Prediction of Potential Distribution of Carposina coreana in China under the Current and Future Climate Change.

Carposina coreana is an important pest of Cornus officinalis, distributed in China, Korea, and Japan. In recent years, its damage to C. officinalis has become increasingly serious, causing enormous economic losses in China. This study and prediction of current and future suitable habitats for C. coreana in China can provide an important reference for the monitoring, early warning, prevention, and control of the pest. In this study, the potential distributions of C. coreana in China under current climate and future climate models were predicted using the maximum entropy (MaxEnt) model with ArcGIS software. The distribution point data of C. coreana were screened using the buffer screening method. Nineteen environmental variables were screened using the knife-cut method and variable correlation analysis. The parameters of the MaxEnt model were optimized using the kuenm package in R software. The MaxEnt model, combined with key environmental variables, was used to predict the distribution range of the suitable area for C. coreana under the current (1971-2000) and four future scenarios. The buffer screening method screened data from 41 distribution points that could be used for modeling. The main factors affecting the distribution of C. coreana were precipitation in the driest month (Bio14), precipitation in the warmest quarter (Bio18), precipitation in the coldest quarter (Bio19), the standard deviation of seasonal variation of temperature (Bio4), minimum temperature in the coldest month (Bio6), and average temperature in the coldest quarter (Bio11). The feature class (FC) after the kuenm package optimization was a Q-quadratic T-threshold combination, and the regularization multiplier (RM) was 0.8. The suitable areas for C. coreana under the current climate model were mainly distributed in central China, and the highly suitable areas were distributed in southern Shaanxi, southwestern Henan, and northwestern Hubei. The lowest temperature in the coldest month (Bio6), the average temperature in the coldest quarter (Bio11), and the precipitation in the warmest quarter (Bio18) all had good predictive ability. In future climate scenarios, the boundary of the suitable area for C. coreana in China is expected to shift northward, and thus, most of the future climate scenarios would shift northward.

Selection and Validation of Reference Genes for Gene Expression in Bactericera gobica Loginova under Different Insecticide Stresses.

Insecticide resistance has long been a problem in crop pest control. Bactericera gobica is a major pest on the well-known medicinal plants Lycium barbarum L. Investigating insecticide resistance mechanisms of B. gobica will help to identify pesticide reduction strategies to control the pest. Gene expression normalization by RT-qPCR requires the selection and validation of appropriate reference genes (RGs). Here, 15 candidate RGs were selected from transcriptome data of B. gobica. Their expression stability was evaluated with five algorithms (Delta Ct, GeNorm, Normfinder, BestKeeper and RefFinder) for sample types differing in response to five insecticide stresses and in four other experimental conditions. Our results indicated that the RGs RPL10 + RPS15 for Imidacloprid and Abamectin; RPL10 + AK for Thiamethoxam; RPL32 + RPL10 for λ-cyhalothrin; RPL10 + RPL8 for Matrine; and EF2 + RPL32 under different insecticide stresses were the most suitable RGs for RT-qPCR normalization. EF1α + RPL8, EF1α + ß-actin, ß-actin + EF2 and ß-actin + RPS15 were the optimal combination of RGs under odor stimulation, temperature, developmental stages and both sexes, respectively. Overall, EF2 and RPL8 were the two most stable RGs in all conditions, while α-TUB and RPL32 were the least stable RGs. The corresponding suitable RGs and one unstable RG were used to normalize a target cytochrome P450 CYP6a1 gene between adult and nymph stages and under imidacloprid stress. The results of CYP6a1 expression were consistent with transcriptome data. This study is the first research on the most stable RG selection in B. gobica nymphs exposed to different insecticides, which will contribute to further research on insecticide resistance mechanisms in B. gobica.

The Odorant-Binding Protein 1 Mediates the Foraging Behavior of Grapholita molesta Larvae.

Since eggs are laid directly on fruit skin, it is typically believed that food odor has little impact on the foraging of Grapholita molesta larvae. It is crucial to note that larvae that hatch on twigs and leaves could need some sort of identification system when foraging. Here, 22 GmolOBP genes were identified from the G. molesta larval transcriptome via the comparison of conserved domain and homology in the protein level. GmolOBP1 had strong affinities for important pear-fruit volatiles, which caused larvae strong behavioral responses. However, after GmolOBP1 silencing, the larvae lost their attraction to methyl salicylate, α-farnesene, butyl acetate, ethyl butanoate, and ethyl hexanoate, and the effects of larvae seeking various pears were significantly reduced. Consequently, GmolOBP1 was required for the reception of pear volatiles and was involved in mediating how G. molesta larvae foraged. Our research revealed the GmolOBP1 foraging signal recognition mechanism as well as potential molecular targets for field pest management.

Feeding-induced plant metabolite responses to a phoretic gall mite, its carrier psyllid and both, after detachment.

Phoresy is one of the most distinctive relationships between mites and insects, and the off-host interaction between phoretic mites and their carriers is the most critical factor sustaining the phoretic association. As phoretic associations commonly occur in temporary habitats, little is known about off-host interactions between phoronts and carriers. However, an off-host interaction has been reported, in which the plant-mediated competition between a phoretic gall mite, Aceria pallida, and its psyllid vector, Bactericera gobica, after detachment decreases leaf abscission caused by B. gobica and then directly facilitates their phoretic association. In this obligate phoresy, A. pallida seasonally attaches to B. gobica for overwinter survival and they share the same host plant, Lycium barbarum, during the growing season. It is unknown how the host plant responds to these two herbivores and what plant metabolites are involved in their interspecific interaction. Here, effects of A. pallida and B. gobica on the host plant's transcriptome and metabolome, and on enzymes involved in plant defence, at various infestation stages were studied by inoculating A. pallida and B. gobica either separately or simultaneously on leaves of L. barbarum. Our results showed that (a) A. pallida significantly promoted primary and secondary metabolite accumulation, (b) B. gobica markedly inhibited primary and secondary metabolite accumulation and had little influence on defence enzyme activity, and (c) under simultaneous A. pallida and B. gobica infestation, an intermediate response was predicted. These findings indicate that A. pallida and B. gobica have different effects on host plants, A. pallida inhibits B. gobica mainly by increasing the secondary metabolism of L. barbarum, whereas B. gobica inhibits A. pallida mainly by decreasing the primary metabolism of L. barbarum. In conjunction with our previous research, we speculate that this trade-off in host plant metabolite response between A. pallida and B. gobica after detachment promotes a stable phoretic association.

Integrated Transcriptome and Metabolome Dynamic Analysis of Galls Induced by the Gall Mite Aceria pallida on Lycium barbarum Reveals the Molecular Mechanism Underlying Gall Formation and Development.

Galls have become the best model for exploring plant-gall inducer relationships, with most studies focusing on gall-inducing insects but few on gall mites. The gall mite Aceria pallida is a major pest of wolfberry, usually inducing galls on its leaves. For a better understanding of gall mite growth and development, the dynamics of the morphological and molecular characteristics and phytohormones of galls induced by A. pallida were studied by histological observation, transcriptomics and metabolomics. The galls developed from cell elongation of the epidermis and cell hyperplasia of mesophylls. The galls grew quickly, within 9 days, and the mite population increased rapidly within 18 days. The genes involved in chlorophyll biosynthesis, photosynthesis and phytohormone synthesis were significantly downregulated in galled tissues, but the genes associated with mitochondrial energy metabolism, transmembrane transport, carbohydrates and amino acid synthesis were distinctly upregulated. The levels of carbohydrates, amino acids and their derivatives, and indole-3-acetic acid (IAA) and cytokinins (CKs), were markedly enhanced in galled tissues. Interestingly, much higher contents of IAA and CKs were detected in gall mites than in plant tissues. These results suggest that galls act as nutrient sinks and favor increased accumulation of nutrients for mites, and that gall mites may contribute IAA and CKs during gall formation.

Secondary Rhinaria Contribute to Major Sexual Dimorphism of Antennae in the Aphid Semiaphis heraclei (Takahashi).

Sexual generation is an important generation in the life cycle of host-alternating aphids, and its population size determines the intensity of the peak in the next spring. Although male trapping techniques based on olfactory stimuli have been successfully established in the field, the biological basis of olfactory perception in males is unclear. In this study, we compared the morphology of antennae and the types, sizes, numbers, and distribution of sensilla between males and sexual females in the host-alternating aphid Semiaphis heraclei (Hemiptera: Aphididae). We found that flagellum length differentiation contributed to the majority of the sexual dimorphism of antennae. Most sensillum types or subtypes, including trichoid sensilla subtype I, campaniform sensilla, and primary rhinaria subtypes I and II, were enlarged in males. In addition, males bore more trichoid sensilla subtype I than sexual females. In particular, secondary rhinaria were present in males only and could not be detected in sexual females. These results revealed the structural basis of male olfactory perception. Our findings provide insight into the mechanism underlying chemical communication between sexual aphids and could thus be useful for pest control.

Combined Metabolome and Transcriptome Analysis Highlights the Host's Influence on Cistanche deserticola Metabolite Accumulation.

The medicinal plant Cistanche deserticola Ma (Orobanchaceae) is a holoparasitic angiosperm that takes life-essential materials from Haloxylon ammodendron (C. A. Mey.) Bunge (Amaranthaceae) roots. Although many experiments have been conducted to improve the quality of C. deserticola, little attention has been paid to the host's influence on metabolite accumulation. In this study, transcriptomic and metabolomic analyses were performed to unveil the host's role in C. deserticola's metabolite accumulation, especially of phenylethanoid glycosides (PhGs). The results indicate that parasitism by C. deserticola causes significant changes in H. ammodendron roots in relation to metabolites and genes linked to phenylalanine metabolism, tryptophan metabolism and phenylpropanoid biosynthesis pathways, which provide precursors for PhGs. Correlation analysis of genes and metabolites further confirms that C. deserticola's parasitism affects PhG biosynthesis in H. ammodendron roots. Then we found specific upregulation of glycosyltransferases in haustoria which connect the parasites and hosts. It was shown that C. deserticola absorbs PhG precursors from the host and that glycosylation takes place in the haustorium. We mainly discuss how the host resists C. deserticola parasitism and how this medicinal parasite exploits its unfavorable position and takes advantage of host-derived metabolites. Our study highlights that the status of the host plant affects not only the production but also the quality of Cistanches Herba, which provides a practical direction for medicinal plant cultivation.

Symbiotic Bacteria System of Locusta migratoria Showed Antifungal Capabilities against Beauveria bassiana.

The stability of symbiotic flora is an important indicator of the health of an organism. Symbiotic bacteria have been proven to be closely involved in the immune process of organisms. The pathogenicity of Beauveria bassiana was studied in relation to symbiotic bacteria on the surface and inside of the migratory locust (Locusta migratoria). The results showed that the surface disinfection of test locusts contributed to the pathogenicity of B. bassiana to locusts. Most of the surface bacteria of L. migratoria caused some inhibition of B. bassiana growth, and LM5-4 (Raoultella ornithinolytica), LM5-2 (Enterobacter aerogenes), and LM5-13 (Citrobacter freundii) showed the highest inhibitory effect on the growth of B. bassiana. The inoculation of locusts with additional surface symbiotic bacteria reduced the virulence of B. bassiana to L. migratoria. Infection by different strains of B. bassiana caused similar changes in the symbiotic flora of migratory locusts. The inoculation of locusts with additional intestinal symbiotic bacteria (Enterobacter sp.) reduced the virulence of B. bassiana to L. migratoria. These findings illustrate the effect of bacterial communities on fungal infections in L. migratoria when seen from the perspective of ecology in a microenvironment. The active antifungal substances of such bacteria and their mechanisms of action need further study.

Roles of insect odorant binding proteins in communication and xenobiotic adaptation.

Odorant binding proteins (OBPs) are small water-soluble proteins mainly associated with olfaction, facilitating the transport of odorant molecules to their relevant receptors in the sensillum lymph. While traditionally considered essential for olfaction, recent research has revealed that OBPs are engaged in a diverse range of physiological functions in modulating chemical communication and defense. Over the past 10 years, emerging evidence suggests that OBPs play vital roles in purifying the perireceptor space from unwanted xenobiotics including plant volatiles and pesticides, potentially facilitating xenobiotic adaptation, such as host location, adaptation, and pesticide resistance. This multifunctionality can be attributed, in part, to their structural variability and effectiveness in transporting, sequestering, and concealing numerous hydrophobic molecules. Here, we firstly overviewed the classification and structural properties of OBPs in diverse insect orders. Subsequently, we discussed the myriad of functional roles of insect OBPs in communication and their adaptation to xenobiotics. By synthesizing the current knowledge in this field, our review paper contributes to a comprehensive understanding of the significance of insect OBPs in chemical ecology, xenobiotic adaptation, paving the way for future research in this fascinating area of study.

Transcriptome-Based Selection and Validation of Reference Genes for Gene Expression in Goji Fruit Fly (Neoceratitis asiatica Becker) under Developmental Stages and Five Abiotic Stresses.

Goji fruit fly, Neoceratitis asiatica, is a major pest on the well-known medicinal plant Lycium barbarum. Dissecting molecular mechanisms of infestation and host selection of N. asiatica will contribute to the determination of best management practices for pest fly control. Gene expression normalization by Real-time quantitative PCR (qPCR) requires the selection and validation of appropriate reference genes (RGs). Hence, 15 candidate RGs were selected from transcriptome data of N. asiatica. Their expression stability was evaluated with five algorithms (∆Ct, Normfinder, GeNorm, BestKeeper, and RefFinder) for sample types differing in the developmental stage, sex, tissue type, and in response to five different abiotic stresses. Our results indicated that the RGs ß-Actin + GST for sex, RPL32 + EF1α for tissue type, RPS13+ EF1α for developmental stages along with odor stimulation, color induction, and starvation-refeeding stresses, EF1α + GAPDH under insecticide stress, RPS13 + RPS18 under temperature stress, respectively, were selected as the most suitable RGs for qPCR normalization. Overall, RPS18 and EF1α were the two most stable RGs in all conditions, while RPS15 and EF1ß were the least stable RGs. The corresponding suitable RGs and one unstable RG were used to normalize a target odorant-binding protein OBP56a gene in male and female antennae, different tissues, and under odor stimulation. The results of OBP56a expression were consistent with transcriptome data. Our study is the first research on the most stable RGs selection in N. asiatica, which will facilitate further studies on the mechanisms of host selection and insecticide resistance in N. asiatica.

Odorant degrading carboxylesterases modulate foraging and mating behaviors of Grapholita molesta.

Odorant degrading carboxylesterases (CXEs) play key roles in the process of odor signal reception via degrading ester odorants. But the functional mechanisms of CXEs in modulating insect behaviors are unclear. Herein, we studied the roles that CXEs played in mating, foraging, and signal receptions of sex pheromones and host volatiles in Grapholita molesta. As a result, 23 candidate CXEs were identified by transcriptome analysis of G. molesta. The GmolCXE1 and 5 highly expressed in the antennae of male moths and GmolCXE14 and 21 abundantly expressed in larval heads, were significantly upregulated after exposure with odors from female adults or fresh ripe fruits respectively. After knockdown of GmolCXE1 and 5, or GmolCXE14 and 21 by RNA interference, the behavioral responses of G. molesta to ester sex pheromones or host volatiles were decreased, by exhibiting an inhibited searching behavior of G. molesta for females or fruits, respectively. Then evidence form GC-MS analysis, showed that the protein GmolCXE1 and GmolCXE5 could metabolize the sex pheromone components (Z/E)-8-dodecenyl acetate to their metabolites products (Z/E)-8-dodecenol, and that GmolCXE14 and GmolCXE21 could metabolize ethyl butanoate and ethyl hexanoate of ripe pears. In addition, fluorescent binding assays verified that GmolCXEs could degrade the free ester odor molecules, but not degrade the odor molecules protected by odorant-binding proteins. Our study not only demonstrated CXEs modulated the mating and foraging behaviors of G. molesta through inactivation of ester sex pheromone and host volatiles, but also discovered great potential molecular targets to develop behavioral inhibitors for pest management.

Variability of Gut Microbiota Across the Life Cycle of Grapholita molesta (Lepidoptera: Tortricidae).

Grapholita molesta, the oriental fruit moth, is a serious global pest of many Rosaceae fruit trees. Gut microorganisms play important roles in host nutrition, digestion, detoxification, and resistance to pathogens. However, there are few studies on the microbiota of G. molesta, particularly during metamorphosis. Here, the diversity of gut microbiota across the holometabolous life cycle of G. molesta was investigated comprehensively by Illumina high-throughput sequencing technology. The results showed that the microbiota associated with eggs had a high number of operational taxonomic units (OTUs). OTU and species richness in early-instar larvae (first and second instars) were significantly higher than those in late-instar larvae (third to fifth instars). Species richness increased again in male pupae and adults, apparently during the process of metamorphosis, compared to late-instar larvae. Proteobacteria and Firmicutes were the dominant phyla in the gut and underwent notable changes during metamorphosis. At the genus level, gut microbial community shifts from Gluconobacter and Pantoea in early-instar larvae to Enterococcus and Enterobacter in late-instar larvae and to Serratia in pupae were apparent, in concert with host developmental changes. Principal coordinate analysis (PCoA) and linear discriminant analysis effect size (LEfSe) analyses confirmed the differences in the structure of gut microbiota across different developmental stages. In addition, sex-dependent bacterial community differences were observed. Microbial interaction network analysis showed different correlations among intestinal microbes at each developmental stage of G. molesta, which may result from the different abundance and diversity of gut microbiota at different life stages. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis indicated that most functional prediction categories of gut microbiota were related to membrane transport, carbohydrate and amino acid metabolism, and DNA replication and repair. Bacteria isolated by conventional culture-dependent methods belonged to Proteobacteria, Firmicutes, and Actinobacteria, which was consistent with high-throughput sequencing results. In conclusion, exploration of gut bacterial community composition in the gut of G. molesta should shed light into deeper understanding about the intricate associations between microbiota and host and might provide clues to the development of novel pest management strategies against fruit borers.

The mechanism underlying OBP heterodimer formation and the recognition of odors in Holotrichia oblita Faldermann.

Insect odorant-binding proteins (OBPs) play key roles in transport odors to receptors and contribute to insect survival. The cooperative interaction of HoblOBP1 and HoblOBP2 in Holotrichia oblita Faldermann (Coleoptera: Melolonthidae) could increase their binding capacity for ligands. In present study, molecular docking results showed that OBP1/OBP2 complex formed a large binding pocket and interacted with the ligands by hydrogen bonds and hydrophobic interactions. Then, nine amino acids for single site mutations, three paired for double sites, and negative control were mutated into alanine successfully by site-directed mutagenesis. Finally, fluorescence binding assays of these mutants showed that breaking one or two pairs of hydrogen bonds between HoblOBP1 and HoblOBP2 or formed with the ligands significantly decrease the binding affinity with the ligands. However, hydrophobic site mutants still showed slight binding affinity to the ligands. Therefore, the three pairs of hydrogen bonds involved in heterodimer formation and the five hydrogen bonding sites in binding pocket played a key role in response to odors in H. oblita. Our findings may promote further understanding of the mechanisms underlying OBP dimer formation and the role of OBP dimers in odor perception and discrimination.

Identification and comparison of candidate odorant receptor genes in the olfactory and non-olfactory organs of Holotrichia oblita Faldermann by transcriptome analysis.

A sophisticated olfactory system is part of the explanation for the prominence of insects among animals because of the essential roles of the olfactory system in foraging, host seeking, mating, ovipositing and avoiding toxic substances. In this study, we sequenced and analysed the transcriptomes of olfactory tissue (antennae) and non-olfactory tissue (legs) of the scarab beetle, Holotrichia oblita Faldermann, which is a serious underground pest in China. We obtained approximately 80.2 million 150bp reads that were assembled into 61,038 unigenes with an average length of 890bp. Among the transcripts, 70% of the unigenes were annotated. A total of 44 odorant receptors (ORs) and 9 ionotropic receptors (IRs) were identified based on homology searches. Then, quantitative real-time PCR experiments were performed to investigate the expression patterns of 32 putative chemosensory genes. The results showed that these genes were highly expressed in olfactory organs (antennae) and might play a key role in the olfaction-related behaviours in H. oblita. Based on the results of our phylogenetic analysis and the detailed tissue and sex-biased expression characteristics, the different roles of the receptor proteins in the olfactory system were also indicated. The results of this study will provide the foundation for further understanding of the olfactory odorant receptors of H. oblita at the molecular level and ultimately help to develop novel targets for manipulating this pest.

Identification of candidate chemosensory genes by transcriptome analysis in Loxostege sticticalis Linnaeus.

Loxostege sticticalis Linnaeus is an economically important agricultural pest, and the larvae cause great damage to crops, especially in Northern China. However, effective and environmentally friendly chemical methods for controlling this pest have not been discovered to date. In the present study, we performed HiSeq2500 sequencing of transcriptomes of the male and female adult antennae, adult legs and third instar larvae, and we identified 54 candidate odorant receptors (ORs), including 1 odorant receptor coreceptor (Orco) and 5 pheromone receptors (PRs), 18 ionotropic receptors (IRs), 13 gustatory receptors (GRs), 34 odorant binding proteins (OBPs), including 1 general odorant binding protein (GOBP1) and 3 pheromone binding proteins (PBPs), 10 chemosensory proteins (CSPs) and 2 sensory neuron membrane proteins (SNMPs). The results of RNA-Seq and RT-qPCR analyses showed the expression levels of most genes in the antennae were higher than that in the legs and larvae. Furthermore, PR4, OR1-4, 7-11, 13-15, 23, 29-32, 34, 41, 43, 47/IR7d.2/GR5b, 45, 7/PBP2-3, GOBP1, OBP3, 8 showed female antennae-biased expression, while PR1/OBP2, 7/IR75d/CSP2 showed male antennae-biased expression. However, IR1, 7d.3, 68a/OBP11, 20-22, 28/CSP9 had larvae enriched expression, and OBP15, 17, 25, 29/CSP5 were mainly expressed in the legs. The results shown above indicated that these genes might play a key role in foraging, seeking mates and host recognition in the L. sticticalis. Our findings will provide the basic knowledge for further studies on the molecular mechanisms of the olfactory system of L. sticticalis and potential novel targets for pest control strategies.