We tested the level of mobbing defenses in Red Wattlebirds and the level of egg ejection defenses in Red Wattlebirds, Magpie-larks and Noisy Friarbirds at two sites, Western Sydney and Canberra. At the time of this study (2016) in Sydney, koels had been parasitizing wattlebirds for 38–86 years with a current parasitism rate of 24%, while in Canberra wattlebirds had been a host of the koel for 8–33 years with a current parasitism rate of only 4% (Abernathy and Langmore, 2017). Abernathy and Langmore (2017) found no cases of parasitism of the Magpie-lark or Noisy Friarbird in Western Sydney or in Canberra. However, there has been one anecdotal report of a Noisy Friarbird raising a koel nestling in Canberra in 2014 (Abernathy and Langmore, 2016). Though no formal studies have been conducted on koel parasitism rates of the two old hosts, Brooker and Brooker (2005) found 20 records of koel parasitism of the Magpie-lark on the east coast of Australia from 1909–2008 and 29 records of koel parasitism of the Noisy Friarbird on the east coast from 1893–2002.

All experiments were conducted during the breeding season. The mobbing experiments on Red Wattlebirds were performed from September–October 2015 in Western Sydney and October–November 2015 in Canberra (Table 1). For the egg ejection experiments we searched for Red Wattlebird, Magpie-lark, and Noisy Friarbird nests at several different sites in Canberra from August 2013–January 2014 and August 2015–December 2016, and in Western Sydney from August 2014–January 2015 and August 2015–December 2016 (Table 1). In both Canberra and Sydney, wattlebird and Magpie-lark nests were mostly found in residential areas that included a wetland feature, while friarbird nests were only found in high numbers in nature parks or rural areas with native habitat. Koel parasitism was only found in residential areas or urban parks in both Western Sydney and Canberra and wattlebirds were the only host species for which parasitism was detected at our study sites (Abernathy and Langmore, 2016, 2017). For more information on the koel-wattlebird study system, a map of the study sites and evidence of duration of sympatry between wattlebirds and koels in Sydney and Canberra, see Abernathy and Langmore (2017). In addition to testing these three host species, we informally tested the egg ejection ability of a minor host, the Olive-backed Oriole (Oriolus sagittatus; Brooker and Brooker, 1989), as we occasionally found them nesting near Noisy Friarbirds and their egg ejection capability was unknown. Due to low sample sizes, we only tested orioles with the non-mimetic blue egg and we did not include these nests in any of our statistical analyses.

We compared the aggressive response of wattlebird pairs in the incubation stage of breeding to mounted specimens of a female koel, a common nest predator (Pied Currawong) and a common, harmless parrot (Crimson Rosella). Each of these species have vastly different plumages, making them easy to distinguish, but similar sizes. Crimson Rosellas are around 35–38 cm long and have mainly crimson and blue-colored plumage (Higgins, 1999). Pied Currawongs are slightly larger, about 48 cm long on average, and have mostly black plumage with some white patches (Higgins et al., 2006). Female koels are around 41 cm long and have a dark cap, off-white to buffy front with dark horizontal barring and a dark brown back with white spotting (Higgins, 1999). We used two rosella and currawong mounts, alternating them for each nest, but only had one available female koel mount. Mounts were placed 2–2.5 m from the nest. For low nests (1.5–3 m high), mounts were fastened to the top of a 2.3 m ladder. For higher nests (4–10 m), mounts were hauled up to the appropriate height using a rope hanging over a nearby branch. While this difference in mount presentation has the potential to cause a response bias, we found no effect on aggressive response based on how the mount was positioned (see section “Results”). Mounts were placed in a mesh cage to protect them from damage by the wattlebirds. Observations were made from a blind or car placed four or more meters from the base of the nest tree.

When possible, each pair was presented with all three mounts and mounts were presented in random order to control for order-effects. We only performed two trials per day at a nest to reduce the amount of disturbance and we waited at least 2 h between trials to reduce the chance of carry-over aggression. Before starting the trial, the wattlebirds were allowed at least 30 min to habituate to the cage and hide setup before the mount was placed in the cage. In all but two trials, if the female was on the nest when the mount was placed in the cage, we flushed her off the nest. Trials commenced when the male or female came within 2 m of the mount and continued for 5 min. Observations of the pair’s response to the mount and any vocalizations given by the pair were recorded into a Tascam DR-07 or DR-05 recorder and microphone. We estimated the distances (in meters) of both members of the pair from the mount each time they moved. We counted the number of times they swooped or attacked (physical contact with the cage), the number and type of vocalizations given and we noted whether the female was on the nest.

Male and female wattlebirds are distinguishable in the field by their vocalizations; females give the more musical whistle, while males give harsh clucks and cackles (Higgins et al., 2001, Abernathy personal observation); females give the more musical whistle, while males give harsh clucks and cackles (Higgins et al., 2001). Higgins et al. (2001) mention that males may incubate, but in our study whenever a bird flushed off the nest and vocalized, it gave whistles and never clucks or cackles. Therefore, a bird on the nest was assumed to be a female. For most trials it was possible to distinguish male from female based on these characteristics. We classified wattlebird calls into seven different types by ear and by visual inspection of structural differences in spectrograms generated using Audacity 2.0.5 and Raven Pro 1.4 (alarm calls, growls, cackles, clucks, whistles, contact calls, and single note calls; Higgins et al., 2001). We used Audacity to score the number of each vocalization type. We excluded vocalizations if they were given by a bird not in our view or far away (8–10 m away) or if the bird was clearly responding to another bird.

Studies have shown that many brood parasite hosts use a combination of cues to recognize brood parasite eggs, including ground color, pattern, luminance and size (e.g., Rothstein, 1982; Marchetti, 2000; Spottiswoode and Stevens, 2010; de la Colina et al., 2012). Therefore, to determine how well hosts could discriminate between their eggs and a foreign egg, we created two types of model eggs: a dark blue immaculate egg that differed in two parameters, pattern and ground color, and a spotted egg that was painted to resemble host eggs in pattern and ground color, at least according to the human eye (Longmore, 1991, Figure 1 and see Supplementary Materials). We chose not to test egg size as a variable, so all model eggs came from molds of the same size and were a similar size to all host eggs (see Supplementary Materials).

One of these egg types was placed into a host nest during the laying or incubation periods and the nest was monitored to determine if and when the host ejected the model egg. Nests were typically checked every 1–2 days. For high nests we used an extendable mirror pole to check contents. If nests were accessible with a ladder or by climbing the tree, model eggs were typically placed in the nest by hand. For nests that were inaccessible, we created a device to deposit the egg in the nest using an extendable mirror pole (see Supplementary Figure 1). Even though koels typically remove one host egg during parasitism (Brooker and Brooker, 1989), we did not remove host eggs from the nest during experimental parasitism, as this has not been found to alter host responses in other studies (Davies and Brooke, 1988; Moksnes and Røskaft, 1989).

In our study, 89% of ejections took place within the first 5 days of the experiment. Therefore, an egg was considered accepted if it remained in an active nest (eggs were warm or bird was observed incubating the clutch or defending the nest) for at least 5 days, though we continued checking after this point for 89% of nests to verify the egg was not ejected after this period (see also Rothstein, 1975b). An egg was considered ejected if the model egg was removed and the nest was clearly still active upon discovery of the ejection event. Of the nests where ejection took place, 75% of nests were checked again at least 1 day after ejection and found to still be active, which suggests the model egg was not taken by a predator. Wattlebird nests that were naturally parasitized during the first 5 days of the experiment were not included in the analyses. There were never any naturally parasitized Magpie-lark or Noisy Friarbird nests. We avoided any potential re-nests or territories of breeding pairs that had already been tested successfully in that particular breeding season or in a previous breeding season to minimize the risk of pseudo-replication. How territories in this study were estimated is explained in a previous study (Abernathy and Langmore, 2017, see “Estimating territories” in the “Materials and Methods” section). It was not possible to record data blind because our study involved focal animals in the field.

In our study, we did not include abandoned nests as rejection events for two reasons. First, all the hosts in our study are large (85–122 g; Higgins et al., 2001, 2006) and were capable of grasp-ejecting a foreign egg (see results in Rohwer and Spaw, 1988). Second, a previous study found nest abandonment by wattlebirds was only related to general nest disturbance by researchers and not to the presence of a koel egg in the nest or the study site (Abernathy and Langmore, 2017).

Model eggs were made using a two-part silicone mold and polyurethane resin. After removal from the mold, eggs were smoothed with sand paper and painted with acrylic paint. Spotted eggs had a pinkish ground color with dark reddish-brown and violet-gray spots and were meant to appear similar to the host’s own eggs (Figure 1 and see Supplementary Materials). Even though koel eggs do appear similar to most of their host eggs in color, luminance and spotting, and there is no evidence they exhibit host egg races, koel eggs are significantly more similar in egg pattern and are closer in ground color to Noisy Friarbird and Red Wattlebird eggs than to Magpie-lark eggs (Abernathy et al., 2017). These differences in spotting pattern and ground color could create confounding variables, making our model eggs easier to distinguish for Magpie-larks than for the other hosts. So instead of making model eggs that resembled koel eggs, we attempted to create model eggs that mimicked each host species’ eggs to reduce this potential issue. Spots were created using the pointed end of a plastic dental floss pick, though it was difficult to create the smallest size of spots that occur naturally on the host eggs. Noisy Friarbird and Red Wattlebird eggs are very similar in ground color, luminance and egg pattern, with the majority of spotting at the larger end of the egg, while Magpie-larks typically have a lighter ground color and an obvious ring of spotting around the larger end of the egg (Longmore, 1991; Abernathy et al., 2017, Figure 1). Therefore, we created Magpie-lark spotted model eggs with a ring of spotting at the blunt end and with a lighter ground color (extra white paint mixed with pink) and for Noisy Friarbird and Red Wattlebird spotted model eggs, we created eggs without the distinct spotting ring and a darker ground color (Figure 1). Many passerine eggs have a spectral reflectance pattern with a peak in the UV range (e.g., Cherry and Bennett, 2001; Honza et al., 2007; Cassey et al., 2010; Abernathy and Peer, 2015), including host eggs in this study (Figure 1). In an effort to create more realistic spotted model eggs, we mixed white ultraviolet-reflecting paint (ReelWings) with pink acrylic paint to make the ground color. While this did create a UV-reflecting egg, the spectral pattern of the spotted model eggs did not perfectly match that of a real egg, as there was no peak in the UV range (Figure 1). However, the hosts are presumed to have VS rather than UVS opsins, which will have very low sensitivity to these ∼320 nm peaks (Ödeen and Håstad, 2010; Ödeen et al., 2011). To determine how similar our eggs appeared to host eggs from the host’s visual perspective, we took objective measurements in size, color, luminance and pattern for a subset of fresh host eggs and each model egg type (see Supplementary Materials). While our spotted model eggs did not mimic host eggs perfectly, their ground color was very similar from a bird’s visual perspective and their size and all pattern measurements did fall within the natural range found for each species (see Supplementary Tables 1, 2 and Supplementary Figure 2).

This project was approved by and conducted in accordance with the Australian National University Animal Experimentation Ethics Committee: protocol number A2013/20. Permits from the Territory and Municipal Services of the ACT (license number: LT2013678) and the Office of Environment and Heritage of the NSW National Parks and Wildlife Service (license number: SL101349) were obtained to conduct scientific experiments in Canberra and Western Sydney.

Overall, we tested the behavioral response of 11 wattlebird breeding pairs in Sydney and 12 pairs in Canberra to the mounts, but two koel trials and one rosella trial conducted on three different pairs were not successfully completed and so these were excluded from the statistical analyses. The best REML for predicting wattlebird aggressive response included mount, site and the interaction between mount type and site (Table 2). Overall, wattlebirds responded significantly more aggressively to the koel mount (n = 21 trials, LSM = 0.70 ± 0.55) than to the currawong mount (n = 23 trials, LSM = −0.07 ± 0.53; Tukey HSD: P = 0.03) and the rosella mount (n = 22 trials, LSM = −0.45 ± 0.54; Tukey HSD: P = 0.001), but their response to currawong and rosella mounts was not significantly different (Tukey HSD: P = 0.40). Furthermore, the interaction between mount type and site was important, as wattlebirds in Sydney exhibited a significantly higher aggressive response toward koel mounts than any other group (Figure 3; Tukey HSD: P < 0.03 for all comparisons). More pairs attacked and/or swooped the koel mount in Sydney (60%, n = 10 pairs), than in Canberra (9%, n = 11 pairs; Fisher’s Exact Test, P = 0.02). The type of mount was a borderline significant predictor of whether a pair actually physically attacked the cage containing the mount (Table 2), with the koel mount eliciting a significantly higher number of attacks (LSM = −0.97 ± 1.38) than the rosella mounts (LSM = −2.91 ± 1.62; Tukey HSD, P = 0.045), but not the currawong mounts (LSM = −1.46 ± 1.38; Tukey HSD, P = 0.72). The number of attacks on currawongs was not significantly different from the number of attacks on rosellas (Tukey HSD, P = 0.16).

Wattlebirds produced significantly more alarm calls in the presence of the koel (LSM = 5.01 ± 2.38) than the currawong (LSM = 0.48 ± 2.27; REML, Tukey HSD: P = 0.02) and the rosella mounts (LSM = 0.05 ± 2.33; REML, Tukey HSD: P = 0.01) and this was the only call type that was significantly predicted by mount type (Table 2 and Figure 2). There was no evidence of a cuckoo-specific vocalization in wattlebirds (e.g., Langmore et al., 2012). Only one call type, the growl call, was given exclusively during the koel trial by one pair, but this call type was also given by multiple other pairs toward researchers checking nests (Abernathy personal observation).

The best GLMM model for predicting how long female wattlebirds sat on the nest included mount type, whether the cage was hanging or attached to the ladder and whether the male attacked the mount or not (Table 2). The results suggest that females do not appear to use passive nest defense against brood parasitism, as females sat for a significantly longer period in the presence of the rosella mounts (LSM = 1.85 ± 1.14) than in the presence of the koel mount (LSM = −0.24 ± 1.01; Tukey HSD, P = 0.001) or currawong mounts (LSM = −0.27 ± 0.95; Tukey HSD, P < 0.001). There was no difference in how long females sat in the presence of the koel or currawong mounts (Tukey HSD, P = 1.00). Females also sat for longer on the nest when the cage was attached to the ladder (LSM = 1.28 ± 1.38) than when it was hanging from a branch (LSM = −0.39 ± 0.88) and when the male was not attacking the mount (male not attacking: LSM = 1.20 ± 0.83; male attacking: LSM = −0.31 ± 1.32).

The three host species differed in their response to the different egg types (best model: host × egg type: χ² = 7.4, df = 2, P = 0.02), and this was the only interaction that was significant. Magpie-larks ejected a similar number of blue eggs (91%, n = 22) and spotted eggs (86%, n = 21; Tukey HSD: z = −0.2, P = 1.00; Figure 4). Friarbirds ejected a similar number of spotted eggs to Magpie-larks (94%, n = 17; Tukey HSD: z = 0.4, P = 1.00), but they ejected significantly fewer blue eggs (38%, n = 24; Tukey HSD: z = −3.6, P = 0.003). Wattlebirds were consistently poor egg ejecters, ejecting significantly fewer blue (3%, n = 35) and spotted eggs (4%, n = 25) than Magpie-larks (blue: Tukey HSD: z = −4.9, P < 0.001; spotted: z = −4.4, P < 0.001) and friarbirds (blue: Tukey HSD: z = −2.9, P = 0.045; spotted: z = −4.3, P < 0.001). When all hosts are combined, ejection was more likely to occur when the model egg was added closer to or after clutch completion (best model: Wald’s test: SE = 0.39, z = −2.0, P = 0.04). In addition, we successfully tested 7 Olive-backed Oriole nests using the non-mimetic blue model egg (3 in Canberra and 4 in Sydney). Orioles showed 100% ejection of the blue egg and all ejected in less than 4 days.

Wattlebirds recognized koels as a special nest threat, responding significantly more aggressively to the koel mount than to a harmless control and a nest predator, but their aggressive response did not differ between the harmless species and nest predator. Presentations of koel mounts elicited significantly more alarm calls than presentations of the other 2 mounts and this vocalization was the only one that was influenced by mount type. Wattlebirds were also more likely to physically attack koel mounts than rosella mounts. Furthermore, Sydney wattlebirds were more aggressive toward the koel, where parasitism rate is higher (24%) than in Canberra (4%) (Abernathy and Langmore, 2017). This might indicate that brood parasite recognition may take longer to spread throughout an entire population when parasitism rates are low. Overall, our results seem to suggest that wattlebirds in Sydney viewed the threat posed by koels as different from the threat posed by currawongs. Considering the extra energetic cost associated with raising a cuckoo nestling, these results do make sense and similar results have been found in other mount experiments where both an obligate brood parasite and nest predator mount were used (e.g., Gill and Sealy, 1996; Li et al., 2015; Noh et al., 2021).

Our results are consistent with a learned response because the behavior was acquired rapidly (within 38–86 years of exploitation by the parasite). Moreover, the lower aggressive response and attack rate in Canberra is consistent with learning because a learned response to a threat requires either personal experience of the threat or observation of other species or conspecifics responding to the threat (e.g., Feeney and Langmore, 2013), and this would be less common at the site with lower exposure to koels. Multiple studies have shown that animals can learn anti-predator behaviors toward novel predators over the course of their life through cultural transmission by watching how other individuals respond to those species (reviewed in Griffin et al., 2000; Davies and Welbergen, 2009; Feeney and Langmore, 2013). Anti-predator responses can be further generalized to other novel threatening species that are morphologically similar to the known predator, allowing individuals to respond quickly to potential threats from species they have not previously encountered (Griffin et al., 2001; Ferrari et al., 2007, 2009). The process of cultural transmission and the generalization of anti-predator responses could allow rapid acquisition of the ability to recognize and mob a brood parasite in a naïve host population. This process may be facilitated further by the resemblance of many brood parasitic cuckoos to hawks (Davies and Welbergen, 2008; Welbergen and Davies, 2011). The rapidity of the spread of this defense, however, is likely to be dependent upon how many individuals in the population are actually exposed to the threatening species and the perceived cost of engaging in an aggressive encounter with the threatening species (e.g., Forsman and Mönkkönen, 2001; Davies and Welbergen, 2008; Welbergen and Davies, 2008, 2011). While brood parasites are not a threat to their adult hosts, they will often remove or damage host eggs when laying their own (Sealy, 1992; Davies, 2000; Soler and Martínez, 2000; Gloag et al., 2013) and will sometimes depredate nests late in incubation or in the nestling phase in order to force the host to re-nest (Elliott, 1999; Davies, 2000; Granfors et al., 2001). Therefore, hosts may quickly learn that brood parasites are a threat to their nest and may even view them as nest predators (Mcleod, 1997). Interestingly, our results show wattlebirds may view koels as a greater threat to their nest than a common nest predator, the currawongs. A previous study conducted on wattlebirds and koels at our study sites indicated that predation rate of wattlebird nests does increase once koels have arrived to the breeding area (from 19%, n = 27 before koels arrive to 40%, n = 102 after koels arrive) (Abernathy and Langmore, 2017). This does not necessarily mean koels are the main predator of wattlebird nests, as the currawong is also nesting during this same time period, but it could mean wattlebirds are more vigilant during this time and more likely to mob koels if they view them as a greater threat to their nest.

Contrary to some other studies (Gill and Sealy, 2004; Canestrari et al., 2009; Medina and Langmore, 2016), we found no evidence that wattlebird females exhibit passive nest defense as they were more likely to sit on the nest in the presence of the harmless mount than in the presence of the koel and currawong mounts. They also seemed more disturbed and less likely to sit on the nest when the cage was hanging from a branch, possibly due to more movement of the cage, especially in windier conditions. And male behavior influenced how females responded to the mount, as they were less likely to sit if the male was attacking the mount, which was more likely to happen when the mount was either the koel or currawong.

The two old primary hosts, the Noisy Friarbird and Magpie-lark, as well as the minor host, the Olive-backed Oriole, all ejected eggs at a higher rate than the recent host, the Red Wattlebird, demonstrating that the old hosts have retained egg ejection in the virtual absence of brood parasitism, while this defense has yet to spread throughout the wattlebird population. Indeed, wattlebird egg ejection rates did not differ significantly between Canberra (6%, a site with 8–33 years of parasitism) and Sydney (0%, a site with 38–86 years of parasitism) despite the longer length of sympatry between wattlebirds and koels in Sydney. Many other studies have found that host egg ejection is often maintained after parasitism has ceased (e.g., Avilés, 2004; Peer et al., 2005; Soler, 2014; Yang et al., 2014) and the adaptation may even be retained from a common ancestor after speciation events (Peer et al., 2013). This suggests that once egg ejection evolves and spreads throughout a population, it may pose little cost to the host to maintain it even in the absence of parasitism.

We did not find higher ejection rate of blue eggs compared to spotted model eggs in any of the host species. Surprisingly, friarbirds showed only intermediate ejection of blue model eggs, but ejected almost 100% of spotted model eggs. This could be a consequence of the extremely atypical appearance of the blue model eggs; friarbirds may have failed to associate them with the intended context (Lahti, 2015) and in some species egg recognition is specifically tuned to the natural gradient of eggshell colors such that artificial colors are not rejected in a predictable way (Hanley et al., 2017). Alternatively, friarbirds may have a pre-existing bias toward blue eggs, as several other studies have found that other brood parasite hosts tend to accept bluer eggs over browner eggs (Soler et al., 2012; Hanley et al., 2017; Abolins-Abols et al., 2019; Hanley et al., 2019; Manna et al., 2020). This suggests that, for Noisy Friarbirds, the spotted model eggs provided a better test of egg discrimination ability.

Hosts were more likely to eject model eggs that were added after their clutch was complete. This supports other findings that hosts are more likely to eject an odd egg once they have learned the appearance of their own eggs (Lotem et al., 1995; Rodríguez-Gironés and Lotem, 1999, but see Soler et al., 2013).

Even though wattlebirds are a fairly recent host and may be considered naïve to brood parasitism prior to being utilized by the koel as a host (Abernathy and Langmore, 2017), they were able to recognize koels as a nest threat and most pairs in Sydney (60%) exhibited a stronger aggressive response toward the koel than wattlebirds in Canberra. However, wattlebirds showed little to no egg ejection response to model eggs at either site and a previous study showed no evidence that wattlebirds reject naturally laid koel eggs or nestlings, unless the koel eggs are laid in the nest before the wattlebird has started laying (Abernathy and Langmore, 2017). This lack of egg ejection could be due to various factors constraining the evolution of this defense in the wattlebird, despite the high costs of parasitism. A previous study found that, while unparasitized wattlebird nests fledged significantly more young than parasitized wattlebird nests, fledging success rate of koels in parasitized wattlebird nests was relatively low (26%, n = 38) (Abernathy and Langmore, 2017). One reason was due to koels laying eggs at inopportune times (too early or too late to be successful), which may have been exacerbated by the fact that wattlebirds tend to have small clutch sizes compared to the older hosts (1–2 eggs, as opposed to 3–5 eggs) and their incubation period is similar to the koel’s, making it more difficult for the koel nestling to hatch out before the wattlebird nestling in order to evict it from the nest. The study also points out that at least 41% of wattlebird pairs in Sydney avoided parasitism of at least one of their nests by initiating nesting before koels arrived to the area. Thus, parasitism rate and the impacts of koel parasitism on wattlebird breeding success appear to be low (Abernathy and Langmore, 2017). This, coupled with the extreme similarity in egg appearance between wattlebird and koel eggs could make egg ejection more costly to evolve in wattlebirds (Davies et al., 1996; Abernathy et al., 2017). We found evidence that the koel’s egg phenotype may have evolved as a way to mimic the appearance of the Noisy Friarbird egg, but not likely the Red Wattlebird egg, as 94% of friarbirds ejected spotted model eggs, but only 4% of wattlebirds ejected this same egg type.