Hymenoptera: Cynipidae) in North America

11 April 2000 PROC.ENTOMOL.SOC.WASH. 102(2),2000, pp. 360-373

LIVE OAKS, NEW HOSTS FOR ODONTOCYNIPS NEBULOSA KIEFFER (HYMENOPTERA: CYNIPIDAE) IN NORTH AMERICA

A. D. WILSON, D. G. LESTER, AND R. E. EDMONSON

(ADW, DGL) Forest Insect and Disease Research, U.S. Department of Agriculture, Forest Service, Southern Research Station, Southern Hardwoods Laboratory, Stoneville, MS 38776-0227, U.S.A. (e-mail: dwilson/[email protected]); (REE) Texas Forest Service, Kerrville, TX 78029-3127, U.S.A. (e-mail: [email protected])

Abstract.-A study of root-feeding insects as potential vectors of the oak wilt fungus Cerutocystis figaceurum (T. W. Bretz) J. Hunt in live oaks, revealed root galls induced by the cynipid gall wasp Odontocynips nebulosu Kieffer. The incidence of the wasp on roots of four oak species and natural live oak hybrids at 14 root excavation sites in 7 counties throughout the Hill Country of central Texas was surveyed. The study was limited to sites within and adjacent to oak wilt infection centers of the live oak-Ashe juniper ecotype where oak wilt infected live oaks were being uprooted and rogued for disease suppression by the Texas Oak Wilt Suppression Project. This is the first report of this root-galling wasp on live oaks, Q. jiusiformis Small and Quercus virginiana Miller X Quercus fusiformis natural hybrids, in North America. The incidence of root-galling by the wasp occurred at relatively low levels among trees examined at excavation sites in each county, indicating a sporadic distribution throughout the region. However, examinations of root-colonization and gall induction by 0. nebulosu in uprooted live oaks showed relatively high levels of root infestations in some trees, including trees exhibiting symptoms of oak wilt disease. This pattern suggests high population densities in small localized areas. Examinations of individual gall clusters formed by the wasp on live oak roots revealed new details of gall morphology and developmental stages of the insect within galls. The significance of this wasp as a potential vector of the oak wilt fungus is discussed. Key Words: host-parasite relationships, Odontocynips nebulosu, Quercus fusiformis, Quercus virginiana, cynipid gall wasps, Cerutocystis fuguceurum, oak wilt fungus

The cynipid wasps comprise a large from 8 of 37 Nearctic genera listed in the group of phytophagous Hymenoptera (Fam- subfamily Cynipinae induce galls on plants ily Cynipidae) that inhabit angiospermous from at least 35 additional plant genera (see ? plants either as gall-makers (subfamily Cy- Burks 1979). Gall induction results from nipinae) or as inquinones (subfamily Sy- the reactions of host tissues to morphogens nerginae). Of the more than 800 Nearctic secreted by larvae during feeding (Roh- , species recognized in the Cynipidae, 78% fritsch 1992, Shorthouse and Rohfritsch induce galls on Quercus species (Burks 1992). The Synerginae, accounting for the 1979, Dreger-Jauffret and Shorthouse remaining 12% of recognized species, de- 1992). Approximately 10% of the species velop as inquinones within galls induced by .

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other cynipids, chalcidoids, or dipterous similar multilocular galls induced by this gall-makers in the family Cecidomyiidae species on post oak and overcup oak (Quer- (Burks 1979, Ronquist 1994). cus Zyrata Walt.). Very little information on Cynipids are not only highly host-specif- the life cycle, host range, host-parasite re- ic, but they are remarkably selective in the lationships, and geographical distribution of . types of host tissues that they will colonize. 0. nebulosa has been elucidated since the Most cynipids that induce galls on oak spe- species was described by Kieffer (1910). cies selectively infest above-ground tissues The genus Odontocynips currently is mono- of their hosts, including either main stems typic (Burks 1979). and branches, twigs, buds, petioles, leaves, The majority of cynipids are considered flowers, or fruits (e.g., acorns), but rarely of minor economic importance, although a several of these tissues on the same host. few species such as the gouty oak gall wasp The greater diversity of somatic, reproduc- Callirhytis quercuspunctata (Bassett), the tive, and meristematic host tissues serving homed oak gall wasp C. cornigera (Osten as niches in above-ground plant tissues rel- Sacken), and the oak rough bulletgall wasp ative to root tissues may account for greater Disholcaspis quercusmamma (Walsh) are numbers of gall-inducing species and more destructive pests that can cause significant numerous and varied gall morphology types injury and even mortality to landscape oaks found on above-ground tissues. Although (Johnson and Lyon 1988, Eckberg and many oak gall wasp species have sexual Cranshaw 1994). A field study was initiated and parthenogenic generations that develop in fall of 1993 to survey populations of different gall types on different oak species root-feeding insects of live oaks that might or plant organs (Askew 1984), this rarely serve as potential vectors of the oak wilt occurs in the same generation. fungus, Ceratocystis fagacearum, in the Relatively little is known about cynipids Texas Hill Country. This fungus is the most that cause root galls. Galls produced by serious pathogen causing oak mortality in most subterranean cynipids form on roots Texas. During initial stages of this study, that arise near the crown at or just below root systems of live oak trees that had been the soil surface. Felt (1965) listed only pushed over during oak wilt disease sup- about 40 cynipid species that form galls on pression activities showed heavy infesta- plant tissues below ground. More recently, tions of a root-galling insect. Subsequent Burks (1979) listed approximately 120 spe- investigations showed that the insect re- cies capable of inducing galls on roots. sponsible for these root galls was 0. ne- Fewer than a dozen species have been de- bulosa. The current research stemmed from scribed as capable of colonizing and form- the 1993 survey. The objectives were to de- ing galls deeper in the soil profile on small termine the incidence and severity of 0. ne- fibrous and larger true roots. This small bulosa infestations of Quercus species in group of subcoronal species includes Belen- and around the perimeter of oak wilt infec- ocnema treatae Mayr, Odontocynips nebu- tion centers, elucidate aspects of its biology loss, and Callirhytis species. with respect to host specificity, tissue pref- erences, and distribution on the host, ex- Odontocynips nebulosa is distinguished from other subterranean gall wasps by its amine gall morphology and insect development within root galls on Q. fusiformis ability to induce the formation of large ir- and Q. virginiana Q. fusifomis natural regular multilocular galls on the roots of its X hybrids, and document new hosts of the hosts. It induces single globose galls and wasp in North America. larger irregularly-shaped multilocular galls up to 10 cm in diameter on post oak (Quer- MATERIALS AND METHODS cus stellata Wangenh.) roots up to 1.3 cm Field survey and root excavations.-The in diameter (Felt 1965). Weld (1959) found root excavations and examinations required 362 PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON to conduct most this study were carried out The incidence (frequency of occurrence) from July through December 1995 in co- of root galling by the wasp was calculated operation with oak wilt suppressions activ- per tree for each oak species sampled with- ities of the Texas Oak Wilt Suppression in each county. The number of oak wilt in- Project, a disease suppression program ad- fection centers that were present within a ministered by the Texas Forest Service. The 500 m radial distance of root excavation oak wilt fungus commonly moves from tree sites also was recorded for each county. The to tree through root grafts and common root host parameters measured for individual systems formed between adjacent trees. trees included tree species, diameter at Oak trees around the perimeter of actively breast height (dbh), and distance from the expanding oak wilt infection centers are nearest infected tree within an oak wilt in- routinely extracted and removed (rogued) fection center. The intensity (severity) and using backhoes and bulldozers to create a spatial distribution of root colonization on distance barrier between the advancing trees with galls was recorded as percent front of the infection center and healthy root flare infestation (proportion of major or trees in an attempt to prevent root trans- primary roots arising from root flares that mission of the oak wilt fungus. Four species had root galls), total number of galls per of oaks and natural live oak hybrids, all primary root and per tree, depth of galled highly susceptible to oak wilt, were pushed roots, and distance of galls from the main over during this roguing process which pro- stem or bole. Root galls were collected vided the opportunity to examine and sam- from individual trees at each excavation site ple the root systems of oaks for root-feed- using lopping shears to cut root segments ing insects of potential importance as vec- 2-5 cm on each side of individual galls. tors of the oak wilt fungus. The oak species Measures of gall morphology and root char- surveyed for 0. nebulosa-infestations in- acteristics of galled roots were recorded as cluded plateau live oak, Q. fusiformis (for- unilocular (single-chambered) or multiloc- merly Q. virginiana var. fusiformis), Q. vir- ular (multichambered) galls with multiple giniana X Q. fusifornzis natural hybrids of locules, gall dimensions, and corresponding plateau live oak and coastal live oak, Q. root diameters associated with the galls virginiana, Lacey oak, Quercus gluucoides found on roots from root excavation sites Mart. & Gal. (= Q. Zuceyi Small), Spanish in each county. All data presented are oak or Texas red oak, Q. texunu Buckley (mean + 1 SE). (= Q. buckleyi Dorr & Nixon), and black- Gall morphology and insect develop- jack oak, Q. mariZundicu Mtinchh. merit.-Representative root galls in various The exposed root systems of 1,993 ex- stages of development were collected from cavated oak trees, uprooted along the pe- the root systems of live oaks near Kerrville, rimeter of fourteen oak wilt infection cen- TX during root excavations in the fall (18 ters, were examined for root galls of 0. ne- November) and early spring (11 February). bulosu. Some trees sampled along the pe- The developmental morphology of root rimeter and within oak wilt centers were galls (n = 44 for the fall collection, and n infected with the oak wilt fungus. Oak wilt = 25 for the spring collection) was exam- infection centers were selected from seven ined by exploratory dissections. The outer Texas counties throughout the Hill Country layers of host tissue forming the wall of the on the Edwards Plateau and Balcones fault galls were removed in sections to reveal hy- zone. In all cases, research trees were lo- perplastic and hypertrophic tissues forming cated in scattered stands of mixed hard- within galls during developmental stages of woods of the live oak-Ashe juniper ecotype the wasp from early larval stages until te- bounded on the east side by the central Tex- nerals (callow adults) within gall locules as post oak Savannah region. emerged from exit holes chewed through VOLUME 102, NUMBER 2 363

Table 1. Incidence (frequency of occurrence) of 0. nebulosa galls on root systems of oaks surveyed around the periphery of oak wilt infection centers from seven counties in central Texas.

. Q. Y. X Q. fusiformis’ Q. f: Hybrids’ Q. glaucoides Q. texana Q. marilandica Oak Wilt CSltet3 Examined Galled Examined Galled Examined Galled F&mined Galled Examined Galled

. Bandera 4 293 3 (1.0) 18 0 3.5 0 - - Bell 2 - 20 2(10.0) ------Gillespie 6 1,303 0 (0.0) - - - - 41 0 25 0 Kendall 2 23 2 (8.7) - - - - Kerr 1 37 33 (89.2) - - - - Mills 2 28 3 (10.7) - - Travis 1 - 170 2(1.2) ------Total 18 1,684 41 (2.4) 190 4(2.1) 18 0 76 0 25 0

I Numbers of oak wilt infection centers in the vicinity (500 m) of root excavation sites established in each county. * Total number of trees examined and number having root galls of 0. nebulosa. Values in parentheses indicate the percentage of trees with root galls. r Natural hybrids of coastal live oak and plateau live oak, Q. virginiana X Q. fusiformis.

the walls. Adults used for examination were pal, and teneral developmental stages with- reared from additional galls (n = 23) placed in galls, percent emergence from gall cham- in 7-10 X 6.5 cm plastic insect cages with bers, and percent insect viability and screened lids held at 24 C for 50 days fol- mortality within galls. Means of all mea- lowing the fall gall collection. In the last surements were expressed as (mean + 1 stages of pupation, tenerals were exposed SE). within gall locules by dissection. Represen- tative adult voucher specimens of 0. ne- RESULTS bulosa were deposited 17 February 1994 in Field survey and root excavations.- the National Museum of Natural History, Root galls induced by 0. nebulosa were Smithsonian Institution, Washington, DC found only on live oaks, Q. fusiformis and (Ref.: TSU Lot no. 94-1040). the natural hybrids Q. virginiana X Q. fit- Galls (n = 69) examined from the fall siformis, encountered at the periphery of and spring collections were placed into surveyed oak wilt infection centers (Table three developmental categories including 1). Some live oaks (<5%) within the infec- previous-year mature galls, current-year tion center that were infested with the wasp mature galls, and current-year immature exhibited diagnostic symptoms of oak wilt. galls. Previous-year mature galls were old Other oak wilt infection centers were found black weathered galls from which a prior in the vicinity of surveyed areas as well. generation had emerged the previous year. The infestation rates of individual root sys- Current-year mature galls were light brown tems among all live oak trees at the periph- and contained locules with larvae, pupae, or ery of oak wilt centers were less than 11% tenerals of the next generation to emerge. (range l.O-10.7%) at all but one survey Current-year immature galls were small, site. At the single site in Kerr County, 89% tan-colored galls containing white internal of surveyed trees exhibiting galled root sys- tissue bearing predominantly larval stages. tems. This high level of incidence was as- Galls in each developmental category were sociated with an oak wilt infection center at characterized by measurements of the gall a rural residence where all of the trees with- and insect including mean number of cham- in the center had to be rogued to contain bers per gall, gall chamber size (internal di- the spread of the disease. ameter), percentage of insects in larval, pu- The live oaks rogued and pushed into 364 PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON

Table 2. Host parameters, root infestations, and location of galls on roots of live oaks species in the Texas Hill Country.

Tree Distance Total No. Galls TeEiS Tree dbh (m) from Root Flare Depth (m) Gall Distance (m) County’ (cm) Infection Center 70 Infestation2 Per Root Per Tree of Galled Roots from Bole

Bandera 27.9 2 6.4 4.7 5 3.7 24.5 IT 2.5 1.3 I! 0.1 9.7 2 4.1 0.4 t 0.1 0.8 ? 0.2 Kendall 38.1 -c 2.5 3.6 -c 2.6 18.4 t 1.7 1.5 ” 0.5 4.5 ? 2.5 0.2 ? 0.1 1.1 rt 0.2 Kerr 33.7 t 8.9 7.1 2 3.8 72.3 2 3.6 1.7 + 0.3 12.9 t 5.3 0.3 t 0.1 1.4 ? 0.3 Travis 18.4 ? 2.7 26.6 2 5.5 20.9 t 4.2 1.2 + 0.1 3.7 k 1.8 0.4 ? 0.1 1.2 t 0.2

t Live oaks surveyed in Bandera, Kendall, and Kerr counties were Q. fusiformis, while those in Travis county were natural hybrids of coastal live oak and plateau live oak, Q. virginiana X Q. fusiformis. All data represent only trees with root galls of 0. nebulosa. * Percentage of all primary or major root branches arising from root flares with root galls of 0. nebulosa on their distal root branchlets (mean i 1 SE). piles during this study were small to me- vidually with a single chamber (unilocular) dium-sized trees up to 40 cm dbh. Most of on roots when a single viable larva began these trees occurred within a mean distance feeding following oviposition beneath the less than 10 m from an adjacent oak wilt root cambium, while multichambered (mul- infection center (Table 2). Root infestation tilocular) galls resulted when several viable rates of live oak primary roots arising from eggs were deposited at a single location in root flares of galled trees ranged from 18- the root. Multilocular galls appeared to 25% at surveyed sites adjacent to infection form by the growth and fusion of the outer centers in three counties, but primary roots galls (walls) surrounding individual larval of live oaks at the Kerr County site exhib- chambers. Multilocular galls formed much ited a higher infestation rate. The average more frequently than unilocular galls on number of galls on live oak roots were <2 live oak roots (Table 3). Unilocular galls per individual galled root, although the total tend to be globose and isodiametric, rang- number of galls per tree ranged from 4 to ing in size from 0.3-1.5 cm in diameter. 13. Galled roots predominantly were less Multilocular galls appeared as broadly- than 0.5 m from the soil surface. Galls on fused aggregates of individual globose galls roots deeper than 1 m were seldom ob- and at maturity were considerably larger served even though the bulk of the root and more irregularly-shaped than unilocular mass for most live oaks penetrated down to galls. These multilocular galls expanded 2 m below the surface. The root systems of through cellular proliferations from hyper- most trees were prevented from deeper soil trophy and hyperplasia to average sizes of penetrations due to shallow, rocky soils that 5.4 cm length and 4.0 cm in diameter (n = are prevalent in this region. Primary and 178) and contained an average of 25 cham- secondary feeder roots of medium-sized bers (cells). Exceptionally large multilocu- live oaks (20-40 cm dbh) typically extend- lar galls > 10 cm long and >8 cm wide ed out to 20 m or more from the bole or contained 70 or more chambers. Multiloc- main stem, however galls usually were lo- ular galls increased in size proportional to cated on live oak roots within 2 m of the the number of chambers fusing to form bole. them. However, the size (internal diameter) Root galls induced by 0. nebulosa gen- of individual chambers was not related to erally occurred on small feeder roots (usu- the number of chambers within each gall. ally < 1 cm diameter) that arose from pri- Gall chamber size (x) relative to number of mary roots below root flares near the bole. chambers (y) was sufficiently variable to Both unilocular and multilocular galls were prevent a statistically valid linear correla- observed. Galls presumably formed indi- tion (n = 442, r* = 0.006). Nevertheless, VOLUME 102, NUMDER 2 365

Table 3. Gall morphology and root diameters associated with 0. nebulosa galls collected from roots of Quercus fisiformis and Q. virginiana X Q. fusiformis hybrid trees.

Gall Morphology (%)I Gall Size (cm)’ No. Galls Root Diameter Examined Unilocular Multilcalar Length Width (cm)

Bandera 29 6.9 93.1 4.4 ? 3.1 3.0 2 2.2 0.5 2 0.2 Bell 37 8.1 91.9 4.1 IT 2.5 2.2 2 1.3 0.4 2 0.1 Kendall 9 22.2 77.8 2.3 2 1.0 2.1 2 1.0 0.6 2 0.1 Kerr 67 4.5 95.5 5.4 5 2.2 4.0 2 1.6 0.4 2 0.2 Mills 36 5.6 94.4 4.0 2 1.5 2.5 ? 0.9 0.5 2 0.2

I Percent of sampled galls that were unilocular (single chamber) or multilocular (multichambered). * Gall dimensions are for multilocular galls only (mean 2 1 SE). the linear model and equation (y = caused by gall formation often induced the 6.415+0.007x) describing the relationship production of small root sprouts from galls was highly significant (P < 0.001) and the close to the soil surface (Figs. lc, e). Root correlation was positive. Multilocular galls segments proximal to the galls toward the also were proportionally larger as root di- root apex were sometimes reduced in diameter increased. Galls that formed on ameter or killed by the gall. Mature current- roots in the l-2 cm-diameter range occa- year galls were typically tan to light brown sionally grew to sizes up to 11 cm long X like host roots, while older previous-years 10 cm in width. The rate of gall expansion galls were dark brown to black and weath- was not measured in this study, but some ered as the root tissue died and decayed galls may have attained full size in one (Fig. If). The majority of galls collected on growing season since multilocular galls 18 November were previous-year galls with were formed by the growth of individual exit holes. chambers, and larvae pupated within the All root galls examined in this study con- locules during the following late fall and tained outer galls that were hard, solid, and winter months. However, this does not pre- woody. Larval chambers were completely clude the possibility that the insect could surrounded by woody outer root tissue, but either have two generations per year or re- were not separated from the outer gall by quire l-2 years for gall development since an internal air space. The gall surfaces were immature and mature galls were observed generally smooth, lacking hairs and spines, in both fall and spring collections. and were not coated with a sticky resin. Gall morphology and insect develop- Unilocular single-chambered galls had out- merit.-Ninety-two galls were removed er walls that were morphologically identical from the root systems of excavated live to those of individual chambers within mul- oaks at the Kerrville survey site during the tilocular galls. fall and spring collections. Some of these The majority of galls collected from root galls were collected from trees exhibiting excavations in the fall and spring were dis- the diagnostic veinal necrosis leaf symptom sected immediately to examine concurrent of oak wilt (Fig. la). Root segments with stages of insect and gall development. Oth- galls were taken from feeder roots in the ers from the fall collection were held at main root ball near the bole (Fig. lb). Many 25°C for seven weeks during which galls of the galls close to the soil surface were were dissected at various stages of devel- collected using a hand spade and lopping opment until tenerals began emerging from sheers without extensive excavation (Figs. exit holes in the galls. Gall formation was lc-d). The apparent disruption of normal initiated by early stages of larval feeding plant hormone diffusion down feeder roots beneath root cambial tissue. Host root tissue 366 PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON

Fig. 1. Host-parasite relationships associated with 0. nebulosa-induced galls collected from roots of live oaks. a, Veinal necrosis leaf symptom of oak wilt-infected tree. b, Location of galled root segments collected on feeder roots in the main root ball near the bole. c, Shallow subterranean root galls with root sprouts, collected before emergence. d, Multilocular (multichambered) gall with exit holes collected after emergence. e, Closeup of unilocular (single-chambered) gall with dead (pruned) distal root section and root sprout. f, Multilocular mature galls on root segments comparing the light-colored, current-year living galls (top segment) with black, necrotic previous-year galls (bottom segment). surrounding the larva began aberrant cell nins and tannins) often formed within the division and expansion resulting in the for- swollen tissues immediately around the lar- mation of localized tissue swellings (Fig. va. The localized aberrant tissue was con- 2a). Dark brown necrotic tissues with oxi- sumed during feeding by the developing dized phenolic compounds (presumably lig- larva to form hollowed-out cells or locular VOLUME 102, NUMBER 2 367

Fig. 2. Developmental morphology of gall formation and insect development in 0. nebulosa-infested live oak roots. a, Initial stage of gall development with the formation of localized tissue swellings around larva (arrow) surrounded by brown necrotic areas in response to larval feeding. b, Development of locular initial (arrow) in gall tissue resulting from tissue consumption by larva. c, Pupae within two adjacent locules of a multilocular gall. d, Pupa with wing cases during late stages of pupation. e, Opaque pupa with separating exoskeleton, removed from gall chamber immediately prior to molt to teneral (callow adult) stage. f, Adult female with expanded wings ready for flight. Scale bars = 1.0 mm. initials (Fig. 2b). Larvae continued to feed nerals while still in the galls. Tenerals within developing galls throughout the emerge from the galls on warm days in late summer and early fall months, enlarging the winter to early spring (usually early Feb- locular cavities to form chambers. None of ruary in Texas) by chewing through the the chambers appeared to contain more than wall of their chamber and escaping through one larva. Larvae began pupating within the exit hole or chewing into the locule of chambers of the galls in the fall (Fig. 2~). an adjacent chamber with an exit hole to Pupae (Figs. 2d-e) began molting, breaking escape. Large numbers of adults were ob- free from their wing cases to become te- served emerging from the soil above sub- 368 PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON

Table 4. Gall and insect development, insect viability, and emergence associated with root galls of 0. nebulosa on infested Quercus fusifonnis and Q. virginiana X Q. fusiformis hybrid trees.

Fall Collection’ Spring Collection

Previous-year current-year Previous-year Current-year Mature Galls Mature Galls Immature Galls Mature Galls Mature Galls Immature Galls

Gall development2 Galls in category (%) 52.3 38.6 9.l4) 32.0 52.0 16.0 (n = 23) (n = 17) (n (n = 8) (n = 13) (n = 4) No. chambers (2) 10.3 +- 1.4 9.6 2 1.5 7.3 L 1.3 24.8 2 2.9 21.4 L 3.3 31.5 k 6.5 Chamber size (x) 6.5 t 0.1 6.7 + 0.1 3.5 r 0.1 6.5 + 0.1 6.5 k 0.1 4.0 + 0.1 (n = 236) (n = 164) (n = 29) (n = 198) (n = 278) (n = 126) Insect developme& Larvae (%) - 34.9 t 10.1 96.9 2 3.1 - 8.8 t 3.5 90.3 + 4.0 Pupae (%) - 27.5 + 8.4 3.1 + 3.1 - 0.0 2 0.0 1.7 2 1.7 Tenerals (%) - 0.0 ? 0.0 0.0 t 0.0 - 23.0 2 5.7 0.0 2 0.0 Insect emergence4 Same chamber (%) 57.0 2 7.6 20.8 + 9.4 0.0 t 0.0 53.0 k 7.4 45.3 ir 7.2 3.7 2 2.2 Adjacent chamber (%) 19.6 + 4.6 9.4 ? 5.4 0.0 2 0.0 31.5 t 4.3 15.5 L 3.9 0.0 c 0.0 Total emergence (%) 77.8 ? 6.7 30.2 ? 10.2 0.0 -t 0.0 79.7 _’ 11.3 60.8 _’ 8.9 3.7 2 2.2 Insect viability in galls 77.8 2 6.7 91.2 + 3.3 100.0 ? 0.0 85.0 2 7.1 92.6 L 3.4 95.7 2 2.5 Dead or aborted (%) 22.2 t 6.7 8.8 + 3.3 0.0 + 0.0 14.3 2 6.9 7.4 k 3.4 4.3 2 2.5

’ Root galls examined are representative of a fall (18 November) and early spring (11 February) collection. * Gall developmental categories for each collection include: previous-year mature, current-year mature, and current-year immature galls. Previous-year mature galls refer to those from which adults emerged the previous year. Current-year galls were new galls that formed since emergence of the previous-year generation. Mean values are expressed as (mean 2 1 SE). 3 The sum of percentages in each column for insect developmental stages, total emergence, and dead or aborted (larvae, pupae, and tenerals) is equal to 100%. Tenerals within sealed galls were distinguished from inactive pupae with wing cases by their activity and partially or fully expanded wings. 4 Indicates emergence that had occurred prior to gall collection (probably the previous spring for previous- year galls) as indicated by exit holes. Adults emerged through an exit hole either in the same chamber (within which they developed) or an adjacent chamber. merged galls at the base of live oaks during rearing the adults. All adults reared from the late morning of the spring collection. galls collected in the summer and fall Tenerals tended to emerge from chambers months were females indicating that root on the sides facing the soil surface. Indi- galls give rise to an asexual generation. viduals in chambers facing downward in A comparison of the developmental mor- the soil tended to chew their way through phology of galls and insects in the fall col- the gall to the top layer of chambers that lection with those in the spring collection already had exit holes. Tenerals burrow to yielded different results (Table 4). The the soil surface and emerge as an adult (Fig. spring collection had a higher percentage of 2f). In the laboratory, callow adult females galls in the current-year immature and ma- that were artificially freed from gall locules ture developmental categories than in the after emerging from pupal cases were ca- fall collection. Although gall chamber size pable of flight within 30 min. Tenerals (internal diameter) was comparable for reared from galls without assistance galls in all three developmental categories emerged from chambers with partially to in the fall and spring collections, immature fully expanded wings and could fly almost galls were smaller than previous-year and immediately. No parasites were recovered current-year mature galls. Current-year ma- or observed from galls (n = 23) used in ture galls from the fall collection contained VOLUME 102, NUMBER 2 369 a higher percentage of larvae and pupae, brids (Q. virginiana X Q. fusiformis) that but had a lower percentage of tenerals and form between coastal and plateau live oaks total emergence than current-year mature occurs abundantly in the region between galls from the spring collection. However, eastern parts of the Edwards Plateau and the immature galls contained a much higher Brazes River to the east (Nixon 1984). percentage of larvae than mature galls Consequently, the current study has dou- among current-year galls from both collec- bled the host range to include semiever- tions. Mature previous-year galls from both green oaks and expanded the known habitat collections lacked insect developmental of 0. nebulosu to xeric savannah-wood- stages since all living tenerals had already lands of the southwestern United States. emerged prior to collection. Insect emer- Lyon (1996) recently described seven new gence was highest in previous-year mature cynipid species on leaves and twigs of galls and lowest in current-year immature white oaks from this region. Weld (1960) galls for both collections. The majority of listed 130 species of phytophagous cynipids tenerals emerging from previous-year and from the southwestern United States with current-year mature galls in both collections an additional 117 gall types that were never came from the same chambers within which associated with a specific cynipid. The they developed while the remaining portion specificity with which 0. nebulosu coloniz- emerged from an adjacent or more distant es and forms galls on the roots of its hosts chamber from which they developed. This may be a survival advantage to the species latter type of emergence was achieve by te- in avoiding dessication in xeric habitats nerals chewing their way into an adjacent (see Femandes and Price 1992). chamber from which the occupant had al- This survey indicates that the incidence ready emerged and created an escape route of live oak root-galling by 0. nebulosa to the outside of the gall. Insect viability around oak wilt infection centers is rela- within galls was highest in immature galls tively low (l.O-10.7%) within most of the and higher in current-year mature galls than areas surveyed. However, the small per- in previous-year mature galls. centage of trees that were infested tended to have relatively high infestations of their DISCUSSION root systems based on the percentage of The limited occurrence of 0. nebulosa on major roots that were galled on individual two live oak species and its absence on the trees. These data suggest that the occurred oaks (subgenus Erythrobalanus), in- rence of this wasp is sporadic in the Ed- cluding blackjack oak, Lacey oak, and wards Plateau region, but that it tends to Spanish oak in this survey, suggests host occasionally form relatively high popula- specificity to certain oaks in the white oak tions in small localized areas or in individ- group (subgenus Leucobalunus). Hitherto, ual clumped stands (motts) of live oaks. 0. nebulosu has been reported from Geor- The occurrence and incidence of the wasp gia and Arkansas only on post oak (Q. stel- does not appear to be influenced by the in- Z&u) and overcup oak (Q. Zyrutu) by Weld fection-status of trees since root-galling did (1959), both white oak species. The live not occur at significantly higher frequency oaks are intermediate species having sap- in infected than in healthy live oaks. There- wood anatomical characteristics of both the fore, oak wilt infection of live oaks proba- white oak and red oak groups. However, bly does not predispose live oaks to 0. ne- live oaks are generally classified as white bulosa-infestation. Since the wasp occurs in oaks based on leaf and acorn characters. both oak wilt infected and uninfected live Quercus fusiformis is well established oaks, the presence of C. fuguceurum in the throughout the Edwards Plateau region of root system does not appear to be a nutri- central Texas. The natural continuum of hy- tional requirement for larval development. 370 PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON

Thus, any potential ability to vector the oak roots with their ovipositor during oviposi- wilt fungus would likely be passive and not tion. Although we did not examine the vec- out of necessity in order to complete its life tor potential of this wasp here, the oppor- cycle. However, the introduction by the tunities to acquire inoculum and its inter- wasp of a highly virulent pathogen such as action with live oak roots make it a suitable C. fagacearum that causes a fatal disease in candidate for further investigation. live oaks would not necessarily be disad- The effect of 0. nebulosa-infestations on vantageous to the wasp’s survival. The root development of live oaks may impact above-ground parts of most live oaks that host-pathogen interactions due to feeder- become infected with the oak wilt fungus root mortality. The death of feeder roots as- die within a few months after infection, yet sociated with gall tissue senescence follow- the root systems often survive and are sup- ing adult emergence could reduce the vigor ported by an abundance of root sprouts that of trees making them more susceptible to quickly develop after the top dies and apical oak wilt-infection. The majority of root seg- dominance is lost. These new shoots can ments collected with galls prior to emer- maintain the living root system indefinitely. gence were alive, suggesting that dead root Hence, the wasp would not sacrifice its segments may abscise from infested roots ability to continue colonization of the root after emergence and decay in the soil. In system if it were to introduce a lethal path- this way, infested roots may be effectively ogen. The abundance of new feeder roots pruned from the root system following resulting from the growth of many new root emergence. The death of root segments sprouts may actually increase the availabil- proximal to the galls toward the root apex ity of colonizable root mass. appeared to result from the disruption of The presence of root galls on oak wilt- phloem transport due to the crushing of root infected trees at the advancing front of oak phloem by proliferating gall tissue. wilt infection centers, the occasional very The observed emergence of unisexual- high root-infestation rates, and the occur- generation females of 0. nebulosa from rence of nearby oak wilt infection centers roots galls in this study raises the question indicates the potential opportunity for 0. of whether heterogony occurs in this spe- nebulosa to acquire inoculum of C. faga- cies. Many cynipid gall wasps on oaks cearum from oak wilt-infected live oak commonly have alternation of sexual and roots. Larvae feed directly on root tissue asexual generations with the parthenogenic (new sapwood) known to serve as a reser- all-female generation usually developing voir for C. fagacearum-inoculum in infect- and emerging in the summer and autumn ed trees. Root tissue tends to have the high- months (Lyon 1963, 1969, 1970; Askew est levels of inoculum because most trees 1984; Rey 1992). Lund et al. (1998) dem- within infection centers become infected onstrated heterogony in B. treatae, another through root transmission as a result of root root-galling cynipid of live oak. In this spe- grafting and common root systems often cies, the bisexual generation emerges in the shared by trees within motts (Appel et al. spring from root galls on Q. fusiformis and 1995). Root inoculum is particularly im- females oviposit into the undersides of portant following root transmission since leaves, inducing unilocular foliar galls on the fungus first enters and accumulates the same host. Morphologically distinct most of its inoculum potential within roots, unisexual-generation females, previously which is used for subsequent colonization described as B. kinseyi Weld (1921), of aerial portions of the tree (Wilson 1995). emerge from the leaf galls in the fall and Furthermore, adult 0. nebulosa females induce multilocular galls on the roots. The have the ability to burrow down into the cycle of 0. nebulosa appears to differ in soil and directly penetrate live oak feeder that unisexual-generation females emerge VOLUME 102, NUMBER 2 371 from root galls in the spring instead of from structural types. The larval chambers are leaf galls in the fall. It is important in as- completely surrounded by, and in direct sessing its vector potential to determine contact with, woody outer gall tissue. The whether unisexual-generation females of 0. galls are multichambered, but lack spines nebulosa oviposit into roots, leaves, or on the outer surface. The Stone and Cook some other parts. The ability of this species system was developed to include the com- to vector C. fagacearum would be less like- plex and diverse gall types represented in ly if heterogony occurs with the alternate the genus Andricus and related oak gallers sexual generation arising from galls on on above-ground parts of oak species. Per- leaves or twigs because these are poor in- haps a different or amended system should fection courts and poorer sources of inoc- be devised for oak root gallwasps to ac- ulum for subsequent infections. count for gall morphological characters re- Several important inferences are suggest- sulting from adaptations to roots coloniza- ed by the gall morphology and insect de- tion in soil environments. The morphology velopment results. The positive correlation of root galls described here may be quite of gall chamber size and number of cham- different from asexual galls arising from bers per gall and the corollary increase in unisexual-generation females presumably in overall gall dimensions with increasing the summer or fall. Asexual galls could chamber number indicate that there is no possibly occur on different live oak tissue, evidence for chamber dwarfing due to in- on a different oak species, or on roots. We traspecific competition as galls increase in do not currently know whether galls form- size. The low mortality of the wasps during ing on roots can be sexual galls, asexual development within galls suggests that gall galls, or both. However, a sexual generation numbers on roots may be used to accurately has not yet been confirmed with this species estimate population density. The occurrence since males have not been observed or de- of immature galls in the spring implies that scribed. there are either two generations per year or Root gall wasps generally are considered that some galls may take two years to de- to be less common than above-ground gall- velop. The latter conclusion is more likely makers perhaps because they are rarely ob- since there was no evidence of a fall emer- served and their impact on host biology is gence in galls from the fall collection (no poorly understood. Root galls no doubt es- fresh exit holes), the percentage of imma- cape observation in most surveys for gall ture galls was small, prior emergence from immature galls was low, and there was an insects. Systematic surveys for root gall absence of tenerals within immature galls wasps of oaks in different habitats should from the fall and spring collection. The lead to significantly more information on higher percentage of later insect develop- the biology of previously undescribed and mental stages within current-year mature unidentified root-galling cynipids. Although galls in the spring than in the fall provides recent studies by Shorthouse and Rohfritsch additional support for a single late winter (1992), Askew (1984), Lund et al. (1998), or early spring emergence. Csoka (1997), and Csoka et al. (1998) have The morphology of the woody multiloc- provided new information on the biology of ular root galls observed in 0. nebulosa do some Nearctic root-galling species, addi- not fit cleanly into a single structural type tional work is needed to further elucidate as defined by Stone and Cook (1998). The the biology of root gall-makers. Such work structure of these asexual galls would best may reveal that some root-galling cynipids be described as a cross (combination) be- may have greater significance than is cur- tween the S3 and S5 structural stages ac- rently attributed to members of this obscure cording to their system for classifying gall insect group. PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON

ACKNOWLEDGMENTS dae), as a street tree pest in Colorado. Journal of the Kansas Entomological Society 67: 290-293. We thank Texas Forest Service personnel Felt, E. P. 1965. Plant gall and gall makers. Hafner including entomologists Drs. Ron E Bill- Publishing Company, New York, 364 p. ings and R. Scott Cameron, and Mark Duff Femandes, G. W. and P W. Price. 1992. The adaptive (field forester) for their assistance in orga- significance of insect gall distribution: Survivor- ship of species in xeric and mesic habitats. Oeco- nizing the field collections, and Lisa B. logia 90: 14-20. Forse for skillful dissection of the root Johnson, W. T. and H. H. Lyon. 1988. Insects That galls. We acknowledge Mr. James B. Briggs Feed on Trees and Shrubs, 2nd ed. Comstock Pub- for assistance in collecting specimens and lishing Associates, Cornell University Press, Ith- data in several central Texas counties. The aca, New York. Kieffer, J. J. 1910. Description de nouveaux Hymen- cooperation of Edward H. Barron and opt&es. Bollettino de1 Laboratorio di Zoologia Bruce R. Miles (Texas state forester) in fa- Generale e Agraria della Portici 4: 112-l 13. cilitating formal cooperative agreement no. Lyon, R. J. 1963. The alternate generation of Heter- 19-93-081 between the USDA Forest Ser- oecus pacijcus (Ashmead) (Hymenoptera: Cyni- vice and the Texas Forest Service also is poidea). Proceedings of the Entomological Soci- gratefully acknowledged. We appreciate the ety of Washington 65: 250-254. -. 1969. The alternate generation of Callirhytis help of Arnold S. Menke (Systematic En- quercussuttonii (Bassett) (Hymenoptera: Cynipi- tomology Laboratory, USDA, Washington, dae). Proceedings of the Entomological Society of DC) who confirmed the identity of 0. ne- Washington 7 1: 6 l-65. bulosa specimens. We also appreciate Drs. -. 1970. Heterogony in Callirhytis serricornis Nathan Schiff, Jim Solomon, and Kathy (Kinsey) (Hymenoptera: Cynipidae). Proceedings of the Entomological Society of Washington 72: Schick for their comments in reviewing 176-178. drafts of the manuscript. -. 1996. New cynipid wasps from the southwestern United States (Hymenoptera: Cynipidae). LITERATURE CITED Pan-Pacific Entomologist 72: 181-192. Lund, J. N., J. R. Ott, and R. J. Lyon. 1998. Heter- Appel, D. N., R. S. Cameron, A. D. Wilson, and J. D. ogony in Belonocnema treatae Mayr (Hymenop- Johnson. 1995. How to identify and manage oak wilt in Texas. USDA Forest Service, Southern Re- tera: Cynipidae). Proceedings of the Entomologi- cal Society of Washington. 100: 755-763. search Station, New Orleans, LA. How-To Special Nixon, K. C. 1984. A biosystematic study of Quercus Report SR-1. Askew, R. R. 1984. The biology of gallwasps, pp. series Virentes (the Live Oaks) with phylogenetic 223-271. In Ananthakrishpan, T. N., ed., The Bi- analysis of Fagales, Fagaceae, and Quercus, Ph.D. ology of Galling Insects. Oxford and IBH Pub- Dissertation, University of Texas, Austin. lishing Co., New Delhi. Rey, L. A. 1992. Developmental morphology of two Burks, B. D. 1979. Superfamily Cynipoidea, pp. 1045- types of hymenopterous galls, pp. 87-101. In 1107. In Krombein, K. V., F! D. Hurd, D. R. Shorthouse, J D. and 0. Rohfritsch, eds., Biology Smith, and B. D. Burks, eds., Catalog of Hyme- of Insect-induced Galls. Oxford University Press, noptera in America North of Mexico, Vol. 1. New York. Smithsonian Institution Press, Washington, D. C. Rohfritsch, 0. 1992. Patterns in gall development, pp. Cs6ka, G. 1997. Plant galls. Forest Research Institute 60-87. In Shorthouse, J D. and 0. Rohfritsch, and Agroinform, Budapest. eds., Biology of Insect-induced Galls. Oxford Cs6ka, G., W. Mattson, G. N. Stone, and P. W. Price, University Press, New York. eds. 1998. Biology of gall-inducing arthropods. Ronquist, E 1994. Evolution of parasitism among USDA Forest Service, North Central Research closely related species: phylogenetic relationships Station, St. Paul, MN. General Technical Report. and the origin of inquilinism in gall wasps (Hy- Dreger-Jauffret, E and D. J. Shorthouse. 1992. Diver- menoptera: Cynipidae). Evolution 48: 241-266. sity of gall-inducing insects and their galls, pp. 8- Shorthouse, J. D. and 0. Rohfritsch. 1992. Biology of 33. In Shorthouse, J. D. and 0. Rohfritsch, eds., Insect-induced Galls. Oxford University Press, Biology of Insect-induced Galls. Oxford Univer- New York. sity Press, New York. Stone, G. N. and J. M. Cook. 1998. The structure of Eckberg, T B. and W. S. Cranshaw. 1994. Occurrence cynipid oak galls: Patterns in the evolution of an of the oak rough bulletgall wasp, Disholcaspis extended phenotype. Proceedings of the Royal So- quercusmamma (Walsh) (Hymenoptera: Cynipi- ciety of London 265: 979-988. YOUJME 102,NUMBERZ 373

Weld, L. H. 1921. American gallflies of the family Wilson, A. D. 1995. Future direction of USDA Forest Cynipidae producing subterranean galls on oak. Service oak wilt research, pp. 181-186. In Appel, Proceedings of the United States National Muse- D. N. and R. E Billings, eds., Oak Wilt Perspec- um 59: 238-239, 241-242. tives: The Proceedings of the National Oak Wilt -. 1959. Cynipid Galls of the Eastern United Symposium. June 22-25, 1992, Austin, Texas. In- States. Privately published. Ann Arbor. formation Development, Inc., Houston. -. 1960. Cynipid Galls of the Southwest. Ed- wards Bros., Ann Arbor, Michigan.