Group Title: Annals of the Entomological Society of America, 99 (1). pp. 113-120.
Title: Biology of parasitoids (Hymenoptera) attacking Dasineura oxycoccana and Prodiplosis vaccinii (Diptera: Cecidomyiidae) in Cultivated Blueberries.
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Title: Biology of parasitoids (Hymenoptera) attacking Dasineura oxycoccana and Prodiplosis vaccinii (Diptera: Cecidomyiidae) in Cultivated Blueberries.
Series Title: Annals of the Entomological Society of America, 99 (1). pp. 113-120.
Physical Description: Book
Creator: Sampson, Blair J.
Rinehart, Timothy A.
Liburd, Oscar E.
Stringer, Stephen J.
Spiers, James M.
Affiliation: University of Florida -- Entomology and Nematology Department
Publication Date: 2006
Subject: Diptera   ( lcsh )
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Bibliographic ID: UF00083970
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Biology of Parasitoids (Hymenoptera) Attacking Dasineura oxycoccana
and Prodiplosis vaccinii (Diptera: Cecidomyiidae)
in Cultivated Blueberries


Ann. Entormol. Soc. Arm. 99(1): 113-120 (2006)
ABSTRACT The blueberry gall midge, Dasineura oxycoccana (Johnson), and blueberry tip midge,
Prodiplosis vaccinii (Felt) (Diptera: C. ,1. .. ....]. ), are recurring ... .1 pests of cultivated
blueberries in the southern United States and '. I. 1.1. .. ... I. ,,, Insecticides can give short-term
control, but overlap in parasitoid phenologies indicates the potential for natural control of midge
populations. Using a combination of laboratory rearing and mitochondrial DNA analysis of 1,. i.I
samples, we identified five species of solitary i.1. i .. .:. ..1 that killed 30 40% of midges. These
species include at least three undescribed platygastrids in the genera .: ..... and
Inostemma. An undescribed, .. .1 ..1 1.. \:rostocetus sp. (Eulophidae: Tetrastichinae) was the
only midge parasitoid that was consistently active when rabbiteye blueberries, NVacciniurn ashei Reade,
were in :i Six percent of midge prepupae, half of which already contained platygastrid larvae,
were parasitized by Aprostocetus.

KEY WORDS biological control, Platygastridae, Eulophidae, high-fidelity polymerase chain reac-
tion, parasitism

BLUEBERRY GALL MIDGE, Dasineura oxycoccana (John-
son), and blueberry tip midge, Prodiplosis avaccnii
(Felt), are two species of univoltine gall midges
(Diptera: Cecidomyiidae) that feed exclusively on
Vaccinium buds. Midges are not 3 detected be-
cause their feeding damage only becomes visible 7
14 d after larvae have left the blueberry bush. Fur-
thermore, bud injury is often mistaken for freeze dam-
age, plant disease, or nutrient .. :. .. (Driggers
1926, Lyrene and Payne 1992, Bosio et al. 1998, Samp-
son et al. 2002, Wei 2004). D. oxycoccana is the first of
these species to break winter dormancy in January or
February in the southeastern United States. Females
principally oviposit between the developing scales of
' buds. Newly hatched larvae crawl deeper into
blueberry buds where they feed on the innermost
meristematic tissue and can reduce up I i
production and potential fruit yield (Lyrene and
Payne 1992, Sampson et al. 2002). As damaged I .
buds disintegrate, D. oxycoccana move to leaf buds,
but they are soon superseded by P. vaccinii (Driggers
1926; Gagn 6 1986, 1989; 1. unpublished data).
Crop losses to P. vaccinii have yet to be assessed.

Mention of trade names or commercial products in this article is
solely for the purpose of providing specific information and does not
imply recommendation or endorsement by the U.S. Department of
1 USDA ARS Small Fruit Research Station, Poplarville, MS 39470.
2 Department of Entomology and Nematology. University of Flor-
ida, Gainesville, FL 32611-0620.

However, injury to blueberry leaf buds and meristems
by P. vaccinii often induces excessive suckering, leaf
distortions, and leaf drop, which could result in lighter
bud sets (Gagn6 1986, Williamson and Miller 2000).
Little is known about the interactions between
midges and their parasitoids on fruit crops. Proc-
totrupoid wasps such as those belonging to the family
Ceraphronidae parasitize D. oxycoccana on Wisconsin
cranberries Vaccinium macrocarpon L. (Barnes 1948).
In the southern United States (Florida, Georgia, Ala-
bana, Mississippi, and Louisiana), endoparasitic
Platygastridae seem to replace ceraphronids as the key
natural enemies of midges (Vlug 1976, Yoshida and
Hirashima 1979, Jeon et al. 1985, Son6 1986, Sampson
et al. 2002, Sarzynski and Liburd 2003, Legner 2004).
Chalcids, like tetrastichine eulophids, are also midge
parasitoids found in both midwestern and southern
habitats, where they attack midges on cranberries and
blueberries, respectively (Sampson et al. 2002).
Here, we studied the ecology of parasitoids found in
rabbiteye, Vaccinium ashei Reade, and southern high-
bush blueberry (Vaccinium corymbosuim X Vaccinium
darrowii Camp) ecosystems. Larval development,
adult reproductive behavior and parasitoid host phe-
nology was investigated during 2003 2004. Mitochon-
drial DNA analysis was used to confirm the identity of
adult parasitoids as II as to assist in interpreting
interactions between parasitoids and their midge


Materials and Methods
HostandT' iI,, ;ii i.l....;nl. Host Eggs and Larvae.
Juvenile midges were sampled from two blueberry
nurseries at the USDA-ARS Small Fruit Research Sta-
tion, Poplarville MS (N 30' 50.21', W 89' 32.65'). Nurs-
ery 1 contained 950 2- to 6-yr-old potted bushes rep-
resenting 50 clones of rabbiteye blueberry, southern
highbush blueberry, and wild blueberries, Vaccinium
elliottii ("' .. ... Nursery 2 housed -11,000 second-
and third-year .Ihl, representing 300 clones of
V. ashei and southern highbush blueberry. Additional
midge hosts were .I1. -.. i from 10 commercial rab-
biteve blueberry farms in southern Louisiana, Missis-
sippi, and Alabama.
A single sample consisted of -10 terminals bearing
30 ;i .. .. or leaf buds enclosed in a resealable plastic
bag. Five or 10 samples were taken from random
bushes in each nursery every 3 to 4 d over a 2-yr
period. Smaller samples were collected when bud
:.,i .,1, .... in the winter. Bags were held for 2
to 3 d to allow larvae to abandon buds. The bags were
then partly filled with water and shaken to flush out
remaining host eggs and larvae as II as larvae that
were diseased, heavily parasitized, or dead. Eggs and
larvae were pipetted into clean petri dishes, counted,
and preserved in 1 ", ethanol. The number of host
larval stages (instars) was established from a fre-
quency histogram generated by PROC UNIVARIATE
(SAS Institute 1990) by using the body width mea-
surements of 474 midge larvae. Mature midge larvae
(third instars or prepupae) were identifiable by a dark
Y-shaped ventral sclerite called a spatula (Gagn6
Juvenile Parasitoids. Preparing a parasitoid brood
for slide mounting sometimes required varying a host
larva's exposure to the clearing, staining, and fixing
agents. Normally, host fat tissues were adequately sol-
ubilized with exposure to solutions of 5 and 10% KOH
for 24 h and 30 min, respectively. Two or three drops
of diluted double stain (I '-U ,r' Products, Inc., Ran-
cho Dominguez, CA) or acid fuschin in lactophenol
was sufficient to clear, stain, and ,. !..... .. fix spec-
imens. Smearing host larvae by .: I,.. .... .i pressure
to the '*i- entirely expulsed many parasitoids
from the host or more clearly exposed them. Live
parasitized hosts, compressed by a coverslip, made it
easier to :.i ...: which host organs nourished the
parasitoid brood (e.g., midgut, brain, or ganglia). Us-
ing an ocular micrometer, we measured the cephalo-
thorax and caudal segments of juvenile parasitoids that
were fixed on glass slides.
Adult Parasitoids. Adult wasps were collected from
the plastic bags and from parasitized midge prepupae
and pupae that were raised on moist peat in dark insect
growth chambers at 300C (I-30 BLL, Percival Scien-
tific Inc., Perry. IA). Randomly placed sticky II
traps (16 by 23 cm, Trec6, Salinas, CA) were hung in
the canopies of 10 blueberry bushes to collect foraging
parasitoids. The tip of an insect pin was used to remove
parasitoid adults from the traps. Residual glue that
,. to the insect was dissolved by agitating spec-

imens for 24 -48 h in Histoclear (National Diagnostics,
Atlanta, GA). Wings were removed from adults, la-
beled, and separately slide mounted in water to pro-
tect fine hairs during the clearing process. Adults were
cleared using 10% KOH, neutralized, and stained with
acid fushin in lactophenol, and then slide mounted in
euparal or silicon oil. The identity of parasitoids on
sticky traps that belonged to species capable of par-
asitizing immature midges was confirmed by compar-
ing them with specimens captured among host eggs
and neonates inside blueberry buds or reared from
larval or prepupal hosts inside the insect growth cham-
Host and Parasitoid Identification. The i. i
taxonomic keys and records were used for midge and
:-.. ,. : .1 identification: ( .1 ... ... (Diptera):
Barnes (1948) and Gagn6 (1986, 1989). 1 -i .. ] .
(Hymenoptera): Ashmead (1887, 1893), Marchal
(1906), Fouts (1924), Kieffer (1926), Masner and
Muesbeck (1968), Krombien et al. (1979), Velikan et
al. (1984), Vlug (1984, 1995), Masner and Huggert
(1989), and Buhl (1998, 2001). Eulophidae (Hyme-
noptera): Crawford (1907), Girault (1917), Myers
(1930), Walter (1941), Burks (1943), Piore and Vig-
giani ('- -), Bou6ek (1986), Krombien et al. (1979),
Velikan et al. (1984), LaSalle (1994), and Shauff et al.
(1997). Taxonomic experts affiliated with the USDA
ARS Systematic Entomology Laboratory Communi-
cations and Taxonomic Services Unit (Beltsville, MD)
confirmed our identifications. Vouchers were depos-
ited in the U.S. National It. ., ... by Raymond J.
Gagn6 (C. ...... ... .. ) MichaelE. Shauff (Eulophi-
dae), Michael W. Gates (Eulophidae and Platygastri-
dae), and Terry Nuhn (Platygastridae).
Ecological Data: Analysis of Host Parasitism and
Parasitoid ;, .e! t,.,,. Behavior. We calculated the
average parasitism rate expected for each month of a
single year (Fig. 1). Parasitism rates were expressed as
the percentage of host eggs and larvae with parasitoid
eggs, larvae, or pupae (Figs. 2 and 3). The chi-square
goodness-of-fit analysis (PROC FREQ) was used to
test whether parasitoid eggs, neonates, and older first
instars were randomly or uniformly distributed among
host larvae (SAS Institute 1990).
Foraging activity of adult parasitoids throughout the
day was evaluated using hourly sticky trap (16 by
23-cm) catches. The resulting data were fitted to a
Gompertz function [y = 163 e 49( -0.26x) i, where x is
the cumulative hours that elapsed starting at 0800
hours, and y is hourly cumulative catch expressed as
a percentage of the total daily catch (PROC NLIN,
SAS Institute 1990). The behavior of female parasi-
toids in or around buds and host larvae also was noted.
Polymerase Chain Reaction (PCR) Procedure and
Sequencing Mitochondrial DNA from Adult Para-
sitoids. Randomly chosen adult wasps of both sexes
(Fig. 4a-i) were sorted by taxon and placed into 1.5-ml
...* i. i..n.. tubes filled with 100% ethanolbefore
analysis. Sequencing mitochondrial DNA revealed di-
agnostic mutations useful for calculating genetic sim-
i ',- .... midge endoparasitoids. A bead-beating tech-
nique and Ultraclean soil DNA isolation kit (MoBio,

Vol. 99, no. 1


Host density (eggs & larvae per 30 blueberry buds)
-- % parasitism (SEM) by Platygasteridae
--o-- % parasitism (SEM) by Eulophidae

a-Bo -o-- T
F M A M J J A Stive

Years 2003 2004
Fig. 1. Seasonal changes in host density
percentage of parasitism of midge larvae by
largely Synopeas and Platygaster (*) and Eulo
sively Aprostocetus ( ) sampled from cultiv
blueberry. Phenology of blueberry bud devel
picted as horizontal boxes. SEM is standard
means represented by vertical bars. The low
marizes monthly sampling effort. Numbers insi
the sample sizes for monthly percentage of p

Solana Beach, CA) extracted total DNI
parasitoids from each taxa (see Fig. 5 fo
ignations). The degenerate primer pai
CAATRATTAT) amplified part of the m
cytochrome oxidase I (COI) gene (Simon
Reactions consisted of 4 pl of MasterN
polymerase (Brinkmann-Eppendorf, We
and 5 ng of total insect DNA in 6.4 /l ofw
parameters were 95C for 2 min followed
of 95C for 1 min, 56C for 2 min, 72C fo
then 720C for 10 min with storage at 40
PCR products were purified using a QIAq
(QIAGEN, Valencia, CA), and the res
strands were sequenced using BigDye ver
plied Biosystems, Foster City, CA).
Partial sequences of the COI gene con:
bp were edited using Sequencher (Gene
Arbor, MI) and aligned with MegAligr

Madison, WI). Phylogenies based on parsimony were
generated using PAUP*4.0 (Swofford 2000). Because
our analysis was limited to adult identifications, we
selected the most parsimonious tree (Fig. 5) to depict
the phylogeny among midge parasitoids based on 114
informative characters. Boot strap values from 100
replicates and percentage of sequence similarity are
shown above and below each branch, respectively
(Fig. 5). COI sequences and alignment for the 19 wasp
specimens used to produce a preliminary phylogeny of
our small parasitoid assemblage can be found in Na-
tional Center for Biotechnology Information Gen-
Bank under the accession nos. AY843313-AY843331.


Host-Parasitoid Ecology and Identification. Syno-
peas were the most common midge parasitoids. Adults
of this genus were distinguished from other midge
o N D parasitoids by a posterior scutellar spine; short and
pointed in S. Sactogaster (Fig. 4a, akin to Ashmead's
original description of S. anomaliventre), longer and
blunter in S. Synopeas [Fig. 4c similar to S. pennsyl-
vanicum (Fouts) ]. Ninety percent of adult Synopeas
actively flew between 1100 and 1700 hours. Females
were observed to crawl deep inside blueberry buds.
SFemale Synopeas dispersed their eggs (Fig. 2a) and
young first instar brood (Fig. 2i) uniformly among
O N D hosts (goodness-of-fit test: 2 = 258.8, df = 6, P <
0.005, n = 355 parasitized hosts) and positioned eggs
(0) and the near the host's midgut (Fig. 2j and k; Ganin 1869,
Platygastridae, Marchal 1906). Nine of 10 broods contained only one
phidae, exclu- or two offspring (mean, 1.5 1.0 per host, n = 354
ated southern parasitized hosts). When broods were larger, offspring
opment is de- were typically staggered in the posterior half of the
error of the host (Fig. 2k).
er figure sum- First instars of Synopeas were cyclopiform (Fig.
de the bars are 2i-1), each with a light yellow-brown cephalothorax.
arasitism. Head capsules bore broad sickle-shaped mandibles as
well as maxillae and ligulae spiked with short rasping
A from adult teeth. Newly hatched larvae measured 210 50 1/m
r taxon des- (n = 38) wide at the head and 390 120 /im between
rs C1-J-1763 apices of the head and furca (n = 17; Fig. 2i). Instars
with C1-N- II (Fig. 2n) and III (Fig. 20) were hymenopteriform
VGTTA) and with reduced mandibles, and before pupating (Fig. 2p
1TTACTAT) and q) they grew five-fold. Instar II: (L) 550 110 inm,
GGNACNG- (W): 280 50 im, n = 13). Instar III: (L): 1000 120
itochondrial inm, (W): 440 80 im, n = 10).
i et al. 1994). Female Inostemma sp. (Fig. 4e) likely oviposited in
lix TaqDNA P. vaccinii eggs. Embryonic development occurred in
'stbury, NY) the brain and first five ganglia of third instar hosts
ater. Cycling (Fig. 2t). Early first instars of Inostemma (Fig. 2g and
by 43 cycles h) were recognized by fine serrations along the inner
)r 3 min, and margins of furcae (Marchal 1906, Jeon et al. 1985).
C. Amplified Adults (Fig. 4e) possessed a prominent marginal vein
uick PCR kit in the forewing and a long arching horn that sheathed
ulting DNA the ovipositor. Parasitism by Platygaster (Fig. 4g) was
sion 3.1 (Ap- indicated by the presence of oval eggs (Fig. 2r), man-
dibulate-type first instars with 12 abdominal and tho-
sisting of 364 racic segments (Fig. 2b-d) and a smaller cyclopiform
Codes, Ann larva (Fig. 2e and f). Clutches of one or two progeny
n (DNAstar, typify Platygaster broods, but as many as seven were

January 2006

Vol. 99, no. 1





- i





Vr- "" .-

''' ^$i

2j 2k 21


Sm- 'i


i '"

2f 2g


2n 2o

Fig. 2. Eggs, larvae, and pupae of Platygastrinae found in D. oxycoccana and P. vaccinii hosts. (a-i) Egg and first instars.
(a) Newly inserted Synopeas egg. (b) Early first instar of Platygaster sp.l. (c) Same partially engorged. (d) Same fully
engorged. (e) First instar of Platygaster sp. 2. (f) Same fully engorged. (g) Inostemma first instar. (h) Same partially engorged.
(i) Early first instar of Synopeas sp. Top scale bar represents larva size in micrometers. (j-q) Development of Synopeas brood
inside host larvae. (j) Fully developed embryos. (k) Superparasitism produces nine first instar progeny. (1) Early and late
(engorged) first instars. (m) Early first instar and second instar. (n) Second instar of Synopeas. (o) Third instar. (p) Early
pupa. (q) Late pupa. Scale is provided by brood shown inside their hosts as well as a lower bar forj-m and o-q. (r-u) Immature
stages of other platygastrids, scale not shown, but can be inferred from figures. (r) Platygaster eggs. (s) superparasitism in
Platygater. (t) Early and late (engorged) first instars of Inostemma. (u) Early Synopeas first instar still surrounded by its

possible per host (Fig. 2s). First instars had a mem-
branous cephalothorax that was 120 + 30 u/m (n = 15)
in width and a body 220 + 30 /m in length (n = 9;
Fig. 2e and f). Adult Platygaster lacked a horn or
scutellar spine, but basal tergites were embossed with
prominent longitudinal grooves. A male Synopeas or
Platygaster had an abdomen shorter than the female,
but his antennae were longer and more filiform
(Fig. 4b, d, f, and g). In Inostemma, males lacked horns
and were consistently larger than females.
Mismatching descriptions of our specimens with
those of males and females of previously described

species of Aprostocetus indicated that our eulophid
wasp was newly discovered. Oviposition by female
Aprostocetus principally occurred in prepupal (third
instar) midges (Fig. 3g) and occasionally in younger
host larvae. Ninety-four percent of Aprostocetus
clutches were small with one or two eggs [ (Fig. 3a, g,
and h); mean 1.3 + 0.6, n = 129) ]. Eggs were uniformly
dispersed among hosts and typically inserted in the
lumen of the host midgut (goodness-of-fit test: X =
90.6, df = 6, P < 0.005; Fig. 3g). Larval Aprostocetus
were hymenopteriform, hyaline, and sometimes had a
dark dorsal spot (Fig. 3b). Earlier instars bore 13 cau-

-- -. ,'. ----



J ,







'. :

:i :



3a Ks

3b -






3i 3j 3k

'I 000pm

,,.~U .

Fig. 3. Eggs, larvae, and pupae of Aprostocetus sp. found in D. oxycoccana and P. vaccinii hosts. (a) Detail of egg. (b)
Neonate first instar. (c) First instar. (d) Partially engorged first instar. (e) Second instar. (f) Third instar. (g) One egg and
three first instars in a host larva. (h) Three eggs and one second instar. (i) Three partially engorged second instars. (j) Third
instar. (k) Pupa. Bottom bar represent size scale in micrometers for g-k.

dal segments and a bulbous head laterally divided by
an incomplete line or suture (Fig. 3b d). Older and
engorged instars lacked any obvious surface segmen-
tation (Fig. 3e and f). Only one first instar (Fig. 3b d)
per host survived to undergo two more molts (Fig. 3e,
f, i, and j) before pupating (Fig. 3k). Adult Aprosto-
cetus (Fig. 4h) were -1.0 mm in length with a shiny
black head with hints of green iridescence. A finely
reticulated scutum was subdivided by a median line
and arrayed laterally with three pairs of long bristles.
The scutellum was fully subdivided by two parallel
submedian grooves and flanked by two rows of stout
bristles. The basal area of the antennal scape in the
male (Fig. 4i) was marked by a prominent circular
carina. The annual parasitism rate of Aprostocetus was
6% and stretched from late March to November. Half
of the hosts that received Aprostocetus eggs already
contained platygastrid larvae.
Mitochondrial DNA Analysis of Adult Parasitoids.
Sequence variation in the COI gene depicted as a
parsimonious tree (Fig. 5) supported the generic
and subgeneric categories to which we identified our
adult midge parasitoids (Fig. 4a-i). No insertions or
deletions were present in the sequence alignment
except for a six base deletion in Aprostocetus samples,
consistent with the coding status of this COI region
(GenBank accession nos. AY843313-AY843314). Se-
quence similarity within taxonomic groups was high

and included an abundance of informative sites. Taxa-
specific characters, or diagnostic mutations, were
identified for each group, including 39 substitutions
specific to Aprostocetus, 20 specific to Inostemma, 27
specific to Platygaster, four specific to Synopeas with an
additional nine defining S. Synopeas, and three more
specific to S. Sactogaster.

Midge parasitoids, especially the Platygastrinae and
Inostemmatinae, are endemic to North America and
show a strong specificity for cecidomyiid eggs and
larvae (Ashmead 1893, MacGown 1979). Their occur-
rence in European blueberry plantings has yet to be
confirmed, but two midge hosts seem to have survived
the passage from North America to Europe most likely
as larvae on nondormant blueberry plants or as pupae
in soil (Bosio et al. 1998, Molina 2004). To date, few
accounts detail the ecology and natural histories of
proctotrupoid and chalcidoid wasps associated with
cultivated fruits in North America (Vlug 1976, Yoshida
and Hirashima 1979, Jeon et al. 1985, Son6 1986). Our
research expands the knowledge of ecological and
developmental interactions between parasitoids and
their cecidomyiid hosts on cultivated blueberries.
Five previously undescribed parasitoid wasps were
found to parasitize the eggs and neonates (Platygas-

January 2006


4a 4b

4a 4

7 4f

i,'7 .



7 = ,

Fig. 4. Adult Platygastrinae and Tetrastichinae (Eulophidae) that parasitized D. oxycoccana and P vaccinii in the
southeastern United States. (a) Synopeas (Sactogaster) female and (b) antenna of male. (c) Synopeas (Synopeas) female and
(d) antenna of male. (e) Inostemma female and (f) antenna of male. (g) Platygaster male. (h) Adult Aprostocetus female.
(i) Detail of antenna of male Aprostocetus. Bar represents size scale in micrometers.

trinae) or third instars (Tetrastichinae) of D. oxy-
coccana and P. vaccinii. Aprostocetus (Tetrastichinae)
is the first parasitoid to attack D. oxycoccana infesting
blueberry flower buds and seems to be more widely
distributed than any of the other blueberry parasitoids
in North America (Crawford 1907, Dean 1911, Girault
1917, Myers 1930, Breland 1939, Burks 1943, Barnes
1948, LaSalle 1994, Sampson et al. 2002). At the end of
bloom and as leaf buds break, Platygastrinae, espe-
cially Synopeas, begin to actively parasitize midge eggs
and larvae. Unfortunately, the biology and host affil-
iations of the few described species of related Platy-
gastrinae and Synopeadines in North America remain
obscure or unknown (Ashmead 1893, Kieffer 1926,
Vlug 1995). However, the clustering of both sexes into
each of two subgroups or clades based on 12 different
molecular characters and two morphological traits
(scutellar spine and abdominal pouch) show two dis-
tinct lineages of Synopeas attacking southern popula-
tions of midges. Both Synopeas species were active
during the day and from May to November females
sought newly hatched larvae deep inside unfurling
leaf buds. Eggs were inserted in the host larva's mid-
gut, and brood developed as solitary koinobionts
(Marchal 1906, Vlug 1976, MacGown 1979, Yoshida
and Hirashima 1979). Other solitary koinobionts,
such as Platygaster, initiated larval development in the
host stomach and hemocoel, whereas embryogenesis
and early larval development for Inostemma occurred
inside cyst-like outgrowths of the host's central ner-
vous system (Marchal 1906, Jeon et al. 1985). Mor-

phological differences among first instars and some
divergence in the mitochondrial sequences of adult
Platygaster indicate that two species probably para-
sitize midges, one common (near P. striaticollis) and
the other rare (B.J.S., unpublished data).
Thirty to 40% of larval midges were paralyzed or
killed in the blueberry nurseries; rates considered
very high in cecidomyiid populations (Gagn6 1989).
Aprostocetus was the only midge parasitoid active for
most of the bloom period, but without parasitism from
other wasp species, this wasp may have produced too
few broods to adequately control D. oxycoccana. If
properly applied, prebloom applications of registered
insecticides, including spinosad, malathion, and dia-
zinon, can reduce 50 95% of D. oxycoccana larvae
(Sampson et al. 2003; B.J.S and O.E.L., unpublished
data), and any midge survivors and their descendants
can be later eliminated by Aprostocetus and other
parasitoids (Grover 1986, Sampson et al. 2002).
Knowing exactly when to apply an insecticide for
midge control without harming parasitoids can be
difficult for scientists and impossible for most berry
producers. Visual crop scouting is limited by the small
size of midges and their parasitoids. The presence of
parasitoids and parasitism rate can be obtained from
field sampling, rearing and lengthy host preparation,
as was done in this study. However, we could more
quickly identify parasitoids and derive parasitism rates
from the percentage of eggs and larval hosts that test
positive for the unique DNA signatures of their para-
sitoids. We have partially developed a high-fidelity

Vol. 99, no. 1



Aprostocetus, male
- Aprostocefus, female

Inostemma, male

Inostemma, female
inostemma, male

Inostemma, male

Inosiemma, male

Platygaster, male


Platygaster, female
- Synopeas (Sactogaster), male

Synopeas (Sactogaster), female
Synopeas (Sactogaster), female

S Synopeas (Sactogaster), female

Synopeas (Sactogaster), female

- Synopeas (Synopeas), female
97 Synopeas (Synopeas), female
97% Synopeas (Synopeas), male

Fig. 5. First step of our high-fidelity PCR assay was to establish consistency in our parasitoid clades based on the most
parsimonious tree generated from the alignment of 364 bases of the COI gene. Generic and subgeneric names correspond
to 19 adult wasps shown in Fig. 4a and i, and Aprostocetus served as an outgroup. Bootstrap values >50% are shown above
branches, and percentage of genetic similarity is shown below branches.

PCR assay for rapid parasitoid detection by success-
fully extracting and sequencing picogram quantities of
adult mitochondrial DNA in the COI region. The next
step is to develop taxa-specific or species-specific PCR
primers from these COI sequences, which will give us
the means to amplify and separate brood DNA from
midge DNA, similar to work by Persad et al. (2004)
and Weatherbee et al. (2004). Our DNA sequences
also will aid the systematic study of these midge para-


We are grateful to Yonmee Han and Ray Langlois for
laboratory and field assistance. Special thanks to Lavonne
Stringer for German-English translation and for helpful ad-
vice from David Furth (Smithsonian), Michael Gates and
Edward Grissell (USDA-ARS-SEL), and Lubomir Masner
(Canadian National Collection). We also thank Wei Qiang
Yang and Paul Lyrene for consultation. This research was
partly funded by a USDA Pest Management Alternatives
Grant (PMAP) No.731497113.

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Received 21 April 2005, accepted 29 August 2005.

Vol. 99, no. 1

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