Among potential hosts, the rejection of foreign eggs, which is a common and effective strategy to counter brood parasitism, depends on egg recognition. Multi-modal and multi-component recognition cues of brood parasitic eggs, which include both tactile (size, shape, and texture) and visual (size, shape, color, and maculation) cues, are potentially involved in the perception and discrimination of foreign eggs by hosts. An egg rejection experiment on the host with different types of model eggs can help to accurately assess the relative contribution of different components on egg recognition and constraints to rejection, in which videos can help identify the method of host rejection. Here, we assessed egg recognition and rejection responses by Oriental reed warblers (Acrocephalus orientalis), one of the most common hosts of common cuckoos (Cuculus canorus) which breed in eastern China. We designed six groups of model eggs for rejection experiments in which sensory cues included three grades of size and two categories of visual mimicry. Our experiments confirmed that the multi-modal traits, which included variation in size, were significant predictors of egg rejection: we detected significantly higher rejection rates of mimetic spotted model eggs than of non-mimetic blue eggs. However, large model eggs did not yield higher rejection rates and, instead, these were less likely to be rejected and more likely to be deserted compared with smaller eggs. Further video-recording data showed that there was no significant effect of egg size on the egg recognition rate (percentage of nests with evidence of egg-pecking). No evidence that the egg appearance had an effect on the method of egg rejection (ejection or nest desertion) was found. Only visual signals, such as color and maculation, contributed to the recognition of foreign eggs by Oriental reed warblers as recognizable clues, but not the egg size. The egg size had an impact on the type of egg rejection. It was less feasible for the warblers to eject large eggs and that is why they opted more often for desertion as the mean of model egg rejection. The significantly lower egg rejection rate of large eggs suggested that although some of them were recognized as foreign eggs, hosts failed to reject these eggs and finally the eggs were assumed to being accepted by the commonly-used nest-checking methods.

Study site and species

This study was conducted during 2010-2012 and 2013-2018 in the Yellow River Delta, Shandong Province, and the Liaohe River Delta, Liaoning Province, China, respectively. The distance between these two sites was 400 km. Both sites were typical estuarine wetlands and contained a large area of reed field, a common breeding habitat for Oriental reed warblers and common cuckoos in China (Li et al. 2016b). The mean clutch size of Oriental reed warblers was 4.74±0.52 (SD, n=276) and varied from four to six eggs (Li 2012). Cuckoo parasitism rates across the two sites varied between 17% and 25%. Detailed information about the study sites can be found in Li et al. (2016a, b).

Experimental design, model egg production, and egg reflectance measure

We used a polymer clay (Ai Tao Le, Shenzhen, China) to produce the model eggs rather than using painted real eggs in the experiment because it was difficult to find a sufficient number of natural eggs of different sizes. We designed six groups of model eggs with two different colors and spotting and with three different sizes (Fig. 1). The non-mimetic model egg was pale blue, and the white model egg was painted with a light blue-green background (Sakura, Japan; #236) with dark brown spots (#17) to mimic the true Oriental reed warbler eggs (Fig. 1). The medium size of model eggs was similar to local Oriental reed warblers egg size (21.32mm x 15.51mm, n=70 clutches), large eggs were 67% larger than medium eggs, and small eggs were 50% smaller than medium eggs (Table 1). Egg reflectance spectra (300–700 nm) of the background color and spot color of model eggs and Oriental reed warbler eggs were measured using a miniature fiber optic spectrometer (AVANTES) following the protocol of Li et al. (2016b) (Fig. 2).

Rejection experiments

Each breeding season, reed beds were searched systematically for Oriental reed warbler nests. The number of eggs (empty nest as 0), incubation stage, and whether the nest was parasitized were recorded the first time it was found and when re-checked every 1-3 d. Oriental reed warblers usually lay five eggs and begin incubation after laying the penultimate egg (Li et al. unpublished data). The stage of embryo development was assessed by candling if the nest was found after clutch completion (Weller 1956). Those nests that were close to hatching with an obvious embryo were not used in this experiment. The clutch initiation date was determined by counting eggs (assuming one egg was laid per day, and that no eggs were lost) or calculated when the chick hatching date was recorded; eggs usually needed 12 d to complete incubation. Most cuckoos typically parasitize the warblers during the laying stage (Lotem et al. 1995; Wang et al. 2020) and only nests that did not contain cuckoo eggs were used for egg recognition experiment. As a result, the onset of egg experiment for each nest was divided into three stages: (i) egg-laying stage: when the females rarely visited their nests, (ii) early incubation stage: ≤4 d after clutch completion, and (iii) late incubation stage: 5 – 8 d after clutch completion.

One of the six types of model eggs was introduced randomly into Oriental reed warbler nests and checked every other day until the 6^th day (Moksnes et al. 1991; Bártol et al. 2012). We added the egg model without removing any host eggs. Several studies confirmed that this is a normal procedure for this kind of experiment, which does not alter the rejection rate (Davies and Brooke 1989; Grim and Honza 2001). Only non-mimetic model eggs (sample sizes for the small, medium and large eggs were 33, 40 and 24 respectively) were trialed in Yellow River Delta, and the egg rejection rates of these model eggs were not significantly different from those in Liaohe River delta (Chi-square tests: all P values ≥ 0.212 for three egg sizes).

When the model egg disappeared from the nest and the warbler eggs were still warm, it was classified as an ejection; when the model egg was still in the nest, but the warbler eggs were cool during two times of nest-checking, it was classified as desertion. Both ejection and desertion were pooled as rejection. Otherwise, the model egg in the nest was re-checked if there was a trace on it, which was presumed to form during pecking or piercing attempts made by a host; this was an example of “egg recognition”, but it was not included as “rejection” because it was not rejected successfully (i.e, either ejected or deserted). Only nests where the model egg showed no trace of pecking and were still being incubated were recognized as accepted. We found model eggs buried in nest material in only two nests.

Video-surveillance of host nests

Another subset of nests (n=76) was filmed using Xiaoyi 4K digital cameras (Shanghai, China) for 8 h after the introduction of a model egg during 2014-2018. In addition, we selected another 13 nests randomly in which we marked one egg with a water-proof pen as controls. We also monitored these nests for 6 d to determine the host response.

We checked the videos after the experiment using Baofeng 5.0 digital player. Similar to the closely-related Eurasian great reed warbler (Acrocephalus arundinaceus ). Oriental reed warblers are “puncture ejectors” that usually peck foreign eggs strongly (Honza and Moskát 2008; Požgayová 2011), considering that their grasp index (161.5±15.7 mm² ; n=21; Li 2012) were less than the supposed values to be grasper ejectors (200 mm² ; Rohwer and Spaw 1988). If the warbler strongly pecked the model egg repeatedly using its bill, it was classified as “egg-pecking”, which indicated that the warbler “recognized” the experimental egg. The percentage of nests with evidence of “egg-pecking” was summed for each egg type. To have an accurate time of egg recognition, we calculated the initial time to egg-pecking (h) since the time when the host first appeared at the nest. In addition, egg-pecking frequency (/h) before the model egg was rejected was also counted from the video.

No further information.