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. 2015 Aug 15;81(16):5375-86.
doi: 10.1128/AEM.00553-15. Epub 2015 May 29.

"Wigglesworthia morsitans" Folate (Vitamin B9) Biosynthesis Contributes to Tsetse Host Fitness

Anna K Snyder  1 , Rita V M Rio  2
Affiliations

"Wigglesworthia morsitans" Folate (Vitamin B9) Biosynthesis Contributes to Tsetse Host Fitness

Anna K Snyder et al. Appl Environ Microbiol. .

Abstract

Closely related ancient endosymbionts may retain minor genomic distinctions through evolutionary time, yet the biological relevance of these small pockets of unique loci remains unknown. The tsetse fly (Diptera: Glossinidae), the sole vector of lethal African trypanosomes (Trypanosoma spp.), maintains an ancient and obligate mutualism with species belonging to the gammaproteobacterium Wigglesworthia. Extensive concordant evolution with associated Wigglesworthia species has occurred through tsetse species radiation. Accordingly, the retention of unique symbiont loci between Wigglesworthia genomes may prove instrumental toward host species-specific biological traits. Genome distinctions between "Wigglesworthia morsitans" (harbored within Glossina morsitans bacteriomes) and the basal species Wigglesworthia glossinidia (harbored within Glossina brevipalpis bacteriomes) include the retention of chorismate and downstream folate (vitamin B9) biosynthesis capabilities, contributing to distinct symbiont metabolomes. Here, we demonstrate that these W. morsitans pathways remain functionally intact, with folate likely being systemically disseminated through a synchronously expressed tsetse folate transporter within bacteriomes. The folate produced by W. morsitans is demonstrated to be pivotal for G. morsitans sexual maturation and reproduction. Modest differences between ancient symbiont genomes may still play key roles in the evolution of their host species, particularly if loci are involved in shaping host physiology and ecology. Enhanced knowledge of the Wigglesworthia-tsetse mutualism may also provide novel and specific avenues for vector control.

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Figures

FIG 1
FIG 1
W. morsitans chorismate and folate biosynthetic loci exhibit differential expression within bacteriomes between the tsetse sexes at age 2 weeks (A) and through female adulthood and pregnancy (B). Graphs represent the normalized transcript abundance, with error bars signifying 1 standard error of the mean (SEM). Student's t tests and Mann-Whitney U tests were performed when variances were equal and unequal, respectively, and statistically significant differences are indicated above the bars. aroA, 3-phosphoshikimate 1-carboxyvinyltransferase; pabB, aminodeoxychorismate synthase subunit I; folP, 7,8-dihydropteroate synthase; rpsC, 30S ribosomal protein S3. The sample size (n) is indicated below each group.
FIG 2
FIG 2
Folate content and host transporter expression within G. morsitans bacteriomes. (A) Total folate quantity was determined using an L. rhamnosus microbiological assay. Statistically significant differences between groups, determined using ANOVA (P = 0.0006), are indicated above the bars using different letters. Error bars represent 1 SEM. The sample size (n) is indicated below each group. nd, samples in which folate was not detected. (B) Semiquantitative RT-PCR analysis of G. morsitans RFC expression, with G. morsitans β-tubulin serving as a loading control. The results for representative samples (n ≥ 3) are shown for each group.
FIG 3
FIG 3
Survival curves, created in JMP software (version 7.0) using the Kaplan-Meier method, of WT or aposymbiotic G. morsitans flies maintained on 10 mM glyphosate-supplemented blood for 60 days. Data are for ≥30 individuals per treatment. Significant differences between treatment groups, determined using the log-rank test, are indicated. †, P = 0.035; *, P ≤ 0.0001.
FIG 4
FIG 4
Concentrations of hemoglobin, representing undigested blood, from 2-week-old G. morsitans flies. Significant differences between control and glyphosate-treated flies at each time point are indicated. *, P < 0.05; **, P < 0.01. Error bars indicate 1 SEM (n = 3 per time point and treatment group).
FIG 5
FIG 5
Inhibition of W. morsitans chorismate biosynthesis impacts progeny development. (A) Comparison of uterine areas of age-matched (20-day-old) females (containing developing larva) maintained on the indicated treatments (P < 0.0001, ANOVA). Representative images for each treatment are included at the bottom. (B) Folate content within bacteriomes of 3-week-old mated females maintained on blood with or without 10 mM glyphosate supplementation. (C to E) Pupal weight (P < 0.0001, ANOVA) (C), teneral wing area (for females, P = 0.0002, ANOVA; for males, P < 0.0001, ANOVA) (D), and length of the pupal life stage (E) (for females, P = 0.0035, ANOVA; for males, P < 0.0001, ANOVA) of progeny deposited from females maintained on the specified blood meal supplementation. Statistically significant differences, determined using Student's t test or ANOVA, are indicated above the bars. The different letters indicate statistically significant differences between samples. Error bars signify 1 SEM. n, sample size.
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