AU673930B2 - Synthetic blowfly attractant - Google Patents

Description

WO 94/27441 PCT/A U94/00268 1

TITLE

"SYNTHETIC BLOWFLY ATTRACTANT" FIELD OF INVENTION THIS INVENTION relates to a synthetic blowfly attractant.

BACKGROUND ART Blowflies of the genus Lucilia and in particular the Australian sheep blowfly Lucilia cuprina cause flystrike in sheep which causes heavy financial costs to sheep owners as well as terrible suffering to infected animals. Protective methods at the present time include surgical operations on unanaesthetised animals such as "Mules' operation" and "pizzle dropping" which are relatively expensive and disliked by graziers and are campaigned against by animal welfare groups. Other protective methods depend upon application of chemicals to sheep which is undesirable because of the effect of the chemicals on human health, environmental problems and problems caused by insecticide residues in meat and wool. Also chemicals often select for resistance in blowflies.

In their Final Reports (Australian Government Printer: Canberra November 1991) the Australian Government's "Ecologically Sustainable Development Group on Agriculture" in Section 14.6 recommends the development of approaches for minimising chemical use in agriculture. Thus there is a need to develop sustainable systems for sheep blowfly management and a need to avoid the use of toxic pesticides because of the reasons referred to above, as well as the effect of the pesticides on non-target organisms.

The incidence of sheep flystrike is directly linked to fly population density as described in Wardhaugh and Morton Aust. J. Agric. Res. 41 1155-1167 in 1990. Their study has shown that at densities of near or below a standardised catch rate of one gravid female blowfly per standard trap per hour, the rate WO 94/27441 PCTIAU94/00268 2 and extent of sheep infestation would not generally be perceived as a major problem by graziers. Thus, suppression of female numbers is a prime management objective.

It has been demonstrated that fly population suppression can be achieved with baiting or trapping e.g. for L. cuprina as described by Mackerras et al.

J. Coun. Sci. Ind. Res. Aust. 9 153-162 in 1936 and also by Anderson et al. Aust. Vet. J. 67 93-97 in 1990. Laveissiere et al. in 1990 in a publication entitled "Appropriate Technology in Vector Control" pp 47-74 CRC Press, Boca Raton also came to the same conclusion in relation to tsetse flies. Foster et al.

in an International Symposium on Management of Insect Pests in 1992 under the aegis of the International Atomic Energy Agency, FAO of the UN, Vienna, in a reference entitled "Advances in Sheep Blowfly Genetic Control in Australia" have calculated that an efficient trapping system for Lucilia blowflies would reduce by 57 to 70% the time required for eradication of Lucilia with genetic control.

The attractants used in the existing systems as described in the abovementioned Mackerras and Anderson references and also by Dadour and Cook in J. Aust.

ent. Soc. 31 205-208 in 1992 have the limitations of being based on organic materials e.g. aqueous liver suspensions and various bacteria. They have intrinsic problems, such as the attractiveness of a food type product to vertebrates and the health risk presented by bacterial cultures. These attractants require frequent servicing to overcome problems such as drying out and skin formation. An additional major drawback is their attractiveness for non-Lucilia species of blowflies and for other insects, including beneficial insects, which is considered environmentally undesirable in the abovementioned Dadour and Cook reference.

WO 94/274141 I'CT/AU94/00268 3 Investigations on the improvement of organic attractants or the use of pure chemical attractants have been undertaken for Lucilla in, for example: (i) Freeney Pamph. Coun. Scient. Ind. Res. Aust. 74 1-25 in 1937, (ii) Cragg and Ramage Parasitology 36 168- 175 in 1945, (iii) Eisemann in a PhD thesis on L.

cuprina at The University of Queensland (iv) Mulla in US Patent 3996349, Mulla et al. in J. Chem.

Ecol. 70 644-648 in 1977, (vi) Pickens et al. J. Med.

Entomol. 10 84-88 in 1973 and (vii) Warner US Patent 5008107.

Freeney in reference investigated the use of fatty acids, amines, sulfides and sodium sulfide for Lucilia species. Cragg and Ramage in reference (ii) tested the use of sheep wool as an attractant to which indole, ammonium carbonate and hydrogen sulfide had been added. Pickens in reference (vi) formulated a dry fly bait containing sucrose, sodium bicarbonate, yeast, dried blood, amyl acetate and sodium lactate which became active upon addition of water. The Mulla references (iv) and developed a synthetic attractant for synanthropic flies consisting of triethylamine, an ammonium salt. linoleic acid and indole. This formulation also includes a 2-7 C hydrocarbon carboxylic acid for pH adjustment. Warner in reference (vii) utilised Mulla's formulation plus cis-9-tricosene. Eisemann in reference (iii) evaluated the response of L. cuprina towards a wide range of pure chemicals such as carboxylic acids, alcohols, thiols, amines, sulfides, phenols, hydrogen sulfide and indoles and various combinations thereof.

Some of Eisemann's binary and tertiary mixtures showed good attractancy for L. cuprina, but did not perform better than a standard liver/sodium sulfide bait.

However, none of Eisemann's mixtures used a combination of components (iii) and (iv) in relation to the present invention as hereinafter WO 94/27441 PCT/AU94/00268 4 described.

In regard to the importance of development of traps for L. cuprina, it must be borne in mind the cost of prevention and production losses from blowfly strike in Australia in regard to sheep average $150 million per annum as shown in Beck Q. Rev. Rural Econ.

7 336-343 in 1985. Blowfly strike is also a problem in New Zealand as shown in Dymock et al. in NZ Journal of Agric. Res. 34 311-316 in 1991 and also in Great Britain as reported in Wall et al Bull. Entomol. Res.

82 125-131 in 1992 and also iii South Africa as shown in Zumpt (1965) in a publication entitled "Myiasis in Man and Animals in the Old World" Butterworth, London.

The Australian sheep blowfly L. cuprina initiates greater than 85% of sheep blowfly strike in Australia as shown in Anderson et al. Aust. J. Zool. 36 241-249 in 1988. Lucilia blowflies also cause problems around Australian suburbs as shown by Logan in a 1991 publication entitled "The Association of Blowflies with Wheelie-Bins in Darwin" published by the NT Department of Health and Community Services in Darwin.

Lucilia blowflies also cause problems in American suburbs as discussed in Rice Ento. Soc. Qld. News Bull. 14 29-36 in 1986. Lucilia blowflies have also been implicated in strikes on hospital patients as discussed in Lukin Med. J. Aust. 150, 237-240 in 1989.

Fly attractant compositions are also described in US Patents 4,947,578; 4,959,209; 4,801,448; 4,911,906 and 4,638,592 as well as Japanese Specifications JP 48058126 and 76005448.

Specification EP 582915 describes an attractant for insects and especially Musca domestica which contains butyric or butanoic acid in synergistic combination with cis-9-tricosene which is a sex pheromone.

WO 94/27441 PCT/AU94/00268 SUMMARY OF THE INVENTION It is therefore an object of the invention to provide a blowfly attractant lure formulation which is efficient in attracting blowflies which include blowflies of the genus Lucilia and which alleviates problems of prior art attractant lures as discussed above.

The attractant lure formulation of the invention includes: one or more straight chain and/or branched chain C 2

-C

8 carboxylic acids optionally containing hydroxyl, amino and/or thiol substituents; (ii) one or more unsubstituted or substituted straight or branched chain thiols including cycloaliphatic thiols having an upper limit of 12 carbons where the substituent(s) may be hydroxyl, amino including substituted amino such as NHR (where R is aliphatic), or additional thiol groups; (iii) one or more indoles or substituted indoles wherein there is a alkyl substituent at the 3 position or their analogues having the N replaced by S or the carbons at the 2 and/or 3 position replaced by N or S respectively; and (iv) hydrogen sulfide produced for exarmple from an aqueous sulfide salt.

Each of the abovementioned components may be provided in a fly trap as a mixture with the exception of the sulfide salt solution which must remain separate. Alternatively the components may be provided as separate components in the trap.

Alternatively component may be combined with component (ii) or component (iii) or in another situation components (ii) and (iii) may be combined.

However, an insect trap is only one option for effecting capture or otherwise processing attracted flies in association with the attractant lure formulation of the invention. Other options include toxicants, chemosterilants, growth regulators, pathogens, or electrified targets. These options may WO 94/27441 PCT/AU94100268 6 be used either inside or outside an insect trap depending on contact requirements such as contact time or ingestion for these options to be effective.

An example of component is butanoic acid; an example of component (ii) is 2-mercaptoethanol; an example of component (iii) is indole; and an example of component (iv) is sodium sulfide.

Other chemicals may be included in the four component lure formulation of the invention and these include straight or branched chain C 2

-C

8 alcohols; dialkyl sulfides and/or dialkyl disulfides; phenols and/or substituted phenols; straight chain and/or branched chain C 4 -CIo aldehydes and straight chain or branched chain C 3

-C

8 ketones.

Preferred ranges of proportions of each of components to (iii) are as follows in relation to forming a mixture 10% to 80% of component 10% to 80% of component (ii); 2.5 to 20% of component (iii); whereby in each case the components (ii) and (iii) add up to 100% or 100g of mixture on a weight/weight basis wherein each of the components are expressed in grams.

The preferred proportions of the abovementioned compDnents (ii) and (iii) is as follows: 40-60% of component and more preferably 49%, 30-50% of component (ii) more preferably 42%, and 5-15% of component (iii) and more preferably 9%.

It will be appreciated that the above concentrations of components (ii) and (iii) are calculated on a dry weight basis without taking into consideration the amount of solvent that may be used.

The solvent used may be any appropriate biologically or chemically inert solvent. Examples of suitable solvents are water, glycerol, ethylene glycol, propylene glycol or other suitable solvent.

WO 94/27441 PCTIAU94/00268 7 In relation to component (iv) any sulfide salt that emits H 2 S preferably in the presence of water would be effective. These include calcium sulfide, magnesium sulfide, potassium sulfide, and sodium sulfide.

An effective release of hydrogen sulfide is achieved by means of a 5-50% by weight solution of sodium sulfide in water.

When using technical grade sodium sulfide which has 68% sodium sulfide, suitably 10-25% by weight of technical grade sodium sulfide in water is utilized.

However when using analytical grade sodium sulfide or hydrated sodium sulfide suitably 25-50% by weight of hydrated sodium sulfide in water is utilized.

The above solutions release hydrogen sulfide for example from a wick in a bottle. Hydrogen sulfide released from such a wick into an air stream above the bottle has been measured at approximately 1 ppm.

However more broadly a range of sulfide solutions in water could be from a minimum of 0.5% up to very concentrated solutions i.e. saturated or supersaturated solutions.

However, it will be appreciated that there are other methods for releasing attractant mixtures or components into the atmosphere than the use of a wick.

These include open bottles, solid supports soaked with attractant, microencapsulation or sachet dispensers.

The release rate of attractant has a great effect on the number of flies caught whereby the greater the release rate will mean a higher catch of flies. In a commercial trap the release rate of mixture (ii) and (iii) is approximately 500 mg per day. But this can be varied over wide limits i.e. a maximum release rate may be several grams per day and a minimum release rate may be of the order of a few mg per day.

IWO 94/27441 PCT/AU94/00268 8 BRIEF DESCRIPTION OF DRAWINGS FIG 1 illustrates the synergistic effect between butanoic acid (component and the other attractant components (iii) and (iv) on the attractiveness for L. cuprina. The number of L. cuprina caught by the attractant including butanoic acid is 2.7 x higher than the sum of the flies caught by butanoic acid alone and caught by the attractant mixture without the butanoic acid.

FIG 2 refers to a graph of an experiment .described hereinafter concerning capture of L. cuprina in traps incorporating the attractant lure formulation of the invention.

DETAILED DESCRIPTION In order to enable the invention to be fully understood, a number of preferred embodiments will now be described which are contained in the following Examples.

EXAMPLE 1 500 ml of an aqueous synthetic attractant mixture containing 5% 2-mercaptoethanol, 5% butanoic acid, pentanoic acid, 0.5% indole 64% cf component (i) 33% of component (ii) and 3% of component (iii)] and a separate 20% sodium sulfide solution in water was tested for five days in an insectary against a standard blowfly attractant (500 gm of bovine liver and 500 ml of a 1.5% solution of sodium sulfide). The synthetic attractant attracted significantly more flies (3.3 x more with P<0.05).

EXAMPLE 2 A synthetic attractant mixture containing 0.5% 2mercaptoethanol, 0.1% butanoic acid, 0.05% indole 15% component 78% of component (ii) and 7% of component (iii)] and 0.2% separate sodium sulfide solution was tested over 7 days in an insectary and attracted 24 x as many flies as 500 ml of a commercial "Efekto" bait.

WO 94/27441 PCT/AU94/00268 9 EXAMPLE 3 In sheep paddocks at Cunnamulla using a 6 x 6 Latin square design over 6 days in October 1991 a synthetic attractant containing a mixture of 8 gm 2mercaptoethanol, 2 gm butanoic acid, 1 gm inc-±e [i.e.

18% of component 72% of component (ii) and 10% of component (iii)] and a separate 10 ml saturated sodium sulfide solution caught 2.7 x as many wild Lucilia cuprina as a standard blowfly attractant (10 gm of bovine liver and 10 ml of a 1.5% sodium sulfide solution). The synthetic attractant was ai more highly selective for Lucilia because it c:iuht oxer 8 x less Chrysoma and much less Calliphora -acilia, compared to the liver/sodium sulfide standard.

EXAMPLE 4 The same attractants as in 3. were compared in suburban Brisbane, Australia, in March 1992 by a duplicated 4 x 4 Latin square over 4 days. The synthetic attractant caught 8.4 x as many Lucilia cuprina as the standard liver attractant. It also caught 6.8 x less Chrysomya and 8.6 x less Calliphora than Lucilia, when compared with the liver/sodium sulfide standard.

EXAMPLE The value of including all four different types of chemicals in the synthetic attractant was demonstrated in an automated insectary by pairwise comparisons of the lure mixtures described in Table 1 herein.

EXAMPLE 6 Field tests at Charleville, Australia, in October 1990 using 4 x 4 Latin square replicated comparisons on variations of the base attractant (pentanoic acid, 2-mercaptoethanol, indole; sodium sulfide solution) showed that without sodium sulfide the catch was WO 94/27441 PCT/AU94/00268 reduced more than 6 times and that without pentanoic acid the catch was reduced more than 15 times.

EXAMPLE 7 In addition to the four major attractant chemicals exemplified above other chemicals have proved able to significantly (P<0.05) increase the catch of Lucilia cuprina in an insectary. A 31% increase over the base attractant catch was obtained by the inclusion of butan-2-ol and 2-methylpropane-1ol. An 11% increase was obtained by inclusion of cis- 3-octen-1-ol. A 26% increase was obtained by inclusion of hexanal, 2-octanone and benzaldehyde.

EXAMPLE 8 Urban tests in Brisbane, Australia in duplicated 4 x 4 Latin square comparisons showed that addition to the basic four component synthetic attractant (2mercaptoethanol, indole, butanoic acid, sodium sulfide) of phenol, 4-methylphenol, butan-2-ol, 2methylpropane-1-ol, dimethyl disulfide increased the catch of Lucilia cuprina 2.2 x. Addition to the basic attractant of butan-2-ol, 2-methylpropane-l-ol, pentanoic acid, acetic acid and acetone increased the catch of Lucilia cuprina 1.2 x.

EXAMPLE 9 Small scale field trials were carried out in western Queensland to test the hypothesis that placing traps with a synthetic attractant (2-mercaptoethanol, indole, butanoic acid, sodium sulfide) in a sheep WO 94/27441 PCT/AU94/00268 11 paddock would lower the Australian sheep blowfly population in that paddock. The trial was run with five replicates (Cunnamulla, Charleville, Roma, Longreach [2 each consisting of two paired sites, one as control (no traps) and the other containing traps. In each site five monitoring traps (opened for 24 hours every seven days) were used to measure the fly population. The mean numbers (from all five replicates) of sheep blowflies caught in the monitoring traps in the trapped and the control areas during the 15 week trial are shown in figure 2. The fly population was substantially lower in the trapped area, indicating population suppression by the presence of the synthetic attractant in a trap.

EXAMPLE The use of various formulations as described in Table 2 where also tested in an automated insectary with the results as shown. In an automated insectary the number of flies on a target ware monitored when presented with vapours containing the attractant lure formulation of the invention. This is equivalent to a response and a statistical analysis is carried out on a comparison between different attractant lure formulations.

The automated insectary was used above in Examples 1-2, 5 and 7 and is a part of or associated with apparatus entitled Behavioural Observation Facility for Flies (BOFF) and is well known to the WO 94/27441 PCT/AU94/00268 12 skilled addressee.

EXAMPLE 11 Further trials were carried out using various formulations in accordance with the invention on a 4x4 Latin Square and the results of these trials are reported in Table 3.

WO 94/27441 PCT/AU94/00268 13 TABLE 1 Composition Mean response (number of flies) Lure 1 Lure 2 Lure 1 Lure 2 2-mercaptoethanol 2-mercaptoethanol 38.9 100.2 indole indole sodium sulfide 2-mercaptoethanol 2-mercaptoethanol 11.1 33.0 indole indole pentanoic acid pentanoic acid sodium sulfide 2-mercaptoethanol 2-mercaptoethanol 48.3 57.5 indole indole sodium sulfide sodium sulfide pentanoic acid 2-mercaptoethanol 2-mercaptoethanol 47.5 96.3 butanoic acid butanoic acid sodium sulfide sodium sulfide indole Differences between treatments (P<0.05) in all cases.

were significant WO 94/27441 WO 9427441PCT/AU94/00268 14 TABLE 2 Automated insectary Attractant Response* ;Ratioo esponses 2-mercaptoethanol indole butanoic acid 9123A sodium sulfide 2-mercaptoethanol indole butanoic acid 8780 pentanoic acid sodium sulfide 2-mercaptoethanol indole butanoic acid 38123 A sodium sulfide 2-mercaptoethanol indole 3-methylbutanoic 25 65 7 .4 acid sodium sulfide 2-mercaptoethanol indole butanoic acid 99 sodium sulfide (02%) 1.39 2-mercaptoethanol indole hexanoic acid682 sodium sulfide 2-mercaptoethanol indole butanoic acid 68 sodium sulfide 1.58 2-mercaptoethanol indole thiolactic acid427 sodium sulfide Within one experiment, responses with the same significantly (P>0.05) superscript do not differ WO 94/27441 WO 94/27441 I A U94/0 0268 TABLE 3 4 x 4 Latin square, Brisbane, February 1992 Attractant, Mean number of L. ciprina per trap per day' 2-rnercaptoethanol indole butanoic acid sodium sulfide 2-mercaptoethanol indole butanoic acid propanoic acid pentanoic acid hexanoic acid octanoic acid sodium sulfide Responses with the same superscript do not differ significantly (P>0.05) WO 94/27441 PCT/AU94/00268 16 Legends Figure 1 Synergism with butanoic acid Brisbane field trial December 1992, 6x6 Latin square Attractants: 1. Butanoic acid (100%).

2. 2-Mercaptoethanol indole sodium sulfide solution.

3. 2-Mercaptoethanol indole butanoic acid sodium sulfide solution.

Figure 2 Mean Lucilia cuprina trap catches per trap per day in monitoring traps located in control and treated (trapped) areas during field trials in Western Queensland September-December 1992.

Claims (4)

1. An attractant lure formulation for insects including: one or more straight chain and/or branched chain C 2 -C 8 carboxylic acids optionally bearing hydroxyl, amino and/or thiol substituents; (ii) one or more unsubstituted or substituted straight or 'Thed chain thiols including cycloaliphatic t having an upper limit of 12 carbons where the ,ubstituent(s) may be hydroxyl, amino including substituted amino such as NHR (where R is aliphatic), or additional thiol groups; (iii) one or more indoles or C 3 alkyl (Ci-C 4 substituted indoles or their analogues having the five membered ring N or the C 2 and C 3 carbons of the five members ring replaced by N or S; and (iv) hydrogen sulfide produced for example from an aqueous sulfide salt.

2. An attractant lure formulation as claimed in claim 1 wherein the ranges of proportions of each of components to (iii) are as follows:

10-80% of component (i) 10-80% of component (ii) 2.5-20% of component (iii) whereby in each case the components (ii) and (iii) add up to 100% or 100g of mixture on a weight/weight basis wherein each of the components is expressed in grams. 3. An attractant lure formulation as claimed in claim 2 wherein there is included 40-60% of component WO 94/27441 PCT/AU94/00268 18

30-50% of component (ii) and 5-15% of component (iii). 4. An attractant lure formulation as claimed in claim 2 wherein in regard to component (iv) sulfide solutions in water are used with a concentration from to saturated or supersaturated solutions. An attractant lure formulation as claimed in claim 1 wherein component is butanoic acid or pentanoic acid. 6. An attractant lure formulation as claimed in claim 1 wherein component (ii) is 2-mercaptoethanol. 7. An attractant lure formulation as claimed in claim 1 wherein component (iii) is indole. 8. An attractant lure formulation claim 1 further including butan-2-ol, 1-ol or cis-3-octen-l-ol. 9. An attractant lure formulation claim 1 further including hexanal, benzaldehyde. 10. An attractant lure formulation claim 1 further including phenol, butan-2-ol, 2 methylpropan-1-ol, disulfide. 11. An attractant lure formulation claim 1 further including butan-2-ol, as med in 2-11., .propan- as claimed in 2-octanone and as claimed in 4-methylphenol, and dimethyl as claimed in 2-methylpropan- 1-ol, pentanoic acid (when component is butanoic acid) acetic acid and acetone. WO 94/27441 PCT/AU94/00268 19 12. A method of capturing flies using an insect trap utilizing the attractant lure formulation of claim 1 wherein each of components (ii) and (iii) is in admixture and component (iv) is separate when located in the insect trap.