CO2-level Dependent Effects of Ocean Acidification on Squid,
Doryteuthis pealeii, Early Life History
Thesis by
Casey Zakroff
In Partial Fulfillment of the Requirements
For the Degree of
Master of Science in Marine Science
King Abdullah University of Science and Technology, Thuwal,
Kingdom of Saudi Arabia
Approval Date: December 2013
2
The thesis of Casey Zakroff is approved by the examination committee.
Committee Chairperson: Michael Berumen
Committee Member: Stein Kaartvedt
Committee Member: Xabier Irigoyen
3
ABSTRACT
Ocean acidification is predicted to lead to global oceanic decreases in pH of up to
0.3 units within the next 100 years. However, those levels are already being reached
currently in coastal regions due to natural CO2 variability. Squid are a vital component of
the pelagic ecosystem, holding a unique niche as a highly active predatory invertebrate
and major prey stock for upper trophic levels. This study examined the effects of a range
of ocean acidification regimes on the early life history of a coastal squid species, the
Atlantic longfin squid, Doryteuthis pealeii. Eggs were raised in a flow-through ocean
acidification system at CO2 levels ranging from ambient (400ppm) to 2200ppm. Time to
hatching, hatching efficiency, and hatchling mantle lengths, yolk sac sizes, and statoliths
were all examined to elucidate stress effects. Delays in hatching time of at least a day
were seen at exposures above 1300ppm in all trials under controlled conditions. Mantle
lengths were significantly reduced at exposures above 1300 ppm. Yolk sac sizes varied
between CO2 treatments, but no distinct pattern emerged. Statoliths were increasingly
porous and malformed as CO2 exposures increased, and were significantly reduced in
surface area at exposures above 1300ppm. Doryteuthis pealeii appears to be able to
withstand acidosis stress without major effects up to 1300ppm, but is strongly impacted
past that threshold. Since yolk consumption did not vary among treatments, it appears
that during its early life stages, D. pealeii reallocates its available energy budget away
from somatic growth and system development in order to mitigate the stress of acidosis.
Keywords: Squid, Ocean acidification, Doryteuthis pealeii, statoliths, CO2 stress, energy budget
4
ACKNOWLEDGMENTS
I would like to thank my research advisor, Michael Berumen, for his guidance,
patience, and assistance in this project and in my career as a KAUST student. I‟d like to
note how much I appreciate him taking the time to listen and advise me on my crazy
ideas and for letting me be a part of some fantastic Red Sea fieldwork and research.
I want to thank my WHOI advisor, Aran Mooney, for giving me the opportunity
to pursue this project and for helping me develop and complete it successfully. I could
not have accomplished this without help at WHOI and would like to thank Anne Cohen,
Maxwell Kaplan, Elizabeth Bonk, Meredith White, and Hannah Barkley for their insights
and assistance, and Dan McCorkle, Karen Chan, Andrea Schlunk, Samantha Zacarias,
and Mary Ann Lee for their immense contributions. I also want to thank Roger Hanlon,
the Hanlon lab, and the Gemma crew for the squid and squid-related advice.
I‟d like to show my appreciation to my committee, Stein Kaartvedt and Xabier
Irigoyen, as well as the rest of the faculty of the Marine Science department for their
mentorship. I have gained a lot by working with the community of scientists in the Red
Sea Research Center, especially alongside my colleagues in the Reef Ecology Lab.
I want to thank my friends and family for their help and support, especially the
amazing group of people I‟ve met at KAUST: Sarah Almahdali, Abdullah Kanee, Hatoon
Baazim, Manalle Al-Salamah, Nada Al-Jassim, Mariam Awlia, Aya Hozumi, Noura
Zahran, Jesse Cochran, Maha Khalil, Nabeelah Ali, Hjörtur Jónasson and the Guanners.
Lastly, I would like to thank KAUST and WHOI for facilitating this research and
granting me the opportunity to pursue my interests in marine biology.
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TABLE OF CONTENTS

Examination Committee Approvals Form………………………………...…….p.2

Abstract………………………………………………………………...………..p.3

Acknowledgments…………………………………………………………...…..p.4

Table of Contents……………………………………………………………......p.5

List of Abbreviations………………………………………………………….....p.7

List of Figures………………………………………………………………...…p.8

List of Tables………………………………………………………………….…p.9

1. Introduction……………………………...……………………………..……p.10
o 1.1Background: Ocean Acidification………...………………………...p.10

1.1a Ocean Acidification…………………………………..…..p.10

1.1b Organismal Response to Ocean Acidification…………....p.12
o 1.2 Research Context…...…………………..………………………….p.15

1.2a Stress Ecophysiology……………………………………..p.15

1.2b Squid……………………………………………………...p.17

1.2c Cephalopod Response to Ocean Acidification.…………...p.20
o 1.3 Research Outline…………………………………………………...p.22

2. Methods……………………………………………………………………...p.23
o 2.1 Squid Collection and Management………………………………...p.23
o 2.2 Experimental System……………………………………………....p.24
o 2.3 Experimental Setup………………………………………………...p.25
o 2.4 Trial Set 1…………………………………………………………..p.28
o 2.5 Issues of the Environmental Systems Laboratory………………….p.29
o 2.6 Trial Set 2…………………………………………………………..p.31
o 2.7 Morphometrics & Physiological Measurements…………………...p.32

2.7a Hatching Time…………………………………………….p.32

2.7b Hatching Efficiency……………………………………....p.33

2.7c Mantle Length…………………………………………….p.33

2.7d Yolk Sac Size…………………….……………………….p.34

2.7e Statoliths…………………………………………………..p.35
o 2.8 Statistics……………………………………………………………p.36
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
3. Results……………………………………………………………………….p.37
o 3.1 Water Quality……………………………………………………....p.37
o 3.2 Hatching Time……………………………………………………...p.37
o 3.3 Hatching Efficiency………………………………………………..p.39
o 3.4 Mantle Length……………………………………………………...p.39
o 3.5 Yolk Sac Size………………………………………………………p.41
o 3.6 Statoliths…………………………………………………………...p.43

4. Discussion…………………………………………………………………...p.45

5. Conclusion ………….………………………………………………………p.56

References……………………………………………………………………...p.58
7
LIST OF ABBREVIATIONS

Facilities
o WHOI, Woods Hole Oceanographic Institution
o ESL, Environmental Systems Laboratory
o MBL, Marine Biological Laboratory
o MRC, Marine Resources Center

Experimental Setup
o T1 (2, 3, 4), Trial 1 (2, 3, 4)
o AMB, Ambient level
o L1 (2, 3), Level 1 (2, 3)

Chemistry
o CO2, Carbon dioxide
o MgCl2, Magnesium chloride
o DO, Dissolved oxygen

Biology
o PVF, Perivitelline fluid

Equipment
o CTD, Conductivity, temperature, and depth sensor
o VINDTA, Versatile INstrument for the Determination of Total inorganic
carbon and titration Alkalinity
o SEM, Scanning electron microscopy
8
LIST OF FIGURES
Figure 1 – Proportion of paralarvae hatched per day for each trial (p.38).
Figure 2 – Average mantle length per CO2 treatment (p.40).
Figure 3 – Between trial comparison of mantle length at 2200ppm exposure (p.40).
Figure 4 – Within trial patterns of mantle length decrease with increasing CO2 exposure
(p.41).
Figure 5 – Average yolk sac size per CO2 treatment (p.42).
Figure 6 – Between trial comparison of yolk sac size at 2200ppm exposure (p.42).
Figure 7 – Average statolith surface area per CO2 treatment (p.43).
Figure 8 – Distribution of statolith structural quality across CO2 treatments (p.44).
Figure 9 – Between cup comparison of trial 4 mantle lengths to view potential genetic
variations in CO2 response (p.50).
9
LIST OF TABLES
Table 1 – Water quality and carbonate measurements of each CO2 treatment (p.37).
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INTRODUCTION
1.1
Background: Ocean Acidification
1.1a The Process of Ocean Acidification
Anthropogenic release of carbon dioxide since the Industrial Revolution has led to
a marked increase in atmospheric carbon levels [1]. The most well documented result of
this phenomenon has been the systematic increase in average global temperature. Much
work has been done to examine the potential impacts this climatic change could have on
organisms and ecosystems, while other effects of the carbon problem have been less
explored. It has been recognized for some time that the ocean acts as a significant carbon
sink for the atmosphere, absorbing as much as a third of the anthropogenic output of
carbon since the 1800‟s [2]. The process of the ocean‟s intake of CO2 is also well
understood: a simple reaction of CO2 with water forms carbonic acid, which then
dissociates releasing bicarbonate and, importantly, hydrogen ions. The increased
concentration of hydrogen ions lowers pH, making the ocean more acidic. Ocean
acidification is now seen as a major and relevant factor in anthropogenic climate change,
not only in predicted scenarios, but also as a process already measurably occurring and
impacting the current oceanic system.
Ocean acidification has resulted in an average drop in pH of 0.1 since the preIndustrial period [3]. Atmospheric carbon models based on status quo scenarios, such as
the Intergovernmental Panel on Climate Change A1FI model, project increases to
1000ppm by the year 2100, which would correspond to a further 0.2-0.3 drop in oceanic
pH [4]. This change in pH corresponds to a more than 100% increase in hydrogen ion
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concentration and would have significant impacts on the physical structure of the ocean
as well as its ecological and biological dynamics.
In addition to increasing hydrogen ion concentrations, ocean acidification also
reduces available carbonate ions due to a buffering reaction between CO2, carbonate ions,
and water to produce bicarbonate ions. The reduction in pH also concurrently lowers the
saturation state of soluble minerals in the ocean, calcium carbonate (CaCO3), in
particular. CaCO3 has two crystalline forms, aragonite and calcite, which are differently
soluble in seawater. Many marine organisms utilize CaCO3 as the basis of their shell,
carapace, or skeleton. Calcite is used to build the shells of coccolithophores and
foraminiferans and the ossicles of echinoderms. Aragonite is the skeletal material of
corals and the main component of most mollusk shells. A natural cline exists for each
form, below which conditions (due to temperature, pressure, and ocean chemistry) are
such that the material is soluble [5]. Above this cline, the material can precipitate and
becomes accessible to organisms for use in organic structures. Aragonite is much more
soluble than calcite and thus has a shallower cline, known as the aragonite saturation
horizon. Reduction in oceanic pH contributes to aragonite solubility and shallows its
saturation horizon, which in combination with the reduced carbonate availability,
presents a significant threat to organisms that rely on CaCO3 structures for survival [1,5].
Recent work, as a result of increased oceanic carbon monitoring, has shown that
natural variability in the carbonate cycle of coastal systems can result in sharp increases
in CO2 and decreases in pH. These effects can range temporally from acute, diurnal
patterns to seasonal and annual cycles and can reach levels of pH decrease (0.1-0.3 units)
that are not predicted for 100 years for the open ocean [6]. Open ocean systems do not
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appear to be subject to these types of fluctuations. Wang et al. has shown that coastal
regions, such as the northwest Atlantic, are more susceptible to shifts in CO2 and pH and
are thus at a higher risk for rapid and dramatic impacts due to ocean acidification [7].
Little is known about what these current and projected CO2/pH levels mean for
organisms and ecosystems of the coastal realm.
1.1b Organismal Response to Ocean Acidification
The organisms thought to be under the most direct threat of ocean acidification
are those that incorporate calcified structures into their bodies. Climate change impact
studies on a diversity of taxa, such as corals, coralline algae, and echinoderms, have
shown reductions in calcification rate, weakened structural integrity, and diminished
survival rates [27,28,29]. In a particular strain of coccolithophore, Jones, et al.
demonstrated that calcification rate was not affected by increased CO2, but that growth
rate was diminished, which indicates a potential for mitigation mechanisms [30].
However, compounded with the other increasing stresses of climate change, even species
that can cope with CO2 increases may struggle for survival [31].
Pteropods are quickly becoming the poster child for acidification impacts, due to
the dramatic levels of shell degradation seen with deceasing pH, though many other
molluscs are also at risk [32]. Decreased calcification rates result in impaired shell
development and structural malformations, which impacts individual success and
survival. With shellfish, this means not only an ecological shift, but also an immediate
impact on human food sources and the fisheries that rely on them. Abalone, clams,
oysters, and mussels have all been shown to be energetically taxed by ocean acidification
13
to the point of reduced growth and survival, indicating a severe threat to the seafood
industry [23,33,34,35]. Talmage & Gobler have demonstrated the plethora of effects,
particularly delays and disruptions in early life development, of increased CO2 on a
variety of economically valuable bivalves [36].
Larval responses of calcifying organisms to ocean acidification is of increasing
concern given what these impacts can tell us about potential changes to population
structure. Early life stage effects include, among other things, stunted growth, diminished
fertilization, malformation of larvae, and impaired larval dispersal and settlement [37].
Increased CO2 exposures decreased calcification and growth in sea urchin larvae, and
induced a budding response, which may act as a mitigating response by sacrificing
individual size to increase the size of the cohort [38,39]. Chan et al. has shown that larval
sand dollars undergo a morphological shift under ocean acidification conditions to a
modality of reduced feeding and increased swimming [40]. In such ways, larval dynamics
give insight into the possible ecological responses to ocean acidification.
Research on climate change impacts on corals and other calcifiers is widespread
due to their direct role in the structure and maintenance of ecosystems [41]. Reductions in
coral biomineralization due to the decrease in available carbonate and increased aragonite
saturation affect the morphology, growth, and success of coral recruits, diminishing the
formation of coral reefs in the long term [42]. Diaz-Pulido et al. has shown that climate
change conditions, driven by ocean acidification, can lead to the dissolution and death of
crustose coralline algae [43]. Coralline algae act as the glue of coral reefs, binding coral
structures together, thus overall reef health is threatened by their destruction. Work at
natural CO2 seeps in Papua New Guinea has examined the effects of increased CO2 on an
14
entire operating reef system and has shown severe reductions in coral diversity, reef
structure, resilience to disturbance, and crustose coralline algae, and a corresponding
increase in seagrass densities [44]. The loss of these vital habitat components has strong
implications for the future of the coral reef habitat and its associated organismal
communities.
Calcifiers are not the only organisms that will be affected by the severe changes in
marine chemistry associated with ocean acidification. Recent work in organismal
response to increased carbon dioxide has expanded to examine other ecologically
important species and the potential threat posed to them. Naturally, fish are a significant
priority given their relevance to oceanic trophic webs and fisheries. Studies focus on
larval stage impacts, and several have found species that are resistant to the typical
reductions in growth and increased metabolic stress of ocean acidification [45,46].
However, response is likely species dependent in fish, as Baumann et al. saw increased
mortality rates in post-hatch larvae after eggs had been exposed to increased CO2 levels
[22].
A diversity of sensory effects has been demonstrated in the larval fish response to
CO2. Munday et al. exposed larval clownfish to projected ocean acidification conditions
and noted that these fish could no longer interpret olfactory stimuli correctly, becoming
attracted to habitat cues they had previously avoided [24]. Otoliths, a calcium carbonate
structure in fishes used in hearing, appear to increase in size as a compensatory response
to CO2 exposure [47]. Though Bignami et al. have shown that this increase in otolith size
and density would lead to increased auditory sensitivity in cobia, it is not understood
whether this would be ecologically beneficial or whether this effect would be present in
15
other species [48]. Simpson et al. exposed larval clownfish and did not see a concurrent
increase in otolith size, but there was impairment of the auditory response to reef noise
[25]. Research into ocean acidification exploring a diversity of organisms highlights the
diversity and complexity of potential responses to this environmental threat.
1.2 Research Context
1.2a Stress Ecophysiology
Environmental stress is an occasional problem for most animals given natural
variability and occasional extreme natural events (e.g. severe weather). Part of the
evolutionary process is developing mechanisms to handle periodic stressors [8].
Anthropogenic stress is not a recent phenomenon; as evidenced archaeologically, humans
have been manipulating the environment and causing the extinction of species since the
earliest tool-users. The advent of humans is the leading causal theory for the mass
extinction of megafauna in Australia and the Americas, which massively restructured the
ecology of these regions [9,10]. The magnitude, diversity, and rate of human impacts on
the environment has, however, reached levels never before seen in the life history of
Earth. Research on stress physiology strives to understand not only the mechanisms an
animal has to mitigate natural stressors, but also how well those mechanisms cope with
the panoply of rapidly escalating anthropogenic stressors, and reasonably predict what
these impacts mean for the ecology and life history of the species.
Processes on the scale of ocean acidification have the temporal and spatial scope
to affect not just individual organisms, but entire populations, and thus the ecosystem as a
whole. Widespread stress effects can shift survival rates and alter population structures,
16
which in turn can reorganize the trophic web and other ecosystem functionalities [11].
Many large scale stressors are known and have been well researched, primarily
temperature and eutrophication [12]. Given a certain threshold of stress, an ecosystem
can undertake a phase shift and be completely functionally reorganized [13]. A wellknown example is the shift from coral reefs to algal plains and rocky urchin fields [14].
Most taxa are not as fundamental to ecosystem function as coral, but still have important
roles to play. One of the foundational steps to understanding ecosystem-wide stress
responses, therefore, is to understand the physiological and ecological responses of
individual organisms.
Environmental stressors tax physiological systems and force an organism to spend
precious energy maintaining internal equilibrium rather than use the energy for growth,
activity, or reproduction [15]. For invertebrates and other ectotherms, temperature has a
direct connection to metabolic rate, so increases in temperature can inherently tax an
animal energetically. Temperature and other stressors, especially in concert, often
increase the basal metabolic rate of the animal as more energy is needed for basic
maintenance [16]. If an organism has a limited energy budget, these metabolic shifts can
be severely detrimental.
Often, the adults of a species are capable of mitigating environmental stressors
without great effect. The early life stages of some organisms, however, are shown to be
much more sensitive than their elders and have much more notable impacts due to energy
partitioning [17]. Embryos intrinsically have a limited energy source, the yolk sac, which
they must utilize for successful development. Larval stages typically also have yolk sac
available to consume, but must settle or begin to actively feed by the time it is consumed.
17
The energy budget of these early life stages is devoted to somatic growth, in addition to
basic maintenance [18]. There is very little energy available to allot to the mitigation of
stressors, so this energy often comes at the cost of successful development [19].
Stress impacts on embryos and larvae extend beyond just stymieing somatic
growth. Perhaps the most significant of these is overall survival rates. High exposure to a
consistent environmental stressor can result in extremely low hatch rates [20,21,22].
Individuals that do successfully hatch into larvae have to contend with a diminished
development state and continued ambient stress, as well as natural threats, which results
in a strongly reduced survival rate [23]. There is also evidence to show that
environmental stressors affect the sensory capacity of some organisms, such as the
olfaction and audition of larval fishes [24,25]. Stress effects can also impact larval
dispersal and settlement, which is a major factor in ecosystem structure [26]. When
physiological responses begin to influence the ecology of the organism, the energetic
strain of environmental stress has the potential to impact overall fitness.
1.2b Squid
Squid are an important part of the pelagic food web, since they act as both
predator and prey to link trophic levels [49]. Squid are often compared to fish in terms of
their aquadynamic morphology and predatory efficiency, but the major difference
between the two taxa is the higher energy, faster metabolism, and much shorter lifespan
of squid [50]. They act at extremely high levels of activity in order to compete in their
ecological niche, which is a unique strategy for a marine invertebrate. Squid life cycles
are relatively brief, spanning between eight months and two years for most species, and
18
consist of a lifetime of hunting and growth followed by a spawning period, after which
they die of exhaustion [51]. The variety of mechanisms that allow for this live fast, die
hard lifestyle make squid physiology an interesting system to study in the context of
environmental change and physiological stress.
Squid egg mats are laid on the ocean floor and consist of strings of egg capsules,
each containing tens to hundreds of eggs. Temperature is a key driver of embryonic
cephalopod development. By influencing the metabolic rate of the embryos, temperature
alters development time and thus time to hatching [52]. Squid eggs are clearly sensitive
to their surroundings, to the point that position within the egg mat can effect survival due
to differences in oxygen availability and waste buildup [53]. Cephalopod egg capsule
permeability is also dependent on the condition of the surroundings. Lacoue-Labarthe et
al. demonstrated that climate change impacts, specifically, increased temperature and
CO2, altered the permeability of cuttlefish eggs to physiologically important trace
elements [54]. Gutowska and Melzner measured the pCO2 and pH of cephalopod egg
case perivitelline fluid and found that oxygen decreases and carbon dioxide increases
linearly over the development time of the embryo, resulting in a vastly reduced egg case
pH (compared to the environment) at the end stages of development that may act as a
stress trigger for hatching [55]. Squid embryos depend on the egg case for protection, but
environmental variability has a large impact on these structures and subsequently on the
sensitive physiological structures being constructed within.
Similar to the otoliths of fish, squid have a paired internal calcium carbonate
structure, the statolith, which functions for gravitational sensing and hearing. Statoliths
are aragonitic structures that are initiated during embryological development and then
19
precipitated in layers over the course of the squid‟s lifetime [56]. Malformations or a lack
of the development of the statolith and its component cellular system, the statocyst, can
result in impairment to the squid‟s swimming ability. Colmers et al. has described one
form of this phenomenon, stemming from strontium deficiencies, as “spinner”
cephalopods, due to their apparent lack of directional control [57]. Deposition of calcium
carbonate onto the statolith is affected by the squid‟s life stage and environmental
conditions, including temperature and carbonate availability [58]. These effects are
heightened in the paralarval stage when the squid is less capable of mitigating
physiological stress.
Squid are renowned for their high metabolic activity and the powerful
cardiovascular system that supports it. Part of the efficiency of this system stems from the
cephalopod hemocyanin and its capacity for carrying oxygen [59]. Squid rely on this
system not only to sustain aerobic respiration, but for many species also to take
advantage of oceanic oxygen minimum zones for hunting and predator avoidance [60].
Since many squid are adapted to handling occasional exposure to environments with low
oxygen and high CO2, the oxygen binding capacity of squid hemocyanin does not change
between pH 7-8 [61]. It is likely, however, that this system is not fully established until
squid have developed into their adult form, built for the complexities of the mesopelagic,
and that the epipelagic paralarvae have a greater sensitivity to changes in blood pH [62].
Slight blood acidosis is a natural part of aerobic respiration, particularly after
bursts of heightened activity. pH buffering in the blood and a wide pH range for oxygen
binding of the hemochrome are basic adaptations to dealing with natural acidosis. Squid
exercised under normal conditions exhibited slight levels of acidosis in the blood, which
20
was tied to strong levels of acidosis intracellularly [63]. The environmental reduction in
pH resulting from ocean acidification would serve to exacerbate these effects. Activating
mechanisms to cope with this physiological stress would require a reallocation of energy.
The energy budget of adult squid can allow for these costs without much detriment to the
individual, but early life stages would have to sacrifice development in order to cover the
cost of
1.2c Cephalopod Response to Ocean Acidification
There has been some examination of the effects of increased CO2 and decreased
pH on cephalopod physiology. Gutowska et al. looked at the combined effects of ocean
acidification on cuttlefish, Sepia officinalis, but saw no impacts on early stage growth or
survival and a hypercalcification response in the cuttlebone [65]. A multifactor analysis
by Dorey et al. added temperature to the variable mix, but had the same result, indicating
that S. officinalis, and perhaps other cephalopods, are particularly well adapted to CO2
stress [66]. Work by Hu et al. exposing squid, Loligo vulgaris, and cuttlefish, S.
officinalis, to ocean acidification conditions and looking at the activity of ion-regulating
epithelia demonstrated that squid were potentially more sensitive to acid/base
environmental stress than cuttlefish were [62]. Experiments on L. vulgaris egg cases have
shown that increased CO2 exposure led to increased uptake of certain trace elements,
enhancing the risk of anthropogenic metal contamination [67]. Recent work by Kaplan et
al. has demonstrated there are notable impacts from exposure to strongly increased CO2,
2200ppm, on the development and growth of squid, Doryteuthis pealeii, paralarvae [68].
21
The extent to which ocean acidification can impair squid physiology is not well
understood. Kaplan et al. compared ambient and highly elevated exposures of CO2 and
saw reductions in hatching time and paralarval mantle length, as well as degradation to
the statoliths [68]. These results indicate an energy demand due to CO2 exposure that
must be paid from available resources. Malformations of the statolith are also an indicator
of problems with internal pH balance, and thus acid/base regulatory systems, given they
are maintained within a cellular structure. Though these impacts are important indicators
of the potential threat posed to squid by ocean acidification, the elevated level of CO2
used was a prediction that may not occur naturally for a century or more. It therefore
becomes necessary to understand not only the extent of impacts on paralarval D. pealeii
due to metabolic stress and acidosis, but also at what levels of exposure these responses
first occur and become significant.
The longfin inshore squid, Doryteuthis pealeii, is a mesopelagic species common
in the western North Atlantic [51]. It is a vital prey item for many finfishes and sharks,
diving birds, and marine mammals, as well as a significant predator on smaller fishes,
other squids, and conspecifics [69]. It is also a substantial fishery, totaling more than 28
million pounds in 2012, mainly caught off of New England [70]. Doryteuthis pealeii
spawn year-round, but have a peak breeding season in higher latitudes from April to
October, when breeding schools move inshore, mate, and lay their eggs in mats on the
ocean bottom [71]. Hatchlings immediately swim to the top of the water column and
remain in the surface layer until they become large and developed enough to begin
actively hunting at depth [51]. Though this stage is a vital period of development and
22
survival, and acts to structure successive cohorts of the population, there is little known
about D. pealeii paralarval biogeography, ecology, behavior, or stress physiology.
1.3 Research Outline
The purpose of this study was to elucidate physiological responses of early life
stage Doryteuthis pealeii to ocean acidification stress. A primary focus was to develop a
dose-response relationship for CO2 exposure by expanding the work of Kaplan et al.
across a range of CO2 levels [68]. Further, this research sought to explore the
mechanisms of metabolic stress behind the responses seen by looking at available energy
stores through the paralarval yolk sac. Yolk utilization is increased in early stage
cephalopods under temperature stress due to the increased metabolic demand [72]. Given
the metabolic demand of acidosis, however, energy may be reallocated from somatic
growth or existing stores of energy may be consumed faster, or both processes may
occur. Analyzing yolk sac size in concert with hatching time, mantle length, and statolith
effects allows for a more complete picture of the paralarval energy budget as it is used to
combat the stress of ocean acidification.
23
METHODS
2.1
Squid Collection and Management
Adult Doryteuthis pealeii were acquired from the Marine Biological Laboratory
(MBL) Marine Resources Center (MRC). The Gemma, an MBL ship, regularly obtains
squid through trawls at 10-30 meters depth in Vineyard Sound. Healthy adults, energetic
and without damaged mantles, were hand selected at the MRC dock and placed in coolers
for transport. Reproductively active females were differentiated by their bright orange
nidamental gland and only those with a visible, dark egg mass were selected. Males with
clearly visible, dense packets of sperm were preferentially selected. Squid were selected
with a 2:1 ratio of females to males, the standard count being twelve females & six males
per trip, to support mating success. Selected squid were within the medium size range for
the species (approximately 20-25cm).
Transit was done as quickly as possible, within an hour of capture, to reduce
stress effects on the squid. Upon arrival at the Environmental Systems Laboratory (ESL),
squid were split among two holding tanks (120cm diameter, 70cm depth), six females
and three males per tank. Holding tanks were flow-through systems that used water
pumped directly from offshore (see Section 2 below) that had been sand-filtered and
brought to 14°C. This temperature reflects average natural exposure at depth for the time
of year and reduced thermal and metabolic stress. Squid were fed with locally captured
killifish, Fundulus heteroclitus, twice per day. Squid were maintained in tanks in the ESL
until they died after breeding or of other causes.
24
2.2
Experimental System
Experiments were conducted at the ESL at the Woods Hole Oceanographic
Institution (WHOI), Woods Hole, Massachusetts, USA from April through August of
2013. The experimental aquariums consisted of individual 1-liter PET food service
containers that were incorporated into a flow-through ocean acidification system that was
equilibrated to different CO2 levels for each trial. The containers had been seawater
soaked and rinsed in advance to remove any plastic toxins. One trial system consisted of
15 PET containers placed 3x5 (3 CO2 levels x 3 experimental, 1 control, and 1 holding
cup) into a water bath, which maintained a constant temperature of about 20°C. Cups
were sealed with fitted lids in which two holes were pierced for tubing, one for the gas
mixture and the other for equilibrated water, and had a small (2x4cm), screened (5µm
mesh) hole near the top of the cup to allow for water outflow without losing paralarvae.
The bubbler of the gas line was placed under the screen to create flow away from the
drain and aid in preventing paralarvae from sticking to the screening.
Ambient ocean water is pumped in from the ESL intake (approximately 100 yards
offshore of the ESL) and passed through the facility‟s sand-filtration system. It was then
passed through a subsequent 10µm filtration (Hayward FLV Series industrial filter
equipped with 10µm felt bag) to remove major particulates and a UV sterilizer (Emperor
Aquatics Smart HO UV Sterilizer, Model 025150) to eliminate potentially harmful
protozoans. The cleaned water then flowed into the header tank (32 gallon Trashmaster
plastic garbage can) of the experimental system and was aerated and heated to 20°C using
high flow aquarium heaters. Water flowed out the bottom of the header tank and was split
among four H-shaped PVC equilibration chambers. Each leg of an „H‟ contained 2 air
25
stones, which bubbled in the corresponding CO2 mixture for the level (1 control of
ambient air and 3 experimental mixtures). Equilibrated water then outflowed into a PVC
manifold, from which it entered drip lines connected to aquaria of the corresponding
experimental level. From the cups, water overflowed through the screens into the water
bath and then circulated in the water bath. Water baths maintained a temperature of 20°C
through a system of aquarium heaters and chillers. Water cycled in the water bath until it
exited via overflow hose into the drain.
High pressure air, delivered at 30 psi, from an indoor air compressor was
connected through a six-way manifold in the lab to the header tank aeration, ambient
equilibration chamber air stones, and three Aalborg (GFC17) mass flow controllers set at
5 liters/minute flow. Pure CO2 was delivered from a cylinder at 30 psi through a filter to
three Aalborg (GFC37) mass flow controllers, which were set at various flow rates to
produce the desired concentration of CO2 in the gas mixtures. Experimental air and CO2
lines were connected downstream of the mass flow controllers and allowed to mix before
being split among the equilibration chamber air stones and aquaria bubbling lines. CO2
mixtures covered a range of values between ambient (400ppm) and 2200ppm (see Trial
Descriptions below). CO2 concentrations for each level were measured before the start of
each trial on a Qubit Systems CO2 Analyzer (model s151) calibrated with three
commercial reference standards (0, 362, and 1036ppm).
2.3
Experimental Setup
Two water baths, containing 15 cups each, were set up to allow for the running of
two simultaneous or staggered trials. Each trial consisted of three CO2 levels, with the
26
ideal consisting of the control (400ppm) and two elevated levels. The system was set up
and run for several days in advance of each trial to allow water levels to equilibrate (to
temperature, flow, and CO2). Flow rates to the cups were set at a slow drip,
approximately 20 liters/day, so water was replaced in the cups about 20 times a day,
which kept waste buildup from being a problem. The flow rate also allowed sufficient
time for bubbled gas to equilibrate with water in the „H‟ chambers. Each cup was bubbled
with the same gas mixture as its water in order to maintain correct pH/CO2 levels
throughout the system, to keep the eggs and paralarvae well oxygenated, and to control
for the effect of the atmosphere within the sealed cups. The lab containing the
experimental set up was kept on a 14:10 light:dark photoperiod, reflecting the natural,
average photoperiod of the region, using ceiling mounted fluorescent lighting. Water bath
temperature and ambient light levels were monitored using an Onset HOBO data logger
(pendant model UA-004-64), one in each water bath, with recordings taken every 15
minutes. Temperatures were 20.49 ± 0.69°C in water bath 1 (used for trials 1 and 3) and
20.26 ± 0.49°C in water bath 2 (used for trials 2 and 4).
After squid were acquired from MBL and brought to the ESL, females would
begin laying eggs within two to four days. Small egg mats were typically found at the
bottom of the tank or attached to the air hose. The morning an eggs cluster was
discovered, it was removed to a holding container and examined for quality. Egg sacs
were then randomly selected and randomly assigned to each experimental cup, with two
egg sacs per cup (18 total egg sacs for three cups for each of three CO2 levels), which
would initiate a trial. If there was no trial to start, eggs would just be disposed of. Within
the holding tank, an egg mass could include egg capsules from multiple females;
27
communal egg-laying is an advantageous wild behavior [51]. Female squid can also store
sperm from multiple males, which could include pre-capture contact for captured
specimens [73]. A typical egg capsule (containing 100-200 eggs) can have eggs fertilized
by multiple fathers as well, which can be from held sperm or males in proximity at laying
[74]. Hence, parentage in squid is inherently complex and this study cannot make claims
of genetic fitness in the results.
The fourth cup from each CO2 level contained no eggs sacs and was used as a
control for water quality and carbonate chemistry measurements. During a trial, pH
measurements were taken for samples from each cup every other day using a pH meter
(Orion 3 Star Plus, ThermoScientific) in order to monitor consistency in CO2 levels.
More accurate pH measurements, used for carbonate chemistry calculations, were taken
with a spectrophotometer using methods adapted from Clayton & Byrne & Dickson et al.
[75,76]. A baseline measurement of all cups would occur prior to the initiation of a trial,
followed by weekly readings of just the control cups once a trial had begun. Salinity
samples were taken in 120mL glass bottles in parallel to spectrophotometric pH readings,
and were analyzed in bulk after trials had been completed.
Concurrent to spectrophotometric pH and salinity sampling, total alkalinity (AT)
samples were taken in 20mL acid-washed, glass scintillation vials and poisoned with
10µL saturated mercuric chloride (HgCl2). Alkalinity samples were analyzed post-trial
using an automated small volume titrator to run Gran titrations of 1mL subsamples.
Samples were run in duplicate and calibrated against standards of ESL water of known
alkalinity. For duplicates with a difference of 4 µmol/kg seawater (SW) or greater,
samples were rerun and an average of the four values was taken. Carbonate chemistry
28
metrics (temperature, salinity, pH, and alkalinity) were input into CO2SYS, using
Mehrbach‟s dissociation constants and Dickson‟s sulfate constants, to calculate pCO2 and
aragonite saturation state (Ωarag) for each level of each trial [77,78].
2.4
Trial Set 1
Trial 1 (T1) consisted of an ambient control (AMB), which was equilibrated with
pure air, Level 1 (L1), equilibrated with a CO2 mixture calculated to 2200ppm, and Level
3 (L3), equilibrated with a CO2 mixture calculated to 1300ppm. CO2 concentrations for
gases used in T1 read as follows on the Qubit: AMB, 377ppm; L1, 2042.2ppm; L3,
1205.6ppm. Carbonate chemistry calculations in CO2SYS showed initial in-cup pCO2‟s
to be 565.58 ± 43.90ppm for AMB (see section 5 below), 2199.56 ± 173.47ppm for L1,
and 1350.51 ± 43.55ppm for L3. Egg sacs for T1 were collected the morning of July 3rd
and the trial concluded July 28th.
Trial 2 (T2) consisted of L1, set to 2200 ppm, Level 2 (L2), equilibrated with a
CO2 mixture calculated to 850ppm, and L3, set to 1300ppm. AMB was not used in this
trial since it deviated from atmospheric levels of CO2 and did not effectively act as a
natural control. Comparisons were instead drawn between repetitions of L1 and L3. CO2
concentrations for gases used in T2 read as follows on the Qubit: L1, 2042.2ppm; L2,
802ppm; L3, 1205.6ppm. CO2SYS calculations showed initial in-cup pCO2‟s to be
2380.50 ± 70.62ppm for L1, 987.43 ± 20.30ppm for L2, and 1351.67 ± 34.26ppm for L3.
Trial 2 was initiated the morning of July 11th, L3 was shut down on August 5th so that
CO2 levels could be reset for Trials 3 & 4, and T2 was shut down on August 6th to allow
the system and aquaria be reset for Trial 4.
29
2.5
Issues of the Environmental Systems Laboratory
Spectrophotometric pH readings taken during indicated pH values lower than
would have been expected for seawater at 400ppm CO2. Delays in establishing the small
volume alkalinity titrator protocol led to the decision to omit AMB from experimentation
until the discrepancy could be confirmed and repaired. Given that air was being extracted
from an indoor source, spikes in CO2 were possible depending on the amount of human
and machine activity. Qubit analysis of the air used to equilibrate the AMB level showed,
however, that levels equated to about atmospheric ambient (377.87 ± 1.68ppm). pH meter
testing of the ESL water supply, including every active water treatment line and several
long term operating aquaria indicated a consistent value of about 7.95-8.00 (NBS scale),
which estimated to about 600-650ppm CO2 with corresponding temperature of the line
and set value of 31psu and 2075µmol/kg SW (based on an average value for ESL
standards). After alkalinity samples had been processed, ESL water pCO2 was shown to
be approximately 600ppm throughout the facility.
Demonstration of the ESL pCO2 level led to the question of whether this was
actually a facility effect or rather just a representation of the natural coastal variation in
CO2 at the intake. Thus, AMB could act as a local ambient value rather than an
atmospheric control. In order to explore this hypothesis, a sampling trip was taken to the
ESL intake pumps located 100 yards directly offshore from the facility. Water samples
and CTD readings were taken at the intake site at surface (1 meter) and depth (3 meters)
and another 100 yards offshore at depth (4 meters) at what was, according to WHOI staff
advice, another potential location for the ESL intake. Preliminary estimates based on
CTD temperature, salinity, and pH values gave pCO2 values of approximately 400ppm at
30
all sampling sites. Water samples were run on a VINDTA to acquire alkalinity and DIC
values. CO2SYS calculated values were 430.36 ± 9.01ppm and indicated that the pCO2
level of water coming into the ESL was at equilibrium with atmospheric levels. This
sampling represents only one trip at one time on one day and cannot be considered a
confirmation that local inshore pCO2 values are consistently at 400ppm. However, it does
indicate the source of the ESL pCO2 discrepancy is likely to be the ESL itself.
Regardless of whether the problem stemmed from natural variation or an issue
with the ESL water system, the pCO2 discrepancy could not be resolved at its source and
had to be repaired downstream as part of the experimental system. Adjustment to the
system included two major changes. First, based on the hypothesis that the single
equilibration chamber was not sufficient to lower CO2 levels in the ESL water and
colloquial recognition that equilibrating CO2 levels up is easier than bringing them down,
two additional „H‟ equilibration chambers were added downstream of the original AMB
equilibration chamber (before the manifold) to increase the time of exposure and volume
of water exposed to bubbled air. Early tests of this system at various rates of water flow
and bubbling indicated that it was still ineffective at reducing the pCO2 to ambient levels.
The second augmentation was the refit of the first equilibration chamber in the
AMB line as a nitrogen scrubbing chamber. A cylinder of liquid nitrogen (gaseous
cylinders were consumed too quickly) provided pure N2 gas, which was bubbled into the
two legs of the first AMB equilibration chamber and removed both the CO2 and O2 from
the water. The two additional downstream chambers thus had to re-equilibrate both gases
to appropriate levels in the water before reaching the experimental cups. Measurement of
O2 levels in water output from the AMB line, using a YSI optical DO probe, showed
31
consistent saturation between 95 and 98%. pH meter readings were around 8.1 (NBS
scale), which corresponded to an estimated 400ppm CO2. These values were corroborated
by subsequent testing with the spectrophotometric pH, salinity bottles, and alkalinity
testing, allowing for a consistent ambient control to be incorporated back into the
experimental design. The subsequent pCO2, though not a perfect equilibration (487.10 ±
12.05ppm), is within the realm of the 50-100ppm variance above input gas concentrations
seen in all levels due to the nature of the flow-through system.
2.6
Trial Set 2
Trial 3 (T3) consisted of AMB, now set to 400ppm with the implementation of
the N2 system, L1, still at 2200ppm, and L3, set to 1600ppm. CO2 concentrations for
gases used in T3 read as follows on the Qubit: AMB, 379.8ppm; L1, 2053.4ppm; L3,
1504.8ppm. CO2SYS calculations showed initial in-cup pCO2‟s to be 485.62 ± 13.60ppm
for AMB, 2180.02 ± 130.96ppm for L1, and 1757.40 ± 19.94ppm for L3. Trial 3 eggs
were placed in cups on the morning of August 1st. On August 4th, it was discovered that
the ESL air compressor had broken and the air system had shut down. The malfunction
could have occurred at any point between 11:00am on the 3rd and noon on the 4th when
it was discovered. It took until 5pm on the 4th for the compressor to be replaced and the
air system to recover function. For L1 and L3 this meant only a brief period wherein the
eggs were exposed to the standard 600ppm ESL water instead of their experimental CO2
level, which could skew the data, but was not utterly detrimental. AMB, however, had the
additional problem of being N2 scrubbed without downstream air equilibration. Thus,
eggs in the AMB cups were exposed not only to water of the incorrect CO2 level, but also
were completely deoxygenated for that period. Data from Trial 3 is not being included in
32
comparative analyses, but will be examined for its own value demonstrating impacts to
anoxia and CO2 variability. Trial 3 concluded on August 28th.
Trial 4 (T4) was started after the air system was repaired. It consisted of AMB,
400 ppm, L1, 2200ppm, and L2, set to 1900ppm. CO2 concentrations for gases used in T3
read as follows on the Qubit: AMB, 379.8ppm; L1, 2053.4ppm; L2, 1784.3ppm.
CO2SYS calculations showed initial in-cup pCO2‟s to be 488.58 ± 10.50ppm for AMB,
2130.17 ± 40.31ppm for L1, and 2003.79 ± 12.84ppm for L2. T4 was the first occasion
where egg masses were found concurrently in both holding tanks in the morning. This
allowed for the design of a simple test for differences between genetically different
populations of paralarvae. Mothers of the egg sacs from holding tank A had to be
different than those of holding tank B and it was unlikely, though not impossible, that
embryos would share the same fathers between tanks. Egg sacs were divided among
experimental cups for each CO2 level (one letter represents one egg sac): Cup 1: AA, Cup
2: BB, Cup 3: AB. T4 was initiated on August 7th and concluded on September 6th.
2.7
Morphometrics & Physiological Measurements
2.7a Hatching Time
During T1, experimental cups were checked for hatching four times daily (7am,
11am, 3pm, and 7pm). The bulk of paralarvae hatched overnight, so in T2, T3 and T4, the
time period was reduced to a 12 hour interval (7am and 7pm) in order to just differentiate
paralarvae that hatched overnight from those that hatched during the day. At initial
hatching, time period shifted from hatchling checks to hatchling counts. Counting
consisted of the pipetting of individual paralarvae from the experimental cup to a petri
33
dish. Hatchlings were not returned to the experimental cups, but instead were subsampled
for morphometric measurements. This allowed for counts of total hatching per day. Any
paralarvae not subsampled were fixed in 97% ethanol for later statolith work. Hatchling
counts continued until a cup had two consecutive days with no hatchlings produced, at
which point egg sacs were removed for analysis. When all cups in a trial reached this
point, the trial was concluded and cleaned up. Hatching time was calculated as the
difference between initiation of the trial (earliest known existence of the eggs) and
initiation of hatching.
2.7b Hatching Efficiency
Once hatching had ceased, egg sacs were analyzed for hatching efficiency. Each
egg sac was removed from the experimental cup, photographed, and then placed under a
dissecting microscope for precise counting of the total number of unhatched eggs.
Unhatched eggs were differentiated as unfertilized eggs, early stage embryos, and late
stage embryos. Total number of unhatched eggs for the cup was compared to the total
number hatched as a metric of hatching efficiency. Values for cups were pooled per CO2
level to produce hatching efficiency over the range of CO2 exposures.
2.7c Mantle Length
10 paralarvae per cup, per trial, per day (90 total/day; no trials overlapped) were
subsampled for mantle length measurements. Samples were taken for 6 days from initial
hatching, so as to get a sufficient sample size for variation between cups, days, levels,
and trials (540 total/trial). Individual paralarvae were placed on a watch glass under a
Zeiss SteREO Discovery.V8 dissecting scope in a drop of seawater. Dorsal photographs
34
of the paralarvae were then taken with a Canon G12 attached to the scope. If paralarvae
were too active to be clearly photographed, they were anesthetized with a few drops of
7.5% w/v magnesium chloride (7.5 MgCl2). Only undamaged paralarvae that were not
premature were used in mantle length analysis. Each day‟s photograph set was prefaced
by a photograph of a scale at that day‟s set zoom and focus. Paralarvae images were then
processed in ImageJ to produce the mantle length dataset.
2.7d Yolk Sac Size
10 paralarvae per cup, per trial, per day (90 total/day; no trials overlapped) were
subsampled for yolk sac staining. Staining was done for 6 days from initial hatching in
order to produce a dataset of sufficient sample size for variation analysis as with mantle
lengths (540 total/trial). Lipid staining procedures were adapted from Gallager [79].
Subsampled paralarvae were placed in a well-plate and pooled by cup. Paralarvae were
first strongly anesthetized with 7.5 MgCl2 and then quickly fixed with formalin in order
to prevent contraction of the mantle in preservation. Fixed paralarvae were stained with
Oil Red for at least 6 hours, or until a deep red color appeared, and were then
successively soaked in ethylene glycol to remove excess stain.
Staining results in paralarvae whose posterior and anterior yolk sacs were stained
red, while the mantle and body remained clear (except for the head, which retained a
small amount of the stain. Individual stained paralarvae were placed under the dissecting
scope and photographed on their ventral side. Each day‟s set of photographs was prefaced
by a scale photo at that day‟s scope settings. Yolk sac photos were processed in ImageJ
per the methods of Vidal et al. [80]. Lines of width and length were drawn for both the
35
anterior and posterior yolk sacs and translated into a formula for a three-dimensional
shape (a cone or cylinder for the anterior sac, and an oval for the posterior) in order to get
volumes of the yolk sacs. The volumes of the anterior and posterior yolk sacs were
summed to produce total yolk volume per individual, which was compiled into the yolk
sac size dataset.
2.7e Statoliths
Post-trial, paralarvae that had been preserved in 97% ethanol were dissected for
their statoliths. Preserved paralarvae were separated by cup, level, and day, but given the
time and effort involved in dissecting a clear, 50 micron stone from an opaque, 1-2mm
fixed paralarvae, statoliths were pooled per level for analysis. For each individual
successfully dissected, only one statolith was taken, so as to retain statistical
independence. Statoliths were washed with drops of 97% ethanol in order to remove any
adherent tissue. Dissected out statoliths were mounted on carbon stubs for scanning
electron microscopy (SEM). An extremely fine paintbrush was used to pick up the
statolith from under the Zeiss dissecting scope and transfer them to the mount underneath
a second, less powerful dissecting scope. Statoliths were aligned and placed in as
consistent an orientation as could be managed working on such a small scale. Lines of
statoliths designated samples from the same level, with one stub fitting about 6 lines of
about 10 statoliths. SEM stubs were sputter-coated with 10nm platinum and imaged at
the MBL Central Microscopy Facility using a Zeiss NTS Supra 40VP.
Statoliths were graded following the methods of Kaplan et al. [68], which uses
porosity and shape as the most highly weighted factors. Categories are defined as: 1,
36
standard statolith shape with normal or minimal porosity; 2, standard shape, but either
slightly porous or including some structural abnormalities; 3, abnormally shaped and/or
highly porous. The standard form of a paralarval statolith is droplet-shaped with a slight
doming that sweeps into a shallow wing [81]. 60 individuals in T1 and 45 in T2 were
dissected for statoliths. Variance in the total number of statoliths analyzed per trial owes
to the inherent difficulty of statolith dissection and the loss of statoliths during dissection
and mounting. T1 had additional statoliths extracted as part of a preliminary swimming
behavior study, so that data is also included here. T3 and T4 statolith dissections have not
yet been performed due to time constraints and the setup of additional analyses for those
samples. 77 statoliths were extracted from T1 paralarvae, and 44 were taken from T2, for
a total of 121 statoliths analyzed.
2.8
Statistics
Statistical analyses were run in Statgraphics Centurion XVI (StatPoint
Technologies, Inc.) for Windows. All reported values (including pCO2 concentrations
above) are mean ± standard error. Datasets for all factors were nonparametric, so
Kruskal-Wallis tests were used to look for significant differences between cups, days,
treatments, and/or trials. Differences in the distribution of statolith grades were tested
using a Chi-squared test.
37
RESULTS
3.1
Water Quality
pCO2 was consistent in all experimental levels throughout all trials (Table 1). The
assumption that all cups had equal exposure conditions was confirmed (Kruskal-Wallis,
H = 0.210, df = 3, p = 0.976), so the use of cup 4 to measure water quality for all cups
through each trial was valid. pCO2 in the experimental cups consistently read 50-100ppm
above the calculated level in CO2SYS (Table 1). The reasons for this inconsistency are
uncertain, but may be an effect of insufficient equilibration time or variation in water
quality testing. Temperature, salinity, pH, and alkalinity, were stable throughout the trials
(Table 1).
Table 1. Monitored parameters of water quality for each level of each trial and CO2SYS output for
carbonate chemistry.
3.2
Hatching Time
CO2 treatment had a strong effect on hatching dynamics, with hatching delays of
one to two days seen in all treatments above 1300ppm in T1, T2, and T4 (Figure 1). No
differences were seen between treatments of T3 (Figure 1). Initial proportions of
hatchlings were not substantially different between treatments, ranging from 0.5 to 1.5%.
38
Hatching periods ranged from nine to fifteen days total and did not correlate with trial or
CO2 treatment. It took between five to seven days for 90% of paralarvae to hatch in all
trials at all treatments. Hatching began after 12 days in T1 and T2, and after 15 days in
T3 and T4. The reason for this shift in hatching initiation could be due to the system
malfunctions that occurred in T3, but is uncertain for T4, as conditions were stable and
accurate throughout its running. Though time to hatching initiation varied between the
trial sets, the pattern of hatching delay in high CO2 exposures is consistent.
Figure 1. Total proportion of paralarvae hatched since eggs were laid for T1 (A), T2 (B), T3 (C), and T4(D).
39
3.3
Hatching Efficiency
No significant difference in hatching efficiency was found between CO2
exposures (Kruskal-Wallis, H = 4.267, df = 6, p = 0.641). All treatments resulted in at
least 85% of eggs hatching into paralarvae, with most reaching around 93% hatched. Two
thirds of all unhatched eggs were non-fertilized or undeveloped eggs and paralarvae in
early development stages 1-26 [71]. There was no pattern between treatment and
proportion of any particular type of unhatched paralarvae.
3.4
Mantle Length
There was a significant difference in mantle length seen between treatments in a
pooled sample of T1, T2, and T4 (Kruskal-Wallis, H = 152.95, df = 5, p < 0.001; Figure
2). All trials included a 2200ppm level to allow for replication and comparison over the
experimental period. Similar values were seen in T1, T2, and T4, but T3 had significantly
longer mantle lengths, and was thus excluded from the pooled analysis (Figure 3;
Kruskal-Wallis, H = 79.47, df = 3, p < 0.001). Paralarvae at exposures of 1300ppm and
below had consistently longer mantles, by one tenth of a millimeter, than those at higher
CO2 levels (e.g., 550ppm: 1.64 ± 0.01mm; 2200ppm: 1.54 ± 0.005mm). A consistent
linear pattern was not seen when trials were pooled, though appeared to occur in T1,
while T2 exhibited a step-wise pattern between high and low exposures (Figure 4).
40
Figure 2. Mantle lengths for each CO2 treatment pooled from T1, T2, and T4.
Mantle length was variable over the hatching period, but did not exhibit any clear
or consistent pattern. Paralarvae at 2200ppm in T1 showed no significant differences
across the days of hatching (Kruskal-Wallis, H = 9.15, df = 5, p = 0.103), while those in
T4 showed significant differences randomly assorted over the days (Kruskal-Wallis, H =
41.03, df = 5, p < 0.001). Significant variation between cups was seen in pooled data and
within trials, except T2, though differences were small (0.03mm).
Figure 3. Comparison of average mantle lengths from 2200ppm treatment for each trial.
41
Figure 4. Mantle lengths per treatment for T1 (A) and T2 (B),
showing a linear and step-wise pattern, respectively.
3.5
Yolk Sac Size
Yolk sac volume varied significantly between treatments, but no discernible
pattern emerged in T1, T2, and T4 (Figure 5; Kruskal-Wallis, H = 174.71, df = 5, p <
0.001). Comparison of the 2200ppm treatment across trials revealed no anomalous
behavior in T3, but was excluded for consistency in analysis. T1, however, had
significantly larger yolk sac volumes at 2200ppm than the other trials (Figure 6; KruskalWallis, H = 51.58, df = 3, p < 0.001). T1 paralarvae had much greater yolk sac volume
than any other trial for each exposure level, even at 2200ppm (0.121 ± 0.007mm2 at
2200ppm in T1 compared to 0.086 ± 0.004mm2 in T2).
Within T1, there was a significant difference between treatments, with a sloping
decrease in yolk sac size as CO2 level increased (Kruskal-Wallis, H = 13.86, df = 2, p <
0.001). However, significant differences were not seen within T2 and T4, and T3
42
exhibited a stepwise pattern between low and high CO2 exposures, so no consistent trend
in yolk sac size emerged. Yolk sac size showed significant daily variation, but no
consistent pattern, across CO2 treatments. Variation between cups was not significant
when data was pooled, but was within T1, T2 and T3.
Figure 5. Yolk sac size for each CO2 exposure pooled across T1, T2, and T4.
Figure 6. Between trials comparison of average yolk sac sizes at 2200ppm.
43
3.6
Statoliths
Statoliths were significantly reduced in surface area at 2200ppm compared to
exposures of 1300ppm and lower (Figure 7; Kruskal-Wallis, H = 19.84, df = 3, p <
0.001). Statoliths at 2200ppm averaged an almost 20% reduction in surface area
compared to those from 550, 850, and 1300ppm, which were relatively equal in size.
Statolith grade varied significantly between exposure levels with abnormalities in shape,
crystal structure, crystal deposition, and porosity increasing consistently with increased
CO2 exposure (Figure 8; Χ2 = 31.42, df = 6, p < 0.001).
Figure 7. Surface areas of T1 and T2 statoliths compared across CO2 treatments.
44
Figure 8. Distribution of statolith grades per CO2 exposures in T1 and T2. Grade 1, Green; Grade
2, Yellow; Grade 3, Red.
45
DISCUSSION
Doryteuthis pealeii exhibited a delay in hatching time, reduction in mantle length,
and degradation of the statoliths at strongly elevated CO2 exposures when compared to
ambient and low exposure states, demonstrating a clear effect of ocean acidification on
this species‟ development during early life. The results of these experiments are
consistent with the work of Kaplan et al., but show the variation in response over a range
of CO2 exposures [68]. All CO2 levels used represent both potential open ocean levels
predicted to be reachable within the next century, as well as current values occurring in
coastal systems due to natural variability [4,6]. The potential for impact of these
physiological effects on the modern and near-future D. pealeii population is, therefore,
high.
Hatching time was delayed by at least 24 hours in exposures above 1300ppm in
trials 1, 2, and 4. Delays in development time indicate either an increased strain on the
metabolism, with reduced energy available for the construction of critical systems, or a
potential impact on the hatching trigger mechanism itself. For cephalopods, it is thought
that hatching may be triggered by the reduction in O2 and increase in CO2 concentration,
due to aerobic respiration, of the perivitelline fluid (PVF) in the egg sacs reaching a
critical threshold [54]. Ocean acidification conditions would increase the CO2
concentrations in the PVF, however, and would therefore be expected to reduce hatching
time and increase the proportion of premature hatchlings if it was affecting the trigger
mechanism. Rather, the delays seen indicate a developmental threshold which is not
reached due to a diminished rate of growth due to impacts on paralarval energetics.
46
The 400ppm level in trial 3 was skewed by an approximately day-long period of
deoxygenation due to the air system malfunction. Oxygen availability is an important
component of successful development in natural squid egg masses, with extended periods
of anoxia causing reduced hatching success [82]. The trial 3 400ppm paralarvae were
exposed to anoxic conditions for only a relatively brief period, so it follows that they
were skewed to a later hatching time. The 1600 and 2200ppm levels were oxygenated
during the malfunction, but were only exposed to ESL CO2 levels during that period, thus
these paralarvae skewed to earlier hatching times. It can be assumed, then, that without
the air system malfunction these paralarvae would likely have shown a similar pattern of
acidification response. The 400ppm results indicate the severity of impact of decreases in
environmental O2, a predicted effect of ocean acidification, as well as the potential for
compounding effects. Conversely, the 1600ppm and 2200ppm effects demonstrate the
potential for adaptability in developing embryos if high CO2 exposures are not consistent
over long periods.
Time to initiation of hatching increased from 12 days in trials 1 and 2 to 15 days
in trials 3 and 4. Even accounting for the malfunctions in trial 3, the overall pattern of
hatching changed between these trials sets. A much larger proportion, over 60%, of trial
4‟s 400ppm hatchlings hatched by day 2 compared to 25% of trial 3‟s 400 ppm stock, so
the deoxygenation effect occurred, but did not change the basic intrinsic timing. Hatching
time is driven primarily by ambient temperature, but this was consistent at around 20°C
for all trials [83]. The prime difference, then, between trials 1 and 2, and trials 3 and 4 is
time of year (a difference of about a month between trial sets) indicating the potential for
a seasonal effect on paralarval response. Parental environment can have a major effect on
47
the allocation of resources to gametes and impact the characteristics of the larvae [84].
Given seasonal variation in temperatures and coastal CO2 levels, parental exposure may
shift the adaptability of the paralarvae, but further work is needed to confirm this.
No significant differences were seen in hatching efficiency across CO2 levels or
between trials. Even at the highest CO2 exposures, most paralarvae hatched eventually,
even if it took a fair amount of time (up to fifteen days from initiation). These results are,
however, laboratory-based and do not incorporate the effects of predation, currents, and
other natural factors, which would impact the egg sacs [83]. The longer egg sacs in the
wild are exposed, the longer they have to be eaten or destroyed. 90% of paralarvae
hatched within five to seven days from initiation in all treatments, so the impact in tail
end of hatching is minimal to the population‟s success. However, the delays in overall
hatching time due to ocean acidification exposure represent a significant threat to the
clutch of paralarvae.
Mantle lengths were significantly shorter in paralarvae exposed to high CO2
conditions in trials 1, 2, and 4. The differentiation of trial 3‟s 2200ppm, which had
mantles the length of those exposed to ambient conditions, from that of the other trials
indicates that its period of reduced carbon exposure was sufficient to mitigate the major
physiological impacts of acidosis, or fell at a crucial developmental period. Within those
trials exposed to consistent ocean acidification conditions, impacts were seen at levels
above 1300ppm and included a notable reduction in mantle length of 0.1mm: an
approximate 6% decrease in body size. Predator-prey interactions are complex, and may
become altered under ocean acidification conditions [85]. While smaller paralarvae are
less noticeable to visual predators, they are disadvantaged in swimming speeds and
48
efficiency, since they function at slightly lower Reynold‟s numbers, and are less capable
of predator avoidance [86]. Size and swimming capability also contribute to a paralarva‟s
ability to locate and capture prey, so smaller paralarvae may have a reduced feeding
efficiency, which would impact population success [83].
Yolk sac size was a considerably variable quantity among hatchlings, but had no
trend associated with the CO2 exposures. Yolk consumption and within egg metabolic
rate are driven by temperature and therefore could be expected to not change severely
when temperature is held constant [80]. Except for trial 1‟s 550ppm exposure, which was
significantly larger, most values for yolk sac size were in the same ballpark, which
indicates a general consistency in yolk consumption rate regardless of stress conditions.
A consistent amount of yolk at hatching may also be an adaptation to ensure paralarvae
the energy required to swim to the surface and begin feeding [51]. Within trial 1 there
was a consistent decrease of yolk sac size with increasing CO2 exposure, but no other
trial demonstrated this pattern. This is either a random effect of sampling or there is a
potential effect of acidification on consumption rate that was not shown in the other trials
due to unknown factors. Continuation of this work is warranted to confirm the results
seen here.
Differences in development time between individual embryos could be
exacerbated by acidification conditions and would result in earlier hatchings that are less
impacted than later ones. Though mantle lengths were variable over the hatching period,
no distinct pattern of daily variation was seen. Likewise, yolk sac size showed strong
variability over the days of hatching, but no consistent trends of increase or reduction.
Thus, neither paralarvae that hatched earlier nor those that hatched later had an advantage
49
over the other. The most important factor differentiating groups of paralarvae from the
same egg sac in the wild, given any one particular CO2 exposure, is time and the
vulnerabilities that come from remaining in the egg sac versus entering the epipelagic
[51].
Significant variation was seen between cups for both mantle length and yolk sac
size; however patterns in variation of these two factors did not correlate. Within trial 1,
significant differences were seen between cups for both mantle length and yolk sac size,
though these differences were small compared to those between treatments. Cup 3 had
longer mantle lengths and smaller yolk sacs, while cup 1 was the opposite. This
difference could be a result of paralarvae consuming more yolk in cup 3 to produce
greater size, whereas cup 1 paralarvae reserved their yolk. However, cup 2 had both the
lower yolk sac size of cup 3 and shorter mantle lengths of cup 1, and all cups were
exposed to the same conditions, thus a direct physiological correlation is unlikely. Rather,
it appears that response is just inconsistent from egg sac to egg sac.
Variation among the cups could be explained by genetic differences between egg
sacs. Parentage is tricky among cephalopods. Egg mats can contain egg sacs from
multiple mothers and egg sacs can be fertilized by multiple fathers [73]. The mother‟s
ability to hold on to sperm packets for extended periods also compounds the complexity
of the genetic makeup of a given egg sac [87]. Since these phenomena occur in the wild
and egg sacs are naturally variable, the assumption to pool per treatment still makes sense
regardless of the variations, especially since variations were much smaller between cups
than they were between treatments. As described in the methods, trial 4 cups were set up
with egg sacs that were known to be genetically different since they came from separate
50
tanks. The pattern of variation expected if genetic differences were significant was cup 1
and cup 2 being significantly different, while cup 3 was of an average value between the
two. This pattern did occur in the trial 4 mantle length data (Figure 9), but was not seen
consistently in the hatching time or yolk sac data. This work thus warrants repeating, but
does at least indicate the potential for differing responses to stress based on parentage.
Analysis for parentage using genetic markers could thus be incorporated into future
experiments in order to map paralarvae to their progenitors and further tease out any
variation in stress response due to genetic factors.
Figure 9. Trial 4 mantle length variation between cups (pooled treatments). Egg sacs
laid the same morning in adult squid tanks A and B were distributed in the cups to look
for a potential genetic effect. Cup 1, AA; Cup 2 BB; Cup 3, AB;
Reductions in statolith surface area were prominent in paralarvae exposed to more
than 1300ppm, while statolith structure diminished in quality concurrent to CO2 increase.
Aragonite saturation decreased from supersaturated levels in ambient exposures to
undersaturated in high CO2 exposures (Table 1). Major calcifying organisms, such as
corals, tend to maintain aragonite deposition in acidifying conditions, but are impacted by
51
reductions in deposition rate or improper crystalline structure [42]. The increase in
porosity, calcite deposits, and disorganized crystalline bundles with increased CO2
exposure seen in D. pealeii statoliths follows this type of response. Cuttlefish, however,
have been shown to accrete more aragonite onto their cuttlebone as a response to severely
undersaturated conditions [65, 66]. Loligo vulgaris was shown to elicit a similar response
[67]. The clear reduction in surface area of D. pealeii statoliths therefore demonstrates a
differential response based on species life history and shows that general assumptions
cannot be expected across taxa.
The consistent trend that emerges from the hatching time data, the mantle length
data, and the statolith surface areas is a threshold of effect above 1300ppm. Only slight
effects are seen, excluding some malformation of the statoliths, at exposures of 1300ppm
and below. Whether this pattern is a direct step-wise effect or a linear reduction is not
clear from the current data. Pooled data tends to indicate a step-wise difference between
low exposures, 1300ppm and below, and high exposures, above 1300ppm, while
individual trial data occasionally showed a linear trend with increasing exposure.
Stress thresholds are an important criterion for understanding climate change
impacts as they help inform predictive models. Thermal stress thresholds are a key factor
of coral bleaching and frame the shifts in community structure that occur over warming
periods [88]. Carbonate and carbon dioxide thresholds for coral calcification have also
been explored to establish the points of reef dissolution [89]. The 1300ppm stress
threshold seen in these experiments are an acute threshold, representing the physiological
capacity of paralarvae given exposure over the entirety of embryonic development. This
threshold does not scale to the population level since it does not account for impacts of
52
exposure during juvenile growth and adulthood, or adaptability through successive
generations [90].
The pattern of ocean acidification responses seen in D. pealeii paralarvae
indicates that stress effects are caused due to a reallocation of the animal‟s energy budget.
Consistency in yolk sac size across treatments implies that it is important that some yolk
is preserved for post-hatching. Thus, assuming yolk consumption rate is conserved, the
energy needed to combat acidosis stress must be taken from another source, or rather,
redistributed from another process: anabolism. The reduction in somatic growth and
impaired system construction demonstrated by the reduced mantle lengths, delayed
hatching time, and degraded, malformed statoliths seen at increased CO2 exposures are
evidence of a decreased budget of energy for these processes.
Cephalopod response to increased temperature stress typically involves an
increase in metabolic rates resulting in stress effects through an overtaxing of the
energetic system [91,92]. The stress response to increased CO2 seen here in D. pealeii
appears instead to be a repartitioning of available energy, through the reduction of growth
and development, to mitigate acidosis. From the yolk sac data alone, it is unclear if the
pH/CO2 shifts also impact paralarval metabolic rate, however, so further study should
focus on establishing a more complete energetic picture of ocean acidification stress on
D. pealeii.
Effects on D. pealeii physiology above the 1300ppm threshold are potentially
detrimental to the success of the egg mass mainly in the increased risk of predation and
destruction over time, but the greater threat to D. pealeii populations stems from a
53
decrease in the survival rate of the paralarvae. The effect size reduction would have on
the success rate of predators of D. pealeii paralarvae is uncertain, but smaller,
energetically stressed paralarvae would have less capacity for efficient jet swimming
[86]. Statolith degradation should also have an impact on swimming behavior,
particularly in terms of directional control, which would translate to a reduced efficiency
for predator avoidance and prey capture. Swimming behavior studies on D. pealeii
paralarvae run in parallel with this study were not conclusive and must be expanded to
gain a better picture of the ecological impacts of ocean acidification.
Scaling laboratory-based physiological data to population and ecological scale
impacts is typically error-ridden since it often lacks the proper data to provide context to
the projections. The larger the magnitude of the process the greater the distance of the
scaling and the more uncertainty will be introduced into the analysis [93]. Responses
elicited in a controlled aquarium do not account for the variety or variation of factors
experienced naturally and so cannot be directly extrapolated to impacts on a species in a
wild setting. However, given the inherent complications of access to these animals in an
oceanic setting, the complexity of the system the animal is reacting to, and a limited to
impossible capacity to control for extraneous variables in the system, the best means by
which to examine the effects of individual factors is in the lab. Studies like the one
performed here provide pieces of information that can be compiled to provide a rough
projection, which does have to be viewed with scrutiny, but still allows for a greater
insight into the overall ecological picture.
The reductions seen in size of the mantle and statolith are likely to impact jet
swimming speeds and control, respectively, which could impact survival. Early
54
paralarvae spend their time in the surface layer bobbing up and down using jet
propulsions to catch prey and avoid predators. A reduction in paralarval survival rate can
translate to major decreases in population size, which could have severe ecological and
socioeconomic impacts [94]. D. pealeii are an important trophic web component as well
as a highly successful fishery, so decreases in population size due to climate change
would increase competition between wild predators and humans [95].
Extension of this work would need to complete the energetic picture of D. pealeii
paralarvae by examining the effect of ocean acidification exposure on metabolism. The
impact of reduced size, reduced development, and energetic stress on the initiation and
success of feeding or on the paralarva‟s ability to sense and operate within its
environment are unclear and requires further study. It is important to understand the
effects of ocean acidification on the physiology of D. pealeii and its life history, but no
single factor of climate change will result in shifts to populations and ecosystems; rather
it is the combination of factors that will have the greatest effect. Multifactor analyses of
climate change effects, as well as other relevant anthropogenic stressors, on this species
would better approximate the types of impacts we can expect to see.
Response to ocean acidification has been shown to be variable among the
cephalopod taxa, with adaptability dependent on the ecophysiology of the particular
species. This examination of D. pealeii increases our understanding of the diverse range
of responses to these stressors and demonstrates the need to explore representative
species based on life history as well as taxon. D. pealeii is a pelagic squid that does not
typically utilize low O2/ high CO2 zones in the ocean to hunt and hide as other squids do,
but lays its eggs in a relatively shallow coastal zone that is susceptible to significant CO2
55
variability. Future work exploring the dynamics of ocean acidification on Doryteuthis
pealeii must look not only at the metabolic picture of stress effects, but also elucidate the
current exposure of the wild population to coastal CO2 variability and the potential for the
species to mitigate ocean acidification stress in order to evaluate the potential for impacts
on this key species in the near future.
56
CONCLUSION
The goal of this research was to examine the effects of ocean acidification on the
early life stages of Doryteuthis pealeii, a pelagic squid that breeds in coastal waters.
Paralarvae demonstrated clear effects of ocean acidification stress at CO2 saturations
above 1300ppm. Malformations of the aragonitic statolith occurred at levels below
1300ppm, as an effect of the decrease in aragonite saturation, but the increase in porosity
and poorly structured crystals may not have as significant an impact on paralarval
gravitational sensing as the reduction in statolith size above 1300ppm could. This value
appears to be the upper bound after which developing embryos cannot compensate for the
stress of acidosis.
Above 1300ppm there were significant delays in hatching time as well as
reductions in mantle length and statolith surface area. Yolk sac size and hatching
efficiency were variable, but did not appear to be dependent on CO2 exposure. This
indicates that even at the highest CO2 exposure, 2200ppm, D. pealeii paralarvae are given
sufficient resources to develop and successfully hatch. The yolk sac reserve was
consistent across exposures, showing that some energy stores are conserved for the
hatchling phase of the squid life cycle. The metabolic response to CO2 stress is unclear,
but these results demonstrate that the energy budget has been reorganized to sacrifice
growth and development time in order to mitigate acidosis.
CO2 levels above 1300ppm could potentially occur in coastal waters, but it is
unknown if these levels are reached where the squid eggs are laid naturally. It is also
likely that these levels would not be sustained for the entire embryonic period as they
57
were here. However, given current climate models, inshore CO2 levels are expected to
increase much faster than open ocean levels, so the onset of these impacts on the current
squid population may occur sooner than expected.
Ocean acidification is an active and ongoing modern process, which is slowly
altering the chemical dynamics of the open ocean and rapidly amplifying natural CO2
variability in coastal continental shelf waters. The effects of ocean acidification on
organisms and ecosystems are diverse in type and magnitude, indicating a need for an
extensive study of organismal response and a contextual understanding of how those
effects could scale into ecological impacts. Stress responses depend on the life stage,
sensitivity, and physiological adaptability of the affected organism, as well as the strength
of the change to the physical environment. Having internal aragonitic structures, which
are susceptible to dissolution, increases susceptibility to high CO2 exposures. Stressed D.
pealeii embryos and paralarvae, which are highly sensitive life stages, need to construct
aragonitic statoliths and fundamental physiological systems, but have to spend more
energy mitigating acidosis as the pH lowers and the CO2 rises past a manageable
threshold. Thus, the costs of basic maintenance increase due to the stress effects of ocean
acidification and are expressed in the diminished development of early life stage D.
pealeii.
58
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