The structure and proteomic analysis of byssus in Pteria penguin: Insights into byssus evolution and formation.

Byssus is a unique external structure in sessile bivalves and is critical for settlement and metamorphosis. However, little is known about the stout byssus in Pteria penguin. We explored the byssus structure and proteins using scanning electron microscopy and proteomics, respectively. The results revealed that P. penguin byssus has a dense and highly aligned fiber inner core, and the outer cuticle contains protein granules embedded in the protein matrix. Proteomic analysis revealed 31 proteins in the byssus, among which 15 differentially expressed proteins were mainly enriched in the EGF/EGF-like and laminin EGF-like domains. Foot proteins were enriched in the EF-hand, immunoglobulin, and fibronectin domains. All these domains can participate in protein-protein and/or protein-metal interactions in the extracellular matrix (ECM), which, together with the seven types of ECM proteins detected in the byssus, supports the hypothesis that the byssus is derived from the ECM. We also found that in vitro acellular structures of the byssus and the shell shared commonalities in their formation processes. These results are useful for further understanding byssus evolution and the characterization of byssus-related proteins. SIGNIFICANCE: This manuscript investigates the structure and the origin of Pteria penguin byssus, given that byssus is vital to provide critical protection for reproduction and even against environmental stresses that affect survival. However, there is rare research on byssus protein composition. Hence, though scanning electron microscopy and proteomic analysis, we discovered that P. penguin byssus possesses the dense and highly aligned fiber inner core, and the outer cuticle has protein granules embedded in the protein matrix. Proteomic analysis showed that there were 31 proteins in the byssus, among which 15 proteins were mainly enriched in the EGF/EGF-like and laminin EGF-like domains. Foot proteins closely related to byssus formation were enriched in EF hand, immunoglobulin, and fibronectin domains. These domains are able to participate in protein-protein and/or protein-metal interactions in the extracellular matrix (ECM), which together with the seven types of ECM proteins detected in byssus support the hypothesis that byssus derive from the ECM. We also found in vitro acellular structures the byssus and the shell share commonalities in their formation processes. These results were useful for further understanding the byssus evolution and the characterization of the byssus-related proteins.

Do microplastics accumulate in penguin internal organs? Evidence from Svenner island, Antarctica.

The prevalence of microplastics (MPs, <5 mm) in natural environments presents a formidable global environmental threat MPs can be found from the Arctic to Antarctica, including glaciers. Despite their widespread distribution, studies on MP accumulation in apex predators inhabiting Polar Regions remain limited. The objective of this study was to conduct a comprehensive examination, for the first time, of MP bioaccumulation in various organs and tissue of Adélie penguins. This investigation comprehends the gastrointestinal tract (GIT), scat, internal organ (lung, trachea, spleen, and liver) and tissue (muscle) samples collected from Svenner Island, Antarctica during the 39th Indian expedition to Antarctica in 2019-2020. Our analyses revealed the presence of 34 MPs across the GIT, scat, lung, and trachea samples, with no MPs detected in muscle, spleen, or liver tissues. Blue-colored microfibers (>50 %) and MPs smaller than 1 mm (38 %) in size were prominently observed. Polymer characterization utilizing µ-FTIR spectroscopy identified low-density polyethylene (LDPE) (~63 %) as the predominant polymer type. The accumulation of MP fibers in the gastrointestinal tract and scat of Adélie penguins may originate from marine ambient media and prey organisms. Furthermore, the presence of LDPE fibers in the trachea and lungs likely occurred through inhalation and subsequent deposition of MPs originating from both local and long-range airborne sources. The identification of fibers ranging between 20 and 100 µm within the trachea suggests a plausible chance of cellular deposition of MPs. Overall our findings provide valuable insights into the organ-specific accumulation of MPs in apex predators. Adélie penguins emerge as promising environmental bio-monitoring species, offering insights into the potential trophic transfer of MPs within frigid environments.

Influence of migration range and foraging ecology on mercury accumulation in Southern Ocean penguins.

In order to evaluate mercury (Hg) accumulation patterns in Southern Ocean penguins, we measured Hg concentrations and carbon (δ13C) and nitrogen (δ15N) stable isotope ratios in body feathers of adult Adélie (Pygoscelis adeliae), gentoo (Pygoscelis papua), and chinstrap (Pygoscelis antarctica) penguins living near Anvers Island, West Antarctic Peninsula (WAP) collected in the 2010/2011 austral summer. With these and data from Pygoscelis and other penguin genera (Eudyptes and Aptenodytes) throughout the Southern Ocean, we modelled Hg variation using δ13C and δ15N values. Mean concentrations of Hg in feathers of Adélie (0.09 ± 0.05 µg g-1) and gentoo (0.16 ± 0.08 µg g-1) penguins from Anvers Island were among the lowest ever reported for the Southern Ocean. However, Hg concentrations in chinstrap penguins (0.80 ± 0.20 µg g-1), which undertake relatively broad longitudinal winter migrations north of expanding sea ice, were significantly higher (P < 0.001) than those in gentoo or Adélie penguins. δ13C and δ15N values for feathers from all three Anvers Island populations were also the lowest among those previously reported for Southern Ocean penguins foraging within Antarctic and subantarctic waters. These observations, along with size distributions of WAP krill, suggest foraging during non-breeding seasons as a primary contributor to higher Hg accumulation in chinstraps relative to other sympatric Pygoscelis along the WAP. δ13C values for all Southern Ocean penguin populations, alone best explained feather Hg concentrations among possible generalized linear models (GLMs) for populations grouped by either breeding site (AICc = 36.9, wi = 0.0590) or Antarctic Frontal Zone (AICc = 36.9, wi = 0.0537). Although Hg feather concentrations can vary locally by species, there was an insignificant species-level effect (wi < 0.001) across the full latitudinal range examined. Therefore, feeding ecology at breeding locations, as tracked by δ13C, control Hg accumulation in penguin populations across the Southern Ocean.

Evaluation of the stress state based on fecal corticosterone metabolite concentrations in captive penguins in Japan.

Fecal corticosterone metabolite (FCM) concentrations, which can be determined noninvasively, have recently been explored as a stress indicator in birds. In our study, we measured FCM concentrations in penguins under nonmolting or molting conditions, cool or hot season, diseased condition, and incubation period. These measurements were conducted in an aquarium that housed king penguins, gentoo penguins, and African penguins. This study aimed to investigate the validity of fecal matter as a stress indicator. Our findings revealed that FCM concentrations were significantly higher in molting individuals than in nonmolting individuals. Compared with the cool season, FCM concentrations were significantly higher in penguins housed outdoors during the hot season. However, no differences were observed in penguins housed indoors. Diseased individuals and an incubating individual showed notably higher FCM concentrations than healthy individuals. Interestingly, the FCM concentration in king penguin that underwent cataract surgery was extremely high before the surgery. However, 1 month postsurgery, it decreased to a level similar to that of healthy individuals. We observed increased FCM concentrations in penguins considered to be exposed to stressors. Notably, FCM concentration decreased after removing the stress factor. The FCM concentration was found to be consistent with the stress state of penguins, suggesting its usefulness as a stress indicator.

Assessing mercury contamination in Southern Hemisphere marine ecosystems: The role of penguins as effective bioindicators.

Mercury (Hg) is a global pollutant known for its significant bioaccumulation and biomagnification capabilities, posing a particular threat to marine environments. Seabirds have been recognized as effective bioindicators of marine pollution, and, among them, penguins present a unique opportunity to serve as a single taxonomic group (Sphenisciformes) for monitoring Hg across distinct marine ecosystems in the Southern Hemisphere. In this study, we conducted a comprehensive systematic review of Hg concentrations, and performed a meta-analysis that took into account the various sources of uncertainty associated with Hg contamination in penguins. Beyond intrinsic species-specific factors shaping Hg levels, our results showed that the penguin community effectively reflects spatial patterns of Hg bioavailability. We identified geographic Hg hotspots in Australia, the Indian Ocean, and Tierra del Fuego, as well as coldspots in Perú and the South Atlantic. Furthermore, specific penguin species, namely the Southern Rockhopper (Eudyptes chrysocome) and Macaroni penguin (Eudyptes chrysolophus), are highlighted as particularly vulnerable to the toxic effects of Hg. Additionally, we identified knowledge gaps in geographic areas such as the Galápagos Islands, South Africa, and the coast of Chile, as well as in species including Fiordland (Eudyptes pachyrhynchus), Snares (Eudyptes robustus), Erect-crested (Eudyptes sclateri), Royal (Eudyptes schlegeli), Yellow-eyed (Megadyptes antipodes), and Galápagos (Spheniscus mendiculus) penguins. Overall, our study contributes to the growing body of literature emphasizing the role of penguins as bioindicators of Hg pollution, but it also highlights areas where further research and data collection are needed for a more comprehensive understanding of Hg contamination in marine ecosystems in the Southern Hemisphere.

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.

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.

Mercury biomagnification in an Antarctic food web of the Antarctic Peninsula.

Under the climate change context, warming Southern Ocean waters may allow mercury (Hg) to become more bioavailable to the Antarctic marine food web (i.e., ice-stored Hg release and higher methylation rates by microorganisms), whose biomagnification processes are poorly documented. Biomagnification of Hg in the food web of the Antarctic Peninsula, one of the world's fastest-warming regions, was examined using carbon (δ13C) and nitrogen (δ15N) stable isotope ratios for estimating feeding habitat and trophic levels, respectively. The stable isotope signatures and total Hg (T-Hg) concentrations were measured in Antarctic krill Euphausia superba and several Antarctic predator species, including seabirds (gentoo penguins Pygoscelis papua, chinstrap penguins Pygoscelis antarcticus, brown skuas Stercorarius antarcticus, kelp gulls Larus dominicanus, southern giant petrels Macronectes giganteus) and marine mammals (southern elephant seals Mirounga leonina). Significant differences in δ13C values among species were noted with a great overlap between seabird species and M. leonina. As expected, significant differences in δ15N values among species were found due to interspecific variations in diet-related to their trophic position within the marine food web. The lowest Hg concentrations were registered in E. superba (0.007 ± 0.008 µg g-1) and the highest values in M. giganteus (12.090 ± 14.177 µg g-1). Additionally, a significant positive relationship was found between Hg concentrations and trophic levels (reflected by δ15N values), biomagnifying nearly 2 times its concentrations at each level. Our results support that trophic interaction is the major pathway for Hg biomagnification in Southern Ocean ecosystems and warn about an increase in the effects of Hg on long-lived (and high trophic level) Antarctic predators under climate change in the future.

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.

Foraging dive frequency predicts body mass gain in the Adélie penguin.

Quantifying food intake in wild animals is crucial to many ecological and evolutionary questions, yet it can be very challenging, especially in the marine environment. Because foraging behavior can be inferred from dive recordings in many marine creatures, we hypothesized that specific behavioral dive variables can indicate food intake. To test this hypothesis, we attached time-depth recorders to breeding Adélie penguins also implanted with RFID tags that crossed a weighbridge as they traveled to and from the ocean to feed their chicks. The weighbridge reported how much mass the penguin had gained during a foraging trip. The variables that explained a significant amount of the change in body mass while at sea were the number of foraging dives per hour (46%) and the number of undulations per hour (12%). Most importantly, every increment of 1 in the rate of foraging dives per hour equated to a penguin gaining an average 170 g of mass, over the course of a 6-60 h foraging trip. These results add to a growing understanding that different metrics of foraging success are likely appropriate for different species, and that assessing the types and frequencies of dives using time-depth recorders can yield valuable insights.

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.

Validating accelerometry-derived proxies of energy expenditure using the doubly labelled water method in the smallest penguin species.

Understanding energy use is central to understanding an animal's physiological and behavioural ecology. However, directly measuring energy expenditure in free-ranging animals is inherently difficult. The doubly labelled water (DLW) method is widely used to investigate energy expenditure in a range of taxa. Although reliable, DLW data collection and analysis is both financially costly and time consuming. Dynamic body acceleration (e.g. VeDBA) calculated from animal-borne accelerometers has been used to determine behavioural patterns, and is increasingly being used as a proxy for energy expenditure. Still its performance as a proxy for energy expenditure in free-ranging animals is not well established and requires validation against established methods. In the present study, the relationship between VeDBA and the at-sea metabolic rate calculated from DLW was investigated in little penguins (Eudyptula minor) using three approaches. Both in a simple correlation and activity-specific approaches were shown to be good predictors of at-sea metabolic rate. The third approach using activity-specific energy expenditure values obtained from literature did not accurately calculate the energy expended by individuals. However, all three approaches were significantly strengthened by the addition of mean horizontal travel speed. These results provide validation for the use of accelerometry as a proxy for energy expenditure and show how energy expenditure may be influenced by both individual behaviour and environmental conditions.

Acceleration predicts energy expenditure in a fat, flightless, diving bird.

Energy drives behaviour and life history decisions, yet it can be hard to measure at fine scales in free-moving animals. Accelerometry has proven a powerful tool to estimate energy expenditure, but requires calibration in the wild. This can be difficult in some environments, or for particular behaviours, and validations have produced equivocal results in some species, particularly air-breathing divers. It is, therefore, important to calibrate accelerometry across different behaviours to understand the most parsimonious way to estimate energy expenditure in free-living conditions. Here, we combine data from miniaturised acceleration loggers on 58 free-living Adélie penguins with doubly labelled water (DLW) measurements of their energy expenditure over several days. Across different behaviours, both in water and on land, dynamic body acceleration was a good predictor of independently measured DLW-derived energy expenditure (R2 = 0.72). The most parsimonious model suggested different calibration coefficients are required to predict behaviours on land versus foraging behaviour in water (R2 = 0.75). Our results show that accelerometry can be used to reliably estimate energy expenditure in penguins, and we provide calibration equations for estimating metabolic rate across several behaviours in the wild.

Biological factors affecting total mercury and methylmercury levels in Antarctic penguins.

Penguins in Antarctica occupy high trophic levels, thus accumulating high amounts of mercury (Hg) through bioaccumulation and biomagnification. Blood reflects the current levels of contaminants circulating in the body, while feathers are known as the main route of Hg elimination in birds. Studies sampling chicks and adults can provide a comprehensive picture of bioaccumulation and local contamination. Three pygoscelid species (Pygoscelis adeliae, Pygoscelis antarcticus and Pygoscelis papua) have circumpolar distributions being the ideal sentinels of Antarctic environmental pollution. This study aimed to assess Hg contamination of the pristine Antarctic region using non-destructive penguin samples. Fieldwork was carried out during the austral summer of 2013/2014 in the South Shetland Islands, off the north-west Antarctic Peninsula. Concentrations of total Hg (ng.g-1 dw) in blood ranged from 39 to 182 in chicks and 45 to 581 in adults, while concentrations in feathers ranged from 73 to 598 in chicks and 156 to 1648 in adults. Most Hg in feathers (about 70%) is accumulated in the form of methylmercury. Differences were demonstrated in mercury bioaccumulation were related to species and age, but not to sex. To our knowledge this is the first study to report MeHg levels in both juvenile and adult pygoscelid penguins.

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.

Adélie penguin colonies as indicators of brominated flame retardants (BFRs) in East Antarctica.

While persistent organic pollutant (POP) contamination within Antarctica is largely caused by long-range atmospheric transport (LRAT), Antarctic research bases have been shown to be local sources of POPs such as brominated flame retardants (BFRs). This study compared concentrations of seven polybrominated diphenyl ethers (PBDE) congeners and five novel flame retardants (NBFRs) found in Adélie penguin (Pygoscelis adeliae) colony soils near the Australian research stations, Mawson and Davis, to assess the stations as local sources of these contaminants and provide a much needed baseline for contamination of BFRs in East Antarctica. Soil samples (n = 46) were collected from Adélie colonies at close proximity to the research stations as well as further afield during the 2016-17 austral summer. Samples were analysed using selective pressurised liquid extraction (S-PLE) and gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). PBDEs (BDE-28, -47, -99, -100, -153, -154 and -183) were detected in 45/46 samples with ∑7PBDE concentrations ranging from <0.01 to 1.63 ng/g dry weight (dw) and NBFRs (2,3,4,5,6-pentabromotoluene (PBT), 2,3,4,5,6-pentabromoethylbenzene (PBEB), hexabromobenzene (HBB), 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB) and bis(2,4,6-tribromophenoxy) ethane (BTBPE)) detected in 20/46 samples, with a range of ∑5NBFR from not detected (ND) to 0.16 ng/g dw. Soils taken from around the Davis and Mawson research stations were more highly contaminated (n = 10) than penguin colonies (n = 27) and control areas not affiliated with breeding seabirds (n = 8). The most common congener detected was BDE-99, reflecting inputs from LRAT. However, the congener profiles of station soils supported the hypothesis that research stations are a local source of PBDEs to the Antarctic environment. In addition, the NBFR pentabromoethylbenzene (PBEB) was quantified for the first time in Antarctic soils, providing essential information for baseline contamination within the region and highlighting the need for ongoing monitoring as global regulations for the use of BFRs continuously change.

Feathers of Humboldt penguin are suitable bioindicators of Rare Earth Elements.

Rare earth elements (REEs), also called lanthanides, are emerging contaminants worldwide, due to their unique physical and chemical characteristics that make them essential in a variety of industrial applications. However, there is still a gap in the knowledge of occurrence and accumulation of REEs in biota, and no investigations have yet been performed in penguin feathers, which have already been widely utilized as a non-invasive tool for the biomonitoring of trace elements. The concentrations of 16 REEs were investigated in a colony of Humboldt penguins (Spheniscus humboldti) housed at the Acquario di Cattolica (Italy). Multielement determination of REEs was performed by an Inductively Coupled Plasma-Mass Spectrometer after a microwave digestion of feathers. As this colony lives indoors in a controlled environment, it was the ideal choice for studying lanthanide occurrence in penguin feathers. Since there is a strict link between metal levels in feathers and the diet of penguins, their food (capelin) was also tested for REEs. Chondrite normalized values revealed the same pattern for REEs in feathers and fish, but REE concentrations were an order of magnitude higher in penguin feathers, demonstrating the suitability of this tissue as a bioindicator of REEs.

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.

Humboldt penguins' feathers as bioindicators of metal exposure.

Avian feathers have the potential to accumulate trace elements originating from contaminated food and polluted environments. In fact, in feathers, metals bind to keratin, a sulphur-containing protein for which several metals have a strong affinity. Here, the concentrations of 18 essential and non-essential elements were investigated in a Humboldt penguin (Spheniscus humboldti) colony housed at the Acquario di Cattolica (Italy). This species is listed as vulnerable in the Red List of the International Union for Conservation of Nature. According to the literature, there is usually a link between metal levels in the diet of birds and levels detected in their feathers. Thus, metals were also determined in the penguins' food (capelin, Mallotus villosus). We hypothesize that the controlled conditions in which birds are kept in captivity, and the homogeneous diet that they follow could allow a better understanding of metal bioaccumulation (such as mercury) or bio-dilution (such as arsenic) in the marine food chain, indicated by penguins' feathers. Moreover, comparisons with our previous investigations performed on an ex-situ African penguin (Spheniscus demersus) colony suggest that penguins living indoors have lower body burden of metals than those living outdoors. Indeed, environmental contaminants usually found in areas subjected to anthropogenic impact, where zoos and aquaria are often located, are not accumulated to levels of concern.