Structural and functional analysis of aquaporins in Bombus terrestris.

Bombus terrestris are efficient pollinators in forestry and agriculture, with higher cold tolerance than other bees. Yet, their cold tolerance mechanism remains unclear. Aquaporins (AQPs) function as cell membrane proteins facilitating rapid water flow, aiding in osmoregulation. Recent studies highlight the importance of insect AQPs in dehydration and cold stress. Comparative transcriptome analysis of B. terrestris under cold stress revealed up-regulation of four AQPs, indicating their potential role in cold tolerance. Seven AQPs-Eglp1, Eglp2, Eglp3, DRIP, PRIP, Bib, and AQP12L-have been identified in B. terrestris. These are widely expressed in various tissues, particularly in the alimentary canal and Malpighian tubules. Functional analysis of BterAQPs in the Xenopus laevis oocytes expressing system showed distinct water and glycerol selectivity, with BterDrip exhibiting the highest water permeability. Molecular modeling of BterDrip revealed six transmembrane domains, two NPA motifs, and an ar/R constriction region (Phe131, His256, Ser265, and Arg271), likely contributing to its water selectivity. Silencing BterDRIP accelerated mortality in B. terrestris under cold stress, highlighting the crucial role of BterDRIP in their cold tolerance and providing a molecular mechanism for their cold adaptation.

Molecular Basis of CO2 Sensing in Hyphantria cunea.

Carbon dioxide (CO2) released by plants can serve as a cue for regulating insect behaviors. Hyphantria cunea is a widely distributed forestry pest that may use CO2 as a cue for foraging and oviposition. However, the molecular mechanism underlying its ability to sense CO2 has not been elucidated. Our initial study showed that CO2 is significantly attractive to H. cunea adults. Subsequently, 44 H. cunea gustatory receptors (GRs) were identified using transcriptome data, and 3 candidate CO2 receptors that are specifically expressed in the labial palps were identified. In vivo electrophysiological assays revealed that the labial palp is the primary organ for CO2 perception in H. cunea, which is similar to findings in other lepidopteran species. By using the Xenopus oocyte expression system, we showed that the HcunGR1 and HcunGR3 co-expressions produced a robust response to CO2, but HcunGR2 had an inhibitory effect on CO2 perception. Finally, immunohistochemical staining revealed sexual dimorphism in the CO2-sensitive labial pit organ glomerulus (LPOG). Taken together, our results clarified the mechanism by which H. cunea sense CO2, laying the foundation for further investigations into the role of CO2 in the rapid spread of H. cunea.

Rational Design of Triazinone Derivatives with Low Bee Toxicity Based on the Binding Mechanism of Neonicotinoids to Apis mellifera.

Bees, one of the most vital pollinators in the ecosystem and agriculture, are currently threatened by neonicotinoids. To explore the molecular mechanisms of neonicotinoid toxicity to bees, the different binding modes of imidacloprid, thiacloprid, and flupyradifurone with nicotinic acetylcholine receptor (nAChR) α1ß1 and cytochrome P450 9Q3 (CYP9Q3) were studied using homology modeling and molecular dynamics simulations. These mechanisms provided a basis for the design of compounds with a potential low bee toxicity. Consequently, we designed and synthesized a series of triazinone derivatives and assessed their bioassays. Among them, compound 5a not only displayed substantially insecticidal activities against Aphis glycines (LC50 = 4.40 mg/L) and Myzus persicae (LC50 = 6.44 mg/L) but also had low toxicity to Apis mellifera. Two-electrode voltage clamp recordings further confirmed that compound 5a interacted with the M. persicae nAChR α1 subunit but not with the A. mellifera nAChR α1 subunit. This work provides a paradigm for applying molecular toxic mechanisms to the design of compounds with low bee toxicity, thereby aiding the future rational design of eco-friendly nicotinic insecticides.

Odorant Receptors Expressing and Antennal Lobes Architecture Are Linked to Caste Dimorphism in Asian Honeybee, Apis cerana (Hymenoptera: Apidae).

Insects heavily rely on the olfactory system for food, mating, and predator evasion. However, the caste-related olfactory differences in Apis cerana, a eusocial insect, remain unclear. To explore the peripheral and primary center of the olfactory system link to the caste dimorphism in A. cerana, transcriptome and immunohistochemistry studies on the odorant receptors (ORs) and architecture of antennal lobes (ALs) were performed on different castes. Through transcriptomesis, we found more olfactory receptor genes in queens and workers than in drones, which were further validated by RT-qPCR, indicating caste dimorphism. Meanwhile, ALs structure, including volume, surface area, and the number of glomeruli, demonstrated a close association with caste dimorphism. Particularly, drones had more macroglomeruli possibly for pheromone recognition. Interestingly, we found that the number of ORs and glomeruli ratio was nearly 1:1. Also, the ORs expression distribution pattern was very similar to the distribution of glomeruli volume. Our results suggest the existence of concurrent plasticity in both the peripheral olfactory system and ALs among different castes of A. cerana, highlighting the role of the olfactory system in labor division in insects.

The dual coding of a single sex pheromone receptor in Asian honeybee Apis cerana.

In Asian honeybees, virgin queens typically only mate during a single nuptial flight before founding a colony. This behavior is controlled by the queen-released mandibular pheromone (QMP). 9-oxo-(E)-2-decenoic acid (9-ODA), a key QMP component, acts as sex pheromone and attracts drones. However, how the queens prevent additional mating remains elusive. Here, we show that the secondary QMP component methyl p-hydroxybenzoate (HOB) released by mated queens inhibits male attraction to 9-ODA. Results from electrophysiology and in situ hybridization assay indicated that HOB alone significantly reduces the spontaneous spike activity of 9-ODA-sensitive neurons, and AcerOr11 is specifically expressed in sensilla placodea from the drone's antennae, which are the sensilla that narrowly respond to both 9-ODA and HOB. Deorphanization of AcerOr11 in Xenopus oocyte system showed 9-ODA induces robust inward (regular) currents, while HOB induces inverse currents in a dose-dependent manner. This suggests that HOB potentially acts as an inverse agonist against AcerOr11.

Circabidian rhythm of sex pheromone reception in a scarab beetle.

Animals have endogenous clocks that regulate their behavior and physiology. These clocks rely on environmental cues (time givers) that appear approximately every 24 h due to the Earth's rotation; thus, most insects exhibit a circadian rhythm. One notable exception is the scarab beetle, Holotrichia parallela, a severe agricultural pest in China, Japan, South Korea, and India. Females emerge from the soil every other night, reach the canopy of host plants, evert an abdominal gland, and release a pheromone bouquet comprising l-isoleucine methyl ester (LIME) and l-linalool. To determine whether this circa'bi'dian rhythm affects the olfactory system, we aimed to identify H. parallela sex pheromone receptor(s) and study their expression patterns. We cloned 14 odorant receptors (ORs) and attempted de-orphanizing them in the Xenopus oocyte recording system. HparOR14 gave robust responses to LIME and smaller responses to l-linalool. Structural modeling, tissue expression profile, and RNAi treatment followed by physiological and behavioral studies support that HparOR14 is a sex pheromone receptor-the first of its kind discovered in Coleoptera. Examination of the HparOR14 transcript levels throughout the adult's life showed that on sexually active days, gene expression was significantly higher in the scotophase than in the photophase. Additionally, the HparOR14 expression profile showed a circabidian rhythm synchronized with the previously identified pattern of sex pheromone emission. 48 h of electroantennogram recordings showed that responses to LIME were abolished on non-calling nights. In contrast, responses to the green leaf volatile (Z)-3-henexyl acetate remained almost constant throughout the recording period.

Discovery of piperonyl-tethered sulfoximines as novel low bee-toxicity aphicides targeting Amelα1/ratß2 complex.

Nicotinic acetylcholine receptor (nAChR) is recognized as a significant insecticide target for neonicotinoids and some agonists. In this study, the nAChR α1 subunit from Apis mellifera was first found to be narrowly tuned to different bee toxicity insecticides, namely, sulfoxaflor (SFX) and flupyradifurone (FPF). Hence, novel sulfoximine derivatives 7a-h were rationally designed and synthesized by introducing a benzo[d][1,3]dioxole moiety into a unique sulfoximine skeleton based on the binding cavity characteristics of Amelα1/ratß2. The two electrode voltage clamp responses of 7a-h were obviously lower than that of SFX, indicating their potentially low bee toxicity. Besides, representative compounds 7b and 7g exhibited low bee toxicity (LD50 > 11.0 µg/bee at 48 h) revealed by acute contact toxicity bioassays. Molecular modelling results indicated that Ile152, Ala151, and Val160 from honeybee subunit Amelα1 and Lys144 and Trp80 from aphid subunit Mpα1 may be crucial for bee toxicity and aphicidal activity, respectively. These results clarify the toxic mechanism of agonist insecticides on nontargeted pollinators and reveal novel scaffold sulfoximine aphicidal candidates with low bee toxicity. These results will provide a new perspective on the rational design and highly effective development of novel eco-friendly insecticides based on the structure of the nAChR subunit.

The evolution of borneol repellency in culicine mosquitoes.

Insects have developed remarkable adaptations to effectively interact with plant secondary metabolites and utilize them as cues to identify suitable hosts. Consequently, humans have used aromatic plants for centuries to repel mosquitoes. The repellent effects of plant volatile compounds are mediated through olfactory structures present in the antennae, and maxillary palps of mosquitoes. Mosquito maxillary palps contain capitate-peg sensilla, which house three olfactory sensory neurons, of which two are mainly tuned to either carbon dioxide or octenol - two animal host odorants. However, the third neuron, which expresses the OR49 receptor, has remained without a known ecologically-relevant odorant since its initial discovery. In this study, we used odorant mixtures and terpenoid-rich Cannabis essential oils to investigate the activation of OR49. Our results demonstrate that two monoterpenoids, borneol and camphor, selectively activate OR49, and OR9-expressing neurons, as well as the MD3 glomerulus in the antennal lobe. We confirm that borneol repels female mosquitoes, and knocking out the gene encoding the OR49 receptor suppresses the response of the corresponding olfactory sensory neuron. Importantly, this molecular mechanism of action is conserved across culicine mosquito species, underscoring its significance in their olfactory systems.

Essential role for SNMP1 in detection of sex pheromones in Helicoverpa armigera.

The sensory neuron membrane protein, SNMP1, was initially discovered in moths and is associated with sex pheromone sensitive neurons, suggesting a role in the detection of these semiochemicals. Although DrosophilaSNMP1 has been reported to be involved in detecting of the sex pheromone cis-vaccenyl acetate (cVA), the role of this protein in moths in vivo is still largely unexplored. In this study we developed a SNMP1-/- homozygous mutant line of Helicoverpa armigera using CRISPR/Cas9. Wind-tunnel behavioral experiments showed that HarmSNMP1-/- males could not be attracted by sex pheromones (Z11-16:Ald/Z9-16:Ald = 97/3), while mating behavior obvervations revealed that the SNMP1 mutant males didn't react much to calling females and the rate of copulation was significantly decreased. The electrophysiological results indicated that HarmSNMP1 contributes to the detection of 16-carbon liner sex pheromones, (Z)-11-hexadecenal (Z11-16:Ald), (Z)-9-hexadecenal (Z9-16:Ald), (Z)-11-hexadecanol (Z11-16:OH) and 16-carbon acetate (Z)-11-hexadecenyl acetate (Z11-16:OAc), but is not required for detecting the 14-carbon sex pheromone component (Z)-9-tetradecenal (Z9-14:Ald) an analogue of Z11-16:Ald, (Z)-9-tetradecen-1-yl formate (Z9-14:OFor), which can activate the Z11-16:Ald-responsive neuron. Taken together, our studies indicated that HarmSNMP1 has an important role in the detection of long-chain sex pheromones, but is not essential for detecting shorter chain sex pheromone in vivo.

Genome of the webworm Hyphantria cunea unveils genetic adaptations supporting its rapid invasion and spread.

BACKGROUND: The fall webworm Hyphantria cunea is an invasive and polyphagous defoliator pest that feeds on nearly any type of deciduous tree worldwide. The silk web of H. cunea aids its aggregating behavior, provides thermal regulation and is regarded as one of causes for its rapid spread. In addition, both chemosensory and detoxification genes are vital for host adaptation in insects. RESULTS: Here, a high-quality genome of H. cunea was obtained. Silk-web-related genes were identified from the genome, and successful silencing of the silk protein gene HcunFib-H resulted in a significant decrease in silk web shelter production. The CAFE analysis showed that some chemosensory and detoxification gene families, such as CSPs, CCEs, GSTs and UGTs, were expanded. A transcriptome analysis using the newly sequenced H. cunea genome showed that most chemosensory genes were specifically expressed in the antennae, while most detoxification genes were highly expressed during the feeding peak. Moreover, we found that many nutrient-related genes and one detoxification gene, HcunP450 (CYP306A1), were under significant positive selection, suggesting a crucial role of these genes in host adaptation in H. cunea. At the metagenomic level, several microbial communities in H. cunea gut and their metabolic pathways might be beneficial to H. cunea for nutrient metabolism and detoxification, and might also contribute to its host adaptation. CONCLUSIONS: These findings explain the host and environmental adaptations of H. cunea at the genetic level and provide partial evidence for the cause of its rapid invasion and potential gene targets for innovative pest management strategies.

Molecular Characterization and Key Binding Sites of Sex Pheromone-Binding Proteins from the Meadow Moth, Loxostege sticticalis.

The meadow moth, Loxostege sticticalis, is a typical agricultural pest that uses sex pheromones to mediate mating behavior; however, the mechanism underlying the selectivity of its pheromone-binding proteins (PBPs) remains unknown. In this study, LstiPBP1 and LstiPBP3 were cloned, expressed, and purified, and the fluorescence binding assay showed that LstiPBP1 binds to the major sex pheromone component, E-11-tetradecenol (E11-14:OH), with high affinity; moreover, E11-14:OH could evoke a significant antennal electrophysiological response and attract L. sticticalis males. After LstiPBP1 was silenced, both the antennal response and attractiveness of E11-14:OH decreased significantly. Molecular docking predicted that a hydrogen bonding site, Leu37, played key role in the binding of LstiPBP1 to E11-14:OH. After Leu37 was mutated, the E11-14:OH-binding affinity decreased drastically. These results suggest that LstiPBP1 participates in E11-14:OH recognition and could be used as a target gene to disturb the mating behavior of L. sticticalis and develop new odorants for pest control.

A herbivore-induced plant volatile of the host plant acts as a collective foraging signal to the larvae of the meadow moth, Loxostege sticticalis (Lepidoptera: Pyralidae).

The meadow moth Loxostege sticticalis is a serious agricultural pest that feeds on the leaves of many economic crops, such as sugar beet, soybean, sunflower, and potato. In addition to the rapid migration of adult moths, the collective foraging behavior of the larvae is also thought to be involved in the search for new food sources and substantially contributes to the expansion of the infested area. However, whether and how the chemical signals take part in this process remains unknown. In this study, two larva-specific expressed odorants, LstiOR5 and LstiOR6, were successfully cloned and deophanized. A heterologous study on Xenopus laevis oocytes showed that several host plant volatiles could evoke LstiOR responses in a dose-dependent manner. One herbivore-induced plant volatile (HIPV) of soybean leaves, methyl salicylate (MeSA), exerted attractive effects on the L. sticticalis larvae at all tested concentrations. Further foraging choice assays showed that the L. sticticalis larvae preferred foraged soybean leaves over unforaged leaves. When MeSA was artificially added to unforaged leaves, the unforaged leaves were preferred over the foraged leaves. In addition, GC-MS analysis demonstrated that MeSA was induced by the foraging behavior of the larvae and acted as a collective food signal in L. sticticalis. Moreover, in situ hybridization showed that LstiOR5 was highly expressed in larval antenna neurons. When LstiOR5 was silenced, both the electrophysiological response of the antenna to MeSA and the preference for foraged leaves were significantly decreased, suggesting that LstiOR5 is involved in the collective foraging behavior of L. sticticalis. Our results clarified the chemical signals that trigger the collective foraging behavior of L. sticticalis and provided more evidence for the molecular mechanism underlying the expansions of their infested areas at a peripheral olfactory sensing level. These findings could facilitate the development of potential control strategies for controlling this pest and provide a potential gene target that correlates with the collective foraging behavior of L. sticticalis, which might lead to better pest management.

Involvement of heat shock protein 40 in the wing dimorphism of the house cricket Acheta domesticus.

Wing dimorphism is a common phenomenon in a wide range of insect taxa. In most insects, the two morphs are macropterous and micropterous, in extreme cases of the latter, wing shedding can occur. Wing dimorphism contributes significantly to the ecological success of many insect species. However, the molecular basis of wing dimorphism is not fully understood, especially for wing-shed. Here, differentially expressed genes over eight developmental stages of the house cricket Acheta domesticus, which undergoes wing-shed dimorphism, were studied. The results revealed a wing-shed peak during adult development in which many DEGs were highly upregulated and it's influenced by cricket population density. A weighted correlation network analysis (WGCNA) grouped 21,922 DEGs among 141,456 unigenes into 18 modules of different expression patterns. The module in which the gene expression pattern correlated with the wing-shed phenotypic data was selected for further analyses with STEM and Cytoscape, and three candidate genes (AdomHSP40: Heat shock protein 40, AdomCFDP: Craniofacial development protein, AdomDIS3L: DIS3 Like 3'-5' Exoribonuclease) were identified by gene network analysis as the DEGs most relevant to wing-shed occurrence. The RNA interference of these genes together with an insulin receptor and Nylanderia fulva virus showed that the silencing of AdomHSP40 significantly decreased wing-shed occurrence, whereas the silencing of other candidate genes did not, suggesting that AdomHSP40 plays a crucial role in the wing-shed of Acheta domesticus. These findings provide insights into the molecular mechanisms underlying wing dimorphism in the house crickets, which differ from those found in other insects such as the planthopper.

Phylogeny and acoustic signal evolution of a pure tone song katydid Pseudophyllus titan (Orthoptera: Tettigoniidae) based on the complete mitogenome.

Ensifera is an extremely diverse group in the order Orthoptera, and it has been the subject of considerable research, especially in terms of phylogeny and bioacoustics. Katydids are known for using high-frequency broadband signals whereas most cricket songs are pure tones with a frequency range of 2-8 kHz. Pseudophyllus titan is a special katydid emits low-frequency pure tone songs similar to that of crickets. Acoustic communication of Ensifera were widely studied, but song evolution could deserve new examination taking into account the newly available phylogenies. To examine the song evolution of P. titan in the Ensifera, the mitogenomes of P. titan and three other ensiferan species (Sphagniana ussuriana, Oecanthus sinensis, Truljalia hibinonis) were obtained by high-throughput sequencing. In addition, the phylogeny of Ensifera was reconstructed including 63 in-group taxa, and the divergence time was further estimated for major ensiferan lineages. Lastly, the evolutions of song frequency were evaluated based on an ancestral character state reconstruction (ACSR). Moreover, we have studied the calling songs of 78 katydids and performed ACSR based on five genes. All phylogeny analyses confirmed that the superfamily Schizodactyloidea was placed basally to the non-grylloid clade. P. titan was in the Pseudophyllinae clade, and this clade had a distant relationship form other lineages in Phaneropteridae. The ACSR showed that P. titan evolved low-frequency pure tone songs independently after ancestors of katydids evolved high-frequency broadband noise. Referring to the geologic time scale, we suggest that the song frequency evolution in the Ensifera to some extent caused by adaptations to the changing habitat under conditions of climate change and plant evolution.

Identification and evolution of olfactory genes in the small poplar longhorn beetle Saperda populnea.

Saperda populnea is a serious pest of poplar and willow trees in the Palaearctic region, causing extensive damage to forests and the lumber industry. Until recently, there is no safe and effective chemical method to control this pest due to the lack of sufficient knowledge on the molecular basis of its olfactory genes, moreover, the evolutionary history of the olfactory gene family in subfamily Lamiinae is still fully unknown. Our RNA sequencing of the antennae of S. populnea identified 43 odorant binding proteins (OBPs), 15 chemosensory proteins (CSPs), 56 odorant receptors (ORs) and 24 inotropic receptors (IRs) in S. populnea. The RT-PCR results showed several genes were expressed in a sex specific manner, suggesting that these genes might play key role in their olfactory-sensing and sex-related behaviors. Further evolutionary studies were performed on these olfactory genes, overall comparison of the Ka/Ks values of orthologous genes in S. populnea and two other Lamiinae species showed three main conclusions: 1. olfactory genes have evolved more rapidly than the non-olfactory genes in the tested long horn beetles; 2. the IR gene family are under a strong purifying selection; 3. the OBPs of Monochamus alternatus evolved more rapidly than the other two species, which is speculated to be correlated with differentiation of selective pressure in different geographic origins. Detailed evolutionary studies on each olfactory genes showed that several OBPs and ORs are under significantly purifying/relaxed selective pressure, and several positive selection sites were also detected, modeling of SpopOR14 and SpopOBP4/5 showed that most of the positive selection sites were distributed at the N-terminus of SpopOR14, while the positive selection sites in SpopOBP4/5 were located in H-bond donors, results suggest that these sites are more likely to be linked with the selectivity of modeled olfactory genes. The research provided a better understanding of the molecular basis and evolutionary history of the olfactory genes in Lamiinae, through elaborating the mechanism whereby amino structural evolution affects specific variants in OBPs and ORs.

Modeling of methane formation in gravity sewer system: the impact of microorganism and hydraulic condition.

Sewer system is an important source of methane formation and emission. Although some models were developed to predict methane production in sewers, the impact of microorganism amount was indicated indirectly. Here, seven laboratory scale sewers with varied wall-shear stresses were established. The biofilm thickness, microorganism amount, DO distribution, microorganism community in the biofilms and methane production in the sewers were measured. Based on experimental data, an empirical model was developed to directly describe the relationship between methane production, microorganism amount and wall-shear stress. The results showed that DO concentration decreased significantly along the biofilm depth under varied wall-shear stress, and the DO reduction rate was positively related to the intensity of wall-shear stress. The dominant archaea species in mature biofilms were similar whereas the proportions showed remarkable differences. The abundance of Methanospirillum in biofilms cultured at 2.0 Pa wall-shear stress was 53.08% more than that at 1.29 Pa. The maximum methane production rate, 2.04 mg/L wastewater day, was obtained when the wall-shear stress kept at 1.45 Pa, which was 1.2-fold higher than the minimum in sewer at 0.5 Pa. The R2 value of the established model was 0.95, the difference between the measurement and simulation was in the rage of 1.5-13.0%.

Suitable flow pattern increases the removal efficiency of nitrogen in gravity sewers: a suitable anoxic and aerobic environment in biofilms.

The sewers have the function of carbon removal, which has been proven. But if the effect of nitrogen removal can be enhanced at the same time of carbon removal, it can lay a foundation for the realization of "sewer's working as a reactor." This paper investigated the effects of shear stress and C/N ratio on nitrogen removal through biofilms on the sewer inner wall and nitrogen transfer. The main conclusions are as follows: (1) nitrogen could be partially removed in sewers after a series of reactions; (2) the anaerobic, anoxic, aerobic environment and some bacteria related to nitrogen metabolism, which exist in the biofilm, promote the nitrification and denitrification; (3) a total of 722 functional genes involved in nitrogen metabolism were detected in the biofilm (C/N ratio of 10, shear stress of 1.4 Pa), accounting for 0.67% of all genes, and the functional genes related to denitrification were dominant. Graphical abstract ᅟ.

Selectivity and ligand-based molecular modeling of an odorant-binding protein from the leaf beetle Ambrostoma quadriimpressum (Coleoptera: Chrysomelidae) in relation to habitat-related volatiles.

In this study, the most abundant and antenna-specific odorant-binding protein (OBP) of the elm pest A. quadriimpressum, AquaOBP4, was expressed and purified. The selectivity of AquaOBP4 was investigated by screening against a panel of 40 habitat-relevant compounds. Based on the obtained results, a homologous model of AquaOBP4 was established. This model indicated that AquaOBP4 is highly homologous to DmelOBP LUSH and includes two main binding sites. A docking analysis showed that four of five active ligands bound at Site 1, whereas the other ligand was situated at Site 2. Furthermore, new ligands were docked in the model, and the results of fluorescence-based binding assays of these compounds were highly consistent with the binding conformation and binding affinity predicted by our model. Additionally, three binding odorants derived from elm leaves elicited a strong electroantennogram response and exerted a significant attractive effect on adult A. quadriimpressum. All of the results showed that AquaOBP4 is likely linked to the foraging behavior of A. quadriimpressum. This study provides a new reliable tool for future large-scale compound screenings and revealed several functional chemicals that might aid the development of a better pest management approach for A. quadriimpressum.

Molecular basis of peripheral olfactory sensing during oviposition in the behavior of the parasitic wasp Anastatus japonicus.

Anastatus japonicus is a parasitic wasp and natural enemy of the litchi pest Tessaratoma papillosa, and for decades in China, A. japonicus has been mass-reared inside the eggs of Antheraea pernyi to control T. papillosa. A series of experiments was performed to explore the olfactory mechanism underlying the oviposition behavior of A. japonicus. First, a transcriptomic analysis was performed on the antennae of A. japonicus, and the resulting assemblies led to the generation of 70,473 unigenes. Subsequently, 21,368 unigenes were matched to known proteins, 48 odorant receptors (ORs) (including Orco) and 13 antennal ionotropic receptors (IRs) (including the co-receptors IR8a and IR25a) were identified and predicted to form complete open reading frames (ORFs). The FPKM (fragments per Kb per million reads) values and RT-PCR results showed that AjapOrco, AjapOR10, AjapOR27, AjapOR33 and AjapOR35 were either highly abundant or expressed specifically in the olfactory organs. Furthermore, AjapOrco silencing resulted in a significant decrease in both the parasitism rate and the host-seeking time of A. japonicus, whereas dsRNA injection showed that IR8a and IR25a did not produce significant behavioral changes, suggesting that the oviposition behavior of A. japonicus is more reliant on OR-based pathways than IR-based pathways. Our previous GC-MS data derived twenty-nine compounds which were abundent from these host plants and host insects. We performed electrophysiological and oviposition assays on A. japonicus, and eight odorants were found to elicit a significant electroantennogram (EAG) response. Among these odorants, ß-Caryophyllene, Undecane, (E)-α-Farnesene (+)-Aromadendrene and Cis-3-Hexen-ol had strong attractant effects on oviposition, whereas 2-Ethyl-1-Hexan-ol, Ethyl Acetate and α-Caryophyllene had a strong repellant effects. Thus, these chemicals might influence oviposition guidance/repulsion behavior in A. japonicus. To further explore the target ORs that are tuned to the functional odorants, the nine candidate ORs described above were silenced by RNA interference, and the results showed that a large decrease in the EAG response of all the tested functional odorants in the AjapOrco-silencing group. In addition, the AjapOR35-silencing group showed a significant decrease in the EAG response to ß-Caryophyllene and (E)-α-Farnesene, indicating that AjapOR35 is tuned to these two oviposition attractants ß-Caryophyllene and (E)-α-Farnesene. Further binary-choice oviposition assays showed that the oviposition attractant effect of ß-Caryophyllene and (E)-α-Farnesene vanished after AjapOR35 was silenced, indicating that the emission of these attractants from host plants can guide A. japonicus to locate eggs for ovipositioning and indicated that AjapOR35 is correlated with the olfactory detection oviposition behavior of this species. This study provides a better understanding of the molecular basis and functional chemicals underlying the oviposition behavior of A. japonicus, and the results may help improve biocontrol approaches.

Micromorphological differentiation of left and right stridulatory apparatus in crickets (Orthoptera: Gryllidae).

The present study compared micromorphological differentiation of stridulatory apparatus between the functional right tegmen and non-functional left tegmen, analyzed under scanning electron microscope (SEM), among eight Gryllidae species. The results showed that the main differences were found on the length and shape of files and teeth. The length of stridulatory file and teeth number were lower on the left stridulatory apparatus than that on right stridulatory apparatus in all species. However, the ratio between the length of stridulatory teeth and the interval of stridulatory teeth was significantly higher on the left stridulatory apparatus than that on right stridulatory apparatus in Dianemobius fascipes, Polionemobius taprobanensis, Pteronemobius gifuensis, Teleogryllus occipitalis and Oecanthus longicauda. In addition, the length of stridulatory teeth was positively related to number of stridulatory teeth, however, the interval of stridulatory teeth was negatively related to the ratio between the length of stridulatory teeth and the interval of stridulatory teeth for left and right stridulatory apparatus. Our result illustrated that the length of left and right stridulatory file and teeth length could be an effective character to distinguish species. Left stridulatory apparatus was not entirely degraded than right stridulatory apparatus.