Syntaxin5 is essential for survival by ensuring midgut epithelial homeostsis and regulating feeding in Locusta migratoria.

Syntaxin5 (Syx5) belongs to SNAREs family, which play important roles in fusion of vesicles to target membranes. Most of what we know about functions of Syx5 originates from studies in fungal or vertebrate cells, how Syx5 operates during the development of insects is poorly understood. In this study, we investigated the role of LmSyx5 in the gut development of the hemimetabolous insect Locusta migratoria. LmSyx5 was expressed in many tissues, with higher levels in the gut. Knockdown of LmSyx5 by RNA interference (RNAi) considerably suppressed feeding in both nymphs and adults. The dsLmSyx5-injected locusts lost body weight and finally died at a mortality of 100%. Furthermore, hematoxylin-eosin staining indicated that the midgut is deformed in dsLmSyx5-treated nymphs and the brush border in midgut epithelial cells is severely damaged, suggesting that LmSyx5 is involved in morphogenesis of the midgut. TEM further showed that the endoplasmic reticulum of midgut cells have a bloated appearance. Taken together, these results suggest that LmSyx5 is essential for midgut epithelial homeostsis that affects growth and development of L. migratoria. Thus, Syx5 is a promising RNAi target for controlling L. migratoria, and even other pests.

RNAi-mediated silencing of the neverland gene inhibits molting in the migratory locust, Locusta migratoria.

7-dehydrocholesterol (7-DHC) is a key intermediate product used for biosynthesis of molting hormone. This is achieved through a series of hydroxylation reactions catalyzed by the Halloween family of cytochrome P450s. Neverland is an enzyme catalyzes the first reaction of the ecdysteroidogenic pathway, which converts dietary cholesterol into 7-DHC. However, research on the physiological function of neverland in orthopteran insects is lacking. In this study, neverland from Locusta migratoria (LmNvd) was cloned and analyzed. LmNvd was mainly expressed in the prothoracic gland and highly expressed on days 6 and 7 of fifth instar nymphs. RNAi-mediated silencing of LmNvd resulted in serious molting delays and abnormal phenotypes, which could be rescued by 7-DHC and 20-hydroxyecdysone supplementation. Hematoxylin and eosin staining results showed that RNAi-mediated silencing of LmNvd disturbed the molting process by both promoting the synthesis of new cuticle and suppressing the degradation of the old cuticle. Quantitative real-time PCR results suggested that the mRNA expression of E75 early gene and chitinase 5 gene decreased and that of chitin synthase 1 gene was markedly upregulated after knockdown of LmNvd. Our results suggest that LmNvd participates in the biosynthesis process of molting hormone, which is involved in regulating chitin synthesis and degradation in molting cycles.

A neural m6A pathway regulates behavioral aggregation in migratory locusts.

RNA N6-methyladenosine (m6A), as the most abundant modification of messenger RNA, can modulate insect behaviors, but its specific roles in aggregation behaviors remain unexplored. Here, we conducted a comprehensive molecular and physiological characterization of the individual components of the methyltransferase and demethylase in the migratory locust Locusta migratoria. Our results demonstrated that METTL3, METTL14 and ALKBH5 were dominantly expressed in the brain and exhibited remarkable responses to crowding or isolation. The individual knockdown of methyltransferases (i.e., METTL3 and METTL14) promoted locust movement and conspecific attraction, whereas ALKBH5 knockdown induced a behavioral shift toward the solitary phase. Furthermore, global transcriptome profiles revealed that m6A modification could regulate the orchestration of gene expression to fine tune the behavioral aggregation of locusts. In summary, our in vivo characterization of the m6A functions in migratory locusts clearly demonstrated the crucial roles of the m6A pathway in effectively modulating aggregation behaviors.

Alternative relay regulates the adenosine triphosphatase activity of Locusta migratoria striated muscle myosin.

Locust (Locusta migratoria) has a single striated muscle myosin heavy chain (Mhc) gene, which contains 5 clusters of alternative exclusive exons and 1 differently included penultimate exon. The alternative exons of Mhc gene encode 4 distinct regions in the myosin motor domain, that is, the N-terminal SH3-like domain, one lip of the nucleotide-binding pocket, the relay, and the converter. Here, we investigated the role of the alternative regions on the motor function of locust muscle myosin. Using Sf9-baculovirus protein expression system, we expressed and purified 5 isoforms of the locust muscle myosin heavy meromyosin (HMM), including the major isoform in the thorax dorsal longitudinal flight muscle (FL1) and 4 isoforms expressed in the abdominal intersegmental muscle (AB1 to AB4). Among these 5 HMMs, FL1-HMM displayed the highest level of actin-activated adenosine triphosphatase (ATPase) activity (hereafter referred as ATPase activity). To identify the alternative region(s) responsible for the elevated ATPase activity of FL1-HMM, we produced a number of chimeras of FL1-HMM and AB4-HMM. Substitution with the relay of AB4-HMM (encoded by exon-14c) substantially decreased the ATPase activity of FL1-HMM, and conversely, the relay of FL1-HMM (encoded by exon-14a) enhanced the ATPase activity of AB4-HMM. Mutagenesis showed that the exon-14a-encoded residues Gly474 and Asn509 are responsible for the elevated ATPase activity of FL1-HMM. Those results indicate that the alternative relay encoded by exon-14a/c play a key role in regulating the ATPase activity of FL1-HMM and AB4-HMM.

A Core Structural Protein That Builds the Locust Mandible with a Mechanical Gradient.

Natural materials, such as locust mandibles and squid beaks, define significant mechanical gradients that have been attributed to the chemical gradients of their specialized structural proteins (SPs). However, the mechanism by which SPs form chemical gradients in these materials remains unknown. In this study, a highly abundant histidine-rich structural protein (LmMHSP) was identified in the mandible of a migratory locust (Locusta migratoria). LmMHSP was proven by both in vivo and in vitro evidence to act as a core building block of the mandible with a variety of synergistic functions including chitin binding, matrix formation via liquid-liquid phase separation, chemical cross-linking, and metal coordination. Furthermore, we found that the SP gradient in the locust mandible stems from the chitin-binding activity of LmMHSP and different microstructures of chitin scaffolds in different regions. These findings advance our understanding of the formation mechanisms of natural biomaterials and have implications for the fabrication of biomimetic materials.

Transcription factor CncC regulates the expression of antennal CYP6MU1 gene responsible for trans-2-hexen-1-al and nonanal recognition in Locusta migratoria.

Cytochrome P450 monooxygenases (P450s) are a superfamily of multifunctional heme-containing proteins and could function as odorant-degrading enzymes (ODEs) in insect olfactory systems. In our previous study, we identified a P450 gene from the antennal transcriptome of Locusta migratoria, LmCYP6MU1, which could be induced by a variety of volatiles. However, the regulatory mechanisms of this gene in response to volatiles remain unknown. In current study, we investigated the tissues and development stages expression patterns of LmCYP6MU1 and determined its olfactory function in the recognition of the main host plant volatiles which induced LmCYP6MU1 expression. The results showed that LmCYP6MU1 was antenna-rich and highly expressed throughout the antennal developmental stages of locusts. LmCYP6MU1 played important roles in the recognition of trans-2-hexen-1-al and nonanal. Insect CncC regulates the expression of P450 genes. We tested whether LmCncC regulates LmCYP6MU1 expression. It was found that LmCncC knockdown in the antennae resulted in the downregulation of LmCYP6MU1 and repressed the volatiles-mediated induction of LmCYP6MU1. LmCncC knockdown reduced the electroantennogram (EAG) and behavioral responses of locusts to volatiles. These results suggested that LmCncC could regulate the basal and volatiles-mediated inducible expression of LmCYP6MU1 responsible for the recognition of trans-2-hexen-1-al and nonanal. These findings provide an original basis for understanding the regulation mechanisms of LmCncC on LmCYP6MU1 expression and help us better understand the LmCncC-mediated olfactory plasticity.

Transcription factors, cap 'n' collar isoform C regulates the expression of CYP450 genes involving in insecticides susceptibility in Locusta migratoria.

BACKGROUND: The cap 'n' collar (Cnc) belongs to the Basic Leucine Zipper (bZIP) transcription factor super family. Cap 'n' collar isoform C (CncC) is highly conserved in the animal kingdom. CncC contributes to the regulation of growth, development, and aging and takes part in the maintenance of homeostasis and the defense against endogenous and environmental stress. Insect CncC participates in the regulation of various kinds of stress-responsive genes and is involved in the development of insecticide resistance. RESULTS: In this study, one full-length CncC sequence of Locusta migratoria was identified and characterized. Upon RNAi silencing of LmCncC, insecticide bioassays showed that LmCncC played an essential role in deltamethrin and imidacloprid susceptibility. To fully investigate the downstream genes regulated by LmCncC and further identify the LmCncC-regulated genes involved in deltamethrin and imidacloprid susceptibility, a comparative transcriptome was constructed. Thirty-five up-regulated genes and 73 down-regulated genes were screened from dsLmCncC-knockdown individuals. We selected 22 LmCncC-regulated genes and verified their gene expression levels using RT-qPCR. Finally, six LmCYP450 genes belonging to the CYP6 family were selected as candidate detoxification genes, and LmCYP6FD1 and LmCYP6FE1 were further validated as detoxification genes of insecticides via RNAi, insecticide bioassays, and metabolite identification. CONCLUSIONS: Our data suggest that the locust CncC gene is associated with deltamethrin and imidacloprid susceptibility via the regulation of LmCYP6FD1 and LmCYP6FE1, respectively.

The Effect of Abamectin on Locusta Migratoria Neurosecretory Cells and Mid Gut, Using Ultrastructure Examination, Oxidative Stress Study, and In-Silico Molecular Docking.

(1) Background: Few studies have been carried out to appraise abamectin toxicity toward Locusta migratoria nymphs. (2) Methods: This study aimed to evaluate the cytotoxic effect of abamectin as an insecticide through examining the changes and damage caused by this drug, in both neurosecretory cells and midgut, using L. migratoria nymphs as a model of the cytotoxic effect. Histopathological change in the brain was examined in both normal and abamectin-treated fifth-instar nymphs. Neurosecretory cells (NSCs) were also examined where there were loosely disintegrated cells or vacuolated cytoplasm. (3) Results: The results showed distinct histological changes in the gastrointestinal tract of L. migratoria nymphs treated with abamectin, with significant cellular damage and disorganization, i.e., characteristic symptoms of cell necrosis, a destroyed epithelium, enlarged cells, and reduced nuclei. The observed biochemical changes included an elevation in all measured oxidative stress parameters compared to untreated controls. The malondialdehyde activities (MDAs) of the treated nymphs had a five- to six-fold increase, with a ten-fold increase in superoxide dismutase (SOD), nine-fold increase in glutathione-S-transferase (GST), and four-fold increase in nitric oxide (NO). (4) Conclusions: To further investigate the theoretical method of action, a molecular docking simulation was performed, examining the possibility that abamectin is an inhibitor of the fatty acid-binding protein Lm-FABP (2FLJ) and that it binds with two successive electrostatic hydrogen bonds.

Genome-Wide Characterization and Gene Expression Analysis of TRP Channel Superfamily Genes in the Migratory Locust, Locusta migratoria.

The TRP channel superfamily was widely found in multiple species. They were involved in many extrasensory perceptions and were important for adapting to the environment. The migratory locust was one of the worldwide agricultural pests due to huge damage. In this study, we identified 13 TRP superfamily genes in the locust genome. The number of LmTRP superfamily genes was consistent with most insects. The phylogenetic tree showed that LmTRP superfamily genes could be divided into seven subfamilies. The conserved motifs and domains analysis documented that LmTRP superfamily genes contained unique characteristics of the TRP superfamily. The expression profiles in different organs identified LmTRP superfamily genes in the head and antennae, which were involved in sensory function. The expression pattern of different life phases also demonstrated that LmTRP superfamily genes were mainly expressed in third-instar nymphs and male adults. Our findings could contribute to a better understanding of the TRP channel superfamily gene and provide potential targets for insect control.

NADPH-cytochrome P450 reductase knockdown decreases the response to precocene I in the migratory locust Locusta migratoria.

Precocene I is a juvenile hormone antagonist that needs to be activated via oxidative biotransformation catalyzed by cytochrome P450 (CYP). NADPH-cytochrome P450 reductase (CPR) supplies CYP with electrons in the oxidation-reduction process; however, its functional role in the activation of precocene I remains unexplored. Here, the representative characteristics of CPRs were analyzed in the CPR gene of Locusta migratoria (LmCPR), the result of model docking indicated that the hydrogen bonds were formed between reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and NADPH-, FAD-, FMN-domains of LmCPR, respectively. Treating the fourth-instar nymphs with precocene I decreased the juvenile hormone titers of nymphs to 0.55-fold of that in acetone-treated controls, and extended the interval time between fourth- and fifth-instar nymphs. 68.75% of the treated fourth-instar nymphs developed into precocious adults in the fifth-instar. LmCPR knockdown decreased the response to precocene I in the nymphs, the occurrence rate of precocious adults induced by precocene I treatment reduced by 23.11%. Therefore, LmCPR may be involved in the activation of precocene I in L. migratoria. In addition, we generated an active recombinant LmCPR protein using a prokaryotic expression system, its activity in reducing cytochrome c was 33.13 ± 11.50 nmol CytCred/min/µg protein. This study lays the foundation for further research on the role of LmCPR in precocene I activation.

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.

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.

Synthetic Nanoscale RNAi Constructs as Pesticides for the Control of Locust Migratoria.

The low efficiency of RNA interference (RNAi) in insects via the oral administration of double-stranded RNA (dsRNA) is a considerable obstacle preventing its application in insect pest control. The instability of dsRNA and insufficient dsRNA uptake are known to limit the RNAi efficiency. To overcome these limitations, the block copolymer poly(ethylene glycol)-polylysine(thiol) [PEG-PLys(SH)] was designed in this study to form well-defined, core-shell nanoparticles to protect dsRNA from premature degradation and to facilitate its movement through various physiological barriers. The developed material had excellent structural stability and dsRNA-protecting capacity, thereby enabling the prolonged survival of dsRNA in the digestive tract for endocytosis into the midgut cells of the migratory locust, Locusta migratoria. After encapsulation of a dsLmCHS2 payload (a midgut gene), a 60% down-regulation of LmCHS2, accompanied with observations of amorphous and discontinuous linings of the peritrophic matrix and abnormal phenotypes, was observed. In addition, the elaborated nanoscale dsRNA condensates appeared to readily extravasate through the narrow fenestrations in the linings of midgut epithelial cells into the hemolymph and be distributed throughout the body. After encapsulation of a dsLmCHS1 payload (a cuticle gene), a distinctive lethal phenotype with molting failure was observed as a result of a 50% down-regulation in LmCHS1. The persistent leaf adherence of these dsRNA constructs was also capable of resisting continuous rinsing. Therefore, these dsRNA constructs represent a robust type of RNAi pesticide, which has potential as a versatile pesticide against a variety of molecular targets for the control of destructive insects and insects resistant to conventional pesticides.

Electroantennographic activity of 21 aliphatic compounds that bind well to a locust odorant-binding protein.

Odorants that bind well to odorant-binding proteins (OBPs) often trigger olfactory responses and have important biological significance. The locust Locusta migratoria (Meyen) (Orthoptera: Acrididae) is a serious agricultural pest. Twenty-one saturated aliphatic compounds with carbon-oxygen bonds and straight chains of 10-17 carbon atoms bind well to an L. migratoria OBP. In this study, olfactory activities of these aliphatic compounds on L. migratoria adult males were tested by electroantennography (EAG) and comparatively analyzed. Four alcohols (undecanol, dodecanol, tridecanol, and tetradecanol), two ketones (2-dodecanone and 2-tridecanone), and two esters (ethyl octanoate and ethyl nonanoate) triggered strong EAG responses, and there was no significant difference between them. The results suggest that the eight compounds are more likely to have important biological significance than the other compounds. Moreover, we found that there is not necessarily a positive correlation between the olfactory activity of odorants and their binding ability with OBP. The study contributes to understanding the odorants with biological significance for L. migratoria and the molecular mechanism of the locust's olfaction.

Effect of genetically modified maize expressing the Cry1Ab and EPSPS proteins on growth, development, and gut bacterial diversity of the non-target arthropod Locusta migratoria.

The widespread cultivation of genetically modified (GM) crops has raised concerns for their safety. Here, we evaluated the effects of a GM maize variety expressing the Cry1Ab (14.76 ± 0.87 µg/g FW) and EPSPS proteins (191.55 ± 15.69 µg/g FW) on the life-history traits and gut bacterial community of a non-target arthropod, Locusta migratoria, in the laboratory. We found that GM maize had no significant effect on the survival or body weight of different development stages of L. migratoria. The midgut and hindgut bacterial diversities and compositions were determined using high-throughput sequencing targeting the V3-V4 regions of the 16S rRNA. No significant changes were found in the species diversity or abundance between insects in the GM-fed treatment and the non-GM control. Furthermore, the concentration of Cry1Ab and EPSPS in the gut was determined after digestion of GM maize. Results showed that the contents of Cry1Ab/EPSPS rapidly decreased and were hard to detect after 72 h. Based on the parameters assessed, we can conclude that the GM maize variety examined has no significant adverse effect on L. migratoria.

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.

Imidacloprid-induced pathophysiological damage in the midgut of Locusta migratoria (Orthoptera: Acrididae) in the field.

Neonicotinoids are modern insecticides widely used in agriculture worldwide. Their impact on target (nervous system) and non-target (midgut) tissues has been well studied in beneficial insects including honeybees under controlled conditions. However, their detailed effects on pest insects on the field are missing to date. Here, we have studied the effects of the neonicotinoid imidacloprid on the midgut of the pest insect Locusta migratoria caught in the field. We found that in the midgut of imidacloprid-exposed locusts the activity of enzymes involved in reactive oxygen metabolism was perturbed. By contrast, the activity of P450 enzymes that have been shown to be activated in a detoxification response and that were also reported to produce reactive oxygen species was elevated. Probably as a consequence, markers of oxidative stress including protein carbonylation and lipid peroxidation accumulated in midgut samples of these locusts. Histological analyses revealed that their midgut epithelium is disorganized and that the brush border of the epithelial cells is markedly reduced. Indeed, microvilli are significantly shorter, misshapen and possibly non-functional in imidacloprid-treated locusts. We hypothesize that imidacloprid induces oxidative stress in the locust midgut, thereby changing the shape of midgut epithelial cells and probably in turn compromising their physiological function. Presumably, these effects reduce the survival rate of imidacloprid-treated locusts and the damage they cause in the field.

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.

A midgut-specific lytic polysaccharide monooxygenase of Locusta migratoria is indispensable for the deconstruction of the peritrophic matrix.

Lytic polysaccharide monooxygenases (LPMOs) are important enzymes that boost the hydrolysis of recalcitrant polysaccharides, such as chitin. They are found extensively in different insect species and are classified as auxiliary activities family 15 (AA15) LPMOs (LPMO15). Some of them were identified from the insect midgut and proven to act on chitin. However, knowledge about their physiological roles during insect growth and development remains limited. Here, we found that midgut-specific LPMO15s are widely distributed in different insect orders, such as the orthopteran Locusta migratoria and the lepidopteran Bombyx mori. Using L. migratoria as a model insect, the function of midgut-specific LmLPMO15-3 during development was investigated. Double-stranded RNA-mediated downregulation of LmLPMO15-3 expression at the 4th or 5th instar nymph stage severely decreased the survival rate and resulted in lethal phenotypes. Hematoxylin and eosin staining results indicated that the deficient individuals exhibited incompletely digested peritrophic matrix (PM), which suggested that LmLPMO15-3 is essential for the deconstruction of the PM during molting. This study provides direct evidence of the physiological importance of a midgut-specific LPMO15 during insect development. As L. migratoria is one of the most destructive agricultural pests, LmLPMO15-3 is a potential target for pest management.

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.