Retracing the evolutionary history of the Trypanosomatidae: the use of kinetoplast DNA in molecular systematics, species identification and diagnostics

University of Technology Sydney. Faculty of Science.The Trypanosomatidae (class Kinetoplastida) are a diverse and widespread group of protists characterised by their possession of a unique and highly specialised mitochondrial homologue known as the kinetoplast. All trypanosomatid parasites are exclusively parasitic, with the majority of genera being restricted to a single invertebrate host (i.e. monoxenous lifecycle). However, they are primarily known for their pathogenic dixenous members (i.e. having a two-host life cycle), that serve as the aetiologic agents of several important neglected tropical diseases (NTDs) including leishmaniasis, Chagas disease and human African Trypanosomiasis. Recent advancements in molecular biology have improved our knowledge of evolutionary relationships between trypanosomatid species by revolutionising the genetic approach to trypanosomatid systematics. The kinetoplast DNA, and more specifically, the maxicircle genome represents a valuable taxonomic marker given its unique presence across all Kinetoplastids. The research described in this thesis was performed to explore the suitability of the kinetoplast DNA as a much-needed standardised framework for the taxonomic classification and species identification of protozoans falling within the Trypanosomatidae family. The main research outcome provides the most in-depth analysis of the trypanosomatid family to date, demonstrating extensive evidence for the superiority of the maxicircle for the taxonomic resolution of the Trypanosomatidae. Chapter 1 presents a comprehensive review outlining the important developments that have been made in the field of trypanosomatid taxonomy, advancing our current knowledge over the relationships between members of the trypanosomatid family. Chapter 2 demonstrates the superiority of the maxicircle genome for the phylogenetic inference of the Leishmaniinae. Phylogenetic analyses provided support imperative towards the Supercontinents hypothesis of dixenous parasitism within the Trypanosomatidae. The comprehensive analysis of the entire family of trypanosomatid parasites in Chapter 3 revealed strong support for the multiple origin and independent evolution of dixenous parasitism within the trypanosomatid family. Kinetoplast DNA is an organelle exclusive to the Kinetoplastids, unique in its structure, function and mode of replication and thus represents a unique diagnostic target, with the potential to differentiate different species and strains of . The implementation of novel diagnostic procedures for leishmaniasis such as the one reported in Chapter 4 is intended to establish a gold standard practice for the diagnosis and treatment of leishmaniasis. Ultimately, Chapter 5 reviews the completion of this thesis, demonstrating that use of the maxicircle genomes provide an excellent benchmark for future studies involving the phylogenetic analyses, taxonomic classification and species identification of the Trypanosomatidae

A new subspecies of Trypanosoma cyclops found in the Australian terrestrial leech Chtonobdella bilineata

Previously it was suggested that haemadipsid leeches represent an important vector of trypanosomes amongst native animals in Australia. Consequently, Chtonobdella bilineata leeches were investigated for the presence of trypanosome species by PCR, DNA sequencing, and in vitro isolation. Phylogenetic analysis ensued to further define the populations present. PCR targeting the 28S rDNA demonstrated that over 95% of C. bilineata contained trypanosomes; diversity profiling by deep amplicon sequencing of 18S rDNA indicated the presence of four different clusters related to the Trypanosoma (Megatrypanum) theileri. Novy-MacNeal-Nicolle (NNN) slopes with liquid overlay were used to isolate trypanosomes into culture that proved similar in morphology to Trypanosoma cyclops in that they contained a large numbers of acidocalcisomes. Phylogeny of 18S rDNA/GAPDH/ND5 DNA sequences from primary cultures and subclones showed the trypanosomes were monophyletic, with T. cyclops as a sister group. Blood meal analysis of leeches showed thatleeches primarily contained blood from Swamp Wallaby (Wallabia bicolour), human (Homo sapiens) or horse (Equus sp.). The leech C. bilineata is a host for at least five lineages of Trypanosoma sp. and these are monophyletic with T. cyclops; we propose Trypanosoma cyclops australiensis as a subspecies of T. cyclops based on genetic similarity and biogeography considerations.This study was funded in-part by the University of Technology Sydney, St Vincent’s Hospital Sydney and ICPMR Westmead. Prof Zhao-Rong Lun was supported by the UTS KTP visiting fellow programme to work on the clone isolation at UTS. Electron microscopy was performed at the Westmead Scientific Platforms, which are supported by the Westmead Research Hub, the Cancer Institute New South Wales, the National Health and Medical Research Council and the Ian Potter Foundation

The evolution of trypanosomatid taxonomy

Abstract Trypanosomatids are protozoan parasites of the class Kinetoplastida predominately restricted to invertebrate hosts (i.e. possess a monoxenous life-cycle). However, several genera are pathogenic to humans, animals and plants, and have an invertebrate vector that facilitates their transmission (i.e. possess a dixenous life-cycle). Phytomonas is one dixenous genus that includes several plant pathogens transmitted by phytophagous insects. Trypanosoma and Leishmania are dixenous genera that infect vertebrates, including humans, and are transmitted by hematophagous invertebrates. Traditionally, monoxenous trypanosomatids such as Leptomonas were distinguished from morphologically similar dixenous species based on their restriction to an invertebrate host. Nonetheless, this criterion is somewhat flawed as exemplified by Leptomonas seymouri which reportedly infects vertebrates opportunistically. Similarly, Novymonas and Zelonia are presumably monoxenous genera yet sit comfortably in the dixenous clade occupied by Leishmania. The isolation of Leishmania macropodum from a biting midge (Forcipomyia spp.) rather than a phlebotomine sand fly calls into question the exclusivity of the Leishmania-sand fly relationship, and its suitability for defining the Leishmania genus. It is now accepted that classic genus-defining characteristics based on parasite morphology and host range are insufficient to form the sole basis of trypanosomatid taxonomy as this has led to several instances of paraphyly. While improvements have been made, resolution of evolutionary relationships within the Trypanosomatidae is confounded by our incomplete knowledge of its true diversity. The known trypanosomatids probably represent a fraction of those that exist and isolation of new species will help resolve relationships in this group with greater accuracy. This review incites a dialogue on how our understanding of the relationships between certain trypanosomatids has shifted, and discusses new knowledge that informs the present taxonomy of these important parasites

Morphology of a female <i>Simulium</i> (<i>Morops</i>) <i>dycei</i>, Colbo 1976.

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

Inferred evolutionary relationship between <i>Zelonia australiensis</i> and other trypanosomatids using concatenated <i>18S rDNA</i> and <i>gGAPDH</i> sequences.

<p>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 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005215#pntd.0005215.ref036" target="_blank">36</a>] and the NJ method [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005215#pntd.0005215.ref037" target="_blank">37</a>]. 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) [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005215#pntd.0005215.ref102" target="_blank">102</a>]. 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 <i>Z</i>. <i>australiensis</i>. The bar represents the number of substitutions per site.</p

Morphology of trypanosomatid cells in axenic cultures.

<p>(A) Photomicrographs of Leishman stained <i>Zelonia australiensis</i> 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 <i>Zelonia australiensis</i> promastigotes viewed under phase contrast microscopy (400X magnification) showing several forms. (D) Photomicrographs of the various <i>Z</i>. <i>australiensis</i> 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 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005215#pntd.0005215.ref016" target="_blank">16</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005215#pntd.0005215.ref101" target="_blank">101</a>].</p

Precise coordinates of insect trap sites and trapping times.

<p>Precise coordinates of insect trap sites and trapping times.</p

Effect of haemoglobin on promastigote growth.

<p>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.</p

Transmission electron micrographs of promastigotes showing fine detail.

<p>(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).</p

Inferred evolutionary relationship between <i>Zelonia australiensis</i> and other trypanosomatids using concatenated <i>18S rDNA</i>, <i>gGAPDH</i>, <i>RPOIILS</i> and <i>HSP70</i> sequences.

<p>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 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005215#pntd.0005215.ref036" target="_blank">36</a>], and the NJ method [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005215#pntd.0005215.ref037" target="_blank">37</a>]. 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) [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005215#pntd.0005215.ref102" target="_blank">102</a>]. A solid diamond indicates a node that obtained a value of 100% for all three methods. The star highlights the phylogenetic position of <i>Z</i>. <i>australiensis</i>. The bar represents the number of substitutions per site.</p