Integrating physiology, behaviour and molecular mechanisms to understand impacts of ocean warming on Southern Calamari (Sepioteuthis australis)

Accelerating climate change will strongly influence marine species across the world, altering the composition, stability and function of ecosystems, and ultimately affecting the wellbeing of the human communities dependent on them. Ectotherms could be particularly vulnerable to temperature changes, as temperature is a major factor influencing their internal regulation (e.g. cellular or physiological mechanisms). Not surprisingly, increases in water temperatures have already been documented shown to have an impact on marine ectotherms with changes in body size, reproduction and geographical distribution. Understanding species responses to ocean warming is a key challenge for contemporary ecology, evolution and conservation. Ocean temperatures are expected to increase by 2 ¬∞C to 4 ¬∞C by the end of the century, yet the rate and magnitude of change is not the same across the world. In south-eastern Australia, ocean warming is increasing almost four times faster than the global average; this region can therefore function as a 'natural laboratory' where temperature effects might be accelerated. Research to date has focused on how species within this region may shift geographically as waters warm; however, little is known about how species might perform and interact in response to thermal challenges within their existing ranges. Southern calamari (Sepioteuthis australis) are an ecologically and economically important endemic species of southern Australia and northern New Zealand. To date, studies examining the effect of ocean warming on cephalopods have been under-represented in the literature in comparison with other group (e.g., fish or marine invertebrates), despite their important role in marine ecosystems as both predator and prey. They are highly plastic and fast-growing species with short generation times, which may allow them to adapt faster than other species to changing environmental conditions, improving their chances of survival. Consequently, by studying rapidly responding species such as squid, in fast-changing regions of the world, we can examine the mechanistic links between ocean warming and the biological responses in advance of the broader scale impacts predicted for the future. The main aim of this thesis is to understand how temperature affects the physiological (oxygen consumption and upper thermal limits) and behavioural performance (thermal preferences and predatory behaviour) of adult southern calamari, as well as the molecular pathways (transcriptomics) underpinning this performance. Physiological and behavioural responses to changes in water temperature can provide important insights regarding species sensitivity and vulnerability to ocean warming. Currently, it is still not clear if changes in temperature will induce shifts in the aerobic metabolism and thermal preferences of squid. The first component of this study aimed to examine the effects of different acclimation temperature on the metabolic activity and behavioural thermal preferences of southern calamari in adult stages (Chapter 2). To do this, intermittent-flow respirometry was used to measure standard, routine, maximum and excess post-exercise oxygen consumption rates, as well as aerobic scope. In addition, a custom-made shuttle box system was utilised to evaluate the thermal preferences of individuals. The results suggested that acclimation temperatures influence squid metabolism and preferences, and that individuals seek out thermal environments that are more favourable for their metabolic capacities. Overall, metabolic rates (standard, routine, and maximum) increased at higher acclimation temperatures, with a simultaneous reduction in the aerobic scope at 22 ¬∞C and 25 ¬∞C. These results suggest that metabolism in southern calamari could be limited by water temperatures above 22 ¬∞C. Coincidentally, squid actively seek temperatures between 15 to 22 ¬∞C, which seem to be temperatures more beneficial for squid physiological capacities. In addition to altering physiological responses, warming may also impact predator‚-prey dynamics in various ways. To further determine an individual's performance under changing environmental conditions, predatory behaviour of southern calamari across temperature treatments (13, 16, 19, 22 and 25 ¬∞C) (Chapter 3) was also examined by way of the interaction of individual squid with a common prey species, Australian salmon (Arripis trutta). Here, squid behaviour differed between treatments, with the ability and decision-making processes (e.g. motivation, capture success) to capture the prey influenced by temperature. At elevated temperatures, southern calamari showed an increase in predation rate, capture success and number of strikes, meanwhile the latency time was greatly reduced. However, handling time increased with temperature. In addition to behavioural thermoregulation, species have another major mechanism to cope with changes in environmental conditions ‚- acclimation. The 'live fast, die young' life history strategy of squid, with fast-paced life histories and multiple generations within short time windows, might facilitate or favour a fast acclimation or adaptation in squid. To examine this, phenotypic plasticity (upper thermal limits) and genome-wide gene expression (RNA-seq) were used (Chapter 4). Squid were exposed to rapid increases in temperature to establish the effect of acclimation on their upper thermal limit (CTmax), and RNA-seq was used to examine the potential mechanisms involved in individual adaptation to temperature. Southern calamari demonstrated a plastic response to temperature, increasing their CTmax significantly. The transcriptomic study suggested that squid used post-transcriptional RNA modifications such as splicing activity, as well as apoptotic and immune responses, to adjust to temperature changes. This thesis demonstrates for the first time, how acclimation temperature affects southern calamari performances, by integrating physiological (metabolic activity and critical thermal limits), behavioural (thermal preferences and predatory performance), as well as molecular (RNA-seq analysis) responses to understand mechanisms of temperature adaptation. Overall, the results indicate that this species could maintain phenotypic plasticity in some traits (CTmax) and behavioural performance measures (predatory strategies) under ocean warming conditions, possibly associated with the highly plastic transcriptional response found in this study. However, other important traits such as aerobic scope, survivorship and the ability to capture prey could be negatively impacted by an increase in ocean temperatures. As squid are key components of trophic webs, transferring energy across trophic levels, the findings of this study might have implications at the community and ecosystem levels, by way of cascading effects. Consequently, a more holistic approach integrating different measures of performance, as well as exploration of the mechanisms involved in adaptation, is needed to better predict future responses. Greater understanding of species climate change responses will be critical for underpinning development of more appropriate management and climate adaptation strategies to better support healthier marine ecosystems, fisheries and aquaculture now and into the future.