Positional cloning of a cyromazine resistance gene in Drosophila melanogaster.

Cyromazine is an effective insecticide used to control dipteran insects. Its precise mode of action is yet to be determined, although it has been suggested that it interferes with the hormone system, sclerotization of the cuticle, or nucleic acid metabolism. To understand the way in which cyromazine acts, we have positionally cloned a cyromazine resistance gene from Drosophila melanogaster. Six cyromazine resistance alleles had previously been generated by ethyl methanasulphonate treatment. Two of these failed to complement each other and here we identify them as having independent non-sense mutations in CG32743, which is an ortholog of Smg1 of worms and mammals and encodes a phosphatidylinositol kinase-like kinase (PIKK). RNAi experiments confirm that cyromazine resistance can be achieved by knocking down CG32743. These are the first cyromazine resistant mutations identified at the nucleotide level. In mammals Smg1 phosphorylates P53 in response to DNA damage. This finding supports the hypothesis that cyromazine interferes with nucleic acid metabolism.

World-wide survey of an Accord insertion and its association with DDT resistance in Drosophila melanogaster.

Previous work showed that insecticide resistance in Drosophila melanogaster is correlated with the insertion of an Accord-like element into the 5' region of the cytochrome P450 gene, Cyp6g1. Here, we study the distribution of the Accord-like element in 673 recently collected D. melanogaster lines from 34 world-wide populations. We also examine the extent of microsatellite variability along a 180-kilobase (kb) genomic region of chromosome II encompassing the resistance gene. We confirm a 100% correlation of the Accord insertion with insecticide resistance and a significant reduction in variability extending at least 20 kb downstream of the Cyp6g1 gene. The frequency of the Accord insertion differs significantly between East African (32-55%) and nonAfrican (85-100%) populations. This pattern is consistent with a selective sweep driving the Accord insertion close to fixation in nonAfrican populations as a result of the insecticide resistance phenotype it confers. This study confirms that hitchhiking mapping can be used to identify beneficial mutations in natural populations.

The insecticidal toxin Makes caterpillars floppy (Mcf) promotes apoptosis in mammalian cells.

Photorhabdus bacteria produce a number of toxins to kill their insect hosts. The expression of one of these, Makes caterpillars floppy (Mcf), is sufficient to allow Escherichia coli to persist within and kill caterpillars. Mcf causes shedding of the insect midgut epithelium and destructive blebbing of haemocytes suggesting it may trigger apoptosis. To investigate this hypothesis, here we examine the effects of E. coli-expressed Mcf on the mammalian cell lines COS-7, NIH 3T3 and HeLa cells. Cells treated with Mcf show apoptotic nuclear morphology, active caspase-3, DNA laddering after 6 h, and the presence of cleaved PARP after 16 h. These effects are prevented by the apoptosis inhibitor zVAD-fmk. Transfection of cells with constructs expressing only the NH2-terminal 1280 amino acids of Mcf, as a fusion with Myc, also triggered cell destruction. The expressed fusion protein was concentrated into the Golgi apparatus before cell death. These results confirm that the novel insecticidal toxin Mcf induces apoptosis but the precise intracellular pathway remains obscure.

Microarray analysis of cytochrome P450 mediated insecticide resistance in Drosophila.

Insecticide resistance in laboratory selected Drosophila strains has been associated with upregulation of a range of different cytochrome P450s, however in recent field isolates of D. melanogaster resistance to DDT and other compounds is conferred by one P450 gene, Cyp6g1. Using microarray analysis of all Drosophila P450 genes, here we show that different P450 genes such as Cyp12d1 and Cyp6a8 can also be selected using DDT in the laboratory. We also show, however, that a homolog of Cyp6g1 is over-expressed in a field resistant strain of D. simulans. In order to determine why Cyp6g1 is so widely selected in the field we examine the pattern of cross-resistance of both resistant strains and transgenic flies over-expressing Cyp6g1 alone. We show that all three DDT selected P450s can confer resistance to the neonicotinoid imidacloprid but that Cyp6a8 confers no cross-resistance to malathion. Transgenic flies over-expressing Cyp6g1 also show cross-resistance to other neonicotinoids such as acetamiprid and nitenpyram. We suggest that the broad level of cross-resistance shown by Cyp6g1 may have facilitated its selection as a resistance gene in natural Drosophila populations.

A single p450 allele associated with insecticide resistance in Drosophila.

Insecticide resistance is one of the most widespread genetic changes caused by human activity, but we still understand little about the origins and spread of resistant alleles in global populations of insects. Here, via microarray analysis of all P450s in Drosophila melanogaster, we show that DDT-R, a gene conferring resistance to DDT, is associated with overtranscription of a single cytochrome P450 gene, Cyp6g1. Transgenic analysis of Cyp6g1 shows that overtranscription of this gene alone is both necessary and sufficient for resistance. Resistance and up-regulation in Drosophila populations are associated with a single Cyp6g1 allele that has spread globally. This allele is characterized by the insertion of an Accord transposable element into the 5' end of the Cyp6g1 gene.

A single Photorhabdus gene, makes caterpillars floppy (mcf), allows Escherichia coli to persist within and kill insects.

Photorhabdus luminescens, a bacterium with alternate pathogenic and symbiotic phases of its lifestyle, represents a source of novel genes associated with both virulence and symbiosis. This entomopathogen lives in a "symbiosis of pathogens" with nematodes that invade insects. Thus the bacteria are symbiotic with entomopathogenic nematodes but become pathogenic on release from the nematode into the insect blood system. Within the insect, the bacteria need to both avoid the peptide- and cellular- (hemocyte) mediated immune response and also to kill the host, which then acts as a reservoir for bacterial and nematode reproduction. However, the mechanisms whereby Photorhabdus evades the insect immune system and kills the host are unclear. Here we show that a single large Photorhabdus gene, makes caterpillars floppy (mcf), is sufficient to allow Esherichia coli both to persist within and kill an insect. The predicted high molecular weight Mcf toxin has little similarity to other known protein sequences but carries a BH3 domain and triggers apoptosis in both insect hemocytes and the midgut epithelium.

Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli.

Previous attempts to express the toxin complex genes of Photorhabdus luminescens W14 in Escherichia coli have failed to reconstitute their oral toxicity to the model insect Manduca sexta. Here we show that the combination of three genes, tcdA, tcdB, and tccC, is essential for oral toxicity to M. sexta when expression in E. coli is used. Further, when transcription from native toxin complex gene promoters is used, maximal toxicity in E. coli cultures is associated with the addition of mitomycin C to the growth medium. In contrast, the expression of tcdAB (or the homologous tcaABC operon) with no recombinant tccC homolog in a different P. luminescens strain, K122, is sufficient to confer oral toxicity on this strain, which is otherwise not orally toxic. We therefore infer that P. luminescens K122 carries a functional tccC-like homolog within its own genome, a hypothesis supported by Southern analysis. Recombinant toxins from both P. luminescens K122 and E. coli were purified as high-molecular-weight particulate preparations. Transmission electron micrograph (TEM) images of these particulate preparations showed that the expression of tcdAB (either with or without tccC) in E. coli produces visible approximately 25-nm-long complexes with a head and tail-like substructure. These data are consistent with a model whereby TcdAB constitutes the majority of the complex visible under TEM and TccC either is a toxin itself or is an activator of the complex. The implications for the potential mode of action of the toxin complex genes are discussed.

Measuring virulence factor expression by the pathogenic bacterium Photorhabdus luminescens in culture and during insect infection.

During insect infection Photorhabdus luminescens emits light and expresses virulence factors, including insecticidal toxin complexes (Tcs) and an RTX-like metalloprotease (Prt). Using quantitative PCR and protein assays, we describe the expression patterns of these factors both in culture and during insect infection and compare them to the associated bacterial growth curves. In culture, light and active Prt protease are produced in stationary phase. Tca also appears in stationary phase, whereas Tcd is expressed earlier. These patterns seen in a culture flask are strikingly similar to those observed during insect infection. Thus, in an infected insect, bacteria grow exponentially until the time of insect death at approximately 48 h, when both light and the virulence factors Prt protease and Tca are produced. In contrast, Tcd appears much earlier in insect infection. However, at present, the biological significance of this difference in timing of the production of the two toxins in unclear. This is the first documentation of the expression of Tcs and Prt in an insect and highlights the malleability of Photorhabdus as a model system for bacterial infection.

A genomic sample sequence of the entomopathogenic bacterium Photorhabdus luminescens W14: potential implications for virulence.

Photorhabdus luminescens is a pathogenic bacterium that lives in the guts of insect-pathogenic nematodes. After invasion of an insect host by a nematode, bacteria are released from the nematode gut and help kill the insect, in which both the bacteria and the nematodes subsequently replicate. However, the bacterial virulence factors associated with this "symbiosis of pathogens" remain largely obscure. In order to identify genes encoding potential virulence factors, we performed approximately 2,000 random sequencing reads from a P. luminescens W14 genomic library. We then compared the sequences obtained to sequences in existing gene databases and to the Escherichia coli K-12 genome sequence. Here we describe the different classes of potential virulence factors found. These factors include genes that putatively encode Tc insecticidal toxin complexes, Rtx-like toxins, proteases and lipases, colicin and pyocins, and various antibiotics. They also include a diverse array of secretion (e.g., type III), iron uptake, and lipopolysaccharide production systems. We speculate on the potential functions of each of these gene classes in insect infection and also examine the extent to which the invertebrate pathogen P. luminescens shares potential antivertebrate virulence factors. The implications for understanding both the biology of this insect pathogen and links between the evolution of vertebrate virulence factors and the evolution of invertebrate virulence factors are discussed.

A genetic analysis of cyromazine resistance in Drosophila melanogaster (Diptera: Drosophilidae).

Flies resistant to the insect growth regulator cyromazine were selected in the F1 generation from a cyromazine-susceptible strain of Drosophila melanogaster (Meigen) treated with ethyl methanesulfonate. Four resistant strains were isolated by screening with cyromazine at a concentration > LC100 of susceptibles. In each strain, resistance is conferred by a single gene mutation. Cyromazine resistance in two of the mutants (rst(1a)cyr1 and rst(1a)cyr2) localizes to map position 17 of the X chromosome. Evidence is presented that these mutations are alleles of the gene rst(1a)cyr. Cyromazine resistance in another of the mutants (Rst(1b)Cyr) is also X-linked, and localizes to map position 49 of the X chromosome. The location of the gene conferring cyromazine resistance in the other mutant (Rst(2b)Cyr) is map position 66 of chromosome II. This is possibly an allele of a previously characterized cyromazine resistance gene, Rst(2)Cyr. Dosage-mortality analyses demonstrate a low level of cyromazine resistance is conferred in all strains.