Characterization of MHC class I in a long distance migratory wader, the Icelandic black-tailed godwit.

The major histocompatibility complex (MHC) encodes proteins that are central for antigen presentation and pathogen elimination. MHC class I (MHC-I) genes have attracted a great deal of interest among researchers in ecology and evolution and have been partly characterized in a wide range of bird species. So far, the main focus has been on species within the bird orders Galliformes and Passeriformes, while Charadriiformes remain vastly underrepresented with only two species studied to date. These two Charadriiformes species exhibit striking differences in MHC-I characteristics and MHC-I diversity. We therefore set out to study a third species within Charadriiformes, the Icelandic subspecies of black-tailed godwits (Limosa limosa islandica). This subspecies is normally confined to parasite-poor environments, and we hence expected low MHC diversity. MHC-I was partially characterized first using Sanger sequencing and then using high-throughput sequencing (MiSeq) in 84 individuals. We verified 47 nucleotide alleles in open reading frame with classical MHC-I characteristics, and each individual godwit had two to seven putatively classical MHC alleles. However, in contrast to previous MHC-I data within Charadriiformes, we did not find any evidence of alleles with low sequence diversity, believed to represent non-classical MHC genes. The diversity and divergence of the godwits MHC-I genes to a large extent fell between the previous estimates within Charadriiformes. However, the MHC genes of the migratory godwits had few sites subject to positive selection, and one possible explanation could be a low exposure to pathogens.

Laridae: A neglected reservoir that could play a major role in avian influenza virus epidemiological dynamics.

Avian influenza viruses (AIVs) are of great concern worldwide due to their economic impact and the threat they represent to human health. As wild birds are the natural reservoirs of AIVs, understanding AIV dynamics in different avian taxa is essential for deciphering the epidemiological links between wildlife, poultry and humans. To date, only the Anatidae (ducks, geese and swans) have been widely studied. Here, we aim to shed light on the current state of knowledge on AIVs in Laridae (gulls, terns and kittiwakes) versus that in Anatidae by setting forth four fundamental questions: how, when, where and to which host species are AIVs transmitted? First, we describe ecological differences between Laridae and Anatidae and discuss how they may explain observed contrasts in preferential transmission routes and the evolution of specific AIV subtypes. Second, we highlight the dissimilarities in the temporal patterns of AIV shedding between Laridae and Anatidae and address the role that immunity likely plays in shaping these patterns. Third, we underscore that Laridae may be key in promoting intercontinental exchanges of AIVs. Finally, we emphasize the crucial epidemiological position that Laridae occupy between wildlife, domestic birds and humans.

Differential effects of specific carotenoids on oxidative damage and immune response of gull chicks.

Micronutrients are essential for normal metabolic processes during early development. Specifically, it has been suggested that diet-derived carotenoids can play a key role in physiological functions because of their antioxidant and immunostimulant properties. However, their role as antioxidants remains controversial. Additionally, it is also unclear whether oxidative stress mediates their immunostimulatory effects. In this field study, we separately supplemented yellow-legged gull (Larus michahellis) chicks with two carotenoids (lutein and ß-carotene) with different molecular structures and different transformation pathways into other oxidative forms of carotenoids. We quantified their effect on the oxidative status and the immune response of chicks before and after an oxidative challenge with paraquat, a pro-oxidant molecule. Prior to oxidative challenge, none of the carotenoid treatments affected the oxidative status of chicks, but they enhanced the inflammatory response to an antigen compared with controls. The oxidative challenge enhanced plasma vitamin E levels (but not in ß-carotene-supplemented chicks) and the antioxidant capacity in the short term. Interestingly, lutein-supplemented chicks showed lower oxidative damage to proteins than non-lutein-supplemented chicks. After the oxidative challenge, the positive effect of carotenoid supplementation on the immune response disappeared. Thus, these results suggest differential effects of two carotenoids with different molecular structures on the oxidative status. Lutein but not ß-carotene helps to combat oxidative damage after a free-radical exposure. Additionally, the results indicate that the immunostimulatory effects of carotenoids are linked to oxidative status during early life.

Diversity of major histocompatibility complex of II B gene and mate choice in a monogamous and long-lived seabird, the Little Auk (Alle alle).

The major histocompatibility complex (MHC) plays a key role in the adaptive immune system of vertebrates, and is known to influence mate choice in many species. In birds, the MHC has been extensively examined but mainly in galliforms and passerines while other taxa that represent specific ecological and evolutionary life-histories, like seabirds, are underexamined. Here, we characterized diversity of MHC Class II B exon 2 in a colonial pelagic seabird, the Little Auk (or Dovekie Alle alle). We further examined whether MHC variation could be maintained through balancing selection and disassortative mating. We found high polymorphism at the genotyped MHC fragment, characterizing 99 distinct alleles across 140 individuals from three populations. The alleles frequencies exhibited a similar skewed distribution in both sexes, with the four most commonly occurring alleles representing approximately 35% of allelic variation. The results of a Bayesian site-by-site selection analysis suggest evidence of balancing selection and no direct evidence for MHC-dependent disassortative mating preferences in the Little Auk. The latter result might be attributed to the high overall polymorphism of the examined fragment, which itself may be maintained by the large population size of the species.

Characterization of MHC class I in a long-distance migrant shorebird suggests multiple transcribed genes and intergenic recombination.

The major histocompatibility complex (MHC) includes highly polymorphic gene families encoding proteins crucial to the vertebrate acquired immune system. Classical MHC class I (MHCI) genes code for molecules expressed on the surfaces of most nucleated cells and are associated with defense against intracellular pathogens, such as viruses. These genes have been studied in a few wild bird species, but have not been studied in long-distance migrating shorebirds. Red Knots Calidris canutus are medium-sized, monogamous sandpipers with migratory routes that span the globe. Understanding how such long-distance migrants protect themselves from disease has gained new relevance since the emergence of avian-borne diseases, including intracellular pathogens recognized by MHCI molecules, such as avian influenza. In this study, we characterized MHCI genes in knots and found 36 alleles in eight individuals and evidence for six putatively functional and expressed MHCI genes in a single bird. We also found evidence for recombination and for positive selection at putative peptide binding sites in exons 2 and 3. These results suggest surprisingly high MHC diversity in knots, given their demographic history. This may be a result of selection from diverse pathogens encountered by shorebirds throughout their annual migrations.

Flight performance of western sandpipers, Calidris mauri, remains uncompromised when mounting an acute phase immune response.

Migratory birds have been implicated in the spread of some zoonotic diseases, but how well infected individuals can fly remains poorly understood. We used western sandpipers, Calidris mauri, to experimentally test whether flight is affected when long-distance migrants are mounting an immune response and whether migrants maintain immune defences during a flight in a wind tunnel. We measured five indicators of innate immunity in 'flown-healthy' birds (flying in a wind tunnel without mounting an immune response), 'flown-sick' birds (flying while mounting an acute phase response, which is part of induced innate immunity), and a non-flying control group ('not-flown'). Voluntary flight duration did not differ between flown-healthy and flown-sick birds, indicating that mounting an acute phase response to simulated infection did not hamper an individual's ability to fly for up to 3 h. However, in comparison to not-flown birds, bacterial killing ability of plasma was significantly reduced after flight in flown-sick birds. In flown-healthy birds, voluntary flight duration was positively correlated with bacterial killing ability and baseline haptoglobin concentration of the blood plasma measured 1-3 weeks before experimental flights, suggesting that high quality birds had strong immune systems and greater flight capacity. Our findings indicate that flight performance is not diminished by prior immune challenge, but that flight while mounting an acute phase response negatively affects other aspects of immune function. These findings have important implications for our understanding of the transmission of avian diseases, as they suggest that birds can still migrate while fighting an infection.

Effects of salinity on the immune response of an 'osmotic generalist' bird.

Salt stress can suppress the immune function of fish and other aquatic animals, but such an effect has not yet been examined in air-breathing vertebrates that frequently cope with waters (and prey) of contrasting salinities. We investigated the effects of seawater salinity on the strength and cost of mounting an immune response in the dunlin Calidris alpina, a long-distance migratory shorebird that shifts seasonally from freshwater environments during the breeding season to marine environments during migration and the winter period. Phytohaemagglutinin (PHA)-induced skin swelling, basal metabolic rate (BMR), body mass, fat stores, and plasma ions were measured in dunlins acclimated to either freshwater or seawater (salinity: 0.3 and 35.0 ‰, respectively). Seawater-acclimated dunlins mounted a PHA-induced swelling response that was up to 56 % weaker than those held under freshwater conditions, despite ad libitum access to food. Freshwater-acclimated dunlins significantly increased their relative BMR 48 h after PHA injection, whereas seawater-acclimated dunlins did not. However, this differential immune and metabolic response between freshwater- and seawater-acclimated dunlins was not associated with significant changes in body mass, fat stores or plasma ions. Our results indicate that the strength of the immune response of this small-sized migratory shorebird was negatively influenced by the salinity of marine habitats. Further, these findings suggest that the reduced immune response observed under saline conditions might not be caused by an energy or nutrient limitation, and raise questions about the role of osmoregulatory hormones in the modulation of the immune system.

Independence among physiological traits suggests flexibility in the face of ecological demands on phenotypes.

Phenotypic flexibility allows animals to adjust their physiology to diverse environmental conditions encountered over the year. Examining how these varying traits covary gives insights into potential constraints or freedoms that may shape evolutionary trajectories. In this study, we examined relationships among haematocrit, baseline corticosterone concentration, constitutive immune function and basal metabolic rate in red knot Calidris canutus islandica individuals subjected to experimentally manipulated temperature treatments over an entire annual cycle. If covariation among traits is constrained, we predict consistent covariation within and among individuals. We further predict consistent correlations between physiological and metabolic traits if constraints underlie species-level patterns found along the slow-fast pace-of-life continuum. We found no consistent correlations among haematocrit, baseline corticosterone concentration, immune function and basal metabolic rate either within or among individuals. This provides no evidence for constraints limiting relationships among these measures of the cardiovascular, endocrine, immune and metabolic systems in individual red knots. Rather, our data suggest that knots are free to adjust individual parts of their physiology independently. This makes good sense if one places the animal within its ecological context where different aspects of the environment might put different pressures on different aspects of physiology.

[Morpho-functional changes of cloacal bursa of the herring gull in experimental infection with gull tapeworm].

Using the methods of light microscopy, the quantitative changes in the cells of the cloacal bursa of herring gull (Larus argentatus mongolicus) chicks were studied during experimental infection with gull-tapeworm Diphyllobothrium dendriticum. The area of the follicles within the bursa and the number of eosinophils in the infected chicks were found to increase. In the follicles the number of blast cells and large lymphocytes was elevated. The reduction of small lymphocytes and plasma cells numbers in the infected gulls may be due to a slower cell cycle or cell migration into the inflammatory focus. Overall, the changes in the bursa indicate the suppression of the B-link of the host immune system in tapeworm invasion.

Characterization and locus-specific typing of MHC class I genes in the red-billed gull (Larus scopulinus) provides evidence for major, minor, and nonclassical loci.

A major challenge facing studies of major histocompatibility complex (MHC) evolution in birds is the difficulty in genotyping alleles at individual loci, and the consequent inability to investigate sequence variation and selection pressures for each gene. In this study, four MHC class I loci were isolated from the red-billed gull (Larus scopulinus), representing both the first characterized MHCI genes within Charadriiformes (shorebirds, gulls, and allies) and the first full-length MHCI sequences described outside Galloanserae (gamebirds + waterfowl). Complete multilocus genotypes were obtained for 470 individuals using a combination of reference-strand conformation analysis and direct sequencing of gene-specific amplification products, and variation of peptide-binding region (PBR) exons was surveyed for all loci. Each gene is transcribed and has conserved sequence features characteristic of antigen-presenting MHCI molecules. However, higher allelic variation, a more even allele frequency distribution, and evidence of positive selection acting on a larger number of PBR residues suggest that only one locus (Lasc-UAA) functions as a major classical MHCI gene. Lasc-UBA, with more limited variation and PBR motifs that encompass a subset of Lasc-UAA diversity, was assigned a putative minor classical function, whereas the divergent and largely invariant binding-groove motifs of Lasc-UCA and -UDA are suggestive of nonclassical loci with specialized ligand-binding roles.

[The generation and characteristics of reassortant influenza A virus with H5 hemagglutinin and other genes from the apathogenic virus H6N2].

The experimental reassortant vaccine strain VN-gull (H5N2) containing H5 hemagglutinin (HA) with a removed polybasic site in the connecting peptide and other genes from the apathogenic H6N2 virus A/gull/Moscow/3100/2006 (gull/M) was obtained using a two-step protocol. At Step 1, the reassortant with HA of A/Vietnam/1203/04-PR8/ CDC-RG and other genes from cold-adapted A/Leningrad/17/47 (VN-Len) viruses was generated due to selection with antibody to H2N2 at 26 degrees C. At Step 2, the reassortant VN-gull was obtained by replacing all genes from Len with those from gull/M due to selection with antibody to H6N2 at 39 degrees C. The reassortant VN-Len was apathogenic and the reassortant VN-gull was weakly virulent in mice. Both gave rise to specific antibodies and 4 weeks after single inoculation they provided complete protection against further challenge with highly pathogenic HSN1 virus A/chicken/Kurgan/3/05 (H5N1) (Ku-Len). The chickens infected with live VN-gull virus showed neither clinical symptoms, nor fecal virus excretion; nevertheless, they gave rise to antibodies and were protected from the further challenge with A/chicken/Kurgan/3/2005. The high yield, safety, and protectivity of VN-Len and Ku-Len made them promising strains for the production of inactivated and live vaccines against H5N1 viruses.

Evolution of innate and adaptive immune genes in a non-model waterbird, the common tern.

Toll-like receptors (TLRs) and the Major Histocompatibility Complex (MHC) are the key pathogen-recognition genes of vertebrate immune system and they have a crucial role in the initiation of innate and adaptive immune response, respectively. Recent advancements in sequencing technology sparked research on highly duplicated MHC genes in non-model species, but TLR variation in natural vertebrate populations has remained little studied and comparisons of polymorphism across both TLRs and MHC are scarce. Here, we aimed to compare variation across innate (four TLR loci) and adaptive (MHC class I and class II) immune genes in a non-model avian species, the common tern Sterna hirundo. We detected relatively high allelic richness at TLR genes (9-48 alleles per locus), which was similar to or even higher than the estimated per locus allelic richness at the MHC (24-30 alleles at class I and 13-16 alleles at class II under uniform sample sizes). Despite this, the total number of MHC alleles across all duplicated loci (four class I and three class II) was much higher and MHC alleles showed greater sequence divergence than TLRs. Positive selection targeted relatively more sites at the MHC than TLRs, but the strength of selection (dN/dS ratios) at TLRs was higher when compared to MHC class I. There were also differences in the signature of positive selection and recombination (gene conversion) between MHC class I and II (stronger signature at class II), suggesting that mechanisms maintaining variation at the MHC may vary between both classes. Our study indicates that allelic richness of both innate and adaptive immune receptors may be maintained at relatively high levels in viable avian populations and we recommend a transition from the traditional gene-specific to multi-gene approach in studying molecular evolution of vertebrate immune system.

[The reaction of the bursa and thymus eosinophils in the herring gull after the experimental infection with gull-tapeworm].

Using the methods of light microscopy, eosinophil topography, quantitative and qualitative changes (degranulation level of and a cationic protein content) were studied in the thymus and bursa of 36 herring gull nestlings Larus argentatus mongolicus (Suskin, 1925) 2 weeks after experimental infestation with gull-tapeworm Diphyllobothrium dendriticum (Nitsch, 1824). Eosinophils in the thymus were located in trabecules, mainly close to the blood vessels, thymic (Hassall's) corpuscles and also directly inside them, while in the bursa they were found within the internodular space. As compared with the control bird counts, relative eosinophil count in the birds with an average invasion intensity was increased 3.8 times in the thymus and 2.5 times in the bursa. In birds with high invasion intensity, these counts were increased 4 times in the thymus and 1.2 times in the bursa.

Relationship between blood test values and blood lead (Pb) levels in Black-headed gull (Chroicocephalus ridibundus: Laridae).

Few studies have evaluated immunosuppression due to lead accumulation below the overt toxicity threshold. If low levels of lead accumulation cause immunosuppression in birds, those birds could become more susceptible to pathogens. We aimed to determine if low levels of lead accumulation lead to immunosuppression in Black-headed gulls (Chroicocephalus ridibundus). Gulls were captured in Tokyo-bay and Mikawa-bay from January to April 2019. Their blood samples were analyzed for eight items. The data were analyzed to evaluate the correlation between lead concentrations and the variables from each bay. Lead was positively correlated with the percentage of heterophils and heterophil and lymphocyte ratio and negatively with lymphocytes. Thus, low lead accumulation levels may induce changes in percentage of the heterophils and lymphocyte.

No evidence for melatonin-linked immunoenhancement over the annual cycle of an avian species.

The winter immunoenhancement hypothesis associates long nights and increased exposure to melatonin with enhanced immune function in winter when resource availability is low and the chances of becoming ill are high. Thus, increased exposure to melatonin in the winter could be adaptive for species facing difficult winter conditions. This idea has found some support in studies of resident mammals. In birds, the link between day length and melatonin over the annual cycle is weaker, and contributions of melatonin to seasonal timing are unclear. Furthermore, many species, especially migrants, do not experience the most difficult conditions of their annual cycle in winter. In this study, we tested whether the winter immunoenhancement hypothesis holds in an avian species, the red knot Calidris canutus. We found that melatonin duration and amplitude varied significantly over the annual cycle with the highest values occurring in winter. However, peaks did not correspond to the winter solstice or with annual variation in immune function. Our findings do not support the winter immunoenhancement hypothesis in knots and question whether the idea that immune function should be bolstered in winter can be generalized to systems where winter is not the most difficult time of the year.

The toxic effects of multiple persistent organic pollutant exposures on the post-hatch immunity maturation of glaucous gulls.

This study tested whether the immune system of the glaucous gull (Larus hyperboreus) chicks became affected by existing environmental contaminants. An experimental group was given food that mimicked the natural contaminant mixture found in food from the North Atlantic marine environment, while the control group was given the equivalent of nearly clean food. All chicks were immunized with herpes virus (EHV), reovirus (REO), influenza virus (EIV), and tetanus toxoid (TET) in order to test their ability to respond to foreign specific antigens. At 8 wk, the experimental group had 3- to 13-fold higher concentrations of hexachlorobenzene (HCB), oxychlordane, p,p'-DDE, and total polychlorinated biphenyls (Sigma PCB) than did the control. The experimental group produced significantly lower antibody titer against EIV and had lower concentrations of immunoglobulin-G (IgG) and -M (IgM) in blood. Hematocrit percent and leukocyte numbers did not differ between the two groups. The ability of lymphocytes to proliferate in vitro was tested with three mitogens, phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM), and three antigens, keyhole limpet hemocyanin (KLH), TET, and Mycobacterium avium subsp. paratuberculosis tuberculin purified protein derivative (PPD). The experimental group had a significantly higher peripheral blood lymphocyte response to PHA and to spleen lymphocytes in vitro stimulated with Con A and PCB congeners 99 or 153, while the Con A, PWM, KLH, TET, PPD, and Con A plus PCB-156 or -126 showed nonsignificant differences between groups. Data indicate that the combined effect of multiple persistent organic pollution exposures occurring naturally in the Arctic negatively affect the immune system of the glaucous gull chick.

Seasonal redistribution of immune function in a migrant shorebird: annual-cycle effects override adjustments to thermal regime.

Throughout the annual cycle, demands on competing physiological systems change, and animals must allocate resources to maximize fitness. Immune function is one such system and is important for survival. Yet detailed empirical data tracking immune function over the entire annual cycle are lacking for most wild animals. We measured constitutive immune indices once a month for a year on captive red knots (Calidris canutus). We also examined temperature as an environmental contributor to immune variation by manipulating ambient temperature to vary energy expenditure. To identify relationships among immune indices, we performed principal-component analysis. We found significant repeatability in immune indices over the annual cycle and covariation of immune indices within and among individuals. This covariation suggests immune strategies as individual traits among individuals and the use of different immune strategies during different annual-cycle stages within individuals. Over the annual cycle, both higher-cost phagocyte-based immunity and lower-cost lymphocyte-based immunity were high during mass change, but there was a clear shift toward lower-cost lymphocyte-based immunity during peak molt. Experimental manipulation of temperature had little effect on annual variation in immune function. This suggests that other environmental factors, such as food availability and disease, should also be examined in the future.

Variable exposure and immunological response to Lyme disease Borrelia among North Atlantic seabird species.

Colonial seabirds often breed in large aggregations. These individuals can be exposed to parasitism by the tick Ixodes uriae, but little is known about the circulation of pathogens carried by this ectoparasite, including Lyme disease Borrelia. Here we investigated the prevalence of antibodies (Ab) against Borrelia burgdorferi sensu lato in seabird species sampled at eight locations across the North Atlantic. Using enzyme-linked immunosorbent assay tests, we found that the prevalence of anti-Borrelia Ab in adult seabirds was 39.6% on average (over 444 individuals), but that it varied among colonies and species. Common guillemots showed higher seroprevalence (77.1%+/-5.9) than black-legged kittiwakes (18.6%+/-6.7) and Atlantic puffins (22.6%+/-6.3). Immunoblot-banding patterns of positive individuals, reflecting the variability of Borrelia antigens against which Ab were produced, also differed among locations and species, and did not tightly match the prevalence of Borrelia phylogroups previously identified in ticks collected from the same host individuals. These results represent the first report of the widespread prevalence of Ab against Borrelia within an assemblage of seabird species and demonstrate that Borrelia is an integrated aspect in the interaction between seabirds and ticks. More detailed studies on the dynamics of Borrelia within and among seabird species at different spatial scales will now be required to better understand the implications of this interaction for seabird ecology and the epidemiology of Lyme disease.

Maternal allocation strategies and differential effects of yolk carotenoids on the phenotype and viability of yellow-legged gull (Larus michahellis) chicks in relation to sex and laying order.

Egg quality may mediate maternal allocation strategies according to progeny sex. In vertebrates, carotenoids have important physiological roles during embryonic and post-natal life, but the consequences of variation in yolk carotenoids for offspring phenotype in oviparous species are largely unknown. In yellow-legged gulls, yolk carotenoids did not vary with embryo sex in combination with egg laying date, order and mass. Yolk lutein supplementation enhanced the growth of sons from first eggs but depressed that of sons from last eggs, enhanced survival of daughters late in the season, and promoted immunity of male chicks and chicks from small eggs. Lack of variation in egg carotenoids in relation to sex and egg features, and the contrasting effects of lutein on sons and daughters, do not support the hypothesis of optimal sex-related egg carotenoid allocation. Carotenoids transferred to the eggs may rather result from a trade-off between opposing effects on sons or daughters.

[Hematological and immunological properties of herring gull (Larus argentatus) nestlings experimentally infected with Diphyllobothrium dendriticum (Cestoda: Pseudophyllidae)].

The morphological structure of the blood in herring gull nestlings and their immune system response to experimental infection with tapeworm Diphyllobothrium dendriticum have been studied. The leukocyte reaction pattern and the dynamics of immunoglobulin synthesis and populations of immunocompetent cells in the blood of nestlings during early invasion indicated a short duration of the cellular immune response and an insignificant humoral immune response. A pronounced trend to decrease in the hemoglobin level in the blood of infected nestlings has been revealed and this reduction was in part compensated by intensified erythropoiesis. It is shown, that the changes in hematologic and immune responses of the obligate definitive host provide for the mutual host-parasite adaptation.