Sex-specific effects of handling time on an index of immune activity in zebra finches

Recently, there has been considerable interest in the role of the immune system in shaping life-history evolution, sexual selection strategies, and indexes of individual quality. The most frequently used assay of immune function, particularly in avian field studies, is the phytohemagglunitin (PHA) skin test. PHA is injected subcutaneously into the wing web, and the magnitude of the resultant swelling has traditionally been interpreted as an index of an individual's cell-mediated immunocompetence. The test follows one of two protocols: the traditional two-wing injection protocol, with one wing web injected with PHA and the other with phosphate-buffered saline (PBS), or the simplified one-wing protocol that omits the PBS injection. In this technical comment, we alert researchers to the importance of considering handling time when performing the PHA test. We show that zebra finches (Taeniopygia guttata) subjected to the two-wing protocol had a lower wing-web swelling than individuals injected in one wing. In males, handling time explained over 50% of the variation in an individual's skin swelling response; females were relatively unaffected by handling time. We suggest that caution should be exercised when comparing the magnitude of wing-web swelling across studies in which the alternate protocol was followed. In addition, the recording of handling time, and its inclusion in subsequent statistical analyses, may aid in the detection of subtle differences across treatments.     

Reproductive state, but not testosterone, reduces immune function in male house sparrows (Passer domesticus)

The immune system requires energetic and nutritional resources to optimally defend organisms against pathogens and parasites. Because resources are typically limited, immune function may require a trade-off with other physiologically demanding activities. Here, we examined whether photoperiodically induced seasonal states (breeding, molting, or nonbreeding) affected the cutaneous immune response of captive male house sparrows (Passer domesticus). To assess immune function in these birds, we injected the mitogen phytohemagglutinin (PHA) into the patagium and measured the resulting wing web swelling. Molting and nonbreeding birds had similar immune responses to PHA injection. However, males in a breeding state showed lower immune responses than both molting and nonbreeding birds even though they did not actually breed. We tested whether this decrease in the PHA swelling response in birds in a breeding state was due to elevated plasma concentrations of testosterone (T) by administering T to birds in a nonbreeding state. Contrary to some evidence in the literature, T did not suppress the response to PHA in house sparrows. Our data show that passerine birds show seasonal modulation in immune function, even in benign environmental conditions. However, even though T is often cited as a strong immunosuppressant, it is not fully responsible for this seasonal modulation.     

No effect of blood sampling or phytohaemagglutinin injection on postfledging survival in a wild songbird

The injection of phytohaemagglutinin (PHA) and sampling of blood are widely used in studies of wild vertebrates to assess components of immune and endocrine function and health state and to obtain genetic material. Despite the pervasive use of these techniques in the life sciences, their potential effects on survival are rarely considered. For example, whether injection of the immunogen PHA into body parts critical for locomotion (e.g., the prepatagium, or wing web, in birds) affects survival has not been tested. Here, we test whether injection of PHA into the wing web and blood sampling from nestling house wrens affects their subsequent recruitment and survival as breeding adults. Capture‐mark‐recapture analysis on a large sample of young (N = 20,152 fledglings from 3959 broods) treated over 10 years revealed that neither PHA injection nor blood sampling affected individual survival and detection probability. Recruitment as a breeder varied among years, but this variation was not attributable to sampling effort, or the percent of all adults identified at the nest during a given year. Variation in the percent of adults identified was primarily attributable to the effect of nest depredation on our ability to capture nesting pairs. Our results indicating lack of an effect of blood sampling and immune stimulation on survival are encouraging, but we recommend further work to assess the potential negative effects of all commonly used techniques on the survival of study subjects in the wild, including the potential costs associated with mounting various immunological responses.     

Immune activity elevates energy expenditure of house sparrows: a link between direct and indirect costs?

The activation of an immune response is beneficial for organisms but may also have costs that affect fitness. Documented immune costs include those associated with acquisition of special nutrients, as well as immunopathology or autoimmunity. Here, we test whether an experimental induction of the immune system with a non-pathological stimulant can elevate energy turnover in passerine birds. We injected phytohaemagglutinin (PHA), a commonly used mitogen that activates the cell-mediated immune response, into the wing web of house sparrows, Passer domesticus. We then examined energetic costs resulting from this immune activity and related those costs to other physiological activities. We found that PHA injection significantly elevated resting metabolic rate (RMR) of challenged sparrows relative to saline controls. We calculated the total cost of this immune activity to be ca. 4.20 kJ per day (29% RMR), which is equivalent to the cost of production of half of an egg (8.23 kJ egg(-1)) in this species. We suggest that immune activity in wild passerines increases energy expenditure, which in turn may influence important life-history characteristics such as clutch size, timing of breeding or the scheduling of moult.     

Secondary phytohaemagglutinin (PHA) swelling response is a good indicator of T‐cell‐mediated immunity in free‐living birds

The validity of using the phytohaemagglutinin (PHA) test to measure acquired immunity, one of the most widely used methods, is currently being debated due to new knowledge on the complex physiology of the process. As a greater secondary response to repeated challenges linked to increases of circulating lymphocyte levels would be indicative of a T‐cell‐mediated immune response, we performed for the first time an experiment under natural conditions with repeated PHA challenges in free‐living adult birds and chicks to shed light on this topic. We found significantly stronger secondary response to PHA injection independent of sex or age, while controlling for body condition, the second response being on average 90% larger than the first. Likewise, lymphocyte counts were significantly higher in the second PHA challenge, whereas no significant differences were found among untreated birds. Significant positive correlations between the PHA response and both lymphocyte counts and plasma protein levels (mainly albumin, globulin precursor) were recovered, whereas no significant differences were recovered in plasma protein levels between challenges. Our results are consistent with those from captive birds, supporting the validity of the PHA skin‐swelling test as an accurate gauge of acquired T‐cell‐mediated immunity in birds.     

Social environment and immunity in male red jungle fowl

We examined the relationship between social dominance, immuneresponse, and ornamentation in captive red jungle fowl by comparingthese variables in males housed individually with a singlefemale to those in the same males after they were placed inflocks with an unfamiliar male and three unfamiliar females. Males with larger combs before being placed in the flocks weremore likely to become dominant, and dominant males' combs grewafter flock formation, whereas subordinate males' combs shrank.Immune response as reflected in hematocrit, immunoglobulinlevels, and wing web swelling (a measure of cell-mediated immunity)was stronger in males that later became dominant, both beforeand after flock formation, although the difference betweendominant and subordinate birds was more pronounced after maleswere housed in the multi-male groups. Dominant and subordinatemales also differed in the relationship between comb lengthand wing web swelling. Among dominant males, individuals withlarger combs had significantly larger swellings after flock formation, whereas within the subordinate males, those withrelatively larger combs had worse cell-mediated immunity thanthose with smaller combs. These results suggest that malesof different quality pay different costs to maintain both ornamentationand immune defense.     

Cytokine expression in phytohaemagglutinin‐induced skin inflammation in a galliform bird

Researchers interested in ecological immunology face substantial methodological problems: 1) most immunological approaches are difficult to perform in free‐living animals, 2) in some of the applicable methods the immunological background of the test remains unclear. The latter is also true for the phytohaemagglutinin (PHA) skin‐swelling test, a trait of cell‐mediated immunity commonly measured in ecology. A lack of direct evidence documenting the immunological processes in the tissue limits our understanding of the mechanism triggering the response to PHA. Understanding of this mechanism is, nonetheless, crucial for us to uncover the nature of ecological costs and benefits of investments into the response. As knowledge of cytokine signalling in the tissue may clarify the response mechanism, in our study we investigated the association between the PHA‐induced skin‐swelling and tissue cytokine expression in males of grey partridge Perdix perdix. In PHA‐challenged birds we assessed expression of nine cytokines (IL‐1β, IL‐2, IL‐4, IL‐6, IL‐10, IL‐12, IL‐17, TGF‐β, IFN‐γ) in wing‐web skin during an early stage of the immune response. We examined the relationship between the magnitude of tissue swelling and cytokine expression. Contrary to some earlier expectations we did not find any differential expression of T‐cell growth factor, IL‐2, in the tissue. Hence, T‐cell proliferation at the time of the swelling measurement is unlikely. We detected differential expression in Th17 pro‐inflammatory (IL‐1β, IL‐6) and anti‐inflammatory (TGF‐β) cytokines. The PHA‐induced swelling response was only weakly linked to the expression of TGF‐β. We also found relationships between the PHA‐induced swelling response and phenotypic traits of the birds; the PHA swelling was positively associated with the extent of melanin‐based breast ornamentation and negatively related to body size. Our results might suggest that variation in swelling is influenced by total numbers of responding cells rather than by differences in signalling. Moreover, we revealed significant correlations in expression of IL‐1β, IL‐6 and TGF‐β. These findings are the first to show on the molecular level that the PHA skin‐swelling test actually measures inflammation process which is part of innate immune defence and not the adaptive immune response (as assumed if the test was the reflection of T‐cell proliferation).     

不同繁殖状态雌性黑线仓鼠对植物血凝素的反应模式

由植物血凝素（Phytohemagglutinin,ＰＨＡ） 诱导产生的肿胀反应是生态免疫学中被广泛使用的免疫学参数,通过注射部位的肿胀程度大体上可反映细胞介导的免疫和天然免疫的综合变化情况。为探讨小型哺乳动物不同繁殖状态对ＰＨＡ 的反应模式,以非繁殖期、妊娠期、哺乳期和断乳期的雌性黑线仓鼠为研究对象,测定了注射ＰＨＡ 和生理盐水前（０ ｈ）及注射后６ ｈ、１２ ｈ 和２４ ｈ 注射部位皮肤组织的增厚程度。结果显示：（１）黑线仓鼠对ＰＨＡ 的反应有两种模式：非繁殖期、妊娠期和断乳期对ＰＨＡ 的反应模式相似,都在注射后６ ｈ 最高,１２ ｈ 和２４ ｈ 后逐渐下降;哺乳期对ＰＨＡ 的反应在注射后６ ｈ、１２ ｈ 和２４ ｈ 接近,彼此间的差异均不显著。（２）哺乳期黑线仓鼠的ＰＨＡ 反应高峰值有下降趋势,但与非繁殖期、妊娠期和断乳期高峰值之间的差异不显著。结果表明,黑线仓鼠在哺乳期对ＰＨＡ 的反应模式不同于其他繁殖阶段,且反应的高峰值被延迟,这对其存活可能有害,但有助于繁殖进程的延续和后代的生长发育。     

Sex differences in circulating antibodies in nestling Pied Flycatchers Ficedula hypoleuca

Sex differences in immune function are relatively well studied in vertebrate animals, although the patterns are not always clear in birds. The study of immune responses in nestlings of wild bird populations may constitute an appropriate way to investigate inherent intersexual differences while controlling for environmental conditions such as parasitism that affect male and female individuals growing in the same nest. We studied whether the cell‐mediated immune response, as measured by phytohaemagglutinin (PHA) injection, and the levels of circulating antibodies differ between sexes of Pied Flycatcher nestlings Ficedula hypoleuca. No sex differences in nestling cell‐mediated immune response were found, but females showed significantly higher levels of plasma immunoglobulins than males did. Although nestling birds may not have a fully functional humoral immune defence, our study indicates that sex differences in the humoral component exist at this early stage of life. Given the importance of antibodies in the fight against parasite, bacterial and viral infections, the intrinsic sex disparity in circulating antibodies may have important implications for the life history of each sex.     

Application of Concanavalin A during immune responsiveness skin‐swelling tests facilitates measurement interpretation in mammalian ecology

The skin‐swelling test is a simple and widespread method used in field ecological research to estimate cellular immune responsiveness in animals. This immunoecological test is based on measuring the magnitude of tissue swelling response at specific times following subcutaneous application of an experimental pro‐inflammatory stimulant. In the vast majority of studies across vertebrate taxa, phytohemagglutinin (PHA) is used as a universal stimulant. Given the complexity of immune response activation pathways of PHA, however, interpretation of test results can be ambiguous. Goal of this study was to improve methodology of the skin‐swelling test to decrease this ambiguity. Here, we present an alternative protocol aimed at facilitating interpretation of skin‐swelling data for mammals. Based on previous evidence suggesting that mammalian T cells are readily activated by Concanavalin A (ConA) in vitro, we compared cellular immune responses in vivo to PHA and ConA as an alternative pro‐inflammatory stimulant in mice. We measured magnitude of tissue swelling and compared it with intensity of blood cell infiltration into tissue over a 72‐hour interval. Our results corroborate that PHA and ConA show important differences in both dynamics and response amplitude in rodents. ConA induces stronger swelling with a distinct leukocyte activity pattern and higher pro‐inflammatory cytokine (interleukin 6 [IL‐6] and interferon gamma[IFN‐γ]) expression than PHA during peak response (24‐h post‐treatment). Furthermore, unlike PHA, magnitude of swelling was positively associated with cellular activity (number of neutrophils infiltrating tissue) following ConA injection. We conclude that ConA is the more suitable stimulant for skin‐swelling tests in mammals. This is because of the molecular binding specificity in the two lectins, that is, ConA specifically activates T cells while PHA also triggers erythroagglutination. We propose that ConA be used in all future ecological testing in mammals as it exhibits better performance and its application facilitates immunological interpretation of skin‐swelling test results.     

Immunity to transplantable carcinogen induced fibrosarcomas in B2/B2 chickens. II. Macrophage dependency of adoptive T cell mediated tumor immunity.

The nature of immunity to transplantable chemically induced fibrosarcomas in SC (B2/B2) chicken was examined using Winn tests performed in the wing web. Immunity in spleen cell donors was induced by pretreatment with C. parvum or BCG followed by tumor cells + bacterial adjuvant in one and tumor cells alone in the other wing web. The T cells mediating the adoptive immunity were sensitive to anti-T + C, nylon wool nonadherent, mitomycin resistant and radiation (1000 R) sensitive. The adoptive immunity could not be expressed in heavily irradiated recipients or in hosts pretreated with trypan blue or silica. The host contribution could be reconstituted by iv injection of spleen or bone marrow cells from agamma-globulinemic (Aγ) unimmunized donors 2 days prior to Winn tests or by the local injection into wing webs of spleen cells or purified peripheral blood monocytes from Aγ donors. It was concluded that cooperation between immune donor T cells and normal monocytes of host origin mediated the inhibition of SCFS growth in Winn tests.     

Does the strength of an immune response reflect its energetic cost?

The energetic cost of immune responses has been proposed to be an important basis for trade-offs between life-history traits, such as between survival and reproduction. A critical assumption of this hypothesis is that the magnitude of the energetic cost increases with the strength of an immune response, so that energy can be saved by partly suppressing a response. Here, we test this assumption experimentally. The immune system of great tits Parus major was experimentally activated by injecting different doses of phytohemagglutinin (PHA) in the wing web. We found the resting metabolic rate of immune challenged birds to increase by 5%. However, although great tits injected with a high dose had a stronger immune response, this was not paralleled by a higher metabolic rate. Thus, we found the energetic cost of the immune response to be relatively low and not dose-dependent. This suggests to us that the energetic cost of immune responses cannot form the basis for trade-offs between life-history traits.     

UV plumage color is an honest signal of quality in male budgerigars

Elaborate and colorful feathers are important traits in female mate choice in birds. Plumage coloration can result from pigments deposited in feathers such as carotenoids and melanins, or can be caused by nano-scale reflective tissues (structurally based coloration), usually producing ultraviolet (UV) coloration. Structural colorations remain the least studied of the three most important feather colorations. Previous studies have found a female preference for UV color in the budgerigar, Melopsittacus undulatus, but it is not clear what information this ornament conveys, nor what is the possible cost associated with its production. We investigated possible correlations between immune response and plumage color of wild-type (green) male budgerigars. In particular we measured the wing web swelling resulting from injection of phytohaemagglutinin (PHA). We did not detect any correlation between the sedimentation rate and morphological and color variables. We found that UV chroma is the best predictor for the cutaneous immune activity. Indeed, male budgerigars with high UV reflectance in the breast feathers showed stronger immune responses. These results are consistent with the idea that UV colors are special signals conveying information about a bird’s condition.     

Cell-mediated immunosenescence in birds

The phytohaemagglutinin (PHA) skin test response, used to assess cell-mediated immunity, is known to vary with many social and energetic factors, but the effects of age have received little attention. We found that the PHA response of immature birds was lower than those of the youngest breeding adults and were decreased in adults. Whenever possible, age should be included as a covariate when the PHA skin test is used to assess immunocompetence in ecological immunology. The rate of decline in PHA response differed between species and was inversely correlated with survival. The decrease in the PHA response averaged 57% over an average 80% of the maximum life span, but the absolute rate varied with species lifespan such that the short-lived species showed a greater loss per year than the long-lived species. This link between declining immune function and survival may reflect differences in resource partitioning between species, and suggests that selection may act on investment in immune function to influence maximum life span.     

Cross-fostering reveals an effect of spleen size and nest temperatures on immune responses in nestling European starlings

Immunocompetence may be a good measure of offspring quality, however, factors affecting variation in immune responses are not clear. Research suggests that immune function can vary due to differences in genetics, development conditions and individual quality. Here, I examined factors affecting variation in immune response among nestling European starlings through a split-nest cross-fostering brood manipulation that included two important covariates: spleen size and nest temperatures. Immunocompetence was assessed via a cell-mediated immune response to phytohaemagglutinin (PHA). This paper provides the first direct evidence that individuals with large spleens also mount strong immune responses. Exposure to PHA did not cause splenomegaly, as there was no difference in spleen size between control birds and those injected with PHA. Offspring immune function was affected by common origin and by rearing environment, though rearing environment appeared to exert its influence only through nest temperatures. A comparison of the immune performance of siblings reared in their home nest versus those reared in other nests revealed a strong effect of maternal quality. As the difference in natal clutch size increased, the magnitude of the difference in immune performance between home-reared nestlings versus out-reared nestlings increased. Overall, nestling immune function appears to be determined by the combination of genetic, maternal and environmental effects.     

The novel application of an immunological technique reveals the immunosuppressive effect of phytoestrogens in Betta splendens

The novel application of a standard technique to assess cell‐mediated immune response to phytohaemagglutinin (PHA) injected in the caudal peduncle in a small fish (<2 g) to test the immunosuppressive effect of three phytoestrogens: genistein, equol and β‐sitosterol is described. Individual Betta splendens exposed to these oestrogenic chemicals produced weaker inflammatory responses to PHA than did control individuals, suggesting that phytoestrogens are immunosuppressive. This technique should enable immune function in fish species, too small to provide sufficient blood for conventional immunological measures, to be assessed.     

Immune challenge of female great tits at nests affects provisioning and body conditions of their offspring

The trade-off between animal’s parental reproductive effort and survival is still poorly understood. Parental allocation between the workload during breeding attempts and the parents’ own body conditions can be assessed through the offspring quality. Here, I questioned whether the immune responsiveness of female great tits may be considered as a mediator of this trade-off. Specifically, I tested whether (1) the parental reproductive effort decreases, (2) the food composition provided to chicks changes, and (3) whether the nestling immunocompetence and body mass decrease after experimental immunisation. Two populations of great tit Parus major occupying nest boxes were studied in Niepo?omice Forest and Krzyszkowice Forest (Southern Poland) in 2011 and 2012, respectively. Three days after hatching, half of the females were challenged with sheep red blood cells (SRBC), while other females were injected with phosphate-buffered saline PBS (control). Six days later, food provided by the parents was collected from nestlings. After another 2 days, the offspring’s body mass was measured and wing web swelling in response to an additional phytohaemagglutinin (PHA) injection. In both years, immunocompetence and in 2012 also body mass in the offspring of SRBC-immunised mothers were lower than in control nestlings, indicating a cost of mounting the immune response in the female. Six days after the start of the female treatment, the number of caterpillars and the volume of food items provided by parents to chicks were higher, whereas the number of spiders was lower in nests with SRBC treatment than in control ones. This might be explained by compensational parental feeding after recovery from the inflammation of a female. Thus, the trade-off between parental effort and survival of parents is mediated by the costs incurred for their immunity and can be assessed by the amount and quality of food provided to the nestlings and the offspring condition.     

Maternal and dietary carotenoids interactively affect cutaneous basophil responses in growing chickens (Gallus gallus domesticus)

This trial examined effects of lutein supplied from maternal (i.e., in ovo) and dietary routes on cutaneous basophil hypersensitivity responses in chickens. Chicks hatched from one of two in ovo carotenoid levels (n=100/level; carotenoid-replete or carotenoid-deplete eggs) were fed one of two diet lutein levels (0 or 40 mg diet lutein/kg diet). At 14 d post-hatch, phytohemagglutinin (PHA) was injected into the wing web, and thickness and leukocyte populations were measured at 0 to 48 h. PHA increased wing web thickness at 4 to 48 h post-PHA (p<0.05), and the swelling response was most related to macrophage numbers in the wing web. Swelling occurred more rapidly in chicks from carotenoid-deplete eggs (p<0.05), but eventually reached a greater thickness in chicks from carotenoid-replete eggs (p<0.05). Differences in leukocyte infiltration occurred due to diet and in ovo carotenoid exposure, and indicate that pre- and post-hatch carotenoid exposure had additive or synergistic effects on the PHA-induced wing web response. Evaluation of the cellular contents of the injection site is a much better indicator of the immunomodulatory effects of lutein than measurements of the amount of swelling.     

Phytohemagglutinin response and immunoglobulin index decrease during incubation fasting in female common eiders

To maximize their fitness, long-lived species face trade-offs between survival and reproduction. The cost of reproduction, which is defined as the negative effect of current parental investment on chances of adult survival and future reproduction, may affect immune function, possibly through hormonal changes. In this study, components of acquired immunity and plasma corticosterone levels of female eiders (Somateria mollissima) have been measured throughout the incubation period as a function of clutch size. These precocial birds lay up to six eggs and fast completely during incubation. Birds were sampled early and late in the incubation period, with clutches ranging from one to four eggs. T-cell-mediated immune response and humoral immunity were assessed by phytohemagglutinin (PHA) skin tests (a challenging method) and measurements of serum immunoglobulins (a monitoring method), respectively. During incubation, responses to PHA injection and immunoglobulin index significantly decreased, by about 40% and 25%, respectively. These observed decreases occurred independently of the number of eggs laid by the females. Corticosterone did not vary significantly during incubation, whatever the clutch size. We conclude that female eiders seem to reallocate their resources from immune function to reproductive effort independently of clutch size or corticosterone levels.     

Inhibiting aphid wing development: is immune challenge important?

Aphids show inhibition of wing development when parasitized by hymenopteran parasitoids, especially when parasitism takes place early in the host's development. The present study tests the hypothesis that the aphid itself redirects the resources needed for the development of wings to support the immune response, rather than the parasitoid directly promoting wing inhibition. When the aphid's immune system is challenged with bacterial antigens and a fungal pathogen, neither immune challenge alters wing development, indicating that perhaps there is no redirection by the aphid of resources required for wing development to cover possible costs of mounting an immune response.