Optimal dsRNA Concentration for RNA Interference in Asian Citrus Psyllid.

The Asian citrus psyllid (ACP) is a citrus pest and insect vector of "Candidatus Liberibacter asiaticus", the causal agent of citrus greening disease. Double-stranded RNA (dsRNA) biopesticides that trigger RNA interference (RNAi) offer an alternative to traditional insecticides. Standardized laboratory screening of dsRNA requires establishing the minimal effective concentration(s) that result in effective RNAi "penetrance" and trigger RNAi, resulting in one or more measurable phenotypes, herein, significant gene knockdown and the potential for mortality. In this study, knockdown was evaluated for a range of dsRNA concentrations of three ACP candidate genes, clathrin heavy chain (CHC), vacuolar ATPase subunit A (vATPase-A), and sucrose non-fermenting protein 7 (Snf7). Gene knockdown was quantified for ACP teneral adults and 3rd instar nymphs allowed a 48 h ingestion-access period (IAP) on 10, 50,100, 200, and 500 ng/µL dsRNA dissolved in 20% sucrose followed by a 5-day post-IAP on orange jasmine shoots. Significant gene knockdown (p < 0.05) in ACP third instar nymphs and adults ranged from 12-34% and 18-39%, 5 days post-IAP on dsRNA at 10-500 and 100-500 ng/µL, respectively. The threshold concentration beyond which no significant gene knockdown and adult mortality was observed post-48 h IAP and 10-day IAP, respectively, was determined as 200 ng/µL, a concentration indicative of optimal RNAi penetrance.

UV-LASER adjuvant-surfactant-facilitated delivery of mobile dsRNA to tomato plant vasculature and evidence of biological activity by gene knockdown in the potato psyllid.

BACKGROUND: Double-stranded RNA (dsRNA) biopesticides are of interest for the abatement of insect vectors of pathogenic bacteria such as 'Candidatus Liberibacter', which infects both its psyllid and plant hosts. Silencing of genes essential for psyllids, or for Liberibacter, is anticipated to lead to mortality or impeded bacterial multiplication. Foliar delivery is preferred for biopesticide application; however, the cuticle impedes dsRNA penetration into the vasculature. Here, conditions were established for wounding tomato leaves using ultraviolet light amplification by stimulated emissions of radiation (UV-LASER) to promote dsRNA penetration into leaves and vasculature. RESULTS: UV-LASER treatment with application of select adjuvants/surfactants resulted in vascular delivery of 100-, 300- and 600-bp dsRNAs that, in general, were correlated with size. The 100-bp dsRNA required no pretreatment, whereas 300- and 600-bp dsRNAs entered the vasculature after UV-LASER treatment only and UV-LASER adjuvant/surfactant treatment, respectively. Of six adjuvant/surfactants evaluated, plant-derived oil combined with an anionic organosilicon compound performed most optimally. Localization of dsRNAs in the tomato vasculature was documented using fluorometry and fluorescence confocal microscopy. The biological activity of in planta-delivered dsRNA (200-250 bp) was determined by feeding third-instar psyllids on tomato leaves post UV-LASER adjuvant/surfactant treatment, with or without psyllid cdc42- and gelsolin dsRNAs. Gene knockdown was quantified by quantitative, real-time polymerase chain reaction with reverse transcription (RT-qPCR) amplification. At 10 days post the ingestion-access period, knockdown of cdc42 and gelsolin expression was 61% and 56%, respectively, indicating that the dsRNAs delivered to the tomato vasculature were mobile and biologically active. CONCLUSION: Results indicated that UV-LASER adjuvant/surfactant treatments facilitated the delivery of mobile, biologically active dsRNA molecules to the plant vasculature. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

RNA interference-mediated knockdown of genes involved in sugar transport and metabolism disrupts psyllid Bactericera cockerelli (Order: Hemiptera) gut physiology and results in high mortality.

Introduction: The causal agent of zebra chip of potato and vein-greening diseases of tomato is "Candidatus Liberibacter solanacearum" (CLso), a fastidious bacterium transmitted by the potato psyllid. In the absence of disease-resistant cultivars, disease management has relied on minimizing vector population size to reduce CLso transmission, which requires frequent insecticide applications. There is growing interest in the use of RNA interference (RNAi) technology to supplant traditional insecticides with biopesticides. This requires knowledge of genes essential for insect livelihood whose knockdown leads to significant mortality or other phenotypes. Such candidate genes can be evaluated by reverse genetics approaches to further corroborate predicted gene function. Methods: Here, five potato psyllid genes involved in sugar homeostasis in the potato psyllid gut, α-glucosidase1 (AGLU1), aquaporin2 (AQP2), facilitated trehalose transporter1 (TRET1), Trehalase1 (TRE1), and Trehalase2 (TRE2), were investigated as candidates for effective gene silencing. Potato psyllid dsRNAs were designed to optimize knockdown of gene targets. Third instar PoP nymphs were given a 48-hr ingestion-access period (IAP) on individual or groups of dsRNA in 20% sucrose. Mortality was recorded 0, 3, 5, 7, and 9 days post-IAP. Gene knockdown was analyzed 9 days post-IAP by quantitative real-time reverse-transcriptase polymerase chain reaction amplification. Results: The individual or stacked dsRNA combinations resulted in 20-60% and 20-40% knockdown, respectively, while subsequent psyllid mortality ranged from 20-40% to >60% for single and stacked dsRNA combinations, respectively. Reverse genetics analysis showed that simultaneous knockdown of the five selected candidate genes with predicted functions in pathways involved in sugar-homeostasis, metabolism, and -transport yielded the highest mortality, when compared with single or combinations of targets. Discussion: Results confirmed the functions afforded by psyllid gut genes responsible for osmotic homeostasis and sugar metabolism/transport are essential for livelihood, identifying them as potentially lucrative RNAi biopesticide targets and highlighted the translational relevance of targeting multiple nodes in a physiological pathway simultaneously.

Characteristics of environmental RNAi in potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Psylloidea: Triozidae).

RNA interference (RNAi) has potential to become a major tool for integrated management of insect pests of agricultural crops based on sequence-specificity and low doses of rapidly biodegradable dsRNA. Deploying 'environmental RNAi' for control of insect vectors of plant pathogens is of increasing interest for combatting emerging plant diseases. Hemipteran insect vectors, including psyllids, are vascular feeders, making their development difficult to control specifically by targeting with pesticidal chemistries. Psyllids transmit "Candidatus Liberibacter solanacearum" the causal organism of potato zebra chip and tomato vein greening diseases, transmitted, respectively, by the potato or tomato psyllid (PoP). Until now, the optimal effective concentration(s) of double-stranded RNA (dsRNA) required for significant gene knockdown and RNAi persistence in PoP have not been determined. The objective of this study was to optimize RNAi in young PoP adults and 3rd instars for screening by oral delivery of dsRNAs. The minimal effective dsRNA concentrations required for robust knockdown and persistence were evaluated by delivering seven concentrations spanning 0.1 ng/µL to 500 ng/µL over post ingestion-access periods (IAP) ranging from 48 h to 12 days. The PoP gene candidates evaluated as targets were vacuolar ATPase subunit A, clathrin heavy chain, and non-fermenting protein 7, which were evaluated for knockdown by qPCR amplification. The minimum and/or the second most effective dsRNA concentration resulting in effective levels of gene knockdown was 100 ng/µL for all three targets. Higher concentrations did not yield further knockdown, indicating potential RISC saturation at the higher doses. Gene silencing post-IAP of 100 ng/µL dsRNA persisted for 3-5 days in adults and nymphs, with the PoP 3rd instar, followed by teneral and mature adults, respectively, exhibiting the most robust RNAi-response.

Environmental RNAi pathways in the two-spotted spider mite.

BACKGROUND: RNA interference (RNAi) regulates gene expression in most multicellular organisms through binding of small RNA effectors to target transcripts. Exploiting this process is a popular strategy for genetic manipulation and has applications that includes arthropod pest control. RNAi technologies are dependent on delivery method with the most convenient likely being feeding, which is effective in some animals while others are insensitive. The two-spotted spider mite, Tetranychus urticae, is prime candidate for developing RNAi approaches due to frequent occurrence of conventional pesticide resistance. Using a sequencing-based approach, the fate of ingested RNAs was explored to identify features and conditions that affect small RNA biogenesis from external sources to better inform RNAi design. RESULTS: Biochemical and sequencing approaches in conjunction with extensive computational assessment were used to evaluate metabolism of ingested RNAs in T. urticae. This chelicerae arthropod shows only modest response to oral RNAi and has biogenesis pathways distinct from model organisms. Processing of synthetic and plant host RNAs ingested during feeding were evaluated to identify active substrates for spider mite RNAi pathways. Through cataloging characteristics of biochemically purified RNA from these sources, trans-acting small RNAs could be distinguished from degradation fragments and their origins documented. CONCLUSIONS: Using a strategy that delineates small RNA processing, we found many transcripts have the potential to enter spider mite RNAi pathways, however, trans-acting RNAs appear very unstable and rare. This suggests potential RNAi pathway substrates from ingested materials are mostly degraded and infrequently converted into regulators of gene expression. Spider mites infest a variety of plants, and it would be maladaptive to generate diverse gene regulators from dietary RNAs. This study provides a framework for assessing RNAi technology in organisms where genetic and biochemical tools are absent and benefit rationale design of RNAi triggers for T.urticae.

Exploiting somatic piRNAs in Bemisia tabaci enables novel gene silencing through RNA feeding.

RNAi promises to reshape pest control by being nontoxic, biodegradable, and species specific. However, due to the plastic nature of RNAi, there is a significant variability in responses. In this study, we investigate small RNA pathways and processing of ingested RNAi trigger molecules in a hemipteran plant pest, the whitefly Bemisia tabaci Unlike Drosophila, where the paradigm for insect RNAi technology was established, whitefly has abundant somatic piwi-associated RNAs (piRNAs). Long regarded as germline restricted, piRNAs are common in the soma of many invertebrates. We sought to exploit this for a novel gene silencing approach. The main principle of piRNA biogenesis is the recruitment of target RNA fragments into the pathway. As such, we designed synthetic RNAs to possess complementarity to the loci we annotated. Following feeding of these exogenous piRNA triggers knockdown as effective as conventional siRNA-only approaches was observed. These results demonstrate a new approach for RNAi technology that could be applicable to dsRNA-recalcitrant pest species and could be fundamental to realizing insecticidal RNAi against pests.

siRNAs and piRNAs collaborate for transposon control in the two-spotted spider mite.

RNAi has revolutionized genetic research, and is being commercialized as an insect pest control technology. Mechanisms exploited for this purpose are antiviral and therefore rapidly evolving. Ideally, RNAi will also be used for noninsect pests; however, differences in RNAi biology make this uncertain. Tetranychus urticae (two-spotted spider mite) is a destructive noninsect pest, which has a proclivity to develop pesticide resistance. Here we provide a comprehensive study of the endogenous RNAi pathways of spider mites to inform design of exogenous RNAi triggers. This effort revealed unexpected roles for small RNAs and novel genome surveillance pathways. Spider mites have an expanded RNAi machinery relative to insects, encoding RNA dependent RNA polymerase (Rdrp) and extra Piwi-class effectors. Through analyzing T. urticae transcriptome data we explored small RNA biogenesis, and discovered five siRNA loci that appear central to genome surveillance. These RNAs are expressed in the gonad, which we hypothesize to trigger production of piRNAs for control of transposable elements (TEs). This work highlights the need to investigate endogenous RNAi biology as lessons from model organisms may not hold in other species, impacting development of an RNAi strategy.

Guanidinium-Functionalized Interpolyelectrolyte Complexes Enabling RNAi in Resistant Insect Pests.

RNAi-based technologies are ideal for pest control as they can provide species specificity and spare nontarget organisms. However, in some pests biological barriers prevent use of RNAi, and therefore broad application. In this study we tested the ability of a synthetic cationic polymer, poly-[ N-(3-guanidinopropyl)methacrylamide] (pGPMA), that mimics arginine-rich cell penetrating peptides to trigger RNAi in an insensitive animal- Spodoptera frugiperda. Polymer-dsRNA interpolyelectrolyte complexes (IPECs) were found to be efficiently taken up by cells, and to drive highly efficient gene knockdown. These IPECs could also trigger target gene knockdown and moderate larval mortality when fed to S. frugiperda larvae. This effect was sequence specific, which is consistent with the low toxicity we found to be associated with this polymer. A method for oral delivery of dsRNA is critical to development of RNAi-based insecticides. Thus, this technology has the potential to make RNAi-based pest control useful for targeting numerous species and facilitate use of RNAi in pest management practices.

Rewired RNAi-mediated genome surveillance in house dust mites.

House dust mites are common pests with an unusual evolutionary history, being descendants of a parasitic ancestor. Transition to parasitism is frequently accompanied by genome rearrangements, possibly to accommodate the genetic change needed to access new ecology. Transposable element (TE) activity is a source of genomic instability that can trigger large-scale genomic alterations. Eukaryotes have multiple transposon control mechanisms, one of which is RNA interference (RNAi). Investigation of the dust mite genome failed to identify a major RNAi pathway: the Piwi-associated RNA (piRNA) pathway, which has been replaced by a novel small-interfering RNA (siRNA)-like pathway. Co-opting of piRNA function by dust mite siRNAs is extensive, including establishment of TE control master loci that produce siRNAs. Interestingly, other members of the Acari have piRNAs indicating loss of this mechanism in dust mites is a recent event. Flux of RNAi-mediated control of TEs highlights the unusual arc of dust mite evolution.