Hemlock Woolly Adelgid and Elongate Hemlock Scale Induce Changes in Foliar and Twig Volatiles of Eastern Hemlock

Eastern hemlock (Tsuga canadensis) is in rapid decline because of infestation by the invasive hemlock woolly adelgid (Adelges tsugae; ‘HWA’) and, to a lesser extent, the invasive elongate hemlock scale (Fiorinia externa; ‘EHS’). For many conifers, induced oleoresin-based defenses play a central role in their response to herbivorous insects; however, it is unknown whether eastern hemlock mobilizes these inducible defenses. We conducted a study to determine if feeding by HWA or EHS induced changes in the volatile resin compounds of eastern hemlock. Young trees were experimentally infested for 3 years with HWA, EHS, or neither insect. Twig and needle resin volatiles were identified and quantified by gas chromatography/mass spectrometry. We observed a suite of changes in eastern hemlock’s volatile profile markedly different from the largely terpenoid-based defense response of similar conifers. Overall, both insects produced a similar effect: most twig volatiles decreased slightly, while most needle volatiles increased slightly. Only HWA feeding led to elevated levels of methyl salicylate, a signal for systemic acquired resistance in many plants, and benzyl alcohol, a strong antimicrobial and aphid deterrent. Green leaf volatiles, often induced in wounded plants, were increased by both insects, but more strongly by EHS. The array of phytochemical changes we observed may reflect manipulation of the tree’s biochemistry by HWA, or simply the absence of functional defenses against piercing-sucking insects due to the lack of evolutionary contact with these species. Our findings verify that HWA and EHS both induce changes in eastern hemlock’s resin chemistry, and represent the first important step toward understanding the effects of inducible chemical defenses on hemlock susceptibility to these exotic pests.


INTRODUCTION 64
Conifers in the family Pinaceae are among the largest and longest-living organisms on earth. Their striking 65 longevity means that individual trees face an imposing array of biotic and abiotic challenges. They respond 66 to these challenges via complex constitutive and inducible defenses that enable them to survive under 67 highly diverse and taxing conditions and dominate vast areas of the earth's temperate and alpine forests 68 (Trapp andCroteau 2001, Dudareva et al. 2006).

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Conifers commonly use oleoresin-based chemical defenses to combat herbivorous insects and 70 pathogens (Zulak and Bohlmann 2010). Oleoresin, or simply 'resin,' is a complex and species-specific 71 mixture of phytochemicals that is usually dominated by volatile monoterpenoids and non-volatile 72 diterpenoid acids but also contains smaller amounts of volatile organic chemicals such as sesquiterpenoids, 73 benzenoids (including phenolics), and fatty acid derivatives. These compounds are produced in resin-cells 74 of buds, needles and woody tissue, and in some conifers (such as Pinus species) they accumulate in 75 intercellular ducts or canals either constitutively or in response to trauma (Keeling and Bohlmann 2006).

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Many conifers can respond to insect and microbial challenges via inducible increases in the biosynthesis 77 and accumulation of resin (Hudgins et al. 2004). These defenses variously act to physically engulf and 78 expel insects from the tree by the force of resin flow, seal off infected regions from surrounding tissue, 79 deter herbivory or oviposition, chemically interfere with insect developmental pathways, ATP production 80 and nervous system functioning, and disrupt microbial cell membranes causing cell leakage and death 81 (Langenheim 1994 1950s, and appears to be of Japanese origin (Havill et al. 2006 Lagalante et al. (2006) measured spatial and temporal variability in resin volatiles and hypothesized that 124 these phytochemical fluctuations drive the HWA's unusual annual patterns of settlement, aestivation, and 125 feeding. European silver fir (Abies alba), a conifer of a genus related to Tsuga, showed increased levels of 126 monoterpenoid accumulation in bark naturally infested with Adelges piceae, the balsam woolly adelgid 127 (Hain et al. 1991). In addition, western hemlock (Tsuga heterophylla) responded to simulated herbivory

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In twig tissue, 16 monoterpenoids, five sesquiterpenoids, and six benzenoid or phenolic 298 compounds were present in quantities sufficient for identification and quantification ( Fig 1A); in needle 299 tissue, the corresponding numbers were 18 monoterpenoids, five sesquiterpenoids, one benzenoid, and 300 three fatty acid derivatives (i.e. green leaf volatiles or 'GLVs'; Fig 1B). Qualitatively, needle and twig 301 volatile profiles were overlapping but different (Table 1). Monoterpenoids dominated in terms of both 302 diversity and mass contribution, and had the greatest effect on the induced changes of total volatiles.

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Sesquiterpenoids, present at somewhat lower abundance, generally increased in both twigs and needles.

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GLVs were detected only in needle tissue, and were consistently increased by both insects, especially by 305 EHS-the total amount of these compounds had nearly doubled from June to October; Fig. 2.

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The effects of insect feeding on volatile concentration were larger in twigs than in needles (Table   307   1

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In contrast to the modest changes in terpenoid levels, a number of the non-terpenoids were sharply 347 increased by HWA feeding, in what may reflect a hemlock defense response (Table 1) Fig. 3). In screening studies, benzyl 351 alcohol deterred feeding by the greenbug aphid Schizaphis graminum, reducing fecundity and causing 352 substantial mortality (Formusoh et al. 1997). MeSA, which was also induced by HWA (Fig. 4), has been 353 found in the volatile mix released after aphid feeding and identified as a deterrent to aphid settling and 354 fecundity in a number of plant-insect systems (Hardie et al. 1994, Quiroz et al. 1998).

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The sharp increase of these two compounds in HWA-infested trees (Table 1)

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It is also possible that increased production of these volatiles reflects the tree's detection of a 367 microbial associate of HWA rather than of the insect itself. An endosymbiont was recently found 368 throughout the body of the HWA and appears essential to the insect's survival (Shields and Hirth 2005

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The HWA-driven increases we observed in levels of benzyl alcohol and MeSA may also help 387 explain previously noted changes in the primary chemistry of the hemlock saplings of the present study 388 (Gomez et al. 2012). Although much of the biosynthetic pathway for the benzenoids has yet to be 389 determined, radio-labeling experiments show they are derived from L-phenylalanine (Dudareva et al.