Proactive resistance management for sustaining the efficacy of RNA interference for pest control.

Biopesticides based on RNA interference (RNAi) took a major step forward with the first registration of a sprayable RNAi product, which targets the world's most damaging potato pest. Proactive resistance management is needed to delay the evolution of resistance by pests and sustain the efficacy of RNAi biopesticides.

The mechanoreceptor Piezo is required for spermatogenesis in Bombyx mori.

BACKGROUND: The animal sperm shows high diversity in morphology, components, and motility. In the lepidopteran model insect, the silkworm Bombyx mori, two types of sperm, including nucleate fertile eupyrene sperm and anucleate unfertile apyrene sperm, are generated. Apyrene sperm assists fertilization by facilitating the migration of eupyrene spermatozoa from the bursa copulatrix to the spermatheca. During spermatogenesis, eupyrene sperm bundles extrude the cytoplasm by peristaltic squeezing, while the nuclei of the apyrene sperm bundles are discarded with the same process, forming matured sperm. RESULTS: In this study, we describe that a mechanoreceptor BmPiezo, the sole Piezo ortholog in B. mori, plays key roles in larval feeding behavior and, more importantly, is essential for eupyrene spermatogenesis and male fertility. CRISPR/Cas9-mediated loss of BmPiezo function decreases larval appetite and subsequent body size and weight. Immunofluorescence analyses reveal that BmPiezo is intensely localized in the inflatable point of eupyrene sperm bundle induced by peristaltic squeezing. BmPiezo is also enriched in the middle region of apyrene sperm bundle before peristaltic squeezing. Cytological analyses of dimorphic sperm reveal developmental arrest of eupyrene sperm bundles in BmPiezo mutants, while the apyrene spermatogenesis is not affected. RNA-seq analysis and q-RT-PCR analyses demonstrate that eupyrene spermatogenic arrest is associated with the dysregulation of the actin cytoskeleton. Moreover, we show that the deformed eupyrene sperm bundles fail to migrate from the testes, resulting in male infertility due to the absence of eupyrene sperm in the bursa copulatrix and spermatheca. CONCLUSIONS: In conclusion, our studies thus uncover a new role for Piezo in regulating spermatogenesis and male fertility in insects.

N(alpha)-acetyltransferase 40-mediated histone acetylation plays an important role in ecdysone regulation of metamorphosis in the red flour beetle, Tribolium castaneum.

Histone acetylation, a crucial epigenetic modification, is governed by histone acetyltransferases (HATs), that regulate many biological processes. Functions of HATs in insects are not well understood. We identified 27 HATs and determined their functions using RNA interference (RNAi) in the model insect, Tribolium castaneum. Among HATs studied, N-alpha-acetyltransferase 40 (NAA40) knockdown caused a severe phenotype of arrested larval development. The steroid hormone, ecdysone induced NAA40 expression through its receptor, EcR (ecdysone receptor). Interestingly, ecdysone-induced NAA40 regulates EcR expression. NAA40 acetylates histone H4 protein, associated with the promoters of ecdysone response genes: EcR, E74, E75, and HR3, and causes an increase in their expression. In the absence of ecdysone and NAA40, histone H4 methylation by arginine methyltransferase 1 (ART1) suppressed the above genes. However, elevated ecdysone levels at the end of the larval period induced NAA40, promoting histone H4 acetylation and increasing the expression of ecdysone response genes. NAA40 is also required for EcR, and steroid-receptor co-activator (SRC) mediated induction of E74, E75, and HR3. These findings highlight the key role of ecdysone-induced NAA40-mediated histone acetylation in the regulation of metamorphosis.

Cyp6g2 is the major P450 epoxidase responsible for juvenile hormone biosynthesis in Drosophila melanogaster.

BACKGROUND: Juvenile hormones (JH) play crucial role in regulating development and reproduction in insects. The most common form of JH is JH III, derived from MF through epoxidation by CYP15 enzymes. However, in the higher dipterans, such as the fruitfly, Drosophila melanogaster, a bis-epoxide form of JHB3, accounted most of the JH detected. Moreover, these higher dipterans have lost the CYP15 gene from their genomes. As a result, the identity of the P450 epoxidase in the JH biosynthesis pathway in higher dipterans remains unknown. RESULTS: In this study, we show that Cyp6g2 serves as the major JH epoxidase responsible for the biosynthesis of JHB3 and JH III in D. melanogaster. The Cyp6g2 is predominantly expressed in the corpus allatum (CA), concurring with the expression pattern of jhamt, another well-studied gene that is crucial in the last steps of JH biosynthesis. Mutation in Cyp6g2 leads to severe disruptions in larval-pupal metamorphosis and exhibits reproductive deficiencies, exceeding those seen in jhamt mutants. Notably, Cyp6g2-/-::jhamt2 double mutants all died at the pupal stage but could be rescued through the topical application of JH analogs. JH titer analyses revealed that both Cyp6g2-/- mutant and jhamt2 mutant lacking JHB3 and JH III, while overexpression of Cyp6g2 or jhamt caused a significant increase in JHB3 and JH III titer. CONCLUSIONS: These findings collectively established that Cyp6g2 as the major JH epoxidase in the higher dipterans and laid the groundwork for the further understanding of JH biosynthesis. Moreover, these findings pave the way for developing specific Cyp6g2 inhibitors as insect growth regulators or insecticides.

CRISPR-Cas9 mediated dsRNase knockout improves RNAi efficiency in the fall armyworm.

Lepidopteran insects are refractory to RNA interference (RNAi) response, especially to orally delivered double-stranded RNA (dsRNA). High nuclease activity in the midgut lumen is proposed as one of the major reasons for RNAi insensitivity. We identified three dsRNase genes highly expressed in the midgut of fall armyworm (FAW), Spodoptera frugiperda. The genomic region harboring those three dsRNase genes was deleted using the CRISPR-Cas9-mediated genome editing method. A homozygous line with deletion of three dsRNase genes was produced. dsRNA degradation by midgut lumen contents of mutant larvae was lower than in wild-type larvae. Feeding dsRNA targeting the inhibitor of apoptosis (IAP) gene increased knockdown of the target gene and mortality in mutants compared to wild-type larvae. These results suggest that dsRNases in the midgut contribute to RNAi inefficiency in FAW. Formulations that protect dsRNA from dsRNase degradation may improve RNAi efficiency in FAW and other lepidopteran insects.

Transcript Level and Sequence Matching Are Key Determinants of Off-Target Effects in RNAi.

Double-stranded RNA (dsRNA) pesticides, those based on RNA interference (RNAi) technology utilizing dsRNA, have shown potential for pest control. However, the off-target effects of dsRNA pose limitations to the widespread application of RNAi and raise concerns regarding potential side effects on other beneficial organisms. The precise impact and underlying factors of these off-target effects are still not well understood. Here, we found that the transcript level and sequence matching jointly regulate off-target effects of dsRNA. The much lower expressed target genes were knocked down to a lesser extent than genes with higher expression levels, and the critical sequence identity of off-target effects is approximately 80%. Moreover, off-target effects could be triggered by a contiguous matching sequence length exceeding 15 nt as well as nearly perfectly matching sequences with one or two base mismatches exceeding 19 nt. Increasing the dosage of dsRNA leads to more severe off-target effects. However, the length of mismatched dsRNA, the choice of different RNAi targets, and the location of target sites within the same gene do not affect the severity of off-target effects. These parameters can be used to guide the design of possibly selective sequences for RNAi, optimize the specificity and efficiency of dsRNA, and facilitate practical applications of RNAi for pest control.

Knockdown of the glutamate-gated chloride channel gene decreases emamectin benzoate susceptibility in the fall armyworm, Spodoptera frugiperda.

Emamectin benzoate (EB), a derivative of avermectin, is the primary insecticide used to control the fall armyworm (FAW) in China. However, the specific molecular targets of EB against FAW remain unclear. In this study, we cloned the glutamate-gated chloride channel (GluCl) gene, which is known to be a primary molecular target for avermectin. We first investigated the transcript levels of SfGluCl in FAW and found that the expression level of SfGluCl in the head and nerve cord was significantly higher than that in other tissues. Furthermore, we found that the expression level of SfGluCl was significantly higher in eggs than that in other developmental stages, including larvae, pupae, and adults. Additionally, we identified three variable splice forms of SfGluCl in exons 3 and 9 and found that their splice frequencies remained unaffected by treatment with the LC50 of EB. RNAi mediated knockdown of SfGluCl showed a significant reduction of 42% and 65% after 48 and 72 h of dsRNA feeding, respectively. Importantly, knockdown of SfGluCl sifgnificantly reduced LC50 and LC90 EB treatment induced mortality of FAW larvae by 15% and 44%, respectively, compared to the control group feeding by dsEGFP. In contrast, there were no significant changes in the mortality of FAW larvae treated with the control insecticides chlorantraniliprole and spinetoram. Finally, molecular docking simulations revealed that EB bound to the large amino-terminal extracellular domain of SfGluCl by forming five hydrogen bonds, two alkyl hydrophobic interactions and one salt bridge. These findings strongly suggest that GluCl may serve as one of the molecular targets of EB in FAW, shedding light on the mode of action of this important insecticide.

Juvenile hormone receptor Methoprene tolerant: Functions and applications.

During the past 15years, after confirming Methoprene tolerant (Met) as a juvenile hormone (JH) receptor, tremendous progress has been made in understanding the function of Met in supporting JH signal transduction. Met role in JH regulation of development, including metamorphosis, reproduction, diapause, cast differentiation, behavior, im`munity, sleep and epigenetic modifications, have been elucidated. Met's Heterodimeric partners involved in performing some of these functions were discovered. The availability of JH response elements (JHRE) and JH receptor allowed the development of screening assays in cell lines and yeast. These screening assays facilitated the identification of new chemicals that function as JH agonists and antagonists. These new chemicals and others that will likely be discovered in the near future by using JH receptor and JHRE will lead to highly effective species-specific environmentally friendly insecticides for controlling pests and disease vectors.

N6-adenosine (m6A) mRNA methylation is required for Tribolium castaneum development and reproduction.

Gene expression is regulated at various levels, including post-transcriptional mRNA modifications, where m6A methylation is the most common modification of mRNA. The m6A methylation regulates multiple stages of mRNA processing, including splicing, export, decay, and translation. How m6A modification is involved in insect development is not well known. We used the red flour beetle, Tribolium castaneum, as a model insect to identify the role of m6A modification in insect development. RNA interference (RNAi)-mediated knockdown of genes coding for m6A writers (m6A methyltransferase complex, depositing m6A to mRNA) and readers (YTH-domain proteins, recognizing and executing the function of m6A) was conducted. Knockdown of most writers during the larval stage caused a failure of ecdysis during eclosion. The loss of m6A machinery sterilized both females and males by interfering with the functioning of reproductive systems. Females treated with dsMettl3, the main m6A methyltransferase, laid significantly fewer and reduced-size eggs than the control insects. In addition, the embryonic development in eggs laid by dsMettl3 injected females was terminated in the early stages. Knockdown studies also showed that the cytosol m6A reader, YTHDF, is likely responsible for executing the function of m6A modifications during insect development. These data suggest that m6A modifications are critical for T. castaneum development and reproduction.

Inefficient uptake of small interfering RNAs is responsible for their inability to trigger RNA interference in Colorado potato beetle cells.

There has been limited success in the usage of exogenous small interference RNA (siRNA) or small hairpin RNA (shRNA) to trigger RNA interference (RNAi) in insects. Instead, long double-stranded RNAs (dsRNA) are used to induce knockdown of target genes in insects. Here, we compared the potency of si/sh RNAs and dsRNA in Colorado potato beetle (CPB) cells. CPB cells showed highly efficient RNAi response to dsRNA. However, si/sh RNAs were inefficient in triggering RNAi in CPB cells. Confocal microscopy observations of Cy3 labeled-si/sh RNA cellular uptake revealed reduced si/sh RNA uptake compared to dsRNA. si/sh RNAs were stable in the conditioned media of CPB cells. Although in a small amount, when internalized by CPB cells, the si/sh RNAs were processed by the Dicer enzyme. Lipid-mediated transfection and chimeric dsRNA approaches were used to improve the delivery of si/sh RNAs. Our results suggest that the uptake of si/sh RNAs is inefficient in CPB cells, resulting in ineffective RNAi response. However, with the help of effective delivery methods, si/sh RNA could be a useful option for developing target-specific RNAi-mediated biopesticides.

Expression and functional analysis of an odorant binding protein PopeOBP16 from Phthorimaea operculella (Zeller).

Odorant binding proteins (OBPs) are essential proteins in the peripheral olfactory system, responsible for odorant recognition and transport to olfactory receptors. Phthorimaea operculella (potato tuber moth) is an important oligophagous pest on Solanaceae crops in many countries and regions. PopeOBP16 is one of the OBPs in potato tuber moth. This study examined the expression profiles of PopeOBP16. The results of qPCR indicated that PopeOBP16 was highly expressed in the antennae of adults, especially in males, suggesting that it may be involved in odor recognition in adults. The electroantennogram (EAG) was used to screen candidate compounds with the antennae of P. operculella. The relative affinities of PopeOBP16 to 27 host volatiles and two sex pheromone components with the highest relative EAG responses were examined with competitive fluorescence-based binding assays. PopeOBP16 had the strongest binding affinity with the plant volatiles: nerol, 2-phenylethanol, linalool, 1,8-cineole, benzaldehyde, ß-pinene, d-limonene, terpinolene, α-terpinene, and the sex pheromone component trans-4, cis-7, cis-10-tridecatrien-1-ol acetate. The results provide a foundation for further research into the functioning of the olfactory system and the potential development of green chemistry for control of the potato tuber moth.

Potential effects of RNA interference of Asian longhorned beetle on its parasitoid.

BACKGROUND: It is important to understand how non-target insects such as parasitoids may be impacted directly or indirectly by RNA interference with double-stranded RNA (dsRNA) that has emerged as a novel pest control tool. We examined the potential effects of a dsRNA targeting an inhibitor of apoptosis (IAP) of the Asian longhorned beetle Anoplophora glabripennis on its gregarious larval ectoparasitoid Ontsira mellipes, directly on adult wasp's survival via injection of 4 µg of dsIAP per wasp, and indirectly on the detectability and suitability of host larvae injected with 2, 4 or 8 µg of dsIAP per larva. RESULTS: Compared with no injection or injection with a control dsGFP targeting a region of gene coding for a green fluorescence protein (GFP), dsIAP did not affect adult wasp's survival. Ontsira mellipes locates hosts in the wood by sensing their movement. Host larvae did not completely cease movement after the injection of dsIAP and were still detected and parasitized. Clutch size was reduced and only 3.8% of the parasitoid offspring developed into adults on host larvae treated at the highest dose. However, clutch size was not affected and 25.5% of the parasitoid offspring developed into adults on host larvae treated at the lowest dose. The fitness of developed wasps (development time, sex ratio, body size, and fecundity) was not affected when compared to the control treatments. No dsIAP was detected in parasitoid larvae. CONCLUSION: The results show no direct effect of the dsRNA on its parasitoid, but the potential indirect effect of dsRNA-affected host on the parasitoid, which may be minimized through optimizing dsRNA dosage to promote compatible applications of both management options for this invasive forest pest. © 2022 Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Gene expression in Verson's glands of the fall armyworm suggests their role in molting and immunity.

Verson's glands are segmental pairs of dermal glands attached to the epidermis in lepidopteran larvae. They produce macromolecules during intermolt period and empty them during each molt. Morphological, histochemical, developmental, and protein analysis studies have been conducted to determine the functions of Verson's glands. However, the exact role of Verson's glands remains unclear. In our previous study, a strain of transgenic fall armyworm, Spdoptera frugiperda expressing green fluorescence protein (GFP) and Systemic RNA interference defective protein 1 (SID1) from Caenorhabditis elegans was established to improve RNA interference (RNAi) efficiency. Unexpectedly, we found that GFP fluorescence was significantly brighter in Verson's glands than in other tissues. Also, RNAi efficiency improved more in Verson's glands than in other tissues. We took advantage of improved RNAi efficiency to explore the function of Verson's glands. RNA-seq analysis revealed that genes highly expressed in Verson's glands code for cuticular proteins, molting fluid proteins, hemolymph proteins, and antimicrobial peptides. Injection of dsRNA targeting essential genes, inhibitor of apoptosis (IAP), Actin, and vacuolar-type ATPase (VATPase) interfered with Verson's glands growth. These results revealed that Verson's glands may contribute to hemolymph, cuticle, molting fluid, and immune response during molting. This study also provide useful tools for future research in identifying the physiological role of Verson's glands in lepidopteran insects.

Autophagy genes AMBRA1 and ATG8 play key roles in midgut remodeling of the yellow fever mosquito, Aedes aegypti.

The function of two autophagy genes, an activating molecule BECN1 regulated autophagy (AMBRA1) and autophagy-related gene 8 (ATG8) in the midgut remodeling of Aedes aegypti was investigated. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of RNA samples collected from the last instar larvae and pupae showed that these two genes are predominantly expressed during the last 12 h and first 24 h of the last larval and pupal stages, respectively. Stable ecdysteroid analog induced and juvenile hormone (JH) analog suppressed these genes. RNA interference (RNAi) studies showed that the ecdysone-induced transcription factor E93 is required for the expression of these genes. JH-induced transcription factor krüppel homolog 1 (Kr-h1) suppressed the expression of these genes. RNAi-mediated silencing of AMBRA1 and ATG8 blocked midgut remodeling. Histological studies of midguts from insects at 48 h after ecdysis to the final larval stage and 12 h after ecdysis to the pupal stage showed that ATG gene knockdown blocked midgut remodeling. AMBRA1 and ATG8 double-stranded (dsRNA)-treated insects retained larval midgut cells and died during the pupal stage. Together, these results demonstrate that ecdysteroid induction of ATG genes initiates autophagy programmed cell death during midgut remodeling. JH inhibits midgut remodeling during metamorphosis by interfering with the expression of ATG genes.

dsRNase1 contribution to dsRNA degradation activity in the Sf9 cells conditioned medium.

RNA interference (RNAi) is inefficient in lepidopteran insects, including Spodoptera frugiperda. RNase activity in the lumen and hemocoel is known to contribute to low RNAi efficiency in these insects. Conditioned medium from Sf9 cells developed from ovaries of S. frugiperda shows high dsRNA degradation activity. But the enzymes responsible for this activity have not been identified. The nuclease genes that are highly expressed in Sf9 cells, REase, RNaseT2, and dsRNase1, were identified. Knockdown of dsRNase1 in Sf9 cells resulted in a reduction of dsRNA degradation activity in the Sf9 cells conditioned medium. Knockdown of dsRNase1 also increased RNAi efficiency in Sf9 cells. The results from these studies identified a major player in dsRNA degradation activity in the Sf9 cells conditioned medium. We also describe an efficient system that can be used to identify other genes responsible for dsRNA degradation and RNAi efficiency in Sf9 cells.

RNAi turns 25:contributions and challenges in insect science.

Since its discovery in 1998, RNA interference (RNAi), a Nobel prize-winning technology, made significant contributions to advances in biology because of its ability to mediate the knockdown of specific target genes. RNAi applications in medicine and agriculture have been explored with mixed success. The past 25 years of research on RNAi resulted in advances in our understanding of the mechanisms of its action, target specificity, and differential efficiency among animals and plants. RNAi played a major role in advances in insect biology. Did RNAi technology fully meet insect pest and disease vector management expectations? This review will discuss recent advances in the mechanisms of RNAi and its contributions to insect science. The remaining challenges, including delivery to the target site, differential efficiency, potential resistance development and possible solutions for the widespread use of this technology in insect management.

Chromosomal-level genome assembly of potato tuberworm, Phthorimaea operculella: a pest of solanaceous crops.

The potato tuberworm, Phthorimaea operculella Zeller, is an oligophagous pest feeding on crops mainly belonging to the family Solanaceae. It is one of the most destructive pests of potato worldwide and attacks foliage and tubers in the field and in storage. However, the lack of a high-quality reference genome has hindered the association of phenotypic traits with their genetic basis. Here, we report on the genome assembly of P. operculella at the chromosomal level. Using Illumina, Nanopore and Hi-C sequencing, a 648.2 Mb genome was generated from 665 contigs, with an N50 length of 3.2 Mb, and 92.0% (596/648.2 Mb) of the assembly was anchored to 29 chromosomes. In total, 16619 genes were annotated, and 92.4% of BUSCO genes were fully represented. The chromosome-level genome of P. operculella will provide a significant resource for understanding the genetic basis for the biological study of this insect, and for promoting the integrative management of this pest in future.

Identification of species-specific juvenile hormone response elements in the fall armyworm, Spodoptera frugiperda.

Juvenile hormones (JH) regulate insect development and reproduction. The JH analogs (JHA) are used as insecticides. However, JHAs are rarely used in managing pests such as the fall armyworm, Spodoptera frugiperda that cause damage during larval stages. The insecticides that antagonize JH action and induce stoppage of feeding and precocious metamorphosis might work better to control these pests. Treating insects with JHA insecticides induces the expression of an early JH response gene, Krüppel homolog 1 (Kr-h1) by working through JH response elements (JHRE) present in its promoter. In this study, we identified JHREs present in the promoter of Kr-h1 gene of a global pest, S. frugiperda, and used them to develop a JHRE-reporter cell platform to screen for JH analogs. JHA, methoprene induced the expression of SfKr-h1 both in vitro and in vivo. JHRE present in the promoters of two SfKr-h1 isoforms, SfKr-h1α and SfKr-h1ß were identified. In Sf9 cells, the knockout of isoform-specific JHRE affected JH response in an isoform-specific manner. We also found that S. frugiperda JHRE (SfJHRE) did not function in the mosquito Aedes aegypti Aag2 cells and Tribolium castaneum TcA cells. Similarly, Ae. aegypti AaJHRE and T. castaneum TcJHRE were only functional in cells derived from these insects. The nucleotide sequence at the 3'end to the conserved core JHRE E-box sequence seems to be responsible for the species specificity observed. Two stable cell lines expressing the luciferase and enhanced green fluorescent protein genes under the control of SfJHRE were established. These cell lines responded well to JHA; these two JHRE-reporter cell lines could be used in screening assays to identify insecticides to manage S. frugiperda and other major pests.

CRISPR-Cas9 Genome Editing Uncovers the Mode of Action of Methoprene in the Yellow Fever Mosquito, Aedes aegypti.

Methoprene, a juvenile hormone (JH) analog, is widely used for insect control, but its mode of action is not known. To study methoprene action in the yellow fever mosquito, Aedes aegypti, the E93 (ecdysone-induced transcription factor) was knocked out using the CRISPR-Cas9 system. The E93 mutant pupae retained larval tissues similar to methoprene-treated insects. These insects completed pupal ecdysis and died as pupa. In addition, the expression of transcription factors, broad complex and Krüppel homolog 1 (Kr-h1), increased and that of programmed cell death (PCD) and autophagy genes decreased in E93 mutants. These data suggest that methoprene functions through JH receptor, methoprene-tolerant, and induces the expression of Kr-h1, which suppresses the expression of E93, resulting in a block in PCD and autophagy of larval tissues. Failure in the elimination of larval tissues and the formation of adult structures results in their death. These results answered long-standing questions on the mode of action of methoprene.

Mitochondria dysfunction impairs Tribolium castaneum wing development during metamorphosis.

The disproportionate growth of insect appendages such as facultative growth of wings and exaggeration of beetle horns are examples of phenotypic plasticity. Insect metamorphosis is the critical stage for development of pupal and adult structures and degeneration of the larval cells. How the disproportionate growth of external appendages is regulated during tissue remodeling remains unanswered. Tribolium castaneum is used as a model to study the function of mitochondria in metamorphosis. Mitochondrial dysfunction is achieved by the knockdown of key mitochondrial regulators. Here we show that mitochondrial function is not required for metamorphosis except that severe mitochondrial dysfunction blocks ecdysis. Surprisingly, various abnormal wing growth, including short and wingless phenotypes, are induced after knocking down mitochondrial regulators. Mitochondrial activity is regulated by IIS (insulin/insulin-like growth factor signaling)/FOXO (forkhead box, sub-group O) pathway through TFAM (transcription factor A, mitochondrial). RNA sequencing and differential gene expression analysis show that wing-patterning and insect hormone response genes are downregulated, while programmed cell death and immune response genes are upregulated in insect wing discs with mitochondrial dysfunction. These studies reveal that mitochondria play critical roles in regulating insect wing growth by targeting wing development during metamorphosis, thus showing a novel molecular mechanism underlying developmental plasticity.