Fluorescent proteins reveal what trypanosomes get up to inside the tsetse fly

Abstract

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.

Keywords: Fluorescent proteins; Gametes; Glossina; Meiosis; Sexual reproduction; Trypanosoma brucei; Tsetse.

Fig. 1

Design of experimental crosses. Sequential experimental designs (a-c) used to investigate mating in Trypanosoma brucei. Diploid parental trypanosomes were genetically engineered to contain the genes indicated: GFP, gene for green fluorescent protein; GFP_TET, GFP gene under control of the TET repressor; TET, gene for bacterial TET repressor; GFP_T7, GFP gene driven by phage T7 promotor; T7 POL, gene for phage T7 polymerase; RFP, gene for modified red fluorescent protein. Parental trypanosome clones were co-transmitted through tsetse flies and the expected genotypes of hybrid progeny, assuming Mendelian inheritance, are as indicated

Fig. 2

Meiotic dividers in the salivary gland. Live phase contrast and epifluorescence images of trypanosomes of Trypanosoma brucei brucei strain J10 expressing the fusion protein YFP::DMC1 inside a tsetse salivary gland. Trypanosomes expressing the fluorescent fusion protein have the nucleus very near the posterior end. a Phase contrast. b Fluorescence. c Merge. Scale-bar: 5 μm

Fig. 3

Interactions and cytoplasmic exchange between gametes. Red fluorescent (J10 RFP) and green fluorescent (1738 GFP) trypanosomes separately derived from tsetse salivary glands at day 20 post-infection and mixed in vitro. The cluster contains several gametes characterised by their small, pear-shaped bodies and relatively long flagella; the righthand trypanosome (arrowhead) is a dividing epimastigote. The cluster contains five trypanosomes, three of which show both red and green fluorescence (arrows), indicating that they have exchanged cytoplasm. a Phase contrast. b Red and green fluorescence. c Green fluorescence. d Red fluorescence. Scale-bar: 10 μm

Fig. 4

Fluorescent trypanosomes within the bloodmeal inside the midgut. Green fluorescent trypanosomes (Trypanosoma brucei gambiense strain TH2) visualised in the bloodmeal of a tsetse fly 48 hours after the infected bloodmeal. a Brightfield image showing the upper extent of the bloodmeal in the anterior midgut. Scale-bar: 100 μm. b Fluorescence image revealing small numbers of green fluorescent trypanosomes distributed throughout the bloodmeal. Copyright: Creative Commons Attribution 4.0 License (https://creativecommons.org/licenses/by/4.0/). Citation: Gibson & Bailey (2003) The development of Trypanosoma brucei in the tsetse fly midgut observed using green fluorescent trypanosomes. Kinetoplastid Biology and Disease. 2003;2:1 [24]

Fig. 5

Attrition of trypanosome infection in midgut. Numbers of trypanosomes (Trypanosoma brucei gambiense strain TH2) present in individual tsetse flies (Glossina morsitans) on days 1–6 after infection. N = number of individual flies examined at each timepoint. Midgut infections have been divided into 5 categories according to the number of trypanosomes. Data from [24]

Fig. 6

Invasion of the ectoperitrophic space. Green fluorescent trypanosomes (Trypanosoma brucei gambiense strain TH2) visualised in the bloodmeal of a tsetse fly 72 hours (a) and 96 hours (b) after the infected bloodmeal. Each panel shows the brightfield image (left) and fluorescence image (right). In a the bloodmeal is held within the peritrophic matrix (PM, arrowed) and trypanosomes are restricted to the endoperitrophic space. In b the trypanosomes have invaded the ectoperitrophic space (es). Scale-bar: 50 μm. Copyright (panel b): Creative Commons Attribution 4.0 License (https://creativecommons.org/licenses/by/4.0/). Citation: Gibson & Bailey (2003) The development of Trypanosoma brucei in the tsetse fly midgut observed using green fluorescent trypanosomes. Kinetoplastid Biology and Disease. 2003;2:1 [24]

Fig. 7

Fluorescent trypanosomes in salivary gland. Paired salivary glands from a single fly dissected 4 weeks after infection with red and green fluorescent trypanosomes. In panel a the salivary gland contains only green fluorescent trypanosomes, while in panel b the gland has a mixed infection of red and green fluorescent trypanosomes. Scale-bar: 500 μm. Copyright: Creative Commons Attribution 4.0 License (https://creativecommons.org/licenses/by/4.0/). Citation: Peacock et al. (2007). Dynamics of infection and competition between two strains of Trypanosoma brucei brucei in the tsetse fly observed using fluorescent markers. Kinetoplastid Biology and Disease. 2007;6:4 [29]

Fig. 8

Life-cycle diagram. Diagram of the life-cycle of Trypanosoma brucei, including sexual stages