Creb2 involved in innate immunity by activating PpMitf-mediated melanogenesis in Pteria penguin.

cAMP response element binding protein 2 (CREB2) acts as an intracellular transcriptional factor and regulates many physiological processes, including melanogenesis and melanocyte differentiation. In our previous research, the Creb2 gene has been characterized from Pteria penguin (P. penguin), but its role and regulatory mechanism in P. penguin are still unclear. In this study, first, the function of PpCreb2 in melanogenesis and innate immunity were identified. PpCreb2 silencing significantly decreased the tyrosinase activity and melanin content, indicating PpCreb2 played an important role in melanogenesis. Meanwhile, PpCreb2 silencing visibly suppressed the antibacterial activity of hemolymph supernatant, indicating that PpCreb2 was involved in innate immunity of P. penguin. Second, the PpCreb2 was confirmed to perform immune function by regulating the melanogenesis. The decreased melanin oxidation product due to PpCreb2 silencing triggered the declining of antibacterial activity of hemolymph supernatant, which then could be rescued by adding exogenous melanin oxidation products. Third, the regulation pathway of PpCreb2 involved in innate immunity was analyzed. The promoter sequence analysis of PpMitf discovered 5 conserved cAMP response element (CRE), which were specifically recognized by basic Leucine zipper domain (bZIP) of upstream activation transcription factor. The luciferase activities analysis showed that PpCreb2 could activate the CRE in PpMitf promoter via highly conserved bZIP domain and regulate the expression of PpMitf, which further regulated the PpTyr expression. Therefore, the results collectively demonstrated that PpCreb2 participated in innate immunity by activating PpMitf-mediated melanogenesis in P. penguin.

Penguin: A tool for predicting pseudouridine sites in direct RNA nanopore sequencing data.

Pseudouridine is one of the most abundant RNA modifications, occurring when uridines are catalyzed by Pseudouridine synthase proteins. It plays an important role in many biological processes and has been reported to have application in drug development. Recently, the single-molecule sequencing techniques such as the direct RNA sequencing platform offered by Oxford Nanopore technologies have enabled direct detection of RNA modifications on the molecule being sequenced. In this study, we introduce a tool called Penguin that integrates several machine learning (ML) models to identify RNA Pseudouridine sites on Nanopore direct RNA sequencing reads. Pseudouridine sites were identified on single molecule sequencing data collected from direct RNA sequencing resulting in 723 K reads in Hek293 and 500 K reads in Hela cell lines. Penguin extracts a set of features from the raw signal measured by the Oxford Nanopore and the corresponding basecalled k-mer. Those features are used to train the predictors included in Penguin, which in turn, can predict whether the signal is modified by the presence of Pseudouridine sites in the testing phase. We have included various predictors in Penguin, including Support vector machines (SVM), Random Forest (RF), and Neural network (NN). The results on the two benchmark data sets for Hek293 and Hela cell lines show outstanding performance of Penguin either in random split testing or in independent validation testing. In random split testing, Penguin has been able to identify Pseudouridine sites with a high accuracy of 93.38% by applying SVM to Hek293 benchmark dataset. In independent validation testing, Penguin achieves an accuracy of 92.61% by training SVM with Hek293 benchmark dataset and testing it for identifying Pseudouridine sites on Hela benchmark dataset. Thus, Penguin outperforms the existing Pseudouridine predictors in the literature by 16 % higher accuracy than those predictors using independent validation testing. Employing penguin to predict Pseudouridine sites revealed a significant enrichment of "regulation of mRNA 3'-end processing" in Hek293 cell line and 'positive regulation of transcription from RNA polymerase II promoter involved in cellular response to chemical stimulus' in Hela cell line. Penguin software and models are available on GitHub at https://github.com/Janga-Lab/Penguin and can be readily employed for predicting Ψ sites from Nanopore direct RNA-sequencing datasets.

Fecal glucocorticoid analysis as a health monitoring tool for endangered African penguins (Spheniscus demersus).

African penguins (Spheniscus demersus) are an endangered species, with approximately 70,000 mature adults remaining in the wild. Population loss is linked to a combination of environmental and anthropogenic stressors. The aim of the study was to validate a commercially available enzyme immunoassay (EIA) to assess adrenal activity and measure the response to stressors in the feces of African penguins. Fecal samples (n = 609) were collected from 12 African penguins housed at Mystic Aquarium throughout their natural lifecycle, including breeding and molt, where measurable changes in fecal glucocorticoid metabolite (FGM) levels are predicted to occur. Fecal samples collected post-veterinary exam were used for biological validation. Longitudinal analysis shows a significant difference (p = <0.0001) between the average FGM levels during baseline and breeding season, 33.97 ± 1.30 ng/g and 50.21 ± 3.18 ng/g, respectively. Females displayed significantly higher FGM levels than males during both baseline (p = 0.0386; females = 38.80 ± 2.19 ng/g; males = 29.34 ± 1.37 ng/g) and breeding periods (p = 0.0175; females = 57.53 ± 4.84 ng/g; males = 42.69 ± 3.95 ng/g). Average FGM levels decreased significantly over the three-week molting period, from 85.40 ± 20.35 ng/g at week one to 20.23 ± 5.30 ng/g at week three. A seasonal difference in FGM levels was observed in both male and female fecal samples, with Fall having the highest average FGM levels, 54.38 ± 3.64 ng/g, and Summer the lowest, 30.87 ± 2.21 ng/g. General linear mixed model analysis determined that lifecycle (females) and visitor presence (males) were the two factors which best explained the variation in FGM levels observed, however neither factor was found to be significant. These results show FGM analysis can detect physiologically meaningful changes in endocrine activity in African penguins and can be used to monitor health for penguins in aquaria and in the wild, thus contributing to conservation efforts for the survival of the species.

PHARMACOKINETICS OF PRIMAQUINE PHOSPHATE AFTER A SINGLE ORAL ADMINISTRATION TO AFRICAN PENGUINS (SPHENISCUS DEMERSUS).

Primaquine is an 8-aminoquinolone drug commonly used for the chemoprophylaxis and treatment of avian malarial infections in managed penguin populations worldwide. Little is known about its pharmacokinetic properties in avian species. The objective of this study was to describe the disposition of primaquine phosphate after a single oral dose in 15 healthy African penguins (Spheniscus demersus). A single tablet containing 26.3 mg of primaquine phosphate (equivalent to 15 mg primaquine base) was administered orally to each bird in a herring fish. Blood samples were collected prior to drug administration and at predetermined timepoints through 144 hr postadministration. Plasma was analyzed for drug concentration by high-performance liquid chromatography with ultraviolet detection. Mean maximum plasma concentration of primaquine phosphate was 277 ± 96 ng/ml at approximately 3.1 hr following oral administration. The mean disappearance half-life was 3.6 ± 1.6 hr. Plasma concentrations were below detectable limits in all but one penguin by 36 hr. A single oral administration of 26.3 mg of primaquine phosphate in African penguins resulted in a pharmacokinetic profile comparable to those attained in human studies. These results suggest that a dosing interval similar to human regimens may be of potential use in the prevention and treatment of avian malaria in penguins. Additional clinical studies are needed to determine the efficacy and safety of this regimen.

Antarctic Penguins as Reservoirs of Diversity for Avian Avulaviruses.

Wild birds harbor a huge diversity of avian avulaviruses (formerly avian paramyxoviruses). Antarctic penguin species have been screened for avian avulaviruses since the 1980s and, as such, are known hosts of these viruses. In this study, we screened three penguin species from the South Shetland Islands and the Antarctic Peninsula for avian avulaviruses. We show that Adelie penguins (Pygoscelis adeliae) are hosts for four different avian avulavirus species, the recently described avian avulaviruses 17 to 19 and avian avulavirus 10-like, never before isolated in Antarctica. A total of 24 viruses were isolated and sequenced; avian avulavirus 17 was the most common, and phylogenetic analysis demonstrated patterns of occurrence, with different genetic clusters corresponding to penguin age and location. Following infection in specific-pathogen-free (SPF) chickens, all four avian avulavirus species were shed from the oral cavity for up to 7 days postinfection. There was limited shedding from the cloaca in a proportion of infected chickens, and all but one bird seroconverted by day 21. No clinical signs were observed. Taken together, we propose that penguin species, including Antarctic penguins, may be the central reservoir for a diversity of avian avulavirus species and that these viruses have the potential to infect other avian hosts.IMPORTANCE Approximately 99% of all viruses are still to be described, and in our changing world, any one of these unknown viruses could potentially expand their host range and cause epidemic disease in wildlife, agricultural animals, or humans. Avian avulavirus 1 causes outbreaks in wild birds and poultry and is thus well described. However, for many avulavirus species, only a single specimen has been described, and their viral ecology and epidemiology are unknown. Through the detection of avian avulaviruses in penguins from Antarctica, we have been able to expand upon our understanding of three avian avulavirus species (avian avulaviruses 17 to 19) and report a potentially novel avulavirus species. Importantly, we show that penguins appear to play a key role in the epidemiology of avian avulaviruses, and we encourage additional sampling of this avian group.

Monitoring glucocorticoid metabolite concentrations as a proxy of environmental stress across important life-history stages in captive African penguins.

Due to considerable global decline in wildlife population numbers and species diversity, because of various anthropogenic activities, conservationists increasingly rely on captive and managed populations as important reservoirs to ensure the survival of endangered and vulnerable species. However, very few of these facilities implement robust, non-invasive monitoring techniques to confirm the effectiveness of their management practices to address animal welfare challenges. This study assessed adrenocortical activity as an indication of environmental stress by investigating the effects of both natural (climate, life-history stages) and anthropogenic (visitor presence) factors on captive-housed African penguins. Seven male-female African penguin breeding pairs were housed in a large, naturalistic outside enclosure at the National Zoological Garden (NZG), South Africa. Weekly urofaecal samples were collected from all individuals over one-year to measure urofaecal glucocorticoid metabolite (ufGCM) concentrations. General linear mixed model analysis determined that visitor presence (for males) and rainfall (for females) were the two factors which best explained the variation in ufGCM concentrations of the study population; however, none of the environmental and anthropogenic factors monitored were found to be significant. A posthoc graphical analysis showed considerable individual variation in terms of ufGCM concentrations within and between sexes when comparing life-history stages. This study confirms that non-invasive steroid monitoring can be an effective tool set for defining and assessing environmental stressors for African penguins and potentially other captive seabirds. However, conservationists and wildlife managers should also consider that individual-, sex-, and population-specific differences in the response to environmental stressors can exist. As such, a generalized management protocol for a specific species may not be sufficient and should be customized according to the specific captive population and/or individual.

Preliminary Pilot Study of Itraconazole After a Single Oral Dose of a Veterinary Formulation Solution in African Penguins (Spheniscus demersus).

Aspergillosis is a common cause of morbidity and mortality in captive penguins. Itraconazole, an antifungal drug, is commonly used to treat aspergillosis infections in avian species; however, commercially available human formulations are costly, and studies have shown the effectiveness of compounded formulations to be unreliable. The US Food and Drug Administration (FDA) recently approved a veterinary formulation of itraconazole, Itrafungol, for use in cats. This study provides preliminary results from limited sampling evaluating whether this veterinary formulation is suitable for future studies in the African penguin (Spheniscus demersus). A 20 mg/kg PO itraconazole dose was administered to 9 African penguins. Blood samples were taken over the course of 24 hours; each sample was collected from a different bird to minimize stress to the animals. Plasma was analyzed by high-performance liquid chromatography for concentrations of itraconazole. The drug was absorbed in all penguins, and plasma concentrations in 5 of 9 penguins (56%) were found to be greater than the established therapeutic dose of 1.0 µg/ mL. To our knowledge, this is the first study that has investigated a 20 mg/kg dose of itraconazole in a penguin species. The small sample size limits the conclusions that can be drawn from this preliminary study. Nonetheless, we demonstrate encouraging evidence that the FDA-approved formulation of oral itraconazole solution should be considered for future study as a cost-effective treatment for aspergillosis in African penguins and other avian species.

Application of different pharmacokinetic models to describe and predict pharmacokinetics of voriconazole in magellanic penguins following oral administration.

Aspergillosis is a condition causing serious morbidity and mortality in captive penguins and other bird species. It can be treated with antifungal drugs, such as voriconazole. However, the pharmacokinetics of voriconazole are variable between different animal and bird species. Therefore, the pharmacokinetics of voriconazole were investigated in this study in Magellanic penguins. Pharmacokinetic models were constructed and applied to predict the pharmacokinetics of voriconazole during long-term treatment in Magellanic penguins, since the voriconazole treatment duration in chronic aspergillosis cases can last up to several months. Plasma voriconazole concentration-time data from adult Magellanic penguins (Spheniscus magellanicus; n = 15) following a single oral (PO) dose of either 2.5 mg/kg or 5 mg/kg in a herring in three separate study periods 7-12 months apart were collected. Mean plasma voriconazole concentrations were above the targeted MIC for Aspergillus fumigatus for 2 hr following a single 2.5 mg/kg voriconazole dose while the plasma concentrations exceeded the MIC for least 24 hr following a 5 mg/kg dose. Nonlinear mixed-effects modeling was used to fit two pharmacokinetic models, one with first-order and another with saturable elimination, to the single-dose data. Fits were good for both, as long as dose was included as a covariate for the first-order model so that clearance was lower and the half-life longer for animals receiving the 5 mg/kg dose. Although the single-dose data suggested saturated elimination at higher concentrations, the model with saturable elimination did not predict plasma voriconazole concentrations well for a clinical aspergillosis case receiving long-term treatment, possibly because of induction of metabolizing enzymes with chronic exposure. Pharmacokinetic models should accurately predict plasma drug concentrations for different dosage regimens in order to be applicable in the field. Future studies should focus on determining clearance at steady-state to be able to refine the pharmacokinetic models presented here and improve model performance for long-term oral voriconazole administration in Magellanic penguins.

Independent pseudogenization of CYP2J19 in penguins, owls and kiwis implicates gene in red carotenoid synthesis.

Carotenoids have important roles in bird behavior, including pigmentation for sexual signaling and improving color vision via retinal oil droplets. Yellow carotenoids are diet-derived, but red carotenoids (ketocarotenoids) are typically synthesized from yellow precursors via a carotenoid ketolase. Recent research on passerines has provided evidence that a cytochrome p450 enzyme, CYP2J19, is responsible for this reaction, though it is unclear if this function is phylogenetically restricted. Here I provide evidence that CYP2J19 is the carotenoid ketolase common to Aves using the genomes of 65 birds and the retinal transcriptomes of 15 avian taxa. CYP2J19 is functionally intact and robustly transcribed in all taxa except for several species adapted to foraging in dim light conditions. Two penguins, an owl and a kiwi show evidence of genetic lesions and relaxed selection in their genomic copy of CYP2J19, and six owls show evidence of marked reduction in CYP2J19 retinal transcription compared to nine diurnal avian taxa. Furthermore, one of the owls appears to transcribe a CYP2J19 pseudogene. Notably, none of these taxa are known to use red carotenoids for sexual signaling and several species of owls and penguins represent the only birds known to completely lack red retinal oil droplets. The remaining avian taxa belong to groups known to possess red oil droplets, are known or expected to deposit red carotenoids in skin and/or plumage, and/or frequently forage in bright light. The loss and reduced expression of CYP2J19 is likely an adaptation to maximize retinal sensitivity, given that oil droplets reduce the amount of light available to the retina.

A Global Overview of Exposure Levels and Biological Effects of Trace Elements in Penguins.

Trace elements are chemical contaminants that can be present almost anywhere on the planet. The study of trace elements in biotic matrices is a topic of great relevance for the implications that it can have on wildlife and human health. Penguins are very useful, since they live exclusively in the Southern Hemisphere and represent about 90% of the biomass of birds of the Southern Ocean. The levels of trace elements (dry weight) in different biotic matrices of penguins were reviewed here. Maps of trace element records in penguins were included. Data on exposure and effects of trace elements in penguins were collected from the literature. The most reported trace elements in penguins are aluminum, arsenic, cadmium, lead, mercury, copper, zinc, and manganese. Trace elements have been measured in 11 of the 18 species of penguins. The most studied biotic matrices are feathers and excreta. Most of the studies have been performed in Antarctica and subantarctic Islands. Little is known about the interaction among metals, which could provide better knowledge about certain mechanisms of detoxification in penguins. Future studies of trace elements in penguins must incorporate other metals such as vanadium, cobalt, nickel, and chromium. Data of metals in the species such as Eudyptes pachyrhynchus, Eudyptes moseleyi, Eudyptes sclateri, Eudyptes robustus, Eudyptes schlegeli, Spheniscus demersus, Spheniscus mendiculus, and Megadyptes antipodes are urged. It is important to correlate levels of metals in different biotic matrices with the effects on different species and in different geographic locations.

An integrative appraisal of the hormonal and metabolic changes induced by acute stress using king penguins as a model.

A large number of studies have focused on the reactivity of the hypothalamic-pituitaryadrenal (HPA) axis and the consequences of glucocorticoids (GC) in mediating life-history trade-offs. Although short-term increases in GCs are viewed as adaptive, mobilizing energy substrates allowing animals to deal with impending threats (e.g. stimulating hepatic gluconeogenesis, stimulating lipolysis, mobilizing amino acids), few studies have actually measured the exact time-course of substrate mobilisation in response to acute stress in natural conditions. We evaluated the hormonal and metabolic components of the stress response to acute stress in 32 free-living king penguins (Aptenodytes patagonicus). We monitored changes in blood GCs (corticosterone, CORT), glucose, lactate, ketone bodies (ß-hydroxybutyrate), non-esterified fatty acids, and uric acid in response to a standardized capture-restraint protocol lasting for up to 90min. Furthermore, we tested whether the vigilance status of the animal (alert or asleep) affected its perception of the capture, thereby modulating the hormonal and metabolic stress responses. The time course of energy mobilisation followed the characteristic pattern expected from laboratory and theoretical models, with a rapid depletion of those energy stores linked to rapid adrenergic responses (i.e. glucose and ketone bodies), followed by a mobilisation of energy stores associated with the sustained longer-term GC response (i.e. fats and protein stores). HPA reactivity was generally slower than reported in other birds, and there was high inter-individual variability. Sleeping birds had higher GC and glucose responses to acute stress, suggesting a more rapid mobilization of energy stores. Our results highlight the importance of considering HPA and metabolic responses to acute stress against species-specific life history and ecological relevant backgrounds.

Metals in Feathers of African Penguins (Spheniscus demersus): Considerations for the Welfare and Management of Seabirds Under Human Care.

Bird feathers have been proven to be reliable indicators of metal exposure originating from contaminated food and polluted environments. The concentrations of 15 essential and non-essential metals were investigated in African penguins (Spheniscus demersus) feathers from a Northwestern Italian zoological facility. These birds are exclusively fed with herring from the northeast Atlantic Ocean. Certain elements, such as Hg and Cd, reflected the bioaccumulation phenomena that occur through the marine food chain. The levels of Cr, Mn, and Ni were comparable to those registered in feathers of birds living in polluted areas. These results are important for comparative studies regarding the health, nutrition and welfare of endangered seabirds kept under human care.

Individual variation and repeatability of corticosterone responses of little penguins (Eudyptula minor) sampled in two successive years at Oamaru, New Zealand.

Plasma corticosterone concentrations increase when birds experience a stressor, and plasma corticosterone responses to a capture and handling stressor have been measured in many species of birds. Whilst it is assumed that the reported corticosterone responses reflect the inherent sensitivity of each bird to the stressor, responses of the same birds have rarely been measured at intervals of one or more years. The current study was conducted to measure the repeatability in two successive years of corticosterone responses of little penguins (Eudyptula minor) at Oamaru, New Zealand. There was a wide range of individual corticosterone responses to capture and restraint in 96 little penguins in 2012 and 50 penguins sampled at the same time of year in 2013. There were significant repeatabilities for the ranks of corticosterone at 15, 30 and 60min (r=0.416±0.160, r=0.636±0.115 and r=0.380±0.166 respectively) and for the ranks of integrated corticosterone responses (r=0.594±0.126) for 23 birds sampled in both years. There were no significant relationships between the size of corticosterone responses and age, body weight or condition index. Mean corticosterone concentrations at 60min were 114.22±6.65ng/ml in 2012 and 116.94±6.42ng/ml in 2013. Mean corticosterone responses did not differ between two successive years and were greater than responses of other penguin species. Penguins are well suited to long term studies in which corticosterone responses are measured annually as potential measures of changing marine environmental conditions.

Uncoupling effect of palmitate is exacerbated in skeletal muscle mitochondria of sea-acclimatized king penguins (Aptenodytes patagonicus).

In king penguin juveniles, the environmental transition from a terrestrial to a marine habitat, occurring at fledging, drastically stimulates lipid catabolism and the remodelling of muscle mitochondria to sustain extensive swimming activity and thermoregulation in the cold circumpolar oceans. However, the exact nature of these mechanisms remains only partially resolved. Here we investigated, in vitro, the uncoupling effect of increasing doses of fatty acids in pectoralis muscle intermyofibrillar mitochondria isolated, either from terrestrial never-immersed or experimentally cold water immersed pre-fledging king penguins or from sea-acclimatized fledged penguins. Mitochondria exhibited much greater palmitate-induced uncoupling respiration and higher maximal oxidative capacity after acclimatization to marine life. Such effects were not reproduced experimentally after repeated immersions in cold water, suggesting that the plasticity of mitochondrial characteristics may not be primarily driven by cold exposure per se but by other aspects of sea acclimatization.

Sex- and age-related variation in metal content of penguin feathers.

The presence of xenobiotics, such as metals, in ecosystems is concerning due to their durability and they pose a threat to the health and life of organisms. Moreover, mercury can biomagnify in many marine food chains and, therefore, organisms at higher trophic levels can be adversely impacted. Although feathers have been used extensively as a bio-monitoring tool, only a few studies have addressed the effect of both age and sex on metal accumulation. In this study, the concentrations of trace elements were determined in the feathers of all members of a captive colony of African Penguins (Spheniscus demersus) housed in a zoological facility in Italy. Tests were performed by inductively coupled plasma-mass spectrometry to detect aluminum, arsenic, cadmium, cobalt, chromium, copper, iron, manganese, nickel, lead, selenium, tin, vanadium, and zinc. Mercury was detected by a direct mercury analyzer. Sexing was performed by a molecular approach based on analyzing the chromo-helicase-DNA-binding1 gene, located on the sex chromosomes. Sex- and age-related differences were studied in order to investigate the different patterns of metal bioaccumulation between male and female individuals and between adults and juveniles. Juvenile females had significantly higher arsenic levels than males, while selenium levels increased significantly with age in both sexes. Penguins kept in controlled environments-given that diet and habitat are under strict control-represent a unique opportunity to determine if and how metal bioaccumulation is related to sex and age.

Structural basis for the ligand-binding specificity of fatty acid-binding proteins (pFABP4 and pFABP5) in gentoo penguin.

Fatty acid-binding proteins (FABPs) are involved in transporting hydrophobic fatty acids between various aqueous compartments of the cell by directly binding ligands inside their ß-barrel cavities. Here, we report the crystal structures of ligand-unbound pFABP4, linoleate-bound pFABP4, and palmitate-bound pFABP5, obtained from gentoo penguin (Pygoscelis papua), at a resolution of 2.1 Å, 2.2 Å, and 2.3 Å, respectively. The pFABP4 and pFABP5 proteins have a canonical ß-barrel structure with two short α-helices that form a cap region and fatty acid ligand binding sites in the hydrophobic cavity within the ß-barrel structure. Linoleate-bound pFABP4 and palmitate-bound pFABP5 possess different ligand-binding modes and a unique ligand-binding pocket due to several sequence dissimilarities (A76/L78, T30/M32, underlining indicates pFABP4 residues) between the two proteins. Structural comparison revealed significantly different conformational changes in the ß3-ß4 loop region (residues 57-62) as well as the flipped Phe60 residue of pFABP5 than that in pFABP4 (the corresponding residue is Phe58). A ligand-binding study using fluorophore displacement assays shows that pFABP4 has a relatively strong affinity for linoleate as compared to pFABP5. In contrast, pFABP5 exhibits higher affinity for palmitate than that for pFABP4. In conclusion, our high-resolution structures and ligand-binding studies provide useful insights into the ligand-binding preferences of pFABPs based on key protein-ligand interactions.

Reassessment of the cardio-respiratory stress response, using the king penguin as a model.

Research in to short-term cardio-respiratory changes in animals in reaction to a psychological stressor typically describes increases in rate of oxygen consumption (V̇(O2)) and heart rate. Consequently, the broad consensus is that they represent a fundamental stressor response generalizable across adult species. However, movement levels can also change in the presence of a stressor, yet studies have not accounted for this possible confound on heart rate. Thus the direct effects of psychological stressors on the cardio-respiratory system are not resolved. We used an innovative experimental design employing accelerometers attached to king penguins (Aptenodytes patagonicus) to measure and thus account for movement levels in a sedentary yet free-to-move animal model during a repeated measures stress experiment. As with previous studies on other species, incubating king penguins (N = 6) exhibited significant increases in both V̇(O2) and heart rate when exposed to the stressor. However, movement levels, while still low, also increased in response to the stressor. Once this was accounted for by comparing periods of time during the control and stress conditions when movement levels were similar as recorded by the accelerometers, only V̇(O2) significantly increased; there was no change in heart rate. These findings offer evidence that changing movement levels have an important effect on the measured stress response and that the cardio-respiratory response per se to a psychological stressor (i.e. the response as a result of physiological changes directly attributable to the stressor) is an increase in V̇(O2) without an increase in heart rate.

Mercury Stable Isotopes in Ornithogenic Deposits As Tracers of Historical Cycling of Mercury in Ross Sea, Antarctica.

Production of methylmercury (MeHg) in ocean waters and its bioaccumulation in marine organisms are critical processes controlling the fate and toxicity of mercury (Hg). However, these processes are not well understood in the Antarctic, where high levels of MeHg are observed in the subsurface ocean (100-1000 m). We explored the use of Hg stable isotope compositions in historical and modern biological deposits as a new approach for discerning Hg sources and tracing MeHg cycling in the ocean and bioaccumulation in marine biota. We found similar mass independent isotope fractionation (MIF) of Hg between a sediment profile containing historical penguin and seal feces deposits from coastal Antarctica and modern penguin and seal feces, suggesting that penguin and seal feces were the dominant sources of Hg to the sediments at different time periods. Furthermore, sediments dominated by seal feces displayed a significantly lower MIF slope (Δ(199)Hg/Δ(201)Hg) than those dominated by penguin feces despite similar extents of MIF. Since seals forage at greater depths (>400 m) than penguins (<100 m), the high MIF values and lower Δ(199)Hg/Δ(201)Hg in seal feces suggest that a significant fraction of MeHg accumulated by seals was produced in situ in the subsurface ocean from residual inorganic Hg(II) that sank from the euphotic zone after partial photoreduction. Our results suggest that in situ Hg methylation can be an important source of MeHg for marine biota, and Hg isotope compositions in biological archives can be valuable tracers of MeHg cycling.

Relationships between isotopic values and oxidative status: insights from populations of gentoo penguins.

Feeding strategies can affect the balance between the production of reactive oxygen species and antioxidant defences (i.e. oxidative status). This is ecologically relevant, as variation in oxidative status can in turn strongly affect fitness. However, how animals regulate their oxidative status through their feeding behaviour under natural conditions remains poorly understood. Thus, relating the isotopic values of free-ranging animals to their oxidative status may prove useful. Here, we considered three colonies of gentoo penguins (Pygoscelis papua) in which we measured (1) δ(13)C and δ(15)N values, and (2) antioxidant defences and oxidative damage. We found that colonies with the highest δ(13)C and δ(15)N values also had the highest levels of antioxidant defences and oxidative damage, resulting in positive relationships between isotopic values and markers of oxidative status. As a result, colony segregation in terms of isotopic values was reflected by segregation in terms of oxidative markers (although more markedly for oxidative damage than for antioxidant defences). Interestingly, variation in the estimated contribution of krill in the diet of penguins followed an opposite pattern to that observed for markers of oxidative status, providing evidence that inter-population differences in terms of foraging strategies can result in inter-population differences in terms of oxidative status. More studies examining simultaneously oxidative status, isotopic signature, foraging behaviour and food allocation between parents and young are, however, needed to understand better the interplay between the foraging strategies adopted by animals in their natural habitat and their oxidative status.

Non-invasive monitoring of adrenocortical activity in captive African Penguin (Spheniscus demersus) by measuring faecal glucocorticoid metabolites.

Measurement of faecal glucocorticoid metabolites (FGMs) has become a useful and widely-accepted method for the non-invasive evaluation of stress in vertebrates. In this study we assessed the adrenocortical activity of five captive African Penguins (Spheniscus demersus) by means of FGM evaluation following a biological stressor, i.e. capture and immobilization. In addition, we detected individual differences in secretion of FGMs during a stage of the normal biological cycle of penguins, namely the breeding period, without any external or induced causes of stress. Our results showed that FGM concentrations peaked 5.5-8h after the induced stress in all birds, and significantly decreased within 30 h. As predictable, the highest peak of FGMs (6591 ng/g) was reached by the youngest penguin, which was at its first experience with the stressor. This peak was 1.8-2.7-fold higher compared to those of the other animals habituated to the stimulus. For the breeding period, our results revealed that the increase in FGMs compared to ordinary levels, and the peaks of FGMs, varied widely depending on the age and mainly on the reproductive state of the animal. The bird which showed the lowest peak (2518 ng/g) was an old male that was not in a reproductive state at the time of the study. Higher FGM increases and peaks were reached by the two birds which were brooding (male: 5552%, 96,631 ng/g; female: 1438%, 22,846 ng/g) and by the youngest bird (1582%, 39,700 ng/g). The impact of the reproductive state on FGM levels was unexpected compared to that produced by the induced stress. The EIA used in this study to measure FGM levels proved to be a reliable tool for assessing individual and biologically-relevant changes in FGM concentrations in African Penguin. Moreover, this method allowed detection of physiological stress during the breeding period, and identification of individual differences in relation to the reproductive status. The increase in FGM levels as a response to capture and immobilization suggests that the measured metabolites are appropriate indicators of adrenal activity in these birds.