Jana Ilgová

Ultrastructure of the digestive tract of Paradiplozoon homoion (Monogenea)

Paradiplozoon homoion is a representative of blood-feeding ectoparasites from the family Diplozoidae (Polyopisthocotylea, Monogenea). Although these worms have been the subject of numerous taxonomical, phylogenetic and ecological studies, the ultrastructure of the alimentary system and related structures, as well as the mechanisms of essential processes like fish blood digestion, remain mostly unknown. Our observation of P. homoion using a transmission electron microscopy (TEM) revealed two main types of digestive cells—U-shaped haematin cells and connecting syncytium. Particular structures such as mouth cavity with specialised receptors, two oval-shaped muscular buccal suckers, pharynx surrounded with the glandular cells, oesophagus, the intestinal caeca with intact erythrocytes in the lumen, the apical pinocytotic fibrous surface complex and haematin vesicles of U-shaped cells have been shown in detail. According to our results, the P. homoion is degrading the blood components predominantly intracellularly.

Life cycle of Cryptosporidium muris in two rodents with different responses to parasitization.

This study focuses on mapping the life cycle of Cryptosporidium muris in two laboratory rodents; BALB/c mice and the southern multimammate rat Mastomys coucha, differing in their prepatent and patent periods. Both rodents were simultaneously experimentally inoculated with viable oocysts of C. muris (strain TS03). Animals were dissected and screened for the presence of the parasite using a combined morphological approach and nested PCR (SSU rRNA) at different times after inoculation. The occurrence of first developmental stages of C. muris in stomach was detected at 2.5 days post-infection (dpi). The presence of Type II merogony, appearing 36 h later than Type I merogony, was confirmed in both rodents. Oocysts exhibiting different size and thickness of their wall were observed from 5 dpi onwards in stomachs of both host models. The early phase of parasitization in BALB/c mice progressed rapidly, with a prepatent period of 7.5-10 days; whereas in M. coucha, the developmental stages of C. muris were first observed 12 h later in comparison with BALB/c mice and prepatent period was longer (18-21 days). Similarly, the patent periods of BALB/c mice and M. coucha differed considerably, i.e. 10-15 days vs chronic infection throughout the life of the host, respectively.

Major acid endopeptidases of the blood-feeding monogenean Eudiplozoon nipponicum (Heteronchoinea: Diplozoidae)

In parasitic flatworms, acid endopeptidases are involved in crucial processes, including digestion, invasion, interactions with the host immune system, etc. In haematophagous monogeneans, however, no solid information has been available about the occurrence of these enzymes. Here we aimed to identify major cysteine and aspartic endopeptidase activities in Eudiplozoon nipponicum, an invasive haematophagous parasite of common carp. Employing biochemical, proteomic and molecular tools, we found that cysteine peptidase activities prevailed in soluble protein extracts and excretory/secretory products (ESP) of E. nipponicum; the major part was cathepsin L-like in nature supplemented with cathepsin B-like activity. Significant activity of the aspartic cathepsin D also occurred in soluble protein extracts. The degradation of haemoglobin in the presence of ESP and worm protein extracts was completely inhibited by a combination of cysteine and aspartic peptidase inhibitors, and diminished by particular cathepsin L, B and D inhibitors. Mass spectrometry revealed several tryptic peptides in ESP matching to two translated sequences of cathepsin L genes, which were amplified from cDNA of E. nipponicum and bioinformatically annotated. The dominance of cysteine peptidases of cathepsin L type in E. nipponicum resembles the situation in, e.g. fasciolid trematodes.

An ultrastructural study of the surface and attachment structures of Paradiplozoon homoion (Bychowsky & Nagibina, 1959) (Monogenea: Diplozoidae)

BackgroundSpecies of Diplozoon Palombi, 1949 (Monogenea: Diplozoidae) are blood-feeding ectoparasites mainly parasitising the gills of cyprinid fishes. Although these parasites have been the subject of numerous taxonomic, phylogenetic and ecological studies, the ultrastructure of the surface and haptor attachment structures remains almost unknown. In this study, we used transmission electron microscopy to examine the ultrastructure of attachment clamps and neodermal surface of Paradiplozoon homoion (Bychowsky & Nagibina, 1959), family Diplozoidae Palombi, 1949, thereby broadening our knowledge of platyhelminth biology.ResultsThe hindbody surface of P. homoion is distinctly ridged, each ridge being supported by several muscle fibers and equipped with scales on the surface plasma membrane. Such structures have not been recorded previously in species of the family Diplozoidae. Comparisons of the surface structure of different body parts revealed slight differences in the thickness and number of organelles. Each of the clamps has a flattened bowl-like structure composed of sclerites, movable skeletal-like structures that are anchored by robust, radially oriented muscle bundles. The base of the posterior median plate sclerites is equipped with glandular cells possessing secretory vesicles.ConclusionThis study brings detailed ultrastructural data for the surface and haptoral attachment clamps of P. homoion and provides new insights into the ultrastructure of Diplozoidae. Glandular cells at the base of the attachment clamps responsible for sclerite development in diplozoid species were observed for the first time. Our findings support the hypothesis that the structure of particular neodermal compartments is similar within the Platyhelminthes. On the other hand, the diplozoid glandular system and the mechanism of sclerite development clearly merits further attention.

Redescription of Paradiplozoon hemiculteri (Monogenea, Diplozoidae) from the type host Hemiculter leucisculus, with neotype designation

Paradiplozoon hemiculteri (Ling, 1973), a member of the Diplozoidae, parasitizes the gills of Asian fish. Not only is the type material unavailable for this species, the original description was poor and somewhat conflicting, and adequate molecular data were not available. What is more, the available morphological and molecular data are inconsistent and fluctuate significantly. Here, we present a redescription of P. hemiculteri based on morphological and molecular data from new isolates collected from the type host, the sharpbelly Hemiculter leucisculus (Basilewsky, 1855), captured at the neotype locality (Shaoguan, Guangdong Province, southern China); a neotype for P. hemiculteri was designated from this collection. The length and width of the body, buccal suckers, pharynx, attachment clamps, sickle and the central hook handle were all measured and the shape of the anterior and posterior part of the median plate and anterior and posterior joining sclerites accurately documented. Phylogenetic analyses based on the sequences of the second rDNA internal transcribed spacer (ITS2) indicated that all new samples clustered together and differed clearly from sequences attributed to P. hemiculteri, which are deposited in GenBank. Our results confirm that P. hemiculteri is the only diplozoid that has demonstrably been found on the gills of H. leucisculus to date.

A novel type I cystatin of parasite origin with atypical legumain-binding domain

Parasite inhibitors of cysteine peptidases are known to influence a vast range of processes linked to a degradation of either the parasites’ own proteins or proteins native to their hosts. We characterise a novel type I cystatin (stefin) found in a sanguinivorous fish parasite Eudiplozoon nipponicum (Platyhelminthes: Monogenea). We have identified a transcript of its coding gene in the transcriptome of adult worms. Its amino acid sequence is similar to other stefins except for containing a legumain-binding domain, which is in this type of cystatins rather unusual. As expected, the recombinant form of E. nipponicum stefin (rEnStef) produced in Escherichia coli inhibits clan CA peptidases – cathepsins L and B of the worm – via the standard papain-binding domain. It also blocks haemoglobinolysis by cysteine peptidases in the worm’s excretory-secretory products and soluble extracts. Furthermore, we had confirmed its ability to inhibit clan CD asparaginyl endopeptidase (legumain). The presence of a native EnStef in the excretory-secretory products of adult worms, detected by mass spectrometry, suggests that this protein has an important biological function at the host-parasite interface. We discuss the inhibitor’s possible role in the regulation of blood digestion, modulation of antigen presentation, and in the regeneration of host tissues.

Excretory system of representatives from family Diplozoidae (Monogenea)

Diplozoons are representatives of blood-feeding ectoparasites from the family Diplozoidae (Polyopisthocotylea, Monogenea). Although these worms have been the subject of numerous taxonomical, phylogenetic, and ecological studies, the detailed study of their excretory system has remained relatively neglected. Our observations focused on the morphological and ultrastructural features of the excretory apparatus of four diplozoid species: Diplozoon paradoxum, Eudiplozoon nipponicum, Paradiplozoon bliccae, and Paradiplozoon homoion. Observations were obtained using two microscope methods: light microscopy, equipped with differential interference contrast (Nomarski DIC) and transmission electron microscopy (TEM). The ultrastructure of two basic compartments which forms the excretory apparatus, flame cells with filtration apparatus, and canal cells forming the protonephridial ducts is revealed in this study. A unique consecutive sequence of longitudinal semi-thin sections of the excretory pore of E. nipponicum is visualized there for the first time.