Tegument and apical end organ fine structure in the metacestode and adult Proteocephalus ambloplitis

The tegument of plerocercoid and adult P. ambloplitis was examined. Differences in tegument structure existed between these two stages. Plerocercoids of P. ambloplitis lacked extensive vacuolization and unicellular gland cells characteristic of adult tegument. Plerocercoid microtriches were short and conoid; adult microtriches were lenticular with an extended, whip-like shaft. An inclusion, not previously reported from proteocephalid cestodes, is described. Adult tegument had ducts, originating from underlying unicellular glands, extending through the distal cytoplasm and opening to the exterior between microtriches.

The apical end organ cavity of P. ambloplitis contained numerous labyrinth-like spherical bodies. These structures appeared to be synthesized and secreted into the end organ by a thin cellular lining of the end organ. This lining was composed of discrete, filamentous cells believed to be modified subtegumental cell bodies. Spherical structures identical to those observed within the end organ cavity occurred within this cellular lining. The spherical bodies may be associated with enzymes necessary for tissue migration by the metacestode.     

Microtriches of tetraphyllidean metacestodes from Western Mediterranean striped dolphins (Stenella coeruleoalba)

The tegumental structures of two types of tetraphyllidean plerocercoids and two types of merocercoids (Phyllobothrium delphini and Monorygma grimaldii) from Mediterranean striped dolphins, Stenella coeruleoalba, are described for the first time using scanning electron microscopy. The tegument of all of the specimens was fully covered with microtriches. Four basic types were found: filitriches, blade-like spinitriches of different sizes and shapes, cone-shaped spinitriches (with two parallel small projections of equal length at the apex), and crowned cylindrical spinitriches (with 6-7 small papillae forming a crown at the apex); the two latter types are newly described. The two types of plerocercoids had a similar morphology and distribution of microtriches; in addition, cilium-like projections appeared interspersed among the microtriches on the apical sucker, accessory suckers, and distal bothridial loculus. Merocercoids exhibited a greater variety of tegumental structures, especially on the distal bothridial loculus. Both merocercoid types had regularly spaced papillae or "buttons" on the accessory suckers and the distal bothridial loculus that were composed of a central cilium-like projection surrounded by numerous filitriches. However, crowned cylindrical spinitriches were specific to P. delphini and cone-shaped spinitriches were specific to M. grimaldii. Differences in the morphology and distribution of scolex microtriches of adult cestodes have been considered useful for species identification. A previous molecular study has suggested that P. delphini and M. grimaldii are actually different congeneric species. Our study has shown that significant differences in the morphology and distribution of microtriches occur between these species at the merocercoid stage.     

A new method for concentration of proteins in the calcareous corpuscles separated from the spargana of Spirometra erinacei

Calcareous corpuscles are a characteristic structure found in larval and adult stage cestodes. These corpuscles are known to contain several protein components and to possess protein-binding activity. However, the proteins bound to calcareous corpuscles in situ have not been studied. The present study was undertaken to identify the proteins on calcareous corpuscles. Calcareous corpuscles were purified from the plerocercoids (= spargana) of Spirometra erinacei, and serially dissolved using 0.1 M sulfamic acid solution. Collected supernatants were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The results showed that only the fraction remaining after the 19th dissolved fraction contained proteins. A total of 20 protein molecules were detected in gel, with major bands at 56, 53, 46, 40, 35, 29, 28, 24.5, 21, 19, 16, 13, 10 and 8 kDa. In particular, the proteins corresponding to the 21 and 16 kDa bands were most abundant. Our results demonstrated for the first time the protein contents of the calcareous corpuscles of spargana. Further studies on the functions of these proteins are required.     

Schistocephalus solidus and Ligula intestinalis: Pinocytosis by the tegument

Using ruthenium red as a macromolecule, endocytosis was demonstrated in the plerocercoid of Ligula intestinalis and adult Schistocephalus solidus. Uptake, transport across the tegument, and exocytosis across the basal plasma membrane occurred within 6 min. The types of vesicles in the tegument of L. intestinalis are redescribed and their former classification is modified. The vertical and longitudinal distribution of pinosomes in the tegument of adult S. solidus and L. intestinalis plerocercoids were determined. The possible role of macromolecular uptake in the economy of pseudophyllidean tapeworms is discussed with particular reference to growth and defence of an unencysted larval stage in the tissues of the intermediate host.     

Genetic Identification of Spirometra decipiens Plerocercoids in Terrestrial Snakes from Korea and China

Human sparganosis is a zoonotic disease caused by infection with larval forms (procercoid/plerocercoid) of Spirometra spp. The purpose of this study was to identify Spirometra spp. of infected snakes using a multiplex PCR assay and phylogenetic analysis of mitochondrial DNA sequence data from the spargana of terrestrial snakes obtained from Korea and China. A total of 283 snakes were obtained that included 4 species of Colubridae comprising Rhabdophis tigrinus tigrinus (n=150), Dinodon rufozonatum rufozonatum (n=64), Elaphe davidi (n=2), and Elaphe schrenkii (n=7), and 1 species of Viperidae, Agkistrodon saxatilis (n=60). The snakes were collected from the provinces of Chungbuk, Chungnam, and Gyeongbuk in Korea (n=161), and from China (n=122). The overall infection rate with spargana was 83% (235/283). The highest was recorded for D. rufozonatum rufozonatum (100%), followed by A. saxatilis (85%) and R. tigrinus tigrinus (80%), with a negative result for E. davidi (0%) and E. schrenkii (0%). The sequence identities between the spargana from snakes (n=50) and Spirometra erinaceieuropaei ({"type":"entrez-nucleotide","attrs":{"text":"KJ599680","term_id":"646228164","term_text":"KJ599680"}}KJ599680) or S. decipiens ({"type":"entrez-nucleotide","attrs":{"text":"KJ599679","term_id":"646228151","term_text":"KJ599679"}}KJ599679) control specimens were 90.8% and 99.2%, respectively. Pairwise genetic distances between spargana (n=50) and S. decipiens ranged from 0.0080 to 0.0107, while those between spargana and S. erinaceieuropaei ranged from 0.1070 to 0.1096. In this study, all of the 904 spargana analyzed were identified as S. decipiens either by a multiplex PCR assay (n=854) or mitochondrial cox1 sequence analysis (n=50).     

Development of the plerocercoid I of Ophiotaenia europaea in reptiles

The existence of a two-host life-cycle in ophiotaeniid proteocephalideans was tested experimentally using Ophiotaenia europaea as a model. Three species of reptiles, Natrix natrix, Natrix tessellata and Lacerta viridis, were fed with experimentally infected copepods containing a large number of infective plerocercoids I. A few plerocercoids, most of which were dead, corresponding morphologically to the plerocercoid II developmental stage of O. europaea, were found encysted in the intestinal wall of N. natrix (8 days p.i.), N. tessellata (5 and 150 days p.i.) and L. viridis (40 days p.i.), while no plerocercoids or adult worms were recovered from their intestines. The results indicate that the infective plerocercoid I of O. europaea cannot undergo further development when ingested directly by the final host (a reptile), and that environmental temperature stimuli cannot initiate a reverse plerocercoid migration to the gut followed by strobilization.     

Ligula intestinalis (Cestoda: Pseudophyllidae): Studies on the properties of proteolytic and protease inhibitor activities of plerocercoid larvae

The somatic extract of L. intestinalis plerocercoids reveals hydrolytic activity against N-Benzoyl-l-tyrosine ethyl ester (BTEE) and Azocoll, and inactivates the esterolysis by mammalian trypsin and chymotripsin. The proteolytic enzyme activity and the inhibitory effect were completely separated by Sephadex G-100 column chromatography. Gel chromatography of the somatic extract revealed two peaks of proteolytic activity : one is bound to macromolecular substances, the other appears to be in free form and has a molecular weight of approx 60,000–65,000. The proteolytic activity showed the following characteristics : Tris-HCl buffer provided the highest activity against BTEE, the pH optimum was 7·4–7·8; the enzyme was activated by 10^?5m-Ca²⁺, Mg²⁺ or Mn²⁺, it was inhibited by 10^?5m-Cu²⁺, but not by 10^?5m-Zn²⁺. 0.001% soybean trypsin inhibitor, 2 × 10^?3m-EDTA, 1 mm-tosyl-l-phenylalanyl chloromethane, 1000 KIU/ml Trasylol did not inhibit the proteolytic activity, but it was inhibited by 1 mm-phenylmethyl-sulphonyl fluoride. The enzyme activity completely ceased upon 5 % TCA treatment or incubation at 56°C for 30 min. The trypsin and chyrnotrypsin inhibitor activities were eluted from the Sephadex G-100 column in a single peak with an estimated molecular weight of 6700–7200. The inhibitory effect was not sensitive to pH changes, and treatment by 5% TCA or incubation at 80°C for 15 min was ineffective. The proteolytic activity of plerocercoid extract was not effected ‘in vitro’ by the inhibitors isolated from this parasite.     

Partial purification and characterization of a cysteine protease inhibitor from the plerocercoid of Spirometra erinacei

Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.     

Comparison of surface topography of three species of Diphyllobothrium (Cestoda,pseudophyllidea) by scanning electron microscopy

Plerocercoids of different sizes as well as adult worms of D. dendriticum, D. latum and D. ditremum were studied using scanning electron microscopy (S.E.M.). In the plerocercoids there were found distinct differences in appearance and length of microtriches between these three species, while the microtriches of adult worms were more similar. A regional difference in microtrix appearance was found in the larvae of D. ditremum and D. dendriticum. This was not apparent with S.E.M. in adult worms. The length of ‘body’ microtriches in D. dendriticum varied with the length of the larvae. The topography of the genital atrium of mature and gravid proglottids in adult worms of these three species is also described.     

Mass Death of Predatory Carp,Chanodichthys erythropterus,Induced by Plerocercoid Larvae of Ligula intestinalis (Cestoda: Diphyllobothriidae)

We describe here the mass death of predatory carp, Chanodichthys erythropterus, in Korea induced by plerocercoid larvae of Ligula intestinalis as a result of host manipulation. The carcasses of fish with ligulid larvae were first found in the river-edge areas of Chilgok-bo in Nakdong-gang (River), Korea at early February 2016. This ecological phenomena also occurred in the adjacent areas of 3 dams of Nakdong-gang, i.e., Gangjeong-bo, Dalseong-bo, and Hapcheon-Changnyeong-bo. Total 1,173 fish carcasses were collected from the 4 regions. To examine the cause of death, we captured 10 wondering carp in the river-edge areas of Hapcheon-Changnyeong-bo with a landing net. They were 24.0-28.5 cm in length and 147-257 g in weight, and had 2-11 plerocercoid larvae in the abdominal cavity. Their digestive organs were slender and empty, and reproductive organs were not observed at all. The plerocercoid larvae occupied almost all spaces of the abdominal cavity under the air bladders. The proportion of larvae per fish was 14.6-32.1% of body weight. The larvae were ivory-white, 21.5-63.0 cm long, and 6.0-13.8 g in weight. We suggest that the preference for the river-edge in infected fish during winter is a modified behavioral response by host manipulation of the tapeworm larvae. The life cycle of this tapeworm seems to be successfully continued as the infected fish can be easily eaten by avian definitive hosts.