EAFP 2005 Myxozoan Session Summary

In view of the growing worldwide interest in Myxozoa, a day-long meeting dedicated to these fish parasites was held on September 14, 2005. The special gathering included two sessions of oral presentations (Biology, Development and Phylogeny; and Myxozoan-host interactions) and a myxozoan poster session, all of which provided an updated overview on contemporary topics of myxozoan research. The oral sessions included 17 presentations, encompassing a diverse range of Myxozoan research topics. A total of 20 posters dealing with Myxozoan topics participated in the poster session; one of which won a best poster prize. A round-table discussion forum that dealt with various current topics, new tools and approaches with the objective of stimulating new areas of Myxozoan research and encouraging international collaboration, concluded the day.

The round-table,session was chaired by Dr Arik Diamant (Eilat, Israel) and Dr Jerri Bartholomew (OSU, USA)several topics were discussed. The first dealt with the idea of creating Tissue microarrays (TMAs) of Myxozoan parasites. This concept deals with production and analysis of serial paraffin histology sections of myxosporean/fish specimens of special interest, processed for histochemistry, immunocytochemistry, ISH, etc. The idea was presented Dr Ariadna Sitja-Bobadilla (CSIC, Spain), who, after introducing the new approach, raised the question whether myxozoan research can benefit from TMAs? Basically, TMAs convert the convenience of DNA microarrays to tissues: a TMA is a paraffin block that includes tens to hundreds of minute tissue samples organized side by side. The TMA is produced by arranging cylinder shaped cores containing tissue areas of particular interest from a selection of "donor" paraffin blocks. Regions within the donor blocks are identified and assembled in an array to form the TMA block, which can then be sectioned and mounted on glass slides. The thickness of the cut sections is determined by the nature of the tissues, and determines how many slides can be generated. The mounted sections can then be treated or stained as required. The approach was initially introduced for cancer research but rapidly expanded to new fields. Thus, slides may be used for morphology or histology studies, immunohistochemistry (IHC), cytochemistry, in-situ hybridization (ISH) and tissue banking, as well as multiple tasks such as comparing gene expression of protein in normal and pathological conditions, or drug targeting.

The advantages of TMA are obvious: hundreds of core samples can be condensed onto a single glass slide. The procedure is economical in amounts of stains, reagents, antibodies or probes, and saves considerably time as well. Maximum uniformity and consistency in staining can be achieved. An additional advantage is that old archive blocks may be used. The innovative TMA approach has not yet been used with Myxozoa, possibly because the average laboratory needs to have not only a skilled technician trained for the task, but also a microarrayer, a piece of equipment that is rather expensive. There are, however, commercial companies that will construct these arrays, but prices are quite high (e.g. a TMA consisting of 140 cores 1mm in diameter would cost around 1200 Euro for the first unit and 600 Euro for each additional block). Discussion was invited to establish who is interested in collaborating to produce an array, what archived tissues are available in the various laboratories and to what purposes could these arrays be used.

A suggested protocol for standardised preparation and preservation of actinosporean spore samples was outlined by Stephen Atkinson and Dr Sascha Hallett (OSU, USA). The protocol consists of a checklist for preservation of actinospores for facilitating morphometrical/comparative work. The various stages were detailed: identification of an infected host using the well plate system technique and measurement of spores using Nomarski interference contrast and/or phase contrast photos of at least 10 fresh spores. Several methods for preservation were presented that would allow sharing of samples between collaborators. Air dried slides (at least 10) provide a good archival record and can be stained with Giemsa, Hematoxylin and eosin, or Phloxine B for resolution of internal structures and valve cells. For maintaining spores over a period of several days, refrigeration is sufficient. For permanent spore preservation, 10% formalin is an excellent fixative; spores will retain shape but may shrink ~10%. Attempts to preserve spores by freezing or placing in ethanol will yield poor phenotypic results, as spores tend to distort, although this is appropriate for genetic studies. At least 2 frozen samples should be taken for 18S rDNA sequencing. One should be stored in a frozen archive (-80�C) for future reference, the other for immediate use: for DNA extraction, sequencing and comparison with sequences in GenBank. Finally, hosts (e.g. annelids) should be appropriately fixed for subsequent taxonomic identification, preparation of histological material and EM observations. Researchers were encouraged to deposit representative samples in the International Reference Tissue Depository for Myxozoans, Queensland Museum, Australia.

Another topic of interest was presented by Dr Oswaldo Palenzuela (CSIC, Spain): Myxozoan systematics. The presentation dealt with the standardization of procedures for obtaining and analyzing SSU rDNA sequences and the ultimate goal of a complete revision of the systematics of the group. The talk dealt with the pitfalls of data acquisition, obtaining reliable sequences, and importance of depositing unambiguous and well annotated sequences. The significance of precise alignment and analysis of sequences was highlighted. Also mentioned was the idea of creating a network/working group on molecular phylogeny, possibly through a Myxozoa website.

The Myxozoa website www.myxozoa.org,which was established in 2004, was introduced and its objectives discussed. The core of the network was a group of researchers working on different aspects of myxozoan biology. Any person interested in becoming part of the network whose research interests include fish parasites and myxozoans was (and is) invited to visit the site and join the online contact list. The purpose of the website is 1) to link myxozoan researchers for the purposes of sharing knowledge and encouraging collaboration, thereby advancing understanding of myxozoan biology and ecology on a worldwide scale, and 2) to serve as a directory and information source for aquaculture professionals affected by myxozoan-related problems. Several possibilities for future expansion of the site were discussed, including addition of images and descriptions of myozoan spores and the diseases they can cause, and construction of an online database or archive of myxozoans and myxozoan literature.

At the end of the day, the panel invited suggestions, thoughts and comments from the attending audience regarding ideas whether a hands-on session in future workshops would be useful, and what type of Myxozoan samples would be considered particularly helpful. Finally, a general discussion with suggestions from participants on what is expected from future meetings and workshops was held.

Arik Diamant

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