Figure 1.
Detection of Wolbachia in larval B. malayi by immunohistology or by in situ hybridization.
A Clusters of Wolbachia (arrow) in single cells of a stretched intrauterine microfilaria labeled by immunohistology using mab Bm WSP. B Consecutive section to A, but Wolbachia (arrow) were detected by in situ hybridization. C Wolbachia (arrow) in single cells of a microfilaria within the midgut of A. aegypti 2 h after the blood meal. D Clustered Wolbachia in a cell of a fragment of an L2 larva in the thorax of A. aegypti 3 d.p.i. E Cross-section of L2 larvae in the thorax of A. aegypti showing a cluster of Wolbachia (arrow) in the hypodermis (7 d.p.i.) F Cross section of isolated infective L3 larvae showing only a single Wolbachia cluster (arrow) in the cells of lateral chord (14 d.p.i). G Several clusters of Wolbachia (arrows) in the lateral chord in a longitudinal section of an infective L3 larva migrating through the abdomen of A. aegypti 14 d.p.i. H Cross-section of the anterior end of a 4th stage larva (14 d.p.i. of a jird). No Wolbachia were detected. I Cross-section of the midbody region of a 4th stage larva showing many Wolbachia (arrow) in one lateral chord. J Cross-section of the midbody region of another 4th stage larva (wide field fluorescence microscopy) showing highly condensed DNA (blue DAPI stain) in the genital primordium and numerous Wolbachia (arrow) in the lateral chord similar to I. K Longitudinal section of the midbody region of a 4th stage larva with many Wolbachia in the lateral chords (arrows). Cells in the pseudocoelomic cavity were free of Wolbachia. Ph, pharynx; gp, genital primordium; I, intestine; m, muscle. Scale bar 10 µm.
Figure 2.
Schematic drawing the anatomy of adult stage B. malayi and distribution of Wolbachia.
Hypodermis, muscles, median chords as well as nerve and secretory-excretory system are not shown. Proportions are estimates. Tissues and organ systems are simplified and the midbody region is interrupted for clarity. The lateral chords are shown dorsally and ventrally instead of laterally. A Adult microfilaria producing female (12 weeks p.i.). The body length is about 4 cm. Wolbachia (red dots) are localized mainly in the lateral chords, ovary and developing embryos. The lateral chords in the head (up to the vulva) of the worm rarely contain Wolbachia. B Adult stage, immature female (5 weeks p.i.) a few days after the 4th molt. The body length is approximately 1.8 cm. Wolbachia are mainly localized in the lateral chords. Occasionally Wolbachia are attached to the ovaries or single endobacteria can be found within the ovary. C Adult spermatozoa producing male worm (12 weeks p.i.) with a total body length of about 2.5 cm. Spiculae are not shown. Wolbachia are localized in the lateral chords; Wolbachia remnants can be detected in parts of the vas deferens but not within spermatids or spermatozoa.
Figure 3.
Detection of Wolbachia in immature female B. malayi at 5 weeks p.i.
A–D, H–J immunohistology using mab Bm WSP. A Cross-section in the posterior part of the female showing massive accumulation of Wolbachia (arrow) in the lateral chord. B Longitudinal section of the distal tip showing Wolbachia-free growing ovaries and numerous clusters of Wolbachia (arrow) in the lateral chord. C Cross-section showing asymmetric distribution of large amounts of Wolbachia (arrow) in the lateral chord and Wolbachia-free ovaries. D Large numbers of Wolbachia (arrow) in close proximity to one ovary branch. Ovaries are still not infected. E–G Consecutive sections to D. E 16S rRNA in situ hybridization. F 16S oligonucleotide FISH. G DAPI stain showing highly condensed chromatin in the ovary and in Wolbachia in the lateral chord (arrow). H Cross-section showing not only Wolbachia clusters in the lateral chords (arrow) but also at the edge of one ovary (arrow head). I Sagital section through lateral chord and ovary showing large numbers of Wolbachia clusters (arrows) in the lateral chords aligned at the ovary and some Wolbachia clusters in the middle of the chord (arrow head). J Cross-section of the midbody region showing large numbers of Wolbachia in the lateral chord (arrow) and empty uterus branches. i, intestine; u, uterus; ov, ovary. Scale bar 25 µm.
Figure 4.
Detection of Wolbachia in female B. malayi at 5 weeks p.i. by advanced microscopy techniques.
Confocal laser scanning microcopy (LSM) and wide field fluorescence microscopy (WFFM) were used together with mab Bm WSP (green) to label Wolbachia and a WGA 633 stain (red) to visualize membranes. WFFM contained also a filter for DAPI to detected concentrated DNA. A Cross-section showing large amounts of Wolbachia in the lateral chords close to the ovaries (arrow). Some Wolbachia were also detected in the hypodermis. For full scans of LSM images and 360° rotation see suppl. material (LSM). B Another cross-section showing a more advanced stage of infection with endobacteria attached to the ovary membrane (arrow head) and already in the ovaries (arrow) (LSM). C Longitudinal section showing Wolbachia lining up in the lateral chords in the vicinity of the ovary (arrow) or within one ovary branch (arrow head)(LSM). D WFFM showing Wolbachia approaching ovaries with highly condensed chromatin (blue DAPI stain). E Another cross-section showing Wolbachia (arrows) attached to the ovaries (blue DAPI stain). One ovary branch is already Wolbachia (arrow head) infected while the other is still free (WFFM). F Close-up of a longitunial section showing Wolbachia (arrow) directly at the ovary membrane (red) (WFFM). G Another close-up showing Wolbachia in the lateral chords close to the ovary (arrow) or already in the somatic cells of the ovary (arrow head) (WFFM). Staining for WSP provides a characteristic donut shaped pattern of a Wolbachia surface protein. Ov, ovary; i, intestine. Scale bar 30 µm.
Figure 5.
Detection of Wolbachia in adult female B. malayi at 8 and 12 weeks p.i.
In A, B, E, F and H Wolbachia were labeled by mab Bm WSP. A Cross-section of a distal end of a worm 8 weeks p.i. showing Wolbachia infected ovaries and some Wolbachia clusters in the lateral chord. B–H B. malayi 12 weeks p.i. B Several cross-sections of the ovaries showing massive Wolbachia staining (arrow), but only weak labeling in the adjacent lateral chord (arrow head). C Consecutive section to B, but in situ 16S rRNA detection confirmed the large number of Wolbachia in the ovaries with much stronger labeling in the adjacent lateral chord (arrow head). D Cross-section of the midbody region shows both uterus branches full of morula stage embryos and Wolbachia (arrow, 16S rRNA). E Longitudinal section in the region of the transition of the ovaries to the tubular oviduct. Large numbers of Wolbachia are visible in the growth zone of the ovaries (arrow heads) and the adjacent lateral chords (arrow) but not in the oviduct. F Cross-section showing one uterus branch with Wolbachia positive (arrow) morula stage embryos and numerous spermatozoa in the seminal receptacle surrounding a few egg cells. Thin lateral chords show only scattered Wolbachia. G Consecutive section to F stained with polyclonal Brugia pahangi WSP antisera [39] shows Wolbachia in the egg cells (arrowhead). H Cross-section of the region close to the female genital opening. Some Wolbachia are visible in the lateral chord (arrow) and in the stretched microfilariae (arrowhead) in the vagina uterine, but the muscular vagina vera is free of microfilariae and Wolbachia. Ov, ovary; u, uterus; od, oviduct; sp, spermatozoa; vv, vagina vera; vu, vagina uterina. Scale bar 25 µm.
Figure 6.
Detection of Wolbachia in immature male B. malayi at 5 weeks p.i.
In A and C Wolbachia were labeled by mab Bm WSP). A This cross-section of the midbody region shows primary spermatogonia in the testis and numerous Wolbachia (arrow) in the lateral chords. B LSM of a consecutive section of A. Individual clusters of Wolbachia (arrow) can be identified. C Two cross-sections more distal compared to A with Wolbachia in the lateral chords (arrow) and in tissue attached to the testis (arrow head). D Consecutive section to C examined by LSM. E Consecutive section to C stained by in situ rRNA hybridization confirming the staining pattern. F–J Serial cross-sections showing many Wolbachia in the lateral chords (arrowheads) and in the spermatogonia (arrows). F mab Bm WSP. G Also mab Bm WSP but LSM H Same staining as G but WFFM with DAPI stain of testis. I 16S in situ rRNA hybridization. J 16S oligonucleotide FISH. te, testis. Scale bar 25 µm.
Figure 7.
Detection of Wolbachia in male B. malayi at 12 weeks p.i.
In A and D Wolbachia were labeled by mab Bm WSP). A Two cross-sections demonstrating Wolbachia in the lateral chords (arrows). One section is in the midbody region showing developed spermatogonia and numerous Wolbachia in the lateral chord, while the other section is in the more muscular distal end of the worm showing spherical spermatids in the vas deferens and fewer Wolbachia in the lateral chord. B Consecutive section to A, but stained with 16S rRNA in situ hybridization. In contrast to A some staining for Wolbachia rRNA is also detected in the testis (arrowheads) and at the border of the vas deferens (arrow heads). C Cross-section of the distal part of another male worm showing Wolbachia 16S rRNA labeling at the epithelium of the vas deferens (arrow heads) and in the lateral chord (arrow). Spermatozoa were never labeled. D–G Consecutive cross-section through the terminal end showing the vas deferens containing fully developed spermatids in transition to spermatozoa. Wolbachia are detected in the lateral chord (arrow), but not in the spermatids. D mab Bm WSP. E 16S rRNA in situ hybridization. Similarly to B, staining is also observed at the border of the vas deferens (arrowheads). F 16S oligonucleotide FISH. Although Wolbachia can be easily identified in the lateral chord, granular staining at the membrane of the vas deferens is difficult to recognize. G DAPI stain, again Wolbachia can be easily identified in the lateral chord, but granular staining at the membrane of the vas deferens is hard to differentiate. Sp, spermatozoa; te, testis; vd, vas deferens. Scale bar 25 µm.
Figure 8.
Transmission electron microscopy of young adult female B. malayi 5 weeks p.i.
A Numerous Wolbachia (arrow) in the lateral chord in the vicinity of developing ovaries. A number of small, electron dense Wolbachia can be detected in more loosened tissue (arrow heads). B Vacuolized epithelium surrounding the ovary with electron dense Wolbachia (arrow heads) on both sides of the basal lamina. C Numerous Wolbachia in the lateral chord (arrow) and a bacilli-shaped, extracellular Wolbachia in the pseudocoelomic cavity (arrow head). D Magnification of C, showing the Wolbachia cell membrane (arrow head). E Wolbachia (arrow head) docking to the outer ovary epithelium. F Magnification of the endobacterium in E showing loosened cell membrane at the apical end (arrow head) and a dense central inclusion (arrow). G Small electron dense Wolbachia (arrow heads) of the oogonia in the proximal part of the ovary. H Single and clusters of electron dense, extracellular Wolbachia (arrow heads) in the ovary. I Electron dense Wolbachia (arrow heads) in vacuolized ovary tissue. Bl, basal lamina; lc, lateral chord nu, nucleus; ov, ovary; ps, pseudocoelomic cavity. Scale bar 0.5 µm.
Figure 9.
Transmission electron microscopy of young adult male B. malayi 5 weeks p.i.
A Numerous Wolbachia (arrows) are observed in the lateral chords close to the testis. Single dark Wolbachia (arrow head) are found in the testis close to the membrane. Mitochondria (asterisk) are found in the periphery of the lateral chord. B Wolbachia (arrow) in the inner testis epithelium in the vicinity of a spermatid. Large amounts of membranous material (arrow heads) can be observed in the testis lumen in the vicinity of the testis epithelium. C Magnification of B showing intracellular Wolbachia (arrow). D Another sample showing Wolbachia (arrows) in the inner testis epithelium. E–G Pleomorphic Wolbachia (arrows) in vacuolized testis tissue. Membranous material (arrow head) can be seen in extracellular spaces. Ps, pseudocoelomic cavity; lc, lateral chord; sp, spermatide; mo, membranous organelle; nu, nucleus; bl, basal, lamina; te, testis. Scale bar 0.5 µm.