Introduction
The subfamily Proterodiplostominae Reference DuboisDubois, 1936 comprises a relatively small yet evolutionarily significant lineage of digenean trematodes that primarily parasitise reptiles, with the highest species richness recorded in crocodilians (Tkach et al. Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and de Vasconcelos Melo2020), although some species also infect snakes and turtles (Dubois Reference Dubois1979; Niewiadomska Reference Niewiadomska, Gibson, Jones and Bray2002), whereas fishes (Díaz Pernett et al. Reference Díaz Pernett, Brant and Locke2022; Stoyanov et al. Reference Stoyanov, Georgieva, Pankov, Kudlai, Kostadinova and Georgiev2017) and amphibians (Queiroz et al. Reference Queiroz, López-Hernández, Locke, Pinto and Anjos2020) may serve as second intermediate or paratenic hosts, harbouring the metacercarial stages required for transmission to definitive hosts. At present, the group comprises 23 valid genera and slightly more than 50 described species worldwide (WoRMS 2026), predominantly distributed in tropical and subtropical regions (Tkach et al. Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and de Vasconcelos Melo2020).
Historically, members of Proterodiplostominae were originally established by Dubois (Reference Dubois1936) at the family level (Proterodiplostomidae Dubois, Reference Dubois1936) to accommodate diplostomids parasitizing reptiles and characterized by the presence of a distinctive glandular structure associated with the male reproductive system, referred to as a paraprostate (Niewiadomska, Reference Niewiadomska, Gibson, Jones and Bray2002; Tkach et al. Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and de Vasconcelos Melo2020). More recently, Achatz and Tkach (Reference Achatz and Tkach2025) conducted a molecular phylogenetic analysis of Diplostomoidea and showed that Diplostomidae and Strigeidae, two of the most species-rich families within the superfamily, are not monophyletic. Furthermore, Achatz and Tkach (Reference Achatz and Tkach2025) showed that Proterodiplostomidae and Strigeidae are nested within Diplostomidae and, consequently, proposed their inclusion within this family to promote greater taxonomic stability within Diplostomoidea.
In Peru, records of Proterodiplostominae are scarce and largely restricted to adult specimens of Proterodiplostomum longum (Brandes, 1888) recovered from Caiman crocodilus Linnaeus, 1758 (Sauropsida: Alligatoridae) in the vicinity near Iquitos, in the Peruvian Amazon (Tkach et al. Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and de Vasconcelos Melo2020). Despite the exceptional diversity of freshwater fishes in the Peruvian Amazon Basin (Dávila et al. 2018), records of Proterodiplostominae metacercariae are lacking (Luque et al. Reference Luque, Pereira, Alves, Oliva and Timi2016), and molecular data linking larval stages in fishes to adult Proterodiplostomids remain virtually unavailable.
As part of parasitological surveys conducted on siluriform fishes from the Peruvian Amazon Basin, metacercariae of Proterodiplostominae were recovered from Hoplosternum littorale (Hancock, 1828) (Callichthyidae) and Pterygoplichthys pardalis (Castelnau, 1855) (Loricariidae), two benthic catfish species widely distributed throughout tropical South America (Orfinger Reference Orfinger2015; Reis Reference Reis1997). The present study aimed to characterize these metacercariae using an integrative approach combining detailed morphological analyses based on light and scanning electron microscopy (SEM) with nuclear (28S rDNA) and mitochondrial (COI) DNA sequences. This work provides the first genetic data for metacercarial stages of Proterodiplostominae from Peru and also represents the first record of Proterodiplostominae metacercariae infecting freshwater fishes in the country, contributing novel insights into the diversity, host associations, and phylogenetic placement of Diplostomidae trematodes in this megadiverse region.
Materials and methods
Collection of samples and morphological study
Thirty-two specimens of siluriform fishes, 28 of H. littorali and 4 of P. pardalis, were collected by artisanal fishermen in November 2024 and March 2025 from the Shapajal seasonal lagoon (08°46′S, 74°08′W), near the Tamaya River, Ucayali Region, and Santa Clara beach (03°49′S, 73°21′W), near the Nanay River, Loreto region, Peru. Host identification followed Dávila et al. (2018). Fish hosts were examined for helminth infections. Digeneans were rinsed in 0.9% saline solution and fixed in hot 70% ethanol. Digeneans were stained following Chero et al. (2025). Observations and photographs were taken using a Nikon™ Eclipse NI-U compound microscope equipped with bright-field and differential interference contrast (DIC) optics, and drawings were prepared with the aid of a drawing tube. All measurements are expressed in micrometres (μm), unless otherwise stated, and correspond to straight-line distances between the outermost limits of each structure. Data are reported as ranges, followed by the mean value and the number of structures examined (n) in parentheses. Nomenclature and classification of Diplostomidae and Proterodiplostominae follow WoRMS (2026). For scanning electron microscopy (SEM), specimens were post-fixed in 10% buffered formalin, dehydrated in a graded ethanol series, and dried using a carbon dioxide critical point dryer. Subsequently, samples were sputter-coated with gold and analyzed with an Inspect S50-FEI scanning electron microscope operated at an accelerating voltage of 5–10 kV.
Specimens were deposited in the Helminthological Collection of the Museum of Natural History at the San Marcos University (MUSM-HEL), Lima, Peru.
Molecular analysis
Genomic DNA from metacercariae was isolated using the TIANamp Genomic DNA Kit, following the manufacturer’s instructions. A partial fragment of the 28S rDNA gene was amplified by polymerase chain reaction (PCR) employing the forward primer digl2 (5′−AAG CAT ATC ACT AAG CGG−3′) and the reverse primer 1500R1 (5′−GCT ACT AGA TGG TTC GAT TAG−3′) as described by Tkach et al. (2003). A partial region of the mitochondrial cytochrome c oxidase subunit I gene (cox1) was amplified using the primers Cox 5_Schisto (5′−TCT TTR GAT CAT AAG CG−3′) and JB5 (5′−AGC ACC TAA ACT TAA AAC ATA ATG AAA ATG−3′), according to previous studies (Derycke et al. 2005; Fernandes et al. 2021; Lockyer et al. 2003). Each PCR reaction was performed in a final volume of 25 μl, containing 12.5 μl of PCRBIO Taq Mix Red, 0.3 μl of each primer (10 μM final concentration), 1.2 μl of template DNA, and ultrapure water to complete the total volume. The annealing temperature was set at 55°C for 28S rDNA and 45°C for cox1 amplification. Amplified products were purified using sparQ PureMag Beads according to the manufacturer’s protocol. Purified amplicons were subsequently sequenced by Macrogen Inc. using the Sanger sequencing method.
Raw sequences were manually inspected, edited, and assembled into consensus contigs using Geneious (Filatov 2002). Homologous sequences were obtained from the National Center for Biotechnology Information (NCBI) database, hereafter referred to as GenBank, through searches conducted with the Basic Local Alignment Search Tool (BLAST) (Altschul et al. 1990). The 28S rDNA and cox1 sequences generated in the present study were aligned with corresponding reference sequences representing recognized species and putative lineages of Proterodiplostominae available in GenBank (see Table 1). Multiple sequence alignments were performed using Clustal W (Larkin et al. 2007). For phylogenetic analyses of both the 28S and cox1 datasets, sequences of Alaria mustelae Bosma, 1931 were designated as outgroup taxa (GenBank accession numbers provided in Table 1). The best-fitting nucleotide substitution models were selected using MEGA X (Kumar et al. 2018) under the corrected Maximum Likelihood (AICc; Posada and Buckley, 2004). The selected models were TN93+I+G for the cox1 dataset and HKY+I+G for the 28S rDNA dataset. Bayesian inference (BI) analyses were performed in MrBayes v3.2.6 (Ronquist et al. 2012), using Metropolis-coupled Markov chain Monte Carlo (MCMC) simulations starting from random trees. The MCMC was run for 2,000,000 generations, with trees sampled every 100 generations, resulting in 500,000 trees per analysis. Convergence and stationarity were assessed by visual inspection of the log-likelihood scores over generations using Tracer v1.7.2. Since likelihood values stabilized before generation 100,000, the first 1,000 trees (10% burn-in) were discarded. The remaining 45,000 trees were used to construct a 50% majority-rule consensus tree. The trees were visualized and edited in FigTree v. 1.4.4 (Rambaut Reference Rambaut2010).
Proterodiplostominae species, including outgroup, used in phylogenetic analyses based on 28S rDNA and cox1 mDNA sequences
Pairwise genetic distances between the sequences of cox1 mDNA and 28S rDNA were calculated in MEGA X (Kumar et al. 2018) using the p-distance (Kimura 1980).
Results
Diplostomoidea Poirier, 1886
Diplostomidae Poirier, 1886
Proterodiplostominae Reference DuboisDubois, 1936
Proterodiplostomum longum (Brandes, 1888) (Isolate M1-M3)
Host: Pterygoplichthys pardalis.
Locality: ‘Calleria sector’ near the Ucayali River (08°26′S, 74°15′W), Ucayali, Peru.
Specimens deposited: 1 voucher specimen (MUSM-HEL 5668).
Representative DNA sequences: PZ316282–84 (28S rDNA).
Description (based on two specimens, Figs. 1A, 2A–B).
Line drawing of Proterodiplostominae metacercarie from siluriform fishes from the Peruvian Amazon Basin. A. Proterodiplostomum longum (Brandes, 1888) from Pterygoplichthys pardalis. B. Proterodiplostominae gen. sp4. from Pterygoplichthys pardalis. C. Proterodiplostominae gen. sp5. from Hoplosternum littorale.
Scanning electron micrographs of Proterodiplostominae metacercarie recovered from Pterygoplichthys pardalis. A. Whole body of Proterodiplostomum longum (Brandes, 1888) (ventral view). B. Whole body of Proterodiplostomum longum (Brandes, 1888) (dorsal view). C. Whole body of Proterodiplostominae gen. sp4. (ventral view).
Body elongated, flattened, broader in the anterior region and tapering posteriorly, 476–524 (500; n = 2) long, greatest width 165–167 (166; n = 2) at anterior region. Body length/body width ratio 2.88–3.14 (3.02; n = 2). Forebody 279–329 (304; n = 2) long. Hindbody 195–196 (196; n = 2) long. Oral sucker small, subspherical, muscular, terminal, 28–30 × 23–24 (29 × 23; n = 2). Oral sucker width/body width ratio 0.137–0.139 (0.138; n = 2). Prepharynx absent. Pharynx elongated, 19–22 × 15–17 (21 × 16; n = 2). Oesophagus long, tapering. Intestinal caeca long, tapering, lacking diverticula, blind-ending posterior to tribocytic organ at level of primordial genital organs. Ventral sucker subspherical, 27–28 × 28–31 (27 × 30; n = 2), located posterior to the equatorial region of body. Ventral sucker width/body width ratio 0.170–0.187 (0.179; n = 2). Ventral sucker width/oral sucker width ratio 1.237–1.353 (1.295; n = 2). Tribositic organ 66–68 × 44–53 (67 × 48; n = 2). Distance between ventral sucker and tribositic organ 64–78 (71; n = 2). Genital primordium in posterior third of body. Two primordial testes, tandem, poorly defined. Primordial ovary oval, post-testicular, adjacent to the testes. Genital pore terminal.
Proterodiplostominae gen. sp4. (Isolates M4-M5)
Host: Pterygoplichthys pardalis.
Locality: ‘Calleria sector’ near the Ucayali River (8°26′S, 74°15′W), Ucayali, Peru.
Specimens deposited: 1 voucher specimen (MUSM-HEL 5669).
Representative DNA sequences: PZ310479 (cox1 mDNA) and PZ316279–80 (28S rDNA).
Description (based on seven specimens, Figs. 1B, 2C).
Body elongated, flattened, fusiform, 0.79–1.18 (1.01; n = 7) mm long, greatest width 257–369 (323; n = 7) at level of ventral sucker. Body length/body width ratio 2.64–3.58 (3.15; n = 7). Forebody 533–811 (658; n = 7) long. Hindbody 259–367 (346; n = 7) long. Oral sucker small, subspherical, muscular, terminal, 40–55 × 31–50 (48 × 42; n = 7). Oral sucker width/body width ratio 0.12–0.13 (0.129; n = 7). Prepharynx absent. Pharynx elongated, 31–40 × 22–33 (37 × 26; n = 6). Oesophagus long, tapering. Intestinal caeca long, tapering, lacking diverticula, blind-ending posterior to tribocytic organ at level of primordial genital organs. Ventral sucker subspherical, 79–87 × 70–97 (84 × 91; n = 7), located posterior to the equatorial region of body. Ventral sucker width/body width ratio 0.25–0.36 (0.29; n = 7). Ventral sucker width/oral sucker width ratio 1.89–2.73 (2.25; n = 7). Tribositic organ 90–130 × 61–82 (103 × 76; n = 7). Distance between ventral sucker and tribositic organ 84–177 (146; n = 7). Genital primordium in posterior third of body. Two primordial testes, tandem, poorly defined. Primordial ovary oval, post-testicular, adjacent to the testes. Genital pore terminal.
Proterodiplostominae gen. sp5. (Isolate M6)
Host: Hoplosternum littorale.
Locality: Zungarococha Lagoon and Santa Clara beach (3°49′S, 73°21′W), near the Nanay River, Loreto, Peru.
Representative DNA sequences: PZ310480 (cox1 mDNA) and PZ316281 (28S rDNA).
Description (based on one specimen, Fig. 1C).
Body elongate, 1.504 mm long; greatest width 0.496 mm at the level between the ventral sucker and the holdfast organ. Body length/body width ratio 3.030. Forebody 903 long. Hindbody 601 long. Oral sucker terminal, oval, smaller than ventral sucker, 46 × 45. Oral sucker width/body width ratio 0.090. Prepharynx absent. Pharynx not observed. Intestinal caeca long, tapering, lacking diverticula, blind-ending posterior to tribocytic organ. Ventral sucker 112 × 116. Ventral sucker width/body width ratio 2.606. Ventral sucker width/oral sucker width ratio 0.235. Tribositic organ 114.19 × 100.93. Distance between ventral sucker and tribositic organ 312. Genital primordium not observed.
Molecular analyses
In this study, we generated partial 28S rDNA sequences from five metacercariae and cox1 mtDNA sequences from two metacercariae of Proterodiplostominae infecting the hosts P. pardalis and H. littorale. Specimens were collected in Ucayali from P. pardalis (n = 4 for 28S; n = 1 for cox1) and in Loreto from H. littorale (n = 1 for each marker).
Cox1: The final dataset comprised 425 bp and 34 terminals, including the newly generated sequences and representatives of Proterodiplostominae, with Alaria mustelae designated as outgroup (Fig. 3). The sequence obtained from Loreto (Proterodiplostominae gen. sp5.) (isolate M6) formed a sister lineage to Crocodillicola pseudostoma Willemoes-Suhm, 1870 (MT398317) with moderate support (PP = 0.86) (Fig. 3). Genetic divergence between Proterodiplostominae gen. sp5. and C. pseudostoma was 4.95%. The Ucayali isolate (Proterodiplostominae gen. sp4.) (M4) clustered in a clade comprising C. pseudostoma and Proterodiplostominae gen. sp5., which in turn was recovered as sister to Paradiplostomum abbreviatum (MT603606) (Fig. 3). The genetic divergence between Proterodiplostominae gen. sp4. and Proterodiplostominae gen. sp5. was 9.882% (42 bp), indicating moderate differentiation and supporting their recognition as closely related but distinct evolutionary lineages within Proterodiplostominae.
Phylogenetic tree of species/lineages of Proterodiplostominae through the cox1 gene inferred using Bayesian Inference (BI). Sequences from Proterodiplostominae species obtained in this study are in blue bold. Scale-bar: average number of nucleotide substitutions per site.
28S: The final alignment comprised 1234 bp and included 42 terminals. In the BI tree (Fig. 4), the three sequences generated in the present study (M1–M3) nested within a fully supported lineage (PP = 1) that included two sequences of P. longum (MT622341 and MT187589) recovered from crocodilian hosts, three sequences identified as Proterodiplostomum sp. 3 (MT187594–MT187596) obtained from fish hosts, and an additional sequence of Proterodiplostomum sp. 3 (MT187589) isolated from a bird host (Fig. 4). The genetic divergence between isolated M2 (PZ316283) and M3 (PZ316284) was 0.174% (2 bp), indicating negligible differentiation and confirming that both sequences belong to the same lineage. Likewise, the remaining sequences within this clade exhibited very low pairwise distances relative to this pair (0.433%–0.536%; 5–6 bp), further supporting their conspecificity and assignment to P. longum.
Phylogenetic tree of species/lineages of Proterodiplostominae through the 28S rRNA gene inferred using Bayesian Inference (BI). Sequences from Proterodiplostominae species obtained in this study are in blue bold. Scale-bar: average number of nucleotide substitutions per site.
Proterodiplostominae gen. sp5. was recovered as sister to Proterodiplostomidae gen. sp. 3 (MT187600) with full support (PP = 1). This pair formed the sister group to a moderately supported clade (PP = 0.81) comprising our two isolated species identified as Proterodiplostominae gen. sp. 4 (PZ316279 and PZ316280) together with Proterodiplostomidae gen. sp. 2 (MT187599), all recovered from fish hosts. Genetic distances among these taxa ranged from 0.4% to 0.7% (4–10 bp), indicating low levels of divergence within this lineage.
Discussion
The present study provides the first integrative morphological and molecular characterization of Proterodiplostominae metacercariae infecting siluriform fishes in the Amazon Basin. Although the morphological features of the metacercariae are insufficient for reliable species identification, molecular evidence based on partial 28S rDNA and mitochondrial cox1 sequences allowed us to confidently assign isolates M1–M3 to P. longum. In addition, we identified two distinct evolutionary lineages, Proterodiplostominae gen. sp. 4 (isolates M4 and M5) and Proterodiplostominae gen. sp. 5 (isolate M6), within the subfamily. These findings expand current knowledge of the diversity, host associations, and phylogenetic relations of Proterodiplostominae in the Neotropical region.
Proterodiplostomum longum was originally described as Diplostomum longum Brandes, 1888 (Dubois Reference Dubois1979). Since its original description, the species has been reported parasitizing several crocodilian hosts within the Alligatoridae, including C. crocodilus; Caiman yacare Daudin, 1802; Caiman sp.; Melanosuchus niger Spix, 1825; Paleosuchus palpebrosus (Cuvier, 1807) and Paleosuchus sp. (Fernandes & Kohn, Reference Fernandes and Kohn2014; Tkach et al. Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and de Vasconcelos Melo2020). These records are distributed throughout the Neotropical region, particularly in Brazil, Paraguay, Peru, and Venezuela. Our 28S rDNA analyses showed that isolates M1–M3 clustered within a strongly supported clade comprising reference sequences of P. longum from crocodilian hosts, as well as metacercarial sequences previously deposited in GenBank as Proterodiplostomum sp. 3 from the fishes Hemiodontichthys acipenserinus (Kner, 1853) and Hemiodus unimaculatus Bloch, 1794, and from the bird Phalacrocorax brasilianus (Gmelin, 1789), considered an accidental host. Although these records have not been formally published in a peer-reviewed journal, they originate from the doctoral thesis of Taboada (2020), and the corresponding GenBank entries include detailed metadata on host identity and site of infection. Genetic distances within this clade were extremely low (≤0.536%), well within the range of intraspecific variation typically observed for 28S in digenean trematodes. The negligible divergence between our isolates and published sequences of adults of P. longum strongly supports their conspecificity. Furthermore, the phylogenetic placement of the sequence labelled as Proterodiplostomum sp. 3 indicates that they most likely correspond to P. longum. Our study provides the first confirmed record of P. pardalis serving as a host of P. longum metacercariae, thereby clarifying a previously undocumented component of its life cycle. Additionally, our findings complement earlier Brazilian records of P. longum metacercariae (reported as Proterodiplostomum sp. 3) infecting two freshwater fishes and a bird, expanding the known host range of this species in the Neotropical region.
The isolates designated here as Proterodiplostominae gen. sp. 4 (M4 and M5) and Proterodiplostominae gen. sp. 5 (M6) were recovered as distinct lineages within Proterodiplostominae, exhibiting a mitochondrial divergence of 9.882% (42 bp) in the cox1 dataset. This level of divergence is generally considered with species-level differentiation in digenean trematodes of the order Diplostomida (Achatz et al. Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022); however, formal taxonomic conclusions require additional morphological evidence. In contrast, analyses of the 28S rDNA marker revealed low levels of divergence among closely related taxa (0.4–0.7%), reflecting the more conserved nature of nuclear ribosomal markers. The 28S phylogeny recovered Proterodiplostominae gen. sp. 5 as sister to Proterodiplostomidae gen. sp. 3 (MT187600) from H. acipenserinus in Brazil. This pair formed a clade that was, in turn, sister to an unresolved assemblage comprising our two sequences of Proterodiplostominae gen. sp. 4 and a single sequence of Proterodiplostomidae gen. sp. 2 (MT187599) from Cichla monoculus Spix & Agassiz, 1831 in Brazil. The entire group was recovered as sister to an adult specimen of Mesodiplostomum gladiolum (MT622332) from C. yacare in Brazil. Conversely, the cox 1 topology placed Proterodiplostominae gen. sp. 5 as sister to Crocodillicola pseudostoma (MF398317) from Rhamdia guatemalensis (Quoy & Gaimard, 1824) in Mexico, and together these taxa formed a clade sister to Proterodiplostominae gen. sp. 4. This topological discordance is not unexpected and may partly reflect the limited taxon sampling available for cox1, as sequences of Proterodiplostomidae gen. sp. 2 and Proterodiplostomidae gen. sp. 3 are currently unavailable. The differing levels of resolution between mitochondrial and nuclear datasets underscore the complementary value of multilocus approaches for species delimitation. The observed mitochondrial differentiation may represent distinct evolutionary lineages within Proterodiplostominae in the Peruvian Amazon.
This study represents the first record of Proterodiplostominae metacercariae infecting freshwater fishes in Peru and provides the first genetic data for larval stages of the group in the country. The recovery of metacercariae from P. pardalis and H. littorale expands the known spectrum of second intermediate hosts for Proterodiplostominae in the Neotropics.
Although the present study provides new insights into the diversity of Proterodiplostominae in Peru, it is based on a limited number of host specimens and genetic markers. Broader sampling of fish hosts, as well as examination of potential definitive hosts such as crocodilians in the Peruvian Amazon, will be necessary to confirm life-cycle links and assess host-parasite specificity. Future studies incorporating additional molecular markers or complete mitochondrial genomes may improve resolution among closely related lineages.
Data availability
All data that support the findings of this study are available in the main text and supplementary material.
Acknowledgements
The authors would like to thank Edson Cacique Molero and Nicolas Tarmeño Rojas, staff at the Laboratory of Invertebrate Zoology, Faculty of Biological Sciences (FCB), National University of San Marcos (UNMSM), Lima, Peru, for their technical support during the study.
Author contribution
All authors contributed to the study conception and design. Material preparation and data collection: NH, CLC, GMM, JYA, JLL, and JDC. Morphological and molecular analyses were performed by NH, JFS, NM, DL, and LA. The first draft of the manuscript was written by NH, LÑ, CLC, and JDC, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Financial support
This work was funded by the Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica (CONCYTEC) and the Programa Nacional de Investigación Científica y Estudios Avanzados (PROCIENCIA) under the call ‘E041-2024-03 Proyectos de Investigación Básica’ (PE501087644-2024).
Competing interests
The authors declare that they have no conflict of interest.
Ethical statement
No ethical statement was reported.
