Introduction
Diplostomids represent one of the most diverse groups of parasitic digeneans worldwide, including approximately 370 species distributed across 42 genera (Achatz et al. Reference Achatz, Von Holten, Kipp, Fecchio, LaFond, Greiman and Tkach2023; Blasco-Costa and Locke Reference Blasco-Costa and Locke2017; Dubois Reference Dubois1937, Reference Dubois1970; GBIF 2023a; Heneberg et al. Reference Heneberg, Sitko and Těšínský2020; Shoop Reference Shoop1989; Travassos et al. Reference Travassos, Teixeira de Freitas and Kohn1969). The genus Neodiplostomum Railliet, 1919, includes approximately 73 species of digeneans that parasitize the gastrointestinal tract of their hosts (Achatz et al. Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022a; Cribb and Pearson Reference Cribb and Pearson1993; Dubois Reference Dubois1937, Reference Dubois1983; GBIF 2023b; Izrailskaia and Besprozvannykh Reference Izrailskaia and Besprozvannykh2023; Noble Reference Noble1936). Recent phylogenetic studies have revealed that Neodiplostomum is not a monophyletic group (Achatz et al. Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022a; Izrailskaia and Besprozvannykh Reference Izrailskaia and Besprozvannykh2023).
Although the majority of species in this genus are found in birds, some have also been described in mammals and, occasionally, in reptiles (Achatz et al. Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022a; Bisseru Reference Bisseru1957; Dubois Reference Dubois1982; Lunaschi and Drago Reference Lunaschi and Drago2005). Sixteen species have been reported as infecting mammals of the orders Rodentia, Carnivora, Didelphimorphia, Dasyuromorphia, Eulitophyla, Primates, and Chiroptera (Achatz et al. Reference Achatz, Chermak, Martens, Woodyard, Rosser, Pulis, Weinstein, Kinsella and Tkach2022b; Asakawa Reference Asakawa1989; Beveridge and Spratt Reference Beveridge and Spratt1996; Chai and Shin Reference Chai and Shin2002; Ching et al. Reference Ching, Leighton and Stephen2000; Cribb and Pearson Reference Cribb and Pearson1993; Dubois Reference Dubois1983; Pfaffenberger and de Bruin Reference Pfaffenberger and de Bruin1988; Smales and Cribb Reference Smales and Cribb1997; Spratt et al. Reference Spratt, Beveridge and Walter1991; Tkach Reference Tkach1993). Among these species, Neodiplostomum vaucheri Dubois, Reference Dubois1983 is the only one known to parasitize a bat host. Neodiplostomum vaucheri was originally described from a single specimen found infecting the woolly false vampire bat Chrotopterus auritus (Peters, 1856) in the Pacaya-Samiria National Reserve, Peru (Southwestern Amazonia) (Dubois Reference Dubois1983). Recently, this species was reported parasitizing the fringe-lipped bat Trachops cirrhosus (Spix, 1823) in Ecuador (Northwestern Amazonia) (Achatz et al. Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022a).
Although Oxford Nanopore Technologies (ONT) has traditionally been applied to long-read sequencing tasks – such as whole-genome assembly and structural variant detection – recent studies have highlighted its potential for amplicon-based sequencing strategies (Patiño et al. Reference Patiño, Ballesteros, Muñoz, Jaime, Castillo-castañeda, Madingan, Paniz-Mondolfi and Ramíez2023). This approach, which involves the targeted amplification of specific genetic markers via PCR, offers a cost-effective and portable alternative for molecular identification. Amplicon-based ONT sequencing has proven effective across a broad range of organisms, particularly for distinguishing closely related taxa (Maia de Souza et al. Reference Maia de Souza, Ruedas Martins, Moyses Franco, Tuon, de Oliveira Junior, Maia da Silva, Imamura and Amato2022; Patiño et al. Reference Patiño, Ballesteros, Muñoz, Jaime, Castillo-castañeda, Madingan, Paniz-Mondolfi and Ramíez2023). The versatility of the ONT platform enables its deployment not only in well-equipped laboratories but also under field conditions or in resource-limited settings. As a result, it represents a valuable tool for molecular surveillance, phylogenetic inference, and biodiversity monitoring in both clinical and ecological contexts.
During a parasitological study of phyllostomid bats at the Kawsay Biological Station in the Madre de Dios region of Peru, diplostomid digeneans were recovered from the small intestine of T. cirrhosus. A detailed morphological comparison between our specimens and the holotype of N. vaucheri confirms their conspecificity. In this study, we provide new data on surface ultrastructure of N. vaucheri using scanning electron microscopy (SEM), along with revised morphometric measurements and illustrations. Furthermore, we present the genetic characterization of this species using a partial sequence of the mitochondrial cytochrome c oxidase subunit I (cox1) gene, obtained through an amplicon-based sequencing approach using the MinION™ platform (Oxford Nanopore Technologies-ONT). This third-generation sequencing method proved effective in generating high-confidence consensus sequences, reinforcing its value for molecular taxonomy and phylogenetic analysis of helminths. To our knowledge, this represents the first application of ONT sequencing in digeneans, highlighting the novelty and potential of this approach for studying this group of parasitic flatworms.
Materials and methods
Collection, processing, and morphological analyses of parasites
In September 2023, two adult individuals of T. cirrhosus were captured using mist nets at the Kawsay Biological Station in Madre de Dios region (12°31′S, 69°W), Peru. The bats were handled, identified in situ by a specialist, and euthanized following Sikes (Reference Sikes2016). The bat collection was authorized by the Servicio Nacional Forestal y de Fauna Silvestre (SERFOR) under Directorial Resolution N°D000011-2024-MIDAGRI-SERFOR-DGGSPFFS-DGSPFS. For parasitological examination, the guts were removed, placed in a petri dish with 0.9% saline solution, and examined under a stereoscopic microscope. The digeneans found were transferred to petri dishes with 0.9% saline solution to be cleaned of organic residues and mucus, fixed with hot 70% ethanol for staining, hot 4% formaldehyde for SEM, and 95% ethanol for molecular analysis. In the laboratory, the digeneans were stained with iron–acetocarmine and Meyer’s paracarmine, dehydrated through a graded ethanol series, cleared in Eugenol, and mounted in Entellan. The specimens were examined and photographed using a NikonTM Eclipse SI compound photomicroscope, and drawings were made with the aid of a drawing tube. Measurements were taken in micrometers, unless otherwise indicated; straight-line distances between extreme points of the structures measured and are expressed as the range, followed by the mean and the number (n) of structures measured in parentheses. Anatomical terms (prosoma and opisthosoma) for diplostomids follow Tkach et al. (Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and de Vasconcelos Melo2020) and Achatz et al. (Reference Achatz, Martens, Kostadinova, Pulis, Orlofske, Bell, Fecchio, Oyarzún-Ruiz, Syrota and Tkach2022c). For SEM observation, specimens were post-fixed in 10% formaldehyde, dehydrated through a graded ethanol series, critical-point dried with carbon dioxide, coated with gold, and examined using an Inspect S50-FEI at an accelerating voltage of 5kV to 10kV. High-resolution images and videos of the holotype of N. vaucheri (MHNG–980.537) from the Muséum d’Histoire Naturelle in Geneva, provided by Dr. Isabel Blasco Costa, were examined. Voucher specimens were deposited in the Helminthological Collection of the Museum of Natural History at the San Marcos University (MUSM-HEL), Lima, Peru.
Genetic procedures
Genomic DNA of the digeneans was isolated using an Extracta DNA Prep for PCR kit (Quantabio, USA) following the manufacturer’s protocol. The mitochondrial DNA cytochrome c oxidase subunit I gene (cox1, partial fragment) was amplified using the primers JB3 (5′-TTT TTT GGG CAT CCT GAG GTT TAT-3′) and JB4.5 (5′-AAA GAA AGA ACA TAA TGA AAA TG-3′) (Bowles et al. Reference Bowles, Blair and McManus1992). PCR assays were carried out in a total volume of 25 μl containing 0.125 μl of GoTaq® G2 Flexi DNA Polymerase (5 u/μl concentration), 5 μl of Green GoTaq® Flexi Buffer (5X concentration), 0.5 μl of dNTPs (10 nM concentration), 7.5 μl of 2× GoTaq® Colorless Master Mix (Promega), 3 μl MgCl2+ (25 mM concentration), 0.5 μl of each primers with final concentration at 10 μM, 3.0 μl of DNA sample and ultrapure water to complete. PCR amplification was carried out using an annealing temperature of 55°C. Amplified PCR products were purified with sparQ PureMag Beads (Quantabio, USA) following the manufacturer’s instructions and sequenced in Umbrella Genomic Company (Lima, Peru) with the Oxford Nanopore Technology (ONT).
Bioinformatics
Raw POD5 data were basecalled using Dorado v0.4.3 (https://github.com/nanoporetech/dorado) with the super-accuracy (sup) model, without demultiplexing or barcode trimming. High-quality reads were subsequently demultiplexed and merged by barcode. PCR primer sequences (JB3 and JB4.5) were subsequently removed using Cutadapt v4.6 (https://cutadapt.readthedocs.io) by specifying known primer motifs at the 5’ and 3’ ends of the reads. Filtered reads were aligned to the mitochondrial cox1 gene from Diplostomum ardeae Dubois, 1969 (NC_049068) using BWA-MEM (https://github.com/lh3/bwa), followed by BAM conversion, sorting, and indexing with SAMtools (Li et al. Reference Li, Handsaker, Wysoker, Fennell, Ruan, Homer, Marth, Abecasis and Durbin2009). Depth of coverage was assessed across all positions, and consensus sequences were generated using iVar (https://github.com/andersen-lab/ivar) with a minimum base quality of 25, a threshold frequency of 0.6, and a minimum coverage depth of 10×. Genomic positions with zero coverage were recorded, and average coverage per sample was calculated and visualized to evaluate mapping uniformity (Supplementary material 1).
Phylogenetic analyses of molecular data
To determine the phylogenetic relationships of the specimen found in the fringe-lipped bat, the consensus sequence was integrated with fourteen additional sequences from the Diplostomidae family, retrieved from GenBank (Table 1). Partial cox1 sequences were aligned using MAFFT v7.525 (Katoh et al. Reference Katoh, Misawa, Kuma and Miyata2002). The alignment was then trimmed to the region covered by the cox1 sequence obtained in the present study using a custom Python script, ensuring that phylogenetic inference was restricted to homologous regions. Sequences lacking coverage in this region were excluded, and the alignment was refined with trimAl v1.4 (Capella-Gutiérrez et al. Reference Capella-Gutiérrez, Silla-Martínez and Gabaldón2009) to remove poorly aligned positions. Maximum likelihood phylogenetic analysis was conducted in IQ-TREE v2 (Minh et al. Reference Minh, Schmidt, Chernomor, Schrempf, Woodhams, Von Haeseler and Lanfear2020). The best-fit substitution model was automatically selected by ModelFinder, which identified HKY+F+I+G4 as the optimal model based on the Bayesian Information Criterion (BIC). Node support was estimated with 1,000 ultrafast bootstrap replicates and 1,000 SH-aLRT tests. Bayesian phylogenetic inference was performed using MrBayes v3.2.7 (Ronquist et al. Reference Ronquist, Teslenko, Van Der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012). The GTR substitution model with gamma-distributed rate variation among sites (nst=6, rates=gamma) was specified. Two independent MCMC runs were performed for 10 million generations each, with four chains per run, sampling every 100 generations. The first 25% of samples were discarded as burn-in. Convergence between runs and adequacy of sampling were assessed using Tracer v1.7 (Rambaut et al. Reference Rambaut, Drummond, Xie, Baele and Suchard2018), confirming average standard deviation of split frequencies < 0.01 and effective sample sizes (ESS) > 200. Posterior probabilities were estimated from the post-burn-in trees. Strigea magnirostris was included as outgroups. Phylogenetic trees were visualized and edited in Figtree 1.4.4 (Rambaut Reference Rambaut2018). Genetic distances were estimated using the Kimura 2-parameter (K2P) model and uncorrected p-distances in MEGA v12.0.12 (Kumar et al. Reference Kumar, Stecher, Suleski, Sanderford, Sharma and Tamura2024), with pairwise deletion applied (Supplementary material 2).
Hosts, locality, GenBank accession numbers of Neodiplostomum spp. and outgroup used in this study (new sequences in bold)
Results
Family Diplostomidae Poirier, 1886
Genus Neodiplostomum Railliet, 1919
Neodiplostomum vaucheri Dubois, Reference Dubois1983
Line drawing of Neodiplostomum vaucheri. A: Specimen from Trachops cirrhosus, ventral view. B: Specimen (holotype, MHNG–980.537) from Chrotopterus auritus, eggs not illustrated.
Scanning electron micrographs of Neodiplostomum vaucheri. A: Ventral view of an adult. B: Oral sucker. C: Digitiforme spines. D: Acicular spines. E: Ventral sucker. F: Holdfast organ. G: Posterior end of opisthosoma showing the genital pore (asterisk) and excretory pore (black arrow).
Description (based on 8 specimens): Body elongate (Figure 1A), 979–1575 (1228; n=8) long. Prosoma spatulate, with a ventral concavity, 442–658 (564; n=8) long, greatest width 214–370 (304; n=8) at level of holdfast organ, with folded margins ventrally. Tegument with fine spines, restricted to prosoma (Figure 2A); ventral spines digitiform (Figure 2C), dorsal spines acicular (Figure 2D). Opisthosoma sub-cylindrical, 533–995 × 226–377 (658 × 287; n=8), with rounded posterior end. Prosoma length:opisthosoma length ratio, 0.57–1.23 (0.91; n=8). Forebody 191–381 (276; n=8). Forebody 14%–32% (23%; n=8) of the body length. Hindbody 404–1218 (797; n=8) long. Oral sucker subterminal, subspherical, smaller than ventral sucker, 47–63 × 41–47 (56 × 44; n=8). Oral sucker length:body length ratio 0.033–0.060 (0.047; n=8). Ventral sucker subspherical, 87–100 × 77–98 (93 × 89; n=8), located in middle of prosoma. Small papillae on outer edges of ventral sucker (Figure 2E). Holdfast organ elliptoid, with median longitudinal slit, 131–203 × 71–126 (170 × 109; n=8), located immediately posterior to ventral sucker. Prepharynx elongate, 12–54 (30; n=8) long. Pharynx oval, 56–65 × 32–43 (59 × 36; n=8). Pharynx length:oral sucker length 0.88–1.19 (1.04; n=8). Esophagus short to absent, 12–16 (14; n=8) long. Caecal bifurcation in anterior third of prosoma. Caeca slender, extends to near posterior end of body. Holdfast organ length:prosoma length ratio 0.22–0.39 (0.31; n=8). Holdfast organ length: width ratio 1.33–1.85 (1.57; n=8). Body length:holdfast organ length ratio 6.28–9.06 (7.23; n=8). Testes two, tandem, asymmetrical, different in size; anterior testis oval, with slightly irregular margins, 98–223 × 111–225 (156 × 172; n=8); posterior testis bilobed, 101–267 × 113–216 (164 × 173; n=8), with asymmetrical lobes, larger lobe 57–171 × 69–140 (102 × 99; n=8), smaller lobe 53–104 × 58–129 (81 × 87; n=8). Seminal vesicle, globular U-shaped, post-testicular. Ovary oval, median, pre-testicular, 44–85 × 76–127 (64 × 106; n=8), situated close to borderline between prosoma and opisthosoma. Mehlis’ gland not observed. Uterus occupying middle field of opisthosoma. Egg oval, operculated, with thick shell, 83–86 × 46–57 (85 × 51; n=3). Vitellarium consists of small irregular follicles, extending from anterior edge of ventral sucker to posterior end of seminal vesicle. Vitelline reservoir rounded, intertesticular (Figure 1A). Genital pore dorsal, located near posterior end of body, surrounded by numerous small papillae, distributed randomly (Figure 2G). Excretory pore terminal (Figure 2G).
Taxonomic summary
Host: Trachops cirrhosus (Spix) (Chiroptera: Phyllostomidae), fringe-lipped bat
Locality: Kawsay Biological Station, Madre de Dios, Peru (12°31’44’’S, 69° 0’ 26’’W)
Site in host: Small intestine
Infection levels: 21 worms observed, 14 in one host, 7 in the other
Deposited specimens: 8 voucher specimens (MUSM-HEL 5519–5526)
Remarks
Neodiplostomum vaucheri was originally described by Dubois (Reference Dubois1983), based on a single specimen found parasitizing Chrotopterus auritus in the Loreto region of northeastern Peru. Since its original description, no new occurrences of N. vaucheri have been reported in Peru, and no redescriptions of this species have been made. However, a recent study by Achatz et al. (Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022a) reported the presence of N. vaucheri in T. cirrhosus, a phyllostomid bat, in Ecuador. While the authors provided genetic data for two specimens of N. vaucheri, they did not include any morphological information on their specimens. We attempted to access the Achatz specimens (QCAZI264292) at the Museo de Zoología, Pontificia Universidad Católica del Ecuador, Quito, Ecuador, but the specimens had not yet arrived. Although our specimens were smaller than the holotype of N. vaucheri (Figure 1B), the relative position of important features such as the gonads, vitellarium, and genital pore, as well as the ratio between prosoma and opisthosoma, suckers, holdfast organ and prosoma, and the egg sizes, were similar when comparing both materials. Therefore, given the similarities in morphometric features (Table 2) and the fact that our specimens were collected from a host belonging to the same subfamily (Phyllostominae) as the type host, there is no strong evidence to suggest that represent a different species.
Morphometric comparison of Neodiplostomum vaucheri and the most similar species in South América
* measurements of the holotype.
The SEM micrographs revealed several previously undescribed features, including a tegument with fine spines (digitiform ventral spines and acicular dorsal spines) limited to the prosoma, small papillae on outer edges of ventral sucker and surrounding the genital pore, and a terminal excretory pore (Figure 2G).
Molecular identification and phylogenetic analyses
Oxford Nanopore sequencing generated a partial cox1 sequence (396 bp) with a mean coverage of 211.36× and no positions with zero coverage, supporting the reliability of the data for downstream phylogenetic analyses (Supplementary material 3). The sequence was derived from a specimen closely related to Neodiplostomum vaucheri (GenBank accession number: PQ665378). BLAST analysis revealed 95.73% identity and 95% query coverage with N. vaucheri (GenBank: OL770065). Phylogenetic reconstruction using both Bayesian inference (BI) and maximum likelihood (ML) yielded identical topologies with varying support values. The specimen clustered in a well-supported clade (BI posterior probability = 0.9587; ML bootstrap = 90) comprising eleven species of Neodiplostomum clade I (Achatz et al. Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022a) (Figure 3). The genetic distance between the partial cox1 gene sequence obtained in this study, and its closest available reference, N. vaucheri (OL770065.1), was 4.47% based on the Kimura two-parameter (K2P) model and 4.27% using uncorrected p-distances. The analysis revealed no differences in the translated amino acid sequences (125 amino acids); all observed nucleotide substitutions were synonymous.
Relationships between Neodiplostomum spp. inferred from Maximum Likelihood (ML) and Bayesian Inference (BI) analyses of the partial cox1 gene dataset. Support values are shown at the nodes as ML/BI. Only support values with ML ≥ 70% and BI ≥ 80% are displayed. The new sequence is indicated in bold. The scale bar represents the expected number of substitutions per site.
Discussion
In the present study, we provide detailed morphological information on N. vaucheri, including line drawings, morphometric data, and SEM illustrating the morphology of spines and papillae. In addition, we added molecular data based on the partial mitochondrial cox1 gene from specimens collected in Madre de Dios, Peru (Southern Peruvian Amazon). Given that N. vaucheri was originally described from a single specimen, the incorporation of both morphological and genetic evidence contributes to a more robust characterization of the species and facilitates its distinction from other closely related taxa potentially occurring in bats.
Achatz et al. (Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022a) conducted a phylogenetic analysis of the genera Neodiplostomum and Fibricola Dubois, 1932, incorporating specimens from various regions worldwide, including N. vaucheri collected from T. cirrhosus in Ecuador. Given the limited morphological data available for the Ecuadorian specimens, Dr. Vasyl Tkach provided virtual access to the specimens identified as N. vaucheri (QCAZI264292). A comparative morphological assessment revealed a high degree of similarity between these specimens and those from Peru examined in this study. However, the Peruvian specimens exhibited a higher oral sucker width to ventral sucker width ratio and more pronounced lobes in the posterior testis. Despite these differences, the limited number of distinguishing morphological features prevents us from conclusively determining whether these specimens represent distinct morphotypes.
The morphological characteristics of Neodiplostomum species provide limited resolution for distinguishing closely related taxa, a pattern observed in other digenean groups. In this context, molecular data, particularly cox1, have proven valuable for species delimitation (de Mello et al. Reference De Mello, Rabelo and da Silva2019; Tkach et al. Reference Tkach, Achatz, Hildebrand and Greiman2018; Woodyard et al. Reference Woodyard, Rosser and Griffin2017). In the present study, phylogenetic reconstruction (ML = 90; BI = 0.0.9587) corroborated the morphological identification, grouping the Peruvian isolate with one Neodiplostomum vaucheri sequence from another South American region (Ecuador). The two methods used to estimate genetic divergence in the partial cox1 gene between the Peruvian specimen and the Ecuadorian specimen (N. vaucheri, OL770065.1) yielded values of 4.47% (Kimura 2-parameter) and 4.22% (uncorrected p-distances). These estimates indicate a moderate level of mitochondrial divergence between the two specimens. Such levels of divergence in cox1 are often observed between closely related but distinct trematode species, suggesting that the Peruvian specimen may represent either a divergent population or a potential cryptic species within Neodiplostomum. While this level of divergence is lower than the interspecific divergence previously reported for Neodiplostomum species (8.6–13.4%) (Achatz et al. Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022a), it is considerably higher than the intraspecific divergence observed in other members of the genus, such as N. reflexum Chandler & Rausch, 1947 (0.2–0.7%) (Achatz et al. Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022a). A similar pattern was observed in N. cratera (Barker and Noll 1915), specifically in N. cratera 3 (isolate VT7457), where specimens collected from Minnesota and Mississippi, USA, exhibited a genetic divergence of 2.6% in cox1. Notably, specimens from Minnesota were reported to be markedly smaller than those from Mississippi. This combination of genetic and morphological differences led Achatz et al. (Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022a) to suggest that the validity of N. laruei Miller, 1940, originally described from a locality near Minnesota and later synonymized with N. cratera, should be reassessed. The similarity between this case and our findings may suggests that the Peruvian and Ecuadorian specimens of N. vaucheri could represent distinct lineages, potentially indicating cryptic diversity within this taxon. However, as our inference is based solely on a partial cox1 sequence and no amino acid differences were detected, further multilocus or genomic data would be needed to confirm species-level distinctions. The collection of N. vaucheri from the type host at the type locality is necessary to evaluate its morphological and morphometric variability. Additionally, incorporating multiple genetic markers or analyzing the complete mitogenome would provide a more comprehensive assessment of genetic differentiation and improve the resolution of its taxonomic placement.
Currently, T. cirrhosus is the only recognized species in the genus Trachops (Burgin et al. Reference Burgin, Jane, Liphart, Parker, Becker, Rochon, Huckaby and Zijlstra2024; Simmons and Cirranello Reference Simmons and Cirranello2024), with a broad distribution ranging from southern Mexico to southeastern Brazil (Cramer et al. Reference Cramer, Willig and Jones2001; Gardner Reference Gardner2008; Medellín Reference Medellín, Wilson and Mittermeier2019; Simmons Reference Simmons, Wilson and Reeder2005). The fringe-lipped bat comprises three identified subspecies: T. c. coffini Goldman, 1925, found in Central America from Mexico to Panama; T. c. ehrhardti Felten, 1956, occurring in the Atlantic Forest along the Brazilian coast; and T. c. cirrhosus Spix, 1823, distributed between the other two subspecies across various South American countries linked to the Amazon Basin (Cramer et al. Reference Cramer, Willig and Jones2001; Medellín Reference Medellín, Wilson and Mittermeier2019; Simmons Reference Simmons, Wilson and Reeder2005). According to Da Silva Fonseca et al. (Reference Da Silva Fonseca, Soto-Centeno, Simmons, Ditchfield and Leite2024), these three subspecies should be elevated to species status. Furthermore, ecological niche modeling analyses indicate evidence for up to five clades within Trachops cirrhosus (sensu Da Silva Fonseca et al. Reference Da Silva Fonseca, Soto-Centeno, Simmons, Ditchfield and Leite2024). Notably, two of these clades, T. cirrhosus from northwestern Amazonia and T. cirrhosus from southwestern Amazonia, are separated by the Amazon river in a north–south arrangement (Da Silva Fonseca et al. Reference Da Silva Fonseca, Soto-Centeno, Simmons, Ditchfield and Leite2024). This geographic structure could provide insights into the presence of two distinct species of Neodiplostomum infecting T. cirrhosus – namely N. vaucheri in Southwestern Amazonia (Peru) (Dubois Reference Dubois1983, present study) and Neodiplostomum vaucheri in Northwestern Amazonia (Ecuador) (Achatz et al. Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022a). This correlation suggests that the geographical distribution of T. cirrhosus clades may play a crucial role in shaping the diversity of Neodiplostomum species associated with this host.
The digenean N. vaucheri has been reported infecting the bats C. auritus (type host) and T. cirrhosus (Achatz et al. Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022a; Dubois Reference Dubois1983; present study). The occurrence of the same digenean species in both hosts is unsurprising, given the dietary overlaps between these bat species (Bonato et al. Reference Bonato, Facure and Uieda2004; Gual-Suárez and Medellín Reference Gual-Suárez and Medellín2021; Hemingway et al. Reference Hemingway, Dixon, Page, Fleming, Dávalos and Mello2020; Leal et al. Reference Leal, Chaves, Neto, Filho, Ramalho, Guerra Filho and Telino-Junior2018; Medellín Reference Medellín, Wilson and Mittermeier2019). It is well established that amphibians serve as the principal secondary intermediate hosts for Neodiplostomum species (Blasco-Costa and Locke Reference Blasco-Costa and Locke2017; Izrailskaia and Besprozvannykh Reference Izrailskaia and Besprozvannykh2023), and both C. auritus and T. cirrhosus include tree frogs in their diets (Gual-Suárez and Medellín Reference Gual-Suárez and Medellín2021; Hemingway et al. Reference Hemingway, Dixon, Page, Fleming, Dávalos and Mello2020). This dietary convergence likely explains the observed co-infection in these two bat species.
Our successful implementation of Oxford Nanopore Technologies (ONT) amplicon sequencing in a local laboratory underscores its potential as a reliable, faster, and more cost-effective alternative to traditional Sanger sequencing, particularly when HPLC purification is required. Beyond confirming species identity, this approach provides a powerful tool for broader biodiversity assessments and molecular surveillance, especially in regions where access to conventional sequencing platforms remains limited. Importantly, the ability to perform in-house sequencing significantly reduces turnaround time and per-sample costs, especially when processing multiple samples in parallel through multiplexing. This highlights ONT as a strategic investment for laboratories seeking to evolve toward more autonomous, locally driven, and data-intensive molecular workflows.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X25100564.
Data availability statement
All data that support the findings of this study are available in the main text and supplementary material.
Acknowledgements
We would like to thank Dr. Isabel Blanco-Costa and Dr. Vasyl Tkach for providing us with access to the type material of N. vaucheri and Achatz’s specimens named as N. vaucheri, respectively. We thank the Kawsay Biological Station for their support in the present investigation. The authors are grateful to Sergio Santillán and Jordan Gómez, both from the National University of San Marcos (UNMSM), who assisted with the collection of bats in Peru.
Author contributions
EC: Investigation, processed the methodology of collecting parasites, formal analysis, writing – original draft, drawing pictures, Writing – review and editing, prepared figures. NB: Investigation, collected the hosts and processed the methodology of collecting parasites. RB: Investigation, collected the hosts and processed the methodology of collecting parasites. JP: Investigation, formal analysis, writing – original draft. LS: Data curation, performed molecular analyses, software. LÑ: Investigation, data curation, performed molecular analyses, software, writing – original draft. AM: data curation, performed molecular analyses, software CC: Investigation, formal analysis, drawing pictures, prepared figures. JC: Investigation, formal analysis, writing – original draft, prepared figures, Writing − review & editing, supervision. All authors have read and agreed to the published version of the manuscript.
Financial support
Financial support was from Universidad Nacional Mayor de San Marcos (UNMSM) through internal resources, under R.R. 006081-R-23 and code B23100321. Edson Cacique Was supported by CONCYTEC- PROCIENCIA for the project through the ‘Tesis de Postgrado y Pregrado’ competition E073-2023-01, under contract No. PE501085512-2023-PROCIENCIA.
Competing interests
The authors declare that they have no conflict of interest.
Ethical standard
No ethical statement was reported
