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Background Many animals rely heavily on olfaction to navigate their environment. Among rodents, olfaction is crucial for a wide range of social behaviors. The vomeronasal olfactory system in particular plays an important role in mediating social communication, including the detection of pheromones and recognition signals. In this study we examine patterns of vomeronasal type-1 receptor (V1R) evolution in the house mouse and related species within the genus Mus. We report the extent of gene repertoire turnover and conservation among species and clades, as well as the prevalence of positive selection on gene sequences across the V1R tree. By exploring the evolution of these receptors, we provide insight into the functional roles of receptor subtypes as well as the dynamics of gene family evolution. Results We generated transcriptomes from the vomeronasal organs of 5 Mus species, and produced high quality V1R repertoires for each species. We find that V1R clades in the house mouse and relatives exhibit distinct evolutionary trajectories. We identify putative species-specific gene expansions, including a large clade D expansion in the house mouse. While gene gains are abundant, we detect very few gene losses. We describe a novel V1R clade and highlight candidate receptors for future study. We find evidence for distinct evolutionary processes across different clades, from largescale turnover to highly conserved repertoires. Patterns of positive selection are similarly variable, as some clades exhibit abundant positive selection while others display high gene sequence conservation. Based on clade-level evolutionary patterns, we identify receptor families that are strong candidates for detecting social signals and predator cues. Our results reveal clades with receptors detecting female reproductive status are among the most conserved across species, suggesting an important role in V1R chemosensation. Conclusion Analysis of clade-level evolution is critical for understanding species’ chemosensory adaptations. This study provides clear evidence that V1R clades are characterized by distinct evolutionary trajectories. As receptor evolution is shaped by ligand identity, these results provide a framework for examining the functional roles of receptors.

The vomeronasal organ (VNO) plays a key role in sensing pheromonal cues, which elicits social and reproductive behaviors. Although the VNO is highly conserved across mammals, it has been lost in some species that have evolved alternate sensing systems during diversification. In this study, we investigate a newly identified VNO-specific gene, ancV1R, in the extant 261 species of mammals to examine the correlation between genotype (ancV1R) and phenotype (VNO). As a result, we found signatures for the relaxation of purifying selection (inactivating mutations and the elevation of dN/dS) on ancV1Rs in VNO-lacking mammals, such as catarrhine primates, cetaceans, the manatees, and several bat lineages, showing the distinct correlation between genotype and phenotype. Interestingly, we further revealed signatures for the relaxation of purifying selection on ancV1R in true seals, otters, the fossa, the owl monkey, and alcelaphine antelopes in which the existence of a functional VNO is still under debate. Our additional analyses on TRPC2, another predictive marker gene for the functional VNO, showed a relaxation of purifying selection, supporting the possibility of VNO loss in these species. The results of our present study invite more in-depth neuroanatomical investigation in mammals for which VNO function remains equivocal.

The Tremellomycetes are a species-rich group within the basidiomycete fungi; however, most analyses of this group to date have focused on pathogenic Cryptococcus species within the order Tremellales. Recent genome-assisted studies of other Tremellomycetes have identified interesting features with respect to biotechnological applications as well as the evolution of genes involved in mating and sexual development. Here, we report genome sequences of two strains of Filobasidium floriforme, a species from the order Filobasidiales, which branches basally to the Tremellales, Trichosporonales, and Holtermanniales. The assembled genomes of strains CBS6241 and CBS6242 are 27.4 Mb and 26.4 Mb in size, respectively, with 8314 and 7695 predicted protein-coding genes. Overall sequence identity at nucleic acid level between the strains is 97%. Among the predicted genes are pheromone precursor and pheromone receptor genes as well as two genes encoding homedomain (HD) transcription factors, which are predicted to be part of the mating type (MAT) locus. Sequence analysis indicates that CBS6241 and CBS6242 carry different alleles for both the pheromone/receptor genes as well as the HD transcription factors. Orthology inference identified 1482 orthogroups exclusively found in F. floriforme, some of which were involved in carbohydrate transport and metabolism. Subsequent CAZyme repertoire characterization identified 267 and 247 enzymes for CBS6241 and CBS6242, respectively, the second highest number of CAZymes among the analyzed Tremellomycete species. In addition, F. floriforme contains five CAZymes absent in other species and several plant-cell-wall degrading CAZymes with the highest copy number in Tremellomycota, indicating the biotechnological potential of this species.

Sensory gene families are of special interest for both what they can tell us about molecular evolution and what they imply as mediators of social communication. The vomeronasal type-1 receptors (V1Rs) have often been hypothesized as playing a fundamental role in driving or maintaining species boundaries given their likely function as mediators of intraspecific mate choice, particularly in nocturnal mammals. Here, we employ a comparative genomic approach for revealing patterns of V1R evolution within primates, with a special focus on the small-bodied nocturnal mouse and dwarf lemurs of Madagascar (genera Microcebus and Cheirogaleus, respectively). By doubling the existing genomic resources for strepsirrhine primates (i.e. the lemurs and lorises), we find that the highly speciose and morphologically cryptic mouse lemurs have experienced an elaborate proliferation of V1Rs that we argue is functionally related to their capacity for rapid lineage diversification. Contrary to a previous study that found equivalent degrees of V1R diversity in diurnal and nocturnal lemurs, our study finds a strong correlation between nocturnality and V1R elaboration, with nocturnal lemurs showing elaborate V1R repertoires and diurnal lemurs showing less diverse repertoires. Recognized subfamilies among V1Rs show unique signatures of diversifying positive selection, as might be expected if they have each evolved to respond to specific stimuli. Furthermore, a detailed syntenic comparison of mouse lemurs with mouse (genus Mus) and other mammalian outgroups shows that orthologous mammalian subfamilies, predicted to be of ancient origin, tend to cluster in a densely populated region across syntenic chromosomes that we refer to as a V1R “hotspot.”

In mammals, social and reproductive behaviors are mediated by chemical cues encoded by hyperdiverse families of receptors expressed in the vomeronasal organ. Between species, the number of intact receptors can vary by orders of magnitude. However, the evolutionary processes behind variation in receptor number, and its link to fitness-related behaviors are not well understood. From vomeronasal transcriptomes, we discovered the first evidence of intact vomeronasal type-1 receptor (V1r) genes in bats, and we tested whether putatively functional bat receptors were orthologous to those of related taxa, or whether bats have evolved novel receptors. Instead of lineage-specific duplications, we found that bat V1rs show high levels of orthology to those of their relatives, and receptors are under comparative levels of purifying selection as non-bats. Despite widespread vomeronasal organ loss in bats, V1r copies have been retained for >65 million years. The highly conserved nature of bat V1rs challenges our current understanding of mammalian V1r function and suggests roles other than conspecific recognition or mating initiation in social behavior.

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.

Sensory neuron membrane proteins (SNMPs), homologs of the human fatty acid transport protein CD36 family, are observed to play a significant role in chemoreception, especially in detecting sex pheromone in Drosophila and some lepidopteran species. In the current study, two full‐length SNMP transcripts, MmedSNMP1 and MmedSNMP2, were identified in the parasitoid Microplitis mediator (Hymenoptera: Braconidae). Quantitative real‐time polymerase chain reaction analysis showed that the expression of MmedSNMP1 was significantly higher in antennae than in other tissues of both sexes. In addition, the MmedSNMP1 transcript was increased dramatically in newly emerged adults and there were no significant differences between adults with or without mating and parasitic experiences. However, compared with MmedSNMP1, the expression of MmedSNMP2 was widely found in various tissues, significantly increased at half‐pigmented pupae stage and remained at a relatively constant level during the following developmental stages. It was found that MmedSNMP1 contained eight exons and seven introns, which was highly conserved compared with other insect species. In situ hybridization assay demonstrated that MmedSNMP1 transcript was distributed widely in antennal flagella. Among selected chemosensory genes (odorant binding protein, odorant receptor, and ionotropic receptor genes), MmedSNMP1 only partially overlapped with MmedORco in olfactory sensory neurons of antennae. Subsequent immunolocalization results further indicated that MmedSNMP1 was mainly expressed in sensilla placodea of antennae and possibly involved in perceiving plant volatiles and sex pheromones. These findings lay a foundation for further investigating the roles of SNMPs in the chemosensation of parasitoids.

Chemoreception systems play a crucial role in regulating key behavioral activities of insects, such as mating, oviposition, and foraging. Odorant receptors (ORs) trigger the transduction of chemical signals into electric signals, and are involved in the corresponding responses associated with odorant guidance behaviors. Pheromone receptors (PRs) of male adult insects are generally thought to function in the recognition of female sex pheromones, and are also important molecular targets for the development of behavioral inhibitors and insecticides. In this study, we successfully expressed and functionally analyzed four AlepPRs of Athetis lepigone in Xenopus oocytes using the two-electrode voltage-clamp method. The results demonstrated that AlepOR3 responded exclusively to the sex pheromone compound of A. lepigone, (Z)-7-dodecenyl acetate (Z7-12:Ac) (EC50 = 8.830 × 10−6 M), while AlepOR4 responded to all five compounds [(Z7-12:Ac, (Z)-8-dodecenyl acetate (Z8-12:Ac), (Z)-9-tetradecenyl acetate (Z9-14:Ac), (Z,E)-9,11-tetradecadienyl acetate (Z9,E11-14:Ac), and (Z,E)-9,12-tetradecadienyl acetate (Z9,E12-14:Ac)] and had a higher response to Z9-14:Ac (EC50 = 2.243 × 10−5 M) than to Z7-12:Ac. However, AlepOR6 displayed a significantly higher response to a non-pheromone of A. lepigone, Z9,E12-14:Ac (EC50 = 7.145 × 10−6 M), than to the other four compounds. AlepOR5 displayed no responses to any of the pheromone compounds of A. lepigone, but responded exclusively to (Z)-11-hexadecenyl acetate (Z11-16:Ac) (EC50 = 7.870 × 10−6 M), a sex pheromone compound of other Noctuidae species. These findings can help explore the molecular mechanisms of sex pheromone recognition in A. lepigone and other moths, and develop broad-spectrum behavioral inhibitors and insecticides against different maize moths in future.

In the European honey bee (Apis mellifera), the olfactory system is essential for foraging and intraspecific communication via pheromones. Honey bees are equipped with a large repertoire of olfactory receptors belonging to the insect odorant receptor (OR) family. Previous studies have indicated that the transcription level of a few OR types including OR11, a receptor activated by the queen-released pheromone compound (2E)-9-oxodecenoic acid (9-ODA), is significantly higher in the antenna of males (drones) than in female workers. However, the number and distribution of antennal cells expressing male-biased ORs is elusive. Here, we analyzed antennal sections from bees by in situ hybridization for the expression of the male-biased receptors OR11, OR18, and OR170. Our results demonstrate that these receptors are expressed in only moderate numbers of cells in the antennae of females (workers and queens), whereas substantially higher cell numbers express these ORs in drones. Thus, the reported male-biased transcript levels are due to sex-specific differences in the number of antennal cells expressing these receptors. Detailed analyses for OR11 and OR18 in drone antennae revealed expression in two distinct subsets of olfactory sensory neurons (OSNs) that in total account for about 69% of the OR-positive cells. Such high percentages of OSNs expressing given receptors are reminiscent of male-biased ORs in moths that mediate the detection of female-released sex pheromone components. Collectively, our findings indicate remarkable similarities between male antenna of bees and moths and support the concept that male-biased ORs in bee drones serve the detection of female-emitted sex pheromones. This article is protected by copyright. All rights reserved.

Palm trees are of immense economic, sociocultural, touristic and patrimonial significance all over the world, and date palm-related knowledge, traditions and practices are now included in UNESCO’s list of the Intangible Cultural Heritage of Humanity. Of all the pests that infest these trees, the red palm weevil (RPW),Rhynchophorus ferrugineus(Olivier) is its primary enemy. The RPW is a category-1 quarantine insect pest that causes enormous economic losses in the cultivation of palm trees worldwide. The RPW synchronizes mass gathering on the palm tree for feeding and mating, regulated by a male-produced pheromone composed of two methyl-branched compounds, (4RS,5RS)-4-methylnonan-5-ol (ferrugineol) and 4(RS)-methylnonan-5-one (ferrugineone). Despite the importance of odorant detection in long-range orientation towards palm trees, palm colonization and mating, nothing regarding the molecular mechanisms of pheromone detection in this species is known. In this study, we report the identification and characterization of the first RPW pheromone receptor,RferOR1. Using gene silencing and functional expression inDrosophilaolfactory receptor neurons, we demonstrate thatRferOR1is tuned to both ferrugineol and ferrugineone and binds five other structurally related molecules. We reveal the lifetime expression ofRferOR1, which correlates with adult mating success irrespective of age, a factor that could explain the wide distribution and spread of this pest. As palm weevils are challenging to control based on conventional methods, elucidation of the mechanisms of pheromone detection opens new routes for mating disruption and the early detection of this pestviathe development of pheromone receptor-based biosensors.

Mirid bugs are a group of important insect pests that cause large annual losses in agricultural production. Many studies have focused on the isolation and identification of sex pheromones in mirid bugs, and the components and biological activity of the sex pheromones have also been studied as a way to control these pests. However, few studies have focused on the mechanisms of pheromone perception. In this study, we identified the odorant receptor repertoire in three mirid bug species, Apolygus lucorum, Adelphocoris lineolatus, and Adelphocoris suturalis using antennal transcriptome sequencing and bioinformatics analysis. The candidate pheromone receptor (PR) genes were then identified by comparative transcriptomic and expression pattern analysis. Importantly, in vitro functional studies have shown that the candidate PRs have robust responses to the main mirid bug sex pheromone components (E)-2-hexenyl butyrate (E2HB) and hexyl butyrate (HB). Our study uncovered the mechanism of pheromone peripheral coding in these three species and elucidated the mechanism by which mirid bugs can specifically recognize a mate. Moreover, the results of our study will provide a theoretical basis for screening effective sex attractants or mating disturbance agents at the molecular and neural levels for enhanced control of these destructive pests.

Sexual reproduction in fungi relies on proteins with well-known functions encoded by the mating-type (MAT) loci. In the Basidiomycota,MATloci are often bipartite, with theP/Rlocus encoding pheromone precursors and pheromone receptors and theHDlocus encoding heterodimerizing homeodomain transcription factors (Hd1/Hd2). The interplay between different alleles of these genes within a single species usually generates at least two compatible mating types. However, a minority of species are homothallic, reproducing sexually without an obligate need for a compatible partner. Here we examine the organization and function of theMATloci ofCystofilobasidium capitatum, a species in the order Cystofilobasidiales, which is unusually rich in homothallic species. We determinedMATgene content and organization inC. capitatumand found that it resembles a mating type of the closely related heterothallic speciesCystofilobasidium ferigula. To explain the homothallic sexual reproduction observed inC. capitatumwe examined HD-protein interactions in the twoCystofilobasidiumspecies and determinedC. capitatum MATgene expression both in a natural setting and upon heterologous expression inPhaffia rhodozyma, a homothallic species belonging to a clade sister toCystofilobasidium. We conclude that the molecular basis for homothallism inC. capitatumappears to be distinct from that previously established forP. rhodozyma. Unlike the latter species, homothallism inC. capitatummay involve constitutive activation or dispensability of the pheromone receptor and the functional replacement of the usual Hd1/Hd2 heterodimer by an Hd2 homodimer. Overall, our results suggest that homothallism evolved multiple times within the Cystofilobasidiales.ImportanceSexual reproduction is important for the biology of eukaryotes because it strongly impacts the dynamics of genetic variation. In fungi, although sexual reproduction is usually associated with the fusion between cells belonging to different individuals (heterothallism), sometimes a single individual is capable of completing the sexual cycle alone (homothallism). Homothallic species are unusually common in a fungal lineage named Cystofilobasidiales. Here we studied the genetic bases of homothallism in one species in this lineage,Cystofilobasidium capitatum, and found it to be different in several aspects when compared to another homothallic species,Phaffia rhodozyma, belonging to the genus most closely related toCystofilobasidium. Our results strongly suggest that homothallism evolved independently inPhaffiaandCystofilobasidium, lending support to the idea that transitions between heterothallism and homothallism are not as infrequent as previously thought. Our work also helps to establish the Cystofilobasidiales as a model lineage in which to study these transitions.

The B mating-type locus of Lentinula edodes, a representative edible mushroom, is highly complex because of allelic variations in the mating pheromone receptors (RCBs) and the mating pheromones (PHBs) in both the B&alpha, and B&beta, subloci. The complexity of the B mating-type locus, five B&alpha, subloci with five alleles of RCB1 and nine PHBs and three B&beta, subloci with 3 alleles of RCB2 and five PHBs, has led us to investigate the specificity of the PHB&ndash, RCB interaction because the interaction plays a key role in non-self-recognition. In this study, the specificities of PHBs to RCB1-2 and RCB1-4 from the B&alpha, sublocus and RCB2-1 from the Bb sublocus were investigated using recombinant yeast strains generated by replacing STE2, an endogenous yeast mating pheromone receptor, with the L. edodes RCBs. Fourteen synthetic PHBs with C-terminal carboxymethylation but without farnesylation were added to the recombinant yeast cells and the PHB&ndash, RCB interaction was monitored by the expression of the FUS1 gene&mdash, a downstream gene of the yeast mating signal pathway. RCB1-2 (B&alpha, 2) was activated by PHB1 (4.3-fold) and PHB2 (2.1-fold) from the B&alpha, 1 sublocus and RCB1-4 (B&alpha, 4) was activated by PHB5 (3.0-fold) and PHB6 (2.7-fold) from the B&alpha, 2 sublocus and PHB13 (3.0-fold) from the B&alpha, 5 sublocus. In particular, PHB3 from B&beta, 2 and PHB9 from B&beta, 3 showed strong activation of RCB2-1 of the B&beta, 1 sublocus by 59-fold. The RCB&ndash, PHB interactions were confirmed in the monokaryotic S1&ndash, 10 strain of L. edodes by showing increased expression of clp1, a downstream gene of the mating signal pathway and the occurrence of clamp connections after the treatment of PHBs. These results indicate that a single PHB can interact with a non-self RCB in a sublocus-specific manner for the activation of the mating pheromone signal pathways in L. edodes.

Gonadotropin-releasing hormone (GnRH) neurons control mammalian reproduction and migrate from their birthplace in the nasal placode to the hypothalamus during development. Despite much work on the origin and migration of GnRH neurons, the processes that control GnRH lineage formation are not fully understood. Here, we demonstrate that Nhlh genes control vomeronasal receptor expression in the developing murine olfactory placode associated with the generation of the first GnRH neurons at embryonic days (E)10-12. Inactivation of s2-microglobulin (s2-m), which selectively affects surface expression of V2Rs, dramatically decreased the number of GnRH neurons in the Nhlh2 mutant background, preventing rescue of fertility in female Nhlh2 mutant mice by male pheromones. In addition, we show that GnRH neurons generated after E12 fail to establish synaptic connections to the vomeronasal amygdala, suggesting the existence of functionally specialized subpopulations of GnRH neurons, which process pheromonal information.

Sensory neuron membrane proteins (SNMPs) play a critical role in insect chemosensory system. Previously, three SNMPs were identified, characterized and functionally investigated in a lepidopteran model insect, Bombyx mori. However, whether these results are consistent across other lepidopteran species are unknown. Here genome and transcriptome data analysis, expression profiling, quantitative real-time PCR (qRT-PCR) and the yeast hybridization system were utilized to examine snmp genes of Helicoverpa armigera, one of the most destructive lepidopteran pests in cropping areas. In silico expression and qRT-PCR analyses showed that, just as the B. mori snmp genes, H. armigera snmp1 (Harmsnmp1) is specifically expressed in adult antennae. Harmsnmp2 is broadly expressed in multiple tissues including adult antennae, tarsi, larval antennae and mouthparts. Harmsnmp3 is specifically expressed in larval midguts. Further RNAseq analysis suggested that the expression levels of Harmsnmp2 and Harmsnmp3 differed significantly depending on the plant species on which the larvae fed, indicating they may be involved in plant-feeding behaviours. Yeast hybridization results revealed a protein-protein interaction between HarmSNMP1 and the sex pheromone receptor, HarmOR13. This study demonstrated that SNMPs may share same functions and mechanisms in different lepidopteran species, which improved our understanding of insect snmp genes and their functions in lepidopterans.

Sparassis latifolia is a valuable edible fungus cultivated in East Asia that is rich in β-glucans. Understanding the mating system and sexual life cycle is important not only for breeding programs to improve strains but also for studies on speciation and population structures. In the present study, mating experiments using monokaryons derived from two different parental strains were performed. Chi-squared test indicated satisfied Mendel segregation, which supported a tetrapolar mating system. A search in the genome for homologs to the well-defined homeodomain and pheromone/receptors, as well as frequently found flanking genes, resulted in the identification of known mating-type loci previously identified in tetrapolar basidiomycetes, each represented by two idiomorphic alleles on separate contigs. Deficiency of the β-flanking protein in S. latifolia and S. crispa around the MAT-A locus may be explained by the locus being rich in transposable elements adjacent to HD genes. Monokaryotic mycelia are characterized by a slower growth rate and a relative lack of aerial mycelia compared with the parental strain. Chlamydospores can be produced in both monokaryotic and dikaryotic mycelial stages. We provide genetic and molecular evidence for the mating system of S. latifolia, a finding that will be helpful for the cross-breeding of this mushroom.

Chemical senses are crucial for insect behaviors such as host preference, mate choice and oviposition site selection. Various protein families are involved in these processes, including odorant receptors (ORs), ionotropic receptors (IRs), gustatory receptors (GRs), chemosensory proteins (CSPs), odorant binding proteins (OBPs) and sensory neuron membrane proteins (SNMPs). To better understand the olfactory mechanism in Spodoptera exigua, we conducted transcriptome analysis of adult antennae and identified a total of 157 candidate chemosensory genes encoding 51 ORs, 20 IRs, 7 GRs, 32 CSPs, 45 OBPs and 2 SNMPs. Quantitative real time PCR (qPCR) analysis of the tissue- and sex-specific expression profiles of ORs, GRs and IRs revealed that these genes could be detected in at least one tissue tested. SexiOR6, 11, 13 and 16, which were predicted to be pheromone receptors based on phylogenetic analysis, exhibited male-specific antennae expression. SexiOR18, 26, 28, 30, 34, 39, and 40 exhibited female-biased expression. SexiGR1, SexiGR2 and SexiGR3, are predicted carbon dioxide receptors, and the former was expressed specifically in antennae, and the latter two were expressed both in antennae and labial palps. SexiIRs had diverse expression profiles. SexiIR8a and SexiIR25a were quite conserved and expressed at high levels. This work will greatly facilitate the understanding of olfactory system in S. exigua and provides valuable information for further functional studies of the chemoreception mechanism in Lepidopteran moths.

Insect pheromone-binding proteins (PBPs) have been proposed to capture and transport hydrophobic sex pheromone components emitted by con-specific insects to pheromone receptors in the hemolymph of male antennal sensilla. In this study, field trapping results indicate that a mixture of E11–16: Ald and Z11–16: Ald can effectively attract a great number of male Tryporyza intacta. Real-time PCR results suggest that the transcript levels of three TintPBP1-3 genes are mainly expressed in the adult antennae. Fluorescence competitive binding experiments show that TintPBP1-3 proteins have great binding affinities to their major sex pheromones. Moreover, TintPBPs clearly cannot bind to other four kinds of sex pheromone components released by another sugarcane borer, Chilo venosatus and Chilo infuscatellu, which have the same host plant and live in similar habitats like T. intacta. The molecular docking results demonstrate that six amino acid residues of the three TintPBPs are crucial for the specific perception of the sex pheromone components. These results will provide a foundation for the development of novel sex pheromone analogues and blocking agents for biological control of sugarcane pests, improving their efficient monitoring and integrated management strategies in the sugarcane field.

Pheromones are crucial for eliciting innate responses and inducing social and sexual behaviors in mammals. The vomeronasal receptor 1 genes, V1Rs, encode members of a pheromone receptor family that are mainly expressed in the vomeronasal organ (VNO). The V1R family shows extraordinary variety in gene number among vertebrates owing to successive gene gains and losses during evolution. Such diversity is believed to reflect a degree of dependence on the VNO. We investigated V1R evolution in primate lineages closely related to humans because these VNOs show a trend toward degeneration. We performed extensive phylogenetic analyses for V1Rs from a broad range of primate species. Although the decline of intact genes was evident in anthropoids (hominoids, Old World monkeys and New World monkeys), we found that a certain number of intact genes persist in New World monkeys. In one New World monkey species, the common marmoset (Callithrix jacchus), we examined seven putatively functional V1Rs using in situ hybridization and reverse transcription-PCR. Based on their mRNA expression patterns in the VNO and other organs, two types of V1Rs emerged: the canonical class with VNO-specific expression, and a second group having more ubiquitous expression in various organs as well as VNO. Furthermore, phylogenetic analysis revealed that the class with the more widespread expression had been retained longer in evolution than the VNO-specific type. We propose that the acquisition of a novel non-VNO-related function(s) may have led to the survival of a small but persistent number of V1Rs in anthropoid primates.

The release and sensation of sex pheromone play a role in the reproductive success of vertebrates including fish. Previous studies have shown that the weather loach Misgurnus anguillicaudatus perceives sex pheromones by olfaction to stimulate courtship behavior. It was speculated that weather loaches use smell to recognize intraspecific mates. However, the identification of loach pheromone receptor has not been reported. By comparative transcriptomic approach, we found that the olfactory receptor gene or114-1 was male-biasedly expressed in the olfactory epithelium of M. anguillicaudatus, M. bipartitus and the closely related species Paramisgurnus dabryanus. This sex-biased expression pattern implicated that or114-1 presumably encoded a sex pheromone receptor in loaches. M. bipartitus and P. dabryanus, like zebrafish, possess one or114-1 only. However, in M. anguillicaudatus, or114-1 has two members: Ma_or114-1a and Ma_or114-1b. Ma_or114-1a, not Ma_or114-1b, showed sex-differential expression in olfactory epithelium. Ma_or114-1b has base insertions that delayed the stop codon, causing the protein sequence length to be extended by 8 amino acids. Ma_or114-1a was subject to positive selection resulting in adaptive amino acid substitutions, which indicated that its ligand binding specificity has probably changed. This adaptive evolution might be driven by the combined effects of sexual selection and reinforcement of premating isolation between the sympatric loach species.

Bacteria use diffusible chemical messengers, termed pheromones, to coordinate gene expression and behavior among cells in a community by a process known as quorum sensing. Pheromones of many gram-positive bacteria, such as Bacillus and Streptococcus, are small, linear peptides secreted from cells and subsequently detected by sensory receptors such as those belonging to the large family of RRNPP proteins. These proteins are cytoplasmic pheromone receptors sharing a structurally similar pheromone-binding domain that functions allosterically to regulate receptor activity. X-ray crystal structures of prototypical RRNPP members have provided atomic-level insights into their mechanism and regulation by pheromones. This review provides an overview of RRNPP prototype signaling; describes the structure–function of this protein family, which is spread widely among gram-positive bacteria; and suggests approaches to target RRNPP systems in order to manipulate beneficial and harmful bacterial behaviors.

Pheromone receptors (PRs) are essential in moths to detect sex pheromones for mate finding. However, it remains unknown from which ancestral proteins these specialized receptors arose. The oldest lineages of moths, so-called non-ditrysian moths, use short-chain pheromone components, secondary alcohols, or ketones, so called Type 0 pheromones that are similar to many common plant volatiles. It is, therefore, possible that receptors for these ancestral pheromones evolved from receptors detecting plant volatiles. Hence, we identified the odorant receptors (ORs) from a non-ditrysian moth, Eriocrania semipurpurella (Eriocraniidae, Lepidoptera), and performed functional characterization of ORs using HEK293 cells. We report the first receptors that respond to Type 0 pheromone compounds; EsemOR3 displayed highest sensitivity toward (2S, 6Z)-6-nonen-2-ol, whereas EsemOR5 was most sensitive to the behavioral antagonist (Z)-6-nonen-2-one. These receptors also respond to plant volatiles of similar chemical structures, but with lower sensitivity. Phylogenetically, EsemOR3 and EsemOR5 group with a plant volatile-responding receptor from the tortricid moth Epiphyas postvittana (EposOR3), which together reside outside the previously defined lepidopteran PR clade that contains the PRs from more derived lepidopteran families. In addition, one receptor (EsemOR1) that falls at the base of the lepidopteran PR clade, responded specifically to β-caryophyllene and not to any other additional plant or pheromone compounds. Our results suggest that PRs for Type 0 pheromones have evolved from ORs that detect structurally-related plant volatiles. They are unrelated to PRs detecting pheromones in more derived Lepidoptera, which, in turn, also independently may have evolved a novel function from ORs detecting plant volatiles.

Agrocybe salicacola is a delicious and cultivable mushroom. It is important to understand this species' inherent characteristics, especially to elucidate the constitution and segregation of mating genes. In this study, two compatible B mating loci in strain YAASM0711 of A. salicacola were cloned from the monokaryons, and sequence and phylogeny analyses showed two conserved genes encoding pheromone receptors maybe lost mating activity, which determined by comparing with those of other mushrooms. In the conserved regions of mating loci, partial insertion/deletion fragments made non-coding regions posses polymorphisms, and monokaryotic strains of different mating types were distinguished from each other according to the amplification profile of variable regions, which suggested mating loci were integrally assigned to offspring strains during mitosis in A. salicacola. As our known, it is the first to develop molecular markers for B mating-type identification using variable non-coding fragments of mating loci in basidiomycetes.

Pheromone receptor-like genes (PRLGs) belong to the G protein-coupled receptors (GPCRs) family that interacts with biotic and abiotic stimulants and transmits signals to intracellular downstream pathways in eukaryotic cells. In this study, we investigated the structure and expressions patterns of PRLGs in Winter Mushroom Flammulina filiformis. Based on the alignment analysis, the structure of PRLGs was found conserved in F. filiformis strains expect few single-nucleotide polymorphism (SNP) sites. Six PRLGs were found at five different unlinked loci, scattered in the genomes of F. filiformis strains. These genes contain 2-5 introns; however, the introns were not found in the same relative positions regarding the encoded protein sequences in tested strains of F. filiformis. Three conserved motifs were identified in peptides structures of PRLGs, however, FfSte3.s6 contained only two types, suggests its difference in evolution and function. We have further analyzed the expression patterns of each PRLGs in different developmental stages of the fruiting body in F. filiformis by quantitative real-time polymerase chain reaction (qRT-PCR). The results exhibited expression variation of PRLGs at different developmental stages of the F. filiformis. Especially, FfSte3.s1 and FfSte3.s2 exhibited maximum expression level in mycelia stage. Other PRLGs exhibited high expression level in fruiting body stages. This study suggests that PRLGs could be vital genes involving in fruiting body development in F. filiformis. However, further studies could be performed to reveal their specific functional pathways in the fruiting body development.

Dendrolimus punctatus Walker (Lepidoptera: Lasiocampidae) is a pine caterpillar moth distributed in most areas of southern China and is an economically important pest of pine, due to its defoliation activity. Understanding fundamental sex pheromone perception mechanisms in D. punctatus may provide effective and sustainable options for novel control strategies. However, the identification and function of pheromone receptors, key genes that receipt the pheromone of this pest, are both unclear now. Previous researches suggested several candidate pheromone receptors whose expression levels were male antennae bias in D. punctatus. In this study, we cloned six candidate pheromone receptors (DpunOR 20/45/46/51/54/58) and Orco from D. punctatus. Phylogenetic tree analysis showed that lepidopteran PRs tend to be conserved and clustered together; however, D. punctatus candidate PRs were located in a distinct clade. Motif analysis of PRs showed clear sequences differences between Dendrolimus spp. and other tested moth species. To illustrate the ligand response properties of the candidate PRs of D. punctatus, each of the six genes was expressed with an Orco gene in Xenopus oocytes and using two-electrode voltage-clamp recordings. Finally, we successfully identified two sex pheromone receptors (PR45 and PR46). Our study, which identified a novel lineage of PRs tuned to Type I pheromones in Lepidoptera, provides evidence for the new evolution origin of sex pheromone communication in moths, and lays a foundation for the development of novel control strategies of D. punctatus.

Identification of pheromone receptors plays a central role for uncovering signaling pathways that underlie chemical communication in animals. Here, we describe the synthesis and bioactivity of photoaffinity probes for the ascaroside ascr#8, a sex-pheromone of the model nematode, Caenorhabditis elegans. Structure-activity studies guided incorporation of alkyne- and diazirine-moieties and revealed that addition of functionality in the sidechain of ascr#8 was well tolerated, whereas modifications to the ascarylose moiety resulted in loss of biological activity. Our study will guide future probe design and provides a basis for pheromone receptor identification via photoaffinity labeling in C. elegans.

Sex pheromones serve a critical role in Lepidopterans finding mates. Male moths perceive and react to sex pheromones emitted by conspecific females through a delicate pheromone communication system. Pheromone receptors (PRs) are the key sensory elements at the beginning of that process. The codling moth (Cydia pomnonella) is an important pome fruit pest globally and a serious invasive species in China. Pheromone-based techniques have been used successfully in monitoring and controlling this species. We conducted ribonucleic acid sequencing analysis of the codling moth antennal transcriptome and identified 66 odorant receptors (ORs) in a population from Xinjiang province, China, of which 14 were PRs, including two novel PRs (CpomOR2e and CpomOR73). Four PRs that contain full-length open reading frames (CpomOR1, OR2a, OR5, OR7) and four PRs with ligands that have not been reported previously (CpomOR1, OR2a, OR5, OR7) were selected to deorphanize in the heterologous Xenopus oocyte expression system. Specifically, we found that CpomOR2a and CpomOR5 responded to (E,E)-8, 10-dodecadien-1-yl acetate (codlemone acetate). Furthermore, CpomOR5 (EC50 = 1.379 × 10-8 mol/L) was much more sensitive to codlemone acetate than CpomOR2a (EC50 = 1.663 × 10-6 mol/L). Since codlemone acetate is an important component of C. pomonella sex pheromone, our results improve the current understanding of pheromone communication in codling moths and will be helpful for the development of pest management strategies.

Mitochondria are inherited uniparentally during sexual reproduction in the majority of eukaryotic species studied, including humans, mice, nematodes, as well as many fungal species. Mitochondrial uniparental inheritance (mito-UPI) could be beneficial in that it avoids possible genetic conflicts between organelles with different genetic backgrounds, as recently shown in mice; and it could prevent the spread of selfish genetic elements in the mitochondrial genome. Despite the prevalence of observed mito-UPI, the underlying mechanisms and the genes involved in controlling this non-mendelian inheritance are poorly understood in many species. InCryptococcus neoformans, a human pathogenic basidiomyceteous fungus, mating types (MATα andMATa) are defined by alternate alleles at the singleMATlocus that evolved from fusion of the twoMATloci (P/Rencoding pheromones and pheromone receptors,HDencoding homeodomain transcription factors) that are the ancestral state in the basidiomycota. Mitochondria are inherited uniparentally from theMATaparent inC. neoformansand this requires theSXI1α andSXI2aHD factors encoded byMAT. However, there is evidence additional genes contribute to control of mito-UPI inCryptococcus. Here we show that inCryptococcus amylolentus, a sibling species ofC. neoformanswith unlinkedP/RandHD MATloci, mitochondrial uniparental inheritance is controlled by theP/Rlocus, and is independent of theHDlocus. Consistently, by replacing theMATα alleles of the pheromones (MF) and pheromone receptor (STE3) with theMATaalleles, we show that theseP/Rlocus defining genes indeed affect mito-UPI inC. neoformansduring sexual reproduction. Additionally, we show that during early stages ofC. neoformanssexual reproduction, conjugation tubes are always produced by theMATα cells, resulting in unidirectional migration of theMATα nucleus into theMATacell during zygote formation. This process is controlled by theP/Rlocus and could serve to physically restrict movement ofMATα mitochondria in the zygotes, and thereby contribute to mito-UPI. We propose a model in which both physical and genetic mechanisms function in concert to prevent the coexistence of mitochondria from the two parents in the zygote and subsequently in the meiotic progeny, thus ensuring mito-UPI in pathogenicCryptococcus, as well as in closely related non-pathogenic species. The implications of these findings are discussed in the context of the evolution of mito-UPI in fungi and other more diverse eukaryotes.

Accurate detection and recognition of chemical signals play extremely important roles for insects in their survival and reproduction. Chemosensory receptors, including odorant receptors (ORs), ionotropic receptors (IRs) and gustatory receptors (GRs), are involved in detection of volatile signals. In the present study, we aimed to identify candidate chemosensory receptors, and RNA-seq technology was employed to sequence the antennal transcriptome of Protaetia brevitarsis (Coleoptera: Scarabaeidae: Cetoniinae), a native agricultural and horticultural pest in East-Asia. According to the sequence similarity analysis, we identified 72 PbreORs, 11 PbreGRs and eight PbreIRs. Among PbreORs, PbreOR2, PbreOR33 and PbreOR53 were preliminarily classified into pheromone receptors. Further qRT-PCR analysis indicated that 11 PbreORs were specifically expressed in the antennae of male P. brevitarsis, whereas 23 PbreORs were specifically expressed in the female antennae. Our results laid a solid foundation for further functional elucidations of insect chemoreceptors, which could be used as the potential targets of pest management.

Sex pheromones are crucial for communication between females and males in moths, and pheromone receptors (PRs) play a key role in peripheral coding of sex pheromones. During the last decade, many PR candidates have been identified based on transcriptome sequencing and bioinformatic analysis, but their detailed functions remain mostly unknown. Here, focusing on four PR candidates of Athetis dissimilis (AdisOR1, AdisOR6, AdisOR11 and AdisOR14) identified in a previous study, we first cloned the full-length cDNAs and determined the tissue expression profiles by quantitative real-time PCR (qPCR). The results revealed that expression of three of these genes were male antennae-specific, while AdisOR11 was similar in expression between male and female antennae. Furthermore, the expression level of AdisOR1 was much higher than those of the other three genes. Then, functional analysis was conducted using Xenopus oocyte system. AdisOR1 responded strongly to the sex pheromone component Z9–14:OH and the potential pheromone component Z9,E12–14:OH, suggesting its important role in the sex pheromone perception; AdisOR14 showed specificity for Z9,E12–14:OH; while AdisOR6 and AdisOR11 did not respond to any of the pheromone components and analogs tested. Taken together, this study contributes to elucidate the molecular mechanism of sex pheromone reception and provides potential targets for development of OR based pest control techniques in A. dissimilis.

A new track in molecular communication is using pheromones which can scale up the range of diffusion-based communication from $\mu $ meters to meters and enable new applications requiring long range. Pheromone communication is the emission of molecules in the air which trigger behavioral or physiological responses in receiving organisms. The objective of this paper is to introduce a new end-to-end model which incorporates pheromone behavior with communication theory for plants. The proposed model includes both the transmission and reception processes as well as the propagation channel. The transmission process is the emission of pheromones from the leaves of plants. The dispersion of pheromones by the flow of wind constitutes the propagation process. The reception process is the sensing of pheromones by the pheromone receptors of plants. The major difference of pheromone communication from other molecular communication techniques is the dispersion channel acting under the laws of turbulent diffusion. In this paper, the pheromone channel is modeled as a Gaussian puff, i.e., a cloud of pheromone released instantaneously from the source whose dispersion follows a Gaussian distribution. Numerical results on the performance of the overall end-to-end pheromone channel in terms of normalized gain and delay are provided.

Sclerotinia sclerotiorum (Lib.) de Bary is a phytopathogenic fungus that relies on the completion of the sexual cycle to initiate aerial infections. The sexual cycle produces apothecia required for inoculum dispersal. In this study, insight into the regulation of apothecial multicellular development was pursued through functional characterization of mating-type genes. These genes are hypothesized to encode master regulatory proteins required for aspects of sexual development ranging from fertilization through fertile fruiting body development. Experimentally, loss-of-function mutants were created for the conserved core mating-type genes (MAT1-1-1, and MAT1-2-1), and the lineage-specific genes found only in S. sclerotiorum and closely related fungi (MAT1-1-5, and MAT1-2-4). The MAT1-1-1, MAT1-1-5, and MAT1-2-1 mutants are able to form ascogonia but are blocked in all aspects of apothecium development. These mutants also exhibit defects in secondary sexual characters including lower numbers of spermatia. The MAT1-2-4 mutants are delayed in carpogenic germination accompanied with altered disc morphogenesis and ascospore production. They too produce lower numbers of spermatia. All four MAT gene mutants showed alterations in the expression of putative pheromone precursor (Ppg-1) and pheromone receptor (PreA, PreB) genes. Our findings support the involvement of MAT genes in sexual fertility, gene regulation, meiosis, and morphogenesis in S. sclerotiorum.

The sensory neuron membrane protein, SNMP1, was initially discovered in moths and is associated with sex pheromone sensitive neurons, suggesting a role in the detection of these semiochemicals. Although DrosophilaSNMP1 has been reported to be involved in detecting of the sex pheromone cis-vaccenyl acetate (cVA), the role of this protein in moths in vivo is still largely unexplored. In this study we developed a SNMP1−/− homozygous mutant line of Helicoverpa armigera using CRISPR/Cas9. Wind-tunnel behavioral experiments showed that HarmSNMP1−/− males could not be attracted by sex pheromones (Z11-16:Ald/Z9-16:Ald = 97/3), while mating behavior obvervations revealed that the SNMP1 mutant males didn't react much to calling females and the rate of copulation was significantly decreased. The electrophysiological results indicated that HarmSNMP1 contributes to the detection of 16-carbon liner sex pheromones, (Z)-11-hexadecenal (Z11-16:Ald), (Z)-9-hexadecenal (Z9-16:Ald), (Z)-11-hexadecanol (Z11-16:OH) and 16-carbon acetate (Z)-11-hexadecenyl acetate (Z11-16:OAc), but is not required for detecting the 14-carbon sex pheromone component (Z)-9-tetradecenal (Z9-14:Ald) an analogue of Z11-16:Ald, (Z)-9-tetradecen-1-yl formate (Z9-14:OFor), which can activate the Z11-16:Ald-responsive neuron. Taken together, our studies indicated that HarmSNMP1 has an important role in the detection of long-chain sex pheromones, but is not essential for detecting shorter chain sex pheromone in vivo.

Insect courtship and mating depend on integration of olfactory, visual, and tactile cues. Compared to other insects, Bombyx mori, the domesticated silkworm, has relatively simple sexual behaviors as it cannot fly. Here by using CRISPR/Cas9 and electrophysiological techniques we found that courtship and mating behaviors are regulated in male silk moths by mutating genes in the sex determination cascade belonging to two conserved pathways. Loss of Bmdsx gene expression significantly reduced the peripheral perception of the major pheromone component bombykol by reducing expression of the product of the BmOR1 gene which completely blocked courtship in adult males. Interestingly, we found that mating behavior was regulated independently by another sexual differentiation gene, Bmfru. Loss of Bmfru completely blocked mating, but males displayed normal courtship behavior. Lack of Bmfru expression significantly reduced the perception of the minor pheromone component bombykal due to the down regulation of BmOR3 expression; further, functional analysis revealed that loss of the product of BmOR3 played a key role in terminating male mating behavior. Our results suggest that Bmdsx and Bmfru are at the base of the two primary pathways that regulate olfactory-based sexual behavior., Author summary The fundamental insect sexual behaviors, courtship and mating, result from successful integration of olfactory, vision, tactile and other complex innate behaviors. In the widely used insect model, Drosophila melanogaster, the sex determination cascade genes fruitless and doublesex are involved in the regulation of courtship and mating behaviors; however, little is known about the function of these sexual differentiation genes in regulating sex behaviors of Lepidoptera. Here we combine genetics and electrophysiology to investigate regulation pathway of sexual behaviors in the model lepidopteran insect, the domesticated silk moth, Bombyx mori. Our results support the presence of two genetic pathways in B. mori, named Bmdsx-BmOR1-bombykol and Bmfru-BmOR3-bombykal, which control distinct aspects of male sexual behavior that are modulated by olfaction. This is the first comprehensive report about the role of sex differentiation genes in the male sexual behavior in the silk moth.

The G protein cascade amplification system couples with several receptors to sense/amplify the cellular signal, implying universal application. In order to explore whether GPCRs can trigger G protein signal amplification in tissues/cells from different species, bombykol receptor was isolated and purified from antennas of male Bombyx mori, which subsequently self-assembled on the cell membrane in rat taste buds/rat vomeronasa/catfish tentacles/taste bud tissues of rabbits/pig/cattle in those lacking endogenous bombykol receptor, followed by immobilization between two sheets of nucleopore membranes fixed by sodium alginate-starch gel, forming the sandwich-type sensing membrane, which in turn was immobilized on the glass-carbon electrode. Thus, bombykol receptor sensors were established with different tissues. The response current of bombykol receptor sensor toward bombykol was measured with an electrochemical workstation. Every bombykol receptor sensor could sense bombykol based on enzyme-substrate kinetics. The double reciprocal plot and the activation constant values of bombykol receptor sensors assembled with rat taste buds, rat vomeronasa, catfish tentacles, rabbit taste buds, pig taste buds, and cattle taste buds were calculated. Approximately 2-3 receptors could trigger the G protein cascade amplification system and achieve the maximum signal output. Moreover, the detection lower limit indicated that the bombykol receptor self-assembled on the cell membranes of different tissues that transmitted and amplified the bombykol signal with hypersensitivity. Also, cattle taste bud tissues served as an ideal system for heterogeneous GPCRs self-assembly and signal sensing/amplification. This sensing technique and method had promising potential in studies of biological pest control, sex pheromone detection, and receptor structure and function.

In fungi, mating between partners depends on the molecular recognition of two peptidyl mating pheromones by their respective receptors. The fission yeast Schizosaccharomyces pombe (Sp) has two mating types, Plus (P) and Minus (M). The mating pheromones P-factor and M-factor, secreted by P and M cells, are recognized by the receptors mating type auxiliary minus 2 (Mam2) and mating type auxiliary plus 3 (Map3), respectively. Our recent study demonstrated that a few mutations in both M-factor and Map3 can trigger reproductive isolation in S. pombe. Here, we explored the mechanism underlying reproductive isolation through genetic changes of pheromones/receptors in nature. We investigated the diversity of genes encoding the pheromones and their receptor in 150 wild S. pombe strains. Whereas the amino acid sequences of M-factor and Map3 were completely conserved, those of P-factor and Mam2 were very diverse. In addition, the P-factor gene contained varying numbers of tandem repeats of P-factor (4–8 repeats). By exploring the recognition specificity of pheromones between S. pombe and its close relative Schizosaccharomyces octosporus (So), we found that So-M-factor did not have an effect on S. pombe P cells, but So-P-factor had a partial effect on S. pombe M cells. Thus, recognition of M-factor seems to be stringent, whereas that of P-factor is relatively relaxed. We speculate that asymmetric diversification of the two pheromones might be facilitated by the distinctly different specificities of the two receptors. Our findings suggest that M-factor communication plays an important role in defining the species, whereas P-factor communication is able to undergo a certain degree of flexible adaptation–perhaps as a first step toward prezygotic isolation in S. pombe., An asymmetric pheromone/receptor system in the fission yeast Schizosaccharomyces pombe might allow flexible adaptation of pheromones to mutational changes while maintaining stringent recognition for mating partners, perhaps as a first step toward prezygotic mating isolation., Author summary The emergence of a new species might occur when two groups can no longer mate. Although such reproductive isolation is considered a key evolutionary process, the mechanisms by which it actually occurs have been confined to conjecture. The two sexes (Plus [P] and Minus [M]) of S. pombe each secrete a pheromone (P-factor and M-factor), which binds to a corresponding receptor (mating type auxiliary minus 2 [Mam2] and mating type auxiliary plus 3 [Map3]) on cells of the opposite sex. The interaction between a pheromone and its receptor is essential for successful mating. Here, we explored conservation of the mating pheromone communication system among 150 wild S. pombe strains of different geographical origins and the closely related species S. octosporus. We found that 1) the M-factor/Map3 interaction was completely conserved, whereas the P-factor/Mam2 interaction was very diverse in the strains investigated, and 2) most of the P-factor variants were functional across species. Thus, we have revealed an asymmetric pheromone/receptor system in fungal mating: namely, whereas M-factor communication operates extremely stringently, P-factor communication has the flexibility to create variations, perhaps facilitating prezygotic isolation in S. pombe.

The odorant receptors (ORs) of insects are crucial for host and mate recognition. In moths (Lepidoptera), specialized ORs are involved in male detection of the sex pheromone produced by females. Most moth sex pheromones are C10-C18 acetates, alcohols, and aldehydes (Type I pheromones), and most pheromone receptors (PRs) characterized to date are from higher Lepidoptera (Ditrysia), responding to these types of compounds. With few exceptions, functionally characterized PRs fall into what has been called the "PR-clade", which also contains receptors that have yet to be characterized. While it has been suggested that moth PRs have evolved from plant odor-detecting ORs, it is not known when receptors for Type I pheromones arose. This is largely due to a lack of functionally characterized PRs from non-ditrysian Lepidoptera. The currant shoot borer moth, Lampronia capitella (Prodoxidae), belongs to a non-ditrysian lineage, and uses Type I pheromone compounds. We identified 53 ORs from antennal transcriptomes of this species, and analyzed their phylogenetic relationships with known lepidopteran ORs. Using a HEK293 cell-based assay, we showed that three of the LcapORs with male-biased expression (based on FPKM values) respond to Type I pheromone compounds. Two of them responded to pheromone components of L. capitella and one to a structurally related compound. These PRs are the first from a non-ditrysian moth species reported to respond to Type I compounds. They belong to two of the more early-diverging subfamilies of the PR-clade for which a role in pheromone detection had not previously been demonstrated. Hence, our definition of the monophyletic lepidopteran PR-clade includes these receptors from a non-ditrysian species, based on functional support.

SummarySex with another species can be disastrous, especially for organisms that mate only once, like yeast [1–3]. Courtship signals, including pheromones, often differ between species and can provide a basis for distinguishing between reproductively compatible and incompatible partners [4–6]. Remarkably, we show that the baker’s yeast Saccharomyces cerevisiae does not reject mates engineered to produce pheromones from highly diverged species, including species that have been reproductively isolated for up to 100 million years. To determine whether effective discrimination against mates producing pheromones from other species is possible, we experimentally evolved pheromone receptors under conditions that imposed high fitness costs on mating with cells producing diverged pheromones. Evolved receptors allowed both efficient mating with cells producing the S. cerevisiae pheromone and near-perfect discrimination against cells producing diverged pheromones. Sequencing evolved receptors revealed that each contained multiple mutations that altered the amino acid sequence. By isolating individual mutations, we identified specific amino acid changes that dramatically improved discrimination. However, the improved discrimination conferred by these individual mutations came at the cost of reduced mating efficiency with cells producing the S. cerevisiae pheromone, resulting in low fitness. This tradeoff could be overcome by simultaneous introduction of separate mutations that improved mating efficiency alongside those that improved discrimination. Thus, if mutations occur sequentially, the shape of the fitness landscape may prevent evolution of the optimal phenotype [7, 8]—offering a possible explanation for the poor discrimination of receptors found in nature.

Chemical senses 43(9), 667 - 695 (2018). doi:10.1093/chemse/bjy061, Published by Oxford Univ. Press, Oxford

Homothallism (self-fertility) describes a wide variety of sexual strategies that enable a fungus to reproduce in the absence of a mating partner. Unisexual reproduction, a form of homothallism, is a process whereby a fungus can progress through sexual reproduction in the absence of mating genes previously considered essential for self-fertility. In this study, we consider the molecular mechanisms that allow for this unique sexual behaviour in the saprotrophic ascomycete; Huntiella moniliformis. These molecular mechanisms are also compared to the underlying mechanisms that control sex in Huntiella omanensis, a closely related, but self-sterile, species. The main finding was that H. omanensis displayed mating-type dependent expression of the a- and α-pheromones. This was in contrast to H. moniliformis where both pheromones were co-expressed during vegetative growth and sexual development. Furthermore, H. moniliformis also expressed the receptors of both pheromones. Consequently, this fungus is likely able to recognize and respond to the endogenously produced pheromones, allowing for self-fertility in the absence of other key mating genes. Overall, these results are concomitant with those reported for other unisexual species, but represent the first detailed study considering the unisexual behaviour of a filamentous fungus.

International audience; Sex pheromone communication in Lepidoptera has long been a valuable model system for studying fundamental aspects of olfaction and its study has led to the establishment of environmental-friendly pest control strategies. The cabbage moth, Mamestra brassicae (Linnaeus) (Lepidoptera: Noctuidae), is a major pest of Cruciferous vegetables in Europe and Asia. Its sex pheromone has been characterized and is currently used as a lure to trap males; however, nothing is known about the molecular mechanisms of sex pheromone reception in male antennae. Using homology cloning and rapid amplification of cDNA ends-PCR strategies, we identified the first candidate pheromone receptor in this species. The transcript was specifically expressed in the antennae with a strong male bias. In situ hybridization experiments within the antennae revealed that the receptor-expressing cells were closely associated with the olfactory structures, especially the long trichoid sensilla known to be pheromone-sensitive. The deduced protein is predicted to adopt a seven-transmembrane structure, a hallmark of insect odorant receptors, and phylogenetically clustered in a clade that grouped a majority of the Lepidoptera pheromone receptors characterized to date. Taken together, our data support identification of a candidate pheromone receptor and provides a basis for better understanding how this species detects a signal critical for reproduction.

The highly specialized olfactory receptor neurons (ORNs) on the antennae of male moths can recognize blends of several pheromone components. In previous studies, a total of six candidate pheromone receptor (PR) genes were cloned and functionally characterized in the diamondback moth, Plutella xylostella. In the present work, we report on three novel candidate pheromone receptor genes: PxylOR8, PxylOR41, and PxylOR45 in the same species. Gene expression analysis revealed that PxylOR8 is specifically expressed in female adult antennae, while PxylOR41 and PxylOR45 are expressed in antennae in both sexes, but with a male bias. In situ hybridization revealed that PxylOR8, PxylOR41 and PxylOR45 are localized in long trichoid sensilla. Functional analyses on the three pheromone receptor genes were then performed using the heterologous expression system of Xenopus oocytes. PxylOR41 was tuned to two minor pheromone components Z9-14:Ac, Z9-14:OH, and their analog Z9-14:Ald. PxylOR8 and PxylOR45 did not respond to any tested pheromone components and analogs. These results may contribute to clarifying how pheromone detection works in P. xylostella.

The piercing fruit moth Oraesia emarginata is an economically significant pest; however, our understanding of its olfactory mechanisms in infestation is limited. The present study conducted antennal transcriptome analysis of olfactory genes using real-time quantitative reverse transcription PCR analysis (RT-qPCR). We identified a total of 104 candidate chemosensory genes from several gene families, including 35 olfactory receptors (ORs), 41 odorant-binding proteins, 20 chemosensory proteins, 6 ionotropic receptors, and 2 sensory neuron membrane proteins. Seven candidate pheromone receptors (PRs) and 3 candidate pheromone-binding proteins (PBPs) for sex pheromone recognition were found. OemaOR29 and OemaPBP1 had the highest fragments per kb per million fragments (FPKM) values in all ORs and OBPs, respectively. Eighteen olfactory genes were upregulated in females, including 5 candidate PRs, and 20 olfactory genes were upregulated in males, including 2 candidate PRs (OemaOR29 and 4) and 2 PBPs (OemaPBP1 and 3). These genes may have roles in mediating sex-specific behaviors. Most candidate olfactory genes of sex pheromone recognition (except OemaOR29 and OemaPBP3) in O. emarginata were not clustered with those of studied noctuid species (type I pheromone). In addition, OemaOR29 was belonged to cluster PRIII, which comprise proteins that recognize type II pheromones instead of type I pheromones. The structure and function of olfactory genes that encode sex pheromones in O. emarginata might thus differ from those of other studied noctuids. The findings of the present study may help explain the molecular mechanism underlying olfaction and the evolution of olfactory genes encoding sex pheromones in O. emarginata.

Traps baited with female-produced sex pheromones have been very effective in the monitoring and management of mirid bugs in numerous field trials. However, none of the target odorant receptors for sex pheromone components in Apolygus lucorum have been identified. Here, we identified one candidate sex pheromone receptor, AlucOR4, from A. lucorum. Quantitative real-time PCR (qPCR) analysis revealed that AlucOR4 was antennae-enriched and male-biased in adult A. lucorum. Xenopus oocyte expression system assays demonstrated that AlucOR4/AlucOrco was sensitive to two major sex pheromone constituents and exhibited high sensitivity to (E)-2-hexenyl butyrate (E2HB) and lower sensitivity to hexyl butyrate (HB). The expression level of target mRNA was significantly reduced (>80%) in dsAlucOR4-injected bugs after five days. The electroantennogram (EAG) responses of male antennae to E2HB and HB were also reduced significantly (~40%). Our findings suggest that AlucOR4 is essential to sex pheromone perception in A. lucorum.

The development of rapid and reliable assays to characterize insect odorant receptors (ORs) and pheromone receptors (PRs) remains a challenge for the field. Typically ORs and PRs are functionally characterized either in vivo in transgenic Drosophila or in vitro through expression in Xenopus oocytes. While these approaches have succeeded, they are not well suited for high-throughput screening campaigns, primarily due to inherent characteristics that limit their ability to screen large quantities of compounds in a short period of time. The development of a practical, robust and consistent in vitro assay for functional studies on ORs and PRs would allow for high-throughput screening for ligands, as well as for compounds that could be used as novel olfactory-based pest management tools. Here we describe a novel method of utilizing human embryonic kidney cells (HEK293) transfected with inducible receptor constructs for the functional characterization of ORs in 96-well plates using a fluorescent spectrophotometer. Using EposOrco and EposOR3 from the pest moth, Epiphyas postvittana as an example, we generated HEK293 cell lines with robust and consistent responses to ligands in functional assays. Single-cell sorting of cell lines by FACS facilitated the selection of isogenic cell lines with maximal responses, and the addition of epitope tags on the N-termini allowed the detection of recombinant proteins in homogenates by western blot and in cells by immunocytochemistry. We thoroughly describe the methods used to generate these OR-expressing cell lines, demonstrating that they have all the necessary features required for use in high-throughput screening platforms.

The olfactory system of moth species in subfamily Heliothinae is an attractive model to study the evolution of the pheromone reception because they show distinct differentiation in sex pheromone components or ratios that activate pheromone receptors (PRs). However, functional assessment of PRs in closely related species remains largely untried. Here we present a special cloning strategy to isolate full-length cDNAs encoding candidate odorant receptors (ORs) from Helicoverpa armigera (Harm) and Helicoverpa assulta (Hass) on the basis of Heliothis virescens ORs, and investigate the functional properties of PRs to determine how the evolution of moth PRs contribute to intraspecific mating choice and speciation extension. We cloned 11 OR orthologs from H. armigera and 10 from H. assulta. We functionally characterized the responses of PRs of both species to seven pheromone compounds using the heterologous expression system of Xenopus ooctyes. HassOR13 was found to be highly tuned to the sex pheromone component Z11-16:Ald, and unexpectedly, both HarmOR14b and HassOR16 were specific for Z9-14:Ald. However, HarmOR6 and HassOR6 showed much higher specificity to Z9-16:OH than to Z9-16:Ald or Z9-14:Ald. HarmOR11, HarmOR14a, HassOR11 and HassOR14b failed to respond to the tested chemicals. Based on our results and previous research, we can show that some PR orthologs from H. armigera, H. assulta and H. virescens such as OR13s have similar ligand selectivity, but others have different ligand specificity. The combined PR function and sex pheromone component analysis suggests that the evolution of PRs can meet species-specific demands.

The Drosophila wing was proposed to be a taste organ more than 35 years ago, but there has been remarkably little study of its role in chemoreception. We carry out a differential RNA-seq analysis of a row of sensilla on the anterior wing margin and find expression of many genes associated with pheromone and chemical perception. To ask whether these sensilla might receive pheromonal input, we devised a dye-transfer paradigm and found that large, hydrophobic molecules comparable to pheromones can be transferred from one fly to the wing margin of another. One gene, Ionotropic receptor (IR)52a, is coexpressed in neurons of these sensilla with fruitless, a marker of sexual circuitry; IR52a is also expressed in legs. Mutation of IR52a and optogenetic silencing of IR52a+ neurons decrease levels of male sexual behavior. Optogenetic activation of IR52a+ neurons induces males to show courtship toward other males and, remarkably, toward females of another species. Surprisingly, IR52a is also required in females for normal sexual behavior. Optogenetic activation of IR52a+ neurons in mated females induces copulation, which normally occurs at very low levels. Unlike other chemoreceptors that act in males to inhibit male-male interactions and promote male-female interactions, IR52a acts in both males and females, and can promote male-male as well as male-female interactions. Moreover, IR52a+ neurons can override the circuitry that normally suppresses sexual behavior toward unproductive targets. Circuit mapping and Ca2+ imaging using the trans-Tango system reveals second-order projections of IR52a+ neurons in the subesophageal zone (SEZ), some of which are sexually dimorphic. Optogenetic activation of IR52a+ neurons in the wing activates second-order projections in the SEZ. Taken together, this study provides a molecular description of the chemosensory sensilla of a greatly understudied taste organ and defines a gene that regulates the sexual circuitry of the fly.

Two distinct but interrelated pheromone-signaling systems, LuxI/LuxR and AinS/AinR, positively control bioluminescence in Vibrio fischeri . Although each system generates an acyl-homoserine lactone (AHL) signal, the protein sequences of LuxI/LuxR and AinS/AinR are unrelated. AinS and LuxI generate the pheromones N -octanoyl-AHL (C 8 -AHL) and N -3-oxo-hexanoyl-AHL (3OC 6 -AHL), respectively. LuxR is a transcriptional activator that responds to 3OC 6 -AHL, and to a lesser extent to C 8 -AHL. AinR is hypothesized to respond to C 8 -AHL and, based on homology to Vibrio harveyi LuxN, to mediate the repression of a Qrr regulatory RNA. However, a Δ ainR mutation decreased luminescence, which was not predicted based on V. harveyi LuxN, raising the possibility of a distinct regulatory mechanism for AinR. Here we show that ainR can complement a luxN mutant, suggesting functional similarity. Moreover, in V. fischeri , we observed ainR -dependent repression of a P qrr - lacZ transcriptional reporter in the presence of C 8 -AHL, consistent with its hypothesized regulatory role. The system appears quite sensitive, with a half-maximal effect on a P qrr reporter at 140 pM C 8 -AHL. Several other AHLs with substituted and unsubstituted acyl chains between 6 and 10 carbons also displayed an AinR-dependent effect on P qrr - lacZ ; however, AHLs with acyl chains of four carbons or 12 or more carbons lacked activity. Interestingly, 3OC 6 -AHL also affected expression from the qrr promoter, but this effect was largely luxR dependent, indicating a previously unknown connection between these systems. Finally, we propose a preliminary explanation for the unexpected luminescence phenotype of the Δ ainR mutant.

The process of mating in Basidiomycota is regulated by homeodomain-encoding genes (HD) and pheromones and G protein-coupled pheromone receptor genes (P/R). Whether these genes are actually involved in determining mating type distinguishes mating systems that are considered tetrapolar (two locus) from bipolar (one locus). Polyporales are a diverse group of wood-decay basidiomycetes displaying high variability in mating and decay systems. Many of the bipolar species appear to be brown-rot fungi, and it has been hypothesized that there is a functional basis for this correlation. Here we characterize mating genes in recently sequenced Polyporales and other Agaricomycete genomes. All Agaricomycete genomes encode HD and pheromone receptor genes regardless of whether they are bipolar or tetrapolar. The HD genes are organized into a MAT-HD locus with a high degree of gene order conservation among neighboring genes, with the gene encoding mitochondrial intermediate peptidase consistently syntenic but no linkage to the P/R genes. To have a complete dataset of species with known mating systems we determined that Wolfiporia cocos appears to be bipolar, using the criterion that DNA polymorphism of MAT genes should be extreme. Testing the correlation of mating and decay systems while controlling for phylogenetic relatedness failed to identify a statistical association, likely due to the small number of taxa employed. Using a phylogenetic analysis of Ste3 proteins, we identified clades of sequences that contain no known mating type-specific receptors and therefore might have evolved novel functions. The data are consistent with multiple origins of bipolarity within the Agaricomycetes and Polyporales, although the alternative hypothesis that tetrapolarity and bipolarity are reversible states needs better testing.

In this study, we show that two splice variants of the pheromone receptor gene (pre-1) transcript coexist in vegetative and reproductive tissues of the filamentous ascomycete fungus Neurospora crassa. The two splice variants differ by intron retention of the last intron, which is predicted to result in a premature stop codon and loss of 322 amino acids in the C-terminal cytosolic region of PRE-1. Using quantitative PCR, we show that expression of the variants is influenced by mating type (mat), with a higher proportion of intron-spliced transcripts in a mat A strain and a higher proportion of the intron-retained variant in a mat a strain. The intron-retained PRE-1 variant is predicted to lack 6 ubiquitination sites that may influence receptor function. In conclusion, N. crassa produce two pre-1 splice variants that display different transcription profiles.