Stress Response In Fish Research Articles

Netting the Stress Responses in Fish.

In the last decade, the concept of animal stress has been stressed thin to accommodate the effects of short-term changes in cell and tissue physiology, major behavioral syndromes in individuals and ecological disturbances in populations. Seyle's definition of stress as “the nonspecific (common) result of any demand upon the body” now encompasses homeostasis in a broader sense, including all the hierarchical levels in a networked biological system. The heterogeneity of stress responses thus varies within individuals, and stressors become multimodal in terms of typology, source and effects, as well as the responses that each individual elicits to cope with the disturbance. In fish, the time course of changes after stress strongly depends on several factors, including the stressful experiences in early life, the vertical transmission of stressful-prone phenotypes, the degree of individual phenotypic plasticity, the robustness and variety of the epigenetic network related to environmentally induced changes, and the intrinsic behavioral responses (individuality/personality) of each individual. The hierarchical heterogeneity of stress responses demands a code that may decrypt and simplify the analysis of both proximate and evolutionary causes of a particular stress phenotype. We propose an analytical framework, the stressotope, defined as an adaptive scenario dominated by common environmental selective pressures that elicit common multilevel acute stress-induced responses and produce a measurable allostatic load in the organism. The stressotope may constitute a blueprint of embedded interactions between stress-related variations in cell states, molecular mediators and systemic networks, a map of circuits that reflect the inherited and acquired stress responses in an ever-changing, microorganismal-loaded medium. Several features of the proposed model are discussed as a starting point to pin down the maximum common stress responses across immune-neuroendocrine relevant physiological levels and scenarios, including the characterization of behavioral responses, in fish.

Variation in scale cortisol concentrations of a wild freshwater fish: Habitat quality or seasonal influences?

A significant body of literature suggests that aquatic pollutants can interfere with the physiological function of the fish hypothalamic-pituitary-interrenal (HPI) axis, and eventually impair the ability to cope with subsequent stressors. For this reason, development of accurate techniques to assess fish stress responses have become of growing interest. Fish scales have been recently recognized as a biomaterial that accumulates cortisol, hence it can be potentially used to assess chronic stress in laboratory conditions. We, therefore, aimed to evaluate the applicability of this novel method for cortisol assessment in fish within their natural environment. Catalan chub (Squalius laietanus) were sampled from two sites; a highly polluted and a less polluted (reference) site, in order to examine if habitat quality could potentially influence the cortisol deposition in scales. We also evaluated the seasonal variation in scale cortisol levels by sampling fish at three different time points during spring-summer 2014. In each sampling, blood was collected to complement the information provided by the scales. Our results demonstrated that blood and scale cortisol levels from individuals inhabiting the reference site were significantly correlated, therefore increasing the applicability of the method as a sensitive-individual measure of fish HPI axis activity, at least in non-polluted habitats. Since different environmental conditions could potentially alter the usefulness of the technique, results highlight that further validation is required to better interpret hormone fluctuations in fish scales. Scale cortisol concentrations were unaffected by habitat quality although fish from the polluted environment presented lower circulating cortisol levels. We detected a seasonal increase in scale cortisol values concurring with an energetically costly period for the species, supporting the idea that the analysis of cortisol in scales reveals changes in the HPI axis activity. Taken together, the present study suggests that cortisol levels in scales are more likely to be influenced by mid-term, intense energetically demanding periods rather than by long-term stressors. Measurement of cortisol in fish scales can open the possibility to study novel spatio-temporal contexts of interest, yet further research is required to better understand its biological relevance.

Effect of cold stress on genomic DNA methylation in zebrafish ZF4 cells

PDF HTML阅读 XML下载 导出引用 引用提醒 低温对斑马鱼ZF4细胞基因组DNA甲基化水平的影响 DOI: 作者: 作者单位: 1. 水产种质资源发掘与利用教育部重点实验室, 上海海洋大学, 上海 201306;2. 水产科学国家级实验教学示范中心, 上海海洋大学, 上海 201306;3. 海洋生物科学国际联合研究中心, 中国科学技术部, 上海海洋大学, 上海 201306 作者简介: 侯艳雯(1993-),女,硕士研究生,研究方向为分子生物学.E-mail:471708976@qq.com 通讯作者: 中图分类号: S917 基金项目: 国家自然科学基金项目（31372516，81770165）；上海市自然科学基金项目（13ZR1419500）；上海市教育委员会"东方学者"计划支持项目. Effect of cold stress on genomic DNA methylation in zebrafish ZF4 cells Author: Affiliation: 1. Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education;Shanghai Ocean University, Shanghai 201306, China;2. National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China;3. International Research Center for Marine Biosciences at Shanghai Ocean University, Ministry of Science and Tech-nology, Shanghai 201306, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:低温压力会导致鱼类生理功能失调、机体损伤甚至死亡，鱼类会产生各种适应性变化来应对低温压力，其中涉及的表观遗传学机制越来越受到重视。为了探讨鱼类低温应激压力下的表观遗传学调控机制，本研究对斑马鱼（）胚胎成纤维细胞ZF4进行短期低温胁迫（18℃处理3 d、5 d和10℃处理3 d、5 d）和长期低温胁迫（18℃处理30 d），然后用具有不同甲基化敏感性的同裂酶Ⅰ对细胞基因组DNA进行酶切以监测细胞基因组DNA甲基化水平变化。结果显示短期低温胁迫下ZF4细胞生长受到抑制甚至死亡，而经过长期低温胁迫的ZF4细胞对低温压力产生了一定适应性。并且低温胁迫下DNA甲基化水平呈现动态变化，短期低温培养细胞的基因组DNA甲基化水平明显增高，但是长期低温培养细胞的DNA甲基化水平反而下降。此外，研究发现抗氧化剂N-乙酰半胱氨酸（N-acetylcysteine，NAC）或者共济失调-毛细血管扩张突变蛋白（ataxia telangiectasia mutated，ATM）抑制剂KU-55933可以抑制18℃ 5 d低温处理后的ZF4细胞DNA甲基化水平的增高，说明活性氧（reactive oxygen species，ROS）的产生和ATM的激活介导了DNA甲基化水平的增高。本研究结果显示，短期低温刺激下ZF4细胞ROS的产生导致DNA损伤，激活DNA损伤修复机制，进而导致基因组DNA甲基化水平上升，该研究为后期斑马鱼低温胁迫分子机制的研究奠定基础。 Abstract:Cold stress causes physiological dysfunction, tissue damage, and finally death in fishes, and increasing studies have suggested that epigenetic mechanisms play essential roles in the cold stress response in fishes. Our previous study showed that cold stress induced the production of reactive oxygen species (ROS) in zebrafish -derived ZF4 cells in a temperature and time-dependent manner and that the genomic DNA methylation level was increased under short-term (18℃ for 5 days) cold exposure and decreased under long-term cold exposure (18℃ for 30 days). However, the relationship among DNA methylation, ROS production, and cold acclimation in fishes remains poorly understood. In the present study, zebrafish ZF4 cells were exposed to short-term (18℃ or 10℃, for 3 or 5 days) and long-term (18℃ for 30 days) cold stress, and then the global DNA methylation level was detected by digestion with the methylation-sensitive enzyme I. The results showed that short-term cold stress caused remarkable growth arrest and cell death in ZF4 cells, and cold acclimation was observed under long-term cold stress. Additionally, global DNA methylation increased remarkably under short-term cold stress ( < 0.05). Co-treatment of ZF4 cells with -acetylcysteine inhibited global DNA methylation induced by short-term cold stress ( < 0.05), suggesting that ROS affects short-term cold stress-induced global DNA methylation levels. Co-treatment of ZF4 cells with the ataxia telangiectasia mutated (ATM) inhibitor KU-55933 also inhibited the induction of global DNA methylation under short-term cold exposure, indicating the involvement of DNA damage repair pathways in this process. Our data indicate that short-term cold stress resulted in ROS production and ataxia telangiectasia mutated activation, which then up-regulated global DNA methylation in ZF4 cells. The present study improves our understanding of the role of DNA methylation under cold stress in fishes. 参考文献 相似文献 引证文献

Development of a novel LED color-switching type biosensor system for the visualization of fish stress responses

A stress visualization system was developed that can make a natural judgment of fish stress responses by visual observation on three different levels (green, yellow, red) depends on the concentration of glucose (stress indicator).

Xenobiotic biotransformation, oxidative stress and obesogenic molecular biomarker responses in Tilapia guineensis from Eleyele Lake, Nigeria

Despite the important ecosystem and hydrological roles of coastal and inland waters, there are no established biomonitoring protocols for evaluating environmental, wildlife and human health for these coastlines in Nigeria. In the present study, contaminants tissue burden and effects at molecular and physiological levels, were investigated in Tilapia guineensis from a man-made lake (Eleyele Lake) that is used for municipal domestic water supply and compared to a reference site (Igboho Lake). Gene expression of phase I and II biotransformation systems, oxidative stress and obesogen responses were analyzed using real-time PCR, and these results were related to general health index (condition factor: CF) and muscle burden of trace metals, aliphatic and polycyclic aromatic hydrocarbons (PAHs). We observed a significant increase in phase I and II biotransformation systems, oxidative stress and obesogen responses in male and female fish from Eleyele lake compared with the reference site. Overall, our data showed significant relationships between biological responses and tissue concentrations of metals and PAHs for the Eleyele lake compared with the reference site. Given that a positive influence on genes and pathways associated with metabolic status has been previously associated with peroxisome proliferator activated receptors (PPARs), xenobiotic compounds that activate PPARs may produce changes in energy and metabolic processes, leading to obesity. The high CF (>1 = good health condition) observed, coupled with the high muscle burden of inorganic and organic contaminants in fish from Eleyele lake, suggest a potential obesogenic effect in these fishes. These findings represent co-relational evidence that the Eleyele lake is contaminated and consequently affecting biological and physiological integrity of organisms inhabiting the lake. These findings also suggest potential health risks for humans, since the lake is extensively used for domestic water supply and fisheries.

Habituation and individual variation in the endocrine stress response in the Trinidadian guppy (Poecilia reticulata)

The vertebrate stress response enables individuals to react to and cope with environmental challenges. A crucial aspect of the stress response is the elevation of circulating glucocorticoids. However, continued activation of the stress response under repeated exposure to stressors can be damaging to fitness. Under certain circumstances it may therefore be adaptive to habituate to repeated exposures to a particular stressor by reducing the magnitude of any associated release of glucocorticoids. Here, we investigate whether Trinidadian guppies (Poecilia reticulata) habituate to repeated exposure to a mild stressor, using a waterborne hormone sampling approach that has previously been shown to elicit a stress response in small fish. We also test for individual variation in the extent of habituation to this stressor. Concentrating on freely circulating cortisol, we found that the first exposure to the assay induced high cortisol release rates but that guppies tended to habituate quickly to subsequent exposures. There were consistent differences among individuals in their average cortisol release rate (after accounting for effects of variables such as body size) over repeated exposures. Our analyses did not find evidence of individual differences in habituation rate, although limitations in statistical power could account for this finding. We repeated the analysis for free 11-ketotestosterone, which can also respond to stressors, but found no obvious habituation pattern and no among-individual variation. We also present data on conjugated forms of both hormones, which were repeatable but did not show the expected time-lagged habituation effect. We discuss consistent individual differences around the general pattern of habituation in the flexible stress response, and highlight the potential for individual variation in habituation to facilitate selection against the deleterious effects of chronic stress.

Real-time fish stress visualization came true：A novel multi-stage color-switching wireless biosensor system

An optical communication type biosensor system has been developed which can measure blood glucose concentration, which is a stress indicator of fish, in real-time while fish swimming freely. However, this system is hard to make instant acknowledgment of fish stress level which has to contain an unavoidable delay in the judgment. In this research, we aimed to develop a novel stress visualization system which can quickly judge the levels for fish stress response instantly based on a color changeable LED while another LED was designed to send data. The present system is based on the principle of converting the output current value measured by the glucose biosensor corresponding to the stress response into a voltage value. Then, the color and stress switching points of the LED (Red, Yellow, Green) were decided based on the voltage value gained from the biosensor which mentioned above. Furthermore, we attempted to use our biosensor system to make real-time monitoring of fish stress in vivo. As results, the proposed sensor can make real-time measurement of glucose and shows a great response to those of actual fish sample in the range from 35.36 to 300 mg dl−1 (R = 0.9899). When the glucose concentration in the collected sample was switched to the concentration pre-sett, it was successful to switch the LED color according to the gained voltage value both in vitro and in vivo. Furthermore, when monitoring the stress responses of the fish in vivo, color switching corresponding to the sensor output current value was observed successfully.

Stress up-regulates oxidative burst in juvenile Chinook salmon leukocytes

When fish perceive stressful scenarios, their hypothalamus-pituitary-interrenal axis is activated resulting in the release of corticotropin releasing hormone, adrenocorticotropic hormone (ACTH), and finally cortisol. The physiologic stress response of fish has most often been linked to the reduced performance of the immune system, with a few exceptions where the immune system is activated. In this report, we tested the hypothesis that oxidative burst activity levels in juvenile Chinook salmon (Oncorhynchus tshawytscha) are altered when the fish is presented with a stressor. Fish were subjected to a stressor for 3 h and then allowed to recover for 20 h following the stressor. Plasma and spleens were collected from euthanized fish before the stressor, at the end of a 3 h stressor, and 23 h after the start of the experiment. Plasma was held at −80 °C until cortisol radioimmunoassay analysis was performed to confirm stress. Spleens were held in Dulbecco's Modified Eagle Medium overnight and analyzed the day following collection. Oxidative burst activity was measured in splenic leukocytes after being stimulated with phorbol 12-myristate 13-acetate. We found a significant increase in activated oxidative burst from fish subjected to the stressor as compared to unstressed fish. Speculation is given to ACTH being the leukocyte priming agent in this experiment rather than the cortisol itself.

Transcriptome analysis demonstrates that long noncoding RNA is involved in the hypoxic response in Larimichthys crocea.

The large yellow croaker (Larimichthys crocea) has low hypoxia tolerance compared with other fish species, and the mRNA levels of hypoxia-inducible factor (HIF)-1α in its brain do not change markedly under hypoxic conditions. In this study, we investigated noncoding transcription in the hypoxic response mechanism of L. crocea. We generated a catalog of long noncoding RNAs (lncRNAs) from the brain of L. crocea individuals under hypoxic stress, investigated lncRNA expression patterns, and analyzed the HIF signaling pathway by RNA sequencing. Prolyl hydroxylase domain 2 (PHD2) expression significantly increased after 6 and 12h of hypoxia, and a lncRNA (Linc_06633.1) was found in the upstream, antisense region of PHD2. Linc_06633.1 may be an important regulator that promotes PDH2 expression under hypoxia in L. crocea, and we constructed a regulatory profile of L. crocea under hypoxic conditions. To the best of our knowledge, it is the first study that has been conducted on hypoxia signaling pathway regulation by lncRNAs in L. crocea and elucidates the role played by lncRNAs in the regulation of the hypoxia stress response in teleost fish.

Chronic exposure to increased water temperature reveals few impacts on stress physiology and growth responses in juvenile Atlantic salmon

Fish are subjected to a variety of stressors under common cage aquaculture conditions. While short-term exposure to a stressor often results in an adaptive response to cope with stress, repeated and/or chronic exposure to stress can result in negative impacts on fish welfare and production. In fish, little is known about the impact of long-term exposure to stressors, including elevated water temperature. In this study we examined and developed temporal response profiles of physiological indicators of stress and growth in juvenile Atlantic salmon (Salmo salar) exposed to 12 °C, 16 °C, and 20 °C for 99 days. Five times throughout the study we quantified plasma cortisol, glucose and cholesterol levels, and growth. Fish body mass and fork length were not significantly different amongst temperatures after 99 days. Plasma cortisol was significantly elevated at 16 °C when comparing day 8 with 99, while at 12 °C plasma cortisol increased from day 1 to day 8, then returned to initial levels (day 1 and 8) after 99 days. Plasma glucose and cholesterol were not significantly different amongst the temperatures throughout the experiment. In addition, at the end of the experiment we quantified eye darkening, and identified the development of a bimodal growth distribution in all temperatures. Fish with a fork length ≤ 240 mm were categorised as lower mode (LM) and those with a fork length > 240 mm as in the upper mode (UM) of growth. Plasma cholesterol was significantly lower in the LM group in all three temperatures, but plasma cortisol and glucose levels did not differ between modes. Eye darkening also did not differ between modes, but increased significantly in the groups exposed to 16 °C and 20 °C when compared with 12 °C. This study showed a clear physiological stress response (elevated levels of cortisol) and eye darkening in fish maintained at 16 °C but not at 12 °C or 20 °C, suggesting that some aspects of the physiological responses available to deal with chronic stress are affected by temperature.

Short-term exposure to repeated chasing stress does not induce habituation in Senegalese sole, Solea senegalensis

Animals can habituate to certain repeated stressors and reduce the physiological response that such stressor evoked initially. Studies related to stress habituation in fish are scarce and the available data differ depending on the species and on the type, duration and severity of the stressor. The main objective of this study was to investigate the stress response of juvenile Senegalese sole (Solea senegalensis) submitted to repeated chasing stress for 3days previous to the experiment in order to evaluate the occurrence of habituation to those stress conditions in this fish species. Thus, five different experimental groups were evaluated: not stressed fish (control, C), fish stressed only on the experimental day (ST/naïve), and fish stressed on the experimental day and on the 3 previous days: during the day (ST/Dt), at night (ST/Nt) or both (ST/Dt+Nt). Parameters related to primary and secondary responses to chasing were evaluated in plasma, liver and brain. Chasing in ST/naïve group induced incremented values of plasma cortisol, glucose and lactate but no changes in catecholamine levels compared to controls. In trained fish, higher cortisol but decreased glucose, lactate and catecholamine levels were observed after stress compared to controls and to ST/naïve groups. In the liver, stress did not induce any changes with respect to controls whereas ST/Dt and ST/Dt+Nt showed lower values of glucose and glycogen than stressed naïve fish. In the brain, ST/naïve group presented no significant changes in serotonergic activity. However, incremented serotonergic activity was detected in fish previously trained. Furthermore, dopaminergic activity decreased in diurnal trained and nocturnal trained groups with respect to ST/naïve fish. Crh expression in hypothalamus was higher in ST/naïve fish but not in fish submitted to repeated stress compared to controls. In summary, it seems that there was no habituation to the repeated acute stress protocol in Solea senegalensis in terms of serotonergic activity and cortisol release during the physiological stress response. However, the decreased levels of plasma catecholamines and energy metabolites, and of the hypothalamic crh mRNA abundance and dopaminergic activity, indicate a modulation of the stress response in trained fish. Altogether, the results suggest that either the chasing stressor was too strong or the training period too short for the animals to habituate, indicating that repeated chasing within short periods should be avoided when manipulating fish in order to keep proper welfare conditions in this species.

ACOUSTIC NOISE POLLUTION FROM MARINE INDUSTRIAL ACTIVITIES: EXPOSURE AND IMPACTS

The improvements in marine technological developments propagate urbanization in the ocean environment. The construction or operational activities of marine structures such as energy plants, oil platforms, pipe-lines, sea-tunnel passages, or cable-stayed suspension bridges, and vessel traffic are sources of underwater noise pollution. How underwater sounds such as piling, pole drilling, or machinery noises may affect the marine live is mostly ignored in marine construction, and there is lack of information regarding underwater sound effects on marine live in the oceans. Recently, a remarkable interest is developing concerning underwater sound effects, especially in aquaculture facilities, with experimentation of musical stimuli or various noises caused by pumps or filter systems on behavior and stress responses of fish in culture conditions. With the increase of urbanization and progressive development of marine industries, more and more pressure from human-generated (anthropogenic) underwater sound pollution may threaten marine mammals, fish species and invertebrates from underwater noises that in terms might be called as “Underwater Noise Pollution”. The future of marine life and that of human being, and the dramatic increase of underwater sound pollution is a new debate that needs to be controlled in a sustainable way with environment-sound approaches. Therefore the potential effects of various sound sources derived from marine industrial activities have been reviewed in this study.

Use of anaesthetic agents in farmed fish

Anaesthetic agents are routinely used in farmed fish to permit the performance of painful procedures, ease handling and reduce stress during grading, tagging, transport, blood sampling, artificial breeding and vaccination of fish. An ideal anaesthetic for fish should induce anaesthesia rapidly, allow a rapid recovery, be safe to both fish and user, leave low tissue residues and be inexpensive and easy to use. A variety of factors, including species, size, body weight, gill surface area to body weight ratio, lipid content, sex, sexual maturity, physical condition and health state of the fish, as well as temperature, pH, salinity and oxygen and mineral content of the water may affect the anaesthetic process in fish. The most commonly used anaesthetics are tricaine, benzocaine, phenoxyethanol, quinaldine, quinate, clove oil and metomidate. Tricaine is the only anaesthetic licensed for farmed fish use in the USA. It is easy to use and safe, but also expensive. Benzocaine is cheaper, but poorly water-soluble and has to be prepared in either ethanol or acetone. Phenoxyethanol is considered by most fish farmers as the anaesthetic of choice for farmed fish, because of its easy preparation, low price, rapid action and bactericidal and fungicidal properties. Quin aldine and quinate are effective and easy to use anaesthetic agents, however, a number of adverse effects have been reported to both fish and users. The active ingredients of clove oil are eugenol and isoeugenol and have been introduced only recently as fish anaesthetics. Apart from being inexpensive and easy to use, clove oil has also bactericidal and fungicidal properties. Metomidate may have a certain advantage as it probably evokes a less severe stress response in fish than the other anaesthetic agents.

Periodic bacterial control with peracetic acid in a recirculating aquaculture system and its long-term beneficial effect on fish health

Fish in a recirculating aquaculture system (RAS) live with abundant microorganisms. These can become a health threat when the fish immune system cannot counterbalance the pathogenic microbial colonization. Therefore, microbial control in a RAS can potentially reduce the risk of infections and hence improve fish health. In the present study, a periodic microbial control was performed in a RAS with 16 tanks stocked with mirror carp (Cyprinus carpio) for 3months. Half of the fish culture tanks were treated with 1mgL−1 peracetic acid (PAA) twice per week, while the other half remained untreated. The water circulation was interrupted immediately before each PAA-treatment, and resumed after 3h. The total aerobic bacterial density was similar in all culture tanks, except during the PAA-treatments and the concurrent circulation interruptions. During these periods, the bacterial density decreased up to 90% in PAA-treated water, while a 6-fold bacterial increase was observed in untreated water. In the first 2months of treatment, PAA-exposed fish showed lower plasma cortisol concentration than the unexposed fish. Subsequently, the trunk kidney leukocytes of PAA-exposed fish showed stronger respiratory burst than the unexposed fish. By the end of the experiment, the PAA-exposed fish had better gill morphology, compared to the unexposed fish. The present study indicates that periodic disinfection of culture water in a RAS with PAA could transiently reduce the suspended bacteria density, modulate the fish stress response, and have an overall beneficial effect on fish health in the long term.

Impacts of wastewater treatment plant effluent on energetics and stress response of rainbow darter (Etheostoma caeruleum) in the Grand River watershed.

The objective of this study was to assess the effects of municipal wastewater treatment plant effluent on the energetics and stress response of rainbow darter (Etheostoma caeruleum). Male and female rainbow darter were collected upstream and downstream of the Waterloo WWTP in the Grand River watershed, ON, Canada. To assess the effects of wastewater treatment plant effluent on whole-body and tissue specific metabolic capacity, closed-chamber respirometry and muscle-enzyme activity analyses were performed. Plasma cortisol was also collected from fish before and after an acute air-exposure stressor to evaluate the cortisol stress response in fish exposed to additional stressors. Male and female rainbow darter collected downstream of the effluent had higher oxygen consumption rates, while differences in enzyme activities were primarily associated with sex rather than collection site. No impairment in the cortisol stress response between downstream and upstream fish was observed, however baseline cortisol levels in female fish from the downstream site were significantly higher compared to other baseline groups. Stress-induced cortisol levels were also higher in female fish from both sites when compared to their male counterparts. Overall, this study demonstrates that chronic exposure to WWTP effluent impacts whole-body metabolic performance. This study was also able to demonstrate that sex-differences are a key determinant of various metabolic changes in response to physiological stress, thereby, providing a novel avenue to be considered and further explored.

High environmental ammonia exposure has developmental-stage specific and long-term consequences on the cortisol stress response in zebrafish

The capacity for early life environmental stressors to induce programming effects on the endocrine stress response in fish is largely unknown. In this study we determined the effects of high environmental ammonia (HEA) exposure on the stress response in larval zebrafish, assessed the tolerance of embryonic and larval stages to HEA, and evaluated whether early life HEA exposure has long-term consequences on the cortisol response to a novel stressor. Exposure to 500–2000μM NH4Cl for 16h did not affect the gene expression of corticotropin-releasing factor (CRF) system components in 1day post-fertilization (dpf) embryos, but differentially increased crfa, crfb and CRF binding protein (crfbp) expression and stimulated both dose- and time-dependent increases in the whole body cortisol of 5dpf larvae. Pre-acclimation to HEA at 1dpf did not affect the cortisol response to a subsequent NH4Cl exposure at 5dpf. In contrast, pre-acclimation to HEA at 5dpf caused a small but significant reduction in the cortisol response to a second NH4Cl exposure at 10dpf. While continuous exposure to 500–2000μM NH4Cl between 0 and 5dpf had a modest effect on mean survival time, exposure to 400–1000μM NH4Cl between 10 and 14dpf decreased mean survival time in a dose-dependent manner. Moreover, pre-acclimation to HEA at 5dpf significantly decreased the risk of mortality to continuous NH4Cl exposure between 10 and 14dpf. Finally, while HEA at 1dpf did not affect the cortisol stress response to a novel vortex stressor at 5dpf, the same HEA treatment at 5dpf abolished vortex stressor-induced increases in whole body cortisol at 10 and 60dpf. Together these results show that the impact of HEA on the cortisol stress response during development is life-stage specific and closely linked to ammonia tolerance. Further, we demonstrate that HEA exposure at the larval stage can have persistent effects on the capacity to respond to stressors in later life.

Stress responses to conspecific visual cues of predation risk in zebrafish.

Chemical communication relating to predation risk is a trait common among fish species. Prey fish under threat of predation can signal risk to conspecific fish, which then exhibit defensive responses. Fish also assess predation risk by visual cues and change their behavior accordingly. Here, we explored whether these behavioral changes act as visual alarm signals to conspecific fish that are not initially under risk. We show that shoals of zebrafish (Danio rerio) visually exposed to a predator display antipredator behaviors. In addition, these defensive maneuvers trigger antipredator reactions in conspecifics and, concomitantly, stimulate the hypothalamus-pituitary-interrenal axis, leading to cortisol increase. Thus, we conclude that zebrafish defensive behaviors act as visual alarm cues that induce antipredator and stress response in conspecific fish.

The impact of acute PAH exposure on the toadfish glucocorticoid stress response

The objective of the present study was to determine whether the polycyclic aromatic hydrocarbons (PAHs) associated with the Deepwater Horizon (DWH) oil spill impacted the stress response of teleost fish. The hypothesis was that intraperitoneal (IP) treatment with PAHs associated with the DWH oil spill or waterborne exposure to DWH oil high energy water-accommodated fraction (HEWAF) would result in the downregulation of the stress response of Gulf toadfish, Opsanus beta, a benthic marine teleost fish that resides in the Gulf of Mexico. In vivo plasma cortisol levels and adrenocorticotropic hormone (ACTH)-mediated cortisol secretion by in vitro isolated kidney tissue were measured. Toadfish at rest IP-treated with naphthalene had higher plasma cortisol compared to fluorene-treated and control fish; phenanthrene-treated fish tended to have higher plasma cortisol levels that fluorene-treated and controls. When subjected to an additional crowding stress, naphthalene and phenanthrene-treated fish were no longer able to mount a stress response compared to fluorene-treated and control fish, suggesting exhaustion of the stress response. Supporting this in vivo data, there tended to be less cortisol released by the kidney in vitro from naphthalene and phenanthrene-treated fish in response to ACTH compared to controls. In contrast, toadfish at rest exposed to 3% Slick A HEWAF did not have significantly different plasma cortisol levels compared to controls. But, exposed fish did have significantly less cortisol released by the kidney in vitro in response to ACTH. When toadfish were subjected to an additional stress, there were no significant differences in plasma cortisol or ACTH, suggesting the action of a secondary secretagogue to maintain plasma cortisol in vivo. Combined, these data suggest that in response to acute PAH exposure, there may be internalization or downregulation of the melanocortin 2 receptor (MC2R) that mediates the action of ACTH.

Effects of subchronic exposure to waterborne cadmium on H-P-I axis hormones and related genes in rare minnows (Gobiocypris rarus)

The H (hypothalamic)-P (pituitary)-I (interrenal) axis is critical in the stress response and other activities of fish. To further investigate cadmium (Cd) toxicity on the H-P-I axis and to identify its potential regulatory genes in fish, the adult female rare minnows (Gobiocypris rarus) were exposed to subchronic (5weeks) levels of waterborne Cd in the present study. This kind of treatment caused dose-dependent decline in fish growth, with significance in the high dose group (100μg/L). Correspondingly, low dose (5–50μg/L) waterborne Cd disrupted the endocrine system of H-P-I axis just at the secretion level, while high dose Cd disrupted both the secretion and synthesis of cortisol and its downstream signals in rare minnows, revealed by the significantly upregulation and positive correlation of corticosteroidogenic genes including MC2R, StAR, CYP11A1, and CYP11B1 in the kidney (including the interrenal tissue) (P<0.05), and the significant alteration of Glcci1, Hsp90AA and Hsp90AB in the hepatopancreas, gill and intestine as well (P<0.05). The expression of Glcci1 was significantly decreased in hepatopancreas, gill and intestine of tested fish following treatment, and its positive correlation with GR (Glucocorticoid receptor) suggested its potential regulation on the cortisol and/or H-P-I axis in fish. The expression of FKBP5 in the intestine was positively and significantly correlated with that of Hsp90AA (P<0.05), and the Hsp90AB transcript in the hepatopancreas was positively correlated with that of Hsp90AA (P<0.05), which indicated that Hsp90AA and Hsp90AB were more likely to serve as cofactors of GR and FKBP5 in response to Cd exposure.

In vitro study on the regulation of cellular mRNA levels by changes in transcription rate and transcript stability in fish red blood cells.

The interplay of transcriptional and post-transcriptional processes in the regulation of gene expression has been extensively studied in mammals but little is known in other vertebrates so far. Most non-mammalian vertebrates are faced with environmental cues and stressors distinct from those experienced by mammals and thus it is likely that the gene expression strategies differ from those of mammals. Here we performed experiments to study in vitro the various levels of gene expression regulation in nucleated fish red blood cells. Three critical environmental cues frequently experienced by fish were chosen: exposure to hypoxia (2.5%), ambient water temperature increase by 10°C (from 10°C to 20°C), and exposure to stress hormones (represented by the β-adrenergic agonist isoproterenol). We found that β-adrenergic stimulation increases the stability of the β3b-adrenergic receptor (β3b-ar) mRNA, suggesting that mRNA stability can play a role in the regulation of hormonal stress responses in fish. The β3b-ar gene encodes a unique β-adrenergic receptor subtype in fish red blood cells which controls the β-Na+/H+ exchanger activity - an important component of responses to oxygen limitations. Our results furthermore show a yet undescribed link between the Hif1a signaling pathway and the β-adrenergic receptor response. After β-adrenergic stimulation, the transcription of hif1a was activated significantly after 4 hours of exposure. So far, such a response has only been described from mammalian species. This indicates that the β-AR is fundamental to the molecular and physiological responses to hypoxia and that Hif1a might have additional functions than those already known.