<b>Precise targeting host </b><b>activity </b><b>for parasitism by common cuckoos</b>

This study was carried out from March to August 2018–2025 in the Liuzhi area (26°13′ N, 105°42′ E), Guizhou, southwestern China. The study area mainly consists of a karst landscape, with a mosaic of villages, farmland, shrubland, and barren slopes. The preservation of traditional agricultural practices may play an important role in the distribution and reproduction of birds in the area (Zhong et al., 2023).The redstart is a small passerine songbird belonging to the family Muscicapidae (Zheng 2023). Previous monitoring in this population indicates that its breeding season lasts from late March to mid-August. This species typically raises 1–2 broods per year, with a clutch size of five eggs (ranging from 3 to 6) (Wan et al., 2025). Eggs are pink or pale blue with brown spots; however, there is substantial phenotypic variation among clutches (Wan et al., 2025). Nest sites are highly variable, spanning a continuum from open nests to secondary cavities. Moreover, most breeding individuals build nests on artificial objects—such as buildings, household items and even cars—reflecting a unique adaptation to cohabitation with humans (Figure 1, Video S2-S8). Common cuckoos parasitizing redstarts lay pure blue eggs and pale blue eggs with brown spots (Figure 2). The latter closely resembles the pink eggs of the redstart in phenotype (Wan et al., 2025). Additionally, other major secondary cavity-nesting species in the area include the Oriental magpie-robin (<i>Copsychus saularis</i>)—which lays blue, brown-spotted eggs, distinct from the cuckoo's pink eggs (Figure 3a)—and the Eurasian tree sparrow (<i>Passer montanus</i>).<br><b>Field data collection</b>We monitored the breeding activities of redstarts within the study area (∼1800 ha) by locating nests and recording parasitism events by the common cuckoo. When finding the host's nest, we recorded the breeding status and types of the breeding site. We then conducted regular checks and video‑recorded cuckoo visits to these nests. Given that human-adapted Daurian redstarts may gain anti-parasitic benefits from nesting in anthropogenic shelters, we classified natural nest sites along two dimensions: (1) based on the possibility that cuckoos avoid approaching buildings (Liang et al. 2013; Zhang et al. 2023), nests were recorded as either "on houses" or "non-houses"; (2) given that cuckoos do parasitize indoor nests and that some hosts prefer breeding indoors (Figure 1), we further categorized nest sites as "indoors" or "outdoors." This classification was designed to investigate what cues cuckoos use to locate indoor target nests. Furthermore, cases in which a cuckoo removed a host egg but did not lay its own were also classified as parasitism events rather than simple predation, as they may reflect a significant parasitic strategy known as “farming behavior” (Zhong et al., 2019; Zhang et al., 2024).To test the relationship between cuckoo selection of redstarts and nest concealment as well as host activity cues, we conducted a nestbox attraction experiment from 2019 to 2023 and in 2025 by attracting some redstarts and other species to breed in nestboxes. Compared to natural nest sites, these nestboxes, with their unobstructed host activity, were more easily detected and parasitized by cuckoos. The internal dimensions of the nestbox were 12 cm × 12 cm × 26 cm (length × width × height). To ensure that the cuckoo can successfully enter, the nest entrance was 10 cm × 8 cm (length × width). The nestboxes were placed in or near residential areas, on utility poles, trees, or the walls of houses, at a height of at least 3 meters above the ground. Nests were checked every 5–6 days during the early breeding season. When breeding pairs were observed in the nestbox, we checked for egg laying and cuckoo parasitism every 2–3 days depending on the nesting progress. Due to the strong nestbox occupancy behavior of the Eurasian tree sparrow, we removed nest material from some sparrow nests during the early nesting period to increase occupancy by redstarts and oriental magpie-robins (Wan et al., 2023). In addition, sparrows typically fill the interior of nestboxes with nesting material and leave small entrances and exits immediately adjacent to the walls of the nestbox, making it virtually impossible for cuckoos to enter the interior of the nest to lay eggs (and natural sparrow nests are not accessible to cuckoos for egg laying); accordingly, we did not consider sparrows as a potential host for cuckoos in this study.The evolution of parasitic strategies through tracking host life‑history patterns represents a key adaptive trait in avian brood parasites. However, when hosts successfully exploit human‑modified environments – such as diverse and concealed nesting sites – the mechanisms enabling cuckoos (<i>Cuculus</i> spp<i>.</i>) to accurately locate and parasitize such nests remains unclear. The Daurian redstart (<i>Phoenicurus auroreus</i>), a human‑commensal secondary cavity‑nester, often breeds in diverse artificial structures—leading to variable and concealed nest locations—making it an ideal system to study this question. Using long‑term field monitoring and a nestbox attraction experiment, we investigated how common cuckoos (<i>Cuculus canorus</i>) locate and parasitize such concealed nests. Our results show no significant variation in common cuckoo parasitism rates across different natural nesting sites. However, nestboxes that were more exposed experienced higher parasitism rates than natural nests. Furthermore, cuckoos targeted exclusively those boxes with active host attendance, rather than boxes occupied by other species. This study provides the first evidence of cuckoo parasitism on a human‑associated host nesting indoors, underscoring the key role of host activity cues and suggesting that cuckoos, like their hosts, are also adapting to anthropogenic refuges.本研究于2018–2025年3月至8月在中国西南部贵州省六枝地区（北纬26°13′，东经105°42′）开展。研究区域以喀斯特地貌为主，镶嵌分布着村落、农田、灌丛与荒坡。传统农耕实践可能对当地鸟类的分布与繁殖发挥重要作用（Zhong等，2023）。 红尾鸲是鹟科（Muscicapidae）的小型雀形目鸣禽（郑光美，2023）。该种群此前的监测结果显示，其繁殖期为3月下旬至8月中旬，每年通常繁殖1–2窝，每窝产卵5枚（范围3–6枚）（Wan等，2025）。卵呈粉色或淡蓝色，布有褐色斑点，但不同窝之间存在显著的表型差异（Wan等，2025）。巢址具有高度多样性，涵盖从开放式巢到次生树洞的连续谱系。此外，多数繁殖个体将巢筑于人工设施上，如建筑物、家居用品甚至汽车，体现出与人类共生的独特适应性（图1，视频S2–S8）。 寄生红尾鸲的普通杜鹃（common cuckoo）所产卵为纯蓝色或带褐色斑点的淡蓝色卵（图2），后者在表型上与红尾鸲的粉色卵极为相似（Wan等，2025）。该区域其他主要次生洞巢鸟类包括鹊鸲（<i>Copsychus saularis</i>）——其卵为蓝色带褐色斑点，与杜鹃的粉色卵截然不同（图3a）——以及树麻雀（<i>Passer montanus</i>）。 <b>野外数据采集</b> 我们通过定位巢址并记录普通杜鹃的寄生事件，对研究区域（约1800公顷）内红尾鸲的繁殖活动进行监测。发现宿主巢后，我们记录其繁殖状态与巢址类型，随后开展定期检查，并通过视频记录杜鹃到访这些巢的过程。 鉴于适应人类活动的达乌里红尾鸲（Daurian redstart）在人工遮蔽处筑巢可能获得抗寄生益处，我们从两个维度对天然巢址进行分类：（1）基于杜鹃可能回避靠近建筑物的假设（Liang等，2013；Zhang等，2023），将巢分为“房屋上”与“非房屋处”两类；（2）鉴于杜鹃确实会寄生室内巢且部分宿主偏好于室内繁殖（图1），我们进一步将巢址划分为“室内”与“室外”。该分类旨在探究杜鹃定位目标室内巢所依赖的线索。 此外，杜鹃移除宿主卵但未产卵的案例也被归类为寄生事件而非简单的捕食行为，因为这可能反映了一种被称为“耕作行为”的重要寄生策略（Zhong等，2019；Zhang等，2024）。 为验证杜鹃对红尾鸲的选择与巢隐蔽性以及宿主活动线索之间的关系，我们于2019–2023年及2025年开展了巢箱（nestbox）招引实验，以吸引部分红尾鸲及其他物种在巢箱内繁殖。与天然巢址相比，这些巢箱宿主活动无遮挡，更易被杜鹃发现并寄生。巢箱内部尺寸为12 cm × 12 cm × 26 cm（长×宽×高），为保证杜鹃可顺利进入，巢入口尺寸为10 cm × 8 cm（长×宽）。巢箱被放置于居民区内部或附近、电线杆、树木或房屋墙壁上，高度至少距地面3米。 繁殖早期阶段，每5–6天检查一次巢。当观察到繁殖对入驻巢箱时，根据筑巢进度每2–3天检查一次产卵情况与杜鹃寄生情况。由于树麻雀具有强烈的巢箱占据行为，我们在筑巢早期移除了部分麻雀巢内的巢材，以提高红尾鸲与鹊鸲的巢箱占有率（Wan等，2023）。此外，麻雀通常会用巢材填满巢箱内部，仅在紧邻巢箱墙壁处留下狭小的出入口，使得杜鹃几乎无法进入巢内产卵（且天然麻雀巢也无法被杜鹃接触产卵）；因此，本研究未将麻雀视为杜鹃的潜在宿主。 鸟类巢寄生生物通过追踪宿主生活史模式演化出寄生策略，这是其关键适应性特征。然而，当宿主成功利用人类改造的环境（如多样且隐蔽的巢址）时，杜鹃（<i>Cuculus</i> spp.）精准定位并寄生此类巢的机制仍不明确。达乌里红尾鸲（<i>Phoenicurus auroreus</i>）作为与人类共生的次生洞巢鸟类，常于各类人工结构中繁殖，巢址多样且隐蔽，成为研究该问题的理想系统。 本研究通过长期野外监测与巢箱招引实验，探究了普通杜鹃（<i>Cuculus canorus</i>）如何定位并寄生此类隐蔽巢。结果显示，不同天然巢址间的普通杜鹃寄生率无显著差异，但暴露度更高的巢箱寄生率显著高于天然巢。此外，杜鹃仅靶向有活跃宿主入驻的巢箱，而非被其他物种占据的巢箱。本研究首次提供了杜鹃寄生与人类共生、且在室内筑巢的宿主的证据，强调了宿主活动线索的关键作用，并表明杜鹃与其宿主一样，也在适应人工庇护所环境。