Free-living ciliates as potential reservoirs for eukaryotic parasites: occurrence of a trypanosomatid in the macronucleus of Euplotes encysticus

Abstract

Background: Flagellates of the family Trypanosomatidae are obligate endoparasites, which can be found in various hosts. Several genera infect insects and occur as monoxenous parasites especially in representatives of Diptera and Hemiptera. These trypanosomatid flagellates probably share the worldwide distribution of their hosts, which are often infested by large numbers of endoparasites. Traditionally, their taxonomy was based on morphology, host origin, and life cycle. Here we report the characterization of a trypanosomatid infection detected in a protozoan, a ciliate collected from a polluted freshwater pond in a suburb of New Delhi (India).

Methods: Live observations and morphological studies applying light, fluorescence and transmission electron microscopy were conducted. Molecular analyses of host and parasite were performed and used for phylogenetic reconstructions and species (host) or genus level (parasite) identification.

Results: Although the morphological characteristics were not revealing, a high similarity of the trypanosomatids 18S rRNA gene sequence to Herpetomonas ztiplika and Herpetomonas trimorpha (Kinetoplastida, Trypanosomatidae), both parasites of biting midges (Culicoides kibunensis and Culicoides truncorum, respectively) allowed the assignment to this genus. The majority of the host population displayed a heavy infection that significantly affected the shape of the host macronucleus, which was the main site of parasite localization. In addition, the growth rate of host cultures, identified as Euplotes encysticus according to cell morphology and 18S rRNA gene sequence, was severely impacted by the infection.

Conclusions: The host-parasite system described here represents a recent example of free-living protists acting as environmental reservoirs for parasitic eukaryotic microorganisms.

Figure 1

1–6. General morphology and nuclear apparatus of infected and uninfected Euplotes encysticus Ind3. 1. Ventral view of the ciliate, fixed unstained specimen, DIC contrast. 2. Ventral view of the ciliate, silver-nitrate impregnation. 3. Dorsal view of the ciliate, fixed unstained specimen, DIC contrast. 4. Dorsal view of the ciliate, silver-nitrate impregnation. 5. Feulgen-staining of uninfected macronucleus. 6. Feulgen-staining of infected macronucleus. Dramatic changes in organelle shape are well visible. Ma, macronucleus; Mi, micronucleus. Bars = 15 μm (1, 3), 10 μm (2, 4, 5, 6).

Figure 2

7–10. Macronucleus of Euplotes encysticus infected with trypanosomatid flagellate Herpetomonas sp. Ind3. 7. General view of a heavily infected nucleus. 8. Part of the nucleus with several trypanosomatids (arrowheads). 9. Promastigote trypanosomatid released from the crushed macronucleus. 7–9. Living cells, DIC contrast. 10. Feulgen-staining. Highlighted are the host’s micronucleus (Mi) and macronucleus (Ma), the latter is infected with numerous flagellates; dark spots (arrows) represent nuclei and kinetoplasts of the parasites. Bars = 10 μm (7, 10), 5 μm (8, 9).

Figure 3

11–14. Living infected specimens of Euplotes encysticus Ind3 and its parasite Herpetomonas sp. Ind3 stained with ethidium bromide. 11. Host cell with a number of parasites released from the infected macronucleus into the cytoplasm (double arrow). 12. Isolated macronucleus harbouring numerous parasites. 13. Promastigote stages of the parasite in the medium. 14. Kinetoplast (small arrow) and nucleus (large arrow) of the parasite. 11, 12, 14. Fluorescent microscopy. 13. DIC contrast. Bars = 20 μm (11), 15 μm (12), 10 μm (13), 3 μm (14).

Figure 4

15–19. Ultrastructure of trypanosomatid flagellate Herpetomonas sp. Ind3 infecting Euplotes encysticus. 15–18. Flagellates harbored in the host cytoplasm. 15, 18. Longitudinal sections of flagellates. 16. Cross-sectioned flagellate through the flagellar pocket; the flagellum, which already bears a paraflagellar rod, is visible. Regularly distributed subpellicular microtubules are also well visible (diagonal arrow). 17. Cross section of two flagellate cells after fission. 19. Parasites in the macronucleus of the host. N, nucleus; horizontal arrow, kinetoplast; double arrows, flagellum. Bars = 1 μm.

Figure 5

20. Bayesian tree of members of Herpetomonas and Phytomonas. The tree was built on the unmodified character matrix of 23 Herpetomonas and 3 Phytomonas sequences (1,904 columns) employing the GTR + I + G model. Numbers associated to each node correspond to posterior probability and Maximum Likelihood bootstrap values. The scale bar represents an estimated sequence divergence of 4%. Underlined is the sequence of the trypanosomatid flagellate of Euplotes encysticus Ind3.