DOI: 10.5061/dryad.3tx95x6d6
title: Contrasting effects of egg size and appearance on egg recognition and rejection response by Oriental reed warblers

description: 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.
description: 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<sup>th </sup>
 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
 (<i>Acrocephalus
 arundinaceus </i>). 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 mm<sup>2 </sup>;
 n=21; Li 2012) were less than the supposed values to be grasper ejectors
 (200 mm<sup>2
 </sup>; 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.
description: No further information.