Wolbachia - arthropod mind control

I'm working on developing additional markers for my mosquito projects. We have several microsatellite markers that work well, but in some places the extent of hybridization between taxa is so extensive that it would be nice to have a maternally inherited marker, to get more information as to what subspecies was originally the mother.

Enter Wolbachia. What's Wolbachia, you ask? The link goes to a nice Wikipedia article about it, but it's an endosymbiotic bacteria that is present in many arthropod species. Saying it's an endosymbiont is being kind of generous, though. It's more of a parasite, because it's able to control the reproductive efforts of the species it inhabits.

It was first discovered in a species that I work with, Culex pipiens, so it's full name is Wolbachia pipientis. Ha, it's kind of like Madonna, so famous that it doesn't need a last name!

One of the most striking effects of a Wolbachia infection is CI, or cytoplasmic incompatibility. This happens when infected males mate with unifected females, and the eggs die. CI can also happen if two individuals are infected with different strains of Wolbachia.

Other things that can happen as a result of Wolbachia infection include the killing of males due to infection, the feminization of males into sterile pseudo-females, and parthogenesis, where females can produce fertile eggs without fertilization from a male.

As you might imagine, this scenario sets the stage for some very interesting evolutionary biology. Patterns observed may not make sense unless one views them in light of possible Wolbachia infection. The rate of infection is high in Culex mosquitoes, but work is still being done to quantify all the strains.

My interest in Wolbachia at this point is that new maternally-inherited marker. A paper came out recently that detailed a protocol to amplify, with the same primer set, two different sized (and therefore subspecies-diagnostic) fragments from a gene for a surface protein. It is interesting because it's not mosquito DNA that I'm using for a mosquito marker; it's the Wolbachia. I have followed the protocol, but alas get results opposite of the paper. P is supposed to have the smaller fragment and Q the larger, but they are switched.

So I'm working on cloning and sequencing the fragment to figure out what's going on. If I can duplicate the results of the paper, it would be a useful marker, but if I can't, I'd like to know what's being amplified.

UPDATE: 8-13-08 It looks like this marker is not going to be as great as I thought. I ran the same set of samples with a known marker and again with this Wolbachia marker and got lots of drop out. That's when there's just no amplification. My known marker amplified in every individual, so I know the DNA's OK, but the Wolbachia one isn't going to cut it. Too bad, and the search for a maternally interited marker continues.