Taxonomic, molecular and ecological approach reveals high diversity of vector sand flies, varied blood source supply and a high detection rate of Leishmania DNA in Colombian Amazon region

Abstract

Background: The Amazon region is home to more than 30% of the sand flies species in Colombia, including vectors of Leishmania mainly in the genus Lutzomyia and Psychodopygus. Advances in morphological and molecular taxonomy of sand flies facilitate the development of updated and robust species inventories in understudied areas, such as the departments of Amazonas and Caquetá. Currently, integrating the detection of blood meal sources and Leishmania DNA represents a key approach under the "One Health" concept by providing insights into human and animal health and the dynamics of different ecosystems.

Methodology/principal findings: This study characterized the sand flies fauna in Amazonas and Caquetá using an integrative taxonomic approach that included DNA detection from blood meal and Leishmania sources. Sand flies were collected using CDC, Shannon, Prokopack traps and mouth aspirators. DNA was analyzed by conventional PCR targeting COI, Cytb, 12S rDNA and HSP-70N markers, respectively. A total of 1,104 specimens were collected, representing 12 genera and 30 species, 10 are recognized vectors of Leishmania, including Nyssomyia antunesi and Psychodopygus amazonensis. Our findings include new reports of regional distribution, particularly the first report of Sciopemyia fluviatilis in Colombia. Homo sapiens (28.8% Cytb; 18.6% 12S) and Sus scrofa (16.9% Cytb; 6.8% 12S) were the main food sources detected. While Nyssomyia fraihai (2.6%), Trichophoromyia cellulana (1.3%), Nyssomyia yuilli pajoti (1.3%) and Evandromyia (Aldamyia) walkeri (1.0%) grouped the highest detection rate of Leishmania DNA (9.0%).

Conclusions/significance: The integration of molecular tools for the confirmation of phlebotomine species allowed the resolution of taxonomic conflicts, especially in the genus Trichophoromyia. These findings provide key information on ecological interactions (vectors-ingesta-Leishmania) related to leishmaniasis in the Colombian Amazon, suggesting a high diversity of sand flies and a significant zoonotic potential.

Fig 1. Geographic location of the study area and sand flies collection sites: (A) Departments of Amazonas and Caquetá, Colombia.

(B) Municipality of Florencia, locality Jericó, Macagual, Santo Domingo and Sebastopol. (C) Municipality of Leticia, locality Leticia, San Pedro de los Lagos and Tanimboca. Source of layers [–42] and Qgis, v. 3.34.12 [43].

Fig 2. Relative abundances of sand flies species collected in the departments of Amazonas (Tanimboca and San Pedro de los Lagos) and Caquetá (Sebastopol, Jericó, Macagual and Santo Domingo).

Dotted line: most abundant genera, N: Total specimens by species and by genus, %: Percentage abundance by species and by genus.

Fig 3. Principal component analysis (PCoA) indicates the distribution of sand flies species from Bray-Curtis similarity and standardized species abundance index (SISA).

SD: super dominant, D: dominant, ND: non-dominant; SA: super abundant, MA: Very abundant, C: common, A: Accidental, W: Constant.

Fig 4. Genetic divergence analysis from sand flies sequences collected in the departments of Amazonas and Caquetá.

(A) Nucleotide heterogeneity according to FINGERPRINT, (B) ABGD “barcode gap” K2P, (C) ABGD “barcode gap” uncorrected p distances. (D) Transitions/transversions rates versus genetic distance.

Fig 5. Neighbor-joining gene tree and morphology of sand flies COI barcode sequences, with emphasis on Psychodopygus.

(A) NJ Dendrogram. (B-C) Male and female genitalia of Ps. davisi. (D-E) Male and female genitalia of Ps. chagasi. (F-G) Male and female genitalia of Ps. paraensis.

Fig 6. Neighbor-joining gene tree and morphology of sand flies COI barcode sequences, with emphasis on Nyssomyia.

(A) NJ Dendrogram. (B-C) Male and female genitalia of Ny. antunesi. (D-E) Male and female genitalia of Ny. yuilli pajoti. (F-G) Male and female genitalia of Ny. fraihai.

Fig 7. Neighbor-joining gene tree and morphology of sand flies COI barcode sequences, with emphasis on Evandromyia and Psathyromyia.

(A) NJ Dendrogram. (B) female genitalia of Ev. (Eva.) georgii. (C) female genitalia of Ev. (Eva.) saulensis. (D-E) male and female genitalia of Ev. (Ald.) walkeri. (F) male genitalia of Pa. (For.) aragaoi. (G) male genitalia of Pa. (Psa.) dendrophyla.

Fig 8. Neighbor-joining gene tree and morphology of sand flies COI barcode sequences, with emphasis on Trichophoromyia and Trichopygomyia.

(A) NJ Dendrogram. (B-C) male and female genitalia of Th. cellulana. (D-E) male and female genitalia of Th. howardi. (F) male genitalia of Ty. witoto.

Fig 9. Neighbor-joining gene tree and morphology of sand flies COI barcode sequences, with emphasis on Lutzomyia and Sciopemyia.

(A) NJ Dendrogram. (B-C) male and female genitalia of Lu. (Trl.) sherlocki. (D-E) male and female genitalia of Lu. (Hel.) tortura.

Fig 10. Neighbor-joining gene tree and morphology of sand flies COI barcode sequences, with emphasis on genera of lesser abundance.

(B) male genitalia of Br. mesai. (C) male genitalia of Mi. (Mic.) pilosa. (D) male genitalia of Pi. (Pif.) serrana. (E) female genitalia of Vi. tuberculata. (F) female genitalia of Vi. caprina.

Fig 11. Neighbor-joining gene tree and blood food sources detected in sand flies collected in the Colombian Amazon region, using the molecular marker Cytochrome B (Cytb).

(A) Dendrogram NJ. (B) Vertebrate species identified.

Fig 12. Neighbor-joining gene tree and blood food sources detected in sand flies collected in the Colombian Amazon region, using the molecular marker 12S.

(A) Dendrogram NJ. (B) Vertebrate species identified.

Fig 13. Relationship between sources of blood ingestion and the detection of Leishmania DNA in sand flies from the department of Amazonas and Caquetá.

Ny.a: Ny. antunesi, Ny.u: Ny. umbratilis, Ny. yp: Ny. yuilli pajoti, Ev. w: Ev. (Ald.) walkeri, Ny. fr: Ny. fraihai, Th. ce: Th. cellulana, Pi. ne: Pi. (Pif.) nevesi, Sc. so: Sc. sordellii, Ps. da: Ps. davisi, Ps.pr: Ps. paraensis.

Fig 14. The ecological dynamics of the tripartite relationship among sand flies, blood meal sources and Leishmania in forest and peridomicialiary areas in the Amazon region from Colombia.