1. Introduction
As human-induced environmental changes progress, establishing how animals respond to projected future environmental conditions, and why these responses occur, is critical (Fuller et al., 2010). A thorough understanding of why biological responses are occurring is especially useful for gaining insight into why some individuals or species are more sensitive to environmental change than others, and improving predictions of how organisms and populations will respond over the time scales at which environmental change is occurring (Cooke et al., 2013). The nervous system forms the fundamental link between the environment and an animal’s responses (Kelley et al., 2018; O’Donnell, 2018). Thus, the neurobiological impacts of anthropogenic environmental change are key to understanding how animals will respond as environmental change progresses, yet the role of the nervous system in biological responses to environmental change has been little explored (Kelley et al., 2018).
The uptake of anthropogenic carbon dioxide (CO2) by the ocean is causing seawater CO2 levels to rise, decreasing seawater pH and altering the concentration of carbonate ions, in a process known as ocean acidification (OA) (Bindoff et al., 2019). These chemical changes can fundamentally affect marine organisms and the ecosystems they inhabit (Doney et al., 2009). OA affects a wide variety of physiological processes, life history traits and behaviours of marine invertebrates (Durant et al., 2023; Kroeker et al., 2010; Nagelkerken & Connell, 2015; Pörtner et al., 2004; Thomas et al., 2020). Invertebrates are vital components of marine ecosystems, comprising over 92% of species in the ocean, are essential to the function of ecosystem processes, and support the livelihoods of human societies across the globe (Bertness et al., 2001; Chen, 2021). Animal behaviour influences an individual’s own fitness, complex interactions with other individuals and species, and key ecological processes that shape the structure of marine communities and ecosystems (Nagelkerken & Munday, 2015). Consequently, any behavioural effects of elevated CO2 on marine invertebrates could potentially have wide-ranging ecological, social and economic consequences.
Despite many studies assessing the behavioural responses of marine invertebrates to OA the link between the environment and behavioural responses, the nervous system, has been largely understudied. The work that has addressed the neurobiological impacts of OA has focused on the functioning of GABAA receptors. The GABA hypothesis was first proposed in fish and suggests acid-base regulatory mechanisms occurring at elevated CO2 conditions alter ionic gradients across neuronal membranes, consequently disturbing GABAA receptor function and causing behavioural alterations (Nilsson et al., 2012). A range of research has supported the GABA hypothesis in fish (reviewed in Heuer et al. (2019)), and more recently pharmacological studies have also supported the GABA hypothesis in molluscs (Clements et al., 2017; Thomas et al., 2021; Watson et al., 2014), but not a crustacean (Charpentier & Cohen, 2016). However, OA may also have a range of other neurobiological impacts, including altering the function of other ligand-gated ion channels that are similar to the GABAreceptor (Thomas et al., 2021) and affecting synaptic plasticity (Lai et al., 2017; Porteus et al., 2018).
Transcriptomics provides a powerful non-targeted, holistic approach to identify functional responses to environmental change. Indeed, transcriptomics has widely been taken up by the OA research community to understand the response of marine animals to elevated CO2 (Strader et al., 2020). However, there is less research assessing the transcriptomic response of nervous tissue to elevated CO2. Recently, studies have examined the transcriptomic response of the fish nervous system to elevated CO2 conditions, including in coral reef fishes (Kang et al., 2022; Schunter et al., 2021; Schunter et al., 2019; Schunter et al., 2018; Schunter et al., 2016), temperate marine fishes (Cohen-Rengifo et al., 2022; Porteus et al., 2018; Toy et al., 2022) and ocean-phase salmon (Williams et al., 2019). In marine invertebrates, two transcriptomic studies assessing the whole-body response of pteropod molluscs to elevated CO2 identified altered expression of genes involved in nervous system function (Johnson & Hofmann, 2017; Moya et al., 2016). However, whole body measurements cannot determine if non-tissue-specific transcripts are responding to elevated CO2 in a system-wide manner, or only within specific tissues. Furthermore, due to the heterogeneity and complexity of gene expression, measurements at the whole-body level may mask transcriptomic responses in specific tissues, such as the nervous system.
Here, we investigated the transcriptomic response to OA in the central and peripheral nervous system of a cephalopod, the two-toned pygmy squid (Idiosepius pygmaeus ), and then correlated the molecular responses with behavioural changes recorded in the same individuals. Cephalopods have complex nervous systems and behaviours rivalling those of fishes (Hanlon & Messenger, 2018), making them a useful taxon to investigate the neurobiological impacts of elevated CO2I. pygmaeus is a diurnal, tropical squid inhabiting shallow, inshore waters of the Indo-Pacific, including Northern and North-eastern Australia (Moynihan, 1983; Reid, 2005). They are a small, short-lived squid growing to a maximum mantle length of 2 cm (Reid, 2005), and have a lifespan of up to 80 days (Jackson, 1988). I. pygmaeus is an ideal species to use as previous research in this species found elevated CO2 alters a range of behaviours (Spady et al., 2018; Spady et al., 2014; Thomas et al., 2021).
In this study, we used RNA from the central nervous system (CNS) and eyes (peripheral sense organ) from squid exposed to current-day (~400 µatm) or elevated (~1,000 µatm) CO2 levels for 7 days in a previous study by Thomas et al. (2021). In these squid, elevated COexposure increased activity levels as well as visually-guided, conspecific-directed attraction and aggression (Thomas et al., 2021). Here, we created a de novo transcriptome assembly, providing a reference which we used to determine the transcriptomic response of the squid CNS and eyes to elevated CO2. We used the eyes because cephalopods, including squid, are highly visual animals with many visually-guided behaviours (Chung et al., 2022; Mather, 2006; Muntz, 1999). Furthermore, we have shown elevated CO2-induced disturbances of visually-guided behaviour in the same squid used in this study (Thomas et al., 2021). As we had transcriptomic and behavioural data from the same individual squid, we also correlated patterns of gene expression with COtreatment levels and OA-affected behaviours to determine key genes and processes in the cephalopod CNS and eyes potentially contributing to OA-induced behavioural changes. The results from this study help us understand, at a molecular level, the neurobiological impacts of ocean acidification in a marine invertebrate with a complex nervous system.