Engineering DMNT emission in cotton enhances direct and indirect defense against mirid bugs.

INTRODUCTION: As an important herbivore-induced plant volatile, (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) is known for its defensive role against multiple insect pests, including attracting natural enemies. A terpene synthase (GhTPS14) and two cytochrome P450 (GhCYP82L1, GhCYP82L2) enzymes are involved in the de novo synthesis of DMNT in cotton. We conducted a study to test the potential of manipulating DMNT-synthesizing enzymes to enhance plant resistance to insects. OBJECTIVES: To manipulate DMNT emissions in cotton and generate cotton lines with increased resistance to mirid bug Apolygus lucorum. METHODS: Biosynthesis and emission of DMNT by cotton plants were altered using CRISPR/Cas9 and overexpression approaches. Dynamic headspace sampling and GC-MS analysis were used to collect, identify and quantify volatiles. The attractiveness and suitability of cotton lines against mirid bug and its parasitoid Peristenus spretus were evaluated through various assays. RESULTS: No DMNT emission was detected in knockout CAS-L1L2 line, where both GhCYP82L1 and GhCYP82L2 were knocked out. In contrast, gene-overexpressed lines released higher amounts of DMNT when infested by A. lucorum. At the flowering stage, L114 (co-overexpressing GhCYP82L1 and GhTPS14) emitted 10-15-fold higher amounts than controls. DMNT emission in overexpressed transgenic lines could be triggered by methyl jasmonate (MeJA) treatment. Apolygus lucorum and its parasitoid were far less attracted to the double edited CAS-L1L2 plants, however, co-overexpressed line L114 significantly attracted bugs and female wasps. A high dose of DMNT, comparable to the emission of L114, significantly inhibited the growth of A. lucorum, and further resulted in higher mortalities. CONCLUSION: Turning down DMNT emission attenuated the behavioral preferences of A. lucorum to cotton. Genetically modified cotton plants with elevated DMNT emission not only recruited parasitoids to enhance indirect defense, but also formed an ecological trap to kill the bugs. Therefore, manipulation of DMNT biosynthesis and emission in plants presents a promising strategy for controlling mirid bugs.

Knockout of the delta11-desaturase SfruDES1 disrupts sex pheromone biosynthesis, mating and oviposition in the fall armyworm, Spodoptera frugiperda.

Moth insects rely on sex pheromones for long distance attraction and searching for sex partners. The biosynthesis of moth sex pheromones involves the catalytic action of multiple enzymes, with desaturases playing a crucial role in the process of carbon chain desaturation. However, the specific desaturases involved in sex pheromone biosynthesis in fall armyworm (FAW), Spodoptera frugiperda, have not been clarified. In this study, a Δ11 desaturase (SfruDES1) gene in FAW was knocked out using the CRISPR/Cas9 genome editing system. A homozygous mutant of SfruDES1 was obtained through genetic crosses. The gas chromatography-mass spectrometry (GC-MS) analysis results showed that the three main sex pheromone components (Z7-12:Ac, Z9-14:Ac, and Z11-16:Ac) and the three minor components (Z9-14:Ald, E11-14:Ac and Z11-14:Ac) of FAW were not detected in homozygous mutant females compared to the wild type. Furthermore, behavioral assay demonstrated that the loss of SfruDES1 resulted in a significant reduction in the attractiveness of females to males, along with disruptions in mating behavior and oviposition. Additionally, in a heterologous expression system, recombinant SfruDES1 could introduce a cis double bond at the Δ11 position in palmitic acid, which resulted in the changes in components of the synthesized products. These findings suggest desaturase plays a key role in the biosynthesis of sex pheromones, and knockout of the SfruDES1 disrupts sex pheromone biosynthesis and mating behavior in FAW. The SfruDES1 could serve as tool to develop a control method for S. frugiperda.

n-octyl acrylate is a candidate sex pheromone component involved in courtship in parasitoid wasp Microplitis mediator.

Sex pheromones are considered to play critical roles in partner communication of most parasitic Hymenoptera. However, the identification of sex pheromone components remains limited to a few families of parasitoid wasps. In this study, we functionally characterized a candidate sex pheromone component in Microplitis mediator (Hymenoptera: Braconidae), a solitary parasitoid of Noctuidae insects. We found that the body surface extract from female wasps could significantly stimulate courtship behavior of males. Gas chromatography-electroantennographic detection (GC-EAD) analysis revealed that a candidate semiochemical from extract triggered significant electrophysiological response of antennae of males. By performing gas chromatography-mass spectrometer (GC-MS) measurement, GC-EAD active compound was identified as n-octyl acrylate, a candidate sex pheromone component in female M. mediator. In electroantennogram (EAG) tests, antennae of male wasps showed significantly higher electrophysiological responses to n-octyl acrylate than those of females. Y-tube olfactometer assays indicated that male wasps significantly chose n-octyl acrylate compared with the control. Furthermore, male wasps showed a remarkable preference for n-octyl acrylate in a simulated field condition behavioral trial; simultaneously, n-octyl acrylate standard could also trigger significant courtship behavior in males. We propose that n-octyl acrylate, as a candidate vital sex pheromone component, could be utilized to design behavioral regulators of M. mediator to implement the protection and utilization of natural enemies.

Loss of Binding Capabilities in an Ecologically Important Odorant Receptor of the Fall Armyworm, Spodoptera frugiperda, by a Single Point Mutation.

Olfaction plays a crucial role in locating food sources, mates, and spawning sites in the fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae). In the current study, SfruOR14, a highly conserved odorant receptor (OR) in lepidopteran species, was newly uncovered in S. frugiperda. In two-electrode voltage clamp recordings, the SfruOR14/Orco complex was narrowly tuned to six volatile compounds including phenylacetaldehyde (PAA), benzaldehyde, heptaldehyde, (E)-2-hexen-1-al, cinnamaldehyde, and 2-phenylethanol, among which PAA showed the strongest binding affinity. Subsequent homology modeling and molecular docking revealed that Phe79, His83, Tyr149, Pro176, Gln177, Leu202, and Thr348 in SfruOR14 were the key binding residues against the six ligands. Finally, as a result of site-directed mutagenesis, the SfruOR14His83Ala mutant completely lost its binding capabilities toward all ligands. Taken together, our findings provide valuable insights into understanding the interaction between SfruOR14 and the chemical ligands including PAA, which can help to design novel olfactory modulators for pest control.

Long-term insect censuses capture progressive loss of ecosystem functioning in East Asia.

Insects provide critical ecosystem services such as biological pest control, in which natural enemies (NE) regulate the populations of crop-feeding herbivores (H). While H-NE dynamics are routinely studied at small spatiotemporal scales, multiyear assessments over entire agrolandscapes are rare. Here, we draw on 18-year radar and searchlight trapping datasets (2003-2020) from eastern Asia to (i) assess temporal population trends of 98 airborne insect species and (ii) characterize the associated H-NE interplay. Although NE consistently constrain interseasonal H population growth, their summer abundance declined by 19.3% over time and prominent agricultural pests abandoned their equilibrium state. Within food webs composed of 124 bitrophic couplets, NE abundance annually fell by 0.7% and network connectance dropped markedly. Our research unveils how a progressive decline in insect numbers debilitates H trophic regulation and ecosystem stability at a macroscale, carrying implications for food security and (agro)ecological resilience during times of global environmental change.

Odorant-Binding Protein HvarOBP5 in Ladybird Hippodamia variegata Regulates the Perception of Semiochemicals from Preys and Habitat Plants.

A total of 12 OBPs were identified in the antennae of ladybird Hippodamia variegata. HvarOBP1/2/5/6/10/11 were highly expressed in adults, whereas HvarOBP3/4/7/8/9/12 had higher expression levels in larvae. In adults, HvarOBP1/2/4/5/6/7/8/11/12 were highly expressed in antennae. Among these, recombinant HvarOBP5 strong bound with (E)-ß-farnesene (EßF), (R)-(+)-limonene, (E,E)-4,8,12-trimethyltrideca-l,3,7,11-tetraene (TMTT), (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), hexyl hexanoate, and geranyl acetate. Molecular docking indicated that Leu42, Lys43, and His64 were the key binding sites of HvarOBP5. All six ligands evoked electroantennography (EAG) responses in ladybirds. Moreover, (R)-(+)-limonene and hexyl hexanoate were attractive to both sexes. After RNA interference for 72 h, the EAGs of dsRNA-injected ladybirds to DMNT and hexyl hexanoate were significantly decreased by 73.8 and 78.6%, respectively. Both dsRNA-injected males and females showed significantly lower behavioral preferences for DMNT and hexyl hexanoate. These findings suggest that HvarOBP5 in H. variegata plays an important role in the perception of semiochemical cues from preys and habitat plants.

Identification and functional analysis of odorant-binding proteins provide new control strategies for Apolygus lucorum.

The green bug Apolygus lucorum is a notorious pest that feeds on multiple crops, including fruit trees, vegetables, and cotton. The odorant-binding proteins (OBPs) are considered to perform crucial roles in regulating A. lucorum behaviors such as mating and feeding. In this study, we first identified OBPs in the A. lucorum genome. Then, we calculated the expression levels of these OBP genes in different tissues and stages. Thereafter, we conducted ligand-binding assay to test the interactions between nine selected AlucOBPs and multiple chemical compounds. The result showed that there were 31 OBP genes encoding 39 transcripts in the A. lucorum genome, and several OBP clusters were found. Comprehensive expression profiling revealed the tissue-specific expression of some OBP genes. The results of fluorescence competitive binding assays showed that these nine AlucOBPs could specifically bind to plant volatiles, nonvolatile compounds, and synthetic analogs thereof. Additionally, AlucOBP19 was suggested to function in gustatory sensing to avoid deleterious plant secondary metabolites, as AlucOBP19 showed high expression in the mouthparts and legs and could interact with quercetin. Our findings highlight the potential biotechnological application of plant volatiles and their synthetic analogs as ecological attractants and provide new gene targets for control of A. lucorum.

A female-biased odorant receptor tuned to the lepidopteran sex pheromone in parasitoid Microplitis mediator guiding habitat of host insects.

INTRODUCTION: The parasitoid wasp Microplitis mediator is an important natural enemy of the turnip moth Agrotis segetum and other Noctuidae pests. In our field observation, it was fortuitously discovered that sex pheromone traps used for A. segetum also attract female wasps, verified by a simulated field condition dual-choice laboratory assay. Therefore, it was hypothesized that olfactory recognition could be crucial in this process. In this regard, a female-biased odorant receptor of the wasp, MmedOR49, attracted our attention. OBJECTIVES: To unravel the significance of the female-biased MmedOR49 regulating host pheromone recognition. METHODS: Expression analysis (fluorescence in situ hybridization; quantitative realtime PCR), in vitro (two-electrode voltage-clamp recordings) and in vivo (RNAi combined with behavioral assessments) functional studies, and bioinformatics (structural modeling and molecular docking) were carried out to investigate the characteristics of MmedOR49. RESULTS: MmedOR49 expression was detected in the antennae of females by FISH. Quantification indicated that the expression level of MmedOR49 increased significantly after adult emergence. In vitro functional study revealed that MmedOR49 was specifically tuned to cis-5-decenyl acetate (Z5-10:Ac), the major sex pheromone component of A. segetum. Molecular docking showed that Z5-10:Ac strongly bound to the key amino acid residues His 80, Ile 81, and Arg 84 of MmedOR49 through hydrogen bonding. Behavioral assays indicated that female wasps were significantly attracted by Z5-10:Ac in a three-cage olfactometer. RNAi targeting further confirmed that MmedOR49 was necessary to recognize Z5-10:Ac, as female wasps lost their original behavioral responses to Z5-10:Ac after down-regulation of the MmedOR49 transcript. CONCLUSION: Although M. mediator is a larval endoparasitoid, female wasps have a behavioral preference for a sex pheromone component of lepidopteran hosts. In this behavior, for female M. mediator, MmedOR49 plays an important role in guiding the habitat of host insects. These data provide a potential target for enhancing natural enemy utilization and pest control.

Volatiles from cotton aphid (Aphis gossypii) infested plants attract the natural enemy Hippodamia variegata.

The Aphis gossypii is a major threat of cotton worldwide due to its short life cycle and rapid reproduction. Chemical control is the primary method used to manage the cotton aphid, which has significant environmental impacts. Therefore, prioritizing eco-friendly alternatives is essential for managing the cotton aphid. The ladybird, Hippodamia variegata, is a predominant predator of the cotton aphid. Its performance in cotton plantation is directly linked to chemical communication, where volatile compounds emitted from aphid-infested plants play important roles in successful predation. Here, we comprehensively studied the chemical interaction between the pest, natural enemy and host plants by analyzing the volatile profiles of aphid-infested cotton plants using gas chromatography-mass spectrometry (GC-MS). We then utilized the identified volatile compounds in electrophysiological recording (EAG) and behavioral assays. Through behavioral tests, we initially demonstrated the clear preference of both larvae and adults of H. variegata for aphid-infested plants. Subsequently, 13 compounds, namely α-pinene, cis-3-hexenyl acetate, 4-ethyl-1-octyn-3-ol, ß-ocimene, dodecane, E-ß-farnesene, decanal, methyl salicylate, ß-caryophyllene, α-humulene, farnesol, DMNT, and TMTT were identified from aphid-infested plants. All these compounds were electrophysiologically active and induced detectable EAG responses in larvae and adults. Y-tube olfactometer assays indicated that, with few exceptions for larvae, all identified chemicals were attractive to H. variegata, particularly at the highest tested concentration (100 mg/ml). The outcomes of this study establish a practical foundation for developing attractants for H. variegata and open avenues for potential advancements in aphid management strategies by understanding the details of chemical communication at a tritrophic level.

Plant volatile ligands for male-biased MmedOBP14 stimulate orientation behavior of the parasitoid wasp Microplitis mediator.

As an important class of chemosensory-associated proteins, odorant binding proteins (OBPs) play a key role in the perception of olfactory signals for insects. Parasitoid wasp Microplitis mediator relies on its sensitive olfactory system to locate host larvae of Noctuidae and Geometridae. In the present study, MmedOBP14, a male-biased OBP in M. mediator, was functionally investigated. In fluorescence competitive binding assays, the recombinant MmedOBP14 showed strong binding abilities to five plant volatiles: ß-ionone, 3,4-dimethylacetophenone, 4-ethylacetophenone, acetophenone and ocimene. Homology modeling and molecular docking results indicated that the binding sites of all five ligands were similar and concentrated in the binding pocket of MmedOBP14. Except acetophenone, the remaining four ligands at 1, 10 and 100 µg/µL caused strong antennal electrophysiological responses in adults M. mediator, and males showed more obvious EAG responses to most ligands than females. In behavioral trials, males were attracted by low concentrations of MmedOBP14 ligands, whereas high doses of ß-ionone and acetophenone had a repellent effect on males. Moreover, 1 µg/µL of 3,4-dimethylacetophenone showed the strongest attractiveness to female wasps. These findings suggest that MmedOBP14 may play a more important role in the perception of plant volatiles for male wasps to locate habitat, supplement nutrition and search partners.

The microRNAs in the antennae of Apolygus lucorum (Hemiptera: Miridae): Expression properties and targets prediction.

MicroRNAs (miRNAs) regulate gene expression and contribute to numerous physiological processes. However, little is known about the functions of miRNAs in insect chemosensation. In this study, nine small RNA libraries were constructed and sequenced from the antennae of nymphs, adult males, and adult females of Apolygus lucorum. In total, 399 (275 known and 124 novel) miRNAs were identified. miR-7-5p_1 was the most abundant miRNA. Altogether, 69,708 target genes related to biogenesis, membrane, and binding activities were predicted. In particular, 15 miRNAs targeted 16 olfactory genes. Comparing the antennae of nymphs and adult males and females, 94 miRNAs were differentially expressed. Alternatively, a subset of differentially expressed miRNAs was verified by qPCR, supporting the reliability of the sequencing results. This study provides a global miRNA transcriptome for the antennae of A. lucorum and valuable information for further investigations of the functions of miRNAs in the regulation of chemosensation.

Identification and Tissue Expression Profiles of Odorant Receptor Genes in the Green Peach Aphid Myzus persicae.

The green peach aphid Myzus persicae (Hemiptera: Aphididae) relies heavily on its olfactory system to locate plant hosts, find mates, and avoid parasitoids or predators. The insect odorant receptors (ORs) have been proven to play a critical role in the perception of odorants from the environment. In the present study, 33 odorant receptor candidate genes including the Orco gene were identified from the antennal, head, legs and body transcriptomes of M. persicae. Phylogenetic analysis of ORs from seven different orders of insect species suggests that ORs from different insect species are highly divergent and most ORs from the same species formed monophyletic groups. In addition, the aphid ORs were clustered into six different sub-clades in the same clade. Furthermore, the genomic structure of the OR genes also tends to be consistent, suggesting that ORs from the family Aphididae have a relatively close evolutionary relationship. Reads per kilobase per million (RPKM) and tissue expression profiles analyses revealed that 27 out of the 33 MperORs were uniquely or primarily expressed in the antennae, indicating their putative roles in chemoreception. This work provides a foundation to further investigate the molecular and ecological functions of MperORs in the aphid-aphid, aphid-plant and aphid-natural enemy interactions.

Antenna-Biased Odorant Receptor HvarOR25 in Hippodamia variegata Tuned to Allelochemicals from Hosts and Habitat Involved in Perceiving Preys.

Odorant receptors (ORs) of ladybird Hippodamia variegata play vital chemosensory roles in searching and locating preys. In the current study, 37 ORs were initially identified from the antennal transcriptome of H. variegata. The quantitative polymerase chain reaction demonstrated that several HvarORs including HvarOR25 were specific or enriched in ladybird antennae. In two-electrode voltage clamp recordings, recombinant HvarOR25 was narrowly tuned to six chemical ligands including aphid-induced, aphid-derived, and plant-derived volatiles. In electroantennogram assays, all six volatiles elicited electrophysiological responses. Among the six volatiles, cis-3-hexenyl acetate, hexyl butyrate, hexyl hexanoate, and 3-methyl-3-buten-1-ol were attractive for both sexes of H. variegata. Additionally, molecular docking indicated that HvarOR25 was bound to all ligands with high binding affinities. Taken together, HvarOR25 facilitates perception of preys by recognizing relevant allelochemicals from hosts and habitat. Our findings provide valuable insights into understanding biological functions of HvarORs and help to develop a novel biocontrol strategy based on olfactory-active compounds.

Functional Characterization of a Candidate Sex Pheromone Receptor AlinOR33 Involved in the Chemoreception of Adelphocoris lineolatus.

Sex pheromones are deemed to play a significant role in sexual communication of most insects. Although many sex pheromone components in mirid bugs have been identified, the roles of odorant receptors in sex pheromone perception in Adelphocoris spp. (Hemiptera: Miridae) remain unknown so far. Here, AlinOR33, a candidate sex pheromone receptor in Adelphocoris lineolatus was functionally characterized. Phylogenetic analysis showed that AlinOR33 clustered with the sex pheromone receptor AlucOR4 fromApolygus lucorum. Quantitative real-time PCR measurement revealed that the expression of AlinOR33 increased gradually from nymph to adult stage and reached its peak in the antennae of 3-day-old mated male bugs. The subsequent in situ hybridization demonstrated that AlinOR33 was mainly expressed in sensilla trichoid on the antennae of A. lineolatus. In the two-electrode voltage clamp recordings, AlinOR33/AlinOrco was specifically tuned to four sex pheromone components including butyl butyrate, hexyl hexanoate, trans-2-hexenyl butyrate and hexyl butyrate, and especially most sensitive to the major component trans-2-hexenyl butyrate. After dsAlinOR33 injection, the electroantennogram responses of males to four sex pheromone components were reduced significantly (∼50%). Compared to control bugs, dsAlinOR33-injected male bugs almost lost behavioral preference for trans-2-hexenyl butyrate. Furthermore, the wingbeat frequency of dsAlinOR33-injected male bugs notably declined. Therefore, we conclude that as a candidate sex pheromone receptor, AlinOR33 plays essential roles in the sexual behavior of A. lineolatus.

Identification and Evaluation of Suitable Reference Genes for RT-qPCR Analysis in Hippodamia variegata (Coleoptera: Coccinellidae) Under Different Biotic and Abiotic Conditions.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is an accurate and convenient technique for quantifying expression levels of the target genes. Selection of the appropriate reference gene is of the vital importance for RT-qPCR analysis. Hippodamia variegata is one of the most important predatory natural enemies of aphids. Recently, transcriptome and genome sequencings of H. variegata facilitate the gene functional studies. However, there has been rare investigation on the detection of stably expressed reference genes in H. variegata. In the current study, by using five analytical tools (Delta Ct, geNorm, NormFinder, BestKeeper, and RefFinder), eight candidate reference genes, namely, Actin, EF1α, RPL7, RPL18, RPS23, Tubulin-α, Tubulin-ß, and TufA, were evaluated under four experimental conditions including developmental stages, tissues, temperatures, and diets. As a result, a specific set of reference genes were recommended for each experimental condition. These findings will help to improve the accuracy and reliability of RT-qPCR data, and lay a foundation for further exploration on the gene function of H. variegata.

Host plants transfer induced regulation of the chemosensory genes repertoire in the alfalfa plant bug Adelphocoris lineolatus (Goeze).

The alfalfa plant bug Adelphocoris lineolatus, an economically important pest, has representative behavioral characteristics with host plants transfer. Olfactory system is essential for insects to perceive ever-changing chemical signals in the external environment, and chemosensory genes play crucial roles in signals reception and transduction. In this work, we compared the differences in chemosensory genes expression before and after host plants transfer by constructing 12 antennal transcriptomes of male and female bugs, respectively. The results showed that the expression levels of most chemosensory genes in A. lineolatus changed to adapt to the transformation of the hosts plant. More remarkable, female bugs had more up-regulated chemosensory genes than males. Differentially expressed genes (DEGs) analysis revealed three odorant binding proteins (OBPs), three chemosensory proteins (CSPs), eight odorant receptors (ORs) and one ionotropic receptor (IR) showed significant differences when the host plant transferred. There were complex characteristics of up- and down- regulated genes in male and female adults, among which OBP19 showed higher expression in females exposing to the new host plant alfalfa, suggesting this OBP may be associated with the localization of the oviposition site. The OR54 and OR82 were up-regulated in both genders, indicating their possible roles in recognizing some alfalfa-specific volatiles. These findings will provide valuable insights in biological functions of chemosensory genes in A. lineolatus and facilitate the development of new targets for novel strategies to control the alfalfa plant bug and other herbivores.

Characterization and target gene analysis of microRNAs in the antennae of the parasitoid wasp Microplitis mediator.

MicroRNAs (miRNAs), a class of non-coding single-strand RNA molecules encoded by endogenous genes, are about 21-24 nucleotides long and are involved in the post-transcriptional regulation of gene expression in plants and animals. Generally, the types and quantities of miRNAs in the different tissues of an organism are diverse, and these divergences may be related to their specific functions. Here we have identified 296 known miRNAs and 46 novel miRNAs in the antennae of the parasitoid wasp Microplitis mediator by high-throughput sequencing. Thirty-three miRNAs were predicted to target olfactory-associated genes, including odorant binding proteins (OBPs), chemosensory proteins, odorant receptors (ORs), ionotropic receptors (IRs) and gustatory receptors. Among these, 17 miRNAs were significantly highly expressed in the antennae, four miRNAs were highly expressed both in the antennae and head or wings, while the remaining 12 miRNAs were mainly expressed in the head, thorax, abdomen, legs and wings. Notably, miR-9a-5p and miR-2525-3p were highly expressed in male antennae, whereas miR-1000-5p and novel-miR-13 were enriched in female antennae. The 17 miRNAs highly expressed in antennae are likely to be associated with olfaction, and were predicted to target one OBP (targeted by miR-3751-3p), one IR (targeted by miR-7-5p) and 14 ORs (targeted by 15 miRNAs including miR-6-3p, miR-9a-5p, miR-9b-5p, miR-29-5p, miR-71-5p, miR-275-3p, miR-1000-5p, miR-1000-3p, miR-2525-3p, miR-6012-3p, miR-9719-3p, novel-miR-10, novel-miR-13, novel-miR-14 and novel-miR-28). These candidate olfactory-associated miRNAs are all likely to be involved in chemoreception through the regulation of chemosensory gene expression in the antennae of M. mediator.

Prior Experience with Food Reward Influences the Behavioral Responses of the Honeybee Apis mellifera and the Bumblebee Bombus lantschouensis to Tomato Floral Scent.

Bee responses to floral scent are usually influenced by both innate biases and prior experience. Honeybees are less attracted than bumblebees to tomato flowers. However, little is known about how tomato floral scent regulates the foraging behaviors of honeybees and bumblebees. In this study, the foraging behaviors of the honeybee Apis mellifera and the bumblebee Bombus lantschouensis on tomato flowers in greenhouses were investigated. Whether the two bee species exhibit different responses to tomato floral scent and how innate biases and prior experience influence bee choice behavior were examined. In the greenhouses, honeybees failed to collect pollen from tomato flowers, and their foraging activities decreased significantly over days. Additionally, neither naïve honeybees nor naïve bumblebees showed a preference for tomato floral scent in a Y-tube olfactometer. However, foraging experience in the tomato greenhouses helped bumblebees develop a strong preference for the scent, whereas honeybees with foraging experience continued to show aversion to tomato floral scent. After learning to associate tomato floral scent with a sugar reward in proboscis extension response (PER) assays, both bee species exhibited a preference for tomato floral scent in Y-tube olfactometers. The findings indicated that prior experience with a food reward strongly influenced bee preference for tomato floral scent.

Broadly Tuned Odorant Receptor AlinOR59 Involved in Chemoreception of Floral Scent in Adelphocoris lineolatus.

Plant volatiles such as floral scent compounds play a crucial role in mediating insect host locating, mate search, and oviposition sites selection. The alfalfa plant bug, Adelphocoris lineolatus (Goeze), is a seriously polyphagous herbivore of alfalfa and cotton that has an obvious preference for flowering host plants. In this study, we focused on the role of an odorant receptor AlinOR59 in the perception of plant volatiles in A. lineolatus. In situ hybridization showed that AlinOR59 was coexpressed with the coreceptor AlinORco in the ORNs cell located in the long curved sensilla trichodea on antennae of both genders. The Xenopus oocytes expression coupled with two-electrode voltage clamp recordings demonstrated that AlinOR59 responded to 15 plant volatiles. In electroantennogram assays, all of the above 15 compounds could excite electrophysiological responses in the antennae of adult bugs. Furthermore, an important floral scent compound, methyl salicylate, was utilized to evaluate the behavioral responses of A. lineolatus. It was found that adult bugs of both genders were significantly attracted to methyl salicylate. Taken together, our findings suggest that AlinOR59 plays a crucial role in the perception of floral scents in A. lineolatus and could be used as a potential target to design novel olfactory regulators for the management of bugs.

Identification of Leg Chemosensory Genes and Sensilla in the Apolygus lucorum.

Apolygus lucorum (Hemiptera: Miridae), one of the main insect pests, causes severe damage in cotton and many other economic crops. As is well-known, legs play important roles in the chemoreception of insects. In this study, the putative chemosensory proteins in legs of A. lucorum involved in close or contact chemical communication of adult bugs were investigated using RNA transcriptome sequencing and qPCR methods. Transcriptome data of forelegs, middle legs and hind legs of adult bugs demonstrated that 20 odorant binding protein (OBP) genes, eight chemosensory protein (CSP) genes, one odorant receptor (OR) gene, one ionotropic receptor (IR) gene and one sensory neuron membrane protein (SNMP) gene were identified in legs of A. lucorum. Compared to the previous antennae transcriptome data, five CSPs, IR21a and SNMP2a were newly identified in legs. Results of qPCR analysis indicated that all these putative chemosensory genes were ubiquitously expressed in forelegs, middle legs and hind legs of bugs. Furthermore, four types of sensilla on legs of A. lucorum including sensilla trichodea (subtypes: long straight sensilla trichodea, Str1; long curved sensilla trichodea, Str2), sensilla chaetica (subtypes: sensilla chaetica 1, Sch1; sensilla chaetica 2, Sch2; and sensilla chaetica 3, Sch3), sensilla basiconca (subtypes: medium-long sensilla basiconca, Sba1; short sensilla basiconca, Sba2) and Böhm bristles (BB) were found using scanning electron microscopy. Additionally, the largest number of sensilla was observed on hind legs, while the forelegs had the smallest number of sensilla. Our data provide valuable insights into understanding the chemoreception of legs in A. lucorum.