Transgenerational effects enhance specific immune response in a wild passerine

Introduction

Vertebrate mothers may provide a favourable growing environment and care, but additionally can transmit diverse components such as hormones (Groothuis & Schwabl, 2008), antioxidants (e.g., Royle, Surai & Hartley, 2003) or immunoglobulins (Grindstaff, Brodie & Ketterson, 2003; Boulinier & Staszewski, 2008; Hasselquist & Nilsson, 2009) that can have important phenotypic consequences on the developing embryo (Mousseau et al., 2009). These maternal effects constitute a major source of transgenerational phenotypic plasticity that can vary according to environmental heterogeneity (e.g., Morosinotto et al., 2013) and maternal condition (Boulinier & Staszewski, 2008). However, the way these transferred components interact with each other, and modulate the development of the offspring phenotype remains unclear (Mousseau et al., 2009).

The vertebrate humoral immune response targets specific infectious agents by means of antibodies (Ab), and persists as immune memory allowing a faster response upon re-exposure to the same antigen. While adult birds can develop a humoral response in a few days (Iseri & Klasing, 2013), newborn individuals require some time to fully develop such capabilities and may rely on maternally transferred Ab while they develop their own endogenous response (Hasselquist, Tobler & Nilsson, 2012). Altricial avian small species are particularly vulnerable at hatching as they need to develop fast enough to be able to fledge in a few days time, and thus could be expected to rely preferentially on maternally transmitted Ab to develop their specific immunity. However, studies on the influence of maternal Ab on host development and fitness are scarce (Boulinier & Staszewski, 2008), despite the important influence individual immune condition exerts on pathogen susceptibility in wild organism populations (e.g., Hochachka & Dhondt, 2000). The influence of maternal Ab on the fitness and disease epidemiology of neonates in humans and domestic mammals is well documented, providing the offspring with transient immunity against the microbial infections that the mother has encountered (Hasselquist & Nilsson, 2009). In avian species, yolk-transmitted maternal Ab detectability ranges from days to few months after hatching, depending on the species (Garnier et al., 2012) and the initial amount transferred (Grindstaff, 2010). However, whether maternally transmitted Ab are enhancing the chick’s subsequent response by a priming mechanism (e.g., Grindstaff et al., 2006; Reid et al., 2006), or blocking the chick’s endogenous response (e.g., Staszewski et al., 2007; Elazab et al., 2009; Staszewski & Siitari, 2010) remains poorly understood.

Individuals exhibit substantial differences in their immune responses and rarely develop it maximally, which suggests there are some costs associated that individuals manage differently (Viney, Riley & Buchanan, 2005). Such individual variation in immune investment may arise from a variety of factors, not only adaptive adjustments but also constraints resulting from allocation conflicts with other physiological and life-history traits (Ardia, Parmentier & Vogel, 2011). Furthermore, individuals may invest differentially in various components of the immune system (Lazzaro & Little, 2009), depending on the pathogen identity (Adamo, 2004) or constraining nutritional or energetic factors (Hõrak et al., 2006), or alternatively these could vary in concert (Ardia, 2005; Ahmed et al., 2007), but see (Forsman et al., 2008).

Oxidative balance and carotenoids have been acknowledged among the factors mediating variation in the immune responses (Hasselquist & Nilsson, 2012). The intensity of the immune response is generally coupled with a shift in the oxidative balance and a decrease in carotenoid concentration that are considered detrimental for the individual (Ardia, Parmentier & Vogel, 2011). Oxidative stress is generated as a metabolic by-product resulting in damage to cell macromolecules (Dowling & Simmons, 2009). Organisms balance their oxidative stress by acquiring and producing antioxidants, and although most antioxidative activity is enzymatic, non-enzymatic antioxidants also play a relevant role in maintaining oxidative balance, particularly in blood (Cohen & McGraw, 2009). Immunity costs in terms of oxidative balance have been argued to underlie trade-offs between immunity and life-history traits, either by directly increasing oxidative stress (Costantini & Møller, 2009) or by competing for antioxidants (Monaghan, Metcalfe & Torres, 2009), but see Speakman & Garratt (2014).

Carotenoids are a diverse group of lipophilic molecules important for immunity and individual fitness, and since they cannot be synthesized de-novo by animals, necessarily need to be ingested or acquired during embryogenesis through maternal transfer (Pérez-Rodríguez, 2009). Avian mothers transmit carotenoids and antioxidants through the egg yolk, and after hatching through diet (Blount et al., 2003; McGraw & Ardia, 2003). These components interact synergistically (Bédécarrats & Leeson, 2006; Koutsos, García López & Klasing, 2007) and likely stimulate the development of the offspring own immune phenotype (Simons, Cohen & Verhulst, 2012). However, little is known on how such maternal effects may interact with each other, especially on wild and non-model species (Hasselquist & Nilsson, 2012).

In this study we explored whether maternal effects modulate offspring specific immune response in a wild breeding house sparrow (Passer domesticus, Linnaeus 1758) population. Using a vaccine to elicit an immune response to a viral antigen, we analysed how maternal exposure during first brood affects transmission of antibodies to the following brood, their offspring development and immune response when exposed to the same antigen during the critical post-hatching period before fledging. We additionally measured chick PHA-induced inflammatory response, to find out whether mother immune condition affected their offspring specific humoral response, or affected more aspects of offspring immunity. Furthermore, we studied the effect of other maternally-transmitted components (antioxidative and carotenoids) which are likely to affect the development of the offspring immune phenotype. We predict vaccinated mothers to positively affect their offspring specific immunity, and this effect to be enhanced by higher levels of maternal antioxidants, antioxidative capacity and carotenoids in blood.

Materials and Methods

Results

Female NDV-Ab concentration before challenge were similar between treatments (F_1,14 = 0.86; P = 0.37), and no differences between experimental and control groups were found before challenge for any of the physiological (Table 1) or reproductive parameters studied. As expected, female NDV-Ab concentration increased significantly in vaccinated individuals with respect to control ones (Control: 0.00 ± 0.38 vs. Vaccinated: 4.70 ± 0.41; F_1,20 = 70.67; P < 0.0001) (Fig. 1). Neither pre-challenge NDV-Ab concentration (F_1,12 = 0.39; P = 0.54), days between vaccination and re-sampling (F_1,13 = 0.07; P = 0.80), the physiological nor the reproductive parameters considered (all P > 0.5) were related to post-challenge NDV-Ab concentration.

Chick NDV-Ab concentration at 11 days of age was independent of maternal treatment (F_1,17 = 0.06; P = 0.81). Surprisingly, chick NDV-Ab concentration was independent from chick treatment (F_1,19 = 3.83; P = 0.07), and the interaction with maternal treatment (F_1,19 = 0.52; P = 0.48). Time between chick vaccination and sampling was not significant (F_1,17 = 0.26; P = 0.615). However, when considering female NDV-Ab concentration as a covariate, the interaction between female NDV concentration and chick’s treatment appeared significant (F_1,53 = 6.61; P = 0.01), together with days between chick and mother sampling (Estimate 0.324 ± 0.121; F_1,17 = 7.20; P = 0.02). NDV-Ab concentration of vaccinated chicks tended to increase with female NDV-Ab concentration, whereas this relation was not significant in control chicks (Control: −0.11 ± 0.14; t₁₈ = − 0.81; P = 0.43 vs. Vaccinated: 0.23 ± 0.13; t₁₈ = 1.75; P = 0.09). Neither sex, biometric measurements nor the breeding parameters were associated with NDV-Ab concentration in chicks (all P > 0.2). Vaccinated chicks from vaccinated mothers developed higher specific humoral response, but only when originating from mothers exhibiting high NDV-Ab titres (Fig. 1).

On the other hand, when including blood metabolites as covariates on the previous model, only carotenoids and TAC were related to chick NDV-Ab. Carotenoids decreased with increasing NDV-Ab concentration in chicks, independently of the treatment (slope: −1.35 ± 0.47; F_1,39 = 8.11; P < 0.01). However, the relation between NDV-Ab and TAC changed slightly according to the chick treatment, the interaction being significant (F_1,50 = 5.95; P = 0.02). NDV-Ab concentration of vaccinated chicks decreased with TAC, whereas the relation was not significant in control chicks (Control: −0.34 ± 0.59; t₅₀ = − 0.59; P = 0.56 vs. Vaccinated: −2.08 ± 0.45; t₅₀ = − 4.66; P < 0.01). Chick’s concentration of NDV-Ab was negatively related to carotenoids in blood, and in the case of vaccinated chicks also to TAC in blood (Fig. 2 in Supplemental Information 1). Uric acid or total protein were unrelated to chick’s NDV-Ab concentration (all P > 0.1).

PHA in chicks was unrelated to maternal (F_1,9 = 0.01; P = 0.93) or chick (F_1,21 = 0.02; P = 0.88) treatments, even when accounting for chick body mass or change in body mass between challenge and measurement (hereafter body mass change), and time between such measurements (all P > 0.2). Furthermore, none of the breeding parameters, biometric measurements nor blood metabolites considered were significantly related to PHA response (all P > 0.09). As expected, PHA variation in chicks was only significantly explained by body mass (slope: −0.06 ± 0.02; F_1,29 = 5.05; P = 0.03), as PHA response was lower the heavier the chick.

Body mass change in chicks from vaccination to sampling date (4–11 days of age) was independent of mother’s and chick’s NDV-Ab titres (all P > 0.30). Likewise, neither treatment, sex nor the blood metabolite variables were related to body mass change (all P > 0.2). As expected, elapsed days between vaccination and sampling (slope: 1.20 ± 0.28; F_1,20 = 18.23; P < 0.01) and tarsus length had a significant influence on body mass change (slope: 0.60 ± 0.27; F_1,63 = 4.96; P = 0.03) (Table 1). House sparrow chicks with larger tarsi grew heavier independently of their mother’s or their own level of NDV-Ab, once the period between measurements was accounted for.

Chick’s survival from 4th day of age until fledging was unrelated to neither the maternal (F_1,22 = 0.76; P = 0.39), nor the chick’s treatments (F_1,23 =0.31; P = 0.59), and only body mass at the time of vaccination was related to fledging success (slope: 0.49 ± 0.11; F_1,89 = 18.29; P < 0.01).

Discussion

House sparrow mothers developed a significant specific humoral response when challenged with NDV vaccine before egg-laying, and as a result their offspring were more likely to develop a specific humoral response when challenged with the same antigen. However, inter-individual variation in maternal NDV-Ab was the main determinant of chick specific immune response to NDV, as only chicks from mothers with high NDV-Ab concentration significantly increased their response to NDV vaccine (Fig. 1).

Maternal Ab transfer may reflect a dynamic balance between the benefits of providing an early specific protection and the costs of blocking the nestling’s immune development (Garnier et al., 2012). In our study, maternal Ab could not be detected in 11-day-old chicks by means of standard diagnostic techniques, as no differences were found between control chicks from vaccinated and control mothers. The results suggest that maternal Ab transmission in house sparrows has a priming beneficial effect on the specific response of pre-fledging chicks (11 days old), by stimulating the endogenous production of Ab when early exposed to same pathogens as their mothers. This priming effect may be explained by a higher and/or faster antibody production in chicks exposed to maternal Abs. However, disentangling such non-mutually exclusive processes would require a detailed temporal monitoring of NDV-Ab response after fledging, which is beyond the focus of this study.

On the other hand, mothers influence offspring immunity in other ways than transmission of specific Ab, by providing other components through the yolk or by modulating parental investment. Therefore, it is possible that the enhanced specific immune response in offspring could result from other maternal effects than the transmission of specific Ab, or a combination of them. However, the fact that chicks did not experience any change in growth rate or PHA-response which is a complex inflammatory and immune response suggests that maternal effects were antigen-specific and not condition dependent (Ahmed et al., 2007).

Maternal treatment per-se was ineffective in explaining offspring specific response, and only when considering inter-individual variation in specific Ab levels such relationship was apparent. This result can be explained by several non exclusive reasons. First, the different response among breeding females to the experimental challenge, which led to a significant inter-individual variation in circulating Ab level, could arise from the usual individual variation to experimental vaccinations (Zinkernagel, 2003). Second, some breeding females could have been previously exposed to the virus in the wild (see Broggi et al., 2013). Previous exposure would lead to different maternal level of circulating Ab and in turn affect passive transmission of Ab to the offspring. In fact, one control chick originating from a control mother presented NDV-Ab, implying a natural exposure to the antigen (Fig. 1). Second, it is possible that laying females effectively transfer Ab only when their own systemic levels are above certain threshold (Grindstaff, 2010). Alternatively, it could be that maternally transmitted Ab do not persist long in the nestling blood, and after 11 days of age they are not detectable anymore (Grindstaff, Brodie & Ketterson, 2003; Nemeth, Oesterle & Bowen, 2008). The fact that breeding females were evaluated a few weeks after being challenged, while their chicks were challenged and sampled after a few days may rend our results conservative as it may be that maternal effects are most effectively transmitted earlier after their challenge, or the effects appear after 11 days of chick age, but see Garnier et al. (2012). The present results add to the accumulating evidence that exposure at different times during the course of a decay of maternal antibodies, which can be short-lived in altricial species like sparrows may affect chick immune response differently (Zinkernagel, 2003).

The few studies on maternally transmitted Ab on offspring specific immunity on wild avian species are not conclusive as the effects often vary among host species, pathogens and timing of exposure (Zinkernagel, 2003; Hasselquist & Nilsson, 2012). The limited knowledge on the persistence of maternal Ab, the temporal pattern of Ab production and the long-term consequences of maternal transmission limits our capacity to interpret these differences (Boulinier & Staszewski, 2008). Some studies report short-term enhancing effects (Grindstaff et al., 2006; Pihlaja, Siitari & Alatalo, 2006), while others have revealed a blocking effect (Gasparini et al., 2009; Staszewski et al., 2007; Staszewski & Siitari, 2010; Garnier et al., 2012; Merrill & Grindstaff, 2014). Interestingly, same host species can respond differently to maternally transmitted Ab when exposed to varying antigens (Gasparini et al., 2006; Addison, Ricklefs & Klasing, 2010). Other studies have found maternal transmission of Ab to buffer costs of an immune response (Grindstaff, 2008), whereas others found the effect of maternal Ab to be negligible (Nemeth, Oesterle & Bowen, 2008; King, Owen & Schwabl, 2010). Finally, long-term effects of maternal Ab-transmission on the specific immune response of chicks to antigens administrated on the previous breeding season to pre-laying mothers have been reported blocking (Staszewski et al., 2007), or enhancing the offspring specific response (Reid et al., 2006). In our study we found house sparrow mothers to enhance their offspring specific humoral response during the pre-fledging period, but not their PHA response, implying that specific Ab are transmitted without conditioning other components of their immune system like those involved in the response to the PHA test. These contrasting results highlight the varying effects that maternal Ab may have among hosts exposed to different pathogens, and the need to analyse differences in the dynamics of antibody circulation, timing of exposure to pathogens and the interaction with other maternally transferred components.

Interspecific comparisons suggest that larger and longer lived species, which also experience slower developmental times, may rely more strongly on the maternal effects that may be transmitted in larger amounts and persist for longer periods (Garnier et al., 2012; Ramos et al., 2014). In line with this suggestion and in contrast with our results, it has been argued that maternal transfer of Ab in fast-developing altricial species (e.g., house sparrow) may be limited or not even play any relevant role as endogenous Ab production is achieved soon after hatching, leaving maternal Ab a too short time-frame to be effective (King, Owen & Schwabl, 2010). Alternatively, maternal Ab may be effectively transmitted and still remain undetected, as they may persist for a too short time period or because of practical constraints as good samples are difficult to obtain from hatchlings of those small-sized species (Lozano & Ydenberg, 2002; Nemeth, Oesterle & Bowen, 2008). Furthermore, methodological differences among studies may partly explain the varying results on the effects of the maternal transmission of Ab on offspring specific immunity. Although most studies tested the effects by studying both mothers and offspring exposed to the same antigen, this was not always the case (e.g., Gasparini et al., 2006; Pihlaja, Siitari & Alatalo, 2006; King, Owen & Schwabl, 2010). Our results suggest that maternal transfer of antibodies in the house sparrow is important enough to prime chick specific immunity, despite not being detectable by usual immunological techniques. The effect of maternally transmitted Ab appears to be host-pathogen specific, and dependent on the development of host humoral immunity at the time of infection. Furthermore, such effect could be of a hormetic nature (Costantini, Metcalfe & Monaghan, 2010), irrelevant under certain threshold and priming or blocking according to the different concentration of Ab transmitted and the individual condition (Lemke, Coutinho & Lange, 2004).

The ontogeny of the constitutive immunity is complex, influenced by both maternal and endogenous factors, with long term implications for the individual development and overall immunity (Butler & McGraw, 2011; Van der Most et al., 2011; Arriero, Majewska & Martin, 2013). Maternal transfer of specific Ab could benefit offspring by enhancing the specific humoral response while permitting growth rate to be maintained (Grindstaff, 2008), as found in this study. However, we found chicks developing stronger specific humoral response experienced decreased carotenoid levels and antioxidative capacity in blood. In addition to Ab, parent females transfer carotenoids and antioxidants to their eggs according to diverse endogenous and environmental factors such as mate attractiveness (Saino et al., 2002), own condition (Hammouda et al., 2012), pathogen abundance (Gasparini et al., 2001) or predation risk (Morosinotto et al., 2013). We found no differences in blood carotenoids or TAC measured in females either at challenge or post-challenge sampling times in relation to any of the treatments or NDV-Ab concentration, suggesting that variation in chick’s levels arose from chick’s own physiological adjustments rather than differential maternal transmission. Several studies have shown costly aspects of immunity in terms of antioxidative balance and carotenoid levels. Interestingly, other studies found carotenoid levels to be positively related to humoral response but unrelated to PHA response (Bédécarrats & Leeson, 2006). Carotenoids appear to compensate costly immune responses to pathogens (Ewen et al., 2009), or become depleted the stronger an immune response (Saino et al., 2003). In our study, house sparrow offspring fledged independently of their treatment or NDV-Ab concentration, suggesting that supposed advantages of enhanced specific humoral response, or costs of decreased antioxidative capacity and carotenoids in blood are to be experienced on a longer-term. However, the final cost-benefits balance derived from maternal transmission of antibodies will depend on the rate of exposure to pathogens, and the fitness costs derived from pathogen exposure in chicks with and without maternal antibodies.

In summary, we found that the specific humoral response to NDV in 11-day-old house sparrow chicks is enhanced by maternal exposure to NDV, most likely through passive transmission of specific Ab. However, nestlings investing in specific NDV-Ab production present lower carotenoid concentration and impaired antioxidative capacity in blood, suggesting that maternal priming of specific humoral response can be beneficial but may come to a physiological cost in pre-fledging small altricial species.

Supplemental Information