Oriental Fruit Moth Grapholita Molesta Research Articles

Molecular characterization and functional analysis of the ecdysone receptor isoform (EcR) from the oriental fruit moth Grapholita molesta (Lepidoptera: Tortricidae).

20-Hydroxyecdysone (20E) plays a vital role in a series of biological processes, via the nuclear receptors, EcR/USP by activating the ecdysone regulatory cascade. To clarify the role of EcR during the development of Grapholita molesta, the complementary DNA of ecdysone receptor isoform B1 (GmEcR-B1) was obtained from the transcriptome of G. molesta and verified by PCR. Alignment analysis revealed that the deduced protein sequence of GmEcR-B1 was highly homologous to EcR proteins identified in other lepidopteran species, especially the EcR-B1 isoform in Spodoptera litura. Quantitative real-time PCR showed that GmEcRs was expressed at all test developmental stages, and the expression level of GmEcRs was relatively higher during the period of the 3rd day of fifth instar larvae to 2nd of pupa than those in other stages. Moreover, the messenger RNA of GmEcRs was much more strongly expressed in the Malpighian tubule and epidermis than those in other tissues, which suggests that this gene may function in a tissue-specific manner during larval development. Silencing of GmEcRs could significantly downregulate the transcriptional level of ecdysone-inducible genes and result in increased mortality during metamorphosis and prolonged prepupal duration. Taken together, the present results indicate that GmEcRs may directly or indirectly affect the development of G. molesta.

Fine structure of the compound eyes of the crepuscular moth Grapholita molesta (Busck 1916) (Lepidoptera: Tortricidae).

Morphological organization, ultrastructure and adaptational changes under different light intensities (10000, 100, 1, and 0.01mW/m2) of the compound eye of the oriental fruit moth Grapholita molesta (Busck 1916) were investigated. Its superposition type of eyes consist of approximately 1072 ommatidia in males and 1029 ommatidia in females with ommatidial diameters of around 15μm. Each ommatidium features a laminated corneal lens densely covered by corneal nipples of 256nm in height. Crystalline cones are formed by four cone cells, proximally tapering to form a narrow crystalline tract with a diameter of 1.5μm. Eight retinula cells, two primary and six secondary pigment cells per ommatidium are present. The 62.3μm long rhabdom is divided into a thin 1.8μm wide distal and a 5.2μm wide proximal region. Distally the fused rhabdom consists of the rhabdomeres of seven retinula cells (R1-R7) and connects with the crystalline cone. In the proximal rhabdom region, the pigment-containing retinula cell R8 occupies a position in centre of the rhabdom while R1-R7 cells have taken peripheral positions. At this level each ommatidial group of retinula cells is surrounded by a tracheal tapetum. In response to changes from bright-light to dim-light adaptations, the pigment granules in the secondary pigment cells and retinula cells migrate distally, with a decrease in the length of crystalline tract.

Molecular characterization and functional analysis of a beta-1,3-glucan recognition protein from oriental fruit moth Grapholita molesta (Lepidoptera: Tortricidae).

The beta-1,3-glucan recognition protein (BGRP) is an important pattern recognition protein (PRP), which plays an important role in immune recognition and signaling pathway of insect innate immunity. Herein, a BGRP gene was obtained from the transcriptome of Grapholita molesta and its expression was verified by PCR. The full cDNA of the GmBGRP gene was 1691 bp encoding 486 amino acid residues. The calculated molecular mass of the mature protein was 54.83 kDa with an estimated pI of 6.14. The amino acid sequence of GmBGRP was highly homologous to BGRPs of other lepidopterans including Leguminivora glycinivorella BGRP-3. Expression profile of GmBGRP at different developmental stages and different tissues of 5th instar larvae showed that the expression level of this gene tends to slightly increase and then decrease at the adult stage, with the highest at the pupa stage; and mainly expressed in the epidermis, fat body and hemocytes compared with other tissues. In addition, we investigated the transcription levels of other immune-related genes, such as Serine-1, Serine-2, Serine-3, Serpin, SRCB (scavenger receptor gene), Toll, PPO (prophenoloxidase) upon GmBGRP gene silencing, indicating that GmBGRP expression is associated with immune responses of G. molesta. This was further supported by the upregulation of the mRNA level of GmBGRP following fungal infection. Taken together, these results provide experimental evidence for the role of GmBGRP gene in immune defense in G. molesta larvae.

Prediction of seasonal population dynamics of Grapholita molesta (Busck) and Adoxophyes orana (Fischer von Röslerstamm) in peach orchards using sex pheromone trap and degree-days and its implications in pest management

The successful management of lepidopteran moths in orchards usually depends on the precise forecast of adult activity. However, the seasonal phenology of moths varies between crop cultivars and years, making it difficult to schedule the control measures. Here, we monitored male flight activity of oriental fruit moth Grapholita molesta and summer fruit tortrix moth Adoxophyes orana by using sex pheromone traps in peach orchards of three different cultivars for three successive years. We developed a logistic multiple-peaks model to fit data and then calculated degree-days (DD) required for male activity and neonate emergency. Results show that G. molesta and A. orana males had 4–5 and 3 flight peaks per year, respectively. The seasonal phenology of G. molesta or A. orana was quite stable with an identical timing of each flight peak between cultivars in a year. The flight activity was usually higher in the second and third peaks for both moths, with a higher cumulative number of G. molesta males captured than that of A. orana. Compared to A. orana, G. molesta emerged early in spring and required lower degree-days to reach the subsequent flight peaks and for neonate emergency. Our results suggest that to decline the possibility of outbreaks of moths during the growing seasons, pheromone traps should be scheduled in April with a cumulative DD between 49.6 and 207.1 for G. molesta and in mid-May–early June with a cumulative DD between 450.4 and 866.7 for A. orana, aiming to trap the newly emerged male adults or disrupting female mating success of overwintered moths in orchards. Based on the thermal requirement for egg hatching (i.e., 79.4 DD for G. molesta and 90.0 DD for A. orana), insecticide treatments would be applied in late-April–early May and late May–early June to reduce the field population density of neonates of G. molesta and A. orana, respectively, to reduce fruit damage in orchards. Furthermore, pheromone traps set up in late July–early August (573.8–1025.2 DD) for G. molesta and in mid-September (1539.7–1788.9 DD) for A. orana may suppress overwintering populations and thus decrease pest infestation in next year.

Symbiotic bacteria on the cuticle protect the oriental fruit moth Grapholita molesta from fungal infection

The oriental fruit moth, Grapholita molesta (Lepidoptera: Tortricidae), is a destructive orchard pest native to the Asian subcontinent that has spread throughout many fruit-growing regions of the world. The entomopathogenic fungus Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Hypocreales: Cordycipitaceae) is widely employed for control of insect pests, including G. molesta. Various studies have shown that microorganisms present on the insect cuticle can inhibit fungal spore germination and growth, thus inhibiting their pathogenicity and biological control efficacy. We conducted bioassays on larvae pre-treated with antibiotics to test whether cuticular microbiota could reduce the pathogenicity of B. bassiana to G. molesta. Control larvae (untreated with antibiotics) had higher survival than antibiotic-treated larvae following inoculation with B. bassiana, indicating that cuticular microbiota inhibited infection of G. molesta by B. bassiana. The bacterial diversity present on the cuticle of G. molesta larvae was characterized by 16S rDNA amplicon sequencing. The dominant bacterium isolated was Pantoea sp. and its antagonism toward B. bassiana was assayed in vitro using the inhibition zone method. Inhibitory effects on the pathogenicity of Pantoea sp. against B. bassiana were demonstrated by reintroducing the bacterium to G. molesta larvae that had been pre-treated with antibiotic, which increased their survival in comparison to antibiotic-treated larvae following inoculation with B. bassiana. This study provides potentially valuable insights for maximizing the efficacy of B. bassiana applications for biological control of G. molesta.

Coordinated transcriptomics and peptidomics of central nervous system identify neuropeptides and their G protein-coupled receptors in the oriental fruit moth Grapholita molesta

The oriental fruit moth Grapholita molesta is a cosmopolitan pest of orchard, which causes serious economic losses to the fruit production. Neuropeptides and their specific receptors (primarily G protein-coupled receptors, GPCRs) regulate multiple biological functions in insects and represent promising next-generation pest management strategy. Here, we generated a transcriptome of the central nervous system (CNS) of G. molesta. Overall, 57 neuropeptide precursor genes were identified and 128 various mature peptides were predicted from these precursors. Using peptidomic analysis of CNS of G. molesta, we identified total of 28 mature peptides and precursor-related peptides from 16 precursors. A total of 41 neuropeptide GPCR genes belonging to three classes were also identified. These GPCRs and their probable ligands were predicted. Additionally, expression patterns of these 98 genes in various larval tissues were evaluated using quantitative real-time PCR. Taken together, these results will benefit further investigations to determine physiological functions and pharmacological characterization of neuropeptides and their GPCRs in G. molesta; and to develop specific neuropeptide-based agents for this tortricid fruit pest control.

Remote monitoring of Cydia pomonella adults among an assemblage of nontargets in sex pheromone-kairomone-baited smart traps.

BACKGROUNDCaptures of codling moth, Cydia pomonella (L.), in traps are used to establish action thresholds and time insecticide sprays. The need for frequent trap inspections in often remote orchards has created a niche for remote sensing smart traps. A smart trap baited with a five‐component pheromone‐kairomone blend was evaluated for codling moth monitoring among an assemblage of other nontargets in apple and pear orchards.RESULTSCodling moth captures did not differ between the smart trap and a standard trap when both were checked manually. However, the correlation between automatic and manual counts of codling moth in the smart traps was low, R 2 = 0.66 ÷ 0.87. False‐negative identifications by the smart trap were infrequent <5%, but false‐positive identifications accounted for up to 67% of the count. These errors were primarily due to the misidentification of three moth species of fairly similar‐size to codling moth: apple clearwing moth Synanthedon myopaeformis (Borkhausen), oriental fruit moth Grapholita molesta (Busck), and carnation tortrix Cacoecimorpha pronubana (Hübner). Other false‐positive counts were less frequent and included the misidentifications of dipterans, other arthropods, patches of moth scales, and the double counting of some moths.CONCLUSIONCodling moth was successfully monitored remotely with a smart trap baited with a nonselective sex pheromone‐kairomone lure, but automatic counts were inflated in some orchards due to mischaracterizations of primarily similar‐sized nontarget moths. Improved image‐identification algorithms are needed for smart traps baited with less‐selective lures and with lure sets targeting multiple species.

Improved Monitoring of Grapholita molesta (Lepidoptera: Tortricidae) in Stone Fruit Orchards with a Pheromone-Kairomone Combination Lure.

Monitoring oriental fruit moth Grapholita molesta Busck (Lepidoptera: Tortricidae), populations based on counts in sex pheromone-baited traps within sex pheromone-treated orchards for mating disruption (MD) is challenging since male orientation to traps is disrupted. In this study, we evaluated a new commercial pheromone–kairomone combination lure for G. molesta (Pherocon® OFM Combo™ Dual™), which combines the G. molesta and Cydia pomonella L. sex pheromones with a blend of acetic acid and terpinyl acetate. Trap comparisons were performed in 33 trials in peach and nectarine orchards under MD (24) or non-MD (9) during the 2018–2019 period in Italy. Male and total moth captures in traps baited with the combination lure were significantly greater than in traps with G. molesta pheromone in both years and across both pheromone treatments. The proportion of females in the total moth captures using the combination lure averaged ca. 7% across all trials. The by-catch of non-targets, i.e., Apis mellifera L., was low in traps baited either with the combination and the sex pheromone lure, while trap color (white) affected the capture of beneficials but not of the target pest. Our study proves that this combination lure can improve the monitoring and management for G. molesta in stone fruits. New studies are needed to define action thresholds to trigger supplemental control methods to MD. Secondly, female-based monitoring lures should be further developed to improve management strategies.

Brightness mediates oviposition in crepuscular moth, Grapholita molesta

Plant colouration is known to be used by some nocturnal flower-visiting insects to foraging among colourful flowers in dim light. However, it remains unknown whether crepuscular species possibly reliably discriminate plant colouration (i.e. colour and/or brightness) for oviposition even driven by challenges of variably light conditions and similar leaf colouration. Here, we explored this question in the oriental fruit moth Grapholita molesta, a major pest of stone fruits worldwide, using coloured squares under changing illuminants (1000, 100, 1 and 0.01 mW/m2). G. molesta female preferred to oviposit on green and orange over yellow, red, blue and purple with a lower discrimination at starlight (0.01 mW/m2), whereas the preferential brighter white can be discriminated clearly from those achromatic grey and black at all tested light intensities. When both cues were presented in conflicting for oviposition, the moths were more attracted to brightness than colour cues irrespective of light intensity they are exposed to. Interestingly, there was a decrease in the reflectance between young and old leaves of P. persica at similar wavelengths between 500 and 550 nm, providing an apparent brightness. Ovipositing females preferred the brighter light green over the dark green. Our results indicate that as a more reliable cue, brightness of leaves rather than colour plays a more important role for mediating crepuscular moth G. molesta to lay eggs on young leaves in microhabitats in changing light environments. We discuss that the brightness oriented in oviposition task in complex orchards is relevant to leaf apparency.

Performance of Trichogramma pintoi when parasitizing eggs of the oriental fruit moth Grapholita molesta

Performance of Trichogramma pintoi when parasitizing eggs of the oriental fruit moth Grapholita molesta

Synergistic Effects of Volatiles from Host-Infested Plants on Host-Searching Behavior in the Parasitoid Wasp Lytopylus rufipes (Hymenoptera: Braconidae).

Herbivore-induced plant volatiles (HIPVs) are important cues for natural enemies to find their hosts. HIPVs are usually present as blends and the effects of combinations of individual components are less studied. Here, we investigated plant volatiles in a tritrophic system, comprising the parasitoid wasp Lytopylus rufipes Nees (Hymenoptera: Braconidae), the Oriental fruit moth Grapholita molesta (Busck) (Lepidoptera: Tortricidae), and Japanese pear, Pyrus pyrifolia 'Kosui', so as to elucidate the effects of single components and blends on wasp behaviors. Bioassays in a four-arm olfactometer, using either shoots or their isolated volatiles collected on adsorbent, revealed that female wasps preferred volatiles from host-infested shoots over those from intact shoots. Analyses identified (Z)-3-hexenyl acetate (H), linalool (L), (E)-β-ocimene (O), (E)-3,8-dimethyl-1,4,7-nonatriene (D), and (E,E)-α-farnesene (F). Among them, only F was induced by infestation with G. molesta. When tested singly, only O and D elicited positive responses by L. rufipes. Binary blends of HO and DF elicited a positive response, but that of HD elicited a negative one, even though D alone elicited a positive response. Remarkably, wasps did not prefer either the ODF or HL blends, but showed a highest positive response to a quinary blend (HLODF). These results show that synergism among volatiles released from host-infested plants is necessary for eliciting high behavioral responses in L. rufipes, enabling L. rufipes to find its host efficiently.

Functional Analysis of the Chemosensory Protein GmolCSP8 From the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae).

Chemosensory proteins (CSPs) belong to a family of small water-soluble proteins that can selectively bind and transport odorant molecules for olfactory communication in insects. To date, their definite physiological functions in olfaction remain controversial when compared with odorant binding proteins (OBPs). To investigate the functions of CSPs in the oriental fruit moth Grapholita molesta, we determined the tissue expression patterns and binding properties of the CSP, GmolCSP8. The key binding sites of GmolCSP8 with a representative ligand were evaluated using molecular flexible docking, site-directed mutagenesis and ligand-binding experiments. Multiple sequence alignment and phylogenetic analysis showed that GmolCSP8 possesses a typical conserved four cysteines motif and shares high sequence identity with some CSP members of other Lepidopteran insects. GmolCSP8 was predominantly expressed in the wings and antennae of both male and female adults and may be involve in contact chemoreception. Recombinant GmolCSP8 (rGmolCSP8) exhibited specific-binding affinities to small aliphatic alcohols (C4–12) and had the strongest binding affinity to 1-hexanol. The three-dimensional structure of GmolCSP8 was constructed using the structure of sgCSP4 as a template. Site-directed mutagenesis and ligand-binding experiments confirmed that Thr27 is the key binding site in GmolCSP8 for 1-hexanol binding, because this residue can form hydrogen bond with the oxygen atom of the hydroxyl group in 1-hexanol, and Leu30 may play an important role in binding to 1-hexanol. We found that pH significantly affected the binding affinities of rGmolCSP8 to ligand, revealing that ligand-binding and -release by this protein is related to a pH-dependent conformational transition. Based on these results, we infer that GmolCSP8 may participate in the recognition and transportation of 1-hexanol and other small aliphatic alcohols.

Multiple refugia from penultimate glaciations in East Asia demonstrated by phylogeography and ecological modelling of an insect pest

BackgroundRefugial populations in Quaternary glaciations are critical to understanding the evolutionary history and climatic interactions of many extant species. Compared with the well-studied areas of Europe and Northern America, refugia of species in eastern Asia remain largely unknown. Here, we investigated the phylogeographic history of a globally important insect pest, the oriental fruit moth Grapholita molesta, in its native range of China.ResultsGenetic structure analyses unveiled three distinct groups and a set of populations with admixture. Approximate Bayesian Computation (ABC) analyses support range expansion of this moth from southwest groups of Yunnan and Sichuan to northern and eastern China. A set of admixed populations was found around these two ancestral groups. This pattern of genetic structure points to two refugia located in the Yunnan region and Sichuan Basin. The split of the two refugia was dated to 329.2 thousand years ago in the penultimate glacial period. One of the lineages was exclusively found around the Sichuan Basin, indicating the formation of endemic populations in this refugium. Ecological niche model analysis suggested a shrinking distribution from the LIG period to the MID period in the Sichuan lineage but a wide and stable distribution in the other lineage.ConclusionsOur results for the first time suggest that Yunnan and Sichuan jointly served as two large-scale refugia in eastern Asia in Quaternary glaciations, helping to maintain genetic diversity overall.

Resistance of oriental fruit moth (Lepidoptera: tortricidae) to insecticides in apple orchards in southern Brazil

Abstract Oriental fruit moth (Grapholita molesta) is the most important pest of apple orchards in Brazil. For many years, insecticides have been the only tool used for its control. The aim of this work was to characterize the management of apple orchards by the system for Assessment of Environment Impact of Agricultural Technology Innovations (AMBITEC-AGRO) and to relate it with the resistance status of G. molesta populations. Insecticide resistance was estimated by toxicology and enzymatic activities of glutathione S-transferase (GST), esterases (a-ß EST), and acetylcholinesterase (AChE). The phytosanitary strategies adopted by the four apple production orchards in southern Brazil were analyzed in: Vacaria (RS population), Fraiburgo (SC1 population), São Joaquim (SC2 population), and Porto Amazonas (PR population). Oriental fruit moth field populations were reared in laboratory for bioassays with chlorpyriphos, carbaryl, deltamethrin, and tebufenozide insecticides and were compared with a reference susceptible population. Larval mortality rates were recorded for seven days. GST, a-ß EST, and AChE assays were performed with third- and fourth-instar larvae. Results indicated that oriental fruit moth populations collected in Vacaria, Fraiburgo, and São Joaquim showed tolerance to carbaryl and chlorpyriphos, whereas the population collected in Porto Amazonas was more susceptible. SC2 and PR populations were more tolerant to deltamethrin, while RS and SC1 populations were more susceptible. No population was tolerant to tebufenozide. RS population showed the highest activity for AChE, GST, and a-EST. The findings of this study suggest that the AMBITEC-AGRO system was a suitable method to evaluate the phytosanitary activity of apple orchards and to relate it to the resistance status of G. molesta populations.

De novo characterization of microRNAs in oriental fruit moth Grapholita molesta and selection of reference genes for normalization of microRNA expression

MicroRNAs (miRNAs) are a group of endogenous non-coding small RNAs that have critical regulatory functions in almost all known biological processes at the post-transcriptional level in a variety of organisms. The oriental fruit moth Grapholita molesta is one of the most serious pests in orchards worldwide and threatens the production of Rosacea fruits. In this study, a de novo small RNA library constructed from mixed stages of G. molesta was sequenced through Illumina sequencing platform and a total of 536 mature miRNAs consisting of 291 conserved and 245 novel miRNAs were identified. Most of the conserved and novel miRNAs were detected with moderate abundance. The miRNAs in the same cluster normally showed correlated expressional profiles. A comparative analysis of the 79 conserved miRNA families within 31 arthropod species indicated that these miRNA families were more conserved among insects and within orders of closer phylogenetic relationships. The KEGG pathway analysis and network prediction of target genes indicated that the complex composed of miRNAs, clock genes and developmental regulation genes may play vital roles to regulate the developmental circadian rhythm of G. molesta. Furthermore, based on the sRNA library of G. molesta, suitable reference genes were selected and validated for study of miRNA transcriptional profile in G. molesta under two biotic and six abiotic experimental conditions. This study systematically documented the miRNA profile in G. molesta, which could lay a foundation for further understanding of the regulatory roles of miRNAs in the development and metabolism in this pest and might also suggest clues to the development of genetic-based techniques for agricultural pest control.

Novel microsatellite markers for the oriental fruit moth Grapholita molesta (Lepidoptera: Tortricidae) and effects of null alleles on population genetics analyses.

The oriental fruit moth (OFM) Grapholita molesta (Lepidoptera: Tortricidae) is an important economic pest of stone and pome fruits worldwide. We sequenced the OFM genome using next-generation sequencing and characterized the microsatellite distribution. In total, 56,674 microsatellites were identified, with 11,584 loci suitable for primer design. Twenty-seven polymorphic microsatellites, including 24 loci with trinucleotide repeat and three with pentanucleotide repeat, were validated in 95 individuals from four natural populations. The allele numbers ranged from 4 to 40, with an average value of 13.7 per locus. A high frequency of null alleles was observed in most loci developed for the OFM. Three marker panels, all of the loci, nine loci with the lowest null allele frequencies, and nine loci with the highest null allele frequencies, were established for population genetics analyses. The null allele influenced estimations of genetic diversity parameters but not the OFM's genetic structure. Both a STRUCTURE analysis and a discriminant analysis of principal components, using the three marker panels, divided the four natural populations into three groups. However, more individuals were incorrectly assigned by the STRUCTURE analysis when the marker panel with the highest null allele frequency was used compared with the other two panels. Our study provides empirical research on the effects of null alleles on population genetics analyses. The microsatellites developed will be valuable markers for genetic studies of the OFM.

Electrophysiological and behavioral response of the oriental fruit moth Grapholita molesta (Busck, 1916) (Lepidoptera: Tortricidae) to the volatiles of apple fruits at different stages of development

The oriental fruit moth, Grapholitamolesta, occurs in Southern Brazil throughout the year, and migrates from peach to apple orchards. Because moths rely on volatile organic compounds (VOCs) during the host-location process, variations in the emission of these compounds during fruit maturation can influence the time of infestation and preference of the moths for a particular genotype. The aim of this work was to identify VOCs emitted by the apples “Eva” and “Gala” at different stages of development and to determine the behavioral and electrophysiological responses of G. molesta to these compounds. For this purpose, VOCs from immature, maturing, and mature fruits of both cultivars were collected and analyzed by gas chromatography and gas chromatography-mass spectroscopy. The response of the antennae of virgin males and females and mated females to volatiles released by the three fruit stages was registered by gas chromatography coupled to an electroantennography. A dual-choice behavioral test for the different combinations of insect groups and fruit stage was also performed. Amongst the volatiles released by mature fruits, twelve compounds elicited a response. The antennae of the oriental fruit moth did respond to isoamyl hexanoate and α-farnesene emitted by “Eva” maturing fruits. In general, virgin females did not respond to volatiles in olfactometer bioassays and mated females were attracted to volatiles released by mature fruits. Our results show that the variation in the emission during the maturation of fruits can influence the orientation of G. molesta.

Genetic variation and population structure of the oriental fruit moth Grapholita molesta in Shanxi, a major pome fruits growing region in North China

Grapholita molesta, commonly known as the oriental fruit moth, is one of the most destructive fruit pests both in China and in other regions of the world. We examined the genetic diversity and population structures of G. molesta populations from 11 locations in a major pome fruit production region (Shanxi province) in North China using seven microsatellite markers and sequence variations in two mitochondrial genes. We found that G. molesta in the studied region exhibited high levels of genetic diversity. Among 271 individual insects examined using microsatellite markers, mean number of alleles ranged from 5.86 to 9.00 with average 8.01. Population structure analysis revealed that populations from the eleven sampling sites formed two clusters without apparent distance isolation. Cluster 1 consisted of eight populations distributed in five regions of the sampling sites, whereas Cluster 2 consisted of three populations distributed in two distant locations of the sampling sites. Genetic exchange analysis revealed that about half of the haplotypes were shared among over 90% individual insects from different regions. Our results indicated that the population structure and genetic exchange of G. molesta in the studied region might have been affected by limited dispersal ability of the insect, environmental condition, colonization history and human-mediated dispersal. These findings should provide useful information for the implementation of area-wide pest management and for further characterization of genetic variation and population structure of this economically important pest.

Toxicity and residual activity of spinetoram to neonate larvae of Grapholita molesta (Busck) and Cydia pomonella (L.) (Lepidoptera: Tortricidae): Semi-field and laboratory trials

Spinetoram is a fermentation insecticide, derived from the actinomycete Saccharopolyspora spinosa. It works by disrupting the GABA-gated chloride channels and by causing persistent activation of insect nicotinic acetylcholine receptors. This study aimed to evaluate the efficacy of spinetoram for control of neonate larvae of both oriental fruit moth (OFM) Grapholita molesta (Busck) and codling moth (CM) Cydia pomonella (L.) in semi-field and laboratory trials. OFM and CM neonate larvae responded similarly to spinetoram, which showed high efficacy on both species. In semi-field experiments, regression analysis of the percentage of damaged fruits as a function of days after treatment showed a better performance of the highest spinetoram dose (10 g a.i./hl) in comparison with the maximum recommended field dose of the reference product emamectin benzoate (2.85 g a.i./hl). Surface-treated diet assays revealed LC50 values of 6.59 and 8.44 ng a.i./cm2 for neonate larvae of OFM and CM larvae, respectively. High percentages of mortality were recorded on both species after 24-h exposure to treated diet. For these reasons spinetoram could be considered a valuable tool in IPM strategies for OFM and CM control.

Binding Properties of General Odorant Binding Proteins from the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae).

BackgroundThe oriental fruit moth Grapholita molesta is a host-switching pest species. The adults highly depend on olfactory cues in locating optimal host plants and oviposition sites. Odorant binding proteins (OBPs) are thought to be responsible for recognizing and transporting hydrophobic odorants across the aqueous sensillum lymph to stimulate the odorant receptors (ORs) within the antennal sensilla and activate the olfactory signal transduction pathway. Exploring the physiological function of these OBPs could facilitate understanding insect chemical communications.Methodology/Principal FindingTwo antennae-specific general OBPs (GOBPs) of G. molesta were expressed and purified in vitro. The binding affinities of G. molesta GOBP1 and 2 (GmolGOBP1 and 2) for sex pheromone components and host plant volatiles were measured by fluorescence ligand-binding assays. The distribution of GmolGOBP1 and 2 in the antennal sensillum were defined by whole mount fluorescence immunohistochemistry (WM-FIHC) experiments. The binding sites of GmolGOBP2 were predicted using homology modeling, molecular docking and site-directed mutagenesis. Both GmolGOBP1 and 2 are housing in sensilla basiconica and with no differences in male and female antennae. Recombinant GmolGOBP1 (rGmolGOBP1) exhibited broad binding properties towards host plant volatiles and sex pheromone components; rGmolGOBP2 could not effectively bind host plant volatiles but showed specific binding affinity with a minor sex pheromone component dodecanol. We chose GmolGOBP2 and dodecanol for further homology modeling, molecular docking, and site-directed mutagenesis. Binding affinities of mutants demonstrated that Thr9 was the key binding site and confirmed dodecanol bonding to protein involves a hydrogen bond. Combined with the pH effect on binding affinities of rGmolGOBP2, ligand binding and release of GmolGOBP2 were related to a pH-dependent conformational transition.ConclusionTwo rGmolGOBPs exhibit different binding characteristics for tested ligands. rGmolGOBP1 has dual functions in recognition of host plant volatiles and sex pheromone components, while rGmolGOBP2 is mainly involved in minor sex pheromone component dodecanol perception. This study also provides empirical evidence for the predicted functions of key amino acids in recombinant protein ligand-binding characteristics.