Identification of the Avian Tracheal Trematode Typhlocoelum

Parasitology Identification of the avian tracheal trematode Typhlocoelum cucumerinum cambridge.org/par (Trematoda: Cyclocoelidae) in a host–parasite–environment system: diagnosis, Research Article life cycle and molecular phylogeny Cite this article: Assis JCA, López-Hernández D, Favoretto S, Medeiros LB, Melo AL, Martins 1 1 2 NRS, Pinto HA (2021). Identification of the Jordana C. A. Assis , Danimar López-Hernández , Samantha Favoretto , avian tracheal trematode Typhlocoelum 3 1 3 1 cucumerinum (Trematoda: Cyclocoelidae) in a Lilian B. Medeiros , Alan L. Melo , Nelson R. S. Martins and Hudson A. Pinto host–parasite–environment system: diagnosis, 1 life cycle and molecular phylogeny. Laboratório de Biologia de Trematoda, Department of Parasitology, Institute of Biological Sciences, Universidade Parasitology 148, 1383–1391. https://doi.org/ Federal de Minas Gerais, Belo Horizonte, Brazil; 2Department of Veterinary Medicine, Universidade Federal de 10.1017/S0031182021000986 Lavras, Lavras, Brazil and 3Laboratório de Doenças das Aves, Department of Preventive Veterinary Medicine, Veterinary School, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil Received: 27 April 2021 Revised: 1 June 2021 Accepted: 1 June 2021 Abstract First published online: 9 June 2021 Typhlocoelum cucumerinum is a tracheal parasite of birds widely distributed across the globe. Key words: Nevertheless, aspects of the biology of this cyclocoelid are still poorly understood. Herein, we Typhlocoelinae; life cycle; domestic waterfowl; report the finding of T. cucumerinum in definitive and intermediate hosts from an urban phylogeny waterbody of Brazil. The parasite was initially detected during the necropsy of domestic Muscovy ducks (Cairina moschata) found dead in the locality. Coproparasitological tests in Author for correspondence: Jordana C. A. Assis, live animals revealed that 12/47 (25.53%) Muscovy ducks and 2/8 (25%) mallards (Anas E-mail: [email protected] platyrhynchos platyrhynchos) were infected with T. cucumerinum. Moreover, rediae and metacercariae morphologically similar to T. cucumerinum were found in 3/248 (1.33%) Biomphalaria straminea collected in the same waterbody frequented by the birds. The conspecificity between the adult and the larval stages was confirmed molecularly (100% similarity in Cox-1). Moreover, the phylogenetic position of T. cucumerinum was determined for the first time based on partial fragments of the 28S, Cox-1 and Nad-1 genes. The species grouped with other members of the subfamily Typhlocoelinae with sequences available, but the data obtained do not support the distinctiveness of the genera Typhlocoelum and Tracheophilus. Further studies involving a broader range of species can result in taxonomic rearrangements in Typhlocoelinae.

Introduction Typhlocoelum cucumerinum (Rudolphi, 1809) is a trematode of the upper respiratory system (mainly trachea) of waterfowls with worldwide distribution (Asia, Europe, Oceania and Americas) and currently included in the family Cyclocoelidae Stossich, 1902 (Gower, 1939; Barry, 1959; Schafranski et al., 1975; Soulsby, 1982; Tkach et al., 2016). Infection with these tracheal parasites can cause airway obstruction, resulting in dyspnoea, suffocation and even death (Schafranski et al., 1975; Soulsby, 1982; Taylor et al., 2017). Despite the potential veterinary importance of typhlocoelosis in domestic anatids, aspects of the biology, epizoology and transmission dynamics of T. cucumerinum are still poorly understood. In fact, the countless reports of infection of birds with this cyclocoelid are based on post-mortem findings (Kinsella and Forrester, 1972; Schafranski et al., 1975; Hoyos et al., 2017). To the best of our knowledge, studies involving the in vivo diagnosis of this trematode are lacking, despite the potential importance of this approach to subside the treatment of infection. Moreover, no study reported the occurrence of T. cucumerinum in naturally infected snails so far. Studies on life cycles of species of the family Cyclocoelidae revealed that these trematodes have a single intermediate host, in which there is the development of rediae, followed by the production of cercariae (Cercariaeum type) and the formation of encysted metacercariae in the same mollusc (Taft, 1973, 1975, 1986; Schafranski et al., 1975; McLaughlin, 1976, 1986;Taft and Heard, 1978; Scott et al., 1982). Infection of birds occurs with the ingestion of infected molluscs (Schafranski et al., 1975; McLaughlin, 1976, 1986; Galaktionov and Dobrovolskij, 2003), a common food habit known to several species of waterfowls (Lavery, 1970; Middleton and van der Valk, 1987). Information on the molluscs that act as intermediate hosts of T. cucumerinum is scarce and restricted to the experimental life cycle studies carried out several decades ago (Schafranski et al., 1975; Scott et al., 1982). © The Author(s), 2021. Published by In another aspect, recent molecular studies have contributed to the advancement of knowl- Cambridge University Press edge on taxonomy and phylogeny of avian trematodes, including species of the family Cyclocoelidae (Sitko et al., 2017; Gomez-Puerta et al., 2018; López-Jiménez et al., 2018; Galosi et al., 2019;Liet al., 2020; Urabe et al., 2020). Despite this, molecular data are scarce for tracheal parasites belonging to the subfamily Typhlocoelinae, which are limited to sequences of isolates identified as Typhlocoelum sp. and Tracheophilus cymbium (Diesing,

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1850) from North America and Asia, respectively (Tkach et al., with the aid of a D-shaped hand net (50 × 30 cm, 1 mm mesh, 2016;Liet al., 2020). The acquisition of new molecular 20 cm depth bag) and sent to the laboratory, where they were information for members of the Typhlocoelinae, especially washed in running tap water, quantified and placed individually T. cucumerinum, the type species of the genus, is necessary to in 24-well polystyrene plates with approximately 2 mL of water. access taxonomic issues related to the validity of the genera cur- After a routine artificial photostimulation test to detect the emer- rently included in this subfamily. Moreover, such molecular gence of larval trematodes, the gastropods, recognized experimen- data can be useful to confirm the wide geographical distribution tal intermediate hosts of T. cucumerinum (Schafranski et al., 1975; previously reported to the species based on morphological data. Scott et al., 1982), were pressed between glass slides inside a Petri In the current study, a host–parasite–environment system dish and dissected under a stereomicroscope for the search of involving T. cucumerinum was identified in an urban lake from non-emergent larval stages. The taxonomic identification of the Brazil. Coproparasitological analysis was used for the first time molluscs was based on morphological traits according to different for the in vivo diagnosis of the infection in free-living domestic authors (Paraense, 1970, 1994; Paraense and Pointier, 2003). The waterfowl. Moreover, different parasite developmental stages, sampling of molluscs was made under the permission of the including larval forms in molluscs, were subjected to morpho- Brazilian Institute of Environment and Renewable Natural logical and molecular studies. This integrative taxonomic Resources (IBAMA, Biodiversity Authorization and Information approach resulted in insights and advances in taxonomy, System – SISBIO permit number 52870-1). phylogeny and biology of this avian tracheal trematode. Morphological study of developmental stages and taxonomic Materials and methods identification Study area, animals and adult parasites A live adult specimen recovered from C. moschata found dead in the locality was processed for morphological study. This trema- The study area comprises an urban lake located at the tode was placed in a Petri dish containing 0.9% saline to obtain ‘ Administrative Center of the State of Minas Gerais ( Cidade eggs. Then, the trematode was pressed between glass slides, killed ’ Administrativa Presidente Tancredo Neves ), Belo Horizonte with water at 70°C and fixed in 10% formalin. Subsequently, the city, Minas Gerais, Southeastern Brazil (19°47′06.20′′S and 43° ′ ′′ parasite was stained with alum acetocarmine, dehydrated in 57 11.41 W), where a population of about 150 domestic water- ascending ethanol series, diaphanized with beechwood creosote, fowl was maintained at the study time. During the necropsy of and mounted on a permanent slide with Canada balsam. The some specimens that died due to an outbreak of botulism between morphological study of the trematode was performed under a March and April 2016 (Silva et al., 2017), the presence of trema- light microscope. Photographs of the specimen in toto were todes in the trachea of Cairina moschata (L.) was verified for the taken using a Samsung ES 70® digital compact camera coupled first time (Fig. 1A). Based on this finding, a study was proposed to to a Leica EZ4® stereomicroscope. Details of internal structures – – characterize the host parasite environment system present in the were photographed with the aid of a Leica ICC50 HD® digital locality. camera attached to a microscope of the same brand. The captured images were analysed using the software Leica Application Suite® Coproparasitological study (LAZ EZ) version 2.0. The identification of the parasite was based on morphological criteria according to different authors (Dubois, Fecal samples from 61 domestic Anseriformes found in the 1959; Travassos et al., 1969; Yamaguti, 1971; Schafranski et al., locality [47 C. moschata (domestic Muscovy ducks), 8 Anas 1975; McDonald, 1981; Kanev et al., 2002). The adult specimen platyrhynchos (L.) (mallards) and 6 Anser cygnoides (L.) was deposited in the Collection of Trematodes of the (African geese)] were collected between June and August 2018. Universidade Federal de Minas Gerais (UFMG-TRE 120). The nomenclature of the birds followed Clements et al. (2019). The larval stages of trematodes recovered after mechanical For the collection of feces, the birds were attracted by offering compression of the molluscs were aspirated with the aid of a food, manually contained and identified with numbered rings Pasteur pipette, transferred to another Petri dish containing on the right leg. After identification, the specimens were individu- water. After this, they were wet-mounted between glass slides ally placed in steel cages (1 m in length and width, 0.6 m in and coverslip and evaluated with the aid of a light microscope. height), with the floor lined with paper for retaining feces. They The photographic record and image analyses were made as remained restricted until defecation, usually within no more described above for the adult. After the morphological study, than 10 min, when samples were collected and placed in plastic the larval forms were removed from the slides with the aid of a containers, fixed in 10% formalin, and sent to the laboratory. Pasteur pipette, transferred to 1.5 ml microtubes, and fixed in The parasitological examination of feces was performed using 95% ethanol for molecular study. the spontaneous sedimentation technique (Hoffman et al., 1934). For each animal, five slides containing aliquots of fecal sediment were examined under a light microscope. Samples of Molecular phylogenetic study eggs found were photographed and subjected to morphometric For the molecular study, we used ethanol-fixed eggs shed by the analysis to confirm the presence of T. cucumerinum. The proce- adult parasite obtained from C. moschata and rediae recovered dures followed ethical principles approved by the Local Ethics from the molluscs. DNA extraction was performed with the Committee in Animal Experimentation (CEUA-UFMG protocol Mini QIAamp® Qiagen® kit (eggs) and Micro QIAamp® Qiagen® 68/2017). (rediae) as recommended by the manufacturer. The concentration of extracted DNA was determined using a spectrophotometer (NanoDrop® ND-1000). For the sample of eggs obtained from Malacological survey an adult, partial regions of the genes 28S (Dig12/1500R primers) A malacological survey was carried out in the same waterbody (Tkach et al., 2003), Cox-1 (JB3/COI-R Trema primers) (Miura frequented by domestic waterfowls. Seven samplings were carried et al., 2005) and Nad- 1 (NDJ1/NDJ2A primers) (Morgan and out between June 2018 and May 2019, with a sampling effort of Blair, 1998) were amplified by polymerase chain reaction approximately 1 h on each occasion. The molluscs were collected (PCR). The amplification reactions were carried out in a final

Fig. 1. Typhlocoelum cucumerinum found in naturally infected Cairina moschata from Brazil. (A) Alive trematodes found in the trachea during necropsy. (B) Whole view of carmine stained worm. (C) Cirrus sac (arrow). (D) Egg of the parasite containing a developed miracidium. Scale bars: A = 15 mm, B–C = 200 μm, D = 50 μm.

volume of 25 μl. The PCR mixture consisted of 2.5 μl of DNA Carlo method via Markov chains, in two runs of four chains, solution (≈30 ng), 12.5 μl of Go Taq Green Master Mix®, 8.75 μl for 106 generations, with sampling of topologies every 100 genera- of ultrapure water and 1.25 μl of a mix of primers (10 μM). The tions, discarding the first 25% of the trees sampled as ‘burn-in’.The PCR reactions were performed in a Labcycler® thermocycler program Fig Tree 1.4.2 (Rambaut, 2012) was used to visualize the (Sensoquest, Germany) using the PCR conditions previously topologies of the trees obtained by BI. The final phylogenetic trees described (Morgan and Blair, 1998; Tkach et al., 2003; Miura were edited in PowerPoint® (Windows 7®) and Photoshop CS5 et al., 2005). The PCR products were subjected to 1% agarose Adobe®. Molecular sequences generated in this study were depos- gel electrophoresis and those samples with the expected band ited in the GenBank under the accession numbers: MZ262701 were purified with 20% polyethylene glycol. Sequencing, in both (28S); MZ254996–MZ254998 (Cox-1) and MZ268020 (Nad-1). directions, was carried out in ABI 3730 capillary sequencer, using POP7 polymer and Big Dye v3.1 Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems®, Foster, USA). Results The chromatograms obtained were edited using the Identification of the parasite from birds ChromasPro program version 2.0.1 (Technelysium Pty Ltd, Australia) and the contigs compared with other sequences avail- The morphological analysis of the adult parasite recovered from able in GenBank, initially by a Blast search (https://blast.ncbi. C. moschata in 2016 enables the identification of this tracheal nlm.nih.gov/Blast.cgi) (Supplementary Table S1). The sequences trematode as T. cucumerinum. The morphology of this parasite for each marker were aligned with others available for members is described below. of the family Cyclocoelidae through the Clustal W program, Adult (N = 1): linguiform, pink-coloured, with very slow implemented in MEGA7.0 (Kumar et al., 2016). Outgroups vermiform movements when alive (Fig. 1A). Body developed, were selected based on previously published phylogenies of tre- elliptical, 15 mm long and 7 mm wide, tegument with tiny scales matodes (Pérez-Ponce de León and Hernández-Mena, 2019). (Fig. 1B). Oral opening subterminal, pharynx rounded, muscular, Phylogenetic analyses were made by maximum likelihood (ML) 461 μm in length by 503 μm wide. Ventral sucker not visualized. and Bayesian inference (BI) using the programs MEGA7.0 and Caecum long, with several diverticula internally, united poster- MrBayes 3.1.2 (Ronquist et al., 2012), respectively. The best iorly. Genital pore in the pharynx region. Cirrus sac median, at nucleotide substitution models were estimated by the lowest the level of the oesophagus, containing a seminal vesicle inside score obtained by the Bayesian information criterion in (Fig. 1C). Testes in tandem, extremely lobulated, in the posterior MEGA 7.0. The selected models were GTR + G, TN93 + G and region of the body. Anterior testis on the left side, 2.13 mm long HKY + G for 28S, Cox-1 and Nad-1 datasets, respectively. For and 1.53 mm wide. Posterior testis 1.27 mm long and 2.60 mm phylogenetic inferences, the CIPRES computational analysis wide. Ovary round, lateral to the anterior testis, 510 μm long platform (phylo.org) was used (Miller et al., 2010). The few and 496 μm wide, in the posterior quarter on the right side. gaps present in the alignment were eliminated from the analysis. Ootype post-ovarian. Vitelline follicles lateral, from the intestinal In the ML analysis, the trees were generated by the IQ-TREE bifurcation up to the posterior end, not united posteriorly. Uterus platform (phylo.org) and clade supports were estimated by the intercecal, sinuous, from the posterior testis to the oesophageal Bootstrap method (10 000 replications). In the BI analysis, the bifurcation. Eggs elliptical to oval-shaped, yellow in coloration, posterior probability values were determined by the Monte with an operculum (Fig. 1D), 157 ± 8 (131–170) μm long and

87 ± 4 (77–93) μm wide (n = 20) in the terminal uterus of the described for T. cucumerinum by Schafranski et al. (1975) after mounted specimen, presenting a miracidium with eyespot and a experimental infections of molluscs. Considering other species formed redia inside. of Cyclocoelidae for which intramolluscan stages are known, the rediae analysed here are significantly smaller than those described for Cyclocoelum mutabile and do not have spines in Remarks the anterior third (Ginetsinskaia, 1949). The absence of ventral The adult specimen recovered from C. moschata has morpho- sucker, here confirmed in the cercariae of T. cucumerinum,is logical characteristics compatible with the genus Typhlocoelum, also known for larvae of other cyclocoelids, as as described by different authors (Dubois, 1959; Schafranski Ophthalmophagus spp. (Taft and Heard, 1978; Taft, 1986) and et al., 1975; Kanev et al., 2002; Hoyos et al., 2017). Unlike the Morishitium dollfusi (Timon-David, 1950) (Yamaguti, 1975). diagnosis of the genus presented by Kanev et al. (2002), the pres- On the other hand, the presence of this structure was well ence of a ventral sucker was not observed in the specimen studied described to the cercariae of Tracheophilus sisowi Skrjabin, 1913 here. However, the morphology, including the complete absence (16 μm) and Cyclocoelum mutabile (Zeder, 1800) (34 × 37 μm) of a ventral sucker, as well as the measurements obtained for (Szidat, 1932; Stunkard, 1934; Ginetsinskaia, 1949; Yamaguti, the evaluated specimen, are in agreement with those reported 1975). In Brazil, a larva found in Biomphalaria glabrata (Say, for T. cucumerinum by different authors (Dubois, 1959; 1818) in the same locality from the current study (Belo Travassos et al., 1969; Schafranski et al., 1975; Hoyos et al., 2017). Horizonte) was described as Cercaria acaudata by Ruiz (1952). It differs from T. cucumerinum by the presence of a ventral sucker, and possibly a member of the genus Cyclocoelum. Coproparasitological study Recently, larval stages have been found in B. glabrata from The analysis of fecal samples of the anatids evaluated in the cur- Brazil were tentatively identified as T. cucumerinum (Oliveira rent study revealed the infection of 12/47 (25.53%) C. moschata et al., 2020). Despite the measures of larvae reported by these and 2/8 (25%) A. platyrhynchos. None of the six specimens of authors are similar to those here described, experimental or A. cygnoides evaluated were found infected. The eggs obtained molecular evidence is still necessary for unequivocal identification from formalin-fixed fecal samples measured 163 ± 6 (152–174) of the parasite. μm long by 84 ± 4 (77–90) μm wide (N = 20). They are similar to the eggs present in the uterus of the adult parasite or described by different authors (Schafranski et al., 1975; Hoyos et al., 2017). Molecular phylogenetic study The analysis of sequences 28S (1170 bp) obtained for T. cucumer- Identification of the first intermediate host inum from C. moschata in the current study revealed high simi- In the malacological analysis, specimens of Biomphalaria strami- larities with other members of the Typhlocoelinae subfamily, nea (Dunker, 1848) (n = 248), Physa acuta (Draparnaud, 1805) Typhlocoelum sp. (KT956960) (99.3%) and T. cymbium (n = 100) and Pseudosuccinea columella (Say, 1817) (n = 39) (MK355447) (99.1%), originated from North America and were evaluated for the presence of larval trematodes after mechan- China, respectively. Regarding the recent Chinese isolate of T. ical compression. Rediae and metacercariae of T. cucumerinum cymbium, it is important to note that there is doubt on specific were found in 3 (1.2%) specimens of B. straminea. Of the seven identification of the parasites, given this species is currently con- collections made, two were made in 2018 (June and October) sidered as belonging to the genus Neivaia Travassos, 1929 and five in 2019 (two collections in March, one in April and (Dubois, 1959; Kanev et al., 2002). Li et al. (2020) did not present 2 in May). Of these, samples of molluscs positive for infection morphological data but mention that the parasite was identified belong to the intervals of March, April and May 2019. according to Tang and Tang (1978). In this last study, the para- Regarding the rediae, few specimens (1, 2 and 3) were found in sites represented have intestinal diverticula, a morphological the infected mollusc. The larval stages obtained are described trait absent in Neivaia cymbium (Diesing, 1850). We opt by indi- below. Measures obtained for T. cucumerinum in the present cating this inconsistency from now on as Tracheophilus sp. (=‘T. and previous studies and other similar larvae reported in molluscs cymbium’ of Li et al., 2020). Considering other cyclocoelids with are shown in Table 1. sequences available, the percentages of similarity were lower Mature rediae (N =3)(Fig. 2A, B): elongated, with locomotor [Morishitium polonicum malayense Urabe, Nor Hashim & Uni appendages in the posterior portion. Mouth in the anterior por- (LC520231) (94.7%) and C. mutabile (AY222249) (92.4%)]. In tion, followed by an oval muscular pharynx. Caecum long, the phylogenetic analysis, the Brazilian isolate of T. cucumerinum large, with dark-coloured material inside. Presence of cercariae grouped in a well-supported clade (1 BI/100 ML) with at different stages of development, with those more developed Typhlocoelum sp. and Tracheophilus sp. (=‘T. cymbium’ of Li in the anterior half. Germinative masses in the posterior portion. et al., 2020). Although belonging to different genera, these two Cercariae (N = 10) (Fig. 2D): Body oval-shaped, flattened last isolates formed a subclade (0.81 BI/94 ML) (Fig. 3A). dorsoventrally. Larvae without tail, with a small protuberance at Concerning mitochondrial markers, Cox-1 (802 bp) sequences the posterior end. Anterior organ subterminal. Pharynx rounded, were obtained for the adult from C. moschata and larval stages immediately below the anterior organ. Ventral sucker absent. from B. straminea, which showed 100% similarity, confirming Caecum with two branches, united in the posterior part of the the conspecificity between these developmental stages. body, without reaching the extremity. Excretory vesicle oval, Considering the other two members of the family Cyclocoelidae terminal. with data available for comparison, T. cucumerinum showed Metacercariae (N = 10) (Fig. 2C): encysted form, round to 82.9 and 92.8% similarity to Uvitellina sp. (NC042722) and spherical, with a thick cystic wall. Tracheophilus sp. (=‘T. cymbium’ of Li et al., 2020) (NC044135), respectively. In the phylogenetic analysis, based on a 343 bp dataset containing eight members of the family Remarks Cyclocoelidae, the isolates were partitioned into representative In general, the morphology and measurements of the larval forms clades containing species from three subfamilies: Cyclocoelinae, recovered from B. straminea in the current study agree with those Haematotrephinae and Typhlocoelinae. It was verified the

Table 1. Morphometric and morphologic data of rediae, cercariae and metacercariae of Typhlocoelum cucumerinum obtained from Biomphalaria straminea from Brazil

Tracheophilus Cyclocoelum Cercaria T. cucumerinum sisowi mutabile acaudata

Host B. straminea Biomphalaria glabrata and Helisoma trivolvis Lymnaea ovata B. glabrata Biomphalaria tenagophila Locality Brazil Brazil USA Russia Brazil Reference Current study Schafranski et al. (1975) Stunkard (1934) Ginetsinskaia Ruiz (1952) (1949) Mature rediae (n =3) Body L 1214 ± 253 (1003– 934 ± 55 860–900 2000–3000 ≈5000 1493) W 430 ± 68 (353–484) 269 ± 44 260–270 –– Ventral sucker L 58 ± 9 (52–68) 62 ± 3 50–65 160 – W 55 ± 10 (52–68) 57 ± 3 – 180 – Cercariae (n = 10) Body L 241 ± 17 (214–259) 351 ± 49 265 340–370 – W 104 ± 5 (98–112) 135 ± 15 100 90–100 – Ventral sucker Absent Absent Present Present Present (16) (34 × 37) (47 × 62) Pharynx L 20 ± 2 (18–23) –––– W 23 ± 2 (21–25) –––– Metacercariae (n = 10) L 157 ± 17 (130–169) 151 175 230 ≈200 W 157 ± 17 (130–169) 141 180

n, number; L, length; W, width. Data reported for larval cyclocoelids by other authors are shown for comparison. Values are given in μm by the mean followed by standard deviation and range between parenthesis.

Fig. 2. Larval stages of Typhlocoelum cucumerinum found in Biomphalaria straminea from an urban lake from Brazil. (A) Redia. (B) Details of anterior end redia. (C) Metacercaria. (D) Cercaria. Scale bars: A = 200 μm, B–D=50μm.

Fig. 3. Phylogenetic relationships between Typhlocoelum cucumerinum and selected members of the family Cyclocoelidae inferred from partial 28S (1170 bp) (A), Cox-1 (343 bp) (B) and Nad-1 (395 bp) (C) sequences analysed by BI and ML methods. Nodal supports are indicated as ML/BI; values smaller than 0.90 for BI and 70 for ML are indicated by a dash. Asterisks indicate nodes that were not found in ML. New sequences from the current study are shown in bold.

grouping of T. cucumerinum with Tracheophilus sp. (=‘T. cym- et al., 1969; Yamaguti, 1971; Schafranski et al., 1975; Kanev bium’ of Li et al., 2020) (NC044135) from China (1 BI/100 et al., 2002). The only divergence found is about the presence ML) (Fig. 3B). of a poorly developed ventral sucker, a characteristic presented Sequences of the gene Nad-1 (395 bp) were obtained for adult in the diagnosis of the genus Typhlocoelum by Kanev et al. T. cucumerinum and compared with 12 species of cyclocoelids. (2002). We observed the complete absence of this character in Again, the Brazilian isolate here characterized showed higher T. cucumerinum, as reported by most other authors that previ- similarity (91.6%) with Tracheophilus sp. (=‘T. cymbium’ of Li ously identified this parasite (Dubois, 1959; Travassos et al., et al., 2020) from China (MK355447 and NC044135). 1969; Schafranski et al., 1975; Hoyos et al., 2017). The absence Considering other genera of the Cyclocoelidae, the percentage of a ventral sucker in the adult stage seems to be a characteristic of similarity was lower [82.9% to Harrahium tringae (Brandes, common to the almost absolute majority of the species of 1892) (KU877898); 80.8% to C. mutabile (MH091808-09, Cyclocoelidae. Thus, its presence in Typhlocoelum spp. and KX097823, KU877891); 80.4% to Uvitellina sp. (NC042722) and Manterocoelum americanum (Manter & Williams, 1928), as below of 80% in relation to Harrahium obscurum (Leidy, 1887) described by Kanev et al. (2002), needs to be reassessed. It is (KU877897), Hyptiasmus oculeus Kossack, 1911 (KU877896) important to comment that M. americanum was originally and Morishitium polonicum (Machalska, 1980) (KU877893)]. In described as a species of Typhlocoelum and illustrated without a phylogenetic analysis, T. cucumerinum also clustered in a well- ventral sucker (Manter and Williams, 1928), and previously con- supported clade with Tracheophilus sp. (‘T. cymbium’ of Li sidered a synonym for T. cucumerinum (Dubois, 1951). Scanning et al., 2020) (1 BI/98 ML) (Fig. 3C). electron microscopy can be an illuminating approach for the unambiguous determination of the presence or absence of ventral sucker in these parasites. Discussion Despite the existence of several reports on the occurrence of T. The occurrence of the tracheal trematode T. cucumerinum in cucumerinum in birds worldwide (Krull, 1940; Branton et al., birds and mollusc present in an urban lake from Brazil is 1985; Hoyos et al., 2017; Galosi et al., 2019), these studies are described in the current study based on an integrative approach. restricted to post mortem evaluations, which also occurred at The data obtained from the analysis of biological samples from the first moment in this study. This fact reveals gaps in the knowl- anatids and the finding of larval stages in molluscs made it pos- edge on the typhlocoelosis. The impact of parasitism with T. cucu- sible to elucidate the natural life cycle of the parasite. The taxo- merinum in domestic anatids, either in their natural environment nomic identification of T. cucumerinum was based on or in creations for consumption, is unknown given the lack of morphological characteristics of the adult parasite obtained studies involving in vivo detection of the parasite. from C. moschata and agrees with data described by different The sensibility of the spontaneous sedimentation technique for authors (Manter & Williams, 1928; Dubois, 1959; Travassos the parasitological diagnosis of T. cucumerinum is demonstrated

here for the first time. Among the 61 birds evaluated, eggs of which can hide the diversity of cyclocoelids. However, this T. cucumerinum were found in the feces of 14 anatids approach does not enable the detection of larval cyclocoelid, sug- (12 ducks and 2 mallards). We believe that the low cost and gesting that the diversity of these parasites in molluscs from nat- speed related to the spontaneous sedimentation method favours ural environments is underestimated. In fact, the knowledge on its use in future surveys involving not only T. cucumerinum but the interaction between cyclocoelids and their first intermediate also other avian parasites worldwide. Additional studies are hosts is practically restricted to experimental data obtained in pla- needed to characterize the performance of coproparasitological norbids [Biomphalaria spp., Planorbis sp. and H. trivolvis], phy- methods in the diagnosis of bird parasites and so direct control sids [P. gyrina] and lymnaeid (Lymnaea spp.) (Kossak, 1911; measures, especially the treatment. Stunkard, 1934; Ginetsinskaia, 1949; Schafranski et al., 1975; The prevalence of infection with T. cucumerinum (≈25%) here Yamaguti, 1975; Scott et al., 1982). Thus, aiming to highlight reported in anatids by coproparasitological exams was close to this hidden parasite diversity, we suggest that the crushing tech- that verified by post mortem studies in the USA (30.5%, 30/78) nique be included in future studies aiming to search for trema- (Kinsella and Forrester, 1972). On the other hand, a lower preva- todes in molluscs. lence of infection with T. cucumerinum was found, also by nec- Among the few studies reporting close-related larval trema- ropsy, in Canada [3.8% (53/534) in Aythya marila (Linnaeus, todes in molluscs from Brazil, Ruiz (1952) described C. acaudata 1761) and 16.7% (90/534) in A. platyrhynchos] (Scott et al., from B. glabrata also in the Minas Gerais State. The prevalence of 1980). More recently, this parasite has been found in 57.1% infection reported by this author ranging from 3.8% (1/16) to (24/42) of C. moschata necropsied in Colombia (Hoyos et al., 9.04% (19/210). This larva was also registered in B. tenagophila 2017). The variability verified in the reported prevalence of infec- in the state of São Paulo, Brazil (prevalence of infection: 0.66%) tion can be related to differences in the transmission dynamics in (Ohlweiler et al., 2013). The cercariae described by these authors each location, host susceptibility, besides ecological variables. differ from T. cucumerinum in the presence of a ventral sucker. In the current study, the occurrence of domestic anatids raised Moreover, the rediae of C. acaudata are approximately five in a semi-extensive system in an urban lake may have favoured times greater than those described for T. cucumerinum. More the occurrence of the parasite. The restriction and long stay of recently, larvae morphologically similar to T. cucumerinum have infected hosts in a single aquatic collection containing an abun- been found in B. glabrata in the state of Rio de Janeiro, Brazil dant population of susceptible intermediate hosts can have poten- (Oliveira et al., 2020). The unequivocal conspecificity between cialized the infection of anatids and snails with T. cucumerinum. these isolates still demands further experimental or molecular However, more in-depth studies are needed for elucidate aspects studies. related to parasite transmission dynamics. Molecular sequences to species of the genus Typhlocoelum are Tracheal trematodes are considered agents that cause respira- scarce and until then limited to one 28S sequence obtained for an tory disorders in birds (Barry, 1959). Although clinical evaluation isolate identified as Typhlocoelum sp. in the USA. In the current was not the focus of the current study, no animal presented study, sequences of the type species of the genus, T. cucumerinum, respiratory failure, characterizing subclinical infection probably were obtained for the first time. Sequences of the Cox-1 gene related to low worm burden. However, some anatids were obtained for larval stages recovered from B. straminea and adult observed performing clearing throat movements during field parasites were 100% similar, supporting the link between these interventions. Further studies aiming at a quantitative analysis different developmental stages. Also, the phylogenetic position of the eggs released in the feces of birds and its correlation with of T. cucumerinum in the family Cyclocoelidae was here evaluated worm burden can allow us inferences on this aspect. On the by three different molecular markers (28S, Cox-1 and Nad-1). other hand, the impact of a subclinical infection cannot be Overall, phylogenetic data here revealed the clustering, in well- neglected given the existence of reports of growth retardation in supported clades, of T. cucumerinum with two other members chicks infected with N. cymbium in Australia (Barry, 1959). of the subfamily Typhlocoelinae with data available for compari- This study represents the first report of a mollusc acting as nat- son. Interestingly, in the different markers evaluated, it was veri- ural intermediate host to T. cucumerinum. The morphology and fied that T. cucumerinum grouped in well-supported clades measures of the larval stages found in B. straminea in the current containing a Chinese identified originally as ‘Tracheophilus cym- study are in accordance with that described for T. cucumerinum bium’ but here considered as Tracheophilus sp. (=‘T. cymbium’ after experimental infection of B. glabrata and Biomphalaria tena- of Li et al., 2020). The percentages of similarity verified between gophila (d’Orbigny, 1835) (Schafranski et al., 1975). It is interest- T. cucumerinum and this Chinese isolate in the analysis of differ- ing to mention that, although the susceptibility of some species of ent markers (28S: 99.8%; Cox-1: 92.5% and Nad-1: 91.2%) are the families Physidae and Lymnaeidae to T. cucumerinum was higher than those verified in relation to other genera of family previously reported (Scott et al., 1982), no specimen of P. acuta Cyclocoelidae included in the analysis (28S: ≤94.7%; Cox-1: or P. columella evaluated in our study was found harbouring lar- ≤86.6% and Nad-1: ≤76.2%). These results, associated with the val stages of the parasite. However, it is important to note that the topology of the phylogenetic trees, point out that these isolates experimental susceptibility of molluscs to infection by T. cucu- of Typhlocoelum and Tracheophilus analysed are distinct species merinum is relatively high [≥90% in Physella gyrina (Say, 1821), but probably belong to the same genus. ≥70% in Helisoma trivolvis (Say, 1817) and ≥50% in Lymnaea The isolate of Tracheophilus included in the phylogenetic ana- elodes (Say, 1821) (Scott et al., 1982)]. In a previous experimental lyses was identified as T. cymbium (Li et al., 2020). However, the study involving other species of Biomphalaria, the rate of infec- cyclocoelid species described as Monostomum cymbium by tion was not evaluated (Schafranski et al., 1975). The biology of Diesing (1850) also has a complex taxonomic history. This spe- the asexual phase of members of Cyclocoelidae, in which the for- cies, described from Brazil, has already been considered to belong mation of the infective stage occurs inside the first intermediate to the genus Typhlocoelum (as Typhlocoelum cymbium) (Gower, host (without environmental emergence of cercariae), makes it 1939) and Tracheophilus (as T. cymbium) (Yamaguti, 1971, challenging to detect the infection. In fact, most studies involving 1975), but it is currently allocated in the genus Neivaia trematode larvae found in molluscs worldwide are carried out by (Dubois, 1959, 1965; Kanev et al., 2002). The Chinese isolate analysing the active emergence of cercariae, as recommend for sequenced by Li et al. (2020) was identified as T. cymbium follow- most of the species of trematodes with medical and veterinary ing the taxonomic concept presented in Tang and Tang (1978). importance (Dawes, 1968; Frandsen and Christensen, 1984), The lack of illustrations and voucher specimens deposited in a

museum makes it impossible for us to check the identity of this Ethical standards. The procedures followed ethical principles approved by isolate. However, the parasites represented in Tang and Tang the Local Ethics Committee in Animal Experimentation (CEUA-UFMG (1978) present intestinal diverticula and so clearly do not corres- protocol 68/2017). pond to the current concept of N. cymbium (Dubois, 1959; Kanev et al., 2002). Until new studies are carried out with this Chinese isolate, we tentatively considered it as Tracheophilus sp. References The genus Tracheophilus Skrjabin, 1913 was rescued and considered distinct from Typhlocoelum by Kanev et al. (2002) based Barry MR (1959) Flukes in the respiratory tract of ducks. 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London, UK: Cambridge University Press. ily Typhlocoelinae, the data presented herein point to the future Diesing KM (1850) Systema Helminthum, vol. 1. Braumüller: Voindobonae. re-synonymization between these two genera, as previously pro- Dubois G (1959) Revision des Cyclocoelidae Kossack 1911. Revue Suisse de posed by Dubois (1959). In this case, Typhlocoelum (the senior Zoologie 66,67–147. Dubois G (1965) Notes sur les Cyclocoelidae Kossack, 1911 (Trematoda). synonym) should be considered the valid name under the priority – law. As an additional example of the taxonomic complexity Revue Suissse de Zoologie 72, 413 427. Frandsen F and Christensen NO (1984) An introductory guide to the iden- related to these parasites, after the literature analysis, we noted tification of cercariae from African freshwater snails with special reference that some characteristics of Typhlophilus shovellus Lal, 1936, to cercariae of trematode species of medical and veterinary importance. such as the presence of an evident ventral sucker, pre-pharyngeal Acta Tropica 41, 181–202. genital pore, the size of eggs and the infection site (small intes- Galaktionov KV and Dobrovolskij AA (2003) The Biology and Evolution of tine) do not support the previous synonymization between Trematodes. Dordrecht, Boston, London: Kluwer Academic Publishers. Typhlophilus Lal, 1936 and Typhlocoelum (Dubois, 1959; Galosi L, Heneberg P, Rossi G, Sitko J, Magi GE and Perrucci S (2019) Air Yamaguti, 1971). 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