Plin-amiR, a pre-microRNA-based technology for controlling herbivorous insect pests.

The cotton bollworm, Helicoverpa armigera, is a major insect pest for a wide range of agricultural crops. It causes significant yield loss through feeding damage and by increasing the crop's vulnerability to bacterial and fungal infections. Although expression of Bacillus thuringiensis (Bt) toxins in transgenic crops has been very successful in protecting against insect pests, including H. armigera, field-evolved resistance has occurred in multiple species. To manage resistant populations, new protection strategies must be continuously developed. Trans-kingdom RNA interference (TK-RNAi) is a promising method for controlling herbivorous pests. TK-RNAi is based on delivering dsRNA or hairpin RNA containing essential insect gene sequences to the feeding insect. The ingested molecules are processed by the insect's RNAi machinery and guide it to silence the target genes. Recently, TK-RNAi delivery has been enhanced by expressing the ds- or hpRNAs in the chloroplast. This compartmentalizes the duplexed RNA away from the plant's RNAi machinery, ensuring that it is delivered in an unprocessed form to the insect. Here, we report another alternative approach for delivering precursor anti-insect RNA in plants. Insect pre-microRNA (pre-miR) transcripts were modified to contain artificial microRNAs (amiRs), targeting insect genes, and expressed in transgenic Nicotiana benthamiana plants. These modified pre-miRs remained largely unprocessed in the plants, and H. armigera feeding on leaves from these plants had increased mortality, developmental abnormalities and delayed growth rates. This shows that plant-expressed insect pre-amiRs (plin-amiRs) are a new strategy of protecting plants against herbivorous insects.

Knockdown of RNA interference pathway genes in western corn rootworm, Diabrotica virgifera virgifera, identifies no fitness costs associated with Argonaute 2 or Dicer-2.

The use of transgenic crops that induce silencing of essential genes using double-stranded RNA (dsRNA) through RNA interference (RNAi) in western corn rootworm, Diabrotica virgifera virgifera, is likely to be an important component of new technologies for the control of this important corn pest. Previous studies have demonstrated that the dsRNA response in D. v. virgifera depends on the presence of RNAi pathway genes including Dicer-2 and Argonaute 2, and that downregulation of these genes limits the lethality of environmental dsRNA. A potential resistance mechanism to lethal dsRNA may involve loss of function of RNAi pathway genes. Howver, the potential for resistance to evolve may depend on whether these pathway genes have essential functions such that the loss of function of core proteins in the RNAi pathway will have fitness costs in D. v. virgifera. Fitness costs associated with potential resistance mechanisms have a central role in determining how resistance can evolve to RNAi technologies in western corn rootworm. We evaluated the effect of dsRNA and microRNA pathway gene knockdown on the development of D. v. virgifera larvae through short-term and long-term exposures to dsRNA for Dicer and Argonaute genes. Downregulation of Argonaute 2, Dicer-2, Dicer-1 did not significantly affect larval survivorship or development through short and long-term exposure to dsRNA. However, downregulation of Argonaute 1 reduced larval survivorship and delayed development. The implications of these results as they relate to D. v. virgifera resistance to lethal dsRNA are discussed.

Rapid and persistent RNAi response in western corn rootworm adults.

RNA interference (RNAi) has proven effective for controlling pest insects such as western corn rootworm (WCR), Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). Previous studies have shown that WCR adults display a robust RNAi response to orally-administered double-stranded RNA (dsRNA). However, it is unclear how quickly the response occurs after ingestion or how long RNAi effect lasts after WCR stop ingesting diet containing dsRNA. In the current study, WCR adult females were provided with diet treated with dsRNAs of Laccase 2 and Argonaute 2, two nonessential genes, for 8 days. RNAi response in WCR females commenced as early as 10 h after exposure to dsRNA and lasted up to 40 days after exposure to dsRNA ended. Our results show that dsRNA-mediated RNAi response in WCR females is rapid and long-lasting. These findings suggest that even a short-term ingestion of transgenic plants expressing dsRNA by WCR may have a sustained impact on this insect.

Toxicity and Binding Studies of Bacillus thuringiensis Cry1Ac, Cry1F, Cry1C, and Cry2A Proteins in the Soybean Pests Anticarsia gemmatalis and Chrysodeixis (Pseudoplusia) includens.

Anticarsia gemmatalis (velvetbean caterpillar) and Chrysodeixis includens (soybean looper, formerly named Pseudoplusia includens) are two important defoliating insects of soybeans. Both lepidopteran pests are controlled mainly with synthetic insecticides. Alternative control strategies, such as biopesticides based on the Bacillus thuringiensis (Bt) toxins or transgenic plants expressing Bt toxins, can be used and are increasingly being adopted. Studies on the insect susceptibilities and modes of action of the different Bt toxins are crucial to determine management strategies to control the pests and to delay outbreaks of insect resistance. In the present study, the susceptibilities of both soybean pests to the Bt toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa have been investigated. Bioassays performed in first-instar larvae showed that both insects are susceptible to all these toxins. Competition-binding studies carried out with Cry1Ac and Cry1Fa 125-iodine labeled proteins demonstrated the presence of specific binding sites for both of them on the midgut brush border membrane vesicles (BBMVs) of both A. gemmatalis and C. includens Competition-binding experiments and specific-binding inhibition studies performed with selected sugars and lectins indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites in the midguts of both insects. Also, the Cry1Ac- or Cry1Fa-binding sites were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity and midgut toxin binding sites in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops.IMPORTANCE In the present study, the toxicity and the mode of action of the Bacillus thuringiensis (Bt) toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa in Anticarsia gemmatalis and Chrysodeixis includens (important defoliating pests of soybeans) have been investigated. These studies are crucial for determining management strategies for pest control. Bioassays showed that both insects were susceptible to the toxins. Competition-binding studies demonstrated the presence of Cry1Fa- and Cry1Ac-specific binding sites in the midguts of both pests. These results, together with the results from binding inhibition studies performed with sugars and lectins, indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites, and that they were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops.

Safety considerations derived from Cry34Ab1/Cry35Ab1 structure and function.

Insecticidal proteins developed for in-plant protection against crop pests undergo extensive safety testing during the product development process. Safety considerations for insecticidal proteins expressed in crops follow recommended, science-based guidelines and specific studies are conducted on a case by case basis. Corn events expressing Bacillus thuringiensis (Bt) Cry34Ab1 and Cry35Ab1 were developed to protect maize from Diabrotica virgifera virgifera (western corn rootworm) feeding damage. The protein crystal structures of Cry34Ab1 and Cry35Ab1 are different from the more common three-domain Cry or Vip3 proteins expressed in insect resistant maize varieties. Cry34Ab1 is a single domain protein that folds into a beta sandwich structure that resembles membrane-active proteins, including several cytolysins, from a variety of natural sources. Cry35Ab1 has two domains, one domain with structural relatedness to sugar binding motifs and a second domain with an extended beta sheet structure that is clearly related to beta pore forming proteins, some of which are insecticidal, e.g. B. sphaericus BinA/BinB. In this review we discuss Cry34Ab1/Cry35Ab1 structure and function in the context of protein safety studies for insect resistant crops.

The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins.

BACKGROUND: The Cry6 family of proteins from Bacillus thuringiensis represents a group of powerful toxins with great potential for use in the control of coleopteran insects and of nematode parasites of importance to agriculture. These proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. This has inhibited our understanding of these toxins and their mode of action, along with our ability to manipulate the proteins to alter their activity to our advantage. To increase our understanding of their mode of action and to facilitate further development of these proteins we have determined the structure of Cry6Aa in protoxin and trypsin-activated forms and demonstrated a pore-forming mechanism of action. RESULTS: The two forms of the toxin were resolved to 2.7 Å and 2.0 Å respectively and showed very similar structures. Cry6Aa shows structural homology to a known class of pore-forming toxins including hemolysin E from Escherichia coli and two Bacillus cereus proteins: the hemolytic toxin HblB and the NheA component of the non-hemolytic toxin (pfam05791). Cry6Aa also shows atypical features compared to other members of this family, including internal repeat sequences and small loop regions within major alpha helices. Trypsin processing was found to result in the loss of some internal sequences while the C-terminal region remains disulfide-linked to the main core of the toxin. Based on the structural similarity of Cry6Aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in Caenorhabditis elegans was demonstrated. A non-toxic mutant was also produced, consistent with the proposed pore-forming mode of action. CONCLUSIONS: Cry6 proteins are members of the alpha helical pore-forming toxins - a structural class not previously recognized among the Cry toxins of B. thuringiensis and representing a new paradigm for nematocidal and insecticidal proteins. Elucidation of both the structure and the pore-forming mechanism of action of Cry6Aa now opens the way to more detailed analysis of toxin specificity and the development of new toxin variants with novel activities.

Reduced stability and intracellular transport of dsRNA contribute to poor RNAi response in lepidopteran insects.

RNA interference (RNAi) has become a widely used reverse genetic tool to study gene function in eukaryotic organisms and is being developed as a technology for insect pest management. The efficiency of RNAi varies among organisms. Insects from different orders also display differential efficiency of RNAi, ranging from highly efficient (coleopterans) to very low efficient (lepidopterans). We investigated the reasons for varying RNAi efficiency between lepidopteran and coleopteran cell lines and also between the Colorado potato beetle, Leptinotarsa decemlineata and tobacco budworm, Heliothis virescens. The dsRNA either injected or fed was degraded faster in H. virescens than in L. decemlineata. Both lepidopteran and coleopteran cell lines and tissues efficiently took up the dsRNA. Interestingly, the dsRNA administered to coleopteran cell lines and tissues was taken up and processed to siRNA whereas the dsRNA was taken up by lepidopteran cell lines and tissues but no siRNA was detected in the total RNA isolated from these cell lines and tissues. The data included in this paper showed that the degradation and intracellular transport of dsRNA are the major factors responsible for reduced RNAi efficiency in lepidopteran insects.

Retargeting of the Bacillus thuringiensis toxin Cyt2Aa against hemipteran insect pests.

Although transgenic crops expressing Bacillus thuringiensis (Bt) toxins have been used successfully for management of lepidopteran and coleopteran pest species, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. To overcome this limitation, we demonstrate that addition of a short peptide sequence selected for binding to the gut of the targeted pest species serves to increase toxicity against said pest. Insertion of a 12-aa pea aphid gut-binding peptide by adding to or replacing amino acids in one of three loops of the Bt cytolytic toxin, Cyt2Aa, resulted in enhanced binding and toxicity against both the pea aphid, Acyrthosiphon pisum, and the green peach aphid, Myzus persicae. This strategy may allow for transgenic plant-mediated suppression of other hemipteran pests, which include some of the most important pests of global agriculture.

Insecticidal activity of Bacillus thuringiensis Cry1Bh1 against Ostrinia nubilalis (Hubner) (Lepidoptera: Crambidae) and other lepidopteran pests.

Bacillus thuringiensis is an important source of insect resistance traits in commercial crops. In an effort to prolong B. thuringiensis trait durability, insect resistance management programs often include combinations of insecticidal proteins that are not cross resistant or have demonstrable differences in their site of action as a means to mitigate the development of resistant insect populations. In this report, we describe the activity spectrum of a novel B. thuringiensis Cry protein, Cry1Bh1, against several lepidopteran pests, including laboratory-selected B. thuringiensis-resistant strains of Ostrinia nubilalis and Heliothis virescens and progeny of field-evolved B. thuringiensis-resistant strains of Plutella xylostella and Spodoptera frugiperda. Cry1Bh1 is active against susceptible and B. thuringiensis-resistant colonies of O. nubilalis, P. xylostella, and H. virescens in laboratory diet-based assays, implying a lack of cross-resistance in these insects. However, Cry1Bh1 is not active against susceptible or Cry1F-resistant S. frugiperda. Further, Cry1Bh1 does not compete with Cry1Fa or Cry1Ab for O. nubilalis midgut brush border membrane binding sites. Cry1Bh1-expressing corn, while not completely resistant to insect damage, provided significantly better leaf protection against Cry1Fa-resistant O. nubilalis than did Cry1Fa-expressing hybrid corn. The lack of cross-resistance with Cry1Ab and Cry1Fa along with independent membrane binding sites in O. nubilalis makes Cry1Bh1 a candidate to further optimize for in-plant resistance to this pest.

RNAi for Western Corn Rootworm Management: Lessons Learned, Challenges, and Future Directions.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is considered one of the most economically important pests of maize (Zea mays L.) in the United States (U.S.) Corn Belt with costs of management and yield losses exceeding USD ~1-2 billion annually. WCR management has proven challenging given the ability of this insect to evolve resistance to multiple management strategies including synthetic insecticides, cultural practices, and plant-incorporated protectants, generating a constant need to develop new management tools. One of the most recent developments is maize expressing double-stranded hairpin RNA structures targeting housekeeping genes, which triggers an RNA interference (RNAi) response and eventually leads to insect death. Following the first description of in planta RNAi in 2007, traits targeting multiple genes have been explored. In June 2017, the U.S. Environmental Protection Agency approved the first in planta RNAi product against insects for commercial use. This product expresses a dsRNA targeting the WCR snf7 gene in combination with Bt proteins (Cry3Bb1 and Cry34Ab1/Cry35Ab1) to improve trait durability and will be introduced for commercial use in 2022.

Elucidation of the MicroRNA Transcriptome in Western Corn Rootworm Reveals Its Dynamic and Evolutionary Complexity.

Diabrotica virgifera virgifera (western corn rootworm, WCR) is one of the most destructive agricultural insect pests in North America. It is highly adaptive to environmental stimuli and crop protection technologies. However, little is known about the underlying genetic basis of WCR behavior and adaptation. More specifically, the involvement of small RNAs (sRNAs), especially microRNAs (miRNAs), a class of endogenous small non-coding RNAs that regulate various biological processes, has not been examined, and the datasets of putative sRNA sequences have not previously been generated for WCR. To achieve a comprehensive collection of sRNA transcriptomes in WCR, we constructed, sequenced, and analyzed sRNA libraries from different life stages of WCR and northern corn rootworm (NCR), and identified 101 conserved precursor miRNAs (pre-miRNAs) in WCR and other Arthropoda. We also identified 277 corn rootworm specific pre-miRNAs. Systematic analyses of sRNA populations in WCR revealed that its sRNA transcriptome, which includes PIWI-interacting RNAs (piRNAs) and miRNAs, undergoes a dynamic change throughout insect development. Phylogenetic analysis of miRNA datasets from model species reveals that a large pool of species-specific miRNAs exists in corn rootworm; these are potentially evolutionarily transient. Comparisons of WCR miRNA clusters to other insect species highlight conserved miRNA-regulated processes that are common to insects. Parallel Analysis of RNA Ends (PARE) also uncovered potential miRNA-guided cleavage sites in WCR. Overall, this study provides a new resource for studying the sRNA transcriptome and miRNA-mediated gene regulation in WCR and other Coleopteran insects.

First Sprayable Double-Stranded RNA-Based Biopesticide Product Targets Proteasome Subunit Beta Type-5 in Colorado Potato Beetle (Leptinotarsa decemlineata).

Colorado potato beetle (CPB, Leptinotarsa decemlineata) is a major pest of potato and other solanaceous vegetables in the Northern Hemisphere. The insect feeds on leaves and can completely defoliate crops. Because of the repeated use of single insecticide classes without rotating active ingredients, many chemicals are no longer effective in controlling CPB. Ledprona is a sprayable double-stranded RNA biopesticide with a new mode of action that triggers the RNA interference pathway. Laboratory assays with second instar larvae fed Ledprona showed a dose-response where 25×10-6g/L of dsPSMB5 caused 90% mortality after 6days of initial exposure. We also showed that exposure to Ledprona for 6h caused larval mortality and decreased target messenger RNA (mRNA) expression. Decrease in PSMB5 protein levels was observed after 48h of larval exposure to Ledprona. Both PSMB5 mRNA and protein levels did not recover over time. Ledprona efficacy was demonstrated in a whole plant greenhouse trial and performed similarly to spinosad. Ledprona, currently pending registration at EPA, represents a new biopesticide class integrated pest management and insecticide resistance management programs directed against CPB.

Control of western corn rootworm via RNAi traits in maize: lethal and sublethal effects of Sec23 dsRNA.

BACKGROUND: RNA interference (RNAi) triggered by maize plants expressing RNA hairpins against specific western corn rootworm (WCR) transcripts have proven to be effective at controlling this pest. To provide robust crop protection, mRNA transcripts targeted by double-stranded RNA must be sensitive to knockdown and encode essential proteins. RESULTS: Using WCR adult feeding assays, we identified Sec23 as a highly lethal RNAi target. Sec23 encodes a coatomer protein, a component of the coat protein (COPII) complex that mediates ER-Golgi transport. The lethality detected in WCR adults was also observed in early instar larvae, the life stage causing most of the crop damage, suggesting that WCR adults can serve as an alternative to larvae for dsRNA screening. Surprisingly, over 85% transcript inhibition resulted in less than 40% protein knockdown, suggesting that complete protein knockdown is not necessary for Sec23 RNAi-mediated mortality. The efficacy of Sec23 dsRNA for rootworm control was confirmed in planta; T0 maize events carrying rootworm Sec23 hairpin transgenes showed high levels of root protection in greenhouse assays. A reduction in larval survival and weight were observed in the offspring of WCR females exposed to Sec23 dsRNA LC25 in diet bioassays. CONCLUSION: We describe Sec23 as RNAi target for in planta rootworm control. High mortality in exposed adult and larvae and moderate sublethal effects in the offspring of females exposed to Sec23 dsRNA LC25 , suggest the potential for field application of this RNAi trait and the need to factor in responses to sublethal exposure into insect resistance management programs. © 2019 Society of Chemical Industry.

Modification of Vip3Ab1 C-Terminus Confers Broadened Plant Protection from Lepidopteran Pests.

Vegetative insecticidal proteins (Vips) from Bacillus thuringiensis (Bt) are unique from crystal (Cry) proteins found in Bt parasporal inclusions as they are secreted during the bacterial vegetative growth phase and bind unique receptors to exert their insecticidal effects. We previously demonstrated that large modifications of the Vip3 C-terminus could redirect insecticidal spectrum but results in an unstable protein with no lethal activity. In the present work, we have generated a new Vip3 protein, Vip3Ab1-740, via modest modification of the Vip3Ab1 C-terminus. Vip3Ab1-740 is readily processed by midgut fluid enzymes and has lethal activity towards Spodoptera eridania, which is not observed with the Vip3Ab1 parent protein. Importantly, Vip3Ab1-740 does retain the lethal activity of Vip3Ab1 against other important lepidopteran pests. Furthermore, transgenic plants expressing Vip3Ab1-740 are protected against S. eridania, Spodoptera frugiperda, Helicoverpa zea, and Pseudoplusia includens. Thus, these studies demonstrate successful engineering of Vip3 proteins at the C-terminus to broaden insecticidal spectrum, which can be employed for functional expression in planta.

RNAi targeting of rootworm Troponin I transcripts confers root protection in maize.

Western corn rootworm, Diabrotica virgifera virgifera, is the major agronomically important pest of maize in the US Corn Belt. To augment the repertoire of the available dsRNA-based traits that control rootworm, we explored a potentially haplolethal gene target, wings up A (wupA), which encodes Troponin I. Troponin I, a component of the Troponin-Tropomyosin complex, is an inhibitory protein involved in muscle contraction. In situ hybridization showed that feeding on wupA-targeted dsRNAs caused systemic transcript knockdown in D. v. virgifera larvae. The knockdown of wupA transcript, and by extension Troponin I protein, led to deterioration of the striated banding pattern in larval body muscle and decreased muscle integrity. Additionally, the loss of function of the circular muscles surrounding the alimentary system led to significant accumulation of food material in the hind gut, which is consistent with a loss of peristaltic motion of the alimentary canal. In this study, we demonstrate that wupA dsRNA is lethal in D. v. virgifera larvae when fed via artificial diet, with growth inhibition of up to 50% within two days of application. Further, wupA hairpins can be stably expressed and detected in maize. Maize expressing wupA hairpins exhibit robust root protection in greenhouse bioassays, with several maize transgene integration events showing root protection equivalent to commercial insecticidal protein-expressing maize.

Improved insect-proofing: expressing double-stranded RNA in chloroplasts.

RNA interference (RNAi) was discovered almost 20 years ago and has been exploited worldwide to silence genes in plants and animals. A decade later, it was found that transforming plants with an RNAi construct targeting an insect gene could protect the plant against feeding by that insect. Production of double-stranded RNA (dsRNA) in a plant to affect the viability of a herbivorous animal is termed trans-kingdom RNAi (TK-RNAi). Since this pioneering work, there have been many further examples of successful TK-RNAi, but also reports of failed attempts and unrepeatable experiments. Recently, three laboratories have shown that producing dsRNA in a plant's chloroplast, rather than in its cellular cytoplasm, is a very effective way of delivering TK-RNAi. Our review examines this potentially game-changing approach and compares it with other transgenic insect-proofing schemes. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Systemic RNAi in western corn rootworm, Diabrotica virgifera virgifera, does not involve transitive pathways.

Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) is highly sensitive to orally delivered double-stranded RNA (dsRNA). RNAi in WCR is systemic and spreads throughout the insect body. This raises the question whether transitive RNAi is a mechanism that functions in WCR to amplify the RNAi response via production of secondary siRNA. Secondary siRNA production is achieved through RNA-dependent RNA polymerase (RdRP) activity in other eukaryotic organisms, but RdRP has not been identified in WCR and any other insects. This study visualized the spread of the RNAi-mediated knockdown of Dv v-ATPase C mRNA throughout the WCR gut and other tissues using high-sensitivity branched DNA in situ hybridization. Furthermore, we did not detect either secondary siRNA production or transitive RNAi in WCR through siRNA sequence profile analysis. Nucleotide mismatched sequences introduced into either the sense or antisense strand of v-ATPase C dsRNAs were maintained in siRNAs derived from WCR fed with the mismatched dsRNAs in a strand specific manner. The distribution of all siRNAs was restricted to within the original target sequence regions, which may indicate the lack of new dsRNA synthesis leading to production of secondary siRNA. Thus, the systemic spread of RNAi in WCR may be derived from the original dsRNA molecules taken up from the gut lumen. These results indicate that the initial dsRNA dose is important for a lethal systemic RNAi response in WCR and have implications in developing effective dsRNA traits to control WCR and in resistance management to prolong the durability of RNAi trait technology.

Clathrin-dependent endocytosis is associated with RNAi response in the western corn rootworm, Diabrotica virgifera virgifera LeConte.

The cellular uptake of dsRNA after dietary exposure is critical for RNAi efficiency; however, the mechanism of its uptake in many insects remains to be understood. In this study, we evaluated the roles of the endocytic pathway genes Clathrin heavy chain (Chc), Clathrin adaptor protein AP50, ADP ribosylation factor-like 1 (Arf72A), Vacuolar H+ ATPase 16 kDa subunit (Vha16), and small GTPase Rab7 and putative sid-1-like genes (silA and silC) in RNAi response in western corn rootworm (WCR) using a two-stage dsRNA exposure bioassay. Silencing of Chc, Vha16, and AP50 led to a significant decrease in the effects of laccase2 dsRNA reporter, indicating that these genes are involved in RNAi response. However, the knockdown of either Arf72A or Rab7 did not suppress the response to laccase2 dsRNA. The silencing of the silC gene did not lead to a significant reduction in mortality or increase in the expression of V-ATPase A reporter. While the silencing of the silA gene significantly decreased insect mortality, significant changes in V-ATPase A expression were not detected. These results suggest that clathrin-dependent endocytosis is a biological mechanism that plays an important role during RNAi response in WCR adults. The fact that no definitive support for the roles of silA or silC in RNAi response was obtained support the idea that RNAi response varies greatly in different insect species, demanding additional studies focused on elucidating their involvement in this mechanism.