- mRNA transcript copy numbers for selected gene targets show that differential mRNA transcript abundance in the brain appears to be partially conserved across species relative to personality type. - We further enriched this dataset by incorporating a zebrafish brain transcriptome dataset specific to the proactive phenotype. - Conclusions: Our data supports the proposition that highly polygenic clusters of genes, with small additive effects, likely support the underpinning molecular variation related to the animal personalities in the fish used in this study.. - The polygenic nature of the proactive brain transcriptome across all three species questions the existence of specific molecular signatures for proactive behaviour, at least at the granularity of specific regulatory gene modules, level of genes, gene networks and molecular functions.. - 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.. - 1 Institute of Aquaculture, University of Stirling, Stirlingshire FK9 4LA, UK Full list of author information is available at the end of the article Rey et al. - It should however be kept in mind that some trait correlations are flexible and can be dissociated during development and modulation of the environment [7]. - Developing tools to reliably identify individuals with contrasting personality traits facilitates the exploration of the underlying molecular and physiological regulation that in turn facilitates efforts to understand adaptation and the evolution of behavioural traits. - Tran- scriptomics provides the ideal platform to interrogate the organisation of the molecular processes underpinning studies in animal behaviour [18–20].. - We previously provided evidence that variation in the transcriptome between individuals in a zebrafish (Danio rerio) population could be partially resolved by a priori screening for animal personality and this accounted for. - 9% of observed variation in the brain transcriptome [14]. - These differences could be mapped to distinct regions of the brain and provide a foundation toward understanding the coordination of underpinning adaptive molecular events within populations [14, 22]. - From this curated gene set, we identified mRNAs that were specific to proactive behaviour and quantified their mRNA transcript copy numbers in the brains of Atlantic salmon (Salmo salar) and European sea bass (Dicen- trarchus labrax) screened a priori for personality by using a behavioural test. - Best BLAST results yielded 3738 and 1734 homologues with high BLAST scores (in- cluding high identities, low e-values and high coverage) for sea bass and salmon in respect to the zebrafish data.. - labrax, GAPDH mRNA transcript copy number contributed 70.4% to the grouping of individuals with different behavioural phenotypes (Fig. - Of these two assemblies, 63 and 88% of the total contig sequences were inspected to be “Good” by TransRate for S. - Accord- ing to the criteria of TransRate metrics, a ‘good’ assem- bly is defined as how contigs are aligned in a way that is consistent with the contig assembly. - (iv) without overlapping either end of the contig. - As evaluated by BUSCOs, most of the anticipated genes are present as single copies in all vertebrates (BUSCOs), are mostly expressed in both S. - salar are relatively high due to the extra genome du- plication event in this species (Table S4). - Subsequently, we combined this data with Illumina Sequencing data from the same tissue [24] to obtain a comprehensive profile of the D. - 2a), which consists of and 67.5% of the whole transcriptomes from D.. - rerio data resulting from microarray analyses (hybridisa- tion) showed a significantly lower dynamic range (fold changes) in comparison to the RNA-Seq data from the other two species, as expected. - The percentage of the variation explained by PC1/2 is shown in coordinates. - The arrow represents the original variable, where the direction represents the correlation between the original variable and the PC, and the length represents the contribution of the original variables to the PC. - UniProt identifiers of all three species were used for comparison and the Pfam (protein family) ID numbers and GO (gene ontology) terms were obtained based on their UniProt identifiers by Biomart (http://www.ensembl.. - c) histogram shows GO annotations and associated numbers of genes of the brain transcriptomes. - In order to facilitate the species-wise comparisons of proactive related DEGs, sequence annotations of the two non-model species were obtained by mapping against D.. - S.salar Volcano plot D.labrax Volcano plot D.rerio Volcano plot. - S .P 2P S .P 3P S.salar Proactive. - S.P1P S .P 2P S .P 3P S.salar Proactive 253 Up. - P 1C L.P3C D.Labrax Proactive. - P1 P2 P3 P4 P8 P9 P5P10 P6 P7C1C2 C3 C6 C5 C7 C8 C4 C9C10 D.Rerio Proactive. - a-i ~ iii) Volcano plots show DEGs, up-regulated DEGs in the proactive group are shown in red and the control group in blue (log2 (Fold Change. - of up-regulated DEGs in the proactive group from each cluster is shown in red, control group DEGs are blue. - For ppfibp1, transcript abundance was signifi- cantly higher in the proactive group in sea bass (P <. - Aiming to understand the functional significance of the correlated transcripts and identify regulatory pathways of proactive behaviour GO network analysis was carried. - a) both up- and down- regulated DEG clusters in the proactive group are combined from each species (based upon annotation of gene names in D. - out with all three species datasets (Fig. - Animal behaviour is shaped by the interaction between genes and the environment. - Such com- plexity may confound the value of molecular data to the evolutionary context of the question where trait. - The name of each group is named using the most significant term in the group. - Group sections represent the number of the terms included in each group. - Recognising individual variation within and across spe- cies has been brought to the fore by the field of animal personality/behavioural syndromes [6]. - In this study, a common behavioural test was designed for both species in order to harmonize the AP screening and obtain the same behavioural outputs regardless of the species. - Therefore, the intensity of O 2 deprivation and its relationship with the underlying species-specific physiology may influence the selectivity of the test par- ticularly to differentiate intermediate and reactive per- sonality types [38]. - Understanding the eco-physiological constraints in the design and development of high- resolution behavioural screening protocols for different species and their associated personalities with emphasis upon oxygen, temperature and light remains a challenge for comparative studies.. - and the remarkable gaps in knowledge remaining. - salar in the measured population. - Such observation suggests the possibility of retrofitting data using personality-specific threshold mRNA abundances to explore individual differences in the huge array of ex- istent experimentation and is an intriguing prospect for future studies.. - Our data revealed strong cor- relations between brain transcriptomes and the proactive personality in each of the species. - highlights, through GO enrichment analyses, that mul- tiple core functions across the three species are indeed conserved in the proactive brain although a set of spe- cific gene-based circuits common to all cannot be identi- fied (Table S10). - Observed inter-individual variation in zebrafish is significantly higher between re- active individuals [14] potentially highlighting the diffi- culties in designing behavioural assays to characterise the complexities of the reactive personality. - Our results support a role for convergent evolution for animal per- sonalities across the Teleostei as observed in the results from the behavioural assays deployed.. - However, the poly- genic nature of the proactive phenotype herein measured questions the existence of specific molecular signatures for proactive behaviour, at least at a granularity of specific regulatory gene modules. - Average weight 2 weeks prior to the experiment was g. - Tag manager soft- ware was used to identify fish leaving to the normoxia tank and those staying (independent of the decreasing oxygen level). - In the hypoxia tank the inlet was connected to a N 2 gas exchanger (15 mg N 2 L − 1. - Control tests prior to the experiment demonstrated that oxygen de- pletion was homogenous throughout the water column.. - Two video cameras were mounted outside the tube on each side of the opening and over water, in order to monitor fish movement. - Prior to the test, the tanks were cleaned, water temperature regulated if ne- cessary and the water flow in each tank set to 3.5 L min − 1 . - Fish were transferred to the experimental set-up as carefully as possible and released into the hypoxia tank. - The two tanks were left undisturbed behind an opaque curtain for the duration of the trial. - The moni- toring of the fish started immediately, and the fish were allowed to acclimate for 2 h prior to the change in the oxygen level. - During the decline in oxygen, the water flow in the hypoxia tank was directed through the gas exchanger, and a door between the hypoxic tank and the normoxic tank was opened so fish could freely swim be- tween the tanks. - After the oxygen level in the hypoxia tank reached 25% O 2 saturation, the screening was ter- minated and the fish transferred back to their holding. - All fish was sampled for blood plasma cortisol to confirm the activity levels of the HPI axis corresponding to proactive and reactive personalities. - For the hypoxia test, oxy- gen concentration was decreased in one chamber of a two-chamber tank and escape from the hypoxic to the normoxic chamber was assessed. - Sixty fish were placed in one chamber of the tank, subsequently the hypoxia tank, and were allowed to acclimate to the conditions for 30 min. - The second chamber of the tank was maintained under nor- mal conditions. - when two thirds of the fish had escaped from the hyp- oxia tank or when 8% oxygen saturation was reached (water temperature 20 °C, salinity 26.9). - In silico cloning in the target species was carried out using genomic resources. - 2.5 K sequences) and NCBI (86,801 ESTs) were added to sequence collections for sea bass held in the AquaSea server which forms part of the Aquagenomics website (http://www.aquagenomics.es). - This was also supported by network building in the Cytoscape platform aiming to identify interactions between the mRNAs identified.. - Target genes were validated for each species using thermal gradient RT-PCR and the products that met the. - Absolute quantification was performed and the copy number of each transcript, derived from the standard di- lution curve obtained from target plasmids was analyzed using a Thermocycler Stratagene Mx3005P (Agilent, USA). - The reaction mix (20 μl final volume) consisted of 10 μ of SYBR Green mix (Aligent, USA), 0.5 μl of each primer (20 μM), 7 μl of H2O and 2 μl of a 1/10 dilution of the cDNA sample. - Exploratory analysis of the gene expression data in rela- tion to the behavioural data was performed with specific software (AutoDiscovery®, Butler Scientifics). - A GLM ANCOVA was used to test for sig- nificant differences in gene expression between all individuals screened for AP in the populations studied.. - The adapters (indexers) in the paired-end raw reads were trimmed out by the quality control tool Trim-Galore (a wrapper tool based on Cutadapt Version 1.4.2 and FastQC Version 0.10.1) for high throughput sequence data, as set up by default quality threshold of Q20. - For each species, all ‘left’ reads were combined into a single file, and the same was applied for the ‘right’ reads. - labrax, both transcriptome assem- blies were compared to the D. - In order to estimate sequence conservation and the consistency of expression patterns in each species the following comparisons were conducted. - rerio (Taxonomy ID:7955) for all three species.. - Numbers of associated genes for each GO term were setup differently according to the number of DEGs iden- tified in each species (1% for D. - The name of each functional group (Overview term) was given by the leading term with the smallest P-value in the group. - The whole-transcriptome sequencing service was pro- vided by the Norwegian Sequencing Centre (www.sequencing.uio.no) hosted by the University of Oslo and supported by the “ Functional Genom- ics ” and “ Infrastructure ” programs of the Research Council of Norway and the Southeastern Regional Health Authorities. - 222378) and the Fundamental Research Funds for the Central Universities (Project No. - SM, SR, MLB, and BD participated in the experimental design. - SR and XJ carried out molecular analysis of the samples and posterior statistical analysis. - The work described in this manuscript was previously approved for ethical considerations in the following publications. - The work was performed under the approved protocol entitled ‘ Behavioural sorting in Atlantic salmon ’ at the Aquaculture Research Station in Tromsø (H KuF), Norway, providing authorization according to the Norwegian ‘ Forskrift om bruk av dyr i forsøk ’ (regulation of the use of animals in experiments), under the Animal Welfare Act. - Experiments were conducted following approval of the Animal Care Committee of France under the official license of M.L. - Contribution of genetics to the study of animal personalities: a review of case studies. - Advances in the Study of Behavior, Vol 30. - Ecological Genomics: Ecology and the Evolution of Genes and Genomes. - Lost in the map
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