TECHNIQUES FOR PRESERVING LIFE CYCLE STAGES

SAVING, STORING AND PRESERVING OOCYSTS FOR OBSERVATION

Before oocysts can be studied critically, they must be properly maintained to keep them viable so that their structural integrity remains intact. in our experience, oocysts from different vertebrate host species fall into two groups, which, of necessity, need to be handled differently when collected under field conditions.

• Oocysts from birds, mammals, and terrestrial invertebrates and reptiles

These oocysts keep best when fresh feces are placed directly in 2-2.5% aqueous (w/v) potassium dichromate (K₂Cr₂O₇) in a ratio of 1 volume of feces : greater than or equal to 5 volumes postassium dichromate. In field collections, either snap-cap or screw-cap 16-25 ml vials work well, but one should not fill the vial all the way to the top; leave a layer of air between the top of the feces-dichromate mixture and the cap to allow the oocysts some atmospheric oxygen. Unfortunately, other solutions for feces, for example, 2% aqueous sulfuric acid (see Wash et al., 1985) or common laboratory fixatives for oocysts (see Duszynski and Gardner, 1991) have proven unsatisfactory either for keeping oocysts viable or for preserving them as types.

• Oocysts from amphibians, fish and aquatic invertbrates and reptiles

These oocysts often are very thin-walled and fragile and sometimes prove difficult to sporulate. When examining hosts from freshwater environments, fresh mucus and feces from the intestinal tract should be placed in vials with tap water or with filtered river water at room temperature. Likewise, mucus and gut contents of marine animals should be placed in containers with filtered seawater. These fecal-water solutions must be supplemented with 200 IU penicillin g/ml, 200 ug streptomycin/ml, and 0.5 ug Fugizone/ml (see Upton et al., 1988; Molnár, 1996).

• Laboratory processing

Upon return to the laboratory, the fecal-dichromate or fecal-water-antibiotic mixtures should be placed into a petri dish, any fecal pellets should be broken up, and the fecal material spread out in the dish and covered (Duszynski and Conder, 1977). The petri dishes generally should be maintained at room temperature (20-23 º C) for 7-10 days, which will allow any oocysts present to sporulate. Fecal-dichromate mixtures (terrestrial hosts) should not be refrigerated prior to the sporulation process as, in our experience, this will interfere with sporulation success. However, oocysts of some marine fishes were found to sporulate adequately only when the fecal-supplemented seawater mixture was placed on ice for 7-8 days (Upton et al., 1988); in this instance, the oocyst wall ruptured shortly after sporulation, releasing free sporocysts. In most species, however, after about 7-10 days, the mixture can be washed from the petri dish with clean potassium dichromate into a screw-cap jar (disposable baby bottle jars work well) filled only about half way and then put into a standard refrigerator (4-7 º C) until the material can be examined (sugar flotation) for the presence of oocysts. In our experience, oocysts of terrestrial vertebrates can remain viable, or at least stucturally intact, in the refrigerator for 3-4 years, whereas oocysts of certain fish coccidia (Upton et al., 1988, Molnár, 1996) may deteriorate soon after sporulation and die within a few days or weeks. Thus, it is probably best to study and document the structure of sporulated oocysts as soon as possible after they are sporulated.

Sporulated oocysts are best separated from the dichromate-fecal mixture by suspending an aliquot (1-3 ml) from the sample in 14-12 ml of modified Sheather's (Sheather, 1923) sugar flotation solution (500 g sucrose, 350 ml tap water, 5 ml phenol) via centrifugation (5 min at 2,000 rpm). It is important to use only number 1, 18 mm² coverslips on top of the 15 ml centrifuge tubes (those with a smooth, beaded edge work best) as this reduces the surface area that needs to be scanned for oocysts. After centrifugation, lift the coverslip carefully from the centrifuge tube, place onto a glass slide, and set aside for 5-10 minutes; this allows the sugar along the edges of the coverslip to harden and minimizes movement of the oocysts during observation, measurement, and photography. The coverslip should be scanned systematically (100-400x total magnification) until oocysts are located. Measuring and detailing the structure of sporulated oocysts should always be done only under an oil immersion objective (Neofluor and Nomarski optics are both useful). Apochromatic lenses are superior to achromats and the higher the numerical aperture on the objective lens, the more accurate will be the measurements.

We strongly suggest that the following criteria be presented to allow accurate evaluation of a proposed new species description for coccidia (family Eimeriidae). In the list of features below, we have followed the example of Lom and Arthur (1989) by marking those features that are indispensable with a solid circle, while those recommended for inclusion are marked with an open circle.

The Host

• Make sure that the host has been reliably identified by a knowledgable taxonomist who works with the host group and use the most up-to-date scientific name and its authority for the species.
• Host life stage infected (larva, juvenile, adult); this may be more important for some host groups (e.g., fish) than for others (e.g., mammals).
• Locality(ies) where infected hosts were collected; supply GIS coordinates whenever possible.
• Prevalence of infection by locality; include seasonal prevalences, if possible.
• Whenever possible, deposit the actual host specimen from which the new species was described (=symbiotype specimen, see Frey et al., 1992) into an appropriate, accredited museum.
• Give any ecological data, habitat data, or host genetic data that may seem relevant (for examples, see Couch et al., 1993; Wilber, Hanelt, et al., 1994; Wilber, McBee, et al., 1994).

The Sporulated Oocyst

• Use only sporulated oocysts (Fig.1, below) for mensural data.
• Supply measurements (means [plus/minus SD] and ranges) of at least 30-50 sporulated oocysts (100 would be best) to include: oocyst length (ol), oocyst width (ow), sporocyst length (sl), sporocyst width (sw), oocyst and sporocyst length : width (L:W) ratios (Figs. 1, 3).
• Note characteristic features of the outer oocyst wall and any inner layers to include: rough (r) or smooth (s) outer surface texture (row, Fig. 1; sow, Fig. 2); spines or conical projections (see McAllister and Upton, 1989); and relative number of layers and approximate thickness(es).
• Note presence/absence of the following structures in/on the sporulated oocyst and, if present, their size, approximate location, and a description: micropyle (m) and its width (mw, Fig. 2): micropyle cap (mc), its width and depth (mcw x mcd, Fig. 2); residuum (or), its diameter and description (or, Fig. 1); polar granule(s) (pg), its/their size, shape (pg, Fig. 1) or if they attach in a unique manner to the inner surface of the oocyst wall (see Parker and Duszynski, 1986).
• Note presence/absence of the following structures in/on the sporocyst: surface features such as sporopodia (spop, Fig 4); adhering membranes (mem, Fig. 4); ridges (see Box et al., 1980) or sutures (see Molnár, 1996); residuum (sr), its diameter and description (sr, Fig. 3); Stieda body (sb, Fig. 3) and associated filaments (fil, Fig. 4); substieda body (ssb, Fig. 3) and/or parastieda body (psb, Fig. 3).
• Note presence/absence of the following structures in/on the sporozoite: refractile body (srb, Fig. 3) and its/their number, diameter, and shape; nucleus (n, Fig. 4); andother defining features such as anterior striations (str, Fig. 4), if visible.
• Deposit at least 1 phototype (see Bandoni and Duszynski, 1988) of a sporulated oocyst into an accredited or appropriate national/regional museum. In the USA, these would include the United States National Parasite Collection (USNPC), Beltsville, Maryland, or the Manter Parasitology Laboratory (MPL), Lincoln, Nebraska.
• Provide a composite line drawing with the new species description that shows all of the structural features that make the new species unique; this should be drawn exactly to scale using the mean ol, ow, sl, and sw measurements and include all distinctive structural features mentioned in the description.
• Be sure that the published manuscript includes at least 1 photomicrograph of a sporulated oocyst and the USNPC, MPL, or other museum accession number in addition to the composite line drawing.
• Minimally, the new coccidian species should be compared in detail to the coccidian species that is most structurally similar to it within the same host genus; however, it would be even better to compare it to all described species found in the host family to avoid naming a new species based solely on host species.
• Assuming that the collected sample of oocysts used in the species description was "pure," (i.e., had only one putative species [morphotype]), then some oocysts should be preserved in 70% ethanol and archived in an accredited museum in the event that future workers choose to amplify and sequence the parasite's DNA (Relman et al., 1996).
• The organ(s) and which part was infected; state if any organs were examined or whether oocysts were collected only from fecal material.
• Pathogenicity and histopathological observations.

Also see Duszynski and Wilber, 1997.

Literature Cited

Bandoni, S.M. and D.W. Duszynski. 1988. A plea for improved presentation of type material for coccidia. Journal of Parasitology 74: 519-523.

Box, E.D., A.A. Marchiondo, D.W. Duszynski, and C.P. Davis. 1980. Ultrastructure of Sarcocystsis sporocysts from passerine birds and opossums: Comments on classification of the genus Isospora. Journal of Parasitology 66: 68-74.

Couch, L., D.W. Duszynski and E. Nevo. 1993. Coccidia (Apicomplexa), genetic diversity, and environmental unpredictability of four chromosmal species of the subterranean superspecies Spalax ehrenbergi (mole-rat) in Israel. Journal of Parasitology 79: 181-189.

Duszynski, D.W. 1986. Host specificity in the coccidia of small mammals: Fact or fiction? In Advances in protozoological research. M. Bereczky (ed.). Symposia Biologica Hungarica, Vol. 33. Akademiai Kiado, Budapest, Hungary, p. 325-337.

Duszynski, D.W. and G.A Conder. 1977. External factors and self-regulating mechanisms which may influence the sporulation of oocysts of the rat coccidium, Eimeria nieschulzi. International Journal of Parasitology 7: 83-88.

Duszynski, D.W. and S.L. Gardner. 1991. Fixing coccidian oocysts is not an adequate solution to the problem of preserving protozoan type material. Journal of Parasitology 77: 52-57.

Frey, J.K., T.L. Yates, D.W. Duszynski, W.L. Gannon and S.L. Gardner. 1992. Designation and curatorial management of type host specimens (symbiotypes) for new parasite species. Journal of Parasitology 78: 930-932.

Lom, J. and J.R. Arthur. 1989. A guideline for the preparation of species descriptions in Myxosporea. Journal of Fish Diseases 12: 151-156.

McAllister, C.T. and S.J. Upton. 1989. The coccidia (Apicomplexa: Eimeriidae) of testudines, with descriptions of three new species. Canadian Journal of Zoology 67: 2459-2467.

Molnár, K. 1996. Eimerian infection in the gut of the tube-nosed goby Proterorhinus marmoratus (Pallas) of the River Danube. Systematic Parasitology 34: 43-48.

Parker, B.B. and D.W. Duszynski. 1986. Coccidiosis of sandhill cranes (Grus canadensis) wintering in New Mexico. Journal of Wildlife Diseases 22: 25-35.

Relman, D.A., T.M. Schmidt, A. Gajadhar, M. Sogin, J. Cross, K. Yoder, O. Sethabutr and P. Echeverria. 1996. Molecular phylogenetic analysis of Cyclospora, the human intestinal pathogen, suggests that it is closely related to Eimeria spp. The Journal of Infectious Diseases 173: 440-445.

Sheather, A.L. 1923. The detection of intestinal protozoa and mange parasites by a flotation technique. Journal of Comparative Pathology 36: 266-275.

Upton, S.J., S.L. Gardner and D.W. Duszynski. 1988. The round stingray, Urolophus halleri (Rajiformes: Dasyatidae), as a host for Eimeria chollaensis sp. nov. (Apicomplexa: Eimeriidae). Canadian Journal of Zoology 66: 2049-2052.

Wash, C.D., D.W. Duszynski and T.L. Yates. 1985. Eimerians from diffrent karyotypes of the Japanese wood mouse (Apodemus spp.), with descriptions of two new species and a redescription of Eimeria montgomeryae Lewis and Ball, 1983. Journal of Parasitology 71: 808-814.

Wilber, P.G., B. Hanelt, B. Van Horne and D.W. Duszynski. 1994. Two new species and temporal changes in the prevalence of eimerians in a free-living population of Townsend's ground squirrels Spermophilus townsendii) in Idaho. Journal of Parasitology 80: 251-259.

Wilber, P.G., K. McBee, D.J. Hafner and D.W. Duszynski. 1994. A new coccidian (Apicomplexa: Eimeriidae) in the northern pocket gopher (Thomomys talpoides) and a comparison of oocyst survival in hosts from radon-rich and radon-poor soils. Journal of Wildlife Diseases 30: 359-364.