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1.
Insect Mol Biol ; 22(5): 532-40, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23834845

RESUMO

Cytochrome P450s are important both in the metabolism of xenobiotics and the production of compounds such as cyanogenic glucosides, which insects use in their defence. In the present study, we use transcriptomic and genomic information to isolate and name P450-encoding genes from the butterfly Heliconius melpomene. We classify each of the putative genes into its appropriate superfamily and compare the distribution of P450s across sequenced insects. We also identify homologues of two P450s known to be involved in cyanogenesis in the six-spot Burnet moth, Zygaena filipendulae. Classification of Heliconius P450s should be an important step in the dissection of their role in the exploitation of their host plant, the passion vine Passiflora.


Assuntos
Borboletas/genética , Sistema Enzimático do Citocromo P-450/genética , Genes de Insetos , Glicosídeos/genética , Receptores Odorantes/genética , Transcriptoma , Animais , Borboletas/enzimologia , Glicosídeos/biossíntese , Família Multigênica , Passiflora/enzimologia , Passiflora/parasitologia , Filogenia
3.
J Evol Biol ; 24(6): 1351-62, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21507117

RESUMO

In Drosophila melanogaster, the DDT resistance allele (DDT-R) is beneficial in the presence of DDT. Interestingly, DDT-R also elevates female fitness in the absence of DDT and existed in populations before DDT use. However, DDT-R did not spread regardless of DDT-independent selective advantages in females. We ask whether sexual antagonism could explain why DDT-R did not spread before pesticide use. We tested pre- and post-copulatory male fitness correlates in two genetic backgrounds into which we backcrossed the DDT-R allele. We found costs to DDT-R that depended on the genetic background in which DDT-R was found and documented strong epistasis between genetic background and DDT-R that influenced male size. Although it remains unclear whether DDT-R is generally sexually antagonistic, or whether the fitness costs noted would be sufficient to retard the spread of DDT-R in the absence of DDT, general fitness advantages to DDT-R in the absence of DDT may be unlikely.


Assuntos
DDT , Drosophila melanogaster/genética , Epistasia Genética , Resistência a Inseticidas/genética , Inseticidas , Preferência de Acasalamento Animal , Alelos , Animais , Tamanho Corporal/genética , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/efeitos dos fármacos , Feminino , Masculino , Espermatozoides/fisiologia
4.
Insect Mol Biol ; 20(2): 267-78, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21205278

RESUMO

Three genes encoding proteins showing sequence similarity and features typical of insect APNs were characterized in C. tremulae and designed as CtAPN1, CtAPN2 and CtAPN3. Expression analysis of the three C. tremulae APN genes showed that CtAPN2 transcript is more abundant in the fat body, whereas both CtAPN1 and CtAPN3 are specifically expressed in the midgut. Despite a similar genomic organization, lepidopteran and coleopteran APNs are phylogenetically distant, suggesting that APN gene duplication events occurred after these two insect orders split. Sequence and expression comparisons of CtAPN1, CtAPN2 and CtAPN3 cDNAs in a C. tremulae Bacillus thuringiensis (Bt)-susceptible and in a Bt-resistant strain did not show any polymorphism at the amino acid level or difference at the transcription level.


Assuntos
Proteínas de Bactérias , Antígenos CD13/genética , Besouros/enzimologia , Besouros/genética , Endotoxinas , Proteínas Hemolisinas , Controle Biológico de Vetores , Sequência de Aminoácidos , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Sistema Digestório/enzimologia , Corpo Adiposo/enzimologia , Feminino , Perfilação da Expressão Gênica , Estágios do Ciclo de Vida , Filogenia , Plantas Geneticamente Modificadas/genética , Populus/genética , Alinhamento de Sequência , Tribolium/genética
5.
Parasitology ; 137(11): 1695-706, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20500922

RESUMO

SUMMARY: 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.


Assuntos
Imunidade Celular , Imunidade Humoral , Manduca , Photorhabdus/imunologia , Rhabditoidea/imunologia , Animais , Regulação da Expressão Gênica/imunologia , Hemócitos/imunologia , Interações Hospedeiro-Parasita , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Manduca/crescimento & desenvolvimento , Manduca/imunologia , Manduca/microbiologia , Manduca/parasitologia , Photorhabdus/patogenicidade , Rhabditoidea/microbiologia , Rhabditoidea/patogenicidade , Simbiose/imunologia , Virulência
6.
Insect Mol Biol ; 19(1): 61-75, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19909380

RESUMO

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.


Assuntos
Perfilação da Expressão Gênica , Proteínas de Insetos/metabolismo , Mariposas/metabolismo , beta-Frutofuranosidase/metabolismo , Sequência de Aminoácidos , Animais , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Digestão , Etiquetas de Sequências Expressas , Trato Gastrointestinal/metabolismo , Transferência Genética Horizontal , Inativação Metabólica , Larva/imunologia , Larva/metabolismo , Dados de Sequência Molecular , Mariposas/genética , Mariposas/imunologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , beta-Frutofuranosidase/genética
7.
Genet Res (Camb) ; 91(4): 237-42, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19640319

RESUMO

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.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Variação Genética , Peptídeos/genética , Animais , Drosophila melanogaster/microbiologia , Drosophila melanogaster/fisiologia , Feminino , Interações Hospedeiro-Patógeno , Masculino , Análise de Regressão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Comportamento Sexual Animal , Especificidade da Espécie , Wolbachia/fisiologia
8.
J Exp Biol ; 212(Pt 12): 1840-8, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19483002

RESUMO

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.


Assuntos
Escherichia coli/imunologia , Imunidade Celular/fisiologia , Proteínas de Insetos/fisiologia , Manduca/microbiologia , Peptídeos/fisiologia , Photorhabdus/imunologia , Animais , Corpo Adiposo/imunologia , Corpo Adiposo/metabolismo , Hemócitos/imunologia , Hemócitos/metabolismo , Proteínas de Insetos/genética , Peptídeos/genética , Interferência de RNA
9.
Insect Mol Biol ; 18(4): 507-16, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19538546

RESUMO

Injecting the insect pathogenic bacterium Photorhabdus luminescens into the blood system of the model lepidopteran insect Manduca sexta induces nitric oxide synthase (NOS) expression in the fat body and blood cells (haemocytes), whereas following oral ingestion of bacteria NOS expression is limited to the gut. We used RNA interference to knock-down expression of NOS throughout the insect. Preventing NOS induction in this way adversely affected the survival of orally infected insects and caused a significant increase in the number of bacteria crossing into the haemolymph. By contrast, knock-down of NOS had no effect on the mortality rate of insects infected with P. luminescens by injection. Pharmacological inhibition of NOS decreased both nitric oxide (NO) levels in the gut wall and survival of orally infected insects, whereas elevation of gut wall NO using an NO donor increased survival of NOS silenced caterpillars. Together, our results imply that induced synthesis of NO is important in mediating insect immune defence against the pathogen by inhibiting transfer of bacteria across the gut wall.


Assuntos
Trato Gastrointestinal/metabolismo , Manduca/metabolismo , Manduca/microbiologia , Óxido Nítrico/biossíntese , Photorhabdus/fisiologia , Animais , Corpo Adiposo/metabolismo , Trato Gastrointestinal/microbiologia , Hemócitos/metabolismo , Larva , Interferência de RNA
10.
J Insect Physiol ; 54(1): 309-18, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18001766

RESUMO

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.


Assuntos
Regulação da Expressão Gênica/imunologia , Manduca/imunologia , Manduca/microbiologia , Photorhabdus/imunologia , Fatores Etários , Animais , Hemócitos/imunologia , Larva/imunologia , Microscopia Confocal , Monofenol Mono-Oxigenase/sangue , Monofenol Mono-Oxigenase/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sobrevida
11.
Insect Mol Biol ; 16(5): 591-600, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17725800

RESUMO

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.


Assuntos
Caderinas/genética , Besouros/genética , Sequência de Aminoácidos , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Sequência de Bases , Caderinas/química , Caderinas/metabolismo , Clonagem Molecular , Besouros/metabolismo , DNA Complementar , Endotoxinas/metabolismo , Trato Gastrointestinal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hemolisinas/metabolismo , Larva/metabolismo , Dados de Sequência Molecular , Filogenia
12.
J Bacteriol ; 188(6): 2254-61, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16513755

RESUMO

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.


Assuntos
Toxinas Bacterianas/toxicidade , Mariposas/microbiologia , Photorhabdus/patogenicidade , Fatores de Virulência/toxicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Citoesqueleto/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Escherichia coli/genética , Escherichia coli/metabolismo , Ordem dos Genes , Hemócitos/efeitos dos fármacos , Larva/microbiologia , Substâncias Macromoleculares , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Glicoproteínas de Membrana/toxicidade , Microscopia Eletrônica de Transmissão , Perforina , Proteínas Citotóxicas Formadoras de Poros , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/toxicidade , Serratia/genética , Fatores de Virulência/genética
13.
Insect Mol Biol ; 14(2): 137-43, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15796746

RESUMO

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.


Assuntos
Caderinas/genética , Catepsinas/genética , Besouros/genética , Etiquetas de Sequências Expressas , Trato Gastrointestinal/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Análise por Conglomerados , Besouros/metabolismo , Biologia Computacional , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA
14.
Mol Ecol ; 13(8): 2491-504, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15245421

RESUMO

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


Assuntos
Sistema Enzimático do Citocromo P-450/genética , DDT/intoxicação , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Resistência a Medicamentos/genética , Seleção Genética , Análise de Variância , Animais , Primers do DNA , Drosophila melanogaster/efeitos dos fármacos , Eletroforese em Gel de Ágar , Frequência do Gene , Triagem de Portadores Genéticos , Variação Genética , Repetições de Microssatélites/genética , Mutação/genética , Reação em Cadeia da Polimerase
15.
Cell Microbiol ; 6(4): 345-53, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15009026

RESUMO

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


Assuntos
Apoptose , Toxinas Bacterianas/toxicidade , Photorhabdus , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Células COS , Caspase 3 , Inibidores de Caspase , Caspases/metabolismo , Tamanho Celular , Inibidores de Cisteína Proteinase/farmacologia , Ativação Enzimática , Escherichia coli/genética , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Photorhabdus/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Estaurosporina/farmacologia , Transfecção
16.
Cell Microbiol ; 6(1): 89-95, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14678333

RESUMO

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.


Assuntos
Hemócitos/fisiologia , Manduca/imunologia , Manduca/microbiologia , Fagocitose , Photorhabdus/patogenicidade , Citoesqueleto de Actina/ultraestrutura , Animais , Sobrevivência Celular , Citoesqueleto/ultraestrutura , Escherichia coli/imunologia , Hemócitos/imunologia , Hemócitos/microbiologia , Hemócitos/ultraestrutura , Larva/imunologia , Larva/microbiologia , Photorhabdus/crescimento & desenvolvimento , Photorhabdus/imunologia
17.
Insect Biochem Mol Biol ; 33(7): 701-8, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12826097

RESUMO

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.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/farmacologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/farmacologia , Drosophila/genética , Perfilação da Expressão Gênica , Resistência a Inseticidas/genética , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Animais Geneticamente Modificados , Drosophila/fisiologia , Regulação da Expressão Gênica , Reação em Cadeia da Polimerase , Regulação para Cima
18.
Science ; 297(5590): 2253-6, 2002 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-12351787

RESUMO

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


Assuntos
Sistema Enzimático do Citocromo P-450/genética , DDT , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genes de Insetos , Resistência a Inseticidas/genética , Inseticidas , Alelos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Mapeamento Cromossômico , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Inseticidas/metabolismo , Íntrons , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Especificidade por Substrato , Transcrição Gênica , Transgenes
19.
Proc Natl Acad Sci U S A ; 99(16): 10742-7, 2002 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-12136122

RESUMO

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


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Cosmídeos/fisiologia , Genes Bacterianos/fisiologia , Controle Biológico de Vetores , Photorhabdus , Sequência de Aminoácidos , Animais , Apoptose , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Sequência de Bases , Sequência Consenso , DNA Complementar , Sistema Digestório/citologia , Escherichia coli/patogenicidade , Hemócitos/citologia , Manduca/microbiologia , Dados de Sequência Molecular , Fases de Leitura Aberta , Photorhabdus/genética
20.
Med Vet Entomol ; 16(4): 452-5, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12510900

RESUMO

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.


Assuntos
Anopheles/genética , Cromossomos/genética , Proteínas de Drosophila , Genes de Insetos/genética , Mapeamento Físico do Cromossomo , Receptores de GABA-A/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Feminino , Hibridização In Situ , Infertilidade/genética , Resistência a Inseticidas/genética , Malária/prevenção & controle , Malária/transmissão , Controle de Mosquitos , Controle Biológico de Vetores , Filogenia , Receptores de GABA-A/química , Homologia de Sequência de Aminoácidos
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