Introduction
Osteogaster aenea (Gill) [syn. Corydoras aeneus (Gill)] (Siluriformes, Callichthyidae) is a small armoured catfish widely distributed in tropical freshwater streams of South America, ranging from Colombia and Trinidad to the La Plata River basin, east of the Andes (Burgess Reference Burgess1989; Froese and Pauly Reference Froese and Pauly2025). This species is known for its tolerance to abrupt temperature fluctuations, poor habitat quality, and low dissolved oxygen concentrations (De Araujo and Garutti Reference De Araujo and Garutti2003; Mol Reference Mol and Ouboter1993), which makes it common in degraded urban streams (Casatti et al. Reference Casatti, Romero, Teresa, Sabino and Langeani2010). In addition, O. aenea is heavily traded as an ornamental fish worldwide, further increasing its commercial relevance (Diatin et al. Reference Diatin, Suprayudi, Budiardi and Surawidjaja2015).
Despite its ecological and commercial importance, information on the parasite fauna of O. aenea is still scarce. Philocorydoras corydori (Molnar, Hanek & Fernando, Reference Molnar, Hanek and Fernando1974) [syn. Urocleidoides corydori Molnar, Hanek & Fernando, Reference Molnar, Hanek and Fernando1974] and Philocorydoras margolisi (Molnar, Hanek & Fernando, Reference Molnar, Hanek and Fernando1974) [syn. Urocleidoides margolisi Molnar, Hanek & Fernando, Reference Molnar, Hanek and Fernando1974] were the first helminths described in O. aenea in specimens from rivers and freshwater swamps in Trinidad (Molnar et al., Reference Molnar, Hanek and Fernando1974). A further report on the occurrence of helminths in this host species was published by Tozato (Reference Tozato2011), who examined populations from streams in São Carlos municipality, São Paulo State, Tietê River basin, in Brazil, and found monopisthocotylids (Philocorydoras spp. and Gyrodactylus spp.), nematodes (Capillariidae spp., Hysterothylacium spp., and Spiroxys spp.), plerocercoid larvae of proteocephalid cestodes, the crustacean Minilernaea floricapitella Thatcher & Huergo, 2005, an unidentified acanthocephalan, and the metacercariae of the digenean Diplostomum sp. infecting this host species. This scarcity of data highlights the need for more comprehensive investigations on the parasite fauna of O. aenea.
During a helminthological survey of O. aenea from localities of the State of São Paulo, Brazil, digeneans were collected from the intestine of some individual hosts. A thorough morphological evaluation, including scanning electron microscopy (SEM) observations, along with the assessment of phylogenetic affinities based on two molecular markers, made it possible to identify the sampled digeneans as a new genus and species within the family Plagiorchiidae, which is described herein.
Material and methods
Host collection and parasitological processing
Fifty specimens of O. aenea (mean total length: 5.55 ± 0.68 (3.4–6.7) cm; standard length 4.21 ± 0.56 (3.2–5.4) cm, and weight 3.25 ± 1.07 (1.6–5.2) g) were collected in the Capivarinha River, Tietê River basin, Vitoriana district, municipality of Botucatu, São Paulo state, Brazil (22°45′22.58″ S; 48°21′57.70″ W) between April and July 2024. The fish were collected using a sieve net, brought alive to the laboratory in oxygenized water tanks, and then killed by pithing the spinal cord. During necropsy, the internal organs were removed, placed in petri dishes with 0.6% saline, and examined under a stereomicroscope. The collected digeneans were placed in saline and gently cleaned of the intestinal debris. Some of the specimens were preserved in molecular-graded ethanol for molecular analyses; others were fixed in a hot 4% formaldehyde solution, while a subset of specimens was fixed under slight coverslip pressure using the same fixative, for morphological analyses by light microscopy or SEM.
For morphological analyses, specimens were stained with chloride carmine, cleared in creosote, and mounted as permanent slides using Canada balsam. Morphometrical analyses were performed using a computerized image analysis system with differential interference contrast (DIC) (Leica Application Suite, V3; Leica Microsystems, Wetzlar, Germany). All measurements are presented in micrometres (μm) and are expressed as the mean followed by the range in parentheses. Illustrations were produced with the aid of a drawing tube mounted on a Leica DMLS microscope with phase-contrast optics and edited in CorelDraw V6.
For SEM, specimens were dehydrated in a graded ethanol series (80%, 90%, 96%, 100%), immersed in hexamethyldisilazane (HMDS) for 15 min, air-dried for 15 min, coated with gold and examined in a FEI Quanta 200 scanning electron microscope at the Electron Microscopy Center, Institute of Biosciences, São Paulo State University ‘Júlio de Mesquita Filho’ (UNESP), Botucatu, Brazil.
Some ecological parameters (prevalence and mean abundance of infection) were calculated following Bush et al. (Reference Bush, Lafferty, Lotz and Shostak1997).
Molecular data acquisition
Genomic DNA was extracted from five digeneans collected from different O. aenea hosts, using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA), following the manufacturer’s protocol and adapted to a final volume of 50 μL. Fragments of partial large subunit of the nuclear ribosomal DNA (28S rDNA; D1–D3 domains) and mitochondrial DNA cytochrome c oxidase subunit I (COI mtDNA) genes were amplified as follows: for the 28S rDNA, the primer pair LSU5’ (5′-TAGGTCGACCCGCTGAAYTTAAGCA-3′) (Lockyer et al. Reference Lockyer, Olson and Littlewood2003) and 1500R (5′-GCTATCCTGAGGGAAACTTCG-3′) (Olson et al. Reference Olson, Cribb, Tkach and Littlewood2003) were used, with amplification cycling conditions of initial denaturation of 5 min at 95 °C followed by 35 cycles of 95 °C for 45 s, 59 °C for 45 s, 72 °C for 1 min and 30 s and a final extension of 10 min at 72 °C; the primer 1200R (5′-GCATAGTTCACCATCTTTCGG-3′) (Olson et al. Reference Olson, Cribb, Tkach and Littlewood2003) was used as internal primer for sequencing; for the COI mtDNA, the primer pairs JB3 (5′-TTTTTTGGGCATCCTGAGGTTTAT-3′) and JB4 (5′-TAAAGAAAGAACATAATGAAAATG-3′) (Morgan and Blair Reference Morgan and Blair1998) were used, with the amplification cycling conditions of initial denaturation of 5 min at 95 °C followed by 35 cycles of 94 °C for 45 s, 47 °C for 30 s, 72 °C for 1 min and 30 s and a final extension of 10 min at 72 °C. Conventional PCR amplifications for both genes were performed on a final volume of 20 μL containing 10 μL of 2× MyFi™ Mix (Bioline, Taunton, MA, USA), 1 μL of extracted DNA, 6.6 μL of UltraPure™ DNase/RNase-Free Distilled Water (Invitrogen), and 1.2 μL of each PCR primer. PCR products (2.0 μL) were run on an agarose gel (1.5%) using GelRed™ fluorescent nucleic acid dye added to BlueJuiceTM Gel Loading Buffer (6X) to confirm amplicon size and yield. PCR amplicons were purified using magnetic beads Agencourt AMPure XP (Beckman Coulter, Inc.), following the manufacturer’s instructions. Automated sequencing was performed directly on purified PCR products using a BigDye v.3.1 Terminator Cycle Sequencing Ready Reaction kit on an ABI3730xl Genetic Analyzer (Applied Biosystems). Contiguous sequences were assembled and edited in Sequencher v. 5.2.4 (Gene Codes, Ann Arbor, MI, USA) and identity checked through the BLAST tool.
Phylogenetic analyses
For the 28S rDNA, an initial dataset was constructed combining the newly obtained sequences and the previously published FASTA file provided by Pérez-Ponce de León and Hernández-Mena (2019), which included all digenean sequences available in GenBank at that time, as well as some aspidogastrean sequences as outgroup. This was employed to assess the phylogenetic placement of the newly obtained sequences within Digenea and to determine their potential familial affiliation. The dataset was aligned using the MUSCLE algorithm implemented in Geneious 7.1.3 (Kearse et al. Reference Kearse, Moir, Wilson, Stones-Havas, Cheung, Sturrock, Buxton, Cooper, Markowitz, Duran, Thierer, Ashton, Meintjes and Drummond2012), with default parameters, and analysed under the Maximum Likelihood (ML) method using RAxML (Stamatakis Reference Stamatakis2014) via the CIPRES online platform (Miller et al. Reference Miller, Schwartz, Pickett, He, Klem, Scheuermann, Passarotti, Kaufman and O’Leary2015), with 1,000 bootstrap replicates. Subsequently, a second dataset was created by including selected sequences from most closely related clades to those in which the newly obtained sequences were grouped in the previous alignment; this refinement allowed for more detailed analyses of the new sequences’ phylogenetic relationships. The final alignment consisted of 35 sequences, including a single representative sequence of the new species and sequences representing members of the families Plagiorchiidae, Glypthelminthidae, Haematoloechidae, Ochetosomatidae, Telorchiidae, Dicrocoeliidae, and Omphalometridae, along with three sequences representing the families Microphallidae, Pleurogenidae, and Prosthogonimidae as the outgroup (Supplementary Table S1); after trimming to the shortest sequence, the final alignment was 1,266 bp long.
For the COI mtDNA, a dataset comprising 20 sequences was assembled, including one isolate of the new species, 18 species belonging to the Plagiorchiidae, Glypthelminthidae, Ochetosomatidae, Telorchiidae, and Dicrocoeliidae, and Prosthogonimus pellucidus (von Linstow 1873) Braun, 1901 (MZ169556; Prosthogonimidae) as outgroup (Supplementary Table S1). The ingroup and outgroup sequences were selected after the obtained results from the BLAST search and the 28S DNA analyses conducted previously; after trimming to the shortest sequence, the final COI mtDNA alignment was 421 bp long.
The final datasets of the 28S rDNA and COI mtDNA were separately aligned using the MUSCLE algorithm implemented on Geneious 7.1.3 (Kearse et al. Reference Kearse, Moir, Wilson, Stones-Havas, Cheung, Sturrock, Buxton, Cooper, Markowitz, Duran, Thierer, Ashton, Meintjes and Drummond2012) with default settings. The best-fitting evolutionary model estimated in the JModelTest software (Posada Reference Posada2008) was GTR + I + G for both datasets. Phylogenetic relationships were then reconstructed for each alignment under Bayesian inference (BI) and Maximum Likelihood (ML) methods. The BI analyses were performed using MrBayes 3.2 (Ronquist et al. Reference Ronquist, Teslenko, der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012) at the online platform CIPRES (Miller et al. Reference Miller, Schwartz, Pickett, He, Klem, Scheuermann, Passarotti, Kaufman and O’Leary2015). The Markov chain Monte Carlo (MCMC) was run with 106 generations, saving one tree every 100 generations, with a burn-in set to the first 25% of the trees. Only nodes with posterior probabilities (pp) greater than 0.95 were considered well-supported. The ML analyses were run in RAxML at the online platform CIPRES (Miller et al. Reference Miller, Schwartz, Pickett, He, Klem, Scheuermann, Passarotti, Kaufman and O’Leary2015) with 1,000 bootstrap replicates. Only nodes with bootstrap values greater than 90 were considered well-supported. The BI and ML trees were visualized in FigTree v. 1.3.1 software (Rambaut Reference Rambaut2009) and edited in CorelDRAW 2024. Pairwise genetic distances among and between sequences were calculated using the Kimura-2-parameter (K2P) model and a bootstrap procedure with 1000 replicates (Kimura Reference Kimura1980) in the MEGA11 program (Tamura et al. Reference Tamura, Stecher and Kumar2021).
Results
Description
Class Trematoda
Order Plagiorchiida La Rue, 1957
Family Plagiorchiidae Lühe, 1901
Atriumlateralis n. gen.
Type species, host, and locality: Atriumlateralis capivarensis n. sp. Kameyama, Ebert, Pérez-Ponce de León, Delgado & Silva from Osteogaster aenea (Gill) (Siluriformes: Callichthyidae), Capivarinha River (22°45′22.58″ S. 48°21′57.70″ W), Vitoriana district, municipality of Botucatu, São Paulo state, Brazil.
Diagnosis: Body elongate, foliaceous, slightly tapered ends, unspined tegument. Oral sucker round and subterminal. Ventral sucker ovoid. Forebody occupies c.1/3 body length. Prepharynx short. Pharynx small, globular, longer than wide. Oesophagus inconspicuous. Intestinal bifurcation immediately anterior to vitelline follicles. Intestinal caeca sinuous, terminating in the posterior third of the body. Testes two, entire, oblique, intercaecal, postequatorial. Anterior testis on the right side and posterior on the left side. Cirrus sac anterior to ventral sucker, seminal vesicle occupying c.1/2 of cirrus sac. Common genital pore immediately anterior to the ventral sucker, opening on the left margin of the body. Ovary entire, dextral, anterior and oblique to anterior testis. Mehlis gland present; Laurer’s canal not observed. Vitelline follicles forming two lateral groups, distributed from the caecal bifurcation to the posterior region of the posterior testis. Uterine coils restricted to the hindbody. Excretory vesicle not observed; in callichthyid fish (Callichthyidae) from Brazil.
Etymology: The name Atriumlateralis is derived from the Latin Atrium, meaning genital pore, and lateralis, meaning flank or side, referring to the position of the genital pore immediately anterior to the ventral sucker, opening on the left margin of the body.
Differential diagnosis: Atriumlateralis n. gen. resembles Choledocystus spp. by the combination of characters such as a foliaceous body, vitelline follicles in bunches, in two lateral groups, oblique testes, and a dextral ovary. However, the new genus differs from Choledocystus spp. by the lateral opening of the genital atrium; in all species of Choledocystus, the genital atrium is intercaecal; by the absence of small papillae or a sucker-like structure surrounding the genital atrium; and by having a smooth tegument. Species of Choledocystus possess triangular tegumental spines. Furthermore, species of Choledocystus are parasites of amphibians, whereas the new genus and species is described from a freshwater fish species.
Atriumlateralis capivarensis n. sp.
(https://zoobank.org/NomenclaturalActs/1e7b1046-6ceb-4879-9a43-04e7cf37f8a7)
Description (based on 9 adult specimens); Figures 1–3: Body elongated, foliaceous, longer than wide, slightly tapered ends, 4,043.8 ± 572.9 (3,451.1–4,982.1) by 1,253.4 ± 219.1 (960–1,568); length/width body ratio 3.3 ± 0.4 (2.8–3.8). Tegument smooth. Oral sucker round, ventral, subterminal, 271.5 ± 32.1 (215.6–303.6) by 306.07 ± 38.21 (226–356.5). Ventral sucker ovoid, wider than long, 382.7 ± 39.9 (306.3–436.0) long and 452.0 ± 67.0 (325–532.1) wide. Oral/ventral sucker length ratio 0.7 ± 0.1 (0.6–0.9) and width 0.7 ± 0.1 (0.6–0.8). Forebody 1,246.4 ± 196.6 (864.5–1,523.0), hindbody 2,817.5 ± 428.5 (2,198.4–3,590.7). Forebody occupying 30.8 ± 3.3 (25.0–36.4) of body length. Prepharynx short, 37.9 ± 5.5 (34.0–41.8). Pharynx globular, muscular, 150.2 ± 31.5 (101.5–189.7) by 124.9 ± 19.9 (91.8–150.3). Oesophagus short, 72.2 ± 31.1 (42.1–141.8). Intestinal bifurcation immediately anterior to vitelline follicles. Caeca narrow, sinuous, ending 457.3 ± 215.5 (304.9–609.7) from the posterior end. Common genital pore immediately anterior to the ventral sucker, opening on the left margin of the body. Testes two, entire, oblique, round, intercaecal, posterior to the ovary, postequatorial. Anterior testis dextral, 398.87 ± 82.28 (274.5–557) by 414.53 ± 89.91 (305.1–573). Posterior testis sinistral, 499.37 ± 90.67 (393.6–704.7) by 464.74 ± 83.84 (323–590.2). The vas efferens from each testis joins forming vas deferens, which connects to cirrus sac base. Cirrus sac anterior to ventral sucker, lacking spines, 571.5 ± 55.4 (489.7–656.8) by 109.6 ± 18.3 (71.9–129.1). Seminal vesicle in the proximal end of the cirrus sac, 326.3 ± 42.7 (272.9–384.8) by 87.6 ± 17.6 (64.3–114.4). Ovary rounded, dextral, posterior to ventral sucker and oblique to anterior testis, 394.8 ± 31.1 (357.3–462.6) by 293.9 ± 35.7 (244.5–342.3). Mehlis gland present. Laurer’s canal not observed. Vitelline follicles forming bunches, in two lateral groups, distributed in intracaecal, caecal, and extracaecal area, extending from caecal bifurcation level, 563.9 ± 88.0 (440.4–676.3) from anterior body end to posterior region of posterior testis, 1,044.2 ± 25.5 (1,026.2–1,062.2) of body end. Uterus in the hindbody, descendent loops at the right side of the body and ascendent loops at the left side, extending from the posterior body end to the ventral sucker. Metraterm slightly muscular, running anteriorly to the cirrus sac, opening into the genital atrium. Eggs, thick-shelled, unoperculated, 32.8 ± 3.4 (26–44) by 12.9 ± 1.9 (9.1–17.7). Excretory vesicle not observed.
Schematic drawing of Atriumlateralis capivarensis n. gen. et n. sp. collected from the intestine of Osteogaster aenea (Gill) from the Capivarinha River, Vitoriana district, municipality of Botucatu, São Paulo state, Brazil. (A) Whole worm, ventral view; (B) Detail of cirrus sac and metraterm; (C) Detail of ovary region highlighting some vitelline follicles, vitelline ducts, Mehlis gland, ovary, and the beginning of uterus (eggs were not represented inside the uterus); and (D) Detail of egg, highlighting the presence of the miracidium. Scales in micrometres.
Figure 1. Long description
Panel A shows the whole worm in ventral view with a scale bar of 1000 micrometres. At the anterior end is an oral sucker followed by a pharynx. Two intestinal caeca extend down the sides of the body. The central region contains a cirrus sac, a ventral sucker, and two large testes. The posterior half is filled with horizontal uterine loops. Dark vitelline follicles are clustered along the lateral margins. Panel B provides a detail of the cirrus sac and metraterm with a scale bar of 200 micrometres, showing the internal seminal vesicle and the muscular walls of the terminal genitalia. Panel C illustrates the ovary region with a scale bar of 500 micrometres. It features a large oval ovary adjacent to a smaller, granular Mehlis gland. Vitelline ducts connect to this complex from clusters of dark follicles on either side. Panel D shows a single oval egg with a scale bar of 10 micrometres, containing a shaded miracidium.
Atriumlateralis capivarensis n. gen. et n. sp. collected in the intestine of Osteogaster aenea (Gill) from the Capivarinha River, Vitoriana district, municipality of Botucatu, São Paulo state, Brazil. (A) Whole worm, ventral view; (B) Anterior region, highlighting the oral sucker, pharynx and beginning of the intestinal cecum; (C) Detail of the cirrus pouch and metraterm; (D) Detail of the ventral sucker and ovary; (E) Details of the vitelline duct containing vitelline cells; (F) Detail of the eggs, highlighting the presence of the miracidium; and (G) Details of the testes. cs, cirrus sac; e, eggs; gp, genital pore; in, intestinal cecum; m, miracidium; me, metraterm; os, oral sucker; ov, ovary; ph, pharynx; t, testis; u, uterus; vi, vitelline follicles; vs, ventral sucker.
Figure 2. Long description
The figure consists of seven panels labeled A through G.
* Panel A: A full-body ventral view of the worm with a 1 millimeter scale bar. The body is elongated and tapered at both ends.
* Panel B: A close-up of the anterior region. At the top is the oral sucker (o s), followed by the pharynx (p h) and the branching intestinal cecum (i n). Vitelline follicles (v i) are visible on the sides. Scale bar is 200 micrometers.
* Panel C: Detail of the reproductive structures. Labels indicate the metraterm (m e), eggs (e), cirrus sac (c s), genital pore (g p), ventral sucker (v s), and vitelline follicles (v i). Scale bar is 200 micrometers.
* Panel D: Detail of the mid-body showing the circular ventral sucker (v s), the dark oval ovary (o v), and several eggs (e). Scale bar is 200 micrometers.
* Panel E: High-magnification view of a vitelline duct containing a row of distinct vitelline cells. Scale bar is 20 micrometers.
* Panel F: High-magnification view of an oval egg (e) containing a miracidium (m). Scale bar is 20 micrometers.
* Panel G: Detail of the posterior region showing two large, dark, rounded testes (t). Scale bar is 200 micrometers.
Atriumlateralis capivarensis n. gen. et n. sp. collected from the intestine of Osteogaster aenea (Gill) from the Capivarinha River, Vitoriana district, municipality of Botucatu, São Paulo state, Brazil. (A) Whole worm, ventral view, showing the lack of spines on the tegument; (B) Detail of the oral sucker; and (C) Ventral sucker region, highlighting the position of the genital pore. gp, genital pore; os, oral sucker; vs, ventral sucker. Scales in micrometres.
Taxonomic summary
Type host: Osteogaster aenea (Gill) (Siluriformes: Callichthyidae).
Type locality: Capivarinha River (22°45′22.58″ S. 48°21′57.70″ W), Vitoriana district. municipality of Botucatu, São Paulo state. Brazil.
Site of infection: Intestine.
Infestation parameters: Prevalence 54% (27 of 50 fish studied); Mean abundance 0.34 ± 0.09 (0–3) trematodes per analysed host; Mean intensity of infection 1.30 ± 0.17 (1–3) trematodes per infected host.
Deposited specimens: Holotype (CHIOC 41110) and paratypes (CHIOC 41111, 41112, 41113a-b, 41114) in Helminthological Collection of the Oswaldo Cruz Institute, Rio de Janeiro State, Brazil. Paratypes (CHIBB 997L - 1000L) and Hologenophores (CHIBB 994L - 996L) in the Helminthological Collection of the Institute of Biosciences, São Paulo State University, UNESP, Botucatu municipality, São Paulo State, Brazil.
Representative DNA sequences: GenBank accession numbers: 28S rDNA – PX927347 to PX927351; COI mtDNA – PZ261577 to PZ261579.
Etymology: The specific epithet capivarensis refers to the locality where the new species was first discovered, the ‘Capivarinha’ River.
Molecular analyses
Five partial sequences of the 28S rDNA gene of Atriumlateralis capivarensis n. gen. et n. sp. (1,129 to 1,238 bp in length) were successfully amplified. The BLAST search showed that the newly obtained sequences presented the closest identity percentage with Choledocystus hepaticus Lutz, 1928 (AY875679; 97.93%) found infecting Rhinella marina Linnaeus, 1758 (= Bufo marinus) in Mexico (Razo-Mendívil et al., Reference Razo-Mendívil, León-Regagnon and Pérez-Ponce de León2006), followed by Infidum infidum (Gomes de Faria, 1910) Travassos, 1916 (MW317230; 97.40%) found infecting Bothrops moojeni Hoge, 1966 in Brazil (Müller et al. Reference Müller, Emmerich, de Alcantara, Ungari, Ebert, Morais, O’Dwyer and da Silva2021).
The 28S rDNA phylogenetic tree showed that the family Plagiorchiidae was resolved as non-monophyletic (Figure 4). The new sequence of Atriumlateralis capivarensis n. gen. et n. sp. was placed as the sister species of Choledocystus spp. with high nodal support value; the sequences of Infidum spp. were yielded as a sister group of the latter two. The interspecific divergence between the new species and C. hepaticus was 2.1% and varied from 2.5% to 3.3% in relation to Infidum spp.
Bayesian topology based on partial 28S rDNA sequences of Plagiorchoidea species. GenBank accession numbers are after species names. Newly sequenced isolates are in bold. The support values are included before the nodes as posterior probabilities for BI analysis, followed by bootstraps for the ML analysis. Only nodes with posterior probabilities >0.95 and bootstrap scores >90 are considered well-supported. Branch length scale bar indicates the number of substitutions per site.
Figure 4. Long description
A horizontal phylogenetic tree diagram. A legend in the top-left corner defines node support: black circles for P P greater than or equal to 0.95 and Boot greater than or equal to 90; grey circles for P P greater than or equal to 0.95 or Boot greater than or equal to 90; and white circles for P P less than or equal to 0.95 and Boot less than or equal to 90.
From top to bottom, the major clades are:
* Dicrocoeliidae: Including Infidum similis and Infidum infidum.
* Plagiorchiidae: Including Atriumlateralis capivarensis n. gen. et n. sp. and Choledocystus hepaticus.
* Telorchiidae: Including Telorchis assula and Telorchis bonnerensis.
* Cephalogonimidae: Cephalogonimus americanus.
* Ochetosomatidae: A large cluster including Ochetosoma kansense, Dasymetra nicolli, and Lechriorchis tygarti.
* Haematoloechidae: Haematoloechus complexus and Haematoloechus meridionalis.
* Glypthelminthidae: Glypthelmins quieta, Glypthelmins facioi, and Glypthelmins californiensis.
* A mixed section containing Choanocotylidae (Choanocotyle nematoides), Plagiorchiidae (Aptorchis megacetabulus), Brachycoeliidae (Parabrachycoelium longicaecum and Brachycoelium salamandrae), and Dicrocoeliidae (Mesocoelium).
* Macroderoididae: Rauschiella tineri and Rauschiella poncedeleoni.
* A second Plagiorchiidae cluster including Dolichosaccus rastellus, Haplometra cylindracea, and Plagiorchis species.
* Omphalometridae: Neoglyphes obolevi and Rubenstrema exasperatum.
* Outgroup: Maritrema neomi, Pleurogenes claviger, and Prosthogonimus ovatus at the bottom.
A scale bar at the bottom left indicates 0.03 substitutions per site.
Three COI mtDNA partial sequences were successfully amplified (436 to 448 bp in length). The BLAST search showed the highest identity percentage of the new species with Cephalogonimus sp. (PV605655; 82.23%), represented by a sequence from a cercaria found in the snail Ampullaceana balthica (Linnaeus, 1758) in Germany (Hüsken et al. Reference Hüsken, Schwelm, Rückert and Sures2025), followed by C. hepaticus (HM137635; 82.00%) found infecting R. marina in Mexico (Razo-Mendívil et al. Reference Razo-Mendívil and Pérez-Ponce de León2011).
The maximum likelihood (ML) and Bayesian inference (BI) analyses of the final COI mtDNA alignment (Figure 5) yielded different phylogenetic topologies, with most clades exhibiting low nodal support values. The representative sequence of Atriumlateralis capivarensis n. gen. et n. sp. occupied different positions within the reconstructions. In the ML analysis, Atriumlateralis capivarensis n. gen. et n. sp. was resolved as the sister species of C. hepaticus, forming a sister clade to a group comprising Ochetosoma spp., Paralechriorchis syntomentera (Sumwalt, 1926) Byrd & Denton, 1938, Telorchis sp., Infidum similis Travassos, 1916, and Cephalogonimus americanus Stafford, 1902 (Figure 5). In contrast, in the BI analysis, Atriumlateralis capivarensis n. gen. et n. sp. did not cluster with C. hepaticus. Instead, C. hepaticus was recovered as the earliest diverging lineage relative to a clade comprising Atriumlateralis capivarensis n. gen. et n. sp., I. similis, C. americanus, Telorchis sp., P. syntomentera, and species of Ochetosoma, albeit with a very low nodal support value (tree not shown). As in the 28S rDNA phylogenetic analyses, the Plagiorchiidae were resolved as non-monophyletic, with Plagiorchis multiglandularis Semenov, 1927 and a sequence identified as Choledocystus incurvatum (Nasir, 1966) forming a separate clade from the one comprising Atriumlateralis capivarensis n. gen. et n. sp. and C. hepaticus. Interestingly, the two sequences of Choledocystus used in the analyses, C. incurvatum and C. hepaticus, did not cluster together, suggesting that these sequences are not congeneric.
Maximum likelihood topology based on partial COI mtDNA sequences of Plagiorchoidea species. GenBank accession numbers are after species names. The newly generated sequence is shown in bold. The support values are included before the nodes as posterior probabilities for BI analysis, followed by bootstraps for the ML analysis. Only nodes with posterior probabilities >0.95 and bootstrap scores >90 are considered well-supported. Branch length scale bar indicates the number of substitutions per site.
Figure 5. Long description
A phylogenetic tree with a root on the left and branches extending to the right. A legend at the top left defines node support: black circles for P P greater than or equal to 0.95 and Boot greater than or equal to 90, grey circles for P P greater than or equal to 0.95 or Boot greater than or equal to 90, and white circles for P P less than or equal to 0.95 and Boot less than or equal to 90.
From top to bottom, the clades are:
- Ochetosomatidae: Includes Ochetosoma kansense L C 8 1 1 3 8 0, Ochetosoma heterocoelium P V 5 2 2 1 8 9, Paralechriorchis syntomentera M W 0 4 2 9 8 2, and Ochetosoma elongatum L C 8 3 1 8 0 7.
- Telorchiidae: Telorchis species L C 8 1 8 8 9 2.
- Dicrocoeliidae: Infidum similis K U 7 2 6 8 8 3.
- Cephalogonimidae: Cephalogonimus americanus P P 9 9 3 5 9 6.
- Plagiorchiidae: Includes Atriumlateralis capivarensis n. gen. et n. sp. P Z 2 6 1 5 7 7 in bold, Choledocystus hepaticus H M 1 3 7 6 3 6, Choledocystus incurvatum M H 0 9 4 8 0 9, and Plagiorchis multiglandularis P V 5 6 3 7 8 2.
- Dicrocoeliidae (second instance): Mesocoelium species L C 6 2 6 4 7 8 and Mesocoelium species L C 6 3 3 5 4 5.
- Glypthelminthidae: Includes Glypthelmins species M W 1 3 9 2 5 5, Glypthelmins quieta L C 7 9 4 2 0 6, Glypthelmins pennsylvaniensis H M 1 3 7 6 2 3, Glypthelmins brownorumae H M 1 3 7 6 2 7, Glypthelmins tuxtlasensis H M 1 3 7 6 2 9, and Glypthelmins facioi H M 1 3 7 6 2 5.
- Outgroup: Prosthogonimus pellucidus M Z 1 6 9 5 5 6 at the bottom.
A scale bar at the bottom left indicates 0.6 substitutions per site.
The COI mtDNA interspecific genetic divergence between the new species and C. hepaticus, I. infidum, and C. incurvatum was 26.2%, 25.6%, and 31.3%, respectively. The genetic divergence between C. hepaticus and C. incurvatum was 22.2%.
Discussion
Atriumlateralis capivarensis n. gen. et n. sp. represents the first record of an adult digenean infecting O. aenea. However, for other Corydoradinae fishes, to which O. aenae belongs, few reports are available. Barros et al. (Reference DMA, Virgilio and LJS2024) identified two adult digenean species in the intestine of Corydoras multiradiatus (Orcés) [syn. Chaenothorax multiradiatus (Orcés)] from the Juruá River, in the western Amazon; although the authors assigned those specimens to Prosthenystera obesa (Diesing, 1850) and Digenea gen. sp., they did not provide any taxonomic descriptions or molecular data of those helminths. Recently, Creptotrema ocloya (Liquin et al. Reference Liquin, Gilardoni, Cremonte, Saravia, Cristóbal and Davies2022) (Syn. Auriculostoma ocloya Liquin et al., Reference Liquin, Gilardoni, Cremonte, Saravia, Cristóbal and Davies2022) was reported in Corydoras micracanthus Regan, 1912 from a creek affluent to the Lesser River, Salta, Argentina (Liquin et al. Reference Liquin, Gilardoni, Cremonte, Saravia, Cristóbal and Davies2022). No other study has reported adult digeneans in any Corydoradinae fishes (see Ferreira et al. Reference Ferreira, Passador and Tavares-Dias2019 for a review).
Interestingly, species of Choledocystus, which were retrieved in most analyses as the sister taxa of the new genus and species herein described, are parasites of amphibians, particularly in some species of Neotropical toads and frogs (see Razo-Mendívil et al. Reference Razo-Mendívil, León-Regagnon and Pérez-Ponce de León2006). Morphologically, Atriumlateralis capivarensis n. gen. et n. sp. is readily distinguished from Choledocystus and from other members of Plagiorchiidae by its foliaceous body, vitelline follicles arranged in clustered groups forming two lateral fields that extend through both intra and extracaecal regions, a ventral sucker larger than the oral sucker, oblique testes, dextral ovary, laterally opening genital atrium, and a smooth, unspinous tegument. Moreover, phylogenetic analyses based on both 28S rDNA and COI mtDNA sequences yielded Atriumlateralis capivarensis n. gen. et n. sp. as a well-supported monophyletic lineage, positioned as sister to the clade containing sequences of Choledocystus. The genus Choledocystus comprises species parasitizing the hepatic system and intestine of South American amphibians (Razo-Mendívil and Pérez-Ponce de León Reference Razo-Mendívil and Pérez-Ponce de León2008; Tkach Reference Tkach, Bray, Gibson and Jones2008); species in this genus are diagnosed by having traits such as a spined tegument, a submedian genital pore located near the caecal bifurcation or immediately anterior to the ventral sucker and surrounded by a small sucker-like structure, absence of a seminal receptacle, a uterus extending laterally beyond the intestinal caeca and vitellaria, and an elongate to sacculate cirrus sac containing a bipartite or coiled seminal vesicle (Hamann et al. Reference Hamann, Leon-Regagnon, Fernández and Gonzalez2020; Razo-Mendívil and Pérez-Ponce de León Reference Razo-Mendívil and Pérez-Ponce de León2008; Tkach Reference Tkach, Bray, Gibson and Jones2008). These characteristics are entirely incongruent with the morphology described here for Atriumlateralis capivarensis n. gen. et n. sp.
Overall, the phylogenetic relationships uncovered in this study are consistent with previous proposals, especially related to the non-monophyletic status of Plagiorchiidae (Emmerich et al. Reference Emmerich, Alcantara, Úngari, O’Dwyer, Pérez-Ponce de León and Silva2025; Martínez-Salazar et al. Reference Martínez-Salazar, Rosas-Valdez, Gregory and Violante-González2016; Müller et al. Reference Müller, Emmerich, de Alcantara, Ungari, Ebert, Morais, O’Dwyer and da Silva2021; Pérez-Ponce de León and Hernández-Mena Reference Pérez-Ponce de León and Hernández-Mena2019), in which the new species was placed. Despite its systematic relevance, the Plagiorchiidae yet remains one of the least resolved and explored digenean families (Ndiaye et al. Reference Ndiaye, Quilichini, Tkach, Greiman and Marchand2013). As a consequence, numerous uncertainties and debates persist regarding the placement of several genera and their evolutionary relationships (Ndiaye et al. Reference Ndiaye, Quilichini, Tkach, Greiman and Marchand2013; Tkach Reference Tkach, Bray, Gibson and Jones2008; Tkach et al. Reference Tkach, Pawlowski and Mariaux2000). The prevalence of homoplastic traits, the remarkable taxonomic diversity, the absence of consistent diagnostic features, the poorly defined intraspecific variation, and the generally low host specificity have all contributed to the challenges surrounding the systematics of Plagiorchiidae (Suleman et al. Reference Suleman, Khan, Tkach, Muhammad, Zhang and Zhu2019; Tkach Reference Tkach, Bray, Gibson and Jones2008; Zikmundová et al. Reference Zikmundová, Georgieva, Faltýnková, Soldánová and Kostadinova2014). The addition of sequence data of more representative species of plagiorchiids, and particularly of members of Plagiorchiidae, will enhance our understanding of the evolution of this group of digeneans.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X26101953.
Funding
This study was supported by the Brazilian National Research Council – CNPq (311635-2021/0); São Paulo Research Foundation – FAPESP (2024/15651-1; 2024/05688-5; 2022/12376-4); and PROPG/PROPe – UNESP (04/2022).
Competing interests
The authors declare that they have no conflict of interest.
Ethical standard
All applicable institutional and national guidelines for the care and use of animals were followed. Fishes were collected under the authorization of the Instituto Chico Mendes de Conservação da Biodiversidade (SISBIO #60640–1). All procedures followed the recommendations and approval of the Ethical Commission for Animal Experimentation from the São Paulo State University (UNESP), Institute of Biosciences, Botucatu, Brazil (CEUA 9415260520). According to Brazilian laws, species registration for scientific research purposes was carried out at SisGen (A63C106).
