Characterizing potential repelling volatiles for "push-pull" strategy against stem borer: a case study in Chilo auricilius.

BACKGROUND: Massive techniques have been evaluated for developing different pest control methods to minimize fertilizer and pesticide inputs. As "push-pull" strategy utilizes generally non-toxic chemicals to manipulate behaviors of insects, such strategy is considered to be environmentally friendly. "Push-pull" strategy has been extraordinarily effective in controlling stem borers, and the identification of new "pushing" or "pull" components against stem borers could be significantly helpful. RESULTS: In this study, the results of field trapping assay and behavioral assay showed the larvae of C.auricilius, one kind of stem borers, could be deterred by rice plant under tilling stage, its main host crop. The profiles of volatiles were compared between rice plants under two different developmental stages, and α-pinene was identified as a key differential component. The repelling activity of α-pinene against C.auricilius was confirmed by Y-tube olfactometer. For illuminating the olfactory recognition mechanism, transcriptome analysis was carried out, and 13 chemosensory proteins (CSPs) were identified in larvae and 19 CSPs were identified in adult of C.auriciliu, which was reported for the first time in this insect. Among these identified CSPs, 4 CSPs were significantly regulated by α-pinene treatment, and CSP8 showed good binding affinity with α-pinene in vitro. CONCLUSIONS: Overall, C.auricilius could be repelled by rice plant at tilling stage, and our results highlighted α-pinene as a key component in inducing repelling activity at this specific stage and confirmed the roles of some candidate chemosensory elements in this chemo-sensing process. The results in this study could provide valuable information for chemosensory mechanism of C.auricilius and for identification of "push" agent against rice stem borers.

Identification of multi-insecticide residues using GC-NPD and the degradation kinetics of chlorpyrifos in sweet corn and soils.

Because more than one insecticide is applied to crops to protect plants from pests, an analytical multi-residue determination method was developed using gas chromatography with a nitrogen phosphorus detector (GC-NPD). The retention time for 12 insecticides was 3.7-27.7min. Under the selected conditions, the limits of detection (LOD) and quantification (LOQ) were below the maximum residue limits (MRLs) and in the range of 0.00315-0.05µgmL(-1) and 0.01-0.165µgmL(-1), respectively. Using GC-NPD, we investigated the dissipation dynamics and final residual levels of chlorpyrifos in sweet corn and soil and determined that the half-lives was 4-7days, that is, that chlorpyrifos is safe to use on sweet corn with a pre-harvest interval of 16-22days before harvest. These results provide new insights into chlorpyrifos degradation in plants and its environmental behavior.

Azadirachtin-induced apoptosis involves lysosomal membrane permeabilization and cathepsin L release in Spodoptera frugiperda Sf9 cells.

Azadirachtin as a kind of botanical insecticide has been widely used in pest control. We previously reported that azadirachtin could induce apoptosis of Spodoptera litura cultured cell line Sl-1, which involves in the up-regulation of P53 protein. However, the detailed mechanism of azadirachtin-induced apoptosis is not clearly understood in insect cultured cells. The aim of the present study was to address the involvement of lysosome and lysosomal protease in azadirachtin-induced apoptosis in Sf9 cells. The result confirmed that azadirachtin indeed inhibited proliferation and induced apoptosis. The lysosomes were divided into different types as time-dependent manner, which suggested that changes of lysosomes were necessarily physiological processes in azadirachtin-induced apoptosis in Sf9 cells. Interestingly, we noticed that azadirachtin could trigger lysosomal membrane permeabilization and cathepsin L releasing to cytosol. Z-FF-FMK (a cathepsin L inhibitor), but not CA-074me (a cathepsin B inhibitor), could effectively hinder the apoptosis induced by azadirachtin in Sf9 cells. Meanwhile, the activity of caspase-3 could also be inactivated by the inhibition of cathepsin L enzymatic activity induced by Z-FF-FMK. Taken together, our findings suggest that azadirachtin could induce apoptosis in Sf9 cells in a lysosomal pathway, and cathepsin L plays a pro-apoptosis role in this process through releasing to cytosol and activating caspase-3.

Ligands binding and molecular simulation: the potential investigation of a biosensor based on an insect odorant binding protein.

Based on mimicking biological olfaction, biosensors have been applied for the detection of various ligands in complex environment, which could represent one of the most promising research fields. In this study, the basic characters of one insect odorant binding protein (OBP) as a biosensor were explored. To explore the molecular recognition process, the tertiary structure of the protein was modeled and the protein-ligand interactions with 1,536,550 chemicals were investigated by the molecular docking. The availability of large amount of recombinant SlitOBP1 overcame the difficulty to obtain biological sensing material. After obtained the purified recombinant protein, the result of fluorescence binding assays proved the candidate protein has good affinities with the majority of the tested chemicals. With the aid of simulation docking, the key conserved amino acids within the binding site were identified and then mutated to alanine. After mutation, the protein-ligand binding characteristics were recorded, and the competitive binding assays were carried out to provide experimental verification. The detailed information on its structure and affinities investigated in this study could allow the design of specific mutants with desired characteristics, which provides a solid base for tailoring OBP for biosensor and provides a role model for screening the other elements in olfactory system for different applications.

Bdor\Orco is important for oviposition-deterring behavior induced by both the volatile and non-volatile repellents in Bactrocera dorsalis (Diptera: Tephritidae).

Several studies have shown that the selections of gravid females to potential oviposition sites from a distance were mediated by volatile signals, however, the means by which the sensory cues from non-volatile chemicals affected the insect behavior were still a controversial subject. Chemosensory in insect is a complex process, which is mediated by multigene families of chemoreceptors, including olfactory receptors, olfactory co-receptors, and odorant-binding proteins. To elucidate the chemoreception mechanism of volatile and non-volatile chemicals, the roles of Orco and OBP in oviposition-deterrent activities induced by citronellal and Rhodojaponin-III were investigated. Our results suggested that RNAi-mediated expression inhibition was successfully achieved by feeding dsRNA in Bactrocera dorsalis. High levels of Bdor\Orco expression were essential for recognizing two chemicals of different physical properties, whereas the expression of Bdor\OBP was only imperative in perception of volatile chemical. The results suggested that volatile and non-volatile chemicals may evoke distinct molecular basis for chemosensory in the flies, while Orco was essential in the perception of both chemicals. The study highlighted that the central role of Orco in chemical recognition, which enabled it to be the universally applied target of designing new botanical pesticide.

Contacting is essential for oviposition deterrence of Rhodojaponin-III in Spodoptera litura.

In Lepidoptera, choosing the right site for egg laying is particularly important, because the small larvae cannot forage for alternate host plants easily. Some secondary compounds of plants have the ability to deter oviposition behaviors of insects. Rhodojaponin-III, a botanical compound, has been reported to have intense deterring-oviposition activity against many insects, which have important implications for agricultural pest management. This study provided evidence for elucidating the perception mechanism underlying Rhodojaponin-III as oviposition deterrent. In this study, the antennas of moths could not elicit notable electroantennogram responses to Rhodojaponin-III, which suggested the Rhodojaponin-III could not exert effects like those volatile compounds. The results of physiological experiments confirmed the Rhodojaponin-III could produce the oviposition deterrence effect against moths without depending on antennas, while the physical contact was essential for perceiving the compound, which suggested that the sensilla on tarsus and ovipositor could be chemoreceptor for Rhodojaponin-III. Therefore, these sensilla were investigated by scanning electron microscopy to explore their potential functions in detecting Rhodojaponin-III. This study highlighted the contacting mechanism in deterring oviposition behaviors of moths by Rhodojaponin-III and provided new insight for development of contact-based pest management.

Involvement of a specific chemosensory protein from Bactrocera dorsalis in perceiving host plant volatiles.

Insects have evolved many physiological and behavioral adaptations to recognize external complex chemicals. Olfaction plays an important role in perceiving volatile chemicals, utilizing them to locate host sites, conspecifics, and enemies. Chemosensory proteins (CSPs) are present in high concentrations within the sensory sensilla of insects and are endowed with a heterogeneous range of functions. However, direct evidence for the involvement of CSPs in olfactory function is still lacking. In this study, a fluorescence-based ligand binding assay using Bdor-CSP2 illustrated its ability to bind the majority of the selected ligands of different shapes and chemical structures that are ecologically significant, host plant volatiles of Bactrocera dorsalis. RNAi-mediated silencing coupled with electrophysiological tests showed lower electrophysiological responses to (3Z)-hex-3-en-1-ol, trans-2-hexenal, 6-methylhept-5-en-2-one, and 3-methylbutyl acetate in dsBdor CSP2 treated flies compared with the untreated controls. The reduced expression of Bdor-CSP2 by RNA interference was confirmed by semi-quantitative PCR, real-time quantitative PCR and Western blot, which suggested the RNAi-treatment was responsible for the observed reduction of antennal responses in EAG recordings. These data suggest that the expression of Bdor-CSP2 is necessary for the recognition of antennal responses to some plant host volatiles by B. dorsalis.

Identification of a novel interacting partner of the chemosensory protein 1 from Plutella xylostella L.

Chemosensory proteins (CSPs) are small soluble proteins endowed with heterogeneous functions. The information so far available for CSPs suggested these well-defined and conserved proteins were involved in diverse activities, including chemical communication, feeding, development, mating, immune regulation, as well as circadian rhythms. However, the detailed mechanisms of these physiological functions remain elusive. To explore the underlying mechanisms of CSPs and their interaction partners, a cDNA library from the head of Plutella xylostella was screened against CSP1 to identify proteins involved in the PxylCSP1-related physiological activities. Protein kinase C (PKC) was screened out as a putative interacting protein of PxylCSP1. The full length of PxylPKC cDNA was obtained, and the results of semi-quantitative real-time PCR and quantitative real-time PCR revealed that PxylPKC showed similar expression pattern as PxylCSP1. In vivo and in vitro interactions between PxylCSP1 and PxylPKC were further confirmed by co-immunoprecipitation and GST pull-down assays, respectively. These findings extended our knowledge on the mechanisms of CSP-regulated functions, and providing new target proteins to facilitate the design of novel intervention strategies against the pest.

BdorCSP2 is important for antifeed and oviposition-deterring activities induced by Rhodojaponin-III against Bactrocera dorsalis.

Rhodojaponin-III is a nonvolatile botanical grayanoid diterpene compound, which has antifeedant and oviposition deterrence effects against many kinds of insects. However, the molecular mechanism of the chemoreception process remains unknown. In this study, the important role of BdorCSP2 in the recognition of Rhodojaponin-III was identified. The full length cDNA encoding BdorCSP2 was cloned from legs of Bactrocera dorsalis. The results of expression pattern revealed that BdorCSP2 was abundantly expressed in the legs of adult B. dorsalis. Moreover, the expression of BdorCSP2 could be up-regulated by Rhodojaponin-III. In order to gain comprehensive understanding of the recognition process, the binding affinity between BdorCSP2 and Rhodojaponin-III was measured by fluorescence binding assay. Silencing the expression of BdorCSP2 through the ingestion of dsRNA could weaken the effect of oviposition deterrence and antifeedant of Rhodojaponin-III. These results suggested that BdorCSP2 of B. dorsalis could be involved in chemoreception of Rhodojaponin-III and played a critical role in antifeedant and oviposition behaviors induced by Rhodojaponin-III.