Native microbiome impedes vertical transmission of Wolbachia in Anopheles mosquitoes

Abstract

Over evolutionary time, Wolbachia has been repeatedly transferred between host species contributing to the widespread distribution of the symbiont in arthropods. For novel infections to be maintained, Wolbachia must infect the female germ line after being acquired by horizontal transfer. Although mechanistic examples of horizontal transfer exist, there is a poor understanding of factors that lead to successful vertical maintenance of the acquired infection. Using Anopheles mosquitoes (which are naturally uninfected by Wolbachia) we demonstrate that the native mosquito microbiota is a major barrier to vertical transmission of a horizontally acquired Wolbachia infection. After injection into adult Anopheles gambiae, some strains of Wolbachia invade the germ line, but are poorly transmitted to the next generation. In Anopheles stephensi, Wolbachia infection elicited massive blood meal-induced mortality, preventing development of progeny. Manipulation of the mosquito microbiota by antibiotic treatment resulted in perfect maternal transmission at significantly elevated titers of the wAlbB Wolbachia strain in A. gambiae, and alleviated blood meal-induced mortality in A. stephensi enabling production of Wolbachia-infected offspring. Microbiome analysis using high-throughput sequencing identified that the bacterium Asaia was significantly reduced by antibiotic treatment in both mosquito species. Supplementation of an antibiotic-resistant mutant of Asaia to antibiotic-treated mosquitoes completely inhibited Wolbachia transmission and partly contributed to blood meal-induced mortality. These data suggest that the components of the native mosquito microbiota can impede Wolbachia transmission in Anopheles. Incompatibility between the microbiota and Wolbachia may in part explain why some hosts are uninfected by this endosymbiont in nature.

Keywords: competitive exclusion; dysbiosis; holobiome; malaria; microbe–microbe interactions.

Fig. 1.

Determining the optimal Wolbachia strain for vertical transmission in Anopheles mosquitoes. (A) Wolbachia density in microinjected mosquitoes assessed by qPCR at 7 dpi indicates the wAlbB strain infects Anopheles at significantly higher levels in both somatic and germ-line tissue. Approximately 9.3 × 10⁶ bacterial cells were injected per mosquito. (B) The titer of wAlbB increases over time in A. gambiae somatic and germ-line tissue. Data for A and B were analyzed by Kruskal–Wallis test using the Dwass method for pairwise contrasts. The different letters (a–e) denote statistical significance [P < 0.05 in A and P < 0.004 in B]. Error bars in A and B represent SEM. (C) FISH performed on mosquitoes 17 dpi localizes the wAlbB infection in diverse tissues within the abdomen. O, ovaries; FB, fat body; G, gut; MT, malpighian tubule. Higher magnification of the (D) midgut, (E) hemocytes, (F) brain, and (G) ovarian follicles. The wAlbB strain infects the follicular epithelium of the oocyte and the nurse cells with the oocyte (white arrow heads in G). Wolbachia also infects the secondary follicles (asterisk in G). For ovarian follicles (G) mosquitoes were assessed 20 dpi. Red represents Wolbachia, blue the mosquito DNA, and green the mosquito tissue autofluorescence. (Scale bars: 300 μm in C and 60 μm in E–G.) FISH controls are available in Fig. S8.

Fig. 2.

Antibiotic perturbation of the microbiome enables vertical transmission of Wolbachia in Anopheles mosquitoes. (A) In A. gambiae, Wolbachia is transmitted poorly to mosquitoes reared on conventional sugar (no antibiotics) but the infection frequency (Fisher’s exact test, P < 0.0001) and density (Mann–Whitney U test, **P < 0.006) increases significantly when mosquitoes are administered an antibiotic mixture. (B) In A. stephensi, no progeny (NP) were obtained from mosquitoes microinjected with Wolbachia reared on conventional sugar due to the severe fitness costs associated with infection. In mosquitoes administered an antibiotic mixture, Wolbachia was transmitted to 90% of offspring. Fractions represent the number of infected offspring over the total number. Green represents conventionally reared mosquitoes, whereas orange denotes antibiotic-treated mosquitos. Box and whiskers represent data quartiles and range, respectively.

Fig. 3.

Microbiome analysis of A. stephensi mosquitoes reared on conventional sugar (−) compared with those on an antibiotic mixture (+). OTUs were grouped by genus (where possible) or higher rank, and the relative abundance in individual samples calculated. The mean relative abundance per treatment is also shown. Asterisks denote presence of OTUs within that taxon grouping that significantly change in frequency (read count) between treatments (nonparametric t test with Monte Carlo simulation; see Table S1 for OTU-specific P values). A maximum likelihood phylogenetic tree was constructed based on the alignment of representative 75-nt OTU sequences for each taxonomic group. See Fig. S3 for a comparison of the relative abundance of bacterial taxa in A. gambiae.

Fig. 4.

Asaia density and mosquito longevity in conventionally reared, antibiotic-treated, and Asaia-supplemented antibiotic-treated A. stephensi. (A) Levels of Asaia in control mosquitoes, antibiotic-treated mosquitoes, and antibiotic-treated mosquitoes supplemented with Asaia^R. Treating mosquitoes with an antibiotic mixture eliminates Asaia, whereas supplementation with Asaia^R in a sugar meal reestablishes the infection (ANOVA, ***P < 0.0001). Asaia levels in conventionally reared and Asaia^R supplemented mosquitoes are not significantly different (ANOVA, P = 0.06). Fractions represent the number of infected mosquitoes over the total number. Box and whiskers represent data quartiles and range, respectively. (B and C) Mosquito mortality trajectories after Wolbachia injection in mosquito lines pre- (B) and post-blood meal (C). Conventionally reared mosquitoes suffer slight fitness costs after injection and severe mortality after a blood meal, whereas antibiotic-treated mosquitoes do not suffer elevated mortality. Antibiotic-treated mosquitoes supplemented with Asaia^R exhibit a modest increase in mortality post-blood meal (ANOVA; letters a–c denote statistical significance P < 0.05). Green represents conventionally reared mosquitoes, orange the antibiotic-treated mosquitos, and purple the antibiotic-reared mosquitos supplemented with Asaia^R. Error bars in B and C represent SEM.

Fig. 5.

Bacterial densities are modulated by blood feeding in A. stephensi. (A) By qPCR, Wolbachia levels in the ovary and the carcass significantly decrease after a blood meal in mosquitoes reared on conventional sugar (CS) [Mann–Whitney U test, **P = 0.0015 (ovary) and **P = 0.0178 (carcass)]. This reduction is abolished when mosquitoes are reared on an antibiotic mixture (Anti) [Mann–Whitney U test, P = 0.18 (ovary) and P = 0.93 (carcass)]. Error bars represent SEM. (B) In conventionally reared mosquitoes, blood feeding significantly increases Asaia levels compared with non-blood fed mosquitoes (Mann–Whitney U test, *P < 0.01). BF, blood fed (red); NBF, non-blood fed (blue). Box and whiskers represent data quartiles and range, respectively.

Fig. 6.

Asaia inhibits Wolbachia transmission in A. stephensi mosquitoes. Supplementing Asaia^R to antibiotic-treated A. stephensi abolishes Wolbachia transmission to offspring. Transmission is significantly different between nonsupplemented (orange) and supplemented mosquito lines that were reared on antibiotics (Fisher’s exact test, ***P < 0.0001). Fractions represent the number of infected offspring over the total number. Box and whiskers represent data quartiles and range, respectively.