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.

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 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.

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.

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.

The adipokinetic hormone signaling system regulates the sensitivity of Bactrocera dorsalis to malathion.

The neuropeptide adipokinetic hormone (AKH) binds to the AKH receptor (AKHR) to regulate carbohydrate and lipid metabolism. It also participates in the insect anti-stress response. We used RT-qPCR to detect the expression levels of 39 neuropeptides in malathion-susceptible (MS) and malathion-resistant (MR) strains of Bactrocera dorsalis. AKH and AKHR were highly expressed in the MR strain. Using a malathion bioassay and RNA interference (RNAi), we demonstrated that AKHR is involved in the susceptibility of B. dorsalis to malathion. We found significantly reduced expression of two detoxification enzyme genes (glutathione-S-transferase, GST and α-esterase, CarE) after AKHR RNAi. Based on our previous data, GSTd10 and CarE6 participate the direct metabolism of malathion in this fly, which is also verified by a malathion metabolism assay by HPLC using the crude enzymes in the current study. These results suggest that AKHR plays an important role in affecting malathion susceptibility via detoxification enzyme genes.

An odorant-binding protein of Asian citrus psyllid, Diaphorina citri, participates in the response of host plant volatiles.

BACKGROUND: Odorant-binding proteins (OBPs) in insects contribute to the sensitivity of the olfactory system and connect external odorants to olfactory receptor neurons. Determination of the chemosensory functions in Diaphorina citri, a vector of the citrus Huanglongbing pathogen, may help in developing a potential target for pest management. RESULTS: Diaphorina citri showed dose-dependent electroantennogram recording (EAG) responses to 12 host plant volatiles. A two-choice behavioral trap experiment showed that four compounds (methyl salicylate, linalool, citral and R-(+)-limonene) that elicited high EAG responses also had significant attraction to adults. The expression profiles induced by these compounds were detected in nine OBP genes, DcitOBP1-9. DcitOBP3, DcitOBP6 and DcitOBP7 commonly showed significant upregulation or downregulation compared with the control. Microscale thermophoresis (MST) showed that the recombinant protein DcitOBP7 had high in vitro binding affinities (Kd < 10 µm) to methyl salicylate, linalool and R-(+)-limonene, and moderate binding affinity to citral with a Kd value of 15.95 µm. Furthermore, RNA interference (RNAi)-suppressed messenger RNA (mRNA) expression of DcitOBP7 resulted in a significant reduction in EAG activity and in adult D. citri behavioral responses to tested volatiles and the preferred host, Murraya paniculata. The hydrophilic residue Arg107 of DcitOBP7 may have a key role in binding odorants via formation of hydrogen bonds. CONCLUSION: These results show that DcitOBP7 plays an important role in the olfactory response. This finding may provide new insight into the functions of OBP families in D. citri and aid in the development of safe strategies for managing D. citri populations. © 2021 Society of Chemical Industry.

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.

Genome-wide and expression-profiling analyses of the cytochrome P450 genes in Bactrocera dorsalis (Hendel) and screening of candidate P450 genes associated with malathion resistance.

BACKGROUND: Bactrocera dorsalis (Hendel) is a notorious agricultural pest worldwide, and its resistance to insecticides is a major obstacle in successful control. Cytochrome P450s (P450s) are major metabolic enzymes associated with insecticide resistance. The genome of B. dorsalis was sequenced recently, allowing an integrated genome-wide analysis of P450 genes (P450s) and the analysis of correlations between these genes and insecticide resistance in this pest. RESULTS: Totally, 101 P450s were identified in the B. dorsalis genome and classified into four clans, 25 families and 57 subfamilies. Quantitative reverse transcription polymerase chain reaction results showed that most of these genes were highly expressed in adults (46) and in metabolic tissues, including the fatbody (63), midgut (61) and Malphagian tubules (66). In a malathion-resistant strain, 13 and 9 genes were significantly upregulated and downregulated, respectively, compared with a susceptible strain, and these genes were screened as candidate genes associated with malathion resistance. CONCLUSION: This study provides useful information for understanding the evolution and potential functions of P450s in B. dorsalis, and the results lay the foundation for further studies on the correlations between P450s and malathion resistance in B. dorsalis. © 2020 Society of Chemical Industry.

A glutathione S-transferase (BdGSTd9) participates in malathion resistance via directly depleting malathion and its toxic oxide malaoxon in Bactrocera dorsalis (Hendel).

BACKGROUND: The oriental fruit fly, Bactrocera dorsalis (Hendel), is a widespread agricultural pest that has evolved resistance to many commonly used insecticides including malathion. Glutathione S-transferases (GSTs) are multifunctional enzymes that metabolize insecticides directly or indirectly. The specific mechanism used by GSTs to confer malathion resistance in B. dorsalis is unclear. RESULTS: BdGSTd9 was identified from B. dorsalis and was expressed at twice the level in a malathion-resistant strain (MR) than in a susceptible strain (MS). By using RNAi of BdGSTd9, the toxicity of malathion against MR was increased. Protein modelling and docking of BdGSTd9 with malathion and malaoxon indicated key amino acid residues for direct binding in the active site. In vitro assays with engineered Sf9 cells overexpressing BdGSTd9 demonstrated lower cytotoxicity of malathion. High performance liquid chromatography (HPLC) analysis indicated that malathion could be broken down significantly by BdGSTd9, and it also could deplete the malathion metabolite malaoxon, which possesses a higher toxicity to B. dorsalis. Taken together, the BdGSTd9 of B. dorsalis could not only deplete malathion, but also react with malaoxon and therefore enhance malathion resistance. CONCLUSION: BdGSTd9 is a component of malathion resistance in B. dorsalis. It acts by depleting both malathion and malaoxon. © 2020 Society of Chemical Industry.

Divergent molecular evolution in glutathione S-transferase conferring malathion resistance in the oriental fruit fly, Bactrocera dorsalis (Hendel).

Insect glutathione S-transferases (GSTs) are important in insecticide detoxification and Insect-specific GSTs, Epsilon and Delta, have largely expanded in insects. In this study, we functionally expressed and characterized an epsilon class GST gene (BdGSTe8), predominant in the adult Malpighian tubules of Bactrocera dorsalis. This gene may be associated with malathion resistance based on transcriptional studies of resistant and susceptible strains. RNA interference-mediated knockdown of this gene significantly recovered malathion susceptibility in the adults of a malathion-resistant strain, and overexpression of BdGSTe8 enhanced resistance in transgenic Drosophila. Analysis of BdGSTe8 polymorphism showed that several point mutations may be associated with metabolic resistance to malathion. A cytotoxicity assay in Escherichia coli indicated that both of the recombinant BdGSTe8 proteins may play a functional role in protecting cells from toxicity. The allele of BdGSTe8-B conferred higher levels of malathion detoxification capability. Liquid chromatography and ultra-performance liquid chromatography-tandem mass spectrometry analysis showed that the BdGSTe8-A allele did not metabolize malathion directly. However, the BdGSTe8-B allele was involved in the direct metabolism of malathion, which was caused by a mutation in V128A. Further analysis of the sequence suggests that BdGSTe8 evolved rapidly. It maybe play the role of a backup gene and could become a new gene in the future in order to retain the ability of detoxification of malathion, which was driven by positive selection. These results suggest that divergent molecular evolution in BdGSTe8 has played a role in metabolic resistance to malathion in B. dorsalis.

Two delta class glutathione S-transferases involved in the detoxification of malathion in Bactrocera dorsalis (Hendel).

BACKGROUND: The oriental fruit fly Bactrocera dorsalis (Hendel), a widespread agricultural pest, has evolved resistance to many insecticides, including organophosphorus compounds. Glutathione S-transferases (GSTs) are involved in xenobiotic detoxification and insecticide resistance in many insects. However, the role of delta class GSTs in detoxifying malathion in B. dorsalis is unknown. Here, we evaluated the roles of two delta class GSTs in malathion detoxification in this species. RESULTS: Two delta class GSTs genes, BdGSTd1 and BdGSTd10, were characterized in B. dorsalis. They were highly expressed in 5-day-old adults, as well as in midgut and Malpighian tubules. Upon malathion exposure, the two genes were upregulated by 2.63- and 2.85-fold, respectively. Injection of double-stranded RNA targeting BdGSTd1 or BdGSTd10 significantly reduced their mRNA levels in adults and also significantly increased adult susceptibility to malathion. The expression of these two GSTs in Escherichia coli helped the host to endure malathion stress at a concentration of 10 µg mL-1 according to a Cell Counting Kit-8 assay. High-performance liquid chromatography analyses indicated that malathion could be significantly depleted by the two delta GSTs. The role of BdGSTd10 in malathion sequestration was also discussed. CONCLUSION: BdGSTd1 and BdGSTd10 play important roles in the detoxification of malathion in B. dorsalis. © 2019 Society of Chemical Industry.

Functional characterization of BdB1, a well-conserved carboxylesterase among tephritid fruit flies associated with malathion resistance in Bactrocera dorsalis (Hendel).

There are many evidences that insect carboxylesterase possess important physiological roles in xenobiotic metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the ongoing resistance development in the oriental fruit fly, Bactrocera dorsalis (Hendel), the molecular basis of carboxylesterase and its ability to confer OP resistance remain largely obscure. This study was initiated to provide a better understanding of carboxylesterase-mediated resistance mechanism in a tephritid pest fly. Here, we narrow this research gap by demonstrating a well-conserved esterase B1 gene, BdB1, mediates malathion resistance development via gene upregulation with the use of a laboratory selected malathion-resistant strain (MR) of B. dorsalis. No sequence mutation of BdB1 was detected between MR and the susceptible strain (MS) of B. dorsalis. BdB1 is predominantly expressed in the midgut, a key insect tissue for detoxification. As compared with transcripts in MS, BdB1 was significantly more abundant in multiple tissues in the MR. RNA interference (RNAi)-mediated knockdown of BdB1 significantly increased malathion susceptibility. Furthermore, heterologous expression along with cytotoxicity assay revealed BdB1 could probably have the function of malathion detoxification.

Functional characterization of an α-esterase gene involving malathion detoxification in Bactrocera dorsalis (Hendel).

Extensive use of insecticides in many orchards has prompted resistance development in the oriental fruit fly, Bactrocera dorsalis (Hendel). In this study, a laboratory selected strain of B. dorsalis (MR) with a 21-fold higher resistance to malathion was used to examine the resistance mechanisms to this organophosphate insecticide. Carboxylesterase (CarE) was found to be involved in malathion resistance in B. dorsalis from the synergism bioassay by CarE-specific inhibitor triphenylphosphate (TPP). Molecular studies further identified a previously uncharacterized α-esterase gene, BdCarE2, that may function in the development of malathion resistance in B. dorsalis via gene upregulation. This gene is predominantly expressed in the Malpighian tubules, a key insect tissue for detoxification. The transcript levels of BdCarE2 were also compared between the MR and a malathion-susceptible (MS) strain of B. dorsalis, and it was significantly more abundant in the MR strain. No sequence mutation or gene copy changes were detected between the two strains. Functional studies using RNA interference (RNAi)-mediated knockdown of BdCarE2 significantly increased the malathion susceptibility in the adult files. Furthermore, heterologous expression of BdCarE2 combined with cytotoxicity assay in Sf9 cells demonstrated that BdCarE2 could probably detoxify malathion. Taken together, the current study bring new molecular evidence supporting the involvement of CarE-mediated metabolism in resistance development against malathion in B. dorsalis and also provide bases on functional analysis of insect α-esterase associated with insecticide resistance.

[Managements of small thyroid nodules with contralateral papillary thyroid microcarcinoma].

OBJECTIVE: To study the diagnoses and treatments of small thyroid nodules (maximum diameter < 1 cm) with contralateral papillary thyroid microcarcinoma (PTMC). METHODS: A total of 253 patients with unilateral PTMC and contralateral thyroid benign nodules identified by ultrasound before thyroidectomy was retrospectively analysed. All patients underwent near-total or total thyroidectomy. Chi-square test was used for univariate analysis and logistic regression test for multivariate analysis. RESULTS: In 53 (20.9%) of 253 patients with unilateral PTMC, the contralateral thyroid benign nodules identified by ultrasound were confirmed pathologically as PTMC. Univariate analysis showed multifocality of the primary tumor and Hashimoto's thyroiditis were correlated with contralateral PTMC (χ(2) = 24.834, χ(2) = 5.182, P < 0.05). However, there were no significant differences for the existence of contralateral PTMC in age, sex, tumor size, capsule invasion, lymph node metastasis, the number of nodules and Tg-level. Multivariate analysis showed only multifocal PTMC was an independent predictive factor for contralateral PTMC (OR = 5.352, P < 0.05). CONCLUSIONS: The patients with unilateral multifocal PTMC have a high rate of PTMC in contralateral small thyroid nodules. However, it is very difficulty to define by ultrasonography preoperatively. The total thyroidectomy maybe serve as a useful treatment.