Taxonomia molecular do complexo Bichromomyia flaviscutellata (Diptera: Psychodidae) e estrutura genética de Bichromomyia flaviscutellata sensu stricto, principal vetor da Leishmania amazonensis (Kinetoplastea: Trypanosomastidae)

Abstract

Phlebotomines are small insects belonging to the order Diptera, family Psychodidae, subfamily Phlebotominae that are subdivided into several genera. Some species of this group are considered transmitters (vectors) of pathogens that cause Leishmaniasis in some vertebrate animals, including man. Bichromomyia flaviscutellata is a complex composed of at least three species: B. flaviscutellata s.s., B. reducta and B. olmeca. The latter is composed of three subspecies: B. olmeca olmeca, B. olmeca bicolor and B. olmeca nociva, which are distributed from Central America to South America. Of these species, B. flaviscutellata s.s. is the main vector of Leishmania amazonesis in Brazil. The present study aimed to molecularly identify the species and subspecies of the B. flaviscutellata complex using the 5' region of the COI gene (known as DNA Barcode) and to verify the genetic structure of B. flaviscutellata s.s. using the 3' region of the gene COI to the variable region. The sequences of the Barcode DNA of the specimens (n = 49) were from six localities, five in the State of Amazonas (Autazes, Manaus, Pitinga, Novo Airão and Rio Preto da Eva) and one (1) in the State of Pará while the sequences of the Variable Region (n = 86) were from the State of Amazonas (Autazes, Manaus, Pitinga, Novo Airão and Rio Preto da Eva). In the Barcode database were added sequences of B. olmeca olmeca and B. olmeca bicolor from Genbank. The consensus sequences of the Barcode DNA showed 649 base pairs (bp) that generated 29 haplotypes and five unconnected networks. Intraspecific genetic distances ranged from 0.000 to 0.037, while interspecific distances ranged from 0.11 to 0.13, with a barcoding gap of 0.087. Phylogenetic analyzes using the algorithms and evolutionary models Neighbor - Joining (NJ - K2P), Maximum Likelihood (ML - HKY + I + G) and Bayesian Inference (BI - HKY + I + G) resulted in topologies with highly supported clades . All trees presented the same topology, but with variations in support values. The relationship between B. olmeca olmeca and B. olmeca bicolor was better clarified in NJ analysis; however, these data, together with those of genetic distances, confirm the position of B. olmeca bicolor as a subspecies of B. olmeca olmeca. Curiously, the topologies of the trees showed the B. olmeca nociva most phylogenetically closer of B. flaviscutellata s.s., as well as the haplotype network separated B. olmeca nociva of B. olmeca olmeca and B. olmeca bicolor. The RV consensus sequences presented 1141 bp and generated 59 haplotypes, showing great variability within B. flaviscutellata s.s. Intraspecific genetic distance between the populations Rio Preto da Eva and Autazes presented the highest value (0.0166). The intrapopulational analysis of the B. flaviscutellata s.s samples from the city of Rio Preto da Eva showed a higher level of genetic differentiation with a value of 0.0167 (1.67%). The Tajima D test was negative but non-significant for all populations, indicating no significant deviation from the neutral evolution model. However, the Fu Fs test was negative and significant for four of the five populations (Autazes, Manaus, Novo Airão and Pitinga), indicating a recent population expansion. The greatest genetic differentiation, using the Fst coefficient and the lowest gene flow, using the Nm coefficient, was between Pitinga and Rio Preto da Eva (Fst = 0.3535; Nm = 0.91). The genetic structure estimated by AMOVA was high and significant, proving that this species is genetically structured. However, the Mantel test was negative and non-significant, which means that this structuring is not related to the Isolation-by-Distance (IBD) model. The presence of shared haplotypes between the populations of Manaus (situated on the left side of the Rio Negro) and Novo Airão (situated on the right side of the Negro River) and the populations of Manaus / Pitinga (situated on the left side of the Solimões River) and Autazes located on the right side of the Solimões River) indicate that the Amazonas / Negro and Solimões River systems are not acting as geographical barriers that impede the gene flow between the populations of B. flaviscutellata s.s. The results of phylogenetic analyzes, together with the haplotype network, genetic distances and morphological characters, suggest that the B. olmeca nociva subspecies should be elevated to the species category.