Isolation of Novel Trypanosomatid, Zelonia australiensis sp. nov. (Kinetoplastida: Trypanosomatidae) Provides Support for a Gondwanan Origin of Dixenous Parasitism in the Leishmaniinae

Abstract

The genus Leishmania includes approximately 53 species, 20 of which cause human leishmaniais; a significant albeit neglected tropical disease. Leishmaniasis has afflicted humans for millennia, but how ancient is Leishmania and where did it arise? These questions have been hotly debated for decades and several theories have been proposed. One theory suggests Leishmania originated in the Palearctic, and dispersed to the New World via the Bering land bridge. Others propose that Leishmania evolved in the Neotropics. The Multiple Origins theory suggests that separation of certain Old World and New World species occurred due to the opening of the Atlantic Ocean. Some suggest that the ancestor of the dixenous genera Leishmania, Endotrypanum and Porcisia evolved on Gondwana between 90 and 140 million years ago. In the present study a detailed molecular and morphological characterisation was performed on a novel Australian trypanosomatid following its isolation in Australia's tropics from the native black fly, Simulium (Morops) dycei Colbo, 1976. Phylogenetic analyses were conducted and confirmed this parasite as a sibling to Zelonia costaricensis, a close relative of Leishmania previously isolated from a reduviid bug in Costa Rica. Consequently, this parasite was assigned the name Zelonia australiensis sp. nov. Assuming Z. costaricensis and Z. australiensis diverged when Australia and South America became completely separated, their divergence occurred between 36 and 41 million years ago at least. Using this vicariance event as a calibration point for a phylogenetic time tree, the common ancestor of the dixenous genera Leishmania, Endotrypanum and Porcisia appeared in Gondwana approximately 91 million years ago. Ultimately, this study contributes to our understanding of trypanosomatid diversity, and of Leishmania origins by providing support for a Gondwanan origin of dixenous parasitism in the Leishmaniinae.

Fig 1. Morphology of a female Simulium (Morops) dycei, Colbo 1976.

(A) Habitus of S. (M.) dycei female. (B) Mandible and lacinia of S. (M.) dycei female. (C) Genital fork of S. (M.) dycei female. (D) Anepisternal (pleural) membrane of S. (M.) dycei female. (E) Antenna of S. (M.) dycei female. (F) Wing of S. (M.) dycei female. (G) Hind leg tarsomeres of S. (M.) dycei female showing the pedisulcus and calcipala.

Fig 2. Effect of haemoglobin on promastigote growth.

Promastigotes were cultured in triplicate in three media differing in haemoglobin content; M1 (0.0099 g/L), M2 (0.495 g/L) and M3 (0.99 g/L). These media were accompanied by a negative control medium containing no haemoglobin (M0). Promastigote growth seems related to haemoglobin concentration, with the most rigorous growth and highest cell densities observed in M3; the media with the highest haemoglobin concentration. The slowest growth and lowest cell densities were observed in M0, the negative control.

Fig 3. Morphology of trypanosomatid cells in axenic cultures.

(A) Photomicrographs of Leishman stained Zelonia australiensis promastigotes cultured in M3, viewed under oil emersion microscopy (1000X magnification). (B) Photomicrograph of a round promastigote with gross morphological characteristics indicated including the nulcleus (N), kinetoplast (K), flagellar pocket (FP), and flagellum (Fl). (C) Wet mount photomicrograph of live axenically cultured Zelonia australiensis promastigotes viewed under phase contrast microscopy (400X magnification) showing several forms. (D) Photomicrographs of the various Z. australiensis forms as seen in Leishman stained slides, prepared from axenically cultured parasites. The parasite shows a high degree of pleomorphism in culture. This has been reported for other trypanosomatids, and limits the use of morphology for classification of these organisms [16, 101].

Fig 4. Transmission electron micrographs of promastigotes showing fine detail.

(A) Fine structure closely associated with the flagellum (fl) including the kinetoplast (K), basal body (bb), flagella pocket (fp), axonemes (ax), kinetoplast disk (kD) and a multivesicular body (mvb). (B) Fine cell structures including the golgi body (gb), glycosomes (gl) and mitochondria (mt). Mitochondrial DNA (mD) is visible within the mitochondria and kinetoplast (K). (C) Longitudinal cross-section of promastigote showing the nucleus (Nu), elongated mitochondria (mt), karyosome (Ka) and pellicle (Pe). (D) Example of striated pattern cause by sectioning of promastigote across the subpellicular microtubules (s).

Fig 5. PCR-RFLP analysis of the newly isolated parasite and other Leishmaniinae.

Comparison of PCR products and Hae III restriction fragments generated for several Leishmaniinae, including Leptomonas seymouri and Wallacemonas collosoma. Stars indicate the PCR products and restriction fragments generated for Zelonia australiensis. Samples were run against a 50 bp Hyperladder molecular weight marker (Bioline). An additional gel image (far right) includes the Hae III digested PCR product from Z. australiensis compared to that of Leishmania donovani.

Fig 6. Inferred evolutionary relationship between Zelonia australiensis and other trypanosomatids using concatenated 18S rDNA and gGAPDH sequences.

This tree was constructed using sequences from 23 trypanosomatids, aligned to a total of 1302 positions with all gaps and missing data eliminated. The structure of this tree was inferred using three statistical methods; the ML method based on the Tamura-Nei model, the ME method [36] and the NJ method [37]. The same tree structure was predicted using each method. The first value at each node is the percentage of trees in which the associated taxa clustered together using the ML method (1000 replicates). The second and third number at each node is the percentage of replicate trees obtained for the ME and NJ methods respectively, in which the associated taxa clustered together in the bootstrap test (1000 replicates) [102]. A solid diamond indicates a node that obtained a value of 100% for all three methods. An open diamond indicates a node that obtained a value of at least 99% for each method. The star highlights the phylogenetic position of Z. australiensis. The bar represents the number of substitutions per site.

Fig 7. Inferred evolutionary relationship between Zelonia australiensis and other trypanosomatids using concatenated 18S rDNA, gGAPDH, RPOIILS and HSP70 sequences.

This phylogenetic tree was constructed using sequences from 15 trypanosomatids, aligned to a total of 2344 positions with all gaps and missing data eliminated. The structure of this tree was inferred using three statistical methods; the ML method based on the Tamura-Nei model, the ME method [36], and the NJ method [37]. The same tree structure was predicted using each method. The first value at each node is the percentage of trees in which the associated taxa clustered together using the ML method (1000 replicates). The second and third number at each node is the percentage of replicate trees obtained for the ME and NJ methods respectively, in which the associated taxa clustered together in the bootstrap test (1000 replicates) [102]. A solid diamond indicates a node that obtained a value of 100% for all three methods. The star highlights the phylogenetic position of Z. australiensis. The bar represents the number of substitutions per site.

Fig 8. Phylogenetic Time Tree inferring the evolutionary relationships between Zelonia australiensis and other trypanosomatids using concatenated 18S rDNA and RPOIILS sequences.

This tree was constructed using sequences from 29 trypanosomatids, aligned to a total of 784 positions with all gaps and missing data eliminated. The structure of this tree was inferred using three statistical methods; the ML method based on the Tamura-Nei model, the ME method [36], and the NJ method [37]. The same tree structure was predicted using each method. The predicted minimum divergence times for each node i.e. the values outside the brackets, were predicted using the RelTime method [40]. Estimated divergence times greater than 1 MYA are rounded to the nearest whole number. The error calculated for the divergence time at each node is shown in S2 Fig. Regarding values within brackets, the first number is the percentage of trees in which the associated taxa clustered together using the ML method (1000 replicates). The second and third number is the percentage of replicate trees obtained for the ME and NJ methods respectively, in which the associated taxa clustered together in the bootstrap test (1000 replicates) [102]. An open diamond indicates a node that obtained a value of 99% or greater for each method. A solid diamond indicates a node that obtained a value of 100% for all methods. A solid circle represents nodes that obtained a value of 60% or less for each method. A solid square represents a collapsed node. The star highlights the phylogenetic position of Z. australiensis. Branches are colour coded to indicate the current dispersion pattern for the different species. Note that Leishmania infantum is also found in European countries flanking the Mediterranean basin. This time tree was calibrated by setting the node depicting the divergence of Z. australiensis and Zelonia costaricensis at 41 to 36 (mean ~39) million years ago; a minimum time estimate for the vicariance event that separated these taxa.