Improving the COI DNA barcoding library for Neotropical phlebotomine sand flies (Diptera: Psychodidae).

Sand fly species are traditionally identified using morphological traits, though this method is hampered by the presence of cryptic species. DNA barcoding is a widely used tool in the case of insects of medical importance, where it is necessary to know quickly which species are present in a transmission area. Here, we assess the usefulness of mitochondrial cytochrome c oxidase subunit I (COI) DNA barcoding as a practical tool for species identification, correct assignment of isomorphic females, and to evaluate the detection of cryptic diversity that occurs in the same species. A fragment of the COI gene was used to generate 156 new barcode sequences for sand flies from different countries of the Neotropical region, mainly Colombia, which had been identified morphologically as 43 species. The sequencing of the COI gene allowed the detection of cryptic diversity within species and correctly associated isomorphic females with males identified by morphology. The maximum intraspecific genetic distances ranged from 0 to 8.32% and 0 to 8.92% using uncorrected p distances and the Kimura 2-parameter (K2P) model, respectively. The minimum interspecific distance (nearest neighbor) for each species ranged from 1.5 to 14.14% and 1.51 to 15.7% using p and K2P distances, respectively. Three species had more than 3% maximum intraspecific distance: Psychodopygus panamensis, Micropygomyia cayennensis cayennensis, and Pintomyia evansi. They also were split into at least two molecular operational taxonomic units (MOTUs) each, using different species delimitation algorithms. Regarding interspecific genetic distances, the species of the genera Nyssomyia and Trichophoromyia generated values lower than 3% (except Nyssomyia ylephiletor and Ny. trapidoi). However, the maximum intraspecific distances did not exceed these values, indicating the presence of a barcode gap despite their proximity. Also, nine sand fly species were DNA barcoded for the first time: Evandromyia georgii, Lutzomyia sherlocki, Ny. ylephiletor, Ny. yuilli pajoti, Psathyromyia punctigeniculata, Sciopemyia preclara, Trichopygomyia triramula, Trichophoromyia howardi, and Th. velezbernali. The COI DNA barcode analysis enabled the correct delimitation of several Neotropical sand fly species from South and Central America and raised questions about the presence of cryptic species for some taxa, which should be further assessed.

Ecological interactions of sand flies, hosts, and Leishmania panamensis in an endemic area of cutaneous leishmaniasis in Colombia.

BACKGROUND: The transmission dynamics of leishmaniasis are complex. There is also a lack of information about the ecological relationships between the vector/host/parasite at a more local and specific level. The Andean region concentrates more than 50% of Colombia's cutaneous leishmaniasis (CL) cases. The study of the ecological interactions of sand flies through the identification of blood sources has provided information on the female's opportunistic behavior, feeding on various hosts. Therefore, this study aimed to determine sand flies' ecological interactions with Leishmania parasites and their blood sources in an endemic area of CL. RESULTS: A total of 4,621 sand flies were collected, comprising 20 species, in which the most abundant were Nyssomyia yuilli yuilli (55.4%), Psychodopygus ayrozai (14.5%) and Ps. panamensis (13.4%). Sequences of 12S gene fragment were analyzed using the BLASTn search tool. Blood-meal source identification was successfully performed for 47 sand flies, detecting seven vertebrate species, human and armadillo being the most frequent. Leishmania DNA was amplified in four female pools, constituted by Ny. yuilli yuilli and Ps. ayrozai, and the identification through RFLP detected Leishmania (Viannia) panamensis in the positive pools. CONCLUSIONS: The interactions between the sand fly species, local mammalian fauna and the Leishmania parasite in this active focus of CL, provide evidence of the potential role of two different species in the maintenance of the parasite transmission, important information for the understanding of the ecoepidemiology and transmission dynamics of the disease in Andean endemic areas. However its necessary further evaluations of the vector and host competence in the transmission and maintenance of Leishmania spp, in these complex and diverse areas.

Diversity and temporal distribution of sand flies in an endemic area of cutaneous leishmaniasis in Centre-West Colombia.

The community structure of sand flies indicates the level of adaptation of vector species in a region, and in the context of vector management and control, this information allows for identifying the potential risks of pathogen transmission. This study aimed to analyze sand fly diversity and spatial-temporal distribution in an endemic area of cutaneous leishmaniasis. The study was carried out in the Carrizales hamlet (Caldas), between September 2019 and October 2021. The monthly distribution of sand fly species was evaluated through collections with CDC traps. Shannon and evenness indices were calculated and used to compare species frequencies at each house. The association between climatic variables and the frequency of sand flies was evaluated using Spearman's correlation. A total of 6,265 females and 1,958 males belonging to 23 species were found. Low diversity and evenness were observed, with the dominance of Nyssomyia yuilli yuilli (Young & Porter). Ecological and diversity indices did not reveal differences between the houses. The sand fly community was composed of 3 dominant species, Ny. yuilli yuilli, Psychodopygus ayrozai (Barretto & Coutinho), and Ps. panamensis (Shannon), representing 75.8% of the total catches. No statistical association was found between the absolute frequency of sand flies, rainfall, and temperature. The results show one dominant species, this fact has epidemiological relevance since density influences parasite-vector contact. The high densities of sand flies recorded in peri- and intradomiciliary areas highlight the necessity of periodic monitoring of vector populations and control activities to reduce the risk of Leishmania transmission in this endemic area.

Vetores de agentes de Leishmania spp. na Colômbia: evidências baseadas em parâmetros de capacidade vetorial / Vectors of Leishmania spp. agents in Colombia: evidence based on vectorial capacity parameters

Introdução. A leishmaniose cutânea (LC) representa um desafio significativo na Colômbia, especialmente na região andina. O departamento de Caldas, tem um registro constante de casos e Victoria está entre os municípios mais afetados, apresentando uma média anual de 300 casos por 100.000 habitantes nos últimos cinco anos. No entanto, a identificação das espécies de flebotomíneos responsáveis pela transmissão dos agentes das leishmanioses, geralmente é baseada apenas em evidências epidemiológicas, sendo necessário avaliar os parâmetros de incriminação vetorial para essas espécies. Objetivo. Analisar as evidências de vetores de Leishmania spp. na Colômbia, com base em parâmetros da capacidade vetorial. Material e métodos. Foi conduzida uma revisão narrativa e análise crítica dos estudos de flebotomíneos na Colômbia, seguindo os critérios de incriminação vetorial propostos por Killick-Kendrick. Para avaliar os parâmetros da capacidade vetorial em um foco endêmico de LC no município de Victoria (Caldas), foram realizadas capturas mensais de flebotomíneos durante 18 meses, para analisar a distribuição espaço-temporal desses insetos. A estrutura ecológica da comunidade foi analisada utilizando índices ecológicos de diversidade de Shannon e equitabilidade de Pielou, bem como o índice padronizado de abundância de espécies. A investigação da circulação de Leishmania e a identificação das fontes sanguíneas foram feitas usando métodos moleculares, com análise descritiva das interações. Para comparar as capturas entre diferentes ambientes (intra e peridomiciliar) e a proporção de fêmeas ingurgitadas para cada espécie, foi realizado um teste de χ2. Resultados. Foram analisados 49 artigos relacionados ao estudo de vetores de Leishmania spp., o que permitiu classificar as espécies como vetores comprovados e vetores suspeitos. No estudo do foco endêmico de LC, foram coletados 8.223 flebotomíneos, 7.999 em armadilha tipo CDC (75,8% fêmeas e 24,2% machos) e 198 em armadilha Shannon (88,4% fêmeas e 11,6% machos). Os espécimes pertencem a 23 espécies distribuídas em 13 gêneros. De acordo com o índice padronizado de abundância de espécies, Nyssomyia yuilli yuilli foi a espécie mais abundante, seguida de Psychodopygus ayrozai e Ps. panamensis, que juntas representaram 75,8% dos flebotomíneos capturados. Leishmania panamensis foi identificada como o parasita circulante no foco, com uma taxa mínima de infecção (IMR) de 1,2%, detectada tanto em Ny. yuilli yuilli quanto em Ps. ayrozai. A identificação das fontes sanguíneas permitiu estabelecer a preferência de Ny. yuilli yuilli, por humanos e animais domésticos. Ps. ayrozai apresentou preferência por humanos e tatus. Conclusão. O estudo fornece uma atualização no status dos vetores dos agentes das leishmanioses na Colômbia. As evidências obtidas no foco endêmico de LC sugerem a existência de diferentes padrões de interação vetor-Leishmania-hospedeiro, sugerindo Ny. yuilli yuilli como seu principal vetor no ambiente domiciliar, porém, com Ps. ayrozai atuando na transição entre o ciclo silvestre e o peridomiciliar.

Introduction. Cutaneous leishmaniasis (CL) represents a significant challenge in Colombia,especially in the Andean region. The department of Caldas has a constant record of cases, and Victoria is among the most affected municipalities, with an annual average of 300 cases per 100,000 inhabitants in the last five years. However, the identification of sand fly species responsible for transmitting leishmaniasis agentsis often based solely on epidemiological evidence, highlighting the need to evaluate vector incrimination parameters for these species. Objective. To analyze the evidence of Leishmaniaspp. vectors in Colombia based on the vectorial capacity parameters. Material and methods. A narrative review and critical analysis of sand flystudies in Colombia were conducted, following the vector incrimination criteria proposed by Killick-Kendrick. To evaluate the vectorial capacity parameters in an endemic focus of CL in the municipality of Victoria (Caldas), monthly sand fly captures were carried out for 18 months to analyze the spatiotemporal distribution of these insects. The ecological structure of the community was analyzed using Shannon's diversity and Pielous equitability indices, as well as the standardized index of species abundance.Investigation of Leishmania circulation and identification of blood sources were performed using molecular methods with a descriptive analysis of interactions.A χ22 test was madeto compare captures between different environments (intra and peridomiciliary) and the proportion of engorged females for each species. Results. A total of 49 articles related to the study of Leishmaniaspp. vectors were analyzed, enabling the classification of species as proven vectors and suspected vectors. In the study of the CLendemic focus, 8,223 sand flieswere collected, of which7,999 in CDC traps (75.8% females and 24.2% males) and 198 in Shannon traps (88.4% females and 11.6% males). The specimens belong to 23 species distributed across 13 genera. According to the standardized index of species abundance, Nyssomyiayuilli yuilliwas the most abundant species, followed by Psychodopygusayrozai andPs. panamensis,accounting for 75.8% of the total sand flies. Leishmania panamensiswas identified as the circulating species in the focus, with a minimum infection rate (MIR) of 1.2% detected in both Ny. yuilli yuilliand Ps. ayrozai. The identification of blood sources allowed for establishing the preference of Ny. yuilli yuillifor humans and domestic animals, while Ps. ayrozaishowed a preference for humans and armadillos. Conclusion. The study provides an update on the status of leishmaniasis vectors in Colombia. Our findings in the CL endemic focus suggeststhe existence of different patterns of vector-Leishmania-host interaction, with Ny. yuilli yuillibeing the main vector in the domestic environment, but with Ps. ayrozaiplaying a role in the transition between the sylvatic and peridomestic cycles.