Shi-Guang Li

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.

Identification and characterisation of seventeen glutathione S-transferase genes from the cabbage white butterfly Pieris rapae

Insect glutathione S-transferases (GSTs) play essential roles in the detoxification of insecticides and other xenobiotic compounds. The cabbage white butterfly, Pieris rapae, is an economically important agricultural pest. In this study, 17 cDNA sequences encoding putative GSTs were identified in P. rapae. All cDNAs include a complete open reading frame and were designated PrGSTd1-PrGSTz2. Based on phylogenetic analysis, PrGSTs were divided into six classes (delta, epsilon, omega, sigma, theta and zeta). The exon-intron organizations of these PrGSTs were also analysed. Recombinant proteins of eight PrGSTs (PrGSTD1, PrGSTD2, PrGSTE1, PrGSTE2, PrGSTO1, PrGSTS1, PrGSTT1 and PrGSTZ1) were heterologously expressed in Escherichia coli, and all of these proteins displayed glutathione-conjugating activity towards 1-chloro-2,4-dinitrobenzene (CDNB). Expression patterns in various larval tissues, at different life stages, and following exposure to sublethal doses of abamectin, chlorantraniliprole or lambda-cyhalothrin were determined by reverse transcription-quantitative PCR. The results showed that PrGSTe3, PrGSTs1, PrGSTs2, and PrGSTs4 were mainly transcribed in the fat body, while PrGSTe2 was expressed predominantly in the Malpighian tubules. Four genes (PrGSTe2, PrGSTo4, PrGSTs4 and PrGSTt1) were mainly expressed in fourth-instar larvae, while others were ubiquitously expressed in egg, larval, pupa and/or adult stages. Abamectin treatment significantly upregulated ten genes (PrGSTd1, PrGSTd3, PrGSTe1, PrGSTe2, PrGSTo1, PrGSTo3, PrGSTs1, PrGSTs3, PrGSTs4 and PrGSTt1). Chlorantraniliprole and lambda-cyhalothrin treatment significantly upregulated nine genes (PrGSTd1, PrGSTd2, PrGSTe1, PrGSTe2, PrGSTe3, PrGSTs1, PrGSTs3, PrGSTs4 and PrGSTz1) and ten genes (PrGSTd1, PrGSTd3, PrGSTe1, PrGSTe2, PrGSTo1, PrGSTo2, PrGSTs1, PrGSTs2, PrGSTs3 and PrGSTz2), respectively. These GSTs are potentially involved in the detoxification of insecticides.

Selection of Beauveria isolates pathogenic to adults of Nilaparvata lugens

Abstract The brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae), is a destructive invasive pest and has become one of the most economically-important rice pests in China. Effective control measures are desperately needed. Entomopathogenic fungi, such as Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Hypocreales: Clavicipitaceae) and B. brongniartii (Saccardo), have shown great potential for the management of some sucking pest species. In this study, to explore alternative strategies for sustainable control of the sucking pest population, nine isolates of Beauveria from different pests were bioassayed under the concentrated standard spray of 1000 conidia/mm 2 in laboratory. The cumulative mortalities of adults ranged from 17.2 to 79.1% 10 days after inoculation. The virulence among all tested isolates exhibited significant differences (at p = 0.05). The highest virulent isolate was Bb09, which killed 79.1% of the treated insects and had a median lethal time of 5.5 days. Its median lethal concentration values were estimated as 134 conidia/mm 2 on day 10. The chitinase activities of nine isolates were also assayed. The results showed that the chitinase activity (18.7 U/mg) of isolate Bbr09 was the highest among all tested isolates. The biological characteristics of these strains, including growth rate, sporulation, and germination rate, were further investigated. The results showed that strain Bbr09 exhibited the best biological characteristics with relatively higher hyphal growth rate, the highest spore production, and the fastest spore germination. The isolate of Bbr09 had strong pathogenicity and exhibited great potential for sustainable control of N . lugens .

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.

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.

Identification of putative cytochrome P450 monooxygenase genes from the small white butterfly, Pieris rapae (Lepidoptera: Pieridae), and their response to insecticides

The small white butterfly, Pieris rapae (Lepidoptera: Pieridae), is an important pest on Brassicaceae plants, causing heavy crop loss each year. Cytochrome P450 monooxygenase (CYP) is a superfamily of enzymes involved in the detoxification of various xenobiotic compounds, including insecticides. However, little is known about the role of CYP genes in P. rapae. In this study, we identified 63 CYP genes in P. rapae, and analyzed their phylogenetic relationships, exon-intron structures and genomic locations. Moreover, our insecticide-response transcription profiling showed that LD5 doses of lambda-cyhalothrin, chlorantraniliprole, and abamectin significantly increased expression of five (CYP4M59, CYP6AE119, CYP6AE120, CYP6AE121, and CYP6BD18), three (CYP4AU1, CYP6AE120, and CYP6AW1), and five (CYP4L40, CYP4AU1, CYP6AE119, CYP6AW1, and CYP6BD19) CYP genes, respectively; and LD20 doses of the three pesticides significantly upregulated six (CYP4M59, CYP6AE119, CYP6AE120, CYP6AE121, CYP4AU1, and CYP6BD18), six (CYP4G168, CYP4L40, CYP4AU1, CYP6AE120, CYP6AW1, and CYP6BD19), and five (CYP4L40, CYP4AU1, CYP6AB108, CYP6AE119, and CYP6BD19) genes, respectively. When we used LD50 doses of the three insecticides, we reported significantly elevated expression levels of five (CYP4M59, CYP6AE119, CYP6AE120, CYP6BD17, and CYP6BD18), eight (CYP4G168, CYP4L40, CYP4AU1, CYP6AE120, CYP6AE121, CYP6AW1, CYP6BD18, and CYP6BD19), and six (CYP4L40, CYP4S34, CYP6AB108, CYP6AE119, CYP6AE120, and CYP6BD19) genes, respectively. Our expression analysis also revealed that five (CYP4G168, CYP4G169, CYP4S34, CYP6AW1, and CYP6CT3) and three (CYP4L40, CYP6AN33, and CYP6BD17) CYP genes were mainly expressed in the midgut and fat body, respectively, and one CYP gene (CYP6AE119) in the Malpighian tubules. This is the first large-scale report into the characterization of CYP genes in P. rapae.

Transcriptome sequencing reveals abundant olfactory genes in the antennae of the rice leaffolder, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

The rice leaffolder, Cnaphalocrocis medinalis Guenée, is an important lepidopteran pest of rice in Asia. Insect olfactory proteins, including olfactory receptors (ORs), ionotropic receptors (IRs), odorant‐binding proteins (OBPs), chemosensory proteins (CSPs) and sensory neuron membrane proteins (SNMPs), are responsible for perception of sex pheromones and host plant volatiles, and thus regulate insect behavior. In the present study, transcriptome sequencing was conducted for C. medinalis antennae to identify genes involved in olfaction. A total of 45800 unigenes were assembled from the transcriptome dataset. Of these, 19696 (43.0%) unigenes were annotated by searching against the NCBI non‐redundant database. Functional classification of these unigenes were also conducted by using the Gene Ontology (GO) and Cluster of Orthologous Groups (COG) databases. We identified 90 putative olfactory genes (including 37 novel ones): 46 ORs, 15 IRs, 12 OBPs, 15 CSPs and two SNMPs. BLASTX best hit results indicated that these genes were most identical to their respective orthologs from other lepidopteran insects. Quantitative reverse transcription‐PCR (qRT‐PCR) assays were performed to investigate the expression profiles of newly identified OR genes. All of the OR genes were antennae‐specific or antennae‐enriched. Of these, CmedOR28 and CmedOR31 were mainly expressed in male antennae, while CmedOR27 and CmedOR32 were enriched in female antennae. Our results establish a solid foundation for future functional studies of these genes.

Molecular characterization of a typical 2-Cys thioredoxin peroxidase from the Asiatic rice borer Chilo suppressalis and its role in oxidative stress

In insects, thioredoxin peroxidase (TPX) plays an important role in protecting against oxidative damage. However, studies on the molecular characteristics of TPXs in the Asiatic rice borer, Chilo suppressalis, are limited. In this work, a cDNA sequence (CsTpx3) encoding a TPX was identified from C. suppressalis. The deduced CsTPX3 protein shares high sequence identity and two positionally conserved cysteines with orthologs from other insect species, and was classified as a typical 2-Cys TPX. CsTpx3 was expressed most highly during the fifth-instar larval stage, and transcripts were most abundant in the midgut. Recombinant CsTPX3 protein expressed in Escherichia coli displayed the expected peroxidase activity by removing H2 O2 . Furthermore, CsTPX3 protected DNA from oxidative damage, and E. coli cells overexpressing CsTPX3 exhibited long-term resistance to oxidative stress. Exposure to various oxidative stressors, such as cold (8°C), heat (35°C), bacteria (E. coli), and two insecticides (chlorpyrifos and lambda-cyhalothrin), significantly upregulated transcription of CsTpx3. However, exposure to abamectin had no such effect. Our results provide valuable information for future studies on the antioxidant mechanism in this insect species.

Molecular characterization of a NADPH–cytochrome P450 reductase gene from the rice leaffolder, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

NADPH–cytochrome P450 reductase (CPR) is the most important redox partner of various cytochrome P450 monooxygenases (P450s) and plays a central role in multiple metabolic reactions. In this paper, a full-length cDNA encoding CPR (designated as CmCPR) was characterized in the rice leaffolder, Cnaphalocrocis medinalis (Guenée), a serious lepidopteran rice pest. The complete open reading frame of CmCPR was 2046 bp, encoding a protein consists of 681 amino acid residues. The secondary structure of CmCPR protein showed marked features of classical CPRs such as N-terminal anchor, conserved functional domains, and catalytic residues. Phylogenic analysis showed that CmCPR was clustered together with CPRs from other lepidopteran species. Recombinant CmCPR protein was expressed in E. coli, and the activity and kinetic parameters of the enzyme were determined. Quantitative reverse transcription-PCR showed that the highest expression levels of CmCPR were detected in fourth- and fifth-instar larvae, and the transcriptional level in larval midgut tended to be higher than those in other tissues. Exposure to sublethal concentrations of three insecticides, abamectin, chlorpyrifos, and chlorantraniliprole, led to upregulated expression of CmCPR and several P450 genes. This work is the first report of molecular characterization of CPR gene in Cn. medinalis.