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Molecular ecology of Triatoma dimidiata in southern Belize reveals risk for

human infection and the local differentiation of Trypanosoma cruzi parasites

Article  in  International journal of infectious diseases: IJID: official publication of the International Society for Infectious Diseases · June 2021
DOI: 10.1016/j.ijid.2021.05.083

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 International Journal of Infectious Diseases 108 (2021) 320–329

Contents lists available at ScienceDirect

International Journal of Infectious Diseases

journal homepage: www.elsevier.com/locate/ijid

Molecular ecology of Triatoma dimidiata in southern Belize reveals

risk for human infection and the local differentiation of
Trypanosoma cruzi parasites
Roy Polonioa , Jaime López-Domínguezb,c,d , Claudia Herrerab , Eric Dumonteilb,*
a
University of Belize, Punta Gorda, Toledo, Belize
b
Department of Tropical Medicine, School of Public Health and Tropical Medicine, Vector-Borne and Infectious Disease Research Center, Tulane University,
New Orleans, LA, USA
c
LADISER Inmunología y Biología Molecular, Facultad de Ciencias Químicas, Universidad Veracruzana, Orizaba, Veracruz, Mexico
d
Centro de Investigaciones Biomédicas, Universidad Veracruzana, Xalapa, Veracruz, Mexico

A R T I C L E I N F O A B S T R A C T

Article history: Objective: In Belize, the main vector for Trypanosoma cruzi, the agent of Chagas disease, is Triatoma
Received 21 April 2021 dimidiata, but transmission cycles and the risk for human infection are unclear. Therefore, the aim of this
Received in revised form 21 May 2021 study was to identify T. dimidiata blood feeding sources and its parasite and microbial diversity, in order
Accepted 31 May 2021
to reconstruct T. cruzi parasite transmission ecology in southern Belize.
Methods: A metabarcoding approach based on deep sequencing of markers was used for bug taxonomy,
Keywords: blood meal sources, T. cruzi genotypes, and microbiota composition. Bugs were collected in 13 villages of
Hematophagous
Toledo district.
Blood meal
Transmission network
Results: Bugs fed on at least 13 species, from domestic hosts such as humans, dogs, cows, and pigs, to
Chagas disease synanthropic species such as mice, rats, and opossums, and sylvatic species such as deer, peccary, and
Microbiome kinkajou, in agreement with an opportunistic feeding behavior. Nonetheless, most feeding focused on a
Parasite diversity few species, including humans. Infection with T. cruzi was detected in 24 of 39 bugs (62%), and the
analysis of 242 T. cruzi mini-exon sequences (average 10  5 haplotypes per bug) indicated the presence
of TcI and TcIV parasite discrete typing units (DTUs). However, for both DTUs, sequences from Belize
mostly clustered apart from sequences from North and South America, suggesting the local
differentiation of parasites. T. dimidiata also harbored a diverse bacterial microbiota, with ontogenic
changes suggesting microbiota maturation during nymphal development.
Conclusions: Together, these results indicate a significant risk for T. cruzi infection in humans. They also
highlight the need to better characterize the diversity of T. cruzi strains in the region and its impact on
disease epidemiology.
© 2021 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-
nd/4.0/).

Introduction situation is unclear. A seroprevalence of 2.7% was reported in Cayo

Chagas disease is a neglected tropical disease in the Americas
caused by the vector-borne protozoan parasite Trypanosoma cruzi.
There are an estimated 6–8 million infected persons in the
continent, resulting in a burden of 29 000 000 disability-adjusted
life years (DALYs) and a health care cost of US$ 24.73 billion (Hotez
et al., 2008, 2013; Lee et al., 2013). In Belize, the epidemiological
* Corresponding author at: Department of Tropical Medicine, School of Public
Health and Tropical Medicine, Vector-Borne and Infectious Disease Research Center,
Tulane University, 1440 Canal St., New Orleans, LA 70112, USA.
E-mail address: edumonte@tulane.edu (E. Dumonteil).
district in 1967, with some of the seropositive cases presenting
cardiac arrhythmias (Coura and Petana, 1967). In 1997, a study of
several populations reported a variable seroprevalence ranging
from 0 to 6.1% (Jaramillo et al., 1997). Currently, the Ministry of
Health of Belize considers that there are no confirmed cases of
Chagas disease, and maintains a continuous screening program for
blood donors (Belize Ministry of Health, 2014).
The main vector in Belize is Triatoma dimidiata, which was first
described in the country in 1932, and found widespread in sylvatic
areas, with limited domiciliation (Petana, 1972). Seasonal infesta-
tion of houses by T. dimidiata has been detected in multiple villages
in Cayo and Toledo districts, suggesting some risk for human
transmission (Polonio et al., 2009). Such a seasonal infestation

https://doi.org/10.1016/j.ijid.2021.05.083
1201-9712/© 2021 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
R. Polonio, J. López-Domínguez, C. Herrera et al.
pattern is very similar to that observed in southern Mexico and
parts of Guatemala (Dumonteil et al., 2002, 2013; Waleckx et al.,
2015b). It is associated with a human seroprevalence of at least 1–
5% in Yucatan, Mexico (Gamboa-León et al., 2014; Buekens et al.,
2018). In addition, T. dimidiata is rather a species complex, and
several haplogroups have been described based on specific
markers, some of these potentially representing species or
subspecies (Dorn et al., 2009; Justi et al., 2018). However, the
epidemiological relevance of these taxonomic distinctions remains
unclear, as some haplogroups and their hybrids are found in
sympatry in the Yucatan Peninsula of Mexico and follow a similar
seasonal house infestation pattern (Herrera-Aguilar et al., 2009).
A high genetic diversity is also present in T. cruzi parasites,
which are currently classified into seven main discrete typing units
(DTUs) (Zingales et al., 2009, 2012). TcI was thought to predomi-
nate in Mexico (Bosseno et al., 2002), but recent studies have
detected several other DTUs (Ramos-Ligonio et al., 2012; Lopez-
Cancino et al., 2015; Dumonteil et al., 2018; Villanueva-Lizama
et al., 2019), and infections with TcII/TcV/TcVI may predominate in
humans (Herrera et al., 2019). In Belize, limited information is
available on T. cruzi diversity (Breniere et al., 2016). A study of
T. dimidiata vectors, including two from Toledo, detected TcI and
TcIV parasites, and phylogenetic analysis grouped Central and
North American TcI parasites in a single cluster (Dorn et al., 2017).
However, a significant geographic structuration of parasite strains
within DTUs is emerging (Llewellyn et al., 2009; Majeau et al.,
2021), pointing to the likely local differentiation of parasite strains,
which may be reflected in differences in transmission cycles or
pathogenesis.
These observations raise the question of the nature of T. cruzi
transmission cycles in Belize and the potential for human
infections. Therefore, the aim of this study was to identify T.
dimidiata blood feeding sources as well as its parasite and
Figure 1. Map of the study area. (A) General map of southern Mexico and part of Central America. (B) Map of Toledo district. Villages for bug collections are indicated, and the
size of each yellow circle is proportional to their population. The insert shows the location of Toledo district in Belize.
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International Journal of Infectious Diseases 108 (2021) 320–329
microbial diversity, in order to reconstruct T. cruzi parasite
transmission ecology. A metabarcoding approach was used, based
on deep sequencing of selected markers from bugs; this has proven
powerful in reconstructing the ecological interactions within these
complex parasite transmission cycles (Dumonteil et al., 2018,
2020a,b; Murillo-Solano et al., 2021).
Methods
Study area and triatomine collection
Toledo district is located in the southern part of Belize (Figure 1),
between latitude 15.8 and 16.7 North and longitude 88.3 and 89.2
West, and has a population of 35 800 inhabitants. Forty-two bugs
derived from previous collections were analyzed (Polonio et al.,
2009); these bugs were obtained from 13 rural villages across Toledo
district (Figure 1), collected inside or around houses, and included
adults and nymphs (Supplementary material Table S1).
DNA extraction, PCR amplification of markers, and sequencing
The inside of the abdomen was washed twice with 0.2 ml of
sterile water using a 1-ml syringe, and DNA was extracted from the
wash solution using the Qiagen DNeasy Blood and Tissue Kit.
Specific markers were amplified for the simultaneous identifica-
tion of T. dimidiata haplogroup, their vertebrate blood meals,
T. cruzi parasites, and gut microbiota, as described previously
(Dumonteil et al., 2018, 2020a,b). DNA extraction, PCR reaction
preparation, PCR amplification, and gel electrophoresis were
performed in separate laboratory rooms, to prevent cross-
contaminations, and all PCR reactions included positive and
negative controls. PCR products for all markers were pooled for
each bug and sequenced on a MiSeq (Illumina) platform. Raw
R. Polonio, J. López-Domínguez, C. Herrera et al. International Journal of Infectious Diseases 108 (2021) 320–329

sequence data are available from the NCBI SRA database discarded from the analysis. Phylogenetic analysis of ITS-2
under BioProject #PRJNA719290, BioSamples #SAMN18594627- sequences allowed the identification of the haplogroup present
SAMN18594665. (Figure 2). Most collected bugs belonged to haplogroup 2 (30/39),
followed by haplogroup 3 (5/39) and haplogroup 1A (1/39). Three
Sequence and data analysis bugs harbored ITS-2 sequences from haplogroups 2 and 3, and
were considered hybrids (Herrera-Aguilar et al., 2009).
Raw FASTQ sequences files were mapped to reference sequences
from each DNA marker. Sequence variants of the respective markers Blood feeding sources and parasite transmission networks
were identified using FreeBayes SNP/variant tool (Garrison and
Marth, 2012). Triatomine ITS-2 haplotypes were then aligned with Analysis of 12S RNA gene sequences allowed the identification
reference sequences from haplogroups 1A, 1B, 2, and 3 (Dorn et al., of blood feeding sources from 13 vertebrate species. These
2009) and a maximum likelihood phylogenetic tree was elaborated included domestic species such as dogs, cows, and pigs,
using PhyML as implemented in Geneious, with 100 bootstrap to synanthropic species such as mice, rats, and opossums, and
assess branch support. ITS-2 sequences were deposited in GenBank sylvatic species such as deer, peccary, and kinkajou, in agreement
under accession numbers MW856024–MW856063. with an opportunistic feeding behavior of T. dimidiata (Dumonteil
For blood meal sources, 12S sequences were analyzed by et al., 2018). Nonetheless, feeding was focused on a limited number
MegaBLAST against the ‘nr’ GenBank database. A sequence match of hosts, which accounted for the majority of blood meals. Analysis
with >97% identity was used for species/genus identification. of alpha diversity of feeding sources indicated that while male and
Sequences were further filtered to remove low abundance female bugs had a similar diversity of feeding sources, nymphs
sequences (<0.5% of reads) and normalized using the total sum tended to have a different feeding diversity, although this did not
scaling (TSS) method, to account for variability in sequencing reach statistical significance (F = 1.37, P = 0.27 and F = 1.38, P = 0.27
depth among samples. Alpha diversity (within bugs) of blood for Chao1 and Shannon indices, respectively) (Figure 3A, B).
feeding sources was assessed by calculating Chao1 and Shannon However, further comparison of feeding profiles indicated signifi-
indices, which were compared by analysis of variance (ANOVA). cant differences among nymphs and adult bugs, with nymphs
Beta diversity of feeding sources among T. dimidiata subpopula- feeding frequently on dogs rather than on humans, and adults
tions was analyzed by non-metric dimensional scaling (NMDS), feeding predominantly on humans rather than on dogs (PERMA-
based on Bray–Curtis index distance measures, and statistical NOVA; F = 4.31, P = 0.011) (Figure 3C, D). Finally, no significant
significance of differences in feeding hosts among groups was differences in blood meal sources were found among adult bugs
evaluated by permutational multivariate ANOVA (PERMANOVA). from the different ITS-2 haplogroups (PERMANOVA; F = 0.90;
Data on multiple blood meal sources in single bugs were used to P = 0.44), suggesting extensive sympatry and their involvement in a
construct feeding networks and parasite transmission pathways in common feeding network.
Cytoscape 3.8 (Dumonteil et al., 2018, 2020a,b). Multiple blood meal sources were detected in most bugs, with
Parasite sequences from the mini-exon gene were aligned with an average of 4.6  1.5 hosts per bug, confirming frequent host-
sequences from reference strains, and maximum-likelihood switching, and allowing the reconstruction of a feeding and
phylogenetic trees were constructed. Sequences were deposited parasite transmission network. The network was found to be
in the GenBank database under accession numbers MW861761– biased towards domestic species that are frequent blood meal
MW862002. Similarity among TcI sequences was further assessed
by principal component analysis using PAST 4.0 software (Hammer
et al., 2001). Statistical significance of differences among groups
was evaluated by PERMANOVA. Sequences from TcI and TcIV DTUs
were further analyzed with additional sequences from other
geographic regions. For this, only the two most frequent T. cruzi
haplotypes from each bug and DTU were included in the analyses.
Sequences were analyzed in Beast 2.6.2 (Bouckaert et al., 2019)
based on an HKY substitution model for a constant coalescent
population, and the molecular clock was set based on a
substitution frequency of 7.1  10 8 (Torres-Silva et al., 2018).
The model was run for 10 million iterations to obtain maximum
credibility trees.
For microbiota composition, bacterial 16S sequences were
analyzed using a Bayesian classifier from the Ribosomal Database
Project (Cole et al., 2014). Taxonomic identification of bacteria was
made at a threshold of >97% sequence identity of bacterial families.
Alpha diversity of the microbiota was assessed with Chao1 and
Shannon indices in Microbiome Analyst (Dhariwal et al., 2017),
which were compared by Student t-test or ANOVA. Beta diversity
was compared by NMDS, based on Bray–Curtis index distance
measures, and statistical significance of differences was evaluated
by PERMANOVA.

Results

T. dimidiata ITS-2 haplogroup diversity
From the 42 analyzed bugs, good amplification of markers and
quality sequences were obtained for 39 bugs and three were
Figure 2. Phylogenetic analysis of Triatoma dimidiata ITS-2 sequences. Partial ITS-2
sequences were analyzed using maximum likelihood, and the bootstrap branch
support is indicated. Bugs from Belize (BEL) clustered with reference sequences
from haplogroups 2, 1A, and 3. Three bugs (BEL160, BEL181, and BEL212) presented
sequences from haplogroups 2 (-A) and 3 (-B), and were considered hybrids.

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R. Polonio, J. López-Domínguez, C. Herrera et al.
Figure 3. Blood feeding profiles of Triatoma dimidiata. Chao1 (A) and Shannon (B) alpha diversity indices were compared among males, females, and nymphs from Toledo
district, but differences did not reach statistical significance (F = 1.37, P = 0.27 and F = 1.38, P = 0.27, respectively). Feeding profiles differed between adults and nymphs (C), with
a significant difference in beta diversity according to developmental stage. (D) NMDS analysis of beta diversity (PERMANOVA; F = 4.31, P = 0.011).
sources in association with humans (Figure 4), as expected for bugs
collected inside/around houses. Chickens, although present in the
feeding network, do not seem to be a major host and were the only
avian species detected. Humans were a major node in the network
and their infection may have been derived from T. cruzi strains
present in multiple domestic animals such as dogs, cats, cows, and
pigs, which are frequently associated in multiple blood meals,
confirming a significant risk for human infections. In addition,
several sylvatic species were also involved in this network,
emphasizing the connectivity between the sylvatic and domestic
habitats, in agreement with the seasonal infestation of houses by
bugs from sylvatic areas (Dumonteil et al., 2007, 2009; Polonio
et al., 2009; Barbu et al., 2010). This connectivity may allow for
strains of T. cruzi infecting sylvatic animals to reach domestic hosts
and humans.
T. cruzi parasite diversity
The diversity of T. cruzi parasite strains in T. dimidiata was
assessed by the mini-exon marker, which provides extensive
information on genetic diversity both among and within parasite
DTUs (Majeau et al., 2019, 2021). Infection with T. cruzi was
detected in 24 of 39 bugs (62%), corresponding to 19 of 30 (63%) for
ITS-2 haplogroup 2, three of five (60%) for haplogroup 3, one of
three (33%) for hybrids (haplogroups 2/3), and one of one (100%)
for haplogroup 1A. A total of 242 mini-exon sequences were
obtained, ranging from 1 to 21 sequences per infected bug (average
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International Journal of Infectious Diseases 108 (2021) 320–329
10  5 haplotypes per bug). Phylogenetic analysis of these
sequences indicated that they clustered with TcI and TcIV DTUs,
while the other parasite DTUs were not detected in these bugs
(Figure 5A). TcI sequences were detected in 24 bugs, and two bugs
presented co-infections with TcIV. Phylogenetic comparison of TcI
sequences indicated that some haplotypes were shared among
bugs, suggesting that identical/similar strains were infecting these
bugs, while other haplotypes were found in single bugs (Figure 5B).
Principal component analysis of TcI haplotypes indicated that there
was no significant difference in mini-exon sequence diversity
among T. dimidiata ITS-2 haplogroups (PERMANOVA; F = 1.34,
P = 0.21) (Figure 5C), suggesting that similar parasites circulate
among these bug subpopulations. On the other hand, significant
differences in TcI parasite haplotypes were found according to the
developmental stages of the bugs (PERMANOVA; F = 14.01,
P = 0.0001) (Figure 5D), likely in agreement with their different
feeding profiles. In addition, significant differences in TcI parasite
haplotypes were found among villages, pointing to geographic
differences among TcI strains (PERMANOVA; F = 18.79, P = 0.011)
(Figure 5E).
TcI and TcIV sequences from Belize were then compared with
sequences from other geographic regions in further phylogenetic
analyses. For TcIV sequences, these clearly differed from the South
American CanIII reference, but they also clustered separately from
sequences from the USA, indicating the local differentiation of the
TcIV strains from Belize (Figure 6A). For TcI strains, sequences from
Belize clustered in a group of sequences that mostly included
R. Polonio, J. López-Domínguez, C. Herrera et al. International Journal of Infectious Diseases 108 (2021) 320–329

Figure 4. Triatoma dimidiata blood feeding and Trypanosoma cruzi parasite transmission network. This network integrates feeding frequency on the distinct hosts and the co-
occurrence of hosts in multiple blood meals from individual bugs. The size of vertebrate hosts nodes is proportional to the frequency of blood meals on each host, and the
width of the edges connecting hosts is proportional to the frequency of their co-occurrence in single bug blood meals. Solid edges link mammalian hosts (green circles) that
are competent hosts for T. cruzi, while dotted edges link non-competent hosts (birds: blue hexagons).

sequences from Panama, although a few sequences from Colombia
and the USA were also present, pointing to the local differentiation
of TcI strains in Central America (Figure 6B). Most of the sequences
from the USA and Mexico were found in a distinct cluster,
previously identified as belonging to the TcIa subgroup, while
sequences from Colombia belonging to the TcIb subgroup formed
another cluster, as well as sequences from South America
corresponding to subgroups TcId and TcIe. These results indicate
that TcI and TcIV sequences from Belize are distinct from sequences
from North and South America.
T. dimidiata gut microbiota
Analysis of bacterial 16S sequence indicated a diverse microbial
diversity in these bugs, with over 36 microbial families detected at
a frequency of at least 0.5%. Microbial diversity tended to be lower
in nymphs compared to male and female adult bugs, as indicated
by Chao1 and Shannon diversity indices (Figure 7A and B,
respectively), but this did not reach statistical significance
(F = 1.63, P = 0.21 and F = 1.56, P = 0.23, respectively). However,
at the level of beta diversity, there was a significant difference in
microbiota composition according to the developmental stage of
the bugs (PERMANOVA; F = 1.82, P = 0.04). In nymphs, Staph-
ylococcaceae, Brevibacteriaceae, and Bacillaceae comprised the large
majority of bacteria present, while in adult males and females a
larger diversity of bacterial families was detected (Figure 7C, D).
In adult bugs, microbiota composition among ITS-2 hap-
logroups tended to be different (Figure 8A), but this did not reach
statistical significance (PERMANOVA; F = 0.55, P = 0.84), likely
because of limited sample sizes. Similarly, microbiota composition
seemed to vary according to T. cruzi infection status of ITS-2
haplogroups 2 and 3 bugs, without reaching statistical significance
(PERMANOVA; F = 0.38, P = 0.94 and F = 0.52, P = 0.9 for
haplogroups 2 and 3, respectively) (Figure 8B, C). Nonetheless,
Bacillaceae, Moraxellaceae, and Enterobacteriaceae were more
abundant in uninfected bugs from haplogroup 2 compared to
T. cruzi-infected ones, in which Nocardiaceae and Pseudomonada-
ceae were more abundant (Figure 8B). In bugs from haplogroup 3,
Staphylococcaceae, Comamonadaceae, Flavobacteriaceae, and Enter-
obacteriaceae were more abundant in uninfected bugs, while
Pseudomonadaceae were more abundant in T. cruzi-infected bugs
(Figure 8C).

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R. Polonio, J. López-Domínguez, C. Herrera et al. International Journal of Infectious Diseases 108 (2021) 320–329

Figure 5. Analysis of Trypanosoma cruzi diversity in Triatoma dimidiata. (A) Maximum likelihood phylogenetic analysis of mini-exon sequences from T. dimidiata from Toledo
district with reference sequences from multiple DTUs, which indicated the clustering of Belize sequences with TcI and TcIV DTUs. (B) Maximum likelihood analysis of TcI
sequences from Belize. Sequences are color-coded by individual bugs, which carried an average of 10  5 mini-exon haplotypes per bug. Principal component analysis of this
TcI diversity indicated that there was no significant difference in mini-exon sequence diversity among T. dimidiata ITS-2 haplogroups (C) (PERMANOVA; F = 1.34, P = 0.21).
Significant differences in TcI parasite haplotypes were found among sex/developmental stages of the bugs (D) (PERMANOVA; F = 14.01, P = 0.0001). Significant differences
were found among villages (E) (PERMANOVA; F = 18.79, P = 0.011).

Discussion domestic transmission of the parasite. The role of dogs as a major

The aim of this study was to better characterize T. cruzi
transmission cycles in southern Belize, and the potential risk for
human infection, as limited information is available on these
aspects. The metabarcoding approach used provided a detailed
snapshot of Chagas disease ecology and epidemiology in this
region.
The presence of several T. dimidiata haplogroups in the region
was first confirmed, in agreement with previous observations
(Dorn et al., 2009). However, the epidemiological relevance of their
taxonomic distinction remains questionable, as the sympatry of
these vectors seems to be associated with indistinguishable blood
feeding profiles, so that they are involved in a shared host-feeding
network. The identification of humans as a major feeding host
further indicates a significant risk for infection, which contrasts
with the lack of current reporting of human cases by the Ministry
of Health (2014). Several common domestic animal species were
found to be involved as feeding hosts and may play a role in
host in this transmission network is in agreement with observa-
tions in other regions (Cohen and Gürtler, 2001; Gurtler et al.,
2007; Dumonteil et al., 2018; Flores-Ferrer et al., 2019), and
strengthens the idea of reducing infection in dogs as part of T. cruzi
control interventions (Reithinger et al., 2005; Gurtler et al., 2009).
Furthermore, the identification of blood meals taken on sylvatic
hosts in bugs collected inside/around houses highlights the strong
connectivity between domestic and sylvatic habitats through the
dispersal of bugs (Dumonteil et al., 2007; Polonio et al., 2009;
Barbu et al., 2010) and/or the proximity of these hosts. Frequent
host-switching provides opportunities for cross-species T. cruzi
transmission among sylvatic and domestic hosts and eventually
humans. Thus the transmission network observed here in southern
Belize is similar to that observed in the Yucatan, Mexico
(Dumonteil et al., 2018).
Differences were observed in the feeding profile of nymphs and
adults, with nymphs feeding on dogs rather than humans, while
adults fed more on humans than on dogs. Such differences may

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R. Polonio, J. López-Domínguez, C. Herrera et al.
Figure 6. Geographic diversity of Trypanosoma cruzi. Maximum clade credibility trees were constructed for TcIV (A) and TcI (B) sequences. Sequences are color-coded
according to country of origin. Uncertainty in time of divergence is indicated for major nodes. Note the clustering of sequences from Belize/Central America compared to
sequences from Mexico/USA.
reflect differences in mobility, as nymphs are unable to fly. Indeed,
it has previously been found that sleeping habits in southern
Mexico could differentially affect host availability by nymphs and
adults (Waleckx et al., 2016), and nymphs may have easier access to
dogs compared to humans in southern Belize.
The analysis of T. cruzi diversity was in agreement with previous
reports identifying TcI and TcIV in bugs from Cayo and Toledo
districts (Dorn et al., 2017), as well as in T. dimidiata from
Guatemala (Orantes et al., 2018). Other DTUs found in vectors from
southern Mexico (Lopez-Cancino et al., 2015; Dumonteil et al.,
2018) were not detected so far in Belize, but due to the limited
sample size, their presence at low frequency cannot be excluded.
Sequence analysis further indicated that identical or highly similar
TcI parasites from Toledo district circulated among the distinct T.
dimidiata ITS-2 haplogroups, strengthening the conclusion that
these haplogroups are involved in a shared parasite transmission
cycle. Thus, ITS-2 haplogroups would play a similar role in the
epidemiology of T. cruzi transmission to humans, as proposed
before in the Yucatan Peninsula (Herrera-Aguilar et al., 2009).
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International Journal of Infectious Diseases 108 (2021) 320–329
Genetic differences in TcI parasites were nonetheless detected
between nymphs and adults, which may reflect their different
blood sources. The analysis of a larger sample size would help
clarify this point. Differences in TcI parasites among villages were
also observed, suggesting the local differentiation of strains. This is
further supported by the analysis of strains from a broader
geographic range, which indicated a remarkable differentiation of
both TcI and TcIV parasites from Toledo, Belize. Central American
TcI strains clustered separately from North American, as well as
from Colombian and other South American strains. This differs
from a previous study in which North and Central American TcI
parasites clustered together (Dorn et al., 2017). This discrepancy is
likely due to the use in the present study of the mini-exon marker
rather than the 18S, which provides increased resolution of within-
DTU genetic diversity of T. cruzi (Majeau et al., 2019, 2021). While a
subdivision of TcIV between North and South American clades had
been proposed (Yeo et al., 2011), the present study results suggest
further geographic substructuring of TcIV between Central and
North America. The Maya Mountains in western Toledo (Figure 1),
R. Polonio, J. López-Domínguez, C. Herrera et al. International Journal of Infectious Diseases 108 (2021) 320–329

Figure 7. Diversity of Triatoma dimidiata microbiota. Comparison of Chao1 (A) and Shannon (B) alpha diversity of the microbiota from males, females, and nymphs (F = 1.64,
P = 0.21 and F = 1.56, P = 0.22, respectively). (C) Beta diversity of microbiota composition according to sex/developmental stage. Numbers in parenthesis next to bars indicate
group sample size. (D) NMDS analysis of microbiota diversity according to sex/developmental stage (F = 1.82, P = 0.04).

which culminate at 1120 m above sea level, may serve as a natural
barrier isolating southern Belize. As noted before, T. cruzi strain
differentiation may interfere with serological diagnostic perfor-
mance (Majeau et al., 2021), and lead to variations in the clinical
presentation and progression of Chagas disease (Zingales, 2017).
The diverse microbiota identified in T. dimidiata from Belize
mirrored that found in bugs from Yucatan, Mexico (Dumonteil
et al., 2018), with Bacillales, Actinomycetales, Enterobacteriales, and
Burkholderiales accounting for the majority of the microbiota.
Nymphs presented a simplified microbial community, predomi-
nated by only Bacillales and Actinomycetales, and multiple
additional families were found in adults. This contrasts with
previous observations that also found ontogenic changes in
microbiota in T. dimidiata from Colombia, in which nymphs had
a more diverse microbiota compared to adults (Murillo Solano
et al., 2021). Nonetheless, these ontogenic changes are consistent
with a maturation of the microbiota during nymphal development
through coprophagy, but also potentially through contacts with
host skin or other environmental contamination (Eichler and
Schaub, 2002; Dumonteil et al., 2018; Rodriguez-Ruano et al.,
2018). Thus, interfering with microbiota development may provide
alternative strategies for vector control. In a similar manner, the
study data also hint at possible differences in microbiota
composition between T. cruzi-infected and uninfected bugs, but
the limited sample size did not allow clear differences to be
established. Associations between T. cruzi infection and bacterial
communities have been detected before in triatomines (Orantes
et al., 2018; Waltmann et al., 2019; Dumonteil et al., 2020a,b;
Murillo Solano et al., 2021), and may also be used to interfere with
vectorial capacity. Thus, improving our understanding of the
interactions between gut microbiota and T. cruzi in triatomines
should be key for the development of transmission-blocking
interventions.
This study has some limitations, the major ones being the
limited bug sample size and the biased sampling of bugs associated
with human housing, so that it provides only a general view of

327
R. Polonio, J. López-Domínguez, C. Herrera et al.
Figure 8. Changes in microbiota composition in Triatoma dimidiata. Microbiota
composition is compared among ITS-2 haplogroups of adult T. dimidiata (A), and
between Trypanosoma cruzi-positive and uninfected bugs from ITS-2 haplogroup 2
(B) and haplogroup 3 (C). Numbers in parenthesis next to bars indicate the group
sample size.
T. dimidiata ecology in the domestic habitat in southern Belize.
These bug populations are, however, the most relevant to assess
the risk for human infection. On the other hand, multiple other
factors, including differences in host availability among villages
and habitats (intradomicile, peridomicile, and sylvatic), may lead
to local differences in T. dimidiata ecology, which could not be
assessed here. This study nonetheless provides a valuable
framework for the further exploration of these differences.
In conclusion, this study showed that T. dimidiata from multiple
ITS-2 haplogroups are involved in a complex network of feeding
host species in southern Belize, with extensive connections
between sylvatic and domestic hosts, and a significant risk for
human infection. Thus, increased disease surveillance is needed,
and house infestation by triatomines should be targeted for control
(Waleckx et al., 2015a,b). Both TcI and TcIV parasites were
identified in these vectors, and sequence analysis showed the
local differentiation of these strains, which may interfere with
serological diagnosis (Majeau et al., 2021) and lead to variable
disease manifestations (Zingales, 2017). The analysis of triatomine
microbiota further suggested that T. cruzi parasite interactions
with bacteria should be better characterized, as this may lead to
novel transmission-blocking strategies to mitigate the impact of
Chagas disease.
Ethical approval
Not applicable.
328
International Journal of Infectious Diseases 108 (2021) 320–329
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This work was funded in part by the Louisiana Board of
Regentsthrough the Board of Regents Support Fund (#LESASF
(2018-21)-RD-A-19) and grant #632083 from Tulane University
School of Public Health and Tropical Medicineto ED. JLD was a
recipient of a Ph.D. Fellowship from CONACyT, Mexico (No.457722)
and a PROMUV Fellowship from Universidad Veracruzana, Mexico
(PROMUV-2019,S16017347). The funders had no role in the study
design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in
the online version, at doi:https://doi.org/10.1016/j.ijid.2021.05.083.
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