Field Notes: There is something in the water.

In kindergarten, one of my favorite activities was playing at the water table on a sunny day. We used dump trucks, buckets, and water wheels to send water splashing around a big trough. There was likely a goal of filling some container at the end, but I just remember splashing each other and squealing loudly.

Most of the time, my current science feels like a glorified version of this.

 Now I use siphons and elaborate PVC pipe structures, but it turns out that studying fish behavior involves a lot of moving water from one place to the next. Especially when you are doing your science in a lab. This is maybe not the typical “Field Notes,” because my “field” is a windowless room where I do my best to mimic as many of the complicated aspects of nature as I can in white plastic aquariums. This enables me to test very specific questions that would be impossible in the field. Most of my research focuses on the effect of changes in the temperature of water and predator presence on the behavior of small fish. I am trying my best to make my fish think they are just swimming around in a normal stream, but there are countless differences between my arenas and the world outside. One major one is that my water is cleaner than many of the local waterways (and, lucky for me, okay to splash around in a little).

It is this difference between the quality of water that my lab-raised fish experience and the water that many wild fish are exposed to that inspired a project I collaborated on led by Dr. Isaac Ligocki. Pesticides are an incredibly complex topic—their application can help to support large-scale agriculture needed to feed a growing population, but they can also have several unintended consequences. Many of these pesticides end up in waterways where they can affect morphology, behavior, and even the expression of genes (i.e. how heritable information in a gene turns into something functional) in animals that were never the target of these pesticides. Due to several troubling headlines about the far-reaching effects of organophosphate pesticides on mammals and birds, pyrethroid pesticides like Bifenthrin, have become more popular as “safer” alternatives. These pesticides are promising because they are still lethal to their insect targets but are much less harmful to mammals and birds than traditional pesticides. 

Unfortunately, the effects are not as benign for fish. There is growing evidence that Bifenthrin is an “endocrine disrupting chemical” for fish. Chemicals, like Bifenthrin, interfere with the normal endocrine functioning (i.e. how hormones help cells communicate to regulate processes in the body) in fish by either being perceived as a hormone or interfering with the normal function of hormones. This is where my fish come in. 

I happily watch fish all day!

I work with Western mosquitofish (Gambusia affinis). Mosquitofish are not particularly exciting looking fish—they are beige colored fish smaller than a fun size candy bar. But for such a bland little fish they are incredibly cool. Most fish have external fertilization—the males shoot sperm and the females shoot eggs into the water where the fertilized eggs develop (outside of the female). Not mosquitofish! Male mosquitofish have a modified fin that essentially acts like a penis and delivers the sperm right to the female. This means that the babies can develop inside the mom where they are protected from becoming caviar to any number of bigger fish that would like to snack on them. Despite their small size they have a huge global impact. Originally from the Mississippi River basin they are now found on every continent except Antarctica. Not only are they hardy and able to spread out when they get to a new place, but they have benefited from a good name. Despite being generalists that will eat just about anything they can fit in their mouths, their name has led them to be widely introduced to control mosquito populations. 

Gambusia affinis [Source]

All of these characteristics make them ideal for studying the impact of pesticides like Bifenthrin. Their wide range would make them a useful global indicator species. Scientists can use the health of species like mosquitofish to compare water quality in different areas. Additionally, because of their unique mating system they are particularly interesting for studying the effects of pollutants that potentially interfere with hormones. There are already researchers studying the effect of pesticides on the development of the penis-like structure in males, but there is less work studying the effects on females. 

All of that is why I found myself in the lab wearing a full lab coat, safety glasses and gloves, along with a dedicated team of undergraduate research assistants. It turns out that when you are working with potentially harmful pesticides, studying fish feels a lot less like kindergarten. Isaac and I divided female fish into groups of five in large pickle jars (fun fact–the silicon sealant in normal aquariums contains chemicals which can react with the pesticides!) After two weeks with their new social groups, each fish went through a series of baseline behavior assays- meaning we looked at their behaviors under their usual environmental conditions. This allowed us to determine how they behaved before being exposed to pesticides. Each lovely lady fish was first exposed to two males—one big, sexy guy and one smaller, less attractive one—and we compared the time that she spent with each one. Then we gave her access to a group of unknown females and recorded how active she was and how much time she spent in a group. We then exposed the groups to different amounts of the pesticide and retested the same behaviors two weeks later.

Dr. Isaac Ligocki models proper PPE–personal protective equipment–for working with pesticides.

Following the behavior assays we sacrificed the fish in order to dissect out the brain and liver. I spend a lot of time taking care of my fish and this is always my least favorite part of the study, but it is important to figure out exactly how the pesticide is affecting the fish’s morphology. The impacts of pesticides are still so poorly understood that we need to collect as much information as possible from every fish.  In this care, we collaborated with other scientists (like fellow Ethogram editor Victoria Farrar!) to look for differences in gene expression in the brains and livers of fish exposed to pesticides. 

It is a good thing we collected this data because it ended up being an important part of the story. While we didn’t see any behavioral differences between our fish that were exposed to pesticides, there ended up being differences in the expression of estrogen receptor and glucocorticoid receptor genes in the brain in response to exposure to Bifenthrin. 

So, what exactly does this mean? It could actually mean lots of things. It could be that in response to pesticides, gene expression changes to buffer against changes in behavior. It could also be that the amount of pesticide we exposed our fish to was enough to elicit changes in gene expression, but not enough to change the behavior. Our behavior tests also may not have picked up on subtle differences in behavior. But it could also be that we were looking at the wrong behaviors. As someone who spends a lot of time thinking about responses to predators, I found the differences in glucocorticoid receptor expression particularly interesting. Glucocorticoids are involved in lots of processes in the body, but they play a big role in responses to stress. Perhaps exposure to pesticides affects the way that fish respond to stressors…like predators!

But that is a question for another project! And that is what I love about science. Each project that you complete opens up more questions and what you are thinking about for one project may be the key to understanding another project. It is that quest to figure out just one more thing that keeps me in science. Well that and the ability to basically play with water all day and call it a job. 

Check out our recent publication summarizing this work below!

Ligocki, I. Y., Munson, A., Farrar, V., Viernes, R., Sih, A., Connon, R. E., & Calisi, R. M. (2019). Environmentally relevant concentrations of bifenthrin affect the expression of estrogen and glucocorticoid receptors in brains of female western mosquitofish. Aquatic toxicology, 209, 121-131.

Amelia Munson is a fifth year PhD student in the Animal Behavior graduate group at UC Davis in Dr. Andrew Sih’s lab. Her research interests focus broadly on acknowledging that the world is complicated and stressful, often for multiple reasons all at once, and understanding why individuals respond to the challenges differently. She responds to the multiple stressors in her own life by hiking, drawing and singing off tune. 

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