Dissertation Submitted to the Department of Biological Sciences of the Florida Institute of Technology in Partial Fulfillment of the Requirements for the Degree Of

Home , Menippe adina, Nephrops norvegicus

THE EFFECTS OF ELEVATED pCO2 AND TEMPERATURE ON

EMBRYONIC DEVELOPMENT, LARVAL SURVIVORSHIP, CONDITION,

CALCIFICATION, MORPHOLOGY, AND BEHAVIOR OF THE FLORIDA

STONE CRAB, MENIPPE MERCENARIA

PHILIP MICHAEL GRAVINESE

B.S., Florida Institute of Technology M.S., Florida Institute of Technology

A dissertation submitted to the Department of Biological Sciences of the Florida Institute of Technology in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY in BIOLOGICAL SCIENCES

Melbourne, Florida November 2016

THE EFFECTS OF ELEVATED pCO2 AND TEMPERATURE ON

EMBRYONIC DEVELOPMENT, LARVAL SURVIVORSHIP, CONDITION,

CALCIFICATION, MORPHOLOGY, AND BEHAVIOR OF THE FLORIDA

STONE CRAB, MENIPPE MERCENARIA

A DISSERTATION

PHILIP MICHAEL GRAVINESE

Approved as to style and content by:

______Robert van Woesik, Ph.D., Andrew Palmer, Ph.D., Member Chairperson Assistant Professor Professor Department of Biological Sciences Department of Biological Sciences

______Richard B. Aronson, Ph.D., Member John Windsor, Ph.D., Member Professor and Head Professor Emeritus, Department of Biological Sciences Florida Institute of Technology

______Ralph G. Turingan, Ph.D., Member Professor Department of Biological Sciences

November 2016

THE EFFECTS OF ELEVATED pCO2 AND TEMPERATURE ON EMBRYONIC DEVELOPMENT, LARVAL SURVIVORSHIP, CONDITION, CALCIFICATION, MORPHOLOGY, AND BEHAVIOR OF THE FLORIDA STONE CRAB, MENIPPE MERCENARIA

by Philip Michael Gravinese, B.S. Florida Institute of Technology M.S., Florida Institute of Technology

Chairperson of Advisory Committee: Robert van Woesik, Ph.D.

ABSTRACT

The emission of greenhouse gases into the atmosphere over the last century has increased atmospheric and oceanic temperatures, and has led to a decrease in oceanic pH. The increased ocean temperatures and reduced pH have been detrimental to marine life. In addition, Florida’s watersheds have suffered from decades of disrupted hydrology and diverted runoff, which has increased nutrients and changed coastal carbonate chemistry. Although the adult stages of many marine species are capable of tolerating fluctuations in environmental conditions (i.e., elevated pCO2 and temperature), marine larvae that hatch in coastal habitats may not have the ability to detect, respond to, or even tolerate such conditions. This study examined the response of the Florida stone crab, Menippe mercenaria, as a model for coastal crustaceans, to determine the impacts of elevated temperature and ocean acidification

(OA) on embryonic development and hatching success. The study determined the impacts of simultaneous exposure to both elevated temperature and OA on stone crab survival, larval growth, larval condition, and larval morphology throughout

iii

development. The study also examined whether changes in environmental conditions affected the ability of larvae to orient vertically. More specifically, the geotactic swimming behavior of larval stone crabs was tested to determine if elevated temperature and pCO 2 impacted geotaxis orientation, and if geotactic responses change throughout larval development. The impacts of OA on stone crab embryonic development and hatching success was determined by maintaining ovigerous females in conditions that mimicked present-day (p CO2 ~ 360 ppm, pH = 8.1) and future carbonate conditions (pCO 2 ~ 1500 ppm, pH = 7.5). To determine the effects of simultaneous exposure of stone-crab larvae to elevated temperature and pCO2, larvae were raised in a fully crossed experiment with two treatments, temperature, and pCO 2, each with two levels. The two temperature levels were 30°C and 32°C, and the two pCO 2 levels were ~450 ppm and ~1100 ppm. This study also synthesized the experimental work by parameterizing a matrix-population model using the larval survivorship data to predict the population densities of larval stone crabs under future temperature and OA scenarios.

The elevated pCO 2 treatment (1000 ppm) significantly reduced the rate of embryonic development (i.e., time to hatching) by ~32%, but had no effect on the size of developing embryos (i.e., embryonic volume). Larvae that successfully hatched were not morphologically different among treatments, although hatching success was reduced by 38% in the elevated p CO2 treatment. Exposure to elevated p CO2 and ambient temperatures significantly reduced larval survivorship, which was exacerbated by elevated temperature. Indeed, exposure to elevated temperatures had iv

the greatest effect on larval survivorship and development. Larvae raised in the combined treatments (i.e., both elevated pCO 2, and temperature) exhibited a ~79% decrease in survivorship relative to the control. The molt-stage duration was ~1.2 days shorter when larvae were exposed to elevated temperatures. However, stage V larvae reared in the elevated pCO 2 and ambient temperature exhibited a significantly longer molt-stage duration than stage V larvae in controls. Larval condition (i.e., ash free dry weight), Ca and Mg content, Mg/Ca ratios, and larval morphology showed no significant differences among treatments. Geotactic responses varied throughout ontogeny and directional movement was dependent on p CO2 level, rather than on temperature. Stage III larvae, which swam upwards under ambient pCO2 conditions, showed a significant reversal of their swimming orientation (i.e., downward swimming), when exposed to elevated pCO 2.

These results indicate that future changes in seawater p CO2 may reduce the reproductive success of stone crabs. The significant decrease in survival at elevated temperatures may also have a negative effect on larval supply, which will be detrimental to the stone-crab populations. Reductions in reproductive output and larval supply could have potential socioeconomic implications for the stone-crab fishery, unless the crabs are able to acclimatize or adapt to future seawater conditions.

Geotactic responses were more adversely affected by OA rather than by temperature. Typically, early to mid-stage larvae of brachyuran crab larvae elicit swimming behaviors that position them at or near the surface, where currents facilitate transport offshore. When exposed to elevated pCO 2, the swimming v

behavior of stage III larvae changed, suggesting that mid-stage stone crab larvae may be positioned deeper in the water column, which may reduce their advection potential to offshore areas. Reducing advection may result in individuals being exposed to different conditions and different predators than typically experienced when larvae are transported offshore.

The population model, using the stage-specific larval survivorship data from the experimental treatments, showed that ~14.8% of the control population survived to post-larval stage. Elevated pCO 2, elevated temperature, and the combined treatment

(elevated pCO2 and elevated temperature) resulted in 11%, 6.5%, and 6.8% survival to the post-larval stage, respectively. The sensitivity analysis indicated that early- stage larvae (stage-II) were most sensitive to mortality, particularly in control simulations. Elevated pCO2 and temperature shifted the sensitivities to late-larval stages (i.e., stages IV and V). This shift was potentially a consequence of greater effects of ocean acidification on more developed larval stages, with more pronounced anatomical features. The sensitivity analyses indicated that elevated pCO 2 and temperature had the greatest impact on late-stage larvae. These results suggest that new recruits into the stone-crab fishery will be affected by both exposure to elevated p CO2 and temperature, which are expected by the end of the century, unless larvae tolerances to such conditions improve.

TABLE OF CONTENTS ABSTRACT. …………………………………………………………….……...... iii TABLE OF CONTENTS………….………………………………………………vii LIST OF TABLES………………………………………………………………….x

LIST OF FIGURES……………………………………………………………... xiii ACKNOWLEDGEMENTS.…. …………………………………………………xxv DEDICATION………………………………………………………………….xxvii CHAPTER I: BACKGROUND ...... 1

INTRODUCTION ...... 1 SEA SURFACE TEMPERATURES ...... 2 IMPACT OF ELEVATED SEA-SURFACE TEMPERATURES AND PCO2 ...... 3

IMPACT OF ELEVATED PCO2 ON CRUSTACEAN LARVAL DEVELOPMENT ...... 4 IMPACT OF ELEVATED TEMPERATURE ON LARVAL CRUSTACEANS ...... 5 THE FLORIDA STONE CRAB, MENIPPE MERCENARIA ...... 7 RESEARCH OBJECTIVES ...... 8 DISSERTATION STRUCTURE ...... 10 CHAPTER II: EMBRYONIC DEVELOPMENT ...... 11 INTRODUCTION ...... 11 MATERIALS AND METHODS ...... 14 STUDY SITE, COLLECTION, AND MAINTAINENCE OF EXPERIMENTAL ANIMALS ...... 14 EXPERIMENTAL SETUP AND WATER CHEMISTRY ...... 15 EMBRYONIC DEVELOPMENT AND HATCHING SUCCESS ...... 16 MORPHOLOGICAL ANALYSIS OF LARVAE ...... 19 RESULTS ...... 20 WATER CHEMISTRY AND EXPERIMENTAL CONDITIONS ...... 20 EMBRYONIC DEVELOPMENT AND HATCHING SUCCESS ...... 20 LARVAL MORPHOLOGY ...... 23

vii

DISCUSSION ...... 27 CHAPTER III: LARVAL DEVELOPMENT ...... 34 INTRODUCTION ...... 34 MATERIALS & METHODS ...... 37 OVIGEROUS FEMALE STONE CRAB COLLECTION ...... 37 EXPERIMENTAL DESIGN AND OA SYSTEM SET-UP ...... 37 SEAWATER CARBONATE CHEMISTRY ...... 38 STONE CRAB LARVAL SURVIVORSHIP AND MOLT STAGE DURATION ...... 40 ASH FREE DRY WEIGHT OF LARVAE ...... 41 LARVAL CA AND MG CONTENT, AND MG/CA RATIOS ...... 42 MORPHOMETRIC ANALYSIS ...... 43 RESULTS ...... 45 CARBONATE CHEMISTRY DURING SURVIVORSHIP AND MOLT STAGE DURATION EXPERIMENTS ...... 45 CARBONATE CHEMISTRY DURING SURVIVORSHIP AND MOLT STAGE DURATION EXPERIMENTS ...... 48 SURVIVAL AND DEVELOPMENT ...... 48 ASH FREE DRY WEIGHT ...... 60 MG AND CA CONTENT AND MG/CA RATIOS ...... 63 LARVAL MORPHOLOGY ...... 68 DISCUSSION ...... 78 CHAPTER IV: BEHAVIOR ...... 85 IN;;TRODUCTION ...... 85 MATERIALS AND METHODS ...... 89 STONE CRAB COLLECTION ...... 89 EXPERIMENTAL DESGN AND SET-UP ...... 89 SEAWATER CARBONATE CHEMISTRY ...... 91 STONE CRAB COLLECTION AND LARVAL REARING ...... 92 SINKING RATES FOR STAGE III AND V LARVAE ...... 93 GEOTACTIC SWIMMING RESPONSES FOR STAGE III AND V LARVAE ...... 93 RESULTS ...... 95 viii

SEAWATER CARBONATE CHEMISTRY ...... 95 PASSIVE SINKING RATES AND DOWNWARD SWIMMING SPEEDS ...... 96 GEOTACTIC SWIMMING BEHAVIOR ...... 100 DISCUSSION ...... 103 CHAPTER V: MATRIX MODEL ...... 110 INTRODUCTION ...... 110 MATERIALS AND METHODS ...... 113 PARAMETER ESTIMATION AND MODEL DESIGN ...... 113 RESULTS ...... 119 LARVAL POPULATION SIZE AND INTRINSIC RATE OF GROWTH ...... 119 SURVIVORSHIP TO POST-LARVAE ...... 122 MODEL SENSITIVITIES ...... 125 DISCUSSION ...... 128 SUPPLEMENTARY TABLES ...... 135 CHAPTER VI: SYNTHESIS AND CONCLUSIONS ...... 136 LITERATURE CITED ...... 141

LIST OF TABLES

Table II.1. Classification scheme used to monitor the development of M.

mercenaria embryos. Embryonic stages were based on yolk content,

the presence and shape of the eyespot, and egg mass coloration.

Descriptions were adapted from Pandian (1970), Helluy and Beltz

(1991), and Garcia-Guerrero and Hendrickx (1991)...... 18

Table II.2. Mean (± SD) treatment conditions for total alkalinity (TA), pCO2,

inorganic carbon dioxide (DIC), pH, salinity, and temperature in

control and experimental aquaria...... 21

Table II.3. Results of a principal component analysis on seven morphological

characters of newly hatched M. mercenaria larvae. Three orthogonal

principal components (PCs) representing 72.9% of the total variation

were retained by the analysis. The derived component scores were

analyzed using separate nested ANOVAs to test for the effects of

elevated pCO2 on larval morphology. The larger factor loadings were

used to determine the variables contribution to each PC...... 26

Table III.1. Seawater carbonate chemistry, temperature, and salinity for the 2014

and 2015 summer laboratory experiments. The A30 treatment

represents the control with ambient temperature and ambient pCO2.

The H30 represents ambient temperature and elevated pCO 2. The

A32 treatment was held at elevated temperature and ambient pCO2, x

while the H32 treatment was maintained at both elevated temperature

and elevated pCO 2...... 47

Table III.2. Results of the repeated measures ANOVAs for stage-III and V ash

free dry weight. Significant values are in bold...... 62

Table III.3. Results of the repeated measures ANOVARs for stage-III Ca and Mg

concentration, and Mg/Ca ratio...... 65

Table III.4. Results of the repeated measures ANOVARs for stage-V Ca and Mg

concentration, and Mg/Ca ratio. The Mg/Ca ratios were rank

transformed...... 70

Table III.5. Results of a principal component analysis on seven morphological

characters of stage-III M. mercenaria larvae . Three orthogonal

principal components (PCs) representing 79.4% of the total variation

were retained by the analysis. The derived component scores were

analyzed using separate ANOVAR to test for the effects of elevated

pCO2 on larval morphology. The largest factor loadings were used to

determine the variables contribution to each PC...... 73

Table III.6. Results of a principal component analysis on seven morphological

characters of Stage-V M. mercenaria larvae . Two orthogonal

principal components (PCs) representing 71.4% of the total variation

were retained by the analysis. The derived component scores were

analyzed using separate ANOVAR’s to test for the effects of elevated

pCO2 on larval morphology. The largest factor loadings were used to

determine the variables contribution to each PC...... 76

Table IV.1. Results of the repeated measures ANOVAs for stage III and V

passive sinking speeds. The ANOVA’s were performed using ranked

data. Significant values are in bold...... 97

Table IV.2. Results of the ANOVAR for stage III and V geotactic responses. The

ANOVAR was performed using ranked data for stage III larvae.

Significant values are in bold...... 101

Table V.1. Summary of the estimated natural larval mortality reported in the

literature for a variety of plankton invertebrates and crustaceans,

including rates for blue crab, Callinectes sapidus...... 115

Table V.2. Assumed stage-specific natural mortality rates for stone crab larvae.

The mortality rates for stage II–V were estimated using the

exponential decay function, assuming that the mortality of early

larval stages is greater than later stages...... 117

Table V.3. Eigenvalues (λ) for each model simulation across all treatments. The

model assumed that natural mortality remained constant and would

not experience increases in survivorship or hatching success...... 123

xii

LIST OF FIGURES

Figure II.1. Change in embryo stage as a function of time for crabs subjected to

elevated pCO2 and control conditions. A linear function (Control: y =

2 2 1.65 + 0.62x, R = 0.77; elevated pCO2: 푦 = 1.06 + 0.4 7x, R = 0.78)

was used to determine the rate of change in development among both

treatments...... 22

Figure II.2. Change in egg volume at each embryo stage for crabs subjected to

elevated pCO2 and control conditions. A linear function (Control: y =

2 2 0.11 + 0.01x, R = 0.70; elevated pCO2: 푦 = 0.11 + 0.009x, R =

0.79) was used to determine the rate of change in embryo volume

among both treatments...... 24

Figure II.3. A box and whiskers plot showing the hatching success of crabs

maintained under control and elevated pCO 2 conditions. Horizontal

lines within the box indicates the median...... 25

Figure III.1. Mean pCO2 (ppm, ± SD) for the experimental treatments and field

collection site. Experimental tanks were not sampled on days 6, 17,

22 and 24 (2014). Higher pCO2 values on day 27 are likely due to a

clog in ambient air deliver system to the elevated pCO2 reservoir. The

shapes in the figure represent the means and the whiskers represent

the standard deviation...... 46

xiii

Figure III.2. Mean pCO2 (ppm, ± SD) for the experimental treatments and for

field collection site Experimental tanks were not sampled on days 4,

6, 13, and 17. The shapes in the figure represent the means and the

whiskers represent the standard deviation...... 49

Figure III.3. Cumulative survivorship of Menippe mercenaria larvae throughout

larval development during exposure to different combinations of

pCO2 and temperature. A log-rank test was used for pairwise

comparisons. Curves with different letters are significantly different

at α = 0.05. A30 (i.e., the control) represents the ambient pCO2 and

ambient temperature treatment. H30 is the elevated pCO2 and

ambient temperature treatment. A32 is the ambient pCO 2 and

elevated temperature treatment, and H32 is the elevated pCO2 and

elevated temperature treatment...... 50

Figure III.4. Box and whiskers plot of the survivorship of stage-I Menippe

mercenaria larvae during exposure to different combinations of pCO2

and temperature. An ANOVAR was performed on the ranked data

with temperature and CO2 as the main effects and brood as the within

subject factor. Boxes with similar letters above them are not

significantly different from each other. H30 is the elevated pCO2 and

ambient temperature treatment. A32 is the ambient pCO 2 and

elevated temperature treatment, and H32 is the elevated pCO2 and

elevated temperature treatment...... 52 xiv

Figure III.5. Box and whiskers plot of the survivorship of stage-II Menippe

mercenaria larvae during exposure to different combinations of pCO2

and temperature. An ANOVAR was performed on the ranked data

with temperature and CO2 as the main effects and brood as the within

subject factor. Boxes with similar letters above them are not

significantly different from each other. H30 is the elevated pCO2 and

ambient temperature treatment. A32 is the ambient pCO2 and

elevated temperature treatment, and H32 is the elevated pCO2 and

elevated temperature treatment...... 54

Figure III.6. Box and whiskers plot of the survivorship of stage-III Menippe

mercenaria larvae during exposure to different combinations of pCO2

and temperature. An ANOVAR was performed on the ranked data

with temperature and CO2 as the main effects and brood as the within

subject factor. Boxes with similar letters above them are not

significantly different from each other. H30 is the elevated pCO2 and

ambient temperature treatment. A32 is the ambient pCO2 and

elevated temperature treatment, and H32 is the elevated pCO2 and

elevated temperature treatment ………………………….... 55

Figure III.7. Box and whiskers plot of the survivorship of stage-IV Menippe

mercenaria larvae during exposure to different combinations of pCO2

and temperature. An ANOVAR was performed on the ranked data

with temperature and CO2 as the main effects and brood as the within xv

subject factor. Boxes with similar letters above them are not

significantly different from each other. H30 is the elevated pCO2 and

ambient temperature treatment. A32 is the ambient pCO2 and

elevated temperature treatment, and H32 is the elevated pCO 2 and

elevated temperature treatment…………………………..……. …..56

Figure III.8. Box and whiskers plot of the survivorship of stage-V Menippe

mercenaria larvae during exposure to different combinations of pCO2

and temperature. An ANOVAR was performed on the ranked data

with temperature and CO2 as the main effects and brood as the within

subject factor. Boxes with similar letters above them are not

significantly different from each other. H30 is the elevated pCO2 and

ambient temperature treatment. A32 is the ambient pCO2 and

elevated temperature treatment, and H32 is the elevated pCO2 and

elevated temperature treatment and whiskers plot of the stage-

specific survivorship of stage V …...………………………………57

Figure III.9. Mean (days, ±S.E.) molt stage duration of Menippe mercenaria

larvae throughout larval development during exposure to different

combinations of pCO2 and temperature. Letters above the bars

represent differences between the treatments at α = 0.05...... 59

Figure III.10. Mean ash free dry weight (µg individual-1, ± S.E.) for stage-III and V

M. mercenaria larvae during exposure to different combinations of

pCO2 and temperature. There were no significant differences among xvi

the main effects. The control (A30) was maintained at both ambient

pCO2 (~450 ppm) and ambient temperature (30 °C). The elevated

pCO2 treatment was maintained at ~1100 ppm whereas the elevated

temperature treatment target was 32 °C...... 61

Figure III.11. Box and whiskers plot of the Ca concentration (mg L-1) for stage-III

M. mercenaria larvae during exposure to different combinations of

pCO2 and temperature. An ANOVAR was performed on the ranked

data with temperature and CO2 as the main effects and brood as the

within subject factor. The horizontal lines in the figure represents the

median, the boxes represent the lower and upper quartiles (25% and

75%), and the vertical lines (whiskers) represent the minimum and

maximum values...... 64

Figure III.12. Box and whiskers plot of the Mg concentration (mg L-1) for stage-III

M. mercenaria larvae during exposure to different combinations of

pCO2 and temperature. An ANOVAR was performed on the ranked

data with temperature and CO2 as the main effects and brood as the

within subject factor. The horizontal lines in the figure represents the

median the boxes represent the lower and upper quartiles (25% and

75%), and the vertical lines (whiskers) represent the minimum and

maximum values...... 66

Figure III.13. Box and whiskers plot of the Mg/Ca ratio for stage-III M. mercenaria

larvae during exposure to different combinations of pCO2 and xvii

temperature. An ANOVAR was performed on the ranked data with

temperature and CO2 as the main effects and brood as the within

subject factor. The horizontal lines in the figure represents the

median the boxes represent the lower and upper quartiles (25% and

75%), and the vertical lines (whiskers) represent the minimum and

maximum values...... 67

Figure III.14. Ca concentration (mg L-1 individual -1) for stage-V M. mercenaria

larvae during exposure to different combinations of pCO2 and

temperature. An ANOVAR was performed on the Ca concentration,

with temperature and CO2 as the main effects and brood as the

within-subject factor. The bars represent the means, and the vertical

lines represent the standard errors...... 69

Figure III.15. Mg concentration (mg L-1 individual -1) for stage-V M. mercenaria

larvae during exposure to different combinations of pCO2 and

temperature. An ANOVAR was performed on with temperature and

CO2 as the main effects and brood as the within-subject factor. The

bars represent the means, and the vertical lines represent the standard

errors...... 71

Figure III.16. Box and whiskers plot of Mg/Ca for stage-V M. mercenaria larvae

during exposure to different combinations of pCO2 and temperature.

An ANOVAR was performed on the ranked data with temperature

and CO2 as the main effects and brood as the within subject factor. xviii

The horizontal lines in the figure represents the median the boxes

represent the lower and upper quartiles (25% and 75%), and the

vertical lines (whiskers) represent the minimum and maximum

values...... 72

Figure III.17. Principal component plot of M. mercenaria stage-III larval

morphometrics during exposure to different combinations of pCO2

and temperature. There were no significant treatment effects on

stage-III larval morphology...... 75

Figure III.18. Principal component plot of M. mercenaria stage-V larval

morphometrics during exposure to different combinations of pCO2

and temperature. There were no significant treatment effects on

stage-V larval morphology. Each data point represents an individual

larva...... 77

Figure IV.1. Box and whiskers plot (cm sec-1) showing the passive sinking (white

bars) and downward swimming speeds (grey bars) of stage III larvae

for exposure to different combinations of pCO2 and temperature. The

asterisks represent passive sinking responses that were significantly

greater than the downward swimming speeds...... 98

Figure IV.2. Box and whiskers plot (cm sec-1) showing the passive sinking (white

bars) and downward swimming speeds (grey bars) of stage V larvae

for exposure to different combinations of pCO2 and temperature.

xix

Asterisks represent passive sinking responses that were significantly

greater than the downward swimming speeds...... 99

Figure IV.3. Box plot of stage III larvae that swam up vs. down among

experimental treatments. Different letters above the boxes indicate

significant differences. Because it was expected that stage III larvae

would swim upward, larvae that maintained position upon

stimulation (i.e., neutral swimming) were included in the upward

response for stage III larvae...... 102

Figure IV.4. Mean percent (± SE) larvae that swam up vs. down Z5 larvae among

experimental treatments. Because it was expected that stage V larvae

would swim downwards, larvae that maintained position upon

stimulation (i.e., neutral swimming) were included in the downward

response for stage V larvae. There were no significant differences

among treatment in the stage V response...... 104

Figure V.1. A) Model schematic where ‘H’ represents hatching success for

females exposed to OA conditioned seawater (Chapter M2). M1- 5

represent stage-specific larval survivorship. Stone crab larval stages

are represented by ‘Z1-Z5’, B) Representation of the stage-class

population matrix-model where n represents the number of

individuals per stage (i.e., n1= stage 1). The model runs for 5 weeks

and assumes a larval supply at each stage of 1x106 individuals...... 118

Figure V.2. Larval stone-crab population densities during a 5 week matrix model

simulation. The model assumes a larval supply at each stage of 1x106

individuals and an initial starting population of 100 ovigerous

females. The black line represents mortality estimates assumed from

the literature. A30 represents the control with ambient pCO2 and

ambient temperatures. H30 is the elevated pCO2 and ambient

temperature treatment. A32 is the ambient pCO 2 and elevated

temperature treatment, and H32 is the elevated pCO2 and elevated

temperature treatment. The solid lines represent hatching success

under ambient pCO 2 conditions, whereas the dotted lines represent a

reduction in hatching success experienced during exposure to

elevated pCO 2...... 120

Figure V.3. The intrinsic rate of population decrease. Natural mortality was

estimated from the literature. A30 represents the control with ambient

pCO2 and ambient temperatures. H30 is the elevated pCO2 and

ambient temperature treatment. A32 is the ambient pCO 2 and

elevated temperature treatment, and H32 is the elevated pCO2 and

elevated temperature treatment. H30R is the elevated pCO2 and

ambient temperature treatment with a reduction in hatching success.

H32R is the elevated pCO2 and elevated temperature treatment with a

reduction in hatching success...... 121

xxi

Figure V.4. The percentage of larvae (±S.E.) surviving to megalopae during a

five week matrix model simulation. Natural mortality was estimated

from the literature and represents the model run with stage-specific

larval mortality, which was estimated using an exponential decay

function. The natural mortality assumed that earlier larval stages

experience greater mortality. The model also assumes a larval supply

at each stage of 1x106 individuals, and an initial starting population

of 100 ovigerous females. A30 represents the control with ambient

pCO2 and ambient temperatures. H30 is the elevated pCO2 and

ambient temperature treatment. A32 is the ambient pCO 2 and

elevated temperature treatment, and H32 is the elevated pCO2 and

elevated temperature treatment. H30R is the elevated pCO2 and

ambient temperature treatment with a reduction in hatching success.

H32R is the elevated pCO2 and elevated temperature treatment with a

reduction in hatching success...... 124

Figure V.5. Sensitivity analysis of population growth rate for stone crab larval

stages I-V, at each experimental simulation. Mortality rates from

each experimental treatment were superimposed onto the natural

mortality rate estimated from the literature. The model also assumed

a larval supply at each stage of 1x106 individuals and an initial

starting population of 100 ovigerous females. A30 represents the

control with ambient pCO2 and ambient temperatures. H30 is the xxii

elevated pCO2 and ambient temperature treatment. A32 is the

ambient p CO2 and elevated temperature treatment, and H32 is the

elevated pCO2 and elevated temperature treatment. The H30R and

the H32R treatments represent model simulations that incorporate the

reduction in hatching success reported in Chapter 2...... 126

Figure V.6. Sensitivity analysis of population growth rate for stone crab larval

stages I-V at each experimental simulation. Mortality rates from each

experimental treatment were superimposed onto the natural mortality

rate estimated from the literature. The model also assumes a larval

supply at each stage of 1x106 individuals and an initial starting

population of 100 ovigerous females. A30 represents the control with

ambient pCO2 and ambient temperatures. H30 is the elevated pCO2

and ambient temperature treatment. A32 is the ambient pCO 2 and

elevated temperature treatment, and H32 is the elevated pCO2 and

elevated temperature treatment...... 127

Figure V.7. Sensitivity analysis of population growth rate for stone crab larval

stages I-V. Mortality rates from each experimental treatment were

superimposed onto the natural mortality rate estimated from the

literature. The model also assumes a larval supply at each stage of

1x106 individuals and an initial starting population of 100 ovigerous

females. A30 represents the control with ambient pCO2 and ambient

temperatures. H30 is the elevated pCO2 and ambient temperature xxiii

treatment. H30R is the elevated pCO2 and ambient temperature

treatment under reduced fecundity from Chapter 2...... 129

Figure V.8. Sensitivity analysis of population growth rate for stone crab larval

stages I-V. Mortality rates from each experimental treatment were

superimposed onto the natural mortality rate estimated from the

literature. The model also assumes a larval supply at each stage of

1x106 individuals and an initial starting population of 100 ovigerous

females. A30 represents the control with ambient pCO2 and ambient

temperatures. H32 is the elevated pCO2 and elevated temperature

treatment. H32R is the elevated pCO2 and elevated temperature

treatment under reduced fecundity from Chapter 2...... 130

xxiv

ACKNOWLEDGEMENTS

This dissertation was supported in part by two separate Mote Protect Our Reef

License Plate Grants (POR-2011-27, and POR-2013-10). I am appreciative for the support and technical assistance provided by Mote staff members including Drs. E.

Hall, E. Muller, D. Vaughn and the Mote Marine Laboratory Chemical Ecology staff during this project. I would also like to acknowledge of Florida Institute of

Technologies InSTEP program for their financial support and professional development training they provided during my dissertation. I am also grateful for the Graduate Student Assistantship provided by the department of biological sciences, and the Research Assistantship provided by the department of Marine and

Environmental Systems.

I thank R. Gandy and the staff of the Florida Fish and Wildlife Stone crab monitoring program, specifically Stephanie Kronstadt, for assistance with animal collection. All ovigerous females were collected in compliance with Scientific

Activity License number SAL-12-0520-SR. I am grateful to Dr. I. Enochs, Dr. D.

Manzello, and Dr. P. Krimsky from NOAA’s AOML laboratory in Miami for the use of their ocean acidification experimental laboratory and carbonate chemistry analytical laboratory. I would also like to acknowledge those that helped with data analysis throughout my dissertation including, K. McCaffrey, A. Smith, and A.

Folcik and countless other volunteers that dedicated their time and assistance.

xxv

I am also grateful of the assistance that rest the Department of Biological

Sciences Faculty and Staff provided, including Dee Dee van Horn, Gayle

Duncombe, Jenn Hobbs, and Glenn Miller. I am also grateful for Dr. R. Aronson’s guidance and support during both my teaching responsibilities and research obligations. Additionally, I am extremely appreciative for Dr. R. Tankersley’s feedback, assistance, and advice throughout my dissertation. His guidance over the years has helped prepare me for the world beyond Florida Tech, and has helped me grow into a well-rounded scientist. I will always be grateful for his direction, and support.

I am extremely thankful to Dr. L. Toth for her editorial assistance, laboratory help, and constant feedback while performing this research. More importantly, for her unwavering support during both good times and bad. I could not have done it without you by my side!

Finally, completion of this dissertation would not be possible without the constant guidance and feedback of my doctoral committee including Dr. R.

Tankersley, Dr. C. Pruett, Dr. R. Turingan, Dr. D. Palmer, Dr. R. Aronson, and Dr.

J. Windsor, and especially Dr. R. van Woesik, for both his council, editorial assistance, and analytical guidance with the R statistical software. Thank you all!

xxvi

DEDICATION

I would like to dedicate this dissertation to both my parents and grandparents whose constant support and encouragement allowed me to pursue my dreams.

Thank you!!! I am forever grateful for the countless sacrifices they made over the years. I would also like to dedicate my dissertation to my grandfather. The many summers we spent fishing and crabbing in Sea Isle helped to instill my love for the oceans. I miss you pop, but I know you are always with me.

xxvii

CHAPTER I: BACKGROUND

INTRODUCTION

Carbon emissions are increasing p CO2 levels in the atmosphere at an alarming rate. Since 1750, ocean pH has declined by 0.1 units (Orr et al. 2005), which is equivalent to a 30% increase in the concentration of H+ ions (Widdicombe and

Spicer 2008). At the current rate of fossil-fuel emissions, pCO2 concentrations are expected to increase from the current level of 400 ppm to 700–1000 ppm by the year 2100, and 2000 ppm by the year 2300 (IPCC 2013). By the end of the 21st century, oceanic pH is expected to decrease by 0.41 units, which will result in a

+ 150% increase in H concentrations (Caldeira and Wickett 2003). The high CO2 in the atmosphere is simultaneously accelerating the rate of global warming. The projected sea-surface warming rate of 3°C for the 21st century is five times faster than the 0.6°C warming rate documented in the 20th century (Kerr 2004). The predicted rates of increase in pCO2 and temperature are unprecedented in Earth’s history (IPCC 2013). The Paleocene-Eocene thermal maximum (PETM), which occurred 55 million years ago, was characterized by “sudden” CO2 increases in both the atmosphere and oceans, elevating global temperatures by 5°C over an interval of 10,000 years (Kennett and Stott 1991, Zachos et al. 2001). Under current emission rates, the atmosphere will exceed the magnitude of changes that occurred in the PETM within a few hundred years.

Such drastic changes in Earth’s climate will threaten the physiology, reproduction, and survivorship of many marine organisms, which could have cascading effects on community composition and sustainability of populations.

More specifically, several studies have already detected negative effects of elevated pCO2 on various physiological processes in marine crustaceans, including influences on the acid-base balance (Pane and Barry 2007), on thermal tolerance

(Metzger et al. 2007), and on growth and survivorship (Kurihara et al. 2008).

SEA SURFACE TEMPERATURES

Recently we have experienced the hottest years on record (Hansen et. al. 2006,

Trenberth et al. 2007), and mean global sea-surface temperatures are expected to rise 2–4.5°C (depending on latitude) by the year 2100 (IPCC 2013). Increased temperature accelerates metabolism in some marine organisms until a threshold is reached beyond which metabolic rates decline (Byrne 2011). As temperatures increase, protein stability and function (e.g., enzymes) begin to fail (Portner 2008).

Energy is then invested in maintenance, resulting in decreased growth (Hofmann and Todgham 2010). The upper thermal threshold for a given taxon’s physiological performance is determined by how close the organisms live to their thermal limits

(Portner et al. 2006). Thermal intolerance at extreme temperatures limits the capacity of oxygen supply mechanisms to compensate for the organisms oxygen demands, and can decrease metabolism (Portner et al. 2006). Highlighting the limitations of individual organisms to environmental stressors will identify a

3 species’ tolerance and help forecast the likely impacts climate change may have on future populations (Helmuth et al. 2006).

IMPACT OF ELEVATED SEA-SURFACE TEMPERATURES AND PCO2

Elevated temperature and pCO2 concentrations are frequently correlated and have been associated with climatic changes throughout Earth’s history (Knoll et al.

1996, 2007). Changes in both temperature and pCO2 likely contributed to the extinction of ~90% of marine life during the Permian (Benton and Twitchett 2003).

Changes in ocean pCO2 and temperature are occurring simultaneously and at accelerated rates when compared with historical climate trends (IPCC 2013).

Making generalizations about the effects of climate change on marine communities from single-stressor studies results in unrealistic assumptions (Byrne 2011), since the magnitude of the organism’s response is likely to differ when exposed to combined scenarios. For example, the interactive effect of ocean warming and acidification could result in decreased carbonate saturation state and hypercapnia

(i.e., high CO2 co ncentration in the blood), which may impair calcification and suppress metabolism (Byrne 2011). Alternatively, elevated temperatures could also enhance developmental processes (until a threshold is reached), but may be associated with certain physiological costs, such as lower lipid stores for postsettlement growth (Byrne 2011). Given the significant ecological and economic role crustaceans play in many ecosystems, it is crucial that we identify the potential detrimental effects that elevated temperature and pCO2 may have on species

4 physiology throughout ontogeny. For instance, physiological compensation to elevated pCO2 could result in reallocation of energy reserves (Wood et al. 2008), reducing survival, growth and reproductive performance (Kurihara et al. 2008,

Egilsdottir et al. 2009, Keppel et al. 2012). Determining the effects of ocean acidification and elevated temperature during early life history stages is a critical step to determine a species sensitivity and ability to tolerate future climate change.

IMPACT OF ELEVATED PCO2 ON CRUSTACEAN LARVAL

DEVELOPMENT

Crustaceans display a wide range of responses to acidified conditions (Kroeker et al. 2013) that vary with habitat (Wantanabe et al. 2006), taxonomic grouping

(Ries et al. 2009), and between populations of the same species (Walther et. al.

2010). This suggests that some species may be able to tolerate changes in ocean pH

(Carter et al. 2013). In general, acidified oceans compromise the ability of some crabs to invest energy toward growth, can decrease fertilization, and can reduce survivorship, especially during early life stages (Garrard et al. 2012). Studies evaluating the impacts of elevated pCO2 on embryonic development and physiology in crab species reported smaller yolk volume (Paralithodes camtschaticus; Long et al. 2013), longer hatch duration (P. camtschaticus; Long et al. 2013), increased development time (Hyas araneus; Walther et al. 2010), and lower embryonic heart rates (Petrolisthes cinctipes, Ceballos-Osuna et al. 2013).

During larval development, crustaceans undergo several molts that require energy. Some crustacean larvae, such as the lobster Homarus gammarus (Arnold et al. 2009), and the barnacles Amphibalanus amphitrite and Semibalanaus balanoides (Findlay et al. 2010), exhibit reduced calcification rates in seawater conditions of 1000 ppm. The effects of acidified conditions on calcification in crustaceans are variable (Whiteley 2011), because some species show positive effects in calcification (Kroeker et al. 2010). However, lower ocean pH could influence CaCO3 precipitation by reducing the exoskeleton compartment (Wood and Cameron 1985). Additionally, elevated p CO2 could impact post-molt calcification of new exoskeleton, which is dependent upon a large uptake of both

2+ - Ca and HCO3 across the gills (Neufield and Cameron 1992, Wheatly 1997).

Larvae may be more susceptible to predation under elevated pCO2 scenarios, because of their small body size (Kurihara et al. 2008), and short carapace length

(Keppel et al. 2012).

IMPACT OF ELEVATED TEMPERATURE ON LARVAL CRUSTACEANS

Temperature is one of the most critical environmental factors that can impact larval survival, molt stage duration, development, and metabolism of crustaceans

(Costlow et al. 1960, Naylor 1965). Crustaceans (i.e., Sesarma, Callinectes,

Menippe spp.) exhibit shorter molt stage durations and reduced survivorship with increasing temperatures (Costlow et al. 1960, Ong and Costlow 1970, Mohamedeen and Hartnoll 1989). For example, early stage C. sapidus larvae exhibited a 15%

6 decrease in survivorship when exposed to increased temperatures (Costlow and

Bookhout 1959). Elevated temperatures also result in individuals with reduced size, despite shorter molt-stage durations (Leffler 1972). Small individuals at higher temperatures are a result of larvae passing through larval development too quickly to accumulate sufficient lipid reserves to sustain additional growth (Swingle et al.

2013). Furthermore, certain enzymes within larvae may only be active at certain temperatures, and at elevated temperatures these pathways may be not operating or may be working less efficiently (Costlow and Bookhout 1971).

Elevated temperature also increase the extent of larval swimming in many crustacean larvae, until a threshold temperature is reached (Sulkin et al. 1980).

Upon reaching the thermal threshold, inactivity (sinking) occurs (Yule 1984). Blue crab larvae actively swim until temperatures exceed 27.5°C, upon which passive sinking occurs (McConnaughey and Sulkin 1984). High temperatures also resulted in a reversal of the phototactic response in newly hatched larvae of

Rhithropanopeus harrisii, from a positive phototaxis (upward swimming) to a negative phototaxis (downward response) (Ott and Forward 1976). Furthermore, elevated temperatures elicited an increase in the geotactic response of stage IV larvae (Ott and Forward 1976). In general, larval behavioral responses are sensitive to higher temperatures (McConnaughey and Sulkin 1984). Temperature increases at or above the upper thermal limit can evoke negative phototaxis, positive geotaxis, and sinking, all of which lead to downward movement (Forward 1990).

Therefore, future changes in sea surface temperature could alter larval vertical

7 position in the water column and result in transport into environments not favorable for development or settlement.

THE FLORIDA STONE CRAB, MENIPPE MERCENARIA

Menippe mercenaria, is a coastal species that supports an important fishery throughout the southeastern U.S. In Florida, the stone crab fishery contributes ~$25 million a year to local economies (Florida Fish and Wildlife Stock Assessments,

2000–2014). Stone crab life history is similar to other brachyuran crab species.

Females extrude embryos onto their abdomen during the summer months and brooding takes ~2 weeks before larval release. Larvae undergo five stages of development in offshore habitats before molting into post-larvae and recruiting back to settlement habitats in estuaries (Porter 1960). Estuaries and coastal habitats regularly experience seasonal fluctuations in pCO2 ranging from 200-700 µmol/mol

(from NOAA PMEL: http://www.pmel.noaa.gov/co2/story/Coastal+Moorings;

Bauer et al. 2013), whereas most oceanic surface waters have less variable CO2 levels (Hofmann et al. 2011). Crustacean species that live in such dynamic habitats can serve as unique models for climate change studies. Species adapted to low-pH environments likely possess the physiological tolerance for higher pCO 2 levels that are expected by the end of the century (Whiteley 2011). However, later larval stages may not have the compensatory mechanisms to physiologically tolerate pH conditions predicted for the end of the century.

Previous studies suggest that larval stone crabs have the ability to tolerate changes in other environmental factors such as salinity and temperature. For instance, stone crab larvae had the highest larval survival (Stage I = 56%, Stage V

= 84%) at a salinity of 30, compared with other salinity levels ranging from 20-40

(Brown et al. 1992). Survivorship can be further reduced by high temperatures. For example, Brown et al. (1992) showed that survival significantly decreased to 18% and 50% for stage I and V larvae respectively at 35°C. Increased temperatures can compromise cellular processes, proteins, and enzymes (Sanford 1999, Hofmann and Todgham 2010). Sensitivity to temperature thresholds is determined by how close an organism lives to its thermal limits (Portner et al. 2006).

RESEARCH OBJECTIVES

My dissertation identifies the potential impacts of changes in pCO2 and temperature on embryonic development, larval condition, growth, morphology, survivorship, and geotactic swimming behavior of Menippe mercenaria. I addressed the following objectives by asking these research questions.

Objective 1: Determine the impact of elevated pCO2 on embryonic development, and hatching success.

1) Does elevated pCO2 impact the embryonic development time, egg

volume, or hatching success of M. mercenaria embryos?

2) Does elevated pCO2 impact morphology (i.e., size and spination) in

newly hatched larvae brooded under ocean acidification conditions?

Objective 2: Determine the effects of elevated pCO2 and elevated temperature on larval survivorship, molt-stage duration, condition, calcification, and morphology.

3) Does elevated pCO2, elevated temperature, or the combined effects of

both stressors negatively impact survivorship, and molt-stage duration

throughout larval development?

4) Does elevated pCO2, elevated temperature, or the combined effects of

both stressors, impact the larval condition (i.e., ash free dry weight),

calcification (i.e., Mg/Ca ratios), and morphology (i.e., size) in stage III

and V larvae?

Objective 3: Determine how elevated pCO2 and temperature impacts larval geotactic swimming behaviors.

5) Does elevated pCO2, elevated temperature, or the combined effect of

both stressors impact passive sinking, downward swimming speed, or

swimming behavior in stage III and V larvae?

Objective 4: Parameterize a population model using stage-specific survivorship from objective 2 to assess how elevated pCO2 and temperature may impact stone crab larval population density.

6) What are the effects of elevated pCO2 and temperature on stone crab

population density?

DISSERTATION STRUCTURE

This dissertation has been organized into six chapters to address the aforementioned research questions. Chapters Two–Five were written as independent manuscript. Chapter One includes the background information with a literature review on the impacts of ocean acidification (OA) and elevated temperature on crustaceans. This chapter also provides the rationale and research questions for my dissertation. Chapter Two focuses on determining whether OA had any negative effects on the embryonic development and hatching success of the

Florida Stone crab, Menippe mercenaria. This chapter also determined if there were morphological differences in newly hatched larvae that were brooded in OA conditions. In Chapter Three I investigate the impacts of multiple stressors

(elevated pCO2 and temperature) on different components of the larval development of stone crabs including survivorship, molt-stage duration, condition, calcification, and morphology. Chapter Four reports on the effects elevated pCO2 and temperature had on the geotactic swimming behavior of stage III and V stone crab larvae. In Chapter Five I used the stage-specific survivorship from chapter three to parameterize a population-matrix model for larval stone crabs to determine how larval population densities may change under future OA and temperature scenarios. Chapter Six provides an overall summary of the significant findings from each chapter.

CHAPTER II: EMBRYONIC DEVELOPMENT

THE IMPACT OF OCEAN ACIDIFICATION ON THE EMBRYONIC DEVELOPMENT AND HATCHING SUCCESS OF THE FLORIDA STONE CRAB, MENIPPE MERCENARIA

INTRODUCTION

Since the preindustrial era, ocean acidification (OA) has resulted in a 0.1-unit decrease in oceanic pH, and forecast models predict an additional decline of 0.14–

0.35 pH units by the end of the century (Calderia and Wickett 2003, Meehl et al.

2007). In coastal areas, anthropogenic activities, including eutrophication, land-use modification, deforestation, sewage inputs, and wetland degradation contribute to increases in the baseline pCO2 (Millero et al. 2011, Bauer et al. 2013, Zhang and

Fischer 2014, Ekstrom et al. 2015), and influence the frequency, magnitude, and duration of extreme pH events (Harris et al. 2013, Hauri et al. 2013). Consequently, marine ecosystems along the east and Gulf coasts of the United States are expected to experience the effects of OA earlier than projected by global-scale models

(Ekstrom et al. 2015). Although nearshore systems, especially estuaries, experience considerable diurnal and tidal fluctuations in pCO2 (Hofmann et al. 2011), future extremes in pH may exceed the critical ecological and physiological thresholds of organisms that have adapted to less extreme environments (Attrill et al. 1999,

Ringwood and Keppler 2002, Hauton et al. 2009).

Ocean acidification causes a myriad of adverse effects on the behavior, physiology, morphology, and survivorship of calcifying marine organisms (Kuihara

2008, Kroeker et al. 2010, de la Haye et al. 2011, Hilmi et al. 2012, Munday et al.

2012). The response of organisms to OA, however, differs among taxonomic groups (Ries et al. 2009) and among populations (Walther et al. 2009). Therefore, broad generalizations about the impacts of OA on marine biota are problematic as future oceans will likely have ‘winners’ and ‘losers’, with some species being more tolerant to OA than other species (Carter et al. 2013, Hilmi et al. 2012). Generally, adult stages of the marine invertebrates inhabiting coastal ecosystems are well- adapted to fluctuations in pH because they possess the physiological mechanisms necessary to tolerate shifts in pCO2 (Kroeker et al. 2010, Ries et al. 2009, Whiteley

2011). However, early life stages (i.e., embryonic and early-larval development) may not possess, or are still developing (i.e., throughout ontogeny), the compensatory mechanisms needed to detect, respond, or tolerate environmental changes associated with shifts in oceanic carbonate chemistry (Waldbusser and

Salisbury 2014). The negative impacts of OA exposure, such as reduced survivorship, impaired development, and reduced embryonic activity, have been reported for several taxa, including the brittle star Ophiothrix fragilis (Dupont et al.

2008), the abalone Haliotis kamtschatkana (Crim et al. 2011), and the oyster

Saccostrea glomerata (Parker et al. 2009). Among crustaceans, exposure to OA conditions results in reduced hatching success (ranging from 30–95%) in the intertidal porcelain crab Petrolisthes cinctipes (Ceballos-Osuna et al. 2013), the copepod Acartia erythraea (Kurihara and Ishimatsu 2008), and the barnacles

Semibalanus balanoides (Findlay et al. 2009) and Calanus finmarchicus (Mayor et

13 al. 2007) while decreases in growth and survival are reported for the juvenile shrimp, Palaemon pacificus (Kurihara et al. 2008), and larvae of the spider crab

Hyas araneus (Walther et al. 2010).

The Florida stone crab, Menippe mercenaria, occurs in subtidal coastal habitats from North Carolina to the Gulf of Mexico and the Caribbean and is an important fishery in the southeastern United States. In Florida alone, stone crab landings support a US$25-million-a-year commercial and an active recreational fishery

(FWC 2000–2014). The continued deterioration of Florida’s coastal habitats threatens the future sustainability of stone-crab populations because embryonic development, larval release, and post-larval recruitment occur within coastal regions (Lindberg and Marshall 1984, Krimsky and Epifanio 2008, Krimsky et al.

2009, Gandy et al. 2010) where anthropogenic inputs have caused an increase in the baseline pCO2 and have accelerated the rate of OA (Bauer et al. 2013, Ekstrom et al. 2015). Florida Bay, which supports a significant stone-crab fishery, has experienced a decline in pH since 2005 that is estimated to be three-times faster than the rate observed in the open ocean (Zhang and Fischer 2014). Given the potential impacts of OA on the ability of crustacean larvae to complete development or successfully hatch, shifting baselines in seawater pCO2 in the coastal habitats of south Florida could have economic ramifications for Florida’s stone-crab fishery.

In this Chapter I examine the effects of future OA conditions on the embryonic development and hatching success of M. mercenaria. I also tested the hypothesis

14 that exposure to elevated p CO2 during embryonic development results in morphological abnormalities in newly-hatched larvae. These abnormalities include reduced body size and spine deformities, which may negatively impact larval survival by altering a range of physiological and behavioral responses, including swimming, the maintenance of vertical position, and predator defense.

MATERIALS AND METHODS

STUDY SITE, COLLECTION, AND MAINTAINENCE OF EXPERIMENTAL

ANIMALS

All experiments were conducted from June-August 2012 at the Mote Tropical

Research Laboratory in Summerland Key, Florida, USA (24° 39.69’ N, 81° 27.25’

W). Ovigerous M. mercenaria (carapace width 휇̅ = 9.56 cm ± 1.06 SD) were collected from Florida Bay within 17 km of the coast using commercial stone crab traps. Following transport to the laboratory, crabs were randomly assigned to either the control (present-day pCO2 levels) or elevated pCO2 treatments (8 broods per treatment). Only females with egg masses containing early-stage embryos (i.e., orange egg masses) were used in experiments. Ovigerous crabs were maintained in individual (40.6 x 20.3 x 25.4 cm) aquaria containing 19 L of seawater (salinity 35) under a light:dark cycle (14 hours day: 10 hours night) that approximated the photoperiod at the time of collection. Water temperatures were maintained within a narrow range (±0.26–0.28 SD) by partially submerging the aquaria in a

15 thermostatically controlled water bath. Crabs were fed frozen shrimp every other day, and aquaria were cleaned daily to remove wastes and uneaten food.

EXPERIMENTAL SETUP AND WATER CHEMISTRY

Experiments were performed using a flow-through ocean-acidification system at the Mote Tropical Research Laboratory in Summerland Key, Florida. The system consisted of 10 experimental aquaria (40.6 x 20.3 x 25.4 cm) that were sealed with acrylic lids. Treatment conditions within each aquarium were maintained by pumping seawater through 0.6- cm-diameter inlet and outlet holes that were drilled into the acrylic lids. Seawater for the control (~350 ppm) and elevated pCO2 treatment (~1500 ppm) were obtained from a deep-well aquifer. The well water contained naturally high levels of CO2, which results in a source of seawater with a pH of 7.43 (± 0.04 SD) (Hall et al. 2012). Before being pumped into the acidification system, seawater was first passed through a sand filter to remove particulates and then maintained in a holding reservoir where aeration was used to lower hydrogen sulfide concentrations to levels comparable to those in adjacent near-shore habitats (Hall et al. 2012). Seawater was then pumped into a second set of holding reservoirs (1000 L) where CO2 was degassed with ambient air until near-present day (control) or future pCO2 levels were achieved. The target level for the elevated pCO 2 treatment (~1500 ppm) was based on IPCC predictions for the next century assuming “business-as-usual” burning of fossil fuels (IPCC 2013, model RCP8.5; > 1000 ppm). Seawater pH within each experimental chamber was

16 monitored daily using a pH probe (Mettler-Toledeo). A daily three-point calibration was performed on the pH probe using NBS buffers. Daily flow rates (ml min-1) were measured by sampling the volume of seawater flowing into each experimental aquarium at 10 second intervals. Temperature, and salinity of each aquarium for both treatments were monitored daily using a Mettler-Toledo probe (temperature) and a refractometer (salinity; LW Scientific). Water samples for total alkalinity

(TA) measurements were collected from each experimental aquaria at the start (day

0), middle (~ day 7), and at the end (after hatching, day 10–12) of each trial. These water samples were refrigerated (~4.4°C; for TA) and were analyzed within 14 days of sampling. All seawater samples were processed by the Mote Marine

Laboratory Analytical Chemical Ecology facility in Sarasota, FL. A gran-titration method [EPA approved method (SM 2320 B-1997)] was used to estimate TA values. Total alkalinity and pH values were used to calculate pCO2 levels for the control and elevated treatments using the CO2calc program (Robbins et al. 2010).

EMBRYONIC DEVELOPMENT AND HATCHING SUCCESS

To monitor and compare the rate of embryonic development of stone crab embryos between the two treatments, a 7-stage classification system was used based on visual inspection of yolk content, eyespot size and shape, presence of abdominal segments, and presence of pigment cells (Table II-1; Pandian 1970,

Helluy and Beltz 1991, Garcia-Guerrero and Hendrickx 2004). The embryonic stage of development of each female was estimated daily by removing a subset of

20 embryos from the egg mass. Embryos were then photographed at 25x with a digital camera (Nikon Coolpix 995) attached to a dissecting microscope (Meiji

Techno), and their development stage was recorded (Table II-1). A generalized linear mixed model was used to determine if the rate of development differed among treatments, with brood treated as a nested factor. An F-test was then used to determine if the treatments had a significant effect on the embryonic development.

The effects of ocean acidification on embryo size, throughout development, were compared by measuring the diameter (d) near the center axis of each egg (20 eggs per day) using digital analysis software (Image J). Embryo volume (V) was then estimated using the formula for a sphere:

4 푑 푉 = 휋( )2 3 2 (1).

A mixed model was also used to compare the rate of embryonic development between treatments.

Hatching success was determined by removing a sample of eggs (~ 100) from the egg mass of females < 24 h prior to the anticipated time of larval release.

Clusters of embryos were maintained in clear acrylic chambers (3 cm x 3 cm x 4 cm) in the same treatment conditions as the brooding female. Hatching success was determined by counting the total number of hatched larvae one hour after the first larvae hatched from the isolated egg cluster. Hatching success did not meet

Table II.1. Classification scheme used to monitor the development of M. mercenaria embryos. Embryonic stages were based on yolk content, the presence and shape of the eyespot, and egg mass coloration. Descriptions were adapted from Pandian (1970), Helluy and Beltz (1991), and Garcia-Guerrero and Hendrickx (1991).

Stage Egg mass color Description 1 Bright orange 90-100% yolk 2 Dark orange Yolk composes 80–90% of egg, clear Pole at one end of egg, blastocyst 3 Rusty orange-light 60-80% yolk present, eyespots appear brown 4 Rusty orange-light Eyespots elongate, ~50% yolk present brown 5 Light-dark brown Eyespot triangular, <50% yolk, abdominal segments and pigment cells appear, heart beat visible 6 Dark brown Eyespots become oval, ~10-20% yolk adjacent to eyespot, abdominal segments defined along periphery of egg 7 Green Large bean shaped eyespots, <10% yolk present, pigment cells present, less than 24 hrs. from hatching

19 the assumption of homogeneity of variance and so the data were ranked.

Differences among treatments were determined using a randomized-block ANOVA on the ranked, data with pCO2 as a fixed factor and brood as a random factor (i.e., blocks).

MORPHOLOGICAL ANALYSIS OF LARVAE

To determine the potential effects of ocean acidification on larval morphology, twenty newly hatched larvae from each brood were collected within 24 hours of hatching and were preserved in 5% buffered formalin following the protocols of

Webb et al. (2006). Each larva was photographed at 25x using a digital camera

(Nikon CoolPix 995) attached to a dissecting microscope (Zeiss 2000-C). Seven morphological features, including the carapace width (CW), carapace height (CH), lateral spine length (LSL), rostral spine length (RSL), rostro-dorsal length (RDL), dorsal spine length (DSL), and terminal spine length (TSL), were measured from the digital micrographs using Image J software. Because of the high degree of shared variability among morphological features, Principle Component Analysis

(PCA) was used to establish a new set of orthogonal variables that were compared among treatment groups. The point of inflection in the scree-plot was used to determine the number of principle components (PCs) to retain. The derived component scores were then analyzed using separate nested ANOVAs (again, with brood as a nested factor) to determine if larval morphology differed among treatments.

RESULTS

WATER CHEMISTRY AND EXPERIMENTAL CONDITIONS

Seawater flow rates (휇 ±SD) through the control and elevated pCO2 aquaria were 12.7 L hour-1 ±2.08 SD and 12.7 L hour-1 ±2.28 SD respectively. The elevated

-1 pCO2 treatment (휇̅ ±SD: 2060 ±18 µmol kg ) had higher DIC than the control

(1829 ±38 µmol kg-1; Table II-2). The coefficient of variation (CV) showed that temperature, salinity, total alkalinity (TA) and pH were maintained within narrow ranges in both the elevated pCO2 (CV in elevated pCO2: temperature = 1.04%, salinity = 0.40%, TA = 1.12%, pH = 0.27%) and the control (CV in control: temperature = 1.01%, salinity = 0.49%, TA = 1.17%, pH = 0.62%). Both TA and

DIC in the experimental setup were within ranges reported for stone crab habitats within Florida Bay, which range from 2000–5600 µmol kg-1, and from 1466–2968

µmol kg-1 for TA and DIC respectively (Millero et al. 2011, Dufroe 2012).

EMBRYONIC DEVELOPMENT AND HATCHING SUCCESS

Embryos exposed to the elevated pCO2 treatment developed significantly slower (~32%) than the control conditions (F3, 134 = 20.4, P < 0.001; Control: y =

2 2 1.65 + 0.62x, R = 0.765; Elevated pCO2: y = 1.06 + 0.47x, R = 0.775, Figure II-

1).

Table II.2. Mean (± SD) treatment conditions for total alkalinity (TA), pCO2, inorganic carbon dioxide (DIC), pH, salinity, and temperature in control and experimental aquaria.

Parameter Elevated pCO2 Control TA (µmol kg-1) 2131 ±24 2140 ±25 pCO2 (ppm) 1514 ±97 349 ±50 DIC (µmol kg-1) 2057 ±32 1829 ±38 pH 7.50 ±0.02 8.05 ±0.05 Salinity 35.0 ±0.14 35.0 ±0.17 Temperature (°C) 26.7 ±0.28 26.6 ±0.27

Figure II.1. Change in embryonic stage as a function of time for crabs subjected to elevated pCO2 and control conditions. A linear function (Control: y = 2 2 1.65 + 0.62x, R = 0.77 ; elevated pCO2: 푦 = 1.06 + 0.47x, R = 0.78) was used to determine the rate of change in development among both treatments.

Embryonic development took on average 8.3 days ±1.3 SD to complete in the control, and 10.9 days ±1.7 SD to complete in the elevated pCO2 treatment.

The slower development in the elevated pCO2 treatment resulted in embryos hatching ~2.5–3.5 days later than embryos in the control. The size of embryos and the rate at which the embryo volume increased, at each developmental stage, was not statistically different among treatments (F3, 92 = 3.69, P = 0.787; Control: y =

2 2 0.11 + 0.01x, R = 0.703; Elevated pCO2: y = 0.11 + 0.009x, R = 0.792, Figure II-

2). Hatching success in the elevated p CO2 treatment varied among broods and ranged from 18%–85% in the elevated pCO2 treatment compared with 66%–87% in the control. Median hatching success in the elevated pCO 2 treatment was 46.3% (±

16.05 MAD) and was significantly reduced compared with the control 74.7% (± 5.1

MAD; F1, 15 = 11.5, P = 0.004; Figure II-3). The decrease among treatments was representative of a 38% reduction in hatching success.

LARVAL MORPHOLOGY

PCA analysis on the seven morphological measurements of newly hatched larvae resulted in three principle components (PC’s) representing 73% of the variation (Table II-3). PC 1 loadings were associated with the carapace width and lateral spine length were interpreted as representing overall larval width. PC 2 loadings were associated with rostro-dorsal spine length, rostral spine length and dorsal spine length, and were interpreted as representing overall animal size, whereas the loadings for PC 3 were interpreted as being associated with carapace

Figure II.2. Change in egg volume at each embryonic stage for crabs subjected to elevated pCO2 and control conditions. A linear function (Control: y = 2 2 0.11 + 0.01x, R = 0.70; elevated pCO2: 푦 = 0.11 + 0.009x, R = 0.79) was used to determine the rate of change in embryo volume among both treatments.

Figure II.3. A box and whiskers plot showing the hatching success of crabs maintained under control and elevated pCO2 conditions. Horizontal lines within the boxes indicate medians.

Table II.3. Results of a principal component analysis on seven morphological characters of newly hatched M. mercenaria larvae. Three orthogonal principal components (PCs) representing 72.9% of the total variation were retained by the analysis. The derived component scores were analyzed using separate nested ANOVAs to test for the effects of elevated pCO2 on larval morphology. The larger factor loadings were used to determine the variables contribution to each PC. PC Eigenvalues % Variation % Cumulative Variation 1 2.04 26.2 26.2 2 1.83 29.7 55.9 3 1.21 17.0 72.9 Loadings Variable PC1 PC2 PC3 Carapace Width 0.36 0.41 0.17 Lateral Spine (right) 0.95 0.01 0.05 Lateral Spine (left) 0.83 0.09 0.48 Rostral Spine 0.12 0.98 0.01 Dorsal Spine 0.22 0.24 0.05 Rostro-Dorsal Spine -0.05 0.92 0.09 Carapace Height 0.20 0.05 0.97

27 height. The derived component scores showed no significant differences among treatments for larval morphology (PC 1: F1,15 = 1.4 P = 0.25; PC 2: F1,15 = 1.1, P =

0.32; PC 3: F1,15 = 0.33, P = 0.67). There was also no significant brood effect on larvae (PC1: F1,15 = 3.2, P = 0.07; PC2: F1,15 = 1.2, P = 0.29; PC3: F1,15 = 0.08, P =

0.79).

DISCUSSION

The Florida stone crab, M. mercenaria, was used as a model to determine the effects of increases in seawater pCO2, projected for the next century, on embryonic development, hatching success, and larval morphology. We anticipated that stone crab embryos would be tolerant of ocean acidification (OA) since brooding occurs in coastal areas where seawater carbonate chemistry and pH vary naturally.

However, our results indicate that elevated pCO2 negatively impacts both embryonic development and larval hatching success. Species, like stone crabs, that live in environments that experience frequent fluctuations in abiotic factors are expected to be more tolerant of variations in environmental conditions (Stillman

2002, 2003, Hofmann and Todgham 2010), including seawater pH (Whiteley et al.

2011). For example, some crustaceans that inhabit coastal areas and experience natural variations in seawater pCO2 exhibit a greater tolerance of ocean acidification (Spicer et al. 2007, Hauton el al. 2009, Melzner et al. 2009, Ries

2009). However, other species, including the copepod Calanus finmarchicus, spider crab Hyas araneus, and porcelain crab Petrolisthes cinctipes, are sensitive to elevated pCO 2, especially during periods of embryonic and larval development

(Mayor et al. 2007, Walther et al. 2009, Carter et al. 2013). These results suggest that earlier life-history stages may be less tolerant of future increases in seawater acidity than juvenile or adult stages. The implications of the study are that under future oceans conditions, this sensitivity to OA may reduce larval supply and create a potential bottleneck for the stone crab fishery, especially if p CO2 levels in coastal habitats continue to increase.

The development rate of stone crab embryos was significantly slower (32%), or about 3 days longer, under elevated pCO2. The only other study to show that elevated pCO2 had a direct effect on the time to hatching in the Brachyura was a study on the Dungeness crab, Cancer magister, which reports a similar delay in the embryonic-development rate (delay by 24%; Miller et al. 2016). Other work on crustaceans has demonstrated that elevated pCO2 results in a similar delay on embryonic development in the amphipod Echinogammarus marinus (~8.5%;

Egilsdottir et al. 2009), and the barnacle Semibalanus balanoides (~19%; Findlay et al. 2009). OA-induced hypercapnia has been shown to reduce metabolism throughout embryogenesis (Ceballos-Osuna et al. 2013). The hypercapnic conditions, associated with OA, may impair the ability of embryos to exchange ions or gases (O2 and CO2) with the surrounding environment, resulting in extracellular acidosis and lower oxygen consumption rates. For instance, Carter et al. (2013) found that oxygen consumption of the porcelain crab, Petrolisthes cinctipes, was 11% lower under OA conditions. Therefore, exposure of stone crab

29 embryos to OA may reduce metabolic rates of oxygen consumption, which would contribute to the slower developmental rates reported here.

Hatching success of stone-crab larvae declined by nearly 28% in the elevated pCO2 treatment. This reduction is less than what has been reported for other crustaceans under similar OA conditions, including the barnacle S. balanoides

(60%; Findlay et al. 2009), the copepod Calanus finmarchicus (86%; Mayor et al.

2007), and the Tanner crab, Chionoecetes bairdi (40%; Swiney et al. 2015). Our hatching success results differ from what has been reported for other crab species

(P. cinctipes, and C. magister), which did not show a decrease in the percentage of larvae that successfully hatched under OA exposure (Ceballos-Osuna et al. 2013,

Miller et al. 2016). Normal embryonic development and hatching in crabs are typically associated with an increase in embryo volume as a result of water intake, metabolite production, and an increase in the size of the developing larva (Pandian

1970, Petersen and Anger 1997). In contrast, more variable hatching success, and a lack of increase in egg volume were observed under OA conditions in P. cinctipes, however, Ceballos-Osuna (2013) did not observe a difference in hatching success among treatments. In this study, the embryos appeared to develop normally (i.e., formation of the limbs and the eye spot), albeit slower in the OA treatment.

Moreover, the embryo size was similar between treatments suggesting that the reduction in hatching success was not linked to the embryonic development.

Although we are not certain of the underlying causes for the lower hatching success, other researchers have suggested that embryos could be undergoing

30 extracellular acidosis or CO2 toxicity (Ceballos-Osuna et al. 2013). During normal development, the oxygen consumption of crustacean embryos increases toward hatching (Brante et al. 2003, Naylor et al. 1999). Therefore, if embryos closer to hatching require more oxygen and are unable to effectively exchange respiratory

+ gases, then higher H concentrations and/or CO2 toxicity associated with OA may limit successful hatching.

Another potential factor contributing to the slower embryonic development and lower hatching success could be the impact of low pH on egg-mass care and maintenance by the gravid female. Environmental stressors (like OA) are known to negatively impact normal physiological function and reproduction in some adult crustacean species, including the lobster Nephrops norvegicus (Hernroth et al.

2012), the shrimp Palaemon pacificus (Kurihara et al. 2008), and the crabs

Chionoecetes tanneri (Pane and Barry 2007), Necora puber (Spicer et al. 2007), and P. cinctipes (Paganini et al. 2014). Additionally, other abiotic factors experienced during brooding, such as extremes in salinity, temperature, and pH are also known to slow hatching and reduce the rate of embryonic development

(Petersen 1995, Gimenez 2000). Brood care in crustaceans already comes at a high cost to the female, resulting in a decrease in energetic reserves (i.e., carbohydrates, lipids, and proteins; Fernandez and Brante 2003, Guadagnoli et al. 2005). Despite the ability of stone crab females to maintain their internal acid-base balance, continued regulation most likely comes with an increase in energetic cost, with less energy being allocated to other physiological functions (Whiteley 2011, Miller et

31 al. 2016). For instance, active brood maintenance by the female crabs during embryonic development facilitates gas exchange and hatching (Wheatly 1981,

Fernandez et. al. 2000). Previous work has shown that unventilated eggs during brooding (Naylor et al. 1999), and a lack of brood maintenance by the female

(Forster and Baeza 2001), results in an extended development period, reduced oxygen uptake by the embryos, and a lower hatching rate. Therefore, if OA results in a reduction in the female’s physiological condition (or activity), then lower seawater pH could have a negative impact on brood maintenance, including a decrease in abdominal pumping and egg mass preening, which could reduce the development and hatching success. However, this hypothesis has not been tested experimentally.

The morphology of newly hatched stone crab larvae was not impacted by lower pH. This result is similar to other studies on early life-history stages of larval crustaceans which have shown that the carapace length of newly hatched larvae in the European lobster, Homarus gammarus, and in C. magister were similar regardless of pCO2 exposure (Arnold et al. 2009, Miller et al. 2016). Walther et al.

(2010) also showed that after exposure to elevated pCO2 conditions newly hatched

Hyas araneus larvae exhibited no morphological change after hatching, a trend which continued throughout larval development. The results of the morphology experiment are in contrast with Long et al. (2013), who showed that Paralithodes camtschaticus larvae increased their carapace size by 4% when exposed to OA conditions. Our negative result may stem from the fact that planktonic decapod

32 larvae have unmineralized exoskeletons, thereby reducing the potential adverse effects of OA on calcification and growth throughout ontogeny (Anger 2001,

Whiteley 2011). However, there is potential for OA to impact the shape, size, or shell thickness of later stage larvae as more calcium is incorporated into the exoskeleton (Richards 1958, Neufeld and Cameron 1992, Arnold et al. 2009,

Whiteley 2011). For example, CO2-induced acidification caused a reduction in the concentration and in the percentage of calcium found in the exoskeleton in late stage (stage IV) H. gammarus larvae (Arnold et al. 2009).

Significant reductions in hatching success and slower embryonic development under OA conditions could have a damaging effect on the sustainability and management of the stone-crab fishery, especially if exposure to multiple stressors

(i.e., OA, salinity, temperature, and hypoxia) intensifies the responses reported here. For example, although anthropogenic increases in seawater temperature should increase the rate of embryonic development (Patel and Crisp 1960, Wear

1974), temperature extremes (>30°) have been shown to prevent hatching entirely in other brachyurans, including the blue crab Callinectes sapidus (Sandoz and

Rogers 1944). Additionally, over the last 4 decades, the trend of rising global sea surface temperatures has resulted in an increase in salinity (+0.1–0.4 psu) at lower latitudes (25°S and 35°N) of the Atlantic basin (Curry et al. 2003). It is not clear how an increase in salinity would impact stone crab hatching success or embryonic development, however, blue crab embryos were highly sensitive to salinity increases resulting in a longer embryonic development time (i.e., 4 days or 40%

33 longer), and no hatching occurred above salinities of 33 (Costlow and Bookhout

1959; Sandoz and Rogers 1944).

This is the first study to determine how OA may impact the embryonic development and hatching success of the Florida stone crab. The conditions experienced during embryonic development may result in carry-over effects and indirectly affect performance in later life stages (juveniles or adults; Gimenez 2002,

Gimenez and Anger 2001). If the negative impacts found in our study are exacerbated by carry-over effects, such as high post-hatch mortality, or impaired physiology throughout larval development, then larval supply may become limited

(Schiffer et al. 2013). These potential impacts are concerning for the stone-crab fishery, especially since anthropogenic inputs are causing coastal regions to experience shifts in the baseline pCO2 earlier than those projected for the open ocean (Bauer et al. 2013, Hauri et al. 2013, Ekstrom et al. 2015).

CHAPTER III: LARVAL DEVELOPMENT

THE EFFECTS OF ELEVATED PCO2 AND TEMPERATURE ON STONE- CRAB LARVAL SURVIVAL, CONDITION, CALCIFICATION, AND MORPHOLOGY

INTRODUCTION

Over the last 100 years, the unprecedented increase in atmospheric CO2 has caused the oceans to become a net CO2 sink (Bauer et al. 2013). At the current rates of fossil fuel emissions surface waters are predicted to increase in pCO2 from current levels of 400 ppm to 700–1000 ppm by the end of the century (IPCC 2013).

This process of increasing pCO2 in the ocean is now referred to as ocean acidification (OA). Increasing atmospheric CO2 is simultaneously warming sea surface temperatures, which are expected to rise by 2–4.5°C by 2100 (IPCC 2013).

OA will occur over multiple decades, which may give some species the ability to adapt to the conditions over multiple generations. However, human activities like increased urbanization, coastal development, and wetland degradation are diverting runoff which is resulting in excess nutrients (eutrophication) and increasing the frequency and magnitude of elevated seawater pCO2 events in coastal marine habitats (Bauer et al. 2013). The high nutrient input from land-based runoff and the degradation of organic material can act synergistically in coastal systems, to increase acidity (Melzner et al. 2013). Some coastal ecosystems are already experiencing a shift in frequency, magnitude, and duration of extreme events, which has led to either conditions that exceed critical thresholds for organisms, or

35 has moved outside the range of normal pH conditions (Hauri et al. 2013, Harris et al. 2013). The combined effects of elevated temperature and pCO2 are likely to constitute severe challenges for future benthic coastal organisms (Portner et al.

2005).

The adult stages of coastal species possess the physiological mechanisms and behavioral responses that are necessary to tolerate and adjust to frequent environmental fluctuations (Whiteley 2011). However, many coastal benthic invertebrates have distinct pelagic larval stages (Waldbusser and Salisbury 2014).

Larval, post-larval, and juvenile stages, have different sensitivities to changing environmental conditions than adult stages. These early developmental stages may not have, or are still developing, the compensatory mechanisms to physiologically tolerate environmental changes associated with unfavorable carbonate chemistry, and elevated temperatures (Waldbusser and Salisbury 2014, Pane and Barry 2007).

Therefore, determining the effects of multiple stressors (elevated pCO2 and temperature) on early life stages is critical to define a species’ tolerance to climate- change scenarios (Carter et al. 2013), and identify potential population bottlenecks, especially for coastal resources that have valued socio-economic importance.

In Florida, marine habitats are estimated to provide over $400 billion in revenue per year ($56 billion from tourism alone), and provide 900,000 jobs to the economy through recreation, tourism, and fishing (Kildow 2008). The stone crab,

Menippe mercenaria, contributes over $25 million a year to Florida’s economy

(Florida Fish and Wildlife Stock Assessments, 1998–2014). Since 1998, the mean

36 annual commercial catch has declined from 3.5 to 2.7 million pounds of claws per year (Florida Fish and Wildlife Conservation Commission Stock Assessments

1998-2014). The declining condition of Florida’s coastal habitats is a major concern for the future sustainability of stone crab populations, and understanding environmental effects on their early life stages may help prevent their decline.

Female stone crabs extrude embryos onto their abdomens during the summer months, and embryos take ~2 weeks to develop before larval release. After completing the five stages of larval development in shelf waters, stone-crab post- larvae are transported shoreward where they metamorphose into juveniles and settle in shallow coastal habitats. Some coastal stone crab habitats are experiencing higher concentrations of dissolved organic matter than previous periods which is resulting in a pH decline estimated to be three times faster than the rate observed in the open ocean (Briceno et al. 2013, Zhang and Fischer 2014). Therefore, tolerance to fluctuations in seawater pCO2 throughout development could ultimately impair the ability of larvae to complete development, which could potentially have economic ramifications for the Florida stone crab.

I tested the hypotheses that elevated pCO2 (~1100 ppm), temperature (32°C), or the combined effect of both stressors simultaneously would result in reduced survivorship and slower development of stone-crab larvae. I also tested the hypothesis that elevated pCO2, temperature, or the combined effect of both stressors simultaneously would reduce larval calcium content, and negatively

37 impact larval condition (ash free dry weight), which could result in smaller and morphologically deformed larvae.

MATERIALS & METHODS

OVIGEROUS FEMALE STONE CRAB COLLECTION

Ovigerous females were collected by Florida Fish and Wildlife using commercial stone crab traps near Pavilion Key, Florida. Ovigerous females were immediately transported back to the University of Miami’s Rosenstiel School’s

Ocean Acidification Laboratory and were maintained in ambient seawater conditions until larval release. Upon larval release, newly hatched larvae were randomly assigned into each of the experimental treatments described below.

Larvae from the same brood were divided among the treatments levels throughout all experiments.

EXPERIMENTAL DESIGN AND OA SYSTEM SET-UP

All experiments consisted of two treatments: 1) temperature, and 2) pCO 2, each with two levels. The two temperature levels were set at 30°C and 32°C. The lower

(control) temperature was based on the mean summer sea surface temperature for the Florida Keys (NOAA, http://www.ndbc.noaa.gov/view_climplot.php?station=lonf1&meas=st). The upper temperature limit was based on IPCC (2013) sea-surface temperature projections for the end of the century. The two pCO2 levels were set at ~450 and ~1100 ppm and were based on current IPCC (2013) projections. To achieve the control pCO2

38 level, the water was pumped into a holding reservoir and vigorously aerated until the reservoir was maintained at ~450 ppm. To achieve the experimental pCO2 levels, seawater was pumped into a separate holding reservoir. The elevated pCO2 levels were achieved by the addition of ultra-pure CO2 gas, which was regulated using mass flow controllers (SmartTrak 100, Sierra). The CO2 gas was slowly introduced through the MFCs into the holding reservoir using a venturi pump injection system.

All larvae were raised within the experimental aquaria which were maintained within a temperature controlled water bath. Once the pCO2 levels were established in each reservoir, the control and treatment water were pumped into each of the experimental aquaria. Probes within each aquaria were set-up to monitor temperatures using a digitally controlled temperature feedback system (Apex

System, Neptune). Individual heaters were placed within each aquaria. Temperature probes triggered the heater to turn on or off relative to set conditions. To avoid shock to the larvae, the use of MFC and the digitally controlled temperature system allowed a gradual increase in the experimental parameters (~200 ppm & ~0.4 °C per day) to the desired treatment levels over the first 5 days of each experiment.

SEAWATER CARBONATE CHEMISTRY

To monitor the carbonate chemistry of the OA system, seawater samples were collected from both the holding reservoirs and from each experimental aquaria in

150 mL borosilicate bottles and were then fixed with 100 μL of mercuric chloride.

Total alkalinity (TA) and dissolved inorganic carbon (DIC) were measured at

NOAA’s Atlantic Oceanographic and Metrological Ocean Acidification Laboratory using automated Gran titration (Apollo Instruments) and checked for accuracy with

Dickson standards (Scripps Institution of Oceanography, La Jolla, CA). Carbonate parameters were monitored every other day during the first week of the experiment, and every 5–7 days thereafter. The holding reservoirs were monitored every 4–5 days after the ramp-up period. The pHtotal within each experimental aquarium was also measured using a handheld pH meter (Oakton) and Ross electrode (Orion

9102BWNP; Thermoscientific), which was calibrated using Tris buffer (Dickson

Standard, Scripps Institution of Oceanography, La Jolla, CA). The pHtotal was used because the uncertainty of a potentiometric measurements of seawater pH can be less than 0.02 when compared with the National Bureau of Standards scale

(pHNBS), which can have uncertainty ranging from 0.5–0.1 units (Riebesell et al.

2010).

To calculate pCO2, both TA and DIC were measured during survivorship experiments (2014), while TA and pHtotal were measured during the larval condition, calcium content, morphology, and behavior experiments (2015). The change in the carbonate parameter being measured between the 2014 and 2015 research season was the result of NOAA’s DIC analyzer malfunctioning during the

2015 research season (until the last week of the experiments). Using TA, DIC, of pH, the remaining carbonate parameters (DIC, and/or p CO2) were determined using

CO2SYS software (Robbins et al. 2010). Temperature and salinity of each experimental aquarium were also monitored twice daily throughout all experiments

(Orion Ecostar). The carbonate chemistry of seawater samples collected at the site of ovigerous female collection were also analyzed for DIC and TA.

STONE CRAB LARVAL SURVIVORSHIP AND MOLT STAGE DURATION

Experiments examining the effects of elevated temperature and pCO2 on molt- stage duration and survivorship were conducted on larvae reared individually in plastic compartmentalized boxes (80 ml), with the plastic bottoms replaced with nylon mesh (190 µm). Each box was kept in its own water bath to maintain constant experimental temperatures. Forty-six larvae per ovigerous female were monitored in the boxes to determine the treatment effects on survivorship and molt- stage duration (MSD). Prior to feeding larvae, Artemia (40 per chamber) were enriched with a lipid diet (Selco, Brine shrimp direct, UT) and fed enriched rotifers.

Rotifers that were fed to Artemia were also enriched with a high protein lipid diet

(One Step, Rotigrow, CA). Larvae were kept on a 14-light:10-dark photoperiod that approximated conditions during the time of collection.

Survivorship and molt-stage duration were monitored by counting exuvia (i.e., molts) and dead larvae at the same time each day. In fact, survivorship was defined as the proportion of individuals that survive from birth to the post-larvae stage, and survival was defined as the chance that an individual will survive to the next stage.

The effect of different treatments on survivorship was determined using a failure-

41 time analysis (Cox Proportional Hazard Model), with larval death serving as the

“event”, and time since the beginning of the experiment as the “time until an event occurs”. Eight broods per treatment combination were used. To control for variation among broods, larvae from the same female were treated as covariates in the analysis. Comparisons of survivorship among treatments were made using a

Log-rank (LR) test.

Stage-specific survival was calculated by dividing the number of larvae surviving at each stage by the initial number of larvae that started each stage. An

Analysis of Variance using Repeated measures (ANOVAR) was performed with temperature and CO2 as the main effects, and brood as the within subject factor.

Because the stage-specific analysis required five separate tests each, the results were Bonferroni corrected to set the alpha level at 0.01. Molt-stage duration was also compared among treatments ANOVAR. All statistical analyses were performed using R (R Core Team, 2016).

ASH FREE DRY WEIGHT OF LARVAE

Larval weights (stage III and V) were determined using a protocol adapted from

Nates and McKenney (2000). The number of larvae used for determining dry weight (DW) and ash free dry weight (AFDW) differed among stages, with 50 individuals per sample being used for stage III larvae, and 10 individuals per sample being used for stage V larvae. Stage I larvae never experienced the experimental treatment set points, and therefore were not used in AFDW analyses.

The larval samples were used from multiple broods (i.e., 13 broods for stage III, and 8 broods for stage V). After harvesting, larvae were rinsed with distilled water, blotted dry on filter paper, and then oven-dried at 60°C for 30 hrs. The DW per group of larvae was determined using an ultra-microbalance (precision = 0.1 µg;

Mettler Toledo UMX2). After measuring DW, each sample was combusted (>

450 °C) for 12 hrs and reweighed. The AFDW was calculated by subtracting the mass of the ash from the total DW. Differences in the mean AFDW for each treatment combination were tested using an ANOVAR with temperature and CO2 as the main effects, and brood as the within subject factor.

LARVAL CA AND MG CONTENT, AND MG/CA RATIOS

To determine Ca and Mg content, larvae were mass reared in the experimental treatments, and harvested upon reaching stage III and V (i.e., stage III: 50 larvae from 11 broods per treatment; stage V: 10 larvae from 8 broods per treatment).

Stage I larvae never experienced the experimental treatment set points, and therefore were not sampled for analysis. Estimates of the Ca and Mg content per individual were determined for each treatment by dividing the concentration of Ca and Mg of the group of larvae, by the number of individuals present in the sample.

The Mg/Ca ratios were determined for larvae using a PerkinElmer 7300 dual view inductively coupled plasma atomic emission spectrometer (ICP-AES) at the United

States Geological Survey analytical laboratory in St. Petersburg, FL. All organic material was removed from the larvae via oxidation in a hot alkali-buffered solution

(~90°C; 100 µl+10 ml NaOH) for 10 minutes. The sample was then sonicated, washed 3x using milliQ water, and desiccated for 48 hours. To ensure larvae were completely digested, the sample was then dissolved by sonication in a warm 70% nitric acid solution for 15 minutes. Samples were centrifuged (7000 rpm for 5 minutes) to allow any remaining organic material to settle at the bottom of the vial.

Each sample was then diluted into a 2% nitric acid solution to match the nitric acid concentration of the standards used to calibrate the instrument. The accuracy of the

ICP-AES was determined from replicate ICP-AES runs of a standard Mg and Ca solution (PlasmaCal). The ICP-AES was calibrated using a standard solution before and after each experimental unit was run following the methods of Schrag (1999).

Differences in the Ca content, Mg content, and Mg/Ca ratio for each larval stage were determined using an ANOVAR with temperature and CO2 as the main effects, and brood as the within subject factor in R (R Core Team 2016).

MORPHOMETRIC ANALYSIS

To determine the potential effect of elevated pCO2 and temperature on larval size (n ~ 10), at each larval developmental stage (III and V), the larvae were harvested and fixed in 3% glutaraldehyde in 0.1 M phosphate buffer at room temperature (Felgenhauer and Abele 1983). Stage I larvae never experienced the experimental treatment set points, and therefore were not used in morphological analyses. After preservation, a Scanning Electron Microscope (SEM; JEOL JSM-

6380LV) was used to take digital images of larvae using methods described by

Felgenhauer and Abele (1983). To determine if any differences exist in spination or size among treatments, larvae were photographed so that the rostrum spine length

(RSL), dorsal spine length (DSL), telson spine length (TSL), rostro-dorsal length

(RDL), whole length (WL), carapace width (C W), and carapace width (CH) could be measured from digital SEM micrographs (using ImageJ software). Prior to measurement, digital images of stage III and V larvae were calibrated in ImageJ by determining the number of pixels within the micrometer scale provided by the

SEM. The CW was defined as the distance from the base of the rostral spine to the midpoint of the posterior lateral margin of the carapace (Long et al. 2013). The CH was defined as the distance from the base of the dorsal spine to the ventral edge of the carapace (Long et al. 2013). Because of the high degree of shared variability among morphological features, principle component analysis (PCA) was used to establish a new set of orthogonal variables that were compared among treatment groups. The contribution of the new variables to each principle component (PC) was determined based on the largest factor loadings for each PC. The point of inflection on the scree-plot was used to determine the number of PCs to retain. The derived component scores were then analyzed using separate ANOVAR’s (with brood as a within subject factor) to determine if larval morphology differed among treatments.

RESULTS

CARBONATE CHEMISTRY DURING SURVIVORSHIP AND MOLT STAGE

DURATION EXPERIMENTS

After pCO2 and temperature were gradually increased to the experimental set points, the control’s (i.e., ambient temperature and ambient pCO 2) mean pCO2 level was maintained at 484 ppm ±SD 28.5 (mean ± SD). The control pCO2 and temperature levels were maintained at ambient set points. The elevated temperature/ambient pCO2 treatment (A32) was held at 524 ppm ±26.3 (mean ±

SD). The mean elevated pCO2 in the ambient temperature/high pCO2 (H30) treatment was 1149 ppm ±SD 103.8 (mean ± SD), whereas the elevated temperature/elevated pCO2 (H32) was held at 1199 ppm (±SD 156.1; Figure III-1).

Temperature, salinity, and total alkalinity (TA) were controlled within a narrow range after the gradual increase to the experimental set points, for the 2014 and

2015 summer research seasons (Table III-1). Changes in dissolved inorganic carbon (DIC) drove the different pCO2 levels within each treatment. As expected, the DIC was higher in the elevated pCO2 treatment (Table III-1). The control pCO2 levels were similar to the pCO 2 of field samples, collected by Florida Fish and

Wildlife, at crab collection sites, and were within the ranges reported for other stone crab habitats (Millero et al. 2011, Dufroe 2012; Figure III-1). During the

2014 summer, the holding reservoirs for the ambient and elevated pCO2 levels were the only tanks that were monitored on days 6, 17, 22, and 27, whereas the holding

Figure III.1. Mean pCO2 (ppm, ± SD) for the experimental treatments and field collection site. Experimental tanks were not sampled on days 6, 17, 22 and 24 (2014). Higher pCO2 values on day 27 are likely due to a clog in ambient air deliver system to the elevated pCO2 reservoir. The shapes in the figure represent the means and the whiskers represent the standard deviation.

Table III.1. Seawater carbonate chemistry, temperature, and salinity for the 2014 and 2015 summer laboratory experiments. The A30 treatment represents the control with ambient temperature and ambient pCO2. The H30 represents ambient temperature and elevated p CO2. The A32 treatment was held at elevated temperature and ambient pCO2, while the H32 treatment was maintained at both elevated temperature and elevated pCO2. 2014 Temperature (°C) TA DIC Salinity Treatments (µequiv kg-1) (µmol kg1) A30 30.1 ± 0.26 2469.0 ± 33.3 2139.3 ± 32.0 35.5 ± 0.19 H30 30.0 ± 0.33 2463.6 ± 30.9 2305.1 ± 30.1 35.5 ± 0.15 A32 32.0 ± 0.16 2468.5 ± 34.9 2140.9 ± 32.2 35.5 ± 0.17 H32 32.0 ± 0.17 2465.5 ± 29.7 2300.6 ± 34.6 35.4 ± 0.18

2015 Temperature (°C) TA pH total Salinity Treatments (µequiv kg-1) A30 30.0 ± 0.23 2286.1 ± 38.4 8.05 ± 0.02 37.7 ± 0.47 H30 30.0 ± 0.39 2285.9 ± 34.7 7.79 ± 0.07 37.7 ± 0.46 A32 31.9 ± 0.16 2282.6 ± 34.4 8.00 ± 0.02 37.8 ± 0.50 H32 31.8 ± 0.28 2284.9 ± 35.2 7.74 ± 0.05 37.8 ± 0.51

48 reservoirs for the ambient and elevated pCO2 levels were only monitored on days 4,

6, 13 and 17 during the 2015 summer. Higher pCO2 values on day 27 (2014) are likely due to a clog in the ambient air delivery system to the elevated pCO2 reservoir.

CARBONATE CHEMISTRY DURING SURVIVORSHIP AND MOLT STAGE

DURATION EXPERIMENTS

After pCO2 and temperature were gradually increased to the experimental set points the control’s mean pCO2 was maintained at 467.4 ppm ± 25.9 (mean ± SD), whereas the A32 treatment was held at 568.7 ppm ±12.8 (mean ± SD; Figure III-2).

The mean pCO2 level in the H30 treatment was 947.6 ppm ±47.4 (mean ± SD), whereas the H32 treatment was held at 1085.0 ppm ±50.4 (mean ± SD).

Temperature, salinity, and total alkalinity (TA), were controlled within a narrow range after the gradual increase to treatment levels 1(Table III- ). The pHtotal was lower in the elevated pCO2 treatments (Table III-1). The ambient pCO2 levels were within the range of the pCO2 at field collection sites and were within ranges reported for stone crab habitats (Millero et al. 2011, Dufroe 2012; Figure III-2).

SURVIVAL AND DEVELOPMENT

When larvae were raised in elevated pCO2 and temperature treatments, survivorship to megalopae was significantly reduced in all treatments relative to the control (LR7 = 272.3, P < 0.001; Figure III-3). The mean percentage of individuals

49 9

Figure III.2. Mean pCO2 (ppm, ± SD) for the experimental treatments and for field collection site Experimental tanks were not sampled on days 4, 6, 13, and 17. The shapes in the figure represent the means and the whiskers represent the standard deviation.

Figure III.3. Cumulative survivorship of Menippe mercenaria larvae throughout larval development during exposure to different combinations of pCO2 and temperature. A log-rank test was used for pairwise comparisons. Curves with different letters are significantly different at α = 0.05. A30 (i.e., the control) represents the ambient pCO2 and ambient temperature treatment. H30 is the elevated pCO2 and ambient temperature treatment. A32 is the ambient p CO2 and elevated temperature treatment, and H32 is the elevated pCO2 and elevated temperature treatment.

51 surviving to megalopae was 51.4% ± 3.2 (mean ±SE) in the control, 32.3% ± 2.8

(mean ±SE) in the H30, 14.9% ±2.0 (mean ±SE) in the A32, and 9.8% ± 1.6 (mean

±SE) in the H32 treatments respectively. Pairwise comparisons (log-rank test) indicated that survivorship was significantly lower than the control (A30) in all treatments (A30 vs. H30: LR1 = 21.7, P < 0.001; A30 vs. A32: LR1 = 152.2, P <

0.001; A30 vs. H32: LR1 = 195.7, P < 0.001). Larval survivorship in the A32 and

H32 were not significantly different from each other (LR1 = 1.9, P = 0.16). A comparison of the hazard ratios indicated that mortality was 3.3 and 3.7 times more likely in the A32 and H32 conditions, respectively, than larvae raised in A30.

Larvae raised in the H30 treatment were only 1.5 times more likely to die than larvae raised in the A30 treatment. Female brood (covariate) had a significant effect on survivorship (Wald χ2 = 45.2, d.f. = 7, P < 0.001).

Comparisons were also made to determine if there were differences in the stage-specific survivorship among treatments. The stage-specific survival did not meet the assumptions of normality and were therefore rank transformed. An

ANOVAR was then run on the ranked data, with temperature and CO2 as the main effects, and brood as the within subject factor. The two main effects showed no significant impact on the stage-I survivorship (Temperature: F1,7 = 1.8, P = 0.19;

CO2: F1,7 = 0.13, P = 0.72; Figure III-4). The median-stage-specific survivorship for stage I larvae, ranged from 92.3–94.5% among all treatments. Newly hatched

Figure III.4. Box and whiskers plot of the survivorship of stage-I Menippe mercenaria larvae during exposure to different combinations of pCO2 and temperature. An ANOVAR was performed on the ranked data with temperature and CO2 as the main effects and brood as the within subject factor. Boxes with similar letters above them are not significantly different from each other. H30 is the elevated pCO2 and ambient temperature treatment. A32 is the ambient pCO 2 and elevated temperature treatment, and H32 is the elevated pCO2 and elevated temperature treatment.

53 larvae also showed no significant interaction among the treatments (F1,7 = 0.69, P <

0.42), however, there was a significant within-subject effect (F7, 21 = 2.7, P < 0.03).

Stage II larvae had a significantly lower median stage-specific survivorship in the A32 and H32 treatments (both elevated temperature treatments were ~5.5% lower than the ambient temperature treatments; Temperature: F1,7 = 15.5, P <

0.001; CO2: F1,7 = 1.01, P = 0.32; Figure III-5). Relative to the control, the median stage-specific survivorship for stage III larvae was also significantly lower by

16.6% and 31.4% in the A32 and H32 treatments respectively (Temperature: F1,7 =

56.2, P < 0.001; CO2: F1,7 = 5.7, P = 0.03; Figure III-6). There were no significant interactions for stage II or stage III larvae (stage II: F1,7 = 1.83, P = 0.19; F1,7 =

0.18, P = 0.67), nor was there any significant effect within subject treatment (stage

II: F7, 21= 1.9, P = 0.13; stage III: F7, 21= 1.79, P = 0.14).

Stage IV larvae exhibited significant differences in both main effects

(Temperature: F1,7 = 15.6, P < 0.001; CO2: F1,7 = 80.4, P < 0.001; Figure III-7).

Stage IV larvae raised in the H30, A32, and H32 treatments showed decreases in survivorship of 11.9%, 31.1%, and 43.2% respectively when compared to the control. The stage-specific survivorship of stage-V larvae showed significant differences among the main effects, with the greatest overall decrease in survivorship compared with the other larval stages (Temperature: F1,7 = 15.3, P <

0.001; CO2: F1,7 = 108.4, P < 0.001; Figure III-8). Relative to the control, the stage-

V larvae showed a decrease in survivorship in the H30, A32, and H32 treatments

Figure III.5. Box and whiskers plot of the survivorship of stage-II M. mercenaria larvae during exposure to different combinations of pCO2 and temperature. An ANOVAR was performed on the ranked data with temperature and CO2 as the main effects and brood as the within subject factor. Boxes with different letters above them are significantly different from each other. H30 is the elevated pCO2 and ambient temperature treatment. A32 is the ambient pCO2 and elevated temperature treatment, and H32 is the elevated pCO2 and elevated temperature treatment.

Figure III.6. Box and whiskers plot of the survivorship of stage-III M. mercenaria larvae during exposure to different combinations of pCO2 and temperature. An ANOVAR was performed on the ranked data with temperature and CO2 as the main effects and brood as the within subject factor. Boxes with different letters above them are significantly different from each other. H30 is the elevated pCO2 and ambient temperature treatment. A32 is the ambient pCO 2 and elevated temperature treatment, and H32 is the elevated pCO2 and elevated temperature treatment.

Figure III.7. Box and whiskers plot of the survivorship of stage-IV M. mercenaria larvae during exposure to different combinations of pCO2 and temperature. An ANOVAR was performed on the ranked data with temperature and CO2 as the main effects and brood as the within subject factor. Boxes with different letters above them are significantly different from each other. H30 is the elevated pCO2 and ambient temperature treatment. A32 is the ambient pCO2 and elevated temperature treatment, and H32 is the elevated pCO2 and elevated temperature treatment.

Figure III.8. Box and whiskers plot of the survivorship of stage-V M. mercenaria larvae during exposure to different combinations of pCO2 and temperature. An ANOVAR was performed on the ranked data with temperature and CO2 as the main effects and brood as the within subject factor. Boxes with different letters above them are significantly different from each other. H30 is the elevated pCO2 and ambient temperature treatment. A32 is the ambient pCO2 and elevated temperature treatment, and H32 is the elevated pCO2 and elevated temperature treatment.

58 by 19.4%, 45.9%, and 53.3% respectively. Stage IV and V larvae did not show any interactions among treatments (stage IV: (stage IV: F1,7 = 1.16, P = 0.29; stage V:

F1,7 = 1.16, P = 0.29), nor was there any within-subject effects (stage IV: F7, 21 =

1.7, P = 0.16; stage V: F7, 21 = 2.18, P = 0.07).

The molt-stage duration (MSD) data met the normality assumptions for an

ANOVAR (stage I: W = 0.97, P = 0.58; stage II: W = 0.94, P = 0.09; stage III: W =

0.96, P = 0.27; stage IV: W = 0.95, P = 0.13; stage V: W = 0.97, P = 0.54; Figure

III-9). Differences among treatments in the MSD for each larval stage were determined using an ANOVAR with temperature and CO2 as the main effects and brood as the within subject factor. Elevated temperature (A32 and H32) had a significant effect on development resulting in shorter molt stage durations (stage I:

Temperature: F1,7 = 44.1, P < 0.001; stage II: Temperature: F1,7 = 132.4, P < 0.001; stage III: Temperature: F1,7 = 81.9, P < 0.001; stage IV: Temperature: F1,7 = 44.8, P

< 0.001; stage V: Temperature: F1,7 = 32.7, P < 0.001; Figure III-9). Larvae in the elevated temperature treatment molted ~0.8–1.2 days earlier than larvae raised at ambient temperature. There was no effect of elevated pCO2 on larval molt-stage duration until stage V (stage I: CO2: F1,7 = 4.1 P = 0.06, stage II: CO2: F1,7 = 3.7 P =

0.06; stage III: CO2: F1,7 = 0.43 P = 0.52, stage IV: CO2: F1,7 = 0.19 P = 0.66, stage

V: CO2: F1,7 = 4.7, P = 0.04; Figure III-9). There were no significant treatment interactions for larval stage I (stage I: F1,7 = 3.4, P = 0.08), however, there was a within-subject effect (stage I: F7, 21 = 3.2, P = 0.01). Stage II–IV larvae also did not show any significant treatment interactions (stage II: F1,7 = 0.011, P = 0.92; stage

Figure III.9. Mean (days, ±S.E.) molt stage duration of Menippe mercenaria larvae throughout larval development during exposure to different combinations of pCO2 and temperature. Letters above the bars represent differences between the treatments at α = 0.05.

II: F1,7 = 0.011, P = 0.92; stage III: F1,7 = 0.11, P = 0.74; stage IV: F1,7 = 3.9, P =

0.06), nor were there any significant within-subject effects for stages II–IV (stage

II: F7, 21 = 1.1, P = 0.39; stage III: F7, 21 = 1.6, P = 0.18; stage IV: F7, 21 = 0.81, P =

0.59).

Stage V larvae exhibited a significant increase in their development time in elevated pCO2 (H30) relative to the control (Figure III.9). The H30 treatment extended the stage V MSD by ~ 0.78 days compared with the control. Stage V larvae also had a significant interaction among the treatments (F1,7 = 7.9, P = 0.01), but there was no within-subject effect (F7,21 = 0.44, P = 0.87).

ASH FREE DRY WEIGHT

The stage III ash free dry weight (AFDW; µg individual-1) met the normality assumption for an ANOVAR (AFDW Shapiro test: W = 0.97, P = 0.34). The mean

AFDW for stage-III larvae (13 broods) was within a narrow range (5.6–5.9 µg

-1 individual ), and did not differ among treatments (Temperature: F1, 12 = 0.012, P =

0.91; pCO 2: F1, 12 = 0.39, P = 0.53; Figure III-10). There was no interaction between temperature and pCO 2 for stage III AFDW (F1, 12 = 0.06, P = 0.81), however, there was a significant within-subject effect (F12, 36 = 6.8, P < 0.001; Table III.2). The

AFDW (µg individual-1) for stage V larvae also met the parametric assumptions for an ANOVAR (AFDW Shapiro test: W = 0.96, P = 0.37). No significant differences were present among the main effects (Temperature: F1, 7 = 0.074, P = 0.78; CO2: F1,

7 = 0.072, P = 0.79; Figure III-10 ). There was no interaction (F1, 7 = 1.25, P = 0.28),

Figure III.10. Mean ash free dry weight (µg individual-1, ± S.E.) for stage-III and V M. mercenaria larvae during exposure to different combinations of pCO2 and temperature. There were no significant differences among the main effects. The control (A30) was maintained at both ambient pCO2 (~450 ppm) and ambient temperature (30 °C). The elevated pCO2 treatment was maintained at ~1100 ppm whereas the elevated temperature treatment target was 32 °C.

Table III.2. Results of the repeated measures ANOVAs for stage-III and V ash free dry weight. Significant values are in bold.

Source of df SS MS F P Variation Stage III Temperature 1 0.01 0.011 0.012 0.91 CO2 1 0.36 0.36 0.39 0.53 Temperature x 1 0.05 0.053 0.059 0.81 CO2 Brood 12 74.2 6.18 6.84 <0.001 Residuals 36 32.6 0.90

Stage V Temperature 1 0.79 0.79 0.074 0.78 CO2 1 0.78 0.77 0.072 0.79 Temperature x 1 13.43 13.42 1.25 0.28 CO2 Brood 7 192.4 27.5 2.55 0.04 Residuals 21 226.0 10.7 62

63 however, there was a significant within subject effect (F7, 21 = 2.55, P = 0.04).

MG AND CA CONTENT AND MG/CA RATIOS

The stage III Ca content, Mg content and Mg/Ca did not meet the normality assumptions and were therefore rank transformed. An ANOVAR was then performed on the ranked data to test for differences among treatments using temperature and CO2 as the main effects, and brood as the within-subject factor.

There were no significant main effects for stage III Ca concentration (Temperature:

F1, 10 = 0.18, P = 0.67; CO2: F1, 10 = 0.57, P = 0.46; Figure III-11; Table III-3), no interaction effects (F1, 10 = 2.2, P = 0.15), and no within subject effects (F10, 30 =

1.05, P = 0.43). There were also no significant main effects in the stage III Mg concentration (Temperature: F1,10 = 0.002, P = 0.96; CO2: F1,10 = 1.1, P = 0.29;

Figure III-12), nor was there any interaction (F1,10 = 0.26, P = 0.61), or within- subject effects (F10,30 = 0.71, P = 0.70; Table III-3). The Mg/Ca ratios also showed no significant difference in the main effects (Temperature: F1,10 = 0.23 P = 0.64;

CO2: F1,10 = 0.03, P = 0.86; Figure III-13), nor was there any interaction effect

(F1,10 = 4.1, P = 0.05). There were also no within-subject effects for the Mg/Ca ratios (F10,30 = 1.5, P = 0.19; Table III-3).

The Stage V Ca and Mg content data met the assumptions of normality (Ca content: W = 0.96, P = 0.47; Mg content: W = 0.95, P = 0.26), therefore, an

ANOVAR was performed with temperature and pCO2 as the main effects and brood as the within-subject factor. There were no significant treatment effects in

Figure III.11. Box and whiskers plot of the Ca concentration (mg L-1) for stage-III M. mercenaria larvae during exposure to different combinations of pCO2 and temperature. An ANOVAR was performed on the ranked data with temperature and CO2 as the main effects and brood as the within subject factor. The horizontal lines in the figure represents the median, the boxes represent the lower and upper quartiles (25% and 75%), and the vertical lines (whiskers) represent the minimum and maximum values.

Table III.3. Results of the repeated measures ANOVARs for stage-III Ca and Mg concentration, and Mg/Ca ratio.

Source of Variation df SS MS F P Stage III Ca Concentration Temperature 1 29 29.5 0.18 0.67 CO2 1 93 93.1 0.57 0.46 Temperature x CO2 1 361 360.8 2.2 0.15 Brood 10 1718 171.8 1.05 0.43 Residuals 30 4894 163.1 Stage III Mg Concentration Temperature 1 0.36 0.36 0.002 0.96 CO2 1 209 209.5 1.14 0.29 Temperature x CO2 1 48 48.1 0.26 0.61 Brood 10 1312 131.2 0.71 0.70 Residuals 30 5525 184.2 Stage III Mg/Ca Temperature 1 33 32.8 0.23 0.64 CO2 1 4 4.5 0.031 0.86 Temperature x CO2 1 596 596.5 4.14 0.05 Brood 10 2137 213.7 1.48 0.19 Residuals 30 4324 144.1

Figure III.12 Box and whiskers plot of the Mg concentration (mg L-1) for stage-III M. mercenaria larvae during exposure to different combinations of pCO2 and temperature. An ANOVAR was performed on the ranked data with temperature and CO2 as the main effects and brood as the within subject factor. The horizontal lines in the figure represents the median the boxes represent the lower and upper quartiles (25% and 75%), and the vertical lines (whiskers) represent the minimum and maximum values.

Figure III.13. Box and whiskers plot of the Mg/Ca ratio for stage-III M. mercenaria larvae during exposure to different combinations of pCO2 and temperature. An ANOVAR was performed on the ranked data with temperature and CO2 as the main effects and brood as the within subject factor. The horizontal lines in the figure represents the median the boxes represent the lower and upper quartiles (25% and 75%), and the vertical lines (whiskers) represent the minimum and maximum values.

68 the stage V Ca concentration per individual (Temperature: F1,7 = 2.6, P = 0.13;

CO2: F1, 7 = 1.5, P = 0.24; Figure III-14), nor was there any interaction (F1, 7 = 0.09,

P = 0.77; Table III-4).

The Mg concentration for stage V larvae also showed no significant treatment effects (Temperature: F1, 7 = 2.9, P = 0.11; CO2: F1, 7 = 0.17, P = 0.69; Figure III-

15), nor was there any interaction (F1, 7 = 0.19, P = 0.66; Table III-4). There were no within-subject effects for stage V Ca or Mg concentrations (Ca: F7, 21 = 2.3, P =

0.06; Mg: F7, 21 = 1.4, P = 0.26).

The Stage V Mg/Ca content did not meet the normality assumptions and were rank transformed. The ANOVAR was then performed on the ranked data with temperature and pCO2 as the between subjects and brood as the within subject factor. There were no significant differences in Mg/Ca among treatments

(Temperature: F1,7 = 3.2, P = 0.09; CO2: F1,7 = 0.03, P = 0.87; Figure III-16), interaction (F1,7 = 0.09, P = 0.77), and no within-subject effect (F7, 21 = 1.9, P =

0.11).

LARVAL MORPHOLOGY

PCA analysis on the morphological measurements of stage III larvae resulted in three principle components (PC’s) representing 79.4% of the variation in the data

(Table III-5). The PC 1 loadings were associated with the carapace height and width, and were interpreted as representative of the overall larval size. The loadings

Figure III.14. Ca concentration (mg L-1 individual -1) for stage-V M. mercenaria larvae during exposure to different combinations of pCO2 and temperature. An ANOVAR was performed on the Ca concentration, with temperature and CO2 as the main effects and brood as the within-subject factor. The bars represent the means, and the vertical lines represent the standard errors.

Table III.4. Results of the repeated measures ANOVARs for stage-V Ca and Mg concentration, and Mg/Ca ratio. The Mg/Ca ratios were rank transformed.

Source of Variation df SS MS F p Stage V Ca Concentration Temperature 1 0.73 0.73 2.6 0.12 CO2 1 0.42 0.42 1.46 0.24 Temperature x CO2 1 0.03 0.03 0.09 0.76 Brood 7 4.5 0.65 2.3 0.06 Residuals 21 5.9 0.29 Stage V Mg Concentration Temperature 1 0.04 0.04 2.9 0.10 CO2 1 0.002 0.002 0.17 0.69 Temperature x CO2 1 0.003 0.003 0.19 0.66 Brood 7 0.14 0.019 1.39 0.26 Residuals 21 0.301 0.014 Stage V Mg/Ca Temperature 1 231.1 231.1 3.2 0.09 CO2 1 2.0 2.0 0.03 0.87 Temperature x CO2 1 6.1 6.1 0.09 0.77 Brood 7 976.0 139.4 1.9 0.11 Residuals 21 1512.8 72.0

Figure III.15. Mg concentration (mg L-1 individual -1) for stage-V M. mercenaria larvae during exposure to different combinations of pCO2 and temperature. An ANOVAR was performed on with temperature and CO2 as the main effects and brood as the within-subject factor. The bars represent the means, and the vertical lines represent the standard errors.

Figure III.16. Box and whiskers plot of Mg/Ca for stage-V M. mercenaria larvae during exposure to different combinations of pCO2 and temperature. An ANOVAR was performed on the ranked data with temperature and CO2 as the main effects and brood as the within subject factor.

Table III.5. Results of a principal component analysis on seven morphological characters of stage-III M. mercenaria larvae. Three orthogonal principal components (PCs) representing 79.4% of the total variation were retained by the analysis. The derived component scores were analyzed using separate ANOVAR to test for the effects of elevated pCO2 on larval morphology. The largest factor loadings were used to determine the variables contribution to each PC.

PC Eigenvalues % Variation % Cumulative Variation

1 4.1 59.2 59.2

2 0.85 12.2 71.4

3 0.56 8.0 79.4

Loadings

Variable PC1 PC2 PC3

Telson spine 0.52 -0.19 -0.22

Dorsal spine -0.26 -0.09 0.92

Rostral spine 0.35 -0.19 0.08

Carapace height -0.39 0.92 -0.09

Carapace width -0.08 0.49 -0.03

Tail length 0.55 -0.23 -0.19

Whole length 0.11 0.01 0.29

74 for PC 2 were associated with the dorsal spine, whereas the loadings for PC 3 were interpreted as being the rostral spine. The derived component scores were compared using an ANOVAR, and showed no significant differences for larval morphology (PC 1 temperature: F1,406 = 0.97 P = 0.43, PC 1 CO2: F1,406 = 0.02, P =

0.89 ; PC 2 temperature: F1,406 = 20.5 P = 0.05, PC 2 CO2: F1,406 = 15.5, P = 0.06;

PC 3 temperature: F1,406 = 0.88 P = 0.45, PC 3 CO2: F1,406 = 0.06, P = 0.83 Figure

III-17). There was also no interaction effect (PC 1: F1,406 = 1.9, P = 0.29; PC 2:

F1,406 = 0.01, P = 0.91; PC 3: F1,406= 6.5, P = 0.12), however, there was a significant within-subject effect (PC 1: F5,406 = 61.5, P < 0.001; PC 2: F5,406 = 39.2, P < 0.001;

PC 3: F5,406= 15.2, P < 0.001).

PCA analysis on the morphological measurements of stage V larvae resulted in two PC representing 71.4% of the variation (Table III-6). The PC 1 loadings were associated with larval tail, telson spine, and rostral spine lengths, and were interpreted as representing animal length. The loadings for PC 2 were associated with the carapace height and width, and were interpreted as representing overall animal size. The derived component scores were compared among the main effects using an ANOVAR, and showed no significant differences for larval morphology

(PC 1: temperature: F1,616 = 1.9 P = 0.19, CO2: F1,616 = 3.3, P = 0.07; PC 2: temperature: F1,616 = 0.97 P = 0.32 CO2: F1,7 = 0.39 P = 0.53; Figure III-18). There was also no interaction effect (PC 1: F1,616 = 0.63, P = 0.43; PC 2: F1,7 =2.7 P =

0.09), however, there was a significant brood effect (PC 1: F6, 616 = 58.5, P < 0.001;

PC 2: F6, 616= 28.9, P < 0.001).

Figure III.17. Principal component plot of M. mercenaria stage-III larval morphometrics during exposure to different combinations of pCO2 and temperature. There were no significant treatment effects on stage-III larval morphology.

Table III.6. Results of a principal component analysis on seven morphological characters of Stage-V M. mercenaria larvae. Two orthogonal principal components (PCs) representing 71.4% of the total variation were retained by the analysis. The derived component scores were analyzed using separate ANOVAR’s to test for the effects of elevated pCO2 on larval morphology. The largest factor loadings were used to determine the variables contribution to each PC.

PC Eigenvalues % Variation % Cumulative Variation

1 4.1 59.1 59.1

2 0.85 12.2 71.4

Loadings

Variable PC1 PC2

Telson spine 0.52 -0.19

Dorsal spine -0.26 -0.09

Rostral spine 0.35 -0.19

Carapace height -0.39 0.92

Carapace width -0.08 0.49

Tail length 0.55 -0.23

Whole length 0.11 0.01

Figure III.18. Principal component plot of M. mercenaria stage-V larval morphometrics during exposure to different combinations of pCO2 and temperature. There were no significant treatment effects on stage-V larval morphology. Each data point represents an individual larva.

DISCUSSION

The results reported here suggest that stone crab larval survivorship was significantly impacted by elevated pCO2 and temperature conditions, however, larval condition, skeletal content, and morphology did not appear to be affected by elevated pCO2 and temperature treatments. Although coastal species in general are considered to be more tolerant to ocean acidification (OA) and elevated temperature due to their exposure to fluctuating environmental changes (Hauton et al. 2009, Whiteley 2011 for review), some coastal crustaceans are sensitive to elevated pCO2 and temperature conditions, especially during larval development

(Farby et al. 2008, Widdicombe and Spicer 2008, Kurihara et al. 2008, Walther et al. 2010, Melzner et al. 2009, Ceballos-Osuna et al. 2013, Carter et al. 2013, Long et al. 2013).

Elevated pCO2 and temperature had the greatest effect on larval survival.

Elevated temperature in particular reduced survival of stone crab larvae, suggesting that larvae are highly sensitive to the effects of elevated temperature. There was also a decrease in survival in the elevated pCO2 (H30) treatment, however, this influence was less detrimental than the temperature treatments. Similar adverse effects of elevated temperature and pCO2 have been reported for the red king crab,

Paralithodes camtschaticus, the Tanner crab, Chionoecetes bairdi (100% higher mortality than the control; Long et al. 2013), and the spider crab, Hyas araneus

(Walther et al. 2010). The low survival observed here agrees with previous stone crab work that reported higher larval mortalities when larvae experienced

79 temperature extremes around 35 °C (Brown and Bert 1992). Elevated temperature is one of the most critical environmental factors that directly influences metabolic rates, molt-stage duration, and development time (Costlow et al. 1960, Naylor

1965). Although the physiological mechanisms contributing to the decrease in survival are not known, elevated temperatures are certainly known to increase metabolism. For example, the northern shrimp Pandalus borealis, showed a metabolic increase (20%) when exposed to both higher temperatures and OA conditions (Arnberg et al. 2013). Additionally, molting in crustaceans is often accompanied by a large increase in oxygen consumption, resulting in a 2-fold increase in metabolism (Roberts 1957, Leffler 1972). Therefore, the low survival at high temperatures may be associated with a higher metabolic rate (Leffler 1972).

Furthermore, higher mortality at elevated temperatures could also be the result of larvae experiencing heat stress, which is known to disrupt enzymatic or hormonal systems that regulate the molt cycle (Anger 1987). Certain enzymes may not function at elevated temperatures, resulting in some pathways either not operating effectively, or working less efficiently (Costlow and Bookhout 1971).

Larval development across all larval stages was also temperature dependent, which was indicated by a 12.7 and 13.5% shorter molt-stage duration (MSD) in the

H32 and A32 levels respectively. A shorter MSD was expected, as the effects of higher temperature have been documented to accelerate molting in both larval and juvenile coastal and estuarine crustacean species, including Paralithodes camtschaticus (Long et al. 2013a), Carcinus maenas, Callinectes sapidus (Leffler

1972), Hyas araneus (Anger 1983), Cancer irroratus (Johns 1981), and Cancer magister (Kondzela and Shirley 1993). Coastal and estuarine crustaceans (i.e.,

Sesarma, Callinectes, Menippe spp.) exposed to elevated temperatures experience an increase in metabolic processes, resulting in larvae progressing through each stage more quickly (Costlow et al. 1960, Ong and Costlow 1970, Mohamedeen and

Hartnoll 1989). For example, increased seawater temperatures are known to accelerate growth (i.e., until a threshold is reached), however, the faster rate of growth is often associated with certain physiological costs, such as lower lipid stores or depletion of energy reserves that may be required in later stages (Byrne

2011, Long et al. 2013).

The present study also showed that exposure to elevated pCO2 also resulted in a

~12% (~0.7 days) longer molt-stage duration in stage-V larvae, therefore prolonging the transition into the post-larval stage. Slower developmental under

OA conditions has been previously reported for the larvae of the spider crab, H. araneus (Walther et al. 2010), and for the shrimp P. pacificus (Kurihara et al.

2008). However, both of these studies observed significant delays in larval development only when OA levels were well above pCO2 projections for the end of the next century (Walther et al. 2010; ~3000 ppm; Kurihara et al. 2008, > 2000 ppm). The lack of any reported delay (i.e., several days or weeks) in larval development, at elevated pCO2 levels (< 1100 ppm), suggests that OA conditions forecast for 2100 will not likely have significant biological impacts on crustacean larval development.

The ash free dry weight (AFDW) results reported here contradict the hypothesis that larval condition was impacted by OA or elevated temperatures. This contradictory result was unexpected, and the reason behind the lack of difference in

AFDW is unknown. Previous work that has attempted to quantify larval condition under elevated temperatures and OA scenarios for other Brachyurans report similar patterns in both larval condition and in survival as the results in the present study.

For instance, H. araneus larval survivorship decreased but was not impacted by

OA, and larval lipid ratios showed no change under elevated pCO2 (710 ppm), and elevated temperature (Walther et al. 2010). Additionally, the Tanner crab,

Chionoecetes bairdi, also exhibited no significant change in its larval condition index, however, larvae did elicit a 130% increase in larval mortality at elevated pCO2 (~800 ppm; Long et al. 2013b). Previous work has suggested that prior to molting, larval metabolism most likely switches from storing lipids to increasing the production of structural proteins needed for molting, which is indicated by a decrease in larval condition (Anger 1987, 2001). Typically, decreases in larval condition and survivorship are associated with OA and elevated temperature, affecting certain metabolic processes (i.e., interference with proper function of certain pH-dependent enzymes or hormones necessary to complete molting) as CO2 diffuses into the larval body to acidify the haemolymph (Anger 1987, Portner et al.

2004). Such changes were hypothesized to occur in post-larvae of H. araneus that were exposed to OA and elevated temperatures, however, the AFDW results reported here do not agree with this hypothesis.

There was no difference in the Ca content, Mg content, and Mg/Ca ratio of stone crab larval raised at elevated pCO2 and elevated temperatures. The results reported here are in contrast to other studies that have found significant differences in the skeletal content of larval crustaceans exposed to both OA and temperature conditions. For instance, larvae of the red king crab Paralithodes camtschaticus showed a 5% increase in Ca concentration (Long et. al. 2013a), whereas larvae of the spider crab H. araneus (Walther et al. 2009) and C. bairdi (Long et al. 2013b) both exhibited reduced calcification under OA and elevated temperatures. The lack of any differences among treatments in larval skeletal content is likely attributed to the molting process in larval crustaceans. During molting, crustacean larvae inflate their body with the surrounding seawater, which permits Ca+ ions to permeate across the thin exoskeleton of the larvae via diffusion (Anger 2001, Walther et al.

2011). Once larvae molt, and develop into post-larvae stages, a greater amount of

Ca+ begins to be embedded into the carapace progressively with each molt, with the highest Ca+ content being found in older post-larvae stages and in juveniles

(Walther et al. 2010, Arnold et al. 2009, Walther et al. 2011). Alternatively, the lack of difference in Ca could also be attributed to aragonite saturation states that were not corrosive within the experimental aquaria. Seawater that has Ω arag > 1.0 favors the formation of shells and skeletons (Feeley et al. 2008). Below a value of

1.0, the water becomes corrosive and dissolution of pure aragonite and unprotected aragonite shells occurs (Feeley et al. 1988; Gazeau et al. 2007). Although the mean pCO2 levels were higher, and the pH was lower, in the OA treatments for this

83 study, the aragonite saturation levels remained supersaturated (Ω ≥ 1.8). The lack of differences in Ca and Mg concentration and Mg/Ca ratios suggest that stone crab larvae may not have difficulty calcifying in future oceans, at least until the year

2100.

Stone crab larval morphology (Stage III or V) was also not affected by OA and elevated seawater temperatures. This result is similar to other studies on crustaceans, which have shown that the larval morphology of red king crab larvae

P. camtschaticus (Long et al. 2013a) were similar regardless of pCO2 exposure.

The results reported here suggest that stone crab larval morphology will not be impacted by future changes in seawater pCO2 or temperature. However, there is potential for OA and elevated temperatures to impact the size, shape, and shell thickness of post-larval and juvenile stage stone crabs, given that crustaceans typically incorporate greater amounts of calcium into the exoskeleton during later life stages (Richards 1958, Neufeld and Cameron 1992, Arnold et al. 2009,

Whiteley 2011).

Elevated seawater temperatures appear to have a greater impact on stone crabs than the effects of elevated pCO2, suggesting that some components of larval development may be tolerant to future OA. Stone crabs are a subtropical species and already live close to their thermal limit, especially during the summer reproductive season. The significant decline in survivorship observed at elevated seawater temperatures is especially concerning considering that predictions forecast seawater temperatures will increase at a faster rate than OA (IPCC 2013). The

84 susceptibility of larval stone crabs to elevated temperatures could therefore promote a range expansion above the population’s northern limit (North Carolina) as ocean temperatures continue to increase (Brown and Bert 1992). Furthermore, the projected increases (+2°C) in seawater temperatures predicted for the end of the century may serve as a potential bottleneck for the population by reducing the number of larvae that survive to become new recruits into the fishery. Therefore, elevated seawater temperatures are likely to cause a decline in the stone crab larval population in the absence of phenotypic or evolutionary adaptation (Long et al.

2013).

CHAPTER IV: BEHAVIOR

IMPACTS OF ELEVATED PCO2 AND TEMPERATURE ON THE GEOTAXIS

OF STONE CRAB LARVAE

INTRODUCTION

Behavioral responses of crustacean larvae to exogenous cues, such as salinity, temperature, pressure, light, and gravity can influence the vertical distribution of the larvae (reviewed in Sulkin 1984; Queiroga and Blanton 2005, Epifanio and

Cohen 2016). Sensitivity to exogenous cues, such as environmental stimuli, can result in directed movements toward or away from the surface, altering their orientation and level of locomotor activity (Sulkin 1984). Sulkin (1984) first proposed a generalized model for depth regulation, suggesting vertical positioning requires larva to compensate for negative buoyancy by active movement, maintaining an individual above the bottom. Downward movement can occur either by cessation of locomotor activity, or by a behavior that complements negative buoyancy, whereas upward movement can only be achieved by actively swimming

(Sulkin 1984).

Since most external stimuli vary with depth, the responses outlined in Sulkin’s

(1984) model—buoyancy, orientation, and level of locomotor activity—may interact to create a positive or negative feedback. For instance, gravity is a directional-environmental cue that is constant with depth (Sulkin 1984).

Brachyuran larvae usually elicit a negative geotactic response that results in

86 movement toward the surface, or a positive geotaxic response that promotes downward movement (Sulkin 1984). As negatively buoyant larvae passively sink, they experience increases in hydrostatic pressure. Increases in pressure during a descent usually induce an increase in locomotor activity (i.e., barokinesis), resulting in corrective movements by either a negative geotaxis, or positive phototaxis (Sulkin 1984; Forward 1989). As larvae move toward the surface they experience a decrease in pressure. Decreases in pressure can elicit a reduction in locomotor activity (i.e., low barokinesis), passive sinking, positive geotaxis, and/or a negative phototaxis, thus allowing larvae to adjust their vertical position (Sulkin

1984). Generally, stimuli that elicit a negative-feedback behavior will result in depth regulation at mid-water, whereas stimuli that induce positive-feedback behavior will position larvae either at the surface or near the bottom (Sulkin 1984).

The responses of larvae to cues such as gravity may be further modified by additional responses to other variables including light, salinity, temperature, and other hydrological variables, all of which can vary with season, time of day, and water quality (Sulkin 1984; Forward 1989, Epifanio and Cohen 2016). Such stimuli can further modify a larva’s ability to overcome or complement negative buoyancy by altering their level of locomotor activity and by changing their orientation (i.e., geotaxis response). For instance, elevated temperatures can influence the direction and speed of a larva’s vertical position. Higher temperatures have been shown to increase the swimming speeds of larval crustaceans until a threshold is reached, beyond which inactivity (i.e., sinking) occurs (Yule 1984; see Sulkin 1984 for

87 review). Additionally, several studies have indicated that the responses of larvae

(i.e., switching from active swimming to passive sinking) to high temperatures is more likely the result of larvae experiencing an absolute upper temperature, rather than a response to rates of temperature change (Ott and Forward 1976;

McConnaughey and Sulkin 1984). Forward (1990) summarized the behavioral responses of crustacean larval, and suggested that increases in temperature at or above an individual’s absolute upper limit evokes negative phototaxis, positive geotaxis, and inactivity, all of which can lead to sinking.

Low pH could also influence the swimming behavior of crustacean larvae by degrading or modifying sensory structures. Anthropogenic inputs are changing the baseline pCO 2 of many coastal habitats, which are accelerating the rate of ocean acidification (OA) in coastal areas (Bauer et al. 2013). As a result, recent studies have attempted to characterize whether future changes in seawater pCO 2 might alter the orientation and swimming behavior of some marine species (Munday et al. 2012, de la Haye 2012, Zittier et al. 2013). For example, juvenile reef fish reared in OA conditions were unable to avoid predators (Munday et al. 2012). Low pH significantly reduced the ability of hermit crabs to find a food source (~50% lower antennular flicking and ~40% increase in time to locate food sources; de la Haye

2012), and also decreased the swimming ability (30% reduction) in shrimp

(Dissanayake et al. 2011). Despite the aforementioned studies, no studies have attempted to determine whether the combined anthropogenic stressors of OA and elevated temperature have an impact on the capacity of crustacean larvae to maintain

88 their vertical position in the water column. Stone crab larvae raised in OA conditions do not appear to have any morphological abnormalities that may impede their buoyancy or vertical swimming (see Chapter 3). However, orientation to external stimuli is controlled by sensory organs, such as the statocyst, which forms during crustacean larval development, dorsal to the antennule basal joint (Prentiss 1901,

Epifanio and Cohen 2016). The statocyst is composed of a calcareous statolith embedded in a gelatinous matrix (Cohen and Dijkgraff 1961). Since the statolith is more dense then the surrounding medium it is displaced by inertia when the animal moves (Montgomery et al. 2006). Movement of the statolith triggers sensory hairs that line the chamber, which in turn help the animal maintain equilibrium by stimulating righting movements (Budelmann 1992). Therefore, any degradation of the statocyst organ, by OA conditions, may impair the larvae’s ability to orient vertically.

The Florida stone crab, M. mercenaria, inhabits sublittoral coastal habitats from

North Carolina to Texas, with isolated populations occurring throughout the

Caribbean. Stone crabs support an important fishery throughout the southeastern

United States; in Florida alone the stone crab fishery contributes ~$25 million a year to local economies (Florida Fish and Wildlife Stock Assessments 2000–2014).

Despite the socioeconomic importance of stone-crab populations in Florida, little is known about the behavior of early-stage larvae, their distribution in the water column, and their settlement behaviors. This study tested the hypothesis that elevated pCO2 and elevated temperature alters the geotaxic response of larvae.

Determining the effects of OA and elevated temperature on the position of stone crab larvae will provide insight into the mechanics of larval dispersal, which is particularly relevant as anthropogenic inputs threaten to alter the carbonate chemistry of future oceans (Sulkin et al. 1980; Epifanio and Garvine 2001; Park et al. 2004).

MATERIALS AND METHODS

STONE CRAB COLLECTION

Ovigerous females were collected by Florida Fish and Wildlife using commercial stone crab traps, 17 km from the coast in Florida Bay near Pavilion

Key, Florida. Ovigerous females were immediately transported back to the

University of Miami’s Rosenstiel School’s Ocean Acidification laboratory and were maintained in ambient seawater conditions until larval release. Upon larval release, newly hatched larvae were randomly assigned into each of the experimental treatments described below. Larvae from the same brood were divided among the treatments levels throughout all experiments.

EXPERIMENTAL DESGN AND SET-UP

Geotactic swimming experiments consisted of two treatments: 1) temperature, and 2) pCO2, each with two levels. The two temperature levels were set at 30°C and 32°C. The lower (control) temperature was based on the mean summer sea surface temperature for the Florida Keys (NOAA,

90 http://www.ndbc.noaa.gov/view_climplot.php?station=lonf1&meas=st). The upper temperature limit was based on IPCC (2013) sea-surface temperature projections for the end of the century. The two pCO2 levels were set at ~450 and ~1100 ppm, and were based on current IPCC (2013) projections. To achieve the experimental pCO2 levels, seawater was pumped into two holding reservoirs. To achieve the control p CO2 level, the water was vigorously aerated until the reservoir was maintained at ~450 ppm. The elevated pCO2 levels were achieved with the addition of ultra-pure CO2 gas, which was regulated using mass flow controllers (MFC,

SmartTrak 100, Sierra). The CO2 gas was slowly introduced through the MFCs into the holding reservoir using a venturi pump injection system.

All larvae were raised within the experimental aquaria, which were maintained within a temperature controlled water bath. Once the pCO2 levels were established in each reservoir, the control and treatment water were pumped into each of the experimental aquaria. Probes within each aquarium were set-up to monitor temperatures using a digitally controlled temperature feedback system (Apex,

Neptune). Individual heaters were placed within each aquaria. Temperature probes triggered the heater to turn on or off relative to the treatment level set-up points, described below. To avoid shock to the larvae, the use of MFC’s and digitally controlled temperature system gradually increasing the experimental parameters

(~200 ppm & ~0.4C° per day) to the desired treatment levels over the first 5 days of each experiment.

SEAWATER CARBONATE CHEMISTRY

To monitor the carbonate chemistry of the OA system, seawater samples were collected from both the holding reservoirs and from each of the experimental aquaria, placed in 150 mL borosilicate bottles, and were then fixed with 100 μL of mercuric chloride. Total alkalinity (TA) and dissolved inorganic carbon (DIC) were measured at NOAA’s Atlantic Oceanographic and Metrological Ocean

Acidification Laboratory using automated Gran titration (Apollo Instruments) and calibrated with Dickson standards (Scripps Institution of Oceanography, La Jolla,

CA). Carbonate parameters were monitored every other day during the first week of the experiment, and every 5–7 days thereafter. The holding reservoirs were monitored every 4–5 days after the ramp-up period. The pHtotal was also measured within each of the experimental aquarium using a handheld pH meter (Oakton) and

Ross electrode (Orion 9102BWNP; Thermoscientific), which was calibrated using

Tris buffer (Dickson Standard, Scripps Institution of Oceanography, La Jolla, CA).

The pHtotal was used because of the uncertainty of a potentiometric measurements of seawater pH can be less than 0.02 when compared with the national bureau of standards scale (pHNBS), which can have an uncertainty ranging from 0.5–0.1 units

(Riebesell et al. 2010).

To calculate pCO2, both TA and pHtotal were measured during geotaxis experiments. Using TA, and pHtotal, the remaining carbonate parameters (DIC, and p CO2) were determined using CO2SYS software (Robbins et al. 2010). During the

92 experiments, temperature, pHtotal, and salinity of the seawater in each of the experimental aquarium were monitored every 10-12 hour using a pH probe (Orion

Ecostar). Seawater samples that were collected from the same field sites as where the ovigerous females were collected, to ensure that the experimental conditions were within the range of naturally occurring carbonate chemistry. The carbonate chemistry of seawater samples collected at the site of ovigerous female collection were analyzed for DIC and TA similar to methods reported in Chapter 2.

STONE CRAB COLLECTION AND LARVAL REARING

After hatching, larvae from the same brood were transferred into each of the experimental treatment combinations (i.e., temperature and pCO. 2) Larvae were reared in 7 L containers, with a stock density ~250 individuals L-1, with 80% of the containers sides being composed of nylon mesh (24 cm diameter x 24 cm depth;

190 µm). Venturi pumps positioned toward the nylon mesh facilitated exchange of water across the mesh. Each container was kept in its own digitally controlled water bath, described above, in order to maintain experimental temperatures within a narrow range. Larvae were fed enriched Artemia daily (~1 ml-1). Artemia were enriched with Selco (lipid diet) and rotifers that were enriched with a micoralgae

(Rotigrow Onestep). Prior to harvesting from each chamber, a subsample of 50 larvae were assessed to determine the developmental stages (in accordance with

Porter, 1960). Larvae were kept on a 14 h-light:10h-dark photoperiod that

93 approximated conditions during the time of collection. To minimize the buildup of nutrients, dead larvae and wastes were removed from each chamber twice daily.

SINKING RATES FOR STAGE III AND V LARVAE

Passive sinking rates were used to determine whether the downward movement, during geotaxis trials, was an active (i.e., swimming) or a passive response (i.e., passive sinking). Stage III and V larvae (n = 20 per treatment combination) were harvested and anesthetized using a 10% MgCl2 solution. After larvae were anesthetized, individual larvae were pipetted into a vertical acrylic chamber (21.6 x

8.3 x 8.3 cm) and allowed to sink for 10 cm. Sinking rates were calculated by determining the rate at which the anesthetized larvae traversed the 10 cm section of the chamber during each trial (Arana and Sulkin 1993). To determine whether there were differences in sinking rates, broods were compared among treatments using an

Analysis of Variance with Repeated Measures (ANOVAR), with temperature and pCO2 as the main effects and brood as the within subject factor. The stage V passive sinking rates did not meet the assumptions of normality and therefore, the data were ranked transformed. The ranked data was then used to determine if sinking rates were different among treatments using an ANOVAR with temperature, and pCO2 as the main effects, and brood as the within subject factor.

GEOTACTIC SWIMMING RESPONSES FOR STAGE III AND V LARVAE

The geotactic responses of larvae were determined by comparing the upward/downward movement of stage III and V larvae to the passive sinking rates of anesthetized individuals. Larvae from multiple broods (stage III = 7 broods;

94 stage V = 5 broods) were monitored for upward or downward movement among treatments. The vertical movements of larvae were determined using a closed circuit video system (Panasonic BP334 B/W video camera attached to a Panasonic

Model AG 1980 video recorder) illuminated with infrared light (775 nm).

Individual larvae were placed in the center of a clear acrylic tube (16 x 3 x 3 cm), which was oriented horizontally and kept in dark conditions. After an acclimation period of 2 minutes, the tube was rotated 90°, vertically, and the directional movements (up or down) of larvae were recorded. Upward or downward movements were recorded for the first 10 seconds after rotation. Larvae that did not move up or down (i.e., maintained position) were recorded as exhibiting a neutral response. Downward swimming speeds were determined by measuring the time that larvae took to traverse the length of the chamber. If downward swimming speeds exceeded the rate of passive sinking rates then the downward response was attributed to positive geotaxis. Larvae that had a net upward response were considered to have negative geotaxis. To determine if sinking rates were significantly faster than the downward swimming speeds of larvae, independent t- tests were used to compare passive versus active responses within each treatment combination.

The number of larvae that swam up, down, or remained neutral was determined and compared among treatments. Stage III larvae were expected to swim upwards and stage V larvae were expected to swim downwards, based on the control responses. Upward and neutral responses for stage III larvae were compared, to

95 determine whether there were differences among treatments. Data were tested for normality assumptions and if the residuals were not normal then the data were rank transformed and the analysis was repeated. Since the stage III geotactic response did not meet the normality assumptions an ANOVAR was performed on the ranked data with temperature and pCO2 as the main effects, and brood as the within subject factor. Stage V larvae did meet the normality assumptions. Therefore, stage-V larvae that swam downward or maintained their positions upon stimulation were compared using an ANOVAR with temperature and pCO2 as the main effects, and brood as the within subject factor. R (R Core Team, 2016) was used for all statistical analyses.

RESULTS

SEAWATER CARBONATE CHEMISTRY

After pCO2 and temperature were gradually increased to the experimental set points the pCO2 within the control chambers was maintained at 467.4 ppm ±25.9

(mean, ±SD), whereas the A32 treatment was held at 568.7 ppm ±12.8 (mean,

±SD) (Figure II-1). The mean pCO2 level in the H30 treatment was 947.6 ppm ±

47.4 (mean, ±SD), whereas H32 treatment was held at 1085.0 ppm ± 50.4 (mean,

±SD). Temperature, salinity, and total alkalinity (TA) were controlled within a narrow range. The pHtotal was lower in the elevated pCO2 treatments (Table II-1).

The ambient pCO2 levels were within the range of the pCO2 at field collection sites

96 and within ranges reported for stone crab habitats (Millero et al. 2011, Dufroe

2012 ; See chapter 2).

PASSIVE SINKING RATES AND DOWNWARD SWIMMING SPEEDS

The stage III passive sinking rates were not significantly different among treatments (temperature: F1, 4 = 0.59, P = 0.44; CO2: F1, 4 = 0.09, P = 2.8), nor were there any interaction effects (F1, 4 = 0.60, P = 0.44; Table IV-1). However, there was a significant brood effect in the sinking rates of stage III larvae (F4, 392 = 7.4, P

< 0.001). The passive sinking rates did not meet the normality assumptions and were compared within treatments using the Mann-Whitney test. The passive sinking rates were significantly greater than the downward swimming speeds within all treatment combinations (A30: U = 173, df = 35,100, P < 0.001; H30: U =

364, df = 99,100, P < 0.001; A32: U = 251, df = 44,100, P < 0.001; H32: U = 613, df = 100,110, P < 0.001; Figure IV-1).

The passive sinking rates of stage V larvae were not significantly different among treatments (temperature: F1, 4 = 2.2, P = 0.14; CO2: F1, 4 = 0.07, P = 0.79;

Table IV.1), nor was there any interaction effect (F1, 4 = 0.05, P = 0.82). However, there was a significant brood effect for stage V larval passive sinking rates (F4, 392 =

37.0, P < 0.001). The stage V passive sinking rates were significantly faster than the downward swimming speeds (A30: U = 50, df = 33, 100, P < 0.001; H30: U =

121, df = 38,100, P < 0.001; A32: U = 63, df =36,100, P < 0.001; H32: U = 65, df =

41,100, P < 0.001; Figure IV.2).

Table IV.1. Results of the repeated measures ANOVAs for stage III and V passive sinking speeds. The ANOVA’s were performed using ranked data. Significant values are in bold.

Source of Variation df SS MS F P Stage III Temperature 1 7405 7405 0.59 0.44 CO2 1 34838 34838 2.8 0.09 Temperature x CO2 1 7517 7517 0.60 0.44 Brood 4 372177 93044 7.4 <0.001 Residuals 392 4911199 12529

Stage V Temperature 1 21934 21934 2.2 0.14 CO2 1 640 640 0.07 0.79 Temperature x CO2 1 493 493 0.05 0.82

Brood 4 1455418 363854 37.0 <0.001 Residuals 392 3854633 9833

Figure IV.1. Box and whiskers plot (cm sec-1) showing the passive sinking (white bars) and downward swimming speeds (grey bars) of stage III larvae for exposure to different combinations of pCO 2 and temperature. The asterisks represent passive sinking responses that were significantly greater than the downward swimming speeds.

Figure IV.2. Box and whiskers plot (cm sec-1) showing the passive sinking (white bars) and downward swimming speeds (grey bars) of stage V larvae for exposure to different combinations of pCO2 and temperature. Asterisks represent passive sinking responses that were significantly greater than the downward swimming speeds.

100

GEOTACTIC SWIMMING BEHAVIOR

Stage III larvae were expected to swim upwards (based on the A30 treatment), therefore, any larvae that swam upwards, or maintained position during geotaxis experiments were compared for differences among treatments using an ANOVAR.

Temperature did not have a significant effect on the stage III geotactic swimming response (F1, 6 = 0.26 P = 0.62; Figure IV-3; Table IV-2). Larvae in the ambient pCO2 treatments had a greater percentage of individuals that maintained their position (i.e., no change of direction), or swam upward, than larvae exposed to elevated p CO2. Elevated pCO2 resulted in a significant reduction in the number of larvae swimming upward (F1, 6 = 43.3, P < 0.001). There was no interaction effect among temperature and pCO2 (F1, 6 = 0.05, P = 0.83), nor were there any brood effects (F6, 18 = 0.69, P = 0.65).

Based on the control (A30) response, stage V larvae were expected to orient downwards (A30 = 68% swam down, or elicited a neutral response). Therefore, the proportion of larvae that swam down, or maintained position, during geotaxis experiments were compared among treatments using a general linear model

(Shapiro test: W = 0.93, P = 0.13), with temperature and pCO2 as the main effects and brood as the blocking factor. There were no significant differences in the

101

Table IV.2. Results of the ANOVAR for stage III and V geotactic responses. The ANOVAR was performed using ranked data for stage III larvae. Significant values are in bold.

Source of Variation df SS MS F P Stage III Temperature 1 7.0 7.0 0.26 0.62 CO2 1 1183 1183 43.3 <0.001 Temperature x CO2 1 1.29 1.29 0.05 0.83 Brood 6 113.5 18.9 0.69 0.65 Residuals 18 491.7 27.3

Stage V Temperature 1 125 125 0.50 0.49 CO2 1 405 405 1.6 0.22 Temperature x CO2 1 5 5 0.02 0.89

Brood 4 2770 692.5 2.8 0.07 Residuals 12 2990 249.2

102

Figure IV.3. Box plot of stage III larvae that swam up vs. down among experimental treatments. Different letters above the boxes indicate significant differences. Because it was expected that stage III larvae would swim upward, larvae that maintained position upon stimulation (i.e., neutral swimming) were included in the upward response for stage III larvae.

103 percentage of stage V larvae that swam down or maintained position among the main treatment effects (Temperature: F1, 4 = 0.50, P = 0.49; CO2: F1, 4 = 1.6, P =

0.23; Figure IV-4; Table IV-2). There was no significant interaction effect among treatments (F1, 4 = 0.02, P = 0.89), nor was there a significant brood effect (F4, 12 =

2.8, P = 0.07).

DISCUSSION

The vertical position of negatively buoyant larvae is the net result of a complex combination of responses, including an individual’s sinking rate, directional orientation, and swimming speed (Sulkin et al. 1980). Sulkin’s (1984) feedback model for depth regulation posits that early larval stages should elicit behaviors that position them near the surface, whereas ontogenetic changes in responses to abiotic factors, in later larval stages, usually results in less precise depth regulation. For instance, gravity, hydrostatic pressure, and light are often the primary cues to which larvae respond. However, these responses can interact with a suite of secondary cues (e.g., salinity and temperature), resulting in further adjustments in behavior

(Naylor 2006). Under ambient seawater conditions, stage III stone crab larvae exhibit vertical swimming behaviors that position them at or near the surface

(Gravinese 2007). These responses change through ontogeny, with stage-V larvae eliciting responses that are more consistent with a deeper distribution (Gravinese

2007).

104

Figure IV.4. Mean percent (± SE) larvae that swam up vs. down Z5 larvae among experimental treatments. Because it was expected that stage V larvae would swim downwards, larvae that maintained position upon stimulation (i.e., neutral swimming) were included in the downward response for stage V larvae. There were no significant differences among treatment in the stage V response.

105

This shift in vertical position through ontogeny is ultimately responsible for modulating their dispersal and distribution. The results reported here are the first to evaluate how brachyuran crab larvae swim may respond to climate change, particularly OA and elevated temperature, and suggests that OA conditions that are forecast for future oceans may adjust the swimming response of mid-stage stone crab larvae, which could limit larval dispersal.

Stage III stone crab larvae showed a negative geotactic swimming response when raised in the elevated temperature (A32), and in control treatments (A30).

The observed negative geotaxis (i.e., upward swimming) response of mid-stage stone crab larvae is consistent with the responses reported for other early and mid- stage brachyuran crabs, including the blue crab, C. sapidus (Sulkin et al. 1980), the mud crab, Rhithropanopeus harrisii (Ott and Forward 1976, Forward et al. 1984), the shore crab, Hemigrapsus sanguineus (Park et al. 2004), the rubble crab

Cataleptodius floridanus (Sulkin 1975), and the Atlantic rock crab, Cancer irroratus (Bigford 1977). A negative geotactic response predicts that early-stage larvae should be positioned closer to the surface, where they would be subject to offshore advection by surface currents (Epifanio 1988, Gravinese 2007). However, larvae raised in the elevated pCO2 (H30), and in the combined treatment (H32), showed the opposite response, with a significantly greater number of larvae eliciting a controlled descent relative to the control. The controlled descent elicited by stage III larvae was not related to the presence of any morphological

106 abnormalities, changes in calcification, or differences in weights (see chapter 3 results), suggesting that elevated pCO2 is likely impairing some other mechanism used for larval orientation. One possible explanation for the drastic change in swimming direction, observed in stage III larvae, could be the result of statocyst degradation by low pH conditions (i.e., H30 and H32 treatments). No other work has tested, or reported changes in, crustacean larval swimming abilities when exposed to OA and elevated temperature conditions. The only other OA work to cite behavioral impairment, or changes in an individual’s directional orientation, was conducted on damsel and cardinal fishes. A change in swimming orientation by the fishes was driven by a breakdown of the olfaction abilities and in their ability to effectively regulate their acid-base balance (Munday et al. 2014). The drastic change observed in the swimming direction of stone crab larvae under OA conditions could result in less precise depth regulation among mid-stage larvae.

The change in depth regulation could potentially result in a deeper distribution, which could have negative consequences for larval dispersal and advection to inhospitable habitats.

Compared with stage III larvae, there was a distinct ontogenetic shift in the stage V swimming response to geotaxis stimuli. Stage V larvae did not conform to behaviors that promote a negative feedback model for depth regulation, as proposed by Sulkin (1984). Instead, the swimming response of Stage V larvae in all treatments appeared to be controlled by behaviors that would promote a deeper distribution in the water column. Previous work has shown that Stage V larvae

107 have a downward response to gravity (Gravinese 2007), and the results reported here align with those observations. The larger size of Stage V larvae resulted in sinking rates that were ~43% faster than Stage III larvae. As a result of being heavier, Stage V larvae have to overcome negative buoyancy to be able to swim upward in the water column. The rapid sinking rates of Stage V larvae make it likely that individuals were experiencing changes in hydrostatic pressure (i.e., pressure increases) during their descent. The strong descent by Stage V larvae during geotaxis experiments was characterized by periods of passive sinking that were punctuated by occasional intermittent swimming in all treatments. Such a response could be the result of larvae adjusting to changes in hydrostatic pressure during their descent. Late stage, stone crab larvae regulate their depth via kinetic responses (barokinesis) that vary with different rates of pressure change (Gravinese

2007). By controlling their descent, the magnitude of pressure change experienced by the individual is less abrupt, which subjects individuals to gradual changes in pressure, and therefore allowing larvae to acclimate and adjust their locomotor activity. Thus, the descent observed could be the result of a negative geotaxis, and a positive barokinetic response. A true positive geotaxis would require active downward swimming speeds greater than the passive sinking rates, which was not the case in the present study.

There were no significant impacts of elevated temperature on the geotactic behavior of stage III or of V stone crab larvae. Laboratory experiments have demonstrated that seawater temperature can play a role in mediating brachyuran

108 crustacean swimming behavior by serving as a cue for vertical positioning. For instance, some experiments have shown that larvae tend to migrate upward, toward warmer waters when experiencing cold water (Sulkin et al. 1983, McConnaughey and Sulkin 1984). Alternatively, other work provides evidence that larvae will limit their upward movement to avoid water temperatures associated with very warm surface layers (McConnaughey and Sulkin 1984). In the present study, stage III stone crab larvae consistently swam down when exposed to elevated pCO2 regardless of temperature, suggesting that OA and not temperature were impairing vertical movement. Stage V stone crab larvae swam down in all treatments, including conditions that exposed individuals to elevated temperatures. Therefore, larval vertical swimming was not impacted by temperature in this study.

Larval release in stone crabs occurs on a diel cycle (Krimsky et al. 2009), within nearshore habitats, where circulation patterns facilitate larval transport away from adult populations. The swimming ability of brachyuran crab larvae is not strong enough for an individual to overcome horizontal currents (Epifanio and

Garvine 2001). However, the capability of crab larvae to adjust their swimming speeds, and orient to specific external cues, plays a critical role in their depth maintenance (Epifanio and Cohen 2016). Orienting to cues to adjust their vertical position also influences the capacity of larvae to disperse in estuarine and coastal environments (Epifanio and Cohen 2016). For instance, Epifanio and Cohen (2016) estimated that a crab larva sinking at 0.2 cm s-1 would traverse a vertical distance of

~45 m during a 6 hr ebb-tide. Therefore, the interaction between larval vertical

109 swimming behavior, and the frequency at which currents vary at any given depth will control the net horizontal displacement of larvae (Epifanio and Garvine 2001).

If OA and elevated temperature result in deeper mid-stage stone crab larvae, then they may have reduced dispersal because they would have limited exposure to surface currents.

Reducing larval dispersal could result in individuals being transported into habitats that are not ideal for completing their development (Sulkin 1984, Epifanio

1988). Furthermore, if less precise depth regulation results in larvae being entrained close to shore, then individuals may be subjected to greater predation, which would significantly reduce larval supply (Morgan and Christy 1996). However, response to gravity is just one cue that contributes to a larva’s vertical position. The vertical position of brachyuran larval crabs is also controlled by other exogenous stimuli, including hydrostatic pressure and light. Therefore, future work should evaluate how larvae respond to these other stimuli, and when exposed to multiple anthropogenic stressors, including OA, hypoxia, and elevated temperatures.

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CHAPTER V: MATRIX MODEL

A MATRIX MODEL SIMULATION OF STONE CRAB LARVAL SURVIVORSHIP DURING EXPOSURE TO OCEAN ACIDIFICATION AND ELEVATED TEMPERATURE

INTRODUCTION

Since the preindustrial era, ocean acidification (OA) has resulted in a 0.1 unit decrease in oceanic pH, and forecast models predict an additional decline of 0.14-

0.35 pH units by the end of the century (Calderia and Wickett 2003, Meehl et al.

2007). In coastal areas, anthropogenic activities, including eutrophication, land-use modification, deforestation, sewage inputs, and wetland degradation are accelerating changes (i.e., increasing) in baseline pCO2 (Millero et al. 2011, Bauer et al. 2013, Zhang and Fischer 2014, Ekstrom et al. 2015), which influence the frequency, magnitude, and duration of extreme pH events (Harris et al. 2013, Hauri et al. 2013). Consequently, marine ecosystems along the east and Gulf coasts of the

United States are expected to experience the effects of OA earlier than projected by global-scale models (Ekstrom et al. 2015). Additionally, OA is occurring simultaneously with a rise in seawater temperatures, which is increasing at accelerated rates when compared with historical climate trends (IPCC 2013).

Future extremes in seawater pH and temperature may, therefore, exceed the critical ecological and physiological thresholds of organisms that have adapted to less extreme nearshore environments (Attrill et al. 1999, Ringwood and Keppler 2002,

Hauton et al. 2009).

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Coastal marine species live in dynamic habitats that experience fluctuations in environmental variability. As a result, many species possess the compensatory mechanisms needed tolerate changing abiotic factors, which include shifts in pCO2, and temperature forecasted for future oceans (Whiteley 2011). However, many coastal benthic invertebrates have distinct pelagic larvae and benthic developmental stages, and larvae often have different sensitivities to unfavorable environmental factors than the adult stages (Waldbusser and Salisbury 2014). Therefore, it is necessary to conduct experimental studies to help determine the impacts of OA and temperature on different life stages, which can be then be used to project population responses using a modeling approach.

Information from reproductive output, growth, and survivorship from laboratory experiments can be integrated into predictive models to forecast how a species may most likely respond to a warmer more acidified ocean (Harvey and

Menden-Deuer 2011). However, most OA models to date have focused on projecting changes in future carbon cycles (Zondervan et al. 2002, Gehlen et al.

2007, Ribesell et al. 2007, Hofmann and Schellnhuber 2009), or have attempted to predict shifts in species distributions with changing climate (Peterson et al. 2002,

Thomas et al. 2004, Poloczanska et al. 2008). Matrix population models, which are useful tools to estimate population densities, express the life cycle of a population as a sequence of interconnected, non-overlapping groups, which might represent size or age-classes (Caswell 1989, Caswell 2001, Eckman 1996). Such models have been useful in serving as a first step toward understanding the population dynamics

112 of a variety of marine organisms, including sea turtles (Crouse et al. 1987), cetaceans (Brault and Caswell 1993), sharks (Cortes 2002), fishes (Quinlan and

Crowder 1999), and crustaceans (Miller 2001). Currently, no published work has attempted to incorporate matrix models into estimating a species population density under different climate change scenarios. This is surprising since such models have frequently been used as important management tools to assist in projecting population dynamics, and to determine the projected population growth under different anthropogenic stresses, including fishing pressure and pollution (Crouse et al. 1987, Miller 2001, Mollet and Cailliet 2002). Highlighting potential bottlenecks through ontogeny will help forecast the likely impacts that climate change may have on future populations, especially for highly valued socioeconomic species

(Helmuth et al. 2006).

The Florida stone crab, Menippe mercenaria, occurs in subtidal coastal habitats from North Carolina to the Gulf of Mexico, and the Caribbean and is an important fishery in the southeastern United States. In Florida alone, stone crab landings support a US$25-million-a-year commercial and an active recreational fishery

(FWC 2000–2014). Florida Bay, which supports a significant stone crab fishery, has experienced a rate of decline in pH since 2005 that is estimated to be three- times faster than the rate observed in the open ocean (Zhang and Fischer 2014). As a result, larval stages may be physiologically constrained in their ability to adapt to climate stressors. Reductions in survivorship or growth of crustacean larvae could lead to potential population bottlenecks resulting in economic ramifications for

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Florida’s stone-crab fishery (Langenbuch and Portner 2004). No studies have attempted to forecast stone crab population densities under changing environmental conditions. Previous models of stone crab populations have been limited to estimates of adult populations within the Everglades (Ehrhardt 1990, Ehrhardt et al.

1990). Sensitivity analysis of matrix models highlight which life stages are most influential in population growth. This will be the first stone crab population model to incorporate stage-specific survivorship rates throughout larval development

(from Chapter 3) in an attempt to: 1) forecast the effects of future OA and elevated temperature scenarios on the stone crab larval population density, and 2) determine the contribution of various changes in survivorship and hatching success to the overall population density of larvae.

MATERIALS AND METHODS

PARAMETER ESTIMATION AND MODEL DESIGN

Stone crab hatching success, and larval stage-specific survivorship from the different experimental treatment combinations (OA and temperature) were used to parameterize a population model to project larval densities. The model assumes stage-specific larval survivorship based on the Chapter 3 results. During larval survivorship experiments, the control (A30) was maintained at ambient temperature

(30°C) and ambient pCO2 (~450 ppm). The H30 treatment had an elevated pCO2

(~1100 ppm) and ambient temperature (30°C). The ambient pCO 2 (~450 ppm) and elevated temperature treatment (32°C) combination was labeled as A32, whereas

114 the H32 treatment experienced both an elevated pCO2 (~1100 ppm) and elevated temperature (32°C).

The model was performed for all treatment combinations (A30, H30, A32, and

H32) using the hatching success observed under ambient pCO2 conditions in

Chapter 2. The effect of elevated pCO2 on the hatching success of stone crabs

(Chapter 2) was also incorporated into the model for the H30 and H32 treatment combinations, and was labeled as H30R and H32R. The model projects the population density using the equation:

푡 Nt = (A )×n0 (eq.1)

Where Nt is a vector of the number of individuals in each larval stage at time t, A is a transition matrix for larval stone crabs, and no is the initial population size. Since there have been no published studies on stone-crab larval survival in the field, natural mortality rates throughout the entire larval duration were based on estimates from the literature (Table V-1).

The density of the larval population was initially estimated using a the mortality described by White et al. (2014), which served as a “control” for comparisons among the other treatment scenarios. White et al. (2014) estimated mortality for species with comparable larval stages to stone crabs. The model assumes that natural larval mortality is greater during earlier larval stages, and exponentially decreases with developmental stage.

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Table V.1. Summary of the estimated natural larval mortality reported in the literature for a variety of plankton invertebrates and crustaceans, including rates for blue crab, Callinectes sapidus.

Species Estimated mortality Reference Planktonic invertebrates 90% Rumrill 1990 Planktonic crustaceans 98.5% White et al. 2014 Callinectes sapidus 88.3% Miller 2001 Callinectes sapidus 97.9-99.8% McConaugha 1992

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The stage-specific survivorship natural mortality rates were estimated using the exponential decay function:

푦 = 푎(1 − 푟)푥 (eq.2), where y is mortality, x is the larval stage, and a and r = are constants that were solved by assuming stage-I stone crab larvae experience a 75% mortality rate under natural conditions. The assumed stage-specific natural mortality rates are defined in

Table V-2.

The model assumes an initial starting population of 100 ovigerous females, which is comparable with the mean monthly number of ovigerous females caught according to Florida Fish and Wildlife’s stone crab fishery-independent monitoring program’s published annual reports (Gandy et al. 2006-2010). The stone crab reproductive output (1.0 x 10 6) was an assumed constant representing the mean output of embryos for stone crabs (Lindberg and Marshall 1984). The mean hatching success (H) under OA conditions was also a constant from Chapter 2, for females exposed to OA and ambient pCO2 scenarios (Figure V-1). Because stone crabs are a subtropical species and experience warm seawater temperatures throughout the brooding season, the model assumes that hatching success was similar among temperature conditions. The stage-specific survivorship values (M1 through M5) recorded in Chapter 3 were superimposed onto the mortality estimate to determine the number of larvae that survive to the next larval stage (until megalopae) for each treatment combination (A30, H30, A32, H32, H30R, and

H32R; Figure V-1). Since stone crab larvae do not stay within a particular larval

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Table V.2. Assumed stage-specific natural mortality rates for stone crab larvae. The mortality rates for stage II–V were estimated using the exponential decay function, assuming that the mortality of early larval stages is greater than later stages.

Larval Stage Estimated mortality I 75% II 18% III 4% IV 1% V 0.5%

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Figure V.1. A) Model schematic, where ‘H’ represents hatching success for females exposed to OA conditioned seawater (Chapter 2). M1-M5 represent stage-specific larval survivorship. Stone crab larval stages are represented by ‘Z1-Z5’, B) Representation of the stage-class population matrix-model where n represents the number of individuals per stage (i.e., n1= stage 1). The model runs for 5 weeks and assumes a larval supply at each stage of 1x106 individuals.

119 stage indefinitely, the model assumes that larvae either transition into the next stage or die. The model also assumes a closed population, with no immigration or emigration. Changes in population growth were determined by matrix multiplication (Figure V-1B, Eckman 1996). The eigenvalues (λ) were determined using the equation:

λ = 푁푡+1/푁푡 (eq.3).

The intrinsic rate of population increase was then determined using:

log λ (eq.4).

Finally, the sensitivities, which are the partial derivatives (휕) of λ, were determined using the equation:

휕휆 푆𝑖𝑗 = (eq. 5). 휕푎𝑖𝑗

Because the model was only run for five weeks, or one reproductive event, larvae within the model do not reach maturity to reproduce. Therefore, there are no fecundity inputs aside from the initial starting population of ovigerous females. For each simulation, the population size, intrinsic rate of growth, and the proportion of megalopae that survived were quantified. The dominant eigenvalues (λ) were also examined for each simulation.

RESULTS

LARVAL POPULATION SIZE AND INTRINSIC RATE OF GROWTH

The stone crab larval population (Figures V-2), and the intrinsic rate of population growth (Figure V-3) decreased in all of the experimental simulations

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Figure V.2. Larval stone-crab population densities during a 5 week matrix model simulation. The model assumes a larval supply at each stage of 1x106 individuals and an initial starting population of 100 ovigerous females. The black line represents mortality estimates assumed from the literature. A30 represents the control with ambient pCO2 and ambient temperatures. H30 is the elevated pCO2 and ambient temperature treatment. A32 is the ambient pCO 2 and elevated temperature treatment, and H32 is the elevated pCO2 and elevated temperature treatment. The solid lines represent hatching success under ambient pCO2 conditions, whereas the dotted lines represent a reduction in hatching success experienced during exposure to elevated pCO2.

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Figure V.3. The intrinsic rate of population decrease. Natural mortality was estimated from the literature. A30 represents the control with ambient pCO2 and ambient temperatures. H30 is the elevated pCO2 and ambient temperature treatment. A32 is the ambient pCO 2 and elevated temperature treatment, and H32 is the elevated pCO2 and elevated temperature treatment. H30R is the elevated pCO2 and ambient temperature treatment with a reduction in hatching success. H32R is the elevated pCO2 and elevated temperature treatment with a reduction in hatching success.

122 compared with the controls. There was an 88.6% and 91.5% decline in the natural mortality and A30 simulations respectively. When elevated pCO2 was added into the model the H30 simulation exhibited an additional 5.7% decrease in the larval population relative to the control (A30). The elevated temperature, combined treatment, and reduced hatching success treatments all showed population declines relative to the control that ranged from 7.0–8.2%.

The eigenvalues also showed decreases in all experimental simulations relative to the natural mortality estimate (Table V-3). Reducing hatching success from exposure to OA resulted in a further reduction in larval supply relative to the same treatment simulations that did not experience a reduction in hatching success

(Figure V-2).

SURVIVORSHIP TO POST-LARVAE

The simulation of natural mortality showed the highest post-larval survival, with ~16% of the individuals surviving to megalopae (Figure V-4). Superimposing the stage-specific survival from Chapter 3 onto the natural mortality estimate resulted in a 5.2% decrease (i.e., 14.8% survivorship to megalopae) in the proportion of surviving megalopae in the A30 simulation (Figure V-4). The H30 simulation showed an 11% survival to the megalopal stage. The greatest decrease

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Table V.3. Eigenvalues (λ) for each model simulation across all treatments. The model assumed that natural mortality remained constant and would not experience increases in survivorship or hatching success.

Treatment Eigenvalues Natural Mortality 0.54 A30 0.47 H30 0.40 A32 0.33 H32 0.30 H30R 0.34 H32R 0.27

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Figure V.4. The percentage of larvae (±S.E.) surviving to megalopae during a five week matrix model simulation. Natural mortality was estimated from the literature and represents the model run with stage-specific larval mortality, which was estimated using an exponential decay function. The natural mortality assumed that earlier larval stages experience greater mortality. The model also assumes a larval supply at each stage of 1x106 individuals, and an initial starting population of 100 ovigerous females. A30 represents the control with ambient pCO2 and ambient temperatures. H30 is the elevated pCO2 and ambient temperature treatment. A32 is the ambient pCO 2 and elevated temperature treatment, and H32 is the elevated pCO2 and elevated temperature treatment. H30R is the elevated pCO2 and ambient temperature treatment with a reduction in hatching success. H32R is the elevated pCO2 and elevated temperature treatment with a reduction in hatching success.

125 in post-larval survival was observed in the A32 and H32 treatments, which were two fold lower than the A30 scenario, with 6.5% and 6.8%, respectively, of the larval population surviving to post-larvae (Figure V-4). Incorporating the effect of elevated pCO2 on hatching success resulted in the H30R simulation having a similar proportion of surviving megalopae (12%) to the H30 treatment (11%). The

H32R simulation, resulted in 7.7% survival. This was similar to the H32 treatment simulation, which showed a 6.8% increase in megalopae survival (Figure V-4).

MODEL SENSITIVITIES

The natural mortality and A30 simulation showed that larval stage-II was the most sensitive stage through the development of megalopae (Figure V-5,1 SV- ).

The sensitivities shifted once elevated pCO2 and temperature were incorporated into the model. The H30 and A32 treatments showed the greatest sensitivity was among stage-V larvae (Figure V-5). This shift in the sensitivities to later larval stages was similar in the H32 simulation, which showed that stage-IV had an almost 2-fold increase in sensitivity relative to the other treatment simulations

(Figure V-5). The model also showed that larval stages I-II had similar sensitivities among all treatment simulations (Figure V-6), except for the H32 scenario.

Elevated pCO2 and temperature shifted the sensitivities, with the greatest impact on late-larval stages (Stage IV-V) in the H30, A32, and H32 treatment combinations

(Figure V-6).

When reduced hatching success was incorporated into the model, the sensitivity for the H30R treatment shifted to stage-IV larvae, relative to the H30 treatment

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Figure V.5. Sensitivity analysis of population growth rate for stone crab larval stages I-V, at each experimental simulation. Mortality rates from each experimental treatment were superimposed onto the natural mortality rate estimated from the literature. The model also assumed a larval supply at each stage of 1x106 in dividuals and an initial starting population of 100 ovigerous females. A30 represents the control with ambient pCO2 and ambient temperatures. H30 is the elevated pCO2 and ambient temperature treatment. A32 is the ambient pCO 2 and elevated temperature treatment, and H32 is the elevated pCO2 and elevated temperature treatment. The H30R and the H32R treatments represent model simulations that incorporate the reduction in hatching success reported in Chapter 2.

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Figure V.6. Sensitivity analysis of population growth rate for stone crab larval stages I-V at each experimental simulation. Mortality rates from each experimental treatment were superimposed onto the natural mortality rate estimated from the literature. The model also assumes a larval supply at each stage of 1x106 individuals and an initial starting population of 100 ovigerous females. A30 represents the control with ambient pCO2 and ambient temperatures. H30 is the elevated pCO2 and ambient temperature treatment. A32 is the ambient pCO 2 and elevated temperature treatment, and H32 is the elevated pCO2 and elevated temperature treatment.

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(Figure V-7, SV-1). The H32R treatment showed the highest sensitivity in stage-IV larvae (Figure V-8, SV-1). The sensitivities in both the H32 and H32R simulations were more than double the sensitivities observed in the control simulation.

DISCUSSION

Successful management of a species requires understanding how the various components of their life history may change relative to environmental perturbations

(Crouse et al. 1987). Population models can therefore, be useful to make predictions and inform management decisions, so that conservation efforts can focus on environmental factors that may improve population survival (Miller

2001). Currently, there are no population models that incorporate how changes in seawater pCO2 and elevated temperature will impact larval supply throughout development. The simulations reported here suggest that elevated pCO2 and temperature may not only reduce the overall larval supply of stone crabs, but also decrease the number of individuals surviving to post-larvae stages.

The stone crab larval population was lower in all treatments relative to the natural mortality estimate, with the lowest populations being in treatments that had elevated temperatures (A32, H32, and H32R; Figures V-3 and V-4). The larval populations were 94%, 93%, and 96%, lower relative to the control respectively.

Although not as great, the elevated pCO2 simulation also showed a substantial decrease in population, with a 76% and 67% reduction relative to the natural mortality simulation and A30 simulation respectively. These results suggest that

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Figure V.7. Sensitivity analysis of population growth rate for stone crab larval stages I-V. Mortality rates from each experimental treatment were superimposed onto the natural mortality rate estimated from the literature. The model also assumes a larval supply at each stage of 1x106 individuals and an initial starting population of 100 ovigerous females. A30 represents the control with ambient pCO2 and ambient temperatures. H30 is the elevated pCO2 and ambient temperature treatment. H30R is the elevated pCO2 and ambient temperature treatment under reduced fecundity from Chapter 2.

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Figure V.8. Sensitivity analysis of population growth rate for stone crab larval stages I-V. Mortality rates from each experimental treatment were superimposed onto the natural mortality rate estimated from the literature. The model also assumes a larval supply at each stage of 1x106 individuals and an initial starting population of 100 ovigerous females. A30 represents the control with ambient pCO2 and ambient temperatures. H32 is the elevated pCO2 and elevated temperature treatment. H32R is the elevated pCO2 and elevated temperature treatment under reduced fecundity from Chapter 2.

131 conservation efforts might be best directed at policies that would reduce anthropogenic inputs to coastal waterways which will assist in preventing drastic fluctuations in seawater carbonate chemistry, and could help mitigate the effects of

OA on stone crab larval survival. These mitigation measures are particularly relevant to the pCO2 and temperature sensitive pelagic phases of stone crabs.

However, rising seawater temperatures, which are more challenging to manage, are more concerning and appear to have a larger impact on the survival of stone crab larval. The current IPCC (2013) projections suggest that seawater temperature will increase at a faster rate than OA resulting in a +2–3°C rise by the end of the century, which may not give stone crab larvae time to adapt to the rate of increase in temperature.

The model sensitivities indicated that earlier stage larvae (stage-II) were most sensitive to changes in model parameters in the natural mortality and A30 trials.

Other population models have shown similar trends in sensitivities of earlier life stages. For instance, sardine and anchovy matrix models show that without any effects of environmental perturbation, survival through early life stages are important determinants of recruitment and population growth (Lo et al. 1995). The sensitivities shifted to later larval stages (stage-IV or V) in the H30, A32, H32,

H32R, suggesting that elevated pCO2 and temperature have a greater impact on late stage larval survivorship. This shift is likely a consequence of greater effects of ocean acidification and elevated temperatures on more developed larval stages, with more pronounced anatomical features.

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There was a significant reduction in the number of surviving post-larvae in the elevated temperature scenarios relative to the control suggesting that elevated pCO2 and temperature predicted for the end of the century may result in lower recruitment potential for stone crab populations. Although informative, a major caveat of previous stone crab models is that they have focused only on adult stage mortality (Ehrhardt 1990), leaving out important vital rates associated with earlier- life history stages like post-larval and juveniles. This is primarily because there are no published in situ estimates of annual stone crab post-larval survivorship or juvenile recruitment. The significant lack of data on various vital rates associated with stone crab post-larval and juvenile survival, and recruitment, is a drawback for the model presented here. Therefore, attempting to make comparisons to other crustacean fisheries like the blue crab, Callinectes sapidus, is difficult as incorporating the success of new recruits (survival and growth) allows for a more precise estimation of the fisheries sustainability on annual scales (Miller 2001). The potential decrease in stone crab post-larvae under future elevated pCO2 and temperature scenarios and coupled with the lack of post-larvae and juvenile stone crab data is concerning considering the sustainability of Florida’s stone-crab industry is largely dependent upon the survival of new recruits into the fishery during the subsequent years after settlement (Ehrhardt et al. 1990). Furthermore, the tolerance of stone crab post-larvae and juveniles to OA and elevated temperature is largely unknown. Other crustacean species, like the spider crab Hyas araneus (Walther et al. 2010), and the Tanner crab Chionoecetes bairdi (Long at al.

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2013), have shown sensitivity to OA and elevated temperatures resulting in a decrease in both megalopal and juvenile survivorship. These studies also demonstrate that OA can extend development time during these life stages, which could result in individuals being more susceptible to predation (Walther et al. 2010,

Long et al. 2013). If stone crab post-larvae and juveniles exhibit a similar sensitivity to anthropogenic stressors, then the predicted changes in seawater conditions for the end of the century could result in additional bottlenecks for stone-crab populations.

The primary management practice for the stone crab fishery includes closing the fishing season during the summer reproductive months which is a successful strategy that protects ovigerous females and acts as a buffer against fishing exploitation (Ehrhardt et al. 1990). Despite the aforementioned management strategy, the annual stone crab commercial harvest has dramatically declined from

3.2 million pounds in 2007 to 1.9 million pounds in 2014 (FWC 2000–2014). This decline has occurred despite statewide fishing efforts remaining relatively stable over the same period (FWC 2000–2014). Therefore extrapolating the model to include all life stages over multiple reproductive seasons would be beneficial to incorporate fishing pressure mortality since previous work has indicated that both fishing mortality and adult natural mortality can be substantial (75% and 94% respectively; Ehrhardt et al. 1990). The decreases in the annual catch are concerning for the fishery, and suggest that other environmental factors are likely at play. However, until estimates of survivorship are reported on all components of

134 the stone crabs life history, a true estimation of the stone crab fisheries sustainability will be uncertain (Miller 2001).

Future stone crab population models should attempt to couple the tolerance of different life stages to fine-scale environmental perturbations with in situ stock estimates, especially if the population estimates reported here are representative of larval survivorship under anthropogenic changes that are likely to occur within the next 50–100 years. Lack of such data makes extrapolating population abundancies over multiple seasons difficult and limits the ecological relevance of any population estimation. Annual recruitment data, size at harvest, variability in the stone crab molt increment, and fishing mortality are important fishery-related population parameters that should be incorporated into a comprehensive model to increase our understanding of the population’s sustainable yield (Bergh and Johnson 1992).

Until estimates of survivorship are reported on all components of the stone crabs life history relative to real time environmental disturbances, a true estimation of the stone crab fisheries sustainability will not be determinate (Miller 2001).

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135 SUPPLEMENTARY TABLES

Table SV.1. The sensitivities for the subdiagonal of the main matrix for each larval stage among all treatment simulations. The values highlighted in yellow indicate the stage with the highest sensitivity for each treatment simulation.

Treatment Natural A30 H30 A32 H32 H30R H32R Mortality Stage-I 0.2133 0.2563 0.1533 0.1199 0.2002 0.2135 0.0788 Stage-II 0.4420 0.4111 0.4263 0.4935 0.2704 0.2388 0.2031 Stage-III 0.3369 0.2091 0.1464 0.4012 0.1617 0.3720 0.5228 Stage-IV 0.2050 0.2910 0.3225 0.3403 0.7607 0.5015 0.3057 Stage-V

0.2645 0.2491 0.5333 0.5497 0.4241 0.3190 0.6686

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CHAPTER VI: SYNTHESIS AND CONCLUSIONS

Decades of urbanization have disrupted the hydrology and diverted nutrient- rich runoff into Florida’s coastal habitats. These local-scale perturbations have increased the baseline pCO2 along Florida’s coastal waters (Bauer et al. 2013,

Ekstrom et al. 2015). Coupled with the increase in CO2 and other greenhouse gases in the atmosphere that are leading to global-scale ocean acidification and elevated seawater temperatures, the alteration of Florida’s coastal habitats is likely to have damaging ramifications for some of Florida’s regional fisheries. The Florida stone crab, Menippe mercenaria, contributes ~$25 million annually to Florida’s economy; however, since 2000, the mean annual commercial catch has declined by

~25%. One of the greatest challenges for the management of crustacean fisheries is the inability to adequately relate the success rate of larvae recruiting to the fishery with fine-scale environmental changes. This dissertation attempted to enhance the functional understanding of trends that may modulate the success of stone crab larvae under future pCO2 and temperature conditions. A more comprehensive understanding of how larvae respond to combined environmental stressors will provide additional tools for managers to use in their interpretation of crustacean fishery data, and how they relate to critical water chemistry changes over time. This is the first study to determine how future changes in seawater conditions may impact the success of the Florida stone crab during embryonic and larval development. Larval release and embryonic development of stone crabs occur

137 within coastal regions, some of which are experiencing declines in pH, which are estimated to be three-times faster than that of the open ocean (Zhang and Fischer

2014). This dissertation shows that despite living in environments that experience frequent fluctuations in environmental conditions, several components of the stone crab life history are sensitive to OA and elevated temperature.

Reductions in hatching success, slower embryonic development, and reduced larval survivorship were observed under OA conditions, which could have a damaging effect on the sustainability of the stone-crab fishery, especially if the larvae are simultaneously exposed to multiple stressors (i.e., OA, salinity, temperature, and hypoxia). Larval survivorship was reduced under OA conditions, however, the effect on survivorship was greater when larvae were exposed to elevated temperatures. This suggests that some components of larval development may be tolerant, and therefore able to adapt to, fluctuations in seawater carbonate chemistry by the end of the century. However, the highly significant reduction in larval survivorship at elevated seawater temperatures is concerning, and may serve as a potential bottleneck for the stone-crab population, especially in the absence of phenotypic plasticity or adaptation (Long et. al. 2013a). The current IPCC (2013) projections suggest that seawater temperature will increase at a faster rate than OA resulting in a +2–3°C rise by the end of the century. Therefore, the thermal limits of stone crabs will likely reduce the larval supply in the tropics, and potentially the subtropics, but may shift the stone crab’s distribution to higher latitudes (Brown and Bert 1992).

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Brachyuran crab larvae cannot swim fast enough for an individual to overcome strong, horizontal currents (Epifanio and Garvine 2001). Larval crabs can adjust their swimming speeds and vertical position to specific cues, including gravity, hydrostatic pressure, and light. Controlling their depth and swimming speed, in response to certain environmental cues, affords larvae the ability to position themselves in favorable currents that can facilitate transport toward favorable nursery habitats (Epifanio and Cohen 2016). For instance, the interaction between vertical swimming behavior and the frequency at which currents vary, at any given depth, can control the net horizontal displacement of larvae (Epifanio and Garvine

2001). Here, I show that elevated pCO2 disrupts the capacity of larvae to maintain their position in the water column, which could result in a deeper distribution with limited advection potential. Reducing larval dispersal could result in individuals being transported into habitats that are not ideal for completing larval development

(Sulkin 1984, Epifanio 1988). Furthermore, if less precise depth regulation results in larvae not being transported away from coastal regions then larvae may be subjected to higher predation pressure nearshore, which could negatively feedback to reduce larval supply (Morgan and Christy 1996). The inability of mid-stage larvae to orient toward the surface, under elevated pCO2, could serve as another bottleneck for stone crab populations, by limiting the population’s ability to find refuge from increasing seawater temperatures in more northern habitats. Therefore, strategies to minimize the impact of anthropogenic input on coastal systems at the

139 northern range of the stone crabs might help mitigate population declines in the future.

The model sensitivities indicated that earlier stage larvae (stage-II) were most sensitive to changes in the natural mortality and control simulations. Elevated pCO2 and temperature shifted the sensitivities to later larval stages (stage-IV and V). This shift was potentially a consequence of greater effects of ocean acidification and temperature stresses on more developed larval stages, with more pronounced anatomical features. Additionally, the matrix-model showed lower larval populations relative to the control, and a reduction of surviving post-larvae at elevated temperatures, suggesting that future changes in climate may result in additional bottlenecks for stone crab populations. Given that only <1% of larvae typically survive to post-larvae, a slight increase in survivorship likely represents a substantial increase in population density. These results indicate that conservation efforts directed at policies that would reduce anthropogenic inputs into nearshore waterways would mitigate drastic fluctuations in coastal seawater pCO2, however, rising seawater temperatures, which are more challenging to manage, appear to have a large impact on stone crab larval survival.

Future work on stone crabs could examine the interaction between OA, elevated seawater temperatures, and hypoxia throughout ontogeny. Especially since the shallow and low-water flow characteristics of some stone crab habitats (i.e.,

Florida Bay) make coastal areas increasingly susceptible to oxygen depletion from excess input of organic matter (Briceño et al. 2013). Evaluating how post-larvae

140 and juveniles respond to multiple anthropogenic perturbations will be necessary to create a more precise predictive model designed to estimate population trajectories as anthropogenic disturbances continue to drive environmental shifts into the future. If these negative impacts are exacerbated by carry-over effects, such as high post-hatch mortality, or impaired physiology throughout juvenile development, then recruitment into the fishery may become limited (Schiffer et al. 2013). In addition, the vertical position of brachyuran crab larvae is also controlled by other exogenous stimuli, including hydrostatic pressure and light. Therefore, future work should determine how responses to the other components of the depth model may change when larvae are exposed to multiple anthropogenic stressors, such OA, hypoxia, and elevated temperatures. Moreover, comparisons among stone-crab populations, which inhabit a range of pH conditions, may provide insights into the geographical range in phenotypic plasticity, and thereby determine the scope for adaptation to future changes in seawater conditions (Long et. al. 2013a). Given the significant impact of temperature on larval survival, efforts to minimize carbon emissions should remain a priority in order to help minimize the costs of adaptation

(Goldberg and Wilkinson 2004), and ensure the sustainability of future stone crab populations.

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LITERATURE CITED

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