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Tissue and stage-specific distribution of Wolbachia in Brugia malayi.

Fischer K, Beatty WL, Jiang D, Weil GJ, Fischer PU - PLoS Negl Trop Dis (2011)

Bottom Line: In inseminated females (8 weeks p.i.) Wolbachia were observed in the ovaries, embryos and in decreasing numbers in the lateral chords.Immunohistology and in situ hybridization show distinct tissue and stage specific distribution patterns for Wolbachia in B. malayi.Extensive multiplication of Wolbachia occurs in the lateral chords of L4 and young adults adjacent to germline cells.

View Article: PubMed Central - PubMed

Affiliation: Infectious Diseases Division, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

ABSTRACT

Background: Most filarial parasite species contain Wolbachia, obligatory bacterial endosymbionts that are crucial for filarial development and reproduction. They are targets for alternative chemotherapy, but their role in the biology of filarial nematodes is not well understood. Light microscopy provides important information on morphology, localization and potential function of these bacteria. Surprisingly, immunohistology and in situ hybridization techniques have not been widely used to monitor Wolbachia distribution during the filarial life cycle.

Methods/principal findings: A monoclonal antibody directed against Wolbachia surface protein and in situ hybridization targeting Wolbachia 16S rRNA were used to monitor Wolbachia during the life cycle of B. malayi. In microfilariae and vector stage larvae only a few cells contain Wolbachia. In contrast, large numbers of Wolbachia were detected in the lateral chords of L4 larvae, but no endobacteria were detected in the genital primordium. In young adult worms (5 weeks p.i.), a massive expansion of Wolbachia was observed in the lateral chords adjacent to ovaries or testis, but no endobacteria were detected in the growth zone of the ovaries, uterus, the growth zone of the testis or the vas deferens. Confocal laser scanning and transmission electron microscopy showed that numerous Wolbachia are aligned towards the developing ovaries and single endobacteria were detected in the germline. In inseminated females (8 weeks p.i.) Wolbachia were observed in the ovaries, embryos and in decreasing numbers in the lateral chords. In young males Wolbachia were found in distinct zones of the testis and in large numbers in the lateral chords in the vicinity of testicular tissue but never in mature spermatids or spermatozoa.

Conclusions: Immunohistology and in situ hybridization show distinct tissue and stage specific distribution patterns for Wolbachia in B. malayi. Extensive multiplication of Wolbachia occurs in the lateral chords of L4 and young adults adjacent to germline cells.

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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.
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pntd-0001174-g007: 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.

Mentions: The genital opening of the male worm lies at the posterior end and forms with the anus a cloaca (Fig. 2C). This is in stark contrast to the anatomy of females. A single vas deferens leads into a seminal vesicle that is connected to the testis; this can be subdivided into a growth zone, a maturation zone, and a germinative zone. In parallel to the distribution of Wolbachia in females, large numbers of endobacteria were observed in the lateral chords of 5 week old males, while the growing sections of the testes in the midbody region were free of Wolbachia (Fig. 6A). However, Wolbachia were present in 5 week males near the testes (Fig. 6B–E) and in the middle part of the testis itself (Fig. 6 F–J). No Wolbachia were detected within the vas deferens by immunohistology (Fig. 7A, D). In contrast, Wolbachia 16S rRNA was detected by in situ hybridization in the testis tissue surrounding the spermatocytes and in the periphery of the vas deferens that contained spermatids (Fig. 7B, C, E). Wolbachia were never observed in the spermatids or the spermatozoa.


Tissue and stage-specific distribution of Wolbachia in Brugia malayi.

Fischer K, Beatty WL, Jiang D, Weil GJ, Fischer PU - PLoS Negl Trop Dis (2011)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101188&req=5

pntd-0001174-g007: 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.
Mentions: The genital opening of the male worm lies at the posterior end and forms with the anus a cloaca (Fig. 2C). This is in stark contrast to the anatomy of females. A single vas deferens leads into a seminal vesicle that is connected to the testis; this can be subdivided into a growth zone, a maturation zone, and a germinative zone. In parallel to the distribution of Wolbachia in females, large numbers of endobacteria were observed in the lateral chords of 5 week old males, while the growing sections of the testes in the midbody region were free of Wolbachia (Fig. 6A). However, Wolbachia were present in 5 week males near the testes (Fig. 6B–E) and in the middle part of the testis itself (Fig. 6 F–J). No Wolbachia were detected within the vas deferens by immunohistology (Fig. 7A, D). In contrast, Wolbachia 16S rRNA was detected by in situ hybridization in the testis tissue surrounding the spermatocytes and in the periphery of the vas deferens that contained spermatids (Fig. 7B, C, E). Wolbachia were never observed in the spermatids or the spermatozoa.

Bottom Line: In inseminated females (8 weeks p.i.) Wolbachia were observed in the ovaries, embryos and in decreasing numbers in the lateral chords.Immunohistology and in situ hybridization show distinct tissue and stage specific distribution patterns for Wolbachia in B. malayi.Extensive multiplication of Wolbachia occurs in the lateral chords of L4 and young adults adjacent to germline cells.

View Article: PubMed Central - PubMed

Affiliation: Infectious Diseases Division, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

ABSTRACT

Background: Most filarial parasite species contain Wolbachia, obligatory bacterial endosymbionts that are crucial for filarial development and reproduction. They are targets for alternative chemotherapy, but their role in the biology of filarial nematodes is not well understood. Light microscopy provides important information on morphology, localization and potential function of these bacteria. Surprisingly, immunohistology and in situ hybridization techniques have not been widely used to monitor Wolbachia distribution during the filarial life cycle.

Methods/principal findings: A monoclonal antibody directed against Wolbachia surface protein and in situ hybridization targeting Wolbachia 16S rRNA were used to monitor Wolbachia during the life cycle of B. malayi. In microfilariae and vector stage larvae only a few cells contain Wolbachia. In contrast, large numbers of Wolbachia were detected in the lateral chords of L4 larvae, but no endobacteria were detected in the genital primordium. In young adult worms (5 weeks p.i.), a massive expansion of Wolbachia was observed in the lateral chords adjacent to ovaries or testis, but no endobacteria were detected in the growth zone of the ovaries, uterus, the growth zone of the testis or the vas deferens. Confocal laser scanning and transmission electron microscopy showed that numerous Wolbachia are aligned towards the developing ovaries and single endobacteria were detected in the germline. In inseminated females (8 weeks p.i.) Wolbachia were observed in the ovaries, embryos and in decreasing numbers in the lateral chords. In young males Wolbachia were found in distinct zones of the testis and in large numbers in the lateral chords in the vicinity of testicular tissue but never in mature spermatids or spermatozoa.

Conclusions: Immunohistology and in situ hybridization show distinct tissue and stage specific distribution patterns for Wolbachia in B. malayi. Extensive multiplication of Wolbachia occurs in the lateral chords of L4 and young adults adjacent to germline cells.

Show MeSH
Related in: MedlinePlus