Field evidence and grower perceptions on the roles of an

Field evidence and grower perceptions on the roles of an omnivore, European earwig, in apple orchardsBiological Control
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Field evidence and grower perceptions on the roles of an omnivore, European earwig, in apple orchards
Robert J. Orpeta,, Jessica R. Goldbergerb, David W. Crowderc, Vincent P. Jonesa
aWashington State University, Department of Entomology, Tree Fruit Research and Extension Center, 1100 N Western Ave, Wenatchee, WA 98801, United States bWashington State University, Department of Crop and Soil Sciences, 263 Johnson Hall, PO Box 646420, Pullman, WA 99164, United States cWashington State University, Department of Entomology, 166 FSHN Bldg, PO Box 646382, Pullman, WA 99164, United States
A R T I C L E I N F O
Keywords: Inundation biological control Diet breadth Gut content analysis Interviews Predator behavior
A B S T R A C T
In agroecosystems, omnivores can be beneficial predators or harmful herbivores. In apple orchards, the omnivorous European earwig (Forficula auricularia) is thought to be a key predator of woolly apple aphid (Eriosoma lanigerum), but has also been implicated in feeding on apple fruit. Assessing the effects of earwigs in orchards is difficult because they are nocturnal, and their damage to fruits can resemble other wounds. Apple orchardists thus may manage earwigs as either predators or pests based on subjective opinions. To understand current 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 insecticides 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
Omnivores have been called “plant bodyguards” because they can suppress pests and also persist during periods when pests are not abundant (gren et al., 2012). However, an omnivore’s benefits can be offset if they damage marketed plant structures (Puentes and Björkman, 2017) or consume other predators (Prasad and Snyder, 2006). To ap- propriately manage omnivore species, it is necessary to understand their potential effects as both an herbivore and as a predator. For example, the omnivore Campylomma verbasci (Meyer-Dür) (Hemiptera: Miridae) is an apple pest when fruits are young and susceptible to feeding damage, but it is considered a beneficial predator later in the year and in pears (Reding et al., 2001).
The omnivorous European earwig (Forficula auricularia L.) has been implicated in suppressing woolly apple aphids (Eriosoma lanigerum [Hausmann]) in apple orchards (Mueller et al., 1988; Quarrell et al.,
2017), but has also been implicated as a pest. European earwigs can damage apples when confined together with them (Carroll et al., 1985; Croxall et al., 1951; Crumb et al., 1941; Glen, 1975; Nicholas et al., 2004), but do not always do so, even after up to seven days with no alternative food (Nicholas et al., 2004). Earwig damage to apples can resemble wounds caused by other organisms or mechanical injury, making it difficult to assess in the open field, where earwig damage might be uncommon because other preferable foods are available. Two field studies found no evidence that excluding European earwigs from trees with physical barriers decreased apple damage (Crumb et al., 1941; Nicholas et al., 2004). One field study using insecticidal barriers suggested the opposite (Croxall et al., 1951), but earwig damage was measured indirectly as the incidence of brown rot, which could infest wounds caused by other organisms.
Although researchers in many regions worldwide are interested in promoting biological control of woolly apple aphid by European
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
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).
Biological Control 132 (2019) 189–198
Available online 22 February 2019 1049-9644/ © 2019 Elsevier Inc. All rights reserved.
earwigs (Lordan et al., 2015; Mueller et al., 1988; Nicholas et al., 2004; Quarrell et al., 2017) and in conserving earwig populations in orchards (Fountain and Harris, 2015; Moerkens et al., 2012), commercial orchardists may consider European earwig a pest rather than a beneficial predator. European earwigs are nocturnal, so they are difficult to observe attacking pests. In addition, European earwigs shelter in tight spaces during the day, so they are relatively easy to observe in damaged fruit, which could have been injured by a different source. Therefore, agricultural professionals, who commonly make their management decisions in response to direct observations (Carolan, 2006; Sherman and Gent, 2014), may be interested in promoting or suppressing earwigs depending on subjective opinions.
In this study, we addressed knowledge gaps on both the subjective and objective roles of earwigs in apple orchards in Washington State, USA. We assessed the opinions of apple orchard decision-makers through in-depth interviews. We also conducted field experiments where we manipulated earwig abundance, then measured apple fruit damage, woolly apple aphid abundance, and evaluated earwig feeding habits through molecular analysis of their gut contents. Previous experiments addressing the positive or negative roles of earwigs in apple orchards manipulated earwig abundance with barriers preventing earwig movement into trees (Crumb et al., 1941; Croxall et al., 1951; Mueller et al., 1988; Nicholas et al., 2004) or with insecticide applications (Ravensberg, 1981). Because these treatments could affect other natural enemies, we directly released or collected earwigs in experimental plots rather than using barriers or pesticides to manipulate earwig abundance. Overall, our study combined interview data with experimental data to assess the perceived and objective effects of an omnivore, and to address whether it should be managed as a beneficial predator or harmful herbivore.
2. Methods
2.1. Interviews
We recorded semi-structured interviews with 15 apple pest management decision-makers from Washington State, USA. These individuals were solicited at Washington extension meetings in 2016. The group included eight pest management consultants, three field managers, and four private owners; 13 were male and two were female. Interviewees ranged from 26 to 77 years old (median= 58) and had between 1 and 40 years of experience in the fruit industry (median=30). They worked with between 0.4 and 3560 ha of apple (median=364), and all but one consultant and one manager worked with both organic and conventional apples at the time of the interview.
Interviews typically lasted 1 h and were structured to learn about
experiences and opinions related to management of woolly apple aphids. For this study, we only analyzed responses from a subset of questions about biological control and specifically the effects of lacewings (Neuroptera: Chrysopidae), syrphids (Diptera: Syrphidae), la- dybugs (Coleoptera: Coccinellidae), Aphelinus mali (the specialist parasitoid of woolly apple aphid; Hymenoptera: Aphelinidae), Daraeocoris brevis (Uhler) (a generalist predator; Hemiptera: Miridae), ground beetles (Coleoptera: Carabidae), and earwigs (European earwig). These natural enemies were chosen because they are thought to comprise the main woolly apple aphid natural enemy taxa in Washington State (Gontijo et al., 2012), except for ground beetles. Ground beetles have not been specifically studied as a woolly apple aphid predator in the field, but can consume woolly apple aphids in the laboratory (Orpet, personal observations). Interviewees were also asked about what kinds of damage (if any) earwigs caused to apples and whether they ever took any management actions meant to promote or suppress earwigs. In addition, because we know of no widely estab- lished economic thresholds for woolly apple aphid management, we analyzed responses about interviewees’ woolly apple aphid action thresholds to assess whether aphid counts in our field study would have been considered below or above acceptable levels. A list of the relevant questions can be found in Supplementary Document S1.
Similar to other studies involving in-depth interviews (e.g., Sherman and Gent, 2014), we used a qualitative approach to analyzing responses, placing emphasis on understanding the experiences and rea- soning behind the opinions of interviewees. In addition, to summarize our interview data, we categorized opinions on the role of biological control, and of each natural enemy species, as: ‘important’ (having a considerable role), ‘potentially important’ (having a role, but with uncertainty of its importance, or having an inconsistent role), ‘unknown’ (lack of knowledge of its existence, or uncertainty that a potential role exists), or ‘not important’ (an assertion that there is no considerable role). We rated interviewees’ importance of earwigs as a pest as ‘yes’ (caused considerable economic damage), ‘slight’ (caused slight economic damage, but there was minimal concern about it), or ‘no’ (had not caused economic damage).
2.2. Experimental design and treatments
To collect objective data to compare with interviews, we monitored European earwig and woolly apple aphid populations within one Gala cultivar orchard block in 2016 and 2017, and three Fuji cultivar blocks in 2017 (Table 1). The Gala block was managed by one of the interviewed managers, Fuji block 1 was managed by one of the interviewed owners, and Fuji blocks 2 and 3 were managed by one of the interviewed 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.
R.J. Orpet, et al. Biological Control 132 (2019) 189–198
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following organic certification standards. Within orchard blocks, we assigned plots to treatments where we removed, increased (inundation), or did not manipulate earwig populations (control). All plots were separated by 30m from other plots and block edges, as European earwigs do not often travel more than 30m in a month (Moerkens et al., 2010). The Gala block had 5 replicates of inundation and removal areas alternatingly interspersed, each consisting of three rows of 13 trees per row. In 2017, we studied the same areas but made no manipulations. The Fuji blocks had 4 replicates each of earwig removal, control (no manipulation), and earwig inundation plots assigned arbitrarily to en- sure interspersion. Plots at all Fuji blocks consisted of two rows of eight trees per row.
2.3. Earwig monitoring and manipulation
Every other tree in the Gala block plots and each tree in Fuji block plots received one 7.6×36 cm rolled strip of single-face corrugated cardboard attached 1m from the ground by rubber band to a tree limb. Thus, each plot at the Gala block received 21 shelters, and each plot at Fuji blocks received 16 shelters. On each visit, earwigs were shaken out of shelters into a bucket, counted, and either collected into plastic bags (removal plots) or released onto the orchard floor (control/inundation plots). In 2016, earwigs were monitored between 27 May and 10 Nov; in 2017 earwigs were monitored between 25 Apr and 14 Nov.
Earwigs collected from removal plots, and from other orchards, were released into inundation plots in variable amounts across time. In the Gala block in 2016 we attempted to increase earwigs to 10 per shelter, an amount associated with woolly apple aphid biological control (Mueller et al., 1988; Nicholas et al., 2005; Quarrell et al., 2017). In the Fuji blocks in 2017 we attempted to increase earwig counts to twice the average observed in control plots. Releases were conducted at multiple times throughout the season, from 3 Jun to 4 Aug at the Gala block in 2016, and from 31 May to 11 Jul at the Fuji blocks in 2017. Because of variation in the initial earwig abundance between orchards, and variation in shelter counts observed during the study, the total number of earwigs released in inundation plots differed between orchard blocks (Table 1). Earwigs were released by shaking them out of plastic bags across the orchard floor. The Gala block received no manipulation in 2017, but we continued monitoring earwigs in the same plots used in 2016. We did not quantify earwig life stages in this study, as phenology of this univoltine species has been studied in detail else- where: the third instar is expected to occur starting in June to May and is the earliest European earwig life stage to spend significant time in tree canopies, and adults are expected to appear by the end of July (Moerkens et al., 2011).
2.4. Woolly apple aphid monitoring
Plots were monitored for woolly apple aphid colonies from 20 May to 10 Nov 2016, and from 25 Apr to 14 Nov 2017. On each visit, every other tree in Gala plots and every tree in Fuji plots was inspected for aphids by counting the number of infested leaf axils on ten random shoots, and the number of infested pruning cuts and other wounds on trunks and woody branches up to a height of 1.5 m.
2.5. Apple damage
Fruit damage was assessed during the 2017 apple harvest period of the Gala, Fuji 1, and Fuji 2 orchards by inspecting up to 30 apples per tree in each earwig removal and inundation plot (or each former removal and inundation plot in the Gala block). The Fuji 3 orchard was not assessed because apples were harvested before we could assess them. The following types of damage were noted: depressions (limb-rub symptoms), russet, rounded holes, irregular holes, bitter bit symptoms, rot, splits (an oblong exposure of internal tissue) on sides of apples, stem bowl splits, and stem bowl splits with rounded holes in them.
Reference photos of limb-rub, russet, bitter pit symptoms, and rot can be found on the Washington State University apple disorders guide (Jones et al., 2007) and Pacific Northwest Extension Pest Management Handbooks (Pscheidt and Ocamb 2018) internet resources. The rounded holes were consistent with descriptions of earwig damage by Nicholas et al. (2004) and Croxall et al. (1951) in laboratory trials. Reference photos of stem bowl splits can be found in Opara (1993). The irregular holes were up to 5 cm in length and had multiple jagged straight edges.
2.6. Primers for PCR assay
We designed woolly apple aphid specific primers for amplification of mitochondrial COI DNA using the following sequences from GenBank: KR034578.1 (woolly apple aphid), KR580369.1 (green apple aphid, Aphis pomi De Geer), MG170854.1 (Spirea aphid, A. spiraecola Patch), MG169207.1 (rosy apple aphid, Dysaphis plantaginea Passerini), EU701888.1 (apple grain aphid, Rhopalosiphum insertum [Walker]), and KY323034.1 (potato aphid, Macrosiphum euphorbiae [Thomas]). We aligned these sequences using Geneious software (version 11.0.4, Biomatters Limited, Auckland, New Zealand), and ordered oligonu- cleotides (Sigma-Aldrich, St. Louis, MO) corresponding to 20 to 30 base-pair length regions specific to woolly apple aphid to use as primers. After initial testing of several pairs, we selected ‘35F’ (5′-GGAA TAATTGGTTCATCCTTA-3′) and ‘300R’ (5′-CTACAAATTATTATTATTA AAGAAGGG-3′) for PCR assays.
To assess the specificity of our primer pair, we tested it with DNA extracted from woolly apple aphid, green apple aphid, rosy apple aphid, and apple grain aphid. We extracted DNA from single aphids using QIAGEN DNeasy blood and tissue extraction kits following the in- structions, but with a final elution of 100 µL of buffer. We ran PCRs with a pre-made master mix (High Yield PCR EcoDry™ Premix, TaKaRa Bio USA), adding 19.75 µL water, 1.25 µL 20X EvaGreen (Biotium), 1 µL forward primer (5 µM), 1 µL reverse primer (5 µM), and 2 µL of eluent from DNA extraction per reaction. EvaGreen permitted real-time detection of DNA amplification and determination of amplicon melting temperatures with an iQ5 Multicolor Realtime PCR Detection System with iCycler (Bio-Rad, Hercules, CA). The PCR program used was 3.5 min at 95 °C for initial denaturation, 38 cycles of [15 s at 95 °C for denaturation, 30 s at 60.8 °C for annealing, 45 s at 70 °C for extension], and 3min at 72 °C for final extension.
We also ran PCRs with DNA extracted from guts of 20 field-collected earwigs and sequenced amplicons to check for non-target amplification. The earwigs were collected within an hour of sunrise on 27 Jun from cardboard shelters placed at Washington State University Sunrise Orchard (Rock Island, WA) and stored in a −20 °C freezer. Earwigs were surface sterilized for 1min in 90% ethanol, then 1min in 5% bleach, and blotted dry on paper towel. Then, guts were dissected using forceps under a stereoscope and placed into 1.5mL centrifuge tubes containing 1.0 mm diameter glass beads (Bio Spec Products, Inc., Bartlesville, OK) and lysis buffer (from QIAGEN DNeasy Blood and Tissue kit). Forceps were kept in 5% bleach and wiped clean with paper towel between dissections. Dissections were made in six total sets on 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 no tissue. After a set of dissections, tubes were shaken at 4.0 m/s for 1min using a FastPrep-24 homogenizer (MP Biomedicals, LLC, Santa Ana, CA). After PCR as above, but with water in place of EvaGreen, we checked for reaction products by gel electrophoresis and sent samples with positive detections to MCLAB (South San Francisco, CA) for cleanup and Sanger sequencing with our 35F primer.
2.7. PCR assay sensitivity
We used PCR assays to determine the detectability of woolly apple aphid DNA 1 and 16 h after ingestion of a single aphid nymph by an
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earwig in the laboratory. Earwigs were collected on 3 Aug from a commercial orchard and brought to a controlled temperature room (20 °C, 8L:16D). The earwigs were divided into groups of 10 in Ziploc bags with corrugated cardboard and no food until 11 Aug, when they were transferred to individual 50× 9mm petri dishes and given one woolly apple aphid adult at 21:00 (start of night cycle). Each individual earwig was set aside and frozen 1 or 16 h at −20 °C after it was observed to begin feeding. Unfed earwigs used for negative controls were also frozen. We dissected earwig guts, extracted DNA, and conducted PCR as described earlier. Reaction product melting temperatures of 75.9 (± 1) °C prompted gel electrophoresis, and visualizations of the expected 265 base-pair amplicon were scored positive for woolly apple aphid.
2.8. Molecular analysis of field-collected earwig guts
On each of four dates per orchard, from 28 June to 3 October 2017, we collected up to 20 earwigs from one earwig inundation area (or former inundation area at the Gala orchard) for molecular analysis of gut contents. Collections were made at least 7 d after any inundative release. We collected, stored, dissected, extracted DNA, and did PCRs as in Section 2.6, with EvaGreen. DNA extractions and PCR assays were carried out in sets of up to 20 individuals plus two negative controls (as in Section 2.6) per set.
We prepared DNA for next-generation sequencing of gut contents by pooling 5 µL of eluent from each DNA extraction for each collection date and location. One negative control was prepared by pooling 10 µL of DNA extraction eluent from unfed earwigs, and a second with 10 µL from each blank negative control from each dissection set. The samples (4 time points× 4 orchards= 16 samples plus two negative controls) were sent to RTLGenomics (Lubbock, Texas) for sequencing with Illumina MiSeq. Primer pairs were selected for sequencing of arthropod (mlCOIintfF and Fol-degen-rev; Krehenwinkel et al., 2017), plant (trnL c-1 and trnL h-1, Lamb et al., 2016), and fungus (ITS1F, Gardes and Bruns, 1993; ITS2, White et al., 1990) DNA. Sequence data was pro- cessed by RTLGenomics following their standard methods as of Aug 2016, which includes denoising, chimera detection, and quality checking before clustering into operational taxonomic units (OTUs) using the UPARSE algorithm (Edgar, 2013). Taxonomic assignments by RTLGenomics were based on finding the top 6 matches for each sequence 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.”
2.9. Statistical analyses
∑ − +
∑ −
+ +
+
( )[( )/2] ( )
1
where Di and Di+1 are adjacent observation time points (days), and Yi
and Yi+1 are insect counts (per tree) on those days. The numerator of this equation is cumulative insect-days, representing the magnitude and duration of insect abundance (Ruppel, 1983). We divided this by the 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 calculations because earwigs move underground to nest during this time (Moerkens et al., 2011). Thus, 28 Oct was the last observation used in the earwig calculations, whereas the last for woolly apple aphids was 17 Nov. We compared the average earwig counts between removal versus inundation plots at the Gala block in 2016, and the former removal versus former inundation plots in 2017 with two-sample t-tests. For the Fuji blocks in 2017, we used ANOVA followed by Tukey tests to compare season-long average earwig counts between removal, control, and inundation plots. For each block (and year for the Gala block), we used Spearman’s rank correlation to assess the relationship between season-long earwig and woolly apple aphid colony counts. We used JMP® statistical software (Student Edition 12. SAS Institute Inc., Cary, NC, 1989–2007) for these analyses.
To compare apple damage between earwig removal and inundation plots, we pooled all apple observations between plots of the three observed blocks and conducted Fisher’s exact tests in 2×2 tables (earwig removal or inundation by fruit damaged or not damaged) for each damage type using the ‘fisher.test’ function in R (RCore Team, 2018).
To assess whether PCR assay detection rates of woolly apple aphid in field-collected earwig guts varied between blocks and with woolly apple aphid density, we conducted logistic regression using the ‘glm’ function in R: the response variable was binary count data (yes or no detection), block was a categorical explanatory variable, and the number of woolly apple aphids found per tree in the collection plot (on the same day as the collection or averaged between the most recent and nearest future collection) was a continuous explanatory variable.
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).
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3. Results
3.1. Interviews
Biological control was considered important for woolly apple aphid management by almost all interviewees (Fig. 1a). Although most interviewees thought earwigs may contribute to woolly apple aphid biological control, responses were usually rated as ‘potentially important’ rather than ‘important’ (Fig. 1a). This was due to uncertainty or a lack of experience: no interviewee reported directly observing earwigs feeding on aphids, whereas other ‘important’ predators were often observed in aphid colonies, as were aphid mummies from the parasitoid A. mali.
Fourteen of the 15 people interviewed had seen earwigs on damaged apples, but only five considered earwigs to be pests (Fig. 1b). Two people thought earwigs were a considerable pest and three were involved in a decision to suppress earwigs (Fig. 1b). People who did not consider earwigs as pests either thought it was likely that earwigs fed mostly in existing damage rather than initiating it, or they observed low incidence of damage. Ten people observed earwigs in splitting apples. Several people noted that any split apple is culled, so unless earwigs caused the splits, earwig presence was inconsequential. One person said that earwigs entered and damaged open-calyx variety apples, but such varieties are rare. Only two people described earwig damage as small rounded holes, consistent with Nicholas et al. (2004), although one of them doubted earwigs caused economic damage. Honeycrisp cultivar apples were most commonly reported as associated with earwigs (they were mentioned as a particular concern by the two interviewees who considered earwigs pests, and by eight others), followed by Gala (three people). Leaf feeding was mentioned by three people but was not considered important.
Only six of the 15 interviewees could give a specific action threshold for woolly apple aphid management. These thresholds included: three to four woolly apple aphid colonies per tree, five nearby trees with many colonies, 20 to 30 percent of shoots infested in a block, most trees in a block infested, or any detection of the aphid (two people). A decision to spray for woolly apple aphid was always based on a visual assessment of colony presence or abundance, but often depended on context, such as: the consultant’s understanding of the orchard owner’s tolerance to aphid abundance (1 person), a higher level of abundance tolerated on older trees (1 person), reduced urgency to suppress aphids after apple harvest (2 people), and less concern about the aphid if natural enemy abundance seemed high (6 people).
3.2. Field study
Earwigs were more abundant in inundation than removal plots at the Gala orchard in 2016, a difference that held in 2017 without further manipulation (Table 2). Few earwigs (mean=0.05 per shelter) were found at removal plots in 2016. At each Fuji orchard, control and removal plots had similar earwig abundance, and inundation plots had significantly greater abundance (Table 2).
Season-long average earwig counts were negatively correlated to average woolly apple aphid colony counts (Fig. 2). Across all blocks and
years, woolly apple aphid population dynamics followed a similar pattern of a mid-season peak and crash during summer, and a recovery in fall (Fig. 3). Compared to control and removal plots, aphid counts were lower and less variable in earwig inundation plots, which had lower mid-season peaks and less late-season recovery (Fig. 3). At each orchard, control and plots with the lowest earwig populations (< 2 earwigs per shelter) (Fig. 2) were prone to the greatest summer peaks and fall increases (Fig. 3). For each orchard and year, earwigs were rarely found in tree canopies until June (Fig. 4), as expected for this univoltine species whose early instars mainly forage on the ground (Moerkens et al., 2011). Therefore, we were not able to mass-collect and release enough earwigs to greatly elevate their abundance in inundation plots until mid-June (Fig. 4), and aphid abundance tended to be similar between treatments until July (Fig. 3). In addition, in Oct through Nov, when earwigs move to underground nests, woolly apple aphid abundance increased notably in some inundation plots (Fig. 3). Earwig abundance across time was similar between control and removal plots (Fig. 4), as removal of all earwigs found in shelters on each visit did not greatly reduce their abundance (Table 2). In inundation plots, earwig abundance remained high until the end of the season in October, even though our latest releases were 4 Aug in 2016 (Gala only) and 11 Jul in 2017 (Fujis only).
There was no difference in incidence of all fruit damage types between inundation and removal plots (Fisher’s exact tests, all P > 0.15), except for rotting apples (P= 0.062; 0 apples in inundation plots, 5 in removal plots) and marginally more stem bowl splits with holes in earwig inundation plots (Table 3). However, the frequency of any stem bowl split (with or without holes) was similar between treatments (Table 3).
3.3. Primers and PCR assay sensitivity
Woolly apple aphid DNA was detected in all 18 earwig guts 1 h after aphid ingestion and 12 out of 18 guts 16 h after aphid ingestion in the laboratory. Out of 20 earwigs collected from Sunrise Orchard, 15 tested positive for aphid DNA, and all sequences obtained matched woolly apple aphid. Woolly apple aphid DNA was not detected in any negative control.
3.4. Molecular gut content analysis of field-collected earwigs
The frequency of woolly apple aphid detection in earwig guts differed 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 rates could be found during periods when woolly apple aphid abundance was low (Fig. 5).
From next generation sequencing, the raw list of taxonomic assignments and counts of reads for each sample of earwigs is in Table S1, and a summarized list of taxa detected in each sample after removal of taxa with≤4 reads (Pompanon et al., 2012) and Dermaptera is in Table S2. Fungi had greater diversity in the dataset (196 taxa) compared with animals (61 taxa) and plants (37 taxa) (Table S2). Pooled across all sites and sampling days, apple pests Lygus sp, and E. lanigerum, were detected, 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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convergens, Psyllobora borealis, and Stethorus punctillum), Phalangium opilio, and a variety of innocuous Diptera, Coleoptera, and Hemiptera. The plant Sorbus sp was detected in all 16 field samples, but this taxonomic assignment may have been an artifact of the RTLGenomics standard method. The COI region of Sorbus amplified by our primer pair
has 100% similarity to apple (Malus domestica) and several other Ro- saceae family plants. We believe these reads most likely correspond to apple.
Within our summarized list (Table S2), 10 taxa (7 fungi and 3 plant) were found in the sample of negative control earwig guts, and no taxa were found in the blank negative control. One of the plants was the putative M. domestica discussed above, but only 10% as many reads (468) were found compared to the minimum found in any field sample (range: 4767 to 38,072), suggesting that M. domestica consumption occurred in the field. The other plants and fungi in the negative control
Fig. 2. Season-long averages of woolly apple aphid colony counts and earwigs 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 negative correlation between aphids and earwigs (Gala, 2016: ρ=−0.84, P=0.002; Gala, 2017: ρ=−0.76, P=0.01; Fuji 1: ρ=−0.93, P < 0.0001; Fuji 2: ρ=−0.96, P < 0.0001; Fuji 3: ρ=−0.71, P=0.009).
Fig. 3. Summary of woolly apple aphid population dynamics 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). Points show mean colonies per tree for each treatment type and error bars show 1 standard deviation, N= 5 replications per point (a, b) or 4 per point (c–e).
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4. Discussion
We found that apple orchard decision-makers usually thought earwigs were biological control agents of potential importance, and some thought earwigs were pests. Our field study provided evidence that earwigs can suppress woolly apple aphids, and we found no evidence that earwigs damaged apples. Woolly apple aphid pest pressure was not extremely high during our study, but colony counts reached a
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 interviewees 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, active 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 present regardless of the density of any single prey species (Piñol et al., 2009). Because they can be present during periods of low pest density, resident predators can quickly exhibit a functional response to limit pest population increases (Murdoch et al., 1985; Piñol et al., 2009). Our results match this expectation, as earwig presence and consumption of aphids while the aphid populations were at low-densities may have prevented rapid aphid population increases in the summer. Rapid aphid population growth with a mid-season peak was observed in some control and earwig removal plots, but not in inundation plots. Fur- thermore, our molecular analysis confirmed that earwigs consumed woolly apple aphids when densities of this pest were very low. This finding was similar to molecular gut content analyses showing Eur- opean earwig consumption of aphids in citrus orchards, even during periods when aphids were rare (Romeu-Dalmau et al., 2012).
Although we have suggested that earwigs can prevent aphid
Fig. 4. Summary of earwig population dynamics 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). Points show mean earwigs per shelter for each treatment type and error bars show 1 standard deviation, N= 5 replications per point (a, b) or 4 per point (c–e).
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
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
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populations from increasing, they may fail to decrease the abundance of high-density aphid populations (Dib et al., 2016a), as univoltinism and low dispersal ability of European earwig precludes them from re- sponding numerically to pest outbreaks within a season. To comple- ment earwigs, other predators such as coccinellids, syrphids, and chrysopids, and the specialist A. mali can suppress woolly apple aphids, but they might not always be present because of narrower windows of phenology or greater density-dependence on prey (Gontijo et al., 2012; Lordan et al., 2015; Quarrell et al., 2017). A build-up of these species, in addition to high summer temperatures above woolly apple aphid’s upper developmental threshold of 32 °C (Asante et al., 1991), may have important role in the mid-season woolly apple aphid population crashes observed in our study and others (Beers et al., 2010). Therefore, a di- verse natural enemy community will likely result in the most consistent and effective biological control across the season. For example, Quarrell et al. (2017) showed that a combination of earwigs with A. mali can provide greater woolly apple aphid suppression than either taxa alone, in part because earwigs can be present earlier in the season before A. mali population levels can build. Similarly, in a cage-exclusion study in the field, Gontijo et al. (2012) showed that woolly apple aphid suppression was greatest when both A. mali and generalist predators were present.
We found no evidence that earwigs caused economic damage to apples. Possible earwig feeding in stem bowl splits (evidenced by holes in splits) was marginally more frequent in inundation plots, but splitting has a physiological cause (Opara, 1993), and interviewees reported that split apples are culled, regardless of earwigs. We found particular concern about earwig damage to Honeycrisp cultivar apples, which are susceptible to stem bowl splitting, have short stems, and grow in tight clusters which may be more vulnerable to physical rubbing and poking damage from limbs (Wargo and Watkins, 2004). These factors create 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 earwigs are a pest. Chewing damage to apple leaves, if severe, could be
economically damaging, particularly on young trees, but the only re- ports of leaf damage by earwigs we are aware of have considered it insignificant (Carroll et al., 1985; Nicholas et al., 2004). Although our results suggest earwigs are not apple pests, the cost versus benefit of earwigs could vary with context. For example, one interviewee suggested that earwigs can be pests of open-calyx apple cultivars, and it remains possible that earwigs could more readily damage some apple cultivars which were not included in our study or others.
We found DNA from other predators in earwig guts, but intraguild predation did not appear to disrupt biological control, as greater earwig abundance resulted in lower woolly apple aphid abundance. This is consistent with other studies suggesting that a combination of earwigs with other natural enemies increases aphid pest suppression (Dib et al., 2011, 2016b; Quarrell et al., 2017). Although coccinellid DNA was found in earwig guts, the assays used in our study cannot determine the number consumed and whether they were depredated or were sca- venged (Pompanon et al., 2012). Moreover, apple tree DNA in earwig guts could have come from leaves, decaying fruit on the orchard floor, or consumption of apple herbivores. Using a species-specific PCR assay similar to what we used for woolly apple aphid detection, Unruh et al. (2016) commonly detected DNA of the key lepidopteran pest of apple, codling moth (Cydia pomonella; Tortricidae), in field-collected earwig guts. We did not find codling moth DNA and rarely detected pest DNA in earwig guts through our next-generation sequencing assays. How- ever, absence of detection does not prove that consumption did not occur, as amplification efficiency of the universal primers used in our assays varies between species (Krehenwinkel et al., 2017; Lamb et al., 2016; Pompanon et al., 2012). Overall, our next-generation sequencing results highlight that earwigs are generalists capable of consuming many foods in apple orchards, and more focused studies will be needed to assess the importance of specific trophic links.
5. Conclusion
Biological control agents that are difficult to observe could often
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 collections.
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have underappreciated benefits in agriculture, and omnivores with context-specific roles might sometimes be incorrectly determined to be pests rather than natural enemies. In our study, experimental manipulations combined with molecular analyses provided evidence that earwigs suppressed woolly apple aphids and did not damage apples. Our interview results suggested that apple pest management decision- makers would be receptive to learning how to exploit earwigs for improved pest management. To conserve earwigs and potentially increase pest suppression, apple growers could time tillage to avoid disturbing earwigs during their nesting phase from late fall to spring, and certain insecticide sprays capable of harming earwigs should be avoided (Moerkens et al., 2011, 2012; Fountain and Harris, 2015; Beers et al., 2016).
CRediT authorship contribution statement
Acknowledgements
We thank our interviewees, collaborating orchardists, field assis- tants Sam Martin and Dayna Dinius, and Tom Unruh and Kelly Archer.
Competing interest’s statement
Declaration of interest
Funding sources
This work was supported by a grant from the Washington Tree Fruit Research Commission, and grants from USDA National Institute of Food and Agriculture: Hatch project accession numbers 1014754, 1016563, and under award number 2016-38640-25383 through the Western Sustainable Agriculture Research and Education Program under suba- ward number 200592-445.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biocontrol.2019.02.011.
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Introduction
Methods
Interviews
Statistical analyses
Molecular gut content analysis of field-collected earwigs
Discussion
Conclusion
mk:H1_19
Acknowledgements

Documents

Field evidence and grower perceptions on the roles of an