Molecular mechanisms of cytochrome P450-mediated detoxification of tetraniliprole, spinetoram, and emamectin benzoate in the fall armyworm, Spodoptera frugiperda (J.E. Smith).

The fall armyworm (FAW) Spodoptera frugiperda (J.E. Smith) is a highly damaging invasive omnivorous pest that has developed varying degrees of resistance to commonly used insecticides. To investigate the molecular mechanisms of tolerance to tetraniliprole, spinetoram, and emamectin benzoate, the enzyme activity, synergistic effect, and RNA interference were implemented in S. frugiperda. The functions of cytochrome P450 monooxygenase (P450) in the tolerance to tetraniliprole, spinetoram, and emamectin benzoate in S. frugiperda was determined by analysing changes in detoxification metabolic enzyme activity and the effects of enzyme inhibitors on susceptibility to the three insecticides. 102 P450 genes were screened via transcriptome and genome, of which 67 P450 genes were differentially expressed in response to tetraniliprole, spinetoram, and emamectin benzoate and validated by quantitative real-time PCR. The expression patterns of CYP9A75, CYP340AA4, CYP340AX8v2, CYP340L16, CYP341B15v2, and CYP341B17v2 were analysed in different tissues and at different developmental stages in S. frugiperda. Silencing CYP340L16 significantly increased the susceptibility of S. frugiperda to tetraniliprole, spinetoram, and emamectin benzoate. Furthermore, knockdown of CYP340AX8v2, CYP9A75, and CYP341B17v2 significantly increased the sensitivity of S. frugiperda to tetraniliprole. Knockdown of CYP340AX8v2 and CYP340AA4 significantly increased mortality of S. frugiperda to spinetoram. Knockdown of CYP9A75 and CYP341B15v2 significantly increased the susceptibility of S. frugiperda to emamectin benzoate. These results may help to elucidate the mechanisms of tolerance to tetraniliprole, spinetoram and emamectin benzoate in S. frugiperda.

Responses of keratinocytes to Trichophyton mentagrophyte infection based on whole transcriptome analysis.

BACKGROUND: Dermatophytosis is an intractable superficial mycosis in humans and animals mainly caused by Trichophyton mentagrophytes (T. mentagrophytes), with a global prevalence of about 20%. Keratinocytes are the most abundant participants in skin immunity, and they also play a role in the first-line defence against T. mentagrophytes. However, no studies of keratinocyte responses against T. mentagrophytes infection based on the whole transcriptome have been reported. OBJECTIVES: Here, we systematically analysed changes in keratinocytes infected with T. mentagrophytes using whole transcriptome sequencing technology. METHODS: The phenotypic changes in keratinocytes after infection with 1 × 105 conidia/mL T. mentagrophytes were observed by light microscopy, scanning electron microscopy, transmission electron microscopy and terminal deoxynucleotidyl transferase dUTP nick end labeling. RNA-sequencing (RNA-seq), small RNA-seq technology and related bioinformatics methods were used to systematically analyse the whole transcriptome changes in keratinocytes upon T. mentagrophytes stimulation. RESULTS: We found that T. mentagrophytes infection caused morphological changes, membrane damage, the formation of irregular organelles and keratinocyte apoptosis. A total of 204 differentially expressed (DE) circular RNAs (circRNAs), 868 DE long noncoding RNAs (lncRNAs), 2973 DE mRNAs and 209 DE micro RNAs (miRNAs) were identified between noninfected and T. mentagrophytes-infected keratinocytes. The expression level of selected RNAs was validated by quantitative real-time polymerase chain reaction (qRT-PCR). Functional enrichment analysis revealed that the parental genes of DE circRNAs were related to cell response, cell death and establishment of the skin barrier. Genes targeted by miRNA were involved in regulating the initiation of the immune response. Based on the expression level of circRNAs, lncRNAs, mRNAs and miRNAs, circRNA-miRNA-mRNA competing endogenous (ceRNA) networks comprised of 159 DE miRNAs, 141 DE circRNAs and 2307 DE mRNAs, and lncRNA-miRNA-mRNA ceRNA networks comprised of 790 DE lncRNAs, 190 DE miRNAs and 2663 DE mRNAs were constructed. The reliability of two selected ceRNA networks was verified using qRT-PCR. Further functional enrichment analysis revealed that the DE mRNAs interacting with circRNAs and lncRNAs in the ceRNA network mainly participated in fungal recognition, inflammation, the innate immune response and the death of keratinocytes. CONCLUSIONS: Our findings might provide new evidence on the pathogenesis of T. mentagrophytes-induced dermatophytosis, which is essential for identifying new therapeutic targets for dermatophytosis treatment.

MicroRNA PC-5p-3991_515 mediates triflumezopyrim susceptibility in the small brown planthopper through regulating the post-transcriptional expression of P450 CYP417A2.

BACKGROUND: Cytochrome P450 monooxygenases (P450s) are recognized as a major contributor to metabolic resistance in insects to most insecticides, through gene overexpressions and protein mutations. MicroRNA (miRNA), an important post-transcriptional regulator, has been reported to promote insecticide resistance by mediating the expression of detoxification enzyme genes. RESULTS: In the present study, we reported that a novel microRNA PC-5p-3991_515 was involved in the post-transcriptional regulation of CYP417A2 and mediated the triflumezopyrim susceptibility in the small brown planthopper (SBPH), Laodelphax striatellus (Fallén). The tissue expression profiles showed that CYP417A2 was highly expressed in fat body. CYP417A2 was significantly up-regulated at 12, 36, 60, 84 and 108 h after the triflumezopyrim treatment. RNA interference (RNAi) against CYP417A2 significantly increased triflumezopyrim susceptibility in SBPH. According to the prediction by miRanda and TargetScan software, three miRNAs were indicated to bind to CYP417A2. However, when oversupply of agomir, only two miRNAs, PC-3p-625_4405 and PC-5p-3991_515, significantly increased the susceptibility to triflumezopyrim and decreased CYP417A2 levels. Furthermore, PC-5p-3991_515 was confirmed to be involved in the post-transcriptional regulation of CYP417A2 by dual luciferase reporter assay. Meanwhile, PC-5p-3991_515 was co-localized with CYP417A2 in the midgut in situ hybridization. CONCLUSION: Our findings revealed that the novel microRNA, PC-5p-3991_515, post-transcriptionally regulated CYP417A2 expression, which then mediated the triflumezopyrim susceptibility in SBPH. © 2023 Society of Chemical Industry.

Plant volatiles mediate Aphis gossypii settling but not predator foraging in intercropped cotton.

BACKGROUND: The cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae) is an important pest of cotton and horticultural crops globally. In China, smallholder farmers regularly intercrop cotton with garlic or onion. Aside from higher farm-level revenue, cotton intercrops are typified by lower Aphis gossypii abundance than monocrops. So far, the mechanistic basis of this lowered pest pressure has not been empirically assessed. RESULTS: Field trials showed that Aphis gossypii abundance is lower and (relative) abundance of aphid predators higher in early-season cotton intercrops than in monocrops. Cage trials and Y-tube olfactometer tests further indicated that garlic and onion volatiles repel Aphis gossypii alates. Electrophysiological bioassays and gas chromatography-mass spectrometry (GC-MS) identified two physiologically active volatiles, that is, diallyl disulfide and propyl disulfide from garlic and onion respectively. Next, behavioral tests confirmed that both sulfur compounds exert a repellent effect on alate Aphis gossypii. CONCLUSION: Garlic and onion volatiles interfere with Aphis gossypii settling, but do not affect its main (ladybird) predators. Meanwhile, early-season cotton/onion intercrops bear higher numbers of Aphis gossypii predators and fewer aphids. By thus unveiling the ecological underpinnings of aphid biological control in diversified cropping systems, our work advances non-chemical management of a globally-important crop pest. © 2023 Society of Chemical Industry.

A microRNA, PC-5p-30_205949, regulates triflumezopyrim susceptibility in Laodelphax striatellus (Fallén) by targeting CYP419A1 and ABCG23.

MicroRNAs (miRNAs) are known to be important post-transcriptional regulators of gene expression and have been shown to be associated with insecticide resistance in insects. In this research, we show that a miRNA, PC-5p-30_205949, is involved in triflumezopyrim susceptibility via regulating expressive abundance of cytochrome P450 CYP419A1 and ATP-binding cassette transporters ABCG23 in the small brown planthopper (SBPH), Laodelphax striatellus (Fallén). Triflumezopyrim treatment significantly reduced the abundance of PC-5p-30_205949, feeding of agomir-PC-5p-30_205949 significantly increased the sensitivity of SBPH to triflumezopyrim, and its spatiotemporal expression profiles showed that PC-5p-30_205949 were expressed at all developmental stages and were highly expressed in head tissue. By software prediction and dual luciferase reporter assay, the target genes of PC-5p-30_205949 were identified as two detoxification metabolism genes CYP419A1 and ABCG23. The relative expressions of CYP419A1 and ABCG23 were significantly up-regulated after 24 h, 48 h and 72 h with triflumezopyrim exposure. CYP419A1 was highly expressed in the 4th-instar nymphs and male adults, with the highest expression level in fat body. ABCG23 was highly expressed in female adults, and had the highest expression in head. Furthermore, silencing of CYP419A1 and ABCG23 by RNA interference significantly increased the mortality of SBPH to triflumezopyrim, and molecular docking showed that CYP419A1 and ABCG23 could stably bind to triflumezopyrim with binding free energies of -171.5622 and - 103.3402 kcal mol-1, respectively. These results suggest that SBPH has a strategy to enhance the resistance to triflumezopyrim by attenuating the expression of PC-5P-30_205949, thereby activating the detoxification metabolic pathway by targeting CYP419A1 and ABCG23.

The effects and interrelationships of intercropping on Cotton Verticillium wilt and soil microbial communities.

BACKGROUND: Cotton Verticillium wilt, causing by Verticillium dahliae, has seriously affected the yield and quality of cotton. The incidence of Verticillium wilt in cotton fields has been on the rise for many years, especially after straw has been returned to the fields. Intercropping can reduce the incidence of soil borne diseases and is often used to control crop diseases, but the relationship between the effects of intercropping on microbial communities and the occurrence of plant diseases is unclear. This research explored the relationship between soil microbial community structure and Cotton Verticillium wilt in interplanting of cotton-onion, cotton-garlic, cotton-wheat and cotton monocultures. Amplicon sequencing applied to the profile of bacterial and fungal communities. RESULTS: The results showed that the disease index of Cotton Verticillium wilt was significantly reduced after intercropping with cotton-garlic and cotton-onion. Chao1 and Sobs indices were not significantly different in the rhizosphere soil and pre-plant soils of the four planting patterns, but the pre-plant fungal shannon index was significantly lower in the cotton-onion intercropping plot than in the other three plots. PCoA analysis showed that the soil microbial communities changed to a certain extent after intercropping, with large differences in the microbial communities under different cropping patterns. The abundance of Chaetomium was highest in the cotton-garlic intercropping before planting; the abundance of Penicillium was significantly higher in the cotton-wheat intercropping than in the other three systems. CONCLUSION: Cotton-garlic and cotton-onion interplanting can control Cotton Verticillium wilt by affecting the soil microbial community. Fungi of the genera Chaetomium and Penicillium may be associated with plant disease resistance.

Activating pathway of three metabolic detoxification phases via down-regulated endogenous microRNAs, modulates triflumezopyrim tolerance in the small brown planthopper, Laodelphax striatellus (Fallén).

Frequent use of insecticide causes an environmental hazard, and also leads pest to develop insecticide resistance. Enhancement of metabolic detoxification and reduction of target sensitivity are the primary mechanism of insecticide resistance. Clarifying the regulatory pathway of resistance mechanism states a pivotal theoretical foundation of delaying insecticide resistance development. Here, we show that three endogenous microRNAs, PC-3p-2522_840, PC-3p-446_6601 and PC-5p-3096_674, are required for the small brown planthopper (SBPH) to modulate triflumezopyrim tolerance via activating pathways of three metabolic detoxification phases. Twenty-one down-regulated miRNAs were acquired, and PC-5p-3096_674, PC-3p-446_6601 and PC-3p-2522_840 were the three most significantly down-regulated miRNAs during triflumezopyrim exposure. The mortality of SBPH was significantly increased after over-supplementation of PC-5p-3096_674, PC-3p-446_6601 and PC-3p-2522_840, with triflumezopyrim exposure. Moreover, the interactions between PC-3p-2522_840 and cytochrome P450 CYP6FL1, PC-3p-446_6601 and glutathione S-transferase GSTD2, UDP-Glycosyltransferase UGT386F1, PC-5p-3096_674 and ATP-binding cassette transporters ABCA3 were systematically demonstrated through the dual luciferase reporter assay. Besides, the mortality of SBPH was significantly increased after knockdown of CYP6FL1, GSTD2, UGT386F1 and ABCA3 with triflumezopyrim exposure. These findings uncover a strategy whereby the SBPH weakens three endogenous microRNAs to activate pathways of three metabolic detoxification phases via targeting CYP6FL1, GSTD2, UGT386F1 and ABCA3 and promotes its tolerance to triflumezopyrim.

Overexpression of ATP-binding cassette transporters ABCG10, ABCH3 and ABCH4 in Aphis craccivora (Koch) facilitates its tolerance to imidacloprid.

Aphis craccivora (Koch), a globally pest that causes significant threat to the legumes, has developed different degrees of resistance to a variety of insecticides. The ATP-binding cassette (ABC) transporters comprise a multifunctional transporter protein superfamily which play important roles in the transport and detoxification of xenobiotic compounds in insects. However, whether ABC transporters take part in the tolerance of imidacloprid in A. craccivora is still unknown. In order to investigate the functions of ABC transporters in the imidacloprid tolerance, fifty- eight ABC transporters were identified in the transcriptome and genome of A. craccivora and the toxicity of imidacloprid against A. craccivora was significantly increased after application the inhibitors of verapamil and Ko143. The relative expression levels of ABCG5, ABCG6, ABCG10, ABCH3, ABCH4, ABCH8 and ABCH10 were significantly up-regulated in response to imidacloprid treatment with LC15, LC50 and LC85 concentrations, and the expression patterns of these seven ABC transporters were further analyzed at different developmental stages and in different tissues of A. craccivora by quantitative real-time PCR (RT-qPCR). Furthermore, knockdown of ABCG10, ABCH3 and ABCH4 significantly increased the mortality of A. craccivora to imidacloprid. Our results reveal the key functions of ABC transporters in the tolerance of imidacloprid and provide valuable information regarding the development of improved management strategies in A. craccivora.

ATP-binding cassette transporters ABCF2 and ABCG9 regulate rice black-streaked dwarf virus infection in its insect vector, Laodelphax striatellus (Fallén).

The majority of plant viral disease is transmitted and spread by insect vectors in the field. The small brown planthopper, Laodelphax striatellus (Fallén), is the only efficient vector for rice black-streaked dwarf virus (RBSDV), a devastating plant virus that infects multiple grain crops, including rice, maize, and wheat. Adenosine triphosphate (ATP)-binding cassette (ABC) transporters participate in various biological processes. However, little is known about whether ABC transporters affect virus infection in insects. In this study, RBSDV accumulation was significantly reduced in L. striatellus after treatment with verapamil, an effective inhibitor of ABC transporters. Thirty-four ABC transporter genes were identified in L. striatellus and expression analysis showed that LsABCF2 and LsABCG9 were significantly upregulated and downregulated, respectively, after RBSDV infection. LsABCF2 and LsABCG9 were expressed during all developmental stages, and LsABCG9 was highly expressed in the midgut of L. striatellus. Knockdown of LsABCF2 promoted RBSDV accumulation, while knockdown of LsABCG9 suppressed RBSDV accumulation in L. striatellus. Our data showed that L. striatellus might upregulate the expression of LsABCF2 and downregulate LsABCG9 expression to suppress RBSDV infection. These results will contribute to understanding the effects of ABC transporters on virus transmission and provide theoretical basis for virus management in the field.

Composition and characteristics of soil microbial communities in cotton fields with different incidences of Verticillium wilt.

Soil microorganisms could affect the growth of plants and play an important role in indicating the change of soil environment. Cotton Verticillium wilt is a serious soil borne disease. This study aimed to analyze the community characteristics of soil microorganisms in cotton fields with different incidences of Verticillium wilt, so as to provide theoretical guidance for the prevention and control of soil borne diseases of cotton. Through the analysis of soil microbial communities in six fields, the results showed that there was no difference in fungal and bacterial alpha-diversity index before cotton planting, while there were differences in rhizosphere of diseased plants. For fungal beta diversity indexes, there were significant differences in these six fields. There was no significant difference for bacterial beta diversity indexes before cotton planting, while there was a certain difference in the rhizosphere of diseased cotton plants. The composition of fungi and bacteria in different fields was roughly the same at the genus level, but the abundances of the same genus varied greatly between different fields. Before cotton planting, there were 61 fungi (genera) and 126 bacteria (genera) with different abundances in the six fields. Pseudomonas, Sphingomonas and Burkholderia had higher abundances in the fields with less incidence. This study will provide a theoretical basis for microbial control of Cotton Verticillium wilt.

Systematic identification and characterization of differentially expressed microRNAs under tetraniliprole exposure in the fall armyworm, Spodoptera frugiperda.

The fall armyworm, Spodoptera frugiperda, is a worldwide agricultural pest and causes huge losses of crop production each year. Tetraniliprole is a novel diamide insecticide with high efficacy against even the insecticide resistant pests of Lepidoptera, Coleoptera, and Diptera. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level and play an important regulatory role in the insecticide resistance in insects. However, the effects of miRNAs on the tetraniliprole tolerance in S. frugiperda are poorly understood. In the present research, the miRNAs response to tetraniliprole application in S. frugiperda were systematically investigated by high-throughput sequencing. A total of thirty differentially expressed miRNAs were identified under tetraniliprole treatment in S. frugiperda. The functions of the target genes of these differentially expressed miRNAs were further predicted by Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes database pathway, and the most significantly enriched pathway was MAPK signaling pathway. The expression changes of six differentially expressed miRNAs were validated by quantitative real-time polymerase chain reaction. Furthermore, miR-278-5p had the highest expression in the hemolymph and malpighian tubule and the lowest expression in the gut. Oversupply of miR-278-5p significantly increased the mortality of S. frugiperda following exposure to tetraniliprole. These results will provide the basis for understanding the regulatory roles of miRNAs regarding to tetraniliprole tolerance in S. frugiperda.

Cytochrome P450 Monooxygenases CYP6AY3 and CYP6CW1 Regulate Rice Black-Streaked Dwarf Virus Replication in Laodelphax striatellus (Fallén).

The small brown planthopper, Laodelphax striatellus (Fallén), is an important agricultural pest that causes significant losses by sucking and transmitting multiple plant viruses, such as rice black-streaked dwarf virus (RBSDV). Insecticides are commonly used to control planthoppers and cause the induction or overexpression of cytochrome P450 monooxygenases (P450s) from the CYP3 and CYP4 clades after insecticide application. However, little is known about the roles of insecticides and P450s in the regulation of viral replication in insects. In this study, RBSDV-infected L. striatellus were injected with imidacloprid, deltamethrin, pymetrozine, and buprofezin, respectively. The insecticide treatments caused a significant decrease in RBSDV abundance in L. striatellus. Treatment of piperonyl butoxide (PBO), an effective inhibitor of P450s, significantly increased the RBSDV abundance in L. striatellus. Fourteen P450 candidate genes in the CYP3 clade and 21 in the CYP4 clade were systematically identified in L. striatellus, and their expression patterns were analyzed under RBSDV infection, in different tissues, and at different developmental stages. Among the thirty-five P450 genes, the expression level of CYP6CW1 was the highest, while CYP6AY3 was the lowest after RBSDV infection. Knockdown of CYP6CW1 and CYP6AY3 significantly increased the virus abundance and promoted virus replication in L. striatellus. Overall, our data reveal that CYP6CW1 and CYP6AY3 play a critical role in the regulation of virus replication in L.striatellus.

Four MicroRNAs, miR-13b-3p, miR-278-5p, miR-10483-5p, and miR-10485-5p, Mediate Insecticide Tolerance in Spodoptera frugiperda.

Spodoptera frugiperda is the world's major agricultural pest and has the distinctive features of high fecundity, strong migratory capacity, and high resistance to most insecticides. At present, the control of S. frugiperda in China relies mainly on the spraying of chemical insecticides. MicroRNAs (miRNAs) are a class of small, single-stranded, non-coding RNAs and play crucial regulatory roles in various physiological processes, including the insecticide resistance in insects. However, little is known about the regulatory roles of miRNAs on the resistance of S. frugiperda to insecticides. In the present research, the miRNAs that were differentially expressed after cyantraniliprole, spinetoram, and emamectin benzoate treatment were analyzed by RNA-Seq. A total of 504 miRNAs were systematically identified from S. frugiperda, and 24, 22, and 31 miRNAs were differentially expressed after treatments of cyantraniliprole, spinetoram, and emamectin benzoate. GO and KEGG enrichment analyses were used to predict the function of differentially expressed target genes of miRNAs. Importantly, ten miRNAs were significantly differentially expressed among the treatments of three insecticides. miR-278-5p, miR-13b-3p, miR-10485-5p, and miR-10483-5p were significantly downregulated among the treatments of three insecticides by RT-qPCR. Furthermore, the overexpression of miR-278-5p, miR-13b-3p, miR-10485-5p, and miR-10483-5p significantly increased the mortality of S. frugiperda to cyantraniliprole and emamectin benzoate. The mortality was significantly increased with spinetoram treatment after the overexpression of miR-13b-3p, miR-10485-5p, and miR-10483-5p. These results suggest that miRNAs, which are differentially expressed in response to insecticides, may play a key regulatory role in the insecticide tolerance in S. frugiperda.

Function Analysis of P450 and GST Genes to Imidacloprid in Aphis craccivora (Koch).

Aphis craccivora (Koch) is an economically important pest that affects legumes in worldwide. Chemical control is still the primary efficient method for A. craccivora management. However, the mechanism underlying insecticide resistance in A. craccivora has not been elucidated. A previous study observed that piperonyl butoxide (PBO) and diethyl maleate (DEM) significantly synergized imidacloprid in A. craccivora field populations, indicating that cytochrome P450 (P450) and glutathione S-transferase (GST) genes may play pivotal roles in imidacloprid resistance. In this study, 38 P450 genes and 10 GST genes were identified in A. craccivora through transcriptomic analysis. The expression levels of these P450 and GST genes were measured in susceptible (SUS) strains of A. craccivora under imidacloprid treatment with LC15, LC50, and LC85 doses. The expression levels of CYP18A1, CYP6CY21, CYP6DA1, CYP6DA2, CYP4CJ1, CYP4CJ2, and CYP380C6 were up-regulated in the three treatments. Most of these genes belong to CYP3 and CYP4 Clans. In addition, the expression levels of all P450 and GST genes in A. craccivora were also measured in the Juye (JY) and Linqing (LQ) field populations. The expression levels of CYP6DA2, CYP4CJ1, and CYP380C6 were up-regulated in the SUS strain after imidacloprid treatment at three doses, and these genes were overexpressed in the JY population. Furthermore, the sensitivity of A. craccivora to imidacloprid was significantly increased after knockdown of CYP380C6 and CYP6DA2 through RNA interference. These results may help to elucidate the mechanisms underlying of imidacloprid resistance in A. craccivora.

New insight into foregut functions of xenobiotic detoxification in the cockroach Periplaneta americana.

The physiological functions of insect foregut, especially in xenobiotic detoxification, are scarcely reported because of unimportance in appearance and insufficient molecular information. The cockroach Periplaneta americana, an entomological model organism, provides perfect material to study physiological functions of foregut tissue due to its architectural feature. Through Illumina sequencing of foregut tissue from P. americana individuals (control) or insects treated with cycloxaprid, as a novel neonicotinoid insecticide, 54 193 166 clean reads were obtained and further assembled into 53 853 unigenes with an average length of 366 bp. Furthermore, the number of unigenes involved in xenobiotic detoxification was analyzed, mainly including 70 cytochrome P450s, 12 glutathione S-transferases (GSTs), seven carboxylesterases (CarEs) and seven adenosine triphosphate-binding cassette (ABC) transporters. Compared to control, the expression of 22 xenobiotic detoxification unigenes was up-regulated after cycloxaprid application, mainly containing 18 P450s, one GST, two CarEs and one ABC adenosine triphosphate transporter, indicating that the oxidation-reduction was the major reactive process to cycloxaprid application. Through quantitative real-time polymerase chain reaction analysis, the expression of selected unigenes (six P450s, one GST and one CarE) was up-regulated at least two-fold following cycloxaprid treatment, and was generally in agreement with transcriptome data. Compared to the previous midgut transcriptome of P. americana, it looks like the expressive abundance of the xenobiotic detoxification unigenes might be important factors to the detoxifying functional differences between foregut and midgut. In conclusion, insect foregut would also play important roles in the physiological processes related to xenobiotic detoxification.

Induction of P450 genes in Nilaparvata lugens and Sogatella furcifera by two neonicotinoid insecticides.

Nilaparvata lugens and Sogatella furcifera are two primary planthoppers on rice throughout Asian countries and areas. Neonicotinoid insecticides, such as imidacloprid (IMI), have been extensively used to control rice planthoppers and IMI resistance consequently occurred with an important mechanism from the over-expression of P450 genes. The induction of P450 genes by IMI may increase the ability to metabolize this insecticide in planthoppers and increase the resistance risk. In this study, the induction of P450 genes was compared in S. furcifera treated with IMI and nitromethyleneimidazole (NMI), in two planthopper species by IMI lethal dose that kills 85% of the population (LD85 ), and in N. lugens among three IMI doses (LD15 , LD50 and LD85 ). When IMI and NMI at the LD85 dose were applied to S. furcifera, the expression changes in most P450 genes were similar, including the up-regulation of nine genes and down-regulation of three genes. In terms of the expression changes in 12 homologous P450 genes between N. lugens and S. furcifera treated with IMI at the LD85 dose, 10 genes had very similar patterns, such as up-regulation in seven genes, down-regulation in one gene and no significant changes in two genes. When three different IMI doses were applied to N. lugens, the changes in P450 gene expression were much different, such as up-regulation in four genes at all doses and dose-dependent regulation of the other nine genes. For example, CYP6AY1 could be induced by all IMI doses, while CYP6ER1 was only up-regulated by the LD50 dose, although both genes were reported important in IMI resistance. In conclusion, P450 genes in two planthopper species showed similar regulation patterns in responding to IMI, and the two neonicotinoid insecticides had similar effects on P450 gene expression, although the regulation was often dose-dependent.

Metabolic imidacloprid resistance in the brown planthopper, Nilaparvata lugens, relies on multiple P450 enzymes.

Target insensitivity contributing to imidacloprid resistance in Nilaparvata lugens has been reported to occur either through point mutations or quantitative change in nicotinic acetylcholine receptors (nAChRs). However, the metabolic resistance, especially the enhanced detoxification by P450 enzymes, is the major mechanism in fields. From one field-originated N. lugens population, an imidacloprid resistant strain G25 and a susceptible counterpart S25 were obtained to analyze putative roles of P450s in imidacloprid resistance. Compared to S25, over-expression of twelve P450 genes was observed in G25, with ratios above 5.0-fold for CYP6AY1, CYP6ER1, CYP6CS1, CYP6CW1, CYP4CE1 and CYP425B1. RNAi against these genes in vivo and recombinant tests on the corresponding proteins in vitro revealed that four P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, played important roles in imidacloprid resistance. The importance of the four P450s was not equal at different stages of resistance development based on their over-expression levels, among which CYP6ER1 was important at all stages, and that the others might only contribute at certain stages. The results indicated that, to completely reflect roles of P450s in insecticide resistances, their over-expression in resistant individuals, expression changes at the stages of resistance development, and catalytic activities against insecticides should be considered. In this study, multiple P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, have proven to be important in imidacloprid resistance.

Synergistic and compensatory effects of two point mutations conferring target-site resistance to fipronil in the insect GABA receptor RDL.

Insecticide resistance can arise from a variety of mechanisms, including changes to the target site, but is often associated with substantial fitness costs to insects. Here we describe two resistance-associated target-site mutations that have synergistic and compensatory effects that combine to produce high and persistent levels of resistance to fipronil, an insecticide targeting on γ-aminobytyric acid (GABA) receptors. In Nilaparvata lugens, a major pest of rice crops in many parts of Asia, we have identified a single point mutation (A302S) in the GABA receptor RDL that has been identified previously in other species and which confers low levels of resistance to fipronil (23-fold) in N. lugans. In addition, we have identified a second resistance-associated RDL mutation (R300Q) that, in combination with A302S, is associated with much higher levels of resistance (237-fold). The R300Q mutation has not been detected in the absence of A302S in either laboratory-selected or field populations, presumably due to the high fitness cost associated with this mutation. Significantly, it appears that the A302S mutation is able to compensate for deleterious effects of R300Q mutation on fitness cost. These findings identify a novel resistance mechanism and may have important implications for the spread of insecticide resistance.

Expression induction of P450 genes by imidacloprid in Nilaparvata lugens: A genome-scale analysis.

The overexpression of P450 monooxygenase genes is a main mechanism for the resistance to imidacloprid, a representative neonicotinoid insecticide, in Nilaparvata lugens (brown planthopper, BPH). However, only two P450 genes (CYP6AY1 and CYP6ER1), among fifty-four P450 genes identified from BPH genome database, have been reported to play important roles in imidacloprid resistance until now. In this study, after the confirmation of important roles of P450s in imidacloprid resistance by the synergism analysis, the expression induction by imidacloprid was determined for all P450 genes. In the susceptible (Sus) strain, eight P450 genes in Clade4, eight in Clade3 and two in Clade2 were up-regulated by imidacloprid, among which three genes (CYP6CS1, CYP6CW1 and CYP6ER1, all in Clade3) were increased to above 4.0-fold and eight genes to above 2.0-fold. In contrast, no P450 genes were induced in Mito clade. Eight genes induced to above 2.0-fold were selected to determine their expression and induced levels in Huzhou population, in which piperonyl butoxide showed the biggest effects on imidacloprid toxicity among eight field populations. The expression levels of seven P450 genes were higher in Huzhou population than that in Sus strain, with the biggest differences for CYP6CS1 (9.8-fold), CYP6ER1 (7.7-fold) and CYP6AY1 (5.1-fold). The induction levels for all tested genes were bigger in Sus strain than that in Huzhou population except CYP425B1. Screening the induction of P450 genes by imidacloprid in the genome-scale will provide an overall view on the possible metabolic factors in the resistance to neonicotinoid insecticides. The further work, such as the functional study of recombinant proteins, will be performed to validate the roles of these P450s in imidacloprid resistance.

No cross-resistance between imidacloprid and pymetrozine in the brown planthopper: status and mechanisms.

Cross-resistance between insecticides, especially from different groups, can be extremely unpredictable, and it has been a serious concern in pest control. Pymetrozine has been widely used to control Nilaparvata lugens with the suspension of imidacloprid for the resistance, and N. lugens has showed obvious pymetrozine resistance in recent years. To investigate the possible cross-resistance between imidacloprid and pymetrozine is very important to avoid the adverse effects on resistance development and pest control. Bioassays of two field populations in five consecutive years showed that imidacloprid resistance decreased greatly, while pymetrozine resistance increased significantly. The synergist piperonyl butoxide (PBO) could synergize both imidacloprid and pymetrozine in all field populations, which indicated the importance of P450s in the resistance to two insecticides. Imidacloprid resistance was reported to be associated with two P450s, CYP6AY1 and CYP6ER1, which could metabolize imidacloprid efficiently. However, the recombinant proteins of these two P450s did not show any enzymatic activity to metabolize pymetrozine. The pymetrozine susceptibility did not change when CYP6AY1 and CYP6ER1 mRNA levels were reduced by RNA interference (RNAi), although which could obviously decrease imidacloprid resistance. In vivo and in vitro studies provided evidences to demonstrate that there was no cross-resistance between imidacloprid and pymetrozine in N. lugens, which was different from the findings in Bemisia tabaci.