RESUMO
Sandflies are insects of public health interest due to their role as vectors of parasites of the genus Leishmania, as well as other pathogens. Psychodopygus carrerai carrerai is considered an important sylvatic vector of Leishmania (Viannia) braziliensis in Amazonia. In this study, sandflies were collected in a forested area in the Xapuri municipality, in the State of Acre (Northern Brazil). Two Ps. carrerai carrerai females were found parasitized with a larval form of a filarial worm, one in the labium of the proboscis, the other after the head was squashed, suggesting they were infective larvae. Sandflies were identified through morphological characters as well as amplification and sequencing of the cytochrome oxidase gene (COI). This was the first sequence obtained for Ps. carrerai carrerai for this marker. The obtained nematodes were also characterized through direct sequencing of a fragment of COI and 12S genes, both mitochondrial, and ITS1, a nuclear marker. Phylogenetic analyses revealed that the filarial nematodes belong to a species without sequences for these markers in the database, part of family Onchocercidade and closely related to genus Onchocerca (12S tree). Although sandfly infection with nematodes including members of the Onchocercidae has been reported in the Old World, this is the first report of sandfly infection by a member of the Onchocercidae family in the New World, to the best of our knowledge. Considering that the phylogenetic relationships and location in the insect, it can be expected that this is a parasite of mammals and the transmission cycle should be clarified.
Assuntos
Filarioidea/patogenicidade , Insetos Vetores/parasitologia , Leishmania braziliensis , Leishmaniose Cutânea/transmissão , Psychodidae/parasitologia , Animais , Brasil , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Filarioidea/classificação , Filarioidea/genética , Genes de Helmintos , Genes de Insetos , Humanos , Leishmaniose Cutânea/parasitologia , Masculino , Filogenia , Psychodidae/enzimologia , Psychodidae/genéticaRESUMO
In sandflies, males and females feed on carbohydrates but females must get a blood meal for egg maturation. Using artificial blood meals, this study aimed to understand how galactosamine interferes with sandfly digestive physiology. We also used galactosamine to manipulate the digestive physiology of Lutzomyia longipalpis to investigate its influence on sandfly digestion and Leishmania development within their insect vectors. Galactosamine was capable to reduce Lu. longipalpis trypsinolytic activity in a dose-dependent manner. This effect was specific to galactosamine as other similar sugars were not able to affect sandfly trypsin production. An excess of amino acids supplemented with the blood meal and 15 mM galactosamine was able to abrogate the reduction of the trypsinolytic activity caused by galactosamine, suggesting this phenomenon may be related to an impairment of amino acid detection by sandfly enterocytes. The TOR inhibitor rapamycin reduces trypsin activity in the L. longipalpis midgut. Galactosamine reduces the phosphorylation of the TOR pathway repressor 4EBP, downregulating TOR activity in the gut of L. longipalpis. Galactosamine reduces sandfly oviposition, causes an impact on sandfly longevity and specifically reduces sandfly gut proteases whereas increasing α-glycosidase activity. The administration of 15 and 30 mM galactosamine increased the number of promastigote forms of Le. mexicana and Le. infantum in galactosamine-treated L. longipalpis. Our results showed that galactosamine influences amino acid sensing, reduces sandfly gut protease activity through TOR downregulation, and benefits Leishmania growth within the Lu. longipalpis gut.
Assuntos
Galactosamina/administração & dosagem , Proteínas de Insetos/metabolismo , Leishmania/fisiologia , Peptídeo Hidrolases/metabolismo , Psychodidae/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Regulação para Baixo , Feminino , Galactosamina/farmacologia , Trato Gastrointestinal/parasitologia , Trato Gastrointestinal/fisiologia , Psychodidae/enzimologia , Psychodidae/parasitologiaRESUMO
BACKGROUND: The sand fly Lutzomyia longipalpis is the main vector of American visceral leishmaniasis, a disease caused by parasites of the genus Leishmania. Adults of this insect feed on blood (females only) or sugar from plant sources, but their digestion of carbohydrates is poorly studied. Beta-glycosides as esculin and amygdalin are plant compounds and release toxic compounds as esculetin and mandelonitrile when hydrolyzed. Beta-glucosidase and trehalase are essential enzymes in sand fly metabolism and participate in sugar digestion. It is therefore possible that the toxic portions of these glycosides, released during digestion, affect sand fly physiology and the development of Leishmania. RESULTS: We tested the oral administration to sand flies of amygdalin, esculin, mandelonitrile, and esculetin in the sugar meal. These compounds significantly decreased the longevity of Lutzomyia longipalpis females and males. Lutzomyia longipalpis adults have significant hydrolytic activities against esculin and feeding on this compound cause changes in trehalase and ß-glucosidase activities. Female trehalase activity is inhibited in vitro by esculin. Esculin is naturally fluorescent, so its ingestion may be detected and quantified in whole insects or tissue samples stored in methanol. Mandelonitrile neither affected the amount of sugar ingested by sand flies nor showed repellent activity. Our results show that mandelonitrile significantly reduces the viability of L. amazonensis, L. braziliensis, L. infantum and L. mexicana, in a concentration-dependent manner. Esculetin caused a similar effect, reducing the number of L. infantum and L. mexicana. Female L. longipalpis fed on mandelonitrile had a reduction in the number of parasites and prevalence of infection after seven days of infection with L. mexicana, either by counting in a Neubauer chamber or by qPCR assays. CONCLUSIONS: Glycosides have significant effects on L. longipalpis longevity and metabolism and also affect the development of parasites in culture and inside the insect. These observations might help to conceptualize new vector control strategies using transmission blocking sugar baits.
Assuntos
Glicosídeos/toxicidade , Controle de Insetos/métodos , Insetos Vetores/enzimologia , Insetos Vetores/parasitologia , Leishmania/crescimento & desenvolvimento , Psychodidae/enzimologia , Psychodidae/parasitologia , Acetonitrilas/toxicidade , Amigdalina/toxicidade , Animais , Esculina/toxicidade , Feminino , Glicosídeos/administração & dosagem , Leishmaniose/prevenção & controle , Leishmaniose/transmissão , Masculino , Trealase/efeitos dos fármacos , Umbeliferonas/administração & dosagem , Umbeliferonas/toxicidade , beta-Glucosidase/efeitos dos fármacosRESUMO
BACKGROUND The insect chitinase gene family is composed by more than 10 paralogs, which can codify proteins with different domain structures. In Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil, a chitinase cDNA from adult female insects was previously characterized. The predicted protein contains one catalytic domain and one chitin-binding domain (CBD). The expression of this gene coincided with the end of blood digestion indicating a putative role in peritrophic matrix degradation. OBJECTIVES To determine the occurrence of alternative splicing in chitinases of L. longipalpis. METHODS We sequenced the LlChit1 gene from a genomic clone and the three spliced forms obtained by reverse transcription polymerase chain reaction (RT-PCR) using larvae cDNA. FINDINGS We showed that LlChit1 from L. longipalpis immature forms undergoes alternative splicing. The spliced form corresponding to the adult cDNA was named LlChit1A and the two larvae specific transcripts were named LlChit1B and LlChit1C. The B and C forms possess stop codons interrupting the translation of the CBD. The A form is present in adult females post blood meal, L4 larvae and pre-pupae, while the other two forms are present only in L4 larvae and disappear just before pupation. Two bands of the expected size were identified by Western blot only in L4 larvae. MAIN CONCLUSIONS We show for the first time alternative splicing generating chitinases with different domain structures increasing our understanding on the finely regulated digestion physiology and shedding light on a potential target for controlling L. longipalpis larval development.
Assuntos
Processamento Alternativo/genética , Quitinases/genética , Sistema Digestório/enzimologia , Psychodidae/enzimologia , Animais , Quitinases/fisiologia , Feminino , Filogenia , Psychodidae/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The genetic divergence caused by genetic drift and/or selection is suggested to affect the vectorial capacity and insecticide susceptibility of sand flies, as well as other arthropods. In the present study, cytochrome b (cyt b) gene sequences were determined in 13 species circulating in Peru to establish a basis for analysis of the genetic structure, and the intraspecific genetic diversity was assessed in the Lutzomyia (Lu.) peruensis, a main vector species of Leishmania (Viannia) peruviana in Peruvian Andes. Analysis of intraspecific genetic diversity in the cyt b gene sequences from 36 Lu. peruensis identified 3 highly polymorphic sites in the middle region of the gene. Haplotype and gene network analyses were performed on the cyt b gene sequences of 130 Lu. peruensis in 9 Andean areas from 3 Departments (Ancash, Lima and La Libertad). The results showed that the populations of La Libertad were highly polymorphic and that their haplotypes were distinct from those of Ancash and Lima, where dominant haplotypes were observed, suggesting that a population bottleneck may have occurred in Ancash and Lima, but not in La Libertad. The present study indicated that the middle region of the cyt b gene is useful for the analysis of genetic structure in sand fly populations.
Assuntos
Citocromos b/genética , Variação Genética , Insetos Vetores/genética , Leishmania/fisiologia , Leishmaniose/transmissão , Psychodidae/genética , Animais , Sequência de Bases , Genética Populacional , Genótipo , Geografia , Haplótipos , Insetos Vetores/enzimologia , Insetos Vetores/parasitologia , Leishmaniose/epidemiologia , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Peru/epidemiologia , Filogenia , Psychodidae/enzimologia , Psychodidae/parasitologia , Análise de Sequência de DNARESUMO
The low dispersal capacity of sand flies could lead to population isolation due to geographic barriers, climate variation, or to population fragmentation associated with specific local habitats due to landscape modification. The phlebotomine sand fly Lutzomyia cruciata has a wide distribution throughout Mexico and is a vector of Leishmania mexicana in the southeast. The aim of this study was to evaluate the genetic diversity, structure, and divergence within and among populations of Lu. cruciata in the state of Chiapas, and to infer the intra-specific phylogeny using the 3' end of the mitochondrial cytochrome b gene. We analyzed 62 sequences from four Lu. cruciata populations and found 26 haplotypes, high genetic differentiation and restricted gene flow among populations (Fst=0.416, Nm=0.701, p<0.001). The highest diversity values were recorded in populations from Loma Bonita and Guadalupe Miramar. Three lineages (100% bootstrap and 7% overall divergence) were identified using a maximum likelihood phylogenetic analysis which showed high genetic divergence (17.2-22.7%). A minimum spanning haplotype network also supported separation into three lineages. Genetic structure and divergence within and among Lu. cruciata populations are hence affected by geographic heterogeneity and evolutionary background. Data obtained in the present study suggest that Lu. cruciata in the state of Chiapas consists of at least three lineages. Such findings may have implications for vector capacity and hence for vector control strategies.
Assuntos
Insetos Vetores/genética , Psychodidae/genética , Animais , Análise por Conglomerados , Citocromos b/genética , Feminino , Genes de Insetos/genética , Variação Genética , Haplótipos/genética , Insetos Vetores/classificação , Leishmania mexicana/isolamento & purificação , Leishmaniose Cutânea/transmissão , México , Filogenia , Psychodidae/classificação , Psychodidae/enzimologiaRESUMO
Lutzomyia longipalpis is the principal species of phlebotomine incriminated as vector of Leishmania infantum, the etiological agent of visceral leishmaniasis in the Americas. Despite its importance as vector, almost nothing related to the larval biology, especially about its digestive system has been published. The objective of the present study was to obtain an overview of carbohydrate digestion by the larvae. Taking in account that phlebotomine larvae live in the soil rich in decaying materials and microorganisms we searched principally for enzymes capable to hydrolyze carbohydrates present in this kind of substrate. The principal carbohydrases encountered in the midgut were partially characterized. One of them is a α-amylase present in the anterior midgut. It is probably involved with the digestion of glycogen, the reserve carbohydrate of fungi. Two other especially active enzymes were present in the posterior midgut, a membrane bound α-glucosidase and a membrane bound trehalase. The first, complete the digestion of glycogen and the other probably acts in the digestion of trehalose, a carbohydrate usually encountered in microorganisms undergoing hydric stress. In a screening done with the use of p-nitrophenyl-derived substrates other less active enzymes were also observed in the midgut. A general view of carbohydrate digestion in L. longipalpis was presented. Our results indicate that soil microorganisms appear to be the main source of nutrients for the larvae.
Assuntos
Metabolismo dos Carboidratos , Glicosídeo Hidrolases/metabolismo , Psychodidae/metabolismo , Animais , Trato Gastrointestinal/enzimologia , Glicólise , Larva/metabolismo , Psychodidae/enzimologia , Trealase/metabolismo , alfa-Amilases/metabolismo , alfa-Glucosidases/metabolismoRESUMO
Lutzomyia longipalpis is the most important vector of visceral leishmaniasis in Brazil. When female sandflies feed on blood, a peritrophic matrix (PM) is formed around the blood bolus. The PM is secreted by midgut cells and composed of proteins, glycoproteins and chitin microfibrils. The PM functions as both a physical barrier against pathogens present in the food bolus and blood meal digestion regulator. Previous studies of mosquitoes and sandflies have shown that the absence of a PM, resulting from adding an exogenous chitinase to the blood meal, accelerates digestion. In the present study, we analysed biological factors associated with the presence of a PM in L. longipalpis females. Insects fed blood containing chitinase (BCC) accelerated egg-laying relative to a control group fed blood without chitinase. However, in the BCC-fed insects, the number of females that died without laying eggs was higher and the number of eggs laid per female was lower. The eggs in both groups were viable and generated adults. Based on these data, we suggest that the absence of a PM accelerates nutrient acquisition, which results in premature egg production and oviposition; however, the absence of a PM reduces the total number of eggs laid per female. Reduced fecundity in the absence of a PM may be due to inefficient nutrient conversion or the loss of the protective role of the PM.
Assuntos
Quitinases/farmacologia , Sistema Digestório/enzimologia , Oviposição/fisiologia , Psychodidae/enzimologia , Animais , Feminino , Fertilidade/efeitos dos fármacos , Fertilidade/fisiologia , Oviposição/efeitos dos fármacos , Psychodidae/fisiologia , Fatores de TempoRESUMO
Lutzomyia longipalpis is the most important vector of visceral leishmaniasis in Brazil. When female sandflies feed on blood, a peritrophic matrix (PM) is formed around the blood bolus. The PM is secreted by midgut cells and composed of proteins, glycoproteins and chitin microfibrils. The PM functions as both a physical barrier against pathogens present in the food bolus and blood meal digestion regulator. Previous studies of mosquitoes and sandflies have shown that the absence of a PM, resulting from adding an exogenous chitinase to the blood meal, accelerates digestion. In the present study, we analysed biological factors associated with the presence of a PM in L. longipalpis females. Insects fed blood containing chitinase (BCC) accelerated egg-laying relative to a control group fed blood without chitinase. However, in the BCC-fed insects, the number of females that died without laying eggs was higher and the number of eggs laid per female was lower. The eggs in both groups were viable and generated adults. Based on these data, we suggest that the absence of a PM accelerates nutrient acquisition, which results in premature egg production and oviposition; however, the absence of a PM reduces the total number of eggs laid per female. Reduced fecundity in the absence of a PM may be due to inefficient nutrient conversion or the loss of the protective role of the PM.
Assuntos
Animais , Feminino , Quitinases/farmacologia , Sistema Digestório/enzimologia , Oviposição/fisiologia , Psychodidae/enzimologia , Fertilidade/efeitos dos fármacos , Fertilidade/fisiologia , Oviposição/efeitos dos fármacos , Psychodidae/fisiologia , Fatores de TempoRESUMO
Phlebotomine sand flies are the vectors of medically important Leishmania. The Leishmania protozoa reside in the sand fly gut, but the nature of the immune response to the presence of Leishmania is unknown. Reactive oxygen species (ROS) are a major component of insect innate immune pathways regulating gut-microbe homeostasis. Here we show that the concentration of ROS increased in sand fly midguts after they fed on the insect pathogen Serratia marcescens but not after feeding on the Leishmania that uses the sand fly as a vector. Moreover, the Leishmania is sensitive to ROS either by oral administration of ROS to the infected fly or by silencing a gene that expresses a sand fly ROS-scavenging enzyme. Finally, the treatment of sand flies with an exogenous ROS scavenger (uric acid) altered the gut microbial homeostasis, led to an increased commensal gut microbiota, and reduced insect survival after oral infection with S. marcescens. Our study demonstrates a differential response of the sand fly ROS system to gut microbiota, an insect pathogen, and the Leishmania that utilize the sand fly as a vehicle for transmission between mammalian hosts.
Assuntos
Imunidade/imunologia , Leishmania mexicana/imunologia , Psychodidae/imunologia , Espécies Reativas de Oxigênio/imunologia , Serratia marcescens/imunologia , Sequência de Aminoácidos , Animais , Antioxidantes/administração & dosagem , Antioxidantes/farmacologia , Catalase/classificação , Catalase/genética , Catalase/metabolismo , Feminino , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/parasitologia , Técnicas de Inativação de Genes , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Peróxido de Hidrogênio/metabolismo , Imunidade/efeitos dos fármacos , Proteínas de Insetos/classificação , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Insetos Vetores/imunologia , Insetos Vetores/microbiologia , Insetos Vetores/parasitologia , Leishmania mexicana/fisiologia , Dados de Sequência Molecular , Peroxirredoxinas/classificação , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Filogenia , Psychodidae/enzimologia , Psychodidae/genética , Espécies Reativas de Oxigênio/metabolismo , Homologia de Sequência de Aminoácidos , Serratia marcescens/fisiologia , Superóxido Dismutase/classificação , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Ácido Úrico/administração & dosagem , Ácido Úrico/farmacologiaRESUMO
Control of the midgut pH in Lutzomyia longipalpis enables the insect's digestive system to deal with different types of diet. Phlebotomines must be able to suddenly change from a condition adequate to process a sugar diet to one required to digest blood. Prior to blood ingestion, the pH in the midgut is maintained at â¼6 via an efficient mechanism. In the abdominal midgut, alkalization to a pH of â¼8 occurs as a consequence of the loss of CO(2) from blood (CO(2) volatilization) and by a second mechanism that is not yet characterized. The present study aimed to characterize the primary stimuli, present in the blood, that are responsible for shutting down the mechanism that maintains a pH of 6 and switching on that responsible for alkalization. Our results show that any ingested protein could induce alkalization. Free amino acids, at the concentrations found in blood, were ineffective at inducing alkalization, although higher concentrations of amino acids were able to induce alkalization. Aqueous extracts of midgut tissue containing putative hormones from intestinal endocrine cells slightly alkalized the midgut lumen when applied to dissected intestines, as did hemolymph collected from blood-fed females. Serotonin, a hormone that is possibly released in the hemolymph after hematophagy commences, was ineffective at promoting alkalization. The carbonic anhydrase (CA) enzyme seems to be involved in alkalizing the midgut, as co-ingestion of acetazolamide (a CA inhibitor) with proteins impaired alkalization efficiency. A general model of alkalization control is presented.
Assuntos
Sistema Digestório/metabolismo , Ingestão de Alimentos , Hormônios/metabolismo , Psychodidae/metabolismo , Acetazolamida/farmacologia , Álcalis/metabolismo , Animais , Anidrases Carbônicas/metabolismo , Bovinos , Sistema Digestório/anatomia & histologia , Sistema Digestório/efeitos dos fármacos , Sistema Digestório/enzimologia , Ingestão de Alimentos/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Feminino , Hemolinfa/efeitos dos fármacos , Hemolinfa/metabolismo , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Psychodidae/anatomia & histologia , Psychodidae/efeitos dos fármacos , Psychodidae/enzimologia , Serotonina/farmacologia , Soroalbumina Bovina/farmacologia , Soluções , Extratos de TecidosRESUMO
BACKGROUND: Midgut enzymatic activity is one of the obstacles that Leishmania must surpass to succeed in establishing infection. Trypsins are abundant digestive enzymes in most insects. We have previously described two trypsin cDNAs of L. longipalpis: one (Lltryp1) with a bloodmeal induced transcription pattern, the other (Lltryp2) with a constitutive transcription pattern. We have now characterized the expression and activity of trypsin-like proteases of Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil. METHODOLOGY AND PRINCIPAL FINDINGS: In order to study trypsin expression profiles we produced antibodies against peptides specific for Lltryp1 and Lltryp2. The anti-Lltryp1-peptide antibody revealed a band of 28 kDa between 6 and 48 hours. The anti-Lltryp2 peptide antibody did not evidence any band. When proteinaceous substrates (gelatin, hemoglobin, casein or albumin) were co-polymerized in polyacrylamide gels, insect midguts obtained at 12 hours after feeding showed a unique proteolytic pattern for each substrate. All activity bands were strongly inhibited by TLCK, benzamidine and 4-amino-benzamidine, indicating that they are trypsin-like proteases. The trypsin-like activity was also measured in vitro at different time points after ingestion of blood or blood containing Leishmania infantum chagasi, using the chromogenic substrate BArhoNA. L. longipalpis females fed on blood infected with L. i. chagasi had lower levels of trypsin activity after 12 and 48 hours than non-infected insects, suggesting that the parasite may have a role in this modulation. CONCLUSIONS AND SIGNIFICANCE: Trypsins are important and abundant digestive enzymes in L. longipalpis. Protein production and enzymatic activity followed previously identified gene expression of a blood modulated trypsin gene. A decrease of enzymatic activity upon the parasite infection, previously detected mostly in Old World vectors, was detected for the first time in the natural vector-parasite pair L. longipalpis-L. i. chagasi.
Assuntos
Leishmania infantum/fisiologia , Psychodidae/enzimologia , Psychodidae/parasitologia , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Ensaios Enzimáticos , Gelatina/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peptídeos/imunologia , Alinhamento de Sequência , Serina Endopeptidases/química , Soluções , Especificidade por SubstratoRESUMO
It has been proposed that the natural cysteine peptidase inhibitor ICP of Leishmania mexicana protects the protozoan parasite from insect host proteolytic enzymes, thereby promoting survival. To test this hypothesis, L. mexicana mutants deficient in ICP were evaluated for their ability to develop in the sand fly Lutzomyia longipalpis. No significant differences were found between the wild-type parasites, two independently derived ICP-deficient mutants, or mutants overexpressing ICP; all lines developed similarly in the sand fly midgut and produced heavy late-stage infections. In addition, recombinant L. mexicana ICP did not inhibit peptidase activity of the midgut extracts in vitro. We conclude that ICP has no major role in promoting survival of L. mexicana in the vectorial part of its life cycle in L. longipalpis.
Assuntos
Inibidores de Cisteína Proteinase/fisiologia , Leishmania mexicana/patogenicidade , Proteínas de Protozoários/fisiologia , Psychodidae/parasitologia , Animais , Cisteína Endopeptidases/fisiologia , Inibidores de Cisteína Proteinase/genética , Feminino , Interações Hospedeiro-Parasita , Proteínas de Insetos/fisiologia , Leishmania mexicana/genética , Proteínas de Protozoários/genética , Psychodidae/enzimologiaRESUMO
Genetic structure in five Phlebotominae (Lutzomyia spp.), townsendi series, verrucarum group, in Colombia (Diptera: Prychodidae). Sixteen isoenzyme patterns were analyzed for five Colombian Lutzomyia species. The average unbiased expected heterozygosity levels ranged from 0.098 (Lu. youngi) to 0.215 (Lu. torvida). The five species samples, taken all the isoenzymes employed, were significantly deviated from the Hardy-Weinberg equilibrium by homozygous excess with classical as well as Markov chain exact tests. Possible causes: (1) Wahlund effect within populations due to subdivision and/or sampling. Endogamy could be discarded because these loci were affected by highly different levels of homozygous excess. (2) Null alleles could be not discarded, at least for some isoenzymes. The hierarchical Wright´s F analysis showed high and significant values for each parameter. The average F IT value was 0.655 with a conspicous homozygous excess at a global level (all species taken together); the average F IS value was significantly positive (0.515) as well, with homozygous excess within each species. The genetic heterogeneity between the fives species was noteworthy (F ST = 0.288), indicating clear genetic differentiation. The more related species pairs were Lu. longiflocosa-Lu. torvida (0.959) and Lu torvida-Lu. spinicrassa (0.960); while Lu. torvida-Lu. youngi (0.805) and Lu. quasitownsendi-Lu. youngi (0.796) were the most divergent (Nei´s genetic identity matrix). UPGMA and Wagner algorithms showed that the most divergent species was Lu. youngi, whereas the most related were Lu. longiflocosa-Lu. torvida and Lu torvida-Lu. spinicrassa. A spatial autocorrelation analysis (Moran´s I index) revealed a very weak, or inexistent spatial structure, which means that the speciation events between these species were independent from the geographic distances from where they currently live. Rev. Biol. Trop. 56 (4): 1717-1739. Epub 2008 December 12.
Se analizaron 16 sistemas isoenzimáticos para cinco especies colombianas del género Lutzomyia. Los niveles de heterocigosis media esperada insesgada oscilaron entre 0.098 (Lu. youngi) y 0.215 (Lu. torvida). Las cinco muestras estudiadas de forma global, para todos los marcadores analizados, presentaron desviación respecto al equilibrio Hardy-Weinberg por un exceso de homocigotos, tanto al utilizar algunas pruebas clásicas como tests exactos con cadenas de Markov. Este hecho puede estar favorecido por diversas causas: (1) la más probable es la existencia de efecto Wahlund en el seno de cada población debido a subdivisión y/o a la técnica de muestreo empleada. La endogamia puede descartarse ya que no todos los loci están afectados por el mismo tipo de exceso de homocigotos. (2) Sin embargo, no se puede descartar la existencia de alelos nulos, al menos, para algunos de los marcadores isoenzimáticos utilizados. El análisis jerarquizado con las F de Wright mostró valores elevados y significativos para cada uno de los estadísticos. El estadístico promedio F IT mostró un valor de 0.655 existiendo un conspicuo exceso de homocigotos a nivel total de todas las especies, el estadístico promedio F IS fue altamente positivo (0.515) mostrando exceso de homocigotos a nivel individual en cada una de las especies estudiadas. La heterogeneidad genética entre las cinco especies fue notable (F ST = 0.288). Esto muestra que esas especies están bien diferenciadas a nivel isoenzimático y que en el interior de cada especie también hay una subdivisión genética. La matriz de identidades genéticas de Nei muestra que las especies más relacionadas fueron Lu. longiflocosa-Lu. torvida (0.959) y Lu torvida-Lu. spinicrassa (0.960) mientras que las genéticamente más distantes fueron Lu. torvida-Lu. youngi (0.805) y Lu. quasitownsendi-Lu. youngi (0.796). Con los algoritmos UPGMA y Wagner, se observó que la especie más divergente fue Lu. youngi, mientras que las relaciones más conspicuas se observaron entre Lu. longiflocosa-Lu. torvida y Lu torvida-Lu. spinicrassa. Adicionalmente, con un análisis de autocorrelación espacial (índice de Moran) la mayoría de los alelos utilizados presentaron una estructura espacial muy débil o inexistente, lo que significa que los eventos de especiación entre las especies estudiadas se dieron en forma independiente de las distancias geográficas existentes actualmente entre ellas.
Assuntos
Animais , Frequência do Gene/genética , Genes de Insetos/genética , Variação Genética/genética , Isoenzimas/genética , Psychodidae/genética , Colômbia , Eletroforese em Gel de Poliacrilamida , Genética Populacional , Psychodidae/enzimologiaRESUMO
Sixteen isoenzyme patterns were analyzed for five Colombian Lutzomyia species. The average unbiased expected heterozygosity levels ranged from 0.098 (Lu. youngi) to 0.215 (Lu. torrvida). The five species samples, taken all the isoenzymes employed, were significantly deviated from the Hardy-Weinberg equilibrium by homozygous excess with classical as well as Markov chain exact tests. Possible causes: (1) Wahlund effect within populations due to subdivision and/or sampling. Endogamy could be discarded because these loci were affected by highly different levels of homozygous excess. (2) Null alleles could be not discarded, at least for some isoenzymes. The hierarchical Wright's F analysis showed high and significant values for each parameter. The average F(IT) value was 0.655 with a conspicous homozygous excess at a global level (all species taken together); the average F(IS) value was significantly positive (0.515) as well, with homozygous excess within each species. The genetic heterogeneity between the fives species was noteworthy (F(ST) 0.288), indicating clear genetic differentiation. The more related species pairs were Lu. longiflocosa-Lu. torvida (0.959) and Lu torvida-Lu. spinicrassa (0.960); while Lu. torvida-Lu. youngi (0.805) and Lu. quasitownsendi-Lu. youngi (0.796) were the most divergent (Nei's genetic identity matrix). UPGMA and Wagner algorithms showed that the most divergent species was Lu. youngi, whereas the most related were Lu. longiflocosa-Lu. torvida and Lu torvida-Lu. spinicrassa. A spatial autocorrelation analysis (Moran's I index) revealed a very weak, or inexistent spatial structure, which means that the speciation events between these species were independent from the geographic distances from where they currently live.
Assuntos
Frequência do Gene/genética , Genes de Insetos/genética , Variação Genética/genética , Isoenzimas/genética , Psychodidae/genética , Animais , Colômbia , Eletroforese em Gel de Poliacrilamida , Genética Populacional , Psychodidae/enzimologiaRESUMO
Trypsins constitute some of the most abundant midgut digestive proteases expressed by hematophagous insects upon blood feeding. In addition to their role in the digestion of the blood meal, these proteases also have been implicated in the ability of certain pathogens to infect their natural vector. In sand flies, digestive proteases including trypsins were associated with early killing of Leishmania and are believed to play a role in the species-specificity dictating sand fly vectorial capacity. Our group is involved in studies of midgut digestive proteases in the sand fly Lutzomyia longipalpis, the principal vector of visceral leishmaniasis in Brazil. Here we report on the identification of two cDNAs, Lltryp1 and Lltryp2, which code for putative midgut trypsins in L. longipalpis. Analyses of RNA abundance using semi-quantitative RT-PCR show a different pattern of expression between the two genes. Lltryp1 expression remains undetected until blood feeding and reaches a peak at 12 h post-blood meal (PBM), returning to pre-blood meal levels at 72 h PBM. Additionally, Lltryp1 expression is undetected during larval development. Lltryp2, on the other hand, is constitutively expressed as high levels in the non-blood fed female, but is reduced upon blood feeding. At the end of the digestive cycle, Lltryp2 regains its pre-blood meal levels. This cDNA also is present in all developmental stages and in adult males. This pattern of expression is reminiscent of what is seen in mosquitoes and Old World sand flies, but has characteristics that are unique to L. longipalpis.
Assuntos
Sangue , Regulação da Expressão Gênica no Desenvolvimento , Psychodidae/genética , Tripsina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Comportamento Alimentar/fisiologia , Feminino , Dados de Sequência Molecular , Filogenia , Psychodidae/enzimologia , Psychodidae/fisiologia , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Acetylcholinesterase (AChE) plays a key role in cholinergic impulse transmission, and it is the target enzyme for organophosphorus and carbamate insecticides. Two genes, AceI and AceII, have been characterized from different insect species, and point mutations in either gene can lead to significant resistance to these classes of insecticides. In this report, we describe the partial characterization of the AceI gene from Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae), and we show that the possibility exists for the development of a resistant phenotype to organophosphates and carbamates in sand flies. Our results point to the presence of a single AceI gene in L. longipalpis (LlAce1) and that AChE activity is inhibited by organophosphorus at a concentration of 5 x 10(-5) M. Regarding insecticide resistance, analysis of the truncated LlAce1 cDNA suggests that a single missense mutation leading to a glycine-to-serine substitution at amino acid position 119 (G119S) may arise in L. longipalpis, similar to what has been detected in Anopheles gambiae s.s. Another missense mutation involved in resistant phenotypes, F331W, detected in Culex tritaeniorhynchus Giles, is less likely to occur in L. longipalpis, because it faces codon constraint in this sand fly species. Comparison of the three-dimensional structures of the deduced amino acid sequence of the truncated LLAChE1 with that of An. gambiae and Cx. tritaeniorhynchus also suggests that similar structural modifications due to the missense amino acid changes in the active site gorge are detected in all three insects.
Assuntos
Acetilcolinesterase/genética , Psychodidae/enzimologia , Acetilcolinesterase/química , Acetilcolinesterase/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência Conservada , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Psychodidae/classificação , RNA/genética , RNA/isolamento & purificação , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Visceral leishmaniasis (VL) is a serious tropical disease that affects approximately 500 thousand people worldwide every year. In the Americas, VL is caused by the parasite Leishmania (Leishmania) infantum chagasi mainly transmitted by the bite of the sand fly vector Lutzomyia longipalpis. Despite recent advances in the study of interaction between Leishmania and sand flies, very little is known about sand fly protein expression profiles. Understanding how the expression of proteins may be affected by blood feeding and/or presence of parasite in the vector's midgut might allow us to devise new strategies for controlling the spread of leishmaniasis. In this work, we report the characterization of a vacuolar ATPase subunit C from L. longipalpis by screening of a midgut cDNA library with a 220 bp fragment identified by means of differential display reverse transcriptase-polymerase chain reaction analysis. The expression of the gene varies along insect development and is upregulated in males and bloodfed L. longipalpis, compared to unfed flies.
Assuntos
Comportamento Alimentar/fisiologia , Insetos Vetores/genética , Psychodidae/genética , ATPases Vacuolares Próton-Translocadoras/genética , Animais , Sequência de Bases , Southern Blotting , Clonagem Molecular , Cricetinae , Sistema Digestório/enzimologia , Sistema Digestório/parasitologia , Insetos Vetores/embriologia , Insetos Vetores/enzimologia , Leishmaniose Visceral/transmissão , Masculino , Dados de Sequência Molecular , Subunidades Proteicas , Psychodidae/embriologia , Psychodidae/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Visceral leishmaniasis (VL) is a serious tropical disease that affects approximately 500 thousand people worldwide every year. In the Americas, VL is caused by the parasite Leishmania (Leishmania) infantum chagasi mainly transmitted by the bite of the sand fly vector Lutzomyia longipalpis. Despite recent advances in the study of interaction between Leishmania and sand flies, very little is known about sand fly protein expression profiles. Understanding how the expression of proteins may be affected by blood feeding and/or presence of parasite in the vector's midgut might allow us to devise new strategies for controlling the spread of leishmaniasis. In this work, we report the characterization of a vacuolar ATPase subunit C from L. longipalpis by screening of a midgut cDNA library with a 220 bp fragment identified by means of differential display reverse transcriptase-polymerase chain reaction analysis. The expression of the gene varies along insect development and is upregulated in males and bloodfed L. longipalpis, compared to unfed flies.
Assuntos
Animais , Masculino , Cricetinae , Comportamento Alimentar/fisiologia , Insetos Vetores/genética , Psychodidae/genética , ATPases Vacuolares Próton-Translocadoras/genética , Sequência de Bases , Southern Blotting , Clonagem Molecular , Sistema Digestório/enzimologia , Sistema Digestório/parasitologia , Insetos Vetores/embriologia , Insetos Vetores/enzimologia , Leishmaniose Visceral/transmissão , Dados de Sequência Molecular , Subunidades Proteicas , Psychodidae/embriologia , Psychodidae/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
The genetic relationships among male Lutzomyia (Nyssomyia) intermedia (Lutz & Neiva) (Diptera: Psychodidae) from three populations from the same endemic area of American cutaneous leishmaniasis (ACL) in the state of Rio de Janeiro, Brazil, were compared. The sandflies were collected in three ecologically different habitats: domestic, extra-domestic and sylvatic over a total range of 800 m. Three molecular markers were employed to assess population variation. Based on MLEE markers, it could not be concluded that the three populations do not belong to the same gene pool (F(st) = 0.005). No within-population departure from Hardy-Weinberg equilibrium was detected (P < 0.05) and they presented the same level of gene variation. The number of migrants (Nm) indicated that at least 50 individuals per generation migrated between the three habitats. RAPD-PCR markers revealed that, except for the primer five, all were polymorphic. Phenetic analysis of the genotypes showed the presence of two principal clusters corresponding to: (1) domestic plus extra-domestic and (2) sylvatic. Unique genotypes were observed in each population. The sylvatic population was the most polymorphic, showing the largest number of genotypes and low level of similarity between them. Three mtDNA gene markers were studied by SSCP analysis. The most frequent haplotype for each marker ranged in frequency from 60 to 87% and individuals with unique haplotypes varied from 1 to 5%. Interestingly, the SSCP analysis showed a low level of polymorphism within populations. The disagreement between the different molecular markers observed and the hypothesis that L. intermedia could be participating in the transmission cycle of Leishmania (Viannia) braziliensis in environments ranging from the interior of human dwellings to the forest, are discussed.