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

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

Three chemosensory proteins enriched in antennae and tarsi of Rhaphuma horsfieldi differentially contribute to the binding of insecticides.

Antennae and legs (primarily the tarsal segments) of insects are the foremost sensory organs that contact a diverse range of toxic chemicals including insecticides. Binding proteins expressed in the two tissues are potential molecular candidates serving as the binding and sequestering of insecticides, like chemosensory proteins (CSPs). Insect CSPs endowed with multiple roles have been suggested to participate in insecticide resistance, focusing mainly on moths, aphids and mosquitos. Yet, the molecular underpinnings underlying the interactions of cerambycid CSPs and insecticides remain unexplored. Here, we present binding properties of three antenna- and tarsus-enriched RhorCSPs (RhorCSP1, CSP2 and CSP3) in Rhaphuma horsfieldi to eight insecticide classes totaling 15 chemicals. From the transcriptome of this beetle, totally 16 CSP-coding genes were found, with seven full-length sequences. In phylogeny, these RhorCSPs were distributed dispersedly in different clades. Expression profiles revealed the abundant expression of RhorCSP1, CSP2 and CSP3 in antennae and tarsi, thus as representatives for studying the protein-insecticide interactions. Binding assays showed that the three RhorCSPs were tuned differentially to insecticides but exhibited the highest affinities with hexaflumuron, chlorpyrifos and rotenone (dissociation constants <13 µM). In particular, RhorCSP3 could interact strongly with 10 of tested insecticides, of which four residues (Tyr25, Phe42, Val65 and Phe68) contributed significantly to the binding of six, four, three and four ligands, respectively. Of these, the binding of four mutated RhorCSP3s to a botanical insecticide rotenone was significantly weakened compared to the wildtype protein. Furthermore, we also evidenced that RhorCSP3 was a broadly-tuned carrier protein in response to a wide variety of plant odorants outside insecticides. Altogether, our findings shed light on different binding mechanisms and odorant-tuning profiles of three RhorCSPs in R. horsfieldi and identify key residues of the RhorCSP3-insecticide interactions.

Comparative transcriptomics reveals the conservation and divergence of reproductive genes across three sympatric Tomicus bark beetles.

Three tree-killing bark beetles belonging to the genus Tomicus, Tomicus yunnanensis, Tomicus brevipilosus and Tomicus minor (Coleoptera; Curculionidae, Scolytinae), are serious wood-borers with larvae feeding on the phloem tissues of Pinus yunnanensis. The three Tomicus beetles, in some cases, coexist in a same habitat, providing a best system for exploring the conservation and divergence of reproductive genes. Here, we applied comparative transcriptomics and molecular biology approaches to characterize reproductive-related genes in three sympatric Tomicus species. Illumina sequencing of female and male reproductive systems and residual bodies generated a large number of clean reads, representing 185,920,232 sequences in T. yunnanensis, 169,153,404 in T. brevipilosus and 178,493,176 in T. minor that were assembled into 32,802, 56,912 and 33,670 unigenes, respectively. The majority of the genes had detectable expression in reproductive tissues (FPKM >1), particularly those genes in T. brevipilosus accounting for 76.61 % of the total genes. From the transcriptomes, totally 838 genes encoding 463 detoxification enzymes, 339 chemosensory membrane proteins and 36 ionotropic glutamate receptors (iGluRs) were identified, including 622 reproductive tissue-expressed genes. Of these, members of carboxylesterases (COEs), ionotropic receptors (IRs), sensory neuron membrane proteins (SNMPs) and iGluRs were highly conserved in gene numbers and sequence identities across three Tomicus species. Further, expression profiling analyses revealed a number of genes expressed in reproductive tissues and the diverse expression characteristics in these beetles. The results provide evidence for the conservation and differences of reproductive genes among three sympatric closely related beetles, helping understand their different reproductive strategies and the maximization of the reproductive success.

Functional characterization of four antenna-biased chemosensory proteins in Dioryctria abietella reveals a broadly tuned olfactory DabiCSP1 and its key residues in ligand-binding.

The orientation of the oligophagous cone-feeding moth Dioryctria abietella (Lepidoptera: Pyralidae) to host plants primarily relies on olfactory-related proteins, particularly those candidates highly expressed in antennae. Here, through a combination of expression profile, ligand-binding assay, molecular docking and site-directed mutagenesis strategies, we characterized the chemosensory protein (CSP) gene family in D. abietella. Quantitative real-time PCR (qPCR) analyses revealed the detectable expression of all 22 DabiCSPs in the antennae, of which seven genes were significantly enriched in this tissue. In addition, the majority of the genes (19/22 relatives) had the expression in at least one reproductive tissue. In the interactions of four antenna-dominant DabiCSPs and different chemical classes, DabiCSP1 was broadly tuned to 27 plant-derived odors, three man-made insecticides and one herbicide with high affinities (Ki < 6.60 µM). By contrast, three other DabiCSPs (DabiCSP4, CSP6 and CSP17) exhibited a narrow odor binding spectrum, in response to six compounds for each protein. Our mutation analyses combined with molecular docking simulations and binding assays further identified four key residues (Tyr25, Thr26, Ile65 and Val69) in the interactions of DabiCSP1 and ligands, of which binding abilities of this protein to 12, 15, 16 and three compounds were significantly decreased compared to the wildtype protein, respectively. Our study reveals different odor binding spectra of four DabiCSPs enriched in antennae and identifies key residues responsible for the binding of DabiCSP1 and potentially active compounds for the control of this pest.

Expression profile and functional characterization of odorant binding proteins in a forest pest, Dioryctria abietella (Lepidoptera: Pyralidae).

In the forest ecosystem dominated by the Pinaceae plants, this boring pest Dioryctria abietella is subject to a variety of odorants derived from host and nonhost plants, in which olfactory-related proteins enriched in antennae are key behavioral modulators for the orientation of feeding and ovipositing hosts. Here, we addressed the odorant binding protein (OBP) gene family in D. abietella. Expression profiles revealed that the majority of OBPs were abundantly expressed in the antennae at a female-biased level. A male-antenna-biased DabiPBP1 was a strong candidate for detecting type I and type II pheromones of D. abitella female moths. Using a prokaryotic expression system combined with affinity chromatography, we harvested two antenna-dominant DabiOBPs. In the ligand-binding assays, the two DabiOBPs exhibited different odorant response spectra, as DabiOBP17 was tuned to most odorants with higher affinities compared to DabiOBP4. Of these, DabiOBP4 could strongly bind syringaldehyde and citral (dissociation constants (Ki) < 14 µM). A floral volatile, benzyl benzoate (Ki = 4.72 ± 0.20 µM), was the best ligand for DabiOBP17. Remarkably, several green leaf volatiles were found to strongly interact with DabiOBP17 (Ki < 8.5 µM), including Z3-hexenyl acetate, E2-hexenol, Z2-hexenal and E2-hexenal that may mediate a repellent response to D. abietella. Structural analyses of ligands revealed that the binding of the two DabiOBPs to odorants was associated with carbon-chain lengths and functional groups. Molecular simulations identified several key residues involved in the interactions of DabiOBPs and ligands, suggesting specific binding mechanisms. This study highlights olfactory roles of two antennal DabiOBPs in D. abietella, helping the identification of potentially behavioral compounds for the population control of this pest.

Identification and characterization of candidate detoxification genes in Pharsalia antennata Gahan (Coleoptera: Cerambycidae).

The wood-boring beetles, including the majority of Cerambycidae, have developed the ability to metabolize a variety of toxic compounds derived from host plants and the surrounding environment. However, detoxification mechanisms underlying the evolutionary adaptation of a cerambycid beetle Pharsalia antennata to hosts and habitats are largely unexplored. Here, we characterized three key gene families in relation to detoxification (cytochrome P450 monooxygenases: P450s, carboxylesterases: COEs and glutathione-S-transferases: GSTs), by combinations of transcriptomics, gene identification, phylogenetics and expression profiles. Illumina sequencing generated 668,701,566 filtered reads in 12 tissues of P. antennata, summing to 100.28 gigabases data. From the transcriptome, 215 genes encoding 106 P450s, 77 COEs and 32 GSTs were identified, of which 107 relatives were differentially expressed genes. Of the identified 215 genes, a number of relatives showed the orthology to those in Anoplophora glabripennis, revealing 1:1 relationships in 94 phylogenetic clades. In the trees, P. antennata detoxification genes mainly clustered into one or two subfamilies, including 64 P450s in the CYP3 clan, 33 COEs in clade A, and 20 GSTs in Delta and Epsilon subclasses. Combining transcriptomic data and PCR approaches, the numbers of detoxification genes expressed in abdomens, antennae and legs were 188, 148 and 141, respectively. Notably, some genes exhibited significantly sex-biased levels in antennae or legs of both sexes. The findings provide valuable reference resources for further exploring xenobiotics metabolism and odorant detection in P. antennata.

Genome-Wide Analysis of Odorant-Binding Proteins in Papilio xuthus with Focus on the Perception of Two PxutGOBPs to Host Odorants and Insecticides.

In this study, we annotated 49 odorant-binding proteins (OBPs) in Papilio xuthus, with four novel genes and seven improved sequences. Expression profiles identified numerous OBPs in antennae or reproductive tissues. Using two antenna-enriched general OBPs (PxutGOBP1 and PxutGOBP2) as targets, we screened three key compounds by a reverse chemical ecology strategy. Of these, an oviposition stimulant vicenin-2 could strongly interact with PxutGOBP1, representing a dissociation constant (Ki) value of 10.34 ± 0.07 µM. Molecular simulations and site-directed mutagenesis revealed the importance of His66, Thr73, and Phe118 between PxutGOBP1 and vicenin-2 interactions. Two other compounds, an ordinary floral scent ß-ionone and a widely used insecticide chlorpyrifos, exhibited high affinities to PxutGOBPs (Ki < 13 µM). Furthermore, two mutations His66Ala and Thr73Ala of PxutGOBP1 significantly reduced the binding to chlorpyrifos. Our study provides insights into the putative roles of PxutGOBPs in odorant perception and identifies key binding sites of PxutGOBP1 to vicenin-2 and chlorpyrifos.

The ionotropic receptor gene family in Lepidoptera and Trichoptera: Annotation, evolutionary and functional perspectives.

Lepidoptera (moths and butterflies) and Trichoptera (caddisflies), belonging to the superorder Amphiesmenoptera, are the most diverse insect orders as representatives of the terrestrial and aquatic insects, respectively. The insects of the two orders possess different biological and behavioral characteristics, especially their larvae, presumably resulting in the differences of the ionotropic receptor (IR) genes in numbers, sequence characteristics or gene structure. Here, we employed genomics, transcriptomics, bioinformatics, phylogenetics and molecular biology strategies to characterize the IR gene repertoire in Lepidoptera and Trichoptera. Genome and transcriptome analyses with exhaustive homology-based searches and manual efforts, in 32 lepidopterans and five trichopterans, led to the identification of 1449 genes encoding IRs with 1170 full-length sequences, representing the most comprehensive set of chemoreceptor superfamilies across the Amphiesmenoptera. Analysis of gene gains and losses in orthologous groups implied that some IRs were lost in related species, and multiple gene copies occurred mainly in divergent IRs (D-IRs) by gene duplications. Phylogenetic analysis of 2442 IR proteins from 67 species revealed that Lepidoptera and Trichoptera IRs could be classified into three subfamilies, i.e., 14 antennal IRs (A-IRs), five Lepidoptera-specific IRs (LS-IRs) and four D-IRs. Of the three subfamilies, A-IRs and LS-IRs members within orthologous groups exhibited high conservation of gene structure, but D-IRs shared extremely low amino acid identities (below 30%). Expression profiles revealed functional diversities of IRs from Bombyx mori and Papilio xuthus involving smell, taste or reproduction, in which some genes displayed sex-biased expression in antennae associated with specific chemosensory behaviors of female or male adults. Our current study has provided insights into the evolution, conservation and divergence of IRs between/within Lepidoptera and Trichoptera, and allows for further experiments to investigate IR functions.