It’s all about the Z’s and the W’s

In humans it is the combination of X and Y chromosomes that determine sex; females have two of the same sex chromosomes, XX, and males have two distinct sex chromosomes, XY. In birds it is Z and W chromosomes which determine sex but with the assignment the other way round to humans, females contain ZW sex chromosomes and males contain ZZ. It is the presence of these Z and W chromosomes in breast feathers, collected last summer, which I have been trying to determine these last few weeks at the NBAF Facility in Sheffield.

Working in a lab is a completely new experience for me and the complete antithesis of working on Bass Rock. The lab, as you would expect, is spotlessly clean, tidy, calm and quiet. Bass is noisy, smelly, dirty (filthy if it’s been raining) and a little bit crazy!

 

The process to get from feather to sex probably takes 3-4 days and involves 4 stages which I’ll attempt to outline below.

Firstly the DNA needs to be extracted from the feather. The end of the feather, or quill, which was attached to the bird, needs to be cut off and cut into tiny fragments. Working with small breast feathers requires a modicum of patience as small fragments of feather have a frustrating habit of pinging off if you fail to hold them down adequately!

Preparing feathers for DNA extraction.

A process involving the use of ammonia acetate is then carried out to leave you with a miniscule (so small you can’t see it with the naked eye) pellet of DNA in the bottom of a 1.5ml tube. To be able to assess and use the DNA it needs to be in liquid form and so is then dissolved in a buffer solution. There is a substantial difference in the quantity of DNA that you can extract from feathers and bird blood. This is because bird blood is nucleated and so contains much more DNA. The photo below of two 1.5ml tubes shows the difference.

 

The quantity of DNA in the sample can then be assessed using an agarose gel.

 

The next step is to run a PCR , or polymerase chain reaction, on the sample to see if it contains two identical Z alleles (male) or a Z and W allele (female). The Z and W alleles differ in size and this allows us to distinguish between them. A primer set that targets the Z/W alleles and an enzyme (Taq DNA polymerase) are added to the sample followed by a process of heating and cooling to three different temperatures. This allows the Z or W alleles to be identified and replicated millions of times. The Z and W alleles are then separated based on size using a DNA Analyser. The use of PCR in this technique means that even with a very small quantity of starting DNA, you can still get a sexing result.

 

Then it’s time for the exciting bit, it’s time to put the samples into the DNA Analyser and see whether my pipetting skills have been up to scratch and if ZZ or ZW alleles have been identified from the samples.

So after much pipetting and spinning down I have managed to successfully  sex all 40 of the birds tested, which is a third of the birds we tagged last summer. That might not sound very many but I’m quite pleased with how it’s gone especially given how much more difficult it is to extract DNA from feathers than blood. Apparently mouth swabs are also pretty reliable but we’ll have to see whether we’re brave enough to get any of those!

 

Finally I’d just like to say a huge thank you to Gavin Horsburgh and Deborah Dawson at the NBAF Facility (NERC Biomolecular Analysis Facility) who have both been incredibly patient with me and taken me through every step. Hopefully when I go back next year I’ll be a little more self-sufficient now I know what’s in store!

 

 

 

Who’s the daddy?

As you may have noticed the gannet research blog has been quiet over the last 6 months … the gannets left the colony at the end of the summer and I returned to Leeds to make a start analysing some of the data we collected. Until now there hasn’t really been too much to blog about, my days have mostly been filled with the joy and frustration of learning how to use a piece of very useful open source software called R which is integral for my analysis. However, for the last few weeks I’ve been enjoying a change of scenery working at the University of Sheffield.

What am I doing in Sheffield? Well, with another few months before I can get back out on the Bass to work with the gannets, I’m spending some time at the NERC (Natural Environment Research Council) Biomolecular Analysis Facility establishing whether the birds we tagged last summer were male or female.

Despite the fact that gannets, like many seabirds, are a monomorphic species (by this I mean male and females look identical) differences in their foraging behaviour have been discovered. Researchers from the University of Exeter found that male gannets from the Grassholm colony are more likely to forage off fisheries discards than females, whilst a recent paper published on the Bass Rock gannets demonstrated that female gannets make longer (in time and distance) foraging trips than males and that they are more likely to be associated with tidal mixing fronts. This is fascinating stuff but we don’t know the reason for these differences, especially considering the two genders are so physically similar.

 

So given that males and females look identical, how is it that we are able to undertake these sex based comparisons? Work by a number of authors in the 60’s, 70’s and 80’s suggested that the sexes could be told apart through a number of subtle morphological and behavioural differences. These included the colour of the head plumage, the colour of the web-lines on the foot, the collection of nest material and a behaviour known as nape-biting.

 

 

In 2000, these criteria were evaluated alongside DNA profiling from blood samples. The results of over 500 hours of observations at each nest found the only reliable non-molecular method for sex determination is nape-biting. It is only the males which will display this behaviour. You can find the paper here if you’d like to read more about what they did.

 

So we can reliably tell males and females apart by observing nape-biting. The only down side to this is that it only occurs when there is a change over at the nest. With foraging trips averaging around 24 hours in length you would have to live on Bass all summer in order to have a chance of observing this at every nest you are monitoring. This is why I am now in the Molecular Ecology lab in Sheffield learning how to determine sex using a DNA-based method.

If you’re interested, my next post will give you a bit more detail about the how we go about doing this.