4. Discussion
As the nervous system forms the fundamental link between animals and the
environments they inhabit (Kelley et al., 2018; O’Donnell, 2018),
understanding the neurobiological impacts of environmental change is key
to predicting how and why animals will respond to anthropogenic climate
change. In this study, we sought to understand how projected
end-of-century CO2 levels alter the nervous system at a
molecular level, and how such changes may affect behaviour of the whole
animal. To do this, we investigated the transcriptomic response to ocean
acidification (OA) of the central and peripheral nervous system of a
marine invertebrate with a complex nervous system, the two-toned pygmy
squid Idiosepius pygmaeus. We then correlated patterns of gene
expression with CO2 treatment levels and OA-affected
behaviours in the same individuals. The central nervous system (CNS) and
eyes of I. pygmaeus responded to elevated CO2 with significant differential expression (DE) of a small number of
genes, and widespread small, coordinated changes of genes belonging
to important functional categories between CO2conditions. Furthermore, we identified 169 genes in the CNS, six genes
in the eyes and ten genes in both tissues that were correlated with
CO2 treatment and one or more behaviours affected by OA,
indicating these genes potentially contribute to OA-induced behavioural
changes.
The GABA hypothesis is the predominant mechanistic explanation for
OA-induced behavioural changes in fish (Nilsson et al., 2012;
Tresguerres & Hamilton, 2017) and may also apply to marine
invertebrates (Thomas et al., 2020). Pharmacological work has
supported the GABA hypothesis in marine molluscs (Clements et
al., 2017; Watson et al., 2014), including in I. pygmaeus (Thomas et al., 2021). In the whole-body of a pteropod mollusc, a
GABAA receptor transcript was upregulated after OA
exposure (Moya et al., 2016). In fish nervous tissue, OA exposure
has variable effects on GABAA R subunit transcript
expression, causing upregulation in some species (Cohen-Rengifo et
al., 2022; Schunter et al., 2018) but not another (Williamset al., 2019). Furthermore, differences in OA exposure duration
(Kang et al., 2022; Schunter et al., 2018; Schunter et al., 2016) and magnitude (Toy et al., 2022) are
associated with variable effects on the expression of genes coding for
GABAA R subunits within species. In I. pygmaeus, we found small, coordinated downregulation in the CNS and upregulation
in the eyes of genes for ion-channel receptors, which included
GABAA receptor subunit transcripts. In the CNS, there
was also upregulation of syvn1-b (implicated in
GABAAα1 receptor subunit degradation (Crider et
al., 2014; Jiao et al., 2017)), regulators of GABAergic
neurotransmission were positively correlated with CO2treatment and behaviours (phf24 , rac1), and aldh5a1(involved in the final degradation step of GABA (Kim et al.,
2009)) was negatively correlated with CO2 treatment and
behaviours. Together, this data suggests an effect of OA on GABAergic
signalling may be widespread, occurring not only in fish but also marine
molluscs, however, effects may be species and tissue-specific.
Recent research suggests that various other types of neurotransmission
may also be affected by OA and potentially contribute to consequent
behavioural responses. Pharmacological research has identified altered
function of a range of different types of ligand-gated
Cl- channels in I. pygmaeus (Thomas et
al., 2021) and the dopamine D1 receptor in a damselfish (Hamilton et al., 2023) contributing to OA-induced behavioural changes.
Elevated CO2 upregulated glycinergic, cholinergic and
glutamatergic transcripts in the whole body of a pteropod mollusc (Moya et al., 2016), upregulated glutamatergic transcripts in the
non-nervous tissue of oysters (Ertl et al., 2016; Wang et
al., 2020), and altered acetylcholine receptor transcript expression in
the whole body of another pteropod mollusc (Johnson & Hofmann, 2017).
In fish nervous tissue, exposure to OA conditions caused upregulation of
genes coding for glutamatergic and cholinergic neurotransmission of some
species (Cohen-Rengifo et al., 2022; Schunter et al.,
2018; Williams et al., 2019), but downregulation in others (Kanget al., 2022; Porteus et al., 2018). In I.
pygmaeus, we identified small, coordinated downregulation in the CNS
and upregulation in the eyes of genes involved in neurotransmission
mediated by ligand-gated ion channels, including transcripts for
subunits of ionotropic glutamate, glycine and acetylcholine receptors.
There was also small, coordinated downregulation in the CNS of G
protein-coupled receptor (GPCR)-mediated neurotransmission, including
genes for subunits of metabotropic glutamate, serotonin, dopamine and
GABA (GABAB) receptors. Key genes for glutamatergic and
monoaminergic signalling were DE, and a subunit of nicotinic
acetylcholine receptors (chrna10) was correlated with
CO2 treatment and behaviours in both tissues. Overall,
this suggests that OA not only impacts GABAA R function,
but various different types of neurotransmission mediated by
ligand-gated ion channels and GPCRs. However, as with GABAergic
signalling, OA effects on other types of neurotransmission may vary by
species and tissue type, and possibly other factors such as the
magnitude and duration of OA exposure. Further experimentation is needed
to understand how OA-induced transcriptomic responses translate to
neurotransmission function and behaviour.
Genes involved in the general processes required for synaptic
neurotransmission were attenuated in the CNS of I. pygmaeus after
OA exposure, including Ca2+, K+ and
Na+ ion channels required for maintenance of membrane
potential, action potential generation and neurotransmitter release.
There was also a negative correlation between the expression of genes
regulating synaptic neurotransmission with CO2 treatment
and activity traits. Genes for Ca2+ and
K+ transporters and the regulation of neurotransmitter
release were also downregulated in the brain of spiny damselfish
collected from CO2 seeps (Kang et al., 2022), but
upregulated after acute and developmental OA exposure (Schunter et
al., 2018), and in fish olfactory tissue after short-term (Williams et al., 2019) and transgenerational OA exposure (Cohen-Rengifo et al., 2022). These transcriptomic signatures suggest an even
more widespread effect of OA on neurotransmission, potentially altering
the general processes required for synaptic neurotransmission to occur.
However, experimentation is required to determine functional effects.
Neuroplasticity is the ability of the nervous system to change. Both
neurogenesis (the process by which new neurons are generated and
integrated into existing neural circuits) and synaptic plasticity
(changing of synaptic strength over time) contribute to neuroplasticity
(Costandi, 2016). In the CNS of I. pygmaeus we found small,
coordinated upregulation and positive correlation with
CO2 treatment and activity traits of genes involved in
all the stages required for neurogenesis (re-entering and exiting the
cell cycle, cell proliferation and differentiation to form new neurons,
and neural wiring involving cell migration and adhesion for new neurons
to be incorporated into existing circuits). Widespread upregulation and
positive correlations with CO2 treatment and behaviours
of genes in the CNS of I. pygmaeus involved in transcription, RNA
processing, and protein processing could potentially be a response to
deal with the changed protein demand required due to increased
neuroplasticity. Notably, cdk10, which plays an important role in
neurogenesis (Yeh et al., 2013) was identified as correlated with
the OA-induced increase in exploratory interactions in both the CNS and
eyes. Genes involved in synaptogenesis (the formation of new synapses)
and synaptic plasticity were also positively correlated with
CO2 treatment and OA-affected behaviours in the CNS of I. pygmaeus .
Despite not assessing the nervous tissue specifically, transcripts
involved in neuronal cell adhesion, neuronal differentiation and
survival and synaptic plasticity were also upregulated in the whole body
of a pteropod mollusc after OA exposure (Moya et al., 2016). In
fish nervous tissue, genes involved in neurogenesis were upregulated in
some species but not others (Lai et al., 2017), and genes
involved in synaptic plasticity were upregulated in some species
(Cohen-Rengifo et al., 2022; Schunter et al., 2018), but
downregulated in another species (Porteus et al., 2018). Thus,
OA-induced transcriptomic responses related to neuroplasticity may be
widespread, occurring in fish and marine molluscs. However, this
response may be taxa-specific and/or could be affected by differences in
CO2 exposure duration and magnitude that have differed
between studies.
Elevated CO2 alters the molluscan immune response, with
most research focusing on bivalves (Bibby et al., 2008; Liet al., 2015; Liu et al., 2016; Su et al., 2018; Wu et al., 2016)), though the immune response of an octopus was also
affected by elevated CO2 (Culler-Juarez & Onthank,
2021). Here, we found DE of genes that regulate immune signal
transduction pathways and which are also implicated in the molluscan
immune response (map4k5/3, syvn1-b, psenen , cbs) (Canesi et al., 2006; De Zoysa et al., 2010; Goodson et
al., 2005; Salazar et al., 2015). There were also changes in
expression of a range of genes that code for immune effectors, including
iron sequestration (tf and cbs), autophagy
(map1l3ca/b), controlling the pool of available nucleoside
triphosphates (nme6), and phagocytosis (‘cell adhesion’ and
multiple cytoskeleton functional categories). Previous research has also
indicated altered phagocytosis in molluscs at elevated
CO2; adhesion capacity of haemocytes was decreased in a
clam and expression of integrin (involved in cell adhesion for
phagocytosis) was decreased in an oyster species (Ivanina et al.,
2014) and increased in another oyster (Ertl et al., 2016).
Furthermore, the phagocytic rate and cytoskeleton component abundance
was decreased, and the expression of cytoskeleton genes was upregulated,
in a clam at elevated CO2 (Su et al., 2018). Our
results show an effect of OA on the transcriptional profile of genes
implicated in the immune response suggesting OA-induced alterations in
immune function may also occur in molluscan nervous tissue, though
further research directly measuring immune function within the nervous
system is required.
Cross-talk between the neuroendocrine and immune systems coordinates
appropriate physiological and behavioural responses to environmental
change (Demas et al., 2011). In molluscs, neuronal release of
norepinephrine regulates immune responses through a
neuroendocrine-immune axis-like pathway (Liu et al., 2017).
Specifically, changes in the expression and activity of maoa (upregulated in I. pygmaeus eyes here) plays a key role in immune
functioning via norepinephrine in molluscs (Liu et al., 2018; Sunet al., 2021; Zhou et al., 2011). Immune-derived factors
can also feedback to alter the nervous system and behaviour (Adamo,
2006; Dantzer & Kelley, 2007). Indeed, tf, which is a key
component of the molluscan innate immune response (Lambert et al., 2005;
Ong et al., 2006; Herath et al., 2015; Salazar et al., 2015; Li et al.,
2019) was upregulated and positively correlated with CO2 treatment and activity traits in the CNS of I. pygmaeus. We also
found the expression of genes coding for integrins itga4 and
itga9, cell adhesion molecules playing a key role in invertebrate
immune responses (Johansson, 1999; Terahara et al., 2006), were
positively correlated in the CNS with CO2 treatment and
activity traits. Therefore, it is possible that OA-induced changes in
neurotransmission could have consequences on immune function, and
changes in immune function could also feedback on the nervous system to
alter behaviours at elevated CO2. However, the potential
links between OA, neurotransmission, immune function and behaviour
remain to be experimentally tested.
Oxidative stress occurs when there is an imbalance between the
production of reactive oxygen species (ROS) and protection by
antioxidant mechanisms (Halliwell & Gutteridge, 2015). Elevated
CO2 induces oxidative stress in molluscs, increasing ROS
and altering antioxidant defences inducing DNA damage, lipid
peroxidation and apoptosis (Cao, Liu, et al., 2018; Cao,
Wang, et al. , 2018; Kim et al., 2023; Tomanek et
al., 2011; Wang et al., 2016; Zhang et al., 2021). In I. pygmaeus , we found DE of genes implicated in the production of
antioxidants, including ascorbate and glutathione. We also identified
upregulation in the CNS of genes involved in DNA damage and repair,
protein damage and endoplasmic reticulum stress, and cellular
stress-induced apoptosis. In molluscs, OA exposure has previously been
shown to result in DNA damage (Cao, Liu, et al., 2018; Nardi et al., 2018) and increased apoptosis (Cao, Liu, et al.,
2018; Zhang et al., 2021).
The nervous system is particularly vulnerable to oxidative stress
(Halliwell, 2006; Valko et al., 2007) and oxidative
stress-induced damage within the nervous system can disrupt
neurotransmission and neuronal function (Bouayed et al., 2009;
Halliwell, 2006; Halliwell & Gutteridge, 2015; Lebel & Bondy, 1991).
In mammals, a link between oxidative stress in the nervous system and
changes in behaviour has been demonstrated (Bhatt et al., 2020;
Bouayed, 2011; Rammal et al., 2010). In the CNS of I.
pygmaeus , we identified a positive correlation between the expression
of genes implicated in oxidative stress and CO2 treatment and OA-affected behaviours. In the eyes, two genes
(crb , zranb1) potentially correlated with OA-induced
behavioural alterations of I. pygmaeus are implicated in
oxidative-stress induced retinal degeneration (Chartier et al.,
2012; Wang et al., 2018). In particular, crb prevents
photoreceptor degeneration by limiting the production of ROS and the
resultant oxidative damage (Chartier et al., 2012). A recent
study in a cuttlefish found the behavioural effects of OA were
associated with an altered retinal structure and an increase in
apoptotic cells within the eyes (Xie et al., 2023). Thus, it’s
possible that OA-induced oxidative stress could contribute to
behavioural alterations at elevated CO2, potentially
through central and peripheral mechanisms, but further
electrophysiological and whole-animal behavioural experimentation is
required.
When interpreting our results, there are a few important things to
consider. Firstly, despite the reasonable assumption that changes in
gene expression driving behavioural responses occur prior to behavioural
production (Fischer et al., 2021), we measured gene expression
immediately after the OA-induced behavioural responses in I.
pygmaeus due to the necessity of terminal sampling to obtain nervous
tissue. Furthermore, the process of transcribing genes is far too slow
to mediate rapid behavioural responses, which are instead mediated by
fast electrical signals passed along and between neurons (Fischeret al., 2021). Thus, our transcriptomic results do not describe
the neuronal mechanisms driving the immediate behavioural responses to a
stimulus, but rather those that likely contribute to longer-term changes
in behaviour (Clayton et al., 2020; Fischer et al., 2021)
in OA conditions. Secondly, organism responses to OA can be sex-specific
(Ellis et al., 2017), including marine invertebrate behavioural
responses (Marčeta et al., 2020; Richardson et al., 2021).
We used males only in this study. Future research could consider using
both sexes to determine whether behavioural responses to OA are
sex-specific, and if so whether differing transcriptional profiles
underly these sex-specific responses.