DOI: 10.6084/m9.figshare.29552144.v1
title: <b>How do oriental reed warblers recognize cuckoo eggs?</b>

description: The Sifangtuozi Farm (46°00′–46°22' N, 123°46'–123°57' E) is located in the Zhenlai County section of the Nenjiang River Basin. The area is under the jurisdiction of Jilin Province in Northeast China and has abundant water resources. Vast patches of reeds and bulrushes grow along the ridges, aqueducts, creeks, ponds, and other areas around the farmlands. During the annual breeding season (May–August), numerous ORWs migrate to the reed marshes in this area to breed (Trnka et al. 2023). ORWs are the main hosts of CCs in Asia. The CC parasitism rate in northern China is as high as 34.3–65.5% (Li et al. 2015; Wang et al. 2020, 221, 2022; Yang et al. 2016, 2017).We searched for new nests in the reed swamps at least twice a week from May to August 2022. Upon identification, the status of the nests was checked every morning. The ORWs of this population typically lay eggs in the early mornings, whereas CCs can lay eggs at anytime of day.With reference to the methods of Moskát and Hauber (2007) and Wang et al. (2015), we used the eggs of the budgerigar (<i>Melopsittacus undulatus</i>) as the foreign eggs and ORW eggs from the same breeding population as the highly-mimetic (conspecific) eggs. The experimental groups were established by removing one of the host eggs from their nest and replacing with the following: (i) budgerigar egg (non-mimetic egg group [NM]; n = 37 nests, Figure 1A, D, G); (ii) budgerigar egg with black spots painted on the surface (poorly-mimetic egg group [PM]; n = 37 nests, Figure 1B, E, H); and (iii) highly-mimetic (conspecific) egg group [HM]; n = 30 nests, Figure 1C, F, I).Next, the three experimental groups were further subdivided into the following stages: (i) pre-egg-laying (PE): n = 12 for NM and PM groups, and n = 10 for HM group; (ii) single host egg (SHE): n = 10 for all groups; and (iii) multiple host eggs (MHE): n = 15 for NM and PM groups, and n = 10 for HM group. For the PE stage (Figure 1A, B, C), a foreign egg was placed into each nest after nest building was completed but before egg laying. For the SHE stage (Figure 1D, E, F), a foreign egg was introduced to replace the only host egg in the nest 1 day after the host had laid the egg. To reduce human interference for this group, we consistently limited our operation time to the morning after the host had laid the egg; that is, we tried our utmost to replace the host egg as soon as possible. For the MHE stage (Figure 1G, H, I), a foreign egg was introduced to replace one of the host eggs after the host had laid 2–6 eggs (the maximum clutch size for ORWs is six eggs, see Figure 1H).We monitored the nests under the parasitism experiments daily for six consecutive days to confirm the hosts’behavioral responses. The status was classified as rejection if the foreign eggs were thrown away or buried or the nests were abandoned; the status was classified as acceptance if the foreign eggs remained intact in the host nest after 6 days or were hatched (Moksnes et al. 1991; Moskát and Hauber 2007; Wang et al. 2015).Oriental reed warblers (<i>Acrocephalus orientalis</i>) are favorite hosts of common cuckoos (<i>Cuculus canorus</i>). However, the cognitive basis and underlying mechanisms of the host egg rejection behavior remain not well understood. In this study, we conducted egg recognition experiments using three egg mimicry levels (non-, poorly-, and highly-mimetic eggs) to observe Oriental reed warbler behavior at three breeding stages (pre-egg-laying, single host egg, and multiple host eggs). The results showed that oriental reed warbler s did not reject highly-mimetic eggs (conspecific eggs) at all breeding stages but rejected non- and poorly-mimetic eggs. During the pre-egg-laying and single-egg stages, the rejection rate for non-mimetic eggs was 100% for both, and that for poorly-mimetic eggs was 83.3% and 70%, respectively. With multiple host eggs, the rejection rate for non- and poorly-mimetic eggs was 86.6% and 66.6%, respectively. That is, oriental reed warblers can identify foreign eggs and conspecific eggs at the pre-egg-laying stage without any comparison. This suggests that oriental reed warblers use a memory-based template as a recognition mechanism, meaning that they rely on innate or long-term memory templates derived from previous reproduction attempts for egg recognition. Rejection of most foreign eggs, but none of conspecific eggs, at the pre-egg-laying stage without any references indicates true egg recognition in response to parasitism, not a behavioral response triggered by nest sanitation. Therefore, oriental reed warblers have a complex cognitive basis and multiple mechanisms for egg recognition.