The ecdysis triggering hormone system is essential for reproductive success in Mythimna separata (Walker).

Ecdysis triggering hormone (ETH) was originally discovered as a key hormone that regulates insect moulting via binding to its receptor, ETH receptor (ETHR). However, the precise role of ETH in moth reproduction remains to be explored in detail. ETH function was verified in vivo using Mythimna separata (Walker), an important cereal crop pest. RT-qPCR analysis revealed that transcriptional expression profiles of MsepETH showed evident sexual dimorphism in the adult stage. MsepETH expression increased in the females on day 3 and persisted thereafter till day 7, consistent with female ovarian maturation, and was merely detectable in males. Meanwhile, MsepETH expression levels were significantly higher in the trachea than in other tissues. MsepETHR-A and MsepETHR-B were expressed in both sexes and were significantly higher in the antennae than in other tissues. MsepETH and MsepETHR knockdown in females by RNA interference significantly reduced the expression of MsepETH, MsepETHR-A, MsepETHR-B, MsepJHAMT, and MsepVG, which delayed egg-laying and significantly reduced egg production. RNAi 20-hydroxyecdysone (20E) receptor (EcR) decreased MsepETH expression whereas injecting 20E restored egg production that had been disrupted by MsepETH interference. Meanwhile, RNAi juvenile hormone (JH) methoprene tolerant protein (Met) also decreased MsepETH expression and smearing JH analog methoprene (Meth) restored egg production. In conclusion, the reproduction roles of ETH, JH, and 20E were investigated in M. separata. These findings will lay the foundation for future research to develop an antagonist that reduces female reproduction and control strategies for pest insects.

Functional analysis of the odorant receptor coreceptor in odor detection in Grapholita molesta (lepidoptera: Tortricidae).

The olfactory system must detect and discriminate various semiochemicals in the environment. In response to such diversity, insects have evolved a family of odorant-gated ion channels composed of a common receptor (coreceptor, Orco) and a ligand-binding tuning odorant receptor (OR) that confers odour specificity. This study aims to examine the expression pattern of Orco gene of Grapholita molesta (GmolOrco) and to elucidate the role of GmolOrco in detecting G. molesta sex pheromone and green leaf volatiles by using gene silencing via RNA interference (RNAi) coupled antennal electrophysiological (EAG). Multiple sequence alignment showed that GmolOrco shared high sequence similarities with the Orco ortholog of lepidopterans. The results of real-time quantitative PCR detection demonstrated that GmolOrco was predominantly expressed in adult antennae and had the highest expression quantity in adult period among the different developmental stages. Compared with the noninjected controls, GmolOrco expression in GmolOrcodouble-stranded RNA (dsRNA)-injected males was reduced to 39.92% and that in females was reduced to 40.43%. EAG assays showed that the responses of GmolOrco-dsRNA injected males to sex pheromones (Z)-8-dodecenyl acetate (Z8-12:OAc) and (Z)-8-dodecenyl alcohol (Z8-12:OH) were significantly reduced, and the GmolOrco-dsRNA-injected female to green leaf volatile (Z)-3-hexenyl acetate also significantly declined. We inferred that Orco-mediated olfaction was different in male and female G. molesta adults and was mainly involved in recognizing the sex pheromones released by female moths.

Demography of Mythimna separata (Lepidoptera: Noctuidae) at outdoor fluctuating temperatures.

The oriental armyworm Mythimna separata (Walker) (Lepidoptera: Noctuidae) is a major migratory pest of cereal crops in East Asia, South Asia and Australia. To comprehensively understand the ecological tolerance of M. separata, we collected life table data of individuals from four consecutive generations reared under outdoor natural fluctuating temperatures from 15 April to 17 October 2018 in Yangling, Shaanxi, China. The results showed that the immature stage in early summer and summer were shorter than in spring and autumn. High mortality in late larval instar and pupal stages was observed in the summer generation. The adult pre-oviposition period in autumn was longer than the other seasons. The population in the earlier two seasons had heavier pupae and higher fecundity than the population in the latter two seasons. The intrinsic rate of increase and the finite rate of increase was the highest in early summer (r = 0.1292 day-1, λ = 1.1391 day-1), followed by spring (r = 0.1102 day-1, λ = 1.1165 day-1), and was the lowest in summer (r = 0.0281 day-1, λ = 1.0293 day-1). The results of this study would be useful to predict the population dynamics of M. separata and deepen our standing of the adaptiveness of this migratory pest in natural fluctuating ambient environments.

Molecular and functional characterization of three odorant binding proteins from the oriental fruit moth Grapholita molesta (Busck) (Lepidoptera: Tortricide).

Odorant binding proteins (OBPs) act in recognizing odor molecules and their most well-studied functions are transporting odors across the sensillum lymph to olfactory receptor neurons within the insect antennal sensillum. The adults of Grapholita molesta highly depend on olfactory cues in locating host plants and selecting oviposition sites, in which OBPs play an important role in perceiving and recognizing host plant volatiles. Exploring the physiological function of OBPs could facilitate our understanding of their importance in insects' chemical communication. In this study, three OBP genes were cloned and named GmolOBP4, GmolOBP5, and GmolOBP10. Quantitative real-time PCR results indicated that GmolOBP4 and GmolOBP10 were predominantly expressed in adult antennae and GmolOBP5 was expressed in multiple tissues, including head, legs, and wings in addition to antennae. The binding affinities of the three recombinant GmolOBPs (rGmolOBPs) with four sex pheromone components and twenty-nine host plant volatiles were measured using 1-N-Phenyl-naphthylamine as a fluorescence probe. The three rGmolOBPs exhibited specific binding properties to potential ligands, GmolOBP4 and GmolOBP10 bound to minor sex pheromone components, such as (Z)-8-dodecenyl alcohol and dodecanol, respectively. rGmolOBP4 showed intermediate binding ability with hexanal, benzyl alcohol, and pear ester, rGmolOBP5 had a weak affinity for benzaldehyde, pear ester and, methyl jasmonate, and rGmolOBP10 showed strong binding capacity toward hexanol, decanol, and α-ocimene. We speculate that the GmolOBP4 and GmolOBP10 have dual functions in perception and recognition of host plant volatiles and sex pheromone components, while GmolOBP5 may serve other function(s).

Functional disparity of four pheromone-binding proteins from the plum fruit moth Grapholita funebrana Treitscheke in detection of sex pheromone components.

Grapholita funebrana, also known as the plum fruit moth, is an oligophagous pest species that causes enormous economic losses of the fruits of Rosaceae. An eco-friendly method for the control of G. funebrana besides chemical control has not yet been developed. The sex pheromone communication system plays an important role in moth courtship and mating, in which pheromone-binding proteins (PBPs) are critical. In this research, we identified four PBPs, namely, GfunPBP1.1, GfunPBP1.2, GfunPBP2, and GfunPBP3, from the antennae of G. funebrana. The results of real-time quantitative PCR (RT-qPCR) showed that all four GfunPBPs were overwhelmingly expressed in the antennae and that GfunPBP1.2 and GfunPBP2 showed male-biased expression patterns, whereas GfunPBP1.1 and GfunPBP3 were equally expressed between sexes. The results of ligand-binding assays illustrated that although all four recombinant GfunPBPs (rGfunPBPs) had binding activity with the tested sex pheromone compounds, their preferred ligands were significantly different. rGfunPBP2 had the strongest binding affinity to Z8-12:Ac and Z8-12:OH; rGfunPBP1.1 preferred to bind Z8-14:Ac, Z10-14:Ac, and 12:OH more than to the other three GfunPBPs; and rGfunPBP1.2 exhibited stronger binding affinity to E8-12:Ac than to the other rGfunPBPs. Molecular docking results demonstrated that hydrophobic forces, especially van der Waals forces and hydrogen bonds, were the most important forces that maintained GfunPBP-pheromone ligand complexes. This study will improve our understanding of the sex pheromone recognition mechanisms of G. funebrana and promote the development of novel strategies for controlling G. funebrana.

'Candidatus Phytoplasma ziziphi' Changes the Metabolite Composition of Jujube Tree Leaves and Affects the Feeding Behavior of Its Insect Vector Hishimonus hamatus Kuoh.

Hishimonus hamatus Kuoh is a leafhopper species native to China that feeds on Chinese jujube leaves. This leafhopper species has been verified to transmit jujube witches' broom (JWB) disease, caused by phytoplasma, a fatal plant pathogen, which belongs to the phytoplasma subgroup 16SrV-B. The transmission of JWB phytoplasma largely relies on the feeding behavior of piercing-sucking leafhoppers. However, the specific mechanisms behind how and why the infection of JWB influences the feeding behavior of these leafhoppers are not fully understood. To address this, a study was conducted to compare the feeding patterns of H. hamatus when feeding JWB-infested jujube leaves to healthy leaves using the electrical penetration graph (EPG) technique. Then, a widely targeted metabolome analysis was performed to identify differences in the metabolite composition of JWB-infected jujube leaves and that of healthy jujube leaves. The results of EPG analyses revealed that when feeding on JWB-infected jujube leaves, H. hamatus exhibited an increased frequency of phloem ingestion and spent longer in the phloem feeding phase compared to when feeding on healthy leaves. In addition, the results of metabolomic analyses showed that JWB-infected leaves accumulated higher levels of small-molecular carbohydrates, free amino acids, and free fatty acids, as well as lower levels of lignans, coumarins and triterpenoids compared to healthy leaves. The above results indicated that the H. hamatus preferentially fed on the phloem of infected leaves, which seems to be linked to the transmission of the JWB phytoplasma. The results of metabolomic analyses partially imply that the chemical compounds might play a role in making the infected leaves more attractive to H. hamatus for feeding.

Different Binding Affinities of Three General Odorant-Binding Proteins in Grapholita funebrana (Treitscheke) (Lepidoptera: Tortricidae) to Sex Pheromones, Host Plant Volatiles, and Insecticides.

Insect general odorant-binding proteins (GOBPs) play irreplaceable roles in filtering, binding, and transporting host odorants to olfactory receptors. Grapholita funebrana (Treitscheke) (Lepidoptera: Tortricidae), an economically important pest of fruit crops, uses fruit volatiles as cues to locate host plants. However, the functions of GOBPs in G. funebrana are still unknown. Three GOBP genes, namely, GfunGOBP1, GfunGOBP2, and GfunGOBP3, were cloned, and their expression profiles in different tissues were detected by the method of real-time quantitative PCR (RT-qPCR). The binding properties of recombinant GfunGOBPs (rGfunGOBPs) to various ligands were investigated via fluorescence binding assays. The three GfunGOBPs were mainly expressed in the antennae of both male and female moths. All these three rGfunGOBPs could bind to sex pheromones, while having varying affinities toward these pheromones. The three rGfunGOBPs also displayed a wide range of ligand-binding spectrums with tested host odorants. The rGfunGOBP1, rGfunGOBP2, and rGfunGOBP3 bound to 34, 33, and 30 out of the 41 tested odorants, respectively. Three rGfunGOBPs had overlapping binding activities to ß-myrcene, (-)-α-phellandrene, and ethyl isovalerate with the Ki less than 3.0 µM. The rGfunGOBP1 and rGfunGOBP3 could selectively bind to several insecticides, whereas rGfunGOBP2 could not. Three rGfunGOBPs had the dual functions of selectively binding to sex pheromones and host odorants. Moreover, the rGfunGOBP1 and rGfunGOBP3 can also serve as 'signal proteins' and bind to different insecticides. This study contributed to elucidating the potential molecular mechanism of the olfaction for G. funebrana, and thereby promotes the development of effective botanical attractants or pheromone synergists to control G. funebrana.

Microsatellite-based analysis of genetic structure and gene flow of Mythimna separata (Walker) (Lepidoptera: Noctuidae) in China.

The oriental armyworm, Mythimna separata, is a serious agricultural pest in China. Seasonal and roundtrip migration has recently led to sudden, localized outbreaks and crop losses. To evaluate genetic differentiation between populations in eastern and western China and elucidate gene flow, the genetic structure of 20 natural populations from nine provinces was examined using seven microsatellite markers. The results indicated high genetic diversity. However, little to moderate (0 < F ST < 0.15) genetic differentiation was detected, and there was no correlation between genetic distance and geographical distance. Bayesian clustering analysis identified three groups whereas discriminant analysis of principal components identified ten clusters that were considered as two clear-cut clusters and one admixed group. Gene flow occurred frequently in most population pairs, and an asymmetrical migration rate was detected in several pairwise population comparisons. The bottleneck test showed that few populations had experienced recent bottlenecks. Correspondingly, large-scale and long-distance migration of M. separata has caused low genetic differentiation and frequent gene exchange. Our findings are important for studying genetic evolution and help to improve predictions of M. separata outbreaks in China.

Morphology and distribution of antennal sensilla of female Phyllotreta striolata (Fabricius) (Coleoptera: Chrysomelidae).

Phyllotreta striolata (Fabricius) is an important pest of Brassicaceae in Southeast Asia and North America. Using scanning electron microscopy, we observed the external structure, number, and distribution of the antennal sensilla in P. striolata females to discuss the putative function of these sensilla in host location and oviposition behaviors. The antenna of female P. striolata is filiform, composed of a scape, a pedicel, and a flagellum with 9 flagellomeres. Five types of sensilla were identified, including sensilla cheaetica, sensilla trichodea, Böhm bristles, sensilla auricillica, and sensilla basiconica (five subtypes, SB1-SB2). External structure and distribution of antennal sensilla are compared with data from other insect species. In addition, we discuss the possible functions of antennal sensilla based on their characteristics.