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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/srs_stoneville @fs.fed.us); (REE) Texas Forest 
Service, Kerrville, TX 78029-3127, U.S.A. (e-mail: tfskerr@ktc.com) 


Abstract.—A study of root-feeding insects as potential vectors of the oak wilt fungus 
Ceratocystis fagacearum (T. W. Bretz) J. Hunt in live oaks, revealed root galls induced 
by the cynipid gall wasp Odontocynips nebulosa 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. fusiformis 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, exami- 
nations of root-colonization and gall induction by O. nebulosa 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 nebulosa, Quercus fusiformis, 
Quercus virginiana, cynipid gall wasps, Ceratocystis fagacearum, oak wilt 


fungus 


The cynipid wasps comprise a large 
group of phytophagous Hymenoptera (Fam- 
ily Cynipidae) that inhabit angiospermous 
plants either as gall-makers (subfamily Cy- 
nipinae) or as inquinones (subfamily Sy- 
nerginae). Of the more than 800 Nearctic 
species recognized in the Cynipidae, 78% 
induce galls on Quercus species (Burks 
1979, Dreger-Jauffret and Shorthouse 
1992). Approximately 10% of the species 


from 8 of 37 Nearctic genera listed in the 
subfamily Cynipinae induce galls on plants 
from at least 35 additional plant genera (see 
Burks 1979). Gall induction results from 
the reactions of host tissues to morphogens 
secreted by larvae during feeding (Roh- 
fritsch 1992, Shorthouse and Rohfritsch 
1992). The Synerginae, accounting for the 
remaining 12% of recognized species, de- 
velop as inquinones within galls induced by 


VOLUME 102, NUMBER 2 


other cynipids, chalcidoids, or dipterous 
gall-makers in the family Cecidomyiidae 
(Burks 1979, Ronquist 1994). 

Cynipids are not only highly host-specif- 
ic, but they are remarkably selective in the 
types of host tissues that they will colonize. 
Most cynipids that induce galls on oak spe- 
cies selectively infest above-ground tissues 
of their hosts, including either main stems 
and branches, twigs, buds, petioles, leaves, 
flowers, or fruits (e.g., acorns), but rarely 
several of these tissues on the same host. 
The greater diversity of somatic, reproduc- 
tive, and meristematic host tissues serving 
as niches in above-ground plant tissues rel- 
ative to root tissues may account for greater 
numbers of gall-inducing species and more 
numerous and varied gall morphology types 
found on above-ground tissues. Although 
many oak gall wasp species have sexual 
and parthenogenic generations that develop 
different gall types on different oak species 
or plant organs (Askew 1984), this rarely 
occurs in the same generation. 

Relatively little is known about cynipids 
that cause root galls. Galls produced by 
most subterranean cynipids form on roots 
that arise near the crown at or just below 
the soil surface. Felt (1965) listed only 
about 40 cynipid species that form galls on 
plant tissues below ground. More recently, 
Burks (1979) listed approximately 120 spe- 
cies capable of inducing galls on roots. 
Fewer than a dozen species have been de- 
scribed as capable of colonizing and form- 
ing galls deeper in the soil profile on small 
fibrous and larger true roots. This small 
group of subcoronal species includes Belen- 
ocnema treatae Mayr, Odontocynips nebu- 
losa, and Callirhytis species. 

Odontocynips nebulosa is distinguished 
from other subterranean gall wasps by its 
ability to induce the formation of large ir- 
regular multilocular galls on the roots of its 
hosts. It induces single globose galls and 
larger irregularly-shaped multilocular galls 
up to 10 cm in diameter on post oak (Quer- 
cus stellata Wangenh.) roots up to 1.3 cm 
in diameter (Felt 1965). Weld (1959) found 


361 


similar multilocular galls induced by this 
species on post oak and overcup oak (Quer- 
cus lyrata Walt.). Very little information on 
the life cycle, host range, host-parasite re- 
lationships, and geographical distribution of 
O. nebulosa has been elucidated since the 
species was described by Kieffer (1910). 
The genus Odontocynips currently is mono- 
typic (Burks 1979). 

The majority of cynipids are considered 
of minor economic importance, although a 
few species such as the gouty oak gall wasp 
Callirhytis quercuspunctata (Bassett), the 
horned oak gall wasp C. cornigera (Osten 
Sacken), and the oak rough bulletgall wasp 
Disholcaspis quercusmamma (Walsh) are 
destructive pests that can cause significant 
injury and even mortality to landscape oaks 
(Johnson and Lyon 1988, Eckberg and 
Cranshaw 1994). A field study was initiated 
in fall of 1993 to survey populations of 
root-feeding insects of live oaks that might 
serve as potential vectors of the oak wilt 
fungus, Ceratocystis fagacearum, in the 
Texas Hill Country. This fungus is the most 
serious pathogen causing oak mortality in 
Texas. During initial stages of this study, 
root systems of live oak trees that had been 
pushed over during oak wilt disease sup- 
pression activities showed heavy infesta- 
tions of a root-galling insect. Subsequent 
investigations showed that the insect re- 
sponsible for these root galls was O. ne- 
bulosa. The current research stemmed from 
the 1993 survey. The objectives were to de- 
termine the incidence and severity of O. ne- 
bulosa infestations of Quercus species in 
and around the perimeter of oak wilt infec- 
tion centers, elucidate aspects of its biology 
with respect to host specificity, tissue pref- 
erences, and distribution on the host, ex- 
amine gall morphology and insect devel- 
opment within root galls on Q. fusiformis 
and Q. virginiana X Q. fusiformis natural 
hybrids, and document new hosts of the 
wasp in North America. 


MATERIALS AND METHODS 


Field survey and root excavations.—The 
root excavations and examinations required 


362 


to conduct most this study were carried out 
from July through December 1995 in co- 
operation with oak wilt suppressions activ- 
ities of the Texas Oak Wilt Suppression 
Project, a disease suppression program ad- 
ministered by the Texas Forest Service. The 
oak wilt fungus commonly moves from tree 
to tree through root grafts and common root 
systems formed between adjacent trees. 
Oak trees around the perimeter of actively 
expanding oak wilt infection centers are 
routinely extracted and removed (rogued) 
using backhoes and bulldozers to create a 
distance barrier between the advancing 
front of the infection center and healthy 
trees in an attempt to prevent root trans- 
mission of the oak wilt fungus. Four species 
of oaks and natural live oak hybrids, all 
highly susceptible to oak wilt, were pushed 
over during this roguing process which pro- 
vided the opportunity to examine and sam- 
ple the root systems of oaks for root-feed- 
ing insects of potential importance as vec- 
tors of the oak wilt fungus. The oak species 
surveyed for O. nebulosa-infestations in- 
cluded plateau live oak, Q. fusiformis (for- 
merly Q. virginiana var. fusiformis), Q. vir- 
giniana X Q. fusiformis natural hybrids of 
plateau live oak and coastal live oak, Q. 
virginiana, Lacey oak, Quercus glaucoides 
Mart. & Gal. (= Q. laceyi Small), Spanish 
oak or Texas red oak, Q. texana Buckley 
(= Q. buckleyi Dorr & Nixon), and black- 
jack oak, Q. marilandica Münchh. 

The exposed root systems of 1,993 ex- 
cavated oak trees, uprooted along the pe- 
rimeter of fourteen oak wilt infection cen- 
ters, were examined for root galls of O. ne- 
bulosa. Some trees sampled along the pe- 
rimeter and within oak wilt centers were 
infected with the oak wilt fungus. Oak wilt 
infection centers were selected from seven 
Texas counties throughout the Hill Country 
on the Edwards Plateau and Balcones fault 
zone. In all cases, research trees were lo- 
cated in scattered stands of mixed hard- 
woods of the live oak-Ashe juniper ecotype 
bounded on the east side by the central Tex- 
as post oak savannah region. 


PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON 


The incidence (frequency of occurrence) 
of root galling by the wasp was calculated 
per tree for each oak species sampled with- 
in each county. The number of oak wilt in- 
fection centers that were present within a 
500 m radial distance of root excavation 
sites also was recorded for each county. The 
host parameters measured for individual 
trees included tree species, diameter at 
breast height (dbh), and distance from the 
nearest infected tree within an oak wilt in- 
fection center. The intensity (severity) and 
spatial distribution of root colonization on 
trees with galls was recorded as percent 
root flare infestation (proportion of major or 
primary roots arising from root flares that 
had root galls), total number of galls per 
primary root and per tree, depth of galled 
roots, and distance of galls from the main 
stem or bole. Root galls were collected 
from individual trees at each excavation site 
using lopping shears to cut root segments 
2-5 cm on each side of individual galls. 
Measures of gall morphology and root char- 
acteristics of galled roots were recorded as 
unilocular (single-chambered) or multiloc- 
ular (multichambered) galls with multiple 
locules, gall dimensions, and corresponding 
root diameters associated with the galls 
found on roots from root excavation sites 
in each county. All data presented are 
(mean + 1 SE). 

Gall morphology and insect develop- 
ment.—Representative root galls in various 
stages of development were collected from 
the root systems of live oaks near Kerrville, 
TX during root excavations in the fall (18 
November) and early spring (11 February). 
The developmental morphology of root 
galls (n = 44 for the fall collection, and n 
= 25 for the spring collection) was exam- 
ined by exploratory dissections. The outer 
layers of host tissue forming the wall of the 
galls were removed in sections to reveal hy- 
perplastic and hypertrophic tissues forming 
within galls during developmental stages of 
the wasp from early larval stages until te- 
nerals (callow adults) within gall locules 
emerged from exit holes chewed through 


VOLUME 102, NUMBER 2 363 


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


County Centers! Examined Galled Examined Galled Examined Galled Examined Galled Examined Galled 
Bandera 4 293 3 (1:0) = = 18 0 35 0 — -— 
Bell 2 — 20 21.0.0) 

Gillespie 6 1,303 0 (0.0) =a = = = 41 0 25 0 
Kendall 2 23 2 (8.7) = = 
Kerr l 37 33109972) = — 
Mills 2 28 3 (10.7) — — 
Travis l — — 170 21.2) 
Total 18 1,684 4l (2.4) 190 4(2.1) 18 0 76 0 25 0 


1 Numbers of oak wilt infection centers in the vicinity (500 m) of root excavation sites established in each 
county. 
2 Total number of trees examined and number having root galls of O. nebulosa. Values in parentheses indicate 


the percentage of trees with root galls. 


* Natural hybrids of coastal live oak and plateau live oak, Q. virginiana X Q. fusiformis. 


the walls. Adults used for examination were 
reared from additional galls (n = 23) placed 
in 7-10 X 6.5 cm plastic insect cages with 
screened lids held at 24 C for 50 days fol- 
lowing the fall gall collection. In the last 
stages of pupation, tenerals were exposed 
within gall locules by dissection. Represen- 
tative adult voucher specimens of O. ne- 
bulosa were deposited 17 February 1994 in 
the National Museum of Natural History, 
Smithsonian Institution, Washington, DC 
(Ref.: TSU Lot no. 94-1040). 

Galls (n = 69) examined from the fall 
and spring collections were placed into 
three developmental categories including 
previous-year mature galls, current-year 
mature galls, and current-year immature 
galls. Previous-year mature galls were old 
black weathered galls from which a prior 
generation had emerged the previous year. 
Current-year mature galls were light brown 
and contained locules with larvae, pupae, or 
tenerals of the next generation to emerge. 
Current-year immature galls were small, 
tan-colored galls containing white internal 
tissue bearing predominantly larval stages. 
Galls in each developmental category were 
characterized by measurements of the gall 
and insect including mean number of cham- 
bers per gall, gall chamber size (internal di- 
ameter), percentage of insects in larval, pu- 


pal, and teneral developmental stages with- 
in galls, percent emergence from gall cham- 
bers, and percent insect viability and 
mortality within galls. Means of all mea- 
surements were expressed as (mean + | 
SE). 


RESULTS 


Field survey and root excavations.— 
Root galls induced by O. nebulosa were 
found only on live oaks, Q. fusiformis and 
the natural hybrids Q. virginiana X Q. fu- 
siformis, encountered at the periphery of 
surveyed oak wilt infection centers (Table 
1). Some live oaks (<5%) within the infec- 
tion center that were infested with the wasp 
exhibited diagnostic symptoms of oak wilt. 
Other oak wilt infection centers were found 
in the vicinity of surveyed areas as well. 
The infestation rates of individual root sys- 
tems among all live oak trees at the periph- 
ery of oak wilt centers were less than 11% 
(range 1.0-10.7%) at all but one survey 
site. At the single site in Kerr County, 89% 
of surveyed trees exhibiting galled root sys- 
tems. This high level of incidence was as- 
sociated with an oak wilt infection center at 
a rural residence where all of the trees with- 
in the center had to be rogued to contain 
the spread of the disease. 

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 
Texas Tree dbh (m) from Root Flare Depth (m) Gall Distance (m) 
County’ (cm) Infection Center % Infestation? Per Root Per Tree of Galled Roots from Bole 
Bandera 2T OIGA AEB BL SEIS) leg) Be (05 97+ 4.1 0.4 + 0.1 Oy se (OLY 
Kendall Shell Be 25) B.0)== 216 18.4 + 1.7 ear a (Obs) 4.5 = 2.5 OA s Ol ei EE OL 
Kerr EPT Be ei) Wall == Ses) IN == SE Eg e5 US PAS) 22 3.3} 0.3 + 0.1 1.4 + 0.3 
Travis S4 Ea 20 05S 2010 2 42 12500 Soi a= tee} 0.4 + O.1 12 $02 


' 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 O. nebulosa. 


* Percentage of all primary or major root branches arising from root flares with root galls of O. nebulosa on 


their distal root branchlets (mean + ] SE). 


piles during this study were small to me- 
dium-sized trees up to 40 cm dbh. Most of 
these trees occurred within a mean distance 
less than 10 m from an adjacent oak wilt 
infection center (Table 2). Root infestation 
rates of live oak primary roots arising from 
root flares of galled trees ranged from 18- 
25% at surveyed sites adjacent to infection 
centers in three counties, but primary roots 
of live oaks at the Kerr County site exhib- 
ited a higher infestation rate. The average 
number of galls on live oak roots were <2 
per individual galled root, although the total 
number of galls per tree ranged from 4 to 
13. Galled roots predominantly were less 
than 0.5 m from the soil surface. Galls on 
roots deeper than 1 m were seldom ob- 
served even though the bulk of the root 
mass for most live oaks penetrated down to 
2 m below the surface. The root systems of 
most trees were prevented from deeper soil 
penetrations due to shallow, rocky soils that 
are prevalent in this region. Primary and 
secondary feeder roots of medium-sized 
live oaks (20—40 cm dbh) typically extend- 
ed out to 20 m or more from the bole or 
main stem, however galls usually were lo- 
cated on live oak roots within 2 m of the 
bole. 

Root galls induced by O. nebulosa gen- 
erally occurred on small feeder roots (usu- 
ally < 1 cm diameter) that arose from pri- 
mary roots below root flares near the bole. 
Both unilocular and multilocular galls were 
observed. Galls presumably formed indi- 


vidually with a single chamber (unilocular) 
on roots when a single viable larva began 
feeding following oviposition beneath the 
root cambium, while multichambered (mul- 
tilocular) galls resulted when several viable 
eggs were deposited at a single location in 
the root. Multilocular galls appeared to 
form by the growth and fusion of the outer 
galls (walls) surrounding individual larval 
chambers. Multilocular galls formed much 
more frequently than unilocular galls on 
live oak roots (Table 3). Unilocular galls 
tend to be globose and isodiametric, rang- 
ing in size from 0.3-1.5 cm in diameter. 
Multilocular galls appeared as broadly- 
fused aggregates of individual globose galls 
and at maturity were considerably larger 
and more irregularly-shaped than unilocular 
galls. These multilocular galls expanded 
through cellular proliferations from hyper- 
trophy and hyperplasia to average sizes of 
5.4 cm length and 4.0 cm in diameter (n = 
178) and contained an average of 25 cham- 
bers (cells). Exceptionally large multilocu- 
lar galls > 10 cm long and >8 cm wide 
contained 70 or more chambers. Multiloc- 
ular galls increased in size proportional to 
the number of chambers fusing to form 
them. However, the size (internal diameter) 
of individual chambers was not related to 
the number of chambers within each gall. 
Gall chamber size (x) relative to number of 
chambers (y) was sufficiently variable to 
prevent a statistically valid linear correla- 
tion (n = 442, °? = 0.006). Nevertheless, 


VOLUME 102, NUMBER 2 


36 


aA 


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


Gall Morphology (%)' 


Texas No. Galls 


Gall Size (cm) i 
Root Diameter 


County Examined Unilocular Multilocular Length Width (cm) 
Bandera 29 6:9 99.1 44+ 3.1 3:0 212 05- = 02 
Bell 37 8.1 ees) elise oes) PORE 0.4 + 0.1 
Kendall 9 222 77.8 2 ERO PI 35; M30) 0:6 +10] 
Kerr 67 4.5 95:5 a4 22 40+ 1.6 0.4 + 0.2 
Mills 36 5.6 94.4 LOES ely se (02) 0:502 


! Percent of sampled galls that were unilocular (single chamber) or multilocular (multichambered). 
? Gall dimensions are for multilocular galls only (mean + 1] SE). 


the linear model and equation (y = 
6.415+0.007x) describing the relationship 
was highly significant (P < 0.001) and the 
correlation was positive. Multilocular galls 
also were proportionally larger as root di- 
ameter increased. Galls that formed on 
roots in the 1-2 cm-diameter range occa- 
sionally grew to sizes up to 11 cm long X 
10 cm in width. The rate of gall expansion 
was not measured in this study, but some 
galls may have attained full size in one 
growing season since multilocular galls 
were formed by the growth of individual 
chambers, and larvae pupated within the 
locules during the following late fall and 
winter months. However, this does not pre- 
clude the possibility that the insect could 
either have two generations per year or re- 
quire 1—2 years for gall development since 
immature and mature galls were observed 
in both fall and spring collections. 

Gall morphology and insect develop- 
ment.—Ninety-two galls were removed 
from the root systems of excavated live 
oaks at the Kerrville survey site during the 
fall and spring collections. Some of these 
galls were collected from trees exhibiting 
the diagnostic veinal necrosis leaf symptom 
of oak wilt (Fig. la). Root segments with 
galls were taken from feeder roots in the 
main root ball near the bole (Fig. 1b). Many 
of the galls close to the soil surface were 
collected using a hand spade and lopping 
sheers without extensive excavation (Figs. 
Ic—d). The apparent disruption of normal 
plant hormone diffusion down feeder roots 


caused by gall formation often induced the 
production of small root sprouts from galls 
close to the soil surface (Figs. 1c, e). Root 
segments proximal to the galls toward the 
root apex were sometimes reduced in di- 
ameter or killed by the gall. Mature current- 
year galls were typically tan to light brown 
like host roots, while older previous-years 
galls were dark brown to black and weath- 
ered as the root tissue died and decayed 
(Fig. 1f). The majority of galls collected on 
18 November were previous-year galls with 
exit holes. 

All root galls examined in this study con- 
tained outer galls that were hard, solid, and 
woody. Larval chambers were completely 
surrounded by woody outer root tissue, but 
were not separated from the outer gall by 
an internal air space. The gall surfaces were 
generally smooth, lacking hairs and spines, 
and were not coated with a sticky resin. 
Unilocular single-chambered galls had out- 
er walls that were morphologically identical 
to those of individual chambers within mul- 
tilocular galls. 

The majority of galls collected from root 
excavations in the fall and spring were dis- 
sected immediately to examine concurrent 
stages of insect and gall development. Oth- 
ers from the fall collection were held at 
25°C for seven weeks during which galls 
were dissected at various stages of devel- 
opment until tenerals began emerging from 
exit holes in the galls. Gall formation was 
initiated by early stages of larval feeding 
beneath root cambial tissue. Host root tissue 


366 PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON 


Fig. |. Host-parasite relationships associated with O. 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 


Fig. 2. 


367 


Developmental morphology of gall formation and insect development in O. nebulosa-intested 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 
within developing galls throughout the 
summer and early fall months, enlarging the 
locular cavities to form chambers. None of 
the chambers appeared to contain more than 
one larva. Larvae began pupating within 
chambers of the galls in the fall (Fig. 2c). 
Pupae (Figs. 2d—e) began molting, breaking 
free from their wing cases to become te- 


nerals while still in the galls. Tenerals 
emerge from the galls on warm days in late 
winter to early spring (usually early Feb- 
ruary in Texas) by chewing through the 
wall of their chamber and escaping through 
the exit hole or chewing into the locule of 
an adjacent chamber with an exit hole to 
escape. Large numbers of adults were ob- 
served emerging from the soil above sub- 


368 


PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON 


Table 4. Gail and insect development, insect viability, and emergence associated with root galls of O. ne- 
bulosa on infested Quercus fusiformis and Q. virginiana X Q. fusiformis hybrid trees. 


Fall Collection! 


Previous-year 


Current-year 


Spring Collection 


Previous-year Current-year 


Mature Galls Mature Galls Immature Galls Mature Galls Mature Galls Immature Galls 
Gall development? 
Galls in category (%) 5p 38.6 9.1 32.0 52.0 16.0 
(n = 23) (n = 17) (n = 4) (n = 8) (n = 13) (n = 4) 
No. chambers (*) HOS Bales: OS = IS) TS ae I3 ey ae PSP eh EnS 28 (5 
Chamber size (%) 6.5 + 0.1 67 £01 S 25 Ol CS0 5 20l T A0 E0 
(n = 236) (n = 164) m = 29) (m= 198) (n = 278) (n = 126) 
Insect development 
Larvae (%) -— 34.9 + 10.1 96.9 + 3.1 — 8.8 ea 3.5 90.3 + 4.0 
Pupae (%) — I BE tess ale = Sl — 0.0 = 007 
Tenerals (%) — OKO == ONO 0.0 + 0.0 — 230 £57 CO0 
Insect emergence* 
Same chamber (%) STOET Ome Oona: 00200 530 £74 45.35 7-25 3 eae 
Adjacent chamber (%) 19.6 + 4.6 g4 E54 OOc OO Biles) as G3) 15.5 = 39 (0}(0) == (0.0) 
Tiotalvemercencel(7o) ee 77-50" 0.7 ee 50.25 lO (OHO) 28 (00) 77 se Wlesh lO} a2 GS) SL se AA, 
Insect viability in galls 77.82 6.7 91.2 + 3.3 100 0T OOM s:0sael 926 as 3A Wij) ae 5) 
Dead or aborted (%) 22.2 + 6.7 ss 2E Shoa 0.0 + 0.0 14.3 + 6.9 7434 43225 


! 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 + 1 SE). 


* 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. 

+ 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 
the late morning of the spring collection. 
Tenerals tended to emerge from chambers 
on the sides facing the soil surface. Indi- 
viduals in chambers facing downward in 
the soil tended to chew their way through 
the gall to the top layer of chambers that 
already had exit holes. Tenerals burrow to 
the soil surface and emerge as an adult (Fig. 
2f). In the laboratory, callow adult females 
that were artificially freed from gall locules 
after emerging from pupal cases were ca- 
pable of flight within 30 min. Tenerals 
reared from galls without assistance 
emerged from chambers with partially to 
fully expanded wings and could fly almost 
immediately. No parasites were recovered 
or observed from galls (n = 23) used in 


rearing the adults. All adults reared from 
galls collected in the summer and fall 
months were females indicating that root 
galls give rise to an asexual generation. 

A comparison of the developmental mor- 
phology of galls and insects in the fall col- 
lection with those in the spring collection 
yielded different results (Table 4). The 
spring collection had a higher percentage of 
galls in the current-year immature and ma- 
ture developmental categories than in the 
fall collection. Although gall chamber size 
(internal diameter) was comparable for 
galls in all three developmental categories 
in the fall and spring collections, immature 
galls were smaller than previous-year and 
current-year mature galls. Current-year ma- 
ture galls from the fall collection contained 


VOLUME 102, NUMBER 2 


a higher percentage of larvae and pupae, 
but had a lower percentage of tenerals and 
total emergence than current-year mature 
galls from the spring collection. However, 
immature galls contained a much higher 
percentage of larvae than mature galls 
among current-year galls from both collec- 
tions. Mature previous-year galls from both 
collections lacked insect developmental 
stages since all living tenerals had already 
emerged prior to collection. Insect emer- 
gence was highest in previous-year mature 
galls and lowest in current-year immature 
galls for both collections. The majority of 
tenerals emerging from previous-year and 
current-year mature galls in both collections 
came from the same chambers within which 
they developed while the remaining portion 
emerged from an adjacent or more distant 
chamber from which they developed. This 
latter type of emergence was achieve by te- 
nerals chewing their way into an adjacent 
chamber from which the occupant had al- 
ready emerged and created an escape route 
to the outside of the gall. Insect viability 
within galls was highest in immature galls 
and higher in current-year mature galls than 
in previous-year mature galls. 


DISCUSSION 


The limited occurrence of O. nebulosa on 
two live oak species and its absence on the 
red oaks (subgenus Erythrobalanus), in- 
cluding blackjack oak, Lacey oak, and 
Spanish oak in this survey, suggests host 
specificity to certain oaks in the white oak 
group (subgenus Leucobalanus). Hitherto, 
O. nebulosa has been reported from Geor- 
gia and Arkansas only on post oak (Q. stel- 
lata) and overcup oak (Q. lyrata) by Weld 
(1959), both white oak species. The live 
oaks are intermediate species having sap- 
wood anatomical characteristics of both the 
white oak and red oak groups. However, 
live oaks are generally classified as white 
oaks based on leaf and acorn characters. 
Quercus fusiformis is well established 
throughout the Edwards Plateau region of 
central Texas. The natural continuum of hy- 


369 


brids (Q. virginiana X Q. fusiformis) that 
form between coastal and plateau live oaks 
occurs abundantly in the region between 
eastern parts of the Edwards Plateau and the 
Brazos River to the east (Nixon 1984). 
Consequently, the current study has dou- 
bled the host range to include semiever- 
green oaks and expanded the known habitat 
of O. nebulosa to xeric savannah-wood- 
lands of the southwestern United States. 
Lyon (1996) recently described seven new 
cynipid species on leaves and twigs of 
white oaks from this region. Weld (1960) 
listed 130 species of phytophagous cynipids 
from the southwestern United States with 
an additional 117 gall types that were never 
associated with a specific cynipid. The 
specificity with which O. nebulosa coloniz- 
es and forms galls on the roots of its hosts 
may be a survival advantage to the species 
in avoiding dessication in xeric habitats 
(see Fernandes and Price 1992). 

This survey indicates that the incidence 
of live oak root-galling by O. nebulosa 
around oak wilt infection centers is rela- 
tively low (1.0-10.7%) within most of the 
areas surveyed. However, the small per- 
centage of trees that were infested tended 
to have relatively high infestations of their 
root systems based on the percentage of 
major roots that were galled on individual 
trees. These data suggest that the occur- 
rence of this wasp is sporadic in the Ed- 
wards Plateau region, but that it tends to 
occasionaily form relatively high popula- 
tions in small localized areas or in individ- 
ual clumped stands (motts) of live oaks. 
The occurrence and incidence of the wasp 
does not appear to be influenced by the in- 
fection-status of trees since root-galling did 
not occur at significantly higher frequency 
in infected than in healthy live oaks. There- 
fore, oak wilt infection of live oaks proba- 
bly does not predispose live oaks to O. ne- 
bulosa-infestation. Since the wasp occurs in 
both oak wilt infected and uninfected live 
oaks, the presence of C. fagacearum in the 
root system does not appear to be a nutri- 
tional requirement for larval development. 


370 


Thus, any potential ability to vector the oak 
wilt fungus would likely be passive and not 
out of necessity in order to complete its life 
cycle. However, the introduction by the 
wasp of a highly virulent pathogen such as 
C. fagacearum that causes a fatal disease in 
live oaks would not necessarily be disad- 
vantageous to the wasp’s survival. The 
above-ground parts of most live oaks that 
become infected with the oak wilt fungus 
die within a few months after infection, yet 
the root systems often survive and are sup- 
ported by an abundance of root sprouts that 
quickly develop after the top dies and apical 
dominance is lost. These new shoots can 
maintain the living root system indefinitely. 
Hence, the wasp would not sacrifice its 
ability to continue colonization of the root 
system if it were to introduce a lethal path- 
ogen. The abundance of new feeder roots 
resulting from the growth of many new root 
sprouts may actually increase the availabil- 
ity of colonizable root mass. 

The presence of root galls on oak wilt- 
infected trees at the advancing front of oak 
wilt infection centers, the occasional very 
high root-infestation rates, and the occur- 
rence of nearby oak wilt infection centers 
indicates the potential opportunity for O. 
nebulosa to acquire inoculum of C. faga- 
cearum from oak wilt-infected live oak 
roots. Larvae feed directly on root tissue 
(new sapwood) known to serve as a reser- 
voir for C. fagacearum-inoculum in infect- 
ed trees. Root tissue tends to have the high- 
est levels of inoculum because most trees 
within infection centers become infected 
through root transmission as a result of root 
grafting and common root systems often 
shared by trees within motts (Appel et al. 
1995). Root inoculum is particularly im- 
portant following root transmission since 
the fungus first enters and accumulates 
most of its inoculum potential within roots, 
which is used for subsequent colonization 
of aerial portions of the tree (Wilson 1995). 
Furthermore, adult O. nebulosa females 
have the ability to burrow down into the 
soil and directly penetrate live oak feeder 


PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON 


roots with their ovipositor during oviposi- 
tion. Although we did not examine the vec- 
tor potential of this wasp here, the oppor- 
tunities to acquire inoculum and its inter- 
action with live oak roots make it a suitable 
candidate for further investigation. 

The effect of O. nebulosa-infestations on 
root development of live oaks may impact 
host-pathogen interactions due to feeder- 
root mortality. The death of feeder roots as- 
sociated with gall tissue senescence follow- 
ing adult emergence could reduce the vigor 
of trees making them more susceptible to 
oak wilt-infection. The majority of root seg- 
ments collected with galls prior to emer- 
gence were alive, suggesting that dead root 
segments may abscise from infested roots 
after emergence and decay in the soil. In 
this way, infested roots may be effectively 
pruned from the root system following 
emergence. The death of root segments 
proximal to the galls toward the root apex 
appeared to result from the disruption of 
phloem transport due to the crushing of root 
phloem by proliferating gall tissue. 

The observed emergence of unisexual- 
generation females of O. nebulosa from 
roots galls in this study raises the question 
of whether heterogony occurs in this spe- 
cies. Many cynipid gall wasps on oaks 
commonly have alternation of sexual and 
asexual generations with the parthenogenic 
all-female generation usually developing 
and emerging in the summer and autumn 
months (Lyon 1963, 1969, 1970; Askew 
1984; Rey 1992). Lund et al. (1998) dem- 
onstrated heterogony in B. treatae, another 
root-galling cynipid of live oak. In this spe- 
cies, the bisexual generation emerges in the 
spring from root galls on Q. fusiformis and 
females oviposit into the undersides of 
leaves, inducing unilocular foliar galls on 
the same host. Morphologically distinct 
unisexual-generation females, previously 
described as B. kinseyi Weld (1921), 
emerge from the leaf galls in the fall and 
induce multilocular galls on the roots. The 
cycle of O. nebulosa appears to differ in 
that unisexual-generation females emerge 


VOLUME 102, NUMBER 2 

from root galls in the spring instead of from 
leaf galls in the fall. It is important in as- 
sessing its vector potential to determine 
whether unisexual-generation females of O. 
nebulosa oviposit into roots, leaves, or 
some other parts. The ability of this species 
to vector C. fagacearum would be less like- 
ly if heterogony occurs with the alternate 
sexual generation arising from galls on 
leaves or twigs because these are poor in- 
fection courts and poorer sources of inoc- 
ulum for subsequent infections. 

Several important inferences are suggest- 
ed by the gall morphology and insect de- 
velopment results. The positive correlation 
of gall chamber size and number of cham- 
bers per gall and the corollary increase in 
overall gall dimensions with increasing 
chamber number indicate that there is no 
evidence for chamber dwarfing due to in- 
traspecific competition as galls increase in 
size. The low mortality of the wasps during 
development within galls suggests that gall 
numbers on roots may be used to accurately 
estimate population density. The occurrence 
of immature galls in the spring implies that 
there are either two generations per year or 
that some galls may take two years to de- 
velop. The latter conclusion is more likely 
since there was no evidence of a fall emer- 
gence in galls from the fall collection (no 
fresh exit holes), the percentage of imma- 
ture galls was small, prior emergence from 
immature galls was low, and there was an 
absence of tenerals within immature galls 
from the fall and spring collection. The 
higher percentage of later insect develop- 
mental stages within current-year mature 
galls in the spring than in the fall provides 
additional support for a single late winter 
or early spring emergence. 

The morphology of the woody multiloc- 
ular root galls observed in O. nebulosa do 
not fit cleanly into a single structural type 
as defined by Stone and Cook (1998). The 
structure of these asexual galls would best 
be described as a cross (combination) be- 
tween the S3 and S5 structural stages ac- 
cording to their system for classifying gall 


371 


structural types. The larval chambers are 
completely surrounded by, and in direct 
contact with, woody outer gall tissue. The 
galls are multichambered, but lack spines 
on the outer surface. The Stone and Cook 
system was developed to include the com- 
plex and diverse gall types represented in 
the genus Andricus and related oak gallers 
on above-ground parts of oak species. Per- 
haps a different or amended system should 
be devised for oak root gallwasps to ac- 
count for gall morphological characters re- 
sulting from adaptations to roots coloniza- 
tion in soil environments. The morphology 
of root galls described here may be quite 
different from asexual galls arising from 
unisexual-generation females presumably in 
the summer or fall. Asexual galls could 
possibly occur on different live oak tissue, 
on a different oak species, or on roots. We 
do not currently know whether galls form- 
ing on roots can be sexual galls, asexual 
galls, or both. However, a sexual generation 
has not yet been confirmed with this species 
since males have not been observed or de- 
scribed. 

Root gall wasps generally are considered 
to be less common than above-ground gall- 
makers perhaps because they are rarely ob- 
served and their impact on host biology is 
poorly understood. Root galls no doubt es- 
cape observation in most surveys for gall 
insects. Systematic surveys for root gall 
wasps of oaks in different habitats should 
lead to significantly more information on 
the biology of previously undescribed and 
unidentified root-galling cynipids. Although 
recent studies by Shorthouse and Rohfritsch 
(1992), Askew (1984), Lund et al. (1998), 
Csóka (1997), and Csóka et al. (1998) have 
provided new information on the biology of 
some Nearctic root-galling species, addi- 
tional work is needed to further elucidate 
the biology of root gall-makers. Such work 
may reveal that some root-galling cynipids 
may have greater significance than is cur- 
rently attributed to members of this obscure 
insect group. 


ACKNOWLEDGMENTS 


We thank Texas Forest Service personnel 
including entomologists Drs. Ron F Bill- 
ings and R. Scott Cameron, and Mark Duff 
(field forester) for their assistance in orga- 
nizing the field collections, and Lisa B. 
Forse for skillful dissection of the root 
galls. We acknowledge Mr. James B. Briggs 
for assistance in collecting specimens and 
data in several central Texas counties. The 
cooperation of Edward H. Barron and 
Bruce R. Miles (Texas state forester) in fa- 
cilitating formal cooperative agreement no. 
19-93-081 between the USDA Forest Ser- 
vice and the Texas Forest Service also is 
gratefully acknowledged. We appreciate the 
help of Arnold S. Menke (Systematic En- 
tomology Laboratory, USDA, Washington, 
DC) who confirmed the identity of O. ne- 
bulosa specimens. We also appreciate Drs. 
Nathan Schiff, Jim Solomon, and Kathy 
Schick for their comments in reviewing 
drafts of the manuscript. 


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