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BackgroundThe identification of DNA methyltransferases (Dnmt) expression patterns during development and their regulation is important to understand the epigenetic mechanisms that modulate larval plasticity in marine fish. In this study, dnmt1 and dnmt3 paralogs were identified in the flatfish Solea senegalensis and expression patterns in early developmental stages and juveniles were determined. Additionally, the regulation of Dnmt transcription by a specific inhibitor (5-aza-2′-deoxycytidine) and temperature was evaluated.ResultsFive paralog genes of dnmt3, namely dnmt3aa, dnmt3ab, dnmt3ba, dnmt3bb.1 and dnmt3bb.2 and one gene for dnmt1 were identified. Phylogenetic analysis revealed that the dnmt gene family was highly conserved in teleosts and three fish-specific genes, dnmt3aa, dnmt3ba and dnmt3bb.2 have evolved. The spatio-temporal expression patterns of four dnmts (dnmt1, dnmt3aa, dnmt3ab and dnmt3bb.1) were different in early larval stages although all of them reduced expression with the age and were detected in neural organs and dnmt3aa appeared specific to somites. In juveniles, the four dnmt genes were expressed in brain and hematopoietic tissues such as kidney, spleen and gills. Treatment of sole embryos with 5-aza-2′-deoxycytidine down-regulated dntm1 and up-regulated dntm3aa. Moreover, in lecithotrophic larval stages, dnmt3aa and dnmt3ab were temperature sensitive and their expression was higher in larvae incubated at 16 °C relative to 20 °C.ConclusionFive dnmt3 and one dnmt1 paralog were identified in sole and their distinct developmental and tissue-specific expression patterns indicate that they may have different roles during development. The inhibitor 5-aza-2′-deoxycytidine modified the transcript abundance of dntm1 and dntm3aa in embryos, which suggests that a regulatory feedback mechanism exists for these genes. The impact of thermal regime on expression levels of dnmt3aa and dnmt3ab in lecithotrophic larval stages suggests that these paralogs might be involved in thermal programing.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-017-0154-0) contains supplementary material, which is available to authorized users.

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In this study, the short-and long-term effects of embryo incubation temperatures (16, 18 and 20ºC) on development and growth of the flatfish Senegalese sole (Solea senegalensis) was determined by investigating the expression patterns of the epigenetic regulators DNA methyltransferases (dnmt) and histone 3 (H3) and genes belonging to the retinoic acid (RA), insulin-like growth factors (IGFs) and hypothalamic-pituitary-thyroid (HPT) axes. Results indicated that egg incubation temperature affected embryo development, but not survival, and incubation at 16ºC significantly delayed development. Coincident with these effects, levels of muscle-specific dnmt3aa transcripts and histone H3 protein levels were significantly different between the 16 and 20ºC groups at hatch. The larvae from eggs incubated at 20ºC relative to the 16ºC group had significantly higher transcript levels of four genes belonging to the HPT axis (trhr1a, tshr, thrb and dio2), four genes of the RA axis (aldh1a2, cyp26a1, rara2, rarg), igfbp1 and the glycolytic enzyme gapdh2. Taken together the data suggest that higher egg incubation temperatures enhance energy production, which accelerates cell proliferation and larval development and that hatching is a key moment for the regulation of epigenetic mechanisms.Long-term effects of egg and larval incubation temperatures were revealed by a higher mRNA abundance of the thyroid-related genes tgb and tpo and the RA degrading enzyme cyp26a1 in preand metamorphic larvae when they were incubated at 20ºC as embryos and may be related to the earlier initiation of metamorphosis in the pelagic larval stages. Evaluation of growth in pelagic larvae and juveniles after weaning (one trial from 42 to 119 and another from 164 to 247 days post-hatch using a longitudinal approach) revealed that juveniles from embryos incubated at 20ºC had a higher growth rate. All these data demonstrate that the thermal regime during embryogenesis modulated mechanisms that regulate larval plasticity and caused imprinting evident in juvenile sole as persistent changes in key endocrinological pathways and growth performance.

Somatic growth is a balance between protein synthesis and degradation, and it is largely influenced by nutritional clues. Antioxidants levels play a key role in protein turnover by reducing the oxidative damage in the skeletal muscle, and hence promoting growth performance in the long-term. In the present study, Senegalese sole postlarvae (45 days after hatching, DAH) were fed with three experimental diets, a control (CTRL) and two supplemented with natural antioxidants: curcumin (CC) and grape seed (GS). Trial spanned for 25 days and growth performance, muscle cellularity and the expression of muscle growth related genes were assessed at the end of the experiment (70 DAH). The diets CC and GS significantly improved growth performance of fish compared to the CTRL diet. This enhanced growth was associated with larger muscle cross sectional area, with fish fed CC being significantly different from those fed the CTRL. Sole fed the CC diet had the highest number of muscle fibers, indicating that this diet promoted muscle hyperplastic growth. Although the mean fiber diameter did not differ significantly amongst treatments, the proportion of large-sized fibers (>25 μm) was also higher in fish fed the CC diet suggesting increased hypertrophic growth. Such differences in the phenotype were associated with a significant up-regulation of the myogenic differentiation 2 ( myod2 ) and the myomaker ( mymk ) transcripts involved in myocyte differentiation and fusion, respectively, during larval development. The inclusion of grape seed extract (GS diet) resulted in a significant increase in the expression of myostatin1 . These results demonstrate that both diets (CC and GS) can positively modulate muscle development and promote growth in sole postlarvae. This effect is more prominent in CC fed fish, where increased hyperplastic and hypertrophic growth of the muscle was associated with an upregulation of myod2 and mymk genes.

Pseudo-albinism is a pigmentation disorder observed in flatfish aquaculture with a complex, multi-factor aetiology. We tested the hypothesis that pigmentation abnormalities are an overt signal of more generalised modifications in tissue structure and function, using as a model the Senegalese sole and two important innate immune barriers, the skin and intestine, and their microbiomes. Stereological analyses in pseudo-albino sole revealed a significantly increased mucous cell number in skin (P < 0.001) and a significantly thicker muscle layer and lamina propria in gut (P < 0.001). RNA-seq transcriptome analysis of the skin and gut identified 573 differentially expressed transcripts (DETs, FDR < 0.05) between pseudo-albino and pigmented soles (one pool/tissue from 4 individuals/phenotype). DETs were mainly linked to pigment production, skin structure and regeneration and smooth muscle contraction. The microbiome (16 S rRNA analysis) was highly diverse in pigmented and pseudo-albino skin but in gut had low complexity and diverged between the two pigmentation phenotypes. Quantitative PCR revealed significantly lower loads of Mycoplasma (P < 0.05) and Vibrio bacteria (P < 0.01) in pseudo-albino compared to the control. The study revealed that pseudo-albinism in addition to pigmentation changes was associated with generalised changes in the skin and gut structure and a modification in the gut microbiome.

Umbilical artery Doppler velocimetry waveform notching has long been associated with umbilical cord abnormalities, such as distortion, torsion, and/or compression (i.e., constriction). The physical mechanism by which the notching occurs has not been elucidated. Flow velocity waveforms (FVWs) from two-dimensional pulsatile flows in a constricted channel approximating a compressed umbilical cord are analyzed, leading to a clear relationship between the notching and the constriction. Two flows with an asymmetric, semi-elliptical constriction are computed using a stabilized finite-element method. In one case, the constriction blocks 75% of the flow passage, and in the other the constriction blocks 85%. Channel width and prescribed flow rates at the channel inflow are consistent with typical cord diameters and flow rates reported in the literature. Computational results indicate that waveform notching is caused by flow separation induced by the constriction, giving rise to a vortex (core) wave and associated eddies. Notching in FVWs based on centerline velocity (centerline FVW) is directly related to the passage of an eddy over the point of measurement on the centerline. Notching in FVWs based on maximum cross-sectional velocity (envelope FVW) is directly related to acceleration and deceleration of the fluid along the vortex wave. Results show that notching in envelope FVW is not present in flows with less than a 75% constriction. Furthermore, notching disappears as the vortex wave is attenuated at distances downstream of the constriction. In the flows with 75 and 85% constriction, notching of the envelope FVW disappears at ∼3.8 and ∼4.3 cm (respectively) downstream of the constriction. These results are of significant medical importance, given that envelope FVW is typically measured by commercial Doppler systems.