Introduction
Avian schistosomatids (Digenea: Schistosomatidae) are blood flukes inhabiting the bloodstream of aquatic birds of different orders (Horák and Kolářová Reference Horák and Kolářová2011; Horák et al. Reference Horák, Mikeš, Lichtenbergová, Skála, Soldánová and Brant2015). Within this family, there are 17 named genera, although several taxa remain to be described. Remarkably, 13 of the 17 named genera use aquatic birds as their definitive hosts, most of them included as part of the avian clade DAS (derived avian schistosomatids), although certain taxa are part of early-diverging clades associated with marine birds (Brant and Loker Reference Brant and Loker2013; Ebbs et al. Reference Ebbs, Loker, Bu, Locke, Tkach, Devkota, Flores, Pinto and Brant2022). These trematodes have been recorded parasitizing 16 families of gastropods within Caenogastropoda and Heterobranchia, alternating between freshwater and marine life cycles (Flores et al. Reference Flores, Viozzi, Casalins, Loker and Brant2021; Horák et al. Reference Horák, Bulantová, Mikeš, Toledo and Fried2024; Lorenti et al. Reference Lorenti, Brant, Gilardoni, Diaz and Cremonte2022; Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Collado, Muñoz and Moreno2022, Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a, Reference Oyarzún-Ruiz, Navarro, Moreno and Landaeta-Aquevequeb). Nevertheless, an important proportion of these schistosomatids have no data about their life cycles or evolutionary relationships, especially those from developing countries in South America and Africa (Horák et al. Reference Horák, Bulantová, Mikeš, Toledo and Fried2024).
These parasites are relevant to human health, causing a seasonal illness called cercarial dermatitis or ‘swimmer’s itch’, which is characterized by a cutaneous inflammation and itching lasting from a few hours to several days (Brant and Loker Reference Brant and Loker2013; Horák and Kolářová Reference Horák and Kolářová2011; Horák et al. Reference Horák, Mikeš, Lichtenbergová, Skála, Soldánová and Brant2015). Besides, a systemic clinical presentation has been suggested because of organic migration by schistosomula, even causing neurological impairment in murine models (Lichtenbergová et al. Reference Lichtenbergová, Lassmann, Jones, Kolářová and Horák2011). This condition is mostly associated with freshwater ecosystems such as lakes and ponds (Horák and Kolářová Reference Horák and Kolářová2011; Lashaki et al. Reference Lashaki, Teshnizi, Gholami, Fakhar, Brant and Dodangeh2020), although there are additional reports in brackish and marine waterbodies (Brant et al. Reference Brant, Cohen, James, Hui, Hom and Loker2010; Horák et al. Reference Horák, Mikeš, Lichtenbergová, Skála, Soldánová and Brant2015; Kolářová Reference Kolářová2007). Depending on certain environmental conditions, this affection could occur as outbreaks, especially in disturbed waterbodies such as eutrophic areas (Horák and Kolářová Reference Horák and Kolářová2011; Selbach et al. Reference Selbach, Soldánová and Sures2016). Species of the genus Trichobilharzia have been reported as the main agents of these outbreaks (Horák et al. Reference Horák, Mikeš, Lichtenbergová, Skála, Soldánová and Brant2015; Kolářová Reference Kolářová2007).
Genus Trichobilharzia is the richest species taxon within Schistosomatidae family, with over 40 species, also considered as the main causal agent of cercarial dermatitis (Horák et al., Reference Horák, Mikeš, Lichtenbergová, Skála, Soldánová and Brant2015). In South America, there are three named species related to this genus: Trichobilharzia jequitibaensis Leite, Costa, and Costa, Reference Leite, Costa and Costa1978; Trichobilharzia physellae (Talbot 1936) McMullen and Beaver, Reference McMullen and Beaver1945; and Trichobilharzia querquedulae McLeod, Reference McLeod1937 (Ebbs et al. Reference Ebbs, Loker, Davis, Flores, Veleizan and Brant2016; Flores et al. Reference Flores, Brant and Loker2015; Leite et al. Reference Leite, Costa and Costa1978; Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a; Szidat Reference Szidat1951). The latter could be considered a low richness; however, there are several other putative new species, mostly from Argentina, Brazil, and Chile, remaining pending to be formally described (Flores et al. Reference Flores, Brant and Loker2015; Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a; Pinto et al. Reference Pinto, Brant and Melo2014, Reference Pinto, Pulido-Murillo, de Melo and Brant2017). Following the available literature in the Neotropical realm, it seems that waterfowl represent the main hosts of these schistosomatids. Apart from a single record from Cuba (Sánchez et al. Reference Sánchez, Alba, García, Cantillo, Castro and Vázquez2018), there is no additional research about schistosomatids in the rest of South American countries despite the richness of waterfowl in the region (Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a). Furthermore, probably because of the recondite sites used by these parasites, e.g., mesenteric and nasal blood vessels, they are often overlooked in helminthological surveys (Flores et al. Reference Flores, Viozzi, Casalins, Loker and Brant2021; Horák et al. Reference Horák, Mikeš, Lichtenbergová, Skála, Soldánová and Brant2015; Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Collado, Muñoz and Moreno2022, Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a). This is coupled with the current requirement of an integrative approach, i.e., morphological and molecular tools, to properly characterize parasites (Horák et al. Reference Horák, Mikeš, Lichtenbergová, Skála, Soldánová and Brant2015; Selbach et al. Reference Selbach, Jorge, Dowle, Bennett, Chai, Doherty, Eriksson, Filion, Hay, Herbison, Lindner, Park, Presswell, Ruehle, Sobrinho, Wainwright and Poulin2019). In consequence, the finding and description of new species of avian schistosomatids appear to be overlooked, a situation which seems exacerbated in the Neotropical region where aquatic birds are, in general, scarcely studied (Flores et al. Reference Flores, Brant and Loker2015; Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a).
In Chile, the study of helminths in waterfowl is restricted to a few reports (e.g., Oyarzún-Ruiz and González-Acuña, Reference Oyarzún-Ruiz and González-Acuña2021; Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a; Salazar-Silva et al. Reference Salazar-Silva, Aravena, Zamorano-Uribe, Andrade-Hernández, Silva-de la Fuente, Cicchino, Mironov, Moreno and Oyarzún-Ruiz2025). Concretely, the mention of avian schistosomatids in this group of birds has been widely neglected until recently (Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a).
Thus, this study aimed to describe a new species retrieved from an endemic duck species, and the importance of such records from the systematics of avian schistosomatids and public health points of view is discussed.
Material and Methods
Sampling and dissection of waterfowl
Between April 2018 and July 2020, and April 2023 to April 2024, 23 Chiloe wigeons (Mareca sibilatrix) from Ñuble region, Chile (Chillán [n = 1], Portezuelo [n = 6], Nebuco [n =16]), and Buenos Aires, Argentina (n = 1), and 3 Cinnamon teals (Spatula cyanoptera) from Chillán, Ñuble region, Chile, were dissected (Figure 1). Twenty-four birds were categorized as adult birds and three as juveniles. The birds from Chile were hunted by certified hunters, following the Chilean hunting law Ley de Caza N° 19.473 (SAG 2018); meanwhile, the bird from Argentina was found dead on the road.
Figure 1. Map showing sampled localities in Chile and Argentina.
The nasal mucosa and turbinates, heart and associated blood vessels, mesenteric blood vessels and their ramifications, liver, gallbladder, lungs, kidneys, and vessels from the cranial vault were examined for the presence of avian schistosomatids following Lutz et al. (Reference Lutz, Tkach, Weckstein and Webster2017) and Oyarzún-Ruiz et al. (Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a). Schistosomatid worms were relaxed in citrated saline, fixed, and preserved in 80% ethanol. Worms considered for molecular analyses were preserved in absolute ethanol and kept at −20°C (Horák et al. Reference Horák, Schets, Kolářová, Brant and Liu2012; Kolářová et al. Reference Kolářová, Horák and Skírnisson2010). An additional pool of worms was preserved in 80% ethanol for scanning electron microscopy (SEM) (Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Collado, Muñoz and Moreno2022). Small fragments of worms aimed for SEM were cut and preserved for molecular studies. Parasitological descriptors such as sample prevalence (P), mean intensity (MI), and mean abundance (MA), including range of infection (R), were estimated and interpreted following Bush et al. (Reference Bush, Lafferty, Lotz and Shostak1997) and Reiczigel et al. (Reference Reiczigel, Marozzi, Fábián and Rózsa2019).
Morphological identification of avian schistosomatids
Adult worms were stained with Alum carmine, dehydrated on increasing concentrations of ethanol (70–100%), cleared in oil clove, and mounted in Canada balsam (Lutz et al. Reference Lutz, Tkach, Weckstein and Webster2017). Avian schistosomatids were measured using Motic Images Plus 2.0 software associated with the light microscope MOTIC BA310. Line drawings of the worms were performed with the aid of a drawing tube on a Leica DM 1000 light microscope. Morphological traits and measurements of these worms were compared with the taxonomic keys and descriptions by McLeod (Reference McLeod1937), McLeod and Little (Reference McLeod and Little1942), McMullen and Beaver (Reference McMullen and Beaver1945), Leite et al. (Reference Leite, Costa and Costa1978), Gibson et al. (Reference Gibson, Jones, Bray, Gibson, Jones and Bray2002), Brant and Loker (Reference Brant and Loker2009), Davis et al. (Reference Davis, Blair and Brant2022), and Oyarzún-Ruiz et al. (Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a). SEM was performed with the scanning electron microscope HITACHI SU 3500 following the settings described by Oyarzún-Ruiz et al. (Reference Oyarzún-Ruiz, Thomas, Santodomingo, Collado, Muñoz and Moreno2022).
DNA extraction, PCR, and phylogenetic analyses
Genomic DNA (gDNA) was extracted using DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. The quantity and quality of gDNA for each extracted sample were measured with an EpochTM Microplate Spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). Samples with values between 1.6 and 2.0 and an absorbency proportion A260/A280 were considered pure and suitable for PCR amplification (Khare et al. Reference Khare, Raj, Chandra and Agarwal2014). gDNA was kept at −20°C until molecular analyses were performed.
A Touchdown PCR was performed to amplify partial sequences of the cytochrome c oxidase subunit I gene (hereafter COI; expected length of band 600–1,000 bp) and 28S rRNA gene (hereafter 28S; expected length of band 1,500 bp) (Brant and Loker Reference Brant and Loker2009; Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Collado, Muñoz and Moreno2022, Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a). Primers for COI; Cox1_schis’_5′ and Cox1_schis’_3′ (Brant and Loker Reference Brant and Loker2009; Lockyer et al. Reference Lockyer, Olson, Østergaard, Rollinson, Johnston, Attwood, Southgate, Horák, Snyder, Le, Agatsuma, McManus, Carmichael, Naem and Littlewood2003), and for 28S; U178, L1642, DIG12 internal, and ECD2 internal (Lockyer et al. Reference Lockyer, Olson, Østergaard, Rollinson, Johnston, Attwood, Southgate, Horák, Snyder, Le, Agatsuma, McManus, Carmichael, Naem and Littlewood2003; Olson et al. Reference Olson, Cribb, Tkach, Bray and Littlewood2003; Tkach et al. Reference Tkach, Pawlowski and Mariaux2000), PCR mixture (Dvořák et al. Reference Dvořák, Vaňáčová, Hampl, Flegr and Horák2002; Horák et al. Reference Horák, Schets, Kolářová, Brant and Liu2012), and PCR thermal conditions (Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a) were stated following the above-mentioned authors. Amplicons were submitted to electrophoresis in 2% agarose gel, stained with GelRed® (Biotum, Tehran, Iran), and visualized in an ENDUROTM GDS UV transilluminator (Labnet International, Eidson, NJ, USA). Amplicons of expected size were purified and sequenced in both directions at Macrogen (South Korea).
The sequences were verified and edited with Geneious Prime® v. 2021.2.2 to get the consensus sequences. A basic local alignment search was performed with the BLASTn tool (https://blast.ncbi.nlm.nih.gov/), and orthologous sequences were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) to build multiple alignments with the MAFFT algorithm using Auto parameters (Katoh and Standley Reference Katoh and Standley2013) (Supplementary Table S1). Informative regions were extracted using BMGE, using default parameters (Criscuolo and Gribaldo Reference Criscuolo and Gribaldo2010).
The phylogenetic reconstructions were performed through the methods of Maximum Likelihood (ML) and Bayesian inference (BI) in IQ-Tree v1.6.12 (Nguyen et al., Reference Nguyen, Schmidt, von Haeseler and Minh2015) and MrBayes v3.2.6 (Ronquist et al. Reference Ronquist, Teslenko, van der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012), respectively. The best nucleotide substitution models for the analysis of ML were selected with the commands in ModelFinder ‘-m MFP+MERGE’ for COI (Kalyaanamoorthy et al. Reference Kalyaanamoorthy, Minh, Wong, von Haeseler and Jermiin2017). To assess the robustness of the phylogenetic tree, 1,000 pseudo-replicates of ultrafast bootstrapping (UFBoot) were performed using a combination of stochastic perturbation and rapid hill-climbing methods. UFBoot values <70 were considered as non-significant, between 70 and 94 as moderate support, and >95 as strong statistical support (Minh et al. Reference Minh, Nguyen and von Haeseler2013). In the BI analysis, the following commands were employed to select the best evolutionary models: the COI dataset was split into three codon positions (position-1, position-2, and position-3) and the command ‘nst=mixed rates=invgamma’ was executed, and for 28S datasets the command ‘nst=mixed rates=gamma’ was used (Huelsenbeck et al. Reference Huelsenbeck, Larget and Alfaro2004; Ronquist et al. Reference Ronquist, Teslenko, van der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012). Two independent tests of 5 × 106 generations (gen) and for MCMC chains were run simultaneously, sampling trees each 1,000 gen, and removing the first 25% as burn-in. The posterior Bayesian probabilities with values ≥0.71 were considered as a strong statistical support (Huelsenbeck and Rannala Reference Huelsenbeck and Rannala2004). Software Tracer v1.7.1. will be used to confirm the correlation and effective sample size (ESS) of the Markov chains (Rambaut et al. Reference Rambaut, Drummond, Xie, Baele and Suchard2018). A consensus tree for ML and BI trees was constructed with Geneious Prime® v. 2021.2.2 following the approach outlined by Santodomingo et al. (Reference Santodomingo, Robbiano, Thomas, Parragué-Migone, Cabello-Stom, Vera-Otarola, Valencia-Soto, Moreira-Arce, Moreno, Hidalgo-Hermoso and Muñoz-Leal2022).
The genetic distances between the sequences generated in the present study and those from GenBank were estimated using MEGA12 (Kumar et al. Reference Kumar, Stecher, Suleski, Sanderford, Sharma and Tamura2024). The sequences obtained in the present study were deposited in the NCBI GenBank database (accession numbers: PX648553-PX648558 for the COI gene, and PX657201-PX657207 for the 28S gene).
The worms, slides, and vouchers, including a symbiotype, were deposited in the collection of Museo de Zoología, Universidad de Concepción, Chile (MZUC-UCCC 48242-48245, 48678), and Museum of Southwestern Biology Division of Parasites, New Mexico, USA (catalogue number MSB:Para:27328).
This paper and the nomenclatural act have been registered in Zoobank (www.zoobank.org), the official register of the International Commission on Zoological Nomenclature. The LSID (Life Science Identifier) number of the publication is: urn:lsid:zoobank.org:act:A4435867-4B47-4B70-95AE-90F614E5A41B.
Results
Nineteen out of 24 Chiloe wigeon (P = 79.2%, MA = 22.22, MI = 28.57, R = 1–57) were parasitized by visceral schistosomatids found mostly in the mesenteric blood vessels and liver, followed by kidneys, and more rarely from heart, lungs, and gallbladder. Only male worms were characterized; meanwhile, female worms were few and in deteriorated condition, which precluded an appropriate description. Both adult (n = 17/21) and juvenile (n = 2/3) birds were found parasitized. Measurements of worms are shown in Table 1. In relation to the dissected Cinnamon teals, all three birds were found parasitized by fragmented worms compatible with the genus Trichobilharzia. Later molecular analyses showed this taxon corresponded to T. querquedulae (Figure 4, Supplementary Table S2). No nasal schistosomatids were recorded.
Table 1. Measurements and morphology of visceral Trichobilharzia species recorded in the Neotropics, including Trichobilharzia kulfu sp. nov. Oyarzún-Ruiz, Flores, Brant, and Tkach. All measurements are expressed in μm, unless otherwise stated
Abbreviations and symbols: Hosts, sample, infection, and country: ?, number of measured worms not detailed; c, intact worms measured; f, fragments of worms measured; M, male worm; n, sample size; Nat, natural; Exp, experimental; ⁑, description and measurements of the taxon recorded in the Neotropics but from other location. Measurements and morphology: Ae, anterior end; Ace, acetabulum; Ace-SV, acetabulum-seminal vesicle area; Ant-SV, anterior seminal vesicle; d, distance; D, diameter; genpapilla, genital papilla; GC, gynaecophoric canal; L, length; Nr, not recorded; OS, oral sucker; Post-SV, posterior seminal vesicle; Prox-SR, proximal end of seminal receptacle; SV, seminal vesicle; SV-GC, seminal vesicle-gynaecophoric canal region; T, testis/testes; W, width. *, when complete worms were not collected, the highest recorded number was mentioned; †, for this species, the seminal vesicle was described as a total length; ‡, authors didn’t mention which host species was based on the description of this taxon, only mention that these avian species are the hosts; §, measurements based on immature worms. Fixatives: ACa, hot corrosive-acetic solution; Bou, Bouin’s fluid; E80, 80% ethanol; Gi, Gilson’s fixative at 60°C. Staining: AC, Alum carmine; ACS, Semichon’s acetocarmine; BC, Borax carmine; TriG, Gomori’s trichrome.
Taxonomic summary
Trichobilharzia kulfu sp. nov. Oyarzún-Ruiz, Flores, Brant, and Tkach, 2025.
Type-definitive host: Chiloe wigeon Mareca sibilatrix (Poeppig, 1829) (Anseriformes, Anatidae).
Site of infection: mesenteric veins, liver, kidneys, heart, lungs, gallbladder.
Type-locality: Nebuco, Ñuble region, Chile (36°38′30.6″S 72°11′39.7″W).
Other localities: Chillán, Portezuelo (Ñuble region, Chile), Buenos Aires (Buenos Aires province, Argentina).
Etymology: The specific epithet is given by the Mapudungún word kül’fü, which is attributed to the Chiloe wigeon, the type-host. Mapudungún is the language of Mapuche, an aboriginal people from Chile and Argentina.
Type specimens: Holotype adult male worm (MZUC-UCCC 48242).
Paratypes: Vouchers with fragments of male worms in ethanol (MZUC-UCCC 48245, MSB catalogue number MSB:Para:27328) and slides (MZUC-UCCC 48243-48244).
Zoobank accession number: urn:lsid:zoobank.org:act:A4435867-4B47-4B70-95AE-90F614E5A41B.
Representative DNA sequences: COI (PX648553-PX648555), 28S (PX657201-PX657204).
Parasite symbiotype (definitive host): Carcass of a parasitized Chiloe wigeon M. sibilatrix (MZUC-UCCC 48678).
Description
Male (Figures 2–3, Table 1): Description based on ten complete worms and four fragments. Long and filamentous worms, slightly flattened dorso-ventrally with well-developed oral sucker and acetabulum. Rounded oral sucker, terminal, opening subterminal, covered with small spines; those surrounding the oral aperture are slightly wider. Posterior third of oesophagus with conic thick-walled bulbous, immediately anterior to acetabulum. Oesophagus bifurcated into two ceca anterior to acetabulum, anastomosing (caecal reunion) posterior to prostatic region to continue as a common cecum or intestine until near posterior end. Oesophagus and caeca on occasions with hyaline content. Rounded acetabulum, slightly wider than oral sucker, covered around its aperture by long and thin spines. Contorted seminal vesicle, divided into two sections: external and internal seminal vesicle. Thin-walled cirrus sac, closely related to internal seminal vesicle, prostatic region, and ejaculatory duct. Prostatic region small and fusiform, closely associated with the internal seminal vesicle. Ejaculatory duct thin, opening into the genital papilla. Genital papilla prominent, anterior to gynaecophoric canal, situated to the right side of longitudinal line of canal in most of the examined worms. Gynaecophoric canal deep, densely covered by fine spines, well-muscled, similar width to the anterior third of the body. First testis in short distance to gynaecophoric canal. Intestine continues posteriorly and sinuously between testes. Complete worms with 60–89 testes in number, tandem, small, rounded, and alternately distributed along the intestine. Spatulated posterior end. At SEM imaging, the body surface shows no spines, net-like appearance, covered with small holes along its surface. Oral sucker filled with small holes on its complete surface too. No papillae were evident on this sucker. Gynaecophoric canal completely covered by short and fine spines on its surface. Border of gynaecophoric canal covered by multiple small papillae alternating with spines.
Figure 2. Morphology of male worm of Trichobilharzia kulfu sp. nov. Oyarzún-Ruiz, Flores, Brant, and Tkach parasitizing Chiloe wigeon from Chile. A. Anterior end of male worm. B. Anterior third of male worm. C. Spatulated posterior end.
Figure 3. SEM images of a male worm of Trichobilharzia kulfu sp. nov. Oyarzún-Ruiz, Flores, Brant, and Tkach parasitizing Chiloe wigeon from Chile. A. Anterior end. Note the porous tegument (*) which extends to the oral sucker, except on the ventral surface. B. Detail of oral aperture where several small, rounded spines are present covering the surface (arrowheads), including a single row of small papilla-like structures (*). C. Region of acetabulum. Note the tegument showing a net-like surface with no spines (*), also the aperture of the acetabulum (arrowhead). D. Close-up of the acetabulum aperture. E. Anterior border of gynaecophoric canal, where several aligned papilla-like structures are bordering the canal (arrowheads), and a few of them are randomly organized (*). F. Inner surface of gynaecophoric canal densely covered by small spines (*). Also, notice the presence of a few papilla-like structures (arrowheads) in this middle segment of the canal.
Figure 4. Phylogenetic tree of COI, positioning Trichobilharzia kulfu sp. nov. Oyarzún-Ruiz, Flores, Brant, and Tkach as a monophyletic group. This phylogeny was inferred using an alignment of 822 bp. Calculated substitution models for ML and BI were the following: HKY+F+I+G4 (position-1 and position-2) and TIM+F+I+G4 (position-3); M 50, M 152, M 125, M 191 (position-1), M 136, M 201, M 200, M 191, M 138, M 125, M 203, and M 166 (position-3), respectively. The best models were chosen using the Bayesian Information Criterion (BIC, Schwarz Reference Schwarz1978). Bootstrap values ≥ 70 (left) and posterior probabilities ≥ 0.71 (right) are presented at every node. An asterisk (*) indicates full support (100/1). The sequences from the present study are highlighted in bold. Abbreviations: AR, Argentina; BR, Brazil; CH, Chile; USA, United States.
Remarks
Trichobilharzia kulfu sp. nov. Oyarzún-Ruiz, Flores, Brant, and Tkach, 2025, could be readily differentiated from other Trichobilharzia species recorded in the Neotropics. Trichobilharzia kulfu sp. nov. is similar to T. jequitibaensis considering a tegument mostly smooth, except for gynaecophoric canal, position of caecal reunion posterior to seminal vesicle, and presence of a bulb-like structure in the posterior third of the oesophagus (Leite et al. Reference Leite, Costa and Costa1978). However, the newly described taxon differs from T. jequitibaensis showing lesser dimensions of the oral sucker, acetabulum, and gynaecophoric canal, plus a reduced number of testes (Leite et al. Reference Leite, Costa and Costa1978; see Table 1). The new species shows spines surrounding the aperture of the acetabulum, which have been described in other species such as T. physellae, T. querquedulae, and T. jequitibaensis. In T. kulfu sp. nov., these spines are numerous, thin, and longer, in comparison to T. physellae, which has thick and shorter spines (McMullen and Beaver Reference McMullen and Beaver1945), T. querquedulae with numerous blunt spines (McLeod Reference McLeod1937; McLeod and Little Reference McLeod and Little1942; Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a), and T. jequitibaensis showing small spines sparsely distributed on the acetabulum (Leite et al. Reference Leite, Costa and Costa1978). Trichobilharzia kulfu sp. nov. differs from T. physellae because the male of the latter has a spinous tegument, longer distance between oral sucker and acetabulum, position of caecal reunion, shorter gynaecophoric canal, and higher number of testes (Brant and Loker Reference Brant and Loker2009; McMullen and Beaver Reference McMullen and Beaver1945). In addition to the above-mentioned differences, T. querquedulae differs from T. kulfu sp. nov. by having a stout body in comparison to the thread-like shape of the newly described species. Furthermore, the number of testes is higher in T. querquedulae, and these are organized as closely packed triplets along the axis of the intestine; meanwhile, in the newly described species there is only one testis on each side. Besides, the cecum/intestine of T. querquedulae is convoluted with acute angles (McLeod Reference McLeod1937, McLeod and Little Reference McLeod and Little1942; Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a), meanwhile, in T. kulfu sp. nov. is a narrow tube with no sharp angles passing subtly between testes. The oesophagus of T. querquedulae also shows a bulb anterior to the acetabulum and caecal reunion between seminal vesicle and gynaecophoric canal, as was seen in T. kulfu sp. nov. (McLeod Reference McLeod1937; Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a). Additional differences between these two species are a greater diameter of acetabulum, longer gynaecophoric canal, and larger testes for T. querquedulae (see Table 1).
The porous tegument of Trichobilharzia kulfu sp. nov. contrasts with other visceral Trichobilharzia, which are generally described as spinous, although only a few of them have been described using SEM imaging, which could accurately describe the distribution of such spines, e.g., Blair and Islam (Reference Blair and Islam1983), Islam (Reference Islam1986), Oyarzún-Ruiz et al. (Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a). However, the tegument of this new taxon is similar, i.e., without spines but porous, to other nasal schistosomatids such as the Australian taxa Trichobilharzia australis Blair and Islam, Reference Blair and Islam1983, and Trichobilharzia arcuata Islam Reference Islam1986, which were also described using SEM (Blair and Islam Reference Blair and Islam1983; Islam Reference Islam1986). Another taxon with a non-spinous tegument is Trichobilharzia longicauda (Macfarlane 1944) Davis, 2006, and Trichobilharzia novaeseelandiae Davis and Brant Reference Davis, Blair and Brant2022, which were described with a rugose tegument only bearing spines on oral sucker, acetabulum, and gynaecophoric canal (Davis et al. Reference Davis, Blair and Brant2022). These descriptions are fairly different from the newly described taxon. The inclusion of SEM imaging in the description of schistosomatid worms has been shown to be relevant to describe previously unnoticed traits under light microscopy (Blair and Islam Reference Blair and Islam1983; Flores et al. Reference Flores, Viozzi, Casalins, Loker and Brant2021; Islam Reference Islam1986; Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a). Thus, the description of the known and newly described taxa would state if this non-spinous, or even porous, tegument is present.
Unfortunately, for the other two phylogenetically related taxa: Trichobilharzia sp. A and Trichobilharzia sp. B, both recorded in the congeneric American wigeon (Mareca americana), there is no formal description available, only genetic sequences, which precluded morphological comparisons related to this monophyletic group.
All type and paratype specimens are deposited at Museo de Zoología (MZUC), University of Concepción, Chile, and Museum of Southwestern Biology Division of Parasites (SBM), New Mexico, USA.
Phylogenetic analyses and pairwise comparisons
The phylogenetic analyses of ML and BI (Figure 4), and genetic distances (Supplementary Table S2) for both COI sequences supported the monophyletic position of this new taxon with robust nodal support (100/1). Besides, both sequences from Chile (A06H) and Argentina (W786) were conspecific (0% genetic distance). An additional sequence also from Chile (Trichobilharzia 5161) proved to be conspecific with the new taxon described. In addition, the COI sequences from S. cyanoptera from Chile (Trichobilharzia 5157, 5158, 5159) were conspecifics (0.7–0.9%) with T. querquedulae (Figure 4, Supplementary Table S2). Trichobilharzia kulfu sp. nov. is closely related to Trichobilharzia sp. A (FJ174525) and Trichobilharzia sp. B (FJ174528) with robust nodal support for BI (0.87) but non-significant for ML (63). The genetic distances between these three taxa were the following: 8.1–8.4% between T. kulfu sp. nov. and Trichobilharzia sp. A, and 8–8.2% for T. kulfu sp. nov. and Trichobilharzia sp. B. In addition, T. kulfu sp. nov. was included as part of Clade Q sensu Brant and Loker (Reference Brant and Loker2009), with robust nodal support (81/1), where T. querquedulae, Trichobilharzia sp. A, and Trichobilharzia sp. B are also included.
Discussion
This study represents a newly described species for genus Trichobilharzia, and the fourth named species of this genus in the Neotropical realm. The two isolates from Chile and Argentina, parasitizing the same host, were conspecifics and named Trichobilharzia kulfu sp. nov. Both isolates showed a notable genetic distance for COI locus with other closely related taxa of genus Trichobilharzia (8–12%), which supported the morphological differences described previously. So far, the schistosomatids recorded in Chile are represented by five taxa: T. querquedulae, Trichobilharzia sp. A46R2/W830, N. melancorhypha, Schistosomatidae gen. sp. II, and Dendritobilharzia sp. Thus, this new species represents the sixth avian schistosomatid recorded in Chile, a country which has recently given relevant data to the diversity of schistosomatids in the region (Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Collado, Muñoz and Moreno2022, Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a, Reference Oyarzún-Ruiz, Navarro, Moreno and Landaeta-Aquevequeb).
This new taxon was included as part of clade Q, with a robust nodal support for COI. Clade Q is comprised of avian schistosomatids of genus Trichobilharzia mainly transmitted by physid snails such as T. querquedulae, T. physellae, Trichobilharzia longicauda, and other still undescribed taxa from the Neotropics, North America, and Europe. An additional taxon included in this clade is T. franki, although this is transmitted by lymnaeid snails (Ashrafi et al. Reference Ashrafi, Sharifdini, Darjani and Brant2021; Brant and Loker Reference Brant and Loker2009; Brant et al. Reference Brant, Bochte and Loker2011; Horák et al. Reference Horák, Mikeš, Lichtenbergová, Skála, Soldánová and Brant2015; Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a; Pinto et al. Reference Pinto, Brant and Melo2014). From the above-mentioned taxa, T. querquedulae, T. physellae, and Trichobilharzia sp. W701 (KJ855994) have been recorded in the Neotropical region (Ebbs et al. Reference Ebbs, Loker, Davis, Flores, Veleizan and Brant2016; Flores et al. Reference Flores, Brant and Loker2015; Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a; Pinto et al. Reference Pinto, Brant and Melo2014). Nevertheless, the record of T. physellae still requires confirmation because it was only mentioned by Szidat (Reference Szidat1951), who recorded it in a S. versicolor duck from Argentina, but with no morphological description. A probable additional taxon of clade Q is T. jequitibaensis from Brazil, which is vectored by the physid snail Stenophysa marmorata, but with no available sequences (Flores et al. Reference Flores, Brant and Loker2015; Leite et al. Reference Leite, Costa and Costa1978, Reference Leite, HMdeA and Costa1979). Meanwhile, two other undescribed taxa, Trichobilharzia sp. W701 and Cercaria I, both retrieved in S. marmorata from Brazil and Argentina, respectively, have been recorded only as larval stages (Ostrowski de Núñez Reference Ostrowski de Núñez1978; Pinto et al. Reference Pinto, Brant and Melo2014). Recently, the life cycle of T. querquedulae was elucidated in South America, with Physa sp. acting as the intermediate host (Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a).
In Chile and Argentina, both Physidae and Lymnaeidae snail families are present: Physidae, conformed by four native and one exotic species, and Lymnaeidae with five native and one exotic species (Correa et al. Reference Correa, Escobar, Noya, Velasquez, Gonzalez-Ramirez, Hurtrez-Bousses and Pointier2011; Cuezzo Reference Cuezzo, Domínguez and Fernández2009; Valdovinos Reference Valdovinos2006). Although both families have been sampled for avian schistosomatids in the Neotropics, it is necessary to identify these snails to a specific taxonomic rank to clarify the role of native or invasive species in the life cycle of these schistosomatids belonging to clade Q (Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Collado, Muñoz and Moreno2022, Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a, Reference Oyarzún-Ruiz, Navarro, Moreno and Landaeta-Aquevequeb). Despite the intermediate host of T. kulfu sp. nov. remaining unknown, we suggest that additional sampling in the geographical range of the type-host could highlight the snail species involved in this life cycle, which probably would be a physid snail, considering its phylogenetic position in clade Q (Brant and Loker Reference Brant and Loker2009; Brant et al. 2011).
Furthermore, the new taxon was closely related (63 ML /0.87 BI) to other two taxa: Trichobilharzia sp. A and B, both recorded in M. americana from the United States (Brant and Loker Reference Brant and Loker2009). This finding would suggest that this small clade could represent, in a first approach, a putative example of host specificity, particularly to ducks of the genus Mareca. Additional sampling of Mareca ducks from other geographical areas, e.g., Africa, Asia, and Europe (Johnsgard Reference Johnsgard2010), would be required to prove there is a ‘wigeon clade’ of avian schistosomatids as occurs with T. querquedulae and ‘blue-winged ducks’ (Spatula spp.) (Brant and Loker Reference Brant and Loker2009; Ebbs et al. Reference Ebbs, Loker, Davis, Flores, Veleizan and Brant2016; Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a). Mareca sibilatrix is a monotypic taxon in South America, so the hypothesis that T. kulfu sp. nov. is specific to this duck would be interesting to test, or, otherwise, if it could be possible to find this trematode in sympatric duck species, e.g., Anas georgica and S. cyanoptera (Johnsgard, Reference Johnsgard2010; Martínez and González Reference Martínez and González2017). There are similar examples of host specificity such as N. melancorhypha parasitizing the black-necked swan (Cygnus melancoryphus), the only swan distributed in the Neotropics (Flores et al. Reference Flores, Viozzi, Casalins, Loker and Brant2021; Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a). On the other hand, there are examples of schistosomatid species parasitizing ecological clades of hosts as occurs with T. physellae parasitizing the ecological clade of diving ducks, which includes different genera (Brant and Loker Reference Brant and Loker2009; Pinto et al. Reference Pinto, Brant and Melo2014). However, considering those examples, such findings of specificity need further exploration, particularly those recently described in the Neotropics (e.g., Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a, Reference Oyarzún-Ruiz, Navarro, Moreno and Landaeta-Aquevequeb).
Bearing in mind that the type-host is restricted to the Neotropical realm, it would not be unexpected that T. kulfu sp. nov. is endemic to this region, as also has been suggested to N. melancorhypha (Flores et al. Reference Flores, Viozzi, Casalins, Loker and Brant2021; Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a).
SEM analysis allowed us to describe more precisely the tegument of adult worms of T. kulfu sp. nov. Its use for the complete description of avian schistosomatids has been suggested as an outstanding tool, e.g., Trichobilharzia, Nasusbilharzia, and Marinabilharzia, although it has been scarcely employed (e.g., Blair and Islam Reference Blair and Islam1983; Flores et al. Reference Flores, Viozzi, Casalins, Loker and Brant2021; Islam Reference Islam1986; Lorenti et al. Reference Lorenti, Brant, Gilardoni, Diaz and Cremonte2022; Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Collado, Muñoz and Moreno2022, Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a). Thus, we reinforce the proposal to consider this tool, in addition to light microscopy and molecular tools, when a new species is described.
Because most species of the genus Trichobilharzia have the potential to accidentally infect people, particularly those belonging to clade Q (Helmer et al. Reference Helmer, Blatterer, Hörweg, Reier, Sattmann, Schindelar, Szucsich and Haring2021; Horák et al. Reference Horák, Bulantová, Mikeš, Toledo and Fried2024), it would be important to assess in future studies the zoonotic potential of this new taxon. For the latter, experimental studies with a murine model would be suitable, as it has been shown with other related taxa (Lichtenbergová et al. Reference Lichtenbergová, Lassmann, Jones, Kolářová and Horák2011).
This new taxon represents an additional species to the rich avian schistosomatid diversity, including at least four endemic genera from the Neotropical region, highlighting the importance of this area as a hotspot for these blood flukes (Flores et al. Reference Flores, Brant and Loker2015, Reference Flores, Viozzi, Casalins, Loker and Brant2021; Lorenti et al. Reference Lorenti, Brant, Gilardoni, Diaz and Cremonte2022; Oyarzún-Ruiz et al. Reference Oyarzún-Ruiz, Thomas, Santodomingo, Collado, Muñoz and Moreno2022, Reference Oyarzún-Ruiz, Thomas, Santodomingo, Zamorano-Uribe, Moroni, Moreno, Muñoz-Leal, Flores and Brant2024a, Reference Oyarzún-Ruiz, Navarro, Moreno and Landaeta-Aquevequeb).
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X25101089.
Acknowledgements
Authors are grateful to N. Martin, L. Aravena, M. Zamorano, V. Aravena, E. Inostroza, and V. Nova for their support in the field and laboratory procedures, also to F. Castro and Ll. Rodríguez for providing us the Epoch™ Microplate Spectrophotometer.
Financial support
ANID Doctorado Nacional fellowship (grant number 21181059) and FONDECYT Postdoctorado fellowship (grant number 3230461) (P.O.-R.).
Competing interests
The authors declare no competing interests.
Ethical standard
This study was approved by the Bioethical Committee of Facultad de Ciencias Veterinarias, Universidad de Concepción (CBE-16-2020), and Vicerrectoría de Investigación y Desarrollo (VRID), Universidad de Concepción (CEBB-14-2023).
Open access
