The first intermediate host of the invasive frog trematode Glypthelmins quieta in Japan.

Glypthelmins quieta is a frog trematode native to North and Central America. This trematode was recently detected in Japan in the American bullfrog Lithobates catesbeianus, which was introduced from North America to Japan. As the first intermediate host of G. quieta, typically a snail, has not yet been identified in Japan, we conducted a snail survey in eastern Japan to screen for an intermediate host using DNA barcoding based on the nuclear 28S ribosomal RNA and mitochondrial cytochrome c oxidase subunit 1. We sampled 3 different snail species, Orientogalba ollula, Physella acuta, and Sinotaia quadrata histrica (157 individuals in total), and only the freshwater snail Physella acuta, which is also believed to have been introduced from North America to Japan, had sporocysts of G. quieta in its hepatopancreas. The introduction of the intermediate and definitive hosts from North America may have facilitated the invasion of G. quieta into Japan.

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.

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.

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.

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.).

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.

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.

Impact of Perkinsus olseni infection on a wild population of Manila clam Ruditapes philippinarum in Ariake Bay, Japan.

Many studies have addressed the production decline of Manila clam, Ruditapes philippinarum, in Japan, but infection of clams with Perkinsus olseni has received scarce attention. To evaluate the impact of P. olseni, infection levels and host density of a wild, unexploited clam population were monitored monthly or bimonthly on a tidal flat from June 2009 to January 2013. Real-time PCR analysis discriminating P. olseni and Perkinsus honshuensis detected only P. olseni in tested clams. The prevalence of P. olseni was 100% or nearly 100% in 7 cohorts throughout the study period, except in newly recruited clams. Infection intensity remained low and seldom reached 106 cells/g wet tissue in newly recruited clams until the month of September. Infection intensity reached 106 cells/g in autumn and remained high at 104-106 cells/g until each cohort of clams disappeared. Clam density began to decrease in the autumn when the infection intensities reached ca. 106 cells/g. Density was relatively stable in winter, increased in spring and decreased again in clams aged 1 year or older during summer and autumn in the following years. Survival of clams experimentally infected with P. olseni at ca. 106 cells/g and placed in a cage in the tidal flat for 1 or 2 months was significantly lower than survival of uninfected control clams. Our results suggested that heavy infection with P. olseni was a major cause of the clam density decrease, although other environmental and biological factors also may have contributed to the decline in density. In addition, uninfected clams were deployed in cages for 1-2 months from June 2010 to January 2013 and prevalence and infection intensity were monitored. Parasite transmission and infection progression increased in summer and autumn.

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.

Experimental challenges of wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams.

Manila clams, Ruditapes philippinarum, are widely harvested in the coastal waters in Japan. However, there have been significant decreases in the populations of Manila clams since the 1980s. It is thought that infection with the protozoan Perkinsus species has contributed to these decreases. A previous study demonstrated that high infection levels of a pure strain of Perkinsus olseni (ATCC PRA-181) were lethal to hatchery-raised small Manila clams, however, the pathogenicity of wild strain Perkinsus species to wild Manila clam is unclear. To address this, we challenged large (30-40 mm in shell length) and small (3-15 mm in shell length) wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams. We report high mortalities among the small clams, but not among the large ones. This is the first report to confirm the pathogenicity of wild isolate of Perkinsus species to wild Manila clams.

Key role of chemical hardness to compare 2,2-diphenyl-1-picrylhydrazyl radical scavenging power of flavone and flavonol O-glycoside and C-glycoside derivatives.

The antioxidant activities of flavonoids and their glycosides were measured with the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH radical, DPPH(·)) scavenging method. The results show that free hydroxyl flavonoids are not necessarily more active than O-glycoside. Quercetin and kaempferol showed higher activity than apigenin. The C- and O-glycosides of flavonoids generally showed higher radical scavenging activity than aglycones; however, kaempferol C3-O-glycoside (astragalin) showed higher activity than kaempferol. In the radical scavenging activity of flavonoids, it was expected that OH substitutions at C3 and C5 and catechol substitution at C2 of B ring and intramolecular hydrogen bonding between OH at C5 and ketone at C3 would increase the activity; however, the reasons have yet to be clarified. We here show that the radical scavenging activities of flavonoids are controlled by their absolute hardness (η) and absolute electronegativity (χ) as a electronic state. Kaempferol and quercetin provide high radical scavenging activity since (i) OH substitutions at C3 and C5 strikingly decrease η of flavones, (ii) OH substitutions at C3 and C7 decrease χ and η of flavones, and (iii) phenol or o-catechol substitution at C2 of B ring decrease χ of flavones. The coordinate r(χ, η) as the electron state must be small to increase the radical scavenging activity of flavonoids. The results show that chemically soft kaempferol and quercetin have higher DPPH radical scavenging activity than chemically hard genistein and daidzein.

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.

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.

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.

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.

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.

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.

Potent 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity of novel antioxidants, double-stranded tyrosine residues conjugating pyrocatechol.

New potent antioxidants conjugating the catechol (=pyrocatechol; pyrCat) group to two N-termini of modified double-stranded tyrosine residues were synthesized and showed radical scavenging activity with 2,2-diphenyl-1-picrylhydrazyl radical (DPPH radical, DPPH˙) as a free radical model, second-order rate constants for the DPPH˙ scavenging reaction, and the results from electron spin resonance (ESR) studies. It was found that the tyrosine (Tyr) residue and pyrCat containing new antioxidants developed in the study have about 3-20 times more potent antioxidative activity than Trolox, pyrCat, and L-ascorbic acid (VC). In order to elucidate the relationship between antioxidant activity and the molecular orbital states, and to design potent antioxidants we present an interesting approach using an absolute hardness (η)-absolute electronegativity (χ) diagram based on chemical hardness. It was shown that quantum chemicals were required to develop potent antioxidants.