Comparisons of microstructure and elemental composition of eggshells among wild plover populations

Langyu Gu, Hanyu Yang, Canwei Xia, Zitan Song, Yahang Cheng, Chenjing Huang,Yuelou Liu, Yang Liu,＊

aState Key Laboratory for Biocontrol,School of Life Sciences,Sun Yat-sen University,Guangzhou,Guangdong,510275,China

bState Key Laboratory for Biocontrol,School of Ecology,Sun Yat-sen University,Shenzhen,518017,China

cMinistry of Education Key Laboratory for Biodiversity and Ecological Engineering,College of Life Sciences,Beijing Normal University,100875,Beijing,China

dDepartment for the Ecology of Animal Societies,Max Planck Institute of Animal Behavior,Konstanz,78467,Germany

Keywords:Eggshell Microstructure Palisade layer Reproduction Shorebird

ABSTRACT

1.Introduction

Reproduction investment is a key trade-off in life-history theory(Stearns, 1989), as it determines individual fitness under strong selection pressure (Flatt and Heyland, 2011).Egg laying is a crucial process in reproduction, where the investment in eggs greatly affects the quality of offspring and reduces resources allocated to the parents themselves.Egg number, cluster size, and egg shape have been reported to reflect parental investment and show intra- and inter-species differences,associated with local adaptation (Stoddard et al., 2017; Song et al.,2020; Liu et al., 2023).In precocial birds, which have limited parental care, egg production is a key form of parental investment (Jetz et al.,2008; Song et al., 2020).The eggshell, an important protective layer between bird embryos and the environment, plays roles in limiting the invasion of external microorganisms (Chien et al., 2008), exchanging oxygen and water conductance (Ar and Rahn, 1985; Hempleman et al.,1993; Solomon, 2010), and providing calcium sources for embryonic development (Karlsson and Lilja, 2008; McClelland et al., 2021; Halgrain et al., 2022).Therefore, the eggshell should be optimized under different selection pressures to ensure successful embryo development and can be particularly important for precocial birds.This is especially true for ground-nesting birds, whose eggs are laid directly on the ground, making the environment directly impact eggshells.Therefore,studying eggshells in wild birds provides an overlooked window to understand local adaptation in avian reproduction.

Avian eggshells can differ in various ways, such as thickness, shape,and color, but their microstructures are generally similar (Dauphin et al., 2018).The three important layers of the bird eggshell, from the inside to the outside, are the mammillary layer, palisade layer, and crystal layer (Chien et al., 2008).The mammillary layer contains important areas called the calcium reserve body, which provide calcium for the skeletal development of the embryo (Dieckert et al., 1989; Chien et al., 2009).The palisade layer, the thickest area of the eggshell, connects the papillae layer and crystal layer.Studies have shown that the breaking strength of eggshells is significantly positively correlated with the thickness of the palisade layer (Kamanl? et al., 2021).The thickness of the crystal layer often varies depending on the species (Dauphin et al.,2018).Most current studies on bird eggshell mainly focus on domestic poultry, for example, by changing feeding ingredient (Liao et al., 2022;Liu et al., 2023), temperature (Sozcu and Ipek, 2015; S¨ozcü et al., 2022),humidity (Peebles et al., 1987; van der Pol et al., 2013), color lighting(Gongruttananun, 2011; Archer, 2019) and lighting time (Xin et al.,2021) to improve egg economic quality.Findings from these studies provide a valuable basis for understanding the mechanism of bird eggshell formation.Although limited, a few studies have already begun to reveal the correlations between eggshell microstructure and environmental factors in wild birds.For example, by analyzing 1350 avian species, Hung et al.(2022) revealed that birds producing eggs with high eggshell stiffness often lay eggs in unstable nests to reduce the risk of egg collision, suggesting the functional importance of eggshells in protecting embryos by adapting to different environments.Changes in temperature and altitude have been reported to lead to changes in eggshell thickness through alterations in maternal investment (Bleu et al., 2019) or oxygen conductance (Hempleman et al., 1993).For instance, environmental pollution has also been reported to affect egg thickness in the Tree Sparrow (Passer montanus) (Ding et al., 2019).Therefore, bird eggshell microstructure has been closely impacted by environmental change.

Bird eggshells contain a large amount of inorganic matter, mainly calcium carbonate, as well as a small amount of organic matter and water.Changes in the elemental composition of the eggshell can affect its quality and are often related to feeding strategy due to maternal effects, which are particularly prominent in poultry research.The most prevalent element in eggshells is calcium (Ca), which is also the major fuel source for embryo growth.Studies have shown that a high calcium diet can improve eggshell quality, such as breaking strength and thickness in Japanese quail hens (Stoewsand et al., 1978).However, other studies have shown a significantly high correlation between high calcium carbonate content and thinner eggshells by analyzing eggshells of 222 bird species (McClelland et al., 2021).The impact of calcium content on eggshell quality thus needs to be analyzed based on specific circumstances.Increasing the contents of zinc (Zn) and manganese (Mn)in the diet has been reported to increase the thickness of the palisade layer and affect eggshell strength in ducks and hens (Zhang et al., 2017;Mayer et al., 2019; Huang et al., 2020).The regulation of phosphorus (P)content can also affect the thickness and quality of eggshell thickness and strength (Liao et al., 2022).Iron (Fe) is required to form the precursor of protoporphyrin, the pigment that affects the color of eggshells.It has been found that diets supplemented with iron result in better eggshell color in hens (Kim et al., 2023).Since elemental composition is largely affected by feeding due to maternal effects, it is reasonable to assume that the heterogeneity of feeding environments in different habitats, with different elemental compositions, can affect the elemental composition of wild bird eggshells due to bioaccumulation in the food web, as has been shown in Rook (Corvus frugilegus) (Or?owski et al.,2016).

In this study, we compared the eggshell microstructure and elemental composition of eggshells during early developmental stage in three shorebird populations that were once treated as one species but are now separated into two closely related plover species (Wang et al.,2019).The Tangshan population and the Qinghai Lake population are Kentish Plovers (Charadrius alexandrinus), and the Zhanjiang population is the White-faced Plover (C.dealbatus).These plovers are ground nesting shorebirds, and their chicks are precocial.They mainly feed on insects from the ground, vegetation, or water (Kosztolányi et al., 2006).They use different microhabitats crossing different climate zones, which vary significantly in temperature, humidity, altitude and wind power(Song et al., 2020, 2023).For instance, Qinghai Lake is an inland soda lake with mud soil, located in the northeast part of the Qinghai-Tibetan Plateau, with a high elevation of about 3000 m.The study sites in Tangshan and Zhanjiang, on the other hand, are coastal regions.However, Zhanjiang is located in tropical region with a sandy beach.The study site in Tangshan, on the other hand, is located in coastal regions with salt evaporation ponds, and has a warm temperate semi-humid continental monsoon climate.Besides, these habitats also have significant differences in element contents.For instance, compared with the national average level of soil element content, the contents of most elements in the surface soil of Qinghai Lake are relatively low, but the Ca content is significantly higher, and the Zn content is significantly lower(Wang et al., 2010).The contents of Fe, Mn, and Zn in the Tangshan coastal environment are within the normal range when compared to the average content of coastal indicator elements (Chang et al., 2020).However, it has been reported that the amount of Zn in sediments exceeds the standard and reaches moderate pollution levels in the Zhanjiang coastal area (Cao et al., 2020).Given such differences in climate,microhabitat and soil element, these three sites are ideal system to study variation of microstructure and elemental composition of eggshells in closely related plovers.

Considering the diverse habitat environments and the groundnesting characteristics of plovers, we propose three predictions.1) The Ca content should be higher in the eggshell of the Qinghai Lake population than others due to its specific soil environment.Although thicker eggshell can help maintain warm and moisture (Arad, 1989; Bebout and Hempleman, 1994), the eggshell should be more prone to thinning due to more deadly hypoxia in the Qinghai-Tibetan Plateau (Hempleman et al., 1993).2) The Zn content should be higher in the eggshell of the Zhanjiang population than others due to the pollution as we mentioned above.Although thinner eggshell help improve heat and water conductance (Ebeid et al., 2012; Yang et al., 2018), the eggshell should be more prone to be thicker to enhance its strenghth to prevent more deadly breaking by frequent typhoons there.3) Compared to populations above, the elemental contents of the Tangshan population should be at the medium level considering its normal soil environment,and the eggshell of Tangshan population can be also thick due to its coastal characters.

2.Materials and methods

2.1.Eggs collection

Sampling was conducted at three breeding sites with different climates during early reproduction period between 2021 and 2022:Tangshan (118?56′ E, 39?16′ N, 200 m above sea level, May to June),Qinghai Lake (100?23′ E, 36?62′ N, 3193 m above sea level, May to July),and Zhanjiang (110?29′ E, 20?54′ N, 10 m above sea level, May to June)in China.

Abandoned eggs were collected during the early developmental stage (A/AB/B) based on standard protocol of egg development (Székely et al., 2008).These codes stand for 0–3 days after hatching, when no obvious chick development were observed from the field.The eggs were packed in egg trays before being sent back to the laboratory, and were stored at 4?C in a refrigerator.To avoid pseudo-replication, we only selected one egg from each nest for corresponding analysis.Totally, 34 eggs were employed in the thinckness measurement, and 27 eggs were employed in the element measurement (Table 1).

2.2.Eggs pre-processing

The eggs were cleaned with distilled water and wiped up with absorbent paper.Afterwards, they were divided into two parts and the contents were removed.The remaining eggshells were cleaned (with 75% ethanol), dried and weighed.

2.3.Eggshell microstructure measurement

We randomly took eggshell fragments (0.5 cm ×0.5 cm) from each egg at the equatorial region multiple times.The removed fragments were etched in a 2% acetic acid solution for about 40 s and then dried.The microstructure was observed under a scanning electron microscopy(SEM) (Phenom-world BV, Phenom Pro).Secondary electron imaging was used for the electron scattering, which can clearly reflect the morphology contrast of the object surface.Based on multiple shots, the eggshell surface was the clearest when the final image scale was between 50 μm and 100 μm.The actual thickness of each layer of the eggshell was calculated based on the scale.

2.4.Eggshell elemental analyses

An inductively coupled plasma emission spectrometer (Thermo Fisher Scientific, X2) was used to determine the elemental composition in the eggshell.The elements selected for measurement were Ca, P, Fe,Mn and Zn.The whole eggshell was grounded into a fine powder and weighed.The standard substance shrub branches and leaves GBW 07602(GSV-1) was used as the reference.2 mL of HNO3(green electron grade),0.5 mL of H2O2(green electron grade), and 0.5 mL of HF (green electron grade) were added to the sample in a polytetrafluoroethylene digestion tank overnight.Afterwards, the samples were placed in a 170?C anticorrosion oven (model JKHF-HOL, Qingdao Jike Experimental Instrument Co., Ltd.) for digestion for 4 h.After cooling, they were taken out and covered with a 100?C anti-corrosion electric heating plate (Qingdao Jike Experimental Instrument Co., Ltd.) to reduce the acid to less than 1 mL.The solution was fixed to a volume of 50 mL using a volumetric flask and filtered with a 0.45 μm inorganic filter.When determining the microelements in the eggshell, such as Ca and P, it needs to be diluted ten times.After processing the solution, a vemammillary layerical fire torch bidirectional observation inductively coupled plasma emission spectrometer (ICP-OES, model 5110VDV) was used to determine the large elements in eggshells.Y021 inductively coupled plasma Mass Spectrometry (model: ICPMS-7900) was used to determine microelements in eggshells.

Table 1Sample size of three wild plover populations in 2021 and 2022.

2.5.Statistical analyses

We randomly took eggshell fragments from each egg at the equatorial region multiple times.The thickness of eggshells from 34 eggs was measured, with 6.00 ±3.46 (mean ±SD,n=34) times of measurements from each egg, depending on the available fragments for each egg.The repeatability of thickness within the eggshell was estimated using the rpt function in the R package rptR (Stoffel et al., 2017), which estimates the repeatability as the proportion of among-egg variance out of the total variance (the sum of among-egg variance and within-egg variance),using a generalized linear mixed model framework with the thickness measurement as the response variable and egg identity as the random effect in independent variable (Nakagawa and Schielzeth, 2017; Stoffel et al., 2017).After confirming the high repeatability in thickness within the eggshell, we performed statistical comparisons of thickness measurements using the average measurements for each egg.

Multivariate analysis of variance (MANOVA) was used to assess the overall differences in thickness measurements among populations and years.Since there was only one egg collected from the Qinghai Lake population in 2022, we performed the analysis after removing the data from the Qinghai Lake population.Then, the variation in thickness measurement among populations was assessed by MANOVA after including the data from the Qinghai Lake population.If a significant difference was detected by MANOVA, analysis of variance (ANOVA) was used to assess each variable, and Tukey test was used to assess the difference between each pair of populations.

For the element variables (Ca, Zn, Fe, Mn and P), Principal Component Analysis (PCA) with varimax rotation was used to compress the five element variables (Ca, Zn, Fe, Mn, P) into independent principal components (with Eigenvalues larger than 1).MANOVA was used to assess the overall differences in element measurements among populations.ANOVA was used to assess each variable, and Tukey test was used to assess the difference between each pair of populations.All analyses were performed using R 4.2.2, andp< 0.05 was considered statistically significant.

3.Results

3.1.Sampling and data pre-processing

Totally, 34 eggs from 34 different nests were employed in the thinckness measurements.We found significant repeatability for the thickness within the eggshell, i.e.the crystal layer (repeatability =0.541± 0.080 (mean ± SE),n= 34; 95% confidence interval ranging from 0.361 to 0.673;p<0.001), mammillary layer (repeatability =0.535 ±0.080,n=34; 95% confidence interval ranging from 0.349 to 0.663;p<0.001), and palisade layer (repeatability =0.706 ±0.063,n=34; 95%confidence interval ranging from 0.557 to 0.800;p< 0.001).This indicates that the variation within the egg is significantly smaller than the variation among eggs.The average thicknesses of the crystal layer,mammillary layer, and palisade layer were therefore calculated for each egg and used for the subsequent analyses.

3.2.Comparisons of eggshell microstructure among populations

The crystal layer, mammillary layer, and palisade layer can be clearly differentiated under SEM (Fig.1).There were no significant differences in thickness measurements between years 2021 and 2022(MANOVA, Pillai’s Trace = 0.177,F3,23= 1.249,p= 0.206), and between the Tangshan population and the Zhanjiang population (MANOVA, Pillai’s Trace =0.025,F3,23=0.201,p= 0.895).Sample size for each population was given in Table 2.However, when the data from the Qinghai Lake population was included, significant differences in thickness measurements were found among populations (MANOVA, Pillai’s Trace =0.412,F6,60=2.595,p=0.027).A nearly significant difference was observed in the palisade layer (ANOVA,F2,31= 3.117,p= 0.058)between the Qinghai Lake population and the Tangshan population(Tukey,p=0.049), while no significant differences were found among populations in both the crystal layer and mammillary layer (Table 2,Fig.1).

Fig.1.(A) Eggshell microstructure of plovers observed under scanning electron microscopy.The crystal layer, palisade layer and mammillary layer are labelled.Left:eggshell from the Tangshan population; middle: eggshell from the Zhanjiang population; right: eggshell from the Qinghai Lake population.(B) Eggs in the wild from different plover populations.Left: Tangshan population; middle: Zhanjiang population; right: Qinghai Lake population.(C) Comparisons of the thickness of the crystal layer, palisade layer, and mammillary layer among the three wild plover populations.＊ indicatesp< 0.05.

Table 2Thickness measurements among populations (compared by ANOVA).

3.3.Comparisons of eggshell elemental composition among populations

PCA analyses (Table 3) revealed that the elemental composition ofsamples from the same geographical population is more similar (Fig.2).

Fig.2.Principal Component Analysis (PCA) of the elemental composition of samples from three plover populations, including the Qinghai Lake population,the Tangshan population, and the Zhanjiang population.Each dot represents an individual sample, and different colors represent different populations.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Table 4Elemental analyses among populations (compared by ANOVA).

The MANOVA analysis revealed significant differences in element measurements among populations (MANOVA, Pillai’s Trace = 1.227,F10,42=6.661,p<0.001).Sample size for each population was given in Table 4.Furthermore, ANOVA results indicated significant differences in the contents of all five elements among populations (Table 4).Specifically, the Ca content was significantly higher in the Qinghai Lake population compared to the Tangshan population (Tukey,p= 0.012).The Zn content was significantly higher in the Zhanjiang population compared to the other two populations (Tukey,p<0.001).Additionally,the Mn content was significantly higher in the Zhanjiang population compared to the Qinghai Lake population (Tukey,p=0.032).Moreover,the Fe content was significantly higher in the Zhanjiang population compared to the Qinghai Lake population (Tukey,p=0.018).Lastly, the P content was significantly higher in the Zhanjiang population compared to the Tangshan population (Tukey,p=0.024) (Fig.3).

4.Discussion

In this study, we compared the microstructure and elemental composition of eggshells among three wild plover populations living in heterogeneous habitats across varied climate conditions for the first time.Our results mostly consistent with our predictions.For example,the palisade layer was significantly thinner in the Qinghai Lake population compared to the Tangshan population.The Ca content was the highest among the three populations, wheares the Zn content was the highest in the Zhanjiang population.We thus propose that these differences in eggshell microstructure and elemental composition among populations could be associated with environmental adaptation.

The relatively thin palisade layer in the Qinghai Lake population could be related to adaptation to the harsh plateau climate.The palisade layer is the thickest layer of the eggshell and contains narrow pores that function in gas exchanges for embryo development.Similarly, previous studies have shown that eggshells of hens at high altitudes are thinner likely due to hypoxia (Hempleman et al., 1993).In low temperature and dry climate conditions, it is easy to assume that the eggshell should be thicker to keep warm and moisturized (Arad, 1989; Bebout and Hempleman, 1994).However, compared to more deadly hypoxia in the Qinghai-Tibetan Plateau, the eggshell should be more prone to thinning.Moreover, parental care of plovers can play a certain role in regulating incubation temperature and humidity (Alrashidi et al., 2010; Vincze et al., 2013).And a thin palisade layer can also facilitate the easy breaking of chicks through their shells during hatching, thereby improving the incubation rate.A significant negative correlation between the hatching rate and the thickness of the palisade layer has been proved in hens (Kamanl? et al., 2021).Coincidentally, our previous studies have shown that the Qinghai Lake population has a higher incubation rate compared to other populations, despite the harsh environment (Song et al., 2020).The thin palisade layer found in the Qinghai Lake population may be one factor, in addition to predation that explain this phenomenon.

Fig.3.Elemental composition comparisons of eggshells among three wild plover populations, including the Qinghai Lake population, the Tangshan population, and the Zhanjiang population.The significance levels are indicated by asterisks: ＊ representsp< 0.05, and ＊＊ representsp< 0.01.

Conversely, the Tangshan population has a relatively thicker palisade layer, which could increase the breaking strength of the eggshell.Under conditions of high temperature and humidity, it is generally assumed that the eggshell should be thinner to increase heat and water conduction (Ebeid et al., 2012; Yang et al., 2018).However, aside from parental incubation which can help regulate temperature and humidity(Alrashidi et al., 2010; Vincze et al., 2013), it is more crucial to strengthen eggs so that they do not get crushed by typhoons, and getting thicker might be an effective adaptaion.Previous studies have demonstrated a significant positive correlation between the breaking strength of eggshells and the thickness of the palisade layer (Kamanl? et al., 2021;Lee et al., 2021).This is particularly useful in the coastal area of Tangshan, where inclement weathers such as heavy rains and winds frequently occurred in summer, posing a threat to ground-nesting eggs.This could also be the case for the Zhanjiang population, which is also located in a coastal area.Indeed, the palisade layer of the Zhanjiang population showed a trend of being thicker than that of the Qinghai Lake population, but the statistical result was not significant, possibly due to limited sample size.

The elemental composition of eggshells among different populations exhibited geographical patterns, indicating their connection to geographical environments.The feeding environment can affect elemental composition of eggshells due to the maternal effect obtained from the female’s diet (Reynolds and Perrins, 2010; Stefanello et al.,2014).The high Ca and low Zn content we detected in the eggshells of the Qinghai Lake population is consistent with reports that the surface soil of Qinghai Lake has significantly higher Ca content and significantly lower Zn content compared to the national average (Wang et al., 2010).Qinghai Lake is an inland lake, and the primary food source for plovers there is worms.Elemental contents in the soil could thus contribute to the elemental composition of eggshell through mother’s diet.In contrast, Zhanjiang is a coastal area that has been reported to have slight pollution of Zn in the coastal sediment (Cao et al., 2020).This could explain the high amount of Zn we identified in their eggshells.These local environmental effects may also explain the high contents of other elements, including Fe, P, and Mn, in the Zhanjiang population.Notably,genetic factors may also have an effect, considering that the Tangshan population and Qinghai Lake population belong to the same species,while the Zhanjiang population is a closely related species to the Kentish plover found in Tangshan and Qinghai Lake.

The elemental composition of the eggshell could also account for its microstructure.For example, it has been reported that a higher content of Zn and Mn in the eggshell can increase the thickness of the palisade layer and improve eggshell strength in ducks and hens (Zhang et al.,2017; Mayer et al., 2019; Huang et al., 2020).This is consistent with our findings that the palisade layer is thin in the Qinghai Lake population with low Zn and low Mn content.A high content of Ca in the eggshell can be beneficial to embryo development, as Ca in the eggshell is the main source to support embryo bone development and must be obtained the female’s diet (Reynolds and Perrins, 2010).Additionally, the calcium carbonate content of the eggshell has been reported to be positively correlated with species that have thinner eggshells, suggesting that more calcium carbonate can form tighter crystals to compensate for the strength loss caused by the thinning of the eggshell (McClelland et al.,2021).The higher contents of both P and Fe in the Zhanjiang population are noteworthy.P has been reported to be related to embryo development, and Fe is an important element affecting eggshell colour.Larger sample sizes and functional experiments can help us better confirm and understand our findings.

To our knowledge, this study is the first to compare microstructure and elemental composition of wild plover populations.There are some limitations in this study, such as a limited sample size and a lack of functional experiments, which warrant further investigation.Our study provides a basis for further research on the mechanism driving eggshell differences and their functions among wild plover populations,contributing to the exploration of biodiversity mechanism in a broader sense.

Funding

LG was funded by the Science and Technology Projects in Guangzhou(202102020231) and YL was funded by the Forestry Administration of Guangdong Province, China (DFGP Project of Fauna of Guangdong-202115 and Science and Technology Planning Projects of Guangdong Province-2021B1212110002).

Ethics statement

All aspects of the fieldwork complied with the Law of the People’s Republic of China on the Protection of Wildlife, and were authorized by Qinghai, Hebei and Guangdong Provincial Forestry and Grassland Administrations.Birds were ringed and handled by trained people aiming to cause as little disturbance to birds as possible.

CRediT authorship contribution statement

Langyu Gu:Conceptualization, Formal analysis, Writing – original draft, Writing – review & editing, Data curation, Funding acquisition,Investigation, Methodology.Hanyu Yang:Formal analysis, Investigation, Methodology.Canwei Xia:Formal analysis, Visualization, Writing– original draft.Zitan Song:Data curation, Formal analysis, Resources.

Yachang Cheng:Data curation, Resources.Chenjing Huang:Data curation, Resources.Yuelou Liu:Data curation, Project administration.

Yang Liu:Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no competing interests.

Acknowledgements

We would like to acknowledge Lei Guan for the valuable suggestions.We also express our gratitude to Prof.Yan Zhao for providing support in element measurement, as well as the Biology Museum of Sun Yat-sen University for the assistance with the ESM.