This lab studies how a certain hormone affects the mating behavior of spadefoot toads.

What is a hormone? The parents of teenagers are often heard complaining about them, but what do they do?

Hormones are chemical messengers in your body. They are usually proteins, but they can also be other types of molecules. Special glands in your body will release lots of hormone molecules when they want to send a message to a different part of your body. The hormones may touch every cell in the body, but only the cells that have the right receptor will be able to receive the message. A receptor is another kind of protein. A hormone will fit into its receptor like a key into a lock. No other protein receptors will be able to recognize it.

Image Courtesy of Wikimedia Commons

This is like having to send a letter to everyone in the world when you only really need to send it to a few friends. To make sure that only your friends get the letter, you put the letter into a special envelope that will only fit into your friends' mailboxes.

Hormones can have lots of different jobs. Leptin is most well known in humans as the hormone that tells you to stop eating when you are full. Remember the fat mouse from earlier? The mouse on the left has had its leptin production blocked. Because it was not triggered by its hormones to stop eating, it became obese. Leptin also has other jobs that are related to that function. It tells your body when you have enough energy to reproduce. One hormone can trigger the production of other hormones that send complex messages all over your body.

Fat Mouse

Image courtesy of Wikimedia Commons

We know that leptin has some similar functions in amphibians, like telling the animal how much to eat. But it also regulates the metamorphosis of a tadpole into a frog or toad. It also seems to have an important role in mate selection, and this role is what is being studied now. We know that it is sending some kind of message. But we don't know where the message started or where it is going.

Because we know that lots of leptin means that the animal has eaten lots of food, you could hypothesize, or make an educated guess, that Plains spadefoot toads would be most likely to mate with their own species if they have a high leptin level. If they have plenty of energy to reproduce, they can afford to be more picky about their mates. So if an animal has low leptin, it has not been eating much and should take the first male she can get.

To test a hypothesis, you have to perform an experiment! We know that female Plains toads will usually choose their own species in deep water and the other species (New Mexico toads) in shallow water. But how does their choice change when they have been injected with leptin?

A group of female Plains toads was divided into two categories. Half of the toads would be placed in deep water for the experiment, and half would be placed in shallow water. Within each of those groups, half of the frogs were injected with leptin, and half were injected with saline.

Saline is just water with a little bit of salt added. Why would we bother injecting it into one group of toads?

You want to test how salt will affect mate choice.
You need to see how a toad will normally behave without leptin.
The toads need to stay hydrated.

Behavioral Experiment

For this experiment, the female Plains spadefoot toads are divided into two groups. One is tested in a pool with lots of water, and the other group is tested in a pool with shallow water. The experiment takes place in a dark closet in the Pfennig lab. The lab contains a pool, two speakers, and a platform on which to place the toad.

Before we can start the experiment, the toads need to be injected with leptin or saline, rinsed off, weighed, and measured. The weight and length of the toads can be used to see if their body conditions are affecting their mate choice. A very well fed toad might choose differently than a hungry toad.

The toad is then put on the platform in the center of the pool under an ultra high-tech flowerpot between two speakers. One speaker plays the Plains toad call, and the other plays the call of the New Mexico toads. The toad is then left under the flowerpot for ten minutes to adjust to its new conditions.

After ten minutes, the flowerpot is lifted off the toad with a string that has been threaded through the wall of the closet. A night-vision camera shows us what is happening in the pool. The toad gets up to thirty minutes to choose a speaker. When the toad chooses, the chosen speaker and the time it took the toad to choose are both recorded. Sometimes the toads don't cooperate. They will sit on their platform for the whole half-hour or just try to escape the pool. Others choose in less than a minute!

This diagram summarizes what Nick found:

What were the results of this experiment?

Leptin makes Plains toads want to mate with the other species.

Which experimental group tells you this?

High water group
Low water group

This experiment had an unexpected result. The toads that had been injected with leptin were more likely to move towards the other species’ calls. This means that the hypothesis was incorrect, and leptin probably has a different function or pathway than what was expected. But this does indicate that leptin is at least part of a series of hormones that are used to make this decision.

Now that Nick knows that leptin has an influence over Plains toads' choices of mates, he wants to find out if it has the same effect on New Mexico toads. If it does, that means that it's not just a quirk of that species' biology; it could apply to other amphibians as well!