Comparison of native and non‐native predator consumption rates and prey avoidance behavior in North America and Europe

Abstract Novel predator–prey interactions can contribute to the invasion success of non‐native predators. For example, native prey can fail to recognize and avoid non‐native predators due to a lack of co‐evolutionary history and cue dissimilarity with native predators. This might result in a competitive advantage for non‐native predators. Numerous lady beetle species were globally redistributed as biological control agents against aphids, resulting in novel predator–prey interactions. Here, we investigated the strength of avoidance behavior of the pea aphid (Acyrthosiphon pisum) toward chemical cues of native lady beetles and non‐native Asian Harmonia axyridis and European Coccinella septempunctata and Hippodamia variegata in North America, hypothesizing that cues of non‐native lady beetles induce weaker avoidance behavior than cues of co‐evolved native lady beetles. Additionally, we compared aphid consumption of lady beetles, examining potential predation advantages of non‐native lady beetles. Finally, we compared cue avoidance behavior between North American and European pea aphid populations and aphid consumption of native and non‐native lady beetles in North America and Europe. In North America, pea aphids avoided chemical cues of all ladybeetle species tested, regardless of their origin. In contrast to pea aphids in North America, European pea aphids did not avoid cues of the non‐native H. axyridis. The non‐native H. axyridis and C. septempunctata were among the largest and most voracious lady beetle species tested, on both continents. Consequently, in North America non‐native lady beetle species might have a competitive advantage on shared food resources due to their relatively large body size, compared to several native American lady beetle species. In Europe, however, non‐native H. axyridis might benefit from missing aphid cue avoidance as well as a large body size. The co‐evolutionary time gap between the European and North American invasion of H. axyridis likely explains the intercontinental differences in cue avoidance behavior and might indicate evolution in aphids toward non‐native predators.

predator species are similar then a similar response can be expected by prey, regardless of predator origin (Sih et al., 2010). Cue similarities between predator species can therefore lower the impact on prey densities compared to dissimilar non-native predators (Sih et al., 2010). In addition, cues of closely genetically-related species tend to be more similar compared to cues of distantly genetically related species, for example, chemical cues within lady beetle genera are more similar than between genera (Magro et al., 2010).
Originally of Palearctic origin, North American populations of pea aphids co-evolved in agricultural fields with native predators for over a century (Thomas, 1878).
Among the introduced lady beetle species, the European species Coccinella septempunctata and Hippodamia variegata have been released for biological control of aphids in North America (Angalet et al., 1979;Ellis et al., 1999). The earliest establishment of C. septempunctata in North America dates back to 1973 (Angalet & Jacques, 1975). The establishment history of H. variegata began in 1984 in North America (Gordon, 1987). Similarly, the Asian Harmonia axyridis was introduced as a biological control agent in North America and Europe (Tedders & Schaefer, 1994;Trouve et al., 1997).
The introduction of the Asian H. axyridis in North America started in 1916 (Gordon, 1985), but its earliest establishment was in 1988 (Chapin & Brou, 1991).
In Europe, H. axyridis was introduced in 1995 and the establishment period started in (Brown et al., 2011. Coccinella septempunctata and the Asian H. axyridis are relatively large and highly voracious compared to common native aphidophagous species (Elliott et al., 1996;Hoki et al., 2014;Ünlü et al., 2020).
Moreover, both species interfere with native trophic interactions associated with a lady beetle species decline in the non-native range, due to resource competition and intraguild predation (Alyokhin & Sewell, 2004;Ware et al., 2009), absence of natural enemies (Roy et al., 2011), high abundance (Horn, 1991;Koch, 2003) and high fecundity (Kajita & Evans, 2010) and are therefore classified as invasive species (Roy & Brown, 2015). The contribution of cue avoidance behavior of aphids confronted with non-native and native chemical lady beetle cues to the invasion success of non-native lady beetles remains to be examined.
In this study, we deployed a multi-species approach to compare differences in cue avoidance behavior of a North American population of pea aphid (Acyrthosiphon pisum) confronted with chemical cues of the Asian lady beetle species Harmonia axyridis, the European lady beetle species Coccinella septempunctata and Hippodamia variegata and three North American lady beetle species, Coleomegilla maculata, Coccinella novemnotata and Hippodamia convergens. Our species set consisted of two native and non-native species of the same genus Coccinella and Hippodamia, referred to as congeneric, expecting similarities between chemical cues (Magro et al., 2010).
In addition, we compared aphid consumption rates between all lady beetles tested. We hypothesized (a) missing avoidance behavior of

| Study species North America
The North American pea aphid Acyrthosiphon pisum (Harris)

| Study species Europe
The European pea aphid colony was obtained from the Julius-Kühn

| Cue avoidance experiments
Beetles were sexed prior to the experiments, to ensure a genderbalanced design (ten male and ten female beetles). Lady beetles were sexed by the morphological differences on the terminal sternites of females and males (Costopoulos et al., 2014;Harmon et al., 2008;Hurst et al., 1999;Nichols & Neel, 1974;Riddick & Schaefer, 2005;Stellwag & Losey, 2014). Double leaflets of Vicia faba were cut in two halves, one control and one treatment leaflet, and separately placed into round Petri dishes (circumference: 3.5 cm × height: 1.0 cm). A single lady beetle adult was placed on the treatment leaflet in the Petri dish for cue deposition (e.g., footprints, feces) and subsequently removed after 12 hr. The control leaflet remained without a lady beetle. The control and the treatment leaflet were randomly assigned and placed into the center of each half of a round Petri dish (circumference: 9.4 cm × height: 1.6 cm); the treatment leaflet was placed into one half of the Petri dish, while the control leaflet was placed into the opposite half.
Ten adult aphids were released into the center of each Petri dish, which were capped to prevent aphids from leaving the experimen-

| Predation experiments
Lady beetles were sexed by morphological differences prior to the experiments, accounting for potential intraspecific predation differences of female and male beetles. A single lady beetle adult was subsequently placed into a small round Petri dish (circumference: 3.5 cm × height: 1.0 cm) and starved for 24 hr prior to the experiment. Thirty pea aphids (second to third nymph stage) were counted and placed with a brush into a round Petri dish (circumference: 9.4 cm × height: 1.6 cm). A single lady beetle was randomly assigned to a Petri dish containing aphids, which was capped. Aphid predation was quantified by counting the remaining aphids in the Petri dish after 6 hr. In North America, we freeze-killed (−7 ± 1°C) lady beetle individuals after the experiments and measured body width (widest horizontal distance of closed elytra) and body length (elytral apex to pronotal apex) of all beetles used for the predation experiments under a stereomicroscope. We followed the procedure of  to obtain elliptical body area for individual beetles (body area (mm 2 ) = (π × 0.5 × body length (mm) × 0.5 × body width (mm)).
Overall, 20 replicates (ten females, ten males) were conducted per species, in the laboratory under 25.41 ± 0.19°C and artificial lightning. The predation experiments in Europe were identical, except that lady beetle individuals were randomly chosen and not sexed prior to the experiments.

| Statistical analysis
For the cue avoidance experiments in North America and the intercontinental comparison, aphid counts on each leaflet were analyzed as proportions (aphids on control leaf versus treatment leaf). We only considered aphids that made a distinct choice of control or the treatment leaflet. We applied a GLMM with a binomial error distribution to analyze differences between cue donator species identity (i.e., different lady beetle species) on aphid leaf choice. We included cue donator species identity as fixed effects and experimental unit (Petri dish identity) and an observation level random effect (OLRE) as random effects (to account for repeated measurements and overdispersion). We obtained statistical parameters for the fixed effects via ANOVA (χ2-test) from the R-package car (Fox & Weisberg, 2019). Pairwise differences between cue donator species identity were analyzed with a Tukey's contrast test for comparison of means with a Holm correction, to account for familywise error rates, using the glht-function from the multcomp R-package (Hothorn et al., 2008). We subsequently tested for equal distribution of aphids on control versus. treatment leaf (i.e., if aphids avoid lady beetle cues of the respective cue donator), by applying a GLMM with binomial error distribution. Our fixed effects included species identity and experimental unit (i.e., repeated measurements) and ORLE (accounting for overdispersion) as random effects.
Differences of predation rates in North America after 6 hr and body area were respectively analyzed with a Games-Howell post hoc test, following a Welch's ANOVA (F test) accounting for heteroscedasticity. To test the effects of lady beetle species identity, gender and body area on predation rates, we conducted a GLM with lady beetle species identity, gender and body area as fixed effects with a quasi-poisson error distribution. Statistical parameters for the fixed effects were obtained via ANOVA (χ2-test). The intercontinental predation differences between lady beetle species were analyzed with a Games-Howell post hoc test, following F I G U R E 1 Proportion of pea aphids on cue-free control leaflets (mean ± SE) in North America. Pea aphids avoided leaflets with chemical cues of the native lady beetle species (unfilled symbols) Coccinella novemnotata (C9), Coleomegilla maculata (C. mac), Hippodamia convergens (Hipc) and the non-native lady beetle species (filled symbols) Harmonia axyridis (Hax), Coccinella septempunctata (C7) and Hippodamia variegata (Hipv) (p ≤ .02, respectively) a Welch's ANOVA (F test). All statistical analyses were performed with the statistical software R, Version 3.4.0 (R Development Core Team, 2017).

| Cue avoidance in North America
Aphid leaf choice did not differ between cues of the different lady beetle species (GLMM; χ 2 = 5.80, df = 5, p = .33). Aphids avoided leaves previously occupied by all species (Figure 1; H
Avoidance behavior of European aphids was weaker when con- F I G U R E 2 Number of pea aphids consumed after 6 hr (mean ± SE) and body area (mm 2 ; mean ± SE) of native (unfilled symbols) and nonnative lady beetle species (filled symbols): Different uppercase letters indicate statistical differences of aphid consumption and different lowercase letters indicate statistical differences in body area between species based on a Games-Howell post hoc test (p < .05, same letters do not differ significantly). Dotted regression line (y = 0.8133x − 0.4305; R 2 = 0.54) shows the linear relationship between aphid consumption and body area American H. variegata (GH; p = .10). Coccinella septempunctata and H. axyridis consumption rates did not differ significantly in North America (GH; p = .97) and in Europe (GH; p = .94; Figure 4).

| D ISCUSS I ON
North American pea aphids avoided chemical cues of all lady beetle species tested, regardless of lady beetle origin. In contrast to North American pea aphids, European pea aphids did not avoid cues of the non-native H. axyridis. Consumption rates were strongly correlated with body size of lady beetles. On both continents, C. septempunctata and H. axyridis were the largest species tested and consumed the most aphids.
Pea aphids in Europe and North America avoided chemical cues of native lady beetles. These findings are in line with previous studies on predator-prey interactions showing that chemical cues of predators induce avoidance behavior in herbivores (Ninkovic et al., 2013;Seo et al., 2018;Ünlü et al., 2020). Chemical cues of predators can persist in the environment and indicate potential predation risk of a nearby predator (Kats & Dill, 1998). By avoiding sides with chemical cues of predators, prey can increase its survival (Lima & Dill, 1990). According to the "landscape of fear," prey shifts to sites with low predation pressure by avoiding sites with high predation risk (Laundré et al., 2001). A previous study on the avoidance behavior of the Asian citrus psyllid, Diaphorina citri, toward lady beetle trail chemicals demonstrated that chemical cues of lady beetles can not only interfere with the feeding activity of prey but additionally reduce oviposition (Seo et al., 2018). Another study confirms that prey can discriminate between risky and suitable feeding sides by demonstrating that the Colorado potato beetle, Leptinotarsa decemlineata reduces feeding on potato leaves covered with predator cues, compared to a cue-free control (Hermann & Thaler, 2014). Moreover, prior studies showed that disturbances in pea aphid behavior induced by predator cues can lead to increased searching behavior for suitable feeding sides and consequently decreased feeding times, resulting in reproductive costs (Nelson, 2007;Nelson et al., 2004).
However, predator cues, covering the surrounding area, can lead to aphid dispersal to sites with less predator pressure and consequently reduce survival costs through immediate predator consumption (Roitberg et al., 1979). Chemical cues deposited by lady beetles were trail chemicals and feces. According to Ninkovic et al. (2013), a similar avoidance response of aphids was observed, when feces were excluded from the experiments and only the trail chemicals of F I G U R E 3 Proportion of aphids on cue-free control leaflets (mean ± SE) in North America (unfilled symbols) and Europe (filled symbols). Asterisk indicates significant avoidance behavior of leaflets with chemical cues of lady beetle species (p < .05). Different letters indicate statistical differences between species based on Tukey's contrast test (p < .05) F I G U R E 4 Number of pea aphids consumed after 6 hr (mean ± SE) of lady beetle species in North America (unfilled symbols) and Europe (filled symbols): Different letters indicate statistical differences of aphid consumption of different lady beetle species based on a Games-Howell post hoc test (p < .05, same letters do not differ significantly) lady beetles were considered, suggesting that the effect of feces on the avoidance behavior of aphids is negligible (Ninkovic et al., 2013).
Thus, our findings suggest that chemical lady beetle cues can equally repel aphids and might subsequently serve as a signal for predation risk. Future field studies should target the effects of lady beetle cues on aphid dispersal and the reproduction of aphids, to shed light on the non-consumptive effects on aphid populations.
The avoidance of non-native H. axyridis cues by North American pea aphids might be explained by the strong selection pressure by non-native predators on native prey, leading to the evolution of antipredator behaviors (Carthey & Blumstein, 2018). In a maritime system, for example, the intertidal snail Littorina obtusata responded to the increasing predation pressure of the intertidal crab (Carcinus maenas), which was expanding its range, with rapid morphological change of shell forms (Seeley, 1986). Moreover, research on cue avoidance behavior in mammals showed that native common ringtail possum (Pseudocheirus peregrinus) recognized and subsequently avoided olfactory cues of the invasive European red fox (Vulpes vulpes), within a few generations of co-evolution (Anson & Dickman, 2013).In addition, our results show that H. axyridis is among the largest and most voracious predators tested; since large predators can pose a greater thread for prey, this can result in stronger antipredator responses by prey (see Binz et al., 2014).
The cue avoidance experiments in North America and Europe revealed differences in avoidance behavior toward cues of the non-native H. axyridis. In contrast to North America, the European pea aphids showed no avoidance behavior toward H. axyridis cues, indicating predator-prey naïveté (sensu Cox & Lima, 2006, Sih et al., 2010. North American and European pea aphid populations used for the experiments were maintained for more than 10 years in the laboratory and were expected to have experienced only low densities of established non-native lady beetle species in the field. However, pea aphids and non-native lady beetles coexisted prior to the establishment of non-native lady beetle species in North America (Gordon, 1985;Harmon et al., 2007). According to regional studies, H. axyridis was first reported in 1992 in Kentucky (Cottrell & Yeargan, 1998). In contrast, pea aphids have a longer history in Kentucky, for example, a study on the parasitation rate of the non-native parasitoid Aphidius smithi on pea aphids was conducted from 1967 to January 1970 confirming the pest status of pea aphids in agricultural environments (Pass & Parr, 1971). Although the regional co-occurrence between H. axyridis and A.pisum might not exceed 10 years, the migration history of non-native H. axyridis as well as pea aphids throughout North America must be considered (Brown et al., 2011;Lamb & MacKay, 1979), suggesting prior co-evolutionary history in agricultural sides beyond Kentucky. Specifically, in North America, H. axyridis was released in multiple agricultural landscapes starting in 1916 (Gordon, 1985) and was repeatedly reintroduced as a biological control agent to control agricultural pest species (Gordon, 1985;Lombaert et al., 2014;Tedders & Schaefer, 1994).
Thus, the frequency of predator-prey encounters and the length of co-evolutionary time since introduction can be decisive for the evolution of antipredator behaviors (Gérard et al., 2014;Nelson, 2007).
In North America, we suggest that North American pea aphid populations might have evolved cue avoidance behavior toward H. axyridis during the longer co-evolutionary time spend in shared agricultural fields (Gordon, 1985). In contrast, co-evolutionary history of A. pisum is shorter with H. axyridis populations in Europe, compared to that of North American populations (Brown et al., 2011;Gordon, 1985). In Europe, H. axyridis was introduced in the 1990s and the establishment period ranged from -2007(Brown et al., 2011Klausnitzer, 2002). The European aphid laboratory colony was established, when spread and establishment of H. axyridis started in Central Europe (Brown et al., 2011). Consequently, the European aphid colony has experienced low H. axyridis densities, if any. Our results thus provide a snapshot of initial interactions between pea aphids and H. axyridis in Europe. To enhance our comprehension of evolutionary changes in non-native predator-prey interactions, long-term studies are required, starting with the initial introduction of the non-native predator (Anton et al., 2020;Mallon et al., 2015).
The repelling substances within the chemical cues of lady beetles, inducing cue avoidance behavior in pea aphids, are unknown and remain to be identified. It might be possible that rather than evolutionary changes, cue similarities between chemical cues of H.
axyridis and native lady beetles might have resulted in avoidance behavior of A. pisum when H. axyridis was introduced to North America.
However, in Europe, chemical cues of the non-native H. axyridis were not avoided, indicating dissimilarity to cues of native lady beetle species. Thus, similarly to chemical cues of H. axyridis in Europe, chemical cues of H. axyridis might differ from cues of native lady beetle species in North America.
In North America, pea aphids avoided chemical cues of non-native C. septempunctata and H. variegata to a similar extend as cues of the congeneric native C. novemnotata and H. convergens. Chemical cue similarities between congeneric non-native C. septempunctata and H. variegata and native C. novemnotata and H. convergens in North America might contribute to the equally strong avoidance behavior in pea aphids. Here, cues of non-native and native lady beetle species of the same genera can be similar (Magro et al., 2010) and consequently a similar response can be expected in prey, regardless of predator origin (Sih et al., 2010).
However, the degree of cue similarity between the tested lady beetle species remains open and needs further attention. In addition, according to the "multipredator hypothesis," prey retains evolved antipredator behaviors toward extinct predators in the presence of remaining predators (Blumstein, 2006). Pea aphids in Europe avoided chemical cues of the co-evolved C. septempunctata and H. variegata to a similar extent, as pea aphids in North America.
Thus, in North America chemical cue avoidance toward non-native European C. septempunctata and H. variegata might be a retained innate antipredator response of pea aphids, which evolved prior to the introduction of pea aphids to North America in the beginning of the 19th century and remained due to the presence of congeneric native predators with potentially similar cues (e.g., C. novemnotata and H. convergens). Overall, the "multipredator hypothesis" in combination with chemical cue similarities of congeneric species might explain cue avoidance of congeneric non-native C. septempunctata and H. variegata and native C. novemnotata and H. convergens in North America, as well as the similar strength of avoidance behavior between European and American pea aphids.
Still further studies are required to evaluate retained antipredator behaviors in non-native insect species beyond their native range.
Next to H. axyridis, C. septempunctata was the largest and most voracious non-native lady beetle species in North America tested.
The regional co-occurrence of pea aphids and C. septempunctata used in the experiments might exceed 10 years, since, for example, Buchele et al. (1992) reported C. septempunctata appearing in research plots in Kentucky in 1987. Similar to H. axyridis, the introduction and establishment of C. septempunctata in North America dates decades back (Angalet & Jacques, 1975;Kajita et al., 2012).
This alternatively suggests that avoidance behavior of pea aphids toward chemical cues of C. septempunctata might have newly evolved in North America, due to the increased predation pressure by this large and voracious predator (see Binz et al., 2014).
In North America, the non-native C. septempunctata and H. axyridis were the largest lady beetles tested and consumed the most aphids, compared to smaller lady beetle species. Food consumption increases with body mass, due to increasing metabolic requirements (Brose et al., 2008), confirming the positive relationship between lady beetle body area and aphid consumption rates. The successful establishment of a large predator can depend on, that is, predator size and prey availability (Crookes et al., 2019). In North America, C. septempunctata was primarily considered as a non-native biological control agent on pest species, due to large size and voraciousness (Elliott et al., 1996). Predation advantages of invasive H. axyridis over the smaller native lady beetle species Cycloneda sanguinea on shared pest species were attributed to a dominance in intraguild interactions, wider dietary range, higher voracity, and larger size (Michaud, 2002). Moreover, we found that the smaller sized, non-native H. variegata consumes a lower number of aphids, compared to C. septempunctata and H. axyirids in North America. Thus, asymmetric competition advantages over smaller native and non-native species can benefit the larger non-native H. axyridis on both continents and non-native C. septempunctata in North America (Hoki et al., 2014;Michaud, 2002). Furthermore, a recent study found that the efficiency of resource utilization was comparatively higher in invasive H. axyridis than in native H. convergens, when allometric scaling was considered. In addition, aphid handling time was lower and maximum consumption rate was higher in H. axyridis, compared to native H. convergens, indicating that the invasive H. axyridis is the dominating competitor (Crookes et al., 2019). Additionally, C. septempunctata and H. axyridis are both successful intraguild predators of native coccinellids in their invaded range (Pell et al., 2008;Snyder et al., 2004), which is not only beneficial in dietary terms, but also reduces competition on shared resources (Yasuda et al., 2004, Dixon andDixon, 2000). Overall, body size and correlated physiological and/ or behavioral traits of invasive species can significantly contribute to a competition advantage toward native and non-native predators (Hemptinne et al., 2012;Kajita & Evans, 2010;Michaud, 2002; Gordon, 1985, Lost Ladybug Project 2020: www.lostl adybug.org). Among other characteristics, we argue that a large body size can contribute to the invasion success of lady beetles due to increased food demands and thus stronger competition. Analyzes of a 24-year dataset in southwestern Michigan revealed only significant declines in the relatively small Col. maculata and A. bipunctata (Bahlai et al., 2015). Likewise, A. bipunctata showed stronger declines in the presence of H. axyridis compared to other native lady beetle species, in Europe (Roy et al., 2012). In contrast, C. novemnotata maintained an ecological foothold in the face of invasion by the equally sized C. septempunctata (Evans, 2017). Thus, we suggest that lady beetle body size might be a good predictor for their invasion potential in areas beyond their native ranges.
In contrast to body size, differences in predator avoidance can diminish with time. Here, North American aphids but not European aphids avoided cues of the Asian H. axyridis. Such evolutionary adaptations can contribute to so called "boom-bust dynamics": invaders go through an initial outbreak before declining to a lower population size (Simberloff & Gibbons, 2004;Strayer et al., 2017). So far, evidence for a decline in H. axyridis populations is restricted to microsatellite effective population estimates (Sethuraman et al., 2018). In the long term, the adaptations of antipredator behaviors by aphids toward non-native predators might result in a stable co-existence within the native community and may consequently lead to a more harmless situation relative to the current impact of H. axyridis.
Evolutionary responses to non-native predators have important consequences for ecological studies aiming to elucidate the underlying mechanism of biological invasion such as a lack of avoidance behavior: Although lacking avoidance behavior toward non-native predators during early stages of biological invasions benefits the non-native predator, the mechanisms might no longer be detectable at later stages due to co-evolution. Our results in concert with lady beetle distribution data in North America and in Europe indicate that relative lady beetle body size is a key predictor of the invasion success of non-native lady beetle species, but also for native lady beetle species that are at particular risk if they co-occur with non-native lady beetles.

| CON CLUS ION
Missing avoidance behavior of European pea aphids toward chemical cues of non-native H. axyridis suggests that non-native predators can benefit from chemical cue novelty resulting in a lack of antipredator behavior of prey, during early stages of biological invasions. In contrast, North American pea aphids showed avoidance behavior toward H. axyridis cues, suggesting adaptations of avoidance behavior against voracious, non-native predators.
Overall, predation advantages of non-native predators due to missing antipredator behaviors of prey might diminish with time, whereas body size-related competition advantages over smaller native and non-native predators could sustainably benefit large, non-native predators.

ACK N OWLED G M ENTS
We thank Nathan Mercer, Denise Bertleff, Jette Diekmann, and Sophia Brand for help with the maintenance and collecting of insects and the assistance with the experiments; the Department of Entomology at the University of Kentucky for the supply with technical equipment. We are grateful to the Julius Kühn-Institut (Braunschweig, Germany) for providing pea aphids. We are thankful to the two anonymous reviewers for their valuable comments on earlier versions of the manuscript. The study was funded by the Deutsche Forschungsgemeinschaft (DFG) under grant number BU3382/1-1 to RB. Open access funding enabled and organized by Projekt DEAL.

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
We archived our data on the Dryad data repository: https://doi. org/10.5061/dryad.47d7w m3bt.