Biological invasions are one of the main causes of biodiversity loss and species extinctions, second only to habitat loss (Bellard et al. 2016). As research progresses, more interactions related to biological invasions are being discovered, providing new insights into global changes. These include interactions between native and invasive species, and among invasive species themselves (Bertolero et al. 2022; Lovas-Kiss et al. 2023; Céspedes et al. 2024). Fundamentally, biological invasions are driven by dispersal, which promotes the spread of organisms at the landscape scale (Reynolds et al. 2015). However, successful invasions also depend on post-dispersal processes, like establishment in new habitats and persistence through biotic interactions and meta-community dynamics, sustained by propagule pressure and repeated dispersal events (Hufbauer et al. 2013; Wu et al. 2023). Alien species are often particularly effective at dispersing, leading to widespread global distribution of certain invasive species (Green 2016).

Freshwater ecosystems provide vital ecosystem services and harbor exceptional biodiversity, yet face disproportionate invasion impacts (Reynolds et al. 2015; Green 2016). Global freshwater systems host hundreds of invasive species—including Eichhornia crassipes, Ludwigia grandiflora, Spartina densiflora, cyprinid fishes and crayfish—with documented ecological disruptions (García-Alvarez et al. 2015; Lovas-Kiss et al. 2018; Lovas-Kiss et al. 2020). Among these invaders, South American apple snails Pomacea canaliculata-complex exemplify severe invasion consequences (Global Invasive Species Database 2024). This thermophilic species invades rice ecosystems globally, from Asian paddies to North American littoral zones (Liu et al. 2018), while establishing European populations as novel prey for glossy ibis Plegadis falcinellus and yellow-legged gull Larus michahellis (Bertolero and Navarro 2018; Bertolero et al. 2022). They are known to damage rice seedlings and other crops, and they feed on submerged plants, impacting nutrient cycling in ponds and streams, altering plant community composition, and subsequently affecting the diversity of zooplankton (Horgan et al. 2014). Rapid maturation (4 months) and high fecundity (thousands of eggs in life), coupled with genetic diversity (Estebenet and Martin 2002), drive their expansion. They lay eggs above the waterline, on stems and leaves of aquatic plants, as well as deposit eggs on human structures like bridges and docks. Eggs deposited on aquatic vegetation and human infrastructure are toxin-protected and considered to be unpalatable for amphibians (Brola et al. 2021), though waterbirds partially suppress populations through predation (Liang et al. 2013). Notably, in Florida, invasive apple snails P. maculate compete with native P. paludosa, and local snail kites have increasingly consumed the more-abundant invasive snails, despite their larger and thicker shells (Machado-Stredel et al. 2024).

While migratory birds are key vectors of long-distance dispersal, short-distance dispersal via repeated stepping-stone events proves equally vital for biological invasions (Coughlan et al. 2017a). Zebra mussel Dreissena polymorpha and Lemna minuta could frequently be transported over short distance, with mallards Anas platyrhynchos acting as typical vectors (Coughlan et al. 2017b, 2017c). Field evidence reveals the role of waterbirds as invasion vectors: gulls and storks disperse alien plant seeds and invertebrates through defecation and regurgitation (Lovas-Kiss et al. 2018; Martín-Vélez et al. 2021), while shorebirds transfer the cysts of North American brine shrimp Artemia franciscana to European wetlands (Green et al. 2005). Crucially, invasive snails can survive avian gut passage (Martín-Vélez et al. 2021), demonstrating waterbirds’ capacity to carry snails across geographical barriers. Despite these documented mechanisms, waterbird-mediated dispersal remains understudied in invasion ecology (Green et al. 2023; Lovas-Kiss et al. 2023). Effective invasive species management requires integrating zoochory mechanisms into control strategies; however, current approaches frequently neglect these pathways (Green 2016; Reynolds et al. 2017).

To investigate the role of waterbirds in endozoochory, researchers have conducted various feeding experiments. In one study, mallards were fed to seeds from congeneric native and invasive plant species, revealing that avian ingestion delays invasive plant germination (Lovas-Kiss et al. 2023). Another experiment demonstrated that mallards disperse fragile invasive cyprinid eggs via defecation, with a subset resisting digestion (Lovas-Kiss et al. 2020). Beyond defecation, ducks can regurgitate propagules under specific conditions. For instance, mallards expel large indigestible seeds from the gizzard up to 10 hours after ingestion (Kleyheeg and van Leeuwen 2015). Regurgitation may be crucial for invertebrates or their propagules that are unable to survive passage through the intestines.

The aim of our study was to experimentally investigate whether waterbirds can facilitate the dispersal of apple snails Pomacea canaliculata by dispersing their eggs through endozoochory. We hypothesized that a fraction of snail eggs would survive passage through mallard guts and be egested in faeces in a viable state. In addition, we expected that a portion of eggs fed to mallards might later be regurgitated and have a higher hatchability than those retrieved from faeces. We discussed the implications of our findings for the spread, impact, and potential control of alien apple snails.

Mallards Anas platyrhynchos are omnivorous birds that are widespread, numerous and highly mobile, serving as vectors for many plants and aquatic invertebrates (Urgyán et al. 2022; Green et al. 2023). Eight six-month-old male mallards were purchased from a poultry farm in Zhejiang Province. In China, some farmers cultivate apple snails Pomacea canaliculata for human consumption. During the apple snail breeding season, farmers sell surplus eggs so that others can rear snails. Consequently, obtaining apple snail eggs from farmers was straightforward. First, we offered a mixture of rice and apple snail eggs as food to the mallards, and confirmed that they fed on the eggs, even when other food was available (Suppl. material 1). This suggests that waterbirds are likely to ingest snail eggs in the wild, and we further investigated their ability to act as dispersal vectors for snail eggs using a controlled experiment.

During the experiment, mallards were kept individually in metal cages (0.40 × 0.40 × 0.60 m) with a mesh floor (2 × 15 cm), and removable plastic trays were placed under each cage to collect droppings. The apple snail eggs we purchased were attached to the stems of aquatic plants. After carefully removing the eggs from the stems, they were separated one by one and counted. This process caused some egg damage, and only the intact eggs were used for feeding and hatching experiments. Separated snail eggs are approximately 2 mm in diameter, and were placed in edible capsules and force-fed to mallards (Fig. 1). The capsules were purchased online from Gaohua Pharmaceutical. Each capsule had a cross section diameter of 8 mm and a length of 22 mm, and was made with glutinous rice flour. At 41 °C, the internal temperature of mallards (Tesson et al. 2018), we confirmed that capsule completely dissolves in water within 10 min when stirred slightly. The capsule may have protected the eggs to some extent, particularly by preventing the mallards’ chewing process and partially withstanding physical and chemical digestion, which may have increased our recovery rate.

A total of 19 feeding trials were conducted over three months. In each trial, all eight mallards were fed 200 eggs separately. The feeding trials were numbered 1 to 19 in chronological order, and each individual mallard was force-fed a total of 3,800 apple snail eggs during the whole experiment. Faeces and regurgitation pellets were collected from the removable trays at 1, 2, 4, 6 and 8 h intervals after force-feeding, and then were immediately filtered through a sieve with a mesh size of 800 μm under tap water to recover intact eggs. Regurgitated and defecated eggs were easily distinguished, the latter being covered in faeces (Fig. 2).

Recovered intact eggs were immediately placed in plastic petri dishes in a hatching chamber at high humidity, a temperature of 25 °C, and 24-hour light for 30 days incubation. In each trial, 150 additional snail eggs from the same batch, not fed to ducks, were divided equally into three control groups, and incubated under the same conditions.

Trials were separated by intervals of 1 to 15 days. Water and whole grain rice were available to the mallards ad libitum throughout the experiment. The ducks did not exhibit any obvious ill effects during the experiment. At the end of the study, the ducks remained in good health and were retained in captivity. The experiment was approved by the laboratory animal welfare and ethics committee of East China Normal University (No. q20240102).

A total of 30,400 apple snail eggs were fed to eight male mallards in 19 feeding trials. In five feeding trials, no egg was recovered from faeces, while 4–6 intact eggs (ca. 0.15% of those ingested, Fig. 1) were recovered from faeces in the remaining 14 trials. Most eggs recovered from faeces were collected between 2 and 6 hours after feeding (Fig. 2). Regurgitation events were observed within 2 h after force-feeding (Fig. 2). In five feeding trials, no egg was regurgitated, while 684 intact eggs (ca. 2.25%, Fig. 1) were recovered in the remaining feeding trials. In the first hour 551 eggs were regurgitated, followed by 133 eggs in the second hour. The frequency of defecation events was higher than that of regurgitation events, but there was no significant difference in their occurrence (Chi-square test, χ² = 1.06, P = 0.3). For a given trial, there was also no significant difference in the number of eggs defecated and regurgitated (Wilcoxon signed-rank test, N = 12 trials, P = 0.15), despite the latter being greater in quantity. The retention time of defecated eggs (3.68 ± 1.33 hours, Mean ± SD, N = 14 trials) and that of regurgitated eggs (1.22 ± 0.42 hours, Mean ± SD, N = 14 trials) was significantly different (Mann-Whitney U test, P < 0.001).

The incubation period for eggs that hatched varied from 5 to 15 days. Egested eggs generally showed lower hatchability than control eggs that had not been ingested. Control egg hatchability ranged from 26% to 94.7% (Fig. 3). Two intact eggs recovered from faeces in the 13^th and 14^th trial hatched successfully. The overall hatching rate of snail eggs from mallard faeces was 4.3% (Fig. 1), and the hatching rate of snail eggs from regurgitations was 10.8% (74 eggs from nine trials were hatched). Mann-Whitney U test showed that control eggs had a significantly higher hatchability than eggs from faeces (N = 14 trials, P < 0.001) and regurgitations (N = 14 trials, P < 0.001). Regurgitated eggs also had a significantly higher hatchability than eggs from faeces (P = 0.02). Overall, 0.007% of ingested eggs hatched after being egested in faeces, and a further 0.243% hatched after being regurgitated (Fig. 1).

Our study provides the first experimental evidence of waterbird-mediated dispersal for invasive apple snail eggs. Crucially, regurgitation enables short-distance dispersal, while intestinal retention facilitates long-range dispersal. This mechanism complements human-mediated dispersal, explaining their rapid colonization of global wetlands within decades. In southern China, waterbird predation may partially suppress their populations (Liang et al. 2013), yet dispersal persists: their eggs can remain in mallard guts for up to 6 hours, and viable eggs could potentially be transported more than 400 km at flight speeds of 60–78 km·h^–1 (Reynolds et al. 2015). Given their body size, adults cannot be dispersed by surviving gut passage in a manner comparable to the alien Physella acuta (Martín-Vélez et al. 2021). Our study showed that the dispersal of apple snail eggs is rare but possible, since mallards actively ingest them and a small but important fraction can survive gut passage. The same may be true for other waterbird species, such as Anatidae, storks and gulls, occurring in rice fields and other habitats invaded by apple snails (Martín-Vélez et al. 2021).

Laboratory and field studies have conclusively demonstrated that waterbirds can disperse a wide range of animal taxa by endozoochory, including chironomid larvae, aquatic beetle eggs, and fish eggs (Green and Sanchez 2006; Laux and Kölsch 2014; Lovas-Kiss et al. 2020). This ecological context supports our finding that apple snail eggs can survive avian gut passage. While regurgitation and defecation both contribute to dispersal (Kleyheeg and van Leeuwen 2015), regurgitation specifically enables short-distance transport of larger quantities of eggs, whereas defecation facilitates long-distance movement of fewer propagules. Such dynamics mirror stepping-stone dispersal, where sequential short-distance movements bridge distant habitats and may transform these localized events into landscape-scale invasions over time, as observed in bivalves spread through waterbirds (Coughlan et al. 2017c). For invasive species, they may undergo secondary dispersal through biotic and abiotic vectors like floodwaters, invertebrates and waterbirds, accelerating invasions in hydrologically connected systems (Lovas-Kiss et al. 2018; Navarro‐Ramos et al. 2021). Furthermore, frequent short-distance movements, such as daily foraging flights, sustain propagule pressure and gene flow between subpopulations. Mallards visiting multiple wetlands within a single day may carry eggs at each site, creating a dispersal network that enhances invasion potential (Kleyheeg et al. 2017). While large egg size reduces survival during gut transit, it promotes local retention and repeated colonization of adjacent habitats through stepping-stone dispersal (Coughlan et al. 2017b).

Our experiment quantified endozoochory of apple snail eggs under controlled conditions, may not fully represent the natural diet, where waterbirds consume mixed plant-animal food. A higher proportion of eggs may potentially survive gut passage, and have longer retention time, when birds feed on a higher fibre diet dominated by plant material, instead of animal-based diet. Crustacean eggs showed higher survival in mallards when mixed with a plant-based than an animal-based diet, and markers had a longer gut retention time on the plant-based diet (Charalambidou et al. 2005). Furthermore, epizoochory, the transport of organisms externally attached to birds via their feathers, feet or bills, provides an additional potential dispersal pathway for apple snails. A review highlights this external dispersal mechanism by various waterbirds (Coughlan et al. 2017a). Molluscs such as bivalves, gastropods, and barnacles can attach to the external surfaces of waterbirds like gulls, shorebirds, and ducks for extended periods (Coughlan et al. 2017c; Green et al. 2023). Recent studies demonstrate that invertebrate eggs also adhere to waterbirds for passive transport as well. For example, cladoceran ephippia can attach to duck legs (Wang et al. 2023), and aquatic insects lay eggs on the leg of waterbirds (Carbonell et al. 2020).

In conclusion, our findings reveal that regurgitation and defecation by waterbirds can facilitate the spread of a highly invasive snail. While waterbirds can reduce the abundance of apple snails through predation, their regurgitation facilitates short-distance dispersal of viable eggs in pellets, and defecation enables long-distance dispersal of fewer eggs within faeces. Waterbird-mediated dispersal of alien species, which has often been an overlooked pathway of biological invasions, should be taken into account if management strategies are to be effective (Reynolds et al. 2015; Green 2016; Reynolds et al. 2017).

YZ: Methodology, Data Curation, Formal analysis, Writing – Original, Visualization; QZ: Methodology; AG: Writing - Review and Editing, Supervision; ST: Supervision, Project administration, Funding Acquisition; XJ: Conceptualization; Resources; Writing - Review and Editing; Supervision; Project administration, Funding Acquisition.

Some assistance was provided by the Yangtze Delta Estuarine Wetland Ecosystem Observation and Research Station, Ministry of Education and the Shanghai Science and Technology Committee, Shanghai, China.

XD was supported by Shanghai Environmental Monitoring Center (HT2024306A2), National Nature Science Foundation of China (32470556, 32301413 and 32401398), Science and Technology Commission of Shanghai Municipality (21DZ1200900, 22DZ1202600, and 19ZR1416200). AJG was supported by the Ministerio de Ciencia e Innovación WaterZoo project (PID2020-112774GB-I00 / AEI / 10.13039/501100011033). YZ was supported by the China Scholarship Council (202206140024).