RESUMO
The Triatoma sordida subcomplex traditionally included four triatomine species, T. sordida, Triatoma garciabesi, Triatoma guasayana, and Triatoma patagonica, distributed in the Southern Cone of South America. These species have a large intraspecific variability together with an overall similarity, making difficult to establish their taxonomic status. Many cytogenetic, morphometric, and molecular markers have been applied to address this. Recent studies have posed concerns on the inclusion of T. guasayana and T. patagonica within the subcomplex. Also, T. sordida from Argentina has been designed as a new species, Triatoma rosai. Using the cuticular hydrocarbon pattern as chemotaxonomic marker, the relationships among several populations of these species were analyzed by capillary gas chromatography and linear discriminant analysis along 25 collection sites in Argentina, Bolivia, Brazil, and Paraguay. T. sordida and T. rosai populations were differentially clustered in two CHC-based groups: "Group 1" included T. sordida from Eastern Brazil, Eastern Paraguay, and the Bolivian populations from La Paz and Izozog G1; "Group 2" included T. rosai, and T. sordida from Izozog G2 (Bolivia), and Western Paraguay. Whereas T. garciabesi remained closely related to T. sordida and T. rosai, T. guasayana, and T. patagonica were clearly separated from the species of the T. sordida subcomplex. Our results agree with those from other several techniques suggesting that the taxonomy of the T. sordida subcomplex should be revised.
Assuntos
Doença de Chagas , Triatoma , Triatominae , Animais , Bolívia , Brasil , HidrocarbonetosRESUMO
Insect resistance to chemical insecticides is attributed to a combination of different mechanisms, such as metabolic resistance, knockdown resistance, and the cuticular resistance or penetration factor. The insect integument offers an efficient barrier against contact insecticides and its role as penetration factor has been previously reported; however, there is no information about its potential function in the metabolic resistance. Cytochrome P450 genes (CYP) are highly expressed in the fat body of several insects and thus play a key role in their metabolic resistance. Here, we describe new members that belong to the highly genome-wide expanded CYP3093A and CYP4EM subfamilies in the Chagas disease vectors Rhodnius prolixus and Triatoma infestans. We modeled the docking of deltamethrin in their active site and detected differences in some amino acids between both species that are critical for a correct interaction with the substrate. We also knocked down the two constitutively most expressed genes in the integument of resistant T. infestans nymphs (CYP3093A11 and CYP4EM10) in order to find clues on their participation in deltamethrin resistance. This is the first report on the role of the insect integument in detoxification events; although these two CYP genes do not fully explain the resistance observed in T. infestans.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Proteínas de Insetos/genética , Insetos Vetores/genética , Inseticidas/farmacocinética , Tegumento Comum/fisiologia , Nitrilas/farmacocinética , Piretrinas/farmacocinética , Triatoma/genética , Animais , Doença de Chagas/patologia , Doença de Chagas/prevenção & controle , Doença de Chagas/transmissão , Sistema Enzimático do Citocromo P-450/metabolismo , Genes de Insetos/genética , Inativação Metabólica/genética , Proteínas de Insetos/metabolismo , Insetos Vetores/metabolismo , Insetos Vetores/parasitologia , Resistência a Inseticidas/genética , Inseticidas/química , Simulação de Acoplamento Molecular , Nitrilas/química , Ninfa , Filogenia , Piretrinas/química , Rhodnius/genética , Rhodnius/metabolismo , Rhodnius/parasitologia , Triatoma/metabolismo , Triatoma/parasitologia , Trypanosoma cruzi/patogenicidadeRESUMO
The chemical control of Triatoma infestans, the major Chagas disease vector in southern South America, has been threatened in the last years by the emergence of pyrethroid-resistant bug populations. As an alternative approach, the efficacy of the entomopathogenic fungus Beauveria bassiana to control T. infestans populations (regardless their pyrethroid susceptibility) has been demonstrated. Growing research efforts on the interaction between T. infestans and B. bassiana by molecular, ecological, biochemical and behavioral traits has allowed framing such interaction as an evolutionary arms race. This review will focus on the relationships established in this particular host-pathogen system, compiling available data on the relevance of fungal pathogenesis, insect behavior, population dynamics and human intervention to favor fungal dissemination in bug populations. The current snapshot shows the fungus ahead in the evolutionary arms race and predicts a promissory landscape for the biological control of Chagas disease vectors.
Assuntos
Beauveria/fisiologia , Evolução Biológica , Interações Hospedeiro-Patógeno , Insetos Vetores/microbiologia , Triatoma/microbiologia , Triatoma/fisiologia , Animais , Agentes de Controle Biológico , Doença de Chagas/transmissão , Humanos , Resistência a Inseticidas , Inseticidas/farmacologia , Característica Quantitativa Herdável , Triatoma/efeitos dos fármacosRESUMO
The insect integument, formed by the cuticle and the underlying epidermis, is essential for insect fitness, regulation of lipid biosynthesis and storage, insect growth and feeding, together with development progress. Its participation in insecticide resistance has also been outlined. Triatoma infestans Klug (Hemiptera: Reduviidae) is one of the major vectors of Chagas disease in South America; however, genomic data are scarce. In this study, we performed a transcriptome analysis of the nymph integument in order to identify which genes are expressed and their putative role. Using the 454 GS-FLX sequencing platform, we obtained approximately 144,620 reads from the integument tissue. These reads were assembled into 6,495 isotigs and 8,504 singletons. Based on BLAST similarity searches, about 8,000 transcripts were annotated with known genes, conserved domains, and/or Gene Ontology terms.The most abundant transcripts corresponded to transcription factors and nucleic acid metabolism, membrane receptors, cell signaling, and proteins related to cytoskeleton, transport, and cell energy processes, among others. More than 10% of the transcripts-encoded proteins putatively involved in the metabolism of fatty acids and related components (fatty acid synthases, elongases, desaturases, fatty alcohol reductases), structural integument proteins, and the insecticide detoxification system (among them, cytochrome P450s, esterases, and glutathione transferases). Real-time qPCR assays were used to investigate their putative participation in the resistance mechanism. This preliminary study is the first transcriptome analysis of a triatomine integument, and together with prior biochemical information, will help further understandthe role of the integument in a wide array of mechanisms.
Assuntos
Triatoma/metabolismo , Animais , Perfilação da Expressão Gênica , Resistência a Inseticidas/genética , Metabolismo dos Lipídeos , Triatoma/genéticaRESUMO
BACKGROUND: Current Chagas disease vector control strategies, based on chemical insecticide spraying, are growingly threatened by the emergence of pyrethroid-resistant Triatoma infestans populations in the Gran Chaco region of South America. METHODOLOGY AND FINDINGS: We have already shown that the entomopathogenic fungus Beauveria bassiana has the ability to breach the insect cuticle and is effective both against pyrethroid-susceptible and pyrethroid-resistant T. infestans, in laboratory as well as field assays. It is also known that T. infestans cuticle lipids play a major role as contact aggregation pheromones. We estimated the effectiveness of pheromone-based infection boxes containing B. bassiana spores to kill indoor bugs, and its effect on the vector population dynamics. Laboratory assays were performed to estimate the effect of fungal infection on female reproductive parameters. The effect of insect exuviae as an aggregation signal in the performance of the infection boxes was estimated both in the laboratory and in the field. We developed a stage-specific matrix model of T. infestans to describe the fungal infection effects on insect population dynamics, and to analyze the performance of the biopesticide device in vector biological control. CONCLUSIONS: The pheromone-containing infective box is a promising new tool against indoor populations of this Chagas disease vector, with the number of boxes per house being the main driver of the reduction of the total domestic bug population. This ecologically safe approach is the first proven alternative to chemical insecticides in the control of T. infestans. The advantageous reduction in vector population by delayed-action fungal biopesticides in a contained environment is here shown supported by mathematical modeling.
Assuntos
Beauveria , Doença de Chagas/prevenção & controle , Controle de Insetos/métodos , Controle Biológico de Vetores , Triatoma/microbiologia , Animais , Doença de Chagas/transmissão , Galinhas , Sinais (Psicologia) , Transmissão de Doença Infecciosa , Feminino , Insetos Vetores , Masculino , Modelos Teóricos , Modelos de Riscos ProporcionaisRESUMO
Entomopathogenic fungi secrete toxic secondary metabolites during the invasion of the insect hemocoel as part of the infection process. Although these compounds have been frequently mentioned as virulence factors, the roles of many of them remain poorly understood, including the question of whether they are expressed during the infection process. A major hurdle to this issue remains the low sensitivity of biochemical detection techniques (e.g., HPLC) within the complex samples that may contain trace quantities of fungal molecules inside the insect. In this study, quantitative reverse transcription real-time PCR (qRT-PCR) was used to measure the transcript levels within the insect fungal pathogen Beauveria bassiana, that encode for the synthetase enzymes of the secondary metabolites tenellin (BbtenS), beauvericin (BbbeaS) and bassianolide (BbbslS) during the infection of Triatoma infestans, a Chagas disease insect vector. Absolute quantification was performed at different time periods after insect treatment with various concentrations of propagules, either by immersing the insects in conidial suspensions or by injecting them with blastospores. Both BbtenS and BbbeaS were highly expressed in conidia-treated insects at days 3 and 12 post-treatment. In blastospore-injected insects, BbtenS and BbbeaS expression peaked at 24h post-injection and were also highly expressed in insect cadavers. The levels of BbbslS transcripts were much lower in all conditions tested. The expression patterns of insect genes encoding proteins that belong to the T. infestans humoral immune system were also evaluated with the same technique. This qPCR-based methodology can contribute to decifering the dynamics of entomopathogenic fungal infection at the molecular level.
Assuntos
Beauveria/patogenicidade , Perfilação da Expressão Gênica/métodos , Regulação Fúngica da Expressão Gênica/fisiologia , Triatoma/parasitologia , Animais , Beauveria/genética , Beauveria/imunologia , Proteínas Fúngicas , Genes Fúngicos , Reação em Cadeia da Polimerase em Tempo Real , Triatoma/imunologiaRESUMO
Entomopathogenic fungi mostly attack their insect hosts by penetration through the cuticle. The outermost insect surface is covered by a lipid-rich layer, usually composed of very long chain hydrocarbons. These fungi are apt to grow on straight chain hydrocarbons (alkanes) as the sole carbon source. Insect-like hydrocarbons are first hydroxylated by a microsomal P450 monooxygenase system, and then fully catabolized by peroxisomal ß-oxidation reactions in Beauveria bassiana. In this review, we will discuss lipid metabolism adaptations in alkane-grown fungi, and how an oxidative stress scenario is established under these conditions. Fungi have to pay a high cost for hydrocarbon utilization; high levels of reactive oxygen species are produced and a concomitant antioxidant response is triggered in fungal cells to cope with this drawback.
Assuntos
Fungos/crescimento & desenvolvimento , Fungos/metabolismo , Hidrocarbonetos/metabolismo , Insetos/microbiologia , Estresse Oxidativo , Alcanos/metabolismo , Animais , Antioxidantes/metabolismo , Metabolismo Energético , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/genética , Interações Hospedeiro-Patógeno , OxirreduçãoRESUMO
BACKGROUND: The chemical control of the Chagas disease vector Triatoma infestans is endangered by the emergence of pyrethroid resistance. An effective alternative control tool is the use of the entomopathogenic fungus Beauveria bassiana. The effect of deltamethrin on fungal growth, gene expression and enzyme activity in relation to detoxification, antioxidant response and oxidative stress levels was studied to evaluate fungal tolerance to deltamethrin. RESULTS: The mean inhibitory concentration (IC50 ) was 50 µg deltamethrin/cm(2). Cytochrome P450 genes were differentially expressed; cyp52X1 and cyp617N1 transcripts were > 2-fold induced, followed by cyp655C1 (1.8-fold). Minor effects were observed on genes encoding for other P450s, epoxide hydrolase and glutathione S-transferase (GST). Superoxide dismutase (SOD) genes showed induction levels ≤ 2, catalase (CAT) and glutathione peroxidase genes were also induced â¼ 2-3-fold and < 2-fold, respectively. The activities of enzymes participating in the antioxidant defense system and phase II detoxification were also evaluated; SOD, CAT and GST activity showed significant differences with deltamethrin concentration. Lipid peroxidation levels and free proline content were also altered. CONCLUSIONS: Beauveria bassiana GHA can be used combined with deltamethrin without significant metabolic detrimental effects. This combination will help optimizing the benefits and increasing the efficacy of vector control tools.
Assuntos
Beauveria/efeitos dos fármacos , Beauveria/fisiologia , Inseticidas/toxicidade , Nitrilas/toxicidade , Piretrinas/toxicidade , Animais , Antioxidantes/metabolismo , Beauveria/genética , Agentes de Controle Biológico , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica/efeitos dos fármacos , Concentração Inibidora 50 , Peroxidação de Lipídeos/efeitos dos fármacos , Prolina/metabolismo , TriatomaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Ulomoides dermestoides (Fairmaire, 1893) is a cosmopolitan tenebrionid beetle reared by Argentine people who consume them alive as an alternative medicine in the treatment of different illnesses such as asthma, Parkinson's, diabetes, arthritis, HIV and specially cancer. AIM OF THE STUDY: To evaluate the cytotoxicity and DNA damage of the major volatile components released by Ulomoides dermestoides on human lung carcinoma epithelial cell line A549. MATERIALS AND METHODS: The defence compounds of Ulomoides dermestoides were extracted with dichloromethane and analyzed and quantified by capillary gas chromatography. The toxicity effects of the beetle's extract against A549 cell line were evaluated. Cytotoxicity was evaluated by MTT test and Trypan blue assay and genotoxicity was evaluated by the comet assay. The synthetic compounds, individually or combined, were also tested in A549 cells and normal mononuclear human cells. RESULTS: The defence compounds of Ulomoides dermestoides extracted with dichloromethane (methyl-1,4-benzoquinones, ethyl-1,4-benzoquinones and 1-pentadecene as major components) showed cytotoxic activity on A549 cells demonstrated by MTT test and Trypan blue assay, with IC(50) values of 0.26equivalent/ml and 0.34equivalent/ml, respectively (1equivalent=amount of components extracted per beetle). The inhibition of A549 cell proliferation with the synthetic blend (1,4-benzoquinone and 1-pentadecene) or 1,4-benzoquinone alone was similar to that obtained with the insect extract. 1-Pentadecene showed no inhibitory effect. Low doses of insect extract or synthetic blend (0.15equivalent/ml) inhibited mononuclear cell proliferation by 72.2±2.7% and induced significant DNA damage both in tumor and mononuclear cells. CONCLUSION: Results of this study demonstrated that defence compounds of Ulomoides dermestoides reduced cell viability and induced DNA damage. We also concluded that the insect benzoquinones are primarily responsible for inducing cytotoxicity and genotoxicity in culture cells.
Assuntos
Antineoplásicos/uso terapêutico , Benzoquinonas/uso terapêutico , Carcinoma/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Besouros/química , Leucócitos Mononucleares/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA , Humanos , Concentração Inibidora 50RESUMO
Triatoma dimidiata Latreille is a major vector of Chagas disease with an extensive geographic distribution from Central Mexico, through Central America, to northern South America. As a result of its variability in phenetic and genetic characters, disagreement concerning its taxonomic status has been raised. In this study, the cuticular hydrocarbon pattern of T. dimidiata populations from Mexico, Belize, Guatemala, Honduras, Costa Rica, and Colombia was analyzed by capillary gas chromatography coupled to mass spectrometry; linear discriminant analysis was used to help elucidate population structure. Vector populations segregated into five distinct groups; specimens from Yucatan Peninsula, together with those from Central Mexico, Central America, and Colombia corresponded to different T. dimidiata subspecies, a putative different species comprising insects from Belize, together with an isolated population collected at bat caves in Guatemala. The analysis revalidates the earlier division of T dimidiata into three subspecies, T. d. maculipennis, T. d. dimidiata, and T. d. capitata; and an additional subspecies and a distinct species are proposed.
Assuntos
Hidrocarbonetos/metabolismo , Tegumento Comum/fisiologia , Triatoma/classificação , Triatoma/fisiologia , Animais , Feminino , Hidrocarbonetos/química , Masculino , Filogenia , Especificidade da Espécie , Triatoma/genéticaRESUMO
Epicuticular lipids are contact cues in intraspecific chemical communication in insects, both for aggregation and sexual behavior. Triatomine bugs are vectors of the parasite Trypanosoma cruzi, the cause of Chagas disease. In Triatoma infestans, the major epicuticular lipids are hydrocarbons, fatty alcohols, and free and esterified fatty acids. Previously, we found that epicuticular lipid extracts, or selected fatty acid components, trigger aggregation and arrestment behavior in this bug. Using headspace solid phase microextraction, we found no sexual dimorphism in epicuticular hydrocarbons, but found female-specific fatty alcohols (eicosanol and docosanol). The role of epicuticular lipids in T. infestans copulation behavior was tested by observing male responses to live or various treatments of freeze-killed females. We report that hexane-soluble contact cues on females trigger copulation by males. Freeze-killed intact females were attractive to males, but no response was observed when males were exposed to hexane-washed females. Responses were partially recovered when epicuticular extract was applied to the dorsal surface of dead, hexane-washed females. One female equivalent of docosanol, evoked similar responses.
Assuntos
Eicosanoides/química , Álcoois Graxos/química , Hidrocarbonetos/química , Comportamento Sexual Animal , Triatoma/fisiologia , Animais , Eicosanoides/isolamento & purificação , Eicosanoides/farmacologia , Álcoois Graxos/isolamento & purificação , Álcoois Graxos/farmacologia , Feminino , Hexanos/química , Hidrocarbonetos/isolamento & purificação , Masculino , Microextração em Fase Sólida , Triatoma/químicaRESUMO
The insect epicuticle or waxy layer comprises a heterogeneous mixture of lipids that include abundant levels of long-chain alkanes, alkenes, wax esters and fatty acids. This structure represents the first barrier against microbial attack and for broad-host-range insect pathogens, such as Beauveria bassiana, it is the initial interface mediating the host-pathogen interaction, since these organisms do not require any specialized mode of entry and infect target hosts via the cuticle. B. bassiana is able to grow on straight chain alkanes up to n-C(33) as a sole source of carbon and energy. The cDNA and genomic sequences, including putative regulatory elements, for eight cytochrome P450 enzymes, postulated to be involved in alkane and insect epicuticle degradation, were isolated and characterized. Expression studies using a range of alkanes as well as an insect-derived epicuticular extract from the blood-sucking bug Triatomas infestans revealed a differential expression pattern for the P450 genes examined, and suggest that B. bassiana contains a series of hydrocarbon-assimilating enzymes with overlapping specificity in order to target the surface lipids of insect hosts. Phylogenetic analysis of the translated ORFs of the sequences revealed that the enzyme which displayed the highest levels of induction on both alkanes and the insect epicuticular extract represents the founding member of a new cytochrome P450 family, with three of the other sequences assigned as the first members of new P450 subfamilies. The remaining four proteins clustered with known P450 families whose members include alkane monooxygenases.
Assuntos
Alcanos/metabolismo , Beauveria/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas Fúngicas/metabolismo , Animais , Beauveria/genética , Sistema Enzimático do Citocromo P-450/genética , DNA Complementar/genética , DNA Fúngico/genética , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Insetos/microbiologia , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNARESUMO
Many species of tenebrionids produce and secrete a defensive volatile blend containing mainly benzoquinones and alkenes. In this study we characterized the volatile organic compounds (VOC) of the beetle Ulomoides dermestoides (Coleoptera: Tenebrionidae). Solid phase microextraction (SPME) coupled to capillary gas chromatography-mass spectrometry (CGC-MS) analysis was used to identify methyl-1,4-benzoquinone (MBQ), ethyl-1,4-benzoquinone (EBQ), 1-tridecene (C(13:1)), and 1-pentadecene (C(15:1)), representing more than 90% of the volatile blend. We also used CGC-MS to analyze the epicuticular hydrocarbons of U. dermestoides. Saturated, unsaturated, and branched structures with chain lengths ranging from 13 to 43 carbons were detected. n-pentacosane (C(25:0)) and 9,11-pentacosadiene (9,11-C(25:2)) were the most abundant components, representing more than 40% of the cuticular hydrocarbons.
Assuntos
Besouros/metabolismo , Hidrocarbonetos/química , Hidrocarbonetos/metabolismo , Animais , Besouros/anatomia & histologia , Besouros/fisiologia , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Hidrocarbonetos/análise , Hidrocarbonetos/isolamento & purificação , Masculino , Extração em Fase Sólida , VolatilizaçãoRESUMO
BACKGROUND: Triatoma infestans-mediated transmission of Tripanosoma cruzi, the causative agent of Chagas disease, remains as a major health issue in southern South America. Key factors of T. infestans prevalence in specific areas of the geographic Gran Chaco region-which extends through northern Argentina, Bolivia, and Paraguay-are both recurrent reinfestations after insecticide spraying and emerging pyrethroid-resistance over the past ten years. Among alternative control tools, the pathogenicity of entomopathogenic fungi against triatomines is already known; furthermore, these fungi have the ability to fully degrade hydrocarbons from T. infestans cuticle and to utilize them as fuel and for incorporation into cellular components. METHODOLOGY AND FINDINGS: Here we provide evidence of resistance-related cuticle differences; capillary gas chromatography coupled to mass spectrometry analyses revealed that pyrethroid-resistant bugs have significantly larger amounts of surface hydrocarbons, peaking 56.2+/-6.4% higher than susceptible specimens. Also, a thicker cuticle was detected by scanning electron microscopy (32.1+/-5.9 microm and 17.8+/-5.4 microm for pyrethroid-resistant and pyrethroid-susceptible, respectively). In laboratory bioassays, we showed that the virulence of the entomopathogenic fungi Beauveria bassiana against T. infestans was significantly enhanced after fungal adaptation to grow on a medium containing insect-like hydrocarbons as the carbon source, regardless of bug susceptibility to pyrethroids. We designed an attraction-infection trap based on manipulating T. infestans behavior in order to facilitate close contact with B. bassiana. Field assays performed in rural village houses infested with pyrethroid-resistant insects showed 52.4% bug mortality. Using available mathematical models, we predicted that further fungal applications could eventually halt infection transmission. CONCLUSIONS: This low cost, low tech, ecologically friendly methodology could help in controlling the spread of pyrethroid-resistant bugs.
Assuntos
Beauveria/fisiologia , Doença de Chagas/prevenção & controle , Insetos Vetores/microbiologia , Inseticidas , Piretrinas , Triatoma/microbiologia , Triatoma/parasitologia , Animais , Doença de Chagas/transmissão , Humanos , Controle de Insetos/métodos , Insetos Vetores/efeitos dos fármacos , Insetos Vetores/parasitologia , Resistência a Inseticidas , Trypanosoma cruzi/fisiologiaRESUMO
BACKGROUND: The triatomine bugs are vectors of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. Aggregation behavior plays an important role in their survival by facilitating the location of refuges and cohesion of aggregates, helping to keep them safely assembled into shelters during daylight time, when they are vulnerable to predators. There are evidences that aggregation is mediated by thigmotaxis, by volatile cues from their faeces, and by hexane-extractable contact chemoreceptive signals from their cuticle surface. The epicuticular lipids of Triatoma infestans include a complex mixture of hydrocarbons, free and esterified fatty acids, alcohols, and sterols. RESULTS: We analyzed the response of T. infestans fifth instar nymphs after exposure to different amounts either of total epicuticular lipid extracts or individual lipid fractions. Assays were performed in a circular arena, employing a binary choice test with filter papers acting as aggregation attractive sites; papers were either impregnated with a hexane-extract of the total lipids, or lipid fraction; or with the solvent. Insects were significantly aggregated around papers impregnated with the epicuticular lipid extracts. Among the lipid fractions separately tested, only the free fatty acid fraction promoted significant bug aggregation. We also investigated the response to different amounts of selected fatty acid components of this fraction; receptiveness varied with the fatty acid chain length. No response was elicited by hexadecanoic acid (C16:0), the major fatty acid component. Octadecanoic acid (C18:0) showed a significant assembling effect in the concentration range tested (0.1 to 2 insect equivalents). The very long chain hexacosanoic acid (C26:0) was significantly attractant at low doses (
RESUMO
The biochemical interaction between fungal pathogens and their insect host epicuticle was studied by examining fungal hydrocarbon degrading ability. As a contact insecticide, entomopathogenic fungi invade their host through the cuticle, covered by an outermost lipid layer mainly composed of highly stable, very long chain structures. Strains of Beauveria bassiana and Metarhizium anisopliae (Deuteromycotina: Hyphomycetes), pathogenic both to the blood-sucking bug Triatoma infestans (Hemiptera: Reduviidae) and the bean-weevil Acanthoscelides obtectus (Coleoptera, Bruchidae), were grown on different carbon sources. Alkane-grown cells showed a lipid pattern different from that of glucose-grown cells, evidenced by a major switch in the triacylglycerol and sterol components. Radiolabelled hydrocarbons were used to investigate the catabolic pathway and the by-product incorporation into fungal cellular components. The first oxidation round is presumably carried out by a cytochrome P450 enzyme system, the metabolites will traverse the peroxisomal membrane, and after successive transformations will eventually provide the appropriate fatty acyl CoA for complete degradation in the peroxisomes, the site of beta-oxidation in fungi. In this review, we will show the relationship between fungal ability to catabolize very long chain hydrocarbons and virulence parameters.
Assuntos
Besouros/microbiologia , Epiderme/microbiologia , Metarhizium/metabolismo , Paecilomyces/metabolismo , Controle Biológico de Vetores/métodos , Triatoma/microbiologia , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Epiderme/metabolismo , Epiderme/ultraestrutura , Insetos Vetores/microbiologia , Metarhizium/patogenicidade , Metarhizium/ultraestrutura , Microscopia Eletrônica , Paecilomyces/patogenicidade , Paecilomyces/ultraestruturaRESUMO
Beauveria bassiana produces acyl-Co oxidase (ACO) in the P(20000 g) fraction of glucose and alkane-grown cultures that catalyze the oxidation of acyl-CoAs of different chain length. The activity was measured indirectly over the formation of H2O2 via the oxidative-coupled assay system. ACO activity was assessed spectrophotometrically in the P(20000 g) fraction of glucose-grown (FS0) and n-alkane grown cultures (FS(alk)), employing acyl-CoAs of 16 to 24 carbons as substrates. A significant increment in the activity was observed in FS(alk) as compared to that of controls (FS0) in all conditions tested. Tetracosane-grown cultures showed the highest activity with lignoceroyl-CoA. The reaction conditions were optimized employing lignoceroyl-CoA as substrate. A variable lag phase was observed when the activity was measured as a function of time. In the presence of 3-amino-1,2,4-triazole (AT) to prevent H2O2 consumption by endogenous catalase, the lag phase became shorter and disappeared when AT concentrations were raised from 40 to 200 mM, thus enhancing acyl-CoA oxidation. Enzyme activity reached its maximal value in the presence of 240 microg peroxidase, 0.08% Triton X-100 and 36 microM bovine serum albumin. The apparent Km using lignoceroyl as substrate was estimated 2.5 microM. ACO showed high activity and stability between 30 and 40 degrees C, as well as between 7.0 and 9.0 pH, for 120 min, being 7.0 the optimum pH.
Assuntos
Acil-CoA Oxidase/metabolismo , Beauveria/enzimologia , Acil-CoA Oxidase/antagonistas & inibidores , Alcanos/metabolismo , Amitrol (Herbicida)/farmacologia , Beauveria/metabolismo , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Espectrofotometria UltravioletaRESUMO
Entomopathogenic fungi adapt to growth in a culture medium containing an insect-like hydrocarbon as the sole carbon source inducing the beta-oxidation pathway during the alkane degradation. The effect of two carbon sources on the catalase activity was studied in the entomopathogenic fungus Beauveria bassiana. Catalase activity was detected both in the peroxisomal and cytosolic fraction. A significant increment in the specific activity of the peroxisomal fraction (12.6-fold) was observed when glucose was replaced by an insect-like hydrocarbon, whereas the specific activity in the cytosol diminished more than 1.2-fold in the same culture condition. After purification to homogeneity by gel filtration and strong anion exchange chromatography, an apparent molecular mass of 54.7 and 84.0 kDa per subunit were determined respectively for the peroxisomal and cytosolic catalase. The enzymes showed different biochemical and kinetic characteristics, but both were inhibited by 3-amino-1,2,4 triazole. Peroxisomal catalase was sensitive to pH, heat and high concentration of the hydrogen peroxide substrate. Inversely the cytosolic isoform exhibited a broad range of optimal pH (6.0-10.0), high thermostability (<55 C) and remained fully active at least up to 70 mM hydrogen peroxide. Measurement of catalase activity is a new approach for evaluating fungal ability to degrade hydrocarbons.
Assuntos
Alcanos/metabolismo , Beauveria/enzimologia , Catalase/metabolismo , Beauveria/metabolismo , Biodegradação Ambiental , Catalase/química , Catalase/isolamento & purificação , Concentração de Íons de Hidrogênio , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Oxirredução , TemperaturaRESUMO
Insect cuticular hydrocarbons are synthesized de novo in integumental tissue through the concerted action of fatty acid synthases (FASs), fatty acyl-CoA elongases, a reductase, and a decarboxylase to produce hydrocarbons and CO2. Elongation of fatty acyl-CoAs to very long chain fatty acids was studied in the integumental microsomes of the German cockroach, Blatella germanica. Incubation of [1-14C]palmitoyl-CoA, malonyl-CoA, and NADPH resulted in the production of 18-CoA with minor amounts of C20, C22, C24, C30, and C32 labeled acyl-CoA moieties. Similar experiments with [1-14C]stearoyl-CoA rendered C20-CoA as the major product, and lesser amounts of C22 and C24-CoAs were also detected. After solubilization of the microsomal FAS, kinetic parameters were determined radiochemically or by measuring NADPH consumption. The reaction velocity was linear for up to 3 min incubation time, and with a protein concentration up to 0.025 microg/microl. The effect of the chain length on the reaction velocity was compared for palmitoyl-CoA, stearoyl-CoA, and eicosanoyl-CoA. The optimal substrate concentration was 10 microM for C16-CoA, between 8 and 12 microM for C18-CoA, and close to 3 microM for C20-CoA. In vivo hydrocarbon biosynthesis was inhibited from 55.5 to 72.5% in the presence of 1 mM trichloroacetic acid, a known inhibitor of elongation reactions.
Assuntos
Acil Coenzima A/metabolismo , Blattellidae/metabolismo , Ácidos Graxos/metabolismo , Animais , Cinética , Microssomos/metabolismo , Ácido Tricloroacético/farmacologiaRESUMO
Capillary gas-liquid chromatography was used to analyse the cuticular hydrocarbons of three triatomine species, Triatoma dimidiata, T. barberi and Dipetalogaster maxima, domestic vectors of Chagas disease in Mexico. Mixtures of saturated hydrocarbons of straight and methyl-branched chains were characteristic of the three species, but quantitatively different. Major methylbranched components mostly corresponded to different saturated isomers of monomethyl, dimethyl and trimethyl branched hydrocarbons ranging from 29 to 39 carbon backbones. Sex-dependent, quantitative differences in certain hydrocarbons were apparent in T. dimidiata.