Zeta class glutathione S-transferase is involved in phoxim tolerance and is potentially regulated by the transcription factor CncC in Agrotis ipsilon (Lepidoptera: Noctuidae).

The black cutworm, Agrotis ipsilon (Lepidoptera: Noctuidae), is an important agricultural pest. Phoxim is an organophosphate insecticide that has been widely used to control A. ipsilon. The extensive application of phoxim has resulted in a reduction in phoxim susceptibility in A. ipsilon. However, the molecular mechanisms underlying phoxim tolerance in A. ipsilon remain unclear. In this work, we report the involvement of AiGSTz1, a zeta class glutathione S-transferase, in phoxim tolerance in A. ipsilon. Exposure to a sublethal concentration (LC50) of phoxim dramatically upregulated the transcription level of the AiGSTz1 gene in A. ipsilon larvae, and this upregulation might be caused by phoxim-induced oxidative stress. The recombinant AiGSTz1 protein expressed in Escherichia coli was able to metabolize phoxim. Furthermore, AiGSTz1 displayed antioxidant activity to protect against oxidative stress. Knockdown of AiGSTz1 by RNA interference significantly increased the mortality rate of A. ipsilon larvae in response to phoxim. In addition, the transcription factor AiCncC can bind to the cap 'n' collar isoform C: muscle aponeurosis fibromatosis (CncC:Maf) binding site in the putative promoter of the AiGSTz1 gene. Silencing of AiCncC resulted in a dramatic downregulation of AiGSTz1. These results indicated that AiGSTz1 is involved in phoxim tolerance and is potentially regulated by AiCncC. These findings provide valuable insights into the defense mechanisms used by A. ipsilon against phoxim.

Clinical analysis of 346 minor patients with maxillofacial fractures.

OBJECTIVES: This study aims to analyze the clinical epidemiology, diagnostic and treatment characteristics of minor patients with maxillofacial fracture and provide a reference for the prevention and treatment. METHODS: The clinical data of minor patients with maxillofacial fracture in Departmentof Traumatic and Plastic Surgery, West China Hospital of Stomatology, Sichuan University, from January 1, 2015 to December 31, 2020 were retrospectively studied and statistically analyzed in terms of age, gender, etiology, anatomic sites and treatment modalities. RESULTS: The mean age of the patients was (10.65±5.15) years, and the male-to-female ratio was 1.91∶1. High fall was the primary cause of maxillofacial fractures in minors aged 0-6 years. Traffic accident injuries were the main cause of maxillofacial fractures in minors aged 7-12 and 13-17 years. About 65.13% of the midface and 83.08% non-condylar fractures were mainly treated by surgery, and condylar fractures were treated conservatively in 74.73% and by surgical treatment in 25.27%. CONCLUSIONS: The etiology of maxillofacial fractures in minors differs at different ages, so prevention strategies should be adjusted according to age. Surgical treatment has become the preferred treatment modality for midface and non-condylar fractures. Conservative treatment is still the main treatment method for condylar fractures, but the proportion of surgical treatment increases.

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.

Time-series gene expression patterns and their characteristics of Beauveria bassiana in the process of infecting pest insects.

Beauveria bassiana has been widely used as an important biological control fungus for agricultural and forest pests, and clarifying the interaction mechanism between B. bassiana and its host will help to better exert the efficacy of the mycoinsecticide. Here, we proposed a novel pattern analysis (PA) method for analyzing time-series data and applied it to a transcriptomic data set of B. bassiana infecting Galleria mellonella. We screened out 14 patterns including 868 genes, which had some characteristics that were not inferior to differentially expressed genes (DEGs). Compared with the previous analysis of this data set, we had three novel discoveries during B. bassiana infection, including overall downregulation of gene expression, the more critical first 24 h, and enrichment of regulatory functions of downregulated genes. Our new PA method promises to be an important complement to DEGs analysis for time-series transcriptomic data, and our findings enrich our knowledge of molecular mechanisms of fungal-host interactions.

Should preoperative computed tomography be routine examination for cervicofacial space infections?

BACKGROUND: Cervicofacial space infections are potentially life-threatening, which require accurate diagnosis, early incision, and adequate drainage. The utilization of computed tomography (CT) in cervicofacial space infections has significantly increased for its advantages in the evaluation of abscesses, its availability, and low cost. However, the clinical value of preoperative CT imaging in cervicofacial space infections remains controversial for its poor specificity, radiation exposure, potential complications, and extra cost. We, therefore, investigated whether CT examination should be used as a routine examination in the treatment of patients with cervicofacial space infections. METHODS: A retrospective study of all patients affected by cervicofacial space infections that received incision and drainage surgery from Jan 2016 to Dec 2020 was performed at West China Hospital of Stomatology at Sichuan University. Patients were divided into two groups: the group with preoperative CT and without preoperative CT. Outcomes, including reoperation rate, missed diagnosis rate, days of symptom relief, length of stay, duration of surgery, and total cost of hospitalization, were analyzed. RESULTS: Out of n = 153 patients, 108 patients underwent surgery with preoperative CT and 45 patients without preoperative CT. The reoperation rate in the preoperative CT group (6/108, 5.6%) was significantly lower (P = 0.00) than that in the group without preoperative CT (10/45, 22.2%). Significant reduction of missed diagnosis rate (P = 0.00), days of symptom relief (P = 0.01), length of stay(P = 0.03), and duration of surgery (P = 0.01) were detected in the preoperative CT group. The results demonstrated that the utilization of preoperative CT can reduce the missed diagnosis rate and repeated surgery complications. CONCLUSIONS: We recommend preoperative CT as a routine examination in cervicofacial space infections.

[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.

Viscozyme L hydrolysis and Lactobacillus fermentation increase the phenolic compound content and antioxidant properties of aqueous solutions of quinoa pretreated by steaming with α-amylase.

In this work, red quinoa was successively subjected to α-amylase steaming, complex enzyme Viscozyme (R) L hydrolysis, and lactic acid bacteria (LAB) fermentation. The total phenolic compound content (TPC), flavonoid content (TFC), and antioxidant capacities of the solvent-extractable (free) and bound fractions and the individual phenolic compounds released were determined. Compared to steaming with α-amylase, enzymatic hydrolysis and fermentation of quinoa resulted in approximately 82.6, 26.9, 36.3, and 45.2% increases in the TPC (the sum of free and bound fractions), TFC, DPPH, and ORAC values, respectively. HPLC-QqQ-MS/MS analysis showed that enzymolysis and fermentation increased the content of protocatechuic acid, catechin, procyanidin B2 , and quercetin by 126.3, 101.9, 524, and 296.3%, respectively. Moreover, a major proportion of individual phenolic compounds existed as bound form. The results indicated that complex enzymatic hydrolysis and LAB fermentation were practical and useful to release promising polyphenols. This research provides a basis for the processing of quinoa beverages rich in phenolic compounds. PRACTICAL APPLICATION: In this work, liquefying with α-amylase, hydrolyzing with cellulolytic enzyme mixture, and fermenting with Lactic acid bacteria (LAB), successively, were exploited to process quinoa. This is an innovative method of quinoa processing to produce beverage products. Complex enzymatic hydrolysis and fermentation with LAB can significantly enhance phenolic compound, especially protocatechuic acid, catechin, procyanidin B2 , and quercetin. In additional, LAB fermentation is very beneficial to improve the antioxidant activity of quinoa. We also found that a major proportion of phenolic compounds existed as bound forms in quinoa.

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.

Effects of Moisture, Temperature, and Salt Content on the Dielectric Properties of Pecan Kernels During Microwave and Radio Frequency Drying Processes.

Dielectric properties of materials influence the interaction of electromagnetic fields with and are therefore important in designing effective dielectric heating processes. We investigated the dielectric properties (DPs) of pecan kernels between 10 and 3000 MHz using a Novocontrol broadband dielectric spectrometer in a temperature range of 5-65 °C and a moisture content range of 10-30% wet basis (wb) at three salt levels. The dielectric constant (ε') and loss factor (ε'') of the pecan kernels decreased significantly with increasing frequency in the radio frequency (RF) band, but gradually in the measured microwave (MW) band. The moisture content and temperature increase greatly contributed to the increase in the ε' and ε'' of samples, and ε'' increased sharply with increasing salt strength. Quadratic polynomial models were established to simulate DPs as functions of temperature and moisture content at four frequencies (27, 40, 915, and 2450 MHz), with R2 > 0.94. The average penetration depth of pecan kernels in the RF band was greater than that in the MW band (238.17 ± 21.78 cm vs. 15.23 ± 7.36 cm; p < 0.01). Based on the measured DP data, the simulated and experimental temperature-time histories of pecan kernels at five moisture contents were compared within the 5 min RF heating period.

Highly virulent Beauveria bassiana strains against the two-spotted spider mite, Tetranychus urticae, show no pathogenicity against five phytoseiid mite species.

Entomopathogenic fungi and predatory mites can independently contribute to suppressing the two-spotted spider mite, Tetranychus urticae Koch. It is important to assess the risk of possible fungal infections in predators when a combination of them are being considered as a tandem control strategy for suppressing T. urticae. The first part of this study tested 12 Beauveria bassiana isolates for virulence in T. urticae. Strains SCWJ-2, SDDZ-9, LNSZ-26, GZGY-1-3 and WLMQ-32 were found to be the most potent, causing 37.6-49.5% adult corrected mortality at a concentration of 1 × 107 m/L conidia 4 days post-treatment. The second part evaluated the pathogenicity of these five strains in five species of predatory phytoseiid mites. The bioassay results indicated that all adult predatory mite mortalities ranged from 7.5 to 9.1% 4 days post-treatment. No viable fungal hyphae were found on predator cadavers. Observations with scanning electron microscopy revealed that conidia were attached to the cuticle of predatory mites within 2-12 h after spraying with strain LNSZ-26, and had germinated within 24-36 h. After 48 h, conidia had gradually been shed from the mites, after none of the conidia had penetrated the cuticular surfaces. In contrast, the germinated conidia successfully penetrated the cuticle of T. urticae, and within 60 h the fungus colonized the mite's body. Our study demonstrated that although several B. bassiana strains displayed a high virulence in T. urticae there was no evident pathogenicity to phytoseiid mites. These findings support the potential use of entomopathogenic fungus in combination with predatory mites in T. urticae control programs.

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.