Digenean Flukes Research Articles

Immune and Oxidative Stress Response of the Fish Xyrichthys novacula Infected with the Trematode Ectoparasite Scaphanocephalus sp. in the Balearic Islands

Global change produces substantial modification to the distribution and rhythm of infection of diseases in fish, favouring the introduction of new pathogens. Recently, the presence of black spot disease, associated with a digenean fluke of the genus Scaphanocephalus, has been observed in specimens of Xyrichthys novacula on the island of Ibiza (Balearic Islands). The aim of the present study was to evaluate the antioxidant and immune response in both the skin mucus and spleen of X. novacula depending on the degree of infection by Scaphanocephalus sp. The specimens were captured in a control area, without the presence of the parasite, and in an affected area, classifying the fish as low infection (1–15 spots) and high infection (>15 spots). As the degree of infection increased, a decrease in the body condition index was observed. The activity of the antioxidant enzymes in the mucus—catalase, superoxide dismutase and glutathione peroxidase—increased progressively with the degree of infection. This activation of antioxidant defences was not enough to prevent an increase in malondialdehyde levels, an indicator of oxidative damage, in the group with the highest infection. Similarly, an increase in immunological parameters—lysozyme, alkaline phosphatase, myeloperoxidase and immunoglobulins—was observed in mucus as infection increased. Regarding the spleen, only an increase in lysozyme activity and alkaline phosphatase in fish with a greater severity of infection was observed. In conclusion, as the severity of Scaphanocephalus sp. infection increased, it induced an immune and oxidative stress response in skin mucus, leading to a decrease in overall body condition. The potential health effects that the ectoparasite may have on X. novacula populations will require follow-up studies.

Familiar snails are also intermediate hosts for pathogenic digenean flukes

Familiar snails are also intermediate hosts for pathogenic digenean flukes

Black spot disease related to a trematode ectoparasite causes oxidative stress in Xyrichtys novacula

Xyricthys novacula is very popular fish species in the Balearic Islands and a main target for recreational fishing. Over the last years, spots were observed on many individuals, which seems to be progressively more common. The aim of the present study was to identify the parasite, determine its abundance in a marine protected area (MPA) and in a non-protected one with more anthropic influence, and to evaluate the antioxidant and immune responses to this parasite presence by studying biomarkers in both liver and epithelial mucus. Analysis of genetic sequences established with 98.6–98.4% certainty that the parasite is a digenean fluke Scaphanocephalus sp. An average abundance of 12.3 ± 11.3 and 1.3 ± 1.3 parasites per individual was found for the non-protected area and the MPA, respectively. The activities of lysozyme and superoxide dismutase and total immunoglobulin concentration were significantly higher in mucus of X. novacula with more parasites. Similarly, in liver, a higher parasite load is related to higher activities of catalase, glutathione peroxidase and glutathione S-transferase, whereas malondialdehyde remained similar. In conclusion, Scaphanocephalus sp. affects X. novacula, inducing an immune and antioxidant response in epithelial mucus and in liver. The potential influence of the environment on parasite transmission, prevalence and abundance require further research to determine whether it makes fish more susceptible to infections.

Descriptions of Two Digenean Trematodes Found from a Chinese Sea Snake, Laticauda semifasciata, in Republic of Korea.

We intended to describe 2 digenean trematodes found from a Chinese sea snake, Laticauda semifasciata, as the new fauna in the Republic of Korea. The snake was caught offshore of Aewol-eup, Jeju-do, in August 2017. Two species of fluke were found in the lung and intestinal tract of the snake in the process of necropsy. They were identified as Pulmovermis cyanovitellosus Coil and Kuntz, 1960 and Harmotrema laticaudae Yamaguti, 1933, respectively based on the morphological characters. Pulmovermis cyanovitellosus showed elongated body with well-developed and elongated male genital system and compact vitelline. And H. laticaudae was characterized by linguiform body with heavily armed cirrus with excretory system. This is the first time both species have been reported and described off the Korean coast. We provide morphological descriptions with some comments on their biology and geographical distribution. In addition, the taxonomic validity of the genus Hydrophitrema Sandars, 1960 was discussed, in terms of morphological descriptions and host ranges. This study provides novel insight into digenean fluke species existing off the coast of Korea.

Eurytrema coelomaticum: updated morphology of adult worms using advanced microscopy experiments.

Eurytrema coelomaticum is a digenean flatworm of ruminants that is the causative agent of eurytrematosis, a disease of veterinary health concern. Although modern techniques of morphological analysis have provided new insights about the morphology and anatomy of parasitic helminths, most studies on E. coelomaticum adults are based on conventional light microscopy. In the present study, a combined approach using brightfield, fluorescence, confocal and scanning electron microscopies (SEMs), together with the cryofracture technique, have updated morphological data on E. coelomaticum recovered from cattle in Rio de Janeiro State, Brazil. Light microscopy confirmed the presence of several structures present in the current description, such as suckers, pharynx, oesophagus, intestinal bifurcation and the cirrus-sac. Fluorescence stereomicroscopy revealed for the first time the cubic crystal protein inclusions in the forebody, which were further detailed by confocal and SEMs. Confocal microscopy provided detailed information of the muscular architecture associated with the attachment structures (suckers), digestive system (pharynx and oesophagus), egg-forming complex (ovary, Mehlis' gland and Laurer's canal) and male reproductive system, which are similar to those found in other digenean flukes. SEM images of cryofractured parasites showed mucus and developing eggs within uterine loops. It was demonstrated that the combination of advanced tools generated complementary information, confirming the importance of experimental morphology in parasitology. Therefore, the knowledge of the adult structural organization of E. coelomaticum was improved and this work has contributed to propose new morphological criteria to evaluate the effects of antiparasitic drugs on flukes of medical and veterinary importance.

Trichobilharzia mergi sp. nov. (Trematoda: Digenea: Schistosomatidae), a visceral schistosome of Mergus serrator (L.) (Aves: Anatidae)

Parasitological investigations on red-breasted mergansers (Mergus serrator L.) in Iceland revealed digenean flukes of the family Schistosomatidae. Adult worms were detected in blood vessels of the large intestine and eggs were deposited in the mucosa and surrounded by granulomatous reactions. Traditional morphological methods showed that the flukes have very slender filiform bodies, males are equipped with a short gynaecophoric canal and both suckers and spatulate ends are present on each sex. Among characteristics of the flukes which render them morphologically distinct from other Trichobilharzia species are: i) males—well developed vesicula seminalis (v.s.) consisting of a short v.s. externa and a significantly longer (approx. 3 times) v.s. interna, unusually well developed genital papilla and localization of the first testis a relatively long distance posterior to the gynaecophoric canal; ii) eggs—small and elongated with slightly rounded poles and a short terminal spine. DNA taxonomic techniques confirmed that a new species had been identified, Trichobilharzia mergi sp. n. The sequence data were deposited in GenBank under the accession numbers JX456151 to JX456172. Comparison of the results with our previously published data on characterization of DNA of cercariae isolated from freshwater lymnaeid snails showed that larval development of T. mergi is associated with Radix balthica L. (=Radix peregra Müller, 1774;=Radix ovata Draparnaud, 1805).

Light and electron microscopy observations of embryogenesis and egg development in the human liver fluke, Opisthorchis viverrini (Platyhelminthes, Digenea)

Eggs of most species digenean flukes hatch in the external environment to liberate larvae that seek and penetrate a snail intermediate host. Those of the human liver flukes, Opisthorchis viverrini, hatch within the gastrointestinal canal of their snail hosts. While adult parasites are primarily responsible for the pathology in cases of human opisthorchiasis, their eggs also contribute by inducing granulomata and in serving as nidi for gallstone formation. In view of the peculiar biology of O. viverrini eggs and their contribution to pathology, we investigated embryogenesis in this species by light and transmission electron microscopy. Egg development was traced from earliest stages of coalescence in the ootype until full embryonation in the distal region of the uterus. Fully mature eggs were generally impermeable to resin and could not be examined by conventional electron microscopy methods. However, the use of high-pressure freezing and freeze-substitution fixation of previously fixed eggs enabled the internal structure of mature eggs, particularly the subshell envelopes, to be elucidated. Fertilization occurs in the ootype, and the large zygote is seen therein with a single spermatozoon wrapped around its plasma membrane. As the zygote begins to divide, the spent vitellocytes are pushed to the periphery of the eggs, where they progressively degrade. The early eggshell is formed in the ootype by coalescing eggshell precursor material released by approximately six vitelline cells. The early eggs have a thinner eggshell and are larger than, but lack the characteristic shape of, mature eggs. Characteristic shell ornamentation, the "muskmelon" appearance of eggs, appears after eggshell polymerization in the ootype. Pores are not present in the shell of O. viverrini eggs. The inner and outer envelopes are poorly formed in this species, with the outer envelope evident beneath the eggshell at the opercular pole of the mature egg. The miracidium has a conical anterior end that lacks the distinctive lamellar appearance of the terebratorium of other digeneans, such as the schistosomes. The miracidium is richly glandular, containing an apical gland in the anterior end, large cephalic gland, and posterior secretory glands. Each gland contains a secretory product with different structure. The paucity of vitelline cells associating with eggs, the reduced size of eggs, and reduced complexity of the extraembryonic envelopes are interpreted as adaptations to the peculiar hatching biology of the miracidia.

Newly discovered parasitic Turbellaria of opisthobranch gastropods

An endoparasitic platyhelminth from six species of sacoglossan opisthobranchs was collected at several localities of temperate to subtropical waters in Japan. Poecilostomatoid copepods (all species of Splanchnotrophidae and several species of Philoblennidae) and a few digenean flukes had been the only endoparasitic metazoans known for opisthobranch hosts. The newly discovered parasite was 1 to 15 mm in length and had no eyes, mouth, pharynx, or intestine. It had no external organs for parasitic life (e.g. attachment organs) and inhabited the haemocoel of the host. When mature, it emerged from the host and secreted a silky substance around itself to form a cocoon. The cocoon contained egg capsules with 19–42 eggs. Larvae, hatched from the capsule, had a ciliated body and a pair of eye spots. They were negatively phototactic and capable of invading suitable hosts. These morphological and life history features suggest this parasitic worm may belong to the family Fecampiidae (Platyhelminthes: Turbellaria), one of a few obligate parasite taxa in Turbellaria. Molluscan hosts which are common for parasitic Platyhelminthes have not previously been known for this family. The newly discovered parasite may be important for understanding the evolution of parasitism in Platyhelminthes.

A case of a facultative life-cycle diversification in the fluke Pleurogenoides sp. (Lecithodendriidae, Plagiorchiida)

Numerous specimens of the native, intestinal digenean fluke Pleurogenoides sp. (Lecithodendriidae, Plagiorchiida), a genus known for the simultaneous co-existence of genuine adults and progenetic, adult-like metacercaria, were found by chance parasitizing in the oesophagus of a recently imported, tropical Bristly Bush Viper (Atheris hispida). The snake had before been force-fed with native water frogs, the assumed definitive host of these flukes. Hence water frogs act as the second intermediate host or as a paratenic host for Pleurogenoides flukes, as they must house progenetic fluke larvae, which develop to genuine adults when transmitted to an appropriate consecutive host, the ancestral definitive host, a reptile. The European Pleurogenoides fluke species seem to display a facultative life-cycle diversification, they can adjust their life-history strategy according to their immediate transmission opportunities. This phenotypic plasticity allows the parasite to respond quickly to any changes in the abundance of a host; usually this biological oddity results in a life-cycle truncation by the elimination of the definitive host.

Parasites, pathological conditions and mortality in QX-resistant and wild-caught Sydney rock oysters, Saccostrea glomerata

Differences in the survival of QX-resistant fifth generation (QXR5) and wild-caught (Wild-Caught) Sydney rock oysters were assessed over the QX-disease risk period in the Pimpama River, SE Queensland. Cumulative mortality of Wild-Caught oysters (31.7%) was significantly greater than QXR5 oysters (0.0%) over the 118 days of the experiment. PCR and histological results showed that Wild-Caught oyster did not die from QX disease. Histology revealed oysters were infected with disseminating hemocytic neoplasia, a Steinhausia-like infection, a Rickettsia-like organism infecting epithelial cells of the gill, digenean flukes encysted in the gonadal tissue and a gill response to an unknown toxin. The cause of mortality is attributed to disseminating hemocytic neoplasia.

Parasites of carp bream, Abramis brama, from Lake Jamno, Poland

Abstract A total of 40 carp bream, Abramis brama (L.) from Lake Jamno were examined for their parasites in May 2002. Lake Jamno is a brackish-water coastal lagoon, strongly eutrophied and polluted by communal and industrial sewage from the city of Koszalin. The survey yielded 15 parasite species, representing different systematic groups, including 7 monogeneans Dactylogyrus auriculatus, D. falcatus, D. wunderi, D. zandti, Gyrodactylus elegans, Gyrodactylus sp., Diplozoon paradoxum; 4 digeneans (at the stage of metacercaria) Diplostomum sp., Tylodelphys clavata, Ichthyocotylurus platycephalus, I. variegatus; 1 cestode Caryophyllaeus laticeps; 2 crustaceans Ergasilus sieboldi, Argulus foliaceus; and 1 hirudinean Caspiobdella fadejewi. Monogeneans constituted the most abundant parasite group, accounting for 61.5 % of all parasite individuals collected. They were followed by the cestodes (20.7 %), digenean flukes (16 %), crustaceans (0.7 %), and leeches (0.2 %).

Allobilharzia visceralis gen. nov., sp. nov. (Schistosomatidae-Trematoda) from Cygnus cygnus (L.) (Anatidae)

In Iceland, the examination of whooper swans ( Cygnus cygnus L.) viscera resulted in the detection of adult digenean flukes of the family Schistosomatidae. The mature worms occurring in the blood vessels of the large intestine and mesenterium caused vascular lesions, around the eggs deposited in the intestinal mucosa and liver granulomatous reactions developed. The morphology of the isolated schistosomes shows certain similarity with the flukes of the genus Trichobilharzia; in males reduced gynecophoral canal, and on both sexes both suckers and spatulate ends are present. However, the Icelandic flukes possess other morphological features which are distinct from the genus: the point of caecal reunion in males takes place posterior to gynecophoral canal and the genital pore is behind acetabulum and anterior to caecal reunion. In order to evaluate the identity of Icelandic schistosomes, sequencing of ITS region of DNA was performed, and the obtained sequence was deposited in GenBank under the accession no. DQ067561. Following phylogenetic analysis of relationship between the sequence of Icelandic flukes and database sequences of other bird schistosome genera ( Trichobilharzia, Gigantobilharzia and Dendritobilharzia) showed different position of Icelandic worms in the phylogenetic tree. In conclusion, our study revealed new genus and species of schistosome flukes— Allobilharzia visceralis gen. et sp. n.

Infestation of Lymnaea stagnalis by digenean flukes in the Jeziorak Lake

The low number of articles on naturally trematode-infected snails results from the difficulty to obtain the quantitatively representative material. The main aim of our study was to check which age (size) groups of snails are the most susceptible to trematode invasion. Furthermore, we examined in which season the parasite prevalence is the highest. We collected Lymnaea stagnalis individuals in a nearshore zone of the Jeziorak Lake (the longest Polish lake located in northern Poland). The shell height of the snails was measured and the infestation by trematode larvae was determined. The logistic regression has shown that parasite prevalence increased significantly with the snail size (with the individuals >30 mm being the most infected), the distance from the beginning of the year (i.e., month) and the vicinity of summer.

Cholecystitis in otters (Lutra lutra) and mink (Mustela vison) caused by the fluke Pseudamphistomum truncatum

Between 1988 and 2004, postmortem examinations were carried out on 445 otters found dead, mostly as a result of road traffic accidents, in southern and south-west England. Thickened, shrunken gall...

The phylogeny of the Schistosomatidae based on three genes with emphasis on the interrelationships of Schistosoma Weinland, 1858.

Schistosomes are digenean flukes, parasitic of birds, mammals and crocodiles. The family Schistosomatidae contains species of considerable medical and veterinary importance, which cause the disease schistosomiasis. Previous studies, both morphological and molecular, which have provided a good deal of information on the phylogenetics of this group, have been limited in the number of species investigated or the type or extent of molecular data used. This paper presents the most comprehensive phylogeny to date, based on the sequences of 3 genes, complete ribosomal small subunit rRNA and large ribosomal subunit rRNA, and mitochondrial cytochrome oxidase 1, sequenced from 30 taxa including at least 1 representative from 10 of the 13 known genera of the Schistosomatidae and 17 of the 20 recognized Schistosoma species. The phylogeny is examined using morphological characters, intermediate and definitive host associations and biogeography. Theories as to the origins and spread of Schistosoma are also explored. The principal findings are that Ornithobilharzia and Austrobilharzia form a sister group to the Schistosoma; mammalian schistosomes appear paraphyletic and 2 Trichobilharzia species, T. ocellata and T. szidati, seem to be synonymous. The position of Orientobilharzia within the Schistosoma is confirmed, as is an Asian origin for the Schistosoma, followed by subsequent dispersal through India and Africa.

Detection of Opisthorchis viverrini in experimentally infected bithynid snails and cyprinoid fishes by a PCR-based method.

A PCR procedure for the detection of Opisthorchis viverrini in experimentally infected bithynid snails and cyprinoid fishes was developed. This procedure was based on primers designed from a pOV-A6 specific probe sequence giving a 330 base-pair product. The detection was accomplished in host tissue homogenates to which a single cercaria or metacercaria was introduced. PCR can detect as little as a single cercaria artificially inoculated in a snail or a single metacercaria artificially inoculated in a fish sample. The method gave a 100% positivity rate for all infected snails or fishes. The method did not yield a 330 base-pair amplified product with other digenean fluke DNAs such as Haplorchis taichui, Centrocestus spp., Echinostoma malayanum, Fasciola gigantica, animal schistosomes, Paragonimus heterotremus or Haplorchoides spp. The assay has great potential for application in epidemiological surveys of both snail and fish intermediate hosts as well as for investigation of foodborne parasites in freshwater fishes.

Sublethal responses of largemouth bass to parasites and organochlorines

Largemouth bass (Micropterus salmoides) at the Rocky Mountain Arsenal National Wildlife Refuge (RMA) experience chronic organochlorine exposures and parasitism by nematodes (Contracaecum spiculigerum) and digenean flukes (Posthodiplostomum minimum centrarchi). We investigated the influences of nematode intensity, fluke intensity, and whole-body organochlorine concentrations on growth of juvenile RMA bass. Lifetime growth, or age-specific lengths, of bass in three RMA lakes were within the range observed for bass in five reference lakes. However, interlake comparisons can be confounded by differing environmental conditions. Therefore, we conducted mesocosm and laboratory studies to compare growth, consumption, and feeding behavior between RMA bass and bass that had little contaminant or parasite exposure. Mean growth rates of RMA bass were 45% lower compared with hatchery bass in experimental ponds. However, regression analysis revealed that parasite and organochlorine burdens were not negatively associated with either short-term growth or age-specific lengths of RMA bass. Hatchery bass growth was likely higher due to their experience with culture pond conditions. In feeding trials, RMA bass exhibited similar food conversion efficiency and consumption rates and significantly elevated feeding activity compared with hatchery bass. This research demonstrates that current parasite and organochlorine loads had benign influences on growth of juvenile RMA bass.

Fine Structure and Sugar Transport Functions of the Tegument in Clinostomum marginatum (Digenea: Clinostomatidae): Environmental Effects on the Adult Phenotype

Digenean flukes can be classified into 3 groups according to their location in the host: the lumen of the alimentary canal or associated organ, body cavity or tissue, and external surfaces. We obtained adults of Clinostomum marginatum that had matured in these 3 habitats and compared the fine structure and glucose transporting capacity of their teguments. Adults from the esophagus of herons, Ardea herodias, had thick, smooth teguments and took up glucose by facilitated diffusion, the type of transport that is Na(+)-independent and insensitive to phlorizin. By contrast, the surfaces of adults cultured from metacercariae in body cavities of laboratory mice were amplified 3-5-fold due to numerous irregular projections of the tegument. Glucose transport by these worms was largely Na(+)-dependent and inhibited by phlorizin, indicating active transport. Ectoparasites from herons' mouths had relatively thick, smooth teguments, but these worms always were encrusted with bacteria and yeast that are known to absorb and metabolize glucose. Most of the attached bacteria, and the apparent glucose uptake associated with their presence, were removed by treating the worms with antibiotics prior to transport assays. As facilitated diffusion and active transport are operational simultaneously in metacercariae, the type of transport function, if any, expressed in the adult is determined by environmental conditions associated with the worm's habitat.

Environmental sodium regulates cutaneous sugar transport in a digenean fluke.

Glucose uptake was examined in adult specimens of Proterometra macrostoma (Trematoda: Digenea) recovered from the stomach (endoparasites) and gills (ectoparasites) of longear sunfish, Lepomis megalotis. The endoparasitic forms transported glucose directly through the external body surface by Na+-independent, facilitated diffusion, but the ectoparasites absorbed glucose by free diffusion alone. To determine how this transport function is regulated, cercariae were incubated in solutions having Na+ concentrations normally found in fish gut (50 mM) and in fresh water (0.5 mM). Glucose transport capacity was retained in 50 mM Na+, but disappeared in worms incubated for 1-3 days in 0.5 mM Na+. Returning worms from the latter solution to one containing 50 mM Na+ fully restored glucose transport within a day. By contrast, incubation up to 5 days in 0.5 mM Na+ had no effect on glucose transport in endoparasitic adults of P. macrostoma and cercariae of P. edneyi. Thus, cutaneous sugar transport function in the migrating larva of P. macrostoma is subject to indirect regulation by environmental Na+.

Niche segregation and sugar transport capacity of the tegument in digenean flukes

Digenean flukes can be broadly classified into three basic groups according to their location within the host: (1) the lumen of the alimentary canal or associated organ, (2) body cavity or tissue and (3) external surfaces. We selected three species of Proterometra that represent these basic groups and tested their capacities for cutaneous transport of glucose. Facilitated diffusion is the type of transport system in the tegument of P. edneyi, which lives in the gut of its host. An activetransport system is present in P. dickermani, the tissue-dwelling species. No cutaneous system for transporting glucose is present in the ectoparasitic P. macrostoma. The known capacities for tegumental sugar transport in other digeneans are correlated with similar habitats. Such associations involving unrelated as well as closely related species suggest that the niche biology of digeneans involves evolutionary specializations of the tegument for absorbing sugar.