Posted on Authorea 17 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158195443.34033633 — This a preprint and has not been peer reviewed. Data may be preliminary. latr ecpino odce n e inl sitmtl necnetdi net RdyadGuerrero and (Reddy insects in interconnected intimately is signals sex Varela and 2004; cues food of perception Olfactory semiochemical, control, Introduction insect sustainable behaviour, Lepidoptera reproductive Tortricidae, olfaction, kairomone, chemicals, behaviour-modifying words the for Key and herbivores insect in attraction behaviour-modifying plant of host identification of efficient control. mechanisms an that insect the volatile confirm for sustainable parallel of plant assays of predictions host development attraction for understanding powerful further and Field improved explanation afford pheromone communication. an an sex receptors chemical Our for offers a insect odorant levels for semiochemicals, PRs in of high channel ester. signals moth investigations olfactory and habitat pear budworm conserved silico ancestry, and volatile A green in social Shared plant compounds. and of the same alliance codling gene. the the and in PR to substantiates In codlemone similarity, orthologous species pheromone sequence both receptors. an sex of and odorant expresses attraction the identity dedicated moth of acid compounds, budworm response co-occurrence amino both behavioural green suggests congruent of to that A species Concomitantly, tuned finds tortricid ester. is analysis two pear males. phylogenetic PR volatile in moth plant volatile one budworm food plant moth, green the and codling attracts to pheromone pheromone attracted also same sex are Codlemone the two the males is of to moth which pomonella. blend codlemone, codling a Cydia produce and also is budworm moth Females Totricidae) green codling attraction. (Lepidoptera, male tortricid, nubiferana elicits Hedya phylogenetic another both and moth of of isolation budworm blend reproductive green a in through only of communication acetates, pheromones chemical pheromone unsaturated of sex sex the role female Investigating and the receptors into The odorants orthologous neurons. insights Studying affords divergence. sensory food chemicals. taxa olfactory relevant sense across behaviourally in ligands Insects of expressed identification their the and are chemicals. instructs which receptors volatile these (PRs), by of receptors range mediated pheromone receptive is and (ORs) food receptors and odorant mates for search The Abstract 2020 5, May 5 4 3 2 1 Unelius moths Gonzalez tortricid Francisco in signals plant sex host for and channels pheromone conserved reveals phylogeny receptor Olfactory wds nvriyo giutrlSciences Kalmar University, Agricultural Linnaeus of Sciences, University University Life Swedish Lund Biology, and Health Dept. of Lab, Faculty Evolution Hungary and Budapest, Ecology HAS, Molecular CAR Institute Protection Plant available not Affiliation 4 ilsToth Miklos , tal. et 01 Rouyar 2011; 1 eieBorrero-Echeverry Felipe , 2 ee Witzgall Peter , tal. et 05 Lebreton 2015; 5 ai Bengtsson Marie , 1 1 tal. et ui Josvai Julia , 07 Borrero-Echeverry 2017; 5 n ila Walker William and , 2 ai Strandh Maria , tal. et 3 Rikard , 08 Conchou 2018; 5 Posted on Authorea 17 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158195443.34033633 — This a preprint and has not been peer reviewed. Data may be preliminary. e hrmns CEDsffr,hwvr rmsrosba n rdcsflepstvswe screening when of positives amounts false trace produces of collections. identification and headspace and bias in detection serious amounts for from large however, tool in antenna suffers, reliable occurring entire GC-EAD and compounds the efficient biases pheromones. of an chromatog- and sex as response gas 1975), designed the is was al. measures attraction GC-EAD et host GC-EAD (Arn mediating their detection. odorants compounds with plant to electroantennographic correlate studying pathways to not for biosynthetic coupled finding. tool does main raphy plant employed headspace from host (Gonzalez widely plant specific stem apples most encode in often The green cannot amounts volatiles from and large of plants, amounts in many abundance trace by released the in shared Compounds that only design released underlines saliency. to and is 2020) behavioural techniques ester pheromone-based al. pear improve et That both (Light pheromone to kairomones sex applications. of with stand-alone combined potential (Knight or the monitoring alone demonstrates population adults, ester Knight and for larvae 2011; efficient moth Knight is pear codling and ester of (E,Z)-2,4-decadienoate, disruption Pear ethyl to behavioural is found for 2005). been compound moth Knight have plant codling and plants for host Light food attractant key larval bisexual such for a characteristic One ester, or unique attractancy. are significant that mediate compounds plant herbi- comparison, pheromones. insect In sex attract with to compared substantial (Najar-Rodriguez faint, found species meets sometimes plant been is many behaviour have across that oviposition found blends compounds al. mediate on volatile build that plant typically Synthetic vores distinctive kairomones, of or difficulties. sequence produced methodological a semiochemicals, are trigger Pheromones of immediately sexes. Identification and been both responses has in 2016), antennal response strong al. and et produce behaviours. production (El-Sayed glands, orders of main insect dedicated coordination current all in mutual a across a therefore pheromones, control. by sex is insect of facilitated for lures hundreds chemicals bisexual many behaviour-modifying or of identified of Identification female been use have Designing widespread semiochemicals more few oviposition. a only for towards mating, challenge species. females for of gravid insects hundreds attract attracting control, pheromones for that (Ridgway population to available achieve attracted even contrast are may becomes stark detection insecticides, In sex or sensitive female pathogens and insect the traps, specific Witzgall when with for 2010; combination Guerrero lures in and communication, lures, Pheromone (Reddy Such sexual insects forest premating 2015). and of Silk orchard disruption key ecol- and few safe chemical for a for environmentally pheromone, against rationale synthetic and used outstanding is with species-specific an Air-permation been a always research. as 2020). therefore 2019; Wagner ogy semiochemicals has severe 2020; al. insecticides other for conventional et al. and to accumulating (Seibold et alternative pheromones is neonicotinoids Longing of the evidence 2019; establishment insecticides, nonethless, al. of The et and family Chmiel 1962) This used 2019; (Carson widely apocalypse. al. most DDT biodiversity et currently the since Yamamuro the of debate The cause of non-target of contributing effects affects 2019). a point side pesticides is synthetic pollination a al. and of Including been overuse et arthropods, The crops. other has Jactel food webs. and 2011; human insects food terrestrial on beneficial al. all feed and to et not integral toxic (Chandler does are most however, insects insecticides the species, services, deregulate efficient insect to few of efforts Recent with majority invasions overwhelming growers 2018). insect and accelerates left Reddy al. that climate has et 2018). 2010a; changing (Deutsch a compounds insecurity 2008, al. of wake food et the aggravating al. Gregg in outbreaks, needed et 2016; and are Silk management Witzgall ecology insect and 1990; for insect Evenden tools New understanding 2014; al. detection to et al. for (Ridgway basic applied et Suckling be is insects can 2010; mates of chemicals Guerrero control and behaviour-modifying safe such food environmentally of encoding knowledge and chemicals the Moreover, the Deciphering evolution. and 2019). al. et 00 rc n ikt 01 Lu 2011; Pickett and Bruce 2010; tal. et 02 ih n ek21;Kih n ih 03.Tedsoeyo pear of discovery The 2013). Light and Knight 2012; Beck and Light 2012; tal. et 05.Teatatn oe fsc bqiospatvolatiles plant ubiquitous such of power attractant The 2015). yi pomonella Cydia tal. et 2 90 El-Sayed 1990; Lpdpea otiia)(Light Tortricidae) (Lepidoptera, tal. et 09 Suckling 2009; tal et tal. et tal. et 03 09 and 2019) 2013, . 00 Evenden 2010; 00 Tasin 2010; tal. et tal. et 2014). 2001; et Posted on Authorea 17 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158195443.34033633 — This a preprint and has not been peer reviewed. Data may be preliminary. n dlswr etudra1: ih-akccei cencgsadwr upidwt rs apple fresh with supplied were and cages screen Pupae in 1984). cycle al. light-dark et h studies, (Rauscher transcriptomic diet 18:6 For agar-based during solution. a semisynthetic sucrose (Sweden) under and a Scania kept branches and in in were leaves orchards buds adults apple apple flower with and in on fed field-collected and were were autumn Larvae larvae May. in last-instar fruit analysis, moth on pheromone codling feed For 1979). with larvae al. co-occurs The et and hemisphere. (Bradley shrubs spring Northern and the the trees throughout Rosacean apple, on on leafroller polyphagous a is moth budworm Green Insects methods and Materials management. insect of how development the demonstrates and This kairomones, (Bengtsson species. insect moth of both codling identification as in the such advance compounds, species these model al. to in et ORs dedicated presence the of channel suggest characterization antennal and olfactory the functional evidence in behavioural conserved ORs the of confirm a analysis moth phylogenetic of codling Comparative and bioassays. budworm field green moth and of laboratory budworm transcriptome in green ester, pear of to 2016). moth response and al. budworm J´osvai the et 2007, investigated Green species have al. related We et volatiles. closely (Schmidt plant a ester in and pear to response to pheromone belongs conserved and which, of which seemingly CpomOR3, interconnection CpomOR3, A nubiferana of rooted of Hedya copies 2019). deeply two ligand al. of this main et presence underscores The (Wan reveals codlemone genome 2019). pheromone, moth sex in (Light codling characterization al. ester the functional et of pear to Wan assembly according volatile recent 2017, plant A the al. 2014). is et clade, Cattaneo PR the 2014, Walker 2012, al. al. et et (Bengtsson gas analysis to transcriptome coupled moth recordings, electrophysiological codling sensillum In single with addressed choice of express be of to (GC-SSR). antenna can is tool chromatography they the approach a where of experimental accordingly 2004), powerful sensilla are A al. defined ORs compounds. Single in bioactive singly 2019). for of ORs Robertson odorscape database plant 2018, accumulating provides the an al. interrogate with annotation et to comparison (Fleischer through and from ORs ligands, ordinary ORs putative extracts insect ligand and the orthologous on deorphaned (PRs) The RNA leads groups from receptors provides analysis antennal OSNs and phylogenetic approach. pheromone Subsequent species between by Sequencing new related differentiation, odorants. a transmitted environmental functional to enables chemicals first brain. responding 1999) a volatile ORs, the and of al. in data expression et spectrum centers OR (Clyne the olfactory ORs determines to insect been ORs antenna of has of hand, code specificity other genetic the binding the on but abundance. of ester, electroantennograms, low discovery pear its produce The as to to such sufficient due compound is recordings active antenna GC-EAD cognate An the in their uncertain. on to overlooked similar is OSNs structurally relevance (OR) many are behavioural receptor from that olfactory its signal the volatiles entire of diffuse by the amounts A generated expressing large or antenna, to ligands. response, acetates the respond antennal aliphatic ORs on Typically, an (OSNs) short neurons repertoire. example elicit sensory invariably for olfactory caryophyllenes, amounts, of and large ensemble in linalools present farnesenes, compounds alcohols, Ubiquitous headspace. plant 06,floe yi iiosuiso nenltasrpoe ntetxnmclyrltdseiswill species related taxonomically the in transcriptomes antennal of studies silico in by followed 2016), yi pomonella Cydia Lpdpea otiia)i trce ocdeoe(r ta.17,E-ae,2019) El-Sayed, 1974, al. et (Arn codlemone to attracted is Tortricidae) (Lepidoptera, ey nubiferana Hedya Lpdpea otiia) pmO3hsbe erhnd following deorphaned, been has OR3 Cpom Tortricidae), (Lepidoptera, aot ( Haworth Xenopus rspiamelanogaster Drosophila dimidioalba oye,rsodt esretn lot oln moth codling to also extent lesser a to respond oocytes, 3 tal. et tal. et .nubiferana H. 01 ih n ngt20,Bntsne al. et Bengtsson 2005, Knight and Light 2001; H 06 n eeooosepeso (Bengtsson expression heterologous and 2016) ezu)(eiotr,Trrcde Fgr 1) (Figure Tortricidae) (Lepidoptera, Retzius) . nubiferana ae eecpue npheromone in captured were males Dbis ta.20;Hle et Hallem 2003; al. et (Dobritsa ocdigmt e pheromone sex moth codling to tal. et yi pomonella Cydia 04 Gonzalez 2014; Posted on Authorea 17 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158195443.34033633 — This a preprint and has not been peer reviewed. Data may be preliminary. h emti smr of isomers geometric The with co-injection by determined trapping 80 was Field from position programmed spectra. bond USA), mass of CA, Double evaluation Folsom, compounds. by Scientific, 7 and synthetic (J&W samples in carrier and synthetic column as 15 natural DB-Wax used to of mm was Helium 5 10 0.25 spectra GC. at of x 5890 min) with Packard m batches 2 instrument, Hewlett 30 (hold B in a the 5970 a with extracted Packard towards interfaced on Hewlett were eV), period, gas a females (70 on calling ionization old done impact the was 4-d electron of (GC-MS) (B to spectrometry onset min 2- chromatography-mass the 1 gas of for at Glands hexane dissected scotophase. redistilled were the glands of pheromone end sex abdominal analysis Female chemical and extraction gland Pheromone ( of 10:1:5-blend ( a acetate with baited traps ihTio eemnal ooeie ihapsl.Tetb a lcdi iudntoe n then another and and pestle nitrogen a liquid with in again placed homogenized was then was tube sample tube The The Eppendorf the temperature. pestle. in room a 500 held at antennae with thaw Briefly, homogenized to Netherlands). allowed manually The Venlo, were (Qiagen, purification Trizol column Kit with spin Mini and protocol RNeasy extraction the Trizol-based following with purified and extracted 500 was tube RNA Thereafter, Total microcentrifuge 1.5-mL nitrogen. a into liquid transferred in antennae. and excised held forceps with Germany) dissected Hamburg, were (Eppendorf, males adult 100 source. of onset extraction the Antennae after behaviour RNA at of h landing and types 3 and following to antennae source, The 1 of a the day, days. towards one Dissection different by cm on on blown 100 tested males), over was were 60 upwind males = Air flying (n 15 flight, individually, times of 2001). taking flown four batches were were Two tested recorded: Males males was were al. stimulus. 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Data may be preliminary. sdt aclt h etfitn oe o oprsni EA otae(auae l 03.Then, 2013). al. et (Tamura software MEGA6 in comparison were for sequences model Aligned fitting parameters. best default the and matrix, calculate scoring to FFT-NS-i JTT200 used the with with method, http://mafft.cbrc.jp/alignment/server/phylogeny.html) of refinement 7.220; iterative PRs (version putative online with MAFFT comparison for 2015), used al. from et receptors (Corcoran pheromone predicted of Sequences analysis kilobase Phylogenetic per 2010). fragments 0.1.19; (Trapnell as (version (FPKM) estimates Samtools reads abundance and transcript mapped 2009) of million measurement al. per allowing et transcript used, 1.2.12; Langmead were (version of package 0.12.6; 2009) software (version RSEM al. analysis. 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Data may be preliminary. so rcia motnefrteipeetto fpeooa oto fcdigmt n efolr in leafrollers and moth which codling 2015), of al. control et pheromonal phe- of Porcel single implementation 1995; with the achieved Minks orchards. for be (Card´e olfactory and European importance may dedicated blends practical moths a pheromone of in via incomplete disruption is codlemone communication or of that effect compounds idea behavioural romonal a the ester corroborating supports of 2), pear This attraction (Table channel. to diminished traps codlemone strongly pheromone of codlemone to addition with les treatments that 5). disruption showed (Schmidt (Table mating and sex ester Orchard either this pear of confirmed to attraction Hungary respond enhance in not to test does reported field been further A also 2016). has moth budworm F(7,72)=4.22, budworm green 4; Green increased (Table F(7,72)=2.62, significantly males moth 4; isomers (Table codling geometric fewer alone three budworm captured codlemone its green over and no captures codlemone and moth moths of codling blend fewer a far Interestingly, codling to attract 3). addition acetate (Table in codlemone all males, trap and moth at the budworm moths codlemone that green confirms of few moth Blends captured pea regularly acetate moth. alone of codlemone Attraction codlemone that with moth. corroborates baited budworm field traps green pea attract a not released to does lures adjacent compound orchard single apple a an as in test trap ester field A a pear by and revealed and be codlemone all, cannot to that at Attraction information released carry are also they may whether compounds show of will blend effluvium test. full female trapping biosynthetic The the be of may ratio. attraction study which on these A at effect Adding apparent no precursors. 2). with or captures. (Table glands by-products catch female significant from trap of identified produce diminished compounds Blends not gland slightly The did blend acetate significant. codlemone, 3-component alcohol, not the analogous was to the compounds difference or the monoenes but 10-12 orchards, apple untreated (F(7,72)=61.95, effect synergistic compound, main The of pheromone sex of major attraction The Field 1979). al. et 1). (Frerot (Table acetates identified ( isomers previously geometric the to addition compound in compounds, moth further budworm eight green of Analysis identification pheromone Sex Results inferred support bootstrap with model JTT+F+G the using replicates. constructed 500 was from Tree Likelihood Maximum a E 8, E E 8, 01A a copne ytemnustrtd8 n 0ddcnlaeae,isthree its acetates, 10-dodecenyl and 8- monounsaturated the by accompanied was 10-12Ac .nubiferana H. E EZ .nubiferana H. 01A thg smrcprt Tbe3 izale l 93 96.I comparison, In 1996). 1993, al. et Witzgall 3; (Table purity isomeric high at 10-12Ac E , ZE 8, E and , 01A yisl a o trcie hl diinof addition while attractive, not was itself by 10-12Ac Z sabedof blend a is P ae ocmonsietfidfo h eaegadcnrsta the that confirms gland female the from identified compounds to males H 8, < Z .01.Adto of Addition 0.0001). . 01A)a ela h nlgu loo codlemone, alcohol analogous the as well as 10-12Ac) nubiferana E 8, E hrmn ln xrcsb CadG-Sshowed GC-MS and GC by extracts gland pheromone 6 01A and 10-12Ac P E 0015 El-Sayed =0.02135; P -2cfrhricesdml trcinin attraction male increased further 8-12Ac 0043.I otat hsioe blend isomer this contrast, In =0.04413). Z -2c(al ;Feo tal et Frerot 2; (Table 8-12Ac E 8, tal et tal. et E Z 01A n the and 10-12Ac -2chdastrong a had 8-12Ac 1998). . .nubiferana H. 07 J´osvai2007; E E 8, 8, E E 10-12OH . 10-12Ac 1979). tal. et ma- [?] Posted on Authorea 17 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158195443.34033633 — This a preprint and has not been peer reviewed. Data may be preliminary. pmR9wspeitdo h ai faioai eunesmlrt Gnae ta.2015). al. et (Gonzalez CpomOR19 similarity sequence orthologs acid receptor amino the of to basis transcripts compares the abundant This on most 2016). predicted the al. was CpomOR3 among et CpomOR19 that Walker are ( show 2B; CpomOR3 SlitOR19 3) (Figure and (Figure and antenna HnubOR3 analysis male addition, similarity the In sequence in close. with are together HnubOR3 lepidopteran 2A), the and (Figure of 2016). moths part al. tortricid is et of Walker compound, by 2014; that volaile 2012, corroborated plant hypothesis a al. been The to et meanwhile tuned (Bengtsson and has albeit clade cells (PR) CpomOR3, and receptor 2019). kidney 2016) al. pheromone embryonic of al. et human Wan neurons et in 2017, Gonzalez sensory al. expression principal 2014; olfactory after its al. in assays as et luminescence CpomOR3 ester (Bengtsson of pear in (SSR) expression sensilla established recordings antennal heterologous has T1 through OR, and achieved moth ab3 CpomOR3 was codling and as This a (McBride such ligand. CpomOR3, selection clades, of taxonomic receptors strong roles. characterization orthologous across adapative under Functional and 2015, play conserved are 2019) likely al. are 2), Robertson genes et which (Figure 2016, (Corcoran OR genes, HnubOR3 al. tortricids Insect ancestral and et other Arguello shared 2018). in 2009; found from fruit al. al. been descending the et et has in Sanchez-Gracia Rochas ORs 2007, ORs of 2015, Arguello number 39 al. similar employs a et perception num- and Steinwender finite olfactory 2016) a al. Peripheral on et signals. depend (Walker odour behaviour, relevant reproductive insect encoding fly of ORs components functional behaviour. of essential olfactory and the of ber driving behavioural finding, semiochemicals transcriptomes by mate of antennal and informed identification the Food the receptors, with for olfactory hypotheses correlates of sound ester, phylogenetics generates pear Molecular data, and species. codlemone two kairomone, these and moth ester pheromone budworm sex pear green that and finding empirical codlemone The to response moth budworm Green magnitude of orders two Discussion or one HnubOR6 were were antennae expressed male in highly detected most PRs the putative 2B). that (Figure 3 showed lower other sequences The 3). predicted HnubOR2. (Figure the and sequences species of protein PRs both the estimation putative of of these Abundance orthologs length between OR3 entire differences acid the the Amino while across respectively. similarity, similarity, observed 66% and are identity and 76% identity and acid 64% amino shared 49% shared HnubOR2 and above values boostrap was HnubOR6 by Notably, supported were in relationships receptors to these homology and displayed 95. CpomOR22) these of and Several CpomOR6 2A). (CpomOR3, (Figure species tortricid other from (including sequences from OR PRs putative lists Predicted (NCBI) Information Biotechnology ”HnubOR##”. for as Center PRs) National The moth. budworm nubiferana Hedya expression antennal and analysis Phylogenetic rspiamelanogaster Drosophila pdpealittoralis Spodoptera aot and Haworth H > . 0 iia oCoO6 eunecmaio nlssrvae htCpomOR1 that revealed analysis comparison Sequence CpomOR6. to similar 50% nubiferana Mnze l 04 rb ta.21) 8Osi oln moth codling in ORs 58 2015), al. et Grabe 2014, al. et (Menuz .nubiferana H. rspiamelanogaster Drosophila ey dimidioalba Hedya .Floigfntoa hrceiaino ltR9 iadant of affinity ligand SlitOR19, of characterization functional Following ). ecie eretrvaHuO3i asmnos Rphylogeny PR a parsimonious: is HnubOR3 via ester pear perceives lsee n4dffrn ufml ldswe oprdwt PRs with compared when clades subfamily different 4 in clustered .nubiferana H. 7 ezu r yoyostxnmcnmsfrgreen for names taxonomic synonymous are Retzius olwdb igesnilmelectrophysiological sensillum single by followed , ae epn ocdigmoth codling to respond males Xenopus oye Ctae et (Cattaneo oocytes yi pomonella Cydia C .pomonella C. . pomonella Posted on Authorea 17 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158195443.34033633 — This a preprint and has not been peer reviewed. Data may be preliminary. hc naibypouea nenlrsos.Rcrig rmsnl eslapoieaslto,but solution, provide compounds, a provide abundant not sensilla most do single the recordings from by Recordings antennal biased response. that antennal also is an are volatiles, produce recordings invariably plant which GC-EAD with activity. (Arn working behavioural identification when pheromone on sex conundrum, information for The conceived coupled 1975). originally recordings, was al. electroantennogram method et GC-EAD on The relies chromatograph. metabolites gas is a plant Light to example active Knudsen 2011, behaviourally Another 2015; on Tasin Knight 2016). research and Light and Current Knudsen 2015, Light 2006; moth Kovanci 2008; al. 2013, fruit et Light al. apple (Bengtsson and for et compounds Knight lure (Schmidt 2012, stand-alone kairomone feeding al. a efficient is et and males an and Knight moth host-finding disruption 2012; codling larval communication Beck monitoring pheromone-based of and for supplement and disruption efficient to adults is for in used example, feeding be tool or for can oviposition ester, it for elicit Pear females, challenge sites, applications. and mating multiple research in to to urgent recognition insects leads attract host and larvae which mediate volatiles bottleneck plant al. metabolites current of et volatile Availability A to plant (Khan 2015). which brought profile al. of be release et understanding insects. et also metabolite phytophagous our Tamiru (El-Sayed can modified is 2015; chemicals trapping advancement with al. behaviour-modifying mass further plants of et and or know-how Stenberg air-permeation techniques The 2014; by 2010a). push-pull control, al. through et insect application Witzgall for 2009; tools al. efficient are Semiochemicals the to silico respond In they that hypothesize moth we fruit Oriental GmolOR11, of and ( compounds GmolOR1 components pheromone to pheromone close acetate are minor which HnubOR7b, and izale l 00) addt Rfor PR candidate A 2010b). al. and et acetate, Witzgall codlemone repertoire. and PR codlemone the of in redundancies by species, by tracked related facilitated subsequently Three are are which shifts production, Such pheromone sex. communication female new male in that the shifts suggesting saltational concepts, through 1992) (Phelan arise to tracking” channels added ”asymmetric and when 2003) 1974; in Dietrich antagonist, al. channels 2002, or pheromone et (Hathaway two synergist respectively of (Catteaneo pheromone amounts, Presence acetate large a codlemone and with is 2001). small to al. acetate together at HnubOR6 et phylogeny, codlemone codlemone Witzgall and PR compound moth, pheromone CpomOR6 1990). codling main and In al. the codlemone, 2016). et to al. probably (Bengtsson tuned 2010b), et groups alcohol 1996, are functional 1993, analogous HnubOR2 1991, differentiate the and al. moths of in et Tortricid levels differences (Witzgall expression 3). resolution steric 2, high to (Tables at due compounds not pheromone is acetate acetate from codlemone while compound, Codlemone channels. olfactory nubiferana separate employs moth pheromone channels budworm moth olfactory green distinct of employs Attraction codlemone and pheromone of sex range to host Attraction broad The 2). 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(Figure HnubOR1 is -codlemone ryeti conjugella Argyresthia E Fgr ;Wle ta.21) ugssta CpomOR1 that suggests 2016), al. et Walker 2; (Figure oismliopnn e hrmn n ocodling to and pheromone sex multicomponent its to 8 and odplants. food ae srmnseto h hpflmntr (Baker monster” ”hopeful the of reminiscent is males --oeey ctt Tbe ,3,wihaemain are which 3), 2, (Tables acetate )-8-dodecenyl .salicella H. E Cree l 1979). al. et (Carde 8, E 01A,snecdeoei ciea single as active is codlemone since 10-12Ac, E r etatatdt the to attracted best are 8, tal. et ae ncaatrsi otplant host characteristic on based , E C 01O osntmmcthe mimic not does 10-12OH . pomonella 08 2017). 2008, hnto than .molesta G. H Z . , E nubifera- overlaps isomers nvivo in H. Posted on Authorea 17 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158195443.34033633 — This a preprint and has not been peer reviewed. Data may be preliminary. n hi eaiua n clgclfntoswl otiuet h td fpyoeei iegnein divergence of phylogenetic development of the drives control. study also insect research the insect sustainable this genes to in and that olfactory efficient contribute isolation perspective of for the reproductive will analysis semiochemicals is to functions rewarding complete rise Equally ecological more gives insects. A and phytophagous selection 2017). behavioural sexual Rosenthal al. their Occurrence and 2002, et 2012). and to natural (Borrero-Echeverry Boughman al. interlinked support of 1978, are et further combination (Paterson communication lends Regier herbivores a sexual attraction 1979; that and plant al. host recognition and et plant and 2018) of (Bradley host finding attraction mate that tribes consistent to concept tortricid contributing for the different genes explanation to olfactory an conserved belong to provides of and but 2019) ester apple, al. pear on to et feed (Wan responds CpomOR3 codlemone that to discovery al. . also recent et extent the (Trona but lesser ester deorphaned, a been pear not and confirmed has the been codlemone HnubOR3 of indeed between imaging has interaction functional to This and synergistic project 2010). neurons these powerful 2013). Benton that projection 2010, a encoding and olfactory OSNs showing Ramdya circuits from 2009, lobe, in recordings the intracellular al. antennal expressed between by et moth, interactions be Krieger codling facilitating 2005, to in al. lobe, tend et antennal similarity (Couto Bengtsson the plant signals sequence 3; in the highest 2A, glomeruli to (Figure with neighbouring clade tuned genes receptor is OR pheromone the CpomOR3 consequences: to association. belongs Walker this it 2014; while confirm ester, context pear phylogenetic volatile evi- and phytophagous further in data provides signals Transcriptome environmental it and because social intriguing, to is dedicated channels codlemone olfactory insects. and of al. ester association et the pear for Tian to dence 2018; attraction 2016, moth al. al. budworm et et pheromones Green Rochas Jia and 2018; 2016; volatiles al. al. plant et et Du of Dong 2017; ParkInteraction 2015; al. 2015; al. al. et et et Yang Li Zhang 2017; 2015; 2015; al. al. al. et et Feng et al. Koenig 2017; 2015; Zeng et 2018) al. al. 2015; Cao et et al. 2013; Corcoran Chang et 2017; al. 2018; 2014, Steinwender et al. 2015; Zhang et al. Zhang (e.g. et 2014; transcriptomes al. antennal et knowledge lepidopteran Jiang thorough 2014; of our of database view accumulating in flies, rapidly Gonzalez fruit 2004; tephritid example, al. for et include, of work Hallem future 2003; for targets al. Promising et (Dobritsa ligands 2016). OR al. of highly et identification highlight of unambiguous expression in transcriptomes for heterologous neurons Furthermore, Antennal approach is sensory targets. ORs. research ligands select prime insect putative in become their of ORs which concerning ORs, database sex-specific hypotheses accumulating or of and Iden- rapidly expressed characterization volatiles. sequencing, the plant functional RNA by behaviour-modifying antennal with about facilitated following combination predictions ORs, powerful in affords of sequences, species, tification model gene in OR ORs of selected analysis Phylogenetic research. silico volatiles In plant not pheromones, investigating when results conclusive (B produced recordings SSR moth, than codling other sensilla from recordings nubiferana cmne l 00 neoe l 2005). al. et Ansebo 2000; al. et ¨ ackman Drosophila dnicto fO iad o mre sa potn dacmn o ln semiochemical plant for advancement opportune an as emerges now ligands OR of identification ocdeoe(als3 ;Ane l 94.Cdigmoth Codling 1974). al. et Arn 4; 3, (Tables codlemone to tal et R (Liu ORs 06.Ta R epn opeooe n ln oaie a vnphysiological even has volatiles plant and pheromones to respond PRs That 2016). . tal et 06 unhadGlza21)o oh rmsvrlfmle,addb a by aided families, several from moths or 2016) Galizia and Muench 2016; . Drosophila s . trichodea nbe igesnilmrcrig sacnein n reliable and convenient a as recordings sensillum single enables otiigpeooesniienuos r eiae In delicate. are neurons, pheromone-sensitive containing , 9 .pomonella C. and .nubiferana H. tal et 2012, . both H Posted on Authorea 17 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158195443.34033633 — This a preprint and has not been peer reviewed. Data may be preliminary. citm sebyaeaalbetruhNB,adaeicue naTasrpoeSognAssembly KY283590.1 Shotgun KY283585.1, Transcriptome numbers a (accession in DDBJ/EMBL/GenBank at included deposited are KY283600.1). been and and the has NCBI, from (Accession that through Archive identified available project Read sequences are Sequence receptor NCBI assembly the pheromone scriptome through Putative available SRX1741573). are data Number: sequence reads raw Transcriptome statement archiving first Data ms, the geometric to the contributed synthesized authors RU All lures. PW. field and of MB MS, PW. by preparation and by JKJ identification including draft by Pheromone PW, Sweden E8,E10-12Ac. in and tests of MB Field isomers MT, WBW. by by supervision supervision under under FB-E, and FG by analysis Transcriptome Contributions Author Infrastructure comments. Genomics infrastructure. valiable National computational and and the constructive sequencing parallel Laboratory, provided massive reviewers Life in Two assistance for providing SLU). for Science (Formas, Uppmax (IC-E3)” from and Evolution (NGI), support and acknowledge Ethology, Ecology, authors Chemical f The “Insect environment Stiftelse Tryggers Linnaeus Carl the by and supported was work This Acknowledgements 10 rVtnkpi osnn,Fra poet2011-1370) (project Formas Forskning, Vetenskaplig ¨ or .nubiferana H. tran- Posted on Authorea 17 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158195443.34033633 — This a preprint and has not been peer reviewed. 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Reports Scientific analysis. transcriptome antennal by n te pl rhr otiisuigamlipce reservoir multispecies a using tortricids orchard apple other and yhmaseparata Mythimna Z ora fPs cec 83139 (doi:10.1007/s10340-014- 88:311-319. Science Pest of Journal . goi segetum Agrotis xrsini aveadaut.SinicRprs6:23518. Reports Scientific adults. and larvae in expression - .(eiotr:Trrcde.Junlo hmclEcology Chemical of Journal Tortricidae). (Lepidoptera: L. -,-eainae edbosaswt oefutvolatiles, fruit pome with bioassays field )-2,4-decadienoate: rpoiamolesta Grapholita Lpdpea otiia) ora fApidEntomology Applied of Journal Tortricidae). (Lepidoptera: eaepeooeeiisseisseicln-ag attrac- long-range species-specific elicits pheromone female rn clEo :4(doi:10.3389/fevo.2016.00024) 4:24 Evol Ecol Front . eet h ln oaiepa se.FotEo vl2:33 Evol Ecol Front ester. pear volatile plant the detects rpoih molesta Grapholitha yi molesta Cydia yatna rncitm nlss nentoa Journal International analysis. transcriptome antennal by yi pomonella Cydia rvdsisgt noceia clg n insecticide and ecology chemical into insights provides edoiu punctatus Dendrolimus noogaEprmnai tApiaa118:77–85. Applicata et Experimentalis Entomolgia . Lpdpea rmia) cetfi eot 6:29067. Reports Scientific Crambidae). (Lepidoptera: fvs ,BntsnM izalP(98.Eetof Effect (1998). P Witzgall M, Bengtsson J, ¨ ofqvist Drosophila epn opeooeadkiooecomponents. kairomone and pheromone to respond ytasrpoi nlss M eois19:518. Genomics BMC analysis. transcriptomic by Lpdpea otia) LSOe12:e0179433. One PLoS Noctuidae). (Lepidoptera: ftd ,Cpj V(90.Structure-activity (1990). SV Copaja C, ¨ ofstedt nenltasrpoe LSOe11:e0147768. One PLoS transcriptome. antennal fvs ,UeisC,Wtgl 20) Anten- (2000). P Witzgall CR, Unelius J, ¨ ofqvist tnpesi obliquana Ctenopseustis ora fCmaaieNuooy523:530-544. Neurology Comparative of Journal . E ae otemi e hrmn component, pheromone sex main the to males npaorcsmedinalis Cnaphalocrocis apsn sasakii Carposina 11 atoeadorsalis Bactrocera ldainterpunctella Plodia , Z ora fCeia clg 16:667-684. Ecology Chemical of Journal . Z nenltasrpoeadepeso pat- expression and transcriptome antennal and ,( ), mueptwy.mi 10:e01395-19. mBio pathways. immune opahadpa oaie.Entomologia volatiles. pear and peach to eoe eelnneta vlto and evolution nonneutral reveal genomes --eey ctt,apeooecom- pheromone a acetate, )-5-decenyl .nubiferana H. Drosophila yi pomonella Cydia Bsk(eiotr:Trrcde pro- Tortricidae) (Busck)(Lepidoptera: Z H L)(eiotr:Trrcde,t the to Tortricidae), (Lepidoptera: (L.) Lpdpea otiia) ora of Journal Tortricidae). (Lepidoptera: , ryeti conjugella Argyresthia ¨ cmnAC etrsnE Witz- E, Pettersson A-C, ackman E edoiu houi Dendrolimus . rb lugens Uraba assulta n ( and ) akr rnir nPhysiology in Frontiers Walker. yi pomonella Cydia ppyspostvittana Epiphyas Lpdpea Carposinidae). (Lepidoptera: nen.Nuo 37:827-841. Neuron antenna. eae yGC/MS by females Z .Po n 11:e0147783. One PloS ). lSOe10:e0117054. One PloS . rspiamelanogaster’s Drosophila E LSOe13:e0189889. One PLoS . , Drosophila Z and , E dnie yantennal by identified , .Junlo Chemical of Journal ). Lpdpea Pyrali- (Lepidoptera: Drosophila )-8,10-dodecadien-1- .herana C. Lpdpea No- (Lepidoptera: Helicoverpa and yi pomonella Cydia eai infer- Sesamia ohs plant host to melanogaster Dendrolimus L) Natur- (L.). Drosophila Drosophila rNatur- ur ¨ Journal . Manduca Neuron . species. PLoS . Hedya Cydia Cydia Posted on Authorea 17 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158195443.34033633 — This a preprint and has not been peer reviewed. Data may be preliminary. pidflgt0b3 3a------* a a 33 17 a 47 a a 39 22 a 51 tested not * b b 0 0 a 48 source at Landing flight Upwind flight Taking pomonella C. nigricana C. nubiferana H. E Z E E Compound moth codling also attracted traps Field test, (Tukey gland. pheromone female the pomonella from identified compounds to males 3 Table F(7,72)=61.95, test, (Tukey different significantly not are letter same the by codlemone, followed with Means P permeated orchards 1997. and July (N=10) to orchards apple untreated in 2 Table -2c55 5 8-12Ac -2c11 1 8, 8-12Ac 8, < E E 0.0001). 01A 01 01 10 10 10-12OH 10 10-12Ac . . il trcinof attraction Field n e moth pea and il rpigi pl rhrs(=0 n idtne trcin(=0 of (N=60) attraction tunnel wind and (N=10) orchards apple in trapping Field E Compound E .pomonella C. nubiferana H. Z Z P 8, 8, 8, 8, Z E E Z < 01O . 2 2 0.5 10-12OH 01O . 2 2 2 0.5 2 0.5 10-12OH 10-12OH 01O 01 01 01 010 10 10 10 10 10 10 10 10-12OH 0.05). ey cae1A . 2 15 0.2 1.0 % ng/female 10Ac 12Ac form Short ( ( ( ( ( ( acetate Dodecyl actate Decyl Compound ( ( ( ( Z Z E Z E Z E E E Z 22a 12.2 0 b 1.4 b 3 0 bc 0.4 0 a 0 53.9 0 a b 57.5 3.4 Male 0 a 20.1 c 0 ubro ae/rpNme fmlsta ubro ae/rpNme fmlsta ubro males/trap of Number males/trap of Number males/trap of Number males/trap of Number males/trap of Number , , , , , -0ddcnlacetate )-10-dodecenyl acetate )-8-dodecenyl acetate )-5-dodecenyl -0ddcnlacetate )-10-dodecenyl acetate )-8-dodecenyl E Z E Z E -,0ddcdey acetate )-8,10-dodecadienyl -,0ddcdey acetate )-8,10-dodecadienyl -,0ddcdey acetate )-8,10-dodecadienyl -,0ddcdey acetate )-8,10-dodecadienyl )-8,10-dodecadienol yi nigricana Cydia .nubiferana H. . . . b1. 19a1. . b32b 3.2 a ab 6.6 6.4 ab a 3.8 10.2 a b 11.9 1.8 ab 2.1 a 10.5 ab ab 4.8 2.3 ab a 3.2 9.0 ab 3.5 a 8.0 b 2.0 H . nubiferana idtne eaiu % Male (%) behaviour tunnel wind en olwdb h aelte r o infiatydifferent significantly not are letter same the by followed Means . ae ocmoet dnie rmtefml hrmn gland, pheromone female the from identified components to males 12 Z Z E Z E Z E E E Z μ g/trap 01A . 2 56 0.1 2 3.6 8, 10-12Ac 0.1 8-12Ac 5-12Ac 8, 01A . 11 10 0.7 0.7 10-12Ac 8-12Ac 8, 8, 8, E Z E Z E 10-12Ac 01A . 4 0.3 10-12Ac 01A . 6 0.4 10-12Ac 01A . 100 6.5 10-12Ac 01:H046 0.4 10-12:OH < .1trace 0.01 .nubiferana H. E 8, E 01O N6,June (N=6), 10-12OH idtne eaiu % Male (%) behaviour tunnel wind H . nubiferana C. μ 01 10 10 10 g/trap .nubiferana H. idtne eaiu % Male (%) behaviour tunnel wind .nubiferana H. idtne eaiu % Male (%) behaviour tunnel wind .nubiferana H. idtne eaiu (%) behaviour tunnel wind Posted on Authorea 17 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158195443.34033633 — This a preprint and has not been peer reviewed. Data may be preliminary. eretr6006006006.000 (:) identity highly indicate as Asterisks indicated (blanks). are substitutions differences non-conservative sequences. sequence as both acid and across conservative, Amino (.) (B). moderately CpomOR3 and and HnubOR3 fragments (A), as CpomOR1 indicated (FPKM). and 3. reads RSEM, Figure million by per quantified 6.000 transcript levels of Expression kilobase antennae. values per male replicates, in bootstrap estimates abundance 500 with assessed was tortricids, other from postvittana sequences PR including (PR), receptors pheromone 2 Figure Lubom´ır Hl´asek. by Foto 6.000 dae). 1 Figure legends Figure 6.000 E ester Pear Compound test, (Tukey different codlemone and decadienoate, 5 Table < 8, 4 Table ae . . . . a a 0.1 0.2 a a 0.04 0.1 a a 0.1 0.3 a a 0.1 0.3 Females Males 0.05). E 8, E 01O N1) en olwdb h aelte r o infiatydffrn Tkytest, (Tukey different significantly not are letter same the by followed Means (N=10). 10-12OH 01O 10 3 1 10-12OH il rpigof trapping Field . il rpigof trapping Field . re uwr moth budworm Green . A aiu ieioduroe hlgntcte fcandidate of tree phylogenetic unrooted likelihood Maximum (A) . mn cdainet fselected of alignments acid Amino E Compound E .pomonella C. nubiferana H. Z Z (Epos), 8, 8, 8, 8, Z E E Z 01O . 2 2 0.5 10-12OH 01O . 2 2 2 0.5 2 0.5 10-12OH 10-12OH 01O 01 01 01 010 10 10 10 10 10 10 10 10-12OH ubro oh/rpNme fmtsta ubro oh/rpNme fmoths/trap of Number moths/trap of Number moths/trap of Number moths/trap of Number rpoiamolesta Grapholita P < 0.05). H . . . b1. 19a1. . b32b 3.2 a ab 6.6 6.4 ab a 3.8 10.2 a 11.9 b 1.8 ab 2.1 a 10.5 ab ab 4.8 2.3 ab a 3.2 9.0 ab a 3.5 8.0 b 2.0 E H . 8, nubiferana . E nubiferana ey nubiferana Hedya 01O N4.Masfloe ytesm etraentsignificantly not are letter same the by followed Means (N=4). 10-12OH (Gmol). and ae n eae ihbed fpa se,ehl(E,Z)-2,4- ethyl ester, pear of blends with females and males ey nubiferana Hedya .pomonella C. obxmori Bombyx > 13 aot ( Haworth 0 r hw.(B) shown. are 70% μ g/trap ae otegoercioeso codlemone of isomers geometric the to males dimidioalba Bo)sre sotru,nd support node outgroup, as served (Bmor) and μ yi pomonella Cydia g/trap yi pomonella Cydia ey nubiferana Hedya ezu)(eiotr,Tortrici- (Lepidoptera, Retzius) ey nubiferana Hedya R.HuO2and HnubOR2 PRs. (Cpom), Rtranscript PR Epiphyias (Hnub) P E Posted on Authorea 17 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158195443.34033633 — This a preprint and has not been peer reviewed. Data may be preliminary. figures/gonzalez-figure2/gonzalez-figure2-eps-converted-to.pdf 14 Posted on Authorea 17 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158195443.34033633 — This a preprint and has not been peer reviewed. Data may be preliminary. figures/gonzalez-figure3/gonzalez-figure3-eps-converted-to.pdf 15