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Phototaxis of Dungeness Crab Zoeae in High-CO2 Seawater: Implications for Coastal Ecosystems in an Acidfied Ocean
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2013
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Creator (dcterms:creator)
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Roberts, Caitlin Payne
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Subject (dcterms:subject)
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Environmental Studies
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extracted text (extracttext:extracted_text)
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PHOTOTAXIS OF DUNGENESS CRAB ZOEAE IN HIGH-CO2 SEAWATER:
IMPLICATIONS FOR COASTAL ECOSYSTEMS IN AN ACIDIFIED OCEAN
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Caitlin Payne Roberts
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A thesis
submitted in partial fulfillment
of the requirements for the degree
Master of Environmental Studies
The Evergreen State College
December 2013
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© 2013 by Caitlin Payne Roberts. All rights reserved.
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This Thesis for the Master of Environmental Studies Degree
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Caitlin Payne Roberts
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has been approved for
The Evergreen State College
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Dr. Erin Martin
Member of the Faculty
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______________________
Date
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ABSTRACT
Phototaxis of Dungeness crab zoeae in high-CO2 seawater:
implications for coastal ecosystems in an acidified ocean.
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Caitlin Payne Roberts
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Anthropogenic carbon dioxide emissions are reducing the global average oceanic
pH in a process known as ocean acidification. High levels of carbon dioxide (CO2) in
seawater are shown to increase phototaxis and impair predator avoidance based on visual
cues in larval fish. However, we currently do not understand the impact of high-CO2
seawater on the phototaxis of any larval crustacean. The present study is the first to
evaluate the phototaxis of a larval crustacean in high-CO2 seawater. Larvae of the
ecologically and economically important Dungeness crab Metacarcinus magister were
reared in three CO2 treatments (400, 1600, 3200 µatm) and exposed individually to a
directional light in a horizontal aquarium in a chronic behavioral bioassay. The response
of larvae to light treatments was video recorded and analyzed for variation in phototaxis
to see if acidification would impact their natural tendency to approach this light stimulus.
Larvae exposed to a light treatment were significantly more likely to swim to the light
than those in the control (i.e., dark) treatment, thus demonstrating positive phototaxis.
Non-significant results indicate that the phototactic behavior of larval M. magister does
not appear to be pH-sensitive. Since benthic Puget Sound organisms are evolutionarily
adapted to withstand large pH fluctuations, it is possible that high-CO2 conditions are not
a threat to M. magister phototactic behavior. However, weak non-significant trends may
suggest that animals reared in high-CO2 seawater swam to the light the faster, spent a
greater proportion of time stationary at the light, and exhibited a lower overall mean
speed in the control treatment than animals from the other two CO2 treatments. The latter
behavior could be explained by a change in metabolism due to CO2-induced acidification.
Info-disruption through overexcitation of the histaminergic photoreceptor cell, which
could affect fitness and survival rates, is proposed as a mechanism for a possible
heightened phototactic response in M. magister larvae reared in high-CO2 seawater.
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TABLE OF CONTENTS
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List of Figures
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List of Tables
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Dedication and Acknowledgements
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1. LITERATURE REVIEW
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1.1. Introduction
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1.2. Ocean acidification
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1.3. The California Current System and Puget Sound
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1.4. Metacarcinus magister natural history
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1.5. Vulnerability of larvae to OA
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1.6. Vulnerability of crustaceans to OA
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1.6.1. Calcification
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1.6.2. Physiology
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1.6.3. Behavior
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1.7. Zoeal phototaxis
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1.8. Phototransduction
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2. INTRODUCTION
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2.1. Identification of the problem
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2.2. Behavioral impacts of OA
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2.3. Vulnerability of the study organism to OA
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2.3.1. Crustaceans
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2.3.2. Marine larvae
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2.3.3. Phototaxis
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2.3.4. Habitat
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2.3.5. Ecosystem
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2.4. Proposed mechanism of behavioral impairment
3. METHODS
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3.1. Experimental system and carbon chemistry measurements
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3.2. Specimen collection and larval rearing
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3.3. Behavioral tests
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3.4. Video analysis
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3.5. Statistical analysis
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4. RESULTS
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4.1. Overall swimming speed
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4.1.1. Control group
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4.1.2. Light treatment group
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4.2. Approach of light
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4.2.1. Control vs. light treatment
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4.2.2. CO2 treatment
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4.3. Speed to light
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4.4. Time to light
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4.5. Proportion of time at light
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4.6. Proportion of time at light once light was reached
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5. DISCUSSION
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5.1. Phototaxis vs. photokinesis
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5.2. No effect of CO2 on behavior
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5.3. Proposed mechanism for increased phototaxis
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5.4. Implications for metabolic rate
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5.5. Recommendations for future research
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6. CONCLUSION
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7. INTERDISCIPLINARY STATEMENT
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References
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List of Figures
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Figure 1: Photograph of behavioral test setup
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Figure 2: Demarcation of rectangular area in aquarium closest to light
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Figure 3: Effect of pCO2 level on overall swimming speed in the control group
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Figure 4: Effect of light treatment on proportion of zoeae that reached light
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Figure 5: Effect of pCO2 level on proportion of zoeae that reached light
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Figure 6: Effect of pCO2 level on speed to light
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Figure 7: Effect of pCO2 level on time to light
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Figure 8: Effect of pCO2 level on proportion of time away from light once
light was reached
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List of Tables
Table 1: Number of zoeae that reached area closest to light by pCO2 treatment
and light treatment
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Dedication and Acknowledgements
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This thesis is lovingly dedicated to the memory of my grandfather, Dr. Don Goodenough,
who taught me algebra on the back of a placemat at a Chinese food restaurant, and to the
memory of my uncles, Jamie Goodenough, who actively motivated me to pursue higher
education, and Chuck Goodenough, who firmly believed that I could accomplish
anything I put my mind to.
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I would like to express deep gratitude to Dr. Paul McElhany, Jason Miller, Michael
Maher, Dr. Schallin Busch, Daniel Bascom, Erin Bohaboy, Dr. Nina Bednaršek, and all
collaborators in the Ocean Acidification Group of the Conservation Biology Division at
the Northwest Fisheries Science Center for offering research facilities and materials, and
encouragement throughout the study period.
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I thank Dr. Steve Sulkin, Retired Director of Shannon Point Marine Center, for offering
invaluable comments on the interpretation of my results, Dr. Brady Olson for showing me
around the OA lab at Shannon Point, and Dr. Danielle Dixson of the Georgia Institute of
Technology for responding to questions about my experimental design. I thank all my
faculty and cohort in undergraduate and graduate coursework at Drexel University, The
Evergreen State College and Friday Harbor Laboratories. Special thanks to Dr. Gerardo
Chin-Leo for encouraging my interest in invertebrates.
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It is with great pleasure that I thank Suquamish Tribe Shellfish Policy Advisor Paul
Williams and animator Charlie Daugherty for inspiring and deepening my interest in
ocean acidification research.
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And to my family, Eric Roberts, Christine Roberts, Kyle Roberts, Mary Payne
Goodenough, Mary Carolyn Roberts, Dr. Shannon Roberts, Alexandra Goodenough, and
August Goodenough, and my enduring friends, I give my love and endless gratitude for
your patience, devotion, wit, and unwaveringly good advice.
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Finally, I would like to deeply thank Dr. Erin Martin, my thesis reader, for her
commitment, expertise, enthusiasm, and spot-on guidance throughout the thesis process.
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1. LITERATURE REVIEW
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1.1 Introduction
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Anthropogenic carbon emissions are increasing the global average concentration
of atmospheric carbon dioxide (CO2) and since the oceans and the atmosphere are
constantly exchanging gases and equilibrating, oceanic CO2 is rising at the same rate as
atmospheric CO2. CO2 and H2O chemically react to decrease the pH of seawater in a
process known as ocean acidification (OA). OA has the potential to impact marine life at
the individual, species, population, and ecosystem level, thereby causing deleterious
socioeconomic repercussions. The survival and fitness of marine organisms may be
affected physiologically by high-CO2 seawater via a number of processes such as
fertilization, growth, and behavior. By determining possible impacts of ocean
acidification on marine species through manipulative experiments exposing marine
organisms to high-CO2 seawater, models may be generated to better predict ecosystemwide impacts.
Accordingly, the research conducted in this thesis examines the behavioral
responses of Dungeness crab Metacarcinus magister zoeae to light, specifically
phototaxis, in high-CO2 conditions by using a chronic bioassay approach. To set the stage
as to why this work is important, this literature review will first review the literature
addressing the process of OA, and then analyze the current state of knowledge about
larval and crustacean behavioral response to elevated levels of CO2 in seawater along
with larval crab visual phototransduction and phototaxis. Finally, it will clarify how the
specific research approach employed in this study advances our understanding of the
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impacts of high-CO2 seawater on larval crustacean phototaxis.
1.2 Ocean acidification
Anthropogenic carbon emissions are changing Earth's chemical balance. Since
pre-industrial times, carbon emissions have elevated the atmospheric CO2 concentrations
from a global average of 287 ppm (Etheridge et al. 1996; Meehl et al. 2007) to an average
in October 2013 of 394 ppm (NOAA 2013, unpublished data). When air and water come
into contact, gases like CO2 are exchanged and equilibrated, and when atmospheric CO2
concentrations rise above oceanic CO2 concentrations, the oceans absorb CO2 to maintain
equilibrium (Doney et al. 2009b). The global ocean is a net sink for anthropogenic CO2
(Sabine et al. 2004). However, this relief of atmospheric CO2 does not come without
problems. Through a process known as “ocean acidification,” global marine life is
threatened with an environment that may be changing too rapidly for adaptation.
Data from 30 years of oceanographic cruises show that surface concentrations of
pCO2 in the North Pacific are increasing at the same rate as atmospheric CO2 levels
(Takahashi et al. 2006). This provides evidence for a stable rate of CO2 mixing between
seawater and the atmosphere. It also provides a solid link between anthropogenic carbon
emissions and the increases in dissolved inorganic carbon. Average global surface ocean
pH has already decreased by about 0.1, which corresponds to a 30% increase in hydrogen
ions, and the ocean pH is predicted to sink by another 0.3-0.4 units by the year 2100
(Feely et al. 2004; Orr et al. 2005; Doney et al. 2009b; Steinacher et al. 2009). Ocean
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acidification (OA) has the potential to change the function of ecosystems worldwide.
Analyses of global measurements of inorganic carbon show that nearly half of
anthropogenic carbon emissions is absorbed by the world's oceans (Sabine et al 2004).
When CO2 enters seawater, it reacts chemically with H2O to create carbonic acid
(H2CO3). Carbonic acid dissociates, increasing the concentration of hydrogen ions (H+) in
seawater and decreasing the concentration of carbonate ions (CO32-) through reactions
described by the chemical equation below (Doney et al. 2009b; Feely et al. 2010).
CO2 + H2O !" H2CO3 !" HCO3- + H+ !" CO32- + H+
This lowered pH is defined as an increase in hydrogen ion activity (Covington et al.
1985). A decrease in pH can cause a decrease in the saturation states of minerals
aragonite and calcite because a decrease in carbonate ions leads to a decrease in
saturation state. Aragonite and calcite are chemically identical since both are forms of
calcium carbonate (CaCO3), and the difference between the two is that the molecules
forming aragonite are less tightly packed than those in calcite, making them more prone
to dissolution in low-pH seawater (Feely and Chen 1982; Mucci 1983). Saturation state
(Ω) is the thermodynamic potential for a mineral to form or dissolve. The following
chemical reaction illustrates the dissolution and precipitation of calcium carbonate:
Ca2+ + CO32- !" CaCO3
The saturation state is defined as the product of calcium and carbonate concentrations
divided by the calcium carbonate concentration:
( [Ca2+] x [CO32-] ) / [CaCO3] = Ω
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A decrease in CO32- makes CaCO3 dissolution more likely. The ratio of the stoichiometric
solubility product (Ksp*) to carbonate (CO32-) primarily dictates the saturation state of
calcium carbonate minerals (Feely et al. 2010):
Ωarg = [Ca2+][ CO32-]/Ksp*arg
Ωcal = [Ca2+][ CO32-]/Ksp*cal
When the saturation state is falls below 1 (Ω < 1), minerals such as aragonite and calcite,
which are vital for the formation of calcified body parts of many marine organisms,
dissolve (Orr et al. 2005, Doney et al. 2009b, Feely et al. 2010). This puts marine
organisms at risk of being able to adequately complete their body plans. A number of
other physiological impacts, some of which may influence behavior, could occur as a
result of ocean acidification.
1.3 The California Current System and Puget Sound
This study focuses on the zooplanktonic larvae of M. magister, which inhabit
Northeast Pacific coastal surface waters within the California Current System (CCS) and
the Puget Sound (Pauley et al. 1989). Puget Sound is a fjordal estuary complex that may
be prone to rapid negative effects of ocean acidification since pH levels are low in
comparison with the global average oceanic pH due to both natural and anthropogenic
factors (Feely et al. 2010). OA in conjunction with existing chemical conditions in the
Puget Sound could have compounding effects on coastal and estuarine ecosystems in the
region (Feely et al. 2010). Natural factors such as coastal upwelling rom the CCS into the
Sound and biotic activity as well as anthropogenic factors such as nutrient inputs and
atmospheric nitric and sulfuric acid emissions can each impact the pH of Puget Sound,
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contributing to its vulnerability to ocean acidification (Doney et al. 2007; Feely et al.
2010). I will review each of these factors in the following paragraphs.
Natural biotic activity can lead to low pH levels in Puget Sound. Phytoplankton
blooms occur with greater frequency during summer months due to nutrient and sunlight
availability (Pitcher et al. 2010). Puget Sound experiences high rates of algal blooms due
to naturally high nutrient concentrations (MacFadyen et al. 2008). When phytoplankton
die, bacteria consume the carbon. With bacterial growth comes increased biotic
respiration, and CO2 seawater concentrations increase, leading to decreases in pH (Feely
et al. 2010). These natural CO2 production mechanisms predispose Puget Sound
ecosystems to vulnerability to ocean acidification.
Anthropogenic nutrient inputs can also contribute to phytoplankton blooms and
consequent increases in acidity. Due to the heavy urbanization of the Puget Sound Basin,
drainage can sweep pollutants, nutrients, and organic matter into the Sound (Feely et al.
2010). Because parts of this inland sea are characterized by sluggish circulation and
constrained flow, local nutrient inputs can have large impacts (Feely et al. 2010). Nutrient
inputs from development can contribute to algal blooms, which may lead to localized
decreases in pH as bacteria consume their biomass, as described above (Khangaonkar et
al. 2012; Feely et al. 2010).
Another key factor contributing to the low average pH of Puget Sound is the
natural acidity of the coastal waters of the CCS that feed this estuary. The CCS is
characterized by coastal seasonal upwelling, which brings to the surface cold, nutrientrich, low-pH, low-oxygen seawater with a low carbonate saturation state (Feely et al.
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2010; Gruber et al. 2012). Deep coastal northeast Pacific waters are low in pH since the
global ocean circulation system brings a deep current that downwells in the North
Atlantic, accumulates CO2 along the way, and upwells in the Northeast Pacific (Broecker
and Peng 1992). Natural conditions in the CCS are already among the most corrosive in
the world, with upwelling events carrying water with pH as low as 7.65 in deep coastal
Washington waters (Feely et al. 2008; Hauri et al. 2013). There are large variations in pH
spatially and temporally in the CCS. The current may rapidly be approaching a departure
from its current pH range (Hauri et al. 2013). Based on models, the average CCS pH
decreased from 8.12 to 8.04 between the years 1750 and 2005, and by the year 2050
under current emission rates, could decrease to a pH of 7.92 (Gruber et al. 2012).
In addition to these stressors to Puget Sound pH, local atmospheric non-CO₂
emissions may further acidify coastal waters. The combustion of biomass and fossil fuels
leads to atmospheric deposition of nitric and sulfuric acid in coastal ecosystems and
could further decrease seawater pH close to shore. This effect is likely to be more
significant in coastal waters than in the open ocean due to proximity to land-based
emissions. In coastal waters, atmospheric nitrogen and sulfur deposition could account
for 10-50% of anthropogenic acidification (Feely et al. 2010). Atmospheric nitrogen
efflux as well as nitrogen carried in freshwater to coastal oceans is estimated to increase
during the next few decades (Doney et al 2009a). These mounting impacts of natural
factors such as biotic activity and coastal upwelling, and anthropogenic factors such as
nutrient input and non-CO2 sources of acidification predispose Puget Sound to the
harmful effects of CO2-driven ocean acidification.
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Although M. magister inhabits the full range of the Pacific coast, the adult
specimen used in this study was collected in Puget Sound, Washington, so it is necessary
to understand the past, current and future pH levels of this estuary, particularly at the
surface. The range of surface pH levels in 2008 in Puget Sound was 7.72 to 7.95 (Feely et
al. 2010). The average overall pH of Puget Sound may be lower than this range since it
does not include deeper waters, which are generally more acidic than surface waters, and
since pH may have decreased in the years since 2008, In Hood Canal, dissolved inorganic
carbon was 54 µmol kg⁻¹ and 18 µmol kg⁻¹ higher than the average Admiralty Inlet
levels in the summer and the winter, respectively (Feely et al. 2010). This corresponds to
a 24-49% decrease in pH that can be accounted for by anthropogenic CO2 input since the
industrial revolution, while the rest is due to natural respiration (Feely et al. 2010).
Modeling future levels of pH in this basin is difficult due to the quantity of unknown
variables at hand (Feely et al. 2010). No comprehensive model of future pH variability
parameters within Puget Sound exists as of December 2013.
1.4 Metacarcinus magister natural history
Marine life is already exhibiting the impacts of ocean acidification (Doney et al.
2009b). Quantifying ocean acidification impacts on ecosystems is a complex task due to
the variability in organismal physiology and the magnitude of diversity. Some species
may proliferate in a low-pH ocean (Le Quesne et al. 2012), while others may become
extinct (Uthicke and Fabricius 2012; Dupont and Thorndyke 2009). In order to determine
the effects of ocean acidification on M. magister, a holistic account of the organism’s
natural history and vulnerability must be considered.
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The Dungeness crab Metacarcinus magister (Decapoda: Brachyura; formerly
known as Cancer magister) is a benthic invertebrate that inhabits the rocky sandy-mud
intertidal zone (Karpov 1983) along the west coast of North America from the Santa
Barbara Channel in California to the Aleutian Islands in Alaska, including Puget Sound
(Pauley et al. 1989). M. magister is distinguishable from crabs within its genus by a
pronounced tenth marginal carapace tooth (MacKay 1934). In Washington, M. magister
females extrude their eggs between October and December (Cleaver 1949). Females
brood the eggs on their pleopods until they hatch, which occurs between January and
April in Washington (Cleaver 1949). Zoeae typically hatch synchronously with high tide
(DeCoursey 1979) and once liberated, swim toward the surface and are transported
seaward by outgoing currents. In the zooplankton, zoeae molt through five different
larval stages, called instars, until they become megalopae and then settle as juvenile crabs
(Poole 1966). Megalopae and zoeae are important prey sources of Chinook salmon, pink
salmon, and coho salmon, as well as rockfish and herring, hence M. magister’s valuable
role in the pelagic ecosystem (Orcutt et al. 1976; Reilly 1983; Prince and Gotshall 1976).
The vulnerability of M. magister zoeae to ocean acidification can be characterized in
terms of larval vulnerability and crustacean vulnerability.
1.5 Vulnerability of larvae to OA
Early developmental stages of marine invertebrates are particularly vulnerable to
the effects of ocean acidification. Since pH tolerance varies by life stage, it is crucial to
test the response of embryonic, larval, and juvenile organisms to ocean acidification
(Kurihara 2008). It is presumably advantageous for larvae to minimize each stage of
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development in order to minimize time spent in the water column where these animals
are vulnerable to predation (Dupont and Thorndyke 2009). Any delays in development
caused by OA could lead to population-level impacts (Dupont and Thorndyke 2009).
Larvae are among the most vulnerable of life stages to predation due to their low trophic
level and small size. Indeed, predation is considered to be the most common cause of
mortality for planktonic larvae (Morgan 1995). During early developmental stages,
marine invertebrates have highly specific environmental requirements and small shifts in
seawater chemistry components such as pH can have large impacts (Thorson 1950;
Kurihara 2008). Many mollusk species are at risk of larval mortality or impaired
development due to ocean acidification (e.g. Timmins-Schiffman et al. 2013). However,
some crab species show enhanced growth and calcification in high-CO2 seawater (e.g.
Long et al. 2013), so calcification may be a lesser concern for crustacean larvae than
other physiological indices (Whiteley 2011).
1.6 Vulnerability of crustaceans to OA
1.6.1 Calcification
One of the most-studied impacts of ocean acidification on marine life is that of
impaired calcification and shell dissolution (e.g. Bednarsek et al. 2012). Studies on
brachyuran crabs have shown that calcification may not be as critical as other
physiological factors that could limit crustacean success in an acidified ocean (Ries et al.
2009). Cancrid crab and other crustacean exoskeletons have a higher ratio of calcite to
aragonite than do those of mollusks and echinoderms (Boßelmann et al. 2007). Aragonite
is more soluble than calcite, so other phyla are more susceptible to population threats
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based on calcification problems than are crustaceans (Whiteley 2011). Blue crab
Callinectes sapidus shows increased calcification with increased acidity (Ries et al.
2009). Barnacles may grow harder shells under highly acidified conditions (McDonald et
al. 2009). Crustacean physiological adjustment to changes in pH, such as internal acidbase regulation and behavioral impacts, may be a factor of greater concern than
calcification (Whiteley 2011). However, post-moult calcification in crustaceans could be
impacted by increases in seawater CO2. Calcification in crustaceans involves the uptake
of calcium (Ca2+) and bicarbonate (HCO3-) across the gills, and with increases in H+
concentration in seawater, HCO3- uptake may be slowed and the period of post-moult
calcification may be extended (Cameron 1985; Whiteley 2011). Delay in post-molt
calcification can leave crustaceans vulnerable to predation; thus, ocean acidification has
the potential to increase crustacean mortality rates (Whiteley 2011).
1.6.2 Physiology
When submerged, crustaceans are constantly in contact with seawater through the
gills, which exchange gases and ions with the surrounding environment (Taylor and
Taylor 1992). Aquatic organisms are more likely to be affected by changes in CO2
concentration than marine organisms since metabolic CO2 and HCO3- levels are generally
much lower in marine organisms than those in terrestrial organisms, leaving a smaller
buffer for changes in the concentrations of CO2 and HCO3- (Nilsson et al. 2012). Changes
in carbonate chemistry in the marine environment lead to a decrease in pH in the
seawater, which then decreases the pH in the extracellular compartment, or the
hemolymph (Whiteley 2011). Acid-base homeostasis is the process by which organisms
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maintain a pH that supports the functioning of processes such as respiration, protein
synthesis, and metabolism (Taylor and Whiteley 1989; Wheatly and Henry 1992). This is
accomplished through compensatory mechanisms, the foremost of which are the
carbonate buffering system and iono-regulation in crustaceans (Whiteley 2011). The
carbonate buffering system is a process in which organisms buffer increases in H+ by
incorporating excess H+ into HCO3- ions (Wheatly and Henry 1992). Iono-regulation,
which is the process by which carbonate buffering occurs, is the ion exchange of HCO3for Cl- and H+ for Na+ across the gill epithelia (Taylor and Taylor 1992; Whiteley 2011).
A slow metabolism is generally correlated with inefficient iono-regulation (Whiteley
2011). Exposure to pH decreases could affect crustacean growth, reproduction, and
behavior by channeling energy away from these functions and towards physiological
compensation for low pH (Whiteley 2011).
Adult M. magister specimens exhibit the ability to compensate for acid-base
disturbance in the haemolymph by efficiently iono-regulating during short-term (24 h)
exposure to extremely low-pH (7.08) seawater (Pane and Barry 2007). This study
suggests that iono-regulation of M. magister adults may not be a limiting factor to
survival. It is possible that M. magister larvae may be more susceptible to changes in
carbonate chemistry. A study on the effect of high-CO2 (1000 ppm) seawater on M.
magister larvae at Day 1 and Day 5 of development shows that these organisms may
exhibit increased swimming speed in CO2-acidified seawater, indicating a possible
metabolic effect (Christmas 2013). Feeding rates and gross growth efficiency were not
impacted by high-CO2 treatment (Christmas 2013). Red king crab Paralithodes
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camtschaticus larvae exhibited decreased yolk size in acidic conditions, longer zoeal
length at hatch, increased eye size, increased calcification, and decreased survival with
greater acidity (Long et al. 2013). In accordance with the suggestion of Doney et al.
(2009b) to administer longer-term laboratory experiments at realistic pH conditions,
additional physiological research is necessary to determine if and what detrimental effects
ocean acidification will bear on M. magister and other crustacean populations.
1.6.3 Behavior
Looking at behavior such as risk assessment and response to visual, olfactory, and
auditory cues is one way of assessing physiological impacts on crustaceans. Through
testing hypotheses about mechanisms for behavioral changes under pH stress, researchers
can elucidate how changes in pH can alter physiological responses, which in turn may
cause shifts in behavior.
One way to study crustacean behavior is through observing antennular flicking
rates, which correspond to olfactory perception. Antennular flicking rates in adult hermit
crabs are reduced at pH 6.8, which could be indicative of decreased metabolic rate or a
disruption to information gathering and processing (De la Haye et al. 2011). Hermit crabs
exposed to this high-CO2 treatment were also less likely to switch into suitable shells, a
behavior that relies on olfactory and visual cues, and to detect prey olfactory cues.
These adverse impacts of high-CO2 seawater indicate a disruption to olfactory
function. Further research indicates that this behavior may be mediated by an inability to
detect odor due to a direct effect of low pH on chemo-receptive function (De la Haye et
al. 2011, De la Haye et al. 2012), which could include neural disruption (Ferrari et al.
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2012). Since the olfactory sensory organs are congruent across the subphylum Crustacea,
similar responses and mechanisms can be tested in other crustacean species (Hallberg et
al. 1992). No studies as of December 2013 have addressed the response of larval
crustaceans to sensory cues.
Crustacean behavior, physiology, and calcification may be affected by ocean
acidification. An examination of the possible behavioral responses to low pH will
elucidate the possible impacts on these organisms. These include avoidance, metabolic
stress, and info-disruption, which is a general term for impairment of perception and
cognition by anthropogenic interference (Briffa et al. 2012, Lurling and Scheffer 2007). I
will demonstrate how these three different responses to low pH could manifest in
crustacean behavior.
One pathway by which organisms may alter their behavior due to increased CO2
concentrations in seawater is avoidance. In the case of a localized low-pH marine habitat,
which may occur naturally or anthropogenically, animals have the option of responding
to this low pH environment by avoiding the area through locomotion (Pörtner and Peck
2010). An example of a possible localized anthropogenic increase in CO2 concentration is
the proposed underwater CO2 storage sites that have the potential to leak and acidify a
pocket of seawater (Hawkins 2004). Intertidal organisms may detect high-CO2 conditions
and leave the water, which can be a costly energetic trade-off (Bibby et al. 2007, Briffa et
al. 2012, Murray et al. 2013; Amaral et al. 2013). On a long-term scale, as habitats
change, organisms adapted to those habitats may leave that region as conditions become
less favorable (Pörtner and Peck 2010), resulting in a restructuring of ecosystems.
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Organisms exposed to high-CO2 conditions have to cope with physiological
pressure, which can lead to changes in behavior. As described above, in high-CO2
conditions, marine organisms exert more energy maintaining acid-base homeostasis
through compensatory mechanisms involving the carbonate buffer system and ionoregulation, thus increasing the metabolic load (Page 10) (Whiteley 2011; Briffa et al.
2012). When energy is consumed by these processes, it is diverted away from vital
behavioral functions like reproduction, feeding, and aggressive behaviors that require a
high circulation rate (Briffa et al. 2012). This decrease in metabolic scope could have
population-level impacts.
A third proposed mechanism for behavioral impact of ocean acidification is
through the disruption of perception and cognition. Info-disruption can be associated with
the processing of visual (Ferrari et al. 2010), auditory (Simpson et al. 2011), and
olfactory (Dixson et al. 2010) cues. Olfactory cues are part of the process known as
chemoreception, in which odor molecules in seawater bind with receptor sites on the
olfactory sensory organs of marine organisms (Tierney and Atema 1988). Disruption to
perception due to ocean acidification may be explained by a change in the charge
distribution at odor molecule receptor sites, a change in the ionic state of odor molecules,
or physical damage to sensory organs (Briffa et al. 2012; De la Haye et al. 2012).
Disruption to cognition due to low pH could occur through damage to neural
mechanisms. The present study focuses on the potential impacts of low pH on the neural
processes of zoeae.
1.7 Zoeal phototaxis
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In the field and in the laboratory, swimming behavior of zoeae is influenced in
part by light. Larvae can respond to light through phototaxis and through photokinesis.
Phototaxis is the active change of the direction of an organism along the axis of a beam of
light (Fraenkel and Gunn 1940). Movement toward the light is known as positive
phototaxis, while movement away from the light is known as negative phototaxis (Diehn
et al. 1977). Photokinesis is a change in velocity of an organism when it is stimulated by
a change in light intensity regardless of direction (Diehn et al. 1977; Fraenkel and Gunn
1940; Crisp and Ghobashy 1971). The present study uses photokinesis to measure
phototaxis.
M. magister larvae, along with the majority of zooplankton, typically migrate
vertically towards the surface of the water column at night and farther down in the water
column during the day in order to avoid predation (Iwasa 1982; Reilly 1983). The depth
and timing of vertical migration varies with larval stage and environmental conditions
(Jacoby 1982; Sulkin 1984). Zoeae are negatively buoyant, meaning they sink when they
are not swimming (Foxon 1934; Spaargaren 1979). In order to regulate their vertical
position in the water column, they orient themselves using light, pressure, and gravity
(Sulkin 1984). The lower threshold for phototaxis, meaning the lowest level at which a
zoea senses light, acts as a barrier to upward migration during the day (Forward et al.
1984). M. magister zoeae phototax negatively at sunrise, and maintain depth during
daylight hours (Forward 1986; Hobbs and Botsford 1992). Although phototaxis likely
contributes to diel vertical migration, the primary influence on vertical migration may not
be phototaxis, but rather geotaxis, which is directional movement along the axis of
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gravitational pull (Sulkin 1984). In order to avoid the confounding factors of gravity and
pressure in the laboratory, researchers use horizontal aquaria with a horizontal beam of
light to isolate phototaxis (e. g. Adams and Paul 1999). Horizontal manipulative
photostimulation, which is the method employed in the present study, may result in
positive phototaxis, but not negative phototaxis (Forward et al. 1984). Observing
phototaxis in the laboratory can be useful for studying photophysiology, which is the
study of how light interacts with physiology, but is not analogous to vertical migration in
the field (Forward et al. 1984). In conditions simulating natural angular light
distribuation, zoeae exhibit negative phototaxis but not positive phototaxis (Forward
1986). Positive phototaxis in zoeae is a product of unnatural laboratory lighting
conditions (Forward 1986). I will describe the utility of chronic bioassay of zoeal
phototaxis in the laboratory setting by describing zoeal phototransduction.
1.8 Phototransduction
When a zoea receives a visual cue, light hits the cornea and lens of the compound
eye and travels through nerve cells to photoreceptors known as rhabdomes, where the
image forms. A series of neural fibers links the rhabdomes to the brain (Litzinger and Del
Rio-Tsonis 2002). Within the brain, synapses serve as information bridges between
neurons. Each neuron has one axon, which outputs information, and each axon has
several axon terminals, which send chemical signals called neurotransmitters through the
synapse from one neuron to the next (Stufflebeam 2008). The process of
phototransduction, or the conversion of a visual stimulus to a neural signal, differs
between invertebrates and vertebrates. Glutamate (Stuart 1999; Kolb et al. 2013), GABA#16
A (Førsgren et al. 2013) and dopamine (Burgess and Fero 2012) are implicated as
neurotransmitters in phototaxis in vertebrate brains, while histamine (Stuart 1999) and
possibly serotonin (Kain et al. 2012; Perrot-Minot et al. 2013) fill this role in arthropod
invertebrate brains. However, little is known about neurotransmitters involved in the
vision of invertebrates (Warrant and Nilsson 2006). Histamine has been identified as the
neurotransmitter associated with photoreception in many arthropod taxa (Stuart 1999).
Histamine is the neurotransmitter associated with photo reception in at least four
arthropod species: juvenile barnacle Balanus amphitrite (Stuart et al. 2007; Stuart et al.
2002), planktonic crustaceans Daphnia magna and Daphnia pulex (McCoole et al. 2011),
and the brown blowfly Calliphora stygia (Hardie 1989). However, some arthropods do
not employ histamine in photoreception, such as the copepod Calanus finmarchus
(Hartline and Christie 2010). Experimental evidence supports the function of serotonin
working in conjunction with histamine as the neurotransmitter involved in phototaxis in
Gammarus pulex, a freshwater amphipod (Perrot-Minot et al. 2013). Thus, the
neurotransmitter associated with photoreception and phototaxis in larval crab has not
been ascertained, but histamine is a likely candidate.
High seawater CO2 levels predicted to occur by the year 2100 clearly
detrimentally impact the behavioral responses of larval fish to olfactory cues as well as
lateralization, which is a direct measurement of brain function, due to the reversal of the
function of neurotransmitter GABA-A (Nilsson et al. 2012, Domenici et al. 2012).
GABA-A plays an important role in both vertebrate and invertebrate behavior (Tsang et
al. 2007) and could be a mechanism for general changes in zoeal behavior. The only
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invertebrate to my knowledge analyzed for the role of GABA-A in phototaxis is
Drosophila, the common fruit fly. It appears that Drosophila does not rely on GABA-A
for phototaxis (Leal et al. 2004), and fellow arthropod M. magister could have
homologous neurology. The proposed mechanism for impairment of vertebrate phototaxis
is the through the GABA-A receptors; however, this may not be applicable in invertebrate
photophysiology. Hence, further research on phototransduction in larval crabs would
benefit an understanding of the mechanism by which phototaxis in M. magister could be
affected by CO2-induced acidification. More information about the type of receptor
associated with photoreception in M. magister would provide researchers with the ability
to determine the mechanism of any effect of high-CO2 seawater on larval phototaxis.
Although no studies to my knowledge have been completed to date examining the
impact of ocean acidification on phototaxis in any invertebrate, studies on neural
disruption due to low pH indicate the possibility that ocean acidification could lead to an
altered behavioral response to light (Ferrari et al. 2012; Førsgren et al. 2013; Nilsson et
al. 2012). A bioassay on patterns of locomotion and phototaxis is a common and useful
manner in which to determine the impact of a physiologically challenging environment
on zooplankton (Whitman and Miller 1982). The bioassay employed in this study will be
chronic, or sublethal (Sprague 1969), as it is intended to investigate behavioral effects of
heightened levels of seawater pCO2 on larval crab phototaxis.
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2. INTRODUCTION
2.1 Identification of the problem
Since the industrial revolution, anthropogenic carbon dioxide (CO2) emissions
have elevated the atmospheric CO2 concentration from a global average of 287 ppm
(Etheridge et al. 1996, Meehl et al. 2007) to an average in October 2013 of 394 ppm
(NOAA 2013, unpublished data). The ocean and the atmosphere exchange gases and
equilibrate, and as atmospheric CO2 concentration increases, oceanic CO2 concentration
increases at the same rate (Doney et al. 2009b). Accordingly, analyses of global
measurements of inorganic carbon show that the world’s oceans absorb approximately
half of anthropogenic carbon emissions (Sabine et al 2004). When CO2 enters seawater, it
reacts chemically with H2O to create carbonic acid. Carbonic acid dissociates, increasing
the concentration of hydrogen ions in seawater (Doney et al. 2009b). This lowered pH,
which is defined as the negative logarithm of the hydrogen ion activity (Covington et al.
1985), can impact the physiology of marine organisms due to dependence on the
chemistry of the seawater in which they are immersed. This process of anthropogenic
reduction of global seawater pH is known as ocean acidification (OA).
Changes in pH may have broad effects on the physiology of marine organisms
(Fabry et al. 2008, Doney et al. 2009b). Numerous physiological indices in marine
organisms show a relationship with tolerance of pH changes, including metabolic rate
(Lannig et al. 2010), calcification (Smith and Buddemeier 1992, Kleypas et al. 1999),
survival (Chambers et al. 2013), reproduction (Miller et al. 2013), fertilization rate
(Barros et al. 2013), growth (Byrne et al. 2013), acid-base homeostasis (Stumpp et al.
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2012), and behavior (Dixson et al. 2010). As evidence from manipulative ocean
acidification simulation experiments is mounting, it is becoming clear that the effects of
OA will show variation within genera (Ferrari et al. 2011) and even among individual
organisms (Kelly et al. 2013). Research initiatives are predicting the ecological and
subsequent socioeconomic effects that ocean acidification will incur on the planet
(Harrould-Kolieb and Herr 2012). In order to make these predictions, researchers are
quantifying species-specific ocean acidification impacts and applying them to wholeecosystem models. For instance, the National Oceanic and Atmospheric Administration
(NOAA) is implementing the Atlantis Ecosystem Model (Kaplan et al. 2010) in order to
better understand the potential consequences of OA.
2.2 Behavioral impacts of OA
Acidified conditions can induce three pathways of behavioral response: infodisruption, avoidance, and metabolic stress (Briffa et al. 2012). Changes in metabolism
due to uptake of hydrogen ions into the interstitial fluid may lead to impaired behavioral
response in marine organisms (Briffa and Sneddon 2007). Acid-base homeostasis, which
maintains pH conditions in the interstitial fluid of invertebrates, demands more metabolic
energy as pH levels become stressful (Whiteley 2011). As metabolic load increases, the
energy available for other activities such as feeding and locomotion decreases, hence
altering behavior (Briffa et al. 2012). A second pathway for changes in behavior due to
shifts in CO2 concentrations is avoidance, since animals exposed to localized pH changes
may move away from these areas (Bibby et al. 2007, Pörtner and Peck 2010).
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Info-disruption, the anthropogenic impairment of sensory perception and cognition
(Lurling and Scheffer 2007), can be associated with the processing of visual (Ferrari et al.
2012), auditory (Simpson et al. 2011), and olfactory (Dixson et al. 2010) cues. Perception
may be disrupted through physical damage to sensory organs (Spicer et al. 2006, Munday
et al. 2009), and cognition may be disrupted through a variety of neural mechanisms,
reviewed below (Briffa et al. 2012).
2.3 Vulnerability of the study organism to OA
The Dungeness crab Metacarcinus magister (Decapoda: Brachyura) is a benthic
invertebrate that inhabits the rocky sandy-mud intertidal zone (Karpov 1983) along the
west coast of North America from the Santa Barbara Channel in California to the
Aleutian Islands in Alaska, including Puget Sound (Pauley et al. 1989). M. magister egg
clutches, which are held in the pleopods of the female crab, hatch between January and
April in Washington State (Cleaver 1949). The larvae, known as zoeae, join the
zooplankton, where they progress through five zoeal instars with a molt between each
stage until they become megalopae and finally settle as juvenile crabs (Poole 1966). The
Dungeness crab zoea is an ideal study organism for a chronic bioassay on phototaxis
since it is vulnerable to ocean acidification as a larval zooplankter, as a crustacean, as an
organism that demonstrates phototaxis, and as an ecologically and economically
important species. I will describe the vulnerability of the study organism to ocean
acidification by examining crustaceans, marine larvae, phototaxis, habitat, and
ecosystem.
2.3.1 Crustaceans
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The impact of ocean acidification on brachyuran crabs is poorly understood.
Acidified water breaks down calcium carbonate molecules like calcite and aragonite,
making these minerals less bioavailable for construction of body parts such as shells (Orr
et al. 2005). The carapace of brachyuran crabs is composed more heavily of calcite than
aragonite, which is a softer mineral than calcite (Boßelmann et al. 2007). Blue crab
Callinectes sapidus shows increased calcification with increased acidity (Ries et al.
2009). Red king crab Paralithodes camtschaticus larvae exhibited decreased yolk size in
acidic conditions, longer zoeal length at hatch, increased eye size, increased calcification,
and decreased survival with greater acidity (Long et al. 2013). There may be shifts in
calcium uptake and thermal tolerance of spider crab Hyas araneus with combined effects
of temperature and low pH (Walther et al. 2009; Walther et al. 2011). The vulnerability of
crustaceans to ocean acidification may lie not in calcification, but in other physiological
and behavioral impacts (Whiteley 2011). For instance, hermit crab Pagurus bernhardus
shows reduced antennular flicking rates in high-CO₂ water, which could indicate impacts
on metabolism or info-disruption (De la Haye et al. 2011).
2.3.2 Marine larvae
Biological ocean acidification impacts can vary with life stage (Fabry et al. 2008).
Larval marine organisms are particularly vulnerable to changes in seawater chemistry,
including pH (Kurihara 2008, Findlay et al. 2008, Chan et al. 2011). Reef fish larvae
swim towards olfactory and visual predator cues instead of away from them at high levels
of CO₂ (Dixson et al. 2010; Ferrari et al. 2012). The feeding behavior and food selection
of larvae of the Chilean abalone are detrimentally affected by decreases in pH (Vargas et
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al. 2013). These studies indicate that the larval stage of development is vulnerable to
behavioral impacts of ocean acidification in marine organisms.
2.3.3 Phototaxis
Larval zooplankton subsist at a low trophic level in the food web and are at high
risk of predation (Nelson 1925). Many species have evolved behavioral adaptations to
avoid this risk (Singarajah 1969, Forward 1977). Diel vertical migration is a behavioral
mechanism by which marine zooplankton avoid predation (Iwasa 1982). M. magister
zoeae migrate up in the water column as the sun sets and sink as the sun rises (Forward
1986). Predators can see the shadow from sunlight cast by these animals, so zoeae
exclude predation by other animals that occupy the surface waters by increasing depth in
the water column (Iwasa 1982). Zoeae sink in the water column in response to shadow
cues, which is thought to be a predator avoidance behavior (Forward 1977; Morgan
1987). Zooplankton respond differently to angular diffused light, which occurs in the
field, than they do to directional light in a laboratory setting (Veirhajen 1958; Forward
1986).
Phototaxis is the directional movement of an organism along the axis of a beam of
light (Fraenkel and Gunn 1940). Movement toward the light is known as positive
phototaxis, while movement away from the light is known as negative phototaxis (Diehn
et al. 1977). The biological function of the phototactic behavior of zoeae demonstrated in
a laboratory setting is unexplained, since zoeae generally demonstrate negative
phototaxis in the field and positive phototaxis in the laboratory (Forward 1986). It is
common to assess the sublethal impacts of a substance on larval crustacean behavior by
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studying phototaxis (Bartolomé and Sanchéz-Fortún 2005; Kolkmeier and Brooks 2013;
Wu et al. 1997), and the behavior of M. magister zoeae in a horizontal light chamber is
well understood (Jacoby 1982), thus providing a reliable index for behavioral change.
Visual cues are important in mediating the behavior of zoeae, in activities such as
predator avoidance, and any alteration in the ways in which zoeae process these cues has
the potential to bear fitness and survival effects. An understanding of zoeal phototaxis
under acidic conditions could gauge the impact of high-CO2 seawater on behavior of M.
magister zoeae and provide information about overall brain function and metabolic rate
through assessments of phototaxis and activity level. Impairment of behavior could lead
to population-level impacts in M. magister populations, which could have ecological and
socioeconomic repercussions.
2.3.4 Habitat
The chemical conditions of the habitat of the study organism, coastal northeast
Pacific and tidal estuaries such as Puget Sound, may have lower average pH than global
average oceanic pH (Feely et al. 2008). This is due to seasonal coastal upwelling, natural
biotic activity, and anthropogenic atmospheric and terrestrial emissions, as outlined
below (Doney et al. 2009a; MacFadyen et al. 2008; Feely et al. 2010). Upwelling of cold,
nutrient-rich, high-CO2 ocean water occurs as the global oceanic conveyer belt, which
downwells in the North Atlantic, accumulates CO2 from organic matter and upwells in
the Northeast Pacific (Broecker and Peng 1992). Based on models, the average CCS pH
decreased from 8.12 to 8.04 between the years 1750 and 2005, and by the year 2050
under current emission rates, could decrease to a pH of 7.92 (Gruber et al. 2012). Natural
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biotic activity in the form of algal blooms is common in Puget Sound, and when the
phytoplankton die off, their biomass is consumed by bacteria, which consume oxygen
and give off CO2, contributing to the acidity of the seawater (Feely et al. 2010). Since
Puget Sound is an estuary, anthropogenic nutrients such as nitrogen flow into the Sound
and often accumulate due to sluggish flow (Feely et al. 2010). Nutrient accumulation can
lead to increases in biotic activity and thereby decrease pH, as described above (Feely et
al. 2010; Khangaonkar et al. 2012). Anthropogenic terrestrial combustion of fossil fuels
near the coast can emit nitric and sulfuric acid, which are absorbed into the seawater and
further acidify the Puget Sound (Feely et al. 2010).
The pH experienced by zoeae may vary temporally during daily vertical
migrations and larval development (Long et al. 2013). The pH range of the seawater
surrounding the substrates in which M. magister broods is currently unknown, but it is
likely that this habitat is low in pH (Mathis et al. 2011). The vulnerability of the study
organism’s habitat to rapid changes in pH creates a need to understand the potential
impacts of ocean acidification on marine ecosystems in this region.
2.3.5 Ecosystem
The Dungeness crab is an ecologically important species as well as a
socioeconomic staple in western North America. M. magister zoeae are important prey
sources of Chinook salmon, pink salmon, and coho salmon, as well as rockfish and
herring, hence this species’ valuable role in the pelagic ecosystem (Orcutt et al. 1976;
Reilly 1983; Prince and Gotshall 1976). A change in the behavior of M. magister zoeae
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could shift population dynamics and have ecosystem-level and socioeconomic
implications.
2.4 Proposed mechanism of behavioral impairment
The neurotransmitter GABA-A, the main inhibitory transmitter in the vertebrate
brain, is implicated in CO2-induced aberrations in larval fish behavior such as excessive
risk-taking (Munday et al. 2010), boldness (Munday et al. 2010), hypersensitivity to light
(Førsgren et al. 2013), and inability to discriminate between ecologically sensitive
olfactory (Dixson et al. 2010), visual (Ferrari et al. 2012), and auditory (Simpson et al.
2011) cues. When seawater pH decreases, marine fish (Brauner and Baker 2009) and
crustaceans (Truchot 1975; Whiteley 2011) maintain acid-base homeostasis by increasing
bicarbonate uptake and releasing chloride into the seawater. An opening of the GABAgated chloride channel causes an influx of chloride ions, leading to a hyperpolarization
and inhibition of the neuron. An outflux of chloride, which may be caused by a decrease
in seawater pH, changes the ion gradient across the membrane of the receptor cell and
leads to depolarization and excitation, sending a neural signal to the brain (Nilsson et al.
2012). Førsgren et al. (2012) propose neural overexcitation as the mechanism for
increased speed to light in goby larvae under high-CO2 treatment. The neurotransmitter
responsible for phototaxis the brain of many amphipods, and is histamine (Stuart 1999).
Thus, overexcitation of the histamine-gated chloride channel in the zoeal brain could lead
to increased phototactic response in zoeae reared in high-CO2 seawater. This
hypersensitivity to light could be maladaptive and lead to expenditure of energy on weak
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or absent light cues (Bradbury and Vehrencamp 2011), with fitness and survival
consequences.
In order to determine the potential impacts of ocean acidification on larval
Dungeness crab phototaxis, I conducted a manipulative experiment simulating potential
future oceanic conditions. This chronic behavioral biossay examined the response of M.
magister zoeae to directional light in a laboratory setting when reared in three levels of
CO2-manipulated seawater. The present study represents the second study addressing the
impact of high-CO2 seawater on the behavior of a larval crustacean (see Christmas 2013),
the second study to address this impact on larval phototaxis (see Førsgren et al. 2013),
and the first to assess this impact on the phototactic behavior of a larval crustacean.
!
3. METHODS
3.1 Experimental system and carbon chemistry measurements
!
Zoeae were reared in an aquarium system at the Northwest Fisheries Science
Center ocean acidification laboratory (Seattle, WA), managed by the National Oceanic
and Atmospheric Administration. Seawater was collected from Elliott Bay in January of
2013 and housed in a reservoir at the laboratory. The reservoir is filtered to 1 µm to
remove organic particles, exposed to a UV filter which kills microbes and viruses, and
degassed using Liqui-Cel membrane contactors (Membrana, Weppertal, Germany), so
that the seawater re-enters the system free of CO2. A protein skimmer removes metabolic
waste compounds such as nitrates and nitrites. To prevent tank isolation, each treatment
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system continuously exchanges water with the system reservoir, with 100% turnover of
each treatment every day, essentially resetting seawater chemistry.
All materials used in the aquarium system were pre-soaked in seawater to remove
trace chemicals or impurities. Seawater flows from the reservoir through PVC pipes to a
holding tank, and then into a header tank containing the equipment necessary to control
seawater chemistry (see below). From each header tank, seawater flows into a series of
large boxes which are fitted with flow meters and water outputs to which rearing jars are
attached, floating in each box. The boxes act as water baths to keep the jars at constant
temperature. Seawater flows through the output of each rearing jar, into each box, and
back to the system reservoir. A 1-µm mesh bag filter was placed in front of the seawater
outflow of each rearing jar to prevent larval escape.
Seawater chemistry, specifically pH, is controlled at the header tanks by a
program built using LabView Software (National Instruments, Austin, Texas) and
maintained by bubbling one of five gas solutions (air, CO₂-free air, pure nitrogen, pure
carbon dioxide, or pure oxygen) through air stones. CO₂-free air is generated with CO₂
adsorbers, which capture the gas with a semi-permeable membrane (Twin Towers
Engineering, Broomfield, Colorado). The pH is controlled to a precision of 0.05 pH units.
Seawater salinity is monitored within header tanks with a Honeywell conductivity
probe and verified with discrete samples. Temperature, pH and dissolved oxygen in each
treatment are continuously measured with temperature probes, a pH probe (Durafet), and
a dissolved oxygen transmitter, respectively (Honeywell Process Solutions). The Durafet
pH probe in each experimental treatment continuously recorded pH and was calibrated
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with a pH-certified Tris buffer (Dickson Laboratory, Scripps Institution of Oceanography)
that is able to measure pH to a precision of 0.01 pH units. We verified pH, alkalinity,
and dissolved organic carbon conditions once daily with a spectrophotometer (Ocean
Optics USB 2000+ Fiber Optic Spectrometer) and m-cresol purple dye (Sigma Aldrich)
in all treatments tanks and 250 mL rearing jars. Verification of pH conditions in 4 L
rearing jars occurred only once and values were found to be accurate.
For this study, tanks were held at three pCO₂ levels: control (pCO₂ = 400 µatm;
pH = 8.2); mid-level (pCO₂ = 1600 µatm; pH = 7.6); and high-level (pCO₂ = 3200 µatm;
pH = 7.17). The temperature was held at 12° C in all treatments. Oxygen was
maintained at a 90% saturation level. Nutrients, bacteria, and phytoplankton were not
added to the system.
3.2 Specimen collection and larval rearing
Saratoga Passage, the site of specimen collection, is located in the Puget Sound, a
fjord in northwest Washington State, USA. The passage is situated between mainland
Washington and Camano Island. The NOAA dive team collected a gravid M. magister
individual on February 13, 2013 and held her at the NOAA Mukilteo Research Station
(Edmonds, WA) in a system consisting of a series of 1-m² lidless aquaria with flowthrough seawater held at ambient conditions from Saratoga Passage. The crab was fed
intertidal bivalves from Saratoga Passage and light conditions were ambient.
Using forceps, egg strands were extracted from the female on March 28, 2013, at
the NWFSC, after 43 days in captivity, using forceps (Wickham 1979). Egg strands were
extracted from haphazardly selected locations within the egg clutch. Eggs were placed
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haphazardly in flow-through jars (250 mL CO₂-impermeable-PET plastic) on PVC
manifolds in the aquarium system and incubated at the three levels of pCO₂ described
above. Zoeae selected for the behavioral experiment hatched on April 4 and April 5, after
a period of 8 to 9 days of incubation. No handling of eggs occurred during incubation
aside from removal of jar lids to determine hatch state. To control for photoperiod
exposure, tanks were covered in black plastic at the end of each work day and uncovered
at the beginning of each work day.
Upon hatching, zoeae were moved to larger plastic flow-through jars (4 L) with a
flow rate of approximately 6 L/hr. A total of 150 newly hatched zoeae were placed in
each jar. There were 2 jars in each CO₂ treatment; one for each hatch day. Zoeae that
hatched on April 4 were held separately from zoeae that hatched on April 5. Every third
day, water in jars was changed and zoeae were fed Artemia salina nauplii (1 nauplius /
mL). Behavioral tests were conducted 21 days after hatch date. Animals that hatched on
April 4 were tested on April 25, and animals that hatched on April 5 were tested on April
26. Molt data during rearing was not recorded, so zoeal instar at time of behavioral test
cannot be ascertained. However, it is likely that the zoeae were in the second instar since
in M. magister, the first zoeal molt occurs at approximately day 18, and the second molt
occurs at approximately day 29 (Poole 1966).
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Photograph of behavioral test setup.
Figure 1. Lateral view of experimental aquarium in behavioral test setup. (A) LED bulb
in PVC housing. (B) PVC pipe used to ensure uniform release of zoeae. (C) String used
to lift PVC and release zoeae. (D) Digital video camera for image recording. Star
indicates position of light.
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3.3 Behavioral test
!
To understand how elevated CO₂ conditions impact zoeal phototaxis, larvae were
exposed to light and control treatments. A total of sixty larvae were tested. Twenty larvae
from each CO₂ treatment were tested, ten of which were exposed to a light treatment and
ten of which were exposed to a control treatment. Each zoea was tested exactly once. In
order to ensure that the researcher remained unbiased during behavioral tests and
analyses, colleagues haphazardly rearranged the lids of the 450 mL jars containing each
individual zoea before each time trial. In addition, a colleague recoded each video file at
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random to ensure the researcher was blind to CO₂ treatment. Larval behavior was
recorded for each 3 minute time trial by using a video camera (see below). Zoeae were
transferred from 4 L cultures to 450 mL plastic jars by selecting zoeae that swam to the
top of the 4 L jar upon removal of the bag filter. This selection criterion increased the
likelihood that the swimming behavior of zoeae was uniform. A single zoea was placed in
each of six jars. Zoeae were tested individually but pulled from the flow-through system
in batches of six. Each batch constituted a time trial. Jars were held in a water bath
between removal from flow-through system and behavioral test at 12° C for no more than
90 minutes during each time trial. The time elapsed between removal and testing ranged
from 6 minutes to 77 minutes. To determine any effect on behavior based on time away
from flow-through system, the time at which each individual zoea was removed from the
flow-through system was recorded and subtracted from the time at which it was tested in
order to calculate time elapsed.
The study took place in a darkened room. To minimize disturbance, the researcher
remotely filmed the trials at a distance of 1.5 m. Video data was collected using a
Chameleon digital camera (Point Grey Research Inc.) and recorded using FlyCapture2
(Point Grey Research Inc.). One LED lightbulb, housed within PVC fittings, was placed
2.3 cm from the edge of the Plexiglass box. Neutral density photo filters were layered to
manipulate brightness. Luminosity was measured at 3.7 lumens using HOBOware Data
Logger software with a PAR sensor placed before the photo filters.
The behavioral test consisted of the exposure of each zoea either to the LED light
treatment or to a dark control treatment. To ensure that each zoea was initially placed at
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an equal distance from the light source, each zoea was placed in a PVC pipe (9 cm long,
1.5 cm diameter) within a Plexiglass box (Figure 1) containing 450 mL of seawater (26.5
cm long, 11.5 cm wide, 1.4 cm deep) from one of the three CO₂ treatments. The LED
light was activated remotely, and the PVC pipe was lifted remotely to release zoea 11.2
cm from the light, slightly offset from the center of the box. Larvae were filmed for 3
minutes after an acclimation period of 30 seconds. The film captured the movement of
each zoea during each test. Each zoea was tested exactly once. Larval preference of side
of aquarium (right side vs. left side) was tested during preliminary trials and found to
have no effect on whether the zoeae swam to the light.
3.4 Video analysis
A total of 59 videos were analyzed. Twenty individuals from each CO₂ treatment
were tested with ten larvae exposed to light treatment and ten larvae exposed to a dark
control treatment. One video (3200 ppm pCO₂ in the light treatment) was lost.
Video analysis was conducted using ImageJ (NIH Image) software. Video files
were decimated by 30 frames to condense files using the program VirtualDub
(VirtualDub.org) yielding a total of 90 frames for each 3-minute video, which were then
analyzed. As a result of this treatment, the temporal difference between consecutive
frames was 1.9987 seconds.
The location of the zoea in each frame was recorded in pixel coordinates using
ROI Manager (ImageJ, NIH Image). Due to glare from the infrared lighting system, there
were blind spots in each video. When zoeae swam into the blind spots, pixel coordinates
were not recorded.
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Using ImageJ and Adobe Photoshop (Adobe Systems Incorporated), a rectangle
was superimposed on top of each frame 1.39 cm left of the wall of the aquarium adjacent
to the LED bulb (Figure 2). The absence or presence of the zoeae in the rectangle in each
frame was recorded to denote proximity to light.
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Demarcation of rectangular area in aquarium closest to light.
Figure 2. Overhead view of experimental aquarium. Fisheye lens in video camera
caused image bowing. Rectangle outlined in red was used to record zoeal position,
providing index of approach of light. Star indicates position of light.
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3.5 Statistical analysis
Pixel coordinate data were processed to determine the overall speed and speed to
light. This study will use the term “light” in reference to “the space closest to the light,”
as a shorthand. Grid cell data were processed for approach of light, speed to light, time to
light, proportion of time at the light, and proportion of time away from the light once the
light was reached.
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Overall swimming speed
Overall swimming speed (cm/s) is defined as the centimeters per second traveled by each
zoea over the course of the 3-minute video.
Control group: A one-way ANOVA was conducted across all three CO2 treatments within
the control group to isolate the overall activity level outside the context of photokinesis.
Light treatment group: A one-way ANOVA was conducted exclusively among zoeae
exposed to the light treatment across CO2 treatments.
Approach of light
Approach of light is defined as a yes or no (0, 1) response to the question of whether the
zoea reached the area closest to the light.
Control vs. light treatment: A one-way ANOVA analyzing entrance of area closest to the
light was run across all zoeae to compare the control treatment against the light treatment.
CO₂ treatments: A one-way ANOVA was run across all zoeae exposed to the light
treatment to compare CO2 treatments.
Speed to light
Speed to light is defined as the centimeters per second traveled from the pixel coordinates
marked on the first frame of each video to those marked on the first frame in which the
zoea entered the area closest to the light. A one-way ANOVA was run to determine
variance across CO2 treatments among zoeae that reached the area closest to the light.
Time to light
Time to light is defined as the time (in seconds) that passed between the first frame of
each video and the first frame in which the zoea entered the area closest to the light. A
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one-way ANOVA was run among all zoeae that were exposed to light treatment and
reached the light.
Proportion of time at light
Proportion of time at light is defined as the ratio of time (in seconds) each zoea spent at
the light to the time each zoea spent elsewhere. A one-way ANOVA was run across CO2
treatments among all zoeae that were exposed to light treatment and reached the light.
Proportion of time spent away from light once light was reached
Proportion of time spent away from light once light was reached is defined as the ratio of
time (in seconds) each zoea spent away from the light after the light was reached to the
time each zoea spent at the light. This metric shows how the tendency to stay at the light
varies across CO2 treatments. A one-way ANOVA was run across CO2 treatments among
all zoeae that were exposed to light treatment and reached the light.
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4. RESULTS
4.1 Overall swimming speed
4.1.1 Control group
There was no significant difference in overall swimming speed among control
CO2 treatments (n = 30, F (30, 3) = 0.4777, p = 0.6253). However, a linear pattern is
exhibited in mean overall swimming speed across CO2 treatments, in which mean overall
swimming speed decreases as CO2 levels increase (Figure 3). The mean overall
swimming speed for the 400, 1600, and 3200 ppm pCO2 treatments in the control group
was 0.23 ± 0.14 cm/s, 0.20 ± 0.09 cm/s, and 0.179 ± 0.12 cm/s, respectively.
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Effect of pCO2 level on overall swimming speed in the control group.
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Figure 3. A one-way ANOVA shows no significant impact of CO₂ treatment on overall
swimming speed in the control group (n = 30, F (30, 3) = 0.4777, p = 0.6253). A very
weak, non-significant trend suggests a decrease in overall mean swimming speed with
increased CO2 level. Green lines indicate mean. Red lines within box plots indicate
median.
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4.1.2 Light treatment group
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There was no significant difference in overall swimming speed among CO2
treatments for zoeae exposed to light (n = 29). However, a linear pattern in mean overall
swimming speed is exhibited across CO2 treatments, in which mean overall swimming
speed decreases as CO2 level rises, similar to the control group (Figure 3). The mean
overall swimming speed for the 400, 1600, and 3200 ppm pCO2 treatments was 0.19 ±
0.08 cm/s, 0.18 ± 0.12 cm/s, and 0.17 ± 0.11 cm/s, respectively. This corresponds to a 5%
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and 14% greater mean overall swimming speed for zoeae in the 400 ppm pCO2 treatment
relative to animals from the 1600 and 3200 ppm pCO2 treatments, respectively.
4.2 Approach of light
4.2.1 Control vs. light treatment
Zoeae exposed to a control treatment (n = 30) were significantly less likely to
swim to the light than those exposed to the light treatment (n = 29) (χ² (1, n = 59) = 26.8,
p < 0.0001) (Figure 4). In 2 out of 30 trials, zoeae exposed to a control treatment swam to
the light. In 24 out of 29 trials, zoeae exposed to the light treatment swam to the light.
Accordingly, zoeae exposed to a control treatment swam to the light 7 ± 25% of the time
and zoeae exposed to the light treatment swam to the light 83 ± 38% of the time.
Effect of light treatment on proportion of zoeae that reached light.
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Figure 4. A one-way ANOVA shows a significant effect of light treatment on proportion
of zoeae that approached the light (χ² (1, n = 59) = 26.8, p < 0.0001). Each error bar was
constructed using 1 standard deviation from the mean.
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4.2.2 CO₂ treatment
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CO2 treatment was not a significant effect on whether the zoea exposed to the
light treatment swam to the light (χ² (2, N = 29) = 0.5518, p = 0.5601) (Figure 5). Zoeae
reared in 400 ppm pCO2 seawater swam to the light 8 out of 10 times, whereas those in
the control treatment swam to the light 1 out of 10 times. Zoeae reared in 1600 ppm pCO2
seawater swam to the light 9 out of 10 times, whereas animals in the control treatment
entered swam to the light 0 out of 10 times. Zoeae reared in 3200 ppm pCO2 seawater
swam to the light 7 out of 9 times, whereasthose in the control treatment swam to the
light 1 out of 10 times (Table 1). Accordingly, zoeae reared in 400 ppm pCO2 seawater
swam to the light 80 ± 12% of the time. Zoeae reared in 1600 ppm pCO2 seawater swam
to the light 90 ± 12% of the time. Zoeae reared in 3200 ppm pCO2 seawater swam to the
light 78 ± 13% of the time.
Effect of pCO2 level on proportion of zoeae that reached light.
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Figure 5. A one-way ANOVA shows no significant impact of CO2 treatment on
whether zoeae reached the light (χ² (2, N = 29) = 0.5518, p = 0.5601). Each error bar was
constructed using 1 standard deviation from the mean.
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Number of zoeae that reached area closest to the light
by CO2 treatment and light treatment.
pCO₂ treatment (ppm)
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# individuals reached
# individuals reached
light out of # videos ana- light out of # videos analyzed (light)
lyzed (control)
400
8 out of 10
1 out of 10
1600
6 out of 10
0 out of 10
3200
7 out of 9
1 out of 10
Table 1. Number of individual zoeae that reached the area closes to the light out of
number of videos analyzed, in light and control (dark) treatments.
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4.3 Speed to light
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A one-way ANOVA was conducted among CO2 treatments to examine the speed
to light. The individuals analyzed were from the non-control group that reached the light
(n = 24). There was no statistically significant difference in speed to light among CO2
treatments (F (24, 2) = 0.8347, p = 0.4479). The mean speed to light for zoeae reared in
400, 1600, and 3200 ppm pCO2 seawater were 0.32 ± 0.24 cm/s, 0.33 ± 0.34 cm/s, and
0.58 ± 0.64 cm/s, respectively. The mean speed to light for zoeae reared in the 3200 ppm
pCO2 treatment was 44% and 42% greater than that of the animals reared in the 400 and
1600 pCO2 treatments, respectively (Figure 6).
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Effect of pCO2 level on speed to light.
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Figure 6. A one-way ANOVA shows no effect of CO2 level on speed to light (F(24, 2) =
0.8347, p = 0.4479). A weak, non-significant trend indicates an increase in speed to light
with increased CO2 level. Green lines indicate mean. Red lines in box plots indicate
median.
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4.4 Time to light
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For zoeae that reached the light (n = 24), pCO2 treatment was not a statistically
significant effect on the time taken for the zoea to reach the light (F (24, 3) = 0.1989, p =
0.8211) (Figure 7). The mean amount of time (in seconds) for zoeae to reach the light for
animals reared in 400, 1600, and 3200 ppm pCO2 seawater was 55 ± 52 s, 68 ± 53 s, and
51 ± 60 s, respectively.
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Effect of pCO2 level on time to light.
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Figure 7. A one-way ANOVA shows no effect of CO2 level on time to light (F (24, 3) =
0.1989, p = 0.8211). Green lines indicate mean. Red lines in box plots indicate median.
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4.5 Proportion of time at light
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For all zoeae exposed to light (n=29), there was no significant effect of CO2
treatment on proportion of time spent at light (F (29, 2) = 0.1429, p = 0.8675). The mean
proportion of time spent at light for zoeae reared in 400, 1600, and 3200 ppm pCO2
seawater was 0.57 ± 0.37, 0.53 ± 0.35, and 0.56 ± 0.37, respectively.
4.6 Proportion of time away from light once light was reached
For all zoeae that reached the light (n=24), there was no significant effect of CO2
treatment on proportion of time spent away from the light once the light was reached (F
(24, 2) = 0.6159, p = 0.5496). However, a relationship is shown in which proportion of
time spent away from the light once the light was reached increases as CO2 treatment
decreases (Figure 8). The mean proportion of time spent away from the light once the
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light was reached for animals reared in 400, 1600, and 3200 ppm pCO2 seawater was
0.15 ± 0.06, 0.07 ± 0.05, and 0.08 ± 0.06, respectively. The mean proportion of time
spent away from the light once the light was reached for zoeae reared in 400 ppm pCO2
seawater was 55% and 50% greater than those reared in 1600 and 3200 ppm pCO2
seawater, respectively.
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Effect of pCO2 level on proportion of time away from light once light was reached.
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Figure 8. A one-way ANOVA shows no effect of CO2 level on proportion of time away
from light once light was reached (F (24, 2) = 0.6159, p = 0.5496). However, very weak,
non-significant variance indicates that proportion of time away from light once the light
was reached may be greatest in the 400 ppm pCO2 treatment. Green lines indicate mean.
Red lines in box plots indicate median.
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5. DISCUSSION
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The results of the present study corroborate preceding data (Jacoby 1982)
supporting positive phototaxis in the second zoeal instar of Metacarcinus magister. No
significant results indicate a relationship between CO2-induced acidification and
phototaxis, lightly suggesting that there is no immediate concern for adverse impacts of
ocean acidification on phototaxis in second instar M. magister zoeae. Non-significant
trends suggest that zoeae under high-CO2 treatment may demonstrate heightened
phototaxis, measured by an increase in mean swimming speed to light. Zoeae in this
treatment may also be more likely to stay at the light once the light is reached. Another
non-significant finding indicates that activity level, as measured by mean overall
swimming speed, may fall with increased acidity. First, I will discuss the literature
supporting a possible change in phototactic response due to CO2-induced seawater
acidification. Then, I will review my findings in the context of the literature.
5.1 Phototaxis vs. photokinesis
Response to light, which is a complex behavior dependent on a number of factors,
can be interpreted as phototaxis and as photokinesis. Phototaxis is the active change of
the direction of an animal along the axis of the source of a beam of light (Fraenkel and
Gunn 1940, Diehn et al. 1977). Movement towards the light is called positive phototaxis,
while movement away from the light is known as negative phototaxis. Phototaxis
determines what direction the zoea will take when it swims. Photokinesis, on the other
hand, is a change in velocity of an organism when it is stimulated by a change in light
intensity regardless of direction (Diehn et al. 1977, Fraenkel and Gunn 1940, Crisp and
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Ghobashy 1971). Light is only one of the internal and external factors that may influence
phototactic and photokinetic behavior (Sulkin 1984). Both phototaxis and photokinesis
may be influenced by light wavelength, intensity, and prior exposure of the study
organism to light (Sulkin 1984). The behavior observed in the present study may be
deemed positive phototaxis based on the statistically significant tendency of zoeae to
swim toward the light more often when the light was activated. This study did not address
photokinesis, which measures a change in swimming speed as response to a change in
light intensity (Sulkin 1984), since the light intensity was static throughout each
behavioral test once the light was activated.
5.2 No effect of CO₂ treatment on behavior
There are several explanations for the observed lack of effect of CO2 treatment on
larval activity and phototaxis. The large variability in natural pH conditions both at the
sea floor (Long et al. 2013) and in the water column (Feely et al. 2010) could predispose
these animals to tolerance of low pH. However, organisms such as brachyuran crabs
(Whiteley 2011) that are well equipped to chemically compensate for acid-base
disruption may experience heightened risk of behavioral impacts due to an uptake of
bicarbonate ions and a reduction of chloride ions, described below (Nilsson et al. 2012,
Munday et al. 2012). My results suggest that there is no effect of heightened CO2-induced
acidity on larval phototaxis; however, this result is non-significant and additional studies
with larger sample sizes are necessary to determine this effect.
5.3 Proposed mechanism for increased phototaxis
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The expected response to directional light in a horizontal chamber for M. magister
zoeae is to swim toward the light in demonstration of positive phototaxis (Jacoby 1982).
A mechanism for possible CO2-induced increase in phototaxis in a larval crustacean has
not yet been proposed. Two studies, both on larval fish, have been conducted on the effect
of high CO2 on the visual information gathering of marine organisms and may elucidate
this mechanism (Ferrari et al. 2012, Førsgren et al. 2013). Larval damselfish
Pomacentrus amboinensis reared in high CO2 (850 ppm) seawater that were exposed to
predators contained in plastic bags, hence a presumed isolated visual cue, swam toward
predators more often than fish larvae reared in control seawater (Ferrari et al. 2012). A
study on the effect of high CO2 on larval temperate goby Gobiusculus flavescens
demonstrates increased phototactic response, via heightened speed to a directional light
source, under increased CO2 at 1400 ppm (Førsgren et al. 2013). The mechanism for any
disruption of response to visual cues could lie in perception or cognition. Disruption of
visual perception due to low pH could occur through physical damage to sensory organs
(Munday et al. 2009); however, physical damage to visual sensory organs in low-pH
seawater has not been assessed. Examination of sensory organs shows that physical
damage is not a source of info-disruption in the olfactory (hermit crab Pagurus
bernhardus, De la Haye et al. 2012) or auditory system (clownfish Amphiprion percula,
Munday et al. 2009) as a result of increased acidity. Larval fish become more likely to
take risk when olfactory, auditory, and visual cues are each isolated, indicating a neural
mechanism rather than physical damage (Ferrari et al. 2012). Impairment of cognition
through neural disruption is more likely than impairment of perception through physical
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damage to sensory organs (Ferrari et al. 2012). Reversal of the function of the
neurotransmitter GABA-A, the main inhibitory transmitter in the vertebrate brain, is
implicated in CO2-induced aberrations in larval fish behavior such as excessive risktaking (Munday et al. 2010), boldness (Munday et al. 2010), hypersensitivity to light
(Førsgren et al. 2013), and inability to discriminate between ecologically sensitive
olfactory (Dixson et al. 2010), visual (Ferrari et al. 2012), and auditory (Simpson et al.
2011) cues. When seawater pH decreases, marine fish (Brauner and Baker 2009) and
crustaceans (Truchot 1975; Whiteley 2011) maintain acid-base homeostasis by increasing
bicarbonate uptake and releasing chloride into the seawater. An opening of the GABAgated chloride channel causes an influx of chloride ions, leading to a hyperpolarization
and inhibition of the neuron. An outflux of chloride, which may be caused by a decrease
in seawater pH, changes the ion gradient across the membrane of the receptor cell and
leads to depolarization and excitation, sending a neural signal to the brain (Nilsson et al.
2012). Førsgren et al. (2012) propose neural overexcitation as the mechanism for
increased speed to light in goby larvae under high-CO2 treatment.
Zoeae reared in high-CO2 seawater exhibited greater speed to light than the other
two treatments. Although this effect was not significant, is reflected in the mean overall
speed of zoeae exposed to light, since the animals in the high-CO2 treatment spent the
largest proportion of time stationary at the light than any other group, driving down the
mean overall speed. The observed non-significant increases in speed to light and
tendency to stay at the light among zoeae in the 3200 ppm pCO2 treatment may be
explained by overexcitation of photoreceptor neurons in the brain of the larval crab. The
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neurotransmitter associated with phototaxis in larval crab has not been ascertained, but
histamine and serotonin are likely candidates (Page 16). Neuronal depolarization causes a
release of histamine, which binds to histamine receptors on the dendrite of the neuron
(Stuart et al. 2007). Continuous light stimulation causes continuous histamine release,
and an increase in light stimulation causes an increase in histamine release (Stuart et al.
2007). Increased influx of chloride into the histamine-gated chloride channel could lead
to overexcitation of the neuron and increased phototactic response. Thus, glutaminergic
or GABAergic neurons in vertebrate brains may be depolarized as a result of high CO2
seawater rather than a light cue, causing overexcitation and behavioral hypersensitivity
(Nilsson et al. 2012). In invertebrate brains, an analogous process may occur in
histaminergic neurons.
Hypersensitivity to light could affect survival and fitness of zoeae. Maladaptive
impulses may lead organisms to exert excessive energy in responding to weak or absent
light stimuli (Bradbury and Vehrencamp 2011), thus detracting from processing
biologically important visual cues necessary for diel vertical migration and predator
avoidance. Failure to avoid predators could lead to increased larval mortality and shifts in
populations (Morgan 1995), thus altering ecosystem dynamics and potentially impacting
commercially important salmon populations. However, the observed increase in speed to
light under high-CO2 conditions is non-significant and further tests are necessary to
elucidate the effect of high-CO2 seawater on zoeal phototaxis.
5.4 Implications for metabolic rate
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A non-significant trend demonstrating reduced overall mean speed with increased
acidity in the control group may be indicative of reduced metabolic rate. Adult hermit
crabs show a reduction in locomotion when exposed to high CO2, which could be due to
an increased metabolic load as the organism puts more energy into acid-base homeostasis
(De la Haye et al. 2012). See Page 10 for a review of the literature on physiological
mechanisms of acid-base regulation in crustaceans. Another explanation for the decreased
locomotion shown in hermit crabs is the lack of olfactory stimulation, which would be
due to info-disruption (De la Haye et al. 2012) (see Page 14). However, info-disruption
could not be a valid explanation for decreased zoeal locomotion at high CO2 levels in the
control treatment since they were not exposed to a visual cue at all. M. magister zoeae
incubated in 1000 ppm pCO2 seawater demonstrated heightened swimming speed in dark
conditions than animals incubated in 400 ppm pCO2 seawater (Christmas 2013),
potentially indicating that high-CO2 seawater may increase larval swimming speed,
which could indicate an increase in metabolic rate rather than a decrease. The nonsignificant finding presented in the present study contradicts the results of Christmas
(2013); however, larvae in the present study were tested 21 days after hatching while
larvae tested by Christmas (2013) were assessed 1 day after hatching. Thus, divergent
trends may be expected due to differential behavior among M. magister zoeal instars. For
instance, the second zoeal instar of M. magister may exhibit reduced phototaxis
compared to the first zoeal instar (Jacoby 1982).
Metabolic depression is a potential mechanism for the decreased locomotion
exhibited by zoeae in the control group in the present study. An analysis of the respiration
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rates of zoeae incubated in varying CO2 treatments would provide more information
about the impact of ocean acidification on zoeal metabolism. The zoeae of northern
shrimp show increased metabolism with the combined effect of temperature increases and
low pH, but not with the sole factor of low pH. This increase in metabolism could lead to
higher maintenance costs and change populations and ecosystems (Arnberg et al. 2013).
The effects of low pH on the metabolism of crab zoeae are currently unknown.
5.5 Recommendations for future research
Temporal restraints during the behavioral bioassay prevented me from using a
larger sample size. This study assessed the behavior of a singular larva at a time, whereas
other studies (Jacoby 1982; Førsgren et al. 2013) on larval phototaxis have observed up
to ten larvae in a single test, which is a method that has a greater potential to yield
significant results in behavioral bioassays.
Information about the pH range to which zoeae are exposed and adapted in the
field, including the benthic zone where crab embryos develop, would inform the pCO2
levels used in future research. Studies on the effect of CO2-induced acidification on the
respiration rate of crab larvae may provide information about the metabolic rate of these
animals and inform future behavioral studies. Examination of the effect of high CO2 on
phototaxis across brachyuran crab species may provide insights as to which species will
be affected behaviorally by high CO2 and predict ecosystem dynamics.
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6. CONCLUSION
The findings of this chronic bioassay of high CO2 seawater on larval phototaxis
lightly suggest that there is no dramatic effect of ocean acidification on the phototaxis
and activity level of Metacarcinus magister zoeae. However, trends that are not
statistically significant indicate that M. magister zoeae reared in 3200 ppm pCO2
seawater may show heightened phototaxis when exposed to directional light in a
horizontal chamber and decreased overall activity level in the dark. Additional studies are
necessary to further assess the effect of high-CO2 seawater on larval crab.
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7. INTERDISCIPLINARY STATEMENT
This thesis research addresses an interdisciplinary problem in which the fields of
physiology and environmental change research intersect with implications for ecology
and socioeconomics. Specifically, the present study provides information about the
potential behavioral impacts of anthropogenic carbon dioxide emissions on the most
economically important crab species on the U. S. west coast, the Dungeness crab.
Biological restrictions prevent this species from being produced in aquaculture
enterprises, so the fishery depends on wild-caught Dungeness crab for revenue. As such,
the changes in wild Dungeness crab population size that may result from impacts on
larval behavior could impact harvesters and consumers throughout the range of this
species. In addition, trophic effects of changes in larval behavior could alter salmon and
other finfish populations, leading to ecological, cultural, and economic shifts.
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The continuing accumulation of anthropogenic atmospheric carbon dioxide is
already drastically altering terrestrial and aquatic ecosystems globally, and models
indicate further change is inevitable. Various collaborative organizations among
scientists, policy makers, economists, tribes, non-governmental organizations, fishers,
and other stakeholders, such as the Blue Ribbon Panel on Ocean Acidification led by
former Governor of Washington Christine Gregoire, are identifying and pursuing
research, policy, and education goals that will best prepare communities in the Pacific
Northwest to adapt to coming marine ecosystem changes. Washington is a progressive
state in terms of climate change awareness and is actively working towards a zero
emissions goal for 2050. Great obstacles remain in the path to ocean acidification
mitigation. Nonetheless, ocean acidification is a growing concern that is gaining
awareness both locally in the Pacific Northwest and globally. Research on ocean
acidification impacts on marine life will allow those who depend on food from the sea to
plan ahead for ecosystem shifts.
The results presented here indicate that there may be no effect of ocean
acidification on the phototactic behavior of Dungeness crab larvae, which bodes well for
those that depend on this species for livelihood. However, further research is needed in
order to assess this impact. This thesis provides information that may be valuable for
future research endeavors on the impacts of ocean acidification on larval phototaxis of
any marine species that exhibits such behavior. This manipulative experiment may be
useful for the Atlantis Ecosystem Model managed by the Ocean Acidification Group at
NOAA’s Northwest Fisheries Science Center that incorporates information about the
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effects of ocean acidification on the Puget Sound food web. The diverse physiological
impacts of ocean acidification on organisms may have far-reaching implications and
further research across disciplines is necessary to predict these impacts.
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References
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Adams, C. F. and A. J. Paul. 1999. “Phototaxis and geotaxis of light-adapted zoeae of the
golden king crab Lithodes aequispinus (Anomura: Lithodidae) in the laboratory.”
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Amaral, V., H. N. Cabral, and M. J. Bishop. 2013. “Prior exposure influences the behav
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Arnberg, M. et al. 2013. “Elevated temperature elicits greater effects than decreased pH
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Bradbury, J. W. and S. L. Vehrencamp. 2011. in: Principles of animal communication. 2nd
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Brauner, C. J. and D. W. Baker. 2009. “Patterns of acid-base regulation during exposure
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Glass and S. C. Wood, eds. pp. 43-63. Berlin: Springer.
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Briffa, M. and L. Sneddon. 2007. “Physiological constraints on contest behavior.”
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