And Rusty Crayfish (Faxonius Rusticus)

AN ABSTRACT OF THE THESIS OF

Adam Bowman for the degree of Master of Science in Biology presented on December 13, 2019

Title: Chemosensory behavior and temperature preferences of northern crayfish (Faxonius virilis) and rusty crayfish (Faxonius rusticus)

Abstract approved:

Kerry L. Yurewicz, Ph.D.

Invasive crayfish can alter aspects of the behavior of native crayfish with consequences that are yet to be fully understood. Crayfish respond to water temperature and chemosensory cues as they navigate, forage, find mates, and encounter predators. Given the disruptive effects invasive species can directly have on native species, it is important to also establish how cues from non-native species may affect the sensory landscape and alter behavior of native species even in the absence of physical contact. We collected a resident species in New Hampshire, the northern crayfish (Faxonius virilis), and quantified the effects of cues (alarm substances and physical presence) from the non-native rusty crayfish (Faxonius rusticus) on its activity. We also quantified the temperature preferences of both species and tested for any interactive effects of temperature and rusty crayfish cues on the behavior of northern crayfish. We found that these two species differed in their temperature preference, with northern crayfish preferring a warmer temperature than the rusty crayfish. The alarm cue from rusty crayfish altered the behavior of northern crayfish, inducing them to move more slowly. We also found that temperature affected how northern crayfish reacted to rusty crayfish cues in terms of movement distance. Our findings suggest that chemosensory cues from non-native crayfish may have an effect on resident species during an invasion process. Future investigation into population- and region- specific chemosensory-induced behavioral changes in native crayfish species is warranted.

Chemosensory behavior and temperature preferences of northern crayfish (Faxonius virilis) and rusty crayfish (Faxonius rusticus)

Adam Bowman B.S. Biological Sciences, Rowan University 2012

A THESIS

Submitted to

Plymouth State University

In partial fulfillment of the requirements for the degree of

Master of Science in Biology

Defended December 13, 2019 Degree Conferred January 2020

Master of Science thesis of Adam Bowman presented on December 13, 2019

APPROVED:

Kerry Yurewicz Ph.D., Thesis Advisor Associate Professor of Ecology, Plymouth State University

Christopher Chabot Ph.D. Professor of Biology, Plymouth State University

Jennifer Guarino M.S. Faculty, Community College of Vermont

I understand that my thesis will become part of the permanent collection of Plymouth State University, Lamson Library. My signature below authorizes release of my thesis to any reader upon request.

Adam Bowman, Author

Acknowledgements

I would like to first and foremost thank my advisor Dr. Kerry Yurewicz; without her

leadership my ability to contribute to Plymouth State University would not have been possible.

Many thanks to Jen Guarino and Dr. Chris Chabot for agreeing to serve on the thesis committee.

My gratitude to Plymouth State University’s Center for the Environment for funding this project.

I would also like to thank Tyler Thomas, Amber Lauchner and Dr. Chris Chabot for their

assistance with and use of their laboratory equipment, and for assistance with video analysis.

Thanks to Jen Guarino for helpful advice on where to collect rusty crayfish, and to NH Fish &

Game for permission to collect and hold crayfish for this study. Special thanks to Amy Ueland,

Dr. Heather Doherty, and Dr. Len Reitsma for their professional support. Thanks to Plymouth

State University, and all the professors and staff members who I have had the fortune of crossing

paths with during my time here.

Personal thanks to my father Robert Bowman, and my mother Christine Bowman for

their unconditional support during my time as a graduate student. Finally, I would like to thank a beautiful, smart, caring woman, who has supported me and kept me whole during troubled times,

Anna Bilz, who I love now and forever.

Contribution of Authors

Dr. Kerry Yurewicz assisted with experimental design, data analysis, and editing of all chapters.

iii

Table of Contents

Chapter 1: General Introduction...……..……………………………….………….……………..1

Crayfish biology………………………………………………….………………….2

Crayfish behavior………………………………………………….………………...4

Chemical sensing in crayfish…………………………………..…………………….4

Crayfish thermal biology…………………………………………..………………...6

Thesis objectives…………………………………………………..…………………7

Chapter 2: Thermal preferences of northern crayfish (Faxonius virilis) and rusty crayfish

(Faxonius rusticus) ……………………………………………………………………………....9

Introduction …………………………………………………………………………9

Methods …………………………………………………………………………....12

Results ……………………………………………………………………………..15

Discussion …………………………………………………………………………16

Chapter 3: Behavioral responses to cues from rusty crayfish (Faxonius rusticus) in a naïve population of northern crayfish (Faxonius virilis) …………...………..…...………………….23

Introduction ………………………………………………………………………23

Methods ……………………………………………………………………...... 26

Results …………………………………………………………………………....30

Discussion ………………………………………………………………………..31

Chapter 4: General Conclusion …………………………………….....……………………….42

Literature Cited………………………………………………………….……………………..45

List of Figures

Figure 2.1 Temperature preference experimental tank ……………….……………………20

Figure 2.2 Results of northern crayfish temperature preference trials …...... 21

Figure 2.3 Results of rusty crayfish temperature preference trials ……….……………...... 22

Figure 3.1 Cue and temperature experimental treatments……….……………………….…36

Figure 3.2 Time spent by northern crayfish in the zone of injection of liquid cue

10min post injection ………………………………………………………….…37

Figure 3.3 Time spent by northern crayfish in the zone of injection of liquid cue

60min post injection …………………………………………………………….38

Figure 3.4 Total distance moved by northern crayfish in treatment combinations of

cue and temperature …………………………...... 39

Figure 3.5 Average velocity of northern crayfish in treatment combinations of

cue and temperature …………………………………………………………….40

Figure 3.6 Times northern crayfish crossed in front of the middle tank zone in

treatment combinations of cue and temperature ………………...……………...41

Chapter 1

General Introduction

In aquatic systems species introductions are common, and some introduced species

become invasive and have detrimental consequences. For example, invasive macrophytes can

deplete aqueous minerals (Richardson & Wilgen 2004, Ehrenfeld 2010), reduce microbial

biodiversity and abundance in sediments (Bugiel et al. 2018, Stefanowicz et al. 2019), and affect

population sizes of benthic invertebrates (Diesburg et al. 2019). In upper trophic levels, invasive

fish species can reduce the abundance, growth, and diversity of native fishes (Britton 2018,

Costantini et al. 2018). Invasive species can have characteristics that increase their fitness over

native species, such as having greater resistance to environmental changes (Jaramillio et al.

2018), greater efficiency in acquiring food (Szela & Perry 2013), and faster growth rates (Britton

2018). Such characteristics can amplify the invasive organism’s disruption to a habitat with limited resources.

Within the benthic zone of freshwater habitats, invasive species can affect nutrient cycling and have widespread impacts on other trophic levels. For example, dreissenid mussels reduce nutrient concentrations, phytoplankton, and zooplankton via their filter-feeding activity

(Higgins & Vander Zanden 2010). Other bottom-dwelling invertebrates can act as a trophic link for nutrients sequestered in detritus and macrophytes by scavenging these resources while acting as food sources for predatory species. Invasive benthic invertebrates can alter these links by growing and taking up nutrients faster than native species, displacing native species, or evading predators more successfully (Zhang et al. 2019). The resulting alteration of nutrient flow can cause changes in ecosystem structure and function (Vanderploeg et al. 2002, 2015).

Crayfish are a highly diverse group of benthic invertebrates with important roles in freshwater systems. North America contains the majority of crayfish diversity with 382 of the 638

(60%) currently known species (Crandall & Buhay 2008), 16% of which are considered

threatened (IUCN 2019). Worldwide, 32% of crayfish are listed as globally threatened; this

percentage exceeds that of birds, mammals, and reptiles. Anthropogenic development, harvesting,

and invasive species rank in the top ten of threats to crayfish (IUCN 2019). Crayfish inhabit both lentic and lotic freshwater habitats and are considered keystone species in these food webs. They connect the benthic and pelagic zones by acting as prey for fish (Weber et al. 2010) while themselves feeding on fish and tadpole eggs (Morse et al. 2013, Ramamonjisoa et al. 2018), benthic macroinvertebrates, and aquatic plants (Lodge et al. 1994). Crayfish break down detritus, affect benthic invertebrate populations (Momot et al. 1978, Reynolds et al. 2013, Manenti et al.

2018), and alter the structure of benthic zone substrate (Albertson & Daniels 2016). Thus, the presence of crayfish affects many aspects of the overall food web in an aquatic ecosystem.

The motivation behind my thesis research was to explore the interactions between a local resident crayfish species (Faxonius virilis: northern crayfish) and a non-native congener

(Faxonius rusticus: rusty crayfish), as well as to gain a better understanding of their thermal biology. Understanding how native and non-native crayfish species interact and how they use

resources is important to understanding how crayfish assemblages, and thus freshwater

community structure and function, may change. In the next section, I review crayfish biology,

including aspects relevant to the spread of non-native species. I then review aspects of crayfish

chemosensory and thermal behavior that may influence interactions between species before summarizing my thesis research objectives.

Crayfish biology

Crayfish have one pair of large front-facing chelae (claws), four pairs of periopods

(walking legs), and a large muscular telson (tail) lined with five pairs of pleopods (smaller abdominal legs). Crayfish can also have intraspecific morphological differences due to sex or

stage. For example, male northern clearwater crayfish (Faxonius propinquus) have larger chelae than females (Berrill & Arsenault 1982, Garvey & Stein 1993), and reproductive (Form I) male rusty crayfish (Faxonius rusticus) have longer hooks on one pair of walking legs than their sexually inactive (Form II) counterparts (Gunderson 2008, Tierney et al. 2008). Crayfish have a hard exoskeleton and they increase in size by shedding this exoskeleton during molting periods.

Their two rows of gills are kept moist by retained water in the branchial chamber when the crayfish is not submerged; larger carapaces allow crayfish to be out of water longer before succumbing to desiccation (Claussen et al. 2000). Crayfish can thus survive and move on land for short periods of time (Grote 1981). When inhabiting small ponds or streams that may dry in summer months, this ability allows crayfish to survive and disperse to other water bodies.

When crayfish mate, males deposit sperm into females’ egg pores (Snedden 1990), and females can store this sperm until they extrude it with their eggs onto the telson. The resulting fertilized eggs are held on the telson, and the pleopods on the telson keep the eggs oxygenated until hatching by constantly rocking the egg pouch back and forth (Vogt 2002). A female crayfish that is carrying eggs is colloquially referred to as “in berry” because the egg pouch is shaped like a clump of raspberries. Clutch size can vary between species, as well as between egg-laying events within species. For example, a single female spiny-cheek crayfish can hold up to 300 eggs

(Kozak et al. 2006). In the crayfish Austropotamobius italicus, clutch size can increase or decrease depending on the size of the male crayfish involved in copulation (mean = 97 eggs/clutch) (Galeotti et al. 2006). The high fecundity and the mobility of females with fertilized eggs highlights the risk of introducing even a single female into a non-native waterway. Some crayfish species are also known to hybridize with each other (Capelli & Capelli 1980, Berrill

1985, Perry et al. 2002), although there is currently no published evidence that our study species of northern and rusty crayfish can produce viable offspring with each other.

Crayfish behavior

Crayfish can exhibit social behaviors that mediate access to food and shelter within

populations. Interactions establish dominant and subordinate individuals, with dominant crayfish

gaining more access to resources. Direct agonistic interactions as well as chemosensory cues can

affect this competitive behavior. For example, larger northern crayfish can occupy and defend

higher quality refuges, displacing smaller subordinates, when shelter is limited (Fero & Moore

2014). Narrow-clawed crayfish (Astacus leptodactylus) excrete signals in urine during

intraspecific fights; the winner of the fight is typically the crayfish that excretes the most urine

(Breithaupt & Eger 2002). The common yabby (Cherax destructor) retains visual memory of

previous agonistic opponents (Van der Velden et al. 2008), allowing these crayfish to recognize dominant conspecifics. Agonistic interactions begin with crayfish touching antennae, then progress to chela and body contact that can result in injury, or even death of the loser followed by cannibalism (Kouba et al. 2011). However, some agonistic interactions end as soon as one crayfish raises its chelae (Fort et al. 2019), showing that some crayfish use cues to avoid physical combat with dominant individuals.

Chemical sensing in crayfish

Chemical cues are ubiquitous in the dynamic environments of lakes (Hazlett 1999, 2000;

Pecor et al. 2010), and chemical cues from crayfish can be derived from multiple sources. An alarm cue originates from a crayfish when its carapace is breached and its internal fluids are released into the water (Chivers & Smith 1998, Ferrari et al. 2010). Alarm cues thus serve as a warning of a predatory threat (Mitchell & Hazlett 1996). There is evidence to suggest that alarm cues in crayfish originate from peptides in the crayfish hemolymph (Acquistapace et al. 2005,

Breithaupt et al. 2016). Disturbance cues originate from crayfish when they are stressed (i.e. sensing danger) or antagonistically interacting with other crayfish (Bergman & Moore 2005,

Bergman et al. 2005); these cues are thought to be primarily from excreted urine (Berry &

Breithaupt 2010). Crayfish can also use chemical cues to locate food and mates (Pecor & Hazlett

2008). To detect chemical cues, crayfish have chemosensory organs on antennules, oral

appendages, and on the tips of their periopods. Crayfish use these organs to detect not only

chemical cues in the water, but also depositions on substrate (Vogt 2002, Hallberg & Skog 2011,

Breithaupt & Thiel 2011, Breithaupt et al. 2016).

Crayfish change their behavior based on chemosensory information. Chemical cues from

northern crayfish (Faxonius virilis) have been shown to attract female northern crayfish to

shelters previously inhabited by females and inhibit males from occupying shelters previously

inhabited by males (Quinn & Graves 1998). Crayfish may avoid foraging when in the presence of

a damaged conspecific (Mitchell & Hazlett 1996) and prioritize shelter over food in the presence

of alarm odor (Jurcak & Moore 2014). Some species can retain memory of novel chemical cues

from potential predators (Hazlett 2003, Beattie & Moore 2018). Chemosensory cues play a role in

crayfish reproductive behavior as well. Females can secrete pheromones that attract male crayfish

(Stebbing et al. 2003, Berry & Breithaupt 2010, Breithaupt & Thiel 2011), and some newly hatched crayfish are attracted to their maternal parent via chemical signaling (Little 1975). Newly hatched crayfish can be preyed upon by fish, but also by non-related conspecific adults (Aquiloni

& Gherardi 2008b, Mathews 2011, Tricarico 2015). Thus, the ability of the offspring to recognize and gather near their maternal parent is crucial for their early survival (Mathews 2011). The introduction of novel cues into a freshwater habitat has the potential to change the behavior of native crayfish.

Some native crayfish change their behavior in response to chemical cues from non-native crayfish (Hazlett 2000). Therefore, the mere presence of a non-native species could alter the

chemosensory landscape early in the establishment of an invasive population. This in turn could

affect many aspects of resident crayfishes’ interactions within the ecosystem and their fitness

(Kubec et al. 2019). Invasive crayfish can sometimes exhibit a greater capacity to respond to chemical cues than native species. In one set of experiments, both the rusty crayfish (F. rusticus) and the red swamp crayfish (Procambarus clarkii) were able to learn to associate a novel chemical cue with predation risk and retain memory of this association longer than native species

(Hazlett et al. 2002). Some invasive crayfish have also been shown to respond more quickly to alarm cues than native species (Gherardi et al. 2002). These factors may provide invasive species with advantages over native species. The study of chemosensory responses by crayfish is a crucial component in understanding the broader impact that invasive crayfish may have on ecosystem function and structure.

Crayfish thermal biology

In addition to sensing and reacting to chemical cues, the ability of crayfish to respond to changing water temperatures is a key aspect of their behavior that relates to fitness. Because crayfish are ectothermic, the temperature of their environment determines their internal temperature. In lakes, crayfish can alter their internal temperature behaviorally by moving to areas with different thermal characteristics. In temperate lakes, thermal stratification in the summer months results in warmer areas closer to the shoreline and cooler areas in deeper benthic habitat. This water temperature gradient is important to key life history events. For example, female northern crayfish move to deeper water during the summer months; this movement is correlated with the maturation of males’ gonads, suggesting that water temperature plays a role in the crayfish reproductive cycle (Momot & Gowing 1972). Because of the importance of water temperature to their physiology, many species gravitate to specific ranges of temperatures within an environment (Westhoff & Rosenberger 2016). These temperatures do not always correlate with the water temperature where some of their metabolic processes (and growth) are optimal

(Westhoff & Rosenberger 2016). For example, rusty crayfish have a preferred temperature of

22○C, but show maximum growth rates from 26○-28○C (Mundahl & Benton 1990). The preferred temperature of some crayfish could represent a tradeoff between optimal metabolic rates and access to shelter and resources.

Water temperature can affect the way crayfish interact, grow, and forage. Northern crayfish individuals segregate along a temperature gradient based on social hierarchy, with larger crayfish taking residence in preferred temperatures and displacing subordinate individuals (Peck

1985). Red swamp crayfish (Procambarus clarkii) will also fight over thermal space, with dominant crayfish gaining more access to the preferred zone (Tattersall et al. 2012). Water temperatures can also affect the growth rates of crayfish feeding on different diets. Red swamp crayfish show reduced growth when feeding on animal protein at high temperatures and reduced growth when feeding on an herbaceous diet at low temperatures (Carreira et al. 2017). If invasive crayfish displace native species from their preferred temperature ranges, this could affect native crayfish foraging efficiency or diet. Given the omnivory of crayfish, this could have consequences to many trophic levels, affecting not only the fitness of native crayfish but the broader assemblage of the lake.

Thesis objectives

There are many subtleties in the chemical and temperature mosaic within aquatic habitats that can affect species interactions and fitness. Understanding this variation could provide insight into mechanisms that enable invasive species to displace native species and disrupt aquatic ecosystems. Data from these types of investigations can aid in understanding how invasive crayfish impact the environment and can help us understand how ecosystems may be affected by crayfish introductions. Crayfish are recognized as model organisms for the ethological study of freshwater invertebrates (Kubec et al. 2019), and they offer exceptional opportunities to

8 investigate chemosensory interactions and impacts of invasive species when introduced into novel benthic environments.

My thesis project focused on two crayfish species: one a long-term resident species in

New Hampshire (northern crayfish), and one a potential invader (rusty crayfish). From their native range in the Ohio River Basin, rusty crayfish have spread as far west as Oregon and

Nevada, and as far east as Vermont and New York (USGS 2019). The dispersal of rusty crayfish into non-native lakes has had serious impacts on local biodiversity. In Trout Lake (Wisconsin), introduced rusty crayfish have reduced native snail populations, macrophyte density, and native crayfish populations significantly. The fishes that share prey with rusty crayfish also declined in numbers (Wilson et al. 2004). Similarly, Lodge et al. (1994) found significant reductions in snail and macrophyte densities in controlled enclosure experiments with rusty crayfish in Plum Lake

(Wisconsin). The range expansion of rusty crayfish also includes the White and Connecticut

Rivers in Vermont (USGS 2019), and the proximity of these locations to the waterways of New

Hampshire makes the rusty crayfish a special concern for possible introduction.

The goal of this thesis project was to explore behavioral changes in northern crayfish when faced with chemosensory input from rusty crayfish, and to find the temperature preferences of both these species. In Chapter 2, I describe an experiment designed to determine the temperature preferences of northern crayfish and rusty crayfish from populations collected in

New Hampshire and Vermont, respectively. In Chapter 3, I highlight an experiment done to determine the behavioral responses of northern crayfish to cues from rusty crayfish, before closing with general conclusions in Chapter 4.

Chapter 2

Thermal preferences of Northern Crayfish (Faxonius virilis)

and Rusty Crayfish (Faxonius rusticus)

Introduction

Temperature selection is crucial to fitness for aquatic ectotherms. In freshwater food webs, organisms can optimize growth and reproduction by avoiding both high temperatures with lower oxygen levels and low temperatures that may slow metabolic rates (Beitinger & Fitzpatrick

1979, Garcia-Guerrero et al. 2013). Temperatures above or below critical thermal limits cause mortality, and these thermal limits are species-specific within aquatic ectotherms (Westhoff &

Rosenberger 2016). For example, the rusty crayfish (Faxonius rusticus) can tolerate temperatures from 0.9○C to 41.5○C (Claussen 1980, Layne et al. 1985), while the blue spring crayfish

(Procambarus horsti) can tolerate temperatures only up to 28.6○C (Caine 1978).

Between their thermal limits, oxygen consumption, nutrient metabolism, and enzymatic activity are most efficient within distinct temperature ranges for ectothermic species (Garcia-

Guerrero et al. 2003, Spanopoulos et al. 2005, Wang et al. 2006). Water temperature also has a direct effect on growth and survival. In the juvenile redclaw crayfish (Cherax quadricarinatus), the highest growth rate and survival were found between 25-28○C when this species was exposed to temperatures spanning a 20-31○C range (Garcia-Guerrero et al. 2013). The preferred temperature of an organism is what it will select when given a choice of varying temperatures.

For some species, these preferred temperatures have been shown to be correlated with the

temperatures at which their metabolic rate and oxygen exchange are optimized (Beitinger &

Fitzpatrick 1979, Gonzalez et al. 2010). For other species (e.g. in the genus Faxonius) this does

not always appear to be the case (Mundahl & Benton 1990, Hellman 1992, Wetzel & Brown

1993, Whitledge & Rabeni 2002).

The mechanisms by which animals sense and move to their preferred temperature can be

complex and varied. Some ectotherms can detect changes in water characteristics such as

dissolved oxygen or salinity in differing temperatures and move based on changes in these

gradients (Craig & Crowder 2005, Kobak et al. 2017). Ectothermic species have also been shown

to exhibit changes in internal cues that could prompt behavioral changes when regulating body

temperature. For example, evidence suggests that neural calcitonin receptors control active body

temperature regulation in flies (Goda et al. 2018). In the crayfish Cherax destructor, higher water

temperatures (increasing from 20○C to 30○C) were found to significantly increase heart rate. This

acute change in heart rate occurred well before any changes in internal temperature of the

crayfish due to the surrounding water (Goudkamp et al. 2004).

In addition to physiological changes, interactions between crayfish can also prompt

movement within temperature gradients. In some cases, the pattern of movement to preferred temperatures may differ between males and females during and after the mating season (Momot

& Gowing 1972). For example, in some northern crayfish (Faxonius virilis) populations the females migrate first to deeper waters during cooler fall months (Momot 1967), presumably in response to maturation of male gonads and corresponding aggressive male behavior (Fast &

Momot 1973). Social hierarchies also affect the selected temperatures of these crayfish, with dominant crayfish able to cause subordinate crayfish to move to a non-optimal temperature zone when crayfish are paired (Peck 1985). Within a population of conspecifics, space within the preferred temperature zone can be considered an important, contested resource.

The preferred temperatures of crayfish can significantly vary between species as well as within species. For example, Astacus astacus crayfish show preferences ranging from 11.9 -18°C, while Orconectes causeyi crayfish have preferences from 14 - 29°C (Loring & Hill 1976). For some species, the water temperature to which they are acclimated can affect their preferred temperature. In Astacus astacus, the preferred temperature is correlated with the temperature to

which the crayfish is acclimated (Kivivuori 1994). This phenomenon could explain some of the

variation between populations of species that occupy warm versus cold geographic locations.

However, some species (e.g. Procambarus clarkii) exhibit preferred temperatures that are

independent of acclimation temperatures (Espina et al. 1993, Buckle et al. 1996). Overall, the

diversity and complex patterns emerging from the study of temperature preferences in crayfish

highlight the need for further elucidation. In a recent review of crayfish thermal ecology,

Westhoff and Rosenberger (2016) were able to locate studies on temperature preferences of just

2.2% of living crayfish species.

The focus of this study was to compare the temperature preferences for New England

populations of two species: the northern crayfish (Faxonius virilis) and the rusty crayfish

(Faxonius rusticus). Northern crayfish were selected for this study because they can be abundant

in lakes, can achieve high biomass in their ecosystems (Momot et al. 1978), and influence other

components of their food webs [e.g. macrophyte density (Roessink et al. 2017), fish egg density

(Dorn & Mittelbach 2004), and benthic invertebrates (Momot et al. 1978)]. A threat to northern

crayfish populations in some regions is the invasive rusty crayfish (USGS 2019). Rusty crayfish

can be detrimental to native species through a variety of mechanisms: they show accelerated

foraging and growth rates (Glon et al. 2017), greater success in avoiding predation (Hill & Lodge

1999), and dominance in antagonistic interactions with other crayfish (Szela & Perry 2013). As rusty crayfish alter the abundance or even extirpate resident species, they cause shifts in the food web (Lodge et al. 1994, Olden et al. 2011), and these impacts can culminate in an overall reduction in biodiversity (Bobeldyk & Lamberti 2010). Understanding both of these species’ preferred temperatures could highlight any overlap in their optimal habitat. This information could help us to better understand the behavior and interactions of these species within lakes where they co-occur.

A few studies have tested aspects of the thermal ecology of northern and rusty crayfish from different habitats. Northern crayfish have been shown to prefer temperatures ranging from

21o-24oC (Peck 1985, Keller & Hazlett 2010), while optimal growth for this species appears to

occur at 25-26○C (Wetzel & Brown 1993, Whitledge & Rabeni 2002). Meanwhile, current

studies show rusty crayfish to prefer 22○C, to be tolerant of temperatures up to 39.5○C, and to

have optimal growth at 26-28○C (Mundahl & Benton 1990, Keller & Hazlett 2010, Westhoff &

Rosenberger 2016). Most studies on the temperature preferences of northern and rusty crayfish have been done on animals collected in the Ohio River basin (Peck 1985, Mundahl & Benton

1990, Keller & Hazlett 2010), and only one study found has directly compared the two species

(Keller & Hazlett 2010). We did not locate any temperature preference assays done on rusty or northern crayfish in New England.

We examined the temperature preferences for northern crayfish collected in New

Hampshire and invasive rusty crayfish currently established in rivers bordering New Hampshire.

Documenting the temperature preferences of local populations of northern and rusty crayfish

helps generate a more complete picture of the preference mosaic of both species. Given how few

studies have been conducted on the thermal ecology of these species, there is still much to learn

about potential genetic differences and local adaptations in different populations across their

geographic range. Adding regionally-specific data on temperature preferences and thermal

physiology may improve the predictive capability of mathematical models of future crayfish

distributional changes (Krause et al. 2019).

Methods

Crayfish collection and handling

Northern and rusty crayfish were collected using Gee® minnow traps baited with raw beef liver. Northern crayfish were collected from Tewksbury Pond, NH (GPS 43.608803, -71.970279)

a 19 Ha mesotrophic lake with a mean depth of 7.62 m (NH Fish and Game). Traps were placed

in areas of low human traffic near woody and vegetative cover. Rusty crayfish were collected

from the White River, a tributary from the main stem of the Connecticut River in Vermont (GPS

43.745699, -72.4194). Traps were set along the riverbank in areas with vegetation and woody

cover.

All traps were set out for 48 hrs and any animals collected were transported to the lab at

Plymouth State University in 22L buckets with polymer rope for the crayfish to crawl on. This

structure was provided to reduce fighting in the buckets during transport. Transport time was approximately 2 hours from collection site to the lab. Crayfish were placed into 40L polypropylene holding bins with approx. 20-30 crayfish per bin immediately upon arrival to the lab. Each bin was filled to a depth of approximately 40cm with tap water. Bins were aerated with aquarium stones and placed on a 12L:12D light cycle. Clay pots and polymer rope were added to the bins for structure and shelter. All crayfish species were fed ad libitum with shrimp pellets

(Wardley©, Secaucus NJ) and algae wafers (PlecoWafers©, Blacksburg VA) while in these

holding bins. The water was fully changed every 7 days and maintained at 21°C.

Temperature preference assays

A vertical temperature gradient was established in a 20L aquarium that contained a

plexiglass ramp with a 13° slope (Figure 2.1). The aquarium was placed inside of a large

styrofoam container to hold ice around the outside perimeter of the tank. Ice was filled to half the height of the aquarium. This was the amount of ice needed to adequately maintain a vertical 18○-

22°C temperature gradient. Two 50-watt Aqueon® submersible heaters were placed at the end of the tank with the top of the ramp. Plexiglass fins (65mm by 80mm) were secured at equal distances bilaterally on the ramp to create uniform structure throughout the tank, and to segment the tank into five temperature zones with 18°C on the lower end leading up to 22°C on the raised end. Using thermal probes connected to an electronic logger (4 channel Gain Express®),

temperatures were continuously monitored in each zone during each 60min assay to verify that

there was a stable temperature gradient. All outside surfaces of the tank were painted flat white

with Rust-Oleum® enamel spray-paint. Inside the tank, the ramp and fins were painted with white

Flexseal® rubber infused waterproof spray-paint to provide a non-slip walking surface for the crayfish. The tank was filled to completely submerge the ramp with tap water.

Fifteen male northern crayfish (range: 26-37mm in carapace length, mean: 32.6 mm), and fifteen male rusty crayfish (range: 23-43mm in carapace length, mean: 32.4 mm) were used in this experiment. Each individual crayfish was tested under each of two treatments. For the temperature gradient treatment the tank was as described above, while in the control treatment the tank lacked ice or heating so that a uniform 21°C temperature was kept throughout the tank. The latter treatment made it possible to correct for any vertical position bias the crayfish might have had in the absence of a temperature gradient. The treatment (control vs. temperature gradient) and species (northern vs. rusty) were randomized for testing order. The Plexiglass ramp system, aquarium, and water were re-set between trials, and all equipment was washed with a mild soap before reuse. Crayfish were placed in individual plastic bins for 24h prior to testing to acclimate the crayfish to a non-communal tank. To standardize hunger levels, crayfish were not fed 24 hours prior to testing. All filming was done in a darkened lab between the hours of 0700 to 1900 from 15 June - 15 August 2018. The crayfish were collected on June 1 2018 and acclimated in the communal tanks for a minimum of 14 days prior to testing. Because only up to six trials were conducted each day, some crayfish were held in the communal tanks for longer periods of time before being used in the experiment. A small amount of ambient light was introduced via a white

60 watt incandescent bulb that was needed to adequately film the experimental trials.

For each trial, a single crayfish was placed in a randomly chosen zone and allowed to acclimate for 15 min. The crayfish was then recorded using a wide-angle camera for 60min. All

footage (30fps, 720p) was transferred to Ethovision© tracking software to quantify the time spent in each zone for the 60m duration by tracking the x,y coordinates of the crayfish in the tank.

Statistical analysis

For each crayfish, its behavior in the control trial was used as its baseline distribution of

time spent per zone. Delta values were then obtained by taking the time spent in the gradient tank

and subtracting the time spent in the control tank for each of the five zones. A positive delta value

in a zone indicates a crayfish spent more time in that zone during the gradient trial compared to

the control trial, i.e. it exhibited a preference for that zone. A negative delta value indicates that a

crayfish avoided that zone in the gradient trial compared to the control trial. For each species,

delta values were analyzed to test for differences in how crayfish distributed their time across the

five temperature zones. Delta value data violated assumptions of normality, so for each species a

non-parametric Friedman test with Friedman-Conover post-hoc comparison was employed using

R© ver. 6.3.1 statistical software and RStudio®. Carapace sizes for rusty crayfish (n = 15), and northern crayfish (n = 15) used in this experiment were compared using a t-test, and no significant difference was found between the species (t = -1.262, df = 28, p = 0.217).

Results

Northern crayfish allocated their time differently in the temperature gradient treatment compared to the control treatment, as shown by a significant difference in delta values between zones (X2 = 18.08, df = 4, p = 0.002, Figure 2.2). Northern crayfish preferred the warmest end of

the temperature gradient (mean positive delta value of 774s in the 22°C zone). The difference in mean delta values between the 18°C and 22°C zone was significant (Friedman-Conover rank pair analysis, p = 0.024). Differences in delta values between any other pairwise combination of zones were not significant (p > 0.05). Northern crayfish showed avoidance of the 19°C zone (mean negative delta value of -308s).

Rusty crayfish also allocated their time per zone differently between the temperature

gradient treatment and the control treatment (X2 = 13.082, df = 4, p = 0.011, Figure 2.3). Rusty

crayfish showed a preference for the 19°C zone (mean positive delta value of 295s) and avoided

the 18°C zone (mean negative delta value of -377s). The mean delta value in the 18°C zone was significantly different from the other four zones (p ≤ 0.001). The only other significant pairwise difference was between the 19°C and 21°C zones (p = 0.045).

Discussion

Northern and rusty crayfish changed their physical location based on water temperature

in an experimental gradient. These results are the first for populations in New England and bolster

the current knowledge about these species’ temperature preferences.

Northern crayfish collected in New Hampshire appeared to prefer the 22°C zone and

avoid the 18°C zone. Our results are similar to the observed preference for 21°C in northern

crayfish from a Michigan population (Keller & Hazlett 2010). That study used the same

temperature range for their trials (18-22°C) as we did. In contrast, Peck (1985) used a temperature gradient of 12-32○C and found that northern crayfish from an Oklahoma population

preferred a water temperature of 24°C. We cannot say definitively that our northern crayfish

would not have chosen higher temperatures given the option. However, it is possible that

populations of crayfish from higher latitudes are adapted to cooler temperatures than more

southern populations.

Rusty crayfish collected in Vermont showed a preference for the 19°C zone (cooler than

the zone preferred by the northern crayfish) and avoided the 18°C zone (as did the northern

crayfish). Our results partially align with previously observed temperature preferences for rusty

crayfish from Michigan (Keller & Hazlett 2010) and Ohio (Mundahl & Benton 1990) in that our

rusty crayfish avoided cooler temperatures. However, the crayfish in those previous studies did prefer a warmer temperature (22○C) compared to the rusty crayfish in our trials (19○C). Data from

populations across a wide latitudinal range would be helpful to better understand how much local

adaptation might influence temperature preferences observed in different populations.

Methodological differences may also have contributed to differences between our results

and previous work on these crayfish species. In our study we were able to record a finer level of

behavioral detail than that found in previous crayfish temperature preference assays. We tracked

the location of crayfish with a level of one observation per second during each 60-min trial. In

contrast, other experimental investigations of northern and rusty crayfish temperature preferences

involved recording the location every 5 minutes (Keller & Hazlett 2010), every 2 minutes (Peck

1985), or every 15 seconds (Mundahl & Benton 1990). Because of this methodological difference

it is possible we captured more temporal variation in thermal behavior for these two species than

previously recorded. The variation seen in crayfish temperature preference assays also presents

statistical challenges, as highlighted by Mathur et al. (1982). Data for thermal preferences usually

show high variability between replicates and deviate from normality, and researchers have taken

varied statistical approaches to analyzing these data. This complicates the ability to make direct

comparisons between studies. Westhoff and Rosenberger (2016) noted in their review of crayfish

optimal and preferred temperatures that a standardized set of criteria for laboratory testing of

these metrics would help biologists make generalized statements about crayfish thermal ecology.

In order to generate robust predictive models of crayfish temperature distributions, more

experimental data on temperature preferences are needed but also more consistency in how those

data are collected and analyzed.

Some studies have suggested that results from temperature preference experiments may

be influenced by the average temperature values that animals experience before testing. Some

crayfish species show changes in preferred temperature dependent upon acclimation temperature,

with lower acclimation temperature resulting in lower preferred temperature (Crawshaw 1983,

Espina et al. 1993, Kivivuori 1994). In our experiment we used an acclimation temperature of

21○C for both crayfish species, and we saw differing temperature preferences between species.

Experiments done by Keller and Hazlett (2010) on northern and rusty crayfish used a constant

20○C acclimation temperature for both species and also found differences between their preferred temperatures (22○C for rusty and 21○C for northern crayfish). Mundahl and Benton (1990) used

rusty crayfish acclimated to field temperatures of 2.5○C and laboratory temperatures of 22○C and found that the respective preferred temperatures were 22.1○C and 22.3○C. Any pattern of

correlation between acclimation temperature and preferred temperature in these experiments on

our study species appears to be very small.

It is possible that the interspecific differences we observed were partly due to the

difference in habitat type (lotic vs. lentic ecosystem) where the two species were collected for our

experiment. When Keller and Hazlett (2010) compared northern crayfish from a stream versus a

lake, they found that the crayfish collected from the lake preferred a warmer temperature. Since

rivers can maintain colder annual temperatures than lakes (Syvitski et al. 2019), perhaps the rusty

crayfish we collected from the White River had an intrinsic or acclimated preference for a lower

temperature compared to the northern crayfish from Tewksbury Pond.

Based on our results, if rusty crayfish were introduced to a lake in New Hampshire, there

could be some partitioning of the thermal environment: rusty crayfish might occupy somewhat

cooler microhabitats than northern crayfish. However, the overlap between these two crayfish

species’ niches could result in contested areas. Previous studies have found that northern crayfish

prefer rocky substrate with macrophyte cover but will associate with sub-optimal sandy substrate

when rusty crayfish are present (Smith et al. 2019). If introduced, rusty crayfish could move into

some of the preferred benthic locations of the northern crayfish and force the northern crayfish

into sub-optimal areas with less cover and greater predation risk. If rusty crayfish exclude

19 northern crayfish from their optimal temperature zones, it may interfere with successful molting, growth, and reproductive behaviors (Momot & Gowing 1972, Wetzel & Brown 1993, Rogowski et al. 2013). In these ways, the non-native species could negatively affect the fitness of the resident species.

Crustaceans are known to be very sensitive to thermal changes and can detect changes in water temperature from 0.2 – 2°C (Lagerspetz & Vainio 2006). Considering rising mean global temperatures (IPCC 2014, NOAA 2018), understanding species’ temperature preferences will be important for predicting changes in species’ distribution patterns with warming temperatures

(Poloczanska et al. 2013, Wernberg et al. 2016). More regionally localized studies of crayfish thermal behavior are needed to generate a more complete picture of crayfish behavior in differing thermal environments, especially given the intraspecific variability seen in crayfish. These types of experiments will also provide crucial metrics in the investigation of crayfish thermal ecology if we wish to extrapolate small-scale temperature preference data into more generalized models of crayfish behavior in fluctuating temperatures.

Figure 2.1. Experimental tank design. In the temperature gradient treatment, the lowest end of the tank was cooled to 18°C while the raised end of the tank was heated to 22°C, resulting in five zones (18, 19, 20, 21, 22°C). Vertical fins were placed to ensure each zone of the tank had the same area and structure for the crayfish to move. In the control treatment, the temperature was a uniform 21°C throughout the tank.

A AB AB AB

Figure 2.2. Northern crayfish mean delta values (± 1 S.E.) comparing time spent in each zone between the control treatment and the temperature gradient treatment (n=15). Positive values denote more time spent in a particular zone during the temperature gradient treatment compared to the control. The distribution of time spent in zones was significantly different comparing the 2 control treatment to the temperature gradient treatment (X = 18.08, df = 4, p = 0.002). Bars sharing letters are not significantly different from each other.

B BC C BC

Figure 2.3. Rusty crayfish mean delta values (± 1 S.E.) comparing time spent in each zone between the control treatment and the temperature gradient treatment (n=15). Positive values denote more time spent in a particular zone during the temperature gradient treatment compared to the control. The distribution of time spent in zones was significantly different comparing the control treatment to the temperature gradient treatment (X2 =

13.08, df = 4, p = 0.011). Bars sharing letters are not significantly different from each other.

Chapter 3

Behavioral responses to cues from rusty crayfish (Faxonius rusticus) in a naïve population

of northern crayfish (Faxonius virilis)

Introduction

Many aquatic organisms use visual and chemical cues to help them evade predators, find cover and spawning habitat, and locate food sources (Chivers & Smith 1998). Such cues can induce distinct behavioral changes (Quinn & Graves 1998), including escape responses (Imre et al. 2014, Miyai et al. 2016), altered foraging and movement (Mitchell & Hazlett 1996, Pecor et al.

2010), and increased sheltering (Jurcak & Moore 2014). For example, escape responses of juvenile damselfish (Ancanthichromis polyacanthus) are amplified in the presence of alarm cues

from damaged conspecifics (Ramasamy et al. 2017). Respiratory rate increases and movement

decreases in Nile tilapia (Oreochromis niloticus) in response to a predatory cue (Miyai et al.

2016). The sea lamprey (Petromyzon marinus) avoids alarm cues from damaged conspecifics,

especially when combined with a predator cue (Imre et al. 2014). Some species of animals and

plants even produce chemical cues that deter foragers from consuming them (Bolser et al. 1998,

Marion & Hay 2011). The amalgam of cues that an organism experiences can be termed the

sensory landscape (Wilson & Weissburg 2013, Jurcak & Moore 2014). This sensory landscape,

by influencing organisms’ behaviors, can affect species interactions and community dynamics.

Crayfish are excellent study organisms in which to investigate the importance of cues to

behavior in the context of interspecific interactions. Crayfish exhibit a variety of behavioral

changes in the presence of chemical cues (Hazlett 1994, Pecor et al. 2010), including reductions

in feeding activity (Mitchell & Hazlett 1996), slowed movement, and increased defensive

posturing (Hazlett 1999). Some of the cues that crayfish react to originate from other crayfish.

There are four of these broad categories of crayfish chemical cues. Alarm cues (odors from

24 injured crayfish) stem from hemolymph proteins released when a crayfish carapace is breached during a predation event (Acquistapace et al. 2005). Cues originating from stressed but undamaged crayfish (Chivers & Smith 1998) can be released from their green glands when a crayfish is threatened by a predator or an aggressive competitor, or faces an environmental stressor such as lack of shelter (Hazlett 1985, 1989, 1990). Urine functions as a signaling cue in agonistic interactions that establish dominance hierarchies in crayfish populations (Zulandt

Schneider et al. 1999, Zulandt Schneider et al. 2001, Breithaupt & Eger 2002). Finally, pheromone signals are released in the context of mating behavior (Stebbing et al. 2003, Simon &

Moore 2007, Berry & Breithaupt 2008, Berry & Breithaupt 2010). Chemical cues can therefore attract or repel other crayfish, depending on the situation.

We investigated crayfish responses to different cues in the context of interactions between a resident species in New Hampshire (the northern crayfish, Faxonius virilis) and a potentially invasive congener (the rusty crayfish, F. rusticus). Where rusty crayfish have invaded habitats outside of their native range, they can displace native crayfish (Hill & Lodge 1999).

Their larger chela size (Hill et al. 1993), dominance over native crayfish when fighting for food

(Szela & Perry 2013), and their behavioral plasticity (Reisinger et al. 2017) – including the ability to respond to novel alarm cues (Hazlett 2000, Hazlett et al. 2002) – all help to facilitate the spread of rusty crayfish populations. It is of interest to understand how native crayfish will behaviorally respond to novel cues from these invaders, as these reactions could affect their fitness.

Northern crayfish are sensitive to alarm cues produced when conspecifics are injured or killed. They have been shown to spend less time near crushed conspecifics compared to live conspecifics stressed by confinement in mesh bags (Mitchell & Hazlett 1996). Northern crayfish also spend less time foraging when food odor is accompanied by alarm cue (Jurcak & Moore

2014), although this effect weakens with prolonged starvation (Hazlett 2003). If alarm cues are reliable indicators of predation risk, then being able to detect and respond appropriately to

heterospecific alarm cues could be advantageous to northern crayfish. Members of some crayfish

populations fail to respond strongly to novel heterospecific alarm cues (Hazlett 2000, Hazlett et

al. 2003), but Pecor et al. (2010) found that northern crayfish from a population in New Jersey

responded similarly to alarm cues from conspecifics, and co-occurring and novel heterospecifics.

Northern crayfish are also sensitive to visual and chemical cues generated by other

individual crayfish. What is not well understood is how such cues from rusty crayfish will affect

the behavior of northern crayfish. For example, northern crayfish establish dominance hierarchies

for shelter, with younger crayfish avoiding shelter in the presence of conspecific chemical cues

(Quinn & Graves 1998). If cues from rusty crayfish also displace subordinate northern crayfish

from shelter, the northern crayfish could be exposed to higher predation risk. This could increase

the advantage of rusty crayfish, which are already less vulnerable than northern crayfish to

predators like largemouth bass (Hill & Lodge 1999). Northern crayfish also move more slowly

and spend more time sheltering in the presence of cues from predators (Hazlett 1999). If this

reaction were induced by visual or chemical cues from introduced rusty crayfish, this could help

northern crayfish avoid risky agonistic interactions, but at a cost to their foraging activity and

growth. Thus, trade-offs involving habitat use, predation risk, and growth could all be influenced by responses to heterospecific cues.

In our study we investigated how northern crayfish responded behaviorally to the physical presence of rusty crayfish versus their alarm cues in a controlled laboratory setting. We tested these responses at three different temperatures in order to better understand how environmental conditions can alter reactions to cues: as ectotherms, crayfish exhibit many behavioral and physiological changes with temperature. The northern crayfish is the most abundant resident crayfish in New Hampshire (NH) lakes and ponds (Aiken 1965, Ramberg Pihl et al. 2018). The rusty crayfish is not known to be established in NH but is reported in the

Connecticut River bordering NH and Vermont (USGS 2019). Thus, the northern crayfish we

tested had presumably never been exposed to cues from rusty crayfish. We hypothesized that the

physical presence of the rusty crayfish and the presence of alarm cues from rusty crayfish would

each significantly change northern crayfish movement and the amount of time they spent in the

area where the cue was introduced. We also predicted that cue treatment and temperature would

have an interactive effect on the behavior of the crayfish, with thermally stressful environments

(i.e. temperatures outside of their preferred range) potentially compromising their reactions to

visual and chemical cues.

Methods

Crayfish collection and housing

Northern and rusty crayfish were collected from July 4 – 6, 2018 using Gee® minnow traps with enlarged openings. Northern crayfish were collected from Tewksbury Pond (GPS

43.608803, -71.970279), a 19 Ha mesotrophic lake in Grafton, NH with a mean depth of 7.62 m

(NH Fish and Game). Traps were placed in the littoral zone in areas with low shoreline development. Rusty crayfish were collected from the White River, a tributary from the main stem of the Connecticut River, in White River Junction, VT (GPS 43.7201, -72.4199). Traps were set along the riverbank in areas with vegetation and woody cover.

All crayfish were transported to the lab at Plymouth State University in 22L buckets within 2 hours of collection. Structure in the form of polymer rope was added to each bucket to minimize the likelihood of antagonistic interactions between crayfish during transport. The two species of crayfish were kept separated from each other from the time of their collection until their use in the experiment. Crayfish were placed into large polypropylene bins (20-30 individuals per bin) immediately upon arrival to the lab. Bins were aerated with aquarium airstones and

placed on a 12L:12D light cycle. Half clay pots and polymer rope provided structure and shelter.

Crayfish were fed shrimp pellets and algae wafers ad libitum while housed in the lab. Bins were

kept at 21○C and water was completely changed every 7 days or as needed.

All experimental trials were run between the hours of 0700 and 1900 from July 20 -

August 15, 2018. Prior to each experimental trial, crayfish were chosen at random from the

communal tanks and isolated in individual plastic bins filled with tap water for 24 hours. The

order in which treatments were tested was completely randomized (using a random number

generator in R that assigned a number to each treatment-species combination), and for each trial a crayfish was chosen at random from the individuals that had been held in isolation for 24 hours.

After being tested, crayfish were placed into a separate holding tank and were not re-used for any trials.

The sex ratio of male to female northern crayfish used in this experiment (on average

73% male: 27% female per treatment) reflected the sex ratio observed in the trapped sub-sample of the population from Tewksbury Pond. Northern crayfish carapace length ( = 31.0 ± 3.8mm) did not significantly differ across treatments (one-way ANOVA: F8,81 = 0.417,𝑥𝑥 p = 0.91). The average carapace length of the rusty crayfish used for the physical presence treatment was 35.2 ±

4.2mm, and only male rusty crayfish were used both in this experiment to avoid any confounding

effects of rusty crayfish sex.

Experimental design

This experiment to test the response of northern crayfish to rusty crayfish cues fully

crossed three cue treatments (alarm cue, physical presence, and control) with three ambient water

temperature treatments (17°C, 21°C, 30°C). These temperatures were chosen to include values above and below both species’ preferred temperatures, and the acclimation temperature for this experiment (21°C). There were ten replicates for every treatment combination, for a total of 90 individual trials. All trials were conducted in a 20L (41L cm x 21W cm x 25.5D cm) aquarium with a vertical plexiglass divider spanning the length of the tank and a permeable mesh-lined

barrier secured to the bottom 3cm of the plexiglass divider. The outside surface of the tank was

painted flat white with Rust-Oleum® enamel spray-paint. The aquarium was filled with tap water

to the top of the divider (20 cm). The divider was used to separate the focal northern crayfish

individual on one side of the tank and manipulate the cue treatment on the other side (this side

was segmented into three equally sized zones, the center one of which was used in one treatment

to hold a live rusty crayfish) (Figure 3.1). A small space (45 mm wide) at one end of the

aquarium left room to place two submersible 50-watt Aqueon® heaters (to establish the

temperature treatments) and a single tube for aeration. Three 5 mm diameter aquarium tubes were

secured to the side of the tank leading down into each of the segmented zones to within 10mm of

the bottom of the aquarium. This allowed us to inject the alarm cue closest to wherever the northern crayfish was located after the acclimation period and to later examine whether the northern crayfish then avoided this area.

To prepare the alarm cue, a rusty crayfish was macerated and homogenized in a volume of distilled water equal to 20x the mass of the rusty crayfish, allowed to sit for 5min, and then filtered through cheesecloth (Pecor et al. 2010). The alarm cue was prepared immediately prior to its use in a trial. For each trial of the alarm cue treatment, a northern crayfish was placed in the aquarium and allowed to acclimate for 15min. A 75mm x 40mm square hand net was inserted and removed from the middle of the treatment side of the plexiglass divider as a sham control for the physical presence treatment (to simulate the water displacement of placing a live rusty crayfish in the tank in that treatment). The alarm cue (60 ml) was then injected (at a rate of 1mL per second) via the aquarium tube closest to the northern crayfish using a 60mL syringe.

For each trial of the physical presence treatment, a northern crayfish was placed in the aquarium and allowed to acclimate for 15 min. A live rusty crayfish was then introduced in the middle zone of the tank on the other side of the plexiglass divider. Distilled water (60 ml) was

injected into the tube closest to the northern crayfish to serve as a sham control for the effects of

injecting a fluid into the aquarium in the alarm cue treatment.

The control treatment consisted of a 15min acclimation period for the northern crayfish

followed by insertion of a dip net in the center zone, and then injection of distilled water into the

tube closest to the northern crayfish. After each treatment was established as described above, the

crayfish were recorded for 60 min via a wide-angle overhead camera connected to a laptop computer.

Data capture and statistical analysis

For all trials, all video footage was transferred to Ethovision®. This software tracked a virtual point on each northern crayfish’s carapace via the recorded video and converted its position into x,y coordinates, allowing for the estimation of total movement distance (cm), movement velocity (cm/s), the amount of time (s) spent near the zone of injection (the location where either alarm cue or water was introduced via a tube) in the tank, and the number of times the northern crayfish moved in front of the middle zone of the tank (where the live rusty crayfish

was contained in the physical presence treatment). The zones were set up virtually in the program

for the side of the tank with the northern crayfish and corresponded to the three physically

divided sections of the opposite side of the tank (Figure 3.1). These variables were selected so

that we could examine whether the experimental treatments affected the overall movement of the

northern crayfish, whether they avoided the alarm cue, and whether they avoided the sequestered

rusty crayfish.

Since the data were not normally distributed, a nonparametric Kruskal-Wallis test was

used to assess possible differences in time spent in the zone of injection across treatments. Total

movement and velocity data for the 60min trials were able to be normalized via square root

transformation, and the residuals were homoscedastic across treatments. These variables were

analyzed using 2-way ANOVAs on the transformed data set. A Kruskal-Wallis test was also used

to analyze the number of times northern crayfish crossed in front of the middle zone of the tank

between treatments due to violations of normality.

Results

The amount of time that northern crayfish spent in the zone of injection did not differ

significantly between treatments for the first 10 min post injection (Figure 3.2) (H = 6.509, df = 8,

p = 0.590), or for the full 60 min post-injection period (Figure 3.3) (H = 15.039, df = 8, p =

0.058). For total movement distance, there was a significant interactive effect of cue and

temperature treatment (F2,81 = 5.695, p < 0.001). Tukeys post-hoc testing showed that at 30°C, crayfish moved farther in the physical presence and alarm cue treatments than in the control treatment (p ≤ 0.02) (Figure 3.4).

Average velocity was significantly different between cue treatments (F2,81 = 4.856, p =

0.010) and between temperature treatments (F2,81 = 3.469, p = 0.036). The interaction term (cue x

temperature) was non-significant (p = 0.684). The walking speed of the crayfish increased with temperature and decreased in the presence of the alarm cue. Velocity was lower in the 17°C treatment than in the 30°C treatment (Tukey test, p = 0.030), and velocity was lower in the alarm cue treatment when compared to the control (Tukey test, p = 0.013) (Figure 3.5).

There was a significant difference between the temperature treatments in the number of times the crayfish crossed in front of the middle zone of the tank (H5 = 13.33, p = 0.001). Focused comparisons on mean ranks between treatment levels showed that the difference was between the

30°C and the 17°C treatment (Figure 3.6). Thus, temperature increased the number of times crayfish traversed the tank, but the presence of a live rusty crayfish did not have a significant impact on this aspect of northern crayfish behavior.

Discussion

We observed that northern crayfish behavior was influenced by alarm odors derived from

the rusty crayfish as well as by an interaction between rusty crayfish cues and water temperature.

The strongest response of northern crayfish to rusty crayfish cues was slower movement in the

presence of alarm odors. Interestingly, northern crayfish did not react strongly to the physical

presence of rusty crayfish. Crayfish often take a defensive posture (e.g. chelae flaring), engage in

an escape response (e.g. tailflip), or reduce their activity in the presence of a threatening hetero- or conspecific crayfish (Bergman & Moore 2003). We expected to see northern crayfish decrease movement and spatially avoid the rusty crayfish, but this was not the case. However, at 30°C, northern crayfish moved significantly longer distances in both the alarm cue and physical presence treatments than in the control; this may be a more subtle pattern of recognition and escape response by the northern crayfish in the presence of the rusty crayfish. Behavioral changes with temperature were also less prevalent than we anticipated, although movement speed did increase with temperature. These results suggest that both rusty crayfish invasions of New

Hampshire lakes and future climate change could alter the behavior of the resident northern crayfish.

Alarm odors released when crayfish are injured or killed typically induce conspecifics to decrease their activity in ways that can reduce their risk of predation. For example, some crayfish respond with a lowered posture (Hazlett 2000, Hazlett et al. 2006) and reduce the time they spend foraging near a perceived threat (Mitchell & Hazlett 1996). Crayfish may also spend significantly more time in shelter when faced with alarm odors (Gherardi et al. 2002). In our experiment, northern crayfish reacted to alarm cues from heterospecifics by decreasing movement speed. This could be indicative of a guarded – and potentially adaptive – response to a perceived threat (i.e. minimizing risk of detection from an unseen predator in the environment). Movement speed is a key variable related to both growth and mortality risk. Other experiments testing the response of

crayfish to alarm and disturbance cues have looked at whether crayfish spend less time in the area

of cue introduction (Pecor & Hazlett 2008), or how they position themselves spatially in relation

to the physical location of the cue (Quinn & Graves 1998, Jurcak & Moore 2014). We did not

find evidence in our experiment that northern crayfish spatially avoided the alarm cue, as

discussed later in this section.

The ability of the northern crayfish in our study to react in a potentially adaptive way to

heterospecific alarm odors is notable, because individuals from the population we tested had

never had any exposure to rusty crayfish. A previous study found that native populations of

crayfish did not respond to novel heterospecific alarm odors, while invasive populations did show

adaptive responses (Hazlett et al. 2003). When Pecor et al. (2010) studied an introduced

population of northern crayfish, they found them capable of responding to alarm cues from novel

heterospecifics. Being able to use a broader range of chemical cues to interpret the level of risk in

an environment is likely to be advantageous. In this sense, northern crayfish may share some

adaptive characteristics with other crayfish species that have become invasive. This may

contribute to the invasion success of northern crayfish elsewhere in North America and in Europe

(Ahern et al. 2008, USGS 2019), and may even help explain how this species came to dominate

New Hampshire lakes.

Surprisingly, northern crayfish showed little behavioral response to visual (and any chemical, e.g. urine) cues associated with the physical presence of a sequestered rusty crayfish.

We did observe an increase in movement distance (compared to the control treatment), but only at the highest of the three water temperatures tested. This observation of increased movement in the presence of the live rusty crayfish contradicts some findings of decreased movement when crayfish are confronted with predatory cues (Blake & Hart 1993, Hazlett & Schoolmaster 1998,

Hazlett 1999). We saw the same increased movement distance in the alarm cue treatment relative to the control at this temperature as well. It is difficult to account for this interactive effect of

33 temperature and cue, which seems largely driven by an unusually low activity level in the control treatment at the highest temperature.

We do know from other studies that crayfish can recognize threats visually and respond to them (Aquiloni & Gherardi 2008, Van der Velden et al. 2008). In our experiment, perhaps the physical separation between the live rusty crayfish and northern crayfish served to interrupt any visual recognition. Some species of crayfish recognize visual characteristics when engaged in close agonistic interactions (Van der Velden et al. 2008); in the absence of face-to-face engagement, visual cues may not have been enough to trigger a response in the northern crayfish.

This nocturnal species may be much more likely to rely heavily on chemosensory cues. Urine excretion plays a clear role in crayfish agonistic interactions, with dominant crayfish releasing more urine than subordinate individuals (Breithaupt & Eger 2002). Our experimental aquaria would have allowed for urine to diffuse into the side of the tank with the northern crayfish.

However, rusty crayfish urine excretion was either unrecognized, or too weak or variable in our trials to induce any clear response. Finally, previous work by Dresser et al. (2016) showed considerable variation in the behavioral responses of native crayfish to invasive rusty crayfish, with aggressiveness to the invader differing between species and between sexes within species.

The lack of response in the northern crayfish we tested could be a result of this population not showing any defensive reaction to novel live crayfish cues, but other populations could show different results.

Northern crayfish also showed no evidence of spatial avoidance of either the live rusty crayfish or the alarm cue. In terms of the alarm cue, Hazlett (1994) noted that when northern crayfish were presented with cues from crushed conspecifics they ceased movement. If this were the case in our experiment we would have expected the time spent in the zone of injection to be significantly higher than the control. While there was some indication of more time spent in this zone, at least at the lowest temperature, the differences between treatments were non-significant.

Given the relatively small dimensions of our test aquaria, perhaps the active substance in the alarm cue spread through the entire tank relatively quickly, making spatial avoidance impossible.

Finally, we did observe significant increases in activity (movement speed and number of

times traversing the middle of the tank) as temperature increased. We also noted an interactive

effect of temperature and cue on total movement distance. However, overall the northern crayfish

were not as sensitive to temperature as we had expected. Crayfish are ectotherms and often

increase locomotion with temperature (Claussen et al. 2000); temperature also plays a critical role

in life events of crayfish such as reproduction (Portlance & Dube 1995), molting (Momot 1967,

Weagle & Ozburn 1972), and growth (Mundahl & Benton 1990). The temperatures in this experiment (17-30°C) were broader than both the preferred temperatures of northern crayfish (21-

24°C), and the range at which they show optimal growth (25-26°C) (Westhoff & Rosenberger

2016). The limited sensitivity to temperature we noted in our trials may indicate an innate adaptability of this species when faced with temperature changes. There is also evidence that crayfish will occupy temperatures well below their preferred range when aggressive dominant crayfish are present (Peck 1985). A more extreme range of temperatures may be necessary to see any strong patterns of interaction between chemosensory cues and crayfish behavior. More work is needed to better understand the relative importance of the chemosensory landscape versus temperature on northern crayfish behavior, and under what conditions these factors may interact.

To conclude, this experiment provided evidence that the northern crayfish decreased walking speed in the presence of novel alarm cues from rusty crayfish. This could be advantageous in the event of a rusty crayfish invasion if the same pattern occurred in natural environments. However, northern crayfish were mostly unresponsive to visual and chemical cues associated with live rusty crayfish. More investigation is needed to better understand how the behavior of northern crayfish in the presence of the potential invaders’ visual and chemical cues may translate into changes in fitness. Our results provide a foundation for future research into

35 how changes to the chemosensory landscape could affect populations of resident crayfish in New

Hampshire lakes if the rusty crayfish invades.

Figure 3.1. Experimental design. Leftmost tank represents the alarm cue treatment, center tank represents the physical presence (live rusty crayfish) treatment, and the rightmost tank represents the control treatment. The smaller crayfish on the right side of each tank represents a focal northern crayfish. All three cue treatments were tested at three temperature levels (17°C, 21°C, 30°C) in a fully crossed 3x3 design with 90 total observations (n = 10 for each treatment combination).

Figure 3.2. Time spent in the zone of injection for the 10 min after injection of either alarm cue or control water shown by temperature (17°C, 21°C, 30°C) and cue treatment (alarm cue, physical presence, and control). There were no significant differences in the amount of time spent in the injection zone between treatment combinations (H = 6.509, df = 8, p = 0.590).

Figure 3.3. Time spent in the zone of injection for the full 60 min period after injection of either alarm cue and control water, shown by temperature (17°C, 21°C, 30°C) and cue treatment (alarm cue, physical presence, and control). There were no significant differences in the amount of time spent in the zone of injection between treatment combinations (H = 15.039, df = 8, p = 0.058).

Figure 3.4. Average total distance moved by crayfish shown by temperature (17°C, 21°C, 30°C) and cue treatment (alarm cue, physical presence, and control). Significant effects were found for cue (F2,81 = 5.3901, p = 0.0039), and for the interaction of cue and temperature (F2,81 = 5.6951, p < 0.001). Tukeys post-hoc testing showed that at 30°C, crayfish moved less in the control treatment than the other two cue treatments (p ≤ 0.02).

○ ○ Figure 3.5. Average velocity of crayfish shown by temperature (17 C, 21 C, 30○C) and cue treatment (alarm cue, physical presence, and control). Significant main effects were found for cue (F2,81 = 4.856, p = 0.010), and for temperature (F2,81 = 3.469, p = 0.036). Tukeys post-hoc testing showed that average velocity was higher at 30°C than at 17°C (p = 0.03), and velocity was lower in the alarm cue treatment than the control (p = 0.013).

Figure 3.6. Number of times the northern crayfish crossed in front of the middle zone of the tank, shown by temperature (17○C, 21○C, 30○C) and cue treatment (alarm cue, physical presence, and control). There were no significant differences based on cue treatment (H = 4.829, p =0.089), but there was a significant difference between temperature treatments (H = 13.33, p = 0.001). Post-hoc mean rank comparisons showed that the number of times crossing the middle zone was significantly lower at 17°C than at 30°C.

Chapter 4

General Conclusion

North America is a biodiversity hotspot for crayfish, home to more than half of the known species worldwide (IUCN 2019). Like many other freshwater fauna, crayfish species face many threats, including impacts of invasive species and climate change (Reid et al. 2019). Since crayfish act as keystone species in freshwater environments, it is crucial we understand crayfish behavior and physiology so that we can create management plans for threatened crayfish. To best predict the risk and consequences of invasive crayfish establishment, we need to explore many variables, including temperature preferences and complex chemosensory landscapes. Studying chemosensory behaviors will help us uncover mechanisms by which the presence of invasive crayfish may affect native crayfish populations. Understanding thermal behavior in crayfish is useful for considering habitat overlap between invasive and native species and how their interactions may change with the environment.

The risk of an introduction of rusty crayfish (Faxonius rusticus) into New Hampshire lakes and ponds is high, given the proximity of this non-native species on the Vermont side of the

Connecticut River. This risk provided the motivation for exploring the behavior of resident northern crayfish (F. virilis) collected in New Hampshire and the potential invader collected in

Vermont. We explored their respective thermal preferences and how the northern crayfish reacted to alarm cues and the physical presence of rusty crayfish.

Northern and rusty crayfish exhibited different thermal preferences in a controlled experimental setting (Chapter 2). We found that northern crayfish had a temperature preference of

22○C, which falls within the range of some previous findings for this species (21-24○C) (Westhoff

& Rosenberger 2016). Rusty crayfish preferred a temperature of 19○C in our experiment; this

does contrast with some previous findings for rusty crayfish (21-22○C) (Mundahl & Benton 1990,

Keller & Hazlett 2010). If these two species exhibit different thermal preferences in natural

environments, it may result in some habitat partitioning that could reduce the frequency of their

interactions. When northern and rusty crayfish do physically interact, rusty crayfish tend to

dominate. However, there is a paucity of data on these two species’ thermal preferences to date,

and the variation seen in crayfish thermal preferences highlights a need for more localized testing.

It is possible that much of the variation seen in temperature preferences in the published literature

can be attributed to geographic location, testing methodology, and intraspecific differences in

crayfish populations. Conducting more comparative temperature presence assays from

conspecific crayfish collected from regions with differing annual temperatures (e.g. northern

versus southern latitudes) may help reveal correlations between crayfish life history and

temperature preferences.

The behavior and interactions between northern and rusty crayfish can also be dependent

on temperature. We conducted the first experiment, to our knowledge, to test how northern crayfish react to chemical cues from rusty crayfish (Chapter 3). We present evidence that northern crayfish altered their movement distance and velocity in the presence of an alarm cue from rusty crayfish, and water temperature was a factor in their response. We tested the behavior of the northern crayfish at three discrete temperatures (17, 21, and 30○C) and found that they

walked further in the presence of rusty crayfish alarm cue (compared to a control) at 30○C.

Northern crayfish also reduced their walking speed in the presence of the rusty crayfish alarm

cue. This is evidence that rusty crayfish can alter northern crayfish behavior even without direct

contact between the two species. It also demonstrates for the first time that northern crayfish can

detect cues from rusty crayfish even if they have never been exposed to them before.

The consequences of the introduction of non-native crayfish chemical cues to a new

lentic waterbody are difficult to predict. Alarm cues from rusty crayfish could warn northern

crayfish of predatory threats. Chemical cues from rusty crayfish could also displace northern

crayfish from their preferred niche space. These new biochemical stimuli in the water column

could even cause the northern crayfish to move into microhabitats with non-optimal water

temperatures when lakes are thermally stratified during the summer. These possibilities warrant

further investigation, which could be useful in predicting the behavior of native crayfish if non-

native species were to be introduced.

Rusty crayfish are known to affect native crayfish through multiple pathways (e.g. lower vulnerability to predators, physical dominance in direct fights, higher growth rates: Hill & Lodge

1999, Hazlett et al. 2002, Szela & Perry 2013). The findings in this study suggest a new mechanism: rusty crayfish may alter the fitness of the native species by chemosensory means.

This impact of this mechanism on fitness will depend on whether the native species can use these chemical cues to recognize and avoid the invader. Northern crayfish seem to be able to respond to alarm cues from several heterospecifics with altered movement (Hazlett 1994, Pecor et al. 2010), and this study adds rusty crayfish to that list. However, if chemical cues from rusty crayfish conflict with or confuse northern crayfishes’ reaction to conspecifics, this could be detrimental to their fitness. Models of rusty crayfish invasions risk aim to predict possible dispersal routes, at- risk waterways, and effects of this invasive on native crayfish (Olden et al. 2011). The types of temperature and chemosensory data obtained in this research project could prove valuable within the framework of large multivariable prediction models for invasion prevention and mitigation

(Peters & Lodge 2013).

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