Comparative karyological analysis of four diplozoid species (Monogenea, Diplozoidae), gill parasites of cyprinid fishes.

The present study has revealed new data on chromosome complements of diplozoid parasites, namely Diplozoon paradoxum from freshwater bream Abramis brama, Paradiplozoon bliccae from white bream Blicca bjoerkna, Paradiplozoon sapae from white-eye bream Ballerus sapa, and Paradiplozoon nagibinae from zope Ballerus ballerus. Particularly, D. paradoxum is characterized by four pairs (2n=8) of long (up to 22.1 µm) chromosomes: pairs 1, 2, and 3 are metacentric and pair 4 acrocentric. Karyotypes of three Paradiplozoon species are nearly identical in number and morphological classification of chromosomes, each comprising diploid number of 14 one-armed chromosomes of very similar length ranging up to 12.5 µm in P. bliccae, 9.2 µm in P. sapae, and 9.9 µm in P. nagibinae. All four species are similar in their total complement length, ranging from 64.4 to 50.4 µm. Interspecific differences were found in location of secondary constriction: it is situated on short arm of the 1st chromosome pair in D. paradoxum, on long arm of the 7th pair in P. bliccae and on long arm of the 4th pairs in P. sapae and P. nagibinae. Phylogenetic interrelationship within the diplozoids and hypothetic karyotype evolution is here discussed.

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

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.