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2021

Down in arms: Marine climate stress inhibits growth and

calcification of regenerating Asterias forbesi (Echinodermata:
Asteroidea) arms
Hannah L. Randazzo
Bowdoin College

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Recommended Citation
Randazzo, Hannah L., "Down in arms: Marine climate stress inhibits growth and calcification of
regenerating Asterias forbesi (Echinodermata: Asteroidea) arms" (2021). Honors Projects. 222.
https://digitalcommons.bowdoin.edu/honorsprojects/222

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 Down in arms: Marine climate stress inhibits growth

and calcification of regenerating Asterias forbesi (Echinodermata: Asteroidea) arms

An Honors Paper for the Department of Biology

By: Hannah L. Randazzo

Bowdoin College, 2021

© 2021 Hannah L. Randazzo

 ABSTRACT
Anthropogenic CO2 is changing the pCO2, temperature, and carbonate chemistry of
seawater. These processes are termed ocean acidification (OA) and ocean warming. Previous
studies suggest two opposing hypotheses for the way in which marine climate stress will
influence echinoderm calcification, metabolic efficiency, and reproduction: either an additive or
synergistic effect. Sea stars have a regenerative capacity, which may be particularly affected while
rebuilding calcium carbonate arm structures, leading to changes in arm growth and calcification.
In this study, Asterias forbesi were exposed to ocean water of either ambient, high temperature,
high pCO2, or high temperature and high pCO2 for 60 days, and the regeneration length of the
amputated arm was measured weekly. Ocean acidification conditions (pCO2 ~1180 μatm) had a
negative impact on regenerated arm length, and an increase in temperature of +4°C above ambient
conditions (Fall, Southern Gulf of Maine) had a positive effect on regenerated arm length, but the
additive effects of these two factors resulted in smaller regenerated arms compared to ambient
conditions. Sea stars regenerating under high pCO2 exhibited a lower proportion of calcified mass,
which could be the result of a more energetically demanding calcification process associated with
marine climate stress. These results indicate that A. forbesi calcification is sensitive to increasing
pCO2, and that climate change will have an overall net negative effect on sea star arm
regeneration. Such effects could translate into lower predation rates by a key consumer in the
temperate rocky intertidal of North America.
Keywords: Echinoderm; Ocean Acidification; Ocean Warming; Arm Regeneration; Calcification

INTRODUCTION
Due to the burning of fossil fuels, atmospheric levels of carbon dioxide (CO2) have been
steadily rising since the industrial revolution. Since water is polar, it readily dissolves carbon
dioxide, making the oceans a natural carbon sink (Siegenthaler and Sarmiento 1993; Feely et al.
2004). Between 30 and 40% of anthropogenic carbon emissions have entered the oceans in the
last two centuries (Sabine et al. 2004; Zeebe et al. 2008). The resulting chemical reaction between
CO2 and water in the ocean results in the formation of hydrogen ions, bicarbonate, and carbonate.
These excess free hydrogen ions increase acidity, leading to a decrease in pH from the typical
North America sea surface pH. Recent projections have predicted that ocean pH will decrease by
approximately 0.3–0.4 by 2050 and 0.5–0.6 by 2100 (Gulf of Maine RCP8.5 and BNAM
simulation; Caldeira and Wickett, 2003). This phenomenon is called ocean acidification.
Simultaneously, increased atmospheric CO2 emits absorbed heat from sunlight, warming air
temperatures worldwide and subsequently warming the oceans. IPCC predictions suggest a North
America Atlantic average sea surface temperature increase of 3 to 4°C by the year 2100 from a
current autumn average of 11°C regardless of seasonal changes in temperature (Sen Gupta et al.
2009; Alexander et al. 2018). Marine climate stress refers to the combined impacts of ocean
warming and ocean acidification.
Although the separate impacts of ocean acidification and ocean warming on marine life have
been widely studied, particular concern revolves around the capacity for marine climate stress and
carbonate ion availability to inhibit the ability of marine calcifiers to construct calcium carbonate
(CaCO3) compounds (Fabry et al. 2008). Recent studies have documented decreases in
calcification in a variety of plant and animal species due to ocean acidification (Gattuso et al.
1998; Riebesell et al. 2000; Gazeau et al. 2007; Miller et al. 2009; Ries et al. 2009; Kroeker et al.
2010). In particular, echinoderms, which have calcium carbonate endoskeletons, have been shown
to experience a variety of negative effects in response to reduced pH (Dupont et al. 2010).
Decreasing acidity reduces the concentration of carbonate in the water and promotes the
dissolution of calcium carbonate by reacting with the carbonate anion. Additionally, ocean

1
acidification conditions have been shown to disrupt the acid-base balance of internal calcifiers,
resulting in a more energetically demanding calcification process (Michaelidis et al. 2005;
Catarino et al. 2012).
Since echinoderms form their skeletons from high magnesium-calcite (>4% mol MgCO3), the
most water-soluble form of calcium carbonate, calcification will be more energetically demanding
under marine climate stress conditions, causing echinoderms to be particularly vulnerable to the
effects of ocean acidification (Morse et al. 2006; Gilbert and Wilt 2011). Conversely, metabolic
efficiency has been shown to increase in marine organisms in response to increased oceanic
temperatures (Byrne et al. 2013; Carey et al. 2016). Specifically, Dupont et al. (2010), suggesting
that calcification and growth rates of sea stars will increase under ocean warming conditions due to
the higher metabolic rate. Therefore, the confounding variable of temperature may compensate for
the negative effects of ocean acidification, so it is important to examine the combined effects of
ocean acidification and ocean warming on marine calcification processes.
The vulnerability of echinoderms to climate change variables may be enhanced while
echinoderms are regenerating, a developmental process unique to echinoderms, which involves
simple tissue repair or replacement of lost limbs following trauma (Mladenov et al. 1989).
Calcification rates increase exponentially during sea star (Asteroidea) and brittlestar
(Ophiuroidea) arm regeneration (Hu et al. 2014), and while regenerating, echinoderms reform
entirely new calcium carbonate endoskeletons. Various studies have analyzed the separate effects
of either ocean acidification or ocean warming on the echinoderm arm regeneration process, and a
few have analyzed both factors in simultaneously (Bingham et al. 2000; Dupont and Thorndyke
2006; Wood et al. 2008; Wood et al. 2010; Wood et al. 2011; Hu et al. 2014). Counterintuitively,
Wood et al. (2008) found an increase in the regeneration rate and the percentage of calcium
carbonate in regenerating arms of the temperate brittle star Amphiura filiformis exposed to ocean
acidification conditions. Yet, these authors also found that muscle tissue volume decreased in
regenerating arms with increasing pCO2, suggesting an important ecological tradeoff in the
metabolic support of faster regeneration rates. Brittle stars have also been used to simultaneously
study the effects of ocean acidification and temperature on regeneration, with different results that
appear to depend on the species and the native environment. For the temperate brittle star
Ophiura ophiura, Wood et al. (2010) found high temperatures resulted in faster regeneration rates,
with ocean acidification playing a relatively minor (but positive) role in arm regeneration. In
contrast, for the Arctic brittle star Ophiocten sericeum, Wood et al. (2011) found a strong negative
effect of high ocean acidity on arm regeneration that counteracted a positive effect of temperature.
Negative effects of ocean acidification on arm regeneration have also been found in sea stars
(Asteroidea). For example, Schram et al. (2011) found that Luidia clathrata regenerated at a faster
rate under ambient pCO2 conditions than under ocean acidification conditions. These results
suggest the interactions between temperature and ocean acidification on arm regeneration are
complex and species specific, impacting both net calcification rates and tissue structure and
composition.
The present study analyzed the combined effects of marine climate stress on Asterias forbesi
(Asteroidea) arm regeneration by measuring changes to the length of regenerating arms under
ambient conditions in the Gulf of Maine, high temperature conditions, high pCO2 conditions, and
high pCO2 and high temperature conditions. A proxy for calcium carbonate content of the
regenerating arm in comparison to the established arms was also measured between the treatments
to determine the way in which marine climate stress affects the proportion of calcified to tissue
mass. Due to the decrease in carbonate availability and the energetic costs of calcification under
ocean acidification conditions and the increased metabolic efficiency under ocean warming
conditions, I hypothesized that sea stars regenerating in the high pCO2 condition would have the
lowest regeneration and calcification rates in comparison to sea stars regenerating in the high
2
temperature condition. However, it was unclear as to whether combining these two variables
would result in an overall positive or negative effect on calcification and regeneration rates
compared to present-day conditions. Although recent projections suggest that the effects of
marine climate stress will worsen within the next century, the combined impacts of these
processes on sea star arm regeneration are still relatively unexplored. This leaves gaps in
knowledge regarding how a top predator in the intertidal community will be impacted at its most
vulnerable stage (Menge et al. 2003). Thus, this study will provide further insight into how marine
climate stress impacts the fundamental regeneration process of the sea star.

METHODS
Organism collection
Asterias forbesi (N=24) were collected from Rockland Breakwater in Rockland, ME
(44°6′14″N 69°4′41″W) on September 8th, 2020. All experimental organisms had no observable
history of regeneration and ranged between 30 - 70 mm in average arm length. Sea stars were
maintained in the running seawater laboratory of the Schiller Coastal Studies Center in
Harpswell, ME for one week prior to the experiment which ran from September 10th, 2020 to
November 11th, 2020. Organisms were transported from the collection site in coolers.
Throughout the experiment, sea stars were fed ~0.2g of a thawed scallop (Singleton’s Faroe
Island 40/80 brand) once a week to sustain growth (e.g., Schram et al. 2011).
Arm amputation
The length (in mm) of each organism’s established arm was measured from the center of
the organism’s oral disc to the tip of each arm using Image J software. An average of the
established arm lengths was taken to determine the mean arm length for each organism. One arm
of each organism was removed with a scalpel along the natural plane of autonomy. Gender was
determined by the presence or absence of gonads at the time of amputation.
Assignment to treatments
Sea stars were grouped based on the average initial length of the established arms. The
average length of the established arms was used as a proxy for the size of the sea star. The groups
consisted of the smallest sea stars (<40 mm), medium-sized sea stars (between 40 and 60 mm),
and large sea stars (>60 mm). A representative sample from these three size groups were
assigned to different treatments. Each treatment had at least one sea star from each size group.
Reepithelialization period
The arm regeneration process can be broken down into several stages: (1) wound closure
and reepithelialization, (2) formation of new tissue and healing of damaged tissue, and (3)
growth of new dermis layers (Mladenov et al. 1989). Stage one (reepithelialization) takes
approximately two weeks. Post amputation, sea stars were maintained in seawater for their
assignment treatment for reepithelialization (15 days), or the restoration of epithelium over a
wounded area by natural regrowth.
Marine climate stress experiment
In this study, pCO2 was manipulated by changing the carbon dioxide concentration and
temperature of the seawater. A CO 2 system, containing four header tanks, was used to mirror
current and marine climate stress conditions (Figure 1). As per four models run under
business-as-usual carbon dioxide emissions (RCP8.5) and a Bedford Institute of Oceanography
North Atlantic Model (BNAM) simulation showing projected sea surface pH and temperature
values for the Gulf of Maine by the year 2050, we chose our marine climate stress setpoints to be
reflective of this model: a pH of 7.50 and a temperature of 4°C above current sea water

3
temperatures. The ambient treatment was the pCO2 of the incoming seawater with no CO2
treatment and no temperature treatment (see Table 1 for averages and ranges). The high
temperature treatment was the pCO2 of the incoming seawater with no CO2 treatment and a
temperature setpoint of +4°C above the ambient seawater temperature. Temperature control was
achieved by InkBird controllers and submersible heaters. The high pCO2 treatment was achieved
by bubbling CO2 gas into an equilibrator to achieve a pH setpoint of 7.50 (see Table 1 for pCO2
conversions) through a Neptune Apex control system (Morgan Hill, California). Finally, the high
pCO2 and high temperature treatment seawater used both setpoints described previously. Each
header tank had a continuous flow-through of filtered seawater and fed six experimental units of
the same pH and temperature treatment containing one sea star. The pH and temperature were
measured and recorded continuously every ten minutes throughout the experimental period. Sea
stars were exposed to their respective treatments for 60 days.

Figure 1. Experimental setup depicting the manipulation of seawater pCO2 and temperature for each of the four
experimental treatments. (A) incoming unfiltered seawater from Gulf of Maine, (B) CO2 (g) mixing tank, (C)
pressurized CO2 storage tank, (D) addition of treated water into header tanks, (E) pH probe and water heaters, (F)
recorded measurements of pCO2, temperature, and dissolved oxygen, (G) header tanks, (H) PVC piping connecting
header tanks to (J) individual experimental units via (I) pliable piping tubes. Header tank treatments labeled above
tank with (-) indicating no added treatment and (+) indicating increased temperature or pCO2.

Table 1. Temperature, pH, and estimated pCO2 of seawater used in the marine climate stress treatments for A.
forbesi (mean and SE). Each treatment (ambient, increased temperature, or increased pCO2) was measured in two
header tanks (a and b). Estimated pCO2 was calculated assuming a TA of 2222 μmol/kg SW, an average
measurement for the Gulf of Maine (https://data.noaa.gov/).
Treatment Parameter Tank a Tank b Average
Ambient CO2 pH 8.02 ±0.05 8.04 ± 0.06 8.03
pCO2 (μatm) 487.2 ± 42.7 494.3 ± 45.2 490.7
High CO2 pH 7.50 ± 0.08 7.51 ± 0.07 7.50
pCO2 (μatm) 1195.2 ± 55.2 1169.1 ± 62.1 1182.2
Ambient Temperature Temp. (°C) 11.3 ± 3.1 11.5 ± 2.9 11.4
High Temperature Temp. (°C) 16.2 ± 3.2 16.3 ± 3.5 16.2

4
pCO2 calculation from TA and pH
Four commonly measured parameters of the aqueous carbonate system are total alkalinity
(TA), dissolved inorganic carbon (DIC), the partial pressure of carbon dioxide (pCO2), and pH.
The measurement of any two of these parameters allows for the calculation of the remaining
carbonate system parameters using equilibrium constants and in-situ temperature data. We
calculated pCO2 from pH values measured by the Apex system, total alkalinity (TA) assumed to
be constant over the course of the experiment (2222 μmol/kg SW average daily TA over the last
ten years; US Department of Commerce, NOAA Data Catalog, Total Alkalinity 2010-2020), and
temperature measurements using carbonic acid dissociation constants of Millero (1979) and the
CO2 solubility from Weiss (1974). U.S. Geological Survey Florida Shelf Ecosystems Response
to Climate Change Project (Robbins et al. 2010) provided conversion software using Equation i.
Hunt et al. (2011) reported a discrepancy lower than 2 % for pCO2 computed this way.
TA [H +]2
(i) [CO2] = K1[H+] + 2K1K2

Data Collection
The wet weight, buoyant weight, and the length of each established arm were measured
twice weekly throughout the experiment. The wet weight (accuracy 0.001 g) of each organism
was taken on a top-loading balance following a brief blotting period on a towel. The buoyant
weight (accuracy 0.001 g) was taken using a water- submerged basket that was suspended from
the top-loading balance (Davies 1989). These measurements served as a proxy for the sea star’s
growth rate over the course of the experiment. The length of the regenerating arm was also
measured twice weekly over the course of the experiment. This measurement was used as a
proxy for each sea star’s regeneration rate.
Measurement of arm calcification
While buoyant weight has been used as a proxy for the proportion of calcified compounds
in previous studies, this study found no relationship between buoyant weight and calcified mass
measured as in Equation ii (Palmer 1982; Appendix A). Therefore, buoyant weight cannot be
used as a proxy for the proportion of calcified mass in the regenerating arm. Shilling and
Manahan (1994) found that ash weight (AW)—the total inorganic mass—can be used as a proxy
for the proportion of calcium carbonate compounds in a sample. Therefore, ash weight was used
as a proxy for calcified mass in this study. At 60 days post amputation, the regenerating arm and
a similarly sized portion of one of the established arms were amputated with a scalpel, covered in
aluminum foil, and weighed using a top loading balance. The samples were then frozen at -24°C
for twelve hours. Dry weight (DW) was determined by drying the arm portions for 24 hours in a
muffle furnace at 60°C and weighing these portions. The ash weight was determined by
combusting the dried arms in the muffle furnace for three hours at 550°C and weighing these
portions. Results were expressed as the proportion of the arm mass that was calcified.
AW
(ii) Proportion inorganic (calcified) mass =
DW

Statistical analysis
A two-way analysis of variance (ANOVA) was used to assess the effects of seawater
temperature and pCO2 on sea star arm regeneration length, proportion of calcified mass in the
regenerated arm, and proportion of calcified mass in the established arm. A three-way analysis of
variance (ANOVA) was used to assess the effects of seawater temperature, seawater pCO2, and
gender on sea star arm regeneration length. A two-way analysis of covariance (ANCOVA) with
sea star size as the covariate was used to assess the effects of seawater temperature and pCO2 on

5
sea star arm regeneration length. All statistical analysis and visualizations were conducted in R
studio version 1.2.1578 (R Core Team 2019). Regeneration length data was normalized for sea
star size, as determined by the average established arm length. Data points for regeneration
length during the reepithelialization period (0 - 15) days post amputation were omitted from the
dataset due to differences in energetics associated with wound closure and healing in comparison
to that of normal regeneration. The number of days post amputation for the buoyant weight data
were transformed (y = x2) since this data were not linear (Appendix B).

RESULTS
Seawater Temperature and pCO2
Ambient temperature data from the header tanks revealed a steady decline from a
maximum of 14.3 °C on September 11th, 2020 to a minimum of 7.69 °C at the end of the
experiment on November 11th, 2020. The mean difference in temperature between the ambient
and high treatments was 3.23°C and showed a declining trend as the experiment progressed
(Appendix C). This decline was due to difficulty with providing enough heating wattage as the
setpoint diverged from the cooling ambient temperature. The mean difference in pCO2
throughout the experiment was 695.2 μatm pCO2, with a few anomalous values mostly due to
periods of low environmental pCO2 (Appendix C). Both header tanks with treated pCO2 and
temperature showed very little variation from each other (Figure 2).

Figure 2. (A) ΔpCO2 and (B) ΔTemperature throughout the experimental period. The black line indicates ambient
pCO2 or temperature, the blue line indicates the difference between the ambient value and the setpoint value in tank
a, and the red line indicates the difference between the ambient value and the setpoint value in tank b.

Arm Regeneration
Sea stars regenerating in high temperatures and at ambient pCO2 had the highest growth
rates, followed by sea stars at ambient temperature and pCO2 (Figure 3). The high pCO2
treatment had suppressed arm regeneration at both temperatures. A two-way ANOVA of relative
mean regeneration length at 60 days post amputation revealed a significant interaction between
6
pCO2 and temperature (Table 2), indicating that temperature only influenced arm regeneration
length at ambient pCO2 values. Additionally, both temperature and pCO2 individually had a
significant effect on sea star arm regeneration rates (p<0.001).

Figure 3. Mean amputated arm regeneration length (mm) normalized for sea star size (average established arm
length) as a function of the number of days post amputation for A. forbesi (N=24) exposed to marine climate stress
conditions. Blue points correspond to ambient temperature treatments, red points correspond to high temperature
treatments, closed circles correspond to high pCO2 treatments, and open circles correspond to ambient pCO2
treatments. Each point represents average measurements for sea stars in the same treatment. Error bars represent
±SEM. The lines depict the line of best fit (exponential) (Table 2).

Table 2. Results from two-way analysis of variance (ANOVA) statistical analysis evaluating the effect of CO2 and
temperature treatments on relative sea star arm regeneration length. (*) indicated significant interactions (p<0.05).
Regeneration Length (mm)
Source of variation Df Mean Sq F value P
CO2 1 3.79 107.44 <0.001*
Temperature 1 0.88 24.94 <0.001*
Temperature : CO2 1 0.53 15.03 <0.001*
Residuals 428 0.035

Smaller sea stars generally had the fastest rates of arm regeneration, and this effect appeared
to be greatest in the high temperature and ambient pCO2 treatment (Figure 4). However, the effect
of sea star size does not significantly change the effects of pCO2 and temperature on the
regeneration rate. This reinforces the need to normalize regeneration rates according to sea star
size. A two-way ANCOVA with size as the covariate revealed a significant interaction between
7
temperature and CO2, further indicating that the effect of temperature is only significant under
ambient pCO2 conditions (Table 3). Further, none of the interactions with size (the covariate) were
significant, indicating that the experimental treatments did not impact the relationship between
size and regeneration rate (e.g. the slopes).

Figure 4. Amputated arm regeneration length (mm) as a function of average established arm length (mm) for A.
forbesi exposed to marine climate stress conditions at 60 days post amputation. Blue points correspond to ambient
temperature treatments, red points correspond to high temperature treatments, closed circles correspond to high
pCO2 treatments, and open circles correspond to ambient pCO2 treatments. Dashed vertical lines represent sea star
size ranges: small (left), medium (middle), and large (right). Each point represents average measurements for an
individual sea star (N=24). The lines depict the line of best fit (linear regression) (Table 3).

Table 3. Results from two-way analysis of covariance (ANCOVA) statistical analysis evaluating the effect of CO2
and temperature treatments with sea star size as a covariate on sea star arm regeneration length. (*) indicated
significant interactions (p<0.05).
Regeneration Length (mm)
df Mean Sq F value P
Size 1 0.80 1.25 0.26
CO2 1 57.20 89.39 <0.001*
Temperature 1 13.62 21.28 <0.001*
Size : Temperature 1 0.02 0.037 0.85
Size : CO2 1 0.26 0.41 0.53
CO2 : Temperature 1 8.11 12.67 <0.001*
Size : CO2 : Temperature 1 0.50 0.76 0.38
Residuals 424 0.64

Arm Calcification
Marine climate stress also impacted the proportion of inorganic (calcified) mass present
in the regenerating and established arms. This study found a significant decrease in the
proportion of calcified mass in both the regenerating and established A. forbesi arms exposed to
the high pCO2 conditions in comparison to the ambient condition (Table 4). This decrease in

8
calcified mass was accompanied by a decrease in buoyant weight for sea stars regenerating in the
high pCO2 treatment (Appendix A). Additionally, there was a significant increase in the
proportion of calcified mass in only the regenerating A. forbesi arms exposed to the high
temperature treatment in comparison to the ambient treatment. The pattern in the proportion of
calcified mass in both regenerating and established arms was approximately the same among all
treatments (Figure 5). However, there was a significant interaction between temperature and
pCO2 in the analysis of calcified mass in the regenerating arm (Figure 5, Table 4). Indicating, as
in the overall regeneration rates, that high pCO2 mediates temperature effects in the growth of the
established arms.

Figure 5. Mean proportion calcified mass (A) in the sampled regenerating arm and (B) in the sampled established
arm portions as a function of treatment for A. forbesi (N=24) exposed to marine climate stress conditions at 60 days
post amputation. In the box plots, the boundary of the box closest to zero indicates the 25th percentile, the line within
the box marks the mean, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers above
and below the box indicate the 10th and 90th percentiles. Points above and below the whiskers indicate outliers outside
the 10th and 90th percentiles (Table 4).

Table 4. Results from two-way analysis of variance (ANOVA) statistical analysis evaluating the effect of CO2 and
temperature treatments on (A) the proportion of calcified mass in the regenerating arm and (B) the proportion of
calcified mass in the established arms. (*) indicated significant interactions (p<0.05).
A Proportion calcified mass in B Proportion calcified mass in
regenerating arm established arm
Source of variation df Mean Sq F value P df Mean Sq F value P
CO2 1 0.28 28.09 <0.001* 1 0.17 41.74 <0.001*
Temperature 1 0.064 6.35 0.020* 1 0.0042 1.042 0.32
CO2 : Temperature 1 0.0089 0.88 0.36 1 0.032 7.77 0.011*
Residuals 20 0.010 20 0.0041

9
Gender
A. forbesi are a dioecious species, indicating that male and female reproductive organs
occur in separate individuals. There has been no observed evidence of hermaphroditism in this
species (Vevers 1952; Mercier and Hamel 2013). The gender of the sea stars as determined
during dissection post amputation appears to be correlated to variation in regeneration length
across marine climate stress conditions (Figure 6). While the gender factor was not significant in
the three-way ANOVA (Table 5), there was a pattern of female sea stars regenerating longer arm
lengths than male sea stars (Figure 6). The only factor significant in this analysis was CO2
treatment, and no interactions were significant (Table 5).

Figure 6. Mean amputated arm regeneration length (mm) as a function of the experimental treatment with A. forbesi
(N=24) separated according to gender at 60 days post amputation. In the box plots, the boundary of the box closest
to zero indicates the 25th percentile, the line within the box marks the mean, and the boundary of the box farthest
from zero indicates the 75th percentile. Whiskers above and below the box indicate the 10th and 90th percentiles.
Points above and below the whiskers indicate outliers outside the 10th and 90th percentiles. Red indicates female sea
stars and blue bars indicate male sea stars in each (Table 5).

Table 5. Results from three-way analysis of variance (ANOVA) statistical analysis evaluating the effect of gender,
CO2 treatment, and temperature treatment on sea star arm regeneration length. (*) indicated significant interactions
(p<0.05).
Regeneration Length (mm)
Source of variation Df Mean Sq F value P
Temperature 1 3.04 2.04 0.172
CO2 1 6.96 4.67 0.046*
Gender 1 0.13 0.09 0.773
Temperature : CO2 1 0.49 0.33 0.573
Temperature : Gender 1 3.31 2.22 0.156
CO2 : Gender 1 1.27 0.85 0.370
Temperature : CO2 : Gender 1 0.67 0.31 0.584
Residuals 16 1.49

DISCUSSION
Arm regeneration
The role of calcium carbonate as a building block for structures such as shells, skeletons and
spines is well recognized. Although decreased calcification under marine climate stress conditions
is not universal in echinoderms (e.g. Wood et al. 2008; Wood et al. 2010; Wood et al. 2011), much
of the concern regarding ocean acidification stems from evidence suggesting the increased
seawater pCO2 will impair function in affected structures. Various studies suggest that the ability
10
of sea stars (Asteroidea) and brittle stars (Ophiuroidea) to form their calcified endoskeletons is
compromised under marine climate stress conditions (Bingham et al. 2000; Dupont and
Thorndyke, 2006; Wood et al. 2008; Dupont et al. 2010; Schram et al. 2011; Hu et al. 2014). The
present study found a reduction in overall arm regeneration length and calcification of Asterias
forbesi regenerating under climate change conditions. As in other studies of echinoderms (Wood
et al. 2010; Wood et al. 2011; Schram et al. 2011; Catarino et al. 2012; Byrne et al. 2013; Carey et
al. 2016), ocean warming increased the rate at which sea stars are able to regenerate and calcify,
but ocean acidification equivalent to a change of 0.5 pH units (or ~700 μatm pCO2) overrode this
positive effect resulting in lower regeneration than in present ambient conditions in the Gulf of
Maine. This result indicates that ocean acidification will suppress the positive effects of ocean
warming and that ocean acidification may be the dominant factor influencing regeneration and
calcification success under near future ocean conditions. Since arm loss and regeneration is a
typical phenomenon experienced by over a third of sea stars throughout their lifetime (Bingham et
al. 2000) and has been shown to negatively impact foraging rates (Campbell 1987; Diaz-Guisado
et al. 2006; Appelhans et al. 2014) ocean acidification is likely to result in functional changes that
can potentially re-structure New England intertidal communities (Lubchenco and Menge 1978).
In analyzing the effects of current and near future ocean conditions on A. forbesi arm
regeneration, this study found that sea stars regenerating at a lower pH experienced reduced arm
regrowth in comparison to those regenerating under ambient pCO2, regardless of the temperature
variable. This is most likely due to a disruption in the sea star’s internal acid-base balance as a
result of ocean acidification. The maintenance of extracellular pH is essential for protecting
invertebrate species against hypercapnia induced disturbances. One of the main acid–base
regulation mechanisms is associated with osmoregulation of active ion transport across cell
membranes (Catarino et al. 2012). Due to a lack of carbonate availability, which is the normal
building block of calcium carbonate endoskeletons, sea stars utilize the high abundance of
bicarbonate ions to form calcium carbonate endoskeletons (Dupont and Thorndyke 2012). In
order to do this, sea stars remove the excess hydrogen from the bicarbonate ion and release this
proton from their ossicles (calcifying cells). This reduces the pH at the site of calcification
(Appelhans et al. 2012). Although this process allows sea stars to regulate the internal pH, this
process is active, meaning that it takes energy to pump the protons out against the concentration
gradient. Over time, the concentration of hydrogen ions outside of the ossicles increases, leading
to an increased energetic cost that could be too high to overcome in order to regenerate an arm
quickly (Fabry et al. 2008). Uthicke et al. (2013) found that the underlying mechanism
responsible for dealing with ocean acidification in calcifying Acanthaster planci larvae was
directly responsible for an increased metabolism associated with the internal acid-base regulation.
In other words, A. planci larvae redirected energy from other processes in order to calcify more
effectively. This regulation was also correlated with a reduction in energy availability and the total
scope for growth. Overall, the mechanism through which sea stars internally calcify could explain
inhibited growth of the regenerating arm for A. forbesi exposed to the high pCO2 condition and
the high temperature and high pCO2 condition.
Arm Calcification
Sea stars regenerating in the ambient condition were found to have a higher proportion of
inorganic compounds in both the regenerating and the established arms in comparison to sea stars
regenerating in the marine climate stress conditions. This can act as a proxy for the proportion of
calcified structures (inorganic) in the arms (Davies 1989). This result suggests that sea stars
regenerating in the high pCO2 conditions, regardless of temperature, had an overall lower
proportion of calcified structures in the regenerating arm than the ambient condition. For sea
stars, forming organic structures is thought to be more energetically costly than the energy

11
required to form calcium carbonate structures (Lawrence 2010). Under ocean acidification
conditions, though, the energy required to regulate the internal acid-base balance would be greater
than the normal energy required to form calcium carbonate structures (Lawrence and Moran
1992). The results of this study show that the increased metabolic efficiency induced by ocean
warming in the high temperature and high pCO2 condition was not sufficient to maintain the
regeneration and calcification rates seen under current ocean chemistry conditions. Therefore,
regenerating calcium carbonate compounds under ocean acidification conditions is more
energetically costly than regenerating these structures under ambient conditions, which could
explain why the sea stars in this study regenerated at lower rates and a lower proportion of
calcified mass.
Furthermore, a lower proportion of calcified mass was accompanied by a higher proportion
of tissue mass in sea stars regenerating under combined marine climate stress conditions (data not
shown). This suggests that sea stars exposed to the increased pCO2 conditions may have been
allocating energy to tissue growth rather than calcification. Sea stars store nutrients in tissue
organs, particularly the pyloric caeca (Lawrence 2010). Schram et al. (2011) noted an increase in
pyloric caeca mass in sea stars regenerating under ocean acidification conditions, which was
attributed to the way in which sea stars can improve energy storage while the calcification process
is energetically unfavorable. However, a larger tissue to calcified mass ratio has the potential to
inhibit locomotor and foraging abilities as well as making the sea star more susceptible to
predation due to reduced arm strength.
The results of this study contradict Wood et al.'s (2008) findings that brittle stars
regenerating under ocean acidification conditions have greater percentage of calcium carbonate in
their arms. Since Ophiuroid arms are structurally and functionally different from Asteroid arms,
brittle stars structurally lose a smaller volume of tissue and calcified mass during amputation than
sea stars. Additionally, brittle star nutrient storage organs are located in the oral disc whereas sea
star nutrient storage organs are located in their arms (Ezhova et al. 2015). Energetically, sea stars
lose approximately 1475 kJ of energy per arm lost (Lawrence and Moran 1992), whereas brittle
stars only lose approximately 4.48 kJ per arm (Lawrence 2010). This could explain why brittle
stars are able to regenerate a larger proportion of calcified mass than sea stars. Therefore, since
brittle stars do not lose a significant portion of their energy storage during arm amputation, they
may be able to invest more energy in compensating for the disruption in the acid-base balance
allowing them to regenerate lost arms more quickly and efficiently than sea stars under near
future marine climate stress conditions (Catarino et al. 2012). Sea stars, on the other hand, cannot
compensate for these changes. This could potentially explain differences observed between
different Echinoderm genera.
Functional ecological costs of reduced arm regeneration
The cost of echinoderm arm loss includes decreased locomotor performance, loss of nutrient
reserves, reduced reproductive output and growth, and altered social interaction (Bourgoin and
Guillou 1994; Pomory and Lawrence 1999; Fleming et al. 2007; Bateman and Fleming 2009;
Hernroth et al. 2010). Since sea star arms can function in feeding, locomotion, and reproduction
(Appelhans et al. 2014), these processes will be directly impacted by the loss of these arms. One
of the most significant changes will be to foraging behavior. Diaz-Guisado et al. (2006) found that
Stichaster striatus (Asteroidea) experienced a 67% decrease in food consumption that persisted up
to five months post amputation. They concluded that the observed reduction in stomach contents
was most likely due to difficulty foraging while regenerating an arm. A reduction in calorie intake
would mean that more energy would need to be redirected from other processes in order for the
sea star to regenerate quickly. This problem would be further exacerbated by the unfavorable
energetics involved in internal calcification under marine climate stress conditions. Since the sea

12
stars in this study consistently consumed approximately 0.2 g of scallops each week throughout
the study, reduction in calories due to difficulty foraging was not a factor in the regeneration rate.
However, due to results indicating that marine climate stress negatively impacts the overall
regeneration rate of sea stars, the aforementioned processes will be affected for a longer period of
time. This will leave sea stars more vulnerable in the intertidal.
In addition to specific functional costs of arm loss, the regeneration process has also been
shown to affect overall life history strategies of echinoderms (Scheibling 1980; Lawrence and
Moran 1992; Shilling and Manahan 1994; Gilbert and Wilt 2011). The regeneration process
creates added stress⁠—the decreased acquisition and deposition of energy⁠—and disturbance⁠—the
removal of energy from the organism⁠(Lawrence 2010). According to Grime’s (1977) life-history
strategies, organisms develop a competitive strategy to cope with the added stress and disturbance
during regeneration. This primarily involves reinvestment in the regeneration of the lost structure,
such as an arm. If the organism is able to return to its ‘stable’ state by regenerating more quickly,
even if this involves the redirection of energy, the organism will be more competitively fit. One
way that organisms have evolved to deal with this stress is to decrease the frequency and length of
the arm lost. Many species, not including Asterias, have developed multiple planes of autonomy
to decrease the length of arm loss so that the organism can regenerate more quickly. Another
solution involves the deterrence of predation by hiding or developing chemical defenses
(Lawrence 2010). Evolutionarily, the physiology of the organism could be altered so that
regeneration rates may be enhanced. Scheibling (1980) found that O. reticulatus (Asteroidea),
which have large, broad arms, were often not able to reepithelialize their open wound, causing
them to die. He suggests that, evolutionarily, sea stars will most likely develop thinner arms to
cope with wound closure. Since marine climate stress in general will exacerbate the current
problems of arm regrowth and wound closure, as shown by this study, sea stars will need to
evolve to cope with these changes to seawater chemistry. If this evolution does not occur, this
could result in a lower abundance of sea stars in the intertidal, which would have functional
impacts for the entire intertidal community structure (Lubchenco and Menge 1978).
Gender
One specific energetic trade off that was examined in this study was the relationship
between the gender of the sea star and its regeneration rate. This study found a trend of male sea
stars regenerating a smaller arm length than females in the same experimental condition, however,
this relationship was not significant. The observed trend could be explained by the energetic costs
of producing eggs versus sperm (Raymond et al. 2004; Lawrence 2010). The energetic investment
in reproduction is three times higher for males during the reproductive season than females due to
the production of a larger volume of gametes. However, the energetic investment of producing a
larger gamete volume is not entirely outweighed by the female’s production of larger eggs at a
smaller volume.
Conclusions
In summary, the present study found that the common sea star, Asterias forbesi, exposed to
the additive effects of near-future marine climate stress conditions (+4°C above ambient
conditions (Fall, Southern Gulf of Maine) and a pCO2 ~1180 μatm) regenerated lost arms more
slowly with a smaller proportion of calcified mass than under current sea water chemistry
conditions. These significant differences in calcification and regeneration rates have the potential
to negatively affect locomotor ability, reproductive output, foraging ability, and predatory
avoidance, leaving a once keystone predator more vulnerable in the intertidal. Therefore, given
the current trends of marine climate stress projections for the next century, we can expect to
observe changes to the overall intertidal community structure.

13
 ACKNOWLEDGEMENTS
This research is part of a Biology Department Honors project completed at Bowdoin
College. Bowdoin College’s Schiller Coastal Studies Center provided facilities. Funding was
provided by the Grua/O’Connell Independent Research Grant from Bowdoin College. I thank my
project advisor, D. Carlon with other notable contributors including O. Ellers, A. Johnson, S.
Allen, B. Whalon, J. Baumann, and H. Franklin.

14
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APPENDIX

Appendix A. Total calcified mass (g) as a function

of buoyant weight (g) for A. forbesi (N=24)
exposed to marine climate stress conditions. Blue
points correspond to ambient temperature
treatments, red points correspond to high
temperature treatments, closed circles correspond
to high pCO2 treatments, and open circles
correspond to ambient pCO2 treatments. Each point
represents average measurements for sea stars in
the same treatment. Error bars represent ±SEM.

Appendix B. Mean buoyant weight

(g) normalized for sea star size
(based on averaged established arm
length) as a function of the number
of days post amputation for A.
forbesi exposed to marine climate
stress conditions. Blue points
correspond to ambient temperature
treatments, red points correspond to
high temperature treatments, closed
circles correspond to high pCO2
treatments, and open circles
correspond to ambient pCO2
treatments. Each point represents
average measurements of sea stars
in the same treatment.

19
 Appendix C. Properties of
incoming seawater
throughout the
experimental period: daily
average (A) pCO2 and (B)
temperature (°C). Blue
lines correspond to
ambient treatments and
red lines correspond to
either CO2 or temperature
treated measurements.
Black lines depict the line
of best fit (linear
regression).

20