Inhibition Effect of Non-Host Plant Volatile Extracts on Reproductive Behaviors in the Diamondback Moth Plutella xylostella (Linnaeus).

The pest management of Plutella xylostella, the global pest of cruciferous plants, is primarily dependent upon continued applications of insecticides, which has led to severe insecticide resistance and a series of ecological concerns. The essential oils (EOs) of non-host plants are considered to have a high application potential in pest behavioral control. In P. xylostella, the insecticidal properties, antifeeding activities, and oviposition inhibition effects of many EOs have been studied in larvae and female moths. However, less focus has been placed on the inhibitory effect on sex pheromone communication during courtship, which is vital for the reproduction of the offspring. In this study, by combining electrophysiological studies, laboratory behavioral assays, and field traps, we demonstrated that non-host plant EOs significantly inhibited the reproductive behaviors of both sexes. Notably, the calamus (Acorus gramineus) EO inhibited the preference of male moths for synthetic sex pheromone blends and reduced the egg-laying number of female moths on host plants, with the highest inhibition rates of 72% and 100%, respectively, suggesting a great application prospect of calamus and its EO on the behavioral control strategies of P. xylostella.

A female-specific odorant receptor mediates oviposition deterrence in the moth Helicoverpa armigera.

Finding ideal oviposition sites is a task of vital importance for all female insects. To ensure optimal conditions for their progeny, females of herbivorous insects detect not only the odors of a relevant host plant but also chemicals released by eggs, named oviposition-deterring pheromones (ODPs). It is reported that such chemicals play critical roles in suppressing female oviposition behavior; however, the molecular mechanism underlying the detection of egg-derived ODPs remains elusive. Here, we have identified three specific fatty acid methyl esters from the surface of eggs of Helicoverpa armigera serving as ODPs-methyl oleate (C18:1ME), methyl palmitate (C16:0ME), and methyl stearate (C18:0ME). We demonstrated that these ODPs are detected by the receptor, HarmOR56, exclusively expressed in sensilla trichodea on female antennae. To assess the significance of this receptor, we disrupted HarmOR56 in H. armigera using CRISPR-Cas9 and found that mutant females did not respond to the ODPs, neither in behavioral nor in electrophysiological tests. We therefore conclude that HarmOR56 is indispensable for identifying the ODPs. This study explores, for the first time, how a female-specific odorant receptor detects chemicals from conspecific eggs. Our data elucidate the intriguing biological phenomenon of repulsion to conspecific eggs during oviposition and contribute new insight into a female-specific olfactory pathway linked to reproduction.

An Odorant Receptor from the Proboscis of the Cotton Bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) Narrowly Tuned to Indole.

Helicoverpa armigera is a serious agricultural pest with polyphagous diets, widespread distribution, and causing severe damage. Among sixty-five candidate ORs in H. armigera, the co-receptor HarmOrco and three specific ORs with partial sequences were identified to be expressed in the proboscis by our previous work, whereas their exact function is not known yet. In this study, we first confirmed the expression of these ORs in the proboscis by full-length cloning, which obtained the complete coding region of HarmOrco, OR24, and OR30. We then performed functional identification of HarmOR24 and OR30 by co-expressing them respectively with HarmOrco in Xenopus oocytes eukaryotic expression system combined with two-electrode voltage-clamp physiology. By testing the response of HarmOR24/OR30-expressing oocytes against eighty structural-divergent compounds, respectively, HarmOR30 was characterized to narrowly tune to indole and showed a specific tuning spectrum compared to its ortholog in Spodoptera littoralis. As indole is a distinctive herbivore-induced plant volatile and floral scent component, HarmOR30 might play roles in foraging and mediating the interactions between H. armigera with its surrounding environment.

Optimization of a pheromone lure by analyzing the peripheral coding of sex pheromones of Spodoptera frugiperda in China.

BACKGOUND Sex pheromones of the fall armyworm, Spodoptera frugiperda, show differences in composition and proportions in different geographical populations, but always contain Z9-14:OAc as the major component. Odorant receptor neurons (ORNs) housed in the long trichoid sensilla (TS) of male antennae are essential to detect female-released sex pheromones in moths. RESULTS: In this study, we identified seven components from pheromone gland extracts of female S. frugiperda in the Yunnan population from China, including (Z)-7-dodecen-1-yl acetate (Z7-12:OAc), (Z)-9-tetradecenal (Z9-14:Ald), (Z)-9-dodecen-1-yl acetate (Z9-12:OAc), (Z)-9-tetradecen-1-yl acetate (Z9-14:OAc), (E)-11-tetradecen-1-yl acetate (E11-14:OAc), (Z)-11-tetradecen-1-yl acetate (Z11-14:OAc) and (Z)-11-hexadecen-1-yl acetate (Z11-16:OAc) at a ratio of 1.2:4:0.8:79.1:1.6:1.6:11.7 by gas chromatography coupled with mass spectrometry. Gas chromatography-electroantennographic detection showed that Z9-14:OAc, Z7-12:OAc and Z11-16:OAc are the male antennal active components. Peripheral coding of pheromones in males was investigated by single sensillum recording. Five functional neurons housed in three types of TS were identified based on profiles of neuronal responses, which are responsible for attractive component Z9-14:OAc, synergistic components Z7-12:OAc, Z11-16:OAc, interspecific pheromones (Z)-9-tetradecen-1-ol (Z9-14:OH) and (Z,E)-9,12-tetradecadien-1-yl acetate (Z9,E12-14:OAc), respectively. Wind tunnel and field tests demonstrated that a ternary combination of Z9-14:OAc, Z7-12:OAc and Z11-16:OAc at a ratio of 88:1:11 shows the strongest attractiveness to males. CONCLUSION: An optimized pheromone blend of Z9-14:OAc, Z7-12:OAc and Z11-16:OAc in an 88:1:11 ratio was identified for monitoring the invasive pest S. frugiperda in China. Five functional ORNs encoding intra- and interspecific pheromones were identified in male antennae, of which three neurons encode attractive component Z9-14:OAc, synergistic components Z7-12:OAc and Z11-16:OAc, respectively, and the other two neurons encode interspecific pheromones Z9-14:OH and Z9,E12-14:OAc, separately. © 2022 Society of Chemical Industry.

Unusual (2R,6R)-bicyclo[3.1.1]heptane ring construction in fungal α-trans-bergamotene biosynthesis.

Bergamotenes are bicyclo[3.1.1]heptane sesquiterpenes found abundantly in plants and fungi. Known bergamotene derivatives all possess (2S,6S)-bergamotene backbone. In this study, two (+)-α-trans-bergamotene derivatives (1 and 2) with unusual (2R,6R) configuration were isolated and elucidated from marine fungus Nectria sp. HLS206. The first (+)-α-trans-bergamotene synthase NsBERS was characterized using genome mining and heterologous expression-based strategies. Based on homology search, we characterized another (+)-α-trans-bergamotene synthase LsBERS from Lachnellula suecica and an (+)-α-bisabolol synthase BcBOS from Botrytis cinerea. We proposed that the cyclization mechanism of (+)-α-trans-bergamotene involved endo-anti cyclization of left-handed helix farnesyl pyrophosphate by (6R)-bisabolyl cation, which was supported by molecular docking. The biosynthesis-based volatiles (3-6) produced by heterologous fungal expression systems elicited significant electroantennographic responses of Helicoverpa armigera and Spodoptera frugiperda, respectively, suggesting their potential in biocontrol of these pests. This work enriches diversity of sesquiterpenoids and fungal sesquiterpene synthases, providing insight into the enzymatic mechanism of formation of enantiomeric sesquiterpenes.

Ionotropic receptor 8a is involved in the attraction of Helicoverpa armigera to acetic acid.

Ionotropic receptors (IRs) were first found in Drosophila melanogaster, and derive from ionotropic glutamate receptors (iGluRs), which are implicated in detecting acids, ammonia, amine, temperature and humidity. Although IRs are involved in sensing acid odors in a few insects, such as D. melanogaster, Aedes aegypti, and Manduca sexta, the function of IRs in Helicoverpa armigera is still unknown. IR8a was confirmed to be a co-receptor associated with acid detection. From the results of phylogenetic analysis, HarmIR8a displayed high similarity compared to homologs in D. melanogaster, M. sexta, and A. aegypti, suggesting that HarmIR8a might have a consistent function as a co-receptor for acid detection. In this study, clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9)-mediated genome editing was implemented to knockout HarmIR8a for in vivo functional analysis. Electrophysiological and behavioral assays were performed to compare the differences between HarmIR8a knockout mutants and wild type individuals. From electroantennogram (EAG) analysis, we found that wild type H. armigera adults could detect short-chain carboxylic acids. In addition, wind tunnel experiments showed that 1% acetic acid attracted wild type H. armigera adults. However, acid sensing and attraction were reduced or abolished in the HarmIR8a knockout mutants. Our data suggest that HarmIR8a is important for H. armigera to detect short-chain carboxylic acids and mediate attraction behavior to acetic acid.

Fine Structure and Olfactory Reception of the Labial Palps of Spodoptera frugiperda.

The olfactory system of insects is essential in many crucial behaviors, such as host seeking, mate recognition, and locating oviposition sites. Lepidopteran moths possess two main olfactory organs, including antennae and labial palps. Compared to antennae, the labial palps are relatively specific and worthy of further investigation due to the labial-palp pit organ (LPO), which contains a large number of sensilla located on the tip segment. The fall armyworm, Spodoptera frugiperda, is a worldwide lepidopteran pest, which can damage more than 350 plants and cause significant economic losses. In this study, we surveyed the structure of the labial palps and LPO of S. frugiperda using a super-high magnification lens zoom 3D microscope. Then, the distribution and fine structure of sensilla located in the LPO of S. frugiperda were investigated using scanning electron microscopy. Subsequently, the electrophysiological responses of labial palps to CO2 and 29 plant volatiles were recorded by using electrolabialpalpography. Our results showed the fine structure of labial palps, the LPO, and the sensilla located in the LPO of S. frugiperda. Moreover, we demonstrated that the labial palps are olfactory organs that respond to both CO2 and other volatile compounds. Our work established a foundation for further study of the roles of labial palps in insect olfactory related behaviors. Further investigations on the function of labial palps and their biological roles together with CO2 and volatile compound responses in S. frugiperda are necessary, as they may provide better insect behavioral regulators for controlling this pest.

Odorant Receptors for Detecting Flowering Plant Cues Are Functionally Conserved across Moths and Butterflies.

Odorant receptors (ORs) are essential for plant-insect interactions. However, despite the global impacts of Lepidoptera (moths and butterflies) as major herbivores and pollinators, little functional data are available about Lepidoptera ORs involved in plant-volatile detection. Here, we initially characterized the plant-volatile-sensing function(s) of 44 ORs from the cotton bollworm Helicoverpa armigera, and subsequently conducted a large-scale comparative analysis that establishes how most orthologous ORs have functionally diverged among closely related species whereas some rare ORs are functionally conserved. Specifically, our systematic analysis of H. armigera ORs cataloged the wide functional scope of the H. armigera OR repertoire, and also showed that HarmOR42 and its Spodoptera littoralis ortholog are functionally conserved. Pursuing this, we characterized the HarmOR42-orthologous ORs from 11 species across the Glossata suborder and confirmed the HarmOR42 orthologs form a unique OR lineage that has undergone strong purifying selection in Glossata species and whose members are tuned with strong specificity to phenylacetaldehyde, a floral scent component common to most angiosperms. In vivo studies via HarmOR42 knockout support that HarmOR42-related ORs are essential for host-detection by sensing phenylacetaldehyde. Our work also supports that these ORs coevolved with the tube-like proboscis, and has maintained functional stability throughout the long-term coexistence of Lepidoptera with angiosperms. Thus, beyond providing a rich empirical resource for delineating the precise functions of H. armigera ORs, our results enable a comparative analysis of insect ORs that have apparently facilitated and currently sustain the intimate adaptations and ecological interactions among nectar feeding insects and flowering plants.

A chromosome-level genome assembly of Cydia pomonella provides insights into chemical ecology and insecticide resistance.

The codling moth Cydia pomonella, a major invasive pest of pome fruit, has spread around the globe in the last half century. We generated a chromosome-level scaffold assembly including the Z chromosome and a portion of the W chromosome. This assembly reveals the duplication of an olfactory receptor gene (OR3), which we demonstrate enhances the ability of C. pomonella to exploit kairomones and pheromones in locating both host plants and mates. Genome-wide association studies contrasting insecticide-resistant and susceptible strains identify hundreds of single nucleotide polymorphisms (SNPs) potentially associated with insecticide resistance, including three SNPs found in the promoter of CYP6B2. RNAi knockdown of CYP6B2 increases C. pomonella sensitivity to two insecticides, deltamethrin and azinphos methyl. The high-quality genome assembly of C. pomonella informs the genetic basis of its invasiveness, suggesting the codling moth has distinctive capabilities and adaptive potential that may explain its worldwide expansion.

Chemoreception of Mouthparts: Sensilla Morphology and Discovery of Chemosensory Genes in Proboscis and Labial Palps of Adult Helicoverpa armigera (Lepidoptera: Noctuidae).

Siphoning mouthparts, consisting of proboscis and labial palps, are the exclusive feeding organs and important chemosensory organs in most adult Lepidoptera. In this study, the general morphology of the mouthpart organs and precision architecture of the proboscis was described in adult Helicoverpa armigera. Three major sensilla types with nine subtypes including three novel subtypes were identified. The novel sensilla styloconica subtype 2 was the only one having a multiporous structure, which may play olfactory roles. For further understanding of the chemosensory functions of mouthpart organs, we conducted transcriptome analysis on labial palps and proboscises. A total of 84 chemosensory genes belonging to six different families including 4 odorant receptors (ORs), 6 ionotropic receptors (IRs), 7 gustatory receptors (GRs), 39 odorant binding proteins (OBPs), 26 chemosensory proteins (CSPs), and 2 sensory neuron membrane proteins (SNMPs) were identified. Furthermore, eight OBPs and six CSPs were identified as the novel genes. The expression level of candidate chemosensory genes in the proboscis and labial palps was evaluated by the differentially expressed gene (DEG) analysis, and the expression of candidate chemosensory receptor genes in different tissues was further investigated by quantitative real-time PCR (qRT-PCR). All the candidate receptors were detected by DEG analysis and qRT-PCR, but only a small part of the OR or IR genes was specifically or partially expressed in proboscis or labial palps, such as HarmOR58 and HarmIR75p.1, however, most of the GRs were abundantly expressed in proboscis or labial palps. The reported CO2 receptors such as HarmGR1, GR2, and GR3 were mainly expressed in labial palps. HarmGR5, GR6, and GR8, belonging to the "sugar receptor" clade, were mainly expressed in proboscis or antenna and were therefore suggested to perceive saccharide. The results suggest that the mouthparts are mutually cooperative but functionally concentrated system. These works contribute to the understanding of chemical signal recognition in mouthpart organs and provide the foundation for further functional studies.

A receptor-neuron correlate for the detection of attractive plant volatiles in Helicoverpa assulta (Lepidoptera: Noctuidae).

Plant volatiles are vital cues in the location of hosts for feeding and oviposition for Lepidoptera moths. The noctuid Helicoverpa assulta is a typical polyphagous moth, regarded as a good model for studying the olfactory reception of plant volatiles. In this study, four full-length genes encoding odorant receptors HassOR24, HassOR40, HassOR41, and HassOR55 expressed in antenna in H. assulta were functionally characterized. The highly expressed HassOR40 was narrowly tuned to a few structurally-related plant volatiles: geranyl acetate, geraniol and nerolidol. By systematically analyzing responses of single neuron in both trichoid sensilla and basiconic sensilla using single sensillum recording, the specific neuron B in one type of short trichoid sensilla was found to be mainly activated by the same chemicals as HassOR40 with high sensitivity, and with no significant difference between male and female neurons. Thus, a clear "receptor-neuron" relationship in H. assulta was demonstrated here, suggesting that HassOR40/HassOrco are expressed in neuron B of short trichoid sensilla. The active tobacco volatile nerolidol, recognized by this receptor-neuron line, elicits significant behavioral attraction of both sexes in H. assulta adults. The results indicate that we identified a receptor-neuron route for the peripheral coding of a behaviorally relevant host volatile in H. assulta.

A Conserved Odorant Receptor Tuned to Floral Volatiles in Three Heliothinae Species.

Odorant receptors (ORs) play an important role in insects to monitor and adapt to the external environment, such as host plant location, oviposition-site selection, mate recognition and natural enemy avoidance. In our study, we identified and characterized OR12 from three closely-related species, Helicoverpa armigera, Helicoverpa assulta, Heliothis virescens, sharing between 90 and 98% of their amino acids. The tissue expression pattern analysis in H. armigera showed that HarmOR12 was strongly expressed both in male and female antennae, but not in other tissues. Functional analysis performed in the heterologous Xenopus expression system showed that all three OR12 were tuned to six structurally related plant volatiles. Electroantennogram recordings from male and female antennae of H. armigera closely matched the data of in vitro functional studies. Our results revealed that OR12 has a conserved role in Heliothinae moths and might represent a suitable target for the control of these crop pests.

Sensillar expression and responses of olfactory receptors reveal different peripheral coding in two Helicoverpa species using the same pheromone components.

Male moths efficiently recognize conspecific sex pheromones thanks to their highly accurate and specific olfactory system. The Heliothis/Helicoverpa species are regarded as good models for studying the perception of sex pheromones. In this study, we performed a series of experiments to investigate the peripheral mechanisms of pheromone coding in two-closely related species, Helicoverpa armigera and H. assulta. The morphology and distribution patterns of sensilla trichoidea are similar between the two species when observed at the scanning electron microscope, but their performances are different. In H. armigera, three functional types of sensilla trichoidea (A, B and C) were found to respond to different pheromone components, while in H. assulta only two types of such sensilla (A and C) could be detected. The response profiles of all types of sensilla trichoidea in the two species well matched the specificities of the pheromone receptors (PRs) expressed in the same sensilla, as measured in voltage-clamp experiments. The expressions of PRs in neighboring olfactory sensory neurons (OSNs) within the same trichoid sensillum were further confirmed by in situ hybridization. Our results show how the same pheromone components can code for different messages at the periphery of two Helicoverpa species.

Narrow tuning of an odorant receptor to plant volatiles in Spodoptera exigua (Hübner).

Olfaction plays an important role in insects in recognizing volatile compounds, which are used to find food and mates, as well as avoid danger, predators and pathogens. The key players in the detection of volatile compounds are olfactory receptor (OR) proteins, which are located within the dendritic membrane of sensory neurons and extend into the lymph of sensilla on insect antennae. In the present study, we identify an OR gene, named SexiOR3, which is expressed in adult antenna in both sexes, but with female bias, in the beet armyworm moth Spodoptera exigua. Further in situ hybridization analysis revealed that SexiOR3 was mainly located in short trichoid sensilla. In in vitro heterologous expression experiments, SexiOR3 was narrowly tuned to E-ß-farnesene and several compounds of related structure, among 62 different compounds tested in this study. Furthermore, SexiOR3 responds to E-ß-farnesene at a low concentration of 10(-9) M, comparable to that of pheromone receptors (PRs) responding to the pheromones. This is a very interesting finding for a general OR, indicating that high specificity is a feature of at least one general OR and not only the PRs. The results suggest that the OR3 gene may play an important role in the moth olfactory system, and underpins important insect behaviour.

Molecular identification and differential expression of sensory neuron membrane proteins in the antennae of the black cutworm moth Agrotis ipsilon.

The insect sensory neuron membrane proteins (SNMPs) SNMP1 and SNMP2 are transmembrane domain-containing proteins and are homologs of the vertebrate CD36 transmembrane proteins. It has been suggested that SNMPs play a significant role in insect chemoreception. Previous studies have demonstrated that SNMP1 is expressed in the pheromone-sensitive olfactory receptor neurons (ORNs), whereas SNMP2 is expressed in the supporting cells. In this study, we identified two full-length SNMP transcripts, AipsSNMP1 and AipsSNMP2, in the black cutworm moth Agrotis ipsilon (Hufnagel). The qRT-PCR results indicated that the AipsSNMP1 and AipsSNMP2 transcripts were expressed significantly higher in the antennae than in other tissues of both sexes. The expression of AipsSNMP1 and AipsSNMP2 in the antennae from different development stages of both sexes was investigated and was shown to begin to express in the pupae stage from 3days before emergence and then increased dramatically at the day of the emergence, and the high expression levels were maintained during the following 4days after the emergence in both sexes. The mating status had no effect on the expression levels of the AipsSNMP1 and AipsSNMP2 transcripts. Consistent with previous in situ hybridization studies in other Lepidoptera insects, our immunolocalization results at protein level demonstrated that both AipsSNMP1 and AipsSNMP2 were expressed in pheromone-sensitive sensilla trichodea but with a completely different expression profile. AipsSNMP1 is more uniformed and highly expressed along the membrane of the ORN dendrites, whereas AipsSNMP2 is widely distributed at the bottom of the sensilla trichodea and highly localized in the sensillum lymph. Our studies provide further detailed evidence for the involvement and general functional role of insect SNMPs in the detection of sex pheromones and general odorant molecules.