Patent Application: US-59971105-A

Abstract:
this invention relates to a composition and procedure for manipulating the behaviour of codling moth larvae , cydia pomonella . in particular , this invention relates to the use of specific pheromone components for manipulating the behaviour of c . pomonella larvae . a composition of chemicals for manipulating the behaviour of cydia pomonella larvae , said composition comprising two or more chemicals in all possible combinations and ratios selected from the group consisting of : 1 ) heptanal ; 2 ) 6 - methyl - 5 - hepten - 2 - one ; 3 ) myrcene ; 4 ) octanal ; 5 ) 3 - carene ; 6 ) - limonene ; 7 ) - 2 - octenal ; 8 ) nonanal ; 9 ) - 2 - nonenal ; 10 ) decanal ; 11 ) geranylacetone .

Description:
throughout the following description , specific details are set forth in order to provide a more thorough understanding of the invention . however , the invention may be practiced without these particulars . in other instances , well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention . accordingly , the specification and drawings are to be regarded in an illustrative , rather than a restrictive , sense . 1 . response of male and female cydia pomonella larvae to cocoon - spinning male or female larvae response of 5 th instar c . pomonella larvae seeking pupation sites was tested in 2 - choice petri dish olfactometers ( duthie et al ., 2003 ), with modified eppendorf tubes in pitfall devices preventing physical contact of larvae with test stimuli . for each of at least 30 replicates per experiment , one male or female larva was released in the center of the olfactometer , and its pupation site recorded 18 - 24 hours later . test stimuli consisted of a control corrugated cardboard ( cb ) strip ( 2 . 5 × 2 . 5 cm ) or a treatment cb strip carrying five 1 - day - old cocoons each containing either a male or female c . pomonella larva / prepupa . male and female c . pomonella larvae preferred to spin cocoons in pitfall devices baited with cb carrying five 1 - day - old cocoons , with no preference for cocoons containing either male or female larvae / prepupae ( fig1 ). fig1 illustrates graphical data of responses of male or female cydia pomonella larvae to pitfall devices baited with control corrugated cardboard ( cb ) strips or treatment cb strips carrying five 1 - day - old cocoons containing either male or female c . pomonella larvae / prepupae . asterisks on bars indicate a significant response to a particular treatment ; chi - square goodness of fit test with yates &# 39 ; correction for continuity ; * p & lt ; 0 . 025 ; ** p & lt ; 0 . 001 . 2 . acquisition , analyses and bioassays of pheromone components produced by cocoon - spinning cydia pomonella larvae to capture airborne pheromone components from cocoon - spinning larvae , three - hundred 5 th instar male and female larvae were placed in a cylindrical pyrex glass chamber ( 15 . 5 × 20 cm ). an empty chamber served as control . a water aspirator drew charcoal - filtered air at ˜ 2 l / min through each chamber and through a glass column ( 140 × 1 . 3 mm od ) containing porapak q ( 50 - 80 mesh , waters associates , inc ., milford , mass . 01757 ). after 72 hours , the filters were desorbed with 3 ml of pentane and ether ( 95 : 5 ). extracts were concentrated under a nitrogen stream so that 1 μl was equivalent to ca . 10 cocoon - spinning larvae hour equivalents ( 10 cslhe = volatiles released from 10 cocoon - spinning c . pomonella larvae during 1 hour ). aliquots of 20 cslhe of porapak q - captured volatiles were subjected to analysis by coupled gas chromatographic - electroantennographic detection ( gc - ead ) ( arm et al . 1975 ) using an antenna of mastrus ridibundus , a specialist parasitic wasp of c . pomonella prepupae , as the electroantennographic detector . using antennae from female m . ridibundus , instead of c . pomonella larvae , in these analyses was necessary because the antennae of c . pomonella larvae are too small for electrophysiolocial studies . it was also justified because host - seeking female m . ridibundus respond to the pheromone produced by cocoon - spinning c . pomonella larvae . in these gc - ead analyses , 10 components ( 8 visible in fig2 ) elicited responses from m . ridibundus antennae . fig2 illustrates flame ionization detector ( fid ) and electroantennographic detector ( ead : female mastrus ridibundus antenna ) responses to 10 cslhe of porapak q extract . chromatography : hewlett packard ( hp ) 5890a gas chromatograph equipped with a fused silica column ( 30 m × 0 . 32 mm id ) coated with db - 23 ( j & amp ; w scientific , folsom , calif . 95630 , usa ); linear flow velocity of carrier gas : 35 cm / sec ; injector and fid detector temperature : 240 ° c . ; temperature program : 1 min at 50 ° c ., 10 ° c ./ min to 220 ° c . full scan electron impact ( e1 ) and chemical ionization ( ci ) mass spectra of ead active compounds were obtained by gc - mass spectrometry ( ms ) using a varian saturn ii ion trap gc - ms and a hp 5985b gc - ms , respectively , each fitted with the db - 210 or db - 5 column . candidate pheromone components were identified as follows : 1 . heptanal ( 0 . 85 ); 2 . 6 - methyl - 5 - hepten - 2 - one ( sulcatone ) ( 0 . 81 ); 3 . myrcene ( 0 . 84 ); 4 . octanal ( 0 . 94 ); 5 . 3 - carene ( 0 . 95 ); 6 . (+)- limonene ( 13 . 00 ); 7 . ( e )- 2 - octenal ( 0 . 41 ); 8 . nonanal ( 4 . 10 ); 9 . ( e )- 2 - nonenal ( 1 . 00 ); 10 . decanal ( 1 . 40 ); 11 . geranylacetone ( 0 . 50 ). note : 1 ) 10 cslhe = volatiles released from 10 cocoon - spinning larvae during 1 hour ; 2 ) number in brackets refers to nanogram quantities present in 10 cslhe ; 3 ) (+)- limonene was not antenally active but was included in bioassay experiments as the most abundant component in extracts . 3 . response of cydia pomonella larvae to blends of natural or synthetic candidate pheromone components in olfactometer experiments in olfactometer experiments ( following the general protocol as described above ), c . pomonella larvae preferred porapak q volatile extract of cocoon - spinning c . pomonella larvae , and a synthetic blend ( sb ) of 11 candidate pheromone components , over a pentane control stimulus ( fig3 ). fig3 illustrates graphical data of responses of cydia pomonella larvae to pitfall devices baited with porapak q volatile extract of cocoon - spinning larvae ( 180 cslhe ) or a synthetic blend ( sb ) of 11 candidate pheromone components . asterisks on bars indicate a significant response to a particular treatment ; chi - square goodness of fit test with yates &# 39 ; correction for continuity ; *** p & lt ; 0 . 001 . note : 1 ) 180 cslhe = volatiles released from 180 cocoon - spinning c . pomonella larvae during 1 hour ; 2 ) components in sb consisted of : 1 . heptanal ; 2 . sulcatone ; 3 . myrcene ; 4 . octanal ; 5 . 3 - carene ; 6 . (+)- limonene ; 7 . ( e )- 2 - octenal ; 8 . nonanal ; 9 . ( e )- 2 - nonenal ; 10 . decanal ; 11 . geranylacetone . similar attractiveness of porapak q extract containing natural cocoon volatiles and the synthetic 11 - component blend ( sb ) strongly suggested that all essential pheromone components were present in sb . of the 4 doses of sb ( 1 , 10 , 100 , 1 , 000 cslhe ) bioassayed , 100 sb elicited the strongest response by c . pomonella larvae ( fig4 ). fig4 illustrates graphical data of responses of cydia pomonella larvae in pitfall olfactometer experiments to 4 doses of a synthetic blend ( sb ) containing 11 candidate pheromone components . bars with an asterisk indicate a significant preference for a particular treatment ; chi - square goodness of fit test with yates &# 39 ; correction for continuity ; ** p & lt ; 0 . 005 . note : components in sb consisted of : 1 . heptanal ; 2 . sulcatone ; 3 . myrcene ; 4 . octanal ; 5 . 3 - carene ; 6 . (+)- limonene ; 7 . ( e )- 2 - octenal ; 8 . nonanal ; 9 . ( e )- 2 - nonenal ; 10 . decanal ; 11 . geranylacetone . to determine the critically important components in sb , individual or groups of pheromone components were deleted and such reduced blends bioassayed in olfactometer experiments ( following the protocol described above ). sb lacking monoterpenes [(+)- limonene , myrcene , 3 - carene ] or ketones ( sulcatone , geranylacetone ) still elicited responses from c . pomonella larvae , whereas sb lacking aldehydes [ heptanal , octanal , nonanal , decanal , ( e )- 2 - octenal , ( e )- 2 - nonenal ] was completely unattractive ( fig5 ). fig5 illustrates graphical data of responses of cydia pomonella larvae to pitfall devices baited with synthetic blends ( sb ) lacking either ketones [ sulcatone , geranylacetone ], monoterpenes [(+)- limonene , myrcene , 3 - carene ], or aldehydes [ heptanal , octanal , nonanal , decanal , ( e )- 2 - octenal , ( e )- 2 - nonenal ]. asterisks on bars indicate a significant preference for a particular treatment ; chi - square goodness of fit test with yates &# 39 ; correction for continuity ; * p & lt ; 0 . 05 ; ** p & lt ; 0 . 01 . sbs lacking saturated aldehydes [ heptanal , octanal , nonanal , decanal ] still elicited significant responses from c . pomonella larvae , whereas sbs lacking either unsaturated aldehydes [( e )- 2 - octenal , ( e )- 2 - nonenal ], sulcatone , or geranylacetone were as ineffective as pentane controls in eliciting response from c . pomonella larvae ( fig6 ). fig6 illustrates graphical data of responses of cydia pomonella larvae to synthetic blends ( sb ) lacking either saturated aldehydes [ heptanal , octanal , nonanal , decanal ], unsaturated aldehydes [( e )- 2 - octenal , ( e )- 2 - nonenal ], or individual components . the asterisk on bars indicates a significant preference for a particular treatment ; chi - square goodness of fit test with yates &# 39 ; correction for continuity ; * p & lt ; 0 . 01 . a rudimentary synthetic blend ( rsb ), comprising ( e )- 2 - octenal , ( e )- 2 - nonenal , sulcatone and geranylacetone , did not elicit a behavioural response by c . pomonella larvae . however , rsb in combination with either the monoterpene 3 - carene , saturated aldehydes ( octanal , nonanal , decanal ), or 3 - carene plus said aldehydes , induced significant attraction / arrestment by c . pomonella larvae ( fig7 ). fig7 illustrates graphical data of responses of cydia pomonella larvae to a 4 - component rudimentary synthetic blend ( rsb ) [( e )- 2 - octenal , ( e )- 2 - nonenal , sulcatone , geranylacetone ] alone or in combination with either 3 - carene , saturated aldehydes [ octanal , nonanal , decanal ], or both . asterisks on bars indicate a significant preference for a particular treatment ; chi - square goodness of fit test with yates &# 39 ; correction for continuity ; * p & lt ; 0 . 01 ; ** p & lt ; 0 . 005 . the 11 - component synthetic blend ( sb ), when tested at the low dose of 10 cocoon - spinning larval hour equivalents and at natural compound ratios ( fig2 ), did not induce behavioural responses by c . pomonella larvae . however , a 10 - fold increase of both ( e )- 2 - octenal and ( e )- 2 - nonenal , but not of either component singly , in sb resulted in significant attraction / arrestment of c . pomonella larvae ( fig8 ). fig8 illustrates graphical data of responses by cydia pomonella larvae to a synthetic blend ( sb ) of 11 components at natural ratios ( fig2 ), or at a 10 - fold increase of either or both ( e )- 2 - octenal and ( e )- 2 - nonenal . the asterisk indicates a significant preference for a particular treatment ; chi - square goodness of fit test with yates &# 39 ; correction for continuity ; * p & lt ; 0 . 05 . note : sb was tested at 10 cocoon - spinning larval hour equivalents . in on - tree experiments , maple ( acer spp .) trees ( 10 - 16 cm diameter at a height of 45 cm ) were banded with corrugated cardboard strips ( 5 cm wide ) 45 cm above ground . strips were divided into two halves , with test stimuli applied to the waxed , center portion of each half . for each replicate in all experiments , twenty 5 th instar c . pomonella larvae were released from a thin , circular platform affixed to the base of the tree &# 39 ; s main branch crotch (˜ 1 . 50 m above ground ). experiments were initiated after dusk , and numbers of c . pomonella larvae cocooning in treatment or control halves of corrugated cardboard strips recorded 10 - 12 hours later . in apple orchards ( experiments 39 - 40 ), trees were banded with cardboard band prototype traps ( 4 in . wide ) ( phero tech , inc ., delta , canada ) impregnated with synthetic pheromone or left unbaited . traps comprised a continuous and central ployurethane film (˜ 3 cm wide ) impreganted with the 8 - component pheromone blend at various doses on corrugated cardboard strips . traps were placed on tree trunks or primary branches ≧ 1 m above ground (≧ 1 ft . circumference ). experiments were conducted in kelowna , bc between aug . 23 - 27 , 2004 just before mature , 5 th instar larvae began exiting apple fruit . traps were collected on oct . 8 - 9 , 2004 and numbers of c . pomonella larvae cocooning in traps were recorded . in experiment 31 , significantly more c . pomonella larvae cocooned in treatment halves of corrugated cardboard strips , bearing twenty - five 1 - day - old c . pomonella cocoons with female larvae / prepupae inside , than in unbaited control halves . similarly , in experiment 32 significantly more c . pomonella larvae cocooned in halves of corrugated cardboard strips baited with a synthetic blend ( sb ) of pheromone components than in halves treated with a solvent control ( fig9 ). fig9 illustrates graphical data of responses by cydia pomonella larvae in on - tree experiments 31 ( 12 replicates ) and 32 ( 18 replicates ) to corrugated cardboard ( cb ) strips . treatment halves of cb strips carried twenty - five 1 - day - old cocoons containing female c . pomonella larvae / prepupae ( exp . 31 ), or were baited with a synthetic blend ( sb ) of 11 components at 1 , 000 cocoon - spinning larval hour equivalents ( exp . 32 ). control halves were bare ( exp . 31 ), or were impregnated with the equivalent amounts of solvent ( exp . 32 ). asterisks indicate a significant preference for a particular stimulus ; wilcoxon paired - sample test ; * p & lt ; 0 . 01 ; ** p & lt ; 0 . 005 . in on - tree experiments 33 - 35 , the synthetic blend ( sb ) at 1 , 000 cocoon - spinning larvae hour equivalents ( cslhe ), but not at 100 or 10 , 000 cslhe , attracted / arrested significantly more c . pomonella larvae than did a solvent control stimulus ( fig1 ). fig1 illustrates graphical data of responses by cydia pomonella larvae in on - tree experiments 33 ( 18 replicates ), 34 ( 18 replicates ), and 36 ( 12 replicates ) to corrugated cardboard ( cb ) strips impregnated with a synthetic blend ( sb ) of 11 components at 100 , 1 , 000 , or 10 , 000 cocoon spinning larvae hour equivalents , or a solvent control . the asterisk indicates a significant preference for a particular stimulus ; wilcoxon paired - sample test ; * p & lt ; 0 . 01 . in on - tree experiment 36 , significantly more c . pomonella larvae cocooned in halves of corrugated cardboard ( cb ) strips impregnated with a synthetic blend ( sb ) of 11 components , tested at 100 cocoon - spinning larval hour equivalents with increased amounts (× 10 ) of ( e )- 2 - octenal and ( e )- 2 - nonenal , than on halves impregnated with a solvent control ( fig1 ). fig1 illustrates graphical data of responses by cydia pomonella larvae in on - tree experiment 36 ( 12 replicates ) to corrugated cardboard ( cb ) strips impregnated with a synthetic blend ( sb ) of pheromone components or a solvent control . the asterisk indicates a significant preference for a particular stimulus ; wilcoxon paired - sample test ; * p & lt ; 0 . 02 . note : sb was tested at 100 cocoon - spinning larval hour equivalents with increased amounts (× 10 ) of ( e )- 2 - octenal and ( e )- 2 - nonenal . in concurrently run on - tree experiments 37 and 38 , significantly more c . pomonella larvae cocooned in halves of corrugated cardboard strips impregnated with a synthetic blend ( sb ) of 11 components at doses of 1 , 000 cocoon - spinning larval hour equivalents ( cslhe ), or a modified synthetic blend at 100 cslhe , than in halves impregnated with the equivalent amount of a solvent ( pentane ) control ( fig1 ). fig1 illustrates graphical data of responses by cydia pomonella larvae in concurrently run on - tree experiments 37 ( 24 replicates ) and 38 ( 24 replicates ) to halves of corrugated cardboard ( cb ) strips impregnated with synthetic blends ( sb ) of 11 components . an asterisk indicates a significant preference for a particular stimulus ; wilcoxon paired - sample test ; * p & lt ; 0 . 005 . note : in experiment 38 , the sb at 100 cocoon - spinning larvae hour equivalents contained increased amounts (× 10 ) of ( e )- 2 - octenal and ( e )- 2 - nonenal . in experiment 39 in an apple orchard , the mean number of cydia pomonella larvae that cocooned in corrugated cardboard traps affixed to tree trunks and baited with the 8 - component pheromone blend at 10 , 000 cocoon - spinning larvae hour equivalents ( cslhe ) was significantly greater than the mean number of larvae that cocooned in traps left unbaited ( fig1 ). fig1 illustrates graphical data obtained in an apple orchard in experiment 39 of mean (± sem ) numbers of cydia pomonella larvae captured in cardboard band prototype traps impregnated with the 8 - component synthetic blend ( sb ) of pheromone or left as unbaited controls . bars with different letter superscripts indicate statistical significance between treatments ; student &# 39 ; s t ; p & lt ; 0 . 02 . note : components in sb consisted of : 1 . sulcatone ; 2 . octanal ; 3 . 3 - carene ; 4 . ( e )- 2 - octenal ; 5 . nonanal ; 6 . ( e )- 2 - nonenal ; 7 . decanal ; 8 . geranylacetone . in experiment 40 in an apple orchard , the mean number of cydia pomonella larvae that cocooned in traps baited with the 8 - component pheromone blend at 100 , 000 cocoon - spinning larvae hour equivalents ( cslhe ) was significantly greater than the mean number of larvae that cocooned in traps baited with 1 , 000 cslhe or in traps left unbaited . moreover traps baited with 10 , 000 cslhe captured on average significantly more larvae than traps baited with 1 , 000 cslhe ( fig1 ). fig1 illustrates graphical data obtained in an apple orchard in experiment 40 of mean (± sem ) numbers of cydia pomonella larvae captured in cardboard band prototype traps impregnated with various doses of the 8 - component synthetic blend ( sb ) of pheromone . bars with different letter superscripts indicate statistical significance between treatments ; anova with student &# 39 ; s t mcp ; p & lt ; 0 . 005 . note : components in sb consisted of : 1 . sulcatone ; 2 . octanal ; 3 . 3 - carene ; 4 . ( e )- 2 - octenal ; 5 . nonanal ; 6 . 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