Wolbachia, an intracellular endosymbiont of insects, is increasingly being seen as an effective biological control agent that can interfere with the transmission of pathogens that are vectored by insects. Fluorescent in situ hybridization is used to qualitatively determine the presence or absence of Wolbachia in fly tissues across the five fly-Wolbachia pairings. Wolbachia is observed in many of the tissues including the head, gut, Malpighian tubules, thoracic ganglion, fat body, and muscle. The Wolbachia distribution differed between the fly-Wolbachia pairings, and Wolbachia is  not detected in the tissues of some fly lines. Although Wolbachia distribution is not ubiquitous across the five fly-Wolbachia combinations, the presence or absence of Wolbachia from a particular tissue did not correlate with the ability to mediate antiviral protection Although Wolbachia tissue distribution did not correlate with antiviral protection, a difference in intensity of fluorescence between fly lines was observed. Quantitative PCR is  used to determine Wolbachia density in the head, gut, Malpighian tubules, testes, thoracic ganglion, accessory glands, fat body, and muscle dissected from protective and nonprotective fly lines. A correlation can be established between the Wolbachia density and the ability to mediate antiviral protection in tissues.

Hi, thanks for copying some text from Osborne et al. 2012, it is a good paper and I met some people from the group recently. However I am not sure how this directly answers my question. Have you tried staining wolbachia in situ with dapi? Unfortunately most of these papers do not show images of infected tissues such as Malpighian tubules, and I have no experience on what to expect.

I have just stained several tissues of my ants with dapi which are wsp-positive by pcr, but I observed no apparent wolbachia among cell nuclei. I am not sure what this could mean?

Thanks 

Not specifically related to DAPI (but FISH, which works), but have you seen this paper?

http://www.sciencedirect.com/science/article/pii/S0092867409015001

Thanks for an interesting paper, however I am aware of the widespread use of fish yet I was suggested that often wolbachia can be detected with direct staining without embedding into blocks. This would make the procedure much easier and cheaper for monitoring. Have you tried with only dapi?

Hi Eduardo,

you should have a look at this paper:

Casper-Lindley et al (2011): Rapid Fluorescence-Based Screening for Wolbachia Endosymbionts in Drosophila Germ Line and Somatic Tissues. Appl Environ Microbiol (link attached)

The authors used a nucleic acid staining (Syto-11) to detect Wolbachia and it seems to work fine. When using a short incubation time, only Wolbachia cells are stained. We tried the same thing with SYBR Green and it also works out. We did not try DAPI, but the first author of the paper told me some time ago that it should work just as well. Maybe ask her for more details.

Dear Michael: this is an excellent insight. Many thanks! One issue I have been suggested is that often Wolbachia are so close to the insect cell nuclei that it might be hard to discern between the two. I will give it a try and let you know. Up to now, my attempts with DAPI returned beautiful images of nuclei but nothing that was obviously an endosymbiont, in wsp+ samples by pcr.

We are also not being successful in identifying Wolbachia, although today was our first trial. we tried with both syto11 and Dapi. Could you please anyone add any valuable suggestions into it?