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      Detection and biochemical characterization of insecticide resistance in field populations of Asian citrus psyllid in Guangdong of China

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          Abstract

          The Asian citrus psyllid, Diaphorina citri Kuwayama, is one of the most damaging pests of citrus-producing regions throughout the world. The use of insecticides is the main strategy for controlling psyllid and has increased year by year. In this study, four field populations of D. citri were evaluated for resistance to nine different insecticides using the leaf-dip method. The results showed that the highest level of resistance for D. citri was found in imidacloprid with a resistance ratio of 15.12 in the Zengcheng population compared with the laboratory susceptible population. This was followed by chlorpyriphos (6.47), dinotefuran (6.16), thiamethoxam (6.04), lambda-cyhalothrin (4.78), and bifenthrin (4.16). Piperonyl butoxide (PBO) and triphenyl phosphate (TPP) showed significant synergism on imidacloprid effects in the Zengcheng population (3.84- and 2.46-fold, respectively). Nevertheless, diethyl maleate (DEM) had no synergism on imidacloprid. Biochemical enzyme assays suggested that general esterase, glutathione S-transferase and cytochrome P450 monooxygenase activities were higher in the field-collected populations than in the laboratory susceptible population. However, glutathione S-transferase may play a minor role in the resistance of adult D. citri to insecticides. At the molecular level, resistance of D. citri to imidacloprid is mainly related to the increased expression of CYP4C68 and CYP4G70 (>5-fold).

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          RNA interference-mediated knockdown of a cytochrome P450, CYP6BG1, from the diamondback moth, Plutella xylostella, reduces larval resistance to permethrin.

          We have previously reported that a cytochrome P450, CYP6BG1, from Plutella xylostella was found to be overexpressed in 4th instars of a permethrin resistant strain and inducible in the susceptible counterpart. The findings suggested potential involvement of CYP6BG1 in permethrin resistance, hence warranted a functional analysis. To assess the functional link of the gene to permethrin resistance, we adopted RNA interference-mediated gene silencing (RNAi) by dsRNA droplet feeding. Here, real time PCR analyses show that oral delivery of dsRNA can efficiently reduce the expression of CYP6BG1. Knockdown of CYP6BG1 transcript was evident in midgut and larval tissues enclosed in carcass. As a consequence of knockdown, a significant reduction in resistance of larvae fed CYP6BG1 dsRNA was observed after 24 and 48h of exposure to permethrin. In addition, CYP6BG1 dsRNA feeding to larvae led to reduced total P450 activities of microsomal preparations toward model substrates p-nitroanisole and benzyloxyresorufin. These results indicate that the overexpressed CYP6BG1 participate in enhanced metabolism of permethrin, thereby, resistance. The knockdown of a non-overexpressed P450, CYP6BF1v4, from the same resistant P. xylostella strain did not lead to changes in the level of resistance to permethrin, supporting further the specific involvement of CYP6BG1 in the resistance.
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            Double-Stranded RNA Uptake through Topical Application, Mediates Silencing of Five CYP4 Genes and Suppresses Insecticide Resistance in Diaphorina citri

            Silencing of genes through RNA interference (RNAi) in insects has gained momentum during the past few years. RNAi has been used to cause insect mortality, inhibit insect growth, increase insecticide susceptibility, and prevent the development of insecticide resistance. We investigated the efficacy of topically applied dsRNA to induce RNAi for five Cytochrome P450 genes family 4 (CYP4) in Diaphorina citri. We previously reported that these CYP4 genes are associated with the development of insecticide resistance in D. citri. We targeted five CYP4 genes that share a consensus sequence with one dsRNA construct. Quantitative PCR confirmed suppressed expression of the five CYP4 genes as a result of dsRNA topically applied to the thoracic region of D. citri when compared to the expression levels in a control group. Western blot analysis indicated a reduced signal of cytochrome P450 proteins (45 kDa) in adult D. citri treated with the dsRNA. In addition, oxidase activity and insecticide resistance were reduced for D. citri treated with dsRNA that targeted specific CYP4 genes. Mortality was significantly higher in adults treated with dsRNA than in adults treated with water. Our results indicate that topically applied dsRNA can penetrate the cuticle of D. citri and induce RNAi. These results broaden the scope of RNAi as a mechanism to manage pests by targeting a broad range of genes. The results also support the application of RNAi as a viable tool to overcome insecticide resistance development in D. citri populations. However, further research is needed to develop grower-friendly delivery systems for the application of dsRNA under field conditions. Considering the high specificity of dsRNA, this tool can also be used for management of D. citri by targeting physiologically critical genes involved in growth and development.
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              Insecticidal Suppression of Asian Citrus Psyllid Diaphorina citri (Hemiptera: Liviidae) Vector of Huanglongbing Pathogens

              Diaphorina citri vectors pathogens that cause ‘huanglongbing’ or citrus greening disease which poses a serious threat to citrus production worldwide. Vector suppression is critical to reduce disease spread. Efficacy is a main concern when choosing an insecticide. Insecticidal treatments of 49 products or 44 active ingredients (a.i) labeled or experimental were field tested between 2005–2013 as foliar sprays (250 treatments, 39 a.i) or soil applications (47 treatments, 9 a.i) to control D. citri in citrus. A combined effect of nymphal and adult suppression in response to sprays of 23 insecticides representing 9 modes of action (MoA) groups and 3 unknown MoA provided more than 90% reduction of adult D. citri over 24–68 days. Observable effects on nymphs were generally of shorter duration due to rapid maturation of flush. However, reduction of 76–100% nymphs or adults over 99–296 days was seen on young trees receiving drenches of the neonicotinoids imidacloprid, thiamethoxam or clothianidin (MoA 4A) and a novel anthranilic diamide, cyantraniliprole (MoA 28). Effective products identified for foliar sprays to control D. citri provide sufficient MoA groups for rotation to delay evolution of insecticide resistance by D. citri and other pests. However, cyantraniliprole is now the only available alternative for rotation with neonicotinoids in soil application to young trees. Sprays of up to eight of the most effective insecticides could be rotated over a year without repetition of any MoA and little or no recourse to neonicotinoids or cyantraniliprole, so important for protection of young trees. Other considerations effecting decisions of what and when to spray include prevalence of huanglongbing, pest pressure, pre-harvest intervals, overall budget, equipment availability, and conservation of beneficial arthropods. Examples of spray programs utilizing broad-spectrum and relatively selective insecticides are provided to improve vector management and may vary depending on individual or regional assessment of all factors.
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                Author and article information

                Contributors
                zengxn@scau.edu.cn
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                22 August 2018
                22 August 2018
                2018
                : 8
                : 12587
                Affiliations
                ISNI 0000 0000 9546 5767, GRID grid.20561.30, College of Agriculture, , South China Agricultural University, ; Guangzhou, 510642 China
                Article
                30674
                10.1038/s41598-018-30674-5
                6105715
                30135479
                a6e6f73d-1470-4404-a423-222352f16814
                © The Author(s) 2018

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 25 September 2017
                : 30 July 2018
                Funding
                Funded by: FundRef https://doi.org/10.13039/501100001809, National Natural Science Foundation of China (National Science Foundation of China);
                Award ID: 31572314
                Award Recipient :
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