https://orcid.org/0000-0003-3275-2648
Figures
Abstract
Background
The fluke Schistosoma mansoni is the causative agent of intestinal schistosomiasis, a neglected tropical disease, and remains prevalent in certain regions of Brazil. In the municipality of Sumidouro, state of Rio de Janeiro, Brazil, a low-endemic area for S. mansoni, water rats (Nectomys squamipes) are naturally infected by this trematode. The S. mansoni populations infecting humans and water-rats in Sumidouro exhibit distinct patterns of cercarial emergence (chronotypes) and phenotypic differences between hosts. Previous studies have shown that the adaptation of S. mansoni populations to human hosts (diurnal chronotype) and water rats (nocturnal chronotype) could result in prezygotic isolation. To test this hypothesis, we employed the mitochondrial cytochrome c oxidase subunit 1 gene (MT-CO1) and microsatellite loci as genetic markers.
Principal findings
We assessed the population structure between the definitive host species and geographically distant isolates collected from two endemic localities (Pamparrão–PAM and Encanto-Soledade–ENC-SOL) in Sumidouro. Additionally, we evaluated the phylogenetic relationships between S. mansoni from Sumidouro and those from other countries. Five haplotypes of the MT-CO1 gene were identified, with haplotypes 3 and 4 exclusive to ENC-SOL, and haplotypes 1, 2, and 3 were shared between humans and water rats. Haplotype 1 was also shared with other Brazilian localities, South American countries and a single locality in West Africa. The remaining haplotypes were exclusive to Sumidouro, indicating local genetic diversity. Population structure analysis revealed no genetic differentiation associated with host species but rather geographical structuring, probably due to the sedentary habits of rodents and the limited movement of humans between localities. This finding indicates that S. mansoni populations with different chronotypes are not genetically isolated and that significant gene flow occurs between them.
Author summary
The parasite Schistosoma mansoni causes intestinal schistosomiasis in humans, resulting in abdominal pain, diarrhea, and blood in the stool. In the municipality of Sumidouro, state of Rio de Janeiro, Brazil, in addition to human transmission, we found wild rodents infected by S. mansoni. In this work, we described and compared the genetic diversity of S. mansoni isolates from wild rodents and humans at two localities in Sumidouro. We did not observe genetic differences between S. mansoni isolates from rodents and humans, indicating the sharing of the parasite. However, we observed two genetic clusters corresponding to the two localities. These results reveal the importance of wild rodents in maintaining the parasite cycle in the Sumidouro region, which contributes to transmission in humans.
Citation: Varella K, Gentile R, Vilela RdV, Thiengo SC, dos Santos Moreira A, Machado-Silva JR, et al. (2025) Genetic analysis of Schistosoma mansoni in a low-transmission area in Brazil suggests population sharing between wild-hosts and humans and geographical isolation. PLoS Negl Trop Dis 19(8): e0013379. https://doi.org/10.1371/journal.pntd.0013379
Editor: Feng Xue, Zhejiang Wanli University, CHINA
Received: December 5, 2024; Accepted: July 18, 2025; Published: August 11, 2025
Copyright: © 2025 Varella et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The sequences of the five MT-CO1 gene haplotypes found for S. mansoni in Sumidouro were deposited in NCBI GenBank under accession numbers PQ738167-PQ738171.
Funding: This work was supported by the National Council for Scientific and Technological Development (CNPq) (303643/2022-6 to RG, 315844/2023-0 to AMJ), Oswaldo Cruz Institute (IOC, FIOCRUZ), and the Carlos Chagas Filho Foundation for Research Support in Rio de Janeiro (E-26/010.001597/2019 to RG, E-26/210.194/2019 to AMJ).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Schistosomiasis is a neglected tropical disease caused by digenetic trematodes of the genus Schistosoma Weiland, 1858 [1,2]. According to the World Health Organization (WHO) [3], schistosomiasis is transmitted in more than 78 countries, predominantly in tropical and subtropical regions with low socioeconomic status, inadequate access to safe water, and poor sanitation. This disease is responsible for approximately 280,000–500,000 thousand deaths annually [2] and poses a significant global health burden, with an estimated 1.75 million disability-adjusted life years (DALYs) [4].
The WHO has set a target to eliminate schistosomiasis as a public health problem by 2030 through a multifaceted approach. This includes preventive chemotherapy for risk groups, improved access to WASH (safe water, sanitation, and hygiene) and education, environmental management, and snail control. However, the transmission dynamics, disease pathology, and effectiveness of control strategies vary by geographic region and are influenced by factors such as environmental conditions, intermediate host habitat, age at infection and treatment, exposure patterns and presence of reservoir hosts [3].
In the Americas, Schistosoma mansoni Sambon, 1907, is the species responsible for intestinal schistosomiasis, with 10 countries and territories considered endemic. In northeastern Brazil and central Venezuela, approximately 25 million people are at risk [5]. Recent studies have provided evidence of the zoonotic nature of S. mansoni in West Africa [6,7], where parasite lineages are shared between humans and wild rodent reservoirs at transmission sites, potentially contributing to human reinfection following therapeutic treatment [7].
In Brazil, some wild rodent species, particularly those in the genera Nectomys Peters, 1861, and Holochilus Brandt, 1835, have been identified as natural reservoirs of S. mansoni [8–10]. These semi-aquatic rodents are highly prone to S. mansoni infection because of their habitat preferences [11–13]. The water rat Nectomys squamipes Brants, 1827, is of particular interest, as it has been found to be naturally infected with S. mansoni. This rodent has crepuscular or nocturnal habits and occurs near streams, rivers, and flooded areas [11,14].
The municipality of Sumidouro is a low-endemic area for S. mansoni, with a prevalence in humans that is close to 10%, where the rural population remains infected despite regular treatment efforts [9,13]. In this region, N. squamipes populations present high prevalence rates (≅ 30%) of S. mansoni infection across different localities [13,15–18]. These localities include small rural areas where water bodies are used for agriculture, consumption, and leisure, creating a conducive environment for transmission [13].
Schistosomiasis surveys in Sumidouro date back to 1959, with research intensifying in the 1980s to investigate the epidemiological importance of water rat populations [13,19]. Since the 1990s, multidisciplinary studies have been conducted to understand the role of N. squamipes in schistosomiasis transmission dynamics [9,13], as well as the population biology of the intermediate host snail Biomphalaria glabrata (Say, 1818) [20] and health education and treatment of the human population [21,22].
Nectomys squamipes often inhabits areas close to human dwellings [23,24], is highly susceptible to S. mansoni infection [12,15,24], and can shed viable eggs throughout its lifespan by infecting B. glabrata [8,25]. These facts contribute for maintaining high infection rates, which are often higher than those of the human population that continues to harbor the parasite [13]. Nectomys squamipes populations do not appear to suffer significant fitness costs from infection, as reproductive females are infected, and individuals exhibit high longevity with no observable impact on recruitment or survival rates due to S. mansoni infection [12]. Furthermore, the low occurrence of granulomatous inflammatory processes in infected N. squamipes is associated with hepatic lipid accumulation, which may serve as a protective mechanism against the parasite [10]. These findings highlight the role of N. squamipes as a wild reservoir for S. mansoni, contributing to the persistence of the parasite in endemic areas and facilitating transmission [12,23,24].
Chronobiological studies have shown that the cercariae emergence rhythms of S. mansoni isolates in Sumidouro are synchronized with the activity patterns of their definitive hosts [26]. Laboratory experiments with B. glabrata snails exposed to S. mansoni miracidia from naturally infected N. squamipes and humans revealed a diurnal pattern (diurnal chronotype) in human isolates and a crepuscular/nocturnal pattern (nocturnal chronotype) in rodent isolates [26]. Freire et al. [27] further observed biological differences between S. mansoni isolates from humans and rodents through experimental infection in snails, with rodent isolates showing a shorter pre-cercarial period and greater infectivity, suggesting the existence of host-specific populations.
Morphological and morphometric studies have also revealed significant phenotypic differences between adult S. mansoni from N. squamipes and those from human isolates in Sumidouro [28,29]. These findings suggest that the presence of different hosts creates environmental heterogeneity, which favors the emergence of new S. mansoni phenotypes [30].
Some researchers have proposed that the adaptation of S. mansoni populations to different hosts could lead to prezygotic isolation [31,32]. Based on the distinct chronotypes [26] and phenotypic differences [28,29] observed in S. mansoni populations infecting humans and water rats in Sumidouro, we tested the hypothesis of prezygotic isolation using the mitochondrial cytochrome c oxidase subunit 1 (MT-CO1) gene and microsatellite loci as genetic markers.
In this study, we investigated the genetic profile of S. mansoni populations from Sumidouro by considering (i) the phylogenetic relationships between S. mansoni in Sumidouro and other countries, (ii) the spatial distance between isolates, (iii) temporal variation up to 22 years of difference between samples, and (iv) representativeness of the genetic sequence data from parasite eggs compared with adult schistosomes. Given the importance of N. squamipes as a reservoir, this research will contribute to a deeper understanding of the complex epidemiology of schistosomiasis in a multi-host context and inform the development of targeted control strategies in areas where water rats are present.
Materials and methods
Ethics statement
Authorization to obtain epidemiological data and collect human biological material was granted under number CAAE: 22322619.3.0000.5248, by the Research Ethics Committee of the Oswaldo Cruz Institute–CEP/Fiocruz. The euthanasia of the rodents followed the guidelines of the Animal Care and Use Committee of the American Society of Mammalogists [33] and the Brazilian Guide to Good Practices for Euthanasia in Animals by the Ethics, Bioethics, and Animal Welfare Committee of Brazil’s Federal Council of Veterinary Medicine [34]. Permits for rodent capture and handling were issued by the Chico Mendes Institute for Biodiversity Conservation (ICMBio/SISBIO, license numbers 13373–1, 45839–1, and 63023–4); procedures were approved by the ethics committees on animal use of the Oswaldo Cruz Foundation and the Oswaldo Cruz Institute (CEUA-FIOCRUZ, license number LW-39/14; and CEUA-IOC, license number L-036/2018).
Study area
This study was conducted in three localities within the municipality of Sumidouro (22º02’59“S, 42º40’29”W) situated at an altitude of 355 meters, in the state of Rio de Janeiro, Brazil. Sumidouro is an endemic area for S. mansoni located in the Centro Fluminense mesoregion. The specific study sites included I - Encanto (22°02’37.5”S, 42°37’13.3”W), II - Soledade (22°3’10”S, 42°35’58”W) and III - Pamparrão (22°02’02.5”S, 42°38’59.1”W) (Fig 1). The local population is 15,206 inhabitants [35], and the municipality covers an area of 413,407 km². Sumidouro is located within the Piabanha Hydrographic Basin, which is part of Hydrographic Region IV and spans 4,484 km². The Piabanha Basin, along with the Paquequer and Preto sub-basins, are among the major sub-basins forming the Paraíba do Sul River.
The symbols represent the sampling sites, and the colors represent positive (red) and negative (green) hosts (humans or N. squamipes) for Schistosoma mansoni. Software: QGIS 3.22. Source: Instituto Brasileiro de Geografia e Estatística (IBGE). Continuous cartographic base (2020). Available from: https://www.ibge.gov.br/geociencias/downloads-geociencias.html?caminho=cartas_e_mapas/bases_cartograficas_continuas/bc250/versao2023/; https://www.ibge.gov.br/geociencias/downloads-geociencias.html?caminho=cartas_e_mapas/bases_cartograficas_continuas/bc25/rj/versao2018_edgv_3.0/.
In the localities of Encanto/Soledade (ENC-SOL), human stools and rodent samples were collected from 2001 to 2003 and in 2022 and 2023. In Pamparrão (PAM), human samples were collected in 2019. Only one stool sample was collected from each resident per study locality and period. Rodent samples were collected in 2021 and 2022. ENC-SOL was treated as a single locality in all analyses because they are part of the same water basin and drained by the same river, therefore, in the context of disease transmission, they cannot be considered separately. Only one specimen of N. squamipes positive for S. mansoni was collected in the Santa Cecília locality (22°01’14.3“S, 42°39’24.3”W) in Sumidouro, thus it was included only in the phylogenetic analyses.
Sampling methods of S. mansoni populations
Human stool samples were collected from residents in each locality who voluntarily participated in the research. All individuals over 18 years of age who agreed to participate by signing an informed consent form were included in the study. For those under 18 years of age, consent was obtained from parents or legal guardians by signing an informed consent form. Stool samples were collected in labeled plastic containers provided to the participants. The stool samples were analyzed using the Kato-Katz method [36], standard method recommended by WHO [3], with three slides prepared for each stool sample, as well as the Lutz method [37], with three slides per sample. The same human stool samples were subjected to both coprological methods to increase the sensitivity of the diagnosis since the studied localities are of low endemicity. The Kato-Katz method was used to quantify eggs only in human stools. All coprological examinations were performed on the day of collection. Subsequently, the sediment from the Lutz method of the positive samples was examined under a stereoscopic microscope to isolate individual parasite eggs, which were then stored in 0.85% saline solution at -20°C for molecular analyses.
To sample rodents, we established 150-meter linear transects with six transects in Pamparrão and five transects in Encanto and Soledade. Each transect contained 15 capture point stations 10 m apart. Tomahawk Live Traps (Model 201 traps 16” × 5” × 5”, Hazelhurst, Wisconsin), baited with a mixture of peanut butter, banana, oats, and bacon, were placed on the ground, along streams and flooded areas, the natural habitat of N. squamipes [11]. For a comprehensive overview of the rodent study conducted from 2001 to 2006, refer to Gentile et al. [24].
Nectomys squamipes specimens captured were identified by their external morphology and transported to a field laboratory. Subsequently, the rodents were euthanized using a lethal dose of anesthetic and subjected to perfusion of the portal-hepatic system, according to Smithers and Terry [38], to recover adult S. mansoni in the mesenteric and portal veins. The anesthetic protocols included ketamine (100 mg/mL) combined with acepromazine (10 mg/mL) at a ratio of 9:1 (dose of 0.15 mL/100 g body weight). The perfusion pressure was provided by a rotary peristaltic pump with a foot-operated switch. Following euthanasia, the abdominal and thoracic cavities of the rodents were opened, and the rib cage was removed. Next, the hepatic portal vein was incised, the perfusing needle was positioned in the left ventricle of the heart, and the pump was initiated. After all procedures, the rodents were taxidermized. Biosafety protocols and personal safety equipment were utilized during all procedures involving animal handling and biological sampling. All N. squamipes specimens were deposited at the Integrated Collection of Wild Reservoir Mammals (COLMASTO), of the Laboratory of Biology and Parasitology of Wild Reservoir Mammals (LABPMR), formally the Laboratory of Biology and Control of Schistosomiasis (LBCE).
Positive cases were confirmed by the presence of adult S. mansoni after the perfusion technique. The recovered adult schistosomes were washed in 0.85% saline solution to remove tissue debris, counted and sexed under a stereoscopic microscope, and stored in 70% ethanol solution for molecular analyses. The stools of positive rodents for S. mansoni were subjected to coprological examinations (Lutz method). The sediment of the positive samples was examined under a stereoscopic microscope to isolate parasite eggs individually with the aid of a micropipette. All coprological examinations were performed on the day of collection. Each isolated egg was placed in a Petri dish and subjected to successive washings in 0.85% saline solution to remove fecal debris and stored in the same solution at -20°C for molecular analyses. Before being subjected to molecular techniques, each egg was placed in a Petri dish and washed in autoclaved distilled water.
Genomic DNA isolation
We isolated genomic DNA from S. mansoni using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions for adult schistosomes and according to Webster et al. [39] for eggs. We used only adult schistosomes obtained from rodents and eggs obtained from humans and rodents. Before DNA isolation, each adult schistosome and egg were washed in autoclaved distilled water to remove residual ethanol or saline solution. The eggs were placed in a Petri dish with the aid of a micropipette and subjected to successive washings with autoclaved distilled water.
MT-CO1 analyses
Amplification and sequencing.
Mitochondrial cytochrome c oxidase subunit I gene (MT-CO1) partial region, with ≅ 600 bp, was amplified from 146 S. mansoni specimens, including eggs and adult schistosomes from 65 rodents, and 82 S. mansoni eggs from 20 human feces samples. Amplifications of the MT-CO1 by polymerase chain reaction (PCR) were performed individually for each adult schistosome and egg using the primer pair described by Morgan et al. [40], NHMMOR F (GCT TAG GTA GAG TAG TAT GGG GTC) and NHMMOR R (CTT AGC CAC CCA CAA CTT AG). Amplifications were performed using T100 PCR thermal cycler in a 96-well plate (Bio-Rad, Hercules, USA). Each reaction contained 12.5 μL of PCR Master Mix, 2X (Promega Corporation) (50 units/mL Taq DNA polymerase, 400 μM dATP, 400 μM dGTP, 400 μM dCTP, 400 μM dTTP, 3 mM MgCl2), 0.5 μL of each primer (10 μM), and 1 μL of genomic DNA (≅ 10–20 ng), and ultrapure water in a total volume of 25 μL. The thermal cycling conditions were 95°C for 2 min; 40 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and a final extension at 72°C for 10 min [41]. Successfully amplified amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Health care, Little Chalfont, Bucks, UK), following the manufacturer’s protocol, and cycle sequenced using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, California, USA). Cycle-sequenced product precipitation, formamide resuspension, and sequencing were conducted on the Fiocruz capillary sequencing (SANGER) platform P01-001A-RPT/FIOCRUZ (https://plataformas.fiocruz.br/). Sequencing was performed using an ABI 3730xl DNA Analyzer (Applied Biosystems).
Molecular phylogenetic and phylogeographic analyses.
The resulting DNA sequencing reads were assembled into contigs and edited for ambiguities using Geneious version 2023.0.3 [42], resulting in consensus sequences. All haplotype sequences were deposited in GenBank (accession numbers PQ738167-PQ738171).
Three MT-CO1 datasets were used in this study: (1) for the phylogenetic analysis, we used unique sequences obtained in the present study and others deposited in Genbank representing Sumidouro haplotypes, and sequences deposited in Genbank representing across the geographic distribution of S. mansoni, with Schistosoma rodhaini (accession numbers: AY446142 and AY446143) as an outgroup (S1 Table); (2) for genetic structure analysis, we used sequences of S. mansoni obtained in the present study from two studied localities (PAM and ENC-SOL); and (3) for temporal analysis (22 years apart), we used sequences obtained in the present study from the ENC-SOL locality.
For each dataset, MT-CO1 sequences were aligned using the MUSCLE algorithm [43] through Geneious. The resulting alignments were manually trimmed of poorly aligned extremities using Mesquite, version 3.81 [44]. Substitution saturation was assessed via the Xia test [45] using DAMBE, version 6.4.79 [46].
Phylogenetic analyses were performed only for the dataset that included our five haplotype sequences of S. mansoni from Sumidouro and other sequences throughout its geographic distribution on different continents and outgroups that are deposited in Genbank. Phylogenetic reconstructions using maximum likelihood (ML) as the optimality criterion were generated using the IQ-TREE, version 2.3.6 online web server [47]. Evolutionary model selection was implemented with ModelFinder [48] in IQ-TREE using the Bayesian information criterion (BIC). Node support was assessed by the SH-like approximate likelihood ratio test (SH-aLRT) [49] and ultrafast bootstrap (UFBoot) [50] after 1,000 replicates.
Bayesian inference (BI) phylogenetic analyses were performed using MrBayes, version 3.2.6 [51], executed on XSEDE through the CIPRES Science Gateway [52]. Independent GTR + I + G (general time-reversible nucleotide substitution model, with a proportion of invariable sites and a gamma distribution of rates among sites) models were used for each codon position, with unlinking of base frequencies and parameters. Markov chain Monte Carlo (MCMC) sampling was performed for 10,000,000 generations with four simultaneous chains in two runs. Node support was assessed by Bayesian posterior probabilities (BPP), which were calculated from trees sampled every 100 generations, after removing the first 25% ‘burn-in’ generations. Sampling adequacy was assessed using the program Tracer, version 1.7.2 [53], to calculate the effective sample sizes (ESSs) of the parameters. Values above 200 effectively independent samples were considered robust.
Haplotype networks were inferred only for S. mansoni sequences from Sumidouro using the program PopART, version 1.7 [54], under the median-joining method (Bandelt et al., 1999). We used DNAsp, version 5.10.1 [55], to organize S. mansoni sequences from Sumidouro into groups according to geographic locality. Additionally, using DNAsp, the genetic diversity of each group was calculated in terms of the number of haplotypes (H), number of polymorphic sites (S), haplotype diversity (Hd), and nucleotide diversity (π).
Genetic structure analyses.
Analysis of molecular variance (AMOVA) [56] and the fixation index (FST) [57] were calculated only for S. mansoni sequences from Sumidouro using the Arlequin software package, version 3.5.2.2 [58]. We used AMOVA to analyze genetic variability between and within previously defined groups and FST to measure levels of genetic differentiation between groups (between hosts, between localities and separately, considering hosts by localities). We also used the program Arlequin to assess deviation from neutrality through Tajima’s D [59] and Fu’s Fs [60] tests for each of the previously determined groups.
Discriminant analysis of principal components.
Haplotype variations between different groups (between hosts, between localities and separately, considering hosts by localities) were analyzed only for S. mansoni sequences from Sumidouro using discriminant analysis of principal components (DAPC), as exemplified in Varella et al. [61]. This analysis makes it possible to investigate variations between groups and select principal components (PCs) to explain the existing variation in the dataset [62,63]. The number of PCs to be used in the analysis was determined by a cross-validation optimization procedure [63]. The percentage of correctly classified haplotypes for their original group was also determined, and differences between groups were tested using Wilk’s Lambda statistics. DAPC was performed using the adegenet package [64], and Wilk’s lambda statistics were performed using the rrcov package [65] in the R software environment, version 4.1.3 [66].
Microsatellite analyses
Amplification and genotyping.
Seven microsatellite loci were amplified from 124 S. mansoni specimens, including eggs and adult schistosomes from 21 rodents, and 34 S. mansoni eggs from eight human feces samples. The amplification of the microsatellite alleles of seven loci via PCR was performed individually for each adult schistosome and egg of S. mansoni from Sumidouro using the primer pairs described in Table 1. Amplifications were performed using a T100 PCR thermal cycler in a 96-well plate (Bio-Rad, Hercules, USA).
The PCRs were standardized at the Fiocruz Fragment Analysis Platform, P01-001D-RPT/FIOCRUZ (https://plataformas.fiocruz.br/), and performed in two multiplex groups. For each multiplex, the forward primers were fluorescently labeled with 6-FAM, VIC, NED, and PET dyes (Applied Biosystems, Foster City, USA) (Table 1).
Each reaction contained 25 μL of PCR Master Mix, 2X (Promega Corporation) (50 units/mL Taq DNA polymerase, 400 μM dATP, 400 μM dGTP, 400 μM dCTP, 400 μM dTTP, 3 mM MgCl2), 1 μL of each primer (10 μM), and 2 μL of genomic DNA (≅ 10–20 ng), and ultrapure water in a total volume of 50 μL. The thermal cycling conditions were 95°C for 2 min; 35 cycles at 95°C for 30 s, 50°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min.
All PCR multiplex products were run with a standard size of GS500 LIZ on an ABI 3130xl Genetic Analyzer at the Fragment Analysis Platform, P01-001D-RPT/FIOCRUZ. Allele fragment lengths were quantified using the GeneMapper software, version 4.1 (Applied Biosystems).
Micro-Checker software, version 2.2.3 [67], was used to verify scoring errors, large allele dropout and the presence of null alleles. The presence of linkage disequilibrium was verified by a likelihood ratio test [68] that was performed in each group using Arlequin software package.
Genetic diversity analyses.
The number of individuals in each group and the number of alleles per locus of each group were calculated using GenAIEx, version 6.503 [69]. The observed (HO) and expected (HE) heterozygosities were calculated using Arlequin. The inbreeding coefficient (FIS) was generated using FSTAT 2.9.4 [70]. Deviations from Hardy–Weinberg equilibrium per locus for each group were estimated with GenAIEx.
Genetic structure analyses.
Three SSR datasets were used in this study: (1) for temporal analysis evaluation (22 years apart), we used microsatellite loci amplified of S. mansoni from the ENC-SOL locality between 2001–2003 and 2019–2023 periods; (2) for genetic structure analysis between definitive host species (humans and N. squamipes isolates), we used microsatellite loci amplified of S. mansoni from the two studied localities (PAM and ENC-SOL) separately; (3) for genetic structure analysis between localities (ENC-SOL and PAM), we used microsatellite loci amplified of S. mansoni using only the 2019–2023 period considering the two definitive hosts together (humans and N. squamipes).
We used AMOVA to analyze genetic variability between and within previously defined groups and FST to measure levels of genetic differentiation between groups for three datasets. AMOVA and FST were calculated using Arlequin. We examined the patterns of connectivity among S. mansoni localities (PAM and ENC-SOL) through network analysis, which was based on FST values [71] without a priori premise of genetic groups, using EDENetworks, version 2.18 [72]. The nodes on the network corresponded to the populations, and the threshold was the maximum distance considered as a link in the network. All links with distances above this threshold value were removed. The number of edges connected to a node, i.e., the degree of connectivity, indicates how strongly a population is connected to other populations. In contrast, the value of the shortest paths between other nodes passing through a node was defined as the betweenness centrality.
The program Structure, version 2.3.4 [73], which implements a Bayesian model-based clustering method for inferring population structure, was used to identify distinct genetic populations and to assign individuals to populations. We evaluated whether the genetic clusters (K) corresponded to the definitive hosts (human or water rat) or to the localities studied. We performed the runs for K = 1–10 with 10 replicates, each with 106 iterations by Markov Chain Monte Carlo (MCMC–2.5 × 105 burn-in), using correlated allele frequencies and assuming the admixture model of ancestry. To find the optimal K, we used Structure Selector, which applies the ΔK method from Evanno et al. [74]. The consensus matrix Q was generated from ten optimal K replicates and visualized in Distruct, version 1.1 [75]. Major and minor modes of clustering patterns at each K were determined using Clumpak [76].
Results
MT-CO1 analyses
Phylogenetic analysis—Sumidouro and other endemic areas of the world.
The dataset for phylogenetic analyses comprised 124 sequences of the MT-CO1 gene (555 bp), 122 of which refer to S. mansoni from Brazil and other countries, as well as two sequences from S. rodhaini as an outgroup. The localities were from Africa, represented by Kenya, Tanzania, Uganda, Zambia, Cameroon, Nigeria, Ghana, Liberia, Mali, and Senegal; the Americas, represented by Brazil, Guadeloupe, Puerto Rico and Venezuela; and Madagascar, Oman and Egypt. Xia’s test provided no evidence for substitution saturation in the data matrix for phylogenetic analyses.
The ML best-fit model chosen by ModelFinder in IQ-TREE under the BIC was TPM3 + F + G4, with four substitution rate categories and a gamma shape parameter of α = 0.1404, resulting in an ML phylogenetic tree with a lnL = -2076.7829 score. The BI MCMC sampling, after 25% ‘burn-in’, resulted in a mean estimated marginal likelihood of phylogenetic trees = –2112.8715 (standard deviation = 12.8003; median = −2112.516). The ESSs were robust for all the parameters.
The tree topologies were similar, with some variations in branch support (Fig 2; S1 Fig). Five S. mansoni clades were found. Clade 1 (UFBoot = 97.5; SH-Alrt = 99; BPP = 0.78) included sequences from Madagascar and Zambia. Clade 2 (UFBoot = 100; SH-Alrt = 100; BPP = 1) included sequences from Cameroon, Egypt and Nigeria. Clade 3 (UFBoot =95.2; SH-Alrt = 97; BPP = 0.99) included sequences from Kenya, Mali, Tanzania, and Uganda. Clade 4 (UFBoot = 100; SH-Alrt = 100; BPP = 1) included sequences from Kenya, Madagascar, Tanzania, and Zambia. Clade 5 (UFBoot = 98.4; SH-Alrt = 99; BPP = 0.91) grouped sequences from Cameroon, Ghana, Liberia, Senegal, Mali, Puerto Rico, Venezuela, Guadaloupe, Brazil and Oman.
The node values are UFBoot, SH-aLRT and BPP supports.
Among the 122 S. mansoni sequences studied, 78 haplotypes were obtained. Among them, only haplotype Hap_1, found in the municipality of Sumidouro, was shared with other localities in Brazil, such as the states of Minas Gerais, Pernambuco, Ceará and Pará; in the Americas, such as Venezuela and Puerto Rico; and in West Africa, such as Liberia. The other haplotypes (Hap_2 to Hap_5) were exclusive to Sumidouro, indicating their local diversity. Haplotype 5 was represented by an S. mansoni specimen from N. squamipes collected in the Santa Cecília locality, so this haplotype was not included in the population genetic analysis of Sumidouro.
Genetic diversity of Sumidouro.
We obtained MT-CO1 gene consensus sequences from 243 S. mansoni specimens, including eggs and adult schistosomes (S2 Table; Fig 3). We built two different datasets: the first dataset included all sequences of different localities, resulting in 243 taxa and 555 bp, and the second dataset for temporal analysis included only sequences of ENC-SOL, resulting in 139 taxa and 555 bp.
Illustration from NIAID NIH BIOART Source (https://bioart.niaid.nih.gov/bioart/54) and Wikimedia Commons Source (https://commons.wikimedia.org/wiki/File:Schistosoma_mansoni_female.png; https://commons.wikimedia.org/wiki/File:Schistosoma_mansoni_egg_(01).png; https://commons.wikimedia.org/wiki/File:Man_(13779)_-_The_Noun_Project.svg).
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Haplotype network of the PAM and ENC-SOL localities.
Among 243 partial MT-CO1 gene sequences (555 bp) of S. mansoni, we identified four haplotypes with three polymorphic sites, separated by genetic distances of one mutational step. The results of the neutrality deviation tests were not significant, indicating that, this genetic marker, populations are not undergoing population expansion or retraction (Table 2). We observed haplotypic sharing between humans and rodents. Haplotype 1 (Hap_1) was found in all the studied localities and was predominant in ENC-SOL. Haplotype 2 (Hap_2) was found in the localities of PAM and ENC-SOL. In the latter locality, this haplotype was found in only three different sources (two eggs and one adult schistosome) recovered from three N. squamipes individuals. Haplotypes 3 and 4 (Hap_3 and Hap_4) were found only in ENC-SOL. Hap_3 was found both in humans and rodents, and Hap_4 was found only in humans (Fig 4).
Circle sizes are proportional to haplotype frequencies, and colors represent the definitive hosts of each geographical locality in which haplotypes occur. Each hatch mark along the branches represents one mutation at a step separating haplotypes.
Population structure between localities.
Considering the fixation indices for different localities, the FST values revealed significant genetic differences between localities when each host type was considered separately. Between rodents and humans within and between localities, significant FST values were observed in all comparisons, except between humans and rodents in ENC-SOL (Table 3).
The AMOVA revealed the greatest variation between localities, which represented 76.41% of the total variation; however, it was not significant (FCT = 0.7636, p = 0.345). The genetic variation between humans and rodents within localities was only 1.52% (FSC = 0.06495, p = 0.006). The genetic variation between humans and rodents from different localities was 21.85% (FST = 0.78146, p = < 0.01). Considering the comparisons between the dataset of eggs and S. mansoni adult schistosomes recovered from rodents, the FST values and the AMOVA results revealed no significant genetic differences at any locality.
Haplotype network and population structure of the ENC-SOL locality: temporal analysis.
When performing a temporal analysis with only S. mansoni specimens from the ENC-SOL locality, we found the same haplotypes recovered for the two localities together (PAM and ENC-SOL) (Fig 5). There was no difference in haplotype frequency across collections (22 years apart). Considering the fixation index for the ENC-SOL dataset, the FST values did not reveal genetic differences between S. mansoni specimens at a 22-year interval in the ENC-SOL locality. The AMOVA results revealed greater variation within each sampling period, which represented 97.47% of the total variation (FST = 0.03534, p = 0.07).
Circle sizes are proportional to haplotype frequencies; colors represent the definitive hosts. Each hatch mark along the branch haplotypes represents one mutation at a step separating the haplotypes.
Discriminant analysis of principal components.
In the DAPC for localities, two PCs were retained, explaining approximately 99% of the data. All the haplotypes were correctly classified into their original group. Variation in haplotype frequency between localities was observed (Wilks’ Lambda = 0.19, df = 4, p < 0.01), with low overlap between the PAM and ENC-SOL localities (Fig 6A). The haplotype that best discriminated the localities was Hap_2 (Fig 6B).
Regarding hosts, similar to the DAPC for localities, two PCs were retained in the analysis, explaining approximately 99% of the variation in the data. The proportion of haplotypes classified correctly to their original group was approximately 50%. No variation in haplotype frequency between hosts was observed (Wilks’ Lambda = 0.86, df = 4, p = 0.22), with a marked overlap observed between humans and rodents (Fig 7A). The haplotype that best discriminated the hosts was Hap_1 (Fig 7B).
When considering both hosts and localities, two PCs were retained in the DAPC, similar to the previous analyses, representing approximately 99% of the data variation. The proportion of haplotypes classified correctly to their original group was approximately 57%. Variation in the haplotype frequency between hosts in each locality was observed (Wilks’ Lambda = 0.14, df = 12, p < 0.01), with a greater difference in the haplotype frequency of humans in the PAM than in that of rodents in the same locality and in ENC-SOL (Fig 8A). However, it should be taken into account that the sample number of infrapopulations from humans in the PAM was lower than the number of infrapopulations from rodents (S2 Table). There was also low overlap in the frequency of schistosome haplotypes from rodents between PAM and ENC-SOL, with only one rodent from ENC-SOL having a haplotype frequency highly similar to that of schistosomes collected from rodents in the PAM. Schistosomes collected from humans in ENC-SOL presented high similarity in haplotype frequency with those collected from rodents in this locality but low similarity in haplotype frequency with schistosomes collected from rodents in the PAM. Haplotypes 1 and 2 were considered the haplotypes that best discriminated the studied groups (Fig 8B).
Microsatellite analyses
Genetic diversity.
Seven microsatellite loci were used for genotyping 158 S. mansoni specimens, among adult schistosomes and egg samples (S3 Table; Fig 9). Null alleles were detected for locus 15J15 only in the ENC-SOL rodent group, for locus SM13–478 in all groups and for locus SMMS16 in the PAM human and ENC-SOL rodent groups. We detected significant linkage disequilibrium between some pairs of loci (S4 Table) and observed deviation from HWE in some loci in all studied groups (S5 Table). The descriptive statistics of the seven microsatellite loci of Schistosoma mansoni are presented in Table 4. All the loci were polymorphic, and the number of alleles ranged from 3 to 13 (Table 4). The FIS values were positive in all the groups studied, except in the Human PAM group (Table 4).
Illustration from NIAID NIH BIOART Source (https://bioart.niaid.nih.gov/bioart/54) and Wikimedia Commons Source (https://commons.wikimedia.org/wiki/File:Schistosoma_mansoni_female.png; https://commons.wikimedia.org/wiki/File:Schistosoma_mansoni_egg_(01).png; https://commons.wikimedia.org/wiki/File:Man_(13779)_-_The_Noun_Project.svg).
Population structure between localities.
Considering the fixation index for the different localities, the microsatellite FST values revealed significant genetic differences in all pairwise comparisons (p < 0.01) (Table 5). The AMOVA results revealed that the greatest variation observed was between humans and rodents from different localities, which represented 87.89% of the total variation (FST = 0.121, p < 0.01). The genetic variation between localities was 9.44% (FCT = 0.094; p < 0.01). The genetic variation between humans and rodents within localities was 2.66% (FSC = 0.03; p < 0.01). Considering the fixation index between localities in the 2019–2023 period, gathering the data of the two definitive hosts, the FST value revealed significant genetic differences between localities (FST = 0.15885; p = < 0.01).
EDENetwork analysis revealed thicker edges in the network between the Rodent PAM and Human PAM groups and between the Rodent ENC-SOL and Human ENC-SOL groups, denoting a connectedness between these groups (Fig 10C). Conversely, the network revealed narrow edges between the PAM and ENC-SOL groups, suggesting restricted gene flow between these two localities (Fig 10C).
Bayesian analysis of population structure clustered individuals from the four studied groups of S. mansoni into two clusters. The ΔK method revealed two clusters (Fig 10B) as the most likely population structure, with individuals clustered according to these localities (PAM and ENC-SOL) (Fig 10A). Structure cluster graphs of the studied groups of S. mansoni in Sumidouro, with minor and major modes for K = 3–10, are presented in the (S2 Fig.).
Population structure of the ENC-SOL locality: temporal analysis.
Considering the fixation index for the ENC-SOL dataset, the FST value revealed a very low genetic difference between S. mansoni specimens over a 22-year interval in the ENC-SOL locality (FST = 0.04045, p = < 0.01) indicating no genetic structure over time.
Discussion
Phylogenetic relationships between Sumidouro and other endemic areas
Phylogenetic analyses of MT-CO1 suggest that the evolutionary origin of S. mansoni lies in East Africa, where the greatest genetic diversity is observed [40,80]. The phylogenetic reconstructions that include our S. mansoni sequences from Sumidouro and sequences deposited in Genbank corroborate these hypotheses, identifying five lineages associated with the geographic distribution of samples without associations with specific host species. The data support the hypotheses that S. mansoni was introduced into South America via the transatlantic slave trade between the 15th and 19th centuries, with haplotypes from Sumidouro closely related to those from West Africa [40,80]. Hap_1, found in Sumidouro, was shared with other South American localities and a single West African (Liberia) locality. The absence of Hap_1 in other African localities may reflect genetic differentiation due to low gene flow between African and South American S. mansoni populations or local differentiation of other haplotypes in Africa. However, most of the sequences obtained from these locations were from strains kept in the laboratory, which have low genetic diversity and may justify haplotype sharing [40]. Additionally, four haplotypes unique to Sumidouro were identified, with three being less frequent. Studies suggest that rare haplotypes may result from local differentiation [80,81].
Although new haplotypes have been identified in Sumidouro, genetic diversity is still low in Brazil compared with the genetic diversity rates observed in African countries [40,80]. This low diversity has been attributed to the founder effect resulting from the recent introduction of S. mansoni into South America [40].
Population structure
This study investigated the population structure of S. mansoni in the municipality of Sumidouro, focusing on two endemic localities. The aim of this study was to assess genetic differentiation between the definitive hosts (humans and water-rats) using the MT-CO1 gene and seven microsatellite loci. The genetic structure analyses, which were based on both the MT-CO1 gene and microsatellite loci used, were consistent and indicated an absence of isolation between S. mansoni populations from humans and water rats. This was evidenced by haplotypic sharing between isolates from the two host species, suggesting multi-host transmission in this region. These findings support the role of N. squamipes in maintaining the parasite life cycle, which contributes to human infections.
The results indicated significant gene flow between S. mansoni populations in different hosts, with no isolation observed between chronotypes. Among the 243 individuals sequenced for the MT-CO1 gene, four haplotypes were identified in the PAM and ENC-SOL localities, with haplotypes 1, 2, and 3 being shared between humans and water-rats and haplotype 4 being found only in humans. Haplotypes 3 and 4 were exclusive to the ENC-SOL locality, whereas haplotypes 1 and 2 occurred in both localities. AMOVA of the MT-CO1 gene and microsatellite loci revealed low genetic variation between definitive hosts within each locality. The consumption of the intermediate host, the snail Biomphalaria glabrata, by rodents, as observed in laboratory experiments, may contribute to parasite homogenization across chronotypes in the definitive host.
However, when comparing hosts from different localities, genetic differentiation was detected through DAPC and FST analyses, with significant values indicating a geographical structure in the S. mansoni populations. Bayesian population structure analysis confirmed the existence of two distinct genetic groups based on locality, with minimal admixture between them.
In ENC-SOL, the FST values were low for both genetic markers between humans and rodents. Similarly, the PAM showed significant, although low, FST values, suggesting gene flow between human and rodent S. mansoni populations. DAPC analysis further supported these findings, showing no variation in haplotypes between humans and rodents within localities. Temporal analysis over a 22-year interval in ENC-SOL did not indicate population differentiation, suggesting that the observed geographic structure between ENC-SOL and PAM was not due to temporal factors.
A study by Catalano et al. [7] in Senegal, West Africa, demonstrated the zoonotic nature of S. mansoni by showing haplotype sharing between humans and wild rodents, similar to the present findings in Sumidouro. Both studies support the role of wild rodents as reservoirs, contributing to transmission in these different regions. Similarly, Long et al. [82] identified genetic structure among S. mansoni populations in human communities in Bahia, Brazil, with limited gene flow between isolated populations. These findings align with our study, where geographic proximity did not prevent low gene flow, likely because of human movement between localities rather than rodent dispersal, which is confined to riverbanks [11,83]. In contrast, Standley et al. [81] did not observe geographic genetic structure among S. mansoni isolates from humans across Lake Victoria in Kenya, Tanzania, and Uganda, attributing the lack of differentiation to human migration, which homogenized S. mansoni populations along the lake’s shorelines.
No genetic differences were detected between the egg and adult stages of S. mansoni, confirming that eggs accurately represent the gene pool of the parasite infrapopulations. This is consistent with findings by Shrivastava et al. [84], who reported no genetic differentiation between larval and adult stages in S. japonicum from wild rodents.
Although microsatellite markers used in this study and others discussed here allow inferences about fine-scale kinship patterns among schistosomes, whole-genomic approaches have been used to improve the resolution of evolutionary aspects and population structure of schistosomes [85–87]. These population genomic studies involving schistosomes have provided a more detailed resolution of the population structure of these parasites, clarifying the transmission patterns and the response to the treatment with praziquantel because it is possible to estimate the degree of kinship between larval stages of the parasite between hosts and among transmission sites. This genomic approach highlighted hotspots of persistent transmission and reemergence of the disease in endemic areas where mass drug administration programs are adopted [86–88]. In addition, the presence of wild reservoir hosts highlights attention for studying the genetic variation in S. mansoni populations that may be associated with different chronotypes from humans and wild reservoir [88].
In conclusion, this study confirmed that wild rodents, particularly N. squamipes, contribute to the maintenance of the S. mansoni cycle in Sumidouro, acting as a wild reservoir of schistosomiasis. Despite possible pre-zygotic isolation, no genetic evidence of host isolation was observed.
These results highlight the importance of considering rodents in disease control programs and support the adoption of the One Health approach. The presence of infected wild rodents in endemic areas should be further investigated, as they can serve as indicators of local transmission hotspots.
Supporting information
S1 Fig. Maximum likelihood phylogenetic tree of Schistosoma mansoni partial MT-CO1 sequences from this study from GenBank and the outgroup.
The node values are UFBoot, SH-aLRT and BPP supports.
https://doi.org/10.1371/journal.pntd.0013379.s001
(TIF)
S2 Fig. Structure cluster graphs of three Schistosoma mansoni groups in Sumidouro with minor and major modes for the data.
The runs were performed for K = 1–10 with 10 replicates, and division for runs by mode is provided below the graphs.
https://doi.org/10.1371/journal.pntd.0013379.s002
(TIF)
S1 Table. GenBank accession number, geographical locality, host, and reference sequences of the GenBank MT-CO1 partial sequences of Schistosoma mansoni and the outgroup (Schistosoma rodhaini) used in this study.
https://doi.org/10.1371/journal.pntd.0013379.s003
(DOCX)
S2 Table. Number of MT-CO1 sequences of Schistosoma mansoni eggs and adult schistosomes collected from Nectomys squamipes and Schistosoma mansoni eggs collected from human feces over time at each locality in Sumidouro, Rio de Janeiro state, Brazil.
https://doi.org/10.1371/journal.pntd.0013379.s004
(DOCX)
S3 Table. Number of genotyping Schistosoma mansoni specimens for seven microsatellite loci among eggs and adult schistosomes collected from Nectomys squamipes and Schistosoma mansoni eggs collected from human feces over time at each locality in Sumidouro, Rio de Janeiro state, Brazil.
https://doi.org/10.1371/journal.pntd.0013379.s005
(DOCX)
S4 Table. Probability of linkage disequilibrium for pairs of loci per population of Schistosoma mansoni generated by Arlequin 3.5.2.2.
https://doi.org/10.1371/journal.pntd.0013379.s006
(DOCX)
S5 Table. Results of the Hardy–Weinberg equilibrium (HWE) test evaluated with GenAIEx version 6.503 using chi-square test.
https://doi.org/10.1371/journal.pntd.0013379.s007
(DOCX)
Acknowledgments
We would like to thank countless professionals and students of the Laboratory of Biology and Parasitology of Wild Reservoir Mammals at the Oswaldo Cruz Foundation who helped in the field work during the 1990s and 2021 collections.
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