AU2016201206B2 - Pest control - Google Patents

Abstract

A method of controlling mosquitoes, the method comprising contacting the mosquitos with a pesticidally effective amount of a composition comprising eremophilone (EM1), 8 hydroxy-1 1 -erenophilen-9-one (EM2) and 9-hydroxy-7(11),9-eremophiladiene-8-one (EM3,).

Description

PEST CONTROL

The present disclosure relates to compositions and methods for the control of mosquitos.

BACKGROUND

The importance of mosquito control and management is recognised worldwide. Mosquitos are controlled at the individual and public level. In some countries and areas, mosquito bites are mostly a nuisance. However, in other areas, mosquitos are responsible for significant health problems. Mosquito borne diseases are responsible for over one million deaths worldwide. Mosquito borne diseases include malaria, dengue, yellow fever, St. Louis encephalitis, West Nile virus, Ross River fever, Murray Valley encephalitis and LaCrosse encephalitis. Mosquitoes also transmit diseases and parasites to dogs and horses.

Personal mosquito repellents are widely used. The most effective and long lasting mosquito repellent is a synthetic chemical, N,N-Diethyl-m-toluamide, commonly known as DEET. DEET is believed to work by blocking the chemical receptors for carbon dioxide and lactic acid that are mosquito attractants. DEET does not kill mosquitos.

However, there are some health concerns with application of DEET to the skin and there is a desire in the industry to provide an alternative that may have similar efficacy and duration of action to DEET.

To this end, there has been significant interest in finding a plant derived repellent with low mammalian toxicity. Thousands of plant-derived essential oils have been tested as potential repellents. However, none show the effectiveness or duration of DEET.

There is clearly both an economic and health benefit to providing such an alternative. Despite such a desire in the industry and active research to provide an alternative, the present inventor is unaware of any product that can so provide.

Mosquitos are also controlled by spraying. Spraying may be done on a personal level using a hand held spray or on a public health level in the form of aerial or truck based spraying.

Aerial spraying is considered an important public health measure in for the control of mosquito borne diseases in some areas.

Aerial spraying targets either the larvae (larviciding) or the adults (aldulticiding). Larviciding uses agents that specifically target mosquito larvae. Methoprene is an insect growth regulator that stops the larvae from becoming adults and has little impact on the environment. Larvicides are applied to bodies of water harbouring the larvae. However, since larvae do not usually occupy the entire body of water, larvicides are applied where the larvae are, usually the areas near the shoreline of the lake, stream or ditch.

Adulticides are used to rapidly reduce infected mosquito numbers or to control pest and nuisance mosquitoes from inaccessible breeding areas. Alduticides are generally of the synthetic pyrethroid family. The pyrethroids have low mammalian and avian toxicity. They are however toxic to bees and fish. For this reason they are sprayed at dawn, dusk or night when there is low bee activity and are generally not sprayed on water ways.

There is therefore a recognised need for a more environmentally acceptable mosquito adulticide that has a similar efficacy for contact killing of mosquitoes as the pyrethroids.

SUMMARY

According to a first aspect of the disclosure there is provided a method of controlling mosquitoes, the method comprising contacting the mosquitos with a pesticidally effective amount of a composition comprising eremophilone (EM1), 8-hydroxy-11-eremophilen-9-one (EM2) and 9-hydroxy-7(11),9-eremophiladiene-8-one (EM3).

Mosquito” is understood to refer to any specie of the 3,500 species of the insect that is commonly associated with and given the common name “mosquito.” Mosquitoes span 41 insect genera, including the non-limiting examples of Aedes, Culex, Anopheles (carrier of malaria), Coquillettidia, and Ochlerotatus. In embodiments described herein, a mosquito can refer to an adult mosquito or a larval mosquito, or both. Thus, some embodiments encompass methods or compositions wherein the insecticidal activity is as a mosquito “adulticide” or alternatively a mosquito “larvicide.”

The disclosed method may be active on mosquito species, such as, for example, Aedes aegypti, Aedes albopictus, Anopheles quadrimaculatus, Anopheles earlei, Anopheles punctipennis, Anopheles walkeri, Ciilex pipiens, Culex quinquefasciatus, Culex resluans, Culex salinarius, Aedes cinereus, Aedes vexans, Ochlerotatus japonicus, Ochlerotatus abserratus, Ochlerotatus atropalpus, Ochlerotatus decticus, Ochlerotatus implicatus, Ochlerotatus intrudens, Ochlerotatus sollicitans, and Ochleortatus excrucians

The present disclosure is particularly directed towards a method for controlling Aedes aegypti as this is the species responsible for the spread of most mosquito borne diseases including dengue.

Dengue is the most common vector-borne viral disease in the world, causing an estimated 50-100 million infections and 25,000 deaths each year. Dengue is carried by Aedes aegypti, the same mosquito that transmits yellow fever and chikungunya. The viruses of dengue fever include serotypes 1, 2, 3, and 4. Recovery from an infection of dengue-1 provides lifelong immunity to that serotype, but does not provide immunity to serotypes 2, 3 or 4. In fact, if a person has had dengue -1 and later contracts a dengue -2, -3, or -4 infection it likely will be more serious and may result in the more severe hemorrhagic form which can be deadly. There is presently is no vaccine available for the treatment of dengue. Aside from personal protective measures, the most effective preventive action that can be taken is to eliminate the mosquito vector that causes the disease.

The composition may also be dispersed on a household or domestic basis such as known pesticide spray method such as hand held aerosol sprays that may dispense the composition into the environment. Other types of automated sprays that can be programmed to periodically and disperse compositors into the environment are also suitable.

In some aspects, the composition can be formulated for application or delivery as an aerosol or a fog wherein the composition allows for the formation of droplets having an average diameter of about 1 pm to about 30 pm. Suitable compositions for such a formulation typically should have a viscosity that allows for the composition to atomize, but not be so thick as to clog a spray nozzle. Such viscosities can vary and be readily determined by one of skill in the art.

The disclosed method can comprise any known route, apparatus, and/or mechanism for the delivery or application of the compositions and formulations thereof.

The composition may also be dispersed or dissolved in an environmentally acceptable carrier. Suitably the composition is dispersed in water for application as an aerosol. The concentration of the composition in the carrier may be varied depending upon the method of dispersal, mosquito population and the like. Suitable concentrations may be determined by those of skill in the art.

Suitably the composition is dispersed in water at a concentration of between 0.1% to 10%, suitably 0.2% - 5%, suitably 0.5% - 4%, suitably 0.5% - 2%.

Adult mosquito control is often conducted from the ground via truck-mounted spray systems, and from the air via helicopters and fixed-wing aircraft. Adulticides can be applied only when meteorological conditions are exactly right. In particular, if the wind speed is too high the spray will move out of the target area too rapidly, or if too low, effective dispersal of the spray will not be achieved. Spray trucks are deployed in the evening usually starting one half hour after sunset, to catch the mosquitoes as they begin their evening flight activity.

Aircraft are generally deployed in the early morning, when temperature inversion conditions ensure the tiny spray droplets will reach the ground in order to contact mosquitoes as they finish their nightly flight activities. In both cases, ultra low volume (ULV) spray technology is used. This allows a very small amount of adulticide, amounting to fractions of an ounce per hectare, to be used.

Control of adult mosquitoes is generally necessary when mosquito populations cannot be treated in their larval stage. Use of mosquito aldulticides is an integral part of a modern, integrated mosquito management program. It also is the most effective way to eliminate adult female mosquitoes that are infected with human pathogens.

When mosquitoes are concentrated in small areas, truck- mounted ULV spray systems may be used. Ground treatments may be done in the evenings beginning approximately one hour before sunset. Trucks may be dispatched to those areas with high numbers of biting mosquitoes, allowing area-specific treatments to control problem populations. Wind can play an important role during spray missions because of the minute size of the insecticide droplets. If the wind is too great, the insecticide will not be distributed properly throughout the air column and will be blown away from the target area. Therefore, spray missions will be conducted during times when wind conditions are optimal, usually between 1 to 15kph.

In another embodiment, the disclosed compositions can be impregnated into for example, fabrics, tents and nettings. The compositions may be dispersed in a volatile solvent such as alcohol to which the fabrics are treated.

Also disclosed in another aspect is a method of repelling mosquitos that comprises applying to the surface of a mammal a pest repelling effective amount of a composition comprising eremophilone (EM1), 8-hydroxy-11-eremophilen-9-one (EM2) and 9-hydroxy-7(11),9-eremophiladiene-8-one (EM3,) and a continuous phase pharmaceutically acceptable carrier.

By continuous phase carrier is meant that the carrier is a single phase and is not a water in oil or oil in water emulsion.

The single phase may be a mixture of components provided the components are miscible.

The composition may be dispersed in water, or water and alcohol, for pump spray applications.

Alternatively, the composition may be dispersed in a suitable carrier oil. Carrier oils for essential oils are suitable, Carrier oils are generally cold-pressed or macerated vegetable oils and include Apricot oil, Sweet almond oil, Grape seed oil, Avocado oil, Olive oil, Sesame oil, Evening primrose, Canola (Rapeseed), Sunflower oil, Jojoba oil, Emu oil, Castor oil, Borage seed oil, Walnut oil, Peanut oil, Pecan oil, Macadamia oil, coconut oil, Hazelnut oil and Cocoa Butter.

The concentration of the composition may vary according to the desired duration of repellency. Stronger concentrations will provide for longer periods of protection.

Suitable concentrations may be between about 0.1 wt% to about 50wt%, 0.5wt% to about 40wt%, about 1wt% to about 30wt%, about 2wt% to about 25wt%, about 3wt% to about 20wt%.

The composition may also be formulated for aerosol delivery.

Essential oils are complex mixtures of large numbers of compounds. Many oils have broad spectrum antimicrobial and therapeutic properties. It is believed that such properties are the result of the complex mixtures and synergistic combinations. In view of the complexity of the mixtures and it has not yet been possible to identify the complex interactions that occur between the compounds in essential oils.

The present inventor has made the surprising and unexpected discovery that when essential oils are prepared in an emulsion form, the efficacy of the oil is severely compromised. Whilst not wishing to be bound by theory, it is believed that when a complex mixture of components such as found in essential oils, the polar and nonpolar compounds fractionate into the respective polar and nonpolar phases of an emulsion that this adversely affects the activity of the composition.

The composition may be formulated to include additives such as atural antioxidants, which can be used advantageously to reduce the effect of oxidation of the compounds of the composition. An example of a suitable naturally-occurring antioxidant is a-tocopherol.

Other additives, such as naturally-occurring stabilisers, are also contemplated, which may desirably be added to improve the stability and shelf life of the composition. Examples of suitable natural stabilisers include gum arabic, guar gum, sodium caseinate, polyvinyl alcohol, locust bean gum, xanthan gum, kelgum, and mixtures thereof. In an alternate embodiment, the naturally-occurring compounds derived from a volatile oil may be modified or derivatised to improve, for instance, their shelf-life, stability, activity and/or bioavailability.

The disclosed compositions may be used singularly or in combination with other mosquito controlling compounds of the invention. By "controlling" is meant preventing, combating, eradicating, destroying, repelling, or mitigating mosquitos or increasing the mortality or inhibiting the growth and/or development of mosquitos.

By "effective amount" is meant the administration or application of that amount of active compound, either in a single dose or as part of a series, that is effective for controlling a significant number of mosquitos. Thus, for example, a "pesticidally-effective" amount is the amount of the composition that is effective for increasing the mortality a significant number of mosquitos.

Alternatively, a "pest-repelling" effective amount is the amount of composition that is noxious to, and/or induces behavioral changes in, a significant number of mosquitos. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

The present inventor has surprisingly and unexpectedly discovered that the disclosed formulations have both mosquito killing and repellence properties. This is unexpected as the mechanisms for repellence and killing are completely different. For example DEET, the gold standard for mosquito repellence does not kill mosquitos but blocks the chemical receptors that detect carbon dioxide and lactic acid. The insecticidal properties of pyrethrins are derived from ketoalcoholic esters of chrysanthemic and pyrethroic acids. These acids are strongly lipophilic and rapidly penetrate many insects and paralyse their nervous system.

Eremophilone is a terpenoid natural product isolated from Eremophila oil, which is an essential oil obtained from the trees of the genus Eremophila in the Myoporaceae family. Eremophilone was first isolated from E. mitchellii in 1932 (Bradfield et al, J. Chem. Soc, 1932) along with other oxygenated derivatives reported six years later (Bradfield et al, 1938). A number of synthetic methods for preparing eremophilone are known. McMurray et al (McMurray, J.E., Musser, J.H., Ahmad, M.S. and Blaszczak, L.C. (1975) J. Org. Chem., 40 (12), 1829-1832) prepared eremophilone from β-pinene as outlined in Scheme 1.

eremophilone Scheme 1

Ziegler et al (Ziegler, F.E., Reid, G.R., Studt, W.L. and Wender, P.A. (1977), J. Org. Chem. 42(11) 1991 -200) prepared eremophilone by an alternative synthesis from a cyclohexanone compound as outlined in Scheme 2.

eremophilone

Scheme 2

Eremophilone may also be prepared from a cyclohexenone compound as outlined in Scheme 3.

The compounds that are present in the disclosed composition are in particular found naturally in the steam distilled wood oil from Eremophilla mitchelli. Eremophila mitchelli, known commonly as false sandalwood, is a shrub or small tree native to Australia. The trees are wild harvested and the timber is steam distilled for around 48 hours. The essential oil is naturally amber red to reddish brown colour. The oil is commercially used in aromatherapy for grounding or assisting in meditation.

Eremophilla mitchelli essential oil is known to have antifeedant activity against termites in which it disrupts the gut microflora so that the termites starve to death. Such activity is completely irrelevant for mosquito control. Still further, E. mitchelli essential oil has been reported to be virtually non-toxic as a fly spray.

The oil contains the compounds EM1, EM2 and EM3 as the major components in about 50% to 90% (as measured by GC analysis). These compounds are eremophilane sesquiterpenes whose structures are illustrated below.

The oil typically contains 20-60% EM1, 11-30% EM2 and 6-25% EM3. E. mitchelli wood oil also contains small amounts (ca less than about 5-10%) of the further ermophilane sesquiterpenes 9-hydroxy-1,7(11),9-eremophilatrien-8-one EM4, 8-hydroxy -1,11-eremophiladien-9-one (EM5) and 8-hydroxy-1,11-eremophiladien -9-ne (EM6).

The balance of the oil is made up of a-selinene, β-selinene (about 1 to 3%) and other sesquiterpenes and oxygenated derivatives thereof.

The disclosed composition in the neat form, i.e. without a diluent or carrier suitably comprises between about 15% to about 70%, suitably between about 30% to about 60%, suitably between about 40 to about 50% EM1. The disclosed composition may comprise between about 5% to about 50%, suitably between about 8% to about 40%, suitably between about 8 to about 30% EM2. The disclosed composition may comprise between 2% to about 40%, suitably between about 5% to about 60%, suitably between about 5 to about 25% EM3.

The amount expressed in % is as measured by gas chromatographic analysis. Unless otherwise indicated in the present specification and claims % will refer to the amount as determined by GC analysis. GC analysis is standard in the art of essential oils.

Eremophila essential oil has passed mammalian and environmentally toxicology testing prior to its registration in Australia as a temiticide.

DETAILED DESCRIPTION

Example 1

Evaluation of the efficacy of the disclosed formulation against mosquitoes using a direct spray method.

Test Organism - Adult female Aedes aegypti mosquitoes, 3-8 days old.

Test Chamber - A 6 x 6 x 6 foot (216 ft3) (0.3 x 0.3 x 0.3 meters (20 meters3) stainless steel Peet Grady Chamber equipped with fluorescent lights, a 17 x 20 inch (43 x 51 cm) viewing window and an exhaust system that provides 50 air changes per hour.

Tests Cages - Cages (cardboard cylinders with open ends covered with cloth screen). The cages have a diameter of 3.5 (8.9 cm) inches and a height of 2.25 inches (5.7 cm). The screen mesh size is 24 openings/inch (24 openings /2.5cm).

Delivery - The CSMA (US Chemical Specialties Manufacturers Association) accepted pesticide atomizer (DeVilbis Atomozier No. 5004), air compressor delivering contaminant free air at 12.5 psi.

Preparation of chamber - The Peet Grady chamber was washed (interior surfaces only) with a trisodium phosphate water solution prior to the test. The wind tunnel stand was placed in the centre of the chamber.

Handling of mosquitos - Ten mosquitoes were aspirated ad released in each cage. The mosquitos were provided sucrose until just prior to their release. The mosquitos were allowed to acclimate for at least 20 minutes prior to treatment.

Replication - There were five replicates with ten mosquitos per replicate (cage) for each formulation tested.

Treatment - The test formulations were evaluated at 6ml per replicate. The test formulations were dispensed with a CSMA accepted DeVilbis atomizer. Horizontal spray distance was used, typically 1 to 3 feet.

The atomizer was operated at a constant pressure of 12.5psi. The atomizer delivered formulations at a rate of 6 ml per 12 seconds. The atomizer remained activated until emission ceased. After spraying, the atomizer and the vial were washed with a suitable solvent and sufficient solvent was sprayed through the atomizer to remove any remaining formulation.

The mosquitos remained in the cages. Sucrose-soaked cotton balls were placed on the screen of the cages to supply food. The cages were maintained in the laboratory until the 24 hour mortality observation.

Recording data - After treatment, the mosquitos were observed for knockdown at 5,15, 30 and 60 minutes. At 24 hours the number of live, moribund and dead mosquitos were counted.

Controls - There were five replicates of 10 mosquitos as untreated controls. These replicates were treated in the same manner as the test replicates, except they were not sprayed.

Data analysis - the average knockdown and mortality resulting from direct application was calculated after adjusting for control mortality with Abbott’s formula.

Formulations - 271 - 272 - 0.8% E. mitchelli wood oil in water 273-

Results

The control is pyrethrums for which the average 24 mortality under this protocol is 90%. Formulation 272 is therefore effective for the control of mosquitos.

Example 2

Evaluation of the reoellencv of the disclosed formulation against mosquitos Materials

Exposure container - a 30.5 cm x 30.5 cm x 30.5 cm cage (rigid plastics frame and screen panels and a sleeved entry on one side and a sliding trap door on the bottom). The cage is supported by four (4cm high) legs.

Membrane Feeder - five wells (3cm in diameter x 8 mm in depth) in line on a hollow plastic block (6cm wide x 22 cm long x 3 cm deep). The plastic block fits into the bottom of the treatment cage. Hoses mounted on either end of the membrane feeder carry water at blood temperature which is from a water bath. The sliding plastic trap door I the bottom of the screened cage covers and uncovers the wells in the membrane feeder, allowing mosquitos to access to the membrane-covered, blood filled wells.

Membrane - Baudruche membrane

Test Organisms - approximately 250 female mosquitos, Aedes aegypti, per replicate (5 replicates).

Methods

Five replicates of 250 adult female mosquitos were placed in a screened cage. The cage was placed in such a way that mosquitos had access to five warmed, blood-filled, membrane-covered wells. One membrane was treated with a 1% DEET solution (positive control), one was treated with isopropyl alcohol (negative control) and the other three wells were treated with the provided test formulation. There were five replicates with a positional rotation of the repellent at each replicate. Each test formulation was tested on each of the five wells.

Water was added to a water bath to warm the membranes to skin temperature (37eC). The sliding door on the bottom of the cage was closed and approximately 250 female mosquitos were added to the cage. Hoses were connected from the water bath to a circulating pump and to the membrane feeder. A thin film of vacuum grease was placed around the top edges of the wells to hold the membranes in place. 72mg of ATP (disodium salt) was added to 26 ml of bovine blood. Sodium citrate was added to the blood to prevent clotting. The blood completely filled each well.

Circular pieces (3cm in diameter) of Baudruche membrane were cut and placed over each of the wells being tested. Care was taken to press the membranes down into the vacuum grease and to eliminate all air bubbles from between the membrane and the surface of the blood. The circulating pump was turned on, allowing the blood to be warmed until the temperature reached 37 +/-2eC. Temperature was recorded using the IP surface thermometer. The sections of membrane were treated by pipetting 25 μΙ of the test formulation, 1% DEET or IPA onto the appropriate membrane and spreading it evenly with the tip of the pipette.

Exposure to mosquitos

The membranes were allowed to dry for approximately five minutes and then the cage was placed on the membrane feeder. The trap door in the floor of the cage was slid back, exposing the mosquitos to the membranes below. The number of mosquitos probing the membrane covering each well was recorded every 2 minutes for twenty minutes.

The above procedures were repeated for five times with five positional rotations of the repellent. Thus, the repellent, negative and positive controls were tested in each of the five well positions so as to cancel any positional bias. Each replicate was run with a new group of mosquitos and newly treated membrane sections.

Data analysis

The average number of probes recorded for each variable were calculated. The percentage repellency was calculated using these totals with the following formula:

Where C = the total number of probes on the untreated control well for the 10 observations and T = the total number of probes on the treated well for the 10 observation.

The data was analysed by analysed by analysis of variance to determine the significance of any differences between the numbers of probes received by each membrane treatment.

Formulations

Sample 232 - 1 % EM in soy oil Samples 233 - 2% EM in soy oil Sample 234 - 3% EM in soy oil

Results

Some treatments had replicates where no probes occurred within the twenty minutes observation time. For the purposes of calculating the mean, these were counted as 20 minutes.

The results show that samples 232, 233, and 234 were all effective against Aedes aegypti mosquitos. Examples 233 and 234 provided complete repellency and 232 provided nearly complete (99.8%) repellency. All three test samples were more effective than 1% DEET. In addition, the average time to first probe was greater and mosquitos probed less on the membranes that were treated with test samples than those treated with 1% DEET.

In vivo human studies

Subjects will place treated forearms into a cage 92 x 2 x 2x ft (60 cm x 60 cm x 60 cm) of 25 - >1500 starved, even age (female) mosquitos or (mixed sex) stable flies for 5 minutes every half hour for up to 8 hours. Protection time is based on time until 2 bites are received within half an hour on each treated arm.

It is expected that the formulations of the present invention will provide similar repellency to the gold standard DEET for at least 2-3 hours.

It will be appreciated that various changes and modifications may be made to the invention as disclosed and claimed herein without departing from the spirit and scope thereof.

Claims (13)

1. A method of controlling mosquitoes, the method comprising contacting the mosquitos with a pesticidally effective amount of a composition comprising eremophilone (EM1), 8-hydroxy-11-eremophilen-9-one (EM2) and 9-hydroxy-7(11),9-eremophiladiene-8-one (EM3,).

2. The method of claim 1, wherein the composition without a diluent or carrier comprises between about 15% to about 70%, EM1, between about 5% to about 50%, EM2 and between 2% to about 40% EM3.

3. The method of claim 1, wherein the composition without a diluent or carrier comprises between about 30% to about 60% EM1, between about 8% to about 40% EM2 and between about 5% to about 60% EM3.

4. The method of claim 1, wherein the composition without a diluent or carrier comprises between about 40 to about 50% EM1 between about 8 to about 30% EM2 and between about 5 to about 25% EM3.

5. The method of claim 1, wherein the composition is the wood oil from Eremophila mitchelli.

6. The method of any one of claims 1 to 5, wherein the composition is dispersed in water and formulated for spray application.

7. A method of repelling mosquitos that comprises applying to the surface of a mammal a pest repelling effective amount of a composition comprising eremophilone (EM1), 8-hydroxy-11-eremophilen-9-one (EM2) and 9-hydroxy-7(11),9-eremophiladiene-8-one (EM3,) and a continuous phase pharmaceutically acceptable carrier.

8. The method of claim 7, wherein the composition without a diluent or carrier comprises between about 15% to about 70%, EM1, between about 5% to about 50%, EM2 and between 2% to about 40% EM3.

9. The method of claim 8, wherein the composition without a diluent or carrier comprises between about 30% to about 60% EM1, between about 8% to about 40% EM2 and between about 5% to about 60% EM3.

10. The method of claim 8, wherein the composition without a diluent or carrier comprises between about 40 to about 50% EM1 between about 8 to about 30% EM2 and between about 5 to about 25% EM3.

11. The method of claim 8, wherein the composition is the wood oil from Eremophila mitchelli.

12. The method of any one of claims 8 to 11, wherein the composition is dispersed in water and formulated for spray application.

13. The method of any one of claims 8 to 11, wherein the composition is dispersed in a carrier oil.