Citrus-mediated gene silencing of cytochrome P450 suppresses insecticide resistance and increases mortality in Diaphorina citri.

BACKGROUND: Asian citrus psyllid, Diaphorina citri, is a hemipteran that vectors the causal pathogen of citrus greening disease, or huanglongbing (HLB). HLB is a tree killing disease that has severely limited citrus production globally. Unfortunately, there is no cure for this disease, and mitigation depends on multiple insecticide applications to reduce vector populations. Silencing of cytochrome P450 expression associated with detoxification enzymes by RNA interference is known to increase susceptibility of D. citri to insecticides. However, dsRNA was previously introduced into psyllids by topical applications. The possible application of this technology for pest management will require effective field delivery of the dsRNA. Therefore, we evaluated a virus vector (Citrus tristeza virus; 'mild strain' T36) to deliver gene silencing directly to this sap-sucking insect via plant phloem. Citrus macrophylla plants inoculated with CTV expressing a truncated consensus sequence of CYP450 (CTV-tCYP450) constantly produced small interfering RNA in the plant phloem that targeted five cytochrome p540 (CYP450) genes in D. citri. RESULTS: Insecticide susceptible D. citri reared on citrus infected with CTV-tCYP450 were subsequently more susceptible to imidacloprid, fenpropathrin, carbaryl, and chlorpyrifos than those reared on citrus infected with wildtype CTV or non-infected negative controls. Additionally, nymph survival and adult lifespan were significantly reduced when psyllids were reared on CTV-tCYP450 citrus plants compared with controls. Interestingly, similar results were obtained after one and two generations of rearing. Finally, field-collected psyllids from areas with known broad-spectrum insecticide resistance were rendered more susceptible to imidacloprid and fenpropathrin after feeding on CTV-tCYP450 citrus trees as compared with those reared on controls. CONCLUSION: The integration of citrus-mediated RNA inference targeting psyllid detoxification enzymes could function as a resistance management tool and reduce insecticide input in an integrated pest management program for HLB. © 2024 Society of Chemical Industry.

The beta pore-forming bacterial pesticidal protein Tpp78Aa1 is toxic to the Asian citrus psyllid vector of the citrus greening bacterium.

The Asian citrus psyllid (ACP) Diaphorina citri transmits the causative agent of huanglongbing, or citrus greening disease, that has decimated global citrus production. Pesticidal proteins derived from bacteria such as Bacillus thuringiensis (Bt) can provide effective and environmentally friendly alternatives for management of D. citri, but few with sufficient toxicity to D. citri have been identified. Here, we report on the toxicity of 14 Bt-derived pesticidal proteins from five different structural groups against D. citri. These proteins were selected based on previously reported toxicity to other hemipteran species and on pesticidal protein availability. Most of the proteins were expressed in Escherichia coli and purified from inclusion bodies or His-tag affinity purification, while App6Aa2 was expressed in Bt and purified from spore/crystal mixtures. Pesticidal proteins were initially screened by feeding psyllids on a single dose, and lethal concentration (LC50) then determined for proteins with significantly greater mortality than the buffer control. The impact of CLas infection of D. citri on toxicity was assessed for selected proteins via topical feeding. The Bt protein Tpp78Aa1 was toxic to D. citri adults with an LC50 of approximately 204 µg/mL. Nymphs were more susceptible to Tpp78Aa1 than adults but no significant difference in susceptibility was observed between healthy and CLas-infected nymphs or adults. Tpp78Aa1 and other reported D. citri-active proteins may provide valuable tools for suppression of D. citri populations.

Influence of 'Candidatus Liberibacter asiaticus' infection on the susceptibility of Asian citrus psyllid, Diaphorina citri to Bacillus thuringiensis pesticidal proteins, Mpp51Aa1 and Cry1Ba1.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae) transmits the Gram-negative bacterium 'Candidatus Liberibacter asiaticus' that causes citrus greening disease. While chemical control has been the main management strategy for limiting D. citri, the widespread usage of chemical sprays has decreased the susceptibility of D. citri to most insecticides. Pesticidal proteins produced by the bacterium Bacillus thuringiensis (Bt) are active against a wide variety of insects and provide a more sustainable approach to insect control. Herein, we investigated the impact of 'Ca. L. asiaticus' infection of D. citri on the toxicity of two Bt proteins (Mpp51Aa1 and Cry1Ba1). Proteins were delivered to healthy and 'Ca. L. asiaticus'-infected D. citri via topical feeding application. The LC50 values of Mpp51Aa1 and Cry1Ba1 were calculated for both nymphs and adults. Additionally, we evaluated the effect of each protein on the survival probability and life span of healthy and 'Ca. L. asiaticus'-infected D. citri. The LC50 values indicated that adults and nymphs were more susceptible to Mpp51Aa1 than to Cry1Ba1 in both healthy and 'Ca. L. asiaticus'-infected D. citri. 'Ca. L. asiaticus'-infected adults and nymphs were more susceptible to Mpp51Aa1 and Cry1Ba1 than healthy insects, and nymphs were more susceptible to Mpp51Aa1 and Cry1Ba1 than adults. Moreover, we found that Mpp51Aa1 had a greater impact than Cry1Ba1 on the survival and lifespan of adults, and 'Ca. L. asiaticus'-infected insects were more affected by these pesticidal proteins than healthy adults. These results have important implications for the use of pesticidal proteins in D. citri management in Florida and elsewhere given the widespread presence of 'Ca. L. asiaticus' in the D. citri population. In this era of eco-friendly control strategies, Bt-derived pesticidal proteins provide a promising avenue to reducing the application of chemical insecticides for D. citri management.

Metabolic Profiling of Hybrids Generated from Pummelo and Citrus latipes in Relation to Their Attraction to Diaphorina citri, the Vector of Huanglongbing.

The citrus industry at present is severely affected by huanglongbing disease (HLB). HLB is caused by the supposed bacterial pathogen "Candidatus Liberibacter asiaticus" and is transmitted by the insect vector, the Asian citrus psyllid, Diaphorina citri Kuwayama. Developing new citrus hybrids to improve HLB management is much needed. In this study, we investigated the metabolomic profiles of three new hybrids produced from the cross of C2-5-12 Pummelo (Citrus maxima (L.) Osbeck) × pollen from Citrus latipes. The hybrids were selected based on leaf morphology and seedling vigor. The selected hybrids exhibited compact and upright tree architecture as seen in C. latipes. Hybrids were verified by simple sequence repeat markers, and were subjected to metabolomic analysis using gas chromatography-mass spectrometry. The volatile organic compounds (VOCs) and polar metabolites profiling also showed that the new hybrids were different from their parents. Interestingly, the levels of stored VOCs in hybrid II were higher than those observed in its parents and other hybrids. The level of most VOCs released by hybrid II was also higher than that released from its parents. Additionally, the preference assay showed that hybrid II was more attractive to D. citri than its parents and other hybrids. The leaf morphology, compact and upright architecture of hybrid II, and its attraction to D. citri suggest that it could be used as a windbreak and trap tree for D. citri (double duty), once its tolerance to HLB disease is confirmed. Our results showed that metabolomic analysis could be successfully used to understand the biochemical mechanisms controlling the interaction of D. citri with its host plants.

RNA interference of acetylcholinesterase in the Asian citrus psyllid, Diaphorina citri, increases its susceptibility to carbamate and organophosphate insecticides.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Lividae) transmits the Candidatus Liberibacter asiaticus, which causes citrus greening disease or Huanglongbing, (HLB). To date, there is no efficient cure for HLB disease and the control of D. citri using insecticides became the most important tools for the management of HLB. However, the extensive use of insecticides could increase D. citri resistance to these insecticides. The objective of this study was to investigate the effect of RNA interference of acetylcholinesterase (AChE) on the mortality and susceptibility of D. citri to the four major insecticides used in Florida. In this study, we used a consensus sequence derived from the two AChE genes and cholinesterase 2-like (ChE-2-like) gene to target all of the three genes. Treatment with dsRNA-AChE increased the mortality percentages of both nymphs and adults of D. citri. The mortality percentage increased with the increase in the concentration of applied dsRNA-AChE, and the highest mortality (> 60%) was observed at the highest applied concentration (125ng/µl). Treatments of nymphs or adults with dsRNA-AChE down-regulated the expression of the three targeted genes of D. citri. Silencing of AChE and ChE in D. citri nymphs increased the susceptibility of emerged adults to chlorpyrifos and carbaryl, which act as AChE inhibitors. However, treatment with dsRNA-AChE did not increase the susceptibility of emerged adults to imidacloprid, which acts as an agonist of nicotinic acetylcholine receptors. In the same manner, treatment of adults with dsRNA-AChE increased their susceptibility to chlorpyrifos and carbaryl, but did not affect their susceptibility to imidacloprid. The ANOVA did not show any significant increase in susceptibility of D. citri adults to fenpropathrin after treatment with dsRNA-AChE, either as nymphs or as adults. However, simple linear regression showed that treatment with dsRNA-AChE increased D. citri susceptibility to fenpropathrin, which indicated that AChE could be involved in the metabolism of fenpropathrin. Our results indicated that silencing of AChE and ChE genes in D. citri to increase its susceptibility to insecticides could be a promising tool for the control of this important vector.

Delivery of dsRNA through topical feeding for RNA interference in the citrus sap piercing-sucking hemipteran, Diaphorina citri.

RNA interference (RNAi) is a powerful means to study functional genomics in insects. The delivery of dsRNA is a challenging step in the development of RNAi assay. Here, we describe a new delivery method to increase the effectiveness of RNAi in the Asian citrus psyllid Diaphorina citri. Bromophenol blue droplets were topically applied to fifth instar nymphs and adults on the ventral side of the thorax between the three pairs of legs. In addition to video recordings that showed sucking of the bromophenol blue by the stylets, dissected guts turned blue indicating that the uptake was through feeding. Thus, we called the method topical feeding. We targeted the abnormal wing disc gene (awd), also called nucleoside diphosphate kinase (NDPK), as a reporter gene to prove the uptake of dsRNA via this method of delivery. Our results showed that dsRNA-awd caused reduction of awd expression and nymph mortality. Survival and lifespan of adults emerged from treated nymphs and treated adults were affected. Silencing awd caused wing malformation in the adults emerged from treated nymphs. Topical feeding as a delivery of dsRNA is highly efficient for both nymphs and adults. The described method could be used to increase the efficiency of RNAi in D. citri and other sap piercing-sucking hemipterans.

RNA interference of carboxyesterases causes nymph mortality in the Asian citrus psyllid, Diaphorina citri.

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an important pest of citrus. In addition, D. citri is the vector of Huanglongbing, a destructive disease in citrus, also known as citrus greening disease caused by Candidatus Liberibacter asiaticus. Huanglongbing causes huge losses for citrus industries. Insecticide application for D. citri is the major strategy to prevent disease spread. The heavy use of insecticides causes development of insecticide resistance. We used RNA interference (RNAi) to silence genes implicated in pesticide resistance in order to increase the susceptibility. The activity of dsRNA to reduce the expression of carboxyesterases including esterases FE4 (EstFE4) and acetylcholinesterases (AChe) in D. citri was investigated. The dsRNA was applied topically to the fourth and fifth instars of nymphs. We targeted several EstFE4 and AChe genes using dsRNA against a consensus sequence for each of them. Five concentrations (25, 50, 75, 100, 125 ng/µl) from both dsRNAs were used. The treatments with the dsRNA caused concentration dependent nymph mortality. The highest gene expression levels of both AChe and EstFE4 were found in the fourth and fifth nymphal instars. Gene expression analysis showed that AChe genes were downregulated in emerged adults from dsRNA-AChe-treated nymphs compared to controls. However, EstFE4 genes were not affected. In the same manner, treatment with dsRNA-EstFE4 reduced expression level of EstFE4 genes in emerged adults from treated nymphs, but did not affect the expression of AChe genes. In the era of environmentally friendly control strategies, RNAi is a new promising venue to reduce pesticide applications.