Tolerance of Plant Monoterpenes and Diterpene Acids by Four Species of Lymantriidae (lepidoptera) Exhibiting a Range of Feeding Specificities

Lymantriidae (Lepidoptera) is a family of leaf-feeding insects that includes some of the most damaging forest pests worldwide. Species within this family vary widely in feeding specificity. We evaluated the ability of four species, Douglas fir tussock moth (Orgyia pseudotsugata McDunnough), nun moth (Lymantria monacha L. ), rusty tussock moth (Orgyia antiqua (L.)), and whitemarked tussock moth (Orgyia leucostigma (J. E. Smith)), to contend with one of the most ubiquitous and effective groups of plant defense compounds, terpenoids. We selected these species to provide a range of feeding specificities on conifer hosts, from obligate to occasional. We evaluated the effects of three monoterpenes (bornyl acetate, limonene, and myrcene) and two diterpene acids (isopimaric acid and neoabietic acid) on larval performance. Although these four species differ in their feeding ranges, utilization of conifers as hosts, and other life history processes, each shows a relatively high tolerance for conifer terpenes. The mean relative growth rates, relative consumption rates, and development times were not affected by these monoterpenes and diterpene acids when administered at concentrations present in the foliage of conifers in which they are most abundant. The most likely explanation seems to be metabolism, as a) no limonene or myrcene were recovered from frass or larvae, and b) borneol, an apparent metabolite of bornyl acetate, was recovered from frass of Douglas fir tussock moth, rusty tussock moth, and white-marked tussock moth, and from tissues of Douglas fir tussock moth and white-marked tussock moth. ____________________ Lymantriidae (Lepidoptera) is a family of folivorous insects with worldwide distribution. It contains some of the most important pests affecting trees in forest and urban settings (Schaefer 1989). In addition to native defoliators, several invasive species pose particular challenges to environmental quality and forest resources. This family of over 350 genera and 2500 species contains members ranging from monophagous to polyphagous. This diverse range of host breadths includes both angiosperm and conifer genera. Consequently, lymantriid larvae encounter a broad range of phytochemicals. Terpenes are among the largest groups of defensive chemicals occurring in plants (Gershenzon and Croteau 1991, Langenheim 1994). They occur in both angiosperm and gymnosperm trees (Staudt et al. 2001), but are generally more prevalent in the latter, especially conifers. In particular, monoterpenes and diterpene acids common in conifers exhibit a wide degree of efficacy against a broad range of herbivores, bacteria, and fungi (Trapp and Croteau 2001). Terpenes can negatively impact herbivores through toxic and deterrent effects (Gershenzon and Croteau 1991, Langenheim 1994). Toxicity may result from several mechanisms, including inhibition of ATP formation, interference with hormone production, and binding proteins or sterols in the gut (Langenheim 1994). Mechanisms of deterrence are less well characterized but may involve interaction with sensory receptors (Gershenzon and Croteau 1991). 1University of Wisconsin, 1630 Linden Dr., Department of Entomology, Madison, WI 53706 U.S.A. E-mail: [email protected]. 2Present address: Department of Biology, Portland State University, Portland. OR 97201. 2004 THE GREAT LAKES ENTOMOLOGIST 117 Insect herbivores may metabolize phytochemicals, excrete them unchanged, passively accumulate them in body tissues, or actively sequester them for defense against predators (Blum 1981). Different species employ different mechanisms to process the same phytochemicals. Several characteristics have been proposed to explain this variation, including feeding breadth (Krieger et al. 1971, Gould 1984, Berenbaum 1991, Osborn and Jaffe 1998) and strategies of predator avoidance (Bowers and Puttick 1986). It is difficult to draw conclusions based on feeding breadth because most studies have compared taxonomically distant species, or species that feed on distantly related plants or phytochemicals. Therefore, we evaluated four species within a single family, that have overlapping host ranges, and whose behaviors range from specialist to generalist. In a more detailed study, we conducted similar evaluations of a fifth lymantriid, the highly polyphagous gypsy moth, Lymantria dispar (L. ), (Powell and Raffa 2003). The Douglas fir tussock moth, Orgyia pseudotsugata (McDunnough), is a specialist on conifers, feeding on only 2 genera. It is one of the most important pests of Douglas fir, Pseudotsuga menziesii, white fir, Abies concolor, and grand fir, Abies grandis, in western North America (Wallner 1989). The nun moth, Lymantria monacha (L. ), feeds primarily on conifers, and to a lesser extent on angiosperms. Preferred conifer hosts include Picea, Pinus, Larix, and Abies (Grijpma 1989). Its native range extends from Western Europe to Siberia, and from southern Scandinavia to northern Spain, Portugal, Italy, Greece and Bulgaria (Grijpma 1989). The rusty tussock moth, Orgyia antiqua (L. ), is found worldwide in northern regions. It feeds on all conifer genera except Juniperus, and on over 50 species of angiosperms (Wallner 1989). White-marked tussock moth, Orgyia leucostigma (J. E. Smith), feeds on over 140 tree species. Host trees are primarily angiosperms, but include some conifers. Its geographic range includes most of the central and eastern United States, and southern Canada (Wallner 1989). The purpose of this work was to a) evaluate effects of various terpenes on lymantriid larvae displaying a range of feeding specificities, and b) explore general categories by which lymantriid larvae likely contend with these phytochemicals. MATERIALS AND METHODS Insect sources and rearing. Douglas fir tussock moth egg masses were field collected in Idaho and Oregon, and obtained from a laboratory colony maintained by the Canadian Forest Service in Victoria, British Columbia, Canada. Nun moth egg masses were obtained from laboratory colonies maintained by the USDA Forest Service in Ansonia, Connecticut, USA. Rusty tussock moth and white-marked tussock moth egg masses were obtained from laboratory colonies maintained by the Canadian Forest Service in Sault Sainte Marie, Ontario, Canada. All experiments were performed at the University of Wisconsin, Madison, Wisconsin, USA, except those with nun moth, which were performed at the USDA Forest Service Laboratory in Ansonia due to quarantine restrictions. Upon receipt, egg masses were surface sterilized in a solution of 97% (v/v) deionized water, 1% (v/v) tween (Polyoxy-sorbitan monooleate) and 2% (v/v) bleach (Chlorox: 5% hyperchlorite) for 3 minutes, then rinsed 3 times in deionized water. Egg masses were allowed to air dry for 30 minutes and placed individually in large petri dishes (d = 14.0 cm, h = 3.9 cm; TriState Plastics, Dixon, KY). Upon eclosion, larvae were fed an agarand wheat germ-based artificial (ICN gypsy moth) diet. Larvae were reared in growth chambers at 16:8 (L:D) h and 25° C. Larvae were offered fresh diet every other day until they reached the appropriate stadium. Nun moth larvae were fed agarand wheat germbased artificial diet amended with 3 ml of linseed oil per liter. 118 THE GREAT LAKES ENTOMOLOGIST Vol. 37, Nos. 3 & 4 Bioassays. The monoterpenes bornyl acetate, limonene, and myrcene (Aldrich Chemical Company, Milwaukee, WI) were diluted individually in a 0.75% solution of Triton X 405 (triton) (Aldrich Chemical Company, Milwaukee, WI) in dH2O before being added to artificial diet (Powell and Raffa 1999). The diterpene acids isopimaric acid and neoabietic acid (Helix Biotechnologies, Canada) were dissolved in HPLC grade methanol (MeOH; Fisher Scientific) before being added to artificial diet (Powell and Raffa 1999). Excess methanol was evaporated under a gentle stream of nitrogen. Diet was amended with 0. 75 ml treatment per mg wet weight artificial diet. Larvae showing head capsule “slippage” just before ecdysis were isolated and placed in large petri dishes as above, without food. After 24 h, newly molted larvae were weighed and used in experiments. Individual larvae were placed in 40 ml cups (Polar Plastics, Winston-Salem, NC) and fed amended artificial diet. Newly amended diet was provided every 24 hours for the duration of the stadium. All uneaten diet was collected daily, dried, and weighed. Development time, relative consumption rates, and relative growth rates were calculated for each instar (Waldbauer 1968). Frass was collected daily and frozen until chemical analysis. Larvae and exuviae were collected after each larva molted into the next stadium, and kept frozen until chemical analysis. Three sets of experiments were performed. In the first set of experiments, bornyl acetate, limonene, and myrcene were tested individually at 0.01%, 0.1%, and 1.0%. The controls were distilled water and triton, separately. These experiments were conducted separately with second, third, and fourth instar Douglas fir tussock moths, rusty tussock moths, and white marked tussock moths. An additional group of 10 second instar larvae were tested at 5.0% bornyl acetate. In the second set of experiments, piperonyl butoxide (PBO), a broad inhibitor of P450 enzymes (Brattsten and Metcalf 1970), was added to artificial diet (0.1%) in combination with monoterpenes to explore potential involvement of these enzymes in terpene metabolism. These experiments were performed using Douglas fir tussock moth, nun moth, rusty tussock moth, and white-marked tussock moth. Controls consisted of distilled water, triton, and PBO. The third set of experiments was conducted with the diterpenes isopimaric acid and neoabietic acid at 12.5 mg/ml, 25 mg/ml, and 125 mg/ml. The controls were distilled water and methanol. These experiments were conducted with second instar Douglas fir tussock moths. Due to uneven availability of insects of different species, the exact treatment combinations varied within experiments. Therefore the exact treatments for each insect species – geographic source instar combination are shown at the bottom of Table 1. Likewise, sample sizes also varied depending on insect availability, with the totals being 449 Douglas fir tussock moths, 125 rusty tussock moths, 571 white marked tussock moths, and 88 nun moths. Exact sample sizes for each terpene – dose – instar geographic source-species combination are in Powell (2002). Chemical analyses and fate of phytochemical substances. Larvae were macerated before chemical analysis; frass and exuviae did not require grinding. Monoterpene analyses were performed as described in Powell and Raffa (2003). Briefly, monoterpenes were extracted with hexane from frass or larval tissues for 24 h. The extract was analyzed using a Shimadzu GLC 17A, fitted with an AOC 20i autosampler (Shimadzu Scientific Instruments, Inc. Columbia, MD). Separations were performed on a 25 m x 0. 25 mm bonded fused silica open tubular polyethylene glycol column (Alltech Assoc., Deerfield, IL). Oven temperature was 60°C for the first 10 minutes, and was increased 10°C per minute for 10 minutes, until 160°C. Helium, the carrier gas, was maintained at 30 cm per second. Compounds were quantified by comparing their percentage of the total with the percentage of a known amount of the internal standard para-cymene (Aldrich Chemical Company, Milwaukee, WI). The details of our approach to evaluating the fate of terpenes are in Powell and Raffa (2003). Briefly, terpenes were considered excreted if they 2004 THE GREAT LAKES ENTOMOLOGIST 119 Ta bl e 1. A na ly se s of v ar ia nc e of e ffe ct s of t er pe no id s on f ou r sp ec ie s of t re efe ed in g tu ss oc k m ot hs ( Le pi do pt er a: L ym an tr iid ae ) sp ec ie s: D FT M = D ou gl as F ir t us so ck m ot h; R TM = R us ty t us so ck m ot h; W M TM = W hi te m ar ke d tu ss oc k m ot h; N M = N un m ot h R el at iv e G ro w th R at e St ad iu m D ur at io n R el at iv e C on su m pt io n (g /g /d ay ) a ( da ys t o m ol t) R at e (g /g /d ay ) b E xp . In se ct In st ar df P F P F P F