Anti-Predation Behavior in Response to Conspecific Visual, Olfactory, and Damage Cues in the Three-Spined Stickleback

Claire VanMeter1,2,3

University of Dayton

300 College Park, Dayton, OH 45469

1. Department of Biology

2. Berry Summer Thesis Institute

3. University Honors Program

Thesis Mentor: Jennifer Hellmann, Ph.D.

Department of Biology

Abstract

Predation is a source of mortality for prey, which creates a selective pressure for being able to avoid predators whenever possible. By using alarm cues produced by conspecifics, organisms can be alerted of nearby predators without coming in direct contact with them. However, we do not know whether individuals can distinguish between different kinds of conspecific cues and if some types of cues may indicate a more severe predation threat compared to others. Three-spined stickleback (Gasterosteus aculeatus) are used as a model for behavioral studies because their defense responses have been well studied and identified, and they are known to respond to both predatory and conspecific visual and olfactory cues. I studied the ability of the three-spined stickleback to distinguish between environmental cues by exposing conspecifics to four different types of conspecific alarm cues: cues of predator-naïve conspecifics (control water with unexposed demonstrator), visual cues of predator-exposed conspecifics (control water with predator-exposed demonstrator), visual and conspecific olfactory cues (stress cues and predator-exposed demonstrator), and visual, conspecific olfactory, and conspecific damage cues (stress cues, damage cues, and predator-exposed demonstrator). For 5 minutes before and after exposure to the cues, I watched for four key defensive behaviors: hiding in plants, hiding in the gravel at the bottom of the tank, swimming into the walls of the tank, and shoaling. I assayed 40 conspecifics over 8 weeks, for a total of 160 trials. Directly after each assay, I placed the focal subject in 200ml of RO water in a 600ml beaker for 1 hour to collect waterborne cortisol. I will run this water through ELISA assays to measure the cortisol concentration in the sample. I hypothesize that the conspecifics will be able to distinguish between the severity of the cues, causing an increase in anti-predator behavior response in exposure to all the combined cues in comparison to a few, or none of the cues. I anticipate that through this research, we will gain a better understanding of the influence of conspecific communication, specifically regarding how conspecifics interpret olfactory and visual conspecific cues. Anti-predator behavior is often costly, so being able to determine when such behavior is necessary based on conspecific communication could be a key factor in the survival and success of species.

Introduction

Predation acts as a major source of mortality for prey, so it is evolutionarily advantageous for prey to develop means of avoiding predators without having to come into direct contact with them. This has selected for the evolution of different types of conspecific communication where groups of conspecifics can communicate about their experiences without having to directly contact the stressor. Visual communication is the ability for animals to observe the actions of conspecifics and react accordingly. For example, zebrafish will behave as if a predator is present when a conspecific (a demonstrator fish) behaves defensively, even without direct visual confirmation of a 82 predator being present (Silva et al. 2019). Olfactory communication is the sensing or smelling of chemicals in the environment that convey information. For aquatic organisms, this mechanism is key for successfully navigating, finding a mate, avoiding predation, and finding food. For example, the Iowa darter is able to voluntarily release disturbance pheromones when exposed to a stressor to alert conspecifics of danger (Wisenden, Chivers & Smith, 1995). Brown et al., (2000) also found that fathead minnows show increased antipredator behavior when exposed to the chemicals that are released from conspecific skin when damaged, as it is a sign that there may be a predator in the area. Most animals use a combination of these kinds of cues to communicate both with conspecifics and other organisms in their environment.

Conspecifics use these cues as a means of communicating about the risk of coming in contact with predators, yet it is unknown as to how conspecifics respond according to the severity of these cues. Performing antipredator behaviors are costly, both because they are energetically intensive and reduce time for other activities. It is evolutionarily favorable to be able to determine the minimum level of defensive behaviors needed to maintain safety so an organism's time and energy can be spent on forging and reproduction rather than defense (Tollrian et al. 2015). If individuals can distinguish between the severity of a conspecific cue, they can alter their behavior accordingly, and spend less time performing defensive mechanisms when they do not need to be. By exposing conspecifics to varying degrees of severity of cues, their ability to respond according to the severity of the cues can be measured.

Here, we used the three-spined stickleback, Gasterosteus aculeatus, to understand how individuals alter their anti-predator behavior in response to visual and olfactory conspecific cues. Sticklebacks display a variety of well studied anti-predator behaviors (Landeira-Dabarca et al. 2019), making them an ideal subject for analyzing how conspecifics respond to varying degrees of conspecific cues. A demonstrator was used to create visual conspecific danger cues in combination with stress and damage olfactory cues to create differing levels of perceived danger for the focal subject. The focal subject was then observed both before and after the addition of the cues for defensive behaviors, and waterborne cortisol was collected after the assay. I hypothesize the stickleback will be able to distinguish between the severity of conspecific cues, causing an increase in antipredation behavior and waterborne cortisol in exposure to a combination of visual, olfactory stress, and olfactory damage cues in comparison to a few, or none of the cues.

Methods

Generating Conspecific Visual Cues

I used 40 adult sticklebacks as the focal subjects, with each stickleback going through 4 assays for a total of 160 assays over 8 weeks. The focal subjects were caught in the wild during the summer of 2021, and raised in the lab for the next year in the absence of predators. I assayed each conspecific through 4 different treatments: Control - control olfactory cues with an unexposed demonstrator, Visual Only - control olfactory cues with an exposed demonstrator, Visual and Stress - stress cues with an exposed demonstrator, and Visual, Stress, and Damage - stress and damage cues with an exposed demonstrator. The treatment groups increase in severity of risk of predation for the focal fish, creating different levels of potentially perceived danger. The demonstrator is another adult conspecific in the line of sight of the focal subject to provide conspecific visual cues to the focal subject. The demonstrator was either left predator-naïve or exposed to a predator through three kinds of cues: 1) visual predator cues (a model predator in the line of sight of only the demonstrator and not the focal subject), 2) olfactory predator cues (water from a tank containing a trout that was fed sticklebacks for 4 days; methodology slightly modified from Crane & Ferrari (2014)), and 3) conspecific damage cues. In a trial with a non-predator exposed demonstrator, there is no model predator used in the center tank, and the demonstrator is only given 105 ml of RO water. In a trial with a predator exposed demonstrator, the model trout is in the center tank and the demonstrator is given 100 ml of trout water and 5 ml of conspecific damage cues.

Generating Conspecific Olfactory Cues

Along with the visual cues of the predator exposed or unexposed demonstrator, I gave the focal subjects two different conspecific olfactory cues depending on the trial. For the control conspecific stress cues, I placed 5 sticklebacks in a clean 10-gallon tank, where they sat for one hour, undisturbed. I then removed them, and froze the water until the date of the assay when it was needed. This exposed the focal fish to the scent of unstressed conspecifics, indicating that conspecifics are present but not in danger (Brown & Godin, 1997). 400 ml of these cues were used during the assays for the Control and Visual Only treatment groups. For the conspecific stress cues, the same procedure occurs, except prior to the one hour waiting period, I chased the fish with a 6 in model of a trout for 90 seconds. This caused the conspecifics to excrete stress hormones, which collected in the water (Brown & Godin, 1997). 400 ml of these cues were used for the Visual and Stress, and Visual, Stress, and Damage groups.

I generated the conspecific damage cues by removing the head and organs of adult sticklebacks, grinding the body into fine particles, and allowing the paste to sit in 75ml of water per 1 stickleback for 5 minutes. Once the 5-minute period was over, I removed the large particles through a fine strainer and the liquid was frozen until the date of the assay. This procedure is a modified version of the procedures done by Brown & Godin (1997), and Mathis & Smith (1993). I added these cues to the Visual, Stress, and Damage treatment group, as well as to the demonstrator tank when the demonstrator was predator exposed.

Procedure

I placed both the focal subject and demonstrator into a divided, ten-gallon tank for 20 minutes prior to the assay for acclimation. The tank was divided into two, uneven sections, with the focal section being three times the size of the demonstrator section. A clear, plastic barrier was between the sections, which allowed the conspecifics to see each other but not smell each other. A five-gallon tank was placed perpendicular to the ten-gallon, which contained the removable clay model trout. The ten-gallon tank had blackout paper on select walls to ensure only the demonstrator can see the model trout, and not the focal subject (Figure 1). After the 20-minute acclimation, I observed the actions of both the demonstrator and the focal subject for five minutes prior to the addition of any cues. I observed four key behaviors which demonstrate stress in three-spined sticklebacks: sitting at the bottom in the gravel, hiding in the plants, scototaxis, and shoaling along the barrier (Landeira-Dabarca et al. 2019). Directly after the baseline observation, the cues were added based on the treatment type, and the barrier between the demonstrator and the model trout tank was removed. The demonstrator was exposed to the visual and olfactory cues of the trout predator, or to the control, while the focal fish was exposed to the conspecific visual cues from the demonstrator and the added conspecific olfactory cues. Then, I did another 5-minute observation for the same four key behaviors. After the second observation period, the barrier is put back and the fish are removed. Then, the tank is cleaned and reset with new water for the following assay. Two tanks were set up at once so while one group acclimated another was being observed.

Waterborne Cortisol

This study investigates two different stress responses in the three-spined stickleback: antipredator behavior and waterborne cortisol levels. The observed and recorded anti-predator behavior measures the immediate physical response to the stimuli, while waterborne cortisol records the chemical stress response experienced post-assay. Increased cortisol levels indicate increased stress even when stress-induced behavior is not observed. Cortisol performs an important function in the stress response of organisms and allows for a better chance of survival in high-pressure scenarios by increasing the availability of sugars and promoting the fight or flight response (Ellis et al. 2012). The stress hormone is excreted through the gills, urine, and feces, which accumulates in the water. I collected the cortisol immediately after each assay by placing the focal subject in a clean, 600 ml beaker with 200 ml of RO water, which was left to sit in a dark environment for one hour. After the one-hour period, I removed the conspecific, and collected and froze the water. The week prior to the week of the assays, I acclimated the focal subjects to the cortisol collection process by placing them in 600 ml beakers with 200 ml of RO water in a dark environment for one hour for five days. This acclimates the fish to the stress of being an enclosed space to ensure the stress hormones that collect in the water are from the stress due to the assay and not because they are in an enclosed environment. The collected samples will be thawed and run through the ELISA (Enzo Life Sciences, Farmingdale, NY, U.S.A.) kit by vacuuming the collected water through columns. For detailed methodology reference Dellinger et al. (2018).

Timeline

Moving forward, I will run the frozen cortisol samples through the ELISA assays to measure their cortisol concentrations. Once this is completed, I will organize the raw data and analyze it for significance. Through this analysis, I will get a better understanding of how sticklebacks react,