Appetite suppression is a common response to hypoxia in fish that confers significant energy savings. Yet little is known about the endocrine signals involved in the regulation of food intake during ...chronic hypoxia. Thus, we assessed the impact of chronic hypoxia on food intake, the expression of the potent anorexigenic signal leptin and its receptor (lepr), the mRNA levels of key hypothalamic appetite-regulating genes, and the activity of the hypothalamic-pituitary-interrenal (HPI) axis in common carp, Cyprinus carpio. Fish exposed to 10% O(2) saturation for 8 days were chronically anorexic and consumed on average 79% less food than normoxic controls. Hypoxia also elicited gradual and parallel increases in the expression of liver leptin-a-I, leptin-a-II, lepr and erythropoietin, a known hypoxia-responsive gene. In contrast, the liver mRNA levels of all four genes remained unchanged in normoxic fish pair-fed to the hypoxia treatment. In the hypothalamus, expression of the appetite-regulating genes were consistent with an inhibition and stimulation of hunger in the hypoxic and pair-fed fish, respectively, and reduced feed intake led to a decrease in lepr. Although both treatments elicited similar delayed increases in plasma cortisol, they were characterized by distinct HPI axis effector transcript levels and a marked differential increase in pituitary lepr expression. Together, these results show that a reduction in O(2) availability, and not feed intake, stimulates liver leptin-a expression in common carp and suggest that this pleiotropic cytokine is involved in the regulation of appetite and the endocrine stress response during chronic hypoxia.
To gain a better understanding of the mechanisms by which cortisol suppresses growth during chronic stress in fish, we characterized the effects of chronic cortisol on food intake, mass gain, the ...expression of appetite-regulating factors, and the activity of the GH/IGF axis. Fish given osmotic pumps that maintained plasma cortisol levels at ∼70 or 116 ng/ml for 34 days were sampled 14, 28 and 42 days post-implantation. Relative to shams, the cortisol treatments reduced food intake by 40-60% and elicited marked increases in liver leptin (lep-a1) and brain preoptic area (POA) corticotropin-releasing factor (crf) mRNA levels. The cortisol treatments also elicited 40-80% reductions in mass gain associated with increases in pituitary gh, liver gh receptor (ghr), liver igfI and igf binding protein (igfbp)-1 and -2 mRNA levels, reduced plasma GH and no change in plasma IGF1. During recovery, while plasma GH and pituitary gh, liver ghr and igfI gene expression did not differ between treatments, the high cortisol-treated fish had lower plasma IGF1 and elevated liver igfbp1 mRNA levels. Finally, the cortisol-treated fish had higher plasma glucose levels, reduced liver glycogen and lipid reserves, and muscle lipid content. Thus, our findings suggest that the growth-suppressing effects of chronic cortisol in rainbow trout result from reduced food intake mediated at least in part by increases in liver lep-a1 and POA crf mRNA, from sustained increases in hepatic igfbp1 expression that reduce the growth-promoting actions of the GH/IGF axis, and from a mobilization of energy reserves.
A characteristic feature of the behavioural response to intensely acute or chronic stressors is a reduction in appetite. In fish, as in other vertebrates, the corticotropin-releasing factor (CRF) ...system plays a key role in coordinating the neuroendocrine, autonomic, and behavioural responses to stress. The following review documents the evidence implicating the CRF system as a mediator of the appetite-suppressing effects of stress in fish. Central injections of CRF or the related peptide, urotensin I (UI), or pharmacological treatments or stressors that result in an increase in forebrain CRF and UI gene expression, can elicit dose-dependent reductions in food intake that are at least partially reversed by pre-treatment with a CRF receptor antagonist. In addition, the appetite suppressing effects of various environmental, pathological, physical, and social stressors are associated with elevated levels of forebrain CRF and UI gene expression and with an activation of the hypothalamic–pituitary–interrenal (HPI) stress axis. In contrast, although stressors can also be associated with an increase in caudal neurosecretory system CRF and UI gene expression and an endocrine role for CRF-related peptides has been suggested, the physiological effects of peripheral CRF-related peptides on the gastrointestinal system and in the regulation of appetite have not been investigated. Overall, while CRF and UI appear to participate in the stress-induced changes in feeding behaviour in fish, the role of other know components of the CRF system is not known. Moreover, the extent to which the anorexigenic effects of CRF-related peptides are mediated through the hypothalamic feeding center, the HPI axis and cortisol, or via actions on descending autonomic pathways remains to be investigated.
Fishes respond to different abiotic and biotic stressors through changes in gene expression as a part of an integrated physiological response. Transcriptomics approaches have been used to quantify ...gene expression patterns as a reductionist approach to understand responses to environmental stressors in animal physiology and have become more commonly used to study wild fishes. We argue that non-lethal sampling for transcriptomics should become the norm for assessing the physiological status of wild fishes, especially when there are conservation implications. Processes at the level of the transcriptome provide a “snapshot” of the cellular conditions at a given time; however, by using a non-lethal sampling protocol, researchers can connect the transcriptome profile with fitness-relevant ecological endpoints such as reproduction, movement patterns and survival. Furthermore, telemetry is a widely used approach in fisheries to understand movement patterns in the wild, and when combined with transcriptional profiling, provides arguably the most powerful use of non-lethal sampling for transcriptomics in wild fishes. In this review, we discuss the different tissues that can be successfully incorporated into non-lethal sampling strategies, which is particularly useful in the context of the emerging field of conservation transcriptomics. We briefly describe different methods for transcriptional profiling in fishes from high-throughput qPCR to whole transcriptome approaches. Further, we discuss strategies and the limitations of using transcriptomics for non-lethally studying fishes. Lastly, as ‘omics’ technology continues to advance, transcriptomics paired with different omics approaches to study wild fishes will provide insight into the factors that regulate phenotypic variation and the physiological responses to changing environmental conditions in the future.
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•The transcriptome response is part of an integrated organismal response to stress•Non-lethal sampling can link transcriptomic responses with fitness level endpoints•Tissues that can be sampled non-lethally are not always the ideal tissue for a specific stressor•Transcriptomics approaches are valuable tools in fish conservation•Pairing transcriptomics with other omics approaches will enhance our understanding of phenotypic variation in wild fishes
Abstract
Harmful algal blooms (HABs) release toxic compounds in water and are increasing in frequency worldwide. The neurotoxin β-methylamino-l-alanine (BMAA) is released by HABs and has garnered ...much attention over the past 20 years due to its association with human neurodegenerative disorders, but its effects on wildlife are still largely unknown. This study characterized the effects of chronic exposure to environmentally relevant concentrations of BMAA on the behavior and brain size of developing zebrafish (Danio rerio). Zebrafish were continuously exposed to 0, 1, 10, or 100 µg/l waterborne BMAA between 0- and 5-days postfertilization (dpf) before the onset of exogenous feeding. At 5 dpf, locomotion and responses to vibrational and visual stimuli were assessed. Following behavioral testing, larvae body and brain size were measured. Survival between 0 and 5 dpf did not differ between treatments. Moreover, BMAA exposure did not affect thigmotaxis, startle response magnitude, habituation to repeated presentation of vibrational startling stimuli, or relative brain size. A moderate increase in overall activity was observed in larvae exposed to 10 μg/l BMAA under light, but this effect was not seen in dark conditions, indicating that visual processing may have been affected by chronic BMAA exposure. Thus, passive continuous exposure to environmentally relevant concentrations of BMAA prior to first feeding in zebrafish did not affect survival or selected measures used to represent brain development, anxiety, and motor reflexes, but a limited light-dependent effect on locomotion suggests targeted neurotoxicity within the visual system.
Fish nursery habitats are increasingly hypoxic and the brain is recognized as highly hypoxia sensitive, yet there is a lack of information on the effects of hypoxia on the development and function of ...the larval fish brain. Here, we tested the hypothesis that by inhibiting brain development, larval exposure to severe hypoxia has persistent functional effects on the cortisol stress response in zebrafish (Danio rerio). Exposing 5 days post-fertilization (dpf) larvae to 10% dissolved O2 (DO) for 16 h only marginally reduced survival, but it decreased forebrain neural proliferation by 55%, and reduced the expression of neurod1, gfap and mbpa, markers of determined neurons, glia and oligodendrocytes, respectively. The 5 dpf hypoxic exposure also elicited transient increases in whole-body cortisol and in crf, uts1 and hsd20b2 expression, key regulators of the endocrine stress response. Hypoxia exposure at 5 dpf also inhibited the cortisol stress response to hypoxia in 10 dpf larvae and increased hypoxia tolerance. However, 10% DO exposure at 5 dpf for 16 h did not affect the cortisol stress response to a novel stressor in 10 dpf larvae or the cortisol stress response to hypoxia in adult fish. Therefore, while larval exposure to severe hypoxia can inhibit brain development, it also increases hypoxia tolerance. These effects may transiently reduce the impact of hypoxia on the cortisol stress response but not its functional capacity to respond to novel stressors. We conclude that the larval cortisol stress response in zebrafish has a high capacity to cope with severe hypoxia-induced neurogenic impairment.
In recent years, natural and anthropogenic factors have increased aquatic hypoxia the world over. In most organisms, the cellular response to hypoxia is mediated by the master regulator ...hypoxia-inducible factor-1 (HIF-1). HIF-1 also plays a critical role in the normal development of the cardiovascular system of vertebrates. We tested the hypothesis that hypoxia exposures which resulted in HIF-1 induction during embryogenesis would be associated with enhanced hypoxia tolerance in subsequent developmental stages. We exposed zebrafish (Danio rerio) embryos to just 4 h of severe hypoxia or total anoxia at 18, 24 and 36 h post-fertilization (hpf). Of these, exposure to hypoxia at 24 and 36 hpf as well as anoxia at 36 hpf activated the HIF-1 cellular pathway. Zebrafish embryos that acutely upregulated the HIF-1 pathway had an increased hypoxia tolerance as larvae. The critical window for hypoxia sensitivity and HIF-1 signalling was 24 hpf. Adult male fish had a lower critical oxygen tension (Pcrit) compared with females. Early induction of HIF-1 correlated directly with an increased proportion of males in the population. We conclude that mounting a HIF-1 response during embryogenesis is associated with long-term impacts on the phenotype of later stages which could influence both individual hypoxia tolerance and population dynamics.
We describe duplicate leptin genes in zebrafish (Danio rerio) that share merely 24% amino acid identity with each other and only 18% with human leptin. We were also able to retrieve a second leptin ...gene in medaka (Oryzias latipes). The presence of duplicate leptin genes in these two distantly related teleosts suggests that duplicate leptin genes are a common feature of teleostean fishes. Despite low primary sequence conservation, we are confident in assigning orthology between mammalian and zebrafish leptins for several reasons. First, both zebrafish leptins share their characteristic gene structure and display key features of conserved synteny with mammalian leptin genes. Secondly, the cysteine residues that make up leptin's single disulphide bridge are equally spaced in mammalian and zebrafish leptins and are unique among all members of the class-I helical cytokine family. Thirdly, the zebrafish leptins cluster with other fish leptins and mammalian leptins in phylogenetic analysis, supported by high bootstrap values. Within the leptin cluster, leptin-b forms a separate clade with the leptin-b orthologue from medaka. Finally, our prediction of the tertiary structures shows that both leptins conform to the typical four alpha-helix bundle structure of the class-I alpha-helical cytokines. The zebrafish leptins are differentially expressed; the liver shows high leptin-a expression (in concordance with what we observed for carp leptins), while leptin-b is expressed at much lower levels, which are downregulated further upon fasting. The finding of duplicate leptin genes in teleosts adds to our understanding of the evolution of leptin physiology in the early vertebrate lineage.
The maternal match hypothesis predicts that maternal exposure to a stressor may help prepare offspring to cope with the same disturbance in later life. Although there is support for this hypothesis, ...the signals involved in non-genetic inheritance are unclear. In this study, we tested how adult zebrafish exposure to diel cycles of thermal stress (27-36°C), hypoxia (20-85% dissolved oxygen) or the combined treatment affects maternal and embryonic levels of cortisol and heat shock proteins (HSPs). While parental exposure to the thermal, hypoxic or combined treatment for 2 weeks did not affect whole-body cortisol levels, the combined exposure increased ovarian cortisol levels by 4-fold and reduced embryonic cortisol content by 60%. The combined treatment also elicited 3- and 19-fold increases in embryo transcripts involved in cortisol breakdown (11bhsd2) and export (abcb4), respectively. The thermal stress and combined exposure also elicited marked increases in ovary and embryo hsp70a (20- to 45-fold) and HSP70 (3- to 7-fold), and smaller increases in ovary and embryo hsp90aa and hsp47 (2- to 4-fold) and in embryo HSP90 and HSP47 (2- to 6-fold). In contrast, except for increases in ovary hsp90aa (2-fold) and embryo HSP90 (3-fold), the hypoxia treatment had little effect on HSP expression and transfer. Overall, while the embryonic deposition of HSPs largely paralleled the ovarian cellular stress response, the inverse relationship between ovary and embryo cortisol levels suggests the existence of barriers against cortisol deposition in response to environmental stressors. We conclude that the endocrine and cellular stress responses make stressor-specific and distinct contributions to non-genetic inheritance.
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