Abstract
Emerald ash borer (EAB, Agrilus planipennis) is an invasive phloem feeder from East Asia that has killed millions of ash trees in North America. Currently, effective options for individual tree protection are limited to systemic insecticides, in particular neonicotinoids, which have come under increased scrutiny for their nontarget effects. In this study, green ash (Fraxinus pennsylvanica) trees were treated with two neonicotinoid insecticides, imidacloprid and dinotefuran, at full and half label rates based on trunk diameter to evaluate residues and efficacy. Analyzing the leaf, stem, and root tissues, there was no difference in insecticide residues between application rates within each tissue type. However, there were significantly higher residues of imidacloprid in root tissue compared to other plant tissues, and dinotefuran applied at the full label rate resulted in lower residues in stem phloem tissue. Additionally, insecticide-treated stems were artificially infested with EAB eggs to measure larval success (survival and growth). EAB larvae consumed less phloem in treated trees compared to untreated controls. These findings suggest that, in small-diameter ash, lower than label-recommended doses may be a viable component of an integrated management plan for EAB.
INTRODUCTION
Emerald ash borer (EAB, Agrilus planipennis Coleoptera: Buprestidae), an invasive pest of forest, shade, and ornamental ash (Fraxinus spp.), has caused extensive tree mortality throughout its invaded range in North America (USDA APHIS 2020) and is expected to have caused $10 billion in losses by the year 2019 (Kovacs et al. 2011).
EAB has a one- or two-year life cycle, depending on latitude (Herms and McCullough 2014). In summer, adults mate and each female oviposits about 70 eggs on stems and large branches of ash hosts (Rutledge and Keena 2012). Eggs hatch and larvae feed on phloem, creating serpentine galleries beneath the bark (Figure 1a). Mature larvae overwinter before pupating and emerging as adults the following or subsequent summer (Cappaert et al. 2005). When beetle populations are high, larval feeding girdles trees and causes rapid tree mortality (Herms and McCullough 2014)(Figure 1b).
Emerald ash borer larvae feed on phloem beneath bark, creating (a) serpentine galleries that expand as the larvae grow that (b) eventually coalesce and completely girdle infested trees.
All North American ash are susceptible to EAB colonization (Liu et al. 2007), though white and green ash (F. americana and F. pennsylvanica) are highly preferred, and blue ash (F. quadrangulata) has some putative resistance (Tanis and McCullough 2012; Spei and Kashian 2017). In the eastern US, ash trees are a significant component of wildland forests (Wharton and Barbour 1973) and are also prevalent as street, park, and landscape trees. Because of its pervasiveness, efforts at EAB management in the USA have focused on classical biological control (Duan et al. 2012; Duan et al. 2013). Four hymenopteran parasitoids, Spathius agrili (Braconidae), S. galinae (Braconidae), Tetrastichus planipennisi (Eulophidae), and Oobius agrili (Encyrtidae), discovered in EAB’s native range of China, have been intensively screened and are now laboratory reared in the USA for incorporation into biological control releases throughout the invaded range. Additionally, endemic natural enemies have been recruited to EAB-invaded forests and may be helping to suppress EAB populations (Duan et al. 2013; Davidson and Rieske 2016; Savage and Rieske 2018).
Classical biological control has had some success suppressing EAB populations (Duan et al. 2011) and has also been deployed in concert with chemical suppression, again with some success. In theory, interspersing insecticide-protected trees throughout an EAB-infested area could facilitate establishment of introduced biological control agents and lead to slower ash mortality (McCullough and Mercader 2012; Davidson and Rieske 2016; Graziosi and Rieske 2017). However, the only way to assure complete protection of individual trees of North American ash from EAB is through chemical means (McCullough et al. 2011).
Following the initial discovery of EAB in the USA, numerous insecticides were screened for efficacy against both larval and adult EAB (McCullough et al. 2005; Herms et al. 2009). Emamectin benzoate, an avermectin derivative applied through trunk injections at approximately 2- to 3-year intervals, is considered the gold standard (Smitley et al. 2010); injections protect trees and cause up to 100% EAB mortality. However, some formulations of emamectin benzoate are restricted use insecticides, require specialized equipment to apply, and are expensive.
As alternatives, imidacloprid or dinotefuran applied as a soil drench, trunk spray, or foliar spray have been employed. Imidacloprid is metabolized by plants into byproducts with varying toxicities (Nauen et al. 1998), including imidacloprid olefin and dihydroxy imidacloprid. Imidacloprid metabolites have demonstrated efficacy against some pests with piercing sucking mouthparts, including aphids (Nauen et al. 1998) and adelgids, and is found in eastern hemlock trees (Tsuga canadensis) treated for hemlock woolly adelgid (Adelges tsuga)(Coots et al. 2013; Eisenback et al. 2014; Benton et al. 2016). Imidacloprid olefin in particular persists in treated hemlock and is highly toxic against hemlock woolly adelgid, whereas dihydroxy imidacloprid is transitory and appears unimportant for insect control in the hemlock system (Benton et al. 2016). While imidacloprid translocation has been well studied in the hemlock woolly adelgid–hemlock system (Cowles 2009; Coots et al. 2013; Eisenback et al. 2014; Benton et al. 2016), gymnosperms as a group use tracheids exclusively, rather than tracheids and vessels, for transport (Sperry et al. 2006). Thus, movement of imidacloprid and its metabolic byproducts through ash, an angiosperm, is expected to differ from its movement through hemlock, a gymnosperm.
Imidacloprid and dinotefuran do not provide the same level of tree protection against EAB as emamectin benzoate trunk injections, but both can provide adequate protection when applied annually (Herms et al. 2009; Smitley et al. 2015). They require no specialized equipment, are more accessible, and therefore are widely used. These chemicals are translocated to the foliage, where adult EAB encounter them as they feed prior to mating and oviposition (Mota-Sanchez et al. 2009), but surprisingly little is known about which ash plant tissues concentrate or retain these chemicals following treatments (but see Harrell 2006; Mota-Sanchez et al. 2009; Tanis et al. 2012).
Application rates and treatment frequency necessary for tree protection against EAB vary and are influenced by season, tree size, canopy condition, site and soil conditions, and overall tree health (Smitley et al. 2015). Consequently, there is some confusion among homeowners and tree care specialists as to optimal application rates and frequency.
Unfortunately, as neonicotinoid insecticides, both imidacloprid and dinotefuran are under scrutiny due to concerns over their potential nontarget effects. In particular, the effects of neonicotinoids on honeybees and other pollinators has caused considerable alarm (Copping 2013; Goulson 2013; Vanbergen et al. 2013). Although deployment of imidacloprid soil drenches at label rates and lower for EAB management in forested situations has shown no negative effects on native hymenopteran abundance and diversity (Davidson and Rieske 2016), even short-term exposure to this chemical class could have long-term implications on pollinator colony fitness (Larson et al. 2013; Stanley et al. 2015).
The focus of this study is to evaluate within-plant insecticide distribution and effects on EAB survival for plants treated with imidacloprid and dinotefuran, two commonly used chemicals for EAB management. Given the scrutiny that neonicotinoids are under, it is essential that we have a full understanding of how these compounds distribute in the plant following application. Using imidacloprid soil drenches and dinotefuran trunk sprays applied at the full label rate and at half the label rate based on trunk diameter, EAB survival and insecticide residues within ash tissue were measured. Specifically, the objectives were to: (1) assess insecticide concentrations in various plant tissues 5 weeks following application; (2) assess insecticide concentrations in trees receiving applications at the full and half label rates based on trunk diameter; and (3) evaluate any differences in EAB survival and phloem consumption.
MATERIALS AND METHODS
This work was conducted at Taylor Fork Ecological Area, a 24.3 ha abandoned pasture in Madison County, KY, situated at the interface of the outer Bluegrass and eastern Knobs regions of Kentucky. Ash thrive on the moist and fertile soils that predominate in the Bluegrass region (Campbell 1989) and were historically a significant component of these forests (Wharton and Barbour 1973). Taylor Fork is owned and administered by Eastern Kentucky University and is characterized by open cane fields, trees in fencerows, small patches of early- to mid-succession woodlands, and sporadically occurring, large, open-grown trees with areas of dense regeneration. At the onset of the study, EAB was present in Madison County but was not yet reported at Taylor Fork, and ash showed no signs of EAB-induced stress. In summer 2016, the large ash at Taylor Fork were treated prophylactically with bark sprays of dinotefuran applied at label rates to protect against EAB.
In spring 2017, 3 discrete sites were established, separated by distinct topographic features and set more than 1,000 m apart. Within each site, 3 plots containing significant green ash regeneration were designated (n = 9 replicates), and in each plot 5 green ash trees (approximately 5 cm diameter at 1.37 m above ground level) were selected for treatment using a randomized block design. On May 9, selected trees in each plot received either: (i) a soil drench (1 L volume) of imidacloprid (Imidacloprid 2F I/T, Prime Source LLC, Evansville, IN, USA) applied at the “full rate” of 2.64 mL/L of water in 0.94 L of total solution, equivalent to 0.64 gai/2.5 cm diameter at 1.37 m, or DBH; (ii) a soil drench (1 L volume) of imidacloprid applied at a “half rate” of 1.32 mL/L of water in 0.94 L of total solution, equivalent to 0.32 gai/2.5 cm DBH; (iii) a basal bark spray of dinotefuran (Safari 20SG, Valent, Walnut Creek, CA, USA) applied to runoff (approximately 52 mL) at the “full rate” of 90 g/L of water, or 0.93 gai/2.5 cm DBH; (iv) a basal bark spray of dinotefuran applied to runoff (approximately 52 mL) at a “half rate” of 45 g/L of water, or 0.47 gai/2.5 cm DBH; or (v) an untreated control.
Seven days after treatment, the trees (n = 15 per treatment for a total of 45 trees) were artificially infested with 3 laboratory-reared emerald ash borer eggs at heights of 25 and 50 cm above ground level for a total of 6 eggs per tree. Eggs were screened with fine polyester mesh to prevent predation, and larvae were allowed to hatch and develop for 30 days (Olson and Rieske 2018), after which plant material was processed.
For processing, plants were excavated, roots were clipped and placed in sample bags, and all foliage was removed from a single south-facing branch on each tree. Samples were then stored on ice for transport. In the laboratory, tap water was used to wash soil from roots, followed by rinsing with distilled water. A sample of root tissue < 4 mm in diameter was then designated for chemical analysis of insecticide residues. The main stem of the tree was sectioned, and phloem tissue from the top 12.7 cm was removed for chemical analysis; the remainder was used to evaluate EAB colonization success. Thus, for each experimental tree there were root, stem phloem, and foliar samples to evaluate for insecticide residues, and stem sections to evaluate for EAB colonization.
EAB Colonization
When neonate larval emergence holes were evident, or larval galleries were present on the stem, EAB survival was scored as positive. Larval galleries were measured by tracing transparent film on each stem, then using ImageJ (Rasband 2018) software to quantify the amount of phloem tissue consumed by each larva (Olson and Rieske 2018).
Insecticide Residues
Root, stem phloem, and foliar tissues were processed within 24 hours of harvest. Plant tissue was placed in liquid nitrogen and ground into a powder with a mortar and pestle and stored at −20 °C prior to analysis. For analysis, a 1:10 ratio of plant tissue to acetonitrile is placed on a rotary table for extraction over 24 hours, after which approximately 300 μL is filtered through a 0.2 μm PTFE syringe filter directly into an autosampler vial for analysis using liquid chromatography mass spectrometry (LC/MS/MS). Imidacloprid and its metabolites, and dinotefuran, were quantified at Villanova University Department of Chemistry using a Shimadzu Prominence HPLC (Shimadzu, Colombia, MD, USA) with a Phenomenex Gemini NX C-18 column (5 μm particle, 4.6 mm ID, 250 mm in length) fitted with a corresponding Gemini guard column using a 10 μL injection volume. The aqueous phase was 10 mM ammonium formate in water, and the organic phase was 10 mM ammonium formate in acetonitrile. Mass spectrometry was performed using a positive electrospray ionization mode; multiple reaction monitoring transitions were used for optimization. Limits of detection (LOD) were 1.36 ppb for imidacloprid, 1.41 ppb for imidacloprid olefin, 6.69 ppb for dihydroxy imidacloprid, and 0.43 ppb for dinotefuran.
Statistical Analysis
For analysis, samples with concentrations of dinotefuran less than the limits of detection (LOD < 0.43) were considered left censored observations and thus were analyzed by the Cox proportional hazards model. However, the Cox model can only handle right censoring, so dinotefuran concentrations, y, were transformed using y*= 40 – y. The choice of 40 is arbitrary, but is chosen so that y* > 0. Therefore, a y that was left censored becomes right censored under the transformation y*, where now an observation is right censored if y* > 39.57. The Cox model was then fit to y* by site, plot, treatment, tissue type, and the interaction of treatment by tissue. The goodness of fit was assessed by the Cox-Snell and Schoenfeld residuals. Tissue samples of imidacloprid olefin were handled similarly using LOD < 1.41, except that the stem tissue was removed from the analysis due to a high percentage of censored observations. Both analyses were conducted in PROC PHREG (SAS 9.3, SAS Institute Inc. 2011). Dihydroxy imidacloprid was also not analyzed due to the number of missing values. Finally, imidacloprid concentrations were log transformed due to right-skewness. Imidacloprid concentrations had no censoring and were analyzed via a mixed model ANOVA using Proc GLIMMIX, where treatment, tissue type, and treatment by tissue type were designated as fixed effects, and site and plot were designated as random effects. The data for EAB larval phloem consumption have many zeroes, and the residuals of a linear regression are not normally distributed, so a nonparametric Kruskal-Wallis test was used to evaluate differences in EAB larval phloem consumption among the 5 treatments using Proc NPAR1WAY. Pairwise differences were assessed using the Dwass, Steel, Critchlow-Fligner method.
RESULTS
Residue concentrations in trees treated with the full rate of imidacloprid were above the limit of detection (1.36 ppb), and ranged from 8.77 ppb to 567 ppb. For trees receiving the half rate, imidacloprid residues ranged from 1.83 to 598 ppb. Concentrations of the metabolite imidacloprid olefin ranged from below the limit of detection (1.41 ppb) to 142 ppb in trees receiving the full rate of imidacloprid; trees receiving the half rate had imidacloprid olefin concentrations ranging from 1.41 (the LOD) to 17.3 ppb. Of the imidacloprid-treated trees (full and half rate), 17 had imidacloprid olefin levels above the limit of detection, including 9 leaf tissue samples, 6 root tissue samples, and 3 stem tissue samples. The range of the metabolite dihydroxy imidacloprid was below the limit of detection (6.69) to 18.9 ppb for trees receiving a full rate, and 6.69 (the LOD) to 16.3 ppb for trees receiving the half rate. Only 2 of the full rate and 2 of the half rate imidacloprid-treated trees had dihydroxy imidacloprid concentrations above the limit of detection, in leaf tissue and root tissue, respectively.
Imidacloprid residues differed significantly based on treatment (full or half application rate) and on tissue type (leaves, stems, or roots), and there was no significant treatment × tissue interaction (Table 1a). Residues of the metabolite imidacloprid olefin did not differ based on treatment or on tissue type, and again, there was no interaction (Table 1b). The metabolite dihydroxy imidacloprid was below the LOD and so was not analyzed.
Analysis of insecticide residues in parts per billion (ppb) 5 weeks post-treatment in leaf, stem, and root tissues of ash trees treated with (a) imidacloprid applied as a soil drench at full rate (2.64 mL/L H2O) and half rate (1.32 mL/L H2O), its metabolite (b) imidacloprid olefin, and (c) dinotefuran applied as a trunk spray at full rate (90 g/L H2O) and half rate (45 g/L H2O).
Root tissue from trees treated with a soil drench of imidacloprid contained over 7 times the residue found in leaf tissue, regardless of application rate, and although leaf and stem tissue did not differ statistically, leaf tissue contained 3 to 5 times higher residues than did stem tissue, again regardless of application rate (Table 2a).
Insecticide residue in parts per billion (ppb)(raw mean [SE]) in ash foliage, stem, and root tissues 5 weeks following application of (a) imidacloprid applied as a soil drench at full rate (2.64 mL/L H2O) and half rate (1.32 mL/L H2O)(means within columns followed by the same letter do not differ) and (b) dinotefuran applied as a bark spray at full rate (90 g/L H2O) and half rate (45 g/L H2O)(n = sample size used to calculate raw means, where values below the limits of detection have been dropped from calculations).
For dinotefuran, 33 of the 55 samples had residue concentrations above the limit of detection (0.43 ppb), ranging from 0.47 to 33.3 ppb. Of the 18 leaf and 18 root samples, 16 had measureable dinotefuran residues. Of the stem samples, only 2 had dinotefuran residues above detectable limits.
In contrast to imidacloprid, dinotefuran applied at full or half rates resulted in no significant differences in residues 5 weeks post-application (Table 1c). There were, however, significant differences based on tissue type, but no interaction between the two factors.
Dinotefuran applied as a trunk spray at the full rate resulted in significantly higher residues in both leaf and root tissue relative to stem tissue; leaves and roots of dinotefuran-treated trees contained over 40 times the residues found in stem tissue. Dinotefuran applied at the half rate was similar (Table 2b).
Phloem tissue consumption by EAB larvae in treated trees was lower than in untreated trees, regardless of chemical insecticide and application rate (χ2 = 26.26; df = 4; P < 0.0001)(Figure 2). Trees treated with either insecticide applied at either the full or half label rates had significantly lower EAB phloem consumption relative to the untreated controls, but there were no differences among treated trees (Table 3).
Phloem tissue consumed (mm2, mean + SE) by EAB larvae developing in green ash trees treated with imidacloprid and dinotefuran applied at full and half strength (ImidFull, ImidHalf, DinoFull, DinoHalf) and untreated controls. Kruskal-Wallis non-parametric test (χ2 = 26.26; df = 4; P < 0.0001).
Comparison of larval EAB phloem consumption on trees treated with imidacloprid applied as a soil drench at full rate (2.64 mL/L H2O) and half rate (1.32 mL/L H2O) and dinotefuran applied as a bark spray at full rate (90 g/L H2O) and half rate (45 g/L H2O) relative to untreated controls.
DISCUSSION
Systemic neonicotinoids, including imidacloprid and dinotefuran, have been used extensively in agricultural settings since the 1990s (Elbert et al. 2008; Jeschke and Nauen 2008) and have also been employed against tree pests (Ahern et al. 2005; Cowles 2009; Faulkenberry et al. 2012), including the emerald ash borer (Herms et al. 2009; Smitley et al. 2015). However, neonicotinoid use is facing public and regulatory scrutiny with concerns over pollinator conservation (Godfray et al. 2014; Karahan et al. 2015; Stanley et al. 2015), human toxicity (Cimino et al. 2017; Zhang et al. 2018; and references therein), and environmental contamination (Morrissey et al. 2015; Wood and Goulson 2017; Hladik et al. 2018) stemming from potential misuse and overuse in both agricultural systems and urban landscape care. Given their utility in managing landscape and agricultural pests, prudent stewardship of these useful insecticides is essential, and understanding the chemicals’ behavior within plant tissues is key to this stewardship.
In this study, within-plant distribution of two neonicotinoids used for emerald ash borer management, imidacloprid and dinotefuran, applied at two rates (full and half), were evaluated in green ash, a common urban and forest tree. Five weeks following conventional application (soil drenching for imidacloprid and basal bark spray for dinotefuran), insecticide residues were analyzed in leaf, stem, and root tissue of small green ash trees (< 5 cm DBH) to determine where these compounds are retained following application and their relative efficacy for EAB suppression.
Within-plant movement of insecticide depends on several factors, including concentration, solubility, tree size, soil moisture, and timing of application (Jeschke and Nauen 2008; McCullough et al. 2011; Faulkenberry et al. 2012; Coots et al. 2013). Additionally, tree health affects insecticide translocation; disrupted vascular tissue, due either to insect infestation or other factors, can compromise insecticide uptake. Similarly, the effects of these neonicotinoids on insect survival and development also depends on several factors. Again, insecticide concentration is key, as is temperature. Temperature affects insect development rate, and therefore the length of time the insects are exposed to toxins, which is important, as both acute and chronic effects of neonicotinoids play a role (McCullough et al. 2007; McCullough et al. 2011; Poland et al. 2016). Woody plants translocate neonicotinoids from the point of application to the leaves, where they act as strong antifeedants (Tanis et al. 2012; Poland et al. 2016). In addition to functioning as antifeedants, both imidacloprid and dinotefuran are lethal to EAB larvae if present at sufficiently high concentrations (Herms et al. 2009; Smitley et al. 2015; Poland et al. 2016).
Plants metabolize imidacloprid into the byproducts imidacloprid olefin and dihydroxy imidacloprid with varying toxicities (Nauen et al. 1998). There were high levels of imidacloprid olefin in the imidacloprid-treated green ash in this study where the compound could be encountered by feeding beetles, and if so, would contribute to suppression. In contrast, dihydroxy imidacloprid was below detectable levels in most of our samples, suggesting that it is a transitory compound that may be inconsequential for EAB suppression in green ash, similar to its role in the hemlock woolly adelgid–hemlock system (Benton et al. 2016).
At both application rates in this study (full vs. half), imidacloprid residues in leaf and root tissues exceeded the LC50s determined by Poland et al. (2016) for second and third instar EAB larvae reared on artificial diet in a laboratory study, and in stem tissue equaled the LC50 when applied at the full rate (Table 2). Residues of imidacloprid were concentrated in green ash roots at the point of application relative to leaves and stems, regardless of the application rate. The high concentrations remaining in root tissues suggests that translocation to foliar tissues was incomplete after 5 weeks. Nevertheless, imidacloprid was found in leaves and stems in concentrations adequate to cause EAB mortality (Poland et al. 2016). EAB larvae in imidacloprid-treated trees consumed measurable amounts of phloem, suggesting both chronic and acute toxicity. Given that EAB suppression using imidacloprid requires annual applications (Herms et al. 2009; McCullough et al. 2011), these data suggest that lower rates may be efficacious for EAB suppression, at least for small-diameter trees. This point warrants further study.
Residues of dinotefuran did not differ based on application rate, but there were differences in residue concentrations between plant tissues. Dinotefuran was applied to the stem as a basal bark spray and was effectively and rapidly transported to leaf and root tissues where it was found in roughly equal amounts (Table 2). This rapid translocation is due, in part, to its solubility, which at 39.8 g/L (EPA 2004) is considerably more soluble than imidacloprid (0.61 g/L, NPIC 2017). Dinotefuran residues in stem tissue were lower than the LC50 of 30 ppb determined by Poland et al. (2016) and were below the limits of detection in all but one sample. Nevertheless, EAB gallery formation was minimal, and no larvae were recovered, suggesting that neonate larvae succumbed very rapidly to acute dinotefuran toxicity.
Although this was a short-term study utilizing small trees, and foliage from only one branch per tree was evaluated, both neonicotinoid insecticides tested were translocated to foliar tissue, regardless of the rate of application (full or half rate). Residual concentrations present in plant tissue 5 weeks post-application did not differ between doses. Additionally, insecticides applied at the half rate effectively reduced larval EAB phloem consumption relative to untreated controls (Figure 2 and Table 3). These findings suggest that, for trees of the size used in this experiment, reduced rates of neonicotinoids may be efficacious as a management strategy for EAB, may reduce the risk of nontarget concerns, and may allow more trees to be treated in a given area. Clearly more research is warranted. Landscape managers, tree care specialists, and homeowners utilizing these products as components of an EAB management plan could reduce costs, minimize environmental exposure, and potentially reduce unwanted collateral damage by reducing insecticide application rates. These findings can assist land managers in making more informed decisions about ash protection as the emerald ash borer continues to devastate urban forests and landscape plants in North America.
ACKNOWLEDGMENTS
The authors thank Eastern Kentucky University for allowing us to conduct this project and access sampling sites at Taylor Fork Ecological Area. We also thank Katherine Hagan, Mitch Hughes, Beth Kyre, Hannah Moore, and Samuel Rivers for assistance with field and laboratory work. Anthony Lagalante (Villanova University) analyzed plant tissue samples. Matthew Rutledge and Eva Loveland assisted with the statistical analyses. Kenneth Haynes and Daniel Potter provided feedback on early versions of the manuscript. This is publication number 18-08-060 of the Kentucky Agricultural Experiment Station and is published with the approval of the director. This work is supported by the Kentucky Division of Forestry and the USDA Forest Service through a Landscape Scale Restoration Grant, and by McIntire Stennis Funds under 2351197000.
Footnotes
Conflicts of Interest:
The authors reported no conflicts of interest.
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