Evidence for haploidy in metacyclic forms of Trypanosoma brucei.

Abstract

The parasitic flagellate Trypanosoma brucei undergoes a series of morphologic and metabolic changes during its passage in the digestive organs of its insect vector, a Glossina or tsetse fly. This morphogenesis ends by the differentiation, in the salivary gland of the fly, of the metacyclic form, which will be transmitted in the bloodstream of the mammalian host. On the basis of DNA microfluorometric measurements, we propose that these metacyclic trypanosomes have a haploid amount of DNA, compared to that of bloodstream forms and also of the proventricular forms, which initiate the invasion of the salivary glands. It can be inferred that trypanosomes undergo meiosis during their developmental cycle in the tsetse fly's salivary glands and syngamy shortly after cyclic transmission.During its life cycle, Trypanosoma brucei differentiates into a series of cytologically and metabolically distinct forms. The principal forms identified in the blood of the mammalian host are the slender form, which rapidly divides and can undergo antigenic variation, and the stumpy, nondividing form. After the tsetse fly has fed on blood from an infected mammalian host, the ingested trypanosomes differentiate into the procyclic and proventricular forms in the digestive tract and subsequently into the epimastigote and metacyclic forms in the salivary glands of the fly. Although many aspects of this parasitic cycle are now well documented (see ref. 1 for a recent review), the existence of a sexual cycle in trypanosomes has been and still remains an open question (2). The identification of mitosis, meiosis, and the ploidy of these parasites is not possible by direct cytogenetic analysis because their genome does not condense into defined chromosomes at any stage of the cell cycle. However, evidence based on isoenzyme studies (2, 3) and on the recent demonstration of hybrid formation between two clones of T. brucei (4) shows that genetic exchange does take place in trypanosomes. Analysis of restriction site polymorphisms (5) and direct measurements of DNA content (6) and genome complexity (7) indicate that T. brucei is diploid. Borst et al. (6) measured the nuclear DNA content of T. brucei using quantitative absorption and fluorescence cytophotometry of individual Feulgen reaction-pararosaniline-stained cells. They performed their analyses on bloodstream and procyclic culture forms and concluded that both forms have the same DNA content. However, the amount of DNA has never been measured in trypanosomes during their developmental cycle in the tsetse fly vector. Therefore, we estimated by direct microfluorometry the nuclear DNA content, after Feulgen reaction-pararosaniline staining of proventricular and metacyclic trypanosomes. These forms are found in the saliva of flies just before and after development of the parasite in the salivary glands, respectively. The quantitative cytochemical method we used is based on the observation of a linear relationship between the amount of fluorescence and the DNA content of Feulgen-stained nucl...

Chromosome organization of the protozoan Trypanosoma brucei.

“…The trypanosome minichromosomes are likely to constitute a separate repertoire, since they do not appear to follow Mendelian inheritance (33), and we therefore assume that the trypanosome genome is aneuploid for these minichromosomes which might represent amplified DNA (M. Weiden and L. H. T. Van der Ploeg, unpublished data).…”

“…Different models to explain genetic exchange in trypanosomes have been proposed. Some models propose cell fusion generating a tetraploid heterokaryon which, by random chromosome loss, reestablishes a neardiploid genome content, while other models propose haploid gametes (8,12,17,21,22,(24)(25)(26)33).…”

Evidence for diploidy in metacyclic forms of African trypanosomes.

“…Recently the same conclusion, as far as T. congolense is concerned, has been reached from Feulgen microfluorometry measurements on two clones (29), in spite of the fact that in one of these clones a difference between MET and BSF was found of the same order as that on which it was previously concluded that T. brucei MET are haploid (5).…”

“…Not a single MET with only half the fluorescence intensity of BSF of the same species was detected. This also applies to the MET of T. brucei STIB 247 [a clone of which was used as parent in the initial genetic study (1)], reported in a previous study (5) Fig. 1) is considered to be the mean value of the cells in G1 phase.…”

“…The hybridization experiments (1) indicated that the metacyclic stages (MET), found in the salivary glands of tsetse flies infected with T. brucei, could not be haploid, as has also been concluded from flow cytometric studies (3) and from the observation of hybrid genotypes in clones derived from single MET (4). Almost simultaneously, however, contradictory results were published, based on direct microfluorometry of total nuclear DNA of T. brucei life cycle stages, which suggested that MET were haploid and that they should be considered as gametes (5). In an attempt to bridge this apparent contradiction between the genetic and microfluorometric results, it was postulated that the hybrids might be tetraploid (6).…”