 25%, 20%, 15% or 10% to a reference 30 quantity, level, value, dimension, size, or amount.

WO 2014/053016 PCT/AU2013/001131 8 As used herein, the term "attractant" refers to a volatile compound containing composition or other stimulus, such as a visual stimulus, that causes, either directly or indirectly, a fruit fly to displace itself toward the source of the stimuli. 5 The term "transparent" as used herein refers to a material having the property of transmitting rays of light through its substance so that bodies situated behind the material can be distinctly seen. The term "semi-transparent" indicates that while the material is imperfectly transparent and the bodies are not as distinct as with transparent material, shapes and colours behind the material can be visually 10 distinguished. The term "alkyl" as used herein, refers to a straight chain or branched hydrocarbon having 1 to 10 carbon atoms. Where appropriate, the alkyl group may have a specified number of carbon atoms, for example, C1- 6 alkyl refers to an alkyl group having 1, 2, 3, 15 4, 5 or 6 carbon atoms in a linear or branched arrangement. Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i butyl, t-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 2-methyl-pentyl, 3 methylpentyl, 4-methylpentyl, 2-ethylbutyl and 3-ethylbutyl. 20 The term "environment" may be a "horticultural environment" where crops of plants that may be infested by fruit flies are being grown. For example a horticultural environment may include where above ground vegetable or fruit crops are grown, such as orchards of fruit trees or single fruit trees in a garden, garden beds of vegetables or fruits such as tomatoes, or commercial enterprises growing large 25 quantities of fruits or vegetables. The environment may also include the location in which vegetables or fruit are stored post-harvest for example, before being transported to market, during transport to market or during storage before sale. As used herein the term "vicinity" refers to an apparatus being placed in a location 30 that will trap a population of fruit flies, reducing or preventing infestation of fruit. In this case, the vicinity refers to a position from which the composition.can emanate and be detected by the population of fruit flies to be trapped. The term "vicinity" is also WO 2014/053016 PCT/AU2013/001131 9 used with reference to borders such as international and interstate borders. The term vicinity used here may refer to a quarantine point where imported or interstate horticultural products are inspected, including sea ports and airports, or vicinity may refer to areas surrounding ports of entry of horticultural products to detect fruit flies 5 not detected at quarantine points: Vicinity of a border may also refer to a defined location known to be fruit fly free which is located close to a location which is not fruit fly free. In this case, the spread of a fruit fly population may be monitored and control measures used if the population of fruit flies approaches the fruit fly free border. 10 2. Attractant compositions In a first aspect of the invention there is provided a fruit fly attractant composition comprising at least two lower alkyl esters of formula (I):

C

1 4 alkylC(O)OC1ialkyl. 15 In some embodiments, the lower alkyl ester of formula (I) is a compound of the formula: Ci.

3 alkylC(O)OCI.

3 alkyl. 20 In some embodiments, the composition comprises at least three lower alkyl esters of formula (I). In other embodiments the composition comprises at least four lower alkyl esters of formula (I). In yet other embodiments, the composition comprises at least five lower alkyl esters of formula (I). In some embodiments, the compounds of formula (I) are selected from the group consisting of methyl acetate, ethyl acetate, 25 ethyl propionate, propyl acetate, ethyl butanoate, methyl butanoate and isobutyl acetate. In some embodiments, the composition comprises methyl acetate and ethyl acetate. In some embodiments, the composition comprises methyl acetate, ethyl acetate and ethyl propionate. In some embodiments, the composition comprises ethyl butanoate, ethyl acetate, methyl butanoate, ethyl propionate and isobutyl acetate. 30 In some embodiments, the composition comprises at least one lower alkyl alcohol of formula (II) is selected from methanol and ethanol or mixtures thereof. In particular WO 2014/053016 PCT/AU2013/001131 10 embodiments, the compound of formula (II) is ethanol. In some emdodiments, the composition may further comprise one or more other esters such as isopentyl acetate (isoamyl acetate) and ethyl hexanoate. 5 In some embodiments, the composition comprises methyl acetate, ethyl acetate, ethyl propionate and ethanol. In some embodiments, the composition consists of methyl acetate, ethyl acetate, ethyl propionate and ethanol. 10 In some embodiments, the ratio of methyl acetate, ethyl acetate, ethyl propionate and ethanol is in the range of 0.1 to 1.5 : 2 : 01 to 1.5 : 0.5 to 2.5. In a particular embodiment, the ratio is about 1 : 2: 1 : 2. In some embodiments, the composition comprises I to 20% methyl acetate, 30 to 90% 15 ethyl acetate, I to 20% ethyl propionate and 7 to 40% ethanol. In some embodiments, the composition comprises ethyl butanoate, ethyl acetate, methyl butanoate, ethyl propionate and isobutyl acetate. In some embodiments, the composition consists of ethyl butanoate, ethyl acetate, methyl butanoate, ethyl 20 propionate and isobutyl acetate. In some embodiments, the ratio of ethyl butanoate, ethyl acetate, methyl butanoate, ethyl propionate and isobutyl acetate is in the range of 0.5 to 1.5 : 1 : 0.5 to 1.5 : 0.5 to 1.5 : 0.5 to 1.5. In a particular embodiment, the ratio is about 1:1:1:1:1. 25 In some embodiments, the composition comprises 15 to 25% ethyl butanoate, 15 to 25% ethyl acetate, 15 to 25% methyl butanoate, 15 to 25% ethyl propanoate and 15 to 25% isobutyl acetate. 30 In some embodiments, the attractant composition is insecticidal towards fruit flies without additional insecticides being added. Without wishing to be bound by theory, the components of the attractant composition are attractive to fruit flies at low WO 2014/053016 PCT/AU2013/001131 11 concentrations, however, once inside a confined space such as a trap apparatus, the concentration of components may be higher or may be increased at regular intervals to toxic concentrations resulting in killing of fruit flies in the trap. One means of achieving the increase in concentration of the attractant composition at regular 5 intervals is to use an aerosol dispenser which dispenses a given amount of attractant into the trap container providing a toxic dose to fruit flies inside the trap which then decreases to an attractant concentration as the attractant composition disperses from the trap to the environment. 10 In other embodiments, the composition may further comprise an insecticide. Suitable insecticides include organophosphates such as acephate, azinphos-methyl, bensulide, chlorethoxyfos, chlorpyrifos, chlorpyrifos-methyl, diazinon, dichlorfos, dicrotophos, dimetoate, disulfoton, ethoprop, fenamiphos, fenitrothion, fenthion, fosthiazate, malathion, methamidophos, methidathion, mevinphos, monocrotophos, naled, 15 omethoate, oxydemeton-methyl, parathion, parathion-methyl, phorate, phosalone, phosmet, phostebuprim, phoxim, pirimiphos-methyl, profenofos, terbufos, tetrachlorvinphos, tribufos and trichlorfon; organochlorides such as aldrin, chlordane, chlordecone, dieldrin, endosulfan, endrin, heptachlor, hexachlorobenzine, lindane, methoxychlor, mirex and pentachlorophenol, neonicotinoids such as acetamiprid, 20 clothiandin, imidacoprid, nitenpyram, nithiazine, thiacloprid and thiamethoxam, pyrethroids such as allethrin, bifenthrin, cyhalothrin, lambda-cyhalothrin, cypermethrin, cyfluthrin, deltamethrin, etofenprox, fenvalerate, permethrin, phenothrin, prallethrin, resmethrin, tetramethrin, tralamethrin and trasfluthrin, carbamates such as aldicarb, bendiocarb, carbofuran, carbaryl, dioxacarb, fenobucarb, 25 fenoxycarb, isoprocarb, methomyl and 2-(1-methylpropyl)phenyl methylcarbamate; insect growth regulators such as benzoylureas including .diflubenzuron and flufenoxuron, methoprene, hydroprene and tebufenozide; and plant derived insecticides such as anabasine, anethole, annonin, asimina, azidirachtin, caffeine, carapa, cinnamaldehyde, citral, deguelin, eugenol, linalool, myristicin, pyrethrin and 30 spinosad. In particular embodiments, the insecticide is one that is registered or will be registered for the purpose of controlling fruit flies.

WO 2014/053016 PCT/AU2013/001131 12 In some embodiments, the composition further comprising an insecticide also comprises at least one food source such as sugar and/or a source of protein. In some embodiments, the food source comprises one or more of glucose and yeast. Suitable sources of yeast include bakers yeast. 5 In some embodiments, the composition is used in neat form without other inactive components. In some embodiments, the composition may further comprise an agriculturally acceptable carrier. The composition may be formulated as a solution, emulsion, adhesive, foam, gel, paste, granules, aerosol or the composition may be 10 impregnated into natural and synthetic materials. Suitable liquid carriers include aromatic hydrocarbons such as xylene, toluene and alkyl naphthalene, chlorinated aromatic or chlorinated aliphatic hydrocarbons such as chlorobenzenes, chloroethylenes and methylene chloride, aliphatic hydrocarbons such 15 as cyclohexane or paraffins, alcohols such as butanol, glycol as well as their esters and ethers, ketones, such as cyclohexanone, polar solvents such as dimethylformamide, dimethylsulfoxide and water. Emulsifiers for emulsions and foams include polyoxyethylene-fatty acid esters, 20 polyoxyethylene-fatty alcohol ethers such as alkylaryl polyglycol ethers, alkylsulfonates and arylsulfonates, and albumin hydrolysis products. Dispersing agents include methyl cellulose. Suitable aerosol propellants include halogenated hydrocarbons, butane, propane, 25 nitrogen and carbon dioxide. Suitable solid carriers include ground natural minerals such as kaolins, clays, talcs, quartzs, plaster of paris, attapulgites, montmorillonites or kieselguhrs, ground synthetic minerals such as highly dispersed silicic acid, alumina and silicate, crushed 30 natural rocks such as calcite, marble, pumice, sepiolite and dolomite, synthetic granules of inorganic or organic coarse powders and organic materials such as sawdust, coconut shells, maize cobs, tobacco stalks and the like.

WO 2014/053016 PCT/AU2013/001131 13 Suitable adhesives and gel or foaming agents include carboxymethylcellulose and natural and synthetic polymers such as gum arabic, polyvinyl alcohol, and polyvinylacetate. In some embodiments, the composition of the invention may be 5 carried in a gel matrix suitable for controlling release of odours. A suitable matrix is the Specialized Pheromone and Lure Application Technology (SPLAT). In general the composition comprises I to 100% by weight active volatile compounds, especially 10 to 100%, 20 to 100%, 30 to 100%, 40 to 100%, 50 to 100%, 60 to 100%, 10 70 to 100%, 80 to 100% or 90 to 100% by weight active volatile compounds. 2. Dispensing Devices In one aspect, the invention relates to a dispensing device comprising an attractant composition of the invention. 15 In one embodiment, the dispensing device comprises the composition impregnated into a solid material. The solid material may be a powder or solid carrier from which the composition of the invention slowly evaporates providing slow release of the attractant composition. Suitable powder or solid carriers include ground natural 20 minerals such as kaolins, clays, talcs, quartzs, plaster of paris, attapulgites, montmorillonites or kieselguhrs, ground synthetic minerals such as high dispersed silicic acid, alumina and silicate, crushed natural rocks such as calcite, marble, pumice, sepiolite and dolomite, synthetic granules of inorganic or organic coarse powders, organic materials such as sawdust, coconut shells, maize cobs, tobacco 25 stalks and wicks such as cotton wicks, and the like. In some embodiments, the solid carrier is plaster of paris. Optionally the powder or solid carrier is in a container that has apertures for release of the composition of the invention. In some embodiments the powder or solid may be compressed into a tablet. 30 In some embodiments, the dispensing device comprises the composition impregnated into a solid inert carrier such as a filter paper, a sintered glass disc or a polymeric disc. This embodiment also provides slow release of the attractant composition.

WO 2014/053016 PCT/AU2013/001131 14 In some embodiments, the dispensing device comprises the composition of the invention carried in a gel matrix suitable for controlling release of odours. A suitable matrix is the Specialized Pheromone and Lure Application Technology (SPLAT). 5 In other embodiments, the dispensing device is designed to release a predetermined amount of composition of the invention at predetermined intervals. Automated and metered aerosol dispensers are known in the art. For example, those in 10 current use as room deoderisers and insect repellants. An example of a suitable aerosol dispenser is shown in Figures 5A and 5B. The dispenser (1) includes a housing (2) and an aerosol can (3) containing the composition of fruit fly attractant. The aerosol can (3) include a depressable nozzle 15 (4), whereby the attractant composition can be released from the aerosol can (3) via the nozzle (4) when nozzle (4) is depressed. The dispenser (1) can further include a control arrangement which is configured to control the release of the attractant composition from the aerosol can (3). In this 20 example the control arrangement includes a cam follower (5) and a rotating cam (6) which is configured to periodically depress the nozzle (4) of the aerosol can (3) once for each complete rotation of the cam (6). The cam (6) is driven by a DC motor (7) via drive shafts (8) and drive gears (9). The motor (7) is coupled to a battery (10) via an electronic timer (11), such that power from the battery (10) is only supplied to the 25 motor (7) to thereby cause the cam (6) to rotate and depress the nozzle (4) at predetermined intervals of time, under the control of the electronic timer (11). This arrangement therefore allows a controlled of release of the attractant composition at predetermined intervals of time. 30 A vapour seal (12) may be provided within the housing (2) to provide a barrier between the motor (7), electronic timer (11) and battery (10) components and the nozzle (4), for reducing exposure of those components to vapours of the attractant WO 2014/053016 PCT/AU2013/001131 15 composition. Usefully the dispenser (1) may have a removable housing portion (13), for allowing replacement of the battery (10) and the aerosol can (3). Optionally, the removable housing portion (13) may be provided in the form of a ventilation screen, for ventilating the electronic components whilst the removable housing portion (13) is 5 fitted. In some embodiments, the aerosol dispenser comprises a pressurized aerosol can containing the composition of the invention, wherein the composition comprises an aerosol propellant as described above. The can is designed to carry an amount of 10 composition to last the duration of the fruit fly infestation season, for example, from before fruit ripens until harvest. Exemplary amounts are in the range of 40 to 150 mL, especially 50 to 100 mL, more especially about 70 mL. The aerosol dispenser is designed to release an amount of composition in the range of 15 0.01 mL to 5.0 mL, especially 0.01 to 1 mL, 0.05 mL to 0.7 mL, 0.08 mL to 0.5 mL or 0.1 mL to 0.3 mL. The aerosol dispenser is designed to release composition at intervals of 10 minutes to 12 hours, for example, every 30 minutes to 10 hours, 1 hour to 8 hours and the like. 20 3. Fruit Fly Trap Apparatus In another aspect of the invention there is provided an apparatus for trapping fruit flies comprising an attractant composition of the invention. The apparatus may comprise any known trap that is suitable for use as a fruit fly trap. 25 For example, the apparatus may be a plastic trap currently used in fruit fly surveillance such as those used with Cue-Lure@ and methyl eugenol. However, the apparatus contains the composition of the invention and not Cure-Lure@ and methyl eugenol. 30 In some embodiments, the apparatus may comprise an at least partially transparent or semi-transparent container.

WO 2014/053016 PCT/AU2013/001131 16 In some embodiments, the at least partially transparent or semi-transparent container has a volume of 0.5 L to 3.5 L, especially 1 L to 3 L, 1.5 L to 2.5 L, more especially about 2 L. 5 The container may be any shape suitable to trap fruit flies, for example, cylindrical, bowl shaped, spherical, cubic and the like. In some embodiments, the container is bowl shaped. The container is also enclosed, optionally with a removable lid to allow access to the container to place attractant composition, insecticide and food sources inside the container and to allow removal of trapped flies and cleaning of the 10 apparatus. The container may be at least partially transparent or semi-transparent. The proportion of the container that is transparent or semi-transparent is sufficient to allow light into the container, and when a visual attractant is present, to allow the visual attractant to 15 be' seen from outside the container. In particular embodiments at least 40% of the container, especially at least 50%, 60%, 70%, 80% or 90% is transparent or semi transparent. The container may be made from any transparent or semi-transparent material, 20 transparent or semi-transparent plastic is particularly useful. In particular embodiments, the container is made from transparent material such as transparent plastic. In some embodiments, the container has one aperture. In other embodiments, the 25 container has a plurality, of apertures, for example, 2 to 6 apertures. The number of apertures may depend on the size of the container. Typically, a 2 L container will have 2 to 4 apertures, especially 2 apertures. In some embodiments, the apertures are located evenly around the container, for example, when two apertures are present, they may be located opposite one another. In some embodiments, an aperture may be 30 located in the top or bottom of the container. The apertures may be any suitable size and shape. The size and shape of the apertures WO 2014/053016 PCT/AU2013/001131 17 may depend on the size of the container being used on the trap and the species being trapped. For example, the apertures may be round and be 5 mm to 120 mm in diameter, especially 5 mm to 55 mm in diameter, 6 mm to 35 mm in diameter, 7 mm to 25 mm in diameter, 8 mm to 18 mm in diameter, most especially about 8 mm to 15 5 mm in diameter. In some embodiments, the apertures are fitted with elongated tubes, the tubes being I cm to 5 cm in length, especially 2 cm to 4 cm in length, more especially about 3 cm in length. The elongated tubes have a diameter to fit snugly within the aperture, for 10 example, having an internal diameter of 5 mm to 100 mm, especially 5 mm to 50 mm, in diameter, 6 mm to 30 mm in diameter, 7 mm to 20 mm in diameter, 8 mm to 15 mm in diameter, especially an internal diameter of about 10 mm. Without wishing to be bound by theory, it is thought that the elongated tubes reduce 15 the likelihood of the fruit flies being able to exit the trap after entry. In some embodiments, the elongated tubes are tapered, for example, the tubes are narrow near the aperture of the container or in a portion of the tube that extends into the container, and wider at the end through which the fruit flies enter for example, a 20 portion of the tube that extends outside the container. In some embodiments, the outer or external end of the elongated tube flares out to a funnel shape. In some embodiments, the elongated tube extends outwardly from the aperture such that the internal end of the tube is attached to the aperture and the external end is 25 outside of the container. In other embodiments, the elongated tube extends into the container from the aperture such that the internal end of the tube is inside of the container and the external end of the tube is attached to the aperture. In yet other embodiments, the elongated tube extends through the aperture such that the external end of the tube is outside the container and the internal end of the tube is inside the 30 container. In some embodiments, the external end of the elongated tube has a cap to close off entry into the trap while it is not in use, for example, the tube may include a thread and a removable screw cap. The cap is removed when the trap is in use.

WO 2014/053016 PCT/AU2013/001131 18 In some embodiments, the apparatus contains a dispensing device as described above. In other embodiments, the composition is located on the inner surface of the container or the bottom of the container. In yet other embodiments, the composition is in an 5 open dish in the bottom of the container. In some embodiments, the apparatus further contains an insecticide composition which may be associated with the composition of the invention or may be a separate composition, The insecticide composition may include suitable insecticides including 10 organophosphates such as acephate, azinphos-methyl, bensulide, chlorethoxyfos, chlorpyrifos, chlorpyrifos-methyl, diazinon, dichlorfos, dicrotophos, dimetoate, disulfoton, ethoprop, fenamiphos, fenitrothion, fenthion, fosthiazate, malathion, methamidophos, methidathion, mevinphos, monocrotophos, naled, omethoate, oxydemeton-methyl, parathion, parathion-methyl, phorate, phosalone, phosmet, 15 phostebuprim, phoxim, pirimiphos-methyl, profenofos, terbufos, tetrachlorvinphos, tribufos and trichlorfon; organochlorides such as aldrin, chlordane, chlordecone, dieldrin, endosulfan, endrin, heptachlor, hexachlorobenzine, lindane, methoxychlor, mirex and pentachlorophenol, neonicotinoids such as acetamiprid, clothiandin, imidacoprid, nitenpyram, nithiazine, thiacloprid and thiamethoxam, pyrethroids such 20 as allethrin, bifenthrin, cyhalothrin, lambda-cyhalothrin, cypermethrin, cyfluthrin, deltamethrin, etofenprox, fenvalerate, permethrin, phenothrin, prallethrin, resmethrin, tetramethrin, tralamethrin and trasfluthrin, carbamates such as aldicarb, bendiocarb, carbofuran, carbaryl, dioxacarb, fenobucarb, fenoxycarb, isoprocarb, methomyl and 2 (1-methylpropyl)phenyl methylcarbamate; insect growth regulators such as 25 benzoylureas including diflubenzuron and flufenoxuron, methoprene, hydroprene and tebufenozide; and plant derived insecticides such as anabasine, anethole, annonin, asimina, azidirachtin, caffeine, carapa, cinnamaldehyde, citral, deguelin, eugenol, linalool, myristicin, pyrethrin and spinosad. In particular embodiments, the insecticide is one that is registered or will be registered for the purpose of controlling 30 fruit flies. In some embodiments, the apparatus further comprises a food source, optionally WO 2014/053016 PCT/AU2013/001131 19 mixed with insecticide composition, wherein the food source comprises a sugar and/or a protein source. In some embodiments, the apparatus comprises a visual attractant. The visual 5 attractant may be any suitable shape, such as fruit shape, substantially spherical or spherical and may be coloured. The colour of the visual attractant depends on the species of fruit fly to be trapped as described in Drew et al. 2003, Drew et al. 2006 and Vargas et al. 1991. For example, Bactrocera tryoni responds to cobalt blue spheres. Bactrocera minax responds to orange and green spheres of about 5 cm in 10 diameter and Bactrocera dorsalis responds to yellow and white spheres of about 4 cm in diameter. In some embodiments, the visual attractant may be placed on the bottom of a container. In other embodiments, the visual attractant may be supported inside a container. For instance the visual attractant may be placed on a stand, such as a wire rack or a spike allowing it to be positioned above the bottom of a container, for 15 example, centrally located in a container. In yet another embodiment, the visual attractant is fixed to the top of a container or is hung from the top of a container, optionally centrally located in a container. In other embodiments, the apparatus comprises a visual attractant coated with an adhesive such as TanglefootTm adhesive, particularly in the absence of a container. 20 In some embodiments, the apparatus further has a means of hanging the apparatus above the ground, for example, a suspension rig, a hook or a loop that may be attached or hung from a pole in the vicinity of the host tree or crop or tree such as a host tree or a tree in the vicinity of the host tree. 25 In other embodiments, the apparatus comprises a container with a flat bottom and it may be placed on the ground in the vicinity of the host tree or crop. Exemplary fruit fly trap apparatuses (20), (30), (40) of the invention are shown in 30 Figures 6, 7 and 8. In the exemplary apparatus (20) depicted in Figure 6 there is provided a clear plastic WO 2014/053016 PCT/AU2013/001131 20 container (21) having two apertures (22). Each aperture is fitted with an elongated tube (23) through which fruit flies enter the container (21). For transport or when the apparatus (20) is not in use, the elongated tubes (23) may be capped with a cap (24). 5 Optionally an external end of each tube (23) has a threaded portion (25) and the cap (24) may screw on to the threaded portion (25). The apparatus (20) may be fitted with a dispensing device (28) as described above for releasing attractant composition into the container (21). In this example, the apparatus (20) is fitted with a device (28) that allows the attractant composition to be released into the container (21). The device 10 (28) may be a container in which the composition is placed or in which a slow release solid composition is placed. In other embodiments, the device (28) may be a solid or gel composition comprising the attractant composition. Optionally, the apparatus (20) contains a suspension rig (27) that allows the apparatus (20) to be suspended in a host plant or in the vicinity of a host plant. 15 In Figure 7, the apparatus (30) is similar to the previously described apparatus (20) shown in Figure 6 and is further fitted with a visual attractant (26), the colour of which is determined by the species of fruit fly being trapped as discussed above. 20 Figure 8 provides another exemplary apparatus (40) similar to the apparatus (30) of Figure 7, in which the device (28) has been replaced by an aerosol dispensing device (1) as shown in Figures 5A and 5B. In some embodiments, the trap apparatus comprises or consists of both an olfactory 25 attractant composition and a visual attractant. In another aspect of the present invention there is provided an apparatus for trapping fruit flies comprising: i) an olfactory attractant composition of the invention; and 30 ii) a visual attractant. The olfactory attractant may be released by evaporation in a slow release composition WO 2014/053016 PCT/AU2013/001131 21 or by aerosol, for example by using the exemplary apparatus shown in Figures 7 and 8, respectively. 4. Methods of the Inventions 5 In one aspect of the invention there is provided a method of attracting fruit fly pests comprising exposing at least one fruit fly to a composition of the invention. In yet another aspect of the invention there is provided a method of trapping fruit fly pests comprising exposing at least one fruit fly to an apparatus of the invention. 10 In some embodiments of these methods, the at least one fruit fly is a population of fruit flies infesting an environment. Suitable environments include horticultural environments such as fruit crops and vegetable crops and harvested fruit and vegetable commodities. 15 In some embodiments, the at least one fruit fly is in the vicinity of a border, such as a national border or an interstate border. Therefore in another aspect of the invention there is provided a method of monitoring for the presence of at least one fruit fly comprising positioning an apparatus of the invention in the vicinity of a border. 20 In some embodiments, the apparatus of the invention is used in a quarantine surveillance system, for example, at an airport, or sea port, or in the area surrounding an environment surrounding an airport or sea port. 25 The fruit fly pest is a pest from the family Tephritidae, especially from the subfamily Dacinae. For example, the fruit flies may be from a tribe selected from Ceratitidini including Ceratitis such as Ceratitis capitata, Dacini including Bactrocera, Dacus and Monacrostichus; In particular embodiments, the fruit fly pest is selected from the Genus Bactrocera, especially B. tryoni, B. cucumis, B. oleae, B. cucurbitae, B. 30 invadens, B. zonata, B. latifrons, B. aquilonis, B. neohumeralis, B. jarvisi, B. papayae, B. philippinensis, B. dorsalis, B. minax, B. umbrosa, B. xanthodes, B. melanotus, B. trivialis, B. carambolae, B. decipiens, B. atrisetosa, B. albistrigata, B. bryoniae, B.

WO 2014/053016 PCT/AU2013/001131 22 caryeae, B. caudata, B. correcta, B. curvifera, B. curvipennis, B. diversa, B. facialis, B. frauenfeldi, B. kandiensis, B. kirki, B. kraussi, B. musae, B. obliqua, B. occipitalis, B. passiflorae, B. psidii, B. pyrifoliae, B. scutellaris, B. scutellata, B. strigifinis, B. tau, B. trilineola, B. tsuneonis and B. tuberculata. In other embodiments, the fruit fly 5 is from the Genus Dacus, especially D. ciliatus and D. solomonensis. The apparatus may be placed in any desired environment. In some embodiments of the methods of the invention, the apparatus is placed in the environment of a fruit or vegetable crop. In some embodiments, the apparatus is hung from a tree or a pole in 10 the environment of a fruit or vegetable crop. The number of apparatus deployed in a crop, will depend on many factors such as the crop and the species of fruit fly being trapped. The number required may be easily determined by those skilled in the art by routine field trial. In some embodiments, 15 where the crop is an orchard crop, the number of apparatus deployed may range from 1 per tree to I every five trees. In those trees which have dense foliage, more apparatus will be required, for example I per tree. In particular embodiments, there is between 1 apparatus per tree and I apparatus every four trees, especially 1 apparatus per tree and 1 apparatus every three trees, more especially either 1 apparatus per tree 20 or 1 apparatus every second tree. Many modifications will be apparent to those skilled in the art without departing from the scope of the present invention. 25 Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

WO 2014/053016 PCT/AU2013/001131 23 EXAMPLES Example 1: Fruit Volatile Compounds As ripe peaches and guava are subject to fruit fly infestation, analysis of head space volatiles of unripe green and ripe peaches and guava to ascertain the identity of 5 volatiles that are produced by ripe fruit was performed. Field collection of fruit Field collections of fruit for chemical analysis were carried out in two stages. Firstly, ripe guava were sampled from wild trees growing in the Brisbane area and forwarded 10 to the Australian Phytochemicals Ltd laboratories at Southern Cross University, Lismore, for chemical analyses of volatile emissions. These fruits contained fruit fly larvae from natural field infestation. Secondly, peach trees on the Boonah-Beaudesert Road and guava trees in a plantation at Redland Bay were selected. Some hard green fruit of both types were sampled for chemical analyses and, at the same time, other 15 green fruits of the same stage on the same trees were enclosed on the trees in brown paper bags. When ripe, the fruits in the bags were sampled for chemical analyses. This field sampling strategy provided data on the chemicals that were present and/or in large concentrations in ripe fruit that attract adult fruit flies in contrast to green fruit that do not attract the flies. 20 Chemical analyses At the Australian Phytochemicals laboratory, six fresh ripe guava fruit were held in a 2L glass jar and the headspace volatiles absorbed on to fibre Solid Phase Microextraction units (SPME). The fibre was desorbed into the GCMS inlet and 25 GCMS analyses conducted. Additional volatile samples were taken by (a) boiling fresh ripe guava fruit and undertaking GCMS analyses of the distillation extracts and (b) homogenising fresh ripe guava fruit in a 2L glass jar with a Bamix blender, extracting chemical components with hexane and then analysing by GCMS. 30 The green and ripe peach and guava fruit were processed at Advanced Analytical laboratories in Sydney, Australia as follows: WO 2014/053016 PCT/AU2013/001131 24 1) Fruit were held in a glass jar and volatiles collected by Thermal Desorption. 2) Analyses of volatiles were conducted by GCMS. 3) GCMS results were compared against the Advanced Analytical's chemical 5 standards library and/or the NIST library. The results are shown in Table 1. Table 1: Fruit volatile compounds in parts per million Chemical Green Ripe Green Ripe Guava Ripe Guava Ripe Guava Peach Peach Guava (6 samples) (11 samples) (7 samples) Carbon 4.116 8.050 5.325 37.250 85.418 87.000 dioxide Ethyl acetate 0.011 9.090 48.405 28.381 39.113 Acetaldehyde 0.112 0.390 0.002 2.588 4.081 5.534 Ethanol 0.247 0.570 0.001 4.003 2.565 3.594 Methyl 0.930 0.902 0.787 0.991 acetate Ethyl 0.507 0.391 0.470 butanoate Ethyl 0.247 0.207 0.230 propionate Isoamyl 0.348 0.120 0.246 acetate Isobutyl 0.260 0.318 0.099 0.128 acetate Methyl 0.140 0.081 0.085 butanoate Dimethyl 0.020 0.047 0.025 disulfide Ethyl 0.031 0.033 WO 2014/053016 PCT/AU2013/001131 25 hexanoate Methanol 0.044 0.980 0.010 Acetone 0.073 0.060 0.100 Propyl 0.024 acetate Methyl ethyl 0.026 ketone Pinene 0.008 Limonene 0.004 Total 4.602 20.354 5.474 94.772 122.207 137.350 From these analyses, it was identified that a number of volatile compounds were not emitted by green unripe fruit but were emitted from ripe fruit and a number of volatile compounds were emitted at low levels by unripe fruit but emitted in significantly 5 increased concentration in ripe fruit. A combination of methyl acetate, ethyl acetate, ethyl propionate and ethanol in a ratio of 1 : 2 : 1 : 2 was selected for testing for attractant activity with female fruit flies. 10 Example 2: Attractant Testing - Screening A series of three interconnected clear acrylic cube cages, 40 cm x 40 cm x 40 cm, were used as cages to perform bench-top tests of 4 hours duration. One cage was used as a target cage and contained 1 mL attractant composition comprising 3.33% ethyl acetate, 3.33% ethanol, 1.66% methyl acetate, 1.66% ethyl propionate and 90% water 15 (10% solution in water). The attractant composition was placed on a filter paper in a glass petrie dish immediately before the commencement of the trial. A second cage was used as the source cage where the fruit flies were released. Fifty to sixty fruit flies (B. tryoni) were released into the source cage at the commencement of each trial. Separate trials were carried out mature males, mature and unmated females and 20 mature mated females. The third cage was a blank cage. The three interconnected cages were set up with the source cage in the middle and the target and blank cages on WO 2014/053016 PCT/AU2013/001131 26 opposite sides of the source cage i.e. either in the order; target cage, source cage, blank cage or blank cage, source cage, target cage. This order was alternated between trials to eliminate any effect of responses to uneven lighting in the laboratory. 5 All cages were thoroughly washed with Decon90 between trials and dried. The number of fruit flies in each cage was counted every 15 minutes over the 4 hours of the trial. The results are shown in Figure 1 and are results from 6 replicate experiments ± SEM. 10 The cage trials showed that 20% of mature and mated females were attracted by the attractant composition, 2% of males were attracted by the attractant composition and 25% of immature females were attracted by the attractant composition over the 4 hour trial. 15 Example 3: Field Cage Testing Experiments were conducted in a large synthetic mesh field cage, approximately 9 m 3 in volume, erected in a lockable room. The traps used in the field cage trials were as shown in Figure 6. 20 Before the commencement of each trial sugar, water and protein were provided in the cage to sustain the flies during the trial. The protein source used was 2 to 3 g of dried yeast powder provided in petri dish. Sugar was provided as 2 to 3 sugar cubes in a petri dish. Water was provided in a sealed plastic container with a small section of 25 sponge-cloth provided as a wick to the outer surface of the container. Approximately 300 flies of either Bactrocera tryoni (Queensland Fruit Fly), Bactrocera cucumis (a major pest of cucurbit and fruit crops) or Bactrocera cacuminata (a non-pest species) were then released into the cage. All flies used in the 30 trials were taken from a single colony at 14 to 21 days post emergence and were assumed to be mature and mated at this stage. Flies were selected for the trials at WO 2014/053016 PCT/AU2013/001131 27 random from the colony and it was assumed that approximately equal numbers of males and females would.be included in the trials. A trap of the design shown in Figure 6 (without suspension rig (27)) was then placed 5 on a platform of approximately 1 m height in the cage. 0.1 mL of undiluted attractant (i.e. 33.3% Ethyl acetate, 33.3% Ethanol, 16.6% Methyl acetate and 16.6% Ethyl propionate) was injected into the body of the trap using a syringe and allowed to evaporate passively. 10 On the following morning, caps were placed over the entry ports of the trap and any flies remaining alive (the majority having died in the trap) were euthanized by placing the trap in a freezer for 20 minutes. The captured flies were preserved in 95% ethanol. The trap was then cleaned and dried with paper towel and an additional 0.1 mIL of the attractant was added to the trap before it was placed back in the field cage. The 15 captured flies were then indentified as either male or female and counted. Where trials ran over weekends or public holidays no additional attractant was added nor were the flies counted on these days. This explains the periods shown in the graphs where no additional flies were trapped over a 2 or 3 day period but the number 20 of trapped flies increased again on the last day of the trial when more attractant was added. At the conclusion of the trial all flies remaining in the field cage were captured with a vacuum aspirator and euthanized by freezing. These flies were then indentified as 25 either male or female and counted. This allowed calculation of the proportion of male and female flies which had been captured on each day of the trial. The results of two separate experiments with B. tryoni are shown in Figure 2. 30 Figure 2A shows that over the 7 day trial, 32 of 80 (40%) male fruit flies were trapped and 56 of 116 (48%) female fruit flies were trapped (88 fruit flies were trapped out of 196 fruit flies).

WO 2014/053016 PCT/AU2013/001131 28 Figure 2B shows that over the 7 day trial, 49 of 78 (63%) males were trapped and 117 of 121 (97%) female fruit flies were trapped (166 fruit flies were trapped out of 199 fruit flies). 5 The results of two separate experiments with B. cucumis are shown in Figure 3. Figure 3A shows that over the 7 day trial, 38 of 92 (41%) males were trapped and 52 of 141 (37%) females fruit flies were trapped (90 fruit flies were trapped out of 233 10 fruit flies). Figure 3B shows that over the 7 day trial 33 of 102 (32%) males were trapped and 64 of 148 (43%) females were trapped (250 fruit flies were trapped out of 403 fruit flies). 15 The results of a single experiment with B. cacuminata is shown in Figure 4. Figure 4 shows that over the 7 day trial, 2 of the 152 male fruit flies and 2 of the 12 female fruit flies were trapped (4 fruit flies were trapped out of 304 fruit flies). 20 The results show that the attractant composition attracted significant numbers of both female and male fruit fly pests (B. tryoni and B. cucumis) but was not an attractant to the non-pest fruit fly (B. cacuminata). Example 4: Field Cage Trials with a Visual Attractant 25 The field cage trial of Example 3 was repeated with approximately 1000 mature three week old B. tryoni fruit flies. However, two trap apparatus were placed in the field cage. One trap container included only a visual attractant that was a cobalt blue sphere 60 mm in diameter, no attractant composition was used. The other trap container included a cobalt blue sphere as a visual attractant and 5 mL of attractant composition 30 absorbed into plaster of paris within a plastic container with a small adjustable pore.

WO 2014/053016 PCT/AU2013/001131 29 The fruit flies in the traps were counted at 24 hours and 5 days. The results are shown in Table 2. 5 Table 2 24 hr count 5 day count TOTAL FRUIT FLIES Males Females Males Females Blue Sphere 73 82 39 34 228 Blue Sphere + 112 241 221 277 851 composition The combination of visual attractant and olfactory attractant composition in a slow release formulation was effective in trapping the fruit flies in the field cage and was significantly improved over the use of the visual attractant alone. 10 Example 5: Field Cage Testing with Eight Attractant Compounds Trials described below were conducted in a large field-cage (2.4m height, 2.4m diameter) erected in an air conditioned room (temperature maintained at 23 0 C +/ 1*C) within the laboratory. 15 A rotary olfactometer was used to allow simultaneous, multiple-choice tests to be carriedsout within the same cage of flies while eliminating any positional effects. The rotary olfactometer was constructed in a similar manner to those described by 20 Gow (1954, J. Econ. Entomol., 47:153-160) and Jang and Nishijima (1990, Environ. Entomol., 19:1726-1731). The design was such that the rotating part of the olfactometer was mounted on a guyed mast (1.9m height) which was fixed to a 20kg concrete base-plate. The rotating part of the olfactometer consisted of a turntable with an electric motor (rotation rate 1.565 RPM) which formed a hub for eight equally 25 spaced radial-arms of 0.8m length. The various trap attractants being tested were fixed to the ends of these arms. An electronic timer was used to activate the turntable WO 2014/053016 PCT/AU2013/001131 30 for a period of 60 seconds every 100 minutes over a period of 24 hours. This had the effect of changing the rotational position of each trap by 203.4* every 100 minutes during the trial period. 5 A culture of 300 to 400 flies (Bactrocera tryoni unless otherwise stated) were released into the field cage I hour prior to the commencement of the trial. All flies used in the trial were aged between 21 and 28 days post-emergence and all were assumed to be sexually mature and mated. In all cases, the flies in the field cage were provided with free access to sugar, water and yeast-protein. 10 At the conclusion of the trial, the traps were sealed and placed in a freezer to euthenase the trapped flies. All flies remaining in the field cage were then captured and similarly euthenaised. The numbers and sex of all flies in the traps and remaining in the cage were then determined with a stereomicroscope. 15 Results This trial was conducted to determine the relative attractancy of the eight most abundant chemical components of the headspace volatiles of ripe peach and guava. These were ethanol, ethyl acetate, methyl acetate, ethyl propionate, ethyl butanoate, 20 isoamyl acetate, isobutyl acetate and methyl butanoate. The results of four replicated trials are illustrated in Figures 9A, 9B and 9C. These results show that in terms of their attractiveness to female flies, the five most attractive of these chemicals, in descending order were; ethyl butanoate, ethyl acetate, 25 methyl butanoate, ethyl propionate and isobutyl acetate (Figure 9A). For male flies there were some slight differences in the ranking of the attractiveness of these chemicals (Figure 9B) and consequently there were also some differences in the attractiveness ranking when the data for males and females were pooled (Figure 9C). However, despite these differences in ranking, the same five chemicals were 30 consistently the most attractive among those tested.

WO 2014/053016 PCT/AU2013/001131 31 Example 6: Combined Attractant Formulation Compared to Ethyl butanoate This trial was conducted as described in Example 5 to determine the relative attractancy of the Attractant composition (i.e. a mixture of equal parts of: ethyl 5 butanoate, ethyl acetate, methyl butanoate, ethyl propionate and isobutyl acetate) compared to ethyl butanoate alone. Four replicated experiments were performed. In each experiment, eight traps were placed on the rotary olfactometer which were alternately baited with either the Attractant composition or ethyl butanoate as an attractant. The numbers of flies from the four traps baited with the same attractant 10 were tallied to give a single datum. This produced a data set of four paired samples which was analysed using a t-test for paired samples. The results of these analyses are presented in Tables 3, 4 and 5 below. These results show that significantly more females were trapped using the Attractant composition 15 than with ethyl butanoate (Table 3). There was no significant difference in the numbers of males trapped using Attractant composition or ethyl butanoate (Table 4). However, there was a significant difference in the total number of flies trapped such that significantly more flies were trapped using the Attractant composition (Table 5). 20 Table 3: percent females trapped Attractant composition Methyl Butanoate % Replicate 1 25..39 16.79 % Replicate 2 23.98 16.26 % Replicate 3 21.73 13.48 % Replicate 4 15.58 11.69 Table 4: percent males trapped Attractant composition Methyl Butanoate % Replicate 1 27.22 25.55 % Replicate 2 22.10 21.05 % Replicate 3 19.72 7.34 WO 2014/053016 PCT/AU2013/001131 32 % Replicate 4 10 2.94 Table 5: percent total flies trapped Attractant composition Methyl Butanoate % Replicate 1 26.15 20.41 % Replicate 2 23.16 18.35 % Replicate 3 20.76 10.49 % Replicate 4 12.658 7.10 Example 7: Attractant Formulations Comparison 5 This trail was conducted in the same manner as Example 5. The attractancy of two Attractant formulations, Attractants 1 and 2 was compared. The formulations were: Attractant 1: 10 1/3 Ethanol 1/3 Ethyl acetate 1/6 Ethyl propionate 1/6 Methyl acetate Attractant 2: 15 1/5 Ethyl butanoate 1/5 Ethyl acetate 1/5 Methyl butanoate 1/5 Ethyl propionate 1/5 Isobutyl acetate 20 A single trial in which eight traps were used in total was performed; four of which were baited with Attractant 1 and four with Attractant 2. The traps baited with different attractants were positioned alternately on the arms of the rotary olfactometer. The four traps baited with the same attractant were treated as replicates in this case. 25 Analyses of these data showed that significantly more females were trapped in traps baited with Attractant 2 than in traps baited with Attractant I (Figure 1 OA). There was WO 2014/053016 PCT/AU2013/001131 33 no significant difference in the numbers males trapped by either attractant (Figure I OB), nor was there a significant difference in the overall number of flies trapped by either attractant (Figure 1 OC). Although this last result was not significant at the 95% probability level there was a clear trend such that more flies were trapped overall by 5 Attractant 2, and this result would be significant if the less conservative significance threshold of 90% was used. Example 8: Trap configuration trials In this trial, the trapping efficiency of eight different trap configurations was 10 compared. In all cases the traps were baited with Attractant 1 from Example 7. Four replicated experiments were performed in which a trap of each form was attached to an arm of the rotary-olfactometer. The trap configurations tested were: o 1 - Standard configuration with wick attractant + blue ball + entry tubes 15 o 2 - Modified configuration with wick attractant + blue ball + entry tubes in lower position o 3 - Modified configuration with wick attractant + blue ball + no entry tubes o 4 - Modified configuration with wick attractant + no blue ball + entry tubes o 5 - Modified configuration with no attractant + blue ball + entry tubes 20 o 6 - Standard configuration i.e. with pulsed attractant + blue ball + entry tubes o 7 - Modified configuration i.e. with no attractant + no blue ball + entry tubes o 8 - Modified configuration i.e. with no attractant + no blue ball + no entry tubes The results of these trials are illustrated in Figures 11 A, 1 B and I IC below and show 25 that there were significant differences in the numbers of flies captured in traps of different configurations. These results show that the most effective design for trapping females was configuration 6, with a blue ball, entry tubes and a pulsed delivery of the attractant 30 (Figure 11 A). This was closely followed by configuration 3 which featured a blue ball, a wick attractant-dispenser and no entry tubes. For males configuration 3 was WO 2014/053016 PCT/AU2013/001131 34 most efficient followed by configuration 6 and this same ranking occurred when considering the overall number of flies captured. Example 9: Trap Designs 5 Different trap designs with or without attractant composition 2 from Example 7 were used in the trial. Four trap designs were used. All traps were 100 mm cobalt blue balls with the bottoms removed. Each ball was coated on the outer surface with insect TanglefootTM. 10 Traps: 1. 4 rows of 6 x 2 mm vertical holes and 25 mL of Attractant 2 lure. 2. 4 rows of 6 x 2 mm vertical holes and no attractant. 3. No holes, 25 mL of Attractant 2 lure. 4. No holes, no lure. 15 Each trap was suspended and rotated in a pepperina tree area. About 200 B. tryoni were released each day. The flies were about 3 weeks old. The flies were removed from the trap and counted each day. The results are shown in 20 Table 6. Table 6: Trap Female Male Total Ball, holes, lure 11 148 159 Ball, holes, no lure 24 29 63 Ball, lure 38 25 63 Ball, no lure 25 19 44

Claims (31)

1. A fruit fly attractant composition comprising at least two lower alkyl esters of 5 formula (I): C 14 alkylC(O)OCi. 4 alkyl.

2. The attractant composition according to claim I wherein the composition comprises at least three lower alkyl esters of formula (I).

3. The attractant composition according to claim 1, wherein the composition 10 comprises at least four lower alkyl esters of formula (I).

4. The attractant composition according to claim 1, wherein the composition comprises at least five lower alkyl esters of formula (I).

5. The attractant composition according to any one of claims I to 4 wherein the compounds of formula (I) are selected from the group consisting of methyl 15 acetate, ethyl acetate, propyl acetate, ethyl propionate, ethyl butanoate, methyl butanoate and isobutyl acetate.

6. The attractant according to any one of claims 1 to 5 comprising methyl acetate and ethyl acetate.

7. The attractant composition according to any one of claims 1 to 6 comprising 20 methyl acetate, ethyl acetate and ethyl propionate.

8. The attractant composition according to any one of claims 1 to 5 comprising ethyl butanoate.

9. The attractant according to any one of claims 1 to 5, comprising ethyl butanoate, ethyl acetate, methyl butanote, ethyl propibnate and isobutyl 25 acetate.

10. The attractant composition according to any one of claims I to 9 further comprising a compound of formula (II): C1-alkylOH (II).

11. The attractant composition according to claim 10 wherein the compound of 30 formula (II) is methanol, ethanol or mixtures thereof.

12. The attractant composition according to any one of claims 1 to 11 further comprising one or more of isopentyl acetate and ethyl hexanoate. WO 2014/053016 PCT/AU2013/001131 36

13. The attractant composition according to any one of claims I to 6 comprising methyl acetate, ethyl acetate, ethyl propionate and ethanol.

14. The attractant composition according to claim 13 wherein the ratio of methyl acetate : ethyl acetate : ethyl propionate : ethanol is in the range of 0.1 to 1.5 5 2 : 0.1 to 1.5 : 0.5 to 2.5.

.15. The attractant composition according to claim 14 wherein the ratio of methyl acetate : ethyl acetate : ethyl propionate : ethanol is about 1 : 2 : 1 : 2.

16. The attractant composition according to claim 13 wherein the composition comprises 1 to 20% methyl acetate, 30 to 90% ethyl acetate, I to 20% ethyl 10 propionate and 7 to 40% ethanol.

17. The attractant composition according to claim 9 wherein the ratio of ethyl butanoate: ethyl acetate : methyl butanoate : ethyl propionate : isobutyl acetate is in the range of 0.5 to 1.5 : 1 : 0.5 to 1.5 : 0.5 to 1.5 : 0.5 to 1.5.

18. The attractant composition according to claim 9 wherein the ratio of ethyl 15 butanoate: ethyl acetate: methyl butanoate : ethyl propionate : isobutyl acetate is in the range of about 1:1:1:1:1.

19. A dispensing device comprising the composition of any one of claims I to 18.

20. The dispensing device according to claim 19 which provides slow release of the composition. 20

21. The dispensing device according to claim 19 which provides release of a predetermined amount of the composition at predetermined intervals.

22. An apparatus for trapping fruit flies comprising an attractant composition according to any one of claims 1 to 18 or a dispensing device according to any one of claims 19 to 21. 25

23. The apparatus according to claim 22 further comprising a visual attractant.

24. The apparatus according to claim 23 wherein the visual attractant is a coloured sphere.

25. The apparatus according to any one of claims 22 to 24 further comprising an insecticide composition. 30

26. A method of attracting fruit fly pests comprising exposing at least one fruit fly to a composition according to any one of claims 1 to 18.

27. A method of trapping fruit fly pests comprising exposing at least one fruit fly WO 2014/053016 PCT/AU2013/001131 37 to an apparatus of any one of claims 22 to 25.

28. A method according to claim 27 wherein the at least one fruit fly is a population of fruit flies infesting an environment.

29. A method according to claim 28 wherein the population of fruit flies is in a 5 fruit producing environment.

30. A method of monitoring for the presence of at least one fruit fly pest comprising positioning an apparatus of any one of claims 22 to 25 in an environment that requires monitoring for the presence of fruit flies.

31. A method according to any one of claims 26 to 30 wherein the fruit fly pests 10 are female fruit fly pests.