THE EFFECT OF CLIMATE CHANGE ON DEVELOPMENT, GROWTH AND METABOLISM OF EMBRYONIC ELASMOBRANCHS, Scyliorhinus sp.

A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health

2019

MUHAMMAD SYAFIQ BIN MUSA

School of Medical Sciences Division of Cardiovascular Sciences

LIST OF CONTENTS

LIST OF TABLES 7 LIST OF FIGURES 8 LIST OF ABBREVIATIONS 10 ABSTRACT 11 DECLARATION 12 COPYRIGHT STATEMENT 13 THE AUTHOR 14 ACKNOWLEDGEMENTS 16

CHAPTER I GENERAL INTRODUCTION 17 1.1 Climate Change 17 1.1.1 Temperature 17 1.1.2 Hypoxia 19 1.1.3 Predicted future climate 20 1.2 Elasmobranchs 20 1.2.1 Importance of elasmobranchs 21 1.2.2 Reproduction of elasmobranchs 22 1.2.3 Egg cases of oviparous elasmobranchs 23 1.2.4 The plight of sharks 24 1.2.5 Vulnerability of early life stages oviparous elasmobranchs 25 1.2.6 Greater spotted catshark and small-spotted catshark 26 1.2.7 Embryological study of oviparous elasmobranchs 29 1.3 Climate Change on Elasmobranchs and Other Ectothermic Fish 29 1.3.1 Temperature 29 1.3.2 Hypoxia 31 1.3.3 Climate change on intertidal organisms 32 1.4 Aim and Objectives 33

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CHAPTER II GENERAL METHODOLOGY 36 2.1 Introduction 36 2.2 Animal Species 36 2.2.1 Small-spotted catshark (Scyliorhinus canicula) 36 2.2.2 Greater spotted catshark (Scyliorhinus 37 stellaris) 38 2.2.3 Egg case shaving 2.3 Experimental Treatments 39 2.3.1 Temperature 39 2.3.2 Hypoxia 39 2.3.3 Experimental rearing conditions 40 2.4 Water Quality 42 2.5 Energy Budget 42 2.6 Growth and Survival 43 2.7 Metabolism 43 2.8 Respirometry 44 2.8.1 Experimental respirometer setup 44 2.8.2 Oxygen consumption calculations 46

CHAPTER III OVIPAROUS ELASMOBRANCH DEVELOPMENT INSIDE THE EGG CASE IN 7 KEY STAGES 48 Abstract 49 3.1 Introduction 49 3.2 Materials and Methods 53 3.2.1 Egg cases source and maintenance 53 3.2.2 Developmental staging 54 3.2.3 Growth pattern and average daily body length gain (ADL) 55 3.2.4 Yolk consumption rate 56 3.2.5 Development time (embryo age and the duration of each developmental stage) 56 3.2.6 Hatching process 57 3.2.7 Effect of air exposure on egg cases 57 3.2.8 Statistical analysis 57 3.3 Results 58

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3.3.1 The egg case 58 3.3.2 Stage 1 60 3.3.3 Stage 2 60 3.3.4 Stage 3 62 3.3.5 Stage 4 64 3.3.6 Stage 5 66 3.3.7 Stage 6 68 3.3.8 Stage 7 70 3.3.9 Growth pattern and average daily body length gain (ADL) 75 3.3.10 Yolk consumption rate 77 3.3.11 Development time (embryo age and the duration of each developmental stage) 79 3.3.12 Hatching process 82 3.3.13 Effect of air exposure on S. stellaris egg cases with jelly and without jelly 83 3.4 Discussion 84 3.4.1 Early embryogenesis 85 3.4.2 Egg case structure and function 86 3.4.3 Jelly as protection 87 3.4.4 Opening of seawater slits 87 3.4.5 Gills and respiration 88 3.4.6 Fin and body morphology 89 3.4.7 Yolk consumption during development 90 3.4.8 Conclusions 91 Acknowledgements 91 Funding 91 References 92 Supporting Information 96

CHAPTER IV THE EFFECTS OF TEMPERATURE AND HYPOXIA ON GROWTH AND SURVIVAL OF EMBRYONIC SMALL-SPOTTED CATSHARK (Scyliorhinus canicula) 97 Abstract 98 4.1 Introduction 98 4.2 Materials and Methods 100 4.2.1 Supply of egg cases and care of animals 100 4.2.2 Experiment (biospheres) tanks and mimic of different climatic conditions 101

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4.2.3 Developmental staging 101 4.2.4 Growth pattern and growth rate 104 4.2.5 Yolk consumption 105 4.2.6 Survival rate 105 4.2.7 Development time and hatchling size 106 4.2.8 Statistical analysis 106 4.3 Results 106 4.3.1 Growth of S. canicula embryos 106 4.3.2 Yolk consumption of S. canicula embryos 108 4.3.3 Survival rate 110 4.3.4 Development time and hatchling size 111 4.4 Discussion 112 4.4.1 Growth rate 113 4.4.2 Yolk consumption 114 4.4.3 Survival of S. canicula under future ocean warming and hypoxia 115 4.4.4 Development time and hatchling size 116 4.4.5 Conclusions and conservation implications 118 Acknowledgements 119 Funding 119 References 120

CHAPTER V THE EFFECTS OF OCEAN WARMING AND HYPOXIA ON METABOLISM OF SMALL- SPOTTED CATSHARK, Scyliorhinus canicula DURING EMBRYONIC DEVELOPMENT 127 Abstract 128 5.1 Introduction 128 5.2 Materials and Methods 131 5.2.1 Animal husbandry and experimental rearing conditions 131 5.2.2 Developmental stages investigated 132 5.2.3 Assessment of metabolic rate during development in different climatic rearing environments 134 5.2.4 Excess post-exercise oxygen consumption (EPOC) 135 5.2.5 Statistical analysis 136 5.3 Results 137

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5.3.1 Effects of different climatic conditions on metabolism at each developmental stage 137 5.3.2 Effects of development on metabolism under different climatic conditions 139 5.3.3 Effects of rearing environment and developmental stage on EPOC 139 5.3.4 Temperature sensitivity (Q10) of metabolism 141 5.3.5 Developmental plasticity and metabolism 142 5.4 Discussion 144 5.4.1 Effect of temperature on embryonic and early post-hatch metabolism 145 5.4.2 Effect of dissolved oxygen levels on embryonic and early post-hatch metabolism 146 5.4.3 Ontogenetic changes in metabolism 148 5.4.4 Developmental environment does not have a lasting impact on metabolism 148 5.4.5 Implications for future oceans 149 5.4.6 Conclusion 151 Acknowledgements 151 Funding 152 References 152

CHAPTER VI GENERAL DISCUSSION AND CONCLUSION 162 6.1 Introduction 162 6.2 Contributions of Current PhD Work 162 6.3 Summary of PhD Findings 165 6.4 Conclusion 171

REFERENCES 172 APPENDIX 187

Final word count: 41,227

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LIST OF TABLES

Table 3.1 Comparison of 7 developmental stages inside the egg case from S. stellaris and S. canicula. 73

Table 5.1 Temperature sensitivity (Q10) of S. canicula oxygen consumption rates (MO2) at different developmental stages developed under normoxic and hypoxic conditions. 141

Table 5.2 Temperature sensitivity (Q10) of S. canicula EPOC recovery (τ values) at different developmental stages under normoxic and hypoxic conditions. 142

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LIST OF FIGURES

Figure 1.1 Scyliorhinus sp. adult. 27 Figure 1.2 Egg cases and hatchlings of S. stellaris and S. canicula. 28 Figure 2.1 The experimental rearing tanks of S. canicula egg cases. 41 Figure 2.2 The respirometer of S. canicula egg cases. 45 Figure 2.3 A summary of the metabolism study on S. canicula embryos. 47 Figure 3.1 External features of the S. stellaris egg case at stage 1. 59 Figure 3.2 The inside of the S. stellaris egg case at stage 2. 61 Figure 3.3 The inside of the S. stellaris egg case at stage 3. 63 Figure 3.4 The inside of the S. stellaris egg case at stage 4. 65 Figure 3.5 The inside of the S. stellaris egg case at stage 5. 67 Figure 3.6 The inside of the S. stellaris egg case at stage 6. 69 Figure 3.7 The inside of the S. stellaris egg case at stage 7. 70 Figure 3.8 7 key developmental stages of S. stellaris inside the egg case. 72 Figure 3.9 S. stellaris body length and external yolk sac volume across the 7 developmental stages. 75 Figure 3.10 Average daily body length gain (ADL) during each of the 7 developmental stages. 76 Figure 3.11 Yolk consumption rate during the 7 developmental stages. 78 Figure 3.12 Development time (age) to reach 7 developmental stages and hatch. 80 Figure 3.13 Duration of development time of the 7 developmental stages. 81 Figure 3.14 The hatching process. 82 Figure 3.15 Shrinking rate of S. stellaris egg cases (% in area h-1) with and without jelly. 84 Figure 4.1 Oviparous elasmobranch development inside the egg case in 7 key stages. 103 Figure 4.2 Summary of specific developmental stages that were investigated in this study based on the 7 key stages of development laid out in Musa et al. (2018). 104

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Figure 4.3 Growth of S. canicula embryos developed under different climatic conditions. 107 Figure 4.4 Yolk consumption of S. canicula embryos developed under different climatic conditions. 109 Figure 4.5 Survival rate (SR) of S. canicula embryos developed under different climatic conditions. 110 Figure 4.6 Development time (age) of S. canicula embryos under different climatic conditions. 111 Figure 4.7 Hatchling size of S. canicula embryos under different climatic conditions. 112 Figure 5.1 Oviparous elasmobranch development in 7 key stages. 133

Figure 5.2 Oxygen consumption rates (MO2) of S. canicula embryos at stage 4 and 6 of development and at 1-day post-hatch under different treatments. 138 Figure 5.3 Time constant of recovery of excess post-exercise oxygen consumption (EPOC) of S. canicula embryos at stage 4 and 6 of development and at 1-day post-hatch under different treatments. 140

Figure 5.4 Oxygen consumption rates (MO2) of S. canicula hatchlings under different treatments and hatchlings acclimated (a week) to control treatment (normoxia at 15 °C). 143 Figure 5.5 EPOC (τ values) of S. canicula hatchlings under different treatments and hatchlings acclimated (a week) to control treatment (normoxia at 15 °C). 144 Figure 6.1 Summary of PhD findings from Results Chapters III, IV and V of this thesis. 170

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LIST OF ABBREVIATION

N15 Normoxia at 15 °C N20 Normoxia at 20 °C H15 Hypoxia at 15 °C H20 Hypoxia at 20 °C TL Total body length V Volume ADL Average daily body length gain SGR Specific growth rate YC Yolk consumption rate SR Survival rate SD Standard deviation MO2 Oxygen consumption rate MMR Maximum metabolic rate RMR Routine metabolic rate SMR Standard metabolic rate AS Aerobic scope EPOC Excess post-exercise oxygen consumption Pcrit Critical oxygen tension Q10 Rate of change over a 10 °C temperature change OCLTT Oxygen- and capacity-limited thermal tolerance Tp Pejus temperature CTmax Maximum critical temperature CTmin Minimum critical temperature [O2] Oxygen concentration CO2 Carbon dioxide ATP Adenosine triphosphate NADH Nicotinamide adenine dinucleotide FADH2 Flavin adenine dinucleotide ppt Parts per thousand ppm Parts per million °C Degree Celsius cm Centimetre mL Millilitre g Gram kg Kilogram % Percentage

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ABSTRACT

Commonly addressed as the apex predator in the oceans, elasmobranchs play an important role in keeping the balance of a healthy marine ecosystem. About a quarter of all elasmobranchs are threatened by extinction mainly due to human activities, such as overfishing, destructive fisheries and shark finning. Elasmobranchs are vulnerable to extinction due to their slow growth, late sexual maturity, and low offspring production. Some oviparous elasmobranch species lay their sessile egg cases around shallow waters of the intertidal zone, where they are exposed to extreme environments, e.g. high temperatures and low dissolved oxygen levels. Climate models predict that CO2 levels will increase to 1100 ppm by 2100, which leads to ocean warming, as well as the extent and frequency of low dissolved oxygen zones. However, only few studies have considered the effect of elevated temperatures and low dissolved oxygen levels on biological fitness and physiological performances of elasmobranchs, especially during crucial embryonic stage. We developed an alternative scale for staging embryonic development non-invasively in living embryos (Chapter III), and then used this scale in later work to determine biological fitness, physiological performances and survival at specific developmental stages. This PhD research is therefore aimed to investigate the effect of both current extreme environments and predicted future climatic conditions, specifically ocean warming and hypoxia, on growth and survival (in Chapter IV), and metabolism (in Chapter V) of small-spotted catshark, Scyliorhinus canicula during early ontogeny. Our results revealed that exposure to elevated temperatures increase the embryonic growth, yolk consumption and metabolic rates of S. canicula, while shrinking the hatchling size. Under hypoxic conditions, S. canicula embryos showing a steady growth, but metabolic rates were significantly reduced, suggesting that S. canicula may have incorporated anaerobic metabolism during this period. As the incubation period was longer in hypoxic treatments, embryonic survival was negatively affected as the animals trapped longer inside the sessile egg cases under low dissolved oxygen conditions. The synergistic effects of both ocean warming and hypoxia increased mortality rate of S. canicula during early embryonic stages. To mimic the free-living hatchling stage that are free to swim in a preferred environment, S. canicula hatchlings were then transferred into a common environment (normoxia, 15 °C), where metabolic performances were re-measured after 1-week. Our results revealed that developmental environment does not have a lasting impact on metabolism of S. canicula hatchlings. Understanding the impacts of future predicted climate change on ecological and physiological aspects of elasmobranchs is essential from a conservation standpoint in order to accurately determine, predict and eventually reduce the consequences of human activity on marine habitats as well as the species within them.

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DECLARATION

No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning.

2nd January 2019 MUHAMMAD SYAFIQ BIN MUSA 9632644

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COPYRIGHT STATEMENT i. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. iii. The ownership of certain Copyright, patents, designs, trademarks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. iv. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=24420), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.library.manchester.ac.uk/about/regulations/) and in The University’s policy on Presentation of Theses

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THE AUTHOR

Education and Employment

2014 – Present: University Lecturer. Marine Science Programme, School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Malaysia.

2015 – 2018: PhD, Adaptive Organismal Biology, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom.

2012 – 2013: Master of Science (Marine Science), Major in Marine Biology. Universiti Kebangsaan Malaysia, Bangi, Malaysia. CGPA : 3.15 Research Title: The Study of Biological and Behaviour of Mudskippers Boleophthalmus boddarti and Periophthalmodon schlosseri.

2009 – 2012: Bachelor of Science with Honours (Marine Science), Major in Marine Biology. Universiti Kebangsaan Malaysia, Bangi, Malaysia. CGPA: 3.51 Dean’s list in 1st Semester of 2010/2011 (CGPA : 3.65) Dean’s list in 2nd Semester of 2010/2011 (CGPA : 3.75) Dean’s list in 1st Semester of 2011/2012 (CGPA : 3.74) Research Title: The Study of Biological and Environmental of Mudskippers Periophthalmus argentilineatus and Periophthalmodon schlosseri.

2008 – 2009: Kedah Matriculation College, Kedah, Malaysia. (Biology) CGPA: 3.47 Dean’s list in 1st Semester of 2008/2009 (CGPA : 3.80)

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Publication

Musa, S. M., Czachur, M. V., & Shiels, H. A. (2018). Oviparous elasmobranch development inside the egg case in 7 key stages. PLoS One, 13(11), e0206984.

International Conferences and Presentations

2017 – Society of Experimental Biology Annual Meeting (Gothenburg, Sweden) (Oral presentation) 2016 – Society of Experimental Biology Annual Meeting (Brighton, UK) 2016 – Cracking the Egg - SEB Satellite Conference (Brighton, UK) (Poster presentation) 2016 – Postgraduate Summer Research Showcase (Manchester, UK) (Poster presentation) 2016 – Manchester Organismal Biology Symposium (Manchester, UK) 2016 – The Heart - New Horizons Symposium (Manchester, UK) 2015 – FSBI Annual Symposium: Biology, Ecology and Conservation of Elasmobranchs (Plymouth, UK) 2015 – Ocean Acidification: What’s It All About? (London, UK)

Public Engagement with Research

Video uploaded by Faculty of Biology, Medicine and Health UoM on YouTube titled “Minute Lectures: Saving Sharks from Climate Change”. Link provided as below. https://www.youtube.com/watch?v=DZ3JSxQtI4M

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ACKNOWLEDGEMENTS

Alhamdulillah, first and foremost, praises and thanks to the God, the Almighty, for His showers of blessings, and for giving me the strength and patience throughout my PhD research. I would like to take this opportunity to express my deepest expression to my supervisor, Dr Holly Shiels for her constant support, guidance and superb supervision. Her motivation and sincerity have deeply inspired me. It was a great privilege and honour to work and study under her guidance. I also would like to thank her for her friendship, empathy, and great sense of humour. As a supervisor and a mentor, she has taught me more than I could ever give her credit for here. She has shown me, by her example, what a good scientist (and person) should be. Thank you so much, Dr Holly. I am extremely grateful to my very supportive parents, Prof. Dato’ Dr. Musa Bin Ahmad and Salmi Binti Abd. Rahman for their love, prayers, caring and sacrifices for educating and preparing me for my future. They are my ultimate role models. Thank you for all your guidance, and continuous support on my interests in animals since I was young. To my other half, my loving and supportive wife, Noraziani Binti Shaari, thank you so much for your endless loves, sacrifices, support through the hardship, and for always being there for me. Thank you for all your hard work and efforts with our little ones, Abdullah Isa and Aisya Sophia. I dedicate this PhD thesis to my wife, and to our growing little Isa and Aisya. Thanks and sorry for everything, ummi sayang. I thank the Shiels Laboratory members, who have helped me along this wonderful PhD journey. Special thanks to Molly Czachur, Miriam Fenkes, Karlina Ozolina, Oliver Wearing, Thomas Sheard, Alexander Holsgrove, Adam Keen, James Ducker, Terry Garner, Ilan Ruhr, James Marchant and Ainerua Martins for all their help and friendship throughout my PhD. I am grateful to all of those with whom I have had the pleasure to work during this and other related projects. “Once a Shiels Lab, always a Shiels Lab”. My sincere thanks also go to my co-supervisor, Dr John Fitzpatrick (Stockholm University, Sweden) and advisor, Dr Robert Nudds for their guidance and ideas for this research project. Finally, I also would like to thank the Ministry of Higher Education (KPT, Malaysia) and Universiti Kebangsaan Malaysia (UKM) for funded this study and for this wonderful opportunity. ‘Terima kasih banyak-banyak’.

Muhammad Syafiq Bin Musa

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CHAPTER I

GENERAL INTRODUCTION

1.1 CLIMATE CHANGE

1.1.1 Temperature

The Earth’s surfaces, including atmospheric and oceanic, are experiencing gradually increasing temperatures in a process known as global warming (Chang et al., 2000). This is caused by the greenhouse effect, involving the interactions between incoming solar radiation and the Earth’s atmosphere (Alley et al., 2003). Radiation from the sun passes through the atmosphere to the Earth’s surface, where atmospheric greenhouse gases absorb and emit infrared thermal radiation in all directions, including towards the Earth’s surface. The infrared thermal radiation is reradiated back from the Earth’s surface as heat energy and trapped in the area between the atmosphere’s surface and the Earth’s surface. This is due to a layer of atmospheric greenhouse gases which exist in our atmosphere, acting as a blanket under which heat is trapped. This causes warming of the planet, since the heat radiation cannot penetrate the greenhouse gases layer and therefore cannot move to outer space (Joos and Spahni, 2007; Norby and Luo, 2004). It is well documented that an increase of human activities, such as burning fossil fuels, open burning, and industrial pollution, leads to an increase of greenhouse gas release into the atmosphere, thus contributing to, and accelerating the greenhouse effect (Norby and Luo, 2004).

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The oceans play an important role in controlling the Earth’s climate with ~70 % of the Earth’s surface and ~90 % of the Earth’s volume consisting of seawater (Bigg et al., 2003; Trenberth, 2009). The oceans regulate the global temperature in a number of different ways. Firstly, the oceans regulate and influence global warming by providing a large surface area for heat absorption and gaseous exchange between the ocean and atmosphere (Chang et al., 2000). Furthermore, the high heat capacity of seawater allows for efficient absorption, storage and transfer of heat from the sun. The heat capacity of the global atmosphere is equivalent to only the top 3.5 meter depths of the global oceans (Trenberth and Stepaniak, 2004). When considering that some ocean depths reach almost 11,000 meters, the oceans are a significant component of global temperature regulation. Due to these characteristics, the oceans ultimately absorb more than 97 % of the Earth’s heat radiation and the temperature of the seawater is subsequently increasing, leading to large-scale ocean warming (Bigg et al., 2003).

The oceans also actively circulate and transfer heat through the processes of convection and advection, where heat is widely distributed by ocean currents (Trenberth and Stepaniak, 2004). Some ocean currents transport warmer tropical and subtropical waters towards colder areas, while other currents transport colder water in the opposite direction. For example, the Gulf Stream transports warm water across the North Atlantic Ocean, originating in Florida and moving towards Northern Europe. Before reaching Europe, the Gulf Stream breaks up into several other currents, one of which flows to the British Isles and Norway. The heat carried in these warmer currents facilitates the warming of winds that blow over these regions, which maintains air temperatures in winter and prevent these areas from becoming much colder (Halkin & Rossby, 1985; Lee et al., 1981). Therefore, ocean temperatures exist in a complex array of oceanographic regimes, and it is clear that constant changes in temperature must be strongly considered when addressing the impacts of climate change on a global scale.

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1.1.2 Hypoxia

Marine ecosystems are varied and diverse, and provide many habitat niches with different energetic demands. Large proportions of the coastal zone, especially shallow- water intertidal areas, experience high fluctuations in temperature and dissolved oxygen levels (Clark et al., 2005; Diaz and Rosenberg, 2008). During low tide, seawater levels become shallow, and some areas are fully exposed to direct sunlight, causing the temperature to increase rapidly in tidal pools and shallow waters. An increase in water temperature decreases the oxygen solubility in seawater, which results in coastal waters becoming more hypoxic (Farrell and Richards, 2009; Frölicher et al., 2009; Pörtner and Knust, 2007).

Coastal upwelling occurs along the coast, whereby nutrient rich waters from deeper areas of the oceans are brought to the surface (Grantham et al., 2004). The deep ocean contains a large concentration of nutrients mainly from the decomposition of phytoplankton biomass and other organic material that has slowly sunk to the bottom of the ocean. As the nutrient concentrations increase, many oceanic processes are altered including excessive algal growth and depletion of oxygen in the water, leading to more frequent hypoxic events as well as spreading of coastal hypoxic zones (Bakun et al., 2015; Grantham et al., 2004).

Increases in nitrogen and phosphorus are attributed to the cause of these hypoxic zones, and this is in part accelerated by the rapid increase in human activities near coastal waters. There has been a surge in anthropogenic contents being introduced into coastal waters, primarily due to intensive agricultural and industrial activities along with human population growth (Bennett et al., 2001; Doney, 2010). Nutrient-rich sewage and polluted waters are therefore entering coastal waters, leading to eutrophication (Bennett et al., 2001; Doney, 2010). It is well known that eutrophication causes algal blooming, thus starving the coastal waters of oxygen and resulting in more hypoxic waters (Bennett et al., 2001; Diaz and Rosenberg, 2008; Doney, 2010). In the more severe conditions, where this leads to oceanic dead zones, the low dissolved oxygen content in an area has

19 become an environmental stressor for native marine organisms (Diaz and Rosenberg, 2008; Farrell and Richards, 2009). Hypoxic events are naturally occurring due to climate change, and for the past 50 years, dissolved oxygen content in the oceans has dropped by ~2 % (Schmidtko et al., 2017).

1.1.3 Predicted Future Climate

The global average surface temperature is expected to increase over the upcoming decades, mainly because of gradual increases in anthropogenic CO2 and other greenhouse gases concentrations in the atmosphere (Caldeira and Wickett, 2003; Fabry et al., 2008; Meehl et al., 2007). This will cause a growing greenhouse effect, which will lead to future climate change with warming oceans (Bigg et al., 2003; Norby and Luo, 2004). For the past 3 decades, the global surface temperature, which includes sea surface temperature (SST) has risen to about 0.6 °C (Hansen et al., 2006). Climate models predict that by 2100, CO2 levels will increase to 1100 ppm and global average surface temperature will rise about 5 °C more than today’s measurements (IPCC, 2001a; 2001b; Joos and Spahni, 2007; Meehl et al., 2007). This additional heat will be absorbed by the oceans, resulting in the coastal waters becoming warmer, and thus more hypoxic (Bigg et al., 2003; Trenberth, 2009; Trenberth and Stepaniak, 2004).

1.2 ELASMOBRANCHS

The term ‘elasmobranch’ refers to a group of fishes in the subclass Elasmobranchii, which consists of sharks, rays, skates, guitarfish and sawfish (Bonfil 1994; Compagno 1984; 2001; Serena 2005). In Greek, ‘elasmo’ means metal plate and in Latin, ‘branchus’ means gill. Apart from Elasmobranchii subclass, Holocephali is another subclass in the class Chondrichthyes, which consists of chimaeras and elephant fish, but the elasmobranchs are the dominant group in Chondrichthyes (Compagno, 1984). There are approximately 60 families, 185 genera, and 929 - 1164 species of living fishes from the class Chondrichthyes and about 96 % of the species are elasmobranchs and only 4 % are holocephalans (Compagno, 1984; Serena 2005).

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Elasmobranch fishes can be distinguished from the true, bony fish (teleost) by their specific characteristics. These mainly include the possession of cartilaginous endoskeletons, the presence of five to seven separate gill openings, and lack of a swim bladder. Elasmobranchs also have rigid dorsal fins or spines, their primary upper jaw is not fused to the skull, their external body is covered with small dermal denticles or placoid scales, and their teeth are present in several series of replicating rows, which allows them to replace their teeth from the inside of their mouths (Bonfil 1994; Compagno, 1984).

1.2.1 Importance of Elasmobranchs

Elasmobranchs play an ecologically important role in the oceans. Elasmobranch species, such as sharks, are the top predators in the oceans, commonly addressed as holding the ‘apex’ position in the marine food web (Stevenson et al., 2007). Elasmobranchs keep the marine ecosystem in balance mainly by predation to control prey populations from the arctic to the tropics (Heithaus et al., 2008; Heupel et al., 2014; Roff et al., 2016). Removing elasmobranchs from the oceans can lead to negative consequences on the food web in marine ecosystems (Friedlander and DeMartini, 2002; Heithaus et al., 2008; Prugh et al., 2009). The mesopredator release hypothesis suggests that due to the declining numbers of apex predators in the oceans, middle sized predators will become abundant, thus resulting in a rapid decline of smaller sized prey (Ferretti et al., 2010; Prugh et al., 2009). This will then lead to decrease in biodiversity of marine species, which transforms the community structure of the food web and leads to an unbalanced marine ecosystem (Ritchie and Johnson, 2009).

Elasmobranch fishes, such as sharks are also used as a good indicator for healthy marine ecosystems. The lack in abundance of sharks in a marine habitat can indicate that the marine ecosystem is out of balance (Baum et al., 2003; Reynolds at al., 2005). Elasmobranchs are therefore suitable bioindicator for the health of marine environments as they are widely distributed globally and contain a high diversity of species (Bonfil 1994; Compagno, 1984).

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1.2.2 Reproduction of Elasmobranchs

The elasmobranch fishes reproduce by sexual reproduction with internal fertilization (Compagno 1984), even though there are several reports of parthenogenesis in sharks (Chapman et al., 2008; Edwards, 2007; Robinson et al., 2011). Approximately, 40 % of elasmobranch species have the ability to lay egg cases (term oviparous), which mainly are benthic sharks (family Hemiscylliidae, Heterodontidae and Scyliorhinidae) and skates (Compagno 1984; Dulvy and Reynolds, 1997; Hamlett & Koob 1999; Hoff, 2009). The other 60 % of elasmobranch species, which consisting mainly of larger pelagic sharks and rays, have the ability to give birth to live young (term viviparous) or hatch eggs prior to the birth of non-placental pups (term ovoviviparous) (Compagno 1984; Francis and Mace, 1980; Hamlett & Koob 1999).

In oviparous elasmobranchs, during ovulation, the oocytes from both ovaries will migrate to the oviducal gland through the oviduct tube and then the fertilized ovum will be enclosed by the egg case (Hamlett et al. 2005). Two egg cases are produced, where one egg case is produced in each oviduct (Hamlett & Koob 1999; Hamlett et al. 2005). The tough egg case membrane that encloses the fertilized ovum is made up by the multilamellar collagenous and non-collagenous proteins that are imperfectly arranged in orthogonal order (Knight and Hunt, 1974; 1976; Knupp and Squire, 1998; Wourms and Sheldon, 1972). These collagen proteins are secreted by the nidamental or the shell gland inside mature females (Cox et al. 1984, 1987; Hamlett et al. 2005; Wourms and Sheldon, 1972). Several rare findings from previous studies have reported on double-yolked eggs (egg case with two yolks) and wind eggs (egg case without a yolk) on some oviparous and ovoviviparous elasmobranch species (Castro, 2000; Powter and Gladstone, 2008). During our study, we have found one double-yolked and 3 wind egg cases of S. stellaris, and a double-yolked egg case of S. canicula (double- yolked and wind egg cases photographs are provided in Appendix section of this thesis; Figs 7.1, 7.2 and 7.3).

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1.2.3 Egg Cases of Oviparous Elasmobranchs

Once elasmobranch females lay and deposit the egg cases, there will be no maternal care and the embryo inside the egg case is entirely dependent on the attached external yolk sac for energy and nutrient supply until most of the yolk is absorbed (Ballard et al., 1993; Rodda and Seymour, 2008). This incubation period can remain for several months to more than a year until the embryo is strong enough to hatch and leave the egg case (Carrier et al., 2004; Hamlett and Koob, 1999). In their natural habitat, some elasmobranch egg cases experience physical stresses, including being washed up onto the beach or being directly exposed to air during low tide. When the egg case is exposed for long periods outside of water, the size and shape of the egg case will be affected by the shrinking of the drying egg case, where for some cases it can shrink to about 30 % of its original size (Treloar et al., 2006).

The elasmobranch egg cases can be identified to different species by their specific external characteristics. Different species’ egg cases have different sizes, shapes and morphological structures. This includes a variety of egg case sizes, different forms of horn and apron length at the end of the egg cases and the presence or absence of tendrils and fibers on the edge of lateral keel (Cox et al., 1987; Ishiyama, 1958). For instance, an egg case with horns that are enclosed in the apron can be found in Dipturus gudgeri egg cases, and this characteristic is special to the subgenus in the genus Dipturus (Ishiyama, 1958; Treloar et al., 2006). This suggests that egg case morphology can be used to identify the phylogenetic relationships in elasmobranch species. The differences in egg case morphology, size and shape may allow different elasmobranch species to successfully adapt to their specific habitats. (Ebert, 2005; Ebert and Davis, 2007). Egg cases can be observed in a variety of colours, even within the lifespan of an individual egg case. Catechol oxidation reactions on the egg case lead to changes in both the colour and transparency of the egg cases, and therefore colour variation in elasmobranch egg cases cannot be used for egg case identification (Koob, 1987).

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1.2.4 The Plight of Sharks

Elasmobranchs are long lived animals (12 – 50 years) and because of their long generation time, there is a lower scope for genetic adaptation and phenotypic plasticity, an important consideration over the next 100 years of predicted climate variability (Bradshaw and Holzapfel, 2006; Compagno, 1990; Hoffmann and Sgrò, 2011). Slow growth, low fecundity rates, long gestation or incubation period and late sexual maturity will all lead to the susceptibility of elasmobranchs extinction (Carrier et al., 2004; Cortés, 2000; Field et al., 2009; Myers and Worm, 2005). When considering the likelihood of elasmobranchs being able to adapt and respond to climate change, it is likely that, the death of an individual will lead to increasingly significant impacts due to the reproductive contribution each individual holds for future shark populations (Herndon et al., 2010).

Overfishing has increasingly threatened sharks as human demand increases for seafood as a major protein source. Recently, high developments in fisheries technology, destructive commercial fishing practices and poor fisheries management strategies have all resulted in the rapid decline of shark populations (Baum and Myers, 2004; Myers and Worm, 2005; Stevens et al., 2000; Stevenson et al., 2007). Even though shark populations are already severely impacted, they are still being killed in large numbers, and are unsustainably targeted for their fins due to the increased market demand for Asian delicacies, such as shark fin soup (Worm et al., 2013). Compared to other marine fishes, elasmobranchs are at a higher risk of extinction (Field et al., 2009). Around a quarter of shark species across the globe are threatened with extinction and many species have been reported as critically endangered, where about 52 % of chondrichthyans have been listed under IUCN Red List compared to only 8 % of teleost species (Dulvy et al., 2014; Field et al., 2009; Fowler et al., 2005). This is indeed, the lowest fraction of species populations of all vertebrates ever been reported worldwide (Dulvy et al., 2014; Simpfendorfer and Dulvy, 2017).

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1.2.5 Vulnerability of Early Life Stages Oviparous Elasmobranchs

Embryonic developmental is considered as the most crucial and vulnerable period for survival during the life cycle of an organism, thus is an important phase in determining the overall fitness of a species (Leonard et al., 1999; Pimentel et al., 2014). The developmental stages of oviparous elasmobranchs, hosted inside a characteristic egg case, can be found widely distributed along the coastal areas. They are usually found attached to seaweed or other sessile organisms, and following storms or highly exposed conditions can be washed up on beaches after becoming dislodged from the substrate that they are attached to. They are also often found, inside crevices between or under rocks, around shallow waters of the intertidal zone, within rockpools and sometimes in deeper water depending on the species, habitat structure and environmental adaptation (Harahush et al., 2007; Jagadis and Ignatius, 2003; Orton, 1926). Due to this, the oviparous elasmobranch embryos are forced to develop inside the sessile egg cases, where they are exposed to extreme environments with high temperatures, low dissolved oxygen levels, high fluctuations in salinity and strong wave exposure (Diaz and Rosenberg, 2008; Harahush et al., 2007; Last & Stevens, 1994; Orton, 1926). Furthermore, it is predicted in the near future that the oceans will warm; the increase of coastal water temperatures will also reduce the solubility of dissolved oxygen, thus making the water more hypoxic (Bigg et al., 2003; Frölicher et al., 2009).

The main function of oviparous elasmobranch egg cases is to act as a barrier between the embryo and the seawater, thereby protecting the developing embryo from the surrounding pathogens (Evans and Kormanik, 1978). During the early embryonic developmental stages of oviparous elasmobranchs, embryos begin to form and the external yolk sac membrane is still fragile and not fully developed (Ballard et al., 1993). During this crucial period, the seawater slits of the egg cases are fully sealed, ensuring no seawater exposure to the vulnerable and developing embryos. Previous studies reported that early embryonic exposure to seawater caused mortality in oviparous elasmobranch embryos (Ballard et al., 1993; Mellinger et al., 1986). This suggests that the elasmobranch embryos are vulnerable to seawater exposure, likely due to the

25 environmental changes and bacterial contamination at this stage. Elasmobranch egg cases are also at risk of predation in their natural habitat, mainly from marine gastropods (Cox and Koob, 1993; Lucifora and García, 2004; Powter and Gladstone, 2008). Therefore, oviparous elasmobranchs are exposed to a high risk of mortality in coastal areas, and future climate change may present an unforeseen threat to the survivability of oviparous elasmobranch during early life stages.

1.2.6 Greater Spotted Catshark and Small-Spotted Catshark

The greater spotted catshark (also known as nursehound, bull huss or large-spotted dogfish), Scyliorhinus stellaris and the small-spotted catshark (also known as lesser- spotted dogfish or sandy dogfish), Scyliorhinus canicula are both shark species from genus Scyliorhinus in the family Scyliorhinidae (Compagno, 1984). They are oviparous, bottom dwelling (benthic) catshark, widely distributed around the continental shelf area of Europe including the Northeast Atlantic and Mediterranean Sea, and can be found from sandy to rocky substrates at depths ranging from 20-100 metres (Compagno, 1984; Serena, 2005). Scyliorhinus sps. are considered as one of the most abundant elasmobranch species in the British Isles and often caught on fishing nets and during trawl surveys (Ellis and Shackley, 1997; Ellis et al., 2005; Gibson et al., 2006).

The body has two dorsal fins with entire skin covered with tiny denticles or placoid scales (Compagno, 1984). Body colour of Scyliorhinus sps. adults are varies from light brown to brown and are covered with various sizes of dark spots on the lateral and dorsal skin (Fig. 1.1). S. stellaris can be differentiated from S. canicula by their relatively larger body size and the existence of small anterior nasal flaps that do not reach the mouth (Compagno, 1984; Serena, 2005). The maximum total body length ever recorded for S. stellaris is 162 cm, while the total body length of S. canicula can reach up to a maximum of 100 cm but usually they were found less than 80 cm in their natural habitat (Compagno, 1984; Serena, 2005).

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Figure 1.1: Scyliorhinus sp. adult. Body of Scyliorhinus sp. has two dorsal fins, where the first dorsal fin is larger than the second. Body colour varies from pale brown to brown with dark brown to black spots of various sizes on the sides and dorsal surface.

Scyliorhinus sps. lay egg cases (commonly known as the mermaid’s purses) in pairs with a single egg case being produced per oviduct (Compagno, 1984; Dulvy and Reynolds, 1997; Hamlett and Koob, 1999). During breeding season, gravid females swim to the shallow waters with high vegetation to lay their egg cases. The egg cases can be found throughout the year, with high abundance during warmer seasons of spring and early summer, and are usually found attached to seaweeds (Compagno, 1984; Orton, 1926; Serene, 2005). Egg cases from both species have a similar egg shape and morphology, but can be easily distinguished by larger size of S. stellaris egg cases compared to S. canicula (Fig. 1.2A). The egg cases are equipped with long tendrils at each corner of the egg case, which help the egg cases to easily tangle and properly attach to the seaweed or other sessile object, ensuring them to stay in protective areas and not be washed away by strong currents (Knight et al., 1996; Mellinger et al., 1986). The incubation period of Scyliorhinus sps. embryos are around 5 to 11 months depending on water temperature, but usually are around 8 to 9 months duration (Compagno, 1984; Mellinger et al., 1986). As the hatchlings from both species shared similar body morphology and skin colour with similar spots pattern, the S. stellaris hatchlings can be easily differentiated by their larger size compared to the S. canicula hatchlings (Fig. 1.2B).

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Figure 1.2: Egg cases and hatchlings of S. stellaris and S. canicula. (A) S. stellaris egg case (larger) and S. canicula egg case (smaller). (B) S. stellaris hatchling (larger) and S. canicula hatchling (smaller).

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1.2.7 Embryological Study of Oviparous Elasmobranchs

In order to understand how oviparous elasmobranchs have successfully survived during the early ontogeny in the extreme environments, the embryological stages of elasmobranch embryos developing inside the egg cases need to be studied. Previous studies have produced a detailed embryological scale, where they have described 34 different embryonic stages of S. canicula (Ballard et al., 1993), a staging scale based on days of development of batoid oviparous elasmobranch, the clearnose skate, Raja eglanteria (Luer et al., 2007), and a more recent oviparous elasmobranch embryonic developmental study on the tropical species, brownbanded bamboo shark, Chiloscyllium punctatum (Onimaru et al., 2018). These studies provide an excellent embryological detail but the slight differences that exist between these developmental stages make them hard to be identified without removing the embryo from the egg case or using specialized equipment. Furthermore, for early staging, the oviparous elasmobranch embryo need to be anaesthetized, exposed to fixative or euthanized (Ballard et al., 1993; Luer et al., 2007; Onimaru et al., 2018). Therefore, available oviparous elasmobranch staging scales are not suitable to be applied on living, developing embryos in order to understand the developmental adaptations which allow oviparous elasmobranchs to survive in the intertidal zone during embryonic stage.

1.3 CLIMATE CHANGE ON ELASMOBRANCHS AND OTHER ECTOTHERMIC FISH

1.3.1 Temperature

Temperature is the key factor that is closely related to metabolism of an organism. In the oceans, marine ectotherms depend on their thermal tolerance in order to maintain aerobic performance (Nilsson et al., 2009). Previous studies suggest that metabolic energy expenditure of an organism is significantly affected by the increase in temperature as it can be seen that metabolic rates of leopard shark, Triakis semifasciata, as well as heart rate of sandbar sharks, Carcharhinus plumbeus increased when being

29 exposed to warmer temperatures (Dowd et al., 2006; Miklos et al., 2003). The oxygen- and capacity-limited thermal tolerance (OCLTT) hypothesis explained that in specific favourable temperature range of species, an ectothermic organism can maximize its capacity of aerobic scope in order to support fitness performance, such as growth, reproduction and locomotion, while the aerobic scope is reduced as critical temperatures are approached (Clark et al., 2013; Pörtner and Knust, 2007; Pörtner and Farrell, 2008). Previous studies reported that acute exposure to elevated temperatures has reduced aerobic scope of coral reef fishes and Pacific salmon (Farrell et al., 2008; Nilsson et al., 2009).

Apart from that, temperature affecting fish growth. Study conducting on young sockeye salmon, Oncorhynchus nerka shows that optimum growth occurred at specific preferred temperature range, while exposure to extreme elevated temperature causing negative impact on growth (Brett et al., 1969). Similar findings have been reported on embryonic oviparous elasmobranch, brownbanded bamboo shark, Chiloscyllium punctatum, where embryos developed under warmer temperatures show faster specific growth rates, and higher routine metabolic rates compared to embryos developed under colder water (Rosa et al., 2014). Therefore, these results suggest that due to the increase in growth when exposed to high temperature, the energetic expenditure also increased, thus increase the metabolic capacity of ectotherms. Under warmer waters, the survival rates or hatching success of marine ectothermic fish eggs are also negatively affected, while making the incubation time faster (Houde, 1989; Pepin, 1991; Rosa et al., 2014).

Apart from studies on biological fitness, temperature also influences migration of ectothermic elasmobranchs. Seasonal changes affect the distribution of Atlantic sharpnose shark, Rhizoprionodon terraenovae, as they were found in high abundance around coastal waters between spring and fall compared to warmer months, where they swim into deeper oceans to avoid warmer shallow waters during summer (Parsons and Hoffmayer, 2005). Due to diel vertical migration which is also affected by temperatures, the blue shark, Prionace glauca migrate into colder deep waters to hunt for prey (Campana et al., 2011). Contrastingly, study conducted on the world’s second largest

30 fish of megaplanktivorous elasmobranch, the basking shark, Cetorhinus maximus were encountered in significantly higher numbers during warmer months in southwest of UK waters, where the data recorded was more correlated to sea surface temperatures (SST) compared to plankton biomass (Cotton et al., 2005; Sims et al., 2005). Therefore, these results suggest that temperature has a significant effect on the ectothermic elasmobranch migrations in the oceans.

1.3.2 Hypoxia

Oxygen is an important element to support life, thus reduced dissolved oxygen condition is considered as an environmental stressor for aquatic organisms. In the oceans, marine organisms rely on the availability of environmental oxygen to maximize physiological efficiency (Gray et al., 2002; Oschmann, 1993). Previous studies reported that fish respond in several ways when exposed to hypoxic conditions; where some species were found to frequently use surface respiration, reducing their oxygen consumption, increase ventilation rate and reducing movement (Chapman and McKenzie, 2009; Pollock et al., 2007). Furthermore, long exposure to hypoxia can reduced growth, reproductive performance and fish survival rates (Landry et al., 2007; Pichavant et al., 2001; Shang and Wu, 2004; Wu, 2009). In response to hypoxic environment, fish also can either physiologically respond by equally maintaining the oxygen consumption rates (oxyregulatory response) or decreasing the oxygen consumption rates (oxyconformity response) in order to conform to hypoxic environments (Di Santo et al.,

2016). The critical oxygen tension (Pcrit) has been widely used as an indicator of hypoxic tolerance in fish, where at Pcrit, fish can no longer regulates the oxygen consumption rates (MO2), thus the MO2 depends on the availability of ambient oxygen in an environment (Rogers et al., 2016).

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As for the elasmobranch fishes, in response to hypoxia, most of the pelagic species need to rely on their ability to avoid and escape hypoxic environments, while most of the benthic species are able to tolerate under hypoxic conditions (Carlson and Parsons, 2003). Hypoxia tolerance is likely to be correlated with species specific and habitat preferences, as benthic elasmobranchs living in coastal areas show strong tolerance and resilience to hypoxia compared to pelagic species as they have adapted to oxygen reduced habitat. (Carlson and Parsons, 2003; Diez and Davenport, 1987). For instance, benthic elasmobranchs, such as S. canicula are able to survive in severe hypoxic condition and long exposure, up to 12 hours of anoxia (Diez and Davenport, 1987). This suggests that benthic elasmobranchs are able to acclimate to extreme environmental conditions by minimizing their energetic costs (Butler and Taylor, 1975).

In more severe hypoxic conditions, where there is no adequate dissolved oxygen availability in the surrounding waters, the aquatic organisms can also shift their energy production from aerobic to anaerobic pathways (Nilsson and Östlund-Nilsson, 2008). Benthic elasmobranchs, such as epaulette shark, Hemiscyllium ocellatum shows an improved anaerobic energy capacity, where this species can tolerate to extreme hypoxia in tropical shallow reef, being able to survive and directly exposed out of water, and is well known for its ability to walk on land during low tide (Goto et al., 1999; Heinrich et al., 2014; Pridmore, 1994). Previous studies reported epaulette shark physiological performance is affected when exposed to severe hypoxic concentrations; while the oxygen consumption rates were successfully maintained, the ventilation rates of buccal pumping were significantly decreased, and blood lactate concentrations increased due to the increase in anaerobic metabolism (Routley et al., 2002; Wise et al., 1998).

1.3.3 Climate Change on Intertidal Organisms

Life of marine ectotherms living in the coastal areas can be challenging as they are forced to adapt and survive in a harsh environment (Chin et al., 2010; Diaz and Rosenberg, 1995; Pistevos et al., 2015). Apart from being exposed to high fluctuations of salinity/rain, intertidal organisms also may be exposed to high temperatures and low

32 dissolved oxygen levels during low tide (Di Santo, 2015; Diaz and Rosenberg, 2008). Within a specific range of preferred temperatures, metabolism of a species can be performed in optimum. Previous studies reported that many species have been reported to have a significant decline in metabolic performance as they been exposed to their maximum and minimum critical temperatures (CTmax and CTmin) (Pörtner and Knust, 2007; Pörtner and Farrell, 2008).

Marine organisms, such as pelagic fishes have an option to swim into deeper water to search for water with preferred, colder temperatures (Block et al., 2011; Wilson et al., 2001). Unfortunately, that will not be the case for the sessile organisms (Cox and Koob, 1993; Lucifora and García, 2004; Powter and Gladstone, 2008). Some intertidal organisms already reached their thermal tolerance limits (Helmuth et al., 2006; Hoegh- Guldberg, 1999; Stillman, 2002). The future ocean warming will result in additional increases in water temperature, as well as the further decrease of dissolved oxygen levels in coastal waters, which will negatively impact the development, growth, metabolism and survival rates of marine organisms (Butler and Taylor, 1975; Diaz and Rosenberg, 2008; Farrell and Richards, 2009).

1.4 AIM AND OBJECTIVES

The aim of this PhD thesis was to investigate the impacts of current and predicted future climate change, specifically the effects of temperature and hypoxia on oviparous elasmobranchs during early life stages using non-invasive methods. This aim was achieved through the specific objectives and approaches outlined below.

In Chapter III, the objective was to produce a developmental scale for embryonic oviparous elasmobranchs that can be applied non-invasively to living, developing embryos without removing them from their egg cases in order to monitor development. This developmental scale was intended to be recognizable by morphological observations with the naked eye using a simple candling method. The key stages were hence intended to be identifiable without the need for any specialized equipment or

33 additional procedures. This chapter is largely descriptive but set the framework for the subsequent results chapters.

In Chapter IV, the objective was to study the effects of temperature and hypoxia on growth and survival of embryonic oviparous elasmobranchs. This chapter was designed to understand the resistance, adaptability and survivability of marine species to current environmental conditions and those predicted in the next 100 years. In this experiment, the S. canicula egg cases were exposed to different climatic conditions and the growth and survival of developing embryos were closely monitored and measured from early life to hatch. The 7 key stages (outlined in Chapter III) was used to identify growth and yolk consumption rates at specific developmental stages. We hypothesized that by exposing S. canicula embryos to elevated temperatures, there would be an increased growth and mortality rate, whereas exposure to reduced oxygen levels would negatively impact the growth and survival of embryos.

In Chapter V, the objective was to investigate the metabolic response of oviparous elasmobranchs to chronic elevated temperature, low dissolved oxygen concentration and development. The 7 key developmental stages scale (outlined in Chapter III) was used to monitor development of S. canicula embryos and oxygen consumption rates were measured at different developmental stages in order to fully understand the effects of development on metabolism of oviparous elasmobranchs reared under different climatic conditions. We hypothesized that with warmer temperature exposure, there would be an increased metabolism, and that low dissolved oxygen exposure would decrease metabolism, while overall the metabolic rates would increase with increasing age of S. canicula.

We also began to address the question of whether development under a specific environmental condition has lasting impact at post-hatch. Thus, in this study we also employed non-invasive measurements of metabolic rate in newly hatched juveniles. Once hatched, oxygen consumptions of S. canicula hatchlings were re-measured after a week being exposed to normoxic condition at 15 °C to investigate the impact of long

34 exposure to different rearing conditions during incubation period. We hypothesized that after a week exposure to normoxic conditions at 15 °C, the metabolic rates of the hatchlings would be less affected by the rearing conditions (during incubation period of S. canicula).

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CHAPTER II

GENERAL METHODOLOGY

2.1 INTRODUCTION

This thesis was written and presented in an alternative format, thus the detailed materials and methods for each results chapter were described in depth in their relevant sections. In this general methodology chapter, an overview is provided, including any additional information that was not deemed suitable for inclusion in the individual results chapters, further justifying the rationale for the methods used in this PhD research.

2.2 ANIMAL SPECIES

The effects of climate change on elasmobranchs were studied here using the oviparous elasmobranch species. Two target species being used in the present study due to the specific reasons as described below.

2.2.1 Small-spotted Catshark (Scyliorhinus canicula)

The small-spotted catshark, Scyliorhinus canicula is an endemic European species that can be found widely distributed throughout the Northeast Atlantic and Mediterranean Sea, and is considered as one of the most abundant species in UK waters (Compagno, 1984; Ellis et al., 2005). The species is categorised as being of Least Concern under the IUCN Red List of Threatened Species, and S. canicula egg cases can be found throughout the year, except during autumn (Ellis et al., 2009; Mellinger, 1983). Because of this, we selected S. canicula as our candidate species because the animal is abundant

36 and native in UK waters, the egg cases can be easily sourced for the research, and can be used without any restrictions often posed on threatened species.

Apart from that, S. canicula is one of the most well described elasmobranchs in terms of their biological reproduction, geographical distribution, embryological development, physiological performance and survival (Ballard et al., 1993; Butler and Taylor, 1975; Ellis and Shackley, 1997; Mellinger et al. 1986; Sumpter and Dodd, 1979; Tort and Torres, 1988). This is mainly due to the fact that both the animals and the egg cases can be easily obtained in high abundances in the wild. Many previous studies have also reported that S. canicula is an environmentally tolerance species and the egg cases have a long incubation period (Butler and Taylor, 1975; Diez and Davenport, 1987; Mellinger, 1983). Therefore, S. canicula is the ideal species for our study as it allows a long incubation period over which measurements of different developmental stages can be observed, allowing us to disentangle the effects of different environmental challenges on a fine-scale resolution. Furthermore, our results are comparable to the wealth of literature available for S. canicula, and accelerates our understanding of how this species may physiologically and physically react towards climate change.

2.2.2 Greater Spotted Catshark (Scyliorhinus stellaris)

The greater spotted catshark, Scyliorhinus stellaris has been selected for oviparous elasmobranch embryonic developmental stages study (in Chapter III) as this species is closely related to the main species that was used for this PhD research, S. canicula and for its several other reasons. The classic oviparous elasmobranch embryological study by Ballard et al. (1993) was conducted on S. canicula (the species investigated in Chapters IV and V of this thesis). We wanted to conduct a novel investigation of oviparous elasmobranch embryonic development that had relevance to the Ballard’s study (Ballard et al., 1993) but that was applicable to living, developing elasmobranch embryos, without needing to remove the embryo from the egg case. Thus, we used S. stellaris to construct a complementary yet alternative oviparous elasmobranch

37 embryonic scale that can be applied non-invasively and still have relevance to S. canicula.

Both S. stellaris and S. canicula are from the same genus Scyliorhinus which belongs to the same family Scyliorhinidae, and share similar morphological features, whereby these two species can only be identified by the small anterior nasal flaps, distinguished between species by their extension towards the mouth (Compagno, 1984; Serena, 2005). For S. stellaris, they do not reach the mouth, whereas the nasal flaps reach the mouth on S. canicula (Compagno, 1984; Serena, 2005). Both species also share similar egg case structures with similar shapes and provided with four long tendrils at each corner of the egg case. In addition, they are both found in similar habitats with similar geographical distribution (Ellis et al., 2005; Serena, 2005). As the egg cases and embryos of S. stellaris are larger than S. canicula, it is beneficial to conduct our embryological developmental study on this similar species with larger animal size.

2.2.3 Egg Case Shaving

All egg cases were closely monitored and observed during the incubation period of all experiments. The egg cases of S. stellaris were larger in size than S. canicula, and thus exhibited thicker egg case membranes. This prevented visualisation of S. stellaris embryos through the egg case membrane, and so the outer pigmented layer of the egg cases were carefully shaved off using a razor blade. Once the pigmented layer was removed, the inner membrane created a semi-transparent window for clearly observing the embryos inside the egg cases using the candling method. This egg case shaving method has been widely used in previous studies conducted on elasmobranch egg cases (Foulley & Mellinger 1980a; 1980b; His 1897). No egg case shaving was performed on the egg cases of S. canicula as the egg case membrane was thin enough for the embryonic development to be clearly seen through the egg case with a light source.

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2.3 EXPERIMENTAL TREATMENTS

We chose experimental parameters that were inclusive of both future predicted climate scenarios and the natural extremes experienced by this species in the wild. Temperature and dissolved oxygen were the abiotic parameters manipulated in this research, as they represent a realistic influence that will affect this species both in current abiotic variations within the intertidal environment, and under future predicted climate change.

2.3.1 Temperature

Before the experiments were conducted, we carried out preliminary observations at several places across coastal areas in the UK, where the egg cases of S. canicula could be found in their natural habitat. Water temperatures were measured during the breeding season and found to consistently measure ~15 °C. This is an agreement with previous studies conducted on S. canicula that have also reported water temperatures between 15 °C to 16 °C as the normal experimental rearing conditions of S. canicula (Ballard et al., 1993; Kearn, 1965; Metcalfe and Butler, 1984; O’neill et al., 2007). Thus, this thesis considers 15 °C as the ambient temperature for control treatments. Leading climate models predict that global average surface temperature will increase by 5 °C by 2100 (IPCC, 2001a; 2001b; Joos and Spahni, 2007; Meehl et al., 2007), thus we have chosen 20 °C as a suitable representation of elevated temperature for future scenarios (+5 °C from the control treatments).

2.3.2 Hypoxia

In order to understand the impacts of reduced oxygen conditions on the physiological and physical responses of S. canicula, we chose an oxygen concentration that reduced the likelihood of animal mortality, thus the concentration used could not be too low and lethal to the experimental animals. S. canicula has being considered as one of the benthic elasmobranch species that is highly tolerable of reduced oxygen environments with the ability to survive up to 12 hours of anoxia exposure (Butler and Taylor, 1971; Diez and

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Davenport, 1987). Thus, we wanted to choose a value that was suitable for triggering a response in the animals, to understand physiological responses to hypoxia out of the window of ‘normoxic’ conditions, where normoxia was considered as a condition where no alteration in physiological response can be observed. Previous studies reported that S. canicula egg cases survived under 50 % air saturation while exhibiting altered physiological performance, whereas exposure to 20 % air saturation was lethal to S. canicula egg cases (Butler and Taylor, 1975; Diez and Davenport, 1990). Therefore, we have chosen to use 50 % air saturation as our hypoxic experimental treatment, and 100 % air saturation as normoxic condition.

2.3.3 Experimental Rearing Conditions

We conducted experiments under 4 different climatic treatments; normoxia at 15 °C (N15/control), normoxia at 20 °C (N20), hypoxia at 15 °C (H15), and hypoxia at 20 °C (H20). Fig. 2.1 shows the rearing conditions of holding tanks for S. canicula egg cases. Each 55 L experimental holding tank was fully sealed to mimic the simulated climate biosphere on a miniaturised scale. For the artificial atmospheres inside the holding tanks, a pre-mixed hypoxic biosphere gas (11 % O2, 0.04 % CO2 balanced with N2) from BOC gas cylinders was used for this purpose. The biosphere gas was pumped directly from the BOC gas cylinder via air stones, for a total of two hypoxic holding tanks with two different temperatures (15 and 20 °C). Gas was pumped into the holding tanks until the biosphere space was fully filled and the water was 100 % concentrated with the hypoxic gas to create ~50 % air saturation hypoxic treatments (H15 and H20). For animals held in normoxic conditions, an air pump was used to create ambient normoxic experimental treatments (N15 and N20).

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Figure 2.1: The experimental rearing tanks of S. canicula egg cases. An air pump introducing ambient air into two holding tanks to create 100 % air saturation normoxic conditions; normoxia at 15 °C (N15/control) and normoxia at 20 °C (N20) treatments. Pre-mixed biosphere gas introduced into two holding tanks to create 50 % air saturation hypoxic conditions; hypoxia at 15 °C (H15) and hypoxia at 20 °C (H20) treatments.

The cold water temperatures were maintained by holding the tanks in a 15 °C temperature controlled room. For elevated temperatures, water heaters were placed inside the relevant holding tanks to maintain the water temperature at 20 °C. Each holding tank was filtered with a filtration pump, which also provided water circulation inside the holding tank, and facilitating the mixing of biosphere gas into the water. Both holding tanks and lids were transparent and exposed to a 12:12 hour light-dark cycle. All holding tanks were constructed and hosted in the Saltwater Facility at the Biological Services Facility (BSF), Stopford Building in the University of Manchester, Manchester, UK.

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2.4 WATER QUALITY

Water parameters (temperature, dissolved oxygen, pH, salinity, nitrite, nitrate and ammonia) were regularly measured for all holding tanks. A digital thermometer was used daily to measure water temperature, while separate sensor probes were used to measure pH and dissolved oxygen concentrations of the water. Water samples were taken twice a week from each holding tank and the nitrite, nitrate and ammonia contents were measured using the API Saltwater Master Test Kit. Due to the closed biosphere set-up, 10 – 30 % water changes were carried out three times a week for each holding tank to ensure the nitrogenous waste contents was maintained at safe levels for the marine organisms. If the nitrogenous waste content was slightly higher than usual in the holding tank, a greater amount of water was used for the water change. A refractometer was used to measure the water salinity to 35 ppt. To assure the accuracy of our readings, a YSI ProDSS (digital sampling system) handheld multiparameter water quality instrument was also used three times a week to measure water quality parameters.

2.5 ENERGY BUDGET

In general, an energy budget can be explained by the simple formula as given:

C = G + R + F + U, where C is the total energy intake provided by the consumed food/yolk, G is the total difference of energy retained for body system (such as for growth), R is the total energy value of metabolism (standard metabolism of resting animal + active metabolism, such as energy needed for swimming + metabolism for digestion), F is the total energy value of faecal production, and U is the total energy from other excretory products (Elliott, 1976; Ricker, 1971; 2015; Sun et al., 2006). Environmental factors, such as temperature play a significant role in the energy budget that an animal allocates to growth and metabolism, and also has implications for the survival of the species (Elliott, 1976; Malloy and Targett, 1994). Therefore, for this PhD research, it is necessary to

42 investigate the effects of different climatic conditions on growth, metabolism and survival of oviparous elasmobranchs during the crucial embryonic development to determine whether this species exhibits altered energetic allocations under different abiotic conditions.

2.6 GROWTH AND SURVIVAL

In order to measure the growth of S. canicula embryos developed under different climatic conditions, several parameters were assessed in Chapter IV of this PhD research. The yolk consumption rate was measured to inform on how quickly the yolk mass (i.e. food) was transferred to the embryo and digested during the early ontogeny, while average daily body length gain (ADL) and specific growth rate were calculated to measure embryonic growth in terms of body length (in cm) and in percentage of growth per day. Additionally, survival rate, developmental time and hatchling size were also measured weekly on S. canicula embryos to support our findings.

2.7 METABOLISM

Both aerobic and anaerobic processes require the production of adenosine triphosphate (ATP) as the energy supply for metabolism in living organisms (Campbell et al., 2008). In most metabolic reactions of animals, oxygen is required by the mitochondria to oxidize NADH and FADH2 via electron transport during oxidative phosphorylation, in order to produce more energy in the form of ATP (Campbell et al., 2008). Therefore, aerobic metabolism is considered as the main energy source for metabolism in animals. Because of this, the oxygen consumption of an organism has been widely considered in previous literature to represent the metabolic processes in a species function, as the ATP production cannot be fully and accurately measured in vivo (Beamish, 1964; Iles et al., 1985; Spoor, 1946). Thus, in Chapter V of this thesis, oxygen consumption rates were measured to study the effects of different climatic conditions on metabolism of S. canicula embryos.

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2.8 RESPIROMETRY

In recent decades, scientists have used different experimental respirometry setups to measure the oxygen consumption rates of fishes in relation to various abiotic and biotic factors including: temperature, dissolved oxygen, salinity, body weight, swim speed, food consumption and comparison across species (Beamish, 1964; Brett, 1965; Muir and Niimi, 1972; Muusze et al., 1998). Before the metabolism experiments in this thesis were performed, we conducted experimental trials on S. canicula egg cases using different respirometry setups (with different respirometry flow/closed settings, using several different oxygen meters with different programs and using different size and shape of custom-built respirometry chambers) in order to optimise a method suitable for this study, and to choose the most optimal method for measuring the metabolic rates of oviparous elasmobranch embryos. The respirometry setup used in this thesis was therefore based on the conclusions drawn from these preliminary experimental trials.

2.8.1 Experimental Respirometer Setup

We used a 350 mL custom-built intermittent flow respirometer (Fig. 2.2) to measure the aerobic metabolic rate of S. canicula embryos developed under different climatic conditions. The respirometer setup that was used for experimental measurements has been described in detail in Chapter V of this thesis. The respirometer needed to be built to a specific size that was suitable for the size of the experimental animals used, and compliant with an accepted ratio of fish mass to water volume ranging between 1:20 and 1:100 for static respirometers (Clark et al., 2013). Our 350 mL respirometry chamber is suitable for S. canicula egg cases, which are about 4 g in mass. An intermittent flow design was chosen for our respirometer to ensure that the aerobic metabolic rates of S. canicula embryos were measured at a specific dissolved oxygen concentration (normoxia of 100 % and hypoxia of 50 % air saturated waters) that could be maintained over a long time period (24 hours per individual).

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Figure 2.2: The respirometer of S. canicula egg cases. The detail of the respirometer setup is fully described in Chapter V of this PhD thesis. This experimental respirometer setup consists of a respirometry chamber (RC), water bath (WB), water chiller (C), flushing water pump (Pf), circulation water pump (Pc), and a gas analyser (G) that is connected to a computer (PC). Arrows represent the direction of water flows during intermittent flush.

Before a respirometry experiment began, the respirometer was sealed and oxygen sensor probe was used to measure the background respiration of the seawater- filled chamber for at least an hour period before introducing the embryo. This allowed the quantification of background respiration to be recorded, which could have been present due to microorganism or other activity that may contribute to the results of respiration rate recorded in the chamber. Only when there was no background respiration, as indicated by a lack of oxygen reduction in the chamber, the respirometer was ready to be used for the experiment. When an individual was transferred into the respirometer, any air bubbles were removed whilst submerged underwater before the respirometer was tightly sealed.

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2.8.2 Oxygen Consumption Calculations

In order to measure the oxygen consumption rates of S. canicula, each individual was weighed using digital weighing scale prior of being introduced into the respirometry chamber. The fish body mass was used to calculate the mass specific oxygen consumption (MO2), where the fish body volume is assumed to be equal to the body mass of the fish (Clark et al., 2013). The aerobic metabolic rate (MO2) of a fish can be calculated by using this formula provided by Clark et al. (2013):

MO2 = [(Vr - Vf) x ΔCwo2] / (Δt x Mf),

where Vr is the total volume of swim tunnel respirometer, Vf is the volume of fish and is usually assumed to be equaled as the mass of fish, where 1 g of fish is equivalent to

1 ml of water. The ΔCwo2 is the difference of dissolved oxygen concentration inside the swim tunnel respirometer, and Δt is the time duration when ΔCwo2 is measured (Clark et al., 2013). This formula has been used in Chapter V of this thesis, where the aerobic metabolic rates of S. canicula embryos have been automatically calculated by AutoRespTM software (version 2.2.0 developed by Loligo® Systems, Denmark).

In order to calculate the aerobic scope of S. canicula embryos, maximum (MMR) and routine (RMR) metabolic rates need to be calculated (Fry, 1971). Standard metabolic rate (SMR) is defined as the minimum amount of energy needed for an animal to sustain life while in post-absorptive mode (no digestion occur) (Fry, 1957). Because of this, we have chosen the term routine metabolic rate (RMR) to describe the basal metabolic level of S. canicula embryos in Chapter V of this PhD research, as the yolk was transferred into the embryos and digestion occurred during the experiment. The way we measured MMR, RMR, aerobic scope, and excess post-exercise oxygen consumption (EPOC) on S. canicula has been fully described in detail in Chapter V of this thesis. A summary on how MMR, RMR, aerobic scope and EPOC were obtained is shown in Fig. 2.3.

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Figure 2.3: A summary of the metabolism study on S. canicula embryos. This metabolism study has been fully described in detail in Chapter V of this PhD thesis. (A) Air saturation measurements during flushing time (F), waiting time (W) and measuring time (M). (B) For each complete loop (F + W + M) measurement, one metabolic rate (MO2) was calculated as shown in the red circle. (C) MMR was obtained from the maximum MO2 point of an individual, RMR was calculated by averaging the lowest 10 % of MO2 values, and aerobic scope was obtained by calculating the difference between MMR and RMR. (D) EPOC was measured by calculating the tau (τ) value of 63 % recovery time.

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CHAPTER III

OVIPAROUS ELASMOBRANCH DEVELOPMENT INSIDE THE EGG CASE IN 7 KEY STAGES

Syafiq M. Musa1,2, Molly V. Czachur1,3, Holly A. Shiels1,*

1 Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom 2 School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia 3Current Address: Department of Botany and Zoology, Stellenbosch University, Stellenbosch, South Africa

*Corresponding author: E-mail: [email protected] (HS)

This chapter has been published in PLoS One and should be cited as, Musa, S. M., Czachur, M. V., & Shiels, H. A. (2018). Oviparous elasmobranch development inside the egg case in 7 key stages. PLoS One, 13(11), e0206984.

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ABSTRACT

Embryological stages of oviparous elasmobranch during development can be difficult to identify, requiring magnification and/or fixation of an anaesthetized embryo. These restrictions are poorly suited for monitoring the development of living elasmobranchs inside their egg cases. There are two major aims of this study. The first was to observe elasmobranch embryonic development non-invasively and produce a non-invasive developmental key for identifying the life stages for an elasmobranch inside the egg case. To this end, 7 key developmental stages were identified for the greater spotted catshark, Scyliorhinus stellaris, and are provided here with diagrams from multiple perspectives to demonstrate the key features of each stage. The physiological and ecological relevance of each stage are discussed in terms of structure and function for embryonic survival in the harsh intertidal zone. Also discussed is the importance of the egg case membrane and the protective embryonic jelly. The second aim of the study was to understand the applicability of the 7 developmental stages from S. stellaris to other oviparous elasmobranchs. Thus, changes in embryonic body size and egg yolk volume at each stage were measured and compared with those of the closely related, lesser spotted catshark, Scyliorhinus canicula. We find nearly identical growth patterns and yolk consumption patterns in both species across the 7 developmental stages. Thus, although the 7 developmental stages have been constructed in reference to the greater spotted catshark, we suggest that it can be applied to other oviparous elasmobranch species with only minor modification.

3.1 INTRODUCTION

Elasmobranchs are a group of cartilaginous fishes from the subclass Elasmobranchii, which consists of sharks, rays, skates, guitarfish and sawfish (Bonfil, 1994; Compagno, 1984; 2001; Serena, 2005). They can be differentiated from teleost fish (true fish with bony endoskeleton) by several characteristics; their cartilaginous endoskeleton, the possession of five to seven gill openings, rigid dorsal fins or spines, lack of swim bladder, and a body that is covered in denticles or placoid scales (Compagno, 1984;

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2001). Elasmobranchs are widely distributed globally and exist in high species diversity (Bonfil, 1994; Compagno, 1984; 2001; Serena, 2005). Elasmobranch species, such as sharks, play an important role in marine ecosystems. They are apex predators and thus are important for ecosystem balance by controlling prey populations (Friedlander and DeMartini, 2002; Heithaus et al., 2008; Stevenson et al., 2007). A reduction of shark abundance in marine environments suggests that an ecosystem is out of balance (Baum et al., 2003; Baum and Myers, 2004; Reynolds et al., 2005). Therefore, shark abundance could be used as an indicator of the health of an aquatic ecosystem.

Embryonic development is a vulnerable period during the life cycle of an organism, important for determining survivability and thus influencing population viability (Leonard et al., 1999; Pimentel et al., 2014). Therefore, to fully understand how oviparous elasmobranchs survive their intertidal embryonic life stage, it is necessary to observe and study the development of the embryo inside the egg case. Ballard et al. (1993) produced the first complete embryonic developmental table for an elasmobranch. They identified 34 different embryonic stages for the lesser spotted dogfish/catshark, Scyliorhinus canicula, under normal developmental conditions (i.e. at 16 °C, with aerated seawater). These stages mark important points in embryological development, but are not easily identifiable with the naked eye or under low magnification, even through cleared egg case membranes. It is not possible to distinguish between the 34 stages without removing the embryo from the egg case. More recently, studies have been conducted on the tropical species, brownbanded bamboo shark, Chiloscyllium punctatum (Onimaru et al., 2018), and developmental stages of a batoid oviparous elasmobranch, the clearnose skate, Raja eglanteria, have been described and classified based on days of development (Luer et al., 2007). Similar to Ballard et al. (1993), this study provides excellent embryological detail but the developmental stages are not easily recognizable without specialized knowledge and equipment. In addition, in both studies, the embryo was anaesthetized, euthanized and exposed to fixative for staging (Ballard et al., 1993; Luer et al., 2007; Onimaru et al., 2018), which is not suitable for studying living, developing embryos.

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There is a growing interest in shark conservation leading to aquarists, naturalists and the general public identifying shark egg cases on beaches worldwide. In the UK, organizations such as the Shark Trust (http://www.sharktrust.org/) educate the public in species identification of elasmobranch egg cases, and encourage observations to be recorded in citizen science databases. A user-friendly key that could allow scuba divers, snorkelers, non-embryological researchers and the general public to stage the age of the embryo, may aid elasmobranch monitoring and conservation efforts by providing egg case laying and hatching time to be estimated in the field. Additionally, there is a growing demand for monitoring of elasmobranch development inside the egg cases for use in studies linking development to climate change and metabolism (Johnson et al., 2016; Rodda, 2000; Rosa et al., 2014). Thus, our study aims to produce a developmental scale which can be used without specialized equipment, and applied non-invasively to living, developing elasmobranch embryos.

We used the oviparous elasmobranch, the greater spotted catshark, Scyliorhinus stellaris to construct our developmental stages. About 40% of elasmobranch species are oviparous, predominantly the benthic sharks (family Scyliorhinidae, Hemiscylliidae and Heterodontidae) and all skate species (Compagno, 1984; Dulvy and Reynolds, 1997; Hamlett and Koob, 1999; Hoff, 2009). S. stellaris is a benthic catshark from the family Scyliorhinidae (Compagno, 1984), is widely distributed and is one of the most abundant elasmobranch species in the British Isles (Compagno, 1984; Serena, 2005). The male S. stellaris will mature at total body length of ~77 cm, while the female will mature at ~79 cm body length; 162 cm is the longest body length recorded (Compagno, 1984; Serena, 2005). The body has two dorsal fins, where the first dorsal fin is larger than the second, and does not have spines. The whole body is covered with tiny denticles or placoid scales. Body colour varies from pale brown to brown and is covered with dark brown to black spots of various sizes on the sides and dorsal surface, whereas the ventral surface is pale yellow to white. They can be found around the continental shelf area of Europe including the Atlantic and Mediterranean Sea at depths ranging from 20-100 m (Compagno, 1984; Serena, 2005). S. stellaris can be distinguished from closely related species, the small spotted catshark, Scyliorhinus canicula by its larger body size and

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small anterior nasal flaps that do not reach the mouth (Compagno, 1984; Serena, 2005). S. canicula is one of the most common elasmobranch species in UK waters (Compagno, 1984; Serena, 2005) and is the elasmobranch species best studied in terms of embryological development (i.e. used in Ballard’s scale) (Ballard et al., 1993). Thus, we have also employed S. canicula in the current study to make reference with earlier embryological works and as a point of comparison with S. stellaris. Indeed, our aim is to create a parallel scale to the current detailed but invasive embryological scale, that can be used when investigating embryos still remaining in their egg cases.

Scyliorhinus sps. lay egg cases (sometimes referred to as mermaid’s purses) in pairs with a single egg case being produced per oviduct (Compagno, 1984; Serena, 2005). S. stellaris egg cases can be found throughout the year, with the peak breeding season in the spring to summer months (Serena, 2005). During egg maturation, the gravid females of S. stellaris swim to the shallow intertidal zone to lay their egg cases in areas with rocky substrate and high vegetation. In this habitat, S. stellaris egg cases are usually found attached to seaweeds, to substrate with coralline algae (Compagno, 1984; Orton, 1926) or associated with other sessile intertidal zone organisms. The intertidal zone is a harsh marine environment with egg cases and their developing embryos encountering fluctuations in temperature, salinity, wave exposure and high predation (Cox and Koob, 1993; Harahush et al., 2007; Last and Stevens, 1994). However, questions still remain about the developmental adaptations which allow oviparous embryos to flourish in the intertidal zone. Thus, the aim of our study was to produce a simplified developmental scale which could be used without specialized equipment, and applied non-invasively to living embryos. In addition to developing a table of growth in 7 key stages, we have paid particular attention to lesser described features of life inside the elasmobranch egg case including the protective jelly, the opening of seawater slits and other changes in the egg case as they may be key for surviving early in the intertidal zone. Finally, to demonstrate the applicability of our 7 key stages based on S. stellaris to other oviparous elasmobranchs, and to be able to correlate our non-invasive scale with earlier embryological studies (Ballard et al., 1993),

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we have examined how each stage identified for S. stellaris compares with that of S. canicula in terms of growth and yolk consumption.

3.2 MATERIALS AND METHODS

3.2.1 Egg Cases Source and Maintenance

All the work contained in this study is on embryos. All protocols including transport, holding conditions and imaging of embryos were approved by the Animal Welfare and Ethics Review Board of the University of Manchester, UK. A total of 46 S. stellaris egg cases used in this study were supplied by the Native Marine Centre, Isle of Portland, Dorset, UK. All egg cases were laid in captivity from wild caught female adults. The egg cases were usually laid over night, collected in the morning and then shipped in small batches to the University of Manchester, Manchester, UK between November 2015 and August 2016. Additionally, a total of 8 Scyliorhinus canicula egg cases were supplied by Ozeaneum, Stralsund, Germany.

Once in the laboratory, the egg cases were transferred into quarantine tanks containing well aerated saltwater (temperature 15 °C, salinity 35 ppt, dissolved oxygen > 95 %). Health of the egg cases was checked using ‘candling’ to assess the state of the yolk and in some cases, the embryo. Candling is a method for visualizing the egg yolk and/or the developing embryo as a silhouette by shinning a high intensity light through the back of the egg case.

An egg case was considered healthy if it had an intact, ellipsoid egg yolk located at the centre of the egg case. The jelly inside the egg case, which surrounds the egg yolk and holds it in place, is healthy when it is cleared and transparent. There should be no yolk mass escaping the thin egg yolk membrane, which turns the jelly murky. If an embryo had already developed upon arrival at the University of Manchester, it could be easily identified by the vigorous movement (wiggling) of its tail, the movement of its external gill filaments and the slow and slight movement associated with buccal

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pumping. A rotten egg case or a dead embryo was easily detected from its bad, rotten fish-like odour and/or lack of embryo movement.

After two days in quarantine, egg cases were transferred to aerated holding tanks (temperature 15 °C, salinity 35 ppt, dissolved oxygen > 95 %) incorporating a water filtration system. The temperature, dissolved oxygen, salinity, nitrogen levels and pH of the saltwater were recorded daily. The egg cases were vertically positioned on strings underwater inside the holding tanks such that the egg case opening end pointed downward. Egg cases were tagged with plastic bird ring tags, which have different colours and numbers for individual identification.

3.2.2 Developmental Staging

To observe the developmental stages of S. stellaris, each egg case was observed three times a week by candling, and morphology and behavior recorded. Most egg cases had a thin, clear egg case membrane (shell/exterior casing) through which the egg yolk and the developing embryo could be easily observed by candling. However, some had a thicker and more strongly pigmented egg case membrane which was gently shaved down using a scalpel blade to create a thinner and more transparent window for observation without cutting deeply or damaging the egg case membrane.

Each embryo was photographed weekly using Canon PowerShot G16. Hand drawings that were digitally coloured were made from these photos from multiple perspectives and angles, so that key features of each developmental stage were clearly visible. Each drawing was based on live samples and photos of 5 – 7 different individuals at the same developmental stage. Original photos for each drawing are supplied in the supplemental files.

During development, the seawater slits of the egg case open and the developing embryo is naturally exposed to saltwater. At this stage, 12 embryos were carefully removed from their egg cases and transferred into clear, permeable containers (artificial

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egg cases) where the oxygenated saltwater could freely move in and out of the container. This allowed morphological details of the later stages of embryonic development to be documented in detail to facilitate comparison between the features of our 7 key stages and those identified in embryological studies (i.e. Ballard et al. 1993). For earlier developmental stages, where the seawater slits were not fully opened, a total of 5 embryos were euthanized by using lethal dosage 0.5 g L-1 Tricaine methanesulfonate (MS-222), buffered with 0.5 g L-1 sodium bicarbonate, removed from the egg cases and photographed. By doing this, each developmental stage could be drawn in detail from different perspectives, showing all the important key features. The higher level of detail was required to make a side-by-side comparison between our non-invasive scale and Ballard’s embryological scale. However, we must emphasize that removal of the embryo from the egg case is not required for our 7 key developmental stages. The 7 key stages were specifically chosen as they are recognizable by the naked eyes via candling, and because they are ecologically and physiologically important to metabolism, growth and thus survivability.

3.2.3 Growth Pattern and Average Daily Body Length Gain (ADL)

A total of 8 S. stellaris egg cases were used to examine the growth pattern of the species across the 7 developmental stages. Each embryo was photographed weekly using Canon PowerShot G16, and total body length (cm) was measured weekly using ImageJ software (http://imagej.nih.gov/ij). The mean ± standard deviation (SD) of total body length (n = 8) were calculated to study the growth pattern of S. stellaris.

Eight S. canicula egg cases were also used to study the average daily body length gain (ADL) at each of the 7 developmental stages to test the applicability of our staging scale in two different species. ADL (cm day-1) was calculated as, ADL = (final TL at given stage – initial TL at the same stage)/duration (in days) of same developmental stage; where TL is total embryo body length (measured from tip of snout to tip of the tail), and stage is a given stage between stage 1 and stage 7. ADL measurements (in cm

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day-1) were also converted to percentages to aid comparison of ADL patterns between S. stellaris and S. canicula at each developmental stage.

3.2.4 Yolk Consumption Rate

Using the same photographs as above, length, width and depth (in cm) of the external yolk sac were measured weekly using ImageJ software (http://imagej.nih.gov/ij) to calculate the volume (V) of the external yolk sac as, V = (4/3)πLWD; where L, W and D are (1/2)length, (1/2)width and (1/2)depth of egg yolk (in cm) respectively. From this, yolk consumption (YC) rate (cm3 day-1) across different developmental stages was calculated for S. stellaris and S. canicula (n = 8 for each species) as, YC = (initial V at given stage – final V at same stage)/duration (in days) of same developmental stage. Yolk consumption rate measurements (in cm3 day-1) were also converted to percentages to compare the yolk consumption rates between S. stellaris and S. canicula at each developmental stage.

3.2.5 Development Time (Embryo Age and The Duration of Each Developmental Stage)

Age of S. stellaris and S. canicula embryos (n = 8 for each species) at different developmental stages was calculated by summing development time (in weeks) starting from lay (week 0) until any given stage, or hatch. Development time was also converted to percentages to compare the age of embryos from each species at each developmental stage and at hatch. Duration of development time for each stage was calculated by summing the total time (in weeks), where the embryo was still in a given developmental stage. This was also converted to percentages to compare the development time in each stage between S. stellaris and S. canicula.

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3.2.6 Hatching Process

Once the embryos reached their final developmental stage, behavior was closely monitored to study the hatching process. Videos of hatching behavior were recorded using Canon PowerShot G16 and GoPro HERO4 Session. Details of the hatching process were illustrated step-by-step. Once hatched, whole animal wet mass (± 0.01 g) was recorded.

3.2.7 Effect of Air Exposure on Egg Cases

Because elasmobranch egg cases are often washed up onto beaches where they are exposed to air and high temperatures, we additionally wanted to understand the function of the egg case jelly and the egg case membrane in embryo survival. A total of 6 egg cases were identified as unfertilized eggs (egg cases contained unfertilized egg yolks and filled with jelly). These egg cases were removed from water and aerially exposed (at room temperature). Photographs were taken every hour for 50 hours. These photographs were used to measure egg cases size (in cm2) using ImageJ software (http://imagej.nih.gov/ij) to estimate the rate of shrinking, and therefore dehydration, over time. As the sharks hatched, 7 empty egg cases were used to measure the desiccation rate of the egg cases without the jelly by using the same method as described earlier. The tendrils of the egg cases were stretched out to their maximum length before they were measured using a measuring tape (± 0.01 cm) before the egg cases were dried.

3.2.8 Statistical Analysis

Linear regressions, correlations and R2 coefficient for embryo growth and yolk consumption were calculated. Statistical significance was assessed using one-way repeated measures analysis of variance (ANOVA), followed by Tukey’s multiple comparisons post-hoc test (P < 0.05). Percentage data between species was compared using Mann-Whitney U test (P < 0.05). Details are provided in each figure legend. All statistical analysis was calculated and graphed using GraphPad Prism 7.

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3.3 RESULTS

3.3.1 The Egg Case

An elasmobranch egg case consists of the egg case membrane (shell/casing), jelly, an egg yolk and the developing embryo. The S. stellaris egg cases were oval to rectangular in shape with colours ranging from yellowish green to brownish green when wet, to darker shades of the same colours when dry. The egg case mean length was 11.58 ± 0.33 cm, mean width 4.32 ± 0.17 cm and mean height 2.88 ± 0.22 cm (n = 12) when wet. The incubation period of the fertilized egg cases varied depends on water temperature with an average incubation period at 15 °C of 27.25 ± 0.89 weeks (n = 8), and a range of 26 to 29 weeks.

The S. stellaris egg case ends were distinguishable due to one having a flatter edge and the other a more rounded and arched end (Fig. 3.1). The egg cases consisted of four long, spring-like tendrils, located on each corner of the egg case. Tendril mean length was 153 ± 3.98 cm (n = 10) when wet. The tendrils were thicker where they joined the egg case and gradually became thinner towards the ends. The collagenous egg case membrane was flexible but not elastic, so it could easily bend but not stretch. The egg cases had a smooth surface with a leathery to rubbery-like texture. At all edges of the egg case membrane, except the flat egg case opening, there was a much tougher structure present that allowed the egg case to maintain its shape and rigidness.

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Figure 3.1: External features of the S. stellaris egg case at stage 1. The internal ellipsoid egg yolk and blastodisc visible at stage 1 of development. The egg case membrane was filled with jelly surrounding the yolk. There were four seawater slits located at each corner of the egg case, two can be seen from the front (as shown in Fig. 3.1), and another two at the back of the egg case (not shown). The flat end of the egg case will be the site of opening during hatching, whereas the arched end remains firm and closed during hatching. There are four tendrils attached at each corner of the egg case. The key feature for stage 1 was no visible embryo on top of the egg yolk. See supplemental S1 File for original photographs of Fig. 3.1 illustration.

During the early developmental stage of S. stellaris, there was an ellipsoid, yellowish egg yolk which was surrounded by a jelly-like substance. This jelly was softer closest to the egg yolk and the developing embryo, and had a tougher consistency as it approached the edges of the egg case. Each S. stellaris egg case had a total of four seawater slits, which were located towards all four corners of the egg case membrane. These seawater slits could only be found on one side of the egg case, when the egg case was positioned as in Fig. 3.1. These seawater slits were open but blocked by the mucous plugs and the tough jelly, which tightly held the egg case closed from the inside. Due to this, seawater was unable to freely enter into the egg case during the early developmental stages.

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3.3.2 Stage 1

All S. stellaris egg cases began in stage 1. At stage 1, there was an ellipsoid egg yolk and no visible embryo on top of the egg yolk membrane; this can be observed through the egg case with candling (Fig. 3.1). If there was still no visible embryo on the egg yolk after four weeks, the egg case was usually unfertilized and therefore did not develop further. Also possible at this stage are freshly laid, fully sealed egg cases with no egg yolk visible inside, known as ‘wind eggs’. We observed 3 wind eggs out of 88 S. stellaris egg cases in this study.

During stage 1, it was necessary to monitor the egg cases closely and more frequently as the embryo developed rapidly. During this stage, the blastodisc can be seen as a small spot, white to light yellow in colour, which can be seen on the top of the egg yolk membrane (Fig. 3.1). Many important, but not visible, developmental processes are occurring during this stage, which outlined in the discussion.

Some egg cases had thin and translucent outer egg case membranes, where the blastodisc was clearly visible, whereas some individuals had thicker or more pigmented outer egg case membranes, and the blastodisc was more difficult to locate. This pigmented outer layer could be carefully shaved to create a translucent inner layer window, so that the blastodisc and embryo development could be more readily observed. The position of the blastodisc was influenced by the egg case positioning as it rotated and changed against the gravitational force. This has been described previously for Scyliorhinus canicula egg cases (Ballard et al., 1993; Mellinger et al., 1986).

3.3.3 Stage 2

The key feature of stage 2 that can be observed non-invasively through the egg case was the small embryo which was completely connected to the membrane of the ellipsoid- shaped egg yolk (Fig. 3.2). At ~3 weeks old (at 15 °C), embryos reached stage 2 and the

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total body length ranged from 0.31 cm to 0.98 cm, with mean length of 0.62 ± 0.2 cm (n = 8). As gastrulation continues (see discussion under early embryogenesis for details), the stage 2 embryo looked like an embedded membrane located on the surface of the egg yolk membrane. At this stage, the embryo had two major distinguishable prenatal body regions, the larger sized anterior region, which later develops into the head part and trunk primordia, and the smaller sized posterior region (tail bud), which later develops into the tail of the embryo (Fig. 3.2B). Movement of the embryo could be observed, including regular movements in the head-end of the body from right to left.

Figure 3.2: The inside of the S. stellaris egg case at stage 2. (A) The egg yolk mass and the associated embryo to scale. (B) The magnified embryo with indication of key morphological features (lateral view). The embryo consists an anterior (head and trunk primordia) and posterior (tail bud), where the somites (segmented mesoderm) can be found. The key feature for stage 2 was the visible embryo developed on top of the egg yolk membrane without a long tail. See supplemental S2 File for original photographs of Fig. 3.2 illustrations.

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There were many somites (segmented mesoderm) on the body towards the posterior of the embryo (Fig. 3.2B), which were difficult to identify or count through the egg case due to the small size and the movement of the embryo. The embryo could also be difficult to locate in the earlier phases of stage 2, as it could be completely flat against the surface of the egg yolk. Similarly to the blastodisc in stage 1, the embryo was most commonly found on top of the egg yolk surface against the gravitational pull. If not in this position, slowly rotating the egg case while searching for the embryo improved the chance of identification.

3.3.4 Stage 3

Stage 3 of embryonic development occurred ~4 weeks after laying at 15 °C, and was characterized by the development of long tail (can be observed non-invasively through the egg case) and more than 60 % of the embryo body is not connected to the surface of the egg yolk membrane (Fig. 3.3). The embryo was connected to the external yolk sac by the short, wide developing yolk stalk (Fig. 3.3A). The total body length of stage 3 embryos ranged from 0.97 cm to 3.39 cm, with mean length of 1.17 ± 0.16 cm (n = 8). At this stage, most of the organs and body parts began to develop and more somites were visible. The heart is known to have formed (Ballard et al., 1993), but it was still difficult to directly observe through the egg case membrane at this stage as it is small and colourless.

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Figure 3.3: The inside of the S. stellaris egg case at stage 3. (A) The ellipsoid egg yolk mass and the associated embryo to scale. (B) The magnified embryo with indication of key morphological features (lateral view). (C) The diamond-shape mouth opening (ventral view). The key feature for stage 3 was the growth of the long tail. See supplemental S3 File for original photographs of Fig. 3.3 illustrations.

Early stage 3 embryos exhibited a blunt, bent tail tip which pointed downward. Throughout stage 3, the tail became sharper, more slender and straighter as it grew longer. In addition, as the tail grew longer, the tail movement inside the egg case increased. A pair of lens placodes (located in the eye cup), were visible in stage 3 (Fig. 3.3B). The fourth ventricle (located in the brain) was also visible, resembling an empty space on the top of the head. 3 – 6 pairs of pharyngeal arches (later to support gills) appeared slowly and gradually throughout stage 3. When embryos were viewed ventrally (Fig. 3.3C), it was possible to identify a diamond-shaped opening (later to be the mouth).

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At the end of stage 3, small buds of gill filaments begin to develop and located between the pharyngeal arches. The top of the external yolk sac, close to the yolk stalk, changed colour from orange to red as blood vessels developed. These blood vessels grew larger and longer throughout stage 3.

3.3.5 Stage 4

Between 7 and 10 weeks of age (mean 8.13 ± 0.99 weeks, n = 8) at 15 °C, the embryo reached stage 4 characterized by clearly visible long, red external gill filaments, which can be observed non-invasively through the egg case (Fig. 3.4). Gill filaments length was dynamic; they grew in the early phase of stage 4 and began to shrink near the end of this stage. Total body length ranged from 3.24 cm to 8.32 cm, with mean length of 3.64 ± 0.28 cm (n = 8). Embryo length often exceeded the length of the external yolk sac (Fig. 3.4B), and long gill filaments were arranged into six pairs of filament groups. One pair of the shorter gill filaments were located in the developing spiracles, while the other 5 pairs of longer gill filaments (later to be gill arches) were horizontally positioned, located between the pharyngeal arches (Fig. 3.4A). When closely observed, at this stage each gill filament consisted of a single blood capillary that had been folded up (Fig. 3.4C).

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Figure 3.4: The inside of the S. stellaris egg case at stage 4. (A) Six pairs of filament groups, and more apparent blood vessels on surface of spheroid external yolk sac (dorsal view). (B) Developing yolk stalk, eye, and fins (lateral view). (C) The magnified head and gill filaments (ventral view). The key feature for stage 4 was gill filament development. See supplemental S4 File for original photographs of Fig. 3.4 illustrations.

The embryo developed a pair of pectoral fins, a pair of small pelvic fins and numerous large and long fin-folds during stage 4 (3 dorsal fin-folds, later develop into two dorsal fins and the dorsal caudal fin; and 2 ventral fin-folds, later develop into the anal fin and the ventral caudal fin) (Fig. 3.4B). The mouth became more rounded in shape (Fig. 3.4C) and dark pigment was observed in the eye. As more blood vessels developed, the embryo body surface colour changed from translucent to pinkish and the skin was thin and non-pigmented. The heart could clearly be seen beating through the body wall from ventral view (Fig. 3.4C) and variable colour intensity occurred (flashing of red) as the heart beat propelled blood. It was possible to measure heart rate at this

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stage from counting changes in colour intensity and we calculated resting heart rate at 15 °C to be ~20 bpm.

The shape of the external yolk sac changed from ellipsoid to spheroid during stage 4. The yolk became surrounded by many long blood vessels and the yolk stalk grew longer and narrower as the embryo grew larger in size. By the end of stage 4, the amount of yolk mass that had been transferred into the embryo was less than 28.05 ± 9.03 % (n = 8) of the total egg yolk volume at stage 1. Although, the embryo had started to consume the egg yolk, the shrinking of the external egg yolk was not easily appreciable during this stage. At the end of stage 4, more jelly was degraded from the inside of the egg case by the embryo activity, and all four seawater slits were fully opened, exposing the embryo to the surrounding seawater, which freely circulated into and out of the egg case.

3.3.6 Stage 5

The embryo usually reached stage 5 between 14 and 17 weeks after laying at 15 °C, with a mean age of 15.63 ± 1.19 weeks (n = 8). Stage 5 was characterized by the shrinking of the gill filaments, which could be observed non-invasively through the egg case; they were completely absent by the end of this stage (Fig. 3.5). The total body length of stage 5 embryos ranged from 6.37 cm to 10.26 cm, with mean length of 7.57 ± 0.77 cm (n = 8). The embryo began to resemble an adult shark in body form and appearance. There were two rows of placoid denticles which can be seen in dorsal view (Fig. 3.5A), and four rows of small caudal denticles located at the tail tip of the embryo. These small caudal denticles were uniformly positioned horizontally to the right, left, top and bottom of the tail tip (Fig. 3.5A,B). More yolk mass was consumed to fuel growth and the external yolk sac was reduced to 38.79 ± 9.37 % (n = 8) of its original size at stage 1 during stage 5. Due to this significant decrease, the reduction in external egg yolk size was now clearly visible. The embryo stomach was visibly filled with the ingested yolk mass, and the ventral stomach area changed to appear yellowish in colour.

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Figure 3.5: The inside of the S. stellaris egg case at stage 5. (A) Position of placoid denticles and caudal denticles (dorsal view). (B) Body shape began to resemble an adult shark and the external yolk sac shrank visible in size (lateral view). (C) The magnified head and reduced gill filaments (ventral view). The key feature for stage 5 was nearly complete loss of external gill filaments. See supplemental S5 File for original photographs of Fig. 3.5 illustrations.

From the lateral view (Fig. 3.5B), differences in the size of the fins were visible. Some of these fins were developing and growing larger, while others were decreasing in size, becoming completely absent by the end of stage 5. The gill filaments continued to shorten in length; one pair developed into spiracles and five pairs into gill openings toward the end of stage 5. The eyes had darker pigmentation at the center of each eye (Fig. 3.5B) and the mouth was almost fully developed (Fig. 3.5C).

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From the ventral view of the embryo head (Fig. 3.5C), it was possible to observe a pair of nostrils that had recently developed at the snout. More blood vessels developed around the snout during stage 5. The skin and flesh of the embryo grew thicker, and the whole embryo changed in colour to pale pink, with more pigment developing at the later end of stage 5 as the skin thickened further. The heart beat was still visible near the yolk stalk, but the colour intensity of the pumping blood appeared less intense than in the previous stage due to thickening of skin (Fig. 3.5C).

3.3.7 Stage 6

After 18 to 20 weeks (mean 19.25 ± 0.71 weeks, n = 8) post-laying at 15 °C, the embryo reached stage 6 which was characterized by the disappearance of fin-folds, which could be observed non-invasively through the egg case, and a significant increase in body size which ranged from 8.84 cm to 17.02 cm (mean length 10.84 ± 1.22 cm, n = 8). At this stage, the body was curled to maintain occupancy in the limited space inside the egg case. The size of the external yolk sac rapidly decreased during this stage and by the end of stage 6, the embryo had consumed 98.79 ± 1.05 % (n = 8) of the original egg yolk volume measured at stage 1.

At stage 6, the embryo had two rows of placoid denticles on top of its body, and two rows of small caudal denticles, which were located at the right and left side of the tail tip (Fig. 3.6A). From the lateral view, clearly visible were two dorsal fins, a pair of pectoral fins, a pair of pelvic fins, an anal fin and a large dorsal and ventral caudal fin (Fig. 3.6B). A pair of spiracles and five pairs of gill openings were fully developed as the gill filaments were completely shrunk (Fig. 3.6B). More pigment had visibly developed on the skin by stage 6, resulting in banded patterns along the whole body that was yellowish peach with brownish grey to black bands, increasing in colour intensity as the embryo developed further.

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Figure 3.6: The inside of the S. stellaris egg case at stage 6. (A) Position of placoid denticles, caudal denticles and banded patterns on the body (dorsal view). (B) A pair of spiracles, five pairs of gill openings, and fins (lateral view). (C) The magnified head (ventral view). The key feature for stage 6 was the loss of fin-folds. See supplemental S6 File for original photographs of Fig. 3.6 illustrations.

From the ventral view of the head (Fig. 3.6C), the mouth was visible, with newly developed teeth inside the mouth opening. Furthermore, the ampullae of Lorenzini which had developed in earlier stages (Ballard et al., 1993) were now visible with the naked eye (Fig. 3.6C). The skin and flesh continued to grow thicker, the embryo became fully covered in small placoid scales, and the heart beat was no longer visible.

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3.3.8 Stage 7

Between 26 and 29 weeks (mean 27 ± 0.93 weeks, n = 8) post-lay at 15 °C the embryo reached stage 7 (Fig. 3.7). This is the final stage of development before hatching out of the egg case. The total body length of stage 7 embryos ranged from 14.48 cm to 17.72 cm, with a mean length of 16.39 ± 0.93 cm (n = 8). Almost all of the yolk mass had been transferred from the external yolk sac into the internal yolk sac inside the embryo body. The yolk stalk and the external yolk sac membrane shrunk, which could be observed non-invasively through the egg case, and which was completely absent at the end of stage 7, leaving a smooth surface on the ventral side of the embryo (Fig. 3.7C).

Figure 3.7: The inside of the S. stellaris egg case at stage 7. (A) Position of placoid denticles and banded patterns on the body (dorsal view). (B) Fully developed eye and greater pigmentation on skin (lateral view). (C) The magnified head and fully consumed yolk (ventral view). The key feature for stage 7 embryo was a fully consumed external yolk. See supplemental S7 File for original photographs of Fig. 3.7 illustrations.

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At stage 7, more pigmentation was visible, and the whole body colour changed to yellowish brown, with brown banding and many dark spots (Fig. 3.7A,B). As the small denticle scales developed, they gradually covered the whole body, and the embryo had a rougher textured skin. The eyes and other sensory organs were completely developed by this stage (Fig. 3.7). At stage 7, the jelly inside of the egg case was fully degraded, and the flat end of the egg case membrane was weakened and could be easily opened fully.

The developmental phases of S. stellaris in the 7 key stages presented to scale for size comparison in Fig. 3.8. Furthermore, a summary of the 7 developmental stages is presented in Table 3.1, which includes the key features of each stage, the approximate age when the embryo reaches each stage (at 15 °C) and values for body length and yolk volume. Importantly, Table 3.1 also provides the same data for the closely related species, S. canicula which was the focus of Ballard’s embryological scale (Ballard et al., 1993). This allowed for a comparison between the 34 stages from Ballard et al. (1993) of S. canicula and the two species categorized using our 7 developmental stages here.

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Figure 3.8: 7 key developmental stages of S. stellaris inside the egg case. Stage 1 was characterized by no visible embryo. Stage 2 was identified as an early embryo formed on the surface of the egg yolk membrane. Stage 3 embryos had long tails and stage 4 embryos had externalized gill filaments. These filaments had shrunk by stage 5. In stage 6 the key feature was the loss of fin-folds, and in stage 7 the key feature was the fully consumed external yolk.

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Table 3.1: Comparison of 7 developmental stages inside the egg case from S. stellaris and S. canicula. 7 34 stages Key Age S. Age S. Body Body Length Length Yolk Yolk develop of S. features stellaris canicula length S. length S. range S. range S. consump consump mental canicula, of each (week) (week) stellaris canicula stellaris canicula tion S. tion S. stages Ballard stage (cm) (cm) (cm) (cm) stellaris canicula et al. (%) (%) (1993) 1 4 – 16 No visible 0 0 - - - - 0 % 0 % embryo 2 17 – 18 Embryo 3 3 0.62 ± 0.2 0.47 ± 0.31 – 0.98 0.29 – 0 % 0 % early 0.11 0.82 formed 3 19 - 27 Long tail 4 4.25 ± 1.17 ± 1.1 ± 0.09 0.97 – 3.39 0.96 – 0 % 0 % developed 0.46 0.16 2.09 4 28 – 32 External 8.13 ± 7 ± 0.53 3.64 ± 2.47 ± 3.24 – 8.32 2.18 – < 28.05 ± < 22.21 ± gill 0.99 0.28 0.21 5.75 9.03 % 5.42 % filaments developed 5 33 External 15.63 ± 14.13 ± 7.57 ± 6 ± 0.67 6.37 – 5.23 – < 38.79 ± < 34.22 ± gill 1.19 0.64 0.77 10.26 8.78 9.37 % 7.23 % filaments shrunk 6 33 Fin-folds 19.25 ± 16.25 ± 10.84 ± 7.85 ± 8.84 – 6.44 – < 98.79 ± < 98.35 ± absent 0.71 0.46 1.22 0.91 17.02 10.63 1.05 % 0.89 % 7 34 Fully 27 ± 0.93 21.38 ± 16.39 ± 10.3 ± 14.48 – 9.78 – ~100 % 100 % consumed 0.74 0.93 0.42 17.72 12.31 external yolk

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Key features are shown for each species developing inside the egg case. Data for both species (S. stellaris and S. canicula) are from this study (n = 8 for each species). The 34 stages of Scyliorhinus canicula (Ballard et al., 1993) was used to compare the detailed embryological staging to our non-invasive 7 developmental stages.

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3.3.9 Growth Pattern and Average Daily Body Length Gain (ADL)

S. stellaris growth inside the egg case was linear between the beginning of stage 2 and the end of stage 6 (Fig. 3.9) however, the average daily body length gain (ADL) differed between developmental stages (Fig. 3.10A). Slowest growth rate was observed in stage 1 whereas stage 5 had the highest growth rate (Fig. 3.10A). A similar pattern was observed for ADL of S. canicula (Fig. 3.10B). Indeed, no difference in ADL values (as % growth day-1) was found between species (Fig. 3.10C).

Figure 3.9: S. stellaris body length and external yolk sac volume across the 7 developmental stages. Total body length (cm, green, Y = 0.6416X - 1.674, R2 = 0.9898, r = 0.9949) and total external yolk sac volume (cm3, orange, Y = -0.7397X + 23.23, R2 = 0.9105, r = -0.9542) of S. stellaris embryo inside the egg case over development time (in weeks). Data are mean ± SD (n = 8). The beginning of each developmental stage is demarked with a vertical line.

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Figure 3.10: Average daily body length gain (ADL) during each of the 7 developmental stages. (A) ADL (cm day-1) of S. stellaris embryos during each developmental stage. Lines show mean ± SD (n = 8), while points show measurements from each individual. Dissimilar letters indicate statistical differences between stages (RM one-way ANOVA, P < 0.05). (B) ADL (cm day-1) of S. canicula embryos during each developmental stage. Lines show mean ± SD (n = 8), while points show measurements from each individual. Dissimilar letters indicate statistical differences between stages (RM one-way ANOVA, P < 0.05). (C) Comparison of ADL (% day-1) between S. stellaris (purple) and S. canicula (pink) embryos at each developmental stage. Values represent mean ± SD (n = 8). There was no statistically significant difference between species at any stage (Mann-Whitney U test, P < 0.05).

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3.3.10 Yolk Consumption Rate

The decrease in the volume of the S. stellaris external yolk sac was visibly noticeable between stage 4 until stage 7 (Fig. 3.9) with a variation in the rate of yolk consumption between stages, being the highest (0.2 ± 0.05 cm3 day-1) at stage 6 (Fig. 3.11A). A similar pattern was observed for S. canicula (Fig. 3.11B), and like growth rate (ADL), yolk consumption rate (as % day-1) at each stage did not differ between species (Fig. 3.11C).

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Figure 3.11: Yolk consumption rate during the 7 developmental stages. (A) Yolk consumption rate (cm3 day-1) of S. stellaris embryos during the 7 developmental stages. Lines show mean ± SD (n = 8), while points show measurements from each individual. Dissimilar letters indicate statistical differences between stages (RM one-way ANOVA, P < 0.05). (B) Yolk consumption rate (cm3 day-1) of S. canicula embryos during the 7 developmental stages. Lines show mean ± SD (n = 8), while points show measurements from each individual. Dissimilar letters indicate statistical differences between stages (RM one-way ANOVA, P < 0.05). (C) Comparison of yolk consumption rate (% day-1) between S. stellaris (purple) and S. canicula (pink) embryos during 7 developmental stages. Values represent mean ± SD (n = 8). There was no statistically significant difference between species at any stage (Mann-Whitney U test, P < 0.05).

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3.3.11 Development Time (Embryo Age and The Duration of Each Developmental Stage)

It took longer for S. stellaris (Fig. 3.12A) to fully develop and hatch (27.25 ± 0.89 weeks) compared with S. canicula (24 ± 0.93 weeks) at the common rearing temperature of 15 °C (Fig. 3.12B). However, the relative amount of time spent in each stage was consistent across species as expressed as the percentage of each species total developmental time spent in each stage (Fig. 3.12C).

The amount of time (in weeks) that S. stellaris spent at each developmental stage is shown in Fig. 3.13A. Embryos spent the least amount of time in stage 2 (a week) and the longest developmental period in stage 6 (7.75 ± 0.89 weeks). A similar pattern was observed for S. canicula (Fig. 3.13B), which each species spending a similar proportion of their development in a given stage (Fig. 3.13C).

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Figure 3.12: Development time (age) to reach 7 developmental stages and hatch. (A) Development time (in weeks) for S. stellaris embryos to reach each developmental stage and hatch. Values represent mean ± SD (n = 8), whilst points show measurements from each individual. (B) Development time (in weeks) for S. canicula embryos to reach each developmental stage and hatch. Values represent mean ± SD (n = 8), whilst points show measurements from each individual. (C) Comparison of time spent in each developmental stage (as % of total developmental time) from lay to hatch for S. stellaris (purple) and S. canicula (pink). Values represent mean ± SD (n = 8). There was no statistically significant difference between species (Mann-Whitney U test, P < 0.05).

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Figure 3.13: Duration of development time of the 7 developmental stages. (A) Duration of development time (in weeks) spent in each of the 7 developmental stages for S. stellaris embryos. Lines show mean ± SD (n = 8), while points show measurements from each individual. Dissimilar letters indicate statistical differences between stages (RM one-way ANOVA, P < 0.05). (B) Duration of development time (in weeks) spent in each of the 7 developmental stages for S. canicula embryos. Lines show mean ± SD (n = 8), while points show measurements from each individual. Dissimilar letters indicate statistical differences between stages (RM one-way ANOVA, P < 0.05). (C) Comparison of duration of development time (as % of total time) between S. stellaris (purple) and S. canicula (pink) embryos at 7 developmental stages. Values represent mean ± SD (n = 8). There was no statistically significant difference between species (Mann-Whitney U test, P < 0.05).

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3.3.12 Hatching Process

The embryo positioned itself, immobilized inside the egg case, with half of its body curled to fit the limited space (Fig. 3.14A). Prior to hatching, it rotated such that the head was pointed toward the flat end opening of the egg case (Fig. 3.14B). Next, the tail struggled and pushed against the rigid, arched wall of the egg case, which forced the head through the flat end opening of the egg case (Fig. 3.14C).

Figure 3.14: The hatching process. (A): Embryo with curled body inside the egg case. (B) Embryo facing the flat end of the egg case. (C) Embryo tail pushed against arched wall of the egg case. (D) Embryo pushed its body through the flat end of the egg case. (E) Embryo swam out of the egg case.

The embryo slowly and gradually pushed from the inside of the egg case, struggling until most of the abdomen was successfully out of the egg case membrane (Fig. 3.14D). Once the abdomen had emerged from the egg case, the tip of the tail touched the surface of the arched wall end of the egg case to push the body forward enough that the embryo could then swim out through the egg case opening (Fig. 3.14E).

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The duration of the hatching process varied greatly between the observed individuals, ranging from a few minutes to more than one hour to completely escape the egg case. At hatch, determining the sex of S. stellaris hatchlings was possible because the male hatchlings had a pair of short claspers between the pelvic fins, whereas the females did not.

3.3.13 Effect of Air Exposure on S. stellaris Egg Cases with Jelly and without Jelly

Unfertilized S. stellaris egg cases which were filled with jelly were able to maintain their shape and size for 1-2 days when aerially exposed. In contrast, the egg cases without jelly, which were discarded from hatched individuals, retained their shape and size for only one hour before starting to shrink. Thus, S. stellaris egg cases without jelly had much faster dehydration rate (2.27 ± 0.35 % day-1) compared to the egg cases with jelly (0.29 ± 0.09 % day-1) (Fig. 3.15) suggesting that the jelly protects air exposed embryos by slowing dehydration/shrinking rate by ~7.8 times.

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Figure 3.15: Shrinking rate of S. stellaris egg cases (% in area h-1) with and without jelly. Values represent mean ± SD, while points show individual measurements. Asterisk indicates statistically significant effect of jelly on the rate of egg case shrinking (Mann-Whitney U test, P < 0.05).

3.4 DISCUSSION

Here we produced the first oviparous elasmobranch embryonic developmental scale that can be applied non-invasively, without specialized equipment, to help approximate embryo age, lay time and hatch time, whilst in the field. The 7 developmental stages were identified based on the greater spotted catshark, Scyliorhinus stellaris, and validated against the closely related elasmobranch species, S. canicula (the same species used in Ballard et al., 1993 embryonic elasmobranch developmental scale). More distantly related oviparous elasmobranch species, such as the tropical, brownbanded bamboo shark (Onimaru et al., 2018), and the batoid elasmobranch species, the

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clearnose skate (Luer et al., 2007) show similar key features during their embryonic development inside the egg cases, as presented here in our 7 key stages scale. Thus, we suggest it can be extended to other oviparous shark species with only slight modification.

The 7 specific developmental stages were chosen because they are recognizable by the naked eye, can be observed non-invasively (without the needing to remove embryo from the egg case), and also are ecologically and physiologically important in allowing the elasmobranch inside the egg case to survive in intertidal zone. Stage 1 was chosen as it did not have a visible embryo, while stage 2 had an embryo at the earliest stage of development. Stage 3 embryos had developed a tail and the embryo movement was nearly continuous. During stage 4, long gill filaments developed and respiratory process beyond cutaneous diffusion occurred. At stage 5, gill filaments shrunk and major changes in shape and body morphology occurred as the animal grew. The stage 6 embryo was characterized by the loss of fin-folds, and an increase in yolk mass transfer from the external yolk sac. The stage 7 embryo was fully developed, with a completely consumed external yolk and ready to hatch. We also were able to determine some unique features of the egg case and the protective jelly encasing the developing embryo that may contribute to survival in the harsh intertidal zone environment.

3.4.1 Early Embryogenesis

Although not visible with the naked eye, after fertilization, embryos at stage 1 undergo several important development processes. The rapid division of cells (cleavage) from one large cell (the zygote) into many smaller cells (called blastomeres) produce a three- dimensional cell mass cluster (the morula), which later develops into a hollow spherical cell mass with a fluid-filled cavity, the blastocoel (the blastula). During gastrulation (embryo during this process called a gastrula), the epiboly of blastoderm occurred, where the cells migrate to form germ layers (the outer layer of ectoderm, the inner layer of endoderm, and the middle layer between these two, called mesoderm), which will allow the later formation of tissues and organs as the embryo develops (Ballard et al.,

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1993). These changes are difficult to be observed with the naked eye, thus during stage 1, the embryo (the blastodisc) can only be seen as a small spot located on the surface of the yolk membrane as shown in Fig. 3.1. As gastrulation continues, more cells migrate into the interior of the embryo forming the primitive streak at the blastoderm’s midline (Ballard et al., 1993). The thickening of primitive streak gives rise to the elevation of the embryo on the surface of the yolk in stage 2 (Fig. 3.2).

3.4.2 Egg Case Structure and Function

The shape and morphology of elasmobranch egg cases are unique and diverse. Different oviparous elasmobranch species have egg cases with different shapes and morphological features, thus the elasmobranch egg case can be used for species identification (Compagno, 1984; 2001). There are many colour variations due to the catechol oxidation reaction (Koob, 1987). We found different colour variations of the same elasmobranch egg case species (for S. stellaris and S. canicula), hence egg case colour cannot be used for species identification.

Features of the S. stellaris egg case increase embryonic survival in the extreme habitats where they are laid. The tough egg case membrane acts as the primary protection for the developing embryo against marine predators and parasites (Cox and Koob, 1993; Harahush et al., 2007; Last and Stevens, 1994) and provides protection from seawater exposure during early development (stages 1-4) (Ballard et al., 1993). The egg cases are also used as camouflage as the egg case itself resembles blades of kelp and the tendrils are similar in form to kelp holdfasts (Compagno, 1984; Orton, 1926). The tendrils, which we found to be easily tangled, are sturdy, flexible, and have a spring-like structure which helps in securing egg cases underwater against the strong currents of the intertidal zone (Orton, 1926; Rodda, 2000).

The S. stellaris egg case shape also appears to help the hatchling escape during hatch (Fig. 3.14). The curvature of the arched end of the egg case may provide increased surface for the tail push against and force the embryo through the small, thin egg case

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opening. Similar interaction between the hatching process and the egg case has also been observed in the clearnose skate, Raja eglanteria (Luer and Gilbert, 1985), whose egg case has a similar morphology.

3.4.3 Jelly as Protection

The embryonic jelly provides secondary protection to the developing embryo with the egg case membrane being the primary. The jelly acts as a shock absorber, protecting the embryo from strong waves in the intertidal zone (Koob and Straus, 1998; Rodda and Seymour, 2008). The tougher outer layers provide support and sealing of the egg case membrane preventing seawater entry, while the centre of the jelly is softer and is thought to providing cushioning from wave action. Indeed, these layers are known to differ in carbohydrate composition (Koob and Straus, 1998).

We also found that egg cases containing jelly maintained their shape and size nearly 8 times longer than those without jelly during air exposure (Fig. 3.15). This suggests that the jelly prevents the egg case membrane from rapidly drying and shrinking when washed up on a beach or in shallow rockpools. By slowing down the dehydration rate of the egg case, the jelly protects the embryo during short term air exposure.

3.4.4 Opening of Seawater Slits

During the earliest developmental stages (stages 1-3), the egg yolk membrane of S. stellaris is thin and fragile. As the epiboly of blastoderm occurred during gastrulation, the formation of germ layers (ectoderm and mesendoderm) spreading around the yolk (Ballard et al., 1993) resulting the external yolk sac became thicker and tougher during stage 4, before the seawater slits open at the end of stage 4. This fragility of the yolk membrane during the early developmental stages has been reported in other elasmobranch species (Ballard et al., 1993; McLaughlin and Morissey, 2005; McLaughlin and O’Gower, 1971; Rodda and Seymour, 2008). Ballard et al. (1993)

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suggested that embryos directly exposed to seawater during early developmental stages could die due to bacterial exposure from seawater (Ballard et al., 1993).

At stage 4, (equivalent to stage 28 from Scyliorhinus canicula, Ballard et al., 1993), enzymes from the hatching gland gradually digest the jelly, starting with the soft central jelly and, then gradually working towards the edge of the egg case with its tougher texture, near to the seawater slits (Ballard et al., 1993). Once most of the tough jelly near the edge of the egg case has been digested, the mucous plugs degrade and the seawater slits open exposing the embryo to seawater. Similar observations had also been reported in the Port Jackson shark, Heterodontus portusjacksoni egg case (Rodda and Seymour, 2008).

In this study, by the end of stage 4, all four seawater slits were fully opened and the seawater could freely enter into the egg case. This confirms that S. stellaris is tolerant to seawater exposure by this stage, similar to previous descriptions for S. canicula and H. portusjacksoni (Ballard et al., 1993; Rodda and Seymour, 2008). Some studies initiated earlier seawater exposure for other elasmobranch species (Ballard et al., 1993), however when replicated in this study, S. stellaris survived for only a few weeks or months, with none surviving beyond 3 months if exposed to seawater before stage 4. This suggests that for this species, stage 4 is the earliest developmental stage that is suitable for seawater exposure.

3.4.5 Gills and Respiration

During early development (stages 1-3), while the egg case membrane is fully sealed, the jelly provides adequate medium for gas exchange across the body wall (Castro and Wourms, 1993; Hamlett and Wourms, 1984; Rodda, 2000; Rombough, 1998). At the end of stage 3, capillaries begin to develop on the surface of the large external yolk sac providing a surface area larger than the body wall for gaseous exchange (Hamlett and Wourms, 1984; Rombough, 1989).

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During stage 4, metabolic requirements begin to increase and the inside of the egg case can become hypoxic (Rodda, 2000). The internal gills extend out from the gill openings, becoming the thin walled external gill filaments (Hamlett et al., 1985; Rodda, 2000), which together with the degradation of the jelly provide increased access to oxygen. S. stellaris and S. canicula embryos spend many weeks in stage 4 (Fig. 3.13), where changes in gill morphology allow them to cope with the growing hypoxia inside the egg case and prepare them for the opening of the seawater slits at the end of this stage.

After the slits open and the inside of the egg case returns to normoxia, the length of external gill filaments gradually shrink during stage 5 and the external gill buds have completely disappeared at the beginning of stage 6. By this stage, buccal pumping of the embryo gill openings was clearly observable. The internal gills are completely formed in stage 6 and the gills are fully functional for respiration.

3.4.6 Fin and Body Morphology

Fin morphology changed during embryonic development with fins emerging and then regressing. During stage 4, the embryo was equipped with a pair of pectoral fins, a pair of pelvic fins and large, long dorsal and ventral fin-folds along the tail. As the embryo grew in size, the added surface area provided by the fin-folds may have improve fluid mixing of enzyme and jelly inside the egg case (Ballard et al., 1993; Rodda, 2000). Lastly, the fin-folds may be important in adding surface area for ventilation of seawater through all four fully opened seawater slits to supply fresh oxygenated seawater to the developing embryo.

In stage 5, the embryo has the greatest ADL (Fig. 3.10) and thus body and tail movements alone may facilitate adequate ventilation inside the egg case, thus the excessive fin-folds are reduced dramatically during this time. Some of these fin-folds extended outwards becoming fins, while most of the fin-folds gradually shrunk and completely disappeared by stage 6. At stage 6, the embryo was fully equipped with two

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dorsal fins, a pair of pectoral fins, a pair of pelvic fins, an anal fin as well as a large dorsal and ventral caudal fin which are typical of the adult shark. The large caudal fin will be used for forward propulsion (Lingham-Soliar, 2005a), the dorsal fins function as stabilizers (Lingham-Soliar, 2005b), and the flexible pectoral and pelvic fins help in steering while swimming and in holding on to the substrate during resting (Wilga and Lauder, 2000; 2001).

3.4.7 Yolk Consumption During Development

There was no visible yolk consumption by the embryo during the early developmental stages (stages 1-3) for both S. stellaris and S. canicula (Fig. 3.11). The external yolk membrane was thin and not fully developed, making the external yolk sac an ellipsoid (Ballard et al., 1993). The development of blood capillaries and thickening of external yolk sac membrane resulted in an apparent increase egg yolk volume during early development as the shape changed from ellipsoid to spheroid (Figs 3.8 and 3.9). By stage 3, the yolk stalk began to develop as the embryo grew larger and more energy was required to support development.

The yolk stalk was completely developed by stage 4, and yolk mass was transferred from the external yolk sac into the embryo’s internal yolk sac and digested, however changes in yolk volume were not appreciable visually at this stage (Ballard et al., 1993; Hamlett et al, 1993; Lechenault et al., 1993). This has been previously described for S. canicula (Ballard et al., 1993); the yolk mass starts to be transferred from external yolk sac to internal yolk sac at stage 31 in Ballard et al. (1993), which is equivalent to our stage 4. By stage 5, the decreased size of the external yolk sac was noticeable by the naked eye and yolk shrank most rapidly during stage 6 (Fig. 3.11). At stage 7, the yolk mass had been fully transferred from the external yolk sac into the embryo for growth, though some of the yolk was transferred into the internal yolk sac inside the body of embryo (Ballard et al., 1993; Hamlett et al., 1993; 2005; Lechenault et al., 1993). Once hatched, the embryo is able to utilize the yolk mass stored inside the internal yolk sac until it is successful in hunting prey (Lechenault et al., 1993).

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3.4.8 Conclusions

7 key stages of embryonic development were identified for S. stellaris that can be identified by the naked eye in living elasmobranch embryos. As elasmobranchs are sentinels for ecosystem health, and because embryonic life stages are particularly vulnerable, being able to monitor key developmental stages non-invasively is vital for eco-physiology and conservation studies. Our developmental stages are provided alongside the gold standard embryological key developed by Ballard et al. (1993), to provide in depth information on features that cannot be seen non-invasively. Our 7 key developmental stages scale can thus be applied non-invasively to living elasmobranch egg cases, without specialized equipment or additional fixation procedures. It is designed to be user-friendly to non-embryological researchers and the general public for development staging in the field. Thus, we hope that this will allow a wider audience to contribute towards elasmobranch conservation efforts and research.

Acknowledgements

We would like to thank Amy Callaghan and Native Marine Centre, UK for supplying S. stellaris egg cases, and Dr Bianka Grunow, Dr Timo Moritz and the Ozeaneum, Germany for supplying S. canicula egg cases. Dr John L. Fitzpatrick, Stockholm University, Sweden, Dr Kathy Hentges, University of Manchester, UK, Noraziani Shaari, Ben O’Neill and James Ducker are thanked for useful discussions, observations and for providing comments on earlier drafts of this paper. Authors would like to thank five anonymous reviewers for their helpful comments on the submitted manuscript.

Funding

Syafiq Musa is supported by the Ministry of Higher Education (KPT, Malaysia) and Universiti Kebangsaan Malaysia (UKM). Molly Czachur was a summer intern student from University of Bangor, UK supported by a University of Manchester Sustainability Summer Studentship. The project was funded by the Higher Education Innovation Fund

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through The University of Manchester’s Knowledge and Innovation hub for Environmental stability, and by the Ministry of Higher Education (KPT, Malaysia).

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Rosa, R., Baptista, M., Lopes, V. M., Pegado, M. R., Paula, J. R., Trübenbach, K., ... & Repolho, T. (2014). Early-life exposure to climate change impairs tropical shark survival. Proc. R. Soc. B, 281(1793), 20141738.

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SUPPORTING INFORMATION

All supplemental files can be found in the Appendix section of this PhD thesis.

Supplemental S1 File. Original photographs of Fig. 3.1 illustration. External features of the S. stellaris egg case at stage 1.

Supplemental S2 File. Original photographs of Fig. 3.2 illustrations. The inside of the S. stellaris egg case at stage 2.

Supplemental S3 File. Original photographs of Fig. 3.3 illustrations. The inside of the S. stellaris egg case at stage 3.

Supplemental S4 File. Original photographs of Fig. 3.4 illustrations. The inside of the S. stellaris egg case at stage 4.

Supplemental S5 File. Original photographs of Fig. 3.5 illustrations. The inside of the S. stellaris egg case at stage 5.

Supplemental S6 File. Original photographs of Fig. 3.6 illustrations. The inside of the S. stellaris egg case at stage 6.

Supplemental S7 File. Original photographs of Fig. 3.7 illustrations. The inside of the S. stellaris egg case at stage 7.

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CHAPTER IV

THE EFFECTS OF TEMPERATURE AND HYPOXIA ON GROWTH AND SURVIVAL OF EMBRYONIC SMALL-SPOTTED CATSHARK (Scyliorhinus canicula)

Syafiq M. Musa1,2, Holly A. Shiels1,*

1Faculty of Biology, Medicine and Health, The University of Manchester, 3.15d Core Technology Facility, 46 Grafton Street, Manchester M13 9NT, United Kingdom 2School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

*Corresponding author: Faculty of Biology, Medicine and Health, The University of Manchester, 3.15d Core Technology Facility, 46 Grafton Street, Manchester M13 9NT, United Kingdom. Tel: +44 161 275 5092. Email: [email protected]

This chapter has been prepared for submission to Conservation Physiology

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ABSTRACT

Elasmobranchs are important in keeping the balance of a healthy marine ecosystem, but are under worldwide threat due to human activities, like destructive fisheries and shark finning. Additionally, embryos of oviparous shallow water chondrichthyans may be further challenged during development by rising temperatures and falling dissolved oxygen levels concomitant with climate change in their intertidal rearing environments. However, the impact of climate change on survival and growth of oviparous elasmobranchs during early ontogeny is still poorly understood. This study investigated the effects of temperature and hypoxia, on growth and survival of embryos of the small spotted catshark, Scyliorhinus canicula. Our study revealed that rearing at warmer temperatures (20 °C) increased growth rate and yolk consumption, and decreased embryonic survival rate and body size of the hatchlings in comparison to rearing at lower temperatures. Under low oxygen conditions (50 % air saturation), S. canicula embryos took longer to hatch and had greater embryonic mortality. The synergistic effect of both high temperature and hypoxia resulted in the highest mortality rates of S. canicula embryos. Thus, this study gives some insight for the understanding of effects of future predicted climate change on elasmobranchs survival and growth during early ontogeny – a factor so far virtually neglected in the urgently needed efforts for the conservation of elasmobranch species.

4.1 INTRODUCTION

Living in coastal areas can be challenging for some marine ectotherms as they are forced to survive in an extreme environment (Chin et al., 2010; Diaz and Rosenberg, 1995; Pistevos et al., 2015). During low tide, some areas are very shallow or even emersed leading to warming. It is predicted that in 2100 the average global surface temperature will increase by about 5 °C (IPCC, 2001a, 2001b; Meehl et al., 2007). Warming reduces oxygen solubility in seawater (Farrell and Richards, 2009; Frölicher et al., 2009; Pörtner and Knust, 2007) which can be compounded by eutrophication from urban run-off leading to coastal hypoxia (low oxygen) and even anoxia (zero oxygen) (Bennett et al.,

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2001; Diaz and Rosenberg, 2008; Doney, 2010). Some marine organisms that live in the intertidal zone are already close to their thermal and oxygen tolerance limits, thus future ocean warming and hypoxia may impact on growth, developmental time and survival rate (Butler and Taylor, 1975; Diaz and Rosenberg, 2008; Farrell and Richards, 2009; Pörtner and Farrell, 2008; Pörtner and Knust, 2007).

Mobile invertebrates and pelagic fishes are able to move into deeper, cooler waters (Block et al., 2011; Wilson et al., 2001). But 40 % of elasmobranchs are sessile during embryonic and larval development as they live inside egg cases (mermaid’s purses) attached to rocks and seaweed in the intertidal zone deposited by their oviparous mother (Compagno, 1984; Dulvy and Reynolds, 1997; Hamlett and Koob, 1999). The sessile nature of development inside the egg case means that these animals cannot escape ambient conditions during development (Cox and Koob, 1993; Lucifora and García, 2004; Powter and Gladstone, 2008).

Elasmobranchs play an important role as the apex predators in the ocean, but they are vulnerable. Currently, about one quarter of all sharks worldwide are already threatened with extinction with many more species critically endangered (Dulvy et al., 2014; Field et al., 2009; Fowler et al., 2005). The reasons include overfishing, destructive fishing methods and shark finning (Baum et al., 2003; Baum and Myers, 2004; Myers et al., 2007; Stevens et al., 2000). Recovery of elasmobranch stocks is difficult due to a low rate of fecundity, slow growth and late sexually maturity (Carrier et al., 2004; Cortés, 2000; Field et al., 2009; Myers and Worm, 2005; Stevenson et al., 2007). For similar reasons, there is limited scope for genetic adaptation and phenotypic plasticity over the next 100 years where significant warming and hypoxia of intertidal zones are anticipated (Baum et al., 2003; Compagno, 1984). Embryonic development is a crucial and yet vulnerable period of life history which may be decisive for the survival rate of individuals and eventually species (Leonard et al., 1999; Pimentel et al., 2014). A loss of reduction in elasmobranch numbers is thought to have negative implications for the whole marine ecosystem (Heithaus et al., 2008; Reynolds et al., 2005; Stevens

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et al., 2000; Stevenson et al., 2007). Thus, it is important to study the impact of climate change on elasmobranch development, growth and survival.

In this study, we aim to investigate the impact of water temperatures and levels of dissolved oxygen on growth and survival of the small-spotted catshark, Scyliorhinus canicula embryos inside the egg case throughout development until hatching. S. canicula is a benthic oviparous shark species from the family Scyliorhinidae (Compagno, 1984; Serena, 2005). The maximum recorded total body length of an adult S. canicula is ~100 cm, but they rarely surpass 80 cm (Compagno, 1984; Serena, 2005). S. canicula egg cases are usually 4 cm in length and 2 cm in width, equipped with long tendrils at each corner for attachment on hard substrates or vegetation in coastal waters (Ballard et al., 1993; Mellinger et al., 1986; Musa et al., 2018; Serena, 2005). The egg cases can be found throughout the year with high abundance during spring and early summer (Compagno, 1984; Serena, 2005). Depending on water temperature, the embryonic incubation period is between 5 and 11 months (Compagno, 1984; Mellinger et al., 1986; Serena, 2005).

Here we measure growth pattern, average daily body length gain (ADL), specific growth rate, external yolk consumption rate, survival rate, developmental time and hatchling size in S. canicula reared under 4 different climatic conditions; normoxia 15 °C, normoxia 20 °C, hypoxia 15 °C and hypoxia 20 °C. We hypothesized that increased temperature would increase growth rate and decrease the incubation period of the embryos, whereas hypoxia would slow growth and development of the embryos.

4.2 MATERIALS AND METHODS

4.2.1 Supply of Egg Cases and Care of Animals

Scyliorhinus canicula egg cases were laid in captivity at the Ozeaneum, Stralsund, Germany, in normoxic seawater maintained at 15 °C. A total of 81 egg cases were transported to the University of Manchester, Manchester, UK in several batches

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between March 2016 and February 2017. On arrival, the egg cases were dipped into dechlorinated tap water for several minutes to remove marine microorganisms that may be attached to the egg cases before being transferred into quarantine tanks containing aerated saltwater (35 ppt salinity, > 95 % air saturation, with 15 °C water temperature). For individual identification and monitoring, each S. canicula egg case was tagged using coloured plastic tags with numbers fixed on the tendrils of the egg case. Candling (see Musa et al., 2018) was used to monitor the health condition of S. canicula embryos in egg cases.

4.2.2 Experiment (Biospheres) Tanks and Mimic of Different Climatic Conditions

After two days quarantine, the S. canicula egg cases were transferred into 1 of 4 biosphere tanks (55 L). Two biosphere tanks were fully aerated to create a normoxic environment (> 95 % air saturation) at either 15 °C (N15/control) or 20 °C (N20). The other two biosphere tanks were treated with special compressed gas from BOC containing 11 % oxygen, 0.04 % carbon dioxide, balanced with nitrogen gas to create a hypoxic environment (~50 % air saturation) with water temperatures at 15 °C (H15) or 20 °C (H20).

Each biosphere tank was fully sealed and equipped with a filtration system, creating circulation inside the tank and mixing the biosphere gas into the water. The egg cases were hung vertically throughout the incubation period. Saltwater changes were done 3 times a week at which time water temperature, dissolved oxygen, pH, salinity, ammonia, nitrite, and nitrate were monitored to ensure good water quality.

4.2.3 Developmental Staging

Musa et al. (2018) identified 7 key developmental stages of oviparous elasmobranch development inside the egg case (Fig. 4.1A) which can be clearly identifiable by the naked eye through the egg case using non-invasive candling. In the current study we monitored growth and yolk consumption across all stages for embryos in each of the 4

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biospheres. We were also particularly interested in certain stages of development due to the appearance of disappearance of certain morphological and physiological features. For example, stage 5 of oviparous elasmobranch development is characterized by the shortening of external gill filaments (Fig. 4.1B), while stage 6 of development is associated by a rapid decreased in external yolk sac size (Fig. 4.1C). A timeline of these stages is provided in Fig. 4.2. Development can also be broadly grouped into early and late stages of development. The early stages are when the seawater slits of the egg cases are still fully sealed, and the later stages of development are when the seawater slits are opened and seawater can freely enter the egg case. Based on our 7 key stages (Musa et al., 2018), the early stages lie between stage 1 and the midpoint of stage 4, while later stages lie between the end of stage 4 and the end of stage 7 (Fig. 4.2). S. canicula embryo mortality during development was classified as occurring in either the early or later stage.

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Figure 4.1: Oviparous elasmobranch development inside the egg case in 7 key stages. (A) Seven developmental key stages. Red circles showing developmental stages 5 and 6 (B) Stage 5 of development. (C) Stage 6 of development. Black lines show scale of 1 cm. Figures are adapted from Musa et al. (2018).

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Figure 4.2: Summary of specific developmental stages that were investigated in this study based on the 7 key stages of development laid out in Musa et al. (2018). The beginning of each developmental stage is demarked with a vertical line. Blue rectangle is the duration between stage 5 and stage 6 that was used to study the S. canicula growth slopes. Purple square is the duration of stage 6 embryos used to study the S. canicula yolk consumption slopes. Red rectangle is the duration of early stages, while green rectangle is the duration of later stages that were both used for survival rate study.

4.2.4 Growth Pattern and Growth Rate

Egg cases were taken out from the biosphere tanks and photographed (Canon PowerShot G16). Total body length (TL) of embryos inside the egg cases were measured from these photographs using ImageJ (http://imagej.nih.gov/ij). Mean ± standard deviation (SD) of total body length was calculated weekly. Growth rate for each individual were determined. As the duration between stage 5 and stage 6 of development showed the

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highest growth rates (Musa et al., 2018), additionally growth rate for each embryo at this specific developmental stage was calculated. The average daily body length gain (ADL, in cm day-1) was calculated as, ADL = (final TL – initial TL)/development time (in days). Specific growth rate (SGR, in % day-1) was calculated as, SGR = [(Ln final TL – Ln initial TL)/development time (in days)] × 100.

4.2.5 Yolk Consumption

Digital photographs were also used to calculate the volume of the external yolk sac for each embryo weekly by measuring diameter length, width and depth of the external yolk sac (in cm) with ImageJ software (http://imagej.nih.gov/ij). The volume (V) of the external yolk sac was calculated as, V = (4/3)π*L*W*D; where L, W and D are (1/2)length, (1/2)width and (1/2)depth of external yolk sac respectively. The mean ± standard deviation (SD) of external yolk sac volume was calculated weekly and the yolk consumption rate for each individual were determined. As S. canicula embryos have the fastest yolk consumption rate at stage 6 (Musa et al., 2018), the yolk consumption rate of each embryo at stage 6 was also been calculated. Yolk consumption (YC) rate (cm3 day-1) was calculated as, YC = (initial V – final V)/development time (in days).

4.2.6 Survival Rate

Mortality was closely monitored and recorded. A dead embryo or a rotten egg case was detected from its lack of embryo movement or/and rotten fish-like odour. The deaths of embryos were classified into early stages and later stages as described earlier. Survival rate (in percentage) of S. canicula embryos across different climatic conditions was calculated as, survival rate = (total number of survived individuals from the same climatic condition/total number of egg cases from the same climatic condition) × 100.

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4.2.7 Development Time and Hatchling Size

Duration of development time was calculated by summing the development time (in week) from lay until hatch. Hatchlings size was calculated by measuring the total body length from digital photographs taken directly after hatching with ImageJ (http://imagej.nih.gov/ij).

4.2.8 Statistical Analysis

Correlations, R2 coefficient and linear regressions for embryo growth pattern and yolk consumption from different climatic conditions were calculated. One-way analysis of variance (ANOVA) was performed to determine any statistical significance in different climatic conditions, followed by Tukey’s multiple comparisons post-hoc test (P < 0.05). Log-rank (Mantel-Cox) test was calculated to compare the survival distributions of S. canicula embryos under different treatments. All statistical analysis was performed using GraphPad Prism 7 (https://www.graphpad.com/scientific-software/prism/).

4.3 RESULTS

4.3.1 Growth of S. canicula Embryos

Embryonic growth of S. canicula showed a linear pattern with different growth slopes across different treatments (Fig. 4.3A). Both average daily body length gain (ADL, as cm day-1) and specific growth rate (SGR, as % day-1) of S. canicula embryos were faster at 20 °C but were unaffected by dissolved oxygen levels (Fig. 4.3B,C). Interestingly, at stage 5 and stage 6 where S. canicula embryos had the fastest growth rate, ADL results showing all treatments were not different to control (N15) treatment (Fig. 4.3D).

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Figure 4.3: Growth of S. canicula embryos developed under different climatic conditions. (A) Growth pattern. Each point is a total body length mean ± SD [green, normoxia at 15 °C (control/N15), n = 17, r = 0.9942, R2 = 0.9884; yellow, normoxia at 20 °C (N20), n = 18, r = 0.9863, R2 = 0.9727; blue, hypoxia at 15 °C (H15), n = 12, r = 0.9941, R2 = 0.9882; red, hypoxia at 20 °C (H20), n = 13, r = 0.9885, R2 = 0.9771]. (B) Average daily body length gain (ADL). (C) Specific growth rate (SGR). (D) ADL at embryonic developmental stages 5 and 6. Columns show mean ± SD (N15, n = 17; N20, n = 18; H15, n = 12; H20, n = 13), while points show measurements from each individual. Dissimilar letters indicate statistical differences between treatments (ordinary one-way ANOVA, P < 0.05).

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4.3.2 Yolk Consumption of S. canicula Embryos

Yolk consumption of S. canicula embryos showed a linear pattern with different yolk consumption slopes across different climatic conditions (Fig. 4.4A). Yolk consumption rates were faster at 20 °C with no significant difference was found in embryos developed under different oxygen levels (Fig. 4.4B). At stage 6, where S. canicula embryos had the fastest yolk consumption rate during the embryonic development showing similar pattern to Fig. 4.4B, however embryos developing under hypoxia at 20 °C (H20) treatment showed no significant difference to control (N15) treatment (Fig. 4.4C).

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Figure 4.4: Yolk consumption of S. canicula embryos developed under different climatic conditions. (A) Yolk volume. Each point is a yolk volume mean ± SD [green, normoxia at 15 °C (control/N15), n = 17, r = -0.966, R2 = 0.9332; yellow, normoxia at 20 °C (N20), n = 18, r = -0.9813, R2 = 0.9629; blue, hypoxia at 15 °C (H15), n = 12, r = -0.959, R2 = 0.9197; red, hypoxia at 20 °C (H20), n = 13, r = -0.9736, R2 = 0.9478]. (B) Yolk consumption rate (YC). (C) YC at embryonic developmental stage 6. Columns show mean ± SD (N15, n = 17; N20, n = 18; H15, n = 12; H20, n = 13), while points show measurements from each individual. Dissimilar letters indicate statistical differences between treatments (ordinary one-way ANOVA, P < 0.05).

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4.3.3 Survival Rate

Survival rate of S. canicula embryos reared under normoxia at 15 °C (control/N15) was 100 % (n = 20), while 85.71 % (n = 21) for embryos reared under normoxia at 20 °C (N20) with 3 embryos dying in the early developmental stages (Fig. 4.5). Under hypoxia at 15 °C (H15), survival rate was 63.16 % (n = 19), with 4 embryos dying in the early stages and 3 deaths at later stages (Fig. 4.5). Hypoxia at 20 °C (H20) had the lowest survival rate of 61.9 % (n = 21) with 6 embryos dying at early stages and 2 deaths at later stages (Fig. 4.5).

Figure 4.5: Survival rate (SR) of S. canicula embryos developed under different climatic conditions. N15, SR = 100 %, n = 20; N20, SR = 85.71 %, n = 21, 3 embryos died at early stages; H15, SR = 63.16 %, n = 19, 4 embryos died at early stages, 3 deaths at later stages; H20, SR = 61.9 %, n = 21, 6 embryos died at early stages, 2 deaths at later stages. Dissimilar letters indicate statistical differences between treatments (Log- rank (Mantel-Cox) test, P < 0.05).

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4.3.4 Development Time and Hatchling Size

Embryos developed under hypoxia at 15 °C (H15) took the longest time to hatch with a mean incubation period of 25.92 ± 0.9 weeks, whilst individuals reared under normoxia at 20 °C (N20) had the fastest development time at 15.44 ± 1.29 weeks (Fig. 4.6). Individuals reared at 15 °C had larger body sizes at hatch, whilst those reared at 20 °C had smaller body sizes at hatch (Fig. 4.7). Dissolved oxygen levels during rearing did not affect hatchling size (Fig. 4.7).

Figure 4.6: Development time (age) of S. canicula embryos under different climatic conditions. Bars show mean ± SD (N15, n = 17; N20, n = 18; H15, n = 12; H20, n = 13), while points show measurements from each individual. Dissimilar letters indicate statistical differences between treatments (ordinary one-way ANOVA, P < 0.05).

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Figure 4.7: Hatchling size of S. canicula embryos under different climatic conditions. Columns show mean ± SD (N15, n = 17; N20, n = 18; H15, n = 12; H20, n = 13), while points show measurements from each individual. Dissimilar letters indicate statistical differences between treatments (ordinary one-way ANOVA, P < 0.05).

4.4 DISCUSSION

Embryonic development is a crucial part of an animal’s life-cycle. Life in coastal areas can be environmentally challenging, especially for sessile organisms (Hoegh-Guldberg,

1999; Leonard et al., 1999; Pimentel et al., 2014). With more anthropogenic CO2 been released into the atmosphere, ocean warming will increase, forcing marine organisms to endure temperatures that may negatively affect their growth and survival (Di Santo, 2015; Diaz and Rosenberg, 2008; Pistevos et al., 2015). Thermal challenges for intertidal organisms are compound by coastal hypoxia which is on the rise in populated coastal areas due to nutrient loading from human populations (Bennett et al., 2001; Diaz and Rosenberg, 2008; Hoegh-Guldberg, 1999). As elasmobranchs are vulnerable with about a quarter of all sharks critically endangered and threatened with extinction (Fowler et al., 2005; Myers et al., 2007; Stevens et al., 2000), we wanted to understand the separate and combined effects of warm temperature and low oxygen on development of a ubiquitous oviparous shark, the small-spotted catshark, S. canicula.

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4.4.1 Growth Rate

Different climatic conditions affected embryonic growth of S. canicula during incubation period. Based on results from both ADL and specific growth rate (SGR) studies, a similar pattern was noticed as embryos developed at 20 °C had faster growth rates, compared to individuals developed at 15 °C (Fig 4.3B,C). These results suggest that future ocean warming (+ 5 °C) will increase the growth rate of S. canicula during embryonic development. Similar result has been reported on embryonic growth of tropical brownbanded bamboo shark, Chiloscyllium punctatum, where individuals exposed to higher temperature showed faster growth rates (Rosa et al., 2014).

Oxygen is the key element to support life, thus animals rely on the availability of ambient oxygen in the environment to support their biological performances (Gray et al., 2002; Oschmann, 1993). Previous studies reported that organisms exposed to oxygen reduced environments showed retarded growth (Mallya, 2007; Weber and Kramer, 1983). However, S. canicula embryos that developed under hypoxic conditions had steady growth rates at both temperatures compared to individuals developed under normoxia (Fig. 4.3B,C). This result suggests that S. canicula embryos may have shift their energy production from aerobic to anaerobic pathways when reared under hypoxic conditions in order to support embryonic growth (Nilsson and Östlund-Nilsson, 2008; Routley et al., 2002; Wise et al., 1998). Comparing S. canicula to other elasmobranchs, this species was known for their well ability to cope with severe hypoxic condition, thus the effects of hypoxia on growth may has been countered (Diez and Davenport, 1987).

Further analysis has been done to calculate ADL at specific developmental stages, which were between stages 5 and 6, where S. canicula embryos had the greatest growth rate value under normal condition (Musa et al., 2018). Interestingly, at stages 5 and 6, ADL results revealed that embryos developed at elevated temperature (20 °C) were not different from control (N15) treatment (Fig. 4.3D). The embryos were expected to have higher growth rate at higher temperature (as shown earlier in Fig. 4.3B,C), but as the measurements were taken at a developmental stage where S. canicula

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embryos had the fastest growth (thus having higher metabolism at this stage), therefore we speculate that the animals could not cope with this high metabolic demands at a given temperature. This finding suggests that at temperature 20 °C, S. canicula embryonic growth was not aligned with the elevated temperature (therefore, begin to show a reducing growth rate), suggesting that 20 °C may be near to Pejus temperature

(Tp) (Pörtner et al., 2017). Oxygen- and capacity-limited thermal tolerance (OCLTT) hypothesis suggests that the metabolic performance of an ectothermic animal can optimally function at a specific range of temperature, exceeding this range (Pejus temperatures, Tp of either side of cold or warm) will results in degraded biological fitness performances, e.g. growth (Pörtner et al., 2017; Pörtner and Farrell, 2008).

4.4.2 Yolk Consumption

Our study revealed that predicted future climate change affected the rate of yolk consumption of S. canicula embryos. Embryos developed at 20 °C had faster yolk consumption rate, compared to individuals developed at 15 °C (Fig. 4.4B). Similar result has been reported on tropical brownbanded bamboo shark, Chiloscyllium punctatum, where embryos developed at elevated temperature showed higher yolk consumption rates (Rosa et al., 2014). These results suggest that under future ocean warming (+ 5 °C) the rate of yolk consumption of S. canicula embryos increase. As growth rate increased with the increase in temperature (Fig. 4.3), more yolk was needed to be consumed to support the fast growth and high metabolism (Lechenault et al., 1993; Rodda and Seymour, 2008; Rosa et al., 2014). During the embryonic development of oviparous elasmobranch, the yolk is the only source of energy for growth, metabolism and development (Ballard et al., 1993; Harahush et al., 2007; Lechenault et al., 1993; Luer et al., 2007; Rodda, 2000).

Again, further analysis has been carried out to calculate the yolk consumption slopes at stage 6, where S. canicula embryos had the fastest yolk consumption rates under normal condition (Musa et al., 2018). Surprisingly, even though at warmer temperature, the yolk consumption rates of stage 6 embryos developed under hypoxic

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condition at 20 °C (H20) was not significantly different to embryos developing under normoxia at 15 °C (N15/control) (Fig. 4.4C). This result suggests that hypoxia does affects the yolk consumption rate of embryos, however the low dissolved oxygen level (11 % oxygen, which is equal to ~50 % of air saturation) that was used in this study may inadequate to strongly reveal the true impacts of hypoxia on yolk consumption rate of S. canicula during embryonic development. Previous study shown that S. canicula embryos has a strong hypoxia tolerance, thus were able to counteract the oxygen reduced condition (Diez and Davenport, 1987).

4.4.3 Survival of S. canicula Under Future Ocean Warming and Hypoxia

Early ontogeny of an organism has always been considered as the most critical period of life and as the determination of survivability of an organism living in the extreme environments (Leonard et al., 1999; Pimentel et al., 2014). No mortality of S. canicula embryos developed under normoxic condition at 15 °C (N15/control) was recorded during the study been conducted (Fig. 4.5). The survival rate of S. canicula embryos was decreased under normoxia at 20 °C (N20) (Fig. 4.5). A similar trend was reported on tropical brownbanded bamboo shark, Chiloscyllium punctatum (Rosa et al., 2014) and little skate, Leucoraja erinacea (Di Santo, 2015). This result suggests that the survival rate of oviparous elasmobranchs during early ontogeny is negatively impacted by the predicted temperature of future ocean warming (+ 5 °C). Oxygen is an important element to support life and is the key of hatching success of an organism (Davis, 1975; Jones and Reynolds, 1999; Keckeis et al., 1996; Wieland et al., 1994). Previous studies reported hypoxia negatively affected mortality rate (Keckeis et al., 1996; Weber and Kramer, 1983). Our study showed that under oxygen reduced conditions, the survival rate of S. canicula dropped to ~60 % (Fig. 4.5B). The highest mortality rate of S. canicula embryos at early stages was recorded from embryos developed under hypoxia at 20 °C (H20) (Fig. 4.5). These results suggest that under future anthropogenic climate change, the synergistic effect of both ocean warming and hypoxia making the condition even worse by increasing the mortality rate of the elasmobranch during early embryonic development.

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Based on the 7 key stages (Musa et al., 2018), the early stages (between stage 1 and mid of stage 4) were considered as a crucial period of development, where the egg cases were fully sealed from seawater exposure, the membrane of the external yolk sac is not fully developed, thin and fragile, embryos were at early formed, small in size and are vulnerable to seawater exposure, environmental changes and bacterial contamination (Ballard et al., 1993; Mellinger et al., 1986). On the other hand, during later stages (between end of stage 4 and stage 7), the seawater slits of the egg cases were fully opened, the membrane of the external yolk sac has been fully developed, the embryos were fully exposed to seawater, have a much larger body size and actively moved inside the egg cases and developing towards hatching (Musa et al., 2018). An interesting pattern was noticed, where under normoxic condition (at 20 °C), the death of S. canicula embryos only occurred at the early developmental stages, while under hypoxic conditions (at both temperatures), the embryo died at both early and later developmental stages (Fig. 4.5). Our result showed that even at later developmental stages, where the embryos have developed into large in size, actively moving and reaching hatching stage, the deaths of S. canicula embryos were still occurred at the experimental hypoxic condition (11 % oxygen, which is equal to ~50 % of air saturation). Even though, S. canicula is a catshark species that are known for their ability to tolerate oxygen reduced condition and long exposure of anoxia (up to 12 hours anoxic exposure; Diez and Davenport, 1987), these results suggest that oxygen is essential requirement for life of an organism, especially during the crucial period of early ontogeny even for a hypoxia tolerance species (Davis, 1975; Keckeis et al., 1996).

4.4.4 Development Time and Hatchling Size

Our results showed that S. canicula embryos developed under hypoxia at 15 °C (H15) took the longest incubation period (Fig. 4.6). This result suggests that in the natural habitat, the oviparous elasmobranch embryos will be trapped longer inside the sessile egg cases under cold, hypoxic waters. This may lead to other negative implication towards the survival of the living egg cases, as the longer the incubation period of the embryos taken to fully develop inside the sessile egg cases, the longer the egg case been

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exposed to predation (Cox and Koob, 1993; Lucifora and García, 2004; Powter and Gladstone, 2008). Furthermore, our survival rate study results suggest that the embryos developing under hypoxic condition had also been exposed to higher risk of death during whole incubation period (at both early and later developmental stages) (Fig. 4.5).

Based on our hatchling size study, S. canicula embryos developing at 20 °C produced smaller hatchlings at hatch (Fig. 4.7). Similar evidences have been reported in other ectotherms (Angilletta et al., 2004; Forster et al., 2011, 2012). This finding suggests that, under future climate change, the elasmobranch egg cases develop under future ocean warming (+ 5 °C) will produce hatchlings with smaller body size and this may affect the swimming performance of the hatchlings (Bainbridge, 1958; Forster et al., 2012; Videler and Wardle, 1991). Our yolk consumption rate study showed that embryos developed at warmer waters had the fastest rate of yolk consumption (Fig. 4.4B), thus we speculate that all of the yolk mass from the external yolk sac has been fully consumed during development inside the egg cases, which includes the small amount of yolk mass that has been transferred into the internal yolk sac (that functioning as a yolk storage for hatchling) inside the body of embryos prior to hatch (Ballard et al., 1993; Lechenault et al., 1993; Rosa et al., 2014). Due to this, the starving hatchlings developed under warmer water are required to have a great swimming performance to swim against the strong current into deeper water to immediately searching for prey to support life (Blaxter and Staines, 1971). As the hatchlings produced under warmer water are smaller, this may affect their prey catching performance and decrease in range of food item availability, thus the survival in the harsh environment (Ferry-Graham, 1998; Juanes and Conover, 1994; Mittelbach, 1981; Sempeski and Gaudin, 1996). Besides that, smaller body size of hatchlings may also make them become an easy prey for larger marine predators (Scharf et al., 2000).

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4.4.5 Conclusions and Conservation Implications

Previous studies reported many marine ectotherms were negatively impacted by climate change and worse condition is predicted in the near future (Chin et al., 2010; Diaz and Rosenberg, 1995; 2008; Hoegh-Guldberg, 1999). Little is known about the effect of these human induced environmental changes on elasmobranchs survival and growth, especially during the crucial period of early ontogeny. Our data from this study suggests that future ocean warming (+ 5 °C) and hypoxic condition will negatively impact the survivability and growth of future oviparous shark population. Increase in water temperature affects oviparous elasmobranchs during early ontogeny by increase the growth and yolk consumption rates, decrease the survival rate of the embryos and decrease the hatchling size. In oxygen reduced condition, the survival rate of oviparous elasmobranchs is negatively affected and the embryos have significantly longer development time, trap inside the sessile egg cases. Whereas, the combination of both ocean warming and hypoxia create the worst environment for the elasmobranch egg cases development as the highest mortality rate were recorded from this treatment. We hope that this study will help to provide us more information to add on the available data from previous studies in a better understanding on the impacts of future climate change on elasmobranchs survivability, especially for sharks. We are strongly believe that more efforts need to be done on conservation work to save more elasmobranch species from extinction and to increase more public awareness on the importance of this apex predators of the ocean and its role in keeping the marine ecosystems in balance.

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Acknowledgements

We would like to thank the Ozeaneum, Germany for supplying S. canicula egg cases. Dr John Fitzpatrick (Stockholm University, Sweden), Dr Bianka Grunow (Leibniz Institute for Farm Animal Biology, Germany), and Dr Timo Moritz (Friedrich-Schiller- Universität Jena, Germany) are thanked for their thoughtful discussions on this research project. Authors also would like to thank Molly Czachur for her help on the biospheres and experimental set up and the Shiels Laboratory, University of Manchester for their useful discussions and help on the project, especially Miriam Fenkes for helping the authors on shark tagging for individual identification.

Funding

S.M.M. is supported by the Ministry of Higher Education (KPT, Malaysia) and Universiti Kebangsaan Malaysia (UKM). The project was funded by the Higher Education Innovation Fund through The University of Manchester’s Knowledge and Innovation hub for Environmental stability, and by the Ministry of Higher Education (KPT, Malaysia).

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Oschmann, W. (1993). Environmental oxygen fluctuations and the adaptive response of marine benthic organisms. Journal of the Geological Society, 150(1), 187-191.

Pimentel, M. S., Faleiro, F., Dionísio, G., Repolho, T., Pousão, P., Machado, J., & Rosa, R. (2014). Defective skeletogenesis and oversized otoliths in fish early stages in a changing ocean. Journal of Experimental Biology, jeb-092635.

Pistevos, J. C., Nagelkerken, I., Rossi, T., Olmos, M., & Connell, S. D. (2015). Ocean acidification and global warming impair shark hunting behaviour and growth. Scientific reports, 5, 16293.

Pörtner, H. O., Bock, C., & Mark, F. C. (2017). Oxygen-and capacity-limited thermal tolerance: bridging ecology and physiology. Journal of Experimental Biology, 220(15), 2685-2696.

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Pörtner, H. O., & Knust, R. (2007). Climate change affects marine fishes through the oxygen limitation of thermal tolerance. science, 315(5808), 95-97.

Powter, D. M., & Gladstone, W. (2008). Embryonic mortality and predation on egg capsules of the Port Jackson shark Heterodontus portusjacksoni (Meyer). Journal of Fish Biology, 72(3), 573-584.

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Rodda, K. R., & Seymour, R. S. (2008). Functional morphology of embryonic development in the Port Jackson shark Heterodontus portusjacksoni (Meyer). Journal of Fish Biology, 72(4), 961-984.

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CHAPTER V

THE EFFECTS OF OCEAN WARMING AND HYPOXIA ON METABOLISM OF SMALL-SPOTTED CATSHARK, Scyliorhinus canicula DURING EMBRYONIC DEVELOPMENT

Syafiq M. Musa1,2, James Ducker1,3 and Holly A. Shiels1,*

1Faculty of Biology, Medicine and Health, The University of Manchester, 3.15d Core Technology Facility, 46 Grafton Street, Manchester M13 9NT, UK 2School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia 3School of Biological and Marine Sciences, University of Plymouth, Drake Circus, Plymouth PL4 8AA, UK

*Author for correspondence ([email protected])

This chapter has been prepared for submission to Journal of Experimental Biology

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ABSTRACT

Anthropogenic climate change is increasing the temperature of marine ecosystems as well as the extent and frequency of low oxygen (hypoxic) zones. Understanding the impact of these changes on metabolism of sessile embryonic animals that develop in coastal areas is particularly important as early life stages are known to be vulnerable. In this study, we used intermittent flow respirometry to measure routine and maximum metabolic rate (RMR and MMR respectively) in small-spotted catsharks, Scyliorhinus canicula, during embryonic development inside sessile egg cases under 1 of 4 environmental conditions: (1) normoxia (100 % air saturated saltwater, 35 ppt) at 15 °C (N15); (2) normoxia at 20 °C (N20); (3) hypoxia (50 % air saturated saltwater, 35 ppt) at 15 °C (H15); (4) hypoxia at 20 °C (H20). Once hatched, RMR and MMR of the S. canicula hatchlings were recorded, first under their development conditions (i.e. N15, N20, H15 or H20) and secondly after one week living under ‘common garden’ conditions at 15 °C and normoxia (N15). Our results show that metabolic rate is depressed by hypoxia at all developmental stages. However, in normoxic embryos, warming elevates RMR and MMR across developmental stages. In contrast, developmental hypoxia lowers RMR and MMR than those developed under normoxic with similar oxygen consumption values at 15 and 20 °C. Importantly, 1-week in the ‘common garden’ environment normalized oxygen consumption across the different developmental groups. This suggests a high degree of metabolic plasticity for this intertidal catshark.

5.1 INTRODUCTION

Oceans cover over 70 % of the world’s surface and contain the greatest natural diversity of life on Earth (Alley et al., 2003). However, human activity is threatening marine ecosystems through the overexploitation of resources, introduction of invasive species, habitat destruction and degradation, human-induced climate change and pollution (Doney, 2010; Griffith et al., 2012; Halpern et al., 2008; Mora et al., 2013; Myers and Worm, 2003). The burning of fossil fuels has led to an elevation in atmospheric carbon

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dioxide (CO2), resulting in a rise in global temperatures (Cox et al, 2000; Washington and Meehl, 1989). Climate models suggest that carbon levels will reach up to 1100 parts per million (ppm) by the year 2100, decreasing ocean pH and causing warming ~5 °C (Metz, 2001; Van Vuuren et al., 2011).

Rising temperatures reduced dissolved oxygen levels in the oceans, termed hypoxia. This physical process is exacerbated by human activities such as agriculture and urban run-off, which cause nutrient loading and eutrophication in coastal areas (Bennett et al., 2001; Doney, 2010; Schmidtko et al., 2017). Indeed, the duration, frequency and severity of coastal hypoxic events has increased across the planet in recent years (Diaz and Rosenberg, 2008; Melzner et al., 2013). Oxygen availability is a key driver of animal physiology, behaviour and ecology and reduced oxygen availability negatively impacts fish, affecting their growth (Chabot and Dutil, 1999), sex ratio (Thomas and Rahman, 2011), predator-prey relationships (Domenici et al., 2007), swimming activity (Killen et al., 2012), niche utilization (Chapman and McKenzie, 2009), and embryonic development (Robertson et al., 2014).

The co-occurrence of lower oxygen availability and increasing temperatures may be particularly challenging for fish: warmer temperatures increase metabolic rate while reducing oxygen availability in the water (Levin and Breitburg, 2015; McBryan et al., 2016; Munday et al., 2009; Nilsson et al., 2010; Schurmann and Steffensen, 1992). Many studies have investigated the effect of temperature and dissolved gasses on metabolic rate in fishes in an attempt to understand how physiology is limited by such abiotic factors. However, evaluating the synergistic impacts of multiple stressors in defining the physiological vulnerability of species to environmental change is not straightforward. Many recent studies have focused on understanding the effect of the environment on an animal’s aerobic scope (AS), defined as the difference between standard/resting/routine metabolic rate (RMR), and active/maximum metabolic rate (MMR) (Fry and Hart, 1948; Pörtner and Farrell, 2008). Aerobic scope is thought to represent the energy an organism has to invest in non-essential functions, such as growth, migration and reproduction, and should thus be intrinsically related to

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biological fitness (Claireaux and Lefrancois, 2007; Pörtner and Knust, 2007). This conceptual framework has provided mix results with some studies showing a temperature-induced reduction in aerobic scope leading to a reduction in other related performance measures (Eliason et al., 2011; Pörtner and Knust, 2007) while others show that reduced aerobic scope has little effect on other performance measures such as growth (Gräns et al., 2014). Surprisingly, very few studies have investigated these relationships in embryonic animals. This is surprising as early life-stages are particularly vulnerable to environmental changes and recent epigenetic studies show that the environmental conditions animals experience during development can have lasting and in some cases, transgenerational effects on latter life and/or offspring fitness (Hicken et al., 2011; Pankhurst and Munday, 2011; Scott and Johnston, 2012).

In this study we have investigated the effect of different environments on AS, MMR and RMR of the small-spotted catshark, Scyliorhinus canicula, during embryonic development and within a week post-hatch. The small-spotted catshark (also known as the lesser-spotted catshark or dogfish) is a benthic and oviparous species, which develops inside egg cases (mermaid’s purses) in coastal waters of the Northeast Atlantic and Mediterranean (Compagno, 1984; Ellis and Shackley, 1997; Ellis et al., 2005; Gibson et al., 2008; Serena, 2005). The sessile eggs are found in the coastal intertidal zone, where they are exposed to changeable and often hostile ambient conditions (Ballard et al., 1993; Di Santo, 2016; Diaz and Rosenberg, 2008; Mellinger et al., 1986; Serena, 2005). Nevertheless, rising ocean temperatures and coastal hypoxia presents a threat to embryonic survival as these abiotic factors are known to alter metabolic processes and energy expenditure during development (Butler and Taylor, 1975; Di Santo et al., 2016; Nilsson and Östlund-Nilsson, 2008).

Elasmobranchs (sharks, skates and rays) are ecologically important species as they maintain the balance of prey populations in marine ecosystems from the arctic to the tropics (Heupel et al., 2014; Roff et al., 2016). However, elasmobranchs are particularly vulnerable to environmental changes due to low fecundity rates, slow growth as well as delayed sexual maturity (Carrier et al., 2012; Sims, 2010; Stevens et

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al., 2000). They also face pressure from overfishing, by-catch, marine pollution, and shark-finning resulting in a quarter of all elasmobranch populations being threatened by extinction (Dulvy et al., 2014; Field et al., 2009; Fowler et al., 2005; Simpfendorfer and Dulvy, 2017). This is the lowest fraction of safe species of all extant vertebrates (Dulvy et al., 2014). Metabolism underlies the physiological effectiveness of individuals and subsequently affects population-wide dynamics from reproduction to distribution, and can therefore influence the survival and reproductive success of any species (Landry et al., 2007; Perry et al., 2005; Wu, 2009). The aim of this study is therefore to determine the metabolic consequences of temperature (15 and 20 °C) and dissolved oxygen (100 and 50 % air saturation) on metabolic rate of S. canicula during embryonic development and within a week post-hatch.

5.2 MATERIALS AND METHODS

5.2.1 Animal Husbandry and Experimental Rearing Conditions

Sixty small-spotted catsharks, Scyliorhinus canicula egg cases were transported from the Ozeaneum, Stralsund, Germany to the University of Manchester, Manchester, UK. In Manchester, the S. canicula egg cases were maintained inside rearing tanks containing aerated saltwater (35 ppt salinity, 100 % air saturation, at 15 °C). In order to differentiate between individuals, differently colored and numbered tags were attached to the tendrils of the egg cases for individual identification. Candling was used for health monitoring of the embryo inside the egg case. Water quality checks (water temperature, dissolved oxygen level, pH, salinity, ammonia, nitrite, and nitrate) were performed daily and water changes were done 3 times per week (further details can be found in Musa et al., 2018).

Following a brief quarantine (see Musa et al., 2018), S. canicula egg cases were divided into 4 developmental biospheres (55 L) providing the various environmental rearing conditions; normoxic (100 % air saturation) conditions at 15 °C (N15) or 20 °C (N20) and mild hypoxic conditions (50 % air saturation) at either 15 °C (H15) or 20 °C

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(H20). The normoxic conditions at 15 °C reflect preferred conditions for S. canicula however, egg cases from this species are found in waters ranging from the north-western coast of Norway to the Southern tip and Senegal (Ellis et al., 2005; Ellis et al., 2009). Throughout the developmental incubation period, the hypoxic treatments were maintained by bubbling water with special tanks of compressed gas (BOC limited) containing 11 % oxygen (O2), 0.04 % carbon dioxide (CO2), balanced with nitrogen gas

(N2). Temperature was maintained by holding tanks in a 15 °C temperature-controlled room with submerged heaters and circulating water pumps. Once hatched, hatchlings remained in their incubation tanks for 1 day (and metabolic rate was measured, as outlined below). After this, they were transferred into a common garden holding tank under normoxia at 15 °C. This experimental design was modelled to resemble free- living stage of S. canicula where they are free to move to a preferred environment. The study was carried out under Project License 40/3584 and approved by the Animal Welfare and Ethics Review Board of the University of Manchester, Manchester, UK.

5.2.2 Developmental Stages Investigated

Musa et al. (2018) identified 7 key developmental stages of oviparous elasmobranchs inside the egg case (Fig. 5.1). Each developmental stage was recognizable by features that were clearly identifiable with the naked eye using candling (Musa et al., 2018). The 7 key stages represent ecologically and physiologically important stages in development and are characterized by features that enhance survival in the intertidal zone.

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Figure 5.1: Oviparous elasmobranch development in 7 key stages. (A) Seven developmental key stages and hatchling (H); red circles denote the developmental stages investigated in the current study. (B) Stage 4 of development (dorsal view). (C) Stage 6 of development (dorsal view). Black lines show scale of 1 cm. Figures are adapted from Musa et al. (2018).

Embryos at stage 4 and 6, and hatchlings within 1-week post-hatch were chosen for this study (Fig. 5.1). Stage 4 is characterized by the development of long, red external gill filaments whose appearance coincides with the opening of seawater slits on each corner of the egg case, exposing the embryo to ambient environmental conditions (Fig. 5.1B). Stage 6 is characterized by rapid yolk utilisation and growth (Fig. 5.1C). During the first week post-hatch, the catsharks do not externally feed as they still consume yolk contained within their internal yolk sac.

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5.2.3 Assessment of Metabolic Rate During Development in Different Climatic Rearing Environments

In all experiments (except the final 1-week measurement), the metabolic rate of each animal was measured under its own environmental rearing conditions. Oxygen consumption rates (MO2) were measured as a proxy for metabolic rate (Chabot et al., 2016; Jette et al., 1990; Steffensen et al., 1994; Svendsen et al., 2016). Oxygen -1 -1 consumption MO2 (in mgO2 kg h ) was calculated as, MO2 = [(Vr – Vf) × ([O2]t0 –

[O2]t1)]/[(t1 – t0) × Mf]; where Vr is respirometer volume, Vf is experimental animal volume (assuming that 1 g of animal mass is equivalent to 1 mL of animal volume),

[O2]t0 is oxygen concentration at time t0, [O2]t1 is oxygen concentration at time t1, t0 is initial time (in hour), t1 is final time (in hour) and is mass of experimental animal (in kg) (Clark et al., 2013).

Each S. canicula egg case containing the embryo, or the hatchling, was weighed in water. Maximum metabolic rate was achieved at stages 4 and 6 (inside the egg cases) by repeated tapping the egg case for 3 minutes, to resemble a predator investigating the egg case. During tapping, the embryos ‘wiggled’ vigorously before coiling their tail around their body and minimizing external gill filaments or buccal movement. This ‘freeze response’ has been reported in another oviparous elasmobranch species including the brownbanded bamboo shark, Chiloscyllium punctatum (Kempster et al., 2013) and is considered to be an antipredator strategy. Maximum metabolic rate in hatchlings was achieved by swimming them for 3 minutes in a small tank by repeatability touching the tail. After completion of these stressors designed to elevate metabolic rate, S. canicula embryos/hatchlings were immediately placed inside a 350 mL volume custom-built intermittent flow respirometer (320 mL respirometry chamber and 30 mL of associated tubing) for 24 hours and the highest recorded oxygen consumption was considered the maximum metabolic rate (MMR) (Norin and Clark, 2016; Reidy et al., 1995). Routine metabolic rate (RMR) was calculated by averaging the lowest 10 % of MO2 values, and represent the oxygen requirements necessary for routine survival. Aerobic scope (AS) was calculated by subtracting the RMR from the

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MMR values for each individual (Norin and Clark, 2016). Each animal’s metabolic rate was measured under the temperature and dissolved oxygen conditions of its rearing environment, with the exception of the hatchling measurements after a week in the common environment (N15). They were measured in 15 °C under normoxic conditions.

While in the respirometer, each 10 minute loop consisted of a flush period (150 seconds) where fresh, aerated/hypoxic saltwater was flushed into the respirometry chamber, a wait period (50 seconds) where oxygen was uniformly distributed throughout the respirometer before the record period where dissolved oxygen levels were measured (400 seconds) via a fiber optic oxygen transmitter (Witrox 1 developed by Loligo® Systems, Denmark). The temperature and oxygen probes (Oxy-4 oxygen meter developed by PreSens Precision Sensing GmbH, Germany) were connected to the DAQ-M instrument, relaying information to AutoRespTM (version 2.2.0 developed by Loligo® Systems, Denmark), which then calculated the oxygen consumption rates -1 -1 (measured in mgO2 kg hr ) inside the respirometry chamber whilst simultaneously recording temperature levels.

The circulation pump prevented oxygen stratification. A water bath was used to accurately control the temperature of respirometry chamber. The background respiration was tested after each experiment. If there was a significant background respiration inside the respirometry chamber, the microbial oxygen consumption rate was subtracted from the experimental animal’s metabolic rate. In order to minimise microbial interference, the saltwater inside the experimental tank was changed 3 times a week and the respirometry chamber and tubing was cleaned weekly.

5.2.4 Excess Post-Exercise Oxygen Consumption (EPOC)

The recovery curve of oxygen consumption during the 24 hours in the respirometry chamber was used to calculate the time constant associated with excess post-exercise oxygen consumption (EPOC) of S. canicula embryos under the different environmental conditions and developmental stages (Ozolina et al., 2016). pClamp 10 software was

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used to calculate a first order exponential equation fit to EPOC generating a time constant, tau (τ), for each individual. Tau provides an index of the time required for an individuals’ metabolic rate to return from MMR towards RMR; i.e. for the rate to decrease by 63 % of its initial value (Bennett, 1972; Nishimura et al., 1993).

5.2.5 Statistical Analysis

One-way and two-way analysis of variance (ANOVA) were performed, followed by Tukey’s or Sidak’s multiple comparisons post-hoc tests (P < 0.05) to determine differences in MMR, RMR, aerobic scope and EPOC across developmental stages and 10/(T2 – T1) rearing environments. Q10 coefficients (Q10 = (R2/R1) ; where R1 is oxygen consumption rate at T1, R2 is oxygen consumption rate at T2, while T1 and T2 are experimental temperatures; T2 > T1) were calculated to determine the thermal sensitivity of MMR, RMR, aerobic scope and EPOC of S. canicula embryos at different developmental stages. All figures and statistical analysis were carried out using Prism 7 software (GraphPad Software, Inc., USA). Statistical details are reported in each figure legend.

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5.3 RESULTS

5.3.1 Effects of Different Climatic Conditions on Metabolism at Each Developmental Stage

Oxygen consumption rate (MO2) of S. canicula embryos is affected by the temperature and dissolved oxygen levels in their rearing environment (Fig. 5.2). In general, and regardless of developmental stage, S. canicula embryos developed under normoxic conditions resulted in higher maximum and routine metabolic rates (MMR and RMR, respectively) compared with hypoxic conditions (Fig. 5.2). Additionally, warm temperatures increased MMR and RMR in normoxia across developmental stages, but this pattern was lost under hypoxia. Aerobic scope (AS) of S. canicula embryos was generally lower under hypoxia than normoxia, and was not affected by temperature under a given dissolved oxygen environment (Fig. 5.2C,F,I).

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Figure 5.2: Oxygen consumption rates (MO2) of S. canicula embryos at stage 4 and 6 of development and at 1-day post-hatch under different treatments. (A) Maximum metabolic rate (MMR) of stage 4. (B) Routine metabolic rate (RMR) of stage 4. (C) Aerobic scope of stage 4. (D) MMR of stage 6. (E) RMR of stage 6. (F) Aerobic scope of stage 6. (G) MMR of hatchlings. (H) RMR of hatchlings. (I) Aerobic scope of hatchlings. Columns show mean ± SD, while dots show individual measurements; stage 4 (N15, n = 9; N20, n = 6; H15, n = 6; H20, n = 7); stage 6 (N15, n = 8; N20, n = 7; H15, n = 6; H20, n = 6); hatchlings (N15, n = 9; N20, n = 9; H15, n = 7; H20, n = 8). Dissimilar letters indicate statistical differences between treatments at the same developmental stage, while same symbols indicate statistical differences between different developmental stages under the same treatment (two-way ANOVA, P < 0.05).

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5.3.2 Effects of Development on Metabolism under Different Climatic Conditions

S. canicula oxygen consumption rates (MO2) increased as the animals progressed through development (Fig. 5.2). MMR (Fig. 5.2A,D,G) and aerobic scope (Fig. 5.2C,F,I) was highest at 1-day post-hatch hatchlings. RMR showing less of a trend, where increased of MO2 across development can only be observed under normoxic conditions (Fig. 5.2B,E,H).

5.3.3 Effects of Rearing Environment and Developmental Stage on EPOC

The time constant of the recovery curve (τ) was used as a measure of the effect of the environment on excess post-exercise oxygen consumption (EPOC) of S. canicula (Fig. 5.3). In normoxia, stage 4 S. canicula embryos had the slowest τ values and this was not influenced by temperature, whilst stage 4 embryos developed under hypoxia at 20 °C had the fastest τ values, indicating that hypoxic animals recovered towards resting levels more quickly (Fig. 5.3A). Similar patterns were found in stage 6 embryos and in 1-day post-hatch hatchlings (Fig. 5.3B,C). Developmental stage alone had no effect on the time constant of EPOC (Fig. 5.3).

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Figure 5.3: Time constant of recovery of excess post-exercise oxygen consumption (EPOC) of S. canicula embryos at stage 4 and 6 of development and at 1-day post- hatch under different treatments. (A) τ values of stage 4. (B) τ values of stage 6. (C) τ values of hatchlings. Bars show mean ± SD, while dots show individual measurements; stage 4 (N15, n = 9; N20, n = 6; H15, n = 6; H20, n = 7); stage 6 (N15, n = 8; N20, n = 7; H15, n = 6; H20, n = 6); hatchlings (N15, n = 9; N20, n = 9; H15, n = 7; H20, n = 8). Dissimilar letters indicate statistical differences between treatments at the same developmental stage, while there were no statistical differences between different developmental stages under the same treatment (two-way ANOVA, P < 0.05).

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5.3.4 Temperature Sensitivity (Q10) of Metabolism

Individuals developed and tested under hypoxia had lower Q10 coefficient values for MMR, RMR and aerobic scope compared with individuals developed under normoxia at same developmental stage (Table 5.1). Similarly, the Q10 of EPOC recovery (τ values) from individuals developed under hypoxia were lower than those developed under normoxia at same developmental stage (Table 5.2). These results suggest that hypoxia reduced the thermal sensitivity of metabolic rate in S. canicula regardless of developmental stage.

Table 5.1: Temperature sensitivity (Q10) of S. canicula oxygen consumption rates

(MO2) at different developmental stages developed under normoxic and hypoxic conditions. Normoxia Hypoxia

Q10 (stage 4, MMR) 1.7683 0.3850

Q10 (stage 6, MMR) 1.3180 0.5744

Q10 (hatchlings, MMR) 1.6660 1.2733

Q10 (stage 4, RMR) 3.7551 0.9514

Q10 (stage 6, RMR) 1.4587 0.7896

Q10 (hatchlings, RMR) 2.9878 1.9992

Q10 (stage 4, aerobic scope) 0.5554 0.2597

Q10 (stage 6, aerobic scope) 1.1633 0.4949

Q10 (hatchlings, aerobic scope) 1.1825 1.1487

The Q10 coefficient of S. canicula MMR, RMR and aerobic scope at stage 4 and 6 of developmental stages and 1-day post-hatch hatchlings under each environmental rearing condition.

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Table 5.2: Temperature sensitivity (Q10) of S. canicula EPOC recovery (τ values) at different developmental stages under normoxic and hypoxic conditions. Normoxia Hypoxia

Q10 (stage 4, EPOC) 1.1330 0.2839

Q10 (stage 6, EPOC) 0.5923 1.4040

Q10 (hatchlings, EPOC) 1.2177 0.4861

The Q10 coefficient of S. canicula τ values at stage 4 and 6 of developmental stages and 1-day post-hatch hatchlings under each environmental rearing condition.

Table 5.1 shows that under normoxic conditions, the Q10 coefficient values for MMR and RMR of S. canicula embryos were declined as the embryos growing.

Whereas under hypoxic conditions, the Q10 coefficient values for MMR and RMR of S. canicula embryos were increased as the embryos growing. As S. canicula embryos developed, the Q10 coefficient values for aerobic scope increased under both different oxygen level conditions. Similar patterns were shown on temperature sensitivity towards EPOC (τ values) of S. canicula embryos, as the Q10 coefficient values increased under both different oxygen level conditions as the embryos growing (Table 5.2).

5.3.5 Developmental Plasticity and Metabolism

To understand if rearing embryos under a given environment had lasting effects on metabolism, we compared the MMR, RMR, aerobic scope and EPOC measured in hatchlings directly after hatch in their environment, with that measured in the same individuals, after 1-week held (and tested) under normoxia at 15 °C environment. After 1-week in a common environment of normoxia at 15 °C, and when measured under normoxia at 15 °C, MMR, RMR and aerobic scope were the same across all hatchlings, regardless of their rearing environment (Fig. 5.4). Less of a trend was observed with EPOC (τ values) (Fig. 5.5). The EPOC values of S. canicula hatchlings that were exposed to 1-week of a common environment (normoxia, 15 °C) were significantly different than hatchlings measured under hypoxia at both temperatures (15 and 20 °C) (Fig. 5.5). There were no differences between EPOC across different treatments once they were acclimated to normoxia at 15 °C.

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Figure 5.4: Oxygen consumption rates (MO2) of S. canicula hatchlings under different treatments and hatchlings acclimated (a week) to control treatment (normoxia at 15 °C). (A) MMR of hatchlings. (B) RMR of hatchlings. (C) Aerobic scope of hatchlings. Columns show mean ± SD (N15, n = 9; N20, n = 9; H15, n = 7; H20, n = 8). Dissimilar letters indicate statistical differences between rearing treatments at the same group (hatchlings under different treatments or hatchlings acclimated 1- week to control treatment), while asterisks indicate statistical differences between groups (hatchlings under different treatments and hatchlings acclimated 1-week to control treatment) that were from the same rearing treatment (two-way repeated measures ANOVA, P < 0.05). The 1-day post-hatch (rearing treatments) hatchling data in Fig. 5.4 is the same data presented in Fig. 5.2.

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Figure 5.5: EPOC (τ values) of S. canicula hatchlings under different treatments and hatchlings acclimated (a week) to control treatment (normoxia at 15 °C). Bars show mean ± SD (N15, n = 9; N20, n = 9; H15, n = 7; H20, n = 8). Dissimilar letters indicate statistical differences between treatments at the same developmental stage, while asterisks indicate statistical differences between groups (hatchlings under different treatments and hatchlings acclimated 1-week to control treatment) that were from the same rearing treatment (two-way repeated measures ANOVA, P < 0.05).

5.4 DISCUSSION

The key finding from this study is that ambient environment directly affects embryonic oxygen consumption rates in S. canicula. Oxygen consumption is greater in normoxia than hypoxia across all developmental stages and warmer temperatures increase oxygen consumption under normoxic conditions but not under hypoxic conditions. Interestingly, after developing for 5-7 months in a given environment, and then spending one week in a common environment, oxygen consumption rates measured in that common environment are the same regardless of rearing environment. This reinforces the known phenotypic plasticity of this ubiquitous benthic elasmobranch.

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5.4.1 Effect of Temperature on Embryonic and Early Post-Hatch Metabolism

Rearing at 20 °C elevates RMR to a greater extent than MMR but as there is a trend in both measures, aerobic scope (difference between RMR and MMR) is unaffected (Fig. 5.2). Similarly, temperature did not affect the capacity for metabolic recovery during development in a normoxic environment (Fig. 5.3). This suggests that warmer ocean conditions during development (at least up to 20 °C) will not be a significant metabolic stress for this species. These results are akin to earlier studies, showing higher temperature increases metabolic rates across elasmobranchs. For instance, bamboo sharks (Chiloscyllium punctatum), nurse sharks (Ginglymostoma cirratum), and leopard sharks (Triakis semifasciata), all report heightened energetic costs when exposed to increased temperatures (Miklos et al., 2003; Rosa et al., 2014; Tullis and Baillie, 2005; Whitney et al., 2016).

Temperature is a key factor in defining the energetic costs and demands for a species such as S. canicula, which experience seasonal or even daily variations in thermal conditions (Butler and Taylor, 1975; Norin and Clark, 2016). However, our findings suggest that 20 °C is not warm enough to induce thermal limitations in aerobic scope. With rising temperatures, the oxygen- and capacity-limited thermal tolerance (OCLTT) hypothesis predicts that oxygen delivery will be unable to match the heightened oxygen demand, resulting in a decline of the aerobic scope (AS) due to a greater increase in RMR relative to MMR (Farrell et al., 2008; Pörtner and Knust, 2007; Norin and Clark, 2016). We speculate that at temperatures approaching critical thermal maximum (CTmax) for S. canicula embryos, aerobic scope will narrow in line with OCLTT.

Although it is recognized that temperature strongly affects metabolism, the sensitivity to increased temperature varies across and sometimes within species

(McNab, 2002). The Q10 for oxygen consumption rate in elasmobranchs typically falls between 2 and 3 (Butler and Taylor, 1975; Carrier et al., 2012; McNab, 2002; Schmidt- Nielsen, 1984). We found a similar range for S. canicula embryonic RMR, but lower

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values for MMR (between ~1.3 and ~1.7; Table 5.1). We did not find a clear effect of developmental stage on the Q10 for metabolic rate in our study which is in contrast to earlier work on brownbanded bamboo sharks, which found Q10 of routine metabolic rates to decrease from ~5.5 to ~3 during ontogeny (Rosa et al., 2014).

5.4.2 Effect of Dissolved Oxygen Levels on Embryonic and Early Post-Hatch Metabolism

Oxygen consumption in a hypoxic environment caused reduced MMR and RMR compared with oxygen consumption in a normoxia at the same temperature (Fig. 5.2). This aligns with earlier work showing that low oxygen levels induce a hypoxic reflex to reduce energetic costs, such as a reduced heart rate (Butler and Taylor, 1975; Taylor et al., 1977). Indeed, S. canicula thrive in coastal areas subject to large fluctuations in environmental conditions such as oxygen (Ellis et al., 2005). Moreover, previous studies report that S. canicula can survive for 12 hours in total anoxia, supporting the presence of an innate resilience to low oxygen levels, and the anaerobic capacity to maintain vital processes under reduced oxygen conditions (Diez and Davenport, 1987). Likewise, the epaulette shark (Hemiscyllium ocellatum), a species that is often exposed to hypoxia during nocturnal low tides, exhibits a high tolerance to oxygen reduction (Heinrich et al., 2014). This tolerance is primarily provided by the capacity to limit fluctuations in cardiac function and respiration rate during hypoxia (Routley et al., 2002; Soderstrom et al., 1999; Speers-Roesch et al., 2012). Additionally, during total anoxia, the epaulette shark can temporarily shut down its cerebellum and suppress retinal light response to conserve the maximum of energy (Renshaw et al., 2002; Stensløkken et al., 2008). Recent research has also uncovered the genes responsible for the hypoxic tolerance of the epaulette shark (eef1b and ubq), which are upregulated in response to oxygen stress levels (Rytkönen et al., 2010). Future studies should investigate whether S. canicula share similar patterns of gene expression.

Living in a hypoxic environment, reduced the thermal dependence of MMR, RMR and aerobic scope (Fig. 5.2). This is unexpected as metabolic processes require

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more oxygen at higher temperatures (Nilsson et al., 2010), perhaps there was active suppression of metabolism under hypoxia, or that under hypoxia, animals only able to increase metabolism at warmer temperatures via anaerobic means. Unfortunately, we did not measure indices of anaerobic metabolism in this study. Of interest is the observation that the capacity of S. canicula to recover from elevated metabolism (EPOC) is positively affected by hypoxic environment (Fig. 5.3). This may be due to the MMR being significantly reduced by hypoxia. Indeed, aerobic scope is reduced in individuals reared in hypoxia compared with normoxic conditions (Fig. 5.2). Oxygen limitation is a considerable challenge to overcome for aquatic ectotherms, particularly at high temperatures where metabolic rate is increased. When exposed to hypoxia, fish can respond behaviorally in several ways such as moving to a different area, increasing ventilation, reducing swim speed or using surface respiration (Chapman and McKenzie, 2009; Pollock et al., 2007). Physiologically, fish can respond by decreasing oxygen consumption in order to conform to environmental oxygen levels (oxyconformity response) or can maintain equal levels of consumption despite low dissolved oxygen in the water (oxyregulatory response) (Di Santo et al., 2016). When there is no longer sufficient oxygen present in the surrounding water, organisms can also shift energy production from aerobic to anaerobic pathways (Nilsson and Östlund-Nilsson, 2008). Furthermore, benthic species are thought to have an improved anaerobic energy capacity, as they are able to tolerate moderate to severe levels of hypoxia, perhaps reflecting their adaption to low oxygen environments (Heinrich et al., 2014; Seibel, 2011). During development, S. canicula are constrained inside the sessile egg case, with limited available oxygen supply, S. canicula have few available strategies in response to chronic hypoxia. Therefore, the resilience of individuals to hypoxic conditions may be a consequence of evolving within an environment of variable oxygen levels combined with limited adaptive strategies.

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5.4.3 Ontogenetic Changes in Metabolism

Oxygen consumption rates (MO2) increased during ontogeny independent of rearing environments (Fig. 5.2). During ontogeny between stage 4 and 6, S. canicula develop external gill filaments that progressively retract to become gills in stage 7 (pre-hatch stage). As a result of the development of gills as well as the process of hatching from the egg case S. canicula embryos experience heightened energetic demands over time. Being no longer limited by the egg case, S. canicula hatchlings experience greater metabolic demands due to greater activity and may also be more susceptible to stress as they no longer have the protective boundary to shelter from predators hence requiring a longer time to recover (Bouyoucos et al., 2018). The action required to sustain buccal ventilation could also heighten potential energetic costs, indicating that hatchlings may require a more favorable environment, in order to maintain suitable metabolic exchanges, which we have demonstrated through the ‘common garden’ conditions. Nonetheless, it is apparent that metabolic activity varies throughout development. Therefore, environmental conditions could differentially impact individuals across developmental stages, particularly when constrained in harmful conditions including warmer temperatures or reduced oxygen availability.

5.4.4 Developmental Environment Does Not Have a Lasting Impact on Metabolism

Climate change not only increases ocean temperatures but also generates more extreme and frequent heat waves, threatening thermally adapted species across marine ecosystems (Meehl and Tebaldi, 2004; Perkins et al., 2012). Aquatic ectotherms in particular rely on the optimality of environmental conditions to which they have adapted in order to adjust metabolic processes including energy expenditure, cardiac and respiration rates (Ern et al., 2015). Consequently, thermal acclimation is increasingly considered as a key factor in determining species resilience to climate change, as it reflects the ability to maintain aerobic scope across a range of temperatures (Sandblom et al., 2014; Seebacher et al., 2015). However, after a week in a common garden environment, we found no evidence of thermal acclimation in our measures of MMR,

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RMR, aerobic scope and EPOC; these indices of metabolism were the same regardless of previous rearing environment (Figs 5.4 and 5.5). Therefore, rearing environment did not have a lasting effect on our indices of metabolism, as may be expected in a species with wide environmental tolerances. It is clear that S. canicula possess a capacity to thrive in variable environmental conditions through physiological plasticity as well as adaptation to their environment.

5.4.5 Implications for Future Oceans

As the oceans get warmer, marine organisms possess few strategies to adapt or escape the temperatures exceeding their natural thermal range. During development, S. canicula and other oviparous elasmobranchs cannot escape warmer water, and therefore rely on developmental plasticity to adapt to the change in surrounding conditions (Donelson et al., 2011). For instance, developmental plasticity provides the capacity to optimize individual weight in response to changing environments (Donelson et al., 2011). The winter skate, for example, reduced its body size in response to 2 °C increase in water temperature in order to reduce metabolic costs, as weight is a determining influence with a positive effect on metabolic rate (Clarke and Johnston, 1999; Gillooly, 2001; Lighten et al., 2016; Sims, 1996). Plasticity therefore allows individuals to optimize both physiology and behavior, and potentially improve the resilience of populations to climatic stressors. Additionally, once S. canicula have fully developed, they would be able to fine tune and control their thermal environment by venturing towards colder waters or deeper waters to maximize growth and limit energy expenditure (Sims et al., 2006). In response to elevated temperatures, S. canicula could then move northward towards novel colder habitats as well as increase their vertical migrations in order to be exposed more frequently to preferred temperatures (Sims et al, 2006). Similar to our results, individuals acclimated to preferred conditions were able to recover from the climatic treatments and restore typical metabolic rates, indicating that S. canicula are able to limit metabolic consequences if they evade the detrimental conditions. Ultimately, increased temperatures have the potential to induce broad

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changes in S. canicula populations such as changes in distribution and life history traits, including body size, in order to adapt to temperatures exceeding their thermal range.

Throughout this study, S. canicula have displayed a strong tolerance and resilience to hypoxia, demonstrating the extent to which S. canicula has adapted to life in low oxygen environments. Nevertheless, research has previously reported that hypoxia can induce long-term detrimental consequences on fitness, including reduced reproductive success, growth, and survival (Landry et al., 2007; Pichavant et al., 2001; Shang and Wu, 2004; Wu, 2009). In addition, the ability to tolerate hypoxia is not widespread across elasmobranchs, but is limited to some benthic and coastal species, such as S. canicula (Carlson and Parsons, 2003). Therefore, it cannot be assumed that most species are able to respond accordingly to limited oxygen as it depends on habitat use, life history traits and mode of ventilation. Indeed, many elasmobranchs ventilate by moving through the water to pass oxygen across their gills, termed ram ventilation. Ram ventilators require increased mouth gape as well as swim speed to respond to hypoxic conditions, as seen in the bonnethead shark (Sphyrna tiburo), but would generally prefer avoiding oxygen limited zones (Carlson and Parsons, 2003). Buccal ventilating species on the other hand, are able to reduce their activity levels to preserve energy expenditure, as seen in the catshark, little skate and shovelnose ray (Di Santo et al., 2016; Speers-Roesch et al., 2012). Consequently, comparative studies between elasmobranchs need to consider and weigh the differences in many features, such as ventilation, when evaluating the vulnerability of species to hypoxia.

Elasmobranchs therefore have several strategies to respond to the effects of climate change. S. canicula in particular appear to be suited for zones of reduced oxygen, but it is not the case for many other species of sharks, skates and rays that must rely on their capacity to avoid and escape adverse conditions. The persisting effects of exposure to warmer temperature can therefore result in the widespread displacement and alter the distribution of elasmobranch populations (Perry et al., 2005; Rijnsdorp et al., 2009; Robinson et al., 2015). Many new elasmobranch species have been recorded in new habitats including the temperate waters surrounding the UK as well as northern

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regions stretching up to Alaska (Ellis et al., 2005; Wright and Hulbert, 2000). Such a displacement leads to wide-ranging consequences on native elasmobranch populations, as increased competition, introduction of diseases and novel predator pressure would have consequences across marine species and habitats (Robinson et al., 2015).

5.4.6 Conclusion

Modern elasmobranchs have evolved over the past 420 million years, and have survived extreme environmental conditions in order to thrive in their current habitats (Wilga et al., 2007). However, anthropogenic climate change presents an unforeseen threat to sharks, skates and rays due to the synergistic effects of environmental stressors on metabolism. In this study, results indicate that S. canicula experienced heightened energetic costs associated with chronic exposure to warmer conditions throughout development. Contrastingly, S. canicula displayed a persistent tolerance to hypoxia from the beginning of ontogeny by minimizing energy expenditure. Our results suggest that adaptation to variable environmental conditions is necessary for S. canicula to survive and thrive in future oceans. In addition, elasmobranchs are further susceptible to environmental change due to severe overfishing, as the depletion of populations across the globe reduces population turnover and resilience when faced with adverse conditions (Dulvy et al., 2014). Nevertheless, understanding the physiological impacts of climate change is essential from a conservation standpoint in order to accurately determine, predict and eventually reduce the consequences of human activity on marine habitats as well as the species within them.

Acknowledgements

We would like to thank the Ozeaneum, Germany for the S. canicula egg cases used in this study. Dr John Fitzpatrick, Stockholm University, Sweden is thanked for his useful discussions during the beginning of the project. Authors also would like to thank Molly Czachur for her help on setting up the biospheres and S. canicula rearing tanks and the Shiels Laboratory, University of Manchester, especially Karlina Ozolina for her

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thoughtful discussions for the project and helping the authors on setting up the oxygen consumption experimental set-up. Noor Haziq Saliman is thanked for his assistance with the statistics used in this study.

Funding

S.M.M. is supported by the Ministry of Higher Education (KPT, Malaysia) and Universiti Kebangsaan Malaysia (UKM). The project was funded by the Higher Education Innovation Fund through The University of Manchester’s Knowledge and Innovation hub for Environmental stability, and by the Ministry of Higher Education (KPT, Malaysia).

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CHAPTER VI

GENERAL DISCUSSION AND CONCLUSION

6.1 INTRODUCTION

As this PhD thesis is presented in alternative format, the experimental results were separately discussed in each relevant results chapter (Chapters III, IV, and V). In this chapter, the general discussion is separated into two major sections; firstly, the experimental results from all experiments conducted for this PhD thesis are discussed in relation to the aim and objectives of study. Here, the PhD work are discussed in relation to previously available literatures in order to highlight the novel findings and contributions of our current work in providing new additional information towards the field of study. Secondly, the findings from all experiments conducted for this PhD thesis were integrated to summarize all the findings in order to fully understand the effects of different climatic conditions on growth, metabolism and survival of oviparous elasmobranchs during early ontogeny.

6.2 CONTRIBUTIONS OF CURRENT PHD WORK

As stated earlier, the broad aim of this PhD thesis is to study the effects of current and predicted future climate change, specifically the effects of temperature and hypoxia on oviparous elasmobranchs during early ontogeny using non-invasive methods.

In Chapter III, the main objective was to produce an alternative developmental scale for oviparous elasmobranch embryos that could be applied non-invasively. Before this PhD work was conducted, researchers usually staged the development of

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elasmobranch embryos using the embryological scale provided by the detailed, classic reference of Ballard et al. (1993). Unfortunately, most of the morphological details described by Ballard et al. (1993) to stage development of S. canicula embryo could only be done invasively, where the embryo was removed from the egg case. This often required specialized equipment such as a microscope, and embryonic exposure to fixative or anaesthetic in order to identify the developmental stage of an embryo. These features made it challenging to use these embryological scales for tracking changes in the development of living, developing embryos. Tracking live embryos is important for answering more ecological scientific questions (Johnson et al., 2016; Rodda, 2000; Rosa et al., 2014), and may be relevant for aquarists, and the elasmobranch enthusiasts of the general public. The published research paper (Musa et al., 2018) that makes up Chapter III of this thesis, responds to this need. Based on this novel work, oviparous elasmobranch embryonic development can now be non-invasively monitored and tracked in living embryos without the need of specialized equipment, can be used by a wider audience, and also in the field. The non-invasively developmental scale presented in Chapter III was built on the greater spotted catshark, Scyliorhinus stellaris. However, another important feature of this work is that we also mapped back to the Ballard’s scale (Ballard et al., 1993) for the lesser spotted catshark. This shows the broad applicability of our scale which we go on to use in subsequent results chapters to understand more ecologically and physiologically relevant questions about embryonic development including the effects of environmental factors (such as temperature, dissolved oxygen, pH, pollution and salinity) on biological fitness, physiological performances and survival of elasmobranch embryos at specific developmental stages.

In Chapters IV and V, the objectives of study were to investigate the effects of temperature and hypoxia on growth and survival (Chapter IV of thesis), and metabolism (Chapter V of thesis) of S. canicula embryos. Before this PhD research was performed, most of the previous works that investigated the impacts of climate change on embryonic oviparous elasmobranchs were solely focused on predicted future environments (such as global warming and ocean acidification phenomenon) and not on the current environmental stressor (such as hypoxic environments). For instance, from

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previous studies, the effects of temperature and pH on growth, survival and metabolism of brownbanded bamboo shark, Chiloscyllium punctatum embryos (Rosa et al., 2014), effects of temperature and pH on growth and hunting behaviour of Port Jackson shark, Heterodontus portusjacksoni (Pistevos et al., 2015), and the effects of pH on growth, ventilation and survival of epaulette shark, Hemiscyllium ocellatum embryos (Johnson et al., 2016) were assessed. In relation to hypoxia, most of the previous studies conducted on elasmobranchs were assessed on adult or juveniles; e.g., the effects of hypoxia on metabolic rate of epaulette shark (Routley et al., 2002), effects of temperature and hypoxia on respiration performance and cardiac function of S. canicula adults (Butler and Taylor, 1975), effects of hypoxia on physiological performance and heart rate of spiny dogfish, Squalus acanthias (Sandblom et al., 2009), and the effects of hypoxia on blood oxygen transport of epaulette shark and eastern shovelnose ray, Aptychotrema rostrata (Speers-Roesch et al., 2012). Therefore, in comparison to these previous studies mentioned above, we believe that this PhD work could be considered as one of the early study aiming to investigate both environmental factors; temperature and hypoxia on biological fitness and metabolic performance of oviparous elasmobranchs during crucial embryonic stages. These environmental factors are interrelated as increases in water temperature will decrease the ability of oxygen solubility in water, thus studying both factors in this PhD work has true relevance for both predicted future climate change and today’s environmental challenges on marine species. In Chapter V, we also believe that this is one of the first study that measured maximum metabolic rate (MMR), aerobic scope, and excess post-exercise oxygen consumption (EPOC) performances of elasmobranch embryos developed under different environmental condition, and re-assessed the metabolic performances of oviparous elasmobranch hatchlings under preferred environment (common garden experiment). This study provides evidence for the plasticity of this elasmobranch to respond and adjust metabolism in a changing environment.

One study has been conducted that has almost a similar intention to our work by using tropical species, the Port Jackson shark, where Rodda (2000) has investigated the effects of temperature and hypoxia on growth and metabolism, and has constructed a

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developmental staging scale of the species. Compared to this previous work, similar to Ballard et al. (1993), in order to staging the development of oviparous elasmobranch embryos, Rodda (2000) has described that the embryos need to be removed from the egg cases and being euthanize by exposing to lethal dose of anaesthetic in order to stage them. Therefore, previous described developmental staging methods are not suitable to be applied on living, developing embryos to monitor growth and metabolism under different treatments as the embryos developed inside the egg cases. Although Rodda (2000) has studied the effects of both temperature and hypoxia on biological fitness and physiological performances on elasmobranch embryos, these important abiotic parameters were studied separately (synergistic effect of both high temperature and low dissolved oxygen was not assessed), and the study was solely focused on the effects of the parameter itself, instead of relating them in the sense to climate change. Additionally, for this PhD work, we have used temperate oviparous elasmobranch species, both S. stellaris and S. canicula, compared to tropical species that was used in Rodda (2000) work, thus the comparison data between these elasmobranch species from two different habitat preferences is always the interest of science within the field of study. Therefore, we strongly believe that the novel findings from this PhD work will contributes in adding and supporting available literatures within the scope of research, in order to help us in understanding on how oviparous elasmobranchs living in the extreme habitat such as intertidal zone survive during the crucial embryonic period in current and future climate change.

6.3 SUMMARY OF PHD FINDINGS

In this section, the experimental data from all experiments of this PhD thesis are integrated and discussed in relation to the wider literature and the future of elasmobranchs in general. Here, the experimental findings were discussed in a broader way to connect the ecological and physiological aspects of this research in order to help us understand the whole impacts of different water temperatures and dissolved oxygen levels on development, growth, metabolism and survival of oviparous elasmobranch during early ontogenetic life.

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The influence of environmental abiotic factors towards biological fitness of an organism need to be studied as previous literatures reported that biological fitness and survival of many marine organisms were negatively impacted by these abiotic changes (Chin et al., 2010; Diaz and Rosenberg, 1995; Pistevos et al., 2015). Climate models suggest that the future climate change will elevate the ocean temperatures, making the intertidal zone becomes more hypoxic, which will further impact the biological fitness and survival of intertidal organisms (Butler and Taylor, 1975; Diaz and Rosenberg, 2008; Farrell and Richards, 2009; Pörtner and Farrell, 2008; Pörtner and Knust, 2007). While many species have been declared as critically endangered, sharks are particularly vulnerable to extinction due to their slow sexual maturity and growth, low offspring production, and being overfished or shark-finned by humans (Carrier et al., 2004; Cortés, 2000; Field et al., 2009; Myers and Worm, 2005; Stevenson et al., 2007). Therefore, it is critically important to increase our knowledge in order to understand the responses of these animals to climatic conditions, which may negatively impact biological fitness and contribute to the decline in shark populations in the near future.

Pörtner et al. (2010) has come up with an integrative concept, the oxygen- and capacity-limited thermal tolerance (OCLTT) in order to explain the relevance of physiological responses of an organism in relation to the ecological aspect. The OCLTT concept hypothesised that the metabolism of an aquatic animal will function optimally over a specific range of temperatures (between low and high pejus temperatures, Tp), and exposure beyond Tp of either side of this optimum results in decline in biological performances of a species (Pörtner et al., 2001; 2010; 2017; Pörtner and Farrell, 2008; Pörtner and Knust, 2007). Our experimental findings indicate that under normoxic conditions, the growth rate of S. canicula embryos increased at warmer temperatures, where this result was supported with the elevated metabolic rates as the animals were fully relying on aerobic metabolism and increased in yolk consumption rates. This can be explained through the energy budget formula (Elliott, 1976; Ricker, 1971; 2015; Sun et al., 2006), where under normoxia at warmer temperature, S. canicula embryos required more energy, thus the yolk was consumed faster in order to support faster

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growth with higher metabolism (Elliott, 1976; Malloy and Targett, 1994). Due to this, more oxygen was needed during cellular respiration in order to produce more energy in the form of adenosine triphosphate (ATP) that were produced aerobically via oxidative phosphorylation to support growth at elevated temperature (Campbell et al., 2008).

Although the routine metabolic rates (RMR) of S. canicula embryos were increased at future predicted temperature (20 °C) under normoxic conditions, the maximum metabolic rates (MMR) were also increased, thus aerobic scope was not different between animals developed at 15 and 20 °C (Fig. 5.2). Pörtner et al. (2017) suggests that animals developed at extreme temperatures will have a decrease in aerobic power budget (the usage of oxygen to generate energy in the form of ATP), which is usually measured as a decline in aerobic scope, which may translate to a decline in biological fitness, such as growth. Thus, our study of temperature, metabolism and growth under normoxic conditions is therefore does not supported the OCLTT hypothesis, as aerobic scope was maintained (Fig. 5.2), while embryonic growth rates increased (Fig. 4.3B,C), indicating no decline in aerobic power budget or biological fitness of S. canicula embryos developed at 20 °C compared with 15 °C. These findings may also suggest that 20 °C is not warm enough to be outside the optimal temperature range for this species. However, it may be that 20 °C is near to the pejus temperature

(Tp) of the species as growth rates at stage 5 and 6 of S. canicula embryos (Fig. 4.3D) and aerobic scopes across different developmental stages (Fig. 5.2C,F,I) were not different to the control treatments (not having higher values although these individuals developed at higher temperatures).

The OCLTT concept may be applicable in bridging the ecology and physiology aspects of some species in some contexts, but it is far from being universally acceptable in predicting the impacts of environmental factors on biological performances of a species. Jutfelt et al. (2018) has a contradict points of view towards the OCLTT theory where they have pointed several issues against this biased hypothesis as there were many similar studies reported having different results that were not aligned with the earlier predictions of OCLTT hypothesis. The range of optimal temperatures for aerobic

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metabolism of marine ectotherms were difficult to be determined and usually its effects on aerobic scope were not aligned to other biological performance, e.g. growth (Gräns et al., 2014; Lefevre, 2016). Additionally, the influence of hypoxia as the key parameter which reducing the biological performances of a species was not being the main focused in consideration of the OCLTT hypothesis, and the theory was only being tested under acute ‘non-steady state’ experiments (Jutfelt et al., 2018; Pörtner et al., 2017). Therefore, these reported contradict findings and the results from long-term ‘more steady-state’ experiments need to be equally considered in order to prevent any misinterpreted hypothesis, inaccurate predictions, and to provide a balanced assessment for future researches of the similar study.

Under hypoxic conditions, S. canicula embryonic growth rates were steady (i.e. not different to animals developed under normoxia at the same temperature; Fig. 4.3B,C). This is despite the finding of lower metabolic rates compared with normoxic individuals at the same temperature (Fig. 5.2). This result supports previous studies conducted on the same species that S. canicula have a high ability to cope and adapt with low dissolved oxygen environments (Diez and Davenport, 1987) however, the mechanisms underlying this ability are still not clear. At elevated temperatures, the embryonic growth of S. canicula increased (Fig. 4.3B,C), thus they were able to grow faster at warm temperatures even when oxygen availability was limited. This could be explained by a number of things. In hypoxic conditions, S. canicula embryos may have incorporated anaerobic metabolism to provide energy to support growth, which resulted in the lower aerobic metabolic rates observed under hypoxic conditions (Fig. 5.2). Many previous studies reported a similar observation of ectothermic fishes shifted to anaerobic metabolic pathways when exposed to hypoxic conditions (Cooper et al., 2002; Farrell and Richards, 2009; Xiao, 2015).

Although growth rates were ‘steady’ under hypoxic conditions, the incubation period of S. canicula embryos were longer at 15 °C (significantly different to the individuals developed under control treatment; Fig. 4.6), thus having higher risk to survive in their natural habitat due to longer exposure time to predation and hypoxic

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environments, which later resulting in higher mortality rate (Fig. 4.5). As the animals have lower metabolic rates to support their biological requirements (e.g. growth and digestion), they were struggling to survive and adapt with low dissolved oxygen environments during these long incubation periods, thus explaining the deaths occurrence at later stages of development in embryos developed under hypoxia (no death of S. canicula embryo at later stages of development under normoxic conditions).

After hatched, although both aerobic metabolic rates (MO2) and excess post-exercise oxygen consumption (EPOC) of S. canicula hatchlings were ‘normalised’ after being exposed to control treatment (normoxic condition at 15 °C) for a week (Figs 5.4 and 5.5), the smaller sized hatchlings from elevated temperature treatments (Fig. 4.7), may negatively affecting their swimming performance, thus their survivability of the hatchlings in the wild (see Discussion section, Chapter IV), and the individuals developed under hypoxic conditions were forced to adapt and survive the extreme environments with longer incubation period before hatching (Fig. 4.6). Fig. 6.1 was presented here to summarise the findings of the current PhD experimental research studies.

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Figure 6.1: Summary of PhD findings from Results Chapters III, IV and V of this thesis. The effects of temperature and dissolved oxygen on embryonic growth, metabolism, survival, development time and hatchling size of Scyliorhinus canicula.

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6.4 CONCLUSION

This PhD research aimed to provide information to understand how oviparous shark living in the extreme habitat of intertidal zone survive during the crucial embryonic period in current and future climate change. Both from a physiological standpoint as well as for wider conservation and modeling purposes, investigating the impacts of human induced climate change is essential for understanding the effects and potential consequences of environmental change on the physiology of elasmobranchs. We hope that this study will help to provide us more information to add on the available data from previous studies in a better understanding on the impacts of future climate change on elasmobranchs biological responses and survivability, especially for sharks. We believe more efforts need to be done on conservation work to save more elasmobranch species from extinction and to increase more public awareness on the importance of this apex predators of the ocean and its role in keeping the marine ecosystems in balance, and eventually reduce the consequences of human activity on marine environments.

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APPENDIX

Supplemental S1 File: Original photographs of Fig. 3.1 illustration. External features of the S. stellaris egg case at developmental stage 1.

Fig. 3.1

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Supplemental S2 File: Original photographs of Fig. 3.2 illustrations. The inside of the S. stellaris egg case at developmental stage 2.

Fig. 3.2A

Fig. 3.2B

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Supplemental S3 File: Original photographs of Fig. 3.3 illustrations. The inside of the S. stellaris egg case at developmental stage 3.

Fig. 3.3A

Fig. 3.3B

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Fig. 3.3C

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Supplemental S4 File: Original photographs of Fig. 3.4 illustrations. The inside of the S. stellaris egg case at developmental stage 4 with the egg yolk mass and the associated embryo.

Fig. 3.4A

Fig. 3.4B

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Fig. 3.4C

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Supplemental S5 File: Original photographs of Fig. 3.5 illustrations. The inside of the S. stellaris egg case at developmental stage 5 with the egg yolk mass and the associated embryo.

Fig. 3.5A

Fig. 3.5B

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Fig. 3.5C

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Supplemental S6 File: Original photographs of Fig. 3.6 illustrations. The inside of the S. stellaris egg case at developmental stage 6 with the egg yolk mass and the associated embryo.

Fig. 3.6A

Fig. 3.6B

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Fig. 3.6C

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Supplemental S7 File: Original photographs of Fig. 3.7 illustrations. The S. stellaris embryo inside of the egg case at the end of developmental stage 7 with the egg yolk mass completely absorbed.

Fig. 3.7A

Fig. 3.7B

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Fig. 3.7C

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Figure 7.1: Double-yolked (twins) egg case of greater spotted catshark, Scyliorhinus stellaris. (A) Size comparison of double-yolked S. stellaris egg case (yellow tag, number 11), its pair egg case with a single yolk (yellow tag, number 21), and normal S. stellaris egg case (green tag, number 10). (B) Close-up of double-yolked S. stellaris egg case. Black bars represent 1 cm.

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Figure 7.2: Double-yolked (twins) egg case of small-spotted catshark, Scyliorhinus canicula. (A) Size comparison of S. canicula double-yolked with twin embryos inside an egg case (below), and normal S. canicula egg case (above). (B) S. canicula twins removed from the egg case. Black bars represent 1 cm.

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Figure 7.3: Wind egg case of greater spotted catshark, Scyliorhinus stellaris. A total of 3 wind eggs (egg case without any yolk) of S. stellaris were found during this study.

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