Evaluation of Trypanosoma cruzi hybrid stocks based on chromosomal size variation
Authors: Elisa Cupolillo
Aurélio Pedroso
Bianca Zingales
Journal: Molecular and Biochemical Parasitology (2003)
Discipline: MICROBIOLOGY
Keywords: Trypanosoma cruzi; hybridisation events; chromosomal polymorphism; evolutionary information; karyotype data

Abstract:  Although all classical lines of evidence point to the fact that Trypanosoma cruzi has a predominantly clonal evolution, accumulating data show that some T. cruzi stocks are the result of hybridisation events. We evaluated whether chromosomal polymorphism would give evolutionary information on hybrid isolates. Twenty-three coding sequences were mapped on the chromosomes of nine parasite stocks, four of which are putative hybrids (CL Brener and rDNA group 1/2). Phenetic analyses of karyotype data were based on the absolute chromosomal size difference index (aCSDI), a method that assumes that the genomic distance between two organisms is the sum of the size differences between their homologous chromosomes. aCSDI-based dendrograms obtained from a variable number of probes (3-18 probes) defined in all the cases three clusters: two corresponding, respectively, to T. cruzi I and T. cruzi II groups; and a third one, to rDNA group 1/2. CL Brener was alternatively positioned in T. cruzi II or rDNA group 1/2 clusters. Three clusters were also observed in the dendrogram constructed with restriction fragment length polymorphism (RFLP) data from 18 probes. The topology of the chromosome and RFLP dendrograms is similar, with a significant correlation coefficient (r=0.86062; P&lt;0.0001), supporting a strong structuring of the clusters. This study also revealed that hybrid stocks have a larger proportion of two different-sized homologous chromosomes, as compared with non-hybrid strains. Overall, our results show that chromosomes are valuable characters for identification of evolutionary groups, in particular, T. cruzi hybrid organisms. 

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PubPeer comments:
 Figure 3. * Colored boxes highlight some  CL Brener lanes or parts of lanes that appear to look similar to other lanes in the same blot. ![file] , 
 The observation of Dr. Bike is correct: Colored boxes highlight some  CL Brener lanes or parts of lanes that appear to look similar to other lanes (u201cNRcl3u201d and u201cSO3cl5u201d) in the same blot. The justification follows: The samples loaded on the gel are from strains of Trypanosoma cruzi, a diploid protozoan that causes Chagas disease in humans. T. cruzi presents great genetic diversity, basically resulting from its clonal model of evolution. On the other hand, some strains are capable of recombination, originating hybrid strains. The lanes indicated by Dr. Bik correspond to the hybrid strains - CL Brener, NRcl3, SO3cl5. Data of the Southern blot, where the genomic DNA of several T. cruzi strains was hybridized with different probes, illustrates that the hybrid strains have identical or very similar genomic organization. Difference in the hybridization intensity of the bands of CL Brener, NRcl3 and SO3cl5 strains is likely a result of the amount of DNA loaded in the lanes and or differences in the number of gene copies in the DNA band. This observation, as well as reports from other authors, served as a basis for establishing the evolutionary origin of T. cruzi hybrid strains. Theoretical support The genetic diversity of Trypanosoma cruzi, the existence of hybrid strains and the models for the origin of hybrid strains have been widely documented in two reviews authored by one of the authors of the article in question (Zingales et al., Inf. Gen. Evol. 12, 240u2013253, 2012, doi:10.1016 j.meegid.2011.12.009; Zingales, Acta Tropica 184, 38-52, 2018, http: dx.doi.org 10.1016 j.actatropica.2017.09.017). The putative genetic origin of DTU TcV (represented by NRcl3, SO3cl5 and Sc43cl1 strains) and DTU TcVI (represented by CL Brener strain) is illustrated in Figure 1 in Zingales et al., 2012 (Inf. Gen. Evol. 12, 240u2013253, 2012, doi:10.1016 j.meegid.2011.12.009). Phylogeny inference and network genealogies support the genetic proximity of DTU TcV and TcVI strains. At the time we published the article by Pedroso et al. (2003) it had not yet been defined that T. cruzi strains should be referred to by six discrete typing units (DTUs TcI to TcVI) (see Zingales et al., Mem. Inst. Oswaldo Cruz 104: 1051-1054, 2009; https: doi.org 10.1590 S0074-02762009000700021). Our study established an important conclusion: u201cOverall, our results show that chromosomes are valuable characters for identification of evolutionary groups, in particular, T. cruzi hybrid organismsu201d (Pedroso et al., 2003; https: doi.org 10.1016 S0166-6851(03)00096-3). , 
 Dear Dr. Zingales, Thank you so much for your reply. It is always great if authors reply to questions raised about their work here on PubPeer. However, you might have missed the reason for my concern. I am not concerned that the CL Brener lanes show that the genomic organization of that strain are similar to that of other strains. I am concerns that the CL Brener lanes look identical to that of other lanes. I am worried that these lanes or parts thereof might have been digitally or physically copied to represent different strains. In the panels in which I am worried that this might have happened, sharp transitions are visible between the first lane and the second lane, suggestive of digital or physical splicing. It is not just the bands that are present at the same location, it is also stains and spots that appear to look the same. These splices are not visible in e.g. the Actin 2 or RNA Helicase blot, so this splicing appears to not be caused by a lack of room on the gels blots. I am afraid your explanation has not taken away my concerns. Would you still have the original blots available? If so, would you have higher resolution images available? , 
 #3 Dear Dr. Bink, Thank you for your message. Now, I understand what you mean in your query. I will look up the original blots with my former doctoral student Dr. Aurelio Pedroso. However, I emphasize that the study was carried out 20 years ago. At that time, hybridization images were obtained after exposure to X-ray films with intensifying screens at u221280 u00b0C. I believe that, as a function of time, the X-ray films must be oxidized. I will also verify with my ex-student if he still has the higher resolution images and will let you know. Thank you for doing this important check-up job. Best regards Bianca Zingales Professor Bianca Zingales, Ph.D. Department of Biochemistry Institute of Chemistry University of Su00e3o Paulo Su00e3o Paulo Brazil zingales@iq.usp.br bszodnas@iq.usp.br ,