Professor Richard Ffrench-Constant
Professor of Molecular Natural History

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Publications by category



Journal articles
Smee, M.R., Pauchet, Y., Wilkinson, P., Wee, B., Singer, M.C., ffrench-Constant, R.H., Hodgson, D.J., Mikheyev, A.S. (2013). Microsatellites for the marsh fritillary butterfly: de novo transcriptome sequencing, and a comparison with amplified fragment length polymorphism (AFLP) markers. Plos One, 8(1).

Abstract:
Microsatellites for the marsh fritillary butterfly: de novo transcriptome sequencing, and a comparison with amplified fragment length polymorphism (AFLP) markers.

Until recently the isolation of microsatellite markers from Lepidoptera has proved troublesome, expensive and time-consuming. Following on from a previous study of Edith's checkerspot butterfly, Euphydryas editha, we developed novel microsatellite markers for the vulnerable marsh fritillary butterfly, E. aurinia. Our goal was to optimize the process in order to reduce both time and cost relative to prevailing techniques. This was accomplished by using a combination of previously developed techniques: in silico mining of a de novo assembled transcriptome sequence, and genotyping the microsatellites found there using an economic method of fluorescently labelling primers.
 Abstract.  Author URL
Dasmahapatra, K.K., Walters, J.R., Briscoe, A.D., Davey, J.W., Whibley, A., Nadeau, N.J., Zimin, A.V., Hughes, D.S.T., Ferguson, L.C., Martin, S.H., et al (2012). Butterfly genome reveals promiscuous exchange of mimicry adaptations among species. Nature, 486(7405), 94-98.
Karatolos, N., Williamson, M.S., Denholm, I., Gorman, K., ffrench-Constant, R.H., Bass, C. (2012). Over-expression of a cytochrome P450 is associated with resistance to pyriproxyfen in the greenhouse whitefly trialeurodes vaporariorum. Plos One, 7(2).
Joron, M., Frezal, L., Jones, R.T., Chamberlain, N.L., Lee, S.F., Haag, C.R., Whibley, A., Becuwe, M., Baxter, S.W., Ferguson, L., et al (2011). Chromosomal rearrangements maintain a polymorphic supergene controlling butterfly mimicry. Nature, 477(7363), 203-U102. Author URL
Pauchet, Y., Wilkinson, P., Chauhan, R., Ffrench-Constant, R.H. (2010). Diversity of beetle genes encoding novel plant cell wall degrading enzymes. Plos One, 5(12).
Dowling, A.J., Wilkinson, P.A., Holden, M.T., Quail, M.A., Bentley, S.D., Reger, J., Waterfield, N.R., Titball, R.W., Ffrench-Constant, R.H. (2010). Genome-wide analysis reveals loci encoding anti-macrophage factors in the human pathogen Burkholderia pseudomallei K96243. Plos One, 5(12).

Abstract:
Genome-wide analysis reveals loci encoding anti-macrophage factors in the human pathogen Burkholderia pseudomallei K96243.

Burkholderia pseudomallei is an important human pathogen whose infection biology is still poorly understood. The bacterium is endemic to tropical regions, including South East Asia and Northern Australia, where it causes melioidosis, a serious disease associated with both high mortality and antibiotic resistance. B. pseudomallei is a Gram-negative facultative intracellular pathogen that is able to replicate in macrophages. However despite the critical nature of its interaction with macrophages, few anti-macrophage factors have been characterized to date. Here we perform a genome-wide gain of function screen of B. pseudomallei strain K96243 to identify loci encoding factors with anti-macrophage activity. We identify a total of 113 such loci scattered across both chromosomes, with positive gene clusters encoding transporters and secretion systems, enzymes/toxins, secondary metabolite, biofilm, adhesion and signal response related factors. Further phenotypic analysis of four of these regions shows that the encoded factors cause striking cellular phenotypes relevant to infection biology, including apoptosis, formation of actin 'tails' and multi-nucleation within treated macrophages. The detailed analysis of the remaining host of loci will facilitate genetic dissection of the interaction of this important pathogen with host macrophages and thus further elucidate this critical part of its infection cycle.
 Abstract.  Author URL
Counterman, B.A., Araujo-Perez, F., Hines, H.M., Baxter, S.W., Morrison, C.M., Lindstrom, D.P., Papa, R., Ferguson, L., Joron, M., Ffrench-Constant, R.H., et al (2010). Genomic Hotspots for Adaptation: the Population Genetics of Mullerian Mimicry in Heliconius erato. Plos Genetics, 6(2). Author URL
Baxter, S.W., Nadeau, N.J., Maroja, L.S., Wilkinson, P., Counterman, B.A., Dawson, A., Beltran, M., Perez-Espona, S., Chamberlain, N., Ferguson, L., et al (2010). Genomic hotspots for adaptation: the population genetics of Müllerian mimicry in the Heliconius melpomene clade. Plos Genet, 6(2).

Abstract:
Genomic hotspots for adaptation: the population genetics of Müllerian mimicry in the Heliconius melpomene clade.

Wing patterning in Heliconius butterflies is a longstanding example of both Müllerian mimicry and phenotypic radiation under strong natural selection. The loci controlling such patterns are "hotspots" for adaptive evolution with great allelic diversity across different species in the genus. We characterise nucleotide variation, genotype-by-phenotype associations, linkage disequilibrium, and candidate gene expression at two loci and across multiple hybrid zones in Heliconius melpomene and relatives. Alleles at HmB control the presence or absence of the red forewing band, while alleles at HmYb control the yellow hindwing bar. Across HmYb two regions, separated by approximately 100 kb, show significant genotype-by-phenotype associations that are replicated across independent hybrid zones. In contrast, at HmB a single peak of association indicates the likely position of functional sites at three genes, encoding a kinesin, a G-protein coupled receptor, and an mRNA splicing factor. At both HmYb and HmB there is evidence for enhanced linkage disequilibrium (LD) between associated sites separated by up to 14 kb, suggesting that multiple sites are under selection. However, there was no evidence for reduced variation or deviations from neutrality that might indicate a recent selective sweep, consistent with these alleles being relatively old. of the three genes showing an association with the HmB locus, the kinesin shows differences in wing disc expression between races that are replicated in the co-mimic, Heliconius erato, providing striking evidence for parallel changes in gene expression between Müllerian co-mimics. Wing patterning loci in Heliconius melpomene therefore show a haplotype structure maintained by selection, but no evidence for a recent selective sweep. The complex genetic pattern contrasts with the simple genetic basis of many adaptive traits studied previously, but may provide a better model for most adaptation in natural populations that has arisen over millions rather than tens of years.
 Abstract.  Author URL
Jones, R.T., Bakker, S.E., Stone, D., Shuttleworth, S.N., Boundy, S., McCart, C., Daborn, P.J., ffrench-Constant, R.H., van den Elsen, J.M. (2010). Homology modelling of Drosophila cytochrome P450 enzymes associated with insecticide resistance. Pest Manag Sci, 66(10), 1106-1115.

Abstract:
Homology modelling of Drosophila cytochrome P450 enzymes associated with insecticide resistance.

Overexpression of the cytochrome P450 gene Cyp6g1 confers resistance against DDT and a broad range of other insecticides in Drosophila melanogaster Meig. In the absence of crystal structures of CYP6G1 or complexes with its substrates, structural studies rely on homology modelling and ligand docking to understand P450-substrate interactions.
 Abstract.  Author URL
Wilkinson, P., Paszkiewicz, K., Moorhouse, A., Szubert, J.M., Beatson, S., Gerrard, J., Waterfield, N.R., Ffrench-Constant, R.H. (2010). New plasmids and putative virulence factors from the draft genome of an Australian clinical isolate of Photorhabdus asymbiotica. Fems Microbiol Lett, 309(2), 136-143.

Abstract:
New plasmids and putative virulence factors from the draft genome of an Australian clinical isolate of Photorhabdus asymbiotica.

Clinical isolates of Photorhabdus asymbiotica have been recovered from patients in both the United States of America and Australia, and the full sequence of P. asymbiotica ATCC43949 from the United States has been reported recently. In contrast to other bacteria in the genus that only infect insects, P. asymbiotica strains are able to infect both insects and humans. Using a combination of Solexa (Illumina) and 454 Life Sciences (Roche) sequence data in different assembly pipelines, we report on a draft genome sequence of a strain of P. asymbiotica recovered from a patient from Kingscliff, Australia. The best assembly yielded an N50 scaffold size of 288 627 base pairs (bp) with >88.6% of the predicted genome covered by scaffolds over 100 000 bp. One of the central differences found between this Australian isolate and the US isolate is the presence of an additional plasmid, pPAA3. This plasmid is similar to pCRY from Yersinia pestis, the causative agent of bubonic plague, and the presence of pPAA3 may account for the increased virulence of Australian isolates both against tissue culture cells and infected patients. The genome of the Kingscliff strain also contains several genomic differences from the US isolate, whose potential significance in virulence against both humans and insects is discussed.
 Abstract.  Author URL
Jones, R.T., Sanchez-Contreras, M., Vlisidou, I., Amos, M.R., Yang, G., Muñoz-Berbel, X., Upadhyay, A., Potter, U.J., Joyce, S.A., Ciche, T.A., et al (2010). Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment. Bmc Microbiol, 10.

Abstract:
Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment.

Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts.
 Abstract.  Author URL
Eleftherianos, I., Joyce, S., Ffrench-Constant, R.H., Clarke, D.J., Reynolds, S.E. (2010). Probing the tri-trophic interaction between insects, nematodes and Photorhabdus. Parasitology, 137(11), 1695-1706.

Abstract:
Probing the tri-trophic interaction between insects, nematodes and Photorhabdus.

Photorhabdus sp. are entomopathogenic bacteria which, upon experimental infection, interact with the insect immune system, but little is known about the roles of their symbiotic nematode partners Heterorhabditis sp. in natural infections. Here, we investigated the respective contributions of nematodes and bacteria by examining humoral and cellular immune reactions of the model lepidopteran insect Manduca sexta against Heterorhabditis carrying Photorhabdus, nematodes free of bacteria (axenic nematodes) and bacteria alone. Insect mortality was slower following infection with axenic nematodes than when insects were infected with nematodes containing Photorhabdus, or the bacteria alone. Nematodes elicited host immune responses to a lesser extent than bacteria. Transcription of certain recognition and antibacterial genes was lower when insects were naturally infected with nematodes carrying no bacteria compared to insects that received bacteria, either with or without nematodes. Axenic nematodes also did not elicit such high levels of phenoloxidase activity and haemocyte aggregates as did treatments involving Photorhabdus. By contrast, the phagocytic capability of host haemocytes was decreased by both axenic and bacteria-associated nematodes, but not by Photorhabdus alone. These results imply that both bacteria and nematodes contribute separately to the pathogenic modulation of host immune responses during natural infections by the mutualistic Heterorhabdus-Photorhabdus complex.
 Abstract.  Author URL
Pauchet, Y., Wilkinson, P., Vogel, H., Nelson, D.R., Reynolds, S.E., Heckel, D.G., ffrench-Constant, R.H. (2010). Pyrosequencing the Manduca sexta larval midgut transcriptome: messages for digestion, detoxification and defence. Insect Mol Biol, 19(1), 61-75.

Abstract:
Pyrosequencing the Manduca sexta larval midgut transcriptome: messages for digestion, detoxification and defence.

The tobacco hornworm Manduca sexta is an important model for insect physiology but genomic and transcriptomic data are currently lacking. Following a recent pyrosequencing study generating immune related expressed sequence tags (ESTs), here we use this new technology to define the M. sexta larval midgut transcriptome. We generated over 387,000 midgut ESTs, using a combination of Sanger and 454 sequencing, and classified predicted proteins into those involved in digestion, detoxification and immunity. In many cases the depth of 454 pyrosequencing coverage allowed us to define the entire cDNA sequence of a particular gene. Many new M. sexta genes are described including up to 36 new cytochrome P450s, some of which have been implicated in the metabolism of host plant-derived nicotine. New lepidopteran gene families such as the beta-fructofuranosidases, previously thought to be restricted to Bombyx mori, are also described. An unexpectedly high number of ESTs were involved in immunity, for example 39 contigs encoding serpins, and the increasingly appreciated role of the midgut in insect immunity is discussed. Similar studies of other tissues will allow for a tissue by tissue description of the M. sexta transcriptome and will form an essential complimentary step on the road to genome sequencing and annotation.
 Abstract.  Author URL
Vlisidou, I., Eleftherianos, I., Dorus, S., Yang, G., ffrench-Constant, R.H., Reynolds, S.E., Waterfield, N.R. (2010). The KdpD/KdpE two-component system of Photorhabdus asymbiotica promotes bacterial survival within M. sexta hemocytes. J Invertebr Pathol, 105(3), 352-362.

Abstract:
The KdpD/KdpE two-component system of Photorhabdus asymbiotica promotes bacterial survival within M. sexta hemocytes.

Many bacteria persist within phagocytes, deploying complex sets of tightly regulated virulence factors to manipulate and survive within host cells. So far, no single factor has been identified that is sufficient to allow intracellular persistence of an otherwise non-pathogenic bacterium. Here we report that the two-component KdpD/KdpE sensor kinase/response regulator of the insect and human pathogen Photorhabdus asymbiotica (Pa) is sufficient to allow a harmless laboratory strain of E. coli to resist phagocytic killing and persist within insect hemocytes, ultimately killing the insect. Screening of a cosmid library of Pa in E. coli by injection into the moth Manduca sexta, previously identified three overlapping clones which caused the insect to cease feeding and subsequently die. Transposon mutagenesis revealed a cosmid encoded kdp high affinity potassium pump regulon was responsible for this phenotype. Gentamycin protection assays and confocal microscopy revealed the cosmid clones were persisting inside insect hemocytes far longer than control bacteria. Cloning and expression of PakdpD/kdpE alone into E. coli recapitulated the phenotype. Bioassay results and transcriptional analysis of various E. coli kdp mutants harboring the Pa kdp genes confirmed that Pa KdpD/KdpE was able to induce the E. coli kdp pump structural genes in response to exposure to insect hemocytes but not blood plasma alone. The finding that Pa KdpD/KdpE can facilitate resistance of E. coli to phagocytic killing suggests a central role for potassium in this process, supporting previous work implicating potassium sensing in virulence of other bacteria and also in the normal process of protease killing of engulfed bacteria by neutrophils.
 Abstract.  Author URL
Eleftherianos, I., Waterfield, N.R., Bone, P., Boundy, S., ffrench-Constant, R.H., Reynolds, S.E. (2009). A single locus from the entomopathogenic bacterium Photorhabdus luminescens inhibits activated Manduca sexta phenoloxidase. Fems Microbiol Lett, 293(2), 170-176.

Abstract:
A single locus from the entomopathogenic bacterium Photorhabdus luminescens inhibits activated Manduca sexta phenoloxidase.

Insect blood (hemolymph) contains prophenoloxidase, a proenzyme that is activated to protective phenoloxidase when the insect is damaged or challenged with microorganisms. The Gram-negative bacterium Photorhabdus luminescens kills the lepidopteron insect Manduca sexta by using a variety of toxins. We screened P. luminescens and Photorhabdus asymbiotica cosmid libraries in an Escherichia coli host against previously activated M. sexta hemolymph phenoloxidase and identified three overlapping cosmid clones from P. luminescens and five from P. asymbiotica that suppressed the activity of the enzyme both in vitro and in vivo. Genome alignments of cosmid end sequences from both species confirmed that they contained orthologous loci. We examined one of the cosmids from P. luminescens in detail: it induced the formation of significantly fewer melanotic nodules, proliferated faster within the insect host and was significantly more virulent towards fifth-stage larvae than E. coli control bacteria. Insertional mutagenesis of this cosmid yielded 11 transposon mutants that were no longer inhibitory. All of these were insertions into a single 5.5-kb locus, which contained three ORFs and was homologous to the maltodextrin phosphorylase locus of E. coli. The implications of this novel inhibitory factor of insect phenoloxidase for Photorhabdus virulence are discussed.
 Abstract.  Author URL
Wilkinson, P., Waterfield, N.R., Crossman, L., Corton, C., Sanchez-Contreras, M., Vlisidou, I., Barron, A., Bignell, A., Clark, L., Ormond, D., et al (2009). Comparative genomics of the emerging human pathogen Photorhabdus asymbiotica with the insect pathogen Photorhabdus luminescens. Bmc Genomics, 10.

Abstract:
Comparative genomics of the emerging human pathogen Photorhabdus asymbiotica with the insect pathogen Photorhabdus luminescens.

The Gram-negative bacterium Photorhabdus asymbiotica (Pa) has been recovered from human infections in both North America and Australia. Recently, Pa has been shown to have a nematode vector that can also infect insects, like its sister species the insect pathogen P. luminescens (Pl). To understand the relationship between pathogenicity to insects and humans in Photorhabdus we have sequenced the complete genome of Pa strain ATCC43949 from North America. This strain (formerly referred to as Xenorhabdus luminescens strain 2) was isolated in 1977 from the blood of an 80 year old female patient with endocarditis, in Maryland, USA. Here we compare the complete genome of Pa ATCC43949 with that of the previously sequenced insect pathogen P. luminescens strain TT01 which was isolated from its entomopathogenic nematode vector collected from soil in Trinidad and Tobago.
 Abstract.  Author URL
Vlisidou, I., Dowling, A.J., Evans, I.R., Waterfield, N., ffrench-Constant, R.H., Wood, W. (2009). Drosophila embryos as model systems for monitoring bacterial infection in real time. Plos Pathog, 5(7).

Abstract:
Drosophila embryos as model systems for monitoring bacterial infection in real time.

Drosophila embryos are well studied developmental microcosms that have been used extensively as models for early development and more recently wound repair. Here we extend this work by looking at embryos as model systems for following bacterial infection in real time. We examine the behaviour of injected pathogenic (Photorhabdus asymbiotica) and non-pathogenic (Escherichia coli) bacteria and their interaction with embryonic hemocytes using time-lapse confocal microscopy. We find that embryonic hemocytes both recognise and phagocytose injected wild type, non-pathogenic E. coli in a Dscam independent manner, proving that embryonic hemocytes are phagocytically competent. In contrast, injection of bacterial cells of the insect pathogen Photorhabdus leads to a rapid 'freezing' phenotype of the hemocytes associated with significant rearrangement of the actin cytoskeleton. This freezing phenotype can be phenocopied by either injection of the purified insecticidal toxin Makes Caterpillars Floppy 1 (Mcf1) or by recombinant E. coli expressing the mcf1 gene. Mcf1 mediated hemocyte freezing is shibire dependent, suggesting that endocytosis is required for Mcf1 toxicity and can be modulated by dominant negative or constitutively active Rac expression, suggesting early and unexpected effects of Mcf1 on the actin cytoskeleton. Together these data show how Drosophila embryos can be used to track bacterial infection in real time and how mutant analysis can be used to genetically dissect the effects of specific bacterial virulence factors.
 Abstract.  Author URL
Ahantarig, A., Chantawat, N., Waterfield, N.R., ffrench-Constant, R., Kittayapong, P. (2009). PirAB toxin from Photorhabdus asymbiotica as a larvicide against dengue vectors. Appl Environ Microbiol, 75(13), 4627-4629.

Abstract:
PirAB toxin from Photorhabdus asymbiotica as a larvicide against dengue vectors.

We have evaluated Photorhabdus insect-related protein (Pir) from Photorhabdus asymbiotica against dengue vectors. PirAB shows larvicidal activity against both Aedes aegypti and Aedes albopictus larvae but did not affect the Mesocyclops thermocyclopoides predator. PirAB expressed the strongest toxicity compared to PirA, PirB, or the mixture of PirA plus PirB. Whether the presence of an enterobacterial repetitive intergenic consensus sequence in PirAB, but not in PirA, PirB, or the mixture of PirA plus PirB, has any impact on biological control efficacy needs further investigation.
 Abstract.  Author URL
Eleftherianos, I., Xu, M., Yadi, H., Ffrench-Constant, R.H., Reynolds, S.E. (2009). Plasmatocyte-spreading peptide (PSP) plays a central role in insect cellular immune defenses against bacterial infection. J Exp Biol, 212(Pt 12), 1840-1848.

Abstract:
Plasmatocyte-spreading peptide (PSP) plays a central role in insect cellular immune defenses against bacterial infection.

Insect hemocytes (blood cells) are a central part of the insect's cellular response to bacterial pathogens, and these specialist cells can both recognize and engulf bacteria. During this process, hemocytes undergo poorly characterized changes in adhesiveness. Previously, a peptide termed plasmatocyte-spreading peptide (PSP), which induces the adhesion and spreading of plasmatocytes on foreign surfaces, has been identified in lepidopteran insects. Here, we investigate the function of this peptide in the moth Manduca sexta using RNA interference (RNAi) to prevent expression of the precursor protein proPSP. We show that infection with the insect-specific bacterial pathogen Photorhabdus luminescens and non-pathogenic Escherichia coli induces proPSP mRNA transcription in the insect fat body but not in hemocytes; subsequently, proPSP protein can be detected in cell-free hemolymph. We used RNAi to silence this upregulation of proPSP and found that the knock-down insects succumbed faster to infection with P. luminescens, but not E. coli. RNAi-treated insects infected with E. coli showed a reduction in the number of circulating hemocytes and higher bacterial growth in hemolymph as well as a reduction in overall cellular immune function compared with infected controls. Interestingly, RNAi-mediated depletion of proPSP adversely affected the formation of melanotic nodules but had no additional effect on other cellular responses when insects were infected with P. luminescens, indicating that this pathogen employs mechanisms that suppress key cellular immune functions in M. sexta. Our results provide evidence for the central role of PSP in M. sexta cellular defenses against bacterial infections.
 Abstract.  Author URL
Pauchet, Y., Wilkinson, P., van Munster, M., Augustin, S., Pauron, D., ffrench-Constant, R.H. (2009). Pyrosequencing of the midgut transcriptome of the poplar leaf beetle Chrysomela tremulae reveals new gene families in Coleoptera. Insect Biochem Mol Biol, 39(5-6), 403-413.

Abstract:
Pyrosequencing of the midgut transcriptome of the poplar leaf beetle Chrysomela tremulae reveals new gene families in Coleoptera.

The insect midgut is the primary target site for Bt-derived insecticides and Bt alternatives. However, despite extensive recent study, the precise role and nature of different Bt receptors remains a subject of considerable debate. This problem is fuelled by a lack of understanding of the genes expressed in the insect midgut and their physiological roles. The poplar leaf beetle, Chrysomela tremulae, is an important model for understanding the mode of action of, and resistance to, coleopteran-specific Bt toxins and currently shows the only known naturally occurring case of resistance to Cry3A toxins. Moreover it belongs to the same family as the corn rootworm, Diabrotica virgifera, an economically important beetle pest and target of hybrid corn expressing Cry3 toxins. Pyrosequencing is a fast and efficient way of defining the transcriptome of specific insect tissues such as the larval midgut. Here we use 454 based pyrosequencing to sample the larval midgut transcriptome of C. tremulae. We identify candidate genes of putative Bt receptors including transcripts encoding cadherin-like proteins, aminopeptidase N and alkaline phosphatase. We also describe a wealth of new transcripts predicting rapidly evolving gene families involved in plant tissue digestion, which have no homologs in the genome of the stored product pest the Red Flour beetle, Tribolium castaneum.
 Abstract.  Author URL
Smith, D.T., Hosken, D.J., Ffrench-Constant, R.H., Wedell, N. (2009). Variation in sex peptide expression in D. melanogaster. Genet Res (camb), 91(4), 237-242.

Abstract:
Variation in sex peptide expression in D. melanogaster.

Male Drosophila melanogaster transfers many accessory-gland proteins to females during copulation. Sex peptide (SP) is one of these and one of its main effects is to decrease female remating propensity. To date, there has been no investigation of genetic variation in SP-gene expression levels, or if such potential variation directly influences female remating behaviour. We assessed both these possibilities and found significant variation in expression levels of the SP gene across D. melanogaster isolines. A non-linear association between SP expression levels and female remating delay suggestive of disruptive selection on expression levels was also documented. Finally, while some isolines were infected with the endosymbiont Wolbachia, no association between Wolbachia and SP expression level was found.
 Abstract.  Author URL
Baxter, S.W., Papa, R., Chamberlain, N., Humphray, S.J., Joron, M., Morrison, C., ffrench-Constant, R.H., McMillan, W.O., Jiggins, C.D. (2008). Convergent evolution in the genetic basis of Müllerian mimicry in heliconius butterflies. Genetics, 180(3), 1567-1577.

Abstract:
Convergent evolution in the genetic basis of Müllerian mimicry in heliconius butterflies.

The neotropical butterflies Heliconius melpomene and H. erato are Müllerian mimics that display the same warningly colored wing patterns in local populations, yet pattern diversity between geographic regions. Linkage mapping has previously shown convergent red wing phenotypes in these species are controlled by loci on homologous chromosomes. Here, AFLP bulk segregant analysis using H. melpomene crosses identified genetic markers tightly linked to two red wing-patterning loci. These markers were used to screen a H. melpomene BAC library and a tile path was assembled spanning one locus completely and part of the second. Concurrently, a similar strategy was used to identify a BAC clone tightly linked to the locus controlling the mimetic red wing phenotypes in H. erato. A methionine rich storage protein (MRSP) gene was identified within this BAC clone, and comparative genetic mapping shows red wing color loci are in homologous regions of the genome of H. erato and H. melpomene. Subtle differences in these convergent phenotypes imply they evolved independently using somewhat different developmental routes, but are nonetheless regulated by the same switch locus. Genetic mapping of MRSP in a third related species, the "tiger" patterned H. numata, has no association with wing patterning and shows no evidence for genomic translocation of wing-patterning loci.
 Abstract.  Author URL
Eleftherianos, I., Baldwin, H., ffrench-Constant, R.H., Reynolds, S.E. (2008). Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus. J Insect Physiol, 54(1), 309-318.

Abstract:
Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus.

In insect pathogen interactions, host developmental stage is among several factors that influence the induction of immune responses. Here, we show that the effectiveness of immune reactions to a pathogen can vary markedly within a single larval stage. Pre-wandering fifth-stage (day 5) larvae of the model lepidopteran insect Manduca sexta succumb faster to infection by the insect pathogenic bacterium Photorhabdus luminescens than newly ecdysed fifth-stage (day 0) caterpillars. The decrease in insect survival of the older larvae is associated with a reduction in both humoral and cellular defence reactions compared to less developed larvae. We present evidence that older fifth-stage larvae are less able to over-transcribe microbial pattern recognition protein and antibacterial effector genes in the fat body and hemocytes. Additionally, older larvae show reduced levels of phenoloxidase (PO) activity in the cell-free hemolymph plasma as well as a dramatic decrease in the number of circulating hemocytes, reduced ability to phagocytose bacteria and fewer melanotic nodules in the infected tissues. The decline in overall immune function of older fifth-stage larvae is reflected by higher bacterial growth in the hemolymph and increased colonization of Photorhabdus on the basal surface of the insect gut. We suggest that developmentally programmed variation in immune competence may have important implications for studies of ecological immunity.
 Abstract.  Author URL
Long, P.R., Zefania, S., Ffrench-Constant, R.H., Szekely, T. (2008). Estimating the population size of an endangered shorebird, the Madagascar plover, using a habitat suitability model. Animal Conservation, 11(2), 118-127. Author URL
Waterfield, N.R., Sanchez-Contreras, M., Eleftherianos, I., Dowling, A., Yang, G., Wilkinson, P., Parkhill, J., Thomson, N., Reynolds, S.E., Bode, H.B., et al (2008). Rapid Virulence Annotation (RVA): identification of virulence factors using a bacterial genome library and multiple invertebrate hosts. Proc Natl Acad Sci U S A, 105(41), 15967-15972.

Abstract:
Rapid Virulence Annotation (RVA): identification of virulence factors using a bacterial genome library and multiple invertebrate hosts.

Current sequence databases now contain numerous whole genome sequences of pathogenic bacteria. However, many of the predicted genes lack any functional annotation. We describe an assumption-free approach, Rapid Virulence Annotation (RVA), for the high-throughput parallel screening of genomic libraries against four different taxa: insects, nematodes, amoeba, and mammalian macrophages. These hosts represent different aspects of both the vertebrate and invertebrate immune system. Here, we apply RVA to the emerging human pathogen Photorhabdus asymbiotica using "gain of toxicity" assays of recombinant Escherichia coli clones. We describe a wealth of potential virulence loci and attribute biological function to several putative genomic islands, which may then be further characterized using conventional molecular techniques. The application of RVA to other pathogen genomes promises to ascribe biological function to otherwise uncharacterized virulence genes.
 Abstract.  Author URL
Hares, M.C., Hinchliffe, S.J., Strong, P.C., Eleftherianos, I., Dowling, A.J., ffrench-Constant, R.H., Waterfield, N. (2008). The Yersinia pseudotuberculosis and Yersinia pestis toxin complex is active against cultured mammalian cells. Microbiology, 154(Pt 11), 3503-3517.

Abstract:
The Yersinia pseudotuberculosis and Yersinia pestis toxin complex is active against cultured mammalian cells.

The toxin complex (Tc) genes were first identified in the insect pathogen Photorhabdus luminescens and encode approximately 1 MDa protein complexes which are toxic to insect pests. Subsequent genome sequencing projects have revealed the presence of tc orthologues in a range of bacterial pathogens known to be associated with insects. Interestingly, members of the mammalian-pathogenic yersiniae have also been shown to encode Tc orthologues. Studies in Yersinia enterocolitica have shown that divergent tc loci either encode insect-active toxins or play a role in colonization of the gut in gastroenteritis models of rats. So far little is known about the activity of the Tc proteins in the other mammalian-pathogenic yersiniae. Here we present work to suggest that Tc proteins in Yersinia pseudotuberculosis and Yersinia pestis are not insecticidal toxins but have evolved for mammalian pathogenicity. We show that Tc is secreted by Y. pseudotuberculosis strain IP32953 during growth in media at 28 degrees C and 37 degrees C. We also demonstrate that oral toxicity of strain IP32953 to Manduca sexta larvae is not due to Tc expression and that lysates of Escherichia coli BL21 expressing the Yersinia Tc proteins are not toxic to Sf9 insect cells but are toxic to cultured mammalian cell lines. Cell lysates of E. coli BL21 expressing the Y. pseudotuberculosis Tc proteins caused actin ruffles, vacuoles and multi-nucleation in cultured human gut cells (Caco-2); similar morphology was observed after application of a lysate of E. coli BL21 expressing the Y. pestis Tc proteins to mouse fibroblast NIH3T3 cells, but not Caco-2 cells. Finally, transient expression of the individual Tc proteins in Caco-2 and NIH3T3 cell lines reproduced the actin and nuclear rearrangement observed with the topical applications. Together these results add weight to the growing hypothesis that the Tc proteins in Y. pseudotuberculosis and Y. pestis have been adapted for mammalian pathogenicity. We further conclude that Tc proteins from Y. pseudotuberculosis and Y. pestis display differential mammalian cell specificity in their toxicity.
 Abstract.  Author URL
McCart, C., Woods, D.J., Terhzaz, S. (2007). A Drosophila systems approach to xenobiotic metabolism. Physiological Genomics, 30, 223-231.
Ffrench-Constant, R.H., Eleftherianos, I., Reynolds, S.E. (2007). A nematode symbiont sheds light on invertebrate immunity. Trends Parasitol, 23(11), 514-517.

Abstract:
A nematode symbiont sheds light on invertebrate immunity.

Photorhabdus bacteria live in a 'symbiosis of pathogens' with nematodes that invade and kill insects. Recent work has begun to use the power of the model insect Drosophila to dissect the molecular basis of the invertebrate immune response to the combined insult of the worms and their symbiotic bacterial pathogens. By using RNA interference, it is now also possible to dissect this complex tripartite interaction in a range of both model and non-model hosts.
 Abstract.  Author URL
Sayed, A., Nekl, E.R., Siqueira, H.A., Wang, H.C., Ffrench-Constant, R.H., Bagley, M., Siegfried, B.D. (2007). A novel cadherin-like gene from western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), larval midgut tissue. Insect Mol Biol, 16(5), 591-600.

Abstract:
A novel cadherin-like gene from western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), larval midgut tissue.

A cadherin-like gene associated with larval midgut tissues was cloned from western corn rootworm (Diabrotica virgifera virgifera: Coleoptera), an economically important agricultural pest in North America and Europe and the primary target pest species for corn hybrids expressing Cry3 toxins from Bacillus thuringiensis (Bt). The full-length cDNA (5371 bp in length) encodes an open reading frame for a 1688 amino acid polypeptide. The putative protein has similar architecture to cadherin-like proteins isolated from lepidopteran midguts that have been shown to bind to Cry1 Bt toxins and have been implicated in Bt resistance. The D. v. virgifera cadherin-like gene is expressed primarily in the larval midgut and regulated during development, with high levels of expression observed in all instars and adults but not pupae. The corresponding genomic sequence spans more than 90 kb and is interspersed with 30 large introns. The genomic organization of the cadherin-like gene for this coleopteran species bears strong resemblance to lepidopteran cadherins suggesting a common molecular basis for susceptibility to Cry3 toxins in Coleoptera.
 Abstract.  Author URL
Boundy, S., Joyce, S., Aslam, S. (2007). An antibiotic produced by an insect-pathogenic bacterium suppresses host defences through phenoloxidase inhibition. Proceedings of the National Academy of Sciences Usa, 104, 2419-2424.
Bogwitz, M.R., McCart, C., Andrianopoulos, A. (2007). Cis-regulatory elements of the Accord retrotransposon result in tissue-specific expression of the Drosophila melanogaster insecticide resistance gene Cyp6g1. Genetics, 175, 1071-1077.
Chamberlain, N., Baxter, S., Jiggins, C., Ffrench-Constant, R.H. (2007). Dissecting butterfly wing pattern formation in Batesian and Mullerian mimicry. Journal of Insect Science, 7Author URL
Lumb, C., Boey, A., Wong, W. (2007). Evaluating the insecticide resistance potential of eight Drosophila melanogaster cytochrome P450 genes by transgenic over-expression. Insect Biochemistry and Molecular Biology, 37, 512-519.
Baxter, S.W., Chamberlain, N., Papa, R., Humphray, S.J., Ffrench-Constant, R.H., McMillan, W.O., Jiggins, C.D. (2007). Identifying DNA markers close to quantitative traits in lepidopteran genomes: Using wing colour variation in Heliconius butterflies as a model. Journal of Insect Science, 7Author URL
ffrench-Constant, R.H., Dowling, A., Waterfield, N.R. (2007). Insecticidal toxins from Photorhabdus bacteria and their potential use in agriculture. Toxicon, 49(4), 436-451.

Abstract:
Insecticidal toxins from Photorhabdus bacteria and their potential use in agriculture.

Most of the insecticidal toxins used in agriculture come from a single bacterium Bacillus thuringiensis or 'Bt'. Here we review our work on the array of toxins produced by Photorhabdus and Xenorhabdus bacteria that are symbiotic with entomopathogenic nematodes, and discuss their potential for use in agriculture as alternatives to Bt. Despite the fact that both Photorhabdus and Xenorhabdus are introduced directly into the insect blood stream by their nematode vectors, they produce a range of toxins with both oral and injectable insecticidal activity. The toxin complexes (Tc's) are large orally active toxins that are displayed on the outer surface of the bacterium. They require three components (A-C) for full toxicity and one 'A' component has been successfully expressed in transgenic Arabidopsis to confer insect resistance. One such group of Tc's, the PirAB binary toxins, have oral activity against mosquitoes and some caterpillar pests. Their mode of action is not known but they show significant sequence similarity to a recently described neurotoxin beta-leptinotarsin-h isolated from the blood of the Colorado potato beetle. Other toxins such as 'makes caterpillars floppy' (Mcf) and proteins encoded by the 'Photorhabdus virulence cassettes' (PVCs) only show injectable activity. Mcf1 promotes apoptosis in a wide range of cells and appears to mimic mammalian BH3 domain-only proteins in the mitochondrion whereas the mode of action of the PVCs remains undetermined. The likely biological reasons for the massive functional redundancy in Photorhabdus insecticidal toxins are discussed.
 Abstract.  Author URL
Eleftherianos, I., Millichap, P.J., Felfoldi, G., Gokcen, F., Waterfield, N., Clarke, D.J., Ffrench-Constant, R.H., Reynolds, S.E. (2007). Probing the insect immune system with the entomopathogen Photorhabdus. Journal of Insect Science, 7Author URL
Dowling, A.J., Waterfield, N.R., Hares, M.C., Le Goff, G., Streuli, C.H., ffrench-Constant, R.H. (2007). The Mcf1 toxin induces apoptosis via the mitochondrial pathway and apoptosis is attenuated by mutation of the BH3-like domain. Cell Microbiol, 9(10), 2470-2484.

Abstract:
The Mcf1 toxin induces apoptosis via the mitochondrial pathway and apoptosis is attenuated by mutation of the BH3-like domain.

Photorhabdus are Gram-negative, nematode-vectored bacteria that produce toxins to kill their insect hosts. The expression of one of these, Makes caterpillars floppy 1 (Mcf1), is sufficient to allow Escherichia coli to survive within, and kill, caterpillars which are otherwise able to clear E. coli infection. Mcf1 treated caterpillars show rapid loss of body turgor (the 'floppy' phenotype) and death is associated with massive apoptosis of both the midgut epithelium and insect phagocytes. Mammalian tissue culture cells treated with Mcf1 also display key features of apoptosis including zeiosis, apoptotic nuclear morphology, DNA laddering, activation of the effector caspase-3 and PARP cleavage. As Mcf1 carries a single BH3-like domain, here we investigate the hypothesis that this toxin promotes apoptosis via the mitochondrial pathway by mimicking a BH3 domain-only protein. Consistent with this hypothesis, a double mutant within the BH3-like domain causes a dramatic decline in apoptosis. Mcf1 also alters mitochondrial membrane potential and triggers the release of cytochrome c. Cells overexpressing Bcl-x(L), an anti-apoptotic Bcl-2 family member, are resistant to Mcf1-mediated apoptosis, as are cells deficient in Bax. In addition, translocation of Bax to the mitochondrion is observed in response to Mcf1 treatment. Together, these results show that Mcf1 mediates apoptosis via the mitochondrial pathway, and are consistent with the hypothesis that the BH3-like domain in Mcf1 is a functional requirement for the pro-apoptotic activity of Mcf1.
 Abstract.  Author URL
Eleftherianos, I., Gökçen, F., Felföldi, G., Millichap, P.J., Trenczek, T.E., ffrench-Constant, R.H., Reynolds, S.E. (2007). The immunoglobulin family protein Hemolin mediates cellular immune responses to bacteria in the insect Manduca sexta. Cell Microbiol, 9(5), 1137-1147.

Abstract:
The immunoglobulin family protein Hemolin mediates cellular immune responses to bacteria in the insect Manduca sexta.

Bacterial recognition in the lepidopteran insect, Manduca sexta, is mediated by pattern recognition proteins including Hemolin, Peptidoglycan recognition protein (PGRP) and Immulectin-2. These proteins bind to molecular patterns present on the surface of bacteria and trigger a protective response involving humoral and cellular reactions. Cellular mechanisms mediated by haemocytes include phagocytosis, encapsulation, and the formation of melanotic nodules. Here, we show that a non-pathogenic strain of Escherichia coli induces mRNA transcription and protein expression of Hemolin and PGRP but not Immulectin-2 in Manduca haemocytes. This upregulation can be effectively prevented (knocked-down) using RNA interference (RNAi) following injection of double-stranded (ds) RNA. Knock-down of Hemolin significantly decreased the ability of insects to clear E. coli from the haemolymph and caused a reduction in the number of free haemocytes. RNAi of Hemolin reduced the ability of haemocytes to engulf bacteria through phagocytosis and to form melanotic nodules in vivo. Importantly, washed haemocytes taken from RNAi-treated insects showed reduced ability to form microaggregates around bacteria in vitro. This shows that the immune function affected by RNAi knock-down of Hemolin is intrinsic to the haemocytes. In contrast, RNAi of PGRP had no effect on any of these cellular immune functions. These results demonstrate the vital role of Hemolin in Manduca cellular immune responses.
 Abstract.  Author URL
Waterfield, N., Hares, M., Hinchliffe, S., Wren, B., ffrench-Constant, R. (2007). The insect toxin complex of Yersinia. Adv Exp Med Biol, 603, 247-257.

Abstract:
The insect toxin complex of Yersinia.

Many members of the Yersinia genus encode homologues of insect toxins first observed in bacteria that are insect pathogens such as Photorhabdus, Xenorhabdus and Serratia entomophila. These bacteria secrete high molecular weight insecticidal toxins comprised of multiple protein subunits, termed the Toxin Complexes or Tc's. In Photorhabdus three distinct Tc subunits are required for full oral toxicity in insects, that include the [A], [B] and [C] types, although the exact stochiometry remains unclear. The genomes of Photorhabdus strains encode multiple tc loci, although only two have been shown to exhibit oral and injectable activity against the Hawk Moth, Manduca sexta. The exact role of the remaining homologues is unclear. The availability of bacterial genome sequences has revealed the presence of tc gene homologues in many different species. In this chapter we review the tc gene homologues in Yersinia genus. We discuss what is known about the activity of the Yersinia Tc protein homologues and attempt to relate this to the evolution of the genus and of the tca gene family.
 Abstract.  Author URL
Felfoldi, G., Eleftherianos, I., Ffrench-Constant, R.H., Venekei, I., Reynolds, S.E. (2007). The role of serineprotease homolgue 3 (SPH-3) in Manduca sexta shown by RNA interference. Journal of Insect Science, 7Author URL
Ffrench-Constant, R.H. (2007). Which came first: insecticides or resistance?. Trends Genet, 23(1), 1-4.

Abstract:
Which came first: insecticides or resistance?

Mutations that confer resistance to insecticides are well documented. However, so far, we have been unable to determine whether these mutations arose before or after the introduction of insecticides. Recently, a landmark study showed that resistance to Malathion can be detected in pinned specimens of Australian sheep blowflies that were collected before the introduction of the insecticide. This finding has numerous implications for our understanding of the prevalence of resistance to new compounds. It also indicates that pre-existing resistance alleles might not carry the fitness cost that is associated with new mutations.
 Abstract.  Author URL
Ffrench-Constant, R.H., Chamberlain, N., Joron, M., Papa, R. (2006). A conserved supergene locus controls colour pattern diversity in Heliconus butterflies. Plos Biology, 4(10), 1831-1840.
Ffrench-Constant, R., Waterfield, N. (2006). An ABC guide to the bacterial toxin complexes. Adv Appl Microbiol, 58, 169-183. Author URL
ffrench-Constant, R.H., Waterfield, N.R. (2006). Ground control for insect pests. Nat Biotechnol, 24(6), 660-661. Author URL
Jenkins, A.T., Dash, H.A., Boundy, S., Halliwell, C.M., ffrench-Constant, R.H. (2006). Methoxy-resorufin ether as an electrochemically active biological probe for cytochrome P450 O-demethylation. Bioelectrochemistry, 68(1), 67-71.

Abstract:
Methoxy-resorufin ether as an electrochemically active biological probe for cytochrome P450 O-demethylation.

This paper describes the utilisation of methoxy-resorufin ether as an electrochemical probe for studying cytochrome P450 CYP6G1. Methoxy-resorufin ether is well established as a versatile substrate for cytochrome P450, as its demethylated product, resorufin, is a fluorophore. We show that in addition to these established properties, methoxy-resorufin ether also exhibits reversible two electron transfer on glassy carbon and edge plane graphite electrodes. Cyclic voltammetry measurements and differential pulse voltammetry measurements show that methoxy-resorufin ether can be easily detected at low concentrations (down to 200 nM) in a conventional three electrode electrochemical cell. These properties of methoxy-resorufin ether mean that it could be used as an electrochemical probe, to follow the rate of its demethylation by CYP6G1. We show that electrochemical measurements could discriminate between the enzyme activity of protein microsomes taken from two strains of Drosophila melanogaster (fruit fly).
 Abstract.  Author URL
Gerrard, J.G., Joyce, S.A., Clarke, D.J., ffrench-Constant, R.H., Nimmo, G.R., Looke, D.F., Feil, E.J., Pearce, L., Waterfield, N.R. (2006). Nematode symbiont for Photorhabdus asymbiotica. Emerg Infect Dis, 12(10), 1562-1564.

Abstract:
Nematode symbiont for Photorhabdus asymbiotica.

Photorhabdus asymbiotica is an emerging bacterial pathogen that causes locally invasive soft tissue and disseminated bacteremic infections in the United States and Australia. Although the source of infection was previously unknown, we report that the bacterium is found in a symbiotic association with an insect-pathogenic soil nematode of the genus Heterorhabditis.
 Abstract.  Author URL
Yang, G., Dowling, A.J., Gerike, U., ffrench-Constant, R.H., Waterfield, N.R. (2006). Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth. J Bacteriol, 188(6), 2254-2261.

Abstract:
Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth.

Two recently sequenced genomes of the insect-pathogenic bacterium Photorhabdus and a large Serratia entomophila plasmid, pADAP, have phage-related loci containing putative toxin effector genes, designated the "Photorhabdus virulence cassettes" (PVCs). In S. entomophila, the single plasmid PVC confers antifeeding activity on larvae of a beetle. Here, we show that recombinant Escherichia coli expressing PVC-containing cosmids from Photorhabdus has injectable insecticidal activity against larvae of the wax moth. Electron microscopy showed that the structure of the PVC products is similar to the structure of the antibacterial R-type pyocins. However, unlike these bacteriocins, the PVC products of Photorhabdus have no demonstrable antibacterial activity. Instead, injection of Photorhabdus PVC products destroys insect hemocytes, which undergo dramatic actin cytoskeleton condensation. Comparison of the genomic organizations of several PVCs showed that they have a conserved phage-like structure with a variable number of putative anti-insect effectors encoded at one end. Expression of these putative effectors directly inside cultured cells showed that they are capable of rearranging the actin cytoskeleton. Together, these data show that the PVCs are functional homologs of the S. entomophila antifeeding genes and encode physical structures that resemble bacteriocins. This raises the interesting hypothesis that the PVC products are bacteriocin-like but that they have been modified to attack eukaryotic host cells.
 Abstract.  Author URL
Eleftherianos, I., Marokhazi, J., Millichap, P.J., Hodgkinson, A.J., Sriboonlert, A., ffrench-Constant, R.H., Reynolds, S.E. (2006). Prior infection of Manduca sexta with non-pathogenic Escherichia coli elicits immunity to pathogenic Photorhabdus luminescens: roles of immune-related proteins shown by RNA interference. Insect Biochem Mol Biol, 36(6), 517-525.

Abstract:
Prior infection of Manduca sexta with non-pathogenic Escherichia coli elicits immunity to pathogenic Photorhabdus luminescens: roles of immune-related proteins shown by RNA interference.

Prior infection of Manduca sexta caterpillars with the non-pathogenic bacterium Escherichia coli elicits effective immunity against subsequent infection by the usually lethal and highly virulent insect pathogen Photorhabdus luminescens TT01. Induction of this protective effect is associated with up-regulation of both microbial pattern recognition protein genes (hemolin, immulectin-2 and peptidoglycan recognition protein) and anti-bacterial effector genes (attacin, cecropin, lebocin, lysozyme and moricin). We used RNA interference to knock down over-transcription of members of both these sets of genes one at a time. Interfering with expression of individual recognition proteins had a drastic adverse effect on the E. coli elicited immunity. RNAi knock-down of immulectin-2 caused the greatest reduction in immunity, followed by hemolin and peptidoglycan recognition protein (PGRP) in that order, to the extent that knock-down of any one of these three proteins left the insects more susceptible to P. luminescens infection than insects that had not experienced prior infection with E. coli. Interfering with the expression of individual antibacterial effector proteins and peptides had a much less marked effect on immunity. Knock-down of attacin, cecropin or moricin caused treated insects to be more susceptible to P. luminescens infection than controls that had been pre-infected with E. coli but which had not received the specific RNAi reagents, but they were still less susceptible than insects that had not been pre-infected with E. coli. RNAi knock-down with expression of lebocin or lysozyme had no effect on E. coli-induced immunity to P. luminescens, indicating that these effectors are not involved in the response. By bleeding pre-infected caterpillars and growing the pathogen directly within cell-free insect haemolymph, we showed that at least part of the protection elicited by previous exposure to E. coli is due to the presence of factors within the blood plasma that inhibit the growth of P. luminescens. The production of these factors is inhibited by RNAi treatment with ds-RNA reagents that knock down hemolin, immulectin-2, and PGRP. These results demonstrate that the insect immune system can be effectively primed by prior infection with non-pathogenic bacteria against subsequent infection by a highly virulent pathogen. Given the continuous normal exposure of insects to environmental and symbiotic bacteria, we suggest that prior infection is likely to play a significant and underestimated role in determining the level of insect immunity found in nature.
 Abstract.  Author URL
Eleftherianos, I., Millichap, P.J., ffrench-Constant, R.H., Reynolds, S.E. (2006). RNAi suppression of recognition protein mediated immune responses in the tobacco hornworm Manduca sexta causes increased susceptibility to the insect pathogen Photorhabdus. Developmental and Comparative Immunology, 30(12), 1099-1107. Author URL
Eleftherianos, I., Millichap, P.J., ffrench-Constant, R.H., Reynolds, S.E. (2006). RNAi suppression of recognition protein mediated immune responses in the tobacco hornworm Manduca sexta causes increased susceptibility to the insect pathogen Photorhabdus. Dev Comp Immunol, 30(12), 1099-1107.

Abstract:
RNAi suppression of recognition protein mediated immune responses in the tobacco hornworm Manduca sexta causes increased susceptibility to the insect pathogen Photorhabdus.

Bacterial pathogens either hide from or overcome the immune response of their hosts. Here we show that two different species of insect pathogenic bacteria, Photorhabdus luminescens TT01 and Photorhabdus asymbiotica ATCC43949, were both recognized by the immune system of their host Manduca sexta, as indicated by a rapid increase in the levels of mRNAs encoding three different inducible microbial recognition proteins, Hemolin, Immulectin-2 and peptidoglycan recognition protein. RNA interference (RNAi)-mediated inhibition of expression ("knock-down") of each of these genes at the level of both mRNA and protein was achieved through injection of double-stranded RNA (dsRNA). Knock-down of any one of these genes markedly decreased the ability of the insects to withstand infection when exposed to either species of Photorhabdus, as measured by the rate at which infected insects died. RNAi against Immulectin-2 caused the greatest reduction in host resistance to infection. The decreased resistance to infection was associated with reduced hemolymph phenoloxidase activity. These results show not only that Photorhabdus is recognized by the Manduca sexta immune system but also that the insect's immune system plays an active, but ultimately ineffective, role in countering infection.
 Abstract.  Author URL
ffrench-Constant, R., Daborn, P., Feyereisen, R. (2006). Resistance and the jumping gene. Bioessays, 28(1), 6-8.

Abstract:
Resistance and the jumping gene.

Transposons are well-known architects of genetic change but their role in insecticide resistance has, until recently, only been speculated upon. Transposon insertion, or transposon-mediated transposition, could alter either metabolic enzymes capable of degrading pesticides or could change the functionality of insecticide targets. The recent work of Aminetzach and coworkers suggests an exciting alternative, that transposon insertion can cause resistance by altering gene product function. This hypothesis is discussed in the light of other examples in which transposons have been implicated in insecticide resistance.
 Abstract.  Author URL
Le Goff, G., Hilliou, F., Siegfried, B.D., Boundy, S., Wajnberg, E., Sofer, L., Audant, P., Ffrench-Constant, R.H., Feyereisen, R. (2006). Xenobiotic response in Drosophila melanogaster: Sex dependence of P450 and GST gene induction. Insect Biochemistry and Molecular Biology, 36(8), 674-682. Author URL
Le Goff, G., Hilliou, F., Siegfried, B.D., Boundy, S., Wajnberg, E., Sofer, L., Audant, P., ffrench-Constant, R.H., Feyereisen, R. (2006). Xenobiotic response in Drosophila melanogaster: sex dependence of P450 and GST gene induction. Insect Biochem Mol Biol, 36(8), 674-682.

Abstract:
Xenobiotic response in Drosophila melanogaster: sex dependence of P450 and GST gene induction.

The effect of xenobiotics (phenobarbital and atrazine) on the expression of Drosophila melanogaster CYP genes encoding cytochromes P450, a gene family generally associated with detoxification, was analyzed by DNA microarray hybridization and verified by real-time RT-PCR in adults of both sexes. Only a small subset of the 86 CYP genes was significantly induced by the xenobiotics. Eleven CYP genes and three glutathione S-transferases (GST) genes were significantly induced by phenobarbital, seven CYP and one GST gene were induced by atrazine. Cyp6d5, Cyp6w1, Cyp12d1 and the ecdysone-inducible Cyp6a2 were induced by both chemicals. The constitutive expression of several of the inducible genes (Cyp6a2, Cyp6a8, Cyp6d5, Cyp12d1) was higher in males than in females, and the induced level similar in both sexes. Thus, the level of induction was consistently higher in females than in males. The female-specific and hormonally regulated yolk protein genes were significantly induced by phenobarbital in males and repressed by atrazine in females. Our results suggest that the numerous CYP genes of Drosophila respond selectively to xenobiotics, providing the fly with an adaptive response to chemically adverse environments. The xenobiotic inducibility of some CYP genes previously associated with insecticide resistance in laboratory-selected strains (Cyp6a2, Cyp6a8, Cyp12d1) suggests that deregulation of P450 gene expression may be a facile way to achieve resistance. Our study also suggests that xenobiotic-induced changes in P450 levels can affect insect fitness by interfering with hormonally regulated networks.
 Abstract.  Author URL
Ffrench-Constant, R., Waterfield, N. (2005). An ABC Guide to the Bacterial Toxin Complexes. Adv Appl Microbiol, 58C, 169-183. Author URL
Buckling, A., McGhee, M., ffrench-constant, R.H. (2005). DDT resistance in flies carries no cost. Current Biology, 15(15), 587-589.
McCart, C., Buckling, A., Ffrench-Constant, R.H. (2005). DDT resistance in flies carries no cost. Curr Biol, 15(15), R587-R589. Author URL
Siegfried, B.D., Waterfield, N., ffrench-Constant, R.H. (2005). Expressed sequence tags from Diabrotica virgifera virgifera midgut identify a coleopteran cadherin and a diversity of cathepsins. Insect Molecular Biology, 14(2), 137-143. Author URL
Siegfried, B.D., Waterfield, N., ffrench-Constant, R.H. (2005). Expressed sequence tags from Diabrotica virgifera virgifera midgut identify a coleopteran cadherin and a diversity of cathepsins. Insect Mol Biol, 14(2), 137-143.

Abstract:
Expressed sequence tags from Diabrotica virgifera virgifera midgut identify a coleopteran cadherin and a diversity of cathepsins.

The Western corn rootworm is the major pest of corn in the USA and has recently become the target for insect-resistant transgenic crops. Transgenic crops have switched the focus for identifying insecticide targets from the insect nervous system to the midgut. Here we describe a collection of 691 sequences from the Western corn rootworm midgut, 27% of which predict proteins with no matches in current databases. of the remaining sequences, most predict proteins with either catalytic (62%) or binding (19%) functions, as expected for proteins expressed in the insect midgut. The utility of this approach for the identification of targets for novel toxins is demonstrated by analysis of the first coleopteran cadherin gene, a putative Bt receptor, and a large class of cysteine-proteases, the cathepsins.
 Abstract.  Author URL
Eleftherianos, I., ffrench-Constant, R.H., Reynolds, S.E. (2005). Modulating lepidopteran immunity to bacteria using pre-infection and RNA interference. Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, 141(3), S109-S109. Author URL
Waterfield, N., Hares, M., Yang, G., Dowling, A., ffrench-Constant, R. (2005). Potentiation and cellular phenotypes of the insecticidal Toxin complexes of Photorhabdus bacteria. Cell Microbiol, 7(3), 373-382.

Abstract:
Potentiation and cellular phenotypes of the insecticidal Toxin complexes of Photorhabdus bacteria.

The toxin complex (tc) genes of bacteria comprise a large and growing family whose mode of action remains obscure. In the insect pathogen Photorhabdus, tc genes encode high molecular weight insecticidal toxins with oral activity against caterpillar pests. One protein, TcdA, has recently been expressed in transgenic plants and shown to confer insect resistance. These toxins therefore represent alternatives to toxins from Bacillus thuringiensis (Bt) for deployment in transgenic crops. Levels of TcdA expression in transgenic plants were, however, low and the full toxicity associated with the native toxin was not reconstituted. Here we show that increased activity of the toxin TcdA1 requires potentiation by either of two pairs of gene products, TcdB1 and TccC1 or TcdB2 and TccC3. Moreover, these same pairs of proteins can also cross-potentiate a second toxin, TcaA1B1. To elucidate the likely functional domains present in these large proteins, we expressed fragments of each 'toxin' or 'potentiator' gene within mammalian cells. Several domains produced abnormal cellular morphologies leading to cell death, while others showed specific phenotypes such as nuclear translocation. Our results prove that the Tc toxins are complex proteins with multiple functional domains. They also show that both toxin genes and their potentiator pairs will need to be expressed to reconstitute full activity in insect-resistant transgenic plants. Moreover, they suggest that the same potentiator pair will be able to cross-potentiate more than one toxin in a single plant.
 Abstract.  Author URL
Ffrench-Constant, R.H. (2005). Something old, something transgenic, or something fungal for mosquito control?. Trends Ecol Evol, 20(11), 577-579.

Abstract:
Something old, something transgenic, or something fungal for mosquito control?

In spite of the current research emphasis on the use of transgenic mosquitoes, insecticides are still the main method for controlling malarial mosquitoes. Although pyrethroids are the compounds of choice, insecticide resistance is now threatening the effective life of these invaluable compounds. Two recent studies have re-focused interest on entomopathogenic fungi as useful alternatives to conventional insecticides, suggesting that these fungi could be used as alternative control methods, which would thus also prolong the effective lifetime of pyrethroids.
 Abstract.  Author URL
Jenkins, A.T., Buckling, A., McGhee, M., ffrench-Constant, R.H. (2005). Surface plasmon resonance shows that type IV pili are important in surface attachment by Pseudomonas aeruginosa. J R Soc Interface, 2(3), 255-259.

Abstract:
Surface plasmon resonance shows that type IV pili are important in surface attachment by Pseudomonas aeruginosa.

Type IV pili have been shown to play a role in the early stages of bacterial biofilm formation, but not in initial bacterial attachment. Here, using the surface analytical technique, surface plasmon resonance (SPR), we follow the attachment of the bacterium Pseudomonas aeruginosa in real time. In contrast to previous studies, we show that type IV pili mutants are defective in attachment. Both mutants lacking pili (pilA), and those possessing an overabundance of pili (pilT), showed reduced SPR measured attachment compared with the wild-type PAO1 strain. Both pil mutants also showed reduced pathogenicity in a model insect host, as measured by percentage mortality after 24h. SPR revealed differences in the kinetics of attachment between pilA and pilT, differences obscured by endpoint assays using crystal violet stain. These results highlight the power of SPR in monitoring bacterial attachment in real time and also demonstrate an additional role for type IV pili beyond bacterial aggregation and micro-colony formation.
 Abstract.  Author URL
Waterfield, N., Kamita, S.G., Hammock, B.D., ffrench-Constant, R. (2005). The Photorhabdus Pir toxins are similar to a developmentally regulated insect protein but show no juvenile hormone esterase activity. Fems Microbiol Lett, 245(1), 47-52.

Abstract:
The Photorhabdus Pir toxins are similar to a developmentally regulated insect protein but show no juvenile hormone esterase activity.

The genome of the insect pathogen Photorhabdus luminescens strain TT01 contains numerous genes predicting toxins and proteases. Within the P. luminescens TT01 genome, the products of two loci, plu 4093-plu 4092 and plu 4437-plu 4436, show oral insecticidal activity against both moth and mosquito larvae. The proteins encoded by these loci, here termed 'Photorhabdus insect related' (Pir) proteins a and B, show similarity both to delta-endotoxins from Bacillus thuringiensis (Bts) and a developmentally regulated protein from a beetle, Leptinotarsa decemlineata. The beetle protein has been inferred to possess juvenile hormone esterase (JHE) activity due to its developmentally regulated pattern of expression and the Photorhabdus proteins PirA and PirB have been proposed to be mimics of insect JHEs that can disrupt insect metamorphosis by metabolizing the insect growth regulator juvenile hormone (JH) [Nat. Biotechnol. 21 (2003) 1307-1313]. Here we confirm that, when injected together, PirA and PirB from two different Photorhabdus strains have insecticidal activity against caterpillars of the moth Galleria mellonella but show no oral activity against a second moth species Manduca sexta. Direct measurement of JHE activity, however, shows that the Pir proteins are not able to metabolise JH. These data show that the Pir proteins have no JHE activity, as suggested, but leave the mode of action of these interesting proteins uncertain.
 Abstract.  Author URL
Daborn, P., McCart, C., Woods, D., ffrench-Constant, R.H. (2004). Detection of insecticide resistance-associated mutations in cat flea Rdl by TaqMan-allele specific amplification. Pesticide Biochemistry and Physiology, 79(1), 25-30. Author URL
Au, C., Dean, P., Reynolds, S.E., ffrench-Constant, R.H. (2004). Effect of the insect pathogenic bacterium Photorhabdus on insect phagocytes. Cell Microbiol, 6(1), 89-95.

Abstract:
Effect of the insect pathogenic bacterium Photorhabdus on insect phagocytes.

Photorhabdus are insect pathogenic bacteria that replicate within the insect haemocoel following release from their entomopathogenic nematode symbionts. To investigate how they escape the cellular immune response we examined the effects of two strains of Photorhabdus, W14 and K122, on Manduca sexta phagocytes (haemocytes), in vitro and in vivo. Following injection of Esherichia coli into Manduca larvae, these non-pathogenic bacteria are rapidly cleared from the haemolymph and the number of free haemocytes transiently increases. In contrast, following injection of either strain of pathogenic Photorhabdus, the bacteria grow rapidly while the number of haemocytes decreases dramatically. In vitro incubation of haemocytes with either Photorhabdus supernatant reduced haemocyte viability, and the W14 supernatant caused distinct changes in the actin cytoskeleton morphology of different haemocyte cell types. In phagocytosis assays both Photorhabdus strains can inhibit their own phagocytosis whether the bacterial cells are alive or dead. Further, the supernatant of W14 also contains a factor capable of inhibiting the phagocytosis of labelled E. coli. Together these results suggest that Photorhabdus evades the cellular immune response by killing haemocytes and suppressing phagocytosis by mechanisms that differ between strains.
 Abstract.  Author URL
Massey, R.C., Buckling, A., ffrench-Constant, R. (2004). Interference competition and parasite virulence. Proc Biol Sci, 271(1541), 785-788.

Abstract:
Interference competition and parasite virulence.

Within-host competition between parasites, a consequence of infection by multiple strains, is predicted to favour rapid host exploitation and greater damage to hosts (virulence). However, the inclusion of biological variables can drastically change this relationship. For example, if competing parasite strains produce toxins that kill each other (interference competition), their growth rates and virulence may be reduced relative to single-strain infections. Bacteriocins are antimicrobial toxins produced by bacteria that target closely related strains and species, and to which the producing strain is immune. We investigated competition between bacteriocin-producing, insect-killing bacteria (Photorhabdus and Xenorhabdus) and how this competition affected virulence in caterpillars. Where one strain could kill the other, and not vice versa, the non-killing strain was competitively excluded, and insect mortality was the same as that of the killing strain alone. However, when caterpillars were multiply infected by strains that could kill each other, we did not observe competitive exclusion and their virulence was less than single-strain infections. The ubiquity and diversity of bacteriocins among pathogenic bacteria suggest mixed infections will be, on average, less virulent than single infections.
 Abstract.  Author URL
Waterfield, N.R., Wren, B.W., Ffrench-Constant, R.H. (2004). Invertebrates as a source of emerging human pathogens. Nat Rev Microbiol, 2(10), 833-841.

Abstract:
Invertebrates as a source of emerging human pathogens.

Despite their importance, little is known about the origins of many emerging human pathogens. However, given the age and current predominance of invertebrates, it is likely that bacteria-invertebrate interactions are not only a present source of human pathogens but have also shaped their evolution. Pathogens of invertebrate and unicellular organisms represent an extensive reservoir of bacterial strains equipped with virulence factors that evolved to overcome the innate immune responses of their hosts. This reservoir might represent a source of new human pathogenic strains and might also foster the spread of novel virulence factors into existing human commensal or pathogenic bacteria. This article examines the available evidence for this concept by examining pairs of closely related bacteria, one of which is benign, but insect associated, and one of which is a human pathogen.
 Abstract.  Author URL
Jenkins, A.T., ffrench-constant, R., Buckling, A., Clarke, D.J., Jarvis, K. (2004). Study of the attachment of Pseudomonas aeruginosa on gold and modified gold surfaces using surface plasmon resonance. Biotechnol Prog, 20(4), 1233-1236.

Abstract:
Study of the attachment of Pseudomonas aeruginosa on gold and modified gold surfaces using surface plasmon resonance.

This paper describes how the technique of surface plasmon resonance (SPR) can be utilized to follow (in real time) the attachment of Pseudomonas aeruginosa bacteria on bare gold and gold modified with a self-assembled monolayer (SAM) of mercaptounadecanoic acid. We show that SPR is able to discriminate between the adsorption of live versus dead (thermally shocked) bacteria. Moreover, the SPR distinguishes between the adsorption of wild-type versus mutant bacteria (single gene knockouts), the concentration of the bacterial suspension, and between bacteria adsorbing on SAM-modified and bare gold. SPR is able to measure bacterial adsorption within seconds of the bacterial suspension being introduced. Finally, a qualitative correlation between results from SPR with a crystal violet staining assay for different mutant bacteria was observed.
 Abstract.  Author URL
Ffrench-Constant, R.H., Daborn, P.J., Le Goff, G. (2004). The genetics and genomics of insecticide resistance. Trends Genet, 20(3), 163-170.

Abstract:
The genetics and genomics of insecticide resistance.

The past ten years have seen the elucidation of the molecular basis of insect resistance to many chemical insecticides. Target genes, mostly in the nervous system, have been identified and cloned from Drosophila melanogaster and resistance-associated mutations have been examined in a range of pest insects. More recently, with the advent of annotated insect genomes, resistance mediated by complex multi-gene enzyme systems such as esterases, cytochrome p450s and glutathione-S-transferases has also been elucidated. In this article, we review the impact of Drosophila genetics on the field of insect resistance and focus on the current and future impact of genomics. These studies enable us to address three fundamental questions in the evolution of resistance. How many genes are involved? How many mutations are there within these genes? How often do these mutations arise in natural populations?
 Abstract.  Author URL
Ffrench-Constant, R.H., Daborn, P.J., Dowling, A.J., Steuli, C.H. (2004). The insecticidal toxin Makes caterpillars floppy (Mcf) promotes apoptosis in mammalian cells. Cellular Microbiology, 6(4), 345-353.
Dowling, A.J., Daborn, P.J., Waterfield, N.R., Wang, P., Streuli, C.H., ffrench-Constant, R.H. (2004). The insecticidal toxin Makes caterpillars floppy (Mcf) promotes apoptosis in mammalian cells. Cellular Microbiology, 6(4), 345-353. Author URL
Catania, F., Kauer, M.O. (2004). World-wide survey of an Accord insertion and its association with DDT resistance in Drosophila Melanogaster. Molecular Ecology, 13(8), 2491-2504.
Bowen, D.J., Rocheleau, T.A., Grutzmacher, C.K., Meslet, L., Valens, M., Marble, D., Dowling, A., Ffrench-Constant, R., Blight, M.A. (2003). Genetic and biochemical characterization of PrtA, an RTX-like metalloprotease from Photorhabdus. Microbiology, 149(Pt 6), 1581-1591.

Abstract:
Genetic and biochemical characterization of PrtA, an RTX-like metalloprotease from Photorhabdus.

Proteases play a key role in the interaction between pathogens and their hosts. The bacterial entomopathogen Photorhabdus lives in symbiosis with nematodes that invade insects. Following entry into the insect, the bacteria are released from the nematode gut into the open blood system of the insect. Here they secrete factors which kill the host and also convert the host tissues into food for the replicating bacteria and nematodes. One of the secreted proteins is PrtA, which is shown here to be a repeats-in-toxin (RTX) alkaline zinc metalloprotease. PrtA has high affinity for artificial substrates such as casein and gelatin and can be inhibited by zinc metalloprotease inhibitors. The metalloprotease also shows a calcium- and temperature-dependent autolysis. The prtA gene carries the characteristic RTX repeated motifs and predicts high similarity to proteases from Erwinia chrysanthemi, Pseudomonas aeruginosa and Serratia marcescens. The prtA gene resides in a locus encoding both the protease ABC transporter (prtBCD) and an intervening ORF encoding a protease inhibitor (inh). PrtA activity is detectable 24 h after artificial bacterial infection of an insect, suggesting that the protease may play a key role in degrading insect tissues rather than in overcoming the insect immune system. Purified PrtA also shows cytotoxicity to mammalian cell cultures, supporting its proposed role in bioconversion of the insect cadaver into food for bacterial and nematode development.
 Abstract.  Author URL
Le Goff, G., Boundy, S., Daborn, P.J., Yen, J.L., Sofer, L., Lind, R., Sabourault, C., Madi-Ravazzi, L., ffrench-Constant, R.H. (2003). Microarray analysis of cytochrome P450 mediated insecticide resistance in Drosophila. Insect Biochem Mol Biol, 33(7), 701-708.

Abstract:
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.
 Abstract.  Author URL
ffrench-Constant, R., Waterfield, N., Daborn, P., Joyce, S., Bennett, H., Au, C., Dowling, A., Boundy, S., Reynolds, S., Clarke, D., et al (2003). Photorhabdus: towards a functional genomic analysis of a symbiont and pathogen. Fems Microbiol Rev, 26(5), 433-456.

Abstract:
Photorhabdus: towards a functional genomic analysis of a symbiont and pathogen.

Pathogenicity and symbiosis are central to bacteria-host interactions. Although several human pathogens have been subjected to functional genomic analysis, we still understand little about bacteria-invertebrate interactions despite their ecological prevalence. Advances in our knowledge of this area are often hindered by the difficulty of isolating and working with invertebrate pathogenic bacteria and their hosts. Here we review studies on pathogenicity and symbiosis in an insect pathogenic bacterium Photorhabdus and its entomopathogenic nematode vector and model insect hosts. Whilst switching between these hosts, Photorhabdus changes from a state of symbiosis with its nematode vector to one of pathogenicity towards its new insect host and both the bacteria and the nematode then cooperatively exploit the dying insect. We examine candidate genes involved in symbiosis and pathogenicity, their secretion and expression patterns in culture and in the host, and begin to dissect the extent of their genetic coregulation. We describe the presence of several large genomic islands, putatively involved in pathogenicity or symbiosis, within the otherwise Yersinia-like backbone of the Photorhabdus genome. Finally, we examine the emerging comparative genomics of the Photorhabdus group and begin to describe the interrelationship between anti-invertebrate virulence factors and those used against vertebrates.
 Abstract.  Author URL
Waterfield, N.R., Daborn, P.J., Dowling, A.J., Yang, G., Hares, M., ffrench-Constant, R.H. (2003). The insecticidal toxin makes caterpillars floppy 2 (Mcf2) shows similarity to HrmA, an avirulence protein from a plant pathogen. Fems Microbiol Lett, 229(2), 265-270.

Abstract:
The insecticidal toxin makes caterpillars floppy 2 (Mcf2) shows similarity to HrmA, an avirulence protein from a plant pathogen.

The Photorhabdus luminescens W14 toxin encoding gene makes caterpillars floppy (mcf) was discovered due to its ability to kill caterpillars when expressed in Escherichia coli. Here we describe a homologue of mcf (renamed as mcf1), termed mcf2, discovered in the same genome. The mcf2 gene predicts another large toxin whose central domain, like Mcf1, also shows limited homology to Clostridium cytotoxin B. However, the N-terminus of Mcf2 shows significant similarity to the type-III secreted effector HrmA from the plant pathogen Pseudomonas syringae and no similarity to the N-terminus of Mcf1. HrmA is a plant avirulence gene whose transient expression in tobacco cells results in cell death. Here we show that E. coli expressing Mcf2 can, like E. coli expressing Mcf1, kill insects. Further, expression of the c-Myc tagged N-terminus of Mcf2, the region showing similarity to HrmA, results in nuclear localisation of the fusion protein and subsequent destruction of transfected mammalian cells. The Mcf1 and Mcf2 toxins therefore belong to a family of high molecular mass toxins, differing at their N-termini, which encode different effector domains.
 Abstract.  Author URL
Marokhazi, J., Waterfield, N., LeGoff, G., Feil, E., Stabler, R., Hinds, J., Fodor, A., ffrench-Constant, R.H. (2003). Using a DNA microarray to investigate the distribution of insect virulence factors in strains of photorhabdus bacteria. J Bacteriol, 185(15), 4648-4656.

Abstract:
Using a DNA microarray to investigate the distribution of insect virulence factors in strains of photorhabdus bacteria.

Photorhabdus is an insect-pathogenic bacterium in which oral toxicity to insects is found in two distinct taxonomic groups. Using a DNA microarray and comparative genomics, we show that oral toxicity is associated with toxin complex genes tcaABC and that this locus can be mobilized or deleted within different strains.
 Abstract.  Author URL
Ffrench-Constant, R.H., Bogwitz, M., Daborn, P.J., Yen, J.L. (2002). A single P450 allele associated with insecticide resistance in Drosophila. Science, 297(5590), 2253-2256.
Ffrench-Constant, R.H., Daborn, P.J., Silva, C.P., Waterfield, N. (2002). A single Photorhabdus gene, makes caterpillars floppy (mfc), allows Escherichia coli to persist within and kill insects. Proceedings of the National Academy of Sciences, 99(16), 10742-10747.
Silva, C.P., Waterfield, N.R., Daborn, P.J., Dean, P., Chilver, T., Au, C.P., Sharma, S., Potter, U., Reynolds, S.E., ffrench-Constant, R.H., et al (2002). Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta. Cell Microbiol, 4(6), 329-339.

Abstract:
Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta.

Invertebrates, including insects, are being developed as model systems for the study of bacterial virulence. However, we understand little of the interaction between bacteria and specific invertebrate tissues or the immune system. To establish an infection model for Photorhabdus, which is released directly into the insect blood system by its nematode symbiont, we document the number and location of recoverable bacteria found during infection of Manduca sexta. After injection into the insect larva, P. luminescens multiplies in both the midgut and haemolymph, only later colonizing the fat body and the remaining tissues of the cadaver. Bacteria persist by suppressing haemocyte-mediated phagocytosis and culture supernatants grown in vitro, as well as plasma from infected insects, suppress phagocytosis of P. luminescens. Using GFP-labelled bacteria, we show that colonization of the gut begins at the anterior of the midgut and proceeds posteriorly. Within the midgut, P. luminescens occupies a specific niche between the extracellular matrix and basal membrane (lamina) of the folded midgut epithelium. Here, the bacteria express the gut-active Toxin complex a (Tca) and an RTX-like metalloprotease PrtA. This close association of the bacteria with the gut, and the production of toxins and protease, triggers a massive programmed cell death of the midgut epithelium.
 Abstract.  Author URL
Waterfield, N.R., Daborn, P.J., ffrench-Constant, R.H. (2002). Genomic islands in Photorhabdus. Trends Microbiol, 10(12), 541-545.

Abstract:
Genomic islands in Photorhabdus.

Genomic islands are responsible for unique aspects of bacterial behavior such as symbiosis and pathogenicity. Photorhabdus luminescens is a pathogen of insects that spends part of its lifecycle in symbiosis with a nematode. Here, we describe novel genomic islands from Photorhabdus that are involved in symbiosis and pathogenicity, and discuss the inter-relationship between virulence factors used against invertebrates and vertebrates.
 Abstract.  Author URL
Andreasen, M.H., Ffrench-Constant, R.H. (2002). In situ hybridization to the Rdl locus on polytene chromosome 3L of Anopheles stephensi. Med Vet Entomol, 16(4), 452-455.

Abstract:
In situ hybridization to the Rdl locus on polytene chromosome 3L of Anopheles stephensi.

We are interested in generating a Y-autosome translocation of the Resistance to dieldrin (Rdl) locus in the malaria vector mosquito Anopheles stephensi Liston (Diptera: Culicidae), for use in sterile insect release. To ensure stability of the system, a recombination suppressing inversion can also be induced which encompasses the Rdl locus. As a first step, here we report the cloning of fragments of the Rdl gene from both An. stephensi and An. gambiae Giles using degenerate primers in the polymerase chain reaction. These fragments encode the second membrane-spanning region of the gamma-aminobutyric acid receptor and show high levels of both nucleotide and predicted amino acid identity to other Rdl-like receptors. They confirm that, as in all other arthropod species examined, dieldrin resistance in An. stephensi is associated with replacement of alanine302, in this case with a serine. In situ hybridization of the Rdl probe to polytene chromosomes of An. stephensi localizes the gene to the left arm of chromosome 3 (3L) in region 45C. Rdl localization will enable us to identify chromosomal rearrangements encompassing the Rdl locus and help anchor the genome sequence of An. gambiae to the polytene map.
 Abstract.  Author URL
Sharma, S., Waterfield, N., Bowen, D., Rocheleau, T., Holland, L., James, R., ffrench-Constant, R. (2002). The lumicins: novel bacteriocins from Photorhabdus luminescens with similarity to the uropathogenic-specific protein (USP) from uropathogenic Escherichia coli. Fems Microbiol Lett, 214(2), 241-249.

Abstract:
The lumicins: novel bacteriocins from Photorhabdus luminescens with similarity to the uropathogenic-specific protein (USP) from uropathogenic Escherichia coli.

Bacteriocins are proteins produced by bacteria to destroy other bacteria occupying their ecological niche. Photorhabdus luminescens is an insect pathogenic bacterium carried by an entomopathogenic nematode and occupies several different niches in its life cycle. The nematode enters the insect and releases a single strain of P. luminescens. The bacteria then kill the host and the bacteria and nematodes replicate within the cadaver. Strikingly, at the end of the infection the cadaver is still occupied by a single strain of bacterium, suggesting that P. luminescens can destroy other bacteria entering, or present within, the insect. Here we describe four loci encoding 'lumicins' in P. luminescens subsp. akhurstii strain W14. The lumicins are novel bacteriocins capable of killing other strains of Photorhabdus and Escherichia coli. These loci predict killer proteins and multiple dual type immunity proteins with domains similar to pyocins and colicins. The killer proteins are chimeric in nature with multiple domains, one of which is similar to the uropathogenic-specific protein (USP) described from uropathogenic E. coli. The implications of these novel bacteriocins for the lifestyle of Photorhabdus and the potential role of USP as a bacteriocin in E. coli are discussed.
 Abstract.  Author URL
Daborn, P., Boundy, S., Yen, J., Pittendrigh, B., ffrench-Constant, R. (2001). DDT resistance in Drosophila correlates with Cyp6g1 over-expression and confers cross-resistance to the neonicotinoid imidacloprid. Mol Genet Genomics, 266(4), 556-563.

Abstract:
DDT resistance in Drosophila correlates with Cyp6g1 over-expression and confers cross-resistance to the neonicotinoid imidacloprid.

Mutagenesis can be used as a means of predicting likely mechanisms of resistance to novel classes of insecticides. We used chemical mutagenesis in Drosophila to screen for mutants that had become resistant to imidacloprid, a neonicotinoid insecticide. Here we report the isolation of two new dominant imidacloprid-resistant mutants. By recombinational mapping we show that these map to the same location as Rst(2)DDT. Furthermore, we show that pre-existing Rst(2)DDT alleles in turn confer cross-resistance to imidacloprid. In order to localize the Rst(2)DDT gene more precisely, we mapped resistance to both DDT and imidacloprid with respect to P-element markers whose genomic location is known. By screening for recombinants between these P-elements and resistance we localized the gene between 48D5-6 and 48F3-6 on the polytene chromosome map. The genomic sequence in this interval shows a cluster of cytochrome P450 genes, one of which, Cyp6g1, is over-expressed in all resistant strains examined. We are now testing the hypothesis that resistance to both compounds is associated with over-expression of this P450 gene.
 Abstract.  Author URL
Daborn, P.J., Waterfield, N., Blight, M.A., Ffrench-Constant, R.H. (2001). Measuring virulence factor expression by the pathogenic bacterium Photorhabdus luminescens in culture and during insect infection. J Bacteriol, 183(20), 5834-5839.

Abstract:
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.
 Abstract.  Author URL
Waterfield, N., Dowling, A., Sharma, S., Daborn, P.J., Potter, U., Ffrench-Constant, R.H. (2001). Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli. Appl Environ Microbiol, 67(11), 5017-5024.

Abstract:
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.
 Abstract.  Author URL
Waterfield, N.R., Bowen, D.J., Fetherston, J.D., Perry, R.D., ffrench-Constant, R.H. (2001). The tc genes of Photorhabdus: a growing family. Trends Microbiol, 9(4), 185-191.

Abstract:
The tc genes of Photorhabdus: a growing family.

The toxin complex (tc) genes of Photorhabdus encode insecticidal, high molecular weight Tc toxins. These toxins have been suggested as useful alternatives to those derived from Bacillus thuringiensis for expression in insect-resistant transgenic plants. Although Photorhabdus luminescens is symbiotic with nematodes that kill insects, tc genes have recently been described from other insect-associated bacteria such as Serratia entomophila, an insect pathogen, and Yersinia pestis, the causative agent of bubonic plague, which has a flea vector. Here, recent advances in our understanding of the tc gene family are reviewed in view of their potential development as insect-control agents.
 Abstract.  Author URL
Ffrench-Constant, R.H., Waterfield, N., Burland, V., Perna, N.T., Daborn, P.J., Bowen, D., Blattner, F.R. (2000). A genomic sample sequence of the entomopathogenic bacterium Photorhabdus luminescens W14: potential implications for virulence. Appl Environ Microbiol, 66(8), 3310-3329.

Abstract:
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.
 Abstract.  Author URL
Koch, P.B., Lorenz, U., Brakefield, P.M., ffrench-Constant, R.H. (2000). Butterfly wing pattern mutants: developmental heterochrony and co-ordinately regulated phenotypes. Dev Genes Evol, 210(11), 536-544.

Abstract:
Butterfly wing pattern mutants: developmental heterochrony and co-ordinately regulated phenotypes.

Butterfly wings are colored late in development, when pigments are synthesized in specialized wing scale cells in a fixed developmental succession. In this succession, colored pigments are deposited first and the remaining areas are later melanized black or brown. Here we studied the developmental changes underlying two wing pattern mutants, firstly melanic mutants of the swallowtail Papilio glaucus, in which the yellow background is turned black, and secondly a Spotty mutant of the satyrid Bicyclus anynana, which carries two additional eyespots. Despite the very different pattern changes in these two mutants, they are both associated with changes in rates of scale development and correspondingly, the final color pattern. In the melanic swallowtail, background scales originally destined to become yellow (normally developing early and synthesizing papiliochrome) show delayed development, fail to make papiliochrome, and subsequently melanize at the same time as scales in the wild-type black pattern. In the B. anynana eyespot, scale maturation begins with the central white focus, then progresses to the surrounding gold ring and later finishes with melanization of the black center. Mutants showing additional eyespots display accelerated rates of scale development (corresponding to new eyespots) in wing cells not normally occupied by eyespots. Thus by either delaying or accelerating rates of scale development, the final color, or position, of a wing pattern element can be changed. We propose that this heterochrony of scale development is a basic mechanism of color pattern formation on which developmental mutants act to change lepidopteran color patterns.
 Abstract.  Author URL
Ffrench-Constant, R.H., Anthony, N., Aronstein, K., Rocheleau, T., Stilwell, G. (2000). Cyclodiene insecticide resistance: from molecular to population genetics. Annu Rev Entomol, 45, 449-466.

Abstract:
Cyclodiene insecticide resistance: from molecular to population genetics.

This review follows progress in the analysis of cyclodiene insecticide resistance from the initial isolation of the mutant, through cloning of the resistance gene, to an examination of the distribution of resistance alleles in natural populations. Emphasis is given to the use of a resistant Drosophila mutant as an entry point to cloning the associated gamma-aminobutyric acid (GABA) receptor subunit gene, Resistance to dieldrin. Resistance is associated with replacements of a single amino acid (alanine302) in the chloride ion channel pore of the protein. Replacements of alanine302 not only directly affect the drug binding site but also allosterically destabilize the drug preferred conformation of the receptor. Resistance is thus conferred by a unique dual mechanism associated with alanine302, which is the only residue replaced in a wide range of different resistant insects. The underlying mutations appear either to have arisen once, or multiply, depending on the population biology of the pest insect. Although resistance frequencies decline in the absence of selection, resistance alleles can persist at relatively high frequency and may cause problems for compounds to which cross-resistance is observed, such as the novel fipronils.
 Abstract.  Author URL
ffrench-Constant, R.H., Bowen, D.J. (2000). Novel insecticidal toxins from nematode-symbiotic bacteria. Cell Mol Life Sci, 57(5), 828-833.

Abstract:
Novel insecticidal toxins from nematode-symbiotic bacteria.

The current strategy of using transgenic crops expressing insecticidal protein toxins is placing increasing emphasis on the discovery of novel toxins, beyond those already derived from the bacterium Bacillus thuringiensis. Here we review the cloning of four insecticidal toxin complex (tc) encoding genes from a different bacterium Photorhabdus luminescens and of similar gene sequences from Xenorhabdus nematophilus. Both these bacteria occupy the gut of entomopathogenic nematodes and are released into the insect upon invasion by the nematode. In the insect the bacteria presumably secrete these insecticidal toxins, as well as a range of other antimicrobials, to establish the insect cadaver as a monocultural breeding ground for both bacteria and nematodes. In this review, the protein biochemistry and structure of the tc encoding loci are discussed in relation to their observed toxicity and histopathology. These toxins may prove useful as alternatives to those derived from B. thuringiensis for deployment in insect-resistant transgenic plants.
 Abstract.  Author URL
Bowen, D., Blackburn, M., Rocheleau, T., Grutzmacher, C., ffrench-Constant, R.H. (2000). Secreted proteases from Photorhabdus luminescens: separation of the extracellular proteases from the insecticidal Tc toxin complexes. Insect Biochem Mol Biol, 30(1), 69-74.

Abstract:
Secreted proteases from Photorhabdus luminescens: separation of the extracellular proteases from the insecticidal Tc toxin complexes.

Photorhabdus luminescens secretes both high molecular weight insecticidal toxin complexes and also a range of extracellular proteases into culture broth. Previous studies by others have suggested that insecticidal activity of the broth is associated with these proteases. However, by gene cloning and targeted knock-out, we have previously shown that oral insecticidal activity is associated with high molecular weight 'toxin complexes' (Tc) encoded by toxin complex or tc genes. Here we further clarify this distinction by biochemically separating the protease fractions away from the oral insecticidal activity of the Tc proteins. We purified three distinct protease fractions from the broth: one consisting of a single species of 55 kDa and two of several putatively related species of approximately 40 kDa. All of these clearly separate from the oral insecticidal activity associated with the high molecular weight Tc proteins and also show no effect on insect weight gain following injection into the haemocoel. Here we examine the substrate preferences and inhibitor profiles of these protease fractions and discuss their relationship with those previously described from other P. luminescens strains and phase variants.
 Abstract.  Author URL
Koch, P.B., Behnecke, B., ffrench-Constant, R.H. (2000). The molecular basis of melanism and mimicry in a swallowtail butterfly. Curr Biol, 10(10), 591-594.

Abstract:
The molecular basis of melanism and mimicry in a swallowtail butterfly.

Melanism in Lepidoptera, either industrial or in mimicry, is one of the most commonly cited examples of natural selection [1] [2]. Despite extensive studies of the frequency and maintenance of melanic genes in insect populations [1] [2], there has been little work on the underlying molecular mechanisms. Nowhere is butterfly melanism more striking than in the Eastern Tiger Swallowtail (Papilio glaucus) of North America [3] [4] [5]. In this species, females can be either yellow (wild type) or black (melanic). The melanic form is a Batesian mimic of the distasteful Pipevine Swallowtail (Battus philenor), which is also black in overall color. Melanism in P. glaucus is controlled by a single Y-linked (female) black gene [6]. Melanic females, therefore, always have melanic daughters. Black melanin replaces the background yellow in melanic females. Here, we show that the key enzyme involved is N-beta-alanyl-dopamine-synthase (BAS), which shunts dopamine from the melanin pathway into the production of the yellow color pigment papiliochrome and also provides products for cuticle sclerotization. In melanic females, this enzyme is suppressed, leading to abnormal melanization of a formerly yellow area, and wing scale maturation is also delayed in the same area. This raises the possibility that either reduced BAS activity itself is preventing scale sclerotization (maturation) or, in contrast, that the delay in scale maturation precludes expression of BAS at the correct stage. Together, these data show how changes in expression of a single gene product could result in multiple wing color phenotypes. The implications for the genetic control of mimicry in other Lepidoptera are discussed.
 Abstract.  Author URL
Crennell, S.J., Tickler, P.M., Bowen, D.J., ffrench-Constant, R.H. (2000). The predicted structure of photopexin from Photorhabdus shows the first haemopexin-like motif in prokaryotes. Fems Microbiol Lett, 191(1), 139-144.

Abstract:
The predicted structure of photopexin from Photorhabdus shows the first haemopexin-like motif in prokaryotes.

The insect pathogenic bacterium Photorhabdus luminescens secretes several insecticidal high molecular mass 'toxin complexes'. Analysis of the putative pathogenicity island surrounding the toxin complex a (tca) locus revealed two open reading frames (ORFs) of unknown function. The predicted protein sequences of these ORFs show a repeated motif similar to those found in the vertebrate haem scavenging molecule haemopexin, limunectin (a phosphocholine binding protein from Limulus) and the C-terminal domains of matrix metalloproteinases (MMPs) (where they are thought to be important for cell attachment and adhesion). We have therefore named the operon photopexin AB and the putative encoded proteins 'photopexins' a and B (PpxA and PpxB). The predicted amino acid sequence of PpxA was modelled onto the crystal structure of a MMP. Our model predicts not only that PpxA and PpxB have beta-propeller domains but also that each haemopexin-like repeat corresponds to one blade of a propeller, suggesting the limunectin structure itself may also contain two or three such haemopexin-like propellers. The overall structure of PpxA has striking similarity to that of haemopexin suggesting that it may be used by the bacterium to scavenge iron containing compounds from insects. The implications for the potential role of Ppx proteins in pathogenicity are discussed. This is the first finding of a haemopexin-like repeat outside plants and animals.
 Abstract.  Author URL
Wang, C.T., Zhang, H.G., Rocheleau, T.A., ffrench-Constant, R.H., Jackson, M.B. (1999). Cation permeability and cation-anion interactions in a mutant GABA-gated chloride channel from Drosophila. Biophys J, 77(2), 691-700.

Abstract:
Cation permeability and cation-anion interactions in a mutant GABA-gated chloride channel from Drosophila.

To investigate the structural basis of anion selectivity of Drosophila GABA-gated Cl(-) channels, the permeation properties of wild-type and mutant channels were studied in Xenopus oocytes. This work focused on asparagine 319, which by homology is one amino acid away from a putative extracellular ring of charge that regulates cation permeation in nicotinic receptors. Mutation of this residue to aspartate reduced channel conductance, and mutation to lysine or arginine increased channel conductance. These results are consistent with an electrostatic interaction between this site and permeating anions. The lysine mutant, but not the arginine mutant, formed a channel that is permeable to cations, and this cannot be explained in terms of electrostatics. The lysine mutant had a 25-mV reversal potential in solutions with symmetrical Cl(-) and asymmetrical cations. The permeability ratio of K(+) to Cl(-) was determined as 0. 33 from reversal potential measurements in KCl gradients. Experiments with large organic cations and anions showed that cation permeation can only be seen in the presence of Cl(-), but Cl(-) permeation can be seen in the absence of permeant cations. Measurements of permeability ratios of organic anions indicated that the lysine mutant has an increased pore size. The cation permeability of the lysine-containing mutant channel cannot be accounted for by a simple electrostatic interaction with permeating ions. It is likely that lysine substitution causes a structural change that extends beyond this one residue to influence the positions of other channel-forming residues. Thus protein conformation plays an important role in enabling ion channels to distinguish between anions and cations.
 Abstract.  Author URL
Andreev, D., Kreitman, M., Phillips, T.W., Beeman, R.W., ffrench-Constant, R.H. (1999). Multiple origins of cyclodiene insecticide resistance in Tribolium castaneum (Coleoptera: Tenebrionidae). J Mol Evol, 48(5), 615-624.

Abstract:
Multiple origins of cyclodiene insecticide resistance in Tribolium castaneum (Coleoptera: Tenebrionidae).

The number of origins of pesticide resistance-associated mutations is important not only to our understanding of the evolution of resistance but also in modeling its spread. Previous studies of amplified esterase genes in a highly dispersive Culex mosquito have suggested that insecticide resistance-associated mutations (specifically a single-gene duplication event) can occur a single time and then spread throughout global populations. In order to provide data for resistance-associated point mutations, which are more typical of pesticide mechanisms as a whole, we studied the number of independent origins of cyclodiene insecticide resistance in the red flour beetle Tribolium castaneum. Target-site insensitivity to cyclodienes is conferred by single point mutations in the gene Resistance to dieldrin (Rdl), which codes for a subunit of a gamma-aminobutyric acid (GABA) receptor. These point mutations are associated with replacements of alanine 302 which render the receptor insensitive to block by the insecticide. We collected 141 strains of Tribolium worldwide and screened them for resistance. Twenty-four strains contained resistant individuals. After homozygosing 23 of these resistance alleles we derived a nucleotide sequence phylogeny of the resistant strains from a 694-bp section of Rdl, encompassing exon 7 (which contains the resistance-associated mutation) and part of a flanking intron. The phylogeny also included six susceptible alleles chosen at random from a range of geographical locations. Resistance alleles fell into six clades and three clades contained both resistant and susceptible alleles. Although statistical analysis provided support at only the 5-6% level, the pattern of variation in resistance alleles is more readily explained by multiple independent origins of resistance than by spread of a single resistance-associated mutation. For example, two resistance alleles differed from two susceptible alleles only by the resistance-associated mutation itself, suggesting that they form the susceptible ancestors and that resistance arose independently in several susceptible backgrounds. This suggests that in Tribolium Rdl, de novo mutations for resistance have arisen independently in several populations. Identical alleles were found in geographically distant regions as well, also implying that some Rdl alleles have been exported in stored grain. These differences from the Culex study may stem both from differences in the population genetics of Tribolium versus that of mosquitoes and differences in mutation rates associated with point mutations versus gene duplication events. The Tribolium data therefore suggest that multiple origins of insecticide resistance (associated with specific point mutations) may be more common than the spread of single events. These findings have implications for the way in which we model the evolution and spread of insecticide resistance genes and also suggest that parallel adaptive substitutions may not be uncommon in phyletic evolution.
 Abstract.  Author URL
ffrench-Constant, R., Bowen, D. (1999). Photorhabdus toxins: novel biological insecticides. Curr Opin Microbiol, 2(3), 284-288.

Abstract:
Photorhabdus toxins: novel biological insecticides.

Following concerns over the potential for insect resistance to insecticidal Bacillus thuringiensis toxins expressed in transgenic plants, there has been recent interest in novel biological insecticides. Over the past year there has been considerable progress in the cloning of several alternative toxin genes from the bacteria Photorhabdus luminescens and Xenorhabdus nematophilus. These genes encode large insecticidal toxin complexes with little homology to other known toxins.
 Abstract.  Author URL
ffrench-Constant, R.H. (1999). Target site mediated insecticide resistance: what questions remain?. Insect Biochemistry and Molecular Biology, 29(5), 397-403. Author URL
Blackburn, M., Golubeva, E., Bowen, D., Ffrench-Constant, R.H. (1998). A novel insecticidal toxin from photorhabdus luminescens, toxin complex a (Tca), and its histopathological effects on the midgut of manduca sexta. Appl Environ Microbiol, 64(8), 3036-3041.

Abstract:
A novel insecticidal toxin from photorhabdus luminescens, toxin complex a (Tca), and its histopathological effects on the midgut of manduca sexta

Photorhabdus luminescens is a bacterium which is mutualistic with entomophagous nematodes and which secretes high-molecular-weight toxin complexes following its release into the insect hemocoel upon nematode invasion. Thus, unlike other protein toxins from Bacillus thuringiensis (delta-endotoxins and Vip's), P. luminescens toxin (Pht) normally acts from within the insect hemocoel. Unexpectedly, therefore, the toxin complex has both oral and injectable activities against a wide range of insects. We have recently fractionated the protein toxin and shown it to consist of several native complexes, the most abundant of which we have termed Toxin complex a (Tca). This complex is highly active against the lepidopteran Manduca sexta. In view of the difference in the normal mode of delivery of P. luminescens toxin and the apparent communality in the histopathological effects of other gut-active toxins from B. thuringiensis, as well as cholesterol oxidase, we were interested in investigating the effects of purified Tca protein on larvae of M. sexta. Here we report that the histopathology of the M. sexta midgut is similar to that for other novel midgut-active toxins. Following oral ingestion of Tca by M. sexta, we observed an acceleration in the blebbing of the midgut epithelium into the gut lumen and eventual lysis of the epithelium. The midgut shows a similar histopathology following injection of Tca into the insect hemocoel. These results not only show that Tca is a highly active oral insecticide but also confirm the similar histopathologies of a range of very different gut-active toxins, despite presumed differences in modes of action and/or delivery. The implications for the mode of action of Tca are discussed.
 Abstract.  Author URL
Liu, H.T., Stilwell, G., Anthony, N., Rocheleau, T., ffrench-Constant, R.H. (1998). Analysis of a mosquito acetylcholinesterase gene promoter. Insect Mol Biol, 7(1), 11-17.

Abstract:
Analysis of a mosquito acetylcholinesterase gene promoter.

Insect acetylcholinesterase is the target site for organophosphorus and carbamate insecticides and point mutations in the Ace gene are associated with resistance in Drosophila melanogaster and Musca domestica. However, little is known of the genetic regulation of insect Ace genes. Here we report the isolation of four different cDNAs from an Aedes Ace locus and identification of the gene promoter. Northern analysis reveals two large (>10 kb) transcripts and one smaller transcript of 4 kb. The region containing the initiation of transcription was localized by sequencing the two 5' most cDNAs and by 5' RACE. The transcription start point was subsequently identified by primer extension and is flanked by a perfect arthropod initiator consensus sequence. The promoter lacks a TATA box but contains several matches to other consensus sequences for eukaryotic transcription factors. In common with the Drosophila Ace gene, there are also multiple potential initiators of translation (ATGs) upstream of the main open reading frame. The structure of the 5' leader and promoter is compared to that found in other insect and vertebrate Ace genes and the possibility that this locus is homologous to one of two Ace loci described in another mosquito, Culex pipiens, is discussed.
 Abstract.  Author URL
Vaughan, A., Chadee, D.D., French-Constant, R. (1998). Biochemical monitoring of organophosphorus and carbamate insecticide resistance in Aedes aegypti mosquitoes from Trinidad. Med Vet Entomol, 12(3), 318-321. Author URL
Anthony, N.M., Brown, J.K., Feyereisen, R., ffrench-Constant, R.H. (1998). Diagnosis and characterization of insecticide-insensitive acetylcholinesterase in three populations of the sweetpotato whitefly Bemisia tabaci. Pesticide Science, 52(1), 39-46. Author URL
Anthony, N., Unruh, T., Ganser, D., ffrench-Constant, R. (1998). Duplication of the Rdl GABA receptor subunit gene in an insecticide-resistant aphid, Myzus persicae. Mol Gen Genet, 260(2-3), 165-175.

Abstract:
Duplication of the Rdl GABA receptor subunit gene in an insecticide-resistant aphid, Myzus persicae.

Resistance to cyclodiene insecticides is associated with replacements of a single amino acid (alanine 302) in a gamma-aminobutyric acid (GABA) receptor subunit encoded by the single-copy gene Resistance to dieldrin (Rdl). Alanine 302 is predicted to reside within the second membrane-spanning region of the Rdl receptor, a region that is thought to line the integral chloride ion channel pore. In all cyclodiene-resistant insects studied to date, this same alanine residue is replaced either by a serine, or, in some resistant strains of Drosophila simulans, a glycine residue. Therefore, individuals can carry only two different Rdl alleles. In contrast, here we report the presence of up to four different Rdl-like alleles in individual clones of the green peach aphid, Myzus persicae. In addition to the wild-type copy of Rdl gene (encoding A302 or allele A), M. persicae carries three other alleles with the following amino acid replacements: A302-->Glycine (allele G), A302-->SerineTCG (allele S) and A302-->SerineAGT (allele S'). Evidence from direct nucleotide sequencing and Single Stranded Conformational Polymorphism (SSCP) analysis shows that at least three of these different Rdl alleles (i.e. A, G and S) are commonly present in individual aphids or aphid clones. Southern analysis using allele-specific probes and analysis of sequences downstream of the exon containing the resistance-associated mutation confirm the presence of two independent Rdl-like loci in M. persicae. One locus carries the susceptible alanine (A) and/or resistant glycine (G) allele while the other carries the two serine alleles (S or S'). Whereas resistance levels are correlated with the glycine replacement, the S allele was present in all aphid clones, regardless of their resistance status. These results suggest that target site insensitivity is associated with replacements at the first (A/G) but not the second (S/S') locus. Phylogenetic analysis of nucleotide sequences indicates that both putative aphid Rdl loci are monophyletic with respect to other insect Rdl genes and may have arisen through a recent gene duplication event. The implications of this duplication with respect to insecticide resistance and insect GABA receptor subunit diversity are discussed.
 Abstract.  Author URL
Bowen, D., Rocheleau, T.A., Blackburn, M., Andreev, O., Golubeva, E., Bhartia, R., ffrench-Constant, R.H. (1998). Insecticidal toxins from the bacterium Photorhabdus luminescens. Science, 280(5372), 2129-2132.

Abstract:
Insecticidal toxins from the bacterium Photorhabdus luminescens.

Transgenic plants expressing Bacillus thuringiensis (Bt) toxins are currently being deployed for insect control. In response to concerns about Bt resistance, we investigated a toxin secreted by a different bacterium Photorhabdus luminescens, which lives in the gut of entomophagous nematodes. In insects infected by the nematode, the bacteria are released into the insect hemocoel; the insect dies and the nematodes and bacteria replicate in the cadaver. The toxin consists of a series of four native complexes encoded by toxin complex loci tca, tcb, tcc, and tcd. Both tca and tcd encode complexes with high oral toxicity to Manduca sexta and therefore they represent potential alternatives to Bt for transgenic deployment.
 Abstract.  Author URL
Andreev, D., Breilid, H., Kirkendall, L., Brun, L.O., ffrench-Constant, R.H. (1998). Lack of nucleotide variability in a beetle pest with extreme inbreeding. Insect Mol Biol, 7(2), 197-200.

Abstract:
Lack of nucleotide variability in a beetle pest with extreme inbreeding.

The coffee berry borer beetle Hypothenemus hampei (Ferrari) (Curculionidae: Scolytinae) is the major insect pest of coffee and has spread to most of the coffee-growing countries of the world. This beetle also displays an unusual life cycle, with regular sibling mating. This regular inbreeding and the population bottlenecks occurring on colonization of new regions should lead to low levels of genetic diversity. We were therefore interested in determining the level of nucleotide variation in nuclear and mitochondrial genomes of this beetle worldwide. Here we show that two nuclear loci (Resistance to dieldrin and ITS2) are completely invariant, whereas some variability is maintained at a mitochondrial locus (COI), probably corresponding to a higher mutation rate in the mitochondrial genome. Phylogenetic analysis of the mitochondrial data shows only two clades of beetle haplotypes outside of Kenya, the proposed origin of the species. These data confirm that inbreeding greatly reduces nucleotide variation and suggest the recent global spread of only two inbreeding lines of this bark beetle.
 Abstract.  Author URL
Koch, P.B., Keys, D.N., Rocheleau, T., Aronstein, K., Blackburn, M., Carroll, S.B., ffrench-Constant, R.H. (1998). Regulation of dopa decarboxylase expression during colour pattern formation in wild-type and melanic tiger swallowtail butterflies. Development, 125(12), 2303-2313.

Abstract:
Regulation of dopa decarboxylase expression during colour pattern formation in wild-type and melanic tiger swallowtail butterflies.

The eastern tiger swallowtail butterfly Papilio glaucus shows a striking example of Batesian mimicry. In this species, females are either wild type (yellow and black) or melanic (where most of the yellow colour is replaced by black). In order to understand how these different colour patterns are regulated, we examined the temporal order of wing pigment synthesis via precursor incorporation studies, enzyme assays, and in situ hybridisation to mRNA encoding a key enzyme, dopa decarboxylase. We show that dopa decarboxylase provides dopamine to both of the two major colour pigments, papiliochrome (yellow) and melanin (black). Interestingly, however, dopa decarboxylase activity is spatially and temporally regulated, being utilised early in presumptive yellow tissues and later in black. Further, in melanic females, both dopa decarboxylase activity and early papiliochrome synthesis are suppressed in the central forewing and this normally yellow area is later melanised. These results show that the regulation of enzyme synthesis observed in the yellow/black pattern of a single wing, is similar to that involved in melanism. We infer that dopa decarboxylase activity must be regulated in concert with downstream enzymes of either the melanin and/or the papiliochrome specific pathways, forming part of a developmental switch between yellow or black. This modification of multiple enzyme activities in concert is consistent with a model of melanisation involving coordinate regulation of the underlying synthetic pathways by a single Y-linked (female) factor.
 Abstract.  Author URL
Stilwell, G.E., ffrench-Constant, R.H. (1998). Transcriptional analysis of the Drosophila GABA receptor gene resistance to dieldrin. J Neurobiol, 36(4), 468-484.

Abstract:
Transcriptional analysis of the Drosophila GABA receptor gene resistance to dieldrin.

The Resistance to dieldrin (Rdl) gene encodes a novel subunit of a gamma-aminobutyric acid (GABA)-gated chloride ion channel in Drosophila. We were interested in defining the spatial and temporal expression pattern of this gene and in understanding the basis of its regulation. Rdl is expressed in both the embryonic central and peripheral nervous system. Here, we describe the complete Rdl transcription unit (approximately 50 kb) via localization of the flanking transcripts. The Rdl transcript itself is large (8.8 kb) and is composed of a short open reading frame (2 kb) with exceptionally long 5' (1.8-kb) and 3' (5-kb) untranslated regions (UTRs). The correct spatial and temporal expression of Rdl can be rescued by transformation constructs containing only 3.5 kb of DNA, a region which encompasses the transcription start point (tsp). This region also contains sequences strikingly similar to those found in other ion channel gene promoters. Failure of minigene constructs lacking the long 3' UTR to fully rescue both the lethal and resistance phenotypes associated with the Rdl locus might arise owing to the role of these sequences in message stability or trafficking.
 Abstract.  Author URL
Pittendrigh, B., Aronstein, K., Zinkovsky, E., Andreev, O., Campbell, B., Daly, J., Trowell, S., Ffrench-Constant, R.H. (1997). Cytochrome P450 genes from Helicoverpa armigera: expression in a pyrethroid-susceptible and -resistant strain. Insect Biochem Mol Biol, 27(6), 507-512.

Abstract:
Cytochrome P450 genes from Helicoverpa armigera: expression in a pyrethroid-susceptible and -resistant strain.

The molecular basis of metabolic resistance to pyrethroids in Helicoverpa armigera is currently under debate. Substantial indirect evidence supports a role for both esterase- and cytochrome-P450-mediated metabolism. However, the relative roles played by these two mechanisms in field-based resistance is uncertain. Our understanding of the importance of P450-mediated metabolism is hindered by the paucity of cloned genes from this species, and the corresponding absence of data on rates of insecticide metabolism by functionally expressed P450s. To facilitate P450 gene isolation from H. armigera we used degenerate primers in the reverse transcriptase-polymerase chain reaction (RT-PCR) to clone P450 gene fragments from the RNA of a pyrethroid-resistant strain. Here we report the isolation of eight new P450 genes: seven from the CYP4 family and one CYP9. One of these genes, CYP4G8, is two-fold over-expressed in the resistant strain, whereas the other CYP4s showed either similar or undetectable levels of expression. CYP9A3 appears to be a homolog of the putatively resistance-associated CYP9A1 of Heliothis virescens. However, no difference in expression between the H. armigera strains was detected. CYP6B2, a gene previously reported to be over-expressed in a different pyrethroid-resistant strain of H. armigera, also revealed non-detectable levels of expression in both strains. These observations suggest that different P450s may be over-expressed in different resistant strains, and emphasize that recombinant expression will be necessary in order to define precisely their individual substrate specificities and ability to metabolize pyrethroids. The gene fragments described here represent an important first step in this direction.
 Abstract.  Author URL
Hosie, A.M., Aronstein, K., Sattelle, D.B., ffrench-Constant, R.H. (1997). Molecular biology of insect neuronal GABA receptors. Trends Neurosci, 20(12), 578-583.

Abstract:
Molecular biology of insect neuronal GABA receptors.

Ionotropic gamma-aminobutyric acid (GABA) receptors are distributed throughout the nervous systems of many insect species. As with their vertebrate counterparts, GABAA receptors and GABAC receptors, the binding of GABA to ionotropic insect receptors elicits a rapid, transient opening of anion-selective ion channels which is generally inhibitory. Although insect and vertebrate GABA receptors share a number of structural and functional similarities, their pharmacology differs in several aspects. Recent studies of cloned Drosophila melanogaster GABA receptors have clarified the contribution of particular subunits to these differences. Insect ionotropic GABA receptors are also the target of numerous insecticides and an insecticide-resistant form of a Drosophila GABA-receptor subunit has enhanced our understanding of the structure-function relationship of one aspect of pharmacology common to both insect and vertebrate GABA receptors, namely antagonism by the plant-derived toxin picrotoxinin.
 Abstract.  Author URL
Severson, D.W., Anthony, N.M., Andreev, O., ffrench-Constant, R.H. (1997). Molecular mapping of insecticide resistance genes in the yellow fever mosquito (Aedes aegypti). J Hered, 88(6), 520-524.

Abstract:
Molecular mapping of insecticide resistance genes in the yellow fever mosquito (Aedes aegypti).

Several loci conferring insecticide resistance in the yellow fever mosquito (Aedes aegypti) have previously been mapped by simple recombinational mapping. Here we describe correlation of these resistance phenotypes with molecular gene probes for insecticide target sites by RFLP mapping. The para sodium channel gene homologue and the GABA receptor gene Resistance to dieldrin map to the same genome regions as the DDT/pyrethroid and cyclodiene resistance loci, respectively. Although the acetylcholinesterase (target site of organophosphorus and carbamate insecticides) gene Ace does not map to any known resistance locus, it maps very close to the sex-determining locus. We discuss the possibilities that, if identified, Ace-mediated resistance in A. aegypti will be sex linked or that, as suggested for anopheline mosquitoes, two independent Ace loci may exist, one of which is autosomal. These results support the importance of target site insensitivity as an insecticide resistance mechanism in mosquitoes.
 Abstract.  Author URL
Pittendrigh, B., Reenan, R., ffrench-Constant, R.H., Ganetzky, B. (1997). Point mutations in the Drosophila sodium channel gene para associated with resistance to DDT and pyrethroid insecticides. Mol Gen Genet, 256(6), 602-610.

Abstract:
Point mutations in the Drosophila sodium channel gene para associated with resistance to DDT and pyrethroid insecticides.

The gene para in Drosophila melanogaster encodes an alpha subunit of voltage-activated sodium channels, the presumed site of action of DDT and pyrethroid insecticides. We used an existing collection of Drosophila para mutants to examine the molecular basis of targetsite resistance to pyrethroids and DDT. Six out of thirteen mutants tested were associated with a largely dominant, 10- to 30-fold increase in DDT resistance. The amino acid lesions associated with these alleles defined four sites in the sodium channel polypeptide where a mutational change can cause resistance: within the intracellular loop between S4 and S5 in homology domains I and III, within the pore region of homology domain III, and within S6 in homology domain III. Some of these sites are analogous with those defined by knockdown resistance (kdr) and super-kdr resistance-associated mutations in houseflies and other insects, but are located in different homologous units of the channel polypeptide. We find a striking synergism in resistance levels with particular heterozygous combinations of para alleles that appears to mimic the super-kdr double mutant housefly phenotype. Our results indicate that the alleles analyzed from natural populations represent only a subset of mutations that can confer resistance. The implications for the binding site of pyrethroids and mechanisms of target-site insensitivity are discussed.
 Abstract.  Author URL
Vaughan, A., Rocheleau, T., ffrench-Constant, R. (1997). Site-directed mutagenesis of an acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti confers insecticide insensitivity. Exp Parasitol, 87(3), 237-244.

Abstract:
Site-directed mutagenesis of an acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti confers insecticide insensitivity.

Insecticide resistance is a serious problem facing the effective control of insect vectors of disease. Insensitive acetylcholinesterase (AChE) confers resistance to organophosphorus (OP) and carbamate insecticides and is a widespread resistance mechanism in vector mosquitoes. Although the point mutations that underlie AChE insensitivity have been described from Drosophila, the Colorado potato beetle, and house flies, no resistance associated mutations have been documented from mosquitoes to date. We are therefore using a cloned acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti as a model in which to perform site directed mutagenesis in order to understand the effects of potential resistance associated mutations. The same resistance associated amino-acid replacements as found in other insects also confer OP and carbamate resistance to the mosquito enzyme. Here we describe the levels of resistance conferred by different combinations of these mutations and the effects of these mutations on the kinetics of the AChE enzyme. Over-expression of these constructs in baculovirus will facilitate purification of each of the mutant enzymes and a more detailed analysis of their associated inhibition kinetics.
 Abstract.  Author URL
Dunkov, B.C., Guzov, V.M., Mocelin, G., Shotkoski, F., Brun, A., Amichot, M., Ffrench-Constant, R.H., Feyereisen, R. (1997). The Drosophila cytochrome P450 gene Cyp6a2: structure, localization, heterologous expression, and induction by phenobarbital. Dna Cell Biol, 16(11), 1345-1356.

Abstract:
The Drosophila cytochrome P450 gene Cyp6a2: structure, localization, heterologous expression, and induction by phenobarbital.

The cytochrome P450 gene Cyp6a2 from Drosophila melanogaster is located on the right arm of chromosome 2 at position 43A1-2 and comprises two exons separated by a 69-bp intron. Phenobarbital treatment of flies leads to a rapid increase in the level of CYP6A2 mRNA and to an increased production of the CYP6A2 protein. DNA from the Cyp6a2 promoter region was functional when linked to a luciferase reporter gene and transfected into D. melanogaster Schneider cells. Moreover, a dose-dependent induction of luciferase activity by phenobarbital indicated that elements necessary for phenobarbital induction are located within 428 bp of the translation start site. Heterologous expression of the CYP6A2 protein in lepidopteran cells infected with a Cyp6a2-recombinant baculovirus was observed by Western blotting of cell lysates and by spectral characterization of the reduced-CO complex of the P450. The CYP6A2 protein produced in this system metabolized aldrin and heptachlor to their epoxides and metabolized the insecticide diazinon by desulfuration to diazoxon and by oxidative ester cleavage to 2-isopropyl-4-methyl-6-hydroxypyrimidine. Metabolism in lysates of cells infected with recombinant baculovirus was greatly enhanced by the addition of purified housefly NADPH cytochrome P450 reductase and cytochrome b5. These results show that CYP6A2 catalyzes the metabolism of organophosphorus insecticides and they implicate Cyp6a2 overexpression in metabolic resistance. The Cyp6a2 gene appears to be a suitable model for a genetic analysis of the phenobarbital induction process.
 Abstract.  Author URL
Brotz, T.M., Bochenek, B., Aronstein, K., Ffrench-Constant, R.H., Borst, A. (1997). gamma-Aminobutyric acid receptor distribution in the mushroom bodies of a fly (Calliphora erythrocephala): a functional subdivision of Kenyon cells?. J Comp Neurol, 383(1), 42-48.

Abstract:
gamma-Aminobutyric acid receptor distribution in the mushroom bodies of a fly (Calliphora erythrocephala): a functional subdivision of Kenyon cells?

Antibodies against the Drosophila gamma-aminobutyric acid (GABA) receptor subunit RDL were used to investigate the significance of inhibitory inputs to the mushroom bodies in the blowfly (Calliphora erythrocephala) brain. The pedunculus and the lobes of the mushroom body, which mainly consist of Kenyon cell fibers, revealed strong immunoreactivity against RDL. Pedunculi, alpha- and beta-lobe show characteristic unstained core structures with concentric labeling along the neuropile axis. The gamma-lobes in contrast exhibit a compartmentalized RDL-immunoreactive pattern. These data suggest an important role of GABAergic inhibition in the pedunculus and the lobes of insect mushroom bodies. It is most likely that the RDL-immunoreactivity in the mushroom bodies is closely related to Kenyon cell fibers suggesting that Kenyon cells are an inhomogeneous class of neurons, only part of which receive inhibitory GABAergic input from extrinsic elements. GABAergic inhibition, therefore, may play a substantial role in the process of learning and memory formation in the insect mushroom bodies.
 Abstract.  Author URL
Dunkov, B.C., Rodriguez-Arnaiz, R., Pittendrigh, B., ffrench-Constant, R.H., Feyereisen, R. (1996). Cytochrome P450 gene clusters in Drosophila melanogaster. Mol Gen Genet, 251(3), 290-297.

Abstract:
Cytochrome P450 gene clusters in Drosophila melanogaster.

Twelve cytochrome P450 cDNA fragments were cloned from Drosophila melanogaster by reverse transcriptase/PCR (RT/PCR) using degenerate oligonucleotide primers. The corresponding genes belong to several subfamilies of the CYP4 and CYP9 P450 families. Only two of these genes, Cyp4dl and Cyp4d2, have previously been described. In situ hybridization of each of the cDNA fragments showed two clusters of genes; one near the tip of the X chromosome and the other on the left arm of chromosome 2. Interestingly the latter cluster comprises widely divergent genes belonging both to the CYP9 and CYP4 families and also to the CYP6 family (Cyp6a2). Putative allelic variants of several of the genes were found in different insecticide-resistant and -susceptible strains (Hikone R, Haag 79 and Oregon R). The identification of these genes and alleles will allow us to clarify the involvement of P450s in xenobiotic metabolism and will facilitate a genetic analysis of P450 functions in insects.
 Abstract.  Author URL
Coustau, C., Carkion, Y., Nappi, A., Shotkoski, F., ffrench-Constant, R. (1996). Differential induction of antibacterial transcripts in Drosophila susceptible and resistant to parasitism by Leptopilina boulardi. Insect Mol Biol, 5(3), 167-172.

Abstract:
Differential induction of antibacterial transcripts in Drosophila susceptible and resistant to parasitism by Leptopilina boulardi.

Two well-described elements of the immune response of insects include encapsulation of metazoan parasites (blood-cell-mediated) and the production of antibacterial peptides (humoral and/or cellular). However, the possible functional interrelationship between cellular encapsulation and antibacterial responses, and the extent to which the two components may be co-regulated, are poorly understood. We used a novel approach involving strains of Drosophila resistant (R) or susceptible (S) to the wasp parasitoid Leptopilina boulardi to study the expression of three genes involved in the antibacterial response: Dorsal-related immunity factor (Dif), Cecropin (CecA1) and Diptericin (Dip). Both S and R strains produced high levels of all antibacterial transcripts upon bacterial injection. However, when parasitized the R strain showed no induction whilst the S strain did. This lack of antibacterial transcript induction in the parasitized R strain not only clarifies the separation of these two types of immune response but also raises the fascinating possibility of a link in their genetic regulation.
 Abstract.  Author URL
Aronstein, K., Auld, V., Ffrench-Constant, R. (1996). Distribution of two GABA receptor-like subunits in the Drosophila CNS. Invert Neurosci, 2(2), 115-120.

Abstract:
Distribution of two GABA receptor-like subunits in the Drosophila CNS.

Previously we have described the distribution of the Rdl GABA receptor subunit in the Drosophila CNS. Knowing that Rdl can coassemble with LCCH3 (a Drosophila GABA receptor-like subunit showing sequence similarity to vertebrate beta subunit GABAA receptors) in baculovirus infected insect cells, we compared the localization of these two receptor subunits in order to identify any potential overlap in their spatial or temporal distribution. The two subunits show very different patterns of localization. Early in development LCCH3 is found in the majority of developing neuroblasts and later is localized to the cell bodies of the embryonic nerve cord and brain, and the neuronal cell bodies surrounding the adult brain. In contrast, Rdl receptor subunits appear confined to the neuropil in all developmental stages. These results have two important implications. Firstly, they suggest that although these two subunits can coassemble in heterologous expression systems, they may not be found in the same tissues in the nervous system. Secondly, production of LCCH3 before neuronal differentiation leads us to speculate on the role of that LCCH3 containing receptors in the developing nervous system.
 Abstract.  Author URL
Shotkoski, F., Morris, A.C., James, A.A., ffrench-Constant, R.H. (1996). Functional analysis of a mosquito gamma-aminobutyric acid receptor gene promoter. Gene, 168(2), 127-133.

Abstract:
Functional analysis of a mosquito gamma-aminobutyric acid receptor gene promoter.

A single point mutation in the insect gamma-aminobutyric acid receptor (GABAR)-encoding gene (Rdl) confers high levels of resistance to cyclodienes in Drosophila and other insects. We were interested in studying the promoter of this gene for two reasons. Firstly, to define the elements underlying Rdl expression. Secondly, to identify the minimum set of regulatory elements necessary for construction of a functional Rdl minigene. Such an insecticide-resistance-associated minigene should form a strong selectable marker for use in the genetic transformation of non-drosophilid pest insects, such as mosquitoes. Here, we report the identification of the region containing the rdl promoter, via transient expression of a luc reporter gene following micro-injection into embryos of the mosquito Aedes aegypti. Promoter activity is contained within a 2.53-kb fragment immediately upstream from the rdl start codon. Primer extension shows three closely linked sites for transcript initiation within this region and sequence analysis reveals anumber of putative consensus regulatory sequences shared by other genes expressed in the nervous system. The implications for construction of a functional minigene and the identification of cis-acting control elements underlying ion-channel gene regulation are discussed.
 Abstract.  Author URL
Coustau, C., Rocheleau, T., Carton, Y., Nappi, A.J., ffrench-Constant, R.H. (1996). Induction of a putative serine protease transcript in immune challenged Drosophila. Dev Comp Immunol, 20(4), 265-272.

Abstract:
Induction of a putative serine protease transcript in immune challenged Drosophila.

In an effort to identify serine proteases involved in the insect's immune response, we used a degenerate PCR approach to amplify putative serine protease gene fragments in Drosophila. Sequencing of the cloned PCR products identified one serine protease previously isolated in D. melanogaster (SER1/SER2), as well as two novel putative serine protease gene fragments (SP2, SP3). The involvement of the corresponding genes in the immune response was examined by analyzing their expression in larval mRNA following both parasitic and bacterial exposures. The overexpression of one of the serine proteases-related mRNAs in immune challenged larvae suggests its involvement in the Drosophila immune response.
 Abstract.  Author URL
Shotkoski, F., Zhang, H.G., Jackson, M.B., ffrench-Constant, R.H. (1996). Stable expression of insect GABA receptors in insect cell lines. Promoters for efficient expression of Drosophila and mosquito Rdl GABA receptors in stably transformed mosquito cell lines. Febs Lett, 380(3), 257-262.

Abstract:
Stable expression of insect GABA receptors in insect cell lines. Promoters for efficient expression of Drosophila and mosquito Rdl GABA receptors in stably transformed mosquito cell lines.

We are interested in establishing stably transformed insect cell lines efficiently expressing the insect gamma-aminobutyric acid (GABA) receptor subunit gene Resistance to dieldrin or Rdl. In order to facilitate this we utilized a system based on stable transformation of Aedes albopictus mosquito cell lines using the dihydrofolate reductase (dhfr) gene as a selected marker. Here we report the production of stable mosquito cell lines carrying high copy numbers of Rdl genes from both Drosophila and Aedes aegypti mosquitoes and the subsequent high efficiency expression of functional GABA gated chlorine ion channels. We also used this system to compare the activity of a range of immediate early baculovirus promoters in mosquito cell culture and demonstrate that IE1 promoter constructs work efficiently across insect species. Results are discussed in relation to the potential use of these constructs in the generic transformation of non-Drosophilid insects.
 Abstract.  Author URL
Pittendrigh, B.R., Mocelin, G., Andreev, O., ffrench-Constant, R.H. (1996). The sequence of a Drosophila Cyp4e2 cytochrome P450-encoding cDNA. Gene, 179(2), 295-296.

Abstract:
The sequence of a Drosophila Cyp4e2 cytochrome P450-encoding cDNA.

A composite 1458-bp cDNA that encodes cytochrome P450 (P450) Cyp4e2 has been constructed from clones isolated from two Drosophila embryonic cDNA libraries. The Drosophila cDNA open reading frame encodes a protein of 486 amino acids that is 40% identical and 61% similar to Cyp4d1 from Drosophila. The predicted protein is unusual in that it appears to lack the hydrophobic N-terminus typical of microsomal P450s and also contains a small insertion at its C-terminus.
 Abstract.  Author URL
Stilwell, G.E., Rocheleau, T., ffrench-Constant, R.H. (1995). GABA receptor minigene rescues insecticide resistance phenotypes in Drosophila. J Mol Biol, 253(2), 223-227.

Abstract:
GABA receptor minigene rescues insecticide resistance phenotypes in Drosophila.

A single point mutation within the GABA receptor gene Resistance to dieldrin (Rdl) confers a high level of resistance to cyclodiene insecticides in a wide range of insects. Previous studies have shown partial rescue of the susceptible phenotype via germline transformation of a 36 kb cosmid coding (or all four alternative Rdl splice forms. Here, we describe the construction of two Rdl promoter/cDNA minigenes, each coding for one of the splice forms alone. Single splice forms rescued both the insecticide susceptible and resistant phenotypes associated with the locus as effectively as the complete cosmid. The minigenes also rescue the lethality associated with homozygous re-arrangements disrupting the Rdl gene, and the level of rescue observed is not increased by the addition of more than one splice form. This demonstrates that only a single Rdl splice form is necessary both to confer insecticide sensitivity and also to rescue lethality. Methods by which phenotype rescue could be enhanced and the potential advantages of using Rdl as a selectable marker are discussed.
 Abstract.  Author URL
Aronstein, K., Ffrench-Constant, R. (1995). Immunocytochemistry of a novel GABA receptor subunit Rdl in Drosophila melanogaster. Invert Neurosci, 1(1), 25-31.

Abstract:
Immunocytochemistry of a novel GABA receptor subunit Rdl in Drosophila melanogaster.

Following our recent cloning of a novel gamma-aminobutyric acid (GABA) receptor subunit gene Resistance to dieldrin or Rdl form cyclodiene resistance locus in Drosophila melanogaster, we were interested in defining its pattern of expression during development. Here we report the raising of an anti-Rdl polyclonal antibody that recognizes a single protein of the expected 65 kDa size in immunoblots of Drosophila head homogenates. In situ hybridization using Rdl cDNA probes and the anti-Rdl antibody shows that Rdl message and protein are highly expressed in the developing central nervous system (CNS) of 15-17 h embryos. Interestingly, despite the use of GABA in both the peripheral and CNS of insects, Rdl GABA receptor subunits appear to be confined to the CNS. Detailed immunocytochemistry of Drosophila brain sections showed particularly strong anti-Rdl antibody staining in the optic lobes, ellipsoid body, fan shaped body, ventrolateral protocerebrum and the glomeruli of the antennal lobes. Results are compared with the distribution of staining observed in the insect CNS with antibodies against GABA itself and synaptotagmin, a synaptic vesicle protein.
 Abstract.  Author URL
Zhang, H.G., Lee, H.J., Rocheleau, T., ffrench-Constant, R.H., Jackson, M.B. (1995). Subunit composition determines picrotoxin and bicuculline sensitivity of Drosophila gamma-aminobutyric acid receptors. Mol Pharmacol, 48(5), 835-840.

Abstract:
Subunit composition determines picrotoxin and bicuculline sensitivity of Drosophila gamma-aminobutyric acid receptors.

Few gamma-aminobutyric acid (GABA) receptor subunits have been cloned from insects. These include Resistance to dieldrin, or Rdl, and a homologue of the vertebrate GABAA receptor beta subunit. Unlike most vertebrate GABAA receptor subunits, Rdl forms a highly functional homomultimeric receptor. This receptor is picrotoxin (PTX) sensitive but bicuculline (BIC) insensitive and cannot be readily classified within the known GABAA receptor subtypes. In contrast, functional expression of the beta subunit homologue has not been reported. We report that coinfection of cells with recombinant baculoviruses containing Rdl plus beta subunits induces GABA receptors with distinct pharmacological and kinetic properties. Coinfection produces two separate receptor populations: one highly sensitive to PTX but BIC insensitive (Rdl homomultimers) and the other PTX insensitive and BIC sensitive (Rdl plus beta heteromultimers). Putative Rdl plus beta channels also show reduced GABA sensitivity, slow desensitization, rapid bursting, and shorter mean open time. These studies not only localize PTX and BIC sensitivity to two distinct GABA receptor subunits but also demonstrate assembly of two highly divergent GABA receptor subunits. Furthermore, the difference in channel conductance and gating between in vivo and recombinant channels implies the existence of uncharacterized GABA receptor subunits in Drosophila.
 Abstract.  Author URL
Zhang, H.G., ffrench-Constant, R.H., Jackson, M.B. (1994). A unique amino acid of the Drosophila GABA receptor with influence on drug sensitivity by two mechanisms. J Physiol, 479 ( Pt 1), 65-75.

Abstract:
A unique amino acid of the Drosophila GABA receptor with influence on drug sensitivity by two mechanisms.

1. The Drosophila gene Rdl (resistance to dieldrin) encodes a GABA receptor. An alanine-to-serine mutation in this gene at residue 302 confers resistance to cyclodiene insecticides and picrotoxin. Patch clamp analysis of GABA receptors in cultured neurons from wild type and mutant Drosophila was undertaken to investigate the biophysical basis of resistance. 2. In cultured neurons from both wild type and mutant strains, GABA activated a channel that reversed near 0 mV in symmetrical chloride. GABA dose-response characteristics of wild type and mutant receptors were very similar. 3. GABA responses in neurons from the mutant strains showed reduced sensitivity to the GABA antagonists picrotoxin, lindane and t-butyl-bicyclophosphorothionate. Resistance ratios were 116, 970 and 9 for the three blockers, respectively. Inhibition increased with blocker concentration in a manner consistent with saturation of a single binding site. 4. The mutation reduced the single channel conductance by 5% for inward current and 17% for outward current. The single channel current was approximately 60% lower for outward current than for inward current in both wild type and mutant. 5. Open and closed times were both well fitted by the sum of two exponentials. Resistance was associated with longer open times and shorter closed times, reflecting a net stabilization of the channel open state by a factor of approximately five. 6. The mutation was associated with a marked reduction in the rate of GABA-induced desensitization, and a net destabilization of the desensitized conformation by a factor of 29. 7. The Rdl mutation manifests resistance through two different mechanisms. (a) the mutation weakens drug binding to the antagonist-favoured (desensitized) conformation by a structural change at the drug binding site. (b) the mutation destabilizes the antagonist-favoured conformation in an allosteric sense. The global association of a single amino acid replacement with cyclodiene resistance suggests that the resistance phenotype depends on changes in both of these properties, and that insecticides have selected residue 302 of Rdl for replacement because of its unique ability to influence both of these functions. 8. The location of alanine 302 in the sequence of the Rdl gene product supports a mechanism of action in which convulsants such as picrotoxin bind within the channel lumen, where they induce a rapid conformational change to the desensitized state.
 Abstract.  Author URL
Shotkoski, F., Lee, H.J., Zhang, H.G., Jackson, M.B., ffrench-Constant, R.H. (1994). Functional expression of insecticide-resistant GABA receptors from the mosquito Aedes aegypti. Insect Mol Biol, 3(4), 283-287.

Abstract:
Functional expression of insecticide-resistant GABA receptors from the mosquito Aedes aegypti.

We are interested in cloning insecticide resistance genes from vector mosquitos for use as selectable markers in their genetic transformation. As a first step towards this goal, we here report the functional homomultimeric expression of a gamma-aminobutyric acid (GABA) receptor subunit gene, Resistance to dieldrin (Rdl), from the yellow fever mosquito Aedes aegypti in baculovirus-infected insect cell lines. Replacement of alanine296 with a serine leads to approximately 100-fold insensitivity to picrotoxin as previously observed in Drosophila. This shows not only that the mosquito GABA receptor cDNA is functional but also that it can be simply mutated to resistance. Strategies for incorporation of this cDNA into a minigene for the genetic transformation of mosquitoes are discussed.
 Abstract.  Author URL
Ffrench-Constant, R.H., Steichen, J.C., Shotkoski, F. (1994). Polymerase chain reaction diagnostic for cyclodiene insecticide resistance in the mosquito Aedes aegypti. Med Vet Entomol, 8(1), 99-100. Author URL
Ffrench-Constant, R.H. (1994). The molecular and population genetics of cyclodiene insecticide resistance. Insect Biochem Mol Biol, 24(4), 335-345.

Abstract:
The molecular and population genetics of cyclodiene insecticide resistance.

Cyclodiene resistance has accounted for over 60% of reported cases of insecticide resistance. Understanding of this resistance can therefore help us answer questions relating to the mechanism and origin of representative resistance-associated mutations, questions fundamental to the molecular and populations genetics of pesticide resistance. The cyclodiene resistance gene Rdl (resistance to dieldrin) was cloned from a mutant of the model insect Drosophila resistant to cyclodienes and picrotoxinin. Rdl codes for a subunit of a novel class of GABA gated chloride ion channels and resistance is correlated with replacement of the same amino acid residue in a wide range of species from different insect orders. This single amino acid replacement Ala302 > Ser, within the proposed lining of the chloride ion channel, also confers insensitivity to the blocking action of cyclodienes and picrotoxinin on GABA gated chloride ion channels expressed in Xenopus oocytes. The resistance mechanism involves both changes in cyclodiene binding site affinity and also a change in the rate of receptor desensitization which destabilizes the cyclodiene-favored conformation. Documentation of the resistance associated mutation has allowed for the design of a PCR based molecular monitoring technique. This technique gives more accurate estimates of resistance gene frequency from smaller sample sizes and has shown the frequency of resistance in apparently unselected populations of Drosophila to be as high as 1%. We are still uncertain as to why resistance persists in the apparent absence of selection pressure and any severe reduction in the fitness of resistant strains, besides a paralytic phenotype at high temperature, remains undocumented.
 Abstract.  Author URL
Ffrench-Constant, R.H., Rocheleau, T.A., Steichen, J.C., Chalmers, A.E. (1993). A point mutation in a Drosophila GABA receptor confers insecticide resistance. Nature, 363(6428), 449-451.

Abstract:
A point mutation in a Drosophila GABA receptor confers insecticide resistance.

Vertebrates and invertebrates both have GABA (gamma-aminobutyric acid) as a major inhibitory neurotransmitter. GABAA receptors in vertebrates assemble as heteromultimers to form an integral chloride ion channel. These receptors are targets for drugs and pesticides and are also implicated in seizure-related diseases. Picrotoxinin (PTX) and cyclodiene insecticides are GABAA receptor antagonists which competitively displace each other from the same binding site. Insects and vertebrates showing resistance to cyclodienes also show cross-resistance to PTX. Previously, we used a field-isolated Drosophila mutant Rdl (Resistant to dieldrin) insensitive to PTX and cyclodienes to clone a putative GABA receptor. Here we report the functional expression and novel pharmacology of this GABA receptor and examine the functionality of a resistance-associated point mutation (alanine to serine) within the second membrane-spanning domain, the region thought to line the chloride ion channel pore. This substitution is found globally in Drosophila populations. This mutation not only identifies a single amino acid conferring high levels of resistance to the important GABA receptor antagonist PTX but also, by conferring resistance to cyclodienes, may account for over 60% of reported cases of insecticide resistance.
 Abstract.  Author URL
ffrench-Constant, R.H., Steichen, J.C., Rocheleau, T.A., Aronstein, K., Roush, R.T. (1993). A single-amino acid substitution in a gamma-aminobutyric acid subtype a receptor locus is associated with cyclodiene insecticide resistance in Drosophila populations. Proc Natl Acad Sci U S A, 90(5), 1957-1961.

Abstract:
A single-amino acid substitution in a gamma-aminobutyric acid subtype a receptor locus is associated with cyclodiene insecticide resistance in Drosophila populations.

Resistance to cyclodiene insecticides, documented in at least 277 species, is perhaps the most common kind of resistance to any pesticide. By using cyclodiene resistance to localize the responsible gene, a gamma-aminobutyric acid type a receptor/chloride ion-channel gene was previously cloned and sequenced from an insecticide-susceptible Drosophila melanogaster strain. We now describe the molecular genetics of the resistance allele. A single-base-pair mutation, causing a single-amino acid substitution (Ala-->Ser) within the second membrane-spanning region of the channel, was found to be the only consistent difference between resistant and susceptible strains of D. melanogaster. Some resistant strains of Drosophila simulans show the same mutation, whereas others show an alternative single-base-pair mutation in the same codon, resulting in the substitution of a different amino acid (glycine). These constitute single-box-pair mutations in insects that confer high levels of resistance to insecticides. The presence of the resistance mutations was then tested in a much larger set of strains by the PCR and subsequent digestion with a diagnostic restriction endonuclease. Both resistance-associated mutations cause the loss of a Hae II site. This site was invariably present in 122 susceptible strains but absent in 58 resistant lines of the two species sampled from five continents. PCR/restriction endonuclease treatment was also used to examine linkage of an EcoRI polymorphism in a neighboring intron in D. melanogaster, which was found associated with resistance in all but 3 of 48 strains examined. These PCR-based techniques are widely applicable to examination of the uniqueness of different resistance alleles in widespread populations, the identification of resistance mechanisms in different species, and the determination of resistance frequencies in monitoring.
 Abstract.  Author URL
Thompson, M., Shotkoski, F., ffrench-Constant, R. (1993). Cloning and sequencing of the cyclodiene insecticide resistance gene from the yellow fever mosquito Aedes aegypti. Conservation of the gene and resistance associated mutation with Drosophila. Febs Lett, 325(3), 187-190.

Abstract:
Cloning and sequencing of the cyclodiene insecticide resistance gene from the yellow fever mosquito Aedes aegypti. Conservation of the gene and resistance associated mutation with Drosophila.

In order to examine the conservation of the mechanism of cyclodiene insecticide resistance between species we cloned a cDNA from the yellow fever mosquito Aedes aegypti homologous to the resistance gene Rdl in Drosophila. In D. melanogaster, resistance to cyclodienes and picrotoxinin is caused by a single amino acid substitution (alanine to serine) in the putative channel lining of a gamma-aminobutyic acid gated chloride ion channel. We report that the mosquito gene not only shows high homology to that of Drosophila but also that resistant strains display substitution of the same amino acid. The significance of this result in relation to the evolution of pesticide resistance, the use of Drosophila as a model insect for resistance studies and the potential use of this gene as a selectable marker in the genetic transformation of non-Drosophilids is discussed.
 Abstract.  Author URL
ffrench-Constant, R.H. (1993). Cloning of a putative GABAA receptor from cyclodiene-resistant Drosophila: a case study in the use of insecticide-resistant mutants to isolate neuroreceptors. Exs, 63, 210-223.

Abstract:
Cloning of a putative GABAA receptor from cyclodiene-resistant Drosophila: a case study in the use of insecticide-resistant mutants to isolate neuroreceptors.

This chapter uses the isolation and cloning of cyclodiene resistance from Drosophila melanogaster to illustrate how mutants resistant to a toxicant can be used to study neuroreceptors. Isolation of mutants from the field, mapping of the single gene responsible and its subsequent cloning are described. As confirmation of gene cloning a susceptible allele of the gene has been used to genetically transform resistant individuals to susceptibility. The gene product appears to code for a subunit of a receptor highly similar to vertebrate GABAA receptor/chloride ion channels, and functional expression studies are described which will elucidate its pharmacology. Cyclodiene resistance is extremely widespread, occurring in both invertebrates and vertebrates. Thus examination of resistance-associated mutations in this receptor in a range of species will enhance our understanding of both the binding sites of toxic ligands and the genetic basis of pesticide resistance.
 Abstract.  Author URL
Ffrench-Constant, R.H. (1993). Cloning of the Drosophila cyclodiene insecticide resistance gene: a novel GABAA receptor subtype?. Comp Biochem Physiol C, 104(1), 9-12.

Abstract:
Cloning of the Drosophila cyclodiene insecticide resistance gene: a novel GABAA receptor subtype?

1. gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in both vertebrates and invertebrates. GABAA receptors are composed of a number of different subunits that assemble to form a chloride ionophore. 2. Several subunit types, alpha, beta, gamma, delta and rho have been cloned from vertebrates, but until recently these receptors have remained uncloned from invertebrates. 3. GABAA receptors form the proposed site of action of cyclodiene insecticides. Therefore a Drosophila mutant (Rdl), resistant to cyclodienes and the GABAA receptor ligand picrotoxin (PTX), was used to clone the gene responsible for resistance as a putative invertebrate GABAA receptor. 4. Analysis of the predicted amino acid sequence and gene structure shows that Rdl codes for a receptor subunit similar to vertebrate GABAA receptors, but sufficiently different that it may represent a novel class of GABAA receptor subtype. 5. Cyclodiene insecticide resistance accounts for over 60% of reported cases of insecticide resistance and is also found in vertebrates. 6. Therefore elucidating the molecular basis of cyclodiene resistance is not only important to our understanding of pesticide action on the GABAA receptor but also in examining the conservation of the resistance mechanism between vertebrates and invertebrates.
 Abstract.  Author URL
Thompson, M., Steichen, J.C., ffrench-Constant, R.H. (1993). Conservation of cyclodiene insecticide resistance-associated mutations in insects. Insect Mol Biol, 2(3), 149-154.

Abstract:
Conservation of cyclodiene insecticide resistance-associated mutations in insects.

Cyclodiene insecticide resistance has accounted for over 60% of reported cases of insecticide resistance. In Drosophila melanogaster resistance is associated with a single base pair substitution in the GABA receptor/chloride ion channel gene Rdl. This substitution predicts the replacement of an alanine with a serine in the second membrane spanning domain, the region thought to line the chloride ion channel pore. Here we report, via the use of degenerate primers in the polymerase chain reaction, that precisely the same substitution is present in three pests from three different insect orders: the house fly (Diptera), red flour beetle (Coleoptera) and American cockroach (Dictyoptera). This finding suggests that there are a limited number of mutations that can confer resistance to cyclodienes, putative channel blockers, while still maintaining adequate chloride ion channel function. The conservation of the resistance-associated mutation between Drosophila and pest insects directly validates the approach of using this insect as a model system for isolating and studying resistance genes. The importance of single base pair substitutions in the evolution of pesticide resistance and in the design of molecular monitoring techniques is discussed.
 Abstract.  Author URL
ffrench-Constant, R.H., Rocheleau, T.A. (1993). Drosophila gamma-aminobutyric acid receptor gene Rdl shows extensive alternative splicing. J Neurochem, 60(6), 2323-2326.

Abstract:
Drosophila gamma-aminobutyric acid receptor gene Rdl shows extensive alternative splicing.

The Drosophila gamma-aminobutyric acid (GABA) receptor subunit gene Rdl was isolated on the basis of a mutant phenotype showing high levels of insensitivity to picrotoxinin and cyclodiene insecticides. Following analysis of two dissimilar cDNAs isolated from the locus, we report that Rdl undergoes extensive alternative splicing at two locations in the putative extracellular domain. At each location a choice is made between exons of the same size: "a" or "b" (23 amino acids long with two substitutions) and "c" or "d" (46 residues long with 10 substitutions). The function of these alternative exons remains unclear; however, exon d contains a putative site for casein kinase II phosphorylation. All possible combinations of exons (a with c or d and b with c or d) were found in RNA isolated from early embryos. This is the first demonstration of alternative splicing in a GABA receptor gene from invertebrates.
 Abstract.  Author URL
Lee, H.J., Rocheleau, T., Zhang, H.G., Jackson, M.B., ffrench-Constant, R.H. (1993). Expression of a Drosophila GABA receptor in a baculovirus insect cell system. Functional expression of insecticide susceptible and resistant GABA receptors from the cyclodiene resistance gene Rdl. Febs Lett, 335(3), 315-318.

Abstract:
Expression of a Drosophila GABA receptor in a baculovirus insect cell system. Functional expression of insecticide susceptible and resistant GABA receptors from the cyclodiene resistance gene Rdl.

Recombinant baculoviruses containing two alternative splice forms of the Drosophila Rdl GABA receptor gene were constructed. Spodoptera frugiperda (Sf21) cells infected with either splice form expressed a transcript of expected size (2.5 kb). Western blotting of cell membrane extracts and immunoprecipitation experiments with an anti-Rdl antiserum recognized a protein of the expected size of approximately 65 kDa. Whole cell patch clamp analysis of cells infected with either splice form revealed functional expression of GABA gated chloride ion channels which were blocked by application of 1 microM picrotoxinin. Following replacement of alanine 302 with a serine, a mutation associated with resistance to picrotoxinin and cyclodiene insecticides, mutant channels showed similar levels of insensitivity to picrotoxinin (approximately 100-fold) as those observed in recordings from cultured Drosophila neurons. The significance of the expression of an insect GABA receptor in an insect cell line and the similarity of the results from these functional expression studies to recordings from cultured neurons is discussed.
 Abstract.  Author URL
ffrench-Constant, R.H., Rocheleau, T. (1992). Drosophila cyclodiene resistance gene shows conserved genomic organization with vertebrate gamma-aminobutyric acidA receptors. J Neurochem, 59(4), 1562-1565.

Abstract:
Drosophila cyclodiene resistance gene shows conserved genomic organization with vertebrate gamma-aminobutyric acidA receptors.

Genomic clones from the Rdl locus of Drosophila, whose mutant phenotype is resistant to cyclodiene insecticides and picrotoxin, were characterized by restriction mapping and partial sequencing to determine intron/exon structure. The coding region of the gene comprises nine identified exons and spans greater than 25 kb of genomic DNA. The structure of the Drosophila Rdl receptor subunit was compared with those of vertebrate gamma-aminobutyric acid subtype a (GABAA) receptors and nicotinic acetylcholine receptors (nAChRs). The first six introns in Rdl show positions similar to those in vertebrate GABAA receptors, whereas the last two differ. It is interesting that the last intron appears to be in a position similar to that in nAChRs. These results are examined in relation to the proposal, based on amino acid identities, that Rdl codes for a novel class of GABAA receptor subunit more closely related to glycine receptors, and the possible place of Rdl in the lineage of the receptor superfamily is discussed.
 Abstract.  Author URL
Bloomquist, J.R., Roush, R.T., ffrench-Constant, R.H. (1992). Reduced neuronal sensitivity to dieldrin and picrotoxinin in a cyclodiene-resistant strain of Drosophila melanogaster (Meigen). Arch Insect Biochem Physiol, 19(1), 17-25.

Abstract:
Reduced neuronal sensitivity to dieldrin and picrotoxinin in a cyclodiene-resistant strain of Drosophila melanogaster (Meigen).

Toxicological and neurophysiological studies were performed to characterize the resistance mechanism in a cyclodiene-resistant strain of Drosophila melanogaster (Maryland strain). Dieldrin had an LC50 of 0.058 ppm against the larvae of susceptible D. melanogaster (Oregon-R wild type) when formulated in the rearing media. The LC50 of the resistant Maryland strain was 10.8 ppm, giving a resistance ratio (LC50-Maryland/LC50-susceptible) of 186-fold. Suction electrode recordings were made from peripheral nerves of the larval central nervous system to test whether reduced nerve sensitivity played any role in the observed resistance. In susceptible preparations (n = 5), inhibition of nerve firing by 1 mM gamma-aminobutyric acid (GABA) was effectively antagonized within 3-10 min by 10 microM dieldrin. In contrast, 30 min incubations with 10 microM dieldrin had no effect on preparations from cyclodiene-resistant individuals (n = 5). Similarly, 10 microM picrotoxinin blocked GABA-dependent inhibition in susceptible nerve preparations (n = 3). In recordings from resistant insects (n = 4), picrotoxinin displayed either weak antagonism of GABA or hyperexcitation indistinguishable from susceptible preparations. These results demonstrate that cyclodiene resistance in the Maryland strain of D. melanogaster 1) is expressed in immature stages, 2) is present at the level of the nerve, and 3) extends to picrotoxinin, albeit at a reduced level compared with dieldrin. The possible role of an altered GABA receptor in this resistance is discussed.
 Abstract.  Author URL
Ffrench-Constant, R.H., Roush, R.T. (1991). Gene mapping and cross-resistance in cyclodiene insecticide-resistant Drosophila melanogaster (Mg.). Genet Res, 57(1), 17-21.

Abstract:
Gene mapping and cross-resistance in cyclodiene insecticide-resistant Drosophila melanogaster (Mg.).

Resistance to the cyclodiene insecticide dieldrin maps to a single gene (Rdl) on the left arm of chromosome III in Drosophila melanogaster (Meigen). The gene was further mapped by the use of chromosomal deficiencies to a single letter sub-region, 66F, on the polytene chromosome. The cross-resistance spectrum of a backcrossed strain lacking elevated mixed function oxidase activity, a common resistance mechanism, was examined. Levels of resistance similar to those found in other insects were found to dieldrin, aldrin, endrin, lindane, and picrotoxinin. Strong similarity of this single major gene with that found in other cyclodiene resistant insects is suggested by its cross-resistance spectrum and chromosomal location, via homology with other Diptera. The significance of major genes in insecticide resistance is discussed.
 Abstract.  Author URL
Ffrench-Constant, R.H., Mortlock, D.P., Shaffer, C.D., MacIntyre, R.J., Roush, R.T. (1991). Molecular cloning and transformation of cyclodiene resistance in Drosophila: an invertebrate gamma-aminobutyric acid subtype a receptor locus. Proc Natl Acad Sci U S A, 88(16), 7209-7213.

Abstract:
Molecular cloning and transformation of cyclodiene resistance in Drosophila: an invertebrate gamma-aminobutyric acid subtype a receptor locus.

Cyclodiene resistance represents 60% of the reported cases of insecticide resistance and is also present in vertebrates. Resistance is due to insensitivity of the cyclodiene/picrotoxinin binding site on the gamma-aminobutyric acid subtype a (GABAA) receptor-chloride ionophore complex. Following isolation of cyclodiene-resistant Drosophila mutants, we report the cloning of the locus conferring resistance via a "chromosomal walk" and rescue of the susceptible phenotype by P-element-mediated germ-line transformation. Amino acid sequence analysis of a cDNA from the locus reveals homology with vertebrate GABAA subunits. To our knowledge, this represents the first cloning of an invertebrate GABA receptor and also allows us to manipulate the resistance status of an insect via germ-line transformation. This gene may be useful as a selectable marker in other insect systems.
 Abstract.  Author URL
Ffrench-Constant, R.H., Roush, R.T., Mortlock, D., Dively, G.P. (1990). Isolation of dieldrin resistance from field populations of Drosophila melanogaster (Diptera: Drosophilidae). J Econ Entomol, 83(5), 1733-1737.

Abstract:
Isolation of dieldrin resistance from field populations of Drosophila melanogaster (Diptera: Drosophilidae).

High levels (about 4,000-fold) of resistance to dieldrin were isolated by screening field-collected populations of Drosophila melanogaster (Meigen). The resistance was made homozygous following 2-4 generations of selection. A single, major gene mapping to the left arm of chromosome III was solely responsible for resistance. The implications of the recovery of resistant mutants from field populations of D. melanogaster are discussed.
 Abstract.  Author URL
Lines, J.D., ffRench-Constant, R.H., Kasim, S.H. (1990). Testing Anopheles albimanus for genetic linkage of insecticide resistance genes by combining insecticide bioassay and biochemical methods. Med Vet Entomol, 4(4), 445-450.

Abstract:
Testing Anopheles albimanus for genetic linkage of insecticide resistance genes by combining insecticide bioassay and biochemical methods.

A microtitre-plate assay which distinguishes propoxur-resistant from susceptibles Anopheles albimanus Weidemann was used to test for linkage between the genes for propoxur- and dieldrin-resistance. The adult progeny of a backcross between a doubly-resistant colony and a fully susceptible colony were exposed in conventional test kits to the standard discriminating dose of dieldrin, and kept in the insectary overnight. Both live and dead insects were then assayed individually for propoxur-resistance. The results showed that heterozygotes for propoxur-resistance could be reliably distinguished from susceptibles whether or not they had been killed up to 24 h previously by dieldrin treatment. In this way all the backcross progeny could be scored at both resistance loci, and all four genotypic classes identified. Resistant and susceptible alleles at the two loci were inherited independently, demonstrating the absence of linkage. The usual method of testing for linkage between resistance genes is inefficient and open to bias, because insects have to be exposed to each insecticide in turn, and only half of them can be scored at both loci. The method shown here avoids these drawbacks.
 Abstract.  Author URL
Ffrench-Constant, R.H., Bonning, B.C. (1989). Rapid microtitre plate test distinguishes insecticide resistant acetylcholinesterase genotypes in the mosquitoes Anopheles albimanus, An. nigerrimus and Culex pipiens. Med Vet Entomol, 3(1), 9-16.

Abstract:
Rapid microtitre plate test distinguishes insecticide resistant acetylcholinesterase genotypes in the mosquitoes Anopheles albimanus, An. nigerrimus and Culex pipiens.

A rapid method of distinguishing insecticide insensitive acetylcholinesterase (AChE) genotypes was applied to three species of mosquitoes. This relies on comparing rates of an AChE mediated reaction in the presence and absence of insecticides which are inhibitors, using a kinetic microtitre plate reader. Clearer and more rapid resolution between genotypes was achieved than with previous assays which measure the amount of product formed at a fixed end-point. Results are presented for the F1s from crossing resistant and susceptible Anopheles albimanus Wiedemann and Culex pipiens L., for a strain of An. albimanus with a translocation linking the AChE gene to the Y chromosome and for field collected An. nigerrimus Giles. Propoxur and malaoxon were used as inhibitors. In all three species the enzyme was more insensitive to propoxur than malaoxon. Susceptible enzymes in all species also showed higher uninhibited AChE activity than their resistant counterparts. Presentation of both inhibited and uninhibited activities side by side may be useful to identify insects likely to be misclassified due to abnormally low AChE activities. Estimated frequencies of the three resistance genotypes in field populations of An. nigerrimus conformed to Hardy-Weinberg ratios. The implications of this technique for laboratory and field studies on insects are discussed.
 Abstract.  Author URL
ffrench-Constant, R.H., Devonshire, A.L. (1987). A multiple homogenizer for rapid sample preparation in immunoassays and electrophoresis. Biochem Genet, 25(7-8), 493-499.

Abstract:
A multiple homogenizer for rapid sample preparation in immunoassays and electrophoresis.

A multiple homogenizer is described for preparing samples of small invertebrates or tissue in a flat-bottom immunoplate. Its efficiency was evaluated by immunoassay of carboxylesterase (E4), the enzyme conferring insecticide resistance in the peach potato aphid (Myzus persicae). This equipment was shown to release more enzyme, with less variability, than homogenizing individual aphids and its efficiency allows one person to analyze up to 3000 individual insects per day. It is also suitable for preparing samples for electrophoretic analysis. In the present study samples were loaded onto electrophoresis gels rapidly and accurately by using an eight-channel multipipette.
 Abstract.  Author URL
Conferences
McCart, C., Ffrench-Constant, R.H. (2008). Dissecting the insecticide-resistance- associated cytochrome P450 gene Cyp6g1. , 64(6), 639-645.

Abstract:
Dissecting the insecticide-resistance- associated cytochrome P450 gene Cyp6g1.

The cytochrome P450 gene Cyp6g1 is overtranscribed in all field isolates of DDT-resistant Drosophila melanogaster (Meigen) and confers a fitness advantage when inherited via the female. Overtranscription is associated with the insertion of an Accord transposable element into the 5' end of the resistance allele. Here the authors attempt to dissect the transcription of the P450 gene in order to understand why resistance confers an advantage rather than the expected cost.
 Abstract.  Author URL
Waterfield, N.R., Daborn, P.J., Ffrench-Constant, R.H. (2004). Insect pathogenicity islands in the insect pathogenic bacterium Photorhabdus. , 29(3), 240-250. Author URL
Koch, P.B., Behnecke, B., Weigmann-Lenz, M., Ffrench-Constant, R.H. (2000). Insect pigmentation: activities of beta-alanyldopamine synthase in wing color patterns of wild-type and melanic mutant swallowtail butterfly Papilio glaucus. , 13 Suppl 8, 54-58.

Abstract:
Insect pigmentation: activities of beta-alanyldopamine synthase in wing color patterns of wild-type and melanic mutant swallowtail butterfly Papilio glaucus.

Color pattern formation was studied in wild-type and melanic swallowtails because of their unique pigment system, the papiliochromes, which are derived from the tyrosine as well as from the tryptophan pathway. In a comparative approach we used females of Papilio glaucus which occur in two phenotypes, either wild-type (yellow and black) or melanic. Pigment synthesis in the developing wings starts with formation of yellow papiliochromes followed later by black melanin. From earlier studies we know that dopamine produced from DOPA by the enzyme dopadecarboxylase (DDC), is a precursor of both black melanin and also of N-beta-alanyldopamine (NBAD) in yellow papiliochrome synthesis. Thus, DDC expression and enzyme activity is required in both types of pigment forming scale cells and occurs in a time and pattern specific manner. However, differential activity of DDC alone can not be sufficient to regulate synthesis of different pigments in differently colored scales. Therefore, we tested the hypothesis whether activity of another enzyme, beta-alanyldopamine synthase (BAS), regulates specifically papiliochrome synthesis. BAS transfers beta-alanine to dopamine to give NBAD a component of yellow papiliochrome. We developed a radio-enzyme-assay of BAS activity in which (14C)-beta-alanine is incubated with dopamine, Mg++-ions and ATP together with wing homogenates containing putative BAS activity. In fact, high BAS activity was measured in yellow wings in concert with a high DDC activity. In contrast, in melanic wings almost no BAS activity was found. From this result it is clear, that papiliochrome synthesis in yellow scales is switched on by BAS shifting dopamine into the papiliochrome pathway and out of the melanin pathway or vice versa.
 Abstract.  Author URL
ffrench-Constant, R.H. (1999). Insecticide resistance and nucleotide variability in the coffee berry borer. , 466, 3-5. Author URL
ffrench-Constant, R.H., Pittendrigh, B., Vaughan, A., Anthony, N. (1999). Why are there so few insecticide resistance-associated mutations?. (232), 137-145. Author URL
ffrench-Constant, R.H., Pittendrigh, B., Vaughan, A., Anthony, N. (1998). Why are there so few resistance-associated mutations in insecticide target genes?. , 353(1376), 1685-1693.

Abstract:
Why are there so few resistance-associated mutations in insecticide target genes?

The genes encoding the three major targets of conventional insecticides are: Rdl, which encodes a gamma-aminobutyric acid receptor subunit (RDL); para, which encodes a voltage-gated sodium channel (PARA); and Ace, which encodes insect acetylcholinesterase (AChE). Interestingly, despite the complexity of the encoded receptors or enzymes, very few amino acid residues are replaced in different resistant insects: one within RDL, two within PARA and three or more within AChE. Here we examine the possible reasons underlying this extreme conservation by looking at the aspects of receptor and/or enzyme function that may constrain replacements to such a limited number of residues.
 Abstract.  Author URL
Brun, L.O., Ffrench-Constant, R.H., CAFE, A.S.I. (1997). Insecticide resistance in the coffee berry borer: State of current knowledge. , 665-672. Author URL
Breilid, H., Brun, L.O., Andreev, D., Ffrench-Constant, R.H., Kirkendall, L.R., CAFE, A.S.I. (1997). Phylogeographic patterns of introduced populations of the coffee berry borer Hypothenemus hampei (Ferrari) (Coleoptera : Scolytidae) inferred from mitochondrial DNA sequences. , 653-655. Author URL

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