mir-31 mediated control of bacteriome size in tsetse flies

Abstract

Tsetse flies are the primary vectors of African trypanosomes, which are transmitted through blood feeding. To supplement this nutritionally limited diet, tsetse evolved an obligate mutualism with the bacterium Wigglesworthia glossinidia, housed within a specialized organ called the bacteriome. While the functional contributions of this symbiosis towards tsetse fitness have been studied, host morphological changes that accommodate this relationship remain less understood. In pregnant flies, variable expression of microRNAs (miRNAs) regulates protein expression, but the specific impacts are unknown. During pregnancy, high expression of fatty acyl-CoA reductase (far) within the bacteriome is indirectly correlated with miR-31 abundance and coincides with bacteriome size increase. We explored the roles of far and miR-31 towards this morphological change. Although RNAi effectively reduced far expression, bacteriome size still increased, suggesting its expansion is independent of far. In contrast, disrupting miR-31 activity resulted in significantly enlarged bacteriomes in virgin flies, resembling those of mated females. These results suggest that gene(s) other than far are regulated by miR-31 and may contribute to bacteriome remodeling during pregnancy, potentially to meet increased symbiosis demands. Ultimately, disrupting this obligate mutualism may present a promising target for future vector control strategies.

Keywords: Bacteriome; Glossina; MicroRNA; Symbiosis; tsetse.

Graphical abstract

Fig. 1

Validation of far expression and regulation by miR-31. (A) Representative RT-PCR results of bacteriomes isolated from two-week-old virgin female, mated female, and male tsetse. (B) far expression following tsetse receiving miR-31 antagomirs, missense antagomirs, or blood only. These assays were carried out on a minimum of 3 pools of 5 bacteriomes from each group with representatives shown here. Positive and negative controls were included but not shown. Amplicon sizes are indicated.

Fig. 2

Average anterior midgut measurements of age-matched two-week-old virgin and mated female G. morsitans. Mated females represent early pregnancy during the first gonotrophic cycle. (A) The distance from proventriculus to the bacteriome (outlined in red and labeled I and I’ in D). (B) The length of the bacteriome (outlined in purple and labeled II and II’ in D). (C) The width of the bacteriome at the bulge region (outlined in yellow and labeled III, III’, and III’’ in D). Each dot represents a single fly sample, and all measurements are in micrometers (µm) with error bars representing standard error of the mean (SEM). The P-values correspond to pooled t-tests with a statistical significance threshold of P < 0.05. (D) Tsetse fly with the bacteriome boxed and an inset representing the bacteriome as viewed under a stereoscope. The locations of measurements made in A-C are shown.

Fig. 3

(A) The survival curves of tsetse flies fed dsRNA (dsfar-fed: n = 85, blue; dsgfp-fed: n = 86, magenta) were analyzed using the log-rank test (Mantel-Cox), P > 0.05 upon comparison of survival between the two treatment groups. (B) Representative RT-PCR results of bacteriomes isolated from two-week old, mated females treated with either dsfar or dsgfp. (C) RT-qPCR results demonstrating the successful knockdown of far expression in dsfar but not dsgfp-treated flies (P= 0.0369). These assays were carried out on a minimum of 3 bacteriome pools (i.e. 5 bacteriomes per pool) from each group with representatives shown here. Positive and negative controls were included but not shown. Amplicon sizes are indicated.

Fig. 4

The average bacteriome width of age-matched two-week-old females. Each point represents the width (µm) of a single bacteriome and the error bars represent the standard error of the mean (SEM). (A) Comparison of bacteriome widths of age-matched control, mated dsfar-treated, or mated dsgfp-treated flies. No significant difference was found using a one-way ANOVA with Tukey's multiple comparisons test (P > 0.05). (B) Comparison of bacteriome widths of age-matched virgin flies fed antagomir (ant-31), missense (ms-31), or receiving blood only (virgin, control), (one-way ANOVA with Tukey's multiple comparisons tests; ant-31 vs ms-31; P= 0.02, and ant-31 vs control P< 0.0001).

Fig. 5

Reproductive fitness was not impacted by dsRNA oral administration. (A) No differences were observed between treatment or control groups in pupal weight (Kruskal-Wallis test, P > 0.05). (B) Average time to deposition was similar between dsfar- and dsgfp-treated flies over 60 days (two-tailed t-test, P > 0.05). (C) The mean time between deposition and eclosion (i.e. adult emergence) was not significantly different between dsRNA-treated and control flies (one-way ANOVA with Tukey's multiple comparisons test, P > 0.05). Each point represents a single pupa and the error bars represent the standard error of the mean (SEM). (D) There were no differences in the percentage of pupal eclosion among the larvae deposited by mated females either administered dsfar, dsgfp, or blood only (Fisher’s exact tests, P > 0.05).