Molecular characterization and RNA interference responses of the lethal giant larvae gene in Diabrotica virgifera virgifera adults.

High specificity for silencing target genes and single-copy target genes that yield clear phenotypes are two important factors for the success of RNA interference (RNAi). The lethal giant larvae (Lgl) gene appears to be an ideal gene for RNAi because RNAi can effectively suppress its expression and results in molting defects and mortality in Tribolium castaneum. To investigate the suitability of this gene for RNAi in other insects, we identified and characterized DvLgl from the western corn rootworm, Diabrotica virgifera virgifera, a species exhibiting high RNAi efficiency. DvLgl was expressed in all developmental stages and tissues investigated. The deduced DvLgl protein showed high amino-acid sequence identities and similar domain architecture to Lgls from other insect species. Despite many similarities among insect Lgls, RNAi-mediated suppression of DvLgl failed to produce a phenotype in D. v. virgifera adults. The difference in developing phenotypes could be attributed greatly to the level of gene suppression and the insect developmental stages for RNAi. These results highlight the variability in RNAi response among insects and showcase the importance of screening multiple target genes when conducting RNAi studies. Our findings are expected to help the design of future RNAi studies and future investigations of Lgl in insects.

Stability of double-stranded RNA in gut contents and hemolymph of Ostrinia nubilalis larvae.

RNA interference (RNAi) is a revolutionary technique for silencing gene expression, but the success of this technique is dependent upon the stability of double-stranded RNA (dsRNA) molecules. In many insects, especially lepidopteran species, RNAi efficiency is limited by high instability of dsRNA in the gut and/or hemolymph, preventing the development of RNAi-based strategies for many serious pests. Previous attempts to perform RNAi on Ostrinia nubilalis (ECB, Lepidoptera: Crambidae) indicate low RNAi efficiency with both dsRNA injection and feeding. To investigate the contribution of dsRNA instability to low RNAi efficiency in ECB, a serious of ex vivo incubation experiments were performed where dsRNA integrity was assessed following incubation in larval gut continents and hemolymph using gel electrophoresis or RT-qPCR. DsRNA was less stable in the gut contents from ECB than in gut contents from Diabrotica virgifera virgifera, a coleopteran exhibiting high RNAi efficiency. Furthermore, characterization of dsRNA stability in ECB gut contents and hemolymph revealed that dsRNA was rapidly degraded under physiologically relevant conditions as a result of enzymatic activity that was neither size- nor sequence-dependent. These findings suggest that instability of dsRNA in ECB tissues is a contributing factor to the poor efficiency of RNAi in this pest. This work advances our understanding of mechanisms impacting RNAi efficiency in ECB and related lepidopteran insects for which novel pest management strategies are needed, and may facilitate the development of strategies for enhancing dsRNA stability in ECB tissues.

Multiple Argonaute family genes contribute to the siRNA-mediated RNAi pathway in Locusta migratoria.

Argonautes (Ago) are important core proteins in RNA interference (RNAi) pathways of eukaryotic cells. Generally, it is thought that Ago1, Ago2 and Ago3 are involved in the miRNA (microRNA), siRNA (small interfering RNA) and piRNA (Piwi-interacting RNA)-mediated RNAi pathways, respectively. As a main component of the RNA-induced silencing complex (RISC), Ago2 plays an indispensable role in using siRNA to recognize and cut target messenger RNAs resulting in suppression of transcript levels, but the contributions of Ago1 and Ago3 to the siRNA-mediated RNAi pathway remain to be explored in many insect species. In this study, we investigated the contributions of four Ago genes (named LmAgo1, LmAgo2a and LmAgo2b and LmAgo3) to RNAi efficiency in Locusta migratoria by using both in vivo and in vitro experiments. Our results showed that suppression of each of the Ago genes significantly impaired RNAi efficiency when targeting Lmß-tubulin transcripts, resulting in recovery of 48, 43.3, 61.4 or 26% of Lmß-tubulin transcripts following RNAi-mediated suppression of LmAgo1, LmAgo2a, LmAgo2b, and LmAgo3, respectively. Furthermore, overexpression of LmAgo1, LmAgo2a, LmAgo2b, or LmAgo3 in a PAc5.1-V5/HisB vector and co-transfection with psicheck2 fluorescence vector in S2 cells reduced luciferase fluorescence by 38.3, 58.9, 53.3 or 55.6%, respectively. Taken together, our results showed that LmAgo1, LmAgo2a, LmAgo2b, and LmAgo3 each make significant contributions to RNAi efficiency in L. migratoria and suggest that the involvement of all four enzymes could be one of the major factors supporting robust RNAi responses observed in this species.

Progress and prospects of arthropod chitin pathways and structures as targets for pest management.

Chitin is a structural component of the arthropod cuticular exoskeleton and the peritrophic matrix of the gut, which play crucial roles in growth and development. In the past few decades, our understanding of the composition, biosynthesis, assembly, degradation, and regulation of chitinous structures has increased. Many chemicals have been developed that target chitin biosynthesis (benzoyphenyl ureas, etoxazole), chitin degradation (allosamidin, psammaplin), and chitin regulation (benzoyl hydrazines), thus resulting in molting deformities and lethality. In addition, proteins that disrupt chitin structures, such as lectins, proteases, and chitinases have been utilized to halt feeding and induce mortality. Chitin-degrading enzymes, such as chitinases are also useful for improving the efficacy of bio-insecticides. Transgenic plants, baculoviruses, fungi, and bacteria have been engineered to express chitinases from a variety of organisms for control of arthropod pests. In addition, RNA interference targeting genes involved in chitin pathways and structures are now being investigated for the development of environmentally friendly pest management strategies. This review describes the chemicals and proteins used to target chitin structures and enzymes for arthropod pest management, as well as pest management strategies based upon these compounds, such as plant-incorporated-protectants and recombinant entomopathogens. Recent advances in RNA interference-based pest management, and how this technology can be used to target chitin pathways and structures are also discussed.

Metabolism of selected model substrates and insecticides by recombinant CYP6FD encoded by its gene predominately expressed in the brain of Locusta migratoria.

The migratory locust, Locusta migartoria, is a major agricultural insect pest and its resistance to insecticides is becoming more prevalent. Cytochrome P450 monooxygenases (CYPs) are important enzymes for biotransformations of various endogenous and xenobiotic substances. These enzymes play a major role in developing insecticide resistance in many insect species. In this study, we heterologously co-expressed a CYP enzyme (CYP6FD1) and cytochrome P450 reductase (CPR) from L. migartoria in Sf9 insect cells. The recombinant enzymes were assayed for metabolic activity towards six selected model substrates (luciferin-H, luciferin-Me, luciferin-Be, luciferin-PFBE, luciferin-CEE and 7-ethoxycoumarin), and four selected insecticides (deltamethrin, chlorpyrifos, carbaryl and methoprene). Recombinant CYP6FD1 showed activity towards 7-ethoxycoumarin and luciferin-Me, but no detectable activity towards the other luciferin derivatives. Furthermore, the enzyme efficiently oxidized deltamethrin to hydroxydeltamethrin through an aromatic hydroxylation in a time-dependent manner. However, the enzyme did not show any detectable activity towards the other three insecticides. Our results provide direct evidence that CYP6FD1 is capable of metabolizing deltamethrin. This work is a step towards a more complete characterization of the catalytic capabilities of CYP6FD1 and other xenobiotic metabolizing CYP enzymes in L. migratoria.

Variable gut pH as a potential mechanism of tolerance to Bacillus thuringiensis subsp. israelensis toxins in the biting midge Culicoides sonorensis.

BACKGROUND: Toxins of Bacillus thuringiensis subsp. israelensis (Bti) are safer alternatives for controlling dipteran pests such as black flies and mosquitoes. The biting midge Culicoides sonorensis (Diptera: Ceratopogonidae) is an important pest of livestock in much of the United States and larval midges utilize semi-aquatic habitats which are permissive for Bti product application. Reports suggest that Bti products are ineffective at killing biting midges despite their taxonomic relation to black flies and mosquitoes. Here, we investigate the toxicity of a Bti-based commercial insecticide and its active ingredient in larval Culicoides sonorensis. A suspected mechanism of Bti tolerance is an acidic larval gut, and we used a pH indicator dye to examine larval Culicoides sonorensis gut pH after exposure to Bti. RESULTS: The lethal concentration to kill 90% (LC90) of larvae of the commercial product (386 mg/L) was determined to be almost 10 000 times more than that of some mosquito species, and no concentration of active ingredient tested achieved 50% larval mortality. The larval gut was found to be more acidic after exposure to Bti which inhibits Bti toxin activity. By comparison, 100% mortality was achieved in larval Aedes aegypti at the product's label rate for this species and mosquito larvae had alkaline guts regardless of treatment. Altering the larval rearing water to alkaline conditions enhanced Bti efficacy when using the active ingredient. CONCLUSION: We conclude that Bti is not practical for larval Culicoides sonorensis control at the same rates as mosquitos but show that alterations or additives to the environment could make the products more effective. © 2024 Society of Chemical Industry.

Tiny silver bullets: silver nanoparticles are insecticidal to Culicoides sonorensis (Diptera: Ceratopogonidae) biting midge larvae.

Insecticide formulations with safer environmental profiles and limited off-target effects are desirable to manage medical and veterinary pests. Silver nanoparticles are insecticidal against mosquitos, nonbiting midges, and other insects. The biting midge, Culicoides sonorensis Wirth and Jones, is a vector of agriculturally important pathogens in much of the United States, and this study aimed to examine the insecticidal properties of silver nanoparticles in larvae of this species. Mortality of third-instar larvae was assessed daily for 7 days after exposure to concentrations of silver nanoparticles, sorghum polymer particles, and hybrid silver-sorghum polymer particles. Both silver nanoparticles and silver-sorghum polymer particles were insecticidal, but sorghum polymer particles alone did not significantly contribute to larval mortality. Concentrations of 100 mg/liter of silver nanoparticles achieved >50% mortality at day 7, and 200 mg/liter treatments achieved >75% larval mortality within 24 h. The antimicrobial properties of silver nanoparticles were also examined, and culturable bacteria were recovered from larval-rearing media at 200 mg/liter but not at 400 mg/liter of silver nanoparticles. These data suggest that C. sonorensis larval mortality is primarily caused by silver nanoparticle toxicity and not by the reduction of bacteria (i.e., a larval food source). This work describes the first use of silver nanoparticles in C. sonorensis and shows the potential insecticide applications of these nanoparticles against this agricultural pest. The grain-polymer particles also successfully carried insecticidal silver nanoparticles, and their utility in loading diverse compounds could be a novel toxin delivery system for biting midges and similar pests.

Erratum: AC-DC Electropenetrography as a Tool to Quantify Probing and Ingestion Behaviors of the Yellow Fever Mosquito (Aedes aegypti) on Mice in Biocontainment.

This corrects the article DOI: 10.30802/AALAS-CM-23-000037. When the above article was first published in the Vol 73 No 6 (December 2023) issue of Comparative Medicine, figure images were incorrectly associated with the figure legends. The correct version of this article has been reprinted in full in volume 74, issue 1 of the February issue of Comparative Medicine. The publisher apologizes for this error and any inconvenience caused.

Indoxacarb, Metaflumizone, and Other Sodium Channel Inhibitor Insecticides: Mechanism and Site of Action on Mammalian Voltage-Gated Sodium Channels.

Sodium channel inhibitor (SCI) insecticides were discovered almost four decades ago but have only recently yielded important commercial products (eg., indoxacarb and metaflumizone). SCI insecticides inhibit sodium channel function by binding selectively to slow-inactivated (non-conducting) sodium channel states. Characterization of the action of SCI insecticides on mammalian sodium channels using both biochemical and electrophysiological approaches demonstrates that they bind at or near a drug receptor site, the "local anesthetic (LA) receptor." This mechanism and site of action on sodium channels differentiates SCI insecticides from other insecticidal agents that act on sodium channels. However, SCI insecticides share a common mode of action with drugs currently under investigation as anticonvulsants and treatments for neuropathic pain. In this paper we summarize the development of the SCI insecticide class and the evidence that this structurally diverse group of compounds have a common mode of action on sodium channels. We then review research that has used site-directed mutagenesis and heterologous expression of cloned mammalian sodium channels in Xenopus laevis oocytes to further elucidate the site and mechanism of action of SCI insecticides. The results of these studies provide new insight into the mechanism of action of SCI insecticides on voltage-gated sodium channels, the location of the SCI insecticide receptor, and its relationship to the LA receptor that binds therapeutic SCI agents.

Outlook on RNAi-Based Strategies for Controlling Culicoides Biting Midges.

Culicoides are small biting midges with the capacity to transmit important livestock pathogens around much of the world, and their impacts on animal welfare are likely to expand. Hemorrhagic diseases resulting from Culicoides-vectored viruses, for example, can lead to millions of dollars in economic damages for producers. Chemical insecticides can reduce Culicoides abundance but may not suppress population numbers enough to prevent pathogen transmission. These insecticides can also cause negative effects on non-target organisms and ecosystems. RNA interference (RNAi) is a cellular regulatory mechanism that degrades mRNA and suppresses gene expression. Studies have examined the utility of this mechanism for insect pest control, and with it, have described the hurdles towards producing, optimizing, and applying these RNAi-based products. These methods hold promise for being highly specific and environmentally benign when compared to chemical insecticides and are more transient than engineering transgenic insects. Given the lack of available control options for Culicoides, RNAi-based products could be an option to treat large areas with minimal environmental impact. In this study, we describe the state of current Culicoides control methods, successes and hurdles towards using RNAi for pest control, and the necessary research required to bring an RNAi-based control method to fruition for Culicoides midges.

Evaluation of potential reference genes in the biting midge Culicoides sonorensis for real-time quantitative PCR analyses.

Studies examining differentially expressed genes and gene silencing by RNA interference (RNAi) require a set of stably expressed reference genes for accurate normalization. The biting midge Culicoides sonorensis is an important vector of livestock pathogens and is often used as a model species for biting midge research. Here, we examine the stable expression of six candidate reference genes in C. sonorensis: actin, ß-tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein subunit (RPS) 18, vacuolar ATPase subunit A (VhaA), and elongation factor 1-beta (EF1b). Gene expression was assessed under seven conditions, including cells treated with double-stranded RNA (dsRNA), 3rd and 4th instar larvae treated with dsRNA, six developmental stages, four adult female body parts or tissue groups, and females injected with bluetongue virus or vesicular stomatitis virus. Stable gene expression was assessed using RefFinder, NormFinder, geNorm, and BestKeeper. The ranked results for each analysis tool under each condition and a comprehensive ranking for each condition are presented. The data show that optimal reference genes vary between conditions and that just two reference genes were necessary for each condition. These findings provide reference genes for use under these conditions in future studies using real-time quantitative PCR to evaluate gene expression in C. sonorensis.

An electropenetrography waveform library for the probing and ingestion behaviors of Culex tarsalis on human hands.

Culex tarsalis Coquillett (Diptera: Culicidae) mosquitoes are capable of vectoring numerous pathogens affecting public and animal health. Unfortunately, the probing behaviors of mosquitoes are poorly understood because they occur in opaque tissues. Electropenetrography (EPG) has the potential to elucidate these behaviors by recording the electrical signals generated during probing. We used an AC-DC EPG with variable input resistors (Ri levels) to construct a waveform library for Cx. tarsalis feeding on human hands. Biological events associated with mosquito probing were used to characterize waveforms at four Ri levels and with two electrical current types. The optimal settings for EPG recordings of Cx. tarsalis probing on human hands was an Ri level of 107 Ohms using an applied signal of 150 millivolts alternating current. Waveforms for Cx. tarsalis included those previously observed and associated with probing behaviors in Aedes aegypti L. (Diptera: Culicidae): waveform families J (surface salivation), K (stylet penetration through the skin), L (types 1 and 2, search for a blood vessel/ingestion site), M (types 1 and 2, ingestion), N (type 1, an unknown behavior which may be a resting and digestion phase), and W (withdrawal). However, we also observed variations in the waveforms not described in Ae. aegypti, which we named types L3, M3, M4, and N2. This investigation enhances our understanding of mosquito probing behaviors. It also provides a new tool for the automated calculation of peak frequency. This work will facilitate future pathogen acquisition and transmission studies and help identify new pest and disease management targets.

Diversity of the Bacterial and Viral Communities in the Tropical Horse Tick, Dermacentor nitens, in Colombia.

Ticks are obligatory hematophagous ectoparasites that transmit pathogens among various vertebrates, including humans. The microbial and viral communities of ticks, including pathogenic microorganisms, are known to be highly diverse. However, the factors driving this diversity are not well understood. The tropical horse tick, Dermacentor nitens, is distributed throughout the Americas and it is recognized as a natural vector of Babesia caballi and Theileria equi, the causal agents of equine piroplasmosis. In this study, we characterized the bacterial and viral communities associated with partially fed Dermacentor nitens females collected using a passive survey on horses from field sites representing three distinct geographical areas in the country of Colombia (Bolivar, Antioquia, and Cordoba). RNA-seq and sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene were performed using the Illumina-Miseq platform (Illumina, San Diego, CA, USA). A total of 356 operational taxonomic units (OTUs) were identified, in which the presumed endosymbiont, Francisellaceae/Francisella spp., was predominantly found. Nine contigs corresponding to six different viruses were identified in three viral families: Chuviridae, Rhabdoviridae, and Flaviviridae. Differences in the relative abundance of the microbial composition among the geographical regions were found to be independent of the presence of Francisella-like endosymbiont (FLE). The most prevalent bacteria found in each region were Corynebacterium in Bolivar, Staphylococcus in Antioquia, and Pseudomonas in Cordoba. Rickettsia-like endosymbionts, mainly recognized as the etiological agent of rickettsioses in Colombia, were detected in the Cordoba samples. Metatranscriptomics revealed 13 contigs containing FLE genes, suggesting a trend of regional differences. These findings suggest regional distinctions among the ticks and their bacterial compositions.

Diversity of the bacterial and viral communities in the tropical horse tick, Dermacentor nitens in Colombia.

Ticks are obligatory hematophagous ectoparasites that transmit pathogens among various vertebrates, including humans. The composition of the microbial and viral communities in addition to the pathogenic microorganisms is highly diverse in ticks, but the factors driving the diversity are not well understood. The tropical horse tick, Dermacentor nitens , is distributed throughout the Americas and it is recognized as a natural vector of Babesia caballi and Theileria equi , the causal agents of equine piroplasmosis. We characterized the bacterial and viral communities associated with partially-fed D. nitens females collected by a passive survey on horses from field sites representing three distinct geographical areas in Colombia (Bolivar, Antioquia, and Cordoba). RNA-seq and sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene were performed using the Illumina-Miseq platform. A total of 356 operational taxonomic units (OTUs) were identified, in which the presumed endosymbiotic Francisellaceae/ Francisella spp. was predominantly found. Nine contigs corresponding to six different viruses were identified in three viral families: Chuviridae, Rhabdoviridae, and Flaviviridae. Differences in the relative abundance of the microbial composition among the geographical regions were found to be independent of the presence of Francisella -Like Endosymbiont (FLE). The most prevalent bacteria found on each region were Corynebacterium in Bolivar, Staphylococcus in Antioquia, and Pseudomonas in Cordoba. Rickettsia -like endosymbionts, mainly recognized as the etiological agent of rickettsioses in Colombia were detected in the Cordoba samples. Metatranscriptomics revealed 13 contigs containing FLE genes, suggesting a trend of regional differences. These findings suggest regional distinctions among the ticks and their bacterial compositions.

Expression of pleiotrophin, an important regulator of cell migration, is inhibited in intestinal epithelial cells by treatment with non-steroidal anti-inflammatory drugs.

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely used drugs for the suppression of inflammation and pain. However, the analgesic properties of NSAIDs are also associated with significant negative side effects, most notably in the gastrointestinal (GI) tract. Increasingly, evidence indicates that the ulcerogenic properties of some NSAIDs are not exclusively the result of inhibition of cyclooxygenase isoforms in the GI tract, and other mechanisms, including inhibition of cell migration and epithelial restitution, are being explored. Recently, microarray analysis was used to identify potential novel targets of NSAID activity in intestinal epithelial cells. Treated cells exhibited significant reductions in the gene expression of pleiotrophin (PTN), a cytokine and growth factor known to participate in angiogenesis and bone growth. This report aimed to confirm the microarray results reported previously, and to measure protein expression of PTN in intestinal epithelial cells. Furthermore, we also examined the effects of exogenous PTN on cell migration in the presence and absence of either NSAIDs with variable ulcerogenic potential or PTN-specific siRNA. Our results demonstrated that indomethacin and NS-398, two NSAIDs with ulcerogenic potential significantly decrease both gene and protein expressions of PTN in IEC-6 cells and protein expression in IEC-6-Cdx2 cells. Additionally, cell migration experiments with PTN siRNA showed that PTN is an important mediator of IEC-6 cell migration, and addition of exogenous PTN partially restores the deficits in cell migration caused by treatment with indomethacin and NS-398. Finally, measurement of PTN protein expression in the GI tract of horses treated with phenylbutazone showed that PTN expression is reduced by NSAIDs in vivo. Our results show that PTN is an important mediator of cell migration in IEC-6 cells, and PTN is a potential target through which NSAIDs may inhibit cell migration, epithelial restitution, and wound healing in the GI tract.

Lethal giant larvae gene is required for normal nymphal development and midgut morphogenesis in Locusta migratoria.

Disruption of morphogenesis, an essential process in organismal development, can lead to disruption of biological processes, reduction in fitness, or even death of an organism. The roles of lethal giant larvae (Lgl) protein in maintaining tissue organization have been studied extensively in mammals, but little is known about this gene's roles in promoting correct tissue morphogenesis in insects. In this study, we identified an Lgl ortholog in Locusta migratoria. RT-qPCR results revealed that LmLgl was constitutively expressed during third, fourth, and fifth instar nymphs. Furthermore, LmLgl showed highest expression in the ovary followed by wing pads, midgut, hindgut, Malpighian tubules, and foregut of the third-instar nymphs. To examine the role of LmLgl in L. migratoria development, RNA interference was performed during nymphal stages. Silencing of LmLgl increased body size but decreased bodyweight by 9.0%. Histological sections of the midgut revealed abnormal large masses of disordered epithelial cells in dsLmLgl-injected nymphs. In addition, downregulation of LmLgl transcript levels significantly altered the morphological structure in midgut, resulting in the formation of tumor-like structures. Our results indicated that LmLgl may act as a tumor-suppressor gene, which plays an essential role in maintaining a normal morphological structure in the midgut of L. migratoria. Our results also suggest that LmLgl may be explored as a potential target for developing dsRNA-based biological pesticides for managing insect pests.

Vacuolar (H+ )-ATPase subunit c is essential for the survival and systemic RNA interference response in Locusta migratoria.

BACKGROUND: Vacuolar (H+ )-ATPase (V-ATPase) is a multi-subunit enzyme that hydrolyzes adenosine triphosphate (ATP) to transport protons across a cellular membrane, and it plays an important role in numerous biological processes, including in growth, development and immune responses. The c subunit of V-ATPase is a highly conserved subunit of the rotatory proteolipid ring that is required for binding and transporting protons. To date, there are only a few published reports on V-ATPase-c functions in insects. RESULTS: We identified and characterized the V-ATPase-c gene in Locusta migratoria, one of the most destructive agricultural insect pests in the world. LmV-ATPase-c was predominately expressed in Malpighian tubules of nymphs, followed by the hindgut and ovary, while the other tissues showed relatively low expression levels. Silencing of LmV-ATPase-c caused severe molting defects in nymphs and a high mortality rate of > 90%. Histological staining and microscopic examination of sections from the abdominal cuticle revealed the absence of newly formed cuticle in nymphs that were injected with dsLmV-ATPase-c. In addition, silencing of LmV-ATPase-c transcript levels significantly impaired RNA interference (RNAi) efficiency of a reporter gene. By quantifying double-stranded RNA (dsRNA) amounts by quantitative polymerase chain reaction (PCR), we found that RNAi against LmV-ATPase-c provoked a dramatic accumulation of dsRNA in the endosomes of epidermal and midgut cells of Locusta migratoria. CONCLUSION: Our results indicate that LmV-ATPase-c is indispensable for the formation of new cuticle during the molting process and has pivotal functions in dsRNA escape from endosomes. LmV-ATPase-c might be a valuable target for developing new strategies for insect pest management. © 2022 Society of Chemical Industry.

Identification of Rab family genes and functional analyses of LmRab5 and LmRab11A in the development and RNA interference of Locusta migratoria.

Rab proteins constitute the largest family of small GTPases, which play pivotal roles in intracellular membrane trafficking in all eukaryotes. A number of Rab genes have been identified in eukaryotes; however, very little information about these genes has been reported in insects. In the current study, for the first time we identified and characterized 27 Rab family genes from Locusta migratoria. Phylogenetic analysis and comparison of domain architecture indicated that Rab family genes are highly conserved among insect species. Tissue-dependent expression profiles indicated that expression of Rab genes was highest in the ovary, except for LmRab3, which was most highly expressed in hemolymph. The biological function of each Rab gene was investigated using RNA interference (RNAi). Double-stranded RNA targeting each Rab gene was injected into the hemocoel of nymphs and revealed that suppression of two Rab genes (LmRab5 and LmRab11A) caused 100% mortality. In addition, nymphs injected with dsLmRab5 exhibited severe phenotypic defects in the gastric caeca and midgut, while dsLmRab11A arrested the molting process. We then applied the RNAi of RNAi technique to test if silencing either of these two genes would affect the suppression of the lethal giant larvae (LmLgl) reporter gene and found that suppression of LmRab5 diminished the RNAi efficiency of LmLgl, whereas suppression of LmRab11A enhanced RNAi efficiency of LmLgl. These results indicate that Rab genes contribute differently to RNAi efficiency in different tissues. Our study provides a foundation for further functional investigations of Rab genes and their contributions to RNAi efficiency in L. migratoria.

Strategies for enhancing the efficiency of RNA interference in insects.

Low RNA interference (RNAi) efficiency in many insect pests has significantly prevented its widespread application for insect pest management. This article provides a comprehensive review of recent research in developing various strategies for enhancing RNAi efficiency. Our review focuses on the strategies in target gene selection and double-stranded RNA (dsRNA) delivery technologies. For target gene selection, genome-wide or large-scale screening strategies have been used to identify most susceptible target genes for RNAi. Other strategies include the design of dsRNA constructs and manipulate the structure of dsRNA to maximize the RNA efficiency for a target gene. For dsRNA delivery strategies, much recent research has focused on the applications of complexed or encapsulated dsRNA using various reagents, polymers, or peptides to enhance dsRNA stability and cellular uptake. Other dsRNA delivery strategies include genetic engineering of microbes (e.g. fungi, bacteria, and viruses) and plants to produce insect-specific dsRNA. The ingestion of the dsRNA-producing organisms or tissues will have lethal or detrimental effects on the target insect pests. This article also identifies obstacles to further developing RNAi for insect pest management and suggests future avenues of research that will maximize the potential for using RNAi for insect pest management. © 2021 Society of Chemical Industry.

Characterization, expression patterns, and transcriptional responses of three core RNA interference pathway genes from Ostrinia nubilalis.

RNA interference (RNAi) is commonly used in the laboratory to analyze gene function, and RNAi-based pest management strategies are now being employed. Unfortunately, RNAi is hindered by inefficient and highly-variable results when different insects are targeted, especially lepidopterans, such as the European corn borer (ECB), Ostrinia nubilalis (Lepidoptera: Crambidae). Previous efforts to achieve RNAi-mediated gene suppression in ECB revealed low RNAi efficiency with both double-stranded RNA (dsRNA) injection and ingestion. One mechanism that can affect RNAi efficiency in insects is the expression and function of core RNAi pathway genes, such as those encoding Argonaut 2 (Ago2), Dicer 2 (Dcr2), and a dsRNA binding protein (R2D2). To determine if deficiencies in these core RNAi pathway genes contribute to low RNAi efficiency in ECB, full-length complementary DNAs encoding OnAgo2, OnDcr2, and OnR2D2 were cloned, sequenced, and characterized. A comparison of domain architecture suggested that all three predicted proteins contained the necessary domains to function. However, a comparison of evolutionary distances revealed potentially important variations in the first RNase III domain of OnDcr2, the double-stranded RNA binding domains of OnR2D2, and both the PAZ and PIWI domains of OnAgo2, which may indicate functional differences in enzymatic activity between species. Expression analysis indicated that transcripts for all three genes were expressed in all developmental stages and tissues investigated. Interestingly, the introduction of non-target dsRNA into ECB second-instar larvae via microinjection did not affect OnAgo2, OnDcr2, or OnR2D2 expression. In contrast, ingestion of the same dsRNAs resulted in upregulation of OnDcr2 but downregulation of OnR2D2. The unexpected transcriptional responses of the core machinery and the divergence in amino-acid sequence between specific domains in each core RNAi protein may possibly contribute to low RNAi efficiency in ECB. Understanding the contributions of different RNAi pathway components is critical to adapting this technology for use in controlling lepidopteran pests that exhibit low RNAi efficiency.