Community Associates of an Exotic Gallmaker, Dryocosmus kuriphilus (Hymenoptera: Cynipidae), in Eastern North America

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1 ARTHROPOD BIOLOGY Community Associates of an Exotic Gallmaker, Dryocosmus kuriphilus (Hymenoptera: Cynipidae), in Eastern North America W. R. COOPER AND L. K. RIESKE 1 Department of Entomology, University of Kentucky, S-225 Ag North, Lexington, KY Ann. Entomol. Soc. Am. 100(2): 236Ð244 (2007) ABSTRACT Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) is a potentially devastating exotic invasive gall wasp that is rapidly spreading throughout the eastern United States, and infests all species of chestnut, Castanea spp. We investigated the community associates of D. kuriphilus in three geographic locations: Meadowview, VA, Bowling Green, KY, and Broadview Heights, OH. Dissection of galls and observation of chamber contents revealed that gall wasp larval mortality is approaching 47%, and parasitism accounts for nearly 70% of the mortality. Six parasitoid wasp species emerged from chestnut galls, including one introduced species and Þve of unknown origin. The life histories of each parasitoid and their potential roles within the D. kuriphilus community complex are discussed. Investigation of parasitoid interactions revealed a negative correlation between certain parasitoids, suggesting competition for resources, hyperparasitism, or both. The small chestnut weevil, Curculio sayi (Gyllenhal) (Coleoptera: Curculionidae), caused external damage to galls. Galls also were readily attacked by an unknown fungal pathogen resulting in gall wasp larval mortality. This study is the Þrst to investigate the community associates of D. kuriphilus in North America. Our results have beneþcial implications for commercial chestnut production, blight resistance breeding programs, and restoration of American chestnut. KEY WORDS chestnut, Torymus, Ormyrus, Curculio, endophyte American chestnut, Castanea dentata Marshall (family Fagaceae), was once a codominant canopy species in hardwood forests of the eastern United States (Braun 1950, Nelson 1955). In 1904, the exotic chestnut blight fungus, Cryphonectria (Endothia) parasitica (Murr) Barr (Diaporthales: Valsaceae), was accidentally introduced and spread quickly throughout the natural range of American chestnut, functionally eliminating the species (Keever 1953, Good 1968, Mackey and Sivec 1973, McCormick and Platt 1980). The fungus causes diffuse stem cankers that eventually girdle the tree, killing the aboveground portions. Today, American chestnuts typically exist only as shrubby, nonfruiting sprouts from surviving stumps, and usually die back after reaching 2Ð3 m in height (Paillet 1984, 2002). Tree breeders and geneticists have worked diligently to breed blight resistance from Asian chestnuts (Castanea spp.) into American chestnut, and trees for restoration could be available within the next several years (Anagnostakis 2001). However, another more recent exotic pest, Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae), is now threatening American chestnut in the eastern United States and could impact restoration programs (Anagnostakis 2001, Schlarbaum et al. 2001). 1 Corresponding author, lrieske@uky.edu. D. kuriphilus causes signiþcant damage to chestnut by forming galls on actively growing shoots that reduce tree vigor, prevent normal shoot development and ßowering, and cause branch and tree mortality (Payne et al. 1975, Kato and Hijii 1997). The gall wasp is parthenogenetic and univoltine. Adult wasps oviposit inside vegetative and mixed buds of Castanea spp. during the summer. Eggs hatch soon after, and larvae remain in the neonate stage until the next growing season. Larval growth begins the next spring, concurrent with bud break, when larvae induce the rapid formation of conspicuous stem or leaf galls. These galls provide the wasp larvae protection from desiccation and predation throughout the remaining larval stages and through pupation. Adults are active for 1 mo after emergence from galls and move to newly formed buds for oviposition. After adult emergence, galls desiccate and become woody, and they can remain on the tree for several years. A native of China, D. kuriphilus was a major pest in Japan and Korea after accidental introductions (Yasumatsu 1951, Murakami et al. 1995). Asian chestnut varieties resistant to galling were initially used for management. However, virulent gall wasp isolates soon became dominant, eliminating the efþcacy of host plant resistance as a management tool (Shimura 1972). In the 1970s, the parasitic Torymus sinensis Kamijo (Hymenoptera: Torymidae) was collected /07/0236Ð0244$04.00/ Entomological Society of America

2 March 2007 COOPER AND RIESKE: CHESTNUT GALL WASP COMMUNITY ASSOCIATES 237 from China and released in Japanese orchards (Murakami et al. 1977, 1980). As a result of T. sinensis parasitism, D. kuriphilus populations rapidly declined below tolerable levels (Moriya et al. 1989, 2003). D. kuriphilus was Þrst observed in the United States infesting Chinese chestnut, C. mollissima Blume, near Byron, GA (Peach Co.), in It is thought to have been accidentally introduced on chestnut nursery stock from Asia (Payne et al. 1975). In the decade after its introduction, the gall wasp nearly eliminated the chestnut industry in Georgia (Anagnostakis and Payne 1993). In addition to commercially planted chestnuts, ornamental Chinese chestnuts also serve as suitable hosts and are planted throughout the United States. D. kuriphilus also threatens efforts to restore American chestnut as a viable component of the Appalachian forest (Anagnostakis 1999, Schlarbaum et al. 2001). In 1977, several parasitoids, including Torymus spp., were introduced to Georgia from Japan with hopes of providing biological control against D. kuriphilus (Payne 1978). However, the establishment and efþcacy of these releases and the dispersal of the parasitoids were not tracked. Consequently, we do not fully understand the ability of these parasitoids to establish themselves and disperse with expanding gall wasp populations, nor do we fully understand the role parasitoids may play in regulating chestnut gall wasp populations in the eastern United States. Many cynipid wasps, including other Dryocosmus species, form galls on oaks (Quercus spp.) in eastern North America, and many parasitoids associated with these gall makers could potentially use D. kuriphilus as a host (Payne 1978). Despite its 30 years in the United States, very little is known about the ecological relationships, dispersal, or population regulation of D. kuriphilus in North America, and much of the information known is anecdotal (Aebi et al. 2006). The goal of our work is to characterize the associates of D. kuriphilus in eastern North America, speciþcally focusing on introduced and native natural enemies, to determine what factors regulate gall wasp populations. Our ultimate goal is to gain a more complete understanding of the ecological interactions, dispersal patterns, and mechanisms regulating D. kuriphilus populations in eastern North America. Materials and Methods Collection Sites. Galled plant material was collected from three geographically distinct locations (Fig. 1). The Þrst, in Meadowview, VA (Washington Co.) (36 45 N, W), is located at the American Chestnut FoundationÕs breeding farm. Meadowview is located in the Ridge and Valley physiographic region and is within the original range of American chestnut (Braun 1950) (Fig. 1). Trees growing on the site are used for the FoundationÕs blight resistance breeding efforts, and they are arranged in evenly spaced and age-synchronized blocks of American, Chinese, or hybrid chestnuts. The area is surrounded by farms, pastureland, and hardwood woodlots. The initial gall wasp infestation was noticed in 2001 and rapidly spread Fig. 1. Initial introduction of D. kuriphilus near Bryon, GA, in Galls for current study were collected near Meadowview, VA, Bowling Green, KY, and Broadview Heights, OH. Historic range of American chestnut in eastern North America is in shaded gray (USGS 1999). throughout the farm (Cooper and Rieske-Kinney 2006). The Bowling Green, KY, collection site (Warren Co.) (36 57 N, W) is located on the boundary of the Pennyroyal and Shawnee Hills physiographic regions and is near the western edge of the original range of American chestnut (Braun 1950, McEwan et al. 2005) (Fig. 1). It is also the western-most recorded occurrence of D. kuriphilus. This site is located in a privately owned forest where several tall (5Ð15 m) naturally occurring American chestnut trees persist, despite infection with the blight fungus. The chestnut gall wasp occurs on a single tree at this site and was Þrst observed in winter 2003Ð2004 (Cooper and Rieske- Kinney 2006). The third collection site is in a suburban setting in Broadview Heights, OH (Cuyahoga Co.) (41 20 N, W) located in the Erie Lake Plain physiographic region near the northwest border of the original range of American chestnut (Braun 1950) (Fig. 1). The location is made up mostly of suburban housing, and chestnut in the immediate area is limited to three mature ornamental Chinese chestnuts located on two adjacent privately owned properties in Broadview Heights. The infestation was Þrst recorded in 2003 (Stehli 2003). In addition to the collection sites described above, on 30 April, 31 naturally occurring American chestnut saplings along ridge tops in the Red River Gorge Geological Area, in the Cliff Section of the Cumberland

3 238 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 100, no. 2 Fig. 2. sharp. D. kuriphilus mandibles (A) are large and ßat, whereas parasitoid mandibles (B) are comparatively small and Plateau in eastern Kentucky (Wolfe Co.) (37 47 N, W), were visually examined for galling caused by D. kuriphilus. Gall Collection. Chestnut shoots containing current and previous generation galls were collected from each site at 3-wk intervals from before bud break to after D. kuriphilus adult emergence. In Meadowview, VA, two shoots per tree (one each from the east and west aspect), each containing 20Ð30 galls per tree, were collected from Þve American and Þve Chinese chestnut trees from 30 March to 22 June. In Bowling Green, KY, three shoots, each containing 20Ð30 galls, were collected from American chestnut from 7 April to 2 July. Because the Bowling Green site was made up of a single tree, collections were replicated only by shoot. In Broadview Heights, three shoots per tree, each with 20Ð30 galls, were collected from Chinese chestnut 22 April to 7 July. Shoots were clipped at the second apical growth scar to ensure only 2 yr of growth. Clipped shoots were bagged and kept cool during transport to the laboratory. To separate the current generation from the previous generationõs galls, all shoots were cut at the previous yearõs apical growth scar in the laboratory. Current generation galls (n 10Ð15 per tree) are green, pliable, living galls that developed during the study in spring 2005 and contain within them D. kuriphilus, parasitoids of D. kuriphilus, or both. Previous generation galls (n 10Ð15 per tree) are dark brown, desiccated woody galls that developed in spring 2004 and do not contain D. kuriphilus larva, but they may contain parasitoids or associates of D. kuriphilus. Galled plant material (current and previous) was placed in 500-ml containers with moist sand and placed under separate cylindrical cages (30.5 by 61 cm) darkened with Þne mesh screen, with a transparent inverted cone Þtted with an 8-ml collection vial (Choate and Rieske 2005). Cages were monitored daily during each 3-wk interval for gall wasp or parasitoid emergence. Galled shoot material was exchanged with fresh collections from each site at the end of each 3-wk interval. Wasps and associates were stored in 70% ethanol and identiþed. Voucher specimens were deposited at the Insect Research Collection (Department of Entomology, University of Wisconsin, Madison, WI). The incidence of parasitoid emergence was recorded as the number of wasps per gall per tree; emergence values represent the rates of emerging parasitoid adults, and they may not represent the actual rates of parasitism. Gall Associates and Parasitism Rates. In addition to the sequential collections, 341 current generation galls also were collected from 20 American chestnut trees in Meadowview on 15 June, just before D. kuriphilus pupation. The exterior of each gall was carefully examined for signs of herbivory or pathogen infection. Each gall was then dissected under an 8.82 dissecting scope to determine the number of chambers per gall and the contents of each chamber. D. kuriphilus larvae and parasitoids were identiþed by examining the mandibles. D. kuriphilus larvae have large ßat mandibles specialized for grazing on the inner tissues of the galls (Fig. 2A). In contrast, parasitoids such as T. sinensis have comparatively small sharp mandibles (Fig. 2B) for feeding on the gall former, allowing the parasitoid to subsequently use the gall chamber for its own development (Njentes-Aldry et al. 2005). Statistical Analyses. To determine whether parasitoids show a preference between American and Chinese chestnuts, the incidence of emergence of the two most abundant parasitoids were compared between the two chestnut species in Meadowview, by using a standard t-test. Occurrences of the remaining parasitoids were not great enough to compare statistically. Parasitoid emergence between collection sites was compared using TukeyÕs honestly signiþcant difference (HSD) and paired t-tests derived from contrast statements. To investigate potential interactions between T. sinensis and pooled native parasitoids (n 5 species), we used a multivariate canonical correlation analysis (PROC CANCORR, SAS Institute 1999) and the standardized canonical coefþcients to assess relationships among variables. All data were normalized using a log transformation. Mean abundance, richness, Simpson diversity index, and Pielou evenness index were calculated (PC-ORD, McCune and Mefford 1999) and compared between collection sites using TukeyÕs HSD.

4 March 2007 COOPER AND RIESKE: CHESTNUT GALL WASP COMMUNITY ASSOCIATES 239 Table 1. D. kuriphilus emergence (mean SE) from galls collected from three sites in eastern North America Collection site D. kuriphilus adults/gall No. of D. kuriphilus emerging No. of galls examined Meadowview, VA b 4 63 Bowling Green, KY a Broadview Heights, OH b 5 55 F 42.16, df 2, P Results and Discussion Adult D. kuriphilus was collected at each site and emerged between 7 and 10 July in Meadowview, 21Ð27 June in Bowling Green, and 12Ð17 July in Broadview Heights, corresponding to 770Ð780 C degree-days (DD base 10 C, start date, 1 January) at each location (Johnson et al. 1998). Emergence was signiþcantly greater in Bowling Green and was not different between Meadowview and Broadview Heights (Table 1). Natural dispersal of D. kuriphilus by ßight in the eastern United States occurs at a rate of 24 km/yr (Payne 1981). Nearly 25 yr after its initial introduction in Georgia, D. kuriphilus was reported in chestnut orchards throughout Georgia, and into Alabama, North Carolina, and Tennessee (Anagnostakis et al. 1993; Anagnostakis 1999, 2001). The gall wasp infestation in Meadowview (2001) and Bowling Green (2003) most likely occurred through natural movement of dispersing wasps and is consistent with the projected rate of ßight dispersal. However, as summer 2006, D. kuriphilus has not been recorded from any other location in Kentucky, and naturally occurring chestnut saplings sampled in the Red River Gorge area of eastern Kentucky showed no signs of infestation in April and May In contrast, Broadview Heights is nearly twice the distance from the initial Byron, GA, introduction. This distance exceeds the projected rate of dispersal by ßight and most likely represents a satellite infestation. Before 2001, D. kuriphilus was reported in McMinnville, TN, an area with extensive nursery production and a hub for mail order sales of chestnut trees (Anagnostakis 1999). We suspect that the Broadview Heights gall wasp infestation resulted from movement of infested nursery stock or other infested material, although the possibility of a second U.S. introduction cannot be discounted. To determine the extent of parasitism of D. kuriphilus larvae, galls (n 341) were dissected before adult D. kuriphilus emergence. Chamber contents fell into three categories: living D. kuriphilus larvae (53.2%), living parasitoid larvae (32.6%), or empty chamber (14.3%). Because each gall chamber was formed by a D. kuriphilus larva, the rate of larval mortality in the galls is the number of chambers with living parasitoids plus the empty chambers and was 46.8%. Parasitism (chambers with living parasitoid larvae/total D. kuriphilus mortality) accounted for 69.5% of the mortality, although parasitoid larvae could not be identi- Þed to taxa. Six parasitoid species were collected as adults emerging from D. kuriphilus galls, including the introduced Torymus sinensis (Fig. 3A), Ormyrus labotus Walker (Hymenoptera: Ormyridae) (Fig. 3B), and four less abundant species of unknown origin. Our data suggests that T. sinensis and O. labotus may be abundant enough to affect gall wasp populations (Tables 2 and 3), but the remaining four genera were collected in very low frequencies (Table 3). Only T. sinensis was previously known to parasitize D. kuriphilus, although other species in the genera Ormyrus, Sycophila, Eupelmus and Pteromalus also parasitize D. kuriphilus. The most abundant parasitoid, T. sinensis, was absent from Bowling Green and was reared only from previous generation galls collected at Meadowview and Broadview Heights (Table 2). The incidence of T. sinensis (n 36) differed marginally among collection sites (t 3.43, df 1, P 0.06), with the greatest abundance in Broadview Heights (Table 2). T. sinensis (n 26) emerged from both American and Chinese chestnut galls collected from Meadowview between 5 April and 12 April, and it did not differ signiþcantly between American and Chinese chestnuts (t 0.22, Fig. 3. The two most abundant parasitoids, T. sinensis (A) and O. labotus (B) emerging from D. kuriphilus galls collected near Meadowview, VA, Bowling Green, KY, and Broadview Heights, OH.

5 240 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 100, no. 2 Table 2. The two most abundant parasitoid species emerging (mean SE parasitoids per gall per tree) from current and previous year s D. kuriphilus galls collected from American and Chinese chestnut at three sites Parasitoid adults emerged (no. of galls examined) Meadowview, VA Bowling Green, KY Broadview Heights, OH T. sinensis Previous a ab (68) 0b (14) a (58) O. labotus Current b b (58) a (13) b (58) Previous a a (68) 0a (13) a (55) Total a (126) a (26) a (113) Means within rows with different letters are signiþcantly different at 0.05 (TukeyÕs HSD). a Previous year D. kuriphilus galls. b Current year D. kuriphilus galls. df 1, P 0.65). T. sinensis emerged from Chinese chestnut galls collected from Broadview Heights on 21 April (Table 2). T. sinensis is a common parasitoid of D. kuriphilus in its native range of China, and it was purposefully introduced to Japan in 1980 to help control D. kuriphilus populations (Murakami et al. 1977, Otake et al. 1984). Before the introduction of T. sinensis, the gall wasp had been the most signiþcant pest of chestnut in Japan (Moriya et al. 1989). Within several years after its introduction, parasitism by T. sinensis signiþcantly reduced gall wasp outbreaks and provided sustainable control throughout Japan (Moriya et al. 1989, 2003). Like D. kuriphilus, T. sinensis is univoltine. However, in contrast to its host, T. sinensis adults emerge in the early spring and oviposit inside developing galls. Larvae consume the gall former and remain in the galls until the next spring. Several strains of T. sinensis have been identiþed based on differences in adult emergence. Strains native to Korea and Japan emerge too early to provide effective control of D. kuriphilus, and instead they exploit oak galling cynipids as hosts (Murakami et al. 1995). The T. sinensis strain native to China and released in Japan is phenologically synchronized with the life cycle of D. kuriphilus, enhancing its effectiveness as a population regulator (Moriya et al. 2003). Several Asian Torymus species were released in southeastern Georgia for control of D. kuriphilus (Payne 1978), but this is the Þrst published account of its successful establishment in the United States. Given the success of T. sinensis as a sustainable biological Table 3. Minor parasitoid emergence (mean no. per gall per tree) from D. kuriphilus galls from American and Chinese chestnut trees from three sites Parasitoid species Meadowview, VA Bowling Green, KY Broadview Heights, OH S. mellea 0.01 (1) 0 0 P. minio (1) E. sp (2) 0 Pteromalus sp (1) 0 0 Values in parentheses indicate the total number emerged. control agent in Japan, and the high emergence rate observed in Meadowview and Broadview Heights, T. sinensis seems to be playing a major role in population regulation of D. kuriphilus in the United States. The distribution of T. sinensis in our collections (present in Meadowview and Broadview Heights, absent in Bowling Green) (Table 2) provides insight into the dispersal patterns and ecological relationships between the gall wasp and its introduced parasitoid. The D. kuriphilus infestation in Bowling Green is relatively recent, and establishment of an introduced parasitoid by ßight dispersal would undoubtedly lag behind that of the host insect. Apparently the dispersal of the introduced parasitoid has not caught up with the natural dispersal of its gall wasp host in the Bowling Green area. In addition, the Bowling Green collection site is relatively inconspicuous and is limited to a single tree within a heterogeneous woodlot, which undoubtedly would affect the success of host Þnding by T. sinensis. An additional explanation for the absence of T. sinensis in Bowling Green could be that competition and/or hyperparasitism may negatively impact its establishment at this location (see below). Emergence of T. sinensis from galls collected from the apparent satellite gall wasp infestation in Broadview Heights suggests that the parasitoid was moved with D. kuriphilus on infested plant material. D. kuriphilus can be difþcult to detect throughout most of the year because larvae are contained within normallooking buds from midsummer until the next spring. T. sinensis, however, is only contained within visible current generation galls or withered overwintering galls. Although Þnding T. sinensis at the Broadview Heights site is encouraging from the perspective of a potential population regulator of D. kuriphilus, it strongly implies that plant material was transported with obvious galling present, and highlights the need for precautions in transporting invasive insects (Chornesky et al. 2005). The second most abundant parasitoid, O. labotus, was reared from all three sites, and it was present in both current and previous generation galls (Table 2). Emergence of O. labotus from current generation galls differed between collection sites (F 7.64, df 1, P 0.006) and was greatest in Bowling Green (Table 2). Total emergence, and emergence from previous generation galls, did not differ between collection sites (F 0.87, df 2, P 0.44 and F 0.51, df 2, P 0.61, respectively) (Table 2). O. labotus (n 7) emerged from previous generation chestnut galls collected in Meadowview from 4 to 8 April, but its occurrence did not differ between chestnut species (t 0.31, df 1, P 0.59). Parasitoid emergence (n 7) from current yearõs American and Chinese chestnut galls occurred on 16 May and on 15 July. O. labotus (n 5) only emerged from current generation galls in Bowling Green between 23 and 27 June. In Broadview Heights, emergence from previous seasonõs galls occurred on 23 April (n 1) and from current seasonõs galls on 13 June (n 1). The difference in O. labotus emergence among our sites may be due to habitat variability. O. labotus is a major parasitoid of oak-

6 March 2007 COOPER AND RIESKE: CHESTNUT GALL WASP COMMUNITY ASSOCIATES 241 galling insects, and Quercus spp. is a major component of woodlots surrounding Meadowview and Bowling Green. In contrast, Broadview Heights is a suburban area that may lack signiþcant oak dominated woodlots as a potential source of O. labotus. O. labotus is among the most abundant parasitoids of the horned oak gall, Callirhytis cornigera Osten Sacken (Hymenoptera: Cynipidae), in central Kentucky (Eliason and Potter 2000). It is a generalist parasitoid of gall formers and has been recorded emerging from galls in the cynipid genera Acraspis, Callirhytis, Ceroptres, Diastrophus, Disholcaspis, and Neuroterus (Burks 1979, Washburn and Cornell 1979, Eliason and Potter 2000, Noyes 2003). This is the Þrst report of O. labotus parasitizing Dryocosmus. However, congeneric species have been recorded parasitizing D. kuriphilus in other regions (Murakami et al. 1980, Otake et al. 1982, Kim 1998, Aebi et al. 2006). In addition to the two parasitoid species collected in abundance, we collected several other species in lower frequencies, including Sycophila mellea Walker (Hymenoptera: Eurytomidae), Pnigalio minio Walker (Hymenoptera: Eulophidae), Eupelmus sp. (Hymenoptera: Eupelmidae), and Pteromalus sp. (Hymenoptera: Pteromalidae). S. mellea is represented by two specimens collected in Meadowview on 25 and 27 June from current generation American chestnut galls (Table 3). It is a generalist parasitoid of cecidomyiid ßies (Diptera: Cecidomyiidae), certain aphids (Hemiptera: Aphididae), and eurytomid wasps (Hymenoptera: Eurytomidae) (Burks 1979, Dubbert et al. 1998, Noyes 2003). Several Sycophila spp. also are associated with gall-forming cynipids on oaks in North America (Eckberg and Cranshaw 1994, Zuparko 1996, Eliason and Potter 2001). Other species within this genus, including Sycophila variegata Curtis, Sycophila concinna Boheman, and Sycophila biguttata Swederus, which have been recorded emerging from D. kuriphilus galls in China, Korea, Japan, and Italy (Yasumatsu and Kamijo 1979; Murakami et al. 1980, 1994, 1995; Luo and Huang 1993; Aebi et al. 2006). Given the biology of this genus in other regions infested with D. kuriphilus, it is likely that S. mellea is associated with oakgalling wasps near the Meadowview collection site and is also capable of using D. kuriphilus as a host. P. minio was represented by a single male specimen collected from a current generation gall on 12 June in Broadview Heights (Table 3). It also is a generalist parasitoid that uses hosts from 16 insect families in four orders (Burks 1979, Heyerdahl and Dutcher 1985, Nystrom and Evans 1989, Duncan and Pena 2000, Petrice et al. 2000, Gates et al. 2002, Noyes 2003). P. minio has not been recorded parasitizing cynipid wasps, but it is most commonly associated with leafmining lepidopterans. However, P. nemati Westwood is a primary parasitoid of the gall-forming sawßy Pontania proxima Lepeletier (Hymenoptera: Tenthredinidae) on crack willow, Salix fragilis L. (Salicaceae) (Al-Saffar and Aldrich 1998). P. minio may be associated with D. kuriphilus galls in Bowling Green; however, we cannot fully discount the possibility that this specimen may have emerged from a host other than D. kuriphilus. Eupelmus sp. was represented by a single specimen that could not be identiþed to species. It emerged from a current generation gall collected in Bowling Green on 6 July (Table 3). The genus has a cosmopolitan distribution (Burks 1979, Noyes 2003) and several species parasitize D. kuriphilus, or hyperparasitize T. sinensis in other regions infested with D. kuriphilus (Otake et al. 1982, Murakami et al. 1994, 1995; Aebi et al. 2006). Eupelmus spp. commonly parasitize oak gall formers in North America (Askew 1961, Noyes 2003), and it is likely associated with oak-galling insects. Finally, Pteromalus sp. also was represented by a single specimen that could not be identiþed to species. It emerged from a current generation gall collected in Meadowview on 6 July (Table 3). Pteromalus spp. also has a cosmopolitan distribution, and members of this genus are parasitoids and hyperparasitoids of a large number of insect hosts, including Eupelmus spp. (Noyes 2003). Only one species in Japan, Pteromalus apantelophagus Crawford, is recorded parasitizing D. kuriphilus (Otake et al. 1982). It is not known whether this Pteromalus sp. is a parasitoid associated with D. kuriphilus or a hyperparasitoid in North America. The abundance of D. kuriphilus community associates was signiþcantly greater in Broadview Heights (F 4.97, df 2, P 0.03), most likely due to the relative abundance of T. sinensis. Rapid parasitoid recruitment by D. kuriphilus has been demonstrated in other regions of its range expansion (Aebi et al. 2006); however, we found no differences in diversity, richness, or evenness of D. kuriphilus associates, in spite of habitat differences and differences in D. kuriphilus residence time between our collection sites. The trends we observed in parasitoid emergence provide insight into potential interactions between the parasitoids and the gall wasp, and between parasitoid species. The highest incidence of gall wasp emergence occurred in Bowling Green, which also had the highest occurrence of the native O. labotus, and where the introduced T. sinensis did not occur. Gall wasp occurrence was lower in the satellite Broadview Heights infestation, where the emergence of T. sinensis was greatest, and the emergence of oak galling parasitoids was lowest. Our canonical correlation analysis shows a marginally signiþcant negative correlation between parasitoids of oak-galling insects (O. labotus, S. mellea, P. minio, Eupelmus spp., and Pteromalus spp.) and the introduced D. kuriphilus parasitoid, T. sinensis (F 2.26; df 4, 26; P 0.09). Although our sample sizes were small, these results support our observations and suggest a negative correlation between the introduced T. sinensis and native parasitoids associated with oak-galling insects. The habitat differences among our collection sites, parasitoid interactions caused by adult competition for oviposition sites, larval competition for suitable hosts, or hyperparasitism, may play a role in driving this trend. Whether this trend is widespread on naturally occurring American chestnut saplings in areas of the eastern U.S. where D.

7 242 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 100, no. 2 kuriphilus is established is unknown and warrants further investigation. Our results have implications for managing expanding D. kuriphilus populations. Currently, there is no effective chemical control for D. kuriphilus because it is protected within the gall throughout most of its life cycle. Cultural control recommendations involve pruning and burning visible galls (Payne et al. 1976, Ahn et al. 1984). D. kuriphilus is present in galls only during the spring in which they develop, after which the galls only house parasitoids that could help regulate the next D. kuriphilus generation. Our data demonstrate that the most numerous parasitoids emerged from previous generation galls. Consequently, pruning these galls could have a negative impact on natural enemies of the gall wasp, and a positive impact on D. kuriphilus. Leaving previous generation galls on the tree would allow parasitoids to complete development and promote gall wasp parasitism and could contribute to sustainable control of D. kuriphilus. In addition to the parasitoids collected, we observed gall herbivory by the small chestnut weevil, Curculio sayi (Gyllenhal) (Coleoptera: Curculionidae), which feeds on chestnut seed as larvae and adults (Drooz 1985). Females oviposit inside chestnuts, and larvae feed on the endoderm. We found signs of adult feeding consisting of small puncture wounds that extended deep into the ßeshy outer layers of the gall, and which were accompanied by a brown oxidation streak extending from the exterior wound site. Weevil feeding was evident on 16% of the collected galls. Cynipid galls are chemically similar to seeds (Schonrogge et al. 2000), and these similarities may allow adult weevils to use, or perhaps mistake, cynipid galls for a food and oviposition resource. Although we observed adult feeding on the galls, we did not observe egg deposition or signs of weevil larval development inside the galls. This the Þrst record of a native weevil using a resource produced by an exotic gallmaker as a food source. In addition to the parasitoid and weevil activity, an unidentiþed fungus associated with the gall exterior occurred on 3% of the collected galls. Infected galls had black lesions on the gall exterior and discoloration throughout the inner gall tissues. All gall wasp larvae within these galls were either dead or were yellowed and desiccating, strongly suggesting fungal pathogenicity. Foliar endophytes are known to produce pathogenic lesions on cynipid galls (Butin 1992, Wilson 1995). Endophytic fungi form inconspicuous infections within tissues of healthy plant foliage and are thought to interact with plants symbiotically (Siegal et al. 1987). Some fungal endophytes produce pathogenic fruiting bodies on insect galls that indirectly kill the larval gall former (Butin 1992). Gall-forming cynipid wasps on Oregon white oak, Quercus garryana Dougl (Fagaceae), leaves suffered nearly 100% mortality when galls were injected with an endophyte spore suspension (Wilson 1995), and gall midges, Contarinia spp. (Diptera: Cecidomyiidae), on Douglas-Þr, Pseudotsuga menziesii Mirbel (Pinaceae), needles suffer high mortality when sprayed with spore suspensions of needle endophytes (Carroll 1991). Endophytes also may have antixenotic effects on gall wasps; for example, gall-forming insects avoid endophyte infected tissue during oviposition (Wilson and Carroll 1997). The fungus observed on D. kuriphilus galls may be a naturally occurring foliar endophyte that warrants further investigation. Our data provide insight into the dispersal patterns of D. kuriphilus in North America, the natural enemies potentially regulating these D. kuriphilus populations, and potential interactions between parasitoids associated with chestnut galls. This is the Þrst report of an association between C. sayi and D. kuriphilus, and the Þrst report of a potentially pathogenic fungus that causes death of D. kuriphilus. Our work expands our knowledge base of D. kuriphilus and its associated community in eastern North America, and is essential for minimizing the negative effects of expanding gall wasp populations. Acknowledgments We thank Fred Hebert, Ed Stehli, and Steve Hartman for gall wasp collections. We also thank Steven Krauth (Insect Research Collection, University of Wisconsin) for identiþcation of collected wasps and weevils. Tom Coleman, Aerin Land, Preeyanhut Phumkhem, and Kevin Pitz provided technical assistance. 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