Fluorescent proteins reveal what trypanosomes get up to inside the tsetse fly
The discovery and development of fluorescent proteins for the investigation of living cells and whole organisms has been a major advance in biomedical research. This approach was quickly exploited by parasitologists, particularly those studying single-celled protists. Here we describe some of our experiments to illustrate how fluorescent proteins have helped to reveal what trypanosomes get up to inside the tsetse fly. Fluorescent proteins turned the tsetse fly from a “black box” into a bright showcase to track trypanosome migration and development within the insect. Crosses of genetically modified red and green fluorescent trypanosomes produced yellow fluorescent hybrids and established the “when” and “where” of trypanosome sexual reproduction inside the fly. Fluorescent-tagging endogenous proteins enabled us to identify the meiotic division stage and gametes inside the salivary glands of the fly and thus elucidate the mechanism of sexual reproduction in trypanosomes. Without fluorescent proteins we would still be in the “dark ages” of understanding what trypanosomes get up to inside the tsetse fly.
KeywordsGlossina Tsetse Trypanosoma brucei Sexual reproduction Meiosis Gametes Fluorescent proteins
Green fluorescent protein
Red fluorescent protein
Yellow fluorescent protein
Keith Vickerman’s iconic image of the life-cycle of Trypanosoma brucei in the mammalian and tsetse fly hosts  is well known to parasitologists - indeed, it is the cover illustration of J. D. Smyth’s parasitology textbook . This very detailed diagram was the culmination of several decades of research using light and electron microscopy, at the time the key research tools available to investigate parasite life-cycles. In this review, we want to show how the use of fluorescent proteins, an experimental approach that could not have been foreseen in 1985, has helped move this story forward (and perhaps something to bear in mind when you hit an impasse in your own research: the techniques you need may not yet have been invented).
In 2008, Osamu Shimomura, Martin Chalfie and Roger Tsien were awarded the Nobel prize for Chemistry for their work on the discovery of green fluorescent protein, GFP, and its application to biological research. The efforts of these three scientists made it possible to make living, fluorescent cells and whole organisms, a tremendous boon to many researchers, parasitologists included. Approaches incorporating the use of GFP were quickly adopted by trypanosomatid researchers e.g. [3, 4], and in the context of our research, fluorescent proteins turned the tsetse fly from a “black box” into a bright showcase to track trypanosome migration, development and mating inside the insect. Here we describe some of our results to illustrate how fluorescent proteins have helped to elucidate what trypanosomes get up to inside the tsetse fly.
Design of experimental crosses
An important omission from Vickerman’s diagram of the T. brucei life-cycle are stages involved in sexual reproduction; this is not surprising, as it was believed at the time that T. brucei reproduced asexually by binary fission. The first experimental evidence for genetic exchange in T. brucei appeared in 1986 when it was shown that hybrids were produced after co-transmission of two genetically distinct strains through the tsetse fly . However, because the hybrids were found to have DNA contents higher than expected for a diploid, it was uncertain whether this was true sexual reproduction involving meiosis and haploid gametes, or some kind of fusion creating a polyploid hybrid with subsequent loss of genetic material to return to the diploid state [6, 7]. The precise mechanism remained elusive, because of the complexity of the developmental cycle of T. brucei in the tsetse fly and the small numbers of trypanosomes available for analysis. For genetic exchange, the life-cycle stages of interest are those found in the salivary glands of the fly [8, 9], and as these are difficult to culture in vitro, experiments on genetic exchange involve co-transmission of the parental trypanosome lines through tsetse flies. While many experimental flies develop a midgut infection, particularly if fed substances such as glutathione that suppress their antimicrobial immune responses , few go on to develop a salivary gland infection. This low success rate means it is rare to find flies with a co-infection, a prerequisite for finding hybrids. Hence, for several years research on the mechanism of genetic exchange in trypanosomes made little headway.
Our next approach was to cross recombinant lines where one parent contained GFP driven by the T7 promotor and the other parent supplied the T7 polymerase, so that only hybrids that received transgenes from both parents would fluoresce (Fig. 1b). Although hybrid clones were generated in these crosses, no fluorescent trypanosomes were produced, perhaps because expression from the strong T7 promotor fatally disrupted normal transcription in the ribosomal RNA locus.
The third experimental design was both simple and effective. Red and green fluorescent trypanosomes were crossed, such that a quarter of the progeny would inherit both RFP and GFP genes and appear yellow fluorescent (Fig. 1c) . This experimental design had the major advantage that salivary glands with a mixed infection could easily be identified and taken forward for analysis, while those containing only a single parental trypanosome were discarded. This was a significant improvement in the efficiency of finding hybrids, as time was no longer wasted in futile analysis of single parental infections. In our first red/green cross, nearly every fly with one or both salivary glands containing a mixed infection of red and green fluorescent trypanosomes produced hybrids , leading to the conclusion that mating was not such a rare event as previously believed. We realised that the limiting factor for mating was whether both parental trypanosomes colonised the same salivary gland.
The red/green experimental design guaranteed success and we used it to investigate whether trypanosomes were capable of intraclonal mating as well as outcrossing by attempting to cross red and green fluorescent trypanosomes derived from the same clonal lineage ; we found that intraclonal mating was rare compared to outcrossing, suggesting that trypanosomes can distinguish self and non-self genotypes and might have mating types like other single-celled eukaryotes. This question remains unanswered, despite analysis of a large series of F1, F2 and back crosses, all based on the red/green cross design . We also investigated whether the trait of human infectivity conferred by the serum resistance associated (SRA) gene [16, 17] was inherited by hybrid progeny, creating new genotypes of human-infective trypanosomes. This had been predicted by population genetics analysis of the microsatellite genotypes of a large collection of T. brucei isolates, which produced clear evidence of admixture between human infective (T. b. rhodesiense) carrying the SRA gene and non-human-infective (T. b. brucei) lacking the SRA gene . Experimental crosses of three different strains of T. b. rhodesiense with various T. b. brucei strains yielded hybrid progeny, some of which had inherited the SRA gene and were resistant to lysis by human serum in vitro , confirming that new genotypes of T. b. rhodesiense can be produced by sexual reproduction between human infective and non-human-infective trypanosomes.
Meiosis and gametes
Development of trypanosomes in tsetse
Independent colonisation of the salivary glands. Salivary glands infection profiles of 60 flies infected with red and green fluorescent Trypanosoma brucei. Data from 
Paired salivary glands from individual flies
No. of flies
Trypanosome population identical in both glands (both red, both green or both mixed infection)
Trypanosome population differs between glands (red + green, red + mixed infection, green + mixed infection)
Only one gland infected (red, green or mixed infection)
We are indebted to the International Atomic Energy Agency, Vienna for supporting our experimental work on tsetse-trypanosome interactions through their generous supply of tsetse flies over many years. We thank all the colleagues who have contributed to this work over the years.
The work described in this review was funded by project grants from the UK Medical Research Council, The Wellcome Trust and the UK Biotechnology and Biological Sciences Research Council. The funding bodies played no part in the design of the study, or collection, analysis and interpretation of data, or in writing the manuscript.
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All data generated or analysed during this study are included in this published article.
WG drafted the manuscript. LP contributed additional experiments to those already published. Both authors read and approved the final manuscript.
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