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Experiments examining mercury (Hg) toxicity in Daphnia are usually conducted in highly standardized conditions that prevent the formation of biofilm. Although such standardization has many advantages, extrapolation of results to natural conditions and inference of ecological effects is challenging. This is especially true since biofilms can accumulate metals/metalloids and play a key role in their transfer to higher trophic level organisms. In this study, we experimentally tested the effects of spontaneously appearing biofilm in Daphnia cultures on accumulation of Hg and its natural antagonist selenium (Se) in Daphnia magna. We added Hg (in the form of mercury (II) chloride) at two concentrations (0.2 µg/L and 2 µg/L) to experimental microcosms and measured the uptake of Hg and Se by D. magna in the presence and absence of biofilm. To test for consistent and replicable results, we ran two identical experimental sets one week apart. Biofilm presence significantly reduced the accumulation of Hg, while increasing the tissue Se content in D. magna, and these findings were reproducible across experimental sets. These findings indicate that highly standardized tests may not be adequate to predict the bioaccumulation and potential toxicity of metals/metalloids under natural conditions.

Background Per- and polyfluoroalkyl substances (PFASs) are environmentally persistent and bioaccumulative chemicals. Immunomodulation is among the most concerning of toxic effects linked with PFAS exposure in mammalian models. However, no studies had yet shown this to be true in birds. Thus, we designed and conducted the first study to determine if PFASs could cause immunomodulation in birds. Secondly, we wanted to determine the effects on an avian host when exposed not only to immunomodulating chemicals, but also to a viral challenge. The aim, to determine if PFAS mediated immunmodulation functionally affects a pathogen challenge for a host. As innate immune system signalling pathways initiate crucial responses against a pathogen challenge, and are lesser studied than their adaptive counterparts, we focused on these pathways. To provide the first information on this, an in vitro experiment was designed and performed using chicken embryo fibroblasts exposed to perfluorooctane sulfonate (PFOS) (22 ppm) and immune markers characterised before and after being infected with gallid herpesvirus-2 (GaHV-2). Results The expression of two pro-inflammatory cytokines, namely interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α), the nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NF-κB), and the anti-inflammatory cytokine interleukin 4 (IL-4) were investigated in various scenarios. These results showed that exposure to PFOS decreased immune gene expression in chicken fibroblasts from 36 h post-exposure. Next, it was shown that this decrease could be mitigated by infection with gallid herpesvirus-2, which increased gene expression back to the baseline/control levels. Conclusions Not only is this the first study to provide the expected evidence that PFOS has immunomodulatory potential in birds, it also provides unexpected data that virus infections can mitigate this negative effect. Thereby, further research, including in vivo and in situ studies, on the impact of PFOS on host-virus interactions is now warranted, as it has been overlooked and might contribute to our understanding of recent disease outbreaks in wildlife. The mechanisms by which gallid herpesvirus mitigates immunomodulation were beyond the scope of this study, but are now of interest for future study.

Exposure to pollutants is a potentially crucial but overlooked driver of population declines in shorebirds along the East Asian-Australasian Flyway. We combined knowledge of moult strategy and life history with a standardised sampling protocol to assess mercury (Hg) contamination in 984 individuals across 33 migratory shorebird species on an intercontinental scale. Over one-third of the samples exceeded toxicity benchmarks. Feather Hg was best explained by moulting region, while habitat preference (coastal obligate vs. non-coastal obligate), the proportion of invertebrates in the diet and foraging stratum (foraging mostly on the surface vs. at depth) also contributed, but were less pronounced. Feather Hg was substantially higher in South China (Mai Po and Leizhou), Australia and the Yellow Sea than in temperate and Arctic breeding ranges. Non-coastal obligate species ( Tringa genus) frequently encountered in freshwater habitats were at the highest risk. It is important to continue and expand biomonitoring research to assess how other pollutants might impact shorebirds. Over one-third of the sampled shorebirds along the East Asian-Australasian Flyway are facing Hg risk. Tringa genus in South China was at the highest risk. Feather Hg was best explained by feathers’ moulting region, while habitat preference, diet, and foraging stratum were less important.

Background Capillary electrophoresis of plasma proteins has shown great potential as a complementary diagnostic tool for avian species. However, reference intervals for plasma proteins are sparse or lacking for several free-living avian species. The current study reports electrophoretic patterns and concentrations of plasma proteins determined for 70 free-living white-tailed eagle ( Haliaeetus albicilla ) nestlings from two locations in Norway (Steigen and Smøla) in order to establish reference values for this subpopulation using capillary electrophoresis. The nestlings were between 44 and 87 days of age, and the plasma protein concentrations were investigated for age, sex, year (2015 and 2016) and location differences. To our knowledge, this is the first report of reference intervals of plasma proteins analysed by capillary electrophoresis in free-living white-tailed eagle nestlings. Results The plasma protein concentrations (% of total protein, mean ± SE) were determined for prealbumin (13.7%, 4.34 ± 0.15 g/L), albumin (46.7%, 14.81 ± 0.24 g/L), α 1 -globulin (2.4%, 0.74 ± 0.03 g/L), α 2 -globulin (11.7%, 3.72 ± 0.06 g/L), β-globulin (15.9%, 5.06 ± 0.08 g/L) and γ-globulin (9.6%, 3.05 ± 0.09 g/L). Significant differences were found between the two locations for prealbumin, α 2 - and γ-globulins. No significant differences were found between the two sampling years or sexes, and no effect of age was found for any of the plasma proteins. However, prealbumin levels were several folds higher than previously reported from adults of closely related birds of prey species. There were no other studies on capillary electrophoresis of nestling plasma available for comparison. Conclusion Significant differences were found between sampling locations for prealbumin, α 2 - and γ-globulins, which may indicate differences in inflammatory or infectious status between nestlings at the two locations. Sampling year, sex or age had no significant effect on the plasma protein concentrations. These results provide novel data on plasma protein concentrations by capillary electrophoresis and may be useful for evaluation of health status in free-living white-tailed eagle nestlings.

Background Wild aquatic birds serve as the natural reservoir for avian influenza virus (AIV), a disease with significant implications for avian and mammalian health. Climate change is predicted to impact the dynamics of AIV, particularly in areas such as the Arctic, but the baseline data needed to detect these shifts is often unavailable. In this study, plasma from two species of gulls breeding on the high-Arctic Svalbard archipelago were screened for antibodies to AIV. Results AIV antibodies were found in black-legged kittiwake (Rissa tridactyla) samples from multiple years, as well as in glaucous gulls (Larus hyperboreous) samples. Conclusions Despite small sample sizes, evidence of exposure to AIV was found among Svalbard gulls. A wider survey of Svalbard avian species is warranted to establish knowledge on the extent of AIV exposure on Svalbard and to determine whether active infections are present.

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Birds of prey, owls and falcons are widely used as sentinel species in raptor biomonitoring programmes. A major current challenge is to facilitate large-scale biomonitoring by coordinating contaminant monitoring activities and by building capacity across countries. This requires sharing, dissemination and adoption of best practices addressed by the Networking Programme Research and Monitoring for and with Raptors in Europe (EURAPMON) and now being advanced by the ongoing international COST Action European Raptor Biomonitoring Facility. The present perspective introduces a schematic sampling protocol for contaminant monitoring in raptors. We provide guidance on sample collection with a view to increasing sampling capacity across countries, ensuring appropriate quality of samples and facilitating harmonization of procedures to maximize the reliability, comparability and interoperability of data. The here presented protocol can be used by professionals and volunteers as a standard guide to ensure harmonised sampling methods for contaminant monitoring in raptors.

Neonicotinoids have been detected in farmland-associated birds and exposure to these insecticides has been linked to adverse effects. Even though neonicotinoids are mobile and persistent and have been detected in surface waters and aquatic invertebrates, there is a considerable lack of knowledge on their occurrence in waterbirds. Here we investigated the occurrence of seven neonicotinoids and some of their transformation products (imidacloprid, thiacloprid, thiamethoxam, acetamiprid, clothianidin, dinotefuran, nitenpyram, 6-chloronicotinic acid, hydroxy-imidacloprid, imidacloprid-urea, imidacloprid-olefin, thiamethoxam-urea, thiacloprid-amide, acetamiprid-acetate, and acetamiprid-desmethyl) in blood plasma of 51 incubating female common goldeneyes ( Bucephala clangula ). We collected samples from five different regions from southern to northern Finland encompassing rural and urban settings in coastal and inland areas. Surprisingly, none of the targeted neonicotinoids was found above the limit of detection in any of the samples. As neonicotinoid concentrations in wild birds can be very low, a likely reason for the nil results is that the LODs were too high; this and other possible reasons for the lack of detection of neonicotinoids in the goldeneyes are discussed. Our results suggest that neonicotinoid exposure in their breeding areas is currently not of major concern to female goldeneyes in Finland. Even though this study did not find any immediate danger of neonicotinoids to goldeneyes, further studies including surface water, aquatic invertebrates, and other bird species could elucidate potential indirect food chain effects.

MicroRNAs (miRNAs) are highly conserved small noncoding RNAs that regulate gene expression post‐transcriptionally. Circulating miRNAs – miRNAs that have been released from cells and circulate in the bloodstream – are relatively stable and interesting molecules for wildlife research, where they may form a proxy for gene expression as a function of the animal's state under a variety of environmental challenges. Aiming at providing initial baseline data on the circulating miRNAome in avian wildlife, we assessed the miRNA profiles of wild ruddy turnstones Arenaria interpres on their Australian non‐breeding grounds. The ruddy turnstone is a long‐distant migrant and a significant reservoir species for low pathogenic avian influenza virus (LPAIV). We therefore investigated both LPAIV‐infected and uninfected individuals for their specific miRNA profiles to potentially elucidate the species' molecular mechanisms underlying its response to LPAIV infection. De novo miRNA characterisation in the ruddy turnstone genome identified 161 conserved and two novel, bird‐specific miRNAs, with liver‐enriched miRNA‐122 being the most abundant. Z chromosome‐linked miR‐2954‐3p was significantly more abundant in serum from males (ZZ) than from females (ZW). Furthermore, we found a sex‐ and age‐associated effect of LPAIV infection on miRNA abundance in serum samples, including one novel miRNA. This circulating miRNA signature may reflect sex‐ and age‐specific differences in the host response, indicating that circulating miRNAs could serve as a valuable non‐destructive analytical tool for enhancing our understanding of avian infections in a wildlife context and should be explored further.

Many persistent organic pollutants, such as polychlorinated biphenyls (PCBs), have high immunomodulating potentials. Exposure to them, in combination with virus infections, has been shown to aggravate outcomes of the infection, leading to increased viral titers and host mortality. Expression of immune-related microRNA (miR) signaling pathways (by host and/or virus) have been shown to be important in determining these outcomes; there is some evidence to suggest pollutants can cause dysregulation of miRNAs. It was thus hypothesized here that modulation of miRNAs (and associated cytokine genes) by pollutants exerts negative effects during viral infections. To test this, an in vitro study on chicken embryo fibroblasts (CEF) exposed to a PCB mixture (Aroclor 1260) and then stimulated with a synthetic RNA virus (poly(I:C)) or infected with a lymphoma-causing DNA virus (Gallid Herpes Virus 2 [GaHV-2]) was conducted. Using quantitative real-time PCR, expression patterns for mir-155, pro-inflammatory TNFα and IL-8, transcription factor NF-κB1, and anti-inflammatory IL-4 were investigated 8, 12, and 18 h after virus activation. The study showed that Aroclor1260 modulated mir-155 expression, such that a down-regulation of mir-155 in poly(I:C)-treated CEF was seen up to 12 h. Aroclor1260 exposure also increased the mRNA expression of pro-inflammatory genes after 8 h in poly(I:C)-treated cells, but levels in GaHV-2-infected cells were unaffected. In contrast to with Aroclor1260/poly(I:C), Aroclor1260/GaHV-2-infected cells displayed an increase in mir-155 levels after 12 h compared to levels seen with either individual treatment. While after 12 h expression of most evaluated genes was down-regulated (independent of treatment regimen), by 18 h, up-regulation was evident again. In conclusion, this study added evidence that mir-155 signaling represents a sensitive pathway to chemically-induced immunomodulation and indicated that PCBs can modulate highly-regulated innate immune system signaling pathways important in determining host immune response outcomes during viral infections.