The obligate mutualist Wigglesworthia glossinidia influences reproduction, digestion, and immunity processes of its host, the tsetse fly

Abstract

Tsetse flies (Diptera: Glossinidae) are vectors for trypanosome parasites, the agents of the deadly sleeping sickness disease in Africa. Tsetse also harbor two maternally transmitted enteric mutualist endosymbionts: the primary intracellular obligate Wigglesworthia glossinidia and the secondary commensal Sodalis glossinidius. Both endosymbionts are transmitted to the intrauterine progeny through the milk gland secretions of the viviparous female. We administered various antibiotics either continuously by per os supplementation of the host blood meal diet or discretely by hemocoelic injections into fertile females in an effort to selectively eliminate the symbionts to study their individual functions. A symbiont-specific PCR amplification assay and fluorescence in situ hybridization analysis were used to evaluate symbiont infection outcomes. Tetracycline and rifampin treatments eliminated all tsetse symbionts but reduced the fecundity of the treated females. Ampicillin treatments did not affect the intracellular Wigglesworthia localized in the bacteriome organ and retained female fecundity. The resulting progeny of ampicillin-treated females, however, lacked Wigglesworthia but still harbored the commensal Sodalis. Our results confirm the presence of two physiologically distinct Wigglesworthia populations: the bacteriome-localized Wigglesworthia involved with nutritional symbiosis and free-living Wigglesworthia in the milk gland organ responsible for maternal transmission to the progeny. We evaluated the reproductive fitness, longevity, digestion, and vectorial competence of flies that were devoid of Wigglesworthia. The absence of Wigglesworthia completely abolished the fertility of females but not that of males. Both the male and female Wigglesworthia-free adult progeny displayed longevity costs and were significantly compromised in their blood meal digestion ability. Finally, while the vectorial competence of the young newly hatched adults without Wigglesworthia was comparable to that of their wild-type counterparts, older flies displayed higher susceptibility to trypanosome infections, indicating a role for the mutualistic symbiosis in host immunobiology. The ability to rear adult tsetse that lack the obligate Wigglesworthia endosymbionts will now enable functional investigations into this ancient symbiosis.

FIG. 1.

PCR assay demonstrating the status of Wigglesworthia and Sodalis infections in gut (A) and abdomen (B) tissues from antibiotic-treated mothers (lanes 1 to 6) and from their adult progeny (lanes 7 to 10). Lanes: 1, control; 2, tetracycline fed; 3, ampicillin injected; 4, ampicillin fed; 5, carbenicillin fed; 6, carbenicillin-injected females; 7, progeny of ampicillin-injected females; 8, progeny of ampicillin-fed females; 9, progeny of carbenicillin-fed females; 10, progeny of carbenicillin-injected females. DNA samples were PCR amplified with diagnostic primers for the presence of Wigglesworthia and Sodalis and for input DNA quality. Negative samples were subjected to a second PCR amplification to confirm the absence of symbionts. This assay was carried out for a minimum of 12 individuals from each group, and one representative analysis is shown here.

FIG. 2.

qPCR analysis of Sodalis density in wild-type and Gmm^Wig− females (P value, <0.001). Values were normalized by tsetse tubulin gene copy number.

FIG. 3.

Images of bacteriome sections stained with Giemsa at ×40 magnification (A to C) and taken at ×10 magnification (D and E).

FIG. 4.

Panels A and B show images of cross sections of milk gland tubuli from untreated flies hybridized with rhodamine-labeled Wigglesworthia (A)- and Sodalis (B)-specific probes. Panels C and D show images of cross sections of milk gland tubuli from ampicillin-treated flies hybridized with Wigglesworthia (C)- and Sodalis (D)-specific probes. The top series of images shows the DAPI signal for all DNA; visible are the large nuclei of the milk gland cells along with the bacteria present in the lumen in normal flies but not in antibiotic-treated females. The middle series of images shows the rhodamine signal of the probes, and the bottom series displays the merged images of both DAPI and rhodamine data in color.

FIG. 5.

Host survival-associated fitness cost after elimination of Wigglesworthia. (A) Longevity. Percent survival determined over a 60-day period is shown. *, P value of <0.03. (B) Heat tolerance response. Percent survival determined over a 60-day period is shown. ♦ denotes Gmm^WT females, and indicates Gmm^Wig− females.

FIG. 6.

Time course of hemoglobin digestion in Gmm^WT and Gmm^Wig− females. Data represent three replicate experiments. The first time point in the gut hemoglobin level analysis corresponds to 1 h before the flies were given a blood meal. This time point represents 48 h after their last blood meal. The bars denote the standard deviations of the multiple samples tested for each time point in the experimental and control groups. denotes Gmm^Wig− females, and ♦ indicates wild-type females.