A new species of the genus Paradistomum (Platyhelminthes: Trematoda) from Iwasaki's snail-eating snake Pareas iwasakii, with a note on morphological variations of Paradistomum megareceptaculum (Tamura, 1941).

Dicrocoeliid trematodes were detected from Iwasaki's snail-eating snake Pareas iwasakii in Iriomote Island, Okinawa Prefecture, Japan, and described as a new species Paradistomum dextra n. sp. in the present study. This new species can be distinguished from the type series of the other members of the genus based on size of eggs and morphological characteristics of body, oral and ventral suckers, and reproductive organs. However, the new species was hard to distinguish from Paradistomum megareceptaculum infecting snakes in Japan, including Iriomote Island where is the type locality of the new species, because it is closely similar to some part of the broad range of morphological variations in P. megareceptaculum. On the other hand, a partial sequence of 28S ribosomal DNA clearly distinguished these two species. Moreover, the new species' host snake Pareas iwasakii is reported to exclusively feed on land snails while host snakes of P. megareceptaculum feed on small vertebrates, indicating that the new species is also ecologically different from P. megareceptaculum. We also redescribed P. megareceptaculum based on adults sampled in this study and past studies to record the morphological variations of this species.

A description of a new species of the genus Brachydistomum (Trematode, Dicrocoeliidae) from the Eurasian Tree Sparrow Passer montanus (Linnaeus) (Passeriformes) in Japan, with a report on the first intermediate host.

The trematode Brachydistomum suzume n. sp. (Dicrocoeliidae) was detected in the Eurasian Tree Sparrow Passer montanus, and described as a new species in Japan. This new species can be distinguished from the other members of the genus on the basis of morphological characters of suckers and reproductive organs. A partial sequence of adult mitochondrial cytochrome c oxidase subunit 1 (COI) was used as a DNA barcode, and dicrocoeliid sporocysts and cercariae detected from four camaenid land snail species, Bradybaena pellucida, Brad. similaris, Acusta sieboldiana and Euhadra brandtii, were molecularly identified as the new species. Phylogenetic trees of nuclear 28S ribosomal DNA and COI also showed the new species to be distinct from the other trematode species, including Brachydistomum spp.

Introducing the land snail Bradybaena pellucida increased infection risk of the avian parasite Postharmostomum commutatum in the Kanto region of Japan.

The trematode Postharmostomum commutatum is a parasite of the chicken Gallus gallus domesticus. Its heavy infection can cause inflammation and hemorrhage in the cecum of host birds. We found a severe infection of metacercariae of P. commutatum, which was identified based on DNA barcodes with morphology, in the introduced land snail Bradybaena pellucida and its related species in the Kanto region of Japan. Our field survey revealed that metacercariae were detected in 14 of 69 sampling locations in this region. B. pellucida was thought to be the major second intermediate host of metacercariae of the trematode because this snail was most frequently found in the study area and the prevalence and infection intensity were higher than those of the other snail species. The observed increase in metacercariae in introduced populations of B. pellucida can enhance the infection risk of chickens and wild host birds, probably owing to the spillback effect. Our seasonal field study showed that the prevalence and infection intensity of metacercaria seemed to be high in populations of B. pellucida during the summer and early autumn. Therefore, chickens should not be bred outdoors during these seasons to prevent severe infection. Our molecular analysis, based on cytochrome c oxidase subunit I sequences, showed a significantly negative value for Tajima's D in P. commutatum, suggesting an increase in its population size. Thus, P. commutatum distributed in the Kanto region may have increased its population size with the introduction of the host snail.

Invasion of fish parasite Prosorhynchoides ozakii (Trematoda: Bucephalidae) into Lake Kasumigaura and surrounding rivers of eastern Japan.

In 2019 to 2021, the golden mussel Limnoperna fortunei and several freshwater fishes were sampled from 22 sites of the Tone River system including Lake Kasumigaura, Honshu, Japan, to examine the invasion of bucephalid trematodes. The parasite species identification was performed by morphological observation and DNA barcoding based on the sequences of nuclear 28S rDNA and mitochondrial cytochrome c oxidase subunit 1 (cox1). A total of 1719 mussels were collected from 10 sites, and trematode-infected mussels were detected from 8 sites with prevalences between 0.3 and 42.9%. The sporocysts and cercariae were identified as Prosorhynchoides ozakii, a newly introduced species in the river system. A total of 700 fish individuals belonging to 24 species were collected from 15 sites. Two species of catfishes (Silurus asotus and Ictalurus punctatus) harbored mature or immature adults of Pr. ozakii in the intestine with prevalences between 8.3 and 20% including both host species. The metacercariae of Pr. ozakii were found from the fins and epidermis of 13 fish species from 10 sites (prevalence 4.8-100%). Fishes were heavily infected with metacercariae in fins, which were surrounded by the infiltration of hemocytes and rodlet cells. A population genetic analysis of Pr. ozakii did not show an obvious bottleneck, suggesting the possibility that the parasite was intentionally and repeatedly introduced into the river system.

Redescription of two parasitic feather mites sampled from the last two Crested Ibises, Nipponia nippon (Temminck, 1835) (Pelecaniformes: Threskiornithidae), lived in Japan.

The objective of this paper is to redescribe two feather mites, Compressalges nipponiae Dubinin, 1950 (Caudiferidae) and Freyanopterolichus nipponiae Dubinin, 1953 (Kramerellidae), based on samples collected from the last two individuals of the Crested Ibis Nipponia nippon (Temminck, 1835) from the native Japanese populations, which had been extirpated in 2003. Our study provides detailed description of the leg and idiosomal chaetotaxy, and many morphological structures, which were missed or misinterpreted in the original publications.

Complete mitochondrial genomes of two snail mite: Riccardoella tokyoensis and R. reaumuri (Acariformes, Prostigmata, Ereynetidae).

We determined the mitochondrial genome sequences of two snail mites, Riccardoella tokyoensis and R. reaumuri. The length of the entire mitogenome of these two species is 15,078 bp and 15,148 bp long, respectively. Both of them contain 13 proteins, two rRNAs, and 22 tRNAs for a total of 37 gene products. The gene order of Riccardoella is able to explain by a single rearrangement event from that of other Eupodina species; the whole region, including both rRNA genes and control region (CR), is inverted at the same position. The CR including a tandem repeat region in both of the mitogenomes of Riccardoella species.

Brachylaima phaedusae n. sp. (Trematoda: Brachylaimidae) from door snails in Japan.

The metacercarial infections of door snails (Gastropoda: Clausiliidae) with unknown species of the genus Brachylaima (Trematoda: Brachylaimidae) have recently been reported in eastern Honshu and Kyushu, Japan. A large scale snail survey was carried out to clarify their taxonomic status. From the period of 2015 to 2020, a total of 1239 land snails (768 door snails and 471 others) were collected from 32 localities in Honshu, Shikoku, and Kyushu. The resulting trematode isolates were identified as Brachylaima sp. by mitochondrial DNA barcoding. The sporocysts were found only a few from Megalophaedusa sublunellata (Clausiliidae), Tauphaedusa subaculus (Clausiliidae), and Aegista trochula (Camaenidae), while the metacercariae were frequently detected from 14 species of Clausiliidae and 2 species of other families. Although Brachylaima sp. showed a broad range of intermediate hosts, door snails seem to be very important to drive the life cycle. The gravid adults of Brachylaima sp. was experimentally raised from metacercariae using immunosuppressed mice. Morphological, phylogenetical, and ecological considerations prompted us to propose Brachylaima phaedusae n. sp. for this unknown species. The definitive hosts of the new species are completely unknown. The wide geographic distribution and high genetic diversity of the new species suggest a possibility that the definitive host is ground-foraging birds, which prefer door snails.

An unknown species of Leucochloridium (Trematoda: Leucochloridiidae) from northern Honshu, Japan.

Pulsating broodsacs of Leucochloridium sp. (Trematoda: Leucochloridiidae) were found from amber snails (Succinea lauta) in Iwate, the northern part of Honshu, Japan. A pattern with red-brown vertical stripes was characteristic of the broodsac. Very similar broodsacs were already detected from Okinawa Islands, the southern archipelago of Japan, and tentatively identified as Leucochloridium cf. passeri. A phylogenetic analysis based on DNA sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) showed that Leucochloridium sp. is different at species level from L. cf. passeri and that both species are related to Leucochloridium vogtianum from Europe. In this study the definitive identification of larval Leucochloridium sp. was impossible, but the resulting phylogeny confirmed that at least 4 species of Leucochloridium are distributed in Japan, depending on locality and climate. The DNA barcode generated in this study will be useful in detecting the adult stage of Leucochloridium sp. from birds.

The first detection of Dicrocoelium chinensis sporocysts from the land snail Aegista vulgivaga in Gifu Prefecture, Japan.

Trematodes of the genus Dicrocoelium are one of the most common parasites in ruminant animals; however, their life cycles in Japan are unclear. To find the sporocysts of D. chinensis in the natural field, we sampled 269 land snails (14 species) at a location with high level infection of sika deer in Gifu Prefecture, Honshu Island, Japan in autumn between 2017 and 2019. During the sampling period, we found mother sporocysts in the hepatopancreas of Aegista vulgivaga and Cyclophorus herklotsi. DNA barcoding based on the sequences of cytochrome c oxidase subunit 1 showed that the sporocysts from A. vulgivaga belonged to D. chinensis, indicating that this snail has potential as the first intermediate host of D. chinensis at this location.

Species composition and infection levels of Anisakis (Nematoda: Anisakidae) in the skipjack tuna Katsuwonus pelamis (Linnaeus) in the Northwest Pacific.

Parasites can be used as biological tags to assess stock structures in various marine fish species. In the present study, the species composition and infection levels of parasitic nematodes of the genus Anisakis in the skipjack tuna Katsuwonus pelamis were examined in the Northwest Pacific and adjacent seas. A total of 867 third-stage larvae of Anisakis were collected from 112 skipjack tunas captured around Japan and in other subtropical localities. All larvae were identified as A. berlandi, A. pegreffii, A. simplex (s.s.), A. typica, and A. physeteris (s.l.) by the direct sequencing of the mitochondrial cox2 gene and real-time PCR assays targeting the nuclear ITS region. Anisakis species composition differed among northeastern Japan, the Sea of Japan, and other areas (central Japan, the Nansei Islands, and subtropical region), which is largely concordant with previous stock discrimination of skipjack tuna. Molecular phylogenetic analysis resulted in two intraspecific genetic groups in A. simplex (s.s.), one of which occurred almost exclusively in northeastern Japan. This could be a useful indicator for stock discrimination. Skipjack tunas from northeastern Japan were also characterized by a remarkable variety in the intensity of A. simplex (s.s.), suggesting the commingling of individuals with different migration patterns. This idea might be further justified by the geographic distribution of two genetically distinct groups of A. physeteris (s.l.).

First report on Mermithidae (Mermithida) infection in Ligidium sp. (Isopoda, Ligiidae).

Specimens of Ligidium sp. (Crustacea, Isopoda, Oniscidea) collected from Kanagawa Prefecture in Japan were found to be infected with parasitic nematodes. We have obtained two Mermithidae (Nematoda) by dissecting the fresh specimen and from the dead specimen of Ligidium, which marks the second known discovery of a mermithid infection in the order Isopoda. Herein, a report on the nuclear 28S rDNA and 18S rDNA sequence of the isolated Mermithidae nematodes and on the morphology of the juveniles is provided.

Natural history study on Riccardoella tokyoensis: life history with ontogeny and host distribution records.

Riccardoella tokyoensis is a snail parasite recently recorded in Japan. To understand the basic ecology of this mite, we surveyed the life history of a population, including seasonal dynamics, host range, and geographical distribution in East Japan. Seasonal dynamics of the mite were studied in Rinshi-no-mori Park, Tokyo, Japan, from June 2016 to May 2018; thereafter, the morphology of each stage was described. In this park, the host population was infected with the mite in all seasons; however, the prevalence and infection intensities were highest during the warm seasons. Larvae and nymphs were found in the warm seasons. Proto- and tritonymphs were uncommon. To study the host range and geographical distribution, we sampled 1135 terrestrial mollusks from 16 families at 21 locations in Kanto and east Tokai areas from June 2016 to May 2020. Mites were detected at 13 locations, and only clausiliid snails were infected with the mite. Through a laboratory experiment, our observations confirmed that the mite was transmitted to the host lung via the pneumostome. None of the artificially infected snails died, although we maintained the snails for 2 months. No inflammation was found in naturally infected host tissue, suggesting that their virulence against the host snails was low. Riccardoella tokyoensis was not found in the litter layer in the field surveys, and no mite was observed on the external surface of the hosts, suggesting that the major habitat of this mite was the host lung.

Probable association between Anisakis infection in the muscle of skipjack tuna (Katsuwonus pelamis) and human anisakiasis in Tokyo, Japan.

Anisakiasis is a gastrointestinal disease caused by parasitic anisakid nematodes, mainly Anisakis simplex sensu stricto (A. simplex). Anisakiasis is prevalent in Japan and approximately 40% of anisakiasis cases in Tokyo occur through the consumption of raw or marinated mackerel. However, in 2018, there was a sudden increase in the number of the food poisoning cases in Tokyo caused by consumption of skipjack tuna (Katsuwonus pelamis). Therefore, we investigated anisakiasis cases resulting from ingestion of skipjack tuna in Tokyo, and surveyed the presence of Anisakis larvae in skipjack tuna in 2018 and 2019. Nineteen samples from 15 patients (13 in 2018 and 2 in 2019) with anisakiasis surely caused by ingestion of skipjack tuna were all identified as A. simplex. The higher mean abundance of Anisakis simplex larvae in skipjack tuna muscle in May 2018 (1.30; 13 larvae/10 fishes) compared to that in the other periods was regarded as a contributing factor in the increase in anisakiasis cases by ingesting skipjack tuna in 2018. To verify whether Anisakis larvae migrate from the visceral organs to the muscle during the period from fishing on the boat until processing for sale, the number of Anisakis larvae in skipjack tuna caught from August to November 2018 was investigated by removing the visceral organs at three different timings, i.e., immediately after catching, after landing, and after transport to the laboratory. Anisakis larvae were detected in the muscle irrespective of the timings at which visceral organs were removed. All larvae from the muscle were detected only from the ventral part and were identified as A. simplex. We thus consider that avoiding raw consumption of the ventral muscle should be an effective measure to prevent anisakiasis.

New records of Philometra pellucida (Jägerskiöld, 1893) (Nematoda: Philometridae) from the body cavity of Arothron mappa (Lesson) and Arothron nigropunctatus (Bloch et Schneider) reared in aquariums, with synonymisation of Philometra robusta Moravec, Möller et Heeger, 1992.

We encountered two cases of infection with large female nematodes of the genus Philometra Costa, 1845 in the body cavity of a map puffer Arothron mappa (Lesson) caught off Okinawa, Japan, and a blackspotted puffer Arothron nigropunctatus (Bloch et Schneider) caught off Queensland, Australia, both reared in aquariums in Japan. No morphological difference was observed between the nematodes from A. mappa and A. nigropunctatus. We identified the nematodes as Philometra pellucida (Jägerskiöld, 1893) based on their morphology. The sequences of the nematodes from both hosts were identical to each other (1,643 bp) and formed a clade with other 17 nematodes belonging to the genera Philometra and Philometroides Yamaguti, 1935 with high bootstrap value (bp = 100). It is the first time that the genetic data on P. pellucida are provided. Philometra robusta Moravec, Möller et Heeger, 1992 is synonymised with the former species.

Brachylaima succini sp. nov. (Trematoda: Brachylaimidae) from Succinea lauta, an amber snail in Hokkaido, Japan.

Through a continuous survey of trematodes in land snails of Hokkaido, the northernmost island of Japan, we have discovered four species of the genus Brachylaima (Trematode: Brachylaimidae). Among them, Brachylaima ezohelicis, Brachylaima asakawai, and Brachylaima lignieuhadrae have already been described. Each of the three species is a strict specialist in selecting a particular species of land snail as the first intermediate host. In this report, we propose the fourth species, Brachylaima succini sp. nov., based on ecological, morphological, and phylogenetic considerations. Sporocysts and metacercariae of the new species were found exclusively from Succinea lauta, which is known as an amber snail indigenous to Hokkaido. Phylogenetic trees of nuclear 28S rDNA and mitochondrial cytochrome c oxidase subunit 1 (cox1) demonstrated it to be distinct from the other sympatric species. Although metacercariae of the new species possessed unique morphological characters, adult worms experimentally raised from the metacercariae were similar to those of B. ezohelicis and B. lignieuhadrae. Natural definitive hosts of the new species are unknown, but the existence of common cox1 haplotypes from far-distant localities suggests a possibility that birds are involved as the definitive hosts. Findings of amber snails coinfected with both sporocysts of the new species and Leucochloridium perturbatum also support the involvement of birds.

Brachylaima lignieuhadrae n. sp. (Trematoda: Brachylaimidae) from land snails of the genus Euhadra in Japan.

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 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 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 worms experimentally raised from the metacercariae were morphologically most similar to Brachylaima ezohelicis in Hokkaido, but could be differentiated by the microstructure of the tegumental surface. We propose Brachylaima lignieuhadrae n. sp. for the unknown species, based on the morphology, DNA profile, host specificity, and geographic distribution. Phylogeography of the new species suggests a possibility that migratory birds serve as the definitive hosts.

The effects of environmental and nutritional conditions on the development of Perkinsus olseni prezoosporangia.

Perkinsus olseni is a widely distributed protozoan pathogen that infects a wide range of marine mollusks. Prezoosporulation of P. olseni trophozoites is easily observed in Ray's fluid thioglycollate medium, but in nature, trophozoites within host tissue should be able to develop into prezoosporangia without any additional artificial medium after the host dies. How this process might work in field conditions remains poorly understood, however, partly because of the lack of appropriate in vitro assays. In this study, we observed that trophozoites of P. olseni successfully developed into prezoosporangia when mixed with minced tissue of the Manila clam Ruditapes philippinarum and placed in seawater. We were thus able to establish a new method to examine the development of P. olseni to prezoosporangia under artificially simulated natural environmental conditions. Using this method, we found that low temperatures (5 °C, 15 °C) significantly suppressed prezoosporangia development. In addition, we found that prezoosporangia were developed in a wide range of salinities (10-50 practical salinity unit) and that P. olseni requires some nutrition factors from host tissue for prezoosporulation to occur. Because the transmission of P. olseni among a host population highly depends on the developmental process of prezoosporangia, which leads to production of the infective zoospore stage, these results will help further our understanding of the parasite's infection dynamics in nature.

Distribution records of three species of Leucochloridium (Trematoda: Leucochloridiidae) in Japan, with comments on their microtaxonomy and ecology.

Insectivorous birds serve as definitive hosts for trematodes of the genus Leucochloridium. The parasites exclusively use amber snails of the family Succineidae as intermediate hosts. A pulsating and colorful display of the larval broodsac in the snail's eyestalk seems to be a caterpillar mimic for attracting birds. A colored design of the broodsac is very useful for parasite identification. In Japan, characteristic broodsacs from amber snails have been recorded from 1980's, but their taxonomic discrimination from Asian, European, and North American species has not been achieved. In this study, old scientific records, sighting information on broodsacs from the general public, and direct molecular evidence by DNA barcoding clearly showed that at least three species of Leucochloridium are distributed in Japan. A vertical-striped broodsac found from Succinea sp. in Okinawa, the subtropical island of Japan, were treated as Leucochloridium sp., but being almost identical to that of Leucochloridium passeri in neighboring Taiwan. The European species of Leucochloridium perturbatum and Leucochloridium paradoxum were frequently detected from Succinea lauta in Hokkaido, the northernmost island of Japan. The former species was common in inland areas of Hokkaido, whereas the latter species was frequently seen in the coastal areas. A possible explanation for the parasite distribution pattern is that principal definitive hosts (migratory or resident birds) differ in each parasite. The conspecificity of Leucochloridium variae in North America and L. perturbatum in Europe and the Far East is also discussed.

Angiostoma namekuji n. sp. (Nematoda: Angiostomatidae) from terrestrial slugs on Oshiba Island in the Seto Inland Sea, Japan.

A new species of nematode, Angiostoma namekuji n. sp. (Angiostomatidae: Rhabditida), is described from the intestinal lumen of the terrestrial slug Philomycidae gen. sp. collected from Oshiba Island in the Seto Inland Sea, Hiroshima Prefecture, Japan. The new species is recognized by the following characteristics: body length 2,782-3,599 (mean 3,240) µm (male); 4,666-5,532 (5,030) µm (female); lateral field present; pharyngeal corpus with valves in the bulb; male with short tail, c = 35-57 (48), with one denticle; and seven pairs of genital papillae arranged as 1+2/3+1; female with tail having small denticles on distal tip; uterus c.50% of the body size; each ovary long, starting near vulva, not coiled, reflexed and reaching uterus; ovaries not crossing each other. Our phylogenetic tree based on sequences of the nuclear 28S ribosomal RNA gene supported the generic allocation of the new species in Angiostoma Dujardin, 1845.

MOLECULAR AND MORPHOLOGICAL DISCRIMINATION OF DICROCOELIID LARVAE (TREMATODA: DIGENEA) FROM TERRESTRIAL MOLLUSKS IN JAPAN.

Trematodes of the family Dicrocoeliidae commonly use terrestrial mollusks as the first intermediate host. Despite the abundant studies on the adult worms in birds and mammals, few reports exist on their larval stage in snail intermediate hosts. A present survey of mollusks in Japan led us to the discovery of dicrocoeliid sporocysts with cercariae in 16 out of 303 individuals, encompassing eight snail species and one slug species. A DNA barcoding based on sequencing of mitochondrial cytochrome c oxidase subunit 1 showed that the larvae consisted of five species. Phylogenetic trees of nuclear 18S and 28S ribosomal DNAs confirmed the five species to be members of the Dicrocoeliidae. These were temporarily termed dicrocoeliid spp. 1 to 5, since conclusive identification was impossible without adult worms. These unknown species were phylogenetically related to each other, except sp. 5. The phylogenetic trees demonstrated close genetic relationships between sp. 3 and the genus Lutztrema, and between sp. 5 and the genus Lyperosomum. The phylogenetic analysis also suggested that a division into the subfamilies Dicrocoeliinae and Leipertrematinae is a wrong classification due to the paraphyly of the Dicrocoeliinae. Morphological characterization of the cercariae and their DNA barcodes provide a primary platform for differentiating dicrocoeliids from various mollusks in Japan. The DNA barcodes, in particular, will enable tracing the parasite life cycles, in case of finding metacercariae and adults from presently unknown hosts.