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. 2026 Sep 23;10:100442. doi: 10.1016/j.crpvbd.2026.100442

Ecological origin and natural Trypanosoma cruzi infection are associated with reconfiguration of the Rhodnius pallescens midgut microbiome

Juliana Cuadros a,1, Valentina Tapia-Perdomo a,1, Carlos Nieto a, Bladimiro Rincón-Orozco a,⁎, Jonny E Duque a,b,⁎⁎
PMCID: PMC13636014  PMID: 42835511

Abstract

The intrusion of the secondary vector Rhodnius pallescens into urban and peri-urban environments in Colombia poses an emerging risk for Chagas disease transmission, compounded by rising insecticide resistance. Developing novel control strategies requires the identification of stable gut symbionts; however, high-resolution microbial profiles for urban-intruding vector populations remain poorly defined. To characterize the intestinal microbiome and identify candidate taxa for paratransgenesis, we generated high-resolution taxonomic composition and predicted functional profiles using 16S rRNA amplicon sequencing of individually processed midgut tissues from laboratory-reared and wild-caught adult R. pallescens with and without Trypanosoma cruzi infection collected in the Bucaramanga metropolitan area, Andean region, Santander, Colombia. Beta diversity analyses revealed that ecological origin and infection status significantly drive microbial restructuring (PERMANOVA P = 0.001). The transition from a sylvatic to a laboratory environment induced a severe taxonomic contraction dominated by the genus Dickeya (79.19% relative abundance). From these wild-type insects, non-infected populations maintained a balanced, functionally redundant midgut community featuring Dickeya (46.82%), Williamsia (20.6%), and Corynebacterium (16.76%). On the other hand, natural T. cruzi colonization was associated with a marked taxonomic reorganization in wild vectors, characterized by the marked expansion of Actinobacteriota, specifically Williamsia (51.22%) and Nocardia (29.99%), alongside an enrichment of predicted pathways for oxidative stress management. Therefore, we hypothesize that ecological origin and T. cruzi infection are primary determinants of microbial structure in R. pallescens. In naturally infected insects, we identify the genus Williamsia as a priority candidate for future studies aimed at evaluating its stability, cultivability, functional role, and potential use in paratransgenic strategies.

Keywords: Rhodnius pallescens, Trypanosoma cruzi, Midgut microbiome, Paratransgenesis, Vector ecology, 16S rRNA amplicon sequencing

Graphical abstract

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Highlights

  • •

    Midgut microbiomes of wild and laboratory-reared Rhodnius pallescens were evaluated.

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    Laboratory rearing induced a severe taxonomic contraction with Dickeya dominance.

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    Natural Trypanosoma cruzi infection was linked to selective Actinobacteriota blooms.

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    Williamsia emerges as a candidate bacterial genus for paratransgenic vector control.

1. Introduction

American trypanosomiasis, commonly known as Chagas disease (CD), is one of the most significant neglected tropical diseases in Latin America. Caused by Trypanosoma cruzi and primarily transmitted by triatomine insects, it affects nearly 7 million people across 21 endemic countries. According to the Pan American Health Organization (PAHO, 2025), 84.8% of cases are concentrated in five countries, Brazil, Bolivia, Argentina, Mexico, and Colombia, while the remaining 15.2% are distributed across 16 additional endemic nations (PAHO, 2025; WHO, 2025). However, it is important to emphasize that CD is no longer restricted to its historically recognized endemic areas in Latin America. Recent evidence supports the establishment of autochthonous transmission cycles and emerging endemicity in the USA, demonstrating the capacity of T. cruzi and its triatomine vectors to adapt and persist in new ecological settings favorable for disease transmission (Beatty et al., 2025; Ramírez et al., 2026).

The epidemiology of CD is closely tied to the ecology and distribution of triatomine vectors. In the Andean region, the main domiciliated species include Rhodnius prolixus, R. ecuadoriensis, Triatoma dimidiata, T. maculata, and T. infestans, together with secondary vectors such as R. pallescens, T. venosa, T. carrioni, Panstrongylus herreri, P. chinai, P. geniculatus, and P. rufotuberculatus (Guhl, 2007; Vallejo et al., 2009). In Colombia, R. prolixus has historically been the predominant domiciliary vector. Control programmes have reduced intradomiciliary infestation in departments such as Arauca, Casanare, Boyacá, Santander, and Cundinamarca, with 33 municipalities certified as free of this species (Rincón-Galvis et al., 2020).

Despite these advances, acute outbreaks of CD continue to be reported in the absence of R. prolixus, attributed to oral transmission or the involvement of secondary vectors (Díaz and González, 2014). Among these vectors, R. pallescens has increasingly been detected invading households after dispersing from sylvatic and peri-urban environments, raising the risk of domiciliation and parasite transmission (Reyes et al., 2017). This scenario, combined with emerging insecticide resistance in triatomine populations (Abad-Franch et al., 2013), highlights the need for innovative biotechnology-based approaches capable of interfering with parasite establishment and disrupting the CD transmission cycle.

Paratransgenesis, based on the manipulation of gut-associated bacteria to interfere with T. cruzi colonization, has emerged as a promising alternative strategy for disrupting CD transmission (Ratcliffe et al., 2022). However, the success of such approaches depends on detailed understanding of the intestinal microbiota across different vector species. In this context, the midgut represents a critical microenvironment, as it is the primary site where T. cruzi establishes, proliferates, and differentiates following ingestion during a blood meal (Garcia et al., 1999; Vieira et al., 2018; Ouali et al., 2021, 2024). Therefore, the composition and dynamics of the midgut microbiota may directly modulate parasite survival and development, ultimately influencing vector competence. In this context, the characterization of these microbial communities constitutes a fundamental step toward understanding vector-parasite interactions and may ultimately support the future development of microbiome-based interventions to disrupt transmission (Kieran et al., 2019).

In Colombia, studies addressing the gut microbiota of triatomines remain scarce and geographically limited, with only a few species investigated to date (Montoya-Porras et al., 2018; Arias-Giraldo et al., 2020; Murillo-Solano et al., 2021). Given the emerging epidemiological risk of Chagas disease in urban settings, this study aimed to characterize and compare the taxonomic composition and predicted functional potential of the midgut microbiota of naturally infected and non-infected R. pallescens collected in the metropolitan area of Bucaramanga. In addition, these findings were compared with microbiota profiles from laboratory-reared insects maintained under controlled conditions, offering valuable insights into differences in microbial composition between wild and laboratory-reared populations. Overall, this study highlights the relevant role of the intestinal microbiome in influencing the biology, vector competence, and epidemiological significance of R. pallescens in urban environments. Thus, our findings open new directions in the understanding of T. cruzi infection and persistence in R. pallescens within the metropolitan area of Bucaramanga.

2. Materials and methods

A comparative cross-sectional study was conducted to evaluate the taxonomic composition and predicted functional potential of the midgut microbiota of R. pallescens according to ecological origin and natural T. cruzi infection status. Two primary factors were considered: (i) ecological origin (laboratory-reared versus wild insects); and (ii) infection status within wild populations (infected versus non-infected). All analyses were restricted to adult insects to minimize ontogenetic effects on microbiome composition (Oliveira et al., 2018; Brown et al., 2020).

2.1. Biological material

2.1.1. Laboratory-reared insects

Adult Rhodnius pallescens specimens (n = 10; balanced sex ratio) were obtained from a laboratory colony maintained at the Medical Entomology Laboratory of the Center for Tropical Disease Research (CINTROP), Industrial University of Santander (UIS), Colombia. The colony was originally established from field-collected insects captured in the metropolitan area of Bucaramanga and has been propagated under controlled environmental conditions for more than seven years. Insects were routinely fed on domestic chickens (Gallus gallus) and were selected for analysis after an approximate two-month fasting period following the last blood meal to minimize transient blood meal-associated effects on gut microbial composition.

2.1.2. Wild insects

Wild adult R. pallescens specimens were collected from palm trees (Acrocomia aculeata) located in forest fragments in Bucaramanga, Floridablanca, and Piedecuesta (Santander, Colombia), where the presence of triatomine vectors had been reported. Specimens were captured using Angulo traps (40 × 28 × 32 cm) baited with a live chicken as an attractant, following previously described protocols (Angulo and Esteban, 2011). Traps were installed at 16:00 h and retrieved at 8:00 h the following day. Captured insects were transferred to sterile containers and transported to the laboratory for processing. Species identification was performed through morphological examination using standard taxonomic keys (Lent and Wygodzinsky, 1979). Only adult specimens were included in the study to minimize ontogenetic variation in microbiome composition. Wild specimens were classified according to natural T. cruzi infection status as determined by microscopy and PCR (see Section 2.4.), resulting in two experimental groups (Wild non-infected, n = 14; Wild infected, n = 3).

2.2. Dissection and extraction of the midgut

Prior to dissection, insects were stored at −70 °C. Each specimen was surface sterilized by sequential immersion in 70% ethanol followed by sterile distilled water to minimize external microbial contamination (Azambuja et al., 2004; Montoya-Porras et al., 2018). Dissections were performed under sterile conditions using scissors and straight-tip forceps disinfected with 70% ethanol and UV-irradiated for 10 min. After removing hemi-elytra and legs, the abdomen was opened from posterior to anterior to expose the digestive tract. The midgut was separated from the hindgut, and midgut tissues were used for microbiome analyses. Each midgut was transferred to a sterile microcentrifuge tube containing 180 μl Buffer ATL (DNeasy® Blood & Tissue kit; Qiagen, Hilden, Germany) and maintained on ice until DNA extraction.

2.3. DNA extraction

Total DNA was extracted using the DNeasy® Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocol with minor modifications. Midgut tissues were mechanically homogenized, incubated with 20 μl proteinase K at 56 °C for 30 min, and lysed with Buffer AL followed by ethanol addition. Lysates were purified using silica-based spin columns, washed with Buffers AW1 and AW2, and DNA was eluted in 200 μl Buffer AE. Concentration and purity were assessed using NanoDrop™ One (Thermo Scientific, Wilmington, USA), and integrity was verified by electrophoresis on 1% agarose gels stained with SYBR™ Safe. Extracted DNA was stored at −20 °C until downstream analyses.

2.4. Parasite identification

Natural infection was initially screened by microscopic examination of Giemsa-stained fecal smears (Hoyos et al., 2019). Molecular confirmation was performed by PCR targeting a ribosomal DNA fragment of T. cruzi using primers p18_Ribosomal-F (5′-ATG TTC TCT GTT CCG GCG GCA G-3′) and p18_Ribosomal-R (5′-AAA AAT CAC GGG CGC CCT CTG G-3′). PCR reactions were conducted in a T100™ Bio-Rad thermocycler under the following conditions: 98 °C for 30 s; 38 cycles of 98 °C for 10 s, 65 °C for 30 s, and 72 °C for 30 s; followed by a final extension step at 72 °C for 2 min. Amplicons were visualized on agarose gels, and samples showing the expected 953-bp amplicon were considered positive for T. cruzi DNA. This PCR assay was used exclusively for molecular confirmation of T. cruzi infection. Amplicons were not sequenced, and no DTU-specific or multilocus genotyping assays were performed; therefore, the discrete typing units of the detected parasites could not be determined.

2.5. Sequencing

Samples with DNA concentrations ≥ 10 ng/μl and purity (A260/A280 ratio 1.8–2.0) were selected for sequencing. The V3-V4 hypervariable regions of the 16S rRNA gene (470 bp) were amplified using primers 341F (5′-CCT AYG GGR BGC ASC AG-3′) and 806R (5′-GGA CTA CNN GGG TAT CTA AT-3′). Amplicon libraries were prepared and sequenced on the Illumina NovaSeq 6000 platform (Novogene, Beijing, China) to generate paired-end reads (2 × 250 bp). This region was chosen because the combination of V3 and V4 allows a more comprehensive bacterial community profile than single variable regions (Winand et al., 2020). Additionally, extraction and PCR negative controls (using sterile nuclease-free water in place of biological sample) were processed concurrently with all samples. Because these blanks exhibited undetectable DNA concentrations and no visible amplification on agarose gels, they did not meet the threshold for library preparation and were consequently not sequenced.

2.6. Data processing and statistical analysis

2.6.1. Taxonomic assignment

Raw FASTQ sequences were processed using DADA2 (v.1.26.0; Bioconductor 3.16) (Callahan et al., 2016) to perform quality filtering, dereplication, error modeling, chimera removal, and inference of amplicon sequence variants (ASVs) with single nucleotide resolution (Callahan et al., 2019). Taxonomic assignment was conducted in Quantitative Insights Into Microbial Ecology (QIIME2) software (v.2024.10) (Bolyen et al., 2019) using a naive Bayes classifier trained on the SILVA 138 reference database (Pruesse et al., 2007; Quast et al., 2013). A confidence threshold of 0.9 was applied. Sequences assigned to chloroplasts, mitochondria, or non-bacterial taxa were removed prior to downstream analyses.

Differential microbial taxa among experimental groups were identified using the linear discriminant analysis effect size (LEfSe) method (Segata et al., 2011), implemented through the microbiomeMarker R package (v.1.12.2) in RStudio (v.2024.04.2). The algorithm combines non-parametric Kruskal-Wallis tests to detect taxa with significantly different relative abundances among groups, followed by pairwise Wilcoxon rank-sum tests and linear discriminant analysis (LDA) to estimate effect sizes. Taxa with an LDA score (log10) ≥ 2.0 and P-value < 0.05 were considered significant biomarkers. LEfSe results were visualized as horizontal bar plots representing discriminative bacterial genera across groups.

Diversity analyses were conducted in RStudio (v.2024.04.2) using the vegan package (v.2.6-6) (Oksanen et al., 2022). Alpha diversity indices, including species richness, evenness, Shannon and Simpson indices (Spellerberg and Fedor, 2003; Somerfield et al., 2008), were calculated to evaluate within-sample diversity. Beta diversity was assessed using the Bray-Curtis metric (Bray and Curtis, 1957), followed by ordination analyses such as principal coordinates analysis (PCoA) (Anderson et al., 2006) and non-metric multidimensional scaling (NMDS) to visualize community composition patterns among groups. Group differences were tested with PERMANOVA (Anderson et al., 2006).

2.6.2. Functional prediction

The functional profiles of gut bacterial communities were predicted with Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) (v.2.6.2) (Douglas et al., 2020). ASV sequences were placed into reference phylogenies using EPA-NG, followed by hidden-state prediction with Castor and inference of functional profiles. Functional profiles were generated based on KEGG Orthology (KO) (Caspi et al., 2020). Predicted KOs were organized into KEGG BRITE hierarchical categories, including Metabolism, Genetic Information Processing, Environmental Information Processing, Signaling and Cellular Processes, and Cellular Processes (Kanehisa et al., 2025). Statistical comparisons of predicted functional profiles were conducted using Welch’s two-sided t-test in STAMP (v.2.1.3) (Parks et al., 2014) for pairwise comparisons, with Benjamini-Hochberg false discovery rate correction (FDR) (Benjamini and Hochberg, 1995). For multi-group comparisons, Kruskal-Wallis tests followed by Dunn’s post-hoc tests with FDR correction were performed in R (v.4.4.1).

3. Results

3.1. Gut microbiota composition

The midgut microbiota of R. pallescens was characterized across three distinct ecological and infection states. High-throughput sequencing of the 16S rRNA gene V3-V4 hypervariable regions yielded 1,410,389 high-quality reads, comprising 470,110 from the “Laboratory” group, 662,243 from the “Wild” group (without T. cruzi infection), and 278,036 from the “Wild-T. cruzi-infected” group. Following quality filtering and truncation (forward: 225 bp; reverse: 220 bp), read merging produced representative sequences of approximately 440 bp. A total of 1974 amplicon sequence variants (ASVs) were identified across all cohorts.

Alpha diversity analyses revealed no statistically significant differences in genus richness, evenness, or diversity indices among the laboratory-reared, wild, and wild-T. cruzi-infected groups (Fig. 1A). Genus richness did not vary across groups (Kruskal-Wallis, P = 0.95), and all pairwise comparisons were non-significant. Similarly, Shannon (P = 0.47) and Simpson (P = 0.18) diversity indices did not show statistically significant differences. Genus evenness was also comparable across groups (Kruskal-Wallis, P = 0.22). Furthermore, these alpha diversity patterns remained consistent even when wild populations were geographically sub-stratified by specific collection sites (Bucaramanga versus Floridablanca), with no significant compositional divergence observed between these proximate sylvatic foci (Supplementary Fig. S1).

Fig. 1.

Fig. 1

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Alpha and beta diversity of triatomine gut microbiota across experimental groups. A Alpha diversity indices (Evenness, Richness, Shannon, Simpson) for laboratory-reared (pink), wild (green), and wild-T. cruzi-infected (yellow) insects. B Beta diversity analyses based on Bray-Curtis distances: principal coordinates analysis (PCoA) (left) and non-metric multidimensional scaling (NMDS) (right).

In contrast, beta diversity analyses indicated that microbial community composition was significantly structured by ecological origin (Fig. 1B). Principal coordinates analysis (PCoA) based on Bray-Curtis dissimilarities showed clear separation among groups. The first PCoA axis explained 27% of the total variance, while the second axis explained 18.1%. Laboratory-reared insects formed a compact cluster, reflecting a relatively homogeneous microbiota. Conversely, wild samples exhibited greater inter-individual dispersion, indicating increased community heterogeneity under natural ecological conditions. PERMANOVA confirmed that these overall compositional differences were statistically significant (P = 0.001), with the grouping factor explaining 19.2% of the total variation (R2 = 0.192). Non-metric multidimensional scaling (NMDS) produced a comparable ordination pattern, yielding a stress value of 0.138. Notably, rather than forming a distinct cluster, the wild-T. cruzi-infected samples were entirely nested within the ordination space occupied by the non-infected wild individuals.

Taxonomic profiling revealed pronounced shifts in the midgut microbiota across the three study groups (Fig. 2). In laboratory-reared R. pallescens, the community was characterized by a massive dominance of Proteobacteria (79.9% relative abundance), followed by secondary contributions from Actinobacteriota (10.08%) and Firmicutes (9.2%). All remaining phyla occurred at frequencies below 1%. Genus-level analysis revealed that this Proteobacteria dominance was driven almost entirely by the genus Dickeya, which constituted 79.19% of the relative abundance in the laboratory-reared cohort. Secondary genera in this group included Nocardia (6.39%) and Williamsia (2.18%).

Fig. 2.

Fig. 2

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Relative abundance of bacterial communities at the phylum (A) and genus (B) levels in the gut microbiota of laboratory-reared, wild, and wild-T. cruzi-infected groups.

Wild R. pallescens populations displayed a significantly more balanced phylum-level profile. Proteobacteria (47.4%) and Actinobacteriota (39.5%) occurred in nearly comparable proportions, while Firmicutes represented 11.2% and Bacteroidota emerged as a distinct component at 1.29%. At the genus level, the wild non-infected group showed a reduction in Dickeya (46.82%) compared to the laboratory-reared cohort, accompanied by substantial relative abundances of the actinobacterial genera Williamsia (20.60%) and Corynebacterium (16.76%). Clostridium (sensu stricto) was also identified as a minor but consistent component (2.12%).

Natural infection with Trypanosoma cruzi was accompanied by a marked taxonomic reorganization toward Actinobacteriota dominance. In the wild-T. cruzi-infected group, Actinobacteriota expanded to constitute 82.58% of the total relative abundance, while Proteobacteria and Firmicutes declined to 10.7% and 5.7%, respectively (Fig. 2A). At the genus level, this enrichment was driven by a massive expansion of Williamsia, which reached a relative abundance of 51.22% (Fig. 2B). This result was accompanied by a substantial increase in Nocardia to 29.99%, while Dickeya further declined to 10.5%. Minor contributions in the wild-T. cruzi-infected group were observed from Staphylococcus, Lactobacillus, Blautia, Corynebacterium, and Bifidobacterium.

3.2. Microbial biomarkers associated with ecological origin and infection

Linear discriminant analysis effect size (LEfSe) between laboratory-reared and wild-caught triatomines identified distinct biomarker genera (Fig. 3A). Laboratory-reared insects were enriched in robust, generalist genera including Bacillus, Kroppenstedtia and Virgibacillus. Notably, while the genus Dickeya appeared near the lower significance threshold in the pairwise comparison (Fig. 3A), its identification as a definitive laboratory indicator is consistent with the broader three-group analysis in Supplementary Fig. S2. In this multi-group comparison, Dickeya emerges as the most significant biomarker for the laboratory-reared group. Conversely, wild insects exhibited significantly higher relative abundances of Corynebacterium, Odoribacter, and Clostridium (sensu stricto) 1 (Fig. 3A).

Fig. 3.

Fig. 3

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Differential bacterial genera identified by LEfSe among experimental groups. A Comparison between laboratory-reared and wild R. pallescens revealed bacterial genera with significantly higher relative abundances in either group. B Comparison between laboratory-reared and wild R. pallescens infected with T. cruzi. C Comparison between wild R. pallescens and wild R. pallescens infected with T. cruzi. Bars indicate the linear discriminant analysis (LDA) scores (log10) of bacterial genera significantly enriched in each group (LDA score ≥ 2.0, P < 0.05). Colors denote group-specific enrichment: laboratory-reared (pink), wild (green), and wild-T. cruzi-infected (yellow).

Within wild populations, natural T. cruzi infection was associated with distinct taxonomic signatures characterized by the enrichment of specific bacterial genera. A comparison between the laboratory baseline and wild-T. cruzi-infected individuals revealed an enrichment of Alloprevotella, Rikenellaceae RC9 gut group, Faecalibaculum, and Williamsia (Fig. 3B). Direct comparison within the wild populations isolated the impact of the parasite, identifying Williamsia and Faecalibaculum as the primary discriminative biomarkers of T. cruzi infection (Fig. 3C). Interestingly, uninfected wild bugs were characterized by higher relative abundances of Alloprevotella, Monoglobus, Muribaculaceae, and Anaerostipes compared to their infected counterparts. Due to the limited sample size of the wild-T. cruzi-infected cohort (n = 3), these LEfSe outputs represent preliminary, associative differential features of the T. cruzi-colonized state.

3.3. Predicted functional potential of the gut microbiota

Functional inference based on KEGG Orthology (KO) using PICRUSt2 revealed distinct predicted metabolic profiles across the three experimental groups. The heatmap of the 50 most abundant KOs showed that laboratory-reared insects possessed a comparatively reduced representation of predicted functional features. In contrast, wild insects exhibited a broader and more diverse set of dominant KOs (Fig. 4). Annotation of the predicted pathways according to the KEGG BRITE hierarchy confirmed that wild insects possessed the highest cumulative abundance across major functional categories, particularly in metabolism and signaling and cellular processes (Supplementary Fig. S3). Laboratory-reared insects showed lower cumulative abundance across most categories, especially within Environmental Information Processing. Wild-T. cruzi-infected insects exhibited intermediate profiles, with functional redistribution particularly evident within signaling and cellular processes.

Fig. 4.

Fig. 4

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Predicted functional profiles (KEGG Orthology, KO) in the midgut microbiota of triatomines from laboratory-reared, wild, and wild-T. cruzi-infected groups. The heatmap displays the relative abundance of significantly different KEGG Orthologs across samples, clustered by similarity. Color intensity represents standardized abundance (Z-score), with green indicating higher relative abundance and pink lower abundance. Functional categories are annotated according to KEGG BRITE hierarchies, including metabolism, cellular processes, environmental information processing, genetic information processing, and signaling pathways.

Welch’s t-tests identified specific functional enrichments that statistically distinguished the groups (Fig. 5). In the comparison between laboratory-reared and wild insects (Fig. 5A), wild bugs were significantly enriched in predicted functions related to broad metabolism, including eukaryotic-like serine/threonine-protein kinase (K12132), ABC-2 type transport system permease protein (K01992), undecaprenyl-diphosphatase (K19302), and triacylglycerol lipase (K01046). Conversely, laboratory-reared bugs exhibited an enrichment in nutrient scavenging pathways, including peptide/nickel transport system ATP-binding proteins (K02031, K02032), polar amino-acid transport system permease proteins (K02028, K02029), iron complex outer membrane receptor proteins (K02014), and acyl-CoA thioester hydrolase (K07107).

Fig. 5.

Fig. 5

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Differentially abundant predicted functional pathways of gut microbiota across triatomine groups. Welch’s test comparisons of KEGG Orthology (KO) functions are shown for laboratory-reared vs wild (A), laboratory-reared vs wild-T. cruzi-infected (B), and wild vs wild-T. cruzi-infected groups (C). Bars represent mean proportions of each functional feature with corresponding 95% confidence intervals, while points on the right indicate differences in mean proportions between groups. Corrected P-values are displayed for each feature, with statistically significant differences highlighting group-specific functional enrichments.

Infection-associated functional shifts were characterized by the computational enrichment of specific pathways in wild-T. cruzi-infected insects (Fig. 5B and C). When compared directly to uninfected wild insects, the infected cohort showed significantly higher predicted abundances of K06911 (annotated computationally as quercetin 2,3-dioxygenase) and acyl-CoA thioester hydrolase (K07107), while uninfected wild insects were enriched in methyl-accepting chemotaxis proteins (K03406) (Fig. 5C). Furthermore, infected insects showed higher representation of mobile genetic elements, such as chromosome partitioning proteins (K03496), when compared to the laboratory baseline (Fig. 5B). These pairwise functional enrichments were further corroborated by multi-group Kruskal-Wallis comparisons, which confirmed the robust differential abundance of these key metabolic and signaling pathways across the three ecological states (Supplementary Fig. S4).

3.4. Taxonomic contribution to predicted functions

Analysis of taxonomic contributions was conducted to explore how ecological origin and infection status influence the distribution of taxa associated with predicted midgut functions. Given the predictive nature of 16S-based inference and the limited infected cohort (n = 3), these represent preliminary observational trends. In laboratory-reared insects, predicted functional profiles were heavily centralized within a narrow taxonomic core, primarily dominated by the genus Dickeya, with minor participation from Nocardia, Williamsia, and Kroppenstedtia (Fig. 6). Across most KEGG BRITE categories, Dickeya provided the bulk of the predicted metabolic and signaling capacity. In contrast, wild insects exhibited a more taxonomically heterogeneous functional distribution. While Williamsia and Dickeya remained relevant, additional genera such as Corynebacterium, Clostridium (sensu stricto) 1, and Agathobacter provided notable contributions to Metabolism and Genetic Information Processing. In wild-T. cruzi-infected insects, a further redistribution of functional contribution was observed. While Williamsia and Dickeya remained prominent, there was an increased relative participation from genera such as Staphylococcus, Pseudomonas, Blautia, Enterococcus, and Fusicatenibacter. The contribution of minor taxa (those < 1% in relative abundance) also appeared proportionally elevated in specific functional classes within the infected cohort.

Fig. 6.

Fig. 6

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Taxonomic contributions to predicted functional pathways. Stacked bar plots represent the relative contribution (%) of bacterial genera to specific KEGG Orthologs (KOs) grouped by major KEGG BRITE categories across laboratory-reared, wild, and wild-T. cruzi-infected groups. Taxa with relative abundance < 1% are grouped. Due to the limited sample size of the wild-T. cruzi-infected group (n = 3), these 16S-inferred functional profiles represent preliminary, exploratory trends.

4. Discussion

The characterization of the midgut microbiota of R. pallescens revealed that ecological origin and natural infection with T. cruzi appear to be relevant determinants of microbial structure and functional potential. This study provides the first high-resolution taxonomic and predicted functional profile of the midgut microbiome of this secondary vector in the metropolitan area of Bucaramanga, highlighting a compositional shift in microbial communities during the transition from sylvatic to laboratory environments, alongside specific taxonomic enrichments associated with parasite colonization. These findings establish a baseline for understanding vector-parasite interactions in R. pallescens, which is increasingly detected invading households in urban and peri-urban areas, thereby raising the risk of non-traditional Chagas disease (CD) transmission (Reyes et al., 2017). Given the emerging insecticide resistance and the ongoing reports of acute outbreaks in Colombia (Díaz and González, 2014), identifying stable microbial components such as Dickeya and Williamsia represents a crucial step toward developing microbiome-informed or paratransgenesis-oriented control strategies (Ratcliffe et al., 2022).

The laboratory environment appeared to impose selective pressures associated with a marked simplification of the gut holobiont. Insects reared under controlled conditions were overwhelmingly dominated by the genus Dickeya (79.19%), presenting a stark reduction in overall community evenness compared to their wild counterparts. This dominance of Proteobacteria in controlled environments is a recurrent observation in triatomine research, often attributed to the homogenization of the diet and the lack of environmental microbial influx in insectaries (Rodríguez-Ruano et al., 2018). Studies on laboratory colonies of Triatoma infestans have similarly reported a simplified taxonomic structure compared to their wild counterparts, reflecting the severe selective pressure of standardized rearing (Waltmann et al., 2019). It remains to be determined whether this profound taxonomic contraction is a reversible phenotypic response to sterile insectary conditions, or if long-term domestication and inbreeding have induced an irreversible genetic shift in the host’s capacity to acquire and maintain a complex wild-type microbiome. Indeed, prolonged laboratory rearing is known to drive inbreeding and genetic drift, which can homogenize host innate immune networks, such as the Toll and immune deficiency (IMD) pathways (Mesquita et al., 2015). Because these pathways modulate the expression of antimicrobial peptides (AMPs) critical for maintaining gut microbial homeostasis, their homogenization during domestication could inherently restrict microbiome diversity and alter host-microbe interactions compared to genetically diverse, wild populations (Mesquita et al., 2015; Rodríguez-Ruano et al., 2018; Zeng et al., 2022). Even though the high prevalence of Dickeya is linked to the existence of B-vitamin biosynthetic pathways and the fulfilment of essential symbiotic roles when traditional symbionts such as Rhodococcus rhodnii are in low abundance (Costechareyre et al., 2010; Díaz et al., 2016), this massive dominance of Dickeya must be interpreted critically. Since the laboratory-reared insects in this study underwent a two-month fasting protocol prior to processing, it is highly plausible that Dickeya acted as an opportunistic survivor by using its resilience to endure severe nutrient starvation and outcompete native, blood-meal-dependent flora. Therefore, rather than representing a stable, baseline symbiosis characteristic of the laboratory-reared colony, this proliferation likely reflects an acute adaptive response to the extreme physiological stress of the rearing protocol. Furthermore, our LEfSe analysis identified Bacillus and other generalists as laboratory indicator taxa recognized for their ability to produce antimicrobial compounds and degrade complex substrates, functions frequently selected for in controlled insectary settings (Hu et al., 2020). The functional enrichment of specific transport systems (KEGG K02031, K02032) in laboratory-reared insects further mirrors previous reports stating that controlled rearing favors microbial communities specialized in efficient nutrient scavenging rather than broad metabolic capacity (Ho Viet et al., 2019).

Conversely, wild R. pallescens maintained a far more balanced and functionally redundant gut ecosystem. The wild cohort exhibited substantial relative abundances of Proteobacteria, Actinobacteriota, Firmicutes, and Bacteroidota. These findings are consistent with the microbial ecology of R. pallescens in Panama, where individuals collected from Attalea butyracea palms showed a similar broad taxonomic core (Kieran et al., 2019). The presence of Bacteroidota in wild Colombian specimens further mirrors the diversity observed in other wild South American vectors such as R. ecuadoriensis, where environmental exposure to soil and plant-associated microbes contributes to taxonomic breadth (Salcedo-Porras et al., 2020; Villacís et al., 2024). The prevalence of Williamsia as a core taxon confirms previous observations by Montoya-Porras et al. (2018), who firmly established the dominance of Actinobacteria in natural R. pallescens populations in Colombia. Furthermore, the enrichment of the anaerobes Odoribacter and Clostridium indicates a microbiota optimized for maintaining intestinal homeostasis and producing short-chain fatty acids (SCFAs), which are critical for regulating host immune responses in the wild (Hu et al., 2020). Corynebacterium is also recognized as a beneficial symbiont essential for nymphal maturation in South American vectors (Eichler and Schaub, 2002; Durvasula et al., 2008; Villacís et al., 2024). Ultimately, this complex taxonomic base drives a higher functional redundancy in natural populations, heavily contrasting the pattern of functional reduction seen in laboratory-reared colonies (Villacís et al., 2024).

The most biologically striking observation within the wild cohort was the relative abundance of the actinobacterial genera Williamsia (51.22%) and Nocardia (29.99%) in naturally infected insects with T. cruzi. While alpha diversity was not significantly associated with T. cruzi infection status, a phenomenon similarly observed in T. sanguisuga and T. dimidiata (Kieran et al., 2019), beta diversity differed significantly, matching patterns reported in wild T. infestans populations (Waltmann et al., 2019). The simultaneous enrichment of Williamsia and Nocardia supports the hypothesis that T. cruzi colonization is associated with an altered gut environment that favors certain actinomycetes. Nocardia species are known to act as primary symbionts in several Triatoma species, and their presence in high abundance during T. cruzi infection has been noted in other vectors as a potential compensatory mechanism during host stress (Eichler and Schaub, 2002; Durvasula et al., 2008). On the other hand, despite the absence of statistically significant differences in alpha diversity, this lack of variation represents a valuable biological finding; it indicates that while ecological origin and infectious pressures drive specific taxonomic enrichments, the overall species richness and carrying capacity of the midgut niche remain stable.

In contrast, our LEfSe analysis isolated specific taxa, such as Alloprevotella and Faecalibaculum, enriched in infected cohorts. Although these taxa have been associated with polysaccharide fermentation and short-chain fatty acid production in other biological systems, their role in the triatomine midgut remains unknown and requires experimental validation (He and Dong, 2023; Pi et al., 2024). Nevertheless, such SCFA-producing bacteria likely modulate the host immune environment during the initial stages of parasite colonization (Vieira et al., 2018; Castañeda et al., 2023). Similar parasite-driven shifts have been documented in Rhodnius prolixus, where the presence of trypanosomatids significantly alters the relative abundance of dominant bacterial orders, potentially establishing an environment advantageous for parasite development (Díaz et al., 2016). It remains unclear whether T. cruzi actively drives this restructuring to secure a developmental niche, or if the expansion is a secondary metabolic byproduct of altered physicochemical conditions.

From a parasite-control perspective, bacterial taxa identified in wild, naturally infected R. pallescens, such as Williamsia, Nocardia, and Corynebacterium, may represent promising candidates for future microbiota-mediated control strategies. However, translating these findings into practical applications presents a challenge for paratransgenesis, as the microbiota of insectarium-reared vectors may not fully reflect that of their wild counterparts. Therefore, candidate bacterial taxa identified or evaluated under laboratory conditions should be rigorously validated under natural or semi-natural conditions. An ideal transmission-blocking candidate should exhibit stable baseline colonization in uninfected vectors to effectively intercept initial parasite colonization (Lopez-Ordonez et al., 2018). Beyond transmission-blocking, paratransgenesis offers versatile tools for broader eco-epidemiological applications. From an entomological surveillance perspective, engineered symbionts could potentially be used to express easily detectable biomarker proteins, which could facilitate the identification of local vector populations and monitoring of their spatial distribution across complex urban-sylvatic interfaces (Ratcliffe et al., 2022). Because triatomines are highly coprophagic, engineered bacteria may spread horizontally among vector populations, potentially providing a means to investigate vector dispersal without relying exclusively on extensive trapping (Lopez-Ordonez et al., 2018). Furthermore, paratransgenesis could provide an experimental platform for elucidating intricate vector-parasite relationships. By engineering midgut bacteria to express fluorescent reporters or specific metabolic inhibitors, researchers can dynamically track T. cruzi colonization and metacyclogenesis in vivo under varying environmental stressors (Ferreira et al., 2016). Consequently, establishing a stable paratransgenic chassis in R. pallescens requires comprehensive validation, including bacterial isolation, recolonization assays, and experimental infection models, but such a system could ultimately contribute to the development of applied vector control strategies.

Functional predictions generated via PICRUSt2 suggest that the more diverse taxonomic base of wild insects may reflect a broader metabolic capacity optimized for the sylvatic environment. For instance, wild insects exhibited significantly higher abundances of triacylglycerol lipase (K01046). In triatomines like R. prolixus, midgut triacylglycerol lipases are essential for lipid metabolism and the digestion of complex blood meals (Díaz et al., 2016). The enrichment of this enzyme, alongside ABC-2 type transport systems (K01992), reflects a microbiome structured to process complex, diverse nutritional inputs from various sylvatic hosts (Ho Viet et al., 2019; Villacís et al., 2024). Conversely, the laboratory-reared cohort, which endured a two-month fasting period, showed a significant functional contraction prioritizing survival and nutrient scavenging. This is evidenced by the enrichment of peptide/nickel transport systems (K02031, K02032), polar amino-acid transporters, and iron complex outer membrane receptors (K02014) (Fig. 5A), indicating a microbial adaptation toward extreme physiological starvation and trace mineral acquisition. Another relevant property of the microbial composition and functional profile of R. pallescens found in the metropolitan area of Bucaramanga is the abundance of the genus Enterococcus. Even though this genus shows a low abundance in wild-T. cruzi-infected insects, its contribution to the pathways determined with PICRUSt2 ranks it among the most critical taxa after the prominent Williamsia and Dickeya. This finding contrasts with that of a study conducted in Belo Horizonte (Brazil) with R. prolixus, in which the genus Enterococcus was enriched itself in wild-T. cruzi-infected individuals (Eberhard et al., 2022).

The presence of T. cruzi in the midgut of wild insects might be associated with an additional functional remodeling, highlighted by the predicted enrichment of acyl-CoA thioester hydrolase (K07107) and quercetin 2,3-dioxygenase (K06911) (Fig. 5C). The increase in lipid-metabolizing enzymes like acyl-CoA thioester hydrolase may support the parasite’s high demand for host lipids during its development and metacyclogenesis in the vector gut (Díaz et al., 2016; Carrillo-Martinez et al., 2024). Furthermore, the identification of K06911 (quercetin 2,3-dioxygenase), an enzyme primarily associated with the degradation of plant-derived flavonoids, presents a biological paradox in a strictly hematophagous insect vector such as R. pallescens. Given that 16S rRNA gene-based functional predictions infer metabolic potential rather than active transcription and lack the resolution to confirm specific substrate specificity (Matchado et al., 2024), this assignment likely represents a bioinformatic artifact. In other words, this result comes from mapping our sequencing reads to homologous, generic oxidoreductases prevalent in environmental Actinobacteriota, rather than from true plant-flavonoid degradation pathways. We hypothesize that this artifactual signal is specifically driven by the marked expansion of the genus Williamsia. Species within this genus are documented to possess diverse generic oxidoreductases including catechol 2,3-dioxygenases and robust genomic pathways for neutralizing reactive oxygen species (ROS) (Le Roes-Hill and Prins, 2016). Consequently, Williamsia likely deploys these generic enzymes as a plastic metabolic response to manage the broad, systemic oxidative stress induced by T. cruzi colonization. This functional versatility in oxidative stress mitigation by the resident microbiota is likely a key determinant of vector survival and redox homeostasis under high parasitic pressure (Ouali et al., 2024; Villacís et al., 2024). Furthermore, the increased functional contribution from minor taxa (< 1% relative abundance) in infected vectors suggests that the oxidative stress associated with T. cruzi colonization may disrupt the colonization resistance typically maintained by the dominant microbiome. This environmental shift could temporarily open new metabolic niches, effectively awakening rare taxa that are equipped with specialized ROS-scavenging capabilities and other survival mechanisms (Lynch and Neufeld, 2015; Jousset et al., 2017).

The main limitation of this study is the small number of wild-T. cruzi-infected insects (n = 3), which restricts statistical power and precludes causal inference regarding the effect of T. cruzi infection on microbiome composition. Regarding the detection of T. cruzi in our samples, the PCR assay used to confirm natural infection did not allow characterization of T. cruzi discrete typing units. Therefore, parasite genetic diversity, including potential strain or DTU-dependent effects and the possible occurrence of mixed infections, represents an unmeasured variable that may have contributed to the microbiome heterogeneity observed among infected insects. Future studies should combine larger field cohorts with validated DTU-genotyping approaches and parasite-load quantification to determine whether specific parasite genotypes are associated with distinct microbiome configurations. Considering the low sample size in wild-T. cruzi-infected, infection-associated taxa and predicted functions should be interpreted as preliminary associative signatures requiring validation in larger, longitudinally sampled field cohorts. In addition, since functional profiles were inferred from 16S rRNA gene sequences, they represent predictions of metabolic potential rather than direct evidence of microbial activity; furthermore, this inference is limited by the lack of reference genomes for uncultured bacteria and Neotropical vector isolates. Although low-biomass insect microbiomes are susceptible to reagent contamination (Salter et al., 2014; Eisenhofer et al., 2019), the absence of amplification in our negative controls provides confidence that the observed taxonomic enrichments represent true biological variance rather than laboratory artifacts. Finally, physiological asymmetries between the baseline starved laboratory-reared cohort and actively foraging wild populations introduce unmeasured nutritional covariates that may influence cross-cohort metabolic comparisons. Regardless of the limitations mentioned above, this study provides an essential baseline for the vector ecology of R. pallescens at the urban-sylvatic interface in Bucaramanga. As this secondary vector increasingly bridges sylvatic pathogen cycles to domestic environments, understanding its microbial dynamics is vital. Future studies must employ shotgun metagenomics to validate the functional metabolic interplay between these actinomycetes and T. cruzi, utilizing highly powered, longitudinally sampled field cohorts to advance microbiome-based vector control interventions in endemic regions.

5. Conclusions

Our study showed that ecological origin and natural T. cruzi infection were associated with marked changes in the midgut bacterial community of R. pallescens. Laboratory-reared insects presented a simplified microbiota dominated by Dickeya, whereas wild insects maintained a more heterogeneous bacterial structure. Wild-T. cruzi-infected insects showed increased relative abundance of Actinobacteriota, particularly Williamsia and Nocardia, together with predicted functional shifts related to oxidative and lipid-associated metabolism. Although these findings identify bacterial taxa of interest for future microbiome-based vector-control studies, their functional role and paratransgenic potential require validation using larger field cohorts, bacterial isolation, shotgun metagenomics, and experimental infection models.

Ethical approval

For wild insects, Decree 1376 of June 27, 2013, was considered, which regulates the collection permit for specimens of wild species of biological diversity for non-commercial scientific research purposes. The Medical Entomology Laboratory, part of CINTROP-UIS, operates under Framework Permit for Specimen Collection No. IDB0398, issued by the National Authority of Environmental Licenses. The collection of R. pallescens was conducted following laboratory animal handling protocols and approved by the Scientific Research Ethics Committee (CEINCI) (Comité de ética en investigación científica) of the Industrial University of Santander (acronym in Spanish CEINCI), as recorded in Minutes No. 15, dated August 27, 2021. This study was approved by the CEINCI-UIS (Approval No. 4110 of 13 March 2026).

CRediT authorship contribution statement

Juliana Cuadros: Conceptualization, Investigation, Methodology, Writing - original draft. Valentina Tapia-Perdomo: Formal analysis, Investigation, Methodology, Data curation, Visualization, Writing - original draft, Writing - review & editing. Carlos Nieto: Formal analysis, Methodology, Supervision, Writing - review & editing. Bladimiro Rincón-Orozco: Conceptualization, Data curation, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing - review & editing. Jonny E. Duque: Conceptualization, Data curation, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing - review & editing.

Statement on the use of AI-assisted technologies

During the preparation of this work the authors used Google Gemini in order to improve the English language, grammar, and readability of the manuscript. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Funding

This work was supported by the Ministerio de Ciencia, Tecnología e Innovación (MinCiencias, Colombia) through the project “Soluciones tecnológicas de vanguardia para enfermedades transmitidas por vectores: CRISPR/Cas y Machine Learning” (Code 111690, Grant No. 474, 2025). This funding supported the completion of the experimental work and the final preparation of the manuscript.

Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors would like to express their gratitude to the Ministerio de Ciencia, Tecnología e Innovación (MinCiencias, Colombia) through the project “Soluciones tecnológicas de vanguardia para enfermedades transmitidas por vectores: CRISPR/Cas y Machine Learning” (Code 111690, Grant No. 474, 2025). This funding supported the completion of the experimental work and the final preparation of the manuscript. Carlos Nieto would like to thank Vicerrectoría de Investigación y Extensión (Universidad Industrial de Santander) for their support in the Postdoctoral Fellowship Programme No. 2025000168.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.crpvbd.2026.100442.

Contributor Information

Bladimiro Rincón-Orozco, Email: blrincon@uis.edu.co.

Jonny E. Duque, Email: jonedulu@uis.edu.co.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.pdf (546.3KB, pdf)

Data availability

The data supporting the conclusions of this article are included within the article and its supplementary files.

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