Thermal imprinting during embryogenesis modifies skin repair in juvenile European sea bass (Dicentrarchus labrax).

Fish skin is a multifunctional tissue that develops during embryogenesis, a developmental stage highly susceptible to epigenetic marks. In this study, the impact of egg incubation temperature on the regeneration of a cutaneous wound caused by scale removal in juvenile European sea bass was evaluated. Sea bass eggs were incubated at 11, 13.5 and 16 °C until hatching and then were reared at a common temperature until 9 months when the skin was damaged and sampled at 0, 1 and 3 days after scale removal and compared to the intact skin from the other flank. Skin damage elicited an immediate significant (p < 0.001) up-regulation of pcna in fish from eggs incubated at higher temperatures. In fish from eggs incubated at 11 °C there was a significant (p < 0.001) up-regulation of krt2 compared to fish from higher thermal backgrounds 1 day after skin damage. Damaged epidermis was regenerated after 3 days in all fish irrespective of the thermal background, but in fish from eggs incubated at 11 °C the epidermis was significantly (p < 0.01) thinner compared to other groups, had less goblet cells and less melanomacrophages. The thickness of the dermis increased during regeneration of wounded skin irrespective of the thermal background and by 3 days was significantly (p < 0.01) thicker than the dermis from the intact flank. The expression of genes for ECM remodelling (mmp9, colXα, col1α1, sparc, and angptl2b) and innate immunity (lyg1, lalba, sod1, csf-1r and pparγ) changed during regeneration but were not affected by egg thermal regime. Overall, the results indicate that thermal imprinting of eggs modifies the damage-repair response in juvenile sea bass skin.

Skin damage caused by scale loss modifies the intestine of chronically stressed gilthead sea bream (Sparus aurata, L.).

The present study was designed to test if the damage caused by scale loss provokes a change in other innate immune barriers such as the intestine and how chronic stress affects this response. Sea bream (Sparus aurata) were kept in tanks at low density (16 kg m-3, LD) or exposed to a chronic high density (45 kg m-3, HD) stress for 4 weeks. Scales were then removed (approximately 50%) from the left flank in the LD and HD fish. Intestine samples (n = 8/group) were examined before and at 12 h, 3 days and 7 days after scale removal. Changes in the morphology of the intestine revealed that chronic stress and scale loss was associated with intestinal inflammation. Specifically, enterocyte height and the width of the lamina propria, submucosa and muscle layer were significantly increased (p < 0.05) 3 days after skin damage in fish under chronic stress (HD) compared to other treatments (LDWgut3d or HDgut0h). This was associated with a significant up-regulation (p < 0.05) in the intestine of gene transcripts for cell proliferation (pcna) and anti-inflammatory cytokine tgfß1 and down-regulation of gene transcripts for the pro-inflammatory cytokines tnf-α and il1ß (p < 0.05) in HD and LD fish 3 days after scale removal compared to the undamaged control (LDgut0h). Furthermore, a significant up-regulation of kit, a marker of mast cells, in the intestine of HDWgut3d and LDWgut3d fish suggests they may mediate the crosstalk between immune barriers. Skin damage induced an increase in cortisol levels in the anterior intestine in HDWgut12 h fish and significant (p < 0.05) down-regulation of mr expression, irrespective of stress. These results suggest glucocorticoid levels and signalling in the intestine of fish are modified by superficial cutaneous wounds and it likely modulates intestine inflammation.

Chronic stress impairs the local immune response during cutaneous repair in gilthead sea bream (Sparus aurata, L.).

Scale removal in fish triggers a damage-repair program to re-establish the lost epidermis and scale and an associated local immune response. In mammals, chronic stress is known to delay wound healing and to modulate the cutaneous stress axis, but this is unstudied in teleost fish the most successful extant vertebrates. The present study was designed to test the hypothesis that chronic stress impairs cutaneous repair in teleost fish as a consequence of suppression of the immune response. The hypothesis was tested by removing the scales and damaging the skin on one side of the body of fish previously exposed for 4 weeks to a chronic crowding stress and then evaluating cutaneous repair for 1 week. Scale removal caused the loss of the epidermis although at 3days it was re-established. At this stage the basement membrane was significantly thicker (p=0.038) and the hypodermis was significantly thinner (p=0.016) in the regenerating skin of stressed fish relative to the control fish. At 3days, stressed fish also had a significantly lower plasma osmolality (p=0.015) than control fish indicative of reduced barrier function. Chronic stress caused a significant down-regulation of the glucocorticoid receptor (gr) in skin before damage (time 0, p=0.005) and of star at 3 and 7days (p<0.05) after regeneration relative to control fish. In regenerating skin key transcripts of cutaneous repair, pcna, colivα1 and mmp9, and the inflammatory response, tgfß1, csf-1r, mpo and crtac2, were down-regulated (p<0.05) by chronic stress. Irrespective of chronic stress and in contrast to intact skin many hyper pigmented masses, putative melanomacrophages, infiltrated the epidermis of regenerating skin. This study reveals that chronic stress suppresses the local immune response to scale removal and impairs the expression of key transcripts of wound healing. Elements of the stress axis were identified and modulated by chronic stress during cutaneous repair in gilthead seabream skin.

Thermal imprinting modifies adult stress and innate immune responsiveness in the teleost sea bream.

The impact of thermal imprinting on the plasticity of the hypothalamic-pituitary-interrenal (HPI) axis and stress response in an adult ectotherm, the gilthead sea bream (Sparusaurata, L.), during its development was assessed. Fish were reared under 4 thermal regimes, and the resulting adults exposed to acute confinement stress and plasma cortisol levels and genes of the HPI axis were monitored. Changes in immune function, a common result of stress, were also evaluated using histomorphometric measurements of melanomacrophages centers (MMCs) in the head kidney and by monitoring macrophage-related transcripts. Thermal history significantly modified the HPI responsiveness in adult sea bream when eggs and larvae were reared at a higher than optimal temperature (HT, 22°C), and they had a reduced amplitude in their cortisol response and significantly upregulated pituitary pomc and head kidney star transcripts. Additionally, after an acute stress challenge, immune function was modified and the head kidney of adult fish reared during development at high temperatures (HT and LHT, 18-22°C) had a decreased number of MMCs and a significant downregulation of dopachrome tautomerase. Thermal imprinting during development influenced adult sea bream physiology and increased plasma levels of glucose and sodium even in the absence of an acute stress in fish reared under a high-low thermal regime (HLT, 22-18°C). Overall, the results demonstrate that temperature during early development influences the adult HPI axis and immune function in a teleost fish.