Tyrosine hydroxylase plays crucial roles in larval cuticle formation and larval-pupal tanning in the rice stem borer, Chilo suppressalis.

The striped stem borer, Chilo suppressalis (Walker), a notorious pest infesting rice, has evolved a high level of resistance to many commonly used insecticides. In this study, we investigate whether tyrosine hydroxylase (TH), which is required for larval development and cuticle tanning in many insects, could be a potential target for the control of C. suppressalis. We identified and characterized the full-length cDNA (CsTH) of C. suppressalis. The complete open reading frame of CsTH (MW690914) was 1683 bp in length, encoding a protein of 560 amino acids. Within the first to the sixth larval instars, CsTH was high in the first day just after molting, and lower in the ensuing days. From the wandering stage to the adult stage, levels of CSTH began to rise and reached a peak at the pupal stage. These patterns suggested a role for the gene in larval development and larval-pupal cuticle tanning. When we injected dsCsTH or 3-iodotyrosine (3-IT) as a TH inhibitor or fed a larva diet supplemented with 3-IT, there were significant impairments in larval development and larval-pupal cuticle tanning. Adult emergence was severely impaired, and most adults died. These results suggest that CsTH might play a critical role in larval development as well as larval-pupal tanning and immunity in C. suppressalis, and this gene could form a potential novel target for pest control.

Stemborer-induced rice plant volatiles boost direct and indirect resistance in neighboring plants.

Herbivore-induced plant volatiles (HIPVs) are known to be perceived by neighboring plants, resulting in induction or priming of chemical defenses. There is little information on the defense responses that are triggered by these plant-plant interactions, and the phenomenon has rarely been studied in rice. Using chemical and molecular analyses in combination with insect behavioral and performance experiments, we studied how volatiles emitted by rice plants infested by the striped stemborer (SSB) Chilo suppressalis affect defenses against this pest in conspecific plants. Compared with rice plants exposed to the volatiles from uninfested plants, plants exposed to SSB-induced volatiles showed enhanced direct and indirect resistance to SSB. When subjected to caterpillar damage, the HIPV-exposed plants showed increased expression of jasmonic acid (JA) signaling genes, resulting in JA accumulation and higher levels of defensive proteinase inhibitors. Moreover, plants exposed to SSB-induced volatiles emitted larger amounts of inducible volatiles and were more attractive to the parasitoid Cotesia chilonis. By unraveling the factors involved in HIPV-mediated defense priming in rice, we reveal a key defensive role for proteinase inhibitors. These findings pave the way for novel rice management strategies to enhance the plant's resistance to one of its most devastating pests.

Chilo suppressalis heat shock proteins are regulated by heat shock factor 1 during heat stress.

Heat shock factor 1 (HSF1) functions to maintain cellular and organismal homeostasis by regulating the expression of target genes, including those encoding heat shock proteins (HSPs). In the present study, the gene encoding HSF1 was cloned from the rice pest Chilo suppressalis, and designated Cshsf1. The deduced protein product, CsHSF1, contained conserved domains typical of the HSF1 family, including a DNA-binding domain, two hydrophobic heptad repeat domains, and a C-terminal transactivation domain. Real-time quantitative PCR showed that Cshsf1 was highly expressed in hemocytes. Expression analysis in different developmental stages of C. suppressalis revealed that Cshsf1 was most highly expressed in male adults. RNAi-mediated silencing of Cshsf1 expression reduced C. suppressalis survival at high temperatures. To investigate the regulatory interactions between Cshsf1 and Cshsps, the promoters and expression patterns of 18 identified Cshsps in C. suppressalis were analysed; four types of heat shock elements (HSEs) were identified in promoter regions including canonical, tail-tail, head-head, and step/gap. The expression of Cshsp19.0, Cshsp21.7B, Cshsp60, Cshsp70 and Cshsp90 was positively regulated by Cshsf1; however, Cshsp22.8, Cshsp702, Cshsp705 and Cshsp706 gene expression was not altered. This study provides a foundation for future studies of HSF1 in insects during thermal stress.

Knockdown of Dcr1 and Dcr2 limits the lethal effect of C-factor in Chilo suppressalis.

Dicer is a highly conserved ribonuclease in evolution. It belongs to the RNase III family and can specifically recognize and cleave double-stranded RNA (dsRNA). In this study, the genome and transcriptome of Chilo suppressalis were analyzed, and it was found that there were two members in the Dicer family, named Dcr1 and Dcr2. The dsRNAs of Dcr1 and Dcr2 genes were synthesized and fed to C. suppressalis larvae. The C-factor of C. suppressalis was selected as the marker gene. The results showed that both Dcr1 and Dcr2 genes were significantly knocked down. The larval mortality was significantly reduced by 43.50% (p < 0.05) after feeding on dsC-factor and dsDcr1. The transcription levels of C-factor genes were significantly increased by 33.95% (p < 0.05) and 32.94% (p < 0.05) when the larvae fed with dsDcr2 + dsC-factor for 72 h and 96 h, respectively. Furthermore, the mortality was significantly decreased by 79% (p < 0.05) after feeding dsC-factor and dsDcr2. These findings imply that Dcr1 can decrease the lethal effect of C-factor gene but cannot affect its RNAi efficiency and Dcr2 can decrease the lethal effect of C-factor gene by inhibiting RNAi efficiency.

Identification and characterization of Prx5 and Prx6 in Chilo suppressalis in response to environmental stress.

The antioxidant proteins, peroxiredoxins (Prxs), function to protect insects from reactive oxygen species-induced toxicity. In this study, two Prx genes, CsPrx5, and CsPrx6, were cloned and characterized from the paddy field pest, Chilo suppressalis, containing open reading frames of 570 and 672 bp encoding 189 and 223 amino acid polypeptides, respectively. Then, we investigated the influence of various stresses on their expression levels using quantitative real-time PCR (qRT-PCR). The results showed expression of CsPrx5 and CsPrx6 in all developmental stages, with eggs having the highest level. CsPrx5 and CsPrx6 showed higher expression in the epidermis and fat body, and CsPrx6 also showed higher expression in midgut, fat body, and epidermis. Increasing concentrations of insecticides (chlorantraniliprole and spinetoram) and hydrogen peroxide (H2 O2 ) increased the expression levels of CsPrx5 and CsPrx6. In addition, the expression levels of CsPrx5 and CsPrx6 were almost markedly upregulated in larvae under temperature stress or fed by vetiver. Thus, CsPrx5 and CsPrx6 upregulation might increase the C. suppressalis defense response by reducing the impact of environmental stress, providing a better understanding of the relationship between environmental stresses and insect defense systems.

Cry9A and Vip3A protein-induced transcriptional changes correspond to their synergistic damage to the midgut of Chilo suppressalis.

Cry and Vip3 proteins are both pore-forming toxins produced by Bacillus thuringiensis that show synergistic insecticidal activity against different insect pests. However, the synergistic effect of Cry and Vip3 proteins on the midgut in target insects is still unclear. In this study, faster and more serious damage was observed after treatment with both Cry9A and Vip3A proteins in the Chilo suppressalis midgut compared to single-protein treatment. Through RNA sequencing, midgut transcriptomic comparison was performed between dual- and single-protein treatments according to midgut injury. After 6 h, 609 differentially expressed genes were found with the combined Cry9A and Vip3A treatments, which was much more than that in the single treatment, corresponding to faster and more serious damage. These genes were mainly enriched in similar pathways, such as lipid metabolic, oxidation-reduction and carbohydrate metabolic process, peptide secretion and cell-cell adhesion; however, the number and expression level of differentially expressed genes are increased. For specific genes significantly regulated by induction of Cry9A and Vip3A, lipases, phospholipid scramblase, probable tape measure protein and arylsulfatase J were significantly downregulated after 6 h treatment. In addition, regular genes related to the activation and receptor binding of B. thuringiensis toxins were differentially regulated, such as ATP-binding cassette subfamily G member 1 and serine protease. Validation with RT-qPCR showed agreement with the sequencing results. Overall, our results support that stronger and faster midgut responses at the cellular and transcriptional levels are induced by the synergistic toxicity of Cry9A and Vip3A in C. suppressalis.

Transcriptomic Analysis Reveals the Detoxification Mechanism of Chilo suppressalis in Response to the Novel Pesticide Cyproflanilide.

Chilo suppressalis is one of the most damaging rice pests in China's rice-growing regions. Chemical pesticides are the primary method for pest control; the excessive use of insecticides has resulted in pesticide resistance. C. suppressalis is highly susceptible to cyproflanilide, a novel pesticide with high efficacy. However, the acute toxicity and detoxification mechanisms remain unclear. We carried out a bioassay experiment with C. suppressalis larvae and found that the LD10, LD30 and LD50 of cyproflanilide for 3rd instar larvae was 1.7 ng/per larvae, 6.62 ng/per larvae and 16.92 ng/per larvae, respectively. Moreover, our field trial results showed that cyproflanilide had a 91.24% control efficiency against C. suppressalis. We investigated the effect of cyproflanilide (LD30) treatment on the transcriptome profiles of C. suppressalis larvae and found that 483 genes were up-regulated and 305 genes were down-regulated in response to cyproflanilide exposure, with significantly higher CYP4G90 and CYP4AU10 expression in the treatment group. The RNA interference knockdown of CYP4G90 and CYP4AU10 increased mortality by 20% and 18%, respectively, compared to the control. Our results indicate that cyproflanilide has effective insecticidal toxicological activity, and that the CYP4G90 and CYP4AU10 genes are involved in detoxification metabolism. These findings provide an insight into the toxicological basis of cyproflanilide and the means to develop efficient resistance management tools for C. suppressalis.

Defense Strategies of Rice in Response to the Attack of the Herbivorous Insect, Chilo suppressalis.

Chilo suppressalis is a notorious pest that attacks rice, feeding throughout the entire growth period of rice and posing a serious threat to rice production worldwide. Due to the boring behavior and overlapping generations of C. suppressalis, the pest is difficult to control. Moreover, no rice variety with high resistance to the striped stem borer (SSB) has been found in the available rice germplasm, which also poses a challenge to controlling the SSB. At present, chemical control is widely used in agricultural production to manage the problem, but its effect is limited and it also pollutes the environment. Therefore, developing genetic resistance is the only way to avoid the use of chemical insecticides. This article primarily focuses on the research status of the induced defense of rice against the SSB from the perspective of immunity, in which plant hormones (such as jasmonic acid and ethylene) and mitogen-activated protein kinases (MAPKs) play an important role in the immune response of rice to the SSB. The article also reviews progress in using transgenic technology to study the relationship between rice and the SSB as well as exploring the resistance genes. Lastly, the article discusses prospects for future research on rice's resistance to the SSB.

FAR knockout significantly inhibits Chilo suppressalis survival and transgene expression of double-stranded FAR in rice exhibits strong pest resistance.

Chilo suppressalis is one of the most prevalent and damaging rice pests, causing significant economic losses each year. Chemical control is currently the primary method of controlling C. suppressalis. However, the indiscriminate use of chemical insecticides increases pest resistance, pollutes the environment and poses a significant health threat to humans and livestock, highlighting the need to find safer, more pest-specific and more effective alternatives to pest control. Plant-mediated RNA interference (RNAi) is a promising agricultural pest control method that is highly pest-specific and has less of an impact on the environment. Using multi-sgRNAs/Cas9 technology to delete Fatty acyl-CoA reductase (FAR) of C. suppressalis in the G0 generation, we show that downregulating FAR transcription may significantly increase the mortality rate and darken the epidermis of C. suppressalis compared with the control. Subsequently, we developed dsFAR transgenic rice lines using Agrobacterium-mediated genetic transformation and then screened three strains expressing dsFAR at high levels using transcriptional level analysis. Using transgenic rice stems, a laboratory feeding bioassay indicated that at least one line (L#10) displayed a particularly high level of insect resistance, with an insect mortality rate of more than 80%. In the field trials, dsFAR transgenic rice displayed high levels of resistance to C. suppressalis damage. Collectively, these results suggest the potential of a new environment-friendly, species-specific strategy for rice pest management.

RNA interference knockdown of insulin receptor inhibits ovarian development in Chilo suppressalis.

BACKGROUND: The nutritional signaling pathway regulates an insect's size, development, and lifespan, as well as playing a vital role in reproduction. The insulin/insulin-like growth factor signaling (IIS) pathway plays a key role in the nutrition signaling pathway. As an integral component of the IIS pathway, insulin receptor (InR), a receptor tyrosine kinase, plays a role in the insulin pathway by controlling reproduction in many insect species. However, the precise molecular function of InR in non-model insect reproduction is poorly understood. METHODS: In our study, Chilo suppressalis, a well-known rice pest, was used as a molecular system to determine the role of InR in insect reproduction. Sequencing the InR gene of C. suppressalis, comparing the amino acid sequence-specific structure, and constructing a phylogenetic tree revealed that this gene has four main domains: ligand binding L domain, Furin-like region, fibronectin type III domains, and Tyrosine kinase catalytic domain, which were all highly conserved in insects. RESULTS: By characterizing the spatiotemporal expression profile of InR in different developmental stages and tissues, we found that InR gene expression was highest on the 3-day old in female pupae, 6th instar larvae, and fat body on the 6-day old in female pupae. InR gene expression may promote the molting and pupation of larvae and play a role in reproduction in the fat body. Furthermore, the RNA interference knockdown of InR dramatically reduced yolk deposition and blocked oocyte maturation. After suppression of InR, the expression of several other genes fluctuated to varying degrees. CONCLUSION: In conclusion, InR is vital to reproduction and is expected to become a new target for pest management.

Identification and characterization of glutathione S-transferases and their potential roles in detoxification of abamectin in the rice stem borer, Chilo suppressalis.

The glutathione S-transferases (GSTs) are a kind of metabolic enzymes and participate in the detoxification metabolism of xenobiotics in various organisms. In insects, GSTs play important roles in the development of insecticide resistance and antioxidant protection. The rice stem borer Chilo suppressalis is one of the most damaging pests in rice and has developed high levels of resistance to abamectin in many areas of China, whereas the potential resistance mechanisms of C suppressalis to abamectin are still unclear. In the present study, a total of 23 CsGSTs genes were identified from the C. suppressalis transcriptome and genome, including 21 cytosolic and two microsomal CsGSTs. The cytosolic CsGSTs were further classified into seven categories based on phylogenetic analysis, and their sequence characteristics and genome structures were also analyzed. Synergism study revealed that the susceptibility of C. suppressalis to abamectin was increased significantly when the CsGSTs were inhibited by diethyl maleate (DEM). Sixteen CsGSTs genes were up-regulated in C. suppressalis larvae after treatment with abamectin, among which four CsGSTs genes including CsGSTe2, CsGSTe4, CsGSTo4 and CsGSTu1 were significantly induced in the midgut and fat body tissues. These results indicated that CsGSTs were associated with the detoxification of C. suppressalis to abamectin, and CsGSTe2, CsGSTe4, CsGSTo4 and CsGSTu1 might play important roles in the insecticide detoxification or antioxidant protection in C. suppressalis. Our present study provides valuable information on C. suppressalis GSTs, and are helpful in understanding the contributions of GSTs in abamectin detoxification in C. suppressalis and other insects.

Downregulation of the CsABCC2 gene is associated with Cry1C resistance in the striped stem borer Chilo suppressalis.

Chilo suppressalis is a major target pest of transgenic rice expressing the Bacillus thuringiensis (Bt) Cry1C toxin in China. The evolution of resistance of this pest is a major threat to Bt rice. Since Bt functions by binding to receptors in the midgut (MG) of target insects, identification of Bt functional receptors in C. suppressalis is crucial for evaluating potential resistance mechanisms and developing effective management strategies. ATP-binding cassette (ABC) transporters have been vastly reported to interact with Cry1A toxins, as receptors and their mutations cause insect Bt resistance. However, the role of ABC transporters in Cry1C resistance to C. suppressalis remains unknown. Here, we measured CsABCC2 expression in C. suppressalis Cry1C-resistant (Cry1C-R) and Cry1C-susceptible strains (selected in the laboratory) via quantitative real-time PCR (qRT-PCR); the transcript level of CsABCC2 in the Cry1C-R strain was significantly lower than that in the Cry1C-susceptible strain. Furthermore, silencing CsABCC2 in C. suppressalis via RNA interference (RNAi) significantly decreased Cry1C susceptibility. Overall, CsABCC2 participates in Cry1C mode of action, and reduced expression of CsABCC2 is functionally associated with Cry1C resistance in C. suppressalis.

Differences in susceptibility to chlorantraniliprole between Chilo suppressalis (Lepidoptera: Crambidae) and two dominant parasitic wasps collected from Sichuan Province, China.

Chilo suppressalis Walker (Lepidoptera: Crambidae) is one of the most destructive pests occurring in the rice-growing regions of Asia. Parasitoids, mainly egg parasitoids, have been of interest for several years even with practical used cases. Therefore, the potential impact of insecticides on natural enemies needs great attention. In this study, chlorantraniliprole was evaluated for its impact on C. suppressalis and two dominant parasitic wasps. Bioassays showed that chlorantraniliprole had negligible toxicity to Eriborus terebrans but was significantly toxic to Chelonus munakatae; the mortality exceeded 50% when the concentration reached 46.83 ng/cm2. Enzyme assays suggested that the significantly different carboxylesterase activity may be involved in the high-level detoxification metabolism of E. terebrans. According to the results of enzyme gene correlation analysis, P450s may be the dominant factor in the detoxification metabolism of C. munakatae. In addition, the ryanodine receptor C-terminus of C. suppressalis (CsRyR), C. munakatae (CmRyR) and E. terebrans (EtRyR) were successfully cloned. Different amino acids at resistance mutation I4758 M between susceptible C. suppressalis (I) and parasitic wasps (M) may be related to susceptibility differences. Simulated docking showed that CsRyR and CmRyR can interact with chlorantraniliprole but not EtRyR. More interaction forces were formed between CsRyR and chlorantraniliprole than CmRyR. Furthermore, a Pi-Pi T-shape formed between 73PHE in CsRyR and the benzene ring in chlorantraniliprole. These results indicated that both detoxification metabolism and the target site could mediate the susceptibility difference between C. suppressalis and its parasitic wasps.

Time-Series Transcriptomic Analysis Reveals the Molecular Profiles of Diapause Termination Induced by Long Photoperiods and High Temperature in Chilo suppressalis.

Survival and adaptation to seasonal changes are challenging for insects. Many temperate insects such as the rice stem borer (Chilo suppressalis) overcome the adverse situation by entering diapause, wherein development changes dynamically occur and metabolic activity is suppressed. The photoperiod and temperature act as major environmental stimuli of diapause. However, the physiological and molecular mechanisms that interpret the ecologically relevant environmental cues in ontogenetic development during diapause termination are poorly understood. Here, we used genome-wide high-throughput RNA-sequencing to examine the patterns of gene expression during diapause termination in C. suppressalis. Major shifts in biological processes and pathways including metabolism, environmental information transmission, and endocrine signalling were observed across diapause termination based on over-representation analysis, short time-series expression miner, and gene set enrichment analysis. Many new pathways were identified in diapause termination including circadian rhythm, MAPK signalling, Wnt signalling, and Ras signalling, together with previously reported pathways including ecdysteroid, juvenile hormone, and insulin/insulin-like signalling. Our results show that convergent biological processes and molecular pathways of diapause termination were shared across different insect species and provided a comprehensive roadmap to better understand diapause termination in C. suppressalis.

Mechanisms for multiple resistances in field populations of rice stem borer, Chilo suppressalis (Lepidoptera: Crambidae) from Sichuan Province, China.

Chilo suppressalis Walker (Lepidoptera: Crambidae) is a widely destructive pest occurring in rice, particularly in the rice-growing regions of Asia. In recent years, C. suppressalis has developed resistance to several insecticides because of the extensive use of insecticides. The resistance levels to four insecticides were determined among populations from different regions of Sichuan Province, China, using a drop-method bioassay. Based on LC50 values of a laboratory susceptible strain, all field populations showed moderate level of resistance to triazophos (23.9- to 83.5-fold) and were either susceptible or had a low level of resistance to abamectin (2.1- to 5.8-fold). All field-collected populations had a low or moderate level of resistance to chlorpyrifos (1.7- to 47.1-fold) and monosultap (2.7- to 13.5-fold). The synergism experiment indicated that the resistance of the XW19 to triazophos may be associated with cytochrome P450 monooxygenases (P450s), with the highest synergistic ratio (SR) of 3.05-fold and increased ratio (IR) of 2.28-fold for piperonylbutoxide (PBO). The P450 activity of the TJ19 population was the greatest among the six field populations. Moreover, the relative expression levels of four resistance-related P450 genes were detected with qRT-PCR, and the results indicated that CYP324A12, CYP321F3 and CYP9A68 were overexpressed in the resistant population, especially in the XW19 population (by 1.2-, 3.4 -, and 18.0-fold, respectively). In addition, the relative expression levels of CYP9A68 among the CZ19 and TJ19 populations were also enhanced 10.5- and 24.9-fold, respectively. These results suggested that CYP324A12, CYP321F3 and CYP9A68 may be related to the resistance development of C. suppressalis to triazophos.

Cloning and RNAi-mediated three lethal genes that can be potentially used for Chilo suppressalis (Lepidoptera: Crambidae) management.

RNA interference (RNAi) has gained attention in recent years as a viable pest control strategy. Here, RNAi assays were performed to screen the potential functionality of genes in Chilo suppressalis, a serious pest of rice, and to determine their potential for developing a highly targeted molecular control approach. Potential homologs of NADH dehydrogenase (ND), glycerol 3-phosphate dehydrogenase (GPDH) and male specific lethal 3 (MSL3) were cloned from C. suppressalis, and their spatiotemporal gene expression evaluated. The expression of all three genes was higher in the pupal and adult stages than the larval stages and largely higher in the larval head compared to other tissues. Newly hatched larvae exhibited high mortalities and suppressed growth when fed bacteria producing double-stranded RNAs (dsRNAs) corresponding to the three target genes. This study provides insights into the function of ND, GPDH and MSL3 during C. suppressalis larval development and suggests that all may be candidate gene targets for C. suppressalis pest management.

Differences in midgut transcriptomes between resistant and susceptible strains of Chilo suppressalis to Cry1C toxin.

BACKGROUND: Chilo suppressalis is a widespread rice pest that poses a major threat to food security in China. This pest can develop resistance to Cry toxins from Bacillus thuringiensis (Bt), threatening the sustainable use of insect-resistant transgenic Bt rice. However, the molecular basis for the resistance mechanisms of C. suppressalis to Cry1C toxin remains unknown. This study aimed to identify genes associated with the mechanism of Cry1C resistance in C. suppressalis by comparing the midgut transcriptomic responses of resistant and susceptible C. suppressalis strains to Cry1C toxin and to provide information for insect resistance management. RESULTS: A C. suppressalis midgut transcriptome of 139,206 unigenes was de novo assembled from 373 million Illumina HiSeq and Roche 454 clean reads. Comparative analysis identified 5328 significantly differentially expressed unigenes (DEGs) between C. suppressalis Cry1C-resistant and -susceptible strains. DEGs encoding Bt Cry toxin receptors, aminopeptidase-P like protein, the ABC subfamily and alkaline phosphatase were downregulated, suggesting an association with C. suppressalis Cry1C resistance. Additionally, Cry1C resistance in C. suppressalis may be related to changes in the transcription levels of enzymes involved in hydrolysis, digestive, catalytic and detoxification processes. CONCLUSION: Our study identified genes potentially involved in Cry1C resistance in C. suppressalis by comparative transcriptome analysis. The assembled and annotated transcriptome data provide valuable genomic resources for further study of the molecular mechanisms of C. suppressalis resistance to Cry toxins.

Knockdown of cadherin genes decreases susceptibility of Chilo suppressalis larvae to Bacillus thuringiensis produced Crystal toxins.

The striped rice stem borer, Chilo suppressalis Walker, is one of the most destructive rice pests in Asia. Insecticidal crystal proteins (Cry toxins) produced by Bacillus thuringiensis are widely used as biopesticides or in developing transgenic crops for pest management. In this study, we tested the involvement of two newly cloned C. suppressalis cadherins (CsCAD3 and CsCAD4) in the toxicity of Cry1Ab/Ac, Cry2Aa and Cry1Ca. Our results showed that CsCAD4 was expressed highest in the midgut, whereas CsCAD3 was expressed highest in the epidermis. The feeding of double-stranded RNA specific to CsCAD3 and CsCAD4 respectively significantly suppressed the expressions of target gene. The knockdown of CsCAD3 significantly reduced the mortality of larvae to Cry1Ab/Ac, whereas knockdown of CsCAD4 significantly decreased the larval susceptibility to Cry2Aa. In contrast, reduced expressions of CsCAD3 or CsCAD4 were not interacted with larval susceptibility to Cry1Ca. Our results suggest that CsCAD3 and CsCAD4 function in Cry toxin toxicity and these findings will help us to better understand the action mechanism of Cry toxins in C. suppressalis.

RNA interference-mediated knockdown of the transcription factor Krüppel homologue 1 suppresses vitellogenesis in Chilo suppressalis.

Vitellogenesis in holometabolous insects involves the production and secretion of vitellogenin (Vg) and other yolk protein precursors in developing oocyte by the fat body, all of which is predominantly orchestrated by juvenile hormone (JH). Krüppel homologue 1 (Kr-h1) is a zinc finger transcription factor that has been demonstrated to be a JH-early inducible gene and to contribute to reproduction. However, the exact molecular function of Kr-h1 in insect reproduction is poorly understood. In the current study, we used the notorious pest Chilo suppressalis as a model system to investigate the role of Kr-h1 in female reproduction. Cloning and sequencing C. suppressalis Kr-h1 revealed that it shares high identity with its homologues from other lepidopteran insects. Moreover, RNA interference-mediated knockdown of CsKr-h1 substantially reduced the transcription of Vg in the fat body, dramatically decreased yolk protein deposition and also impaired oocyte maturation and ovarian development, indicating that Kr-h1 is indispensable for normal vitellogenesis in C. suppressalis. Based on these results, we conclude that Kr-h1 is crucial to reproduction in insects and that targeting this gene could potentially be a new way to suppress rice pests.

Molecular characterization and functional analysis of the vitellogenin receptor in the rice stem borer, Chilo suppressalis.

As a member of the low-density lipoprotein receptor (LDLR) superfamily, vitellogenin (Vg) receptor (VgR) is responsible for the uptake of Vg into developing oocytes and is a potential target for pest control. Here, a full-length VgR complementary DNA (named as CsVgR) was isolated and characterized in the rice stem borer, Chilo suppressalis. The composite CsVgR gene contained an open reading frame of 5,484 bp encoding a protein of 1,827 amino acid residues. Structural analysis revealed that CsVgR contained two ligand-binding domains (LBDs) with four Class A (LDLRA ) repeats in LBD1 and seven in LBD2, which was structurally different from most non-Lepidopteran insect VgRs having five repeats in LBD1 and eight in LBD2. The developmental expression analysis showed that CsVgR messenger RNA expression was first detectable in 3-day-old pupae, sharply increased in newly emerged female adults, and reached a peak in 2-day-old female adults. Consistent with most other insects VgRs, CsVgR was exclusively expressed in the ovary. Notably, injection of dsCsVgR into late pupae resulted in fewer follicles in the ovarioles as well as reduced fecundity, suggesting a critical role of CsVgR in female reproduction. These results may contribute to the development of RNA interference-mediated disruption of reproduction as a control strategy of C. suppressalis.