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Brachylaima lignieuhadrae n. sp. (Trematoda: Brachylaimidae) from land snails of the genus Euhadra in Japan
Journal Pre-proof Brachylaima lignieuhadrae n. sp. (Trematoda: Brachylaimidae) from land snails of the genus Euhadra in Japan
Tsukasa Waki, Mizuki Sasaki, Kazuyuki Mashino, Takashi Iwaki, Minoru Nakao PII:
S1383-5769(19)30343-5
DOI:
https://doi.org/10.1016/j.parint.2019.101992
Reference:
PARINT 101992
To appear in:
Parasitology International
Received date:
23 December 2018
Revised date:
1 August 2019
Accepted date:
11 September 2019
Please cite this article as: T. Waki, M. Sasaki, K. Mashino, et al., Brachylaima lignieuhadrae n. sp. (Trematoda: Brachylaimidae) from land snails of the genus Euhadra in Japan, Parasitology International(2019), https://doi.org/10.1016/j.parint.2019.101992
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© 2019 Published by Elsevier.
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Brachylaima lignieuhadrae n. sp. (Trematoda: Brachylaimidae) from land snails of the genus Euhadra in Japan Tsukasa Wakia,* [email protected], Mizuki Sasakib, Kazuyuki Mashinoc, Takashi Iwakid, Minoru Nakaob a
Graduate School of Science, Toho University, Funabashi, Chiba 274–8510,
Japan b
of
Department of Parasitology, Asahikawa Medical University, Asahikawa,
Hokkaido 078-8510, Japan c
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The Firefly Museum of Toyota Town, Shimonoseki, Yamaguchi 750-0441, Japan
d
Meguro Parasitological Museum, Meguro-ku, Tokyo 153-0064, Japan
*
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Correspondence author.
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ABSTRACT Land snails of the genus Euhadra (Gastropoda: Bradybaenidae) are indigenous to the Japanese Archipelago. The larvae of an unknown species, tentatively named as Brachylaima sp. B (Trematoda: Brachylaimidae), have been found from Euhadra brandtii sapporo in Hokkaido, the northernmost island of Japan. In this study, a large-scale snail survey covering a wide area of Japan was conducted to confirm the larval parasite from members of Euhadra and related genera. Sporocysts with cercariae were found only from Eu. brandtii sapporo in
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Hokkaido and Euhadra callizona in central Honshu at low prevalence (1.0–9.6%). The metacercariae were detected widely from 6 species of Euhadra and the
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related genera at high prevalence (7.1–100%). A molecular identification by DNA barcoding demonstrated almost all of the larvae to be Brachylaima sp. B. Adult
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worms experimentally raised from the metacercariae were morphologically most
microstructure
of
the
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similar to Brachylaima ezohelicis in Hokkaido, but could be differentiated by the tegumental
surface.
We
propose
Brachylaima
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lignieuhadrae n. sp. for the unknown species, based on the morphology, DNA profile, host specificity, and geographic distribution. Phylogeography of the new
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species suggests a possibility that migratory birds serve as the definitive hosts.
Japan
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Keywords: Land snail, Euhadra, Trematoda, Brachylaima lignieuhadrae n. sp.,
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1. Introduction
Terrestrial gastropod mollusks, known as land snails and slugs, show patchy distributions over large and small scales due to their low mobility and specific habitat preferences. In the Japanese Archipelago, complicated landscapes promoted the speciation of gastropods due to geographical isolation, resulting in a rich fauna with approximately 800 species described to date [Biodiversity Center of Japan (www.biodic.go.jp)] [1]. Many of the gastropods are endemic
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species in Japan, suggesting that cospeciation may have occurred in their parasitic organisms, which include protozoans, platyhelminths, nematodes, and
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arthropods. However, few studies have been conducted on their host-parasite relationships.
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Terrestrial gastropods are a food source for a wide range of wildlife,
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including vertebrates [2–4]. This food chain plays a fundamental role in life cycles of various parasites. Trematodes of the family Brachylaimidae use
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gastropods as the first and second intermediate hosts and vertebrates as the definitive host [5]. Within this family, life cycle has been the most well studied for
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the genus Brachylaima [6–13]. Land snails (the first intermediate host) ingest parasite eggs from feces of birds or mammals. A reticular sporocyst
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subsequently grows in the hepatopancreas and generates a large number of cercariae by asexual multiplication. When the cercariae reach maturity, they are released to the environment to further parasitize other snails (the second intermediate host). The invading cercariae metamorphose into non-encysted metacercariae in the kidney. When birds or mammals (the definitive host) feed on the metacercaria-infected snails, gravid adults develop in the digestive tract. Each species of Brachylaima uses a particular species or group of snails as the first intermediate host, but a wide range of snail species are involved as the second intermediate host [6, 11–13]. In this manner, the parasites properly play the different roles of specialist and generalist. In Japan, Brachylaima syrmatici [14], Brachylaima eophonae [15], Brachylaima tokudai [16], and Brachylaima ishigakiense [17] have been described formerly, but their larval stages are still unknown. We have recently
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initiated a survey to find the larval stages of Brachylaima from land snails in Japan and described two species from Hokkaido, the northernmost island of Japan [12, 13]. The first species, Brachylaima ezohelicis, uses Ezohelix gainesi (Bradybaenidae) as the first intermediate host, and its metacercaria prevails in several species of land snails including Ez. gainesi [12, 13]. Natural definitive hosts of B. ezohelicis are unclear, although its adult stage has been described through experimental infections of mice [12]. The second species, Brachylaima asakawai, is a well-studied species, whose life cycle is maintained by Discus
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pauper (Discidae) as the first and second intermediate hosts and rodents as the definitive host [13]. Moreover, the metacercaria of an unknown species of
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Brachylaima was found from Allopeas satsumense (Subulinidae) in Tokyo [18]. Our previous report further demonstrated that two additional unknown
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species, tentatively named as Brachylaima sp. A and sp. B, prevail in land snails
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of Hokkaido [13]. The metacercaria of Brachylaima sp. A has been found from Succinea lauta (Succineidae), while that of Brachylaima sp. B was from Euhadra
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brandtii sapporo (Bradybaenidae). Both snails are indigenous to Japan, and the species diversity of Euhadra is high, especially in Honshu, the main island of
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Japan. In this study, we conducted a large scale snail survey of Brachylaima sp. B from members of Euhadra and the related genera in various localities of Japan.
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The main purpose of this study is to erect a new species for Brachylaima sp. B, using the larvae from naturally infected snails and the adults from experimentally infected mice.
2. Materials and methods
2.1. Snail collection and parasite isolation Land snails of Bradybaenidae and Camaenidae [19] were collected in 28 localities of Japan from 2013 to 2018 (Fig. 1, Table 1). Each of the snails collected was dissected in Dulbecco’s phosphate buffered saline (PBS) or 0.4% NaCl solution to find sporocysts, cercariae, and metacercariae. When the larvae were detected, the infected organs were recorded. The number of metacercariae was counted in each infected snail to calculate the intensity of infection (i.e., the
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mean number of metacercariae). Larvae were immediately used for microscopical observations, and the remaining individuals were preserved in 10% neutral-buffered formalin or 70–99% ethanol for morphological and molecular analyses.
2.2. Experimental infection of mice Metacercariae from Eu. brandtii in Hokkaido (nos. 4, 5, 7, and 8 in Fig.1) or from Eu. callizona in central Japan (no. 20) were administered perorally to 11
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female ICR mice. Simultaneously, 0.2 ml Depo-Medrol ® (Pfizer) was subcutaneously injected to each of the mice to suppress inflammatory reaction
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[12, 13]. The numbers of metacercariae administered were 6 to 42 per mouse.
2.3. Morphological observation
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worms from the small intestine.
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The mice were killed between 9 and 14 days post infection to recover adult
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Sporocysts and cercariae were observed under living conditions. These were mounted on glass slides with PBS or 0.4% NaCl containing neutral red, a
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vital dye. Metacercariae and adults were mounted on slides and then fixed under cover glass pressure with 70–99% ethanol or 5–10% neutral-buffered formalin.
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All developmental stages were observed under microscopes with a digital camera (DS-L3, Nikon and AxioVision, Zeiss). Morphometric measurements were taken via digital images. All measurements are in μm as the mean, with minimum-maximum range in parentheses. A camera lucida (Eclipse Ni-U, Nikon) was used for line drawings of the larvae and adults. Some of the specimens were stained with acetocarmine and mounted with Canada balsam. The tegumental surface of adult worms was observed with scanning electron microscopy (SEM). As reported previously [12, 13], adult worms were fixed serially with 1% glutaraldehyde,1% tannic acid, and 1% osmium tetroxide. Fixed worms were coated with platinum-palladium after dehydration. A field emission SEM (S4100, Hitachi) was used for observation.
2.4. DNA sequencing and phylogenetic analyses
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As reported previously [12, 13, 20], alkaline lysates of sporocysts and metacercariae were made from ethanol-preserved specimens for definitive identification of the parasites through DNA sequencing. The lysates were individually used as a template for polymerase chain reaction (PCR) to amplify both nuclear DNA and mitochondrial DNA (mtDNA). The nuclear 28S ribosomal DNA (rDNA) and mitochondrial cytochrome c oxidase subunit 1 (cox1) were selected as target genes. The latter gene is required for DNA barcoding to identify Brachylaima isolates at species-level [13]. We used the primer set digl2
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and 1500R for 28S rDNA [21] and the set JB3 and CO1-R trema for cox1 [22]. The PCR amplification and subsequent DNA sequencing were performed as
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reported previously [12, 13]. Part of the sequencing was outsourced to a Eurofins Genomics Inc. (Tokyo).
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Nucleotide sequences were assembled with BioEdit Sequence Alignment
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Editor [23] and MEGA6 [24]. The 28S rDNA and cox1 datasets were composed of 1268 and 728 nucleotide sites, respectively. The values of mean pairwise
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genetic distance among Brachylaima spp. were computed by MEGA6 under p-distance model. Phylogenetic trees of 28S and cox1 were reconstructed by the
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maximum-likelihood (ML) method in MEGA6 under the best-fit nucleotide substitution models GTR+I for 28S rDNA and HKY+G for cox1. Bootstrapping
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with 1000 replicates was conducted for the ML trees. Using the data set of cox1, a haplotype network was illustrated by TCS1.21 [25], and population genetics indices were calculated by DnaSP [26].
3. Results
3.1. Snail survey The results of the snail survey were summarized in Table 1. A total of 646 land snails belonging to 18 species (Bradybaenidae and Camaenidae) were collected from 28 sampling sites in Japan (Fig. 1). Six sporocyst-infected snails and 196 metacercaria-infected snails were found from the 646 snails. All 6 isolates of sporocysts and selective 61 isolates of metacercariae from each locality were subjected to DNA barcoding. Almost all of the isolates were
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identified as Brachylaima sp. B. Other species of Brachylaima were not found from members of Euhadra and the related genera, except 4 metacercarial isolates of B. ezohelicis from Ez. gainesi in Hokkaido (locality nos. 4, 5, and 8 in Table 1). The reticular sporocysts of Brachylaima sp. B containing nearly tailless cercariae were detected from the hepatopancreas (Supplementary Fig. 1), and its non-encysted metacercariae from the kidney. The sporocyst infections were restricted to members of the genus Euhadra (i.e., Eu. brandtii in Hokkaido and
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Eu. callizona in central Honshu), whereas the metacercarial infections widely occurred in members of the genera Euhadra, Ezohelix and Aegista (i.e. Eu.
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brandtii and Ez. gainesi in Hokkaido and Eu. callizona, Eu. peliomphala, Ae. eumenes, and Ae. hiroshifukudai in the other areas). All of the species are
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tree-climbing snails, excepting Ez. gainesi. The prevalence of sporocyst
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infections was much lower than that of metacercarial infections. Moreover, the metacercaria-infected snails were detected even in sites where no sporocysts
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were found. The prevalence and intensity of the metacercarial infections
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fluctuated according to locality, season, and snail species.
3.2. Experimentally raised adult worms
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A total of 50 adult worms were recovered from the ileum of 9 mice at 9 to 14 days post infection. All of them reached a gravid state. There were no differences in morphology among adults from different localities. The overall morphology of the experimentally raised adults agrees with that of Brachylaima, but its detailed structure was different from those of already known species. The morphology of the adult stage, the host specificity of the larval stage, the unique sequence of DNA barcode (cox1), and the autochthonous distribution of host snails enabled us to propose the following new species.
3.3. A new trematode (Brachylaimidae)
3.3.1. Morphological description Brachylaima lignieuhadrae n. sp. Waki, Sasaki et Nakao (Figs. 2–4)
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Specimens observed were 20 gravid adults from experimentally infected mice, and 8 cercariae and 7 metacercariae from naturally infected Euhadra snails. Specimens were observed ventrally. Adult (Figs. 2 and 3): Body cylindrical, 4452 (3457–5034) in length by 816 (671–963) in maximum width. Entire lateral and ventral surface spinose. Spine half-round in shaped, with very short digitated branches. Spines on posterior region of body gradually becoming sparser in density and smaller in size than anterior region. Oral and ventral suckers almost equal in size. Oral sucker oval,
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subterminal, 263 (191–343) long by 278 (219–341) wide. Ventral sucker oval, located in anterior quarter of body, 305 (243–392) long by 300 (232–370) wide.
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Prepharynx short. Pharynx well-developed, 151 (102–199) long by 194 (155– 243) wide. Esophagus absent. Cecum bifurcation post-pharyngeal. Caeca
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tubular, slightly undulating, running along bilateral borders to posterior extremity.
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Uterus tortuous, ascending from right side of anterior testis to postbifurcal region, then descending to genital pore. Gonads tandem, contiguous, located in
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posterior third of body. Testes large, weakly lobed. Anterior testis 443 (330–568) long by 411 (304–527) wide, posterior testis 419 (278–582) long by 393 (307–
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496) wide. Cirrus pouch elongate-oval, 278 (211–359) long by 102 (73–140) wide. Genital pore just anterior to anterior testis. Seminal vesicle distinctively
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enlarged, extending from left side of anterior testis, weakly coiled, connecting with cirrus pouch. Seminal receptacle spherical, sinistral to ovary. Ovary oval, located between testes, 212 (155–293) long by 276 (211–359) wide. Vitelline reservoir laying on posterior of ovary. Seminal receptacle spherical, located left side of ovary. Metraterm dextral to cirrus pouch. Vitellarium follicular, distributed bilaterally from level of posterior margin of ventral sucker to level of center of anterior testis. Excretory pore located at posterior end of body. Cercaria (Fig. 4A). Body cylindrical, slender posteriorly, 467 (432–498) in length by 136 (125–146) in maximum width. Stylet absent. Oral and ventral suckers equal in size. Oral sucker oval, subterminal, 76 (72–79) long by 73 (70– 78) wide. Ventral sucker oval, located in posterior half of body, 75 (72–80) long by 74 (71–77) wide. Prepharynx short. Pharynx globular, 29 (23–38) long by 39 (33–50) wide. Esophagus very short. Cecum reverse-Y-shaped, terminate in
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mid-body. Genital primordium present between ventral sucker and tail. Tail rudimentary, 36 (32–40) long by 39 (35–41) wide. Metacercaria (Fig. 4B). Body non-encysted, 1048 (849–1275) in length and 625 (526–724) in maximum width. Oral sucker slightly larger than ventral sucker. Oral sucker oval, subterminal, 256 (200–314) long by 217 (180–255) wide. Ventral sucker oval, located in anterior half of body, 172 (147–209) long by 189 (160–225) wide. Prepharynx short. Pharynx globular, 116 (99–135) long by 136 (101–171) wide. Esophagus absent. Cecum bifurcation post-pharyngeal. Ceca
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tubular, undulating, extending bilaterally to posterior end of body. Gonads immature, tandemly located in posterior half of body. Testes irregular in form.
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Anterior testis 69 (59–90) long by 88 (57–109) wide; posterior testis 88 (52–80)
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in diameter. Genital pore unobservable.
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long by 74 (53–108) wide. Ovary spherical, located between testes, 43 (35–55)
3.3.2. Taxonomic summary
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Type host: Euhadra brandtii sapporo Ijima, 1891. Additional second intermediate hosts: Ezohelix gainesi Pilsbry, 1900,
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Euhadra callizona Crosse, 1871, Euhadra peliomphala Pilsbry, 1890, Aegista eumenes Westerlund 1883, Aegista hiroshifukudai Kameda & Chiba, 2015
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Sites of infection: Hepatopancreas (sporocyst) and kidney (metacercaria). Type locality: Ebetsu, Hokkaido, Japan. Additional localities: Hokkaido, Honshu, and Kyushu (see Table 1). Collectors: T. Waki, M. Nakao, and M. Sasaki. Dates of collection: August 15, 2016 (a holotype and paratypes) and May 29, 2017 (paratypes). Definitive hosts: Unknown. The laboratory mouse, Mus musculus L., can be used as an alternative host under immuno-suppressive condition. Type specimens: The type series have been deposited in Meguro Parasitological Museum under the collection numbers MPM 21422 (holotype, adult), MPM 21423A–S (19 paratypes, adults), and MPM 21424 (7 paratypes, metacercariae). DNA differential markers: The parasite DNA sequences of nuclear 28S rDNA
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and
mitochondrial
cox1
(32
haplotypes)
have
been
deposited
into
DDBJ/ENA/Genbank databases under the accession numbers MK286935– 286939 (28S rDNA) and LC438346–438377 (cox1). Etymology: The new species is named after the arboreal behavior of Euhadra snails. The specific name means "of tree-climbing Euhadra" in Latin. Japanese name: Kinobori-maimai-sango-mushi (a coral-like parasite of arboreal snails)
3.4. Molecular phylogeny and haplotype network
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Synonym: Brachylaima sp. B [13]
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Molecular phylogenetic analyses were carried out to confirm the validity of the new species. A ML tree of nuclear 28S rDNA illustrated that Brachylaima spp.,
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including the new species, are monophyletic (Fig. 5A). In this tree, five
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geographic isolates of the new species from distant localities formed a robust clade, which could be differentiated from the other species of Brachylaima.
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Another ML tree of mitochondrial cox1, including the representative isolates of Brachylaima in Japan, clearly demonstrated the new species to be distinct from
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B. ezohelicis, and B. asakawai (Fig. 5B). The mean pairwise genetic distance of cox1 sequences among the three species was highly divergent, ranging from
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0.161 to 0.188.
Forty-nine randomly selected cox1 sequences of the new species were subjected to population genetics analyses. Those were composed of 32 samples from Hokkaido (nos. 1 to 8 in Fig. 1), 9 from central Japan (nos. 18 to 20), and 8 from western Japan (nos. 25 to 28). A total of 32 cox1 haplotypes were found from these samples. The resultant population genetics indices showed that the new species kept a high genetic diversity without bottlenecking (Supplementary Table 1). The cox1 haplotype network of all the samples depicted a highly scattered pattern, showing that there is almost no cline of the regional distribution in Japan (Fig. 6). Only one haplotype was common between Hokkaido (nos. 1, 3, and 5 in Fig. 1) and western Japan (nos. 26 and 27). The former island is approximately 1500 kilometers away from the latter localities in a straight line.
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4. Discussion
Morphological discrimination among various species of Brachylaima is often quite difficult, even for their adult stage. Ecological information about both localities and host animals is, therefore, essential in identifying the species. The lack of data on the definitive hosts of B. lignieuhadrae n. sp. is highly disadvantageous for species identification. However, the experimentally raised
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adult worms showed the following morphological characteristics: 1) almost equal-sized suckers, 2) a ventral sucker located in anterior quarter of body, 3)
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slightly undulating ceca, 4) an uterine tube extending to postpharyngeal position, 5) weakly lobed large testes, 6) an unlobed small ovary, 7) a well-developed
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cirrus pouch, 8) a thick and long seminal vesicle, 9) bilateral vitelline glands
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extending from level of posterior margin of ventral sucker to level of center of anterior testis, 10) the presence of a seminal receptacle, and 11) tegumental
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spines distributed throughout the ventral surface. The new species is morphologically distinguishable from its congeners when compared to them in
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combination with the above-mentioned diagnostic characters. The new species especially resembles B. eophonae [15], B. apoplania [27],
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B. cribbi [8], B. mascomai [11], and B. ratti [28] in morphology. However, the shape and size of their testes are obviously different from those of the new species (Supplementary Table 2). The other differential points are as follows: B. eophonae has shorter fields of bilateral vitelline glands, and B. ratti has a ventral sucker on anterior third of body. The new species most closely resembles B. ezohelicis. Both species were indistinguishable at light microscopic level. A SEM observation, however, revealed that the shape of tegumental spines and their distribution on the ventral surface were dissimilar. In the new species, a typical tegumental spine is half-round in shape and has short digitated branches (Fig. 3C). The spines are distributed throughout the ventral surface, but those on the posterior part become sparse in density and smaller in size (Fig. 3A). Conversely, B. ezohelicis has fine spines only on the ventral surface in the anterior half [12]. Since both of the species are sympatrically distributed in Hokkaido (Table 1), the
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application of distance-based DNA barcoding seems to be a practical and reliable way for the differential diagnosis of the morphologically similar species. The genus Brachylaima distributes worldwide and all species use snails as the intermediate hosts [29]. In Japan, six Brachylaima species had been reported from Mammals, birds and land snails. B. ezohelicis and B. eophonae can be distinguished from the new species as we mentioned above, and the remaining four species can be also distinguished based on morphologies of and uterus, vitelline, seminal receptacle and size of testis.
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The present molecular phylogenetic analyses are insufficient in evaluating the evolutionary history of brachylaimids, because of the lack of taxa
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represented by molecular data. Nevertheless, the present phylogenies based on nuclear 28S rDNA and mitochondrial cox1 are available for assessing the
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specific status of the new species. Even in the conservative 28S rDNA
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sequences, the resultant tree showed that the new species is substantially divergent from the other congeneric species. In the case of cox1, long
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sequences of Brachylaima (>700 bases) have been unpublished, excepting our sequences from the Japanese species. The cox1 tree was, therefore, made by
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using only isolates of the Japanese species to confirm their individuality. The topology of the cox1 tree clearly demonstrated that the Japanese species are
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related at a certain distance to one another. These genetic data strongly support the validity of the new species, together with other objective evidence from morphology, host-parasite ecology, and biogeography. The cox1 haplotype network analysis provides an important implication for the geographic spread of the new species. Many haplotypes from Hokkaido, central Japan, and western Japan were closely related to one another, and one haplotype was common in Hokkaido and western Japan. The high genetic diversity of the haplotypes and their disseminated network suggest that an invasive event and subsequent disturbance have repeatedly occurred in each locality. The possible explanation of the haplotype distribution is that the new species has been randomly shuffled by definitive hosts regularly travelling a long distance. The intermediate host snails of Euhadra and the related genera are excludable from the haplotype shuffling because of their very low mobility. The
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artificial movement of the host snails could be denied because their species diversity and endemism are well conserved in each locality [1]. In the Japanese Archipelago, many birds migrate to Hokkaido in the spring to early summer seasons and return southward in the winter [30]. Although the definitive hosts of the new species are completely unknown, we consider migratory birds as the candidates because of their high mobility. Moreover, the arboreal preference of the intermediate host snails indicates that the candidates could be narrowed down to particular birds, which prefer arboreal foraging and eating. The
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involvement of synanthropic rodents (members of Rattus and Mus) in the parasite dispersal seems to be low, because there is little overlap of ecological
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niche between the rodents and the host snails.
The distribution of trematodes is generally restricted to particular areas due
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to the habitat range of snails and their susceptibility to the parasites [31, 32]. The
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transmission of brachylaimid is ecologically unique because land snails have a dual responsibility for the first and second intermediate hosts. Each species of
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brachylaimids uses only a limited species of land snails for the development of the sporocyst [6, 11], probably due to an evolutionary selection linked to
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cospeciation between parasites and snails associated with co-evolution of host immune systems and sporocysts lethal to their hosts. Our previous field studies
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clearly demonstrated that the sporocyst infection occurs under a strict combination of brachylaimids and snails, namely B. ezohelicis and Ez. gainesi [12] and B. asakawai and Di. pauper [13]. By contrast, metacercarial infections are nonspecific to land snails. Accordingly, brachylaimids must have adapted for indigenous snails in each geographic area, and the detection of the sporocyst from indigenous snails may indicate the discovery of a new species. In this study, the sporocyst of B. lignieuhadrae n. sp. has been found only from members of Euhadra (i.e., Eu. brandtii in Hokkaido and Eu. callizona in central Japan). Most species of Euhadra are strictly endemic to the Japanese Archipelago, but only two species are exceptionally distributed in the southern part of the Korean Peninsula and Jeju Island [33]. Therefore, this species probably does not expand their distribution, resulting in undiscovered before our survey. This endemicity supports the validity of the new species.
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The prevalence of brachylaimid sporocysts in land snails is generally lower than that of metacercariae in various localities [11–13, 34]. This tendency was also observed in this study. The difference of the prevalences between sporocysts and metacercariae seems to be caused by the following factors: 1) the high pathogenicity of sporocyst, 2) the efficient transmission of cercariae, and 3) the densities of snails and definitive hosts. The sporocyst grows in the hepatopancreas, a digestive gland of snails [6, 11–13], and most of the gland is replaced by the reticular tubes containing enormous cercariae. The mortality of
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the sporocyst-infected snails is likely to be high, probably because of nutritional deficiency due to the dysfunction of hepatopancreas [34–36]. Even if the
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mortality is very high, the remaining infected snails must act as a superspreader of cercariae to other normal snails. Land snails generally have the habit of being
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aggregate with other individuals of the same species [37, 38]. Therefore, the
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transmission of cercariae from a sporocyst-infected snail to normal snails occurs probably in a certain space where the aggregation is arising. In this study,
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metacercariae of the new species were detected from 6 species of the genera Euhadra, Ezohelix and Aegista (Eu. brandtii, Ez. gainesi, Eu. peliomphala, Eu.
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callizona, Ae. eumenes, and Ae. hiroshifukudai) (Table 1) belonging to the family Camaenidae or Bradybaenidae, which should be included in Camaenidae by the
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most recent taxonomic revision [19]. The sporocyst-infected snails (Eu. brandtii and Eu. callizona) are included in these species. All the species have the habit of tree-climbing [39–43], excepting Ez. gainesi. It is likely that the tree-climbing behaviors are closely linked to the snail-to-snail transmission. This study showed that the prevalence of metacercarial infections is relatively high in E. brandtii sapporo from Hokkaido (Table 1). In this survey, we noticed that snails belonging to Euhadra are abundant in Hokkaido as compared with other localities. The abundance of the host snails may increase the chance of the sporocyst infections and accelerate the subsequent snail-to-snail transmission. The raccoon (Procyon lotor), an alien species from North America, is widely distributed in Japan, and its food habit is omnivorous, including arboreal land snails of Euhadra [43]. In the type locality of B. lignieuhadrae n. sp. (no. 7 in Fig. 1), the adult worms of Brachylaima sp. were found from raccoons [44, 45]. Given
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that the raccoons feed on arboreal land snails of Euhadra [43], we can speculate that they are accidentally infected with the new species. In Japan, adult worms of Brachylaima spp. have been reported from several mammals and birds [14– 17, 46, 47], although their intermediate hosts are still unclear. Our continuous snail survey will clarify their intermediate hosts.
Acknowledgements
of
This study was supported by LNest Grant Natural history award 2018 and the research project "Studies on Fauna and Flora of the Institute for Nature
ro
Study, National Museum of Nature and Science”. We would like to thank the staffs of the Shells Museum “Palais la mer” and the University of Tokyo Chiba
-p
Forest (UTCBF) for their kind support. Thanks are also due to the office staffs of
re
the Institute for Nature Study, National Museum of Nature and Science for permitting the collection of snails and providing the facilities, and to Mr. Katsuya
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na
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Tsukamoto and Dr. Tatsuo Yabe for providing snails for this study.
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Figure legends Fig. 1. Sampling sites of land snails belonging to Euhadra and the related genera. The sites are serially numbered from 1 to 28. A) Locations in Japan. B) Locations in Hokkaido. C) Locations in Kanto district. Closed and open circles represent locations where Brachylaima lignieuhadrae n. sp. were detected and undetected, respectively. The locality numbers correspond to those given in Table 1. Fig. 2. An experimentally raised adult of Brachylaima lignieuhadrae n. sp. (the
ro
of
ventral view of the holotype). Scale bar 500 μm. Abbreviations: c, cecum; cp, cirrus pouch; ed, excretory duct; ep, excretory pore; ev, excretory vesicle; gp, genital pore; m, metraterm; o, ovary; os, oral sucker; p, pharynx; sr, seminal
re
-p
receptacle; sv, seminal vesicle; t, testis; u, uterus; v, vitellarium; vd, vitelline duct; vr, vitelline reservoir; vs, ventral sucker.
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Fig. 3. Scanning electron microscopic photographs of adult Brachylaima lignieuhadrae n. sp. A) Ventral view of the whole body. The density of tegmental
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na
spines becomes sparse from the posterior (indicated by an arrow with bar), but their distribution continues till the posterior end (an arrow without bar). Scale bar 1 mm. B) Half-round shaped spines (the upside is anterior). Very short digitated branches are included in each of the spines. Scale bar 10 μm. C) Enlargement of the spine. Scale bar 5 μm. Fig. 4. Laval stages of Brachylaima lignieuhadrae n. sp. A) Cercaria. Scale bar 100 μm. B) Metacercaria. Scale bar 200 μm. Fig. 5. Maximum-likelihood phylogenetic trees of the genus Brachylaima. Bootstrap percentages are shown on representative nodes. Scale bars indicate the number of substitutions per nucleotide site. A) The tree of 28S rDNA. Members of Brachylaima and the related genera are included. The tree was rooted by the outgroup taxa of Clinostomum. B) The midpoint-rooted tree of mitochondrial cox1. Fig. 6. A statistical parsimony network inferred from 49 mitochondrial cox1 sequences of Brachylaima lignieuhadrae n. sp. The cox1 haplotypes were
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divided into three geographic populations (Hokkaido, central Japan, and western Japan). The size of ovals indicates the frequency of the haplotypes. Small circles show hypothetical haplotypes. Supplementary Fig. 1. Sporocysts of Brachylaima lignieuhadrae n. sp. A) The reticular development in the hepatopancreas of Euhadra brandtii sapporo. Scale
Jo ur
na
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bar 1 mm. B) Cercariae within the sporocyst. Scale bar 200 μm.
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Table 1. Detection of sporocysts (SC) and metacercariae (MC) of Brachylaima lignieuhadrae n. sp. from members of Euhadra and related land snails in Japan Localities a
Coordinates
Periods
Snail species b
No. snails
No.
No.
MC
No. snails with
examined
snails with
snails with MC
intensity (range) c
(no. examined)
SC (%)
(%)
1, Hokkaido
43.826, 142.613
Jun, 2017
Eu. brandtii*
5
0 (0)
5 (100)
4.2 ± 3.1 (1-8)
4 (4)
2, Hokkaido
43.634, 142.631
May, 2017
Eu. brandtii*
6
0 (0)
3 (50.0)
4.7 ± 1.2 (4-6)
3 (3)
3, Hokkaido
43.945,
May,
Eu. brandtii*
16
0 (0)
2 (12.5)
3 (3)
1 (1)
142.498
2017
43.757,
May,
Eu. brandtii*
11
0 (0)
5 (45.5)
6.6 ± 5.3
4 (4)
142.278
2017
4, Hokkaido 4
(2-14)
May,
e
Ez. gainesi
18
0 (0)
1 (5.6)
18
0 (1)
May, 2017
Eu. brandtii*
34
0 (0)
22 (64.7)
6.5 ± 5.6 (1-20)
8 (8)
May, 2017
Ez. gainesi
12
0 (0)
7 (58.3)
3.1 ± 4.8 (1-14)
5 (7) e
43.732,
Sep,
Eu. brandtii*
97
1 (1.0)
4.6 ± 3.9
2 (2)
142.203
2017
43.054, 141.510
Aug, 2016
Eu. brandtii*
14
Aug,
Ez. gainesi
40
5 6, Hokkaido 7, Hokkaido 7
2016 Aug, 2016
Ai. editha
7
May, 2017
Eu. brandtii*
43.029,
May,
Eu. brandtii*
141.316
2017
52 (53.6) 9 (64.3)
4.2 ± 1.4 (1-6)
6 (6)
0 (0)
4 (10.0)
2.0 ± 1.4
2 (2)
1
re
7
ro
43.680, 142.330
1 (7.1)
-p
5, Hokkaido
of
2017
(1-4)
0 (0)
0 (0)
1 (1.1)
34 (39.1)
6.8 ± 6.5 (1-29)
6 (6)
12 (92.3)
3.3 ± 2.1
4 (4)
0 (0)
9, Ibaraki
36.315, 140.588
Jun, 2017
lP
87
(1-21)
Eu. brandtii*
2
0 (0)
0 (0)
10, Chiba
35.160, 140.148
Oct, 2017
Eu. peliomphala*
43
0 (0)
0 (0)
Oct,
Ae. vulgivaga
6
0 (0)
0 (0)
2
0 (0)
10
13
(1-7)
May, 2017
Ez. gainesi
1
0 (0)
1 (100)
Jo ur
8
na
8, Hokkaido
1
0 (1) e
2017
10
Oct, 2017
Ni. sp.
11, Saitama
35.841, 139.609
Sep, 2017
Eu. quaesita
6
0 (0)
0 (0)
12, Saitama
35.801, 139.676
Sep, 2017
Br. pellucida
12
0 (0)
0 (0)
13, Tokyo
35.638, 139.721
2017
Eu. peliomphala*
15
0 (0)
0 (0)
f
13
2017 f
Br. pellucida
7
0 (0)
0 (0)
13
2017 f
Sa. japonica
4
0 (0)
0 (0)
13
2017 f
Ae. conospira
7
0 (0)
0 (0)
35.626,
Aug,
Eu. quaesita
9
0 (0)
0 (0)
139.703
2016
15, Kanagawa
35.443, 139.647
Mar, 2017
Br. pellucida
7
0 (0)
0 (0)
16, Kanagawa
35.256,
Aug,
Eu.
10
0 (0)
0 (0)
139.740
2016
peliomphala*
17, Tokyo
35.850, 139.035
Apr, 2018
Eu. peliomphala*
1
0 (0)
0 (0)
18, Tokyo
35.623, 139.247
Jun, 2016
Eu. peliomphala*
7
0 (0)
2 (28.6)
14, Tokyo
0 (0)
1.5 (1-2)
2 (2)
d
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19, Tokyo
34.680, 139.438
Feb, 2017
Eu. peliomphala*
14
0 (0)
1 (7.1)
Feb,
Ae. conospira
1
0 (0)
0 (0)
Oct, 2016
Eu. callizona*
29
0 (0)
17 (58.6)
20
Oct, 2016
Eu. scaevola
6
0 (0)
0 (0)
20
Oct,
Ae. vulgivaga
7
0 (0)
0 (0)
Sa. japonica
4
0 (0)
0 (0)
Eu. callizona*
31
3 (9.6)
13 (41.9)
19
2 (2)
1 (1)
6.5 ± 4.8 (1-16)
6 (6)
5.5 ± 7.6
3 (3)
2017 20, Shizuoka
35.077, 138.413
2016 20
Oct, 2016
20
Jun, 2017
21, Gifu
35.261,
Sep,
136.528
2017
21
(1-29) Eu. eoa
13
0 (0)
0 (0)
Sa. japonica
5
0 (0)
0 (0)
Ae. vulgivaga
5
0 (0)
0 (0)
Sep, 2017
Ae. commoda
2
0 (0) 0 (0)
0 (0)
Sep,
21
Sep,
of
2017
35.365, 136.468
Sep, 2017
Eu. eoa
2
23,
33.680,
Oct,
Eu. sigeonis*
13
0 (0)
0 (0)
Wakayama
135.359
2017 Eu. sandai
1
0 (0)
0 (0)
2
0 (0)
0 (0)
23
Oct,
re
21
-p
0 (0)
22, Gifu
ro
2017
33.550, 133.514
Mar, 2018
Eu. subnimbosa*
25, Shimane
34.922,
Sep,
Ae. eumenes*
1
0 (0)
1 (100)
3
1 (1)
132.347
2016
34.489, 131.403
Jun, 2013
Ae. hiroshifukudai*
7
0 (0)
1 (14.3)
9
1 (1)
Nov, 2015
Ae. hiroshifukudai*
2
0 (0)
1 (50.0)
8
1 (1)
May, 2015
Ae. eumenes*
2
0 (0)
1 (50.0)
8
1 (1)
Apr,
Ae. eumenes*
3
0 (0)
1 (33.3)
5
1 (1)
Ae. eumenes*
3
0 (0)
1 (33.3)
22
1 (1)
646
6 (0.9)
196 (30.3)
63 (67)
26, Yamaguchi 26
27
34.178, 131.208
Jo ur
27, Yamaguchi
na
24, Kochi
lP
2017
2017
28, Oita Total
33.010, 131.750
May, 2013
a
Individual numbers of sampling sites were shown in the map of Fig. 1. Abbreviations of genera are as follows: Ae, Aegista; Ai, Ainohelix; Br, Bradybaena; Eu, Euhadra; Ez, Ezohelix; Ni, Nipponochloritis; Sa, Satsuma;. Asterisks indicate tree-climbing snails. c The mean number of MC with standard deviation was computed in infected snails. d All SC and selected MC were subjected to DNA barcoding for species identification. In the case of MC, at least one MC per snail was b
identified. e Some of MC were identified as Brachylaima ezohelicis. f Collection records during March to November were combined.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Tsukasa Waki, Mizuki Sasaki, Kazuyuki Mashino, Takashi Iwaki, Minoru Nakao PII:
S1383-5769(19)30343-5
DOI:
https://doi.org/10.1016/j.parint.2019.101992
Reference:
PARINT 101992
To appear in:
Parasitology International
Received date:
23 December 2018
Revised date:
1 August 2019
Accepted date:
11 September 2019
Please cite this article as: T. Waki, M. Sasaki, K. Mashino, et al., Brachylaima lignieuhadrae n. sp. (Trematoda: Brachylaimidae) from land snails of the genus Euhadra in Japan, Parasitology International(2019), https://doi.org/10.1016/j.parint.2019.101992
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Brachylaima lignieuhadrae n. sp. (Trematoda: Brachylaimidae) from land snails of the genus Euhadra in Japan Tsukasa Wakia,* [email protected], Mizuki Sasakib, Kazuyuki Mashinoc, Takashi Iwakid, Minoru Nakaob a
Graduate School of Science, Toho University, Funabashi, Chiba 274–8510,
Japan b
of
Department of Parasitology, Asahikawa Medical University, Asahikawa,
Hokkaido 078-8510, Japan c
ro
The Firefly Museum of Toyota Town, Shimonoseki, Yamaguchi 750-0441, Japan
d
Meguro Parasitological Museum, Meguro-ku, Tokyo 153-0064, Japan
*
Jo ur
na
lP
re
-p
Correspondence author.
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ABSTRACT Land snails of the genus Euhadra (Gastropoda: Bradybaenidae) are indigenous to the Japanese Archipelago. The larvae of an unknown species, tentatively named as Brachylaima sp. B (Trematoda: Brachylaimidae), have been found from Euhadra brandtii sapporo in Hokkaido, the northernmost island of Japan. In this study, a large-scale snail survey covering a wide area of Japan was conducted to confirm the larval parasite from members of Euhadra and related genera. Sporocysts with cercariae were found only from Eu. brandtii sapporo in
of
Hokkaido and Euhadra callizona in central Honshu at low prevalence (1.0–9.6%). The metacercariae were detected widely from 6 species of Euhadra and the
ro
related genera at high prevalence (7.1–100%). A molecular identification by DNA barcoding demonstrated almost all of the larvae to be Brachylaima sp. B. Adult
-p
worms experimentally raised from the metacercariae were morphologically most
microstructure
of
the
re
similar to Brachylaima ezohelicis in Hokkaido, but could be differentiated by the tegumental
surface.
We
propose
Brachylaima
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lignieuhadrae n. sp. for the unknown species, based on the morphology, DNA profile, host specificity, and geographic distribution. Phylogeography of the new
na
species suggests a possibility that migratory birds serve as the definitive hosts.
Japan
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Keywords: Land snail, Euhadra, Trematoda, Brachylaima lignieuhadrae n. sp.,
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1. Introduction
Terrestrial gastropod mollusks, known as land snails and slugs, show patchy distributions over large and small scales due to their low mobility and specific habitat preferences. In the Japanese Archipelago, complicated landscapes promoted the speciation of gastropods due to geographical isolation, resulting in a rich fauna with approximately 800 species described to date [Biodiversity Center of Japan (www.biodic.go.jp)] [1]. Many of the gastropods are endemic
of
species in Japan, suggesting that cospeciation may have occurred in their parasitic organisms, which include protozoans, platyhelminths, nematodes, and
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arthropods. However, few studies have been conducted on their host-parasite relationships.
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Terrestrial gastropods are a food source for a wide range of wildlife,
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including vertebrates [2–4]. This food chain plays a fundamental role in life cycles of various parasites. Trematodes of the family Brachylaimidae use
lP
gastropods as the first and second intermediate hosts and vertebrates as the definitive host [5]. Within this family, life cycle has been the most well studied for
na
the genus Brachylaima [6–13]. Land snails (the first intermediate host) ingest parasite eggs from feces of birds or mammals. A reticular sporocyst
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subsequently grows in the hepatopancreas and generates a large number of cercariae by asexual multiplication. When the cercariae reach maturity, they are released to the environment to further parasitize other snails (the second intermediate host). The invading cercariae metamorphose into non-encysted metacercariae in the kidney. When birds or mammals (the definitive host) feed on the metacercaria-infected snails, gravid adults develop in the digestive tract. Each species of Brachylaima uses a particular species or group of snails as the first intermediate host, but a wide range of snail species are involved as the second intermediate host [6, 11–13]. In this manner, the parasites properly play the different roles of specialist and generalist. In Japan, Brachylaima syrmatici [14], Brachylaima eophonae [15], Brachylaima tokudai [16], and Brachylaima ishigakiense [17] have been described formerly, but their larval stages are still unknown. We have recently
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initiated a survey to find the larval stages of Brachylaima from land snails in Japan and described two species from Hokkaido, the northernmost island of Japan [12, 13]. The first species, Brachylaima ezohelicis, uses Ezohelix gainesi (Bradybaenidae) as the first intermediate host, and its metacercaria prevails in several species of land snails including Ez. gainesi [12, 13]. Natural definitive hosts of B. ezohelicis are unclear, although its adult stage has been described through experimental infections of mice [12]. The second species, Brachylaima asakawai, is a well-studied species, whose life cycle is maintained by Discus
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pauper (Discidae) as the first and second intermediate hosts and rodents as the definitive host [13]. Moreover, the metacercaria of an unknown species of
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Brachylaima was found from Allopeas satsumense (Subulinidae) in Tokyo [18]. Our previous report further demonstrated that two additional unknown
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species, tentatively named as Brachylaima sp. A and sp. B, prevail in land snails
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of Hokkaido [13]. The metacercaria of Brachylaima sp. A has been found from Succinea lauta (Succineidae), while that of Brachylaima sp. B was from Euhadra
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brandtii sapporo (Bradybaenidae). Both snails are indigenous to Japan, and the species diversity of Euhadra is high, especially in Honshu, the main island of
na
Japan. In this study, we conducted a large scale snail survey of Brachylaima sp. B from members of Euhadra and the related genera in various localities of Japan.
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The main purpose of this study is to erect a new species for Brachylaima sp. B, using the larvae from naturally infected snails and the adults from experimentally infected mice.
2. Materials and methods
2.1. Snail collection and parasite isolation Land snails of Bradybaenidae and Camaenidae [19] were collected in 28 localities of Japan from 2013 to 2018 (Fig. 1, Table 1). Each of the snails collected was dissected in Dulbecco’s phosphate buffered saline (PBS) or 0.4% NaCl solution to find sporocysts, cercariae, and metacercariae. When the larvae were detected, the infected organs were recorded. The number of metacercariae was counted in each infected snail to calculate the intensity of infection (i.e., the
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mean number of metacercariae). Larvae were immediately used for microscopical observations, and the remaining individuals were preserved in 10% neutral-buffered formalin or 70–99% ethanol for morphological and molecular analyses.
2.2. Experimental infection of mice Metacercariae from Eu. brandtii in Hokkaido (nos. 4, 5, 7, and 8 in Fig.1) or from Eu. callizona in central Japan (no. 20) were administered perorally to 11
of
female ICR mice. Simultaneously, 0.2 ml Depo-Medrol ® (Pfizer) was subcutaneously injected to each of the mice to suppress inflammatory reaction
ro
[12, 13]. The numbers of metacercariae administered were 6 to 42 per mouse.
2.3. Morphological observation
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worms from the small intestine.
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The mice were killed between 9 and 14 days post infection to recover adult
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Sporocysts and cercariae were observed under living conditions. These were mounted on glass slides with PBS or 0.4% NaCl containing neutral red, a
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vital dye. Metacercariae and adults were mounted on slides and then fixed under cover glass pressure with 70–99% ethanol or 5–10% neutral-buffered formalin.
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All developmental stages were observed under microscopes with a digital camera (DS-L3, Nikon and AxioVision, Zeiss). Morphometric measurements were taken via digital images. All measurements are in μm as the mean, with minimum-maximum range in parentheses. A camera lucida (Eclipse Ni-U, Nikon) was used for line drawings of the larvae and adults. Some of the specimens were stained with acetocarmine and mounted with Canada balsam. The tegumental surface of adult worms was observed with scanning electron microscopy (SEM). As reported previously [12, 13], adult worms were fixed serially with 1% glutaraldehyde,1% tannic acid, and 1% osmium tetroxide. Fixed worms were coated with platinum-palladium after dehydration. A field emission SEM (S4100, Hitachi) was used for observation.
2.4. DNA sequencing and phylogenetic analyses
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As reported previously [12, 13, 20], alkaline lysates of sporocysts and metacercariae were made from ethanol-preserved specimens for definitive identification of the parasites through DNA sequencing. The lysates were individually used as a template for polymerase chain reaction (PCR) to amplify both nuclear DNA and mitochondrial DNA (mtDNA). The nuclear 28S ribosomal DNA (rDNA) and mitochondrial cytochrome c oxidase subunit 1 (cox1) were selected as target genes. The latter gene is required for DNA barcoding to identify Brachylaima isolates at species-level [13]. We used the primer set digl2
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and 1500R for 28S rDNA [21] and the set JB3 and CO1-R trema for cox1 [22]. The PCR amplification and subsequent DNA sequencing were performed as
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reported previously [12, 13]. Part of the sequencing was outsourced to a Eurofins Genomics Inc. (Tokyo).
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Nucleotide sequences were assembled with BioEdit Sequence Alignment
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Editor [23] and MEGA6 [24]. The 28S rDNA and cox1 datasets were composed of 1268 and 728 nucleotide sites, respectively. The values of mean pairwise
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genetic distance among Brachylaima spp. were computed by MEGA6 under p-distance model. Phylogenetic trees of 28S and cox1 were reconstructed by the
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maximum-likelihood (ML) method in MEGA6 under the best-fit nucleotide substitution models GTR+I for 28S rDNA and HKY+G for cox1. Bootstrapping
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with 1000 replicates was conducted for the ML trees. Using the data set of cox1, a haplotype network was illustrated by TCS1.21 [25], and population genetics indices were calculated by DnaSP [26].
3. Results
3.1. Snail survey The results of the snail survey were summarized in Table 1. A total of 646 land snails belonging to 18 species (Bradybaenidae and Camaenidae) were collected from 28 sampling sites in Japan (Fig. 1). Six sporocyst-infected snails and 196 metacercaria-infected snails were found from the 646 snails. All 6 isolates of sporocysts and selective 61 isolates of metacercariae from each locality were subjected to DNA barcoding. Almost all of the isolates were
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identified as Brachylaima sp. B. Other species of Brachylaima were not found from members of Euhadra and the related genera, except 4 metacercarial isolates of B. ezohelicis from Ez. gainesi in Hokkaido (locality nos. 4, 5, and 8 in Table 1). The reticular sporocysts of Brachylaima sp. B containing nearly tailless cercariae were detected from the hepatopancreas (Supplementary Fig. 1), and its non-encysted metacercariae from the kidney. The sporocyst infections were restricted to members of the genus Euhadra (i.e., Eu. brandtii in Hokkaido and
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Eu. callizona in central Honshu), whereas the metacercarial infections widely occurred in members of the genera Euhadra, Ezohelix and Aegista (i.e. Eu.
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brandtii and Ez. gainesi in Hokkaido and Eu. callizona, Eu. peliomphala, Ae. eumenes, and Ae. hiroshifukudai in the other areas). All of the species are
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tree-climbing snails, excepting Ez. gainesi. The prevalence of sporocyst
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infections was much lower than that of metacercarial infections. Moreover, the metacercaria-infected snails were detected even in sites where no sporocysts
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were found. The prevalence and intensity of the metacercarial infections
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fluctuated according to locality, season, and snail species.
3.2. Experimentally raised adult worms
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A total of 50 adult worms were recovered from the ileum of 9 mice at 9 to 14 days post infection. All of them reached a gravid state. There were no differences in morphology among adults from different localities. The overall morphology of the experimentally raised adults agrees with that of Brachylaima, but its detailed structure was different from those of already known species. The morphology of the adult stage, the host specificity of the larval stage, the unique sequence of DNA barcode (cox1), and the autochthonous distribution of host snails enabled us to propose the following new species.
3.3. A new trematode (Brachylaimidae)
3.3.1. Morphological description Brachylaima lignieuhadrae n. sp. Waki, Sasaki et Nakao (Figs. 2–4)
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Specimens observed were 20 gravid adults from experimentally infected mice, and 8 cercariae and 7 metacercariae from naturally infected Euhadra snails. Specimens were observed ventrally. Adult (Figs. 2 and 3): Body cylindrical, 4452 (3457–5034) in length by 816 (671–963) in maximum width. Entire lateral and ventral surface spinose. Spine half-round in shaped, with very short digitated branches. Spines on posterior region of body gradually becoming sparser in density and smaller in size than anterior region. Oral and ventral suckers almost equal in size. Oral sucker oval,
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subterminal, 263 (191–343) long by 278 (219–341) wide. Ventral sucker oval, located in anterior quarter of body, 305 (243–392) long by 300 (232–370) wide.
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Prepharynx short. Pharynx well-developed, 151 (102–199) long by 194 (155– 243) wide. Esophagus absent. Cecum bifurcation post-pharyngeal. Caeca
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tubular, slightly undulating, running along bilateral borders to posterior extremity.
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Uterus tortuous, ascending from right side of anterior testis to postbifurcal region, then descending to genital pore. Gonads tandem, contiguous, located in
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posterior third of body. Testes large, weakly lobed. Anterior testis 443 (330–568) long by 411 (304–527) wide, posterior testis 419 (278–582) long by 393 (307–
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496) wide. Cirrus pouch elongate-oval, 278 (211–359) long by 102 (73–140) wide. Genital pore just anterior to anterior testis. Seminal vesicle distinctively
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enlarged, extending from left side of anterior testis, weakly coiled, connecting with cirrus pouch. Seminal receptacle spherical, sinistral to ovary. Ovary oval, located between testes, 212 (155–293) long by 276 (211–359) wide. Vitelline reservoir laying on posterior of ovary. Seminal receptacle spherical, located left side of ovary. Metraterm dextral to cirrus pouch. Vitellarium follicular, distributed bilaterally from level of posterior margin of ventral sucker to level of center of anterior testis. Excretory pore located at posterior end of body. Cercaria (Fig. 4A). Body cylindrical, slender posteriorly, 467 (432–498) in length by 136 (125–146) in maximum width. Stylet absent. Oral and ventral suckers equal in size. Oral sucker oval, subterminal, 76 (72–79) long by 73 (70– 78) wide. Ventral sucker oval, located in posterior half of body, 75 (72–80) long by 74 (71–77) wide. Prepharynx short. Pharynx globular, 29 (23–38) long by 39 (33–50) wide. Esophagus very short. Cecum reverse-Y-shaped, terminate in
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mid-body. Genital primordium present between ventral sucker and tail. Tail rudimentary, 36 (32–40) long by 39 (35–41) wide. Metacercaria (Fig. 4B). Body non-encysted, 1048 (849–1275) in length and 625 (526–724) in maximum width. Oral sucker slightly larger than ventral sucker. Oral sucker oval, subterminal, 256 (200–314) long by 217 (180–255) wide. Ventral sucker oval, located in anterior half of body, 172 (147–209) long by 189 (160–225) wide. Prepharynx short. Pharynx globular, 116 (99–135) long by 136 (101–171) wide. Esophagus absent. Cecum bifurcation post-pharyngeal. Ceca
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tubular, undulating, extending bilaterally to posterior end of body. Gonads immature, tandemly located in posterior half of body. Testes irregular in form.
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Anterior testis 69 (59–90) long by 88 (57–109) wide; posterior testis 88 (52–80)
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in diameter. Genital pore unobservable.
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long by 74 (53–108) wide. Ovary spherical, located between testes, 43 (35–55)
3.3.2. Taxonomic summary
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Type host: Euhadra brandtii sapporo Ijima, 1891. Additional second intermediate hosts: Ezohelix gainesi Pilsbry, 1900,
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Euhadra callizona Crosse, 1871, Euhadra peliomphala Pilsbry, 1890, Aegista eumenes Westerlund 1883, Aegista hiroshifukudai Kameda & Chiba, 2015
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Sites of infection: Hepatopancreas (sporocyst) and kidney (metacercaria). Type locality: Ebetsu, Hokkaido, Japan. Additional localities: Hokkaido, Honshu, and Kyushu (see Table 1). Collectors: T. Waki, M. Nakao, and M. Sasaki. Dates of collection: August 15, 2016 (a holotype and paratypes) and May 29, 2017 (paratypes). Definitive hosts: Unknown. The laboratory mouse, Mus musculus L., can be used as an alternative host under immuno-suppressive condition. Type specimens: The type series have been deposited in Meguro Parasitological Museum under the collection numbers MPM 21422 (holotype, adult), MPM 21423A–S (19 paratypes, adults), and MPM 21424 (7 paratypes, metacercariae). DNA differential markers: The parasite DNA sequences of nuclear 28S rDNA
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and
mitochondrial
cox1
(32
haplotypes)
have
been
deposited
into
DDBJ/ENA/Genbank databases under the accession numbers MK286935– 286939 (28S rDNA) and LC438346–438377 (cox1). Etymology: The new species is named after the arboreal behavior of Euhadra snails. The specific name means "of tree-climbing Euhadra" in Latin. Japanese name: Kinobori-maimai-sango-mushi (a coral-like parasite of arboreal snails)
3.4. Molecular phylogeny and haplotype network
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Synonym: Brachylaima sp. B [13]
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Molecular phylogenetic analyses were carried out to confirm the validity of the new species. A ML tree of nuclear 28S rDNA illustrated that Brachylaima spp.,
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including the new species, are monophyletic (Fig. 5A). In this tree, five
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geographic isolates of the new species from distant localities formed a robust clade, which could be differentiated from the other species of Brachylaima.
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Another ML tree of mitochondrial cox1, including the representative isolates of Brachylaima in Japan, clearly demonstrated the new species to be distinct from
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B. ezohelicis, and B. asakawai (Fig. 5B). The mean pairwise genetic distance of cox1 sequences among the three species was highly divergent, ranging from
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0.161 to 0.188.
Forty-nine randomly selected cox1 sequences of the new species were subjected to population genetics analyses. Those were composed of 32 samples from Hokkaido (nos. 1 to 8 in Fig. 1), 9 from central Japan (nos. 18 to 20), and 8 from western Japan (nos. 25 to 28). A total of 32 cox1 haplotypes were found from these samples. The resultant population genetics indices showed that the new species kept a high genetic diversity without bottlenecking (Supplementary Table 1). The cox1 haplotype network of all the samples depicted a highly scattered pattern, showing that there is almost no cline of the regional distribution in Japan (Fig. 6). Only one haplotype was common between Hokkaido (nos. 1, 3, and 5 in Fig. 1) and western Japan (nos. 26 and 27). The former island is approximately 1500 kilometers away from the latter localities in a straight line.
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4. Discussion
Morphological discrimination among various species of Brachylaima is often quite difficult, even for their adult stage. Ecological information about both localities and host animals is, therefore, essential in identifying the species. The lack of data on the definitive hosts of B. lignieuhadrae n. sp. is highly disadvantageous for species identification. However, the experimentally raised
of
adult worms showed the following morphological characteristics: 1) almost equal-sized suckers, 2) a ventral sucker located in anterior quarter of body, 3)
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slightly undulating ceca, 4) an uterine tube extending to postpharyngeal position, 5) weakly lobed large testes, 6) an unlobed small ovary, 7) a well-developed
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cirrus pouch, 8) a thick and long seminal vesicle, 9) bilateral vitelline glands
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extending from level of posterior margin of ventral sucker to level of center of anterior testis, 10) the presence of a seminal receptacle, and 11) tegumental
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spines distributed throughout the ventral surface. The new species is morphologically distinguishable from its congeners when compared to them in
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combination with the above-mentioned diagnostic characters. The new species especially resembles B. eophonae [15], B. apoplania [27],
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B. cribbi [8], B. mascomai [11], and B. ratti [28] in morphology. However, the shape and size of their testes are obviously different from those of the new species (Supplementary Table 2). The other differential points are as follows: B. eophonae has shorter fields of bilateral vitelline glands, and B. ratti has a ventral sucker on anterior third of body. The new species most closely resembles B. ezohelicis. Both species were indistinguishable at light microscopic level. A SEM observation, however, revealed that the shape of tegumental spines and their distribution on the ventral surface were dissimilar. In the new species, a typical tegumental spine is half-round in shape and has short digitated branches (Fig. 3C). The spines are distributed throughout the ventral surface, but those on the posterior part become sparse in density and smaller in size (Fig. 3A). Conversely, B. ezohelicis has fine spines only on the ventral surface in the anterior half [12]. Since both of the species are sympatrically distributed in Hokkaido (Table 1), the
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application of distance-based DNA barcoding seems to be a practical and reliable way for the differential diagnosis of the morphologically similar species. The genus Brachylaima distributes worldwide and all species use snails as the intermediate hosts [29]. In Japan, six Brachylaima species had been reported from Mammals, birds and land snails. B. ezohelicis and B. eophonae can be distinguished from the new species as we mentioned above, and the remaining four species can be also distinguished based on morphologies of and uterus, vitelline, seminal receptacle and size of testis.
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The present molecular phylogenetic analyses are insufficient in evaluating the evolutionary history of brachylaimids, because of the lack of taxa
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represented by molecular data. Nevertheless, the present phylogenies based on nuclear 28S rDNA and mitochondrial cox1 are available for assessing the
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specific status of the new species. Even in the conservative 28S rDNA
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sequences, the resultant tree showed that the new species is substantially divergent from the other congeneric species. In the case of cox1, long
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sequences of Brachylaima (>700 bases) have been unpublished, excepting our sequences from the Japanese species. The cox1 tree was, therefore, made by
na
using only isolates of the Japanese species to confirm their individuality. The topology of the cox1 tree clearly demonstrated that the Japanese species are
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related at a certain distance to one another. These genetic data strongly support the validity of the new species, together with other objective evidence from morphology, host-parasite ecology, and biogeography. The cox1 haplotype network analysis provides an important implication for the geographic spread of the new species. Many haplotypes from Hokkaido, central Japan, and western Japan were closely related to one another, and one haplotype was common in Hokkaido and western Japan. The high genetic diversity of the haplotypes and their disseminated network suggest that an invasive event and subsequent disturbance have repeatedly occurred in each locality. The possible explanation of the haplotype distribution is that the new species has been randomly shuffled by definitive hosts regularly travelling a long distance. The intermediate host snails of Euhadra and the related genera are excludable from the haplotype shuffling because of their very low mobility. The
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artificial movement of the host snails could be denied because their species diversity and endemism are well conserved in each locality [1]. In the Japanese Archipelago, many birds migrate to Hokkaido in the spring to early summer seasons and return southward in the winter [30]. Although the definitive hosts of the new species are completely unknown, we consider migratory birds as the candidates because of their high mobility. Moreover, the arboreal preference of the intermediate host snails indicates that the candidates could be narrowed down to particular birds, which prefer arboreal foraging and eating. The
of
involvement of synanthropic rodents (members of Rattus and Mus) in the parasite dispersal seems to be low, because there is little overlap of ecological
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niche between the rodents and the host snails.
The distribution of trematodes is generally restricted to particular areas due
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to the habitat range of snails and their susceptibility to the parasites [31, 32]. The
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transmission of brachylaimid is ecologically unique because land snails have a dual responsibility for the first and second intermediate hosts. Each species of
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brachylaimids uses only a limited species of land snails for the development of the sporocyst [6, 11], probably due to an evolutionary selection linked to
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cospeciation between parasites and snails associated with co-evolution of host immune systems and sporocysts lethal to their hosts. Our previous field studies
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clearly demonstrated that the sporocyst infection occurs under a strict combination of brachylaimids and snails, namely B. ezohelicis and Ez. gainesi [12] and B. asakawai and Di. pauper [13]. By contrast, metacercarial infections are nonspecific to land snails. Accordingly, brachylaimids must have adapted for indigenous snails in each geographic area, and the detection of the sporocyst from indigenous snails may indicate the discovery of a new species. In this study, the sporocyst of B. lignieuhadrae n. sp. has been found only from members of Euhadra (i.e., Eu. brandtii in Hokkaido and Eu. callizona in central Japan). Most species of Euhadra are strictly endemic to the Japanese Archipelago, but only two species are exceptionally distributed in the southern part of the Korean Peninsula and Jeju Island [33]. Therefore, this species probably does not expand their distribution, resulting in undiscovered before our survey. This endemicity supports the validity of the new species.
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The prevalence of brachylaimid sporocysts in land snails is generally lower than that of metacercariae in various localities [11–13, 34]. This tendency was also observed in this study. The difference of the prevalences between sporocysts and metacercariae seems to be caused by the following factors: 1) the high pathogenicity of sporocyst, 2) the efficient transmission of cercariae, and 3) the densities of snails and definitive hosts. The sporocyst grows in the hepatopancreas, a digestive gland of snails [6, 11–13], and most of the gland is replaced by the reticular tubes containing enormous cercariae. The mortality of
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the sporocyst-infected snails is likely to be high, probably because of nutritional deficiency due to the dysfunction of hepatopancreas [34–36]. Even if the
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mortality is very high, the remaining infected snails must act as a superspreader of cercariae to other normal snails. Land snails generally have the habit of being
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aggregate with other individuals of the same species [37, 38]. Therefore, the
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transmission of cercariae from a sporocyst-infected snail to normal snails occurs probably in a certain space where the aggregation is arising. In this study,
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metacercariae of the new species were detected from 6 species of the genera Euhadra, Ezohelix and Aegista (Eu. brandtii, Ez. gainesi, Eu. peliomphala, Eu.
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callizona, Ae. eumenes, and Ae. hiroshifukudai) (Table 1) belonging to the family Camaenidae or Bradybaenidae, which should be included in Camaenidae by the
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most recent taxonomic revision [19]. The sporocyst-infected snails (Eu. brandtii and Eu. callizona) are included in these species. All the species have the habit of tree-climbing [39–43], excepting Ez. gainesi. It is likely that the tree-climbing behaviors are closely linked to the snail-to-snail transmission. This study showed that the prevalence of metacercarial infections is relatively high in E. brandtii sapporo from Hokkaido (Table 1). In this survey, we noticed that snails belonging to Euhadra are abundant in Hokkaido as compared with other localities. The abundance of the host snails may increase the chance of the sporocyst infections and accelerate the subsequent snail-to-snail transmission. The raccoon (Procyon lotor), an alien species from North America, is widely distributed in Japan, and its food habit is omnivorous, including arboreal land snails of Euhadra [43]. In the type locality of B. lignieuhadrae n. sp. (no. 7 in Fig. 1), the adult worms of Brachylaima sp. were found from raccoons [44, 45]. Given
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that the raccoons feed on arboreal land snails of Euhadra [43], we can speculate that they are accidentally infected with the new species. In Japan, adult worms of Brachylaima spp. have been reported from several mammals and birds [14– 17, 46, 47], although their intermediate hosts are still unclear. Our continuous snail survey will clarify their intermediate hosts.
Acknowledgements
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This study was supported by LNest Grant Natural history award 2018 and the research project "Studies on Fauna and Flora of the Institute for Nature
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Study, National Museum of Nature and Science”. We would like to thank the staffs of the Shells Museum “Palais la mer” and the University of Tokyo Chiba
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Forest (UTCBF) for their kind support. Thanks are also due to the office staffs of
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the Institute for Nature Study, National Museum of Nature and Science for permitting the collection of snails and providing the facilities, and to Mr. Katsuya
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Tsukamoto and Dr. Tatsuo Yabe for providing snails for this study.
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M. Azuma, Colored Illustrations of the Land Snails of Japan (in Japanese), Hoikusha, Osaka, 1982.
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R. Okuma, Predators on land snails (In Japanese), Venus 16 (1985) 22– 27.
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land crab predation in the land snail Cerion, Funct. Ecol. 11 (1997) 464– T. Pojmańska, Parasites as a natural element of any ecosystem, Wiad. Parazytol. 48 (2002) 139–154.
S. Mas-Coma, I. Montoliu, The life cycle of Brachylaima ruminae n. sp.
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(1986) 739–753. [7]
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(Trematoda: Brachylaimidae), a parasite of rodents, Z. Parasitenkd. 72
W.F. Sirgel, P. Artigas, M.D. Bargues, S. Mas-Coma, Life cycle of
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Renylaima capensis, a brachylaimid trematode of shrews and slugs in South Africa: two-host and three-host transmission modalities suggested
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Figure legends Fig. 1. Sampling sites of land snails belonging to Euhadra and the related genera. The sites are serially numbered from 1 to 28. A) Locations in Japan. B) Locations in Hokkaido. C) Locations in Kanto district. Closed and open circles represent locations where Brachylaima lignieuhadrae n. sp. were detected and undetected, respectively. The locality numbers correspond to those given in Table 1. Fig. 2. An experimentally raised adult of Brachylaima lignieuhadrae n. sp. (the
ro
of
ventral view of the holotype). Scale bar 500 μm. Abbreviations: c, cecum; cp, cirrus pouch; ed, excretory duct; ep, excretory pore; ev, excretory vesicle; gp, genital pore; m, metraterm; o, ovary; os, oral sucker; p, pharynx; sr, seminal
re
-p
receptacle; sv, seminal vesicle; t, testis; u, uterus; v, vitellarium; vd, vitelline duct; vr, vitelline reservoir; vs, ventral sucker.
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Fig. 3. Scanning electron microscopic photographs of adult Brachylaima lignieuhadrae n. sp. A) Ventral view of the whole body. The density of tegmental
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na
spines becomes sparse from the posterior (indicated by an arrow with bar), but their distribution continues till the posterior end (an arrow without bar). Scale bar 1 mm. B) Half-round shaped spines (the upside is anterior). Very short digitated branches are included in each of the spines. Scale bar 10 μm. C) Enlargement of the spine. Scale bar 5 μm. Fig. 4. Laval stages of Brachylaima lignieuhadrae n. sp. A) Cercaria. Scale bar 100 μm. B) Metacercaria. Scale bar 200 μm. Fig. 5. Maximum-likelihood phylogenetic trees of the genus Brachylaima. Bootstrap percentages are shown on representative nodes. Scale bars indicate the number of substitutions per nucleotide site. A) The tree of 28S rDNA. Members of Brachylaima and the related genera are included. The tree was rooted by the outgroup taxa of Clinostomum. B) The midpoint-rooted tree of mitochondrial cox1. Fig. 6. A statistical parsimony network inferred from 49 mitochondrial cox1 sequences of Brachylaima lignieuhadrae n. sp. The cox1 haplotypes were
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divided into three geographic populations (Hokkaido, central Japan, and western Japan). The size of ovals indicates the frequency of the haplotypes. Small circles show hypothetical haplotypes. Supplementary Fig. 1. Sporocysts of Brachylaima lignieuhadrae n. sp. A) The reticular development in the hepatopancreas of Euhadra brandtii sapporo. Scale
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bar 1 mm. B) Cercariae within the sporocyst. Scale bar 200 μm.
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Table 1. Detection of sporocysts (SC) and metacercariae (MC) of Brachylaima lignieuhadrae n. sp. from members of Euhadra and related land snails in Japan Localities a
Coordinates
Periods
Snail species b
No. snails
No.
No.
MC
No. snails with
examined
snails with
snails with MC
intensity (range) c
(no. examined)
SC (%)
(%)
1, Hokkaido
43.826, 142.613
Jun, 2017
Eu. brandtii*
5
0 (0)
5 (100)
4.2 ± 3.1 (1-8)
4 (4)
2, Hokkaido
43.634, 142.631
May, 2017
Eu. brandtii*
6
0 (0)
3 (50.0)
4.7 ± 1.2 (4-6)
3 (3)
3, Hokkaido
43.945,
May,
Eu. brandtii*
16
0 (0)
2 (12.5)
3 (3)
1 (1)
142.498
2017
43.757,
May,
Eu. brandtii*
11
0 (0)
5 (45.5)
6.6 ± 5.3
4 (4)
142.278
2017
4, Hokkaido 4
(2-14)
May,
e
Ez. gainesi
18
0 (0)
1 (5.6)
18
0 (1)
May, 2017
Eu. brandtii*
34
0 (0)
22 (64.7)
6.5 ± 5.6 (1-20)
8 (8)
May, 2017
Ez. gainesi
12
0 (0)
7 (58.3)
3.1 ± 4.8 (1-14)
5 (7) e
43.732,
Sep,
Eu. brandtii*
97
1 (1.0)
4.6 ± 3.9
2 (2)
142.203
2017
43.054, 141.510
Aug, 2016
Eu. brandtii*
14
Aug,
Ez. gainesi
40
5 6, Hokkaido 7, Hokkaido 7
2016 Aug, 2016
Ai. editha
7
May, 2017
Eu. brandtii*
43.029,
May,
Eu. brandtii*
141.316
2017
52 (53.6) 9 (64.3)
4.2 ± 1.4 (1-6)
6 (6)
0 (0)
4 (10.0)
2.0 ± 1.4
2 (2)
1
re
7
ro
43.680, 142.330
1 (7.1)
-p
5, Hokkaido
of
2017
(1-4)
0 (0)
0 (0)
1 (1.1)
34 (39.1)
6.8 ± 6.5 (1-29)
6 (6)
12 (92.3)
3.3 ± 2.1
4 (4)
0 (0)
9, Ibaraki
36.315, 140.588
Jun, 2017
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87
(1-21)
Eu. brandtii*
2
0 (0)
0 (0)
10, Chiba
35.160, 140.148
Oct, 2017
Eu. peliomphala*
43
0 (0)
0 (0)
Oct,
Ae. vulgivaga
6
0 (0)
0 (0)
2
0 (0)
10
13
(1-7)
May, 2017
Ez. gainesi
1
0 (0)
1 (100)
Jo ur
8
na
8, Hokkaido
1
0 (1) e
2017
10
Oct, 2017
Ni. sp.
11, Saitama
35.841, 139.609
Sep, 2017
Eu. quaesita
6
0 (0)
0 (0)
12, Saitama
35.801, 139.676
Sep, 2017
Br. pellucida
12
0 (0)
0 (0)
13, Tokyo
35.638, 139.721
2017
Eu. peliomphala*
15
0 (0)
0 (0)
f
13
2017 f
Br. pellucida
7
0 (0)
0 (0)
13
2017 f
Sa. japonica
4
0 (0)
0 (0)
13
2017 f
Ae. conospira
7
0 (0)
0 (0)
35.626,
Aug,
Eu. quaesita
9
0 (0)
0 (0)
139.703
2016
15, Kanagawa
35.443, 139.647
Mar, 2017
Br. pellucida
7
0 (0)
0 (0)
16, Kanagawa
35.256,
Aug,
Eu.
10
0 (0)
0 (0)
139.740
2016
peliomphala*
17, Tokyo
35.850, 139.035
Apr, 2018
Eu. peliomphala*
1
0 (0)
0 (0)
18, Tokyo
35.623, 139.247
Jun, 2016
Eu. peliomphala*
7
0 (0)
2 (28.6)
14, Tokyo
0 (0)
1.5 (1-2)
2 (2)
d
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19, Tokyo
34.680, 139.438
Feb, 2017
Eu. peliomphala*
14
0 (0)
1 (7.1)
Feb,
Ae. conospira
1
0 (0)
0 (0)
Oct, 2016
Eu. callizona*
29
0 (0)
17 (58.6)
20
Oct, 2016
Eu. scaevola
6
0 (0)
0 (0)
20
Oct,
Ae. vulgivaga
7
0 (0)
0 (0)
Sa. japonica
4
0 (0)
0 (0)
Eu. callizona*
31
3 (9.6)
13 (41.9)
19
2 (2)
1 (1)
6.5 ± 4.8 (1-16)
6 (6)
5.5 ± 7.6
3 (3)
2017 20, Shizuoka
35.077, 138.413
2016 20
Oct, 2016
20
Jun, 2017
21, Gifu
35.261,
Sep,
136.528
2017
21
(1-29) Eu. eoa
13
0 (0)
0 (0)
Sa. japonica
5
0 (0)
0 (0)
Ae. vulgivaga
5
0 (0)
0 (0)
Sep, 2017
Ae. commoda
2
0 (0) 0 (0)
0 (0)
Sep,
21
Sep,
of
2017
35.365, 136.468
Sep, 2017
Eu. eoa
2
23,
33.680,
Oct,
Eu. sigeonis*
13
0 (0)
0 (0)
Wakayama
135.359
2017 Eu. sandai
1
0 (0)
0 (0)
2
0 (0)
0 (0)
23
Oct,
re
21
-p
0 (0)
22, Gifu
ro
2017
33.550, 133.514
Mar, 2018
Eu. subnimbosa*
25, Shimane
34.922,
Sep,
Ae. eumenes*
1
0 (0)
1 (100)
3
1 (1)
132.347
2016
34.489, 131.403
Jun, 2013
Ae. hiroshifukudai*
7
0 (0)
1 (14.3)
9
1 (1)
Nov, 2015
Ae. hiroshifukudai*
2
0 (0)
1 (50.0)
8
1 (1)
May, 2015
Ae. eumenes*
2
0 (0)
1 (50.0)
8
1 (1)
Apr,
Ae. eumenes*
3
0 (0)
1 (33.3)
5
1 (1)
Ae. eumenes*
3
0 (0)
1 (33.3)
22
1 (1)
646
6 (0.9)
196 (30.3)
63 (67)
26, Yamaguchi 26
27
34.178, 131.208
Jo ur
27, Yamaguchi
na
24, Kochi
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2017
2017
28, Oita Total
33.010, 131.750
May, 2013
a
Individual numbers of sampling sites were shown in the map of Fig. 1. Abbreviations of genera are as follows: Ae, Aegista; Ai, Ainohelix; Br, Bradybaena; Eu, Euhadra; Ez, Ezohelix; Ni, Nipponochloritis; Sa, Satsuma;. Asterisks indicate tree-climbing snails. c The mean number of MC with standard deviation was computed in infected snails. d All SC and selected MC were subjected to DNA barcoding for species identification. In the case of MC, at least one MC per snail was b
identified. e Some of MC were identified as Brachylaima ezohelicis. f Collection records during March to November were combined.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7