Ethanol-fixed material used for both classical and molecular identification purposes: Eudiplozoon nipponicum (Monogenea: Diplozoidae) as a case parasite species.
ABSTRACT This study is focused on the feasibility of two treatments of alcohol-fixed monogenean parasites which are intended to be use for the combined morphological and molecular characterizations. The monogenean parasite, Eudiplozoon nipponicum, was selected as a model parasite species; however it is expected that these techniques will be suitable for other monogeneans and other parasitic families. The haptor of diplozoid parasites is equipped with sclerotized attachment clamps and central hooks which are utilized for morphological identification. As parasite tissue become very tough and rigid when preserved in ethanol, using these structures for species identification without additional treatment is difficult. We investigated two different techniques to digest the surrounding tissues, the first was treatment with 10% sodium dodecyl sulphate (SDS) and the second treatment was proteinase K. Tissue was successfully digested in both treatments and all clamps, central hook and even individual sclerites of the clamps were clearly visible and well defined. After treatment, the digest was used to extract genomic DNA, and the second internal transcribed spacer of the ribosomal DNA genes (rDNA) was amplified. Nucleic acid sequence was obtained from 90% of parasite specimens processed by both treatments. Treatment of haptors with SDS was proven to be more successful with no visible changes or damage observed to sclerites even after a month. This method represents a useful tool for the combined morphological and molecular studies as the correct sequence can be assigned to the same individual worm from which haptoral parts have been obtained.
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ABSTRACT: The paper presents a description of Paradiplozoon bingolensis sp. n. from the gills of Garra rufa Heckel, 1843 (Cyprinidae) collected from the Göynük Stream, a tributary of the Murat River, Turkey. This is the first diplozoid species to be described from G. rufa. P. bingolensis is distinguished from the other valid species in the genus by the combination of the morphology of the sclerites of its clamps and by the size of the central hooks. Even molecular characterization based on variability of the second internal transcribed spacer (ITS2) of the ribosomal DNA region provided additional support of separation of this new species from the valid ones. The sequences were compared with previously published ITS2 sequences of other diplozoid species. Subsequent analysis demonstrated the uniqueness of this new parasite species and revealed uncertainties in the current taxonomic division of the Diplozoidae that are commented in the text.Parasitology Research 06/2013; DOI:10.1007/s00436-013-3480-6 · 2.33 Impact Factor
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ABSTRACT: Many methods have been proposed for collecting and fixing parasites, but most were written before the molecular age, and were intended to be practised by experienced parasitologists in well-equipped laboratories. We describe here a very simple method, illustrated by photographs, for collecting helminths from the digestive tract of vertebrates. It only requires a few plastic vials, some ethanol and a means to heat water. Basically, the method consists of: (a) the extraction of all organs from the abdominal cavity; (b) opening the digestive system longitudinally; (c) agitate gut and contents in a saline solution (i.e. ca. 9% NaCl or 1/4 sea water in tap water); (d) decant in saline as many times as needed to clean contents; (e) immediately fix parasites in near-boiling saline; (f) discard saline and keep specimens in 95% ethanol. Additional information is given for collecting parasites from fish gills with a similar process. The method will collect most helminths (digeneans, larval cestodes, nematodes, acanthocephalans) from the digestive tract, and monogeneans and isopod and copepod crustaceans from fish gills. The specimens will be suitable for both morphological study and DNA sequencing. The method is simple, fast, inexpensive and can be used by untrained personnel, even in the field without electricity and without a binocular microscope. It can also be used by trained parasitologists who need to expedite treatment of abundant samples.Parasitology Research 02/2012; 111(1):341-51. DOI:10.1007/s00436-012-2845-6 · 2.33 Impact Factor
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ABSTRACT: Tintinnids (Ciliophora: Spirotricha: Tintinnina) are occasionally the dominant ciliates in the marine plankton. The tintinnid loricae are minute artworks fascinating scientists for more than 230 years, but their chemical composition remained unclear, viz., chitinous or proteinaceous substances were discussed. Since sedimenting loricae contribute to the flux of elements and organic compounds in the oceans, knowledge about their nature is necessary in assessing their ecological role. Previous techniques and new methods, e.g. enzymatic digestion and high-resolution transmission electron microscopy, are applied in the present study. A chitinous nature of the loricae is rejected by the Van-Wisselingh test and failure of chitinase digestion. Only proteins might show a resistance against strong hot bases (KOH at 160°C for ~ 40 min. in tintinnid loricae) similar to that of chitin. Actually, the presence of nitrogen in the EDX analyses and the digestion of at least some loricae by proteinase K strongly indicate a proteinaceous nature. Furthermore, the crystal lattice revealed by high-resolution TEM in Eutintinnus loricae is similar to the proteinaceous surface layer (S-layer) of archaea, and the striation recognizable in transverse sections of Eutintinnus loricae has a periodicity resembling that of the crystalline proteins in the extruded trichocysts of Paramecium and Frontonia. The proteolytic resistance of some loricae does not reject the idea of a proteinaceous nature, as proteins in S-layers of some archaea and in most naturally occurring prions show comparable reactions. The data from the present study and the literature indicate proteins in the loricae of thirteen genera. Differences in the proteolytic resistance and staining properties between genera and congeners are probably due to deviations in the protein composition and the additional substances, e.g. lipids, carbohydrates. At the present state of knowledge, correlations between lorica structure, wall texture, ultrastructure of the lorica forming granules, and the histochemical and enzymatic findings are not evident. Therefore, further studies are required to estimate the taxonomic significance of these features and the ecological role of sedimenting loricae.Acta protozoologica 06/2012; 51(1):1-19. · 1.18 Impact Factor