Understanding the role of pigs in the transmission of zoonotic Echinococcus ortleppi in Haryana, India.

The etiological agents of zoonotic cystic echinococcosis comprise the Echinococcus granulosus sensu lato (s.l.) species complex. The present study was aimed at investigating the zoonotic genotypes of Echinococcus granulosus s.l. circulating in the pig population of Haryana, India. Out of 253 slaughtered pigs screened, 5 showed the presence of hydatid cysts. The amplification of the partial mitochondrial NADH dehydrogenase subunit 1 (nad1) gene for the molecular confirmation and phylogenetics of the retrieved metacestodes (n = 2) revealed the presence of E. ortleppi. The sequences generated herein exhibited 99.80% homology to the GenBank archived E. ortleppi sequences. Cladistics targeting genetic diversity and haplotype network analysis involved 37 E. granulosus s.l. GenBank archived sequences from India corresponding to different hosts (large and small ruminants and humans) along with the sequences (n = 2) generated in the present study. Overall, 14 haplotypes with high haplotype (0.780 ± 0.059) and low nucleotide (0.033 ± 0.010) diversities were recorded for the overall data set, which evinced a population expansion. The median-joining haplotype network revealed a stellate shape of E. granulosus sensu stricto (s.s.) sequences, which was indicative of rapid population expansion. High genetic differentiation (FST = 0.840 - 0.983) and low gene flow (Nm = 0.003 - 0.047) were recorded between the pig intermediate hosts infected with E. ortleppi and other hosts infected with E. granulosus s.s. The findings are of paramount significance for the formulation of effective control strategies considering the public health and economic impact of cystic echinococcosis.

Study on the genotypes of Echinococcus granulosus in yaks and sheep from Langkazi County in Tibet Autonomous Region of China based on mitochondrial cox1 and nad1.

To determine the genotypes of the epidemic strains of Echinococcus granulosus in livestock in Tibet, samples of E. granulosus cysts were collected from 11 yaks and 62 sheep. Genomic DNA was extracted from these samples, and gene fragments of mitochondrial cytochrome c oxidase subunit I (cox1) and NADH dehydrogenase subunit I (nad1) were amplified by PCR and sequenced. DNASTAR and MAGA7.0 were employed for homology analysis and phylogenetic tree construction. Echinococcus granulosus cysts were detected in 56.2% (41/73) of the samples screened. Of these, 63.4% (26/41) were identified as E. granulosus G1 genotype (common sheep strain), 24.4% (10 /41) as G3 genotype (buffalo strain), and 12.2% (5/41) were G6 genotype (camel strain). The study concludes that yaks and sheep in Langkazi county, Tibet, carry three E. granulosus genotypes (G1, G3, and G6), with the G1 genotype the predominant genotype in the region. This study clarifies the distribution of E. granulosus genotypes, providing genetic data and insight for the surveillance and prevention of echinococcosis.

Genetic variation and population structure of Fasciola hepatica: an in silico analysis.

Fasciola hepatica is a trematode leading to heavy economic setbacks to the livestock sector globally. The population's genetic information and intimate kinship level are frequently assessed using analysis of mitochondrial DNA. In this analysis, we retrieved cox1 (n = 247) and nad1 (n = 357) sequences of F. hepatica from the NCBI GenBank database and aligned the sequences with the respective reference sequences using MEGA software. The median joining network was drawn using PopArt software while neutrality and diversity indices were estimated with the help of DnaSp software. Neighbor-joining phylogenetic tree was constructed using the MEGA software package. A total of 46 and 98 distinctive haplotypes were observed for cox1 and nad1 genes, respectively. Diversity indices indicated high haplotype and nucleotide diversities in both genes. Positive Tajima's D and Fu's Fs values were found for the entire population of both the genes under study. The cox1 and nad1 gene segments in this study showed high Tajima's D values, suggesting a low likelihood of future population growth. The Tajima's D value of the nad1 gene sequence is lower (2.14910) than that of the cox1 gene sequence (3.40314), which suggests that the former is growing at a slower rate. However, the region-wise analysis revealed that both the cox1 and nad1 genes showed deviation from neutrality suggesting a recent population expansion as a result of an excess of low-frequency polymorphism. Furthermore, the overall host-wise analysis showed positive and significant Tajima's D values for the cox1 and nad1 gene sequences. To the best of our knowledge, this is the first attempt to provide insights into genetic variations and population structure of F. hepatica at a global scale using cox1 and nad1 genes. Our findings suggest the existence of specific variants of F. hepatica in different parts of the world and provide information on the molecular ecology of F. hepatica. The results of this study also mark a critical development in upcoming epidemiological investigations on F. hepatica and will also contribute to understanding the global molecular epidemiology and population structure of F. hepatica.

Dual RNA-Seq Analysis Pinpoints a Balanced Regulation between Symbiosis and Immunity in Medicago truncatula-Sinorhizobium meliloti Symbiotic Nodules.

Legume-rhizobial symbiosis initiates the formation of root nodules, within which rhizobia reside and differentiate into bacteroids to convert nitrogen into ammonium, facilitating plant growth. This process raises a fundamental question: how is plant immunity modulated within nodules when exposed to a substantial number of foreign bacteria? In Medicago truncatula, a mutation in the NAD1 (Nodules with Activated Defense 1) gene exclusively results in the formation of necrotic nodules combined with activated immunity, underscoring the critical role of NAD1 in suppressing immunity within nodules. In this study, we employed a dual RNA-seq transcriptomic technology to comprehensively analyze gene expression from both hosts and symbionts in the nad1-1 mutant nodules at different developmental stages (6 dpi and 10 dpi). We identified 89 differentially expressed genes (DEGs) related to symbiotic nitrogen fixation and 89 DEGs from M. truncatula associated with immunity in the nad1-1 nodules. Concurrently, we identified 27 rhizobial DEGs in the fix and nif genes of Sinorhizobium meliloti. Furthermore, we identified 56 DEGs from S. meliloti that are related to stress responses to ROS and NO. Our analyses of nitrogen fixation-defective plant nad1-1 mutants with overactivated defenses suggest that the host employs plant immunity to regulate the substantial bacterial colonization in nodules. These findings shed light on the role of NAD1 in inhibiting the plant's immune response to maintain numerous rhizobial endosymbiosis in nodules.

Molecular identification of Taenia hydatigena from domestic and free-living animals in Slovakia, Central Europe.

Taenia hydatigena is a cosmopolitan tapeworm that uses canids or felines as definitive hosts, while the larval stage (metacestode), formerly referred to as cysticercus tenuicollis, infects a wide variety of intermediate hosts, in particular ruminants. In the present study, we used partial nucleotide sequences of the cox1 and nad1 genes of T. hydatigena from different animal species to analyse the intraspecies genetic diversity of this economically important parasite. Twenty-four samples of metacestodes or adults of T. hydatigena from infected sheep, chamois, roe deer, fallow deer, wild boar, and dogs from Slovakia were collected and further analysed. Several haplotypes of T. hydatigena were identified with unique mutations that have not been previously recorded in Slovakia. Analysis of nucleotide polymorphism revealed the existence of 9 and 13 haplotypes, with relatively low nucleotide pairwise divergence ranging between 0.3-1.3 and 0.2-1.8% for the Hcox and Hnad haplotypes, respectively. In general, low nucleotide and high haplotype diversities in the overall population of T. hydatigena from the study indicate a high number of closely related haplotypes within the explored population; nucleotide diversity per site was low for cox1 (Pi = 0.00540) and slightly higher for nad1 (Pi = 0.00898). A molecular study confirmed the existence of genetic variation within T. hydatigena isolates from Slovakia. However, further investigations with more samples collected from different intermediate and definitive hosts are required in order to investigate the epidemiological significance of the apparent genetic differences observed in this study.

Assessing the population structure of trematode Metagonimus suifunensis using three mitochondrial markers.

In this work, for the first time, the genetic variability of the Metagonimus suifunensis population in the Russian southern Far East was estimated based on the full-length sequences of the nad1 gene of mitochondrial DNA. In addition, for a sample of the same size, the sequences of cox1 and cytb genes, previously used for population studies for M. suifunensis, were reanalysed. Three markers were combined to a common sequence, and the obtained data were studied. Despite the higher level of variability, nad1 and cox1 mtDNA genes did not reveal subdivisions within the population. The combined dataset made it possible to determine that the sample from the Odyr River was the centre of the species' range formation and clarified the continental migration route of the parasite from south to north. According to the data obtained, it was presumed that piscivorous birds participate in the life cycle of the parasite. The subdivision within population revealed that using all three mitochondrial markers is consistent with the features of differentiation within populations of related species, but the reasons for its formation remain unclear due to the insufficient amount of data and the use of different markers in studies of different species.

First record of Patagifer bilobus (Rudolphi, 1819) Dietz, 1909 (Digenea: Echinostomatidae), with a morphological and molecular characterization from two threskiornithid species in Mexico.

Patagifer Dietz, 1909 is a small genus of echinostomatids, with 12 recognized species, mostly parasitising threskiornithid birds, distributed worldwide. In the current research, adult specimens of the type species, Patagifer bilobus (Rudolphi, 1819) Dietz, 1909 from the white faced ibis (Plegadis chihi) and white ibis (Eudocimus albus) were re-described, providing new metrical data for the number of head collar spines. Those specimens were recorded from eight localities in Mexico and compared morphologically with specimens previously identified as Patagifer lamothei. A total of 19 specimens identified as P. bilobus including two hologenophores were sequenced with three molecular markers: domains D1-D3 of the large subunit (LSU), the internal transcribed spacer (ITS1, ITS2) plus 5.8S from the nuclear rDNA, and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) from mitochondrial DNA. The new sequences were aligned with other sequences of Patagifer spp., downloaded from GenBank. Phylogenetic trees inferred from each data set, placed all the specimens in a clade, confirming that the isolates belonged to the same species. The morphological examination of specimens previously identified as P. lamothei by Ortega-Olivares MP, Hernández-Mena DI, Pérez-Ponce de León G, García-Varela M (2011) Helminths of the white ibis, Eudocimus albus (Aves Therskiornithidae) in Mexico. (Zootaxa 3088, 15-26. 10.11646/zootaxa.3088.1.2) and in combination with molecular data confirms that those specimens should be reassigned to P. bilobus. In addition, this is the first study in P. bilobus using an integrative taxonomy approach.

Nuclear-Encoded Maturase Protein 3 Is Required for the Splicing of Various Group II Introns in Mitochondria during Maize (Zea mays L.) Seed Development.

Splicing of plant organellar group II introns from precursor-RNA transcripts requires the assistance of nuclear-encoded splicing factors. Maturase (nMAT) is one such factor, as its three homologs (nMAT1, 2 and 4) have been identified as being required for the splicing of various mitochondrial introns in Arabidopsis. However, the function of nMAT in maize (Zea mays L.) is unknown. In this study, we identified a seed development mutant, empty pericarp 2441 (emp2441) from maize, which showed severely arrested embryogenesis and endosperm development. Positional cloning and transgenic complementation assays revealed that Emp2441 encodes a maturase-related protein, ZmnMAT3. ZmnMAT3 is highly expressed during seed development and its protein locates to the mitochondria. The loss of function of ZmnMAT3 resulted in the reduced splicing efficiency of various mitochondrial group II introns, particularly of the trans-splicing of nad1 introns 1, 3 and 4, which consequently abolished the transcript of nad1 and severely impaired the assembly and activity of mitochondrial complex I. Moreover, the Zmnmat3 mutant showed defective mitochondrial structure and exhibited expression and activity of alternative oxidases. These results indicate that ZmnMAT3 is essential for mitochondrial complex I assembly during kernel development in maize.

Genetic diversity and population structure analyses based on microsatellite DNA of parthenogenetic Fasciola flukes obtained from cattle and sika deer in Japan.

Understanding the population structure of Fasciola flukes in domestic and wild animals is important for determining the extent of cross-infection between them. Although the parthenogenetic Fasciola flukes in Japan have been shown to comprise five genetic types based on the ribosomal internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase subunit 1 (nad1) regions, these genetic regions are not suitable for analyzing their population structure. In the present study, the genetic diversity and population structure of the parthenogenetic Fasciola flukes in Japan were studied using microsatellite DNA, ITS1, and nad1 regions. A total of 144 parthenogenetic Fasciola flukes, obtained from cattle and sika deer in 16 localities, were individually analyzed using PCR-RFLP for ITS1, PCR-direct sequence analysis for nad1, and post-labeling PCR and capillary electrophoresis for microsatellite DNA regions. The flukes showed higher genetic diversity in the microsatellite DNA regions than ITS1 and nad1. The population structures of parthenogenetic Fasciola flukes were unclear, however, it was suggested that the flukes are more diverse populations. We hypothesized that their distribution throughout Japan is closely related to livestock movement dependent on human activity. Moreover, it is considered that cross-infection of the flukes between cattle and sika deer possibly has occurred in the past.

Echinostoma miyagawai Ishii, 1932 (Echinostomatidae) from Ducks in Aceh Province, Indonesia with Special Reference to Its Synonymy with Echinostoma robustum Yamaguti, 1935.

Adult echinostomes having 37 collar spines collected from the intestine of Pitalah ducks in Aceh Province, Indonesia in 2018 were morphologically and molecularly determined to be Echinostoma miyagawai Ishii, 1932 (Digenea: Echinostomatidae). Among 20 ducks examined, 7 (35.0%) were found to be infected with this echinostome, and the number of flukes collected was 48 in total with average 6.9 (1-17) worms per duck. The adult flukes were 7.2 (6.1-8.5) mm in length and 1.2 (1.0-1.4) mm in width (pre-ovarian or testicular level) and characterized by having a head collar armed with 37 collar spines (dorsal spines arranged in 2 alternating rows), including 5 end group spines, and variable morphology of the testes, irregularly or deeply lobed (3-5 lobes) at times with horizontal extension. The eggs within the worm uterus were 93 (79-105) µm long and 62 (56-70) µm wide. These morphological features were consistent with both E. miyagawai and Echinostoma robustum, for which synonymy to each other has been raised. Sequencing of 2 mitochondrial genes, cox1 and nad1, revealed high homology with E. miyagawai (98.6-100% for cox1 and 99.0-99.8% for nad1) and also with E. robustum (99.3-99.8% for nad1) deposited in GenBank. We accepted the synonymy between the 2 species and diagnosed our flukes as E. miyagawai (syn. E. robustum) with redescription of its morphology. Further studies are required to determine the biological characteristics of E. miyagawai in Aceh Province, Indonesia, including the intermediate host and larval stage information.

Bertiella studeri Infection in Children, Sri Lanka.

We provide a detailed molecular and phylogenetic description of Bertiella studeri tapeworms infecting children in Sri Lanka. Our findings can be used to identify multiple species of Bertiella tapeworms that can infect human hosts in the Old World.

Echinococcus canadensis G8 Tapeworm Infection in a Sheep, China, 2018.

We report a sheep infected with Echinococcus canadensis G8 tapeworm in China in 2018. This pathogen was previously detected in moose, elk, muskox, and mule deer in Europe and North America; our findings suggest a wider host range and geographic distribution. Surveillance for the G8 tapeworm should be conducted in China.

Rice FLOURY ENDOSPERM10 encodes a pentatricopeptide repeat protein that is essential for the trans-splicing of mitochondrial nad1 intron 1 and endosperm development.

Endosperm, the major storage organ in cereal grains, determines grain yield and quality. Despite the fact that a role for P-type pentatricopeptide repeat (PPR) proteins in the regulation of endosperm development has emerged, molecular functions of many P-type PPR proteins remain obscure. Here, we report a rice endosperm defective mutant, floury endosperm10 (flo10), which developed smaller starch grains in starchy endosperm and abnormal cells in the aleurone layer. Map-based cloning and rescued experiments showed that FLO10 encodes a P-type PPR protein with 26 PPR motifs, which is localized to mitochondria. Loss of function of FLO10 affected the trans-splicing of the mitochondrial nad1 intron 1, which was accompanied by the increased accumulation of the nad1 exon 1 and exons 2-5 precursors. The failed formation of mature nad1 led to a dramatically decreased assembly and activity of complex I, reduced ATP production, and changed mitochondrial morphology. In addition, loss of function of FLO10 significantly induced an alternative respiratory pathway involving alternative oxidase. These results reveal that FLO10 plays an important role in the maintenance of mitochondrial function and endosperm development through its effect on the trans-splicing of the mitochondrial nad1 intron 1 in rice.

Identity of Spirometra theileri from a Leopard (Panthera pardus) and Spotted Hyena (Crocuta crocuta) in Tanzania.

In the present study, a Spirometra species of Tanzania origin obtained from an African leopard (Panthera pardus) and spotted hyena (Crocuta crocuta) was identified based on molecular analysis of cytochrome c oxidase I (cox1) and NADH dehydrogenase subunit I (nad1) as well as by morphological observations of an adult tapeworm. One strobila and several segments of a Spirometra species were obtained from the intestine of an African male leopard (Panthera pardus) and spotted hyena (Crocuta Crocuta) in the Maswa Game Reserve of Tanzania. The morphological characteristics of S. theileri observed comprised 3 uterine loops on one side and 4 on the other side of the mid-line, a uterine pore situated posterior to the vagina and alternating irregularly either to the right or left of the latter, and vesicular seminis that were much smaller than other Spirometra species. Sequence differences in the cox1 and nad1 genes between S. theileri (Tanzania origin) and S. erinaceieuropaei were 10.1% (cox1) and 12.0% (nad1), while those of S. decipiens and S. ranarum were 9.6%, 9.8% (cox1) and 13.0%, 12.6% (nad1), respectively. The morphological features of the Tanzania-origin Spirometra specimens coincided with those of S. theileri, and the molecular data was also consistent with that of S. theileri, thereby demonstrating the distribution of S. theileri in Tanzania. This places the leopard (Panthera pardus) and spotted hyena (Crocuta Crocuta) as new definitive hosts of this spirometrid tapeworm.

Suppression of innate immunity mediated by the CDPK-Rboh complex is required for rhizobial colonization in Medicago truncatula nodules.

Suppression of innate immunity is essential for rhizobial infection and colonization in compatible interactions with leguminous plants. In Medicago nad1 mutant plants, innate immunity is excessively activated, resulting in necrotic cell death after rhizobia are released from infection threads into symbiotic cells, suggesting that innate immunity plays a critical role in regulating bacteroid persistence. In this study, we identified three respiratory burst oxidase homologs (Rboh) and one calcium-dependent protein kinase (CDPK) as key factors for the activation of immunity in Medicago nodules using genetic and biochemical methods. Knock-out of either MtRbohB or MtRbohD in nad1-1 mutant plants produced effective nodules with intact symbiotic cells, while knock-out of MtRbohC decreased brown pigment deposition, leading to less necrosis in nad1-1 mutant nodules. MtCDPK5 directly phosphorylated MtRbohB, MtRbohC and MtRbohD, which triggered immune responses in plants. Knock-out of MtCDPK5 in nad1-1 mutant plants partially restored nitrogen-fixing nodules. Overexpression of the constitutively activated variant MtCDPK5VK under the control of the NAD1 promoter elicited strong immune responses, resulting in ineffective nodules in wild-type plants. Our data provide direct evidence that host plants utilize innate immunity to regulate rhizobial colonization in symbiotic cells in Medicago truncatula.

Molecular and Morphologic Identification of Spirometra ranarum Found in the Stool of African Lion, Panthera leo in the Serengeti Plain of Tanzania.

The present study was performed with morphological and molecular analysis (cox1 and nad1 mitochondrial genes) to identify the proglottids of spirometrid tapeworm found in the stool of an African lion, Panthera leo, in the Serengeti plain of Tanzania. A strand of tapeworm strobila, about 75 cm in length, was obtained in the stool of a male African lion in the Serengeti National Park (34˚ 50' E, 02˚ 30' S), Tanzania, in February 2012. The morphological features of the adult worm examined exhibited 3 uterine coils with a bow tie appearance and adopted a diagonal direction in the second turn. The posterior uterine coils are larger than terminal uterine ball and the feature of uteri are swirling rather than spirally coiling. The sequence difference between the Spirometra species (Tanzania origin) and S. erinaceieuropaei (GenBank no. KJ599680) was 9.4% while those of S. decipiens (GenBank no. KJ599679) differed by 2.1% in the cox1 and nad1 genes. Phylogenetic tree topologies generated using the 2 analytic methods were identical and presented high level of confidence values for the 3 major branches of the 3 Spirometra species in the cox1 gene. The morphological and molecular findings obtained in this study were nearly coincided with those of S. ranarum. Therefore, we can know for the first time that the African lion, Panthera leo, is to the definitive host of this tapeworm.

Genetic and Morphologic Identification of Spirometra ranarum in Myanmar.

In the present study, we identified a Spirometra species of Myanmar origin (plerocercoid) by molecular analysis using mitochondrial cox1 and nad1 genes, as well as by morphological observations of an adult tapeworm. Spargana specimens were collected from a paddy-field in Taik Kyi Township Tarkwa Village, Yangon, Myanmar in December 2017. A total of 5 spargana were obtained from 20 frogs Hoplobatrachus rugulosus; syn: Rana rugulosa (Wiegmann, 1834) or R. tigrina (Steindachner, 1867). The plerocercoids were used for experimental infection of a dog. After 4 weeks of infection, an adult tapeworm was recovered from the intestine of the dog. Morphologically, the distinct features of Spirometra sp. (Myanmar origin) relative to S. erinaceieuropaei and S. decipiens include a uterine morphology comprising posterior uterine coils that larger than the terminal uterine ball and coiling of the uteri diagonally (swirling) rather than spirally. The cox1 sequences (1,566 bp) of the Myanmar-origin Spirometra species showed 97.9% similarity to a reference sequence of S. decipiens (GenBank no. KJ599679) and 90.5% similarity to a reference sequence of S. erinaceieuropaei (GenBank no. KJ599680). Phylogenetic tree topologies were identical and presented high confidence level of values for the 3 major branches of the 3 Spirometra species in cox1 and nad1 genes. These results indicated that Myanmar-origin Spirometra species coincided with those of S. ranarum and may be considered as a valid species.

Genetic polymorphism and population structure of Echinococcus ortleppi.

The zoonotic cestode Echinococcus ortleppi (Lopez-Neyra and Soler Planas, 1943) is mainly transmitted between dogs and cattle. It occurs worldwide but is only found sporadically in most regions, with the notable exception of parts of southern Africa and South America. Its epidemiology is little understood and the extent of intraspecific variability is unknown. We have analysed in the present study the genetic diversity among 178 E. ortleppi isolates from sub-Saharan Africa, Europe and South America using the complete mitochondrial cox1 (1608 bp) and nad1 (894 bp) DNA sequences. Genetic polymorphism within the loci revealed 15 cox1 and six nad1 haplotypes, respectively, and 20 haplotypes of the concatenated genes. Presence of most haplotypes was correlated to geographical regions, and only one haplotype had a wider spread in both eastern and southern Africa. Intraspecific microvariance was low in comparison with Echinococcus granulosus sensu stricto, despite the wide geographic range of examined isolates. In addition, the various sub-populations showed only subtle deviation from neutrality and were mostly genetically differentiated. This is the first insight into the population genetics of the enigmatic cattle adapted Echinococcus ortleppi. It, therefore, provides baseline data for biogeographical comparison among E. ortleppi endemic regions and for tracing its translocation paths.

Nuclear and mitochondrial DNA analysis reveals that hybridization between Fasciola hepatica and Fasciola gigantica occurred in China.

The well-known pathogens of fasciolosis, Fasciola hepatica (Fh) and Fasciola Gigantica (Fg), possess abundant mature sperms in their seminal vesicles, and thus, they reproduce bisexually. On the other hand, aspermic Fasciola flukes reported from Asian countries, which have no sperm in their seminal vesicles, probably reproduce parthenogenetically. The aim of this study was to reveal the origin of aspermic Fasciola flukes. The nuclear single copy markers, phosphoenolpyruvate carboxykinase and DNA polymerase delta, were employed for analysis of Fasciola species from China. The hybrid origin of aspermic Fasciola flukes was strongly suggested by the presence of the Fh/Fg type, which includes DNA fragments of both F. hepatica and F. gigantica. China can be regarded as the cradle of the interspecific hybridization because F. hepatica and F. gigantica were detected in the northern and southern parts of China, respectively, and hybrids flukes were distributed between the habitats of the two species. The Chinese origin was supported by the fact that a larger number of mitochondrial NADH dehydrogenase subunit 1 (nad1) haplotypes was detected in Chinese aspermic Fasciola populations than in aspermic populations from the neighbouring countries. Hereafter, 'aspermic' Fasciola flukes should be termed as 'hybrid' Fasciola flukes.

Phylogenetic relationships between Dicrocoelium chinensis populations in Japan and China based on mitochondrial nad1 gene sequences.

We carried out phylogenetic analyses of the relationships between Dicrocoelium chinensis populations in Japan and China using molecular markers. One hundred nine lancet flukes collected from Japan and China were identified as D. chinensis based on their testis orientation and the nucleotide sequences of their ribosomal ITS2. These flukes were analyzed phylogenetically using mitochondrial nad1 gene sequences. An analysis of molecular variance found that the percentage of variation between the countries was extremely high, indicating that the D. chinensis populations in Japan and China are differentiated genetically. D. chinensis mainly parasitizes wild sika deer, which is thought to originate in northeast Asia and to have colonized into Japan from the Eurasia continent in the Pleistocene glaciations. In addition, phylogenic analyses indicated that Japanese sika deer is genetically differentiated from Chinese population; therefore, we hypothesize that D. chinensis might have been introduced into Japan along with the migration of infected wild ruminants in the Pleistocene, and then the population became differentiated from the Chinese population. This study provides the nucleotide sequences of the nad1 gene of D. chinensis in Japan for the first time and shows that these sequences are useful for elucidating the phylogenetic relationships of the Dicrocoelium species prevalent in Asia.