Biological Control 52 (2010) 24–29
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Biological Control
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Host-seeking behavior and parasitism by Spathius agrili Yang (Hymenoptera: Braconidae), a parasitoid of the emerald ash borer
Xiao-Yi Wang a, Zhong-Qi Yang a,*, Juli R. Gould b, Hui Wu c, Jian-Hai Ma d a The Key Laboratory of Forest Protection, State Forestry Administration, Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing 100091, China b Animal and Plant Health Inspection Service, Department of Agriculture, Otis ANGB, MA 02542 5008, USA c Forestry Bureau of Sanming City in Fujian Province, Sanming, Fujian 365000, China d Forest Pest Control Station of Qinghai Province, Xining, Qinghai 810000, China article info abstract
Article history: Spathius agrili Yang (Hymenoptera: Braconidae) is a newly described and important idiobiont ectoparasi- Received 6 November 2008 toid of the emerald ash borer (EAB) that has excellent potential as a biological control agent against EAB Accepted 15 September 2009 populations in the USA. In order to understand the ecological factors involved in the search and discovery Available online 23 September 2009 of concealed hosts by S. agrili, we investigated the behavioral responses of adult female wasps to potential semiochemicals from host plants, hosts, and host frass as well as to vibration signals from host feeding Keywords: and movement. Using a bioassay, we showed that S. agrili first finds the host’s habitats by detecting Spathius agrili the volatile compounds emitted by ash. In the second phase of host location and acceptance, the parasit- Agrilus planipennis oids detect the mechanical vibrations produced by host feeding and movement under the surface of the Host location Concealed insect pest bark and then probe to find the EAB larvae. Contact chemicals seem to play little or no role in short-range Biological control host finding. Female wasps avoided laying eggs on EAB larvae already parasitized and thus paralyzed. We hypothesized that female wasps were not attracted to these larvae due to their lack of feeding or move- ment. While an induced paralysis in the host is instrumental in avoiding superparasitism, we cannot rule out that S. agrili females also use an oviposition pheromone to deter conspecific females. Together, these results suggest that vibration and olfactory cues play significant roles in distinct phases of S. agrili host habitat and host location behaviors. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction is packed inside gallery, which makes detection and control of the insect challenging (Wang et al., in press). EAB was accidentally The emerald ash borer (EAB), Agrilus planipennis Fairmaire (=A. introduced into North America in the late 1990s and subsequently marcopoli Obenberger) (Coleoptera: Buprestidae), is an important killed large numbers of ash trees in the United States and Canada insect pest attacking trees in the genus Fraxinus (Oleaceae). It is na- (Haack et al., 2002). Eradication of the pest appears increasingly tive to Japan, Korea, China, and far eastern Russia. The susceptible unlikely as its distribution continues to expand. host trees include Fraxinus mandshurica Rupr., Fraxinus velutina Spathius agrili Yang (Hymenoptera: Braconidae) is an ectopar- Torr., Fraxinus americana L., Fraxinus pennsylvanica Marshall var. asitoid initially discovered in Tianjin, China that can cause a high subintegerrima (Vahl), and Fraxinus nigra Marshall (Yu, 1992; Pan percentage of parasitism in EAB populations. Parasitism rates are and You, 1994; Zhang et al., 1995; Haack et al., 2002; Zhao et al., typically 40–50%, but can be as high as 85–90% in some areas (Yang 2005). The EAB lays eggs inside bark crevices and the newly et al., 2005). Spathius agrili has also been found in Changchun, Jilin hatched larvae chew through the outer bark layer, later feeding Province. The adult female detects EAB larvae hidden under the on the cambium layer. There are four larval instars (Wang et al., bark, and using its ovipositor, penetrates through the bark to inject 2005). The fully grown fourth-instar larvae excavate galleries in venoms for paralyzing the host. The female then lays a clutch of the outer sapwood, where they pupate the following spring. EAB eggs on the surface of host (Yang et al., in press). Parasitized EAB adults emerge in early summer, leaving a D-shaped exit hole in larvae cease feeding and parasitoid progeny develop on the para- the bark. During immature development, larval frass is not ejected lyzed EAB larvae. The braconid larvae pupate at the end of host gal- outside of the tree as is the case for many wood-boring beetles, but leries after completely consuming the host. Parasitoid females have the ability to evaluate host larval sizes under the bark to opti- mally allocate the size and sex ratio of each clutch of eggs (Wang * Corresponding author. Fax: +86 10 6288 9502. et al., 2008). Female adults usually select late larval instars, which E-mail address: [email protected] (Z.-Q. Yang).
1049-9644/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2009.09.008 X.-Y. Wang et al. / Biological Control 52 (2010) 24–29 25 are larger, to ensure an adequate food supply for their offspring ima leaves, 10 mg of fresh EAB frass or 64 fourth-instar EAB larvae. (Wang et al., 2006). Each material was tested against clean air flowing in the other arm Spathius agrili has good potential as a biological control agent of the tube. Newly eclosed (within 1 week), mated female wasps and was released in the United States to control EAB populations were released individually into the base of the Y-tube and ob- in 2007 (Gould, personal comm.). The use of this parasitoid will re- served for up to 5 min. Choice was recorded when the wasp quire an adequate knowledge of its interactions with EAB, and we crossed a line half-way up the length of a given arm. No response provide here a preliminary investigation of the ecological mecha- was recorded if the wasp failed to make a selection within 5 min of nisms of S. agrili host location. More specifically, we have investi- its release into the tube. Each treatment included at least 30 repli- gated (i) S. agrili host-habitat location behavior (role played by cates (different female wasps), and wasps that failed to make a chemical cues from ash plants), (ii) host location behavior (re- choice were eliminated from the statistical analysis. Each wasp sponses to host vibration or semiochemicals from host larvae and was tested only once and all tests were performed between 0800 frass), and (iii) host-acceptance behaviors (with EAB, non-habitual and 1700 h at a temperature range of 25–29 °C and a relative hosts, and artificial hosts) and occurrence of superparasitism (two humidity of 55–70%. The Y-tube was washed with 95% ethanol egg clutches on one host larva) in a laboratory setting. This knowl- once every 10 runs, and for the testing of treatments (plant, EAB edge should prove useful in several applications related to the use larvae, and frass materials), the positions of the sample bottle of this natural EAB control agent, notably in artificial rearing tech- and the clean air bottle were switched once every five runs. In or- niques and field efficacy tests. der to expose wasps to uniform lighting, the entire Y-tube device was positioned vertically inside a wood box covered with a white cloth. 2. Materials and methods 2.2. Host location Both the parasitoids, S. agrili, and hosts, EAB larvae, tested in this study were collected from the Guangang Forest Park of Dagang 2.2.1. Field and laboratory observations of host-seeking behavior District in Tianjin, China (38°560N, 117°290E). The experimental Adult S. agrili were observed at a study site located in the Guan- forest contained a stand of 12-year-old velvet ash, F. velutina, gang Forest Park of Dagang District in Tianjin, China in 2005–2006. approximately 80% of which were infested with EAB. EAB larvae A stand of 12-year-old velvet ash, F. velutina, infested with EAB was were collected from the ash trees by peeling the bark. Wasp co- selected for the observations. From July to September, the mating coons were obtained from the host galleries and taken to the lab- behavior of adults and the host-seeking behaviors of female para- oratory for adult emergence. Newly emerging adult wasps were sitic wasps were observed on trees selected randomly in the field gregariously maintained within glass vials (2.8 cm dia. 11.5 cm and on twigs in the laboratory. Host-seeking behaviors of 10 fe- length) for group mating and developmental maturation of the fe- males were observed on ash trunks in field, while under laboratory male reproductive systems, which took approximately one week. conditions, 30 observations of mating and host-seeking behaviors All adult wasps were reared in glass vials (2.8 cm dia. 11.5 cm were recorded. length) sealed with cotton plugs and were fed a 20% honey solution applied to a small cotton ball on a piece of filter paper attached to 2.2.2. Response to dead hosts and artificial hosts the inside of the vial. One possible mechanism of S. agrili host location is the detec- Two methods were used to present EAB larvae to S. agrili fe- tion of mechanical vibrations produced by live hosts (feeding or males for parasitization. The ‘‘twig” method involved using small movement). We tested whether female wasps will oviposit on lar- F. velutina twigs (1.0–1.5 cm in diameter) that were cut into 5– vae that are not moving by presenting them with dead or artificial 10 cm long sections and then split in half along their length. A 3- hosts. No response to dead or artificial hosts would suggest that fe- cm-long chamber was excavated inside one half of the twig male wasps may detect vibrations produced by live hosts, while through to the edge of the bark, and an EAB larva was inserted into oviposition on dead hosts and artificial hosts indicates that host the cavity. The two halves were put back together and fastened vibration is not a necessary cue for oviposition. tightly with rubber bands. Both ends of the section were waxed The response of S. agrili toward dead EAB larvae was measured to prevent water loss while the EAB larva was in the chamber. using third- and fourth-instar larvae killed either by exposure to The ‘‘paper-packaging” method made use of toilet tissue that was freezing temperatures ( 20 °C for 30 min) or injected with ethyl cut into 3.5 4.5 cm pieces. An EAB larva was wrapped in a single acetate (0.04 ml/larva). Larvae killed by freezing have a soft body layer of toilet tissue and the two ends were twisted and sealed and are convenient for wrapping but easily deteriorate and become with adhesive tape. moldy. Larvae killed by freezing were, therefore, tested using the paper-packaging method. EAB larvae killed with ethyl acetate are 2.1. Host-habitat location hard-bodied and less prone to deterioration and were appropriate for use with the twig method. Every 3–5 days, the dead host larva The behavioral responses of female wasp towards volatiles was removed from either the paper packaging or the twig to ob- emitted by EAB larvae, frass, and plants were examined by using serve whether the wasp had laid any eggs. Each treatment was rep- a Y-tube olfactometer in a dual choice test (Rutledge and Wieden- licated 30 times, and the wasps were tested until they died. mann, 1999). The base tube of the olfactometer was 18.0 cm in The response of S. agrili to artificial hosts was also measured. length, and arm tubes were 23.5 cm long, with an inside diameter The artificial hosts consisted of either milk, egg fluid (mixture of of 2.0 cm and a 75° angle between the two arms. Each arm was at- egg white and egg yolk), or milk powder mixed with egg yolk tached to a sample bottle and air was pulled into Y-tube using a wrapped in ParafilmÒ, respectively. One ml of the liquid was in- vacuum pump connected to the base tube. The air was run through cluded per ParafilmÒ package which was shaped into a small a charcoal filter and a beaker of clean water before it went into the host-like cylinder. Artificial hosts were coated with EAB larval chamber with the test material. Airflow in each arm was controlled hemolymph, and the parasitization responses of female S. agrili with flow meters to equal 90 ml/min. Two sizes of sample bottles were observed using both the paper-packaging and twig methods. were used; a 2500-ml bottle for testing plant material and a 300- Every 3–5 days, the artificial hosts were removed to record ml bottle for testing EAB larvae and frass. Samples consisted of whether female wasps had laid any eggs. The controls consisted 250 g of freshly cut F. velutina or F. pennsylvanica var. subintegerr- of live third- and fourth-instar EAB larvae. Each treatment (milk, 26 X.-Y. Wang et al. / Biological Control 52 (2010) 24–29 egg fluid, milk powder, live EAB, dead with acetate, dead from was monitored by removing the twigs after the female died and freezing) was replicated 30 times for both presentation methods. counting the number of eggs laid. Each treatment consisted of at least 30 replicates (30 female wasps). We scored repeated oviposi- 2.3. Host acceptance tion as indicating the absence of an oviposition deterrent phero- mone on the surface of the twig, whereas the absence of 2.3.1. Field and laboratory assessment of superparasitism oviposition might indicate the presence of such a pheromone. Field observations on the oviposition behaviors and superpara- sitism assessment of S. agrili female adults were conducted at the 2.3.3. Response of S. agrili to extracts of EAB cuticle and frass same stand mentioned above, during the same period, July to Sep- Fresh EAB larval frass in galleries of live insects was collected tember, 2005–2006. Oviposition behaviors of 10 females were ob- and placed in four closed glass vials (5 g per vial). Fifty ml of ace- served on ash trunks selected randomly in the field. To assess tone (analytical grade reagent) was added to each of two of the parasitization success and superparasitism, the bark of trees on vials and 50 ml of 95% ethanol (analytical grade reagent) was which females were observed to drill with their ovipositor was re- added to each of the remaining two vials. The extraction was con- moved. The presence of EAB larvae, the presence of S. agrili eggs on ducted at room temperature (about 20 °C) for 48 h. Crude extracts EAB and the occurrence of superparasitism were recorded. Super- were concentrated to 5 ml by blowing cold air using a hair dryer parasitism by conspecific females could be determined by the pres- and stored at approximately 5 °C in closed glass vials until ready ence of more than one egg clutch on a single host larva. for use. Cuticular extracts were obtained by washing 50 third- or Superparasitism by S. agrili was measured using both the twig fourth-instar EAB larvae with 50 ml of acetone and 50 larvae with and paper-packaging methods in the laboratory. Larvae were 95% ethanol, and the solutions were concentrated as above. Con- examined every 3–5 days to assess parasitization by female wasps. centrated crude extracts were applied to 1 cm by 2 cm filter paper If a larva was parasitized, it was removed from the twig, and the by complete immersion. The filter papers were air dried for wasp eggs or newly hatched larvae were removed from the body approximately 10 min to remove the solvent and inserted singly surface. The paralyzed larva was reintroduced into the same twig, into a vial along with a mated S. agrili female. The types and fre- which was placed back into the vial containing the same S. agrili fe- quencies of behavioral responses of the wasps were recorded for male. Presence or absence of oviposition was again checked once 5 min after exposure to the filter paper. The specific behaviors re- every 3–5 days until the female parasitoid died. Control females corded included (i) crawling, (ii) antennal drumming, and (iii) ovi- were provided healthy host larvae in fresh twigs that had never positor probing. The treatments included frass in acetone, frass in been exposed to S. agrili. Each treatment consisted of 30 replicates ethanol, cuticle in acetone, cuticle in ethanol and controls where (i.e., 30 vials with female wasps that were exposed to previously the filter paper was treated with solvents only. Each treatment parasitized larvae and 30 vials with females who received healthy had 30 replicates. larvae). To assess the importance of A. planipennis semiochemicals in Five paper packages with the third- or fourth-instar EAB larvae the behavioral response of S. agrili, two non-host insect larvae, were introduced in a glass vial along with 2 females and 1 male. Thyestilla gebleri Faldermann and Agrilus subrobustus Saunders, Packages were examined every 3–5 days to assess parasitization. were coated with the crude EAB extracts using 95% ethanol as a If the EAB larva within a package was parasitized, it was removed solvent. After the solvent volatilized completely, treated non-host and the wasp eggs or newly hatched parasitoid larvae were re- larvae were wrapped with toilet tissue and inserted in a vial with moved as above. The parasitized larva was rewrapped using a dif- a pair of S. agrili (a male and a female). Host-searching and ovipo- ferent piece of toilet tissue and exposed again to the S. agrili sition behaviors were recorded as in the filter paper experiment females. Larvae were checked every 3–5 days to assess parasitiza- above. Live, third- or fourth-instar EAB larvae wrapped in toilet tis- tion until both S. agrili females died. The controls were provided sue were used as controls. Each treatment included 30 replicates. healthy host larvae never exposed to the wasp. Each treatment was replicated 30 times. 2.4. Statistical analysis 2.3.2. Instar preference and assessment of oviposition pheromone All data were analyzed using the SAS software Version 9.1.3 presence (SAS Institute Inc., 2006). Analyses of variance (PROC GLM), fol- The EAB has 4 larval instars, which were determined based on lowed by Tukey’s separation of means test, was used to compare Wang et al. (2005). EAB of each instar were presented to the female wasp oviposition on the different host instars, as well as between wasps using the twig method, and we tested parasitization rates of treatments and controls. The parasitism rates of wasp for various 30 females (replicates) on each EAB instar (treatment). Every 3– EAB instar larvae were compared with a chi-square (v2) tests 5 days, the twigs were disassembled to determine if EAB larvae (PROC FREQ). For the Y-tube choice experimental data, a chi- were parasitized by S. agrili, and the number of eggs or parasitoid square test (one-way) for goodness-of-fit with one degree of free- larvae that were visible on host body surface was recorded. Parasit- dom was used to determine significance. The chi-square values ized host larvae were replaced with healthy ones, whereas unpar- were adjusted for continuity ðv2Þ. asitized larvae were left in place. Egg production by S. agrili was c monitored as described until the death of the female wasp. Parasit- ism rate, mean wasp progeny produced on the first- through 3. Results fourth-instar EAB larvae and the status of host larvae (feeding or non-feeding) were recorded. Larvae were assumed to be feeding 3.1. Host-habitat location when frass or sawdust was present inside the artificial chamber. Spathius agrili females were also observed to determine if they Results of Y-tube olfactometer tests showed that adult S. agrili deposited an oviposition deterrent pheromone on the surface of females were attracted to the volatiles of fresh leaves of F. pennsyl- the bark. A third- or fourth-instar EAB larva was inserted into a vanica and F. velutina, which are the preferred host plants of EAB. twig and exposed to a female wasp in a vial as described above. The wasps displayed a significant positive tropism towards ash If the larva was parasitized, it was removed and replaced with a leaves, but odors released by host larvae, host larval frass, and healthy larva of the same size as the one removed. The acceptance ash bark with host larval frass, did not significantly attract the par- of the new larva inside the twig previously used for oviposition asitoids when compared to clean air (Table 1). X.-Y. Wang et al. / Biological Control 52 (2010) 24–29 27
Table 1 Olfactory response of Spathius agrili to chemical volatiles from emerald ash borer (EAB) larvae and frass and leaves of Fraxinus spp.
2 Test materials Wasps towards stimulus Wasps away from stimulus vc Value t Value P value F. pennsylvanica 29 9 9.5000* 3.77 0.0006 F. velutina 25 9 6.6176* 3.06 0.0043 Bark with frass 21 14 1.0286 1.19 0.2422 EAB larvae 19 13 0.7813 1.06 0.2961 EAB frass 20 11 2.0645 1.66 0.1070
* 2 Showed significant difference at a = 0.05 level, df =1,v0:05 ¼ 3:84.
3.2. Host location ing third-instar larvae (F = 0.65, df = 1, 11, P = 0.4388). However, the average clutch size was significantly higher when feeding 3.2.1. Field and laboratory observations of host-seeking behavior rather than non-feeding fourth-instar larvae were tested In its natural environment in Tianjin, China, S. agrili parasitizes (F = 5.02, df = 1, 63, P = 0.0286) (Fig. 1B). EAB larvae from July to September. Results showed that males usu- ally emerged prior to females and often waited near the exit holes 3.2.3. Oviposition on dead and artificial hosts for mating with newly eclosed females. Some males also searched Spathius agrili adult females never laid eggs on dead host larvae for and pursued females on the trunks. Females began to parasitize or artificial hosts (v2 = 37.4143, df =6,P < 0.0001). Even when the EAB larvae after a week-long pre-oviposition period (based on surface of the artificial hosts was coated with EAB larval hemo- observations in laboratory). Females preferred to search for hosts lymph, placed in the paper package or twig, and exposed to the on bright sunny days with high temperatures (approaching wasps, no egg deposition occurred. In contrast, the live EAB larval 30 °C) and little or no wind. Activity typically peaked between controls had 26.7% and 10% parasitism in the twigs and paper 8:00 and 11:00 and 15:00 and 18:00 h in the summer. Females packages, respectively. The results suggest that S. agrili only para- usually walked on the EAB infested ash trunks back and forth sitizes active host larvae. and sometimes drummed the bark with their antennae. Laboratory observations revealed that the parasitoids only attacked hidden hosts, and females showed no interest in exposed host larvae 3.2.4. Behavioral responses of S. agrili to A. planipennis crude extracts (unpublished observation). When the location of a host larva was Parasitoids introduced into vials along with filter paper contain- ascertained, the female wasp slowly inserted her ovipositor into ing crude frass or cuticular extracts showed poor responses, irre- the bark and then injected venom inside host larva, paralyzing it spective of the origin of the extract. Although most females prior to laying eggs. Female S. agrili were seldom disturbed by walked on the paper surface, only a few individuals drummed 2 other individuals during the 40–90 min required to complete each the surface with their antennae (v = 0.6545, df =5,P = 0.9854) or 2 bout of egg laying (Yang et al., in press). probed the paper with their ovipositors (v = 4.0449, df =5, P = 0.5430). The percentage of antennal drumming among all treat- 3.2.2. Effect of host activity on S. agrili oviposition ments and controls ranged from 6.7% to 10%, while the percentage We hypothesized that EAB feeding activity and/or movement of ovipositor probing was less than 3.3%. could be used as cues to trigger S. agrili oviposition behavior. In- deed, whether or not an EAB larva was feeding affected the ovipo- 3.3. Host acceptance sition behaviors of female wasps (Fig. 1). The parasitism rate on feeding third-instar EAB larvae (61.5%) was significantly greater 3.3.1. Rates of parasitism on different EAB instars than on non-feeding (but potentially moving) third-instar larvae Data from laboratory studies revealed that S. agrili showed the 2 (20.8%) (vc ¼ 4:4746, df =1, P = 0.0344). While feeding fourth-in- highest parasitism rates for third- and fourth-instar EAB larvae at star larvae appeared to have greater levels of parasitism (56.6%) 35.1% and 47.1%, respectively. Wasps did not show a preference than non-feeding fourth-instar larvae (41.2%), the difference was for either third or fourth instar as determined by the v2-test 2 2 not significant (vc ¼ 2:5299, df =1, P = 0.1117) (Fig. 1A). Feeding (vc ¼ 1:2414, df =1, P = 0.2652). Spathius agrili did not parasitize activity did not significantly affect the mean number of eggs first- or second-instar host larvae (v2 = 41.9132, df =3, deposited on the third-instar larvae when compared to non-feed- P < 0.0001). The wasps laid significantly more eggs per host
Fig. 1. Spathius agrili (A) parasitism rates and (B) clutch sizes among the four emerald ash borer (EAB) larval instars. The different letters above the bars show significant differences between treatments at a = 0.05 level. 28 X.-Y. Wang et al. / Biological Control 52 (2010) 24–29
(6.6 ± 0.4) on fourth-instar hosts than they laid on third instars Parasitoids are known to utilize various stimuli besides semio- (3.5 ± 0.5) (F = 10.46, df = 1, 76, P = 0.0018). chemicals to locate hosts, such as cues from vision, sound, touch, and heat (Quicke, 1997). Some parasitic wasps may integrate the 3.3.2. Responses to non-host larvae coated with EAB cuticular and information of several stimuli from different sources for enhancing frass extracts the reliability and accuracy of host locations (Fischer et al., 2001). Spathius agrili did not parasitize larvae of the two non-host spe- Among these multiple cues, semiochemicals play a prominent role, cies, T. gebleri (v2 = 20.4545, df =5,P = 0.0010) and A. subrobustus especially secondary volatile substances derived from hosts, host (v2 = 16.4384, df =4,P = 0.0025), even if these larvae were coated frass, host galleries, and host-infested plants (Vinson, 1991; Mills with EAB larval hemolymph or with ethanol or acetone extracts et al., 1991; McCall et al., 1993; Steinberg et al., 1993; Potting of host larvae and larval frass. Parasitism of the control EAB larvae et al., 1995; de Moraes et al., 1998; Pettersson, 2001; Pettersson was 13.3%. et al., 2001; Gohole and Ngi-Song, 2001; Neveu et al., 2002; Sil- va-Torres et al., 2005; Rojas et al., 2006). In the case of concealed hosts, some parasitic wasps can use the vibratory signals produced 3.3.3. Ability to discriminate against parasitized hosts by the hosts. At present, this host-finding strategy is known for During our 2003–2005 field surveys in Tianjin, 236 EAB larvae parasitoids in the superfamilies, Orussoidea, Ichneumonoidea, parasitized by S. agrili were collected, yet superparasitism was and Chalcidoidea (Quicke, 1997; Meyhöfer and Casas, 1999; never recorded. Results from the laboratory assays on superpara- Fischer et al., 2003). Our research highlights the importance of sitism showed that female parasitoids never laid eggs on hosts lar- semiochemicals and vibration in host habitat and host location vae previously parasitized by conspecific females in either the twig by S. agrili. (v2 ¼ 7:0673, df =1, P = 0.0079) or paper-packaging method c In our study, we showed that S. agrili uses volatile chemicals (v2 ¼ 1:4035, df =1,P = 0.2361) trials. The controls of live EAB lar- c from EAB host plants (ash) for host-habitat location. Previously, vae showed 26.7% and 10% parasitism in the twig and paper pack- Yang et al. (2008) demonstrated the importance of ash volatiles age, respectively. as attractants for S. agrili, where female S. agrili were attracted to leaves of F. velutina and F. pennsylvanica but were not attracted 3.3.4. Assessment of an oviposition deterrent pheromone to 12 other potential host plants (Citrus reticulata Blanco, Malus In this experiment, an EAB larva previously unexposed to a fe- pumila Mill., Zanthoxylum bungeanum Maxim., Juglans regia L., Albi- male parasitoid was placed in a twig that had previously contained zzia julibrissin Durass., Salix babylonica L., Pyrus bretschneideri Reh- a parasitized EAB. The parasitism rate for the unexposed larvae was der, Prunus persica (L.), Populus deltoides Bartr., Crataegus pinnatifida 62.1%, compared with 44.6% parasitism for the control larva, where Fructus, Euonymus japonica L., Ailanthus altissima (Mill.). Although the twig containing an EAB larva had no previous exposure to a fe- volatiles from ash leaves were important stimuli for host-habitat male parasitoid (Fig. 2A). The parasitism rates between the two location, volatiles from EAB larval frass and EAB larvae were not 2 treatments were not significantly different (vc ¼ 2:3952, df =1, attractive to S. agrili females. P = 0.1217). Similarly, the clutch sizes did not differ significantly During the course of host location and host acceptance, contact between groups (F = 0.14, df = 1, 94, P = 0.7141), with 6.4 ± 1.0 for semiochemicals from EAB larvae or frass seem unimportant and the EAB larvae in the twigs previously exposed to the parasitoid we provide evidence that vibration-emitting activities by the host and 6.1 ± 0.4 for the control twigs that were not previously ex- larvae, such as feeding and movement, could play a more impor- posed to the parasitoid (Fig. 2B). tant role. Wang et al. (2008) found that S. agrili can assess the size of the host larva under the bark and apparently deposit the appro- 4. Discussion priate number of eggs relative to the host size. Different sizes of host larvae could emit different frequencies or intensities of vibra- Agrilus planipennis is a highly concealed wood borer that is very tion signals, serving as cues to females who thus regulate clutch difficult to control. The braconid parasitoid wasp, S. agrili, has pow- size. erful host-searching and location abilities, which makes it a prom- In our study, we found no evidence that S. agrili uses a marking ising natural enemy to use as a biological control agent (Wang and or oviposition pheromone to avoid superparasitism. EAB larvae in Yang, 2005). By exploring the behavioral mechanisms that parasit- twigs previously experiencing a bout of oviposition were accepted oids use for discovering hidden host pests, we gain a better under- as hosts. The fact that parasitism was slightly higher in previously standing of why some parasitoids are more effective in biological used twigs is somewhat unexpected. Perhaps some twigs were control programs. particularly suitable for feeding and the test larvae were more
Fig. 2. (A) Percentage parasitism and (B) clutch size of Spathius agrili on healthy emerald ash borer (EAB) larvae in twigs previously used for oviposition by a conspecific female wasp or used for the first time. The different letters on top of bars show significant differences between treatment and control groups at a = 0.05 level. X.-Y. Wang et al. / Biological Control 52 (2010) 24–29 29 likely to feed on the wood within the twigs resulting in the female Meyhöfer, R., Casas, J., 1999. Vibratory stimuli in host location by parasitic wasps. wasps being able to detect and find these host larvae more easily. It Journal of Insect Physiology 45, 967–971. Meyhöffer, R., Casas, J., Dorn, S., 1994. Host location by a parasitoid using leafminer is still possible that a marking pheromone is deposited on the body vibrations: characterizing the vibrational signals produced by the leafmining of the host larva but we consider this possibility remote. However, host. Physiological Entomology 19, 349–359. we cannot eliminate this possibility entirely because the female Mills, N.J., Kruger, K., Schlup, J., 1991. Short-range host location mechanisms of bark beetle parasitoids. Journal of Applied Entomology 111, 33–43. wasp may oviposit more readily when it is given no choice of hosts Neveu, N., Grandgirard, J., Nenon, J.P., Cortesero, A.M., 2002. Systemic release of as in our laboratory tests. We feel that the absence of egg laying on herbivore-induced plant volatiles by turnips infested by concealed root-feeding previously parasitized host larvae was not due to the presence of a larvae Delia radicum L. Journal of Chemical Ecology 28, 1717–1732. Pan, Z.G., You, Y.T., 1994. Growing Exotic Trees in China. Beijing Science & marking pheromone but rather because the host larvae were par- Technology Press, Beijing, pp. 632–643 (in Chinese). alyzed and were unable to produce vibrations. Given that S. agrili Pettersson, E.M., 2001. Volatiles from potential hosts of Rhopalicus tutela a bark triggers a cessation of feeding in its host immediately after parasit- beetle parasitoid. Journal of Chemical Ecology 27, 2219–2231. Pettersson, E.M., Hallberg, E., Birgersson, G., 2001. Evidence for the importance of ization (through the injection of paralyzing venoms), we suggest odour-perception in the parasitoid Rhopalicus tutela (Walker) (Hym., that the absence of vibration makes the parasitized larva undetect- Pteromalidae). Journal of Applied Entomology 125, 293–301. able by conspecific females. Potting, R.J., Vet, L.E.M., Dicke, M., 1995. Host microhabitat location by stem-borer Our study made use of a variety of bioassays, which are indirect parasitoid Cotesia flavipes: the role of herbivore volatiles and locally and systemically induced plant volatiles. Journal of Chemical Ecology 21, 525–539. methods, to investigate the mechanisms by which S. agrili searches Quicke, D.L.J., 1997. Parasitic Wasps. Chapman & Hall, London, UK. for and finds its hidden hosts. In order to strengthen our contention Richerson, J.V., Borden, J.H., 1972. Host finding by heat perception in Coeloides that S. agrili females rely on host-generated vibrations to find suit- brunneri (Hymenoptera: Braconidae). The Canadian Entomologist 104, 1877– 1881. able hosts, direct evaluations with highly sensitive vibration me- Rojas, J.C., Castillo, A., Virgen, A., 2006. Chemical cues used in host location by ters, such as the laser Doppler vibrometer (Meyhöfer et al., 1994; Phymastichus coffea, a parasitoid of coffee berry borer adults, Hypothenemus Wäckers et al., 1998), will be needed. In addition, the parasitoid hampei. Biological Control 37, 141–147. Rutledge, C.E., Wiedenmann, R.N., 1999. Habitat preferences of three congeneric may track concealed hosts through infrared sensilla by detecting braconid parasitoids: implications for host-range testing in biological control. the radiant heat generated from host movements and/or metabo- Biological Control 16, 144–154. Ò lism (Richerson and Borden, 1972). Observations on the ultrastruc- SAS Institute Inc., 2006. SAS OnlineDoc . Version 9.1.3. SAS Institute Inc., Cary, NC, USA. Available from: