A Native Lepidopteran is Impacted by Host Defenses Induced by Hemlock Woolly Adelgid (Adelges tsugae)

Eastern hemlock, Tsuga canadensis, is currently experiencing widespread mortality due to the invasive hemlock woolly adelgid (HWA; Adelges tsugae). Eastern hemlock looper (Lambdina fiscellaria), another hemlock pest, is a native lepidopteran that has reached outbreak levels in the past. While these insects share a host and overlap in range, little is known about their interactions. Research has shown that HWA infestation increases methyl salicylate concentrations (an indicator of the induction of the salicylic acid pathway) in hemlock tissue while hemlock looper, a chewing insect, likely elicits the jasmonic acid pathway. While plants are capable of inducing both of these defensive pathways, they have been found to be mutually antagonistic. We tested the hypothesis that looper performance is affected by prior HWA infestation and plant defense. Specifically, we hypothesized that looper larvae would perform better on HWA infested hemlock foliage. Looper were reared to pupation on hemlock foliage that was either infested or uninfested with HWA. Within those treatments, groups of foliage were sprayed with defensive elicitors to induce either the jasmonicor salicylic-acid pathway. We also analyzed total phenolic content and terpenoid levels in host needle tissue in all treatments. Application of the chemical elicitors negatively affected looper survival, and their impact was altered by HWA presence. Larval survival was significantly lower in treatments where both HWA and elicitor were present. In contrast, larval survival was higher in the HWA-infested control treatment in comparison to the uninfested treatment. This demonstrates a strong interactive effect between HWA and elicitor. The combined effect of elicitor and HWA infestation led to increases in both phenolics and terpenoids, suggesting a major role for these compounds in plant defense. This study demonstrates the complex host-mediated interactions occurring between a native and an invasive insect which may aid in the further study of insect interactions.

We also analyzed total phenolic content and terpenoid levels in host needle tissue in all treatments. Application of the chemical elicitors negatively affected looper survival, and their impact was altered by HWA presence. Larval survival was significantly lower in treatments where both HWA and elicitor were present. In contrast, larval survival was higher in the HWA-infested control treatment in comparison to the uninfested treatment.
This demonstrates a strong interactive effect between HWA and elicitor. The combined effect of elicitor and HWA infestation led to increases in both phenolics and terpenoids, suggesting a major role for these compounds in plant defense. This study demonstrates

INTRODUCTION
Most long-lived plant species must withstand both simultaneous and sequential attacks from multiple herbivores. Interactions between herbivores that share a host can take several forms. Insects that feed at the same time can interact either directly (e.g., interference competition) or via their impact on the shared resource (e.g., exploitative competition) (Park 1948, Mattson 1986, Denno et al. 1995. For herbivores that differ in their phenology, these effects are necessarily asymmetric -feeding by an earlier-arriving insect species, for instance, may affect a later-arriving herbivore via changes in the host plant. Such herbivore-induced changes may negatively impact the later-arriving species by increasing plant defense or decreasing host quality (Faeth 1986, Harrison andKarban 1986); it is also possible, however, for the early-arriving species to benefit the laterarriving one via decreases in plant defense (Rodriguez-Saona et al. 2010, Soler et al. 2012).
The induced production of chemical compounds is a commonly-used defense against herbivory (Karban and Baldwin 1997). Two of the most well-studied plant defense pathways are the salicylic acid (SA) and jasmonic acid (JA) pathways. While the SA pathway is often induced by pathogen infection and piercing-sucking insects (Malamy et al. 1990, Kaloshian andWalling 2005), the JA pathway is more closely associated with defense against chewing herbivores (McCloud andBaldwin 1997, Walling 2000). While plants can induce both the SA and JA pathways, the two pathways are often antagonistic; the induction of one precludes expression of the other (Kunkel and Brooks 2002) . This creates the possibility for defense-signaling crosstalk within plants attacked by multiple herbivores. SA pathway expression in pathogen-attacked plants, for instance, may suppress the JA pathway and increase susceptibility to subsequent herbivory. Cases where JA-SA signaling crosstalk has impacted insect performance have been documented in many plant species (Cipollini et al. 2004, Zhang et al. 2013, Schweiger et al. 2014).
The hemlock woolly adelgid (Adelges tsugae Annand; 'HWA') is a sessile herbivore native to Japan that was first recorded in the eastern United States in the 1950s (Havill et al. 2006). In the northern portion of its invaded range, this aphid-like insect feeds solely on eastern hemlock (Tsuga canadensis [L.] Carriere) by inserting its stylet into the base of a needle and feeding on xylem ray parenchyma cells (Young et al. 1995).
Eastern hemlocks are highly susceptible to HWA (Montgomery et al. 2009) and trees quickly decline in health. HWA can kill even mature trees in as little as four years (McClure 1991), with some HWA-infested stands experiencing over 95% hemlock mortality (Orwig and Foster 1998).
Although HWA poses a major threat, it is not the only insect capable of causing substantial hemlock mortality. The eastern hemlock looper (Lambdina fiscellaria Guenée; 'looper') is a native lepidopteran whose larvae defoliate hemlocks and can cause tree mortality after just four years of severe defoliation (Hudak et al. 1978). Although the looper and HWA share a host and co-occur in southern New England, very little is known about their interplay (Wilson et al. 2015). The potentially dramatic impacts of both herbivores on eastern hemlock forests suggests, however, that their interactions may be extremely important.
The hemlock woolly adelgid and looper differ in a number of respects. While both insects begin feeding in spring, HWA typically settle on foliage several weeks before looper larvae emerge. In addition, HWA are sessile xylem feeders while looper larvae are mobile folivores. These differences create the potential for plant-mediated interactions between HWA and the looper: previous research indicates that looper larvae may perform better on HWA-infested foliage (Wilson et al. 2015). While the mechanistic basis for these results is unknown, it may reflect herbivore-induced changes in plant defense.
Previous research has shown that HWA feeding increases the production of methyl salicylate, an ester of SA, in hemlock (Pezet and Elkinton 2014). By contrast, foliar-feeding looper larvae might be expected to increase expression of the JA pathway (Karban and Baldwin 1997, Kessler and Baldwin 2002, Van Wees et al. 2013). The cooccurrence of herbivores with different feeding modes on the same host suggests the potential for crosstalk between the SA and JA pathways. We hypothesized that looper larvae would have higher survival and perform better on foliage that had been previously infested with HWA. We also assessed whether spraying trees with chemicals designed to elicit either the JA or SA pathway would affect looper performance. We present research addressing the effects of HWA infestation on looper larval performance in the presence and absence of JA and SA pathway elicitors. In addition, an analysis of the phenolic and terpenoid content of needle tissue was performed in order to assess potential mechanisms for treatment differences in larval survival.

Larval Survival
In spring 2015, we purchased 300 T. canadensis saplings (0.5-0.7 m height; 'hemlocks') from Vans Pines Nursery (West Olive, MI; derived from seed collected in PA). All hemlocks were herbivore-free and had not been treated with insecticides. After potting the hemlocks, we randomly selected half of them for HWA inoculation. Trees in the HWA treatment were inoculated in June 2015 by loosely tying two HWA-infested branches to each sapling, a standard protocol (Butin et al. 2007). We collected the HWA- 15-cm piece of foliage from each tree and placed it in a water-filled floral tube (7.6 cm standard; Royal Imports) that we stuck into the foam. Hatchling larvae were randomly assigned to one of the 60 foliage-filled jars until each jar contained five larvae (300 total larvae). Each jar was covered with a fine white mesh (~0.5 mm; nylon) to allow ventilation but prevent escape. All 60 jars were kept in the growth chamber under the conditions described above. All jars were rotated weekly to control for potential microclimatic differences. Each jar was considered a replicate (60 total).
We assessed looper performance by measuring larval survival, mass, and days to pupation. Eight days after the experiment began, we counted the surviving larvae in each jar and weighed them together using a Mettler Toledo scale (±0.001 mg) in order to determine average live larval mass per jar. The larvae were then returned to their respective jar in the growth chamber. We recorded survival 13 times (~weekly) throughout the experiment; larval mass was recorded on days 15, 28, 39, 49, and 62. All larvae were given fresh foliage seven days after the start of the experiment, then as often as necessary throughout the experiment. The experiment ended in late July, when the nine surviving larvae that had failed to pupate were weighed and frozen at -20℃ until autoclaving as per our APHIS permit.

Chemical Defenses of Needle Tissue
On June 6, 2016, three randomly-selected ~10 cm twigs were clipped from each tree, stored in aluminum foil packets, and immediately placed in liquid nitrogen. Packets were subsequently stored at -80°C until extractions were performed. For extractions, needles and twigs were separated and the two tissue types ground to powder in liquid nitrogen. Approximately 100 mg of powdered needle tissue was placed in 1.5 mL microtubes, followed by the addition of 0.5 mL of 100% methanol. Tubes were incubated for 24 hours with occasional vortexing, after which the 9,000 x g supernatant was removed and placed into new 1.5 mL tubes. The process was repeated and supernatants were eventually combined.
Total soluble phenolics were quantified according to Cipollini et al. (2011). For terpenoids, the spectrophotometric procedure described by Ghorai et al.
(2012) was used. A 200 µL aliquot of methanol extract was added to a 2 mL mictrotube containing 1.5 mL chloroform, followed by the addition of 100 µL H 2 SO 4. After the samples were incubated for two hours in the dark, 1 mL of the top layer was removed, replaced with 1 mL 100% methanol, and the tubes were vortexed to solubilize the red precipitate. The absorbance of the solution was quantified at 538 nm against a standard curve of linalool, and terpenoids were expressed as mg/g FW.

Statistical Methods for Analysis of Larval Survival
All statistical analyses were performed using R Statistical Software (R Core Team 2016). Due to the unexpected decline of the HWA-infested trees sprayed with the JAelicitor, we ceased applying elicitors after four weeks, as described previously. Thus, in assessing the effects of the elicitor treatments on larval survival, we analyzed the first six data points (through Julian day 173), which coincides with the last application of the SAand JA-elicitors; larval survival after this point would not have likely been impacted by these applications. To assess the impacts of HWA infestation of host material and elicitor applications, a repeated measures analysis of variance (rm-ANOVA) was employed.
Survival data were arcsine-square root transformed (Lindroth et al. 1990). Percent larval survival in a given jar was the response variable and HWA infestation, elicitor application, and the interaction between the two were the predictor variables. To estimate treatment differences, individual treatment combinations were used as the predictor variable and percent survival as the response variable in a linear mixed-effect model with sampling date as the random effect. A post-hoc separation of means was then performed on this model via a Tukey test using the simultaneous tests for general linear hypotheses ('glht') function, part of the 'multcomp' package (Hothorn et al. 2008) in R.

Statistical Methods for Defense Chemistry and Nutritional Attributes
All statistics were performed using R Statistical Software (R Core Team 2016).
The Shapiro-Wilk normality test was used to confirm data normality and the Dixon test was used to identify and remove outliers using the 'Outliers' package in R (Komsta 2011). An analysis of variance (ANOVA) was used to determine the significance of HWA infestation, hormone application, and the interaction between the two, with these factors as predictor variables and soluble phenolics or terpenoids as response variables. If either the interaction or hormone application predictor variables were significant, posthoc Tukey's HSD was used to determine which treatments differed from each other.

Larval survival
HWA infestation tended to increase larval survival over time (F 1,324 =5.32, P=0.069) according to the results of the repeated measures ANOVA (Table 1) (Figure 1).
There was a significant negative effect of both the chemical elicitor treatment (F 2,324 =10.30, P=0.004) and the interaction between HWA infestation and elicitor (F 2,324 =7.90, P=0.009) on survival. The post-hoc analysis revealed that the JA treatment significantly lowered larval survival (P=0.007) in comparison to both the ethanol and SA treatments.
The post-hoc analysis of the interactive effects revealed that the HWA-infested + JA (P = 0.017) and the HWA-infested + SA (P = 0.016) treatments had significantly lower survival than the HWA-infested + ethanol treatment. No other treatment had a statistically significant effect on survival relative to other treatment combinations. There was also no significant effect of any treatment on larval mass (Table 1).

DISCUSSION
Looper larvae fed HWA-infested foliage tended to have higher survival than those reared on uninfested foliage. The presence of elicitors also affected survival, and their impact was altered by HWA presence (Table 1). Looper larvae fed HWA-infested foliage treated with either the JA or SA elicitor had significantly lower survival rates than all other treatment combinations (Figure 1). We also analyzed phenolic and terpenoid content in needle tissue, both of which have been shown to negatively affect herbivorous insects (Feeny 1968, Bezemer et al. 2003, War et al. 2012) and play a major role in conifer resistance. The fact that both phenolic content and terpenoid levels increased with MeJA and SA application is consistent with our general understanding of the role that both metabolites play in herbivore-induced defense in hemlock.
The application of SA on uninfested foliage did not significantly increase levels of phenolics or terpenoids, and neither did HWA infestation alone. However, the  (Kessler et al. 2006, Hirao et al. 2012. If the initial HWA infestation defensively primed the hemlock trees, this would explain why the subsequent addition of elicitors induced greater defense, i.e., higher phenolic and terpenoid concentrations. While the potential for defensive priming is intriguing, further study is necessary to confirm that it is in fact occurring.
Our JA elicitor increased phenolic, but not terpenoid, concentrations. Applying MeJA to uninfested hemlocks increased foliar phenolics but did not alter larval survival.
As a result, phenolic and terpenoid concentrations do not appear to explain low larval survival in the JA + HWA treatment. It is possible that JA-driven decreases in larval survival are due to factor(s) we did not quantify, such as plant nutritional quality or another defensive compound. Future research might consider analyzing factors such as carbon, nitrogen, and water content, as well as starch and tannin levels, in order to provide additional insight into interplay between plant chemistry and larval survival.
We were surprised that HWA infestation alone did not significantly alter phenolic or terpenoid levels. We did, however, find that the impact of both elicitors was only apparent when applied to HWA-infested foliage. Adelgid infestation increased survival in the absence of elicitors but decreased it in their presence: both the HWA + JA and HWA + SA treatments had significantly lower survival than the HWA + ethanol treatment.
While the strong HWA by elicitor interaction is intriguing, the broad-scale chemical analyses we conducted proved insufficient to provide a clear and convincing explanation. Studying invasive insects, for example, and their impacts on host species in their invaded range is certainly important, however, the resulting effects of invasion often extend beyond these immediate interactions (Kenis et al. 2009). It is increasingly important, therefore, to include potential secondary interactions and impacts when studying invasive species.
Eastern hemlock is an ecologically important forest tree and is considered a foundation species (Ellison et al. 2005). The presence and abundance of T. canadensis not only defines a hemlock forest, but greatly influences both terrestrial and aquatic habitats (Snyder et al. 2002, Tingley et al. 2002. It is currently experiencing widespread mortality due to the invasive hemlock woolly adelgid (Orwig and Foster 1998). The eastern hemlock looper, another hemlock pest, is a native lepidopteran that has reached outbreak levels in the past (Hudak et al. 1978). Little is known about the relationship between these insects, such as how feeding by both may affect the chemical profile of their host, and how this plant-mediated interaction affects each species. While HWA infestation poses a significant threat to North America's hemlock forests, it is not an isolated system: the overlap of hemlock looper and HWA presents an opportunity to study interactions between a native herbivore and an invasive pest. Our results provide insight into the interactions occurring between two ecologically-important insect herbivores and their shared host plant. Further study may reveal additional insights applicable to other systems where native and invasive insect species interact. and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.   Terpenoids (mg/g FW) Figure S1: Images of representative trees from each treatment group. Trees in the HWA+JA group (highlighted in yellow) suffered dramatic needle loss and were visibly more stressed than trees from all other treatment groups.