Experimental genetic crosses in tsetse flies of the livestock pathogen Trypanosoma congolense savannah

Abstract

Background: In tropical Africa animal trypanosomiasis is a disease that has severe impacts on the health and productivity of livestock in tsetse fly-infested regions. Trypanosoma congolense savannah (TCS) is one of the main causative agents and is widely distributed across the sub-Saharan tsetse belt. Population genetics analysis has shown that TCS is genetically heterogeneous and there is evidence for genetic exchange, but to date Trypanosoma brucei is the only tsetse-transmitted trypanosome with experimentally proven capability to undergo sexual reproduction, with meiosis and production of haploid gametes. In T. brucei sex occurs in the fly salivary glands, so by analogy, sex in TCS should occur in the proboscis, where the corresponding portion of the developmental cycle takes place. Here we test this prediction using genetically modified red and green fluorescent clones of TCS.

Methods: Three fly-transmissible strains of TCS were transfected with genes for red or green fluorescent protein, linked to a gene for resistance to the antibiotic hygromycin, and experimental crosses were set up by co-transmitting red and green fluorescent lines in different combinations via tsetse flies, Glossina pallidipes. To test whether sex occurred in vitro, co-cultures of attached epimastigotes of one red and one green fluorescent TCS strain were set up and sampled at intervals for 28 days.

Results: All interclonal crosses of genetically modified trypanosomes produced hybrids containing both red and green fluorescent proteins, but yellow fluorescent hybrids were only present among trypanosomes from the fly proboscis, not from the midgut or proventriculus. It was not possible to identify the precise life cycle stage that undergoes mating, but it is probably attached epimastigotes in the food canal of the proboscis. Yellow hybrids were seen as early as 14 days post-infection. One intraclonal cross in tsetse and in vitro co-cultures of epimastigotes also produced yellow hybrids in small numbers. The hybrid nature of the yellow fluorescent trypanosomes observed was not confirmed by genetic analysis.

Conclusions: Despite absence of genetic characterisation of hybrid trypanosomes, the fact that these were produced only in the proboscis and in several independent crosses suggests that they are products of mating rather than cell fusion. The three-way strain compatibility observed is similar to that demonstrated previously for T. brucei, indicating that a simple two mating type system does not apply for either trypanosome species.

Keywords: Green fluorescent protein; Mating; Red fluorescent protein; Sexual reproduction; Trypanosoma congolense; Tsetse fly.

Fig. 1

Visualization of fluorescent trypanosomes in tsetse fly organs. Red and green fluorescent Trypanosoma congolense savannah (TCS) in proventriculus (A) and midgut (B) from a tsetse fly dissected 25 days post-infection with a mixture of Gam2 RFP and 1/148 GFP. C Autofluorescence of the tsetse proboscis; a portion of an uninfected labrum is shown. Rows top to bottom: phase contrast, green fluorescence, red fluorescence, merge of red and green fluorescence, merge all. Areas of overlapping red and green fluorescence appear yellow in the merged images of the proventriculus (A) and midgut (B), but no yellow fluorescent trypanosomes were seen when the preparations were squashed under the coverslip to separate individual trypanosomes. Scale bar = 50 µm

Fig. 2

Examples of yellow fluorescent trypanosomes from proboscides of flies infected with mixtures of different TCS strains. Flies were dissected 23–39 days post-infection. A, B Gam2 RFP × 1/148 GFP; C, D 1/148 RFP × WG81 GFP; E, F Gam2 RFP × WG81 GFP; G intraclonal cross 1/148 RFP × 1/148 GFP. From left to right: phase contrast, green fluorescence, red fluorescence, merge of red and green fluorescence. Scale bar = 10 µm

Fig. 3

Morphology of yellow fluorescent TCS epimastigotes and trypomastigotes from proboscides. Examples of yellow fluorescent trypanosomes from proboscides of tsetse infected with Gam2 RFP and 1/148 GFP (dissected 28 days post-infection) and stained live with Hoechst 33342. Epimastigotes (A–F) varied greatly in morphology; some cells were replicating as they had two kinetoplasts and one or two nuclei (E, F). Trypomastigotes (G–I) were usually long and often had pronounced undulations at the anterior end. From left to right: phase contrast, Hoechst 33342, merge, green fluorescence, red fluorescence, merge of red and green fluorescence. Arrows indicate kinetoplasts. Scale bar = 10 µm

Fig. 4

Interacting red and green fluorescent trypanosomes. Trypanosomes from proboscides of tsetse infected with the TCS cross Gam2 RFP and 1/148 GFP (dissected 28 days post-infection). Panels A - C show examples of red and green fluorescent trypanosomes in close contact; the interacting cells are clearly of different sizes in B. In Panel D the trypanosome appears to have two anterior ends, suggesting recent cell fusion and exchange of cytoplasm. From left to right: phase contrast, green fluorescence, red fluorescence, merge of red and green fluorescence. Scale bar = 10 µm

Fig. 5

Clumps of red, green and yellow fluorescent trypanosomes. Trypanosomes from proboscides of tsetse infected with the TCS cross Gam2 RFP and 1/148 GFP (dissected 32–39 days post-infection). Panels A - G show groups of red, green and yellow fluorescent trypanosomes, which are mostly of short conformation. From left to right: phase contrast, green fluorescence, red fluorescence, merge of red and green fluorescence, merge all. Yellow fluorescent trypanosomes are arrowed. Scale bar = 10 µm

Fig. 6

Groups of red, green and yellow fluorescent trypanosomes. Trypanosomes from proboscides of tsetse infected with the TCS cross Gam2 RFP and 1/148 GFP (dissected 28 days post-infection). In A one red and one green fluorescent trypanosome are adjacent and appear to be attached together at their anterior ends by debris. In B one yellow fluorescent trypanosome (arrowed) appears joined to two red ones at the anterior. From left to right: phase contrast, Hoechst 33342 merge, green fluorescence, red fluorescence, merge of red and green fluorescence, merge with phase contrast image. Scale bar = 10 µm

Fig. 7

Putative asymmetric division of epimastigotes. Trypanosomes from TCS cross Gam2 RFP and 1/148 GFP (dissected 20 days post-infection). Trypanosomes have both red and green fluorescence (A, C and E), but either green (B) or red fluorescence (D), respectively. From left to right: phase contrast, Hoechst 33,342 merge, green fluorescence, red fluorescence, merge of red and green fluorescence, merge with phase contrast image. Scale bar = 10 µm

Fig. 8

In vitro mixing of red and green fluorescent epimastigotes; 1/148 RFP and Gam2 GFP epimastigotes were mixed in vitro, but very few groups of red and green trypanosomes such as that shown in A were found, most clumps being a single colour. Co-cultures were found to contain a few yellow fluorescent trypanosomes (arrowed) at 21 days post-mixing (B–D), presumably resulting from cytoplasmic exchange between cells of the different strains. From left to right: phase contrast, green fluorescence, red fluorescence, merge of red and green fluorescence. A Scale bar = 20 µm; B–D scale bar = 10 µm