BIOLOGY Biology of () Attacking Dasineura oxycoccana and Prodiplosis vaccinii (Diptera: ) in Cultivated Blueberries

BLAIR J. SAMPSON,1 TIMOTHY A. RINEHART,1 OSCAR E. LIBURD,2 STEPHEN J. STRINGER,1 1 AND JAMES M. SPIERS

Ann. Entomol. Soc. Am. 99(1): 113Ð120 (2006) ABSTRACT The blueberry , Dasineura oxycoccana (Johnson), and blueberry tip midge, Prodiplosis vaccinii (Felt) (Diptera: Cecidomyiidae), are recurring cecidomyiid pests of cultivated blueberries in the southern United States and Mediterranean Europe. Insecticides can give short-term control, but overlap in phenologies indicates the potential for natural control of midge populations. Using a combination of laboratory rearing and mitochondrial DNA analysis of Þeld samples, we identiÞed Þve species of solitary endoparasitoids that killed 30Ð40% of . These species include at least three undescribed platygastrids in the genera , , and . An undescribed prepupal idiobiont, sp. (: Tetrastichinae) was the only midge parasitoid that was consistently active when rabbiteye blueberries, Vaccinium ashei Reade, were in ßower. Six percent of midge prepupae, half of which already contained platygastrid larvae, were parasitized by Aprostocetus.

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

BLUEBERRY GALL MIDGE, Dasineura oxycoccana (John- However, injury to blueberry leaf buds and meristems son), and blueberry tip midge, Prodiplosis vaccinii by P. vaccinii often induces excessive suckering, leaf (Felt), are two species of univoltine gall midges distortions, and leaf drop, which could result in lighter (Diptera: Cecidomyiidae) that feed exclusively on bud sets (Gagne´ 1986, Williamson and Miller 2000). Vaccinium buds. Midges are not easily detected be- Little is known about the interactions between cause their feeding damage only becomes visible 7Ð midges and their parasitoids on fruit crops. Proc- 14 d after larvae have left the blueberry bush. Fur- totrupoid such as those belonging to the family thermore, bud injury is often mistaken for freeze dam- parasitize D. oxycoccana on Wisconsin age, plant disease, or nutrient deÞciency (Driggers cranberries, Vaccinium macrocarpon L. (Barnes 1948). 1926, Lyrene and Payne 1992, Bosio et al. 1998, Samp- In the southern United States (Florida, Georgia, Ala- son et al. 2002, Wei 2004). D. oxycoccana is the Þrst of bama, Mississippi, and Louisiana), endoparasitic these species to break winter dormancy in January or Platygastridae seem to replace ceraphronids as the key February in the southeastern United States. Females natural enemies of midges (Vlug 1976, Yoshida and principally oviposit between the developing scales of Hirashima 1979, Jeon et al. 1985, Sone´ 1986, Sampson ßower buds. Newly hatched larvae crawl deeper into et al. 2002, Sarzynski and Liburd 2003, Legner 2004). blueberry buds where they feed on the innermost Chalcids, like tetrastichine eulophids, are also midge meristematic tissue and can reduce up to 80% of ßower production and potential fruit yield (Lyrene and parasitoids found in both midwestern and southern Payne 1992, Sampson et al. 2002). As damaged ßoral habitats, where they attack midges on cranberries and buds disintegrate, D. oxycoccana move to leaf buds, blueberries, respectively (Sampson et al. 2002). but they are soon superseded by P. vaccinii (Driggers Here, we studied the ecology of parasitoids found in 1926; Gagne´ 1986, 1989; B.J.S., unpublished data). rabbiteye, Vaccinium ashei Reade, and southern high- Crop losses to P. vaccinii have yet to be assessed. bush blueberry (Vaccinium corymbosum ϫ Vaccinium darrowii Camp) ecosystems. Larval development, Mention of trade names or commercial products in this article is adult reproductive behavior and parasitoidÐhost phe- solely for the purpose of providing speciÞc information and does not nology was investigated during 2003Ð2004. Mitochon- imply recommendation or endorsement by the U.S. Department of drial DNA analysis was used to conÞrm the identity of Agriculture. adult parasitoids as well as to assist in interpreting 1 USDAÐARS Small Fruit Research Station, Poplarville, MS 39470. 2 Department of Entomology and Nematology, University of Flor- interactions between parasitoids and their midge ida, Gainesville, FL 32611Ð0620. hosts. 114 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 99, no. 1

Materials and Methods imens for 24Ð48 h in Histoclear (National Diagnostics, Atlanta, GA). Wings were removed from adults, la- Host and Parasitoid Sampling. Host Eggs and Larvae. beled, and separately slide mounted in water to pro- Juvenile midges were sampled from two blueberry tect Þne hairs during the clearing process. Adults were nurseries at the USDAÐARS Small Fruit Research Sta- cleared using 10% KOH, neutralized, and stained with tion, Poplarville MS (N 30Њ 50.21Ј,W89Њ 32.65Ј). Nurs- acid fushin in lactophenol, and then slide mounted in ery 1 contained 950 2- to 6-yr-old potted bushes rep- Ϸ euparal or silicon oil. The identity of parasitoids on resenting 50 clones of rabbiteye blueberry, southern sticky traps that belonged to species capable of par- highbush blueberry, and wild blueberries, Vaccinium Ϸ asitizing immature midges was conÞrmed by compar- elliottii Chapman. Nursery 2 housed 11,000 second- ing them with specimens captured among host eggs and third-year seedlings representing 300 clones of and neonates inside blueberry buds or reared from V. ashei and southern highbush blueberry. Additional larval or prepupal hosts inside the growth cham- midge hosts were collected from 10 commercial rab- bers. biteye blueberry farms in southern Louisiana, Missis- Host and Parasitoid Identification. The following sippi, and Alabama. Ϸ taxonomic keys and records were used for midge and A single sample consisted of 10 terminals bearing parasitoid identiÞcation: Cecidomyiidae (Diptera): 30 ßower or leaf buds enclosed in a resealable plastic Barnes (1948) and Gagne´ (1986, 1989). Platygastridae bag. Five or 10 samples were taken from random (Hymenoptera): Ashmead (1887, 1893), Marchal bushes in each nursery every 3 to 4 d over a 2-yr (1906), Fouts (1924), Kieffer (1926), Masner and period. Smaller samples were collected when bud Muesbeck (1968), Krombien et al. (1979), Velikan et availability waned in the winter. Bags were held for 2 al. (1984), Vlug (1984, 1995), Masner and Huggert to3dtoallow larvae to abandon buds. The bags were (1989), and Buhl (1998, 2001). Eulophidae (Hyme- then partly Þlled with water and shaken to ßush out noptera): Crawford (1907), Girault (1917), Myers remaining host eggs and larvae as well as larvae that (1930), Walter (1941), Burks (1943), Piore and Vig- were diseased, heavily parasitized, or dead. Eggs and giani (1965), Boucˇek (1986), Krombien et al. (1979), larvae were pipetted into clean petri dishes, counted, Velikan et al. (1984), LaSalle (1994), and Shauff et al. and preserved in 100% ethanol. The number of host (1997). Taxonomic experts afÞliated with the USDAÐ larval stages (instars) was established from a fre- ARS Systematic Entomology Laboratory Communi- quency histogram generated by PROC UNIVARIATE cations and Taxonomic Services Unit (Beltsville, MD) (SAS Institute 1990) by using the body width mea- conÞrmed our identiÞcations. Vouchers were depos- surements of 474 midge larvae. Mature midge larvae ited in the U.S. National Collection by Raymond J. (third instars or prepupae) were identiÞable by a dark Gagne´ (Cecidomyiidae), Michael E. Shauff (Eulophi- Y-shaped ventral sclerite called a spatula (Gagne´ dae), Michael W. Gates (Eulophidae and Platygastri- 1989). dae), and Terry Nuhn (Platygastridae). Juvenile Parasitoids. Preparing a parasitoid brood Ecological Data: Analysis of Host Parasitism and for slide mounting sometimes required varying a host Parasitoid Reproductive Behavior. We calculated the larvaÕs exposure to the clearing, staining, and Þxing average parasitism rate expected for each month of a agents. Normally, host fat tissues were adequately sol- single year (Fig. 1). Parasitism rates were expressed as ubilized with exposure to solutions of 5 and 10% KOH the percentage of host eggs and larvae with parasitoid for 24 h and 30 min, respectively. Two or three drops eggs, larvae, or pupae (Figs. 2 and 3). The chi-square of diluted double stain (BioQuip Products, Inc., Ran- goodness-of-Þt analysis (PROC FREQ) was used to cho Dominguez, CA) or acid fuschin in lactophenol test whether parasitoid eggs, neonates, and older Þrst was sufÞcient to clear, stain, and temporarily Þx spec- instars were randomly or uniformly distributed among imens. Smearing host larvae by applying mild pressure host larvae (SAS Institute 1990). to the coverslip entirely expulsed many parasitoids Foraging activity of adult parasitoids throughout the from the host or more clearly exposed them. Live day was evaluated using hourly sticky trap (16 by parasitized hosts, compressed by a coverslip, made it 23-cm) catches. The resulting data were Þtted to a easier to identify which host organs nourished the Gompertz function [y ϭ 163 eϪ49eˆ(Ϫ0.26x)], where x is parasitoid brood (e.g., midgut, brain, or ganglia). Us- the cumulative hours that elapsed starting at 0800 ing an ocular micrometer, we measured the cephalo- hours, and y is hourly cumulative catch expressed as thorax and caudal segments of juvenile parasitoids that a percentage of the total daily catch (PROC NLIN, were Þxed on glass slides. SAS Institute 1990). The behavior of female parasi- Adult Parasitoids. Adult wasps were collected from toids in or around buds and host larvae also was noted. the plastic bags and from parasitized midge prepupae Polymerase Chain Reaction (PCR) Procedure and and pupae that were raised on moist peat in dark insect Sequencing Mitochondrial DNA from Adult Para- growth chambers at 30ЊC (I-30 BLL, Percival Scien- sitoids. Randomly chosen adult wasps of both sexes tiÞc Inc., Perry, IA). Randomly placed sticky yellow (Fig. 4aÐi) were sorted by taxon and placed into 1.5-ml traps (16 by 23 cm, Trece´, Salinas, CA) were hung in microcentrifuge tubes Þlled with 100% ethanol before the canopies of 10 blueberry bushes to collect foraging analysis. Sequencing mitochondrial DNA revealed di- parasitoids. The tip of an insect pin was used to remove agnostic mutations useful for calculating genetic sim- parasitoid adults from the traps. Residual glue that ilarity in midge endoparasitoids. A bead-beating tech- adhered to the insect was dissolved by agitating spec- nique and Ultraclean soil DNA isolation kit (MoBio, January 2006 SAMPSON ET AL.: BIOLOGY OF PARASITOIDS ATTACKING CECIDOMYIID PESTS 115

Madison, WI). Phylogenies based on parsimony were generated using PAUP*4.0 (Swofford 2000). Because our analysis was limited to adult identiÞcations, 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 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. AY843313ÐAY843331.

Results Host–Parasitoid Ecology and Identification. Syno- were the most common midge parasitoids. Adults of this were distinguished from other midge parasitoids by a posterior scutellar spine; short and pointed in S. Sactogaster (Fig. 4a, akin to AshmeadÕs 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 ßew between 1100 and 1700 hours. Females were observed to crawl deep inside blueberry buds. Female Synopeas dispersed their eggs (Fig. 2a) and young Þrst instar brood (Fig. 2i) uniformly among hosts (goodness-of-Þt test: ␹2 ϭ 258.8, df ϭ 6, P Ͻ 0.005, n ϭ 355 parasitized hosts) and positioned eggs Fig. 1. Seasonal changes in host density (Ⅺ) and the near the hostÕs midgut (Fig. 2j and k; Ganin 1869, percentage of parasitism of midge larvae by Platygastridae, Marchal 1906). Nine of 10 broods contained only one largely Synopeas and Platygaster (F) and Eulophidae, exclu- or two offspring (mean, 1.5 Ϯ 1.0 per host, n ϭ 354 sively Aprostocetus (E) sampled from cultivated southern parasitized hosts). When broods were larger, offspring blueberry. Phenology of blueberry bud development is de- were typically staggered in the posterior half of the picted as horizontal boxes. SEM is standard error of the host (Fig. 2k). means represented by vertical bars. The lower Þgure sum- First instars of Synopeas were cyclopiform (Fig. marizes monthly sampling effort. Numbers inside the bars are 2iÐl), each with a light yellow-brown cephalothorax. the sample sizes for monthly percentage of parasitism. Head capsules bore broad sickle-shaped mandibles as well as maxillae and ligulae spiked with short rasping Solana Beach, CA) extracted total DNA from adult teeth. Newly hatched larvae measured 210 Ϯ 50 ␮m parasitoids from each taxa (see Fig. 5 for taxon des- (n ϭ 38) wide at the head and 390 Ϯ 120 ␮m between ignations). The degenerate primer pairs C1-J-1763 apices of the head and furca (n ϭ 17; Fig. 2i). Instars (TATAGCATTCCCACGAATAAATAA) with C1-N- II (Fig. 2n) and III (Fig. 2o) were hymenopteriform 2096D (GANGTATTWARRTTTCGRTCWGTTA) and with reduced mandibles, and before pupating (Fig. 2p C1-J-2090 (AGTTTTAGCAGGAGCAATTACTAT) and q) they grew Þve-fold. Instar II: (L) 550 Ϯ 110 ␮m, with C1-N-2395D (TTAATWCCWGTWGGNACNG- (W): 280 Ϯ 50 ␮m, n ϭ 13). Instar III: (L): 1000 Ϯ 120 CAATRATTAT) ampliÞed part of the mitochondrial ␮m, (W): 440 Ϯ 80 ␮m, n ϭ 10). cytochrome oxidase I (COI) gene (Simon et al. 1994). Female Inostemma sp. (Fig. 4e) likely oviposited in Reactions consisted of 4 ␮l of MasterMix TaqDNA P. vaccinii eggs. Embryonic development occurred in polymerase (Brinkmann-Eppendorf, Westbury, NY) the brain and Þrst Þve ganglia of third instar hosts and 5 ng of total insect DNA in 6.4 ␮l of water. Cycling (Fig. 2t). Early Þrst instars of Inostemma (Fig. 2g and parameters were 95ЊC for 2 min followed by 43 cycles h) were recognized by Þne serrations along the inner of 95ЊC for 1 min, 56ЊC for 2 min, 72ЊC for 3 min, and margins of furcae (Marchal 1906, Jeon et al. 1985). then 72ЊC for 10 min with storage at 4ЊC. AmpliÞed Adults (Fig. 4e) possessed a prominent marginal vein PCR products were puriÞed using a QIAquick PCR kit in the forewing and a long arching horn that sheathed (QIAGEN, Valencia, CA), and the resulting DNA the ovipositor. Parasitism by Platygaster (Fig. 4g) was strands were sequenced using BigDye version 3.1 (Ap- indicated by the presence of oval eggs (Fig. 2r), man- plied Biosystems, Foster City, CA). dibulate-type Þrst instars with 12 abdominal and tho- Partial sequences of the COI gene consisting of 364 racic segments (Fig. 2bÐd) and a smaller cyclopiform bp were edited using Sequencher (Gene Codes, Ann larva (Fig. 2e and f). Clutches of one or two progeny Arbor, MI) and aligned with MegAlign (DNAstar, typify Platygaster broods, but as many as seven were 116 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 99, no. 1

Fig. 2. Eggs, larvae, and pupae of found in D. oxycoccana and P. vaccinii hosts. (aÐi) Egg and Þrst instars. (a) Newly inserted Synopeas egg. (b) Early Þrst instar of Platygaster sp.1. (c) Same partially engorged. (d) Same fully engorged. (e) First instar of Platygaster sp. 2. (f) Same fully engorged. (g) Inostemma Þrst instar. (h) Same partially engorged. (i) Early Þrst instar of Synopeas sp. Top scale bar represents larva size in micrometers. (jÐq) Development of Synopeas brood inside host larvae. (j) Fully developed embryos. (k) Superparasitism produces nine Þrst instar progeny. (l) Early and late (engorged) Þrst instars. (m) Early Þrst instar and second instar. (n) Second instar of Synopeas. (o) Third instar. (p) Early . (q) Late pupa. Scale is provided by brood shown inside their hosts as well as a lower bar for jÐm and oÐq. (rÐu) Immature stages of other platygastrids, scale not shown, but can be inferred from Þgures. (r) Platygaster eggs. (s) superparasitism in Platygater. (t) Early and late (engorged) Þrst instars of Inostemma. (u) Early Synopeas Þrst instar still surrounded by its trophamnion. possible per host (Fig. 2s). First instars had a mem- species of Aprostocetus indicated that our eulophid branous cephalothorax that was 120 Ϯ 30 ␮m(n ϭ 15) wasp was newly discovered. Oviposition by female in width and a body 220 Ϯ 30 ␮m in length (n ϭ 9; Aprostocetus principally occurred in prepupal (third Fig. 2e and f). Adult Platygaster lacked a horn or instar) midges (Fig. 3g) and occasionally in younger scutellar spine, but basal tergites were embossed with host larvae. Ninety-four percent of Aprostocetus prominent longitudinal grooves. A male Synopeas or clutches were small with one or two eggs [(Fig. 3a, g, Platygaster had an abdomen shorter than the female, and h); mean 1.3 Ϯ 0.6, n ϭ 129)]. Eggs were uniformly but his antennae were longer and more Þliform dispersed among hosts and typically inserted in the (Fig. 4b, d, f, and g). In Inostemma, males lacked horns lumen of the host midgut (goodness-of-Þt test: ␹2 ϭ and were consistently larger than females. 90.6, df ϭ 6, P Ͻ 0.005; Fig. 3g). Larval Aprostocetus Mismatching descriptions of our specimens with were hymenopteriform, hyaline, and sometimes had a those of males and females of previously described dark dorsal spot (Fig. 3b). Earlier instars bore 13 cau- January 2006 SAMPSON ET AL.: BIOLOGY OF PARASITOIDS ATTACKING CECIDOMYIID PESTS 117

Fig. 3. Eggs, larvae, and pupae of Aprostocetus sp. found in D. oxycoccana and P. vaccinii hosts. (a) Detail of egg. (b) Neonate Þrst instar. (c) First instar. (d) Partially engorged Þrst instar. (e) Second instar. (f) Third instar. (g) One egg and three Þrst 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 gÐk. dal segments and a bulbous head laterally divided by and included an abundance of informative sites. Taxa- an incomplete line or suture (Fig. 3bÐd). Older and speciÞc characters, or diagnostic mutations, were engorged instars lacked any obvious surface segmen- identiÞed for each group, including 39 substitutions tation (Fig. 3e and f). Only one Þrst instar (Fig. 3bÐd) speciÞc to Aprostocetus, 20 speciÞc to Inostemma, 27 per host survived to undergo two more molts (Fig. 3e, speciÞc to Platygaster, four speciÞc to Synopeas with an f, i, and j) before pupating (Fig. 3k). Adult Aprosto- additional nine deÞning S. Synopeas, and three more cetus (Fig. 4h) were Ϸ1.0 mm in length with a shiny speciÞc to S. Sactogaster. black head with hints of green iridescence. A Þnely reticulated scutum was subdivided by a median line Discussion and arrayed laterally with three pairs of long bristles. The scutellum was fully subdivided by two parallel Midge parasitoids, especially the Platygastrinae and submedian grooves and ßanked by two rows of stout Inostemmatinae, are endemic to North America and bristles. The basal area of the antennal scape in the show a strong speciÞcity for cecidomyiid eggs and male (Fig. 4i) was marked by a prominent circular larvae (Ashmead 1893, MacGown 1979). Their occur- carina. The annual parasitism rate of Aprostocetus was rence in European blueberry plantings has yet to be 6% and stretched from late March to November. Half conÞrmed, but two midge hosts seem to have survived of the hosts that received Aprostocetus eggs already the passage from North America to Europe most likely contained platygastrid larvae. as larvae on nondormant blueberry plants or as pupae Mitochondrial DNA Analysis of Adult Parasitoids. in soil (Bosio et al. 1998, Molina 2004). To date, few Sequence variation in the COI gene depicted as a accounts detail the ecology and natural histories of parsimonious tree (Fig. 5) supported the generic proctotrupoid and chalcidoid wasps associated with and subgeneric categories to which we identiÞed our cultivated fruits in North America (Vlug 1976, Yoshida adult midge parasitoids (Fig. 4aÐi). No insertions or and Hirashima 1979, Jeon et al. 1985, Sone´ 1986). Our deletions were present in the sequence alignment research expands the knowledge of ecological and except for a six base deletion in Aprostocetus samples, developmental interactions between parasitoids and consistent with the coding status of this COI region their cecidomyiid hosts on cultivated blueberries. (GenBank accession nos. AY843313ÐAY843314). Se- Five previously undescribed parasitoid wasps were quence similarity within taxonomic groups was high found to parasitize the eggs and neonates (Platygas- 118 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 99, no. 1

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) 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- phological differences among Þrst instars and some coccana and P. vaccinii. Aprostocetus (Tetrastichinae) divergence in the mitochondrial sequences of adult is the Þrst parasitoid to attack D. oxycoccana infesting Platygaster indicate that two species probably para- blueberry ßower buds and seems to be more widely sitize midges, one common (near P. striaticollis) and distributed than any of the other blueberry parasitoids the other rare (B.J.S., unpublished data). in North America (Crawford 1907, Dean 1911, Girault Thirty to 40% of larval midges were paralyzed or 1917, Myers 1930, Breland 1939, Burks 1943, Barnes killed in the blueberry nurseries; rates considered 1948, LaSalle 1994, Sampson et al. 2002). At the end of very high in cecidomyiid populations (Gagne´ 1989). bloom and as leaf buds break, Platygastrinae, espe- Aprostocetus was the only midge parasitoid active for cially Synopeas, begin to actively parasitize midge eggs most of the bloom period, but without parasitism from and larvae. Unfortunately, the biology and host afÞl- other wasp species, this wasp may have produced too iations of the few described species of related Platy- few broods to adequately control D. oxycoccana. If gastrinae and Synopeadines in North America remain properly applied, prebloom applications of registered obscure or unknown (Ashmead 1893, Kieffer 1926, insecticides, including spinosad, malathion, and dia- Vlug 1995). However, the clustering of both sexes into zinon, can reduce 50Ð95% of D. oxycoccana larvae each of two subgroups or clades based on 12 different (Sampson et al. 2003; B.J.S and O.E.L., unpublished molecular characters and two morphological traits data), and any midge survivors and their descendants (scutellar spine and abdominal pouch) show two dis- can be later eliminated by Aprostocetus and other tinct lineages of Synopeas attacking southern popula- parasitoids (Grover 1986, Sampson et al. 2002). tions of midges. Both Synopeas species were active Knowing exactly when to apply an insecticide for during the day and from May to November females midge control without harming parasitoids can be sought newly hatched larvae deep inside unfurling difÞcult for scientists and impossible for most berry leaf buds. Eggs were inserted in the host larvaÕs mid- producers. Visual crop scouting is limited by the small gut, and brood developed as solitary koinobionts size of midges and their parasitoids. The presence of (Marchal 1906, Vlug 1976, MacGown 1979, Yoshida parasitoids and parasitism rate can be obtained from and Hirashima 1979). Other solitary koinobionts, Þeld sampling, rearing and lengthy host preparation, such as Platygaster, initiated larval development in the as was done in this study. However, we could more host stomach and hemocoel, whereas embryogenesis quickly identify parasitoids and derive parasitism rates and early larval development for Inostemma occurred from the percentage of eggs and larval hosts that test inside cyst-like outgrowths of the hostÕs central ner- positive for the unique DNA signatures of their para- vous system (Marchal 1906, Jeon et al. 1985). Mor- sitoids. We have partially developed a high-Þdelity January 2006 SAMPSON ET AL.: BIOLOGY OF PARASITOIDS ATTACKING CECIDOMYIID PESTS 119

Fig. 5. First step of our high-Þdelity 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 Ͼ50% are shown above branches, and percentage of genetic similarity is shown below branches.

PCR assay for rapid parasitoid detection by success- Barnes, H. F. 1948. Gall midges of economic importance, fully extracting and sequencing picogram quantities of vol. 3, pp. 71Ð80. In Gall midges of fruit. Crosby Lock- adult mitochondrial DNA in the COI region. The next wood & Son Ltd., London, United Kingdom. step is to develop taxa-speciÞc or species-speciÞc PCR Bosio, G. C. Bogetti, G. Brussino, F. Gremo, and F. Scarpelli. primers from these COI sequences, which will give us 1998. Dasineura oxycoccana, a new pest of highbush blue- the means to amplify and separate brood DNA from berry in Italy. Informatore Fitopatologico 11: 36Ð41. midge DNA, similar to work by Persad et al. (2004) Boucˇek, Z. 1986. Taxonomic study of chalcidoid wasps (Hymenoptera) associated with gall midges (Diptera: and Weatherbee et al. (2004). Our DNA sequences Cecidomyiidae) on mango trees. Bull. Entomol. Res. 76: also will aid the systematic study of these midge para- 393Ð407. sitoids. Breland, O. P. 1939. Additional notes on sunßower . Ann. Entomol. Soc. Am. 32: 719Ð726. Acknowledgments Buhl, P. N. 1998. On some new or little known NW Euro- pean species of Platygastridae (Hymenoptera, Proc- We are grateful to Yonmee Han and Ray Langlois for totrupoidea). Fragmenta Entomol. 30: 295Ð334. laboratory and Þeld assistance. Special thanks to Lavonne Buhl, P. N. 2001. Eleven new species of Platygastrinae (Hy- Stringer for German-English translation and for helpful ad- menoptera: Platygastridae). Folia-Entomol.-Hung. 62: vice from David Furth (Smithsonian), Michael Gates and 133Ð149. Edward Grissell (USDAÐARSÐSEL), and Lubomir Masner Burks, B. D. 1943. The North American parasitic wasps of (Canadian National Collection). We also thank Wei Qiang the genus , a contribution to biological con- Yang and Paul Lyrene for consultation. This research was trol of insect pests. Proc. U.S. Natl. Mus. 93: 505Ð608. partly funded by a USDA Pest Management Alternatives Crawford, J. C. 1907. New North American Hymenoptera. Grant (PMAP) No.731497113. J. N.Y. Entomol. Soc. 15: 177Ð189. Dean. 1911. The sorghum midge. U.S. Dep. Agr. Bur. References Cited Entomol. Bull. 85: 55Ð57. Driggers, B. F. 1926. The blueberry tipworm (Contarinia Ashmead, W. H. 1887. Studies of the North American Proc- vaccinii Felt.), a new species of midge attacking culti- totrupidae, with descriptions of new species from Florida. vated blueberries. J. N.Y. Entomol. Soc. 35: 82Ð85. Can. Entomol. 19: 125Ð132. Fouts, R. M. 1924. Revision of the North American wasps of Ashmead, W. H. 1893. Monograph of the North American the subfamily Platygasterinae. Proc. U.S. Natl. Mus. 63: . Bull. U.S. Natl. Mus. 45: 1Ð472. 1Ð145. 120 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 99, no. 1

Gagne´, R. J. 1986. Revision of Prodiplosis (Diptera: Ce- Sampson, B. J., J. L. McLaughlin, and D. E. Wedge. 2003. cidomyiidae) with descriptions of three new species. Paw Paw extract as a botanical insecticide. Arthropod Ann. Entomol. Soc. Am. 79: 235Ð245. Manag. Tests 28: L5. Gagne´, R. J. 1989. The plant-feeding gall midges of North Sarzynski, E. M., and O. E. Liburd. 2003. Techniques for America. Cornell University Press, Ithaca, NY. monitoring cranberry tipworm (Diptera: Cecidomyi- Ganin, M. 1869. Beitra¨ge zur Erkentniss der Entwickelungs- idae) in rabbiteye and southern highbush blueberries. geschichte bei den Insecten. Z. Zool. 19: 381Ð451. J. Econ. Entomol. 96: 1821Ð1827. Girault, A. A. 1917. New chalcid ßies. Insector Inscitiae SAS Institute. 1990. SAS userÕs guide: statistics, version 5th Menstruus. 5: 29Ð37. ed. SAS Institute. Cary, NC. Grover, P. 1986. Integrated control of midge pests. Cecido- Shauff, M. E., J. LaSalle, and L. C. Coote. 1997. Chapter 10. logia-Int. 7: 1Ð28. Eulophidae. In G.A.P. Gibson, J. T. Huber, and J. B. Wool- Jeon, M-J, Lee B-Y, Ko J-H, and Y. Hirashima. 1985. Ecology ley [eds.], Annotated keys to the genera of Nearctic of Platygaster matsutama and Inostemma seoulis (Hyme- Chalcidoidea (Hymenoptera). NRC Research Press, noptera: Platygastridae), egg-larval parasites of the pine Ottawa, Ontario, Canada. Simon, C., F. Frati, A. Beckenbach, B. Crespi, H. Liu, and needle gall midge, Thecodiplosis japonensis (Diptera, P. Flook. 1994. Evolution, weighting and phylogenetic Cecidomyiidae). Esakia 23: 131Ð143. utility of mitochondrial gene sequences and a compila- Kieffer, J. J. 1926. Scelionidae. Das Tierreich 48: 1Ð885. tion of conserved polymerase chain reaction primers. Krombien, K. V., P. D. Hurd Jr., D. R. Smith and B. D. Burks. Ann. Entomol. Soc. Am. 87: 65Ð701. 1979. Catalog of Hymenoptera in American North of Sone´, K. 1986. Ecology of host-parasitoid community in the Mexico: Volume 1 Symphyta and (). pine needle gall midge, Thecodiplosis japonensis Uchida et Smithsonian Institution Press. Washington, DC. Inouye (Diptera: Cecidomyiidae). J. Appl. Entomol. 102: LaSalle, J. 1994. North American genera of Tetrastichinae 516Ð527. (Hymenoptera: Eulophidae). J. Nat. Hist. 28: 109Ð236. Swofford, D. L. 2000. PAUP*: phylogenetic analysis using Legner, E. F. 2004. Homepage. Principal groups of insect parsimony (*and other methods). Sinauer, Sunderland, parasitoids and predators (http://faculty.ucr.edu/ MA. ϳlegneref). Velikan, V. S., A. M. Gegechkori, V. B. Golub, E. M. Danzig, Lyrene, P. M., and J. A. Payne. 1992. Blueberry tipworm: a G. I. Dorokhova, A. F. Emelyanov, V. M. Ermolenko, pest on rabbiteye blueberry in Florida. Proc. Fla. St. M. D. Zerova, and V. S. Kuslitskij. 1984. Key to harmful Hortic. Soc. 105: 297Ð300. and useful insects and mites of fruit and berry cultures in MacGown, M. W. 1979. The Platygastridae (Hymenoptera: the USSR. Kolos, Leningrad, Russia. ) parasitic on midges (Cecidomyiidae) Vlug, H. J. 1976. Synopeas talhouki n.sp. (Hym. Platygastri- found on conifers in Canada and the United States. Info. dae), a parasite of Odinadiplosis amygdale (Anagnosto- Bull. 9, Mississippi Agric. For. Exp. Stn. poulos) (Dipt. Cecidomyiidae) with notes on its distri- Marchal, P. 1906. Recherches sur la biologie et le develop- bution and biology. Z. Entomol. 80: 262Ð266. ment des Hymenopteres parasites. Les Platygasters. Arch. Vlug, H. J. 1984. The types of Platygastrinae (Hymenoptera, Zool. Exp. Gen. 4, Ser. 4. 485Ð640, pl. 17Ð24. Scelionoidea) described by Haliday and Walker and pre- Masner, L., and L. Huggert. 1989. World review and keys to served in the National Museum of Ireland and in the genera of the subfamily Inostemmatinae with reassign- British Museum (Natural History). 2. Keys to species, ment of the taxa to the Platygastrinae and Sceliotrach- redescriptions, synonymy. Tijdschrift-voor-Entomol. 127: elinae (Hymenoptera: Platygastridae). Mem. Entomol. 179Ð224. Soc. Canada 147: 1Ð214. Vlug, H. J. 1995. Catalogue of the Platygastridae (Platygas- Masner, L., and C.W.F. Muesbeck. 1968. The types of Proc- troidea) of the World (Insecta: Hymenoptera). SPB Ac- totrupoidea in the United States National Museum. Bull. ademic Publishing. Amsterdam, The Netherlands. U.S. Natl. Mus. 270: 1Ð145. Walter, E. V. 1941. The biology and control of the sorghum midge. U.S. Dep. Agr. Tech. Bull. 778: 1Ð26. Molina, J. 2004. Prodiplosis vaccinii, a pest of blueberries in Weatherbee, A. A., K. A. Shufran, T. D. Panchal, P. M. Dang, Spain. In Proceedings of the 8th International Symposium and G. A. Evans. 2004. Detection and differentiation of on Vaccinium Culture, 3Ð8 May 2004, Seville, Spain. parasitoids (Hymenoptera: Aphidiidae and ) Myers, I. H. 1930. Notes on parasites of the gall-midge Jat- of the brown citrus aphid (Homoptera: Aphididae): spe- rophobia brasiliensis Rh bs on cassava in Trinidad. Bull. cies-speciÞc polymerase chain reaction ampliÞcation of Entomol. Res. 21: 309Ð313. 18S rDNA. Ann. Entomol. Soc. Am. 97: 286Ð292. Persad, A. B., A. Jeyaprakash, and M. A. Hoy. 2004. High- Wei, Y. 2004. Blueberry gall midge - a new emerging pest? Þdelity PCR assay discriminates between immature Li- Oreg. Blueberry Newsl. 4: 2Ð3. polexis oregmae and Lysiphlebus testaceipes (Hymenop- Williamson, J. G., and E. P. Miller. 2000. Early fall defoli- tera: Aphidiidae) within their aphid hosts. Fla. Entomol. ation of southern highbush blueberry inhibits ßower bud 87: 18Ð24. initiation and retards ßower bud development. Hort- Piore, R., and G. Viggiani. 1965. La Science 35: 505. Coq. (Diptera Cecidomyiidae) ed i suoi parassitti in Italia. Yoshida, N., and Y. Hirashima. 1979. Systematic studies on Lab. Entomol. Agr. Portici Bol. 23: 1Ð36. proctotrupoid and chalcidoid parasites of gall midges Sampson, B. J., S. J. Stringer, and J. M. Spiers. 2002. Inte- injurious to cryptomeria in Japan and Korea. Esekia 14: grated pest management for Dasineura oxycoccana 133Ð133. (Diptera: Cecidomyiidae) in blueberry. Environ. Ento- mol. 31: 339Ð347. Received 21 April 2005; accepted 29 August 2005.