Biological Control 132 (2019) 189–198

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Biological Control

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Field evidence and grower perceptions on the roles of an omnivore, European , in apple orchards T ⁎ Robert J. Orpeta, , Jessica R. Goldbergerb, David W. Crowderc, Vincent P. Jonesa a Washington State University, Department of Entomology, Tree Fruit Research and Extension Center, 1100 N Western Ave, Wenatchee, WA 98801, United States b Washington State University, Department of Crop and Soil Sciences, 263 Johnson Hall, PO Box 646420, Pullman, WA 99164, United States c Washington State University, Department of Entomology, 166 FSHN Bldg, PO Box 646382, Pullman, WA 99164, United States

ARTICLE INFO ABSTRACT

Keywords: In agroecosystems, omnivores can be beneficial predators or harmful herbivores. In apple orchards, the omni- Inundation biological control vorous European earwig (Forficula auricularia) is thought to be a key predator of woolly apple ( Diet breadth lanigerum), but has also been implicated in feeding on apple fruit. Assessing the effects of in orchards is Gut content analysis difficult because they are nocturnal, and their damage to fruits can resemble other wounds. Apple orchardists Interviews thus may manage earwigs as either predators or pests based on subjective opinions. To understand current Predator behavior opinions on earwigs in apple orchards, we interviewed 15 apple pest management decision-makers in Washington State, USA. To compare opinions to objective measurements, we manipulated earwig abundance within plots at four orchards and collected data on fruit damage, woolly apple aphid abundance, and molecular gut contents of earwigs. Most interviewees thought earwigs were aphid predators, but some thought earwigs could be minor pests, and most were more uncertain about earwigs’ effects relative to other aphid natural enemies. In the field, earwig abundance was negatively correlated to woolly apple aphid abundance, and earwig guts regularly contained woolly apple aphid DNA, even when aphid densities were low. We found no evidence earwigs damaged fruits. Overall, our results suggest earwigs improved biological control and were not pests, so discontinuing the occasional use of against earwigs could benefit apple growers. More generally, omnivores and difficult-to-observe natural enemies could often have important underappreciated benefits in agriculture.

1. Introduction 2017), but has also been implicated as a pest. European earwigs can damage apples when confined together with them (Carroll et al., 1985; Omnivores have been called “plant bodyguards” because they can Croxall et al., 1951; Crumb et al., 1941; Glen, 1975; Nicholas et al., suppress pests and also persist during periods when pests are not 2004), but do not always do so, even after up to seven days with no abundant (Ågren et al., 2012). However, an omnivore’s benefits can be alternative food (Nicholas et al., 2004). Earwig damage to apples can offset if they damage marketed plant structures (Puentes and Björkman, resemble wounds caused by other organisms or mechanical injury, 2017) or consume other predators (Prasad and Snyder, 2006). To ap- making it difficult to assess in the open field, where earwig damage propriately manage omnivore species, it is necessary to understand might be uncommon because other preferable foods are available. Two their potential effects as both an herbivore and as a predator. For ex- field studies found no evidence that excluding European earwigs from ample, the omnivore verbasci (Meyer-Dür) (: trees with physical barriers decreased apple damage (Crumb et al., ) is an apple pest when fruits are young and susceptible to 1941; Nicholas et al., 2004). One field study using insecticidal barriers feeding damage, but it is considered a beneficial predator later in the suggested the opposite (Croxall et al., 1951), but earwig damage was year and in (Reding et al., 2001). measured indirectly as the incidence of brown rot, which could infest The omnivorous European earwig (Forficula auricularia L.) has been wounds caused by other organisms. implicated in suppressing woolly apple ( Although researchers in many regions worldwide are interested in [Hausmann]) in apple orchards (Mueller et al., 1988; Quarrell et al., promoting biological control of woolly apple aphid by European

⁎ Corresponding author. E-mail addresses: [email protected] (R.J. Orpet), [email protected] (J.R. Goldberger), [email protected] (D.W. Crowder), [email protected] (V.P. Jones). https://doi.org/10.1016/j.biocontrol.2019.02.011 Received 19 December 2018; Received in revised form 18 February 2019; Accepted 19 February 2019 Available online 22 February 2019 1049-9644/ © 2019 Elsevier Inc. All rights reserved. R.J. Orpet, et al. Biological Control 132 (2019) 189–198 earwigs (Lordan et al., 2015; Mueller et al., 1988; Nicholas et al., 2004; experiences and opinions related to management of woolly apple Quarrell et al., 2017) and in conserving earwig populations in orchards aphids. For this study, we only analyzed responses from a subset of (Fountain and Harris, 2015; Moerkens et al., 2012), commercial orch- questions about biological control and specifically the effects of la- ardists may consider European earwig a pest rather than a beneficial cewings (: Chrysopidae), syrphids (Diptera: Syrphidae), la- predator. European earwigs are nocturnal, so they are difficult to ob- dybugs (Coleoptera: ), (the specialist serve attacking pests. In addition, European earwigs shelter in tight parasitoid of woolly apple aphid; Hymenoptera: Aphelinidae), spaces during the day, so they are relatively easy to observe in damaged Daraeocoris brevis (Uhler) (a generalist predator; Hemiptera: Miridae), fruit, which could have been injured by a different source. Therefore, ground (Coleoptera: Carabidae), and earwigs (European agricultural professionals, who commonly make their management earwig). These natural enemies were chosen because they are thought decisions in response to direct observations (Carolan, 2006; Sherman to comprise the main woolly apple aphid natural enemy taxa in and Gent, 2014), may be interested in promoting or suppressing ear- Washington State (Gontijo et al., 2012), except for ground beetles. wigs depending on subjective opinions. Ground beetles have not been specifically studied as a woolly apple In this study, we addressed knowledge gaps on both the subjective aphid predator in the field, but can consume woolly apple aphids in the and objective roles of earwigs in apple orchards in Washington State, laboratory (Orpet, personal observations). Interviewees were also asked USA. We assessed the opinions of apple orchard decision-makers about what kinds of damage (if any) earwigs caused to apples and through in-depth interviews. We also conducted field experiments whether they ever took any management actions meant to promote or where we manipulated earwig abundance, then measured apple fruit suppress earwigs. In addition, because we know of no widely estab- damage, woolly apple aphid abundance, and evaluated earwig feeding lished economic thresholds for woolly apple aphid management, we habits through molecular analysis of their gut contents. Previous ex- analyzed responses about interviewees ’ woolly apple aphid action periments addressing the positive or negative roles of earwigs in apple thresholds to assess whether aphid counts in our field study would have orchards manipulated earwig abundance with barriers preventing been considered below or above acceptable levels. A list of the relevant earwig movement into trees (Crumb et al., 1941; Croxall et al., 1951; questions can be found in Supplementary Document S1. Mueller et al., 1988; Nicholas et al., 2004) or with appli- Similar to other studies involving in-depth interviews (e.g., Sherman cations (Ravensberg, 1981). Because these treatments could affect other and Gent, 2014), we used a qualitative approach to analyzing re- natural enemies, we directly released or collected earwigs in experi- sponses, placing emphasis on understanding the experiences and rea- mental plots rather than using barriers or pesticides to manipulate soning behind the opinions of interviewees. In addition, to summarize earwig abundance. Overall, our study combined interview data with our interview data, we categorized opinions on the role of biological experimental data to assess the perceived and objective effects of an control, and of each natural enemy species, as: ‘important’ (having a omnivore, and to address whether it should be managed as a beneficial considerable role), ‘potentially important’ (having a role, but with un- predator or harmful herbivore. certainty of its importance, or having an inconsistent role), ‘unknown’ (lack of knowledge of its existence, or uncertainty that a potential role 2. Methods exists), or ‘not important’ (an assertion that there is no considerable role). We rated interviewees’ importance of earwigs as a pest as ‘yes’ 2.1. Interviews (caused considerable economic damage), ‘slight’ (caused slight eco- nomic damage, but there was minimal concern about it), or ‘no’ (had We recorded semi-structured interviews with 15 apple pest man- not caused economic damage). agement decision-makers from Washington State, USA. These in- dividuals were solicited at Washington extension meetings in 2016. The 2.2. Experimental design and treatments group included eight pest management consultants, three field man- agers, and four private owners; 13 were male and two were female. To collect objective data to compare with interviews, we monitored Interviewees ranged from 26 to 77 years old (median = 58) and had European earwig and woolly apple aphid populations within one Gala between 1 and 40 years of experience in the fruit industry cultivar orchard block in 2016 and 2017, and three Fuji cultivar blocks (median = 30). They worked with between 0.4 and 3560 ha of apple in 2017 (Table 1). The Gala block was managed by one of the inter- (median = 364), and all but one consultant and one manager worked viewed managers, Fuji block 1 was managed by one of the interviewed with both organic and conventional apples at the time of the interview. owners, and Fuji blocks 2 and 3 were managed by one of the inter- Interviews typically lasted 1 h and were structured to learn about viewed consultants. All orchards were commercially managed

Table 1 Information on study orchard blocks, and numbers of earwigs released or removed in study plots.

Block Nearest town in WA Spacing (m) Rootstock Year Treatment (replications) Earwigs added or removeda

Trees Rows

Gala Winchester 0.9 3.0 Malling 9 2016 Inundation (5) 47 Removal (5) 0.5 2017 Inundation (5) 0 Removal (5) 0 Fuji 1 Orondo 2.4 4.6 Malling 26 2017 Inundation (4) 215 Control (4) 0 Removal (4) 1.8 Fuji 2 Quincy 1.1 3.7 Malling 9 2017 Inundation (4) 140 Control (4) 0 Removal (4) 9.1 Fuji 3 Quincy 2.1 4.6 Malling 26 2017 Inundation (4) 230 Control (4) 0 Removal (4) 48.5

a Inundation treatment: the total number of earwigs released per tree in each replicate across the year (release rates were identical between replicates); removal treatment: the number of total earwigs removed per tree across the year averaged across replicates.

190 R.J. Orpet, et al. Biological Control 132 (2019) 189–198 following organic certification standards. Within orchard blocks, we Reference photos of limb-rub, russet, bitter pit symptoms, and rot can assigned plots to treatments where we removed, increased (inunda- be found on the Washington State University apple disorders guide tion), or did not manipulate earwig populations (control). All plots were (Jones et al., 2007) and Pacific Northwest Extension Pest Management separated by 30 m from other plots and block edges, as European ear- Handbooks (Pscheidt and Ocamb 2018) internet resources. The wigs do not often travel more than 30 m in a month (Moerkens et al., rounded holes were consistent with descriptions of earwig damage by 2010). The Gala block had 5 replicates of inundation and removal areas Nicholas et al. (2004) and Croxall et al. (1951) in laboratory trials. alternatingly interspersed, each consisting of three rows of 13 trees per Reference photos of stem bowl splits can be found in Opara (1993). The row. In 2017, we studied the same areas but made no manipulations. irregular holes were up to 5 cm in length and had multiple jagged The Fuji blocks had 4 replicates each of earwig removal, control (no straight edges. manipulation), and earwig inundation plots assigned arbitrarily to en- sure interspersion. Plots at all Fuji blocks consisted of two rows of eight 2.6. Primers for PCR assay trees per row. We designed woolly apple aphid specific primers for amplification 2.3. Earwig monitoring and manipulation of mitochondrial COI DNA using the following sequences from GenBank: KR034578.1 (woolly apple aphid), KR580369.1 (green apple Every other tree in the Gala block plots and each tree in Fuji block aphid, Aphis pomi De Geer), MG170854.1 (Spirea aphid, A. spiraecola plots received one 7.6 × 36 cm rolled strip of single-face corrugated Patch), MG169207.1 (rosy apple aphid, Dysaphis plantaginea Passerini), cardboard attached 1 m from the ground by rubber band to a tree limb. EU701888.1 (apple grain aphid, Rhopalosiphum insertum [Walker]), and Thus, each plot at the Gala block received 21 shelters, and each plot at KY323034.1 (potato aphid, Macrosiphum euphorbiae [Thomas]). We Fuji blocks received 16 shelters. On each visit, earwigs were shaken out aligned these sequences using Geneious software (version 11.0.4, of shelters into a bucket, counted, and either collected into plastic bags Biomatters Limited, Auckland, New Zealand), and ordered oligonu- (removal plots) or released onto the orchard floor (control/inundation cleotides (Sigma-Aldrich, St. Louis, MO) corresponding to 20 to 30 plots). In 2016, earwigs were monitored between 27 May and 10 Nov; base-pair length regions specific to woolly apple aphid to use as pri- in 2017 earwigs were monitored between 25 Apr and 14 Nov. mers. After initial testing of several pairs, we selected ‘35F’ (5′-GGAA Earwigs collected from removal plots, and from other orchards, TAATTGGTTCATCCTTA-3′) and ‘300R’ (5′-CTACAAATTATTATTATTA were released into inundation plots in variable amounts across time. In AAGAAGGG-3′) for PCR assays. the Gala block in 2016 we attempted to increase earwigs to 10 per To assess the specificity of our primer pair, we tested it with DNA shelter, an amount associated with woolly apple aphid biological con- extracted from woolly apple aphid, green apple aphid, rosy apple aphid, trol (Mueller et al., 1988; Nicholas et al., 2005; Quarrell et al., 2017). In and apple grain aphid. We extracted DNA from single aphids using the Fuji blocks in 2017 we attempted to increase earwig counts to twice QIAGEN DNeasy blood and tissue extraction kits following the in- the average observed in control plots. Releases were conducted at structions, but with a final elution of 100 µL of buffer. We ran PCRs with multiple times throughout the season, from 3 Jun to 4 Aug at the Gala a pre-made master mix (High Yield PCR EcoDry™ Premix, TaKaRa Bio block in 2016, and from 31 May to 11 Jul at the Fuji blocks in 2017. USA), adding 19.75 µL water, 1.25 µL 20X EvaGreen (Biotium), 1 µL Because of variation in the initial earwig abundance between orchards, forward primer (5 µM), 1 µL reverse primer (5 µM), and 2 µL of eluent and variation in shelter counts observed during the study, the total from DNA extraction per reaction. EvaGreen permitted real-time de- number of earwigs released in inundation plots differed between tection of DNA amplification and determination of amplicon melting orchard blocks (Table 1). Earwigs were released by shaking them out of temperatures with an iQ5 Multicolor Realtime PCR Detection System plastic bags across the orchard floor. The Gala block received no ma- with iCycler (Bio-Rad, Hercules, CA). The PCR program used was nipulation in 2017, but we continued monitoring earwigs in the same 3.5 min at 95 °C for initial denaturation, 38 cycles of [15 s at 95 °C for plots used in 2016. We did not quantify earwig life stages in this study, denaturation, 30 s at 60.8 °C for annealing, 45 s at 70 °C for extension], as phenology of this univoltine species has been studied in detail else- and 3 min at 72 °C for final extension. where: the third instar is expected to occur starting in June to May and We also ran PCRs with DNA extracted from guts of 20 field-collected is the earliest European earwig life stage to spend significant time in earwigs and sequenced amplicons to check for non-target amplification. tree canopies, and adults are expected to appear by the end of July The earwigs were collected within an hour of sunrise on 27 Jun from (Moerkens et al., 2011). cardboard shelters placed at Washington State University Sunrise Orchard (Rock Island, WA) and stored in a −20 °C freezer. Earwigs 2.4. Woolly apple aphid monitoring were surface sterilized for 1 min in 90% ethanol, then 1 min in 5% bleach, and blotted dry on paper towel. Then, guts were dissected using Plots were monitored for woolly apple aphid colonies from 20 May forceps under a stereoscope and placed into 1.5 mL centrifuge tubes to 10 Nov 2016, and from 25 Apr to 14 Nov 2017. On each visit, every containing 1.0 mm diameter glass beads (Bio Spec Products, Inc., other tree in Gala plots and every tree in Fuji plots was inspected for Bartlesville, OK) and lysis buffer (from QIAGEN DNeasy Blood and aphids by counting the number of infested leaf axils on ten random Tissue kit). Forceps were kept in 5% bleach and wiped clean with paper shoots, and the number of infested pruning cuts and other wounds on towel between dissections. Dissections were made in six total sets on trunks and woody branches up to a height of 1.5 m. different days, each including two negative controls: a gut from an earwig starved in the laboratory for over 10 d, and a blank sample with 2.5. Apple damage no tissue. After a set of dissections, tubes were shaken at 4.0 m/s for 1 min using a FastPrep-24 homogenizer (MP Biomedicals, LLC, Santa Fruit damage was assessed during the 2017 apple harvest period of Ana, CA). After PCR as above, but with water in place of EvaGreen, we the Gala, Fuji 1, and Fuji 2 orchards by inspecting up to 30 apples per checked for reaction products by gel electrophoresis and sent samples tree in each earwig removal and inundation plot (or each former re- with positive detections to MCLAB (South San Francisco, CA) for moval and inundation plot in the Gala block). The Fuji 3 orchard was cleanup and Sanger sequencing with our 35F primer. not assessed because apples were harvested before we could assess them. The following types of damage were noted: depressions (limb-rub 2.7. PCR assay sensitivity symptoms), russet, rounded holes, irregular holes, bitter bit symptoms, rot, splits (an oblong exposure of internal tissue) on sides of apples, We used PCR assays to determine the detectability of woolly apple stem bowl splits, and stem bowl splits with rounded holes in them. aphid DNA 1 and 16 h after ingestion of a single aphid nymph by an

191 R.J. Orpet, et al. Biological Control 132 (2019) 189–198 earwig in the laboratory. Earwigs were collected on 3 Aug from a 2.9. Statistical analyses commercial orchard and brought to a controlled temperature room (20 °C, 8L:16D). The earwigs were divided into groups of 10 in Ziploc We calculated the season-long average counts of earwigs and woolly bags with corrugated cardboard and no food until 11 Aug, when they apple aphid colonies within each plot (and year for the Gala block) as were transferred to individual 50 × 9 mm petri dishes and given one woolly apple aphid adult at 21:00 (start of night cycle). Each individual ∑ ()[()/2]DDYYiiii++11−+ earwig was set aside and frozen 1 or 16 h at −20 °C after it was ob- ∑ ()DDii+1 − served to begin feeding. Unfed earwigs used for negative controls were also frozen. We dissected earwig guts, extracted DNA, and conducted where Di and Di+1 are adjacent observation time points (days), and Yi PCR as described earlier. Reaction product melting temperatures of and Yi+1 are counts (per tree) on those days. The numerator of 75.9 ( ± 1) °C prompted gel electrophoresis, and visualizations of the this equation is cumulative insect-days, representing the magnitude and expected 265 base-pair amplicon were scored positive for woolly apple duration of insect abundance (Ruppel, 1983). We divided this by the aphid. number of days between the first and last observation to put orchard blocks on the same scale and to yield a number interpretable as the season-long average. We excluded the final observation from earwig 2.8. Molecular analysis of field-collected earwig guts calculations because earwigs move underground to nest during this time (Moerkens et al., 2011). Thus, 28 Oct was the last observation used On each of four dates per orchard, from 28 June to 3 October 2017, in the earwig calculations, whereas the last for woolly apple aphids was we collected up to 20 earwigs from one earwig inundation area (or 17 Nov. We compared the average earwig counts between removal former inundation area at the Gala orchard) for molecular analysis of versus inundation plots at the Gala block in 2016, and the former re- gut contents. Collections were made at least 7 d after any inundative moval versus former inundation plots in 2017 with two-sample t-tests. release. We collected, stored, dissected, extracted DNA, and did PCRs as For the Fuji blocks in 2017, we used ANOVA followed by Tukey tests to in Section 2.6, with EvaGreen. DNA extractions and PCR assays were compare season-long average earwig counts between removal, control, carried out in sets of up to 20 individuals plus two negative controls (as and inundation plots. For each block (and year for the Gala block), we in Section 2.6) per set. used Spearman’s rank correlation to assess the relationship between We prepared DNA for next-generation sequencing of gut contents by season-long earwig and woolly apple aphid colony counts. We used pooling 5 µL of eluent from each DNA extraction for each collection JMP® statistical software (Student Edition 12. SAS Institute Inc., Cary, date and location. One negative control was prepared by pooling 10 µL NC, 1989–2007) for these analyses. of DNA extraction eluent from unfed earwigs, and a second with 10 µL To compare apple damage between earwig removal and inundation from each blank negative control from each dissection set. The samples plots, we pooled all apple observations between plots of the three ob- (4 time points × 4 orchards = 16 samples plus two negative controls) served blocks and conducted Fisher’s exact tests in 2 × 2 tables (earwig were sent to RTLGenomics (Lubbock, Texas) for sequencing with removal or inundation by fruit damaged or not damaged) for each Illumina MiSeq. Primer pairs were selected for sequencing of damage type using the ‘fisher.test’ function in R (RCore Team, 2018). (mlCOIintfF and Fol-degen-rev; Krehenwinkel et al., 2017), plant (trnL To assess whether PCR assay detection rates of woolly apple aphid c-1 and trnL h-1, Lamb et al., 2016), and fungus (ITS1F, Gardes and in field-collected earwig guts varied between blocks and with woolly Bruns, 1993; ITS2, White et al., 1990) DNA. Sequence data was pro- apple aphid density, we conducted logistic regression using the ‘glm’ cessed by RTLGenomics following their standard methods as of Aug function in R: the response variable was binary count data (yes or no 2016, which includes denoising, chimera detection, and quality detection), block was a categorical explanatory variable, and the checking before clustering into operational taxonomic units (OTUs) number of woolly apple aphids found per tree in the collection plot (on using the UPARSE algorithm (Edgar, 2013). Taxonomic assignments by the same day as the collection or averaged between the most recent and RTLGenomics were based on finding the top 6 matches for each se- nearest future collection) was a continuous explanatory variable. quence within their genomics database (derived from sequences from NCBI), calculating the number of taxa agreeing with the top match divided by the total, and replacing any taxonomic level with a value under 0.51 with the designation “unknown.”

Fig. 1. Interviewees’ opinions on the overall importance of biological control and of specific biological control agents in their overall strategy for woolly apple aphid management in apple orchards (a); and interviewees’ responses on the pest status of earwigs in apple orchards (b).

192 R.J. Orpet, et al. Biological Control 132 (2019) 189–198

3. Results years, woolly apple aphid population dynamics followed a similar pattern of a mid-season peak and crash during summer, and a recovery 3.1. Interviews in fall (Fig. 3). Compared to control and removal plots, aphid counts were lower and less variable in earwig inundation plots, which had Biological control was considered important for woolly apple aphid lower mid-season peaks and less late-season recovery (Fig. 3). At each management by almost all interviewees (Fig. 1a). Although most in- orchard, control and plots with the lowest earwig populations (< 2 terviewees thought earwigs may contribute to woolly apple aphid earwigs per shelter) (Fig. 2) were prone to the greatest summer peaks biological control, responses were usually rated as ‘potentially im- and fall increases (Fig. 3). For each orchard and year, earwigs were portant’ rather than ‘important’ (Fig. 1a). This was due to uncertainty or rarely found in tree canopies until June (Fig. 4), as expected for this a lack of experience: no interviewee reported directly observing earwigs univoltine species whose early instars mainly forage on the ground feeding on aphids, whereas other ‘important’ predators were often ob- (Moerkens et al., 2011). Therefore, we were not able to mass-collect served in aphid colonies, as were aphid mummies from the parasitoid A. and release enough earwigs to greatly elevate their abundance in in- mali. undation plots until mid-June (Fig. 4), and aphid abundance tended to Fourteen of the 15 people interviewed had seen earwigs on da- be similar between treatments until July (Fig. 3). In addition, in Oct maged apples, but only five considered earwigs to be pests (Fig. 1b). through Nov, when earwigs move to underground nests, woolly apple Two people thought earwigs were a considerable pest and three were aphid abundance increased notably in some inundation plots (Fig. 3). involved in a decision to suppress earwigs (Fig. 1b). People who did not Earwig abundance across time was similar between control and re- consider earwigs as pests either thought it was likely that earwigs fed moval plots (Fig. 4), as removal of all earwigs found in shelters on each mostly in existing damage rather than initiating it, or they observed low visit did not greatly reduce their abundance (Table 2). In inundation incidence of damage. Ten people observed earwigs in splitting apples. plots, earwig abundance remained high until the end of the season in Several people noted that any split apple is culled, so unless earwigs October, even though our latest releases were 4 Aug in 2016 (Gala only) caused the splits, earwig presence was inconsequential. One person said and 11 Jul in 2017 (Fujis only). that earwigs entered and damaged open-calyx variety apples, but such There was no difference in incidence of all fruit damage types be- varieties are rare. Only two people described earwig damage as small tween inundation and removal plots (Fisher’s exact tests, all P > 0.15), rounded holes, consistent with Nicholas et al. (2004), although one of except for rotting apples (P=0.062; 0 apples in inundation plots, 5 in them doubted earwigs caused economic damage. Honeycrisp cultivar removal plots) and marginally more stem bowl splits with holes in apples were most commonly reported as associated with earwigs (they earwig inundation plots (Table 3). However, the frequency of any stem were mentioned as a particular concern by the two interviewees who bowl split (with or without holes) was similar between treatments considered earwigs pests, and by eight others), followed by Gala (three (Table 3). people). Leaf feeding was mentioned by three people but was not considered important. 3.3. Primers and PCR assay sensitivity Only six of the 15 interviewees could give a specific action threshold for woolly apple aphid management. These thresholds included: three Woolly apple aphid DNA was detected in all 18 earwig guts 1 h after to four woolly apple aphid colonies per tree, five nearby trees with aphid ingestion and 12 out of 18 guts 16 h after aphid ingestion in the many colonies, 20 to 30 percent of shoots infested in a block, most trees laboratory. Out of 20 earwigs collected from Sunrise Orchard, 15 tested in a block infested, or any detection of the aphid (two people). A de- positive for aphid DNA, and all sequences obtained matched woolly cision to spray for woolly apple aphid was always based on a visual apple aphid. Woolly apple aphid DNA was not detected in any negative assessment of colony presence or abundance, but often depended on control. context, such as: the consultant’s understanding of the orchard owner’s tolerance to aphid abundance (1 person), a higher level of abundance 3.4. Molecular gut content analysis of field-collected earwigs tolerated on older trees (1 person), reduced urgency to suppress aphids after apple harvest (2 people), and less concern about the aphid if The frequency of woolly apple aphid detection in earwig guts dif- natural enemy abundance seemed high (6 people). fered between orchard blocks (Z = 1.5, P = 0.014) but was not affected by woolly apple aphid abundance (Z = 0.9, P = 0.35). High detection 3.2. Field study rates could be found during periods when woolly apple aphid abun- dance was low (Fig. 5). Earwigs were more abundant in inundation than removal plots at From next generation sequencing, the raw list of taxonomic as- the Gala orchard in 2016, a difference that held in 2017 without further signments and counts of reads for each sample of earwigs is in Table S1, manipulation (Table 2). Few earwigs (mean = 0.05 per shelter) were and a summarized list of taxa detected in each sample after removal of found at removal plots in 2016. At each Fuji orchard, control and re- taxa with ≤4 reads (Pompanon et al., 2012) and Dermaptera is in Table moval plots had similar earwig abundance, and inundation plots had S2. Fungi had greater diversity in the dataset (196 taxa) compared with significantly greater abundance (Table 2). (61 taxa) and plants (37 taxa) (Table S2). Pooled across all sites Season-long average earwig counts were negatively correlated to and sampling days, apple pests Lygus sp, and E. lanigerum, were de- average woolly apple aphid colony counts (Fig. 2). Across all blocks and tected, along with predators such as Nabis sp, lady beetles (Hippodamia

Table 2 Mean season-long averages of earwigs found per shelter and SEM for different earwig manipulation treatments at all orchards blocks and years.

Treatment Mean season-long average of earwigs per shelter ± SEM

Gala 2016 Gala 2017a Fuji 1 2017 Fuji 2 2017 Fuji 3 2017

Remove 0.05 ± 0.01a 0.55 ± 0.07a 0.13 ± 0.04a 0.78 ± 0.32a 2.92 ± 0.36a Control 0.69 ± 0.53a 1.79 ± 0.76a 4.04 ± 1.12a Inundate 4.33 ± 0.10a 4.20 ± 0.76b 6.10 ± 1.15b 7.01 ± 0.68b 7.68 ± 0.48b

Values followed by different letters in the same column indicate significant differences (t-tests for Gala plot, Tukey tests for Fuji plots, a = 0.05). a Experimental manipulation at the Gala block (removal and inundation treatments) was conducted in 2016 only.

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Fig. 3. Summary of woolly apple aphid population dynamics in earwig in- undation, removal, and control plots in 2016 and 2017 at a Gala orchard (a, b), and three Fuji orchards in 2017 (c–e). Points show mean colonies per tree for each treatment type and error bars show 1 standard deviation, N = 5 replica- Fig. 2. Season-long averages of woolly apple aphid colony counts and earwigs tions per point (a, b) or 4 per point (c–e). in earwig inundation, removal, and control plots in 2016 and 2017 at a Gala orchard (a, b), and three Fuji orchards in 2017 (c–e). There was always a ne- gative correlation between aphids and earwigs (Gala, 2016: ρ = −0.84, has 100% similarity to apple ( domestica) and several other Ro- P = 0.002; Gala, 2017: ρ = −0.76, P = 0.01; Fuji 1: ρ = −0.93, P < 0.0001; saceae family plants. We believe these reads most likely correspond to Fuji 2: ρ = −0.96, P < 0.0001; Fuji 3: ρ = −0.71, P = 0.009). apple. Within our summarized list (Table S2), 10 taxa (7 fungi and 3 plant) convergens, borealis, and Stethorus punctillum), Phalangium were found in the sample of negative control earwig guts, and no taxa opilio, and a variety of innocuous Diptera, Coleoptera, and Hemiptera. were found in the blank negative control. One of the plants was the The plant Sorbus sp was detected in all 16 field samples, but this putative M. domestica discussed above, but only 10% as many reads fi taxonomic assignment may have been an artifact of the RTLGenomics (468) were found compared to the minimum found in any eld sample standard method. The COI region of Sorbus amplified by our primer pair (range: 4767 to 38,072), suggesting that M. domestica consumption occurred in the field. The other plants and fungi in the negative control

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Table 3 Number of apples inspected, percentage with no damage, and percentage with stem bowl splits or stem bowl splits with holes in them at earwig inundation and removal plots in three orchard blocks in 2017.

Apples inspected

With stem bowl splits

Site Treatment Total (#) Not damaged (%) With holes (%) All (%)

Gala Inundate 1048 93.0 0.5 6.4 Remove 1050 94.3 0.0 5.1 Fuji 1 Inundate 1478 94.3 0.2 0.9 Remove 1482 94.0 0.3 1.0 Fuji 2 Inundate 1835 96.9 0.3 1.0 Remove 1831 96.0 0.1 2.0

Inundate total 4361 95.1 0.3 2.3 Remove total 4363 94.9 0.1 2.4 Fisher’s exact test n/a P = 0.95 P = 0.063 P = 0.78

maximum of an average of ca. three colonies per tree at earwig removal plots of the most-infested orchard. This level of infestation would have been above the level of tolerance for interviewees who reported specific action thresholds. Earwig inundation plots always had an average of less than one woolly apple aphid colony per tree, which would have been considered tolerable to most. Our results support studies which suggested that earwigs are important woolly apple aphid predators (Nicholas et al., 2005; Quarrell et al., 2017; Stap et al., 1987), and also highlight that earwigs are potentially underappreciated by orchard decision-makers, who might benefit from considering European earwig in their integrated pest management strategies. Cause and effect relationships which are difficult to directly observe are expected to be less appreciated in agricultural management (Carolan, 2006). This expectation is consistent with our results, as in- terviewees were more uncertain about beneficial effects of nocturnal earwigs compared with predators which can be active during the day, such as syrphids, coccinellids, and lacewings. Specialized techniques such as video-recording (Frank et al., 2011) and molecular analyses (Petráková et al., 2016; Romeu-Dalmau et al., 2012) can be helpful in providing direct evidence on the roles of difficult-to-observe natural enemies. In Central Washington, researchers have spread information on enhancing biological control to apple industry decision-makers through interactive on-farm experiences and internet resources (Gadino et al., 2016). Incorporation of earwigs into such efforts could increase understanding of their benefits, particularly if direct evidence, such as visual or molecular data, is provided. The role of European earwigs in biological control is related to their phenology and feeding behavior. European earwigs are univoltine, ac- tive from spring to fall (Moerkens et al., 2011), have low dispersal ability (Moerkens et al., 2010), and can survive on many different foods. These traits exemplify a resident predator, one which can be Fig. 4. Summary of earwig population dynamics in earwig inundation, re- present regardless of the density of any single prey species (Piñol et al., moval, and control plots in 2016 and 2017 at a Gala orchard (a, b), and three 2009). Because they can be present during periods of low pest density, – Fuji orchards in 2017 (c e). Points show mean earwigs per shelter for each resident predators can quickly exhibit a functional response to limit treatment type and error bars show 1 standard deviation, N = 5 replications per pest population increases (Murdoch et al., 1985; Piñol et al., 2009). Our point (a, b) or 4 per point (c–e). results match this expectation, as earwig presence and consumption of aphids while the aphid populations were at low-densities may have may have been laboratory contaminants. prevented rapid aphid population increases in the summer. Rapid aphid population growth with a mid-season peak was observed in some 4. Discussion control and earwig removal plots, but not in inundation plots. Fur- thermore, our molecular analysis confirmed that earwigs consumed We found that apple orchard decision-makers usually thought ear- woolly apple aphids when densities of this pest were very low. This wigs were biological control agents of potential importance, and some finding was similar to molecular gut content analyses showing Eur- thought earwigs were pests. Our field study provided evidence that opean earwig consumption of aphids in citrus orchards, even during earwigs can suppress woolly apple aphids, and we found no evidence periods when aphids were rare (Romeu-Dalmau et al., 2012). that earwigs damaged apples. Woolly apple aphid pest pressure was not Although we have suggested that earwigs can prevent aphid extremely high during our study, but colony counts reached a

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Fig. 5. Woolly apple aphid colonies per tree (primary axis, circles with solid lines) and percentage of earwig guts testing positive for woolly apple aphid (secondary axis, open diamonds with dashed lines) within one earwig inundation plot at each of four orchard blocks: Gala 1 (a), Fuji 1 (b), Fuji 2 (c), and Fuji 3 (d) in 2017. The sample sizes for woolly apple aphid gut detection were 19 for the second Fuji 1 collection and the first two Fuji 3 collections, 18 for the third Gala collection, and 20 for all other col- lections.

populations from increasing, they may fail to decrease the abundance of economically damaging, particularly on young trees, but the only re- high-density aphid populations (Dib et al., 2016a), as univoltinism and ports of leaf damage by earwigs we are aware of have considered it low dispersal ability of European earwig precludes them from re- insignificant (Carroll et al., 1985; Nicholas et al., 2004). Although our sponding numerically to pest outbreaks within a season. To comple- results suggest earwigs are not apple pests, the cost versus benefitof ment earwigs, other predators such as coccinellids, syrphids, and earwigs could vary with context. For example, one interviewee sug- chrysopids, and the specialist A. mali can suppress woolly apple aphids, gested that earwigs can be pests of open-calyx apple cultivars, and it but they might not always be present because of narrower windows of remains possible that earwigs could more readily damage some apple phenology or greater density-dependence on prey (Gontijo et al., 2012; cultivars which were not included in our study or others. Lordan et al., 2015; Quarrell et al., 2017). A build-up of these species, in We found DNA from other predators in earwig guts, but intraguild addition to high summer temperatures above woolly apple aphid’s predation did not appear to disrupt biological control, as greater earwig upper developmental threshold of 32 °C (Asante et al., 1991), may have abundance resulted in lower woolly apple aphid abundance. This is important role in the mid-season woolly apple aphid population crashes consistent with other studies suggesting that a combination of earwigs observed in our study and others (Beers et al., 2010). Therefore, a di- with other natural enemies increases aphid pest suppression (Dib et al., verse natural enemy community will likely result in the most consistent 2011, 2016b; Quarrell et al., 2017). Although coccinellid DNA was and effective biological control across the season. For example, Quarrell found in earwig guts, the assays used in our study cannot determine the et al. (2017) showed that a combination of earwigs with A. mali can number consumed and whether they were depredated or were sca- provide greater woolly apple aphid suppression than either taxa alone, venged (Pompanon et al., 2012). Moreover, apple tree DNA in earwig in part because earwigs can be present earlier in the season before A. guts could have come from leaves, decaying fruit on the orchard floor, mali population levels can build. Similarly, in a cage-exclusion study in or consumption of apple herbivores. Using a species-specific PCR assay the field, Gontijo et al. (2012) showed that woolly apple aphid sup- similar to what we used for woolly apple aphid detection, Unruh et al. pression was greatest when both A. mali and generalist predators were (2016) commonly detected DNA of the key lepidopteran pest of apple, present. (Cydia pomonella; Tortricidae), in field-collected earwig We found no evidence that earwigs caused economic damage to guts. We did not find codling moth DNA and rarely detected pest DNA apples. Possible earwig feeding in stem bowl splits (evidenced by holes in earwig guts through our next-generation sequencing assays. How- in splits) was marginally more frequent in inundation plots, but split- ever, absence of detection does not prove that consumption did not ting has a physiological cause (Opara, 1993), and interviewees reported occur, as amplification efficiency of the universal primers used in our that split apples are culled, regardless of earwigs. We found particular assays varies between species (Krehenwinkel et al., 2017; Lamb et al., concern about earwig damage to Honeycrisp cultivar apples, which are 2016; Pompanon et al., 2012). Overall, our next-generation sequencing susceptible to stem bowl splitting, have short stems, and grow in tight results highlight that earwigs are generalists capable of consuming clusters which may be more vulnerable to physical rubbing and poking many foods in apple orchards, and more focused studies will be needed damage from limbs (Wargo and Watkins, 2004). These factors create to assess the importance of specific trophic links. attractive shelter areas for earwigs, so the correlation between earwig presence with damage, and the current high value of Honeycrisp apples (Granatstein and Kirby, 2018), may contribute to perceptions that 5. Conclusion earwigs are a pest. Chewing damage to apple leaves, if severe, could be Biological control agents that are difficult to observe could often

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