Haemoproteus parasites and passerines: the effect of local generalists on inferences of host-parasite co-phylogeny in the British Isles.

Host­parasite associations provide a benchmark for investigating evolutionary arms races and antagonistic coevolution. However, potential ecological mechanisms underlying such associations are difficult to unravel. In particular, local adaptations of hosts and/or parasites may hamper reliable inferences of host­parasite relationships and the specialist­generalist definitions of parasite lineages, making it problematic to understand such relationships on a global scale. Phylogenetic methods were used to investigate co-phylogenetic patterns between vector-borne parasites of the genus Haemoproteus and their passeriform hosts, to infer the ecological interactions of parasites and hosts that may have driven the evolution of both groups in a local geographic domain. As several Haemoproteus lineages were only detected once, and given the occurrence of a single extreme generalist, the effect of removing individual lineages on the co-phylogeny pattern was tested. When all lineages were included, and when all singly detected lineages were removed, there was no convincing evidence for host­parasite co-phylogeny. However, when only the generalist lineage was removed, strong support for co-phylogeny was indicated, and ecological interactions could be successfully inferred. This study exemplifies the importance of identifying locally abundant lineages when sampling host­parasite systems, to provide reliable insights into the precise mechanisms underlying host­parasite interactions.

Epidemiology of protozoan and helminthic parasites in wild passerine birds of Britain and Ireland.

Avian endoparasites play important roles in conservation, biodiversity and host evolution. Currently, little is known about the epidemiology of intestinal helminths and protozoans infecting wild birds of Britain and Ireland. This study aimed to determine the rates of parasite prevalence, abundance and infection intensity in wild passerines. Fecal samples (n = 755) from 18 bird families were collected from 13 sites across England, Wales and Ireland from March 2020 to June 2021. A conventional sodium nitrate flotation method allowed morphological identification and abundance estimation of eggs/oocysts. Associations with host family and age were examined alongside spatiotemporal and ecological factors using Bayesian phylogenetically controlled models. Parasites were detected in 20.0% of samples, with corvids and finches having the highest prevalences and intensities, respectively. Syngamus (33%) and Isospora (32%) were the most prevalent genera observed. Parasite prevalence and abundance differed amongst avian families and seasons, while infection intensity varied between families and regions. Prevalence was affected by diet diversity, while abundance differed by host age and habitat diversity. Infection intensity was higher in birds using a wider range of habitats, and doubled in areas with feeders present. The elucidation of these patterns will increase the understanding of parasite fauna in British and Irish birds.

Evidence for strain-specific virulence of Trichomonas gallinae in African columbiformes.

Infection by parasites or pathogens can have marked physiological impacts on individuals. In birds, infection may affect moult and feather growth, which is an energetically demanding time in the annual cycle. Previous work has suggested a potential link between clinically visible Trichomonas gallinae infection and wing length in turtle doves Streptopelia turtur arriving on breeding grounds. First, T. gallinae infection was characterized in 149 columbids from 5 species, sampled on turtle dove wintering grounds in Senegal during the moulting period, testing whether infection by T. gallinae is linked to moult. Trichomonas gallinae prevalence was 100%, so rather than testing for differences between infected and uninfected birds, we tested for differences in moult progression between birds infected by different T. gallinae strains. Twelve strains of T. gallinae were characterized at the internal transcribed spacer 1 (ITS1)/5.8S/ITS2 region, of which 6 were newly identified within this study. In turtle doves only, evidence for differences in wing length by strain was found, with birds infected by strain Tcl-1 having wings nearly 6 mm longer than those infected with strain GEO. No evidence was found for an effect of strain identity within species on moult progression, but comparisons between infected and uninfected birds should be further investigated in species where prevalence is lower.

Assessing rates of parasite coinfection and spatiotemporal strain variation via metabarcoding: Insights for the conservation of European turtle doves Streptopelia turtur.

Understanding the frequency, spatiotemporal dynamics and impacts of parasite coinfections is fundamental to developing control measures and predicting disease impacts. The European turtle dove (Streptopelia turtur) is one of Europe's most threatened bird species. High prevalence of infection by the protozoan parasite Trichomonas gallinae has previously been identified, but the role of this and other coinfecting parasites in turtle dove declines remains unclear. Using a high-throughput sequencing approach, we identified seven strains of T. gallinae, including two novel strains, from ITS1/5.8S/ITS2 ribosomal sequences in turtle doves on breeding and wintering grounds, with further intrastrain variation and four novel subtypes revealed by the iron-hydrogenase gene. High spatiotemporal turnover was observed in T. gallinae strain composition, and infection was prevalent in all populations (89%-100%). Coinfection by multiple Trichomonas strains was rarer than expected (1% observed compared to 38.6% expected), suggesting either within-host competition, or high mortality of coinfected individuals. In contrast, coinfection by multiple haemosporidians was common (43%), as was coinfection by haemosporidians and T. gallinae (90%), with positive associations between strains of T. gallinae and Leucocytozoon suggesting a mechanism such as parasite-induced immune modulation. We found no evidence for negative associations between coinfections and host body condition. We suggest that longitudinal studies involving the recapture and investigation of infection status of individuals over their lifespan are crucial to understand the epidemiology of coinfections in natural populations.

Fungal microbiomes are determined by host phylogeny and exhibit widespread associations with the bacterial microbiome.

Interactions between hosts and their resident microbial communities are a fundamental component of fitness for both agents. Though recent research has highlighted the importance of interactions between animals and their bacterial communities, comparative evidence for fungi is lacking, especially in natural populations. Using data from 49 species, we present novel evidence of strong covariation between fungal and bacterial communities across the host phylogeny, indicative of recruitment by hosts for specific suites of microbes. Using co-occurrence networks, we demonstrate marked variation across host taxonomy in patterns of covariation between bacterial and fungal abundances. Host phylogeny drives differences in the overall richness of bacterial and fungal communities, but the effect of diet on richness was only evident in the mammalian gut microbiome. Sample type, tissue storage and DNA extraction method also affected bacterial and fungal community composition, and future studies would benefit from standardized approaches to sample processing. Collectively these data indicate fungal microbiomes may play a key role in host fitness and suggest an urgent need to study multiple agents of the animal microbiome to accurately determine the strength and ecological significance of host-microbe interactions.

Epitherapy and immune checkpoint blockade: using epigenetic reinvigoration of exhausted and dysfunctional T cells to reimburse immunotherapy response.

BACKGROUND: Cancer cells subvert natural immunosuppression by upregulating the expression of checkpoint proteins and their ligands. For example, tumor cells expressing programmed death-ligand 1 (PD-L1) induce immune cell tolerance to cancers, thereby facilitating tumor progression. The recent clinical success of immunotherapy, particularly checkpoint blockade, represents a significant advance in cancer therapy. However, many cancers develop resistance to immunotherapies, and the underlying mechanisms and how these might be exploited to overcome resistance still need to be determined. METHODS: T cell dysfunction, in part caused by chronic T cell receptor stimulation, diminishes the capacity for durable responses to checkpoint blockade. Furthermore, T cell populations are phenotypically and functionally heterogeneous, resulting in varying responses to checkpoint blockade. Recent molecular studies of T cell heterogeneity have shown that checkpoint blockade on its own does not alter the epigenetic landscape of T cells, despite epigenetic changes governing T cell phenotype. CONCLUSION: Here we argue that epigenetic modifiers can be used to prime and sensitize T cells to immunotherapy. Administering epitherapy in conjunction with checkpoint blockade could decrease T cell exhaustion and immunotherapy resistance in many cancer types.

Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation.

Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation. This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood. By genome-wide ChIP-sequencing ex vivo human CD4(+) T cells, here, we show that the signaling enzyme, protein kinase C theta (PKC-θ) directly relays stimulatory signals to chromatin by binding to transcriptional-memory-responsive genes to induce transcriptional activation. Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4(+) T cells. Within the nucleus, PKC-θ catalytic activity maintains the Ser536 phosphorylation on the p65 subunit of NF-κB (also known as RelA) and can directly influence chromatin accessibility at transcriptional memory genes by regulating H2B deposition through Ser32 phosphorylation. Furthermore, using a cytoplasm-restricted PKC-θ mutant, we highlight that chromatin-anchored PKC-θ integrates activating signals at the chromatin template to elicit transcriptional memory responses in human memory T cells.

The decline of the Turtle Dove: Dietary associations with body condition and competition with other columbids analysed using high-throughput sequencing.

Dietary changes linked to the availability of anthropogenic food resources can have complex implications for species and ecosystems, especially when species are in decline. Here, we use recently developed primers targeting the ITS2 region of plants to characterize diet from faecal samples of four UK columbids, with particular focus on the European turtle dove (Streptopelia turtur), a rapidly declining obligate granivore. We examine dietary overlap between species (potential competition), associations with body condition in turtle doves and spatiotemporal variation in diet. We identified 143 taxonomic units, of which we classified 55% to species, another 34% to genus and the remaining 11% to family. We found significant dietary overlap between all columbid species, with the highest between turtle doves and stock doves (Columba oenas), then between turtle doves and woodpigeons (Columba palumbus). The lowest overlap was between woodpigeons and collared doves (Streptopelia decaocto). We show considerable change in columbid diets compared to previous studies, probably reflecting opportunistic foraging behaviour by columbids within a highly anthropogenically modified landscape, although our data for nonturtle doves should be considered preliminary. Nestling turtle doves in better condition had a higher dietary proportion of taxonomic units from natural arable plant species and a lower proportion of taxonomic units from anthropogenic food resources such as garden bird seed mixes and brassicas. This suggests that breeding ground conservation strategies for turtle doves should include provision of anthropogenic seeds for adults early in the breeding season, coupled with habitat rich in accessible seeds from arable plants once chicks have hatched.

High rates of infection by blood parasites during the nestling phase in UK Columbids with notes on ecological associations.

Studies of blood parasite infection in nestling birds rarely find a high prevalence of infection. This is likely due to a combination of short nestling periods (limiting the age at which nestlings can be sampled) and long parasite prepatent periods before gametocytes can be detected in peripheral blood. Here we examine rates of blood parasite infection in nestlings from three Columbid species in the UK. We use this system to address two key hypotheses in the epidemiology of avian haemoparasites: first, that nestlings in open nests have a higher prevalence of infection; and second, that nestlings sampled at 14 days old have a higher apparent infection rate than those sampled at 7 days old. Open-nesting individuals had a 54% infection rate compared with 25% for box-nesters, probably due to an increased exposure of open-nesting species to dipteran vectors. Nestlings sampled at 14 days had a 68% infection rate compared with 32% in nestlings sampled at 7 days, suggesting that rates of infection in the nest are high. Further work should examine nestlings post-fledging to identify rates of successful parasite infection (as opposed to abortive development within a dead-end host) as well as impacts on host post-fledging survival and behaviour.

Genomic evidence of demographic fluctuations and lack of genetic structure across flyways in a long distance migrant, the European turtle dove.

BACKGROUND: Understanding how past climatic oscillations have affected organismic evolution will help predict the impact that current climate change has on living organisms. The European turtle dove, Streptopelia turtur, is a warm-temperature adapted species and a long distance migrant that uses multiple flyways to move between Europe and Africa. Despite being abundant, it is categorized as vulnerable because of a long-term demographic decline. We studied the demographic history and population genetic structure of the European turtle dove using genomic data and mitochondrial DNA sequences from individuals sampled across Europe, and performing paleoclimatic niche modelling simulations. RESULTS: Overall our data suggest that this species is panmictic across Europe, and is not genetically structured across flyways. We found the genetic signatures of demographic fluctuations, inferring an effective population size (Ne) expansion that occurred between the late Pleistocene and early Holocene, followed by a decrease in the Ne that started between the mid Holocene and the present. Our niche modelling analyses suggest that the variations in the Ne are coincident with recent changes in the availability of suitable habitat. CONCLUSIONS: We argue that the European turtle dove is prone to undergo demographic fluctuations, a trait that makes it sensitive to anthropogenic impacts, especially when its numbers are decreasing. Also, considering the lack of genetic structure, we suggest all populations across Europe are equally relevant for conservation.

Can hedgerow management mitigate the impacts of predation on songbird nest survival?

Nest predators can have significant impacts on songbird reproductive success. These impacts may be amplified by habitat simplification and here we test whether sympathetic management of farmland hedgerows can reduce nest depredation, especially by corvids. We test whether songbirds select nest sites according to structural features of hedgerows (including nest visibility and accessibility), and whether these features influence nest predation risk. Songbirds selected nesting sites affording higher vegetation cover above the nest, increased visibility on the nest-side of the hedgerow and reduced visibility on the far side of the hedge. Nest survival was unrelated to corvid abundance and only weakly related (at the egg stage) to corvid nest proximity. Nest survival at the chick stage was higher where vegetation structure restricted access to corvid-sized predators (averaging 0.78 vs. 0.53), and at nests close to potential vantage points. Overall nest survival was sensitive to hedgerow structure (accessibility) particularly at low exposure to corvid predation, while the overall impact of corvid exposure was dependent on the relationship involving proximity to vantage points. Nest survival over the chick stage was much higher (0.67) in stock-proof, trimmed and mechanically cut hedgerows, (which tended to provide lower side visibility and accessibility) than in recently laid, remnant or leggy hedgerows (0.18). Long-term reductions in the management of British hedgerows may therefore be exposing nesting songbirds to increased predation risk. We recommend regular rotational cutting of hedgerows to maintain a dense woody structure and thereby reduce songbird nest predation.

The protozoan parasite Trichomonas gallinae causes adult and nestling mortality in a declining population of European Turtle Doves, Streptopelia turtur.

Studies incorporating the ecology of clinical and sub-clinical disease in wild populations of conservation concern are rare. Here we examine sub-clinical infection by Trichomonas gallinae in a declining population of free-living European Turtle Doves and suggest caseous lesions cause mortality in adults and nestlings through subsequent starvation and/or suffocation. We found a 100% infection rate by T. gallinae in adult and nestling Turtle Doves (n = 25) and observed clinical signs in three adults and four nestlings (28%). Adults with clinical signs displayed no differences in any skeletal measures of size but had a mean 3.7% reduction in wing length, with no overlap compared to those without clinical signs. We also identified T. gallinae as the suggested cause of mortality in one Red-legged Partridge although disease presentation was different. A minimum of four strains of T. gallinae, characterized at the ITS/5.8S/ITS2 ribosomal region, were isolated from Turtle Doves. However, all birds with clinical signs (Turtle Doves and the Red-legged Partridge) carried a single strain of T. gallinae, suggesting that parasite spill over between Columbidae and Galliformes is a possibility that should be further investigated. Overall, we highlight the importance of monitoring populations for sub-clinical infection rather than just clinical disease.

Trichomonad parasite infection in four species of Columbidae in the UK.

Trichomonas gallinae is an emerging pathogen in wild birds, linked to recent declines in finch (Fringillidae) populations across Europe. Globally, the main hosts for this parasite are species of Columbidae (doves and pigeons); here we carry out the first investigation into the presence and incidence of Trichomonas in four species of Columbidae in the UK, through live sampling of wild-caught birds and subsequent PCR. We report the first known UK cases of Trichomonas infection in 86% of European Turtle Doves Streptopelia turtur sampled, along with 86% of Eurasian Collared Doves Streptopelia decaocto, 47% of Woodpigeons Columba palumbus and 40% of Stock Doves Columba oenas. Birds were more likely to be infected if the farm provided supplementary food for gamebirds. We found three strains of T. gallinae and one strain clustering within the Trichomonas tenax clade, not previously associated with avian hosts in the UK. One T. gallinae strain was identical at the ITS/5.8S/ITS2 ribosomal region to that responsible for the finch trichomonosis epizootic. We highlight the importance of increasing our knowledge of the diversity and ecological implications of Trichomonas parasites in order further to understand the sub-clinical impacts of parasite infection.

Avian blood parasite infection during the non-breeding season: an overlooked issue in declining populations?

BACKGROUND: Pathogens and parasites can have major impacts on host population dynamics, both through direct mortality and via indirect effects. Both types of effect may be stronger in species whose populations are already under pressure. We investigated the potential for blood parasites to impact upon their hosts at the immunological, physiological and population level during the non-breeding season using a declining population of yellowhammers Emberiza citrinella as a model. RESULTS: Yellowhammers infected by Haemoproteus spp. showed both a reduced heterophil to lymphocyte (H:L) ratio, and an elevated standardised white blood cell (WBC) count compared to uninfected birds, indicating an immunological response to infection. Infected birds had shorter wings during the first winter of sampling but not during the second, colder, winter; survival analysis of 321 birds sampled across four winters indicated that increased wing length conferred a survival advantage. CONCLUSIONS: We suggest that the potential impacts of blood parasite infections on over-wintering birds may have been underestimated. Further research should consider the potential impacts of sub-clinical parasite infections on the dynamics of vulnerable populations, and we suggest using declining populations as model systems within which to investigate these relationships as well as examining interactions between sub-clinical disease and other environmental stressors. JEL CODE: Q5.

Bird species with wider geographical ranges have higher blood parasite diversity but not prevalence across the African-Eurasian flyway.

Avian blood parasites, from the genera Plasmodium, Haemoproteus and Leucocytozoon, are predicted to alter their range and prevalence as global temperatures change, and host and vector ranges shift. Understanding large-scale patterns in the prevalence and diversity of avian malaria and malaria-like parasites is important due to an incomplete understanding of their effects in the wild, where studies suggest even light parasitaemia can potentially cause rapid mortality, especially in naïve populations. We conducted phylogenetically controlled analyses to test for differences in prevalence and lineage diversity of haemoparasite infection (for Plasmodium, Haemoproteus and Leucocytozoon) in and between resident and migratory species along the African-Eurasian flyway. To test whether migratory strategy or range size drives differences in parasite prevalence and diversity between resident and migrant species, we included three categories of resident species: Eurasian only (n = 36 species), African only (n = 41), and species resident on both continents (n = 17), alongside intercontinental migrants (n = 64), using a subset of data from the MalAvi database comprising 27,861 individual birds. We found that species resident on both continents had a higher overall parasite diversity than all other categories. Eurasian residents had lower Plasmodium diversity than all other groups, and both migrants and species resident on both continents had higher Haemoproteus diversity than both African and Eurasian residents. Leucocytozoon diversity did not differ between groups. Prevalence patterns were less clear, with marked differences between genera. Both Plasmodium and Leucocytozoon prevalence was higher in species resident on both continents and African residents than in migrants and Eurasian residents. Haemoproteus prevalence was lower in Eurasian residents than species resident on both continents. Our findings contrast with previous findings in the North-South American flyway, where long-distance migrants had higher parasite diversity than residents and short-distance migrants, although we found contrasting patterns for parasite diversity to those seen for parasite prevalence. Crucially, our results suggest that geographic range may be more important than migratory strategy in driving parasite diversity within species along the African-Palaearctic flyway. Our findings differ between the three parasite genera included in our analysis, suggesting that vector ecology may be important in determining these large-scale patterns. Our results add to our understanding of global patterns in parasite diversity and abundance, and highlight the need to better understand the influence of vector ecology to understand the drivers of infection risk and predict responses to environmental change.

Parasite exchange and hybridisation at a wild-feral-domestic interface.

Interactions between wild, feral, and domestic animals are of economic and conservation significance. The pigeon Columba livia is a synanthropic species in a feral form, but it also includes the rare Rock Dove. Columba livia is an important player at the wild-domestic interface, acting as a carrier of avian diseases, and the feral form threatens Rock Doves with extinction via hybridisation. Despite its abundance, little is known about drivers of disease prevalence in C. livia, or how disease and hybridisation represent synergistic threats to Rock Doves. We focused on infection by the parasite Trichomonas, first collating prevalence estimates in domestic and free-living populations from relevant studies of C. livia. Second, we characterised variation in the diversity and prevalence of Trichomonas among three C. livia populations in the United Kingdom: a feral, a Rock Dove, and a feral-wild hybrid population. Across multiple continents, free-living pigeons had lower Trichomonas infection than captive conspecifics, but the effect was weak. Environmental factors which could impact Trichomonas infection status did not explain variation in infection among populations. Among the British populations, strain diversity varied, and there was lower parasite prevalence in Rock Doves than feral pigeons. Individual infection status was not explained by the available covariates, including hybrid score and site. The drivers of Trichomonas prevalence are unclear, perhaps due to idiosyncratic local-scale drivers. However, given the population-level variation in both infection prevalence and introgressive hybridisation, the potential combined effects could accelerate the extinction of the Rock Dove. Further study of the synergistic effects of multiple types of biotic interactions at the wild-feral-domestic interface is warranted, especially where vagile, globally distributed and superabundant animals are involved.

In vivo inhibition of nuclear ACE2 translocation protects against SARS-CoV-2 replication and lung damage through epigenetic imprinting.

In vitro, ACE2 translocates to the nucleus to induce SARS-CoV-2 replication. Here, using digital spatial profiling of lung tissues from SARS-CoV-2-infected golden Syrian hamsters, we show that a specific and selective peptide inhibitor of nuclear ACE2 (NACE2i) inhibits viral replication two days after SARS-CoV-2 infection. Moreover, the peptide also prevents inflammation and macrophage infiltration, and increases NK cell infiltration in bronchioles. NACE2i treatment increases the levels of the active histone mark, H3K27ac, restores host translation in infected hamster bronchiolar cells, and leads to an enrichment in methylated ACE2 in hamster bronchioles and lung macrophages, a signature associated with virus protection. In addition, ACE2 methylation is increased in myeloid cells from vaccinated patients and associated with reduced SARS-CoV-2 spike protein expression in monocytes from individuals who have recovered from infection. This protective epigenetic scarring of ACE2 is associated with a reduced latent viral reservoir in monocytes/macrophages and enhanced immune protection against SARS-CoV-2. Nuclear ACE2 may represent a therapeutic target independent of the variant and strain of viruses that use the ACE2 receptor for host cell entry.

Selective Targeting of Protein Kinase C (PKC)-θ Nuclear Translocation Reduces Mesenchymal Gene Signatures and Reinvigorates Dysfunctional CD8+ T Cells in Immunotherapy-Resistant and Metastatic Cancers.

Protein kinase C (PKC)-θ is a serine/threonine kinase with both cytoplasmic and nuclear functions. Nuclear chromatin-associated PKC-θ (nPKC-θ) is increasingly recognized to be pathogenic in cancer, whereas its cytoplasmic signaling is restricted to normal T-cell function. Here we show that nPKC-θ is enriched in circulating tumor cells (CTCs) in patients with triple-negative breast cancer (TNBC) brain metastases and immunotherapy-resistant metastatic melanoma and is associated with poor survival in immunotherapy-resistant disease. To target nPKC-θ, we designed a novel PKC-θ peptide inhibitor (nPKC-θi2) that selectively inhibits nPKC-θ nuclear translocation but not PKC-θ signaling in healthy T cells. Targeting nPKC-θ reduced mesenchymal cancer stem cell signatures in immunotherapy-resistant CTCs and TNBC xenografts. PKC-θ was also enriched in the nuclei of CD8+ T cells isolated from stage IV immunotherapy-resistant metastatic cancer patients. We show for the first time that nPKC-θ complexes with ZEB1, a key repressive transcription factor in epithelial-to-mesenchymal transition (EMT), in immunotherapy-resistant dysfunctional PD1+/CD8+ T cells. nPKC-θi2 inhibited the ZEB1/PKC-θ repressive complex to induce cytokine production in CD8+ T cells isolated from patients with immunotherapy-resistant disease. These data establish for the first time that nPKC-θ mediates immunotherapy resistance via its activity in CTCs and dysfunctional CD8+ T cells. Disrupting nPKC-θ but retaining its cytoplasmic function may offer a means to target metastases in combination with chemotherapy or immunotherapy.

High prevalence of the neonicotinoid clothianidin in liver and plasma samples collected from gamebirds during autumn sowing.

Since neonicotinoid insecticides were introduced to the agricultural market, evidence of the negative impacts of these systemic compounds on non-target species has accumulated. Birds are one of the largest groups of species to inhabit farmland, but the extent of neonicotinoid exposure in avian communities is poorly understood and very little is known about how any exposure may affect wild birds. Here, free-living gamebirds were used as a model group to measure the extent of avian exposure to the neonicotinoid clothianidin via seed treatment. During a typical sowing period of winter cereals treated with clothianidin, blood and liver samples were collected simultaneously from individual hunted gamebird carcasses, both pre- (n = 18) and post-sowing (n = 57) and were analysed for clothianidin via LC/MS-MS. Body weight, fat score and faecal parasite load were also quantified in the birds to ascertain whether any of these health parameters were associated with clothianidin exposure under field conditions. Clothianidin was detected in 6% of individuals sampled pre-sowing and 89% of individuals sampled post-sowing. The frequency of clothianidin detection in plasma samples and the concentration of clothianidin in liver and plasma samples decreased significantly between the first week and 2-4 weeks post-sowing. Faecal parasite load was positively associated with concentrations of clothianidin in the liver (but not plasma) of partridge species, but there was no association between clothianidin concentration and fat score or body weight, for either sample type. This study provides clear evidence that treated seed is a source of pesticide exposure for gamebirds following autumn sowing. These findings have implications for gamebirds worldwide where seed treatments are in use, and will aid the design of any future avian biomonitoring studies for agrochemical compounds.