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Endo-parasites of the bathypelagic king of herrings Regalecus glesne and oarfish Regalecus russelii are only known from few specimens opportunistically examined. As a consequence, there are few records of parasites from either Regalecus species. We report plerocercoid larvae of phyllobothriidean cestodes parasitizing an adult R. glesne stranded in Bahia de La Paz, Baja California Sur, Mexico. Sixty-three plerocercoids were obtained from the intestine of R. glesne and characterized using morphological and molecular methods (nuclear 28S rDNA and mitochondrial cytochrome c oxidase I gene sequences). Following the morphological diagnostic criteria of scolex and muscle bands in the strobila, plerocercoids specimens were preliminary assigned to the genus Clistobothrium. Mitochondrial and nuclear DNA sequences indicate these plerocercoids correspond to Clistobothrium montaukensis Ruhnke, 1993. Regalecus glesne is a new host known for C. montaukensis and this report is a new geographical record of C. montaukensis parasitizing species of the genus Regalecus previously known only from California and Florida, USA.

This is the first acute-intensity record of helminths parasitizing the subtropical krill Nyctiphanes simplex Hansen, 1911. We briefly describe the pathology of infection of Phyllobothriidae gen. sp. plerocercoids parasitizing N. simplex in the Gulf of California. Infection occurred with a very low prevalence (P = 0.06%, n = 1563 specimens), although acute-intensity exceeded several hundred plerocercoids crowding the hemocoel in one female host. Nyctiphanes simplex showed inflammatory response of hemocyte-based infiltration, nodule formation, and presumptive melanization. Remarkably, cestodes invade and supplant the gonad, causing atretic oocytes and severe tissue destruction in the gonad likely leading to castration and cell death in connective tissue of the infected organs suggesting that acute-intensity infection exceeds the krill's reaction capacity. Thus, Phyllobothriidae gen. sp. negatively affects the host by depleting its fitness, leading to total castration to prevent/block host reproduction.

The sensory organs in tegument of two trypanorhynchean species--Nybelinia surmenicola (plerocercoid) and adult Parachristianella sp. (Cestoda, Trypanorhyncha)--were studied with the aim of ultrastructural description and a comparative analysis. The Nybelinia surmenicola plerocercoid lacks papillae with sensory cilia on the bothria adhesive surface. We found an unciliated sensory organ within the median bothria fold. This unciliated free nerve ending contains the central electron-dense disc, three dense supporting rings, and broad root. The nerve ending locates in the basal matrix under the tegument. The tegument of N. surmenicola has a number of ultrastructural features which make it significantly different from other Trypanorhyncha: (i) the tegumental cytoplasm has a plicated constitution in a form of high apical and deep basal folds, (ii) numerous layers of the basal matrix are presented in the subtegument, and (iii) the squamiform and bristlelike microtriches N. surmenicola lack the base and the basal plate. In contrast, numerous ciliated and unciliated receptors were found in Parachristianella sp.: six types on the bothria and one type in the strobila tegument. Ultrastructural constitution of sensory organs in the form of ciliated free nerve endings as well as unciliated basal nerve endings of Parachristianella sp. has many common features inside Eucestoda. In comparison with other Trypanorhyncha, all Nybelinia species studied have less quantity of the bothrial sensory organs. This fact may reflect behavioral patterns of Nybelinia as well as phylogenetic position into Trypanorhyncha. Our observations of living animals conventionally demonstrate the ability of N. surmenicola plerocercoids to locomote in forward direction on the Petri dish surface. The participation of the bothrial microtriches in a parasite movement has been discussed.

The Spirometra erinacei casein kinase I (SeCKI) gene was cloned and expressed in Escherichia coli, and its characteristics were investigated in this study. The recombinant SeCP protein (rSeCKI) was purified. The vaccination of mice with rSeCKI induced the Th1/Th2-mixed type of immune response with Th2 predominant (high levels of IgG1). Western blotting analysis showed that rSeCP was recognized by the sera of plerocercoid-infected mice, and anti-rSeCP serum recognized the native SeCP protein of plerocercoid crude antigens. Transcription and expression of SeCP was observed at the plerocercoid and adult stages of S. erinacei. Immunolocalization identified SeCKI in the tegument and parenchymal tissues of plerocercoids and in the teguments of adults. SeCKI appeared to be essential indispensable for the S. erinacei development and survival in host, but its biological functions need to be further investigated.

Spirometra erinacei is a pseudophyllidean tapeworm which inhabits the intestines of cats and dogs. The infections are usually asymptomatic in these animals, but the infection of the plerocercoid larvae of the parasite, spargana, cause sparganosis in other vertebrates, including human. In this study, we identified a gene encoding the copper/zinc-superoxide dismutase of S. erinacei (SeCuZn-SOD) and partially characterized the biochemical and functional properties of the enzyme. The open reading frame of SeCuZnSOD was 465 bp that encodes 154 amino acids. The characteristic amino acid residues and motifs required for coordinating copper and zinc enzymatic function were well conserved. The genomic length of the SeCuZnSOD was 1,985 bp consisting of three exons that are separated by two introns. SeCuZnSOD is a typical cytosolic form which shares similar biochemical properties, including broad pH optima and inhibition profile by KCN and H(2)O(2), with cytosolic Cu/Zn-SODs of other organisms. SeCuZnSOD was functionally expressed in both S. erinacei plerocercoid larvae and adult worms, and its expression level was significantly increased when the plerocercoid larvae were treated with paraquat. The enzyme may play essential roles for survival of the parasite not only by protecting itself from endogenous oxidative stress, but also by detoxifying oxidative killing of the parasite by host immune effector cells.

This study was undertaken to identify genes involved in the growth and development of Spirometra erinacei larvae, an intestinal tapeworm of cats and dogs, within the final host. The differential protein expression at three different stages of S. erinacei, the plerocercoid larvae, 8-day-old juveniles, and adults, was compared using two-dimensional electrophoresis. Specifically or highly expressed proteins in juvenile worms were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MS)/MS. The proteome map of larvae showed fewer protein spots than juveniles or adults, whereas juveniles or adults revealed a similar protein expression profile. Eight juvenile-specific and five upregulated proteins of juveniles were identified and matched to proteins of known biological functions. These were grouped into several categories of functionally related proteins: DNA/RNA metabolism, cell trafficking, cytoskeleton, protein processing and degradation, energy metabolism, and oxidative stress. Our results give an overview of the growth and development mechanisms of cestodes within the final host and extend our understanding of parasite biology in the host-parasite relationship.

Lake Kilpisjarvi was stocked with a total number of 440 000 migratory and benthic whitefish fry during the period of 1959–1964. Thereafter, the Diphyllobothrium ditremum plerocercoid infection became a problem decreasing the feasibility and commercial exploitation of the whitefish stock. Since dense fish populations may cause stunting, all stockings in the study area were stopped in an attempt to enhance the fisheries. The aim of the present study was to explore the effect of the cessation of the stocking on the growth, condition, catches and D. ditremum infection of the whitefish population. A total of 1594 whitefish were sampled with benthic gill nets. Growth was estimated from length-at-age and mass–length relationship was estimated with a non-linear regression in 1982–1983, 1992–1993 and 1997. Parasite infection was studied in 1992–1993 and 1997. Growth and condition were poor in the beginning of the 1980s. In 1992–1993, the growth and condition of the fish improved as the numerical catch per unit effort decreased from 1982 to 1992. The mean catches of other fish species were low. There were no significant changes in whitefish growth or condition from 1992–1993 to 1997. From the 1970s to 1982–1983, during slow growth and poor condition of the fish, the median gillraker count was decreasing to 19 (min–max 14–26). In 1992–1993, the gillraker count had increased significantly to 23 (min–max 19–26). However, the improved growth and condition did not cause decrease in the D. ditremum burden. On the contrary, based on our own results and literature notes, the parasite abundance seems to increase during the observation period of 1964–1997.

Museum specimens of Ligula (Pseudophyllidea, Ligulidae), a fish parasite tapeworm, that have been preserved in ethanol or fixed permanently in formalin up to 24 years were used for DNA extraction and molecular characterization. DNA was amplified via PCR from samples collected from different fish hosts that lived in both salt and fresh water bodies in the Chinese Qinghai–Tibet Plateau, Russia, and England. Phylogenetic analyses based on partial nucleotide sequences of the 5"-end of nuclear 28S rRNA gene and the mitochondrial cytochrome c oxidase subunit I (COI) gene support the morphologically based taxonomy that groups the Chinese Ligula within the same species as their Europe counterpart: Lingula intestinalis. No nucleotide variation was detected in either the 28S rRNA gene or the COI gene among the seven plerocercoid samples, suggesting a considerable genetic homogeneity among Ligula from different regions. Our results show that geographic isolation, affinity of hosts, and host habitats are not reliable taxonomic criteria for Ligula classification. Our data also indicate a low genetic diversity in the Ligula prevalent in China and in Europe. Our experiments demonstrate that endogenous DNA from specimens that were subjected to permanent formalin fixation can be routinely amplified from parasitic tapeworms, suggesting that fixation time in formalin may not be a critical factor affecting DNA degradation in such museum specimens.

The distribution of actin filaments in all developmental stages of the tapeworm Diphyllobothrium dendriticum was studied. It is the first investigation of the placement of microfilaments during the development of a flatworm, and the results show that actin filaments, in all developmental stages, can be found in the subtegument and the flame cells. Muscle fibers possibly corresponding to the longitudinal, transversal, and dorsoventral muscles of the adult and plerocercoid were already detected in the procercoid. Concerning the adult worm, a new set of longitudinal fibers in the peripheral parts of the adult proglottid was found. The ducts of the protonephridial system and the vitellarias were seen to be surrounded by longitudinally oriented actin filaments, while the uterine ducts and the vagina were encircled by microfilaments. Prominent layers of circular muscle fibers surrounded the cirrus and the seminal vesicle, and radial fibers were also detected. Areas faintly stained with TRITC-phalloidin were found in the developing germ cells, the cells of the genital anlage, vitelline cells, the tegument, and the main nerve cords. None of these structures were autofluorescent, which is also true concerning the intensively labeled oncospheral hooks.

The species of the pseudophyllidean genus Bothriocephalus Rudolphi, 1808 parasitising freshwater fishes in America are revised, based on the examination of type and voucher specimens of seven taxa. There are five valid species: Bothriocephalus claviceps (Goeze, 1782), B. cuspidatus Cooper, 1917, B. formosus Mueller & Van Cleave, 1932, B. acheilognathi Yamaguti, 1934, and B. pearsei Scholz, Vargas-Vazquez & Moravec, 1996. B. texomensis Self, 1954 from Hiodon alosoides in the USA, and B. musculosus Baer, 1937 from a cichlid Cichlasoma biocellatum (= C. octofasciatum) which died in an aquarium in Switzerland, are synonymised with B. cuspidatus. B. schilbeodis Cheng & James, 1960 from Schilbeodes insignis in the USA, B. speciosus (Leidy, 1858) Leidy, 1872 from Boleostoma olmstedi in the USA, and B. cestus Leidy, 1885 from Salvelinus sp. in Canada are considered to be species inquirendae until new material for the evaluation of their taxonomic status is available. B. cordiceps (Leidy, 1872) from Salmo (= Salvelinus) fontinalis in North America is in fact a larva (plerocercoid) of a Diphyllobothrium species. The study showed that there have been many misidentifications, mostly of B. cuspidatus erroneously designated as B. formosus or B. claviceps. The five valid species are redescribed and illustrated, with emphasis on scolex morphology. The distribution of individual taxa and the spectrum of their definitive hosts are briefly reviewed and a key facilitating identification of individual species is also provided.

A comparative study of some morphological (segment number, scolex morphology and biometry, length and weight) and biological (maturation in different hosts) features ofSchistocephalus solidus plerocercoids and adults from different geographical regions (Baltic Sea and the British Isles) was carried out. The length of the plerocercoids fromGasterosteus aculeatus was shown to be the variable that best correlated with segment number. A very clear bimodal distribution of segment numbers separated the majority of British and Baltic plerocercoids (British n=21, mean length 25.48, SD 5.63, range 14–34 mm; mean segment number 66.33, SD 8.68, range 51–86. Baltic n=30, mean length 33.23, SD 4.64, range 23–48 mm; mean segment number 117.27, SD 10.30, range 99–138). AdultS. solidus from the intestines of Baltic ringed sealsPhoca hispida botnica and from a Welsh cormorantPhalacrocorax carbo carbo were also compared, and a similar bimodal distribution of segment numbers was found (Baltic n=70, mean segment number 106.16, SD 10.60, range 77–136; Welsh n=98, mean segment number 73.13, SD 8.78, range 54–97). Neither the morphology nor measurements of the scolex from apical-view scanning electron microphotographs provided distinguishing features for taxonomic purposes. Of 580 adult worms from Baltic ringed seals only 2.9% were gravid, 2.1% from spring and 10.5% from autumn samples. By contrast, of 98 adults from the Welsh cormorant 46.7% were gravid. The proportion of gravid worms did not increase with increasing worm numbers in seals. Reasons for poor maturation are discussed. Plerocercoids of BritishS. solidus were fromleiurus (gymnurus) forms ofG. aculeatus, which were relatively small, whereas in the northern Baltic plerocercoids were fromsemiarmatus ortrachurus forms, which were larger. As segment number was definitively established during the growth of the plerocercoid in the stickleback, the hypothesis is proposed that segment number is a phenotypic variable related to stickleback length (size).

In cestode parasites, calcareous corpuscles are thought to be associated with a number of intracellular physiologies by regulating the trafficking of mineral components. We previously separated these particular components by Ficoll, and their binding proteins of 10 kDa and 35 kDa in the Spirometra mansoni plerocercoid (sparganum). In the present study, we purified a 10 kDa protein employing octyl-Sepharose CL-4B affinity chromatography. Anti-serum raised against the purified protein showed specific reactions to the calcareous corpuscles of the worm section by immunohistochemistry, and recognized the 10 kDa protein by immunoblotting.

To investigate the mechanism by which liver weight increases during plerocercoid infections as well as the possible existence of a hepatocyte-growth-factor (HGF)-like substance in the serum of mice infected with Spirometra erinacei plerocercoids, liver DNA synthesis was measured in vivo and in vitro. Infection with S. erinacei plerocercoids significantly stimulated DNA synthesis in mouse parenchymal hepatocytes prior to the increase in liver weight, at least partly by stimulating the induction of the salvage pathways of pyrimidine biosynthesis. Furthermore, infected mouse serum directly stimulated DNA synthesis in cultured mouse parenchymal hepatocytes. These results suggest that an HGF-like substance is present in the serum of mice infected with S. erinacei plerocercoids.

Young, male ICR mice were given tap water or distilled water containing 200 mg/l propylthiouracil (PTU) and were then infected with 10 plerocercoids of Spirometra erinacei to investigate the effect of plerocercoid infection on thyroid hormone in their hosts. Plerocercoid infection stimulated growth in PTU-induced hypothyroid mice as if they had never received PTU treatment: there were increases in weight in the liver, skeletal muscle, and spleen, as well as enhancement of the head and body length, in spite of a greater decrease in serum T4 levels than was observed in PTU-treated controls. Furthermore, the intact mice infected with plerocercoids showed a decrease in serum T4 levels as well as in the concentration of T4-binding globulin. These observations suggest that the growth stimulation and the decrease in concentrations of serum T4 and T4-binding globulin associated with plerocercoid infection in mice probably resulted from secretion of a growth hormone-like substance produced by plerocercoids of S. erinacei.

The excretory system ofDiphyllobothrium dendriticum was studied morphologically and functionally. In living plerocercoids, one of the large excretory ducts was injected with latex-based stain. The excretory system was found to consist of a peripheral network of ducts, a central system of two longitudinal ducts, communicating through transverse ducts, a capillary plexus in the scolex and the excretory bladder and pore in the tail. The central and peripheral duct systems were both found to communicate with the capillary plexus in the scolex. In the peripheral network the stain was mainly spread by those contractions of the plerocercoid, which start in the neck region and are propagated through the body. The flow in the central ducts was usually independent of the contractions. Occasional reverse contractions led to a rapid flush in the central ducts. From the peripheral and central ducts the stain was transported out through the excretory bladder and pore. The distributive role of the excretory system is discussed.

All enzymes of the tricarboxylic acid cycle are present in subcellular fractions of the plerocercoid of Ligula intestinalis but the low activity of aconitase and malate dehydrogenase in the forward direction suggests that the complete cycle may be of questionable importance to the parasite. As in other helminths and intertidal molluscs, succinate, formed via a partial reversal of the tricarboxylic acid cycle, is a major end-product of anaerobic glucose degradation.

Plerocercoids of Diphyllobothrium dendriticum from cisco (Coregonus artedii) and whitefish (C. clupeaformis) and of Diphyllobothrium latum from pike (Esox lucius) and walleye (Stizostedion vitreum) were collected at Quigly Lake, Manitoba. Adult tapeworms were obtained by experimental infection of hamsters (Mesocricetus auratus) and were used to verify species identity. Isozyme analysis showed no differences between adult and plerocercoid stages. The enzymes esterase and malate dehydrogenase differed between species. Comparison of DNA restriction fragment profiles showed species specific differences, as did Southern hybridization analysis using nematode ribosomal gene probes.

Two types of frontal glands were detected in plerocercoids ofDiphyllobothrium dendriticum and their fates during the development from plerocercoid to adult worm were followed. Type I—the green glands—dominate the scolex of the larvae. They are located posteriorly and peripherally in the scolex and in the cortical and medullary parenchyma of the neck. The glands empty into the frontal pit. Type II—the golden glands—have earlier eluded observation or been mingled with the green glands. The golden glands are confined to the scolex and are closely associated with the nervous system. The green glands are activated during the first day of cultivation in vitro and in vivo. Already after 12 h of cultivation most of the green gland material has been secreted. The process continues, rapidly emptying ducts and cells. The green glands disappear completely within three days. The golden glands remain, on the whole, unaltered throughout the maturation of the worm and still occur in the adult worm. The roles of the two glands are discussed.

The systematics ofNybelinia sp. is briefly presented. The percentage incidence and intensity of infection are seen to be high during colder months, and in the smallest and the largest fish. Both sexes are more or less equally susceptible to the infection. The histopathological change in the infected tissue is in the form of a fibrous connective tissue reaction to encapsulate the parasite. A curious mechanism seems to be involved in the formation of ‘Nybelinia cysts’ on the outer wall of the oesophagus. This aspect is discussed in detail.

The histology of plerocercoid ofPenetrocephalus sp. reveal that the body surface consists of tegument, basement membrane, epidermal longitudinal musculature, parenchymatic longitudinal musculature, transverse and dorsoventral muscle fibres. Three types of glands could be recognized from the scolex of the plerocercoid. The musculature of the plerocercoid consists of glycogen, acid, sulfated, neutral and carboxylated mucopolysac-charides: basic proteins containing tyrosine, S-H and S-S groups, protein bound amino groups, sulfhydril groups, glycoprotein and lipid.

The influence of different feeding levels on the growth rate and survival of three-spined sticklebacks (Gasterosteus aculeatus) parasitized by the plerocercoid(s) of Schistocephalus solidus was compared with that of non-parasitized sticklebacks on similar diets. Parasitized fish, on restricted diets, died before non-parasitized ones. The feeding rate required to maintain total body weight (maintenance diet level) was higher for non-parasitized fish than for parasitized sticklebacks: this may be related to the greater gross efficiency of the parasite.

Free amino acids in the plerocercoid ofLigula intestinalis were compared with free amino acids in the plasma and perivisceral fluid of the fish host. In addition, the uptake of [14C]leucine by plerocercoids was investigated. The latter studies showed that the worm is permeable in vitro to leucine and that uptake of the amino acid occurs via a mediated process.

Light and transmission electron microscopy of the strobila of a large old Ligula intestinalis plerocercoid has revealed microcrystals with a morphology similar to that of microapatite crystals from vertebrates. Analysis of the microcrystals with EMMA-4 showed them to contain calcium and phosphorus. The tissue in the immediate vicinity of the microcrystals shows signs of necrosis while further away it is histologically normal.

An electron dense marker, lanthanum nitrate, was injected into the excretory ducts of living plerocercoids of Diphyllobothrium dendriticum and the observations made by electron microscopy. The contractions of the plerocercoid spread the marker into the excretory system, and the distribution was found to be irregular both within the ducts and from one duct to another. Communication between the smallest ducts and the rest of the excretory system was noted. The marker was also introduced from the surrounding medium, in this case the fixative. It was found to flush into the excretory ducts through the pore in the tail and to be distributed into the ducts in the same way as the injected marker. No other pores were observed through the tegument other than the excretory pore in the tail by either method. In ultrastructure the duct wall is similar to that of other cestodes. The distributive role of the excretory system is discussed, including a comparison between the tegument and the duct wall.

The fine structure of the tegument of the plerocercoid and the adult worm differs in several ways. A large increase in surface area, due to a rapid development of microvilli, takes place as the worm switches from a cold-blooded to a warm-blooded host. The plerocercoid has few and short microvilli, approximately 1.5 μ long, while the surface of the adult worm is densely packed with microvilli measuring more than 4 μ in length. Structures described as lamellated bodies from the tegument of the procercoid (Braten, 1968) and from the plerocercoid disappear when the worm enters the final host. In the internal level of the tegument an increase in the number and size of mitochondria at the adult stage is a prominent feature.

IT has been shown that plerocercoid larvae of the cestodes Schistocephalus solidus and Ligula intestinalis may be cultured to maturity in vitro in liquid media at 40° C. under sterile conditions. In the early experiments1,2, although sexually mature tapeworms developed, eggs produced in vitro failed on testing to form embryos. Histological examination revealed that spermatozoa were not present in the receptaculum seminis of the proglottids, suggesting that failure of eggs to form embryos was attributable to non-fertilization. In later experiments with Ligula3, a small number (6 per cent) of embryonated eggs were obtained from larvae cultured in horse serum.

THE plerocercoid of Ligula intestinalis is found in the body cavity of cyprinid fishes, where it causes castration1. Kerr2 in 1948 showed that the presence of the plerocercoid reduces the number of basophils in the pituitary of infested roach, with consequent virtual elimination of gonad stimulating hormone of the pituitary, and this work was afterwards confirmed3,4. More recently, it has been shown that this occurs not only in roach parasitizod by Ligula plerocercoids, but in other cyprinids as well.

PLEROCERCOID larvae of Schistocephalus solidus and Ligula intestinalis, when removed aseptically from their respective fish hosts by means of a special technique, develop into sexually mature tapeworms if cultured in vitro in certain nutrient media at 40° C.1,2. The eggs, which were produced in vitro in large numbers, proved on testing to be infertile. It was tentatively suggested that infertility of such eggs was possibly due to failure of normal copulation to take place within the culture tube, although other factors were also considered.