Olfactory Gene Families in Scopula subpunctaria and Candidates for Type-II Sex Pheromone Detection.

Scopula subpunctaria, an abundant pest in tea gardens, produce type-II sex pheromone components, which are critical for its communicative and reproductive abilities; however, genes encoding the proteins involved in the detection of type-II sex pheromone components have rarely been documented in moths. In the present study, we sequenced the transcriptomes of the male and female S. subpunctaria antennae. A total of 150 candidate olfaction genes, comprising 58 odorant receptors (SsubORs), 26 ionotropic receptors (SsubIRs), 24 chemosensory proteins (SsubCSPs), 40 odorant-binding proteins (SsubOBPs), and 2 sensory neuron membrane proteins (SsubSNMPs) were identified in S. subpunctaria. Phylogenetic analysis, qPCR, and mRNA abundance analysis results suggested that SsubOR46 may be the Orco (non-traditional odorant receptor, a subfamily of ORs) of S. subpunctaria. SsubOR9, SsubOR53, and SsubOR55 belonged to the pheromone receptor (PR) clades which have a higher expression in male antennae. Interestingly, SsubOR44 was uniquely expressed in the antennae, with a higher expression in males than in females. SsubOBP25, SsubOBP27, and SsubOBP28 were clustered into the moth pheromone-binding protein (PBP) sub-family, and they were uniquely expressed in the antennae, with a higher expression in males than in females. SsubOBP19, a member of the GOBP2 group, was the most abundant OBP in the antennae. These findings indicate that these olfactory genes, comprising five candidate PRs, three candidate PBPs, and one candidate GOBP2, may be involved in type II sex pheromone detection. As well as these genes, most of the remaining SsubORs, and all of the SsubIRs, showed a considerably higher expression in the female antennae than in the male antennae. Many of these, including SsubOR40, SsubOR42, SsubOR43, and SsubIR26, were more abundant in female antennae. These olfactory and ionotropic receptors may be related to the detection of host plant volatiles. The results of this present study provide a basis for exploring the olfaction mechanisms in S. subpunctaria, with a focus on the genes involved in type II sex pheromones. The evolutionary analyses in our study provide new insights into the differentiation and evolution of lepidopteran PRs.

Identification of cytochrome P450, odorant-binding protein, and chemosensory protein genes involved in Type II sex pheromone biosynthesis and transportation in the tea pest, Scopula subpunctaria.

Sex pheromone-based pest management technology has been widely used to monitor and control insect pests in the agricultural, forestry, and public health sectors. Scopula subpunctaria is a widespread tea pest in China with Type II sex pheromone components. However, limited information is available on the biosynthesis and transportation of Type II sex pheromone components. In this study, we constructed an S. subpunctaria sex pheromone gland (PG) transcriptome and obtained 85,246 transcripts. Cytochrome P450 monooxygenases (CYPs) thought to epoxidize dienes and trienes to epoxides in the PG and odorant-binding proteins (OBPs) and chemosensory genes (CSPs) thought to be responsible for the binding and transportation of sex pheromone components. In present study, a total of 79 CYPs, 29 OBPs and 17 CSPs were identified. We found that SsubCYP341A and SsubCYP341B_ortholog1 belonged to the CYP341 family and were more highly expressed in the PG than in the female body. Of these, SsubCYP341A was the seventh-most PG-enriched CYP in the PG transcriptome. Two CYP4 members, CYP340BD_ortholog2 and CYP4G, were the top two most PG-enriched CYPs. Tissue expression and phylogenetic tree analysis showed that SsubOBP25, 27, and 28 belonged to the moth pheromone-binding protein family; they were distinctly expressed in the antennae and were more abundant in male antennae than in female antennae. SsubCSP16 was distributed into the same clade as CSPs from other moths that showed high binding affinities to sex pheromone components. It indicated that all the above-mentioned genes could be involved in sex pheromone biosynthesis or transportation. Our study provides large-scale PG sequence information that can be used to identify potential targets for the biological control of S. subpunctaria by disrupting its sex pheromone biosynthesis and transportation pathways.

Different binding properties of two general-odorant binding proteins in Athetis lepigone with sex pheromones, host plant volatiles and insecticides.

Athetis lepigone (Alep) is a polyphagous pest native to Europe and Asia that has experienced major outbreaks in the summer maize area of China since 2011 and has shown evidence of resistance to some insecticides. Insect olfaction is crucial for recognition of sex pheromones, host plant volatiles and even insecticides, in which two general-odorant binding proteins (GOBPs) play important roles. To elucidate the functions of GOBPs in A. lepigone, we first expressed the two AlepGOBP proteins in the E. coli expression system. Then, the results of fluorescence competitive binding assays demonstrated that the high binding affinity of AlepGOBP2 with sex pheromones [(Z)-7-dodecenyl acetate (Z7-12:Ac), Ki = 0.65 µM; (Z)-9-tetradecenyl acetate (Z9-14:Ac), Ki = 0.83 µM], two maize plant volatiles [Ocimene, Ki = 9.63 µM; (E)-ß-Farnesene, Ki = 4.76 µM] and two insecticides (Chlorpyrifos Ki =5.61 µM; Phoxim, Ki = 4.38 µM). However, AlepGOBP1 could only bind Ocimene (Ki = 13.0 µM) and two insecticides (Chlorpyrifos Ki =4.46 µM; Phoxim, Ki = 3.27 µM). These results clearly suggest that AlepGOBP1 and AlepGOBP2 differentiate among odorants and other ligands. The molecular docking results further revealed different key residues involved in the ligand binding of AlepGOBPs. In summary, this study provides a foundation for exploring the olfactory mechanism of A. lepigone and identified two potential target genes for the development of highly effective insecticides in the future.

A Δ9 desaturase (SlitDes11) is associated with the biosynthesis of ester sex pheromone components in Spodoptera litura.

Sex pheromone biosynthesis in moths relies on the activity of multiple enzymes, including Δ9 desaturase, which plays an important role in catalyzing desaturation at the Δ9 position of the carbon chain. However, the physiological function of moth Δ9 desaturase has not been elucidated in vivo. In this study, we used the CRISPR/Cas9 system to knockout the Δ9 desaturase gene (SlitDes11) of Spodoptera litura to analyze its role in sex pheromone biosynthesis. First, through the direct injection of SlitDes11-single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, gene editing was induced in around 30% of eggs 24 h after injection and was induced in 20.8% of the resulting adult moths. Second, using a sibling-crossing strategy, insects with mutant SlitDes11 (bearing a premature stop codon) were selected, and homozygous mutants were obtained in the G5 generation. Third, pheromone gland extracts of adult female homozygous SlitDes11 mutants were analyzed using Gas chromatography (GC). The results showed that titers of all three ester sex pheromone components; Z9, E11-14:Ac, Z9,E12-14:Ac, and Z9-14:Ac; were reduced by 62.40%, 78.50%, and 72.50%, respectively. This study provides the first direct evidence for the role of SlitDes11 in sex pheromone biosynthesis in S. litura, and indicates the gene could be as potential target to disrupt sexual communication in S. litura for developing a new pollution-free insecticide.

Molecular identification of differential expression genes associated with sex pheromone biosynthesis in Spodoptera exigua.

Species-specific sex pheromone is biosynthesized and released in most female moths as a chemical cue in mating communication. However, information on genes involved in this pathway is limited. The beet armyworm, Spodoptera exigua, is a cosmopolitan agricultural pest that causes severe economic losses to many crops. In China, the female sex pheromones in sex pheromone glands (PGs) of S. exigua have been measured which comprises (Z,E)-9,12-tetradecadienyl acetate, (Z)-9-tetradecen-l-ol, (Z)-9-tetradecenyl acetate, and (Z,E)-9,12-tetradecadien-1-ol in a ratio of 47:18:18:17. Fifty-nine putative genes related to sex pheromone biosynthesis were identified in the present study by sequencing and analyzing the sex pheromone gland (PG) transcriptome of S. exigua. Expression profiles revealed that two desaturase (SexiDes5 and SexiDes11) and three fatty acyl reductase (SexiFAR2, 3, and 9) genes had PG-specific expression, and phylogenetic analysis demonstrated that they clustered with genes known to be involved in pheromone synthesis in other moth species. Our results provide crucial background information that could facilitate the elucidation of sex pheromone biosynthesis pathway of S. exigua as well as other Spodoptera species and help identify potential targets for disrupting sexual communication in S. exigua for developing novel environment-friendly pesticides.

Functional Disparity of Three Pheromone-Binding Proteins to Different Sex Pheromone Components in Hyphantria cunea (Drury).

Hyphantria cunea (Drury) is a destructive invasive pest species in China that uses type II sex pheromone components. To date, however, the binding mechanisms of its sex pheromone components to their respective pheromone-binding proteins (HcunPBPs 1/2/3) have not been explored. In the current study, all three HcunPBPs were expressed in the antennae of both sexes. The prokaryotic expression and ligand binding assays were employed to study the binding of the moth's four sex pheromone components, including two aldehydes and two epoxides, and 24 plant volatiles to the HcunPBPs. Our results showed that the abilities of these HcunPBPs to bind to the aldehydes were significantly different from binding to the epoxides. These three HcunPBPs also selectively bind to some of the plant volatiles tested. Our molecular docking results indicated that some crucial hydrophobic residues might play a role in the binding of HcunPBPs to their sex pheromone components. Three HcunPBPs have different selectivities for pheromone components with both major and minor structural differences. Our study provides a fundamental insight into the olfactory mechanism of moths at the molecular level, especially for moth species that use various type II pheromone components.

Identification and Expression Profile of Olfactory Receptor Genes Based on Apriona germari (Hope) Antennal Transcriptome.

Insects' olfactory receptor plays a central role in detecting chemosensory information from the environment. Odorant receptors (ORs) and ionotropic receptors (IRs) are two types of olfactory receptors, and they are essential for the recognition of ligands at peripheral neurons. Apriona germari (Hope) (Coleoptera: Cerambycidae) is one of the most serious insect pests that cause damage to economic trees and landscaping trees, resulting in massive environmental damages and economic losses. Olfactory-based management strategy has been suggested as a promising strategy to control this wood-boring beetle. However, the olfactory perception mechanism in A. germari is now almost unknown. In the present study, RNA sequencing analysis was used to determine the transcriptomes of adult A. germari antennae. Among 36,834 unigenes derived from the antennal assembly, we identified 42 AgerORs and three AgerIRs. Based on the tissue expression pattern analysis, 27 AgerORs displayed a female-biased expression. Notably, AgerOR3, 5, 13, 33, and 40 showed a significant female-biased expression and were clustered with the pheromone receptors of Megacyllene caryae in the phylogenetic tree, suggesting that these AgerORs could be potential pheromone receptors for sensing male-produced sex pheromones in A. germari. The AgerIRs expression profile demonstrated that AgerIR2 had high expression levels in male labial palps, suggesting that this receptor may function to detect female-deposited trail-sex pheromone blend of A. germari. In addition, the phylogenetic tree showed that the Orco gene of five cerambycidae species was highly conservative. These results provide a foundation for further studies on the molecular mechanisms of olfactory chemoreception in A. germari apart from suggesting novel targets for the control of this pest in the future.

Correction: Analysis of the Antennal Transcriptome and Insights into Olfactory Genes in Hyphantria cunea (Drury).

[This corrects the article DOI: 10.1371/journal.pone.0164729.].

A new species of Contarinia (Diptera: Cecidomyiidae) damaging inflorescence of Carya cathayensis (Juglandaceae) in China.

Chinese hickory, Carya cathayensis Sargent (Juglandaceae), is a tree naturally occurring and industrially grown in China for the nuts that are valued for their taste and nutrient content. Larvae of a previously unknown species of gall midge were found feeding on male and female inflorescences of Carya cathayensis in Zhejiang and Anhui Provinces in eastern China, reducing pollination and fruit development, and causing substantial damage to the nut industry. The new species is named Contarinia caryafloralis Jiao, Bu Kolesik, its morphology is described, the basic biology is given, and the Cytochrome Oxidase subunit I (COI) mitochondrial gene segment is sequenced. Contarinia caryafloralis is the first gall midge known to feed on a Carya species native to Asia.

Identification and Expression Patterns of Anoplophora chinensis (Forster) Chemosensory Receptor Genes from the Antennal Transcriptome.

The citrus long-horned beetle (CLB), Anoplophora chinensis (Forster) is a destructive native pest in China. Chemosensory receptors including odorant receptors (ORs), gustatory receptors (GRs), and ionotropic receptors (IRs) function to interface the insect with its chemical environment. In the current study, we assembled the antennal transcriptome of A. chinensis by next-generation sequencing. We assembled 44,938 unigenes from 64,787,784 clean reads and annotated their putative gene functions based on gene ontology (GO) and Clusters of Orthologous Groups of proteins (COG). Overall, 74 putative receptor genes from chemosensory receptor gene families, including 53 ORs, 17 GRs, and 4 IRs were identified. Expression patterns of these receptors on the antennae, maxillary and labial palps, and remaining body segments of both male and female A. chinensis were performed using quantitative real time-PCR (RT-qPCR). The results revealed that 23 ORs, 6 GRs, and 1 IR showed male-biased expression profiles, suggesting that they may play a significant role in sensing female-produced sex pheromones; whereas 8 ORs, 5 GRs, and 1 IR showed female-biased expression profiles, indicating that these receptors may be involved in some female-specific behaviors such as oviposition site seeking. These results lay a solid foundation for deeply understanding CLB olfactory processing mechanisms. Moreover, by comparing our results with those from chemosensory receptor studies in other cerambycid species, several highly probable pheromone receptor candidates were highlighted, which may facilitate the identification of additional pheromone and/or host attractants in CLB.

Molecular identification and expression patterns of odorant binding protein and chemosensory protein genes in Athetis lepigone (Lepidoptera: Noctuidae).

The olfaction system of insects plays an important role in mediating various physiological behaviors, including locating hosts, avoiding predators, and recognizing mates and oviposition sites. Therefore, some key genes in the system present valuable opportunities as targets for developing novel green pesticides. Athetis lepigone, a noctuid moth can feed on more than 30 different host plants making it a serious polyphagous pest worldwide, and it has become one of the major maize pests in northern China since 2011. However, there are no reports on effective and environmentally friendly pesticides for the control of this pest. In this study, we identified 28 genes encoding putative odorant binding proteins (OBPs) and 20 chemosensory protein (CSPs) genes based on our previous A. lepigone transcriptomic data. A tissue expression investigation and phylogenetic analysis were conducted in an effort to postulate the functions of these genes. Our results show that nearly half (46.4%) of the AlOBPs exhibited antennae-biased expression while many of the AlCSPs were highly abundant in non-antennal tissues. These results will aid in exploring the chemosensory mechanisms of A. lepigone and developing environmentally friendly pesticides against this pest in the future.

Likely Aggregation-Sex Pheromones of the Invasive Beetle Callidiellum villosulum, and the Related Asian Species Allotraeus asiaticus, Semanotus bifasciatus, and Xylotrechus buqueti (Coleoptera: Cerambycidae).

During field trials of the two known cerambycid beetle pheromone components 3-hydroxyhexan-2-one and 1-(1H-pyrrol-2-yl)-1,2-propanedione (henceforth "pyrrole") in Guangxi and Anhui provinces in China, four species in the subfamily Cerambycinae were attracted to lures containing one of the two components, or the blend of the two. Thus, the invasive species Callidiellum villosulum (Fairmaire) (tribe Callidiini) and a second species, Xylotrechus buqueti (Castelnau & Gory) (tribe Clytini), were specifically attracted to the blend of 3-hydroxyhexan-2-one and the pyrrole. In contrast, Allotreus asiaticus (Schwarzer) (tribe Phoracanthini) and Semanotus bifasciatus Motschulsky (tribe Callidiini) were specifically attracted to the pyrrole as a single component. In most cases, both males and females were attracted, indicating that the compounds are likely to be aggregation-sex pheromones. The results indicate that the two compounds are conserved as pheromone components among species within at least three tribes within the subfamily Cerambycinae. For practical purposes, the attractants could find immediate use in surveillance programs aimed at detecting incursions of these species into new areas of the world, including the United States.

Analysis of the Antennal Transcriptome and Insights into Olfactory Genes in Hyphantria cunea (Drury).

Hyphantria cunea (Drury) (Lepidoptera: Arctiidae) is an invasive insect pest which, in China, causes unprecedented damage and economic losses due to its extreme fecundity and wide host range, including forest and shade trees, and even crops. Compared to the better known lepidopteran species which use Type-I pheromones, little is known at the molecular level about the olfactory mechanisms of host location and mate choice in H. cunea, a species using Type-II lepidopteran pheromones. In the present study, the H. cunea antennal transcriptome was constructed by Illumina Hiseq 2500TM sequencing, with the aim of discovering olfaction-related genes. We obtained 64,020,776 clean reads, and 59,243 unigenes from the analysis of the transcriptome, and the putative gene functions were annotated using gene ontology (GO) annotation. We further identified 124 putative chemosensory unigenes based on homology searches and phylogenetic analysis, including 30 odorant binding proteins (OBPs), 17 chemosensory proteins (CSPs), 52 odorant receptors (ORs), 14 ionotropic receptors (IRs), nine gustatory receptors (GRs) and two sensory neuron membrane proteins (SNMPs). We also found many conserved motif patterns of OBPs and CSPs using a MEME system. Moreover, we systematically analyzed expression patterns of OBPs and CSPs based on reverse transcription PCR and quantitative real time PCR (RT-qPCR) with RNA extracted from different tissues and life stages of both sexes in H. cunea. The antennae-biased expression may provide a deeper further understanding of olfactory processing in H. cunea. The first ever identification of olfactory genes in H. cunea may provide new leads for control of this major pest.

Antennal Transcriptome Analysis of Odorant Reception Genes in the Red Turpentine Beetle (RTB), Dendroctonus valens.

BACKGROUND: The red turpentine beetle (RTB), Dendroctonus valens LeConte (Coleoptera: Curculionidae, Scolytinae), is a destructive invasive pest of conifers which has become the second most important forest pest nationwide in China. Dendroctonus valens is known to use host odors and aggregation pheromones, as well as non-host volatiles, in host location and mass-attack modulation, and thus antennal olfaction is of the utmost importance for the beetles' survival and fitness. However, information on the genes underlying olfaction has been lacking in D. valens. Here, we report the antennal transcriptome of D. valens from next-generation sequencing, with the goal of identifying the olfaction gene repertoire that is involved in D. valens odor-processing. RESULTS: We obtained 51 million reads that were assembled into 61,889 genes, including 39,831 contigs and 22,058 unigenes. In total, we identified 68 novel putative odorant reception genes, including 21 transcripts encoding for putative odorant binding proteins (OBP), six chemosensory proteins (CSP), four sensory neuron membrane proteins (SNMP), 22 odorant receptors (OR), four gustatory receptors (GR), three ionotropic receptors (IR), and eight ionotropic glutamate receptors. We also identified 155 odorant/xenobiotic degradation enzymes from the antennal transcriptome, putatively identified to be involved in olfaction processes including cytochrome P450s, glutathione-S-transferases, and aldehyde dehydrogenase. Predicted protein sequences were compared with counterparts in Tribolium castaneum, Megacyllene caryae, Ips typographus, Dendroctonus ponderosae, and Agrilus planipennis. CONCLUSION: The antennal transcriptome described here represents the first study of the repertoire of odor processing genes in D. valens. The genes reported here provide a significant addition to the pool of identified olfactory genes in Coleoptera, which might represent novel targets for insect management. The results from our study also will assist with evolutionary analyses of coleopteran olfaction.

Male adaptations to minimize sexual cannibalism during reproduction in the funnel-web spider Hololena curta.

Males of many spider species risk being attacked and cannibalized while searching for, courting, and mating with conspecific females. However, there are exceptions. We show that the funnel-web spider, Hololena curta, has 3 adaptations that minimize risk to males during courtship and mating, and enhance reproductive success. First, males detected chemical or tactile signals associated with webs of virgin females, and differentiated them from webs of mated females, enabling males to increase encounter rates with virgin females and avoid aggressive mated females. Second, males produced stereotyped vibrational signals during courting which induced female quiescence and suppressed female aggression. Third, when touched by males, sexually receptive females entered a cataleptic state, allowing males to safely approach and copulate. Because males can mate multiple times and the sex ratio in natural populations of H. curta is female biased, overall reproductive output is likely increased by males of this species avoiding sexual cannibalism.