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 GABAA receptor (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 CO2. I. 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 CO2 exposure 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 CO2 treatment 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.