Journal of Bacteriology & Parasitology

Abstract

Use of in situ Hybridization to Localize Wolbachia during embryogenesis in Brugia malayi

Daojun Jiang, Peter U. Fischer and Gary J.Weil

Wolbachia are intracellular bacteria that are required for development and reproduction in most filarial nematodes. New approaches are needed to improve understanding of the role of Wolbachia in filarial biology. Recent studies using fluorescent immunohistology revealed asymmetric segregation of Wolbachia in early embryos of Brugia malayi, and this helps to explain the restricted distribution of Wolbachia in adult worms. However, the distribution of Wolbachia in late stages of embryo development or in infective larvae (L3) is largely unknown. In this study we have explored the use of in situ hybridization (ISH) to localize Wolbachia and Wolbachia gene expression during embryogenesis and morphogenesis in B. malayi.

ISH with a 16S rRNA probe revealed Wolbachia in the lateral cords, oocytes, and embryos of B. malayi female orms. This was consistent with prior studies that detected Wolbachia by immunohistology. ISH with probes for Wolbachia surface protein gene (wsp) and Wolbachia heme biosynthetic pathway gene (hemE) showed that these genes were strongly expressed in early embryos but not in later stage embryos (“comma”, “pretzel”, or stretched intrauterine microfilariae). A detailed study of the distribution of Wolbachia in B. malayi embryos using ISH with the 16S rRNA probe documented the asymmetric segregation of Wolbachia in early embryos (morulae) and showed that Wolbachia were only present in hypodermal cord precursor cells in later stage embryos (“comma” stage or later). Progressively restricted localization of Wolbachia during morphogenesis may explain why Wolbachia are absent from the genital primordium in L3.

Wolbachia distribution and gene expression vary dramatically during filarial embryo development and across the worm’s life cycle. Additional research is needed to understand the tissue-specific population dynamics of Wolbachiaand to explore how signals from the nematode host might influence the growth, distribution, and gene expression in these bacteria.