May 10, 2022
Written by: Marissa Maroni
Clownfish, of the scientific subfamily Amphiprioninae, are known for their striking orange and white coloring and for starring in the animated movie series Finding Nemo. But there’s much more to clownfish than their good looks and Hollywood acclaim. Clownfish are protandrous, meaning they change from male to female, based on their current social environment. They live in a group where there is a single monogamous female and male pairing, and the remainder of the fish are non-breeding males. The largest and most dominant fish will develop into a female and the second largest will develop into a breeding male1. Clownfish undergo the process of sex differentiation from male to female when the female of the group dies or is lost. This fascinating socially cued sex differentiation has led a lot of neuroscientists to wonder what is mediating this change. Below we’ll be diving into some studies aimed at understanding this process.
How is the brain altering behavior?
After a female is removed from a clownfish group, the male breeder has a behavioral change in which he no longer takes orders from the female and instead dominates over the rest of the fish in the group. This led researchers to question what brain changes cause this increased aggression in the former male breeder that transitions into the dominant female. Researchers from University of Illinois at Urbana-Champaign aimed to answer this by studying the neuropeptide arginine vasoactin which is important in social behavior and reproduction in nonmammalian vertebrates2. To see if this neuropeptide was involved in the sex-transition behavior as well, the researchers used two size-matched juvenile clownfish and injected one of these two fish with an arginine vasoactin inhibitor, meaning the arginine vasoactin was no longer in the injected fish. They then had this clownfish pair interact in a ten-minute contest where their aggressive behaviors could be quantified. Since size typically determines which fish become the breeding male and female, two juveniles of the same size will “break the tie” by attempting to dominate the other to be of higher social rank. The researchers found that juveniles with inhibited arginine vasoactin had fewer aggressive behaviors (i.e. charging). This shows that arginine vasoactin has a role in establishing aggressive behaviors in juveniles. But what about breeding males that exhibit aggressive behaviors after the dominant female leaves? To test this, researchers used two breeding males of the same size, injected one with the arginine vasoactin inhibitor and put them into a 90-minute contest. Like the juvenile contest, the researchers found the male breeder with inhibited arginine vasoactin had fewer aggressive behaviors! Altogether, this study suggests that arginine vasoactin signaling plays a role in aggressive behaviors seen in both juvenile and male breeding clownfish when attempting to establish dominance to gain higher social rank.
What genes change in the brain during sex differentiation?
From the prior study, we know that arginine vasoactin plays a role in producing aggressive behaviors in breeding male and juvenile clownfish. But what else could be changing in the brain during this process and how could this potentially explain behavioral and physical changes? To test this, researchers used RNA-sequencing, a technique that allows scientists to compare the gene expression profiles, or the amount of each gene in a system, to see if across different groups the amount of specific genes change. The researchers in this study used RNA-sequencing on transitioning males and females along with mature males and females and analyzed what genes differed between these groups3. Various genes were differentially expressed but one gene of interest was cyp19a1b which converts androgens, a group of sex hormones found at higher levels in males (i.e. testosterone), into estrogens, a group of female sex hormones. This gene was found to be more highly expressed in mature and transitioning females in the brain suggesting that brain alterations are mediated by an enzyme that acts to increase the amount of estrogen in a clownfish!
Does the environment impact this process?
These previous studies began to determine how this sex differentiation works in a controlled lab environment but often the environment in nature is far more complex. For example, how is sex differentiation impacted when environmental toxins are present? Bisphenol A (BPA) and 17α-Ethinylestradiol (EE2) are two common endocrine disruptors that pollute the oceans. BPA comes from plastics and EE2 is often found in hormonal birth control pills. A group of researchers was interested in whether these two common pollutants can alter clownfish sex differentiation4. To test this, clownfish were fed these pollutants and their behaviors, gene expression, and gonads were assessed. They found that aggressive behaviors were reduced in clownfish exposed to BPA. Further, BPA was found to increase the expression of the gene mentioned above, cyp19a1b and feminized the gonads of the fish. EE2 did not influence behavior but did have some effects on genes previously found to change in expression during the transition from male to female. This suggests that ocean pollutants can alter sex differentiation and shift the sex differentiation to favor a female result.
Hopefully this article has convinced you that clownfish are an incredibly interesting species. This research illustrates how complex brains are in which they can sense changes in their social environmental and subsequently produce different behavior!
1. Kobayashi, Y., Nagahama, Y., & Nakamura, M. (2013). Diversity and plasticity of sex determination and differentiation in fishes. Sexual Development, 7(1-3), 115-125.
2. Yaeger, C., Ros, A. M., Cross, V., Deangelis, R. S., Stobaugh, D. J., & Rhodes, J. S. (2014). Blockade of arginine vasotocin signaling reduces aggressive behavior and c-Fos expression in the preoptic area and periventricular nucleus of the posterior tuberculum in male Amphiprion ocellaris. Neuroscience, 267, 205-218.
3. Casas, L., Saborido-Rey, F., Ryu, T., Michell, C., Ravasi, T., & Irigoien, X. (2016). Sex change in clownfish: molecular insights from transcriptome analysis. Scientific reports, 6(1), 1-19.
4. Gonzalez, J. A., Histed, A. R., Nowak, E., Lange, D., Craig, S. E., Parker, C. G., … & Rhodes, J. S. (2021). Impact of bisphenol-A and synthetic estradiol on brain, behavior, gonads and sex hormones in a sexually labile coral reef fish. Hormones and behavior, 136, 105043.