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.

Combined transcriptomic, proteomic and genomic analysis identifies reproductive-related proteins and potential modulators of female behaviors in Spodoptera litura.

The common cutworm, Spodoptera litura, is a polyandrous moth with high reproductive ability. Sexual reproduction is a unique strategy for survival and reproduction of population in this species. However, to date available information about its reproductive genes is rare. Here, we combined transcriptomics, genomics and proteomics approaches to characterize reproductive-related proteins in S. litura. Illumina sequencing in parallel with the reference genome led to the yields of 12,161 reproductive genes, representing 47.83% of genes annotated in the genome. Further, 524 genes of 19 specific gene families annotated in the genome were detected in reproductive tissues of both sexes, some of which exhibited sex-biased and/or tissue-enriched expression. Of these, manual efforts together with the transcriptome analyses re-annotated 54 odorant binding proteins (OBPs) and 23 chemosensory proteins (CSPs) with an increase of 18 OBPs and one CSP compared to those previously annotated in the genome. Interestingly, at least 35 OBPs and 22 CSPs were transcribed in at least one reproductive tissue, suggestive of their involvement in reproduction. Further proteomic analysis revealed 2381 common proteins between virgin and mated female reproductive systems, 79 of which were differentially expressed. More importantly, 74 proteins exclusive to mated females were identified as transferred relatives, coupled with their specific or high expression in male reproductive systems. Of the transferred proteins, several conserved protein classes across insects were observed including OBPs, serpins, trypsins and juvenile hormone-binding proteins. Our current study has extensively surveyed reproductive genes in S. litura with an emphasis on the roles of OBPs and CSPs in reproduction, and identifies potentially transferred proteins serving as modulators of female post-mating behaviors.