No More Seashells by the Seashore:The Effects of Ocean Acidification on Shell Resource and Assessment

Behavior of Hermit Crab Pagurus longicarpusJackie Ricca

BMSS Independent Research Project December 13th, 2016

Abstract

The behavior of marine organisms dependent upon sensory-mediated information via chemical,

visual, and tactile cues that guide fundamental decision-making processes. Hermit crabs are rely

on calcified shells that are used as fully armored exoskeletons. The choice of an optimal shell is

of utmost importance to hermit crabs, as shells provide shelter, protection and ensures individual

fitness and survival. While many researchers compile data regarding the physical effects of

ocean acidification on marine organisms, more attention needs to be paid to the potential

behavioral effects of increased acidity. Hermit crabs are excellent model organisms for

investigating the potential effects of reduced pH on behavioral resource allocation. Using the

model organism Pagurus longicarpus, I investigated the effects of reduced sea water pH (7.6) on

the shell assessment, selection behavior, distance traveled, time in locomotion, and shell

investigation time. In low pH, hermit crabs were slightly more likely to switch shells, spent less

time in locomotion, more time investigating shells, and traveled further. The findings of this

study indicate that different responses to an altered environment could be indicative of some

individuals having an advantage over others. Acclimatization of behavior to increasingly

acidified oceans may only be possible through behavioral plasticity and adaptation.

Introduction

A variety of calcifying marine taxa rely on stable chemical conditions and pH levels,

modulated by the ocean’s carbonate-alkalinity pump, to build their calcium based shells,

skeletons, and internal structures. Due to continued anthropogenic input, atmospheric CO2 is

absorbed into the ocean and decreases pH, causing the dissolution of carbonate structures

(Hofmann et al., 2010). This process is known as ocean acidification (OA) and we expect a

decrease of average ocean pH to 7.80 under high emissions by 2100 (IPCC AR5, 2015). We also

expect to see changes in behavioral processes that are pH dependent, or influenced by the

energetic costs of maintaining calcified structures and internal homeostatic chemistry (Dory et

al., 2010). While many researchers compile data regarding the physical effects of OA on marine

organisms, more attention needs to be paid to the potential behavioral effects of increased

acidity. I propose to address how OA changes critical behaviors of the common New England

hermit crab Pagurus longicarpus, a decapod crustacean commonly found inhabiting Littorina

littorea shells along the Atlantic coast in intertidal rock pools.

Hermit crabs are excellent model organisms for investigating the potential effects of

reduced pH on behavioral resource allocation. Marine hermit crabs live in tidal pools as well as

the benthic, subtidal ocean zones. Prior research has focused mostly on the process of shell

acquisition by negotiations of shell exchanges and preferences for size. With increased

atmospheric CO2 levels, ocean pH is dropping putting stress on shell growth. This makes it hard

for hermit crabs, who are dependent on the shells of gastropods, to find suitable shells (de la

Haye et al., 2011). If the pH becomes low enough, this could result in a decreased rate of

calcification and shell dissolution limiting shell choice, which would be detrimental for shell-

switching behavior.

        There are a number of ways in which the effects of OA may impact the behavioral

ecology of hermit crabs as it relates to the shell resource; their life history is dependent upon

adequate calcified shells (Hazlett et al., 1981), and OA will increase the rate of shell dissolution

(Azevedo et al., 2015). To build shells, oversaturated carbonate water is needed in order for

gastropods to build and inhabit the shells that are then used as a fully armored exoskeleton for

hermit crabs. Although shells are deposited all over the ocean floor, hermit crabs are constrained

consumers in the sense that their only supply of shells comes from gastropods and conspecifics.

Hermit crabs do not remove the gastropod from its shell whether the gastropod is alive or dead

(Laidre et al., 2011). Gastropod shells are found with a dependence on chemical cues. Living

gastropods secrete periostracum around their shells, which reduces the amount of calcium lost

from the shell surface. But after predation or death, the olfactory detection of calcium ions,

resulting from dissolution, orients hermit crab individuals toward gastropod shells (Ismail, 2012;

Bibby et al., 2007). Therefore, shells exposed to reduced seawater pH dissolve faster and may be

less preferred by hermit crabs due to reduced fitness (i.e. structural integrity), but may release

stronger olfactory chemical cues.

        Many hermit crabs have evolved elaborate and ritualized social behaviors specifically for

the purpose of shell assessment and acquisition. The choice of an optimal shell is of utmost

importance to hermit crabs, as shells provide shelter, protection and ensures individual fitness

and survival. Hermit crabs show preferences for different shells and the condition of shells

influence shell choice. Most hermit crabs prefer larger shells, clear of any epibionts or cracks

(Rotjan et al., 2010). Among these shell-switching behaviors is the vacancy chain theory, which

describes a unique mechanism from the sequential distribution of a shell resource across multiple

individuals, where each individual benefits from a better quality shell (Rotjan et al., 2010; Chase,

2012). Thus, competition is often intense with agonistic behavior in the form of “piggybacking”

and “tug-of-war” (Rotjan et al., 2010). Shell resources are typically limiting for hermit crab

populations, leading to intense inter- and intraspecific forms of competition as some species tend

to dominate others, and the subordinate species tends to exhibit lower shell quality (Hazeltt,

1981). Competitive interactions and the behavior of shell resource selection and assessment is

important in regards to a continued need for larger, undamaged shells for growth and protection.

Through OA, changes in pH may also affect the behavioral process in shell assessment.

There is some uncertainty regarding how the underlying mechanisms of chemo-responsiveness

and shell acquisition behavior of hermit crabs will respond to the adverse effects of changes in

pH. Although the idea that acidification can influence animal behavior is still being investigated,

there are four hypotheses describing potential ways pH may be influential. Firstly, low pH could

cause a change in the ionic state of the odor molecules, reducing the ability to recognize calcium

ions (Brown et al., 2002). Secondly, low pH could reduce chemoreceptive acuity by changing the

charge distribution so ions do not bind on the odor chemoreceptor cells of antennules (Tierney

and Atema, 1988). Thirdly, low pH could cause ‘info-disruption’ through physical damage to

delicate calcium sensory structures, which are essential information gathering (Lürling and

Scheffer, 2007; Spicer et al,. 2007). Finally, changes in chemo-responsiveness might reflect

reduced activity levels in order to cope with the elevated metabolic load of maintaining an acid-

base balance (Pörtner et al., 2004).

The effects of OA on hermit crab behavior have been studied by de la Haye et al. (2011).

The hermit crab Pagurus bernhardus was exposed to high CO2 (12,000 ppm), and the rate of

antennular flicking and decision making, both aspects of shell-selection behavior, appeared to be

impaired under high CO2, indicating info-disruption. Unanswered questions relate to more subtle

changes in OA expected at the end of this century (e.g. an average decrease of 0.3 pH units) and

the ability of hermit crabs to respond to changes in shell quality related to changes in OA.

  The purpose of this experiment will be to determine the effects of exposure to the

reduced seawater pH of 7.6, as expected by the year 2100, on the ability of Pagurus longicarpus

to perform a standard asynchronous vacancy chain using both chemical and visual cues to exhibit

shell investigation and shell choice behavior. Additionally, this study aims to test if Pagurus

longicarpus can detect shells weakened by exposure to a pH of 7.6, in comparison to a shell in a

pH of 8.0 (normal). I hypothesize the lower pH to cause a reduction in the ability to detect and

investigate shells as well as cause an overall decrease in activity rates as a result of overall

metabolic depression. I expect hermit crabs to reject shells exposed to lowered pH. These

hypotheses are based on results from (de la Haye et al., 2011).Understanding long-term

behavioral responses are critical to test for potential acclimatization through phenotypic

plasticity and adaptation as well as changes to species distribution and ecosystem structure

among hermit crabs and other marine organisms.

Purpose

1.     Assess the effect of exposure to reduced seawater pH on the ability of Pagurus longicarpus

to perform a standard asynchronous vacancy chain using both chemical and visual cues to exhibit

shell investigation and shell choice behavior.

2.     Assess shell-switching behavior in Pagurus longicarpus when given choice between a

degraded shell (exposed reduced pH) versus a normal shell (exposed to normal pH).

3.     Discuss and compare the effects of reduced seawater pH in Pagurus longicarpus to Pagurus

bernhardus (de la Haye et al., 2011) in an effort to assess differential effects upon different

species.

MethodsStudy Organisms

    Hermit crabs P. longicarpus were collected from Wyer’s Island and Mussel Beach at the

Bowdoin Coastal Studies Center on Orr’s Island in Harpswell, ME from mid October to early

November of 2016. Crab size was as standardized as possible by determining a mean weight of

the group. Only crabs inhabiting Littorina littorea shells were collected, since this is the

preferred conspecific gastropod shell for this species. Individuals were kept in two large holding

tanks (6 inches deep, 100 gallon capacity) supplied with flowing seawater. Natural shelters were

included in tanks to prevent agonistic and harmful interactions among hermit crabs. Hermit crabs

were fed shrimp, or grazed on the natural macro algae that grows in the tank. At least 5 days

elapsed between collection and the start of the experiment so hermit crabs could acclimate to

their new conditions. Two days before the start of the experiment, hermit crabs were be

randomly selected for the control or experimental conditions and separated between tanks. Also

during this period, hermit crabs were starved and evicted from their shells using a bench vice.

Only hermit crabs with a complete set of undamaged limbs and free from any disease or parasites

were used. Each crab was then placed into a suboptimal shell, 50% of the mass of an optimal

shell, to induce an asynchronous vacancy chain during the behavioral trials. Hermit crabs could

then recover for at least 24 hours before experimentation begins.

Experimental Design

Pagurus longicarpus were divided into two groups. In Part One of the experiment, both

hermit crab groups A and Group B of were kept in untreated seawater from the ocean for 5 days

(pH=8.0). At the end of these 5 days, both groups underwent behavioral trials in which they were

offered an optimal shell and measurements such as distance traveled during locomotion (distance

traveled in five minutes), the duration of shell investigation (seconds), the duration of

locomotion (seconds), and the occurrence of shell exchange (yes or no) were taken. In Part Two

of the experiment, Group A continued for the next 5 days in the untreated seawater from the

ocean (pH=8.0), while Group B will continue for the next 5 days in reduced pH seawater

(pH=7.6). Then, behavioral trials will commence again with the same measurements. Also, a

subset of shells were exposed to lowered pH, which a subset of Group A2 (9 hermit crab

individuals) were given the option to switch into after both Parts One and Two. A total of 36

individuals will be used in the experiment, 18 in Group A and 18 in Group B.

Table 1. Summary of Experimental Design

Time Part One Part Two

Day 1 - 5 Group A1- Control (18 individuals)

Group B1 -Control (18 individuals)

Day 6 - 10

Group A2- Control(same 18

individuals)

Group B2- Low pH(same 18

individuals)

Sea Water Treatments

There was no need for water changes as the water was constantly flowing through the

holding tanks. Hermit crabs were kept in holding tanks with either untreated seawater from the

ocean (pH=8.0) or reduced pH seawater (pH=7.6). Hermit crabs in Group B1 of the reduced pH

treatment had the pH gradually lowered as not to shock their systems (see appendix Table 2).

Behavioral Observations

Behavioral observations were be carried out at the end of Part One and end of Part Two.

Hermit crabs were be placed in a dish (4.5 inch diameter) filled with the appropriate seawater

from their experimental condition. New water was be added for each trial so as to remove any

chemical cues left behind from hermit crabs. The dish was be placed in a larger arena shaped like

Photograph 1. Behavioral arena

a plus sign (+) along with four other dishes. The walls of the arena (four sides of the + sign)

concealed each hermit crab from each other as well as the observer , so hermit crabs were not

phased. Hermit crabs were placed in the middle of the dish and left undisturbed for five minutes.

Then, an optimal gastropod Littorina littorea shell was be placed in front of the hermit crab and

their behavior was observed for five minutes. The behavioral measures recorded were distance

traveled during locomotion (distance traveled in five minutes), the duration of shell investigation

(seconds), the duration of locomotion (seconds), and the occurrence of shell exchange (yes or

no). Investigatory behavior was be recorded using a Samsung handheld video camera which was

mounted to a tripod over the + arena. The subset of shells that were exposed to the lowered pH of

7.6 were presented to a subset of hermit crabs from Group A2 (9 hermit crab individuals), which

went through the same behavioral observation of the occurrence of shell exchange (yes or no)

following the same procedure as explained above.

Statistical Methods

    Locomotion (distance traveled and duration) was analyzed with the computer software

Tracker Video Analysis and Modeling Tool, a free video analysis and modeling tool built on the

Open Source Physics (OSP) Java framework.

The distance traveled, duration of locomotion, and duration of shell investigation data

was analyzed using Post hoc t tests (paired and unpaired) to make pairwise comparisons between

means to investigate which mean differences (trial, group, trials and group) caused any

significant interaction effects. Significant differences in the effects of trial number between

groups would be indicated by a significant interaction effect between trial number and treatment.

Contingency tables were created based on proportions of the occurrence of shell switching

behavior (yes or no).

Results

There was no overall effect of pH treatment (F=2.0209, P=0.1597) or trial (F=0.5628, P=0.4557) and no significant interaction effect of pH treatment and trial (F=1.2786, P=0.2621) on the time spent in locomotion (Figure 6). The amount of time spent in locomotion was lower for hermit crabs in Acidified (Group B) than it was for hermit crabs in Ambient (Group A) in trial 1. The amount of time spent in locomotion was also lower for hermit crabs in Acidified (Group B) than it was for hermit crabs in Ambient (Group A) in trial 2. The amount of time spent in locomotion was lower for hermit crabs in Ambient (Group A) in trial 2 than for the same group in trial 1. Whereas the amount of time spent in locomotion for hermit crabs in Acidified (Group B) was higher in trial 2 than for the same group in trial 1. Post hoc tests revealed no significant differences between or within groups.

Figure 1. A plot of the means and standard errors of the Time Spent in Locomotion. The dependent variable is proportion time spent in locomotion (s). The independent variable is trial. The first trial was conducted after both groups of crabs had been kept in ambient, untreated sea water (pH=8.0) for 5 days (Trial 1). The second trial was conducted after Group A (Ambient, red bars) had been kept in untreated sea water (pH=8.0) for another 5 days, and Group B (Acidified, blue bars) had been kept in reduced pH sea water (pH=7.6) for the same period. Values are means +SE. Asterisks indicate significant differences between groups.

There was a significant effect of pH treatment (F=4.0929, P=0.0470) on the time spent investigating shells (Figure 12). There was no overall effect of trial (F=1.6268, P=0.2065) or interaction effect of pH treatment and trial (F=0.2739, P=0.6024) on the time spent investigating shells (Figure 6). The amount of time spent investigating shells was higher for hermit crabs in Acidified (Group B) than it was for hermit crabs in Ambient (Group A) in trial 1. The amount of time spent investigating shells was also higher for hermit crabs in Acidified (Group B) than it was for hermit crabs in Ambient (Group A). The amount of time spent investigating shells was lower for hermit crabs in Ambient (Group A) in trial 2 than the same group in trial 1. Similarly, the amount of time spent investigating shells for hermit crabs in Acidified (Group B) was lower in trial 2 than for the same group in trial 1. Post hoc tests revealed no significant differences between or within groups.

Figure 2. A plot of the means and standard errors of The Time Spent Investigating Shell. The dependent variable is proportion time spent investigating (s). The independent variable is trial. The first trial was conducted after both groups of crabs had been kept in ambient, untreated sea water (pH=8.0) for 5 days (Trial 1). The second trial was conducted after Group A (Ambient, red bars) had been kept in untreated sea water (pH=8.0) for another 5 days, and Group B (Acidified, blue bars) had been kept in reduced pH sea water (pH=7.6) for the same period. Values are means +SE. Asterisks indicate significant differences between groups.

There was no overall effect of pH treatment (F=0.1780, P=0.6745) or trial (F=0.1248, P=0.7250) and no significant interaction effect of pH treatment and trial (F=1.4218, P=0.2373) on the distance traveled (Figure 6). Post hoc tests revealed no significant differences between or within groups. The distance traveled was lower for hermit crabs in Acidified (Group B) than it was for hermit crabs in Ambient (Group A) in trial 1. Whereas the distance traveled for hermit crabs in Acidified (Group B) was higher than hermit crabs in Group A (Ambient) in trial 2. The distance traveled for hermit crabs in Ambient (Group A) was lower in trial 2 than the same group in trial 1. The distance traveled for hermit crabs in Acidified (Group B) was higher in trial 2 than the same group in trial 1.

Figure 3. A plot of the means and standard errors of Distance Traveled. The dependent variable is distance traveled (m). The independent variable is trial. The first trial was conducted after both groups of crabs had been kept in ambient, untreated sea water (pH=8.0) for 5 days (Trial 1). The second trial was conducted after Group A (Ambient, red bars) had been kept in untreated sea water (pH=8.0) for another 5 days, and Group B (Acidified, blue bars) had been kept in reduced pH sea water (pH=7.6) for the same period. Values are means +SE. Asterisks indicate significant differences between groups.

Trial 2 Occurrence of Shell Switching Behavior

Acidified Ambient No 15 16 Yes 3 2

Acidified Ambient No 0.8333333 0.8888889 Yes 0.1666667 0.1111111

Hermit crabs in Acidified (Group B2) were more likely to change shells than hermit crabs in Group A (Ambient). Of the 18 hermit crabs in untreated sea water (Group A2) two exchanged shells (11.1%), whereas of the 18 hermit crabs in reduced pH sea water (pH=7.6) (Group B2), three exchanged shells (16.67%).

Reduced pH shells Occurrence of Shell Switching Behavior

Acidified Ambient No 8 16 Yes 1 2

Acidified Ambient No 0.8888889 0.8888889 Yes 0.1111111 0.1111111

Hermit crabs given a reduced pH shell were less likely to change shells than hermit crabs in Ambient (Group A2) from trial 2. Of the 9 hermit crabs given a reduced pH shell one exchanged shells (11.1%), whereas of the 18 hermit crabs in Ambient (Group A2) from trial 2, two exchanged shells (11.1%).

Figure 4. Contingency table for the behavior of switching shells. Ambient hermit crabs (Group A2) had been kept in untreated sea water (pH=8.0) for a total of 10 days at the end of trial 2 and were given optimal shells. Acidified hermit crabs (Group B2) had been kept in untreated sea water (pH=8.0) for 5 days, then reduced pH sea water (pH=7.6) for a total of 5 days, and were also given optimal shells.

Figure 5. Contingency table for the behavior of switching shells. Ambient hermit crabs (Group A2) from trial 2 had been kept in untreated sea water (pH=8.0) for a total of 10 days at the end of trial 2 and were given optimal shells. A subset of ambient hermit crabs (Group A2) had also been kept in untreated sea water (pH=8.0) for 10 days were given suboptimal shells that had been kept in reduced pH sea water (pH=7.6) for a total of 5 days.

Discussion

Reduced seawater pH altered the normal shell assessment and selection behavior of P.

longicarpus. The crabs in the reduced pH treatment were more likely to change shells, however

they took longer to do so than the crabs in the ambient pH treatment, which were less likely to

change shells. This wastes valuable time and energy, and prolonging such a risky activity makes

them more vulnerable to predation. Compared to the control, hermit crabs in the reduced pH

treatment spent less time investigating shells, with the majority remaining in inferior shells that

gives poor protection. This implies a possible disruption to chemosensory function and an effect

of reduced pH on overall activity and duration of locomotion.

In trial 2, hermit crabs in the acidified treatment experienced reduced time spent in

locomotion for hermit crabs compared to the hermit crabs in the ambient treatment. Reduced

time in locomotion has been demonstrated in response to physiological stress in a variety of

crustaceans (Eriksson and Baden 1997; Taylor and Eggleston, 2000). However, this could also

mean a lack of olfactory simulation (Reeder and Ache, 1980). Therefore, physiological stress and

its effects on the metabolic and neurological functions of these hermit crabs may be responsible

for the disruption to decision-making and reduced locomotion under lowered pH conditions. The

change to the crabs’ normal shell assessment and selection behavior could reduce survival, as

finding an optimal shell protects them from predators and increases overall fitness of the

organism (Bertness 1981). The reduced pH level of 7.6 could be an indicative value of the steady

increase in acidity in our world’s oceans as the effects of climate change become clearer. If

hermit crabs aren’t able to switch out of and locate optimal shells on a regular basis (which will

become harder to do as calcified shells will degrade in reduced pH), this could drastically effect

their survival.

Sensory-mediated assessment and decision-making processes in marine organisms is

essential to their existence. This study shows that the disruption to such activities because of low

pH has the potential to affect the behavioral interactions between individuals and their intertidal

environment. In trial 2, the hermit crabs in the reduced pH treatment took longer to investigate

shells compared to the hermit crabs in the ambient pH treatment. While crabs were able to find

the shells, many did not ultimately change. As the results show, two exchanged shells in the

reduced pH treatment (11.1%), whereas of the 18 hermit crabs in reduced pH sea water, three

exchanged shells (16.67%). Although one more hermit crab from the reduced pH treatment

switched shells, this was not found to be significant. In the subset of shells that were exposed to

the lowered pH of 7.6 that were then presented to a subset of hermit crabs from Group A2, one

exchanged shells (11.1%), whereas of the 18 hermit crabs in Ambient (Group A2) from trial 2,

two exchanged shells (11.1%). This shows that hermit crabs in the ambient condition are able to

determine a reduced pH shells is not fit to switch into.

What is interesting to note, is that the reduced pH affected decision-making processes,

and not all individuals were affected in the same way. The different responses to an altered

environment could be indicative of some individuals having an advantage over others. This could

have extreme implications for hermit crabs, which often compete using ritual assessment and

decision-making processes while exhibiting shell finding and contest behavior (Elwood and Neil,

1992). Physiological stress could be the cause of behavioral disruption, as it is known to alter

neurological functions and decision-making in animals (Graham el al., 2010). On the other hand,

animals are also able to alter their behavioral decisions in response to a changing environment

(Inglis and Langton, 2006). Gherardi and Atema demonstrated that hermit crabs are sensitive to

chemical context and are able to tell the differences between chemical odors so they can adjust

their behavior to the conditions accordingly (2005).

Additionally, environmental factors such as hypoxia have been shown to alter shell

investigation and agonistic behavior (Cote et al., 1998). Crabs in hypoxic conditions chose

thinner, lighter, and smaller shells. This decision involved a costly tradeoff; lighter shells are less

energetically costly to carry, yet there isn’t much room to grow leaving individuals vulnerable to

predation. In this study, the decision of the hermit crabs in the reduced pH treatment to not move

to an optimal shell and remain in a smaller, lighter one may have been indicative of an adaptive

response to increased acidity conditions instead of declining to switch because of an inability to

detect. A similar response was seen in the marine gastropod Littorina littorea that were exposed

to low pH water. Unable to thicken their shell in response to a predator because of the reduced

availability of carbonate ions and shell dissolution, when exposed to a predator cue, because their

shells were thinner, the snails exhibited an avoidance response and left the water (Bibby et al.,

2007). Reductions in sea water pH will create complex problems and costly energy tradeoffs for

marine organisms in the future, as different responses of organisms in the same species may

affect overall survivorship and intraspecific interactions.

Despite the reduction in time spent in locomotion for hermit crabs in trial two in the

reduced pH condition, the same hermit crabs exhibited a large distance traveled as compared to

the hermit crabs in the ambient pH condition and the controls. Essentially, hermit crabs spent

less time in locomotion, however when they were moving around they covered a lot of distance.

This differs from a study by Reeder and Ache, which found stillness to be a response to a lack of

olfactory simulation in crustaceans (1980). Similarly, Allison et al. determined that under

physiological stress, responsive behavior is likely to be deemed a lower priority and energetically

costly activities deferred (1992). These findings are in direct opposition to a major conclusion to

this study, which found hermit crabs did indeed move around and at least distance-wise, were not

stifled by stress. Many previous studies on benthic marine invertebrates have reported

hypometabolism (Michaelidis et al., 2007). Here, the opposite is shown, at least with an increase

in metabolic rate to account for an increase in energy expended to travel farther in reduced pH

conditions. For a particular echinoderm species, the ophiuroid brittlestar (Amphiura filiformis),

organisms in an experimental setup increased the rates of their biological processes (in this case,

their metabolism) to compensate for increased sea water acidity (Wood et al., 2008). Whereas it

was previously assumed many marine assemblages would succumb, we now know that is not the

case for every aspect of a species’ behavior.

This study was done in part to discuss and compare the effects of reduced seawater pH in

Pagurus longicarpus to Pagurus bernhardus in an effort to assess differential effects upon

different species (de la Haye et al., 2011). P. bernhardus is also a marine intertidal species.

Intertidal species experience wide and rapidly changing fluctuations in, so crustaceans show a

wide variety of responses. It is predicted that strong iono- and osmo - regulating species are

likely to be the most tolerant to ocean acidification, simply because they have the compensatory

mechanisms to respond to acid–base disruptions. These species tend to inhabit shallow coastal

environments under freshwater influence, where they experience natural variations in seawater.

For instance, when left behind in rock pools during the night, crabs can experience increased

pCO2 and decreased pH levels and pO2 in the seawater (Morris and Taylor, 1983). Using P.

bernhardus (n=74), de la Haye et al. found some different results, albeit at a different pH level.

The reduced pH level used by de la Haye et al. was 6.8, whereas this study used 7.6, in which the

authors were trying to mimic a CO2 storage leak. Hermit crabs are unlikely to be exposed to such

an extreme pH, which may account for more marked behavioral consequences between studies.

There was a significant effect of pH treatment and a significant interaction effect on the latency

to change shells. They also found crabs in the acidified condition were less likely to change

shells than crabs in the ambient condition of trial two. There was a significant effect of pH

treatment and a significant interaction effect between trial and pH treatment on the time spent in

locomotory activities. This study also looked at a new variable, antennular flicking, which was

found vary significantly with pH treatment and trial. The flicking rate was lower for hermit crabs

in the acidified treatment than for hermit crabs in the ambient treatment of trial 2. This may

indicate disruption to olfactory function. The authors concluded that their study implied an

impaired ability to detect the chemical stimulus of calcareous objects. Even though the pH levels

were different in both studies, where 6.8 is extreme and 7.6 is mild, researchers can now see a

gradient-wise comparison of the effects of different levels of reduced pH.

Future work could address a better experimental design in which four distinct groups of

P. longicarpus hermit crabs were assigned to one of 4 groups, inside of which would be

replicates. This would better account for independence between groups. In the same vein, to

decrease variability and increase significance, future studies would include more hermit crabs as

well as account for the antennular flicking variable. Additionally, it would be interesting to study

the haemolymph of hermit crabs to assess the state of the acid-base balance and measure

important processes such as respiration and calcification to better determine whether hermit

crabs are being energetically compromised by the reduced pH. To test for the effects of reduced

pH on the shell itself, future studies could determine the calcium content of seashells to calculate

what percent of the shells is made of CaCO3. This could be effective in determining just how

much the effect of lowered pH has on shells in different pH treatments.

The study of behavioral responses to reduced pH is important, as behavior can be used to

study long-term acclimatization through behavioral plasticity and adaptation. Behavioral

plasticity can also allow animals to respond rapidly to environmental disruption where physical

acclimation might take longer to achieve and/or be too costly (Tuomainen and Candolin, 2010).

Therefore, the study of behavioral responses to environmental disruption is important as

behavior can connect physiological function with ecological processes (Scott and Sloman, 2004).

In reality, ongoing anthropogenic pollution and ocean acidification will not act in isolation;

behavior will also be influenced synergistically with other variables (Hofmann et al., 2012). The

study of behavioral effects of high CO2 is still in its early stages, however hermit crabs show that

there are clear species-specific sensitivities which force marine animals to make costly trade-offs

to mitigate changes to their internal and external environment (Briffa et al., 2012). Changes to

normal behavior could have serious consequences for marine animals that rely on gathering

chemosensitive information to make life-altering decisions that impact their individual and

population fitness and survival.

This study indicates that reduced pH seawater may affect sensory information gathering

and decision-making activities, which could have serious consequences for marine animals that

rely heavily on chemosensory information gathering. The Intergovernmental Panel on Climate

Change predicts that under their worst-case scenario of CO2 emissions, sea water pH will reach

the pH level of this study 7.6-7.7, by 2100. In a future where a more acidic ocean is becoming

more and more likely every day, we must take drastic measures to study, both physically and

behaviorally, how this will alter intertidal and marine communities.

Acknowledgements

I would like to thank my independent project mentor, Dr. David Carlon for his guidance and expertise on experimental design and interpretation of results. Additionally, I thank Dr. Elizabeth Halliday-Walker for discussions on statistical analysis of the data. A thanks also goes out to Dr. Amy Johnson and Dr. Olaf Ellers for taking the time to share their skills with the Tracker Video Analysis and Modeling Tool program. I would also like to acknowledge Nick Keeney for designating holding tanks, lab space, and research equipment for this study. My gratitude is also extended towards Dr. Sarah Kingston, Steve Allen, Dr. Bobbie Lyon, Russ Rymer, Rosie Armstrong, and the students of the Bowdoin Marine Science Semester for their helpful comments and critiques throughout the experimental process.

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Appendix

Figure 6. Results of post hoc paired and unpaired t tests for pairwise comparisons between means. Group A (Ambient) was exposed to untreated sea water (pH=8.0) in trial 1 and trial 2. Group B (Acidified) was exposed to untreated sea water (pH=8.0) in trial 1 and reduced pH (pH=7.6) in trial 2.

Behavior Within-groups paired t tests

Group t df P

Between-groups paired t tests

Group t df P

Time Spent A1 vs A2 1.2947 32.965 0.2044in Locomotion B1 vs B2 -0.27686 33.252 0.7836

Time Spent A1 vs A2 1.05 29.039 0.3024Investigating B1 vs B2 0.96331 25.948 0.343Shell

Distance A1 vs A2 0.58101 34 0.5651Traveled B1 vs B2 -1.1172 27.985 0.2734

A1 vs B1 1.9743 33.948 0.05653 A2 vs B2 0.19057 33.848 0.85

A1 vs B1 -1.4516 21.462 0.1611 A2 vs B2 -1.5613 23.384 0.1319

A1 vs B2 0.61733 30.458 0.5416A2 vs B2 -1.033 33.279 0.3091

Variable Aquaria

Ambient (Group A) pH 8.0 Acidified (Group B) pH 7.6

Measured pH 8.0 ± .065Temperature (°C) 10.79 ± .48Salinity (SAL) 32.21 ± .08

7.6 ± .03 10.79 ± .48

32.21 ± .08

Figure 7. Mean ± for aquaria physical parameters measured three times during the 10 day exposure period. The parameters were calculated with a YSI Sensor.