In the lab!

Hihi are beautifully sexually dimorphic – meaning that males look different to females – so telling them apart is very easy. Not only do males have distinctive yellow wing bars and cute little white “eyebrow tufts”, they are also quite a bit bigger than females, even when they are in the nest.

Male on left (lovely photo by J Graham:; female on right (photo Steve Attwood)
Male on left (lovely photo by J Graham:; female on right (photo Steve Attwood)

Behavioural ecologists are interested in knowing the sex ratio of broods, social groups, and populations for lots of reasons (2). I’m interested in how the sex ratio of the offspring that mothers produce might vary over time – perhaps because one sex is more costly to produce (if one sex is bigger, for example), and mothers in good condition might better be able to “afford” the cost. Or, perhaps because one sex has greater benefits in different circumstances – for example, if female offspring are more likely to stay in the group and help, and the group is low on helpers (3). I’ve been interested in exploring what factors might affect sex ratio in hihi – but quickly came upon a problem. Unfortunately, lots of hihi die as embryos still in their eggs, or as tiny chicks in the nest when they are still too young to see sex differences (3), so determining brood sex ratio (and therefore, getting an idea of why adult sex ratios might differ) is difficult.

Luckily, researchers on Tiritiri Matangi collect as much tissue from dead embryos and chicks as possible, so earlier this year, I was able to hit the lab to extract DNA from some hihi chick samples in order to determine the sex of around 100 chicks that died before they were able to be sexed visually. A great chance to learn some molecular techniques! And also a cool chance to see my study species – as I haven’t made it out to New Zealand yet!

Apologies if the following images are a little grisly – but I was fascinated to see some examples of different stages of hihi development when embryos had been able to be preserved whole! They reminded me perfectly of the early drawings of Haeckel’s embryos (pictured below), with which Ernst Haeckel argued persuasively for a common vertebrate origin.



George Romanes’s 1892 copy of Haeckel’s embryo drawings

From each of the samples, I took a small amount in order to extract DNA (if you’re interested, I was using Qiagen DN-Easy kits), which I then amplified using Polymerase Chain Reaction (this takes a small amount of DNA and copies it over and over, so you end up with much more than you started with – meaning you don’t have to use so much of a sample to begin with!).

I then used gel electrophoresis to allow me to identify whether each sample contained male or female sex chromosomes. This process works by using an electric current to move molecules (which are negatively charged) through a softish medium (most commonly used is agarose). As male and female sex chromosomes (for example, our X and Y) differ in size, they move different distances, so when the DNA is stained on the agarose, you see either one band (indicating the sample contained two X’s), or two (indicating the presence of two chromosomes of different size (in humans, X and Y).  Birds differ in that females are the sex that has two different chromosomes – the “Z” and “W” chromosomes – while males just have one (Z). So, when I ran my hihi samples on the gel, I saw two bands for a female, and one band for a male.

Stained samples visualised using UV - two bands represent a female. On the left is a test column called a ladder.
Stained samples visualised using UV – two bands represent a female. On the left is a test column called a ladder.

I’m writing a paper investigating what factors influence how sex ratio varies in the hihi at the moment – so look out for the results of this lab work soon! In the meantime, big thanks to Wenfei Tong who showed me how to do all this – my first time in a molecular lab! – and to Patricia Brekke who kindly provided the samples.

1. West, S. Sex allocation. 2009. Princeton University Press.

2. Emlen, S. et al. 1986. Sex-Ratio Selection in Species with Helpers-At-The-Nest. Am. Nat. 127: 1-8.

3. Rippon R, Alley M, Castro I. Causes of mortality in a nestling population of free-living hihi (stitchbird— Notiomystis cincta ). New Zeal J Zool. 2011. 38(3):207–22.