Luisa M. Arias Giraldo

Background Triatomines are hematophagous insects that play an important role as vectors of Trypanosoma cruzi, the causative agent of Chagas disease. These insects have adapted to multiple blood-feeding sources that can affect relevant aspects of their life-cycle and interactions, thereby influencing parasitic transmission dynamics. We conducted a characterization of the feeding sources of individuals from the primary circulating triatomine genera in Colombia using amplicon-based next-generation sequencing (NGS). Methods We used 42 triatomines collected in different departments of Colombia. DNA was extracted from the gut. The presence of T. cruzi was identified using real-time PCR, and discrete typing units (DTUs) were determined by conventional PCR. For blood-feeding source identification, PCR products of the vertebrate 12S rRNA gene were obtained and sequenced by next-generation sequencing (NGS). Blood-meal sources were inferred using blastn against a curated reference dataset containing the 12S rRNA sequences belonging to vertebrates with a distribution in South America that represent a potential feeding source for triatomine bugs. Mean and median comparison tests were performed to evaluate differences in triatomine blood-feeding sources, infection state, and geographical regions. Lastly, the inverse Simpsonʼs diversity index was calculated. Results The overall frequency of T. cruzi infection was 83.3%. TcI was found as the most predominant DTU (65.7%). A total of 67 feeding sources were detected from the analyses of approximately 7 million reads. The predominant feeding source found was Homo sapiens (76.8%), followed by birds (10.5%), artiodactyls (4.4%), and non-human primates (3.9%). There were differences among numerous feeding sources of triatomines of different species. The diversity of feeding sources also differed depending on the presence of T. cruzi. Conclusions To the best of our knowledge, this is the first study to employ amplicon-based NGS of the 12S rRNA gene to depict blood-feeding sources of multiple triatomine species collected in different regions of Colombia. Our findings report a striking read diversity that has not been reported previously. This is a powerful approach to unravel transmission dynamics at microgeographical levels.

Triatomines (Hemiptera: Reduviidae) are the insect vectors of Trypanosoma cruzi, the causative agent of Chagas disease. The gut bacterial communities affect the development of T. cruzi inside the vector, making the characterization of its composition important in the understanding of infection development. We collected 54 triatomine bugs corresponding to four genera in different departments of Colombia. DNA extraction and PCR were performed to evaluate T. cruzi presence and to determine the discrete typing unit (DTU) of the parasite. PCR products of the bacterial 16S rRNA gene were pooled and sequenced. Resulting reads were denoised and QIIME 2 was used for the identification of amplicon sequence variants (ASVs). Diversity (alpha and beta diversity) and richness analyses, Circos plots, and principal component analysis (PCA) were also performed. The overall T. cruzi infection frequency was 75.9%, with TcI being the predominant DTU. Approximately 500,000 sequences were analyzed and 27 bacterial phyla were identified. The most abundant phyla were Proteobacteria (33.9%), Actinobacteria (32.4%), Firmicutes (19.6%), and Bacteroidetes (7.6%), which together accounted for over 90% of the gut communities identified in this study. Genera were identified for these main bacterial phyla, revealing the presence of important bacteria such as Rhodococcus, Serratia, and Wolbachia. The composition of bacterial phyla in the gut of the insects was significantly different between triatomine species, whereas no significant difference was seen between the state of T. cruzi infection. We suggest further investigation with the evaluation of additional variables and a larger sample size. To our knowledge, this study is the first characterization of the gut bacterial structure of the main triatomine genera in Colombia.