Xinghui Qiu

Sublethal effects of spinosad on survival, growth and reproduction of Helicoverpa armigera (Lepidoptera: Noctuidae).

BACKGROUNDnHelicoverpa armigera (Hübner) is one of the most important pests in many countries. Spinosad is used widely for the control of pests, but there is sparse information available regarding its sublethal effects on H. armigera. Here, the authors attempt to investigate the sublethal effects of spinosad on H. armigera in order to reveal the negative, non-lethal impact of insecticides on this pest.nnnRESULTSnThe toxicity of spinosad against H. armigera was determined under laboratory conditions by oral exposure of late second-instar larvae to the compound. The 48 h LC(50) and 72 h LC(50) values of spinosad to this pest were found to be 0.41 mg kg(-1) and 0.35 mg kg(-1) respectively. Spinosad at sublethal concentrations significantly extended the developmental time of survivor larvae, and reduced larval wet weight. Post-exposure effects were indicated by decreased pupation ratio and pupal weight, by prolonged prepupal and pupal periods and by decreased emergence ratio, fecundity and longevity of adults.nnnCONCLUSIONnThese results suggest that the combination of lethal and sublethal effects of spinosad might affect pest population dynamics significantly by decreasing its survival and reproduction, and by delaying its development.

CYP9A12 and CYP9A17 in the cotton bollworm, Helicoverpa armigera: sequence similarity, expression profile and xenobiotic response

BACKGROUNDnHelicoverpa armigera (Hübner) causes severe losses in many crops including cotton. Cytochrome P450s play crucial roles in the metabolism of many important compounds in various organisms. The authors attempt to identify new cytochrome P450 genes and investigate their expression profile and xenobiotic response in order to understand the nature and roles of cytochrome P450s in this important pest.nnnRESULTSnA novel P450 cDNA (CYP9A17), encoding a protein of 531 amino acid residues, was isolated from H. armigera. CYP9A17 is a typical microsomal P450, showing the highest (93.9%) amino acid identity with CYP9A12 from H. armigera. The high similarity is not only found between cDNAs, but also between the intron-exon organisation. CYP9A12 is constitutively universally expressed in all four life stages and in all tested larval tissues, whereas CYP9A17 is specifically expressed in the larval midgut and fat body. Real-time quantitative RT-PCR showed that the level of both CYP9A12 and CYP9A17 mRNA is affected in dose-dependent and tissue-specific manners by deltamethrin, gossypol and phenobarbital.nnnCONCLUSIONnCYP9A12 and CYP9A17 showed high sequence identity, but with differential expression patterns, suggesting that CYP9A12 and CYP9A17 genes in H. armigera might diverge via subfunctionalisation after the gene duplication event.

Nrf2/Maf-binding-site-containing functional Cyp6a2 allele is associated with DDT resistance in Drosophila melanogaster

BACKGROUNDnIncreased insecticide detoxification mediated by cytochrome P450s is a common mechanism of insecticide resistance. Although Cyp6a2 has been observed to be overexpressed in many 4,4-dichlorodiphenyltrichloroethane (DDT)-resistant strains of Drosophilau2009melanogaster, how Cyp6a2 is regulated and whether its overproduction confers DDT resistance remain elusive.nnnRESULTSnMolecular analysis identified five Cyp6a2 alleles (Cyp6a2(Canton) (-S-1) , Cyp6a2(Canton) (-S-2) , Cyp6a2(91-C) , Cyp6a2(91-R) and Cyp6a2(Wisconsin) (-) (WD) ) from four D.u2009melanogaster strains, notably differing in the presence or absence of an intact Nrf2/Maf (a transcription factor) binding site in the 5-promoter core region, a G1410 frameshift deletion mutation in the heme-binding region and a long terminal repeat (LTR) of transposable element 17.6 in the 3-untranslated region (UTR). Linkage analysis confirmed that DDT resistance was genetically linked to a Nrf2/Maf-binding-site-containing, LTR-lacking functional allele of Cyp6a2 (Cyp6a2(91-R) ). The qRT-PCR results showed that overexpression of functional Cyp6a2 was consistently associated with DDT resistance. Luciferase reporter gene assays revealed that an intact Nrf2/Maf binding site in the 5-promoter core region enhanced the constitutive transcription of Cyp6a2.nnnCONCLUSIONnThe results suggest that the Nrf2/Maf binding-site-containing functional Cyp6a2u2009allele is associated with DDT resistance in the D.u2009melanogaster strains under study.

Multiple origins of kdr-type resistance in the house fly, Musca domestica.

Insecticide resistance is a model phenotype that can be used to investigate evolutionary processes underlying the spread of alleles across a global landscape, while offering valuable insights into solving the problems that resistant pests present to human health and agriculture. Pyrethroids are one of the most widely used classes of insecticides world-wide and they exert their toxic effects through interactions with the voltage-sensitive sodium channel (Vssc). Specific mutations in Vssc (kdr, kdr-his and super-kdr) are known to cause resistance to pyrethroid insecticides in house flies. In order to determine the number of evolutionary origins of kdr, kdr-his and super-kdr, we sequenced a region of Vssc from house flies collected in the USA, Turkey and China. Our phylogenetic analysis of Vssc unequivocally supports the hypothesis of multiple independent origins of kdr, super-kdr and kdr-his on an unprecedented geographic scale. The implications of these evolutionary processes on pest management are discussed.

Effects of spinosad on Helicoverpa armigera (Lepidoptera: Noctuidae) from China: tolerance status, synergism and enzymatic responses.

BACKGROUNDnSpinosad is increasingly used in pest management programmes, and resistance to it has been detected in recent years. However, there is no report on the susceptibilities of field populations of Helicoverpa armigera (Hübner) from China. Furthermore, the impact of spinosad on metabolic enzymes in this pest remains unknown.nnnRESULTSnFour populations of H. armigera from different locations in China displayed less than 6.5-fold difference in LC(50) to spinosad, the highest being in the Xinjiang population, followed by Xiajin, Taian and Hubei populations, while there was no significant difference at LC(99) level among the four populations. The toxicity of spinosad could be synergised by piperonyl butoxide (PBO) and triphenylphosphate (TPP), but not by diethyl maleate (DEM). Spinosad exposure for 48 h significantly increased the activities of p-nitroanisole O-demethylase (ODM), while no significant changes in glutathione-S-transferase (GST) and carboxyl esterase (CarE) were observed.nnnCONCLUSIONnField populations of H. armigera from China displayed marginally different susceptibilities to spinosad and had a relatively low LC(50). Cytochrome P450 monooxygenase might be involved in the metabolism of, and hence resistance to, spinosad in this pest in China.

Reduced fitness associated with spinosad resistance in Helicoverpa armigera

The fitness cost of spinosad resistance was investigated in the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). Laboratory experiments were conducted to compare relative fitness of H. armigera between the spinosad-susceptible and -resistant strains. During the experiments, the average development periods of the resistant strain were lengthened by 4–5xa0days, reflected in a prolongation of egg, larval and pupal periods. Furthermore, pupal survival, pupal weight, the mean life span of emerged adults, eggs laid and hatched decreased greatly in the resistant strain in comparison with the susceptible strain. Other life-cycle parameters such as larval survival, larval wet weights, prepupal periods, pupation ratio, and sex ratio did not change significantly. As a result, both net replacement rate (R0) and intrinsic rate of increase (rm) were reduced for the resistant strain. Our results clearly indicated that relative fitness of resistant individuals was reduced in the absence of spinosad. Rational measures including pesticide rotations should be expected to delay development of resistance to spinosad in H. armigera field populations from China.

Genome-wide analysis of genes associated with moderate and high DDT resistance in Drosophila melanogaster

BACKGROUNDnModerate to high DDT resistance in generally associated with overexpression of multiple genes and therefore has been considered to be polygenic. However, very little information is available about the molecular mechanisms that insect populations employ when evolving increased levels of resistance. The presence of common regulatory motifs among resistance-associated genes may help to explain how and why certain suites of genes are preferentially represented in genomic-scale analyses.nnnRESULTSnA set of commonly differentially expressed genes associated with DDT resistance in the fruit fly was identified on the basis of genome-wide microarray analysis followed by qRT-PCR verification. More genes were observed to be overtranscribed in the highly resistant strain (91-R) than in the moderately resistant strain (Wisconsin) and susceptible strain (Canton-S). Furthermore, possible transcription factor binding sites that occurred in coexpressed resistance-associated genes were discovered by computational motif discovery methods.nnnCONCLUSIONnA glucocorticoid receptor (GR)-like putative transcription factor binding motif (TFBM) was observed to be associated with genes commonly differentially transcribed in both the 91-R and Wisconsin lines of DDT-resistant Drosophila.

De Novo Transcriptome of the Hemimetabolous German Cockroach (Blattella germanica)

Background The German cockroach, Blattella germanica, is an important insect pest that transmits various pathogens mechanically and causes severe allergic diseases. This insect has long served as a model system for studies of insect biology, physiology and ecology. However, the lack of genome or transcriptome information heavily hinder our further understanding about the German cockroach in every aspect at a molecular level and on a genome-wide scale. To explore the transcriptome and identify unique sequences of interest, we subjected the B. germanica transcriptome to massively parallel pyrosequencing and generated the first reference transcriptome for B. germanica. Methodology/Principal Findings A total of 1,365,609 raw reads with an average length of 529 bp were generated via pyrosequencing the mixed cDNA library from different life stages of German cockroach including maturing oothecae, nymphs, adult females and males. The raw reads were de novo assembled to 48,800 contigs and 3,961 singletons with high-quality unique sequences. These sequences were annotated and classified functionally in terms of BLAST, GO and KEGG, and the genes putatively coding detoxification enzyme systems, insecticide targets, key components in systematic RNA interference, immunity and chemoreception pathways were identified. A total of 3,601 SSRs (Simple Sequence Repeats) loci were also predicted. Conclusions/Significance The whole transcriptome pyrosequencing data from this study provides a usable genetic resource for future identification of potential functional genes involved in various biological processes.

Characterization of chicken cytochrome P450 1A4 and 1A5: Inter-paralog comparisons of substrate preference and inhibitor selectivity

The chicken (Gallus gallus) is one of the most economically important domestic animals and also an avian model species. Chickens have two CYP1A genes (CYP1A4 and CYP1A5) which are orthologous to mammalian CYP1A1 and CYP1A2. Although the importance of chicken CYP1As in metabolism of endogenous compounds and xenobiotics is well recognized, their enzymatic properties, substrate preference and inhibitor selectivity remain poorly understood. In this study, functional enzymes of chicken CYP1A4 and CYP1A5 were successfully produced in Escherichia coli (E. coli). The substrate preference and inhibitor specificity of the two chicken CYP1As were compared. Kinetic results showed that the enzymatic parameters (K(m), V(max), V(max)/K(m)) for ethoxyresorufin O-deethylase (EROD) and benzyloxyresorufin O-debenzylase (BROD) differed between CYP1A4 and CYP1A5, while no significant difference was observed for methoxyresorufin O-demethylase (MROD). Lower K(m) of CYP1A4 for BROD suggests that CYP1A4 has a greater binding affinity to benzyloxyresorufin than either ethoxyresorufin or methoxyresorufin. The highest V(max)/K(m) ratio was seen in BROD activity for CYP1A4 and in MROD for CYP1A5 respectively. These results indicate that substrate preference of chicken CYP1As is more notably distinguished by BROD activity and CYP1A5 prefers shorter alkoxyresorufins resembling its mammalian ortholog CYP1A2. Differential patterns of MROD inhibition were observed between CYP1As and among the five CYP inhibitors (α-naphthoflavone, furafylline, piperonyl butoxide, erythromycin and ketoconazole). α-Naphthoflavone was determined to be a potent MROD inhibitor of both CYP1A4 and CYP1A5. In contrast, no or only a trace inhibitory effect (<15%) was observed by erythromycin at a concentration of 500 μM. Stronger inhibition of MROD activity was found in CYP1A5 than CYP1A4 by relatively small molecules α-naphthoflavone, piperonyl butoxide and furafylline. AROD kinetics and inhibition profiles between chicken CYP1A4 and CYP1A5 demonstrate that the two paralogous members of the CYP1A subfamily have distinct enzymatic properties, reflecting differences in the active site geometry between CYP1A4 and CYP1A5. These findings suggest that CYP1A4 and CYP1A5 play partially overlapping but distinctly different physiological and toxicological roles in the chicken.

Characterization of NADPH-cytochrome P450 reductase gene from the cotton bollworm, Helicoverpa armigera.

A complete cDNA encoding the NADPH-cytochrome P450 reductase (haCPR) and its genomic sequence from the cotton bollworm Helicoverpa armigera were cloned and sequenced. The open reading frame of haCPR codes for a protein of 687 amino acid residues with a calculated molecular mass of 77.4kDa. The haCPR gene spans over 11 kb and its coding region is interrupted by 11 introns. haCPR is ubiquitously expressed in various tissues and at various stages of development. Escherichia coli produced haCPR enzyme exhibited catalytic activity for NADPH-dependent reduction of cytochrome c, following Michaelis-Menten kinetics. The functionality of CPR was further demonstrated by its capacity to support cytochrome P450 (e.g. haCYP9A14 and chicken CYP1A5) mediated O-dealkylation activity of alkoxyresorufins. The flavoprotein-specific inhibitor (diphenyleneiodonium chloride, DPI) showed a potent inhibition to haCPR activity (IC50=1.69 μM). Inhibitory effect of secondary metabolites in the host plants (tannic acid, quercetin and gossypol) on CPR activity (with an IC50 value ranged from 15 to 90 μM) was also observed.