Dynamics of gamete production and mating in the parasitic protist Trypanosoma brucei

Abstract

Background: Sexual reproduction in Plasmodium falciparum and Trypanosoma brucei occurs in the insect vector and is important in generating hybrid strains with different combinations of parental characteristics. Production of hybrid parasite genotypes depends on the likelihood of co-infection of the vector with multiple strains. In mosquitoes, existing infection with Plasmodium facilitates the establishment of a second infection, although the asynchronicity of gamete production subsequently prevents mating. In the trypanosome/tsetse system, flies become increasingly refractory to infection as they age, so the likelihood of a fly acquiring a second infection also decreases. This effectively restricts opportunities for trypanosome mating to co-infections picked up by the fly on its first feed, unless an existing infection increases the chance of successful second infection as in the Plasmodium/mosquito system.

Results: Using green and red fluorescent trypanosomes, we compared the rates of trypanosome infection and hybrid production in flies co-infected on the first feed, co-infected on a subsequent feed 18 days after emergence, or fed sequentially with each trypanosome clone 18 days apart. Infection rates were highest in the midguts and salivary glands (SG) of flies that received both trypanosome clones in their first feed, and were halved when the infected feed was delayed to day 18. In flies fed the two trypanosome clones sequentially, the second clone often failed to establish a midgut infection and consequently was not present in the SG. Nevertheless, hybrids were recovered from all three groups of infected flies. Meiotic stages and gametes were produced continuously from day 11 to 42 after the infective feed, and in sequentially infected flies, the co-occurrence of gametes led to hybrid formation.

Conclusions: We found that a second trypanosome strain can establish infection in the tsetse SG 18 days after the first infected feed, with co-mingling of gametes and production of trypanosome hybrids. Establishment of the second strain was severely compromised by the strong immune response of the fly to the existing infection. Although sequential infection provides an opportunity for trypanosome mating, the easiest way for a tsetse fly to acquire a mixed infection is by feeding on a co-infected host.

Keywords: Competition; Human infectivity; Hybrids; Immune response; Mating; Sequential infection; Sexual reproduction; Trypanosoma; Tsetse.

Fig. 1

Experimental design. Tsetse flies were fed red (R) and green (G) fluorescent T. b. brucei clones in three different regimes: Teneral, together in the first bloodmeal as newly-emerged flies; Non-teneral, together on day 18; or Sequential, R or G on day 0, followed by the other colour on day 18. Flies were assigned to each treatment from the same batch of emergents and all flies received an infected or uninfected bloodmeal on the same day (day 0); non-teneral and sequential groups also received an infected bloodmeal on day 18; otherwise, flies were fed sterile horse blood (black arrows) until dissection

Fig. 2

Timecourse of initial establishment (days 1–5) of midgut infection in teneral, non-teneral and sequentially-fed flies. Flies (Glossina morsitans morsitans) were fed on a bloodmeal containing F1R1 and F1G2 as tenerals, 18 day non-tenerals, or sequentially with the F1R1 infective feed 18 days after the first feed with F1G2. a Percent of midguts that were positive or negative for FIR1 trypanosomes upon dissection; all midguts were positive for F1G2. The sequentially fed flies had significantly more negative midguts than both teneral (Fisher’s exact test: P = 0.02) and non-teneral flies (Fisher’s exact test: P = 0.04) at day 5. N = number of flies. b Average counts (log ± se) of F1R1 trypanosomes per macerated positive midgut. Treatment had a significant effect on the number of trypanosomes in midguts (ANOVA: F _(2,77) = 25.15, P < 0.0001), with significantly lower numbers of trypanosomes in the positive midguts for the sequentially fed flies than either teneral (Post-hoc LSD test: P < 0.0001) or non-teneral flies (Post-hoc LSD test: P = 0.04), and significantly lower numbers for non-teneral than teneral flies (Post-hoc LSD test: P < 0.0001). N = number of flies; trypanosome negative flies have been excluded from the total

Fig. 3

Trypanosome developmental stages from salivary exudate. Trypanosome developmental stages recorded from salivary exudates from flies (Glossina pallidipes) infected with a 1738G and b F1R1. Percentage of flies with trypanosomes in exudates that contained the development stage; n = number of flies with a sample containing trypanosomes on that day. Flies were given an infective feed containing 1738G on day 0, and subsequently fed a further infective feed containing F1R1 on day 18; the experiment continued to day 42. From day 11 onwards, flies were encouraged to probe on warmed glass slides before feeding. Results were initially obtained for 1738G alone (timecourse A, days 11–18), and then for both 1738G and F1R1, starting 11 days after the second infected feed with F1R1 (1738G, timecourse A, days 29–42; F1R1, timecourse B, days 11–24). Abbreviations: trypos proventricular trypomastigotes, asymdiv asymmetric dividers, epis salivary gland epimastigotes, metas metacyclics

Fig. 4

Trypanosome migratory and developmental stages in salivary exudate. Trypanosomes in the salivary probe of a sequentially infected fly (Glossina pallidipes) 35 days after infection with 1738G and 17 days after infection with F1R1. Asymmetric dividers (a) of both trypanosome clones are present as well as metacyclics (m). From left to right, upper row, brightfield, DAPI and merge; lower row, green fluorescence, red fluorescence, merge. Scale-bar: 10 μm

Fig. 5

Sexual stages in salivary exudate. Trypanosomes in the salivary probe of a sequentially infected fly (Glossina pallidipes) 31 days after infection with 1738G and 13 days after infection with F1R1. Cells shown have the typical morphology of a haploid gamete (g) and a trypanosome in meiosis I (m). Both trypanosomes were red fluorescent, i.e. F1R1. From left to right, brightfield, DAPI and merge. Scale-bar: 5 μm