Flow cytometric analysis and microsatellite genotyping reveal extensive DNA content variation in Trypanosoma cruzi populations and expose contrasts between natural and experimental hybrids

Abstract

Trypanosoma cruzi exhibits remarkable genetic heterogeneity. This is evident at the nucleotide level but also structurally, in the form of karyotypic variation and DNA content differences between strains. Although natural populations of T. cruzi are predominantly clonal, hybrid lineages (TcIId and TcIIe) have been identified and hybridisation has been demonstrated in vitro, raising the possibility that genetic exchange may continue to shape the evolution of this pathogen. The mechanism of genetic exchange identified in the laboratory is unusual, apparently involving fusion of diploid parents followed by genome erosion. We investigated DNA content diversity in natural populations of T. cruzi in the context of its genetic subdivisions by using flow cytometric analysis and multilocus microsatellite genotyping to determine the relative DNA content and estimate the ploidy of 54 cloned isolates. The maximum difference observed was 47.5% between strain Tu18 cl2 (TcIIb) and strain C8 cl1 (TcI), which we estimated to be equivalent to approximately 73 Mb of DNA. Large DNA content differences were identified within and between discrete typing units (DTUs). In particular, the mean DNA content of TcI strains was significantly less than that for TcII strains (P<0.001). Comparisons of hybrid DTUs TcIId/IIe with corresponding parental DTUs TcIIb/IIc indicated that natural hybrids are predominantly diploid. We also measured the relative DNA content of six in vitro-generated TcI hybrid clones and their parents. In contrast to TcIId/IIe hybrid strains these experimental hybrids comprised populations of sub-tetraploid organisms with mean DNA contents 1.65-1.72 times higher than the parental organisms. The DNA contents of both parents and hybrids were shown to be relatively stable after passage through a mammalian host, heat shock or nutritional stress. The results are discussed in the context of hybridisation mechanisms in both natural and in vitro settings.

Fig. 1

Flow cytometric analysis of relative DNA content in Trypanosoma cruzi discrete typing units (DTUs). (A–F) Overlaid DNA histograms for multiple cloned T. cruzi strains illustrating levels of variation within different DTUs. (G) DNA histogram for mixed sample population of two TcIIb strains with striking DNA content differences. (H) Box plot summary of variation within and between DTUs; grey boxes, inter-quartile ranges; horizontal lines inside grey boxes, median values; upper and low whiskers are the largest and smallest non-outlying values, respectively; asterisk, outlying value; outlying data were determined by the statistical software (SPSS v.14), additional putative outliers were identified (see Section 3.2). (I) Chart showing spread of estimated genome sizes for all samples across 10 Mb size categories; the number of strains from each DTU in each category is indicated by the split shading of the bars.

Fig. 2

Genotyping of microsatellite locus 10101(TA). The x-axis shows PCR product (allele) size; y-axis indicates fluorescence intensity (arbitrary units). Each panel shows the result from a single sample as indicated. Note the presence of three alleles in P251 cl7 and the presence of both TcIIb and TcIIc alleles in TcIId/IIe hybrid samples. Allele sizes are shown adjacent to corresponding peaks.

Fig. 3

DNA histograms for experimental hybrid and parental clones. The x-axes represent fluorescence intensity (arbitrary units) and the y-axes represent number of events in each fluorescence channel. (A) parent PI; (B) parent PII; (C) hybrid 1C2; (D) hybrid 2F9; (E) mixed sample of parent PI and hybrid 2D9; (F) overlaid histograms of parent PII (P, green or light grey trace) and hybrid 2C1 (Hy, red or dark grey trace).