Biological Control 39 (2006) 225–231 www.elsevier.com/locate/ybcon

Species-speciWc close-range sexual communication systems prevent cross-attraction in three species of Glyptapanteles parasitic (: )

Adela Danci a, Paul W. Schaefer b, Axel Schopf c, Gerhard Gries a,¤

a Department of Biological Sciences, Simon Fraser University, Burnaby, BC, Canada V5A 1S6 b United States Department of Agriculture, Agricultural Research Service, BeneWcial Introduction Research Laboratory, Newark, DE 19713, USA c Institute of Forest Entomology, Forest Pathology and Forest Protection, BOKU—University of Natural Resources and Applied Life Sciences, A-1190 Vienna, Austria

Received 21 February 2006; accepted 21 July 2006 Available online 2 August 2006

Abstract

The braconid Glyptapanteles indiensis (Marsh) and G. liparidis (Bouché) occur in sympatry and allopatry, respectively, with their congener G. Xavicoxis (Marsh). We tested the hypothesis that all three parasitoids, but particularly sympatric G. indiensis and G. Xavicoxis, use species-speciWc sex pheromone blends for close-range sexual communication. In coupled gas chromatographic–electro- antennographic detection (GC–EAD) analyses of body extracts of conspeciWc females, male G. indiensis antennae responded to Wve com- ponents, one of which is speciWc to G. indiensis, and four are in common with G. Xavicoxis. Male G. liparidis antennae responded to six components, two of which are speciWc to G. liparidis, and four are in common with G. Xavicoxis. In Y-tube olfactometer experiments, body extracts of females elicited close-range attraction and wing-fanning responses only by conspeciWc but not by heterospeciWc males, supporting the hypothesis of close-range species-speciWc sex pheromone blends. © 2006 Elsevier Inc. All rights reserved.

Keywords: Glyptapanteles Xavicoxis; Glyptapanteles indiensis; Glyptapanteles liparidis; ; Hymenoptera; Braconidae; ; Species- speciWcity; Pheromone; Close-range pheromonal communication

1. Introduction hymeria intermedia (Nees) and B. lasus (Walker) (Hyme- noptera: Chalcididae) exhibit courtship behavior when Sexual communication in parasitoids is mediated mainly exposed to pheromone extract of conspeciWc but not heter- by pheromones that are emitted by females and induce ospeciWc females, suggesting that they use species-speciWc searching, courtship, and mating behavior by males sex pheromones (Mohamed and Coppel, 1987). Intrigu- (Quicke, 1997). ingly, male Melittobia digitata (Dahms) (Hymenoptera: SpeciWcity of the pheromone blend might serve as a Eulophidae) emit sex pheromone that attracts conspeciWc reproductive isolating mechanism. Male sawXy parasitoids females, but also cross-attracts female M. femorata Syndipnus gaspesianus (Provancher) (Hymenoptera: Ich- (Dahms) and M. australica (Girault), suggesting that all neumonidae) are not attracted to sympatric heterospeciWc three species use similar if not identical long-range phero- female S. rubiginosus (Walley) or their pheromone (Z)-9- mones. However, following antennal contact of prospective hexadecenoate (Eller et al., 1984). Similarly, males of Brac- mates, heterospeciWcs are rejected, likely due to species-spe- ciWc contact pheromones (Cônsoli et al., 2002). Bioacoustic signals constitute alternative reproductive * Corresponding author. Fax: +1 604 291 3496. isolating mechanisms. Both Diachasmimorpha longicaudata E-mail address: [email protected] (G. Gries). (Ashmead) and D. kraussii (Fullaway) (Hymenoptera: Bra-

1049-9644/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2006.07.009 226 A. Danci et al. / Biological Control 39 (2006) 225–231 conidae) use pheromonal, visual and bioacoustic signals. (18 £ 18 £ 12 cm) contained 10–15 mated female parasit- Males are attracted to the females’ cuticular pheromone, oids, Wve L. dispar larvae (3–4 ) (Fuester et al., 1987), and respond with wing vibrational bioacoustic signals and larval diet (Bell et al., 1981). After 1–2 days, parasitized which, in turn, increase the females’ activity. The females’ host larvae were removed and placed on a diet in plastic cuticular chemicals are similar across species, but acoustic cups (192 ml) with tight-Wtting paper lids (Sweetheart Plas- signals of males appear to be species-speciWc (Rungrojwa- tics, Wilmington, Massachusetts). Every second day, larval nich and Walter, 2000). frass was removed, and the diet replenished if needed. The Sympatric Glyptapanteles Xavicoxis and G. indiensis parasitoid cocoons to be used for mass rearing were placed (Hymenoptera: Braconidae) are gregarious and solitary in plastic Petri dishes (14 cm diam.) and food-provisioned parasitoids, respectively, of larval Indian gypsy moth, as described above. Lymantria obfuscata (L.) (Lepidoptera: Lymantriidae), Cocoons of G. indiensis and G. liparidis were provided by which occurs in the northern parts of India, Pakistan and the BeneWcial Insects Introduction Research Laboratory Afghanistan. Allopatric G. liparidis is a gregarious endo- (see above), and the Institute of Forest Entomology, Forest parasitoid attacking mainly 2nd and 3rd of the Pathology and Forest Protection, BOKU—University of gypsy moth, Lymantria dispar (L.), and alternate host spe- Natural Resources and Applied Life Sciences, Vienna, Aus- cies of the lepidopteran families Lymantriidae and Lasio- tria. Parasitoid cocoons with insects to be used in bioassays campidae for further generations during the season and as were transferred individually to capped plastic cups (30 ml) hibernating hosts. It occurs in Japan, Korea, the Kurile provisioned with sugar water-soaked cotton wicks. Rearing Islands, Russia, North Africa, and Europe (Marsh, 1979). took place under a 16L:8D photoregime at 22–25 °C and Sexual communication in G. Xavicoxis is mediated, in 50–70% RH. part, by a four-component close-range pheromone (Danci et al., 2006). Females press their abdominal tip to the 2.2. Acquisition of pheromone extracts substrate depositing pheromone in the process. These “deposits,” or body extract of females, provoke substrate- Groups of 1- to 3-day-old female G. Xavicoxis, G. indien- antennation, wing-fanning and close-range anemotactic sis, and G. liparidis were macerated in three separate hex- attraction responses by conspeciWc males. Pheromonal ane-containing vials (ca. 10 l per female) placed on dry ice. communications in G. liparidis and G. indiensis may be After extracts were kept at room temperature for »15 min, similarly complex but have not yet been investigated. the supernatant was withdrawn, Wltered through glass wool Congeners in the Lepidoptera often share pheromone in a pipette, and quantiWed to determine the volume repre- components. Allopatric congeners may use the very same senting one female body extract equivalent (FBE). pheromone (Gries et al., 2002b), whereas sympatric con- geners typically employ one or more additional phero- 2.3. Acquisition of volatiles mone components to maintain reproductive isolation (Gries et al., 1996). Similarly, the tortricid moths Archips Body extract of female G. Xavicoxis elicited strong close- argyrospilus (Walker) and A. mortuanus (Kearfoot) share range behavioural responses by conspeciWc males, with pheromone components in species-speciWc ratios (Cardé headspace volatiles not signiWcantly contributing to the et al., 1977a). attractiveness of the stimulus (Danci et al., 2006). Nonethe- Our objective was to test the hypothesis that G. Xavic- less, to generate experimental evidence for close-range oxis, G. indiensis, and G. liparidis use species-speciWc com- pheromonal communication in congeneric G. indiensis and ponents to confer speciWcity to their close-range sexual G. liparidis, we decided to bioassay Wrst the strongest possi- communication systems. ble stimulus, a combination of body extracts and headspace volatiles. 2. Materials and methods To acquire headspace volatiles, unmated 1- to 2-day-old female G. indiensis and G. liparidis were placed into vertical 2.1. Experimental insects cylindrical Pyrex glass chambers (10 ID £ 6 cm), and were provisioned with a sugar water-soaked cotton wick. Con- Glyptapanteles Xavicoxis and its host L. dispar were trol chambers contained the same food source, but no para- reared in the Global Forest Quarantine Facility at Simon sitoids. A water aspirator drew humidiWed, charcoal-Wltered Fraser University (SFU), and populations were repeatedly air at a rate of 1.5–2 L/min for two days through the cham- augmented with specimens obtained from the BeneWcial ber and a glass column (14 £ 1.3 cm OD) Wlled with 150 mg Insects Introduction Research Laboratory, United States of Porapak Q (50–80 mesh, Waters Associates Inc., Mil- Department of Agriculture, Agricultural Research Service, ford, Massachusetts, USA). Volatiles were eluted from Newark, Delaware. To facilitate mating in G. Xavicoxis, 10 Porapak Q volatile traps with re-distilled pentane (2 ml). females and 30 males were placed in plastic mesh cages The extracts were concentrated under a stream of nitrogen (10 £ 10 £ 6cm) (Hu et al., 1986), and provisioned with cot- such that 10 l of extract contained one female hour equiv- ton wicks (1 £ 10 cm; Richmond Dental, Charlotte, North alent (FHE) of volatile acquisition ( D amount of volatiles Carolina) soaked in sugar water solution. Oviposition cages released by 1 female during 1 h). A. Danci et al. / Biological Control 39 (2006) 225–231 227

2.4. Y-tube olfactometer bioassays (J&W ScientiWc, Folsom, CA 95630). For GC-EAD record- ings, a male’s head was severed and placed into the opening The Pyrex glass Y-shaped olfactometer (stem: of a glass capillary electrode Wlled with saline solution 20 £ 2.5 cm ID; side arms at 120°: 18 cm long) was posi- (Staddon and Everton, 1980). One antenna with its tip tioned vertically 15 cm below, and with its plane aligned removed by spring microscissors (Fine Science Tools Inc., with, a light source consisting of one tube of Xuorescent North Vancouver, BC, Canada) was placed into the open- “daylight” (F40DX, H118; Osram Sylvania Ltd., Ont., Can- ing of a second (indiVerent) electrode. ada) and one tube of “wide spectrum grow light” (F40GRO/WS, H658; Osram Sylvania Ltd., Ont., Canada). 3. Results Treatment or control stimuli were pipetted in trail like fashion on white strips of paper (15 £ 1 cm) placed in side In GC–EAD analyses of female G. indiensis phero- arms of the Y-tube. A water aspirator drew air at »1l/min mone extracts, male G. indiensis antennae responded to through the Y-tube to test close-range anemotactic Wve components, one of which is speciWc to G. indiensis response, wing-fanning and substrate antennation of para- (Gi-spec), and four are shared with G. Xavicoxis (Fig. 1; sitoids released individually into the stem of the Y-tube. An Table 1). Similarly, in GC–EAD analyses of female G. was considered a responder when it within 10 min traversed the entire paper strip up to the oriWce of the side arm; all others were classiWed as non-responders. We tested 3 20 insects per experiment, and for each insect, a clean (Sparkleen-washed and oven-dried) Y-tube, and a new EAD: Gf antenna to paper strip were used, with test stimuli randomly assigned Gf body extract 4 1 2 to side arms. ~1 mV To demonstrate the presence of pheromonal communi- cation in G. indiensis and G. liparidis, experiments 1 and 2 tested behavioural responses by male G. indiensis and G. liparidis to eZuvia plus body extract of conspeciWc females. X In G. avicoxis, female body extract by itself or combined Gi -spec with the females’ eZuvia were equally attractive to males (Danci et al., 2006). As body extract was easier to produce, 4 experiments in this study deployed body extracts as test EAD: Gi antenna to 3 Gi body extract stimuli. Experiments 3–14 tested body extracts of female G. Xavicoxis, G. indiensis, and G. liparidis for their potential cross-attractiveness to heterospeciWc males. Expecting con- 1 2 sistent strong attraction of males to conspeciWc female Dectector response (mV) pheromone, we tested the response of conspeciWc and heter- ospeciWc males in parallel experiments with alternating rep- licates. Thus, on any given bioassay day the males’ lack of 1 wing-fanning or close-range anemotactic attraction Gl -spec 2 2 responses to heterospeciWc female pheromone would likely Gl -spec 1 be due to the non-eVectiveness of the stimulus rather than the males’ non-responsiveness. EAD: Gl antenna to 4 Gl body extract The number of parasitoids responding to stimuli were 3 analysed with the 2 goodness-of-Wt test using Yates’ cor- rection for continuity ( D 0.05), testing the null hypothesis that insects did not prefer treatment or control stimuli (Zar, 1996). 10 11 12 13 Retention time (min) 2.5. Analyses of G. Xavicoxis, G. indiensis, and G. liparidis Fig. 1. Electroantennographic detector (EAD: conspeciWc male antenna) pheromone extracts responses to aliquots of female Glyptapanteles Xavicoxis body extract (top), female G. indiensis body extract (middle), and female G. liparidis Aliquots of 1 FBE were analyzed by a custom-built cou- body extract (bottom). Chromatography: Hewlett–Packard 5890A gas pled gas chromatographic-electroantennographic detector chromatograph equipped with a DB-23 coated GC column X (GC–EAD) system (Arn et al., 1975; Gries et al., 2002a), (30 m £ 0.25 mm ID); linear ow velocity of carrier gas: 35 ml/min injector and FID detector temperature: 220 °C; temperature program: 1 min at employing a Hewlett–Packard (HP) 5890 A gas chromato- 100 °C, 10 °C/min to 220 °C. Note: corresponding Xame ionization detec- graph equipped with a GC column (30 m £ 0.25 or 0.32 mm tor (FID) traces of the gas chromatograph are omitted because all anten- ID) coated with DB-5, DB-17, DB-210, DB-23 or FFAP nal-stimulatory compounds occurred below FID detection threshold. 228 A. Danci et al. / Biological Control 39 (2006) 225–231

Table 1 In Y-tube olfactometer experiments, female G. indiensis Retention indices (relative to alkane standards) (Van den Dool and Kratz, body extract at 1 FBE in combination with eZuvium (1 FHE) W 1963) of candidate pheromone components speci c to female Glyptapan- elicited signiWcant close-range attraction and wing-fanning teles indiensis (Gi-spec) and G. liparidis (Gl-spec1 and Gl-spec2) (Fig. 1) responses by conspeciWc males (Fig. 3, Experiment 1). Simi- GC column Retention indices of larly, female G. liparidis body extract (1 FBE) plus eZuvium Gi-spec Gl-spec1 Gl-spec2 (1 FHE) elicited signiWcant close-range attraction and wing- DB-5 2038 2019 2041 fanning responses by conspeciWc males (Fig. 3, Experiment 2). DB-17 2306 2289 2308 In Experiments 3–14 (Fig. 4), which were designed to test DB-210 2378 2318 2338 potential pheromonal cross-attraction among species, body DB-23 2547 2519 2536 X FFAP 2508 2469 2495 extract of female G. avicoxis elicited both close-range attrac- tion and wing-fanning responses by conspeciWc males (Exper- iments 3 and 5), but not by heterospeciWc male G. indiensis liparidis pheromone extract, male G. liparidis antennae (Experiment 4) or G. liparidis (Experiment 6). Furthermore, responded to six components, two of which (Gl-spec1 and body extract of female G. indiensis elicited close-range attrac- Gl-spec2) are speciWc to G. liparidis, and four are in com- tion and wing-fanning responses by conspeciWc males (Exper- mon with G. Xavicoxis (Fig. 1; Table 1). The four common iments 7 and 9), but not by heterospeciWc male G. Xavicoxis components were detected by males of all three species in (Experiment 8) or G. liparidis (Experiment 10). Finally, body body extracts of all three heterospeciWc females, when extract of female G. liparidis elicited close-range attraction extracts were analyzed on each of Wve diVerent GC col- and wing-fanning responses by conspeciWc males (Experi- umns. Results of these analyses on a DB-23 GC column ments 11 and 13), but not by heterospeciWc male G. Xavicoxis are shown in Fig. 2. (Experiment 12) or G. indiensis (Experiment 14).

3 3 3

EAD: 2 EAD: 2 EAD: 2 1 1 1 Gf antenna 4 Gf antenna 4 Gf antenna 4 to Gf body to Gf body to Gf body extract extract extract

4 1

2 1 EAD: 3 EAD: EAD: 3 Gf antenna 4 Gl antenna Gi antenna 3 4 to Gi body 2 to Gf body to Gf body extract extract extract 1 2

4

Detector response Detector response (mV) EAD: 1 ~ 2 mV Gi antenna to Gl body extract 3

EAD: 1 4 EAD: 3 2 Gf antenna 2 Gl antenna to Gl body to Gi body 1 extract extract 2 4 3

11.0 11.5 12.0 11.0 11.5 12.0 11.0 11.5 12.0 Retention time (min)

Fig. 2. Electroantennographic detector (EAD: antennae of male Glyptapanteles Xavicoxis, G. liparidis, and G. indiensis) responses to aliquots of body extracts of heterospeciWc females. The top EAD trace in each of the three columns serves as a reference and depicts male G. Xavicoxis antennal responses to pheromone components in body extracts of conspeciWc females. Chromatography: Hewlett–Packard 5890A gas chromatograph equipped with a DB-23 coated GC column (30 m £ 0.25 mm ID); linear Xow velocity of carrier gas: 35 ml/min; injector and FID detector temperature: 220 °C; temperature pro- gram: 1 min at 100 °C, 10 °C/min to 220 °C. A. Danci et al. / Biological Control 39 (2006) 225–231 229

Exp. 1 Gi effluvium (1 FHE) plus Gi body extract (1 FBE) 18 *** 5 Solvent control 2

No. of male Gi attracted No. of male Gi wing fanning

Exp. 2 Gl effluvium (1 FHE) plus Gl body extract (1 FBE) 15 * 12 Solvent control 5

0 5152010 0 51510 20 Treatments No. of male Gl attracted No. of male Gl wing fanning

Fig. 3. Number of male Glyptapanteles indiensis and G. liparidis that were attracted, or wing-fanned, in response to test stimuli tested in Y-tube olfactome- ter experiments 1 and 2. 1 FHE D one female hour equivalent D pheromone component(s) released by one female during 1 h; 1 FBE D one female body extract equivalent D pheromone component(s) contained in extract of one macerated female body. In each experiment, bars with an asterisk (¤) indicate a signiWcant response to a particular treatment; 2 test with Yates’ correction for continuity, treatment versus control; *P < 0.05; ***P <0.001.

4. Discussion Similarly, bark beetle aggregation pheromones contain components that interrupt the pheromonal response of Our data support the hypothesis that G. indiensis, G. competing species. Sympatric Ips paraconfusus (Lanier) and liparidis, and G. Xavicoxis share (candidate) pheromone Ips pini (Say), for example, infest the same host, but compo- components but use additional components to confer speci- nents of their respective pheromones inhibit cross-attrac- Wcity to their sexual communication. The four pheromone tion (Birch and Haynes, 1982). components that are present in body extracts of female G. It is also conceivable that Gi-spec in G. indiensis, and Gl- Xavicoxis and elicit antennal and behavioural responses spec1 and Gl-spec2 in G. liparidis, are non-pheromonal from conspeciWc males (Fig. 1; Danci et al., 2006), are also constituents in their respective communication systems, present in body extracts of female G. indiensis and G. lipari- serving the single role of reducing cross-attraction of het- dis (Figs. 1 and 2). However, whether all of them are phero- erospeciWcs. Such a concept has been proposed for nun mone components in G. indiensis and G. liparidis, as in G. moth, Lymantria monacha (L.), and its sympatric congener Xavicoxis, is yet to be determined. L. dispar, both using (+)-disparlure as a pheromone com- The presence of the same four components in all three ponent. Attraction of male L. dispar to (+)-disparlure is species is indicative of phylogenetic relatedness, and sup- inhibited in the presence of (¡)-disparlure (Klimetzek et al., ports taxonomic placement of the three species as conge- 1976; Cardé et al., 1977b; Plimmer et al., 1977), which is ners. Comparable blends of (candidate) pheromone likely produced as a non-pheromonal constituent by female components of sympatric G. Xavicoxis and G. indiensis were L. monacha to enhance the speciWcity of sexual communica- expected, but the very similar blend of allopatric G. liparidis tion (Hansen, 1984). Similarly, (Z)-11-hexadecen-1-yl ace- is surprising. It is suggestive that all three species might tate which does not appear to be a component of the female have evolved from a common ancestor. H. subXexa sex pheromone (Vickers, 2002), inhibits phero- The complete lack of pheromonal close-range cross- monal attraction responses of male H. zea (Fadamiro and attraction among the three Glyptapanteles species (Fig. 4) is Baker, 1997; Baker et al., 1998). likely due to the species-speciWc components in G. indiensis To assign non-pheromonal or synomonal roles to Gi- (Gi-spec) and G. liparidis (Gl-spec1 and/or Gl-spec2). spec in G. indiensis, and to Gl-spec1 or Gl-spec2 in G. Should these compounds be part of the respective phero- liparidis, will require their isolation and bioassay testing. mone blends, they would be synomones that enhance Although they occur well below GC or GC–mass spectro- attraction of conspeciWcs while simultaneously inhibiting metric detection thresholds, they can be separated from the response of heterospeciWcs. Synomonal activity of pher- other candidate pheromone components by high-perfor- omone components has been well documented in the Cole- mance liquid chromatography (HPLC), carefully moni- optera and Lepidoptera. The heliothine moths Heliothis zea toring HPLC fractions by GC–EAD (see Danci et al., (Boddie), Heliothis virescens (Fabricius), and Heliothis sub- 2006). Xexa (Guenée) share (Z)-11-hexadecenal as a common Reproductive isolating mechanisms in insects operate at pheromone component, whereas (Z)-9-hexadecenal in H. multiple levels (Birch and Haynes, 1982). At the behav- zea, (Z)-9-hexadecenal and (Z)-11-hexadecen-1-ol in H. ioural and physiological level, species-speciWc sexual com- subXexa, and (Z)-9-tetradecenal in H. virescens enhance munication systems contribute to prezygotic reproductive attractiveness or species-speciWcity of the respective phero- isolation of G. Xavicoxis, G. indiensis, and G. liparidis, irre- mone blends (Vetter and Baker, 1983, 1984; Vickers, 2002). spective of their allopatric or sympatric occurrence. 230 A. Danci et al. / Biological Control 39 (2006) 225–231

Attraction Wing fanning Exp. 3 Gf body extract (1 FBE) 13 11 Solvent control 7

Exp. 4 Gf body extract (1 FBE) 7 No wing fanning Solvent control 12

No. of male Gf (Exp. 3) or Gi (Exp. 4) responding

Exp. 5 Gf body extract (1 FBE) 17 ** 10 Solvent control 3

Exp. 6 Gf body extract (1 FBE) 10 No wing fanning Solvent control 10

No. of male Gf (Exp. 5) or Gl (Exp. 6) responding

Exp. 7 Gi body extract (1 FBE) 15 * 7 Solvent control 5

Exp. 8 Gi body extract (1 FBE) 10 No wing fanning Solvent control 8

No. of male Gi (Exp. 7) or Gf (Exp. 8) responding

Exp. 9 Gi body extract (1 FBE) 18 *** 17 Solvent control 1

Exp. 10 Gi body extract (1 FBE) 11 No wing fanning Solvent control 9

No. of male Gi (Exp. 9) or Gl (Exp. 10) responding

Exp. 11 Gl body extract (1 FBE) 14 10 Solvent control 6

Exp. 12 Gl body extract (1 FBE) 8 No wing fanning Solvent control 12

No. of male Gl (Exp. 11) or Gf (Exp. 12) responding

Exp. 13 Gl body extract (1 FBE) 15 * 11 Solvent control 5

Exp. 14 Gl body extract (1 FBE) 10 No wing fanning Solvent control 9

0 5152010 0 51510 20 Treatments No. of male Gl (Exp. 13) or Gi (Exp. 14) responding

Fig. 4. Number of male Glyptapanteles Xavicoxis, G. indiensis, and G. liparidis that were attracted, or wing-fanned, in response to test stimuli tested in Y- tube olfactometer experiments 3–14. 1 FBE D one female body extract equivalent D pheromone component(s) contained in extract of one macerated female body. In each experiment, bars with an asterisk (¤) indicate a signiWcant response to a particular treatment; heterogeneity 2 test with Yates’ correc- tion for continuity, treatment versus control; *P < 0.05; **P < 0.01; ***P < 0.001. Note: (1) Experiments grouped by brackets were run in parallel; (2) one male in each of experiments 4, 9 and 14, and two males in experiment 8 did not respond to test stimuli. A. Danci et al. / Biological Control 39 (2006) 225–231 231

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