Biological Control 114 (2017) 8–13

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Biological Control

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Can galinae attack larvae feeding in large ash MARK trees? ⁎ Theresa C. Murphya, , Roy G. Van Driescheb,1, Juli R. Gouldc,2, Joseph S. Elkintona,b,1 a Organismic and Evolutionary Biology, University of Massachusetts Amherst, MA 10003, United States b Department of Environmental Conservation, University of Massachusetts Amherst, MA 10003, United States c USDA-APHIS-PPQ, Buzzards Bay, MA 02542, United States

GRAPHICAL ABSTRACT

ARTICLE INFO ABSTRACT

Keywords: Agrilus planipennis, the emerald ash borer (EAB), is an invasive forest pest decimating North American ash trees. Bark thickness Population-wide management of EAB is focused on biological control through the introduction of four parasitic Biological control wasps, including the recently approved larval . This species was approved for release Oviposition limit in the north-central and northeastern US in 2015 and its long ovipositor (4–5.3 mm) is expected to allow it to Tetrastichus planipennisi reach EAB larvae in larger ash trees with thicker bark, than the only other successfully established larval Refuge parasitoid, Tetrastichus planipennisi. Using experimentally infested logs of varying diameters in the laboratory, we Diameter measured the effect of bark thickness on oviposition of S. galinae to understand its potential value for controlling EAB in trees of differing diameter and bark thickness. Parasitism by S. galinae was highest when bark was thin (< 4 mm) and dropped significantly as valley bark thickness increased beyond 6.5 mm. We also found that EAB larval feeding on inner bark, reduced the bark thickness directly over the larval galleries by 0.4 mm on average. Our results suggest that S. galinae will be able to reach EAB larvae in > 95% of all ash in the northeastern United States. S. galinae will likely play a vital role in providing additional control and in supporting ash regeneration in aftermath areas of EAB invasions.

Abbreviations: EAB, emerald ash borer; DBH, diameter at breast height (approximately 1.4 m); USFS, United States Forest Service ⁎ Corresponding author at: 250 Natural Resources Road, Ag. Engineering Rm 115, University of Massachusetts, Amherst, MA 01003, United States. E-mail addresses: [email protected] (T.C. Murphy), [email protected] (R.G. Van Driesche), [email protected] (J.R. Gould), [email protected] (J.S. Elkinton). 1 Address: 160 Holdsworth Way, Dept. of Environmental Conservation, University of Massachusetts, Amherst, MA 01003, United States. 2 Address: 1398 West Truck Road, Buzzards Bay, MA 02542, United States. http://dx.doi.org/10.1016/j.biocontrol.2017.07.004 Received 1 March 2017; Received in revised form 5 July 2017; Accepted 5 July 2017 Available online 08 July 2017 1049-9644/ © 2017 Elsevier Inc. All rights reserved. T.C. Murphy et al. Biological Control 114 (2017) 8–13

1. Introduction rates for S. galinae across log sizes tested (Wang et al., 2015). However, in their experiment the large logs did not exceed 10 cm in diameter. The emerald ash borer (EAB), Agrilus plannipennis Fairmaire While this diameter is close to the upper limit (11.2 cm) for T. plani- (Coleoptera: Buprestidae), is one of the most destructive forest invaders pennisi, we hypothesize that S. galinae has a much larger upper size limit in North America (Aukema et al., 2011; Herms and McCullough, 2014). than that of T. planipennisi due to its longer ovipositor (Abell et al., Since its accidental introduction to Michigan in the 1990s, EAB has 2012; Wang et al., 2015). By using larger diameter logs, we examined already spread to 30 states, two Canadian provinces, and has killed over conditions closer to S. galinae’s expected oviposition limit to assess 100 million ash trees, Fraxinus spp., (Herms and McCullough, 2014 whether a similar change in parasitism is noticeable for S. galinae as it emeraldashborer.info, 2016). EAB causes severe environmental and approaches its oviposition limit. economic harm and may result in the functional extinction of several As a relatively recently approved agent in the EAB biocontrol pro- species of Fraxinus from North American forests (Aukema et al., 2011; gram, Spathius galinae’s potential impact is poorly known. Our goal was Gandhi and Herms, 2010; Herms and McCullough, 2014). Eradication to investigate how oviposition success by S. galinae is affected by bark of the emerald ash borer was attempted when the infestation was first thickness over a wider range of tree sizes. We also wanted to study the discovered, but was unsuccessful and discontinued (Herms and effect of EAB larval feeding on bark thickness. EAB larvae feed at the McCullough, 2014). Due to the logistics and high cost of insecticide intersection of the inner bark and cambium (Poland and McCullough, applications, use of systematic insecticides in forests is not practical 2006), and we wanted to determine if this feeding significantly reduced (McCullough and Mercader, 2012). Since 2007, classical biological bark thickness, facilitating parasitism. Knowing S. galinae’s oviposition control (the introduction of non-native natural enemies to regulate an limits would improve EAB management through a greater under- invasive species) has been the dominant management practice for large- standing of the expected impact of S. galinae in the field and would scale control of EAB populations (Bauer et al., 2008). As of 2016, the assist in future modeling of EAB population dynamics. Researchers can USDA has approved four parasitic wasps for EAB biocontrol: an egg use this information to help choose parasitoid release sites, and man- parasitoid (Oobius agrili Zhang and Huang) and three larval agers can possibly choose alternative control methods, such as using ( Yang, Spathius galinae Belokobylskij and Strazenac, and trunk injections of pesticides or selected tree removal, to eliminate Tetrastichus planipennisi Yang) (Belokobylskij et al., 2012; Yang et al., larvae in large trees deemed inaccessible to larval parasitoids. 2005; Yang et al., 2006; Zhang et al., 2005). Tetrastichus planipennisi is the most widely established introduced 2. Materials and methods EAB biocontrol in North America (Bauer et al., 2015); however, Abell et al. (2012) found that this parasitoid species cannot oviposit in larvae Under controlled conditions in the laboratory, white ash, Fraxinus in the lower boles of trees with a bark thickness exceeding 3.2 mm americana L., logs were artificially infested with EAB larvae and then (equal to trees with a DBH [diameter at breast height] > 11.2 cm) due introduced to parasitoids in cages after larvae had reached a suitable to its short ovipositor. This limitation of T. planipennisi creates a large age for parasitism (3rd or 4th instars) (Abell et al., 2012). After ex- refuge for EAB larvae, particularly in stands with more mature, larger posure larvae were reared for 2 weeks to allow parasitoids to develop ash trees. Data from the United States Forest Service (USFS) show that (Duan et al., 2014), and then all logs were debarked to record the fate of as of 2014, over 500 million ash trees or 26% of all Fraxinus spp. on the EAB larvae (alive, dead, parasitized), calculate parasitism rates and forested land in the United States northeastern region (Connecticut, record bark thicknesses (Duan et al., 2010). Maine, Massachusetts, New Jersey, New York, Pennsylvania, Rhode Island, Vermont) were too large at DBH for T. planipennisi (FIDO 2009- 2.1. Log preparation 2014). Limited T. planipennisi parasitism can occur on larger trees, but only where available larvae are under thinner bark (< 3.2 mm) on the Three trials of the experiment were run between July 2015 and upper bole and smaller branches of the tree (Duan, pers. comm.). An- December 2015. Logs were cut within 3 days of starting each trial. other larval parasitoid, S. agrili, was also approved for release in 2007 White ash trees were selected in three size classes based on DBH. and has a longer ovipositor than T. planipennisi, but S. agrili has failed to Small = 3–8 cm DBH, medium = 12–18 cm and large = 25–30 cm. establish north of the 40th parallel (Gould and Duan, 2013; These size ranges were chosen to help ensure a wide range of bark USDA–APHIS/ARS/FS, 2016). With T. planipennisi as the only currently thicknesses. Logs cut from these trees and used in our experiment established introduced larval parasitoid, many EAB larvae in larger ash ranged in diameter from small = 6.4–8.7 cm, to med- trees remain inaccessible to introduced larval parasitoids, highlighting ium = 8.2–15.4 cm, and large = 18.9–32.2 cm. Log lengths and the the need for another biocontrol with a longer ovipositor able to attack number of logs were varied among treatments (four to eight small logs, larvae in larger trees. two to four medium logs, and one to two large logs) such that in ag- Spathius galinae, a third larval parasitoid, which was approved by gregate each treatment had the same bark surface area available to S. the USDA for release in 2015, has a longer ovipositor than T. plani- galinae. To accommodate equal surface area per treatment, logs were pennisi. The ovipositor of S. galinae is 4–5.3 mm in length (Gould and cut anywhere between 10 and 30 cm in length. Duan, 2013), while that of T. planipennisi is only 2.0–2.5 mm (Duan and Oppel, 2012). Given its longer ovipositor length, S. galinae should be 2.2. Egg application and EAB larval development able to attack hosts in larger ash trees that T. planipennisi (Gould and Duan, 2013). Furthermore, climate matching suggests that there is a To inoculate logs with EAB, 20 eggs were applied per replicate for better fit between S. galinae’s native range in the Russian Far East and all treatments (log or group of logs). EAB eggs were produced and that of the north-central and northeastern United States than is true for supplied by the USDA-APHIS EAB Rearing Facility (Brighton, MI, USA) S. agrili, which failed to establish in the north-central region of the and were attached to paper coffee filters. These coffee filters were cut United States (Duan and Oppel, 2012; Gould and Duan, 2013). In ad- into paper squares with one to three eggs per square. An edge of the dition to its long ovipositor and climatic suitability, S. galinae is known filter paper was then glued to the bark, with the eggs facing out. Eggs to cause parasitism rates of up to 63% on EAB in American green ash were then covered with a cotton ball, and the covered eggs secured to (Fraxinus pennsylvanica) in Russia (Duan et al., 2012). The effect of bark the log with breathable quick-drying ribbon, 3.8 mm satin (100% thickness on parasitism has previously been determined using both T. polyester). This egg technique was modified from Abell et al. (2012) planipennisi and S. galinae; Wang et al. (2015) found that parasitism because parafilm did not adhere to the large, rough-barked logs that rates of T. planipennisi on large logs were significantly lower than on were used in our study. Before egg application, any rough bark surfaces small logs. In contrast, there was no significant difference in parasitism were lightly scraped to create a flat, smooth surface for egg placement.

9 T.C. Murphy et al. Biological Control 114 (2017) 8–13

Our method ensured the eggs stayed dry and lay flush on the bark, so that the neonate larvae could successfully enter the log after hatching. Logs were then placed with the lower cut end sitting in plastic trays filled with 2 cm of water in a climate-controlled room or chamber (temperature varied, see below, L16:D8 photoperiod, RH > 60%). After 2 to 3 weeks, the filter paper squares were removed and the number of hatched eggs counted. The first two trials of the experiment were conducted in a climate-controlled room at 30 °C ± 2 °C during larval development and 25 °C ± 2 °C during parasitoid exposure. The third trial was conducted in climate-controlled chambers with both larval development and parasitoid exposures at 25 °C ± 1 °C. The water was changed twice a week and the cut ends of the logs scrubbed to prevent excessive growth of mold or algae. Trial 1 had an egg in- cubation period of 4 weeks (28 July–27 August), Trial 2 had an egg incubation period of 6 weeks (8 September–21 October) and Trial 3 had an egg incubation period of 6 weeks (7 October–23 November). In- cubation period was based on larval development; once larvae had developed to 3rd/4th instars, logs were exposed to parasitoids.

2.3. Parasitoid exposure

Fig. 1. A diagram cross section of bark with an EAB larva feeding underneath. The white A few logs were debarked to check for larval development. When line is bark furrow thickness defined as the depth of the outer bark and inner bark larvae were found to be in instars suitable for parasitism, logs were (phloem) or the distance from the outer bark to the cambium of the tree. The black line is grouped to ensure consistent surface area and approximately the same bark furrow thickness over a larva defined as the depth from the outer bark through the number of larvae per treatment, based on egg hatch rates. Once inner bark to the top of the larval gallery. grouped, logs were placed in mesh cages, large logs in 45 cm3 cages, medium and small logs in 30 cm3 cages, all cages being made of 680 µm measurements were used in our analyses (Abell et al., 2012). When polyester mesh (BioQuip, Rancho Dominguez, CA), during the initial measuring bark thickness where EAB larval feeding galleries were two trials and in similarly sized cages, but with 27 cm3 of volume and present, the thickness was defined as the distance or depth only to the 24 × 24 mesh plastic screen (BioQuip, Rancho Dominguez, CA) during top of the gallery, which is often partially in the inner bark due to EAB the last trial, which was run in a climate-controlled chamber. Logs were larval feeding (Fig. 1). We use the designation “bark thickness over exposed to S. galinae females in approximately a 2:1 wasp: host ratio galleries” to denote these measurements. Whenever possible, we col- based on egg hatch rates. Logs with mature EAB larvae were exposed to lected samples of bark from over parasitized and non-parasitized larvae. 7–14 day-old, mated, naïve S. galinae for 2 weeks to provide ample We then measured furrow bark thickness directly over the gallery opportunity for parasitism. A L16:D8 photoperiod, RH > 60%, and where the larva had been and the bark thickness in the furrow im- 25 °C ± 2 °C was used for all parasitoid exposures. Logs were then mediately adjacent to the larva (Fig. 1.). removed from cages and returned to plastic trays with water for an additional 2 weeks to allow parasitoids to develop under the same 2.6. Measuring bark thickness in the field temperature and light conditions (Duan et al., 2014). Trial 1 was ex- posed to parasitoids for a period of 16 days (27 August–12 September), To help understand how tree size in the field relates to bark thick- Trial 2 was exposed for a period of 14 days (21 October–4 November) ness, bark samples were taken from ten white ash trees of various large and Trial 3 was exposed for a period of 14 days (23 November–7 De- diameters at two sites, one in Pittsfield, Massachusetts, and the other in cember). Hamden, Connecticut. Bark thickness samples and diameter measure- ments were taken from each tree at two heights, 0.5 m and 1.4 m. Tree 2.4. Detection of parasitism diameter was recorded at each height, and then a leather punch (2.5 cm diameter) and mallet were used to collect three bark plug samples (at S, To determine the percentage of EAB larvae successfully parasitized NW and NE-facing sides of the bole) from each height where each in logs of different diameters, logs were debarked with a drawknife or diameter was measured. We measured two furrows for each bark chisel to locate larvae and determine their parasitism status. Three to sample and averaged together all six furrow measurements per dia- five randomly selected, intact, vertical bark segments were kept from meter to calculate average furrow bark thickness. each log, to measure bark thickness as discussed in 2.6. Additional field data (from several sites throughout New York) was provided by USDA-APHIS-PPQ in which average furrow bark thickness 2.5. Measuring bark thickness in experimental logs per one meter tree segment was found by taking furrow measurements at three, equally-spaced, randomly selected points along the edge of To determine the thickness of the bark in the experimental logs, all each cut tree segment. Diameter of each tree segment was also provided bark segments were cut across the grain with a band saw and the cut in the data set. These data were incorporated into our analysis of edge was sanded with an electric sander to more clearly delineate be- average bark furrow thickness versus diameter. tween the inner and outer bark. Unlike Abell et al. (2012), our mea- surements incorporated both outer and inner bark (phloem), because 2.7. Statistical methods larvae were found feeding at the intersection of the inner bark and cambium (Poland and McCullough, 2006). We define bark thickness as Logistic regression was used to evaluate the effect of bark thickness the distance from the outer bark to the cambium of the tree, in- on the probability of parasitism. We used a generalized linear mixed- corporating the depth of both the outer and inner bark (Fig. 1). effects model (GLMM) with binomial error distribution and logit link. Thicknesses (depth) of furrows (valleys) and ridges of inner and outer Proportion of parasitized larvae served as the response variable and bark were recorded, by averaging two thickness measurements per bark thickness and larval density (larvae/m2 surface area) as predictors. piece of bark with digital calipers. However, only furrow thickness To help with model convergence, density was standardized to a z-score

10 T.C. Murphy et al. Biological Control 114 (2017) 8–13

Fig. 2. A fitted logistic regression of the proportion of larvae parasitized by Spathius ga- linae in the laboratory experiments as a function of average bark furrow thickness.

Fig. 3. A histogram of minimum furrow thicknesses over Agrilus planipennis larvae from (where Z-Score = (Y-My)/Sy: Y- the original score, My- mean of the the laboratory experiment that were parasitized by Spathius galinae. sample and Sy- standard deviation of the sample). Use of z-scores helps allow for comparison of values from different distributions by centering and normalizing the distribution (Abdi, 2007). To account for small differences in exposure length and cage type between trials, trial and cage nested-within-trial were incorporated as random effects. Trial was then dropped as a random effect because its variance was almost 0 and did not influence the model. Laplace approximation was used for all GLMM parameter estimation and differences among predictors were compared using Wald Z tests. All GLMM models were tested for over- dispersion. After we built the model we could show the relationship using the visreg package in R (Fig. 2), which allowed us to isolate and show the relationship between bark thickness and parasitism, while controlling for the effect of other predictors in our model (Breheny and Burchett, 2013). Another GLMM was used to test for differences in furrow bark thickness for sites over galleries versus not over galleries, to determine how larval feeding affected thickness of the bark directly over the gallery. To account for overdispersion observed in the data we used a Fig. 4. Effect of galleries on reduction in furrow bark thickness from the laboratory ex- gamma error distribution and inverse link. Log identity was in- ff periment. The black line is for standard bark furrow thickness and the grey line is for bark corporated as a random e ect to account for variability in bark thick- furrow thickness over Agrilus planipennis galleries. nesses between logs. Lastly, the relationship of average bark furrow thickness to diameter 3.2. Comparison of bark thickness over vs not over EAB galleries was analyzed with both a linear and logarithmic line of best fit, in- fi corporating data from all laboratory, eld, and USDA experiments. All Bark furrows directly over EAB galleries had significantly thinner analyses were conducted in R version 3.2.5 (R Core Team, 2013). bark than bark furrows not over EAB galleries, by an average difference of 0.35 mm ± 0.06 SE (T = 6.81, P < 0.001, GLMM, Fig. 4, Table S2). Measurements of galleries over unparasitized EAB larvae and over 3. Results S. galinae-parasitized EAB larvae were grouped before analysis because a Tukey means comparison of our original GLMM model confirmed that ff 3.1. E ect of bark thickness on parasitism the bark thicknesses over S. galinae-parasitized EAB larval galleries did not differ statistically from thicknesses over the galleries of non-para- fi Predicted percentage parasitism declined signi cantly with in- sitized larvae (Z = 2.10, P = 0.084, GLMM). Finally, we also found − creasing furrow bark thickness (Z = 3.84, P < 0.001, GLMM, Fig. 2, that bark furrow thickness over larvae differed significantly from bark see Table S1 Supplementary Material). Predicted percentage parasitism thickness of a furrow directly adjacent to that larvae, by an average of was also moderately positively correlated with the density of larvae −0.41 mm, as determined by a non-parametric lower-tailed Wilcoxon 2 available (larvae/m surface area) (Z = -2.07, P = 0.04, GLMM). signed-rank test (V = 283.5, P < 0.0001). Average larval numbers available to Spathius galinae varied but were similar across size classes, 10.7 ± 4.0, 6.0 ± 3.8, and 8.1 ± 2.6 in our small, medium and large classes respectively. Corresponding den- 3.3. Relation between bark thickness and tree size in the field sities were 0.008 ± 0.005, 0.004 ± 0.004, and 0.005 ± 0.003 larvae/m2 surface area respectively. Minimum mea- Average furrow bark thickness was compared to log diameter/DBH 2 2 sured bark furrow thickness over galleries of parasitized larvae ranged with both a linear (R = 0.55), and logarithmic (R = 0.57) model. The from 0.95 mm to 5.5 mm, with an average of 2.7 mm ± 0.13 SE. best fit was from the logarithmic model, y = 1.898∗ln(x) − 1.064, (Fig. 3). where y is bark thickness (mm) and x is diameter (cm) (Fig. 5). This model shows that bark thickness levels off at 6–8 mm for trees up to 83 cm DBH.

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northeastern region (Connecticut, Maine, Massachusetts, New Jersey, New York, Pennsylvania, Rhode Island, Vermont), S. galinae would be able to attack EAB larvae at DBH in 97.5% of Fraxinus trees in the re- gion (FIDO 2008-2012). Additionally, parasitism by S. galinae could occur even in trees > 39.2 cm DBH, on the upper bole and smaller branches where the bark is thinner. When viewed as a series of life stages, the life of an ash tree can be divided into sapling (< 5 cm DBH), pole size trees (5–12 cm DBH), and mature trees of moderate to large size (15–50 cm DBH). Tetrastichus planipennisi was found to parasitize 36–85% of susceptible larval stages (3rd-JL) in saplings in Michigan (Duan et al. 2017). For pole size ash trees (7–15 cm DBH) in Michigan, T. planipennisi attacked up to 22% of suitably sized larvae (Duan et al. 2013), with a likely upper limit of 11 cm (for the lower bole) (Abell et al. 2012). S. galinae is most needed for biological control of larger pole size and mature trees, although the added parasitism of S. galinae across all ash tree life stages will be helpful for population suppression. Fig. 5. A logarithmic regression comparing average furrow bark thicknesses of a log Our results show that the predicted percentage of parasitism de- (mm) to the natural log of the diameter of the log (cm) for white ash, Fraxinus americana, creases even before S. galinae’s ovipositor limit is reached. At our fi from combined laboratory, eld and USDA datasets. conservative ovipositor limit of 5.9 mm, predicted parasitism is 26% (Fig. 2). This finding suggests that while S. galinae will be an important 4. Discussion control, it alone will not control the population of EAB in large trees. This result is not unexpected, and for this reason the EAB biological fi Our ndings suggest that Spathius galinae is a highly valuable ad- control program is focused on releasing a suite of biological control dition to the emerald ash borer biocontrol program, with the potential agents, including several larval parasitoids and the egg parasitoid Oo- to provide biological control of EAB in ash tree boles up to 39 cm in bius agrili. The need for multiple mortality agents to control the EAB diameter. This is important because T. planipennisi is establishing, dis- population is also why researchers are actively looking at other mor- persing, and causing high mortality of EAB in saplings after EAB po- tality factors, to help understand if the presence of natural control pulations collapse (Duan et al. 2017), however this species cannot agents such as woodpeckers, tree mortality, etc., will work synergisti- oviposit through bark that is over 3.2 mm thick (equal to trees with a cally with the biological controls to provide the comprehensive control DBH > 11.2 cm) (Abell et al. 2012). As these saplings mature, a needed for population suppression and ash regeneration. parasitoid with a longer ovipositor is needed for continued suppression Researchers have suggested that the largest role for these in- of EAB populations. troduced biocontrol agents will be in maintaining EAB populations at ’ Our results suggest that S. galinae s oviposition limit lies between low levels to permit survival and regeneration of ash in the aftermath of – 4 8mm(Fig. 2). Due to poor larval establishment in some of our an EAB invasion (Bauer et al., 2015). For ash trees in aftermath areas, medium class logs, there were not as many replications between 4 and establishing a complex of biological control agents is likely the only 8 mm as desired. However, we can also look at oviposition limit by management strategy that will allow ash to renew itself on the land- measuring the bark thicknesses directly where parasitism did occur in scape (Duan et al., 2013). Therefore, in addition to S. galinae being an our logs. During oviposition S. galinae paralyzes the larvae, so when we important new component to add into existing EAB biocontrol release found parasitized larvae in our experiment, we knew that the adult programs, it may prove vital in allowing ash to reach larger sizes as they female must have pierced the bark directly over the end of EAB larvae age and mature in recovering forests. Additionally, in these study areas gallery where the parasitized brood was found (Yang et al., 2005). our results support the prediction that S. galinae will provide even Taking the minimum furrow measurement over that section of gallery greater parasitism percentages over a wide range of tree sizes, as gave us a conservative estimate of bark thickness through which ovi- needed for comprehensive control of the borer and ash regeneration. position is possible (Fig. 3). If we also account for EAB larval feeding, which significantly reduces the thickness of bark directly over the larval galleries, we can translate our conservative bark thickness estimate into Acknowledgments a diameter limit for oviposition, that can help us understand the po- ff tential oviposition success in various forest stands (with di erent aged This research was funded by cooperative agreements with the USDA trees). FS (State and Private Forestry) and USDA APHIS. We thank J. Podos, M. For example, based on our recorded measurements of minimum Davis, and H. Broadley for reviewing earlier versions of the manuscript, furrow bark thicknesses over galleries of parasitized larvae (Fig. 1), our J. Lelito, K. Abell, and L. Bauer for help with project design, M. Barrett, ’ conservative estimate of S. galinae s oviposition limit is projected to be E Lee, M Salhany for help with data collection, and K. McGarigal, C. ’ around 5.5 mm (Fig. 3). If the barrier of bark to S. galinae s oviposition Sutherland, A. Jordaan, J. Finn, M. Akresh, and M. Warden for pro- is reduced by 0.4 mm on average due to EAB larval feeding (Fig. 1), viding statistical advice. We thank E. Reynolds and F. Andre (MA DCR) ’ then S. galinae s limit of around 5.5 mm would be equivalent to an for providing all the ash logs used in our experiments, D. Burt, S. average furrow thickness of 5.9 mm. Using our regression in Fig. 5,we McMahon, and the USDA EAB rearing facility in Brighton, Michigan for can translate that 5.9 mm limit into a 39.2 cm diameter bole (Fig. 5). producing and providing the eggs used in our experiments, and J. Duan fi This nding suggests that S. galinae would be able to exceed the bole (USDA APHIS) for producing and providing additional S. galinae needed – penetration limit of T. planipennisi (11 12 cm diameter) by fourfold. for our experiments. Using our conservative limit estimate (< 5.9 mm in furrow bark thickness, < 39.2 cm diameter) and data.3 from the USFS for the Appendix A. Supplementary data

3 Measurements are done using US forest service estimates of tree counts and discount Supplementary data associated with this article can be found, in the any saplings under 2.5 cm diameter. online version, at http://dx.doi.org/10.1016/j.biocontrol.2017.07.004.

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