PHEROMONES

Abstract: The present invention relates to the use of the sexual pheromone compounds 2,11-diacetoxy tridecane, 2,12-diacetoxy tridecane, and 2-acetoxy tridecane either as a racemate and/or as a biologically active enantiomer, in a combination, for the prognostication and/or control of peamidge, Contarinia pisi, as well as the compounds as such; method for prognostication; and method for controlling.

Description: TITLE PHEROMONES DESCRIPTION Technical field The present invention relates to sexual pheromones of the pea midge, Contarinia pisi, and their use for the prognostication of the presence of the pea midge, as well as controlling said insect, method for its use, as well as compounds having such an activity. The object of the present invention is to obtain a possibility of determining the quantitative presence of as well as the hatching time point of the pea midge for prognostication purpose, and to obtain an efficient control of the pea midge, C. pisi. A further object of the invention is to obtain an environmentally acceptable control of the pea midge, C. pisi. Background of the invention Cecidomyiidae, gall midges, is a family encompassing about 4460 species (Gagne 1989) of which several are important noxious insects within the agricultural area all over the world. One of these gall midges is the pea midge, C. pisi, which is hardly 2 mm long. In Sweden it hatches from the end of June to the beginning of July. The female pea midge lays its eggs in the flower buds of pea plants, where galls are formed in which the larvae develop. When the larvae have become fully developed they will go down into the soil and form winter cocoons, in which they will spend a dia pause which can be one or two winters long. The larvae will leave the winter cocoon in springtime and create a summer cocoon in which it will pass into the chrysalis stage and then will hatch. The hatching is determined by temperature and moisture (Jόnsson, 1988). Mating will occur immediately after hatching thereafter the females will leave the hatching field (usually a wheat field following a pea field) and look for a pea field to lay its eggs. The pea midge is present all over Europe. The damages can be reflected by the situation in the United Kingdom, where 20,000 ha of peas to be frozen are grown, i.e. peas to be frozen for human consumption. The peas are sprayed once or twice using an insecticide to the pea midge to a cost of about 2 million British pounds. In spite of this massive effort the harvest losses are considerable, up to 40 %. One reason for the high damage level is the lack of a good prognostication method for determining the hatching time point. The prognostication method used today means sifting and floatation of soil samples which are then analysed under microscope to determine in which development stage the chrysalis are. The analyses are carried out by someone skilled in the art and are very time consuming. Soil samples of a few number of fields are, however, only analysed. As the hatching is dependent on the local climate (moisture and temperature) the exactness of prognostication becomes low with regard to other fields. Furthermore, the hatching appears very suddenly and for a short time period, and as the control actions must be carried out prior to egg laying, the method is very uncertain. It requires a very efficient communication system as well, in order to reach all farmers with the results of the prognostication and recommendation concerning the time for controlling action. In pheromone based prognostication methods a few number of traps baited with a pheromone related to the intended insect, in particular a sexual pheromone, in synthetic form, are used. A pheromone trap is the most sensitive prognostication system one knows of today. Using such, even very low population densities can be detected. Noxious insects, as well as all other insects, vary a lot in number from year to year. The difficulty is hereby to know when they need to be controlled. The catch in the traps will however, provide an answer on three essential questions, viz: 1) if the insect is present in the field; 2) when the insect is present in the field; and 3) about how many there are present in the field. The answer to the first question is essential to prevent or eliminate many unnecessary control treatments of the type "as a matter of precaution", using insecticides. Having the answer to question no. 2) an optional control treatment can be made at the right time point when the insect is sensitive to control. Pheromone baited traps do not provide an exact measure of the population density but the catch result denotes if there is a low, an average, or a high risk infestation. Pheromone based prognostication systems are used today for about 200 different noxious insects, in particular moths. Using such prognostication traps the use of insecticides can be lowered with 50 to 75 %. Unnecessary control treatment can thus be avoided, which not only reduces the farmer's costs but also reduces the load on the environment. If the catch in the traps should indicate for a necessary control treatment one will be able to carry out the control for the insect in question at the most sensitive development stage using the catch results. Chemical insecticides to noxious insects will, with regard to the load on the environment and there quite often broad control spectrum, i.e., they hit against many different insects, to be replaced by biological control methods. Thereby, basic knowledge of the ecology and biochemistry of the specific insects is utilized. The precision, i.e., the desired specie specificity can thus become very high. The methods which are the most commercially successful ones, are based upon the sexual pheromones or other behaviour related pheromones of insects. The use of pheromones means a utilization of Nature's own tools for amounts and concentrations commonly present in Nature which means a maximum of consideration of the environment. When controlling noxious insects using sexual pheromones one imitates and utilizes the natural situation at which a female attracts a male by disposing the sexual pheromone which will be spread as a fog in the wind direction. A male who is coming into the pheromone plume responds by flying against the wind and thereby to the female. If a synthetic sexual pheromone or other behaviour pheromone is brought into an area of insect control at a greater number of sources the whole area will become thick with such a pheromone sent. In this sent environment the female's own signal will be drowned and the male will have no possibility of finding her. The technique is called confusion and is commonly spread control method. Other control methods can be to bait traps using a pheromone in order to kill the incoming insect in those traps, often being provided with glue or another "catching" agent. For the great number of gall midge species, among them the pea midge, it has been shown that females use a sexual pheromone in order to attract the males (Wall et al, 1985; Miller & Borden, 1984; Williams & Martin, 1986; Sharma & Vidyasagar, 1991; Pivnick & Labbe, 1992). Hitherto, however, only a part of the sexual pheromone of one gall midge, has been identified, viz a compound of the sexual pheromone of the barley gall midge (Foster et al, 1991). Description of the present invention It has surprisingly been shown possible to be able to prognosticate and control pea midge, C. pisi, by using its sexual pheromones, 2S,l lS-diacetoxy tridecane, 2S,12S-diacetoxy tridecane and 2-acetoxy tridecane, either as a racemate and/or as a biologically active enantiomer. The present invention relates to the following compounds, as well, viz: 2S,l lS-diacetoxy tridecane, of the formulae (I) CH 3 -CH-(CH 2 ) 8 -CH-CH 2 -CH 3 (I) O O CH 3 CO CH 3 CO and/or 2S,12S-diacetoxy tridecane, having the formulae (II) CH 3 -CH-(CH 2 ) 9 -CH-CH 3 (II) 0 O 1 I CH 3 CO CH 3 CO and more specifically their by females of pea midge, C. pisi, stereo isomeric enantiomers, as well as a third pheromone compound produced by the pea midge, viz: 2-acetoxy tridecane, either as a racemate and/or as biologically active enantiomer having the formulae (III) CH 3 -CH-(CH 2 ) 10 -CH 3 (III) O I CH 3 CO In a morphological study of a gall midge Solinas & Isidoro (1991) have described the appearance of the gland that produces the sexual pheromone. The gland is situated at the base of the ovipositor (egg laying tube) of the female. At the present invention this gland has been dissected and extracted using hexane. A gas chromatographic comparison of such an extract and the corresponding extract from the outermost tail body segment of the males shows that the female extract comprises three compounds, viz: 2S,12S - diacetoxy tridecane, 2S,1 IS - diacetoxy tridecane and 2-acetoxy tridecane, either as a racemate and/or as a biologically active enantiomer in a ratio of about 10:7:0,05-0,1. FIG. 1A shows the gas chromatographic deflection of the two first mentioned compounds of the female, while they are lacking at the male which is evident from FIG. IB. In order to obtain an analysable extract at least 800 females of the pea midge are needed. At electro-physiological studies carried out in accordance with Andersson et al., 1993 the antenna of the male exhibited a clear dose-response on the extract of the pheromone gland of the female, which indicates that the extract comprises sexual pheromone. Further, the male antenna gave an evident response at a contact with the racemate of 2S,1 IS - diacetoxy tri decane and 2S,12S - diacetoxy tridecane. In the GC-EAD technique (Arn et al., 1975) the outgoing flow from a gas chromatographic column is split into two flows, whereby one is fed to flame ionization detector of the gas chromatograph and the other is fed over a male antenna which electro-physiologically connected in such a way that EAD can be registered. As the male antenna responds specifically on the pheromone components of the own specie these can be determined from a gland extract composed of a great number of components. GC-EAD analysis of the gland extract of the pea midge showed that the male antenna responded clearly on said two compounds which, above, have been shown to differ female extract from male extract. These two compounds are thereby verified to be the sexual pheromones of the specie complemented with a further compound, as evident from above. Gas chromatography - mass spectrometry (GC-MS) was used to identify the components of the sexual pheromone of the pea midge. The first compound is (a) 2,11-diacetoxy tridecane, or 2S,llS-diacetoxy tridecane as active enantiomer, and the second one is (b) 2,12-diacetoxy tridecane, or 2S,12S-diacetoxy tridecane, as active enantiomer. The present pheromone compounds, (a) 2S,1 lS-diacetoxy tridecane, as active enantiomer, and (b) 2S,12S-diacetoxy tridecane, as active enantiomer, can be prepared in accordance with common synthesis methods known to the one skilled in the art of organic-chemical synthesis work. Thus it is thereby convenient to start with (a) 2,11-dihydroxy tridecane or (b) 2,12- dihydroxy tridecane and esterify these using acetic acid in order to obtain the acetate compounds. These can then be separated into its respective enantiomers using known resolution methods. 1. Determination of the stereoisomery of the female produced compounds of 2S,1 IS-diacetoxy tridecane and 2S,12S-diacetoxy tridecane with means of GC-MS (gas chromatography-mass spectrometry). The pure enantiomers, in total seven: 2S,1 IS; 2S,1 IR; 2R,1 IR; 2R,1 IS-diacetoxy tridecane and 2S,12S; 2R12R; meso-diacetoxy tridecane were synthetised and were separated on a particular (chiral) (Kδnig et al, 1994) under isothermal conditions. The retention times of the synthetic compounds were used as references at the corresponding analyses of female extract. In such away it was determined which of the enantiomers which are produced by the female pheromone gland, viz. 2S,1 IS-diacetoxy tridecane and 2S,12S-diacetoxy tridecane. 2. Determination of further female produced pheromone by optimizing the GC-EAD technology. At column separation of the pheromone produced by the female it was noted that there was an electro-physiologically active compound eluating exactly 5 min prior to 2S,1 IS-diacetoxy tridecane. As evident from FIG. 4A a female extract comprises so little of this component that only the sensitive male antenna, not the gas chromatograph, was able to detect the same. This led to a problem with regard to a GC-MS analysis. However, by using an alternative method, viz. By analysing the retention times of a number of reference compounds it was determined that 2-acetoxy tridecane eluated exactly at the interesting time point. By means of GC-EAD analysis of a synthetic mixture of 10 ng of 2S,1 IS-diacetoxy tridecane, 10 ng of 2S,12S- diacetoxy tridecane, and 0,1 ng of 2-acetoxy tridecane, when the antenna responded likewise to all three components, it was determined that 2-acetoxy tridecane was the compound looked for (FIG. 4A). The structural formulae of 2-acetoxy tridecane is evident from FIG. 5. 3. Behaviour activity of mixtures of electro-physiologically active synthetic compounds, compared with female extract in a wind tunnel test. Behaviour tests in a wind tunnel show that a synthetic mixture of the three electro- physiologically active compounds, 2S,12S-diacetoxy tridecane, 2S,1 IS-diacetoxy tridecane, and 2-acetoxy tridecane in a ratio of 10:7:0.1 (FIG. 6d) is equally attractive to males as a female extract (FIG. 6a) having the corresponding concentrations of these compounds. A mixture of 2S,1 IS-diacetoxy tridecane and 2S,12S-diacetoxy tridecane, only, in the ratio 7:10 (FIG. 6b) did not attract any of the males tested. A mixture of the three components, but in the ratio 10:7:0.5 (FIG. 6c) attracted somewhat less number of males than the female extract and the mixture 10:7:0.1 did. The ratio 10:7:0.05-0.5 seems to be a convenient range within which the best effects are obtained. When applying the pheromone combination of synthetic compounds in the connection to a field, in particular for controlling, the pheromone may conveniently be absorbed by an absorbent, partly to provide a more handable volume at the distribution, as the amount of pure active compounds is small, partly to obtain a delayed release of the active compounds over the time to provide for a maximal confusion effect over the hatching period. At prognostication work the compound mixture or the individual compounds are applied in a dispensing means which is placed in glue trap. The task of the dispensers is to release the compounds during a prolonged time period. The task of the glue trap to catch the males being attracted. The number of males caught indicates of the specie is present in the field when it flies, and an approximate population density. FIGURE LEGENDS. FIG. 1 A.Gas chromatographic response in females to 2,11-diacetoxy tridecane and 2S,12- diacetoxy tridecane is marked with a and b, respectively. FIG. IB. Gas chromatographic response in males to 2,11-diacetoxy tridecane and 2S,12- diacetoxy tridecane is marked with a and b, respectively. FIG. IB shows the lack of these compounds. FIG. 2. Dose-response at the male antenna of an extract of the female pheromone gland. FIG. 3. Gas chromatographic response (FID) and electro antenno graphic response (EAD) to female extract of pea midge. FIG. 4a. Gas chromatographic response (FID) and electro antenno graphic response (EAD) to female extract. Responses 2S,1 IS-diacetoxy tridecane and 2S,12S-diacetoxy tridecane are marked with 2 and 3, respectively. The response to the third component is marked with 1. FIG. 4b. Gas chromatographic response (FID) and electro antenno graphic response (EAD) to a mixture of 0.1 ng of 2-acetoxy tridecane (1) and 10 ng of 2S,1 IS-diacetoxy tridecane (2) and 10 ng of 2S,12S-diacetoxy tridecane (3). FIG. 5. Structural formula of 2-acetoxy tridecane. FIG. 6 Number males (in %) which fly up to the bait consisting of a) female extract; b) 2S.11S- and 2S,12S-diacetoxy tridecane (ratio 7:10); c) 2S.11S- and 2S,12S-diacetoxy tridecane and 2-acetoxy tridecane (ratio 7:10:0,05); d) the same as c) but in the ratio of 7:10:0,1. REFERENCES Anderson, P., Hilker, M., Hansson, B.S., Bombosch, S., Klein, B., and Schildknecht, H., 1993. Oviposition deferring components in larval frass of Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae). A behavioural and elektrophysiological evaluation. J. Insect. Phys. 39:129-137. Am, H., Stadler, E., and Rausher, S., 1975. The electroantennographic detector - a selective tool in the gas chromatographic analysis of insect pheromones. Z. Naturforsch. 30c, 722-725 Jδnsson, B.G., 1988. An ecological approach to management of the pea midge, Contarinia pisi (Winn.) in vining peas. Plant protection reports, Dissertation, 17. Department of plant and forest protection, Swedish University of Agricultural Sciences. Foster, S.P., Harris, M.O., and Millar, J.G., 1991. Identification of the sex pheromone of the Hessian fly, Mayetiola destructor (Say). Naturwissenschaften 78:130-131. Kδnig, W. A., Gehrke, B., Hochmuth, D. H., Mlynek, E., Hopf, H., 1994. Resolution of chiral [2,2]paracyclophanes by enantioselective gas chromatography. Tetrahedron: Assymmetry 5:347-350. Miller, G.E., and Borden, J.E., 1984. Reproductive behaviour of the Douglas-fir cone gall midge, Contarinia oregonensis (Diptera: Cecidomyiidae). Can. Ent. 116:607-618. Pivnick, K.A. and Labbe, E., 1992. Emergence and calling behaviour of the orange wheat blossom midge, Sitodiplosis mosellana (Gehlin) (Diptera: Cecidomyiidae). Can. Ent. 124:501- 507. Sharma, H.C., and Vidyasagar, P., 1991. Orientation of males of the sorghum midge, Contarinia sorghicola, to sex pheromones from virgin females in the field. Entomol.exp. appl. 64:23-29. Solinas, M., and Isidoro, N., 1991. Identification of the female sex pheromone gland in the sorghum midge, Contarinia sorghicola (Coq.) Solinas (Diptera: Cecidomyiidae). Redia, 74:441-446. Wall, C, Pickett, J.A., Garthwaite, D.G., and Morris, N., 1985. A female sex pheromone in the pea midge, Contarinia pisi. Entomol. exp. appl. 39:11-14. Williams, I.H., and Martin, A.P., 1986. Evidence for a female sex pheromone in the brassica pod midge, Dasineura brassicae. Physiol. Entomol. 11:353-356.

Claims: CLAIMS 1. The use of the sexual pheromone compounds 2,11-diacetoxy tridecane, 2,12-diacetoxy tridecane, and 2-acetoxy tridecane in a combination at the preparation of a dispenser for prognostication and/or control of pea midge, Contarinia pisi. 2. Use according to claim 1, of the sexual pheromone compounds 2S,1 IS-diacetoxy tridecane, 2S,12S-diacetoxy tridecane, and 2-acetoxy tridecane in a combination, at the preparation of a dispenser for prognostication and/or control of pea midge, Contarinia pisi. 3. Use according to claim 1-2, whereby 2,12-diacetoxy tridecane, 2,11-diacetoxy tridecane, or their biologically active enantiomers and 2-acetoxy tridecane are present in a weight ratio of about 10:7:0.05-0.5. 4. The compound, 2,11-diacetoxy tridecane, having the formula CH 3 -CH-(CH 2 ) 8 -CH-CH 2 -CH 3 (I) I I 0 O 1 I CH 3 CO CH 3 CO 5. The compound, 2,12-diacetoxy tridecane, having the formula CH 3 -CH-(CH 2 ) 9 -CH-CH 3 (II) O O CH 3 CO CH 3 CO 6. The compound, 2S,1 IS-diacetoxy tridecane. 7. The compound, 2S,12S-diacetoxy tridecane. 8. The compound, 2-acetoxy tridecane, having the formula CH 3 -CH-(CH 2 ) 10 -CH 3 (III) O I CH 3 CO either as a racemate and/or as a biologically active enantiomer. 9.Method for prognostication of pea midge, characterized in that one applies 2,11-diacetoxy tridecane, 2,12-diacetoxy tridecane and 2-acetoxy tridecane either as a racemate and/or as a biologically active enantiomer, in a combination, in a trap in connection with a field to be prognosticated with regard to the presence of pea midge, Contarinia pisi. 10. Method for prognostication of pea midge according to claim 9 characterized in, that one applies 2S,1 IS-diacetoxy tridecane, 2S,12S-diacetoxy tridecane and 2-acetoxy tridecane either as a racemate and/or as a biologically active enantiomer, in a combination. 11. Method for controlling pea midge characterized in, that one applies 2,11-diacetoxy tridecane, 2,12-diacetoxy tridecane and 2-acetoxy tridecane, either as a racemate and/or as a biologically active enantiomer, in a combination, in connection to a field, to be controlled, for confusion spreading of these compounds. 12. Method for controlling pea midge, characterized in, that one applies 2S,1 IS-diacetoxy tridecane, 2S,12S-diacetoxy tridecane and 2-acetoxy tridecane, either as a racemate and/or as a biologically active enantiomer, in a combination. 13. Method according to claims 9-10 or 11-12, whereby 2,12-diacetoxy tridecane, 2,11- diacetoxy tridecane and 2-acetoxy tridecane, either as a racemate and/or as a biologically active enantiomer, are present in a weight ratio of about 10:7:0.05-0.5. 14. Method according to claims 9-10 or 11-12, whereby 2S,12S-diacetoxy tridecane, 2S,1 IS- diacetoxy tridecane and 2-acetoxy tridecane, either as a racemate and/or as a biologically active enantiomer, are present in a weight ratio of about 10:7:0.05-0.5.

Inventors: BIDDLE ANTHONY, FRANCKE WITTKO, FRANKE STEPHAN, HILLBUR YLVA BIRGITTA, LOEFQVIST JAN, PLASS ERNST, BIDDLE, ANTHONY, FRANCKE, WITTKO, FRANKE, STEPHAN, HILLBUR, YLVA, BIRGITTA, LOEFQVIST, JAN, PLASS, ERNST
Applicants: BIDDLE ANTHONY, FRANCKE WITTKO, FRANKE STEPHAN, HILLBUR YLVA BIRGITTA, LOEFQVIST JAN, PLASS ERNST, BIDDLE, ANTHONY, FRANCKE, WITTKO, FRANKE, STEPHAN, HILLBUR, YLVA, BIRGITTA, LOEFQVIST, JAN, PLASS, ERNST
Application year: 1999
Main classification: A01N37
IPC codes: A01N37, C07C69