We showed that a modified epiGBS protocol originally proposed by Van Gurp et al. [33] was applicable to further analyze patterns of cytosine methylation in RBCs of D. labrax. This is the first use of epiGBS in fish and the second in an animal species (Canadian lynx [38]). Overall, RBC’s DNA methylation was shown to respond to the challenge test, but observed changes were found mainly explained by the genetic background of individuals resulting from family-based effects, and involved few sites and DMC-related genes.
Mining the sea bass epigenome
The addition of a second restriction enzyme illustrates the flexibility of the epiGBS originally proposed by Van Gurp et al. [33] and more generally of reduced-representation bisulfite sequencing (RRBS) protocols for data acquisition and impact [103]. The addition of a second restriction enzyme to a RRBS protocol in order to improve coverage and accuracy of CpG methylation profiling was however already shown [39], but hereby proposed in a context of improved multiplexing of samples.
The information provided in this study is based on the analysis of 47,983 distinct methylated sites distributed over all sea bass LGs. The mapping efficiency was high (74.5%) when compared to early values retrieved in human (~65%) [40], or in fish studies screening for genome-wide methylation (e.g. 55-60% in [41]; 40% in [17]). Other studies reported similar mapping efficiencies, but reported percentages of mapping for unique best hits that were generally lower. For example, in Kryptolebias marmoratus, Berbel-Filho et al. [42] reported a mean mapping efficiency of 74.2% but 61.1% unique best hits while, in this study, this latter percentage reached 73.0%. This reflects a more robust mapping of the DMCs we detected and significantly enlarge the breadth of the sites that can confidently exploit to retrieve functional information. Taking advantage of the epiGBS protocol that allow to process more samples [88], the number of individuals considered in this study is rather high (n = 70 distinct individuals), when most epigenomic studies in fish dealt with less than 30 individuals (range: n = 3 in [43]; n = 106 in [44] for a population study). In sea bass, Anastasiadi and Piferrer [26] previously reported a study that used 27 samples and as many libraries to be sequenced while our data were obtained from a unique library preparation. Our modified epiGBS protocol provides a considerable amount of information, certainly at a reasonable cost, to decipher methylation landscapes of sea bass or other species.
The operational and statistical thresholds used in the successive steps of this study are conservative, resulting in the discovery of a rather low total number of methylated sites, but certainly limiting the report of false positives. For example, a threshold of 30X and nominal cut-off value of 0.001 are quite conservative, when some studies might consider a threshold of 5X or 10X for a CpG to be analysed and associated cut-off values of 0.05 or 0.01 (e.g. [26,41,45]). Relaxing thresholds would enable to retrieve more DMCs, but elevated thresholds should normally ensure that access to relevant information is reached. Thus, only 57 DMCs have been found in RBCs of pre- and post-challenge European sea bass. These DMCs were found mostly hypermethylated in post- compared to the pre-challenge individuals, and mostly located in gene bodies (i.e. the transcriptionally active portion of the genome) of fifty-one different genes. Differential methylation in gene bodies may regulate splicing and/or act as alternative promoters to reshape gene expression [46-48].
In addition to DMCs located in gene bodies, a dozen of DMCs were found in intergenic regions (21.0%). Intergenic cytosine methylation has been frequently described, including in response to stress [48], but its role remains poorly understood [50,51]. While numbers of genic vs intergenic DMCs may greatly vary, a ratio of ~80% of DMCs located in gene bodies and ~20% located in other genomic regions has been reported in other fish studies (e.g. [9]).
An epigenomic perspective on stress biomarkers?
Studies looking at the epigenomic landscape of RBCs in fish are scarce, and did not focus on response to a stress challenge [17]. Our study yielded mixed results regarding this issue. Negatively, this study did not considered controls (i.e. un-stressed fish) and it is difficult to assess if cytosines that were shown to respond to the stress challenge test really reflect the impact of stress or other parameters. This notably includes growth and ontogenetic changes in first year juvenile sea bass, together with sexual differentiation. Sexual differentiation occurs between 150 and 250 day post-fertilization in sea bass (e.g. [20]) and differential methylation measured in gonads at few candidate genes has been reported over this period [20,23]. As our challenge test covers this period, results might be partially influenced by sexual differentiation. This has to be investigated further. However, we are not aware of studies that showed that differential methylation recorded in gonads might translate to RBCs, and none of the candidate differentially methylated genes previously studied in sea bass gonads was detected in this study. Methylation variation accompanying ontogeny and/or aging is reported in fish [52,53], and it has been shown to be modified with age in sea bass muscles [27]. Unfortunately, methylation results also concerned candidate genes not detected as differentially methylated in this study. Relevant to our study, BMP3 [54], FURIN [55], NOL4B [56], Myf5 [57,58], NPAS3 [59], and ROBO3 [60] are engaged in the development of the anterior region and/or the craniofacial skeleton which is modified during sea bass farming [26]. Their roles were however studied in early development stages of mammals or zebrafish. As ‘epigenetic programming’ – apart of transgenerational inheritance - is mostly an early-life process that influence late-life effects (in fish, see, e.g., [5,9,61,62]), we thus hypothesize that the epigenetic marks that could affect them would have been already present in six month-old sea bass that initiated the challenge test. Developmentally induced differential methylation acquired during the challenge test seems unlikely. Furthermore, some of them have clear relationships to stress exposure (e.g. FURIN, NPAS3, see below). However, in absence of dedicated study, we cannot totally rule out that methylation patterns observed in sea bass RBCs could also partly reflect the developmental or sexual regulation of a particular phenotype between pre- and post-challenge fish, rather than being directly related to the stress challenge.
Nevertheless - and more positively - some DMC-related genes detected in this study have been shown to be involved in the stress response in fish. Strikingly, 37 over 51 DMC related-genes were reported mainly from few neurotranscriptomics zebrafish studies that dealt either with reactive-proactive behavioural response to stress [63,64] or with changes in social regulation that may promote stressful behaviour among congeners [65,66] (Table 1). While not detected in RBCs but in brain tissues, correspondence across stress studies is interesting in this particular case. Indeed, while not investigated in fish so far, human stress studies have shown that blood cells responded to DNA methylation in the brain [67-69] (but see [70]), and specifically RBCs in birds [71]. Furthermore, several of these DMC-related genes are involved in the maturation of proBDNF to mature BDNF or the regulation of its activities (ABLIM2, ADCY1b, CRTC2, FURIN, NPAS3, PLG, and possibly SLC22A2 and DLG1). BDNF consolidates both the within- and between-generation fear memory owing to epigenetic regulation [72,73]. Its activity is strongly linked to glucocorticoid stress to imprint neurogenesis [74] and it acts as both a regulator and a target of stress hormone signaling [75]. BDNF is one of the target genes in fish stress studies (zebrafish [76], sea bream [77], sea bass [32,78-80]), but its methylation status could not be investigated in this study as no SbfI restriction site is present within or close to this gene. However, the above-mentioned DMC-related genes might be linked to a ‘BDNF network’. The adenyl(ate) cyclase (AC, ADCY1b gene) is a brain-specific signaling enzyme that synthesizes the cyclic AMP [81]. This inducible signaling pathway participates to the synthesis of the active form of BDNF (proBDNF to mature BDNF) [82]. ProBDNF is processed by furin and the plasminogen system [83], including processing steps that necessitate actions of actin-binding LIM kinases (ABLIM) [84]. Stress imprinting at FURIN is likely and it has recently been shown that transgenerational epigenetic effects of furin activity were active in brain of mice [85]. Furin has also been shown to modulate learning abilities and memory [86]. ProBDNF cleavage by furin depends on brain AC and CREB (cAMP response element-binding protein) signaling [87,88], but also plasminogen (PLG) [89]. This activity is modulated by stress hormones (corticosteroids) [90,91]. One interesting supplementary observation is that CREB signaling necessary to furin is associated to CRTC2 - a CREB co-activator. In mice, CRTC2 is known to act as a switch for BDNF and glucocorticoids to direct the expression of corticotropin-releasing hormone (CRH) in the hypothalamus [92]. Additionally, in the brain, plasminogen encoded by PLG is converted to plasmin that cleaves BDNF in the extracellular synaptic domain [83,93]. PLG has also been shown to regulate pro-opiomelanocortin (POMC) in the hypothalamic-pituitary axis, then the production of peptides hormones such as the adrenocorticotropic hormone (ACTH) [94].
Few other DMC-related genes should be mentioned. SLC22A2 - also known as OCT2 (organic cation transporter 2) – associated to the unique 3’UTR DMC found in this study is involved in numerous transmembrane transports [95], including at the blood-brain barrier [96]. It was found involved in memory in mice [97,98] or Drosophila [99]. In this study, PLG and SLC22A2 are associated to the same DMC; the functional significance of this situation needs further investigation. NPAS3 (neuronal PAS domain containing protein 3) has a well-established action in memory [100,101], and participate to a neural network that also includes BDNF [102]. NPAS3 is also associated to the glial cell line-derived neurotrophic factor receptor-alpha2 gene (GFRA2) detected in this study and related to stress and anxiety [103]. Finally, DLG1 (Disk-large homolog 1) plays a critical role in neural synapse formation, insulin secretion and glucose transport that are activated or modulated by stressors [104]. Adrenergic modulation implying DLG1 was also found to correlate with emotional states and stress sensitivity in mice [105] and it indirectly participates to the regulation of BDNF as DLG1 activates the glutamate receptor 1 (GluR1; [104]) that interacts with molecular processing of BDNF [106]. A relationship of DLG1 with NCBP2 (nuclear cap binding protein 2) was detected in protein-protein interaction analyses. NCBP2 protect cellular RNA polymerase II transcripts from degradation and to guide them through the sequence of steps leading from transcription to translation [107]. As the cellular response to environmental challenges requires immediate and precise regulation of transcriptional programs, differences in cytosine methylation among pre- and post-challenge sea bass close the NCBP2 gene could partly reflect the impact of the stress challenge.
While confounding factors may be present, results thus suggest that some DMCs reported in this study did not occur only by chance, are related to processes that regulate the hypothalamus-pituitary-interrenal (HPI) axis and hormones, but also to features that are expected to be developed or regulated during a stress challenge (e.g. anxiety, fear memory, neurogenesis). While presumptive, these DMCs could effectively reflect the impact of acute stress periods developed during the challenge test, and suggest that traditional blood plasma biomarkers could be potentially enriched by epigenetic marks to monitor welfare in cultured fish species. The link between brain and blood epigenomics remains however to be explored more deeply in fish and requires careful evaluation and validation to correct for tissue specificity, as requested in human [108]. Recent results on chicken RBCs are encouraging [71].
Hereby, we focused on possible relationships among DMC-related genes detected in this study and expressed in stress-related studies in brain tissues of fish [63-66]. It should be however important to note that some DMC-related genes could be related to other components of the stress response (e.g., immune response, glucose metabolism). For example, a role of PRKCQ (PKC-theta, a protein kinase C theta type) in the immune response is well-known in vertebrates (e.g. [109]). PRKCQ is also known to participate to glucose metabolism, including glucose homeostasis [110]. One association with DLG1 is reported, also related to the immune response [111]. The role of CRTC2 on glucose homeostasis when facing stress has also been repeatedly reported [112-114], notably in relation to glucocorticoid levels [115]. We cannot expand further on this topic, but this suggests that DMC-related genes detected in this study may integrate several aspects of the stress response in fish.
A family effect, but the possible absence of individual response
As in other fish species [116-118], a family effect imprinting the methylome was detected in this study. In parallel, results showed that the epigenetic profiles of the four individuals that were analyzed in the pre- and post-challenge conditions clustered very closely from each other. While based on few observations of randomly sampled individuals, this suggests that the stress challenge had few impact on the cytosine methylation landscape in sea bass compared to family effects. Nature and strength of family-based epigenomic variation are of considerable importance attention to engage future selection breeding improvements in cultured fish like sea bass, including issues about health and welfare [119]. More generally, how transgenerational and within-generation stress-imprinting events may interact to shape both the plastic and the heritable component of the stress response in relation to environmental stimuli require in depth evaluation [120,121]. To do so, far more complex and rigorous experimental designs that the one followed in the present study as to be adopted and temporal monitoring of the individual response of blood methylome to stress has to be promoted. In sea bass, such a research has been engaged for sex determination [24]. Results showed that some epigenetic marks were more likely engaged in transgenerational inheritance, while others be related to within-generation differences acquired during early development [24].