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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 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.
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pntd-0001174-g005: 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.

Mentions: In order to understand the distribution of Wolbachia in adult worms it is crucial to recall the anatomy and development of reproductive organs of filarial worms [13], [14]. The genital opening (vulva) lies close to the anterior end of the female worm, approximately at the level of the esophagus (Fig. 2A, B). The vagina leads into the bifurcated uterus which ends in the seminal receptacles. Theses organs are linked by oviducts with two ovaries that have an anterior growth zone, a maturation zone in the middle, and a posterior germinative zone. At 5 weeks p.i. in the vertebrate host, young adult female B. malayi worms are approximately 1.8 cm long and still growing. At that time point a massive accumulation of Wolbachia was observed, mainly in the lateral chords. Increased numbers of Wolbachia were observed in the lateral chords in the posterior end of the female which was still free of ovaries (Fig. 3A). Sections of the posterior part of the ovaries showed large numbers of Wolbachia in the adjacent lateral chords, but the ovaries themselves were free of Wolbachia (Fig. 3B, C, 4A). In the oocyte maturation and growth zones of the ovaries, Wolbachia were oriented within the lateral chords towards the pseudocoelomic cavity (Fig. 3D–G), and some sections showed Wolbachia in the periphery of the ovary (Fig. 3H, 4B, D, F). Two distribution patterns of Wolbachia were found in the lateral chords. Scattered Wolbachia were present in the apical part of the chords, and numerous clusters of Wolbachia were present basal border of the hypodermal chords adjacent to the ovaries (Fig. 3I). A similar staining pattern for Wolbachia was observed in the lateral chords in the midbody region of 5 week old females, but their empty uterus branches were always free of Wolbachia (Fig. 3J). In 8 week old female worms, less Wolbachia were detected in the lateral chords, but the mature ovaries in the posterior part of the worms were heavily infected with Wolbachia (Fig. 5A). These worms contained developing microfilariae, and the ovaries showed strong staining of nuclear chromatin (as determined by DAPI). Morula stage embryos were observed in the uterus with many Wolbachia, while in this region the number of endobacteria in the lateral chords was lower than in the distal parts of the lateral chords.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3101188&req=5

pntd-0001174-g005: 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.
Mentions: In order to understand the distribution of Wolbachia in adult worms it is crucial to recall the anatomy and development of reproductive organs of filarial worms [13], [14]. The genital opening (vulva) lies close to the anterior end of the female worm, approximately at the level of the esophagus (Fig. 2A, B). The vagina leads into the bifurcated uterus which ends in the seminal receptacles. Theses organs are linked by oviducts with two ovaries that have an anterior growth zone, a maturation zone in the middle, and a posterior germinative zone. At 5 weeks p.i. in the vertebrate host, young adult female B. malayi worms are approximately 1.8 cm long and still growing. At that time point a massive accumulation of Wolbachia was observed, mainly in the lateral chords. Increased numbers of Wolbachia were observed in the lateral chords in the posterior end of the female which was still free of ovaries (Fig. 3A). Sections of the posterior part of the ovaries showed large numbers of Wolbachia in the adjacent lateral chords, but the ovaries themselves were free of Wolbachia (Fig. 3B, C, 4A). In the oocyte maturation and growth zones of the ovaries, Wolbachia were oriented within the lateral chords towards the pseudocoelomic cavity (Fig. 3D–G), and some sections showed Wolbachia in the periphery of the ovary (Fig. 3H, 4B, D, F). Two distribution patterns of Wolbachia were found in the lateral chords. Scattered Wolbachia were present in the apical part of the chords, and numerous clusters of Wolbachia were present basal border of the hypodermal chords adjacent to the ovaries (Fig. 3I). A similar staining pattern for Wolbachia was observed in the lateral chords in the midbody region of 5 week old females, but their empty uterus branches were always free of Wolbachia (Fig. 3J). In 8 week old female worms, less Wolbachia were detected in the lateral chords, but the mature ovaries in the posterior part of the worms were heavily infected with Wolbachia (Fig. 5A). These worms contained developing microfilariae, and the ovaries showed strong staining of nuclear chromatin (as determined by DAPI). Morula stage embryos were observed in the uterus with many Wolbachia, while in this region the number of endobacteria in the lateral chords was lower than in the distal parts of the lateral chords.

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