See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/284805770

Potential for exploitative competition, not intraguild predation, between invasive harlequin ladybirds and flowerbugs in urban parks

Article in Biological Invasions · November 2015 DOI: 10.1007/s10530-015-1024-y

CITATIONS READS 11 140

4 authors, including:

Andy G Howe Christian Bressen Pipper University of Copenhagen LEO Pharma

23 PUBLICATIONS 286 CITATIONS 145 PUBLICATIONS 3,142 CITATIONS

SEE PROFILE SEE PROFILE

Alex Aebi Université de Neuchâtel

123 PUBLICATIONS 1,363 CITATIONS

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Teaching, supervising, project development, writing! View project

Modelling grouped survival data View project

All content following this page was uploaded by Alex Aebi on 23 February 2016.

The user has requested enhancement of the downloaded file. Potential for exploitative competition, not intraguild predation, between invasive harlequin ladybirds and flowerbugs in urban parks

Andy G. Howe, Hans Peter Ravn, Christian Bressen Pipper & Alexandre Aebi

Biological Invasions

ISSN 1387-3547 Volume 18 Number 2

Biol Invasions (2016) 18:517-532 DOI 10.1007/s10530-015-1024-y

1 23 Your article is protected by copyright and all rights are held exclusively by Springer International Publishing Switzerland. This e- offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”.

1 23 Author's personal copy

Biol Invasions (2016) 18:517–532 DOI 10.1007/s10530-015-1024-y

ORIGINAL PAPER

Potential for exploitative competition, not intraguild predation, between invasive harlequin ladybirds and flowerbugs in urban parks

Andy G. Howe . Hans Peter Ravn . Christian Bressen Pipper . Alexandre Aebi

Received: 31 December 2014 / Accepted: 19 November 2015 / Published online: 25 November 2015 Ó Springer International Publishing Switzerland 2015

Abstract In aphidophagous communities nemoralis DNA. The presence of lime DNA in invaded by the harlequin ladybird Harmonia axyridis predators was higher: 56.5 and 47.9 % of H. axyridis Pallas (Coleoptera: ), intraguild preda- larvae and adults, respectively, contained E. tiliae tion (IGP) is widely implicated in the displacement of DNA, whereas 60.8 % of adult A. nemoralis tested native predators, however, indirect trophic interac- positive for aphid DNA. Incorporating insect densities tions are rarely assessed. Using molecular gut-content revealed that the density of H. axyridis larvae had a analysis, we investigated the relative frequencies of strong negative effect on the likelihood of detecting IGP by H. axyridis on the predatory flowerbug aphid DNA in A. nemoralis. Prey overlap for E. tiliae nemoralis Fabricius (Heteroptera: Antho- was widespread in space (2–13 m height in tree coridae) and prey overlap for a shared prey, the lime crowns) and time (May–October 2011) which sug- aphid tiliae L. (: ), gests that interspecific exploitative competition, medi- in 9 europaea crowns in urban parks. The ated by predator life-stage, more so than IGP, is an frequency of IGP by H. axyridis was low: 2.7 % of important indirect trophic interaction between co- larvae and 3.4 % of adults tested positive for A. occurring H. axyridis and A. nemoralis. Whether exploitative competition equates to displacement of A. nemoralis populations requires further investigation. Electronic supplementary material The online version of Our results emphasize the need to incorporate indirect this article (doi:10.1007/s10530-015-1024-y) contains supple- mentary material, which is available to authorized users. interactions in studies of insect communities follow- ing invasion, not least because they potentially affect A. G. Howe (&) Á H. P. Ravn more species than direct interactions alone. Department of Geosciences and Natural Resource Management, University of Copenhagen, Rolighedsvej 23, 1958 Frederiksberg C, Denmark Keywords Indirect effects Á Molecular gut content Á e-mail: [email protected] Anthocoris nemoralis Á Harmonia axyridis Á Eucallipterus tiliae Á Predator–prey interactions C. B. Pipper Section of Biostatistics, Department of Public Health, University of Copenhagen, Øster Farimagsgade 5, 1014 Copenhagen K, Denmark Introduction A. Aebi Laboratoire de Biologie du Sol, Institut de biologie, Universite´ de Neuchaˆtel, Emile-Argand 11, Invasive generalist insect predators are typically 2000 Neuchaˆtel, Switzerland involved in complex ecological interactions with 123 Author's personal copy

518 A. G. Howe et al. members of invaded communities, often resulting in studies employing molecular gut-content techniques negative impacts on resident native species (Snyder reveal IGP of native coccinellids by invasive H. and Evans 2006; Crowder and Snyder 2010). As axyridis is spatially widespread and relatively intense, generalist invaders often achieve high densities in providing support that this mechanism contributes to introduced communities, and given the relatively short native species declines (Gagnon et al. 2011b; Brown time periods over which invasions occur, trophic et al. 2015). interactions might be particularly vulnerable to change Interspecific exploitative competition involves an by invaders with potential effects extending beyond indirect interaction between two species mediated single trophic levels (Snyder and Evans 2006; Kenis through changes in abundance of a third species et al. 2009). Within trophic communities species are (Lawton and Hassell 1984; White et al. 2006). An linked through a myriad of direct and/or indirect invasive generalist competitor may be superior at trophic interactions, most of which are not readily utilising a limited shared prey through better har- perceptible (White et al. 2006; Tylianakis 2008). By vesting ability and/or resource conversion efficiency, way of their omnivorous diets, invasive generalist and reduced density of the limiting resource can predators interact with multiple trophic levels in food result in indirect negative effects on native species, webs; not only do they consume herbivorous prey, but e.g. reduction in fecundity, growth or survival also other predators, detritivores, plants and/or detritus (Brown et al. 1994; Obrycki et al. 1998; Kasper may comprise parts of their diet (Polis and Strong et al. 2004; Evans et al. 2011). Resource competition 1996; Snyder and Evans 2006). among aphidophages is likely to be widespread due An invasive generalist predatory beetle implicated to the ephemeral distribution of in space and in disruption of trophic interactions is Harmonia time (Hironori and Katsuhiro 1997; Dixon 1998; axyridis Pallas (Coleoptera: Coccinellidae). Origi- Osawa 2000; Ware et al. 2009). Predator niche nally introduced to North America as a biological overlap for shared prey involving H. axyridis pop- control agent (BCA) against aphids and coccids ulations in invaded communities has received limited (Chapin and Brou 1991; Brown et al. 2011b), long- attention (Pell et al. 2008; Kenis et al. 2010), despite term studies from North America have documented that niche overlap can contribute to native species coccinellid declines in agroecosystems (Michaud displacement (Kasper et al. 2004). On the other hand, 2002; Alyokhin and Sewell 2004; Harmon et al. spatial and temporal niche partitioning of competitors 2007), while the spread of H. axyridis in Europe has may reduce effects of interspecific interactions impinged several native coccinellids whose niches involving invasive generalist predators, thereby con- overlap (Adriaens et al. 2008; Brown et al. 2011a; Roy tributing to coexistence (Amarasekare 2003; Crowder et al. 2012). Intraguild predation (IGP) and resource and Snyder 2010). competition by H. axyridis are suspected of contribut- The predatory insect Anthocoris nemoralis F. ing to native species declines (Michaud 2002; Lucas (Heteroptera: ) is common throughout et al. 2007; Alhmedi et al. 2010; Roy et al. 2012). Western Europe (Pe´ricart 1996), and tracks prey on However, while studies of IGP by H. axyridis among leaf surfaces of deciduous trees (Anderson 1962; native coccinellids in agroecosystems have received Lattin 1999; Sigsgaard 2010). Both H. axyridis and A. most attention, indirect trophic interactions such as nemoralis are found on common lime Tilia 9 eu- exploitative and apparent competition have barely ropaea L., Malvaceae (Anderson 1962; Adriaens et al. been explored in the field (Alhmedi et al. 2010; Roy 2003; Brown et al. 2008), which also hosts the and Handley 2012). monophagous aphid and dominant herbivore Eucal- Intraguild predation occurs when predators sharing lipterus tiliae L. (Hemiptera: Aphididae; Dixon a common extraguild (EG) prey engage in predation of 1971a). Eucallipterus tiliae (EG prey; Fig. 1)is each other (Polis et al. 1989), and is often associated shared prey for H. axyridis (IG predator; Fig. 1) and with native species displacement (Reitz and Trumble A. nemoralis (IG prey; Fig. 1), and important for pre- 2002). Laboratory studies confirm that H. axyridis is a overwintering reproduction in A. nemoralis (Anderson strong asymmetric intraguild (IG) predator in encoun- 1962; Tomov et al. 2009). ters with other aphidophages, i.e. intraguild (IG) prey Laboratory investigations of interspecific interac- (for recent summary see Nedved et al. 2013). Field tions between H. axyridis and A. nemoralis revealed a 123 Author's personal copy

Potential for exploitative competition, not intraguild predation, between invasive 519

densities of H. axyridis and low E. tiliae densities. Harmonia axyridis Only H. axyridis adults and larvae (4th instar) were (IG predator) collected for DNA-gut analysis during 2009. In 2011,

Aphidophage five T. 9 europaea in 5 urban parks were sampled 1–3 guild times per park, between May and October 2011, in total 10 sampling dates (Table 2), which covered Anthocoris nemoralis predators’ active period. Both insect community (IG prey) densities and trophic interactions were assessed in tree crowns in 2011. As there was at least a month Eucallipterus tiliae between repeated collections from the same parks, (EG prey) samples were considered to be independent. Fig. 1 Conceptual diagram of species and trophic interactions Bottom-up sampling was conducted to assure that in Tilia 9 europaea crowns in urban parks in Copenhagen, higher up in tree crowns were not disturbed by Denmark. The aphidophage guild comprises invasive Harmonia sampling at lower heights. Within trees, insects were axyridis [intraguild (IG) predator] and A. nemoralis [intraguild sampled at three heights, low (up to 2.5 m), mid (IG) prey], with E. tiliae their shared [extraguild (EG) prey]. Direct trophic interactions solid lines; indirect interaction (5–7 m) and high (10–13 m), although on three dates dashed lines only two heights were sampled (Table 2). Collection heights were based on tree architecture and limitations low level of IGP by coccinellids in potted Tilia of the access technique (single rope technique) cordata Mill. (Malvaceae) microcosms, and a nega- relating to responsible, safe and non-destructive tree tive effect on A. nemoralis weight gain through climbing. competition for E. tiliae (Howe et al. 2015). We were Densities of insects other than aphids were assessed interested in assessing the relative strengths of trophic with a white fabric beating-tray (75 cm 9 75 cm) interactions under field conditions and whether preda- mounted to a telescopic aluminium pole (Ronstan tor niche overlap could explain interaction strengths. Battle stick-tiller extender, Australia) which allowed We hypothesised (1) that IGP of A. nemoralis by H. 4 m extension, adequate to obtain samples from the axyridis is limited on T. 9 europaea in urban parks, outer crown in trees. Ten branches at different orien- but occurs more frequently as E. tiliae populations tations in crowns at each height (in 2009 only at low decline. Furthermore, as predators on T. 9 europaea height), were hit 5 times per branch with a light-weight, share a limiting aphid prey, (2) that interspecific rubber-ended metal pole (2.5 m long). Insects other competition is potentially a more important trophic than aphids were counted and sorted immediately, interaction than IGP as spatiotemporal niche overlap within crowns, for subsequent identification/analysis. for prey occurs in T. 9 europaea crowns. By sampling Aphids were sampled by inspecting 50 leaves at each in tree crowns we hoped to increase the spatial area in height, and wherever possible, leaves in the outer crown which to detect trophic interactions, while also were included by bending branches to within a safe illuminating the spatial distribution of arboreal working distance. Adults included only winged indi- species. viduals (i.e. parthenogenetic virginoparae and males), while nymphs were apterous, but could have included ovipare wingless females (Dixon 1971b). Materials and methods On occasions when no predators were recovered using the aforementioned beating procedure, extra Field collection and assessment of insect density beating was employed to attain predators for subse- quent DNA gut-content analysis. Predators to be used In 2009, Tilia 9 europaea was sampled in seven urban for gut-content analysis were immediately placed parks and green spaces on six dates from October 2 to individually into 1.5 mL Eppendorf tubes filled with 22 [green spaces are situated between 55°47016.600N chilled 85 % EtOH (in 2009 insects were stored dry). 12°29039.100E and 55°40008.500N12°25032.800E; see Individuals were either scooped from the net directly Electronic supplementary material (ESM) for further into Eppendorf tubes to minimise potential contami- descriptions]. This period coincided with high nation between samples, or were transferred by pooter 123 Author's personal copy

520 A. G. Howe et al. for fast-moving species. After collection in trees, testing positive for successful extraction were used for specimens were transferred to an iced cooler box the assays. (5–8 °C) and shaded in the field for 3–6 h before being frozen at -20 °C, until subsequent morphological Primer design identification and DNA analysis. Previously, H. axyridis in Japan has been shown to feed between Following morphological identification of insects 1000 and 1600 hours (Miura and Nishimura 1980). In collected from lime trees (Table 1, ESM Table 1) 2009, collection adhered to this period; during 2011 with keys (Southwood and Leston 1959) and valida- the period was extended from 1000 to 1800 hours to tion by a national Heteroptera expert, DNA was allow for the time demands associated with tree extracted from 1 to 2 individuals of each species (as for climbing. DNA extraction). COI sequences were amplified with the LepF/LepR primer pair (PCR conditions as for Molecular DNA-gut analysis DNA extraction) and shipped to Macrogen (the Netherlands, Europe) for purification and sequencing A PCR-based detection system was developed for in both directions using the same primer pair. primer pairs specific for A. nemoralis and E. tiliae Target primers were designed for mtDNA because mitochondrial DNA (mtDNA) from the cytochrome thousands of mtDNA copies in an invertebrate cell oxidase subunit I (COI) gene. We followed the enhance sensitivity and probability of amplifying procedure described by Aebi et al. (2011, Fig. 1 DNA from a target prey species (Hoy 1994; King therein) to test primer specificity and post-feeding et al. 2008). Primers were designed to amplify detection times. See ESM for details of primer amplicons \150-bp in order to optimise detectability specificity tests. of semi-digested DNA and to contain as high a GC content as possible (King et al. 2008; Aebi et al. 2011). DNA extraction We used Geneious (version 5.5, Biomatters, 2011) to edit sequences, Bioedit for sequence alignment (http:// All stages of H. axyridis, adult Anthocoris and www.mbio.ncsu.edu/bioedit/bioedit.html), designed reference insect species for specificity tests (see primers by sight based on sequence differences in ESM Table 1) were briefly dried of EtOH on paper alignments, while Primer 3 was used to assess primer towel, before being transferred individually to a new properties (Koressaar and Remm 2007; Untergasser 1.5 mL Eppendorf tube containing 180 lL of PBS et al. 2012). Morphological identification of species buffer, and homogenised using a sterile plastic micro- was verified using the BOLD identification system for pestle. DNA was extracted using a DNEasy Blood & COI (Ratnasingham and Hebert 2007). Tissue Kit (Qiagen, Denmark) following the manu- facturer’s specifications. PCR amplification with target primers The success of all extractions at all stages of the study was tested using the LepF/LepR primer pair PCR reactions (25 lL) contained 5 lL59 Promega (Foottit et al. 2008) which amplifies an approximately Green Flexi Buffer, 0.5 lLofdNTPs(200lM each),

700 bp fragment of mtDNA from the COI gene. Each 2 lLof2mMMgCl2,0.35lLofprimer(0.14lM PCR (25 lL) contained 1.25 U of Promega Taq forward/reverse), 1.25 U Promega Taq-polymerase, and polymerase (0.125 lL, Promega, Sweden), 59 Green 14.675 lL MilliQ H20 in addition to 2 lL of raw DNA. Flexi buffer (5 lL, Promega), 200 lM of each dNTP For A. nemoralis primers, 5 lL Q-solution (Qiagen,

(0.5 lL), 2 mM MgCl2 (2 lL), 0.14 lM each primer Denmark) was added per reaction with H20reduced (0.35 lL) and 14.675 lL MilliQ H2O and 2 lLof accordingly. The optimal primer specific thermocycle extracted DNA. Thermocycling conditions followed times were: for A. nemoralis (primer pair Ans-F2/R2), Hebert et al. (2003). PCR was verified with elec- 95 °C for 90 s initial denaturation was followed by 20 trophoresis of 7 lL of product in 1.5 % agarose in touchdown cycles of 95 °C for 30 s, 61–51 °C for 60 s 0.59 TBE buffer run at 65 V for up to 90 min. DNA (0.5 °C reduction per cycle), 72 °C for 30 s; followed by was visualised with GelRed (Biotium, USA) and 20 cycles at 95 °C for 30 s, 51 °C for 60 s and 72 °Cfor photographed on a UV transilluminator. Only samples 30 s and concluded with 72 °C for 10 min; for E. tiliae 123 Author's personal copy

Potential for exploitative competition, not intraguild predation, between invasive 521

Table 1 Densities of non-aphid insects (per 50 hits) and E. tiliae (per 50 leaves), sampled over 10 dates during 2011 in 5 Tilia 9 europaea crowns in inner city parks in Copenhagen, Denmark Family Species Height in canopy (m) No. dates species was sampled 2.5 5–6 10–13 (10 max.) Mean SE Mean SE Mean SE

Aphididae Eucallipterus tiliae L. 17.30 8.19 3.88 1.11 2.87 0.67 10 E. tiliae (nymph) 82.3 38.41 54.33 24.70 29.62 11.24 10 Anthocoridae Anthocoris nemoralis Fabricius 1.8 0.74 2 1.56 0.62 0.50 7 Coccinellidae Harmonia axyridis Pallas 3.40 0.67 2.88 0.63 4.62 2.01 9 H. axyridis (larva) 1.4 0.64 3.22 1.05 0.50 0.38 9 Adalia bipunctata (L.) 0.2 0.2 0.37 0.26 3 A. decempunctata (L.) 0.3 0.21 0.11 0.11 3 Calvia quattuordecimguttata (L.) 0.1 0.1 0.25 0.25 2 Exochomus quadripustulatus (L.) 0.1 0.1 1 Native coccinellids (total) 0.70 0.40 0.11 0.11 0.62 0.32 Miridae Blepharidopterus angulatus (Fallen) 0.10 0.10 0.55 0.55 2 Neolygus viridis (Fallen) 0.9 0.55 0.62 0.62 3 Deraeocoris flavilinea (Costa) 0.50 0.40 1.22 0.66 0.25 0.25 3 D. lutescens (Schilling) 0.50 0.50 1 Phytocoris tiliae (F.) 0.20 0.13 0.11 0.11 2 Mirids (total) 2.2 0.68 1.89 0.77 0.87 0.64 Number of collections per height: up to 2.5 m (10), 5–6 m (9), 10–13 m (8). Numbers denote adult densities unless stated otherwise

(primer pair Eti-F5.1/R2), 95 °C for 90 s was followed ingesting one E. tiliae adult (see ESM for starvation by 45 cycles of 95 °C for 30 s, 54 °C for 60 s and 72 °C times of predators prior to being offered prey). for 30 s prior to final elongation at 72 °C for 10 min. For Predators were offered prey in 1.5 mL Eppendorf both pairs of primers 35 cycles did not produced tubes and observed for an hour until ingestion which sufficient amplification of target DNA and the number was noted to the nearest quarter hour. Six to eight H. of cycles was therefore increased. Four controls were axyridis were frozen as soon as ingestion was included in all PCR reactions: a negative control of the complete (time = 0), whereas for A. nemoralis specific laboratory raised predator (H. axyridis or A. ingestion lasted up to an hour (time = 1). Thereafter nemoralis)fedEphestia kuehniella (Zeller); a negative all predators were transferred to Petri dishes kept in blank with MilliQ H2O substituting DNA; and two climate chambers (20 ± 1 °C, 16:8 L:D and RH positive target prey DNA controls in 1:1 and 1:100 50 ± 20 %). Initially, H. axyridis adults offered E. dilution (E. tiliae: predator) with the appropriate preda- tiliae were given up to 24 h digestion time (follow- tor. PCR was verified as for DNA extractions success, ing Gagnon et al. 2011a), which was not long using a 100 bp DNA ladder. enough to calculate a prey detectability half-life; unfortunately we were unable to extend digestion Post-feeding detection period of prey times beyond 24 h for this combination. However, remaining combinations were given 2, 4, 6, 8, 24 To assess prey detectability times the following four and 36 h to digest prey (see ESM Table 2). At each predator–prey combinations were investigated: 4th time interval 6 live individuals were transferred to a instar H. axyridis larvae ingesting one 2nd instar A. freezer (-20 °C), until PCR of six H. axyridis from nemoralis nymph; 4th instar H. axyridis larvae each digestion time tested for prey DNA digestion. ingesting one E. tiliae adult; H. axyridis adults Six A. nemoralis were tested as for ladybirds, except ingesting one E. tiliae adult and; A. nemoralis adults at times t = 1(n = 5), t = 6(n = 5), t = 8(n = 5)

123 Author's personal copy

522 A. G. Howe et al.

and t = 36 (n = 4). Despite starving A. nemoralis alone to account for potentially different DS50 values for 48 h, we were unable to achieve greater sample (between life stages and predators). sizes due to their reluctance to reliably ingest prey For A. nemoralis, we analysed the explanatory and the constraints this places on available fixed effects: collection height (3 levels), densities resources. See ESM for further details of post- of E. tiliae (adults and nymphs separately), total feeding detection periods and cross-reactivity spec- density of predatory insects (i.e. potential competi- imens (n = 26; ESM Table 1). tors: mirids, coccinellids, A. nemoralis, H. axyridis larvae and adults), densities of H. axyridis larvae Data analysis and adults (separately), and the sex of adult A. nemoralis.ForH. axyridis, explanatory fixed effects Prey detectability half-lives and weighting prey included: collection height, densities of E. tiliae detectability (adults and nymphs separately), densities of mirids, coccinellids and A. nemoralis, total density of

Prey detectability half-lives (DS50, i.e. the time post- predatory insects, and the life stage of H. axyridis feeding at which prey remains are detected in half the (two levels, larva/adult); the latter removed in the assayed predators (reviewed by Greenstone et al. larvae only model. 2014) were calculated using probit regression model Random effects were location and location at a parameters (i.e. -b0/b1) from individual predator– given date to account for heterogeneity between prey digestion periods (following Chen et al. 2000). sampling locations. Model reduction was based on

As mentioned, DS50 value for H. axyridis adults fed E. backwards elimination with a 5 % cut-off value. tiliae was not calculated; for an overview of DS50 P values correspond to likelihood ratio tests (LR). values (see Table 3, ESM Table 2). Standard errors of Effects are recorded as odds ratios accompanied by

DS50 values were estimated using the delta method 95 % confidence intervals in brackets. with the msm package in R (Jackson 2011), while Correlations between aphid, H. axyridis and A. predator DS50 values were compared using Pearson’s nemoralis densities were investigated using Pearson Chi square statistic with Yates continuity correction. correlation coefficients on raw data, while poisson To account for potential differences in prey detectabil- regression accompanied by post hoc pairwise com- ity between predators which may lead to biased parisons (adjusted for multiple testing with the single- interpretations of raw qualitative gut-content data step method) assessed whether species’ densities

(Greenstone et al. 2014), we calculated weighted DS50 differed with height. values and subsequently corrected field-based IGP and In 2009, no explanatory variables were recorded; aphid predation values. therefore only positive detection and weighted values of IGP and E. tiliae DNA in H. axyridis is reported Likelihood of detecting E. tiliae DNA in predators (data not analysed). Statistical analysis was conducted using R, version In 2011, only five positive IGP events occurred in 3.0.0 (R Core Team 2013). GLMMs were fitted using 2011 which precluded statistical analysis. In contrast, the glmer function in the lme4 package (Bates et al. predator gut content data (presence/absence E. tiliae 2014), multiple comparisons with the multcomp DNA) reflecting prey overlap were analysed sepa- package (Hothorn et al. 2008), and figures were rately for each predator. The likelihood of detecting E. produced with ggplot2 (Wickham 2009). tiliae DNA was analysed by logistic regression with random effects (GLMM). In both models, raw response variables (presence/absence E. tiliae DNA) Results were analysed, because DS50 values for digestion of E. tiliae did not reveal differences between predators. Predatory insects and aphids in tree crowns

However, as we did not calculate a DS50 value for H. axyridis adults digesting E. tiliae, we analysed the Within inner city parks, Tilia 9 europaea crowns to likelihood of detecting aphid DNA for H. axyridis life 13 m contained a host of predatory insects, includ- stages combined (i.e. larvae and adults) and for larvae ing several members of the aphidophage guild 123 Author's personal copy

Potential for exploitative competition, not intraguild predation, between invasive 523

(Table 1). Native coccinellids were not abundant There was a marginal significant positive correla- with only 4 species found, while 5 species of mirids tion for densities of E. tiliae nymphs and H. axyridis were caught. Harmonia axyridis (larvae and adults) larvae (r = 0.39, P = 0.04). Stronger positive linear accounted for 59 % of all predators caught, while A. relationships were revealed for E. tiliae nymphs and A. nemoralis was the next most abundant predator nemoralis (r = 0.654, P = 0.0002), and densities of (17 %). In tree crowns, A. nemoralis and H. axyridis A. nemoralis and H. axyridis larvae (r = 0.519, were caught simultaneously on the same date and P = 0.005). height, but not after 20 September (Table 2). Densities of predators were similar between heights, Detectability half-lives and weighting prey suggesting homogenous distribution throughout tree detectability of field-caught predators crowns (pairwise comparisons between H. axyridis adults and larvae, A. nemoralis, coccinellids and The primer pair Ans-F2 (50-GAATGACAGGAGT- mirids: P [ 0.05). TATTTTAGC-30)/Ans-R2 (50-GTGGAAGTGTGC- Aphid nymph and adult densities varied across TACTACG-30) was specific for a 79 bp sequence of sites, dates and heights in trees (Table 1; Fig. 2). Anthocoris nemoralis mtDNA, while aphid predation Aphid nymph densities ranged between 0.09 and 6.20 was detected using the primer pair Eti-F5 (50-T aphids/leaf (mean ± SE: 1.38 ± 0.59 aphids/leaf), TTCTTATTAATAATGGTACAGG-30)/Eti-R2.1 (50- while aphid adults densities ranged from 0.02 to 1.74 TGAGATTCCTGCTAAATGTAGC-30), specific for aphids/leaf (mean ± SE: 0.52 ± 0.16). Both aphid a 134 bp amplicon of E. tiliae mtDNA. Primer pairs adult and nymph densities generally decreased with were specific for target DNA when tested against 26 height (pairwise comparisons: P \ 0.01, except adults insect species occurring on Tilia spp. (ESM Table 1). which had similar densities at mid and high positions: Detectability half-lives decreased significantly with P [ 0.05), although this could reflect the physical time post-digestion for all combinations except H. difficulties associated with counting aphids on leaves axyridis adults fed E. tiliae which could not be in the outer upper crown, more so than the actual calculated (Table 3, ESM Table 2). The DS50 for distribution of aphids. detection of A. nemoralis in H. axyridis larvae (5.6 h)

Table 2 Number of H. axyridis and A. nemoralis testing (Tilia 9 europaea) in five urban parks in Copenhagen, Den- positive for E. tiliae DNA in 2011 at 3 heights (low: up to mark: a = Churchillparken, b = Ørstedsparken, c = Gen- 2.5 m; mid: 5–7 m; high: 10–13 m), in five trees foreningspladsen, d = Bispebjerg Parkalle´,e= Østre Anlæg Height/date H. axyridis (larva) H. axyridis (adult) A. nemoralis (adult) Low Mid High Low Mid High Low Mid High

31/05a 0 (2) 3 (7) 0 (0) 1 (2) 1 (2) 0 (0) 2 (2) 0 (0) 0 (0) 06/06b 8 (13) 3 (6) – 0 (1) 2 (2)* – 1 (2) 0 (0) – 20/06c 1 (1) – 0 (0) 1 (2) – 0 (0) 5 (5) – 0 (0) 22/06b 2 (2) 7 (11) – 3 (5) 2 (3) – 3 (6) 8 (11) – 30/06a 0 (0) 1 (1) 0 (0) 5 (6) 3 (4) 8 (8)* 6 (8) 2 (4) 1 (4) 20/07d 1 (3) 2 (3)* 3 (5) 1 (1) 0 (0) 0 (0) 0 (2) 0 (0) 0 (0) 22/08e 0 (0) 0 (0) 0 (0) 1 (2) 1 (5) 3 (7) 1 (1) 0 (0) 0 (1) 20/09b 1 (5) 1 (5)* 0 (0) 1 (6) 1 (1) 2 (2) 0 (0) 0 (0) 1 (1) 26/09a 0 (0) 3 (4) 1 (1) 3 (6) 1 (2)* 0 (7) 0 (0) 0 (0) 0 (0) 03/10c 1 (1) 2 (3) 0 (0) 0 (4) 0 (6) 0 (3) 0 (0) 0 (0) 0 (0) Total 14 (27) 22 (40) 4 (6) 16 (35) 11 (25) 13 (27) 18 (26) 10 (15) 2 (6) Number in parenthesis: total no. tested individuals – no sample * Locations and heights intraguild predation was detected in H. axyridis

123 Author's personal copy

524 A. G. Howe et al.

Fig. 2 a Mean number of E. tiliae per leaf on sampling dates (SD is based on 2–3 pooled heights). b Percentage of H. axridis adults/larvae and A. nemoralis screening positive for E. tiliae on sampling dates (2011). Each date represents results for pooled heights

was significantly shorter than DS50 values for E. tiliae aphid DNA (54.8 %; Fig. 2; Table 3). Similarly for digested by H. axyridis larvae (32.4 h; d.f. = 1, adult H. axyridis (n = 87: 45 females, 42 males), 3 v2 = 10.322, P = 0.001) and A. nemoralis (24.8 h; adults revealed IGP events (3.4 %; collected: 6/6, 2 d.f. = 1, v = 8.095, P = 0.004). However, DS50 30/6, 26/9) and 45.9 % or 40 individuals (19 males, 21 values for E. tiliae in predators did not differ (d.f. = 1, females) tested positive for E. tiliae (Fig. 2; Table 3). v2 \ 0.0001, P [ 0.05; Table 3, ESM Table 2). All H. axyridis adults, but only one larva, testing In 2009, seventy H. axyridis larvae (4th instar) collected positive for A. nemoralis DNA, also tested positive for in October were screened for prey DNA. A single IGP E. tiliae DNA. event (1.4 %) was detected (caught 19/10), whereas E. Forty-seven A. nemoralis (28 females, 19 males) tiliae DNA was detected in 32 individuals (45.7 %). were collected in 2011. Thirty individuals (9 males, 21 In 2011, 160 H. axyridis were collected from tree females) screened positive for E. tiliae DNA (63.8 %; crowns. Seventy-three H. axyridis larvae (3rd and 4th Fig. 2; Table 3). instars) were screened for prey DNA in which IGP was Applying the DS50 correction revealed that the detected in only two 4th instar larvae (2.7 %; collected relative importance of IGP was between 5.7 and 6.4 20/7, 20/9). In contrast, 40 larvae screened positive for times less than aphid consumption by H. axyridis 123 Author's personal copy

Potential for exploitative competition, not intraguild predation, between invasive 525

Table 3 Calculations of weighted trophic interactions based [intraguild (IG) prey] and the aphid E. tiliae [extraguild (EG) on detectability half-life assays and field-collected predators prey] on Tilia 9 europaea among H. axyridis [intraguild (IG) predator], A. nemoralis

° Predator–prey IGP (%) Aphid DS50 ± SE (h) DS50weighted* IGPweighted Aphid ° DNA (%) DNAweighted

H. axyridis (larva)–A. nemoralis 1.4 /2.7 – 5.6 ± 0.6a 1.00 0.014 /0.027 – H. axyridis (adult)–A. nemoralis 3.4 – DNA decay not ––– tested A. nemoralis (adult)–E. tiliae – 63.8 24.8 ± 3.4b 0.22 – 0.14 H. axyridis (larva)–E. tiliae –45.7 /54.8 32.4 ± 6.8b 0.17 – 0.08 /0.09 H. axyridis (adult)–E. tiliae – 45.9 Not calculated –––

DS50 values followed by a different letter are significantly different

*DS50weighted derived as: the shortest DS50 was assigned a value of 1.00, other DS50weighted values attained by dividing shortest DS50 (numerator) with relevant DS50 (denominator) ° Obtained by multiplying proportion of positive DNA by DS50weighted values Trophic interactions from specimens collected in 2009, otherwise 2011

Post-digestion times did not extend long enough to evaluate DS50 larvae (Table 3). Corrected values for aphid consump- Collection height did not influence the likelihood of tion were markedly less for both predators compared aphid ingestion in either predator, suggesting similar with raw data, but comparatively, relatively more aphid predation levels at all heights. None of the anthocorids tested positive for aphid DNA (Table 3). remaining variables contributed to explaining the Furthermore, aphid consumption by H. axyridis larvae likelihood of detecting E. tiliae in H. axyridis guts was similar in 2009 and 2011 (0.08 and 0.09, compared to null models (both life stages, LR: respectively) compared to proportions of raw data P = 0.12; larvae alone, LR: P = 0.29). testing positive for aphid DNA (Table 3). Interestingly, aphid DNA was detected in predators collected at all heights, however, IGP events in 2011 Discussion were detected in coccinellids collected above 2.5 m (Table 2). IGP and interspecific competition for E. tiliae in urban parks Factors affecting the likelihood of detecting E. tiliae DNA in predators DNA gut-content analysis revealed low levels of IGP by H. axyridis, but higher levels of prey overlap Four variables significantly influenced the likelihood between co-occurring predators in tree crowns (Fig. 2; of aphid ingestion by A. nemoralis (Table 4). The Tables 2, 3). While adjusting raw gut-content data abundance of H. axyridis larvae had a strong negative with the DS50 correction values reduced the relative effect on the likelihood of detecting aphid DNA in A. proportions of predators testing positive for prey nemoralis. The density of aphid nymphs had a weak DNA, the weighted values did not alter the relative positive influence, while a stronger albeit negative importance of trophic interactions in this system. influence of aphid adult density was revealed. Finally, Furthermore, positive correlations of predator density the sex of A. nemoralis was highly influential with the through time, and a negative effect of H. axyridis likelihood of aphid ingestion decreasing markedly for larvae density on aphid predation by A. nemoralis, are male bugs (Table 4). Other variables were not influ- indicative that spatiotemporal niche overlap and ential (LR: height, P = 0.18; H. axyridis adults, exploitation competition with H. axyridis occur in P = 0.84; competitors, P = 0.18). these invaded urban habitats.

123 Author's personal copy

526 A. G. Howe et al.

Table 4 Summary of final model with variables influencing the likelihood of detecting E. tiliae DNA in A. nemoralis Variable Comparison Odds ratio (95 % CI) Variable effect on odds P value

No. of aphid adults One unit change 0.853 (0.754–0.964) Negative 0.01 No. of aphid nymphs One unit change 1.033 (1.006–1.062) Positive 0.01 No. of H. axyridis larvae One unit change 0.495 (0.267–0.531) Negative 0.03 Sex Male versus female 0.083 (0.013–0.531) Negative 0.01 P values correspond to the likelihood ratio test of the effect of a variable in the presence of the other variables in the model. Odds ratios (Wald CI) are adjusted for other variables in the model

The frequency of intraguild predation by H. from predation (Dixon and Russel 1972; Lucas 2005; axyridis usually increases concurrent with declines Howe et al. 2015). in extraguild prey densities (Yasuda et al. 2004; Noia We hypothesised that interspecific competition is et al. 2008; Gagnon and Brodeur 2014). In this system, potentially a more important interaction than IGP aphid densities peaked in late June 2011 and declined between predators, as it may occur over longer thereafter. Indeed, three of the five IGP events in 2011 temporal scales. Potential for prey shortage was took place after aphid populations had peaked in late reflected in decreasing aphid densities from late June June (Table 2; Fig. 2). However, even though preda- and a decreased proportion of H. axyridis adults tors co-occurred temporally and often spatially in testing positive for E. tiliae following the aphid trees, IGP on A. nemoralis was a minor trophic population peak in late June (Fig. 2). Concurrently, interaction (1.4–3.4 %; IGPweighted = 0.014), which detection of aphid DNA in predators remained vastly supports our first hypothesis (Tables 2, 3). higher throughout the year relative to IGP levels, Recent studies detected traces of native coccinellid revealing prey overlap and potential for interspecific in 12.2 and 20.5 % of sampled H. axyridis larvae (but also intraspecific) competition for a limited EG collected on Tilia spp. (Hautier et al. 2011; Thomas prey (Fig. 2; Table 3). Rondoni et al. (2014) recently et al. 2013). In H. axyridis sampled predominantly reveal vastly higher levels of E. tiliae predation by H. from T x europaea across Europe, Brown et al. (2015) axyridis larvae (0.28, derived from DS50 data in report 0–13.7 % of larvae contain A. decempunctata Rondoni et al. 2014) relative to IGP of native L. DNA and 0–11.4 % contain remains of A. bipunc- coccinellids (A. bipunctata: 0.015; Oenopia conglo- tata L. While these results reveal comparatively bata L.: 0.012) over a 2-month sampling period on higher IGP levels against European coccinellids, Tilia spp. Despite these authors did not test the extent Rondoni et al. (2014) report lower IGP levels (1.5 of prey (E. tiliae) overlap with native coccinellids, and 5.0 %) against two native coccinellids in Italy. In their results demonstrate IGP is a much weaker trophic comparison, A. nemoralis in this study apparently interaction compared to EG predation, which lends suffers less direct negative effects (e.g. IGP) than support to the findings of the present study in native coccinellids in similar habitats. Noteworthy, as T. 9 europaea. IGP was detected in individuals collected above 2.5 m The strength of competition may be ameliorated in tree crowns, it is possible we would have missed through niche partitioning in space and time, thereby these events had we only sampled trees from the fostering co-existence (Hutchinson 1959; Armstrong ground. We propose that IGP contributes little to and Mcgehee 1980; Pell et al. 2008; Crowder and mortality of A. nemoralis populations in Tilia 9 eu- Snyder 2010). However, by exploiting multiple ropaea, and probably elsewhere these species co- resources invasive generalist species reduce the extent occur. Mechanisms underlying low IGP levels require of niche partitioning in invaded habitats, which may investigation, but could be related to escape behaviour impinge on native species (Snyder and Evans 2006). afforded by high mobility of large A. nemoralis Our results suggest temporal co-occurrence, as preda- nymphs and adults, dropping behaviour upon encoun- tors were simultaneously collected from tree crowns ters, and utilisation of leaf axils and stipules as refuge until late September in 2011 (Fig. 2; Table 2). In

123 Author's personal copy

Potential for exploitative competition, not intraguild predation, between invasive 527 northern Europe, these predators are bivoltine, becom- Invasive insect generalist predators typically ing active in May, H. axyridis remains active through achieve higher densities than the native predators they to November, whereas A. nemoralis begins hiberna- replace (Snyder and Evans 2006; Crowder and Snyder tion between August and October (Anderson 1962; 2010). Comparable to European studies reporting Adriaens et al. 2008; Sigsgaard 2010). Spatial niche coccinellid densities (Hautier et al. 2011; Brown et al. overlap was further supported by significant positive 2011a) we found H. axyridis densities 2.5–8 times correlations of predator densities, predator and aphid greater than A. nemoralis (Table 2). The density of H. nymph densities, and similar densities of predators axyridis larvae had a strong negative effect on aphid between heights. Moreover, detection of aphid DNA predation by A. nemoralis suggesting that H. axyridis was not influenced by collection height, lending life stage may mediate interspecific competition further support that these predators forage for prey at between these predators. Furthermore, although similar spatial scales in T. 9 europaea crowns. weighted proportions revealed relatively higher inter- Different prey preferences contribute to niche actions between anthocorids and E. tiliae compared to partitioning in aphid/predator systems (Losey and H. axyridis larvae and aphids, the values do not take Denno 1998; Pell et al. 2008), and may occur to an differences in predator voracity into account. For extent between A. nemoralis and H. axyridis. Due to example, adult H. axyridis may eat between 15 and 65 their relatively small body size, anthocorids are aphids per day (ref. in Koch 2003). In contrast, adult A. ineffective at capturing large aphid nymphs/adults nemoralis eat between 3.6 and 12.7 aphids per day (Dixon and Russel 1972). Our analysis showed a (Meyling et al. 2003). While we draw attention to the positive influence of aphid nymph and negative small sample size (n = 47), the negative effect of influence of aphid adult densities on aphid predation larvae density underscores that the extent and strength by A. nemoralis, which could indicate that anthocorids of interspecific competition may be exacerbated by prefer small prey. In contrast, none of the investigated superabundant densities of voracious H. axyridis. variables contributed to explaining aphid predation by The strength of IGP and resource competition is H. axyridis (independent of the ladybird’s life stage), influenced by habitat structure (Hoogendoorn and suggesting the ladybird does not discriminate between Heimpel 2004; Crowder and Snyder 2010). Complex aphid sizes. These results could imply that niche structured habitats may reduce encounter rates, partitioning based on prey size is curtailed (Snyder and impede predator perception, and provide spatial refuge Evans 2006). This effect is potentially amplified for A. for intraguild prey compared to simple structures nemoralis apterous nymph stages, due to their small (Persson and Eklov 1995; Lucas et al. 2000; Raak-van size relative to adults, but also as emigration in Den Berg et al. 2012). However, when an IG prey is an response to dwindling aphid densities in late summer inferior competitor, the benefit of reduced IGP may be is restricted (Dixon 1971b). Furthermore, second outweighed by competition for shared prey (Janssen generation nymphs are likely to contend for a shortage et al. 2007). For example, dramatic declines of of aphids at local scales (Dixon 1971b), subsequently predator assemblages including anthocorids (Orius increasing the intensity of exploitative competition insidious Say) on pecan trees (12–15 m high) and and concurrently the risk of intraguild predation ornamental crape myrtle (2–4 m high) were shown among aphidophages (Obrycki et al. 1998; Ware following invasion by H. axyridis, presumably et al. 2009). through resource competition (Mizell 2007). How- Anthocorid males were significantly less likely to ever, competition with H. axyridis in a structurally contain aphid DNA than females (Table 4). This may complex habitat can be alleviated when co-occurring not necessarily equate to a distinct disadvantage for predators adjust their distribution in response to H. males, as A. nemoralis can utilise various prey axyridis (Hoogendoorn and Heimpel 2004). In our (Anderson 1962). However, as E. tiliae is important study, complex T. 9 europaea crowns may be a factor for A. nemoralis reproduction (Anderson 1962), contributing to low IGP levels, but given the high level female anthocorids are potentially more dependent of spatiotemporal niche overlap of predators, compet- on E. tiliae, and thereby more vulnerable to inter- itive abilities of these species are more likely to drive specific exploitation competition by H. axyridis. outcomes of interspecific interactions in tree crowns.

123 Author's personal copy

528 A. G. Howe et al.

Known characteristics of H. axyridis conferring com- insect communities, which may inflate the potential petitive advantage over native coccinellids include for bias through cross-reactivity. superior foraging behaviour, e.g. prey searching and Secondary predation and scavenging events are attack efficiency, interspecific aggression (Yasuda indistinguishable using DNA-based methods which et al. 2004; Labrie et al. 2006), while recent laboratory may distort detected interaction strengths by yielding microcosm studies reveal H. axyridis superior com- false positives (Sheppard and Harwood 2005; Foltan petition for E. tiliae compared to A. nemoralis (Howe et al. 2005; Hosseini et al. 2008). Laboratory feeding et al. 2015). trials with H. axyridis and A. nemoralis did not reveal scavenging (Howe et al. 2015), but the breadth of Limitations of this study scavenging by both species in natural settings requires more attention, and could improve knowledge of The limited spatial and temporal scales of this study trophic pathways in general (Lattin 1999; Gagnon precluded assessment of whether resource competition et al. 2011a). In addition, IGP potentially overesti- has negative consequences on A. nemoralis popula- mated prey overlap in cases where prey DNA detected tions, or contributes to its competitive displacement. in H. axyridis was actually ingested by A. nemoralis. We had a priori knowledge that parks contained dense While this does little to alter our prey overlap results populations of H. axyridis, which may inflate trophic (aphid DNA detected in 4 of the 5 IGP events), it may interactions quantified here. Assessment of interac- have been a confounding factor were IGP levels tions where invasive H. axyridis is less abundant higher. Similarly, aphid DNA in either predator could would provide a more nuanced picture of interaction be a result of IGP of non-focal predators, but given strengths (Kenis et al. 2009). In addition, although E. densities of other predators were relatively low in tree tiliae is the numerically dominant herbivore on Tilia crowns this effect is probably negligible (Table 1). spp., other insects are found on Tilia spp., and are Finally, a range of factors influence the detection potentially exploited as alternate resources by poly- period of target DNA following ingestion including phagous predators (Lattin 1999; Alford 2002; Van- the effect of temperature on enzyme activity during dereycken et al. 2012; Table 1). Such resources can digesting, time since ingestion, length of the target weaken competition (MacArthur and Levins 1967; DNA sequence, predator species, activity levels, Tilman 1982), which has potential to alter the strength starvation and sample storage, e.g. ethanol versus of interactions demonstrated in this study. dry (Hoogendoorn and Heimpel 2001; King et al. Detection of aphid predation in this study may not 2008; Gagnon et al. 2011a). precisely reflect prey overlap in sampled tree crowns, Aphid DNA decayed at similar rates in predators as we cannot discount that winged adults ingested and as such the respective trophic interactions between aphids in nearby Tilia spp. Dispersal between trees, or predators and E. tiliae are comparable (Gagnon et al. at larger scales between parks, may contribute toward 2011a; Table 3, ESM Table 2). Calculation of DS50 reducing resource competition (Amarasekare 2003); for H. axyridis adults ingesting E. tiliae obviously future studies could reduce this potential bias by requires digestion times beyond 24 h, but assuming focusing on immature life stages of predators. Fur- adults share a DS50 similar to larvae, weighted trophic thermore, testing primer specificity against a greater interactions probably reflect values for H. axyridis number of species/families for cross-reactivity (i.e. adults, which were similar to larvae, at least, for raw [26 species) would reduce the potential for interpre- gut-content data. The relatively short detection period tation of false-positives as a result of amplification of for A. nemoralis DNA likely reflects IGP taking place non-target prey. This is applicable not only to non- on the day samples were collected (i.e. up 6 h after an target species within Tilia spp. crowns, but also IGP event), although a slightly longer detection period species ingested by focal (generalist) predators dis- would enhance detection of a rare event. Our sampling persing from other locations/plants to focal study sites. time extended 2 h beyond H. axyridis primary feeding This may be particularly relevant in cities compared to time (due to setup of tree climbing equipment), which agroecosystems, due to the heterogeneity of urban may equate to underestimation of prey ingestion green spaces within a relatively small area, e.g. diverse because individuals had longer time to digest meals assemblages of vegetation types supporting different in crowns. However, collection height did not affect 123 Author's personal copy

Potential for exploitative competition, not intraguild predation, between invasive 529 the likelihood of aphid detection (high positions were ecosystem services remain intact when functional sampled latest during a collection day), so this diversity is altered by H. axyridis remains a pressing potential error was likely negligible. Furthermore, area of research (Pell et al. 2008; Roy et al. 2012); trophic interactions in this study are a ‘‘snapshot in while H. axyridis is a rare case of a BCA becoming time’’ in that we do not precisely know when or how invasive, environmental risk assessment of future many individuals were ingested (Chen et al. 2000), BCAs can benefit from post-introduction studies which could be addressed by developing a qPCR elucidating effects of BCAs on trophic interactions approach (King et al. 2008). Finally, digestion times in invaded food webs (Aebi et al. 2011). were performed using A. nemoralis reared for biocon- trol, whose populations (origins from Germany and Acknowledgments We are grateful to Lars Skipper (www. Denmark) may differ from those in Copenhagen parks. miridae.dk) for verifying the identity of mirids and EWH BioProduction ApS for provision of A. nemoralis; Gitte Jensen (DK), Renate Zindel and Mario Waldburger (Agroscope ART, CH) for technical assistance; and Peter Brown (UK) and Brecht Conclusion Ingels (BEL) for collection of specificity samples. We also thank three anonymous reviewers for their insightful comments. HPR was financed by Villum Fonden, AGH was supported by a A consequence of invasive generalist insect predators PhD grant from University of Copenhagen. is the competitive displacement of related or func- tionally similar native predators (Snyder and Evans 2006; Crowder and Snyder 2010). A combination of References mechanisms typically contribute to competitive dis- placement (Reitz and Trumble 2002), and specifically Adriaens T, Branquart E, Maes D (2003) The multicoloured for H. axyridis asymmetrical IGP is identified as a Asian ladybird Harmonia axyridis Pallas (Coleoptera: Coccinellidae), a threat for native aphid predators in Bel- driver of native coccinellid displacement (Koch and gium? Belg J Zool 133:195–196 Galvan 2008; Roy et al. 2012). Our results reveal Adriaens T, San M, Maes D (2008) Invasion history, habitat resource competition for E. tiliae is a stronger trophic preferences and phenology of the invasive ladybird Har- interaction relative to IGP between A. nemoralis and monia axyridis in Belgium. Biocontrol 53:69–88 H. axyridis Tilia Aebi A, Brown PM, De Clercq P, Hautier L, Howe A, Ingels B, in x europaea crowns. As both larvae Ravn HP, Sloggett JJ, Zindel R, Thomas A (2011) and adult H. axyridis exploit E. tiliae over multiple, Detecting intraguild predation in the field. Bio- overlapping generations throughout the year, compe- control 56:429–440 tition for E. tiliae is likely to be intense, especially Alford DV (2002) Pests of ornamental trees, shrubs and flowers. Manson Publishing Ltd., London where H. axyridis achieves high densities. Addition- Alhmedi A, Haubruge E, Francis F (2010) Intraguild interac- ally, the negative effect of H. axyridis larvae density tions implicating invasive species: Harmonia axyridis as a on detection of aphid ingestion by A. nemoralis model species. Biotechnol Agron Soc Environ 14:187–201 suggests that interspecific exploitative competition Alyokhin A, Sewell G (2004) Changes in a lady beetle com- H. munity following the establishment of three alien species. may be life stage-mediated, i.e. most intense when Biol Invasions 6:463–471 axyridis are larvae. Amarasekare P (2003) Competitive coexistence in spatially Although interactions between H. axyridis and structured environments: a synthesis. Ecol Lett 6:1109–1122 coccinellids have received most attention to date, this Anderson NH (1962) Bionomics of six species of Anthocoris (Heteroptera: Anthocoridae) in England. Trans R Entomol study revealed numerous non-coccinellid aphi- Soc Lond 114:67–95 dophages co-occur with invasive H. axyridis in Armstrong RA, Mcgehee R (1980) Competitive-exclusion. Am T. 9 europaea. Findings here are indicative that for Nat 115:151–170 the assemblage of native aphidophages sharing Bates D, Maechler M, Bolker B, Walker S (2014) lme4: linear H. mixed-effects models using Eigen and S4. R package resources with, yet avoiding IGP by invasive version 1.0-6 axyridis, indirect trophic interactions are likely to play Brown JS, Kotler BP, Mitchell WA (1994) Foraging theory, an important role in shaping invaded insect commu- patch use, and the structure of a Negev Desert granivore nities. Given H. axyridis eurytopic and polyphagous community. Ecology 75:2286–2300 Brown PMJ, Roy HE, Rothery P, Roy DB, Ware RL, Majerus nature, this has implications for many native insect MEN (2008) Harmonia axyridis in Great Britain: analysis species, especially those whose life stages overlap of the spread and distribution of a non-native coccinellid. with H. axyridis 3rd and 4th larval stages. Whether Biocontrol 53:55–67 123 Author's personal copy

530 A. G. Howe et al.

Brown PMJ, Frost R, Doberski J, Sparks T, Harrington R, Roy Harmon JP, Stephens E, Losey J (2007) The decline of native HE (2011a) Decline in native ladybirds in response to the coccinellids (Coleoptera: Coccinellidae) in the United arrival of Harmonia axyridis: early evidence from Eng- States and Canada. J Insect Conserv 11:85–94 land. Ecol Entomol 36:231–240 Hautier L, Martin GS, Callier P, de Biseau JC, Gregoire JC Brown PMJ, Thomas CE, Lombaert E, Jeffries DL, Estoup A, (2011) Alkaloids provide evidence of intraguild predation Handley LJL (2011b) The global spread of Harmonia on native coccinellids by Harmonia axyridis in the field. axyridis (Coleoptera: Coccinellidae): distribution, disper- Biol Invasions 13:1805–1814 sal and routes of invasion. Biocontrol 56:623–641 Hebert PDN, Cywinska A, Ball SL, de Waard JR (2003) Bio- Brown PMJ, Ingels B, Wheatley A, Rhule EL, de Clercq P, van logical Identifications through DNA Barcodes. Proceed- Leeuwen T, Thomas A (2015) Intraguild predation by ings: Biological Sciences: 270(1512):313–321 Harmonia axyridis (Coleoptera: Coccinellidae) on native Hironori Y, Katsuhiro S (1997) Cannibalism and interspecific insects in Europe: molecular detection from field samples. predation in two predatory ladybirds in relation to prey Entomol Sci 18:130–133 abundance in the field. Entomophaga 42:153–163 Chapin JB, Brou VA (1991) Harmonia axyridis (Pallas), the Hoogendoorn M, Heimpel GE (2001) PCR-based gut content third species of the genus to be found in the United States analysis of insect predators: using ribosomal ITS-1 frag- (Coleoptera: Coccinellidae). Proc Entomol Soc Wash ments from prey to estimate predation frequency. Mol Ecol 93:630–635 10:2059–2067 Chen Y, Giles KL, Payton ME, Greenstone MH (2000) Identi- Hoogendoorn M, Heimpel GE (2004) Competitive interactions fying key cereal aphid predators by molecular gut analysis. between an exotic and a native ladybeetle: a field cage Mol Ecol 9:1887–1898 study. Entomol Exp Appl 111:19–28 Crowder DW, Snyder WE (2010) Eating their way to the top? Hosseini R, Schmidt O, Keller MA (2008) Factors affecting Mechanisms underlying the success of invasive insect detectability of prey DNA in the gut contents of inverte- generalist predators. Biol Invasions 12:2857–2876 brate predators: a polymerase chain reaction-based Dixon AFG (1971a) Role of aphids in wood formation. 2. Effect method. Entomol Exp Appl 126:194–202 of lime aphid, Eucallipterus tiliae L. (Aphididae), on Hothorn T, Bretz F, Westfall P (2008) Simultaneous inference in growth of lime, Tilia x vulgaris Hayne. J Appl Ecol 8:393 general parametric models. Biom J 50:346–363 Dixon AFG (1971b) Role of intra-specific mechanisms and Howe AG, Ransijn J, Ravn HP (2015) A sublethal effect on predation in regulating numbers of lime aphid, Eucal- native Anthocoris nemoralis through competitive interac- lipterus tiliae L. Oecologia 8:179 tions with Harmonia axyridis. Ecol Entomol. 40:639–649 Dixon AFG (1998) Aphid ecology: an optimization approach. Hoy MA (1994) Insect molecular genetics: an introduction to Chapman & Hall, London principals and applications. Academic Press, San Diego Dixon AFG, Russel RJ (1972) Effectiveness of Anthocoris Hutchinson GE (1959) Homage to Santa-Rosalia or why are nemorum and A. Confusus (Hemiptera: Anthocoridae) as there so many kinds of . Am Nat 93:145–159 predators of sycamore aphid, Drepanosiphum platanoides. Jackson CH (2011) Multi-state models for panel data: the msm 2. Searching behavior and incidence of predation in field. package for R. J Stat Softw 38(8):1–29 Entomol Exp Appl 15:35 Janssen A, Sabelis MW, Magalhaes S, Montserrat M, Van der Evans EW, Soares AO, Yasuda H (2011) Invasions by ladybugs, Hammen T (2007) Habitat structure affects intraguild ladybirds, and other predatory beetles. Biocontrol 56: predation. Ecology 88:2713–2719 597–611 Kasper ML, Reeson AF, Cooper SJB, Perry KD, Austin AD Foltan P, Sheppard S, Konvicka M, Symondson WOC (2005) (2004) Assessment of prey overlap between a native The significance of facultative scavenging in generalist (Polistes humilis) and an introduced (Vespula germanica) predator nutrition: detecting decayed prey in the guts of social wasp using morphology and phylogenetic analyses predators using PCR. Mol Ecol 14:4147–4158 of 16S rDNA. Mol Ecol 13:2037–2048 Foottit RG, Maw HEL, Von Dohlen CD, Hebert PDN (2008) Kenis M, Auger-Rozenberg MA, Roques A, Timms L, Pere C, Species identification of aphids (Insecta: Hemiptera: Cock M, Settele J, Augustin S, Lopez-Vaamonde C (2009) Aphididae) through DNA barcodes. Mol Ecol Resour Ecological effects of invasive alien insects. Biol Invasions 8:1189–1201 11:21–45 Gagnon AE, Brodeur J (2014) Impact of plant architecture and Kenis M, Adriaens T, Brown P, Katsanis A Van, Vlaenderen J, extraguild prey density on intraguild predation in an Eschen R, Golaz L, Zindel R San, Martin y Gomez G, agroecosystem. Entomol Exp Appl 152:165–173 Babendreier D, Ware R (2010) Impact of Harmonia Gagnon AE, Doyon J, Heimpel GE, Brodeur J (2011a) Prey axyridis on European ladybirds: which species are most at DNA detection success following digestion by intraguild risk? IOBC/WPRS Bull 58:57–59 predators: influence of prey and predator species. Mol Ecol King RA, Read DS, Traugott M, Symondson WOC (2008) Resour 11:1022–1032 Molecular analysis of predation: a review of best practice Gagnon AE, Heimpel GE, Brodeur J (2011b) The ubiquity of for DNA-based approaches. Mol Ecol 17:947–963 intraguild predation among predatory . Plos One Koch RL (2003) The multicolored Asian lady beetle, Harmonia 6(11):e28061 axyridis: a review of its biology, uses in biological control, Greenstone MH, Payton ME, Weber DC, Simmons AM (2014) and non-target impacts. J Insect Sci 3:32 The detectability half-life in arthropod predator–prey Koch RL, Galvan TL (2008) Bad side of a good beetle: the North research: what it is, why we need it, how to measure it, and American experience with Harmonia axyridis. Biocontrol how to use it. Mol Ecol 23:3799–3813 53:23–35 123 Author's personal copy

Potential for exploitative competition, not intraguild predation, between invasive 531

Koressaar T, Remm M (2007) Enhancements and modifications Pe´ricart J (1996) Family Anthocoridae Fieber. 1836—flower of primer design program Primer3. Bioinformatics bugs, minute pirate bugs. In: Aukema B, Rieger C (eds) 23:1289–1291 Catalogue of the Heteroptera. Ponsen and Looijen, Labrie G, Lucas E, Coderre D (2006) Can developmental and Wageningen, pp 108–140 behavioral characteristics of the multicolored Asian lady Persson L, Eklov P (1995) Prey refuges affecting interactions beetle Harmonia axyridis explain its invasive success? Biol between piscivorous perch and juvenile perch and roach. Invasions 8:743–754 Ecology 76:70–81 Lattin JD (1999) Bionomics of the Anthocoridae. Annu Rev Polis GA, Strong DR (1996) Food web complexity and com- Entomol 44:207–231 munity dynamics. Am Nat 147:813–846 Lawton JH, Hassell MP (1984) Interspecific competition in Polis GA, Myers CA, Holt RD (1989) The ecology and evolu- insects. In: Huffaker CB, Rabb RL (eds) Ecological ento- tion of intraguild predation—potential competitors that eat mology. Wiley, New York, pp 451–495 each other. Annu Rev Ecol Syst 20:297–330 Losey JE, Denno RF (1998) Positive predator–predator inter- R Core Team (2013) R: a language and environment for sta- actions: enhanced predation rates and synergistic sup- tistical computing. R Foundation for Statistical Computing pression of aphid populations. Ecology 79:2143–2152 Raak-van Den Berg CL, De Lange HJ, Van Lenteren JC (2012) Lucas E (2005) Intraguild predation among aphidophagous Intraguild predation behaviour of ladybirds in semi-field predators. Eur J Entomol 102:351–363 experiments explains invasion success of Harmonia axyr- Lucas E, Coderre D, Brodeur J (2000) Selection of molting and idis. Plos One 7(7):e40681 pupation sites by Coleomegilla maculata (Coleoptera: Ratnasingham S, Hebert PDN (2007) BOLD: the barcode of life Coccinellidae): avoidance of intraguild predation. Environ data system (www.barcodinglife.org). Mol Ecol Notes Entomol 29:454–459 7:355–364 Lucas E, Labrie G, Lazarovits G (2007) The multicolour Asian Reitz SR, Trumble JT (2002) Competitive displacement among ladybird beetle: beneficial or nuisance organism? In: Vin- insects and arachnids. Annu Rev Entomol 47:435–465 cent C, Goettel MS, Lazarovits G (eds) Biological control: Rondoni G, Athey KJ, Harwood JD, Conti E, Ricci R, Obrycki a global perspective. CABI Publishing, Wallingford, JJ (2014) Development and application of molecular gut- pp 38–52 content analysis to detect aphid and coccinellid predation MacArthur R, Levins R (1967) Limiting similarity convergence by Harmonia axyridis (Coleoptera: Coccinellidae) in Italy. and divergence of coexisting species. Am Nat 101:377 Insect Sci 00:1–12. doi:10.1111/1744-7917.12165 Meyling NV, Enkegaard A, Brødsgaard H (2003) Two Antho- Roy HE, Handley LJL (2012) Networking: a community coris bugs as predators of glasshouse aphids—voracity and approach to invaders and their parasites. Funct Ecol prey preference. Entomol Exp Appl 108:59–70 26:1238–1248 Michaud JP (2002) Invasion of the Florida citrus ecosystem by Roy HE, Adriaens T, Isaac NJB, Kenis M, Onkelinx T, San Harmonia axyridis (Coleoptera: Coccinellidae) and Martin G, Brown PMJ, Hautier L, Poland R, Roy DB, asymmetric competition with a native species, Cycloneda Comont R, Eschen R, Frost R, Zindel R, Van Vlaenderen J, sanguinea. Environ Entomol 31:827–835 Nedved O, Ravn HP, Gregoire JC, de Biseau JC, Maes D Miura T, Nishimura S (1980) The larval period and predacious (2012) Invasive alien predator causes rapid declines of activity of an aphidophagous coccinellid, Harmonia axyridis native European ladybirds. Divers Distrib 18:717–725 PALLAS. Bull Fac Agric Shimane Univ 14:144–148 Sheppard SK, Harwood JD (2005) Advances in molecular Mizell RF (2007) Impact of Harmonia axyridis (Coleoptera: ecology: tracking trophic links through predator–prey Coccinellidae) on native arthropod predators in pecan and food-webs. Funct Ecol 19:751–762 crape myrtle. Fla Entomol 90:524–536 Sigsgaard L (2010) Habitat and prey preferences of the two Nedved O, Fois X, Ungerova D, Kalushkov P (2013) Alien vs. predatory bugs Anthocoris nemorum (L.) and A. nemoralis predator—the native lacewing Chrysoperla carnea is the (Fabricius) (Anthocoridae: Hemiptera-Heteroptera). Biol superior intraguild predator in trials against the invasive Control 53:46–54 ladybird Harmonia axyridis. Bull Insectol 66(1):73–78 Snyder WE, Evans EW (2006) Ecological effects of invasive Noia M, Borges I, Soares AO (2008) Intraguild predation arthropod generalist predators. Annu Rev Ecol Evol Syst between the aphidophagous ladybird beetles Harmonia 37:95–122 axyridis and Coccinella undecimpunctata (Coleoptera: Southwood TRE, Leston D (1959) Land and water bugs of the Coccinellidae): the role of intra and extraguild prey den- British Isles (CD-ROM version). Pisces Conservation sities. Biol Control 46:140–146 Limited, Lymington Obrycki JJ, Giles KL, Ormord AM (1998) Interactions between Thomas AP, Trotman J, Wheatley A, Aebi A, Zindel R, Brown an introduced and indigenous coccinellid species at dif- PMJ (2013) Predation of native coccinellids by the inva- ferent prey densities. Oecologia 117:279–285 sive alien Harmonia axyridis (Coleoptera: Coccinellidae): Osawa N (2000) Population field studies on the aphidophagous detection in Britain by PCR-based gut analysis. Insect ladybird beetle Harmonia axyridis (Coleoptera: Coccinel- Conserv Divers 6:20–27 lidae): resource tracking and population characteristics. Tilman D (1982) Resource competition and structure. Princeton Popul Ecol 42:115–127 University Press, Princeton Pell JK, Baverstock J, Roy HE, Ware RL, Majerus MEN (2008) Tomov R, Trencheva K, Trenchev G, Kenis M (2009) The Intraguild predation involving Harmonia axyridis:a multicolored invasive Asian ladybird Harmonia axyridis review of current knowledge and future perspectives. (PALLAS, 1773) (Coleoptera: Coccinellidae) new to the Biocontrol 53:147–168 fauna of Bulgaria. Acta Zool Bulg 61:307–311 123 Author's personal copy

532 A. G. Howe et al.

Tylianakis JM (2008) Understanding the web of life: the birds, development of the European coccinellid Adalia bipunc- the bees, and sex with aliens. PLoS Biol 6:224–228 tata and the invasive species Harmonia axyridis. Ecol Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Entomol 34:12–19 Remm M, Rozen SG (2012) Primer3-new capabilities and White EM, Wilson JC, Clarke AR (2006) Biotic indirect effects: interfaces. Nucleic Acids Res 40(15):e115 a neglected concept in invasion biology. Divers Distrib Vandereycken A, Durieux D, Joie E´ , Haubruge E´ , Verheggen FJ 12:443–455 (2012) Habitat diversity of the multicolored Asian lady- Wickham H (2009) Ggplot2: elegant graphics for data analysis. beetle Harmonia axyridis Pallas (Coleoptera: Coccinelli- Springer, New York dae) in agricultural and arboreal ecosystems: a review. Yasuda H, Evans EW, Kajita Y, Urakawa K, Takizawa T (2004) Biotechnol Agron Soc Environ 16(4):553–563 Asymmetric larval interactions between introduced and Ware R, Yguel B, Majerus M (2009) Effects of competition, indigenous ladybirds in North America. Oecologia cannibalism and intra-guild predation on larval 141:722–731

123

View publication stats