Patent Application: US-201614994752-A

Abstract:
the present disclosure discusses dodecan - 12 - olide , and formulations thereof , and its use in detection surveys of , and in mitigation methods for , emerald ash borer beetle infestations of ash trees .

Description:
the natural sex pheromone ( 3z )- dodecen - 12 - olide and its geometrical isomer , ( 3e )- dodecen - 12 - olide , are both electroantennogram ( eag ) active . both compounds , in combination with ( 3z )- hexenol , effectively trap eabs ( mostly males ) in green prism traps deployed in the ash tree canopy . the structure of ( 3e )- dodecen - 12 - olide is shown below : chemoreceptors are very highly compound specific , as discussed by xu et al , in 2012 ( xu p . garczynsk , s f , atungulu e , syed z , choo y - m , vidal d m , zitelli c h , leal , w s ( 2012 ) moth sex pheromone receptors and deceitful parapheromones . plos one 7 : 1 - 9 ). in an effort to identify biologically active analogs of the ( 3z )- lactone , the authors of the present disclosure tested various cyclic lactone esters . examples of tested analogs include : 2e - dodecan - 12 - olide , pentadecan - 15 - olide , and dodecan - 12 - olide . the structures of 2e - dodecan - 12 - olide , pentadecan - 15 - olide , and dodecan - 12 - olide are shown below : while 2e - dodecan - 12 - olide and pentadecan - 15 - olide were found to have electroantennogram activity with male or female eabs that was not significantly different from the controls , it was surprisingly found that dodecan - 12 - olide showed electroantennogram activity with both male and female eabs that was comparable to the corresponding electroantennogram activity both the natural ( 3z )- lactone and the ( 3e )- lactone analog . given the lack of electroantennogram activity with 2e - dodecan - 12 - olide and pentadecan - 15 - olide , it would not have been possible to predict a priori the activity of dodecan - 12 - olide . the activity of dodecan - 12 - olide in bioassays confirmed that the compound could be used as a sex pheromone analog . in some examples , the present disclosure provides an attractant that includes dodecan - 12 - olide . when placed in an ash tree that is suffering from tissue damage , green leaf volatiles released by the ash tree may combine with the released dodecan - 12 - olide to attract male eabs to the attractant . in preferred embodiments , the present disclosure provides an attractant that includes dodecan - 12 - olide and a green leaf volatile , such as ( 3z )- hexenol . a green leaf volatile is a volatile organic compound that is released when a plant suffers tissue damage . green leaf volatiles include aldehydes , esters , and alcohols of 6 - carbon compounds released after wounding or insect feeding . in the context of the current specification , green leaf volatiles refers to volatiles released by ash trees . other examples of green leaf volatiles that may be used in combination with dodecan - 12 - olide include : ( 2e )- hexenol , hexanal , ( 2e )- hexenal , and congeneric acetates of these alcohols . the attractant may include a lure that releases the dodecan - 12 - olide and a source of green leaf volatile that releases the volatile organic compound . the source of green leaf volatile may be a vessel , such as a pouch , containing a volume of the green leaf volatile . the vessel is preferably made of polyethylene since polyethylene is not dissolved by the organic compound . green leaf volatiles effuse through the polyethylene vessel and are released into the environment . the rate of effusion is dependent on , among other things , the surface area of the vessel and the thickness of the walls of the vessel . the walls of the vessel used to release the green leaf volatile may be from about 4 mils ( that is 0 . 004 inches ) to about 8 mils thick . the vessel containing the green leaf volatile is preferably sized and shaped to release the green leaf volatile at a rate from about 40 mg / day to about 400 mg / day . the walls are preferably 6 mils thick , which provides a release rate of the green leaf volatile that is from about 50 to about 100 mg / day , depending on the weather . in attractants that either include or lack the green leaf volatile , the dodecan - 12 - olide may be dissolved in a carrier solvent and applied to the lure . one exemplary carrier solvent is dichloromethane . the dodecan - 12 - olide may be applied to the lure , such as a piece of rubber , resulting in a lure loaded with dodecan - 12 - olide . the lure may be made from the same natural rubber material as is used in rubber suba • seal ™ septa . the natural rubber has a release rate that is substantially constant over time for several months under field conditions . lures with a constant release rate are preferable to lures with an exponential or a first order release rate . the lure may be treated with a sufficiently high concentration of dodecan - 12 - olide to result in a release rate of from about 20 to about 200 μg of dodecan - 12 - olide per day . in particular embodiments , the lure will have a release rate of from about 40 to about 120 μg of dodecan - 12 - olide per day . in still other particular embodiments , the lure will have a release rate of from about 50 to about 90 μg of dodecan - 12 - olide per day . in specific embodiments , the lure will have a release rate of from about 60 to about 80 μg of dodecan - 12 - olide per day . in attractants that include the green leaf volatile , the attractant may include a lure loaded with sufficient dodecan - 12 - olide , and a source of sufficient green leaf volatile to release from ( a ) about 1 μg of dodecan - 12 - olide per about 5 mg of the green leaf volatile released , to ( b ) about 1 μg of dodecan - 12 - olide per about 0 . 1 mg of the green leaf volatile released . in particular embodiments , the attractant may release from about 1 μg of dodecan - 12 - olide per about 2 mg of the green leaf volatile released , to about 1 μg of dodecan - 12 - olide per about 0 . 25 mg of the green leaf volatile released . in still other particular embodiments , the attractant may release from about 1 μg of dodecan - 12 - olide per about 1 . 25 mg of the green leaf volatile released , to about 1 μg of dodecan - 12 - olide per about 0 . 5 mg of the green leaf volatile released . alternatively , the attractant may be a vessel containing a liquid mixture of the dodecan - 12 - olide and the green leaf volatile . as discussed above , the vessel is preferably made of polyethylene and the walls of the vessel may be from about 4 mils to about 8 mils thick . the rate of effusion of the dodecan - 12 - olide is dependent on the concentration of dodecan - 12 - olide in the mixture . the mixture may contain sufficient dodecan - 12 - olide mixed with the green leaf volatile to result in a release rate from the vessel of about 1 μg of dodecan - 12 - olide per about 5 mg of the green leaf volatile released , to about 1 μg of dodecan - 12 - olide per about 0 . 1 mg of the green leaf volatile released . in particular embodiments , the mixture may contain sufficient dodecan - 12 - olide to result in a release rate of from about 1 μg of dodecan - 12 - olide per about 2 mg of the green leaf volatile released , to about 1 μg of dodecan - 12 - olide per about 0 . 25 mg of the green leaf volatile released . in still other particular embodiments , the mixture may contain sufficient dodecan - 12 - olide to result in a release rate of from about 1 μg of dodecan - 12 - olide per about 1 . 25 mg of the green leaf volatile released , to about 1 μg of dodecan - 12 - olide per about 0 . 5 mg of the green leaf volatile released . the vessel containing the green leaf volatile is preferably sized and shaped to release the green leaf volatile at a rate from about 40 mg / day to about 400 mg / day . in other embodiments , the present disclosure provides dodecan - 12 - olide for use as a sex pheromone analog for eabs . in still other embodiments , the present disclosure provides the use of dodecan - 12 - olide as a sex pheromone for eabs . using dodecan - 12 - olide as a sex pheromone analog may attract male eabs to a trap that includes the dodecan - 12 - olide . the trap may be , for example , a three - sided sticky prism trap , a lindgren multifunnel trap , or another trap known in the art . three - sided sticky prism traps are preferable as they provide a large surface on which the dodecan - 12 - olide may be applied . the trap may be green or purple , but green is preferred as green traps have been found to preferably capture males . the trap may preferably be suspended in the canopy of an ash tree , for example from about 20 to about 40 feet above the ground . preferably the trap is suspended about 30 feet above the ground . the trap may preferably be suspended on the south aspect of the tree . in order to attract male eabs in a natural environment , it is desirable to release , on average , at least about 20 μg of dodecan - 12 - olide per day per attractant source . in particular embodiments , it is desirable to release , on average , at least about 40 μg of dodecan - 12 - olide per day per attractant source . in other particular embodiments , it is desirable to release , on average , at least about 60 pa of dodecan - 12 - olide per day per attractant source . dodecan - 12 - olide may be used to attract male eabs to a tree or other location that is not suitable for eab spreading . for example , dodecan - 12 - olide may be used to attract male eabs to a non - host tree ( i . e . a tree other than an ash tree , such as a conifer tree ). mating and laying eggs in the non - host tree may be fatal to at least some of the resulting larva . in yet other embodiments , the dodecan - 12 - olide may be used to overwhelm the receptors on male eab antennae and reduce or disrupt the ability of the male eabs to locate a female eab . such a disruption may reduce the rate of eab spreading , or reduce the rate of ash tree mortality caused by the eabs . in embodiments where it is desirable to overwhelm male eabs ability to locate a mate by substantially saturating the receptors of the male eabs , methods according to the present disclosure may use a plurality of high release - rate lures per tree , where each lure releases on average at least about 0 . 5 mg of dodecan - 12 - olide per day , and preferably from about 1 to about 10 mg of dodecan - 12 - olide per day . the method may use sufficient lures to result in 1 mg of dodecan - 12 - olide released per m 3 of canopy volume per day . alternatively , the methods may use a plurality of high release rate vessels , such as pouches , containing dodecan - 12 - olide . the vessel is preferably made of polyethylene since polyethylene is not dissolved by the dodecan - 12 - olide . the walls of the vessel used to release the dodecan - 12 - olide may be from about 4 mils to about 8 mils thick . the present disclosure provides a method for attracting male eabs . dodecan - 12 - olide may be applied to at least a portion of an ash tree , to a non - host tree that is close to an ash tree , in the canopy of an ash tree , or any combination thereof . for example , the male eabs may be attracted to a trap , such as discussed above , having an attractant that includes the dodecan - 12 - olide where the trap is placed in the canopy of an ash tree . in another example , an attractant that includes the dodecan - 12 - olide could be applied to a non - host tree . attracting the male eabs may disrupt their ability to locate a mate . such a disruption may reduce the rate of eab spreading , or reduce the rate of ash tree mortality caused by the eabs . dodecan - 12 - olide , or an attractant that includes dodecan - 12 - olide , may be used to disrupt eab mating or to kill eabs in a “ push - pull ” method . in an exemplary “ push - pull ” method , a number of ash trees are masked from eab detection , or eab are repelled from the ash trees , by applying a chemical stimulus on , in , or near the ash trees . simultaneously , an attractant that includes dodecan - 12 - olide and a green leaf volatile is used to attract eabs to a trap , or to another ash tree on which , or in which , a systemic insecticide is applied . the insecticide may be azadirachtin , emamectin benzoate , imidacloprid , dinotefuran , permethrin , bifenthrin , cyfluthrin , carbaryl , or a combination thereof . other insecticides known in the art may also be used . treeazin ™ is an injectible commercial insecticide formulated with azadirachtin , an extract of neem tree seeds . alternatively , the attractant may be used to attract eabs to a non - host tree . while the method is conceptually straight - forward , it is not possible to predict which chemical stimuli will “ push ” the eab toward the trap . the authors of the present disclosure believe that conifer volatiles could be used as chemical stimuli since eab larva would not survive if laid in conifer trees . in particular examples , the volatiles from a spruce tree may be used . examples of spruce tree volatiles that may be used as a masking agent in such a method include : α - pinene , β - pinene , 3 - carene , limonene , α - terpinolene , or a combination thereof . in another exemplary “ push - pull ” method , a trap is baited with an attractant that includes dodecan - 12 - olide and a green leaf volatile , such as ( 3z )- hexenol . the trap is positioned in an ash tree , as discussed above , and a plurality of high release - rate lures releasing dodecan - 12 - olide are positioned in the canopy of the ash tree , preferably around the trap and preferably at about the same height as the trap . alternatively , the trap is positioned in a non - host tree and a plurality of high release - rate lures releasing dodecan - 12 - olide are positioned in the canopy of an ash tree close to the non - host tree . in particular examples , the high release - rate lures are polycaprolactone - based lures . high release - rate lures are lures that release at least about 0 . 5 mg dodecan - 12 - olide per day . in some examples , high release - rate lures release from about 1 to about 10 mg / day . preferably , a sufficient number of high release - rate lures are used to result in 1 mg of dodecan - 12 - olide being released per m 3 of canopy volume per day . without wishing to be bound by theory , these high release rate disrupter lures are believed to disrupt eab orientation since they lack the green leaf volatile , and the eabs are instead more attracted to the trap or non - host tree that is baited with an attractant that more closely mimics the natural attractant . this compound was prepared as described by silk et al . in 2011 ( silk p j , et al ( 2011 ) evidence for a volatile pheromone in agrilus planipennis fairmaire ( coleoptera : buprestidae ) that increases attraction to a host foliar volatile . env entomol 40 : 904 - 916 ). the synthesized ( 3z )- dodecen - 12 - olide was & gt ; 98 % pure with ca . 2 % of the e - isomer . the spectral data for the prepared compound corresponded to the data reported by silk et al . in 2011 . this compound was synthesized similarly to the z - isomer , however , a modified julia - kocienski trans - olefination was used to introduce the e - olefin at position 3 . the method is described by silk et al . in 2011 and by magee et al . in 2013 ( magee d i , et al . ( 2013 ) synthesis of ( 3e )- dodecen - 12 - olide , a potential pheromone component of the emerald ash borer . synth comm 43 : 1368 - 1377 ). the synthesized ( 3e )- dodecen - 12 - olide was & gt ; 98 % pure with ca . 2 % z - isomer . the spectral data for the prepared compound corresponded to the data reported by magee et al . in 2013 . this compound was synthesized as illustrated in fig1 and 2 . fig1 illustrates the synthesis via mitsunobu cyclization using the method reported by boden et al . in 1993 ( boden c d , et al ( 1993 ) a concise , efficient and flexible strategy for the synthesis of the pheromones of oryzaephilus and cryptolestes grain beetles . synthesis 4 : 411 - 420 ). this method involves the mitsunobu esterification of ω - hydroxyacid using pph 3 and diad ( diisopropyl azodicarboxylate ) in toluene at rt . this method provided the dodecan - 12 - olide in a 55 % yield and one step from the hydroxyacid . fig2 illustrates the synthesis via baeyer - villiger oxidation ( bvo ) of cyclododecanone . this method provides the dodecan - 12 - olide in one step . reaction of cyclododecanone with meta - chloroperoxybenzoic acid ( mcpba , commercially available ) provided the dodecan - 12 - olide . this reaction ( table 1 , entry 1 ), however , proved to be extremely slow ; this was also reported by van der mee et al . in 2006 ( van der mee l , et al ( 2006 ) investigation of lipase - catalysed ring - opening polymerization of lactones with various ring sizes . kin evaluat macromol 39 : 5021 - 5027 ), who refluxed cyclododecanone and mcpba in ch 2 cl 2 for 10 d and still reported incomplete consumption of cyclododecanone . the authors of the present disclosure found that toluenesulfonic acid monohydrate -( tsoh . h 2 o )- catalyzed mcpba bvo of cyclododecanone proceeds with 99 . 8 % completion in 3 . 5 wks . ( table 1 , entry 2 ). the use of basic conditions ( 2 equiv . of nahco 3 instead of a catalytic amount of tsoh . h 2 o ) gave 98 % completion after 2 . 5 months ( table 1 , entry 1 ). a large - scale synthesis of dodecan - 12 - olide from cyclododecanone using mcpba and a catalytic amount of tsoh . h 2 o was conducted and dodecan - 12 - olide was obtained in 87 % yield . the more reactive trifluoroperoxyacetic acid ( prepared in situ from trifluoroacetic anhydride and hydrogen peroxide ) gave dodecan - 12 - olide from cyclododecanone in 11 d , 72 % yield and with complete consumption of the starting material ( table 1 , entry 3 ). also , the reagent oxone ® ( potassium peroxymonosulfate , 2khso 5 . khso 4 . k 2 so 4 ) was completely inert toward cyclododecanone when stirred at rt in dichloromethane over 2 d , and magnesium monoperphthalate hexahydrate ( mmpp )/ nahco 3 only converted ˜ 0 . 5 % of starting material cyclododecanone to product dodecan - 12 - olide when stirred at room temperature in 1 : 1 meoh : h 2 o over 1 d , and further stirring at this temperature produced no further conversion . the reagent permaleic acid converts cyclododecanone to dodecan - 12 - olide in 1 d , and the authors of the present disclosure found that cyclododecanone is cleanly converted to dodecan - 12 - olide by stirring with a solution of permaleic acid in ch 2 cl 2 at rt for 5 d in 75 % yield ( table 1 , entry 4 ). the spectral data for the prepared dodecan - 12 - olide corresponded to the data reported by taber and qui in 2013 ( taber d f , qiu j ( 2013 ) permaleic acid : baeyer - villiger oxidation of cyclododecanone . j chem educ 90 : 1103 - 1104 ). the spectral data is : r f = 0 . 24 ( 1 : 20 etoac : hexanes ). 1 h nmr ( cdcl 3 , 400 mhz ): δ 4 . 12 ( aa ′ xx ′, 2h ), 2 . 33 ( aa ′ xx ′, 2h ), 1 . 60 - 1 . 66 ( m , 4h ), 1 . 27 - 1 . 41 ( m , 14h ). 13 c nmr ( cdcl 3 , 100 mhz ): δ 174 . 1 , 64 . 5 , 34 . 6 , 27 . 4 , 26 . 6 , 26 . 4 , 26 . 3 , 25 . 35 , 25 . 31 , 24 . 9 , 24 . 5 , 24 . 2 . ir ( neat , cm − 1 ): 2928 ( m ), 2859 ( m ), 1730 ( s ), 1361 ( m ), 1380 ( w ), 1334 ( w ), 1247 ( m ), 1173 ( w ), 1140 ( m ), 1096 ( w ), 1049 ( w ). ms ( el , 70 ev ) ( main peaks ): m / z 55 ( base peak ), 69 , 83 , 98 , 110 , 123 , 125 , 129 , 138 , 151 , 162 , 169 , 180 , 198 ( m + ). hmrs for dodecan - 12 - olide : ([ c 12 h 22 nao 2 + ] calc . 221 . 15133 . found 221 . 1512 , mass measurement error of − 0 . 59 ppm ). the pentadecan - 15 - olide was obtained from sigma - aldrich . the 2e - dodecen - 12 - olide was synthesized following the scheme shown in fig3 . trees with larval eab were felled in lambton and middlesex counties , ontario , and infested logs were transported to the great lakes forestry centre in sault ste . marie , ontario . storage and rearing protocols have been previously reported by silk et al . in 2009 ( silk p j , et al ( 2009 ) a contact sex pheromone component of the emerald ash borer agrilus planipennis fairmaire ( coleoptera : buprestidae ). naturwissenschaften 96 : 601 - 608 ). emerged adults were kept on a 16 : 8 h l : d cycle and supplied with water and foliage of evergreen ash , fraxinus uhdei ( wenzig ) linglesh . these insects were sent to the atlantic forestry centre laboratory ( fredericton , new brunswick ) under a canadian food inspection agency movement certificate , placed in the quarantine facility , and used within 3 - 4 d . electroantennogram analyses ( eag ) were conducted using methods and equipment generally described by silk et al . in 2007 ( silk p j , et al ( 2007 ) evidence for a male - produced pheromone in tetropium fuscum ( f .) ( coleoptera : cerambycidae ). naturwissenschaften 94 : 697 - 701 ). antennae were excised close to the head and mounted using electrode gel ( spectra 360 electrode gel ; parker , fairfield , n . j ., usa ) on an eag probe ( gold ) for electrical contact . eag signals were recorded using syntech recording and analysis software v . 2 . 6 ( syntech , hilversum , the netherlands ). the ( 3z )- and ( 3e )- lactones , and dodecan - 12 - olide were tested using the puff technique described by silk et al . in 2011 ( silk p j , ryall k , mayo p , lemay m , grant g , crook d , cossé a , fraser i , sweeney j d , lyons d b , pitt d , scarr t , magee d ( 2011 ) evidence for a volatile pheromone in agrilus planipennis fairmaire ( coleoptera : buprestidae ) that increases attraction to a host foliar volatile . env entomol 40 : 904 - 916 ). briefly : two microliters of serially diluted solutions ( dichloromethane ; dcm ) of synthetic compounds were applied to filter paper strips ( 0 . 5 cm × 5 cm , whatman no . 1 ). the filter paper strips were placed in 14 cm long pasteur pipettes , hereafter referred to as stimulus cartridges , after 5 min at room temperature . the stimulus dose tested was 1 μg . male and female antennae were exposed to single 0 . 2 s puffs of odor - bearing air at 5 ml / s by placing the tip of a stimulus cartridge into a hole of a glass tube ( 0 . 7 cm id × 20 cm ), 10 cm from the outlet and 11 cm away from the antennal preparation . airflow through the glass tube was passed through a water bubbler and set at 10 ml / s . each antennal preparation was tested with freshly prepared sets of stimuli cartridges using ˜ 6 male and ˜ 10 female antennae / treatment and used once for each treatment . the mean responses of both male and female eab antennae to the ( 3z )- lactone , the ( 3e )- lactone , and dodecan - 12 - olide ( 1 μg source concentration ) were not significantly different ( p & lt ; 0 . 05 ; tukey &# 39 ; s ), but were significantly greater than those to the solvent control ( dichloromethane ) stimulus . the results are illustrated in fig4 , which shows mean eag responses of male and female eab to the compounds ± standard error . a y - tube olfactometer ( analytical research systems inc , micanopy , fla ., usa ) was used to test for short - range attraction of adult eab to the lactones . the glass olfactometer ( 1 . 5 cm i . d .) had an 11 - cm main stem that branched into two 9 - cm arms . each arm was connected to a cylinder that contained the stimulus . charcoal - filtered air was passed into each arm at a flow rate of 1 . 2 l / min . treatments were 1 μg each of ( 3z )- lactone , ( 3e )- lactone , and dodecan - 12 - olide . each stimulus was diluted in dichloromethane , placed on a strip of filter paper , and given 1 min for the solvent to evaporate before being placed in the olfactometer . a filter paper with only solvent ( control ) was placed in the other arm of the olfactometer . the apparatus was rinsed with acetone after each treatment , and the arm attached to the test stimulus was randomized between replicates . for each trial , a single eab ( male or female ) was given 10 min to choose between the two stimuli . a choice was recorded when the beetle passed a “ finish line ”, 7 cm beyond the branching point of each arm . “ no choice ” was recorded if the beetle failed to pass either finish line after the 10 min . beetles were 8 - 14 d old , and 18 - 23 beetles were used per treatment . in the y - tube olfactometer assay at the dosages tested , males were significantly attracted to ( 3e )- lactone ( χ2 = 4 ; df = 1 ; p = 0 . 046 ) but not attracted to the ( 3z )- lactone ( χ2 = 1 ; df = 1 ; p = 0 . 347 ). the opposite was true for females , which were somewhat attracted to the ( 3z )- lactone ( χ2 = 3 . 77 ; df = 1 ; p = 0 . 052 ) but not to the ( 3e )- lactone ( χ2 = 0 ; df = 1 ; p = 1 ). both sexes were attracted to dodecan - 12 - olide ( χ2 = 4 ; df = 1 ; p = 0 . 046 ). among both females and males , there was a high proportion that made no choice in the olfactometer (˜ 50 %). the results are illustrated in fig5 , which shows the results for the males , and in fig6 , which shows the results for the females . the y - axis indicates the proportion of the beetles that chose that arm . for example the results from the ( 3e )- lactone indicate that about 75 % chose the stimulus , and about 25 % chose the control . a chi - square goodness of fit test was used to test whether the ratio of beetles choosing the stimulus vs . the hexane control differed significantly from 1 : 1 ; * represents significant differences . three trapping experiments were conducted to test the effects of ( 3z )- lactone and dodecan - 12 - olide on mean captures of eab on dark green sticky prism traps using protocols discussed by francese et al . in 2010 ( francese j a , crook d j , fraser i , lance d a , a . j . sawyer a j , mastro v c ( 2010a ) optimization of trap color for the emerald ash borer , agrilus planipennis ( coleoptera : buprestidae ). j econ entomol 103 : 1235 - 1241 ) co - baited with ( 3z )- hexenol . urban street trees were used : trees were 20 - 40 cm in diameter , and 10 - 16 m in height . blocks were chosen based on the proximity of known infested trees at low to moderate densities ; trees used for the trapping experiment showed few obvious signs or symptoms of infestation . dark green prism sticky traps ( 0 . 30 × 25 . 00 × 58 . 75 cm ) ( synergy semiochemicals corp ., burnaby , british columbia ) were used in all field trials . traps were spaced ˜ 25 m apart , and lure treatments replicated in a randomized complete block design . traps were deployed using either a limb hook over a lower to mid - canopy branch or a single rope over a mid - canopy branch . in both years , traps were checked every 2 wk and all eab were collected , counted , and sexed . traps were baited with one of three treatments : 1 ) ( 3z )- hexenol alone ; 2 ) 3 . 0 mg dose of ( 3z )- lactone +( 3z )- hexenol ; or 3 ) dodecan - 12 - olide +( 3z )- hexenol . the dodecan - 12 - olide dose was 3 . 0 mg or 5 . 0 mg . a higher dose of the analog was used to attempt to increase its effect on captures of eab relative to the ( 3z )- lactone . ( 3z )- lactone and the analog were each loaded onto rubber septa lures ( wheaton scientific , millville , n . j .). ( 3z )- hexenol lures were obtained from synergy semiochemicals . release rates of the 3 . 0 mg ( 3z )- lactone , 3 . 0 mg and 5 . 0 mg dodecan - 12 - olide lures , and ( 3z )- hexenol were ˜ 66 μg / d , ˜ 60 μg / d and ˜ 80 μg / d . these values were determined as in silk et al . 2011 , and 50 - 100 mg / d ( by weight loss ), respectively , at 25 ° c . the ( 3z )- lactone and analog lures were estimated to maintain sustained and almost constant release rates for a minimum of 6 wk , and thus were not replaced during the experiment . similarly , ( 3z )- hexenol lures were not replaced during the experiment . mean catch of male eab varied significantly among the three treatment trials ( χ2 = 81 . 19 , df = 2 , p & lt ; 0 . 001 ) ( χ2 = 62 . 51 , df = 2 , p & lt ; 0 . 001 ) and ( χ2 = 42 . 135 , df = 2 , p & lt ; 0 . 001 ), as illustrated in fig7 . the dose of the tested compounds was the same in the results shown in fig7 a ( 3 mg per lure ). the dose of the dodecan - 12 - olide was higher ( 5 mg per lure ) than the dose of the ( 3z )- lactone ( 3 mg per lure ) in the results shown in fig7 b and 7 c . the treatment was replicated 11 times in the results shown in fig7 a , and replicated 12 times the results shown in fig7 b and 7 c . in two of three trials , traps baited with ( 3z )- hexenol + dodecan - 12 - olide captured significantly more male eab than traps baited with ( 3z )- hexenol alone ( fig7 a and 6 b ). similarly , in two of three trials , where the dose of the dodecan - 12 - olide 5 mg per lure , mean catch of eab was not significantly different between traps baited with ( 3z )- hexenol + dodecan - 12 - olide versus ( 3z )- hexenol +( 3z )- lactone ( fig7 b and 6 c ). in the trial where the traps all used 3 mg of compound per lure , traps baited with ( 3z )- hexenol + dodecan - 12 - olide captured approximately 50 male beetles while traps baited with ( 3z )- hexenol +( 3z )- lactone captured approximately 60 male beetles ( fig7 a ). dodecan - 12 - olide is about 85 % as active in these trials as the natural ( 3z )- lactone under comparable conditions . mean catch of female eab also varied significantly among the three treatments in two of the three trials ( χ2 = 80 . 693 , df = 2 , p & lt ; 0 . 001 ) and ( χ2 = 22 . 271 , df = 2 , p & lt ; 0 . 001 ). too few females ( two ) were captured in the results shown in fig7 c for analysis . in the results shown in fig7 a , significantly more females were captured on traps baited with dodecan - 12 - olide compared with the other two treatments . in the results shown in fig7 b , more females were captured on traps baited with either dodecan - 12 - olide or ( 3z )- lactone compared with ( 3z )- hexenol alone ( fig7 b ; p & lt ; 0 . 01 ); similar numbers of females were captured on traps baited with dodecan - 12 - olide vs . ( 3z )- lactone ( p = 0 . 102 ). the location of the trap was tested by deploying dark green sticky prism traps in the canopy , on the north and south aspects of the same tree . each trap was baited with 3 . 0 mg ( 3z )- lactone and ( 3z )- hexenol in low - moderate density eag populations . the results are illustrated in fig8 , which indicate that traps placed in the south aspect show increased trapping of both male and female eabs . in the context of the present disclosure , the term “ south aspect ” should be understood to refer to a position that is within 30 ° from due south . similarly , the term “ north aspect ” should be understood to refer to a position that is within 30 ° from due north . these results may be due to the increased amount of light falling on the south facing traps since the tested trees were in the northern hemisphere . different light conditions were tested for eab behaviours that are referred to as “ calling behaviours ,” which are behaviours performed by eab males leading up to mating . the results are illustrated in fig9 , which show that lamps , but not fluorescent lights , increase the number of calls by both male and female eabs . given the broad spectra of incandescent and halogen lamps , in comparison to the narrow peaks in the spectra for fluorescent lights , these data suggest that placing traps in a sunny location in the tree would increase the number of eabs attracted to the trap . a trial was performed to identify whether eabs could be pulled towards a trap baited with ( 3z )- hexenol and ( 3z )- lactone , while being pushed away from high release - rate lures that release only the ( 3z )- lactone . three groups of traps were studied in 13 replications : ( a ) control - only , where the trap was baited with ( 3z )- hexenol alone ; ( b ) attractant - only , where the trap was baited with ( 3z )- hexenol and ( 3z )- lactone ; and ( c ) attractant and disrupter ; where the trap was baited with ( 3z )- hexenol and ( 3z )- lactone and the trap was surrounded by high release rate disrupter lures releasing ( 3z )- lactone only . the results illustrated in fig1 show the total captures using four high release rate disrupter lures . the results illustrated in fig1 show the total captures using seven high release rate disrupter lures . increasing the number of high release - rate lures proportionally increased the number of eabs captured . in view of the results discussed above , the authors of the present disclosure predict that dodecan - 12 - olide could be used as a replacement for the ( 3z )- lactone . a trial was performed to identify whether a blend of volatile spruce compounds could be used to reduce the attraction of traps baited with ( 3z )- hexenol and ( 3z )- lactone . the blend of volatile compounds included : α - pinene , β - pinene , 3 - carene , limonene , and α - terpinolene . control traps baited with ( 3z )- hexenol and ( 3z )- lactone , and test traps baited with ( 3z )- hexenol and ( 3z )- lactone but masked with the blend of spruce compounds , were placed in ash tree canopies . the numbers of eabs captured per trap were measured . the results are illustrated in fig1 , which shows that trap captures for male eabs were significantly reduced on traps masked with the blend of spruce compounds ( t = 3 . 151 , df = 9 , p = 0 . 012 ), as compared to the control traps baited ( using a paired t - test on captures ). this reduction was not observed for females eabs ( t = 1 . 588 , df = 9 , p = 0 . 147 ). the authors of the present disclosure believe that any one of the volatiles could be used separately as a masking compound . for the eag results , male and female eag responses were submitted to analysis of variance ( anova ) and mean responses to each of the tested compounds separated using tukey &# 39 ; s test ( p & lt ; 0 . 05 ). for the olfactometer study , a chi - square goodness of fit test used to test whether the ratio of beetles choosing the stimulus vs . the hexane control differed significantly from 1 : 1 ( minitab ); beetles that did not select either the stimulus or the control ( i . e ., no choice ) were excluded from the analysis . finally , in the trapping study , numbers of male and female eab captured among the three treatments were analyzed separately by fitting generalized linear mixed effect models , with block as a random factor , to the poisson distribution ( glmer function , r development core team 2013 ). chi - square tests were used to determine if a significant proportion of the variation in capture of male and female eab was attributable to treatment . chi - square tests were considered significant at p = 0 . 05 or less and , if significant , the means separated using the tukey &# 39 ; s contrast option in the glht function ( r development core team 2013 ); raw data are presented as means ± standard error . in the preceding description , for purposes of explanation , numerous details are set forth in order to provide a thorough understanding of the embodiments . however , it will be apparent to one skilled in the art that these specific details are not required . accordingly , what has been described is merely illustrative of the application of the described embodiments and numerous modifications and variations are possible in light of the above teachings . since the above description provides example embodiments , it will be appreciated that modifications and variations can be effected to the particular embodiments by those of skill in the art . accordingly , the scope of the claims should not be limited by the particular embodiments set forth herein , but should be construed in a manner consistent with the specification as a whole . bartelt r , cossé a a , zilkowski b w , fraser i ( 2007 ) antennally active macrolide from the emerald ash borer agrilus planipennis emitted predominantly by females . j chem ecol 33 : 1299 - 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