Molecular mechanisms of thermal resistance of the insect trypanosomatid Crithidia thermophila

Abstract

In the present work, we investigated molecular mechanisms governing thermal resistance of a monoxenous trypanosomatid Crithidia luciliae thermophila, which we reclassified as a separate species C. thermophila. We analyzed morphology, growth kinetics, and transcriptomic profiles of flagellates cultivated at low (23°C) and elevated (34°C) temperature. When maintained at high temperature, they grew significantly faster, became shorter, with genes involved in sugar metabolism and mitochondrial stress protection significantly upregulated. Comparison with another thermoresistant monoxenous trypanosomatid, Leptomonas seymouri, revealed dramatic differences in transcription profiles of the two species with only few genes showing the same expression pattern. This disparity illustrates differences in the biology of these two parasites and distinct mechanisms of their thermotolerance, a prerequisite for living in warm-blooded vertebrates.

Fig 1. Light microscopy of the isolate COLPROT 054.

(A) Giemsa-stained pro- and choanomastigotes are shown. Differential interference contrast (B) and fluorescent (C) microscopy of the DAPI-stained slides demonstrate presence of the nucleus and kinetoplast. Scale bars are 2.5 μm.

Fig 2. Electron microscopy of the isolate COLPROT 054.

(A) Scanning electron microscopy, (B) high-pressure freezing transmission electron microscopy. The longitudinal sections reveal typical features of trypanosomatids such as axoneme (a), flagellum (f), glycosomes (g), Golgi apparatus (ga), kinetoplast (k), mitochondrion (m), nucleus (n), and paraflagellar rod (pr). Scale bars are 1 μm.

Fig 3. Maximum likelihood phylogenetic tree of Trypanosomatidae.

This tree is based on concatenated 18S (SSU) rRNA and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene sequences and inferred with separation of model parameters for each of the two genes and for all three codon positions of gGAPDH gene. Bayesian posterior probabilities (5 million generations) and maximum likelihood bootstrap values (1,000 replicates) are shown at the nodes. Dots mark branches with maximal statistical support. Dashes (-) indicate bootstrap support below 50% or different topology. The tree was rooted with sequences of Paratrypanosoma confusum. Double-crossed branches are at 50% of their original lengths. The scale bar denotes the number of substitutions per site.

Fig 4. Comparison of growth of Crithidia thermophila (isolate COLPROT 054) at 23°C and 34°C.

(A) Growth curves; (B) morphology on Giemsa-stained smears, scale bars are 5 μm; (C) morphometry of cells. Boxplots are from three independent biological replicates (50 cells per replicate) and show 1^st quartile, median, and 3^rd quartile, and 1.5 x interquartile range values. All measurements are in μm.