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: In the present work we examined the efficacy of three different chemical solutions (EtOH 70%, DMSO-NaCl solution, and Longmire buffer) in field preservation of fish gills to be subsequently screened for monogenean specimens destined to morphological and molecular analyses. Degree of difficulty in collecting monogeneans from gills, morphological state of parasites, integrity of their DNA and reliability of sequence reading were observed and qualitatively compared to those of gills and parasites stored in 5% formalin and 99% ethanol. Data were collected over a period of 2 months. Storage in Longmire buffer resulted in dissociation of gills and parasites, while both DMSO and 70% ethanol provided a fine physical and molecular preservation of gills and monogeneans, allowing rapid collection of parasites from lamellae, and easy extraction, amplification and sequencing of parasitic DNA.Parasitology International 11/2008; 58(1):51-4. · 2.30 Impact Factor
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ABSTRACT: The present study addresses the effect of varying temperature and host species on the size and shape of the opisthaptoral hard-parts in isogenic strains of Gyrodactylus salaris and G. thymalli. Variation in shape was examined using geometric morphometrics. Since the opisthaptoral hard-parts of Gyrodactylus have few specific landmarks, their shape information mostly being represented by outlines and surfaces, a method based on sliding semi-landmarks was applied. The ventral bars of G. salaris did not follow the previously postulated negative correlation between size and temperature, and the largest hamuli and marginal hooks from G. salaris and the smallest from G. thymalli clearly overlapped in size. Consistent shape differences with temperature were detected for the hard-parts from G. thymalli but not from G. salaris. The hard-parts of G. salaris were similar in size but significantly different in shape when grown on secondary hosts rather than the primary host.Parasitology 09/2009; 136(11):1305-15. · 2.36 Impact Factor
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ABSTRACT: The Gyrodactylus fauna of 274 fish taken from ten salmonid farms in Poland was sampled in 2006. Four fish species were investigated: rainbow trout Oncorhynchus mykiss, brown trout Salmo trutta (morphs fario, lacustris, and trutta), grayling Thymallus thymallus and huchen Hucho hucho. No parasites were observed on huchen. No indications of gyrodactylosis were observed, but an unexpected parasite species diversity was found. A molecular species identification by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of ITS1 + 5.8S + ITS2 was utilized, with addition of morphometric methods. The most frequent parasite was a new record in Poland, G. teuchis. It was present in two molecular forms on brown trout and rainbow trout, which also carried G. derjavinoides and G. truttae. Three molecular forms of G. salaris/G. thymalli were found, the standard type ITS only on grayling. A heterozygous (or heterogenic) G. salaris type described earlier in Denmark was found in seven farms on rainbow trout, and a complementary homozygous clone which differs from the standard by three nucleotides, in two farms. This homozygous form has not been recorded earlier. The PCR-RFLP results were confirmed by sequencing ITS segment from representative specimens of each type and comparing them with all available salmonid-specific Gyrodactylus sequences in GenBank. The Polish fauna with seven different Gyrodactylus clones separated by PCR-RFLP was the most diverse reported in fish farms in any country so far.Acta Parasitologica 52(3):185-195. · 1.00 Impact Factor