Acclimation to warmer temperature reversibly improves high-temperature hypoxia tolerance in both diploid and triploid brook charr, Salvelinus fontinalis.

Rising temperature leads to reduced oxygen solubility and therefore increases the risk of exposure to harmful hypoxic condition for fish in their natural aquatic environments and in aquaculture. The goal of this study was to determine whether acclimation to warmer temperature can improve high-temperature hypoxia tolerance in fish, using sibling diploid and triploid brook charr as the experimental model. Triploid fish are used for aquaculture and fisheries management because they are sterile, but they are known to have reduced thermal and hypoxia tolerance compared to conventional diploids. Fish were pre-acclimated to either 15 °C (optimum temperature for diploids) or 18 °C and then assessed for high-temperature hypoxia tolerance by rapidly increasing temperature to pre-determined levels (up to 30 °C), holding fish at these temperatures for one hour, and then using compressed nitrogen to drive oxygen out of the water. Hypoxia tolerance was expressed as both the oxygen tension at loss of equilibrium and the time taken to reach this endpoint following the start of the trial. Acclimation to 18 °C improved hypoxia tolerance at high temperatures but this advantage was lost after reacclimation to 15 °C. Although 18 °C acclimation improved the hypoxia tolerance of triploids, it remained inferior to that of diploids under identical test conditions. Somatic energy reserves (estimated as condition factor and hepatosomatic index), cardiac output (relative ventricular mass) and oxygen carrying capacity of the blood (hemoglobin concentration and hematocrit) did not markedly affect high-temperature hypoxia tolerance.

Validation of dried blood spot sampling for determining trophic positions of Arctic char using nitrogen stable isotope analyses of amino acids.

RATIONALE: Dried blood spots (DBSs) are gaining popularity for biomarker analyses in ecological research due to their advantages for use in field-based research and in remote settings; however, many DBS biomarkers remain unvalidated. We validated the application of compound-specific stable nitrogen isotope analyses of amino acids (CSIA-AAs) to field-prepared DBSs for determining trophic positions of wild-caught Arctic char (Salvelinus alpinus). METHODS: Whole blood and muscle from Arctic char were collected, and DBSs were created in the field. We measured the stable nitrogen isotope ratios (expressed as δ15 N values) of the amino acids glutamic acid (Glu) and phenylalanine (Phe) isolated from Arctic char samples using CSIA-AAs. We then compared amino acid δ15 N values from DBSs and the other sample types (whole blood and muscle) from the same specimens. We calculated and compared trophic position estimates generated from whole blood, DBSs, and muscle. RESULTS: The δ15 N values of Glu and Phe, as well as trophic position estimates from DBSs, were highly correlated with δ15 N values and estimates from both whole blood and muscle. The DBS amino acid δ15 N values and trophic position estimates agreed well with those from whole blood. Although mean differences between amino acid δ15 N values from DBSs and muscle were noted, the offsets were small and resulted in a 0.2 mean difference between trophic position estimates for DBSs and muscle. CONCLUSIONS: We demonstrate that the application of CSIA-AAs to field-prepared DBSs of Arctic char generates similar trophic position estimates to those from whole blood and muscle. We suggest that DBSs could be developed as a minimally invasive sampling technique to study feeding ecology of wild fish and perhaps other organisms of interest.

Short-term homeostatic regulation of blood/interstitial fluid Ca2+ concentration by the scales of anadromous sea trout Salmo trutta L. during smoltification and migration.

The elasmoid scales of anadromous sea trout Salmo trutta L. represent a significant internal reservoir of Ca2+ . Although more is known about long-term remodelling of scales in response to calciotropic challenges encountered during smoltification and migration, very little is known about the contribution made by scales to the short-term, minute-to-minute regulation of Ca2+ homeostasis in the extracellular fluid (ECF) during these phases of the life cycle. This gap in the knowledge is partly due to the technical challenges involved in measuring small Ca2+ fluxes around the scales of live fish in real time. Here, this study describes exfoliating, mounting and culturing scales and their resident cells from parr, smolt and adult sea trout from a freshwater environment, as well as from adult sea trout caught in sea or brackish water. All the scales were then examined using an extracellular, non-invasive, surface-scanning Ca2+ -sensitive microelectrode. The authors quantified the Ca2+ fluxes, in the absence of any systemic or local regulators, into and out of scales on both the episquamal and hyposquamal sides under different extracellular calcemic challenges set to mimic a variety of ECF-Ca2+ concentrations. Scales from the life-cycle stages as well as from adult fish taken from sea, brackish or fresh water all showed a consistent efflux or influx of Ca2+ under hypo- or hypercalcemic conditions, respectively. What were considered to be isocalcemic conditions resulted in minimal flux of Ca2+ in either direction, or in the case of adult scales, a consistent but small influx. Indeed, adult scales appeared to display the largest flux densities in either direction. These new data extend the current understanding of the role played by fish scales in the short-term, minute-to-minute homeostatic regulation of ECF-Ca2+ concentration, and are similar to those recently reported from zebrafish Danio rerio scales. This suggests that this short-term regulatory response might be a common feature of teleost scales.

Haematological and serum biochemical reference values of snow trout, Schizothorax labiatus habiting in river Sindh of Indian Himalayan region.

Haematological and serum biochemical parameters are a valuable tool in determining the wellbeing status of different fish species and response in close association to various biological and environmental factors, therefore the aim of the present study was to establish sex-wise reference values of haemato-biochemical parameters of S. labiatus. The haematological parameters such as haemoglobin (Hb) concentration, total erythrocyte count (RBC), total leucocyte count (WBC), packed cell volume (PCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV) and serum biochemical parameters such as glucose, cholesterol, urea, total protein, albumin and globulin were analysed. The results revealed that the haematological parameters, i.e., Hb, RBC, WBC and PCV showed significant (P < 0.05) differences with respect to sex. Significantly (P < 0.05) higher values of Hb content (11.18 g dl-1 ), RBC count (1.88 × 106 mm-3 ) and PCV (36.15%) were noted in male fish than females throughout the study period, while significantly (P < 0.05) higher WBC count was noted in females (20.38 × 103 mm-3 ) compared to males. However, no significant (P > 0.05) difference was observed in erythrocyte indices like MCH, MCHC and MCV between sexes. The serum biochemical parameters, i.e., glucose, cholesterol and urea, also showed significant (P < 0.05) difference between the sexes, but no significant (P > 0.05) difference was noticed in total protein, albumin and globulin. Significantly (P < 0.05) highest values of glucose (116.76 mg dl-1 ) and urea (9.01 mg dl-1 ) were recorded in male S. labiatus, while highest value of cholesterol (223.53 mg dl-1 ) was noted in females. The information generated in the present study gives the basic reference values of haematological and serum biochemical parameters of S. labiatus which will be useful in monitoring the wellbeing status of fish populations. Moreover, the data will also be helpful to ascertain the occurrence of different clinical and subclinical diseases, which may in turn help to boost the overall production of this species.

Methemoglobin concentrations in three salmonid species following exposure to benzocaine or tricaine methanesulfonate.

Methemoglobin is hemoglobin containing ferric iron rather than ferrous iron which renders it incapable of binding to oxygen. Blood sampling of fish is done under sedation or general anesthesia. Tricaine methanesulfonate (TMS) or benzocaine is commonly used but both can cause oxidation of hemoglobin to methemoglobin. Our objective was to determine if methemoglobin concentrations in healthy rainbow trout (Oncorhynchus mykiss), brook trout (Salvelinus fontinalis), or Atlantic salmon (Salmo salar) increase during sedation with 25 mg/L of a 10% benzocaine solution or with repeated short anesthetizations by 65 mg/L of 10% benzocaine solution or 65 mg/L of TMS. Sedation by benzocaine caused a significant increase in methemoglobin in all species over time (P < 0.05). The methemoglobin percentage in brook trout increased by 129%, rainbow trout by 42%, and Atlantic salmon by 49%. The methemoglobin in brook trout was significantly greater than the other species at multiple time points. Repeated brief anesthetizing by benzocaine and TMS caused significant methemoglobin by 60 (P < 0.05), 90 (P < 0.01), and 120 min (P < 0.001) in brook trout but no significant change in methemoglobin in rainbow trout or Atlantic salmon except at 120 min in Atlantic salmon (P < 0.05) repeatedly anesthetized with benzocaine. For example, following multiple anesthetizations with benzocaine, the methemoglobin percentage in brook trout increased by 140%, whereas the rise in methemoglobin in rainbow trout and Atlantic salmon was more modest (37% increase in Rainbow trout and 53% increase in Atlantic salmon). Following multiple anesthetizations with TMS, the methemoglobin increased by 90%, 5%, and 1% in brook trout, rainbow trout, and Atlantic salmon, respectively. Methemoglobin may increase significantly over time in fish immersed in a sedating dose of benzocaine or repeatedly anesthetized with benzocaine or TMS. The susceptibility varies with the individual and species with brook trout being more susceptible than Atlantic salmon or rainbow trout.

Comparative evaluation of two anticoagulants used for the analysis of haematological, biochemical parameters and blood cell morphology of himalayan snow trout, Schizopyge plagiostomus.

The aim of the present study is to evaluate the effect of lithium heparin and K2EDTA on haematology, biochemical parameters and blood cell morphology of snow trout Schizopyge plagiostomus inhabiting in Kashmir Himalayan region. The haematological parameters such as hemoglobin (Hb), red blood cellcount (RBC), white blood cell (WBC), haematocrit (Hct), erythrocyte indices; mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), produced certain significant differences in their values with respect to both anticoagulants. Higher values of Hct, MCH and MCV while lower RBC count and MCHC values were recorded with a sample treated with K2EDTA compared to sample treated with lithium heparin. However, no significant change in Hb and WBC count was observed in the values of samples treated with both the anticoagulants. In the present study, the effect of two anticoagulant in biochemical parameters revealed that the alkaline phosphatase (ALP) and aspartate transaminase (AST) produced significant (P < 0.05) differences. Higher AST and lower ALP activity were recorded in the sample treated with K2EDTA compared to overall values in serum. Morphometric analysis showed an increase in the RBC perimeter area in samples treated with K2EDTA as compared to samples treated with lithium heparin. On the basis of above findings, it is recommended that in fish blood analysis heparin as anticoagulant could be useful as compared to K2 EDTA.

The effects of the neurotoxic agent emamectin benzoate on the expression of immune and stress-related genes and blood serum profiles in the Rainbow trout.

Emamectin, a neurotoxic agent, is a semi-synthetic insecticide that belongs to the Avermectin family and is used against helmintic infections in the Salmonidae family. Its secondary effects are not clear; thus, the aim of this study was to investigate the only effects of emamectin benzoate on various biochemical parameters (AST, ALT, GGT, total protein, albumin and glucose) in serum and expressional changes of IL-1ß, TNF-α, HSP70 and IL-8 in liver and spleen. For the purpose stated above, rainbow trout (n = 15) were administered 50 µg EB per kg fish daily for 7, 14 and 21 days. The results indicated that weight gains did not change (p > 0.05), AST increased at day 21 (p < 0.05), while the changes of other biochemical parameters were not significant (p > 0.05). The changes in expression of IL-1ß, TNF-α and HSP70 were significant (p < 0.05), while the changes of IL-8 expressions were not significant (p ˃ 0.05). In a conclusion, EB changed immun and stress-related gene expression in liver and spleen, and furthermore, AST changed in a dose- and time-dependent manner. The results imply that emamectin benzoate cause stress. This study is helpful to understand the effects of avermectin pharmaceutical family.

Sub-lethal temperature thresholds indicate acclimation and physiological limits in brook trout Salvelinus fontinalis.

The upper thermal tolerance of brook trout Salvelinus fontinalis was estimated using critical thermal maxima (CTmax ) experiments on fish acclimated to temperatures that span the species' thermal range (5-25°C). The CTmax increased with acclimation temperature but plateaued in fish acclimated to 20, 23 and 25°C. Plasma lactate was highest, and the hepato-somatic index (IH ) was lowest at 23 and 25°C, which suggests additional metabolic costs at those acclimation temperatures. The results suggest that there is a sub-lethal threshold between 20 and 23°C, beyond which the fish experience reduced physiological performance.

Acute ammonia poisoning in dolly varden char (Salvelinus malma) and effect of methionine sulfoximine.

Ammonia is toxic to most bony fishes. However, little information is available on the toxicology mechanisms induced by ammonia and the means to mitigate the effects by various fishes. In this study, four groups of experiments were designed and carried out to test the response of dolly varden char to ammonia toxicity and their mitigation through methionine sulfoximine (MSO). NaCl group was injected with NaCl, NH3 group was injected with ammonium acetate, NH3+MSO group was injected with ammonium acetate and MSO, MSO group was injected with MSO. Results showed that ammonia toxicity could lead to blood deterioration (elevation in white blood cell and blood ammonia), free amino acid imbalance (elevation in glutamine, glutamate, arginine and ornithine, coupled with reduction of citrulline and aspartate), ammonia metabolism enzyme activity inhibition (reduction in carbamyl phosphate synthetase, ornithine transcarbamylase and arginase), oxidative stress (reduction in superoxide dismutase, catalase and glutathione peroxidase) and immunosuppression (reduction in lysozyme, 50% hemolytic complement, total immunoglobulin and phagocytic index), but the MSO can eliminate fatal effect of oxidative damage. In addition, ammonia poisoning could induce down-regulation of antioxidant enzymes coding genes (SOD, CAT and GPx) and up-regulation of inflammatory cytokine genes (TNFα, IL-1ß and IL-8) transcription, suggesting that immunosuppression and inflammation may relate to oxidative stress in fish.

Dried Blood Spot Sampling of Landlocked Arctic Char (Salvelinus alpinus) for Estimating Mercury Exposure and Stable Carbon Isotope Fingerprinting of Essential Amino Acids.

Dried blood spots (DBS), created by applying and drying a whole blood sample onto filter paper, provide a simple and minimally invasive procedure for collecting, transporting, and storing blood. Because DBS are ideal for use in field and resource-limited settings, we aimed to develop a simple and accurate DBS-based approach for assessing mercury (Hg) exposure and dietary carbon sources for landlocked Arctic char, a sentinel fish species in the Arctic. We collected liquid whole blood (from the caudal vein), muscle, liver, and brains of Arctic char (n = 36) from 8 lakes spanning a Hg gradient in the Canadian High Arctic. We measured total Hg concentrations ([THg]) of field-prepared DBS and Arctic char tissues. Across a considerable range, [THg] of DBS (0.04-3.38 µg/g wet wt) were highly correlated with [THg] of all tissues (r2 range = 0.928-0.996). We also analyzed the compound-specific carbon isotope ratios (expressed as δ13 C values) of essential amino acids (EAAs) isolated from DBS, liquid whole blood, and muscle. The δ13 C values of 5 EAAs (δ13 CEAAs ; isoleucine [Ile], leucine [Leu], phenylalanine [Phe], valine [Val], and threonine [Thr]) from DBS were highly correlated with δ13 CEAAs of liquid whole blood (r2 range = 0.693-0.895) and muscle (r2 range = 0.642-0.881). The patterns of δ13 CEAAs of landlocked Arctic char were remarkably consistent across sample types and indicate that EAAs are most likely of algal origin. Because a small volume of blood (~50 µL) dried on filter paper can be used to determine Hg exposure levels of various tissues and to fingerprint carbon sources, DBS sampling may decrease the burdens of research and may be developed as a nonlethal sampling technique. Environ Toxicol Chem 2020;39:893-903. © 2020 SETAC.

Cortisol predicts migration timing and success in both Atlantic salmon and sea trout kelts.

Kelts - individuals of anadromous fish species which have successfully spawned and may return to sea to repeat the cycle - are perhaps the least studied life stage of iteroparous fish species. To date, our understanding of what makes them successful in their return migration to sea is limited. We investigated the relationship between three physiological parameters (baseline cortisol, baseline glucose and low molecular weight antioxidants) and the timing and success of Atlantic salmon (Salmo salar) and sea trout (Salmo trutta) kelt migration. To do so, we combined blood samples obtained within 3 minutes of capture and acoustic telemetry to track 66 salmon and 72 sea trout as they migrated out of rivers, into fjords and out at sea. We show that baseline cortisol may be a good predictor of migration success. Individuals with high baseline cortisol levels exited the river earlier but were less likely to successfully reach the sea. Similar relationships were not observed with glucose or antioxidants. We provide the first evidence to support the role of physiological status in migration success in Atlantic salmon and sea trout kelts. Our findings contribute to our understanding of the relationship between physiology and fitness in wild animals. Further, we suggest that migration timing is a trade-off between stress and readiness to migrate.

The Combined Efficiency of Dietary Isomaltooligosaccharides and Bacillus spp. on the Growth, Hemato-Serological, and Intestinal Microbiota Indices of Caspian Brown Trout (Salmo trutta caspius Kessler, 1877).

The combined effects of a commercial probiotic, BetaPlus®, and a prebiotic, isomaltooligosaccharides (IMOS) on the growth, survival rate, intestinal microbiota, and hemato-immunological parameters were evaluated in Caspian brown trout (Salmo trutta caspius Kessler, 1877). Caspian brown trout fingerlings (~ 9 g) were fed a control diet (basal diet) or a synbiotic diet (the basal diet + 2 g kg-1 IMOS + 1 g kg-1 BetaPlus®) for 7 weeks. At the end of this trial, fish fed the synbiotic diet showed significant improvements in body weight increase, feed conversion ratio, and survival rate compared with fish fed the control diet (P < 0.05). In addition, fish fed the synbiotic diet had the highest levels of white blood cells, monocytes, and neutrophils (P < 0.05), while the red blood cells, hemoglobin, hematrocrit, mean corpuscular volume, and lymphocytes were significantly higher in the control group (P < 0.05). The serum triglycerides, cholesterol, total protein, albumin, albumin/globulin ratio, and immunoglobulin M levels, as well as alanine aminotransferase and lactate dehydrogenase activities were higher in the synbiotic group than in the control group (P < 0.05). In addition, fish fed the synbiotic diet showed significantly higher gut total viable aerobic bacterial counts and lactic acid bacteria (P < 0.05). The results demonstrated that BetaPlus® in combination with IMOS enhanced the growth, survival rate, intestinal microbiota, and some haemato-immunological parameters in Caspian brown trout fingerlings.

Light intensity and suppression of nocturnal plasma melatonin in Arctic charr (Salvelinus alpinus).

The problem of early sexual maturation among farmed Arctic charr and other salmonids can be effectively reduced by 24 h light overwinter, provided it is bright enough to over-ride interference from the natural daylength cycle. To determine the threshold light intensity to suppress the nocturnal elevation of plasma melatonin, three groups of individually tagged fish (n = 26-28/group ca. 1040 g) were reared on 12 h light: 12 h dark (LD 12:12) and subjected to nighttime light intensities of either 50-65, 0.1-0.3 or 0 (control) lux for five months (November to April). Daytime light intensity was 720-750 lx. Diel plasma melatonin profiles in both November and April were similar; mean daytime levels ranged from 20 to 100 pg/ml, and nighttime levels were inversely proportional to light intensity. In the control group at 0 lx, plasma melatonin increased about four-fold after lights-off, ranging between 320 and 430 pg/ml. Nighttime light intensity of 0.1-0.3 lx halved plasma melatonin levels to 140-220 pg/ml, and 50-65 lx further reduced the levels to one quarter of the control group, 68-108 pg/ml. Among the lit groups, daytime plasma melatonin levels were about 20-30 pg/ml, significantly lower than the nocturnal levels suggesting the diel hormonal rhythm was not completely abolished. Fish grew steadily from about 1100 g to 1600 g between November and April, independent of light intensity (P = .67). Overall, the study demonstrated the sensitivity of pineal melatonin hormone to different light intensities in Arctic charr.

Plasma and tissue disposition of danofloxacin in brown trout (Salmo trutta fario) after intravenous and intramuscular administrations.

Plasma and muscle pharmacokinetics of danofloxacin were investigated after 10 mg/kg intravenous (IV, caudal vein) and intramuscular (IM, right epaxial muscles) administrations in 168 healthy brown trout (Salmo trutta fario) at 10°C-13°C. High-performance liquid chromatography was used to determine its plasma and muscle concentrations. Pharmacokinetic parameters were analysed with a non-compartmental model. After IV administration, elimination half-life (t1/2ʎz), area under the concentration-time curve (AUC0-∞), mean residence time (MRT0-∞), volume of distribution at steady state, total body clearance in plasma and AUCMuscle/AUCPlasma ratio were 22.22 h, 140.66 h*µg/mL, 23.15 h, 2.28 L/kg, 0.07 L/h/kg and 3.79, respectively. After IM administration, t1/2ʎz, AUC0-∞, MRT0-∞, peak concentration (Cmax), time to reach Cmax, bioavailability in plasma and AUCMuscle/AUCPlasma ratio were 28.28 h, 84.39 h*µg/mL, 37.31 h, 4.79 µg/mL, 1 h, 59.99% and 8.46, respectively. Danofloxacin exhibited long t1/2ʎz and good bioavailability after IM administration. Therefore, 10 mg/kg IM administration of danofloxacin in brown trout can provide AUC0-24/MIC of > 125 and Cmax/MIC of > 10 to treat diseases caused by susceptible bacteria with ≤ 0.336 µg/mL MIC.

Comparative Analysis of the Concentrations of Ions (Na+, K+, Ca2+, and Cl‒) in the Blood of the Brown Trout Salmo trutta from Different Rivers.

The contents of ions in the blood have been compared in three ecological groups of brown trout (parr, mature, and resident) living in rives of the basins of Lake Onega and Lake Ladoga. Brown trout from the Alats-oya (the Ladoga basin, remote from two other rivers belonging to the Onega Lake) is characterized by the lowest concentrations of the Na+, Ca2+, and Cl- ions in blood. The probable effect of the ion level in blood of brown trout on selection of life strategy is discussed.

Comparison of vegetable shortening and cocoa butter as vehicles for cortisol manipulation in Salmo trutta.

This study demonstrates that vegetable shortening and cocoa butter are two effective vehicles for intraperitoneal cortisol implants in juvenile teleosts, specifically brown trout Salmo trutta, residing in north temperate freshwater environments. Each vehicle showed a different pattern of cortisol elevation. Vegetable shortening was found to be a more suitable vehicle for long-term cortisol elevation [elevated at 3, 6 and 9 days post treatment (dpt)], while cocoa butter may be better suited for short-term cortisol elevation (only elevated at 3 dpt). Additionally, plasma cortisol levels were higher with cortisol-vegetable shortening than with cortisol-cocoa butter implants. Plasma glucose levels were elevated 6 and 9 dpt for fishes injected with cortisol-vegetable shortening, but did not change relative to controls and shams in cortisol-cocoa butter fishes. In conclusion, vegetable shortening and cocoa butter are both viable techniques for cortisol manipulation in fishes in temperate climates, providing researchers with different options depending on study objectives.

Vitellogenin concentrations in feral Danish brown trout have decreased: An effect of improved sewage treatment in rural areas?

Feminization of male and juvenile fish because of exposure to estrogens or estrogenic chemicals in effluents from central wastewater treatment plants (WWTPs) is a worldwide issue of concern. Intersex and induction of the female yolk protein, vitellogenin, in male and juvenile fish are robust biomarkers for estrogenic exposure, and feminized fish have been observed downstream of WWTP outlets in many countries. Danish central WWTPs reduce effluent estrogenicity effectively by advanced sewage treatment, and feminizations have not been observed downstream of central WWTP outlets. However, between 2000 and 2004, investigations of Danish streams not receiving sewage from central WWTPs revealed a high variation in vitellogenin concentrations of male juvenile brown trout (Salmo trutta); some individuals had high concentrations, probably as a result of estrogenic point sources, and the plasma concentration was >50 ng mL-1 in 79% of the juvenile males. The streams were reinvestigated in 2010 to 2016, and the average male level had decreased to a hitherto unseen baseline level; in 2010 only 0.7% (one individual) of the males had a vitellogenin concentration >50 ng mL-1 , which could indicate that the estrogenicity of the streams decreased after 2004. We examined possible estrogenic sources in streams unaffected by central WWTP effluents, and found that the reduced vitellogenin levels are most likely explained by a national effort to improve on-site wastewater treatment in scattered houses not connected to central WWTPs. Environ Toxicol Chem 2018;37:839-845. © 2017 SETAC.

Morphological, physiological and dietary covariation in migratory and resident adult brown trout (Salmo trutta).

The causes and consequences of trait relationships within and among the categories of physiology, morphology, and life-history remain poorly studied. Few studies cross the boundaries of these categories, and recent reviews have pointed out not only the dearth of evidence for among-category correlations but that trait relationships may change depending on the ecological conditions a population faces. We examined changes in mean values and correlations between traits in a partially migrant population of brown trout when migrant sea-run and resident stream forms were breeding sympatrically. Within each sex and life-history strategy group, we used carbon and nitrogen stable isotopes to assess trophic level and habitat use; assessed morphology which reflects swimming and foraging ability; measured circulating cortisol as it is released in response to stressors and is involved in the transition from salt to freshwater; and determined oxidative status by measuring oxidative stress and antioxidants. We found that sea-run trout were larger and had higher values of stable isotopes, cortisol and oxidative stress compared to residents. Most groups showed some correlations between morphology and diet, indicating individual resource specialization was occurring, and we found consistent correlations between morphology and cortisol. Additionally, relationships differed between the sexes (cortisol and oxidative status were related in females but not males) and between life-history strategies (habitat use was related to oxidative status in male sea-run trout but not in either sex of residents). The differing patterns of covariation between the two life-history strategies and between the sexes suggest that the relationships among phenotypic traits are subjected to different selection pressures, illustrating the importance of integrating multiple phenotypic measures across different trait categories and contrasting life-history strategies.

High-sensitivity determination of estrogens in fish plasma using chemical derivatization upstream UHPLC-MSMS.

This article describes the development and validation of a sensitive LC-MSMS method for determination of estrogen in fish plasma. Dansyl chloride derivatization of the phenol functional group in estrogen was used to enhance the response to atmospheric pressure ionization leading to improve the sensitivity. Individual 13C internal standards were selected after comparison with deuterated standards. Liquid-liquid extraction (ethyl acetate or methyl tert-butyl ether) and protein precipitation (acetonitrile, methanol or acetone) were compared for the extraction and clean-up of estrogens from fish plasma. Ethyl acetate was selected as the best alternative with recovery ranging from 61 to 96% and matrix effect ranging from 88 to 106%. Limits of quantification ranged from 0.5 to 1pg/mL showing a gain in sensitivity of 10,000 times over electrospray ionization of underivatized estrogens. Accuracy and precision were validated over three consecutive days and the method was applied to measure estrogen in sea lamprey (Petromyzon marinus) and lake trout (Salvelinus namaycush) plasma. Estrone and estriol were detected in fish below 1ng/mL in plasma, justifying the need of a highly sensitive LC-MSMS quantification method.

Biomarker response and hypothalamus-pituitary-interrenal axis functioning in Arctic charr from Bjørnøya (74°30' N), Norway, with high levels of organohalogenated compounds.

The populations of Arctic charr (Salvelinus alpinus) residing in Lake Ellasjøen at Bjørnøya Island in the Norwegian Arctic (74° 30'N, 19° 00'E) possess substantially higher levels of organohalogenated compounds (strongly dominated by polychlorinated biphenyls, PCBs) than conspecifics residing in other, proximate lakes on the island. In the present study we sampled large (<400g), immature charr from Lake Ellasjøen (high PCB levels) and Lake Laksvatn (reference lake, low PCB levels) by hook and line for an immediate blood sampling, and blood and tissue sampling after a 1h confinement stressor. This was done in order to investigate possible effects of pollutants on an acute stress performance in a high-latitude fish species by comparing muscle PCB levels, hepatic cytochrome P4501A (CYP1A) biomarker activation and functioning of the hypothalamus-pituitary-interrenal (HPI) axis between these two populations of Arctic charr. As expected sum PCB muscle levels were 8-fold higher on a wet weigh basis, and 19-fold higher on a lipid weight basis, in charr from Ellasjøen than in charr from Laksvatn. This was accompanied by a 3.5-fold higher liver cyp1a mRNA abundance in the Ellasjøen charr compared to Laksvatn charr. Brain transcript levels encoding glucocorticoid receptor 1 and 2 (GR2) and corticotropin-releasing factor, and pituitary transcript levels encoding GR2 and proopiomelanocortin A1 and A2 were higher in Ellasjøen charr than in Laksvatn charr, while interrenal transcript levels encoding melanocortin 2 receptor and steroidogenic acute regulatory protein were lower. There were no differences in plasma cortisol concentration between the two charr populations immediately after capture and one hour after confinement. The strong biomarker response to OHCs and altered mRNA abundances of key genes related to HPI axis functioning in the Ellasjøen charr suggest endocrine disruptive effects of OHCs in this charr population. Possible ecological implications are not known, but it cannot be excluded that a slower growth rate in Ellasjøen charr compared to Laksvatn charr due to an increased metabolic demand associated with the activation of xenobiotic defense and detoxification systems may have contributed to the lower body mass of Ellasjøen charr compared to Laksvatn charr.