The G932C mutation of chitin synthase 1 gene (CHS1) mediates buprofezin resistance as confirmed by CRISPR/Cas9-mediated knock-in approach in the brown planthopper, Nilaparvata lugens.

The brown planthopper (Nilaparvata lugens) is a major destructive rice pest in Asia. High levels of insecticide resistance have been frequently reported, and the G932C mutation in the chitin synthase 1 (CHS1) gene has been found to mediate buprofezin resistance. However, there has been no direct evidence to confirm the functional significance of the single G932C substitution mutation leading to buprofezin resistance in N. lugens. Here, we successfully constructed a knock-in homozygous strain (Nl-G932C) of N. lugens using CRISPR/Cas9 coupled with homology-directed repair (HDR). Compared with the background strain susceptible to buprofezin (Nl-SS), the knock-in strain (Nl-G932C) showed a 94.9-fold resistance to buprofezin. Furthermore, resistant strains (Nl-932C) isolated from the field exhibited a 2078.8-fold resistance to buprofezin, indicating that there are other mechanisms contributing to buprofezin resistance in the field. Inheritance analysis showed that the resistance trait is incomplete dominance. In addition, the Nl-G932C strain had a relative fitness of 0.33 with a substantially decreased survival rate, emergence rate, and fecundity. This study provided in vivo functional evidence for the causality of G932C substitution mutation of CHS1 with buprofezin resistance and valuable information for facilitating the development of resistance management strategies in N. lugens. This is the first example of using CRISPR/Cas9 gene-editing technology in a hemipteran insect to directly confirm the role of a candidate target site mutation in insecticide resistance.

Flight capacity and behavior of Ephestia kuehniella (Lepidoptera: Pyralidae) in response to kairomonal and pheromonal stimuli.

Flight behavior is an important component to understand in the context of pest management. However, because of their small size, little is known about the flight capacity of most stored-product insects, and when a flight has been assessed, it usually consists of a propensity for initiating flight. Despite a priori expectations of the importance of flight for moths, there are no data about the flight capacity and little on the flight behavior of the Mediterranean flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). As a result, the objective of the current study was to (i) characterize the baseline flight capacity of E. kuehniella and (ii) determine how flight capacity is affected by the presence of kairomonal, pheromonal, or no stimuli. We found adult E. kuehniella flew a mean of 24-34 km in a 24-h period, and the distance flown per bout increased from 91 to 207 m in the presence of pheromones but decreased to 41 m when food was nearby compared to a negative control. The total number of flight bouts was 1.6-fold higher in the presence of pheromone compared to the negative control, but E. kuehniella flew significantly slower with pheromone and food cues present, suggesting they may be exhibiting an optimal foraging strategy. Our data on flight capacity results in qualitatively and quantitatively different conclusions about flight than those conclusions formed if only flight initiation is considered. Overall, this novel information is useful for understanding the spread within facilities and in the landscape (between facilities), as well as parameterizing ecological modeling.

Disruption of semiochemical-mediated movement by the immature Trogoderma variabile Baillon and Trogoderma inclusum Le Conte (Coleoptera: Dermestidae) after exposure to long-lasting insecticide-incorporated netting.

BACKGROUND: Highly mobile stored product insects may be able to readily orient in response to food cues and pheromones to attack durable commodities at each link of the postharvest supply chain. A 0.4% deltamethrin-incorporated long-lasting insecticide-incorporated netting (LLIN) is a successful novel preventative integrated pest management (IPM) tactic to intercept dispersing insects after harvest. However, it is unknown whether exposure to LLIN may affect olfaction and orientation to important semiochemicals by immature stored product dermestids, therefore the aim of this study was to assess whether exposure to LLIN disrupts the normal olfactory and chemotactic behavior of warehouse beetle, Trogoderma variabile Ballion (Coleoptera: Dermestidae), and the larger cabinet beetle, T. inclusum Le Conte (Coleoptera: Dermestidae), larval movement in the presence of important semiochemicals, including food kairomones (e.g., flour) and pheromones, e.g., (Z)-14-methyl-8-hexadecenal. RESULTS: The distance moved by the larval population of T. variabile was reduced by 64% after 24-h exposure to LLIN compared to control netting but not immediately after exposure, while T. inclusum larvae movement was reduced by 50% after 24-h exposure to LLIN compared to the control netting. Generally, the olfaction and orientation of larval dermestids were affected after exposure to LLIN compared to control netting. There were species-linked differences in effects on olfaction after the insects were exposed to LLIN. CONCLUSION: Our study suggests the use of LLIN may enhance the effectiveness of other concurrent behaviorally-based strategies such as mating disruption when used as part of a comprehensive IPM program in the postharvest environment. © 2023 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.

Short-term spatial dispersion patterns between the larger grain borer and the maize weevil in grain columns.

The invasive larger grain borer (Prostephanus truncatus) and the maize weevil (Sitophilus zeamais) co-occur in many regions of the world. While competition between these 2 species has been studied extensively, there is little information on spatial dispersion patterns in bulk storage of grain. To evaluate potential overlap in realized niche, we evaluated the short-term spatial dispersion behavior of P. truncatus and S. zeamais in monolayers of maize alone or together for 1 day compared to 7 days. We evaluated competition under three different densities, namely 10-20, 75-150, and 150-300 insects/kg for P. truncatus and S. zeamais. The monolayers were equally divided into 24 zones to track location the abundance of insects and damage to maize. We found that both species generally aggregated together and were correlated to the same location as heterospecifics. After 1 day, most of the insects for both species were near the top of the monolayer, but by 7 days, most individuals were at the bottom of the monolayers. In monolayers, when alone, P. truncatus created a clear path of destruction to the bottom of the monolayer, but when S. zeamais was present, damage was lessened and shifted upwards in the grain column. In an olfactometer assay, P. truncatus preferred maize odors, while S. zeamais exhibited no preference among maize, conspecifics, and heterospecifics. In evaluating relative emissions, each of these treatments emitted unique odors but with significant overlap. These data may improve targeting of chemical control tactics by identifying the position of these insects in the grain mass.

Rolling circle transcription: A new system to produce RNA microspheres for improving RNAi efficiency in an agriculturally important lepidopteran pest (Mythimna separate).

We applied a new RNA interference (RNAi) system using rolling circle transcription (RCT) technology to generate RNA microspheres (RMS) for targeting two key chitin synthetic pathway genes [chitin synthase A (CHSA), chitin synthase B (CHSB)] in the larvae of the oriental armyworm (Mythimna separate), a RNAi-unsusceptible agriculturally important lepidopteran pest. Feeding the third-instar larvae with the RMS-CHSA- or RMS-CHSB-treated corn leaf discs suppressed the expression of CHSA by 81.7% or CHSB by 88.1%, respectively, at 72 h. The silencing of CHSA consequently affected the larval development, including the reduced body weight (54.0%) and length (41.3%), as evaluated on the 7th day, and caused significant larval mortalities (51.1%) as evaluated on the 14th day. Similar results were obtained with the larvae fed RMS-CHSB. We also compared RNAi efficiencies among different strategies: 1) two multi-target RMS [i.e., RMS-(CHSA + CHSB), RMS-CHSA + RMS-CHSB], and 2) multi-target RMS and single-target RMS (i.e., either RMS-CHSA or RMS-CHSB) and found no significant differences in RNAi efficiency. By using Cy3-labeled RMS, we confirmed that RMS can be rapidly internalized into Sf9 cells (<6 h). The rapid cellular uptake of RMS accompanied with significant RNAi efficiency through larval feeding suggests that the RCT-based RNAi system can be readily applied to study the gene functions and further developed as bio-pesticides for insect pest management. Additionally, our new RNAi system takes the advantage of the microRNA (miRNA)-mediated RNAi pathway using miRNA duplexes generated in vivo from the RMS by the target insect. The system can be used for RNAi in a wide range of insect species, including lepidopteran insects which often exhibit extremely low RNAi efficiency using other RNAi approaches.

Three alternative splicing variants of Loquacious play different roles in miRNA- and siRNA-mediated RNAi pathways in Locusta migratoria.

RNA interference (RNAi) is a specific post-transcriptional gene-silencing phenomenon, which plays an important role in the regulation of gene expression and the protection from transposable elements in eukaryotic organisms. In Drosophila melanogaster, RNAi can be induced by microRNA (miRNA), endogenous small interfering RNA (siRNA), or exogenous siRNA. However, the biogenesis of miRNA and siRNA in these RNAi pathways is aided by the double-stranded RNA binding proteins (dsRBPs) Loquacious (Loqs)-PB, Loqs-PD or R2D2. In this study, we identified three alternative splicing variants of Loqs, namely Loqs-PA, -PB, and -PC in the orthopteran Locusta migratoria. We performed in vitro and in vivo experiments to study the roles of the three Loqs variants in the miRNA- and siRNA-mediated RNAi pathways. Our results show that Loqs-PB assists the binding of pre-miRNA to Dicer-1 to lead to the cleavage of pre-miRNA to yield matured miRNA in the miRNA-mediated RNAi pathway. In contrast, different Loqs proteins participate in different siRNA-mediated RNAi pathways. In exogenous siRNA-mediated RNAi pathway, binding of Loqs-PA or LmLoqs-PB to exogenous dsRNA facilitates the cleavage of dsRNA by Dicer-2, whereas in endogenous siRNA-mediated RNAi pathway, binding of Loqs-PB or Loqs-PC to endogenous dsRNA facilitates the cleavage of dsRNA by Dicer-2. Our findings provide new insights into the functional importance of different Loqs proteins derived from alternative splicing variants of Loqs in achieving high RNAi efficiency in different RNAi pathways in insects.

Efficacy of deltamethrin and pirimiphos-methyl in proportionally layer-treated maize against the larger grain borer and the maize weevil.

Two insecticides (deltamethrin and pirimiphos-methyl) were evaluated in laboratory assays as grain protectants when applied as a total percentage of a maize mass to control adult Prostephanus truncatus and Sitophilus zeamais. All experiments were conducted at the University of Thessaly in Greece at 30°C and 65% RH under continuous darkness. Either insecticide was applied to 20 g of maize placed in a vial or to the upper one half, one fourth, or one-eighth layer of the maize, then insects were either added to the vials before or after the maize in a completely randomized block design CRBD with n = 9 replicates. Mortality, progeny production, and insect damaged kernels (IDK) were then evaluated for each vial. Insect introduction method (before or after) did not have any impact on any of the variables. Mortality was nearly 100% for all treatments for both insecticides for P. truncatus. Subsequently, progeny production and the number of insect damaged kernels were very low or zero for P. truncatus. Mortality of S. zeamais remained low across layer treatments for deltamethrin. However, S. zeamais was easily controlled by pirimiphos-methyl. The results of this laboratory study show that while deltamethrin and pirimiphos-methyl have some effectiveness as a layer treatment on a column of maize, efficacy will be dependent on the target species, and the depth of the treated layer, as well as the location on which the insects are present.

Characterization of Iflavirus in the Red Flour Beetle, Tribolium castaneum (Coleoptera; Tenebrionidae).

Iflavirus is a group of viruses distributed mainly in arthropod species. We surveyed Tribolium castaneum iflavirus (TcIV) in different laboratory strains and in Sequence Read Archives (SRA) in GenBank. TcIV is highly specific to only T. castaneum and is not found in seven other Tenebrionid species, including the closely related species T. freemani. The same strains from different laboratories and different strains displayed largely different degrees of infections in the examination of 50 different lines by using Taqman-based quantitative PCR. We found that ~63% (27 out of 43 strains) of T. castaneum strains in different laboratories are positive for TcIV PCR with large degrees of variation, in the range of seven orders of magnitude, indicating that the TcIV is highly fluctuating depending on the rearing conditions. The TcIV was prevalent in the nervous system with low levels found in the gonad and gut. The transovarial transmission was supported in the experiment with surface-sterilized eggs. Interestingly, TcIV infection did not show observable pathogenicity. TcIV offers an opportunity to study the interaction between the virus and the immune system of this model beetle species.

CRISPR/Cas9-mediated knockout of NlCYP6CS1 gene reveals its role in detoxification of insecticides in Nilaparvata lugens (Hemiptera: Delphacidae).

BACKGROUND: The brown planthopper (Nilaparvata lugens) is one of the major rice insect pests in Asia. Recently, high levels of insecticide resistance have been frequently reported and cytochrome P450 monooxygenase (P450)-mediated metabolic detoxification is a common resistance mechanism in N. lugens. However, there has been no persuasive genetic method to prove the role of P450s in insecticide resistance in N. lugens. RESULTS: Here, CRISPR/Cas9 system was used to disrupt the P450 gene NlCYP6CS1 to elucidate its role in insecticide resistance in field populations of N. lugens. We successfully constructed a homozygous strain (Nl6CS1-KO) with a 5-bp deletion and 1-bp insertion mutation of NlCYP6CS1. Compared with a background resistant strain (Nl-R), the susceptibility of knockout strain Nl6CS1-KO to imidacloprid, nitenpyram, thiamethoxam, dinotefuran, and pymetrozine was increased by 2.3-, 3.4-, 7.0-, 4.2- and 3.9-fold, respectively, but not significantly changed to triflumezopyrim, chlorpyrifos and buprofezin. Life table analysis demonstrated that the Nl6CS1-KO strain resembled the Nl-R strain in terms of egg and nymph developmental duration and adult lifespan, but differed from the Nl-R strain in the survival rate of eggs and nymphs, reproduction, and body weight. CONCLUSIONS: Our study demonstrates the effect of functional deletion of NlCYP6CS1 on multiple insecticide resistance in N. lugens. For the first time, we applied CRISPR/Cas9 system to reveal the mechanism of insecticide resistance in N. lugens, which may shed light on similar studies in other hemipteran insects. © 2023 Society of Chemical Industry.

Lipophorin receptor is required for the accumulations of cuticular hydrocarbons and ovarian neutral lipids in Locusta migratoria.

Lipophorin is the most abundant lipoprotein particle in insect hemolymph. Lipophorin receptor (LPR) is a glycoprotein that binds to the lipophorin and mediates cellular uptake and metabolism of lipids by endocytosis. However, the roles of LPR in uptake of lipids in the integument and ovary remain unknown in the migratory locust (Locusta migratoria). In present study, we characterized the molecular properties and biological roles of LmLPR in L. migratoria. The LmLPR transcript level was high in the first 2 days of the adults after eclosion, then gradually declined. LmLPR was predominately expressed in fat body, ovary and integument. Using immuno-detection methods, we revealed that LmLPR was mainly localized in the membrane of oenocytes, epidermal cells, fat body cells and follicular cells. RNAi-mediated silencing of LmLPR led to a slight decrease of the cuticle hydrocarbon contents but with little effect on the cuticular permeability. However, the neutral lipid content was significantly decreased in the ovary after RNAi against LmLPR, which led to a retarded ovarian development. Taken together, our results indicated that LmLPR is involved in the uptake and accumulation of lipids in the ovary and plays a crucial role in ovarian development in L. migratoria. Therefore, LmLPR could be a promising RNAi target for insect pest management by disrupting insect ovarian development.

Clathrin-dependent endocytosis plays a critical role in larval and pupal development, and female oocyte production in the red flour beetle (Tribolium castaneum).

BACKGROUND: Clathrin-dependent endocytosis is a vesicular transport process by which cells take macromolecules from the extracellular space to the intracellular space. It plays important roles in various cellular functions, but its biological significance in insect development and reproduction has not been well studied. RESULTS: We characterized and functionally analyzed four major clathrin-dependent endocytic pathway genes (TcChc, TcAP50, TcVhaSFD, TcRab7) in Tribolium castaneum. RNA interference (RNAi) by injecting double-stranded RNA (dsRNA) targeting each gene at three doses (50, 100, or 200 ng per insect) in 20-day-old larvae led to 100% larval mortality. When the expressions of TcChc, TcVhaSFD, and TcRab7 were suppressed by injecting their respective dsRNAs at each dose in 1-day-old pupae, the adults that emerged from the dsRNA-injected pupae were deformed, with the absence of wing development. The deformed adults died within 2 days after eclosion. When the expression of TcAP50 was suppressed by injecting its dsRNA into 1-day-old pupae, although no apparent deformed adults were observed, all the adults died within 35 days after eclosion. In addition, when the expressions of TcChc and TcVhaSFD were suppressed by injecting their respective dsRNAs at a reduced dose (10 ng per insect) in 5-day-old pupae, the ovarian development and oocyte production in the resultant females were completely inhibited. CONCLUSION: Our results indicate that clathrin-dependent endocytosis is essential for insect development and reproduction. The results from this study can help researchers identify potential molecular targets for developing novel strategies for insect pest management. © 2023 Society of Chemical Industry.

Both LmDicer-1 and two LmDicer-2s participate in siRNA-mediated RNAi pathway and contribute to high gene silencing efficiency in Locusta migratoria.

Dicers belong to a class of large RNase III multidomain ribonucleases and are central components of the RNA interference (RNAi) pathways. In insects, Dicer-2 has been known to cleave long double-stranded RNA (dsRNA) in small interfering RNA (siRNA)-mediated-RNAi pathway. However, Dicer-1 is responsible for cleaving precursor microRNAs (pre28 miRNAs) in miRNA-mediated RNAi pathway. In this study, we identified one LmDicer-1 and two LmDicer-2 (LmDicer-2a and LmDicer-2b) genes in Locusta migratoria. The RNAi of RNAi assay showed that knockdown of each of the Dicer genes reduced RNAi efficiency against a target gene (Lmß-Tubulin), suggesting that all these genes participated in the siRNA-mediated RNAi pathway. Sequence analyses of the siRNAs generated from dsLmß-Tubulin after silencing each LmDicer gene showed no significant difference in the pattern of siRNAs mapped to dsLmß-Tubulin. This result indicated that all the three LmDicers are capable of generating siRNAs from the dsRNA. We then generated recombinant proteins consisting of different domains using Escherichia coli expression system and incubated each recombinant protein with dsLmß-Tubulin. We found that the recombinant Dicer proteins successfully cleaved dsLmß-Tubulin. However, LmDicer-2a-R lacking dsRBD domain lost activity, suggesting that dsRBD domain is critical for Dicer function. Furthermore, overexpression of these proteins in Drosophila S2 cells improved RNAi efficiency. Our siRNA affinity chromatography and LC-MS/MS analysis identified LmDicer-2a, LmDicer-2b, LmR2D2, LmAgo2a, LmAgo1, LmStaufen and LmTARBP2 as constituents of RNA-induced silencing complex. Taken together, these data show that both LmDicer-1 and two LmDicer-2s all participate in siRNA-mediated RNAi pathway and likely contribute to high RNAi efficiency in L. migratoria.

Synergism of deltamethrin with a mixture of short chain fatty acids for toxicity against pyrethroid-resistant and susceptible strains of Tribolium castaneum (Coleoptera: Tenebrionidae).

Deltamethrin is one of the most effective pyrethroid compounds used in stored product protection to control a wide range of pests. However, the development of resistance to deltamethrin in many pest species has been reported and useful research to overcome this problem is required. The present study investigated the possible synergistic effect of a commercial formulation of a mixture of the short chain fatty acids, octanoic, nonanoic and decanoic acid, in a formulation called "C8910" on the lethal activity of deltamethrin against susceptible (Lab-S) and relatively pyrethroid-resistant (Pyr-R) strains of T. castaneum. The possible mechanisms of synergism were studied by investigating the inhibitory effect of C8910 on the activity of detoxification enzymes including cytochrome P450s, esterases, and glutathione S-transferases (GST). In addition, the possible role of C8910 in enhancement of cuticular penetration of deltamethrin through insect cuticle was studied using GC analysis. The results showed that C8910 enhanced the toxicity of deltamethrin at mixing ratios of 1:5 and 1:10 against the Lab-S strain after 24 and 48 h of exposure, and synergistic factors (SF) ranged between 5.69 and 13.59. C8910 also showed greater synergism on the deltamethrin toxicity against the resistant strain than the susceptible one after 24 and 48 h of treatment at 1:5 and 1:10 ratios with SF values ranging from 22.82 and 47.16. C8910 showed strong inhibition of cytochrome P450 of rat microsomal fraction with IC50 value of 6.24 mM. Meanwhile, C8910 inhibited the activity of general esterases in Lab-S and Pyr-R strains with IC50 values of 26.22 and 51.73 mM, respectively. However, weak inhibition of GST activity was observed with inhibition of 52.0 and 22.6% at concentration of 100 mM of C8910 for Lab-S and Pyr-R, respectively. In addition, the results showed no significant difference between the unpenetrated amounts of deltamethrin when insects were treated with deltamethrin alone or with deltamethrin+C8910 (1:20) through the insect cuticle. Results suggested that the synergism between C8910 and deltamethrin could be related to the ability of C8910 to inhibit the detoxification enzymes such as cytochrome P450 and esterases. Therefore, C8910 could be a promising synergist to enhance deltamethrin toxicity and to be a possible natural alternative for conventional synergists such as piperonyl butoxide.

Identification and RNAi-based functional analysis of chitinase family genes in Agrotis ipsilon.

BACKGROUND: Chitin is a major component in the extracellular matrix of insects, and its metabolism largely affects insect development and molting. As essential degradative enzymes, chitinases are encoded by multiple genes that differ in size, expression pattern and function in insects. However, our limited knowledge on the functions of different chitinases in Agrotis ipsilon has prevented our application of new technologies to target these genes as new pest management strategies. RESULTS: We revealed 11 full-length complementary DNA sequences of chitinase genes (AiChts) from A. ipsilon transcriptome. Although the domain architecture of these chitinases varied greatly, they all contained at least one chitinase catalytic domain. Developmental stage- and tissue-dependent expression profiles showed that most AiChts had the highest expression in the pupal stage. Furthermore, AiCht2, AiCht6, AiCht7 and AiCht10 were mainly expressed in the integument, whereas AiCht8 and AiCht-h had the highest expression in the midgut. The RNA interference (RNAi) experiment revealed that knockdown of AiCht10 or the imaginal disc growth factor gene (AiIDGF) induced high larval mortality. Larvae failed to shed the old cuticle during molting after the injection of double-stranded RNA targeting AiCht10 (dsAiCht10), whereas the larval bodies shrunk and blackened after the injection of dsRNA targeting AiIDGF (dsAiIDGF). CONCLUSION: Our results revealed for the first time the important functions of AiCht10 and AiIDGF in A. ipsilon. These genes are essential for larval development, and can potentially serve as new targets for RNAi-based pest management. © 2022 Society of Chemical Industry.

Mobility of Phosphine-Susceptible and -Resistant Rhyzopertha dominica (Coleoptera: Bostrichidae) and Tribolium castaneum (Coleoptera: Tenebrionidae) After Exposure to Controlled Release Materials With Existing and Novel Active Ingredients.

There is interest in developing controlled release materials (CRMs) with novel modes of action to improve resistance management. Long-lasting insecticide-incorporated netting (LLIN) with deltamethrin has been effectively used against stored-product pests. Here, we evaluated the efficacy of different CRMs (LLIN or packaging) with each of four active ingredients (AI) (deltamethrin, permethrin, indoxacarb, and dinotefuran) and compared them to control CRMs in reducing movement and increasing mortality of phosphine-susceptible and -resistant Rhyzopertha dominica and Tribolium castaneum. Adults were exposed for 0.5, 2, or 60 min, and movement was assessed immediately or after 24, or 168 h using video-tracking and Ethovision software. We recorded total distance and velocity traveled by adults. Finally, we tested higher rates of each AI on surrogate netting material (e.g., standardized-sized cheesecloth) and varied exposure time to obtain median lethal time (LT50) for each compound and susceptibility. Exposure to LLIN with deltamethrin significantly reduced the movement of both species compared to the other CRMs regardless of their susceptibility to phosphine. Deltamethrin was the most effective AI for both species, while dinotefuran and indoxacarb were the least effective for R. dominica and T. castaneum adults, respectively. Most AIs resulted in appreciable and approximately equivalent mortality at higher concentrations among phosphine-susceptible and -resistant strains. Our results demonstrate that CRMs can be an additional approach to combat phosphine-resistant populations of stored product insects around food facilities. Other compounds such as permethrin, dinotefuran, and indoxacarb are also effective against phosphine-resistant populations of these key stored product insects except indoxacarb for T. castaneum.

Evaluations of two glutathione S-transferase epsilon genes for their contributions to metabolism of three selected insecticides in Locusta migratoria.

The insect-specific epsilon class of glutathione S-transferases (GSTEs) plays important roles in insecticide detoxification in insects. In our previous work, five GSTEs were identified in Locusta migratoria, and two recombinant GSTEs, rLmGSTE1 and rLmGSTE4, showed high catalytic activity when 1-chloro-2,4-dinitrobenzene (CDNB) was used as a substrate. In this work, we further investigated whether these two GSTEs could metabolize three insecticides including malathion, deltamethrin and DDT. Using ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC/MS) method, we found that rLmGSTE4, but not rLmGSTE1, can metabolize malathion and DDT. Malathion bioassays of L.migratoria after the expression of LmGSTE4 was suppressed by RNA interference (RNAi) showed increased insect mortality from 33.8% to 68.9%. However, no changes in mortality were observed in deltamethrin- or DDT-treated L.migratoria after the expression of LmGSTE4 was suppressed by RNAi. Our results provided direct evidences that LmGSTE4 participates in malathion detoxification in L.migratoria. These findings are important for understanding the mechanisms of insecticide resistance in L.migratoria and developing new strategies for managing the insect populations in the field.

Increased RNAi Efficiency by dsEGFP-Induced Up-Regulation of Two Core RNAi Pathway Genes (OfDicer2 and OfAgo2) in the Asian Corn Borer (Ostrinia furnacalis).

RNA interference (RNAi) is a sequence-specific gene silencing mechanism that holds great promise for effective management of agricultural pests. Previous studies have shown that the efficacy of RNAi varies among different insect species, which limits its wide spread application in the field of crop protection. In this study, we identified and characterized six core RNAi pathway genes including OfDicer1, OfDicer2, OfR2D2, OfAgo1, OfAgo2, and OfAgo3 from the transcriptomic database of the Asian corn borer (Ostrinia furnacalis). Domain analysis showed that the six deduced proteins contained the necessary functional domains. Insect developmental stage- and tissue-specific expression analysis showed that five genes were expressed in all the stages and tissues examined except OfAgo3, which showed low expression in larvae, and high expression in pupae and adults and in the midgut. RT-qPCR was performed to examine the response of these six genes to exogenous double-stranded RNA (dsRNA). Interestingly, the transcript levels of OfDicer2 and OfAgo2 were significantly enhanced after the injection of dsEGFP at different time points and tissues investigated. Consequently, the RNAi efficiency in targeting the insect endogenous genes can be greatly enhanced in the hemolymph or midgut. Taken together, our investigations suggest that RNAi efficiency can be enhanced by pre-injection of dsRNA to induce the RNAi core machinery genes, which could be a useful strategy to improving RNAi efficiency for studying gene functions under laboratory conditions.

Clathrin heavy chain is essential for the development and reproduction of Locusta migratoria.

Clathrin heavy chain (Chc) is a constituent of clathrin-coated vesicles and serves important functions in endocytosis and intracellular membrane trafficking but appears to have physiological roles also at the organismal level. Most of what we know about Chc functions originates from studies performed in fungal or vertebrate cells. However, the physiological functions of Chc in insects remain poorly understood. Here, we identified a Chc ortholog from a Locusta migratoria transcriptome database. RT-qPCR revealed that LmChc was constitutively expressed in fifth-instar nymphs. In this developmental stage, LmChc showed the highest expression in the ovary followed by hemolymph, testis, hindgut, midgut, and foregut. In isolated hemocytes, we detected the Chc protein in patches at the plasma membrane. To examine the role of LmChc in L. migratoria during development, RNA interference was performed by injecting dsRNA into the early fifth-instar nymphs. Silencing of LmChc caused a lethal phenotype with molting defect from nymph to adult. In addition, silencing of LmChc resulted in abnormal development of the ovaries, the size of which was significantly smaller than that in controls. Taken together, our results suggest that LmChc is a vital gene in L. migratoria that plays an important role in growth, development, and reproduction. LmChc may be used as an efficient RNAi target gene for developing dsRNA-based biological insecticides to manage insect pests.

The stability and sequence cleavage preference of dsRNA are key factors differentiating RNAi efficiency between migratory locust and Asian corn borer.

We compared the stability of double-stranded RNA (dsRNA) in each of two body fluids (hemolymph, midgut fluid) and in each of two tissues (integument, midgut), and the uptake of dsRNA in each of two cultured tissues (integument, midgut) between the migratory locust (Locusta migratoria) and the Asian corn borer (Ostrinia furnacalis). We further compared the abundance of putative small interfering RNAs (siRNAs) generated from each of two dsRNAs (dsß-actin, dsEf1α) and the preference of dsRNA cleavages between the two insect species. Our studies showed a rapid degradation of dsRNA in the midgut fluids of both insect species and in O. furnacalis hemolymph. However, dsRNA remained reasonably stable in L. migratoria hemolymph. When nuclease degradation of dsRNA in cultured tissues was inhibited, dsRNA uptake was not significantly different between the two species. We further showed that the silencing efficiency against target genes was consistent with the abundance of putative siRNAs processed from the dsRNA. In addition, O. furnacalis showed a strong preference in cleaving dsRNA when the nucleotide G was in the position of "1" at 5'-end whereas L. migratoria showed broad spectrum in cleavage sites to generate siRNA. Taken together, our study revealed that silencing efficiency of a target gene by RNAi was directly related to the dsRNA degradation by nucleases and the abundance of siRNAs generated from the dsRNA.

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.