Structural differences in gut bacteria communities in developmental stages of natural populations of Lutzomyia evansi from Colombia's Caribbean coast

Abstract

Background: Lutzomyia evansi, a phlebotomine insect endemic to Colombia's Caribbean coast, is considered to be the main vector of visceral and cutaneous leishmaniasis in the region. Although insects of this species can harbor pathogenic and non-pathogenic microorganisms in their intestinal microbiota, there is little information available about the diversity of gut bacteria present in Lutzomyia evansi. In this study, conventional microbiological methods and molecular tools were used to assess the composition of bacterial communities associated with Lutzomyia evansi guts in immature and adult stages of natural populations from the department of Sucre (Caribbean coast of Colombia).

Methods: Sand flies were collected from two locations (peri-urban and jungle biotype) in the Department of Sucre (Caribbean coast of Colombia). A total of 752 Lutzomyia evansi intestines were dissected. In this study, 125 bacterial strains were isolated from different culture media (LB Agar, MacConkey Agar). Different methods were used for bacterial identification, including ribosomal intergenic spacer analysis (RISA) and analysis of the 16S rRNA and gyrB gene sequences. The genetic profiles of the bacterial populations were generated and temporal temperature gradient gel electrophoresis (TTGE) was used to compare them with total gut DNA. We also used PCR and DNA sequence analysis to determine the presence of Wolbachia endosymbiont bacteria and Leishmania parasites.

Results: The culture-dependent technique showed that the dominant intestinal bacteria isolated belong to Acinetobacter, Enterobacter, Pseudomonas, Ochrobactrum, Shinella and Paenibacillus in the larval stage; Lysobacter, Microbacterium, Streptomyces, Bacillus and Rummeliibacillus in the pupal stage; and Staphylococcus, Streptomyces, Brevibacterium, Acinetobacter, Enterobacter and Pantoea in the adult stage. Statistical analysis revealed significant differences between the fingerprint patterns of the PCR-TTGE bands in bacterial communities from immature and adult stages. Additionally, differences were found in bacterial community structure in fed females, unfed females, males and larvae. The intestinal bacteria detected by PCR-TTGE were Enterobacter cloacae and Bacillus thuringiensis, which were present in different life stages of Lu. evansi, and Burkholderia cenocepacia and Bacillus gibsonii, which were detected only in the larval stage. Wolbachia and Leishmania were not detected in gut samples of Lutzomyia evansi.

Conclusions: The analyses conducted using microbiological and molecular approaches indicated significant variations in the bacterial communities associated with the gut of Lu. evansi, depending on the developmental stage and food source. We propose that these elements affect microbial diversity in L. evansi guts and may in turn influence pathogen transmission to humans bitten by this insect.

Keywords: Adults; Gut; Immature; Microbiota; Sand flies; Vector.

Fig. 1

Dissection and obtaining of guts. A1: male; A2: gut of a male; B1: unfed female; B2: gut of an unfed female; C1: fed female; C2: gut of fed female; D1: fourth-instar larvae recovered in natural breeding sites; D2: gut of a fourth-instar larva recovered in natural breeding sites; E1: pupa recovered in natural breeding sites; E2: gut of a pupa recovered in natural breeding sites

Fig. 2

Bacterial counts (CFU) in Lu. evansi guts, obtained in media LB agar and MacConkey. Abbreviations: FFO, fed females from Ovejas; FFC, fed females from Coloso; UFO, unfed females from Ovejas; UFC, unfed females from Coloso; MC, males from Coloso; MV, males from Ovejas; L4, larvae from Ovejas; PP, pupae from Ovejas

Fig. 3

Neighbor-joining dendrogram for partial 16S rDNA sequences of bacteria obtained from adult stages of Lutzomyia evansi collected in the municipalities of Ovejas and Colosó (Sucre Department, Colombia), based on the Kimura 2-parameter model. Numbers at the nodes represent bootstrap values. The robustness of the phylogeny was tested by bootstrap analysis using 1000 iterations. Abbreviations: FFO, fed females from Ovejas; FFC, fed females from Coloso; UFO, unfed females from Ovejas; UFC, unfed females from Coloso; MC, males from Coloso; MV, males from Ovejas

Fig. 4

Neighbor-joining dendrogram for partial 16S rDNA sequences of bacteria obtained from immature stages of Lutzomyia evansi collected in the Ovejas municipality (Sucre Department, Colombia), based on the Kimura 2-parameter model. Numbers at the nodes represent bootstrap values. The robustness of the phylogeny was tested by bootstrap analysis using 1000 iterations. Abbreviations: L4, larvae from Ovejas; PP, pupae from Ovejas

Fig. 5

Neighbor-joining dendrogram for gyrB gene sequences of bacteria obtained from adult and immature stages of Lutzomyia evansi collected in the municipalities of Ovejas and Colosó (Sucre Department, Colombia), based on the Kimura 2-parameter model. Numbers in nodes represent bootstrap values. The robustness of the phylogeny was tested by bootstrap analysis using 1000 iterations. Abbreviations: FFO, fed females from Ovejas; FFC, fed females from Coloso; UFO, unfed females from Ovejas; UFC, unfed females from Coloso; MC, males from Coloso; MV, males from Ovejas; L4, larvae from Ovejas

Fig. 6

Dice-UPGMA clustering analysis of bacterial diversity obtained of PCR-TTGE Profiles of 16S rDNA amplification products (V3–V6 variable regions), through GelCompar II software. Abbreviations: L, larvae; MO, males from Ovejas; MC, males from Coloso; FFO, fed females from Ovejas; FFC, fed females from Coloso; UFO, unfed females from Ovejas; UFC, unfed females from Coloso. Numbers at the nodes represent the percent similarity between the profiles of banding in the samples analyzed, based on migration with respect to temperature melting of ribosomal amplicons, as well the intensity and thickness of the band that symbolizes the dominance of the microbial community

Fig. 7

Neighbor-joining dendrogram of 16S rDNA fragment sequences (~450 bp) obtained from the PCR-TGGE profiles, based on the Kimura 2-parameter model. Numbers in nodes represent bootstrap values. The robustness of the phylogeny was tested by bootstrap analysis using 1000 iterations. FFO, fed females from Ovejas; FFC, fed females from Coloso; UFO, unfed females from Ovejas; UFC, unfed females from Coloso; MC, males from Coloso; MV, males from Ovejas; LO, larvae from Ovejas; PO, pupae from Ovejas

Fig. 8

Simple correspondence analysis of the intestinal bacterial flora identified with partial 16S rDNA sequences of isolates and fragment sequences (~450 bp) obtained from the PCR-TGGE profiles. Abbreviations: UFO, unfed females from Ovejas; UFC, unfed females from Coloso; FFO, fed females from Ovejas; FFC, fed females from Coloso; MC, males from Coloso; MO, males from Ovejas; PP, pupae; L4, larvae. The XLSTAT 3.04 (https://www.xlstat.com/es/) program was used to generate a simple correspondence analysis. The dots symbolize the location of the group of bacteria (consortia) associated with intestines of Lu. evansi, using ellipses of black color