Summary

Diplomonad flagellates belonging to the genera Hexamita and Spironucleus (family Hexamitidae) frequently infect fish from a wide range of salinities in many parts of the world (Becker, 1977; Kulda & Lom, 1964a; Kulda & Nohynkova, 1978). These flagellates are unique in having a double set of organelles, and a primitive metabolism lacking mitochondria and golgi. Most commonly the flagellates are reported from the lumen of the intestine, and their pathogenicity has been disputed. However more recently there have been reports of systemic infections where the diplomonads are believed to be pathogenic (Kent et al., 1992; Poppe et al., 1992).

The identity of diplomonads from fish is poorly known, partly because the small organisms (less than 20 um in length) are difficult to distinguish by light microscopy alone (Kulda & Lom, 1964a). Few have been studied by electron microscopy - the currently accepted method for species determination (Brugerolle, 1974; Brugerolle et al., 1973; Lee, 1985). Two of the consequences of this rudimentary knowledge is that pathogenicity of particular diplomonads, and host-parasite specificity, is poorly understood. Spironucleus is believed to be more highly evolved to to parasitic way of life than is Hexamita (Brugerolle, 1975; Siddall et al., 1992), and it has recently been reported that Spironucleus can attach to, and invade, host tissue, whereas Hexamita can not (Siddall et al., 1992). Thus accurate identification of diplomonads to genus and species level is important for understanding their relationship with invasion and disease.

Although there are numerous reports of diplomonads from fish, and many species have been named, only three species have been studied by ultrastructural methods. Thus only three species are valid according to currently accepted criteria.

The species, their hosts, and source of descriptions are as follows:

 Hexamita salmonis, freshwater salmonids (Ferguson, 1979; Poynton & Morrison, 1990)

 Spironucleus elegans, freshwater cichlids and amphibians (Brugerolle et al., 1973; Kulda & Lom, 1964b)

 Spironucleus torosa, marine gadids (Poynton & Morrison! 1990)

The genera can be distinguished by scanning electron microscopy (SEM) based on the morphology of the posterior end. In Hexamita the two posterior recurrent flagella emerge posteriorly, close together from the blunt end of the organism (Figure). In Spironucleus, the two recurrent flagella emerge posteriolaterally, separated by the caudal projection. Hexamita salmonis has an unadorned body, whereas Spironucleus torosa is characterized by the possession of a ring-like swelling, or torus, around the exit of each recurrent flagellum. The external morphology of S. elegans has never been studied by SEM. Thus SEM offers a valuable technique for distinguishing diplomonads at both the genus and species level.

Figure.

Transmission electron microscopy (TEM) provides further distinctions of genera and species (Figure. 1) (Brugerolle, 1974; Brugerolle et al., 1973; Lee, 1985). Hexamita has anterior flagella bases (kinetosomes) that are anterio-lateral, and spherical nuclei apposed only in their innermost regions. Spironucleus has anterior flagella bases (kinetosomes) that are anterio-medial, and elongate nuclei, that narrow and spiral together anteriorly. TEM can distinguish species of diplomonads, by the presence or absence of such features as microtubules and lamellae, and tufts of microfilaments.

To further our understanding of the significance of diplomonads, we need to be able to identify them, and also culture and preserve them so that experimental infections can be carried out. To date the only diplomonad from fish that has been successfully cultured is H. salmonis (Uzmann & Hayduk, 1963).

Overview of Oral Presentation

Studies of angel fish in Europe, using light microscopy have shown that Spironucleus elegans is present in the intestine of the fish (Kulda & Lom, 1964b). This flagellate species has also been studied by transmission electon microscopy using flagellates from their amphibian hosts in Europe ( Brugerolle et al., 1973). Despite the common occurrence of diplomonads in the intestinal tract of Angel fish Pterophyllum scalare in North America, their morphology, and hence identity, has never been comprehensively studied, and the external morphology of S. elegans has never been reported.

In view of the above, we decided to carry out a comprehensive study of the morphology of diplomonads from the lumen of the intestine of angel fish farmed in Florida. By chance, diplomonads were also found in a culture of lip lesion from the same hosts, and we also looked at these organisms in detail. The techniques we are using to describe the flagellates are light microscopy (both of live organisms and those stained by Protargol silver protein), scanning electron microscopy, and transmission electron microscopy.

The morphological questions we wish to answer are:

1.  What is the identity of the diplomonad from the gut?

2.  What is the identity of the diplomonad from the lip lesion culture?

3.  If S. elegans is present, what is its external morphology?

4.  Are diplomonads present in the lip lesions, or was there contamination?

In addition, we are also studying ways to cryo preserve and culture the diplomonads. When protocols have been successfully established, this will allow other investigators to study the diplomonads from angel fish, without needing to sacrifice additional hosts as is necessary at present.

The culture questions we wish to answer are:

1.  Which media support growth of angel fish diplomonads?

2.  Which temperatures are optimal?

3.  How may they be cryopreserved?

References

1.  Becker, C. D. 1977. Flagellate parasites of fish. In Kreier, J.P. (ed.) Parasitic Protozoa. Academic Press, New York, New York, I, 357 - 416.

2.  Brugerolle, G. 1974. Contribution a l'etude cytologique et phyletique des diplozoaires (Zoomastigophorea, Diplozoa, Dangeard 1910). 111. Etude ultrastructurale du genre Hexamita (Dujardin 1836). Protistologica, X: 83 - 90.

3.  Brugerolle, G. 1975. Contribution a l'etude cytologique et phyletique des diplozoaires (Zoomastigophorea, Diplozoa, Dangeard 1910). VI. Characteres generaux des diplozoaires. Protistologica, IX: 111 - 118.

4.  Brugerolle, G., Joyon, L. & Oktem, N. 1§73. Contribution a l'etude cytologique et phyletique des diplozaires (Zoomastigophorea, Diplozoa, Dangeard 1910). 11. Etude ultrastructurale du genre Spironucleus (Lavier 1936). Protistologica, IX: 495 - 502.

5.  Ferguson, H.W. 1979. Scanning and transmission electron microscopical observations on Hexamita salmonis (Moore, 1922) related to mortalities in rainbow trout fry Salmo gairdneri Richardson. Journal of Fish Diseases 2: 57 - 67.

6.  Kent, M.L., Ellis, J. Fournie, J.W., Dawe, S.C., Bagshaw, J.W., & Whitaker, D.J. 1992. systemic hexamitid (Protozoa: Diplomonadida) infection in seawater pen-reared chinook salmon Oncorhynchus tshawvtscha. Diseases of Aquatic Organisms 14: 81 - 89.

7.  Kulda, J. & Lom, J. 1964a. Remarks on the diplomastigine flagellates from the intestine of fishes. Parasitology, 54: 753 762.

8.  Kulda, J. & Lom, J. 1964b. Spironucleus elegans Lavier, parasite of fish. Cesk. Parasitol, XI: 187 - 192.

9.  Kulda, J. & Nohynkova, E. 1978. Flagellates of the human intestine and of intestine of other species. In: Kreier, J. P. (ed.) Parasitic Protozoa. Academic Press, New York, New York, II: 1 - 138.

10. Lee, J.J. 1985. Mastogophora: Order 7. Diplomonadida. In: Lee, J.J., Hutner, S.H. & Bovee, E.C. (ed.). An Illustrated Guide to the Protozoa, Society of Protozoologists, Lawrence, Kansas, pp. 130 -134.

11. Poppe, T.T., Mo, T.A., & Iversen, L. 1992. Disseminated hexamitosis in sea-cages Atlantic salmon Salmo salar. Diseases of Aquatic Organisms 14: 91 - 97.

12. Poynton, S.L. & Morrison, C.M. 1990. Morphology of diplomonad flagellates: Spironucleus torosa N.Sp. from Atlantic Cod Gadus morhua L., and Haddock Melanogrammus aeglefinus (L.) and Hexamita salmonis Moore from Brook Trout Salvelinus fontinalis (Mitchill). Journal of Protozoology 37: 369 - 383.

13. Siddall, M.E., Hong, H., & Desser, S.S. 1992. Phylogenetic analysis of the Diplomonadida (Wenyon, 1926) Brugerolle, 1975: evidence for heterochrony in protozoa and against Giardia lamblia as a "missing link". Journal of Protozoology 39: 361 - 367.

14. Uzmann, J.R. & Hayduk, S.H. 1963. In vitro culture of the flagellate protozoan Hexamita salmonis. Science 140: 290 - 292.