Ocean acidification (OA), one of the aspects of global change, has been shown to have adverse effects on numerous marine organisms due to its impact on diverse biological processes [1, 2, 3]. While marine fishes were initially thought to be resilient to OA, a plethora of studies have shown that exposure to elevated CO2 can negatively impact various aspects of fish physiology and behaviour [1, 4, 5, 6]. Some of the most concerning detrimental effects of OA on coral reef fish, with significant ecological consequences such as sensory and behavioural abnormalities, have been observed at CO2 levels between 700–1000 µatms [7, 8, 9, 10, 11, 12]. The underlying cause of these abnormalities are changes in brain activity in high CO2 environments, particularly alterations in GABAergic neurotransmission, neurological functioning, and disruption of the circadian rhythm [13, 14, 15, 16, 17, 18, 19, 20]. It has been hypothesized that environmental pH changes may alter the natural circadian rhythm, which governs almost all physiological processes [21]. This perturbation could be linked to variations in synaptic transmission and neuronal activity, potentially leading to physiological and behavioural changes. However, further studies incorporating multiple timepoints throughout a circadian day are needed to gain a more comprehensive understanding of the interplay between environmental pH variability and circadian rhythm and elucidate potential underlying molecular mechanisms.
The majority of OA studies have been conducted at stable, elevated CO2 levels, thereby neglecting the temporal fluctuations in CO2 levels resulting from a variety of biological and physical processes in coral reefs, coastal zones, and shallow habitats [22, 23, 24]. Coral reef habitats are highly dynamic and undergo daily cycles of CO2 variation resulting from photosynthesis/respiration, calcification/dissolution and physical factors like flow rates and trajectory [25, 26, 27]. With the increasing uptake of CO2 by the oceans, these natural CO2 fluctuations will intensify as seawater buffering capacity diminishes [28, 29]. In light of the ongoing amplification of global environmental changes and heightened environmental variability, it is imperative to incorporate these fluctuations, which mirror the natural habitats of species, into experiments to accurately assess the biological and ecological consequences of OA.
Diel CO2 oscillations have been shown to alleviate or reduce the severity of behavioural abnormalities observed in stable elevated CO2 environments [30, 31]. Moreover, ecologically and evolutionarily important traits such as survival, growth, and metabolic function appear to be less affected by elevated CO2 in fluctuating compared to stable environments [31, 32, 33, 34]. This disparity in the impact of elevated CO2 on fish physiology and behaviour in stable versus fluctuating environments may be attributed to differences in the magnitude of circadian control of these biological processes [35]. In fact, the brain transcriptional profile in coral reef fishes differed significantly between fish in stable and fluctuating CO2 conditions with a distinct regulation of circadian rhythm genes in the two environments [35]. Therefore, the increased flexibility of fish species in adjusting physiological processes to pH changes could be attributed to change in expression of circadian rhythm genes.
While alterations in the expression levels of circadian rhythm genes have been repeatedly reported in fish upon exposure to OA conditions, there is limited information on the regulatory mechanisms that drive these patterns in fish. Alternative mRNA splicing, a crucial RNA-processing mechanism that enhances cellular proteomic complexity [36, 37], has been linked to the circadian clock and its modulation [38, 39, 40, 41]. Evidence suggests that clock regulated alternative splicing events play a role in the rhythmic expression of various genes, including neuro-specific genes and neurotransmitters [42, 43, 44]. Consequently, rhythmic splicing events could influence the physiological and behavioural responses of organisms to environmental variations, such as changes in CO2 levels. Indeed, the role of alternative splicing has been highlighted in driving adaptive evolution over both short and long timescales [45] and alternative splicing has been implicated as a mechanism driving the response of coral reef fish to marine heatwaves [46]. Given its potential to rapidly generate phenotypic diversity, alternative splicing may serve as a key molecular mechanism for facilitating phenotypic plasticity in response to rapidly changing environments caused by climate change.
In this study, we examine the transcriptional patterns mediated by alternative splicing in driving the molecular response of fish to OA across their daily cycle. We assess the impact of environmental CO2 variation at four timepoints throughout a circadian (24-hr) day and aim to: (1) determine if the natural rhythmic patterns of splicing events are altered in response to changes in near-future predicted CO2 levels, and (2) examine how exposure to stable and fluctuating elevated CO2 conditions affects the circadian splicing patterns in fish brains. We reveal how splicing events may facilitate regulation of biological responses of fish to environmental CO2 variability across a circadian timeline.