WO2000015034A1

WO2000015034A1 - Use of a composition for controlling and/or attracting insects

Info

Publication number: WO2000015034A1
Application number: PCT/SE1999/001604
Authority: WO
Inventors: Rikard Unelius; Peter Valeur; Junwei Zhu; Christer LÖFSTEDT; Camilla Ryne
Original assignee: Rikard Unelius; Peter Valeur; Junwei Zhu; Loefstedt Christer; Camilla Ryne
Priority date: 1998-09-15
Filing date: 1999-09-14
Publication date: 2000-03-23
Also published as: SE9803130L; AU6378399A; SE9803130D0

Abstract

The present invention relates to the use of a composition comprising (Z,E)-9,12-tetradecadienyl acetate (Z9,E12-14:OAc), (Z,E)-tetradecadienal (Z9,E12-14:A1d), (Z)-9-tetradecenyl acetate (Z9-14:Oac) and (Z,E)-9,12 - tetradecadienol (Z9,E12-14:OH) for controlling and/or attracting insects.

Description

USE OF A COMPOSITION FOR CONTROLLING AND/OR ATTRACTING INSECTS

The present invention relates to the use of a composition for controlling and/or attracting insects.

Background of the invention

The Indian meal moth, Plodia interpunctella, Hϋbner (Lepidoptera: Pyralidae), is considered one of the most serious insect pests infesting stored grains, nuts, dried fruits, legumes, and other food products (Phillips, 1994). Because of increasing environmental and human health concerns, the use of chemical pesticides has been restricted for control of this pest in food storage facilities. However, the recent advances in the use of biorational methods such as pheromones, provide us with a new highly effective way to deal with such a problem. A combination of pheromone-based monitoring systems, mass trapping and mating disruption can be used for the control of pests in food storage facilities, instead of using conventional chemical pesticides with associated health hazards (Muller and Pierce, 1992; Siiss and Trematerra, 1986).

The first pheromone component identified from female P. interpunctella was (Z,E)- 9,12-tetradecadienyl acetate (Z9,E12-14:OAc), and this compound was reported to be very attractive to its conspecific males (Brady et al, 1971; Kuwahara et al., 1971a). Kuwahara and Casida (1973), and Sower et al. (1974) subsequently published (Z,£ -9, 12-tetradecadienol (Z9,E12-14:OH) as an additional pheromone compound in the extract of females and Soderstrom et al. (1980) and Vick et al. (1981) found that the alcohol increased the behavioural responses of male P. interpunctella when presented together with Z9,E12-14:OAc. A third pheromone compound, (Z,£)-9,12-tetradecadienal (Z9,E12-14:Ald), was more recently identified by Teal et al. (1995) in collections of volatiles from calling female P. interpunctella, but no evaluation of its potential behavioural importance was reported. P. interpunctella shares the major pheromone component, Z9,E12- 14:0Ac, with many other phycitine moths, including the Mediterranean flour moth Ephestia kuehmella Zeller (Lepidoptera: Pyralidae) (Kuwahara et al, 1971b; Kuwahara and Casida, 1973) and significant cross attraction has been reported (Phelan & Baker, 1986).

EP-A-0 475 665 describes a device for attracting moths. When the moth is Plodia interpunctella the pheromone may be Z9,E12-14:OAc with or without minor amounts of (Z)-9-tetradecenyl acetate (Z9-14:OAc) and Z9,E12-14:OH. However, this document does neither describe the pheromone properties nor the use of Z9,E12-14:Ald.

Despite the success in identification of pheromone compounds from P. interpunctella, and the efficiency of male capture using the bait with the major component Z9,E12-14:OAc alone, the pheromone system of this species is far from fully understood. The discovery of new, behaviourally important secondary compounds could increase the trapping efficiency, and contribute to the control of this stored product pest by mass trapping or mating disruption. The purpose of the present study was thus to find secondary pheromone component candidates and to investigate their effects on male attraction. Different combinations of secondary pheromone candidates were tested in the flight tunnel along with the previously known primary pheromone component. Pheromone baits were also tested in a pet food storage facility, infested with both P. interpunctella and E. kuehmella.

Pheromone gland extracts from calling female Plodia interpunctella contained at least seven compounds that consistently elicited electroantennographic responses from male antenna upon gas chromatographic analysis. Three of these compounds were found to be the previously identified gland constituents, i.e. (Z,E)-9, 12- tetradecadienyl acetate (Z9,E12-14:OAc), (Z,E)-9, 12-tetradecadιenal (Z9,E12- 14:Ald) and (Z,£)-9, 12-tetradecadιenol (Z9,E12-14:OH). A fourth EAD-active compound was identified as (Z)-9-tetradecenyl acetate (Z9-14:OAc). The homologue (Z)-l 1-hexadecenyl acetate (Zl l-16:OAc) was also identified in the extracts, but showed no EAD activity. The identity of all five compounds was confirmed by comparison of GC retention times and mass spectra with those of synthetic standards. In flight tunnel tests, male P. interpunctella responded to the bait containing the four EAD-active compounds equally well as to female extracts. A behavioural assay of different 2-compound blends in the flight tunnel showed that only addition of the corresponding aldehyde to the major component Z9,E12- 14:OAc raised the male response. A subtractive assay, however, revealed that the exclusion of any of the secondary compounds from the complete 4-compound pheromone blend reduced its activity significantly. We thus conclude that the female-produced sex pheromone of . interpunctella consists of at least four components, i.e. Z9,E12-14:OAc, Z9,E12-14:Ald, Z9,E12-14:OH, and Z9-14:OAc.

In a field trapping test, performed in a storage facility, the 4-component blend attracted significantly more males of P. interpunctella than traps baited with Z9,E12-14:OAc singly. In contrast, the highest number of Ephestia kuehmella males was found in the traps baited with this major component, suggesting that the secondary pheromone components contribute to the species specificity of the blend.

Struble and Richards, 1983 show that extracts from Vitula edmandsae serratilmeella belonging to Pyralidae comprise 16 different pheromone-like components, e.g. Z9,E12-14:Ald and Z9,E12-14:OH, but no mixtures consisting of Z9,E12-14:OAc, Z9,E12-14:Ald, Z9,E12-14:OH, and Z9-14:OAc.

Summary of the invention

It has been shown that Z9,E12-14:Ald shows a synergistic effect when it is added to a mixture of Z9,E12-14:OAc, Z9,E12-14:OH, and Z9-14:OAc (Fig. 3 and 5). There is also a synergistic effect of Z9,E12-14:OH when it is added to a mixture of Z9,E12-14:OAc, Z9,E12-14:AId, and Z9-14:OAc (Fig. 5). Therefore, the present invention relates to the use of a composition for controlling and/or attracting insects. The composition comprises Z9,E12-14:OAc, Z9 E12- 14:Ald, Z9-14:OAc, and Z9,E12-14:OH.

Detailed description of the invention

One object of the present invention is the use of a composition for controlling and/or attracting insects, characterized in that the composition comprises (Z,E)- 9,12-tetradecadienyl acetate (Z9,E12-14:OAc), (Z.y^-tetradecadienal (Z9,E12- 14:Ald), (Z)-9-tetradecenyl acetate (Z9-14:OAc) and (Z.EJ-9, 12-tetradecadienol (Z9,E12-14:OH), pure or in a solvent, with or without a suitable carrier, such as gum, cork, cellulose, plastic, rubber, leather or skin, milled carbon, wood-flour, silicates, pumice, polyvinyl chloride, polyvinyl acetate, chlorinated polyethylene, straw, cane, lignocellulose and silica.

The composition above could also contain an insecticide. Examples of suitable insecticides are DDT, aldrin, dieldrin, chlordan, lindane, heptachlor, toxaphene, carbaryl, carbofuran, propoxur, allethrin, cyfluthrin, and peπnethrin.

In our experiments, the addition of Z9,E12-14:OH decreases male responses significantly, which indicates an antagonistic effect of this compound (Fig. 2), while Vick et al (1981) reported it to enhance the attractiveness to males. When we added this alcohol to a 3 -component blend, however, a synergistic effect was clearly shown (Fig. 5).

In a 4-component composition, the appropriate ratio of Z9,E12-14:OAc: Z9,E12- 14:Ald: Z9-14:OAc and Z9,E12-14:OH ranges from 100:0.3: 1 :0.1 to 100:300:30:30, by weight. The preferred ratio is 100: 16:6:7, by weight.

The 4-component blend, as well as some of the 3- and 2-component blends we propose here is very attractive to male P. interpunctella, which suggests that it represents an improvement when it comes to detecting infestations by this species, especially under low population density. The 4-component blend may also represent an improvement in terms of specificity. The current napping tests suggest that the more complex blends are less attractive to males ofE. kuehmella than the major component alone. Finally, the high attractiveness of the four-component synthetic pheromone of P. interpunctella is promising in terms of future direct control of this insect. An observed high female reproductive potential and a multiple mating ability of males (Ryne et al, unpublished) may limit the success of control via mass trapping, but mating disruption with the complex pheromone blend (see references in Carde and Minks, 1995) could be an efficient method to control infestations of P. interpunctella.

Examples of insects are selected from the group consisting of Plodia interpunctella and Ephestia kuehniella. Plodia interpunctella is preferred.

The synthetic compounds Z9,E12-14:OAc, Z9,E12-14:OH, Z9-14:OAc, and Zl l- 16:OAc are commercially available. The aldehyde Z9,E12-14:Ald was synthesized according to Example 1.

By the expression "comprising" we understand including but not limited to. Thus, other non-mentioned substances, additives or carriers may be present. It is to be understood that the mechanisms according to the invention are symbiotic.

Short description of the Figures

Fig. 1. Simultaneously recorded flame ionisation detector (FID) and electiOantennographic detector (EAD) responses using antennae of male Indian meal moth, Plodia interpunctella in response to pheromone gland extracts from conspecific females (A), and a synthetic reference mixture. ,_„, , PCT/SE

WO 00/15034 g

Fig. 2. Behavioural responses of male Plodia interpunctella in a flight tunnel to different combinations of pheromone component candidates. Data points with the same letters do not differ significantly (P>0.05, Ryan^'s test). * Figures listed under 1FE are the relative proportions, instead of relative amounts listed in the other columns.

Fig. 3. Behavioural responses of male Plodia interpunctella in a flight tunnel to different pheromone stimuli. Data points with the same letters do not differ significantly (P>0.05, Ryan^'s test). * Figures listed under 1FE are the relative proportions, instead of relative amounts listed in the other columns

Fig. 4. Behavioural responses of male Plodia interpunctella in a flight tunnel to a 2- compound blend containing 100 ng of Z9,E12-14:OAc and different doses of Z9,E12-14:Ald. Data points with the same letters do not differ significantly (P>0.05, Ryan^'s test).

Fig. 5. Behavioural responses of male Plodia interpunctella in a subtractive assay in a flight tunnel. Data points with the same letters do not differ significantly (P>0.05, Ryan^'s test).

Fig. 6. Trapping of male Plodia interpunctella and Ephestia kuehniella with different combinations of pheromone compounds (three replicates) in the storage room of a pet food distributor. Error bars indicate standard error. Statistical test: ANOVA with log transformed values and the degrees of freedom adjusted with Satterthwaite^'s formula. Columns labelled with different letters do not differ significantly (P>0.05).

The invention will be illuminated by the following Examples, which are only intended to illuminate and not restrict the invention in any way. Examples

Materials and methods

Insects. A laboratory culture of P. interpunctella was established from fifth instar larvae provided by the Danish Pest Infestation Laboratory at Lyngby, Denmark. Larvae were reared on an artificial diet containing 100 g of wheat germ, 10 g of Brewer^'s yeast and 20 g of glycerol, together with 10 raisins sprinkled on top of the diet. Insects were sexed during the pupal stage. Emerged male and female adults were kept in separate environmental chambers at 23 °C and 60% rH with a 17-h light : 7-h dark photoperiod.

Preparation of extracts. Female P. interpunctella were observed to have a period of maximum calling between two to four hours into the scotophase. Pheromone glands of females were dissected at their peak calling time using hexane as the solvent. Extracts were used for gas chromatography with electroantennographic detection (GC-EAD) and for gas chromatographic-mass spectrometric analysis (GC-MS), as well as for some behavioural flight tunnel tests. Individual female gland extracts were made in 8 μl of solvent with 5 ng heneicosane (C₂ιH₄₄) added as an internal standard.

Electrophysiological and chemical analysis. A Hewlett Packard 5890 Series II GC was equipped with a DB-WAX column (30 m x 0.25 mm i.d., J & W Scientific, Folsom, CA), and an effluent split at a 1 : 1 ratio allowed simultaneous flame ionisation (FID) and electroantennographic detection (EAD) of the separated pheromone components. Hydrogen was used as carrier gas at approximately 50 cm/s flow rate. Samples were injected splitless. The injector temperature was 225°C and the split valve was opened 1 min after injection. The column temperature was maintained at 80 °C for 1 min following the injection and then linearly increased to 230°C at a rate of 10°C /min. The outlet for the EAD was placed in a purified air stream flowing over the antennal preparation at a speed of 0.5 m/s. Two microliters of extracts containing 2-3 female equivalents were injected into the column for the .analysis. The equivalent chain lengths of active pheromone compounds relative to a homologous series of straight-chain acetates (7-22:OAc) were established. The EAD registration setup was purchased from Syntech (Hilversum, The Netherlands). A GC-EAD program developed by Syntech was used to record and analyse both amplified EAD and FID signals on a Pentium PC computer.

Gas chromatographic-mass spectrometric analyses of GC-EAD active compounds were performed by using a Hewlett-Packard 5890 Series II gas chromatograph equipped with a DB-WAX column (30 m x 0.25 mm i.d., J & W Scientific, Folsom, CA), linked with a Hewlett-Packard 5972 Mass Selective Detector (MSD). The GC operating condition was the same as that described for GC-EAD analysis above, except that helium was used as the earner gas. The flow rate of helium through the column was kept at 40 cm s. Spectra were recorded from 40 to 550 amu after electron impact ionization at 70 eV.

Chemicals. The synthetic compounds Z9,E12-14:OAc, Z9,E12-14:OH, Z9-14:OAc and Zl l-16:OAc were purchased from Research Institute for Plant Protection (IPO- DLO, Wageningen, The Netherlands), and 7-22 :OAc from SIGMA, Sweden.

Example 1 - Synthesis of Z9.E12-14:Ald

The aldehyde Z9,E12-14:Ald was synthesized by a hydrolysis of the commercially available Z9,E12-14:OAc followed by a pyridinium chlorochromate oxidation of the alcohol formed.

A 400 mg amount of Z9,E12-14:OAc (SIGMA, Sweden) was reacted with 10% KOH/MeOH (4 ml). After the hydrolysis was completed, the mixture was extracted with hexane (HPLC grade, Merck, Sweden). The combined organic phases was dried over anhydrous MgS0₄ and the solvent was evaporated before oxidation. Pyridinium chlorochromate (1.0 g, 4.58 mmol) was added to a mixture of the Z9,E12-14:OH and silica gel (2.5 g, Merck 60) in 20 ml of CH₂C1₂, and stirred for 150 min. Hexane (10 ml) was added to the mixture before it was filtered. The solution was concentrated to 20 ml and then pumped into a silica gel column and subjected to liquid chromatography. The purity of the product Z9,E12-14:Ald was checked by mass spectrometry and NMR spectroscopy.

The MS fragment ions and their relative intensities of the synthesised Z9,E12- 14: Aid were m/z 208 (M, 24), 179 (16), 166 (18), 151 (24), 109 (25), 98 (28), 95 (66), 93 (36), 82 (63), 81 (96), 79 (100), 68 (83), 67 (98), 55 (52), 44 (55), 41 (82), 39 (43).

Η NMR δ: 9.77 (t, J = 1.8Hz, 1H), 5.36-5.47 (m, 4H), 2.75 (app t, J = 5.4Hz, 2H), 2.42 (dt, J = 7.4, 1.8Hz, 2H), 2.03(q, J = 6.4Hz, 2H), 1.63 (m, 5H), 1.3-1.4 (m, 8H).

¹³C NMR 6: 202.9, 130.3, 129.6, 127.8, 125.1, 43.9, 30.4, 29.5, 29.2, 29.1, 29.0,

27.0, 22.1, 17.9.

All chemical compounds were purer than 99.9% according to analyses by GC- MSD, with no trace amounts of the other isomers. The ratios of different pheromone blends used for the flight tunnel behavioural assays were checked by GC prior to tests.

Example 2 - GC-EAD of seven compounds isolated from female gland extracts and chemical identification.

Seven compounds (I- VII) in the female gland extracts elicited significant EAD responses from conspecific male antennae in all successful runs (Fig. 1). Among these, four were positively identified as Z9,E12-14:OAc(V), Z9-14:OAc(IV), Z9,E12-14:Ald(II) and Z9,E12-14:OH(VII) by comparison of their retention times and mass spectra with those of synthetic standards. The fifth compound (III) was identified as 14:OAc based on retention time, whereas the other two could not be identified. Although female P. interpunctella produced relatively high amounts of Zl l-16:OAc, no EAD response to this compound was observed from the male antenna (Fig. 1). The quantities and relative amounts of the four positively identified, EAD active pheromone compounds are listed in Table 1.

Table 1. Absolute and relative amounts of pheromone component candidates in individual Indian meal moth females, Plodia interpunctella (N=33)

Z9,E12-14:Ald Z9-14:OAc Z9,E12-14:OAc Z9,E12-14:OH

Quantity 0.39 ± 0.07 0.43 + 0.15 3.62 + 0.52 0.66 ± 0.28

(ng/female)

± SE

Relative 11 12 100 18 amounts

Among the seven EAD-active pheromone compounds that we found in pheromone gland extracts of female P. interpunctella, Z9,E12-14:OAc, Z9,E12-14:OH and Z9,E12-14:Ald have been previously identified (Brady et al, \911; Kuwahara et al. ,1971a; Kuwahara and Casida, 1973; Teal et al, 1995). Furthermore, these pheromone compounds were found in quantities and ratios similar to those previously reported by Teal et al (1995). A fourth EAD-active compound was identified as Z9-14:OAc. This compound is known as one of the pheromone components of Ephestia cautella females (Read and Beevor, 1976). but has not previously been reported from female P. interpunctella. We also found the homologue Zl l-16:OAc in the female extracts, but as it did not elicit any EAD response we did not include it in the subsequent behavioural tests. Finally, three additional EAD-active compounds were discovered during the GC-EAD runs, but the amounts of these compounds were too low to allow their positive identification The physiological activity of the unidentified compounds suggests that the pheromone blend of P. interpunctella could be even more complex than the 4- component blend investigated in the present study. Example 3 - Pheromone baits in the flight tunnel and synergistic effect of Z9.E12- 14: Aid.

0.75 cm² triangular pieces of filter paper (KEBO Lab, Sweden) were used as pheromone dispensers for all behavioural tests in the flight tunnel. The filter paper bait was pinned to an insect needle, which was supported by penetrating through a rubber septum (Thomas Scientific, USA). The pheromone bait was set on a wire stand approx. 30 cm above the flight tunnel floor. The bait was prepared 2 min before being tested, .and renewed every 10 min. Synthetic blends and female extracts (lFE/lOμl), were used for the test. All synthetic pheromone baits tested in the flight tunnel contained approx. lOOng of the major pheromone component (Z9,E12-14:OAc), and the secondary compounds were added in the relative amounts indicated in the figures. These amounts were generally based on the GC analyses of extracts from pinned pheromone glands, which in previous wind tunnel tests had been demonstrated to have high behavioural activity. The composition of each synthetic bait was also determined by GC analysis. Minor variation in ratios between some of the baits was unintentional and just reflects the precision of the method used for preparation of the baits.

Male P. interpunctella responded to a 4-compound blend containing Z9,E12- 14:OAc, Z9,E12-14:Ald, Z9-14:OAc and Z9,E12-14:OH equally well compared to female extracts. Among the 2-compound mixtures, the blend containing Z9,E12- 14:Ald in addition to the corresponding acetate, was the only one which did not show significantly lower activity (Fig. 2). The synergistic effect of Z9,E12-14:Ald was coiToborated in a second experiment, in which all baits lacking the aldehyde were significantly inferior to the 4-compound bait (Fig. 3). In both of these experiments as well as in a subsequent experiment, less than 60% of the males were observed to land on the source containing the major pheromone component Z9,E12- 14:OAc alone (Fig. 2, 3 and 4). Addition of Z9,E12-14:OH to the primary pheromone component Z9,E 12- 14:0 Ac reduced its activity significantly (Fig. 2). In a test of the secondary compound Z9,E12-14:Ald in different amounts relative to the primary pheromone component, the best male responses were obtained when the proportions of Z9,E12-14:OAc and Z9,E12-14:Ald were between 100:3 and 100:30 (Fig. 4). A 100:3000 mixture of Z9,E12-14:OAc to Z9,E12-14:Ald (100 ng) caused take off response in 82 % of the males, but no orientation (N=22). In a subtractive assay, exclusion of any of the four compounds caused significant decrease in male responses relative to the 4-compound blend (Fig. 5). Subtraction of the primary component Z9,E 12- 14: OAc caused an almost complete loss of activity.

Phillips (1994) reported that male P. interpunctella were more attracted to caged females than to the single component Z9,E12-14:OAc. Our behavioural tests in the flight tunnel as well as the field trapping test showed that a single component bait is less attractive to male moths than the 4-compound pheromone blend. In the flight tunnel test, the attractiveness of the 4-compound bait towards males was twice as high as that of the single component bait (Fig. 2 and 3). The combination of the corresponding aldehyde with the major component Z9,E12-14:OAc increases the male attraction significantly, compared to the bait with the major component alone. The aldehyde was earlier found by Teal et al. (1995) in solvent extracts and volatile collections from calling females, but its behavioural activity has not been previously reported.

The subtractive flight tunnel assay demonstrated that subtraction of Z9,E12-14:OAc from the 4-compound blend causes almost complete loss of activity (Fig. 5). This evidence supports the role of the acetate as a primary pheromone component. The remaining three compounds, however, all qualify as secondary pheromone components in P. interpunctella, since exclusion of either one of them from the 4- component blend decreases the male response (Fig. 5). Thus, based on the flight tunnel evidence we conclude that the sex pheromone of P. interpunctella consists of at least four components. We find a few discrepancies when we compare our results with those from earlier reports. The increase of the attractivity of a synthetic pheromone blend does not necessarily come along as a linear additive effect. The addition of a single minor component does not always result in a better blend even if this component increases the attractivity in a complete blend.

Example 4 - Pheromone baits in the storage-room test

Synthetic blends of pheromone were prepared in lOOμl hexane. Red rubber septa (Arthur H. Thomas Co. Catalog No. 1780-J07) were used as dispensers. The septa were extracted by hexane before being loaded. Each bait contained lOOμg of the main pheromone compound (Z9,E12-14:OAc) and the secondaiy compounds in the amounts displayed in Fig. 6, as confirmed by GC analysis.

The three baits containing the Z9,E12-14:OAc plus the corresponding aldehyde; i.e. the 4-compound, the 3-compound and the 2-compound baits, were the most attractive and not significantly different from each other (Fig. 6). The 4-compound blend caught a significantly higher number of P. interpunctella males than the Z9,E12-14:OAc or the combination of Z9,E12-14:OAc and Z9-14:OAc did. The major compound Z9,E12-14:OAc alone, trapped high numbers of is. kuehniella males (Fig. 6). A significantly lower number of male E. kuehniella were caught in the trap baited with 2-component blend containing aldehyde, whereas no significant differences in male captures were found among baits with the 1-compound, 3- compound .and 4-compound blend ( ?>0.05).

Example 5 - Flight tunnel tests

A 0.7 m x 0.7 m x 2 m Plexiglas flight tunnel was used. The floor was covered by white paper with painted dots and the downwind end of the tunnel was covered with a metal mesh keeping moths trapped in the flight tunnel while the air could pass freely. An exhaust funnel placed after the mesh removed pheromone-contaminated air from the room. The tunnel was illuminated from above with 8 incandescent lamps (15 'W) above a translucent Plexiglas plate. The temperature in the flight tunnel was maintained at 23 °C ± 2°C during experiments, wind speed was 0.4-0.5 m/s, humidity was 25-40% r.h, and light intensity was regulated to 5 lux. Males of P. interpunctella aged 1 to 3 days were placed in the flight tunnel room during the last hour of the photoperiod. Male moths were tested 2 to 6 hours after the onset of scotophase, which was considered as their most active period of flight (Quartey and Coaker, 1996). Males were transferred to glass tubes (10 cm x 0 3 cm) just before they were introduced into the flight tunnel. Individual males were released from the glass tube approx. 1.8 m downwind of the tested pheromone source. Each male was allowed 1 min to initiate flight after being introduced into the pheromone plume. Once flight was initiated, males were observed (2 min.) until they either landed on the source or stopped responding, e.g. by flying to the ceiling or resting on the screen. A number of behavioural characteristics were scored: taking flight; orientation in the plume; upwind flight half way to the baits, and landing on the source.

The four compounds to be tested could be combined in a large number of ways. For practical reasons, four separate trials with reasonable numbers of treatments, were designed; (1) Different 2-component combinations with the major component and a four-compound blend; (2) Different combinations of pheromone compounds; (3) Different doses of the Z9,E12-14:Ald in a 2-component blend with Z9,E12-14:OAc; (4) A subtractive assay.

Ryan's multiple comparison test for proportions was used for the comparison of all behavioural observations at the 5% level (Ryan, 1960).

Example 6 - Trapping test

A trapping test to evaluate the attractiveness of different combinations of the pheromone compounds, was carried out in the storage room (60 x 80m²) of a local pet food distributor. This facility was infested with both P. interpunctella and E. kuehniella. Delta traps with exchangeable sticky inserts were employed. All traps were hung from the frames of the stock shelves about one and a half meters above the floor. All traps were distributed at least two meters apart. The traps were checked approximately once a month. To minimise the effect of habitat heterogeneity, the trap positions within each series (three series in total) were rotated during each visit. The test was run from 26th of June to 3rd of November 1997, and the site was visited six times. The sticky inserts were changed twice during the period. The baits were not renewed during the test period. Caught individuals were checked under a preparation microscope for sex and species identity.

The number of caught males in the traps were log transformed and tested with an analysis of variance, using the SAS statistical package. Traps were treated as a random factor to model and correct for possible dependence. The degrees of freedom were adjusted with Satterthwaite's formula (Littell et al, 1996).

The field trapping tests, as well as the flight tunnel tests, demonstrated the synergistic effect of Z9,E12-14:Ald.

References

Brady, U.E, J.H. Tumlinson, R.G. Brownlee & R.M. Silverstein, 1971. Sex stimulant and attractant in the Indian meal moth and almond moth. Science 171 : 802-804.

Carde, R.T. & A.K. Minks, 1995. Control of moth pests by mating disruption:

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Claims

1. Use of a composition for controlling and/or attracting insects, characterized in that the composition comprises fZ,i╬╢J-9,12-tetradecadienyl acetate (Z9,E12- 14:OAc), Z,┬ú tetradecadienal (Z9,E12-14:Ald), Zj-9-tetradecenyl acetate (Z9-

14:OAc) and (%P 9, 12-tetradecadienol (Z9,E12-14:OH), pure or in a solvent, with or without a suitable carrier.

2. Use according to claim 1, characterized in that the composition further comprises an insecticide.

3. Use according to claim 1-2, characterized in that the appropriate ratio of Z9,E12-14:OAc: Z9,E12-14:Ald: Z9-14:OAc and Z9,E12-14:OH ranges from 100:0.3: 1:0.1 to 100:300:30:30, by weight.

4. Use according to any of the claims 1-3, characterized in that the insects belong to the family Pyralidae, preferably Plodia interpunctella and Ephestia kuehniella, most preferably Plodia interpunctella.

5. Use according to any of the claims 1-4, characterized in that the suitable carrier is selected from the group consisting of gum, cork, cellulose, plastic, rubber, leather or skin, milled carbon, wood-flour, silicates, pumice, polyvinyl chloride, polyvinyl acetate, chlorinated polyethylene, straw, cane, lignocellulose and silica.