Sublethal and transgenerational effects of broflanilide on the citrus red mite, Panonychus citri.

BACKGROUND: The citrus red mite, Panonychus citri is a serious pest of the citrus industry and has developed resistance to many acaricides. Broflanilide is a novel meta-diamide insecticide that binds to a new site on the γ -aminobutyric acid receptor with high potency against pests. However, little information has been reported about its effect on the citrus red mite. RESULTS: Broflanilide exhibited higher toxicity to female adults and eggs of a laboratory strain of P. citri The median lethal concentration (LC50), 9.769 mg/L and 4.576 mg/L, respectively) than other commonly used acaricides and was also toxic to two P. citri field strains. Broflanilide treatment with LC10, LC20, and LC30 significantly decreased the fecundity and longevity of female adults of F0 P. citri compared with the control. The duration of larva, protonymph, deutonymph and adult, and total life span in the F1 generation were significantly reduced after treatment of F0 with broflanilide. Population parameters, including the intrinsic rate of increase (r) and finite rate of increase (λ), were significantly increased, and the mean generation time (T) of F1 progeny was significantly reduced in the LC20 treatment. The predicted population size of F1 increased when parental female adults were treated with sublethal concentrations. CONCLUSION: Broflanilide had high acaricidal activity toward P. citri, and exposure to a sublethal concentration significantly inhibited the population growth of F0. The transgenerational hormesis effect is likely to cause population expansion of F1. More attention should be paid when broflanilide is applied to control P. citri in citrus orchards. © 2024 Society of Chemical Industry.

Lethal and sublethal effects of fluralaner on the citrus red mite, Panonychus citri (McGregor).

BACKGROUND: The citrus red mite, Panonychus citri (McGregor) is a globally distributed agricultural pest. Of late, resistance to common acaricides has raised concerns that chemical control of P. citri is an inefficient means of control. Fluralaner, a highly toxic isoxazoline insecticide used to treat various ectoparasites, presents one potential alternative. However, little information has been reported about the effect of fluralaner on the citrus red mite. This study aims to evaluate the toxicity, sublethal and transgenerational effects of fluralaner on P. citri. RESULTS: In both laboratory and field populations of P. citri, we found fluralaner to be more toxic than conventional alternatives, including fenpropathrin, bifenazate, azocyclotin and chlorpyrifos. Interestingly, fluralaner proved more toxic to female adults than to the eggs of P. citri, with median lethal concentrations (LC50) of 2.446 and 122.7 mg L-1, respectively. Exposure to sublethal concentrations of fluralaner (LC10, LC20 and LC30) significantly reduced the fecundity and longevity of female adults P. citri individuals. Although concentrations of fluralaner applied to the parental female adults (F0) led to some changes in the developmental parameters, there were no significant changes in most of the life table parameters or population growth of the F1 generation. CONCLUSION: Our results indicate that fluralaner is highly toxic to P. citri, and a significant sublethal effect on F0 could suppress the population growth of P. citri, but not for F1. Fluralaner may be considered as a pesticide for the future management of the citrus red mite. © 2024 Society of Chemical Industry.

Cross-resistance, inheritance and biochemical mechanism of abamectin resistance in a field-derived strain of the citrus red mite, Panonychus citri (Acari: Tetranychidae).

BACKGROUND: The citrus red mite, Panonychus citri (McGregor), a global pest of citrus, has developed different levels of resistance to various acaricides in the field. Abamectin is one of the most important insecticides/acaricides worldwide, targetting a wide number of insect and mite pests. The evolution of abamectin resistance in P. citri is threatening the sustainable use of abamectin for mite control. RESULTS: The abamectin resistant strain (NN-Aba), derived from a field strain NN by consistent selection with abamectin, showed 4279-fold resistance to abamectin compared to a relatively susceptible strain (SS) of P. citri. Cross-resistance of NN-Aba was observed between abamectin and emamectin benzoate, pyridaben, fenpropathrin and cyflumetofen. Inheritance analyses indicated that abamectin resistance in the NN-Aba strain was autosomal, incompletely recessive and polygenic. The synergy experiment showed that abamectin toxicity was synergized by piperonyl butoxide (PBO), diethyl maleate (DEM) and tributyl phosphorotrithiotate (TPP) in the NN-Aba strain, and synergy ratios were 2.72-, 2.48- and 2.13-fold, respectively. The glutathione-S-transferases activity in the NN-Aba strain were significantly increased by 2.08-fold compared with the SS strain. CONCLUSION: The abamectin resistance was autosomal, incompletely recessive and polygenic in P. citri. The NN-Aba strain showed cross-resistance to various acaricides with different modes of action. Metabolic detoxification mechanism participated in abamectin resistance in NN-Aba strain. These findings provide useful information for resistance management of P. citri in the field. © 2023 Society of Chemical Industry.

Genome-wide analysis of Panonychus citri microRNAs with a focus on potential insecticidal activity of 4 microRNAs to eggs and nymphs.

Panonychus citri McGregor (Acari: Tetranychidae), a destructive citrus pest, causes considerable annual economic losses due to its short lifespan and rapid resistance development. MicroRNA (miRNA)-induced RNA interference is a promising approach for pest control because of endogenous regulation of pest growth and development. To search for miRNAs with potential insecticidal activity in P. citri, genome-wide analysis of miRNAs at different developmental stages was conducted, resulting in the identification of 136 miRNAs, including 73 known and 63 novel miRNAs. A total of 17 isomiRNAs and 12 duplicated miRNAs were characterized. MiR-1 and miR-252-5p were identified as reference miRNAs for P. citri and Tetranychus urticae. Based on differential expression analysis, treatments with miR-let-7a and miR-315 mimics and the miR-let-7a antagomir significantly reduced the egg hatch rate and resulted in abnormal egg development. Overexpression or downregulation of miR-34-5p and miR-305-5p through feeding significantly decreased the adult eclosion rate and caused molting defects. The 4 miRNAs, miR-let-7a, miR-315, miR-34-5p, and miR-305-5p, had important regulatory functions and insecticidal properties in egg hatching and adult eclosion. In general, these data advance our understanding of miRNAs in mite biology, which can assist future studies on insect-specific miRNA-based green pest control technology.

CYP4CL2 Confers Metabolic Resistance to Pyridaben in the Citrus Pest Mite Panonychus citri.

The citrus red mite Panonychus citri has developed strong resistance to acaricides. Cytochrome P450 monooxygenases (P450s) can detoxify pesticides and are involved in pesticide resistance in many insects. Here, a pyridaben-resistant P. citri strain showed cross-resistance to cyenopyrafen, bifenazate, fenpyroximate, and tolfenpyrad. Piperonyl butoxide, a P450 inhibitor, significantly increased the toxicity of pyridaben to resistant (Pyr_Rs) and susceptible (Pyr_Control) P. citri strains. P450 activity was significantly higher in Pyr_Rs than in Pyr_Control. Analyses of RNA-Seq data identified a P450 gene (CYP4CL2) that is potentially involved in pyridaben resistance. Consistently, it was up-regulated in two field-derived resistant populations (CQ_WZ and CQ_TN). RNA interference-mediated knockdown of CYP4CL2 significantly decreased the pyridaben resistance in P. citri. Transgenic Drosophila melanogaster expressing CYP4CL2 showed increased pyridaben resistance. Molecular docking analysis showed that pyridaben could bind to several amino acids at substrate recognition sites in CYP4CL2. These findings shed light on P450-mediated pyridaben resistance in pest mites.

Cuticular competing endogenous RNAs regulate insecticide penetration and resistance in a major agricultural pest.

BACKGROUND: The continuously developing pesticide resistance is a great threat to agriculture and human health. Understanding the mechanisms of insecticide resistance is a key step in dealing with the phenomenon. Insect cuticle is recently documented to delay xenobiotic penetration which breaks the previous stereotype that cuticle is useless in insecticide resistance, while the underlying mechanism remains scarce. RESULTS: Here, we find the integument contributes over 40.0% to insecticide resistance via different insecticide delivery strategies in oriental fruit fly. A negative relationship exists between cuticle thickening and insecticide penetration in resistant/susceptible, also in field strains of oriental fruit fly which is a reason for integument-mediated resistance. Our investigations uncover a regulator of insecticide penetration that miR-994 mimic treatment causes cuticle thinning and increases susceptibility to malathion, whereas miR-994 inhibitor results in opposite phenotypes. The target of miR-994 is a most abundant cuticle protein (CPCFC) in resistant/susceptible integument expression profile, which possesses capability of chitin-binding and influences the cuticle thickness-mediated insecticide penetration. Our analyses find an upstream transcriptional regulatory signal of miR-994 cascade, long noncoding RNA (lnc19419), that indirectly upregulates CPCFC in cuticle of the resistant strain by sponging miR-994. Thus, we elucidate the mechanism of cuticular competing endogenous RNAs for regulating insecticide penetration and demonstrate it also exists in field strain of oriental fruit fly. CONCLUSIONS: We unveil a regulatory axis of lnc19419 ~ miR-994 ~ CPCFC on the cuticle thickness that leads to insecticide penetration resistance. These findings indicate that competing endogenous RNAs regulate insecticide resistance by modulating the cuticle thickness and provide insight into the resistance mechanism in insects.

Functional analysis of an overexpressed glutathione S-transferase BdGSTd5 involved in malathion and malaoxon detoxification in Bactrocera dorsalis.

Glutathione S-transferases (GSTs) are one of the three detoxification enzyme families. The constitutive and inducible overexpression of GSTs genes plays an important role in insecticide resistance. Previous study showed that malathion resistance was polygenic, and elevated GSTs activity was one of the important factor participating in malathion resistance of Bactrocera dorsalis (Hendel), a serious economic pest worldwide. BdGSTd5 overexpression was inducible upon exposure to malathion. However, the involvement of BdGSTd5 in malathion resistance has not been clarified. In this study, we found that BdGSTd5 sequence harbored the conserved region of delta class GSTs, which were overexpressed in malathion resistant strain of B. dorsalis compared to malathion susceptible strain. The highest mRNA expression level of BdGSTd5 was found in 1-day-old adult, and the levels decreased with aging. The dsBdGSTd5 injection effectively silenced (73.4% reduction) the expression of BdGSTd5 and caused significant increase in susceptibility to malathion with a cumulative mortality increasing of 13.5% at 72 h post malathion treatment (p < 0.05). Cytotoxicity assay demonstrated that BdGSTd5 was capable of malathion detoxification. Molecular docking analysis further indicated the interactive potential of BdGSTd5 with malathion and its toxic oxide malaoxon. The recombinant BdGSTd5 exhibited glutathione-conjugating activity toward 1-chloro-2, 4-dinitrobenzene and malathion and malaoxon metabolic capacity with significant reduction (p < 0.05) of the peak areas by 90.0% and 73.1%, respectively. Taken together, the overexpressed BdGSTd5 contributes to malathion metabolism and resistance, which detoxify the malathion in B. dorsalis via directly depleting malathion and malaoxon.

Mutations of voltage-gated sodium channel contribute to pyrethroid resistance in Panonychus citri.

Insecticide resistance in Panonychus citri is a major obstacle to mite control in citrus orchards. Pyrethroid insecticides are continually used to control mites in China, although resistance to pyrethroids has evolved in some populations. Here, the resistance to the pyrethroid fenpropathrin was investigated and 7 out of 8 field-collected populations of P. citri exhibited a high level of resistance, ranging from 171-fold to 15 391-fold higher than the susceptible (SS) comparison strain. Three voltage-gated sodium channel (VGSC) mutations were identified in the tested populations: L1031V, F1747L, and F1751I. Amplicon sequencing was used to evaluate the frequency of these mutations in the 19 field populations. L1031V and F1751I were present in all populations at frequencies of 11.6%-82.1% and 0.5%-31.8%, respectively, whereas the F1747L mutation was only present in 12 populations from Chongqing, Sichuan, Guangxi, and Yunnan provinces. Introduction of these mutations singly or in combination into transgenic flies significantly increased their resistance to fenpropathrin and these flies also exhibited reduced mortality after exposure to the pyrethroids permethrin and ß-cypermethrin. Panonychus citri VGSC homology modeling and ligand docking indicate that F1747 and F1751 form direct binding contacts with pyrethroids, which are lost with mutation, whereas L1031 mutation may diminish pyrethroid effects through an allosteric mechanism. Overall, the results provide molecular markers for monitoring pest resistance to pyrethroids and offer new insights into the basis of pyrethroid actions on sodium channels.

An Antenna-Abundant Glutathione S-Transferase BdGSTd8 Participates in Detoxification of Two Organophosphorus Insecticides in Bactrocera dorsalis (Hendel).

The oriental fruit fly, Bactrocera dorsalis, is a damaging insect pest for many vegetable and fruit crops that has evolved severe chemical insecticide resistance, including organophosphorus, neonicotinoid, pyrethroid, and macrolides. Hence, it is important to elucidate its detoxification mechanism to improve its management and mitigate resource destruction. Glutathione S-transferase (GST) is a critical secondary phase enzyme that plays multiple detoxification functions against xenobiotics. In this study, we identified several BdGSTs by characterizing their potential relationships with five insecticides using inducible and tissue-specific expression pattern analyses. We found that an antenna-abundant BdGSTd8 responded to four different classes of insecticides. Subsequently, our immunohistochemical and immunogold staining analysis further confirmed that BdGSTd8 was primarily located in the antenna. Our investigations also confirmed that BdGSTd8 possesses the capability to enhance cell viability by directly interacting with malathion and chlorpyrifos, which clarified the function of antenna-abundant GST in B. dorsalis. Altogether, these findings enrich our understanding of GST molecular characteristics in B. dorsalis and provide new insights into the detoxification of superfluous xenobiotics in the insect antenna.

Identification, expression profiles and involvement in insecticides tolerance and detoxification of carboxylesterase genes in Bactrocera dorsalis.

Carboxylesterases (CarEs) are a multifunctional superfamily of enzymes and play an important role in detoxification of various insecticides in insects. The oriental fruit fly, Bactrocera dorsalis, is one of the most destructive agricultural pests and has developed different degrees of resistance to organophosphates in field. However, the involvement of BdCarEs in tolerance or resistance to other alternative insecticides are still unclear. In the present study, 33 BdCarEs genes were identified based on the genome database of B. dorsalis. Phylogenetic analysis demonstrated that they were classified into nine clades, with abundance of α-esterases. Meanwhile, the sequence characterization and the chromosome distribution were also analyzed. The spatiotemporal expression analysis of BdCarEs genes suggested that the diversity of potential function in different physiological processes. With the exception of BdCarE21, all BdCarEs genes responded to at least one insecticide exposure, and BdCarE20 was found to be up-regulated after exposure to all five tested insecticides individually. Eight BdCarEs genes were overexpressed in MR strain when compared to that in SS strain. Subsequently, knockdown the expression of representative BdCarEs genes significantly increased the susceptibility of the oriental fruit fly to corresponding insecticides, which indicated that the tested BdCarEs genes contributed to one or multiple insecticide detoxification. These findings provide valuable insights into the potential role in respond to tolerance or resistance to insecticides with different mode of action, and will facilitate development of efficiency management strategy for B. dorsalis.

Sublethal and transgenerational effects of pyridaben exposure on the fitness and gene expression of Panonychus citri.

BACKGROUND: Panonychus citri is a globally dominant citrus plant pest mite. Pesticide-induced population resurgence is a concern for mite control. Exposure to sublethal concentrations of pesticides has stimulated reproduction and outbreak risks in many pests. Pyridaben, a mitochondrial electron transport inhibitor, has been frequently used worldwide in mite control. In the study, sublethal and transgenerational effects of pyridaben exposure on Pyr_Rs (resistant) and Pyr_Control (susceptible) strains were systematically investigated in both exposed parental generation (F0 ) and unexposed offspring generations (F1 and F2 ) by evaluating life-table and physiological parameters. RESULTS: After exposure to pyridaben, the fecundity of both strains was significantly reduced in F0 generation while significantly induced in F1 generation. Interestingly, these effects also stimulated the fecundity of the F2 generation in Pyr_Control strain while no significant effects occurred for Pyr_Rs strain. The intrinsic rate of increase (r) and finite rate of increase (λ) were significantly decreased only in F1 generation of Pyr_Control strain after exposure treatment. Meanwhile, the population projection indicated a smaller population size in F1 generation of Pyr_Control strain while a population increase for Pyr_Rs strain after sublethal treatment. Subsequent detoxification enzyme assays indicated that only P450 activities in F0 generation were significantly activated by LC30 exposure to pyridaben in both strains. Significant downregulation of reproduction-related (Pc_Vg) genes was observed in the F0 generations of both strains. Significant upregulation of P450 (CYP4CL2) and Pc_Vg of the F1 generation in both strains suggested the presence of delayed hormesis effects on the reproduction and developed tolerance to pyridaben, although the effects did not last over a longer period (F2 generation). CONCLUSION: These results provide evidence for transgenerational hormesis effects of low concentrations of pyridaben exposure that may lead to population increase and resurgence risks of resistant-mites in natural settings by stimulating reproduction. © 2023 Society of Chemical Industry.

Resistance of Panonychus citri (McGregor) (Acari: Tetranychidae) to pyridaben in China: monitoring and fitness costs.

BACKGROUND: Panonychus citri is a major citrus pest worldwide. The short life cycle and high reproductive potential of P. citri, combined with heavy acaricide use, have led to high levels of resistance to acaricides, posing a threat to global resistance management programs. Here, resistance monitoring was established to determine the pyridaben resistance status of ten P. citri populations in China from 2014 to 2021 using a leaf-dipping assay. Four characterized strains-the susceptible strain (Lab_S), the resistant strain (Pyr_R), as well as the segregated resistant strain (Pyr_Rs) and the segregated susceptible strain (Pyr_Control) derived from the crossing of the Lab_S and Pyr_R strains, were used to evaluate the life-history characteristics using age-stage, two-sex life tables. RESULTS: Most P. citri populations developed high resistance to pyridaben. Resistance levels exceeded 1000-fold in Yuxi, Anyue, Nanning, and Ganzhou populations compared with the Lab_S strain. Compared with Pyr_Control, two key fitness cost criteria, developmental period and fecundity, showed significant differences in Pyr_Rs under consistent conditions. The intrinsic rate of increase, net reproductive rate and gross reproductive rate were lower in the resistant strain compared with the Pyr_Control strain. The Pyr_Rs strain had a lower relative fitness of 0.934 compared with the Pyr_Control. Moreover, the life-history traits and population parameters of the Pyr_R strain also showed significant differences compared with the Lab_S strain. CONCLUSION: The resistance levels to pyridaben varied greatly among the different P. citri populations and showed regional differences. Substantial fitness costs are associated with pyridaben resistance. This study provides potential implications for developing strategies for resistance management in P. citri. © 2022 Society of Chemical Industry.

Identification and characterization of UDP-glycosyltransferase genes and the potential role in response to insecticides exposure in Bactrocera dorsalis.

BACKGROUND: The oriental fruit fly, Bactrocera dorsalis (Hendel) is a worldwide pest damaging a wide range of hosts. Due to the long-term indiscriminate use of insecticides, B. dorsalis has developed serious resistance to several insecticides. UDP-glycosyltransferases (UGTs) are secondary metabolic enzymes involved in biotransformation and play an important role in the metabolism of plant secondary metabolites and synthetic insecticides in insects. Thus, we suspect that UGTs in B. dorsalis play an important role in insecticide tolerance. RESULTS: In this study, 31 UGT genes were identified in the genome of B. dorsalis, belonging to 13 subfamilies. Real-time quantitative polymerase chain reaction (RT-qPCR) results revealed that 12 UGT genes were highly expressed in the antennae, midgut, Malpighian tubule and fat body. The mRNA expressions of 17 UGT genes were up-regulated upon exposure to λ-cyhalothrin, imidacloprid, abamectin and chlorpyrifos. Knockdown of the selected five UGT genes (BdUGT301D2, BdUGT35F2, BdUGT36K2, BdUGT49D2, BdUGT50B5) by RNA interference increased the mortality of B. dorsalis from 9.29% to 27.22% upon exposure to four insecticides. CONCLUSION: The abundance of UGTs in B. dorsalis is similar to other insect species, and 12 out of 31 UGTs were specifically expressed in metabolic tissues, suggesting a key role in detoxification. Down-regulation of five selected UGT genes increased the susceptibility of B. dorsalis to various insecticides, indicating that UGTs may play an important role in tolerance of B. dorsalis to multiple insecticides. © 2022 Society of Chemical Industry.

RNAi-mediated knockdown of juvenile hormone acid O-methyltransferase disrupts larval development in the oriental fruit fly, Bactrocera dorsalis (Hendel).

The oriental fruit fly, Bactrocera dorsalis (Hendel), is a notoriously agricultural pest that causes serious economic losses to fruits and vegetables. Widespread insecticide resistance in B. dorsalis is a major obstacle in successful control. Therefore, new pest control strategies, such as those targeting specific genes that can block pest development, are urgently needed. In the current study, the function of JHAMT in B. dorsalis was systematically investigated. A methyltransferase gene in B. dorsalis (BdJHAMT) that is homologous to JHAMT of Drosophila melanogaster was cloned firstly. The subsequently spatiotemporal expression analysis indicated that BdJHAMT mRNA was continuously present in the larval stage, declined sharply immediately before pupation, and then increased in the adult. Subcellular localization showed that BdJHAMT was localized in the adult corpora allata and larval intestinal wall cells. The JH III titer in B. dorsalis was closely related to the transcription level of BdJHAMT in different developmental stages. The dsBdJHAMT feeding-based RNAi resulted in a greatly decreased JH III titer that disrupted fly development. The slow growth caused by BdJHAMT silencing was partially rescued by application of the JH mimic, methoprene. These results demonstrated that BdJHAMT was crucial for JH biosynthesis and thus regulated larval development in B. dorsalis, indicating it may serve as a prospective target for the development of novel control strategies against this pest.

Knockdown of a Nicotinic Acetylcholine Receptor Subunit Gene Bdorß1 Decreases Susceptibility to Oxa-Bridged trans- instead of cis-Nitromethylene Neonicotinoid Insecticides in Bactrocera dorsalis.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate the fast action of acetylcholine in synaptic cholinergic transmissions. Insect nAChRs are the target of several classes of insecticides. Here, the full-length cDNA encoding a nAChR beta1 subunit (Bdorß1) was identified and characterized from a destructive pest, Bactrocera dorsalis. The amino acid sequence of Bdorß1 shows high identities to other insect nAChRs ß1 subunits. Double injection of dsBdorß1 reduced the expression of Bdorß1 and in turn significantly decreased susceptibility to oxa-bridged trans- instead of cis-nitromethylene neonicotinoids. Our results support the involvement of Bdorß1 in the susceptibility of B. dorsalis to oxa-bridged trans- instead of cis-nitromethylene neonicotinoids and imply that these two classes of neonicotinoids might be acting at different nAChR subtypes.

miR-309a is a regulator of ovarian development in the oriental fruit fly Bactrocera dorsalis.

Fecundity is arguably one of the most important life history traits, as it is closely tied to fitness. Most arthropods are recognized for their extreme reproductive capacity. For example, a single female of the oriental fruit fly Bactrocera dorsalis, a highly invasive species that is one of the most destructive agricultural pests worldwide, can lay more than 3000 eggs during its life span. The ovary is crucial for insect reproduction and its development requires further investigation at the molecular level. We report here that miR-309a is a regulator of ovarian development in B. dorsalis. Our bioinformatics and molecular studies have revealed that miR-309a binds the transcription factor pannier (GATA-binding factor A/pnr), and this activates yolk vitellogenin 2 (Vg 2) and vitellogenin receptor (VgR) advancing ovarian development. We further show that miR-309a is under the control of juvenile hormone (JH) and independent from 20-hydroxyecdysone. Thus, we identified a JH-controlled miR-309a/pnr axis that regulates Vg2 and VgR to control the ovarian development. This study has further enhanced our understanding of molecular mechanisms governing ovarian development and insect reproduction. It provides a background for identifying targets for controlling important Dipteran pests.

CYP4G100 contributes to desiccation resistance by mediating cuticular hydrocarbon synthesis in Bactrocera dorsalis.

The oriental fruit fly Bactrocera dorsalis (Hendel) is expanding its distribution to higher latitudes. Our goal in this study was to understand how B. dorsalis adapts to higher latitude environments that are more arid than tropical regions. Cuticular hydrocarbons (CHCs) on the surface of the epicuticle in insects act as a hydrophobic barrier against water loss. The essential decarbonylation reaction in CHC synthesis is catalysed by CYP4G, a cytochrome P450 subfamily protein. Hence, in B. dorsalis it is necessary to clarify the function of the CYP4G gene and its role in desiccation resistance. CYP4G100 was identified in the B. dorsalis genome. The complete open reading frame (ORF) encodes a CYP4 family protein (552 amino acid residues) that has the CYP4G-specific insertion. This CYP4G gene was highly expressed in adults, especially in the oenocyte-rich peripheral fat body. The gene can be induced by desiccation treatment, suggesting its role in CHC synthesis and waterproofing. Silencing of CYP4G100 resulted in a decrease of CHC levels and the accumulation of triglycerides. It also increased water loss and resulted in higher desiccation susceptibility. CYP4G100 is involved in hydrocarbon synthesis and contributes to cuticle waterproofing to help B. dorsalis resist desiccation in arid environments.

RNA interference of Argonaute-1 delays ovarian development in the oriental fruit fly, Bactrocera dorsalis (Hendel).

BACKGROUND: With the development of rapid resistance, new modes of action for pesticides are needed for insect control, such as RNAi-based biopesticides targeting essential genes. To explore the function of Argonaute-1 (Ago-1) and potential miRNAs in ovarian development of Bactrocera dorsalis, an important agricultural pest, and to develop a novel control strategy for the pest, BdAgo-1 was first identified in B. dorsalis. RESULTS: Spatiotemporal expression analysis indicated that BdAgo-1 had a relatively high transcriptional level in the ovarian tissues of adult female B. dorsalis during the sexual maturation period. RNA interference (RNAi) experiment showed that BdAgo-1 knockdown significantly decreased the expression levels of ovarian development-related genes and delayed ovarian development. Although RNAi-mediated silencing of Ago-1 led to a reduced ovary surface area, a subsequent oviposition assay revealed that the influence was minimal over a longer time period. Small RNA libraries were constructed and sequenced from different ovarian developmental stages of B. dorsalis adults. Among 161 identified miRNAs, 84 miRNAs were differentially expressed during the three developmental stages of the B. dorsalis ovary. BdAgo-1 silencing caused significant down-regulation of seven differentially expressed miRNAs (DEMs) showing relatively high expression levels (>1000 TPM (Transcripts per kilobase of exon model per million mapped reads)). The expression patterns of these seven core DEMs and their putative target genes were analyzed in the ovaries of B. dorsalis. CONCLUSION: The results indicate that Ago-1 and Ago-1-dependent miRNAs are indispensable for normal ovarian development in B. dorsalis and help identify miRNA targets useful for control of this pest.

Genome-wide identification of long non-coding RNAs (lncRNAs) associated with malathion resistance in Bactrocera dorsalis.

BACKGROUND: Long non-coding RNAs (lncRNAs) play important roles in the regulation of biological processes and have been identified in many species including insects. However, the association between lncRNAs and pesticide resistance in insect species such as Bactrocera dorsalis is unknown. RESULTS: RNA-seq was performed on malathion resistant (MR1) and susceptible (MS) strains of B. dorsalis and a total of 6171 lncRNAs transcripts were identified. These included 3728 lincRNAs, 653 antisense lncRNAs, 1402 intronic lncRNAs, and 388 sense lncRNAs. A total of 40 and 52 upregulated lncRNAs were found in females and males of the MR1 strain compared to 54 and 49 in the same sexes of the MS strain, respectively. Twenty-seven of these lncRNAs showed the same trend of expression in both females and males in the MR1 strain, in which 15 lncRNAs were upregulated and 12 were downregulated. RT-qPCR results indicated that the differentially expressed lncRNAs were associated with malathion resistance. The lnc15010.10 and lnc3774.2 were highly expressed in the cuticle of the MR1 strain, indicating that these two lncRNAs may be related to malathion resistance. RNAi of lnc3774.2 and a bioassay showed that malathion resistance was possibly influenced by changes in the B. dorsalis cuticle. CONCLUSION: LncRNAs of B. dorsalis potentially related to the malathion resistance were identified. Two lncRNAs appear to influence malathion resistance via modulating the structure, or components, of the cuticle. © 2021 Society of Chemical Industry.

NADPH-cytochrome P450 reductase mediates the susceptibility of Asian citrus psyllid Diaphorina citri to imidacloprid and thiamethoxam.

BACKGROUND: The Asian citrus psyllid Diaphorina citri has developed high levels of resistance to many insecticides, and understanding its resistance mechanism will aid in the chemical control of this species. Nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) is crucial in cytochrome P450 function, and in some insects CPR knockdown has increased their susceptibility to insecticides. However, the CPR from D. citri has not been characterized and its function is undescribed. RESULTS: The CPR gene of D. citri (DcCPR) was cloned and sequenced. The expression level of DcCPR, determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analysis, was highest in the midgut and in nymphs. After feeding on double-stranded RNA for 72 h, the DcCPR messenger RNA level in D. citri adults decreased by 68.4%, and the susceptibility of D. citri to imidacloprid and thiamethoxam significantly increased. Meanwhile, after DcCPR silencing, the specific activities of DcCPR protein and P450s were significantly reduced by 41.6% and 44.7%, respectively. The subsequent western blot analysis and quantification of band intensity also showed that DcCPR content significantly decreased, consistent with the results of the specific activity test. In a eukaryotic expression assay, the viability of cells expressing DcCPR was significantly higher than the viability of cells expressing green fluorescent protein (GFP) when cells were exposed to imidacloprid or thiamethoxam. CONCLUSION: These results indicate that DcCPR contributes to D. citri susceptibility to imidacloprid and thiamethoxam.