Subclinical peripheral inflammation has systemic effects impacting central nervous system proteome in budgerigars.

Regulation of neuroimmune interactions varies across avian species. Little is presently known about the interplay between periphery and central nervous system (CNS) in parrots, birds sensitive to neuroinflammation. Here we investigated the systemic and CNS responses to dextran sulphate sodium (DSS)- and lipopolysaccharide (LPS)-induced subclinical acute peripheral inflammation in budgerigar (Melopsittacus undulatus). Three experimental treatment groups differing in DSS and LPS stimulation were compared to controls. Individuals treated with DSS showed significant histological intestinal damage. Through quantitative proteomics we described changes in plasma (PL) and cerebrospinal fluid (CSF) composition. In total, we identified 180 proteins in PL and 978 proteins in CSF, with moderate co-structure between the proteomes. Between treatments we detected differences in immune, coagulation and metabolic pathways. Proteomic variation was associated with the levels of pro-inflammatory cytokine mRNA expression in intestine and brain. Our findings shed light on systemic impacts of peripheral low-grade inflammation in birds.

Peripheral inflammation-induced changes in songbird brain gene expression: 3' mRNA transcriptomic approach.

Species-specific neural inflammation can be induced by profound immune signalling from periphery to brain. Recent advances in transcriptomics offer cost-effective approaches to study this regulation. In a population of captive zebra finch (Taeniopygia guttata), we compare the differential gene expression patterns in lipopolysaccharide (LPS)-triggered peripheral inflammation revealed by RNA-seq and QuantSeq. The RNA-seq approach identified more differentially expressed genes but failed to detect any inflammatory markers. In contrast, QuantSeq results identified specific expression changes in the genes regulating inflammation. Next, we adopted QuantSeq to relate peripheral and brain transcriptomes. We identified subtle changes in the brain gene expression during the peripheral inflammation (e.g. up-regulation in AVD-like and ACOD1 expression) and detected co-structure between the peripheral and brain inflammation. Our results suggest benefits of the 3'end transcriptomics for association studies between peripheral and neural inflammation in genetically heterogeneous models and identify potential targets for the future brain research in birds.

Nearly (?) sterile avian egg in a passerine bird.

During early ontogeny, microbiome affects development of the gastrointestinal tract, immunity, and survival in vertebrates. Bird eggs are thought to be (1) initially sterile (sterile egg hypothesis) and (2) colonized after oviposition through horizontal trans-shell migration, or (3) initially seeded with bacteria by vertical transfer from mother oviduct. To date, however, little empirical data illuminate the contribution of these mechanisms to gut microbiota formation in avian embryos. We investigated microbiome of the egg content (day 0; E0-egg), embryonic gut at day 13 (E13) and female faeces in a free-living passerine, the great tit (Parus major), using a methodologically advanced procedure combining 16S rRNA gene sequencing and microbe-specific qPCR assays. Our metabarcoding revealed that the avian egg is (nearly) sterile, but acquires a slightly richer microbiome during the embryonic development. Of the three potentially pathogenic bacteria targeted by qPCR, only Dietzia was found in E0-egg (yet also in negative controls), E13 gut and female samples, which might indicate possible vertical transfer. Unlike in poultry, we have shown that major bacterial colonization of the gut in passerines does not occur before hatching. We emphasize that protocols that carefully check for environmental contamination are critical in studies with low-bacterial biomass samples.

Cannabinoid receptor 2 evolutionary gene loss makes parrots more susceptible to neuroinflammation.

In vertebrates, cannabinoids modulate neuroimmune interactions through two cannabinoid receptors (CNRs) conservatively expressed in the brain (CNR1, syn. CB1) and in the periphery (CNR2, syn. CB2). Our comparative genomic analysis indicates several evolutionary losses in the CNR2 gene that is involved in immune regulation. Notably, we show that the CNR2 gene pseudogenized in all parrots (Psittaciformes). This CNR2 gene loss occurred because of chromosomal rearrangements. Our positive selection analysis suggests the absence of any specific molecular adaptations in parrot CNR1 that would compensate for the CNR2 loss in the modulation of the neuroimmune interactions. Using transcriptomic data from the brains of birds with experimentally induced sterile inflammation we highlight possible functional effects of such a CNR2 gene loss. We compare the expression patterns of CNR and neuroinflammatory markers in CNR2-deficient parrots (represented by the budgerigar, Melopsittacus undulatus and five other parrot species) with CNR2-intact passerines (represented by the zebra finch, Taeniopygia guttata). Unlike in passerines, stimulation with lipopolysaccharide resulted in neuroinflammation in the parrots linked with a significant upregulation of expression in proinflammatory cytokines (including interleukin 1 beta (IL1B) and 6 (IL6)) in the brain. Our results indicate the functional importance of the CNR2 gene loss for increased sensitivity to brain inflammation.

Inter-annual repeatability and age-dependent changes in plasma testosterone levels in a longitudinally monitored free-living passerine bird.

While seasonal trends in testosterone levels are known from cross-cohort studies, data on testosterone inter-annual individual repeatability in wild birds are rare. Also, our understanding of hormonal age-dependent changes in testosterone levels is limited. We assessed plasma testosterone levels in 105 samples originating from 49 repeatedly captured free-living great tits (Parus major) sampled during the nesting to investigate their relative long-term repeatability and within-individual changes. Furthermore, we examined the inter-annual repeatability of condition-related traits (carotenoid- and melanin-based plumage ornamentation, ptilochronological feather growth rate, body mass, and haematological heterophil/lymphocyte ratio) and their relationships to testosterone levels. We show that testosterone levels are inter-annually repeatable in females, with a non-significant pattern in males, both in absolute values and individual ranks (indicating the maintenance of relative status in a population). In males, we found a quadratic dependence of testosterone levels on age, with a peak in midlife. In contrast, female testosterone levels showed no age-dependent trends. The inter-annual repeatability of condition-related traits ranged from zero to moderate and was mostly unrelated to plasma testosterone concentrations. However, males with elevated testosterone had significantly higher carotenoid-pigmented yellow plumage brightness, a trait presumably involved in mating. Showing inter-annual repeatability in testosterone levels, this research opens the way to further understanding the causes of variation in condition-related traits. Based on a longitudinal dataset, this study demonstrates that male plasma testosterone undergoes age-related changes that may regulate resource allocation. Our results thus suggest that, unlike females, male birds undergo hormonal senescence similar to mammals.

Longitudinal evidence for immunosenescence and inflammaging in free-living great tits.

The first-line effector mechanisms of immune defence, including inflammation and oxidative burst, contribute significantly to host-pathogen resistance. Whether these immune responses undergo age-related changes in birds remains unknown. Here, we tracked selected inflammatory parameters in 54 free-living great tits (Parus major) of known age, captured repeatedly over three consecutive years, with the aims to investigate long-term repeatability and age-dependent changes in cellular oxidative burst responsiveness upon in vitro stimulation with bacterial lipopolysaccharide (LPS), and to identify its relationships with leukotriene B4 (LTB4) levels and haematological traits. In addition, we linked these immunological traits to selected physiological markers (antioxidants and oxidative stress markers). LTB4 levels increased with age and we have shown a similar non-significant tendency also for absolute granulocyte counts, indicating propagating chronic inflammation over the bird's lifetime, consistent with the inflammaging hypothesis. In contrast, cellular oxidative burst followed a quadratic trend of dependency on age with a peak in midlife individuals, in line with the immunosenescence hypothesis. Interestingly, LTB4 levels were positively associated with general oxidative damage, but negatively with antioxidant glutathione peroxidase activity, indicating links to redox balance. This longitudinal study demonstrates the contrasting patterns of age-related changes in background and acute markers of pro-inflammatory immunity contributing to immunosenescence in birds and thus provides basis for interpretation of the tested inflammatory markers in cross-cohort datasets.

Longitudinally monitored lifetime changes in blood heavy metal concentrations and their health effects in urban birds.

Urban heavy metal pollution can impair the health of humans and other organisms inhabiting cities. While birds are suggested as one of the appropriate bioindicators for essential and non-essential trace element monitoring, the process of particular elements' accumulation in blood and its possible adverse health effects during ageing of individuals remain unexplored. We have investigated lifetime changes in blood lead (Pb), cadmium (Cd), arsenic (As) and zinc (Zn) concentrations and searched for links to health-related traits in sub-urban free-living great tit (Parus major) population monitored over a long period of time. The blood As concentrations were under the limit of detection in most samples. The blood Pb levels showed a non-linear relationship to individuals age, where the highest Pb concentrations were measured in nestlings and in a very small group of highly senescent birds (over 7 years old), while no clear trend was observed for the majority of the adult age stages. No age-related patterns were found for blood Cd or Zn concentrations. The positive relationship between date of capture and blood Cd and Zn levels may reflect seasonal changes in diet composition. We did not reveal any anaemia-like conditions (decreased total erythrocyte count or increased immature erythrocyte count) in relation to blood heavy metal concentrations in the investigated birds. Total leukocyte counts, heterophil/lymphocyte (H/L) ratio and total heterophil and lymphocyte counts increased with increasing Pb, Cd and Zn concentrations in blood. This study demonstrates the suitability of avian blood for actual heavy metal spatial and temporal biomonitoring even in situations when the precise age of the individuals remains unknown.