Cell shape is one, often overlooked, way in which protozoan parasites have adapted to a variety of host and vector environments and directional transmissions between these environments. Consequently, different parasite life cycle stages have characteristic morphologies. Trypanosomatid parasites are an excellent example of this in which large morphological variations between species and life cycle stage occur, despite sharing well-conserved cytoskeletal and membranous structures. Here, using previously published reports in the literature of the morphology of 248 isolates of trypanosomatid species from different hosts, we perform a meta-analysis of the occurrence and limits on morphological diversity of different classes of trypanosomatid morphology (trypomastigote, promastigote, etc.) in the vertebrate bloodstream and invertebrate gut environments. We identified several limits on cell body length, cell body width and flagellum length diversity which can be interpreted as biomechanical limits on the capacity of the cell to attain particular dimensions. These limits differed for morphologies with and without a laterally attached flagellum which we suggest represent two morphological superclasses, the ‘juxtaform’ and ‘liberform’ superclasses. Further limits were identified consistent with a selective pressure from the mechanical properties of the vertebrate bloodstream environment; trypanosomatid size showed limits relative to host erythrocyte dimensions. This is the first comprehensive analysis of the limits of morphological diversity in any protozoan parasite, revealing the morphogenetic constraints and extrinsic selection pressures associated with the full diversity of trypanosomatid morphology.

Funding: EG is a Royal Society University Research Fellow. Work in KG’s laboratory is funded by The Wellcome Trust. This work was initiated whilst RJW was funded by a Wellcome Trust 4-year PhD studentship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2013 Wheeler et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In order to detect these potential constraints or selective pressures, we performed a literature-based meta-analysis of trypanosomatid morphology in a wide range of species. Morphometric data from 248 isolates of trypanosomatids from the vertebrate bloodstream or invertebrate host were extracted from the literature to analyse cell length, cell width, flagellum length and flagellar pocket positioning. We established the concept of two morphological superclasses, the juxtaforms (trypomastigotes and epimastigotes) and liberforms (promastigotes, choanomastigotes and opisthomastigotes) on the bases of the phylogeny of the species in which they occur and the incapacity of trypanosomatids to transition between these two superclasses in the life cycle as a basis for analysing these morphometric data. The correlations of quantitative morphological measures were used to analyse whether morphological superclasses are a valid concept and determine limits on morphological variation which may be associated with constraints intrinsic to the juxtaforms and liberforms. In juxtaforms, morphological diversity, in combination with correlation of trypanosomatid shape with host erythrocyte sizes, was used to analyse limits on morphological variation in the blood environment. Some of these limits may be associated with selective pressures from the bloodstream and we determine which are consistent with proposed functions of cell shape and motility in T. brucei. We then discuss which mechanisms may apply constraints and selection pressures consistent with our observations which may have driven the evolution of trypanosomatid morphological diversity.

We reasoned that more information about constraints and selective pressures acting on trypanosomatid shape could be inferred by analysing limits on trypanosomatid shape and size quantitatively, and that this could be done most effectively if we considered multiple classes of trypanosomatid morphology in combination. Whether any particular limit in morphological diversity arises from a constraint or a selective pressure could be determined by analysing multiple classes of morphology in different environments. Constraints universal to all trypanosomatids will give rise to limits to cell shape in all species and classes, while constraints restricted to only particular morphological classes will give rise to limits to cell shape within only those classes. Limits in cell shape observed in trypanosomatids of a particular morphological class in one host environment but not another may have arisen through host selective pressures.

The diversity, and limits on diversity, of the morphologies of life cycle stages of any parasite are likely to have arisen through two biological phenomena. Firstly intrinsic biomechanical constraints arising from the cell organisation (and its growth and division) may limit the range of potential viable cell shapes. Secondly selective pressures from the host environment may limit the range of these viable cell shapes actually observed in different host environment. In qualitative terms if occurrence of particular morphological classes are limited to particular environments it is suggestive of a selection pressure; for example in trypanosomatids a selection for trypomastigotes in the vertebrate bloodstream [1] , [5] – [9] and for amastigotes in intracellular life cycle stages [10] – [12] appear to have occurred. In a similar line of reasoning, trypanosomatid morphologies universally have a sub-pellicular microtubule cytoskeleton, flagellum and flagellar pocket, even in immotile amastigotes [10] – [14] , indicating their role in basic cell organisation confers a biomechanical constraint. This limit in basic cell organisation is consistent with the well known and diverse roles of the flagellar pocket and associated structures in trypanosomatids, particularly for kinetoplast division [15] , [16] and endo and exocytosis [17] – [20] amongst others [21] .

Diagrams of six common, easily distinguished trypanosomatid morphologies [3] . A. Morphologies with a flagellum laterally attached to the cell body. B. Morphologies with a free flagellum (no lateral attachment of the flagellum to the cell body extending beyond the flagellar pocket neck). Metrics used to record cell morphology are indicated (cell body length, free flagellum length, kinetoplast–posterior distance (KP) and nucleus–posterior distance (NP)). Genera in which each morphology occurs [28] in are indicated, monophyletic genera [25] are underlined. C. Amastigote morphology, which does not have a long, motile, flagellum. D. Key. Structures associated with the flagellum (the basal body/pro-basal body pair (BB), flagellar pocket (FP), Axoneme (Ax) and paraflagellar rod (PFR)) are indicated.

Life cycle stage adaptation may incorporate many metabolic, biochemical and cell biological adaptations, including adaptation of cell shape. In trypanosomatids large morphological variation occurs both between life cycle stages and between species, despite great ultrastructural similarity which is universally conserved [1] , [2] . This diversity of shape has been catalogued extensively as light microscopy provided the earliest means for classifying these microorganisms, with six major morphological classes commonly defined by the position and depth of the flagellar pocket, flagellum length, and lateral attachment of the flagellum to the cell body ( Figure 1 ) [3] . The function of these cell shapes is largely unknown, although there are examples indicating that correct morphogenesis is vital for pathogenicity [4] . Together these properties mean the trypanosomatids are an excellent model for considering the function of cell shape in a unicellular parasite’s pathogenicity, and how this links to the capacity for morphological change for adaptation to different host environments.

Protozoan parasite life cycles are often characterised by specialised proliferative and transmissive life cycle stages, each of which represents an adaptation to that host environment (for a replicative stage) or a pre-adaptation to the next host environment and any conditions likely to be encountered during transmission (a transmissive stage). It is often the case that transmissive stages are non-proliferative, meaning a parasite life cycle is often made up of several linked proliferative cycles. Trypanosomatids, which are a diverse order of exclusively parasitic protozoa with a monoxonous life cycle in an insect host or a dixenous life cycle between an invertebrate and vertebrate or plant host, include many excellent examples of this life cycle structure. This family includes the human pathogens Leishmania spp., Trypanosoma brucei and Trypanosoma cruzi.

Results

In order to perform the meta-analysis of limits to trypanosomatid cell shape associated with constraints or selective pressures we generated a database of previously published trypanosomatid morphometric data for motile life cycle stages of many different species from different vertebrate and invertebrate hosts (Table S1). The literature describing new species or isolates was systematically surveyed, guided by existing indices of trypanosomes [22], [23] and other trypanosomatids [23], [24], and supplemented by database searches for more recently described species and isolates. This yielded approximately 250 references and each was screened to determine if it included suitable morphometric data. Briefly (for more detail see Materials and Methods), if morphology measurements were available and taken from a sample immediately derived from a host or axenic cultures immediately derived from an isolate from a host, and data were presented as representative of the complete range of trypanosomatid dimensions present in that life cycle stage then it was judged to be of sufficient quality for inclusion in the morphometry database. Cell body length and width, free and total flagellum length and kinetoplast to posterior distance and the ranges of each of these measures (or the available subset) were recorded. All morphological analyses, unless otherwise indicated, are derived from these data which is the first quantitative survey of trypanosomatid shape of this magnitude. Data collection was focused on motile life cycle stages and identified examples of Trypanosoma in the bloodstream and invertebrate hosts, and Blastocrithidia, Leishmania, Phytomonas, Leptomonas, Crithidia, Herpetomonas, Strigomonas, Angomonas and Paratrypanosoma in invertebrate hosts. This represents coverage of the Trypanosoma, Phytomonas, Leishmaniinae, Blastocrithidia, Herpetomonas and the endosymbiont-bearing clades [25] and the newly-identified Paratrypanosoma genus which is the most basal known trypanosomatid lineage [26]. Descriptions of Leishmania, Phytomonas and Trypanosoma morphology in the invertebrate host were comparatively rare, reports were dominated by those of amastigotes from vertebrates, promastigotes from plants and trypomastigotes from vertebrates respectively.

For analysis these morphometric data required placement into morphological classes. There are well established morphological classes (trypomastigote, epimastigote, promastigote, choanomastigote, opisthomastigote and amastigote) for trypanosomatids which have historically been used to define the genera (Figure 1) [24], [27], [28]. Several of these genera have since been shown to be paraphyletic [25] indicating this degree of morphological subclassification is taxonomically deceptive. We therefore aimed to superclassify morphologies in a more biological relevant way guided by the phylogeny of trypanosomatids and the morphological transitions they can undergo through the life cycle. A comprehensive analysis of trypanosomatid morphological class occurrence by phylogeny would have been desirable, however there is little overlap between species description by morphology and by genetic data. Therefore we instead focused on fewer high quality descriptions of species morphology through the whole life cycle where both small subunit (SSU) rRNA and glycosomal glyceraldehydephosphate dehydrogenase (gGAPDH) sequence data (the most commonly sequenced genes for species identification and phylogenetic analysis of trypanosomatids) were available in GenBank [29]. This analysis revealed two distinct classes of life cycle: those which transition between trypomastigotes, epimastigotes and/or amastigotes, and those which transition between promastigotes, choanomastigotes, opisthomastigote and/or amastigotes (Figure 2). These life cycle patterns clustered by both SSU and gGAPDH phylogeny (Figure 2A and D). On this basis we defined two morphological superclasses which we tentatively named for the apparent morphological distinction of whether the trypanosomatid has an extended region of lateral flagellum attachment; ‘juxtaform’ (from the Latin juxta (beside), incorporating trypomastigotes and epimastigotes), or a free flagellum with no lateral attachment extending beyond the flagellar pocket neck region no lateral attachment; ‘liberform’ (from the Latin liber (free), incorporating promastigotes, choanomastigotes and opisthomastigotes).

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larger image TIFF original image Download: Figure 2. Trypanosomatid morphology, life cycle and phylogeny are indicative of two morphological superclasses. A. Phylogeny of 12 representative trypanosomatids inferred from the small subunit (SSU) rRNA gene sequence, rooted with the outgroup B. saltans. Values at nodes indicate bootstrap support. The apparent paraphyly of Trypanosoma is a well documented example of a long branch attraction artefact [212]. B. Morphological classes attained though the 12 trypanosomatid life cycles. a[3], b[1], c[8], d[6], e[9], f[5], g[7], h[213], i[36], j[195], k[214], l[215], m[216], n[217], o[187], p[218], r[219], s[213]. C. Life cycle type and transmission route from the insect host in the 12 trypanosomatid life cycles. Relevance of the L. tarentolae amastigote in the life cycle [220], [221] and the transmission pathway [213] are debated. D. Phylogeny of the 12 trypanosomatids inferred from the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH), rooted with the outgroup B. saltans. Values at nodes indicate bootstrap support. https://doi.org/10.1371/journal.pone.0079581.g002

The differences in morphology of juxtaforms and liberforms suggests there may be significant differences in the presence or molecular composition of major cytoskeletal components, particularly the flagellum attachment zone (FAZ), but also the paraflagellar rod (PFR), sub-pellicular microtubules, the flagellar pocket collar and the bilobe structure. However, a survey of presence or absence of homologs to known proteins in these structures in two representative juxtaforms (T. brucei and T. cruzi) and two representative liberforms (Leishmania mexicana and Crithidia fasciculata) with published genomes [30]–[33] did not reveal any clear groups of absent homologs (Figure 3). Therefore, in the absence of clear molecular markers for these two morphological superclasses, species were assigned to each superclass on the basis of genera for analysis; Trypanosoma and Blastocrithidia being juxtaform and Leishmania, Phytomonas, Leptomonas, Crithidia, Herpetomonas, Strigomonas, Angomonas and Paratrypanosoma being liberform. Genera were taken as those listed by Sergei Podlipaev [23], or any later reclassification. We identified one species with an ambiguous superclass arising from reclassification of its genus over time; Strigomonas culicis, which was previously classified as a Blastocrithidia [34]. Unlike the other Strigomonas species, S. culcis was analysed with the juxtaforms because of its epimastigote morphology.

We analysed trypanosomatid shape and size diversity to determine whether limits to cell shape diversity across many trypanosomatid species correlate with different morphological classes, and what this implies for attainable trypanosomatid morphologies and their morphogenesis. Trypanosoma species have life cycle stages with juxtaform morphology both in and outside of the bloodstream where they may be subject to similar morphogenetic constraints, but different selective pressures. We therefore analysed the morphometric data particularly considering Trypanosoma species and whether there were limits to morphological diversity in different hosts which could be attributed to host selective pressures.