Saprolegnia infection after vaccination in Atlantic salmon is associated with differential expression of stress and immune genes in the host.

This study assessed the impact of routine vaccination of Atlantic salmon pre-smolts on gene expression and the possible link to saprolegniosis on Scottish fish farms. Fish were in 4 different groups 1) 'control' - fish without handling or vaccination 2) 'vaccinated' - fish undergoing full vaccination procedure 3) 'non vaccinated' - fish undergoing full vaccination procedure but not vaccinated and 4) 'vaccinated-MH' - fish undergoing vaccination, but procedure involved minimal handling. A strong increase in cortisol and glucose levels was observed after 1 h in all groups relative to the control group. Only in the non-vaccinated group did the level decrease to near control levels by 4 h. Expression levels of six stress marker genes in general for all groups showed down regulation over a 9-day sampling period. In contrast, expression levels for immune response genes in the head kidney showed significant up-regulation for all eight genes tested for both vaccinated groups whereas the non-vaccinated group showed up-regulation for only MHC-II and IL-6b in comparison to the control. Both the vaccination procedure and the administration of the vaccine itself were factors mediating changes in gene expression consistent with fish being susceptible to natural occurring saprolegniosis following vaccination.

Immunoassays are not immune to errors: Examples from two studies of steroid output from freshwater trout farms.

A "reproducibility crisis" is widespread across scientific disciplines, where results and conclusions of studies are not supported by subsequent investigation. Here we provide a steroid immunoassay example where human errors generated unreproducible results and conclusions. Our study was triggered by a scientific report citing abnormally high concentrations (means of 4-79 ng L-1) of three natural sex steroids [11-ketotestosterone (11-KT), testosterone (T) and oestradiol (E2)] in water samples collected from two UK rivers over 4 years (2002-6). Furthermore, the data suggested that trout farms were a major source because reported steroid concentrations were 1.3-6 times higher downstream than upstream. We hypothesised that the reported levels were erroneous due to substances co-extracted from the water causing matrix effects (i.e. "false positives") during measurement by enzyme-linked immunoassay (EIA). Thus, in collaboration with three other groups (including the one that had conducted the 2002-6 study), we carried out field sampling and assaying to examine this hypothesis. Water samples were collected in 2010 from the same sites and prepared for assay using an analogous method [C18 solid phase extraction (SPE) followed by extract clean-up with aminopropyl SPE]. Additional quality control ("spiked" and "blank") samples were processed. Water extracts were assayed for steroids using radioimmunoassay (RIA) as well as EIA. Although there were statistically significant differences between EIA and RIA (and laboratories), there was no indication of matrix effects in the EIAs. Both the EIAs and RIAs (uncorrected for recovery) measured all three natural steroids at <0.6 ng L-1 in all river water samples, indicating that the trout farms were not a significant source of natural steroids. The differences between the two studies were considerable: E2 and T concentrations were ca. 100-fold lower and 11-KT ca. 1000-fold lower than those reported in the 2002-6 study. In the absence of evidence for any marked changes in husbandry practice (e.g. stock, diet) or environmental conditions (e.g. water flow rate) between the study periods, we concluded that calculation errors were probably made in the first (2002-6) study associated with confusion between extract and water sample concentrations. The second (2010) study also had several identified examples of calculation error (use of an incorrect standard curve; extrapolation below the minimum standard; confusion of assay dilutions during result work-up; failure to correct for loss during extraction) and an example of sample contamination. Similar and further errors have been noted in other studies. It must be recognised that assays do not provide absolute measurements and are prone to a variety of errors, so published steroid levels should be viewed with caution until independently confirmed.

Effects of temperature on amoebic gill disease development: Does it play a role?

A relationship between increasing water temperature and amoebic gill disease (AGD) prevalence in Atlantic salmon (Salmo salar) has been noted at fish farms in numerous countries. In Scotland (UK), temperatures above 12°C are considered to be an important risk factor for AGD outbreaks. Thus, the purpose of this study was to test for the presence of an association between temperature and variation in the severity of AGD in Atlantic salmon at 10 and 15°C. The results showed an association between temperature and variation in AGD severity in salmon from analysis of histopathology and Paramoeba perurans load, reflecting an earlier and stronger infection post-amoebae exposure at the higher temperature. While no significant difference between the two temperature treatment groups was found in plasma cortisol levels, both glucose and lactate levels increased when gill pathology was evident at both temperatures. Expression analysis of immune- and stress-related genes showed more modulation in gills than in head kidney, revealing an organ-specific response and an interplay between temperature and infection. In conclusion, temperature may not only affect the host response, but perhaps also favour higher attachment/growth capacity of the amoebae as seen with the earlier and stronger P. perurans infection at 15°C.

The Influence of Sex, Parasitism, and Ontogeny on the Physiological Response of European Eels (Anguilla anguilla) to an Abiotic Stressor.

Migration of adult European eels (Anguilla anguilla) from freshwater feeding grounds to oceanic spawning grounds is an energetically demanding process and is accompanied by dramatic physiological and behavioral changes. Humans have altered the aquatic environment (e.g., dams) and made an inherently challenging migration even more difficult; human activity is regarded as the primary driver of the collapse in eel populations. The neuroendocrine stress response is central in coping with these challenging conditions, yet little is known about how various biotic factors such as sex, parasites, and ontogeny influence (singly and via interactions) the stress response of eels. In this study, mixed-effects and linear models were used to quantify the influence of sex, parasitism (Anguillicola crassus), life stage (yellow and silver eels), and silvering stage on the stress response of eels when exposed to a standardized handling stressor. The physiological response of eels to a standardized abiotic stressor (netting confinement in air) was quantified through measurements of blood glucose and plasma cortisol. The relationships between biotic factors and the activity of gill Na+/K+-ATPase was also examined. Analyses revealed that in some instances a biotic factor acted alone while in other cases several factors interacted to influence the stress response. Blood glucose concentrations increased after exposure to the standardized stressor and remained elevated after 4 h. Variation in plasma cortisol concentrations after exposure to the stressor were found to be time dependent, which was exacerbated by life stage and parasitism condition. Males and nonparasitized silver eels had the highest Na+/K+-ATPase activity. Silvering stage was strongly positively correlated with Na+/K+-ATPase activity in female eels. Collectively, these findings confirm that the factors mediating stress responsiveness in fish are complicated and that aspects of inherent biotic variation cannot be ignored.

Modulation of the stress response in wild fish is associated with variation in dissolved nitrate and nitrite.

Disruption of non-reproductive endocrine systems in wildlife by chemicals has received little attention but represents a potentially significant problem. Nitrate is a major anthropogenic contaminant in the freshwater aquatic environment and has been identified as a potential disrupter of endocrine function in aquatic animals. This study was conducted to investigate the relationship between the function of the neuroendocrine stress axis in fish and inorganic N loading along reaches of rivers receiving cumulative point source and diffuse chemical inputs. To accomplish this, the responsiveness of the stress axis, quantified as the rate of release of cortisol to water across the gills during exposure to a standardised stressor, was measured in three-spined sticklebacks (Gasterosteus aculeatus L.) resident at three sites on each of four rivers in north-west England. The magnitude of the stress response in fish captured at the sites furthest downstream on all rivers was more than twice that of fish captured at upstream sites. Site-specific variation in stress axis reactivity was better explained by between-site variation in concentrations of dissolved nitrate, nitrite, and ammonia than by the concentration of wastewater treatment works effluent. An increase in the magnitude of the stress response was seen among sticklebacks at sites where long-term averaged concentrations of NH3-N, NO3-N and NO2-N exceeded 0.6, 4.0 and 0.1 mg/L respectively. These data suggest that either (i) inorganic N is a better surrogate than wastewater effluent concentration for an unknown factor or factors affecting stress axis function in fish, or (ii) dissolved inorganic N directly exerts a disruptive influence on the function of the neuroendocrine stress axis in fish, supporting concerns that nitrate is an endocrine-modulating chemical.

Long-term water quality data explain interpopulation variation in responsiveness to stress in sticklebacks at both wastewater effluent-contaminated and uncontaminated sites.

The magnitude of the corticosteroid response to a standardized stressor varied in proportion to the concentration of effluent in three-spined sticklebacks (Gasterosteus aculeatus L.) captured downstream of 10 wastewater-treatment plants (WWTPs). However, at 9 sites with no upstream WWTP input interpopulation variation in the reactivity of the stress axis occurred across a similar range to that seen for fish at impacted sites, suggesting that the factor(s) responsible for modulating stress responsiveness in sticklebacks is not unique to sites receiving WWTP effluent. Physicochemical data from a long-term monitoring program were employed to investigate whether variation in water quality contributed to between-site variation in stress axis reactivity. Between-site variation in 14 water quality determinands explained between 30% and 60% of the variation in stress reactivity and fish size for sticklebacks at both WWTP-contaminated and uncontaminated sites. At uncontaminated sites the mean mass and length of sticklebacks increased with total oxidized nitrogen (N) concentration (as an indicator of anthropogenic input), whereas at WWTP-contaminated sites fish size decreased with increasing effluent concentration, suggesting that factors adversely affecting growth were present predominantly at WWTP-contaminated sites. In contrast, at both contaminated and uncontaminated sites the magnitude of the corticosteroid response to a standardized stressor increased with anthropogenic input (effluent concentration or total oxidized N, respectively), indicating that a factor or factors modulating the reactivity of the stress axis may be present at both WWTP-contaminated and uncontaminated sites. Environ Toxicol Chem 2016;35:3014-3022. © 2016 SETAC.

A comparison of two methods for the assessment of stress axis activity in wild fish in relation to wastewater effluent exposure.

Riverine fish are particularly vulnerable to chemical exposure - rivers receive chemicals of anthropogenic origin from a variety of sources, one of the most significant being the chemically complex effluents discharged by wastewater treatment works (WWTWs). The extent to which non-reproductive components of the endocrine system in fish may be vulnerable to interference by contaminants associated with WWTW effluent is not well understood, but a significant body of evidence does suggest that contaminants present in the aquatic environment may interfere with the normal function of the neuroendocrine stress axis in fish. Field investigations of stress axis function in free-living populations of fish by measurement of hormone concentrations in blood can be confounded by the remoteness of sampling locations and the size of target species. Two methods for assessing stress axis reactivity in situations where blood samples are unavailable were compared in three-spined sticklebacks in relation to their exposure to WWTWs effluent. Sticklebacks were sampled in two successive years at fifteen sites in north-west England impacted by WWTW effluent and the response of each fish to the combined stressor of capture and a brief period of confinement was evaluated using both whole-body immunoreactive cortisol concentrations (WBIC) and the rate of release of cortisol to water (CRTW). A positive relationship between the magnitude of stress-induced CRTW in sticklebacks of both sexes and WWTW effluent concentration at site of capture was observed in both years. However, the relationship between stress-induced WBIC and WWTW effluent concentration was not consistent. These results suggest that components of WWTW effluent can modulate the magnitude of the neuroendocrine stress response in sticklebacks, and by inference in other fish species, but they raise questions about the measurement and interpretation of stress axis responses in fish via endpoints other than blood hormone concentrations. Possible factors underlying the disparity between the CRTW and WBIC results are discussed.

Disruption of the stress response in wastewater treatment works effluent-exposed three-spined sticklebacks persists after translocation to an unpolluted environment.

The hypothalamic-pituitary-adrenal/interrenal (HPA/I) axis plays a key role in responding to biotic and abiotic challenges in all vertebrates. Recent studies have shown that the apical response of the HPI axis to stressors in three-spined sticklebacks varies in proportion to the concentration of wastewater treatment works (WWTW) effluent to which the fish are exposed. This study was conducted to determine whether between-site variation in stress responsiveness among WWTW effluent-exposed sticklebacks is persistent or reversible. Sticklebacks from eight sites in north-west England affected by WWTW effluent and exhibiting between-population variation in HPI axis reactivity, were moved to a clean-water aquarium environment. After five months in the contaminant-free environment the responsiveness of these fish to a standardised stressor was determined, by measuring the rate of stress-induced cortisol release across the gills, and compared with the responses of fish newly sampled from the eight original capture sites. Inter-site differences in the reactivity of the HPI axis, proportional to the effluent concentration at each site, persisted among the translocated female sticklebacks for at least 5 months. In male fish however, the direct relationship between stress responsiveness and site-specific effluent was not evident 5 months post-translocation. These results support previous observations that the HPA/I axis, a non-reproductive endocrine system, is vulnerable to modulation by anthropogenic factors in fish and show for the first time that, in female fish at least, this modulation is not transient. The mechanisms underlying these observations, and the implications for the fitness and resilience of affected populations, requires investigation.

Feather corticosterone content in predatory birds in relation to body condition and hepatic metal concentration.

This study investigated the feasibility of measuring corticosterone in feathers from cryo-archived raptor specimens, in order to provide a retrospective assessment of the activity of the stress axis in relation to contaminant burden. Feather samples were taken from sparrowhawk Accipiter nisus, kestrel Falco tinnunculus, buzzard Buteo buteo, barn owl Tyto alba, and tawny owl Strix aluco and the variation in feather CORT concentrations with respect to species, age, sex, feather position, and body condition was assessed. In sparrowhawks only, variation in feather CORT content was compared with hepatic metal concentrations. For individuals, CORT concentration (pgmm(-1)) in adjacent primary flight feathers (P5 and P6), and left and right wing primaries (P5), was statistically indistinguishable. The lowest concentrations of CORT were found in sparrowhawk feathers and CORT concentrations did not vary systematically with age or sex for any species. Significant relationships between feather CORT content and condition were observed in only tawny owl and kestrel. In sparrowhawks, feather CORT concentration was found to be positively related to the hepatic concentrations of five metals (Cd, Mn, Co, Cu, Mo) and the metalloid As. There was also a negative relationship between measures of condition and total hepatic metal concentration in males. The results suggest that some factors affecting CORT uptake by feathers remain to be resolved but feather CORT content from archived specimens has the potential to provide a simple effects biomarker for exposure to environmental contaminants.

The stress response of three-spined sticklebacks is modified in proportion to effluent exposure downstream of wastewater treatment works.

This study was conducted to investigate whether exposure to wastewater treatment works (WWTW) effluent affects the adaptive stress axis of fish resident within the receiving water. Three-spined sticklebacks (Gasterosteus aculeatus) were sampled from sites downstream of ten WWTWs in north-west England, selected to represent a range of human population equivalents between 1000 and 125,000. Following capture, indices of stress (whole-body cortisol and glucose concentrations) were measured both prior to, and following, the imposition of a standardised stressor to establish both baseline and stress-induced concentrations of cortisol and glucose. There was considerable between-site variation in size, and to a lesser extent condition, of the fish. Pre- and post-stress cortisol and glucose concentrations also varied significantly between-sites. A large proportion of the variation in both the somatic data and the stress response was explained by variation in the proportion of effluent contributing to total river flow at the study sites. Mass (r(2)=0.35, P<0.001) and length (r(2)=0.37, P<0.001) of the fish, and cortisol (r(2)=0.26, P<0.001) and glucose (r(2)=0.12, P<0.01) concentrations in unstressed sticklebacks, were positively related to the concentration of effluent across the sample sites. However, in stressed fish, cortisol (r(2)=0.32, P<0.001) and glucose (r(2)=0.14, P<0.001) concentrations exhibited a negative trend in relation to the effluent concentrations across sites. Individual variation in fish size did not account for the variation in either cortisol or glucose levels. These data provide the first indication that modulation of the stress axis in fish by anthropogenic factors might be widespread and of greater significance than hitherto assumed.

Field surveys reveal the presence of anti-androgens in an effluent-receiving river using stickleback-specific biomarkers.

This study was designed to assess whether the removal of endocrine disrupting chemicals (EDCs) and other substances from a Waste Water Treatment Works (WWTW) effluent (receiving water: R. Ray, Swindon, UK) by granular activated carbon (GAC) affected biomarkers of exposure to EDCs [vitellogenin (VTG) and spiggin] in male and female three-spined sticklebacks in the receiving water. A nearby river (R. Ock), with a negligible effluent loading, was used as a control. On each river fish were sampled from four sites on five occasions both before and after remediation of the WWTW effluent. The results show for the first time in a UK field study a clear seasonality of blood VTG concentrations in wild male fish, following closely the VTG profile in female fish from both rivers. VTG levels in male fish from the R. Ray were significantly reduced after the GAC installation. However, VTG levels in males from the control sites also varied significantly across the same period, reducing the significance of this finding. A laboratory exposure to oestradiol (using site-specific lower and upper levels of oestrogenic activity) failed to elevate VTG concentrations in male sticklebacks suggesting that concentrations in the effluent, even prior to remediation, may not have exceeded a critical sensitivity threshold. Most importantly, a significant increase in female kidney spiggin content (a highly specific biomarker of xeno-androgen exposure) occurred in fish in the R. Ray after the GAC installation to levels comparable with those in fish from the control river. The significance of this finding is strengthened by the fact that during the pre-remediation period in the R. Ray, female spiggin levels increased with increasing distance from the WWTW. Our results provide the first in vivo evidence of the presence of anti-androgens in a UK WWTW effluent. To our knowledge this is the first UK-based comprehensive field study on the effects of a WWTW upgrade on biomarkers of EDC exposure using a sentinel fish species and our findings confirm the value of the stickleback as a model species for studying EDCs both in the laboratory and in the wild.

Plasticity of boldness in rainbow trout, Oncorhynchus mykiss: do hunger and predation influence risk-taking behaviour?

Boldness, a measure of an individual's propensity for taking risks, is an important determinant of fitness but is not necessarily a fixed trait. Dependent upon an individual's state, and given certain contexts or challenges, individuals may be able to alter their inclination to be bold or shy in response. Furthermore, the degree to which individuals can modulate their behaviour has been linked with physiological responses to stress. Here we attempted to determine whether bold and shy rainbow trout, Oncorhynchus mykiss, can exhibit behavioural plasticity in response to changes in state (nutritional availability) and context (predation threat). Individual trout were initially assessed for boldness using a standard novel object paradigm; subsequently, each day for one week fish experienced either predictable, unpredictable, or no simulated predator threat in combination with a high (2% body weight) or low (0.15%) food ration, before being reassessed for boldness. Bold trout were generally more plastic, altering levels of neophobia and activity relevant to the challenge, whereas shy trout were more fixed and remained shy. Increased predation risk generally resulted in an increase in the expression of three candidate genes linked to boldness, appetite regulation and physiological stress responses - ependymin, corticotrophin releasing factor and GABA(A) - but did not produce a significant increase in plasma cortisol. The results suggest a divergence in the ability of bold and shy trout to alter their behavioural profiles in response to internal and exogenous factors, and have important implications for our understanding of the maintenance of different behavioural phenotypes in natural populations.

Analysis of stress-induced hepatic gene expression in rainbow trout (Oncorhynchus mykiss) selected for high- and low-responsiveness to stress.

The production and welfare of intensively reared fish would be improved by reducing stress responsiveness. One approach to achieving this goal is selective breeding utilising stress-responsive genes as direct genetic markers of the desirable trait. As a first step in this process, microarray analysis has been carried out on liver tissues of rainbow trout selectively bred for high (HR) or low (LR) responsiveness to a stressor. Microarray hybridizations provided gene expression profiles for pooled samples of fish confined for 6 h, 24 h and 168 h and for individual fish (168 h only). 161 genes were shown to be differentially regulated in HR and LR fish during confinement exposure and eight of these gene expression profiles were validated by quantitative PCR. Genes of particular interest included intelectin-2 precursor which showed greater than 100-fold higher expression in HR fish compared to LR fish irrespective of whether the fish were confined or not; interferon inducible transmembrane protein 3 which was differentially stress-induced between the two lines; and hepatic pro-opiomelanocortin B (POMC B) which was upregulated during stress in HR fish but downregulated in LR fish. All these offer potential as direct markers of low stress responsiveness in a marker-assisted selection scheme.

Effects of sewage effluent remediation on body size, somatic RNA: DNA ratio, and markers of chemical exposure in three-spined sticklebacks.

Body mass, fork length, RNA:DNA ratio, specific growth rate, and hepatic EROD activity and CYP1A expression, were measured in three-spined sticklebacks in the River Ray (south west England) at sites downstream of an urban waste water treatment works (WWTW) prior to, and following, remediation of the effluent with granular activated carbon (GAC) tertiary treatment. During the same two-year period fish were also sampled from a neighbouring reference river (R. Ock). The WWTW effluent elevated water temperatures and nutrient content in the R. Ray and rendered a direct comparison of fish populations in the two rivers untenable. Instead, the stability of population parameters within each river during matched pre- and post-remediation periods was compared. Stickleback populations in both rivers were annual but fish in the R. Ray spawned earlier and were larger than those in the R. Ock. In the R. Ray fish gained mass throughout the winter months whereas in the R. Ock growth was much reduced during this period. In fish from the R. Ray the somatic RNA:DNA ratio remained elevated during May-November after remediation, rather than declining as in the same period pre-remediation and as was the case for fish in the R. Ock during both periods. The specific growth rate of the first post-remediation generation of sticklebacks in the R. Ray was higher than that of the previous pre-remediation generation. Following remediation there was no decline in hepatic EROD activity or in the abundance of hepatic CYP1A transcripts in fish in the R. Ray suggesting that the primary route of exposure to contaminants for these fish was not via the water column, and that the change in performance of the fish post-remediation was not impeded by continued exposure to contaminants. Both EROD activity and CYP1A expression increased in fish in the R. Ock during the later stages of the study suggesting that the fish in this river were exposed to an unidentified contaminant episode. This may have been linked with the poorer performance of fish in the R. Ock during the post-remediation period. The improved performance of fish in the R. Ray suggest that there may be factors in good quality secondary treated sewage effluent which can adversely influence the performance of fish populations, directly or indirectly, and which can be removed by tertiary treatment.

Physiological and genetic correlates of boldness: characterising the mechanisms of behavioural variation in rainbow trout, Oncorhynchus mykiss.

Bold, risk-taking animals have previously been putatively linked with a proactive stress coping style whereas it is suggested shyer, risk-averse animals exhibit a reactive coping style. The aim of this study was to investigate whether differences in the expression of bold-type behaviour were evident within and between two lines of rainbow trout, Oncorhynchus mykiss, selectively bred for a low (LR) or high (HR) endocrine response to stress, and to link boldness and stress responsiveness with the expression of related candidate genes. Boldness was determined in individual fish over two trials by measuring the latency to approach a novel object. Differences in plasma cortisol concentrations and the expression of eight novel candidate genes previously identified as being linked with divergent behaviours or stress were determined. Bold and shy individuals, approaching the object within 180 s or not approaching within 300 s respectively, were evident within each line, and this was linked with activity levels in the HR line. Post-stress plasma cortisol concentrations were significantly greater in the HR line compared with the LR line, and six of the eight tested genes were upregulated in the brains of LR fish compared with HR fish. However, no direct relationship between boldness and either stress responsiveness or gene expression was found, although clear differences in stress physiology and, for the first time, gene expression could be identified between the lines. This lack of correlation between physiological and molecular responses and behavioural variation within both lines highlights the complexity of the behavioural-physiological complex.

Acute phase gene expression in rainbow trout (Oncorhynchus mykiss) after exposure to a confinement stressor: A comparison of pooled and individual data.

This study set out to investigate whether differential expression of genes for acute phase proteins in rainbow trout (Oncorhynchus mykiss) could be induced by confinement stress, a non-invasive method of activating the neuroendocrine stress response. In addition, a second objective was to assess the variation in gene expression between individual fish within the population of stressed fish in an attempt to identify APP genes having uniform and consistent changes in expression during stress. The liver was chosen for this investigation as it is the primary site of acute phase protein synthesis. Relative expression of the eight genes including transferrin, fibrinogen-like protein 2 (flp2), alpha-1-anti-proteinase-like protein (alpha1-antiprot), leukocyte cell-derived chemotaxin 2 (LECT2), pentraxin, serum amyloid A (SAA), haptoglobin (Hp), and differentially regulated trout protein 1 (DRTP1) was analysed by quantitative real-time PCR (qPCR) over 5 experimental time points spanning the course of a week. The results showed that the expression of three genes, SAA, haptoglobin and DRTP1, were most altered as a result of exposure to confinement stress. A correlation was identified between the expression of haptoglobin and DRTP1. Gene expression analyses in individual fish found that the transcript levels of haptoglobin and DRTP1 genes varied much less between individuals than was the case for SAA. The increase of haptoglobin and DRTP1 gene expression and its uniformity in response to stress make these genes potential biomarkers for stress in trout.

Detection of the anti-androgenic effect of endocrine disrupting environmental contaminants using in vivo and in vitro assays in the three-spined stickleback.

We have previously developed a novel in vitro assay that utilises cultures of primed female stickleback kidney cells for the screening of potential androgenic and anti-androgenic environmental contaminants. Stickleback kidney cells are natural targets for steroid hormones and are able to produce a protein, spiggin, in response to androgenic stimulation. We undertook a combined in vivo/in vitro study where we used the magnitude of spiggin production as an endpoint to test the anti-androgenic properties of the pharmaceutical androgen antagonist flutamide and three environmental contaminants: the organophosphate insecticide fenitrothion, the urea-based herbicide linuron and the fungicide vinclozolin. In vitro, kidney cells were exposed to a range of concentrations [from 10(-14) M (2.5 pg/L) up to 10(-6) M (280 microg/L)] of the test compounds alone for determining agonist activities, or together with 10(-8) M (3 microg/L) dihydrotestosterone (DHT) for determining antagonist activities. An in vivo flow-through aquarium-based study was carried out in parallel. Female sticklebacks were exposed to a range of concentrations of the same chemicals alone or in combination with DHT (5 microg/L) for 21 days. All of the compounds significantly inhibited DHT-induced spiggin production in a concentration-dependent manner in both the in vitro (FN > or = FL > or = LN > VZ) and in vivo (FN > FL > or = VZ > LN) assays. Fenitrothion and flutamide inhibited spiggin production in vitro at a concentration as low as 10(-12) M (P < 0.05), while linuron and vinclozolin inhibited DHT-induced spiggin production at concentrations of 10(-10) M (P < 0.05) and 10(-6) M (P < 0.001) respectively. Similarly, fenitrothion and flutamide were the most potent chemicals in vivo and significantly reduced DHT-induced spiggin production at a concentration of 10 microg/L and 25 microg/L respectively (P < 0.01). Both linuron and vinclozolin induced a significant decrease in DHT-induced spiggin production at a concentration of 100 microg/L when tested in vivo. In addition, kidney cell primary culture was used to test the (anti-)androgenic effects of the major environmental contaminants: oestradiol (E2), nonylphenol (NP) and bisphenol A (BPA) for the first time in teleosts. We observed that these compounds were able to significantly inhibit spiggin production at high doses (E2: 270 microg/L; NP: 2.2 microg/L; BPA: 2.3 microg/L). When tested in the absence of DHT, none of the compounds showed a significant agonistic activity in either in vivo or in vitro assays. Overall, our data further demonstrate that kidney cell primary culture is a reliable and a sensitive screening tool for the detection of (anti-)androgenic compounds. In addition, our study represents the first attempt to develop a combined in vivo/in vitro screening strategy for assessing the effects of (anti-)androgenic endocrine disrupters.

Do suspended sediments modulate the effects of octylphenol on rainbow trout?

A system was devised which allows particles to remain in suspension in a conventional 60 L aquarium without undue disturbance to resident fish. Using this system, juvenile rainbow trout were exposed for one week to 4-tert-octylphenol (OP, 10-1000 microg/L) with or without the presence of suspended sediments (10-20mg/L of natural suspended sediments from the River Calder, UK). About 8% of the added OP partitioned to the solid phase. Vitellogenin levels were determined in the plasma of the exposed rainbow trout and showed a dose-dependent increase with regards to OP exposure concentration. Considerable variation in the vitellogenin response was observed between separate runs with the same OP concentration. There was no statistically significant (at P < 0.05) difference in plasma VTG levels between the OP treatments with or without suspended sediments. This suggests that the dissolved concentration is the key factor and natural suspended sediment neither protects against, nor exacerbates, the endocrine disrupting effect of OP on fish.

Intercalibration exercise using a stickleback endocrine disrupter screening assay.

The Organisation for Economic Cooperation and Development (OECD) is currently validating a short-term fish screening protocol for endocrine disrupters (estrogens, androgens, and their antagonists and aromatase inhibitors), using three core species: fathead minnow, Japanese medaka, and zebrafish. The main endpoints proposed for the first phase of validation of the screen are vitellogenin (VTG) concentration, gross morphology (secondary sexual characteristics and gonado-somatic index), and gonadal histopathology. A similar protocol is concurrently being developed in the United Kingdom using the three-spined stickleback, with identical endpoints to those for the core species and, in addition, a unique androgen-specific endpoint in the form of spiggin (glue protein) induction. To assess the suitability of this species for inclusion in the OECD protocol alongside the core species, an intercalibration was conducted using 17beta-estradiol (a natural estrogen) and trenbolone (a synthetic androgen), thus mimicking a previous intercalibration with the core species. All three participating laboratories detected statistically significant increases in VTG in males after 14 d exposure to nominal concentrations of 100 ng/L 17beta-estradiol and statistically significant increases in spiggin in females after 14 d exposure to nominal concentrations of 5,000 ng/L trenbolone. The stickleback screen is reliable, possessing both relevant and reproducible endpoints for the detection of potent estrogens and androgens. Further work is underway to assess the relevance and suitability of the screen for weakly acting estrogens, anti-androgens, and aromatase inhibitors.

Chronic social stress in rainbow trout: does it promote physiological habituation?

The effect of chronic social stress on growth, energetic substrates and hormones was tested in rainbow trout, Oncorhynchus mykiss. After a 14-day isolation period, the fish were paired for 8 days. In order to expose fish to chronic intermittent social contact during pairing, they were maintained in direct contact with each other during the first day. After that, a black plastic screen partition was introduced in each tank, preventing direct contact between animals. Every day the partition was removed for 30 min, allowing physical interaction between fish. At the end of pairing period, they were isolated again for 13 days. Fish were weighed and blood was sampled frequently during the experiment. Plasma levels of cortisol, growth hormone, glucose, total protein and free amino acids were quantified. Both dominants and subordinates had specific growth rate decreased during the pairing period, but only subordinates increased when the stressor was abolished (dominants: 0.32+/-0.21 and 0.24+/-0.41, subordinates: -0.77+/-0.29 and 0.37+/-0.31, respectively). Dominants showed a higher cortisol level one week after pairing condition had been abolished than subordinates (dominants: 56.76+/-13.26, subordinates: 31.89+/-13.36). We conclude that chronic condition of intermittent social stress represents a stressful condition for animals of both hierarchical ranks and a treatment of one daily short direct contact between conspecifics does not promote habituation in fish, as mentioned for other stressors.