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The shortnose sturgeon ( Acipenser brevirostrum LeSueur, 1818) is a vulnerable species that is found along the eastern coast of North America. Little is known about temperature tolerance in this species and with a rapidly changing global climate, it becomes increasingly important to define the thermal tolerance of this species to better predict population distribution. Using a modified critical thermal maximum test (CTMax), the objectives of this study were to determine the impact of heating rate (0.1, 0.2 and 0.25 °C min − 1 ) on the thermal tolerance, associated hematological responses, and oxygen consumption in juvenile sturgeon. In addition, transcripts associated with physiological stress and heat shock (i.e., heat shock proteins) were also measured. Heating rate did not alter the CTMax values of shortnose sturgeon. Neither heating rate nor thermal stress affected plasma sodium and chloride levels, nor the expression of transcripts that included catalase , glucocorticoid receptor , heat shock protein70 ( hsp70 ), heat shock protein 90α ( hsp90α ) and cytochrome P450 1a ( cyp1a ). However, regardless of heating rate, thermal stress increased both plasma potassium and lactate concentrations. Glucose levels were increased at heating rates of 0.2 and 0.25 °C min − 1 , but not at 0.1 °C min − 1 . Overall, oxygen consumption rates increased with thermal stress, but the response patterns were not affected by heating rate. These data support the hypothesis that shortnose sturgeon can tolerate acute heat stress, as many physiological and molecular parameters measured here were non-responsive to the thermal stress. [ABSTRACT FROM AUTHOR]

Hydropower is the most common form of renewable energy, and countries worldwide are considering expanding hydropower to new areas. One of the challenges of hydropower deployment is mitigation of the environmental impacts including water quality, habitat alterations, and ecosystem connectivity. For fish species that inhabit river systems with hydropower facilities, passage through the facility to access spawning and rearing habitats can be particularly challenging. Fish moving downstream through a hydro facility can be exposed to a number of stressors (e.g., rapid decompression, cavitation, blade strike and collision, turbulence, and shear forces), which can affect fish survival in direct and indirect ways. Many studies have investigated the effects of hydroturbine passage on fish; however, the comparability among studies is limited by variation in the metrics and biological endpoints used. Future studies investigating the effects of hydroturbine passage should focus on using metrics and endpoints that are easily comparable. This review summarizes four categories of metrics that are used in fisheries research that have application to hydroturbine passage (i.e., mortality, injury, physiological markers, behavior) and evaluates them based on several criteria (i.e., resources needed, invasiveness, comparability among stressors and species, and diagnostic properties). Additionally, these comparisons are put into context of study setting (i.e., laboratory versus field). Overall, injury and physiological markers are ideal for studies in which there is a need to understand the mechanisms of effect, whereas behavior and mortality metrics provide information on the whole body response of the fish. The study setting strongly influences the comparability among studies. In laboratory-based studies, stressors can be controlled by type, intensity, and duration, allowing for easy comparisons among studies. In contrast, field studies expose fish to realistic passage environments but the comparability is limited. Based on these results, future studies, whether lab or field-based, should focus on metrics that relate to mortality for ease of comparison, while some diagnostic capability is important for furthering the science. [ABSTRACT FROM AUTHOR]

The response to capture is important in fisheries because it can reveal potential threats to species beyond fishing mortalities resulting from direct harvest. To date, the vast majority of studies assessing shark stress responses have used physiology or biotelemetry to look at sensitivity after capture, leaving a gap in our understanding of the behaviours of sharks during capture. We examined the behavioural responses of sharks to capture by attaching accelerometers to fishing gear and measuring the immediate and prolonged forces they exerted while on the line. We recorded acceleration vectors and derived the rate of intense fighting behaviours of 23 individual sharks comprising three species. Results suggest that blacktip sharks ( Carcharhinus limbatus) exhibited intense bouts of fighting behaviour at the onset of hooking, while nurse ( Ginglymostoma cirratum) and tiger sharks ( Galeocerdo cuvier) displayed more subdued acceleration values during capture. We also obtained plasma lactate from a subset of individuals and detected a strong correlation with maximum acceleration. These results align with previously published values and suggest that shark movement during fisheries capture is an important factor during bycatch and catch-and-release interactions. [ABSTRACT FROM AUTHOR]

Selenoproteins, which contain the selenocysteine (Sec), play crucial roles in antioxidant protection. In the present study, we have identified and characterized three selenoprotein T paralogs (designated as gfSelT1a, gfSelT1b and gfSelT2) in goldfish, Carassius auratus . The SECIS element was found in the 3′-UTRs of gfSelT1a , gfSelT1b and gfSelT2 mRNA. Sequence analysis showed that they all possess the CxxU motif (where U represents Sec) in the N-terminal. Constitutive expressions of gfSelT1a , gfSelT1b and gfSelT2 were observed in all tissues studied. In liver, gfSelT1a , gfSelT1b and gfSelT2 mRNA expression levels significantly decreased when fasting and increased after re-feeding. The expressions of gfSelT1a , gfSelT1b and gfSelT2 were all inducible by cadmium exposure and H 2 O 2 -mediated oxidative stress, except the transcripts of gfSelT1b decreasing with H 2 O 2 stimulation in brain. Furthermore, the expressions of gfSelT1a , gfSelT1b and gfSelT2 consistently increased in spleen but decreased in brain in response to heat stress. However, in liver only gfSelT1a mRNA expression significantly increased. In a word, this is the first report of the presence of three SelT genes in goldfish and their distinct expression patterns in response to environmental stressors. Our results suggest a potential involvement of gfSelT1a , gfSelT1b and gfSelT2 in host protection against environmental stressors including heavy mental, oxidative stress and heat stress in goldfish. [ABSTRACT FROM AUTHOR]

Environmental warming and acute stress increase cardiorespiratory activity in ectothermic animals like fish. While thermal acclimation can buffer the direct thermal effects on basal cardiorespiratory function during chronic warming, little is known about how acclimation affects stress-induced cardiorespiratory responses. We compared cardiovascular and haematological responses to chasing stress in cannulated wild European perch ( Perca fluviatilis) from a reference area at natural temperature (16 °C) with perch from the 'Biotest enclosure'; an experimental system chronically warmed (22 °C) by effluents from a nuclear power plant. Routine blood pressure was similar, but Biotest perch had slightly higher resting heart rate (59.9 ± 2.8 vs 51.3 ± 2.9 beats min), although the Q for heart rate was 1.3, indicating pronounced thermal compensation. Chasing stress caused hypertension and a delayed tachycardia in both groups, but the maximum heart rate increase was 2.5-fold greater in Biotest fish (43.3 ± 4.3 vs 16.9 ± 2.7 beats min). Moreover, the pulse pressure response after stress was greater in reference fish, possibly due to the less pronounced tachycardia or a greater ventricular pressure generating capacity and thermally mediated differences in aortic compliance. Baseline haematological status was also similar, but after chasing stress, the haematocrit was higher in Biotest fish due to exacerbated red blood cell swelling. This study highlights that while eurythermal fishes can greatly compensate routine cardiorespiratory functions through acclimation processes, stress-induced responses may still differ markedly. This knowledge is essential when utilising cardiorespiratory variables to quantify and compare stress responses across environmental temperatures, and to forecast energetic costs and physiological constraints in ectothermic animals under global warming. [ABSTRACT FROM AUTHOR]

The physiological ionoregulatory, metabolic and immune responses of Persian sturgeon, Acipenser persicus, to acute stress were investigated. Water levels were lowered to the fish dorsal scutes, and fish were blood-sampled before stress (pre-stress), and 0, 6, 24 and 72 h after stress. Results showed that serum cortisol rapidly increased after stress, returning to initial levels at 24 h. Serum glucose significantly increased at 6 h, declining to the pre-stress levels at 72 h. Serum triglyceride and cholesterol showed significant decreases at 0 h, then increasing to higher than the initial levels at 72 h. Serum T3 and T4 significantly decreased at 0 h and recovered at 72 h. Serum chloride levels showed no significant changes while serum calcium showed a significant increase at 0 h and a further increase until 72 h poststress. Serum total protein and alternative complement activity showed a significant initial decrease and recovery at 24 h with further increase at 72 h. Serum lysozyme activity increased significantly at 24 and 72 h after stress. Serum total immunoglobulin significantly increased at 0 h and peaked at 24 h. This is the first work showing thyroid hormone and immunological responses in Persian sturgeon subjected to stress, and the results show that this species follows a classical hormonal and energetic stress response, although, in the low range among the sturgeons. Although transient and moderate, confinement stress can induce significant changes in the innate immune response. [ABSTRACT FROM AUTHOR]

Animals typically display among-individual differences in behavior that are consistent over time (i.e., personality). These differences are often triggered by variable individual responses to environmental stress factors experienced during life, such as competition for resources and risk of predation. While the causes underlying animal personality are considered to be an issue of prime importance, it is still unknown whether personality emerges and develops over ontogeny if the main sources of behavioral differentiation are absent. Here, we tested whether personality emerged and was strengthened during the lifetime of Eastern mosquitofish ( Gambusia holbrooki), once intraspecific competition and risk of predation were completely removed and genetic and maternal differences minimized. We found that individual differences in behavior were overall repeatable over ontogeny (i.e., personality was manifested). Personality was, however, not detectable in juvenile individuals but emerged during and after their sexual maturation. The emergence of personality was triggered by the decline in behavioral plasticity of individuals over ontogeny, while differences in behavior among individuals did not vary with age. Our results suggest that animal personality might be inevitable and emerge in fish under laboratory-controlled conditions even in absence of extrinsic factors that typically lead to behavioral differentiation. The decline of behavioral plasticity over lifetime might be a relevant mechanism for the development of personality in animals. Significance statement: Increasing evidence suggests that animals have personality, that is, individuals consistently differ in behavior among each other (e.g., bold and shy or social and non-social individuals). Personality differences among animals should be, by definition, consistent over time and often caused by environmental challenges experienced early in life. In this study, we observed that personality differences were not present at juvenile age in social fish but emerged later in their life, despite the fact that environmental challenges (i.e., predation risk and competition for space, food, and mates) were absent. Personality differences strengthened over lifetime, resulting from declines in individual behavioral plasticity. Our results suggest that the decline in behavioral plasticity with age may represent a relevant mechanism for behavioral differentiation in animals, in agreement with evidences from the human literature on age-related loss in behavioral plasticity. [ABSTRACT FROM AUTHOR]

Aquatic ecosystems are subjected to a variety of man-induced stressors but also vary spatially and temporally due to variation in natural factors. In such complex environments, it remains difficult to detect, dissociate and evaluate the effects of contaminants in wild organisms. In this context, the aim of this study was to test whether the hepatic transcriptome profile of fish may be used to detect in situ exposure to a particular contaminant. Transcriptomic profiles from laboratory-exposed and wild eels sampled along a contamination gradient were compared. During laboratory experiments, fish were exposed during 45 days to different pollutants (Hg, PCBs, OCPs or Cd) or natural factors (temperature, salinity or low food supply) at levels close to those found in the sampling sites. A strong difference was observed between the transcriptomic profiles obtained from wild and laboratory-exposed animals (whatever the sites or experimental conditions), suggesting a general stress induced by captivity in the laboratory. Among the biological functions that were up-regulated in laboratory eels in comparison to wild eels, histone modification was the most represented. This finding suggests that laboratory conditions could affect the epigenome of fish and thus modulate the transcriptional responses developed by fish in response to pollutant exposure. Among experimental conditions, only the transcription profiles of laboratory animals exposed to cold temperature were correlated with those obtained from wild fish, and more significantly with fish from contaminated sites. Common regulated genes were mainly involved in cell differentiation and liver development, suggesting that stem/progenitor liver cells could be involved in the adaptive response developed by fish chronically exposed to pollutant mixtures. [ABSTRACT FROM AUTHOR]

Lectins are a superfamily of carbohydrate-binding proteins that are widely distributed throughout living organisms. In earlier work, we identified lily-type lectin ( Sm LTL) in the skin mucus of turbot Scophthalmus maximus , and we characterized the protein in the present study. Results from qRT-PCR indicated that Sm LTL was expressed highly in skin, intestine and gill tissue. Changes in Sm LTL expression occurred in these tissues in response to environmental stressors including ciliate infection, high temperature and salinity. Recombinant Sm LTL purified from Escherichia coli was able to haemagglutinate mouse erythrocytes in the absence of calcium, and was inhibited by d -mannose. In addition, Sm LTL displayed selective binding to bacterial species including Edwardsiella tarda a nd Vibrio anguillarum , and exhibited toxicity towards Philasterides dicentrarchi, with a mortality of over 60% after 24 h at a concentration of only 100 μgml −1 . To investigate this toxicity further, we measured binding of Sm LTL after incubating the ciliate in FITC- Sm LTL solution. Surface fluorescence decreased substantially in the presence of 400 mM d -mannose. Together these results suggest that lily-type lectins serve as the first line of defence against microbial attack and play a pivotal role in the mucosal immune system. [ABSTRACT FROM AUTHOR]

Several fish species exhibit antipredator responses when exposed to chemicals which indicate risk of predation. One such substance is the scent of a predator (a kairomone) that may induce defensive responses in a potential prey. In the present study, we show that chemical cues (odour) from predator fish induce antipredator and stress responses in Nile tilapia. When exposed to predator odour, Nile tilapia decreased activity and increased ventilation rate (VR), but no increase in plasma levels of cortisol and glucose was found. Although the hypothalamic-pituitary-interrenal axis (HPI axis) was not activated, an increase in ventilation is a fast response which can provide the fish enough oxygen for a ‘fight or flight’ event when facing a predator. Thus, this respiratory response suggests an anticipated adjustment in order to prepare the body for a defensive response, such as escaping, irrespective of HPI axis activation. [ABSTRACT FROM AUTHOR]

To determine whether dietary lipid sources used in regimens to replace fish oil differentially impact the production performance and physiological status of hybrid Striped Bass (White BassMorone chrysops× Striped BassM. saxatilis), the growth performance and stress response of fish raised on diets containing oil from fish, canola, corn, or flaxseed were evaluated. Production performance was unaffected by diet. Net-chasing and handling stress challenges elicited changes in hematology consistent with the generalized stress response, and there was a significant main effect of stressor exposure (P< 0.01) on both plasma cortisol and osmolality, as well as a main effect of lipid source (P< 0.05) on plasma osmolality. Across diets, cortisol and osmolality levels were highest 1 h after stressor exposure (411 ng/mL and 385 mOsm/kg, respectively) but decreased significantly by 4 h poststressor (169 ng/mL and 332 mOsm/kg, respectively). The plasma lipid fatty acid (FA) profile was influenced by diet and generally reflected the FA profile in the feed. However, higher levels of saturated and monounsaturated FAs and lower levels of polyunsaturated FAs were typically observed shortly after stressor exposure. Although differences in hematology were associated with differences in lipid source and stressor exposure, neither obvious impairment of physiological function nor gross effects (i.e., morbidity and mortality) were observed. Received November 16, 2015; accepted March 20, 2016 [ABSTRACT FROM PUBLISHER]

Our study explored the dietary effects of adenosine monophosphate (AMP) to enhance growth, digestibility, innate immune responses and stress resistance of juvenile red sea bream. A semi-purified basal diet supplemented with 0% (Control), 0.1% (AMP-0.1), 0.2% (AMP-0.2), 0.4% (AMP-0.4) and 0.8% (AMP-0.8) purified AMP to formulate five experimental diets. Each diet was randomly allocated to triplicate groups of fish (mean initial weight 3.4 g) for 56 days. The results indicated that dietary AMP supplements tended to improve growth performances. One of the best ones was found in diet group AMP-0.2, followed by diet groups AMP-0.1, AMP-0.4 and AMP-0.8. The Apparent digestibility coefficients (dry matter, protein and lipid) also improved by AMP supplementation and the significantly highest dry matter digestibility was observed in diet group AMP-0.2. Fish fed diet groups AMP-0.2 and AMP-0.4 had significantly higher peroxidase and bactericidal activities than fish fed the control diet. Nitro-blue-tetrazolium (NBT) activity was found to be significantly (P < 0.05) greater in fish fed diet groups AMP-0.4 and AMP-0.8. Total serum protein, lysozyme activity and agglutination antibody titer were also increased (P > 0.05) by dietary supplementation. In contrast, catalase activity decreased with AMP supplementation. Moreover, the fish fed AMP supplemented diets had better improvement (P < 0.05) in body lipid contents, condition factor, hematocrit content and glutamyl oxaloacetic transaminase (GOT) level than the control group. Supplementation also improved both freshwater and oxidative stress resistances. Interestingly, the fish fed diet groups AMP-0.2 and AMP-0.4 showed the least oxidative stress condition. Finally it is concluded that, dietary AMP supplementation enhanced the growth, digestibility, immune response and stress resistance of red sea bream. The regression analysis revealed that a dietary AMP supplementation between 0.2 and 0.4% supported weight gain and lysozyme activity as a marker of immune functions for red sea bream, which is also inline with the most of the growth and health performance parameters of fish under present experimental conditions. [ABSTRACT FROM AUTHOR]

Propranolol (PROP) is a β-blocker prescribed mainly to treat human cardiovascular diseases and as a result of its wide usage and persistence, it is reported in aquatic environments. This study examined whether PROP alters developmental patterns and catecholamine (CA)-regulated processes in the zebrafish ( Danio rerio ) and if exposure during early life alters the stress response and behaviors of adults. The calculated 48 h larva LC 50 was 21.6 mg/L, well above reported environmental levels (0.01–0.59 μg/L). Stressed and PROP-exposed adult zebrafish had reduced testosterone and estradiol levels and exhibited behaviors indicating less anxiety than control fish. Furthermore, adults previously PROP-exposed as embryos/larvae had decreased growth in terms of body length and mass. Finally, these adults showed increased cholesterol and a dose-dependent decrease in testosterone levels compared with unexposed zebrafish. Thus PROP-exposure of zebrafish embryos/larvae alters developmental patterns and CA-regulated processes that may affect normal behaviors and responses to stressors, and at least some of these changes persist in the adult zebrafish. [ABSTRACT FROM AUTHOR]

Remodeling the muscle metabolic machinery in mammals in response to energetic challenges depends on the energy sensor AMP-activated protein kinase (AMPK) and its ability to phosphorylate PPAR γ coactivator 1 α (PGC1α), which in turn coactivates metabolic genes through direct and indirect association with DNA-binding proteins such as the nuclear respiratory factor 1 (NRF1) (Wu et al., 1999). The integrity of this axis in fish is uncertain because PGC1α i) lacks the critical Thr 177 targeted by AMPK and ii) has mutations that may preclude binding NRF1. In this study we found no evidence that AMPK regulates mitochondrial gene expression through PGC1α in zebrafish and goldfish. AICAR treatment of zebrafish blastula cells increased phosphorylation of AMPK and led to changes in transcript levels of the AMPK targets mTOR and hexokinase 2. However, we saw no increases in mRNA levels for genes associated with mitochondrial biogenesis, including PGC1α, NRF1, and COX7C, a cytochrome c oxidase subunit. Further, AMPK phosphorylated mammalian peptides of PGC1α but not the corresponding region of zebrafish or goldfish in vitro . In vivo cold acclimation of goldfish caused an increase in mitochondrial enzymes, AMP and ADP levels, however AMPK phosphorylation decreased. In fish, the NRF1-PGC1α axis may be disrupted due to insertions in fish PGC1α orthologs within the region that serves as NRF1 binding domain in mammals. Immunocopurification showed that recombinant NRF1 protein binds mammalian but not fish PGC1α. Collectively, our studies suggest that fish have a disruption in the AMPK-PGC1α-NRF1 pathway due to structural differences between fish and mammalian PGC1α. [ABSTRACT FROM AUTHOR]

Here we provide an overview of the mechanistic characterization of the hepatic action of cortisol during stress in fish. Cortisol is the main circulating glucocorticoid in fish and its action is mediated through its cytosolic receptor, the glucocorticoid receptor (GR), and regulates the expression of genes involved in growth, metabolism and immune function. When taken together, the data suggests that cortisol may be playing a key role in the energy substrate re-partitioning in hepatocytes to cope with stress. The proposed model is that cortisol upregulates pathways involved in energy substrate mobilization, including gluconeogenesis, while downregulating energy demanding pathways, including growth and immune function. Recent work also points to a role for cortisol in mediating rapid action that is non-genomic and includes modulation of secondary signalling cascades; however, the physiological relevance of these studies remains to be determined. Altogether, studies carried out in hepatocytes are bringing to fore the complex nature of the cortisol signalling pathways in the organismal stress response. The mode of actions and their physiological implications for stress coping awaits further study. [ABSTRACT FROM AUTHOR]

The objectives of this experiment were to (i) determine the efficacy of essential oils of clove ( CO) and Lippia alba ( EOLA) to induce deep anaesthesia in juvenile specimens (49.0 ± 6.2 g body mass, 16.6 ± 0.8 cm; n = 8 per treatment) of meagre ( Argyrosomus regius); and (ii) study the feasibility of these substances, together with 2-phenoxyethanol (2- PHE), as potential sedatives [low concentration: (i) EOLA: 12 mg L−1; (ii) CO: 1 mg L−1; and (iii) 2- PHE: 33 mg·L −1; n = 8 per treatment] for live fish transport of this species. All test were performed at a constant temperature (18°C). Thus, the main primary stress indicator (plasma cortisol) and secondary factors (plasma metabolites) were evaluated. In addition, growth hormone ( GH) mRNA expression was also evaluated in the pituitary gland. The results indicated that EOLA is considered to be effective for deep anaesthesia when the concentration is close to 160 mg L−1, while CO produces the same effect when lower concentrations are added (40-50 mg L−1). Regarding sedative concentrations, a significant ~3-fold increase in plasma cortisol levels was detected in the EOLA group when compared to control specimens. In addition, glucose levels were not reduced and significantly increased (~1.6-fold) for 2- PHE in relation to the control fish. None of the anaesthetics promoted a significant difference for GH expression with respect to the control group, but a significant ~2-fold increase for 2- PHE treatment with respect to the EOLA exposition was found in this gene expression. Results show that none of the anaesthetics analysed, at least in the ranges of concentrations used in this study ( EOLA 12 mg L−1, CO 1 mg L−1, 2- PHE 33 mg L−1), are recommended for live fish transport, as shown by the absence of inhibition on the stress parameters assessed. [ABSTRACT FROM AUTHOR]

This study evaluated effects of graded levels of dietary β-glucan (0, 0.5, 1, 2 and 4 g kg −1 ) on growth performance, haematological parameters, intestinal Vibrio counts, dose requirement and salinity stress resistance in pompano fish Trachinotus ovatus (6.45 g ± 0.06 (SEM)). After 8-weeks of diet feeding, growth was significantly higher in fish fed diets with 0.10% β-glucan compared to fish fed control diet (no β-glucan). Survival increased significantly in fish fed 0.05 and 0.10% β-glucan compared to control diet. There were significant increases in red blood cells (in 0.20% β-glucan diet), in total leukocytes (in 0.05–0.20% β-glucan diet), in both lymphocyte and monocyte count in fish fed 0.10%–0.40% β-glucan diet. However, dietary β-glucan did not affect neutrophil, eosinophil and basophil counts. Intestinal Vibrio counts were reduced in fish fed any level of β-glucan compared to control. In addition, dietary β-glucan levels highly correlated with growth, survival, intestinal Vibrio counts and haematological index. Optimal β-glucan levels for maximal growth of fish were predicted to be 0.122% at day 21 (R 2 = 98.53%), 0.120% at day 28 (R 2 = 78.55%), 0.115% at day 42 (R 2 = 62.21%) and 0.090% at day 56 (R 2 = 75.18%), showing a decreasing β-glucan requirement with increasing fish size. Furthermore, optimal β-glucan levels for maximal haematological parameters based on lymphocyte count, was estimated to be 0.120% (R 2 = 98.53%) at day 56. Also, fish fed 0.05%–0.20% β-glucan showed better resistance against salinity stress. In conclusion, β-glucan supplementation is effective for improving growth, intestinal Vibrio counts and boosted stress resistance of the pompano fish, T. ovatus . [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the effect of ractopamine intake at concentrations of 0 ppm (control), 10 ppm, 20 ppm and 40 ppm in the diet on the physiological and immunological parameters, through the analysis of hematological parameters and serum concentration of cortisol and lactate in 80 Nile tilapia (Oreochromis niloticus) of the GIFT strain, weighing 500 grams, randomly distributed in 16 water tanks with a capacity of 500 liters, with the density of 5 animals. Withdrawing from day 0 to 1 animal in each box, and every 20 days, 2 fish per box, totaling eight fish each treatment. They were collected blood samples for cortisol assay, lactate and haematological parameters (red blood cell count, white blood cells, total protein - PPT, packed cell volume. - VG, hemoglobin, lactate showed a significant decrease (p <0.05) at both times collection with 10 ppm (3.79 mmol / L to 20 days and 1.58 mmol / L at 40 days) and compared with the control (4.61 mmol / L at day 0, 4.78 mmol / L to 20 4.54 days and 40 days) and the same concentration differed compared with 40 ppm of ractopamine at 40 days (3.15 mmol / L) The parameters evaluated, cortisol, blood counts, white blood cells, packed cell volume. - VG, hemoglobin, showed no significant difference (p <0.05) between treatments and collection times. The results of this study did not demonstrate a physiological stress produced by ractopamine intake in concentrations of 10, 20 and 40 ppm for animals. However, more studies on the subject should be carried out so that the additive use is recommended securely on the welfare of fish. [ABSTRACT FROM AUTHOR]

Electrical stunners have been introduced on some Scottish seiners in Norway to facilitate easier handling of fish in connection with the bleeding operation. Evaluation of stunning efficiency of Atlantic cod, haddock and saithe at sea has been successful for cod and haddock. In contrast, electrical stunning of saithe can result in broken spines and blood spots for a certain fraction of a catch. A controlled experiment was therefore carried out with saithe where the goal was to minimize the detrimental effects of electrical immobilisation. Since two configurations of the electrical stunner are in use on different vessels, we studied stunner configuration and voltage (40, 70 and 100 V) as variables. In addition, we hypothesized that post-capture remaining energy for contraction of white muscle (pre-stunning stress), stimulated by electricity, could be a factor to consider related to the occurrence of spinal fractures. Fish behaviour, eye-roll (VOR), and recovery time were used as indices of stunning efficiency. Effects of stress on white muscle were assessed by changes in initial pH, twitch ability, and blood lactate levels. The results showed that regardless of pre-immobilisation stress level, stunner configuration, and voltage, all fish responded instantly upon exposure to electricity. Absence of VOR indicated that the fish were unconscious as assessed immediately after immobilisation. However, after 10 min of recovery in seawater, many fish in all experimental groups regained consciousness. Compared with rested fish (control), blood lactate, white muscle initial pH and twitch ability were affected by pre-immobilisation stress and subsequent fish handling procedures. Neither stunner configuration nor applied voltage fully solved the problem with spinal fractures and blood spots in saithe. To minimize the problem though, the current research showed that the best stunner configuration was to use the conveyor belt as counter electrode rather than using alternating charge on adjacent rows of electrodes. Furthermore, fish subjected to pre-immobilisation stress also reduced the probability for spinal fractures and blood spots. More research is nevertheless necessary to eradicate the problem altogether. [ABSTRACT FROM AUTHOR]

This study looks at how low infestation loads of adult Caligus rogercresseyi and other stressors affect the physiology of Salmo salar. Experimental fish groups were with (infested) or without (control) exposure to the parasite. The parasite cohort was followed for 78 days post-infestation (dpi), and only adult lice were observed. Additional stressors were applied at 60 and 75 dpi. The analysis included measurements of fish physiology and weight. Low-level infestations by adult C. rogercresseyi for more than 50 dpi induced moderate stress in S. salar as well as a high energy demand and increased small skin mucous cells. Threshold lice loads were identified, and above those loads, a high stress response was observed. Additional stressors altered fish physiology, inducing downregulation of the cortisol response after the first stressor and upregulation after the second stressor, but infested fish responded more strongly. Parasitism by C. rogercresseyi is energetically demanding, affecting the primary and secondary responses (e.g. cortisol and glucose levels), as well as the tertiary response (fish weight). [ABSTRACT FROM AUTHOR]

Hyperosmotic stress has often been investigated from osmoregulation perspectives while the effects of such stress on the immune capacity remain largely unexplored. In this study, striped catfish were submitted to a gradual hyperosmotic stress (up to 20 ppt), followed by an infection with a virulent strain of an intracellular bacteria, Edwardsiella ictaluri . Physiological parameters (plasma osmolarity, gill NaK ATPase) have been investigated. Moreover, a label free quantitative proteomics workflow was used to study how salinity affects the proteome of kidney of no infected and infected fish. The flow consisted in initial global profiling of relative peptide abundances (by LC/MS, peak area quantification based on extracted ion currents) followed by identification (by MS/MS). Differentially expressed proteins were analysed in DAVID interface to isolate functional pathways involved in the response to bacterial infection and which are influenced by environmental salinity changes. In response to hyperosmotic stress, plasma osmolarity increased while gill Na + K + ATPase slightly increased. In kidney proteome, 2024 proteins were identified, among which 496 proteins were differentially expressed. Hierarchical clustering analysis leads to isolation of 4 main clusters. Several pathways and functional categories, mostly related to cell metabolism, response to stress, cell structure, immunity and ion homeostasis were highlighted and discussed. Significance The striped catfish is a highly worldwide commercialized species and is mainly produced in the Mekong Delta (Vietnam), which is currently subjected to sea level rise and increasing salt intrusions in these aquacultures resources. Moreover, salt is commonly used as antiseptic in aquacultures to treat microbial diseases. To date, the effect of prolonged exposure to hyperosmotic condition have been mainly looking at changes regarding the growth and the osmoregulation while the effect of such stress on the immune capacity and susceptibility to pathogen have been poorly investigated. This study aims to highlight molecular changes in kidney occurring during acclimation to hyperosmotic conditions, particularly those involved in fish immune defences. [ABSTRACT FROM AUTHOR]

African sharptooth catfish Clarias gariepinus were housed under continuous dim light (1 lx) or 12L: 12D (350-0 lx) cycles. The number of skin lesions, as indicator of aggressive acts, and plasma cortisol levels, as indicator of stress-axis activity, were measured at baseline as well as following a stressor (given in the light or dark phase). Results showed that (1) baseline plasma cortisol levels were not different between photoperiods, (2) the number of baseline skin lesions was highest for C. gariepinus housed under continuous dim light, (3) stressor-induced peak levels of plasma cortisol were highest in the light phase and (4) the number of skin lesions following a stressor was highest in the dark phase. The higher number of stressor-related skin lesions in the dark (active) phase suggests increased stressor-induced aggression while in the active phase. In addition, the data suggest that housing under continuous dim light does not result in higher stress-axis activity, as measured by baseline levels of cortisol, but does result in more stressor-induced aggression, as measured by the higher number of skin lesions. The latter may be related to the fact that the continuous dim light photoperiod has twice the number of dark-phase (active) hours in which stressor-induced aggression is stronger compared to the 12L: 12D photoperiod, which has a light phase in which stressor-induced aggression is lower. [ABSTRACT FROM AUTHOR]

Jundiá ( Rhamdia quelen ) is a suitable species for aquaculture in regions of temperate or subtropical climate. This species has received great attention regarding several aspects of physiology as well as an organism to study the impact of environmental contaminations. However, experiments using validated and objective tests to evaluate the jundiá behavior are scarce. The effects of acute stress have been studied in other fish species, such as zebrafish ( Danio rerio ), however, the effects in jundiá are lacking. Thus, we evaluated the effects of acute stress (net chasing) on anxiety-like and social behavior in jundiá. For these purpose, all behavioral analyses were carried out using automated tracking software. We showed that the acute stress protocol increased cortisol levels and induced anxiogenic-like behavior in the novel tank test, and decreased social behavior in jundiá. The antidepressant fluoxetine was able to prevent the effects of acute stress on social behavior. Here we show a behavioral evaluation of Rhamdia quelen using consolidated tests and computerized analysis, which allows more measurable, reliable and comparable results. [ABSTRACT FROM AUTHOR]

This study was conducted to assess the status of Lake Mainit by using fluctuating asymmetry in the populations of Glosogobius giuris. A total of 200 G. giuris was collected with 100 per sex. All the samples were placed in a flat styrofoam for the pinning of its fins to make it wider and to clearly see the samples point of origin for the land-marking process. 10% Formalin was applied in all the fins of the fish samples to make it hardened using a small brush. Twenty (20) landmarks were used to analyze the body shape of the fish. Several studies proved that FA can be used to directly assess water quality and the overall status of the ecosystem. Using thin-plate spline (TPS) series, landmark analyses were obtained and subjected to Symmetry and Asymmetry in Geometric Data (SAGE) software. Results in Procrustes ANOVA showed that individual symmetry showed a highly significant difference (P = 0.00) as well as Sides (Directional Asymmetry) and Interaction (Fluctuating Asymmetry) in the study area. All the samples showed FA in both sexes. The results of Principal Component Scores displayed a higher percentage in female (69.2797%) than male (63.9214%) from Lake Mainit. Variations are almost found in all the body part of the goby in female and male gobies except for anterior insertion of second dorsal fin and superior margin of the preoperculum. Females have a higher fluctuating asymmetry than in males. Females spawn and males protect the eggs from any predators. In this case, females are more susceptible to stress because they have to forage in order to compensate the energy used up during spawning. Males are less susceptible because they usually stay on the nesting ground, and don't have to cover far distances in finding food. This study validates the use of FA in determining the status of the fishes caught connecting to what the status of the environment is based on the result. [ABSTRACT FROM AUTHOR]

Emodin is an anthraquinone exhibiting several positive benefits of anti-inflammatory, free radical scavenging, and resistance to stress. The goal of this study is to investigate the effects of emodin on the hepatic cell line of grass carp exposed to hyperthermic stress. Cultured cells were treated with various emodin concentrations (0, 0.04, 0.2, and 1.0 µg/ mL) at 27 C for 24 h. Then all cultured cells were exposed to thermal stress by increasing the culture temperature (32 ± 0.5 C) for 0.5 h. Increased temperatures significantly reduced cell viability and increased lactate dehydrogenase ( LDH) release in three of the four experimental groups (0, 0.2, and 1 µg/ mL emodin) compared with the control. Additionally, reactive oxygen species ( ROS) were significantly higher in cells exposed to elevated temperatures and treated with 0.2 or 1 µg/ mL emodin and mitochondrial membrane potential was significantly lower in cells treated with 0, 0.2, or 1 µg/ mL emodin. Expressions of heat shock proteins ( HSP60, HSP70, and HSP90) were significantly higher in all but one of the experimental groups. Our results suggest that 0.04 µg/ mL emodin can increase cell viability and HSP90 mRNA level, reduce LDH release and concentration of ROS, and contribute to enhance the resistance to high temperature stress in the hepatic cells of grass carp. [ABSTRACT FROM AUTHOR]

To study the effect of low temperature stress on hematological parameters and HSP gene expression in the turbot ( Scophthalmus maximus), water temperature was lowered rapidly from 18 to 1°C. During the cooling process, three individuals were removed from culture tanks at 18, 13, 8, 5, 3, and 1°C. Blood samples and tissues were taken from each individual, hematological indices and HSP gene expression in tissues were measured. The red blood cell count, white blood cell count, and hemoglobin concentration decreased significantly ( P < 0.05) as temperature decreased. Enzyme activities of plasma alanine transaminase and creatine kinase increased as temperature decreased, whereas aspartic transaminase and γ-glutamyl transpeptidase activities displayed no obvious changes above 1°C and lactate dehydrogenase activity increased first and then decreased. Blood urea nitrogen and uric acid levels were highest at 8°C, and creatinine concentration was highest at 3°C. The concentrations of plasma cortisol, cholesterol, and triglyceride all increased significantly ( P < 0.05) as temperature decreased. The serum glucose concentration increased first and then decreased to the initial level. The HSP70 mRNA expression showed various patterns in different tissues, whereas HSP90 mRNA expression showed the same tendency in all tissues. Overall, these results indicate that temperature decreases in the range of 8 to 5°C may induce a stress response in S. maximus and that temperature should be kept above 8°C in the aquaculture setting to avoid damage to the fish. [ABSTRACT FROM AUTHOR]

Inosine and inosine monophosphate (IMP) are the most extensively studied specific nucleotide for feeding stimulation research rather than using as functional nutrients for potential growth and health benefit of aquatic species. Therefore, a 10-week feeding trial was conducted to determine comparative effects of inosine and IMP as functional nutrients on growth performance, immune responses, stress resistance and intestinal morphology of juvenile red sea bream ( Pagrus major ). Casein based semi-purified basal diet was formulated to contain 54% protein, 11% lipid, without inosine and IMP supplementation (Control). Four levels of inosine nucleoside (0.2, 0.4, 0.6 and 0.8% for diet groups INO-0.2, INO-0.4, INO-0.6 and INO-0.8 respectively) and IMP nucleotide (0.2, 0.4, 0.6 and 0.8% for diet groups IMP-0.2, IMP-0.4, IMP-0.6 and IMP-0.8 respectively) were added to the basal diet. Each diet was randomly allocated to triplicate groups of fish with initial average weight of 6.6 g. The results indicated that dietary inosine and IMP supplementations tended to improve growth performances, in which one of the best ones was found in diet group IMP-0.6, but the values did not significantly differ from those in diet groups IMP-0.4 and INO-0.4, respectively. Superoxide dismutase, peroxidase and bactericidal activity were significantly influenced by dietary supplementation of inosine and IMP. However, the values of these parameters were not significantly different among diet groups INO-0.4, IMP-0.4 and IMP-0.6. Total serum protein, catalase and lysozyme activity were also improved (P > 0.05) by dietary supplementation of inosine and IMP. Supplementations improved both freshwater stress and oxidative stress resistances. Fish fed diet groups INO-0.4 and IMP-0.6 showed the least oxidative stress condition. Inosine and IMP supplementations significantly increased enterocyte height (hE), fold height (hF) of both anterior and posterior intestine and microvillus height (hMV) of posterior intestine compared to those of the control. The highest hF observed in diet group IMP-0.4 and the mean value was not significantly different from those in other IMP diets, INO-0.4 and INO-0.6, respectively. Significantly highest anterior hE and posterior hF, hE and hMV observed in diet group INO-0.4. However, no significant difference of posterior hF observed between INO-0.4 and IMP supplemented diet groups. Fish fed supplemented diets also showed improved survival compared to control (P > 0.05). Considering overall performances, it can be concluded that diet groups INO-0.4 and IMP-0.4 showed relatively better performance among inosine and IMP supplemented diet groups respectively compared to control. Furthermore, diets supplemented with either inosine or IMP at 0.4% are beneficial to promote growth, immune responses, stress resistance and intestinal health condition of juvenile red sea bream. Statement of relevance In this study inosine and inosine monophosphate has been evaluated as potential functional nutrients for marine fish such as red sea bream. Since supplementations of such immunostimulant-like functional nutrients are very important for the development of functional aquafeeds, I hope findings of this paper will help to develop low fishmeal based functional aquafeeds in near future. [ABSTRACT FROM AUTHOR]

Integrated recirculating aquaculture systems (IRASs) were developed to overcome the problems of nutrient overloading and to address the issues of animal welfare in recirculating aquaculture systems. Barramundi ( Lates calcarifer Bloch) (initial weight of 13.34 ± 0.01 g) were reared for 84 days in 12 (4 × 3 design) independent IRASs at four densities: 10.10, 12.98, 15.86 and 18.75 kg m − 3 . Throughout the trial, the air was the only source of dissolved oxygen to the fish-rearing tank, and duckweed ( Lemna minor Linnaeus) as a biofilter medium was used as an additional macrophytic crop in order to qualify as an IRAS. Therefore, each IRAS consisted of a fish-rearing tank, a waste-collection tank and a biofilter tank that housed the L. minor . At the end of the trial, blood samples were taken to determine levels of stress-related parameters (cortisol, glucose, lactate and whole-blood haemoglobin [Hb]) and thyroid hormones (thyroxine [T 4 ] and triiodothyronine [T 3 ]). In order to evaluate the acute stress response, the remaining fish were further exposed to elevated total ammonia, of which a portion was in the more toxic unionised form followed by blood analysis so as to determine the levels of stress-related parameters and thyroid hormones. The results demonstrated that a high stocking density of 18.75 kg m − 3 of barramundi significantly decreased the specific growth rate, increased the feed conversion ratio and altered plasma levels of total T 4 , total T 3 , cortisol, glucose, lactate and whole-blood Hb. Total plasma concentrations of T 3 were inversely correlated with stocking densities, whereas total plasma T 4 levels of juvenile barramundi were significantly higher when the juvenile barramundi were stocked at a higher density of 18.75 kg m − 3 . These results also suggest that the growth suppression of the juvenile barramundi caused by a high stocking density might be associated with a decrease in circulating levels of thyroid hormones as a consequence of small but significant increased cortisol level. Therefore, a stocking density of lower than 18.75 kg m − 3 of barramundi is recommended for culturing in IRASs. Statement of relevance 1. An IRAS is developed to overcome the problem of nutrient overloading and animal welfare problems in recirculating aquaculture systems (RASs). 2. The study is required to establish an optimum stocking density requirement for juvenile barramundi in IRASs. 3. No studies have investigated the effects of stocking density on growth and physiological responses of juvenile barramundi species reared in integrated recirculating aquaculture systems (IRASs). [ABSTRACT FROM AUTHOR]

Senegalese sole ( Solea senegalensis ) is a highly valued flatfish that grows well with diets containing plant ingredients but their effects on immune competence is still a matter of debate. The current study aimed to examine changes in innate immune parameters and gut microbiota in Senegalese sole fed with 35% or 72% of plant ingredients with or without probiotic or yeast supplementation. Overall, fish fed diets with 72% of plant ingredients showed lower transcript levels of key immune- and stress-related genes in distal intestine, rectum and head-kidney than the 35% diets. In particular, hsp90b mRNA levels in distal intestine were down-regulated by 70% and 60% with the use of high content of plant ingredients in the diet containing the multispecies probiotic and autolyzed yeast, respectively. Denaturing gradient gel electrophoresis showed lower similarity values for distal intestine than rectum. Also fish fed high content of plant ingredients displayed lower similarity values, pointing to a difference in the microbial populations between fish fed different plant ingredient contents on the diet. Our data revealed that inclusion of plant ingredients was associated with differences in gene expression and a more diverse microbiota profile but without a significant effect on growth performance. Moreover, probiotic supplementation resulted in up-regulation of hsp90b , gpx , cat and apoa1 transcript levels in distal intestine concomitantly with a growth rate reduction compared to non-supplemented fish. Statement of relevance There is an increasing trend in the aquaculture industry to replace fishmeal for plant ingredients, as a means of promoting sustainability of the industry. This paper contributes significantly to our limited knowledge of how plant ingredients and supplements affect gut microbiota and immunocompetence. [ABSTRACT FROM AUTHOR]

Yellow catfish ( Pelteobagrus fulvidraco ) has become a commercially important fish species in China and eastern Asia. High-density aquaculture has led to congestion and excessive stress and contributed to bacterial infection outbreaks that have caused high mortality. We investigated the effects of dietary supplementation with astaxanthin and emodin alone and in combination on the growth and stress resistance of yellow catfish. After 60 days of feeding, each group of fish (control, astaxanthin, emodin, and astaxanthin plus emodin (combination) groups) was exposed to acute crowding stress for 24 h, and a subsample of fish from the four groups was challenged with the bacterial septicemia pathogen Proteus mirabilis after the end of the crowding stress experiment. Compared with the control, the astaxanthin and emodin groups showed increases in serum total protein (TP), hepatic superoxide dismutase (SOD) activity and hepatic heat shock proteins 70 (HSP70) mRNA levels at 12 and 24 h after the initiation of crowding stress. The combination group exhibited increases in alanine aminotransferase (ALT) activity, aspartate aminotransferase (AST) activity, serum TP, hepatic SOD activity and hepatic HSP70 mRNA levels within 24 h after the initiation of crowding stress. However, decreases relative to the control were observed in the serum cortisol and glucose contents in the three treatment groups at 12 and 24 h after the initiation of crowding stress, in ALT and AST activity in the astaxanthin and emodin group at 24 h after the initiation of crowding stress, and in the serum lysozyme activity, serum alkaline phosphatase (ALP) activity, and hepatic catalase (CAT) and malondialdehyde (MDA) activity in the combination group at 24 h after the initiation of crowding stress. Additionally, the cumulative mortality after P. mirabilis infection was lower in all three treatment groups (57.00%–70.33%) than in the control (77.67%). Dietary supplementation with astaxanthin and emodin decreased the specific growth rate (SGR) and weight gain (WG) of healthy yellow catfish, although significant differences in mortality were not observed. These results indicate that dietary supplementation with 80 mg/kg astaxanthin and 150 mg/kg emodin can improve the anti-oxidative capabilities, hepatic HSP70 levels, and resistance to acute crowding stress of yellow catfish. Finally, an appropriate strategy for enhance yellow catfish stress resistance and disease resistance is proposed. [ABSTRACT FROM AUTHOR]

The article presents a study conducted to evaluate effects on the life of Hucho taimen fishes reared in coloured tanks. It mentions that study revealed little effects of colour tank on the growth of fishes, physiological effect was noticed but no effects on plasma glucose and lactate levels, Hucho taimen reared in yellow tanks provides favourable climate for food intake, difference in body colour has been noticed in black tank that can reduce conspicuousness and stress in fishes.

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. [ABSTRACT FROM AUTHOR]

The main objective of the present study was to shed light on the effects of 2-phenoxyethanol on possible primary (cortisol level) and secondary (hematological indices and glucose level) stress responses and changes in the activity of metabolic enzymes (AST, ALT and ALP) in bighead carp ( Hypophthalmichthys nobilis). The fish were exposed to 0.1, 0.3, 0.5, 0.7 and 0.9 ml L of 2-phenoxyethanol. 2-Phenoxyethanol induction and recovery times were 115-595 and 29.66-179.3 s, respectively. At a concentration of 0.1 ml L, the anesthetic failed to induce deep anesthesia. The lowest effective concentration of 2-phenoxyethanol was determined to be 0.7 ml L, whereas 0.9 ml L was found to be the most effective one. Data showed that RBC, WBC, hemoglobin and hematocrit values were significantly high in some treatments compared to the control. MCV and MCH contents decreased significantly. MCHC, ALP, AST and ALT did not vary significantly. Plasma glucose and cortisol levels were significantly high compared to the control (in some doses). Moreover, Na, K, Ca, Mg and Cl did not vary significantly. Findings suggest that blood parameters were affected by anesthetizing bighead with 2-phenoxyethanol at 0.9 ml L to the lowest extent. [ABSTRACT FROM AUTHOR]

Structural environmental enrichment, that is, a deliberate addition of physical complexity to the rearing environment, is sometimes utilized to reduce the expression of the undesirable traits that fish develop in captivity. Increasing demands and regulations regarding usage of enrichment to promote fish welfare also make investigations on the effects of enrichment important. Here, we sythesize the current state-of-the-art knowledge about the effects of structural environmental enrichment for fish in captive environments. We find that enrichment can affect several aspects of the biology of captive fish, for example, aggression, stress, energy expenditure, injury and disease susceptibility. Importantly, these effects are often varying in direction and magnitude, and it is clear that just addition of structure is not a solution to all problems in fish rearing. Each species and life stage needs special consideration with respect to its natural history and preferences. A multitude of different enrichment types has been investigated and many studies investigate several enrichment components at the same time, making comparisons among studies difficult. To the present date, the majority of efforts have been directed to investigate salmonid fish in stock-fish hatcheries and cichlids from a basic research perspective. Some contexts are under-studied with respect to environmental enrichment, for instance effects on results in basic research and welfare effects in display aquaria. There are many research opportunities left within this field. However, future studies could utilize experimental designs which make it possible to discriminate between effects of different environmental manipulations to a higher degree than what has been performed to this date. [ABSTRACT FROM AUTHOR]

The stress responses in early growth stages of the wedge sole have been studied to determine whether the high cortisol levels described in juvenile fish are present from early developmental stages. Whole-body cortisol, glucose and lactate contents, as well as biometric parameters in wedge sole larvae were measured at three different stocking densities. Stocking density affected growth-related variables significantly, and larvae in lower stocking densities grew faster. Survival did not significantly differ among treatments. At hatching, the whole-body cortisol concentration was 0.33 ± 0.01 ng g−1 and varied significantly from 0 to 30 days after hatching ( DAH) for each stocking density, though values remained stable for the remaining time in the low-stocking density group. These hormone levels rose significantly (5.17 ± 2.43 to 22.10 ± 4.95 ng g−1) at the end of the experiment, depending directly on the stocking density. Glucose and lactate-body concentrations did not vary among treatments. We conclude that the stress responses of wedge sole larvae are detectable from 45 DAH and that stocking density already can be a stressor at that age. As described for juvenile stage, cortisol content values in wedge sole larvae under non-stressful conditions are one of the highest among those reported in the literature. The captivity conditions could be responsible for this apparently stressful situation, though those values also could be normal in wild specimens. [ABSTRACT FROM AUTHOR]

A 10-week feeding experiment was conducted to investigate the effects of dietary DHA/ EPA ratio on juvenile Japanese seabass reared in sea floating cages. Six practical diets were formulated differing only in DHA/ EPA ratio: 0.55 (Diet D/E0.55), 1.04 (D/E1.04), 1.53 (D/E1.53), 2.08 (D/E2.08), 2.44 (D/E2.44) and 2.93 (D/E2.93). All diets had the same contents of total n-3 long-chain polyunsaturated fatty acid ( LC- PUFA) and arachidonic acid ( ARA). The results showed that the final weight and specific growth rate significantly increased with increasing dietary DHA/ EPA ratio from 0.55 to 2.08 and thereafter declined. Activities of lysozyme and superoxide dismutase in serum in groups with DHA/ EPA of 1.53-2.93 was significantly higher compared to group D/E0.55 while the activity of serum alternative complement pathway in group D/E2.93 was significantly lower compared with group D/E1.53. However, no difference was observed in activities of both respiratory burst of head kidney macrophage and serum catalase among dietary treatments. The per cent survival after air exposure in group D/E1.53, D/E2.08 and D/E2.93 was significantly higher compared with group D/E0.55. The fatty acid composition of whole body and tissues reflected closely those of diets, while fish accumulated more DHA than EPA in fish tissues, especially in muscle. These results suggested that at the same dietary contents of n-3 LC- PUFA (appr. 18% of TFA) and ARA (appr. 0.7% of TFA), moderate dietary DHA/ EPA ratios of 1.53-2.08 significantly enhanced growth performances, certain innate immune responses, and the stress tolerance of Japanese seabass, in accordance with the preferential incorporation of DHA into fish tissues. [ABSTRACT FROM AUTHOR]

The EU Directive on animal experimentation suggests that all protected animals should have enrichment to improve welfare yet relatively little research has been conducted on the impact of enrichment in fish. Studies employing enrichment in zebrafish have been contradictory and all fish species should be provided with species-specific enrichments relevant to their ecology. Salmonids are important experimental models in studies within aquaculture, toxicology and natural ecosystems. This study therefore sought to establish whether an enriched environment in an experimental aquarium may promote improved welfare in rainbow trout ( Oncorhynchus mykiss ) by enhancing their recovery from invasive procedures. Trout were held individually in either barren (no tank ornamentation) or enriched (gravel, plants and an area of cover) conditions. Recovery rates after a noxious stimulus and a standard stressor were investigated by monitoring behaviour, opercular beat rate and plasma cortisol concentrations. Fish were randomly assigned to one of four treatment groups: Control (undisturbed), Sham (handled but not manipulated), Stress (air emersion) and Pain (subcutaneous injection of acetic acid). The results suggest that for rainbow trout environmental enrichment appears to promote recovery and ameliorate adverse effects following a stressor. However, recovery rate did not differ between environments in the pain treatment groups. Thus environmental enrichment may not be an important factor when the fish is responding to a painful stimulus. These results have important implications for the husbandry and welfare of captive rainbow trout and possibly other salmonids and suggest that enriched environments may be preferable to barren environments in experimental studies. [ABSTRACT FROM AUTHOR]

Stress of aquatic animals occurs due to physical and physiological disturbances in the aquatic environment or system when transportation, crowding, handling or changes of physical and chemical factors take place. There are three regulatory systems having a vital role in stress response: the neural, endocrine and immune systems. Fish exhibit multiple genomic and physiological responses to adjust the compensatory or adaptive mechanism that allows them to mitigate the stressors, maintain their haemostasis and survive. In this review, we describe multiple important genes that are associated with responses to environmental and husbandry stressors and that could be used as biomarkers of environmental and husbandry stressors in fish. The described environmental and husbandry stressors include salinity, temperature, hypoxia and hyperoxia, confinement, density and handling. The main role of stress response in aquatic animals is to compensate or adapt their biological systems and arrange the metabolism to afford the energy required by the stressor and a wide array of metabolic processes and pathways are involved. We summarized and discussed highly significant genes in several organs and tissues that are involved and active during this adaption process. The traditional stress biomarkers in some circumstances have some difficulties in interpretation of results and lead to tricky diagnosis and searching and understanding alternative tools is critical for aquaculture, fisheries and fish welfare. Using genomic tools to study the candidate genes associated with stress responses are often unique signatures or imprints of specific stressors and could determine early signs of stressors. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a class of short, evolutionary conserved non-coding RNA molecules, which are shown as the key regulators of many biological functions. External stress can alter miRNA expression levels, thereby changing the expression of mRNA target genes. Here, we show that miR-21 is involved in the regulation of alkalinity tolerance in Nile tilapia. Alkalinity stress results in a marked reduction in miR-21 levels. miR-21 loss of function could affect ion balance regulation, ROS production, and antioxidant enzyme activity in vivo. Moreover, miR-21 knockdown protects cell against alkalinity stress-induced injury in vitro. miR-21 directly regulates VEGFB and VEGFC expression by targeting the 3′-untranslated regions (UTRs) of their mRNAs, and inhibition of miR-21 significantly increases the levels of VEGFB and VEGFC expression in vivo. Taken together, our study reveals that miR-21 knockdown plays a protective role in alkalinity tolerance in tilapia. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effect of live transport of rainbow trout and subsequent live storage in an outlet aquarium on water quality, animal welfare and stress. A total of 40 fish were sampled at the following steps in the process: (1) at the raceway before transport, (2) after transport (arrival at fish outlet), (3) 24 h after fish were transferred to outlet aquarium (market), and finally, (4) after 48 h storage in the aquarium. Blood samples were collected from 10 fish at each stage and the levels of plasma cortisol, electrolytes (Na ⁺, K ⁺, Clˉ), and whole blood lactate were assessed. Twitch ability, initial pH, and temperature were assessed in white muscle. Respiration rate and swimming behavior were also assessed while the fish were kept in the outlet. Water quality (temperature, pH, dissolved oxygen, total ammonium nitrogen, total organic carbon, alkalinity and color) were assessed along with all stress measurements. Compared with fish at the farm, live transport, including effects of loading and unloading, represented the main stressor in our study. Heavy oxygen supersaturation, possibly along with elevated levels of carbon dioxide during transport contributed to this result. Despite sub-optimal water quality and excessive respiration rates, the fish in the aquarium seemed to slowly recover after transport. To reduce the effects of stress and improve animal welfare, it is suggested to minimize air exposure time and to avoid exposing the fish to supersaturated and sub-optimal levels of dissolved oxygen. Statement of relevance In a European context, selling live salmonids in the market is unusual. In this way, the highest possible freshness can be assured. However, live transport in closed systems and the live storage in fish outlets can imply risks related to compromised animal welfare and excessive stress as well as possible mortalities. In the present MS we looked into the how present handling routines and water quality affected the fish (behavior, stress and welfare). Possible improvements of present routines are discussed. We are not familiar with other published papers covering such a value chain for salmonids. [ABSTRACT FROM AUTHOR]

This two-factor experiment incorporating three photoperiods (12L:12D, 16L:8D and 24L:0D) and two feeding methods (manual and self-feeding) was carried out to determine their effect on the growth performance of striped knifejaw, Oplegnathus fasciatus . Thirty juveniles were stocked into each 300 L tanks in triplicate for each treatment and fed a commercial diet for 10 weeks. Two-way ANOVA revealed that both photoperiod and feeding method have significant effect on the final mean weight, weight gain (%) and feed efficiency (FE) (P < 0.05); however, their interaction did not produce any significant effect (P > 0.05). Although the growth parameters didn't have significant difference between manual and self-feeding in fish exposed to 12L:12D photoperiod (P > 0.05), fish under other two photoperiods showed significant improvement under self-feeding system compared to manual feeding (P < 0.05). Final mean body weights in fish under 16L:8D and 24L:0D were 119.7 and 124.0 g at manual feeding; however, increased to 128.0 and 133.7 g, respectively at self-feeding system. While two daily peaks of feed intake were observed in 12L:12D and 16L:8D photoperiods when self-feeders used, 24L:0D photoperiod showed only one morning peak. At the end, there was no significant difference in different stress indicators such as cortisol, glucose and total protein among the treatments (P > 0.05). Finally, the results suggested that the extended (16L:8D) and continuous (24 L:0D) photoperiods, and the provision of self-feeder could significantly improve the growth performance of striped knifejaw without changing the stress response. Statement of relevance This is the first report on the combined application of photoperiod manipulation and self-feeder on striped knifejaw, which revealed that the combination would help to improve the growth performance significantly. The indoor aquaculture industry, at least, will be benefited from the important insights achieved from this study to stimulate the growth performance of this commercially important species, which has great consumer demand in Japanese market. [ABSTRACT FROM AUTHOR]

There is a growing interest in the development of hapuku ( Polyprion oxygeneios ) for aquaculture in New Zealand and Australia. This is driven by the high value of this species prized for its excellent flesh quality, texture and its rapid growth capability. As a relatively new aquaculture candidate, little is currently known about their thermal tolerance and stress response. Juveniles inhabit surface waters, have a high rate of growth and move into a demersal habitat at an age between 3 and 4 years, where water temperature is cooler (7–15 °C) and more stable. The sea surface temperature in New Zealand can reach 22 °C during the summer months in more northerly locations, and captive rearing has indicated that during periods of high temperature, growth is reduced and it is possible that the physiological response is compromised. We examined the effects of two rearing temperatures (18 °C and 22 °C) and three commercial diets on the growth of P. oxygeneios during a 14 week trial. At the end of this trial, fish were exposed to a crowding stressor, and their stress response (plasma cortisol, glucose and cholesterol levels) determined. In addition, we examined the temporal stress response of P. oxygeneios acclimated to 18 °C and 22 °C subjected to a single acute handling stress. Specific growth rate and condition factor significantly increased over time in fish reared at 18 °C, but not at 22 °C. Plasma cortisol levels in hapuku prior to and after application of the stressors were within the range observed in other teleost species and the magnitude of the cortisol response was higher in hapuku subjected to crowding than handling stress. In summary, the results indicated that rearing P. oxygeneios at temperatures of 22 °C compromised their growth and that all three diets tested promoted growth in hapuku reared at 18 °C but not at 22 °C. Statement of relevance Hapuku over 1 kg had better growth rates at 18 °C than 22 °C. [ABSTRACT FROM AUTHOR]

Microcystin (MC) and nodularin are structurally similar cyanobacterial toxins that inhibit protein phosphatases. Additional modes of action are poorly known, in particular for nodularin. In our associated work, we showed that active cellular uptake is mediated by the organic anion transporting polypeptide drOatp1d1 in zebrafish ( Faltermann et al., 2016 ). Here, we assessed the transcriptional expression of three genes encoding three uptake transporters during embryonic development from 24 h post fertilization (hpf) to 168 hpf. Transcripts of drOatp1d1 and drOatp2b1 are present at 24 hpf. The abundance increased after hatching and remained about constant up to 168 hpf. Transcripts of drOatp2b1 were most abundant, while drOapt1f transcripts showed very low relative abundance compared to drOatp1d1 and drOatp2b1 . We further demonstrated the uptake of fluorescent labeled MC-LR in eleuthero-embryos and its accumulation in the glomerulus of the pronephros. An important molecular effect of MC-LR in human liver cells is the induction of endoplasmic reticulum (ER)-stress. Here, we investigated, whether MC-LR and nodularin similarly lead to induction of ER-stress in zebrafish by analyzing changes of mRNA levels of genes indicative of ER-stress. In zebrafish liver organ cultures short- and long-term exposures to 0.15 and 0.3 μmol L −1 MC-LR, and 0.5 and 1 μM L −1 nodularin led to significant transcriptional induction of several ER-stress marker genes, including the chaperone glucose regulated protein 78 ( bip ), the spliced form of x-box binding protein ( xbp-1s ), the CCAAT-enhancer-binding protein homologous protein ( chop ) and activating transcription factor 4 ( atf4 ). Furthermore, strong transcriptional changes occurred for tumor necrosis factor alpha ( tnfa ) and dual specificity phosphatase 5 ( dusp5 ), associated with mitogen activated protein kinase (MAPK) pathway. However, no alterations in transcript levels of pro-apoptotic genes Bcl-2 like protein 4 (bax) and p53 occurred. In contrast to adult liver, MC-LR and nodularin did not result in detectable changes of mRNA levels of selected target genes involved in ER-stress in zebrafish eleuthero-embryos, nor was the abundance of transcripts belonging to the MAPK and pro-apoptosis pathways altered. In conclusion, our data indicate that MC-LR and nodularin have similar transcriptional effects. They lead to changes in mRNA levels of genes that suggest induction of ER-stress, and furthermore, lead to increased level of tnfα mRNA in the adult liver, which suggests a novel (transcriptional) mode of action in fish. However, although taken up by eleuthero-embryos, no transcriptional changes induced by these cyanobacterial toxins were detected. This is probably due to action to specific organs such as liver and kidneys that could not be identified by whole-embryo sampling. [ABSTRACT FROM AUTHOR]

Estuaries are subjected to multiple anthropogenic stressors, which have additive, antagonistic or synergistic effects. Current challenges include the use of large databases of biological monitoring surveys (e.g. the European Water Framework Directive) to help environmental managers prioritizing restoration measures. This study investigated the impact of nine stressor categories on the fish ecological status derived from 90 estuaries of the North East Atlantic countries. We used a random forest model to: 1) detect the dominant stressors and their non-linear effects; 2) evaluate the ecological benefits expected from reducing pressure from stressors; and 3) investigate the interactions among stressors. Results showed that largest restoration benefits were expected when mitigating water pollution and oxygen depletion. Non-additive effects represented half of pairwise interactions among stressors, and antagonisms were the most common. Dredged sediments, flow changes and oxygen depletion were predominantly implicated in non-additive interactions, whereas the remainder stressors often showed additive impacts. The prevalence of interactive impacts reflects a complex scenario for estuaries management; hence, we proposed a step-by-step restoration scheme focusing on the mitigation of stressors providing the maximum of restoration benefits under a multi-stress context. [ABSTRACT FROM AUTHOR]

Land use changes within watersheds can have large effects on stream ecosystems, but the mechanistic basis of those effects remains poorly understood. While changes to population size presumably reflect underlying variation in organismal health and condition, such individual-level metrics are rarely evaluated in the context of ecosystem disturbance. To address this deficiency, we combined physiological sampling with geographic information systems to quantify the effects of land use on physiological indicators of health in largemouth bass. More specifically, we first quantified blood metrics relating to nutrition, oxidative stress, and the glucocorticoid stress response from largemouth bass residing in eight watersheds. We then used Akaike's information criterion to define relationships between these blood metrics and land cover, including forests, agricultural areas, urban areas, and wetlands. The proportion of forest cover in a watershed was the best predictor of blood metrics representing recent feeding and resistance to oxidative stress, whereas the proportion of wetlands was the best predictor of glucocorticoid function; however, further investigation is needed, as the explanatory power of the models was relatively low. Patterns in energy reserves were not influenced by any land use practices. Interestingly, anthropogenic land use categories, such as urban and agricultural areas, were not the best predictor for any blood metrics. Together, our results indicate that fish health is most related to natural features of a landscape rather than anthropogenic land uses. Furthermore, these findings suggest that physiological methods could supplement traditional population and community assessments to develop a more comprehensive understanding of ecosystem interactions and improve stream management. [ABSTRACT FROM AUTHOR]

A common approach used to assess environmental impacts in aquatic environments is to measure indicators of stress (biomarkers) and condition of fish within ecosystems. Particularly in estuarine ecosystems with multiple stressors, it is often desirable to quantify a suite of biological endpoints that (1) reflect fish condition at several levels of biological organization and time scales and (2) are sensitive to a range of environmental stressors. However, established methods of preservation and processing of fish for specific endpoints are often incompatible. Here, we developed a novel flash-freezing approach for assessing the health of a small, sensitive fish, the endangered Delta Smelt ( Hypomesus transpacificus ) after collections from the San Francisco Estuary (SFE). We assess whether flash-freezing the entire fish ensures effective preservation of multiple tissues for subsequent biomarker analyses by comparing measurements of fresh to frozen tissue. Tissues included brain, gill, and liver for enzyme activity, kidney and spleen for pathogens, and gills, liver, and gonads for histopathology and reproduction. Although flash-freezing in liquid nitrogen altered the length, weight, and condition factor of Delta Smelt, the percent changes were small (<1.5%). Histological analyses of the cellular morphology of gills, liver, and gonads were similar between both methods. Freezing artefacts were observed in ovaries, but did not hinder the identification and interpretation of cell types and oocyte stages. Freezing did not alter bacterial isolation or the activities of ethoxyresorufin-0-deethylase (EROD) or acetylcholinesterase (AChE), but had a small, negative influence on sodium potassium adenosine triphosphatase (ATPase) activity. Thus, flash-freezing in the field is a versatile preservation method for Delta Smelt, allowing for multiple tissue collections and bioassays from an individual tiny fish exposed to a wide range of natural and anthropogenic stressors. Similar methodology may be applicable to other species for which a range of biological endpoints and histopathology data are needed. [ABSTRACT FROM AUTHOR]

Knowledge on multi-stressors effects required for environmental and human risk assessments is still limited. This study investigated the combined effects of gold nanoparticles (Au-NP), microplastics (MP) and temperature increase on Pomatoschistus microps , an important prey for several higher level predators, including some species edible to humans. Four null hypotheses were tested: H0 1 : P. microps juveniles do not take up Au-NP through the water; H0 2 : Au-NP (ppb range) are not toxic to juveniles; H0 3 : the presence of MP do not influence the effects of Au-NP on juveniles; H0 4 : temperature increase (20–25 °C) does not change the effects of the tested chemicals on juveniles. Wild juveniles were acclimated to laboratory conditions. Then, they were exposed to Au-NP (≈5 nm diameter) and MP (polyethylene spheres, 1–5 μm diameter), alone and in mixture, at 20 °C and 25 °C, in semi-static conditions. After 96 h of exposure to Au-NP, fish had gold in their body (0.129–0.546 μg/g w.w.) leading to H0 1 refusal. Exposure to Au-NP alone caused a predatory performance decrease (≈−39%, p < 0.05) leading to H0 2 refusal. MP did not change the Au-NP toxicity leading to H0 3 acceptance. Temperature rise significantly increased the concentration of gold in fish exposed to Au-NP (≈2.3 fold), and interacted with chemical effects (e.g. glutathione S -transferases activity) leading to H0 4 refusal. Thus, the results of this study highlight the importance of further investigating the effects of multi-stressors on marine fish, particularly the effects of temperature on the uptake, biotransformation, elimination and effects of nanoparticles and microplastics, either alone or in mixture. This knowledge is most important to improve the basis for environmental and human risk assessments of these environmental contaminants of high concern. [ABSTRACT FROM AUTHOR]

We evaluate the effects of total ammonia nitrogen-N (TAN) exposure for 72 h on (Na + ,K + )- and V(H + )-ATPase activities and on their subunit expressions in gills of the diadromous freshwater shrimp Macrobrachium amazonicum . Specific (Na + ,K + )- and V(H + )-ATPase activities increased roughly 1.5- to 2-fold, respectively, after exposure to 2.0 mmol L −1 TAN. Quantitative RT-PCR analyses revealed a 2.5-fold increase in V(H + )-ATPase B subunit mRNA expression while (Na + ,K + )-ATPase α-subunit expression was unchanged. Immunohistochemical analyses of the gill lamellae located the (Na + ,K + )-ATPase throughout the intralamellar septal cells, independently of TAN concentration, while the V(H + )-ATPase was located in both the apical pillar cell flanges and pillar cell bodies. Systemic stress parameters like total hemocyte count decreased by 30% after exposure to 2.0 mmol L −1 TAN, accompanied by increased activities of the oxidative stress enzymes superoxide dismutase, glutathione reductase and glucose-6-phosphate dehydrogenase in the gills. The stress responses of M. amazonicum to elevated TAN include increases in gill (Na + ,K + )- and V(H + )-ATPase activities that are accompanied by changes in oxidative stress enzyme activities, immune system effects and an increase in gill V(H + )-ATPase gene expression. These findings likely underpin physiological effects in a crustacean like M. amazonicum that exploits multiple ecosystems during its life cycle, as well as under culture conditions that may significantly impact shrimp production by the aquaculture industry. [ABSTRACT FROM AUTHOR]

Although consistent individual-level differences in behaviour are widespread and potentially important in evolutionary and ecological processes, relatively few studies focus on the physiological mechanisms that might underlie and regulate these individual-level differences in wild populations. We conducted experiments to determine whether checkered pufferfish ( Sphoeroides testudineus ), which were collected from a dynamic (in terms of depth and water temperature) tidal mangrove creek environment in The Bahamas, have consistent individual-level differences in locomotor activity and the response to a simulated predator threat, as well as swimming performance and puffing in response to stressors. The relationships between personality and performance traits were evaluated to determine whether they represented stress-coping styles or syndromes. Subsequently, a displacement study was conducted to determine how personality and performance in the laboratory compared to movements in the field. In addition, we tested whether a physiological dose of the stress hormone cortisol would alter individual consistency in behavioural and performance traits. We found that pufferfish exhibited consistent individual differences in personality traits over time (e.g., activity and the duration of a response to a threat) and that performance was consistent between the lab and the natural enclosure. Locomotor activity and the duration of startled behaviour were not associated with swimming and puffing performance. Locomotor activity, puffing performance, and swimming performance were not related to whether fish returned to the tidal creek of capture after displacement. Similarly, a cortisol treatment did not modify behaviour or performance in the laboratory. The results reveal that consistent individual-level differences in behaviour and performance were present in a population from a fluctuating and physiologically challenging environment but that such traits are not necessarily correlated. We also determined that certain individual performance traits were repeatable between the lab and a natural enclosure. However, we found no evidence of a relationship between exogenous cortisol levels and behavioural traits or performance in these fish, which suggests that other internal and external mechanisms may underlie the behaviours and performance tested. [ABSTRACT FROM AUTHOR]

This study examined the effects of anesthesia on the hematological and biochemical parameters as well as the reproductive performance of wild female Persian sturgeon, Acipenser persicus, during controlled spawning. Fourteen mature females were divided into two groups: 'anesthetized' and 'non-anesthetized'. All activities including transportation, catheterizing and handling were performed with both groups: (i) under anesthesia (150 ppm clove oil), and (ii) without anesthesia. After 10 days storage and handling, blood samples were taken from all fish after anesthesia. No signifi- cant differences were found in the reproductive performance of either group. However, differences were found in the hematological parameters, with values being significantly higher in the non-anesthetized group, including neutrophils (34.36 ±6 .33% vs 23.63 ± 5.22%), monocytes (2.84 ± 1.70% vs 1.27 ± 0.64%), mean corpuscular volume (321.3 ± 39.40 pg vs 228.0 ± 24.46 pg) and mean corpuscular hemoglobin (106.9 ± 15.70 fl vs 76.50 ± 7.50 fl). Significantly lower values were found in the non-anesthetized group for lymphocytes (60.68 ± 7.25% vs 73.54 ± 4.80%), Hb (4.62 ± 0.74 mg dl-1 vs 6.28 ± 1.21 mg dl-1), Hct (13.86 ± 1.76% vs 18.84 ± 3.85%), red blood cell (0.43 ± 0.05 cell mm-3 vs 0.85 ± 0.13 9 106 cell mm-3) and white blood cell (22 403 ± 2240 cell mm-3 vs 35 318 ± 084 cell mm-3). The non-anesthetized fish had significantly higher cortisol levels compared to the anesthetized group (62.33 ± 8.85 ng ml-1 vs 46.12 ± 8.07 ng ml-1). There was no difference in plasma glucose levels between groups. It is concluded that the use of clove oil as an anesthetic is suitable for handling of wild female Persian sturgeon in controlled propagation programmes. However, further research is needed to determine a standardized protocol for the safe application of anesthesia for use in sturgeons in general. [ABSTRACT FROM AUTHOR]