Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in Mythimna loreyi (Lepidoptera: Noctuidae).

Quantitative real-time PCR (qRT-PCR) is a widely applied technique for accurately assessing the expression of target genes. In practice, the evaluation of gene expression requires appropriate reference genes. To screen reliable reference genes for evaluating gene expression via qRT-PCR in Mythimna loreyi, a notorious migratory pest across Asia, Africa, Europe, and Australia, we assessed the expression stability of 13 candidate reference genes in M. loreyi using the ΔCt method, BestKeeper, Normfinder, GeNorm, and the web-based comprehensive platform RefFinder. These reference genes include RPL10, RPL27, RPL32, RPS3, TATA-box, GAPDH, AK, Actin, EF, α-tubulin, SOD, 18S rRNA, and FTZ-F1, which is frequently employed in Lepidoptera insects. Our findings revealed that the performance of the candidate reference gene depended on experimental conditions. Specifically, RPL27 and RPL10 were the most suitable for evaluating expression changes across developmental stages, tissues, and adult ages. The optimal reference genes were recommended in specific experiment conditions, for instance, EF and RPS3 were recommended for mating status, AK and RPL10 were recommended for temperature treatments, RPL27 and FTZ-F1 were recommended for larva diet, and EF and RPL27 were recommended for adult diet treatments. Additionally, expression profiles of pheromone-binding protein 2 (MlorPBP2) and glutathione S-transferase (MlorGST1) were used to validate the reference genes. This study provides reference genes for the accurate normalization of qRT-PCR data, laying the groundwork for studying the expression of target genes in M. loreyi.

Identification of sex pheromone in Macdunnoughia crassisigna Warren (Lepidoptera: Noctuidae) and field optimization of the sex attractant.

BACKGROUND: Sex pheromones have proven to be a viable tool for monitoring and controlling pests and is an important part of integrated pest management (IPM). The noctuid moth Macdunnoughia crassisigna Warren poses a significant threat as a defoliator pest, impacting soybean and cruciferous vegetable production and quality in East Asia. However, a lack of comprehensive knowledge about its sexual chemical signaling hampers the development of semiochemical-based IPM approaches for M. crassisigna. RESULTS: We first determined the mating rhythms of M. crassisigna. We then collected pheromones from the sex glands of virgin females at the mating peak and analyzed their components using gas chromatography-electroantennogram detection analysis. The results showed that three components elicited significant electrophysiological responses in male antennae. Gas chromatography-mass spectrometry analysis characterized these components as (Z)-7-dodecene acetate (Z7-12:OAc), (Z)-9-tetradecene acetate (Z9-14:OAc), and (Z)-11-hexadecen-1-ol (Z11-16:OH). Further field experiments indicated that the mixture of Z7-12:OAc and Z9-14:OAc at a ratio of 3:1 displayed significant attractivity to males, confirming its role as a putative sex pheromone of M. crassisigna. Long-term monitoring tests showed that traps baited with these pheromone lures effectively mirrored the population dynamics of M. crassisigna. CONCLUSION: This study successfully identified and validated the sex pheromone released by female M. crassisigna and formulated potent sex lures for field-based pest monitoring. These findings enriched our understanding of chemical communication in Noctuidae and laid a foundation for developing practical monitoring and control methods against M. crassisigna. © 2023 Society of Chemical Industry.

Nano-Micro Core-Shell Fibers for Efficient Pest Trapping.

Insect sex pheromones as an alternative to chemical pesticides hold promising prospects in pest control. However, their burst release and duration need to be optimized. Herein, pheromone-loaded core-shell fibers composed of degradable polycaprolactone and polyhydroxybutyrate were prepared by coaxial electrospinning. The results showed that this core-shell fiber had good hydrophobic performance and thermal stability, and the light transmittance in the ultraviolet band was only below 40%, which provided protection to pheromones. The core-shell structure alleviated the burst release of pheromone in the fiber and extended the release time to about 133 days. In the field, the pheromone-loaded core-shell fibers showed the same continuous and efficient trapping of Spodoptera litura as the commercial carriers. More importantly, the electrospun fibers combined with biomaterials had a degradability unmatched by commercial carriers. The structure design strategy provides ideas for the innovative design of pheromone carriers and is a potential tool for the management of agricultural pests.

Biodegradable electrospun fibers as sustained-release carriers of insect pheromones for field trapping of Spodoptera litura (Lepidoptera: Noctuidae).

BACKGROUND: Insect pheromones are highly effective and environmentally friendly, and are widely used in the monitoring and trapping of pests. However, many researchers have found that various factors such as ultraviolet light and temperature in the field environment can accelerate the volatilization of pheromones, thus affecting the actual control effect. In recent years, electrospinning technology has demonstrated remarkable potential in the preparation of sustained carriers. Moreover, the utilization of biodegradable materials in electrospinning presents a promising avenue for the advancement of eco-friendly carriers. RESULTS: In this study, homogeneous and defect-free pheromone carriers were obtained by electrospinning using fully biodegradable polyhydroxybutyrate materials and pheromones of Spodoptera litura. The electrospun fibers with porous structure could continuously release pheromone (the longest can be ≤80 days). They also had low light transmission, hydrophobic protection. More importantly, the pheromone-loaded electrospun fiber carriers showed stable release and good trapping effect in the field. They could trap pests for at least 7 weeks in the field environment without other light stabilizers added. CONCLUSION: Sustained-release carriers constructed by electrospinning and green materials could improve the efficacy of pheromones and ensure environmental friendliness, and provided a tool for the management of S. litura and other pests and sustainable development of agricultural. © 2023 Society of Chemical Industry.

Optimization and field evaluation of sex-pheromone of potato tuber moth, Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae).

BACKGROUND: The potato tuber moth, Phthorimaea operculella is a key pest of potato and tobacco in China. We compared the sex pheromone titers released by P. operculella female, and the electroantennogram (EAG) responses of male antennae to pheromone compounds under laboratory conditions. Then, the optimal sex pheromone ratio was screened in the field. RESULTS: The P. operculella sex pheromone feeding on potato or tobacco was extracted by solvent-extraction method. Main sex pheromone compounds including E4, Z7-13: AC (PTM1) and E4, Z7, Z10-13: AC (PTM2) were found in gland. The titer and relative ratio of P. operculella sex pheromone compounds secreted by female adult moths differ between host populations, while the relative EAG responses trend of P. operculella males to the pheromone components were the same when directly stimulated. In field trials, PTM1:PTM2 = 8:1-4:1 and PTM1:PTM2 = 1:4-1:7 was the most attractive ratio to P. operculella males in tobacco and potato fields, respectively. During 2019-2020, after 8 weeks of mass trapping for each year, the sex pheromone could reduce the population of P. operculella in the potato and tobacco fields. CONCLUSIONS: In this study, fixed property and quantity analyzing methods were adopted to compare sex pheromones from feeding on potato versus tobacco. EAG tests then were carried out on P. operculella males. Finally, we optimized the ratio of two sex pheromone compounds in potato and examined how this influenced field trapping. By further improving the parameters of sex pheromone application in the field, we demonstrate that deployment of sex pheromones can provide effective control of P. operculella. © 2021 Society of Chemical Industry.

Identification and field verification of an aggregation pheromone from the white-spotted flower chafer, Protaetia brevitarsis Lewis (Coleoptera: Scarabaeidae).

The white-spotted flower chafer (WSFC), Protaetia brevitarsis Lewis, is native to East Asia. Although their larvae are considered a potential resource insect for degrading plant residues, producing protein fodder, and processing to traditional medicine, adult WSFCs inflict damage to dozens of fruit and economic crops. The control of the WSFC still relies heavily on pesticides and the inefficient manual extraction of adults. Here, we report the identification and evaluation of the aggregation pheromone of WSFCs. From the headspace volatiles emitted from WSFC adults, anisole, 4-methylanisole, 2-heptanone and 2-nonanone were identified as WSFC-specific components. However, only anisole and 4-methylanisole elicited positive dose-response relationship in electroantennography tests, and only 4-methylanisole significantly attracted WSFCs of both sexes in olfactometer bioassays and field experiments. These results concluded that 4-methylanisole is the aggregation pheromone of WSFCs. Furthermore, we developed polyethylene vials as long-term dispensers of 4-methylanisole to attract and kill WSFCs. The polyethylene vial lures could effectively attracted WSFCs for more than four weeks. Pheromone-based lures can be developed as an environmentally friendly protocol for monitoring and controlling WSFC adults.

Influence of the neonicotinoid insecticide thiamethoxam on soil bacterial community composition and metabolic function.

Understanding of neonicotinoid insecticides toxicity on non-target organisms, such as bees, has indirectly promoted their soil treatment use. However, their effect on soil ecosystems haven't fully understood. Here, based on 16S rRNA high-throughput sequencing and metagenomics, the effects of neonicotinoid insecticide thiamethoxam on bacterial communities and metabolic functions in two types of soils were studied. Thiamethoxam treatment significantly affected soil bacterial abundance, reduced microbial diversity, and changed the bacterial community structure in the short term, and the structure soon returned to a stable state. Soil type and time were important factors affecting bacterial community structure. Some plant growth-promoting rhizosphere bacteria (PGPR) including Actinobacteria were found, and their populations were reduced, while pollutant-degrading bacteria including Firmicutes were also found, and their populations were increased. Based on metagenomics analysis, thiamethoxam treatment insignificantly promoted or inhibited multiple metabolic processes, but gene abundance of some key processes significantly changed. Subtypes of 18 biodegradation genes (BDGs) and 5 pesticide degradation genes (PDGs) were identified. Thiamethoxam treatment significantly increased the abundance of BDGs and PDGs, including cytochrome P450. Potential hosts of P450 degradation genes, including the genus Rhodococcus, were discovered. Conclusions of this study will promote safety evaluation and degradation-related research on neonicotinoid insecticides in soil.

Spatial and temporal distribution, degradation, and metabolism of three neonicotinoid insecticides on different parts, especially pests' target feeding parts of apple tree.

BACKGROUND: Neonicotinoid insecticides (NIs) have been recently banned in some countries because of increased pest resistance and deleterious risks to non-target organisms. Recent studies considered all parts of crops as a whole part in plant protection. However, there are few reports focused on the distribution and metabolic trends of NIs on target feeding sites of different pests in apple orchards. RESULTS: The spatial and temporal distribution, absorption, degradation, and metabolism of three NIs, imidacloprid, acetamiprid, and thiamethoxam, on different parts of apple trees were studied under foliar spray and root irrigation treatments. In the spray treatment, the initial average concentration ratios (TCRs) were 31.6% for lower shoots, 23.3% for upper leaves, 23.2% for upper shoots, 21.0% for lower leaves, and 0.5% and 0.4% for upper and lower fruits, respectively. The average half-lives of the three NIs were 2.9 days for shoots, 7.4 days for leaves, and 10.8 days for fruits. The degradation rate of shoots was 2.5 times that of leaves, and 3.6 times that of fruits. Imidacloprid olefin and N-methyl acetamiprid were two of the main metabolites. In the root treatment, both roots and soils had high TCRs during the whole sampling period. Only imidacloprid was transmitted to above-ground parts of the plants, with TCRs of 0.38-50.94%. CONCLUSION: This study found significant differences in spatial and temporal distribution, degradation, metabolism, and trends of NIs on different pest target sites of apple trees. The data obtained may help promote scientific control of target pests and evaluation of safety for non-target species in orchards. © 2020 Society of Chemical Industry.

Identification and field evaluation of the sex pheromone of Apolygus lucorum (Hemiptera: Miridae) in China.

BACKGROUND: The plant bug, Apolygus lucorum Meyer-Dür, has begun a resurgence and has become a key pest in cotton in northern China, with the wide-scale adoption of transgenic Bt cotton. We attempted to develop a new approach to the control of this plant bug by identifying and utilizing its sex pheromone. RESULTS: Extracts from A. lucorum adults and nymphs were analyzed using gas chromatography-mass spectrometry (GC-MS) and gas chromatography-electroantennogram detection (GC-EAD) to identify the sex pheromone components. (E)-4-Oxo-2-hexenal and (E)-2-hexenyl butyrate were the major sex pheromone components from females and were also electrophysiology-active towards male antennae. For males, (E)-4-oxo-2-hexenal and hexyl butyrate were the major sex pheromone components. These three components, however, were not detected in nymphs. Field tests showed that binary blends of (E)-4-oxo-2-hexenal and (E)-2-hexenyl butyrate at a ratio of 3:2 resulted in trapping the greatest numbers of A. lucorum males. A long-term dispenser was developed by loading the pheromone components dissolved in sunflower oil into polyethylene vials. Further field evaluation showed that the polyethylene dispensers attracted significantly more bugs even when deployed in the field for 5 weeks. CONCLUSION: We identified the sex pheromone of A. lucorum and developed a type of high-efficiency and long-term lure. Our results could expand knowledge of the pheromones of plant bugs, and provide novel technologies to monitor and control this pest. © 2019 Society of Chemical Industry.

Isotope-labeled internal standards and grouping scheme for determination of neonicotinoid insecticides and their metabolites in fruits, vegetables and cereals - A compensation of matrix effects.

Neonicotinoid insecticides (NIs) are widely used. However they produce high levels of residues and are toxic to non-target organisms, especially when their metabolites exhibit comparable or elevated toxicities. In this study, we established a multi-residue analytical method for analyzing 20 compounds - 8 NIs and their 11 major toxic metabolites. QuEChERS pretreatment and ultra-high-performance liquid chromatography-tandem mass spectrometry were used to detect residues in fruits, vegetables and cereals. Determination time was shortened to 6 min. Average recoveries of all compounds were in the range of 90.1-105.5%, with relative standard deviations lower than 15.0% and the limit of quantitation being 10 µg kg-1. For the first time, 8 NIs and 11 major toxic metabolites were grouped simultaneously, such that the matrix effect of analytes was satisfactorily corrected using their own isotopically labeled molecules; other NIs and metabolites in the same group were also corrected. This is the first report that simultaneously quantified multiple metabolites of eight NIs in multiple matrices.

Distribution, Dissipation, and Metabolism of Neonicotinoid Insecticides in the Cotton Ecosystem under Foliar Spray and Root Irrigation.

The uptake, distribution, metabolism, and degradation of three neonicotinoid insecticides (NIs)-imidacloprid (IMI), acetamiprid (ACE), and thiamethoxam (THI) in different parts of cotton plants were investigated under field conditions. Insecticides were either applied by foliar spraying or root irrigation. Foliar application resulted in high tissue concentration (average tissue concentration ratio, TCR: 46.78-68.61% for leaves and 12.2-31.40% for flowers). The flowers showed high NI residual. The metabolism and trends of NIs in different parts of cotton were reported here for the first time. Metabolites, toxic to bees, were detected in the flowers. The translocation factor was around 0.004 for the spray treatment and 0.2-0.7 for the root irrigation treatment. The average root concentration factors of IMI, ACE, and THI were 0.838, 8.027, and 1.014, respectively, indicating that the three NIs can be transported from the soil to the plant. The high concentrations of NIs and their metabolites in flowers indicate exposure risk to pollinators, such as bees.

Synthesis and antifungal activity of novel pyrazolines and isoxazolines derived from cuminaldehyde.

Two series of novel cuminaldehyde derivatives containing pyrazoline and isoxazoline moieties have been designed and synthesized. All of the compounds were characterized via 1H-NMR,13C-NMR, and HRMS. The antifungal activities were evaluated against six plant-pathogenic fungi. 3-(2-Fluorophenyl)-5-(4-isopropylphenyl) isoxazoline (2d) and 1-acetyl-3-(2-fluorophenyl)-5-(4-isopropylphenyl)-2-pyrazoline (3d) displayed higher antifungal activities than commercial fungicides against Sclerotinia sclerotiorum, Physalospora piricola and Pyricularia oryzae. The title compounds (2d and 3d) with strong antifungal activities are worth being further evaluated in vivo and in the field.

Design, Synthesis and Insecticide Activity of Novel Acetylcholinesterase Inhibitors: Triazolinone and Phthalimide Heterodimers.

Based on the "cluster effect" and the structure characters of acetylcholinesterase (AChE; EC 3.1.1.7), a new series of 1,2,4-triazolin-3-one and phthalimide heterodimers were designed, synthesized, and evaluated as potent dual acetylcholinesterase inhibitors (AChEIs). Most of the synthesized compounds showed good in vitro inhibitory activities towards both Drosophila melanogaster acetylcholinesterase (DmAChE) and Musca domestica acetylcholinesterase (MdAChE). Among them, 5g was found to be the most potent anti-AChE derivate (5g, IC50 = 8.07 µM to DmAChE, IC50 = 32.24 µM to MdAChE). It was 2.31- and 1.35-fold more active than the positive control ethion (CP, IC50 = 18.62 µM to DmAChE, IC50 = 43.56 µM to MdAChE). The docking model study revealed that 5g possessed the fitted spatial structure and bound to the central pocket and peripheral site of DmAChE. Moreover, most compounds demonstrated high insecticidal activity to Lipaphis erysimi and Tetranychus cinnabarinus at the concentration of 300 mg/L.

Identification and expression of candidate chemosensory receptors in the white-spotted flower chafer, Protaetia brevitarsis.

Accurate detection and recognition of chemical signals play extremely important roles for insects in their survival and reproduction. Chemosensory receptors, including odorant receptors (ORs), ionotropic receptors (IRs) and gustatory receptors (GRs), are involved in detection of volatile signals. In the present study, we aimed to identify candidate chemosensory receptors, and RNA-seq technology was employed to sequence the antennal transcriptome of Protaetia brevitarsis (Coleoptera: Scarabaeidae: Cetoniinae), a native agricultural and horticultural pest in East-Asia. According to the sequence similarity analysis, we identified 72 PbreORs, 11 PbreGRs and eight PbreIRs. Among PbreORs, PbreOR2, PbreOR33 and PbreOR53 were preliminarily classified into pheromone receptors. Further qRT-PCR analysis indicated that 11 PbreORs were specifically expressed in the antennae of male P. brevitarsis, whereas 23 PbreORs were specifically expressed in the female antennae. Our results laid a solid foundation for further functional elucidations of insect chemoreceptors, which could be used as the potential targets of pest management.

Modeling of the Phytophthora capsici cellulose synthase 3 and its inhibitors activity assay.

BACKGROUND: Phytophthora capsici is a devastating pathogen for crop. Cellulose synthase 3 (CesA3) is a target for many potential fungicides such as valinamide derivatives. However, the 3-dimensional structure (3-DS) of CesA3 in Phytophthora capsici was still unknown. RESULTS: Here CesA3 protein sequence was retrieved from the NCBI protein sequence database We did the 3-DS structural modeling for CesA3 and used molecular dynamics to optimize the model. The model was further validated by the Ramachandran plot in PROCHECK program. Two series of new valinamide compound were synthesized and tested for its biological activity. The docking data obtained by the model perfectly matched with the biometric data, indicating that the model is valid. Moreover, docking study data revealed the mechanism of action of inhibitors on target enzymes. CONCLUSION: The 3-DS structural model was analyzed from the perspective of the biocide receptor, the structure of the target protein and the mechanism of action of the compound. It provides a new perspective for the design of new fungicides. © 2019 Society of Chemical Industry.

Synthesis and bioactivity studies of sex pheromone analogs of the diamond back moth, Plutella xylostella.

BACKGROUND: Diamondback moth, Plutella xylostella L., is a very important pest of cruciferous vegetables causing excessive economic losses worldwide. Bioactivities of halo-, diazo-, and cyclopropane acetates of P. xylostella sex pheromone have been evaluated using electrophysiology and enzyme inhibition assays. RESULTS: A total of 23 sex pheromone analogs of P. xylostella were designed and synthesized and the result shows that (11Z)-hexadec-11-en-1-yl 2,2,2-trifluoroacetate, (11Z)-hexadec-11-en-1-yl 2,2,3,3,3-pentafluoropropanoate, and (11Z)-hexadec-11-en-1-yl trifluoromethanesulfonate elicited potential inhibitory effects at all doses tested in the electrophysiology and enzyme inhibition assays. Interference of locating the sex pheromone source was found strongest when these three analogs were mixed with the sex pheromone at a 10:1 ratio. In addition, field test showed that the rate of mating disruption was over 90% when (11Z)-hexadec-11-en-1-yl 2,2,2-trifluoroacetate or (11Z)-hexadec-11-en-1-yl 2,2,3,3,3-pentafluoropropanoate was mixed with the sex pheromone at a 10:1 ratio. CONCLUSION: Two sex pheromone antagonists were screen out by electrophysiology, enzyme inhibition assays, wind tunnel and field tests. We believe that these antagonists could be used to establish a novel eco-friendly measure to control P. xylostella and provide evidence for clarifying the specific functions and molecular mechanisms of sex pheromone antagonists. © 2018 Society of Chemical Industry.

Potential exposure to clothianidin and risk assessment of manual users of treated soil.

BACKGROUND: Treated soil is the second most prevalent application technique for all registered pesticides in China. Some developing countries also adopt this method. However, the safety of this scenario has not been reported in the literature. Experiments were therefore conducted to assess exposure using standard whole-body dosimetry and air sampling methodologies. RESULTS: Dermal deposition was the main route of exposure in this scenario. The total dermal unit exposure (UE) of operators to clothianidin-treated soil was 51.7 mg kg-1 AI handled (SD = 20.59, n = 16), and hands accounted for 36%. Inhalation UE was 0.04 mg kg-1 AI handled (SD = 0.02, n = 4), negligible compared with dermal exposure. Using an NOAEL (no observed adverse effect level) of 10 mg kg-1 day-1 , the margin of exposure was 773, i.e. greater than 100. CONCLUSION: For the first time, the scenario of treated soil exposure was assessed and was found to pose less risk than conventional pesticide application. These results can be used as a reference in pesticide management. © 2017 Society of Chemical Industry.

Attraction of Coffee Bean Weevil, Araecerus fasciculatus, to Volatiles from the Industrial Yeast Kluyveromyces lactis.

The coffee bean weevil (CBW), Araecerus fasciculatus (De Geer, 1775) (Coleoptera: Anthribidae) is an important pest of stored products such as grains, coffee beans, cassava, and traditional Chinese medicine materials. In China, CBW causes large losses of Daqu, a traditional Chinese liquor fermentation starter, and, unfortunately, the use of conventional insecticides against CBW is not suitable in Daqu storage. We found CBW to be highly attracted to fermenting yeast cultures, such as Kluyveromyces lactis. Eight volatile compounds, produced by fermenting cultures and not by sterile samples, were identified by gas chromatography coupled with mass spectrometry. Five of these substances elicited significant responses in Y-tube behavioral bioassays. Field trapping experiments revealed 2-phenylethanol and 2-phenylethyl acetate to be crucial for attraction of CBW. Results show that yeast volatiles play an important role in host location, and that 2-phenylethanol and 2-phenylethyl acetate could be utilized as potential attractants in monitoring and control systems against this important pest.

Design, synthesis and bioactivity of novel phthalimide derivatives as acetylcholinesterase inhibitors.

A series of novel phthalimide derivatives related to benzylpiperazine were synthesized and evaluated as cholinesterase inhibitors. The results showed that all compounds were able to inhibit acetylcholinesterase (AChE), with two of them dramatically inhibiting butyrylcholinesterase (BuChE). Most compounds exhibited potent anti-AChE activity in the range of nM concentrations. In particular, compounds 7aIII and 10a showed the most potent activity with the IC50 values of 18.44 nM and 13.58 nM, respectively. To understand the excellent activity of these compounds, the structure-activity relationship was further examined. The protein-ligand docking study demonstrated that the target compounds have special binding modes and these results are in agreement with the kinetic study.

Synthesis and biological activity of novel benzimidazole derivatives as potential antifungal agents.

In the present study, a series of novel benzimidazole derivatives containing chrysanthemum acid moieties was designed and synthesized. Preliminary investigation of biological activity indicated that all of the compounds exhibited lower activity than that of beta-cypermethrin against Plutella xylostella and Lipaphis erysimi; meanwhile, they showed good inhibitory activity against Botrytis cinerea and Sclerotinia sclerotiorum in vitro. The fungicidal activity of compound 8a against B. cinerea was approximately equal to that of thiabendazole and was twice as active against S. sclerotiorum as was thiabendazole. In addition, compound 9e displayed the most potent inhibitory activity against both fungi and was almost twice as potent as thiabendazole.