Vertebrate Stress Research Articles

Effects of cadmium exposure on the gill proteome of Cottus gobio: Modulatory effects of prior thermal acclimation

Temperature and trace metals are common environmental stressors, and their importance is increasing due to global climate change and anthropogenic pollution. The aim of the present study was to investigate whether acclimation to elevated temperature affects the response of the European bullhead (Cottus gobio) to subsequent cadmium (Cd) exposure by using enzymatic and proteomic approaches. Fish acclimated to 15 (standard temperature), 18 or 21°C for 28 days were exposed to 1mg Cd/L for 4 days at the respective acclimation temperature. First, exposure to Cd significantly decreased the activity of the lactate dehydrogenase (LDH) in gills of fish acclimated to 15 or 18°C. However, an acclimation to 21°C suppressed the inhibitory effect of Cd. Second, using a proteomic analysis by 2D-DIGE, we observed that thermal acclimation was the first parameter affecting the protein expression profile in gills of C. gobio, while subsequent Cd exposure seemed to attenuate this temperature effect. Moreover, our results showed opposite effects of these two environmental stressors at protein expression level. From the 52 protein spots displaying significant interaction effects of temperature and Cd exposure, a total of 28 different proteins were identified using nano LC–MS/MS and the Peptide and Protein Prophet algorithms of Scaffold software. The identified differentially expressed proteins can be categorized into diverse functional classes, related to protein turnover, folding and chaperoning, metabolic process, ion transport, cell signaling and cytoskeleton. Within a same functional class, we further reported that several proteins displayed reverse responses following sequential exposure to heat and Cd. This work provides insights into the molecular pathways potentially involved in heat acclimation process and the interactive effects of temperature and Cd stress in ectothermic vertebrates.

Physiological indices of stress in wild and captive garter snakes: Correlations, repeatability, and ecological variation

Glucocorticoids and leukocyte ratios have become the most widespread variables employed to test hypotheses regarding physiological stress in wild and captive vertebrates. Little is known, however, regarding how these two indices of stress covary in response to stressors, their repeatability within individuals, and differences in response time upon capture. Furthermore, few studies compare stress indices between captive and wild populations, to assess potential alteration of stress physiology in captivity. To address these issues, we examined corticosterone (CORT) and heterophil to lymphocyte (H:L) ratios in two ecotypes of the garter snake Thamnophis elegans. We found that CORT and H:L ratios were not correlated within individuals, and both variables showed little or no repeatability over a period of months. CORT levels, but not H:L ratios, were higher for individuals sampled after 10min from the time of capture. However, both variables showed similar patterns of ecotypic variation, and both increased over time in gravid females maintained in captivity for four months. We suggest that CORT and H:L ratios are both useful, but disparate indices of stress in this species, and may show complex relationships to each other and to ecological and anthropogenic variables.

Arginine Vasotocin Treatment Induces a Stress Response and Exerts a Potent Anorexigenic Effect in Rainbow Trout, Oncorhynchus mykiss

The peptide arginine vasotocin (AVT), homologous to mammalian arginine vasopressin, is involved in many aspects of fish physiology, such as osmoregulation, regulation of biological rhythms, reproduction, metabolism or responses to stress, and the modulation of social behaviours. Because a decrease in appetite is a general response to stress in fish and other vertebrates, we investigated the role of AVT as a possible food intake regulator in fish. We used i.c.v. injections for central administration of AVT to rainbow trout (Oncorhynchus mykiss). In a first experiment, we evaluated the temporal response of food intake after AVT treatment. In a second experiment, we investigated the effects of central AVT administration on the response of typical stress markers (plasma cortisol, glucose and lactate), as well as brain serotonergic, noradrenergic and dopaminergic activity. In addition, the mRNA levels of genes involved in food intake regulation [neuropetide Y, pro-opiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART) and corticotrophin-releasing factor (CRF)] and in CRF- (CRF-binding protein) and AVT-signalling (pro-VT and AVT receptor), were also assessed after AVT treatment. Our results showed that AVT is a potent anorexigenic factor in fish. Increases of plasma cortisol and glucose after AVT treatment strongly suggest that AVT administration induced a stress response and that AVT action was mediated by hypothalamic-pituitary-interrenal axis activation, which was also supported by the increase of the serotonergic activity in trout telencephalon and hypothalamus. The increased hypothalamic levels of POMC and CART suggest that these peptides might have a role in the anorexigenic action of AVT, whereas the involvement of CRF signalling is unclear.

β‐Naphthoflavone induces oxidative stress in the intertidal copepod, Tigriopus japonicus

ABSTRACTβ‐Naphtoflavone (β‐NF) is a flavonoid and enhances oxidative stress in vertebrates with little information from aquatic invertebrates as yet. In this study, we investigated the effects of β‐NF on the antioxidant defense systems of the intertidal copepod Tigriopus japonicus. To measure the β‐NF‐triggered changes in oxidative stress markers, such as intracellular reactive oxygen species (ROS), glutathione (GSH) concentration, residual glutathione S‐transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), and superoxide dismutase (SOD) activity, T. japonicus were exposed to β‐NF (0.5 and 1 mg/L) for 72 h. Significant (P < 0.05) induction of the intracellular ROS content (%) was observed in 1 mg/L of β‐NF exposed T. japonicus, compared to the negative control and H2O2‐exposed group. The GSH levels were significantly increased in the 0.5 mg/L of β‐NF‐exposed group for 12 h and 1 mg/L of β‐NF‐exposed groups for 12–24 h. GPx, GST, and GR activities showed a significant increase in the 1 mg/L β‐NF‐exposed group, indicating that β‐NF induces oxidative stress in T. japonicus. To understand the effects of β‐NF at the level of transcript expression, a 6K microarray analysis was employed. Transcript profiles of selected antioxidant‐related genes were modulated after 72 h exposure to 1 mg/L of β‐NF. From microarray data, 10 GST isoforms, GR, GPx, PH‐GPx, and Se‐GPx were chosen for a time‐course test by real‐time RT‐PCR. T. japonicus GST‐S, GST‐O, GST‐M, and GST‐D1 were significantly increased in a 1 mg/L β‐NF‐exposed group. T. japonicus GPx, GR, and Se‐GPx mRNA levels were also significantly increased at both concentrations. Our results revealed that oxidative stress was induced by β‐NF exposure in T. japonicus. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 332–342, 2015.

Signaling stress? An analysis of phaeomelanin-based plumage color and individual corticosterone levels at two temporal scales in North American barn swallows, Hirundo rustica erythrogaster

Sexually selected traits confer greater reproductive benefits to individuals with more elaborate forms of the signal. However, whether these signals convey reliable information about the physiology underlying trait development remains unknown in many species. The steroid hormone corticosterone (CORT) mediates important physiological and behavioral processes during the vertebrate stress response, and CORT secretion itself can be modulated by melanocortins. Thus, sexually selected melanin-based plumage coloration could function as an honest signal of an individual's ability to respond to stressors. This hypothesis was tested in North American barn swallows, Hirundo rustica erythrogaster, where males with darker ventral plumage color exhibit higher phaeomelanin content and are more successful at reproduction. Because reproductive behavior occurs months after plumage signals are developed, we also addressed the potential temporal disconnect of physiological state during trait development and trait advertisement by analyzing three different measurements of CORT levels in adult males during the breeding season (trait advertisement) and in nestling males while they were growing their feathers (trait development). Variation in adult plumage color did not predict baseline or stress-induced CORT, or stress responsiveness. Likewise, there was no relationship between nestling plumage color and any of the CORT measurements, but heavier nestlings had significantly lower baseline CORT. Our finding that a predominantly phaeomelanin-based trait is unrelated to circulating CORT suggests that phaeomelanin and eumelanin signals may convey different physiological information, and highlights the need for further study on the biochemical links between the hypothalamic–pituitary–adrenal (HPA) axis and the production of different melanin-based pigments.

Chronically raised glucocorticoids reduce innate immune function in Belding’s ground squirrels (Urocitellus beldingi) after an immune challenge

The hypothalamic–pituitary–adrenal (HPA) axis releases glucocorticoids (GCs), or stress hormones, during the vertebrate stress response. GCs can both enhance and suppress the immune system depending on whether the experienced stressor is acute or chronic and what aspect of immune function is measured. More research is needed to fully understand how the immune system reacts to stressors. In this study, we examined the effects of chronically raised GCs on innate immune function in Belding’s ground squirrels (Urocitellus beldingi). We measured immune function with a bacteria killing ability (BKA) assay, an integrative and functional assessment of an animal’s ability to clear a bacterial infection. All studies to date have examined how acute stressors or repeated social stressors impact BKA. This study is the first to our knowledge to investigate how chronically raised GCs impact BKA both before and after an immune challenge. We noninvasively raised GCs in treatment squirrels for six days and then gave them, and a group of untreated (control) squirrels, an injection of lipopolysaccharide (LPS) to stimulate their innate immune system. Treatment squirrels exhibited lower BKA after, but not before, being challenged with LPS. These results suggest that experiencing chronic stress may not be detrimental to immune functioning until an individual is challenged with an infection.

Constraints, concerns and considerations about the necessity of estimating free glucocorticoid concentrations for field endocrine studies

Summary We evaluate the utility of measuring corticosteroid‐binding globulins (CBG) and estimating free glucocorticoid (GC) concentrations for field endocrine studies. We assert that for three general reasons, measurement of free GCs might not be more useful than total GCs for many studies. First, estimates of so‐called ‘free’ GCs are likely inaccurate, in part because of the following: (i) other factors in the blood also bind GCs, (ii) CBG binds plasma steroids other than GCs and (iii) CBG binding affinity can vary with local conditions, such as enzymatic activity and tissue temperature. Second, evidence suggests an active role for CBG‐bound GCs, CBG or both, in the vertebrate stress response, calling into question the validity and generality of the free hormone hypothesis. Third, free and total GCs function over different time frames. Free GCs are likely important in the seconds‐to‐minutes time‐scale of interaction with tissues, but total GCs could function at minutes‐to‐hours time‐scales by serving as the reservoir to continue supplying GCs to tissues. As transcription regulators, most GC effects manifest in hours; thus, total GCs would be the most appropriate measure for estimating total biological impact. Our understanding of the biochemistry and the biological actions of both GCs and CBG indicates that total GC concentrations are currently less prone to error and more biologically interpretable than estimates of free hormone. Although further work is necessary, total GC titres currently remain the most accurate and informative estimates of stress hormone levels to address biological questions in nature.

The ecology of stress: a marriage of disciplines

The ecology of stress: a marriage of disciplines

Central corticotropin releasing factor and social stress

Social interactions are a main source of stress in vertebrates. Social stressors, as well as other stressors, activate the hypothalamic–pituitary–adrenal (HPA) axis resulting in glucocorticoid release. One of the main components of the HPA axis is corticotropin releasing factor (CRF). The neuropeptide CRF is part of a peptide family including CRF, urocortin 1–3, urotensin 1–3, and sauvagine. The actions of the CRF family are mediated by at least two different receptors with different anatomical distribution and affinities for the peptides. The CRF peptides affect several behavioral and physiological responses to stress including aggression, feeding, and locomotor activity. This review will summarize recent research in vertebrates concerning how social stress interacts with components of the CRF system. Consideration will be taken to the different models used for social stress ranging from social isolation, dyadic interactions, to group dominance hierarchies. Further, the temporal effect of social stressor from acute, intermittent, to chronic will be considered. Finally, strains selected for specific behavior or physiology linked to social stress will also be discussed.

The response of brain serotonergic and dopaminergic systems to an acute stressor in rainbow trout: a time-course study

The brain monoaminergic neurotransmitter systems are known to be involved in the integrated response to stress in vertebrates. However, present knowledge about the timing of their actions as well as their specific roles in the regulation of the endocrine axes that drive the stress response is incomplete. This is partly because of the complexity of the reciprocal interactions among the monoaminergic systems and other biochemical effectors of the stress response such as corticotropin-releasing factor (CRF), arginine vasotocin (AVT), adrenocorticotropic hormone (ACTH) and corticosteroids. In this study, we show for the first time in teleost fish (rainbow trout) the short- and mid-term time course of the response of the forebrain serotonergic and dopaminergic activities after exposure to an acute stressor. Other stress markers like the plasma levels of cortisol, glucose and lactate were also monitored, providing a context in which to precisely locate the monoaminergic activation within the fish acute stress response. Our results show that acute stress induced a rapid increase in forebrain serotonergic activity, which became elevated after only 15 s of chasing. Several hours after stress, serotonergic activity recovered its basal levels, in parallel with the recovery of other stress markers such as plasma catecholamines and cortisol. Dopaminergic activity was also increased after stress, but only in the telencephalon and only after 20 min. The increase in serotonergic activity happened before the elevation of plasma catecholamines, suggesting that this monoamine system could have a key role in triggering the initial steps of the activation of not only the hypothalamus-pituitary-inter-renal axis but also the brain-sympathetic-chromaffin axis in fish.

Stress-induced tradeoffs in a free-living lizard across a variable landscape: consequences for individuals and populations.

Current life history theory suggests that the allocation of energetic resources between competing physiological needs should be dictated by an individual’s longevity and pace of life. One key physiological pathway likely to contribute to the partitioning of resources is the vertebrate stress response. By increasing circulating glucocorticoids the stress response can exert a suite of physiological effects, such as altering immune function. We investigated the effects of stress physiology on individual immunity, reproduction and oxidative stress, across an urban landscape. We sampled populations in and around St. George, Utah, examining corticosterone in response to restraint stress, two innate immune measures, reproductive output, and the presence of both reactive oxygen metabolites and antioxidant binding capacity, in populations of common side-blotched lizards (Uta stansburiana) experiencing variable levels of environmental stress. Additionally, using capture-mark-recapture techniques, we examined the relationships between these physiological parameters and population-level differences. Our results reveal elevated physiological stress corresponds with suppressed immunity and increased oxidative stress. Interestingly, urban populations experiencing the most physiological stress also exhibited greater reproductive output and decreased survival relative to rural populations experiencing less physiological stress, demonstrating a tradeoff between reproduction and life maintenance processes. Our results suggest that environmental stress may augment life history strategy in this fast-paced species, and that shifts in life history strategy can in turn affect the population at large. Finally, the urban environment poses definite challenges for organisms, and while it appears that side-blotched lizards are adjusting physiologically, it is unknown what fitness costs these physiological adjustments accrue.

Characterization of genes involved in ceramide metabolism in the Pacific oyster (Crassostrea gigas)

BackgroundThe lipid signaling molecule, ceramide, is a key component of the vertebrate stress response, however, there is limited information concerning its role in invertebrate species. In order to identify genes involved in ceramide metabolism in bivalve molluscs, Pacific oyster genomic resources were examined for genes associated with ceramide metabolism and signaling.ResultsSeveral genes were identified including full-length sequences characterized for serine palmitoyltransferase-1, 3-ketodihydrosphingosine reductase, acid ceramidase, and ceramide glucosyltransferase. Genes involved in ceramide synthesis and metabolism are conserved across taxa in both form and function. Expression analysis as assessed by quantitative PCR indicated all genes were expressed at high levels in gill tissue. The role of the ceramide pathway genes in the invertebrate stress response was also explored by measuring expression levels in adult oysters exposed to Vibrio vulnificus. Two genes demonstrated increased expression during the bacterial challenge: a gene involved in hydrolytic breakdown of ceramide (acid ceramidase) and a gene involved in de novo generation of ceramide (3-ketodihydrosphingosine reductase), suggesting a possible role of ceramide in the invertebrate stress and immune responses.ConclusionsIn silico and laboratory results support that Pacific oysters have the basic components of the ceramide metabolism pathway. These results also indicate that ceramide may have analogous functions in vertebrates and invertebrates. The gene expression pattern of acid ceramidase and 3-kethodihydrosphingosine reductase in response to bacterial exposure especially supports that ceramide and sphingolipid metabolism may be involved in the oyster’s stress and/or immune responses.

SEB‐3, a CRF receptor‐like GPCR, regulates locomotor activity states, stress responses and ethanol tolerance in Caenorhabditiselegans

The CRF (corticotropin-releasing factor) system is a key mediator of the stress response. Alterations in CRF signaling have been implicated in drug craving and ethanol consumption. The development of negative reinforcement via activation of brain stress systems has been proposed as a mechanism that contributes to alcohol dependence. Here, we isolated a gain-of-function allele of seb-3, a CRF receptor-like GPCR in Caenorhabditis elegans, providing an in vivo model of a constitutively activated stress system. We also characterized a loss-of-function allele of seb-3 and showed that SEB-3 positively regulates a stress response that leads to an enhanced active state of locomotion, behavioral arousal and tremor. SEB-3 also contributed to acute tolerance to ethanol and to the development of tremor during ethanol withdrawal. Furthermore, we found that a specific CRF(1) receptor antagonist reduced acute functional tolerance to ethanol in mice. These findings demonstrate functional conservation of the CRF system in responses to stress and ethanol in vertebrates and invertebrates.

Maternal adversity and ecological stressors in natural populations: the role of stress axis programming in individuals, with implications for populations and communities

Summary Biomedical researchers have long appreciated that maternal stressors can induce preparative and adaptive programming in offspring via exposure to maternal Glucocorticoids (GCs). However, few ecologists are aware of the capacity for maternal GC exposure to translate ecological and environmental stressors into preparative and adaptive programmed offspring responses in free‐living systems. We review a growing body of experimental work indicating that circulating maternal GCs link ecological stressors with adaptive programming of the stress axis. Throughout, we emphasise that natural and human‐induced ecological stressors play a fundamental role in programming the capacity of individuals, populations and communities to respond to both predictable and unpredictable ecological change via translating maternal adversity into responsive programming of the vertebrate stress axis. To encourage rigorous testing of this paradigm in a broad range of ecological systems, we introduce the principal extrinsic stressors with a recognised potential to alter maternal circulating GC levels. We then review from the biomedical literature regarding the underlying physiological and epigenetic mechanisms of stress‐induced programming of individual phenotypes to predict how variation in ecological stressors can produce individual variation in stress axis management. To appreciate the potential evolutionary inertia (i.e. adaptive value) of maternally programmed individual variation, we review key recent studies in free‐living systems that test its adaptive function, and then discuss how variation in stress‐axis programming may scale up to influence populations and ecological communities. Given the huge potential of this field, it is encouraging that ecologists are beginning to examine how and why maternal GCs translate ecological and environmental stressors into preparative stress axis programming in free‐living systems.

Oxidative stress and detoxification biomarker responses in aquatic freshwater vertebrates exposed to microcystins and cyanobacterial biomass

Cyanobacterial blooms represent a serious threat to the aquatic environment. Among other effects, biochemical markers have been studied in aquatic vertebrates after exposures to toxic cyanobacteria. Some parameters such as protein phosphatases may serve as selective markers of exposure to microcystins, but under natural conditions, fish are exposed to complex mixtures, which affect the overall biomarker response. This review aims to provide a critical summary of biomarker responses in aquatic vertebrates (mostly fish) to toxic cyanobacteria with a special focus on detoxification and oxidative stress. Detoxification biomarkers such as glutathione (GSH) and glutathione-S-transferase (GST) showed very high variability with poor general trends. Often, stimulations and/or inhibitions and/or no effects at GSH or GST have been reported, even within a single study, depending on many variables, including time, dose, tissue, species, etc. Most of the oxidative stress biomarkers (e.g., superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase) provided more consistent responses, but only lipid peroxidation (LPO) seemed to fulfill the criteria needed for biomarkers, i.e., a sufficiently long half-life and systematic response. Indeed, reviewed papers demonstrated that toxic cyanobacteria systematically elevate levels of LPO, which indicates the important role of oxidative damage in cyanobacterial toxicity. In summary, the measurement of biochemical changes under laboratory conditions may provide information on the mode of toxic action. However, comparison of different studies is very difficult, and the practical use of detoxification or oxidative stress biomarkers as diagnostic tools or early warnings of cyanobacterial toxicity is questionable.

Baseline corticosterone is positively related to juvenile survival in a migrant passerine bird

Summary1. Glucocorticoid hormones are an integral part of the vertebrate stress response, and theoretical models argue for a link between glucocorticoid levels and individual fitness. The cort‐fitness hypothesis posits that elevated levels of baseline glucocorticoids are reflective of an individual in poor condition and with a reduced likelihood of survival. Surprisingly, this hypothesis remains virtually untested for the juvenile life‐history stage, a period that is often characterized by high mortality rates.2. To address this issue, we explored whether glucocorticoid levels just prior to fledging were related to survival during the juvenile period in the Swainson's thrush (Catharus ustulatus), a short‐lived, temperate‐breeding passerine bird. Over 2 years, we blood‐sampled nestling thrushes to quantify glucocorticoid levels and then used radio telemetry to assess whether individuals died or survived to emigrate from the study area. Finally, we measured vegetation characteristics at the nest and at locations used by individuals during the juvenile period to quantify the relative importance of habitat features and glucocorticoid levels on survival.3. Predation was the leading cause of death, and overall juvenile survival rate was 34·6%. We found that survival was positively associated with baseline corticosterone and, to a lesser extent, size‐corrected body mass and date of fledging. Contrary to expectations, we found no evidence that the amount of vegetative cover at the nest site or at locations used during the juvenile period was associated with survival.5. Although we cannot completely rule out the cort‐fitness hypothesis, our data appear to support the cort‐activity hypothesis and suggest that elevated baseline corticosterone levels in juvenile thrushes may be linked to enhanced post‐fledging survival via increased locomotor activity that promotes foraging, more effective escape from predators or both.

Interpreting indices of physiological stress in free-living vertebrates

When vertebrate physiological ecologists use the terms 'stress' or 'physiological stress', they typically mean the level of hypothalamus-pituitary-adrenal (HPA-) axis activation. Measurements of stress hormone concentrations (e.g. glucocorticoids in blood, urine or faeces), leukocytes (e.g. the neutrophil-lymphocyte ratio or heterophil equivalent), immunofunction (e.g. innate, cell-mediated or humoral immunity measures) and regenerative anaemia (e.g. mean erythrocyte volume and red blood cell distribution width) have all been used to estimate HPA-axis activity in free-living vertebrates. Stress metrics have provided insights into aspects of autecology or population regulation that could not have been easily obtained using other indices of population wellbeing, such as body condition or relative abundance. However, short- and long-term stress (often problematically termed acute and chronic stress, respectively) can interact in unpredictable ways. When animals experience trapping and handling stress before blood, faeces and/or urine is sampled, the interaction of short- and long-term stress can confound interpretation of the data, a fact not always acknowledged in studies of stress in free-living vertebrates. This review examines how stress metrics can be confounded when estimates of HPA-axis activation are collected for free-living vertebrates and outlines some approaches that can be used to help circumvent the influence of potentially confounding factors.

Regulation of the HPA axis is related to song complexity and measures of phenotypic quality in song sparrows

Regulation of the hypothalamic–pituitary–adrenal (HPA) axis is a key component of the vertebrate stress response. Prior studies have found that variation in HPA responses were correlated to measures of fitness and physiological condition. In addition, sexually-selected traits have also been found to correlate to measures of condition. The proximate mechanisms responsible for such covariation between sexually selected traits and measures of quality are unclear, but could involve variation in HPA regulation. We investigated whether HPA activity is related to song complexity, body size/condition and leukocyte profiles in wild male song sparrows (Melospiza melodia). We characterized three aspects of HPA activity: 1) response to restraint stress; 2) negative feedback, assessed by the ability of exogenous dexamethasone to suppress corticosterone levels; and 3) adrenal sensitivity to exogenous adrenocorticotropic hormone (ACTH). Birds with lower responses to restraint stress had more complex song and more heterophils and higher heterophil to lymphocyte (H:L) ratios. Birds with more effective negative feedback were larger and had fewer heterophils and lower H:L ratios, suggesting lower levels of physiological stress or infection. We observed no relationship between adrenal sensitivity to exogenous ACTH and any of the factors. These findings illustrate important relationships between HPA activity, song complexity, and morphological and physiological traits. Song complexity may thus provide receivers with information about the ability of the singer to cope with stressors.

Faecal 11-ketoetiocholanolone measurement in Iberian red deer (Cervus elaphus hispanicus): validation of methodology using HPLC–MS/MS

A cortisol metabolite, 11-ketoetiocholanolone (11-k), is widely used in monitoring stress in several vertebrates, and can be detected by immunoassay. However, these assays have certain limitations with respect to specificity. Also, differences in the excretion of faecal glucocorticoid metabolites (FGM) among species and even between sexes make validation necessary in each case. Therefore, our aims were, first, to develop and validate a high-pressure liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) methodology for monitoring 11-k in faeces of Iberian red deer (Cervus elaphus hispanicus), and second, to investigate the capability of our method to determine variations of this FGM in a longitudinal study. Third, and finally, we assessed the correspondence between faecal 11-k concentrations and plasma cortisol. An adrenocorticotropic hormone (ACTH) test was performed on six red deer stags translocated and kept in captivity for a week and faecal samples were collected twice a day. One single blood and faecal sample from another seven stags was also collected after 2 weeks in captivity. The results of the longitudinal study showed a first peak in 11-k 36 h after the ACTH test and handling, and a second peak at 120 h of being kept indoors. Maximum concentrations of 11-k ranged from 22.71 to 375.68 ng/g. In the second stag group, 11-k concentrations of 25.09 ± 20.53 ng/g had a correlation of r2 = 0.88 with the concentration of plasma cortisol, which was 54.6 ± 55.1 ng/mL. This technique is capable of detecting changes in the concentrations of faecal 11-k. The values determined have a good correlation with the cortisol concentration in blood, and we also detected differences in different individuals’ responses to the same stressors.

Food odor, visual danger stimulus, and retrieval of an aversive memory trigger heat shock protein HSP70 expression in the olfactory lobe of the crab Chasmagnathus granulatus

Although some of the neuronal substrates that support memory process have been shown in optic ganglia, the brain areas activated by memory process are still unknown in crustaceans. Heat shock proteins (HSPs) are synthesized in the CNS not only in response to traumas but also after changes in metabolic activity triggered by the processing of different types of sensory information. Indeed, the expression of citosolic/nuclear forms of HSP70 (HSC/HSP70) has been repeatedly used as a marker for increases in neural metabolic activity in several processes, including psychophysiological stress, fear conditioning, and spatial learning in vertebrates. Previously, we have shown that, in the crab Chasmagnathus, two different environmental challenges, water deprivation and heat shock, trigger a rise in the number of glomeruli of the olfactory lobes (OLs) expressing HSC/HSP70. In this study, we initially performed a morphometric analysis and identified a total of 154 glomeruli in each OL of Chasmagnathus. Here, we found that crabs exposed to food odor stimuli also showed a significant rise in the number of olfactory glomeruli expressing HSC/HSP70. In the crab Chasmagnathus, a powerful memory paradigm based on a change in its defensive strategy against a visual danger stimulus (VDS) has been extensively studied. Remarkably, the iterative presentation of a VDS caused an increase as well. This increase was triggered in animals visually stimulated using protocols that either build up a long-term memory or generate only short-term habituation. Besides, memory reactivation was sufficient to trigger the increase in HSC/HSP70 expression in the OL. Present and previous results strongly suggest that, directly or indirectly, an increase in arousal is a sufficient condition to bring about an increase in HSC/HSP70 expression in the OL of Chasmagnathus.