General and Comparative Endocrinology

The corticotropin-releasing factor (CRF) system bidirectionally interacts with cytokines and other immune-related components in mammals. However, the nature of these interactions remains poorly characterized in other vertebrates, including teleost fishes. To gain insight into the relationship between immune responses and the CRF system in teleosts, we explored how CRF system components were transcriptionally regulated in immune organs of rainbow trout (Oncorhynchus mykiss). We first characterized the CRF system in the spleen and head kidney--two primary immune organs in teleosts--and found that many CRF system components were present in both tissues, but splenic expression was consistently greater. Changes in the abundance of splenic CRF system components following vaccination (which transiently stimulated inflammatory responses and cytokine production) indicated contrasting and time-dependent regulation of CRF receptor 1 (CRFR1; suppression) and CRFR2 (stimulation) activities in response to an inflammatory challenge. Using spleen explant cultures, we then evaluated whether these effects were mediated by either of nuclear factor kappa B (NF-{kappa}B; a pro-inflammatory transcription factor) or cortisol (an anti-inflammatory hormone). At baseline, cultured spleens increased cytokine production and exhibited transcriptional changes in CRF system components comparable to those observed following vaccination. Cortisol treatment and NF-{kappa}B inhibition both attenuated the rise in cytokine transcription; however, cortisol treatment generally affected transcripts influencing CRFR1 activity, while NF-{kappa}B inhibition reduced CRFR2 activity. Overall, our data provide novel insight into CRF system regulation in the spleen and suggest that cortisol and inflammatory cytokines differentially regulate CRFR1 and CRFR2 activity within this organ.

The yellow-bellied marmot (Marmota flaviventer) study at the Rocky Mountain Biological Laboratory near Crested Butte, Colorado, USA is the worlds second longest study of free-living mammals. Quantifying physiological stress is essential for understanding their health, reproductive success, and survival in a variable environment. Historically, we used a validated radioimmunoassay (RIA) to measure fecal glucocorticoid metabolites (FGMs). Given the costs and risks of working with radioisotopes, we have shifted to a more sustainable method. Here we evaluate the suitability of two competitive enzyme-linked immunosorbent assays (ELISA) from Cayman Chemical Company (CCC) and Arbor Assays (AA) to measure corticosterone levels in FGMs. The findings revealed that the AA ELISA, unlike the CCC ELISA, consistently matched the RIA in terms of accuracy across high and low corticosterone concentrations, demonstrated superior assay parameters, showed the highest correlations with RIA results and effectively captured the annual variations in FGM concentrations, indicative of its reliability for use in longitudinal studies. We further analytically validated the usage of the AA ELISA for FGMs, confirming its efficacy without matrix effects, thus establishing its suitability for ongoing and future studies of FGMs in marmots. The transition to the AA ELISA from the RIA ensures continued data integrity while enhancing safety and environmental sustainability.

The corticotropin-releasing factor (CRF) system is a major neural regulator of stress responses in vertebrates. However, stress-related roles for the CRF system in other tissues--and whether these roles vary between stressor types--remain unclear. To address this gap, we first characterized the CRF system in the gills and intestine of rainbow trout (Oncorhynchus mykiss) and then evaluated how it is transcriptionally regulated following either an immune (vaccination) or osmotic (seawater transfer) stressor. Additionally, since the CRF system is involved in food intake regulation, we also evaluated whether feeding state affects the intestinal CRF system. Vaccination against Vibrio anguillarum reduced CRF system activity in the intestine--as indicated by elevations in CRF binding protein transcripts paired with reductions in ligand (crfa2) and receptor (crfr1b) transcripts--but did not affect the gill CRF system. In contrast, seawater transfer caused the abundance of most CRF system transcripts to increase in the middle (but not posterior) portion of the intestine, while transcript levels of CRF binding proteins and receptors in the gills declined. Finally, levels of CRF system transcripts in the intestine varied with feeding state in a region-specific manner. In the middle intestine, transcript levels of most components declined with fasting and increased when feeding was resumed, whereas the opposite pattern occurred in the posterior intestine. Overall, our results implicate the peripheral CRF system as a stressor- and epithelial tissue-specific modulator of immune and osmoregulatory functions in teleosts.

A general tenet in stress physiology is that the hypothalamic-pituitary-adrenal (HPA) axis predominantly produces one glucocorticoid (GC) in response to stressors. However, most vertebrates produce both cortisol and corticosterone, and these steroids show variation across species in absolute levels, relative proportions, and stress responsivity and regulate much more than just stress physiology. Therefore, focusing on a single GC within a species may not capture the whole story. In the present study, we set out to validate non-invasive waterborne hormone measurements in our focal species, the dyeing poison frog Dendrobates tinctorius. In pursuing this goal, we uncovered unexpected patterns of GC abundance within and across species of poison frogs. D. tinctorius had higher amounts of corticosterone than cortisol in both plasma and waterborne samples, and corticosterone was responsive to adrenocorticotropic hormone (ACTH) as canonically assumed. However, corticosterone and cortisol levels were surprisingly similar in D. tinctorius, and cortisol was more abundant than corticosterone in water samples from four additional poison frog species. These results challenge the broadly accepted assumption that corticosterone is always more abundant in amphibians and add to the growing literature highlighting the importance of measuring both GCs to understand (stress) physiology.

Adipokinetic hormone/corazonin-related peptide (ACP) and adipokinetic hormone (AKH) are two neuropeptides that demonstrate homology to the vertebrate gonadotropin-releasing hormone (GnRH). Despite the structural similarity and the close evolutionary relationship between the ACP and the AKH, their signaling systems function independently. To date, the role of ACP and its receptor (ACPR) remains unclear in the Aedes aegypti mosquito. Structure-activity relationships (SARs) are often carried out on peptide ligands to determine critical residues for bioactivity and receptor activation; however, residues and features necessary for ligand binding and specificity in the receptors themselves are less studied. Herein, this study focuses on the ACP and AKH signaling systems and examines structural features of their receptors critical for conferring activation and ligand specificity. Firstly, to determine the specific ACPR regions most critical for ligand fidelity and specificity, ACPR chimeras were created by singly replacing each of the three ACPR extracellular loops (ECLs) in their entirety and incorporating the corresponding ECLs from the AKH receptor (AKHR). Heterologous functional assays determined that the three ACPR ECL chimera receptors with complete replacement of full individual ECLs showed no response to either ACP or AKH. These results suggest that the complete replacement of each individual extracellular loop is detrimental to ligand binding and recognition. Secondly, through a more targeted approach, we aimed to determine specific residues critical for functional ligand-binding by substituting only select highly conserved residues within the three ECLs of the ACPR with those from the AKHR. Modifications of specific and highly conserved residues in these ACPR ECLs chimeras suggest that the third extracellular loop contains the most critical residues necessary for ACP binding and receptor activation. In addition, the combination of two selectively-modified ACPR ECLs demonstrated a significant decrease in response to ACP compared to the native ACPR response. Interestingly, combining all of the ACPR ECLs chimeras together resulted in a significant decrease in response to ACP compared to native ACPR. Relatedly, a significantly increased response to AKH was observed in the receptor chimera combining selected modifications in all three ECLs compared to native ACPR. Hence, the particular residues essential for ACP ligand interaction were identified due to the detrimental effect that occurred in ACPR activation after the selective modification of crucial residues localized within the three extracellular domains of the receptor. These data provide key insight into how these two closely related neuropeptidergic systems maintain functional independence in the mosquito A. aegypti as well as other insects.

Anuran metamorphosis is characterized by profound morphological changes including remodeling of tissues and organs. This transition is initiated by thyroid hormones (THs). However, the current knowledge of changing levels of THs during metamorphosis relies on pooled samples using methods known for high variability with sparse reporting of measured variation. Moreover, establishing a clear linkage between key gene expression bioindicators and TH levels throughout the metamorphic process is needed. Using state-of-the-art ultra-high performance liquid chromatography isotope-dilution tandem mass spectrometry, we targeted 12 THs and metabolites in the serum of Rana [Lithobates] catesbeiana (n=5-10) across seven distinct postembryonic stages beginning with premetamorphic tadpoles (Gosner stage 31-33) and continuing through metamorphosis to a juvenile frog (Gosner stage 46). TH levels were related to TH-relevant gene transcripts (thra, thrb, and thibz) in back skin of the same individual animals. Significant increases from basal levels were observed for thyroxine (T4) and 3,3,5-triiodothyronine (T3) at Gosner stage 41, reaching maximal levels at Gosner stage 44 (28{+/-}10 and 2.3{+/-}0.5 ng/mL, respectively), and decreasing to basal levels in juvenile frogs. In contrast, 3,5-diiodothyronine (T2) increased significantly at Gosner stage 40 and was maintained elevated until stage 44. While thra transcript levels remained constant and then decreased at the end of metamorphic climax, thrb and thibz were induced to maximal levels at Gosner stage 41, followed by a decrease to basal levels in the froglet. This exemplifies the exquisite timing of events during metamorphosis as classic early response genes are transcribed in anticipation of peak TH concentrations. The distinct T2 concentration profile suggests a biological role of this biomolecule in anuran postembryonic development and an additional aspect that may be a target of anthropogenic chemicals that can disrupt anuran metamorphosis and TH signalling. Hence, as a second aim of the study, we set out to find additional bioindicators of metamorphosis, which can aid future investigations of developmental disruption. Using a sensitive nanoLC-Orbitrap system an untargeted analysis workflow was applied. Among 6,062 endogenous metabolites, 421 showed metamorphosis-dependent concentration dynamics. These potential bioindicators included several carnitines, prostaglandins and some steroid hormones.

Cocaine- and amphetamine-regulated transcript (Cart) is a pleiotropic neuropeptide involved in the regulation of stress and anxiety, depression, reproduction and circadian functions, yet it is mainly known for its metabolic regulation of body weight and appetite. While mammals possess a single cart gene, the genomes of birds may contain up to two carts and fish may possess up to ten cart genes. Furthermore, in some fish species the number of cart paralogues exceeds the number expected according to whole-genome duplication events in actinopterygians, suggesting a species-specific diversification of the cart system. In the current study, we identified multiple cart genes in two fish species with global importance -Nile tilapia and gilthead seabream. Bioinformatics analysis revealed seven cart genes in the tilapia genome and six cart genes in the seabream genome, all of which show high homology with carts of other vertebrates. Additionally, the predicted mature cart peptide sequences contain all the cysteines known to stabilize the tertiary peptide structure in other vertebrates. Nevertheless, protein structure modeling suggested that some carts lost part or all of the cysteine-based disulfide bridges. Quantitative-PCR analyses of all cart genes cloned in this research demonstrated that while all carts are mainly expressed in the brain, some cart genes show wider tissue distribution with significant expression in peripheral tissues including the kidney and gonads. Taken together, these findings emphasize the complexity of the piscine cart system.

Neuropeptide signalling is transversally important in all living animals as it constitutes the basis of cellular communication. The investigation of the functional roles of peptide signalling represents an important route to understanding evolution of specific physiological traits and behaviours in metazoans. Allatostatins and their cognate receptors are classically defined as invertebrate neuropeptide hormones. Among these, allatostatin C was firstly associated with insect development. However, accumulating evidence recognises the presence of allatostatin C as a conserved signalling molecule across all invertebrate lineages, with reported functions spanning from regulation of feeding and digestion to immune responses and modulation of core nociception. Here we combined in silico and experimental approaches to reveal the interacting molecular determinants of the allatostatin C signalling in the cephalopod Octopus vulgaris, a scientifically and culturally interesting invertebrate for its centralised nervous system, capable of top-down modulation of complex behaviours. This resolved a single prepropeptide encompassing allatostatin C peptide (OvAstC), whose conserved mature form (AVITACYFQAVSCY) was shown to differentially activate two identified cognate receptors (OvAstCR1 and OvAstCR2) when heterologously expressed in the recombinant system HEK293G5A. PCR analysis carried out in O. vulgaris tissues, showed a broad distribution of OvAstC and OvAstCRs. This wide expression across nervous, immune and digestive tissues is consistent with a pleiotropic role of this peptidergic system. Together, the opioid/somatostatin-related phylogenetic placement of OvAstCRs and the broad expression of OvAstC components in nervous and sensory tissues nominate this pathway as a candidate for neuromodulatory control of sensory processing, including nociception, with potential welfare relevance in cephalopods. Significance statementCephalopods represent an evolutionarily distinctive molluscan lineage that evolved a centralised nervous system capable of displaying advanced learning and behavioural complexity compared with other invertebrates. These features, speculated to allow elaboration of pain-like states, granted cephalopods inclusion as the only invertebrate taxon requiring protection under European legislations when used in research. Investigation of the neuropeptide signalling in cephalopods is currently understudied despite its crucial role in regulating broad physiological functions in organisms. This study identified for the first time a single allatostatin C peptide and two cognate receptors in Octopus vulgaris. Our characterisation of a putative endogenous allatostatin C system in octopus, the accumulating evidence of its central role in invertebrate antinociception and its evolutionary relationship with the vertebrate-exclusive analgesic opioid family, represent a critical starting point for a more in-depth analysis of the physiological role of allatostatin C in this subclass of molluscs, with important welfare implications.

Male and female rodents experience different selective pressures associated with reproductive costs. Thus, we may expect the expression of different Pace-of-life (POL) strategies between sexes. Further, the pace-of-life syndrome (POLS) hypothesis and anisogamy predict differences in the costs of gamete production, where variation in life history trait expression should follow a fast-slow continuum such that males and females might exist on opposite ends of the spectrum. However, males and females could express a similar POL strategy in systems where the reproductive costs are consistent between sexes or where selective pressures force a similar directionality of traits. We used standardized behavioural tests and fecal glucocorticoid metabolite (FGM) concentrations to measure potential differences in POL strategies among three rodent species in Algonquin Provincial Park. We hypothesized that differences in reproductive costs between males and females would result in differences in POL traits along the fast-slow continuum. We predicted that males would express more explorative behaviours and have a higher level FGMs than female counterparts. We found little support for the POLS hypothesis between sexes. Instead, we suggest that the species observed may express a similar directionality of selection for the observed behavioural traits, where both sexes express similar relationships in POL. Thus, some male rodents may be more explorative to accommodate the increase of energetic stress associated with mate acquisition, while females may share similar trait expressions to accommodate the increased metabolic demand for the care and development of young. Significance statementDocility and exploration are measurements of how species or individuals react to novel and sometimes risky stimuli. Further, reproduction is an energetically expensive and inherently risky process. In seasonally breeding rodents, males often invest in fast pace-of-life behaviours that accommodate an increased necessity to disperse and find a mate. Meanwhile, females often invest in metabolically expensive physiological processes, including pregnancy and lactate development, increased protection of the young and the need for self-maintenance. Using a series of behavioural tests and fecal glucocorticoid metabolites (FGMs), we measured the relationship between metabolic stress, docility and exploration in three species of rodents during the breeding season. We observed consistent relationships in behaviour and FGMs, indicating uniformity between sexes regarding these behaviours. We posit that the costs associated with mate acquisition for males and the care and development of young for females can influence similar behavioural strategies.

In mammals, most sex differences in phenotype are controlled by gonadal hormones, but recent work on transgenic mice have shown that sex chromosomes can have a direct influence on sex-specific behaviors. In this study, we take advantage of the naturally occurring sex reversal in a mouse species, Mus minutoides, to investigate for the first time the relationship between sex chromosomes, hormones and behaviors in a wild species. In this model, a feminizing variant of the X chromosome, named X*, produces three types of females with different sex chromosome complements (XX, XX*, and X*Y), associated with alternative behavioral phenotypes, while all males are XY. We thus compared the levels of three major circulating steroid hormones (testosterone, corticosterone and estradiol) in the four sex genotypes to disentangle the influence of sex chromosomes and sex hormones on behavior. First, we did not find any difference in testosterone levels in the three female genotypes, although X*Y females are notoriously more aggressive. Second, in agreement with their lower anxiety-related behaviors, X*Y females and XY males display lower baseline corticosterone concentration than XX and XX* females. Instead of a direct hormonal influence, this result rather suggests that sex chromosomes may have an impact on the baseline corticosterone level, which in turn may influence behaviors. Third, estradiol concentrations do not explain the enhanced reproductive performance and maternal care behavior of the X*Y females compared to the XX and XX* females. Overall, this study highlights that most of the behaviors varying along with sex chromosome complement of this species are driven by genetic factors rather than steroid hormone concentrations.

Maternal hormones, such as thyroid hormones transferred to embryos and eggs, are key signalling pathways to mediate maternal effects. To be able to respond to maternal cues, embryos must express key molecular machinery of the hormone pathways, such as enzymes and receptors. While altricial birds begin thyroid hormone (TH) production only at/after hatching, experimental evidence suggests that their phenotype can be influenced by maternal THs deposited in the egg. However, it is not understood, how and when altricial birds express genes in the TH-pathway. For the first time, we measured the expression of key TH-pathway genes in altricial embryos, using two common altricial ecological model species (pied flycatcher, Ficedula hypoleuca and blue tit Cyanistes caeruleus). Deiodinase DIO1 gene expression could not be reliably confirmed in either species, but deiodinase enzyme DIO2 and DIO3 genes were expressed in both species. Given that DIO2 coverts T4 to biologically active T3, and DIO3 mostly T3 to inactive forms of thyroid hormones, our results suggest that embryos may modulate maternal signals. Thyroid hormone receptor (THRA and THRB) and monocarboxyl membrane transporter gene (SLC15A2) were also expressed, enabling TH-responses. Our results suggest that early altricial embryos may be able to respond and potentially modulate maternal signals conveyed by thyroid hormones.

ContextEthyl-Iophenoxic acid (Et-IPA) has been widely used as a bait biomarker to determine oral bait consumption by vertebrate wildlife species. Oral bait vaccines have been delivered to numerous wildlife species to protect them from disease. The Tasmanian devil (Sarcophilis harrisii), the largest extant carnivorous marsupial species, is threatened by the transmissible cancers known as devil facial tumour disease (DFTD). Development of a protective DFTD vaccine is underway, and an oral bait has been proposed to deliver the vaccine in the wild. The bait delivery system requires a biomarker that can be detected for several months post-consumption in Tasmanian devils. AimTo determine the suitability of Et-IPA as a bait biomarker in the Tasmanian devil. MethodTwo Tasmanian devils were fed 50 mg Et-IPA (4.5 to 7.1 mg Et-IPA/kg bodyweight). Liquid chromatography with tandem mass spectrometry (LC-MS/-MS) was used to directly measure Et-IPA in baseline serum samples and samples collected on days 1, 14, 26 and 56 post-baiting. Key resultBoth devils retained serum Et-IPA concentrations at two orders of magnitude above negative control sera when this study concluded. ConclusionsEt-IPA is a useful bait biomarker for Tasmanian devils and can be included in future DFTD bait vaccine field trials to determine bait vaccine uptake.

Experience with enriched environments positively impacts the health and wellbeing of nonhuman animals ranging from rodents to primates. Little is known, however, about the specific effects of enhanced cognitive enrichment (ECE) on nonhuman primates. The study reported here used archival samples to provide preliminary analysis of ECE on hormones associated with stress and wellbeing, as well as evaluation of persistent effects of infant social rearing. Hair samples from 24 adult male rhesus macaques were analyzed via LC-MS/MS technique for the main stress response hormones: cortisol, cortisone, and dehydroepiandrosterone. The ECE and care-as-usual (CAU) groups did not significantly differ in any of the mean analyte values. Cortisol and dehydroepiandrosterone were strongly and positively correlated in the ECE group. By contrast, for the CAU group the two analytes were weakly correlated. Within the ECE group, cortisol and dehydroepiandrosterone were significantly and positively correlated in the mother-reared group, but not in the nursery-reared group. Overall, these results provide preliminary evidence that experience with ECE could help to maintain a balanced cortisol:dehydroepiandrosterone ratio, possibly indicative of a healthy stress response. Further examination of this ratio in hair is needed to support this hypothesis. These observations may also suggest that nursery rearing could have persistent effects, including dysregulation of the hypothalamic-pituitary-adrenal axis, apparent in the unbalanced cortisol:dehydroepiandrosterone ratio. Together these findings are consistent with the growing literature that supports the use of ECE to promote nonhuman primate wellbeing and healthy development.

The Fusarium mycotoxin deoxynivalenol (DON) is one of the most frequently occurring food contaminants. Nearly all individuals are exposed to DON, due to it widespread presence in grains and grain-based products. Chronic DON poisoning is associated with growth retardation, immunotoxicity as well as impaired reproduction and fetal development. At the molecular level, DON alters intracellular signaling by activating mitogen-activated protein kinases (MAPKs) that modulate cell growth, differentiation, and apoptosis. Of note, these MAPKs are also critical mediators of gonadotrophin-releasing hormone (GnRH)-induced synthesis and secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by pituitary gonadotrope cells. So far, no research has explored the potential endocrine-disrupting effects of DON on pituitary gonadotropins production. Herein, we show the first evidence that DON affects LH production by the immortalized gonadotrope-like cell line L{beta}T2 in a concentration-dependent manner. Taken together, our experiments demonstrated that low concentrations of DON affect GnRH-induced signaling through a mechanism that, at least in part, involves apoptosis and inhibition of GnRH-induced phosphorylation of ERK-MAPK. Consequently, DON also affects the GnRH-induced expression of Cga and Lhb, two critical genes for LH synthesis and secretion by gonadotrope cells in mammals. This research broadens our knowledge of the toxicity of DON and brings a new depth to the potential neuroendocrine implications for reproduction. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/607800v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@78d077org.highwire.dtl.DTLVardef@1be5bdaorg.highwire.dtl.DTLVardef@804d49org.highwire.dtl.DTLVardef@1520136_HPS_FORMAT_FIGEXP M_FIG C_FIG

Reproduction is a highly demanding biological process that occurs at the optimal time of the year and day to ensure the success of spawn and offspring. Melatonin is a hormone that, secreted mainly by the pineal gland, plays a critical role in the integration of the photoneuroendocrine information from environment (annual and daily variations) to modulate reproductive activity and gonadal development in fish. In this study we assessed the effect of exogenous melatonin on the circadian levels of sex steroids and gonadotropins in pubertal 2 yr-old male sea bass during their reproductive cycle including, pre-spermatogenesis (Pspg), spermiation (Spm) and post-spermiation (PSpm) stages. Our results demonstrated that all reproductive hormones displayed circadian variations along the entire reproductive cycle in pubertal fish. Circulating levels of the luteinizing hormone (Lh) were affected by both melatonin injection and the daily timing of administration during the Spm and PSpm stages, thus evoking variations of Lh levels at night. Melatonin also significantly affected circadian rhythms of Fsh during the Spm stage. Overall, both 11-Kt and T plasma levels displayed circadian variations during the reproductive cycle in the sea bass which were not prevented by melatonin. However, melatonin showed a significant decrease of plasma levels of 11-Kt 1h after dusk during the Pspg stage, while it increased those levels of T 5 h after dusk during the PSpm stage. These findings provide new insights into the role of melatonin in fish reproduction as a key factor in regulation of daily variation of key hormones involved in gonadal development. This circumstance may have implications in the control of gametogenesis and management of fish in aquaculture. HighlightsO_LIPlasma levels of Fsh, Lh, T and 11-Kt show daily rhythms in pubertal male sea bass. C_LIO_LIMelatonin evokes changes in daily rhythms of Lh during the reproductive cycle. C_LIO_LIMelatonin yields Fsh plasma level differences during spermiation stage. C_LIO_LIMelatonin elicits T and 11-Kt plasma level differences depending on reproductive stage and time. C_LI

U.S. quail species are vulnerable to population declines as a result climate change, habitat loss, and habitat fragmentation; all of which can induce physiological stress. Additionally, population restoration techniques (PRTs), like translocations, also induce stress. Traditional stress assessments include capturing and handling birds to extract blood, methods which are inherently stressful and can compound stress analyses. However, the stress hormone corticosterone is metabolized from the blood and excreted in feces as fecal corticosterone metabolites (FCMs). FCMs have been used as a non-invasive measurement of stress in a variety of species, but must be validated for each species. The objective of this study was to biologically validate the use of FCMs as a non-invasive measurement of stress-hormone levels in California valley quail (Callipepla californica). Reference and treatment quail were acclimated for 3 wks in an outdoor aviary. Subsequently, treatment quail were subjected to a simulated, 48-h translocation, a common and stressful PRT. Fecal samples were collected every 4 h and processed using an enzyme immunoassay. Mean FCM concentrations of treatment quail (41.50 {+/-}16.13 ng/g) were higher than reference FCM concentrations (24.07 {+/-}10.4 ng/g). These results biologically validate the use of FCMs as a non-invasive method to assess stress hormone levels in California valley quail, demonstrate diurnal variation in quail stress levels, and confirms that quail translocations are a stressful PRT. Ultimately, this research validates a new non-invasive tool for stress measurement to advance quail research, management, and conservation. Lay summaryThis study biologically validates the use of fecal corticosterone metabolites as a non-invasive method for detecting stress in quail, demonstrates diurnal variation in quail stress levels, confirms that translocations elevate stress which likely impacts success, and establishes a new non-invasive tool for stress measurement in quail research, management, and conservation.

Steroids play a crucial role in modulating brain and behavior. While traditionally it is considered that the brain is a target of peripheral hormones produced in endocrine glands, it has been discovered that the brain itself produces steroids, known as neurosteroids. Neurosteroids can be produced in brain regions involved in the regulation of social behaviors and can act locally regulating behaviors like reproduction and aggression. Here, for the first time in a teleost fish, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify a panel of 8 steroids in both plasma and brain. We use the weakly electric fish Gymnotus omarorum, a species which shows non-breeding aggression in both sexes, to characterize these hormonal profiles in wild non-breeding adults. We show that: 1) systemic steroids in the non-breeding season are similar in both sexes, although only males have circulating 11-KT, 2) brain steroid levels are sexually dimorphic, as females display higher levels of AE, T and E1, and only males had 11-KT, 3) systemic androgens such as AE and T in the non-breeding season are potential precursors for neuroestrogen synthesis, and 4) estrogens, which play a key role in non-breeding aggression, are detectable in the brain (but not the plasma) in both sexes. These data fall in line with previous reports in G. omarorum which show that non-breeding aggression is dependent on the estrogenic pathway, as has also been shown in bird and mammal models. Overall, our results constitute a fundamental groundwork to understanding the complexity of hormonal modulation, its potential sex differences, the role of neurosteroids and the interplay between central and peripheral hormones in the regulation of behaviors.

Glucocorticoids (GC) are prescribed for periods >3 months to 1-3% of the UK population; 10-50% of these patients develop hypothalamus-pituitary-adrenal (HPA) axis suppression, which may last over 6 months and is associated with morbidity and mortality. Recovery of higher nodes of the axis is necessary for recovery of adrenal function. We developed a mouse model of Dexamethasone (DEX)-induced HPA axis dysfunction in order to further explore recovery in the pituitary. Adult male C57BL6/J or those crossed with Pomc-eGFP mice were randomly assigned to receive DEX (~0.4 mg/kg bodyweight/day) or vehicle via drinking water for 4 weeks following which treatment was withdrawn. Tissues were harvested at 0, 1, and 4 weeks following withdrawal of treatment. Corticotrophs were isolated from Pomc-eGFP pituitaries using FACS, and RNA extracted for RNA-seq. DEX treatment suppressed corticosterone production, which remained partially suppressed at least 1 week following DEX withdrawal. In the adrenal, at time 0, Hsd3b2, Cyp11a1, and Mc2r mRNA levels were significantly reduced, with Mc2r and Cyp11a1 remaining reduced 1 week following DEX withdrawal. The corticotroph transcriptome was modified by DEX treatment with some differences between groups persisting 4 weeks following withdrawal. No genes supressed by DEX exhibited ongoing attenuation 1 and 4 weeks following withdrawal, whilst only 2 genes were upregulated and remained so following withdrawal. A pattern of rebound at 1 and 4 weeks was observed in 14 genes that increased following suppression, and 6 genes that were reduced by DEX and then increased. Chronic GC treatment may induce persistent changes in the pituitary that may influence future response to GC treatment or stress.

As a rapidly evolving trait, animal venom exhibits compositional variation even at the intraspecific level between populations, ontogenetic stages and sexes. In extreme cases venom is used for different functional purposes across ontogenetic stages. This phenomenon occurs for example in case of horse flies (Tabanidae), which utilize venom for predation during the larval stage and for blood feeding in the adult stage. While the venom composition and activity of adult tabanids has been investigated for some species, nothing is known about the venom compounds of tabanid larvae. With the current study we provide first insights into the venom composition of larval stages of a tabanid. Besides a plethora of hydrolyzing enzymes, we find the venom of T. autumnalis larvae dominated by peptide toxins. Some of these peptide toxins could be annotated as putative neurotoxins and cytolytic peptides and these compounds likely are the main drivers of fast prey incapacitation. Previous studies identified the main functional constituents utilized by tabanid imagines for blood feeding to be protease inhibitors affecting vasoconstriction and thrombin to promote blood feeding. Interestingly we find highly similar putative protease inhibitors in the larval venom, though with relatively low expression levels. This provides a first indication for a shifted expression profile between the venom of tabanid life stages to fulfill both the predatory needs of the larvae and the blood lust of female imagines.

Bats have deserved much scientific attention due to their biological-ecological properties and increasingly recognized epidemiological significance. Ecdysteroids are insect-molting hormones that (under experimental conditions) have stimulating and anabolic effects in mammals, including humans. Therefore, these biologically active compounds are currently under consideration by the World Anti-Doping Agency (WADA) to become doping-controlled substances. Previously we demonstrated that low to high concentrations of ecdysteroids appear in the blood of insectivorous passerine birds. Since passerine birds and echolocating bats share several adaptive mechanisms in connection with flying, and insectivorism is also among their common traits, we hypothesized that ecdysteroids might also be present in the blood of insectivorous bats. To test this hypothesis, blood samples of eight insectivorous bat species were collected and analyzed for the presence of ecdysteroids with highly sensitive targeted ultra-high-performance liquid chromatography coupled to high-resolution quadrupole-orbitrap mass spectrometry method (UHPLC-HRMS). The results supported our hypothesis, because nine ecdysteroids were detected in bat blood. The spectrum of these ecdysteroids was similar in those bat species which have their most preferred food items from the same insect order, supporting insects as the most likely source of these hormones. It was also shown that the spectrum of blood-borne ecdysteroids was broader in the autumn than in the summer, and higher concentrations of 20-hydroxyecdysone were measured in samples of large size bat species in comparison with small size ones. Based on the known physiologic effects of ecdysteroids, we postulate that these results might have implications on the metabolic rate and parasite burdens of insectivorous bats.