Coccidian oöcysts as type-specimens: long-term storage in aqueous potassium dichromate solution preserves DNA

Abstract

Preservation of the exogenous oöcyst stage of coccidian parasites (phylum Apicomplexa N.D. Levine, 1970) as type-specimens of newly described species has long been problematical. Conventional fixatives have proved unsatisfactory, and compromises such as embedding oöcysts in resin or photographing them are not entirely appropriate for various reasons. As an alternative, chilled potassium dichromate solution (normally used in the laboratory to prevent putrefaction of temporary preparations of live oöcysts) has been tested as a long-term preservative of sporulated oöcysts of Eimeria brunetti P.P. Levine, 1942, E. maxima Tyzzer, 1929, E. mitis Tyzzer, 1929, E. necatrix Johnson, 1930, E. praecox Johnson, 1930 and E. tenella (Railliet & Lucet, 1891) (suborder Eimeriorina Léger, 1911; family Eimeriidae Minchin, 1903). Oöcysts from faeces of chickens Gallus gallus (Linnaeus) were placed in 2.5% w/v aqueous potassium dichromate solution (PDS) and stored in the dark at 4 ± 2°C. After 23 years in storage, oöcysts of each species were administered orally to chickens and failed to initiate infections, indicating that the oöcysts were dead. Nevertheless, after about 24 years, DNA was still recoverable from the oöcysts, and the original species identifications made by classic parasitological methods were confirmed by polymerase chain reaction assays. Furthermore, after almost 25 years, microscopical examination revealed that the walls and internal structures remained well preserved in 83–98% of the oöcysts of the six species investigated. Hence, PDS is potentially suitable for the long-term preservation of sporulated coccidian oöcysts as type-specimens for taxonomic purposes. The samples used in this study are now in the care of the Natural History Museum, London, UK. It is recommended that they be monitored in like manner, by suitably qualified scientists, at intervals of about 5 years to assess their state of preservation and the recoverability of DNA. Enough material is available to monitor it until it is at least 100 years old.

Appendix: PDS-preserved oöcysts available for future study, deposited in the Natural History Museum (NHM), London, UK

Identification details of the material used in the present study and now available for future examination are given in Table 2. The specimens are stored in 2-ml vials, 15 for each species, so that a single vial may be removed for study without the risk of spilling or contamination of the remaining material. If one vial is examined per five years, there is enough material of each species to allow monitoring for 75 years from now, giving a potential total of about 100 years of monitoring to assess the suitability of PDS for preservation of type-specimens.