Jessica Douxfils

Health of farmed fish: its relation to fish welfare and its utility as welfare indicator

This brief review focuses on health and biological function as cornerstones of fish welfare. From the function-based point of view, good welfare is reflected in the ability of the animal to cope with infectious and non-infectious stressors, thereby maintaining homeostasis and good health, whereas stressful husbandry conditions and protracted suffering will lead to the loss of the coping ability and, thus, to impaired health. In the first part of the review, the physiological processes through which stressful husbandry conditions modulate health of farmed fish are examined. If fish are subjected to unfavourable husbandry conditions, the resulting disruption of internal homeostasis necessitates energy-demanding physiological adjustments (allostasis/acclimation). The ensuing energy drain leads to trade-offs with other energy-demanding processes such as the functioning of the primary epithelial barriers (gut, skin, gills) and the immune system. Understanding of the relation between husbandry conditions, allostatic responses and fish health provides the basis for the second theme developed in this review, the potential use of biological function and health parameters as operational welfare indicators (OWIs). Advantages of function- and health-related parameters are that they are relatively straightforward to recognize and to measure and are routinely monitored in most aquaculture units, thereby providing feasible tools to assess fish welfare under practical farming conditions. As the efforts to improve fish welfare and environmental sustainability lead to increasingly diverse solutions, in particular integrated production, it is imperative that we have objective OWIs to compare with other production forms, such as high-density aquaculture. However, to receive the necessary acceptance for legislation, more robust scientific backing of the health- and function-related OWIs is urgently needed.

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.

Evidence that Elevated Water Temperature Affects the Reproductive Physiology of the European Bullhead Cottus Gobio

Climate change is predicted to increase the average water temperature and alter the ecology and physiology of several organisms including fish species. To examine the effects of increased water temperature on freshwater fish reproduction, adult European bullhead Cottus gobio of both genders were maintained under three temperature regimes (T1: 6–10, T2: 10–14 and T3: 14–18°C) and assessed for gonad development (gonadosomatic index—GSI and gonad histology), sex steroids (testosterone—T, 17β-estradiol—E2 and 11-ketotestosterone—11-KT) and vitellogenin (alkali-labile phosphoprotein phosphorus—ALP) dynamics in December, January, February and March. The results indicate that a 8°C rise in water temperature (T3) deeply disrupted the gonadal maturation in both genders. This observation was associated with the absence of GSI peak from January to March, and low levels of plasma sex steroids compared with T1-exposed fish. Nevertheless, exposure to an increasing temperature of 4°C (T2) appeared to accelerate oogenesis with an early peak value in GSI and level of plasma T recorded in January relative to T1-exposed females. In males, the low GSI, reduced level of plasma 11-KT and the absence of GSI increase from January to March support the deleterious effects of increasing water temperature on spermatogenesis. The findings of the present study suggest that exposure to elevated temperatures within the context of climate warming might affect the reproductive success of C. gobio. Specifically, a 4°C rise in water temperature affects gametogenesis by advancing the spawning, and a complete reproductive failure is observed at an elevated temperature of 8°C.

Fish welfare and genomics

There is a considerable public and scientific debate concerning welfare of fish in aquaculture. In this review, we will consider fish welfare as an integration of physiological, behavioral, and cognitive/emotional responses, all of which are essentially adaptative responses to stressful situations. An overview of fish welfare in this context suggests that understanding will rely on knowledge of all components of allostatic responses to stress and environmental perturbations. The development of genomic technologies provides new approaches to this task, exemplified by how genome-wide analysis of genetic structures and corresponding expression patterns can lead to the discovery of new aspects of adaptative responses. We will illustrate how the genomic approach may give rise to new biomarkers for fish welfare and also increase our understanding of the interaction between physiological, behavioral, and emotional responses. In a first part, we present data on expression of candidate genes selected a priori. This is a common avenue to develop molecular biomarkers capable of diagnosing a stress condition at its earliest onset, in order to allow quick corrective intervention in an aquaculture setting. However, most of these studies address isolated physiological functions and stress responses that may not be truly indicative of animal welfare, and there is only rudimentary understanding of genes related to possible cognitive and emotional responses in fish. We also present an overview on transcriptomic analysis related to the effect of aquaculture stressors, environmental changes (temperature, salinity, hypoxia), or concerning specific behavioral patterns. These studies illustrate the potential of genomic approaches to characterize the complexity of the molecular mechanisms which underlies not only physiological but also behavioral responses in relation to fish welfare. Thirdly, we address proteomic studies on biological responses to stressors such as salinity change and hypoxia. We will also consider proteomic studies developed in mammals in relation to anxiety and depressive status which may lead to new potential candidates in fish. Finally, in the conclusion, we will suggest new developments to facilitate an integrated view of fish welfare. This includes use of laser microdissection in the transcriptomic/proteomic studies, development of meta-analysis methods for extracting information from genomic data sets, and implementation of technological advances for high-throughput proteomic studies. Development of these new approaches should be as productive for our understanding of the biological processes underlying fish welfare as it has been for the progress of pathophysiological research.

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.

Dietary β-glucans differentially modulate immune and stress-related gene expression in lymphoid organs from healthy and Aeromonas hydrophila-infected rainbow trout (Oncorhynchus mykiss)

Abstract Although &bgr;‐glucans stimulating effects have already been demonstrated on the immune system of numerous animal species, available data remain relatively variable and more research should be done regarding the complexity of underlying mechanisms. In this context, the present study aimed to evaluate the stress and immune‐related effects of dietary &bgr;‐glucans (i.e. Macrogard®) by considering a number of influencing factors such as the dose (0, 0.1, 0.2 and 0.5% in food), feeding duration (15 versus 30 days), tissue (blood, kidney, spleen, gills) and infection status (healthy or infected). Blood parameters (lysozyme, ACH50 activities, leucocyte populations) and mRNA expression level of several immune‐ and stress‐related genes (TFN‐&agr;1, IL‐1&bgr;, IL10, COX‐2, TGF‐&bgr;, MC2R, HSP70) were measured. Our results suggest that spleen may be a highly responsive organ to dietary &bgr;‐glucans both in healthy or infected fish, and that this organ may therefore significantly contribute to the immune reinforcement induced by such immunostimulatory diet. Our study further reveals that overdoses of &bgr;‐glucans and/or prolonged medication can lead to a non‐reactive physiological status and, consequently, to a poor immune response. All in all, the current data emphasizes the need for further extensive research in the field of dietary &bgr;‐glucans as a preventive method for farmed fish protection. HighlightsImmune and stress related gene expression was studied in &bgr;‐glucans fed trout.Spleen is highly responsive to glucan diet, mainly involving proinflammatory cytokines.Overdose of glucan may lead to non‐reactive status and poor immune response.

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.

First evidence of the possible implication of the 11-deoxycorticosterone (DOC) in immune activity of Eurasian perch (Perca fluviatilis, L.): Comparison with cortisol

Cortisol, the main corticosteroid in fish, is frequently described as a modulator of fish immune system. Moreover, 11-deoxycorticosterone (DOC) was shown to bind and transcriptionally activate the mineralocorticoid receptor and may act as a mineralocorticoid in fish. Immune modulations induced by intraperitoneal injections of these two corticosteroids were assessed in Eurasian perch juveniles. Cortisol and DOC were injected at 0.8 mg kg(-1) and 0.08 mg kg(-1) body weight respectively. Cortisol increased plasma lysozyme activity 72 h post-injection, C-type lysozyme expression in spleen from 1 to 72 h post-injection, and favoured blood neutrophils at the expense of a mixture of lymphocytes and thrombocytes. Moreover, 6 h after injection, cortisol reduced expression levels of the pro-inflammatory cytokine TNF-α in spleen. DOC had no effects on the immune variables measured in plasma, but increased expression levels of C-type lysozyme and apolipoprotein A1 mRNA in both gills and spleen. Meanwhile, DOC stimulated its putative signalling pathway by increasing expression of mineralocorticoid receptor and 11β-hydroxysteroid dehydrogenase-2 in spleen. These results confirmed the role of cortisol as an innate, short term immune stimulator. For the first time, DOC is described as a possible immune stimulator in fish.

In vivo response of some immune and endocrine variables to LPS in Eurasian perch (Perca fluviatilis, L.) and modulation of this response by two corticosteroids, cortisol and 11-deoxycorticosterone.

In fish, the endocrine system, especially corticosteroids pathway, strongly interacts with immune system. On the other hand, in vivo co-stimulation of both systems is not well documented. To better understand this interaction, we decided to evaluate the in vivo effects of both stimulation of the immune system and co-stimulation of both systems in Eurasian perch juveniles. Fish were injected either with 10mgkg(-1) LPS, or with a combination of LPS and 0.8mgkg(-1) cortisol or LPS and 0.08mgkg(-1) 11-deoxycorticosterone (DOC) and sampled 1, 3 or 7days after injection. LPS affected the immune system by increasing plasma lysozyme activity and blood neutrophils populations. During the same time-course, LPS decreased the proportion of a mixture of lymphocytes and thrombocytes in blood and TNF-α expression in spleen. Cortisol modulated the LPS-mediated response in TNF-α mRNA expression levels in spleen. Contrary to LPS alone, the association of LPS with DOC modulated the abundance of complement component 3 (C3) mRNA in spleen. On the other hand, LPS altered the corticotropic axis by decreasing mRNA expression levels of all corticosteroid receptors and of 11β-HSD-2 in spleen. Both corticosteroids injected were not able to balance these LPS-induced suppressive effects on corticosteroid receptors and 11β-HSD-2 expression levels in spleen. Contrary to LPS alone, the association of LPS with DOC modulated GR-1b expression in gills. These results indicated that LPS is a strong modulator of the corticosteroid receptors expression in spleen. Furthermore, we report for the first time a LPS-induced decrease of the mineralocorticoid receptor expression. Finally, corticosteroids were able to modulate the LPS-mediated response at the transcriptional level.