Defense Mechanisms in Spotted Salamanders (Ambystoma maculatum) Induced by Perceived Predation Risk as a Response to Differing Cues BIOS 35502-01: Practicum in Field Biology Carolina Quiles Bengochea Advisor: Jasper Leavitt 2019 Quiles-Bengochea 2 Abstract Predation is among the most important factors to understand the ecology, behavior, and evolution of animal species. The detection of visual cues and kairomones (chemical) released by predators allows the prey to find a way to increase their chances of survival and fitness. Ambystoma maculatum can be top predators in their adulthood but often suffer from high rates of predation as larvae and, therefore, rely on the use of these cues to avoid predation. However, the interaction between two or more cues and how the prey use it as a way to avoid predation is something poorly understood. In this experiment, we studied how the visual and chemical cues induced by dragonfly nymphs and diving beetles influence the anti-predator defenses of A. maculatum. We did not find a difference between the morphological responses of A. maculatum and the type of predation they faced. We found that larvae reduced activity in the presence of predators and that they had no preference when selecting a refuge. We also found that A. maculatum is more susceptible to perceiving chemical signals from predators. This is one of the first studies showing a decrease in the mass and length of A. maculatum during the larval stage when facing predators from its natural habitat. We discuss these results and provide a theoretical framework for future studies of predation perceived risk. Introduction Predator-prey dynamics can influence prey populations through consumptive effects, like hunting and killing, or through increasing the risk of consumption (Preisser and Bolnick 2008). Predation risk is a significant influence on changes in behavior, morphology, development, reproduction, and diet of the species that are a target as preys (Dwyer 2009). Still, these defensive strategies often have significant costs that reduce prey fecundity or survival (Preisser and Bolnick 2008). Those demands are often mediated by the time and resource allocation trade- Quiles-Bengochea 3 offs (Steiner and Pfeiffer 2007). Resource reallocation is often limited by competing traits, such as growth, maintenance, reproduction, and storage (Steiner and Pfeiffer 2007) and that is why many animals do not develop these defensives traits unless they are necessary. Therefore, the effectiveness and benefits of such traits will be dependent on the speed at which they can be expressed relative to the time when the risk began and the duration of it (Mitchell et al. 2017). Behavioral and morphological defenses are often plastic and only appear when the prey senses predation risk (Steiner and Pfeiffer 2007) but once they invest in the development of new characteristics or behaviors to survive, they can be permanent. Behavioral responses are usually associated with the reduction of activity and increased refuge use or hiding, and temporal and spatial avoidance of predators (Shaffery and Relyea 2015) while changes in morphology can include changes in growth or developing secondary structures to improve survival (Urban 2010; Dawidowicz and Loose 1992). Prey can also invest in the alteration of life-history traits like hatching plasticity (Dwyer 2009) and changes in time to metamorphosis (Shaffery and Relyea 2015). Prey utilize a variety of sensory inputs such as visual, chemical, tactile, and auditory cues to detect and avoid predators, and the type of cue can have strong effects on the type of response elicited by the prey (Lönnstedt 2013). In aquatic environments, visual chemical cues have been identified as a primary source of information for both vertebrates and invertebrates (Elvidge and Brown 2012). Visual cues are useful in predator recognition because they can provide information on the predator’s location and whether it is actively hunting or not (Shelton 2016). However, this type of cue is limited by photoperiod and the effectiveness can easily be compromised (Elvidge and Brown 2012). For example, using visual cues in lentic areas such as wetlands and ephemeral ponds can be less effective because they have low light due to turbidity of the water or due to structural visual Quiles-Bengochea 4 disruptions such as vegetation (Shelton 2016; Gardner 2019). Instead, many aquatic amphibians rely on the kairomones to avoid predation because predators and prey often are in close proximity and the exposure to chemical cues tend to be more intense than visual (Murray et al. 2004). Chemical cues can provide information about temporal and spatial predation risk and therefore allow the prey to regulate the expressions of inducible defenses based on proximate risk (Mitchell et al. 2017). These cues are a cocktail of two types of chemical cues: the ones derived by predators (kairomones; odors and diet) and from conspecifics or prey guild member (near attacks) (Mitchell et al. 2017). They have a relatively long duration compared to the other types of cues, and also provide information about the past presence of predators (Shelton 2016). The effectiveness of these cues relies on the fact that they can cross barriers that otherwise would limit the predation risk identification by the preys until it is too late (Shelton 2016). Separately these cues can induce different behavioral responses and many animals are relying on using multiple cues to accurately assess predation risk levels (Lönnstedt 2013), however the interactions between two or more cues together is poorly understood. Even though there are some clear benefits for behavioral responses to predation signals, it also involves a cost for the sustainability and survival of the species, especially since some of these changes are permanent. Spotted salamanders (Ambystoma maculatum) are used in perceived predation risk experiments because they can behave as intermediate or top predators as adults but suffer from high levels of predation in the larval stage from a variety of predators (Shaffery and Relyea 2015). Ambystoma maculatum typically breed in seasonally ephemeral pools and have high fidelity to these breeding ponds (Pfingsten et al. 2013). Once they hatch, larval survival is limited due to competition for food sources, aggression, cannibalism, and predation. Common predators consist of aquatic vertebrates like fish and turtles, or aquatic Quiles-Bengochea 5 insects such as diving beetles and dragonfly nymphs. Predation risk changes based on foraging strategies employed by predators, and larval Ambystoma are known to change their behavioral and morphological responses based on these different strategies (Rack 2016; Urban 2008). For example, A. gracile is active at day at night in lakes that lack predatory fish, but in the presence of trout they are strictly nocturnal (Petranka et al. 1987). Dragonfly nymphs use an ambush technique in which they wait for their prey to pass by so they can catch it (Berger and Hansen 2004; George 2013). Diving beetles, on the other hand, feed themselves by swimming through the water, cling to the prey and use their mandibles to inject a digestive enzyme (George 2013, Johansson and Nilsson 1992). Spotted salamander’s larvae respond uniquely to chemical cues from different predator species and employ different adaptations to escape accordingly (Rack 2016; Urban 2010; Urban 2008). These responses can be genetically innate or learned, which typically arise from exposure to a predator (Albecker and Vance-Chalcraft 2015). However, innate responses are more common in the younger stages because these evolved reactions can help the prey to diagnose the level of threat present in the environment even without any previous experience with the predator (Albecker and Vance-Chalcraft 2015). This study aims to determine how the visual and chemical cues issued by predators influence the defense mechanism of A. maculatum. Additionally, we tested if cue responses of A. maculatum differ according to the identity of two naturally-occurring predators. We hypothesized that the larvae respond differently to different cue-types. An increase in length and body mass is expected as a result of the defense mechanisms developed to avoid predation, and this would be more pronounced when larvae are exposed to chemical cues than visual cues. Being able to see predators will increase the chances of dying due to the energy investment, limitation of food sources, and the immediate danger they face. In order to avoid been detected Quiles-Bengochea 6 by predators, the larvae will reduce the activity and increase the use of refuge. Our objective was to evaluate how the predator-prey interactions affect the development of anti-predator traits and the importance that they have in the evolutionary and developmental level of this species. Methods Study Site – The study was conducted in the University of Notre Dame Environmental Research Center (UNDERC) located between the state line of Wisconsin and Michigan's Upper Peninsula in Vilas County (Wisconsin) and Gogebic County (Michigan). The property comprises over 7500 acres which are divided between a land area of 6150 acres with 30 lakes and bogs and 1350 acres that include a combined area. It has a variety of aquatic and terrestrial habitats that include vernal ponds, marshes, sluggish headwater streams, coniferous forest, mix, and non-mix deciduous forests (Jones 2017). Animal collection and rearing – We collected by hand from May 27, 2019-May 30, 2019 from twelve different ponds (Fig. 1a; Table S1). I used masses from ponds J, A2, N, Wood Duck, and V to ensure genetic diversity. The females show a preference for the same breeding pond, so they travel to it to lay their eggs there every year and only move to others when they have been altered or destroyed (Shoop 1968). We used two types of predators: diving beetles from the family Dytiscidae and dragonfly nymphs from the family Aeshnidae. Predators were collected through dip netting and minnow traps. The first round of predators was caught on May 30, 2019, at Morris Lake and on June 6, 2019 five minnow traps were set in two vernal pools, Wood Duck and Pool 9, for a total of 10 traps (Fig.