Wolbachia symbiont infections induce strong cytoplasmic incompatibility in the tsetse fly Glossina morsitans

Abstract

Tsetse flies are vectors of the protozoan parasite African trypanosomes, which cause sleeping sickness disease in humans and nagana in livestock. Although there are no effective vaccines and efficacious drugs against this parasite, vector reduction methods have been successful in curbing the disease, especially for nagana. Potential vector control methods that do not involve use of chemicals is a genetic modification approach where flies engineered to be parasite resistant are allowed to replace their susceptible natural counterparts, and Sterile Insect technique (SIT) where males sterilized by chemical means are released to suppress female fecundity. The success of genetic modification approaches requires identification of strong drive systems to spread the desirable traits and the efficacy of SIT can be enhanced by identification of natural mating incompatibility. One such drive mechanism results from the cytoplasmic incompatibility (CI) phenomenon induced by the symbiont Wolbachia. CI can also be used to induce natural mating incompatibility between release males and natural populations. Although Wolbachia infections have been reported in tsetse, it has been a challenge to understand their functional biology as attempts to cure tsetse of Wolbachia infections by antibiotic treatment damages the obligate mutualistic symbiont (Wigglesworthia), without which the flies are sterile. Here, we developed aposymbiotic (symbiont-free) and fertile tsetse lines by dietary provisioning of tetracycline supplemented blood meals with yeast extract, which rescues Wigglesworthia-induced sterility. Our results reveal that Wolbachia infections confer strong CI during embryogenesis in Wolbachia-free (Gmm(Apo)) females when mated with Wolbachia-infected (Gmm(Wt)) males. These results are the first demonstration of the biological significance of Wolbachia infections in tsetse. Furthermore, when incorporated into a mathematical model, our results confirm that Wolbachia can be used successfully as a gene driver. This lays the foundation for new disease control methods including a population replacement approach with parasite resistant flies. Alternatively, the availability of males that are reproductively incompatible with natural populations can enhance the efficacy of the ongoing sterile insect technique (SIT) applications by eliminating the need for chemical irradiation.

Figure 1. The effects of antibiotic treatment on G. m. morsitans.

(A) Effect of yeast supplementation on percent larval deposition over two gonotrophic cycles between wild type flies maintained on normal blood supplemented with antibiotics (ampicillin or tetracycline) compared to flies maintained on yeast supplementation. The sample size (n) is above each column, and is represented as the number of females alive at the beginning of each gonotrophic cycle. (B) PCR analysis shows the Gmm^Wt flies are positive for Wigglesworthia (Wig Thic), Sodalis (Sod Chit) and Wolbachia (Wol Groel). In contrast offspring resulting from tetracycline treated females (A and B) lack all three of the symbionts. The bottom panel shows gDNA quality as measured by tsetse β-tubulin. (C) Presence of Wolbachia infections in late developing egg chambers of Gmm^Wt females. Nuclei are indicated by the blue DAPI stain and Wolbachia is shown by the red stain (D&E) Presence and absence of Wolbachia infections in early developing egg chambers of Gmm^Wt and Gmm^Apo females respectively. (F) Comparison of adult longevity between female Gmm^Wt and Gmm^Apo over a forty-day period on yeast supplemented diet. Error bars are reflective of standard error. Data points are offset for clarity.

Figure 2. Wolbachia-induced CI phenotype in G. m. morsitans.

Normal reproduction between Gmm^Wt females and males is discernible by a developing oocyte indicated by the white arrow and the presence of a larva in the uterus indicated by the pink arrow, following the first gonotrophic cycle. (B) Unmated adult female tsetse. Unmated Gmm^Wt females have an empty uterus and multiple developing oocytes indicated by white arrows. Note the transparent nature of the spermatheca reflective of lack of sperm (C) Manifested CI. CI is indicated by Gmm^Apo females mated with Gmm^Wt males by the absence of a larva in the uterus and deformed embryo indicated by the blue arrow. Many of these embryos were aborted without hatching into larva. Orange arrows indicate spermathecae in each image. Images were collected forty days (corresponding to the second gonotrophic cycle) post mating.