The development of an egg-soaking method for delivering dsRNAs into spider mites.

Recently, the first sprayable RNAi biopesticide, Ledprona, against the Colorado potato beetle, Leptinotarsa decemlineata, has been registered at the United States Environmental Protection Agency. Spider mites (Acari: Tetranychidae), a group of destructive agricultural and horticultural pests, are notorious for rapid development of insecticide/acaricide resistance. The management options, on the other hand, are extremely limited. RNAi-based biopesticides offer a promising control alternative to address this emerging issue. In this study, we i) developed an egg-soaking dsRNA delivery method; ii) evaluated the factors influencing RNAi efficiency, and finally iii) investigated the potential mode of entry of this newly developed egg-soaking RNAi method. In comparison to other dsRNA delivery methods, egg-soaking method was the most efficient, convenient/practical, and cost-effective method for delivering dsRNAs into spider mites. RNAi efficiency of this RNAi method was affected by target genes, dsRNA concentration, developmental stages, and mite species. In general, the hawthorn spider mite, Amphitetranychus viennensis, is more sensitive to RNAi than the two-spotted spider mite, Tetranychus urticae, and both of them have dose-dependent RNAi effect. For different life stages, egg and larvae are the most sensitive life stages to dsRNAs. For different target genes, there is no apparent association between the suppression level and the resultant phenotype. Finally, we demonstrated that this egg-soaking RNAi method acts as both stomach and contact toxicity. Our combined results demonstrate the effectiveness of a topically applied dsRNA delivery method, and the potential of a spray induced gene silencing (SIGS) method as a control alternative for spider mites.

Surfactants Improving the Wetting Behavior and Adhesion Mechanism of Pesticide Dilution Droplets on Jujube Leaf Surfaces.

Fruit tree leaves have different chemical compositions and diverse wax layer structures that result in different patterns of wetting and pesticide solution spreading on their surface. Fruit development is a time when pests and diseases occur, during which a large number of pesticides are needed. The wetting and diffusion properties of pesticide droplets on fruit tree leaves were relatively poor. To solve this problem, the wetting characteristics of leaf surfaces with different surfactants were studied. The contact angle, surface tension, adhesive tension, adhesion work, and solid-liquid interfacial tension of five surfactant solution droplets on jujube leaf surfaces during fruit growth were studied by the sessile drop method. C12E5 and Triton X-100 have the best wetting effects. Two surfactants were added to a 3% beta-cyfluthrin emulsion in water, and field efficacy tests were carried out on peach fruit moths in a jujube orchard at different dilutions. The control effect is as high as 90%. During the initial stage when the concentration is low, due to the surface roughness of the leaves, the surfactant molecules adsorbed at the gas-liquid and solid-liquid interfaces reach an equilibrium, and the contact angle on the leaf surface changes slightly. With increasing surfactant concentration, the pinning effect in the spatial structure on the leaf surface is overcome by liquid droplets, thereby significantly decreasing the contact angle. When the concentration is further increased, the surfactant molecules form a saturated adsorption layer on the leaf surface. Due to the existence of a precursor water film in the droplets, surfactant molecules on the interface continuously move to the water film on the surface of jujube tree leaves, thus causing interactions between the droplets and the leaves. The conclusion of this study provides theoretical guidance for the wettability and adhesion of pesticides on jujube leaves, so as to achieve the purpose of reducing pesticide use and improving pesticide efficacy.

Target gene selection for RNAi-based biopesticides against the hawthorn spider mite, Amphitetranychus viennensis (Acari: Tetranychidae).

BACKGROUND: Recently, RNA interference (RNAi)-based biopesticide, a species-specific pest control alternative, has been deregulated and commercialized in the US and Canada. The hawthorn spider mite, Amphitetranychus viennensis Zacher, is a major pest for rosaceous plants, which has been controlled primarily by synthetic pesticides. To address the emerging resistance issues in A. viennensis, we initiated a project to develop RNAi-based biopesticides. RESULTS: In this study, we (i) developed a dietary RNAi system for A. viennensis using leaf disc, (ii) assessed the suitability of multiple control genes to distinguish sequence-specific silencing from non-specific effects within this RNAi system, and (iii) screened for the target gene candidates. As a result, ß-Glucuronidase (GUS), an enzyme derived from E. coli and a broadly used reporter for plants is the appropriate control for A. viennensis RNAi, while green fluorescent protein (GFP), is not suitable due to its significantly higher mortality than the other controls. For target gene screening, suppression was confirmed for all the candidates, including two housekeeping genes (Vacuolar-type H + -ATPase subunit A (V-ATPase A) and Glyceraldehyde 3-phosphate dehydrogenase, (GAPDH)), and three genes associated with development (ATP-dependent RNA Helicase DDX3Y (Belle), CREB-binding protein (CBP), and Farnesoic acid O-methyltransferase (FaMet)). Knocking down of V-ATPase A resulted in the highest mortality (~ 90%) and reduced fecundity (over 90%) than other candidates. As for the genes associated with development, suppression of Belle and CBP, led to approximately 65% mortality, as well as 86% and 40% reduction in fecundity, respectively. Silencing of FaMet, however, had negligible biological impacts on A. viennensis. CONCLUSION: The combined efforts not only establish an effective dsRNA delivery method, but also provide potential target genes for RNAi-based biopesticides against A. viennensis, a devastating invasive pest for fruit trees and woody ornamental plants throughout Asia and Europe. © 2023 Society of Chemical Industry.

Energy Utilization and Flight Muscle Development in Chrysoperla sinica (Neuroptera: Chrysopidae) Female Adults: Relationship With Age and Flight Behavior.

We investigated the accumulation of energy substances, dynamics of flight muscle development, changes in energy substances accumulation, and flight muscle structure after flight activities in female adults of the green lacewing Chrysoperla sinica (Tjeder), a common natural enemy of various insect pests in China. Virgin individuals were chosen at 24, 72, and 120 h after eclosion for energy substance determination and flight muscle observation in this study. Individuals with strong flight ability at 72 h after eclosion were selected for tethered flight assays, followed by detection of energy substances, and flight muscle observation. The results showed that virgin female adults had the highest fat content 72 h after eclosion. Sarcomere length and myofibril diameter changed significantly with age, with the lowest at 24 h after eclosion. With an increase in flight distance, the fat and glycogen contents, sarcomere length, and volume fraction of the transverse tubular system (T-system) decreased and myofibril diameters increased. The volume fraction of the mitochondria did not significantly change, but the structure of the mitochondrial membrane was destroyed, inclusions were reduced, and cavities appeared. The reserves of energy substances, especially lipids, are closely related to the flight ability of C. sinica. The observational results of both flight muscle structure and morphology of mitochondria build a strong relationship with flight behavior. This research should help reveal the regulatory mechanism of flight activity of C. sinica.

Effects of Imidacloprid Applied Alone or in Combination With Organosilicone Surfactants on Biological Traits and Predatory Feeding of Chrysoperla nipponensis (Neuroptera: Chrysopidae).

Organosilicone adjuvants are widely used to increase insecticide application on targeted surfaces. In this work, our aim was to investigate side effects of imidacloprid treatment, either applied alone or in combination with organosilicone compounds, against Chrysoperla nipponensis, an important predator of aphids. Four types of organosilicones were mixed with imidacloprid at different concentrations. The toxicity of the mixture to C. nipponensis was measured under laboratory conditions. The LC50 and LC30 of imidacloprid applied alone and in combination with 0.05% organosilicone were determined. Imidacloprid (LC30) applied alone or in combination with 0.05% organosilicone was used to treat second instar larvae of C. nipponensis; thereafter, its effects on the growth, development, longevity, reproduction, and predatory ability of C. nipponensis were evaluated The results demonstrated that the organosilicone Silwet L-77 reduced the LC50 and LC30 of imidacloprid to 6.09 (95% CI: 2.31-9.42) and 10.95 mg/L (95% CI: 8.16-13.63), respectively, and enhanced imidacloprid toxicity to C. nipponensis, as reflected by the resulting extension of the growth and developmental period, reduction in female longevity, and inhibition of reproduction. When applied alone or in combination with an organosilicone, imidacloprid reduced the consumption of Corcyra cephalonica eggs by C. nipponensis. The functional response of C. nipponensis treated with imidacloprid alone or in combination with organosilicone was type II. Concomitantly, the attack rate was reduced and the handling time of prey increased.

Analysis of Differentially Expressed Genes of Chrysoperla sinica Related to Flight Capacity by Transcriptome.

The lacewing Chrysoperla sinica (Tjeder) is a common natural enemy of many insect pests in China and is frequently employed for biological control programs. Adults make migratory flights after emergence, which reduces their effectiveness as biological control agents. Previously, we proved that 2-d-old unmated females exhibited significantly stronger flight ability than 3-d-old ones. Meanwhile, 3-d-old unmated adults flew significantly longer distances than mated ones. In this study, Illumina RNA sequencing was performed to characterize differentially expressed genes (DEGs) between virgin and mated adults of different ages in a single female strain of C. sinica. In total, 713,563,726 clean reads were obtained and de novo assembled into 109,165 unigenes with an average length of 847 bp (N50 of 1,754 bp), among which 4,382 (4.01%) unigenes matched known proteins. Based on these annotations, many putative transcripts were related to C. sinica's flight capacity and muscle structure, energy supply, growth, development, environmental adaptability, and metabolism of nutritional components and bioactive components. In addition, the differential expression of transcripts between different ages and mating status were analyzed, and DEGs participating in flight capacity and muscles were detected, including glutathione hydrolase, NAD-specific glutamate dehydrogenase, aminopeptidase, and acidic amino acid decarboxylase. The DEGs with functions associated with flight capacity and muscles exhibited higher transcript levels for younger (2 d--old) virgins. This comprehensive C. sinica transcriptomic data provide a foundation for a better understanding of the molecular mechanisms underlying the flight capacity to meet the physiological demands of flight muscles in C. sinica.

Selection of Reference Genes for RT-qPCR Analysis Under Extrinsic Conditions in the Hawthorn Spider Mite, Amphitetranychus viennensis.

Hawthorn spider mite, Amphitetranychus viennensis Zacher, is an economically important arthropod pest for fruit trees and woody ornamental plants. Extensive and repetitive use of synthetic acaricides has led to the development of resistance in A. viennensis. To understand the molecular basis of pesticide resistance, and to develop genetic-based control alternatives (e.g., RNAi-based biopesticides), a standardized protocol for real-time quantitative reverse transcription PCR (RT-qPCR) is needed. In the proceeding phase of this research, we screened for the internal references for RT-qPCR analysis from a pool of A. viennensis housekeeping genes under the intrinsic conditions, including developmental stage, sex, and diapause. Here, we continued our efforts to search for the reference genes under an array of extrinsic conditions, including temperature, humidity, photoperiod, host plant, and dietary RNAi. The stability of these candidate reference genes was investigated using geNorm, NormFinder, BestKeeper, and ΔCt method, respectively. Finally, RefFinder, a statistical platform integrating all four algorisms, provided a comprehensive list of genes for each extrinsic condition: (1) EF1A, α-tubulin and Actin3 were the best candidates for temperature, (2) GAPDH, 18S, and Actin3 were the most stable genes for humidity, (3) V-ATPase B, Actin3, and 18S were the top reference genes for photoperiod, (4) GAPDH, V-ATPase B, and α-tubulin were recommended for host plants, and (5) GAPDH, V-ATPase B, and RPS9 were the top choices for dietary RNAi. Overall, V-ATPase B, GAPDH, and Actin3 were the most commonly selected reference genes in A. viennensis regardless of the experimental conditions, including both intrinsic and extrinsic. Information present here lays the foundation for the genomic and functional genomic research in A. viennensis.

Wetting and adhesion behavior on apple tree leaf surface by adding different surfactants.

OBJECTIVE: This study was conducted to investigate the wetting behavior of different surfactant solutions on the leaf surfaces of apple during the fruit formation stage. METHODS: Five surfactants, including C12E5, Tween-20, Triton X-100, DTAB, and SDS were evaluated in this study. The contact angle, surface tension, adhesion tension, work of adhesion, and solid-liquid interface tension of droplets on the leaf surface were determined by the drop method. RESULTS: The results showed that the nonionic surfactants C12E5 and Triton X-100 had better wetting effects than other surfactants. Moreover, when the concentration of C12E5 and Triton X-100 was 1 × 10-3 mol/L, the leaves reached a completely wet state. Toxicity measurement showed that the incubation rate of Carposina niponensis eggs decreased gradually with increasing content of C12E5 or Triton X-100. Additionally, field efficacy analysis showed that adding C12E5 or Triton X-100 significantly improved the beta-cyfluthrin 3% water emulsion (EW) against C. niponensis. CONCLUSIONS: These results indicate that the surfactants C12E5 and Triton X-100 can significantly improve pesticide application, which will be helpful for reducing pesticide use and developing new pesticides.

Selection of Reference Genes for RT-qPCR Analysis Under Intrinsic Conditions in the Hawthorn Spider Mite, Amphitetranychus viennensis (Acarina: Tetranychidae).

Hawthorn spider mite, Amphitetranychus viennensis Zacher, is one of the most devastating pests of deciduous fruit trees. The overall goal of this research is to develop a standardized protocol for real-time quantitative reverse transcription PCR (RT-qPCR) analysis in A. viennensis following the MIQE (minimum information for publication of Quantitative real time PCR experiments) guidelines. Based on the previous knowledge, we hypothesized that internal references for RT-qPCR analysis reside in housekeeping genes (HKGs). To test this hypothesis, we examined the stability of nine HKGs from A. viennensis, including 18S ribosomal RNA (18S), 28S ribosomal RNA (28S), Elongation factor 1-alpha (EF1A), Actin3, V-ATP vacuolar-type H+-ATPase (V-ATPase), α-tubulin (α-tubulin), Ribosomal protein L13 (RPL13), 40S ribosomal protein S9 (RPS9), and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The expression profile of these candidates under intrinsic conditions was evaluated by a panel of computational programs, including geNorm, Normfinder, BestKeeper, and ΔCt method. Based on RefFinder, a comprehensive software integrating all four above-mentioned algorithms, V-ATPase, Actin3, and GAPDH are the top three reference genes, which are stably expressed across all the intrinsic conditions, including developmental stage, sex, and diapause. In addition, we compared reference genes recommended for different developmental stages among the nine cell-content feeding arthropods, including four spider mites, A. viennensis, Tetranychus urticae, Tetranychus cinnabarinus, and Panonychus citri, and five hemipterans, Myzus persicae, Aphis gossypii, Toxoptera citricida, Lipaphis erysimi, and Sogatella furcifera. Not surprisingly, rRNAs and ribosomal proteins, the most abundant RNA species, is the top choice, and follows by EF1A, Actin, GAPDH, and tubulin. Information present here lays the foundation for the genomic and functional genomic research in cell-content feeding arthropods in general and A. viennensis in particular.

Wettability of pear leaves from three regions characterized at different stages after flowering using the OWRK method.

BACKGROUND: A better understanding of leaf surface wettability is critical to improve the adhesion of liquid pesticides. Leaf surface wettability is dependent on the property of the liquid as well as the physical and chemical properties of the leaf, which vary with climate and growth stage. The aim of this study was to characterize the wettability of pear leaves from three different climatic regions at different stages after flowering. RESULTS: The contact angles of different test liquids were measured on both adaxial and abaxial pear leaf surfaces and the Owens-Wendt-Rabel-Kaelble (OWRK) method was used to calculate surface free energy (SFE) and its polar and non-polar components. The results demonstrated that the SFE of both the adaxial and abaxial surface of the pear leaf, and the proportion of polar component, increased with increasing time after flowering. At early growth stages, pear leaves were highly hydrophobic, similar to a polytetrafluoroethylene surface, whereas at later growth stages, pear leaves were hydrophobic, more similar to a polymethylmethacrylate surface. Also, the SFE differed with climatic region. Factors influencing these changes are discussed. CONCLUSION: Changes in contact angles and SFE correlated with the change of the leaf surface wettability. Leaves became easier to wet (higher SFE), with an overall increasing polar component to the surface, with increasing age after flowering. As expected, changes in wettability were found in pear leaves at different stages after flowering and in different regions (P < 0.05). Pear leaves from Yuanping were easier to wet than leaves from Yuci and Linyi, and adaxial surfaces were easier to wet than abaxial surfaces. These results provide beneficial information for the application of agrochemicals for improved wetting and spreading behavior. © 2018 Society of Chemical Industry.

[Distribution and source apportionment of n-alkanes in atmospheric particle in Taiyuan, China].

The n-alkanes in PM10 and typical emission sources samples collected during heating and non-heating periods in Taiyuan were determined with GC-MS. Meanwhile, the distribution characteristics and source identification of n-alkanes were investigated with diagnostic parameters and principal component analysis (PCA). Concentrations of n-alkanes ranged from 213.74 to 573.32 ng.m-3 and 22.69 to 150.82 ng.m-3 in the heating and non-heating seasons, respectively. The n-alkanes concentrations in suburban districts including JY, JCP, XD and SL were higher than those in urban sites in the heating quarter, and the relative concentration in JS was 7 times higher than that in SL in the other period. The correlation of the total n-alkanes in PM10 with that derived from fossil fuel was higher than the correlation with those from plant in the heating quarter, while the opposite result was detected in the other period, manifesting higher contribution of fossil fuel in the heating days. CPI and % WNA values showed that the contribution from plant wax in the non-heating period was higher than that in the heating period, and the alkanes production rate was elevated along with the increase in environmental pressures. Information on higher organic matter maturity was obtained during the heating period by Cmax and OEP and the existence of UCM bulge confirmed that vehicles were the significant contributor to n-alkanes concentration during the whole year. PCA analysis indicated the major component was the mixture of vehicle emission and higher plant, accounting for 51.28% of the total variances, followed by coal dust, accounting for 43. 14%. Cooperating control of emissions from coal combustions and vehicles would be the effective way to lower the concentrations of the corresponding n-alkanes.