Functional characterization of pheromone receptors in the tobacco budworm Heliothis virescens

Functional analyses of candidate Heliothis virescens pheromone odorant receptors (HvORs) were conducted using heterologous expression in Xenopus oocytes. HvOR6 was found to be highly tuned to Z9-14:Ald, while HvOR13, HvOR14 and HvOR16 showed specificity for Z11-16:Ald, Z11-16:OAc and Z11-16:OH, respectively. HvOR15, which had been considered a candidate receptor for Z9-14:Ald did not respond to any of the pheromone compounds tested, nor to 50 other general odorants. Thus, while HvOR15 is specifically expressed in H. virescens male antennae, its role in pheromone reception remains unknown. Based on our results and previous research we can now assign pheromone receptors in H. virescens males to each of the critical H. virescens agonistic pheromone compounds and two antagonistic compounds produced by heterospecific females.     

Female-biased expression of odourant receptor genes in the adult antennae of the silkworm, Bombyx mori

Olfaction plays an important role in the life history of insects, including key behaviours such as host selection, oviposition and mate recognition. Odour perception by insects is primarily mediated by the large diverse family of odourant receptors (Ors) that are expressed on the dendrites of olfactory neurones housed within chemosensilla. However, few Or sequences have been identified from the Lepidoptera, an insect order that includes some of the most important pest species worldwide. We have identified 41 Or gene sequences from the silkworm (Bombyx mori) genome, more than double the number of published Or sequences from the Lepidoptera. Many silkworm Ors appear to be orthologs of the 17 published tobacco budworm (Heliothis virescens) Ors indicating that many Or lineages may be conserved within the Lepidoptera. The majority of the Or genes are expressed in adult female and male antennae (determined by quantitative real-time PCR analysis), supporting their probable roles in adult olfaction. Several Or genes are expressed at high levels in both male and female antennae, suggesting they mediate the perception of common host or conspecific volatiles important to both sexes. BmOrs 45-47 group together in the same phylogenetic branch and all three are expressed at moderate female-biased ratios, six to eight times higher in female compared to male moth antennae. Interestingly, BmOrs19 and 30 appear to be expressed predominantly in female antennae, opposite to that of the published silkworm pheromone receptors BmOrs 1 and 3 that are specific to male antennae. These results suggest that BmOr19 and 30 may detect odours critical to female behaviour, such as oviposition cues or male-produced courtship pheromones.     

Larval sensilla of the moth Heliothis virescens respond to sex pheromone components

Female‐released sex pheromones orchestrate the mating behaviour of moths. Recent studies have shown that sex pheromones not only attract adult males but also caterpillars. Single sensillum recordings revealed that larval antennal sensilla of the moth Heliothis virescens respond to specific sex pheromone components. In search for the molecular basis of pheromone detection in larvae, we found that olfactory sensilla on the larval antennae are equipped with the same molecular elements that mediate sex pheromone detection in adult male moths, including the Heliothis virescens receptors 6 (HR6) and HR13, as well as sensory neurone membrane protein 1 (SNMP1). Thirty‐eight olfactory sensory neurones were identified in three large sensilla basiconica; six of these are considered as candidate pheromone responsive cells based on the expression of SNMP1. The pheromone receptor HR6 was found to be expressed in two cells and the receptor HR13 in three cells. These putative pheromone responsive neurones were accompanied by cells expressing pheromone‐binding protein 1 (PBP1) and PBP2. The results indicate that the responsiveness of larval sensilla to female‐emitted sex pheromones is based on the same molecular machinery as in the antennae of adult males.     

A garlic substance disrupts odorant‐binding protein recognition of insect pheromones released from adults of the angoumois grain moth,Sitotroga cerealella (Lepidoptera: Gelechiidae)

The angoumois grain moth, Sitotroga cerealella, is one of the most serious stored‐grain pests worldwide. Control of this moth may be achieved by interfering with olfactory pathways to disrupt male–female communication with sex pheromones, using plant volatiles like garlic or its active substances. Here, three odorant‐binding protein (OBP) genes [namely Si. cerealella general OBP 1 (ScerGOBP1), ScerGOBP2 and Si. cerealella pheromone‐binding protein (ScerPBP)] were cloned from Si. cerealella antennae, and quantitative real‐time PCR showed that these genes were predominantly expressed in adult antennae. ScerPBP expression was male‐biased, but ScerGOBP1 and ScerGOBP2 were similar between sexes. The results of competitive binding assays indicated that a garlic substance, diallyl trisulphide (DATS), had similar or even higher binding affinity to ScerPBP than Si. cerealella sex pheromone, 7Z, 11E‐hexadecadien‐1‐ol acetate (HDA). In olfactometer bioassays, DATS significantly reduced the response of adults to HDA when they were exposed to air filled with HDA and DATS. Surprisingly, ScerGOBP2, which is postulated to be involved in the detection of general odours, displayed higher affinity with HDA than did ScerPBP, indicating that ScerGOBP2 may also have a role in pheromone perception. These data suggest that DATS may interfere with recognition of female‐produced sex pheromone, disrupting female and male mating behaviour and resulting in a new idea for controlling stored grain pests.     

Functional characterization of sex pheromone receptors in the purple stem borer,Sesamia inferens (Walker)

The sex pheromone communication system in moths is highly species‐specific and extremely sensitive, and pheromone receptors (PRs) are thought to be the most important factors in males. In the present study, three full‐length cDNAs encoding PRs were characterized from Sesamia inferens antennae. These three PRs were all male‐specific in expression, but their relative expression levels were very different; SinfOR29 was 17‐ to 23‐fold higher than the other two PRs. Phylogenetic and motif pattern analyses showed that these three PRs were allocated to different PR subfamilies with different motif patterns. Functional analysis using the heterologous expression system of Xenopus oocytes demonstrated that SinfOR29 specifically and sensitively responded to the major pheromone component, Z11‐16:OAc [concentration for 50% of maximal effect (EC₅₀) = 3.431 × 10^?7 M], while SinfOR21 responded robustly to a minor pheromone component Z11‐16:OH (EC₅₀ = 1.087 × 10^?6 M). SinfOR27, however, displayed no response to any of the three pheromone components, but, interestingly, it was sensitive to a non‐sex pheromone component Z9,E12‐14:OAc (EC₅₀ = 1.522 × 10^?6 M). Our results provide insight into the molecular mechanisms of specificity and sensitivity of the sex pheromone communication system in moths.     

Expressed sequence tags and proteomics of antennae from the tortricid moth, Epiphyas postvittana

Genomic and proteomic analyses of the antennae of the light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae) were undertaken to identify genes and proteins potentially involved in odorant and pheromone binding and turnover. An EST approach yielded 5739 sequences, comprising 808 contigs and 1545 singletons. InterPro and B last analyses revealed members of families implicated in odorant and pheromone binding (PBPs, GOBPs, ABPXs and CSPs) and turnover (CXEs, GSTs, CYPs). Of the three pheromone binding proteins (PBPs) identified, two were more highly expressed at the RNA and protein levels in adult male antennae (EpPBP1, EpPBP3), while a third was more highly expressed in female antennae (EpPBP2). To identify proteins involved in the detection of sex-specific signals, differential 2D gel electrophoresis (pH 5–8) followed by mass spectrometry was conducted on antennal proteins from males versus females. Identified male-biased proteins included a pheromone binding protein, a porin, a short chain dehydrogenase/reductase, and a member of the takeout family.     

A cytochrome P450 gene plays a role in the recognition of sex pheromones in the tobacco cutworm,Spodoptera litura

Cytochrome P450 (P450 or CYP) genes are involved in fundamental physiological functions, and might be also associated with the olfactory recognition of sex pheromones in beetles and moths. A P450 gene, Spodoptera litura CYP4L4 (SlituCYP4L4), was cloned for the first time from the antennae of S. litura. SlituCYP4L4 was almost exclusively expressed in the adult stage and predominantly expressed in the adult antennae. In situ hybridization showed that SlituCYP4L4 localized mainly at the base of the long sensilla trichoidea, which responds to sex pheromone components. Pretreatment with an S. litura sex pheromone significantly reduced the expression levels of SlituCYP4L4, consistent with other genes involved in sex pheromone recognition. The expression level of SlituCYP4L4 was different in moths collected with different ratios of sex pheromone lures and collected in different geographical locations. After gene knockdown of SlituCYP4L4 in the antennae, the electroantennogram (EAG) responses of male and female moths to (9Z,11E)‐tetradecadienyl acetate or (9Z,12E)‐tetradecadienyl acetate were significantly decreased. In contrast, EAG responses to plant volatiles and sex pheromones of other moth species were not significantly influenced in these moths. SlituCYP4L4 was also expressed in the gustatory tissues and sensilla, which suggests that SlituCYP4L4 may have other functions in the chemosensory system. Our results have shown for the first time the function of a CYP gene with appendage‐specific expression in insect sex pheromone recognition, especially in adult moths.     

A diversity of putative carboxylesterases are expressed in the antennae of the noctuid moth Spodoptera littoralis

Recent studies have suggested that pheromone‐degrading enzymes belonging to the carboxylesterase family could play a role in the dynamics of the olfactory response to acetate sex pheromones in insects. Bioinformatic analyses of a male antennal expressed sequence tag library allowed the identification of 19 putative esterase genes expressed in the antennae of the moth Spodoptera littoralis. Phylogenetic analysis revealed that these genes belong to different insect esterase clades, defined by their putative cellular localization and substrate preferences. Interestingly, two of the 19 genes appeared to be antennal specific, suggesting a specific role in olfactory processing. This high esterase diversity suggested that the antennae are the location for intense esterase‐based metabolism, against potentially a large range of exogenous and endogenous molecules.     

An odorant receptor from the common cutworm (Spodoptera litura) exclusively tuned to the important plant volatile cis‐3‐Hexenyl acetate

Olfaction plays an important role in insect behaviours. The odorant receptor (OR) repertoire, housed within the dendritic membrane of sensory neurons, is one of the primary determinants of odour recognition. ORs in moths could be classified into pheromone receptors (PRs) and non‐pheromone receptors (non‐PR ORs). Much research in the field of insect olfaction recently has been focused on PRs of the male moth, but few Lepidoptera studies have been done on the functional study of non‐PR ORs. In the present study, we identified and characterized four non‐PR ORs from Spodoptera litura (Lepidoptera: Noctuidae) antennae. The tissue expression pattern showed that the four ORs were mainly expressed in adult antennae and further in situ hybridization revealed SlituOR12 was expressed in both long and short sensilla trichodea and sensilla basiconica. A functional analysis of the four SlituORs was conducted in the heterologous expression system Xenopus oocytes. SlituOR12 was exclusively and sensitively tuned to cis‐3‐Hexenyl acetate and SlituOR19 slightly responded to 4′‐Ethylacetophenone; however, SlituOR44 and SlituOR51 did not respond to any chemicals tested in this study. It is proposed that SlituOR12 might partially account for some key behaviours of the female, such as detection of host location and oviposition site.     

Identification and expression profile of odorant‐binding proteins in Halyomorpha halys (Hemiptera: Pentatomidae)

The brown marmorated stink bug, Halyomorpha halys, is a devastating invasive species in the USA. Similar to other insects, olfaction plays an important role in its survival and reproduction. As odorant‐binding proteins (OBPs) are involved in the initial semiochemical recognition steps, we used RNA‐Sequencing (RNA‐Seq) to identify OBPs in its antennae, and studied their expression pattern in different body parts under semiochemical stimulation by either aggregation or alarm pheromone or food odorants. Thirty full‐length putative HhalOBPs were identified, corresponding to 22 ‘classic’ OBPs and eight ‘Plus‐C’ OBPs. The similarity amongst them ranged from 4.95–70.92%, and with another 325 hemipteran OBPs similarity ranged from 1.94–91.51%, the highest levels being with other stink bug OBPs. Phylogenetic analysis confirmed the monophyly of seven groups of stink bug and other hemipteran OBPs. All 30 HhalOBPs were expressed and about 2/3 were expressed primarily in antennae. The expression of 21 HhalOBPs was higher in the antennae under alarm pheromone stimulus, indicating that multiple OBPs may be responding to this pheromone. Two were highest in antennae under aggregation pheromone stimulus. These findings should provide a basis for understanding the physiological functions of HhalOBPs and the chemosensory perception of this pest, which may help to uncover new control targets for behavioural interference.     

cDNA cloning of biotransformation enzymes belonging to the cytochrome P450 family in the antennae of the noctuid moth Mamestra brassicae

The involvement of cytochrome P450 enzymes in olfaction was demonstrated in vertebrates some time ago. In insects these enzymes are well known for their role in insecticide resistance, but the involvement of P450 in pheromone degradation was only recently demonstrated. Using a PCR strategy, we have isolated two cDNAs from the antennae of the cabbage armyworm Mamestra brassicae - CYP4L4 and CYP4S4 - which encode microsomal P450s. CYP4S4 expression is restricted to the antennae, whereas CYP4L4 is also found in the proboscis and legs. Moreover, the two genes are strongly expressed in one type of sensory unit of the antennae - the sensilla trichodea - which are tuned to the detection of odourants. The putative function of the corresponding enzymes is discussed with regard to their respective expression patterns.     

The tetraspanin superfamily in insects

We describe four members of the tetraspanin/TM4SF superfamily of proteins that were identified in expressed sequence tag projects on the antennae of Manduca sexta moths and Apis mellifera honey bees. The three moth genes are expressed in the sensillar epithelium of male antennae, and some are expressed in female antennae, haemocytes, wing scale cell primordia and/or embryonic tissues. These proteins are probably involved in diverse cellular processes, much like their vertebrate homologues. A phylogenetic analysis of all known tetraspanins, including thirty-seven members of the superfamily revealed by the Drosophila melanogaster genome and twenty in the nematode Caenorhabditis elegans genome, reveals some phylum-specific gene amplification, in particular a contiguous array of eighteen genes in the D. melanogaster genome.