Fig 1.
A type 2 immune environment is associated with low Wolbachia titers in adult female filariae.
Type 2-competent (wild-type) and type 2-deficient (Il4rα-/-/Il5) were inoculated with 40 infective larvae (L3) of the filaria Litomosoides sigmodontis. Parasites were harvested at various time points before and after the fourth molt (indicated by the arrow at approximately 30 days post-infection, [dpi]), and Wolbachia density was evaluated in female filariae by qPCR of the bacterial gene ftsZ. (A) Relative quantification of Wolbachia density (ratio between Wolbachia’s ftsZ and filarial actin gene copies) in female filariae (see S1 Fig for actin quantification). (B) Absolute quantification of Wolbachia’s ftsZ counts in female filariae; results were normalized by average worm size in each group (see S1 Fig for worm sizes). Results are expressed as the ± SD of n = 4–6 filariae per group (24–60 dpi), n = 10–15 filariae per group (70 dpi). Two-way ANOVAs followed by Bonferroni’s multiple comparisons tests were performed; *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant difference between filariae from wild-type and Il4rα-/-/Il5-/- hosts. #p < 0.05, ##p < 0.01, ###p < 0.001, indicate significant differences between timepoints within the Il4rα-/-/Il5-/- group.
Fig 2.
Selective germline Wolbachia depletion by type 2 immunity and systemic antibiotic clearance are both associated with impaired filarial reproduction.
Wild type (WT) and Il-4rα-/-/Il-5-/- (KO) BALB/c mice were infected with 40 L. sigmodontis L3 larvae. From 40 dpi, KO mice were treated with a combination of rifampicin (10 mg/kg/day for 5 days) and doxycycline (100 mg/kg/day for 14 days) to deplete Wolbachia in filariae (KO + DR). (A) Absolute quantification of Wolbachia’s ftsZ in female filariae; results were normalized by average worm size in each group. Results are expressed as the mean ± SEM of n = 15 filariae from WT mice, 10 from KO mice and 5 from KO + DR mice. (B) Kinetics of microfilaremia in blood from WT, KO and KO + DR mice. Data points represent mean microfilarial counts per 10 µL of blood at different time points post-infection (dpi). Results are expressed as mean ± SEM for n = 3-23 mice per group and per timepoint. (C-E) Parasites were harvested at 55 and 70 dpi and whole mount female filariae were stained for DNA (DAPI, cyan) and Wolbachia (16S ribosomal subunit, yellow) for analysis by confocal fluorescence imaging. (C-D) Representative confocal images of the proximal uteri of female filariae from WT, KO and KO + DR mice at 55 dpi (C) and 70 dpi (D). (E) Representative confocal z-stack images of the lateral hypodermal chords of female filariae from WT, KO and KO + DR mice at 70 dpi. (F) Quantification of the number of microfilariae (left) and aborted embryos (right) in the uteri images, normalized to 100 µm. (G) Proportion (%) of Wolbachia-positive microfilariae in the uteri. (F-G) Results are expressed as mean ± SD of n = 11 filariae from WT mice, 11 filariae from KO mice and 8 filariae from KO + DR mice. Kruskal-Wallis tests were performed followed by Dunn’s multiple comparison test. ns: no statistical difference, ** p < 0.01, *** p < 0.001. (H) Quantification of Wolbachia density in images of the proximal lateral chords (expressed in µm3/100 µm) in 3D images. Results are expressed as mean ± SEM of n = 10 filariae from WT mice and n = 9 filariae from KO mice and 6 from KO + DR mice. A one-way ANOVA was performed followed by Tukey’s multiple comparison test, *** p < 0.001.
Fig 3.
Host genetic background and antibiotic-induced Wolbachia depletion disrupt filarial ovarian dynamics and oogenesis.
Wild-type (WT) and Il4rα-/-/Il5-/- (KO) BALB/c mice were infected with 40 L. sigmodontis L3 larvae. From 40 days post-infection (dpi), KO mice were treated with a combination of rifampicin (10 mg/kg/day for 5 days) and doxycycline (100 mg/kg/day for 14 days) to deplete Wolbachia (KO + DR). At 70 dpi, female parasites were harvested to analyze Wolbachia dynamics, oogenesis, and apoptosis in the filarial germline. Parasite’s ovaries were dissected and stained for DNA (DAPI, cyan), mitotic nuclei (Phospho Histone 3 – PH3, magenta), and Wolbachia (16S ribosomal subunit, yellow), and analyzed by confocal fluorescence imaging. (A) Linearized confocal images of the PZ in the distal part of filarial ovaries, showing DNA (DAPI, cyan), mitotic nuclei (PH3, magenta), and Wolbachia (yellow) signals. The PZ extends from the ampulla to the last PH3-positive nucleus (indicated by arrowheads). (B) Quantification of the total number of PH3 + nuclei in the PZ. Results are expressed as mean ± SD of n = 8-12 ovaries per group. (C) Quantification of Wolbachia+ coverage in the PZ. Results are expressed as mean ± SD of n = 5-12 ovaries per group. (D) Quantification of pyknotic nuclei (indicative of germline apoptosis) in the PZ. Results are expressed as mean ± SD of n = 5 ovaries per group. (B-D) Kruskal-Wallis tests were performed followed by Dunn’s multiple comparison test. ns = not significant, *p < 0.05, ***p < 0.001. (E-F) Spatial analysis of PH3 + mitotic nuclei (E) and Wolbachia+ area (F) along the ovarian PZ. Results were segmented into 100-µm intervals, with mean data smoothed for 4 neighboring segments and standard deviations (SD) shown for raw data. n = 5-12 ovaries per group. (G) Representative 3D confocal images of the distal ampulla in female filariae from WT, KO, and KO + DR hosts. Wolbachia are localized in the central rachis (arrow) and somatic sheath cells (lines) in parasites from WT and KO mice but are ether absent or present as patches in parasites from KO + DR mice. PH3 + nuclei are indicated by an asterisk (*).
Fig 4.
Wolbachia-deficient microfilariae develop normally in the arthropod vector but exhibit delayed growth and differentiation after the third molt in the mammalian host.
(A) Representative confocal fluorescence images of microfilariae (L1) recovered from the blood of infected Il4rα-/-/Il5-/- mice at 70 dpi. Wb(+) and Wb(-) microfilariae were stained for DNA (DAPI, cyan) and Wolbachia 16S ribosomal RNA (yellow). The yellow arrow highlights Wolbachia clusters in Wb(+) worms, which are absent in Wb(-) worms. Head (H) and tail (T) are indicated to orientate the worms. (B) Representative confocal images of infective L3 larvae recovered from Ornithonyssus bacoti mites fed on Wb(+) or Wb(-) microfilaremic Il4rα-/-/Il5-/- mice. Insets show zooms on the squared areas, confirming the presence of Wolbachia (yellow) in Wb(+) larvae and their absence in Wb(-) larvae. (C-F) To assess the developmental potential of Wolbachia-deficient L3 larvae, 40 Wb(+) or Wb(-) L3 larvae of L. sigmodontis were inoculated into Il-4rα-/-/Il-5-/- mice. Larvae were harvested at 14-, 20- or 33-days post-infection (dpi) for analysis. (C) Quantification of the number of Wb(+) and Wb(-) filariae recovered from the pleural cavity (PC) of Il-4rα-/-/Il-5-/- mice at 20 (dots) and 33 (squares) dpi. (D) Proportion (%) of female filariae in the pleural cavity at 20 (dots) and 33 (squares) dpi. (E) Representative Differential Interference Contrast (DIC) images of the buccal capsule of worms at 20 and 33 dpi, allowing to differentiate L4 larvae and adult worms. L4 larvae have a buccal capsule composed of two thin walls while the capsule of adult worms displays three large segments. At 33 dpi, Wb(+) worms had fully molted into adults, while Wb(-) worms remained arrested in the L4 stage. 4-11 worms were analyzed at day 14, 32-39 at day 20 and 68 Wb(+) filariae and 28 Wb(-) filariae at 33dpi. (F) Growth dynamics of Wb(+) and Wb(-) filariae over time. Worm size (mm) is shown as median for each group, with individual worm data points displayed. n = 6-9 worms were measured at day 0, 4-11 at day 14, 32-39 at day 20. At day 33, 68 Wb(+) filariae and 28 Wb(-) filariae were analyzed. Statistical significance for size differences between groups was assessed using two-way ANOVA followed by Tukey’s post-hoc test, **** p < 0.0001.
Fig 5.
Impact of Wolbachia on larval reproductive development.
Whole-mount confocal images of 33 dpi Wb(+) and Wb(-) Litomosoides sigmodontis worms stained for DNA (DAPI, cyan), Wolbachia (16S ribosomal subunit, yellow). Tissue autofluorescence is displayed in magenta. Head (H) and tail (T) are indicated to orientate the worms. (A) Tail of a Wb(+) female worm, cropped (indicated by\\) to fit the panel due to its length (total length ~4 cm). (B) High-magnification view of the proliferation zone of the Wb(+) ovaries. Note the dense organization of germline nuclei and abundant Wolbachia in the rachis (arrow). (C) Whole Wb(-) female worm, with underdeveloped ovaries. (D) High-magnification view of the proliferation zone of the Wb(-) ovaries, showing the ovarian primordium devoid of Wolbachia. (E) Comparison of Wb(+) and Wb(-) male worms. The Wb(+) male exhibits a fully developed testicle running along the entire body length, terminating near the tail (arrows), while the Wb(delineated by 2 arrows) male shows an underdeveloped testicle. (F) Close-up of the tail of a Wb(+) male worm, showing a fully formed spicule (asterisk) and sperm (bright DAPI foci), indicative of mature reproductive tissues. (G) Close-up of the tail of a Wb(-) male worm, showing the absence of a spicule (arrow) and sperm.
Fig 6.
The key roles of Wolbachia on filarial biology.
In adult female Litomosoides sigmodontis, Wolbachia and the germline maintain a mutual dependency: Wolbachia are required to support oogenesis, while a functional germline environment sustains Wolbachia proliferation. Disruption of this balance—either by type 2 immune environment affecting the ovarian tissue or by direct antibiotic depletion of Wolbachia—initiates a cascade of defects. These include reduced Wolbachia density in the germline, defective oogenesis, stunted parasite growth, and eventually, reproductive failure. Despite these impairments, adult females can still transiently release microfilariae lacking Wolbachia. These Wolbachia-depleted microfilariae can develop into infective L3 larvae within the mite vector. However, once transmitted to the vertebrate host, they arrest at the L4 stage, failing to grow, molt, or differentiate into adults. This indicates the existence of a developmental threshold at which Wolbachia becomes essential for sustaining growth, sexual maturation, and successful progression to adulthood. Illustrations adapted from NIH BioArt Source and CDC PHIL (Public Domain).