Here's Dr. Helen's third instalment in our genetics series, and this time we are starting to see tangible results. Thanks again to Helen, Ilze and all the folks at the genetics department at Aberystwyth University for doing all this work for us. We couldn't get any funding for it so they decided to do it for free. I will write a follow-up blog next weekend, but for now, here's Dr Helen to explain the results we've received during the last few days.

In my previous blog, I mentioned the lab technique that's crucial for all the genetic analysis that we're carrying out on our osprey DNA samples: the polymerase chain reaction (PCR for short). It's a way of turning a tiny quantity of DNA into an amount that's sufficient to work with.

At the same time it ensures that you're only looking at the right part of the DNA from the species you're interested in - in our case, ospreys, of course - rather than contaminating DNA from other plant and animal material. As I said last time, there could be DNA from all kinds of things in an osprey's mouth!

Clarach's saliva - there's more than just osprey DNA in there

The first analysis Ilze and I have been carrying out is to discover the sex of each of our osprey chicks, and see whether our results agree with the sex that the ringing team assigned on the basis of size and weight - females are on average bigger and heavier than males. So I'll talk about PCR using osprey sexing as an example. This is a very brief explanation that leaves out a lot of the detail; if you'd like to know more about PCR, leave a message on Facebook.

An osprey, like all other birds, has a pair of sex chromosomes, one inherited from its mother and one from its father, along with all the other chromosomes. Humans are similar; each of us gets an X chromosome from our mum and either an X or a Y from our dad. People with two X chromosomes, like me, are female, people with an X and a Y are male (there are a few exceptions, because biology is complicated, but generally in humans XX means female, XY means male). Birds do it slightly differently; their sex chromosomes are called Z and W. Birds with two Zs, one from each parent, are male; those with one Z from the father and one W from the mother are female.

Sex chromosone differences between birds and mammals:

So, for example, Clarach, who bred for the first time this year on her nest in Scotland, has inherited a Z chromosome from Monty and a W chromosome from Glesni, and she will pass the W one on to all her female chicks.

Clarach on her nest with her chicks in 2017

To tell the sex of an osprey using genetics, we need something that we know will always be different in females compared to males. Fortunately, there's a small part of the osprey sex chromosomes that meets just this need: it's present in the DNA of both the Z and the W chromosome, but it's a slightly different size in the two cases. Dr Deborah Dawson and her team in Sheffield University discovered this useful bit of the chromosome, and kindly sent us the material we need to check it out for ourselves: the primers.

Primers, which come in pairs, are short lengths of DNA that stick to the DNA in a sample. The primers we're using stick one on either side of the part of the sex chromosome that's different in the Z and W forms - what we call the sex marker. Once the primers have stuck, it's possible for a special enzyme - the polymerase in "PCR" - to come along and make a copy of the DNA in between where the primers have stuck. It doesn't just do it once, either; once it's made one copy, if you heat the DNA up and then cool it down, you can get the primers to stick again, but this time to the original DNA and the copy.

How primers (in red) work:

The polymerase does its job again, and now you have the original and three copies, four in total. Round you go again, giving you eight of the bit of DNA you're interested in. And again... 16... again... 32, and so on. By the time this cycle has run 10 times, there are 1024 copies of the original piece DNA.

In the lab, we actually run the cycle thirty times. That means we end up with over a thousand million copies of the sex marker part of the DNA, which is plenty to work with. The process is called the polymerase chain reaction (PCR) by analogy with a nuclear chain reaction, because the amount of DNA starts out small but gets bigger very fast.

Here's a picture of one of the machines that do the heating and cooling for us: a thermal cycler. This one is Ilze's lucky machine - when she was working on the red kite project it always gave her good results. So of course we had to use it in the osprey research too! In this picture, so far it's run ten of the 30 cycles for us.

Setting up a reaction is quite time consuming and it's delicate work; we have to handle tiny volumes of liquid, containing osprey DNA, primers, the polymerase and a few other things that are needed for the reactions to work. It's like following a recipe, but on a very small scale - the droplets of liquid are so tiny that they're hard to see. Here's me in the lab, setting up a set of PCRs. I look very serious, and that's because I'm concentrating hard so as not to waste any of our precious samples!

Dr. Helen in the lab (Ed - look at the earrings!!!!)

Before we go on to the next step, we need to check that our PCR reactions have run OK. This photograph shows that most of our reactions with the primers for sexing did work: except for the one on the very left, each column in the photo represents DNA multiplied up from one Welsh osprey chick's sample. I was so pleased to see this that I had to message my husband Sid straightaway, with a copy of the picture and a big smiling-face emoji!

Now we give a small amount of each of the successful PCR mixtures to Caron, who runs them through a DNA analyser - a machine that's able to measure the sizes of bits of DNA very accurately. That's what we need, because the sex marker section of the chromosome is only slightly different in size between the Z and the W chromosome.

Here's what the results look like. If there are two blue peaks, that means that both the Z-sized marker and the W-sized marker are present, so the chick that the sample came from must be female. If there's only one peak the chick must be male.

Caron has run the samples from 25 Welsh ospreys through the genetic analyser, and so far we have good results for 20 out of the 25 (we are pretty sure that we can repeat the PCRs and get better results for at least three of the remaining five).

And here's what you've all been waiting for:

In 19 out of the 20 cases, the sex identified by our genetic analysis matches with the sex that the ringing team assigned to the chick. So, for example, at Dyfi last year, Ceri was indeed female, and Tegid is male. This year, yes, Aeron is male and Menai is female (we need to redo the analysis for Eitha). The same applies to the samples from the other Welsh nests. And Ceulan, that very special osprey who survived the 2012 floods but died accidentally caught in fishermen's nets in Africa? We extracted DNA from one of Ceulan's feathers and confirmed that he was male.

Getting ready to extract DNA from feathers

These results show just how good the licensed ringers are.

They weigh and measure the chicks at the time of ringing, and use this information (Ed - and other parameters, more next week) to work out the sex of each chick - and they've got it right for 19 out of 20 of the Welsh chicks they've ringed over the past three years.

Tegid being weighed in 2016

But what about the other chick, the one where the ringing team's assessment wasn't correct?

This is where it gets really interesting - OK, I may be slightly biased! For those relatively new to this site: at the Dyfi Osprey Project in 2015, the first-hatched of Monty and Glesni's chicks, Merin (who was fitted with a blue/white Darvic ring, number W1) was intermediate in weight between what's typical for males, on the one hand, and females, on the other: Merin weighed 1450 grams.

In the end Merin was listed as being possibly female, possibly male, but in the end we decided we had to go for one, so went for female. But it really was 50:50 as Merin was a classic 'inbetweenie".

Merin being weighed in 2015

Now we have the results of the DNA analysis, and...
...
wait for it...
...

Merin is male! So of Monty and Glesni's three chicks hatched in 2015 - all of which grew up to be strong, healthy young ospreys and set off on their migration to Africa - one (Celyn) is female, and two (Brenig and, as we've just learnt, Merin - are male).

Girl-Boy-Boy

The situation for ospreys in Wales is still precarious, with only four breeding pairs so far as we know. Every chick that hatches, fledges and migrates to its winter home is immensely valuable. But in Wales we've been especially short of males in past years, and males tend to return to the patch where they were hatched much more than females do (read Emyr's blog about Clarach, which towards the end says more on philopatry).

And of course this means that, if they form bonds with suitable females, they'll start families themselves in the area. So learning that, in Merin, there's definitely one more Welsh male osprey, is great news, for the osprey population in Wales and beyond.

In my next blog, sometime in the New Year, I'll talk about some of the other genetic analysis we're doing, which we hope will tell us more about the way different Welsh ospreys are related to one another and to ospreys in England, Scotland and mainland Europe. I need to go back into the lab to re-run a few PCRs that didn't work well the first time, and then work out what all the results mean; this part is rather more complicated than the sex identification, so it'll take a few weeks.

In the meantime, best wishes to everyone for the holiday period, and thanks so much for all your kind comments on my postings.

Glesni & Monty - we are finally starting to understand more about their offspring