<Emphasis Type="Italic">Paragonimus skrjabini</Emphasis> Chen, 1959 (Digenea: Paragonimidae) and related species in eastern Asia: a combined molecular and morphological approach to identification and taxonomy

A molecular and morphometric investigation is reported on the species complex of mammalian lung.ukes of which Paragonimus skrjabini Chen, 1959 and P.miyazakii Kamo, Nishida, Hatsushika & Tomimura, 1961 are the best-known examples. This species complex (here called the P.skrjabini complex) is shown to be monophyletic using DNA sequences from the nuclear ITS2 region and the mitochondrial cox1gene. The latter marker permits the discrimination of populations, some previously named as distinct species, from various geographical locations in China and Japan. Morphometric analysis of a number of variables accords remarkably closely with the molecular results. Main findings are that (1) nominal P.skrjabini from Fujian Province in eastern China is phylogenetically very close to P.miyazakii from Japan. It is proposed that both taxa should be referred to the same subspecies as P.skrjabini miyazakii. (2) Populations from Guangdong Province, Guangxi Zhuang Autonomous Region, Sichuan and Hubei should be referred to P.skrjabini skrjabini. Specimens of P.skrjabini from the Yangtze basin in Sichuan and Hubei are remarkably similar to one another according to genetic and morphometric data, but are not so different from Guangdong populations that they should be referred to a different subspecies at this stage. Specimens of P.skrjabini from Yunnan have not been assigned to a subspecies at this stage. (3) Partly following earlier studies, we regard the following as synonyms of P.skrjabini: P.miyazakii (reduced to subspecific status); P.szechuanensis Chung & Tsao, 1962 (probably belongs to P.s.skrjabini); and P.hueitungensis Chung, Hsu, Ho, Kao, Shao, Chiu, Pi, Liu, Ouyang, Shen, Yi & Yao, 1975 (probably belongs to P.s.skrjabini ); P.veocularis (Chen & Li, 1979) (specimens from Fujian regarded as belonging to this species should be referred to P.skrjabini miyazakii: the subspecific status of specimens from the type-locality in northern Sichuan is unclear). A number of questions remain unresolved. The name P. hokuoensis Ho & Chung, 1964 was proposed for two individual metacercariae of distinctive appearance from southern Yunnan. DNA sequences from very similar metacercariae from the same locality place this nominal species within, or sister to, the P. skrjabini complex. As yet, nothing is known regarding adult morphology or biology of this taxon and we retain it here as a distinct species. P. heterorchis (Zhou, Pang & Hsiang, 1982) might be a synonym of P. skrjabini: the form of the metacercaria provides evidence against this view and further work is required. P. macrorchis Chen, 1962 has probably been confused with P. skrjabiniin China. Within China, the former probably occurs only on Hainan Island, although P. fukienensis Tang & Tang, 1962 from Fujian Province could be a synonym.     

Studies on metabolism of lung flukes of the genus Paragonimus. VI. Succinoxidase system in homogenates of eggs, larvae, and adults

The present study was concerned with the succinoxidase system in Paragonimus westermani, Paragonimus ohirai, and Paragonimus miyazakii. Potassium cyanide inhibited the motility of larval and adult forms. Succinate stimulated the reduction of methylene blue by homogenates of embryonated eggs, larvae, and adults, while malonate inhibited the reduction. Reduced cytochrome c was oxidized by the 1,000g supernatant from homogenates of embryonated eggs, larvae, and adults. The supernatant prepared from unembryonated eggs did not oxidize reduced cytochrome c. Succinate stimulated oxygen consumption by the homogenate of adult worms. Oxygen consumption markedly increased in the homogenate of adults when both succinate and cytochrome c were added as substrate to the reaction mixture, while malonate and cyanide inhibited oxygen consumption.     

Characterization and localization of Paragonimus westermani antigen stimulating antibody formation in both the infected cat and rat

Applicability of the adult Paragonimus westermani antigen for detection of anti-immature P. westermani antibodies in experimentally infected rats, a paratenic host of this lung fluke, was examined. The serum antibodies of the cats and rats infected with P. westermani metacercariae were detected by enzyme-linked immunosorbent assay (ELISA) with the adult-fluke antigen. The ELISA titers of serum samples of the rats infected with only immature flukes were as high as those of the cats infected with adult flukes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and the immunoblotting technique showed that a major protein band of 27,000 daltons was recognized in the sera of the infected cats and rats. Immunoperoxidase staining applied on the sectioned flukes provided evidence showing that the antigenic substance was located on the surface of the gut epithelium and in the luminal contents in both adult and immature flukes. The adult-fluke antigen containing the 27,000-dalton substance is applicable as a standard antigen for diagnosis of paragonimiasis westermani in not only definitive hosts but also in paratenic hosts.     

Alternative transmission pathways for guinea worm in dogs: implications for outbreak risk and control

Guinea worm (Dracunculus medinensis) has exerted a high human health burden in parts of Africa. Complete eradication of Guinea worm disease (dracunculiasis) may be delayed by the circulation of the parasite in domestic dogs. As with humans, dogs acquire the parasite by directly ingesting infected copepods, and recent evidence suggests that consuming frogs that ingested infected copepods as tadpoles may be a viable transmission route (paratenic route). To understand the relative contributions of direct and paratenic transmission routes, we developed a mathematical model that describes transmission of Guinea worm between dogs, copepods and frogs. We explored how the parasite basic reproductive number (R₀) depends on parameters amenable to actionable interventions under three scenarios: frogs/tadpoles do not consume copepods; tadpoles consume copepods but frogs do not contribute to transmission; and frogs are paratenic hosts. We found a non-monotonic relationship between the number of dogs and R₀. Generally, frogs can contribute to disease control by removing infected copepods from the waterbody even when paratenic transmission can occur. However, paratenic transmission could play an important role in maintaining the parasite when direct transmission is reduced by interventions focused on reducing copepod ingestion by dogs. Together, these suggest that the most effective intervention strategies may be those which focus on the reduction of copepods, as this reduces outbreak potential irrespective of the importance of the paratenic route.     

Studies on the in vitro effects of bithionol and menichlopholan on flukes of Clonorchis sinensis, Metagonimus takahashii and Paragonimus miyazakii.

The present study was carried out in order to obtain information on the action of bithionol and menichlopholan on Clonorchis sinensis, Metagonimus takahashii and Paragonimus miyazakii. The drugs inhibited the motility of the newly excysted juveniles and adult worms in vitro. Moreover, the in vitro treatment slightly decreased lactate dehydrogenase activity in homogenates of adult worms. The drug treatments of adult worms in vitro also inhibited the activity of fumarate reductase, malic enzyme, phosphogluconate dehydrogenase and succinate dehydrogenase in the homogenate. In addition to this, these drugs caused morphological alterations in the ultrastructure of the tegument of the flukes.     

Molecular identification and larval morphological description of Contracaecum pelagicum (Nematoda: Anisakidae) from the anchovy Engraulis anchoita (Engraulidae) and fish-eating birds from the Argentine North Patagonian Sea

Anisakids use invertebrates as paratenic and/or intermediate hosts as a basic feature of larval transmission. The third-stage larva usually develops in invertebrates which are prey items of finfish paratenic hosts. Contracaecum larvae molt twice inside the egg and hatch as free third-stage larvae ensheathed in the second-stage larval cuticle. Copepods act as paratenic or obligatory hosts, usually ingesting these free L3 larvae, and fish act as intermediate/paratenic or metaparatenic hosts preying on infected copepods. Fish-eating birds acquire L3 larvae by ingesting infected fish where they develop into the fourth-stage larvae and adults. Objectives of this work were to establish the specific correspondence between Contracaecum pelagicum L3 larvae parasitizing the anchovy Engraulis anchoita, and the adults parasitizing the Magellanic penguin Spheniscus magellanicus and the Imperial shag Phalacrocorax atriceps through the use of molecular markers; and, to evaluate the anisakid L3 larval recruitment and infection caused by ingestion of anchovy by S. magellanicus. Sixteen specimens of Contracaecum L3 larvae were analyzed from E. anchoita from Bahía Engaño, Chubut, eight adult nematodes from S. magellanicus and six adult specimens from P. atriceps both from the Valdés Peninsula, Chubut. All nematodes were sequenced for three genes: mitochondrial cytochrome oxidase 2 (mtDNA cox2), mitochondrial ribosomal RNA (rrnS), and the internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA region. Phylogenetic analyses were performed by using Maximum Parsimony (MP) analysis by PAUP. In addition, studies under SEM and LM were carried out on L3 larvae. All L3 individuals from E. anchoita, adults from S. magellanicus, and P. atriceps clustered in the same clade, well supported in the MP tree inferred from the mtDNA cox2, and rrnS gene sequences analyses. Further, the sequence alignments of L3 larvae and adults of C. pelagicum here obtained at the ITS-1 and ITS-2 regions of the rDNA matched the sequences of C. pelagicum previously deposited by us in GenBank. Nematode recruitment (R_o) was equal to 33.07 (7.20–91.14) L3 larvae for C. pelagicum in each penguin's meal of anchovy. The MP tree topologies obtained from mtDNA cox2 and rrnS genes demonstrated that specimens of Contracaecum L3 larvae from E. anchoita and C. pelagicum from S. magellanicus as well as from P. atriceps constitute a unique clade, well-distinct and supported from all the others formed by the Contracaecum spp. sequenced so far for these genes. Molecular markers are considered to be an effective tool to elucidate larval transmission. The Contracaecum L3 larval recruitment value showed that many worms fail to establish in the bird digestive tract, probably because they are below a critical size. Further work is needed to elucidate other factors (e.g., physiological, immunological) that control nematode populations in the penguin digestive tract.     

Dependence of the structure of the capsule surrounding the acanthocephalan Corynosoma strumosum on the species of its natural paratenic host

The fine and ultrafine structure of the capsules surrounding the acanthocephalan Corynosoma strumosum in natural paratenic hosts of three species—the whitespotted greenling Hexagrammos stelleri, Steller’s sculpin Myoxocephalus stelleri, and the saffron cod Eleginus gracilis—was studied. The results of this study, together with earlier data, support the hypothesis that the structure of the capsule depends on the paratenic host species. Three types of capsules differing in the ratio of fibroblasts and inflammatory cells have been identified: fibroblastic (in Japanese smelt, rainbow smelt, and saffron cod), intermediate (in whitespotted greenling), and leukocytic (in Steller’s sculpin and the yellowfin sole). It was assumed that the structure of the capsule is determined by the degree of mutual adaptation of organisms in a given host-parasite system.     

Molecular identification of Paragonimus species by DNA pyrosequencing technology

DNA pyrosequencing for PCR amplicons is an attractive strategy for the identification of microorganisms because of its short time performance for large number of samples. In this study, the primers targeting the fragment of ITS2 region of nuclear ribosomal RNA gene were newly developed for pyrosequencing-based identification of 6 Paragonimus species, Paragonimus bangkokensis, Paragonimus harinasutai, Paragonimus heterotremus, Paragonimus macrorchis, Paragonimus siamensis and Paragonimus westermani. Pyrosequencing determination of 39 nucleotides of partial ITS2 region could discriminate 6 Paragonimus species, and could also detect intra-species genetic variation of P. macrorchis. This DNA pyrosequencing-based identification can be a valuable tool to improve species-level identification of Paragonimus in the endemic areas.     

Paragonimus paishuihoensis Metacercariae in Freshwater Crabs,Potamon lipkei,in Vientiane Province,Lao PDR

Among Paragonimus species, P. paishuihoensis is one of the most mysterious and poorly understood species. Metacercariae are characterized by having a unique dendritically branched excretory bladder. However, the morphology of the adult worm remains unknown. To date, metacercariae of this species have been reported only in China and Thailand. In this study, we first found P. paishuihoensis metacercariae in freshwater crabs, Potamon lipkei, in Hinheub District, Vientiane, Lao PDR, with a prevalence of 77.7% and the average intensity of 10.3 (range 1-28) metacercariae per crab. The molecular data based on ITS2 and CO1 markers indicated that P. paishuihoensis from Laos and Thailand were almost completely identical and were close to members of the Paragonimus bangkokensis/Paragonimus harinasutai complex. Attempts to infect experimental animals (cats, dogs, and rats) with P. paishuihoensis were unsuccessful, suggesting that these animals might be unsuitable definitive hosts for the species. Further studies are necessary to elucidate the taxonomic status and life cycle of P. paishuihoensis.     

To grow or not to grow? Intermediate and paratenic hosts as helminth life cycle strategies

Larval helminths in intermediate hosts often stop growing long before their growth is limited by host resources, and do not grow at all in paratenic hosts. We develop our model [Ball, M.A., Parker, G.A., Chubb, J.C., 2008. The evolution of complex life cycles when parasite mortality is size- or time-dependent. J. Theor. Biol. 253, 202-214] for optimal growth arrest at larval maturity (GALM) in trophically transmitted helminths. This model assumes that on entering an intermediate host, larval death rate initially has both time- (or size-) dependent and time-constant components, the former increasing as the larva grows. At GALM, mortality changes to a new and constant rate in which the size-dependent component is proportional to that immediately before GALM. Mortality then remains constant until death or transmission to the definitive host. We analyse linear increasing and accelerating forms for time-dependent mortality to deduce why there is sometimes growth (intermediate hosts) and sometimes no growth (paratenic hosts). Calling i the intermediate or paratenic host, and j the definitive host, conditions favouring paratenicity are: (i) high values in host i for size at establishment, size-related mortality, expected intensity, (ii) low values in host i for size-independent mortality rate, potential growth rate, transmission rate to j, and ratio of death rate in j/growth rate in j. Opposite conditions favour growth in the (intermediate) host, either to GALM or until death without GALM. We offer circumstantial evidence from the literature supporting some of these predictions. In certain conditions, two of the three possible growth strategies (no growth; growth to an optimal size then growth arrest (GALM); unlimited growth until larval death) can exist as local optima. The effect of the discontinuity in death rate after GALM is complex and depends on mortality and growth parameters in the two hosts, and on the mortality functions before and after GALM.     

The first report of sparganosis (Spirometra sp.) in Eurasian badger (Meles meles)

Spirometra sp. is a diphyllobothriid cestode which reproduces mainly in cat-like carnivores and canids. Several animal species that are not definitive hosts function as paratenic hosts, in which plerocercoids migrate to other tissues causing sparganosis. In this paper we describe the first case of sparganosis (Spirometra sp.) in Eurasian badger (Meles meles). It was found in an adult female Eurasian badger killed on the road in the Białowieża Primeval Forest (north-eastern Poland) in April 2013. At necropsy, 128 complete and 40 fragments of plerocercoids (spargana) were found and were located subcutaneously, mainly on the hind legs and along the spine. The average length of spargana was 87 ± 38 mm. No adult Spirometra sp. tapeworms were found in the animal intestine, indicating that the investigated badger was a paratenic host for Spirometra sp. Analysis of 18S rRNA gene sequences combined with morphological examination confirmed affiliation of the concerned plerocercoids to genus Spirometra.     

Postcyclic transmission and its effect on the distribution of Paulisentis missouriensis (Acanthocephala) in the definitive host Semotilus atromaculatus

The relationship of fish age class to parasitism by Paulisentis missouriensis was determined by sampling at least 29 creek chubs, Semotilus atromaculatus, from Easly Creek, Richardson County, Nebraska, every month from February 1996 to March 1997. In general, mean abundance and prevalence of the acanthocephalans increased with the age or length of chubs. It is unlikely that this distribution is explained by increased consumption of intermediate hosts by older, larger fish or by predatory fish acquiring parasites from paratenic hosts. The intermediate host for P. missouriensis is the cyclopoid copepod Acanthocyclops robustus, and creek chubs do not consume more microscopic crustaceans as they age or grow. Instead, the percentage of fish in the diet of creek chubs increases. Furthermore, P. missouriensis apparently does not use paratenic hosts. In laboratory infections, P. missouriensis survived predation of its original definitive host and transferred to the predator. Postcyclically transmitted P. missouriensis survived at least 14 days in the intestine of creek chubs, where they localized around the first flexure beyond the stomach. All stages of development of both sexes were transferred successfully. Postcyclic transmission is a plausible explanation, in some cases, for the greater worm burden frequently observed in older, larger hosts and for the occurrence in top carnivores of parasites not known to have paratenic hosts. This method of transmission appears to result in distribution of acanthocephalans to groups of animals that otherwise would be inaccessible.     

A molecular perspective on the genera Paragonimus Braun, Euparagonimus Chen and Pagumogonimus Chen

The status of the genera Euparagonimus Chen, 1963 and Pagumogonimus Chen, 1963 relative to Paragonimus Braun, 1899 was investigated using DNA sequences from the mitochondrial cytochrome c oxidase subunit I (CO1) gene (partial) and the nuclear ribosomal DNA second internal transcribed spacer (ITS2). In the phylogenetic trees constructed, the genus Pagumogonimus is clearly not monophyletic and therefore not a natural taxon. Indeed, the type species of Pagumogonimus, P. skrjabini from China, is very closely related to Paragonimus miyazakii from Japan. The status of Euparagonimus is less obvious. Euparagonimus cenocopiosus lies distant from other lungflukes included in the analysis. It can be placed as sister to Paragonimus in some analyses and falls within the genus in others. A recently published morphological study placed E. cenocopiosus within the genus Paragonimus and probably this is where it should remain.     

Natural hybridization between Paragonimus bangkokensis and Paragonimus harinasutai

Paragonimus bangkokensis and Paragonimus harinasutai, which are morphologically distinguishable species, often co-infect in the same crab intermediate hosts. Recent molecular phylogenetic analyses revealed that these two species are genetically close to each other and are considered as the sister species. While we have been studying Paragonimus adult worms obtained from the lungs of a cat experimentally infected with Paragonimus metacercariae which were morphologically identified as P. harinasutai collected from central Viet Nam, one out of 6 adult worms has grouped cuticular spines, which is a feature of P. bangkokensis. By molecular analyses, the CO1 sequence of this specimen was identical with that of P. bangkokensis, but the ITS2 and the D2 region of 28S rDNA sequences showed a two peak pattern. Then, PCR products of the ITS2 and the D2 region of 28S rDNA sequences were ligated to TOPO vector and subcloned to determine the heterozygosity. Two types of sequences were obtained from each ITS2 and D2 region of 28S; one was identical with P. harinasutai and the other with P. bangkokensis. Taking all these morphological and molecular data together, we identified this adult worm as a hybrid specimen of P. bangkokensis and P. harinasutai.     

Comparative ultrastructural topography of the gut epithelia of the lung fluke Paragonimus (Trematoda: Troglotrematidae)

The gut epithelia in the species of Paragonimus known in Japan, P. westermani, P. ohirai, P. iloktsuenemis, P. miyazakii, and P. sadoensis, were observed with the scanning electron microscope. Transmission electron microscopy was employed to substantiate and clarify the results of these topographic studies. Morphological differences in the cytoplasmic projections of the gut epithelial surface were found to be present among some species of Paragonimus. The cytoplasmic projections in this group are of a sheet-like or broad lamellar type, although fine digitiform type projections have never been described in other trematodes.     

斯氏并殖吸虫染色体核型分析

最早研究并殖吸虫染色体的是陈品芝(Chea,1937)系采用石蜡切片法,观察到克氏并殖吸虫(P.kellicotti)染色体2n=16,n=8,但未提到其染色体核型。以后近四十年中,未见有关并殖吸虫染色体的报道。而这些年来,国内外并殖吸虫的分类学研究却有了蓬勃的开展,并殖吸虫新种以及对人体致病的虫种不断发现。分类学研究主要以     

并殖吸虫成虫形态结构特征排序

对9种并殖吸虫成虫形态特征进行主成分分析及聚类分析,结果表明:在并殖吸虫成虫的6项主要特征中,体形因素及卵巢类型是重要的分类依据。在被分析的9个种中,怡乐村并殖吸虫、斯氏狸殖吸虫、巨睾狸殖吸虫、三平正并殖吸虫、异盘并殖吸虫、陈氏并殖吸虫与卫氏并殖吸虫7个种具有种的独立地位,而泡囊狸殖吸虫和扁囊并殖吸虫未能分别从斯氏狸殖吸虫和卫氏并殖吸虫中分开。     

云南省威信县24例肺吸虫病病原追踪调查

目的对云南省威信县肺吸虫病病原进行疫点追踪调查。方法收集2008～2011年确诊的来自威信县的肺吸虫病患者24例,根据病例追踪,自当地疫点采集溪蟹,带回实验室检查并殖吸虫囊蚴,鉴定虫种。结果 24例患者中游走性包块11例,占45.83%,胸腔积液型11例,占45.83%,脑型1例,占4.16%,睾丸鞘膜积液1例,占4.16%。患者主要来自扎西镇、长安乡和双河乡。检查溪蟹278只,斯氏并殖吸虫囊蚴感染率为34.17%(95/278),其中扎西镇溪蟹囊蚴感染率为33.08%(43/130),平均感染度11.63(500/43),长安村溪蟹囊蚴感染率为35.14%(52/148),平均感染度8.98(467/52)。结论云南威信县是肺吸虫病疫源地,第二中间宿主为锯齿华溪蟹,致病虫种为斯氏并殖吸虫,应加强当地肺吸虫病防治。