Coral reef fish exhibit a large variety of behaviours crucial for fitness and survival. The cleaner wrasse Labroides dimidiatus displays cognitive abilities during interspecific interactions by providing services of ectoparasite cleaning, thus serving as a good example to understand the processes of complex social behaviour. However, little is known about the molecular underpinnings of cooperative behaviour between L. dimidiatus and a potential client fish (Acanthurus leucosternon). Therefore, we investigated the molecular mechanisms in three regions of the brain (Fore-, Mid-, and Hindbrain) during the interaction of these fishes. Here we show, using transcriptomics, that most of the transcriptional response in both species was regulated in the Hindbrain and Forebrain regions and that the interacting behaviour responses of L. dimidiatus involved immediate early gene alteration, dopaminergic and glutamatergic pathways, the expression of neurohormones (such as isotocin) and steroids (e.g. progesterone and estrogen). In contrast, in the client, fewer molecular alterations were found, mostly involving pituitary hormone responses. The particular pathways found suggested synaptic plasticity, learning and memory processes in the cleaner wrasse, while the client indicated stress relief.

Coral reef fish exhibit a large variety of behaviours crucial for fitness and survival. The cleaner wrasse Labroides dimidiatus displays cognitive abilities during interspecific interactions by providing services of ectoparasite cleaning, thus serving as a good model to understand the processes of complex social behaviour. However, little is known about the molecular underpinnings of cooperative behaviour between L. dimidiatus and a potential client fish (Acanthurus leucosternon). Therefore, we investigated the molecular mechanisms in three regions of the brain (fore-, mid-, and hindbrain) during the interaction of these fishes. Here we show, using transcriptomics, that most of the transcriptional response in both species was regulated in the hindbrain and forebrain regions and that the interacting behaviour responses of L. dimidiatus involved immediate early gene alteration, dopaminergic and glutamatergic pathways, the expression of neurohormones (such as isotocin) and steroids (e.g. progesterone and estrogen), as well as social decision-making genes. In contrast, in the client, fewer molecular alterations were found, mostly involving pituitary hormone responses. The particular pathways found suggested learning and memory processes in the cleaner wrasse, while the client indicated stress relief and a reduction in aggression.

Mutualistic interactions, which constitute some of the most advantageous interactions among fish species, are highly vulnerable to environmental changes. A key mutualistic interaction is the cleaning service rendered by the cleaner wrasse, Labroides dimidiatus, which involves intricate processes of social behaviour to remove ectoparasites from client fish and can be altered in near-future environmental conditions. Here, we evaluated the neuromolecular mechanisms behind the behavioural disruption of cleaning interactions in response to future environments. We subjected cleaner wrasses and surgeonfish (Acanthurus leucosternon, serving as clients) to elevated temperature (Warming, 32oC), increased levels of CO2 (High CO2, 1000 ppm), and a combined condition of elevated CO2 and temperature (Warming & High CO2, 32oC & 1000 ppm) for 28 days. Each of these conditions resulted in behavioural disruptions concerning the motivation to interact and the quality of interaction (High CO2 -80.7%, Warming -92.6%, Warming & High CO2 -79.5%, p<0.001). Using transcriptomic of the fore-, mid-, and hindbrain, we discovered that most transcriptional reprogramming in both species under warming conditions occurred primarily in the hind- and forebrain. The associated functions under warming were linked to stress, heat shock proteins, hypoxia, and behaviour. In contrast, elevated CO2 exposure affected a range of functions associated with GABA, behaviour, visual perception, and circadian rhythm. Interestingly, in the combined Warming & High CO2 condition, we did not observe any expression changes of behaviour. However, we did find signs of endoplasmic reticulum stress and apoptosis, suggesting not only an additive effect of the environmental conditions but also a trade-off between physiological performance and behaviour in the cleaner wrasse. We suggest that impending environmental shifts can affect the behaviour and molecular processes that sustain mutualistic interactions between L. dimidiatus and its clients, which could have a cascading effect on their adaptation potential and possibly cause large-scale impacts on coral reef ecosystems.

The majority of the transcribed genome does not have coding potential but is composed of non-coding transcripts that are involved in transcriptional and post-transcriptional regulation of protein-coding genes. Regulation of gene expression is important in determining the response of organisms to changes in the environment, and therefore their persistence as population or species under global change. However, lncRNAs are scarcely studied especially in non-model organisms due to the lack of a reliable pipeline for their accurate identification and annotation. Here, we present a pipeline which uses a combination of alignment-dependent and independent methods for the identification of conserved and species-specific lncRNAs from RNA-Seq data. Validation of this pipeline was performed using existing RNA-Seq data from Acanthochromis polyacanthus brain tissue, identifying a total of 4,728 lncRNAs across the whole genome, the majority of which (3,272) are intergenic. To investigate the possible implications of these lncRNAs, we estimated the expression changes of lncRNAs and neighbouring coding genes in response to ocean acidification. We found that intergenic lncRNAs might adjust the expression of neighbouring coding genes related to pH regulation, neural signal transduction and ion transport, which are known to be important in the response to ocean acidification in fish. Overall, this pipeline enables the use of existing RNA sequencing technology to reveal additional underlying molecular mechanisms involved in the response to environmental changes in non-model species by integrating the study of lncRNAs with gene expression.