Dehydration Stress in the Wolf Spider Schizocosa Ocreata (Araneae: Lycosidae): Tolerance, Resistance, and Coping Mechanisms

Home , Anelosimus, Anelosimus studiosus, Dehydration, Larinioides cornutus, Lycosa, Oecobius navus

Dehydration Stress in the Wolf Spider Schizocosa ocreata (Araneae: Lycosidae): Tolerance, Resistance, and Coping Mechanisms

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University.

By: Samantha Kelly Herrmann, BA

Graduate Program in Evolution, Ecology, and Organismal Biology

The Ohio State University

2015

Dissertation Committee:

Dr. J. Andrew Roberts, Advisor

Dr. Richard Bradley

Dr. Roman Lanno

Samantha Kelly Herrmann

2015

Abstract

Dehydration stress is a potential challenge for any terrestrial organism that must seek out free water and limit the amount of water lost to the environment. Water is required for maintaining homeostasis and so dehydration can affect a number of functions in the body, including nutrient transport, structure and mobility, and thermoregulation.

Animals that experience dehydration can respond by tolerating it or by making behavioral and physiological adjustments to evade it or mitigate its effects. We examined the effects of dehydration stress on the brush-legged wolf spider, Schizocosa ocreata (Hentz 1844), by investigating three potential modes of responding to dehydration stress: 1) tolerance 2) behavioral adjustment and 3) stress hormone (octopamine) production.

We investigated dehydration tolerance by examining survivorship under varying humidity regimes, and measuring total body water content, critical water loss mass, and water loss rates. Using controlled humidity chambers, we specifically compared male and female S. ocreata, which we hypothesized to have different dehydration tolerances due to differences in morphology, metabolism, reproductive strategy, and life history traits.

Males and females survived significantly longer at higher relative humidity (>55% RH), but females survived significantly longer than males at each RH level. Females had significantly lower critical water loss mass and lower water loss rates than the males, while the males had higher body water content relative to dry mass. We concluded that

females have a greater dehydration tolerance than the males and would likely be more successful long term in dry environments.

Behavioral responses to dehydration stress can help the individual minimize the effects stress or locate depleted water resources. We investigated the behavioral responses to dehydration stress by examining three aspects of wolf spider behavior: exploratory behavior, response to perceived threat, and microhabitat selection. We used dehydration chambers to stress the spiders and compared their behavioral responses to those of low-stress spiders. While there was no difference between the groups in propensity to explore a novel environment, we found that dehydration-stressed spiders displayed reduced anti-predator behaviors and chose cooler microhabitats than the low- stress spiders. The results from this study provide an understanding of how spiders behaviorally mitigate stress and the tradeoffs they may make in responding to dehydration stress.

Finally, we studied the effects of dehydration stress on production of the stress hormone octopamine. In arthropods and other invertebrates, octopamine modulates many behaviors and mobilizes lipids to help the animal prepare for a “fight or flight” situation.

We used dehydration chambers with and without desiccant to compare dehydration-stress spiders to container-stress spiders, both of which we compared to low-stress, minimally handled control spiders. We hypothesized that wolf spiders demonstrate a graded hormonal response to stress. Both the dehydration-stress spiders and the container-stress spiders had elevated levels of octopamine compared to the low-stress spiders and there was no difference between the dehydration-stress spiders and container-stress spiders, iii

suggesting that octopamine production is an all-or-nothing response to stress. This study confirmed that the neurohormone octopamine is produced in response to stress in a wolf spider, and has provided information critical to understanding how behavioral responses to stress are likely moderated.

Altogether, these studies greatly expand our understanding of dehydration stress response, and stress in general, in an important behavioral model system. This work has significant implications for the design of a wide array of behavioral experiments from predator avoidance, to courtship and mate seeking in an environmentally variable and structurally complex deciduous forest habitat.

Dedication To my incredibly supportive and patient family.

Acknowledgments

I’d like to thank all of my undergraduate researchers, without whom I’d not have been successful on this project, including Bernard Paniccia, Matthew Harcha, Madison

Nashu, Jessica O’Hara, Christina Lehn, Abigail Fresch, and Christa Eyster. Additionally, the advice I received from my committee members Dr. Richard Bradley and Dr. Roman

Lanno was hugely valuable and I appreciate the time they were willing to spend with me.

I also would like to thank The Department of Evolution, Ecology, and Organismal

Biology, The Ohio State University at Newark, and the American Arachnological Society for support. Above all, I am immensely grateful for the support of both my advisor, Dr. J.

Andrew Roberts, and my labmate, Dr. Ryan Bell. Without their patience and encouragement this would not have been possible.

Vita

June 2004……………….St. Charles North High School

May 2008……………….B.A. Zoology and Environmental Sciences, Miami University

August 2008-Present……Graduate Teaching Assistant, Department of Evolution,

Ecology, and Organismal Biology, The Ohio State University

Autumn 2012 & 2013….Instructor of Record, Human Biology, Department of Center for

Life Science Education, The Ohio State University

2013…………………….Center for Life Science Education Graduate Teaching Award

Field of Study

Major Field: Evolution, Ecology and Organismal Biology

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Table of Contents

Abstract………………………………………………………………………..…………..ii

Dedication………..………………………………………..……………………..………..v

Acknowledgements ……….…………………………………………………….………..vi

Vita………………………………………………..………………….…………..……...vii

List of Tables……………………………………...……………………………….……..ix

List of Figures……………………………………..…………………………………..…..x

Chapter 1: Introduction to dehydration stress: what are the challenges and how do

animals respond?...... 1

Chapter 2: Dehydration Tolerance in the Wolf Spider Schizocosa ocreata: A comparison

of survivorship, body water content and critical water loss, and water loss rates

between sexes………………………………………………….………………..10

Chapter 3: Behavioral coping mechanisms to dehydration stress in the wolf spider

Schizocosa ocreata…………………………………………………….…………30

Chapter 4: Physiological responses to water loss in the wolf spider Schizocosa ocreata:

the role of octopamine in dehydration stress………………………...…………..48

References………………………………………………………….…………………….64

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List of Tables

Table 2.1: Post-hoc analysis of survivorship between males and females…………26

List of Figures

Figure 2.1: Mean survival time for males and females in each humidity regime…….....27

Figure 2.2: Survivorship in varying humidity regimes in (A) females and (B) males...... 28

Figure 2.3: Body water content in males and females…………………………….…..…29

Figure 3.1: Aerial view of the exploratory arena…………………………………...……44

Figure 3.2: Time spent in each zone of the exploratory arena…………………..…….…45

Figure 3.3: Freeze time after receiving a puff of air…………………………..…………46

Figure 3.4: Preference index of dehydration-stress and low-stress spiders………...…....47

Figure 4.1 Sample chromatogram of wolf spider hemolymph…………………………..62

Figure 4.2: Normalized octopamine levels among three groups of spiders………..……63

Chapter 1: An Introduction to Dehydration Stress: What Are The

Challenges and How Do Animals Respond?

Abstract

Throughout its lifetime, an animal may encounter a variety of environmental stressors, including thermal stress, dehydration stress, wind stress, etc. All of these have the potential to disrupt homeostasis, and so organisms are expected to respond to stressors behaviorally and physiologically to reduce or mitigate the effects of that stressor.

Dehydration stress receives little attention in the literature, yet water is essential to maintaining proper body functions in all animals. Animals can respond to dehydration stress a number of ways, such as migrating towards environments with more moisture to reducing their respiratory water loss via discontinuous gas exchange. In the following review, I discuss the concept of stress in biology and how animals can respond to stress. I then address dehydration stress and the potential problems it causes individuals. Finally, I cover the ways animals respond both behaviorally and physiologically to avoid or reduce the impact of dehydration.

The Concept of Stress in Biology

Stress has traditionally been broadly defined as the “non-specific response of the body to any demand” (Selye 1978). Unfortunately, the term has come to be used in a myriad of different ways, and might be used to describe the event causing a behavioral or physiological response in an organism, or it might describe the response itself (see

McEwan and Wingfield 2003). In the work presented here, I follow the McEwan and

Wingfield (2010) definition of “stress” as real or implied disruption of homeostasis, or the maintenance of physiological conditions within an optimal range within an organism where the “stressor” is the factor that causes the disruption and the response

(physiological or behavioral) is termed “coping” (Wingfield et al. 2011). There are numerous potential stressors that animals may face within their environment, including food availability, extreme weather conditions, predation, illness, etc. (Wingfield 2003)

These stressors may be short term or infrequent (acute), or these stressors can be long- term or frequently repeated (chronic), and animals respond to these stressors in different ways (Johnson et al. 1992, Romero et al. 2009, Adamo and Baker 2011).

Coping includes behavioral modifications or physiological adjustments in an attempt to return to homeostasis (Johnson et al. 1992). Animals can respond to stressors with three coping mechanisms: evading or reducing stress, resisting or tolerating stress, and/or utilizing recovery mechanisms to quickly return to homeostasis (Huey et al. 2002).

Evading stress, and to a lesser degree, stress reduction, involves behavioral modification(s). In fact, an animal’s first and most rapid response to a stressor is typically a behavioral adjustment (Huey et al. 2002). This can allow the animal to quickly evade

the stressor, such as moving away from an unfavorable environment for a more comfortable one. For example, digger wasps (Phylanthus psyche) face significant thermal challenges during the breeding season because males defend territories of bare ground which heat to temperatures upwards of 50C, but simple behavioral responses such as switching to cooler perches on plants throughout the day help them avoid detrimental effects of overheating (O’Neill and O’Neill 1988).

If escaping or avoiding the stressor is not possible, an animal may find a way to tolerate the stress, that is, they remain in the stressful situation but make adjustments to physiologically resist the stressor or respond through recovery mechanisms (Huey et al.

2002). Some animals can tolerate a certain amount of stress before perishing (reaching a critical limit). The moor frog (Rana arvalis) has a wide range throughout Europe and females rely on water to lay eggs. Unfortunately, many of these bodies of water have relatively low pH due to anthropogenic environmental acidification. Räsänen et al. (2003) compared the survival rate of embryos from acidified lakes to those from lakes with a more neutral pH after placing the embryos in water with either low or neutral pH.

Embryos from populations found in acidified lakes had higher survival rates in low pH water and therefore a greater tolerance for acid exposure than those from environments with lower levels of acidification (Räsänen et al. 2003).

In the final set of coping mechanisms collectively called stress recovery mechanisms, heat shock proteins have been particularly well studied and are probably the quintessential example (Reinhart et al. 2007, Lopez-Martinez et al. 2008, Boardman et al.

2013). Heat shock proteins act as molecular chaperones, so named because they are

induced in organisms in response to a variety of environmental stressors (including thermal stress, hence “heat shock”) to maintain proper protein folding and ensure those proteins continue functioning (Feder and Hoffman 1999). For example, increased temperature results in a corresponding increase in Hsp70 in the fish Trachinotus carolinus. (Cardoso et al. 2015). Dehydration stress also results in increased expression of heat shock proteins. Benoit et al. (2010) found increased expression of the heat shock protein Hsp70 in three species of mosquitos after dehydration. Because of the important role they play, recovery mechanisms such as heat shock proteins may be integral to tolerating a variety of environmental stressors (Feder and Hoffman 1999).

The Challenges of Dehydration Stress

In most habitats around the world, animals will experience variation in their local environment, be it seasonal or acute, because very few environments are unchanging

(Wingfield 2003). This variation includes temperature changes, food or nutrient availability, water availability, light duration, etc. Of these, temperature variation has received much recent attention, particularly due to global climate change concerns, yet, other environmental factors such as water availability are also expected to shift with the changing climate (see Chown et al. 2011). Water availability is particularly important to terrestrial animals who must seek out water sources without losing too much water to the environment in order to maintain a certain level of hydration. Water availability is generally assumed to be a widely important component of an animal’s environment and yet it receives relatively little attention in the literature, perhaps because of this assumed 3

importance (McCluney and Date 2008). This is unfortunate because a better understanding of how animals are impacted by changes in water availability provides a more well-rounded understanding of how animals are likely to respond to a changing environment.

In animals (vertebrates and invertebrates), water makes up approximately 70% of the total body mass (Hadley 1994, Schmidt-Nielsen 1997, Edney 2012), plays a variety of roles crucial in maintaining homeostasis, and is needed for structural support and growth as the majority of a cell is composed of water (Danks 2000). It is a universal solvent and transports nutrients, hormones, and wastes throughout the body (Alpert 2006). Water is also required for proper thermoregulation. Not only does its high specific heat capacity allow animals to maintain some stability in body temperature, the high latent heat of evaporation also allows for effective heat removal via evaporative cooling (Jéquier and

Constant 2010). It is no surprise that dehydration can be greatly detrimental on an animal, leading to changes in osmolality in body fluids (e.g., increased concentrations of Na, K, and Cl; Naidu and Hattingh 1986), reduced metabolism (Alpert 2006), and oxidative stress that can cause lipid and DNA damage, and subsequently death (França et al. 2007).

Because of water’s incredible importance for maintaining homeostasis, dehydration is an excellent example of a stressor impacting a wide range of taxa. There are a number of ways animals can respond to dehydration stress to either avoid it or mitigate its effects. In the remainder of this chapter, I will address the common behavioral and physiological mechanisms of evading or reducing water stress.

Behavioral Responses to Dehydration Stress

One of the more obvious forms of behavioral coping response to dehydration stress is microhabitat selection. The Virgin Islands dwarf gecko (Sphaerodactylus parthenopion) is a small lizard with high water loss rates, and to reduce water loss to the environment, chooses microhabitats with more moisture than those chosen by its larger counterparts (MacLean 1985). The urban wall spider, Oecobius navus, tends to build webs in more sheltered walls, where the relative humidity is higher (Voss et al. 2007).

Depending on how much moisture is available, the cane toad (Rhinella marinus) switches its preferred microhabitat from dense vegetation to hollows under trees and logs

(Seebacher and Alford 1999). In all of these situations, selecting microhabitats that either provide more available moisture or reduces water loss can help individuals avoid dehydration and thus evade the stress it can cause.

Of course, animals can change their habitat on a larger scale by migrating away from a stressful environment. There are many examples among the ungulates, because these large mammals are capable of traveling sometimes vast distances (Cain et al. 1999).

The desert mule deer, Odocoileus hemionus crooki, is typically a non-migratory desert ungulate; however those in Southern California migrate well out of their home range during the dry season to areas that have more available moisture (Rautenstrauch and

Krausman 1989). Similarly in Africa where many large herbivores migrate seasonally, rainfall or moisture content in food are at least partly responsible for the migration of many of these animals (Fryxell and Sinclair 1988). Birds are also capable of migrating large distances. The red-billed quelea (Quelea quelea) is an African migrant who follows

rain fronts in its migration (Dallimer and Jones 2002) and in doing so it avoids the challenges of drought.

Migration from a challenging environment is not always necessary. Animals can also adjust activity times to avoid water stress, largely by avoiding the dryer time of the day. In many areas, temperature decreases at night and relative humidity increases, both of which can help reduce water loss (Cain et al. 1999). Woodlice species (Isopoda) with relatively higher water loss rates due to evaporation are more active at night than those with lower water loss rates (Cloudsley-Thompson 1956). Leptodactylid frogs, such as

Eleutherodactylus coqui (Thomas), will increase time spent performing activities at night and adjusting their posture during dry periods, reducing water lost to the environment

(Pough et al. 1983). The salamander, Desmognathus fuscus, reduces water loss by being active at night but will become less active as the night goes on when moisture becomes limited (Keen 1984).

Physiological Responses to Dehydration Stress

If animals cannot evade or reduce the stressor behaviorally, physiological adjustments can help them tolerate the stressor. Perhaps the most common way animals can reduce the amount of water lost or the damage done by water loss is via control of solutes in the body; for example, excreting highly concentrated feces and urine (Hadley

1994, Cain et al. 1999). In addition to controlling solutes, very often, when animals are dehydrated their metabolism slows (Schmidt-Nielsen 1997, Chown et al. 2011), and this

lower metabolic rate can minimize dehydration by associated reductions in evaporative and respiratory water loss (Anderson and Prestwich 1982, Chown 2002).

Respiration can be a significant source of water loss in animals. Vertebrates may have a tradeoff between thermoregulation and water loss rates. Sweating and panting can be employed for evaporative cooling, yet these can also increase respiratory water loss

(Cain et al. 1999). Interestingly, some desert ungulates are capable of using facultative evaporative cooling, where sweating or cutaneous evaporation increases when the individual is hydrated and decreases when dehydrated, which allows the animal to avoid water loss (Schmidt-Nielsen 1997). Spiders and insects have spiracles, or respiratory openings on their cuticle, that allow for gas exchange (Hadley 1994, Foelix 2011). When spiracles are open, individuals experience greater evaporative water loss (Hadley 1994), but individuals can reduce the amount of water lost to the environment by limiting the amount of time they have their spiracles open (Davies and Edney 1952, Figueroa et al.

2010).

Regardless of other physiological coping mechanisms, both vertebrates and invertebrates produce stress hormones that mediate the individual’s response to stressors such that circulating or systemic hormone concentrations can be used as indicators of stress (Wingfield et al. 2011, Romero and Butler 2007). In vertebrates, corticosteroid hormones (e.g. cortisol and corticosterone) are typically the proxies of chronic stress because they prime the individual to cope with long term stress (Creel et al. 2009, Denver

2009, Tort and Teles 2011), such as by influencing behavior or increasing blood glucose

(Romero 2004). In cases of acute stress, norepinephrine and epinephrine modulate the

“fight or flight” response (Romero and Butler 2007), which allows the animal to quickly respond to an immediate threat by increasing heart rate and metabolism, enhancing olfaction, decreasing pain reception, etc. (Haller et al. 1998). Among invertebrates, octopamine is the analog of norephinephrine with similar functions, and is generally elevated in response to both acute and chronic stress (Orchard 1982, Adamo et al. 1995,

Roeder 1999, Adamo and Baker 2011). Other hormones, such as vasopressin and angiotensin, certainly play a role in maintaining proper body water balance in animals

(Takei 2015), yet few studies have examined stress hormones in the context of dehydration stress. Those few that have focus on cortisol levels in response to stress

(Parrott et al. 1996, Hamadeh et al. 2006), and the results from these studies are strangely conflicting. Dupoué et al. (2014) found that while there was no difference in baseline cortisol levels between dehydrated Children’s pythons (Antaresia childreni) and hydrated individuals, the dehydrated individuals responded to additional capture stress with elevated levels of cortisol compared to stressed hydrated individuals, indicating hydration plays a significant role in the stress response even if dehydration alone does not elicit a hormonal response. It is suggested that dehydration makes other stressful situations, such as the presence of a predator, more difficult to escape from and therefore more stressful than when well-hydrated (Dupoué et al. 2014). At this point, however, studies of the response of stress hormones to dehydration are greatly lacking, especially in terms of acute stress.

Conclusions and Scope of Study

Water is clearly an essential part of maintaining homeostasis for any animal, and terrestrial animals have an added challenge of finding water in their environment or going longer periods of time without it. In spite of this importance and the very real challenges of limited water access, dehydration stress remains relatively poorly studied. With the earth’s climate changing and the expected changes in both temperature and water distribution patterns, dehydration stress deserves attention now more than ever, as we can expect both plants and animals in various environments to experience this with increasing regularity. In the following three chapters, I will examine the dehydration tolerance and physiological and behavioral responses to dehydration stress in a wolf spider commonly found throughout North America, Schizocosa ocreata. This small spider is an ideal candidate for a dehydration stress study because it is subject to seasonal variation in water and periodic drought as well as varying microclimates (Cady 1983, Hanson and

Weltzen 2000), has a high surface area to volume ratio and therefore relatively higher water-loss rates (Hadley 1994), and requires a certain amount of hydration for its partially hydrostatic skeleton to function properly (Foelix 2011). Further, it is an increasingly widely used model system in behavioral ecology (Hebets 2011), yet one where little or nothing is known of its underlying physiology. This information will give insight not only into the biology and physiology of this species, but will give us a greater understanding of how invertebrates respond to dehydration stress in general.

Chapter 2: Dehydration Tolerance in the Wolf Spider Schizocosa ocreata: A comparison of survivorship, body water content and critical

water loss, and water loss rates between sexes.

Abstract

Terrestrial animals face challenges relative to maintaining water reserves which are essential to homeostasis. Wolf spiders (Araneae: Lycosidae), like other small-bodied animals, are especially susceptible to water loss due to a high surface area-to-volume ratio. Wolf spiders may experience dry microclimates as well as seasonal changes within their lifetime, and so dehydration is potentially a common and significant stressor.

Understanding the physiological limits of individuals can help to understand habitat partitioning and other behaviors as they may make choices based on water availability.

This study examines the dehydration tolerance of the brush-legged wolf spider

Schizocosa ocreata, common in the leaf litter of deciduous forests in Ohio, USA. We compared males and females because we hypothesized males and females to have different dehydration tolerances based on their morphology, physiology, reproductive strategy, and life history differences. We used humidity chambers to examine survivorship under varying humidity regimes and dehydration chambers to study water

loss rates, body water content, and critical water mass. Both males and females survived significantly longer under higher humidity regimes (>50% RH) compared to the lowest, even without access to food or water. Females had significantly greater survivorship under all humidity regimes than the males. Females also had lower body loss rates and lower critical mass, while the males had a greater percent body water content. Results indicate that females have greater desiccation tolerance as they are able to withstand a greater loss of their body water content and survive longer in the absence of drinking water. Although the differences in survival time and water loss rates between males and females is likely, at least in part, an effect of body size, this indicates that females of this species may be more successful than their male counterparts during periods of episodic drought, and are probably selected to survive later into the season while caring for offspring.

Introduction

Across all terrestrial organisms, water is an essential component for maintaining proper body function. It is required for ionic regulation in body fluids and tissues (Le

Rudulier et al. 1984), aids in gas exchange (Feder and Burggren 1985), and in some cases is required for structural support and motion (Foelix 2011). Because of these vital roles, access to water and maintenance of water balance (dehydration prevention) are potential challenges for any terrestrial animal. Small-bodied terrestrial invertebrates are particularly susceptible to issues of water balance and rapid dehydration due to their high

surface area to volume ratio (SA:V) (Hadley 1994). They can lose water to their environment via cuticular evaporative water loss (CEWL), respiratory water loss (RWL), and even excretion (Pulz 1984). Given the critical nature of maintaining water balance, it is unsurprising that physiological and behavioral adaptations that prevent water loss are well documented in arthropods. A thick waxy cuticle on many arthropods living in xeric conditions reduces the amount of water lost to the environment via evaporative water loss

(Hadley 1980, Punzo and Jellies 1983). Very small arthropods, such as mites and ticks, have an especially high SA:V and some have evolved the ability to absorb water vapor to compensate for this (Arlian and Wharton 1974, McMullen et al. 1976). Insects can reduce respiratory water loss through controlling the opening and closing of their spiracles

(Loveridge 1968). Many animals also show behavioral responses to reduce water lost to the environment. Both mesic and arid species of spiders choose microhabitats with more moisture or higher humidity (Humphreys 1975, Uetz 1979, Cady 1983). Feeding behavior may also reduce water loss. Mites tend to eat less if they are water stressed, which presumably will reduce any fecal water loss (Arlian 1977).

Despite a reasonably broad understanding of adaptations to minimize water loss, dehydration tolerance (the ability to withstand a non-lethal amount of water loss) remains poorly understood for many arthropods. This is unfortunate since physiological limits can provide crucial information about the biology and ecology of the organism and even help explain emergent behaviors (Lubin and Henschel 1990, Klok et al. 2004). Investigations into critical dehydration limits may help to understand habitat partitioning, food choice,

and responses to acute and chronic environmental disturbance, such as drought (Hadley

1994, DeVito et al. 2004, McCluney and Sabo 2009).

In terms of dehydration, spiders face the same challenges as any small invertebrate due to their large SA:V and therefore comparatively high CEWL. In addition to these challenges, spiders may be especially vulnerable to dehydration due to their hydraulically-mediated leg extension (Foelix 2011). Without sufficient hydration, spiders cannot fully extend their legs, and therefore would have trouble moving about to forage or escape predation (Anderson and Prestwich 1975). The goal of this study is to establish the dehydration limits of the wolf spider Schizocosa ocreata (Araneae: Lycosidae), and as a result better understand how these spiders will respond to dehydration stress both behaviorally and physiologically. Schizocosa ocreata is an ideal species for this study as they are common wolf spiders in deciduous leaf litter of central Ohio (Dondale and

Redner 1990), and individuals are easily maintained in a lab. They are cursorial spiders, potentially traveling large distances and experiencing various microclimates within their lifetime (Cady 1983, Walker et al. 1999, Samu et al. 2003, Roberts and Uetz unpublished). Additionally, deciduous forests in Ohio commonly experience random episodic and/or seasonal drought (Hanson and Weltzin 2000), meaning that these spiders are likely to experience acute or chronic dehydration at some point within their lifetime.

In this study we investigated dehydration tolerance in S. ocreata by examining three key aspects: survivorship under varying relative humidity regimes, water loss rates, and critical water loss, or the lethal amount of water loss. We hypothesized that due to the morphological and ecological differences between males and females, dehydration 13

tolerance would vary between the two sexes reflecting these differences. Males typically have less mass than females (Dondale and Redner 1990), higher metabolic rates (Kotiaho

1998), and different reproductive strategies than the females (Foelix 2011), and we predicted that males would have lower dehydration tolerance than the females due to their larger SA:V. We expected females to survive longer, have lower water loss rates, and greater critical water loss values.

Methods

Animal collection and care

We collected spiders as juveniles in spring and fall of 2011 at The Dawes

Arboretum in Newark, Ohio, USA (N 39.973863, W -82.40128). We returned all individuals to the lab where they were reared to adulthood in individual 500-ml round, plastic containers, each with a moistened peat moss substrate to provide constant access to moisture. We maintained the spiders on a 13 hour light: 11 hour dark cycle. All spiders were fed a mixed diet of three to four Drosophila spp. and/or pinhead crickets (Acheta domestica), as appropriate by individual size, twice a week. Adult spiders no older than four weeks past their final molt were randomly selected to participate in each of the trials.

All of these studies involved destructive sampling of spiders, so experimental methods were designed to minimize total sample sizes where possible.

Humidity Chambers

We used humidity chambers consisting of closed, 6-L clear-plastic containers to establish treatment groups for all experiments. Following the methods of Winston and

Bates (1960), we used DrieRite desiccant or saturated salt solutions (as appropriate) to create and maintain relatively stable humidity levels in each chamber, and we placed a platform within each chamber but above the chemical/solution in order to keep spiders away from treatment solutions. The treatments were as follows: 4% RH (DrieRite), 33%

RH (CaCl2), 55% RH (MgNO3), 75% RH (NaCl), and 90% RH (KNO3). We confirmed that we could establish appropriate humidity levels using these solutions approximately three weeks prior to starting experiments, then periodically checked the humidity levels during the experiment phase using a HOBO data logger (Onset® model # U-DT-2) with a

Temperature/RH Smart Sensor (Onset® model# S-THB-M00x). We used all of these chambers for the survivorship experiment, while we only used the 4% RH chambers for the critical water mass and water loss rate experiments.

Survivorship

In this survivorship study we randomly assigned 100 individuals spiders (50 adult males and 50 adult females) from the lab population to five different humidity treatments such that each treatment had 10 spiders of each sex. Prior to the experiment, we provided spiders with food and water ad libitum for three days. At the start of the experiment, we recorded the mass of each individual, placed them into separate vials with a mesh

covering to prevent escape, and then placed vials on the platform in a humidity chamber.

Once in the chamber, spiders received no food or water for the duration of the experiment. We checked the spiders daily for survival, and considered a spider “dead” if the legs were curled in toward the ventral surface and the individual was unresponsive to light movement of their vial and gentle prodding by a brush. Once we determined a spider to be dead, we removed it from the chamber, weighed it, and then placed it in a

50°C drying oven over night (approximately 12-h). The following day, we recorded a final dry mass for each spider.

Body water content, critical water mass, and water loss rates

In order to investigate body water content, critical water mass, and water loss rates in S. ocreata, we gave 50 spiders (25 adult males and 25 adult females, randomly selected from the lab population) food and water ad libitum for three days prior to the experiment. Before beginning the trials, we recorded the weight of each spider and placed it in a vial with a mesh covering to prevent escape. We placed each of these vials into one of three 4% RH chambers, and for the first 12-h, we recorded the mass of each spider every hour. After 12 hours, we left the spiders relatively undisturbed in the chambers until death. We checked the spiders daily, and when a spider had perished (using the same criteria as for the survivorship study), we removed it from the chamber and weighed it. As described previously, we then put the dead spider in a drying oven overnight and weighed them again the next day to get a dry mass.

We calculated the body water content (% body weight) by subtracting the dry mass from the mass at the start of the experiment and dividing that by the total mass. We calculated the critical water mass by subtracting the amount of water left in the body at death (dead mass-dry mass) from the total body water mass at the beginning of the trial.

To measure water loss rates, we determined the water mass for each spider by subtracting the dry mass from the mass taken each hour during the first 12 hours of the experiment. Using the exponential model described in Wharton (1985),

-kt mt=moe

We calculated the water loss rate by finding the slope of the regression line of the equation ln(mt/mo), where k is the percentage mass lost at time t, mt is the water mass at any time t and mo is the initial water mass of the animal. We used the initial mass, mass at death, and dry mass to examine the critical water mass for spiders. Using the dry mass, we calculated proportion of body mass that consists of water and the proportion of total body water that is lost at the time of death. We analyzed data using JMP version 11.0 and

Microsoft Office Excel 2007.

Results

Survivorship

Survival times for the spiders were not normally distributed according to a

Shapiro-Wilk goodness of fit test, before or after a log transformation, thus we used a

nonparametric analysis to compare survival times for each sex. The statistical significance level of all tests was α=0.05. There was differential survivorship under the varying humidity levels for both females (Kruskal Wallis: 2 =37.94, df=4, p <0.0001,

Figure 2.1a) and males (Kruskal Wallis: 2 =38.39, df=4, p <0.0001, Figure 2.1b). We used the Steel-Dwass method for post-hoc comparison of humidity levels on survivorship within each sex, and we used the Wilcoxan rank sum analysis to compare males and females in each treatment (Table 2.1). Females survived significantly longer than males across all treatments (Figure 2.2). Females had the greatest survival times in the 75% and

90% RH chambers with a mean survival time of 32.4±2.5 days and 26.7±3.5 days, respectively, and there was no significant difference in survival time between these treatments (Steel-Dwass: Z= -1.136, p =0.7874). There was also no difference among the females between 55% and 90% RH treatments (Steel-Dwass: Z=2.389, p=0.1182). The difference among the females between the 33% and 55% RH treatments was marginally significant (Steel-Dwass: Z=2.713, p=0.0520).

Males also had the highest average survival times in the 75% and 90% RH chambers, but with mean survival times of only 7±0.68 days and 8±1.1 days, respectively. As in the females, there was no significant difference between these two treatments for the males (Steel-Dwass: Z=0.5779, p=0.9783). Among the males, there was no difference in survival between the 33% RH and the 55% RH treatments (Steel-

Dwass: Z=1.4177, p=0.6162) or between the 4% and 33% RH treatment (Steel-Dwass:

2.714, p=0.0520). For both sexes, the lowest average survival time was in the 4% RH

chamber, with a mean survival time of 5.8±0.4 days for the females and 1.6±0.2 days for the males.

Body Water Content and Critical Percent Water Mass

During these experiments one male escaped early in the experiment, and there was one outlier in the female group, both of which were removed from the analysis for a final sample size of 24 males and 24 females. The statistical significance level for these tests was α=0.05. Females had a mean body water content of 70.8±0.004%, which was significantly lower than the mean body water content of males at 74±0.004% (t(46)=5.45, p<0.0001; Figure 2.3).

Data for the critical water mass were not normally distributed (Shapiro-Wilk test for goodness of fit), therefore we arcsine square root transformed the data for analysis.

The mean critical percent water mass for females was 37.1±0.86%, which is significantly higher than the mean critical percent water mass for males of 27.8±1.23% (t(40)=-6.23, p<0.001).

Water loss rates

In this analysis it was important that all individuals survive the full 12 hour period, unlike measurements of body water content and critical percent water mass. In addition to the two individuals previously excluded, two males died before the 12 hour period had ended and they were also removed from this analysis, resulting in a final sample size of 24 females and 22 males. The statistical significance level for this test was 19

α=0.05. The females had significantly lower water loss rates (0.64±0.03%/h) than the males (1.04±0.06%/h) (t(32)=5.9, p<0.0001).

Discussion

Females had significantly greater survivorship in all treatments than the males.

This was expected as the larger body size of the females means they have a lower surface area to volume ratio and subsequently lower cuticular water loss. As the humidity increases, evaporative water loss should decrease, thus slowing down the rate of dehydration and allowing longer survival times in higher humidity levels. Both males and females had higher survivorship in the higher humidity treatments (75% RH and 95%

RH). Interestingly, there was no significant difference in survivorship between the 75%

RH treatment and the 95% RH treatment, nor was there a significant difference between survivorship in the 4% RH, 33% RH, and 55% RH treatments for either sex. High humidity clearly provides a benefit to spiders by reducing water loss and thus allowing the spiders to survive for long periods of time without access to food or drinking water.

Males had significantly higher water content as a percent of body weight than females. With males having an average of 74% body water content and females having an average of 71% body water content, both sexes fall within the range typical for spiders, which is about 60-85% (Pulz 1987). The lower water content of females may be explained by egg production. Female wolf spiders develop eggs within their abdomens for several weeks before depositing the eggs into an egg sac (Foelix 2011). Eggs tend to

be lipid-rich, and this may add considerable mass and volume (abdomen size) at the expense of water content, resulting in females having less total water within their bodies than the males (Carrel 1990, Edney 2012).

This tradeoff between lipid content and water content raises an interesting implication of this study. Documenting hydration state and response to dehydration may influence how we measure and interpret the body condition of spiders. Body condition is typically defined as the energy reserves in an organism, is used as a proxy for an individual’s overall quality, and is measured as a body condition index (Jakob et al. 1996,

Moya-Laraño et al. 2008). In wolf spiders, body condition is correlated with size, egg sac production, courtship behaviors, and expression of secondary sexual characteristics

(Kotiaho et al. 1998, Persons et al. 2002, Uetz et al. 2002). Body condition indices are assumed to reflect nutritional history and current reserves of lipids and/or proteins, with hydration state of the measured individuals rarely considered (Rutledge and Uetz unpublished). While there is debate on the best measure of body condition (Jakob et al.

1996, Green 2001), in spiders it often includes some sort of measurement of mass and abdomen size (Moya-Larano et al. 2008, Rutledge and Uetz unpublished). Both of these measures (mass and abdomen size) can be readily affected by the hydration state of an individual, reflecting only recent access to water instead of providing information about historical or recent foraging success. Currently, there is no accepted, non-destructive technique that teases apart the effects of the animal’s hydration state from their overall body condition. Rutledge and Uetz (unpublished) compared starved and dehydrated spiders and found that standard body condition indices did not differ between the two 21

treatments for the first 10 days of the experiments, indicating that typical measurements may not be accurately measuring an animal’s energy reserves but rather may also be confounded by the animal’s hydration state. An understanding of typical hydration levels and dehydration tolerance in spiders may better help us make inferences from and interpret body condition indices in the future.

Females have a significantly lower critical water mass than males, or alternatively, significantly higher dehydration tolerance. While in females an average loss of 38.7% of their water mass resulted in death, males died after an average of only 27.8% of their body water was lost. Both of these values are high compared to other spiders, which range from about 18%-31% (Pulz 1987). Similarly, females had significantly lower water loss rates (.63 %/hour) than males (1.04 %/hour). Although the smaller SA:V may explain the higher survivorship and lower water loss rates of females, it doesn’t explain why they were able to withstand greater loss of water before perishing. The average water-loss rates of S. ocreata males and females are similar to that of other wolf spiders (Davies and Edney 1952, Aspey et al. 1972), although it is important to note that the methods in all of these studies vary. The wolf spiders in these studies are larger, but they are also all mesic species. Water-loss rates may be reflective of the general moisture availability in the animal’s habitat; more so than the body water content or critical water loss amount (Vollmer and MacMahon 1974, Mazer and Appel 2001). The data here supports this hypothesis as the water-loss rates for S. ocreata are quite high compared to desert arachnids (Hadley 1970), yet the body water content is similar (Pulz 1987).

That females have a higher dehydration tolerance than males is intriguing because males tend to travel greater distances than females and thus are more likely to encounter unfavorable environments (Cady 1983). Similar results were found by DeVito and

Formanowicz (2003), when they examined dehydration tolerance in the wolf spider,

Pirata sedentarius. Females of that species have higher survivorship than males when subjected to thermal and dehydration stress. One possible explanation is that male and female spiders have different metabolic rates. A higher metabolic rate leads to higher respiratory evaporative water loss (Anderson and Preswich 1982, Chown 2002); therefore a higher metabolic rate in males could explain the disproportionately lower dehydration tolerance. Although males travel farther (Cady 1983), it is possible that they encounter enough accessible water along the way that they are unlikely to reach their critical hydration limits, and therefore a higher metabolic rate would benefit males by enabling them to travel long distances. Currently, the available data for S. ocreata is not conclusive on which sex exhibits the higher metabolic rate, although in many of the species studied, such as Pardosa milvina, males have do have a higher metabolic rate than females, and this difference seems to be associated with activity (Kotiaho 1998,

Walker and Irwin 2006). Until now, however, most of the literature has focused on metabolic rate in females only for convenience because the relatively low growth and lower activity rate of adult females compared to juveniles or adult males has traditionally been considered to provide more consistency in experimental measures (Kotiaho 1998).

Additionally, the mating strategy of male S. ocreata is generally considered to be scramble competition (Norton and Uetz 2005). The first males to get to females don’t

need to be able to withstand dehydration stress once they’ve mated, so dehydration tolerance would not be under as strong of selection as it is in the females who need to build and carry egg sacs for extended periods after mating (Foelix 2011).

It is also possible that the female’s reproductive state impacts metabolic rate, and therefore may impact dehydration tolerance. Gravid females who have not yet produced an egg sac (the typical condition of females used in this study) may display higher dehydration tolerance due to a temporarily increased SA:V. Additionally, females may have decreased water content after releasing an egg sac as they must invest their own water reserves into that egg sac (Pulz 1987). Females with egg sacs typically have lower metabolic rates than those without egg sacs, and therefore likely have lower evaporative water loss (Canals et al. 2011). Females may have undergone selection towards a higher dehydration tolerance as they must invest their own reserves into producing eggs and carrying egg sacs. Egg sacs require both water and lipids, and females may have to withstand unfavorable conditions in order to maintain the egg sacs at appropriate temperatures (Carrel 1990, Kotiaho 1998). Therefore, the ability to withstand dehydration stress would be particularly beneficial for increased fecundity. Future studies that compare females in different reproductive states can further enhance our understanding of the determinants of dehydration tolerance.

Schizocosa ocreata is an increasingly common model system in behavioral ecology (Hebets 2011) and the data from this study provides critical information into the physiological tolerances of both sexes. Spiders are both predators and prey and so have incredible ecological significance in their habitats (Riechert 1974). Because much of their 24

habitat partitioning is dictated by temperature and water availability (Riechert and Bishop

1990), responses to dehydration will help understand how spiders are likely to respond to a changing environment. In order to further investigate the effects of water availability on various behaviors, from microhabitat selection to courtship and mating, baseline information on dehydration tolerance is necessary to understand the influence this environmental parameter has on spider behavior.

Treatment RH Z value P value

4% -3.834 0.0001*

33% -3.823 0.0001*

55% -3.77 0.0002*

75% -3.753 0.0002*

90% -3.154 0.0016*

Table 2.1: Wilcoxon rank summed test of survivorship to compare males and females at each RH level. Asterisk (*) indicates significant difference in survivorship between the sexes at a statistical significance level of α=0.05.

Figure 2.1: Survivorship over time in (a) female and (b) male spiders in different humidity chambers. Females had the highest survival times at 75% and 90% RH with no significant difference between each treatment (Steel-Dwass: Z=-1.136, p=0.7874) Or between the 55% and 90% RH treatment (Steel-Dwass: Z=2.389, p=0.1182. Males had the highest survival times at 75% and 90% RH with no difference between the two treatments (Steel-Dwass: Z=0.5779: p=0.9783.)

Figure 2.2: Mean (± SE) survival time for female and male spiders in five different humidity regimes. Shared letters above the bars indicates no significance by the Steel- Dwass method. See Table 2.1 for statistical comparison.

Figure 2.3: Body water content (as a proportion of body mass) for males and females. Males had significantly greater body water content of 74±0.004% than the females body water content of 70.8±0.004% (t(40) =-6.23, p<0.001).

Chapter 3: Behavioral coping mechanisms to dehydration stress in the

wolf spider Schizocosa ocreata

Abstract

Maintaining body water balance is essential to all animals as it is required for homeostasis, or keeping internal conditions within an optimal range. Animals that experience dehydration stress may respond, or cope, behaviorally in a way that limits further stress. The wolf spider Schizocosa ocreata is likely to face dehydration stress within its lifetime because it is found in deciduous forest habitats that may experience periods of drought. In this study, we examined the effects of dehydration stress on three aspects of wolf spider behavior: open field exploration, response to perceived threat, and microhabitat selection. Dehydration stress was induced by placing spiders in chambers with DrieRite for 12 hours, while low-stress spiders remained in their home containers with access to water. Dehydration-stressed spiders spent significantly less time performing an anti-predator behavior, and also preferred cooler microhabitats than low- stress individuals. Interestingly, there was no detectable difference in exploratory behavior or activity levels between dehydration-stress individuals and low-stress individuals. The results from this study indicate that spiders can behaviorally

mitigate dehydration stress but may have to make tradeoffs between foraging and anti- predator behaviors when resources, such as water, are low.

Introduction

Throughout its lifetime, an individual organism may face a variety of stressors.

These can be chronic, relatively long-term influences, such as seasonal temperature variation (e.g. cold temperatures in the winter) or consistent threat of predation, or these stressors can be acute, such as an extreme weather event or predator attack. Most animals have the capability to cope physiologically and/or behaviorally with these stressors, either through preemptive adjustments for predictable changes or by rapidly responding to an unexpected disturbance (Wingfield 2003). Behavioral responses allow the animal to quickly respond to that stressor, either by avoiding it or making adjustments to minimize the stress. For example, salinity stress reduces the predatory activity of the starfish

Asterias rubens, who feeds less often and on smaller prey when the salinity of the environment decreases (Agüera et al. 2015). Another example can be found in Dabbling ducks such as the green-winged teal (Anas carolinensis), gadwall (Anas strepera) and northern pintail (Anas acuta), all of which increase their foraging speed on cold days presumably as a way to maintain warmth, reduce the total foraging time, and avoid thermal stress from cold temperatures (Hepp 1985).

Maintaining relative homeostasis, or keeping internal conditions within an optimal range, is critical for all living things, and one significant component of homeostasis is maintenance of water balance (see Chapter 1). Holding body water within physiologically tolerable limits is essential to the proper function of many body structures and/or processes (Hadley 1994, Jequier and Constant 2010). Keeping within these limits is a potential challenge for any terrestrial animal as they must be able to find ingestible water sources, which vary with both environment and season, while limiting losses to the environment through excretion, respiration, and evaporation (Hadley 1994). Small- bodied invertebrates, such as wolf spiders (Araneae: Lycosidae), may be especially susceptible to water balance and dehydration stress. Their relatively high surface area to volume ratio (SA:V) makes them susceptible to high rates of cuticular evaporative water loss (CEWL) (Humphreys 1975, Foelix 2011). This is particularly challenging to spiders because they rely on hydrostatic pressure to extend their legs and are unable to move if they become overly dehydrated (Foelix 2011).

Schizocosa ocreata is a small wolf spider commonly found in deciduous forests of

North America (Dondale and Redner 1990). Similar to other Lycosids, S. ocreata are cursorial spiders and can travel large distances encountering various microhabitats while foraging or seeking mates (Cady 1983, Walker et al. 1999, Samu et al. 2003). Deciduous forests commonly experience random episodic and/or seasonal drought (Hanson and

Weltzin 2000), therefore these animals likely encounter dry environments within their lifetimes both spatially and temporally. A dry environment is likely to be stressful to S. ocreata because these spiders prefer microhabitats with more moisture (Cady 1983) and 32

they have significantly lower survival rates in dry environments than in humid environments with no access to food (Chapter 2). Schizocosa ocreata’s habitat preference for more moisture may help them avoid physiological consequences of dehydration stress, including reduced mobility, lower metabolism, or even death (Pulz 1987).

Spiders can behave in a way that helps them avoid stressful situations, such as avoiding particular microhabitats to limit thermal or dehydration stressors or staying immobile in response to predator cues to limit predation exposure (Lubin and Henschel

1990, Jones et al. 2011), and while avoiding stress is important, it is still critical to respond to stress experiences with mitigating behaviors that lessen the impact of the acute stress event. In this paper, we examine behavioral responses of S. ocreata to dehydration stress by exploring three specific aspects of S. ocreata behavior: open field exploration, response to perceived predator threat, and thermal microhabitat selection. These behaviors are easily quantifiable in a lab and they are representative of an array of natural behaviors that can be critical to survival (Carducci and Jakob 2000).

Methods

Behavioral trials took place in December 2014 and January 2015 using adult spiders that had been wild-caught as juveniles at The Dawes Arboretum in Newark, Ohio,

USA (N 39.973863, W -82.40128), in September 2014. We lab-raised spiders to adulthood in individual 500-ml, round, plastic containers. Each container had a substrate of moist coconut fiber to provide constant access to water. We fed spiders a mixed diet of 33

Drosophila melanogaster, D. hydei, and/or pinhead crickets (Gryllodes sigillatus), as available and appropriate by individual size, twice a week, and all spiders were maintained on a 13 hour light: 11 hour dark cycle. We only used adult female spiders (1 to 4 weeks post-adult molt) from the lab population, randomly selected for one of two treatment groups: dehydration-stress or low-stress (control). We then further randomized the subjects into one of three behavioral test groups (open field exploration, response to perceived threat, or microhabitat selection). Males were not used as they rarely survive the 12-h dehydration treatment (chapter 2).

Stress Treatments

We fed all subjects ad libitum for 3 days prior to initiating stress treatments, and at the end of the third day, removed all food from the individual containers and randomly divided females into the treatments. Low-stress females remained relatively undisturbed in their home containers with access to water but no food, while the other group

(dehydration-stress) was subjected to rapid dehydration, as well as some additional handling and restraint compared to the low-stress group, over a 12-h period. We handled dehydration-stress females repeatedly at the start of the stress period (potential handling stress), primarily while they were placed in small, mesh-topped plastic vials (114 ml) to allow airflow but prevent escape (potential restraint stress). All spiders in the lab population are handled periodically during feeding/watering/etc. over the course of their lives in captivity, so we acknowledge but largely discount the potential of additive effects

of handling and restraint on stress-treatment spiders. We induced the primary stressor

(dehydration) by placing restrained females into 6-L enclosed plastic containers where humidity was maintained between ~0-4% through the use of DrieRite©. We kept spiders in the dehydration chambers 4 or 5 vials to a chamber during the treatment period. Based on water loss rate calculations from Chapter 2, we expect that the 12-h treatment resulted in an average of 7.7% body water content loss.

Both dehydration- and low-stress spiders remained in their respective treatment containers overnight (12 h). In the morning, we returned dehydration-stress spiders to their home containers, giving them access to water for one hour which allowed spiders to rehydrate temporarily.

Open Field Exploration

Open field exploration has been a useful tool in measuring a variety of aspects of animal personality (Archer 1973, Walsh and Cummins 1976), and allows us to quantify wall-seeking behavior or timidness and general activity (Carducci and Jakob 2000). In order to examine exploratory behavior following stress treatment, we tested the hypothesis that stress affects the boldness and activity levels of spiders. We predicted that females that had undergone dehydration stress would have increased activity levels and be bolder or exhibit more exploratory behavior. To test this, we used open arenas with a pre-defined grid drawn in the base allowing us to measure the amount of time spiders spent exploring different areas of the arena and count line crossing as a proxy for

generalized activity level. Animals that spend more time along the wall can be considered more timid or less exploratory than those that spend more time exploring the center of the arena (Walsh and Cummins 1976). The exploratory arenas were polyvinyl chloride

(PVC) boxes measuring 21cm x 21cm x 10cm with a Plaster of Paris substrate. We used a graphite pencil to draw a grid of 36 quadrats on the substrate, each measuring 3.5cm x

3.5cm. This allowed us to establish three zones including an Inner Zone (I), a Middle

Zone (M), and an Outer Zone (O), similar to the methods outlined in Carducci and Jakob

(2000) (Figure 3.1). We gently deposited individual spiders in the center of the arena and allowed them to explore for 20 minutes. A JVC camcorder was mounted above the arena to record the spider’s movement for later analysis using JWatcher (Version 1.0). We recorded the amount of time spent in each zone of the arena and how many lines individuals crossed while moving about the arena to determine relative activity.

Perceived Threat Trials

Trichobothria are hair-like sensory structures concentrated on the legs of many spiders that allow them to detect changes in air current or airborne sound (Foelix 2011), and likely mediate response to attacks by aerial predators (Lohrey et al. 2009). Stress typically involves a tradeoff between foraging for resources and anti-predator behavior

(Huey et al. 2002). Because these spiders would be stressed from depleted resources

(water), we predicted that they would show reduced anti-predator behavior to compensate for increased foraging behavior. We mimicked a potential aerial predator by puffing air

on spiders using an empty 16-ml plastic squeeze bottle for a consistent source of air per puff. Schizocosa ocreata typically respond to the puff of air by remaining motionless for a period of time (based on personal observations). We recorded the spider’s latency to return to normal activity following a puff of air and used this measurement as a proxy for response to perceived threat.

We used round Pyrex® glass containers (15.24 cm diameter) placed under a video camera to conduct and record trials. We used an opaque barrier propped between the glass container and the observer so that spiders would not see any observer movement during approach to the arena. We placed an individual spider in the center of the glass container but kept it under a 118 ml round plastic container for 30 seconds in order to allow the spider to acclimate to the glass bowl. We removed the plastic container and allowed the spider another 60 seconds to explore the glass bowl. At that time we gave the spider a quick double puff of air from the plastic bottle, then left the spider undisturbed for 120 seconds, while recording the time it took for the spider to resume normal activity.

Microhabitat Selection

Stressed animals have been repeatedly demonstrated to alter their thermal preferences following a stress event (Humphreys 1978, Ladyman and Bradshaw 2003,

Cain et al. 1999). In this study we tested the hypothesis that dehydrated spiders shift their thermal preferences to a lower temperature, which would reduce water loss to the environment. We predicted that spiders that had undergone dehydration stress would

choose to spend more time in cooler zones than those that had not undergone dehydration stress. In order to explore the effects of stress on thermal preference, we used a thermoelectric choice chamber with an aluminum floor and PVC walls to enclose the table measuring 122 cm x 13 cm x 10 cm. We established a temperature gradient in the aluminum base using a Peltier device (thermoelectric cooler) with external voltage control to regulate maximum temperature. We were able to maintain a consistent gradient in the aluminum plate from 27C (thermal comfort) to 50C (thermal discomfort)

(Roberts and Mandelstein unpublished). We divided this gradient into 5 zones based on temperature: zone 1 ranged from 27-31C, zone 2 was 31-34C, zone 3 was 34-37C, zone 4 was 37-40C, and zone 5 was 40-50C. We marked the zones with strips of 3M

ScotchBlue™ painters tape to make analysis of the overhead video recordings easier. We checked the temperature of the zones using a Raytek Raynger ST infrared thermometer.

For a given trial, we placed an individual spider in the center of the heating table and then allowed 20 minutes to move about. We took instantaneous scan samples of the individual every 60 seconds, each time marking the position of the spider on the table. We then calculated a preference index as:

Where i is the zone number (1,…,5) and 1200 is the total time (seconds) of the trials, following the methods of Fischer et al. (2014). Therefore, a larger preference index indicates the spider spent more time towards the hot end of the table. All data were analyzed using Microsoft Excel and JMP Version 11. 38

Results

Exploratory Behavior

We used 25 spiders in the control group and 29 spiders in the stress treatment group, for a total of 54 spiders. We compared the amount of time individuals of each treatment spent in the inner, middle, and outer zones of the test plate. The distribution of the time spent in each zone was not normal, so we log transformed the data for analysis.

We used three separate tests to analyze differences in the time spent in the inner, middle, and outer zone between the two treatment groups with a statistical significance level of α=0.05. Because these tests are not independent of each other, we corrected the probability obtained using the Bonferroni sequential correction technique (Rice 1989).

There was no significant difference between stress treatments for the amount time spent in each of the three zones (Figure 3.2). In the outer zone, stressed spiders spent a mean time of 1154.97±5.3s and unstressed spiders spent a meant time of 1147±6.5s (t(48)=0.89, p=0.38). In the middle zone, stressed spiders spent a mean time of 31.0±4.6s and the unstressed spiders were there for a mean time of 30.5±4.1s (t(52)=-0.31, p=0.76). In the inner zone, stressed spiders spent a mean time of 16.65±2.77s and the unstressed spiders spent a mean time of 24.86±4.23s (t(52)=-1.66, p=0.10)

There was also no significant difference in the overall activity levels of the two groups. The dehydration-stress spiders crossed an average of 137.1±13.21 lines over the

20 minute period, while the low-stress spiders crossed an average of 166.9±17.57 (t(46)=-

1.36, p=0.18).

Perceived Threat Response

For the response to perceived threat trials, there was one outlier which we removed from the analysis for a final sample size of 26 dehydration-stress spiders and 26 for the low-stress treatment (52 total spiders). The freeze time of spiders after receiving a puff of air was not normally distributed so we log transformed the data for analysis. The mean freeze time of low-stress spiders was 16.4±2.8s, which was significantly longer than the freeze time of the dehydration-stressed spiders 10.7±2.6s (t(47)=-2.38, p=0.0212)

(Figure 3.3). The statistical significance level of this test was α=0.05.

Microhabitat Selection

The preference index values differed from normality according to a Shapiro

Wilkes goodness of fit test, so we used a Mann Whitney U test with a statistical significance level of α=0.05 to compare the mean indices for the treatment and control groups. There were two outliers in this study so they were removed from further analysis.

Therefore, we calculated the preference index of 29 low-stress females and 28 dehydration-stress females, for a total of 57 individuals. The low-stress individuals had a mean preference index of 0.068±0.002, which was significantly higher than the dehydration-stress individuals’ preference index of 0.054±0.004 (U=257, Z=2.37, p=0.0177) (Figure 3.4).

Discussion

Acute dehydration stress resulted in several detectable behavioral differences in S. ocreata in the hours following recovery, although not always in the manner hypothesized.

In fact, the only time dehydration-stress spiders behaved similarly to low-stress spiders was in the open field exploration trials. Dehydration stress exposure did not result in a detectable difference in the amount of time spiders spent in each zone compared to low- stress control animals. The majority of spiders, both stressed and control, spent most of their time towards the edge of the exploratory arena. This was not what we predicted because acutely-stressed animals tend to have higher activity levels presumably to quickly escape the stressful situation and/or seek out depleted resources (Katz et al. 1981,

Davenport and Evans 1984). This elevated activity level may be modulated by hormones because it does not seem to occur in chronically-stressed animals (Katz et al. 1981). It’s possible in this study that the dehydration-stress spiders did not have significantly different hormone levels from the low-stress group, or that the 12-h treatment was long enough to blur the somewhat ambiguous line between acute and chronic stress. In addition to this, the simple fact that the arena was a novel environment for all animals could have produced an acute stress response that overrode any treatment effects, though this seems unlikely. It’s more likely that the spiders were able to rehydrate enough during the brief recovery period that they did not display any discernible differences in the resource-seeking behavior we expected to see in this portion of the study.

In the perceived threat trials, all spiders remained immobile long enough to observe a “freeze” response to a puff of air. However, stressed spiders returned to normal activity much faster than spiders that were relatively unstressed, indicating that acute stress may reduce antipredator behavior in this species. This is in agreement with Jones et al. (2011) in which they found that the orb weaving spider Larinioides cornutus spent less time huddled in response to perceived threat when the individual was treated with the stress hormone octopamine. Stressed individuals may have to make tradeoffs between basic antipredator behaviors and seeking out depleted resources (Huey et al. 2002), such as water which was the source of the stress as in this study. All of the spiders in this study still displayed anti-predator behavior of “freezing” in response to a potential threat, yet the dehydration-stress spiders reduced the loss in potential foraging time by returning to being active more quickly than the low-stress spiders.

The dehydration-stress spiders had a higher preference for the cooler temperatures

(Figure 3.4) which was the predicted response. Lycosids that avoid hotter habitats have lower water loss rates because there is a direct relationship between temperature and water loss rates, and so a higher temperature is expected to speed up water loss rates

(Davies and Edney 1952, Humphreys 1975). Similar studies done on reptiles indicate dehydrated individuals prefer cooler temperatures because, as ectotherms, they can choose cooler temperatures to lower their body temperature and reduce CEWL (Crowley

1987, Ladyman and Bradshaw 2003). Additionally, metabolism increases with increasing temperature in spiders (Pulz 1987). Increased metabolism also increases water lost to the environment, mostly due to respiratory water loss (Anderson and Preswich 1982, Chown 42

2002). By choosing cooler temperatures, ectotherms like spiders can keep their metabolism lower and reduce respiratory loss.

The spiders in this study showed some clear behavioral responses to dehydration stress. All of these responses could help the individuals minimize further water loss and/or help in water gain. Typically, behavioral responses to stress are mediated by hormones (Johnson et al. 1992). At this point, it is unclear how hormones play a role in the behavioral responses to dehydration stress, seen in this study. Future studies addressing the role of hormones in modulating behavioral responses to stress will elucidate this relationship.

Figure 3.1: Aerial view of exploratory chambers for the open field exploration experiment. The white zone indicates the outer (O) region, the light grey zone indicates the middle (M) region, and the dark grey zone indicates the inner (I) region. Zone colors are for illustrative purposes only; the actual arena was uniform in color with a visible grid drawn on the surface. Quadrats are 3.5 x 3.5 cm.

Figure 3.2: Amount of time (s) spent in each zone of the arena. There was no significant difference between stressed and control spiders in the outer (t(48)=0.89, p=0.38), middle (t(52)=-0.31, p=0.76), or inner zone (t(52)=-1.66, p=0.10)

Figure 3.3: Freeze time (s) in female S. ocreata measured as the time remaining immobile after receiving a puff of air. Low-stress females spent significantly less time “freezing” (16.4±2.8s) than the dehydration-stressed spiders (10.7±2.6s) (t(47)=-2.38, p=0.0212) .

Figure 3.4: Preference index of low-stress and dehydration-stress spiders. A larger preference index indicates greater preference for warmer zones. Low-stress females had a significantly higher preference index than dehydration-stress females (Mann Whitney: U=257, Z=2.37, p=0.0177).

Chapter 4: Physiological responses to water loss in the wolf spider

Schizocosa ocreata: the role of octopamine in dehydration stress.

Abstract

The biogenic amine octopamine is implicated in mediating stress response in invertebrates, yet the influence of environmental stress on octopamine production remains unclear. In this study, we explored the effects of acute dehydration stress

(combining dehydration, restraint, and handling stress) on octopamine levels in the hemolymph of female wolf spiders, Schizocosa ocreata (Araneae; Lycosidae). We tested hemolymph from dehydration-stress individuals that were restrained in small vials and forcibly dehydrated (using chemical desiccant) and compared it to hemolymph drawn from container-stress (restraint and handling stress) females and low-stress (mild handling stress only) females. The levels of biogenic amines in the hemolymph were measured using high performance liquid chromatography with electrochemical detection

(HPLC-ECD). Both dehydration-stress and container-stress females had significantly elevated octopamine levels compared to the low-stress females, though the stress treatments were not different from each other. These results suggest that octopamine production in wolf spiders is a discrete, all-or-nothing response to stress. This study

demonstrates that stress alters circulating octopamine levels in spiders and provides a basis to further understand hormone influence on behavioral and physiological responses of stressed spiders.

Introduction

The term “stress” is commonly used in biology and yet it is rarely defined in the literature. At its most general definition, stress can be defined as any condition that disturbs homeostasis, or the maintenance of a range of optimal internal conditions, while the stressor is the event that leads to this perturbation (McEwan and Wingfield 2003, also see Chapter 1). Stress response can therefore be defined as the adjustments (physiological and behavioral) made in response to the perturbation from the homeostatic state

(McEwan and Wingfield 2003, McEwan and Wingfield 2010). Animals have to deal with a wide variety of potential stressors in their environment, which can include predation, fluctuating resource availability, local environmental disturbance, etc. (Johnson et al.

1992, Wingfield et al. 2011)

Most animals secrete specific hormones in response to chronic or acute stress that moderate the animal’s response to that stress (Wingfield et al. 2011, Romero and Butler

2007). In vertebrates, corticosteroid hormones (such as cortisol) are typically indicative of chronic stress (Creel et al. 2009, Denver 2009, Tort and Teles 2011). For example, the southern toad (Anaxyrus terrestris) had elevated corticosterone levels for several weeks after it was transplanted to a coal burning power plant site with high levels of heavy

metal toxins in the Southeastern United States (Hopkins et al. 1997). These corticosteroids elicit systemic responses that prepare the individual for long term stress

(Romero 2004). These hormonal coping mechanisms are a tradeoff, however, because corticosteroids also inhibit the immune system and reduce growth and reproduction

(Romero 2004, Martin 2009). On the other hand, the response to acute stress (the “fight or flight” response”) is modulated by norepinephrine and epinephrine. Specifically, elevated levels of norepinephrine and epinephrine enhance the senses, cause an increase in heart rate, etc. which prepares animals to rapidly respond to a threat (Haller et al. 1998,

Romero and Butler 2007).

One important mechanism of the invertebrate stress response is releasing the neurohormone octopamine into circulation. Octopamine is an analog of norepinephrine and is widely considered to be the invertebrate “fight or flight” hormone, released in response to a variety of stressors in invertebrates (Roeder 1999). Octopamine mobilizes lipid stores, providing an immediate source of energy for a rapid response to acute stress

(Orchard et al. 1981). House crickets (Acheta domesticus) that were forced to exercise for

5 minutes had double the levels of octopamine in their hemolymph compared to resting levels (Woodring et al. 1988). Davenport and Evans (1984) found that both locusts

(Schistocerca americana gregaria) and cockroaches (Periplaneta americana) respond to mechanical and thermal stress with elevated levels of octopamine. Chemical stress in the form of nonlethal insecticide application also results in elevated octopamine levels in locusts and red flour beetles (Davenport and Evans 1984, Hirashima et al. 1994).

Octopamine also seems to modulate a wide variety of behaviors in invertebrates.

Increased octopamine levels cause increased aggressive behavior in crickets (Gryllus bimaculatus, Stevenson et al. 2000). Interestingly it causes the opposite effect in the squat lobster Munida quadrispina, which displays a characteristic submissive posture once it has received an octopamine application (Livingstone et al. 1980, Antonsen and Paul

1997). Octopamine is known to affect many behaviors in honey bees, such as foraging, division of labor, and waggle dance intensity (Schulz et al. 2002, Barron et al. 2007). In the orb weaving spider (Larinioides cornutus), topical application of octopamine decreased the duration of the huddle response, an anti-predator behavior related to aggression (Pruitt et al. 2008, Jones et al. 2012). In addition to any physiological changes affected by octopamine, such as the mobilization of fat stores, these behaviors may allow the animal cope with an acutely stressful situation (see Chapter 1).

Although environmental stress is well studied, most of the attention is on thermal stress with relatively little focus on dehydration stress (see Chown et al. 2011).

Dehydration is a potential challenge for any terrestrial animal, and as water is essential to body structure and processes, individuals must maintain their body water within physiologically tolerable limits. Animals commonly lose water to the environment through three main routes: excretion, evaporative water loss across the skin or cuticle, and respiratory water loss (Schmidt-Nielsen 1997). If the animal does not effectively replace or reduce the amount of body water lost to the environment, it can disrupt the homeostatic state of the individual and it becomes stressed (Chapter 1). Small-bodied invertebrates may be particularly challenged by dehydration, in large part because they

have a high surface area to volume ratio (SA:V) which could result in relatively high rates of cuticular evaporative water loss (CEWL) (Hadley 1994). Spiders have an added challenge in that, unlike many arthropods, they have partially hydrostatic skeletons in addition to musculature and exoskeletal elements to maintain body posture and mediate movement (Foelix 2011). Essentially, they are unable to properly extend their legs, and therefore move, if they do not have enough water pressure in their bodies.

Studies on dehydration stress in spiders typically focus on xeric species as they must deal with the chronic daily threat of dehydration. Yet these species are likely well- adapted to a dry environment and may not experience acute stress the way a mesic individual will during a drought (McEwan and Wingfield 2003). Because the brush- legged wolf spider, Schizocosa ocreata, is typically found in mesic environments, it is an ideal subject for the effects of dehydration stress on behavior caused by spikes in biogenic amine levels. The species is common in deciduous forests of Ohio (Dondale and Redner 1990), which experience seasonal shifts in rainfall as well as episodic drought

(Hanson and Weltzen 2000). Schizocosa ocreata is similar to many small invertebrates in that it may encounter different microhabitats with varying microclimates within its lifetime (Cady 1983, Walker et al. 1999, Samu et al. 2003). Therefore, it is likely that individuals will unpredictably encounter a dry environment and become stressed. In the previous chapter, we determined dehydration stress impacts the behavior of S. ocreata.

Because octopamine is a stress hormone and affects invertebrate behavior, we hypothesize that octopamine is also responsible for the behavioral differences seen in dehydrated spider compared to their unstressed counterparts, therefore we predict that

octopamine levels will be highest in dehydration-stressed spiders and lowest in low-stress spiders.

Methods

Housing

We collected subadult S. ocreata in April 2015 from deciduous forest habitat in

Fresno, Ohio, USA (N 40.339033, W -81.757291). We housed the spiders individually in

500-ml plastic containers and raised to maturity in the lab. Each container had a moistened coconut fiber substrate to provide an ample source of water, and we fed spiders a mixed diet of size-appropriate crickets (Gryllodes sigillatus) and/or mixed species of fruit flies (Drosophila melanogaster and D. hydei) twice a week. We maintained the spiders on a 13 hour light: 11 hour dark cycle to simulate spring lighting conditions. We randomly selected 45 female spiders from the lab population for use in this experiment, and then arbitrarily divided them into three groups: dehydration stress, container stress, and low stress (control). We only used females due to the substantial amount of hemolymph (4µl) required from each test individual for the assays. Males typically have less mass than females (Dondale and Redner 1990) and we found it impossible to extract consistent volumes of hemolymph (Herrmann unpublished). We provided the spiders ad libitum access to food and water for three days prior to experimentation. Stress experiments were carried out in May 2015.

Experimental Treatment

We created dehydration stress treatment chambers by placing a 3-cm layer of

DrieRite® desiccant in the bottom of a 4-L, square plastic container with tight fitting lid.

Within an airtight chamber, DrieRite® maintains the relative humidity between 0-4%, as measured using a HOBO data logger (Onset® model # U-DT-2) with a Temperature/RH

Smart Sensor (Onset® model# S-THB-M00x). We used a platform placed over the desiccant layer to hold individual treatment vials. We placed individual spiders in the dehydration-stress treatment in small vials with a mesh top to allow for proper air flow between the vial and the chamber. We used this same design for the females in the container-stress treatment, except the containers had a layer of damp coconut fiber

(instead of DrieRite) on the bottom. Container-stress spiders were also allowed access to drinking water. The females in the low-stress group remained in their lab housing with adequate water but we removed any available food. We left the spiders in these conditions for 12 hours (overnight) prior to hemolymph extraction. We chose this time because over 12 hours, S. ocreata in the dry chamber lose approximately 7.7% of their body water content but there are typically no mortalities (calculated from data provided in Chapter 2).

After 12 hours, we returned the spiders from both dehydration- and container- stress treatments to their original housing with access to water for 1 hour. Membrane- bound transporters rapidly clear extracellular octopamine (Farooqui 2012), but the clearance rate of octopamine in spider hemolymph is not documented and sufficient hemolymph could not be extracted from test females without this brief rehydration

period. Preliminary data using the same techniques as described in this paper indicate discernible differences in hemolymph octopamine between a stressed and unstressed group. Therefore, this treatment allows time for rehydration, but not sufficient time to erase the effects of the stress treatment (Herrmann and Roberts unpublished), and likely provides a conservative measure of treatment effects.

Hemolymph Extraction

We placed spiders on a carbon dioxide diffuser plate to rapidly render them immobile and insensate. We then placed spiders onto a small Teflon sheet and cut a front or back leg at the femur with dissection scissors, allowing hemolymph to spill out onto the Teflon. After the hemolymph spilled out we collected 4 µL with a Fisherbrand

Finnpipette® II Adjustable-Volume Pipetters (0.5-10 µL). If insufficient hemolymph spilled out on its own upon removal of a leg, we applied gentle pressure on the abdomen to force out additional fluid. After that, we removed another leg, and if we still could not collect enough hemolymph that female was removed from the study. There was one female eliminated from this study due to insufficient bleeding. We added the hemolymph to chilled buffer (see below) in microcentrifuge tubes and centrifuged each tube. Samples were frozen and stored until analysis. Equipment was cleaned between each spider using

70% ethanol.

Chemical Analysis of Octopamine

The 4µl hemolymph samples from each spider were preserved in 100µl 0.2M perchloric acid buffer containing 1µg/ml synephrine, an internal standard for octopamine.

We thawed and vortexed each sample prior to analysis. Following vortexing, we filtered each sample using a 0.22µm cellulose acetate filter insert in a 2ml centrifuge tube (Costar

Spin-X, Corning). Each filter was wetted with 25µl 0.2M perchloric acid prior to introduction of the sample. We centrifuged each sample/filter tube for 6 minutes at

13,000 rpm, and the resulting filtrate was transferred to a 300ml HPLC vial. We analyzed

5µl of each sample by HPLC-ECD (high performance liquid chromatography with electrochemical detection) (Antec, Netherlands) at 10nA for 70 minutes, using an ALF-

115/C18 column and an ISAAC flow cell, for detection of biogenic amines. Mobile phase composition was as follows: 50 mM phosphoric acid, 50 mM citric acid, 0.1 mM EDTA,

10 % methanol, 500 mg/l OSA, 8 mM KCl, pH 3.25. We ran samples in alternating order, with each control sample followed by a sample from each of the experimental groups throughout the entire batch sequence. The biological sample batch was bracketed by a sequence of blanks and standards which were used to rule out contamination and to identify biogenic amines in samples, respectively. We identified individual peaks by comparison of retention times to known standards. Data from each run were collected and reported by Clarity (HPLC interface software).

Statistical Analysis

Data were analyzed with JMP version 11. Because we are interested in relative comparisons between groups, we used absolute peak areas to estimate the octopamine content from each sample, rather than the estimated concentrations. Octopamine peak areas were determined by dividing the absolute area under the curve by the absolute area under the curve of synephrine, an internal standard for octopamine (see figure 4.1 for sample chromatogram). Peak areas were normally distributed so we used a one-way

ANOVA to compare peak areas between the three groups of individuals, and a Tukey-

Kramer HSD comparison for post-hoc analysis. The level of statistical significance for this analysis was α=0.05.

Results

There were two individuals who were removed from the study due to an insufficient hemolymph sample and one outlier that was removed from the analysis. We therefore used a total of 42 individual females for this study.

Normalized octopamine levels varied significantly between the three treatment groups (ANOVA: F=5.398, df=2, p=0.0085) (Figure 4.2). The low-stress group had significantly lower normalized octopamine levels than either the container-stress group

(Tukey-Kramer HSD: p=0.017) or the dehydration-stress group (Tukey-Kramer HSD: p=0.0184). There was no difference between the dehydration-stress and the container- stress groups (Tukey-Kramer HSD: p=0.9964).

Discussion

In this study we demonstrated a clear, measurable hormonal response in wolf spiders under stress. Spiders from the control group remained in the containers they were raised in and had little handling prior to hemolymph extraction. Their hemolymph octopamine levels were significantly lower than those in either stress treatment.

Interestingly, there was no difference in the octopamine levels in either the dehydration- stress group or in the container-stress group. We had expected the octopamine levels of the dehydration stress group to be significantly elevated over the other treatments given the added stressor of significant dehydration, but found no such evidence. One possible explanation for this is that the stress response may be discrete not graded, i.e. stress either elicits a hormonal response or it doesn’t, with no cumulative effects depending on the severity of the stressor. Many of the studies thus far have tested octopamine levels based on stress and no-stress treatments and have not examined multiple levels of stress as we did here (for example, Adamo et al. 1995 and Hirashima et al. 2000), so there is little basis for comparison to groups with graded hormonal response.

Another possibility is that dehydration alone does not elicit the hormonal stress response, and the hormone levels we see are a response to the handling and container stress. Because of the constraints of this experimental design, we were not able to test for dehydration stress without some additional handling and container stress compared to controls. Dupoué et al. (2014) tested the effects of water deprivation on corticosterone levels in the Children’s python Antaresia childreni. While water deprivation alone did not result in elevated corticosterone levels, it did make the subjects more sensitive to

future stress, as the water deprived group had significantly higher cortisol levels than the control groups after further handling stress. While water depravation or dehydration alone may not be enough to elicit a hormonal stress response, a stressful situation such as being in the presence of a predator, may become more stressful if the individual’s capacity to escape that situation is limited.

Octopamine modulates both physiological and behavioral responses to stress, including lipid mobilization and anti-predatory behavior (Orchard et al. 1981, Jones et al.

2011). This provides insight into the behavioral responses to dehydration we described in

Chapter 3. Individuals that were dehydrated had reduced anti-predator response and preferred a cooler microhabitat. Spiders have sensory setae on their legs that allow them to detect changes in air current and thus detect potential aerial predators (Foelix 2011).

Octopamine affects trichobothria sensitivity and so therefore should make the spiders more alert to predator presence (Widmer et al. 2005). Regardless of this, my study is in agreement with Jones et al. (2011) that elevated octopamine in spiders, either topically- applied or experimentally elicited, results in reduced anti-predator behavior. They suggested that octopamine acts peripherally to make spiders more sensitive to predators and acts centrally to avoid the costs of anti-predator behavior, such as reduced foraging time. This makes sense because an individual that is stressed would benefit through enhanced awareness but also through the propensity to continue seeking resources that may reduce that stress, such as water, food, or even a more suitable microhabitat.

In Chapter 3, we found no difference in the activity levels or the timidity of high stress and low stress spiders. This is also in agreement with Jones et al. (2011) who found

octopamine did not increase the activity levels of L. cornutus. In insects, octopamine enhances excitability, just as norepinephrine does in vertebrates (Roeder 1999). It is fascinating that spiders have not shown a similar response, and suggests that octopamine acts differently in spiders and insects. The notion of differential effects of octopamine across taxa is not new, but is not well understood. In crustaceans, octopamine application results in a submissive response (Antonsen and Paul 1997) while in insects octopamine modulates aggressive behaviors (Stevenson et al. 2005). With this information in mind, it is important that researchers not assume that a) octopamine and norepinephrine act the same way in invertebrates and vertebrates, respectfully, and b) that octopamine affects all invertebrates in the same manner. While there has been an extensive amount of research on octopamine in insects (see Roeder 1999 for review) and can provide a useful comparison, future studies on hormone levels in a variety of invertebrate taxa are necessary to give a clearer understanding into how different species respond to stress.

This study demonstrates clear hormonal differences in response to stress in wolf spiders and the methods we have outlined here provide a useful protocol for examining circulating hormone levels in stressed spiders. Still, there is relatively little known about the actions of octopamine in spiders. It remains unclear as to what conditions specifically cause a hormonal response in spiders. This information is necessary for understanding how elevated levels of octopamine will affect spider physiology and behavior. Future studies examining a variety of conditions (such as temperature, predator presence, etc.) would greatly add to our understanding of how spiders respond to potential stressors.

Additionally, we are unaware of any study addressing the clearing time of octopamine in

spiders or even insects, information that is essential to our knowledge of how spiders are likely to respond to changes in hormones. Further studies into both the hormonal response to stress and the behavioral response to hormones are warranted to elucidate the stress response in spiders generally.

Figure 4.1: Sample chromatogram from a single individual from the container-stress group.

Figure 4.2: Octopamine levels of container-stress (CS), dehydration-stressed (DS) and low-stress (LS) spiders. Levels represent the mean normalized area under the chromatograph curve obtained for each individual. Any bars with the same letter above them indicate they are not statistically significant. The difference in octopamine levels between the three treatments was statistically significant (ANOVA: F=5.398, df=2, p=0.0085). The low-stress group had significantly lower normalized octopamine levels than either the container-stress group (Tukey-Kramer HSD: p=0.017) or the dehydration- stress group (Tukey-Kramer HSD: p=0.0184). There was no difference between the dehydration-stress and the container-stress groups (Tukey-Kramer HSD: p=0.9964).

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