Unclassified glutathione-S-transferase AiGSTu1 confers chlorantraniliprole tolerance in Agrotis ipsilon.

BACKGROUND: Chlorantraniliprole (CAP) is a diamide insecticide with high efficacy against many pest insects, including the black cutworm, Agrotis ipsilon. Agrotis ipsilon is a serious pest causing significant yield losses in crops. Glutathione-S-transferases (GSTs) belong to a family of metabolic enzymes that can detoxify a wide range of pesticides. However, little is known about the functions of GSTs in CAP tolerance in A. ipsilon. RESULTS: A cDNA sequence (designated AiGSTu1) encoding an unclassified GST was identified from A. ipsilon. AiGSTu1 is highly expressed during the 3rd -instar larval and the pupal stages. Most of the mRNA transcripts were found in larval Malpighian tubules. Exposure to CAP strongly enhanced AiGSTu1 expression, GST activity, hydrogen peroxide (H2 O2 ) and malondialdehyde levels in larvae. H2 O2 treatment upregulated the transcription level of AiGSTu1, suggesting that CAP-induced oxidative stress may activate AiGSTu1 expression. The activity of recombinant AiGSTu1 was inhibited by CAP in a dose-dependent manner. Metabolism assay results demonstrated that AiGSTu1 is capable of depleting CAP. Overexpression of AiGSTu1 enhanced the tolerance of Escherichia coli cells to H2 O2 and the oxidative stress inducer, cumene hydroperoxide. Silencing of AiGSTu1 by RNA interference increased the susceptibility of A. ipsilon larvae to CAP. CONCLUSION: The findings of this study provide valuable insights into the potential role of AiGSTu1 in CAP detoxification and will improve our understanding of CAP tolerance in A. ipsilon. © 2023 Society of Chemical Industry.

Epsilon Class Glutathione S-Transferase Confers Phoxim Tolerance in the Black Cutworm Agrotis ipsilon (Lepidoptera: Noctuidae).

The black cutworm Agrotis ipsilon is a serious crop pest. Phoxim, an organophosphorus insecticide, has been widely used to control A. ipsilon. When phoxim is extensively applied, the susceptibility of A. ipsilon to insecticide is reduced. However, the mechanisms of tolerance of A. ipsilon to phoxim remain unclear. Herein, we report that an epsilon class glutathione S-transferase, AiGSTE1, confers phoxim tolerance in A. ipsilon. Exposure to a sublethal concentration (LC50) of phoxim caused oxidative stress and activated the transcription of AiGSTe1 genes in A. ipsilon larvae. Recombinant AiGSTE1 expressed in Escherichia coli could metabolize phoxim. Furthermore, E. coli cells overexpressing AiGSTE1 displayed significant tolerance to oxidative stress. Knockdown of AiGSTe1 by RNA interference significantly increased the mortality of A. ipsilon larvae to phoxim. These results demonstrate that AiGSTE1 confers phoxim tolerance in A. ipsilon by metabolizing the insecticide and preventing phoxim-induced oxidative stress.

Identification of 14 glutathione S-transferase genes from Lasioderma serricorne and characterization of LsGSTe1 involved in lambda-cyhalothrin detoxification.

In insects, glutathione S-transferases (GSTs) play a pivotal role in the detoxification of a wide range of pesticides. The cigarette beetle, Lasioderma serricorne, is an economically important pest insect of stored products. Recently, pyrethroid insecticides have been used to control this pest. However, little is known concerning the responses and functions of GSTs in L. serricorne under pyrethroid exposure. In this study, transcriptome sequencing was performed on L. serricorne, and a total of 14 GSTs were identified by retrieving the unigene dataset. Of these, 13 predicted GSTs fell into six cytosolic classes, namely, delta, epsilon, omega, sigma, theta, and zeta, and one was assigned to an "unclassified" group. The GST genes were differentially expressed in various larval tissues and at different developmental stages. Exposure to the pyrethroid insecticide lambda-cyhalothrin (LCT) caused oxidative stress in L. serricorne larvae and led to significantly elevated expression levels of six genes, among which LsGSTe1 was the most upregulated. Recombinant LsGSTE1 protein displayed LCT-metabolizing activity. Furthermore, LsGSTE1 protects cells against oxidative stress. Moreover, knockdown of LsGSTe1 by RNA interference dramatically increased the susceptibility of L. serricorne larvae to LCT treatment. The results from this study provide sequence resources and expression data for GST genes in L. serricorne. Our findings indicate that LsGSTE1 plays a dual role in LCT detoxification by metabolizing the pesticide and by preventing LCT-induced oxidative stress. Thus, the LsGSTe1 gene could be used as a potential target for sustainable management of the cigarette beetle.

A thioredoxin peroxidase protects Pieris rapae from oxidative stress induced by chlorantraniliprole exposure.

Chlorantraniliprole (CAP) is an insecticide widely used to control the small white butterfly (SWB), Pieris rapae. Exposure to CAP can cause oxidative injury in SWB; however, it is unclear if antioxidant enzymes are involved in the defense process. In this study, a thioredoxin peroxidase (PrTPX1) gene was identified from SWB by using a homology search method. The gene encoded a 195 amino-acid PrTPX1 protein. Sequence characteristics and phylogenetic analysis indicated that PrTPX1 was a typical "2-Cys" TPX, and the PrTPX1 gene consisted of four exons and three introns. Reverse transcription-quantitative polymerase chain reaction analysis indicated that the messenger RNA levels of PrTPX1 were highest in third-, fourth- and fifth-instar larval stages and in the larval midgut. Treatment with sublethal doses (LD20 and LD50 ) of CAP for 6, 12, 18, and 24 h resulted in increased H2 O2 concentration in SWB larvae, indicating insecticide-induced oxidative stress. The transcriptional levels of PrTPX1 were significantly enhanced in larvae exposed to CAP. Recombinant PrTPX1 protein was expressed in Escherichia coli. Enzymatic assay revealed that the protein displayed antioxidant activity and was able to protect against oxidative challenge. These results indicated that PrTPX1 plays an important role in oxidative stress responses and may contribute to the CAP tolerance in SWB.

[Application of Extracellular Vesicles Derived from Vascular Endothelial/Endothelial Progenitor Cells in Tissue Regeneration and Repair].

Extracellular vesicles can be released by almost all types of cells and are important mediators of intercellular signal transmssion. Extracellular vesicles regulate the function and activity of recipient cells by delivering biologically active molecules such as proteins and nucleic acids, which is of great significance in tissue repair and regeneration. According to numerous studies, extracellular vesicles derived from endothelial/endothelial progenitor cells can induce cell proliferation and differentiation, inhibit cell apoptosis, and promote angiogenesis, playing an increasingly important role in regenerative medicine. We reported in this review the latest findings on applying extracellular vesicles derived from endothelial/endothelial progenitor cells in tissue regeneration and repair, and discussed the challenges and future development directions of their application in the field of regenerative medicine.

A heat shock protein protects against oxidative stress induced by lambda-cyhalothrin in the green peach aphid Myzus persicae.

Lambda-cyhalothrin (LCT) is a pyrethroid insecticide widely used to control insect pests. Insect exposure to LCT may cause abnormal accumulation of reactive oxygen species (ROS) and result in oxidative damage. Heat shock proteins (HSPs) may help protect against oxidative stress. However, little is known about the role of HSPs in response to LCT in the green peach aphid, Myzus persicae. This insect is an important agricultural pest causing severe yield losses in crops. In this study, we characterized a cDNA sequence (MpHsp70) encoding a member of the HSP70 family in M. persicae. MpHsp70 encoded a 623 amino acid protein putatively localized in the cytosol. The highest expression level of MpHsp70 occurred in fourth-instar nymphs. Treatment of M. persicae with LCT resulted in oxidative stress and significantly increased H2O2 and malondialdehyde levels. This led to an elevated transcription level of MpHsp70. Injection of H2O2 into M. persicae also upregulated the MpHsp70 expression level, suggesting that MpHsp70 is responsive to ROS, particularly H2O2, induced by LCT. Recombinant MpHSP70 protein was expressed in Escherichia coli. E. coli cells overexpressing MpHSP70 exhibited significant tolerance to H2O2 and the ROS generators, cumene hydroperoxide and paraquat. This indicated that MpHSP70 protects against oxidative stress. Furthermore, knockdown of MpHsp70 by RNA interference resulted in increased susceptibility in apterous adults of M. persicae to LCT. These findings indicate that MpHsp70 plays an important role in defense against LCT-induced oxidative stress and insecticide susceptibility in M. persicae.

Identification and Functional Analysis of Differentially Expressed Genes in Myzus persicae (Hemiptera: Aphididae) in Response to Trans-anethole.

Plant essential oils, with high bioactivity and biodegradability, provide promising alternatives to synthetic pesticides for pest control. Trans-anethole is the major component of essential oil from star anise, Illicium verum Hook. The compound has a strong contact toxicity against the green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae), which is a major insect pest of many vegetables and crops. However, little information is known about how M. persicae responds to trans-anethole at the molecular level. We conducted a comparative transcriptome analysis of M. persicae in response to a LD50 dose of trans-anethole. A total of 559 differentially expressed genes were detected in the treated individuals, with 318 genes up-regulated, and 241 genes down-regulated. Gene ontology (GO) analysis revealed that these genes were classified into different biological processes and pathways. We also found that genes encoding ATP-binding cassette (ABC) transporters, DnaJ, and cuticle proteins were dramatically up-regulated in response to trans-anethole. To study the function of these genes, we performed RNA interference (RNAi) analysis. Knockdown of an ABC transporter gene (ABCG4) and a DnaJ gene (DnaJC1) resulted in a significantly increased mortality rate in M. persicae following trans-anethole exposure, indicating the involvement of these two genes in the toxicity response to trans-anethole. The findings provide new insights into the mechanisms of M. persicae in coping with plant essential oils.

Molecular characterization of a catalase gene from the green peach aphid (Myzus persicae).

The green peach aphid, Myzus persicae (Sulzer), is a serious agricultural pest with a worldwide distribution. Catalase (CAT), which is encoded by the catalase (Cat) gene, is an extremely important antioxidant enzyme that plays a pivotal role in protecting cells against the toxic effects of hydrogen peroxide. The Cat gene has not been characterized in M. persicae; therefore, this study describes the identification of the Cat (MpCat) gene from M. persicae. MpCat contains an open reading frame of 1515 bp and encodes a MpCAT protein consisting of 504 amino-acid residues. MpCAT possesses features typical of other insect catalases, including 7 conserved amino acids involved in binding heme and 15 involved in binding nicotinamide adenine dinucleotide phosphate. Phylogenetic analysis showed that MpCAT was closely related to orthologs from other aphid species. MpCat consisted of nine exons and eight introns, and the number and insertion sites of introns are consistent with those of Cat genes from Acyrthosiphon pisum (Harris) and Aphis gossypii Glover. The mRNA transcripts of MpCat were detected at all tested developmental stages, with the highest mRNA level in alate adults. The expression of MpCat was significantly upregulated when M. persicae was exposed to low and high temperatures, ultraviolet radiation, Beauveria bassiana, and permethrin. The transcription of MpCat and the activity of catalase were suppressed by RNA interference, and knockdown of MpCat significantly reduced the survival rate in M. persicae under heat stress. The results provide valuable information for further study on the physiological functions of MpCat.

Characterisation of a copper/zinc superoxide dismutase from Pieris rapae and its role in protecting against oxidative stress induced by chlorantraniliprole.

Insecticide exposure typically leads to abnormally high levels of reactive oxygen species (ROS) and oxidative damage in insects. Superoxide dismutases (SODs) are potent antioxidant enzymes for ROS scavenging that are essential to protect insects against insecticide-induced oxidative injury. The small white butterfly, Pieris rapae, is an economically important lepidopteran pest of cruciferous crops, and the anthranilic diamide insecticide chlorantraniliprole is widely used to control this organism. However, whether chlorantraniliprole causes oxidative stress, and whether SODs are involved in ROS scavenging, remains unclear in P. rapae. In this study, an intracellular copper/zinc SOD (designated PrSOD1) gene was identified and characterised in P. rapae. The gene consists of four exons and three introns, and the PrSOD1 protein encoded by the gene has typical highly conserved features of CuZnSODs, including two signature motifs and seven Cu/Zn-interacting residues. Transcription of PrSOD1 was highest in the larval fat body and at the fifth-instar larval stage. Recombinant PrSOD1 protein expressed in Escherichia coli displayed antioxidant activity and high thermal and pH stability, confirming that PrSOD1 encodes a functional enzyme. Exposure to three sublethal doses of chlorantraniliprole for 6, 12 or 24 h resulted in significantly increased malondialdehyde concentration in P. rapae larvae, indicating insecticide-induced oxidative stress. Furthermore, both PrSOD1 transcription levels and CuZnSOD activity were quickly (6 and 12 h, respectively) upregulated in larvae subjected to chlorantraniliprole, strongly suggesting that PrSOD1 plays an important role in protecting against oxidative damage and possibly chlorantraniliprole tolerance in P. rapae.

Identification and Expression Profiles of 14 Odorant-Binding Protein Genes From Pieris rapae (Lepidoptera: Pieridae).

The small white butterfly, Pieris rapae (L.), is an important insect pest of Brassica crops. This species utilize olfactory cues to find their hosts and mates. However, the molecular mechanism underlying the olfactory perception in this species remains unclear. Here, we identified 14 odorant-binding proteins (OBP) genes-essential for insect olfaction-in P. rapae by exploring a previously published transcriptome dataset. Proteins encoded by all of these genes contain N-terminal signal peptides and six positionally conserved cysteine residues, which are characteristic of insect OBPs. These OBPs displayed high amino acid identity with their respective orthologs in other lepidopterans, and several conserved motifs were identified within these OBPs. Phylogenetic analysis showed that these OBPs were well segregated from each other and clustered into different branches. PrapOBP1 and PrapOBP2 were clustered into the 'general odorant-binding protein' clade, and PrapOBP3 and PrapOBP4 fall into the 'pheromone-binding protein' clade. The 14 OBP genes were located on seven genomic scaffolds. Of these, PrapOBP1, 2, 3, and 4 were located on scaffold332, whereas PrapOBP5, 6, 7, 8, and 9 were located on scaffold116. Ten of the 14 genes had antenna-biased expression. Of these, PrapOBP1, 2, 4, and 13 were enriched in male antennae, whereas PrapOBP7 and PrapOBP10 were female-biased. Our findings suggest that these OBPs may be involved in olfactory communication. To the best of our knowledge, this is the first report on the identification and characterization of OBPs in P. rapae, and our findings provide a solid foundation for studying the functions of these genes.

Characterization of the active fragments of Spodoptera litura Lebocin-1.

Insects can produce various antimicrobial peptides (AMPs) upon immune stimulation. One class of AMPs are characterized by their high proline content in certain fragments. They are generally called proline-rich antimicrobial peptides (PrAMPs). We previously reported the characterization of Spodoptera litura lebocin-1 (SlLeb-1), a PrAMP proprotein. Preliminary studies with synthetic polypeptides showed that among the four deductive active fragments, the C-terminal fragment SlLeb-1 (124-158) showed strong antibacterial activities. Here, we further characterized the antibacterial and antifungal activities of 124-158 and its four subfragments: 124-155, 124-149, 127-158, and 135-158. Only 124-158 and 127-158 could agglutinate bacteria, while 124-158 and four subfragments all could agglutinate Beauveria bassiana spores. Confocal microscopy showed that fluorescent peptides were located on the microbial surface. Fragment 135-158 lost activity completely against Escherichia coli and Staphylococcus aureus, and partially against Bacillus subtilis. Only 124-149 showed low activity against Serratia marcescens. Negative staining, transmission, and scanning electron microscopy of 124-158 treated bacteria showed different morphologies. Flow cytometry analysis of S. aureus showed that 124-158 and four subfragments changed bacterial subpopulations and caused an increase of DNA content. These results indicate that active fragments of SlLeb-1 may have diverse antimicrobial effects against different microbes. This study may provide an insight into the development of novel antimicrobial agents.

MOLECULAR CHARACTERIZATION OF TWO ACETYLCHOLINESTERASE GENES FROM THE RICE LEAFFOLDER, Cnaphalocrocis medinalis (LEPIDOPTERA: PYRALIDAE).

In this study, two full-length cDNA sequences (Cmace1 and Cmace2) encoding putative acetylcholinesterases (AChEs) were cloned and characterized from the rice leaffolder, Cnaphalocrocis medinalis, an important lepidopteran rice pest in Asia. Cmace1 encodes a CmAChE1 consisting of 689 amino acid residues, while Cmace2 encodes a 639 amino acids CmAChE2. The two CmAChEs both have N-terminal signal peptides and conserved motifs including the catalytic triad, choline-binding sites, oxianion hole, acyl pocket, peripheral anionic subsite, and the characteristic FGESAG motif and conserved 14 aromatic amino acids. Phylogenetic analysis showed that Cmace1 and Cmace2 are clustered into distinct clusters that are completely diverged from each other. Reverse-transcription quantitative PCR analysis revealed that Cmace1 and Cmace2 were predominately expressed in the larval brain and at the fifth-instar larvae stage, and the transcription levels of Cmace1 were significantly higher than those of Cmace2 in all the tested samples. Recombinant CmAChE1 and CmAChE2 were heterologously expressed in baculovirus system. Using acetylthiocholine iodide (ATChI) as substrate, the Michaelis constant (Km ) values of rCmAChE1 and rCmAChE2 were 39.81 ± 6.49 and 68.29 ± 6.72 µmol/l, respectively; and the maximum velocity (Vmax ) values of the two rCmAChEs were 0.60 ± 0.02 and 0.31 ± 0.06 µmol/min/mg protein, respectively. Inhibition assay indicated that rCmAChE1 was more sensitive to the organophosphate insecticides chlorpyrifos and triazophos than rCmAChE2. This study is the first report of molecular cloning and biochemical characterization of two acetylcholinesterase genes/enzymes in C. medinalis.

Aphicidal Activity of Illicium verum Fruit Extracts and Their Effects on the Acetylcholinesterase and Glutathione S-transferases Activities in Myzus persicae (Hemiptera: Aphididae).

This study aims to explore the aphicidal activity and underlying mechanism of Illicium verum Hook. f. that is used as both food and medicine. The contact toxicity of the extracts from I. verum fruit with methyl alcohol (MA), ethyl acetate (EA), and petroleum ether (PE) against Myzus persicae (Sulzer), and the activities of acetylcholinesterase (AChE) and glutathione S-transferases (GSTs) of M. persicae after contact treatment were tested. The results showed that MA, EA, and PE extracts of 1.000 mg/l caused, respectively, M. persicae mortalities of 68.93%, 89.95% and 74.46%, and the LC50 of MA, EA, and PE extracts were 0.31, 0.14 and 0.27 mg/l at 72 h after treatment, respectively; the activities of AChE and GSTs in M. persicae were obviously inhibited by the three extracts, as compared with the control, with strong dose and time-dependent effects, the inhibition rates on the whole reached more than 50.00% at the concentration of 1.000 mg/l at 72 h after treatment. The inhibition of the extracts on AChE and GSTs activities (EA extract > PE extract > MA extract) were correlated with theirs contact toxic effects, so it is inferred that the decline of the metabolic enzymes activities may be one of important reasons of M. persicae death. The study results suggested that I. verum extracts have potential as a eco-friendly biopesticide in integrated pest management against M. persicae.

Identification of Putative Carboxylesterase and Glutathione S-transferase Genes from the Antennae of the Chilo suppressalis (Lepidoptera: Pyralidae).

In insects, rapid degradation of odorants in antennae is extremely important for the sensitivity of olfactory receptor neurons. Odorant degradation in insect antennae is mediated by multiple enzymes, especially the carboxylesterases (CXEs) and glutathione S-transferases (GSTs). The Asiatic rice borer, Chilo suppressalis, is an economically important lepidopteran pest which causes great economic damage to cultivated rice crops in many Asian countries. In this study, we identified 19 putative CXE and 16 GST genes by analyzing previously constructed antennal transcriptomes of C. suppressalis. BLASTX best hit results showed that these genes are most homologous to their respective orthologs in other lepidopteran species. Phylogenetic analyses revealed that these CXE and GST genes were clustered into various clades. Reverse-transcription quantitative polymerase chain reaction assays showed that three CXE genes (CsupCXE8, CsupCXE13, and CsupCXE18) are antennae-enriched. These genes are candidates for involvement in odorant degradation. Unexpectedly, none of the GST genes were found to be antennae-specific. Our results pave the way for future researches of the odorant degradation mechanism of C. suppressalis at the molecular level.

GLUTATHIONE S-TRANSFERASE Genes IN THE RICE LEAFFOLDER, Cnaphalocrocis medinalis (LEPIDOPTERA: PYRALIDAE): IDENTIFICATION AND EXPRESSION PROFILES.

In insects, glutathione S-transferases (GSTs) play critical roles in the detoxification of various insecticides, resulting in insecticide resistance. The rice leaffolder, Cnaphalocrocis medinalis, is an economically important pest of rice in Asia. GST genes have not been largely identified in this insect species. In the present study, by searching the transcriptome dataset, 25 candidate GST genes were identified in C. medinalis for the first time. Of these, 23 predicted GST proteins fell into five cytosolic classes (delta, epsilon, omega, sigma, and zeta), and two were assigned to the "unclassified" subgroup. Real-time quantitative PCR analysis showed that these GST genes were differentially expressed in various tissues, including the midgut, Malpighian tubules, and fat body of larvae, and the antenna, abdomen, and leg of adults, indicating diversified functions for these genes. Transcription levels of CmGSTd2, CmGSTe6, and CmGSTe7 increased significantly in larvae following exposure to chlorpyrifos, suggesting that these GST genes could be involved in the detoxification of this insecticide. The results of our study pave the way to a better understanding of the detoxification system of C. medinalis.

Identification of candidate chemosensory genes in the antennal transcriptome of Tenebrio molitor (Coleoptera: Tenebrionidae).

We present the first antennal transcriptome sequencing information for the yellow mealworm beetle, Tenebrio molitor (Coleoptera: Tenebrionidae). Analysis of the transcriptome dataset obtained 52,216,616 clean reads, from which 35,363 unigenes were assembled. Of these, 18,820 unigenes showed significant similarity (E-value <10(-5)) to known proteins in the NCBI non-redundant protein database. Gene ontology (GO) and Cluster of Orthologous Groups (COG) analyses were used for functional classification of these unigenes. We identified 19 putative odorant-binding protein (OBP) genes, 12 chemosensory protein (CSP) genes, 20 olfactory receptor (OR) genes, 6 ionotropic receptor (IR) genes and 2 sensory neuron membrane protein (SNMP) genes. BLASTX best hit results indicated that these chemosensory genes were most identical to their respective orthologs from Tribolium castaneum. Phylogenetic analyses also revealed that the T. molitor OBPs and CSPs are closely related to those of T. castaneum. Real-time quantitative PCR assays showed that eight TmolOBP genes were antennae-specific. Of these, TmolOBP5, TmolOBP7 and TmolOBP16 were found to be predominantly expressed in male antennae, while TmolOBP17 was expressed mainly in the legs of males. Several other genes were identified that were neither tissue-specific nor sex-specific. These results establish a firm foundation for future studies of the chemosensory genes in T. molitor.