Ethanol-fixed material used for both classical and molecular identification purposes: Eudiplozoon nipponicum (Monogenea: Diplozoidae) as a case parasite species

Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlárská 2, 611 37, Brno, Czech Republic.
Parasitology Research (Impact Factor: 2.1). 09/2010; 107(4):909-14. DOI: 10.1007/s00436-010-1949-0
Source: PubMed


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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    • "The widespread use of sclerites is also related to the fact that, thanks to their chemical composition, they are the least perishable monogenean structures, allowing species identification even for specimens that are not optimally preserved. This is important, because the best way to prepare monogenean slides for long-term preservation is debated, and incorrect preparation may produce serious morphological artefacts (Wong et al. 2006; Strona et al. 2009; Ko skov a et al. 2010; Justine et al. 2012). However, not all sclerotized structures may be clearly visible on prepared materials, because of different reasons, such as a suboptimal orientation of specimen on slide, or the chemical properties of the media used for preservation (Galli et al. 2007). "
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    ABSTRACT: We introduce a new statistical method to select which morphological characters are most useful to identify monogenean species. The method estimates the average size overlap of candidate diagnostic structures among a set of species to individuate those that mostly differ between the species. To dem-onstrate our approach, we report a comprehensive analysis conducted on two of the most species-rich monogenean genera: Dactylogyrus Diesing, 1850 and Gyrodactylus von Nordmann, 1832. We demonstrate that, in contrast to common taxonomic practice, very few but highly diagnostic mea-surements are necessary to correctly identify a specimen. In particular, we found that most of Dactylogyrus and Gyrodactylus species can be classified on the basis of the width of the supplementary connecting bar and of the length of the hook sickle, respectively.
    Journal of Zoological Systematics and Evolutionary Research 10/2013; 52(2). DOI:10.1111/jzs.12050 · 1.68 Impact Factor
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    • "For morphometric analysis, only one opisthaptor of each specimen was cutoff (Khotenovsky 1974) and soaked in 10 % sodium dodecyl sulfate for 30– 60 min. This was done in order to soften rigid tissue and thereby to make the clamps and central hooks clearly visible (Wong et al. 2006; Košková et al. 2010). The treated opisthaptor was washed in distilled water before being mounted on a microscope slide and fixed with a mixture of ammonium picrate and glycerin (Malmberg 1956; Khotenovsky 1974). "
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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; 112(8). DOI:10.1007/s00436-013-3480-6 · 2.10 Impact Factor
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    • "The emergence of molecular methods has prompted a renewed interest in methods which can process material for both morphological and molecular methods (Harris et al. 1999; Košková et al. 2011; Naem et al. 2010; Strona et al. 2009; Toe et al. 1997; Yoder et al. 2006). "
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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 · 2.10 Impact Factor
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