SNMP1 is critical for sensitive detection of the desert locust aromatic courtship inhibition pheromone phenylacetonitrile.

BACKGROUND: Accurate detection of pheromones is crucial for chemical communication and reproduction in insects. In holometabolous flies and moths, the sensory neuron membrane protein 1 (SNMP1) is essential for detecting long-chain aliphatic pheromones by olfactory neurons. However, its function in hemimetabolous insects and its role for detecting pheromones of a different chemical nature remain elusive. Therefore, we investigated the relevance of SNMP1 for pheromone detection in a hemimetabolous insect pest of considerable economic importance, the desert locust Schistocerca gregaria, which moreover employs the aromatic pheromone phenylacetonitrile (PAN) to govern reproductive behaviors. RESULTS: Employing CRISPR/Cas-mediated gene editing, a mutant locust line lacking functional SNMP1 was established. In electroantennography experiments and single sensillum recordings, we found significantly decreased electrical responses to PAN in SNMP1-deficient (SNMP1-/-) locusts. Moreover, calcium imaging in the antennal lobe of the brain revealed a substantially reduced activation of projection neurons in SNMP1-/- individuals upon exposure to PAN, indicating that the diminished antennal responsiveness to PAN in mutants affects pheromone-evoked neuronal activity in the brain. Furthermore, in behavioral experiments, PAN-induced effects on pairing and mate choice were altered in SNMP1-/- locusts. CONCLUSIONS: Our findings emphasize the importance of SNMP1 for chemical communication in a hemimetabolous insect pest. Moreover, they show that SNMP1 plays a crucial role in pheromone detection that goes beyond long-chain aliphatic substances and includes aromatic compounds controlling reproductive behaviors.

Silencing sensory neuron membrane protein RferSNMPu1 impairs pheromone detection in the invasive Asian Palm Weevil.

The red palm weevil (RPW), Rhynchophorus ferrugineus (Olivier), also known as the Asian palm weevil, is an invasive pest that causes widespread damage to palm trees around the globe. As pheromone communication is crucial for their mass attack and survival on palm trees, the olfactory concept of pest control strategies has been widely explored recently. We aim to understand the molecular basis of olfaction in RPW by studying one of the key olfactory proteins in insect pheromone communication, sensory neuron membrane proteins (SNMPs). SNMPs belong to the CD36 (cluster of differentiation 36) family that perform two distinct olfactory roles in insects, either in pheromone (odorant) transfer to the odorant receptors (SNMP1) or in the pheromone clearing process (SNMP2). In this study, we performed antennal transcriptomic screening and identified six SNMPs, mapping them on the R. ferrugineus genome, and confirmed four distinct SNMPs. Both SNMP1 proteins in RPW, viz., RferSNMPu1 and RferSNMPu2, were mapped onto the same scaffold in different loci in the RPW genome. To further understand the function of these proteins, we first classified them using phylogenetic analysis and checked their tissue-specific expression patterns. Further, we measured the relative transcript abundance of SNMPs in laboratory-reared, field-collected adults and pheromone-exposure experiments, ultimately identifying RferSNMPu1 as a potential candidate for functional analysis. We mapped RferSNMPu1 expression in the antennae and found that expression patterns were similar in both sexes. We used RNAi-based gene silencing to knockdown RferSNMPu1 and tested the changes in the RPW responses to aggregation pheromone compounds, 4-methyl-5-nonanol (ferrugineol) and 4-methyl-5-nonanone (ferrugineone), and a kairomone, ethyl acetate using electroantennogram (EAG) recordings. We found a significant reduction in the EAG recordings in the RferSNMPu1 knockdown strain of adult RPWs, confirming its potential role in pheromone detection. The structural modelling revealed the key domains in the RferSNMPu1 structure, which could likely be involved in pheromone detection based on the identified ectodomain tunnels. Our studies on RferSNMPu1 with a putative role in pheromone detection provide valuable insight into understanding the olfaction in R. ferrugineus as well as in other Curculionids, as SNMPs are under-explored in terms of its functional role in insect olfaction. Most importantly, RferSNMPu1 can be used as a potential target for the olfactory communication disruption in the R. ferrugineus control strategies.

Candidate membrane protein gene families related to chemoreception in a wood-boring beetle, Pharsalia antennata Gahan (Coleoptera: Cerambycidae).

The longhorned beetles are key players for the maintenance of biodiversity in the terrestrial ecosystem. As xylophagous cerambycid insects in Coleoptera, the beetles have evolved specialized olfactory and gustatory systems to recognize chemical cues in the surrounding habitats. Despite over 36,000 described species in the Cerambycidae family including a wood-boring pest Pharsalia antennata, only a limited number of them (<1 %) have been characterized regarding their chemical ecology at the molecular level. Here, we surveyed four membrane protein gene families in P. antennata related to chemoreception through transcriptomics, phylogenetics and expression profiling analyses. In total, 144 genes encoding 72 odorant receptors (ORs), 33 gustatory receptors (GRs), 23 ionotropic receptors (IRs), four sensory neuron membrane proteins (SNMPs) and 12 ionotropic glutamate receptors (iGluRs) were harvested from the transcriptome of multiple tissues including antennae and legs of both sexes. The lineage-specific expansion of PantORs possibly implied a diverse range of host plants in this beetle, supporting this correlation between the host range and olfactory receptor repertoire sizes across cerambycid species. Further phylogenetic analysis revealed that Group 2 was contributed mainly to the large OR gene repertoire in P. antennata, representing 18 genes in Group 2A and eight in Group 2B. On the other hand, some key chemosensory genes were identified by applying a phylogenetics approach, such as PantOR21 close to the 2-phenylethanol receptor in Megacyllene caryae, three carbon dioxide GRs and seven Antennal IRs (A-IRs) clades. We also determined sex- and tissue-specific expression profiles of 69 chemosensory genes, revealing the high expression of most PantORs in antennae. Noticeably, 10 sex-biased genes (six PantORs, three PantIRs and PantSNMP1a) were presented in antennae, five sex-biased PantGRs in legs and 39 sex-biased genes (15 PantORs, 13 PantGRs, eight PantIRs and three PantSNMPs) in abdomens. These findings have greatly enhanced our knowledge about the chemical ecology of P. antennata and identify candidate molecular targets for mediating smell and taste of this beetle.

The Molecular and Functional Characterization of Sensory Neuron Membrane Protein 1b (SNMP1b) from Cyrtotrachelus buqueti (Coleoptera: Curculionidae).

Sensory neuron membrane proteins (SNMPs) play important roles in insect chemoreception and SNMP1s have been reported to be essential in detecting sex pheromones in Drosophila and some lepidopteran species. However, SNMPs for Cyrtotrachelus buqueti (Coleoptera: Curculionidae), a major insect pest of bamboo plantations, remain uncharacterized. In this study, a novel SNMP gene, CbuqSNMP1b, from C. buqueti was functionally characterized. The expression of CbuqSNMP1b was significantly higher in antennae than in other tissues of both sexes and the expression level was significantly male-biased. Additionally, CbuqSNMP1b showed significantly higher transcription levels in the adult stage and very low transcription levels in other stages, suggesting that CbuqSNMP1b is involved in the process of olfaction. Fluorescence binding assays indicated that CbuqSNMP1b displayed the strongest binding affinity to dibutyl phthalate (Ki = 9.03 µM) followed by benzothiazole (Ki = 11.59 µM) and phenol (Ki = 20.95 µM) among fourteen C. buqueti volatiles. Furthermore, molecular docking revealed key residues in CbuqSNMP1b that interact with dibutyl phthalate, benzothiazole, and phenol. In conclusion, these findings will lay a foundation to further understand the olfactory mechanisms of C. buqueti and promote the development of novel methods for controlling this pest.

Evidence for a role of SNMP2 and antennal support cells in sensillum lymph clearance processes of moth pheromone-responsive sensilla.

In insect antenna, following the activation of olfactory sensory neurons, odorant molecules are inactivated by enzymes in the sensillum lymph. How the inactivation products are cleared from the sensillum lymph is presently unknown. Here we studied the role of support cells (SCs) and the so-called sensory neuron membrane protein 2 (SNMP2), a member of the CD36 family of lipid transporters abundantly expressed in SCs, in sensillum lymph clearance processes in the moths Heliothis virescens and Bombyx mori. In these species, the sex pheromone components are inactivated to long-chain fatty acids. To approach a role of SNMP2 in the removal of such inactivation products, we analyzed the uptake of a fluorescent long-chain fatty acid analog into a newly generated HvirSNMP2-expressing cell line. We found an increased uptake of the analog into SNMP2-cells compared to control cells, which could be blocked by the CD36 protein inhibitor, SSO. Furthermore, analyses of sensilla from antenna treated with the fatty acid analog indicated that SNMP2-expressing SCs are able to take up fatty acids from the sensillum lymph. In addition, sensilla from SSO-pretreated antenna of B. mori showed reduced removal of the fluorescent analog from the sensillum lymph. Finally, we revealed that SSO pretreatment of male silkmoth antenna significantly prolonged the duration of the female pheromone-induced wing-fluttering behavior, possibly as a result of impaired lymph clearance processes. Together our findings in H. virescens and B. mori support a pivotal role of olfactory SCs in sensillum lymph maintenance processes and suggest an integral role of SNMP2 in the removal of lipophilic "waste products" such as fatty acids resulting from sex pheromone inactivation.

Identification and expression analyses of the olfactory-related genes in different tissues' transcriptome of a predacious soldier beetle, Podabrus annulatus (Coleoptera, Cantharidae).

We sequenced and analyzed the transcriptomes from different tissues of the soldier beetle, Podabrus annulatus (Coleoptera: Cantharidae), and obtained 75.74 Gb clean reads which were assembled into 95,274 unigenes. Among these transcripts, 25,484 unigenes of highly quality were annotated. Based on annotation and tBLASTn results, we identified a total of 101 candidate olfactory-related genes for the first time, including 11 putative odorant-binding proteins (OBPs), 6 chemosensory proteins (CSP), 50 olfactory receptors (ORs), 25 gustatory receptors (GRs), 6 ionotropic receptors (IRs), and 3 sensory neuron membrane proteins (SNMPs). BLASTX best-hit results indicated that these chemosensory genes were most identical to their respective orthologs from Photinus pyralis. Phylogenetic analyses also revealed that the ORs, GRs, and IRs of Podabrus annulatus are closely related to those of Photinus pyralis. The fragment per kilobase per million mapped fragments (FPKM) values showed that the PannOBP2, PannOBP3, and PannOBP10 were predominantly expressed in the antennae, PannOBP1 in the abdomen-thorax, while others were not identified to be tissue-specific. These olfactory-related differentially expressed genes (DEGs) demonstrated different roles in the olfactory system of Podabrus annulatus. This study establishes the groundwork for future research into the molecular mechanism of olfactory recognition in Podabrus annulatus.

Analysis of the Antennal Transcriptome and Identification of Tissue-specific Expression of Olfactory-related Genes in Micromelalopha troglodyta (Lepidoptera: Notodontidae).

Micromelalopha troglodyta (Graeser) has been one of the most serious pests on poplars in China. We used Illumina HiSeq 2000 sequencing to construct an antennal transcriptome and identify olfactory-related genes. In total, 142 transcripts were identified, including 74 odorant receptors (ORs), 32 odorant-binding proteins (OBPs), 13 chemosensory proteins (CSPs), 20 ionotropic receptors (IRs), and 3 sensory neuron membrane proteins (SNMPs). The genetic relationships were obtained by the phylogenetic tree, and the tissue-specific expression of important olfactory-related genes was determined by quantitative real-time PCR (qRT-PCR). The results showed that most of these genes are abundantly expressed in the antennae and head. In most insects, olfaction plays a key role in foraging, host localization, and searching for mates. Our research lays the foundation for future research on the molecular mechanism of the olfactory system in M. troglodyta. In addition, this study provides a theoretical basis for exploring the relationship between M. troglodyta and their host plants, and for the biological control of M. troglodyta using olfactory receptor as targets.

The Sensilla-Specific Expression and Subcellular Localization of SNMP1 and SNMP2 Reveal Novel Insights into Their Roles in the Antenna of the Desert Locust Schistocerca gregaria.

Insect olfactory sensilla house olfactory sensory neurons (OSNs) and supports cells (SCs). The olfactory sensory processes require, besides the odorant receptors (ORs), insect-specific members of the CD36 family, named sensory neuron membrane proteins (SNMPs). While SNMP1 is considered to act as a coreceptor in the OR-mediated detection of pheromones, SNMP2 was found to be expressed in SCs; however, its function is unknown. For the desert locust, Schistocerca gregaria, we previously visualized mRNA for SNMP1 in OSNs and SNMP2 mRNA in cells associated with OSN clusters. Towards an understanding of their functional implication, it is imperative to explore the cellular and the subcellular localization the SNMP proteins. Therefore, we have generated polyclonal antibodies against SNMP1 and SNMP2 and used fluorescence immunohistochemistry (FIHC) to visualize the SNMP proteins. We found SNMP1 in the somata and respective dendrites of all OSNs in trichoid sensilla and in subsets of OSNs in basiconic sensilla. Notably, SNMP1 was also detected in SCs of these sensilla types. In contrast, SNMP2 protein was only visualized in SCs of basiconic and coeloconic sensilla, but not of trichoid sensilla. Exploring the subcellular localization by electron microscopy using anti-SNMP1-ab and anti-SNMP2-ab revealed an immunogold labelling of SC microvilli bordering the sensillum lymph. Together our findings suggest a dual role of SNMP1 in the antenna of S. gregaria, in some OSN subpopulations in odor detection as well as in functions of some SCs, whereas the role of SNMP2 is limited to the functions of support cells.

Identification and expression profiling of chemosensory membrane protein genes in Achelura yunnanensis (Lepidoptera: Zygaenidae).

During the past decade, antennal transcriptome sequencing has been applied to at least 50 species from 16 families of the Lepidoptera order of insects, emphasizing the identification and characterization of chemosensory-related genes. However, little is known about the chemosensory genes in the Zygaenidae family of Lepidoptera. Herein, we report the transmembrane protein gene repertoires involved in chemoreception from Achelura yunnanensis (Lepidoptera: Zygaenidae) through transcriptome sequencing, bioinformatics, phylogenetics and polymerase chain reaction (PCR) approaches. Transcriptome analysis led to the generation of 555.47 million clean reads and accumulation of 83.30 gigabases of data. From this transcriptome, 132 transcripts encoding 69 odorant receptors (ORs), 33 gustatory receptors (GRs), 26 ionotropic receptors (IRs), and four sensory neuron membrane proteins (SNMPs) were identified, 69 of which were full-length sequences. Notably, the number of SNMPs in A. yunnanensis was the largest set in Lepidoptera to date. Phylogenetic analysis combined with sequence homology highlighted several conserved groups of chemoreceptors, including pheromone receptors (a so-called pheromone receptor (PR) clade: AyunOR50 and novel PR members: AyunOR39 and OR40), a phenylacetaldehyde-sensing OR (AyunOR28), carbon dioxide receptors (AyunGR1-3), and antennal IRs (13 A-IRs). In addition, a Zygaenidae-specific OR expansion was observed, including 15 A. yunnanensis members. Expression profiles revealed 99 detectable chemosensory genes in the antennae and 20 in the reproductive tissues, some of which displayed a sex-biased expression. This study identifies potential olfactory molecular candidates for sensing sex pheromones, phenylacetaldehyde or other odorants, and provides preliminary evidence for the putative reproductive function of chemosensory membrane protein genes in A. yunnanensis.

The role of SNMPs in insect olfaction.

The sense of smell enables insects to recognize olfactory signals crucial for survival and reproduction. In insects, odorant detection highly depends on the interplay of distinct proteins expressed by specialized olfactory sensory neurons (OSNs) and associated support cells which are housed together in chemosensory units, named sensilla, mainly located on the antenna. Besides odorant-binding proteins (OBPs) and olfactory receptors, so-called sensory neuron membrane proteins (SNMPs) are indicated to play a critical role in the detection of certain odorants. SNMPs are insect-specific membrane proteins initially identified in pheromone-sensitive OSNs of Lepidoptera and are indispensable for a proper detection of pheromones. In the last decades, genome and transcriptome analyses have revealed a wide distribution of SNMP-encoding genes in holometabolous and hemimetabolous insects, with a given species expressing multiple subtypes in distinct cells of the olfactory system. Besides SNMPs having a neuronal expression in subpopulations of OSNs, certain SNMP types were found expressed in OSN-associated support cells suggesting different decisive roles of SNMPs in the peripheral olfactory system. In this review, we will report the state of knowledge of neuronal and non-neuronal members of the SNMP family and discuss their possible functions in insect olfaction.

Genome-based analysis reveals a novel SNMP group of the Coleoptera and chemosensory receptors in Rhaphuma horsfieldi.

Through an exhaustive homology-based approach, coupled with manual efforts, we annotated and characterized 128 sensory neuron membrane proteins (SNMPs) from genomes and transcriptomes of 22 coleopteran species, with 107 novel candidates. Remarkably, we discovered, for the first time, a novel SNMP group, defined as Group 4 based on the phylogeny, sequence characteristics, gene structure and organization. The lineage-specific expansions in SNMPs occurred mainly in the family Scarabaeidae, harboring 12 representatives in Onthophagus taurus as a typical gene duplication and the most massive set of SNMPs in insects to date. Transcriptome sequencing of Rhaphuma horsfieldi resulted in the yields of approximately 611.9 million clean reads that were further assembled into 543,841 transcripts and 327,550 unigenes, respectively. From the transcriptome, 177 transcripts encoding 84 odorant (ORs), 62 gustatory (GRs), 20 ionotropic (IRs), and 11 ionotropic glutamate (iGluRs) receptors were identified. Phylogenetic analysis classified RhorORs into six groups, RhorGRs into four subfamilies, and RhorIRs into 10 conserved antennal IRs and one divergent IRs. Expression profiles revealed that over 80% of chemosensory genes were specifically or highly transcribed in antennae or tarsi, suggestive of their olfactory and/or gustatory roles. This study has greatly complemented the resources for chemosensory genes in the cerambycid beetles, and most importantly, identifies a novel group of SNMPs in Coleoptera.

Identification and comparative expression profiles of chemosensory genes in major chemoreception organs of a notorious pests, Laodelphax striatellus.

The small brown planthopper, Laodelphax striatellus (Stål) (SBPH), is a notorious rice pest in East Asia and damages the host by feeding on the phloem and transmitting virus particles. Although SBPH relies on chemosensory perception for seeking the host, courtship, selecting oviposition sites and spreading virus particles, a systematic study of chemosensory genes in SBPH is lacking. In this study, we identified multi-gene chemosensory families from the transcriptome of SBPH olfactory organs and analyzed their expression patterns in male and female tissues. Among the chemosensory genes, 14 odorant-binding proteins (OBPs), 12 chemosensory proteins (CSPs), 7 sensory neuron membrane proteins (SNMPs) and 95 odorant receptors (ORs) were identified and annotated in SBPH olfactory organs. Based on expression profile and phylogenetic analysis, LstrOBP1, 2, 5, 6, 7, 10, LstrSNMP1, and most LstrORs showed an antennae-enriched expression pattern, which suggests an olfactory role for these genes. Relative expression of LstrOBPs was validated by quantitative real-time PCR. Our findings provide the genetic information for disrupting the feeding behavior of SBPH, which is essential for developing eco-friendly pest management technologies.

Identification and Characterization of Two Sensory Neuron Membrane Proteins From Onion Maggot (Diptera: Anthomyiidae).

Sensory neuron membrane proteins (SNMPs) in insects are critical peripheral olfactory proteins and act as markers for pheromone detection. However, the SNMPs for onion maggot, Delia antiqua Meigen, a world-wide subterranean pest, have not been previously characterized. In this study, we first report the cloning and characterization of two novel SNMPs from D. antiqua, DantSNMP1 and DantSNMP2. Sequence alignment and phylogenetic analysis showed that DantSNMP1 and DantSNMP2 are very similar to the previously reported SNMP1 and SNMP2 isolated from other dipteran insects but they share low identity with each other. Further expression profile experiments showed that DantSNMP1 is antenna-specific, while DantSNMP2 is expressed both in antennae and nonantennal tissues. Immunocytochemical localization experiments showed that DantSNMP1 was expressed only in sensilla trichodae, which suggests that this protein is involved in pheromone reception in insect olfaction.

SNMP1 and odorant receptors are co-expressed in olfactory neurons of the labial and maxillary palps from the desert locust Schistocerca gregaria (Orthoptera: Acrididae).

In insects, pheromones are detected by olfactory sensory neurons (OSNs) of the antennae that co-express pheromone receptors (PRs) and the "sensory neuron membrane protein 1" (SNMP1). Beyond its relevance for pheromone detection via the antenna, little is known about a potential expression and functional role of SNMP1 in cells of other chemosensory appendages. Here, we report that in the desert locust Schistocerca gregaria, SNMP1 is also expressed in the labial and maxillary palps of the mouthparts. In the palps, the SNMP1-positive cells were situated next to the so-called terminal sensilla that are considered as chemosensory. Moreover, the SNMP1-positive cells of the palps expressed the "odorant receptor co-receptor" (Orco), a marker for OSNs endowed with odorant receptors (ORs), suggesting that these cells are olfactory. With respect to an olfactory function of the SNMP1-positive cells, further analyses examining a possible expression of ORs (notably putative PRs) in the labial and maxillary palps revealed that several members of a particular OR subfamily from S. gregaria, the b-OR group, are co-expressed with SNMP1 in cells of the palps. Interestingly, b-OR types co-expressed with SNMP1 in antennal OSNs were also co-expressed with SNMP1 in cells of the palps, indicating a specific pairing in the expression of SNMP1 and given ORs in both antennae and palps. The co-expression of SNMP1 and certain b-ORs that are regarded as candidate PRs opens up the possibility that chemosensory cells on the palps of the desert locust may contribute to pheromone detection.

Sex- and tissue-specific transcriptome analyses and expression profiling of olfactory-related genes in Ceracris nigricornis Walker (Orthoptera: Acrididae).

BACKGROUND: The sophisticated insect olfactory system plays an important role in recognizing external odors and enabling insects to adapt to environment. Foraging, host seeking, mating, ovipositing and other forms of chemical communication are based on olfaction, which requires the participation of multiple olfactory genes. The exclusive evolutionary trend of the olfactory system in Orthoptera insects is an excellent model for studying olfactory evolution, but limited olfaction research is available for these species. The olfactory-related genes of Ceracris nigricornis Walker (Orthoptera: Acrididae), a severe pest of bamboos, have not yet been reported. RESULTS: We sequenced and analyzed the transcriptomes from different tissues of C. nigricornis and obtained 223.76 Gb clean data that were assembled into 43,603 unigenes with an N50 length of 2235 bp. Among the transcripts, 66.79% of unigenes were annotated. Based on annotation and tBLASTn results, 112 candidate olfactory-related genes were identified for the first time, including 20 odorant-binding proteins (OBPs), 10 chemosensory-binding proteins (CSPs), 71 odorant receptors (ORs), eight ionotropic receptors (IRs) and three sensory neuron membrane proteins (SNMPs). The fragments per kilobase per million mapped fragments (FPKM) values showed that most olfactory-related differentially expressed genes (DEGs) were enriched in the antennae, and these results were confirmed by detecting the expression of olfactory-related genes with quantitative real-time PCR (qRT-PCR). Among these antennae-enriched genes, some were sex-biased, indicating their different roles in the olfactory system of C. nigricornis. CONCLUSIONS: This study provides the first comprehensive list and expression profiles of olfactory-related genes in C. nigricornis and a foundation for functional studies of these olfactory-related genes at the molecular level.

Genomic content of chemosensory genes correlates with host range in wood-boring beetles (Dendroctonus ponderosae, Agrilus planipennis, and Anoplophora glabripennis).

BACKGROUND: Olfaction and gustation underlie behaviors that are crucial for insect fitness, such as host and mate selection. The detection of semiochemicals is mediated via proteins from large and rapidly evolving chemosensory gene families; however, the links between a species' ecology and the diversification of these genes remain poorly understood. Hence, we annotated the chemosensory genes from genomes of select wood-boring coleopterans, and compared the gene repertoires from stenophagous species with those from polyphagous species. RESULTS: We annotated 86 odorant receptors (ORs), 60 gustatory receptors (GRs), 57 ionotropic receptors (IRs), 4 sensory neuron membrane proteins (SNMPs), 36 odorant binding proteins (OBPs), and 11 chemosensory proteins (CSPs) in the mountain pine beetle (Dendroctonus ponderosae), and 47 ORs, 30 GRs, 31 IRs, 4 SNMPs, 12 OBPs, and 14 CSPs in the emerald ash borer (Agrilus planipennis). Four SNMPs and 17 CSPs were annotated in the polyphagous wood-borer Anoplophora glabripennis. The gene repertoires in the stenophagous D. ponderosae and A. planipennis are reduced compared with those in the polyphagous A. glabripennis and T. castaneum, which is largely manifested through small gene lineage expansions and entire lineage losses. Alternative splicing of GR genes was limited in D. ponderosae and apparently absent in A. planipennis, which also seems to have lost one carbon dioxide receptor (GR1). A. planipennis has two SNMPs, which are related to SNMP3 in T. castaneum. D. ponderosae has two alternatively spliced OBP genes, a novel OBP "tetramer", and as many as eleven IR75 members. Simple orthology was generally rare in beetles; however, we found one clade with orthologues of putative bitter-taste GRs (named the "GR215 clade"), and conservation of IR60a from Drosophila melanogaster. CONCLUSIONS: Our genome annotations represent important quantitative and qualitative improvements of the original datasets derived from transcriptomes of D. ponderosae and A. planipennis, facilitating evolutionary analysis of chemosensory genes in the Coleoptera where only a few genomes were previously annotated. Our analysis suggests a correlation between chemosensory gene content and host specificity in beetles. Future studies should include additional species to consolidate this correlation, and functionally characterize identified proteins as an important step towards improved control of these pests.

The expression patterns of SNMP1 and SNMP2 underline distinct functions of two CD36-related proteins in the olfactory system of the tobacco budworm Heliothis virescens.

In insects, male and female pheromone signals are detected by olfactory sensory neurons (OSNs) expressing the "sensory neuron membrane protein type 1". SNMP1 is supposed to function as a co-receptor involved in the transfer of pheromones to adjacent pheromone receptors. In the moth Heliothis virescens, we previously found OSNs that project their dendrites into pheromone-responsive trichoid sensilla and are associated with cells containing transcripts for the HvirSNMP1-related protein HvirSNMP2. Like HvirSNMP1, HvirSNMP2 belongs to the CD36-family of two-transmembrane domain receptors and transporters for lipophilic compounds, but its role in the olfactory system is unknown. Here, we generated polyclonal anti-peptide antibodies against HvirSNMP2 as well as HvirSNMP1 and conducted an in-depth immunohistochemical analysis of their subcellular localization in the antenna of both sexes. In line with a function in pheromone detection, HvirSNMP1 was immunodetected in the somata and the dendrites of distinct OSNs in subsets of trichoid sensilla. These trichoid sensilla contained only one α-SNMP1-positive OSN in males and clusters of 2-3 labeled cells in females. In contrast, experiments with α-SNMP2-antibodies revealed a broad labeling of non-neuronal support cells (SCs) that are associated with OSNs in likely all trichoid and basiconic sensilla of the antenna with no differences between sexes. Detailed confocal microscope examinations of olfactory sensilla revealed SNMP2-like immunoreactivity close to the apical membrane of SCs and interestingly inside the sensillum. Together, these findings indicate a potential function of SNMP2 in pheromone- as well as general odorant-responsive sensilla and a role fundamentally different from SNMP1.

Antennal Transcriptome Analysis of the Chemosensory Gene Families From Trichoptera and Basal Lepidoptera.

The chemosensory gene families of insects encode proteins that are crucial for host location, mate finding, oviposition, and avoidance behaviors. The insect peripheral chemosensory system comprises odorant receptors (ORs), gustatory receptors (GRs), ionotropic receptors (IRs), odorant binding proteins (OBPs), chemosensory proteins (CSPs), and sensory neuron membrane proteins (SNMPs). These protein families have been identified from a large number of insect species, however, they still remain unidentified from several taxa that could provide important clues to their evolution. These taxa include older lepidopteran lineages and the sister order of Lepidoptera, Trichoptera (caddisflies). Studies of these insects should improve evolutionary analyses of insect chemoreception, and in particular shed light on the origin of certain lepidopteran protein subfamilies. These include the pheromone receptors (PRs) in the "PR clade", the pheromone binding proteins (PBPs), general odorant binding proteins (GOBPs), and certain presumably Lepidoptera-specific IR subfamilies. Hence, we analyzed antennal transcriptomes from Rhyacophila nubila (Trichoptera), Eriocrania semipurpurella, and Lampronia capitella (representing two old lepidopteran lineages). We report 37 ORs, 17 IRs, 9 GRs, 30 OBPs, 7 CSPs, and 2 SNMPs in R. nubila; 37 ORs, 17 IRs, 3 GRs, 23 OBPs, 14 CSPs, and 2 SNMPs in E. semipurpurella; and 53 ORs, 20 IRs, 5 GRs, 29 OBPs, 17 CSPs, and 3 SNMPs in L. capitella. We identified IR members of the "Lepidoptera-specific" subfamilies IR1 and IR87a also in R. nubila, demonstrating that these IRs also occur in Trichoptera. Members of the GOBP subfamily were only found in the two lepidopterans. ORs grouping within the PR clade, as well as PBPs, were only found in L. capitella, a species that in contrast to R. nubila and E. semipurpurella uses a so-called Type I pheromone similar to the pheromones of most species of the derived Lepidoptera (Ditrysia). Thus, in addition to providing increased coverage for evolutionary analyses of chemoreception in insects, our findings suggest that certain subfamilies of chemosensory genes have evolved in parallel with the transition of sex pheromone types in Lepidoptera. In addition, other chemoreceptor subfamilies show a broader taxonomic occurrence than hitherto acknowledged.

In Search for Pheromone Receptors: Certain Members of the Odorant Receptor Family in the Desert Locust Schistocerca gregaria (Orthoptera: Acrididae) Are Co-expressed with SNMP1.

Under given environmental conditions, the desert locust (Schistocera gregaria) forms destructive migratory swarms of billions of animals, leading to enormous crop losses in invaded regions. Swarm formation requires massive reproduction as well as aggregation of the animals. Pheromones that are detected via the olfactory system have been reported to control both reproductive and aggregation behavior. However, the molecular basis of pheromone detection in the antennae of Schistocerca gregaria is unknown. As an initial step to disclose pheromone receptors, we sequenced the antennal transcriptome of the desert locust. By subsequent bioinformatical approaches, 119 distinct nucleotide sequences encoding candidate odorant receptors (ORs) were identified. Phylogenetic analyses employing the identified ORs from Schistocerca gregaria (SgreORs) and OR sequences from the related species Locusta migratoria revealed a group of locust ORs positioned close to the root, i.e. at a basal site in a phylogenetic tree. Within this particular OR group (termed basal or b-OR group), the locust OR sequences were strictly orthologous, a trait reminiscent of pheromone receptors from lepidopteran species. In situ hybridization experiments with antennal tissue demonstrated expression of b-OR types from Schistocerca gregaria in olfactory sensory neurons (OSNs) of either sensilla trichodea or sensilla basiconica, both of which have been reported to respond to pheromonal substances. More importantly, two-color fluorescent in situ hybridization experiments showed that most b-OR types were expressed in cells co-expressing the "sensory neuron membrane protein 1" (SNMP1), a marker indicative of pheromone-sensitive OSNs in insects. Analyzing the expression of a larger number of SgreOR types outside the b-OR group revealed that only a few of them were co-expressed with SNMP1. In summary, we have identified several candidate pheromone receptors from Schistocerca gregaria that could mediate responses to pheromones implicated in controlling reproduction and aggregation behavior.

A Sex Pheromone Receptor in the Hessian Fly Mayetiola destructor (Diptera, Cecidomyiidae).

The Hessian fly, Mayetiola destructor Say (Diptera, Cecidomyiidae), is a pest of wheat and belongs to a group of gall-inducing herbivores. This species has a unique life history and several ecological features that differentiate it from other Diptera such as Drosophila melanogaster and blood-feeding mosquitoes. These features include a short, non-feeding adult life stage (1-2 days) and the use of a long-range sex pheromone produced and released by adult females. Sex pheromones are detected by members of the odorant receptor (OR) family within the Lepidoptera, but no receptors for similar long-range sex pheromones have been characterized from the Diptera. Previously, 122 OR genes have been annotated from the Hessian fly genome, with many of them showing sex-biased expression in the antennae. Here we have expressed, in HEK293 cells, five MdesORs that display male-biased expression in antennae, and we have identified MdesOR115 as a Hessian fly sex pheromone receptor. MdesOR115 responds primarily to the sex pheromone component (2S,8E,10E)-8,10-tridecadien-2-yl acetate, and secondarily to the corresponding Z,E-isomer. Certain sensory neuron membrane proteins (i.e., SNMP1) are important for responses of pheromone receptors in flies and moths. The Hessian fly genome is unusual in that it encodes six SNMP1 paralogs, of which five are expressed in antennae. We co-expressed each of the five antennal SNMP1 paralogs together with each of the five candidate sex pheromone receptors from the Hessian fly and found that they do not influence the response of MdesOR115, nor do they confer responsiveness in any of the non-responsive ORs to any of the sex pheromone components identified to date in the Hessian fly. Using Western blots, we detected protein expression of MdesOrco, all MdesSNMPs, and all MdesORs except for MdesOR113, potentially explaining the lack of response from this OR. In conclusion, we report the first functional characterization of an OR from the Cecidomyiidae, extending the role of ORs as long-range sex pheromone detectors from the Lepidoptera into the Diptera.