Sex dimorphism at early age -- nestling male and female Great tits differ in size and immune function

Sex dimorphism at early age -- nestling male and female Great tits differ in size and immune function | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Journal of Avian Biology This is a preprint and has not been peer reviewed. Data may be preliminary. 24 May 2025 V1 Latest version Share on Sex dimorphism at early age -- nestling male and female Great tits differ in size and immune function Authors : Sofia Ventura 0009-0003-2272-1335 [email protected] , Tiancheng Liu , Juli Broggi 0000-0002-1706-4014 , Jan-Åke Nilsson 0000-0001-8982-1064 , and Arne Hegemann 0000-0002-3309-9866 Authors Info & Affiliations https://doi.org/10.22541/au.174806847.77532920/v1 603 views 196 downloads Contents Abstract Introduction Materials and methods Results Discussion Conclusion References Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Birds can show patterns of sexual size dimorphism as early as during the nestling stage. This raises the question of how the faster growing sex might reconcile the energetic and nutritional needs of a faster growth rate with resource allocation to other important life functions, such as the development of innate immune function. Innate immunity represents the main line of defence against diseases, and while some innate immune defences are already present at hatching, substantial development occurs throughout the nestling stage. Hence, this development may compete for resource allocation with growth, potentially affecting nestlings in a sex-specific way in species showing sexual size dimorphism at early age. However, little is known about how sex might shape life-history strategies early into the life cycle. In this two-year study, we molecularly determined the sex of Great tit (Parus major) nestlings. We measured morphometrics (mass, wing and tarsus) and carried out innate immunity assays (Hemolysis-hemagglutination assay, Bacteria Killing Assay, and Haptoglobin assay). We then compared size, mass and immune function among sexes shortly before fledging, likely reflecting the outcome of relative resource allocation during ontogeny. We also carried out a brood size manipulation experiment to simulate resource limitation in the nest. We found that male nestlings grew to a larger size at day 14 than their female siblings. However, we also found some indication that males developed a better immune defense than females albeit their faster growth. Thus, males manage to invest more heavily in both growth rate and immune defence, probably depending on males being dominant to females in the competition for parental feeding, resulting in higher resource acquisition. Abstract Birds can show patterns of sexual size dimorphism as early as during the nestling stage. This raises the question of how the faster growing sex might reconcile the energetic and nutritional needs of a faster growth rate with resource allocation to other important life functions, such as the development of innate immune function. Innate immunity represents the main line of defence against diseases, and while some innate immune defences are already present at hatching, substantial development occurs throughout the nestling stage. Hence, this development may compete for resource allocation with growth, potentially affecting nestlings in a sex-specific way in species showing sexual size dimorphism at early age. However, little is known about how sex might shape life-history strategies early into the life cycle. In this two-year study, we molecularly determined the sex of Great tit ( Parus major ) nestlings. We measured morphometrics (mass, wing and tarsus) and carried out innate immunity assays (Hemolysis-hemagglutination assay, Bacteria Killing Assay, and Haptoglobin assay). We then compared size, mass and immune function among sexes shortly before fledging, likely reflecting the outcome of relative resource allocation during ontogeny. We also carried out a brood size manipulation experiment to simulate resource limitation in the nest. We found that male nestlings grew to a larger size at day 14 than their female siblings. However, we also found some indication that males developed a better immune defense than females albeit their faster growth. Thus, males manage to invest more heavily in both growth rate and immune defence, probably depending on males being dominant to females in the competition for parental feeding, resulting in higher resource acquisition. Key words: life-history theory, eco-immunology, trade-offs, avian nestling development, sexual dimorphism. Introduction Sexual size dimorphisms are widespread among avian species. In such species, the sexes may develop to be morphologically and physiologically divergent already at the nestling stage. Sex-specific effects of the growing environment has been reported in relation to size (Dietrich-Bischoff et al., 2008a; Mainwaring et al., 2012; Nicolaus et al., 2009; Vedder et al., 2005), mortality and fledgling success (Arnold & Griffiths, 2003; Benito et al., 2007; Råberg et al., 2005), differentiation in begging displays (Mainwaring et al., 2012; Saino, de Ayala, et al., 2008), parasite resistance (Tschirren et al., 2003; Stjernman et al., 2008), and immunocompetence (Fargallo et al., 2002). Although birds are born with some functional innate immune function, their innate immunity needs to significantly develop during the nestling stage, a process that continues into early adulthood (Aastrup & Hegemann, 2021; Killpack et al., 2013; Vermeulen et al., 2017). In young birds, this baseline innate immunity is particularly important as it represents a primary defense mechanism against diseases in the absence of an articulated acquired immune system (Grindstaff et al., 2006; Klasing & Leshchinsky, 1999; Pihlaja et al., 2006), and its benefits for pre-and post-fledgling survival have been documented (Cichoń & Dubiec, 2005; Gonzalez et al., 1999; Marri & Richner, 2015). Both somatic growth and development of baseline immune function are resource demanding and might be constrained by resource availability (Bonneaud et al., 2003; Boots & Best, 2018; Chin et al., 2005; Cornelius Ruhs et al., 2020). Therefore, whether the nestlings will be able to grow properly whilst developing a strong baseline immunity may depend on growing conditions. As siblings of both sexes develop in the same environment, that is the nest, they should be exposed to the same pathogen pressure and hence be under the same selection pressure to develop innate immune function. However, in sexually dimorphic species the outcome of this trade-off could also be sex specific. The larger sex, due to the energetic requirements of a faster growth rate, might be more vulnerable to poor growing conditions (Chin et al., 2005; Hõrak et al., 1999; Saino et al., 1997). Alternatively, large size may represent a competitive advantage, meaning that the bigger sex would be able to acquire sufficient resources to support both a faster growth rate and the development of innate immunity regardless of adverse growing conditions (Nicolaus et al., 2009; Oddie, 2000; Råberg et al., 2005; Rowland et al., 2007). Sex-specific differences at the nestling stage might be condition-dependent. Thus, altering resource availability in the nest is a useful approach to test whether the early-life environment might be a driver of sex differences. Brood size manipulations are a common experiment to achieve this (e.g. Santos & Nakagawa, 2012). As parents might fail to properly adjust provisioning effort to a larger brood, resulting in reduced resources per capita, increasing brood size creates a sub-optimal environment for the offspring. Moreover, nestlings may suffer the effects of increased within-brood competition. These adverse conditions might affect the nestlings’ investment in competing trait development, such as bodily growth and innate immune function. In some studies, increased brood size resulted in immunodeficiency but not in stunted growth in nestlings (Dubiec et al., 2006; Hõrak et al., 1999; Ilmonen et al., 2003; Saino et al., 1997), potentially owing to fitness costs of a smaller size post-fledgling (Metcalfe & Monaghan, 2001). Yet, if those effects are sex-specific and whether male and female nestlings invest differentially into immune function development under resource-limited conditions remains unknown. In this two-year study, we investigated whether male and female nestlings of the Great tit ( Parus major), a sexually dimorphic species at adult stage, show differences in sizes and immune function during the nestling stage. To do so, we molecularly sexed Great tit nestlings, measured morphometrics (mass, wing and tarsus) and carried out innate immune function assays (Hemolysis-hemagglutination assay, Bacteria Killing Assay, and Haptoglobin assay). These assays refer to different aspects of innate immunity, thereby providing us a comprehensive view into early development of innate immunity. The hemolyisis-hemagglutination assay measures the plasma’s ability to recognise, clump around (agglutinate) and lyse foreign cells (Matson et al., 2005; Ochsenbein & Zinkernagel, 2000). Through the bacteria killing assay, we quantify the ability of the plasma to remove pathogens (French & Neuman-Lee, 2012). Haptoglobin is an acute phase protein, whose baseline plasma concentration reflects the individual’s ability to respond to an immune challenge (Matson et al., 2012; Quaye, 2008; Thomas, 2000). Through experimental brood size manipulation, we exposed nestlings to various levels of resource availability and within-brood competition. This enabled us to test whether and to what extent any observed differences between the sexes is depending on early-life conditions. Materials and methods This study was performed during spring 2022 and 2023 in a nest box population of Great tits ( Parus major ) breeding in a mixed forest south of Vombsjön, in southern Sweden (55.65°N, 13.56°E). The forest mostly consists of pine ( Pinus sylvestris ) though some deciduous patches, mainly birch ( Betula spp) and oaks ( Quercus robur ) are present. Starting in mid-April, we monitored nest building and egg laying weekly. Tits commonly lay one egg per day, in the morning, while the rest of the day is spent foraging away from the nest. Incubation usually starts once egg laying is complete and lasts for around 12 days. Towards the end of the calculated incubation period, we visited nest boxes daily to record the exact hatching date. We visited the nestboxes six days (2022) or five days (2023) after hatching to ring and weigh all nestlings to the nearest 0.1 g. In 2023, due to time constraints, not all nestlings were ringed on day 5 after hatching (152 not ringed, 216 ringed). Brood size manipulations were carried out in both years by moving five nestlings to create reduced and enlarged broods. Experimental pairs were matched for hatching date and clutch sizes not differing by more than two eggs, and their proximity to minimise manipulation time during nestling exchange. Some broods were chosen as unmanipulated controls following the same criteria. During the study period some predation and partial predation events occurred. Broods that had lost more than two nestlings were excluded from further analysis. That being accounted for, the final experimental setup included 16 reduced, 14 control and 11 enlarged nests in 2022 and 18 reduced, 15 control and 10 enlarged nests in 2023. In neither year, hatching date (ANCOVA 2022: F2,43 = 0.64, p= 0.53, 2023: F2,42= 1.23, p=0.30), clutch size (F2,43 = 0.10, p= 0.90, F2,42= 0.43, p=0.65), nor the mass of nestlings before manipulation (F2,105 = 1.52, p= 0.24, F2,375=0.37, p=0.69) differed among the experimental groups. In 2022, all nestlings were weighed again 14 days after hatching (Great tit nestlings usually fledge at an age of around 20 days). Four nestlings were randomly picked from the reduced and control broods for blood sampling (100 μl from the jugular vein). In enlarged broods, blood samples were taken from eight nestlings: four from the donor brood and four from the original brood. In 2023, at a nestling age of 14 days, previously unringed nestlings were ringed, and we measured tarsus, wing and body mass of all nestlings. Two randomly picked nestlings from each of the reduced and control broods were blood sampled (100 μl from the jugular vein). In enlarged broods, four nestlings were sampled. If broods were ringed on day 5, two moved and two original nestlings were selected for sampling. The blood samples were centrifuged (for 10 minutes at 4000 rpm) to separate the plasma from the blood pellet, and frozen for later analyses. The blood pellet was used for molecular sexing and the plasma for the immune assays. Molecular sexing: DNA extraction DNA from the blood samples was extracted following the NH 4 AC (ammonium acetate) protocol. About 3µl of blood pellet was resuspended in 122 µl of SET buffer mix before adding 3.5 µl of 20% SDS and 2.5µl of Proteinase K. The samples were shaken for about 45 seconds, spun down and incubated in a water bath at 56°C overnight. The following day, after adding 125 µl of 4M NH 4 Ac solution, the samples were left at room temperature for 1 hour and shaken every 15 minutes. After spinning at 13000 rpm for 15 minutes, the supernatant was separated from the blood pellet and 500 µl of 95% ice cold ethanol was added before the samples were again spinned down at 13000 rpm for 15 minutes. The supernatant was discarded and 250 µl of 70% ice cold ethanol were added to the now visible DNA pellet. The supernatant was immediately removed and the samples left to air dry at room temperature overnight. The next day, the extracted DNA was resuspended in 20 µl of 1xTE buffer. After about one hour at room temperature, the samples were moved to the fridge for a minimum of 3 days at 4°C, before they were quantified using a nanodrop. DNA concentrations below 500 ng/µl and with a 260:280 ratio between 1.80 and 2.00 were considered satisfactory. Any samples with concentrations above 500 ng/µl were diluted until the concentration dropped below 500 ng/µl. For the PCR reaction, we made new 25 ng/µl dilutions. Molecular sexing: Polymerase Chain Reaction We prepared a mastermix including ddH 2 0, MgCl 2 , PCR 10x buffer, dNTPs, forward and reverse primers and the template DNA, according to the standard 10µl rxn. We used P8 (5’-CTCCCAAGGATGAGRAAYTG-3’) and P2 (5’-TCTGCATCGCTAAATCCTTT-3’) primers to amplify the homologous genes CHD-W and CHD-Z on the sex chromosomes on birds. Working on ice, the polymerase was added to the mastermix cocktail. 9 µl of the mastermix and 1 µl of sample were pipetted into each PCR well. The protocol used for the PCR reaction was adapted from Kabasakal and Albayrak (2012). As PCR cycles, we used: denaturation at 94°C for 3 minutes, 30 cycles of 30 second denaturation at 94°C, annealing at 50°C for 30 seconds and extension at 72°C for 1 minute. Finally, a 5-minute extension at 72°C. Molecular sexing: gel electrophoresis The PCR products were run on a 2% agarose gel (~20 ml stained with 5µl GelRed) for 30 minutes at 80 V. Each well was filled with 2 µl 1:1 stop mix and 2.5 µl of the sample. 6 µl of 1 kb ladder was added to each row as a reference. The gels were scanned using UV light. Samples showing 2 lines at 340 bp and 380 bp contained one copy of each gene (CHD-W and CHD-Z) and were thus assigned “female”. Samples displaying one line at 340 bp contained two copies of the CHD-Z gene and were assigned “male” (Kabasakal & Albayrak, 2012) Immune assays To quantify baseline (constitutive) innate immune function, we used three immune assays. Lab work for samples collected in 2022 was done in October and November 2022 and for samples collected in 2023 was done in November 2023. All samples were randomised within year but experimental triplets (one control, enlarged and reduced box that hatched on the same day) were always kept next to each other with the order of treatment groups being random. Plasma samples were refrozen between different assays as these parameters are robust to repeated freeze-thaw cycles (Hegemann et al., 2017). Haptoglobin is an acute phase protein that binds with free haemoglobin due to haemolysis, thus rendering nutrients unavailable to pathogens (Quaye, 2008). Haptoglobin is produced by the liver in response to inflammation, trauma or infection. In the absence of an immune challenge, haptoglobin is present constitutively in the blood plasma at low concentrations (Quaye, 2008). Baseline plasma haptoglobin provides a repeatable measure of the strength of the immune response (Matson et al., 2012). To quantify its concentration, we used a commercially available kit (Phase™ Haptoglobin colorimetric assay, Tridelta Development Ltd. Cat. No. TP801). We followed the manufacturer’s instructions. In addition, we did pre-scans at 405 and 450 nm to correct for sample redness (Matson et al., 2012). The haemagglutination-haemolysis assay is used to measure natural antibody activity (by agglutination) and the strength of the complement system (by lysis) (Matson et al., 2005). These two processes are tightly linked: natural antibodies recognise and opsonize foreign cells, which initiates an enzymatic cascade, ending in cell lysis (Matson et al., 2005; Ochsenbein & Zinkernagel, 2000). Thus, this assay quantifies the strength of innate humoral immunity and is particularly well suited for studying immunologically naïve individuals, such as our nestlings, since the presence of natural antibodies is unrelated to previous antigen exposure (Pereira et al., 1986). We followed the method described by Matson et al. (2005) but used 20 µl instead of 25 µl for all liquids. Every morning an RBC working solution was prepared (source: Håtunalab AB, Sweden). Following Matson et al. (2005), we did a scan (Epson perfection 1670™) after 20 minutes (to score agglutination) and one after 90 minutes (to score lysis). Since nestlings showed no lysis after 90 minutes, an additional scan was made after 24 hours (following Matson et al. 2012). Images of the plates were cut into strips, and all samples (2022 and 2023 pooled) were randomised, to allow for blind scoring with respect to sample ID and year. The strips were scored twice during different days by one person (SV). The final score was calculated as the mean between the two measurements. If the scores differed by more than one, the strip was evaluated a third time and the final measurement calculated as the median of the three scores. In 2023, none of the plasma samples from the nestlings showed any response in the agglutination test, hence this parameter was not considered for further analysis in that year. The bacteria killing assay measures the ability of an individual to remove pathogens from the blood and represents an overall evaluation of phagocytes, complements, acute phase proteins and natural antibodies (French & Neuman-Lee, 2012).We followed the protocol by French & Neuman-Lee (2012), with further modifications by Eikenaar & Hegemann (2016). Initial tests showed that a concentration of 1.6 × 10 6 E. coli per millilitre would be optimal for our Great tit nestlings. For every sample, 3 μl plasma was added mixed with 9 μl 1M PBS and 4 μl of E. coli . The plates were then vortexed for 1 minute and were spun down in a plate centrifuge at 300 rpm for 1 minute to ensure all liquids aggregate in the bottom of the well. After that, the plates were incubated at 37°C for 30 minutes. After incubation, the plates were vortexed for 1 minute and 83 μl broth (Tryptic Soy Broth, 15g powder in 500ml) was added to each well on the plate. Then the plates were vortexed again for 1 minute and were measured at 600 nm in a microplate reader (FLUOstar Omega, BMG Labtech) for background absorption. The plates were then incubated at 37°C for 12 hours. After this incubation period, the plates were vortexed for 1 minute and measured again at 600nm. All statistical analyses were carried out in R software, (v4.4.3; R core Team, 2025) using multiway ANOVA testing. We used a multiway ANOVA test (“lme4” package, Bates et al., 2015) to test for differences in nestling body mass, tarsus and wing length as well as immune function (lysis, agglutination, haptoglobin and BKA) between the sexes. The two-way interactions between sex and the variables treatment, hatching date and year were included in all models. In order to control for pseudo-replication, we included nest box as a random effect in all models. Starting with a full model including all two-way interactions, we proceeded by stepwise removing all non-significant interactions. Final models included all main factors (independent of significance) and the significant interactions. For the analysis of haptoglobin, we included absorbance at 450 nm as a covariate to capture variation due to plasma colour. For haptoglobin, we used different standards in 2022 and 2023. Since individual values are calculated based on the standard curves, we are not able to disentangle biological year effects from potential batch effects for haptogloin. Results Fig. 1 Morphometric measurements taken on male and female Great tit nestlings at the age of 14 days (Great tits fledge at around 20 days of age): A) Body mass (g), B) Wing length (mm) C) Tarsus length (mm). Dots show the spread of the data, and the error bars indicate standard error of the mean. At the age of 14 days, male and female nestlings significantly differed in body mass (F 1,250 =27.75, p<0.001) and tarsus length (F 1,96 =14.3, p<0.001), but not in wing length (F 1,96 =0.74, p=0.4). Males were 4.45 % heavier than females (0.8 g difference) and had 2.1% longer tarsi (0.42 mm difference). The difference between the sexes was independent of brood size manipulation for both body mass (F 2,250 = 0.5, P=0.6) and tarsus length (F 2,96 =0.35, p=0.7). For wing length, we found a marginally non-significant interaction between sex and brood size manipulation (F 2,96 =2.8, p=0.06): in the reduced treatment of the experiment, males had 5.7% longer wings (2.9 mm) as compared to females, in the enlarged brood size group, males had 1% (0.5 mm) shorter wings than females, and in the control group, female nestlings had 1.15% (0.6 mm) longer wings than males. The effect of sex was independent of hatch day (interaction hatch day x sex) for both mass (F 1,250 = 0.22, p=0.6), tarsus length (F 1,96 =0.08, p=0.8) and wing length (F 1,96 =0.01, p=0.9). Body mass was the only morphometric measure taken in both 2022 and 2023, and the difference between the sexes was similar between years (interaction sex x year, F 1,250 = 0.6, p=0.4). Table 1 Statistics and coefficients for final linear mixed models showing mass, wing and tarsus length in male and female Great tit nestlings at the age of 14 days. P-values <0.05 are highlighted in bold. Non-significant interactions were removed except if there was a trend. Body mass (g) Sex 1,250 27.8 <0.001 Sex- Male 0.69 (0.43 – 0.95) Treatment 2,250 11.6 <0.001 Treatment - Enlarged -1.21 (-1.82 – -0.59) Treatment - Reduced 0.18 (-0.39 – 0.75) Hatching (Julian) date -0.1 (-0.18 - -0.01) 1,250 4.96 0.03 Year 1,250 0.003 0.95 Year- 2023 -0.01 (-0.5 – 0.48) Wing length (mm) Sex 1,96 0.74 0.39 Sex- Male -0.97 (-2.78 – 0.94) Treatment 2,96 3.93 0.03 Treatment- Enlarged -3.03 (-5.14 – -0.88) Treatment- Reduced -3.07(-5.16 – -1) Hatching (Julian) date -0.03 (-0.26 – 0.19) 1,96 0.07 0.79 Sex x Treatment 2,96 2.8 0.06 Treatment E x Sex M 1.27 (-1.3 – 3.55) Treatment R x Sex M 2.89 (0.55 – 5.42) Tarsus length (mm) Sex 1,96 14.3 <0.001 Sex- Male 0.37 (0.18 – 0.56) Treatment 1,96 1.26 0.29 Treatment - Enlarged -0.06 (-0.38 – 0.26) Treatment- Reduced 0.18 (-0.12 – 0.48) Hatching (Julian) date -0.07 (-0.11 – -0.02) 1,96 8.41 0.006 Differences in innate immune function between male and female nestlings at the age of 14 days Fig. 2 Indices of innate immune function measured in male and female great tit nestlings at the age of 14 days (Great tits fledge at an age of about 20 days): A) Complement activity (measured as lysis), B) Natural antibody activity (measured as agglutination) C) Haptoglobin concentration and D) Bacteria killing capacity against E.coli . Dots show the spread of the data, and the error bars indicate standard error of the mean. Fig.3 Differences in haptoglobin concentration (mg/ml) in Great tit male and female nestlings at the age of 14 days due to hatching (julian) date. Male and female nestlings differed significantly in lysis (F 1,247 =4.9, p=0.03) and marginally in agglutination (F 1, 144 = 3.4, p=0.07) with male nestlings having 12.6% higher lysis and 15.2% lower agglutination. We also found differences between the sexes in haptoglobin concentration, but as an interaction with hatching date (interaction sex x hatching date, F 1,217 =6.4, p=0.01; Figure 3). Haptoglobin concentration decreased with hatching date in female nestlings, but not in males. On average, males had a haptoglobin concentration of 0.48 mg/ml and females of 0.46 mg/ml. We found no difference in BKA between the sexes (F 1,148 = 0.05, p=0.8). The difference between the sexes was marginally dependent on brood size manipulation for lysis (interaction sex x treatment, F 2,247 =2.65, p=0.07) but independent of brood size for haptoglobin concentration (F 2,217 = 0.08, p=0.9), agglutination (F 2,144 = 0.5, p=0.6) and BKA (F 2,148 =0.7, p=0.5). Lysis and haptoglobin were the only immune parameters measured in both 2022 and 2023. The difference between the sexes in lysis was independent of year (interaction sex x year F 1,247 =0.5, p=0.5). In 2023, no agglutination was found and could therefore not be analysed. Since different standards were used for haptoglobin between the years (see methods), we did not test the year x sex interaction for haptoglobin. Table 2 Statistics and coefficients from linear mixed models showing metrics of innate immune function (lysis, haptoglobin concentration, agglutination, bacteria killing activity) measured in male and female Great tit nestlings at the age of 14 days. P-values except if there was a trend. Lysis (titres) Sex 1,247 4.98 0.03 Sex - Male 0.5 (0.16 – 0.83) Treatment 2,247 5.57 0.006 Treatment - Enlarged 0.33 (-0.01 – 0.68) Treatment - Reduced 0.63 (0.28 – 0.98) Hatching (Julian) date -0.03 (-0.07 – 0.002) 1,247 3.31 0.07 Year 1,247 109.4 <0.001 Year 2023 -1.15(-1.36 – -0.94) Sex x Treatment 2,247 2.65 0.07 Sex M x Treatment E -0.47(-0.89 – -0.05) Sex M x Treatment R -0.43(-0.87 – 0.007) Haptoglobin concentration (mg/ml) Sex 1,217 5.66 0.02 Sex - Male 0.62 (0.1 – 1.12) Treatment 2,217 0.73 0.49 Treatment - Enlarged -0.1 (-0.29 – 0.09) Treatment - Reduced -0.1 (-0.28 – 0.08) Hatching (Julian) date 0.008 (-0.02 – 0.04) 1,217 0.78 0.38 Year 1,217 9.1 0.004 Year 2023 -0.25 (-0.4 – - 0.09) Sex x Hatching date 1,217 6.36 0.01 Sex M x Hatching date -0.04 (-0.07 – - 0.009) Absorbance at 450 nm 1 (0.83 –1.17) 1,217 139.2 <0.001 Agglutination (titres) Sex 1,144 3.41 0.07 Sex - Male -0.32 (-0.65 – 0.02) Treatment 2,144 0.15 0.86 Treatment - Enlarged 0.31 (-0.77 – 1.39) Treatment - Reduced 0.15 (-0.84 – 1.15) Hatching (Julian) date -0.01 (-0.16 – 0.13) 1,144 0.02 0.88 Bacteria Killing Assay (Proportion E.coli killed) Sex 1,148 0.05 0.82 Sex – Male -0.004 (-0.04 – 0.03) Treatment 2,148 1.86 0.17 Treatment - Enlarged 0.03 (-0.01 – 0.08) Treatment – Reduced -0.006 (-0.05 – 0.04) Hatching (Julian) date -0.002 (-0.008 – 0.004) 1,148 0.39 0.54 Discussion Male Great tit nestlings were bigger than their female siblings already at the age of 14 days, and showed higher complement activity and higher haptoglobin values, while having lower natural antibody titers. These results were independent of resource variation caused by the experimental brood size manipulation. Sex-specific differences in nestling morphometric growth Males had higher body mass and longer tarsi as compared to females (Figures 1A, 1C), and this result was consistent even after a brood size manipulation. Our results add to the literature documenting sexual size dimorphism in this species at the nestling stage (Nicolaus et al., 2009; Oddie, 2000; Stauss et al., 2005; Tschirren et al., 2003, 2021), and similarly to other tit species (Dietrich-Bischoff et al., 2008b; Dubiec et al., 2006; Mainwaring et al., 2011). The fact that males attained bigger size suggests that they also grew faster as compared to their female siblings, which should come with added nutritional and energetic costs. However, contrary to other studies (Nicolaus et al., 2009; Rosivall et al., 2010; Saino, De Ayala, et al., 2008), we did not find evidence that male growth was constrained by resource availability, as males grew faster than females independently of being reared in a reduced, enlarged or control group, thereby supporting the results by Dubiec et al. (2006 ) and Musgrove & Wiebe (2016). Thus, the observed differences in body mass and tarsus length are supposedly determined by a sexual size dimorphism per se, and are not driven by environmental constraints. Male and female nestlings at 14 days of age did not differ in wing length. Wing length is correlated to fledging date (Kouba et al., 2015; Michaud & Leonard, 2000; Radersma et al., 2011) and post-fledging survival (Morrison et al. 2009, Martin 2014; 2015, Jones et al. 2017, Jones and Ward 2020, Gerritsma et al. 2022, Aastrup et al. 2023) in different avian species. Thus, it might be such an important trait that both sexes invest equally in its development. Only in the reduced brood size treatment, where resources were likely plentiful and within-brood competition moderate, did we find that males had marginally longer wings as compared to females. The sexual dimorphism in body size was also reflected in innate baseline (constitutive) immune function with males showing a somewhat stronger development compared to females. Similarly to morphometrics, this pattern was the same among brood size manipulation categories, and hence independent of access to resources. Males had higher complement activity (measured as lysis; Figure 2A, Table 2) than females regardless of brood size manipulation (Table 2, non-significant interaction sex x treatment). Complement activity has been shown to increase with age in nestlings of different avian species (e.g. Aastrup & Hegemann, 2021; Killpack et al., 2013; Nwaogu et al., 2023), yet those studies did not find differences between male and female nestlings. Our results seem to support the male dominance hypothesis. Due to their competitiveness in resource acquisition, male nestlings were likely able to develop this component of innate immune function whilst simultaneously supporting their faster growth rate. However, our results contrast to other studies where, shortly before fledgling, male nestlings had weaker immune function than females in Great tits (Tschirren et al., 2003), Blue tits Cyanistes caeruleus (Dubiec et al., 2006), European starlings Sturnus vulgaris (Chin et al., 2005) and Eurasian kestrels Falco tinnunculus (Fargallo et al., 2002). More in line with those results are our findings that agglutination was marginally higher in females (Figure 2B, Table 2), regardless of access to resources (non-significant interaction sex x brood size treatment). Natural antibodies, due to their more generalised function, are commonly associated to lower maintenance costs (Ochsenbein & Zinkernagel, 2000; Vinterstare et al., 2019). Thus, female nestlings, who supposedly have a disadvantage in the competition for parental feeding due to their smaller size, might have invested more in this form of innate immunity as compared to the more costly complement activity. Similarly, lighter individuals of a species with asynchronous hatching, i.e. individuals occupying a suboptimal position within the nest hierarchy, had higher agglutination scores (Parejo et al. 2007). This is in line with our hypothesis that male nestlings were dominant within the brood. Hence, we interpret our findings on higher lysis in males and higher agglutination in females as a result of different resource allocation within the immune system following trade-offs between immune function development and growth. Alternatively, and not mutually exclusive, a divergent life-history strategy at the nestling stage might explain the female’s higher agglutination scores. Lobato et al. (2008) proposed that female pied flycatcher ( Ficedula hypoleuca ) nestlings showed higher immunoglobulin concentration in anticipation of higher parasite prevalence during adulthood. Valdebenito et al. (2024), reviewing the literature on sex-specific parasite prevalence, found significantly higher Haemoproteus infections prevalence in females during the breeding season. This result might be the consequence of the trade-off between investing in costly egg production and incubation and investing in immunity, as well as a function of time spent in the nest. Haptoglobin concentrations differed between the sexes depending on hatching date (Figure 3, Table 2): haptoglobin concentration was lower in later-hatched female nestlings compared to earlier hatched ones, while male nestlings had higher haptoglobin concentrations regardless of hatching date. Haptoglobin is an immune parameter that reaches (near) adult levels already early in life (Aastrup & Hegemann, 2021; Ziegler et al., 2021), probably because of its importance during inflammatory responses (Quaye, 2008; Thomas, 2000) and hence for survival (Hegemann et al., 2015). Our results may suggest that males could develop this immune parameter independent of resource availability (either by brood size manipulation or progress of season). We found no differences in haptoglobin concentrations in females among brood size groups, which might suggest that it is not a general resource allocation issue explaining lower haptoglobin concentrations in later hatched females. However, since late hatched females had lower haptoglobin concentrations, we cannot rule out the hypothesis that these differences in immune function development were caused by variation in food quality (Hegemann et al., 2013; Klasing, 2004), which became only evident later in the breeding season and caused a sex-specific competition for high quality food. This would also be in line with the findings of Dubiec & Cichoń (2011), who found environmental quality deterioration over the season to be the most important predictor of nestling immunodeficiency. Since we only considered first clutches in our study, further studies accounting the whole breeding season may further examine these patterns. Conclusion Great tit nestlings showed sexual dimorphism in size and innate (constitutive) immune function already at the age of 14 days. These results did not vary due to brood size manipulation, suggesting that the observed differences were due to a sexual dimorphism per se, and not to any resource constraints. Thereby, our study contributes to the understanding of the early implications of sex on the nestlings’ growth and innate immune function development. We show that distinguishing between males and females already at the nestling stage is crucial to understand resource allocation and its impacts on life-history strategies. Future studies may investigate the consequences of sex-specific development of baseline immune function on the nestling’s ability to respond to immune challenges. Induced components of the immune system were found to be more resource demanding as compared to baseline immune function (Hasselquist & Nilsson, 2012; Scanes & Braun, 2012). Hence, studying the acute phase of an infection might emphasise the importance of access to resources during early development. More broadly, such studies may reinforce the importance of considering sexual differences to understand patterns in ecology and evolution. References 1. Aastrup, C., & Hegemann, A. (2021). Jackdaw nestlings rapidly increase innate immune function during the nestling phase but no evidence for a trade-off with growth. Developmental and Comparative Immunology , 117 . https://doi.org/10.1016/J.DCI.2020.103967 Aastrup, C., Nilsson, J. Å., Hasselquist, D., & Hegemann, A. (2023). Size and immune function as predictors of predation risk in nestling and newly fledged jackdaws. Animal Behaviour , 198 , 73–84. https://doi.org/10.1016/J.ANBEHAV.2023.01.012 Arnold, K. E., & Griffiths, R. (2003). Sex-Specific Hatching Order, Growth Rates and Fledging Success in Jackdaws Corvus monedula. Source: Journal of Avian Biology , 34 (3), 275–281. https://www.jstor.org/stable/3677476 Bates, D., Mächler, M., Bolker, B. M., & Walker, S. C. (2015). Fitting Linear Mixed-Effects Models Using lme4. Journal of Statistical Software , 67 (1), 1–48. https://doi.org/10.18637/JSS.V067.I01 Benito, M. M., Gonza´lez, J., Gonza´lez-Soli´s, G., & Soli´s, S. (2007). Sex ratio, sex-specific chick mortality and sexual size dimorphism in birds. Journal of Evolutionary Biology , 20 (4), 1522–1530. https://doi.org/10.1111/J.1420-9101.2007.01327.X Bonneaud, C., Mazuc, J., Gonzalez, G., Haussy, C., Chastel, O., Faivre, B., & Sorci, G. (2003). Assessing the cost of mounting an immune response. American Naturalist , 161 (3), 367–379. https://doi.org/10.1086/346134 Boots, M., & Best, A. (2018). The evolution of constitutive and induced defences to infectious disease. Proceedings of the Royal Society B: Biological Sciences , 285 (1883). https://doi.org/10.1098/RSPB.2018.0658 Chin, E. H., Love, O. P., Clark, A. M., & Williams, T. D. (2005). Brood Size and Environmental Conditions Sex-Specifically affect Nestling Immune Response in the European Starling Sturnus vulgaris. Source: Journal of Avian Biology , 36 (6), 549–554. Cichoń, M., & Dubiec, A. (2005). Cell-mediated immunity predicts the probability of local recruitment in nestling blue tits. Journal of Evolutionary Biology , 18 (4), 962–966. https://doi.org/10.1111/J.1420-9101.2005.00910.X Cornelius Ruhs, E., Vézina, F., Walker, M. A., & Karasov, W. H. (2020). Who pays the bill? The effects of altered brood size on parental and nestling physiology. Journal of Ornithology , 161 (1), 275–288. https://doi.org/10.1007/S10336-019-01715-1/FIGURES/5 Dietrich-Bischoff, V., Schmoll, T., Winkel, W., & Lubjuhn, T. (2008a). Variation in the magnitude of sexual size dimorphism in nestling Coal Tits (Periparus ater). Journal of Ornithology , 149 (4), 565–577. https://doi.org/10.1007/S10336-008-0302-1/TABLES/4 Dietrich-Bischoff, V., Schmoll, T., Winkel, W., & Lubjuhn, T. (2008b). Variation in the magnitude of sexual size dimorphism in nestling Coal Tits (Periparus ater). Journal of Ornithology , 149 (4), 565–577. https://doi.org/10.1007/S10336-008-0302-1/TABLES/4 Dubiec, A., & Cichoń, M. (2011). Seasonal decline in nestling cellular immunocompetence results from environmental factors — an experimental study. Https://Doi-Org.Ludwig.Lub.Lu.Se/10.1139/Z05-076 , 83 (7), 920–925. https://doi.org/10.1139/Z05-076 Dubiec, A., Cichoń, M., & Deptuch, K. (2006). Sex-specific development of cell-mediated immunity under experimentally altered rearing conditions in blue tit nestlings. Proceedings of the Royal Society B: Biological Sciences , 273 (1595), 1759–1764. https://doi.org/10.1098/RSPB.2006.3510 Eikenaar, C., & Hegemann, A. (2016). Migratory common blackbirds have lower innate immune function during autumn migration than resident conspecifics. Biology Letters , 12 (3). https://doi.org/10.1098/RSBL.2016.0078/SUPPL_FILE/RSBL20160078SUPP1.XLSX Fargallo, J. A., Laaksonen, T., Pöyri, V., & Korpimäki, E. (2002). Inter-sexual differences in the immune response of Eurasian kestrel nestlings under food shortage. Ecology Letters , 5 (1), 95–101. https://doi.org/10.1046/J.1461-0248.2002.00290.X French, S. S., & Neuman-Lee, L. A. (2012). Improved ex vivo method for microbiocidal activity across vertebrate species. Biology Open , 1 (5), 482–487. https://doi.org/10.1242/BIO.2012919, Gerritsma, Y. H., Driessen, M. M. G., Tangili, M., de Boer, S. F., & Verhulst, S. (2022). Experimentally manipulated food availability affects offspring quality but not quantity in zebra finch meso-populations. Oecologia , 199 (4), 769–783. https://doi.org/10.1007/S00442-022-05183-Y/METRICS Gonzalez, G., Sorci, G., & De Lope, F. (1999). Seasonal variation in the relationship between cellular immune response and badge size in male house sparrows (Passer domesticus). Behavioral Ecology and Sociobiology , 46 (2), 117–122. https://doi.org/10.1007/S002650050600/METRICS Grindstaff, J. L., Hasselquist, D., Nilsson, J. Å., Sandell, M., Smith, H. G., & Stjernman, M. (2006). Transgenerational priming of immunity: maternal exposure to a bacterial antigen enhances offspring humoral immunity. Proceedings. Biological Sciences , 273 (1600), 2551–2557. https://doi.org/10.1098/RSPB.2006.3608 Hasselquist, D., & Nilsson, J. Å. (2012). Physiological mechanisms mediating costs of immune responses: what can we learn from studies of birds? Animal Behaviour , 83 (6), 1303–1312. https://doi.org/10.1016/J.ANBEHAV.2012.03.025 Hegemann, A., Marra, P. P., & Tieleman, B. I. (2015). Causes and consequences of partial migration in a passerine bird. American Naturalist , 186 (4), 531–546. https://doi.org/10.1086/682667, Hegemann, A., Matson, K. D., Flinks, H., & Tieleman, B. I. (2013). Offspring pay sooner, parents pay later: Experimental manipulation of body mass reveals trade-offs between immune function, reproduction and survival. Frontiers in Zoology , 10 (1). https://doi.org/10.1186/1742-9994-10-77 Hegemann, A., Pardal, S., & Matson, K. D. (2017). Indices of immune function used by ecologists are mostly unaffected by repeated freeze-thaw cycles and methodological deviations. Frontiers in Zoology , 14 (1). https://doi.org/10.1186/S12983-017-0226-9 Hõrak, P., Tegelmann, L., Ots, I., & Møller, A. P. (1999). Immune function and survival of great tit nestlings in relation to growth conditions. Oecologia , 121 (3), 316–322. https://doi.org/10.1007/S004420050934/METRICS Ilmonen, P., Hasselquist, D., Langefors, Å., & Wiehn, J. (2003). Stress, immunocompetence and leukocyte profiles of pied flycatchers in relation to brood size manipulation. Oecologia , 136 (1), 148–154. https://doi.org/10.1007/S00442-003-1243-2/FIGURES/2 Jones, T. M., & Ward, M. P. (2020). Pre- to post-fledging carryover effects and the adaptive significance of variation in wing development for juvenile songbirds. Journal of Animal Ecology , 89 (10), 2235–2245. https://doi.org/10.1111/1365-2656.13285 Jones, T. M., Ward, M. P., Benson, T. J., & Brawn, J. D. (2017). Variation in nestling body condition and wing development predict cause-specific mortality in fledgling dickcissels. Journal of Avian Biology , 48 (3), 439–447. https://doi.org/10.1111/JAV.01143;WGROUP:STRING:PUBLICATION Kabasakal, B., & Albayrak, T. (2012). Offspring sex ratios and breeding success of a population of the Great Tit, Parus major. Zoology in the Middle East , 57 (1), 27–34. https://doi.org/10.1080/09397140.2012.10648960 Killpack, T. L., Oguchi, Y., & Karasov, W. H. (2013). Ontogenetic patterns of constitutive immune parameters in altricial house sparrows. Journal of Avian Biology , 44 (6), 513–520. https://doi.org/10.1111/J.1600-048X.2013.00239.X Klasing, K. C. (2004). The costs of immunity. Acta Zool. Sin. 50: , 961–969. Klasing, K. C., & Leshchinsky, T. V. (1999). Functions, costs, and benefits of the immune system during development and growth . Ostric. https://www.internationalornithology.org/PROCEEDINGS_Durban/Symposium/S46/S46.4.htm Kouba, M., Bartoš, L., Korpimäki, E., & Zárybnická, M. (2015). Factors Affecting the Duration of Nestling Period and Fledging Order in Tengmalm’s Owl (Aegolius funereus): Effect of Wing Length and Hatching Sequence . https://doi.org/10.1371/journal.pone.0121641 Lobato, E., Merino, S., Morales, J., Tomás, G., Martínez-De La Puente, J., SÁnchez, E., García-Fraile, S., & Moreno, J. (2008). Sex differences in circulating antibodies in nestling Pied Flycatchers Ficedula hypoleuca. Ibis , 150 (4), 799–806. https://doi.org/10.1111/J.1474-919X.2008.00863.X Mainwaring, M. C., Dickens, M., & Hartley, I. R. (2011). Sexual dimorphism and growth trade-offs in Blue Tit Cyanistes caeruleus nestlings. Ibis , 153 (1), 175–179. https://doi.org/10.1111/J.1474-919X.2010.01071.X;WGROUP:STRING:PUBLICATION Mainwaring, M. C., Dickens, M., & Hartley, I. R. (2012). Sexual dimorphism and offspring growth: Smaller female Blue Tit nestlings develop relatively larger gapes. Journal of Ornithology , 153 (4), 1011–1016. https://doi.org/10.1007/S10336-012-0828-0/TABLES/1 Marri, V., & Richner, H. (2015). Immune response, oxidative stress and dietary antioxidants in great tit nestlings. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology , 179 , 192–196. https://doi.org/10.1016/J.CBPA.2014.10.013 Martin, T. E. (2014). A conceptual framework for clutch-size evolution in songbirds. American Naturalist , 183 (3), 313–324. https://doi.org/10.1086/674966/SUPPL_FILE/54693APA.PDF Martin, T. E. (2015). Age-related mortality explains life history strategies of tropical and temperate songbirds. Science , 349 (6251), 966–970. https://doi.org/10.1126/SCIENCE.AAD1173/SUPPL_FILE/MARTIN.SM.PDF Matson, K. D., Horrocks, N. P. C., Versteegh, M. A., & Tieleman, B. I. (2012). Baseline haptoglobin concentrations are repeatable and predictive of certain aspects of a subsequent experimentally-induced inflammatory response. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology , 162 (1), 7–15. https://doi.org/10.1016/J.CBPA.2012.01.010 Matson, K. D., Ricklefs, R. E., & Klasing, K. C. (2005). A hemolysis–hemagglutination assay for characterizing constitutive innate humoral immunity in wild and domestic birds. Developmental & Comparative Immunology , 29 (3), 275–286. https://doi.org/10.1016/J.DCI.2004.07.006 Metcalfe, N. B., & Monaghan, P. (2001). Compensation for a bad start: Grow now, pay later? Trends in Ecology and Evolution , 16 (5), 254–260. https://doi.org/10.1016/S0169-5347(01)02124-3 Michaud, T., & Leonard, M. (2000). The Role of Development, Parental Behavior, and Nestmate Competition in Fledging of Nestling Tree Swallows. The Auk , 117 (4), 996–1002. https://doi.org/10.1093/AUK/117.4.996 Morrison, K. W., Hipfner, J. M., Gjerdrum, C., & Green, D. J. (2009). Wing Length and Mass at Fledging Predict Local Juvenile Survival and Age at First Return in Tufted Puffins. The Condor: Ornithological Applications , 111 (3), 433–441. https://doi.org/10.1525/COND.2009.080099 Musgrove, A. B., & Wiebe, K. L. (2016). Condition-dependent expression of carotenoid- and melanin-based plumage colour of northern flicker nestlings revealed by manipulation of brood size. Journal of Avian Biology , 47 (2), 176–184. https://doi.org/10.1111/JAV.00730 Nicolaus, M., Michler, S. P. M., Ubels, R., Van Der Velde, M., Komdeur, J., Both, C., & Tinbergen, J. M. (2009). Sex-specific effects of altered competition on nestling growth and survival: an experimental manipulation of brood size and sex ratio. Journal of Animal Ecology , 78 (2), 414–426. https://doi.org/10.1111/J.1365-2656.2008.01505.X Nwaogu, C. J., Amar, A., Nebel, C., Isaksson, C., Hegemann, A., & Sumasgutner, P. (2023). Innate immune function and antioxidant capacity of nestlings of an African raptor covary with the level of urbanisation around breeding territories. Journal of Animal Ecology , 92 (1), 124–141. https://doi.org/10.1111/1365-2656.13837 Ochsenbein, A. F., & Zinkernagel, R. M. (2000). Natural antibodies and complement link innate and acquired immunity. Immunology Today , 21 (12), 624–630. https://doi.org/10.1016/S0167-5699(00)01754-0 Oddie, K. R. (2000). Size matters: competition between male and female great tit offspring. Journal of Animal Ecology , 69 (5), 903–912. https://doi.org/10.1046/J.1365-2656.2000.00438.X Parejo, D., Silva, N., & Avilés, J. M. (2007). Within-brood size differences affect innate and acquired immunity in roller Coracias garrulus nestlings. Journal of Avian Biology , 38 (6), 717–725. https://doi.org/10.1111/J.2007.0908-8857.04081.X Pereira, P., Forni, L., Larsson, E. ‐L, Cooper, M., Heusser, C., & Coutinho, A. (1986). Autonomous activation of B and T cells in antigen‐free mice. European Journal of Immunology , 16 (6), 685–688. https://doi.org/10.1002/EJI.1830160616, Pihlaja, M., Siitari, H., & Alatalo, R. V. (2006). Maternal antibodies in a wild altricial bird: effects on offspring immunity, growth and survival. Journal of Animal Ecology , 75 (5), 1154–1164. https://doi.org/10.1111/J.1365-2656.2006.01136.X Quaye, I. K. (2008). Haptoglobin, inflammation and disease. Transactions of the Royal Society of Tropical Medicine and Hygiene , 102 (8), 735–742. https://doi.org/10.1016/J.TRSTMH.2008.04.010 R core Team. (2025). ._R: A Language and Environment for Statistical Computing_. (v4.4.3). R Foundation for Statistical Computing. https://www.R-project.org/ Råberg, L., Stjernman, M., & Nilsson, J. Å. (2005). Sex and environmental sensitivity in blue tit nestlings. Oecologia , 145 (3), 496–503. https://doi.org/10.1007/S00442-005-0133-1/TABLES/3 Radersma, R., Tinbergen, J. M., & Komdeur, J. (2011). Do brood sex ratio, nestling development and sex affect fledging timing and order? An experimental study on great tits. Animal Behaviour , 81 (1), 69–75. https://doi.org/10.1016/J.ANBEHAV.2010.09.007 Rosivall, B., Szöllosi, E., Hasselquist, D., & Török, J. (2010). Males are sensitive - sex-dependent effect of rearing conditions on nestling growth. Behavioral Ecology and Sociobiology , 64 (10), 1555–1562. https://doi.org/10.1007/S00265-010-0969-1/FIGURES/2 Rowland, E., Love, O. P., Verspoor, J. J., Sheldon, L., Williams, T. D., Rowland, E., Love, O. P., Verspoor, J. J., & Williams, T. D. (2007). Manipulating rearing conditions reveals developmental sensitivity in the smaller sex of a passerine bird, the European starling Sturnus vulgaris. Journal of Avian Biology , 38 (5), 612–618. https://doi.org/10.1111/J.0908-8857.2007.04082.X Saino, N., Calza, S., & pape Moller, A. (1997). Immunocompetence of Nestling Barn Swallows in Relation to Brood Size and Parental Effort. The Journal of Animal Ecology , 66 (6), 827. https://doi.org/10.2307/5998 Saino, N., de Ayala, R. M., Boncoraglio, G., & Martinelli, R. (2008). Sex difference in mouth coloration and begging calls of barn swallow nestlings. Animal Behaviour , 75 (4), 1375–1382. https://doi.org/10.1016/J.ANBEHAV.2007.09.011 Saino, N., De Ayala, R. M., Martinelli, R., & Boncoraglio, G. (2008). Male-biased brood sex ratio depresses average phenotypic quality of barn swallow nestlings under experimentally harsh conditions. Oecologia , 156 (2), 441–453. https://doi.org/10.1007/S00442-008-0971-8/FIGURES/3 Santos, E. S. A., & Nakagawa, S. (2012). The costs of parental care: a meta‐analysis of the trade‐off between parental effort and survival in birds. Journal of Evolutionary Biology , 25 (9), 1911–1917. https://doi.org/10.1111/J.1420-9101.2012.02569.X Scanes, C. G., & Braun, E. (2012). Avian metabolism: its control and evolution. Frontiers in Biology 2012 8:2 , 8 (2), 134–159. https://doi.org/10.1007/S11515-012-1206-2 Stauss, M., Segelbacher, G., Tomiuk, J., & Bachmann, L. (2005). Sex ratio of Parus major and P. caeruleus broods depends on parental condition and habitat quality. Oikos , 109 (2), 367–373. https://doi.org/10.1111/J.0030-1299.2005.13333.X Thomas, J. S. (2000). Overview of plasma proteins. In B. F. Feldman, J. G. Zinkl, N. C. Jain, & O. W. Schalm (Eds.), Schalm’s Veterinary Hematology. (pp. 891–1344). Lippincott Williams & Wilkins. Tschirren, B., Fitze, P. S., & Richner, H. (2003). Sexual Dimorphism in Susceptibility to Parasites and Cell-Mediated Immunity in Great Tit. Source: Journal of Animal Ecology , 72 (5), 839–845. https://about.jstor.org/terms Tschirren, B., Romero-Haro, A. Á., Zahn, S., & Criscuolo, F. (2021). Sex‐specific effects of experimental ectoparasite infestation on telomere length in great tit nestlings. Journal of Evolutionary Biology , 34 (3), 584–589. https://doi.org/10.1111/JEB.13744 Valdebenito, J. O., Jones, W., & Székely, T. (2024). Evolutionary drivers of sex-specific parasite prevalence in wild birds. Proceedings of the Royal Society B , 291 (2028). https://doi.org/10.1098/RSPB.2024.1013 Vedder, O., Dekker, A. L., Visser, G. H., & Dijkstra, C. (2005). Sex-specific energy requirements in nestlings of an extremely sexually size dimorphic bird, the European sparrowhawk (Accipiter nisus). Behavioral Ecology and Sociobiology , 58 (4), 429–436. https://doi.org/10.1007/S00265-005-0926-6/FIGURES/4 Vermeulen, A., Eens, M., Van Dongen, S., & Müller, W. (2017). Does baseline innate immunity change with age? A multi-year study in great tits. Experimental Gerontology , 92 , 67–73. https://doi.org/10.1016/J.EXGER.2017.03.011 Vinterstare, J., Hegemann, A., Nilsson, P. A., Hulthén, K., & Brönmark, C. (2019). Defence versus defence: Are crucian carp trading off immune function against predator-induced morphology? Journal of Animal Ecology , 88 (10), 1510–1521. https://doi.org/10.1111/1365-2656.13047 Ziegler, A. K., Watson, H., Hegemann, A., Meitern, R., Canoine, V., Nilsson, J. Å., & Isaksson, C. (2021). Exposure to artificial light at night alters innate immune response in wild great tit nestlings. The Journal of Experimental Biology , 224 (10), jeb239350. https://doi.org/10.1242/JEB.239350 Crossref Google Scholar Information & Authors Information Version history V1 Version 1 24 May 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Journal of Avian Biology Keywords avian nestling development eco-immunology life-history theory sexual dimorphism trade-offs Authors Affiliations Sofia Ventura 0009-0003-2272-1335 [email protected] Lund University View all articles by this author Tiancheng Liu Lund University Faculty of Science View all articles by this author Juli Broggi 0000-0002-1706-4014 Estación Biológica de Doñana, CSIC View all articles by this author Jan-Åke Nilsson 0000-0001-8982-1064 Lund University View all articles by this author Arne Hegemann 0000-0002-3309-9866 Lund University View all articles by this author Metrics & Citations Metrics Article Usage 603 views 196 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Sofia Ventura, Tiancheng Liu, Juli Broggi, et al. Sex dimorphism at early age -- nestling male and female Great tits differ in size and immune function. Authorea . 24 May 2025. DOI: https://doi.org/10.22541/au.174806847.77532920/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. Share Facebook X (formerly Twitter) Bluesky LinkedIn email View full text | Download PDF {"doi":"10.22541/au.174806847.77532920/v1","type":"Article"} Now Reading: Share Figures Tables Close figure viewer Back to article Figure title goes here Change zoom level Go to figure location within the article Download figure Toggle share panel Toggle share panel Share Toggle information panel Toggle information panel Go to previous graphic Go to next graphic Go to previous table Go to next table All figures All tables View all material View all material xrefBack.goTo xrefBack.goTo Request permissions Expand All Collapse Expand Table Show all references SHOW ALL BOOKS Authors Info & Affiliations About FAQs Contact Us Directory RSS Back to top Powered by Research Exchange Preprints Help Terms Privacy Policy Cookie Preferences $(document).ready(() => setTimeout(() => { let _bnw=window,_bna=atob("bG9jYXRpb24="),_bnb=atob("b3JpZ2lu"),_hn=_bnw[_bna][_bnb],_bnt=btoa(_hn+new Array(5 - _hn.length % 4).join(" ")); $.get("/resource/lodash?t="+_bnt); },4000)); (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'9ffd2cec98168650',t:'MTc3OTQ2NzExMg=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();