The effect of daylight exposure on the immune response and body condition of Great Tit nestlings

Natural light plays an important role in many aspects of birds’ biology, ecology and behaviour. Wild birds experience various light conditions, both in terms of its intensity and spectral properties, which vary within the nesting habitat of avian species. Recent study has shown that some bird species prefer to build their nests in bright nest sites. Light is one of the major factors affecting early development and condition of nestlings, but to date, this effect has not been tested in wild conditions. This study aims to answer whether birds could benefit from nesting in brighter light environment. Potential benefits may occur in nestlings’ condition, growth rate and breeding output. The research was carried out in the population of Great Tits (Parus major) breeding in two types nest boxes which differed with internal illumination. In “dark” boxes, the only source of light was the entrance hole. “Bright” boxes have additional side semi-transparent windows. We conducted two experiments in which light environment in nest box was set after (Experiment I) or before (Experiment II) birds settle in. Our study showed that light environment did not significantly affect nestlings body condition and growth. However, females in their first breeding attempt showed strong preference to the brightened nest boxes. In this group, the offspring were heavier and showed stronger immune responses than nestlings reared in dark nest boxes. Nestlings in bright nest boxes fledged about one day earlier. Nesting in brightened nest boxes may provide potential benefit for adult females in their first breeding attempt. The interaction between maternal effect and light environment is being discussed.

The effect of luminance within the nesting habitat of avian species might be critically important. Light is known to influence the development and condition of young birds. Theoretically, this process starts as early as at the embryo stage, due to the fact that eggshells transmit some portion of the light waves to the developing organisms (Maurer et al. 2011(Maurer et al. , 2015. Exposure of eggs to light during incubation is, in general, beneficial because it accelerates embryo development (Cooper et al. 2011;Austin et al. 2014), increases functional brain asymmetry, helps to establish circadian rhythm, activates antimicrobial defense mechanisms on the surface of the eggshell, and plays the role in repairing DNA in the process of photoreactivation (reviewed by Maurer et al. 2011). The positive effect of light continues after hatching and it promotes body mass growth (Robbins et al. 1984) and synthesis of vitamin D3 (Lewis et al. 2009), and also prevents arrhythmia, tibial dyschondroplasia and rickets (Lewis et al. 2009). Evidence for the impact of light on development and conditions of young birds is rare and come mainly from experiments in poultry farms, where the light intensity and wavelength spectrum is beyond its natural range (Fairchild 2000;Olanrewaju et al. 2006Olanrewaju et al. , 2012Lewis 2010). Rare studies on wild species were also confined to laboratory experiments (Cooper et al. 2011;Austin et al. 2014). In wild free-living bird species, a large majority of studies investigate the effect of Artificial Light at Night (ALAN) on various aspects of biology and physiology (e.g. Kempenaers et al. 2010;de Jong et al. 2015;Russ et al. 2017). Although these studies are valuable sources of knowledge on the effect of disturbance of daily light rhythm, however, they do not provide information about natural light variation on avian biology.
Light conditions in an offspring rearing environment may differ even within the same population of a given species (Muñoz et al. 2007;Honza et al. 2011;Maziarz and Wesołowski 2014). Consequently, birds use light characteristics as a cue when choosing the nest sites. This phenomena in known in both open cup nesters (Hartman and Oring 2003;Burton 2007), as well as in cavity nesting birds (Maziarz and Wesołowski 2014;Podkowa and Surmacki 2017;Monti et al. 2019).
The light may be crucial for development of young birds in diurnal cavity nesters. As was shown in an earlier study, natural cavities are very dark, so birds that breed there face light conditions similar to those during moonless night (Wesołowski and Maziarz 2012). On the other hand, especially in secondary cavity nesters, there is a significant variation in the illuminance within the natural cavities due to their depth, and the shape and orientation of the entrance hole (Wesołowski and Maziarz 2012;Maziarz and Wesołowski 2014). Moreover, birds may adjust the amount of light entering the cavity by modifying the nest construction . It can be assumed that due to the deficiency of light in cavities, diurnal species whose life history is closely related to well-lit environments (Kluijver 1951;Austin et al. 2014) should be particularly sensitive to dim light conditions experienced during reproduction.
In the present paper, we made the first attempt to assess the impact of natural daylight on the condition and development of nestlings of a free-ranging birds' species. Our goal was to extract the effect of natural light on birds' biology without interrupting their circadian rhythm nor photoperiod perception. We performed our surveys on Great Tit (Parus major) breeding in standard, dark nest boxes in which the only source of light was the entrance hole (hereafter "dark" nest boxes) and nest boxes in which daylight illuminance was increased by applying semitransparent plastic windows (hereafter "bright" nest boxes). Basing on findings from earlier studies, we expected that birds raised in brighter nest boxes would (i) grow faster, (ii) have better body condition, and (iii) have a greater immune response.

General procedures
The research was carried out between 2015 and 2017 in the Wielkopolski National Park (Western Poland, 52° 18′ N 16° 49′ E). In 2014, 159 wooden breeding boxes were hung in three study sites: Site I (27, 7 ha), Site II (11,2 ha) and Site III (9,45 ha), for more details see Kudelska et al. (2017). Internal dimensions of nest boxes were 12 × 14 × 40 cm and the entrance hole diameter was 3.2 cm. Each nest box was equipped with two translucent synthetic resin windows with a diameter of 5 cm located on side walls, 15 cm above nest box floor. Daylight intensity inside the breeding box was controlled by opening/ closing the windows with external shutters made of black opaque plastic. Over the course of the study, we created two types of boxes: dark with closed shutters, where the only source of light was the entrance hole, and bright, with opened shutters. Because all boxes were equipped with windows, we could freely change the type of nest box type and decide when it happens . Light intensity (lx) in nesting boxes was measured using a Sonopan L-100 luxmeter (Sonopan, Poland). The internal illuminance in bright nest boxes (Me = 52.67 lx, Q25-75% = 42.83-62.28) was about fifty times higher than in dark nest boxes (Me = 1.41 lx, Q25-75% = 1.04-2.19, Podkowa and Surmacki 2017).
At the ceiling of the nest box, we placed a trail camera (Bushnell® HD LiveView), to record the timing of the first egg-laying and hatching. For more details on nest box design and the use of trail cameras see Surmacki and Podkowa (2022). From mid-March to the end of June, the boxes were monitored to determine history of broods.
We performed two experiments, which differed in the time when both experimental groups (dark and bright boxes) were created. In the first approach conducted in 2015 (hereafter Experiment I), at the beginning of the breeding season, shutters in all nest boxes were shut (all boxes were initially dark). Soon after clutch completion, half of nest boxes were randomly assigned to bright group in which we opened the windows. Other boxes in which windows remained shut served as a dark group. In the second approach conducted in 2016-2017 (hereafter Experiment II), we opened windows in a half of the randomly chosen nest boxes before the onset of nest building (the ratio of available bright and dark nest boxes was 1:1). While in Experiment I the effect of light was separated from the effect of nest box choice by the parents, in Experiment II, adult birds could choose between the dark and bright nest boxes. In the studied population, Great Tits showed strong preference to bright nest boxes which were occupied about twice as often comparing to the dark ones . In both experiments, we assessed the effect of light on nestlings' biometrics and breeding success. In Experiment II, we assessed also nestlings' immunocompetence and fledging time. Because more than 50% of nests in year 2017 were abandoned due to weather collapse in mid-April, season 2017 were excluded from further biometric analysis. During seasons 2015-2016 we did not observe any suboptimal weather condition nor between-years differences in daily temperature measured for 19 consecutive days (starting 10 days prior to median egg lying date, t = 1.588, p = 0.130). We did not find any differences in the date of first egg lay-

Biometric measurements of nestlings
We used a standard protocol for biometric measurement of the nestlings (e.g. Mainwaring et al. 2010). Birds were weighed on an electronic scale (0.1 g) on the 2nd and 12th day after hatching (day of hatching being the "0" day). The chicks were individually marked by painting their claws with a black marker (2nd day), followed by ringing (8th day). We obtained biometrical measurements from nestlings hatched in 78 nests (2015: 21 dark and 16 bright boxes; 2016: 14 dark and 27 bright boxes). When nestlings were 12 days old, we took the following measurements: right wing length (ruler, 0.5 mm), head length with beak and right tarsus length (electronic caliper, 0.1 mm). To assess condition of nestlings, we calculated Scaled Mass Index (SMI) proposed by Peig and Green (2009). SMI is an alternative for traditional body mass index (i.e. residuals from body mass-linear measure regression), which might be biased towards larger individuals (Arnold and Green 2007). We calculated SMI from body mass and a linear body measurement which has the strongest correlation with body mass. In our population the highest correlation was observed between body mass and head-beak length (2015: Pearson's r 2 = 0.525, n = 320, P < 0.001; 2016: Pearson's r 2 = 0.672, n = 319, P < 0.001). Body mass index was than scaled using coefficient estimated by standardized major axis (SMA) regression of body mass and beak-head length (following Peig and Green 2009).

Breeding success and timing
To assess the relation between nest box illumination and breeding characteristics, we compared clutch size, number of hatchlings, number of fledglings, hatching success, fledging success, breeding success and duration of nestling phase 1 3 between dark and bright nest boxes. Number of hatchlings were the number of eggs which hatched, while number of fledglings was the number of nestlings which left the nest box. The hatching success was expressed as the hatched eggs/laid eggs ratio, while the fledging success was fledglings/hatchlings ratio. The breeding success was calculated as fledglings/laid eggs ratio.
In 2016 (Experiment II), we investigated also the effect of light on duration of nestling phase. For this purpose, for each brood, we calculated difference (days) between the dates when the first nestling fledged and the first egg hatched. To assess hatching and fledging times we used trail cameras recordings (see Surmacki & Podkowa 2022 for details). To establish egg hatching date, we inserted trail cameras between 10 and 12th day of incubation. In order to record fledging date cameras were installed when nestlings were ~ 15 days old, i.e., about 5 days before fledging. Cameras recorded one 3 MP photo every 5 and 30 min, during the hatching and fledging period, respectively. In total, we used data from 20 bright and 14 dark nest boxes.

Immunocompetence analysis
The immune system is a key defense mechanism against pathogens in birds (Wakelin and Apanius 1997). Studies have confirmed that the test using underskin injection with phytohemagglutinin (PHA) as a pathogenic simulator reliably reflects not only T lymphocytes immunocompetence (Smits et al. 1999) but also assess the potential of multiple immune-cell response (Martin et al. 2006). This test has been successfully used in ecological immunology to evaluate nestlings immunity in response to changing rearing environment, ectoparasite load, hatching order, nutrition condition (Brinkhof et al. 1999;Saino et al. 2001;De Ayala et al. 2007, reviewed in Martin et al. 2006). We performed immunocompetence PHA test in 2016-2017 (Experiment II). When the nestlings were 7 days old, 26 individuals from 13 bright boxes and 22 nestlings from 11 dark nest boxes were tested. We used standard PHA protocol from studies on passerines (e.g. Dubiec et al. 2006). In each brood, two nestlings with a mass closest to the average brood mass were tested. The wing skin (patagium) of each nestling was injected with 0.2 mg of PHA suspended in 0.04 ml of saline. Patagium thickness and body mass were measured just before and 24 h after injection. The patagium thickness was measured with a dial thickness gauge (0.01 mm, Baker Gauges, India). The level of immune response was expressed as a percentage increase in patagium thickness 24 h after injection. No control injection was made following the protocol by Smits et al. (1999). Each tested individual was weighed before and 24 h after injection to measure the daily body mass increase, which was used as a covariate in the analysis to control nestlings' overall body condition.

Molecular sexing
We used standard protocol used in the study of related species (e.g. Dubiec et al. 2006). Blood for analysis was taken from the brachial vein on the 14 th day of life of the nestlings. DNA isolation was performed using the DNeasy Blood & Tissue Kit protocol (Qiagen GmbH, Hilden, Germany). Sex was determined based on the amplification of the CHD1W and CHD1Z genes (Griffiths et al. 1998).

Maternal effect
To assess the condition of parent female, and possible contribution of maternal component in development of nestlings, we collected a set of measurements of 37 females caught during late nestling stage in 2016 (N = 23) and 2017 (N = 14). We used the self-made nest box traps to catch feeding females. Each female entering nest box automatically triggered the trap plate that closes the entrance. Each nest box was observed during the catching trial to shorten the captivity time and reduce the possible stress. Females were aged using wing plumage colors and divided into two groups: second year of life (SY) and after second year of life (ASY). Similarly, to the nestlings' protocol, we measured wing length (0.5 mm), head length with beak, tarsus length (0.1 mm), and body mass (0.1 g). Because of the low correlation between females' body mass and each of linear measures (and all p > 0.05) we were not able to calculate reliable SMI index. Such low correlation may be explained by the rapid changes in caught females' body mass (mean ± SD = 18.14 ± 1.56, Min-Max = 16.3-23.40) during period of intensive parental care effort. Instead in 2016, we have taken blood samples from 17 females to assess the condition index based on proportion of heterophils to lymphocytes (H/L ratio). Such index has been used commonly in passerine birds to assess the magnitude of the stressors, diseases, infections or stress hormones levels (reviewed in Davis et al. 2008;Skwarska 2018). Blood smears were collected in the field and air-dried. Next, we followed the protocol from Hauptmanová et al. (2002) regarding to smear stain and blood cell count procedures. All smears were examined according to the criteria from Campbell (1994).

Statistical analysis
Due to the different experimental approach, we conducted the separate statistical analyses for each experiment. For biometrical analysis, we performed generalized linear mixed models (GLMM) with dependent variables: 'Body mass' (2nd and 12th day after hatch), 'tarsus length' (at 12th day), and 'SMI' (scaled mass index). As independent variables, we used fixed factors: 'Nest box type' (dark/bright), 'Sex' (male/female), Study site (I, II or III), and covariates: 'First 1 3 hatch day', 'Clutch size' and 'Nest height'. Nest height was the distance between the bottom of the nest cup and the nest box floor. We included nest height to analysis because it is positively correlated with illumination of the nest cup (Podkowa and Surmacki 2017). Moreover, clutch size and nest height may be related to parental performance thus potentially affects the growth rate and condition of the offspring. In PHA analysis, we performed Linear Mixed Model (LMM) using dependent variable 'Patagium thickness increase', and explanatory variables: 'Nest box type' (dark/bright), 'Season' (2016/2017) and 'Body mass increase' as a covariate. In all mixed models, nest box identity was added as random factor. The choice of the best fitted models based on the values of the Akaike Information Criteria (AIC), we present final models with ΔAIC < 2.
For between groups comparison of breeding success and females' condition we used Student's t test for normally distributed data (biometrics) and U Mann-Whitney test for other parameters that were non-normally distributed (breeding success and H/L ratio). To assess breeding success, we compared 'Clutch size', 'Number of hatchlings', 'Number of fledglings', 'Hatching success', 'Fledging success', and 'Breeding success' between experimental groups. To assess the relation between females' age and the occupied type of nest box we used Chi-squared test with Fisher's exact test. All tests were two tailed. Analyses were performed using IBM SPSS Statistics statistical package. Scaled mass index was obtained using R studio software with 'SMATR' package (Warton et al. 2012).

Biometric measurements
Experiment I, in which adult females were not able to choose the nest box type, did not reveal any effect of nest box type on nestlings' biometrics nor SMI. We observed the significant effect of sex on body mass and tarsus length (all p < 0.05, Table 1) showing that male nestlings were heavier and bigger than female nestlings but with no effect on body condition (SMI). Nestlings reared in study site I were significantly heavier at day 2 comparing to other sites. We observed statistically significant, but inconsiderable effect of nest height on nestlings' body mass and tarsus length at day 12. We did not find any effect of hatch date on body mass nor tarsus length (Table 1).
In Experiment II, in which light choice was possible, 2 days old nestlings from bright nest boxes were significantly heavier than the nestlings reared in dark nest boxes (F 1,31.18 = 9.628, p = 0.004, Fig. 1A). Similarly, 12-day nestlings reared in bright boxes had longer tarsus (F 1,34.20 = 4.635, p = 0.038, Fig. 1B). We did not find any significant effect nest box type on body mass at 12 th day and SMI (Fig. 1C, D). We also found significant sex dependent variation in body mass at 12 th day, tarsus length and SMI being higher in male nestlings. The study site had a significant effect on mass and tarsus measurements in Experiment I and SMI in Experiment II.

Fledging time and breeding success
The duration of nestling phase in bright nest boxes was almost one day shorter when compared to dark nest boxes (Table 2). We observed the non-significant tendency of breeding success to be higher in bright nest boxes (Fig. 2). However, the only difference that was on the border of statistical significance ( Table 2) was observed within 'Hatching success' during Experiment II (Fig. 2A). The results of comparison of breeding success between nest box types is presented in Table 2.

Immunocompetence analysis
We observed that the magnitude of the response to PHA injections differed between two types of nest boxes. The swelling reaction was stronger in bright nest boxes (F 1,20.05 = 13.851, p = 0.001), what indicates a better response of immune system. During the treatment we did not observe any effect of body mass increase (F 1,25.48 = 3.659, p = 0.067) nor a year (F 1,20.04 = 1.202, p = 0.286) on daily differences in patagium thickness (Table 3). We also did not observe any significant interaction between type of the nest box and body mass increase (F 1,33.27 = 0.024, p = 0.878).

Maternal effect
Comparison of adult females' H/L ratio as well as most of biometrical measurements showed no differences between females breeding in dark and bright nest boxes during Experiment II (Table 4). The only significant difference was observed in female body mass, which was higher in SY females (Table 4). Bright nest boxes were occupied mainly by SY females while the opposite trend was observed in ASY females (Fischer's exact test p = 0.002, Fig. 3). We did not observe any differences in the date of the first egg-lying between females' age groups (t = 1.262, df = 22, p = 0.226). The offspring of SY females was heavier at day 12 comparing to the offspring of ASY females (t = 2.265, df = 15, p = 0.039, Fig. 4). We also found that female age correlated with nestlings' body mass at day 12 (r 2 = -0.505, n17, p = 0.039) and SMI (r 2 = − 0.524, n = 17, p = 0.031).

Biometry, body condition and breeding success
In the light-choice experiment (Experiment II), nestlings reared in bright nest boxes were significantly heavier and had longer tarsus compared to nestlings from dark boxes. On the contrary, in experiment based on random nest box selection (Experiment I), no effect of light regime on biometry was observed. Moreover, in both experiments, offspring from dark and bright nest boxes had similar body condition (SMI). Several lines of evidence suggests that the positive effect of light in Experiment II was related to the maternal effect. First, nest boxes were occupied by females in no random fashion; bright boxes were chosen mainly by second year (SY) females, while the opposite tendency was found for after second year (ASY) females. Second, SY females were significantly heavier than ASY females. Third, there was a positive correlation between females' mass and offspring mass and body condition at day 12. The lack of the effect of light in the Experiment I could be explained by the fact that boxes were occupied randomly with respect to females' age. Although we did not study females' age in 2015, light condition in bright boxes were set after clutch completion; therefore, light could not affect the age females which choose best site.
The age-related difference in a female's mass found between dark and bright nest boxes probably results from fat reservoirs, because both groups were similar with respect to linear body measurements. It is not clear what factor could drive this difference. One possible explanation is the energy expenditures for nest building and chick feeding. Earlier studies in the studied population showed that Great Tit females in dark boxes built over two times higher nest compering to bright box females presumably to compensate for poorer light conditions (Podkowa and Fig. 1 The effect of nest box type effect on biometric measurements. a Body mass at day 2, b body mass at day 12, c tarsus length, and d SMI. Black squares represent dark nest boxes while white circles rep-resents bright nest boxes. Numbers indicate sample size. Error bars shows 95% confidence intervals Surmacki 2017). Moreover, duration of feeding in bright boxes is significantly shorter, probably because a higher illuminance helps females to quickly locate chicks' gapes and decide which one to feed (Podkowa et al. 2019). As a result, young females from bright boxes may have more time for self-maintenance, which in turn may increase their fat reservoirs and body mass. Earlier studies showed that in optimal nutritional conditions, there are significant genetic correlations between mother and nestlings biometric measures in the Great Tit (Gebhardt-Henrich and van Noordwijk 1991). Lower mass of ASY females could be also attributed to senescence processes, which in the Great Tit is especially pronounced after the second year of life (Bouwhuis et al. 2009). Despite some differences in nestlings' characteristics, which could be ascribed to mother's age, the indirect effect of light conditions on breeding output was relatively weak. Although there was some tendency in breeding success to be higher in bright boxes, differences in nestlings' characteristics and breeding success between nest box types were not statistically significant. Further studies are needed to investigate whether nestlings from bright boxes perform better after fledging. For example, our study demonstrated that duration of nestling phase in bright boxes is about one day shorter comparing to dark boxes. This difference could be due to a better tarsus development in bright nest boxes. Results from earlier studies on nest box nesting species suggest that tarsus length determines the time of fledging (Cornell et al. 2017). Shorter duration of nestling phase should be regarded as advantageous, because it is inversely correlated with the exposure to ectoparasites inhabiting nest (Richner et al. 1993). It is possible that tarsus length, as well as other body dimensions, would increase survival chances of young birds. Although we did not find any differences in nestlings' body mass at late stage of nestlings' phase, in other Great Tit populations, it was found that fledgling with a higher body mass had greater survival and recruitment chances (Monrós et al. 2002;Naef-Daenzer et al. 2001). It is also possible that light affected nestlings' behavior displayed just before the fledging and strengthened the interaction between nestlings (Santema et al. 2021). It is not clear why SY females preferred to breed in brightened cavities. Perhaps for young inexperienced females, it is easier to undertake some activities connected with rearing offspring like nest building or food provisioning (see Podkowa et al. 2019), which are generally visually oriented. This result led us to the conclusion that increased internal daylight level may not only affect nestlings' condition by itself, but also may interact with the experience of females in their first breeding attempt.
It is important to remember that, aside from the manner in which "bright" boxes were created, there might be other factors, that could potentially account for differences between different experimental output between years. For example, factor which is strongly related with the accessibility of food and thus affects nestlings body mass is the time in season (Naef-Daenzer and Keller 1999;Kaliński et al. 2019), however, we did not find any significance of the day of hatch on body mass and tarsus length in studied population. Moreover, there was no difference in the date of the first egg lying. Although, in 2016 eggs hatched statistically earlier, but the difference was only 2 days. Also, the nest temperature that could affect the nestlings' growth (Rodríguez and Barba 2016), did not differ between both types of nest boxes used in this study  nor between Exp I and Exp II. We also did not observe any cold or hot spells in both years. Studies on poultry repeatedly show positive effects of light on the growth of young birds (Robbins et al. 1984;Fairchild and Christensen 2000;Olanrewaju et al. 2006;Gharahveysi et al. 2020) which contradicts our results. A possible explanation for this discrepancy is the intensity, duration and the spectrum of light used in lab experiments. In most of poultry experiments light intensity ranged between 5 and 500 lx (Olanrewaju et al. 2006;Khalil et al. 2016;Hofmann et al. 2020), what is considerably greater than differences in illumination between in dark and bright nest boxes used   (Gharahveysi et al. 2020). Another difference between our study and poultry experiments is the spectral property of the light source. In nest boxes, we used natural sunlight slightly filtered by resin windows. On the other hand, in poultry experiments incandescent, fluorescent or LED bulbs were used (Olanrewaju et al. 2006;Khalil et al. 2016;Hofmann et al. 2020). Such a light sources are often characterized by "spikey" spectrum, which is very different from a sunlight (Troscianko and Stevens 2015). Moreover, in some of poultry experiments, birds were exposed only to particular wavelengths, which affected their body weight and general performance (Olanrewaju 2006;Soliman and El-Sabrout 2020). In most of poultry species, the body growth is positively affected by blue and green light, but in turkeys and ducks higher weight gain was observed under red light (Çapar Akyüz and Onbaşilar 2018). Red light increases also the reproduction and possibility of aggressive behavior in hens, while blue light has calming effect (Rozenboim 1999). Only one study used full-spectrum with UV wavelengths to imitate the effect of daylight on hens' behavior, but such effect was rather small (Wichman 2021). Finally, it is important to remember, that the possible effect of light on nestlings' biometry and body condition in our study may have been affected by other factors like presence of ectoparasites (Dufva and Allander 1996) or siblings' competition (Nilsson and Svensson 1996), which were not controlled.

Immune system
Our study supported the hypothesis that light has a positive effect on efficiency of the immune system. Tissue swelling reaction after PHA injections in nestlings reared in bright nest boxes were significantly greater compared to birds from dark nest boxes. It is commonly assumed that greater PHA swelling indicates a better T cell-mediated response, but it is worth to mention that it is rather related to the presence of the numerous classes of immune cells (e.g. macrophages, basophiles and heterophiles), hormones and acute-phase response (Martin et al. 2006). The difference in such complex immune response, observed under control of the age and body condition of the nestlings, leads to question the environment vs genetic effects. All PHA tests were performed under Experiment II, meaning that nestlings who were tested were reared by females that chose the nest box type and probably differed with respect to age and body mass. Nevertheless, we may assume that any potential differences in females' characteristics have no effect on the obtained results. The majority of earlier studies on wild passerines showed that immune response to pathogens is not heritable and depends mainly on rearing conditions (Tella et al. 2000, Kilpimaa et al. 2005, Pitala et al. 2007, but see Cichoń et al. 2006). In addition, previous research has showed no link between parasite infestation and cell-mediated immune response that may result from yolk-carotenoid content (Berthouly et al. 2007). Finally, we tested nestlings that were at the same age, and we controlled the effect of weight gain, which was not significant.
Our study provides the first information about the effect of light intensity on immune system in wild living birds. Earlier investigations on this subject were mostly focused on poultry kept indoor under artificial light and used various methods to characterize immune response (reviewed in Hofmann et al. 2020). Results of these experiments are not conclusive, but most studies in poultry indicate that low light intensity has no negative effect on birds' immune system (Hofmann et al. 2020). However, another study showed that a continuous light phase affected organization of gene expression and hormones involved in immune system in zebra finches (Mishra et al. 2019), but this result is probably due to disruption of circadian clock, rather than to the intensity of natural light (Mishra et al. 2019).
We did not fully confirm our predictions regarding a direct and positive effect of light on body condition and breeding success. However, we found a strong and positive effect of light within the cavity on immune response of young birds. This result warrants further studies to investigate potential link between the light and T cell-mediated immunocompetence. Our study revealed also indirect positive effects of light on nestlings' size, which could be ascribed to females' age. Bright nest boxes were preferably occupied by younger females, which were on average heavier than older female. Probable consequence of these differences was the nestlings' ability to fledge earlier, what could be regarded as advantageous. Future studies are needed to explain preference of young females to the brighter nest sites and potential benefits resulting from that decision.