Estetrol has a lower impact than 17α-ethinylestradiol on the reproductive capacity of zebrafish (Danio rerio).

Natural and synthetic oestrogens are commonly found in aquatic ecosystems. The synthetic oestrogen 17α-ethinylestradiol (EE2) is widely used in oral contraceptives and its ecotoxicological effects on aquatic organisms have been widely reported. The natural oestrogen estetrol (E4) was recently approved for use in a new combined oral contraceptive and, after therapeutic use, is likely to be found in the aquatic environment. However, its potential effects on non-target species such as fish is unknown. In order to characterize and compare the endocrine disruptive potential of E4 with EE2, zebrafish (Danio rerio) were exposed to E4 or EE2 in a fish short-term reproduction assay conducted according to OECD Test Guideline 229. Sexually mature male and female fish were exposed to a range of concentrations, including environmentally relevant concentrations of E4 and EE2, for 21 days. Endpoints included fecundity, fertilization success, gonad histopathology, head/tail vitellogenin concentrations, as well as transcriptional analysis of genes related to ovarian sex steroid hormones synthesis. Our data confirmed the strong impact of EE2 on several parameters including an inhibition of fecundity, an induction of vitellogenin both in male and female fish, an alteration of gonadal structures and the modulation of genes involved in sex steroid hormone synthesis in female fish. In contrast, only few significant effects were observed with E4 with no impact on fecundity. The results suggest that the natural oestrogen, E4, presents a more favorable environmental profile than EE2 and is less likely to affect fish reproductive capacity.

Immunomodulatory effects of graded levels of docosahexaenoic acid (DHA) in common carp (Cyprinus carpio) - In vitro and in vivo approaches.

The D-series resolvins formed from docosahexaenoic acid (DHA) work as anti-inflammatory mediators indicating the role of this long-chain polyunsaturated fatty acid in the immune system of mammals. However, such information is still limited in fish. The current study was conducted to evaluate the immunomodulatory effects of graded levels of DHA in common carp Cyprinus carpio by in vitro and in vivo approaches. In the in vitro experiment, the head kidney leukocytes isolated from common carp (body weight = 120.3 ± 12.4 g) were exposed to DHA at 0, 15, and 75 µM (corresponding to DHA0, DHA15, and DHA75) for 1 h; the cells were then immediately exposed to lipopolysaccharides (LPS) at a dose of 10 µg/ml for 4 or 24 h to stimulate the pro-inflammatory responses. The expression of several target genes involved in the inflammatory response (tlr4, nfkb, il-1, il-6, pge2, pla2, nf-kbi, il-10, and tgf-ß1) and cytoprotection (hsp70, gpx1, and prdx3) was then assessed by RT-qPCR. Results showed that the pro-inflammatory response induced by LPS was confirmed through the upregulations of il-1 and il-6 expressions in the DHA0 group after 4 h of LPS exposure. The downregulations of il-6 in DHA15 and DHA75 cells after 4 h of LPS exposure compared to DHA0 indicated that the free DHA supplementation in the cell culture medium induced an anti-inflammatory response. Decreases of il-10 and nf-kbi expression were also observed in DHA-treated groups and the highest expression of hsp70 in DHA75 cells. In the in vivo experiment, common carp juveniles (21.7 ± 0.9 g) were fed to apparent satiation with a diet supplemented with DHA at 0, 6, and 20 g/kg for 8 weeks. After the feeding trial, the fish were challenged with Aeromonas veronii at 2.1 × 107 CFU/ml and the fish mortality was then recorded during 14 days. At the end of the feeding trial and the second day of bacterial infection, fish blood samples were collected for haematological parameters while liver and head kidney were used for assaying different immune variables. Results showed that the DHA supplementation in fish diet did not influence the fish growth and other husbandry parameters. The lowest value of fish mortality was recorded in DHA20-fed fish. The positive effects of DHA-supplemented diets were also found in myeloperoxidase (MPO), glutathione (GSH), and catalase (CAT) activities. In conclusion, the results suggest that DHA is able to modulate the inflammatory response in the immune celsl at a dose of 75 µM/mL and to boost disease resistance in common carp fed on a diet supplemented with DHA at 20 g/kg of feed.

Intestinal histopathology and immune responses following Escherichia coli lipopolysaccharide challenge in Nile tilapia fed enriched black soldier fly larval (BSF) meal supplemented with chitinase.

This study aimed to determine to what extend the addition of chitinase to black soldier fly larvae (BSF) meals enriched with either PUFA or LC-PUFA could improve the gut health of Nile tilapia and increase its immune status. Two types of BSF meals enriched with either α-linolenic acid (ALA) or ALA + eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) were produced using BSF larvae cultured on vegetable substrates (VGS) or fish offal substrates (FOS), respectively. Seven diets were formulated: a control FMFO diet and two other control diets VGD0 vs FOD0 containing the meals of each type of BSF meal as total replacement for fishmeal (FM) and fish oil (FO), as well as four diets supplemented with chitinase. Two doses of commercial chitinase from Aspergillus niger (2 g/kg and 5 g/kg of feed) were supplemented to the other diets VGD0 and FOD0 to formulate VGD2, VGD5, FOD2 and FOD5. After 53 days of feeding, FOD5 diet induced a similar growth performance as the FMFO control diet, while a significant decrease of growth was observed for the other BSF larval-based diets. BSF/FOS meal led to higher SGR of fish than BSF/VGS, as for the FOD5 compared to VGD5. At day 53, lysozyme values showed an increasing trend in fish fed all the BSF-based diets, especially those fed the VGD5. After the Escherichia coli lipopolysaccharide (LPS) injection (day 54), the same increasing trend was observed in lysozyme activity, and modulation was observed only in the VGD5 fish. ACH50 activity was reduced by the BSF-based diets except for the FOD5 diet at day 53, and LPS modulation was only observed for the VGS-chitinase-based diets at day 54. Peroxidase activity and total immunoglobulin (Igs) blood level were not affected by substrate, chitinase dose or LPS injection. At day 53, the low or high dose of chitinase increased the expressions of tlr2, il-1ß and il-6 genes in the head kidney of fish fed the BSF/VGS diets compared to those fed the VGD0 or FMFO control diets. At day 54 after LPS injection, the high dose of chitinase decreased the expressions of tlr5 gene in the spleen and mhcII-α gene in the head kidney of fish fed FOD5 diets compared to those fed FOD0 diets. BSF/VGS but not BSF/FOS based diets increased fish sub-epithelial mucosa (SM) and lamina propria (LP) thickness and the number of goblet cells (GC) in fish, but dietary chitinase seemed to prevent some of these effects, especially at low dose. Results showed that chitinase supplementation of 5 g/kg of chitinase to a BSF-based diet enriched with LC-PUFA improved growth, prevented histological changes in the proximal intestine and enhanced some innate immune functions of Nile tilapia without any clear booster effect after challenge with E. coli LPS.

Comparative study on accuracy of mucosal estradiol-17ß, testosterone and 11-ketotestosterone, for maturity, and cutaneous vitellogenin gene expression in goldfish (Carassius auratus).

Providing a non-invasive procedure to track fish maturity remains a priority in broodstocks' management. In the present study, the main goal was to assess reproduction status by measuring sex steroids and vitellogenin (VTG) in the skin mucosa, as a non-invasive method. For this purpose, the present study compared the levels of estradiol-17ß (E2 ), testosterone (T), 11-ketotestosterone (11-KT), VTG and calcium (Ca) in skin mucosa and blood plasma of goldfish (Carassius auratus). Skin mucosal and blood samples were collected, as well as gonad tissues, from goldfish, as a seasonal spawner. Histological analysis confirmed the gender and maturity status from females' ovaries (as primary-growth, cortical-alveoli, initial and late-vitellogenesis) and males' testes (as spermatogenesis and spermiation). Furthermore, vitellogenin (vtg) expression was observed in skin, liver and gonads. The results indicate that mucosal E2 concentrations were significantly higher during initial and late vitellogenesis than the other stages. Mucosal 11-KT concentrations significantly increased at spermiation (P < 0.05). E2 /T and 11-KT/E2 ratios significantly increased at early vitellogenesis and spermatogenesis, respectively (P < 0.05). Females' mucosal VTG levels were significantly fluctuated according to the maturity stage. Ca showed a similar trend, but Ca was more accurate for sex identification than the VTG. Although mucus showed high levels of VTG, ovarian vtg expression was strongest while liver and skin had the similar results. These results show that measuring the mucosal androgens could be considered as an accurate, non-invasive method to monitor fish maturity.

Contamination of Water, Sediment and Fish with Residues of Pesticides Used in Cotton Production in Northern Benin.

In Northern Benin, insecticides are used for cotton production. These insecticides can be easily transferred to water ponds close to cotton fields. To monitor insecticides levels in water, sediments and fish samples from water ponds, a GC-MS analytical method was developed to detect residues of endosulfan, DDT and its parent compounds, isomers of HCH, pyrethroids and chlorpyrifos. In addition, the influence of storage conditions of water sample on pesticides determination performance has been studied. The limits of quantification were between 0.16 and 0.32 µg/L in water, 0.5 and 1 µg/kg in sediment and 1 and 2 µg/kg in fish. Twenty samples of water, twenty of sediments and forty of fish were taken in four different water reservoirs at five different times. Alpha-endosulfan, lambda-cyhalothrin and permethrin were identified in sediment while p,p'-DDE, α- and ß-HCH, chlorpyrifos, lambda-cyhalothrin and permethrin were detected in fish. Only organochlorines were determined in water because of the lack of recovery of pyrethroids from water stored in glass. Concentrations of insecticide residues in sediment for all water ponds ranged from non-detected to 101 µg/kg and from non-detected to 36 µg/kg in fish. Preliminary risk assessment for consumers of the North of Benin showed that the Estimated Daily Intakes were lower than the Acceptable Daily Intakes and Acute Reference Doses for all consumers. However, as one fish can be contaminated by five pesticide residues at the same time, it is not possible to exclude a risk for the consumer due to his exposure to mixtures of pesticides.

GAS1: A New ß-Glucan Immunostimulant Candidate to Increase Rainbow Trout (Oncorhynchus mykiss) Resistance to Bacterial Infections With Aeromonas salmonicida achromogenes.

ß-glucans are prebiotic and/or food additives used by the aquaculture industry to enhance the immune response of fish. Their efficiency may vary according to their origin and structure. In this study, the immunostimulant effects of two ß-glucan types extracted from wild-type baker's yeast (Saccharomyces cerevisiae) and its null-mutant Gas1 were investigated. Gas1 has a beta-1,3-glucanosyltransferase activity necessary for cell wall assembly. Using a positive (commercial product MacroGard®) and a negative control (a diet without glucans), we evaluated the immune responses and disease resistance of rainbow trout juveniles (mean weight, ~44 g) fed control, low (0.2%) and high (0.5%) doses of Macrogard®, Gas1, and Wild type-ß-glucan after a short-term (15 days, D15) or mid-term (36 days, D36) feeding periods. We found that ß-glucan supplemented diets did not affect growth performance, mortality, splenic index, or leukocyte respiratory burst activity on D15 nor D36. However, each ß-glucan triggered different immune effectors, depending of the doses or length of exposure compared to others and/or the negative control. Indeed, high dose of MacroGard® significantly increased lysozyme activities at D15 compared with the control and other diets (p<0.05). At D36, MacroGard ß-glucan enhanced the production of lymphocytes in comparison with the control diet (p<0.05). Regarding WT ß-glucan, at D36, WT-ß-glucan, especially the high dose, provided the highest enzymatic activities (lysozyme and ACH50) and Ig level (p<0.01). Furthermore, on D36, Gas1 also increased lysozyme activity, Ig proportion, and some immune genes (mcsfra, hepcidin) compared with MacroGard® (p<0.05). Besides, both doses of Gas1-ß-glucans increased the resistance of juveniles to bacterial infection highlighted by a higher survival rate at 14 days post-challenge compared with the control and other types and doses of ß-glucans (p<0.05). In conclusion, our results suggest that Gas1-ß-glucan could represent a promising immunostimulant that would help to prevent diseases in aquaculture even more efficiently than other ß-glucans already in use. Mode of action and particular efficiency of this new Gas1 mutant are debated.

Interplay between dietary lipids and cadmium exposure in rainbow trout liver: Influence on fatty acid metabolism, metal accumulation and stress response.

The present study aimed at investigating interactive effects between dietary lipids and both short- and long-term exposures to a low, environmentally realistic, cadmium (Cd) concentration. Juvenile rainbow trout were fed four isolipidic diets (31.7 g/kg) enriched in either linoleic acid (LA, 18:2n-6), alpha-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3) or docosahexaenoic acid (DHA, 22:6n-3). From the 4th week of this 10-week experiment, the lipid level of the diet was increased (120.0 g/kg) and half of the fish fed each diet were aqueously exposed to Cd (0.3 µg/L) while the other half were not exposed to Cd (control). Fish were sampled and their liver was harvested for fatty acid profile, hepatic Cd and calcium concentrations, total glutathione level and gene expression assessment, either (i) after 4 weeks of feeding and 24 h of Cd contamination (day 29) (short-term Cd exposure) or (ii) after 10 weeks of feeding and 6 weeks of Cd contamination (day 70) (long-term Cd exposure). We found that both dietary lipids and Cd exposure influenced fatty acid homeostasis and metabolism. The hepatic fatty acid profile mostly reflected that of the diet (e.g. n-3/n-6 ratio) with some differences, including selective retention of specific long chain polyunsaturated fatty acids (LC-PUFAs) like DHA and active biotransformation of dietary LA and ALA into LC-PUFAs. Cd effects on hepatic fatty acid profiles were influenced by the duration of the exposure and the nutritional status of the fish. The effects of diet and Cd exposure on the fatty acid profiles were only sparsely explained by variation of the expression pattern of genes involved in fatty acid metabolism. The biological responses to Cd were also influenced by dietary lipids. Fish fed the ALA-enriched diet seemed to be the least affected by the Cd exposure, as they showed a higher detoxifying ability against Cd with an early upregulation of protective metallothionein a (MTa) and apoptosis regulator BCL2-Like1 (BCLx) genes, an increased long-term phospholipid synthesis and turnover and fatty acid bioconversion efficiency, as well as a lower long-term accumulation of Cd in their liver. In contrast, fish fed the EPA-enriched diet seemed to be the most sensitive to a long-term Cd exposure, with an impaired growth performance and a decreased antioxidant capacity (lower glutathione level). Our results highlight that low, environmentally realistic aqueous concentrations of Cd can affect biological response in fish and that these effects are influenced by the dietary fatty acid composition.

Repeated hormonal induction of spermiation affects the stress but not the immune response in pikeperch (Sander lucioperca).

Hormonal induction of spermiation, previously reported to be immunogenic in fishes, is a common hatchery practice in pikeperch, Sander lucioperca. The aim of the present study was to investigate the effects of repeated induction of spermiation in pikeperch, following application of either human chorionic gonadotropin (hCG) or salmon gonadoliberine analogue (sGnRHa) on sperm quality indices as well as on immune and stress response. Mature males of pikeperch (n = 7 per group) were stimulated twice with five days between injections of either hCG (hCG; 500 IU kg-1), sGnRHa (sGnRHa; 50 µg kg-1) or NaCl (control group; 1 ml kg-1) to assess spermatozoa motility with a computer-assisted sperm analysis (CASA) system. During second sampling, blood plasma was sampled for humoral innate immune (peroxidase and lysozyme activities, ACH50), stress (cortisol, glucose) and endocrine (testosterone) markers. In addition, the head kidney was dissected to assay the expression of several immune genes (such as il1, c3, hamp, tnf-α and lys genes). The results indicate that hormonal treatment significantly increased sperm production. Sperm sampled after the hormonal treatment maintained its quality throughout the study, regardless of the sampling time. However, it appears that the application of hCG induced elevated cortisol and glucose plasma levels compared to the control group. Almost all immune markers, except the relative expression of hepcidin (hamp gene), were unaffected by the two hormones applied. The results showed that the induction treatment of spermiation processes in pikeperch resulted in an important physiological stress response for which the intensity varied according to the hormonal agent used. However, this stress response (more profound following application of hCG) was weakly associated with innate immune functions. On the other hand, a significant negative correlation between the expression of several important immune markers (peroxidase activity, relative expression of c3 and il1 genes) and sperm quality indices indicates significant involvement of immune status on sperm quality. The results obtained shed light on immune-system-induced modifications to sperm quality. The data presented here highlight the need for careful revision of broodstock management and selection practices where welfare status as well as individual predispositions of fish to cope with the stress should be taken under the consideration.

Anti-inflammatory properties of the ethanol extract from Clerodendrum cyrtophyllum Turcz based on in vitro and in vivo studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Clerodendrum cyrtophyllum Turcz, a plant belonging to the Verbenaceae family, has been used in traditional medicine for the treatment of various inflammatory diseases in many Asian countries. AIM OF THE STUDY: The study aimed to evaluate anti-inflammatory properties of the ethanol extract from Clerodendrum cyrthophyllum Turcz leaves (EE-CC) through in vitro and in vivo models. MATERIAL AND METHODS: Total phenolic and flavonoid contents in the extract were determined using colorimetric methods and HPTLC. In red blood cell membrane stabilization model, rat erythrocyte suspension was treated with crude ethanol extract at different concentrations, the hemoglobin content of the supernatant solution released by red blood hemolysis was estimated. We also evaluated the effects of the ethanol extract from this plant on the production of nitric oxide (NO), tumor necrosis factor alpha (TNF-α) in stimulated RAW 264.7 cells. In order to elucidate its anti-inflammatory molecular mechanisms, we further evaluated the effects of the EE-CC on the expression of the inflammatory genes in inflammation-induced zebrafish model by tail-cutting using qPCR analysis. RESULTS: Colorimetric methods and HPTLC revealed high phenolic and flavonoid contents in the extract. In the red blood cell membrane stabilization model, the amount of hemoglobin released by red blood hemolysis significantly decreased in the presence of EE-CC, demonstrating a strong membrane stabilizing activity. EE-CC did not cause any toxic effect on cell viability but strongly inhibited NO and TNF-ɑ release due to LPS induction. The association with EE-CC significantly reduced the expression of cox-2, pla2, c3a, il-1(il1fma), il-8 (cxcl8b.1), tnf-α, and nf-ƙb, while increased the expression of the anti-inflammatory cytokine il-10 gene in cut-tail induced inflammation of zebrafish model. CONCLUSIONS: Taken together, the results suggest that the raw ethanol extract from C. cyrtophyllum Turcz leaves presents potent anti-inflammatory activities and may be useful for the treatment of various inflammatory diseases.

Impact of temperature shift on gill physiology during smoltification of Atlantic salmon smolts (Salmo salar L.).

Exposure to a temperature increase may disrupt smoltification and delay or stop the downstream migration of smolts. Thermal regimes are often different between a river and its tributaries, but the effects of a relative temperature shift are not well described. We used expression of smoltification genes coupled with gill Na+/K+-ATPase activity (NKA) and plasma cortisol and growth hormone (GH) levels to investigate the impact of a 5 °C difference between tributary and river on salmon juveniles. Responses to a temperature challenge were examined at four time points during the smoltification period, with juveniles reared under three regimes including control, early and late temperature increase. The temperature shifts reduced gill NKA, plasma GH and cortisol levels which indicate hypo-osmoregulation impairment and may reduce the survival of smolts. Out of the 22 genes examined, the expression of six genes was influenced by the temperature treatments, while changes in further eleven genes were influenced by the date of sampling. Genes usually known to be upregulated during smoltification were downregulated after the temperature increase, notably nkaα1b, nkcc1a and igf1r. Upregulation of some genes involved in the hormonal regulation and acid-base equilibrium in early June may indicate a switch towards desmoltification. This study gives further insights about the impact of temperature increase on the molecular processes underlying smoltification and possible responses to human-related water temperature increase. The data also suggest dual roles in the smoltification and desmoltification for GH and IGF1 and points to the implication of genes in the smoltification process, that have previously been unstudied (nbc) or with little data available (igf2).

A combined in vivo and in vitro approach to evaluate the influence of linseed oil or sesame oil and their combination on innate immune competence and eicosanoid metabolism processes in common carp (Cyprinus carpio).

This study aimed to evaluate the influence of dietary pure linseed oil or sesame oil or a mixture on innate immune competence and eicosanoid metabolism in common carp (Cyprinus carpio). Carp of 100.4 ±â€¯4.7 g were fed to satiation twice daily for 6 weeks with four diets prepared from three lipid sources (CLO; LO; SO; SLO). On day 42, plasma was sampled for immune parameter analyses, and kidney and liver tissues were dissected for gene expression analysis. On day 45, HKL and PBMCs from remaining fish were isolated and exposed to E. coli LPS at a dose of 10 µg/mL for 24 h. Results show that the SLO diet enhanced feed utilisation (P = 0.01), while no negative effects on growth or survival were observed in plant oil-fed fish compared to those fed a fish-oil based diet. Plant oil diets did not alter lysozyme and peroxidase activities or gene expression levels. Moreover, the diets did not affect the expression levels of some genes involved in eicosanoid metabolism processes (pla, pge2, lox5). Lys expression in HKL in vitro following exposure to LPS was up-regulated in LO-fed fish, while expression levels of pge2 were higher in SLO fish than in other groups (P < 0.05). The highest value for peroxidase activity in HKL exposed to LPS was found in the SLO-fed group (P < 0.05). In conclusion, our results indicate that dietary plant oils did not induce any negative effects on fish growth, survival, and immune competence status. Moreover, a dietary combination of SO and LO improved the feed utilisation efficiency and seemed more effective in inducing a better immunomodulatory response to LPS through a more active eicosanoid metabolism process.

Growth performance and immune status in common carp Cyprinus carpio as affected by plant oil-based diets complemented with ß-glucan.

Omnivorous fish species such as the common carp (Cyprinus carpio) are able to biosynthesise long chain polyunsaturated fatty acids (LC-PUFAs) from plant oil PUFA precursors, but the influence of the amount and quality of the LC-PUFAs biosynthesised from these oils on the immunocompetence status of the fish has received little attention. This study aims to evaluate whether the conversion of PUFA by carp induces a sufficient biosynthesis of LC-PUFA to maintain a good immunocompetence status in this species. Six iso-nitrogenous (crude protein = 39.1%) and iso-lipidic (crude lipids = 10%) diets containing three different lipid sources (cod liver oil (CLO) as fish oil; linseed oil (LO) and sunflower oil (SFO) as plant oils) were formulated with or without ß-glucan supplementation at 0.25 g/kg diet. Juvenile carp (16.3 ±â€¯0.6 g initial body weight) were fed a daily ration of 4% body weight for 9 weeks and then infected at day 64 with the bacteria Aeromonas hydrophyla. No significant differences in survival rate, final body weight, specific growth rate and feed conversion rate were observed between diets. After bacterial infection, mortality rate did not differ between fish fed CLO and plant oil-based diets, indicating that the latter oils did not affect the overall immunocompetence status of common carp. Plant oil-based diets did not alter lysozyme activity in healthy and infected fish. No negative effects of plant oils on complement activity (ACH50) were observed in healthy fish, even if both plant oil-based diets induced a decrease in stimulated fish two days after infection. Furthermore, the levels of various immune genes (nk, lys, il-8, pla, pge, alox) were not affected by plant oil-based diets. The expression of pla and pge genes were higher in SFO-fed fish than in CLO ones, indicating that this plant oil rich in linoleic acid (LA) better stimulated the eicosanoid metabolism process than fish oil. In response to ß-glucan supplementation, some innate immune functions seemed differentially affected by plant oil-based diets. LO and SFO induced substantial LC-PUFA production, even if fish fed CLO displayed the highest EPA and DHA levels in tissues. SFO rich in LA induced the highest ARA levels in fish muscle while LO rich in α-linolenic acid (ALA) sustained higher EPA production than SFO. A significantly higher fads-6a expression level was observed in SFO fish than in LO ones, but this was not observed for elovl5 expression. In conclusion, the results show that common carp fed plant oil-based diets are able to produce substantial amounts of LC-PUFA for sustaining growth rate, immune status and disease resistance similar to fish fed a fish oil-based diet. The differences in the production capacity of LC-PUFAs by the two plant oil-based diets were associated to a differential activation of some immune pathways, explaining how the use of these oils did not affect the overall immunocompetence of fish challenged with bacterial infection. Moreover, plant oil-based diets did not induce substantial negative effects on the immunomodulatory action of ß-glucans, confirming that these oils are suitable for sustaining a good immunocompetence status in common carp.

A temperature shift on the migratory route similarly impairs hypo-osmoregulatory capacities in two strains of Atlantic salmon (Salmo salar L.) smolts.

Anthropogenic use of water systems may cause temperature fluctuations between tributaries and large rivers for which physiological population related-effects on osmoregulatory capacity of Atlantic salmon are not well described. We simulated the downstream route in the case of the River Meuse basin to investigate the impact of a 5 °C temperature shift during smoltification on hypo-osmoregulatory capacities of smolts. Three temperature regimes were tested: control temperature-treatment (T1) without temperature shift, early (T2) or late (T3) temperature shift-treatment. Moreover, fish were subjected to seawater challenge during and after the downstream migration peak time. Two allochtonous strains were used: Loire-Allier (LA) and Cong (CG). Without temperature shift (T1), significant differences between the strains were noticed in the peak date and maximum activity of gill Na+/K+ATPase as well as in plasma sodium and potassium concentrations. For early (T2) and late (T3) temperature shift-treatments, gill Na+/K+ATPase activity, plasma osmolality and ion concentrations were negatively influenced in both strains. After salinity challenge, the highest osmolality was measured in smolts subjected to the temperature shift. Predictably circulating levels of GH and IGF-1 changed over the smolting period but they did not explain the observed modifications in hypo-osmoregulatory abilities whatever the population. The results show a negative impact of a temperature shift on hypo-osmoregulatory capacities of smolts regardless of population differences in smoltification timing under conditions without temperature shift. The resilience of such physiological impact was sustained at least for 1 week, comforting the role of high temperature in influencing the rate of changes occurring during smoltification. Therefore, favouring the downstream migration to help smolts reach the sea faster may mitigate the impact of a rapid temperature increase.

Environmentally-realistic concentration of cadmium combined with polyunsaturated fatty acids enriched diets modulated non-specific immunity in rainbow trout.

Nutrition is crucial to grow healthy fish particularly in a context of pollution, overcrowding and pathogen risks. Nowadays, the search for food components able to improve fish health is increasingly developing. Here, the influence of four dietary polyunsaturated fatty acids (PUFAs) that are alpha-linolenic acid (ALA, 18:3n-3), linoleic acid (LA, 18:2n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) on the sensitivity of rainbow trout (Oncorhynchus mykiss) juveniles to environmentally realistic cadmium (Cd, 0.3 µg/L) concentration was investigated. Fish diets were designed to ensure the specific abundance of one of these individual PUFAs, and were given for a 4-week pre-conditioning period followed by a 6-week Cd exposure period. Focus was put on growth performance and immune responses following a short (24 h) and a long-term (6 weeks) Cd exposure. For each experimental condition, some fish were submitted to a bacterial challenge (24 h) with Aeromonas salmonicida achromogenes at the end of Cd conditioning period. DHA-enriched diet improved growth performances as compared to LA-enriched diet, but also increased ROS production (after short-term exposure to Cd) that could lead to a higher inflammation status, and some immunity-related genes (at short and long-term exposure). We notably highlighted the fact that even a low, environmentally-realistic concentration, Cd can strongly impact the immune system of rainbow trout, and that specific dietary PUFA enrichment strategies can improve growth performance (DHA-enriched diet), provide protection against oxidative stress (ALA- and EPA-enriched diet) and stimulate non-specific immunity.

Influence of strain origin on osmoregulatory and endocrine parameters of two non-native strains of Atlantic salmon (Salmo salar L.).

Non-native strains of Atlantic salmon are used in reinstatement trials where populations are extinct. Environmental cues like photoperiod and temperature are known to influence the smolting process and there is evidence of strain-, stock- or population-specific differences associated with seaward migration or smoltification. The objective of this study was to compare morphological, osmoregulatory and endorcrine features between two strains, one originating from a cold and short river in Ireland (Cong) and another from a long and warm river in France (Loire-Allier), reared under Belgian conditions in order to highlight major differences in restocking adaptability. Comprehensive endocrine profiles, consistent with their interactive role of mediating changes associated with smolting, have been observed. Na+/K+ATPase activity (1.3-10.5 µmol ADP∗mg prot.-1∗h-1) and hormone plasma levels (e.g. 55-122 ng∗mL-1 of cortisol and 4.5-6.4 ng∗mL-1 of GH) were consistent with reported values. We observed strain-related differences of the influence of temperature and daylength on cortisol, GH and sodium plasma levels. These may be related to the respective environmental conditions prevailing in the river of origin, which have impacted the genetic background for smoltification. Using Na+/K+ATPase activity as an indicator, both strains smoltified successfully and simultaneously testifying a prevailing influence of environmental cues over genetic factors for smoltification.

Cadmium exposure exerts neurotoxic effects in peacock blennies Salaria pavo.

Cadmium (Cd) is considered as an important factor involved in several neurological disturbances. The aim of this study was to assess the effects of Cd in the brain of peacock blennies Salaria pavo, a species used as a bioindicator of water pollution. A sublethal contamination of 2mg CdCl2 L-1 was performed over periods of 1, 4, 10 and 15 days. Total Cd accumulation was measured in brains and displayed low concentrations throughout the experiment. Partial-length cDNA of different ATP-binding cassette transporters (abcb1, abcc1, abcc2, abcg2 proteins) and acetylcholinesterase (ache) were characterized. mRNA expressions profiles displayed an up-regulation of abcc2 mRNA after 4 days of Cd exposure only while abcg2 mRNA was down-regulated after 10 days only. For AChE, the mRNA transcription and the activity of the enzyme were followed and highlighted that Cd exerted an inhibitory effect on the nervous information transmission. At the histological level, fish exhibited pathological symptoms in the optic tectum and the cerebellum and results showed that the cerebellum was the most affected organ.

Osmoregulatory and immunological status of the pond-raised striped catfish (Pangasianodon hypophthalmus S.) as affected by seasonal runoff and salinity changes in the Mekong Delta, Vietnam.

In Vietnam, the production of striped catfish Pangasianodon hypophthalmus S. reached more than 1.2 millions of tons in 2014 and is mainly gathered in the Mekong Delta (South Vietnam). A survey was carried out during the dry season 2013 (March-April) and rainy season 2014 (July-August) in 12 fish farms of the lower (Tra Vinh Province) and higher (Can Tho Province) Mekong River Delta. This study allowed drawing up current key osmoregulatory and innate immune parameters of striped catfish in aquaculture depending on the geographical location, particularly the proximity to the River Mekong Estuary and the seasonal surface runoff. In the dry season, plasma osmolality was positively correlated with salinity. Gill Na+K+ ATPase dropped in the rainy season, while kidney Na+K+ ATPase remained stable. Abundance of immune blood cells, especially thrombocytes and monocytes, tended to increase in farms located in tidal area. Production of reactive oxygen species in the spleen, kidney lysozyme activity and plasma complement activity did not vary whatever the season or the proximity to the estuary. Plasma lysozyme activity was 50-fold higher than in kidney and increased in the rainy season as well as in tidal sites. Kidney complement activity decreased in the dry season, especially in tidal sites. In conclusion, regarding key osmoregulatory and immune factors, striped catfish farms located in the Mekong Delta may be affected by seasonal and regional salinity and runoff fluctuations.

Temperature Increase Negatively Affects the Fatty Acid Bioconversion Capacity of Rainbow Trout (Oncorhynchus mykiss) Fed a Linseed Oil-Based Diet.

Aquaculture is meant to provide fish rich in omega-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA). This objective must be reached despite (1) the necessity to replace the finite and limited fish oil in feed production and (2) the increased temperature of the supply water induced by the global warming. The objective of the present paper was to determine to what extent increased water temperature influences the fatty acid bioconversion capacity of rainbow trout (Oncorhynchus mykiss) fed a plant-derived diet. Fish were fed two diets formulated with fish oil (FO) or linseed oil (LO) as only added lipid source at the optimal water temperature of 15°C or at the increased water temperature of 19°C for 60 days. We observed that a temperature increase close to the upper limit of the species temperature tolerance range negatively affected the feed efficiency of rainbow trout fed LO despite a higher feed intake. The negative impact of increased water temperature on fatty acid bioconversion capacity appeared also to be quite clear considering the reduced expression of fatty acid desaturase 2 in liver and intestine and the reduced Δ6 desaturase enzymatic activity in intestinal microsomes. The present results also highlighted a negative impact of increased temperature on the apparent in vivo enzymatic activity of Δ5 and Δ6 desaturases of fish fed LO. Interestingly, this last parameter appeared less affected than those mentioned above. This study highlights that the increased temperature that rainbow trout may face due to global warming could reduce their fatty acid bioconversion capacity. The unavoidable replacement of finite fish oil by more sustainable, readily available and economically viable alternative lipid sources in aquaculture feeds should take this undeniable environmental issue on aquaculture productivity into account.

Biomarkers assessment in the peacock blenny Salaria pavo exposed to cadmium.

Cadmium (Cd) is one of the most toxic metals and is widely distributed in freshwater and marine environments. It has received much attention from a toxicological perspective. The aim of this study was to assess the effect of Cd in the peacock blenny Salaria pavo, a species of the family of blennies that was used as bioindicator of water pollution. We performed a sublethal contamination of fish to 2 mg CdCl2 L(-1) during 1, 4, 10, and 15 days. Cd accumulation was measured in gills and liver and displayed a significant increase of its concentration throughout the experiment, with slightly higher levels in the liver, except after 4 days. Partial-length cDNA of mt1, mt2, mnsod, cuznsod, cat, and gpx were characterized. Results from mRNA expression levels displayed an up-regulation of mt2 and mnsod. Biomarker activities were determined in gills and liver. In gills, data displayed an inhibition of EROD and GST activities. Cd exposure significantly increased GPx activities but did not affect CAT levels throughout the experiment. No LPO induction was observed in gills of exposed fish. Regarding the liver, the activity of all enzymes and MDA levels increased significantly from the beginning of the experiment except EROD that increased after 15 days of contamination only. At the histological level, fish exhibited pathological symptoms in gills and liver with a predominance of circulatory disturbances in gills and regressive changes in the liver. Our results displayed that peacock blennies are able to survive Cd toxicity due to various physiological adaptation mechanisms.

Adaptation of a Bioinformatics Microarray Analysis Workflow for a Toxicogenomic Study in Rainbow Trout.

Sex steroids play a key role in triggering sex differentiation in fish, the use of exogenous hormone treatment leading to partial or complete sex reversal. This phenomenon has attracted attention since the discovery that even low environmental doses of exogenous steroids can adversely affect gonad morphology (ovotestis development) and induce reproductive failure. Modern genomic-based technologies have enhanced opportunities to find out mechanisms of actions (MOA) and identify biomarkers related to the toxic action of a compound. However, high throughput data interpretation relies on statistical analysis, species genomic resources, and bioinformatics tools. The goals of this study are to improve the knowledge of feminisation in fish, by the analysis of molecular responses in the gonads of rainbow trout fry after chronic exposure to several doses (0.01, 0.1, 1 and 10 µg/L) of ethynylestradiol (EE2) and to offer target genes as potential biomarkers of ovotestis development. We successfully adapted a bioinformatics microarray analysis workflow elaborated on human data to a toxicogenomic study using rainbow trout, a fish species lacking accurate functional annotation and genomic resources. The workflow allowed to obtain lists of genes supposed to be enriched in true positive differentially expressed genes (DEGs), which were subjected to over-representation analysis methods (ORA). Several pathways and ontologies, mostly related to cell division and metabolism, sexual reproduction and steroid production, were found significantly enriched in our analyses. Moreover, two sets of potential ovotestis biomarkers were selected using several criteria. The first group displayed specific potential biomarkers belonging to pathways/ontologies highlighted in the experiment. Among them, the early ovarian differentiation gene foxl2a was overexpressed. The second group, which was highly sensitive but not specific, included the DEGs presenting the highest fold change and lowest p-value of the statistical workflow output. The methodology can be generalized to other (non-model) species and various types of microarray platforms.