Journal of Chemical Ecology, Vol. 17, No. 9, 1991 WHITE ALDER AND DOUGLAS-FIR FOLIAGE QUALITY AND INTEREGG-MASS INFLUENCES ON LARVAL DEVELOPMENT OF GYPSY MOTH, Lymantria dispar G. JOSEPH, I. J.C. MILLER, R.E. BERRY, J. WERNZ, A.F. MOLDENKE, and R.G. KELSEY2 Department of Entomology Oregon State University Corvallis, Oregon 97331-2907 (Received February I I, 1991; accepted April 29, 1991) Abstract—Individual families of gypsy moth collected from a single popu- lation exhibited different degrees of fitness when fed diets of white alder, a suitable broadleaf host, and Douglas-fir, an unsuitable conifer host. Members of families on diets of Douglas-fir had significantly lower survival, longer larval periods, lower pupal weights, and shorter pupal periods than members of the same families fed alder. Foliar nutritional quality, including nitrogen level and allelochemical composition (terpenes and phenols), was considered the key factor responsible for these differences. Growth parameters differed significantly for families within diet treatments, indicating that the genetic resources of a family did affect performance somewhat. The influence of a familys genetic resources on larval survival was most notable when larvae were under the greatest nutritional stress. Key Words—Douglas-fir, Pseudotsuga menziesii, gypsy moth, Lymantria dispar. Lepidoptera, Lymantriidae, insect-host plant relations, phenolics, terpenes. white alder, Alnus rhombifolia. INTRODUCTION Developmental rate and survival of the gypsy moth are influenced by many factors, including host species (Hough and Pimentel, 1978; Barbosa et al., To whom correspondence should be addressed. Present address: Department of Forest Science, School of Forestry, Oregon State University, Corvallis, Oregon 97331-2907. Present address: USDA, Forest Service, Pacific Northwest Research Station, Forestry Sciences Laboratory, Corvallis, Oregon 97331-2907. 1783 0098-0331/91/0900- I 783806.50/0 © 1991 Plenum Publishing Corporation 1784 JOSEPH ET AL. 1986); constitutive and induced chemical variation within the host species (Rhoades, 1983; Rossiter et al., 1988); genetic variation among and within insect populations (Leonard, 1966); and environmental variables, such as tem- perature and humidity (Elkinton and Liebhold, 1990). Previous studies relating the suitability of hosts to gypsy moth fitness have suggested that certain alle- lochemicals, such as alkaloids, influence fitness parameters, such as larval development (Doskotch et al., 1981; Barbosa et al., 1983, 1990a,b; Barbosa and Krischik, 1987; Miller and Hanson, 1989a). Plants containing iridoids and alkaloids appear to be poor hosts (Barbosa and Krischik, 1987; Miller and Han- son, 1989a, Barbosa et al., 1990a), but many other plants containing a wide array of allelochemicals permit successful development (Miller and Hanson. 1989a). Lechowicz (1983) suggested that suitable plants are characterized by precipitable (hydrolyzable) tannins and sclerophylly (a combined measure of leaf toughness and water content). Goldschmidt (1934) and Leonard (1966. 1969) noted significant intra- and interpopulation variance in larval development that was related to the parental source (egg mass) of the larvae. Variation in growth among individuals in a population may be influenced by genetic differences between families that also may influence suitability of hosts, particularly those newly encountered. The gypsy moth is not an established pest in the forests of the Pacific Northwest of North America. Isolated infestations were detected in Pacific Coast states in the early 1980s, when egg masses were accidentally transported from sites with major infestations in the northeastern United States. Oregon had a serious problem in 1984 (Daterman et al., 1986). Spraying with Bacillus thu- ringiensis in 1985 and 1986 successfully controlled the outbreak. As long as large infestations exist in the United States, new introductions of gypsy moth into western forests remains a possibility. The objective of this research was to determine the range of certain devel- opmental parameters relative to the parental source of larvae and the allelo- chemical content of the diet. Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco], a conifer, was selected for study because of its prominent status in forests of the Pacific Northwest, and its limited suitability as a host (Miller and Hanson. 1989b; Miller et al., 1991). White alder (Alnus rhombifolia Nutt.) was chosen to represent the highly suitable woody angiosperm hosts that commonly grow intermixed or adjacent to Douglas-fir forests. The differences in host suit- ability of these two species was believed to be due to significant differences in the allelochemical contents of their foliage. METHODS AND MATERIALS Insects. Egg masses were obtained at the end of January from an oak woodland in Seneca Creek State Park, Montgomery County, Maryland, and stored at 4°C until May. Eight large egg masses (each containing ca. 700-1000 FOLIAGE QUALITY AND GYPSY MOTH DEVELOPMENT 1785 eggs) were selected for evaluation of larval performance on Douglas-fir; eggs from four of these masses also were evaluated on alder. Large egg masses were chosen to ensure against selecting two egg masses from the same parents (Doane and McManus, 1981), thus assuring that each egg mass was of a separate family line. Foliage Collection. Foliage was collected from three alder trees initially selected for their differences in nitrogen content after a preliminary analysis; two trees had higher nitrogen levels (tree-type A) than the third (tree-type B). These differences in nitrogen did not persist, however, resulting in nearly iden- tical nitrogen contents for the two alder types when foliage collection began. Douglas-fir foliage was collected from five trees. Two were 10 years old and had high levels of foliar nitrogen resulting from fertilization (tree-type A); the other three, ranging in age from 10 to 15 years old, were unfertilized and had low foliar nitrogen (tree-type B). Collections for laboratory feeding trials began in May and were completed by the end of June, coincident with the natural life cycle of gypsy moth larvae in Oregon. Only one tree of each type was harvested on collection days, with the same tree harvested on subsequent days until the foliage became limited; then a new tree of the same type was selected. The freshly gathered foliage was immediately transported to the laboratory, where it was subsampled for chem- ical analysis and then prepared for feeding by surface sterilizing in 0.25% sodium hypochlorite, rinsing in distilled water, and briefly air-drying. Larval Feeding Experiment. Upon eclosion, 80 larvae from each of eight families were randomly selected for rearing on Douglas-fir foliage; an addi- tional 42 larvae were selected from each of four of these families and reared on alder foliage. Larvae on Douglas-fir diets were reared in 145-m1 cups, 10 per cup until the third instar and three per cup for the remaining instars. Larvae on alder diets were reared in groups of three per cup throughout. Larvae were reared at 24°C, 45-50% relative humidity, and 16: 8 (light— dark) hr photoperiod. Foliage was replaced every two days for early instars and daily for later instars. Larvae were observed every three days to determine per- cent survival. Pupae were weighed 48 hr after pupation. Days to pupation and pupal period also were determined by daily observations. Nitrogen Analysis. Subsamples of foliage for nitrogen analysis were washed in dilute soap solution, rinsed three times with distilled H70, oven-dried at 60°C for 48 hr, and ground in a Wiley mill to pass a 20-mesh screen. Nitrogen content was determined by a micro-Kjeldahl technique with an automated Technicon Autoanalyzer II (Anonymous, 1975). Terpene Analysis. Five to twelve branch tips of Douglas-fir, similar to those used to feed larvae, were selected. One-year-old needles were detached, combined into a composite sample, sealed in double air-tight plastic bags, and stored frozen until processed for analysis. Before analysis, samples were warmed to room temperature inside the bags. 1786 JOSEPH ET AL. A subsample was withdrawn, frozen with liquid N,, and ground with a mortar and pestle. Ground tissue was transferred to a capped scintillation vial. Water content was determined on triplicate samples dried at 105°C overnight. Terpenes were extracted from 0.5 g of the freshly ground subsamples in I ml Me0H-H,0 (2:1) and 2 ml of pentane containing fenchone (0. I m g,/m1) as an internal standard (Brooks et al., 1987). Samples were shaken mechanically for 60 min and then centrifuged at room temperature in a IEC HN-SII centrifuge at approximately 1000 rpm for 3-4 min. The pentane, containing the terpenes, was removed and stored at — 16°C. Terpenes were analyzed on a Hewlett-Packard 5890 gas chromatograph equipped with a flame ionization detector and a Supelcowax 10 fused silica capillary column (30 m x 0.32 mm ID. 0.25-gm film thickness. 1:50 split). Injector and detector were at 250°C. Oven temperature was programmed from 60 to 220°C at 5°/min with a I5-min pause at 220°C. Peak areas were obtained with a Hewlett-Packard 3390A integrator. Compounds were identified by a combination of gas chromatography-mass spectrometry and peak enrichment with standards. Separate response factors for hydrocarbons and monooxygen- ated and dioxygenated compounds were determined with standards relative to fenchone. Total Phenol Anal ysis. A subsample of alder leaves was air-dried after collection and stored in plastic bags at room temperature. A portion of the fresh- frozen Douglas-fir needles analyzed for terpenes was subsampled for phenolic analysis. Before analysis, alder and Douglas-fir foliage were oven-dried for 24 hr at 60°C, ground to pass a 40-mesh screen, and redried overni g ht. Phenols were extracted from a 100-mg subsample in 8 ml (Douglas-fir) or 10 ml (alder) Me0H-H,0 (7:3) on a shaker for 1 hr. The mixture was centrifuged at 1000 rpm in an IEC HN-SII centrifuge at room temperature for 5 min and analyzed with Folin-Ciocalteu phenol reagent as described by Julkunen-Tiitto (1985).