Introduction

Avian obligate brood parasites lay eggs in other birds’ nests, which results in reproduction costs to their hosts. The cost of parasitism is high for the host (Lyu and Liang 2021) because parasites will remove or destroy host eggs during the parasitic process (Davies 2000); the exclusive parasitic nestling, after hatching, will remove all other host eggs or host nestlings in the same nest (Davies 2011; Soler 2014), or the non-exclusive parasitic nestling will win the competition for food (Davies et al. 1998; Kilner 2003); and compared to the host’s own nestlings, they require more time and energy from the host (Rothstein 1990; Feeney et al. 2014; but see Samaš et al. 2018, 2019). The pressure exerted by such high costs forces the host to evolve abilities to recognize and reject parasitic eggs or to take defensive actions such as guarding against and attacking parasites (Davies and Brooke 1989; Soler et al. 1999; Røskaft et al. 2002; Avilés et al. 2004; Moskát 2005). Recognizing and rejecting parasitic eggs, compared to other defensive strategies, is an effective anti-parasitic strategy when parasitism happens because it is time- and effort-saving, and avoids direct confrontation with parasitic birds (Davies and Brooke 1989; Moksnes et al. 1991, 2013; Sealy and Underwood 2012).

It is highly likely for non-mimetic eggs to be recognized and rejected by the host. For their eggs to be accepted by the host, some parasites have evolved to lay eggs that highly match their host’s eggs in appearance. For example, egg mimicry by common cuckoos (Cuculus canorus) occurs in some warbler hosts (Avilés et al. 2006; Cherry et al. 2007; Honza et al. 2014; Li et al. 2016). Furthermore, plaintive cuckoo (Cacomantis merulinus) eggs laid in common tailorbird (Orthotomus sutorius) nests (Yang et al. 2016a, b) and Oriental cuckoo (Cuculus optatus) eggs laid in yellow-bellied prinia (Prinia flavientris) nests (Wang et al. 2015) are both highly mimetic.

For species without egg color mimicry, an alternative strategy would be to lay eggs with cryptic colors. Such eggs are more likely to be overlooked by the host, being less distinguishable in the nest due to low surface brightness, and thus more likely to eventually achieve successful parasitism. This approach apparently possesses similarities with egg mimicry, e.g., several species of Australasian bronze-cuckoos (Chalcites spp.) and their common hosts Gerygone and Acanthiza spp. (Langmore et al. 2009; Gloag et al. 2014), and the shining bronze-cuckoo (Chalcites lucidus) with their sole host the grey gerygone (Gerygone igata) in New Zealand (Thorogood et al. 2017).

It is most suitable to test the egg crypsis hypothesis in darker nests such as the nests of the large-billed gerygone (Gerygone magnirostris) (Gloag et al. 2014), yellow-rumped thornbill (Acanthiza chrsyorrhoa) (Medina and Langmore 2019) and common redstart (Phoenicurus phoenicurus) (Samaš et al. 2016). Unfortunately, these hosts are egg acceptors, and they rarely recognize and reject foreign eggs, including model eggs and parasitic cuckoo eggs. Even when the brightness of the nest is manually increased so that the host is able to clearly see the foreign eggs in the nest, the egg rejection rate does not increase, indicating that a rapid increase of visibility in the nest does not change the host’s rejection behaviors (Avilés et al. 2015; Medina and Langmore 2019). Yang et al. (2016a, b) investigated egg mimicry in common cuckoos, parasites of Oriental reed warblers (Acrocephalus orientalis, hereafter “warblers”), in China using spectrum analysis to analyze similarities in cuckoo and warbler egg color and pattern. The results showed that the cuckoo eggs were not as fully mimetic as the host eggs. Some cuckoo eggs were greyer on the surface (Fig. 1a), with a higher surface brightness than that of the host eggs (Fig. 1b). Although these eggs were dissimilar to the host eggs, they were accepted and hatched anyway. From this point, it seems that cryptic eggs in open nests can also be deceptive to a certain extent. To understand whether cryptic foreign eggs can deceive hosts in the parasitic system of open nests, we conducted a test on the ability of warblers to recognize and reject foreign eggs. We simultaneously devised an experiment with two groups, one with more cryptic model eggs and the other with brighter model eggs, to observe the rejection behaviors of the warblers. According to the egg crypsis hypothesis, we predicted that if the warblers had strong egg recognition ability, they would preferentially reject more distinguishable foreign eggs.

Fig. 1
figure 1

Eggs of the common cuckoo and Oriental reed warbler in the nests (a shows single parasitism with one cuckoo egg and b shows the multiple parasitism case. The yellow arrow pointed eggs are cuckoo eggs)

Full size image

Materials and methods

Study area and study species

We performed this study at the Zhalong National Nature Reserve (46°48′–47°31′N, 123°51′–124°37′E) in Heilongjiang, northeastern China. Field experiments were conducted during the breeding season (June to August) between 2012 and 2019. We systematically searched for Oriental reed warbler nests in reed swamps in our study area and identified nests parasitized by the common cuckoo (Wang et al. 2020a, b). The parasitism rate ranged from 34.3% to 65.5% in our study area (Liang et al. 2014; Yang et al. 2014).

Field experiments

We numbered each warbler nest discovered in the field and recorded the dates, clutch size, and cuckoo parasitism. In the incubation stage (usually after four eggs were laid), we performed the following three sets of experiments to test the warblers’ abilities to recognize and reject different foreign eggs: (1) the black model egg trial, where we inserted one black model egg made of synthetic clay into the natural warbler nest (egg length: 22.71 ± 0.63 mm, egg width: 17.47 ± 0.49 mm, egg mass: 3.56 ± 0.31 g, mean ± SD; n = 15); (2) the white model egg trial, where we inserted one white model egg made of synthetic clay into the nest (22.56 ± 0.82 mm, 17.06 ± 0.83 mm; egg mass: 3.47 ± 0.28 g; n = 15); (3) the common cuckoo egg trial, where we recorded natural cuckoo parasitism and cuckoo egg rejection by the host from years 2012–2019 (22.41 ± 0.88 mm, 17.08 ± 0.56 mm; egg mass: 3.74 ± 0.29 g; n = 15).

In addition to the above egg recognition experiment, we further examined the warblers’ rejection behaviors toward foreign eggs with a cryptic color and with a bright color. To minimize the influence on the reproduction of the warblers, we set up an experimental nest at a spot 0.5 m away from the focal warbler nest. We found this distance was suitable for our experimental design, as the warblers would visit experimental nests quickly and then reject the model eggs, perhaps the alien nests may have encroached on their breeding territory. The experimental nests were natural nests collected during the previous breeding season. Two experimental groups were set up. In group 1, the inside of the nest was painted with black spray paint (Fig. 2, group 1), and the nest was dried and left for more than a week before being used for the experiment to ensure there was no smell. Two black model eggs and two white model eggs were placed in the nest (egg length = 17.68 ± 0.42 mm, egg width = 13.40 ± 0.31 mm, and egg mass = 2.76 ± 0.27 g; n = 15). The set-up of this group was to highlight that the white model eggs were more distinguishable while the black eggs were more cryptic against a black nest lining. The warblers would visit the experimental nest, and then recognize and reject the eggs in it. We predicted that the warblers would first reject the white eggs.

Fig. 2
figure 2

Example of experimental nests and active Oriental reed warbler nests in the field. Group 1 refers to the painted black color nest lining, and the white nest lining in group 2

Full size image

On the contrary, the set-up of group 2 was designed to help black cryptic eggs become more distinguishable and bright white eggs become more cryptic. We painted the nest lining white following the same methods as for group 1 and placed the same set of black and white model eggs within it (Fig. 2, group 2). We predicted that the warblers would first reject black eggs: the opposite result to that observed in group 1.

To determine the levels of concealment of the eggs placed in the black and white nests, we measured the color difference between the experimental eggs and the nest lining. A Canon EOS 20D camera was used to take photos of the experimental nests. Birds can detect ultraviolet (UV) light besides visible light by using the fourth cone cell type in the retina (Bennett et al. 1996; Cuthill et al. 2000; Rajchard 2009; Šulc et al. 2016), however, in this study, all model eggs were made of synthetic clay, which have no UV reflectance spectra (300–400 nm) on egg appearance (i.e., Gloag et al. 2014; Yang et al. 2019), in this case, cuckoo cannot remove the model egg depend on the UV cue. Therefore, we try to use the method of CIEL*a*b* to quantify the degree of crypsis of the eggs in the black and white nests (Robertson 2007; McCormick-Goodhart and Wilhelm 2003).

We used the CIE L*a*b* (International Commission on Illumination) color space in Adobe Photoshop CS6 (Robertson 2007; McCormick-Goodhart and Wilhelm 2003) to measure the colors of the nest lining and the eggs. The L* value represents brightness while a* and b* indicate chromaticity, with values representing the color ranges of red to green, and yellow to blue, respectively. For the measurement of egg colors, we took the entire photo to obtain all the parameters of the egg; as for the value of the nest background color, we took four squares of the same size around the egg to measure and calculate the average (Wang et al. 2021; Xiao et al. 2016). After the values of L*, a* and b* were obtained, we used an equation to calculate the overall color difference (ΔE) between the eggs and the nest lining, using the calculation result to indicate the cryptic level or the distinguishable level. The equation for calculating the overall color difference (ΔE) is as follows:

ΔE = \(\sqrt{{\left({L}_{\mathrm{m}}^{*}-{L}_{\mathrm{n}}^{*}\right)}^{2}+{\left({a}_{\mathrm{m}}^{*}-{a}_{\mathrm{n}}^{*}\right)}^{2}+{\left({b}_{\mathrm{m}}^{*}-{b}_{\mathrm{n}}^{*}\right)}^{2}}\)

where m stands for model egg and n for nest lining. The lower the ΔE, the more cryptic the model egg in the nest. On the contrary, the higher the ΔE, the more distinguishable the model egg. In the end, 15 photos were selected for the black nest and the white nest, respectively, recording a total of 30 black and 30 white model eggs.

All the experimental nests were checked daily to assess the warbler response after five days (Moksnes et al. 1991; Wang et al. 2015; Medina and Langmore 2019). The warbler’s response was classified as rejection or acceptance. If the model egg disappeared during the observation period, the egg was considered to have been rejected. If the model egg remained undamaged and the clutches remained actively incubated by the sixth day, it was deemed to be accepted. There was no interference from the experimenters towards the warblers during the experimental process.

Statistical analysis

Binomial tests (two-tailed test) were used to analyze the frequency of warblers rejecting cryptic model eggs in relation to non-cryptic model eggs. One-way ANOVA was used to test for color differences (ΔE) between the nest linings and the model eggs. Differences were considered to be significant at p < 0.05, highly significant at p < 0.01, and not significant at p > 0.05. Statistical analysis was conducted using IBM SPSS Version 25.0 (IBM Corp., Armonk, NY, USA).

Results

Not only were warblers able to recognize and reject 100% of the model eggs (Table 1), they were also able to reject some of the naturally parasitic cuckoo eggs (4.3%, 13 out of 302). Warblers showed a significant egg rejection response toward white model eggs versus black model eggs in a black nest lining (Binomial test, p < 0.0001, Fig. 3, group 1). However, no difference in egg rejection was seen in response to black and white model eggs in a white nest lining (Binomial test p = 0.405, Fig. 3, group 2).

Table 1 Egg rejection in Oriental reed warblers to model eggs (black and white color) and naturally parasitized cuckoo eggs
Full size table
Fig. 3
figure 3

Rejection frequency of Oriental reed warblers choosing experimental eggs in relation to the different color of model eggs in black nest lining (group 1) and white nest lining (group 2). Numbers above the bars refer to sample size

Full size image

The mean color difference (ΔE) between the black nest lining (Table 2) and the white model egg was 207, while the mean ΔE between the black nest lining and the black model egg was 25. This was a significant difference, indicating that black model eggs are highly significantly cryptic compared to white ones in a black nest lining (F = 2298.514, p < 0.001). Conversely, mean ΔE between the white nest lining (Table 2) and the white model egg was 69, while the mean ΔE between the white nest lining and the black model egg was 78. There was no significant difference in ΔE between the two colors of model eggs, indicating that there is no significant difference in crypsis between the two egg colors in a nest with white lining (F = 3.273, p = 0.081).

Table 2 Color difference (ΔE) between white and black model eggs in artificial painted nest lining (N refers to nest sample size)
Full size table

Additionally, our video-recordings showed that warblers first removed the white model egg in the black nest lining in group 1 (ESM Video S1). However, in group 2, warblers either first removed both the white and the black model eggs (ESM Video S2 and S3).

Discussion

We tested warblers’ abilities to recognize and reject non-mimetic model eggs and further examined their rejection behaviors toward the different cryptic model eggs placed against the black nest lining and the white nest lining. The results showed that warblers were able to recognize and reject all non-mimetic model eggs and a few of the naturally parasitic mimetic cuckoo eggs. Consequently, cuckoos must evolve more mimetic or cryptic eggs to be better accepted by the hosts. In the cryptically colored egg experiment, the warblers rejected almost the white model eggs against the black nest lining, which was in line with our prediction. However, in the group with black model eggs against the white nest lining, the result did not support our hypothesis. In general, during our observations, warblers chose to reject brighter foreign eggs while the eggs with the more cryptic color could be kept and accepted.

In other host species, it has been observed that some natural parasitic eggs will also be rejected. For example, eastern olivaceous warblers (Iduna pallida elaeica) were observed to reject 12% of common cuckoo eggs (Antonov et al. 2007). Similarly, great reed warblers (Acrocephalus arundinace) rejected 57% of cuckoo eggs (66 out of 116) by ejection (n = 45), desertion (n = 20), or by egg burial with nest material (n = 1) (Trnka et al. 2012). In the present study, the rejection rate of cuckoo eggs was 4.3% (13 out of 302). The rejection rate may have been underestimated due to hosts possibly having rejected parasitic eggs prior to being discovered (Davies 2000; Samaš et al. 2014; Li et al. 2016). We observed that accepted eggs tended to be more mimetic or cryptic (i.e., Fig. 1a). From this point of view, producing cryptic eggs is adaptive for parasitic cuckoos. First, it can maximize the possibility of its eggs not being recognized and rejected by the host, and second, it can also prevent a second cuckoo female from removing its eggs in a multi-parasite nest (Gloag et al. 2014). When a mimetic parasitic egg and a host egg are in the nest together, the probability of them being caught by a parasite is identical. However, a cryptic egg is less likely to be detected, achieving a lower probability of being recognized and removed (Wang et al. 2021). Therefore, from a comprehensive point of view, eggs with cryptic colors are more advantageous.

Laying relatively more cryptic eggs may prevent subsequent female cuckoos from noticing the egg during the limited time of the parasitism process. For example, the olive-brown eggs laid by Australian bronze-cuckoos that are cryptic, yet far from being similar to their host eggs, still managed to be accepted by the hosts (Langmore et al. 2009; Gloag et al. 2014), indicating that cryptic eggs are more difficult to be detected. Therefore, in group 1 of our experiment, the color of the black model eggs was almost identical to that of their nest lining, making them highly cryptic. By contrast, the white eggs in the same nest lining stood out and were rejected by the warblers. The results proved to be a good interpretation of the crypsis hypothesis.

Our findings are not consistent with previous work showing that increased egg-nest visual contrasts do not increase parasitic egg discriminability and rejection in hosts eastern phoebes (Sayornis phoebe) and American robins (Turdus migratorius) of brown-headed cowbirds (Molothrus ater). They found that despite successfully increasing egg-nest visual contrast in their treatments, these two hosts did not increase the rate of reject model parasitic eggs (Aidala et al. 2015, 2019). In great reed warblers (Acrocephalus arundinaceus) of common cuckoos, egg-nest contrast was a collateral, not a causal factor in egg rejection (Hauber et al. 2015). It is possible that their treatment reciprocally dyed both eggs and the nest lining with one of two colors: orange and green, and the contrast is not distinct compared with our study: the black and white color are more distinct.

In the white nest lining in group 2, we predicted that the white model eggs should be more cryptic, and the black ones more distinguishable and therefore possibly more likely to be carried off by the warblers. However, the results were not consistent with this prediction. The warblers randomly rejected the model eggs in the nest, and there was no significant difference in rejection between black and white eggs, as there was in group 1. To our surprise, the number of white model eggs picked was slightly higher than the number of black eggs. Some possible explanations are as follows.

Firstly, in the white nest lining, the crypsis of the white model eggs did not achieve our expected level, where its ΔE showed no significant difference, just like that of the black model eggs. As a result, both colors were distinguishable, resulting in similar removal rates. Alternatively, the white spray paint cannot make the nest lining look obviously different in color, therefore, white or black model eggs look no different together within a nest.

Second, in nature, bird eggs are mostly white, blue, and brown, and there are few reports of pure black bird eggs (Kilner 2006). The egg color of this warbler species, and of its host species, common cuckoos, is white (Liang et al. 2014; Li et al. 2016; Yang et al. 2016a, b). It is likely that warblers are more sensitive to white eggs and as a result, the white model eggs were removed first in this experiment.

Finally, in the field, we found that when a small number of warblers lay their last egg, it may be pure pale or lighter coloration in egg appearance, e.g., the eggshell has no black spots, which is a relatively common phenomenon in most birds (Ruxton et al. 2001; Peer and Sealy 2004; Kilner 2006; Huo et al. 2018). Therefore, white eggs may be more likely to be considered as threatening foreign parasitic eggs by warblers, and therefore to be preferentially removed.

In conclusion, the results of the present study support the crypsis hypothesis, revealing that even in open nests, more distinguishable eggs are preferentially removed by the host while more cryptic eggs are spared and kept. This allows us to further understand why parasites choose to lay eggs that are either more mimetic to the host eggs or more cryptic. The advantage of doing so is to better increase the chance of the eggs being accepted by the host. We validated the crypsis hypothesis by conducting experiments on how host birds remove eggs. However, whether a natural parasitic egg that carries cryptic features, but is not mimetic at all to the host eggs in terms of color, can deceive the host in an open nest with relatively good lighting conditions still requires further study.