Changes in heat stress tolerance in a freshwater amphipod following starvation: The role of oxygen availability, metabolic rate, heat shock proteins and energy reserves.

The ability of organisms to cope with environmental stressors depends on the duration and intensity of the stressor, as well as the type of stress. For aquatic organisms, oxygen limitation has been implicated in limiting heat tolerance. Here we examine how starvation affects heat tolerance in the amphipod Gammarus fossarum (Koch, 1836) and whether observed changes can be explained from alterations in oxidative metabolism, depletion of energy reserves, upregulation of heat shock proteins or susceptibility to oxygen limitation. Starved amphipods showed impaired survival compared to fed amphipods during prolonged exposure to mild heat. In contrast, under acute, high-intensity heat exposure they actually showed improved survival. We observed a lower demand for oxygen in starved amphipods which could make them less susceptible to oxygen limitation. Such a role for oxygen in limiting heat tolerance was verified as hypoxia impaired the heat tolerance of amphipods, especially starved ones. Fed amphipods likely rely more on anaerobic metabolism to maintain energy status during heat stress, whereas for starved amphipods aerobic metabolism appears to be more important. The depletion of their energy reserves constrains their ability to maintain energy status via anaerobic metabolism. We did not find evidence that alterations in heat tolerance following starvation were related to the upregulation of heat shock proteins. In conclusion, starvation can have opposite effects on heat tolerance, acting via pathways that are operating on different time scales.

Effects of Pro-Tex on zebrafish (Danio rerio) larvae, adult common carp (Cyprinus carpio) and adult yellowtail kingfish (Seriola lalandi).

Aquaculture practices bring several stressful events to fish. Stressors not only activate the hypothalamus-pituitary-interrenal-axis, but also evoke cellular stress responses. Up-regulation of heat shock proteins (HSPs) is among the best studied mechanisms of the cellular stress response. An extract of the prickly pear cactus (Opuntia ficus indica), Pro-Tex, a soluble variant of TEX-OE(®), may induce expression of HSPs and reduce negative effects of cellular stress. Pro-Tex therefore is used to ameliorate conditions during stressful aquaculture-related practices. We tested Pro-Tex in zebrafish (Danio rerio), common carp (Cyprinus carpio L.) and yellowtail kingfish (Seriola lalandi) exposed to aquaculture-relevant stressors (thermal stress, net confinement, transport) and assessed its effects on stress physiology. Heat shock produced a mild increase in hsp70 mRNA expression in 5-day-old zebrafish larvae. Pro-Tex increased basal hsp70 mRNA expression, but decreased heat-shock-induced expression of hsp70 mRNA. In carp, Pro-Tex increased plasma cortisol and glucose levels, while it did not affect the mild stress response (increased plasma cortisol and glucose) to net confinement. In gills, and proximal and distal intestine, stress increased hsp70 mRNA expression; in the distal intestine, an additive enhancement of hsp70 mRNA expression by Pro-Tex was seen under stress. In yellowtail kingfish, Pro-Tex reduced the negative physiological effects of transport more efficiently than when fish were sedated with AQUI-S(®). Overall, our data indicate that Pro-Tex has protective effects under high levels of stress only. As Pro-Tex has potential for use in aquaculture, its functioning and impact on health and welfare of fish should be further studied.

Stress in African catfish (Clarias gariepinus) following overland transportation.

Of the many stressors in aquaculture, transportation of fish has remained poorly studied. The objective of this study was therefore to assess the effects of a (simulated) commercial transportation on stress physiology of market-size African catfish (Clarias gariepinus). Catfish weighing approximately 1.25 kg were returned to the farm after 3 h of truck-transportation, and stress-related parameters were measured for up to 72 h following return. Recovery from transportation was assessed through blood samples measuring plasma cortisol, glucose and non-esterified fatty acids (NEFA) and gill histology. Also, the number of skin lesions was compared before and after transport. Pre-transport handling and sorting elevated plasma cortisol levels compared to unhandled animals (before fasting). Plasma cortisol levels were further increased due to transportation. In control fish, plasma cortisol levels returned to baseline values within 6 h, whereas it took 48 h to reach baseline values in transported catfish. Plasma glucose and NEFA levels remained stable and were similar across all groups. Transported catfish did not, on average, have more skin lesions than the handling group, but the number of skin lesions had increased compared to unhandled animals. The macroscopic condition of the gills was similar in control, transported and unhandled catfish; however, light microscopy and immunohistochemistry revealed atypical morphology and chloride cell migration normally associated with adverse water conditions. From our data, we conclude that transportation may be considered a strong stressor to catfish that may add to other stressors and thus inflict upon the welfare of the fish.

Sex-dependent and differential responses to acute restraint stress of corticotropin-releasing factor-producing neurons in the rat paraventricular nucleus, central amygdala, and bed nucleus of the stria terminalis.

Male and female rodents respond differently to acute stress. We tested our hypothesis that this sex difference is based on differences in stress sensitivity of forebrain areas, by determining possible effects of a single acute psychogenic stressor (1-hr restraint stress) on neuronal gene expression (c-Fos and FosB immunoreactivities), storage of corticotropin-releasing factor (CRF) immunoreactivity, and CRF production (CRF mRNA in situ hybridization) as well as the expression of genes associated with epigenetic processes (quantitative RT-PCR) in the rat paraventricular nucleus (PVN), the oval and fusiform subdivisions of the bed nucleus of the stria terminalis (BSTov and BSTfu, respectively), and the central amygdala (CeA), in both males and females. Compared with females, male rats responded to the stressor with a stronger rise in corticosterone titer and a stronger increase in neuronal contents of c-Fos, CRF mRNA, and CREB-binding protein mRNA in the PVN. In the BSTov, females but not males showed an increase in c-Fos, whereas the CRF mRNA content was increased in males only. In the BSTfu, males and females showed similar stress-induced increases in c-Fos and FosB, whereas in the CeA, both sexes revealed similar increases in c-Fos and in CRF mRNA. We conclude that male and female rats differ in their reactivity to acute stress with respect to possibly epigenetically mediated (particularly in the PVN) neuronal gene expression and neuropeptide dynamics (PVN and BSTov) and that this difference may contribute to the sex dependence of the animal's physiological and behavioral responses to an acute stressor.

Chronic stress induces sex-specific alterations in methylation and expression of corticotropin-releasing factor gene in the rat.

BACKGROUND: Although the higher prevalence of depression in women than in men is well known, the neuronal basis of this sex difference is largely elusive. METHODS: Male and female rats were exposed to chronic variable mild stress (CVMS) after which immediate early gene products, corticotropin-releasing factor (CRF) mRNA and peptide, various epigenetic-associated enzymes and DNA methylation of the Crf gene were determined in the hypothalamic paraventricular nucleus (PVN), oval (BSTov) and fusiform (BSTfu) parts of the bed nucleus of the stria terminalis, and central amygdala (CeA). RESULTS: CVMS induced site-specific changes in Crf gene methylation in all brain centers studied in female rats and in the male BST and CeA, whereas the histone acetyltransferase, CREB-binding protein was increased in the female BST and the histone-deacetylase-5 decreased in the male CeA. These changes were accompanied by an increased amount of c-Fos in the PVN, BSTfu and CeA in males, and of FosB in the PVN of both sexes and in the male BSTov and BSTfu. In the PVN, CVMS increased CRF mRNA in males and CRF peptide decreased in females. CONCLUSIONS: The data confirm our hypothesis that chronic stress affects gene expression and CRF transcriptional, translational and secretory activities in the PVN, BSTov, BSTfu and CeA, in a brain center-specific and sex-specific manner. Brain region-specific and sex-specific changes in epigenetic activity and neuronal activation may play, too, an important role in the sex specificity of the stress response and the susceptibility to depression.