EFFECTS OF ACIDIFIED SEAWATER ON ASEXUAL

REPRODUCTION AND STATOLITH SIZE IN THE SCYPHOZOAN

CHRYSAORA COLORATA

A Thesis

Presented to the Faculty of

Moss Landing Marine Laboratories

And

California State University Monterey Bay

In Partial Fulfillment

of the Requirements for the Degree

Master of Science

in

Marine Science

by

Thomas S. Knowles

December 2012 iii

Copyright © 2012

by

Thomas S. Knowles

All Rights Reserved

iv

DEDICATION

I dedicate this work to my family and friends, whom I am very fortunate to have in my life.

v

It is advisable to look from the tide pool to the stars and then back to the tide pool again.

– John Steinbeck, The Log from the Sea of Cortez

vi

ABSTRACT

Effects of Acidified Seawater on Asexual Reproduction and Statolith Size in the Scyphozoan Chrysaora colorata by Thomas S. Knowles Master of Science in Marine Science California State University Monterey Bay, 2012

Absorption of anthropogenic atmospheric CO2 into the ocean surface is causing ocean acidification and chemistry changes that reduce calcification in organisms that form calcium carbonate skeletons and shells. Increased acidity also affects aspects of life history other than calcification, such as sexual reproduction and recruitment. Studies of scyphozoan jellyfish abundance have not reached a consensus on the effects of ocean acidification on jellyfish populations, and few laboratory studies have looked at the effects of acidified seawater on jellyfish biology. This study examined the effect of acidity on the benthic and early pelagic stages of the scyphozoan Chrysaora colorata and the formation of a calcium sulfate sensory structure, the statolith. Researchers who model future ocean surface pH levels predict a drop of 0.3-0.5 units from pre-industrial levels by 2100 if fossil fuel consumption continues at its current rate. To understand how these conditions will affect C. colorata, treatments of acidified seawater (pH = 7.85, 7.75, 7.65, and 7.55) and a control (pH = 7.97) were used to test the effects of ocean acidification on asexual reproduction (number of podocysts formed, number of new polyps formed, number of days to begin strobilation, duration of strobilation, number of healthy ephyrae released, and percentage of ephyrae that were healthy) and statolith size. There was no effect of acidity on asexual reproduction in C. colorata, but there was a significant negative effect of acidity on statolith size—this supports previous research on the scyphozoan Aurelia labiata. This study suggests that C. colorata will be able to survive and asexually reproduce from the polyp stage through the ephyra stage in near-future ocean conditions. Previous studies have shown that a lack of statoliths results in swimming abnormalities, but the effect of smaller statoliths is unknown. To fully understand how C. colorata will be affected by ocean acidification, further research needs to be conducted on other stages of the lifecycle. C. colorata and other scyphozoans play important roles in their ecosystems, and if their abundance is negatively affected then their predators, prey, and competitors will be affected as well. However, it is possible that the effects of ocean acidification on C. colorata and other scyphozoans will be subtle and that they could benefit from declines in the abundance of predators and competitors that are more sensitive to the chemistry changes of ocean acidification.

vii

TABLE OF CONTENTS

PAGE

ABSTRACT...... vi LIST OF FIGURES...... viii ACKNOWLEDGEMENTS...... ix INTRODUCTION...... 1 Chrysaora colorata ...... 2 Statoliths...... 4 METHODS ...... 7 Polyp Culture...... 7 Experimental Manipulation of pH ...... 7 Statolith Measurement...... 9 Statistical Analysis...... 9 Treatment Validation ...... 9 RESULTS...... 11 Effects of Acidity on Asexual Reproduction...... 11 Swimming Observations ...... 11 Effects of Acidity on Statolith Size...... 11 DISCUSSION ...... 14 Asexual Reproduction...... 14 Statolith Size...... 14 Implications for Scyphozoans...... 16 Further Research ...... 17 LITERATURE CITED...... 18

viii

LIST OF FIGURES

PAGE

Figure 1. An adult Chrysaora colorata in the pelagic environment (Photo: D. Wrobel)...... 2 Figure 2. (A) Podocysts of Chrysaora colorata photographed using a scanning electron microscope. (B) C. colorata polyp with an emerging stolon and podocysts that were formed by that polyp...... 3 Figure 3. Statoliths of Chrysaora colorata in the statocyst...... 5 Figure 4. (A) One replicate—a 12 L “critter keeper” tank that was divided into 5 sections with 500 µm mesh. (B) Five dishes per replicate, one in each section, each starting with 1 polyp. (C) Styrofoam boxes serving as temperature baths, with filtered Monterey Bay seawater continuously flowing into the boxes, around the critter keepers, and then exiting the boxes...... 8 Figure 5. (A) Mean pH (± 99% confidence intervals) in each replicate throughout the experiment. (B) Mean temperature (± 99% confidence intervals) in each replicate throughout the experiment...... 10 Figure 6. Mean values ±SE of (A) the number of podocysts formed and (B) the number of new polyps formed. Each dot represents a replicate of 5 polyps...... 12 Figure 6 cont. Mean values ±SE of (C) the number of days to begin strobilation, (D) the duration of strobilation, (E) the number of healthy ephyrae that were released, (F) the percentage of total ephyrae that were healthy, and (G) statolith size. Each dot represents a replicate of 5 polyps...... 13 Figure 7. The surface pH at Wharf 2 in Monterey, CA, from June to November 2012 (Moss Landing Marine Laboratories Public Data Portal)...... 16

ix

ACKNOWLEDGEMENTS

Funding for this project was obtained from the Monterey Bay Aquarium and from the The James Nybakken Scholarship and Friends of Moss Landing Marine Laboratories. I would like to thank my thesis committee: Jonathan Geller and Kenneth Coale of Moss Landing Marine Laboratories and Jim Barry of the Monterey Bay Aquarium Research Institute. This project benefited greatly from their world-class expertise, excellent guidance, and moral support. They spent many hours of their time making sure the science was sound and helping me to hone the thesis defense and final manuscript. Thank you especially to Jonathan Geller for being a great and supportive graduate school advisor during my time here at MLML, and thank you for being patient with my unorthodox schedule throughout the years. Ivano Aiello provided critical help with the measurement of the statoliths. He spent many hours helping me figure out how best to isolate and measure the tiny crystals, and he allowed me use of microscopes, cameras, and lab space in the MLML Geological Oceanography Lab. He also facilitated the use of the scanning electron microscope at MLML for photographing the podocysts. Mike Graham and Jim Harvey provided help with the statistical analysis. Both provided elegant solutions to difficult problems that I encountered during analysis of the data. Thank you to the MLML Invertebrate Zoology and Molecular Ecology Lab and the Monterey Bay Aquarium Husbandry staff for all of their support over the years. Thank you also to the Monterey Bay Aquarium Water Quality Lab for keeping the pH probe calibrated and providing much advice along the way. Thank you to Chad Widmer, who inspired me to study jellyfish and who helped me very much in the early stages of this project. Thank you to my parents, Ted and Gretchen Knowles, and my brothers, Sam and Dave, for supporting me since my earliest interest in marine biology when I was catching catfish and getting stung by Chrysaora quinquecirrha on the Eastern Shore of Maryland.

x

Thank you most of all to my wife Meghan who helped me through the hardest parts of this process. She also provided valuable feedback on layout and aesthetics as well as technical computer support. Thanks for always being there for me.

1

INTRODUCTION

Carbon dioxide emissions from burning fossil fuels are being absorbed through the surface of the ocean, causing a decrease in the pH of the ocean surface (Caldeira and Wickett 2003, 2005; Sabine et al. 2004; Cao et al. 2007; Steinacher et al. 2008). Oxides of anthropogenic sulfur and nitrogen in the atmosphere are also absorbed through the surface of the ocean, but the percentage of acidification due to these sources is minimal compared to the acidification caused by carbon dioxide absorption (Doney et al. 2007). The increase in dissolved carbon dioxide causes a decrease in the saturation state of calcium carbonate minerals such as calcite and aragonite (Doney et al. 2009). This results in reduced calcification in organisms that form calcium carbonate minerals to build skeletons and shells, such as pteropods (Orr et al. 2005), shellfish (Gazeau et al. 2007), corals (Kleypas et al. 2006; Silverman et al. 2009), and numerous others. However, because pH is a master variable affecting many aspects of both chemistry and physiology in biological systems, the effects of ocean acidification are not limited to calcification. Reproduction and recruitment of fish and invertebrates are affected as well, as shown by reduced sperm motility and fertilization rate in sea urchins (Havenhand et al. 2008) and reduced ability in clown fish to use olfactory cues to detect suitable habitat (Munday et al. 2009). Few studies have directly examined the effects of ocean acidification on jellyfish biology and ecology. It is important to learn how ocean acidification will affect jellyfish because they play very important roles in their ecosystems. They are prolific predators of fish eggs, larval fish (Purcell and Arai 2001; Purcell 2003), and zooplankton (Purcell 1997), including a wide variety of other gelatinous organisms (Purcell 1991), and are also important prey items for sea turtles, ocean sunfish, and numerous other marine animals (Arai 2005; Blumenthal et al. 2009). They engage in a variety of mutualistic, commensalistic, and parasitic relationships (Brodeur 1998), and they also impact many human activities (Purcell et al. 2007) by competing for resources with commercially

2 important fish species, clogging fishing nets and power plant intakes, killing fish in aquaculture pens, and stinging and occasionally killing beachgoers. The few studies on the impact of acidification on scyphozoans have not found consensus. Attrill et al. (2007) found a correlation between decreases in ocean pH and increases in jellyfish abundance. However, Richardson and Gibbons (2008) found no correlation, and they and Haddock (2008) rejected Attrill’s finding. Few laboratory studies have examined the effects of acidity on scyphozoans. Winans and Purcell (2010) studied Aurelia labiata and found no effect of acidity on asexual reproduction but a negative effect of acidity on statolith volume. This study examined the effect of acidity on the benthic and early pelagic stages of the purple-striped jelly, Chrysaora colorata (Figure 1), and the formation of a calcified sensory structure, the statolith.

Figure 1. An adult Chrysaora colorata in the pelagic environment (Photo: D. Wrobel).

CHRYSAORA COLORATA

The genus Chrysaora represents the sea nettles and is composed of 13 valid species around the globe, all of which possess the iconic feature of four oral arms that, in some species, can grow to tens of meters long (Morandini and Marques 2010). Chrysaora colorata occurs in the northeast Pacific and has a range from Bodega Bay, CA south to the Los Angeles county area (Wrobel and Mills 2003). The adults are easily identified by 16 purple stripes along the sides of the bell that radiate down from purple

3 ring at the apex of the bell and can grow to over 50 cm in bell diameter (Gershwin and Collins 2002). Male and female adult Chrysaora colorata medusae sexually reproduce in the pelagic environment, and the fertilized egg develops into a planula larva that settles on the substrate and develops into a scyphistoma polyp (Gershwin and Collins 2002). Polyps can asexually create new polyps. In the orders Rhizostoma and Semaeostoma (to which C. colorata belongs), polyps of many species deposit chitinous cysts called podocysts (Figure 2A) onto the substrate, and new polyps emerge from those podocysts (Chapman 1968; Blanquet 1972; Gershwin and Collins 2002). The podocysts of C. colorata are 200-500 µm in diameter (Gershwin and Collins 2002) and contain soft tissue with carbohydrate, lipid, and protein reserves (Chapman 1968). The podocyst is formed beneath the pedal disc of the polyp against the substrate. A stolon (Figure 2B) emerges from the stalk of the polyp near the base, attaches to the substrate, and the polyp releases from the podocyst and moves to where the stolon is attached, where it can then form a new pedal disc and a new podocyst (Cargo and Rabenold 1980).

A B

Podocysts Stolon

Figure 2. (A) Podocysts of Chrysaora colorata photographed using a scanning electron microscope. (B) C. colorata polyp with an emerging stolon and podocysts that were formed by that polyp.

The podocyst will lay dormant until a polyp emerges through the top of the cyst. This polyp will grow into a fully developed polyp that is capable of creating more podocysts and strobilating (Cargo and Rabenold 1980). This asexual reproduction results in the formation of dense polyp colonies (Arai 2009). The podocyst also serves as a way

4 for jellyfish to survive through unsuitable conditions (Chapman 1968). The energy reserves allow polyp colonies to survive when food for polyps is scarce, and the chitinous cyst protects the soft tissue from predation and unfavorable seawater conditions (Arai 2009). When conditions improve, a polyp can emerge from the podocyst and begin to reproduce. To create ephyrae (juvenile jellyfish), the Chrysaora polyps undergo another form of asexual reproduction called strobilation (Gershwin and Collins 2002). As with most scyphozoans, this occurs when the polyp metamorphoses into a strobila, a chain of ephyrae which are less developed near the base of the polyp and more developed at the end of the chain. One at a time, the ephyrae are released into the pelagic environment and grow up into adult medusae.

STATOLITHS

Aside from reproduction, other aspects of jellyfish development may be affected by ocean acidification. Of special interest is the one calcified part of a jellyfish, the statolith (Figure 3). Scyphozoan statoliths are made of calcium sulfate hemihydrate

(CaSO4•0.5H2O), otherwise known as bassanite or plaster of paris, and are formed during the strobila stage (Becker et al. 2005). Statoliths are located in the rhopalium, which is an assemblage of sensory structures located between the lappets in ephyrae and at the margin of the bell in adult medusae (Arai 1997). Chrysaora colorata has 8 rhopalia, each alternating with the 8 tentacles around the margin of the bell (Gershwin and Collins 2002). Each rhopalium contains several sensory organs: ocelli, which are simple eyes capable of detecting light and dark; chemosensory pits, which are possibly for detecting food; and one statocyst, the organ that senses gravity (Arai 1997). The statocyst contains numerous concretions, the statoliths, which settle onto sensory cells that line the inside of the statocyst. This enables a jellyfish to detect its orientation and swim normally and is therefore important for the survival of ephyrae and medusae in the pelagic environment. Spangenberg (1968) showed that ephyrae lacking statoliths were not able to swim normally. However, the effect of smaller statoliths on swimming ability is unknown.

5

By studying the effects of low pH on asexual reproduction and statolith development, we can gain an understanding of how acidification affects survival through the polyp, strobila, and ephyra stages of the lifecycle. The numbers of podocysts and new polyps formed in the polyp stage greatly influence the number of ephyrae that are produced. The length of time to begin strobilation and the duration of strobilation could also impact the number of ephyrae released in the strobila stage, which in turn could impact the number of individuals that survive to adulthood. Well-formed statoliths are necessary for normal swimming, which is likely strongly related to survivorship. If ocean acidification affects any of these factors in C. colorata, either positively or negatively, survival into the next stage of the life cycle could be affected, and population numbers could either increase or decrease.

Figure 3. Statoliths of Chrysaora colorata in the statocyst.

The average global ocean surface pH before the industrial revolution was around 8.2, and researchers who model future ocean surface pH levels predict a drop of 0.3-0.5 units from this level by 2100 if fossil fuel consumption continues at its current rate (Caldeira and Wickett 2005; Cao et al. 2007; Steinacher et al. 2008). This study seeks to determine the effects of near-future ocean conditions such as those predicted by these models, and therefore the treatments in this study consist of reduced-pH seawater ranging down to a pH of 7.55. The null hypothesis of this study is that there will be no differences between the reduced-pH treatments and the control in the number of new podocysts formed, the number of new polyps formed, the length of time to begin strobilation, the duration of strobilation, the number of healthy ephyrae released, the percentage of healthy ephyrae

6 released, and statolith size. The alternative hypothesis is that treatments with reduced pH will differ from the control.

7

METHODS

POLYP CULTURE

Mature Chrysaora colorata medusae were collected from Monterey Bay. Four medusae were placed in 60 L of 13º C seawater overnight for 18 hours, and planulae were collected from the tub in the morning. Planulae were placed into glass dishes where they settled and developed into polyps. The polyps were maintained at 20º C in seawater filtered to 15µm in the Monterey Bay Aquarium jelly lab. To select the experimental polyps, polyps were removed from the glass dishes, swirled in a beaker, and selected at random. Each polyp was placed into a 50 mm diameter glass dish and was maintained at 20º C while attaching to the dish.

EXPERIMENTAL MANIPULATION OF PH

The pH experiment consisted of a control of filtered Monterey Bay seawater and 4 treatments of reduced-pH seawater. The average pH of the control seawater during the experiment was 7.97. In the reduced-pH treatments, the targeted pH levels were 7.85, 7.75, 7.65, and 7.55. There were 3 replicates per treatment, each consisting of a 12 L “critter keeper” tank that was divided into 5 sections with 500 µm mesh, so that food and water could pass between the sections but ephyrae could not (Figure 4A). There were 5 polyps per replicate, 1 polyp in each section (Figure 4B). Seawater pH was reduced by bubbling carbon dioxide into a 50 L tub of filtered seawater. To dissolve the carbon dioxide into the seawater, carbon dioxide gas was released from a cylinder through a fine air stone, creating fine bubbles. The seawater was stirred with a rod during the bubbling to promote dissolution of the carbon dioxide gas into the seawater. As the carbon dioxide dissolved into the seawater, the pH of the seawater dropped—pH was measured using a Hach HQ11d portable pH meter. When the pH of the seawater in the tub reached the desired level, the bubbling was stopped. Replicate tanks were filled with the treated seawater, covered with parafilm to reduce gas

8 exchange, and left stagnant. Water changes were performed every 2-3 days, except for 2 5-day intervals, and polyps were fed Artemia salina nauplii after the water change. Because the water was stagnant, the pH in the replicates could change up to 0.07 pH units between water changes. Therefore, the pH was measured before and after each water change in order to determine the range of pH experienced within each replicate. Replicates were kept in temperature baths of filtered Monterey Bay seawater (Figure 4C), which was ~12.5ºC during the experiment, and the temperature in each replicate was measured before and after each water change. This reduction in temperature from 20ºC to 12.5ºC typically induces strobilation in C. colorata within a few weeks.

A C temperature baths

B

seawater inflow

dishes Figure 4. (A) One replicate—a 12 L “critter keeper” tank that was divided into 5 sections with 500 µm mesh. (B) Five dishes per replicate, one in each section, each starting with 1 polyp. (C) Styrofoam boxes serving as temperature baths, with filtered Monterey Bay seawater continuously flowing into the boxes, around the critter keepers, and then exiting the boxes.

Ephyrae were collected and counted at each water change. It is normal for some of the ephyrae released during strobilation to be deformed. Ephyrae that were shriveled or otherwise deformed were recorded as unhealthy. Each healthy ephyra was observed

9 for 1 minute to assess swimming ability. They were evaluated for the ability to pulse symmetrically and at a normal rate and the ability to keep themselves up in the water column. All ephyrae were then preserved in 10% buffered formalin. The experiment was run for 99 days and was ended when most strobilation had stopped or tapered greatly.

STATOLITH MEASUREMENT

Nine preserved ephyrae were taken from each replicate—three ephyrae from three different polyps. The soft tissue of the ephyrae was dissolved in a 10% bleach solution, and the remaining statoliths were photographed at 4X magnification. ImageJ® software was used to measure the area of the statoliths in the photographs.

STATISTICAL ANALYSIS

One-way ANOVAs were performed on the number of podocysts formed, the number of new polyps formed, the number of days to begin strobilation, the duration of strobilation in number of days, the number of healthy ephyrae released, and statolith area. For the analysis of statolith size, 700 data points (statolith area) were randomly chosen from each replicate to achieve an equal sample size in each replicate; then, the data were transformed using the natural log in order to meet the assumptions for ANOVA. A Kruskal-Wallis test was used to analyze the percentage of healthy ephyrae in each replicate because the data did not meet the assumptions for ANOVA.

TREATMENT VALIDATION

To validate the pH of the seawater in each treatment, the average pH in each replicate throughout the experiment was plotted with 99% confidence intervals (Figure 5A). While a target pH was aimed for in each treatment, the actual average pH was off by .01 in some replicates. However, the error bars within each treatment all overlap, so the pH of all of the replicates within each treatment were not significantly different. Also, the graph demonstrates that the pH values in each treatment were distinct from all the other treatments.

10

The mean temperature of each replicate throughout the experiment was also plotted with 99% confidence intervals (Figure 5B). This graph shows that the temperatures in all the replicates were not significantly different, and this is important to show that any observed effects were not due to a difference in temperature.

8.05 A 7.9 5 7.85

pH 7.75

7.65

7.55 Treatment

♦ Control 7.45 ♦ 7.85

13.0 ♦ 7.75

B ♦ 7.65 12.8 ♦ 7.55

12.6

12.4

Temperature (ºC) 12.2

12.0 Replicate

Figure 5. (A) Mean pH (± 99% confidence intervals) in each replicate throughout the experiment. (B) Mean temperature (± 99% confidence intervals) in each replicate throughout the experiment.

11

RESULTS

EFFECTS OF ACIDITY ON ASEXUAL REPRODUCTION

Polyps were able to form podocysts in all pH treatments (Figure 6A). ANOVA showed no effect of pH treatment on the number of podocysts formed (F(4,10) = 2.512, p = 0.108). New polyps emerged from podocysts in all treatments (Figure 6B). ANOVA showed no effect of pH treatment on the number of polyps formed (F(4,10) = 1.249, p = 0.351). In addition, polyps in all treatments were able to transition from the polyp to the strobila stage. ANOVA showed no effect of pH treatment on the number of days to begin strobilation (F(4,10) = 0.882, p = 0.508) (Figure 6C). ANOVA showed no effect of pH treatment on the duration of strobilation (F(4,10) = 3.304, p = 0.057) (Figure 6D). Strobilae in every treatment produced healthy ephyrae (Figure 6E), with no significant differences among treatments (F(4,10) = 1.214, p = 0.364). In all the treatments, a high percentage of the ephyrae produced were healthy and not deformed (Figure 6F). The Kruskal-Wallis test showed no effect of pH treatment on the percentage of healthy ephyrae (KW test statistic = 3.648, p = 0.456). Based upon theses observations, the null hypothesis was not rejected.

SWIMMING OBSERVATIONS

During the 1 minute observations that were conducted on each healthy ephyra, no swimming abnormalities were observed. All ephyrae were able to pulse symmetrically and at a normal rate, and they were all able to keep themselves up in the water column.

EFFECTS OF ACIDITY ON STATOLITH SIZE

ANOVA did show a significant effect of pH treatment on statolith size (F(4,10) = 4.068, p = 0.033) (Figure 6G). Tukey’s post hoc test showed significant differences between the 7.75 and 7.85 treatments, and the Student-Newman-Keuls test showed that the 7.75 treatment was significantly different from all other treatments.

12

One replicate in the 7.85 treatment seemed anomalously high (statolith area = 300.12 µm2), and it seemed that possibly it was driving the data toward a significant result when there was actually no real effect of pH. Performing the ANOVA with this replicate removed resulted in a non-significant result (p = 0.056), but the new p value was only slightly different from the original p value. Since the results did not change dramatically with the removal of this replicate, this replicate alone did not drive the result and the null hypothesis was rejected. There is indeed an effect of pH on statolith size.

14 5 A A B 12

4

10

3 8

6 2

# Podocysts # Polyps New 4 1 2

0 0 7.55 7.65 7.75 7.85 7.97 7.55 7.65 7.75 7.85 7.97

Treatment pH Treatment pH

Figure 6. Mean values ±SE of (A) the number of podocysts formed and (B) the number of new polyps formed. Each dot represents a replicate of 5 polyps.

13 60 C

50

40

30

20

10 # to DaysBegin Strobilation

0

80 90

D E 70 80

70 60

60 50

Ephyrae 50

40 40

30

30

20 # Healthy 20

10 10

Duration of Strobilation (# Days) 0 0

100 350 F G 300

80 ) 2 250

60 200

40 150

100 StatolithSize (µm

% Healthy Ephyrae 20 50

0 0 7.55 7.65 7.75 7.85 7.97 7.55 7.65 7.75 7.85 7.97

Treatment pH Treatment pH

Figure 6 cont. Mean values ±SE of (C) the number of days to begin strobilation, (D) the duration of strobilation, (E) the number of healthy ephyrae that were released, (F) the percentage of total ephyrae that were healthy, and (G) statolith size. Each dot represents a replicate of 5 polyps.

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DISCUSSION

ASEXUAL REPRODUCTION

This study provides insight into how well Chrysaora colorata will survive from the polyp through the ephyra stages in acidified ocean conditions. No negative effects of decreasing pH were found on the ability of C. colorata polyps to survive and asexually produce new polyps and ephyrae down to a pH of 7.55, a range that is representative of possible ocean conditions in the year 2100. These results support the findings of Winans and Purcell (2010) who found no effects of pH on the asexual reproduction of Aurelia labiata.

STATOLITH SIZE

This study did find a significant negative effect of acidity on statolith size. As pH decreased, statolith size also decreased. This also supports the findings of Winans and Purcell (2010) who found a negative effect of acidity on statolith volume in Aurelia labiata. The cause of the decrease in size of the bassanite statoliths in scyphozoans is likely different from the processes that cause reduced calcification in organisms that form calcium carbonate minerals. Unlike calcium carbonate, calcium sulfate is not dependent upon carbonate ion concentrations. While gypsum (calcium sulfate dihydrate,

CaSO4•2H2O) in general is insoluble, studies of bassanite indicate that this mineral's solubility has only a weak dependence upon pH (Shukla et al. 2008). Although there are several studies of bassanite solubility under extreme conditions relevant to industrial processes such as flue gas scaling and power plant cooling water treatment, there are few if any studies of bassanite solubility under the conditions relevant to seawater at natural temperature and pH. In a study of bassanite under industrial conditions, Azimi et al. (2007) showed that even under relatively concentrated sulfuric acid conditions, bassanite was only weakly soluble, even at a pH of 0. This pH is far from any climate change

15 scenario, yet even under such conditions both bassanite and gypsum are extremely insoluble. This suggests that the differences in statolith size in this study are related to processes other than dissolution solubility. Research on marine invertebrates has shown that acidification due to elevated CO2 reduces ion transfer, protein synthesis, and metabolic rates (Pörtner et al. 2005; Lannig et al. 2010). In addition, acidification has been shown to reduce the expression of genes important to biomineralization in sea urchins (O’Donnell et al. 2010; Stumpp et al. 2011; Hammond and Hofmann 2012), cephalopods (Hu et al. 2011), and corals (Moya et al. 2012). Reduced statolith size in Chrysaora colorata is likely the result of the effects of acidification on either the physiological processes involved in the biomineralization of bassanite or the expression of genes that code for proteins that are important to those processes. Previous research has shown that a lack of statoliths results in severe swimming abnormalities (Spangenberg 1968). However, the effects of smaller statoliths on swimming ability are unknown. No swimming abnormalities were observed in the ephyrae in this study, however a big question remains unanswered: how will the statoliths continue to develop and function as the ephyrae grow up into adult medusae in an acidified ocean? If swimming ability is negatively affected, feeding and sexual reproduction in adult medusae could be negatively impacted, and that could affect the abundance of jellyfish populations. If scyphozoan jellyfish populations decline, there will be significant ecosystem- wide effects. Many organisms that consume jellyfish (Arai 2005; Blumenthal et al. 2009) will be negatively impacted by reduced prey availability, and those that engage in commensal relationships with jellyfish (Brodeur 1998) will also suffer. Jellyfish are important predators of zooplankton, fish eggs, and larval fish (Purcell 1997; Purcell and Arai 2001; Purcell 2003), and a decrease in jellyfish abundance could impact the abundance of both their prey and their competitors for that prey.

16

IMPLICATIONS FOR SCYPHOZOANS

Previous studies on the effects of pH on jellyfish abundance disagree regarding how acidification will affect population numbers (Attrill et al. 2007; Richardson and Gibbons 2008; Haddock 2008). It is possible that the direct effects of acidification on the physiology of Chrysaora colorata may be subtle, but that C. colorata will be most affected indirectly by the success or failure of their predators, prey, and competitors in future ocean conditions. Organisms native to coastal upwelling systems experience a wide range of pH levels, as currents from the deep ocean regularly bring more acidic seawater up to the surface. Organisms in Monterey Bay, CA, experience a wide range of pH over the course of a few months, and at times in just a few days (Figure 7). These organisms may have tolerance for more acidic conditions. They may not perform optimally at these conditions, but they are still able to survive and reproduce up to a point.

8.4

8.2

8.0

pH 7.8

7.6

7.4 June 17 July 7 July 27 Aug 16 Sept 5 Sept 25 Oct 15 Nov 4 2012 2012 2012 2012 2012 2012 2012 2012

Date

Figure 7. The surface pH at Wharf 2 in Monterey, CA, from June to November 2012 (Moss Landing Marine Laboratories Public Data Portal).

17

As ocean acidification takes its toll across the ecosystem, there will be winners and there will be losers. If C. colorata survives the direct physiological effects of lower pH, it could fill the ecological void left by organisms that are more sensitive to the chemistry changes of ocean acidification. C. colorata and other scyphozoans could end up being the winners in an acidified ocean.

FURTHER RESEARCH

To fully understand how Chrysaora colorata will be affected by acidification, further research needs to be conducted on the rest of the life cycle. We must know how acidification will affect growth rates of ephyrae and medusae, and the effects on fertilization rate and other aspects of sexual reproduction are especially interesting questions given the reduced fertilization rate in sea urchins (Havenhand et al. 2008). In addition, the effects of acidification on scyphozoan planula settlement and polyp growth have yet to be studied. Holger et al. (2011) found no effect of pH on planula settlement in corals and found that primary polyp growth in corals was only marginally affected. To understand the full range of physiological effects on C. colorata, we will need to understand the effects on survival between all stages of the life cycle.

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LITERATURE CITED

Arai MN. 1988. Interactions of fish and pelagic coelenterates. Canadian Journal of Zoology 66:1913–1927. Arai MN. 1997. A functional biology of Scyphozoa. London: Chapman & Hall. Arai MN. 2005. Predation on pelagic coelenterates: a review. Journal of the Marine Biological Association of the United Kingdom 85:523-536. Arai MN. 2009. The potential importance of podocysts to the formation of scyphozoan blooms: a review. Hydrobiologia 616:241-246. Attrill M, Wright J, Edwards M. 2007. Climate-related increases in jellyfish frequency suggest a more gelatinous future for the North Sea. Limnology and Oceanography 52(1):480-485. Azimi KG, Papangelakis VG, Dutrizac JE. 2007. Modeling of calcium sulfate solubility in concentrated multicomponent sulfate solutions. Fluid Phase Equilibria 260(2):300-315. Becker A, Sotje I, Paulmann C, Beckmann F, Donath T, Boese R, Prymak O, Tiemann H, Epple M. 2005. Calcium sulfate hemihydrate is the inorganic mineral in statoliths of scyphozoan medusae (Cnidaria). Royal Society of Chemistry 1545-1550. Blanquet RS. 1972. Structural and chemical aspects of the podocyst cuticle of the scyphozoan medusa, Chrysaora quinquecirrha. Biological Bulletin 142(1):1-10. Blumenthal JM, Austin TJ, Bell CDL, Bothwell JB, Broderick AC, Ebanks-Petrie G, Gibb JA, Luke KE, Olynik JR, Orr MF, Solomon JL, Godley BJ. 2009. Ecology of Hawksbill Turtles, Eretmochelys imbricata, on a Western Caribbean Foraging Ground. Chelonian Conservation and Biology 8(1):1-10. Brodeur RD. 1998. In situ observations of the association between juvenile fish and scyphomedusae in the Bering Sea. Marine Ecology Progress Series 163:11-20. Caldeira K, Wickett M. 2003. Anthropogenic carbon and ocean pH. Nature 425:365. Caldeira K, Wickett M. 2005. Ocean model predictions of chemistry changes from carbon dioxide emissions to the atmosphere and ocean. Journal of Geophysical Research 110:C09S04. Cao L, Caldeira K, Jain A. 2007. Effects of carbon dioxide and climate change on ocean acidification and carbonate mineral saturation. Geophysical Research Letters 34:L05607. Cargo DG, Rabenold GE. 1980. Observations on the asexual reproductive activities of the sessile stages of the sea nettle Chrysaora quinquecirrha (scyphozoan). Estuaries 3(1):20–27.

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Chapman DM. 1968. Structure, histochemistry and formation of the podocyst and cuticle of Aurelia aurita. Journal of the Marine Biological Association of the United Kingdom 48:187-208. Doney SC, Mahowald N, Lima I, Feely RA, Mackenzie FT, Lamarque J, Rasch PJ. 2007. Impact of anthropogenic nitrogen and sulfur deposition on ocean acidification and the inorganic carbon system. Proceedings of the National Academy of Science 104(37):14580-14585. Doney SC, Balch WM, Fabry VJ, Feely RA. 2009. Ocean acidification: a critical emerging problem for the ocean sciences. Oceanography 22(4):16-25. Gazeau F, Quiblier C, Jansen J, Gattuso J, Middleburg J, Heip C. 2007. Impact of elevated CO2 on shellfish calcification. Geophysical Research Letters 34:L07603. Gershwin L, Collins AG. 2002. A preliminary phylogeny of Pelagiidae (Cnidaria, Scyphozoa), with new observations on Chrysaora colorata comb. nov. Journal of Natural History 36:127-148. Haddock S. 2008. Reconsidering evidence for potential climate-related increases in jellyfish. Limnology and Oceanography 53(6):2759-2762. Hammond LM, Hofmann GE. 2012. Early developmental gene regulation in Strongylocentrotus purpuratus embryos in response to elevated CO2 seawater conditions. The Journal of Experimental Biology 215:2445-2454. Havenhand H, Buttler F, Thorndyke M, Williamson J. 2008. Near-future levels of ocean acidification reduce fertilization success in a sea urchin. Current Biology 18(5):R651. Holger A, D’Croz L, O’Dea A. 2011. A corrosive concoction: the combined effects of ocean warming and acidification on the early growth of a stony coral are multiplicative. Journal of Experimental Marine Biology and Ecology 397(1):13- 20. Hu MY, Tseng Y, Stumpp M, Gutowska MA, Kiko R, Lucassen M, Melzner F. 2011. Elevated seawater pCO2 differentially affects branchial acid-base transporters over the course of development in the cephalopod Sepia officinalis. American Journal of Physiology—Regulatory, Integrative, and Comparative Physiology 300:R1100-R1114. Kleypas JA, Feely RA, Fabry VJ, Langdon C, Sabine CL, Robbins LL. 2006. Impacts of ocean acidification on coral reefs and other marine calcifiers: a guide for future research, report of a workshop held 18-20 April 2005, St. Petersburg, FL, sponsored by NSF, NOAA, and the U. S. Geological Survey, 88 pp. Lannig G, Eilers S, Portner HO, Sokolova IM, Bock C. 2010. Impact of ocean acidification on energy metabolism of oyster, Crassostrea gigas—changes in metabolic pathways and thermal response. Marine Drugs 8:2318-2339. Morandini AC, Marques AC. 2010. Revision of the genus Chrysaora Péron & Lesueur, 1810 (Cnidaria: Scyphozoa). Zootaxa 2464:1-97.

20

Moss Landing Marine Laboratories Public Data Portal. 2012. Monterey Wharf II Monitoring Station. Available from: http://www.ysieconet.com/public/WebUI/Default.aspx?hidCustomerID=265 Moya A, Huisman L, Ball EE, Hayward DC, Grasso LC, Chua CM, Woo HN, Gattuso J- P, Foret S, Miller DJ. 2012. Whole transcriptome analysis of the coral Acropora millepora reveals complex responses to CO2-driven acidification during the initiation of calcification. Molecular Ecology 21(10):2440-2454. Munday PL, Dixson DL, Donelson JM, Jones GP, Pratchett MS, Devitsina GV, Døving KB. 2009. Ocean acidification impairs olfactory discrimination and homing ability of a marine fish. Proceedings of the National Academy of Sciences 106(6):1848-1852. O’Donnell MJ, Todgham AE, Sewell MA, Hammond LM, Ruggiero K, Fangue NA, Zippay ML, Hofmann GE. 2010. Ocean acidification alters skeletogenesis and gene expression on larval sea urchins. Marine Ecology Progress Series 398:157- 171. Orr J, Fabry V, Aumont O, Bopp L, Doney S, Feely R, Gnanadesikan A, Gruber N, Ishida A, Joos F, Key R, Lindsay K, Maier-Reimer E, Matear R, Monfray P, Mouchet A, Najjar R, Plattner G, Rodgers K, Sabine C, Sarmiento J, Schlitzer R, Slater R, Totterdell I, Weirig M, Yamanaka Y, Yool A. 2005. Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms. Nature 437:681-686. Pörtner HO, Langenbuch M, Michaelidis B. 2005. Synergistic effect of temperature extremes, hypoxia, and increases in CO2 on marine animals: from Earth history to global change. Journal of Geophysical Research 110:C09S10. Purcell JE. 1991. A review of cnidarians and ctenophores feeding on competitors in the plankton. Hydrobiologia 216/217:335-342. Purcell JE. 1997. Pelagic cnidarians and ctenophores as predators: selective predation, feeding rates, and effects on prey populations. Annales de l'Institut océanographique 73:125-137. Purcell JE. 2003. Predation on zooplankton by large jellyfish, Aurelia labiata, Cyanea capillata and Aequorea aequorea, in Prince William Sound, Alaska. Marine Ecology Progress Series 246:137-152. Purcell JE. 2005. Climate effects on formation of jellyfish and ctenophore blooms: a review. Journal of the Marine Biological Association of the United Kingdom 85:461-476. Purcell JE. 2007. Environmental effects on asexual reproduction rates of the scyphozoan Aurelia labiata. Marine Ecology Progress Series 348:183-196. Purcell JE, Arai MN. 2001. Interactions of pelagic cnidarians and ctenopores with fish: a review. Hydrobiologia 451:27-44.

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Purcell JE, Uye S, Lo W. 2007. Anthropogenic causes of jellyfish blooms and their direct consequences for humans: a review. Marine Ecology Progress Series 350:153- 174. Richardson A, Gibbons M. 2008. Are jellyfish increasing in response to ocean acidification? Limnology and Oceanography 53(5):2040-2045. Sabine CL, Feely RA, Gruber N, Key RM, Lee K, Bullister JL, Wanninkhof R, Wong CS, Wallace DWR, Tilbrook B, Millero FJ, Peng T, Kozyr A, Ono T, Rios AF. 2004. The ocean sink for anthropogenic CO2. Science 305:367-371.

Shukla J, Mohandas VP, Kumar A. 2008. Effect of pH on the solubility of CaSO4•2H2O in aqueous NaCl solutions and physicochemical solution properties at 35º C. Journal of Chemical Engineering Data 53:2797-2800. Silverman J, Lazar B, Cao L, Caldeira K, Erez J. 2009. Coral reefs may start dissolving when atmospheric CO2 doubles. Geophysical Research Letters. 36:L05606. Spangenberg D. 1968. Statolith differentiation in Aurelia aurita. Journal of Experimental Zoology 169(4):487-499. Spangenberg D, Beck C. 1968. Calcium sulfate dihydrate statoliths in Aurelia. Transactions of the American Microscopical Society 87(3):329-335. Steinacher M, Joos F, Frapglicher T, Plattner G. 2008. Ocean acidification in the Arctic over the 21st century. 2008 Ocean Sciences Meeting: From the Watershed to the Global Ocean. American Geophysical Union, 2000 Florida Ave., N.W. Washington DC 20009 USA. Available from: http://www.agu.org Steinbeck J. 2000 [1951]. The log from the Sea of Cortez. London: Penguin Books.

Stumpp M, Dupont S, Thorndyke MC, Melzner F. 2011. CO2 induced seawater acidification impacts sea urchin larval development II: gene expression patterns in pluteus larvae. Comparative Biochemistry and Physiology Part A: Molecular and Integrative Physiology 160(3):320-330. Winans AK, Purcell JE. 2010. Effects of pH on asexual reproduction and statolith formation of the scyphozoan, Aurelia labiata. Hydrobiologia 645:39-52. Wrobel D and Mills C. 1998. Pacific coast pelagic invertebrates: a guide to the common gelatinous animals. Monterey, CA: Sea Challengers.