All experimental hosts were managed according to the guidelines approved by the Animal Ethics Committee of the Faculty of Science, Chiang Mai University, Thailand, and the relevant document (no. RE004/19) was approved by the committee. This study complied with all the relevant national regulations and institutional policies for human care and use of animals.

The natural hosts (bats and snails) were investigated in a canal in Su Thep subdistrict, Mueang district (in Chiang Mai University), Chiang Mai province (18°48′15.6″N, 98°57′08.4″E) between February 2020 and January 2021 (Fig. 1). The sampling locations were surrounded by water resources and forests. Only one Chinese pipistrelle bat (Hypsugo sp.) was found dead near the canal at Chiang Mai University and was immediately transported to our laboratory. For the recovery of worms, the internal organs, including the intestines, were separated and thoroughly examined with the naked eye and under a stereomicroscope. A total of 35 viviparid snails (F. martensi martensi) were collected near the canal and transported to the laboratory. Each snail was crushed to detect larval trematodes, including xiphidiocercariae. The specimens were fixed in 4% formaldehyde solution on a glass slide for morphological studies and preserved in 95% ethanol in a sample tube for molecular studies. The prevalence of infection was assessed according to Bush et al. [19]. A total of 56 adult specimens of P. longiforme were collected from the intestine of the dead bat, and 3 out of 35 (8.6%) snails infected with the xiphidiocercariae of P. longiforme were examined.

The specimens of adult and xiphidiocercariae were fixed with 4% formaldehyde between a glass slide and a cover slip overnight. The specimens were washed with distilled water and stained with Delafield’s hematoxylin (main stain) and eosin (counterstain). After staining with Delafield’s hematoxylin, the specimens were dehydrated with gradually increasing percentages of ethanol. If the stain was dark, it was removed from the specimen using a destaining solution (hydrochloric acid in 70% ethanol). The specimens were counterstained with a few drops of eosin in 85% ethanol. The specimens were cleared in xylene and transferred to a drop of Permount, which was placed on the glass slide and covered with a cover slip. The morphologies of the specimens were captured with a compound microscope using the software Optika Vision Pro (Optika, Bergamo, Italy) and drawn with the aid of a camera lucida attachment (Olympus, Tokyo, Japan). The muscular arrangement and nervous system were observed using a confocal laser scanning microscope equipped with Leica LAS X software (Leica, Wetzlar, Germany) and OlyVIA software (Olympus). The measurements of the specimens were designated by micrometers (μm) and millimeters (mm).

The DNA of adult P. longiforme and xiphidiocercariae were individually extracted using Chelex solution [20] with 5% Chelex and 3 μl of proteinase K (Sigma-Aldrich, St. Louis, MO, USA). The specimens were heated at 55°C for 1 h, followed by vortexing and heating at 95°C for 30 min. The mixture was then centrifuged at 13,000 rpm for 1 min. The supernatant of each specimen was collected and preserved at −20°C until further use.

ITS2 region amplification was performed using the following primers: a forward 3S primer (5′-GGTACCGGTGGATCACTCGGCTCGTG-3′) and a reverse BD2 primer (5′-TATG CTTAAATTCAGCGGGT-3′) [21]. PCR was performed using the following cycling conditions: 94°C for 2 min, followed by 35 cycles of 94°C for 1 min and 60°C for 1 min, then 72°C for 1 min, and a final extension at 72°C for 7 min.

28S rDNA was amplified using 2 primers: a forward LSU-5 primer (5′-TAGGTCGACC CGCTGAAYTTAAGC A-3′) and a reverse 1500R primer (5′-GCTATCCTGAGGGAAACT TCG-3′), where Y represets C or T [22]. PCR was conducted using the following parameters: 94°C for 2 min, followed by 35 cycles of 94°C for 30 sec and 60°C for 30 sec, then 72°C for 75 sec, and a final extension at 72°C for 7 min.

The PCR products were separated by 1% agarose gel electrophoresis and stained with the nucleic acid stain, SafeView^TM FireRed (Applied Biological Materials, Richmond, British Columbia, Canada), and observed under ultraviolet light. The PCR products were cleaned and sequenced by Ward Medic Ltd., Part. (Bangkok, Thailand). The partial sequences were aligned and edited using MEGA version 10.1.8. The sequencing results were compared and analyzed using the BLAST program in NCBI (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). The phylogenetic trees were constructed using maximum likelihood (ML) and Bayesian inference (BI) methods from ITS2 and 28S rDNA datasets of relative species in the superfamily Microphalloidea. The outgroups included Centrocestus formosanus species (family, Heterophyidae). MEGA version 10.1.8 (Pennsylvania State University, USA) was used to run the ML trees, and node support was estimated using 1,000ML replicate bootstrapping of tree topologies [23]. Bootstrap values of 70 were treated as well supported [24]. MrBayes 3.1.2 was used to create the BI trees. Posterior probabilities <0.95 were deemed significant [25]. The ITS2 and 28S rDNA trees were analyzed using FigTree version 1.4.4. The ITS2 region and 28S rDNA gene sequences determined in this study are available in the NCBI database under the accession numbers OQ402582-83 (adults) and OQ402586-87 (cercariae).

The shape of adult trematode (Figs. 2A, 3A; Table 1) was fusiform and moderate, measuring 0.84–0.98 mm×0.37–0.49 mm (n=10). The body tegument was smooth and without spines. The muscular oral sucker was well-developed and oval, located subterminally at the anteroventral side of the body, and measured 0.17–0.19 mm×0.14–0.15 mm. The prepharynx was short (0.01–0.02 mm in length) and was followed by the circular pharynx (Fig. 3A). The pharynx was 0.04–0.05 in diameter. The esophagus was longer than the prepharynx and was divided into two spacious saculiform intestinal ceca. The acetabulum was globular and well-developed, measured 0.08–0.09 mm in diameter and was situated at the middle line of the body. The acetabulum was smaller than the oral sucker. The pseudocirrus sac was circular, 0.04–0.05 mm in diameter, and was located between the acetabulum and ceca bifurcation area. The single ovary was slightly oval, measuring 0.05–0.06 mm×0.04–0.04 mm, and presented at the preacetabular area. Two circular testes were situated symmetrically at the level of the acetabulum and were 0.06–0.08 mm in diameter. The uterus occupied the entire posterior portion of the body. The vitelline follicle consisted of 20–24 follicles, which were anteriorly distributed to the split ceca on both sides of the body.

The sporocyst (Fig. 2B) was an elongated oval. The tegument was thin and had no spines, measuring 124.6–297.0 μm in length and 113.0–157.9 μm in width (n=10). The matured sporocyst contained 1 to 6 xiphidiocercariae. A stylet was observed at the oral sucker of each xiphidiocercaria. The xiphidiocercaria (Figs. 2C, 3B–G) were developed in the sporocyst. The cercarial body was a small, elongate oval, measuring 117.5–138.3 μm×48.3–52.2 μm (n=10). The tegumental surface of the body was covered with tiny spines. The body wall consisted of an outer layer of circular, diagonal, and longitudinal muscle fibers. The layers of the circular muscle fibers were difficult to observe. The diagonal muscle fiber layers were conspicuously observed below the oral sucker and were widespread to the posterior of the body. The longitudinal muscle fibers were arranged at the extremities of the body and extended to the posterior end of the body. The diagonal muscle fibers were present with spaces wider than those of the longitudinal fibers (Fig. 3B). The well-developed oral sucker was situated at the subterminus, measuring 21.2–25.1 μm in diameter. The stylet was observed at the oral sucker, which was situated dorsally to the mouth opening. The stylet was lanceolate in shape, with a wide basal portion, and tapered upward, measuring 13.1–16.3 μm (Fig. 2D). The prepharynx was short, measuring 3.9–4.9 μm, and was followed by the pharynx. The pharynx was small and circular, measuring 4.7–6.0 μm in diameter. The esophagus was long, narrow, and split into two intestinal ceca at the anterior part of the acetabulum. Two cephalic ganglions occupied the anterior part of the body near the pharynx. The glands were connected by transverse commissures. Many nerve cords originated from the cephalic ganglions and extended toward the anterior and posterior parts of the body. Nerve cords were observed surrounding the oral sucker and the acetabulum (Fig. 3C). The well-developed acetabulum was 14.66–15.50 μm in diameter, which was smaller than the oral sucker. Three pairs of penetration glands were observed in the middle of the body, between the area of the pharynx and the split ceca. Each gland was connected to the penetration duct and laterally extended to the oral sucker area (Fig. 3D). The genital primordium was represented posterior to the acetabulum. The excretory bladder was V-shaped (Fig. 3E). The bladder was continued with the excretory canal of the tail and opened at the tail extremity. The tail was simple and flattened, measuring 100.7–103.7 μm×15.0–18.9 μm. The tail surface was covered with annular transverse striation (Fig. 3G). The excretory duct was also observed in the tail (Fig. 3G).

The ITS2 sequences of adults and xiphidiocercariae were successfully amplified for 399 bp, whereas the fragment of 28S rDNA obtained was 1,238 bp. The ITS2 tree (Fig. 4) revealed four families in the superfamily Microphalloidea: Lecithodendriidae, Collyriclidae, Prosthogonimidae, and Microphallidae. The lecithodendriid clade was separated into three genera: Paralecithodendrium, Lecithodendrium, and Pycnoporus. In this clade, P. longiforme adults isolated from the Chinese pipistrelle bat were clustered together with P. longiforme obtained from a bat (Myotis daubentoni) in Ukraine (identity: 94.5%) with a robustly supported clade (98ML/1BI). The xiphidiocercariae isolated from the snail F. martensi martensi were grouped with P. longiforme adults from this study (identity: 99.3%) with a high support clade (95ML/1BI) (Table 2).

The 28S rDNA tree (Fig. 5) demonstrated five families of the superfamily Microphalloidea: Lecithodendriidae, Phaneropsolidae, Pleurogenidae, Prosthogonimidae, and Microphallidae. From the tree, the lecithodendriid clade was monophyletic. The family Lecithodendriidae represented four genera: Paralecithodendrium, Lecithodendrium, Pycnoporus, and Ochoterenatrema. P. longiforme from the present study was clustered together with P. longiforme obtained from the bat in Ukraine (identity: 98.7%) with a robust support clade (99ML/1BI). The xiphidiocercariae obtained in this study also appeared in the P. longiforme clade (99.6% identity with adults obtained in this study) with a high support clade (100ML/1BI) (Table 2).