The Direct and Indirect Impacts of Ocean Warming and Acidification on Marine Invertebrates

The direct and indirect impacts of ocean warming and acidification on marine invertebrates

Sarah Graham

Thesis submitted in fulfilment of the requirements of the degree of Masters of Research within the University of New South Wales

Evolution and Ecology Research Centre School of Biological, Earth and Environmental Science University of New South Wales Sydney, 2052 New South Wales Australia

March 2013

AKNOWLEDGEMENTS

I would like to sincerely thank my supervisor, Alistair Poore, for his support, guidance and wisdom during my time in the Subtidal Ecology and Ecotoxicology (SEE) Laboratory. If it were not for you, I would have never explored the wonderful world of marine invertebrates and seaweed.

Thank you to my co-supervisor Symon Dworjanyn, who was always there to offer advice, bounce ideas off, for the fun times and practical support in the BEAR Laboratory.

Much of the work described in the thesis could not have been done without the help of both my colleagues and friends in the SEE and BEAR Laboratories. Special thanks to Ceiwen Pease, for your help in the lab and in the field, for your guidance and for your ridiculously positive attitude, you have been a great mentor. Thank you to Hannah Sheppard Brennand, for keeping me sane during the long hours in the lab, but also for the wonderful and memorable fun times. I would also like to say a big thank you to everyone in both labs for being such a great team to work with, in no particular order, Aria Lee, Damon Bolton, Natalie Rivero, Vivian Sim, Katelyn Edge, Jaz Lawes, Melanie Sun, Sally Bracewell, James Lavender, Shinjiro Ushiama, Keryn Bain, Alexia Landry and Pamela Aro Kamya.

This work, however, could not have been done without the financial support of the NSW Environmental Research Trust Grant, The School of Biological Earth and Environmental Science as well as the Evolutionary and Ecology Start Up Research Grant.

Finally, a very big thank you to my family and friends. Thanks to my loving mum, I could not have done this without your unconditional love and support. To my dad and my sister, for always being there. Thank you to my friends for making me laugh and reminding me there is more to life than amphipods and seaweed.

LIST OF FIGURES PAGE 2

Figure 2.1. The maximum daily ocean temperature at Mutton Bird Island, 15 Coffs Harbour, Australia between the 1st of January 2010 to the 1st of January 2011 from a depth of 3 m. Data was obtained from the Australian Institute of Marine Science.

Figure 2.2. The temperature treatments of seawater over the experimental 21 period for P. parmerong in either an ambient thermal environment (23 °C) or the elevated thermal environment of +3 °C (26 °C).

Figure 2.3. Mean a) survival, b) size and c) fecundity of P. parmerong that 22 were raised in either an ambient or elevated temperature environment of +3 °C from infancy to maturity (± s.e). Significant difference between treatments in denoted by (*).

Figure 2.4 Mean a) survival and b) size of the 2nd generation at 28 days of age 23 (± s.e.) from each temperature treatment, descending from mothers that developed in either temperature treatment. (White bars represent offspring that developed in Ambient, black bars represent offspring that developed in +3 °C treatment).

Figure 2.5. Correlation between the size of offspring at 28 days of age and the 24 number of offspring in each family at the same time point among each combinations of treatments across the two generations. Linear Regression analysis of pooled treatments was P=0.000, y = -0.112x + 9.9023, R2 = 0.18

Figure 2.6 Average daily in situ ocean temperature conditions from Mutton 29 Bird Island, Coffs Harbour, between a) April – July and b) August - November from a depth of 13m. Data obtained from the Australian Institute of Marine Science.

Figure 3.1a Variation in thermal tolerance with life history stage and sex. Figure 44 illustrates the mean ( s.e.) CTmax of males, females and juveniles amphipods from ambient temperature conditions, ramped at a rate of 0.06.C.min-1.

Figure 3.1b Correlation between the size (mm) of amphipods and CTmax (C) of 44 all individuals including males, females and juveniles. Linear Regression analysis of pooled life history stage and sex was P = 0.4648, y=0.06887x + 32.09, R2 = 0.01.

Figure 3.2 Comparison of the mean (± s.e) CTmax of amphipods after 45 acclimation to a thermal environment of 20 °C or 26 °C for a period of 28 days, ramped at a rate of 0.02 °C.min-1.

Figure 3.3 Comparison of the thermal tolerance time (mins) of the second 45 generation (F2) of P. pamerong after acclimation for 28 days in a thermal environment of 23°C or 26°C, from parents (F1) that developed in either thermal environment of 23°C or 26°C. Amphipods were exposed to a static temperature of 33°C until amphipods died.

Figure 3.4 Comparison of the mean ( s.e) CTmax of amphipods acclimatised 46 to either thermal environment of 20 C or 26 C, ramped from two starting temperatures ( 20 C or 26 C, dotted line from the y-axis) and a two rates ( 0.02 C per.min-1 or 0.06 C per.min-1). Dotted lines denotes rate of temperature increase from initial starting temperature.

Figure 4.1 Temperature (Ambient, +2 °C and +4 °C) and pH levels 63 (Ambient, 7.8, 6.6) recorded daily in the treatments over the duration of the experiment.

Figure 4.2a-c Growth (mean ± s.e. % increase in mass) of six temperate algal 64 species exposed to three temperature treatments (Ambient (24 °C), +2 °C (26 °C) and +4 °C (28 °C) and three pH treatments (8.1, 7.8 and 7.6) for a period of 2 weeks. The seaweeds investigated include a) Dictyopteris acrostichoides, b) Pterocladia capillacea c) Rhodymenia. Treatments sharing letters do not differ in post- hoc tests.

Figure 4.2d-f Growth (mean ± s.e. % increase in mass) of six temperate algal 65 species exposed to three temperature treatments (Ambient (24 °C), +2 °C (26 °C) and +4 °C (28 °C) and three pH treatments (8.1, 7.8 and 7.6) for a period of 2 weeks. The seaweeds investigated included) Dilophus intermedius, e) Zoneria diesingiana, and f) Sargassum vestitum. Treatments sharing letters do not differ in post-hoc tests.

Figure 4.3a-c Consumption (mean ± SE mass loss) of 6 temperate seaweed 66 species exposed to three temperature treatments (24°C, 26°C and 28°C) and three pH treatments (8.1, 7.8 and 7.6) by the marine snail Phasianotrochus eximius under ambient conditions over a period of 24hrs. The seaweed investigated include a) Dictyopteris acrostichoides, b) Pterocladia capillacea, c) Rhodymenia australis. Treatments sharing letters do not differ in post-hoc tests.

Figure 4.3 d-f Consumption (mean ± SE mass loss) of 6 temperate seaweed 67 species exposed to three temperature treatments (24°C, 26°C and 28°C) and three pH treatments (8.1, 7.8 and 7.6) by the marine snail Phasianotrochus eximius under ambient conditions over a period of 24hrs. The seaweed investigated include d) Dilophus intermedius e) Zonaria diesingiana and f) Sargassum vestitum. Treatments sharing letters do not differ in post-hoc tests.

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LIST OF TABLES PAGE 3

Table 2.1. ANOVA results for size and fecundity of the 1st generation of P. 25 parmerong under the two thermal environments. a) Indicates a three-factor nested analysis of variance contrasting size of amphipods at 28days among tanks and between temperature treatments. A two-factor nested analysis of variance contrasts b) fecundity of the first brood of gravid females between the two temperature treatments and among tanks.

Table 2.2 The analysis of variance results on size and survival of the 2nd 26 generation of P. parmerong that developed in each thermal environment for 28days A four factor-nested analysis of variance contrasts the a) size of offspring from each maternal thermal environment and their own thermal environment. A three-factor nested analysis of variance contrasts b) survival of offspring from their own or their mothers thermal environment.

Table 3.1 An analysis of variance contrasting the critical thermal maximum 47 (CTmax) among males, females and juveniles. Sex & Life History Stage (i.e., males vs females vs juveniles) is a fixed factor and Block a random factor.

Table 3.2 A four-factor nested analysis of variance contrasting the 47 maximum thermal tolerance (time to death) of offspring exposed to a static temperature of 33 °C at day 28 from both the offspring

environmental temperature (F2 Temperature) and the

environmental temperature of the parents (F1 Temperature). F1

Temperature and F2 Temperature were fixed factors, Tank was a

random factor nested in F1, Temperature and Family was a

random factor nested in both F2 Temperature and Tank. * denotes a significant effect (P < 0.05).

Table 3.3. A three-factor analysis of variance contrasting the CTmax of 48 amphipods acclimatised to two temperatures (20 C or 26 C), ramped from two starting temperatures of either 20 C or 26 C, and at two rates of 0.02C per.min-1 or 0.06C per.min-1. Starting Temperature, Acclimation Temperature and Rate were all fixed factors. * denotes a significant effect (P < 0.05).

Table 4.1 Two-factor analyses of variance contrasting the percentage growth 68 (increase in wet mass) of six species of seaweed over a period of two weeks, exposed to three temperature treatments and three pH treatments. Temperature and pH are fixed factors. Probabilities were determined using 9999 permutations

Table 4.2 Two-factor analyses of variance contrasting the consumption (mg) 69 by Phasianotrochus eximius of six different species of algae exposed to three temperature and three pH treatments for a period of 24 hr, under ambient conditions. Temperature and pH are fixed factors with snail weight a covariate. Probabilities were determined using 999 permutations. 4

TABLE OF CONTENTS PAGE 5

Acknowledgements……………………………………………………...... ………...... i 6

List of Figures…………..….………………………...... ………………………………..ii 7

List of Tables………….……………………………………….………………….……….....iv 8

ABSTRACT ...... 1 9

CHAPTER 1. GENERAL INTRODUCTION ...... 2 10 1.1 Variation In thermal tolerance ...... 2 11 1.2 Climate change & thermal adaptation...... 5 12 1.3 Ocean warming and acidification ...... 6 13 1.4 Thesis outline ...... 8 14

CHAPTER 2. PHENOTYPIC PLASTICITY AND TRANS-GENERATIONAL EFFECTS OF A 15 MARINE CRUSTACEAN IN A WARMING ENVIRONMENT ...... 9 16

2.1 ABSTRACT ...... 9 17 2.2 INTRODUCTION ...... 10 18 2.3 METHODS ...... 13 19 2.3.1 Study site and organisms ...... 13 20 2.3.2 Experimental conditions ...... 13 21 2.3.3 Effects of elevated temperature across generations ...... 16 22 2.3.4 Statistical analysis ...... 18 23 2.4 RESULTS ...... 19 24 2.4.1 Experimental conditions ...... 19 25 2.4.2 Size, survival and fecundity after one generation ...... 19 26 2.4.3 Maternal influence on size and survival ...... 19 27 2.5 DISCUSSION ...... 27 28 2.5.1 Survival unaffected by temperature ...... 27 29 2.5.2 Size and fecundity in a warmer environment ...... 30 30 2.5.3 Trans-generational effects with increasing temperatures ...... 31 31 2.5.4 Conclusion ...... 32 32

CHAPTER 3. SHORT TERM ACCLIMATION BUFFERS THERMAL EXTREMES ...... 33 33

3.1 ABSTRACT ...... 33 34 3.2 INTRODUCTION ...... 34 35 3.3 METHODS ...... 38 36 3.3.1 Study organisms ...... 38 37 3.3.2 Variation in CTmax between sex and life history stage ...... 38 38 3.3.3 Variation in CTmax with recent thermal history ...... 39 39 3.3.4 Thermal tolerance and trans-generational effects ...... 40 40 3.3.5 Methodological effects on thermal tolerance ...... 41 41 3.3.6 Statistical analysis ...... 41 42 3.4 RESULTS ...... 42 43 3.4.1 Variation in CTmax between sex and life history stage ...... 42 44

3.4.2 Variation in CTmax with recent thermal history ...... 42 45 3.4.3 Thermal tolerance and trans-generational effects ...... 42 46 3.4.4 Methodological effects on thermal tolerance ...... 42 47 3.5 DISCUSSION ...... 49 48 3.5.1 Thermal sensitivity among individuals with recent exposure to elevated 49 temperatures ...... 49 50 3.5.2 Thermal tolerance and trans-generational effects ...... 51 51 3.5.3 Procedural issues ...... 51 52 3.5.4 Conclusion ...... 52 53

CHAPTER 4. EFFECTS OF PH AND TEMPERATURE ON THE GROWTH AND 54 PALATABILITY OF TEMPERATE MACROALGAE ...... 53 55

4.1 ABSTRACT ...... 53 56 4.2 INTRODUCTION ...... 54 57 4.3 METHODS ...... 57 58 4.3.1 Study organisms and sites ...... 57 59 4.3.2 Experimental conditions ...... 58 60 4.3.3 Effects of temperature and pH on algal growth ...... 58 61 4.3.4 Effects of temperature and pH on algal palatability...... 59 62 4.3.5 statistical analyses ...... 60 63 4.4 RESULTS ...... 61 64 4.4.1 Experimental conditions ...... 61 65 4.4.2 Effects of temperature and pH on algal growth ...... 61 66 4.4.3 Effects of temperature and pH on algal palatability...... 62 67 4.5 DISCUSSION ...... 70 68 4.5.1 Effects of temperature on algal growtH ...... 70 69 4.5.2 Effects of pH on algal growth ...... 71 70 4.5.3 Climatic stressors and algal palatability ...... 72 71 4.5.4 Ecological relevance ...... 73 72

CHAPTER 5. GENERAL DISCUSSION ...... 75 73

5.1 Variation in thermal tolerance ...... 75 74 5.2 Effects of climatic stressors on species interactions ...... 77 75

REFERENCES...... 79 76

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ABSTRACT

Many studies have identified that the direct effects of global warming are going to have detrimental impacts on the growth, development and survival on an array of marine species. Tolerance to stress may however be buffered through acclimation, and adaptive across generations, but our understanding on the role of these mechanisms is limited with respect to global warming. I therefore tested how elevated temperatures may affect the size, survival, fecundity and thermal sensitivity of a marine crustacean Peramphithoe parmerong. The acclimation to their maximum habitat temperature did not affect survival, but it did increase growth, reduce fecundity and increased their tolerance to heat stress. This was however, not adaptive across generations as there were no trans-generational effects in association to survival, size or their capacity to tolerate heat. I also identified that the protocols used to assess thermal sensitivity can have a significant effect on heat tolerance, and future studies should recognise and consider this before assessing the critical thermal limits of an organism, particularly those from aquatic systems. As ocean temperatures rise, so too will the concentration of CO2 (coupled with a lowering pH), and the impact of these stressors, acting simultaneously on species interactions, is not clearly understood. The focus of much of the current research in marine systems is associated with calcifying organisms, however non-calcifying organisms are also likely to be affected, in particular primary producers. The growth rates of five temperate species of macroalgae were significantly reduced by temperatures predicted to occur over the next century. In addition, but independent of temperature, a lower pH also lead to the reduce growth of two species of macroalge. Although, for the one species (Dictyopteris acrostichoides), which was unaffected by either temperature or pH, palatability towards the marine gastropod (Phasianotrochus eximius) increased, when grown under increasingly acidic conditions. Given this, the complexities of both the direct and indirect effects of climate change make it difficult to predict the ‘winner and losers’ in the years to come.

1. Introduction

Chapter 1

GENERAL INTRODUCTION

1.1 Variation in thermal tolerance

All organisms have an optimal temperature range that supports growth, development and reproduction. Temperatures beyond or below this range can interfere with enzymatic efficiency, membrane structures and oxygen demands with subsequent effects on an individual’s growth, reproduction, fitness and ultimately survival (Angilletta 2009). The relationship between temperature and the performance of a specific trait (within the tolerable limits of the organism) is described by a Thermal Performance Curve (Huey 1982, Schulte et al. 2011). As temperatures rise, so too, does the performance of a specific trait until the thermal optimum (Topt) is reached and any further increases in temperature reduces performance (Schulte et al. 2011, Huey et al. 2012). Thermal performance curves are variable across species, and, within a species can also vary among populations, individuals and in association to the history of exposure to stressful temperatures (Tomanek 2008, Schulte et al. 2011). Understanding the variation in thermal tolerance is critical for predicting the effects of changing temperature on the persistence of species under thermal stress.

Within an organism’s lifetime, temperatures will fluctuate on a daily, seasonal and annual basis (depending on the lifespan). Endothermic organisms are able to counteract these changes by maintaining a stable internal body temperature through metabolic processes (Buckley et al. 2012). Whereas the body temperature of ectothermic organisms tends to reflect those of their surrounding environmental conditions and thus are more dependent on other mechanisms to thermoregulate (Huey 1982). To maintain functioning in response to changing temperatures, both ectotherms and endotherms, may alter their thermal performance curves through thermal acclimation via modification in their behaviour or morphology (Huey and Berrigan 2001, Hoffmann 2010, Schulte et al 2011). Behavioural responses to acute or chronic temperature shifts are one of the first responses to reduce thermal stress (Agrawal 2001, Chapperon and Seuront 2011, Smit et al. 2013). These may include shuttling between warm and cool microhabitats (Snucins and Gunn 1995, Kreuger and Potter 2001), shifting posture or orientation to improve heat exchange (Munoz et al. 2005), or panting and tongue-lashing to increase the evaporation of heat (Roberts and Harrison 1998). If behavioural responses are unable to compensate to stressful temperature 2

1. Introduction conditions, physiological changes such as the activation of heat shock proteins (Hsp) or mechanisms to maintain membrane integrity (homeoviscous adaptation), can increase tolerance to thermal stress in both plants and animals (Hazel 1995, Hofmann and Todgham 2010). Change in colour, size or shape may also be influential for some organisms to maintain internal functioning and increase their thermal tolerance (Stevenson 1985, Clusella‚Trullas et al. 2008). All three forms of thermal acclimation may change the breadth of an individual’s thermal range, increase their performance or shift their thermal optima (Angilletta 2009). The capacity to do this is, however, variable among individuals within a population.

Thermal tolerance can vary with age, sex and or size. Several studies have reported differences in thermal tolerance with different ages or life stages of a population (Davison 1969, Klok and Chown 2001, Jensen et al. 2007), but this relationship is variable among species and the mechanisms are not well understood (Bowler and Terblanche 2008). With limited resources, the capacity of an organism to compensate for thermal stress may trade-off with other behavioural or physiological changes (Bowler and Terblanche 2008) such as foraging for food, becoming sexually mature, or seeking a mate (Nyamukondiwa and Terblanche 2009). The cost of maintaining internal functioning in stressful thermal conditions may also play a part in the differences found between sexes (Willett 2010). High temperatures increase metabolism and the production of either heat shock proteins or the restoration of cellular components is costly (Halpin et al. 2002, Somero 2002). This may place a greater toll on the energy budget of sexually mature or reproducing females as opposed to males who theoretically should have a greater net energy budget (Min and Chin, 2012). However, sex commonly covaries with size, which is also a trait that affects the ability to tolerate thermal extremes (Peck et al. 2009). For example, a study conducted on 16 marine species from six phyla showed that smaller individuals consistently survived to higher temperatures (Peck et al 2009). This relationship between temperature and size is regularly seen amongst ectotherms, with individuals being much smaller at maturity in warm environments than cool adapted individuals (Atkinson 1994, Atkinson and Sibly 1997). These differences in size, sex and gender are important to understand, as the variation in vulnerabilities among individuals may in turn affect the dynamics of populations faced with changing temperatures over the long term.

Variation in thermal tolerance is not only present within a lifetime, and among individuals in a population, but can also vary across generations. Species may adapt to thermal variability within their environment over successive generations by genetic change (via selection) or by non-genetic effects whereby changes to parents are expressed in their offspring (i.e.,

1. Introduction maternal or trans-generational effects) (Donelson et al. 2011, Salinas and Munch 2011). Increasingly, there is evidence of rapid evolution in response to climatic change (Franks et al. 2007, Van Doorslaer et al. 2007, Urbanski et al. 2012) and predicting the effects of a changing climate will depend on potential for species to adapt (a function of the strength of selection, levels of genetic variation for thermal tolerance and gene-flow among populations) (Somero 2010, Hoffmann and Sgro 2011). Less well understood, is the potential for variation in tolerance across generations to result from phenotypic change alone, rather than genetic adaptation. Tolerance could be an expression of a phenotype instigated by either the environment of the preceding generation or manipulated by the parents (Marshall et al. 2008, Shimada et al. 2010). These are known as trans-generational effects, and most often are derived from changes to the mothers alone, (known as maternal effects) (Marshall et al. 2008). If the mother is able to anticipate such an environmental change and it does not impede her fitness, then the expression of a particular trait (e.g. egg provisioning) in her offspring may increase their chances of survival in a stressful environment (Marshall and Uller 2007, Rasanen and Kruuk 2007). This expression of phenotypes instigated by either the previous generation or their environment is commonly observed in response to predation and pollution (Gustafsson et al. 2005, Storm and Lima 2010) but also in association to temperature (Burgess and Marshall 2011, Donelson et al. 2011). Understanding the role that trans-generational effects have on thermal tolerance is important for establishing the capacity for populations to persist in a warmer environment.

Given these mechanisms for variation among individuals within populations, we expect to see disparities in the capacity to tolerate different thermal environments across large and small-scale temperature gradients. These patterns have arisen through acclimatisation and adaptation to past and present thermal conditions. There is often a close relationship between upper temperature tolerance and maximum habitat temperatures. Across latitudinal gradients, tropical species are uniformly more heat tolerant than temperate and polar species (Somero 2008). Although due to seasonal variability, the thermal breadth experienced by these organisms is greatest at mid-latitudes and decreases with the encroachment of tropic and polar regions (Sunday et al. 2011). Animals living in the tropics, in which they experience slight variations in temperature across seasons, have adapted to a narrower thermal environment near or above their optima for performance (Tewksbury et al. 2008). Similarly, so too have those living in polar regions. In comparison, animals at mid-latitudes (temperate regions) see greater fluctuations in temperature either on a daily or seasonal basis, but because of this, they experience temperatures fringing onto their maximum thermal capacity less frequently (Tewksbury et al. 2008). Such variation in thermal environments and breadth

1. Introduction also commonly exist across smaller gradient scales. In the littoral zone of the marine environment, there is a variation in breadth and maximum temperatures experienced between the subtidal and intertidal zones (Somero 2002, Bedulina et al. 2010). Congeneric species in the intertidal areas often find more thermally tolerant individuals than those in the subtidal zone (Tomanek and Somero 1999, Bedulina et al. 2010), consistent with the relatively cooler, more static thermal conditions. It has been suggested that species adapted to either a relatively stable environment or an environment in which organisms frequently experience temperature near or above their thermal optimum, may be the most vulnerable as temperature rise (Tewksbury et al. 2008, Somero 2010).This, therefore, raises questions in relation to the variances in the adaptive capacity of both populations and species currently residing in various thermal regimes.

1.2 Climate change and thermal adaptation

Despite its natural variability among days, seasons, years and inter-annual time frames, global temperatures have risen by 0.69 °C - 1.08 °C between the period of 1901–2012 (Solomon et al. 2007). This relatively minute temperature shift has coincided with the earlier onset of spring events in the northern hemisphere (Bradley et al. 1999), the contraction of glaciers and melting of polar icecaps (Solomon et al. 2007, Rignot et al. 2011), as well as the occurrence of more frequent extreme hot events (Allison et al. 2009). Over the turn of the 171 next century, climate change models predict a further rise by 6.4 °C (Solomon et al. 2007). With such a large shift in global mean temperatures, the frequency and intensity of extreme heat events are going to increase. There is a large body of work on how species will respond, on average, to climate change (Parmesan 2006, Calosi et al. 2008, Doney et al. 2011, Poloczanska et al. 2012), but a poorer understanding of how the capacity of many species to tolerate increased or extreme temperatures vary within populations or over time. Through investigations on the mechanisms driving the differences in thermal tolerance, insight into the current capacity for many species to adapt to further increases in temperature may be gained.

Living in a warmer environment will benefit some organisms and be detrimental to others, depending on their thermal tolerance and capacity to adapt. Studies conducted on species and populations across thermal gradients commonly find that individuals living in warmer environments are more thermo-tolerant (Kuo and Sanford 2009, Byrne et al. 2011) and this may give them a greater capacity to cope with rising temperatures, particularly if they have shorter generational time (Sgro et al. 2010, Walters et al. 2012). However, it is possible that those heat-tolerant individuals are living closer to their thermal limits and the capacity for

1. Introduction the species or population to adapt any further is dramatically reduced (Tewksbury et al. 2008). Variation in thermal histories may also influence the likelihood of thermal adaptation. For example, organisms living in static temperature environments with little seasonal variability are believed to have a lower capacity to tolerate fluctuations from their optima through acclimation. Therefore, slight deviations from their normal habitat temperatures can lead to their immediate death. Taking this into consideration, both the past and present thermal environments may provide some insight into the current capacity for many species to adapt to both the average inclines in temperature as well as extreme thermal events.

1.3 Ocean warming and acidification

Changes in mean temperatures and the frequency of extreme heat events are not limited to terrestrial systems. The thermal energy absorbed by the oceans has contributed to its increase in temperature by 0.6 °C over the past 100 years (Hoegh-Guldberg et al 2010). The oceans however, are not warming evenly and some regions are expected to incur temperatures well above average over the coming years (Wernberg et al. 2011). In south eastern Australia, for example, sea-surface temperatures are expected to increase three-fold faster than other areas (Poloczanska et al 2007, Poloczanska et al. 2012). By 2030, temperatures in that region are predicted to be 1 °C above current temperatures and 1.5–3 °C above, by 2070 (Poloczanska et al 2007, Poloczanska et al. 2012). Such temperature increases will be coupled with extreme heat events, as has been observed recently off the west coast of Australia. In the summer of 2010/2011, temperatures increased by 5 °C above average for a period of a week, leading to detrimental impacts on the local marine ecosystem (Smale and Wernberg, 2013, Wernber et al., 2013).

In the coming years, temperature is not the only issue of concern in the marine environment, as the ocean also plays an important role in the global carbon cycle. Over the past 200 years the ocean has absorbed 50% of the CO2 produced by anthropogenic activities (Raven et al, 2005), where it reacts with water and decreases its pH (Orr et al. 2005). This has already lead to a reduction in ocean pH by 0.1 units (Haugan and Drange 1996), and if business continues as usual (under the IS92a Scenario) a further reduction of 0.3-0.4 units is expected by the end of the century (Caldeira and Wickett 2003, Orr et al. 2005). The creation of a more acidic ocean, in which there is a greater concentration of hydrogen ions H+, diminishes the

2- concentration of carbonate ions CO3 , through the production of bicarbonate. Under the following reaction:

- + 2- + CO2 + H2O  H2CO3  HCO3 + H  CO3 +2H

1. Introduction

2- Carbonate ion CO3 , is a vital component for the production of calcium carbonate that is used by many marine organisms (including corals, algae, fish, zooplankton, crustaceans, echinoderms and molluscs) to produce their shells and structures. Recent meta-analyses’ have revealed that ocean acidification as a single stressor are generally detrimental on calcify organisms, whereas non-calcifies are relatively unaffected. (Kroeker et al. 2010). Similarly, ocean warming commonly increases the growth of non-calcifying organism, however, negatively effecting calcifying organisms (Harvey et al, 2013). The responses to these climatic stressors in combination are variable across different taxonomic groups. For example, echinoderms are highly vulnerable to ocean acidification (Dupont et al 2010), which is further altered by increasing temperatures. (Byrne et al 2011). Alternatively, in the case of coral calcification and photosynthesis, which are negatively affected by ocean acidification, are unaffected when in combination with increasing temperatures (Harvey et al 2013). In general, the simultaneous exposure of both ocean warming and acidification more commonly then not leads to a synergistic effect in association to calcification, photosynthesis, reproduction and survival of many marine organisms (Harvey et al 2013). The majority of studies to date, however, have focused on calcifying organisms, with limited research into how changes in ocean temperature and chemistry may affect non-calcifying autotrophic organisms such as many species of macroalgae (Koch et al 2013).

Marine primary producers are fundamental in food-web dynamics and are dependent on both temperature and CO2 for metabolic functioning and growth. If temperatures increase and exceed the threshold of macrophytes, then productivity is likely to decrease. Though elevations in CO2 may provide a buffer (as has been observed in terrestrial systems) it is not clearly understood whether this also applies to seaweeds and seagrasses in the marine environment (Harley et al. 2012, Roleda and Hurd 2012). Furthermore, elevations in both temperature and CO2 are known to affect the chemical defences and nutritional content of macrophytes. Potential shifts in these chemical characteristics may then affect their palatability towards their consumers. Given the fundamental importance of herbivory in the marine environment (Poore et al. 2012), with rates of herbivory up to 3 times greater compared to their terrestrial counterparts (Cyr and Pace 1993), climatic changes to plant- herbivore interactions have the potential to alter the abundance and species composition of entire communities.

1. Introduction

1.4 Thesis outline

As climatic conditions change over the next century, predicting the responses of organisms depends on an understanding of how organisms will respond directly to single climatic stressors, but also the variation in their responses and the degree to which species are able to adapt. Furthermore, it requires an understanding of whether they will be affected by both increases in average conditions, response to extreme events, and how these effects may alter species interactions. To further investigate these concepts, this thesis, including three data chapters, aims to address the following;

The study begins with establishing the direct impacts of increasing temperatures on the marine amphipod crustacean, Perampithoe parmerong. Specifically, in Chapter 2, I assess the capacity for this species to acclimate to increased temperatures that approach their thermal maxima. I assess how exposure to increased temperatures within a single generation, and in the maternal generation, affect growth, survival and fecundity. This was designed to assess the role of phenotypic plasticity in buffering the potential impacts of increasing temperatures as well as how this may be altered through trans-generation effects.

Chapter 3 investigates the responses of Peramphithoe parmerong to extreme thermal conditions, and how tolerance may vary with acclimation, amphipod size and sex, and the methodology used to estimate CTmax (the critical maximum temperature). Exploring how this may be affected by recent thermal history among individuals can establish how close a population may be to their thermal limits, and the capacity they possess to respond to further increases.

Finally, in Chapter 4, I examine the direct effects on both temperature and ocean acidification on growth of six species of abundant macroalgae and subsequently if these effects may indirectly alter the interaction with an abundant consumer of these species, the gastropod, Phasianotrochus eximius. This study examines how the dual exposure to two climatic stressors affects non-calcifying macroalgae, and how these effects could alter important species interactions.

2. Trans-generational effects Chapter 2

Phenotypic plasticity and trans-generational effects of a marine crustacean in a warming environment

2.1 ABSTRACT

Individual organisms that encounter stressful environments may be able to withstand environmental deviations from their optimal conditions through shifts in phenotype within their lifetime (i.e., phenotypic plasticity). Increased tolerance to stress may also arise from changes across generations due to adaptation or non-genetic mechanisms that facilitate tolerance in offspring following environmental change faced by the parental generation. In the coming years, sea-surface temperatures are expected to rise and an understanding of variation in thermal tolerance is required to predict its impacts. We assessed the importance of phenotypic plasticity and maternal environment in an abundant marine amphipod, Peramphithoe parmerong, residing in a global warming hotspot. A first generation was raised in either an ambient temperature (23 ˚C) or a stressful temperature (26 ˚C), and offspring from each of these maternal temperatures then raised in each thermal environment. Survival of the first generation was unaffected by temperature, however amphipods were less fecund and larger in the warmer temperature. In the second generation, maternal environment did not interact with offspring thermal environment, suggesting that trans- generational effects are not a likely source of variation in thermal tolerance for this species over the range of temperatures tested. If temperatures increase beyond thresholds of tolerance that result from phenotypic plasticity, then there will need to be a greater emphasis on their genetic capacity to adapt.

2. Trans-generational effects

2.2 INTRODUCTION

Environmental stress is a key driver of natural selection affecting individual organisms through to entire ecological communities (Bijlsma and Loeschcke 2005, Steinberg 2012). Stressors may be associated with biotic (resources, competition, predation) or abiotic factors (temperature, nutrients, moisture, toxins), and result from natural or anthropogenic sources (Steinberg 2012). Tolerance to stress commonly varies among individuals, populations and species, and thus the impacts of a changing environment may be detrimental for some organisms, but advantageous for others due to increased resource availability or reduced competition (Poloczanska et al. 2008, Hughes 2011a). With rapidly changing climatic conditions, it is becoming increasingly important to predict the ‘winners and losers’ with respect to climatic stressors (Tewksbury et al. 2008, Somero 2010). Predicting impacts depends on both quantifying the effects of changing climate on organisms, but also their capacity to adapt via genetic and non-genetic means (Visser 2008, Chown et al. 2010). Investigating the adaptive capacity of species residing in climate change hotspots provides an opportunity to establish the vulnerabilities that may lie ahead for some species (Hoffmann and Sgro 2011).

Depending on the severity and period of exposure, organisms may respond to a novel or enhanced environmental stressor via genetic or non-genetic means (Badyaev 2005). Sub- lethal environmental stressors may encourage either a population or entire species to shift their geographical distribution and migrate away from their existing habitat to a more compatible one. Range shifts have been the most observed responses to climate change thus far. A global trend in the geographical shift of tropical and temperate species migrating poleward has become increasingly documented over the past decade (Parmesan 2006b, Sunday et al. 2011a). However, species with limited dispersal potential or capacity to migrate (due to contracting habitat e.g. alpine or polar regions) are largely dependent on their ability to acclimatise. Within a single generation, individual organisms may acclimatise to their environment by modifying their phenotype in the form of their behaviour, morphology and physiology (Agrawal 2001, Price et al. 2003). This may include moving to cooler habitats during the hottest parts of the day (Chapperon and Seuront 2011, Smit et al. 2013), alteration in body size to improve thermoregulation (Gardner et al. 2011) or the activation of heat shock proteins (e.g. Hsp70) to improve thermal tolerance (Tomanek 2008b, 2010). If the environmental stressor reduces individual fitness, then selection can result in genetic changes within a population through time. Selection for altered phenological traits or for greater plasticity of a particular trait in response to climatic changes have resulted in genetic changes in several species (Bradshaw and Holzapfel 2001).

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The tolerance of an individual to environmental stress is not purely a response of their genetic makeup, with changes to phenotypes possible due to exposure to stress in the environment faced by the preceding generation or manipulated by the parents themselves (Marshall et al. 2008). Trans-generational plasticity is a non-genetic mechanism that may facilitate increased tolerance to environmental changes over successive generations (Marshall et al 2008). It occurs when the environment experienced by the parents shapes the phenotype of the subsequent generation either in direct response to the environment or indirectly via maternal changes to offspring phenotype (defined as maternal effects) (Mousseau and Fox 1998). This mechanism is commonly overlooked, but has been observed in response to environmental change due to contaminants, food shortages, desiccation and predation (Marshall et al. 2008, Storm and Lima 2010). Maternal effects can be detrimental to the fitness of offspring (to maximise fitness of mothers), but in many instances both parties may benefit if the mothers can anticipate or influence the natal environment (Marshall and Uller 2007). Mothers may manipulate offspring size, brood protection, oviposition site, immune responses, toxicant resistance, offspring competition or sex determination, to buffer the effects of the environment on her progeny (Marshall et al 2008). Several studies have found trans-generational effects from temperature on growth (Salinas and Munch 2011), life history traits (Kvist et al. 2012), metabolic performance (Donelson et al. 2011) as well as metamorphic success and dispersal potential (Burgess and Marshall 2011) and it is possible that this understudied mechanism may contribute to tolerance of climatic stressors.

The aim of this study is to test for the significance of trans-generational plasticity in an abundant marine invertebrate faced with a changing thermal environment in south-eastern Australia. Temperature is one of the most important abiotic environmental factors that can interfere with enzymatic efficiency, membrane structures, oxygen demand, life history traits, fecundity, size, species distribution and ultimately survival (Angilletta 2009). Global average sea-surface temperatures are rising and some marine regions are expected to experience temperatures well above global averages over the next century. The waters off south-eastern Australia have warmed by 0.68 C since 1910 (Poloczanska et al 2012). With the strengthening of the Eastern Australian Current (EAC), which extends warm tropical water south (to higher latitudes), this region is expected to incur sea-surface temperatures 1-2 C above average by 2030 and 2–3 C above average by 2070 (Poloczanska et al. 2007a, Hobday and Lough 2011). This has already lead to the retreat or range extension southward of many species of macroalgae (Wernberg et al. 2011b), zooplankton, phytoplankton, both pelagic and dermsal fish as well as invertebrates (Poloczanska et al. 2012). Poorly

2. Trans-generational effects understood, however, is the capacity for most marine organisms in this region to tolerate any further increases, and what mechanisms may predict variation in this capacity.

A recent study on the abundant amphipod Peramphithoe parmerong Poore & Lowry, 1997 found that an elevated temperature of 3 C above ambient reduced survival of amphipods, and a further increase of 6 C killed all amphipods completely (Poore et al. 2013). While elevated temperatures clearly have a negative effect on this species, the effects of temperature on fecundity and their ability to acclimate to increased temperature are unknown, and thus so too is their likely persistence over the longer term and across generations. It is possible that tolerant individuals may alter the number of offspring or the phenotype of the offspring in some manner to improve survival. This has the potential to buffer the impact to climate change.

In this study, I therefore tested for the effects of a stressful temperature on the survival, growth and fecundity of this marine amphipod and whether variation in the maternal thermal environment affects offspring performance. This species is a generalist herbivore, feeding and residing in the abundant Sargassum spp. algal beds within south-eastern Australian (Poore and Steinberg 1999). Their short generation times and brooding habit make them an ideal species to investigate potential maternal effects associated with increased temperature. I asked the following specific questions: 1) Does the thermal environment that the amphipods develop in affect their survival, size and fecundity and, 2) How does the maternal thermal environment affect the future performance of offspring?

2. Trans-generational effects

2.3 METHODS

2.3.1 Study site and organisms

Amphipods were sourced from Sargassum spp. at low tide from a depth between 0.5–2 m at Charlesworth Bay (30°18’03.61’’S 153°08’31.90’’E) and Mutton Bird Island Nature Reserve (30°18’16.85’’S 153°08’55.20’’E), NSW, Australia on the 4th, 13th and 18th of July 2011. Amphipods were separated from Sargassum spp. in the laboratory by placing the seaweed briefly in fresh water, pouring the freshwater through a sieve and quickly replacing individuals back into clean seawater (methods previously used by Pease et al 2010, Poore and Steinberg 1999). Male, female and juvenile P. parmerong were held in a number of holding tanks for several days prior to experimentation, in a flow through sea water system at the National Marine Science Centre, Coffs Harbour. They were maintained at a similar temperature to the collection site (approx 18 °C) and at a 18:6 h light:dark regime. Fresh Sargassum spp. was provided in excess and tanks were cleaned once a week (Poore and Steinberg 1999). All experiments were conducted within 7 days of collecting amphipods from the field.

2.3.2 Experimental conditions

The effect of increased temperature on P. parmerong was quantified by rearing two generations in an ambient thermal environment (23 °C) or an elevated thermal environment of +3 °C (26 °C) in all four combinations, in a flow through seawater system. Water temperatures were manipulated by allowing ambient UV sterilised and filtered FSW (0.2 µm), to flow into a 60 L header tank. The tanks were continuously bubbled with air to maintain dissolved oxygen (DO) > 90%, which was recorded daily. A constant volume of water was maintained in each header tank using a float valve. This water was then fed into sub-header tanks (20 L) where it was unaltered to maintain ambient conditions (23 °C) or warmed to the required temperature (26 °C) using aquarium heaters (TUNZE temperature controller 7028/3). The water was delivered independently into each individual 60ml rearing container at a rate of 0 9 ml.sec-1. Each container was modified with a section removed from the side and covered with 65 µm mesh to allow for appropriate flow.

The temperatures used in this study are within the range experienced by the population of amphipods collected at the study site (Malcolm et al. 2011), Figure 2.1. The overall maxima in situ temperature recordings for this region are approximately ~26 °C, whilst 23 °C is well within the thermal breadth of the waters of this area (Malcolm et al. 2011). Assuming that SST increase by 2–3 °C over the next century (Poloczanska et al. 2007a, Hobday and Lough

2. Trans-generational effects

2011), a water temperature of 26 °C is likely to occur more frequently during the warmer months, and was thus considered an appropriate temperature.

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Figure 2.1. The maximum1 daily ocean temperature at Mutton Bird Island, Coffs Harbour,

1 1 1 Australia between 1 the 1st of January 2010 to the 1st of January 2011 from a depth of 3 m. Data was obtained from the Australian Institute of Marine Science. (http://www.aims.gov.au/docs/data-centre/seatemperatures.html).

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2.3.3 Effects of elevated temperature across successive generations

To investigate how elevated temperatures affect the condition of P. parmerong, and whether maternal experience altered offspring performance, two generations were allowed to develop in varying thermal environments from infancy to reproductive maturity. The offspring from field-collected amphipods were defined as generation one and exposed to one of two temperatures (ambient or +3 °C). A subsequent second generation were also exposed to either thermal environment in all four combinations of maternal thermal environment and offspring thermal environment.

Prior to the experiment, gravid females collected from the field were removed from the laboratory holding tanks, and placed individually into 60 ml plastic containers in a seawater flow through system set at a temperature of 23 °C. To prevent shock from the change in water temperature, gravid females were kept in holding tank water, and the experimental water temperature was then slowly added. The presence of the female’s exoskeleton was checked daily. Once this was observed, it is considered that all offspring had been expelled from the brood pouch (Poore and Steinberg 1999). The offspring were counted and placed into one of six 600 ml containers. Each container consisted of offspring from 11 females, with a density between ~100 to 200 individuals. Three of these containers were kept in the ambient temperature treatment (23 °C) or placed into the elevated temperature of +3 °C (26 °C). The amphipods in each container developed under these temperature conditions (Ambient or +3 °C) for a period of 28 days, which is sufficient time for this species to reach maturity (Poore and Steinberg 1999). The tanks were cleaned once a week and un-fouled Sargassum sp. was provided to each tank and replenished when required.

All individuals within each of the tanks were counted and sexed after 28 days. Survival of amphipods after one generation between the temperature treatments was established by calculating the remaining number of amphipods at day 28 compared to day 0 as a percentage in each container. One hundred amphipods were randomly chosen from each tank to be sized. They were photographed with a digital camera mounted on top of a compound microscope. Length was measured from first antenna to the tip of the telson (Poore and Steinberg 1999), using Image J (http://rsb.info.nih.gov) The fecundity of females in each thermal environment was quantified by removing between 23 and 30 gravid females from each container. They were then placed individually into 60 ml plastic containers under the same temperature treatment. Once the female had moulted the number of offspring were counted. If females did not release any live young after a period of three weeks they were recorded as having no offspring. This was used to establish the number of offspring per

2. Trans-generational effects female (including those that did not produce any) as well as establishing the number of offspring per female that only released live young. Live offspring subsequently became the second generation and were used to investigate maternal influences on temperature tolerances.

A student t-test contrasted the survival of amphipods between the two temperature treatments with each tank acting as a replicate. The size of the amphipods was contrasted between treatments with a three-factor nested analysis of variance (ANOVA). Temperature and sex were defined as fixed factors and the container in which they developed (from here on in is defined as Tank) as a random factor nested within temperature. A two-factor nested ANOVAs were used to contrast fecundity (i.e., the number of offspring released from all gravid females removed from each tank) and the number of offspring per brood (only those female successful in releasing offspring) across the two temperature treatments, with temperature as fixed factor and tank as a random factor nested within temperature. Due to the large proportion of gravid females that were unsuccessful in releasing any offspring in the elevated temperature of +3 °C, a Student’s t-test was conducted on the proportion of females to successfully release offspring among the tanks.

The response of the successive progeny to the environment of the preceding generation was investigated through the survival and size of each brood. Once fecundity had been established, the brood from each of the maternal environments was allocated a temperature treatment similar to the preceding generation or the alternative option. A total of 98 broods were used, with each brood remaining in the allocated temperature treatment for a period of 28 days. They were counted every seven days and sized at the end of this period.

To establish if either the temperature of the offspring or the temperature of the mother had any influence on the survival of the offspring, a three factor nested ANOVA contrasted proportional survival of the brood at day 28 across treatments, with tank as a random factor nested within maternal temperature and both maternal temperature and offspring temperature as fixed factors. The size of offspring at day 28 was contrasted among treatments with a four-factor nested ANOVA. The factors were the same as the survival analyses with the addition of family, a random factor nested within both tank and offspring temperature. This established if amphipod density influenced the outcome. A linear regression analyses between the number of offspring in each brood and their size at day 28 was also performed. Treatments were pooled for the regression analysis, as there was no correlation found between treatment and size of amphipods

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2.3.4 Statistical analyses

Analyses of variance and linear regression were performed in PASW Statistic (v18) and PERMANOVA Primer (V6). Frequency histograms of the residuals and plots of the residuals were performed to ensure that the assumptions of normality and homogeneity of variance were met. Size of the 1st generation amphipods was log transformed to meet these assumptions.

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2.4 RESULTS

2.4.1 Experimental conditions

The mean (± s.e.) temperatures in Ambient and +3 C during the experiment were 22.7 ±0.03 (pH 8.10 ± 0.002) and 25.5 ± 0.02 (pH 8.08 ± 0.001) respectively, as shown in Figure 2.2.

2.4.2 Size, survival and fecundity after one generation

Temperature had no significant effect on the survival of amphipods at 28 days (P > 0.05, not shown, Figure 2.3a), but it was found that the size of amphipods in the two temperature treatments were significantly different (Figure 2.3b, Table 2.1a). Amphipods that developed in the elevated temperature of +3 °C were significantly larger than those in the ambient temperature treatment.

The temperature that gravid females developed in had a significant effect on the number of offspring per brood (Figure 2.3c, Table 2.1b). There were significantly fewer offspring per family from mothers that developed in the elevated temperature than those in ambient (P < 0.05). There was, however, a difference in fecundity between tanks. The lower fecundity of gravid females in the elevated temperature was associated with the fact that the proportion of gravid females that did not release any live young was significantly higher in the elevated temperature of +3 °C than those in the ambient temperature (t = 0.022, P<0.01, not shown). Of the 69 females that developed in Ambient conditions only 6 females with eggs did not produce live young. In contrast, of the 72 females that developed in +3°C temperature treatment, 40 did not produced any live offspring. For the females that did release offspring, there was no significant difference in the number per brood between treatments (P = 0.53,

F1,91 = 0.03, not shown). A linear regression on mothers size and the number of offspring per brood at Day 0 showed no significant relationship (P=0.670; R2 = 0.0023; not shown).

2.4.3 Maternal influence on size and survival

The temperature in which the mothers developed did not have an effect on survival of her brood after a period of 28 days (Figure 2.4a, Table 2.2b). There was no relationship between the size of mothers and the survival of their offspring (P = 0.808; R2 = 0.0008; not shown). There was also no correlation between the size of the mothers and the number of offspring in each brood at Day 28 (P=0.934; y=0.0035x + 62.226, R2=0.000; not shown).

The size of offspring was also not influenced by the temperature in which their mothers developed, or the temperature in which they developed (Figure 2.4b, Table 2.2a,). This was, however, variable among families. There was a negative correlation between the number of

2. Trans-generational effects offspring per brood at Day 28 and size of amphipods among all four combinations of mothers treatment and offspring treatment was found, which may suggest that density hinders size in some manner (Figure 2.5).

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28 Ambient +3°C

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Figure 2.2. The temperature treatments of seawater over the experimental period for P. parmerong in either an ambient thermal environment (23 °C) or the elevated thermal environment of +3 °C (26 °C).

2. Trans-generational effects

Figure 2.3. Mean a) survival, b) size and c) fecundity of P.parmerong that were raised in either an ambient or elevated temperature environment of +3 °C from infancy to maturity (± s.e). Significant difference between treatments in denoted by (*).

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a) Ambient 11.000 +3°C

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Figure 2.4 Mean a) survival and b) size of the 2nd generation at 28 days of age (± s.e.) from each temperature treatment, descending from mothers that developed in either temperature treatment. (White bars represent offspring that developed in Ambient, black bars represent offspring that developed in +3 °C treatment).

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)

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Figure 2. 5. Correlation between the size of offspring at 28 days of age and the number of offspring in each family at the same time point among each combinations of treatments across the two generations. Linear Regression analysis of pooled treatments was P=0.000, y = -0.112x + 9.9023, R2 = 0.18.

Table 2.1. Analysis of variance results for size and fecundity of the 1st generation of P. parmerong under the two thermal environments. a) Indicates a three-factor nested analysis of variance contrasting size of amphipods at 28days among tanks and between temperature treatments. A two-factor nested analysis of variance contrasts b) fecundity of the first brood of gravid females between the two temperature treatments and among tanks. * denotes significant effects (P<0.05). The mean squares used as denominators for each F test are as follows: Size - Temperature, 0.999MSTank(Temperature) + 0.001MSError; Tank(Temperature), MSsex*Tank(Temperature); Sex, 0.999MSSex*Tank(Temperature) + 0.001MSError; Temperature x Sex, 0.999MSsex*Tank(Temperature) + MSError; Sex x Tank(Temperature), MSError. Fecundity – Temperature, 0.991MSTank(Temperature) + 0.009MSError; Tank(Temperature), MSError.

a) Size b) Fecundity Source df MS F P df MS F P Temperature 1 0.126 8.691 0.042* 1 520.075 7.967 0.047* Tank (temperature) 4 0.015 4.472 0.088 4 65.648 2.474 0.047* Sex 1 0.005 1.448 0.295 -005 Sex*temperature 1 8.83 0.027 0.878 Sex*tank (temperature) 4 0.003 2.475 0.043* Error 605 0.001 134 26.535

Table 2.2. The analysis of variance results on size and survival of the 2nd generation of P.parmerong that developed in each thermal environment for 28days A four factor-nested analysis of variance contrasts the a) size of offspring from each maternal thermal environment and their own thermal environment. A three-factor nested analysis of variance contrasts b) survival of offspring from their own or their mothers thermal environment. * denotes significant effects (P<0.05). The means squares used as denominators for each F test are as follows: Size - Maternal Temp, 0.486MSTank(Maternal Temp) + 0.014MSTank(Maternal Temp)*Offspring Temp + 0.099MSFamily(Tank(Maternal Temp)*(Offspring Temp) + 3.99MSError; Offspring Temp, 0.884MS(Tank(Maternal Temp)*Offspring Temp + 0.045MSFamily(Tank(Maternal Temp) x Offspring Temp) + 0.071 MSError; Tank (Maternal Temp), 0.0814MSTank(Maternal Temp)*Offspring + 0.034MSFamily(Tank(Maternal Temp)*Offspring Temp) + 0.152 MSError; Maternal Temp x Offspring Temp, 0.880MSTank(Maternal Temp)*Offspring Temp + 0.047MS(Family(Tank(Maternal Temp)*Offspring Temp + 0.073MSError; Tank(Maternal Temp) x Offspring Temp, 0.588MSFamily(Tank(Maternal Temp)*Offspring Temp +0.412 MSError; Family(Tank(Maternal Temp) x Offspring Temp), MSError. Survival – Maternal Temp, 0.943MSTank(Maternal Temp) + 0.057 MSError; Offspring Temp, 0.043MSTank(Maternal Temp)*Offspring + 0.057MSError; Tank(Maternal Temp), MSTank(Maternal Temp)*Offspring Temp; Tank(Maternal Temp) x Offspring Temp, 0.943MSTank(Maternal Temp)*Offspring Temp + 0.057MSError; Family(Tank(Maternal Temp) x Offspring Temp), MSError.

a) Size b) Survival Source df MS F P df MS F P

Maternal temp 1 0.832 0.237 0.640 1 0.111 1.870 0.232 Offspring temp 1 6.186 1.640 0.263 1 0.432 1.737 0.255 Tank(maternal temp) 5 5.784 1.641 0.310 4 0.057 0.222 0.913 Maternal temp x offspring temp 1 5.315 1.412 0.294 1 0.115 0.462 0.533 Tank (maternal temp) x offspring temp 4 4.050 1.966 0.105 4 0.258 2.814 0.030* Family (tank (maternal temp) x offspring temp) 75 2.904 3.388 0.000* Error 370 0.857 86 0.092

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2.5 DISCUSSION

While the impacts of climate change on both terrestrial and marine species is becoming increasingly clear (Parmesan 2006b, Byrne et al. 2009, Poloczanska et al. 2012), how species may respond to these changes over the coming years is less well understood (Somero 2010, Hoffmann and Sgro 2011). Temperature is one of the most influential abiotic factors, affecting the growth, development and distribution of species (Angilletta 2009). As temperature changes, so too will the fitness (Byrne et al. 2009, Byrne et al. 2010b) and the range of many species (Ling et al. 2009, Wernberg et al. 2011b). For organisms with a contracting habitat (e.g., alpine and polar regions) or limited mobility and dispersal potential, the capacity to acclimatise or rapidly evolve greater tolerance is vital (Visser 2008). From the experiments conducted in this study, there were two key findings. Firstly, amphipods that had acclimated to an elevated water temperature of +3 °C were larger, but had a significantly lower reproductive success compared to those acclimated to ambient temperatures. Secondly, neither the size, nor survival of offspring was affected by variation in the thermal environments faced by their parents. These results emphasise the importance of evaluating multiple measures of performance (e.g. survival, size and fecundity), to determine the effects of an environmental stressor on a species.

2.5.1 Survival unaffected by temperature

The survival of P. parmerong was unaffected when acclimated to an environment that fringes on its current maximum habitat range of 26 °C for a period of 28 days. This contrasts with the recent study of Poore et al. (2013), in which survival was significantly reduced after seven and 14 days at 27 °C compared to the ambient temperature of 24 °C. Given that the majority of amphipods died at 27°C (Poore et al. 2013), a decrease in temperature of 1°C was considered appropriate in the current study. The contrast in survival could be associated to the long-term variation in temperatures. A chronic exposure of 27°C may exceed the thermal tolerance thresholds of juvenile P. parmerong, whilst 26 °C may not. However, more likely than not, it could be associated to the differences in thermal histories of the species. The current study collected amphipods from the field during July while Poore et al. (2013), collected amphipods during November. Given that in situ ocean temperature conditions were not collected at the time of the study it is difficult to compare with Poore et al. (2013). However, ocean temperature recordings at one of the collection sites (Mutton Bird Island at 13m) obtained during 2009 and 2010 (Figure 2.6) indicate contrasting thermal regimes during the months amphipods were collected between the two studies. (http://www.aims.gov.au/docs/data-centre/seatemperatures.html). Prior to November, ocean

2. Trans-generational effects temperatures between August and October gradually ascend from 18°C to 22°C. In comparison, prior to July, ocean water temperatures between April and June gradually descent from 24 °C to 18°C. Thus, temperatures at the time of collection may have been similar, but the past thermal histories of P. parmerong may influence their current thermal tolerance in some manner. The underlying reasons driving this variation, particularly if it is transcending onto a successive generation, remains unclear.

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Figure 2.6 Average daily in situ ocean temperature conditions from Mutton Bird Island, Coffs Harbour, between a) April – July and b) August - November from a depth of 13 m. Data obtained from the Australian Institute of Marine Science. (http://www.aims.gov.au/docs/data-centre/seatemperatures .html).

2. Trans-generational effects

2.5.2 Size and fecundity in a warmer environment

Although, amphipod survival was unaffected in the elevated water temperature, both their size and fecundity were significantly affected (Figure 3b, 3c, Table1). P. parmerong were, on average, smaller in each of the tanks at 23 °C compared to those in the warmer tanks, except one. The effect of temperature on ectotherms has a strong relationship with size at maturity. A review conducted by (Atkinson 1994) found that 80% of species (including animals, plants, protists, and a bacterium) were larger at maturity in colder temperature environments. The current study did not find this relationship in P. parmerong. At 28 days the majority of amphipods had reached maturity in both treatments, but were larger in the warmer temperature. Exceptions to the temperature-size rule for ectotherms have been found (Atkinson 1995, Gregory et al. 2000, Cabanita and Atkinson 2006, Hosono 2011), but there appears to be no uniform reason for these exceptions. The thermal range of P. parmerong is yet to be established and therefore so too a thorough understanding of the temperature-size rule for this species. Similar results were obtained by Sotka et al. (in review) from an experiment with P. parmerong conducted over six weeks at three temperatures (15 to 25 °C) and two different diets. Amphipods were smaller in the colder temperature irrespective of diet but many had not yet reached maturity. Therefore it is possible the temperature-size rule may still apply to this ectothermic species, but more research is required to establish this specifically.

Females acclimated to the cooler temperature for their entire life were more successful with the release of live offspring. Between 74 and 100% of gravid females removed from each of the tanks under ambient conditions had offspring. In comparison, only 35 to 50% of gravid females taken from the warmer tanks produced any live young. This significant variation in the number of females with eggs and subsequent success at producing live offspring contributes to the large differences in fecundity between the two temperature treatments. When gravid females that did not produce any juveniles were excluded, there was no difference in the fecundity, suggesting that temperature is affecting the likelihood of successful reproduction rather than the number of eggs per female. Similarly, Wiklund and Sundelin (2001) found exposure to increased temperatures before mating of the amphipods, Monoporeia affinis and Pontoporeia femorata, decreased fecundity for these two species. It was established that fertilisation of females decreased as temperatures rose and a greater proportion of females carried undeveloped, unfertilised or dead broods if temperatures were high prior to mating. The causes for this were thought to be associated with the initial stages of fertilisation, with higher temperature potentially affecting gonad maturity, male sperm

2. Trans-generational effects sensitivity or causing amphipods to delay mating. It is possible that P. parmerong are being affected by temperature in this manner as well. The current study did not take into account the sources of variation potentially associated to male contribution (sperm quality), fertilisation or egg development leading to the failure of viable offspring. It does, however, provide an opportunity to further investigate. Increased temperatures caused by climate change may not have a direct effect on the survival of amphipods, but may impede reproductive success and thus result in a declining population over successive years.

2.5.3 Trans-generational effects with increasing temperatures

Neither parental temperature or offspring temperature had a significant effect on the survival or size of offspring after a period of 28 days. Irrespective of the temperature in which the parents developed in, offspring survival in the warmer temperature was lower than in the cooler temperature of 23 °C for only one of the six tanks. The absence of a temperature effect on the survival of offspring is consistent with the findings for the preceding generation. This therefore suggests that an increase of 3 °C from ambient temperature is not lethal on the developmental stages after release from the brood pouch. As a result, it may not be necessary to induce phenotypic changes in offspring such as size, as found in this study. However, with the exclusion of plants only 3 studies have detected thermal trans- generational plasticity in growth, two of which were insects and one an invertebrate fish. In the study conducted by Salinas and Munch (2011), the growth rates of the juveniles fish, Cyprinodon variegates grew faster when their parents were exposed to higher temperatures, and faster growth rates commonly lead to increased survival in fish (Salinas and Munch 2011). Similarly in the milkweed bug, Oncopeltus fasciatus and the butterfuly Bicyclus anynana, offspring growth were also dependent on the temperature experienced by the parents (Groeters and Dingle 1988, Jablonka and Raz 2009). In contrast, a study conducted on the dung fly, found that the effect of maternal thermal environment on offspring growth, was variable among populations (Scharf et al. 2010). Given that, P. parmernong lives in a temperate subtidal environment that encounters a variable temperature regime throughout the year in comparison to the more limited thermal ranges experienced by their tropical or polar counterparts, they maybe relatively tolerable. The manipulation of the offspring’s phenotype to support its survival is only beneficial if mothers can anticipate the environment their offspring will experience (Marshall and Uller 2007). Living in an environment with a relatively wider thermal range, suggests that the environmental temperature is likely to vary from generation to generation and thus there is no incentive to provide any maternal provisioning.

2. Trans-generational effects

2.5.4 Conclusions

The results obtained from this experiment indicate that the increases in temperature that are predicted to occur over the next century by the IPCC, will not have a lethal effect on the marine crustacean P. parmerong. This contradicts the recent findings of Poore et al. (2013), which found elevated temperatures had a significant affect on the survival of this species. The only difference between studies was the times of the year in which the amphipods were collected from the field. It is possible that past thermal histories played a role in the variation. Given that there was no evidence of trans-generational effects of temperature on offspring size or survival, this is difficult to explain. Therefore, this is further investigated in Chapter 3, which focuses on the influence of thermal history to tolerate extreme temperatures over successive generations.

The current study did, however, indicate that elevated temperatures would have a negative effect on P.parmerong reproduction. Females that developed in warmer temperatures produced fewer viable offspring. Investigations, either through modelling or multigenerational experiments may give greater insight into the long-term implications of this on the population dynamics of this species. Similarly, an examination of the effect of temperature on behaviour, gonad maturity, sperm sensitivity, fertilisations and egg development is needed to establish the underlying mechanisms for the reduced fecundity.

Chapter 3

Short term acclimation buffers against thermal extremes in a temperate marine crustacean

3.1 ABSTRACT

The investigation of thermal sensitivity provides an opportunity to evaluate how close a population may be to its thermal limits, and the capacity of individuals to tolerate extreme conditions (e.g. heat waves). It is, however, only through sampling the variation in the tolerance within a population and across generations that a true representation of the susceptibility of a species to global warming can be provided. Survival of the amphipod Peramphithoe parmerong was unaffected when reared at their maximum habitat temperature (~26 ˚C), through physiological acclimation (See Chapter 2). To further explore the vulnerability of this species to increased temperature, a detailed investigation was conducted on the effects of acclimation on their thermal sensitivity to both average increases and extreme thermal events. This study revealed that the critical thermal maximum (CTmax) of P. parmerong did not differ with sex, size or life history stage. Neither did the environmental conditions of the previous generation affect heat tolerance, suggesting that there are no trans- generational effects associated with thermal sensitivity. However, recent exposure to the maximum temperatures typically experienced in its habitat increased their thermal tolerance by approximately ~1.5 ˚C. Acclimatization, may thus buffer individuals against thermal extremes. Furthermore, I demonstrate that the estimation of CTmax is dependent on the procedures used to measure thermal tolerance of individuals. The rate of temperature increase, but not starting temperature, affected the estimation of CTmax, indicating that future investigations should consider realistic rates of temperature increase, particularly for aquatic organisms. Previous studies with high rates of temperature increase are likely to have overestimated the capacity to tolerate thermal extremes

3.2 INTRODUCTION

Temperature is one of the most influential abiotic factors, affecting an organism’s metabolism (Donelson and Munday 2012), growth (Sibly and Atkinson 1994, Atkinson and Sibly 1997), fecundity (Prasad et al. 2011), feeding rates (O'Connor 2009, Vucic-Pestic et al. 2011), behaviour (Huey and Tewksbury 2009) and ultimately survival. All organisms have an optimal thermal range that allows them to maintain functioning under fluctuating temperature conditions (Angilletta, 2009). These fluctuations are, however, encroaching more frequently and with greater intensity into the upper extremes (Jones et al. 2007, Field et al. 2012, Rummukainen 2012). The occurrence of heatwaves has become increasingly obvious in the last decade (Barriopedro et al. 2011, Karoly et al. 2013) coupled with inclines in the mean surface temperature (Perkins et al. 2012). The ocean may have a higher heat capacity, but the marine environment is not except from the occurrence of extreme temperature events (Pearce and Feng. 2012, Abraham et al. 2013). As the predicted averages continue to incline both on land and in the ocean, so too with the prevalence of heatwaves (Field et al. 2012, Rummukainen 2012). Given this, efforts to explore the thermal sensitivity of a species is important (Huey et al. 2012). It not only establishes their relative proximity to their current thermal limits, but it also provides insight into their capacity to respond to further increases (Somero 2009).

Thermal sensitivity is variable among species, populations and individuals (Somero 2010, Huey et al 2012). This can be attributed to genetic adaptation to past environments or acclimation to the current one (Somero 2010, Hoffmann and Sgro 2011). For ecothermic organisms that are unable to thermoregulate, two general patterns have been observed (Tewksbury et al. 2008, Somero 2010). Firstly, those adapted to environments with limited seasonal variability in temperature appear to be more vulnerable than those in a moderately fluctuating thermal environment (Sunday et al. 2011b). This is in response to the thermal breath in which they have adapted to (Richard et al. 2012). Ectotherms genetically evolved to a stable environment (e.g. Tropical, marine polar regions), have a lower acclimation capacity because the need to tolerate temperatures beyond their optima has been negligible in the past (Clark and Peck 2009, Peck et al. 2010). Secondly, species living in warmer environments, which experience temperatures, near or above their optima, are potentially at a greater risk (Overgaard et al 2011). Latitudinal studies on ectothermic organisms have identified that congeners of a species have relatively similar upper thermal limits (Stillman 2003, Deutsch et al. 2008). Thus, individuals of a given species in topical areas are living closer to their maximum thermal tolerance in comparison to their temperate counterparts

3. Thermal extremes

(Teksbury et al., 2008). This has been mirrored for ectotherms residing in the littoral zone of the marine environment (Stenseng et al. 2005, Dong and Somero 2009). Intertidal congeners are believed to be living closer to their thermal limits than those of the subtidal zone (Tomanek and Somero, 1999, Stenseng et al., 2005). Thus any further increases may exceed their absolute capacity. As temperatures rise and extreme events become more frequent, the ability for some organisms to tolerate further increases may be limited due to their previous and current thermal conditions. To quantify the current thermal sensitivity of a species or population, past thermal histories and sources of variation should be assessed. It is only through which a true representation of their current vulnerability to climate change can be revealed.

Within a population or species, many studies have identified that the physiological tolerance of ectotherms to temperature can differ between sexes (Willet 2010), life history stages (Madeira et al. 2012) as well as the size (Peck et al 2009) of individuals. The underlying reasons driving such variation may be attributed to a difference in energetic availability (Nyamukondiwa and Terblanche, 2009, Min and Chin 2012) or capacity to compensate via other means (e.g. morphological or behavioural) (Peck et al 2009, Bowler and Terblanche 2008). If a specific size, sex or life history stage is more thermally sensitive, the vulnerability of the species or population to global warming could be greater than predicted if experiments failed to account for these sources of variation (Bowler and Terblanche 2008, Byrne 2010). If juvenile stages die before they reach maturity, the implications of thermal stress on adult stages may become irrelevant (Byrne 2010). Similarly if a particular sex is more vulnerable, fewer viable mating partners could potentially lead to reductions in abundance (Perrin 1993, Peck et al 2009). Age and size are closely related in most species, and the offspring of larger and older individuals can have greater survival potential than younger, smaller ones (Bireland & Dayton 2005). Therefore, size and age distributions within a population will affect the thermal sensitivity of a population, and predicting thermal limits requires the identification of variance within a population.

Understanding the thermal sensitivity of a species also requires evaluating potential non- genetic transfer of tolerance across generations. As mentioned in Chapter 2, tolerance to environmental stressors may be influenced either by the environmental conditions of the previous generation, or the parents themselves (more often then not, by mothers) through trans-generational effects. These have, in the past, commonly been associated with predation, pollution or starvation (Marshall et al. 2008, Storm and Lima 2010, Fernandez- Gozalez et al. 2011). Although, several studies have identified a relationship between elevated temperatures and trans-generational effects in offspring growth rates (Salinas and

3. Thermal extremes

Munch 2011), dispersal potential (Burgess and Marshall 2011) and metabolic performance (Donelson et al. 2011). There is, however, a limited understanding of how acclimation to elevated temperatures in the previous generation may influence the thermal sensitivity of the subsequent generation.

In the subtidal habitats of the marine environment, temperature fluctuations are not as severe as those experienced in terrestrial systems. The rates of temperature change are slower, and the exposure to extreme temperatures can last for extended periods of time (e.g. days to weeks) (Wernberg et al. 2012). Studies conducted on terrestrial ectotherms find that the rate at which temperatures change can have a significant effect on thermal tolerance (Terblanche et al. 2007b, Chown et al. 2009). Commonly, if temperatures rise at a slower rate, thermal tolerance is significantly reduced (Chown et al. 2009, Terblanche et al. 2011). In the upper subtidal temperate regions (<15m), temperatures commonly fluctuate by only few degrees each day and by 5–10 C over the course of the year (Smale and Wernberg 2009, Malcolm et al. 2011). The relative narrow thermal range and slower rate of temperature change of the ocean environment could potentially put many marine ectotherms at risk with the occurrence of heatwaves. Currently, temperate species have the capacity to tolerate their maximum habitat temperature (see Chapter 2), but the occurrence of marine heat events, adds an additional factor that has yet to be considered. Of particular concern is the influence of their prevalence, duration and rates of temperature change on marine organisms.

Extreme weather events, such as “marine heat-waves”, shift temperatures well beyond the average increases and may push many species over their absolute thermal thresholds. In 2011, unseasonably high temperatures were recorded off the coast of Western Australia with near shore regions experiencing temperatures ~5 C above average for a period of a week (Pearce and Feng 2012). This had a significant impact on many marine organisms including lobsters, abalone, corals and fish (Pearce and Feng 2012, Wernberg et al. 2012). Therefore, it is imperative to establish the capacity of vulnerable species to tolerate thermal extremes. A commonly used method to determine the thermal tolerance of a species is to establish their critical thermal limits (Lutterschmidt and Hutchison 1997, Sunday et al. 2011b). This involves changing the temperature at a constant rate until the onset of either muscular spasms, loss of mobility or death (the latter two commonly applied to ectotherms) (Lutterschmidt and Hutchison 1997, Terblanche et al. 2011). The temperature at which this endpoint occurs establishes the critical thermal maximum (CTmax) or critical thermal mimimum (CTmin) (Lutterschmidt and Hutchison 1997). This method has found to be very effective in establishing the variation in thermal tolerance among species, populations or individuals (Lutterschmidt and Hutchison 1997, Terblanche et al 2011, Sunday et al 2011b).

3. Thermal extremes

The use of this method does, however, come with procedural issues that may influence the end results (Terblanche et al. 2007a, Mitchell and Hoffmann 2010, Rezende et al. 2011). Many studies conducted on the critical thermal limits of aquatic species use rates of increase that represent those experienced in terrestrial systems (Buchanan et al. 1988b, Lagerspetz and Bowler 1993, Cuculescu et al. 1998, Diaz et al. 2002, Hopkin et al. 2006, Dallas and Ketley 2011). The thermal sensitivity of terrestrial ectothermic organisms is, however, strongly influenced by the rate at which the environmental temperature changes (Terblanche et al. 2007, Chown et al. 2009, Peck et al. 2009, Rezende et al.2011). The slower the temperature change, the lower the thermal tolerance. Given that the rates of temperature change are very different between terrestrial and subtidal marine systems, a true representation of the thermal tolerance of many marine ectothermic organisms, may be significantly overestimated. Furthermore, the past thermal histories of organisms are likely to differ. This may be attributed to a controlled variation in environmental temperature to assess acclimation capacity and/or associated to the collection of organisms from different seasons or thermal environments. When establishing the critical thermal limits (e.g CTmax and CTmin), the initial starting temperature, should be considered. Ideally, the temperature from which the organisms are ramped from, among treatments, need to be the same (Chown et al. 2009). This, however, is complicated by the fact that the experimental individuals most recent thermal environments are likely to differ (due to acclimation treatment, season, location). Whether a shift from an individual’s recent thermal exposure to the initial starting temperature, affects their thermal tolerance, remains unclear. Thus, it is important that these procedural issues are verified prior to assessing the thermal sensitivity of a species.

In this study, the specific focus is therefore to assess the CTmax of the amphipod Peramphithoe parmerong and how experimental procedures affect it. Previously, in Chapter 2, it was identified that P. parmerong showed no significant difference in survival between ambient temperature (23 C) and an elevated temperature of +3 C (26 C). However, it is uncertain if this influences their thermal tolerance. We therefore asked the following questions; (1) How does the CTmax vary among sexes and life history stages? (2) Does the thermal environment in which they are acclimated to, or that of the preceding generation, affect their thermal sensitivity? (3) How does the rate of temperature increase and starting temperature used to establish the critical thermal maximum affect thermal tolerance?

3. Thermal extremes

3.3 METHODS

3.3.1 Study organisms

Peramphithoe parmerong were sourced from Sargassum spp. at low tide from a depth between 1–3 m at Charlesworth Bay (3018.036’S 15308.319’E), Diggers Beach (30° 16.396' S 153° 8.709' E) and Mutton Bird Island Nature Reserve (3018.169’S 15308.552’E), NSW, Australia. These sites are less than 5km apart and are expected to face similar thermal regimes. Amphipods were separated from Sargassum spp. in the laboratory using the methods detailed in Chapter 2. Males, females and juveniles were held together in several holding tanks (n>300), under ambient conditions (Salinity; 33.59  0.08ppm, Temperature 20.67  0.31C) in a flowthrough sea water system at the National Marine Science Centre, Coffs Harbour. Amphipods were maintained in these tanks for several months until placed into controlled temperature treatments. Fresh Sargassum vestitum was provided and replenished regularly, and tanks were cleaned once a week.

3.3.2 Variation in CTmax between sex and life history stage

To establish the critical thermal maximum (CTmax), male, female and juvenile amphipods were removed from holding tanks and placed individually into a 60 ml plastic container filled with sea water. Previous studies including Pease et al 2010 and Poore et al l999 have identified that P.parmerong can successfully grow to maturity after a period of approximately 4 weeks in such containers. The water temperature was increased from ambient (20 C) using a digital temperature controlled water bath (Polyscience model 9102), at a rate of 0.06.min-1 until amphipod movement ceased, at which point they were declared dead. Healthy amphipods continually beat their pleopods to maintain water flow over their gills. The temperature that this stopped was recorded using four Thermodata IBTAG’s placed within containers within the water bath. The experiment was repeated three times (blocks) each time using eight individuals per male, female and juvenile group. At the end of the experiment, a photograph of each amphipod was taken to determine size (Image J) (methods as in Chapter 2).

The CTmax was contrasted among males, females and juveniles with a two-factor analysis of variance with the sex/life history stage of the amphipods a fixed factor and experimental block a random factor. The relationship between size and CTmax was analysed with a linear regression, with separate analyses conducted for each of males, females and juveniles.

3. Thermal extremes

3.3.3 Variation in CTmax with recent thermal history

Exposure to increased temperatures often improves tolerance to thermal extremes within ectothermic organisms. To investigate if the thermal environment in which amphipods developed affected their thermal tolerance, the critical thermal maximum was contrasted among groups of amphipods that had been acclimatised to different temperatures. Juvenile amphipods (<3 mm) were placed into a temperature treatment of 20 C (20.75  0.33 (s.e.)) or 26 C (26.04  0.03 (s.e.)) within a temperature manipulated flowthrough seawater system (See Chapter 2 for system setup). For each temperature, ten amphipods were placed into a single 60 ml container with flow through seawater (flow rate c.a ~0 9 ml.sec-1, 300-350 turnovers.day-1) with a total of 30 containers per treatment. Amphipods developed in either temperature for a period of 28 days, which is sufficient time for this species to reach maturity (Poore and Steinberg, 1999). Containers were cleaned once a week and amphipods were fed Sargassum vestitum, which was regularly replaced to ensure amphipods were fed in excess.

The temperatures used in this experiment reflect the upper and lower thermal range of P. parmerong in the location that amphipods were collected (Figure 1.1, Chapter 2). Assuming SST increase by 2-3 C on average over the next century (Poloczanska et al. 2007b, Hobday and Lough 2011) amphipods are likely to experience a water temperature of 26 C more frequently, and during short-term extreme events may be exposed to temperatures well beyond this.

The CTmax of amphipods acclimated to either 20 C or 26 C for 28 days were measured using the methods described above. Amphipods were placed individually into a 60 ml container into a thermally controlled water bath and ramped at a rate of 0.02 C.min-1, until movement ceased, at which point they were declared dead (n = 6-10). Due to the duration of the ramping experiment, amphipods were provided a piece of Sargassum vestitum (~5 mm2) as a source of food. This was large enough (~1 cm2) to ensure amphipods did not starve for the duration of the experiment (~7 hours). At the end of the experiment, all amphipods were photographed and both the size (Image J) and sex were determined.

The effect of the thermal environment in which amphipods developed was analysed with a one-factor analysis of variance with acclimation temperature as a fixed factor. The sex and size of the amphipods was not included in the analyses due to their insignificance in the previous experiment.

3. Thermal extremes

3.3.4 Thermal tolerance and trans-generational effects

To further explore the influence of temperature acclimation on the thermal tolerance across generations and the potential role of trans-generational effect, thermal tolerance of amphipods acclimated over two generations was investigated. This was performed by assessing the thermal sensitivity of the offspring from the experiment outlined in the previous chapter (Chapter 2). By exposing two successive generations to an ambient thermal environment of 23 C or an elevated thermal environment of 26C, in all combinations, the influence of past thermal environments on an organism’s current thermal sensitivity can be assessed. Once the progeny of gravid females (acclimated to either temperature), developed in either thermal environment for a period of 28 days, in each combination of treatments (maternal environment x offspring environment), their capacity to tolerate an acute extreme thermal environment was measured. Although, due to the large quantity of offspring emerging at different time periods, and the duration required to establish the CTmax, thermal tolerance was assessed using a different method.

This was carried out by placing all individuals at 28 days of age into a water bath kept at a constant temperature of 33 C, and recorded the time until death. Prior to being placed into the water bath, 30 ml plastic vials were filled with 20 ml of filtered seawater (0.2 m). Amphipods were individually placed into separate vials once the filtered seawater had reached the required temperature. Temperature was recorded every 15 mins thereafter. Time was started once all amphipods had been placed into the water bath to a maximum of 24 vials at any one time (n = 70-140). The same endpoint as described previously was used. Once this occurred, amphipods were removed from the water bath and the time taken to reach this point was recorded. The thermal assay lasted until all amphipods had died.

The difference in time to reach the endpoint was contrasted among groups of offspring that developed in either temperature (F2), and from parents acclimated to either temperature (F1) with a four-factor nested analysis of variance. The temperatures for the F1 and F2 generation were fixed factors with the tank that the F1 generation developed in a random factor nested within F1 temperature treatment. The family of the offspring (F2) was also a random factor nested within both Tank and F2 temperature treatment. Size of amphipods was contrasted across treatments also with a four-factor analysis of variance to ensure consistency and exclude possible bias. Factors were as mentioned above.

3. Thermal extremes

3.3.5 Methodological effects on thermal tolerance

Thermal tolerance of an organism, established by increasing their thermal environment until they reach an endpoint, may be affected by the protocols used. Exploring the effect of acclimation on the critical thermal maximum of an amphipod highlighted two possible methodological issues. The first is the temperature at which the ramping experiment is conducted at and the second is the rate at which the temperature rises. Using the amphipods acclimated to the two thermal environments of 20 C and 26 C for a period of 28 days (as mentioned above), an experiment was conducted on how variation in these methods may affect the outcome of amphipod thermal tolerance experiments.

The CTmax of amphipods acclimated to 20 C and 26 C was compared between two starting temperatures (of either acclimation temperature) of 20C or 26C, and between two rates of temperature increase (0.02C.min-1 and 0.06 C.min-1) in all combinations. This established how thermal tolerance varied between the acclimation temperature and the starting temperature. Furthermore, it also determined whether a realistic rate of temperature increase (0.02 C.min-1) (Figure 2.1) differed from a generic ramping rate (0.06 C.min-1), commonly used in the literature (Kelly et al 2011, Gonzalez et al. 2010, Dallas and Kelley 2011). Amphipods were randomly allocated to a treatment, and subsequently placed into the water bath once the allocated temperature treatment was reach at a given rate (n = 6-10). The CTmax assay was run as described above, and the time taken for amphipods to die was recorded and so too the temperature at which this occurred (thermodata temperature logger, IBCod22L).

The CTmax of amphipods was contrasting among acclimation temperatures, starting temperatures and rates of temperature increase with a three-factor analysis of variance. Acclimation temperature, starting temperature and rate were all fixed factors. The sex or the size of the amphipods were not included in this analysis due to no effects of these factors on CTmax in the previous experiment.

3.3.6 Statistical analyses

Analyses of variance and linear regression were performed in PASW Statistic (V.20) and Primer+ PERMANOVA. Frequency histograms of the residuals and plots of the residuals were performed to ensure that the assumptions of the normality and homogeneity of variance were met. The thermal assay on the thermal acclimation over successive generations was transformed by the square root to meet these assumptions.

3. Thermal extremes

3.4 RESULTS

3.4.1 Variation in CTmax between sex and life history stage

All amphipods reached their maximum thermal tolerance at approximately 32 C (Figure 3.1a) when ramped from 20 C at a rate of 0.06 C.min-1. There was no significant difference among the thermal tolerance of males, females or juveniles (Table 3.1). There was also no relationship between the size of amphipods and their CTmax (Figure 3.1b) for males (P = 0.63, R2 = 0.014), females (P = 0.93, R2=0.0005) or juveniles (P=0.45, R2= 0.044).

3.4.2 Variation in CTmax with recent thermal history

The thermal environment that amphipods were acclimated to (20 C or 26 C) during their development as juveniles had a significant effect on thermal tolerance (Figure 3.2) Amphipods that developed at 20 C had a lower tolerance to extreme temperatures than in comparison to amphipods that developed at a temperature of 26 C (F1,14 = 7.11, P < 0.01).

3.4.3 Thermal tolerance and trans-generational effects

The thermal environment experienced by amphipods affected tolerance to extreme temperatures, but the thermal environments experienced by their maternal environment did not (Figure 3.3). Amphipods that developed in the lower temperature of 23 C from hatching to maturity had significantly lower thermal tolerance, and thus died sooner, than those that developed at the higher temperature of 26 C (Table 3.2) when exposed to the lethal temperature of 33 C. This acclimation to thermal extremes in the F2 generation was not influenced by the thermal environment of the preceding generation (F1), nor was there any interaction between the thermal environments of either generation (Table 3.2). There was also no significant difference found with size of amphipods among treatments (P>0.05, figure not shown).

3.4.4 Methodological effects on thermal tolerance

The rate at which temperature was raised during the experimental procedure strongly affected the measured thermal tolerance of amphipods (Figure 3.4) The CTmax of amphipods with the experimental ramping rate of 0.06 C.min-1 had significantly higher thermal tolerance than amphipods that underwent a ramping rate of 0.02 C.min-1 (Table 3.23.3). At both experimental ramping rates, the thermal tolerance of amphipods was always significantly higher for those that were acclimated to 26 C in comparison to those reared in 20 C (Table 3.3). The starting temperature at which the experiment began did not affect the

3. Thermal extremes

CTmax of amphipods at either ramping rate, nor was there interactions among any of the treatments (Table 3.3).

3. Thermal extremes

a) 34

) 32

(

x a

m 30

T C

Male Female Juvenile

Figure 3.1a. Variation in thermal tolerance with life history stage and sex. Figure illustrates the mean ( s.e.) CTmax of males, females and juveniles amphipods from ambient temperature conditions, ramped at a rate of 0.06.C.min-1.

) 34

(

a 32

m T

C 30

4 6 8 10 12 14 Size (mm)

Figure 3.1b. Correlation between the size (mm) of amphipods and CTmax (C) of all individuals including males, females and juveniles. Linear Regression analysis of pooled life history stage and sex was P = 0.4648, y=0.06887x + 32.09, R2 = 0.01.

3. Thermal extremes

) C

˚ 32

(

a m

T 30 C

20 26

Acclimation Temperature (˚C)

Figure 3.2. Comparison of the mean ( s.e) CTmax of amphipods after acclimation to a thermal environment of 20 °C or 26 °C for a period of 28days, ramped at a rate of 0.02 °C.min-1.

F2 50 Temperature (°C) 23 26

40 s

e 30

n i

M 20

0 23 26

F1 Temperature (°C)

Figure 3.3. Comparison of the thermal tolerance time (mins) of the second generation (F2) of P. Pamerong after acclimation for 28 days in a thermal environment of 23°C or 26°C, from parents

(F1) that developed in either thermal environment of 23°C or 26°C. Amphipods were exposed to a static temperature of 33°C until amphipods died.

3. Thermal extremes

Figure 3.4. Comparison of the mean ( s.e) CTmax of amphipods acclimatised to either thermal environment of 20 C or 26 C, ramped from two starting temperatures ( 20 C or 26 C, ) and a two rates ( 0.02 C per.min-1 or 0.06 C per.min-1). Dotted lines denotes rate of temperature increase from initial starting temperature.

3. Thermal extremes

Table 3.1. An analysis of variance contrasting the critical thermal maximum (CTmax) among males, females and juveniles. Sex & Life History Stage (i.e., males vs females vs juveniles) is a fixed factor and experimental ‘Block’ a random factor. The mean squares used as denominators for each F test are as follows: Sex, MSSex*Block. Block, MSSex*Block. Sex * Block, MSError.

a) CTmax Source df MS F P Sex & Life History stage 2 0.538 0.239 0.807 Block 1 0.120 0.053 0.849

Sex & Life History stage*Block 2 2.255 1.946 0.160

Error 30 1.158

Table 3.2. A four-factor nested analysis of variance contrasting the maximum thermal tolerance (time to death) of offspring exposed to a static temperature of 33 °C at day 28 from both the offspring environmental temperature (F2 Temperature) and the environmental temperature of the parents (F1 Temperature). F1 Temperature and F2 Temperature were fixed factors, Tank was a random factor nested in F1, Temperature and Family was a random factor nested in both F2 Temperature and Tank. * denotes a significant effect (P < 0.05). The mean squares used as denominators for each F test are as follows: F1 Temperature, 0.492MS Tank(F1 Temperature) + 0.14MSF2 Temperature*Tank(F1 Temperature) + 0.099MSFamily (F2 Temperature*Tank(F1 Temperature)) +0.395MSError; F2 Temperature, 0.885MSF2 Temperature*Tank(F1 Temperature) + 0.45MSFamily(F2 Temperature*Tank(F1 Temperature)) + 0.070MSError; Tank (F1 Temperature), 0.814MSF2 Temperature*Tank(F1 Temperature) + 0.35MSFamily(F2 Temperature*Tank(F1 Temperature)) +0.151MSError; F1 Temperature*F2Temperature, 0.881MS(F2 Temperature*Tank(F1 Temperature)) + 0.47MS(Family(F2Temperature*Tank(F1Treatment))) + 0.072MSError; F2 Temperature*Tank(F1 Temperature), 0.589MSFamily(F2 Temperture*Tank(F1 Temperature)) +0.411MSError; Family(F2 Temperature*Tank(F1Temperature)), MSError.

Source df MS F P

F1 Temperature 1 0.015 0.009 0.926

F2 Temperature 1 149.218 87.693 <0.001*

Tank (F1 Temperature) 5 2.099 1.273 0.390

F1 Temperature * F2 Temperature 1 2.042 1.200 0.323

F2 Temperature * Tank (F1 Temperature) 4 1.717 0.949 0.438

Family (F2 Temperature * Tank (F1 Temperature)) 75 2.279 2.008 <0.001* Error 363 1.135

3. Thermal extremes

Table 3.3. A three-factor analysis of variance contrasting the CTmax of amphipods acclimatised to two temperatures (20 C or 26 C), ramped from two starting temperatures of either 20 C or 26 C, and at two rates of 0.02C per.min-1 or 0.06C per.min-1. Starting Temperature, Acclimation Temperature and Rate were all fixed factors. * denotes a significant effect (P < 0.05). The mean squares as denominators for each F test are as

follows: Starting Temperature, MSError; Acclimation Temperature, MSError.; Rate, MSError.; Starting Temperature* Acclimation Temperature, MSError.; Starting Temperature * Rate, MSError.; Starting Temperature* Acclimation Temperature * Rate, MSError..

CTmax (˚C) Source df MS F P Starting Temperature 1 1.648 2.606 0.111 Acclimation Temperature 1 26.591 42.049 <0.001* Rate 1 119.925 189.639 <0.001* Starting Temperature * Acclimation Temperature 1 0.293 0.204 0.653 Starting Temperature* Rate 1 0.149 0.236 0.629 Acclimation Temperature* Rate 1 0.107 0.169 0.682 Starting Temperature*Acclimation Temperature*Rate 1 0.230 0.364 0.548 Error 66 0.632 Total 74

3. Thermal extremes

3.5 DISCUSSION

This study identified that the current physiological thermal maximum for P. parmerong is about 7–10 C above the maximum habitat temperature. This is, however, dependent on the thermal history of the amphipod, with recent exposure to higher temperatures increasing the heat tolerance of this species. Heat tolerance was found to be uniform across size, sex and life history stage of individuals within this population. There was also no relationship found between the thermal environment of the previous generation and the thermal sensitivity of their offspring. This suggests there are no trans-generational effects and that heat tolerance of an individual is entirely dependent on their current thermal environment. The protocols used to determine the critical thermal limits strongly affect the estimation of thermal tolerance suggesting that caution should be taken when conducting these experiments and interpreting the results of many others.

3.5.1 Variation in thermal sensitivity among individuals and with recent exposure to elevated temperatures

Thermal tolerance among individuals of P. parmerong did not differ between sex or life history stage. This suggests that all individuals within this population have the same vulnerability to increasing temperatures. Therefore, acute temperature increases are unlikely to be a selective force against certain individuals and thus will not alter recruitment or the mean populations thermal sensitivity in this manner. It should, however, be noted that prolonged temperature increases may affect recruitment as identified in Chapter 2 for this species of amphipod.

Consistent with other work, this study found that thermal acclimation increases heat tolerance. In Chapter 2 it was identified that survival of this species of amphipod, is unaffected when they developed under their maximum habitat temperature, however, here it was shown that this does improve their heat tolerance. The mean CTmax for individuals acclimated to the highest thermal temperature, was 33.27 C ( 0.32), whereas amphipods acclimated to 20 C had a mean heat tolerance of 30.58 C ( 0.46). Many studies have identified that both terrestrial and marine ectotherms, increase their CTmax after thermal acclimation (Chown et al. 2009, Dallas and Rivers-Moore 2012, Ravaux et al. 2012). This, increase in tolerance is commonly associated with the activation of heat shock proteins (Tomanek and Somero 1999, Hoffmann et al. 2003, Sorensen et al. 2003). Under normal conditions, these molecular chaperones are used to create protein homeostasis by transporting, refolding, breaking down and replacing denatured proteins (Tomanek 2008a, Hofmann and Todgham 2010). Under stressful situations, as with an increase in temperature

3. Thermal extremes through acclimation or hardening, Hsp are induced and expand the thermal range and thermal limits of an organism (Hoffmann et al. 2003, Sorensen et al. 2003). The relationship between Hsp and thermal tolerance has also been observed in species of amphipods in freshwater and marine systems (Bedulina et al. 2010, Shatilina et al. 2011). This level of expression is, however variable among species with some amphipods having a higher level of Hsp’s under control conditions then others (Shatilina et al. 2011). This is found to correlate to the thermal environment of their habitat and believed to be pre-adaptive responses to past thermal environments (Shatilina et al. 2010). The underlying mechanisms for these response (e.g. trans-generational effects or genetic adaptation across generations) are not well defined.

Comparing the influence of acclimation on thermal tolerance between studies on amphipods is difficult as either different methods are used (e.g. LT50) (Bedulina et al. 2010, Shatilina et al. 2010) or the rate at which temperatures are increased to determine the critical thermal limits, are variable and/or unrealistic (Buchanan et al. 1988a, Lahdes et al. 1993). This study identified that P. parmerong, a temperate species, is able to increase thermal tolerance by approximately 1.5 C after recent exposure to a 6 C increase from ambient conditions. In comparison, two amphipod species from Antarctica, Onisiums sp. and Paraceradocus gibber were unable to increase their thermal tolerance after exposure to temperatures between 3 C and 6 C above ambient for a period of 2 months (Peck et al. 2010, Richard et al. 2012). This variation, between species from different latitudes coincides with the theory that ectotherms adapted to habitats with a relatively small thermal breadth (e.g. polar marine habitats) have a lower acclimation capacity than species that experience larger thermal fluctuations in their environment (i.e., temperate habitats) (Tewksbury et al. 2008, Somero 2010). In which case, in comparison to amphipods in the Antarctic regions, P. parmerong is relatively robust. This will however, depend on the differences in temperature inclines as well as the frequency, intensity and duration of extreme heat events.

Emerging research does, however, suggest that acclimation capacity should be assessed at a population level (Donelson and Munday 2012, Kelly et al. 2012). The ability to increase thermal tolerance in this population of P. parmerong, may not apply to all populations across their species distribution. A study conducted by Kelly et al. (2012), on genetically different populations of the copepod Tigriopus californicus, found that there were large differences in thermal tolerance among populations. In addition, selection over 10 generations on populations adapted to the cooler climate habitats did not result in the copepod expressing the tolerant phenotypes observed in the populations of the warmer environments. This suggests that the tolerant traits expressed by a particular genotype required to adapt to further

3. Thermal extremes increases in temperature, may not be present in all populations within a species. In which case, if a species has low genetic dispersal and long generation times, some populations will be more vulnerable than others. P. parmerong has a large distribution (southern Queensland to Tasmania), and their dispersal potential on large scales is unknown. Therefore, it is possible that some populations are more vulnerable than others and the results of this study should not be used as a generalisation for the entire species.

3.5.2 Thermal tolerance and trans-generational effects

Thermotolerance of early developmental stages in marine species can be affected through trans-generational acclimation (Fujisawa 1995, Bingham et al. 1997, Donelson et al. 2011), but emerging research suggests that this increase in tolerance can be subsequently passed on to successive generations (Donelson and Munday 2012). However, in the current study, heat tolerance appears to be entirely influenced by the environment in which an individual develops, as there was no indication of increased tolerance in association with the thermal conditions faced by the previous generation. Given the results of this study, it implies that the recent environment of offspring is far more influential on their capacity to tolerate thermal extremes than any possible non-genetic provisions provided by the parents. The identification that this is not affected by past thermal conditions indicates that this species of amphipod, is entirely dependent on genetic adaptation if they are required to change. However, further research could investigate the possible trans-generational effects at a physiological level in association with Hsp (Sconzo et al. 1997, Pijanowska and Kloc 2004), antioxidant enzymes (i.e. peroxidase, catalase, glutathione S-transferase) (Ortiz-Rodriguez et al. 2012) or metabolic performance (Donelson et al. 2011), that could decrease heat stress but are not evident in response to acute thermal tolerance.

3.5.3 Procedural issues

The protocols used to determine the critical thermal maximum vary from study to study. Commonly, experiments exploring the effects of heat tolerance in aquatic organisms increase temperatures at a rate well beyond those experienced in the environment (Re et al. 2005, Hopkin et al. 2006, González et al. 2010, Dallas and Ketley 2011, Ravaux et al. 2012). As identified in the procedural experiment in this study, and those conducted on terrestrial (Terblanche et al. 2007b, Chown et al. 2009, Mitchell and Hoffmann 2010), and marine ectotherms (Peck et al. 2009, Nguyen et al. 2011), heat tolerance is influenced by the rate at which temperatures increase. The CTmax of amphipods was lower when the temperature was ramped at a rate of 0.02 C.min-1 in comparison to when it was ramped at a faster rate of 0.06 C.min-1, irrespective of acclimation temperature. This in turn can influence the

3. Thermal extremes estimates of adaptive potential or heritability of thermal tolerance in a species or population (Mitchell and Hoffmann 2010). It also makes it difficult to compare tolerances among studies. However, the variation in rate of temperature change influencing thermal tolerance also has ecological relevance, particularly in association extreme heat events.

The slowest rate used in this study was 0.02 C.min-1, which has been observed in the subtidal environment near the study site (Malcolm et al. 2011). However, these changes can also occur at a much slower rate (Malcolm et al. 2011). In which case, if heat waves occur over a gradual period (e.g. a week), and is held constant over a time frame of days to weeks, this species of amphipod among many other subtidal marine ecotherms, could have a much lower thermal tolerance. The marine heat wave experienced in Western Australia in the summer of 2010/2011 encountered temperatures 2–4 C above average and persisted for more than one week for a given area (Pearce and Feng 2012, Wernberg et al. 2012). Therefore, scientific studies should not only be focusing on the average inclines in temperature with global warming, but also the potential impact of extreme events such as heat waves.

3.5.4 Conclusion

The thermal sensitivity of the amphipod Peramphithoe parmerong living in the temperate subtidal environment appears to be relatively robust. This is because their CTmax is approximately 10 C higher than what they currently experience today. In addition, their heat tolerance can be increased through acclimation to exposure to temperatures close to their current maximum habitat temperature. However, these estimates of thermal tolerance are dependent on the procedural methods used, and the results obtained here should be used as a guideline for their absolute maximum heat tolerance to acute thermal extremes. It should also be noted that fecundity is reduced at their current maximum habitat temperature (e.g. 26 C) (Chapter 2) and that the endpoint of death (as measured in studies of CTmax) is not the only trait affecting the abundance of the population over time. Furthermore, Poore et al. (2012), found that this species was unable to acclimate to 1 C above their maximum habitat temperature (e.g. 27 C), as there was approximately ~70% reduction in survival of juveniles at this temperature after 14 days. Therefore, if they cannot acclimatise to temperatures above their current maximum habitat temperature, they will entirely depend on their ability to genetically adapt.

Chapter 4

Effects of pH and temperature on the growth and palatability of temperate macroalgae

4.1 ABSTRACT

Emerging research suggests that population declines and extinctions associated to climate change will primarily be associated to biotic interactions, rather than physiological intolerances. Phenological shifts, migration or the direct decline in abundance of a species at a population level will have cascading effects on co-dependent organisms. One area of research that has been understudied is how climatic stressors such as temperature and CO2 may directly affect productivity of primary produces and indirectly affects their susceptibility by consumers. Here, I explore how the simultaneous exposure of rising temperatures and increasing CO2 (coupled with a lowering pH) affect the biomass of six commonly occurring temperate macroalgal species. Furthermore, I investigated the influence of how the development of these species of alga under these two stressors affects their palatability to a co-occurring consumer. The main findings of this study identified that five of the six species of macroalge studied experienced reduced growth with elevating temperatures. Two of these alga species also experienced reduced growth with a lowering pH environment. However, there was no interaction between the two treatments. Furthermore, a lowering pH was, also found to increase the susceptibility of Dictyopteris acrostichoides to the marine snail Phasianotrochus eximius, as greater amounts were consumed when grown in an increasingly acidic environment.

4.2 INTRODUCTION

As our climate changes over the next century, the fate of many species remains uncertain. Some will persist by shifting their geographical range, altering their phenonology or evolve greater tolerance to new conditions, while others will be lost to extinction (Bell and Collins 2008, Visser 2008, Chown et al. 2010). These changes, at a population level, in turn, will alter the species interactions and the dynamics of food webs (Doney et al. 2012, Hughes 2012). As temperatures rise and atmospheric carbon dioxide increases (with the associated lowering of pH in the oceans), the direct effects that each of these stressors place on survival, development and reproduction of many organisms is becoming increasingly clear (Orr et al. 2005a, Parmesan 2006a). How these direct effects, however, may cascade through multiple trophic levels is only beginning to be understood (Hoegh-Guldberg and Bruno 2010b, Van der Putten et al. 2010). Emerging research suggests that population declines and extinctions related to climate change are not primarily a consequence of physiological intolerance to the shifting environmental conditions, rather a response to changes in biotic interactions (Cahill et al. 2013). Changing climate may affect the relationship among predators and prey (Graham et al. 2009, Durance and Ormerod 2010, Pearce-Higgins et al. 2010), herbivores and primary producers (McLaughlin et al. 2002, O'Connor 2009), pathogens and their hosts (Rohr and Raffel 2010, Campbell et al. 2011) or competitors (Durance and Ormerod 2010, Diaz-Pulido et al. 2011). By disrupting species interactions, predicting the ecological impacts of climate change may be further complicated by the indirect effects.

The interaction between plants and herbivores are an integral component to food-web dynamics. The dietary choices of herbivores influence the structure and composition of plant communities (Huntly 1991), whilst plants influence herbivore performance through their physical and chemical characteristics (Amsler 2008, Schaller 2008). Shifting climatic conditions may alter plant-herbivore interactions by directly affecting the abundance of either interacting species (Hughes 2011b), or by changes to the traits of either species that affect the per-capita interaction strength (O'Connor 2009). Metabolic theory predicts rising temperatures will generally increase metabolic rates in both plants and their co-occurring consumers (O'Connor 2009). An increase in consumption rates by herbivores should strengthen their top-down control of plant communities, however, this is complicated by possible concurrent shifts in abundance, palatability, or the quality of food (Dury et al. 2002, Murray et al. 2013). Elevated carbon dioxide concentration and increases in temperature may directly alter the biomass of a viable food source or it may alter the chemical defences or

4. Algal Growth & Palatability

nutritional value of plants (Zvereva and Kozlov 2006). This in turn has the potential to feedback onto the fitness of herbivores as well as the structure and dynamics of plant communities.

Our understanding of possible synergistic effects of rising temperatures and atmospheric

CO2 concentrations on the productivity of primary producers and the key traits driving the susceptibility of herbivory is in its early stages. In terrestrial C3 plants (plants that convert

CO2 and ribulose bisphosphate into 3-phosphoglycerate), a warming of 3-4 C increases photorespiration, which reduces the efficiency of photosynthesis (Long 1991, Sage and Kubien 2007). A reduction in photosynthesis in turn will lead to a loss in growth and productivity, although CO2 enrichment can offset this, as it has been shown to raise the thermal optima of C3 plants in terrestrial systems (Sage and Kubien 2007). However, because these two stressors have the capacity to further alter the biochemistry and physiology of plants, sub-lethal effects associated with changes to nutritional content, chemical defences and digestibility may also affect the susceptibility of plants to their herbivores (Harley et al. 2012). Plant susceptibility to herbivores is strongly affected by the presence of chemical defenses (also known as secondary metabolites) in both terrestrial and aquatic systems (McClintock and Baker 2001, Amsler 2008). Secondary metabolites can deter herbivores through unpalatable tastes, smells, or lead to negative effects on fitness once consumed (Paul et al. 2001, Toth et al. 2005). It has been demonstrated that both elevated temperature and carbon dioxide can independently alter the nutritional content of food (e.g. carbohyrdates, nitrogen, nitrogen: carbon ratio) and defensive chemical compounds (e.g. phenolics, terpene) (Zvereva and Kozlov 2006, Sudatti et al. 2011), but few studies have considered how these two climatic signals may interact to affect plant performance and susceptibility to herbivores.

In the marine environment, the direct impact of exposure to rising temperatures and ocean acidification is increasingly well known for many marine organisms (e.g. urchins, molluscs, crustaceans, fish) (Hoegh-Guldberg and Bruno 2010a, Kroeker et al. 2010, Doney et al. 2011) and there is an emerging literature that considers how these two stressors may interact (Byrne et al. 2009, Parker et al. 2009, Brennand et al. 2010, Byrne et al. 2010a). Less well understood, however, is how the two stressors may interact to affect non-calcifying organisms (such as most macroalgae) or their interactions with herbivores. Many species of seagrass and macroalgae appear to be C3 species, but whether increasing concentrations of

CO2 buffer the impacts of elevating temperatures in the same manner to their terrestrial counterparts is unclear (Koch et al. 2012). The few studies that have considered how temperature and pH may alter algal traits that affect their susceptibility to herbivores indicate

4. Algal Growth & Palatability

the potential for these stressors to alter marine plant-herbivore interactions. In the red algal species Laurenica dendroidea, the major secondary metabolite, elatol, known to inhibit herbivory, was found to vary in concentration as temperatures rose (Sudatti et al. 2011). Low ocean pH has also been shown to alter the concentration of defensive phenolic compounds in seagrass (Arnold et al. 2010), and brown macroalgae (Swanson and Fox 2007). Although, a lowering pH may also influence the quality of marine plants and algae to herbivores through an increase in carbohydrate content (Jiang et al. 2010), increased nitrogen uptake (Xu et al. 2010) or decreased calcification (Johnson et al. 2012). With limited knowledge on the effect of the dual exposure of climatic stressors on growth, nutritional content or defensive chemicals in macrophytes, it is difficult to predict how this may affect marine herbivores in the future years to come

Macroalgal communities are one of the most productive and diverse ecosystems supporting a range of sessile invertebrates, epifauna and fish. The loss or growth of productivity of these systems may have significant impacts on co-dependent or competitively interacting species. Marine herbivores strongly affect the abundance of primary producers (Poore et al. 2012) and the rates of grazing upon these ecosystems can be up to three times greater than their terrestrial counterparts (Cyr and Pace 1993). Any shift in palatability or preferences of grazers, therefore, could have a strong influence on the structure of coastal ecosystems (Kleesing 2001, Wright et al. 2005). This study therefore aims to test the effects of warming and lowered pH, and their interaction, on the growth of six abundant species of macroalgae, and their subsequent palatability to a common generalist herbivore, the gastropod Phasinaotrochus eximius. This marine mollusc resides in the macroalgal beds off south- eastern Australia, an area that is predicted to be a hotspot for climate change (Poloczanska et al. 2007a, Hobday and Lough 2011). I address the following specific questions: 1) Is the growth of six species of commonly occurring macroalgae affected by increases in temperature (2 C and 4 C over ambient conditions) and lowered pH (Ambient, 7.8, 7.6)? 2) Do the feeding rates of P. eximius vary with the temperature and pH conditions in which the algae grew prior to the feeding experiment?

4. Algal Growth & Palatability

4.3 METHODS

4.3.1 Study organisms and sites

Algae and snails were collected from Charlesworth Bay (30°18’03.61’’S 153°08’31.90’’E) and Mutton Bird Island Nature Reserve (30°18’16.85’’S 153°08’55.20’’E), Coffs Harbour, Australia. Once collected, algae and gastropods were maintained separately in a flowthrough aquaria (~3500 L) at ambient temperature (~23 C) and under natural light conditions at the National Marine Science Centre. Seaweeds were kept for no longer than 4 days. The snail Phasianotrochus eximius were provided a mixed diet of local seaweeds, but 24 hrs prior to the feeding assay, the gastropods were given Sargassum vestitum, (Edgar 2000, Wright et al. 2004, Taylor and Steinberg 2005), which lacks non-polar secondary metabolites, only. This limited the possible influence of variation in recent diets that vary in seaweed chemistry on the consumption rates of P. eximius (see Sotka and Whalen 2008).

Six commonly occurring species of macroalgae in northern New South Wales were used to test the effects of increased temperature and decreased pH on algal growth, and to determine potential shifts in palatability under different climatic scenarios. This included the brown algae Sargassum vestitum (Fucales); Dictyopteris acrostichoides, Dilophus intermedius. and Zonaria diesingiana (Dictyotales); and two red algae Rhodymenia australis (Rhodymeniales) and Pterocladia capillacea (Gelidiales). Collection of the seaweeds occurred on the 6th and 8th of November, 2012 from the intertidal (P. capillacea) and subtidal regions (S. vestitum, D. arostichoides, Dilophus intermedius and Z. diesingiana, R. australis), at a depth of 1–2 m at low tide. Identification of the seaweeds was determined through visual reference to (Fuhrer et al. 1981, Huisman 2000), except for D. acrostichoides and Z. diesingiana which required a dissection of mature fronds and analysis of the cellular structure with reference to Farrant and King (1989).

The herbivorous gastropod P. eximius Perry 1811 (Mollusca: Gastropoda: Trochoidea) is commonly found in the subtidal algal beds along the southern coastline of Australia (Edgar 2000). These mesograzers are highly abundant throughout the year and consume a wide variety of commonly occurring macroalgae, including the six species chosen for this study (Edgar 2000, Wright et al. 2004, Taylor and Steinberg 2005). The combination of their wide diet breath and their occurrence in a climate change hotspot (Poloczanska et al. 2007b, Hobday and Lough 2011), made them an ideal organism to determine the effect of palatability of seaweeds grown under different climatic scenarios.

4. Algal Growth & Palatability

4.3.2 Experimental conditions

The effect of increased temperature, lowered pH and their interaction on the growth and palatability of the six species of macroalgae was quantified by rearing each in the nine combinations of three temperatures (ambient = 24 °C, +2 °C, +4 °C) and three levels of pH (Ambient = 8.10, 7.8, 7.6) in a flow through seawater system. The water fed into this system was UV sterilised and filtered with an average salinity of 33.59 ppt, which led into three 60

L header tanks. The pH of two header tanks were altered using an automatic CO2 injection system with two pH controllers (Tunze 7070/20) set at 7.8 and 7.6, which are indicative of the predicted ‘business-as-usual’ IS92a scenario (the IPCC base case)(Caldeira and Wickett

2003, Orr et al. 2005b, Solomon .S. et al. 2007, Gosling et al. 2011). The pure CO2 infused into these header tanks were mixed using a vortex mixer (Red Sea) and aerated continuously to aid mixing and maintain dissolved oxygen (>90%). The third header tank was unaltered and aerated only to track ambient pH. A constant volume of water was maintained in each header tank using a float valve.

This water was subsequently fed into sub-header tanks (20 L) where it was unaltered to maintain ambient conditions (24 °C) or warmed to the required temperatures of +2 °C (26 °C) or +4 °C (28 °C), representing the thermal conditions for the near future warming of SST in south-eastern Australia (Poloczanska et al. 2007a, Hobday and Lough 2011). The temperature of the water was increased to the required thermal conditions using aquarium heaters (200 W, Jager) that were automatically regulated with temperature sensors placed within the rearing containers and a temperature controller (Tunze 7028/3) connected to the heaters. The water was delivered independently into each individual rearing container using irrigation dripper valves (flow rate c.a ~0 9 ml.sec-1, 300-350 turnovers.day-1). Temperature, pH and salinity were recorded daily using a Hach Hqd Portable Multiprobe.

A three way analysis of variance (ANOVA) was conducted for each of daily temperature and pH to determine if there were significant differences among the treatments. Temperature and pH were fixed factors and day a random factor in this analysis

4.3.3 Effects of temperature and pH on algal growth

To determine the effect of the simultaneous exposure of temperature and pH on seaweed growth, the six species of macroalgae were cultured in the nine temperature/pH combinations. Collected algae were firstly cleaned of visible epiphytes and sediment, then apical fronds with actively growing meristems were weighed and placed into one of nine temperature (ambient, +2 °C, +4 °C) and pH (ambient, 7.8, 7.6) treatments. Three apical

4. Algal Growth & Palatability

fronds were maintained in a single 50 ml container (defined as Tank in analysis) with six containers per combination of treatments kept within the flow through seawater system as described above. The algae were allowed to grow for a period of two weeks, under a 16:8 hr light dark ratio, after which algae were again weighed. Light levels were maintained between 25–30 μmol m-2 s-1, irradiated from normal fluorescent tubes. The light level was determined by the average light (PAR) meter (Skye) readings taken at midday at approximately two m depth from three locations at Charlesworth Bay and Mutton Bird Island immediately prior to the experiment. The growth per apical frond (increase in wet mass) over the period of two weeks, for each species, was contrasted among treatments using a three-factor nested ANOVA with pH and temperature as fixed factors and container as a random factor nested within Temperature and pH.

4.3.4 Effects of temperature and pH on algal palatability

The palatability of the six seaweeds grown in all combinations of the temperature and pH treatments was determined using a non-choice feeding assay with P. eximius. This independently established the consumption rates of the snail, as opposed to choice feeding assays, which evaluate both behavioural preferences and rates of consumption. After algae were grown for two weeks in each treatment, they were weighed and placed into individual 50 ml containers under ambient temperature and pH conditions with an individual P. eximius. Seaweeds that were either excessively fouled or deteriorated were excluded from the experiment, resulting in 4 to 9 replicates per seaweed treatment combination. The same number of algal pieces from all treatment combinations was also allocated to a second set of containers that lacked P. eximius to control for any changes in algal mass not associated with herbivory. Gastropods were allowed to feed on the allocated treated seaweed for a period of 24 hr, after which both seaweed and snail were weighed.

The effects of temperature, pH or their interaction on the palatability of the seaweeds was analysed with an ANOVA on the mass loss of seaweed fronds in the 24 hr feeding assay. Temperature and pH were fixed factors, with Tank (the container fronds developed in for 2 weeks prior), a random factor, nested within Temperature and pH. Snail weight was also included as a covariate. The autogenic controls were analysed with a similar ANOVA to determine if there were any difference in seaweed weight between treatments in the absence of snails.

4. Algal Growth & Palatability

4.3.5 Statistical analyses

Univariate analyses of variance were carried out using the PERMANOVA routine of Primer V6. If treatments were significantly different, post hoc pairwise tests were conducted. Frequency histograms of the residuals and plots of the residuals were performed to ensure that the assumptions of normality and homogeneity of variance were met. No transformations were required.

4. Algal Growth & Palatability

4.4 RESULTS

4.4.1 Experimental conditions

The mean (± s.e.) temperatures in Ambient, +2 °C, +4 °C treatments within ambient pH, during the experiment were 24.16 °C ± 0.03 (pH 8.10 ± 0.004), 25.76°C ± 0.02 (pH 8.09 ± 0.003), 27.91°C ± 0.03 (pH 8.06 ± 0.003), respectively (n = 150) (Figure 4.1). These three temperature treatments all differed significantly from each other (F2,18 = 1365.4, P = <0.0001, Ambient °C <+2 °C <+4 °C in post-hoc pairwise tests). The mean (± s.e.) temperature of the +2 °C treatments in the two lower pH treatments of 7.8 and 7.6 were 25.80°C ± 0.05 (pH 7.82 ± 0.04) and 25.67 °C ± 0.03 (pH 7.63 ± 0.008), respectively. The mean (± s.e.) temperature of the +4°C treatments in the two lower pH treatments of 7.8 and 7.6 were 28.09°C ± 0.04 (pH 7.80 ± 0.003) and 27.83°C ± 0.05 (pH 7.58 ± 0.005), respectively. The three pH treatments also significantly differed from each other (F2,18 = 1631.2, P = <0.0001, Ambient < 7.8<7.6 in post-hoc pairwise tests).

4.4.2 Effects of pH and temperature on algal growth

All six species of algae grew over the period of two weeks under the nine different temperature and pH treatments (Figure 2), with the proportional increase in mass in ambient conditions ranging from 10 to 70 %. Temperature had a significant effect of the growth of five species, including Dilophus intermedius, P. capillacea, Z. diesingiana, R. australis and S. vestitum (Figure.1 and Table. 1). In each of these cases, the growth of the algae in temperatures increased by 2 °C, did not significantly differ from the growth of algae under ambient temperature conditions (P ≥ 0.05). However, at the highest temperature of +4 °C, all five species experienced a significant reduction in growth in contrast to the +2 °C temperature treatment (Dilophus intermedius, P = 0.031; P. capillacea, P = 0.01; Ambient ≥ +2°C > +4°C in post-hoc pairwise tests) or both ambient and +2 °C (Z. diesingiana, P=0.0001; R. australis P=0.0008; S. vestitum, P=0.0001; Ambient ≥ +2 °C > +4 °C in post- hoc pairwise tests). The only seaweed species to be unaffected by temperature was D. acrostichoides.

Reduced pH significantly affected the growth of two of the six species investigated; Z. diesingiana and R. australis (Figure 2. Table.1). In Z. diesingiana, growth was highest at the intermediate pH of 7.8 in all three temperature treatments, with this treatment differing significant to the pH 7.6 treatment, but not the ambient pH treatment in in post-hoc tests). The growth of R. australis was reduced in the most acidic treatment (pH 7.6), with this treatment differing from the ambient pH (8.1), but not the intermediate pH (7.8) in post-hoc

4. Algal Growth & Palatability

tests). There were no interactions between the temperature or pH treatments for any of the seaweed species (Table 1).

4.4.3 Effects of pH and temperature on algal palatability

The pH environment that the alga D. acrostichoides grew in prior to the feeding assay significantly affected the feeding rates of Phasianotrochus eximius. The gastropods consumed more algae grown at the intermediate pH of 7.8 in comparison to algae grown in an ambient pH environment, irrespective of the temperature (Figure 3, Table 2a). The mass loss of D. acrostichoides grown at ambient pH did not differ from that grown at the lowest pH of 7.6 was not significant, nor was there a difference in feeding rates between algae grown at the two lowest pH levels of 7.8 and 7.6.

For all other species, there were no effects of temperature, pH or the interaction between temperature and pH on the feeding rates of the snails (Table 2). The mass of the snails positively correlated with the amount of seaweed consumed for two seaweed species, D. acrostichoides and R. australis (Table 2). There were no differences in the mass loss among treatments for any species in the absence of snails.

4. Algal Growth & Palatability

30 Ambient +2°C

) 28 C

° +4°C

(

u t

r 26

m e

T 24

22 29 120 122 123 214 215 129 220 221 222 /1 /1 /1 /1 /1 /1 /1 /1 /1 /1 1 1 1 1 1 1 1 1 1 1 /1 /1 /1 /1 /N1 ove/1meb/1er /1 /1 /1 9 0 2 3 4 5 9 0 1 2 1 1 1 1 1 1 2 2 2

8.5 Ambient 7.8 7.6

8.0

H p

7.5

7.0 9 10 12 13 14 15 19 20 21 22 Novemeber 9/11/1210/11/1212/11/1213/11/1214/11/1215/11/1219/11/1220/11/1221/11/1222/11/12

Figure 4.1 Temperature (Ambient, +2 °C and +4 °C) and pH levels (Ambient, 7.8, 6.6) recorded daily in the treatments over the duration of the experiment.

4. Algal Growth & Palatability

a) Dictyopteris arostichoides 80 8.1 7.8 60

) 7.6

(

t 40

r G 20

0 Ambient +2°C +4°C Temperature (°C)

b) Pterocladia capillacia 80 8.1 7.8 60

) 7.6

(

t 40

r G 20

0 Ambientab +2°Ca +4°Cb Temperature (°C)

c) Rhodymenia australis 50 8.1a 40 7.8ab

) 7.6b

% (

t w

o 20

r G 10

0 Ambienta +2°Ca +4°Cb Temperature (°C)

Figure 4.2a-c. Growth (mean ± s.e. % increase in mass) of six temperate algal species exposed to three temperature treatments (Ambient (24 °C), +2 °C (26 °C) and +4 °C (28 °C) and three pH treatments (8.1, 7.8 and 7.6) for a period of 2 weeks. The seaweeds investigated include a) Dictyopteris acrostichoides, b) Pterocladia capillacea c) Rhodymenia. Treatments sharing letters do not differ in post-hoc tests.

4. Algal Growth & Palatability

d) Dilophus intermedius 80 8.1 7.8 60

) 7.6

(

t 40

r G 20

0 Ambientab +2°Ca +4°Cb Temperature (°C)

e) Zonaria diesingiana 20 8.1ab 7.8b 15

) 7.6a

(

t 10

r G 5

0 Ambienta +2°Ca +4°Cb Temperature (°C)

f) Sargassum vestitum 100 8.1 80 7.8

) 7.6

% (

t w

o 40

r G 20

0 Ambient a +2°Ca +4°Cb Temperature (°C)

Figure 4.2d-f. Growth (mean ± s.e. % increase in mass) of six temperate algal species exposed to three temperature treatments (Ambient (24 °C), +2 °C (26 °C) and +4 °C (28 °C) and three pH treatments (8.1, 7.8 and 7.6) for a period of 2 weeks. The seaweeds investigated include d) Dilophus intermedius, e) Zonaria diesingiana, and f) Sargassum vestitum. Treatments sharing letters do not differ in post-hoc tests.

4. Algal Growth & Palatability

a) Dictyopteris arostichoides 40

8.1a ) r 7.8

h b

4 30 2

/ 7.6ab

(

s 20

s 10

a M

0 Ambient +2°C +4°C

Temperature (°C)

b) Pterocladia capillacia 5

8.1 ) r 7.8

h 4

4 2

/ 7.6

g 3

(

s s

o 2

s s

a 1 M

0 Ambient +2°C +4°C Temperature (°C)

c) Rhodymenia australis 8

8.1 )

r 7.8 h

4 6 2

/ 7.6

(

s 4

s 2

a M

0 Ambient +2°C +4°C Temperature (°C)

Figure 4.3a-c. Consumption (mean ± SE mass loss) of 6 temperate seaweed species exposed to three temperature treatments (24°C, 26°C and 28°C) and three pH treatments (8.1, 7.8 and 7.6) by the marine snail Phasianotrochus eximius under ambient conditions over a period of 24hrs. The seaweed investigated include a) Dictyopteris acrostichoides, b) Pterocladia capillacea, c) Rhodymenia australis. Treatments sharing letters do not differ in post-hoc tests.

4. Algal Growth & Palatability

d) Dilophus intermedius 15

8.1 )

r 7.8

4 2

/ 10 7.6

(

o L

a M

0 Ambient +2°C +4°C Temperature (°C)

e) Zonaria diesingiana 15

8.1 )

r 7.8

4 2

/ 7.6

(

o L

a M

0 Ambient +2°C +4°C Temperature (°C)

f) Sargassum vestitum 50

8.1 ) r 7.8

h 40

4 2

/ 7.6

g 30

(

s s

o 20

s s

a 10 M

0 Ambient +2°C +4°C Temperature (°C)

Figure 4.3d-f. Consumption (mean ± SE mass loss) of 6 temperate seaweed species exposed to three temperature treatments (24°C, 26°C and 28°C) and three pH treatments (8.1, 7.8 and 7.6) by the marine snail Phasianotrochus eximius under ambient conditions over a period of 24hrs. The seaweed investigated include d) Dilophus intermedius e) Zonaria diesingiana and f) Sargassum vestitum. Treatments sharing letters do not differ in post-hoc tests.

4. Algal Growth & Palatability

Table 4.1. Three-factor analyses of variance contrasting the percentage growth (increase in wet mass) of six species of seaweed over a period of two weeks, exposed to three temperature treatments and three pH treatments. Temperature and pH are fixed factors. Probabilities were determined using 9999 permutations. The mean squares as denominators for each F test are as follows: pH, MStank; Temperature, MStank.;pH x Temperature, MStank.; Tank, MSError.(* denotes significance <0.05 and ** denotes significance <0.001) Growth (%) Source df MS F P a) Dictyopteris acrostichoides pH 2 881.11 1.45 0.24 Temperature 2 1440.3 2.38 0.10 pH x Temperature 4 905.69 1.49 0.22 Tank(pH x Temperature) 45 609.89 2.13 <0.01** Error 103 286.3 b) Dilophus intermedius. pH 2 71.732 0.167 0.85 Temperature 2 1696.2 3.91 <0.05* pH x Temperature 4 771.91 1.78 0.15 Tank(pH x Temperature) 44 420.04 0.71 0.78 Error 19 590.94 c) Pterocladia capillacea pH 2 2520.2 2.19 0.12 Temperature 2 5870.1 5.10 <0.01** pH x Temperature 4 318.75 0.28 0.89 Tank(pH x Temperature) 44 1152.1 2.36 <0.01** Error 106 488.29 d) Zoneria diesingiana pH 2 293.24 4.21 <0.05* Temperature 2 863.01 12.40 <0.01** pH x Temperature 4 50.031 0.719 0.60 Tank(pH x Temperature) 45 69.578 0.16 0.16 Error 108 56.346 e) Rhodymenia australis pH 2 794.47 3.69 <0.05* Temperature 2 1844.1 8.57 <0.01** pH x Temperature 4 313.06 1.45 0.23 Tank(pH x Temperature) 46 215.38 1.04 0.42 Error 106 206.78 f) Sargassum vestitum pH 2 186.69 0.16 0.86 Temperature 2 22150 18.26 <0.01** pH x Temperature 4 2292.4 1.97 0.11 Tank(pH x Temperature) 44 1162 1.09 0.33 Error 100 1060.2

4. Algal Growth & Palatability

Table 4.2. Two-factor analyses of variance constrasting the consumption (mg) by Phasianotrochus eximius of six different species of algae exposed to three temperature and three pH treatments for a period of 24 hr, under ambient conditions. Temperature and pH are fixed factors with snail weight a covariate. Probabilities were determined using 999 permutations. The mean squares as denominators for each F test are as follows: Snail Weight, MSError; pH, MSTank+MSError;

Temperature, MSTank+MSError;pH x Temperature, MSTank+MSError; Tank, MSError. .(* denotes significance <0.05 and ** denotes significance <0.001)

Consumption (mg/24hr) Source df MS F P a) Dictyopteris acrostichoides Snail Weight 1 475.89 4.69 <0.01** pH 2 307.19 3.78 <0.05* Temperature 2 213.21 2.78 0.07 pH x Temperature 4 126.85 1.65 0.18 Tank(pH x Temperature) 36 76.01 0.91 0.61 b) Dilophus intermedius Snail Weight 1 0.36 0.39 0.59 pH 2 2.00 0.11 0.90 Temperature 2 0.95 0.04 0.96 pH x Temperature 4 15.14 0.71 0.59 Tank(pH x Temperature) 44 23.84 25.96 <0.05* c) Pterocladia capillacea Snail Weight 1 5.45 1.00 0.33 pH 2 6.45 1.34 0.28 Temperature 2 5.51 0.73 0.49 pH x Temperature 4 0.83 0.17 0.95 Tank(pH x Temperature) 29 4.74 0.87 0.65 d) Zonaria diesingiana Snail Weight 1 162.41 12.02 <0.01** pH 2 52.42 1.89 0.16 Temperature 2 48.54 1.79 0.18 pH x Temperature 4 35.90 1.32 0.28 Tank(pH x Temperature) 37 28.88 2.14 <0.05* e) Rhodymenia australis Snail Weight 1 44.36 4.15 <0.05* pH 2 4.06 0.29 0.75 Temperature 2 34.00 2.38 0.10 pH x Temperature 4 27.50 1.93 0.12 Tank(pH x Temperature) 42 14.74 1.38 0.19 f) Sargassum vestitum Snail Weight 1 85.41 0.25 0.62 pH 2 367.24 1.11 0.34 Temperature 2 91.65 0.28 0.76 pH x Temperature 4 331.42 0.46 0.76 Tank(pH x Temperature) 41 341.63 0.97 0.55

4. Algal Growth & Palatability

4.5 DISCUSSION

Both increased temperature and a lowering pH environment affected the growth of five of the six species studied. Exposure to elevated temperatures reduced the growth of Dilophus intermedius, P. capillacea, Z. diesingiana, R. australis and S.vestitum, independent of the pH environment. In addition to reduced growth at elevated temperatures, both R. australis and Z. diesingiana were also affected by a lowered pH. The only species to be unaffected across the range of treatments was D. acrostichoides, but the prior exposure to more acidic treatments changed the palatability of this seaweed to the herbivorous gastropod P. eximius. These results show that alteration in the abundance, or the traits of macroalgae that can affect their susceptibility to herbivores, may shift as climatic conditions change. This has the potential to affect the structure and dynamics of marine communities.

4.5.1 Effects of temperature on algal growth

Temperature is an important determinant of the growth rates of marine primary producers (Raven and Geider 2006). Irrespective of the pH environment, elevated temperatures decreased the growth of five of the seaweed species studied. Only D. acrostichoides showed no relationship. As with all organisms, the optimal performance curve varies among algal species and therefore the relationship between temperature and growth is not uniform (Koch et al. 2012). In general, temperatures that exceed a species optimum are likely to lead to disruptive stress at a cellular or sub-cellular level (Davison and Pearson 1996, Eggert 2012). The reallocation of recourses to repair damage cells or limit the disruptive stress may hinder other primary functioning such as growth and development (Koch et al 2012). This is the likely scenario for the algae in this study that experience reduced growth with elevated temperatures.

High temperatures that exceed the upper thermal tolerance limits of macrophytes commonly lead to the breakdown or disruption of photosynthetic mechanisms (Campbell et al. 2006, Allakhverdiev et al. 2008, Collier et al. 2011). In the green alga, Codium edule, the exposure to higher temperatures for an extended period of time led to the rupturing of vacuoles causing decomposition of chloroplast (Collier et al 2011). This, in turn reduces light- harvesting capacity and ultimately photosynthetic activity (Eggert 2012). Similarly in seagrasses, thermal stress leads to the breakdown of the photosystem II functioning, causing photoinhibition, and also disrupting photosynthetic activities. Predicting the long term responses of algae to increased temperatures, however, requires an understanding of variation in their ability to alter thermal tolerances through physiological acclimation or

4. Algal Growth & Palatability

rapid evolution due to thermal stress potentially selecting for tolerant genotypes (Clark et al. in press). Such adaptation would enable the persistence of populations, but requires studies over a longer time frame than conducted here (Davison 1987, Eggert 2012).

The absence of any effect of elevated temperature on the growth of D. acrostichoides on the other hand, could be associated with the fact that this species is also found in tropical regions which routinely experience temperatures in excess of ~30 °C (Womersley 1987), above the highest temperatures investigated in these experiments. Depending on dispersal potential of this species, it is possible that populations found in the subtropical environment of Coffs Harbour have similar thermal tolerances to those populations found in more northerly, tropical regions. The capacity for this species to tolerate higher temperatures may be associated with the activation of heat shock proteins (Hsp) (Massa et al. 2011) or the ability to minimise damage to membrane structure or proteins through other mechanisms, as found in other heat tolerant macrophytes (Burritt et al. 2002). The ability of this seaweed to tolerate temperatures in tropical regions suggests that elevated temperatures in temperate regions as predicted by climate change are unlikely to affect this species. It does, however, raise competitive interactions between this species and more vulnerable temperate species to be affected by rising ocean temperatures.

4.5.2 Effect of pH on algal growth

Lowered pH had a significant effect on the growth of two of the species studied, irrespective of temperature. The optimal pH environment for the growth of R. australis was under ambient conditions, whilst Z. diesingiana grew faster in the slightly acidic environment of 7.8. Although, a further decrease in pH to 7.6, led to the reduced growth in comparison to ambient conditions (R. australis) or the pH treatment of 7.8 (Z. diesingiana), suggesting that their optimal range had surpassed. Some seaweeds rely soley on CO2 for inorganic carbon, - although some are also able to take up bicarbonate (HCO3 ) and convert it to CO2 via carbon concentrating mechanisms (CCM) (Roleda and Hurd 2012). CCM is found in most seaweeds - and requires the active uptake of HCO3 or CO2, while seaweeds that don’t have this mechanism (mostly red algae) depend on the uptake of CO2 by passive diffusion only. As the ocean chemistry changes, increasing the amount of CO2, there will be little or no advantages - for seaweeds that are able to utilise HCO3 (Beardall et al. 1998). It is hypothesised that that the down regulation of CCM could increase growth but this is yet to be confirmed (Roleda and Hurd 2012). Macroaglae in the orders Fucales (which include Sargassum) and the Rhyodymeniales (which includes, Rhodymenia), have a carbon concentrating mechanism (Cook and Colman 1987, Surif and Raven 1989), although in this study, R. australis

4. Algal Growth & Palatability

experienced reduced growth with lower pH, while S. vestitum was insensitive to pH change. Alternatively, it is hypothesised that seaweeds lacking CCMs are more likely to be carbon limited and thus benefit from additional CO2 (Harley et al. 2012). Algae in the order

Dictyotales, which includes Dilophus, Dictyopteris and Zonaria, passively take up CO2 (Franklin and Badger 2002), although there was no consistent response to elevated carbon dioxide with these species studies. Both Dilophus intermedius. and D. acrostichoides showed no changes, whilst the growth of Z. diesingiana was reduced with higher concentrations of CO2. Evidently from these experiments, and the existing literature, it is difficult to predict responses based on the mechanisms used to take up inorganic carbon

(Hepburn et al. 2011). While elevated CO2 concentrations are likely to increase the productivity of terrestrial plants, it does not have the same response in these temperate macroalgal species. Whether this changes over a longer time frame, is yet to be investigated.

In terrestrial plants, CO2 partially offsets high temperature effects (Sage and Kubien 2007). However, of the six species of macroalgae studied, there was no interaction between temperature and pH. The only species that showed an increase in growth with a lowering pH and an elevated temperature of +2 C was S. vesitium. However, this was not significant and with a further increase in temperature of +4 C, this positive interaction disappeared. Therefore, the responses found in association with the dual exposure of increasing temperature and CO2 concentrations in terrestrial plants cannot be applied to marine macroalgae as well.

4.5.3 Climatic stressors & algal palatability

Changes to the growth rates of macroalgaes under elevated temperatures and a lowered pH did not translate into changes to the palatability of these species to the herbivorous gastropod P. eximius. The only algae where pH altered the consumption was also the only algae whose growth was not altered by any ph or temperature treatments. The consumption of D. acrostichoides was lowest under an ambient pH environment and higher when grown in the intermediate pH of 7.8. The amount of algae consumed did subside at the lowest pH but was not significantly different form either pH treatment. The increased palatability of D. arostichoides when grown in a more acidic environment could result from changes to either the chemical defences present in this algae, or to changes in its nutritional quality for this herbivore.

Emerging research suggests that temperature and ocean acidification may affect key traits that influence susceptibility of macrophytes to herbivory. Temperature is known to affect the chemical defences of macroalages (Lyons et al. 2007, Sudatti et al. 2011), making them

4. Algal Growth & Palatability

more vulnerable to other herbivores or disease (Campbell et al. 2011). Ocean acidification has also been linked to changed concentration of secondary metabolites as well as their nutritional content in algaes. Dictyopteris arostichoides produces a variety of lipophilic secondary metabolites including acetogenins, terpenes, hydrocarbons and ecosanoids (Wirth et al. 2004). It is possible that a lower pH environment changes the concentration or composition of these secondary compounds, which then allow for a greater rate of consumption. Alternatively, increased CO2 can stimulate carbon fixation that in turn reduces the nitrogen content relative to carbon (Bellerby et al. 2008, Rossoll et al. 2012). If the quality of food is reduced then, herbivores will consume more to gain the same amount of nutrients (Cruz-Rivera and Hay 2000). Quantifying the mechanisms that resulted in the altered interaction between P. eximius and D arostichoides requires further analysis on both the nutritional content and concentration of chemical deterrents in this species across a range of pH levels. A similar experiment conducted on the specialist herbivore Perampithoe parmerong found that feeding rates were significantly reduced when their food source Sargassum linearfolium were exposed to an elevated temperature of +3 C in an ambient pH environment (Poore et al. 2012). The underlying reason for the shift in consumption by the amphipod were inconclusive as there were no effects of ph and temperature treatments on the nitrogen, carbon, or phlorotannin (secondary compound) content of the macroalgae. In comparison, in this study, there was the no variation in palatability in the closely related Sargassum vestitum to P. eximuis, and the only variation in palatability observed (for D. acrostichoides) was associated with pH rather than temperature. This suggests that both temperature and pH have the potential to alter the palatability of algaes to marine herbivores, but this is likely to be species specific and will not be uniform across all herbivores.

4.5.4 Ecological relevance

In the last decade, global warming has lead to the poleward shift of many species of macroalage (Wernberg et al. 2011a). The range contraction of temperate species provides an opportunity for more thermally tolerant tropical species to become competitively dominant (Koch et al. 2012). This is likely to affect biodiversity and indirectly affect co-dependent organisms (Van der Putten et al. 2010, Hughes 2011a). Whether the loss of macroalgal biomass in response to either elevated temperatures or a lowering pH, will lead to population level declines of interacting species, is yet to be evaluated.

The change in palatability of macroalgae to commonly occurring marine herbivores has the potential to directly interfere with herbivore fitness as well as the structure and dynamics of the surrounding ecosystem. Grazers play an important role in the abundance of individual

4. Algal Growth & Palatability

seaweeds and the species composition of macroalgal beds, and macrophytes are the fundamental link that transfers energy captured from the sun to higher trophic levels. A shift in the feeding rates or preferences of herbivores has the potential to alter the species composition of macroalgal communities. This coupled with increasing rates of herbivory associated with metabolic functioning (e.g. increase rate of consumption) could lead to significant top-down impacts on this community. This will, however, depend on the direct effects of climatic stressors at a population level on consumers. Alternatively, a change in palatability or abundance of primary producers may lead to bottom-up effects on higher trophic levels. Either the loss of a viable food source or a shift in the concentration of chemical defences or nutritional content of that food sources, may affect the fitness of consumers over the longer term. The impacts of both increasing temperature and a lowering pH are not so straight forward, and this study highlights that it is not only the direct effects on calcifying organisms that are an issue with respect to climate change in our oceans. Elevated temperatures and ocean acidification are likely to change the interaction strength between plants and herbivores, alter the abundance of primary producers as well as their palatability to various marine herbivores. The complexities of this make it difficult to predict the ‘winners and losers’ as the climate changes over the years to come, as both calcifying and non-calcifying organisms are going to be affected directly, but also and indirectly through changes to species interactions.

5. General discussion

Chapter 5

GENERAL DISCUSSION

The overall aim of this thesis was to investigate the direct and indirect impacts of shifting oceanic conditions on vulnerable marine species. Given this, the capacity of a marine herbivore to tolerate ocean warming were assessed by exploring how thermal tolerance varies within a single lifetime and across generations. However, considering that elevated temperatures will be coupled with increasing concentrations of CO2, the combined effects of these stressors on non-calcifying macroalgae and the potential impacts to higher tropic levels, were also evaluated. This study identified several key findings that will increase the knowledge base for research in this area;

5.1 Variation in thermal tolerance

Previous research on the amphipod Peramphithoe parmerong identified that this species is susceptible to global warming (Poore et al. 2012). Thus, using this subtidal marine crustacean as a model species provided an opportunity to assess variation in thermal tolerance and the potential for adaptive acclimation. Exposure to an elevated temperature established that this species is able to successfully develop and survive at their current maximum habitat temperature (Chapter 2), and that acclimation to this temperature increased their tolerance to thermal extremes (Chapter 3). However, this increased tolerance may be associated with reduced fecundity, as has been seen following the induction of heat shock proteins (e.g., in Drosophila melanogaster, Shilbermann and Tater 2000). The proportion of female P. parmerong that released live offspring was lower when they developed in the elevated temperature compared to the ambient environment (Chapter 2). If P. parmerong is required to use additional resources to increase tolerance of elevated temperatures, it could be contributing to their observed reduction in fecundity. This decrease in brood size or the proportion of females successfully reproducing is likely to result in a declining population at elevated temperatures and further research that investigates population growth over multiple generations (either experimentally or through modelling) is needed to predict effects on future population sizes.

Acclimation to elevated temperatures did increase the thermal tolerance of P. parmerong within the lifetime of an individual, however, these effects were not carried over to the successive generation. This suggests that trans-generational effects, such as maternal provisioning that improves offspring survival, do not play a role in the size, survival or

5. General discussion

thermal tolerance of individuals at maturity for the temperatures studied for this species (Chapters 2 & 3). However, it is possible that there could be a transfer of tolerance across generations at higher temperatures or for physiological measures that were undetected by the performance responses examined. Mentioned previously, the elevated temperature of 26 C did not affect survival of P. parmerong. Therefore, it is difficult to determine if there are possible trans-generational effects that increase survival, as the elevated temperature is this study did not appear to be lethal. The size at maturity of amphipods was also not influenced by the maternal environment. With the exclusion of plants, only four studies have detected trans-generational plasticity in growth in response to thermal environments, three of which were with insects and one with a fish (Groeters and Dingle 1988, Jablonka and Raz 2009, Salinas and Munch 2011). Lastly, there does not appear to be any trans-generational effects in response to tolerance to extreme heat. This, cannot rule out potential physiological responses such as changes to metabolic performance as observed in the damselfish Acanthochromis polyacanthus (Donelson et al 2011). From this study, it is clear that the changes associated with thermal acclimation within an individual’s lifetime, outweigh any possible provisions provided by the previous generation for this species.

The assessment of heat tolerance using different protocols did identify some issues, which should be taken into consideration with future studies. Assessing the critical thermal maximum (CTmax), the influence at which the experiment is started does not affect tolerance, however the rate of increase does. Various other studies investigation the critical thermal limits among individuals, population or species of aquatic organisms use increase temperatures at a rate that is unrealistic (e.g. 6 C.hour-1, 12C.hour-1, 20.4C.hour-1, 60C.hour-1) (Re et al. 2005, Hopkins et al. 2006, Gonzalez et al 2010, Dallas and Ketley 2011). The faster the rate, the higher an organisms tolerance, thus the potential overestimation of heat sensitivity (Teranche et al. 2007, Peck et al 2009, Chown et al. 2009, Mitchell and Hoffmann 2010). However, the influence of the rate of temperature change also has an ecological relevance in association with the onset of heat waves. Many organisms may be able to acclimate to the average inclines in temperature, predicted to occur with global warming, but the rate at which the temperatures change with the occurrence of heat waves as well as their frequency and intensity may move beyond their threshold. Therefore both averages temperature shifts and extreme events need to be considered when assessing the impact of climate change

Further research should focus on how the thermal tolerance of P. parmerong may vary across its geographical distribution, and their genetic capacity to adapt to increasing temperatures. The work carried out in this thesis identifies that acclimation does provide a

5. General discussion

buffer to elevated temperatures, but this is not passed on to their progeny. Thus there is still the risk that this species is vulnerable to extreme events, depending on the frequency, intensity and duration of such an event. Therefore, if thermo-tolerant genotypes exist within this species, selection for those individuals over time may increase tolerance at a population level. This is dependent on the levels of genetic variation for thermal tolerance within the population as well as dispersal potential to less tolerant population. Mutigenerational experiments conducted in the laboratory may provide some answers as to whether selection for tolerant genotypes can result in increased tolerance over time in P. parmerong.

5.2 Effects of climatic stressors on species interactions

In addition to a warming ocean, increasing concentrations of CO2 are also affecting many species of organisms in the marine environment (Orr et al. 2005b, Byrne et al. 2010a, Kroeker et al. 2010). The influence of the dual exposure of temperature and lowered pH on non-calcifying species of macroalgae and their interaction with herbivores, is however, less understood (Harley et al. 2012). This thesis identified that increasing temperatures reduced the growth of 5 of the six macroalgal species studied, whilst a lowered pH affected two species (Chapter 4). No cases where temperature and pH interacted to affect algal growth, were found. For these species, there was a general decline in growth with both climatic stressors, but they appear to act independently of each other and the impact on macroalgae will be species specific. The reduced abundance of most of the temperate algal species studied could be compounded by increase in the strength of their interaction with herbivores. As elevated temperatures rise so too will metabolic performance for some grazers thus increasing potential rates of consumption (O'Connor 2009) and the possibility that changed water conditions will affect traits of the algae that determine their palatability to herbivores. Given the strength of herbivory in marine ecosystems, both changes could lead to large impacts on macrolagal communities. In this thesis, potential bottom up effects on herbivores from pH driven changes to a primary producer, were identified. Increased consumption of one of the algal species grown in a more acidic environment by a herbivorous gastropod suggest some change in physiological or morphological characteristic of the seaweed. The mechanism is currently unknown, but if this is associated with lower nutritional content it may lead to detrimental impacts on the consumer fitness (if the herbivore is consuming the alga at a faster rate to compensate for its poor quality). Alternatively, if associated with a lowered concentration of chemical defences, certain species of algae may become more vulnerable to herbivores (coupled with direct declines due to stressors and increased consumption rate due to metabolic responses of herbivores) and well as potential diseases and pathogens (Campbell et al. 2011).

5. General discussion

Further research could investigate the underling mechanisms affecting this change in consumption (e.g. morphology, chemistry, nutrient content), and the effects on changing climate on species interactions for a wider range of marine herbivores. These interactions are likely to be species-specific. and the challenge is to identify how often climatic stressors are likely to alter feeding preferences. Furthermore, longer-term studies could identify whether macroalgae grown in a warmer or more acidic environments impede the fitness of consumers raised on these diets.

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