Molecular And Cellular Basis Of Sex Pheromonc Communication In The Black Cutworm Moth Agrotis Ipsilon

Feeding, courtship and mating are fundamental for insects, and most insects rely on their sensitiveantennae that express specific olfactory proteins to detect survival-and reproductive-related chemicalcues from the environment. Pheromones and plant volatiles diffuse into the sensilla via the multiporesthat penetrate the cuticular surface. When pheromones and plant volatiles enter the sensillum lymph, theantennae-enriched binding proteins （odorant binding proteins （OBPs） and chemosensory proteins（CSPs）） capture these semiochemicals and transport them across the aqueous lumen to themembrane-bound chemosensory receptors （odorant receptors （ORs） and ionotropic receptors （IRs））.Subsequently, the odorant molecules are rapidly degraded by odorant-degrading enzymes （ODEs）. Thehigh specificity and sensitivity of insect to sex pheromones make them as effective biological controlagents for population monitoring and mass trapping of noxious insects in integrated pest management（IPM） programs. Elucidating the mechanism of insect olfactory perception can further facilitate thedesign and implementation of novel intervention strategies against these pests. The black cutworm mothAgrotis ipsilon （Hufnagel）（Lepidoptera: Noctuidae） is a destructive pest affecting many cropsworldwide and the male A. ipsilon moth is high attracted by the sex pheromones released by the femalemoth. However, the molecular and cellular mechanisms of how sex pheromones are perceived by maleA. ipsilon moths are still rudimentary. The identification and functional characterization of antennalolfactory proteins in A. ipsilon will enhance our knowledge of the molecular and cellular basis of insectchemoreception. More importantly, can provide us new methods to control this pest through interringtheir olfaction perception. In this study, several kinds of olfactory genes putatively involved in theolfactory reception in A. ipsilon antennae, and several kinds of genes putatively involved in pheromoneproduction, transport and degradation in the A. ipsilon female moth pheromone gland were identifiedthrough next-generation transcriptome sequencing. We have taken systematic functional studies of threekind of key olfactory proteins in the A. ipsilon antennae, including pheromone binding proteins （PBPs）,pheromone receptors （PRs） and sensory neuron membrane proteins （SNMPs）, we have provideddetailed evidences of PBPs, PRs and SNMPs in A. ipsilon sex pheromone reception at transcriptionallevel, proteins level and cellular level. The main results are as follows:1. Types and ultrastructures of olfactory sensilla in A. ipsilon antennaeThe scanning and transmission electron microscopic examinations revealed that the grossmorphology between male and female A. ipsilon moth antennae was sexually dimorphic: bipectinate inmale antennae, threadlike in female antennae. The mean length of antennae was about12mm andsimilar between male and female moths. There were at least six types of olfactory sensilla in eachantenna: trichodea sensilla （str）, chaetica sensilla （sch）, basiconica sensilla （sba）, coeloconica sensilla（sco）, squamiformia sensilla （ssq） and B hm bristles （bbr）. Trichoid sensilla （str） were most numerous on the antennae with2–3dendritic profiles in each sensillum. Basiconica sensilla （sba） have a thincuticular wall and12–25dendritic profiles. The coeloconica sensilla （sco） have4–7neurons in eachsensillum. The chaetica sensilla （sch） had no neurons but a thick cuticular wall.2. Global transcriptional analysis and chemosensory genes in A. ipsilonantennaeUsing Roche454GS-FLX sequencing platform and particular bioinformatics analysis, we haveidentified several olfactory gene families in A. ipsilon antennal transcriptome, the differential expressionprofiles of these olfactory genes are evaluated by RT-PCR and qRT-PCR. The main results aresummarized as follows:（1） A total of33OBP genes were annotated from the A. ipsilon antennal transcriptome. Among them30have intact ORF with the length from402bp to759bp. The RT-PCR results indicated that22OBPgenes （PBP1, PBP2, PBP3, GOBP1, GOBP2, OBP1, OBP2, OBP4, OBP5, OBP9, OBP11, OBP12,OBP13, OBP15, OBP16, OBP17, OBP19, OBP20, OBP21, OBP22, OBP24and OBP26） areuniquely or mostly expressed in the male and female antennae, this suggested antennae-specific or-enriched expressed OBPs play important roles in sex pheromone detection, suitable host plantsearching and oviposition site location. Interestingly, one OBP （OBP6） was mostly expressed in thepheromone gland （PG）, unlike the common antennae-enriched OBPs, this PG-expressed OBP mayplay completely different role in the odorant and pheromone detection and transportation.（2）12novel CSP genes were identified in the A. ipsilon antennae. The RT-PCR and qRT-PCR resultsindicated that3CSP genes （CSP8, CSP9and CSP10） are highly expressed in the male and femaleantennae, suggesting these three antennae-enriched CSPs may play essential role in the chemicalcommunication process in A. ipsilon. Interestingly, one CSP gene （CSP2） was not expressed in theantennae but specially expressed in the female pheromone gland （PG）, suggesting a possibleinvolvement of these proteins in carrying and releasing sex pheromones as demonstrated for theantennal OBPs and CSPs. The insect may use these female PG-enriched CSPs to auto-detect andmonitor the sex pheromones released by themselves.（3）42OR genes （41typical ORs and the atypical coreceptor） from the A. ipsilon antennaltranscriptome were identified. The RT-PCR and qRT-PCR results indicated that35ORs exclusivelyor mainly expressed in the antennae, among them4ORs （OR1, OR3, OR4and OR14） are maleantennae-specific expression, suggesting they may play essential role in the detection of sexpheromones as pheromone receptors.4ORs （OR6, OR7, OR8and OR23） are femaleantennae-enriched expression, suggesting they may play important roles in the detection of generalodorants such as host plant volatiles.（4）24IRs including two highly conserved coreceptors IR8a and IR25a were identified from the A.ipsilon antennal transcriptome. RT-PCR and qRT-PCR results indicated that14A. ipsilon IRs （IR8a, IR25a, IR21a, IR41a, IR75q.1, IR75q.2, IR76b, IR87a, IR1, IR3, IR4, IR8, IR12and IR13） arehighly expressed in the antennae, particularly, IR12was specially expressed in the male antennae,suggesting it may be a novel pheromone receptor gene and devoted to response to the female sexpheromone.3. Sex pheromone recognition and immunolocalization of threepheromone binding proteins in A. ipsilonThe full-length of three pheromone binding protein （PBP） genes were obtained using homologouscloning combined with RACE PCR strategy. We have undertaken systematic studies of the A. ipsilonPBP proteins, the main results are summarized as follows:（1） The amino acid identity among AipsPBP1–3was about40%. The AipsPBP1and AipsPBP2full-length sequences described here added the missing N-terminal sequences of those reportedpreviously with additional8（MAPHPSVT） and23amino acids（MAASRWCIACLVCVLFAARSVMT）, respectively. There are also several amino acid differencescompared to previously reported sequences, which likely represent sequence diversity between theChinese and French strains of A. ipsilon moth.（2） The expression patterns of AipsPBP1–3transcripts measured by qRT-PCR indicated thatAipsPBP1–3transcripts were highly expressed in antennae than in other tissues but not specific toantennae, low expressions were also detected in the taste organs proboscis and labial palp.AipsPBP1–3expression levels were very low in the heads, thoraxes, abdomens, legs and wings.Furthermore, the transcripts of AipsPBP1–3were about2.2±0.3,4.5±0.4and2.6±0.3times higherin the male antennae than in the female antennae （p<0.01）, respectively.（3） Polyclonal antisera made from each purified AipsPBPs were used for the cellular localization ofAipsPBPs in A. ipsilon antennal sensilla. Our immunocytochemistry results indicated that in maleantennae the trichoid sensilla were strongly labeled by all three anti-PBP antisera, sometimes thegeneral odorant-sensitive basiconic sensilla were also slightly labeled. It was interesting to note thatsome trichoid sensilla were not labeled by any of three anti-PBP antisera. In the female antennae,only a few trichoid and basiconic sensilla were strongly labeled. Other sensilla such as chaetica,coeloconica, squamiformia and B hm bristles were not labeled in both sexes.（4） Fluorescence competitive binding assay results indicated that the dissociation constants of theAipsPBPs/1-NPN complex were4.4±0.3μM,3.2±0.2μM and2.1±0.2μM for AipsPBP1,AipsPBP2and AipsPBP3, respectively. AipsPBP1had high binding affinities with the two majorsex pheromone components Z7-12:Ac and Z9-14:Ac with the IC50values of0.46±0.03μM and0.84±0.08μM, respectively, but showed a weak binding abilities to the remaining sex pheromoneconponents Z11-16:Ac, Z5-10:Ac and Z8-12:Ac. AipsPBP2had high binding affinities withZ7-12:Ac, Z9-14:Ac as well as Z11-16:Ac with the IC50values of0.56±0.06μM,0.45±0.03μM and0.79±0.05μM, respectively. Interestingly, AipsPBP3only had a high binding affinity toZ11-16:Ac with the IC50values of0.62±0.04μM.（5） The results obtained by the two phase binding assays were similar to those of the fluorescencecompetitive binding assay. AipsPBP1bound significantly more with Z7-12:Ac and Z9-14:Ac thanother pheromone components. The amount of Z7-12:Ac and Z9-14:Ac bound with each μgAipsPBP1were2.2±0.5ng and2.0±0.5ng, respectively. AipsPBP2bound equally well withZ7-12:Ac, Z9-14:Ac and Z11-16:Ac, with the amount as1.9±0.4ng,2.1±0.5ng and2.0±0.4ng perμg protein, respectively. AipsPBP3bound significantly more Z11-16:Ac with the amount as1.9±0.4ng per μg AipsPBP3protein than any other pheromone components. However, none of AipsPBPswere specific to any one of the pheromone components.4. Molecular cloning and functional characterization of pheromonereceptors in A. ipsilonThe full-length of atypical olfactory coreceptor Orco and three pheromone receptor PR genes wereobtained using homologous cloning combined with RACE PCR strategy. The differential responses ofpheromone receptors AipsOR1, AipsOR3and AipsOR4to A. ipsilon sex pheromone molecules arecharacterized using heterologous expression system in Xenopus oocytes in vitro and two-electrode,voltage-clamp physiological recordings. The main results are summarized as follows:（1） The qRT-PCR results indicated that the atypical olfactory coreceptor AipsOrco and three pheromonereceptor gene AipsOR1, AipsOR3and AipsOR4were mainly expressed in the antennae. AipsOrcoshowed similar expression level between male and female antennae （p>0.01） and also showed lowexpression levels in the taste organs proboscis and Labial palp, and also can be detected in thewings and legs. The pheromone receptor gene AipsOR1, AipsOR3and AipsOR4mainly expressedin male antennae, with more than20times higher expressed in the male antennae than in the femaleantennae（p<0.01）. The three pheromone receptor genes also showed low expression levels in thetaste organs proboscis and labial palp.（2） The Xenopus oocytes which expressed different pheromone receptors showed completely differentresponse profiles to A. ipsilon five sex pheromone components. The AipsOR3/Orco specificallytuned to the major pheromone component Z7-12:Ac, the mean inward current responses of Xenopusoocytes with co-expresed AipsOR3/Orco in response to10-4Mol/L was5100nA. The Xenopusoocytes expressing AipsOR3/Orco responsed robustly to the second main sex pheromone comnentZ9-14:Ac, with a mean amplitude as1335nA when response to Z9-14:Ac at the concentration of10-4Mol/L. The Xenopus oocytes expressing AipsOR4/Orco specifically responsed the minor sexpheromone comnent Z5-10:Ac, with a mean amplitude as362nA when response to Z5-10:Ac at theconcentration of10-4Mol/L. 5. Differential expression and immunolocalization of sensory neuronmembrane proteins in the antennae of A. ipsilon mothTwo full-length SNMP transcripts in the A. ipsilon, AipsSNMP1and AipsSNMP2, were obtainedusing homologous cloning combined with RACE PCR strategy. We performed extensive expressionprofiling of AipsSNMP1and AipsSNMP2transcripts among different tissues of both sexes, in theantennae at different developmental stages and in the antennae of both sexes before and after mating byquantitative real-time PCR at the transcript level. The specific subcellular distribution of AipsSNMP1and AipsSNMP2proteins in the various olfactory sensilla was also investigated usingimmunocytochemistry techniques. The possible functions of the SNMPs in moth odorant detection arediscussed. The main results are summarized as follows:（1） The qRT-PCR results revealed that both AipsSNMP1and AipsSNMP2genes were expressedsignificantly highly in the male and female antennae than in other tissues （more than100-foldhigher than in the abdomen, p<0.05） with insignificant differences between sexes and between twotranscripts in each tissue type. The relative expression levels of AipsSNMP1and AipsSNMP2transcripts in the male antennae were approximately1.5-fold and2.3-fold higher than in the femaleantennae, respectively （p<0.05）. The expression levels in the proboscis, and labial palp, head, legsand wings were lower than in the antennae but higher than in the thorax, abdomen and accessoryglands, where no transcripts could be detected. Both AipsSNMP1and AipsSNMP2genes began inthe pupae stage about3days before adult emergence, increased dramatically from one day beforethe emergence, doubled at the day of the emergence and maintained at the high level for4daysduring adult life. On the4th day after the emergence, the expression levels began to decrease to alow level similar to that of one day before the emergence and remained at this low level from day5to day7after the emergence. There were no significant differences in the AipsSNMP1andAipsSNMP2transcript expression levels before and after mating in the4-day-old male or femaleantennae （p>0.05）.（2） The immunocytochemistry results at protein level demonstrated that both AipsSNMP1andAipsSNMP2were expressed in pheromone-sensitive sensilla trichodea but with a completelydifferent expression profile. AipsSNMP1is more uniformed and highly expressed along themembrane of the ORN dendrites, whereas AipsSNMP2is widely distributed at the bottom of thesensilla trichodea and highly localized in the sensillum lymph. Furthermore, the AipsSNMP2protein was also expressed in the general odorant-sensitive sensilla basiconica and sensillacoeloconica. The visual observations showed that a higher number of each type of olfactory sensillawas labeled with the SNMP antisera in male antennae compared with female antennae. 6. Transcriptome analysis of A. ipsilon pheromone gland revealedcandidate genes with putative functions in pheromone production,transport and degradationHere we report next-generation sequencing of the A. ipsilon pheromone gland transcriptome,identification and expression profiling of genes putatively involved in pheromone production, transportand degradation. The main results are summarized as follows:（1） Pheromone biosynthesis activating neuropeptide （PBAN） receptor gene. One transcriptUnigene₃821encoding protein highly homologous to PBAN receptor isoform B was identified. Ithad very low abundance in the A. ipsilon transcriptome with the RPKM value as31.（2） Acetyl-CoA carboxylase （ACC）. Two transcripts （Unigene₂338and Unigene₆244） encodingACCs were identified. The RT-PCR and qRT-PCR revealed that both Unigene₂338andUnigene₆244are highly expressed in the pheromone gland as compared to the body. However,they have very low abundance （81and21RPKM） in the transcriptome.（3） Fatty acid synthase （FAS）. We have identified one putative FAS transcript （Unigene₁8120） in the A.ipsilon pheromone gland, containing an ORF of7176bp. The RT-PCR and qRT-PCR revealed thatUnigene₁8120is highly expressed in the pheromone gland （40-fold higher than in bodies） and alsohas a high abundance （343RPKM） in the PG transcriptome.（4） Desaturases （DES）. In the A. ipsilon pheromone gland transctiptome5transcripts have highhomology to genes encoding desaturases. Unigene₆5is encoding an acyl-CoA9-desaturase andUnigene₇41encodes an acyl-CoA11desaturase, these two genes possibly involves in the A.ipsilon sex pheromone biosynthesis. RT-PCR and qRT-PCR results indicated that Unigene₇41andUnigene₇80are highly expressed in the A. ipsilon pheromone gland compared with the body （85and63fold higher, respectively）. One of the transcripts （Unigene₇80） is also highly abundant（1206RPKM） in the pheromone gland transcriptome, suggesting a possible role in A. ipsilon sexpheromone biosynthesis. The remaining three transcripts Unigene₆5, Unigene₁0494andUnigene₁5401showed similar expressed intensity between the PG and the body part.（5） Fatty acyl-CoA reductase （FAR）. In the A. ipsilon pheromone gland transcriptome there are13transcripts homologous to putative FAR genes. RT-PCR and qRT-PCR results indicated that threetranscripts （Unigene₁63, Unigene₄302and Unigene₇344） are highly expressed in thepheromone gland. The other ten transcripts seem equally expressed in the pheromone gland and thebody or highly expressed in the body. All FAR transcripts except two （Unigene₁63andUnigene₂537） have low abundance （from81and16RPKM） in the pheromone glandtranscriptome.（6） Aldehyde reductase （AR）. In the A. ipsilon pheromone gland we identified11transcripts encodingaldo-ketoreductase. The RT-PCR and qRT-PCR results indicated that Unigene₃134andUnigene₄806are mainly expressed in the pheromone gland, while the other9putative aldehyde reductase transcripts have equal expression levels between the pheromone gland and the body or ahigher expression level in the body. All aldehyde reductase transcripts are present at low abundance（from67to10RPKM） in the pheromone gland transcriptome.（7） Acetyltransferase （ATF）. In the A. ipsilon pheromone gland transcriptome5acetyltransferasehomologous transcripts were identified. RT-PCR and qRT-PCR revealed that three transcripts（Unigene₄07, Unigene₂015and Unigene₁73） are mainly expressed in the pheromone gland andhave a relative high abundance of195,155and71RPKM, respectively in the pheromone glandtranscriptome.（8） Pheromone degrading enzymes （PDEs）. We have identified17transcripts predicted to encodeesterases in the A. ipsilon pheromone gland. Our qRT-PCR results revealed that7of the transcripts（AipsCXE3, AipsCXE7, AipsCXE8, AipsCXE9, AipsCXE11, AipsCXE14and AipsCXE20） areantennal-enriched, three （AipsCXE5, AipsCXE10and AipsCXE15） are both antennal-andpheromone gland-enriched and the remaining7（AipsCXE1, AipsCXE2, AipsCXE4, AipsCXE6,AipsCXE12, AipsCXE13and AipsCXE16） have similar expression levels in antennae, body andpheromone gland, suggesting they are not pheromone specific.（9） OBPs and CSPs. We have identified transcripts of7OBPs and8CSPs from the A. ipsilonpheromone gland. There is one OBP transcript （Unigene₁5711） which is highly expressed in thepheromone gland, and3OBPs （Unigene₅20, Unigene₂120and Unigene₈860） are highlyexpressed in the antennae. One CSP transcript Unigene₁704seems to be gland-specific and has anextremely high expression level （>100folds） in the pheromone glands compared with the antennaeand bodies and a relative high abundance in the pheromone gland transcriptome. Another CSPtranscript Unigene₇21shows a higher expression level in the pheromone gland （10-fold higherthan in bodies） and is extremely abundant with1364RPKM in the pheromone gland transcriptome.The high expression levels of OBPs and CSPs in the pheromone gland is interesting because itsuggests a possible involvement in carrying and releasing sex pheromones.