Who’s Really Calling the Shots?

Although we have all heard of hormones, it’s easy to forget how important they are, not only to us, but to every organism. In high school, or maybe an introductory science class you took in college, you probably learned a little bit about hormones, most likely in relation to the physical processes that they joyfully accompany puberty. Let’s hear it for estrogen and testosterone, am I right… However, in reality, estrogen and testosterone are not even the tip of the hormonal iceberg. There are so many hormones! Don’t even get me started on what hormones do for us. They control everything…and I mean everything. When you get hungry and decide to go explore the depths of your fridge (don’t lie, we all do it), you probably think that is a decision of your own volition. Wrong. Hormones made you do it. What about that nightly ‘decision’ you make to go to sleep? Well, it’s not so much a decision as it is hormones telling you what to do. Even the trust you might feel for a significant other or your best friend is a product of hormones! While you can’t feel them or see them with the naked eye, believe me when I say that it is the hormones your body is churning out every second of every day that control everything from your mood to your appetite, heart rate, metabolism, sex drive and the list goes on and on. So now that I have made you feel like you have no free will, what exactly are hormones and why am I talking about them on a fish blog?

Well, let’s start with the basics. Hormones are special chemical messengers that are secreted by endocrine glands, often under the influence of the central nervous system (aka the brain and spinal cord), in every organism. Now don’t be intimidated by the term ‘endocrine.’ It simply refers to glands that secrete hormones directly into the blood. Why couldn’t I have just said that to begin with?! These glands can be distinguished from other bodily glands because they don’t have ducts, and they release their hormone products directly into the bloodstream where they travel, sometimes long distances, to many different target tissues in the body. It’s at these various target tissues that hormones exert their effects. Now, you might be wondering why we are studying hormones in a research lab that claims to be focused on integrative animal behavior. Well, if you think back to what I said about hormones ruling your life and a lot of your behaviors, then it makes sense we would call ourselves an animal behavior lab. Hormones dictate the lives of just about every organism, including our favorite little mangrove rivulus fish. Hormones underlie almost all of the behaviors we study. If you’re not convinced, just let me tell you a little bit about some of the behaviors that hormones control in the mangrove rivulus.

The mangrove rivulus may appear small, and definitely cute, to the naked eye, but these little guys love to fight. Have you ever seen fish fight before? It’s really just a bunch of bumping into each other. When mangrove rivulus get into a fight with another member of its species the two fish will bump into each and even hook their mouths together, which propels them into a sort of spin. It looks almost like a dance, but trust me: it’s a fight. Now, these little guys really care a lot about whether they win or lose. I mean, no one likes losing, right? Well the mangrove rivulus cares so much about the outcome of its fight that research has shown it will change its post-fight behavior based on whether it has lost or won. After a recent victory, the winners of a fight, basking in their own glory, experience a sort of ‘winner effect’ that makes them more likely to initiate and escalate other fights (see the great stuff being done in this area by our long-time collaborator Yuying Hsu) (Chang et al. 2012). Research has indicated that they have a higher chance of winning future fights as opposed to their losing counterparts. The losers of these fish fights, in addition to having to suffer obvious defeat, also become less aggressive, voluntarily retreat and suffer a higher chance of losing future fights (Chang et al. 2012). Talk about getting the short end of the stick. So what does all of this have to do with hormones? Well, it has a lot to do with hormones actually! Levels of hormones, in particular stress and sex steroid hormones, can actually predict how aggressive the mangrove rivulus will be during a fight, which, in turn, allows researchers to gauge how the fish will perform during fights. But wait, there’s more! These levels of hormones are not static. Rather, they change and fluctuate in response to a variety of things, including the fights the fish engage in. The dynamics of the fights these fish get in (that is, how nasty the fights get) influence these hormonal levels, and causes them to fluctuate. In addition, recent victories and defeats also have an affect on the individual’s behavior in future fights, as well as that individual’s tendency to win these future contests (Hsu et al. 2006). Generally, rivulus’ personal experiences with winning or losing fights actually increases, or decreases, the fish’s probability of future wins, or losses (depending on whether it won or lost, of course) (Hsu et al. 2006). Previous fighting experiences, and their outcomes, actually affect the mangrove rivulus’ perception of their fighting ability and also affects their motivation to engage in future fights (Hsu et al. 2006). Now that’s cool! If we allow ourselves to think outside of the box a little bit we can see how this research could even apply to fights between humans, or the way bullies act after they have successfully belittled someone. People often say that bullies have superiority complexes that develop from their ability to successfully make another person feel inferior. Does it seem so far-fetched to blame the increased aggression we observe on hormonal changes they might experience as a result of their previous experiences with fighting? It’s just an idea but it might merit some research. Really it illustrates an interesting point: our research, although done on fish, can have much broader applications.

Speaking of broader applications, several other studies involving the mangrove rivulus and hormones have been conducted that could speak volumes for us humans. One of these studies involved exposing the fish to environmentally relevant doses of ethinyl estradiol. Ethinyl estradiol is not a hormone that is produced endogenously (synthesized by the fish itself). Rather, it is a synthetic derivative of the major endogenous estrogen, estradiol, which is found in both our fish and in humans. Estrogen regulates a lot of developmental processes in humans. Most notably perhaps is its role in the formation of female physical features and its involvement in reproductive processes. Perhaps one of the most common misconceptions is that men do not have estrogen. Well they do, and it is absolutely essential for their health as well. So, what the heck is ethinyl estradiol then? Well, ladies, it’s the active ingredient found in almost all oral contraceptives. That’s right, its birth control! Females that take birth control pills are essentially just taking a big dose of synthetic estrogen (and often progesterone as well-another hormone involved in female reproduction). This daily dose of hormones tricks the female body into thinking it is pregnant, which leads to no more monthly ovulations and, in turn, very little chance of actually getting pregnant. Thank you ethinyl estradiol, right? Wrong. While its ability to stop pregnancy is just fine and dandy, its effects on the environment are not so great. When females taking birth control use the bathroom, the water, along with all their waste (sorry for the visuals) goes to a wastewater treatment plant. There, all the gross chemicals in the water are supposed to be filtered out before it’s released back into the waterways, like rivers. Unfortunately, one of the chemicals that is not effectively filtered by these treatment plants is ethinyl estradiol. So, this lovely synthetic estrogen is allowed to pollute our waterways, and everything that depends on these waterways. Here is where the mangrove rivulus comes in. Our lab has conducted research on the effects that exposure to environmentally relevant doses of ethinyl estradiol has on the reproductive physiology of mangrove rivulus (Johnson et al. 2016). The results were not pretty. Basically, after thirty days of exposure to a super-low dose of ethinyl estradiol, the fish experienced significant changes to their own endogenously produced hormones, which included 17β-estradiol (the most prevalent form of estrogen) and 11-ketotestosterone, which is fish-specific hormone derived from testosterone (Johnson et al. 2016). In addition, hermaphrodites and males experienced changes in the morphology of their gonads. Essentially, males had reduced sperm production and hermaphrodites experienced an increase in the number of early stage eggs (aka primary oocytes) they produced (Johnson et al. 2016). Now if these are the effects this synthetic compound is having on our fish, what do you think it is doing to people or other animals that utilize these same waterways for food or water? It’s not fun to think about but these are the real questions that we need to be asking in order to be more environmentally conscious. These are also the types of questions, and the broader applications, that our research in the Earley lab seeks to investigate.

So, now that you know a little bit about hormones, and the research that is being done with them, I challenge you to go out and explore the field of endocrinology (aka the study of hormones and their targets) a little more in depth. It’s amazing what you will discover and, I won’t lie, it will make you probably question if the decisions you make are of your own volition or of the chemicals that are constantly coursing through your body.

References

Chang C, Li C , Earley RL & Hsu Y (2012) Aggression and Related Behavioral Traits: The Impact of Winning and Losing and the Role of Hormones. Integrative and Comparative Biology 52 (6): 801-813.

Earley RL & Hsu Y (2008) Reciprocity between endocrine state and contest behavior in the killifish, Kryptolebias marmoratus. Hormones and Behavior 53: 442–451.

Earley RL, Hanninen AF, Fuller A, Garcia MJ & Lee EA (2012) Phenotypic plasticity and integration in the mangrove rivulus (Kryptolebias marmoratus): a prospectus. Integrative and Comparative Biology 52: 814–827.

Hsu Y, Early RL & Wolf LL (2006) Modulation of aggressive behavior by fighting experience: mechanisms and contest outcomes. Biological Reviews 81: 33-74.

Johnson EL, Weinersmith KL & Earley RL (2016) Changes in reproductive physiology of mangrove rivulus Kryptolebias marmoratus following exposure to environmentally relevant doses of ethinyl oestradiol. Journal of Fish Biology 88: 774-786.

The Blueprint of Life

While most people have heard of DNA, not everyone knows how important it is. Every living thing on planet Earth has DNA, or deoxyribonucleic acid if we are being formal. DNA is the chemical compound that contains the instructions needed to make proteins that are essential to the survival of just about every organism. An organism’s entire collection of DNA is called its genome. Basically, it’s the blueprint of life and every cell in every organism contains a complete copy of its particular genome. You might be thinking “Wow, that’s a lot of blueprints for just one organism.” Yes, it is, but that’s to be expected. Life is complicated, and it requires a lot of instructions.

So, what does this have to do with the mangrove rivulus? Well, it has everything to do with the mangrove rivulus! In the Earley lab, we care about the genetic makeup, or the “blueprint,” of each of our fish. Why? Because without this information we would not be able to study them the way we do. Before we can get into the nitty gritty of this ‘genetic makeup’ stuff, and why we study it, there are a couple of things you should know. Every organism’s genome is made up of a collection of genes. Simple enough, right? Wrong. In most organisms, these genes are present in two alternative forms, or alleles, that can be identical or different. Organisms, including us, are said to be heterozygous if they have two different alleles for a given gene and homozygous if they have identical alleles for a given gene. In a previous article (see “Let’s Talk About Sex”), I mentioned that the mangrove rivulus, specifically the hermaphrodites, can fertilize themselves. This is especially cool if the hermaphrodite is completely homozygous, a situation where the animal has identical alleles for all of its genes. In this case, self-fertilization will result in offspring that also have identical alleles for all of their genes, and the exact same alleles as the parent. Thus, self-fertilization effectively produces clones! For our experiments, you can imagine why homozygous offspring would be very useful. We can expose individuals of each homozygous clonal lineage to different environmental conditions and see how it affects their behavior, physiology, morphology, or any other trait of interest. Since there is no genetic variation among siblings, the only differences we might see among them are the direct result of environmental factors. Remember, there might not be variation among siblings in one clonal lineage, but there definitely is variation among lineages. One homozygous ‘parent’ might have a different genetic composition than another homozygous ‘parent,’ leading to clonal lineages that are distinct from one another. All this talk of homozygotes might have led you to believe that heterozygotes don’t exist in mangrove rivulus populations. Well, they do. Unlike homozygotes however their offspring are not genetically identical. But, how are there both homozygotes and heterozygotes in natural populations? Instead of fertilizing themselves, hermaphrodites also can mate with male members of the population. This is known as outcrossing and it introduces new, different genetic information, and heterozygosity, to the offspring. Therefore, the kids are not all genetically identical and some might have inherited some different versions of a trait that proves to be advantageous.

Considering our research and how I mentioned that we really like to make clonal lineages from homozygotes, you might be wondering how we can tell if a fish we acquire from a natural population is a heterozygote and homozygote. Well here is where being able to actually figure out the fish’s blueprint, or at least part of it anyways, comes in handy. In order to do these types of studies, we have to decipher each fish’s blueprint before we can actually do anything with it. But how the heck can we do that? Science, duh! It’s complicated, but definitely not impossible.

So, here’s the rundown. The process of deciphering the ‘foreign language’ that makes up an organism’s genome is called “sequencing.” Technically, there are many different types of sequencing methods that can be used to determine the degree of variation between different individuals in a population, but today we will just focus on one: DNA fingerprinting. DNA fingerprinting does not require the researcher to decipher the fish’s entire genome in order to find out if it is a homozygote or a heterozygote. Instead of taking the time and resources to determine an individual fish’s genome, we can instead take only a portion of that genome, sequence it and use it to determine how heterozygous an individual is, and also levels of heterozygosity in an entire natural population. The process involves collecting a little piece of the fish’s fin, 1 millimeter by 1 millimeter in size, which is then used to determine their genotype, as well as if they are homozygous or heterozygous.

Interesting stuff, but how do we know what portion of the genome we should be comparing? Can we just take any random part of the fish’s genome and determine its heterozygosity from there? Well, no. The thing about genomes, and especially genomes from members of the same species, is that there are always parts that are the same within a species. That means that every individual mangrove rivulus, regardless of who its parent or parents are, shares part of their blueprint with other members of the species. This also applies to heterozygotes and homozygotes. Lets take a look at this from a human perspective. Part of my genome is identical to part of your genome. So how are we not exactly identical? Well, only part of our genome is conserved. Other parts of our genome might be very different from each other, and it is these differences that make me uniquely me, and you uniquely you. So with this idea in mind, lets get back to the fish. Researchers of the mangrove rivulus have identified 32 neutral loci, a fancy word for different positions in the fishes genome that do not encode a protein and that, when amplified, studied and compared, can tell us whether these fish are heterozygotes or homozygotes. These loci are called “microsatellites” and, because they do not encode proteins, they are ‘free’ to vary in their sequence without having any adverse effects on the organism. That’s exactly why these regions often have more variation than other loci; the fish can afford it! These microsatellites typically consist of tandemly repeated nucleotide bases. The genetic code is just a bunch of A’s, T’s, C’s, and G’s (nucleotides) strung together in a particular order; microsatellites can be identified because they have repeated sequences of nucleotides. For example, a microsatellite might have ATC repeated multiple times; you might have ATCATC, and I might have ATCATCATC.

Now, remember that this fingerprinting is being done on fish collected in the wild. We use these tests to find homozygotes so we can collect them and create lineages of genetically identical fish (isogenic lineages) for our experiments. The fact that homozygotes as well as heterozygotes exist in natural populations raises a very interesting question: if the homozygotes are able to successfully produce offspring in a completely self-sufficient manner, why should they ever want to deal with a male? Good question, and one that many researchers are interested in answering. One idea is that reproducing with males introduces new combinations of the blueprint into the population. If these new genotypes confer some sort of benefit to the fish in terms of survival or reproduction, then this new genotype will persist, and some combination of self-fertilization and outcrossing with males will be favored evolutionarily. It has also come to light that homozygotes collected in the field are actually more susceptible to parasites than their heterozygote counterparts (Ellison et al. 2011). This could be one possible benefit that heterozygotes have over homozygotes. Other studies have shown that male heterozygotes are often larger than homozygotes. This could be another reason we observe heterozygotes in natural populations, especially if you consider the competitive edge a larger male could have over a smaller male when looking for love or when fighting with other individuals (Molloy et al. 2011).

Wow, while this may seem like a lot of information it is really just the tip of the iceberg when it comes to what we can do with genomes, as well as genome sequencing. Being able to decipher the language of the rivulus’ genetic blueprint affords us endless opportunities for studying these little fish with a lot to offer. For more information about how examining our fish’s blueprint can provide novel insights into its biology, visit the pages of our collaborators. Dr. Andrei Tatarenkov and Dr. John Avise at the University of California, Irvine are experts in identifying microsatellites and using them (as well as an arsenal of population genetic analyses) to address fascinating questions in mangrove rivulus and loads of other organisms! Dr. Joanna Kelley at Washington State University is diving into the mangrove rivulus genome to determine how our fish’s blueprint governs many aspects of its extraordinary biology!

References:

Ellison A, Cable J, Consuegra S (2011). Best of both worlds? association between outcrossing and parasite loads in a selfing fish. Evolution 65: 3021–3026.

Molloy PP, Nyboer EA, Côté IM. Male-male competition in a mixed-mating fish. Ethology. 2011;117:1–11.

Author:

Ana Preda-Naumescu