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Cytochrome P450 monooxygenases (CYPs) play essential physiological functions in insects. CYP303A1 is highly conserved in insect species studied to date, and shows an indispensable role for adult eclosion in both Locusta migratoria and Drosophila melanogaster. However, how CYP303A1 is regulated to control insect developmental process remains uninvestigated. In this study, we discovered functional binding sites for miR-184 in the coding sequence of LmCYP303A1. The luciferase reporter assay showed that miR-184 could target LmCYP303A1 and regulate its expression in vitro. Furthermore, overexpression of miR-184 through microinjection of agomir to locusts reduced the transcripts of LmCYP303A1 and led to the abnormal molting, which is similar to the phenotype of silencing LmCYP303A1 by direct injection of dsLmCYP303A1 to locusts. Meanwhile, down regulation of miR-184 by injection of antagomir increased the LmCYP303A1 transcript and caused molting defects. These findings suggested that miR-184 could target LmCYP303A1 to regulate the molting process in L. migratoria, which might be considered as a novel target for pest control. This article is protected by copyright. All rights reserved.

BACKGROUND Knickkopf (Knk) proteins play crucial roles in the formation of insect cuticle. Recent studies in the holometabolous insect red flour beetle (Tribolium castaneum) have shown that three Knk genes encoding TcKnk, TcKnk2 and TcKnk3 play different but essential roles at different developmental stages and in different tissues. However, the functions of Knk genes had not been fully explored in hemimetabolous insects such as the migratory locust Locusta migratoria. RESULTS We identified three transcripts of LmKnk-like genes LmKnk2 and LmKnk3 with the full-length cDNA sequences, which were named as LmKnk2, LmKnk3-FL and LmKnk3-5'. These three transcripts were highly expressed before molting and mainly expressed in the integument. Among these genes, silencing only LmKnk3-5' by RNA interference (RNAi) caused molting defects and high mortality of the locusts. Injection of dsLmKnk3-5' dramatically decreased chitin content, but did not affect cuticle laminar ultra-structures in the integument. After the knockdown of LmKnk3-5' transcript, lipid deposition on the cuticle surface was impeded, and locusts exhibited increased susceptibility to each of four insecticides in three different classes. However, no visible phenotypic changes were observed after LmKnk2 or LmKnk3-FL was silenced by RNAi. CONCLUSION We demonstrate that LmKnk3-5' is essential for cuticle formation in L. migratoria. This contrasts the findings that the cognate protein in T. castaneum TcKnk3-5' is dispensable for cuticle formation and survival. Hence, we provide some evidence that the function of Knk-type proteins may be species-specific. We therefore think that LmKnk3-5' may be a good target for the application of RNAi-based technologies for species-specific insect pest management. © 2020 Society of Chemical Industry.

Background The aryl hydrocarbon receptor (AhR) belongs to the bHLH-PAS (basic Helix-Loop-Helix - Period/ARNT/Single minded) family of transcription factors. AhR is a ligand-activated transcription factor, which participates in the sensing and transmitting stimuli of endogenous and exogenous chemicals, and subsequently activates the transcription of genes related to various physiological and detoxification functions. Result In this study, a single full-length LmAhR sequence was cloned and characterized. RNA interference (RNAi) and insecticide bioassays showed that LmAhR plays a vital role in chlorpyrifos susceptibility. To better identify aryl hydrocarbon receptor from locusta migratoria (LmAhR)-regulated genes involved in chlorpyrifos susceptibility, a comparative transcriptome analysis was performed using double-stranded (ds)GFP- and dsLmAhR-injected Locusta migratoria. Differential gene expression analysis identified 145 down-regulated and 67 up-regulated genes (P ≤ 0.05 and fold change ≥2) in dsLmAhR-knockdown insects. We selected 27 down-regulated genes and verified their expression levels using reverse transcription quantitative PCR. Finally, one glutathione S-transferase (GST) gene (LmGSTd7) was selected as a candidate detoxification gene and was further validated via RNAi and chlorpyrifos bioassays. Conclusion Our data suggest that AhR is associated with chlorpyrifos susceptibility via the regulation of LmGSTd7 expression in L. migratoria. © 2019 Society of Chemical Industry.

Cells produce an extracellular matrix (ECM) with a stereotypic organization that is important for tissue function. The insect cuticle is a layered ECM that mainly consists of the polysaccharide chitin and associated proteins adopting a quasi-crystalline structure. Our understanding of the molecular mechanisms deployed during construction of the highly ordered protein-chitin ECM so far is limited. In this study, we report on the role of the chitin deacetylase 1 (LmCDA1) in the organization of the protein-chitin ECM in the migratory locust Locusta migratoria, and LmCDA1 localizes predominantly to the apical tier of the protein-chitin ECM, but it is also found in lower regions. Reduction of LmCDA1 function correlates with lower amounts of chitin and impedes conversion of chitin to chitosan by deacetylation. Establishment of the quasi-crystalline architecture of the protein-chitin ECM is, however, independent of LmCDA1 activity, but it is dependent on another chitin deacetylase, LmCDA2, which has no detectable effects on chitin deacetylation and, as shown previously, no influence on chitin content. Our data reveal that LmCDA1 and LmCDA2 act in parallel and independently from each other in defining the dimensions of the cuticle. Both enzymes are non-uniformly distributed within the protein-chitin matrix, suggesting a site-autonomous function.

Purified Brazilin from Sappan wood extract has been reported with significant antitumor effect, especially on human T24 cells and bladder cancer mouse models. Brazilin can significantly induce expression of c-Fos and GADD45β and transfection expression of c-Fos and GADD45β in T24 cells can induce significant cell morphology changes, reduced viability and cell death, while transfection of siRNA-c-Fos and siRNA-GADD45β can reverse the induced cell death. Co-transfection of both c-Fos and GADD45β into T24 cells resulted in a significantly additive effect when compared to single transfection with only c-Fos or GADD45β. Meanwhile, transfection of interfering siRNA-c-Fos or siRNA-GADD45β can partially rescue the cell viability and siRNA co-transfection showed increased rescue rate. The transfection expression and interference with pcDNA3.1-c-Fos/siRNA-c-Fos or pcDNA3.1-GADD45β/siRNA-GADD45β did not affect each other's expression. Moreover, analysis of c-Fos and GADD45β regulated genes and signal pathways showed that no common regulated genes or pathways were present. All the results indicated that c-Fos and GADD45β mediate independent Brazilin-inducible genes and pathways. © 2018 IUBMB Life, 70(11):1101-1110, 2018.

In Locusta migratoria, we found that two chitin biosynthesis genes, UDP N-acetylglucosamine pyrophosphorylase gene LmUAP1 and chitin synthase gene LmCHS1, are expressed mainly in the integument and are responsible for cuticle formation. However, whether these genes are regulated by 20-hydroxyecdysone (20E) is still largely unclear. Here, we showed the developmental expression pattern of LmUAP1, LmCHS1 and the corresponding 20E titer during the last instar nymph stage of locust. RNA interference (RNAi) directed toward a common region of the two isoforms of LmEcR (LmEcRcom) reduced the expression level of LmUAP1, while there was no difference in the expression of LmCHS1. Meantime, injection of 20E in vivo induced the expression of LmUAP1 but not LmCHS1. Further, we found injection-based RNAi of LmEcRcom resulted in 100% mortality. The locusts failed to molt with no apolysis, and maintained in the nymph stage until death. In conclusion, our preliminary results indicated that LmUAP1 in the chitin biosynthesis pathway is a 20E late-response gene and LmEcR plays an essential role in locust growth and development, which could be a good potential target for RNAi-based pest control.

The nuclear receptor-mediated 20-hydroxyecdysone (20E) signalling pathway plays crucial roles in insects by initiating and regulating moulting and metamorphosis. In the present study, we identified and characterized a cDNA encoding a putative nuclear receptor protein (Locusta migratoria hormone receptor 39, LmHR39) based on L. migratoria transcriptomics data. Reverse-transcription quantitative PCR (RT-qPCR) revealed that LmHR39 shows low-level expression in the early days of fifth-instar nymphs, and peak expression occurs on day 5, which is followed by a decrease before ecdysis. LmHR39 transcription could be induced by 20E in vivo and was significantly suppressed by knocking down the expression of the L. migratoria ecdysone receptor gene and early-late gene LmHR3. After RNA interference of LmHR39 with double-stranded RNA (dsRNA), 85% of the insects showed abnormal morphology, with curly wings after moulting and delayed eclosion time. Haematoxylin and eosin staining indicated that apolysis of the integument and wing pad cuticle in the dsLmHR39-treated insects was delayed compared to that in the dsRNA for green fluorescent protein-injected control. Furthermore, RNA-sequencing and RT-qPCR analysis showed the expression level of carboxypeptidase genes (Carboxypeptidase A (CPA) and Carboxypeptidase M (CPM)) and chitin degrading genes (LmChitinase5 (LmCHT5) and LmChitinase10 (LmCHT10)) dramatically declined in the dsLmHR39-treated insects, implying that the LmHR39-mediated 20E signalling pathway is involved in the regulation of carboxypeptidase genes (CPA and CPM) and chitinase genes (LmCHT5 and LmCHT10), and participated in apolysis of the integument and wing pads during locust moulting.

Background Many insect cytochrome P450s (CYPs) play critical roles in detoxification of insecticides. The CYP6 family is unique to the class Insecta, and its biochemical function has essentially been associated with the metabolism of xenobiotics. In this study, we sequenced and characterised the full-length cDNAs of five CYP genes from Locusta migratoria, a highly destructive agricultural pest worldwide. Results The five genes were predominantly expressed in brain, guts, fat bodies or Malpighian tubules. CYP6FE1, CYP6FF1 and CYP6FG1 were expressed at higher levels in fourth-instar nymphs than in other developmental stages. CYPFD2 is specifically expressed in adults, whereas CYP6FD1, CYP6FD2 and CYP6FE1 showed significantly lower expression in eggs than in other developmental stages. Deltamethrin suppressed CYP6FD1 expression in third-instar nymphs and upregulated the expression level of CYP6FD2, CYP6FF1 and CYP6FG1 at the dose of LD10 . Efficient RNA interference-mediated gene silencing was established for four of the five CYP genes. Silencing of CYP6FF1 increased the nymphal mortality from 23 to 50% in response to deltamethrin. Silencing of CYP6FD2 and CYP6FE1 increased the nymphal mortality from 32 to 72 and 66%, respectively, to carbaryl. Conclusion Three of the four CYP6F subfamily genes in L. migratoria were associated with the detoxification of deltamethrin or carbaryl. The role of CYPs in insecticide detoxification appears to be both gene and insecticide specific. © 2016 Society of Chemical Industry.

Background Management of the adverse effects of chemotherapy is essential to improve outcome of children with leukemia. Some genetic polymorphisms can predict treatment-related toxicity, and be used individually in dose modification of 6-mercaptopurine (6-MP) and methotrexate (MTX) in maintenance therapy for childhood acute lymphoblastic leukemia (ALL). We investigated associations between clinical course and candidate gene polymorphisms less evaluated in Japanese patients. Methods Fifty-three children who received maintenance chemotherapy were enrolled in this study. The scheduled dose of oral 6-MP was 40 mg/m2 daily and that of oral MTX was 25 mg/m2 weekly. The doses were adjusted according to white blood cell count (target range, 2.5–3.5 × 109/L) and aspartate aminotransferase and alanine aminotransferase level ( C than in the patients with wild homozygous genotype. The other analyzed polymorphisms were not associated with toxicity, 6-MP, or MTX dose. Conclusions Polymorphism of SLCO1B1 c.521 T > C could be a strong predictor of 6-MP dose reduction in maintenance chemotherapy in childhood ALL.

β-N-acetylglucosaminidases are crucial enzymes involved in chitin degradation in insects. We identified a β-N-acetylglucosaminidase gene (LmNAG1) from Locusta migratoria. The full-length complementary DNA (cDNA) of LmNAG1 consists of 2 667 nucleotides, including an open reading frame (ORF) of 1 845 nucleotides encoding 614 amino acid residues, and 233- and 589-nucleotide non-coding regions at the 5'- and 3'-ends, respectively. Phylogenetic analysis grouped the cDNA-deduced LmNAG1 protein with the enzymatically characterized β-N-acetylglucosaminidases in group I. Analyses of stage- and tissue-dependent expression patterns of LmNAG1 were carried out by real-time quantitative polymerase chain reaction. Our results showed that LmNAG1 transcript level in the integument was significantly high in the last 2 days of the fourth and fifth instar nymphs. LmNAG1 was highly expressed in foregut and hindgut. RNA interference of LmNAG1 resulted in an effective silence of the gene and a significantly reduced total LmNAG enzyme activity at 48 and 72 h after the injection of LmNAG1 double-stranded RNA (dsRNA). As compared with the control nymphs injected with GFP dsRNA, 50% of the dsLmNAG1-injected nymphs were not able to molt successfully and eventually died. Our results suggest that LmNAG1 plays an essential role in molting process of L. migratoria.

Glutathione S-transferases (GSTs) are an important family of detoxifying enzymes and play a key role in pesticide resistance in the insect. Tyrosine is essential for its detoxification function. In the present study, two conserved tyrosine residues are located at positions 108 and 116 in H-site of LmGSTD1. To elucidate how the two residues participate in the catalytic process and keeping structural stability, four mutants, Y108A, Y108E, Y116A, and Y116E, were generated. It was found that the four mutants affected the specific activity of LmGSTD1 in various degrees, depending on the types of substrate and reaction mechanism. Steady-state kinetics assay revealed that Y108E and Y116E had a significant influence on GSH-binding ability, which indicates the two tyrosine residues of H-site contribute to topology rearrangement of G-site. Both Y116A and Y116E exhibited lower CDNB-binding affinity, suggesting that Y116 takes part in hydrophobic substrate binding. The thermostability assay, intrinsic, and 8-anilino-1-naphthalenesulfonic acid (ANS) florescence results showed that the two tyrosine residues were involved in regulation of active-site conformation. Finally, homology modeling provided evidence that the two tyrosines in H-site participate in hydrophobic substrate binding. Furthermore, Y108 is closer to the S atom of S-hexylglutathione. In conclusion, the two tyrosines in LmGSTD1 are important residues in both the catalytic process and protein stability.

The cytochrome P450 monooxygenase (cytochrome P450) gene superfamily comprises many genes that may be involved in the biotransformations of pesticides and otherxenobiotics.Todate,verylittle isknownaboutcytochromeP450genesintheoriental migratory locust, Locusta migratoria manilensis. In this study, we carried out a genome- wide analysisofcytochrome P450genesofthe locusttoidentify putative cytochrome P450 genesandcharacterizetheirexpressionresponsestoinsecticideexposures.Weidentified15 cytochrome P450-like genes from a locust expressed sequence tag database (LocustDB). Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that most cytochrome P450-like genes displayed different tissue and developmental stage expression patterns. However, most of them were predominantly expressed in the midgut, gastric caeca, fatbodies, and/or hindgut. Biochemical analysis showed that cytochrome P450 was differentially affected by three different insecticides. Deltamethrin caused significant inductions in 12 h at LD30 (dose to kill 30% of the tested individuals) in the nymphs, whereasmalathionandcarbaryldidnothavesignificanteffectoncytochromeP450enzyme activity. Further RT-PCR analysis showed significant increases of transcriptions of several cytochrome P450 genes in deltamethrin-treated locusts. Thus, the increased cytochrome P450 enzyme activity is likely due to increased transcriptions of multiple cytochrome P450 genes in response to deltamethrin exposure. These results are expected to help us better understand the interactions between insecticides and major detoxification enzymes, and possible changes of the susceptibility to other insecticides in deltamethrin-treated insects at various molecular levels.

Synthetic pyrethroids are the primary insecticides that are widely used for controlling Locusta migratoria manilensis (Meyen), a major pest in eastern and southern Asia and the Pacific region. In this paper, ten cDNAs encoding glutathione S-transferases (GSTs) were sequenced and characterised in L. migratoria manilensis. The effects of deltamethrin on the ten GST gene expressions were studied.Phylogenetic analysis revealed nine GSTs in three different classes, including seven in sigma, one in delta and one in theta. The remaining GST (LmGSTu1) was unclassified. RT-PCR analysis showed that most GST genes were expressed in all tissues examined, including the foregut, midgut, gastric caecum, hindgut, Malpighian tubules, fat bodies, muscles, spermaries and ovaries, except that LmGSTs2, LmGSTs4, LmGSTs7 and LmGSTu1 were expressed in several tissues. LmGSTu1 appeared to be the only gene whose expressions could not be detected in eggs. Real-time quantitative PCR showed that deltamethrin at 0.08 and/or 0.12 µg mL⁻¹ increased almost all ten GST gene expressions in third-instar nymph locusts. However, deltamethrin at 0.16 and/or 0.2 µg mL⁻¹ decreased the expressions of LmGSTd1, LmGSTs1, LmGSTs5 and LmGSTs6.The increases in GST gene expressions after deltamethrin exposure in L. migratoria manilensis might result in its elevating tolerance to other insecticides and xenobiotics.

BACKGROUND: Previous studies have indicated that increased caboxylesterase (CarE) activity is a major mechanism of malathion resistance in field populations of the oriental migratory locust, Locusta migratoria manilensis (Meyen), in China. The aim of the present study was to screen CarE-like genes from a large locust expressed sequence tag (EST) database and to assess their potential roles in malathion resistance. RESULTS: Twenty-five ESTs derived from different CarE-like genes in the locust EST database were identified, and 12 candidate genes with significantly increased expressions, ranging from 2.6- to 11.6-fold in a field-derived resistant (FR) colony of the locust, were found. These candidate genes were constitutively expressed in all nymph and adult stages, and most of them were predominantly expressed in the gastric caeca and the midgut. Among the 12 genes, two representative genes (LmCarE9 and LmCarE25) were chosen for RNAi followed by malathion bioassay. The nymph mortalities increased from 34.3 to 65.2 and 54.2% respectively after LmCarE9 and LmcarE25 were silenced. These results indicated significant roles of these CarE-like genes in conferring malathion resistance in the locust. CONCLUSION: Multiple CarE-like genes were involved in malathion resistance in the locust. As validated by RNAi followed by malathion bioassay, LmCarE9 and LmcarE25 played a significant role in conferring malathion resistance.

We evaluate comparative toxicity of malathion in the two populations of the grasshopper Oxya chinensis, collected from Daixian and Fanshi of Shanxi province, China. General esterases and acetylcholinesterase (AChE) from the two populations were charac- terized and compared. LD50 of the Daixian population (7.58 ∝g/g body weight) was 2.02-fold higher than that of the Fanshi population (3.75 ∝g/g body weight). General esterase-specific activities in the Daixian population were 1.91, 1.10 and 1.85-fold higher than those in the Fanshi population, when α-NA, α-NB and β-NA were used as a substrate, respectively. Kinetic studies of general esterase showed that Vmax values of general esterases hydrolyzing α-NA, α-NB and β-NA in the Daixian population were 2.15-, 1.12-, and 1.47-fold, respectively, higher than those in the Fanshi population. The AChE activity of the Fanshi population was 1.54-fold higher than that of the Daixian population. Kinetic analysis of AChE showed that significant differences were presented between the two populations in the Km values; and the Vmax value in the Fanshi population was higher than that in the Daixian population. Inhibition studies of AChE indicated that AChE from the Daixian population was 2.56-, 2.80-, and 2.29-fold less sensitive to inhibition by paraoxon, chlorpyrifos-oxon, and demeton-S-methyl, respectively, than that from the Fanshi population. These biochemi- cal characterizations of general esterases and AChE were consistent with malathion bioassay in the two populations. It is inferred that the reduced sensitivity of altered AChE and increased general esterase activities play an important role in the differences of insuscepti- bility of Oxya chinensis to malathion between the two populations.

The chromosomal conventional karyotype and C-banding karyotype of eight species in the genus Oxya (Catantopidae) are analysed. The result shows that O. chinensis (Thunberg), O. shanghaiensis Willemse and O. adentata Willemse all have similar C-banding distribution, but the model of chromosomal group, the chiasma localization data and the total heterochromatin content (THC) value are different. O. agavisa Tsai has its own c-banding feature and is distinguished from other species in the genus. O. bicingula Ma et al. is similar to both the O. chinensis group and O. agavisu in chromosomal marks and morphological characters, indicating that these three species have some relations during the evolutionary process. O. hyla intricata (Stal) is a “sibling species group” as indicated by the variation of the morphological feature and there are also diversiforms in cytotaxonomic marks. It seems that this group has higher differentiational speed and the speciational evolution is more active in modern times. In this group, O. apicocingula sp. nov. and O. flave femura sp. nov. are more specialized than the other populations which have not many terminal C-bands in genome. As a conclusion, we consider that the evolutionary rate of the species in Oxya genus is unbalanced. This status is due to the actions from both the hereditary basis and the environmental condition of the different species.