RESUMO
In the current context of global change and human-induced biodiversity decline, there is an urgent need for developing sampling approaches able to accurately describe the state of biodiversity. Traditional surveys of vertebrate fauna involve time-consuming and skill-demanding field methods. Recently, the use of DNA derived from invertebrate parasites (leeches and blowflies) was suggested as a new tool for vertebrate diversity assessment. Bloodmeal analyses of arthropod disease vectors have long been performed to describe their feeding behaviour, for epidemiological purposes. On the other hand, this existing expertise has not yet been applied to investigate vertebrate fauna per se. Here, we evaluate the usefulness of hematophagous dipterans as vertebrate samplers. Blood-fed sand flies and mosquitoes were collected in Amazonian forest sites and analysed using high-throughput sequencing of short mitochondrial markers. Bloodmeal identifications highlighted contrasting ecological features and feeding behaviour among dipteran species, which allowed unveiling arboreal and terrestrial mammals of various body size, as well as birds, lizards and amphibians. Additionally, lower vertebrate diversity was found in sites undergoing higher levels of human-induced perturbation. These results suggest that, in addition to providing precious information on disease vector host use, dipteran bloodmeal analyses may represent a useful tool in the study of vertebrate communities. Although further effort is required to validate the approach and consider its application to large-scale studies, this first work opens up promising perspectives for biodiversity monitoring and eco-epidemiology.
Assuntos
DNA/sangue , Insetos Vetores/genética , Vertebrados/classificação , Animais , Biodiversidade , Culicidae/genética , Comportamento Alimentar , Guiana Francesa , Psychodidae/genéticaRESUMO
Phlebotomine sand flies are haematophagous dipterans of primary medical importance. They represent the only proven vectors of leishmaniasis worldwide and are involved in the transmission of various other pathogens. Studying the ecology of sand flies is crucial to understand the epidemiology of leishmaniasis and further control this disease. A major limitation in this regard is that traditional morphological-based methods for sand fly species identifications are time-consuming and require taxonomic expertise. DNA metabarcoding holds great promise in overcoming this issue by allowing the identification of multiple species from a single bulk sample. Here, we assessed the reliability of a short insect metabarcode located in the mitochondrial 16S rRNA for the identification of Neotropical sand flies, and constructed a reference database for 40 species found in French Guiana. Then, we conducted a metabarcoding experiment on sand flies mixtures of known content and showed that the method allows an accurate identification of specimens in pools. Finally, we applied metabarcoding to field samples caught in a 1-ha forest plot in French Guiana. Besides providing reliable molecular data for species-level assignations of phlebotomine sand flies, our study proves the efficiency of metabarcoding based on the mitochondrial 16S rRNA for studying sand fly diversity from bulk samples. The application of this high-throughput identification procedure to field samples can provide great opportunities for vector monitoring and eco-epidemiological studies.
Assuntos
Código de Barras de DNA Taxonômico/métodos , Insetos Vetores , Metagenômica/métodos , Psychodidae/classificação , Psychodidae/genética , Animais , Análise por Conglomerados , DNA Mitocondrial/química , DNA Mitocondrial/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Guiana Francesa , Filogenia , Psychodidae/crescimento & desenvolvimento , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Leishmania species of the subgenus Viannia and especially Leishmania Viannia guyanensis are responsible for a large proportion of New World leishmaniasis cases. Since a recent publication on Leishmania Viannia braziliensis, the debate on the mode of reproduction of Leishmania parasites has been reopened. A predominant endogamic reproductive mode (mating with relatives), together with strong Wahlund effects (sampling of strains from heterogeneous subpopulations), was indeed evidenced. To determine whether this hypothesis can be generalized to other Leishmania Viannia species, we performed a population genetic study on 153 human strains of L. (V.) guyanensis from French Guiana based on 12 microsatellite loci. The results revealed important homozygosity and very modest linkage disequilibrium, which is in agreement with a high level of sexual recombination and substantial endogamy. These results also revealed a significant isolation by distance with relatively small neighbourhoods and hence substantial viscosity of Leishmania populations in French Guiana. These results are of epidemiological relevance and suggest a major role for natural hosts and/or vectors in parasite strain diffusion across the country as compared to human hosts.
Assuntos
Genética Populacional/métodos , Leishmania guyanensis/genética , Leishmania guyanensis/fisiologia , Reprodução , Simulação por Computador , DNA de Protozoário/genética , Guiana Francesa , Variação Genética , Técnicas de Genotipagem , Humanos , Leishmaniose Mucocutânea/parasitologia , Desequilíbrio de Ligação , Repetições de Microssatélites , Isolamento ReprodutivoRESUMO
Leishmania species of the subgenus Viannia and especially Leishmania braziliensis are responsible for a large proportion of New World leishmaniasis cases. The reproductive mode of Leishmania species has often been assumed to be predominantly clonal, but remains unsettled. We have investigated the genetic polymorphism at 12 microsatellite loci on 124 human strains of Leishmania braziliensis from 2 countries, Peru and Bolivia. There is substantial genetic diversity, with an average of 12.4 +/- 4.4 alleles per locus. There is linkage disequilibrium at a genome-wide scale, as well as a substantial heterozygote deficit (more than 50% the expected value from Hardy-Weinberg equilibrium), which indicates high levels of inbreeding. These observations are inconsistent with a strictly clonal model of reproduction, which implies excess heterozygosity. Moreover, there is large genetic heterogeneity between populations within countries (Wahlund effect), which evinces a strong population structure at a microgeographic scale. Our findings are compatible with the existence of population foci at a microgeographic scale, where clonality alternates with sexuality of an endogamic nature, with possible occasional recombination events between individuals of different genotypes. These findings provide key clues on the ecology and transmission patterns of Leishmania parasites.
Assuntos
Leishmania braziliensis/genética , Animais , Bolívia , Heterozigoto , Humanos , Desequilíbrio de Ligação , Repetições de Microssatélites/genética , Peru , Polimorfismo Genético , Reprodução/genéticaAssuntos
Doenças dos Bovinos/parasitologia , Criptosporidiose/parasitologia , Criptosporidiose/veterinária , Cryptosporidium/classificação , Cryptosporidium/isolamento & purificação , Doenças das Cabras/parasitologia , Síndrome da Imunodeficiência Adquirida/complicações , Animais , Bovinos , Criptosporidiose/epidemiologia , Criptosporidiose/transmissão , Cryptosporidium/genética , DNA de Protozoário/química , DNA de Protozoário/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Fezes/parasitologia , França/epidemiologia , Genes de Protozoários , Variação Genética , Genótipo , Cabras , Haiti/epidemiologia , Humanos , Epidemiologia Molecular , Filogenia , RNA Ribossômico 18S/genética , Análise de Sequência de DNARESUMO
The southernmost limit of the distribution of endemic Andean cutaneous leishmaniasis (CL), commonly known as Uta, is localized in the western Andean valleys of Ayacucho, Peru. This area is completely isolated from other regions endemic for this disease. Identification of the insect vector for Andean CL was carried out by combining entomologic and parasitologic approaches. Two Lutzomyia species were captured: Lutzomyia ayacuchensis and Lu. noguchii. The former species was considered responsible for transmission of Leishmania because 1) there was a coincidence in space and time between the presence of this insect and the distribution of Andean CL, 2) it was shown to be highly anthropophilic, 3) Leishmania parasites of the subgenus Viannia were detected by a specific polymerase chain reaction assay, 4) promastigotes isolated from this insect were shown by multilocus enzyme electrophoresis and molecular karyotyping to belong to the same deme of Leishmania (Viannia) peruviana as the one circulating in humans living in the study area, and 5) the complete cycle of L. (V.) peruviana was observed in experimental infections of Lu. ayacuchensis. Parasite and vector homogeneity found in Ayacucho contrasted with the heterogeneity reported for other areas endemic for Andean CL. The potential influence of ecologic determinants on this geographically isolated area is discussed.