A novel lineage of candidate pheromone receptors for sex communication in moths
The use of pheromone signals for mate recognition is widespread in animals , and changes in sex pheromone communication are expected to play a major role in the rise of reproductive barriers and the emergence of new species ( Smadja and Butlin , 2009 ) . Since the first chemical identification of such a pheromone in Bombyx mori ( Butenandt et al . , 1959 ) , moths ( Insecta , Lepidoptera ) have been a preferred taxon for pheromone research ( Cardé and Haynes , 2004; Kaissling , 2014 ) . The diversification of pheromone signals has likely played a prominent role in the extensive radiation observed in Lepidoptera , which represents almost 10% of the total described species of living organisms ( Stork , 2018 ) . Female moths generally release a species-specific blend of volatile molecules , which attract males over a long distance ( Cardé and Haynes , 2004 ) . Four types of sex pheromones have been described in moths ( types 0 , I , II and III ) , with distinct chemical structures and biosynthetic pathways ( Löfstedt et al . , 2016 ) . 75% of all known moth sex pheromone compounds belong to type I and are straight-chain acetates , alcohols or aldehydes with 10 to 18 carbon atoms ( Ando et al . , 2004 ) . Type I pheromones have been found in most moth families investigated , whereas the other types are restricted to only a few families ( Löfstedt et al . , 2016 ) . In moth male antennae , pheromone compounds are detected by dedicated populations of olfactory sensory neurons ( OSNs ) . Each type of OSN usually expresses one pheromone receptor ( PR ) responsible for signal transduction . PRs are 7-transmembrane domain proteins belonging to the odorant receptor ( OR ) family and , as ORs , are co-expressed in OSNs together with the conserved co-receptor Orco ( Chertemps , 2017; Fleischer and Krieger , 2018 ) . Since the first discovery of moth PRs ( Krieger et al . , 2004; Sakurai et al . , 2004 ) , numerous pheromone receptors tuned to type I pheromone compounds have been characterized through different hererologous expression systems , and most appeared to be specific to only one compound ( Zhang and Löfstedt , 2015 ) . More recently , a few receptors tuned to type 0 and type II pheromones have also been characterized ( Zhang et al . , 2016; Yuvaraj et al . , 2017 ) . Type I PRs belong to a dedicated monophyletic subfamily of ORs , the so-called ‘PR clade’ , suggesting a unique emergence early in the evolution of Lepidoptera ( Yuvaraj et al . , 2018 ) . Another hallmark of type I PRs is their male-biased expression ( Koenig et al . , 2015 ) . The phylogenetic position and the expression pattern have thus been the main criteria used to select candidate PRs for functional studies . Likewise , we selected PRs from the male transcriptome of the cotton leafworm Spodoptera littoralis ( Legeai et al . , 2011 ) , a polyphagous crop pest that uses type I sex pheromone compounds ( Muñoz et al . , 2008 ) and that has been established as a model in insect chemical ecology ( Ljungberg et al . , 1993; Binyameen et al . , 2012; Saveer et al . , 2012; Poivet et al . , 2012; de Fouchier et al . , 2017 ) . Through heterologous expression , we characterized two PRs tuned to minor components of the S . littoralis pheromone blend ( Montagné et al . , 2012; de Fouchier et al . , 2015 ) , but none of the tested candidate PRs detected the major pheromone component ( Z , E ) -9 , 11-tetradecadienyl acetate ( hereafter referred as ( Z , E ) -9 , 11-14:OAc ) , which is necessary and sufficient to elicit all steps of the male mate-seeking behavioral sequence ( Quero et al . , 1996 ) . In order to identify new type I PR candidates , we focused on male-biased ORs , whether they belong to the PR clade or not . Notably , a preliminary analysis of S . littoralis OR expression patterns led to the identification of such a receptor , SlitOR5 , which was highly expressed in male antennae but did not belong to the PR clade ( Legeai et al . , 2011 ) . Furthermore , a recent RNAseq analysis showed that SlitOR5 was the most abundant OR in S . littoralis male antennae ( Walker et al . , 2019 ) . Here , we demonstrate that SlitOR5 is the receptor for ( Z , E ) -9 , 11-14:OAc using a combination of heterologous expression and in vivo genome editing methods . Based on a comprehensive phylogenetic analysis of lepidopteran ORs , we show that SlitOR5 belongs to an OR subfamily that is distantly related to the PR clade but harbors numerous sex-biased ORs from distinct moth families . Altogether , these results suggest that PRs detecting type I pheromones evolved at least twice in Lepidoptera , which offers a more detailed and complex panorama on moth PR evolution .
We first used quantitative real-time PCR to compare the relative expression levels of the SlitOr5 gene in S . littoralis male and female adult antennae . We found SlitOr5 expressed with a more than 50-fold enrichment in the male antennae ( Figure 1A ) , thus confirming previous observations ( Legeai et al . , 2011; Walker et al . , 2019 ) . We reconstructed a maximum likelihood phylogeny of lepidopteran ORs , based on entire OR repertoires from nine different species . Among the 20 paralogous lineages identified ( each having evolved in principle from an ancestral OR present in the last common ancestor of Lepidoptera ) , SlitOR5 belonged to a lineage distantly related to the type I PR clade , as well as to the lineages containing type 0 and type II PRs ( Figure 1B ) . These four paralogous lineages grouped within a larger clade highly supported by the bootstrap analysis ( highlighted in grey in Figure 1B ) . This clade has been previously shown to contain ORs tuned to terpenes and aliphatic molecules – including sex pheromones – and exhibits higher evolutionary rates compared to more ancient clades that contain many receptors for aromatics ( de Fouchier et al . , 2017 ) . We next used two complementary heterologous systems to characterize the function of SlitOR5 and assess whether it is the receptor to ( Z , E ) -9 , 11-14:OAc , the major component of the S . littoralis sex pheromone blend . First , we expressed SlitOR5 in Drosophila melanogaster OSNs housed in at1 trichoid sensilla , in place of the endogenous PR DmelOR67d ( Kurtovic et al . , 2007 ) . Single-sensillum recordings were performed to measure the response of at1 OSNs to 26 type I pheromone compounds ( Supplementary file 1 ) , including all the components identified in the pheromones of Spodoptera species ( El-Sayed , 2018 ) . SlitOR5-expressing OSNs strongly responded to ( Z , E ) -9 , 11-14:OAc ( 65 ± 15 spikes . s−1 ) , whereas there was no significant response to any other compound ( Figure 2A ) . Then , we co-expressed SlitOR5 with its co-receptor SlitOrco in Xenopus oocytes and recorded the responses to the same panel of pheromone compounds using two-electrode voltage-clamp . A strong current was induced in SlitOR5-expressing oocytes when stimulated with ( Z , E ) -9 , 11-14:OAc ( 3 . 9 ± 0 . 3 µA ) , whereas only minor currents were recorded in response to ( Z , E ) -9 , 12-14:OAc and ( Z ) 9-12:OAc ( Figure 2B ) . SlitOR5 sensitivity was assessed with a dose-response experiment that showed a low detection threshold ( 10−8 M ) and an EC50 of 1 . 707 × 10−7 M ( Figure 2D–E ) . We compared the response spectra of heterologously expressed SlitOR5 with that of S . littoralis male OSNs housed in type one long trichoid sensilla ( LT1A OSNs , Figure 2C ) , known to detect ( Z , E ) -9 , 11-14:OAc ( Ljungberg et al . , 1993; Quero et al . , 1996 ) . When stimulated with the 26 pheromone compounds , LT1A OSNs significantly responded to ( Z , E ) -9 , 11-14:OAc ( 55 ± 4 spikes . s−1 ) and to a lesser extent to its stereoisomer ( Z , Z ) -9 , 11-14:OAc , which is absent from any Spodoptera pheromone . This mirrored the response spectra of heterologously expressed SlitOR5 , especially the one observed in Drosophila OSNs ( Figure 2A ) . To confirm in vivo that SlitOR5 is the receptor for the major sex pheromone component of S . littoralis , we carried out a loss-of-function study by generating mutant insects for the gene SlitOr5 . The CRISPR/Cas9 genome editing system was used to create a mutation in the first exon of SlitOr5 with the aim of disrupting the open-reading frame . Guide RNAs were injected along with the Cas9 protein in more than one thousand eggs . We genotyped 66 G0 hatched larvae and found seven individuals carrying at least one mutation in SlitOr5 . These 7 G0 individuals were back-crossed with wild-type individuals to create 7 G1 heterozygous mutant lines . We selected a line carrying a single mutation that consisted of a 10 bp deletion at the expected location , introducing a premature STOP codon within the SlitOr5 transcript after 247 codons ( Figure 3A ) . We next generated G2 homozygous mutant males ( SlitOr5-/- ) and compared their ability to detect ( Z , E ) -9 , 11-14:OAc to that of wild-type and heterozygous ( SlitOr5+/- ) males , using electroantennogram ( EAG ) recordings ( Figure 3B ) . When stimulated with ( Z , E ) -9 , 11-14:OAc , wild-type and SlitOr5+/- antennae exhibited similar EAG amplitudes ( 0 . 89 mV and 1 . 16 mV , respectively ) , whereas the response was completely abolished in SlitOr5-/- antennae ( 0 . 02 mV ) . Control experiments using a S . littoralis minor pheromone component and plant volatiles known to induce EAG responses in S . littoralis ( Saveer et al . , 2012; López et al . , 2017 ) showed that antennal responses were not impaired in SlitOr5-/- mutants , as these odorants elicited similar responses in wild-type , heterozygous and homozygous moths ( Figure 3B ) . Then , we analyzed the courtship behavior of SlitOr5-/- and wild-type males in the presence of ( Z , E ) -9 , 11-14:OAc , and found a strong behavioral defect in mutants . Whereas more than 80% of wild-type males initiated a movement toward the pheromone source in the first 8 min , only a minority of SlitOr5 mutants initiated such a movement ( even after 30 min of test ) , a response similar to that of control wild-type males not stimulated with the pheromone ( Figure 3C ) . All the steps of the courtship behavior were similarly affected ( Figure 3—figure supplement 1 ) . We also verified whether SlitOr5 knock-out would result in mating inability . When paired with a wild-type virgin female , only 1 out of the 13 SlitOr5-/- males tested was able to mate , compared to ~75% for wild-type males ( Figure 3D ) . This behavioral defect was further confirmed by analyzing the number of eggs laid by the females and the number of offspring ( Figure 3E ) . Overall , these results confirm that SlitOR5 is the receptor responsible for the detection of the major component of the S . littoralis female pheromone blend . In view of these results and the unexpected phylogenetic position of SlitOR5 , we rebuilt the phylogeny of the lepidopteran OR clade containing SlitOR5 and the known receptors for type 0 , type I and type II pheromones ( highlighted in grey in Figure 1B ) , adding all ORs showing a strong sex-biased expression ( at least 10-fold in one sex compared to the other ) and ORs whose ligands were known as of September 2018 ( Supplementary file 2 ) . ORs grouped within eight different paralogous lineages , four of which including PRs ( Figure 4 ) . One was the so-called PR clade that , as previously observed , contained all type I PRs characterized so far ( except SlitOR5 ) as well as two type II PRs ( Zhang et al . , 2016 ) . The other three lineages harboring PRs consisted of one containing SlitOR5 , one containing EgriOR31 ( a type II PR from the geometrid Ectropis grisescens; Li et al . , 2017 ) and one containing EsemOR3 and 5 ( type 0 PRs from the non-dytrisian moth Eriocrania semipurpurella; Yuvaraj et al . , 2017 ) . Interestingly , most sex-biased lepidopteran ORs identified to date clustered within the PR clade and the lineage containing SlitOR5 . While sex-biased ORs within the PR clade were mainly male-biased , the SlitOR5 lineage contained an equal proportion of male and female-biased receptors , identified from species belonging to different families of Lepidoptera . Deep nodes within the phylogeny were highly supported by the bootstrap analysis , enabling us to state that these two PR-containing clades were more closely related to clades harboring receptors for plant volatiles than to each other . This suggests that receptors tuned to type I pheromone compounds emerged twice independently during the evolution of Lepidoptera , and that the clade containing SlitOR5 may constitute a novel lineage of candidate PRs .
While moth sex pheromone receptors have been the most investigated ORs in Lepidoptera , with more than 60 being functionally characterized ( Zhang and Löfstedt , 2015 ) , it remains unclear how and when these specialized receptors arose . Type I PRs have been proposed to form a monophyletic , specialized clade of ORs , the so-called ‘PR clade’ , which emerged early in the evolution of Lepidoptera ( Yuvaraj et al . , 2017; Yuvaraj et al . , 2018 ) . Here , we bring functional and phylogenetic evidence that type I PRs are not restricted to this clade and likely appeared twice independently in Lepidoptera . We focused on an atypical OR , SlitOR5 , which exhibited a strong male-biased expression in antennae of the noctuid moth S . littoralis but did not group with the PR clade . We demonstrated , using a combination of heterologous expression and loss-of-function studies , that this OR is responsible for the detection of ( Z , E ) -9 , 11-14:OAc , the major component of the S . littoralis sex pheromone blend . Due to the unexpected phylogenetic position of SlitOR5 outside of the previously defined PR clade , the question arose whether SlitOR5 is an exception or belongs to a previously unknown clade of moth PRs . This latter hypothesis is supported by the observation that the paralogous lineage containing SlitOR5 harbored many other sex-biased ORs , identified in species from six distinct lepidopteran families . Notably , male-biased ORs have been found in Lasiocampidae , Sphingidae , Noctuidae and Tortricidae . Among these , two ORs from Ctenopseustis obliquana and C . herana ( Tortricidae ) have been functionally studied by heterologous expression in cell cultures , but no ligand could be identified ( Steinwender et al . , 2015 ) . In the Lasiocampidae species Dendrolimus punctatus , these male-biased ORs have been referred to as ‘Dendrolimus-specific odorant receptors’ , with the suspicion that they would represent good PR candidates since in Dendrolimus species , there is no OR clustering in the PR clade ( Zhang et al . , 2014; Zhang et al . , 2017 ) . No functional data yet confirmed this suspicion . Conversely , within the SlitOR5 lineage , almost half of the sex-biased ORs were female-biased ( 13 out of 28 , compared to 4 out of 63 in the classical PR clade ) . Female-biased ORs have been generally proposed to be involved in the detection of plant-emitted oviposition cues , as demonstrated in Bombyx mori ( Anderson et al . , 2009 ) . However , another interesting hypothesis is that they could be tuned to male sex pheromones . In moths , little attention has been put on male pheromones , which are known to be involved in various mating behaviors such as female attraction , female acceptance , aggregation of males to form leks , mate assessment or inhibition of other males ( reviewed in Conner and Iyengar , 2016 ) . The use of male pheromone systems has been selected multiple times in distinct moth families , as reflected by the chemical diversity of male pheromone compounds and of the disseminating structures ( Birch et al . , 1990; Phelan , 1997; Conner and Iyengar , 2016 ) . It is thus expected that this polyphyletic nature of male pheromones would result in a large diversity of female PR types . Accordingly , female-biased ORs were found in different clades within the phylogeny . However , most remain orphan ORs , including BmorOR30 that does belong to the SlitOR5 lineage but for which no ligand could be identified ( Anderson et al . , 2009 ) . Although the most common male-emitted volatiles are plant-derived pheromones ( Conner and Iyengar , 2016 ) , some male courtship pheromones are long-chained hydrocarbons related to type I female pheromone compounds ( Hillier and Vickers , 2004 ) that could be detected by female-biased type I PRs such as those identified within the SlitOR5 lineage . The ancestral protein from which the so-called ‘PR clade’ would have arisen is thought to be an OR tuned to plant-emitted volatiles ( Yuvaraj et al . , 2017; Yuvaraj et al . , 2018 ) . Here , we evidence that SlitOR5 is a new type I PR that belongs to a distinct early diverging lineage for which a role in pheromone detection had never been demonstrated . Together with the findings that PRs for Type 0 ( Yuvaraj et al . , 2017 ) and one PR for type II pheromones ( Li et al . , 2017 ) group in distinct paralogous lineages also unrelated to the PR clade , our data suggest that lepidopteran PRs have evolved four times in four paralogous lineages . Whether the SlitOR5 lineage has evolved from ORs that detected structurally related plant volatiles - as has been proposed for type 0 ( Yuvaraj et al . , 2017 ) and classical type I PRs - remains elusive . Yet , no OR tuned to plant volatiles has been identified in closely related lineages . More functional data on SlitOR5 paralogs and orthologs in different moth species , possibly revealing more type I PRs , will help in understanding evolutionary history of this lineage , as to when and how these receptors have evolved , and confirm that we are facing a novel type I PR clade .
S . littoralis were reared in the laboratory on a semi-artificial diet ( Poitout and Buès , 1974 ) at 22°C , 60% relative humidity and under a 16 hr light:8 hr dark cycle . Males and females were sexed as pupae and further reared separately . D . melanogaster lines were reared on standard cornmeal-yeast-agar medium and kept in a climate- and light-controlled environment ( 25°C , 12 hr light:12 hr dark cycle ) . The 26 pheromone compounds used for electrophysiology experiments ( Supplementary file 1 ) were either synthesized in the lab or purchased from Sigma-Aldrich ( St Louis , MO ) and Pherobank ( Wijk bij Duurstede , The Netherlands ) . Paraffin oil was purchased from Sigma-Aldrich and hexane from Carlo Erba Reagents ( Val de Reuil , France ) . Total RNA from three biological replicates of 15 pairs of antennae of two-day-old virgin male and female S . littoralis was extracted using RNeasy Micro Kit ( Qiagen , Hilden , Germany ) , which included a DNase treatment . cDNA was synthesized from total RNA ( 1 μg ) using Invitrogen SuperScript II reverse transcriptase ( Thermo Fisher Scientific , Waltham , MA ) . Gene-specific primers were designed for SlitOr5 ( Or5up: 5’-TCGGGAGAAACTGAAGGACGTTGT-3’ , Or5do: 5’-GCACGGAACCGCACTTATCACTAT-3’ ) and for the reference gene SlitRpl13 ( Rpl13up: 5’-GTACCTGCCGCTCTCCGTGT-3’ , Rpl13do: 5’-CTGCGGTGAATGGTGCTGTC-3’ ) . qPCR mix was prepared in a total volume of 10 μL with 5 μL of LightCycler 480 SYBR Green I Master ( Roche , Basel , Switzerland ) , 4 μL of diluted cDNA ( or water for the negative control ) and 0 . 5 μM of each primer . qPCR assays were performed using the LightCycler 480 Real-Time PCR system ( Roche ) . All reactions were performed in triplicate for the three biological replicates . The PCR program began with a cycle at 95°C for 13 . 5 min , followed by 50 cycles of 10 s at 95°C , 15 s at 60°C and 15 s at 72°C . Dissociation curves of the amplified products were performed by gradual heating from 55°C to 95°C at 0 . 5 °C . s−1 . A negative control ( mix without cDNA ) and a fivefold dilution series protocol of pooled cDNAs ( from all conditions ) were included . The fivefold dilution series were used to construct relative standard curves to determine the PCR efficiencies used for further quantification analyses . Data were analyzed with the LightCycler 480 software ( Roche ) . Normalized expression of the SlitOr5 gene was calculated with the Q-Gene software ( Joehanes and Nelson , 2008 ) using SlitRpl13 as a reference , considering it displays consistent expression as previously described in Durand et al . ( 2010 ) . The SlitOr5 full-length open-reading frame ( 1191 bp , GenBank acc . num . MK614705 ) was subcloned into the pUAST . attB vector . Transformant UAS-SlitOr5 balanced fly lines were generated by BestGene Inc ( Chino Hills , CA ) , by injecting the pUAST . attB-SlitOr5 plasmid ( Endofree prepared , Qiagen ) into fly embryos with the genotype y1 M{vas-int . Dm}ZH-2A w*; M{3xP3-RFP . attP}ZH-51C ( Bischof et al . , 2007 ) , leading to a non-random insertion of the UAS-SlitOr5 construct into the locus 51C of the second chromosome . The UAS-SlitOr5 balanced line was then crossed to the Or67dGAL4 line ( Kurtovic et al . , 2007 ) to obtain double homozygous flies ( genotype w; UAS-SlitOr5 , w+; Or67dGAL4 ) expressing the SlitOr5 transgene in at1 OSNs instead of the endogenous Drosophila receptor gene Or67d . The correct expression of SlitOr5 was confirmed by RT-PCR on total RNA extracted from 100 pairs of antennae . Single-sensillum extracellular recordings on Drosophila at1 OSNs were performed as previously described ( de Fouchier et al . , 2015 ) . OSNs were stimulated during 500 ms , using stimulus cartridges containing 10 μg of pheromone ( 1 μg/μl in hexane ) dropped onto a filter paper . Single-sensillum recordings on S . littoralis LT1A OSNs were performed using the tip-recording technique , as previously described ( Pézier et al . , 2007 ) . Briefly , the tips of a few LT1 sensilla were cut off using sharpened forceps and a recording glass electrode filled with a saline solution ( 170 mM KCl , 25 mM NaCl , 3 mM MgCl2 , 6 mM CaCl2 , 10 mM HEPES and 7 . 5 mM glucose , pH 6 . 5 ) was slipped over the end of a cut LT1 sensillum . OSNs were stimulated with an air pulse of 200 ms ( 10 L . h−1 ) , odorized using a stimulus cartridge containing 1 μg of pheromone ( diluted at 1 μg/μl in hexane ) . Odorants were considered as active if the response they elicited was statistically different from the response elicited by the solvent alone ( one-way ANOVA followed by a Tukey’s post hoc test ) . Open-reading frames of SlitOr5 and SlitOrco ( GenBank acc . num . EF395366 , Malpel et al . , 2008 ) were subcloned into the pCS2+ vector ( Turner and Weintraub , 1994 ) . Template plasmids were fully linearized with PteI for pCS2+-SlitOr5 and NotI for pCS2+-SlitOrco and capped cRNAs were transcribed using SP6 RNA polymerase . Purified cRNAs were re-suspended in nuclease-free water at a concentration of 2 μg/μL and stored at −80°C . Mature healthy oocytes were treated with 2 mg/ml collagenase type I in washing buffer ( 96 mM NaCl , 2 mM KCl , 5 mM MgCl2 and 5 mM HEPES , pH 7 . 6 ) for 1–2 hr at room temperature . Oocytes were later microinjected with 27 . 6 ng of SlitOr5 cRNA and 27 . 6 ng of SlitOrco cRNA . After 4 days of incubation at 18°C in 1 × Ringer’s solution ( 96 mM NaCl , 2 mM KCl , 5 mM MgCl2 , 0 . 8 mM CaCl2 , and 5 mM HEPES , pH 7 . 6 ) supplemented with 5% dialyzed horse serum , 50 mg/ml tetracycline , 100 mg/ml streptomycin and 550 mg/ml sodium pyruvate , the whole-cell currents were recorded from the injected oocytes with a two-electrode voltage clamp . Oocytes were exposed to the 26 pheromone compounds diluted at 10−4 M in Ringer’s solution , with an interval between exposures which allowed the current to return to baseline . Data acquisition and analysis were carried out with Digidata 1440A and pCLAMP10 software ( Molecular Devices , San Jose , CA ) . Odorants were considered as active if the mean response they elicited was statistically different from 0 ( Wilcoxon signed rank test ) . Dose–response experiments were performed using pheromone concentrations ranging from 10−9 up to 10−5 M and data were analyzed using GraphPad Prism 5 . A guide RNA ( gRNA sequence: AGCATAAATACTGGACCCAG TGG ) was designed against the first exon of the SlitOr5 gene using the CRISPOR gRNA design tool ( crispor . tefor . net; Haeussler et al . , 2016 ) and transcribed after subcloning into the DR274 vector using the HiScribe T7 High Yield RNA Synthesis Kit ( New England Biolabs , Ipswich , MA ) . The Cas9 protein was produced in Escherichia coli as previously described ( Ménoret et al . , 2015 ) . A mix of Cas9 protein and gRNA was injected in freshly laid eggs using an Eppendorf - Transjector 5246 , as previously described ( Koutroumpa et al . , 2016 ) . Individual genotyping at every generation was performed via PCR on genomic DNA extracted from larvae pseudopods ( Wizard Genomic DNA Purification Kit , Promega , Madison , WI ) using gene-specific primers ( Or5_forward: 5’-CCAAAAGGACTTGGACTTTGAA-3’; Or5_reverse: 5’-CCCGAATCTTTTCAGGATTAGAA-3’ ) amplifying a fragment of 728 bp encompassing the target sequence . Mutagenic events were detected by sequencing the amplification products ( Biofidal , Vaulx-en-Velin , France ) . G0 larvae carrying a single mutagenic event were reared until adults and crossed with wild-type individuals . Homozygous G2 individuals were obtained by crossing G1 heterozygous males and females . Electroantennogram recordings were performed as previously described ( Koutroumpa et al . , 2016 ) on isolated male antennae from wild-type animals and from heterozygous and homozygous SlitOr5 mutants . Antennae were stimulated using stimulus cartridges loaded with 10 μg of linalool or benzyl alcohol diluted in paraffin oil , and 1 μg of ( Z , E ) -9 , 11-14:OAc or ( Z , E ) -9 , 12-14:OAc diluted in hexane . Stimulations lasted for 500 ms ( 30 L/h ) . Negative controls consisted of paraffin oil and hexane alone . The maximum depolarization amplitude was measured using the pCLAMP10 software . Normalized mean responses were calculated ( response to the solvent was subtracted ) and data were analyzed using a one-way ANOVA followed by a Tukey’s post hoc test . For courtship monitoring , 2-day-old wild-type or homozygous SlitOr5 mutant males were placed individually in a plastic squared petri dish ( size = 12 cm ) before the onset of the scotophase , and experiments started at the middle of the scotophase ( 22°C , 70% relative humidity ) . Odorant stimulation was performed using Pasteur pipettes containing a filter paper loaded with 100 ng of ( Z , E ) -9 , 11-14:OAc ( 10 ng/μl in hexane ) , or hexane alone as control . The narrow end of the pipette was inserted into the petri dish , and a constant stream of charcoal-filtered , humidified air ( 0 . 2 L . min−1 ) passed through the pipette during all the experiment . Male courtship behavior was recorded under dim red light during 30 min using a webcam ( Logitech QuickCam Pro 9000 ) . The latency of each of the following stereotyped behavioral items was individually screened: antennal flicking , movement toward the odor source , wing fanning , abdomen curving and extrusion of genitalia . Results were presented as cumulative occurrence of each item along the time of the experiment . Statistical analysis of the distributions obtained for each treatment were compared using a log-rank ( Mantel-Cox ) test . For mating experiments , 2-day-old wild-type or homozygous SlitOr5 mutant males were paired with a 2-day-old wild-type virgin female in a cylindrical plastic box ( diameter 8 cm , height 5 cm ) , the walls of which were covered up with filter paper . Experiments started at the onset of the scotophase and lasted over 6 hr . Copulation events were visually inspected every 20 min under dim red light . After the end of the experiment , females were kept in the boxes for 12 hr . Then , females were discarded and filter papers were examined for the presence of egg clutches . When present , eggs were transferred to a rearing plastic box , hatch was monitored every day during 7 days and second-instar larvae were counted , if any . Statistical analysis of mating rates was done using a Fisher’s exact test , and analyses of the number of eggs and offspring were done using a Mann-Whitney U test . The dataset of lepidopteran amino acid OR sequences used to build the phylogeny shown in Figure 1 included entire OR repertoires from the following nine species , each belonging to a different lepidopteran superfamily: Bombyx mori ( Bombycoidea; Tanaka et al . , 2009 ) , Dendrolimus punctatus ( Lasiocampoidea; Zhang et al . , 2017 ) , Ectropis grisescens ( Geometroidea; Li et al . , 2017 ) , Epiphyas postvittana ( Tortricoidea; Corcoran et al . , 2015 ) , Eriocrania semipurpurella ( Eriocranioidea; Yuvaraj et al . , 2017 ) , Heliconius melpomene ( Papilionoidea; Heliconius Genome Consortium , 2012 ) , Ostrinia furnacalis ( Pyraloidea; Yang et al . , 2015 ) , Plutella xylostella ( Yponomeutoidea; Engsontia et al . , 2014 ) and Spodoptera littoralis ( Noctuoidea; Walker et al . , 2019 ) . The dataset used to build the phylogeny shown in Figure 4 contained amino acid sequences from the same nine species falling into that clade , plus all the sequences of ORs within that clade showing a marked sex-biased expression ( threshold of a 10-fold difference in expression rate between male and female antennae , based on RNAseq or qPCR experiments ) and/or for which ligands have been identified as of September 2018 ( Supplementary file 2 ) . Alignments were performed using Muscle ( Edgar , 2004 ) as implemented in Jalview v2 . 10 . 5 ( Waterhouse et al . , 2009 ) . Phylogenetic reconstruction was performed using maximum likelihood . The best-fit model of protein evolution was selected by SMS ( Lefort et al . , 2017 ) and tree reconstruction was performed using PhyML 3 . 0 ( Guindon et al . , 2010 ) . Node support was first assessed using 100 bootstrap iterations , then the file containing bootstrap trees was uploaded on the Booster website ( Lemoine et al . , 2018 ) to obtain the TBE ( Transfer Bootstrap Expectation ) node support estimation . Figures were created using the iTOL web server ( Letunic and Bork , 2016 ) .
Sex pheromone receptors ( PRs ) are key players in chemical communication between mating partners in insects . In the highly diversified insect order Lepidoptera , male PRs tuned to female-emitted type I pheromones ( which make up the vast majority of pheromones identified ) form a dedicated subfamily of odorant receptors ( ORs ) . Here , using a combination of heterologous expression and in vivo genome editing methods , we bring functional evidence that at least one moth PR does not belong to this subfamily but to a distantly related OR lineage . This PR , identified in the cotton leafworm Spodoptera littoralis , is highly expressed in male antennae and is specifically tuned to the major sex pheromone component emitted by females . Together with a comprehensive phylogenetic analysis of moth ORs , our functional data suggest two independent apparitions of PRs tuned to type I pheromones in Lepidoptera , opening up a new path for studying the evolution of moth pheromone communication .
Many animals make use of chemical signals to communicate with other members of their species . Such chemical signals , called pheromones , often allow males and females of the same species to recognize each other before mating . Since the discovery of the very first pheromone in the silkworm moth Bombyx mori at the end of the 1950s , moths have been a model for pheromone research . The sex pheromone communication system in these insects has thus been well described: females emit a mixture of volatile chemicals , which can be detected by the antennae of males up to several hundred meters away . This detection is achieved through neurons with specialized proteins known as pheromone receptors that bind to the chemical signals produced by the females . Recognizing mates by detecting a very specific pheromone signature prevents moths from interbreeding with other species . The evolution of pheromone signals and their corresponding receptors can therefore lead to the rise of new reproductive barriers between populations , and eventually to the emergence of new species . The rate at which sex pheromones have diversified is likely one reason for the existence of over 160 , 000 species of moths . But how did moths’ sex pheromone receptors evolve in the first place ? Previous studies suggested that moth pheromone receptors had appeared just once during evolution . Specifically , they revealed that these receptors belong to the same branch or lineage in the ‘family tree’ of all receptors that detect chemical compounds in moths . This meant that when researchers looked for pheromone receptors in a new species of moth , they always focused on this lineage . But Bastin-Héline et al . have now found that one pheromone receptor from a pest moth called Spodoptera littoralis does not belong to this established group . First , Bastin-Héline et al . inserted this receptor into animal cells grown in the laboratory to confirm that it responds to a specific pheromone produced by S . littoralis . Next , they genetically modified moths of this species and showed that males need this receptor in order to mate . An evolutionary analysis showed that the receptor belongs to a different lineage than all the other known pheromone receptors . Together these results indicate the receptors for sex pheromones must have evolved multiple times independently in moths . These results will open new avenues for deciphering pheromone communication in moths , and lead to further research into this newly discovered lineage of candidate pheromone receptors . Such studies may foster the development of new strategies to control agricultural pests , given that some species of moths can have devastating effects on the yields of certain crops .