Optimization of dietary RNA interference delivery to western flower thrips Frankliniella occidentalis and onion thrips Thrips tabaci

In insect reverse genetics, dietary delivery of interfering RNAs is a practical approach in nonmodel species, such as thrips, whose small size, and feeding behavior restricts the use of other delivery methods. In a laboratory context, an unsuitable diet could confound the interpretation of an RNA interference (RNAi) phenotype, however well-formulated artificial diets can minimize experimental variability, reduce the need for insect handling, and can further be used for roles, such as delivering double-strand RNA (dsRNA)-expressing recombinant bacteria. In this study, artificial diets for oral delivery of dsRNA were developed for two important pest thrips species, western flower thrips (Frankliniella occidentalis) and onion thrips (Thrips tabaci), with the goal of (a) stimulating feeding behavior, (b) supporting optimal growth rates of dsRNA-expressing symbiotic bacteria, and (c) nutritionally supporting the thrips for sufficient periods to observe RNAi phenotypes. The efficacy of artificial diets for ingesting “naked” dsRNA or dsRNA-expressing symbionts and dsRNA delivery via host plant uptake was evaluated. Compared with previously published diet formulations, new combinations based on tryptone, yeast, and soy were superior for enhancing feeding and longevity. However, simply adding “naked” dsRNA to an artificial diet was an unreliable form of RNAi delivery in our hands due to dsRNA degradation. Delivery via host plants was more successful, and the new diet formulation was suitable for symbiont-mediated dsRNA delivery, which we believe is the most convenient approach for large-scale knockdown experiments. This study, therefore, provides alternative methodologies for thrips rearing, dietary RNAi delivery, and insights into the challenges of performing dietary RNAi in nonmodel insects.     

Gene silencing in adult Aedes aegypti mosquitoes through oral delivery of double‐stranded RNA

The induction of the naturally occurring phenomenon of RNA interference (RNAi) to study gene function in insects is now common practice. With appropriately chosen targets, the RNAi pathway has also been exploited for insect control, typically through oral delivery of dsRNA. Adapting current methods to deliver foreign compounds, such as amino acids and pesticides, to mosquitoes through sucrose solutions, we tested whether such an approach could be used in the yellow fever mosquito, Aedes aegypti. Using a non‐specific dsRNA construct, we found that adult Ae. aegypti ingested dsRNA through this method and that the ingested dsRNA can be recovered from the mosquitoes post‐feeding. Through the feeding of a species‐specific dsRNA construct against vacuolar ATPase, subunit A, we found that significant gene knockdown could be achieved at 12, 24 and 48 h post‐feeding.     

The effects of geographic origin and antibiotic treatment on the gut symbiotic communities of Bactrocera oleae populations

The olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), is the major insect pest of olive orchards (Olea europaea L.), causing extensive damages on cultivated olive crops worldwide. Due to its economic importance, it has been the target species for a variety of population control approaches including the sterile insect technique (SIT). However, the inefficiency of the current mass‐rearing techniques impedes the successful application of area‐wide integrated pest management programs with an SIT component. It has been shown that insect mass rearing and quality of sterile insects can be improved by the manipulation of the insect gut microbiota and probiotic applications. In order to exploit the gut bacteria, it is important to investigate the structure of the gut microbial community. In the current study, we characterized the gut bacterial profile of two wild olive fruit fly populations introduced in laboratory conditions using next generation sequencing of two regions of the 16S rRNA gene. We compared the microbiota profiles regarding the geographic origin of the samples. Additionally, we investigated potential changes in the gut bacteria community before and after the first exposure of the wild adult flies to artificial adult diet with and without antibiotics. Various genera – such as Erwinia, Providencia, Enterobacter, and Klebsiella – were detected for the first time in B. oleae. The most dominant species was Candidatus Erwinia dacicola Capuzzo et al. and it was not affected by the antibiotics in the artificial adult diet used in the first generation of laboratory rearing. Geographic origin affected the overall structure of the gut community of the olive fruit fly, but antibiotic treatment in the first generation did not significantly alter the gut microbiota community.     

Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: A review

RNA interference already proved its usefulness in functional genomic research on insects, but it also has considerable potential for the control of pest insects. For this purpose, the insect should be able to autonomously take up the dsRNA, for example through feeding and digestion in its midgut. In this review we bring together current knowledge on the uptake mechanisms of dsRNA in insects and the potential of RNAi to affect pest insects. At least two pathways for dsRNA uptake in insects are described: the transmembrane channel-mediated uptake mechanism based on Caenorhabditis elegans’ SID-1 protein and an ‘alternative’ endocytosis-mediated uptake mechanism. In the second part of the review dsRNA feeding experiments on insects are brought together for the first time, highlighting the achievement of implementing RNAi in insect control with the first successful experiments in transgenic plants and the diversity of successfully tested insect orders/species and target genes. We conclude with points of discussion and concerns regarding further research on dsRNA uptake mechanisms and the promising application possibilities for RNAi in insect control.     

Systemic gene knockdown in Camponotus floridanus workers by feeding of dsRNA

RNA interference (RNAi) technology enables to study specific gene functions also in social insects, which are otherwise difficult to access for genetic manipulations. The recent sequencing of the genomes from seven ant species made these members of the Formicidae available for knockdown studies. However, for this purpose the RNAi technology first needs to be adapted for application in ants. Studies on other insects show that the effectiveness of RNAi is quite species-specific and can depend on several experimental parameters such as the investigated stage of the insect, the target gene and/or the dsRNA delivery method. RNAi in ants through feeding of dsRNA is a preferable approach, since knockdown can be achieved in individuals without interfering with the animal’s physiology in contrast to injection of dsRNA. Here, we present a protocol for gene knockdown in Formicidae by feeding of dsRNA to worker animals. The expression of a peptidoglycan recognition protein gene, PGRP-LB, was efficiently knocked down in the body of Camponotus floridanus worker ants. Moreover, we describe a relatively cheap method to extract dsRNA from bacteria in order to obtain large quantities needed for feeding experiments.     

Symbiont-mediated RNA interference in insects

RNA interference (RNAi) methods for insects are often limited by problems with double-stranded (ds) RNA delivery, which restricts reverse genetics studies and the development of RNAi-based biocides. We therefore delegated to insect symbiotic bacteria the task of: (i) constitutive dsRNA synthesis and (ii) trauma-free delivery. RNaseIII-deficient, dsRNA-expressing bacterial strains were created from the symbionts of two very diverse pest species: a long-lived blood-sucking bug, Rhodnius prolixus, and a short-lived globally invasive polyphagous agricultural pest, western flower thrips (Frankliniella occidentalis). When ingested, the manipulated bacteria colonized the insects, successfully competed with the wild-type microflora, and sustainably mediated systemic knockdown phenotypes that were horizontally transmissible. This represents a significant advance in the ability to deliver RNAi, potentially to a large range of non-model insects.     

Feasibility,limitation and possible solutions of RNAi‐based technology for insect pest control

Abstract Numerous studies indicate that target gene silencing by RNA interference (RNAi) could lead to insect death. This phenomenon has been considered as a potential strategy for insect pest control, and it is termed RNAi‐mediated crop protection. However, there are many limitations using RNAi‐based technology for pest control, with the effectiveness target gene selection and reliable double‐strand RNA (dsRNA) delivery being two of the major challenges. With respect to target gene selection, at present, the use of homologous genes and genome‐scale high‐throughput screening are the main strategies adopted by researchers. Once the target gene is identified, dsRNA can be delivered by micro‐injection or by feeding as a dietary component. However, micro‐injection, which is the most common method, can only be used in laboratory experiments. Expression of dsRNAs directed against insect genes in transgenic plants and spraying dsRNA reagents have been shown to induce RNAi effects on target insects. Hence, RNAi‐mediated crop protection has been considered as a potential new‐generation technology for pest control, or as a complementary method of existing pest control strategies; however, further development to improve the efficacy of protection and range of species affected is necessary. In this review, we have summarized current research on RNAi‐based technology for pest insect management. Current progress has proven that RNAi technology has the potential to be a tool for designing a new generation of insect control measures. To accelerate its practical application in crop protection, further study on dsRNA uptake mechanisms based on the knowledge of insect physiology and biochemistry is needed.     

Studies on host preference and its biological effects on the red palm weevil,Rhynchophorus ferrugineus Olivier in Egypt The fruit trees

A comparative bioassay (choice or non-choice) was developed to evaluate the efficacy of different species of fruit trees as alternative host plants on the morphological and biological aspects of the red palm weevil, Rhynchophorus ferrugineus and also the latent effect of feeding the larvae on pupal and adult stages. Highly significant differences exist between averages of larval body weight of R. ferrugineus larvae fed on fruit trees. The lowest average of larval weight occurred in the larvae fed on the pieces of sour orange, mandarin followed by olive and lime trees. A small increase in body weight appeared in larvae fed on mango, fig and guava in comparison with those fed on date palm trees (control). The larval mortality reached the highest percentages (100%) by obligatory feeding of R. ferrugineus insects on olive, lime, sour orange and mandarin followed by mango (97%) and guava (96%) compared with control (0%), respectively. The latent effect of obligatory feeding on the larvae of R. ferrugineus was highly decreased during pupation (4%, 4% and 8%) when the insect fed on mango, guava and fig, compared with 100% pupation in the case of larvae fed on ornamental palm or date palm, respectively. Most pupae resulting from the treated larvae by R. ferrugineus failed in adult emergence especially those insects fed on olive, lime, sour orange and mandarin trees compared to untreated insects (95.6–96.2%), respectively. Data also proved that if an insect is forced to feed on a non-preferred host plant, this results in death of the insect often during the larval stage, a decrease of pupation or failure of adult emergence. These results led to the conclusion that mango, fig, and guava trees are believed to be vulnerable hosts for red weevil attack. The lowest percentage of food consumption appeared in those larvae fed on mandarin, sour orange, lime and olive trees compared with those fed on palm. The present results emphasise the need to test the efficiency of the compounds extracted from these host plants if it is considered as an insecticide or an alternative host protectant for management of the red palm weevil R. ferrugineus.     

Saliva of Lygus lineolaris digests double stranded ribonucleic acids

The prospects for development of highly specific pesticides based on double stranded ribonucleic acid have been a recent focus of scientific research. Creative applications have been proposed and demonstrated. However, not all insects are sensitive to double stranded RNA (dsRNA) gene knockdown effects; applications in the order Lepidoptera, for example, have met with varied success. Gene knockdown has been demonstrated in several species in the order Hemiptera. In our laboratory, knockdown experiments relied on microinjection of dsRNA into the hemocoel of the tarnished plant bug, Lygus lineolaris. Subsequent experiments delivering dsRNA to insects by feeding were repeatedly unsuccessful in demonstrating knockdown, and a hypothesis was formulated that the dsRNA was digested and degraded by the insect prior to contact with the insect cells. Exposure of dsRNA to insect saliva, insect salivary glands, and insect hemolymph was compared with commercial RNAase III. The saliva of L. lineolaris was found to rapidly digest double stranded RNA. RNAase inhibitor did not affect the activity but heat treatment slowed enzymatic activity.     

Delivery of dsRNA for RNAi in insects: an overview and future directions

Abstract RNA interference (RNAi) refers to the process of exogenous double‐stranded RNA (dsRNA) silencing the complementary endogenous messenger RNA. RNAi has been widely used in entomological research for functional genomics in a variety of insects and its potential for RNAi‐based pest control has been increasingly emphasized mainly because of its high specificity. This review focuses on the approaches of introducing dsRNA into insect cells or insect bodies to induce effective RNAi. The three most common delivery methods, namely, microinjection, ingestion, and soaking, are illustrated in details and their advantages and limitations are summarized for purpose of feasible RNAi research. In this review, we also briefly introduce the two possible dsRNA uptake machineries, other dsRNA delivery methods and the history of RNAi in entomology. Factors that influence the specificity and efficiency of RNAi such as transfection reagents, selection of dsRNA region, length, and stability of dsRNA in RNAi research are discussed for further studies.     

Simple cost-effective larval injection method for dsRNA delivery to induce RNAi response in Helicoverpa armigera (Hübner)

Microinjection is considered as an effective method for dsRNA delivery in insects. It also facilitates the delivery of a precise quantity of dsRNA in the host insect, inducing an efficient RNAi response. However, the microinjection method needs prior optimization of several parameters like concentration of dsRNA, site of injection, developmental stage of insect etc. for inducing effective RNAi response. Moreover, sophisticated microinjection devices are largely expensive with high maintenance cost. The Old-World bollworm, Helicoverpa armigera (Hübner) is known to be a detrimental polyphagous pest with widespread infestations across the globe. In the present study, we demonstrate a low-cost and effective dsRNA delivery method for inducing RNAi response in H. armigera with the aid of basic insulin injection syringe and fabricated micropipette tip. In order to validate the RNAi response following dsRNA injection, we have selected three key genes from the chitin biosynthesis pathway of the insect. Besides these, argonaute 1 (ago1) was also used as an indicator gene for dsRNA-mediated RNAi induction. Delivery of dsRNA using injection with insulin syringe caused significant upregulation of the ago1 gene in the insect irrespective of any of the three target genes concerned viz. HaNAGK (3.9 fold; p < 0.001), HaAGM (6.3 fold; p < 0.001) and HaUAP (5.9 fold; p < 0.01) respectively, as compared to control injected with nuclease-free water. The dsRNA-injected insects showed aberrant developmental symptoms typical of impeded chitin synthesis, eventually leading to anomalous ecdysis with substantial mortality (up to 69.04%), as compared to controls. The described protocol reduces insect injury, enabling easy restraining of larva and quick execution of dsRNA injection with efficient RNAi response.     

Chitosan nanoparticles help double-stranded RNA escape from endosomes and improve RNA interference in the fall armyworm,Spodoptera frugiperda

RNA interference (RNAi) is a promising technology for the development of next-generation insect pest control products. Though RNAi is efficient and systemic in coleopteran insects, it is inefficient and variable in lepidopteron insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda by conjugating double-stranded RNA (dsRNA) with biodegradable chitosan (Chi). dsRNA conjugated with chitosan was protected from degradation by endonucleases present in Sf9 cell-conditioned medium, hemolymph, and midgut lumen contents collected from the FAW larvae. Chi–dsRNA complexes showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing chitosan formulated dsRNA in Sf9 cells and the tissues induced a significant knockdown of endogenous genes. Chi–dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation, and mortality. Processing of dsRNA into small interfering RNA was detected with chitosan-conjugated ³²P-UTP-labeled ds green fluorescent protein in Sf9 cells and FAW larval tissues. Overall, these data suggest that dsRNA conjugated with chitosan helps dsRNA escape from the endosomes and improves RNAi efficiency in FAW cells and tissues.     

RNAi-mediated crop protection against insects

Downregulation of the expression of specific genes through RNA interference (RNAi), has been widely used for genetic research in insects. The method has relied on the injection of double-stranded RNA (dsRNA), which is not possible for practical applications in crop protection. By contrast, specific suppression of gene expression in nematodes is possible through feeding with dsRNA. This approach was thought to be unfeasible in insects, but recent results have shown that dsRNA fed as a diet component can be effective in downregulating targeted genes. More significantly, expression of dsRNA directed against suitable insect target genes in transgenic plants has been shown to give protection against pests, opening the way for a new generation of insect-resistant crops.     

Prevalence of Candidatus Erwinia dacicola in wild and laboratory olive fruit fly populations and across developmental stages

The microbiome of the olive fruit fly, Bactrocera oleae (Gmelin), a worldwide pest of olives (Olea europaea L.), has been examined for >100 yr as part of efforts to identify bacteria that are plant pathogens vectored by the fly or are beneficial endosymbionts essential for the fly's survival and thus targets for possible biological control. Because tephritid fruit flies feed on free-living bacteria in their environment, distinguishing between the transient, acquired bacteria of their diet and persistent, resident bacteria that are vertically transmitted endosymbionts is difficult. Several culture-dependent and -independent studies have identified a diversity of species in the olive fruit fly microbiome, but they have not distinguished the roles of the microbes. Candidatus Erwinia dacicola, has been proposed to be a coevolved endosymbiont of the olive fruit fly; however, this was based on limited samples from two Italian populations. Our study shows that C. Erwinia dacicola was present in all New and Old World populations and in the majority of individuals of all life stages sampled in 2 yr. Olive fruit flies reared on olives in the laboratory had frequencies of C. Erwinia dacicola similar to that of wild populations; however, flies reared on artificial diets containing antibiotics in the laboratory rarely had the endosymbiont. The relative abundance of C. Erwinia dacicola varied across development stages, being most abundant in ovipositing females and larvae. This uniform presence of C. Erwini dacicola suggests that it is a persistent, resident endosymbiont of the olive fruit fly.     

Double-stranded RNAs targeting inhibitor of apoptosis gene show no significant cross-species activity

RNA interference (RNAi) has become an integral part of mainstream research due to its versatility and ease of use. However, the potential nontarget effects associated with double-stranded RNAs (dsRNA) are poorly understood. To explore this, we used dsRNAs targeting the inhibitor of apoptosis (iap) gene from nine insect species and assayed their possible nontarget effects. For each assay, we used a control (dsRNA targeting the gene coding for green fluorescent protein, GFP) and a species-specific dsRNA targeting nine iap genes in insect species to evaluate target gene knockdown efficiency, apoptosis phenotype in cells and mortality in insects. Our results revealed that dsIAP efficiently knocks down iap gene expression and induces apoptosis phenotype and mortality in target insect species. In contrast, no significant knockdown of the iap gene expression, apoptosis phenotypes, or mortality were detected in cell lines developed from nontarget insects or nontarget insects treated with dsIAPs. Interestingly, even among closely related insects such as stink bugs, Nezara viridula, Halyomorpha halys, and Murgantia histrionica, with substantial sequence similarity among iap genes from these insects, no significant nontarget effects of dsIAP were observed under the conditions tested. These data demonstrate no significant nontarget effects for dsIAPs and suggest that the threat of nontarget effects of RNAi technology may not be substantial.     

Orally delivered dsRNA induces knockdown of target genes and mortality in the Asian long-horned beetle,Anoplophora glabripennis

The Asian long-horned beetle (ALB) Anoplophora glabripennis is a serious invasive forest pest in several countries, including the United States. Methods available to manage or eradicate this pest are extremely limited, but RNA interference (RNAi) technology is a potentially effective method to control ALB. In this study, we used sucrose feeding bioassay for oral delivery of double-strand RNA (dsRNA) to ALB larvae. ³²P-labeled dsRNA orally delivered to ALB larvae using the sucrose droplet feeding method was processed to small interfering RNA. Feeding neonate larvae with dsRNA targeting genes coding for the inhibitor of apoptosis (IAP), vacuolar sorting protein SNF7 (SNF7), and snakeskin (SSK) induced knockdown of target genes and mortality. Feeding 2 µg of dsRNA per day for 3 days did not induce a significant decrease in the expression of target genes or mortality. However, feeding 5 or 10 µg of dsRNA per day for 3 days induced a significant decrease in the expression of target genes and 50–90% mortality. Interestingly, feeding 2.5 µg each of dsIAP plus dsSNF7, dsIAP plus dsSSK, or dsSNF7 plus dsSSK per day for 3 days induced a significant decrease in the expression of both target genes and approximately 80% mortality. Our findings demonstrate that orally delivered dsRNA induces target gene knockdown and mortality in ALB neonate larvae and RNAi technology may have the potential for effective ALB control.