Emilie Henrotte

Spleen immune status is affected after acute handling stress but not regulated by cortisol in Eurasian perch, Perca fluviatilis.

The effects of acute stress on immune status and its regulation by cortisol/corticosteroid receptors have received little attention in percids. To address that question, we investigated the physiological and immune responses of Eurasian perch, Perca fluviatilis to acute stress. We exposed immature perch to an 1-min exondation and measured at 1 h, 6 h, 24 h and 72 h post-stress: (1) stress-related parameters including plasma cortisol and glucose levels, (2) immune parameters in the plasma and in the spleen (complement, respiratory burst and lysozyme activity, total immunoglobulins; gene expression of lysozyme, complement unit 3, apolipoprotein A1 and 14 kDa, hepcidin and chemotaxin) (3) the corticosteroid receptors gene expression in the spleen after having cloned them. In addition, the in vitro effects of cortisol on the spleen immune parameters were also investigated. Plasma cortisol and glucose levels increased markedly 1h post-stress and returned at basal levels after 24 h. P. fluviatilis mineralocorticoid receptor, but not glucocorticoid receptors, was significantly up-regulated both in vivo after the stress and in vitro by cortisol at a physiological concentration (100 ng/ml). The plasma immune parameters were not significantly affected by the stress. In contrast, spleno-somatic index, spleen lysozyme activity, lysozyme and hepcidin gene expression were depleted and total immunoglobulins increased along the whole time-course (1-72 h). But, these immune parameters were not regulated in vitro by cortisol at physiological or supra-physiological doses. Our results indicate that handling stress may affect spleen antibacterial defences without clear effects on circulating immune compounds and that the elevation of plasma cortisol after handling stress may not be related to the regulation of this splenic response.

Physiological and proteomic evidences that domestication process differentially modulates the immune status of juvenile Eurasian perch (Perca fluviatilis) under chronic confinement stress.

The current study aimed to evaluate the influence of domestication process on the stress response and subsequent immune modulation in Eurasian perch juveniles (Perca fluviatilis) submitted to chronic confinement. Briefly, F1 and F4 generations were confined into small-size tanks and sampled 7 and 55 days after stocking. Cortisol and glucose levels as well as lysozyme activity and immunoglobulin level were evaluated in the serum. Spleen Somatic Index and spleen ROS production were also measured. A proteomic analysis was performed on serum sampled on day 7. Finally, both generations were genetically characterized using a microsatellite approach. Globally, results revealed that chronic confinement did not elicit a typical stress response but resulted in a prolonged immune stimulation. Proteomic results suggested that domestication process influenced the immune status of perch submitted to chronic confinement as the F1 confined fish displayed lower abundance of C3 complement component, transferrin and Apolipoprotein E. Microsatellite data showed a strong genetic drift as well as reduced genetic diversity, allelic number and heterozygosity along with domestication process. The present work is the first to report that fish under domestication can develop an immune response, assessed by a combined approach, following recurrent challenges imposed by captive environment despite a reduced genetic variation.

Does domestication process affect stress response in juvenile Eurasian perch Perca fluviatilis

The objective was to evaluate the impact of domestication process on the physiological stress response of cultured Eurasian perch confronted to a chronic stress situation. Briefly, F1 and F4 juveniles were submitted to chronic confinement and investigated on days 5, 15 and 30. Capture and 15min-anesthesia were imposed on fish to assess the effect of preceding confinement on acute stress response. On day 30, the fish were finally challenged with Aeromonas hydrophila and sampled after 5 and 10 days for immune parameter measurements. Cortisol and glucose levels were not affected by confinement but increased significantly after acute stressor exposure. Moreover, cortisol rise following capture and anesthesia was higher in F1 confined-fish, suggesting that they have previously been affected by chronic confinement. A higher HSP70 level was also observed on day 30 in F1 confined-juveniles. During bacterial challenge, regardless of confinement level, F4 juveniles displayed higher lysozyme activity and agglutination response than F1 which may indicate a higher immune capacity in domesticated fish. In conclusion, chronic confinement stressor induced few physiological responses but may increase the responsiveness to other aquacultural stressors. Domestication process also seems to improve chronic stress resistance, growth as well as the immune status of the fish.

Physiological and proteomic responses to single and repeated hypoxia in juvenile Eurasian perch under domestication – Clues to physiological acclimation and humoral immune modulations

We evaluated the physiological and humoral immune responses of Eurasian perch submitted to 4-h hypoxia in either single or repeated way. Two generations (F1 and F5) were tested to study the potential changes in these responses with domestication. In both generations, single and repeated hypoxia resulted in hyperglycemia and spleen somatic index reduction. Glucose elevation and lysozyme activity decreased following repeated hypoxia. Complement hemolytic activity was unchanged regardless of hypoxic stress or domestication level. A 2D-DIGE proteomic analysis showed that some C3 components were positively modulated by single hypoxia while C3 up- and down-regulations and over-expression of transferrin were observed following repeated hypoxia. Domestication was associated with a low divergence in stress and immune responses to hypoxia but was accompanied by various changes in the abundance of serum proteins related to innate/specific immunity and acute phase response. Thus, it appeared that the humoral immune system was modulated following single and repeated hypoxia (independently of generational level) or during domestication and that Eurasian perch may display physiological acclimation to frequent hypoxic disturbances.

Influence of domestication process on immune response to repeated emersion stressors in Eurasian perch (Perca fluviatilis L.)

Domestication might be a possible way to reduce the physiological response to long-term stressors and deleterious effects on immunity. The present study aimed to evaluate the chronic immune response induced by repeated emersions and the possible impact of domestication by comparing farmed Eurasian perch with short (F1) and long (F4) captive-life history. In the first experiment, fish were exposed to a single emersion and physiological stress response was measured in the short term to characterize fish sensitivity to the tested stressor. Serum cortisol and glucose elevated within 6h post-stress and splenosomatic index (SSI) decreased within 48h, indicating that the species was affected by emersion stressor. In the second experiment, F1 and F4 generations were submitted to repeated water emersions (3 times/week during 44days). On day 9, 18 and 44, samplings were performed 48h post-stressor to highlight any sustained disruption of immune system. Serum cortisol, glucose, SSI and lysozyme activity were evaluated and serum proteome was analyzed using 2D-DIGE. Any of the tested variables were affected by repeated emersions and proteomic analysis only revealed that alpha-2 macroglobulins (a2Ms) were up-regulated in the serum of stressed individuals. Domestication also resulted in the up-regulation of five a2M isoforms and down-regulation of complement C3 and Ig light chain proteins, independently of any stressor exposure. In conclusion, the results suggested that repeated emersions are not severe stressors for Eurasian perch, probably explaining why domestication had no influence on fish responses. Changes associated with domestication are highly complex and certainly need further investigations.

Nutritional Requirements and Feeding of Broodstock and Early Life Stages of Eurasian Perch and Pikeperch

Up to date, the nutritional requirements of breeders and early life stages of European percid fishes, Perca fluviatilis and Sander lucioperca, have not been defined precisely, and, in fish farms, breeders are still relying on the regular supply of forage fish. It has been demonstrated in both species that the feeding conditions of broodstock largely influence the quality of gametes (especially in females) and hatched larvae. The best results, in terms of hatching rate and survival to challenge tests during the first days post-hatching, have been obtained when breeders were fed on forage fish, either as unique feed source or in combination with dry feed. Experimental diets, based on a suitable supply of phospholipids (PLs) and adequate ratio of essential long chain fatty acids (docosahexaenoic, eicosapentaenoic and arachidonic acids) have been used successfully in Eurasian perch breeders to produce high quality eggs and larvae, comparable to those obtained from perch fed forage fish. Fatty acid composition of broodstock diet significantly influenced the fatty acid composition of eggs. On the other contrary, none of the characteristics of the sperm were significantly modified by the HUFAs ratio, neither in terms of sperm volume and density, spermatozoa motility and velocity, nor in terms of seminal plasma osmolality. During the first weeks of larval rearing, Artemia nauplii are still currently used as starter feed for both Eurasian perch and pikeperch larvae. Enrichment of nauplii with HUFA has proved to be efficient in pikeperch but not in Eurasian perch. At the end of feeding trials with pikeperch larvae, highly significant correlations were achieved between dietary ascorbic acid content and the ascorbic acid content in larval carcass or the reduction of larval deformity. Based on commercial larval feed formulated for marine or freshwater fish, it has been shown that the freshwater feed (containing low Ca/P ratio) was more suitable for pikeperch larvae and juveniles than marine fish diet, in terms of growth, survival and resistance to stress test. Recently, some experimental dry diets varying by their phospholipid content have been tested. The highest survival and growth rates of pikeperch post-larvae (first fed with Artemia nauplii) were obtained in groups fed 9.5 % of PLs but higher levels still need to be tested, eventually at an earlier stage of development.

Immune Status and Immunomodulation in Percid Fish

Knowledge of the adaptability of the immune system is an important issue that can enable a better management of environmental conditions for increasing fish performances and welfare. Unfortunately, the immune defence of percid fish has not yet been characterized, but they should be able to accomplish innate and adaptive immunity as other fishes. Indeed, the available information about molecular characterization of some immune genes of percid fish showed similar features as that of other teleost. But some specific pathways were identified; and it could be interesting to precise their implication for the immune competence. It seems also likely that percid fish are able to adapt their immunocompetence to the seasonal changes in environmental conditions, or to respond positively to some immunomodulatory compounds as other fish species, but more studies are still needed to highlight the specific immune pathways of the relative responses.