Introduction
Bucephalidae Poche, 1907 is a trematode group that typically uses three hosts in its lifecycle: molluscs, fishes, and piscivorous fishes (Baba and Urabe Reference Baba, Urabe, Boltovskoy and fortunei2015; Overstreet and Curran Reference Overstreet, Curran, Gibson, Jones and Bray2002; Shimazu Reference Shimazu2014; Yamaguti Reference Yamaguti1975). Piscivorous fishes, the definitive hosts, harbour adult worms and shed trematode eggs through faeces. When molluscs ingest the eggs, the larvae develop into sporocysts in the host tissues. The sporocyst-infected molluscs, acting as the first intermediate host, release cercariae. The cercariae directly penetrate fishes, the second intermediate host, and develop into metacercariae. The metacercariae mature into adult worms when piscivorous fishes consume the metacercaria-infected fish. Intense bucephalid infections (e.g., from 650 to 9700 worms per fish host (Ogawa et al. Reference Ogawa, Nakatsugawa and Yasuzaki2004)), especially by metacercariae, have been reported to negatively affect host fish, causing haemorrhages (e.g., in fins and head skin) and abnormal swimming, sometimes leading to the host’s death (Baba and Urabe Reference Baba and Urabe2011; Bullard and Overstreet Reference Bullard, Overstreet, Eiras, Segner, Wahil and Kapoor2008; Hoffmann et al. Reference Hoffmann, Körting, Fischer-Scherl and Schäfer1990; MacKenzie Reference MacKenzie1991; Ogawa et al. Reference Ogawa, Nakatsugawa and Yasuzaki2004).
The bucephalid trematode ‘Dollfustrema hefeiense’ (an invalid name for reasons described later; the specific name was changed from ‘Dollfustrema hefeiensis’ for grammatical agreement with the epithet of Gibson (Reference Gibson2020)) is found in Mainland China (Chen Reference Chen2007; Chen et al. Reference Chen, Wang, Yao and Nie2007). Adults have been detected in the intestines of freshwater piscivorous fishes of Bagridae, Cyprinidae, and Sinipercidae, such as an oriental perch Coreoperca whiteheadi Boulenger, 1900; a folktaled bullhead Tachysurus sinensis Lacepède, 1803; the mandarin fish Siniperca chuatsi (Basilewsky, 1855); the big-eye mandarin fish Siniperca kneri Garman, 1912; a Chinese perch Siniperca obscura Nichols, 1930; the Leopard mandarin fish Siniperca scherzeri Steindachner, 1892; and the gills of the Chinese false gudgeon Abbottina rivularis (Basilewsky, 1852) (Chen et al. Reference Chen, Wang, Yao and Nie2007). Although ‘D. hefeiense’ has been reported from Mainland China (e.g., Chen et al. Reference Chen2007; Zhang et al. Reference Zhang, Qiu and Ding1999), its scientific name is considered invalid for the following reasons. Chen et al. (Reference Chen, Wang, Yao and Nie2007) cited Zhang et al. (Reference Zhang, Qiu and Ding1999) as providing the original description of ‘D. hefeiense’; however, Zhang et al. (Reference Zhang, Qiu and Ding1999) used ‘Liu’ as the authority and only provided brief information regarding the trematode’s host and sampling locality without citations, morphological details, or type series. Therefore, based on ICZN13.1.1, 13.1.2 and 16.1 (ICZN 1999), Zhang et al. (Reference Zhang, Qiu and Ding1999) did not provide the original description, making ‘D. hefeiense Liu in Zhang, Qiu & Ding, Reference Zhang, Qiu and Ding1999’ a nomen nudum.
Zhang et al. (Reference Zhang, Qiu and Ding1999) and Li (Reference Li, Qiu, Li and Liu2019) used ‘Liu’ and ‘Liu, Reference Liu1985’ for the authority and description date for this trematode, respectively. Furthermore, Chen (Reference Chen2007), a doctoral thesis, cited Liu (Reference Liu1985) and ‘Wang (1995)’ regarding the morphological features of this trematode and Dollfustrema vaneyi (Tseng, Reference Tseng1930). This suggests that Liu (Reference Liu1985) or ‘Wang (1995)’ was the original description of the trematode. Liu (Reference Liu1985) proposed a description of ‘D. hefeiensis’ as a new species, and there was no information of D. vaneyi. However, Liu (Reference Liu1985) was a handwritten manuscript and/or its copies. The remaining candidate ‘Wang (1995)’ was a doctoral thesis which is not open to the public and we could not obtain, indicating that these publications cannot be considered the original descriptions under ICZN 8.1 and 9.1 (ICZN 1999). Moreover, later publications by the author of ‘Wang (1995)’ (Wang and Wang Reference Wang and Wang1998a, Reference Wang and Wangb, Reference Wang and Wangc) do not mention ‘D. hefeiense’ although D. vaneyi was only provided (Wang et al. 1998b), suggesting that ‘Wang (1995)’ was not the original description. Chen (Reference Chen2007) and Chen et al. (Reference Chen, Wang, Yao and Nie2007) briefly described the trematode’s morphology without providing figures, stating that ‘D. hefeiense’ is morphologically similar to D. vaneyi but distinguishable by genital pore position and DNA barcodes. Li (Reference Li, Qiu, Li and Liu2019) provided a detailed description and illustration of the trematode, which matched information provided by Liu (Reference Liu1985). However, because none of these publications (Chen Reference Chen2007; Chen et al. Reference Chen, Wang, Yao and Nie2007; Li Reference Li, Qiu, Li and Liu2019) explicitly intended to describe a new species, they do not qualify as original descriptions of the parasite based on IZCN 16.1 (ICZN 1999). Consequently ‘D. hefeiense Liu, Reference Liu1985’ is considered an invalid name as per ICZN 10.1 (ICZN 1999). Moreover, Nolan and Cribb (Reference Nolan and Cribb2010) and Nolan et al. (Reference Nolan, Curran, Miller, Cutmore, Cantacessi and Cribb2015) reported that the original description for this species was unavailable. Given that no valid synonym exists (refer to the synonyms listed below), ‘D. hefeiense’ must be described as a new species.
Bucephalid trematodes were not detected in Japanese freshwater until 1998 (Shimazu Reference Shimazu, Otsuru, Kamegai and Hayashi2003; Urabe et al. Reference Urabe, Ogawa, Nakatsugawa, Imanishi, Kondo, Okunishi, Kaji and Tanaka2001). However, in 1999, a heavy infestation of bucephalid metacercariae was suddenly discovered in cyprinid fishes from the Uji River, central Japan (Urabe et al. Reference Urabe, Ogawa, Nakatsugawa, Imanishi, Kondo, Okunishi, Kaji and Tanaka2001). The metacercariae were later identified as two species: Prosorhynchoides ozakii (Nagaty, 1937) and Parabucephalopsis parasiluri Wang, Reference Wang1985, both found across a wide area of the Uji River system (Baba and Urabe Reference Baba and Urabe2011; Ogawa et al. Reference Ogawa, Nakatsugawa and Yasuzaki2004; Urabe et al. Reference Urabe, Ogawa, Nakatsugawa, Nakai, Tanaka and Wang2007). In 2019, Pr. ozakii was detected in freshwater fish from the Tone River system, east Japan (Hayashi et al. Reference Hayashi, Sano, Ishikawa, Hagiwara, Sasaki, Nakao, Urabe and Waki2022). These two species are thought to have been introduced to Japan along with the golden mussel Limnoperna fortunei (Dunker, 1857), which serves as the first intermediate host for both bucephalid trematodes in Japan (Baba and Urabe Reference Baba and Urabe2011; Hayashi et al. Reference Hayashi, Sano, Ishikawa, Hagiwara, Sasaki, Nakao, Urabe and Waki2022). It is thought that the golden mussel was introduced to Japan around 1990 along with the Asian clam Corbicula fluminea (Muller, 1774), imported from Mainland China for freshwater aquaculture (Magara et al. Reference Magara, Matsui, Goto and Yuasa2001; Nishimura and Habe Reference Nishimura and Habe1987). An unidentified bucephalid sporocyst was also detected in golden mussels from the Yodo River system, Japan (Hayakawa et al. Reference Hayakawa, Urabe and Taniguchi2019).
Following the introduction of Pr. ozakii to the Tone River system (Hayahshi et al. 2022), we conducted surveys of molluscs and fishes to monitor bucephalid infections in this system. In 2021, we occasionally found adult trematodes of the genus Dollfustrema in freshwater fishes in the water system. Molecular and morphological analyses confirmed that the Dollfustrema species was identified as ‘D. hefeiense’, as reported in previous studies (Chen Reference Chen2007; Chen et al. Reference Chen, Wang, Yao and Nie2007; Li Reference Li, Qiu, Li and Liu2019). Additionally, a DNA-based survey allowed us to trace the larval stages of the trematode in the water system. The objectives of the present study are to describe this species, document its introduction to Japan, and determine its life cycle in the water system.
Materials and methods
Mussel and fish survey
Golden mussels were sampled from seven sites in the Tone River system from 2021 to 2023 (Figure 1). The mussels were transported to the laboratory, identified following Masuda and Uchiyama (Reference Masuda and Uchiyama2004), and then killed with knives and subsequently dissected to search for sporocysts under a stereomicroscope. When sporocysts were found, they were fixed and preserved in 70% or 99% ethanol. Sporocyst tissues from randomly selected mussels at each site were used for polymerase chain reaction (PCR) to identify species, as described later.
Figure 1. Sampling sites from the present study, numbered from 1 to 28. Site numbers correspond to those given in Table 1. Black circles: only Prosorhynchoides ozakii detected. White circle: only Dollfustrema invadens n. sp. detected. Grey circles: both species detected. Crosses: neither species detected.
Freshwater fishes were either sampled or purchased from fisheries in 28 sites between 2021 and 2023 (Figure 1). They were transported to the laboratory and identified following Nakabo (Reference Nakabo2013) and Fukuchi et al. (Reference Fukuchi, Matsuzawa and Sado2018), after which they were dissected to examine the fins, gills, muscles, and internal organs both with the naked eye and under a stereomicroscope. When metacercariae and adult bucephalid trematodes were detected, the worms were fixed for detailed morphological observations and PCR as described later. The scientific names of the fishes used in this study follow those of Froese and Pauly (Reference Froese and Pauly2024), and common names basically follow Froese and Pauly (Reference Froese and Pauly2024) and Hosoya (Reference Hosoya2019).
Morphological observations
From fish analyses, we detected bucephalid adults and metacercariae of two trematode species: Pr. ozakii, the only species previously reported in the Tone River system (Hayashi et al. Reference Hayashi, Sano, Ishikawa, Hagiwara, Sasaki, Nakao, Urabe and Waki2022), and ‘D. hefeiense’. Notably, ‘D. hefeiense’ and Pr. ozakii were easily distinguished at both adult and metacercaria stages, including under the stereomicroscope, as they differ in body colour (yellow or brown in ‘D. hefeiense’ vs. white in Pr. ozakii), body shape (elongate oval or pear-shaped vs. oval or ellipsoid), and rhynchus shape (truncate with spines vs. rounded without spines) (Figure 2b). Species identifications were confirmed through molecular analyses and detailed morphological observations of slide-mounted specimens.
Figure 2. Dollfustrema invadens n. sp. from the present study. (a) Pectoral fin base of the short-spined Japanese trident goby Tridentiger brevispinis. Black arrowhead: D. invadens n. sp. adults in muscle tissue. (b) D. invadens n. sp. adult (black arrowhead) and Prosorhynchoides ozakii metacercariae (white arrowhead) under a stereomicroscope. Arrowhead position indicates the rhynchus in each species. (c, d) D. invadens n. sp. metacercariae from the bluegill Lepomis macrochirus. c, encysted metacercaria; d, metacercaria and its cyst wall. Black and white arrowheads indicate the metacercaria and cyst wall, respectively.
Figure 3. Maximum-likelihood phylogenetic tree for trematodes of the genus Dollfustrema inferred from the partial nucleotide sequences of ITS1−5.8S−ITS2 (763 bp) (a) and 28S rDNA (909 bp) (b). The trematode isolates from the present study are shown in bold. Bootstrap values >50% are shown, and INSDC accession numbers are provided after scientific names. Telorhynchus sp. KNQ5T (N969625) and Brachylaima spp. (MK286936 and LC626505) comprised the outgroups. Asterisk indicates a sequence identical to LC847294.
For the morphological observations, selected adults and metacercariae were fixed in 70% or 99% ethanol between cover slips and glass slides. These specimens were stained with alum carmine or Heidenhein’s iron hematoxylin, dehydrated in an ethanol series, cleared in xylene or creosote, and mounted on slides with Canada balsam. Some adults and metacercariae were identified as Pr. ozakii through comparisons with morphological data reported by Hayashi et al. (Reference Hayashi, Sano, Ishikawa, Hagiwara, Sasaki, Nakao, Urabe and Waki2022). The remaining adults and metacercariae were morphologically and molecularly identified as ‘D. hefeiense’, as described below. Selected specimens (type series and additional specimens) were measured for body and organ size using a light microscope (BX53, Olympus) and a digital camera unit (AdvanCam-U3II, Advan Vision) to provide morphological descriptions. Observations were made ventrally, and line drawings were created using a camera lucida (U-DA, Olympus) attached to the microscope.
Cercariae from a fixed sporocyst molecularly identified as ‘D. hefeiense’ (see below) were mounted on slides with Hoyer’s medium for several hours and observed under the microscope (as described above). The type series and sporocyst were deposited at Ibaraki Nature Museum, Japan. Selected Pr. ozakii adults and metacercariae were also stained and mounted for museum deposition.
Molecular analysis
Alkaline lysates of sporocysts, metacercariae, and small tissue pieces of adults were used as templates for PCR, targeting nuclear 28S ribosomal RNA (rDNA) and the ITS1−5.8S−ITS2 region. The PCR primer sets were digl2 and LSU1500R (Snyder and Tkach Reference Snyder and Tkach2001; Tkach et al. Reference Tkach, Kudlai and Kostadinova2016) for 28S rDNA and BD1 and BD2 (Chen et al. Reference Chen, Wang, Yao and Nie2007) for the ITS1−5.8S−ITS2 region. DNA amplification and sequencing were performed as described by Nakao et al. (Reference Nakao, Waki, Sasaki, Anders, Koga and Asakawa2017). Alignment datasets were prepared using BioEdit Sequence Alignment Editor (Hall 1999) for data comparison. Sequences from related species were retrieved from the International Nucleotide Sequence Database Collaboration (INSDC: DDBJ/ENA/GenBank). The 28S rDNA and ITS1−5.8S−ITS2 datasets were employed for comparisons between the sporocyst, metacercariae, and adult sequences to identify species via pairwise divergence values (p-distance) using MEGA X (Kumar et al. Reference Kumar, Stecher, Li, Knyaz and Tamura2018). Phylogenetic analyses were performed based on ITS1−5.8S−ITS2 and 28S rDNA sequences of the new species and related species retrieved from INSDC in MEGA X, using the maximum likelihood method with 1,000 bootstrap replicates. MEGA X selected the Kimura 2-parameter model (Kimura Reference Kimura1980) and the HKY model (Hasegawa et al. Reference Hasegawa, Kishino and Yano1985) for ITS1−5.8S−ITS2 and 28S rDNA trees, respectively.
Results
Mussel survey
In total, 730 mussels were sampled from six sites during the survey (Table 1; Figure 1). Golden mussels with sporocysts were found at five of the six sites, with 77 infected mussels in total. From these, sporocysts from randomly selected mussels at each site (12 sporocysts in total) were used for PCR analysis. Results revealed that only one sporocyst was ‘D. hefeiense’, whereas the remaining 11 were confirmed as Pr. ozakii through later molecular analysis (Table 1).
Table 1. Detection of sporocysts of Dollfustrema invadens n. sp. and Prosorhynchoides ozakii from the golden mussels. Site numbers correspond to those given in Figure 1 (only sites where mussels were sampled are shown). Pref.: Prefecture
* Identified as D. invadens n. sp. (the other sporocysts were Pr. ozakii)
Fish survey
In total, 1,237 fish, representing 38 species, were collected from 28 sampling sites (Figure 1; Table 2). Adults of ‘D. hefeiense’ were found in eight fish species (Table 2): yoshinobori goby Rhinogobius sp., the river sleeper Odontobutis potamophila, the short-spined Japanese trident goby Tridentiger brevispinis, the swamp moroko gudgeon Gnathopogon elongatus, the sugo moroko gudgeon Squalidus chankaensis, the Chinese false gudgeon Abbottina rivularis, the channel catfish Ictalurus punctatus, and the bluegill Lepomis macrochirus. Among the species, only the T. brevispinis and Rhinogobius sp. are native to the survey area (Hosoya Reference Hosoya2019). Three species of gudgeon are introduced species from western Japan (Hosoya Reference Hosoya2019), whereas the river sleeper likely originated from East Asia (Fukuchi et al. Reference Fukuchi, Matsuzawa and Sado2018). The channel catfish and the bluegill were introduced to Japan from North America (Hosoya Reference Hosoya2019). Adults were found in the intestines of the channel catfish, the gill tissues of the river sleeper, and the muscles and fin tissues of the other six host fishes. A few encysted metacercariae of ‘D. hefeiense’ were found in the fin tissues and muscles of the swamp moroko gudgeon and the bluegill (Figure 2c, d).
Table 2. Detection of adults and metacercariae of Dollfustrema invadens n. sp. and Prosorhynchoides ozakii from fish. Site numbers correspond to those given in Figure 1. Pref.: Prefecture
* Four metacercariae were included.
** Twenty-five metacercariae were included.
*** Metacercariae infection was found in liver.
**** Adults were only detected.
Regarding Pr. ozakii, metacercariae were detected in the fins and epidermis of 504 individuals from 21 fish species across 18 sites (Table 2). Adults of Pr. ozakii were found only in the intestines of the channel catfish, as noted by Hayashi et al. (Reference Hayashi, Sano, Ishikawa, Hagiwara, Sasaki, Nakao, Urabe and Waki2022). In addition to the 13 second intermediate host species reported by Hayashi et al. (2002), 10 fish species were newly identified as metacercariae hosts in the present study: Cyprinus rubrofuscus, Carassius langsdorfii, Carassius auratus, Zacco platypus, Squalidus chankaensis, Micropterus salmoides, Odontobutis potamophila, Gymnogobius castaneus, Gymnogobius urotaenia, and Tachysurus sinensis. Metacercariae were found in the fins and fin base tissues in all hosts, except Tachysurus sinensis, in which they were found only in the liver. Slide specimens of Pr. ozakii were deposited in the Ibaraki Nature Museum, Japan, as follows: adults (slide specimens), INM-1-123584; metacercariae (slide specimens), INM-1-123585; sporocysts (molecularly identified and preserved in 99% ethanol), INM-1-123586.
Molecular analysis
DNA fragments of the ITS1−5.8S−ITS2, amplified by one primer set, from 10 ‘D. hefeiense’ adults and three Dollfustrema metacercariae were identical (766 bp). The sequence alignment (733 bp) of ITS1−5.8S−ITS2 from the database showed intraspecific divergence values of 0.000−0.016 and interspecific variations of 0.011−0.075 within the genus Dollfustrema (Supplementary Table S1). Divergence between our sequences and those of ‘D. hefeiense’ reported by Chen et al. (Reference Chen, Wang, Yao and Nie2007) was 0.000−0.005 (Supplementary Table S1), indicating that the adults and metacercariae in both studies belong to the same species: ‘D. hefeiense’. In the ITS1−5.8S−ITS2 phylogenetic tree, our sequences formed a single clade with the ‘D. hefeiense’ sequences from the previous study (Chen et al. Reference Chen, Wang, Yao and Nie2007) (Figure 3a), supporting this identification.
For 28S rDNA, DNA fragments (829−1174 bp) from five adults, one metacercaria, and one sporocyst were also identical. Sequence comparisons from online databases (745 bp) revealed intraspecific divergence values of 0.000−0.004 and interspecific variations of 0.033−0.060 within the genus Dollfustrema (Supplementary Table S2). Given the divergence values of zero between our sequences, the sporocyst, metacercariae, and adults were confirmed as a single species. In the 28S rDNA phylogenetic tree, the sporocyst sequence formed a single clade with the other ‘D. hefeiense’ sequences, supporting our molecular identification. Additionally, this new species occupied an independent phylogenetic position, even in this conserved gene.
The remaining 11 sporocysts could not be identified via our analysis, as their 28S rDNA sequences (884−1253 bp) differed from those of Dollfustrema spp. (0.123−0.140; Supplementary Table S2). These unidentified sporocysts were suspected to be Pr. ozakii, so we compared their sequences with those of Prosorhynchoides spp. from online databases. Based on 28S rDNA sequences (784 bp), intraspecific divergence in Prosorhynchoides was zero, whereas interspecific variation was 0.006−0.112 (Supplementary Table S3). Our sporocyst sequences were identical to those of Pr. ozakii from previous studies, confirming that these 11 sporocysts belong to Pr. ozakii.
The sequences of ‘D. hefeiense’ and Pr. ozakii obtained in this study were deposited in the INSDC through DNA Data Bank of Japan under the following accession numbers: new species LC847294 (28S rDNA: 1174 bp) and LC847296−847297 (ITS1−5.8S−ITS2: 763 bp); Pr. ozakii LC847295 (28S rDNA: 1253 bp).
Morphological descriptions of Dollfustrema invadens Saito, Iwata, Nitta & Waki n. sp.
The specimens used for morphological descriptions included five gravid adults (holotype and four paratypes) and one metacercaria (paratype) from tissue of the swamp moroko gudgeon Gnathopogon elongatus, as well as five cercariae from a sporocyst in the golden mussel. For data comparison, morphological characters of four gravid adults from an intestine of the channel catfish Ictalurus punctatus were reported as additional materials. All measurements are presented at averages, with ranges in parentheses, and are in micrometres unless otherwise stated.
Adult (based on holotype and three paratypes, Figure 4a–c, Table 3)
Body elongated oval or pear-shaped, 888 (783–945) in length and 215 (194–215) in width at widest point. Body yellow or brown in living condition. Tegmental spines on body surface. Suckers absent. Rhynchus small, truncate, 75 (56–95) in length and 67 (54–77) in width, with muscular apical disc. Three rows of rhynchal spines circling the anterior portion of rhynchus. Rhynchal spines larger in middle row, 6.3 (5.4–7.0) in length, and shorter in anterior and posterior rows, 3.6 (2.3–4.5) in length. Mouth opening ventral surface, posterior to anterior margin of anterior testis, from middle to two-third of body. Pharynx globular, 56 (50–62) in length and 51 (43–59) in width. Esophagus short, 21 (16–30) in length, extending anteriorly from pharynx. Intestinal cecum oblong, 182 (173–191) in length and 89 (60–99) in width. Testes two, ovoid, slightly lobed, placed obliquely. Anterior testis 118 (99–155) in length and 68 (47–88) in width. Posterior testis 106 (77–135) in length and 71 (59–81) in width. Cirrus pouch cylindrical, 229 (159–253) in length and 59 (53–64) in width. Seminal vesicle ovoid, in proximal part of cirrus pouch. Seminal duct, proximal part of prostatic duct in Bucephalidae (Overstreet and Curran Reference Overstreet, Curran, Gibson, Jones and Bray2002), from distal portion of seminal vesicle. Boundaries of seminal vesicle and seminal duct, and seminal duct and pars prostatica unclear. Pars prostatica well developed, looped at the level of cirrus pouch, surrounded by prostate gland cells. Genital lobe wide but its detail indistinct. Genital atrium posterior to ejaculatory duct. Genital pore opening just posterior to posterior margin of genital atrium. Ovary elliptic, situated between testes, 94 (91–100) in length and 70 (42–87) in width. Uterus extends from anterior fourth of body to nearly posterior end of cirrus pouch. Uterus wall indistinct. Metraterm lateral to cirrus pouch, opening into genital atrium. Eggs elongated oval, 32 (29–36) in length and 15 (13–18) in width. Vitelline follicles distributed dome-shape, extending forward beyond anterior terminal of caecum. Excretory pore at posterior terminal of body.
Figure 4. Dollfustrema invadens n. sp. (a–c) Adult, ventral view. (a) Entire body, holotype. (b) Rhynchal spines, holotype. (c) Cirrus pouch, paratype. (d) Metacercaria, paratype. (e) Cercaria (an additional material). (f) adult (an additional material). Abbreviations: c: caecum; ci, cirrus pouch; e, excretory pore; ed, ejaculatory duct; ga, genital atrium; gl, genital lobe; gp, genital pore; mt, metraterm; oe, oesophagus; ov, ovary; pc, prostatic gland cells; pp, pars prostatica; r, rhynchus; sd, seminal duct; sv, seminal vesicle; t, testis; v, vitellarium.
Table 3. Comparison of morphological measurements of Dollfustrema invadens n. sp. in different references. All measurements, unless indicated otherwise, are in micrometres. Dash represents no description in the cited reference
* Variation described in Chen et al. (Reference Chen, Wang, Yao and Nie2007)
** Length of all spines
*** Sizes of two testes
Metacercaria (based on one paratype, Figure 4d)
Excysted body elongated oval, 533 and 134 in length and greatest width, respectively. Body yellow-brown in living condition. Tiny tegmental spines on surface of body. Suckers absent. Rhynchus with muscular apical disc. Rhynchus small and truncate, 58 in length and 54 in width at widest point, with three rows of tiny rhynchal spines. Mouth opening ventral surface at two-third of body. Pharynx globular, 28 in length and 21 in width. Esophagus 32 in length, extending anteriorly from pharynx. Intestinal caecum oblong and elongated, 83 in length and 29 in width. Testes two, ovoid and slightly lobed. Anterior testis 41 in length and 26 in width. Posterior testis 48 in length and 21 in width. Ovary oblong, between two testes, 26 in length and 23 in width. Cirrus pouch cylindrical, 119 in length and 38 in width. Seminal vesicle oval and lobed, situated in anterior part of cirrus pouch. Prostate gland cells indistinct. Genital pore at nearly posterior end of cirrus pouch. Vitelline follicles immature, distributed dome-shape, extending forward beyond anterior terminal of caecum. Excretory pore at posterior terminal of body. Anterior extent of excretory bladder at level of posterior testis.
Cercaria (based on five additional materials Figure 4e)
Body elliptic, 205 (165–238) in length and 54 (20–79) in width. Suckers absent. Rhynchus with tiny spines, situated anterior terminal of body, 21 (15–26) in length and 27 (26–35) in width at widest point. Mouth opening ventral surface at two-third of body. Pharynx globular, 19 (18–21) in length and 18 (16–22) in width. Caecum oval, extending anteriorly from pharynx, length 40 (26–62) and width 21 (14–30). Tail stem oblong, connected to posterior terminal of body, 57 (29–80) in length and 61 (34–93) in width. Furcae paired, long, very elastic, 352 (194–435) in length and 14 (10–20) in width.
Adult (based on three additional materials, Figure 4f, Table 3)
Shape as in type series. Body slightly larger than the type series, 1041 (980–1148) in length and 300 (292–308) in width at widest point. Rhynchus 118 (92–141) in length and 92 (83–99) in width. Rhynchal spines larger in middle row, 7.2 (6.6–7.6) in length, and shorter in anterior and posterior rows, 5.2 (4.8–5.9) in length. Pharynx 64 (59–70) in length and 65 (61–70) in width. Esophagus 28 (26–30) in length, intestinal cecum 274 (145–365) in length and 97 (68–125) in width. Anterior testis 87 (63–110) in length and 80 (62–99) in width. Posterior testis 88 (75–101) in length and 77 (67–87) in width. Cirrus pouch 263 (241–274) in length and 78 (67–87) in width. Ovary 86 (67–105) in length and 87 (63–110) in width. Eggs elongated oval, 33 (31–35) in length and 18 (16–19) in width.
Remarks
The adults in this study are considered members of the genus Dollfustrema based on the following morphological characters: a rhynchus with a muscular apical disc, a small and truncate rhynchus with three rows of rhynchal spines, and a proximal part of pars prostatica looped in anterior part of cirrus pouch (Overstreet and Curran Reference Overstreet, Curran, Gibson, Jones and Bray2002). Our adult specimens can be distinguished from other members of genus Dollfustrema by the following morphological characters (Table 3): a small, truncate rhynchus with three rows of rhynchal spines (with the middle row having longer spines), a mouth opening located posterior to the anterior margin of the anterior testis, genital openings positioned at the level of the posterior margin of the cirrus pouch, the ovary between the testes, and a dome-shaped vitellarium. The new species resembles D. bagarii, D. bengalense, D. gibsoni, and D. vaneyi, as these species also have a truncate rhynchus and dome-shaped vitellarium. However, D. bagarii differs from our specimens in the position of the mouth opening (anterior to anterior testis in D. bagarii vs. not anterior to anterior testis in our specimens) and the location of the longest rhynchal spines (anterior row in D. bagarii vs. middle row in our specimens). Notably, D. gibsoni has a rhynchus with 4–5 rows of rhynchal spines (vs. 3 rows in our specimens) and genital pores near the posterior margin of the body (vs. the posterior terminal of the cirrus pouch in our specimens), further distinguishing it from the new species. Additionally, D. bengalense differed from our specimens by having 5 rows of spines on the anterior rhynchus (vs. 3 rows in our specimens). Although D. vaneyi has been reported to show morphological variations in body size, rhynchal spine length, and ovary position, our specimens can be distinguished from this species based on the position of the genital opening (at the level of the posterior margin of the cirrus pouch in D. hefeiense vs. the posterior margin of the ventral body in D. vaneyi). The morphological characters of ‘D. hefeiense’ reported by Liu (Reference Liu1985), Zhang et al. (Reference Zhang, Qiu and Ding1999), Chen (Reference Chen2007), and Li (Reference Li, Qiu, Li and Liu2019) closely resemble those of our specimens except numbers of rows of rhynchal spines. However, Chen et al. (Reference Chen2007) mentioned that two or three rows of rhynchal spines were found both in ‘D. hefeiense’ and D. vaneyi. Moreover, the ITS1−5.8S−ITS2 and 28S rDNA sequences of ‘D. hefeiense’ reported by Chen et al. (Reference Chen, Wang, Yao and Nie2007) were identical to those of our specimens as mentioned above. In addition, the distinct of the sampling localities in Chen et al. (Reference Chen, Wang, Yao and Nie2007) were adjacent to that of Liu (Reference Liu1985). These findings confirm that the trematodes from both our study and prior studies belong to the same species – namely, D. invadens n. sp. The additional materials are slightly larger than the type series but identified as D. invadens n. sp. as described above. Additionally, Li (Reference Li, Qiu, Li and Liu2019) classified this species under the genus Neodollfustrema Long & Lee, 1964. However, the distinction between Neodollfustrema and Dollfustrema was based on the position of the ovary (anterior to the testes in Neodollfustrema vs. not anterior in Dollfustrema) (Li Reference Li, Qiu, Li and Liu2019). However, Neodollfustrema had already been synonymised with Dollfustrema (Liu et al. Reference Liu, Peng, Gao, Fu, Wu, Lu, Gao and Xiao2010). Moreover, molecular analyses conducted by Chen et al. (Reference Chen, Wang, Yao and Nie2007) and that in the present study revealed that D. invadens n. sp. and D. vaneyi, which had been placed in the genera Neodollfustrema and Dollfustrema by Li (Reference Li, Qiu, Li and Liu2019), respectively, were closely related, supporting the synonymising of Neodollfustrema with Dollfustrema.
Taxonomical summary
Family Bucephalidae Poche, 1907
Genus Dollfustrema Eckmann, 1934
Species Dollfustrema invadens Saito, Iwata, Nitta & Waki n. sp.
Synonyms
Dollfustrema hefeiensis Liu, Reference Liu1985: 1–6, fig. 1 (invalidly described as new).
Neodollfustrema hefeiensis (Liu, Reference Liu1985): Li, Reference Li, Qiu, Li and Liu2019: 195–196, fig 132 (invalid).
Dollfustrema hefeiensis Liu in Zhang, Qiu & Ding, Reference Zhang, Qiu and Ding1999: 306; Nolan & Cribb, Reference Nolan and Cribb2010: 85; Nolan et al., Reference Nolan, Curran, Miller, Cutmore, Cantacessi and Cribb2015: 563–567; Anglade & Randhawa, Reference Anglade and Randhawa2018: 190; Corner et al., Reference Corner, Cribb and Cutmore2020: 458, 462; Atopkin et al., Reference Atopkin, Shedko, Rozhkovan, Nguyen and Besprozvannykh2022: 783 (nomen nudum).
Dollfustrema hefeiense Liu in Zhang, Qiu & Ding, Reference Zhang, Qiu and Ding1999: de Oliveira et al., Reference de Oliveira, Menezes, Keidel, Mello-Silva and Santos2022: 3 (nomen nudum).
Dollfustrema hefeiensis Zhang, Qiu & Ding, Reference Zhang, Qiu and Ding1999: Chen et al., Reference Chen, Wang, Yao and Nie2007: 791–799 (nomen nudum).
Dollfustrema hefeiensis (without attribution): Chen, Reference Chen, Wang, Yao and Nie2007: 16, 98, 107–109; Bott et al., Reference Bott, Miller and Cribb2013: 2564; Cremonte et al., Reference Cremonte, Pina, Gilardoni, Rodrigues, Chai and Ituarte2013: 86; Choudhary et al., Reference Choudhary, Verma, Swaroop and Agrawal2015: 169; Cremonte et al., Reference Cremonte, Gilardoni, Pina, Rodrigues and Ituarte2015: 203; Hammond et al., Reference Hammond, Cribb and Bott2018: 455; Hammond et al., Reference Hammond, Cribb, Nolan and Bott2020: 5; Shirakashi et al., Reference Shirakashi, Waki and Ogawa2020: 98; Malsawmtluangi, & Lalramliana, Reference Malsawmtluangi and Lalramliana2023: 2–5; Galaktionov et al., Reference Galaktionov, Gonchar, Postanogova, Miroliubov and Bodrov2024: 336, 338.
Dollfustrema hefeiense (without attribution): Curran et al., Reference Curran, Calhoun, Tkach, Warren and Bullard2022: 85.
Japanese name: Dorufusu-fukkou-kyuchu (bucephalid trematode of Dollfus)
Type host: The swamp moroko gudgeon Gnathopogon elongatus (Temminck & Schlegel, 1846)
Infection site: Holotype and adult paratypes, fin and fin-base tissues. Metacercaria paratype, fin.
Type locality: Kasumigaura lake, Ibaraki Prefecture, Japan
Date of collection: August 25, 2022
Additional material: Three adults from an intestine of the channel catfish Ictalurus punctatus (Rafinesque, 1818). A sporocyst from the golden mussel Limnoperna fortune (Dunker, 1857)
Deposition: Ibaraki Nature Museum, Ibaraki Prefecture, Japan. Collection Nos. INM-1-123580 (holotype, adult), INM-1-123581 (paratypes, adults), INM-1-123582 (paratype, metacercaria), INM-1-XXXXXX (additional material, adults), INM-1-123583 (additional material, sporocyst).
Etymology: The new species is named after ‘invasive’ species in Latin because it is an introduced species in Japan, type locality.
DNA markers: LC847294 (28S rDNA, 1174 bp) and LC847296−847297 (ITS1−5.8S−ITS2, 763 bp)
ZooBank identifier: urn:lsid:zoobank.org:act:A2169C5F-35A7-4283-A5EC-2CD67B22BA6A
ZooBank identifer (reference): urn:lsid:zoobank.org:pub:7F83C0D0-56FE-44FB-9C11-6FEB506BFF37
Discussion
In the present study, we described a Dollfustrema species previously reported in Mainland China, the origin of this trematode (Chen Reference Chen2007; Chen et al. Reference Chen, Wang, Yao and Nie2007; Li Reference Li, Qiu, Li and Liu2019; Liu Reference Liu1985; Zhang et al. Reference Zhang, Qiu and Ding1999). Although the exact introduction route is unclear, our hypothesis is that D. hefeiense n. sp. was possibly introduced directly to the Tone River system. From 2019 to 2021, Hayashi et al. (Reference Hayashi, Sano, Ishikawa, Hagiwara, Sasaki, Nakao, Urabe and Waki2022) detected Pr. ozakii in golden mussels and freshwater fishes in the same water system, but they did not find D. invadens n. sp. In contrast, we first detected D. invadens n. sp. in golden mussels and freshwater fishes in 2021 and 2022, respectively. These findings suggest that D. invadens n. sp. was introduced around 2020 and is now expanding its population in the water system.
The golden mussel, the first intermediate host of this new species, is thought to have been introduced to the Tone River system in 2005, likely with Asian clam seeds imported for aquaculture from East Asia (Ito Reference Ito2007), although no official records of these clam imports exist. Multiple haplotypes of golden mussels in this water system suggest repeated introductions (Tominaga et al. Reference Tominaga, Goka, Kimura and Ito2009), and it is possible that that D. invadens n. sp. arrived through such repeated introductions. Additionally, we detected the new species in Odontobutis potamophila, which was likely introduced directly from the East Asia continent to the Tone River system in 2017 (Fukuchi et al. Reference Fukuchi, Matsuzawa and Sado2018). Therefore, the trematode may have been introduced to Japan from Mainland China along with specific fishes, including the O. potamophila.
Bucephalid trematodes typically have three hosts in their life cycle (e.g., Hayashi et al. (Reference Hayashi, Sano, Ishikawa, Hagiwara, Sasaki, Nakao, Urabe and Waki2022)). In the current study, mature adult worms were found in the intestine of channel catfish, indicating that the catfish consumed small fishes infected with metacercariae. However, adult worms with eggs were also found in the fins, fin bases, and gills of small fishes, where both adults and metacercariae co-occurred. This suggests that metacercariae can develop directly into adults within the tissues of second intermediate hosts. Moreover, the adults may reproduce via self-fertilisation within the host tissues, as movement between tissues to find mates is unlikely. Consequently, D. invadens n. sp. appears to use a two- or three-host life cycle (Figure 5). The golden mussel serves as the first intermediate host, harbouring sporocysts that release cercariae, which infect small freshwater fishes, such as the Tamoroko, serving as second intermediate hosts. In these fish, the cercariae develop into encysted metacercariae, which may further develop into adults within the fish tissues or intestines of definitive hosts, such as the channel catfish, after ingestion. Adult worms in the muscles of small fishes may survive in the channel catfish’s intestines when it consumes its prey.
Figure 5. Life cycle of Dollfustrema invadens n. sp. in the Tone River system, Japan.
In the studied water system, the three life stages of D. invadens n. sp. – namely, sporocysts, metacercariae, and adults – primarily use introduced species as hosts. The golden mussel, the first intermediate host, is native to East Asia (Boltovskoy Reference Boltovskoy and Boltovskoy2015). Regarding metacercariae and adults, six of the eight host fish species were introduced from western Japan, Mainland China, and North America (Hosoya Reference Hosoya2019; NatureServe 2013). Notably, the parasite itself is an alien species, and its life cycle is maintained primarily by introduced hosts. The channel catfish and bluegill, both introduced from North America, are common in the water system (Japan Wild Research Center 2008; Ozaki and Miyabe Reference Ozaki and Miyabe2007; Seno and Matsuzawa Reference Seno and Matsuzawa2008) and exhibited heavy infections (approximately 200 worms per host), suggesting that they may be major spreaders of the new species.
The potential negative effects of D. invadens n. sp. infection on fishes, especially native species in Japan, remain unclear. However, the visible brown or yellow worms in fish muscle lower the commercial value of freshwater fish. As the golden mussel expands its distribution, this mussels are currently found in at least eight river systems (Nakano et al. Reference Nakano, Baba, Endo, Nagayama, Fujinaga, Uchida, Shiragane, Urabe and Kobayashi2015). The potential fish hosts of D. invadens n. sp., such as gobies, perches, and catfishes, are also becoming more widespread in these river systems (Hosoya Reference Hosoya2019; Tsuji et al. Reference Tsuji, Doi, Hibino, Shibata and Watanabe2024). Given that a single adult likely generates eggs through self-fertilisation, the population of D. invadens n. sp. can expand rapidly when newly introduced into water systems. To prevent further spread of the species, the introduction of potential hosts from the Tone River system into other water systems should be strictly avoided.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X24000932.
Acknowledgements
The authors are grateful to Hiroshi Eguchi, Takashi Kimura, Tomohiro Koyama, Masahiro Kumagai, Wakana Kuroki, Toshiyuki Matsui, Yasushi Morooka, Takahiro Morosawa, Yuto Sato, Shiro Satoh, Nie Pin, Takuya Ueda, and Shun Watanabe for helping our research.
Financial support
This work was supported by JSPS KAKENHI Grant Number JP 23K05375.
Competing interest
The authors declare none.
Ethical standard
Approval from research ethics committees was not for this study, as the experimental work involved unregulated fish and invertebrate species.
