Human diphyllobothriasis in Argentina: assessing the epidemiological significance from historical records and reports of new cases.

Between 1898 and 1940, eight human cases of diphyllobothriasis were reported in Argentina, always in recently arrived European immigrants. In 1982, the first autochthonous case was detected, and since then, 33 other autochthonous cases have been reported, totaling 42 cases of human diphyllobothriasis in Argentina before the present study. Our aim is to update the information on diphyllobothriasis in Argentina by identifying specimens from new cases using morphometrical and/or molecular methods. We also aim to assess the epidemiological relevance of this food-borne disease in the country. Anamnestic data were obtained from patients or professionals, along with 26 worms identified using morphometrical (21 samples) and molecular techniques (5 samples). All the patients acquired the infection by consuming freshwater salmonids caught in Andean lakes in Northern Patagonia. Morphometrics and DNA markers of worms were compatible with Dibothriocephalus latus. In total, 68 human cases have been detected in Argentina, 60 of which were autochthonous. The human population living North-western Patagonia, whose lakes are inhabited by salmonids, is increasing. Similarly, the number of other definitive hosts for Dibothriocephalus dendriticus (gulls) and for D. latus (dogs) is also increasing. In addition, salmonid fishing and the habit of consuming home-prepared raw fish dishes are becoming widespread. Therefore, it is to be expected that diphyllobothriasis in Argentina will increase further.

A possible circulation of a dominant Dibothriocephalus nihonkaiensis haplotype in Japan revealed by molecular analysis of clinical tapeworm samples.

Human diphyllobothriasis, caused by Dibothriocephalus nihonkaiensis, is prevalent globally, especially in regions where raw fish is consumed. Recent molecular diagnostic techniques have made species identification of tapeworm parasites and the determination of genetic variations among parasite populations possible. However, only a few studies done over a decade ago, have reported on the genetic variation among D. nihonkaiensis in Japan. The present study employed PCR-based mitochondrial DNA analysis to specifically detect D. nihonkaiensis from archived clinical samples, and to determine any genetic variation that may exist among the Japanese broad tapeworms from patients of Kanagawa Prefecture, Japan. Target genes were amplified from DNA extracted from the ethanol- or formaldehyde-fixed samples by PCR. Further sequencing and comparative phylogenetic analyses based on mitochondrial COI and ND1 sequences were also performed. In our results, all PCR-amplified and sequenced samples were identified as D. nihonkaiensis. Analysis of COI sequences revealed two haplotype lineages. However, clustering of almost all COI (and ND1) sample sequences into one of the two haplotype clades, together with reference sequences from different countries worldwide, revealed a common haplotype among D. nihonkaiensis samples in our study. Our results suggest a possible presence of a dominant D. nihonkaiensis haplotype, with a global distribution circulating in Japan. Results from this study have the potential to improve the management of clinical cases and establish robust control measures to reduce the burden of human diphyllobothriasis in Japan.

Genetic characterization of Dibothriocephalus latus and Dibothriocephalus dendriticus (Cestoda: Diphyllobothriidae) from Chile based on haplotype analysis using mitochondrial DNA markers.

Dibothriocephalus latus and Dibothriocephalus dendriticus are found throughout the temperate and sub-arctic zones of the northern hemisphere, but they are also found in the southern core countries of South America, Chile and Argentina. Genetic characteristics of D. latus and D. dendriticus from South America have yet to be fully defined. The present study aimed to understand the genetic characteristics of D. latus and D. dendriticus from Chile by haplotype network analysis of mitochondrial cytochrome c oxidase subunit I gene (cox1) and cytochrome b gene (cob), as well as their origins. Dibothriocephalus latus and D. dendriticus plerocercoid larvae were obtained from feral and/or wild salmonids captured in Lake Llanquihue in Región de Los Lagos, and Lake Panguipulli in Región de Los Ríos, located south of central Chile. Haplotype analysis of D. latus revealed that H1 in cox1 and H2 in cob are the key haplotypes common to D. latus across the world, including Chile, and both genes exhibited limited genetic diversity in D. latus. It was assumed that D. latus was brought into South America by European and Russian immigrants in the 19th century as previously reported. In contrast, both the cox1 and cob of D. dendriticus display considerable genetic diversity, with no common haplotypes between D. dendriticus populations from Chile and the northern hemisphere. More intriguingly, two cob haplotypes (H24, H25) detected in Chilean D. dendriticus were closely linked to haplotypes (H30, H31) detected in North American D. dendriticus, strongly implying that D. dendriticus in Chile was brought by piscivorous migrating birds from North America. It has also been estimated that the D. dendriticus from Chile genetically diverged from the D. dendriticus from the northern hemisphere approximately 1.11 million years ago, long before humans migrated to the southern parts of South America.

Plerocercoids of Adenocephalus pacificus in Argentine hakes: Broad distribution, low zoonotic risk.

Adenocephalus pacificus is a tapeworm parasitic of marine mammals and the main agent of human diphylobothriosis caused by consumption of raw or undercooked marine fishes, being considered as a reemerging disease. Despite having a broad distribution in marine mammals in both hemispheres, plerocercoid larvae in fish have only been reported in the Pacific Ocean, in Peruvian waters, from where most human cases are known. In Argentine waters larval stages of Diphyllobothriidae have been recorded in Merluccius hubbsi, a main fish resource mostly exported frozen, headed and gutted (H&G) or as fillets; therefore, the possible presence of A. pacificus in edible products, and the extent of the risk of parasitism for humans becomes of health and commercial relevance. With the aim of detecting and identifying potentially zoonotic diphyllobothriids and quantifying infection levels in viscera and fillets of hakes, 43 entire fish, 471 H&G, and 942 fillets obtained from research cruises in 2019 and 2021 from the southern Argentine Sea (44-53°S; 63-68°W) were examined by transillumination and under stereoscopic microscopy. Plerocercoids were recovered at low prevalence and mean abundance in entire fish (13.95 % and 0.35) and H&G fish (2.76 % and 0.03) but no larval worms were found adhered to musculature or peritoneum, furthermore, no larvae were found in the fillets. Larvae were genetically identified, based on sequences of the large subunit ribosomal RNA nuclear gene (lsrDNA) and the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene, as members of A. pacificus, representing the first report of this species in a fish host outside South American Pacific coasts. No spatial nor bathymetric patterns in parasite burdens were observed across sampling sites, but prevalence increased with fish size. The recorded low parasite burdens, the absence of infective stages in fillets and the fact that most products are commercialized deeply frozen, diminish the risk of parasitism for consumers to a minimum. However, the identification of this zoonotic agent and the assessment of its distribution in fish products are a first indispensable step for the design of efficient and suitable measures, such as freezing or cooking meet, to ensure the prevention of human infections.

Unique genetic structure of the human tapeworm Dibothriocephalus latus from the Alpine lakes region - a successful adaptation?

Dibothriocephalus latus is the most frequent causative agent of fish-borne zoonosis (diphyllobothriosis) in Europe, where it is currently circulating mainly in the Alpine lakes region (ALR) and Russia. Three mitochondrial genes (cox1, cob and nad3) and 6 microsatellite loci were analysed to determine how is the recently detected triploidy/parthenogenesis in tapeworms from ALR displayed at the DNA level. A geographically distant population from the Krasnoyarsk Reservoir in Russia (RU-KR) was analysed as a comparative population. One or 2 alleles of each microsatellite locus was detected in plerocercoids from RU-KR, corresponding to the microsatellite pattern of a diploid organism. In contrast, 1­3 alleles were observed in tapeworms from ALR, in accordance with their triploidy. The high diversity of mitochondrial haplotypes in D. latus from RU-KR implied an original and relatively stable population, but the identical structure of mitochondrial genes of tapeworms from ALR was probably a consequence of a bottleneck typical of introduced populations. These results indicated that the diploid/sexually reproducing population from RU-KR was ancestral, located within the centre of the distribution of the species, and the triploid/parthenogenetically reproducing subalpine population was at the margin of the distribution. The current study revealed the allelic structure of the microsatellite loci in the triploid tapeworm for the first time.

Population genetic structure of diphyllobothriid tapeworms (Cestoda: Diphyllobothriidea) parasitising fish in the Baikal Rift Zone.

Tapeworms of the genus Dibothriocephalus are widely distributed throughout the world, and some are agents of human diphyllobothriasis, one of the most important fish-borne zoonoses caused by a cestode parasite. Until now, the population genetic structure of diphyllobothriid tapeworms in the Baikal Rift Zone (BRZ) has remained unexplored. The major aim of this study was to analyse the population genetic structure of D. dendriticus and D. ditremus parasitising fish in the BRZ based on internal transcribed spacer 1 (ITS1) and mitochondrial gene cytochrome oxidase subunit I (cox1) sequences. We found that both species had complex population genetic structures. Each species formed 2 clades (D. dendriticus: Clade 1 & 2; D. ditremus Clade A & B) that differed in genetic diversity. D. dendriticus haplotypes in Clade 1 formed a star-like sub-network with a main haplotype, whereas the haplotypes in Clade 2 formed a diffuse network. We assumed that the complex population genetic structure of D. dendriticus was a consequence of populations evolving under different palaeoecological conditions during the Last Glacial Maximum. In contrast to D. dendriticus, both clades in the D. ditremus samples formed a diffuse network. Our findings revealed hypothetical pathways in the formation of the population genetic structure of diphyllobothriids in the BRZ. On one hand, isolation by distance played an important role; on the other hand, lake recolonisation from refugia and a genetic bottleneck after the end of the Last Glacial Maximum had a possible influence.

Diphyllobothrium stemmacephalum (Cestoda: Diphyllobothriidae) found from a harbor porpoise in northern Japan, with comments on a geographical gap with human infection cases in southern Japan.

Even though the cetacean tapeworm Diphyllobothrium stemmacephalum occurs in both cold and warm waters, human infections and final host occurrences have been confined to temperate areas in and near Japan. We recently obtained a strobila of this cestode that was excreted from a harbor porpoise accidentally caught offshore of Hokkaido of northern Japan. Genetic analysis of 28S rDNA and cox1 genes confirmed that the cestode was D. stemmacephalum. Our finding sets the northernmost record of D. stemmacephalum in the western Pacific, suggesting that the risk of human infections by this parasite in northern Japan deserves further attention.

Diphyllobothrium sprakeri n. sp. (Cestoda: Diphyllobothriidae): a hidden broad tapeworm from sea lions off North and South America.

BACKGROUND: The systematic of several marine diphyllobothriid tapeworms of pinnipeds has been revised in recent years. However, 20 species of Diphyllobothrium from phocids and otariids are still recognized as incertae sedis. We describe a new species of Diphyllobothrium from the intestine of California sea lions Zalophus californianus (Lesson) (type-host) and South American sea lions Otaria flavescens (Shaw). METHODS: Zalophus californianus from the Pacific coast of the USA and O. flavescens from Peru and Argentina were screened for parasites. Partial fragments of the large ribosomal subunit gene (lsrDNA) and the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene were amplified for 22 isolates. Properly fixed material from California sea lions was examined using light and scanning electron microscopy. RESULTS: A total of four lsrDNA and 21 cox1 sequences were generated and aligned with published sequences of other diphyllobothriid taxa. Based on cox1 sequences, four diphyllobothriid tapeworms from O. flavescens in Peru were found to be conspecific with Adenocephalus pacificus Nybelin, 1931. The other newly generated sequences fall into a well-supported clade with sequences of a putative new species previously identified as Diphyllobothrium sp. 1. from Z. californianus and O. flavescens. A new species, Diphyllobothrium sprakeri n. sp., is proposed for tapeworms of this clade. CONCLUSIONS: Diphyllobothrium sprakeri n. sp. is the first diphyllobothriid species described from Z. californianus from the Pacific coast of North America, but O. flavescens from Argentina, Chile and Peru was confirmed as an additional host. The present study molecularly confirmed the first coinfection of two diphyllobothriid species in sea lions from the Southern Hemisphere.

Molecular cytogenetic analysis of a triploid population of the human broad tapeworm, Dibothriocephalus latus (Diphyllobothriidea).

The large-sized tapeworm Dibothriocephalus latus is known as the broad or fish-borne cestode of mammals that is capable to infect humans and cause diphyllobothriosis. Recently, molecular data on D. latus has been accumulating in the literature and a complete genome sequence has been published; however, little is known about the karyotype and chromosome architecture. In this study, an in-depth karyological analysis of 2 D. latus specimens was carried out. The plerocercoids originated from a perch caught in subalpine Lake Iseo (Italy) and the tapeworms were reared in hamsters. Both specimens contained cells with a highly variable number of chromosomes ranging from18 to 27. Nevertheless, the largest portion of mitotic figures (47%) showed a number corresponding to the triploid set, 3n = 27. Accordingly, the karyotype of the analyzed specimens consisted of 9 triplets of metacentric chromosomes. Fluorescence in situ hybridization (FISH) with the 18S rDNA probe clearly demonstrated the presence of 3 clusters of hybridization signals on the triplet of chromosome 7, thus confirming the triploid status of the specimens. FISH with a telomeric (TTAGGG)n probe confined hybridization signals exclusively to the terminal chromosomal regions, supporting the earlier findings that this repetitive motif is a conserved feature of tapeworm telomeres.

Endohelminths of European Perch (Perca fluviatilis) from Selected Localities in Poland with an Emphasis on Search of the Broad Fish Tapeworm Dibothriocephalus latus.

INTRODUCTION: A piscivorous fish European perch (Perca fluviatilis) is present in all types of lakes and brackish waters in Poland. Previous ichthyoparasitological surveys revealed broad spectrum of endohelminths in perch from different aquatic environments. Among them, detection of Diphyllobothrium sp. and Dibothriocephalus latus (syn. Diphyllobothrium latum) in the Pomeranian Bay is of particular interest, since D. latus is one of the causative agents of diphyllobothriasis, fish-borne parasitic zoonosis. Besides, D. latus eggs were previously detected in coprological samples of otter, wolf and lynx from the Bialowieza Primeval Forest. AIM: To conduct parasitological examinations of European perch from the Pomeranian Bay in order to detect the spectrum of its endohelminths and to provide a pilot study on helminths of perch from different water bodies in the Bialowieza Primeval Forest. Due to zoonotic character of D. latus, we have focused our attention to this tapeworm. RESULTS: The larvae of tapeworm Triaenophorus nodulosus and thorny-headed worm Acanthocephalus lucii were detected in perch from the Pomeranian Bay. In perch from different localities in the Bialowieza Primeval Forest, T. nodulosus, A. lucii and tapeworm Proteocephalus percae were detected. D. latus plerocercoids were found neither in musculature nor in peritoneal cavity and other internal organs of any of the fish examined from both studied localities in Poland. CONCLUSION: Future screening implementing morphological and molecular markers is needed in order to understand the current distribution of D. latus in Europe.

Dibothriocephalus nihonkaiensis: an emerging foodborne parasite in Brittany (France)?

BACKGROUND: Diphyllobothriosis is an intestinal cestodosis caused by tapeworms of the family Diphyllobothriidae. In France, endemic cases are limited to south-east and due to Dibothriocephalus latus. In this paper, we investigate a series of seven cases of diphyllobothriosis in the non-endemic French region of Brittany. All have been diagnosed between 2016 and 2018 at the University Hospital of Rennes. METHODS: Parasites were identified by their morphological features and by phylogenetic analysis of the cox1 gene. Phylogenetic tree was built using maximum likelihood criterion under the GTR+G+I model and 2000 bootstrap replicates. A form was sent to all patients to collect data concerning clinical signs and possible sources of infection. RESULTS: All cases were due to Dibothriocephalus nihonkaiensis, a species strictly distributed in the North Pacific. Epidemiological investigation showed that the parasite was probably acquired in France, after consumption of Japanese food containing raw salmon. All patients presented with at least abdominal pain and fatigue except for one patient who had no symptoms. CONCLUSIONS: To our knowledge, this case series is the most important cohort of allochthonous diphyllobothriosis described in Europe. This sudden emergence raises concern about foodborne infections, highlighting (i) risky food habits in absence of adequate sanitary control; and (ii) the breaking of the rule of geographical restriction due to globalization and worldwide trades.

Development of microsatellite loci in zoonotic tapeworm Dibothriocephalus latus (Linnaeus, 1758), Lühe, 1899 (syn. Diphyllobothrium latum) using microsatellite library screening.

The broad fish tapeworm Dibothriocephalus latus is a causative agent of human food-borne disease called diphyllobothriosis. Medical importance, scattered geographical distribution and unknown origin of D. latus in Europe and North America make this species to be an interesting model for population genetics. Microsatellite markers were originally designed by library screening using NGS approach and validated as tools for future studies on population genetics of D. latus. Out of 122 candidates selected after NGS analysis, 110 yielded PCR products of the expected size, and in 78 of them, a declared repetitive motif was confirmed by Sanger sequencing. After the fragment analysis, six loci were proved to be polymorphic and tested for observed (Ho) and expected (He) heterozygosity, and deviations from Hardy-Weinberg equilibrium (HWE). They promise future application in studies on genetic interrelationships, origin and migratory routes of this medically important emerging tapeworm.

Diphyllobothriidea in the north area of the Andean Patagonia: Epidemiology in urban dogs, morphometrical and molecular identification, with comments on wild carnivores.

Diphyllobothriidea are the principal agents of Diphyllobothriosis, a widespread food-borne cestodosis. Accurate identification of the species in samples is therefore crucial for diagnosis and epidemiology in wild and domestic animals, and also humans. We aim to identify at specific level the causative agent, and provide an observational, descriptive, and transversal study of the epidemiology of this zoonosis in urban dogs. Also data on wild carnivores from Northwestern Patagonia are presented. Dog feces were collected in thirteen neighborhoods of varying socioeconomic status, and stools were analyzed by two concentration methods. Adult worms were collected and identified by molecular methods. The population of free-roaming dogs in each neighborhood was estimated, and surveys were conducted at all veterinary clinics registered in the Veterinary Medical College of Bariloche city. A total of 36 wild carnivores road killed or found dead in three National Parks were analyzed. Molecular and morphometric analyses of proglottids and eggs from dogs indicate they are infected with D. latum. Twenty out of 118 dog feces were positive for Diphyllobothrium, from 9 out of 13 neighborhoods, with infection values between 10% and 66%. Percentage of infection was correlated positively with the number of free roaming dogs per block, and with Unsatisfied Basic Needs (UBN %), but not with distance to nearest water body. Infection by D. latum in dogs is widely distributed throughout the city. Not all local veterinarians know the occurrence of Diphyllobothriosis in the dogs of the city, and it is evident that this zoonosis is underdiagnosed in relation to the percentage of infection found in this study. None of the analyzed wild carnivores were positive for Diphyllobothrium.

Molecular Identification of Diphyllobothrium latum from a Pediatric Case in Taiwan.

Human diphyllobothriasis is a parasitic disease caused by ingestion of larvae (plerocercoids) in raw or undercooked fish and commonly found in temperate areas. Rare cases were reported in tropical or subtropical areas especially in children. The first documented case of pediatric diphyllobothriasis in Taiwan had been reported 11 years ago. Here, we report another 8-year-old girl case who presented with a live noodle-like worm hanging down from her anus, with no other detectable symptoms. We pulled the worm out and found the strobila being 260 cm in length. Examination of gravid proglottids showed that they were wider than their lengths, containing an ovoid cirrus sac in the anterior side and the rosette-shaped uterus. Eggs extracted from the uterus were ovoid and operculated. Diphyllobothrium latum was confirmed by molecular analysis of the mitochondrial DNA cytochrome c oxidase subunit 1 (cox1) gene. The girl was treated with a single oral dose of praziquantel, and no eggs or proglottids were observed from her stool in the subsequent 3 months. The reemergence of human diphyllobothriasis in non-endemic countries is probably due to prevalent habit of eating imported raw fish from endemic areas. This pediatric case raised our concern that human diphyllobothriasis is likely underestimated because of unremarkable symptoms.

Four Human Cases of Diphyllobothrium nihonkaiense (Eucestoda: Diphyllobothriidae) in China with a Brief Review of Chinese Cases.

We described 4 human infection cases of zoonotic fish-tapeworm, Diphyllobothrium nihonkaiense, identified with morphological and molecular characters and briefly reviewed Chinese cases in consideration of it as an emerging parasitic disease in China. The scolex and mature and gravid proglottids of some cases were seen, a rosette-shaped uterus was observed in the middle of the mature and gravid proglottids, and the diphyllobothriid eggs were yellowish-brown in color and displayed a small knob or abopercular protuberance on the opposite end of a lid-like opening. The average size of the eggs was recorded as 62-67×42-45 µm. The parasitic materials gathered from 4 human cases were morphologically identified as belonging to the genera Diphyllobothrium and Adenocephalus. The phylogenetic analysis based on the nucleotide sequences of cytochrome c oxidase subunit 1 gene of the etiologic agents confirmed that the 4 cases were D. nihonkaiense infection. The finding of 4 additional D. nihonkaiense cases suggests that D. nihonkaiense might be a major causative species of human diphyllobothriasis in China. A combined morphological and molecular analysis is the main method to confirm D. nihonkaiense infection.

Complete sequence and characterization of the mitochondrial genome of Diphyllobothrium stemmacephalum, the type species of genus Diphyllobothrium (Cestoda: Diphyllobothriidae), using next generation sequencing.

We first constructed and characterized the complete mitochondrial genome (mitogenome) sequence of Diphyllobothrium stemmacephalum, the type species of genus Diphyllobothrium, using next generation sequencing (NGS). The mitogenome of D. stemmacephalum was 13,716bp, including 12 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 2 longer intergenic non-coding regions, and has features common to mitogenomes of other cestodes. Although it has been accepted that tRNA for serine (trnS2(UCN)) in Platyhelminthes lacks a D arm, the trnS2(UCN) of D. stemmacephalum was predicted to have a paired D arm as in Diplogonoporus balaenopterae. The non-coding region 2 contained eight tandem repeat units (34nucleotides/unit). This study also corroborated that D. stemmacephalum is phylogenetically more closely related to Dip. balaenopterae than to Diphyllobothrium latum and Diphyllobothrium nihonkaiense. As demonstrated here, mitogenome sequence data obtained using NGS is useful for gaining a better understanding of the systematics, phylogeny and taxonomic revisions involving valuable specimens preserved in museums, universities or research institutes for which sequence data are not yet available, and also for making diagnoses based on clinical samples.

Prevalence of Diphyllobothrium latum (Cestoda: Diphyllobothriidae) plerocercoids in fish species from four Italian lakes and risk for the consumers.

In recent years there has been a re-emergence of diphyllobothriasis by Diphyllobothrium latum (Cestoda: Diphyllobothriidae) in Italy, France and Switzerland, where in the past this fish-borne zoonosis was widespread and then virtually disappeared. A change in eating habits such as the consumption of raw/undercooked freshwater fish, has led to an increased risk for consumers of ingesting infective larvae of D. latum. A survey on the factors responsible for the re-emergence of human diphyllobothriasis in Italy was carried out from March 2013 to December 2014. The aim of this study was to assess the diffusion of D. latum plerocercoids in the fish populations of the sub-alpine lakes of Maggiore, Como, Iseo and Garda, updating the scarce historical data and assessing a preliminary "risk level" of the lacustrine environments and fish species under investigation. A total of 2228 fish belonging to 5 species, 690 from Lake Maggiore, 500 from Lake Como, 655 from Lake Iseo and 383 from Lake Garda were submitted to parasitological examination. The presence of D. latum plerocercoid larvae was detected in 6.6%, 25.4% and 7.6% of perch (Perca fluviatilis) from Lakes Maggiore, Como and Iseo respectively. The parasite was also present in pike (Esox lucius) with prevalence values ranging from 71.4 to 84.2% and in 3.6-3.8% of burbot (Lota lota) from Lakes Iseo and Como. Fish from Lake Garda were negative as well as sampled whitefish (Coregonus lavaretus) and shad (Alosa fallax lacustris). The results of this survey showed a widespread presence of D. latum plerocercoid larvae in Maggiore, Como and Iseo fish populations. Urban fecal contamination of water is still a key issue to be resolved, together with the improvement of communication with consumers regarding the best dietary habits and the most effective processes of parasite inactivation, required for the consumption of raw/undercooked fish caught in high-risk areas.

Invalidation of Diphyllobothrium hottai (Cestoda: Diphyllobothriidae) based on morphological and molecular phylogenetic analyses.

Diphyllobothrium hottai Yazaki, Fukumoto & Abe, 1988 was described based on the morphology of adult worms recovered from golden hamsters that had been experimentally infected with plerocercoids obtained from Japanese surf smelts (Hypomesus pretiosus japonicus) and olive rainbow smelts (Osmerus eperlanus mordax). Although D. hottai was considered to be distinct from Diphyllobothrium ditremum (Creplin, 1825), their taxonomic relationship requires further clarification. In our study, D. hottai and D. ditremum obtained from hamsters experimentally infected with plerocercoids isolated from Japanese surf smelts were compared using morphological and molecular methods. The criterion usually used to differentiate between D. hottai and D. ditremum is the difference in the angle between the long axis of the cirrus sac and that of the seminal vesicle. However, we found variation of the angle within the same individual and, one specimen showed both of the different angles that were supposedly unique to each of the species. Furthermore, phylogenetic analysis of the complete sequences of the mitochondrial cytochrome c oxidase subunit 1 and cytochrome b genes revealed that both species were genetically indistinguishable. Therefore, D. hottai is considered to be a junior synonym of D. ditremum.