Apostolos Kapranas, Jocelyn G. Millar, and Robert F. Luck Department of Entomology, University of California, Riverside CA 92521, U.S.A.

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1 CHEMICAL CUES MEDIATING HOST RECOGNITION BY TWO METAPHYCUS SP. PARASITOIDS OF BROWN SOFT SCALE: INITIAL EXTRACTION, BIOASSAY, AND ROLE IN HOST SELECTION BEHAVIOR Apostolos Kapranas, Jocelyn G. Millar, and Robert F. Luck Department of Entomology, University of California, Riverside CA 9221, U.S.A. ABSTRACT Preliminary observations suggested that the parasitoids Metaphycus sp. nr. flavus and Metaphycus luteolus might use contact kairomones in order to recognize and accept as a host the brown soft scale Coccus hesperidum. Here we test this hypothesis by obtaining extracts of brown soft scales with different solvents and checking if the extracts are biologically active. Both species responded to extracts obtained with water and less to methanol extracts, with M. sp. nr. flavus responding more strongly than M. luteolus. These findings indicate that the kairomones used by these species to recognize their hosts are polar compounds, and they have a primary role in host selection and recognition behavior of these two species. Extraction protocols for preparing crude extracts and bioassay methods are described. INTRODUCTION Metaphycus sp. nr. flavus and M. luteolus Timberlake (Hymenoptera: Encyrtidae) are endoparasitoids of various soft scales (Guerierri and Noyes, 2). The former species was collected from citricola scale Coccus pseudomagnoliarum (Kuwana) (Homoptera: Coccidae) in 1996, at Kozan, Turkey, during exploration for natural enemies of this scale. Metaphycus sp. nr. flavus was introduced into California for augmentative biological control of several soft scales (Bernal, 1999; Schweizer et al., 22, 23), whereas M. luteolus is considered native to California. Brown soft scale, Coccus hesperidum L. (Homoptera: Coccidae) is used as insectary host for rearing these parasitoids. It is a natural host for both parasitoid species (Guerierri and Noyes, 2) and female wasps of both species readily accept brown soft scale as a host. Oviposition behaviors of these wasps have been documented as follows (Kapranas, 22). Upon contact with a host the wasps antennate and then walk over the scale body while continuing to antennate the host. Once their antennae reach the margin of the scale body they turn 18 degrees and repeat this behavior several times. They then engage their ovipositor, drill through the integument of the host, and oviposit. With larger scales the wasps lay two or more eggs depending on scale size and the wasps egg load, with the drumming/turning behavior usually being repeated between consecutive ovipositions. Initial observations suggest that even if a brown soft scale is removed from its host plant, the remaining residue elicits ovipositional behavior by both M. sp. nr. flavus and M. luteolus: the female parasitoids often probe the location from which the scale had recently been removed (Kapranas and Luck, pers. obs.). These observations suggest that the wasps use chemical cues to recognize and accept brown soft scales as hosts (contact kairomones). Moreover this study is motivated by the fact that Metaphycus sp. nr. flavus eggs are encapsulated more frequently by brown soft scale relative to those of M. luteolus (Kapranas and Luck, unpublished data). We hypothesize that the cues used by these wasps to recognize and accept brown soft scale and citricola scale as a host exist in both citricola and brown soft scale, but the suitability of the two host species differs. To test this hypothesis, more knowledge is required concerning the semiochemicals that are involved in the soft scale parasitoid interaction. Our preliminary experiments had three objectives: 1) to document whether contact kairomones mediate host recognition for these two species, 2) to determine the role of these compounds in host recognition, and 3) to determine their chemical properties and establish protocols by which active compounds can be extracted. MATERIALS AND METHODS Metaphycus sp. nr. flavus and M. luteolus are cultured in the insectary facility at University of California, Riverside, on brown soft scale, grown on excised leaves of Yucca recurvifolia Salisb. (Agavaceae) (L. D. Forster, P. Pachecco, P. Flores, and R. F. Luck, unpublished data). Brown soft scale was collected at the University of California, Riverside campus from guava (Psidium guajava L.: Myrtaceae) in October 199. Groups of hosts, days old and in the nymphal early female age, were carefully removed from the yucca leaves and placed in vials. Extracts of these groups were obtained by adding 2 µl of solvent and gently shaking the vials for 3 minutes. The extract was then filtered through a plug of cotton wool, transferred into a clean vial and stored in the refrigerator until needed. We extracted five batches of soft scales with five different solvents per batch: water, methanol (MeOH), ethyl acetate (EtOAc), methylene chloride (CH 2 Cl 2 ) and hexane (C 6 H 14 ). In another set of experiments, we extracted groups of scales with 2 µl water (see results) twice to determine whether one extraction removed most of the active material, or whether multiple extractions were needed. 113

2 Bioassays were conducted by introducing naïve (no experience with healthy hosts) 3-4 day old female wasps into an arena consisting of a modified Pyrex Petri dish (2 mm diameter) with an entrance hole in the middle. To prevent disturbance of the wasps by the experimenter, we covered the inner walls of the dish with green sticky tape. This also prevented the wasps from walking on the walls of the dish and escaping from the bioassay arena. Each wasp was offered four options: two filter discs treated with the soft scale extract and two controls treated with only the corresponding solvent. The extracts and control solvents were spotted on 4mm diameter filter paper discs and air dried before the assays. Fresh discs were prepared for each replicate. Wasp behavior was monitored with a dissecting microscope for minutes. Two behaviors were scored: drumming/turning and drilling. Table 1 summarizes the number of replicates for each wasp species and different solvent extracts. The distribution of the number of drums/turns and drills on treated and untreated discs were analyzed using G-tests with Williams corrections (Sokal and Rohlf, 1997) to test the null hypothesis that the distribution of observed behaviors was independent of treatment. Table 1 Number of wasps of each species tested and number responding to extracts of five different solvents. Water Methanol Ethyl Acetate Methylene chloride Hexane M. sp. nr. flavus # wasps tested # wasps responding M. luteolus # wasps tested # wasps responding 6 2 RESULTS Female parasitoids of both species responded to the filter discs treated with solvent extracts. They were observed drumming and turning on the filter papers and quite frequently probing on them. Metaphycus sp. nr. flavus responded more frequently to discs treated with scale extracts relative to the control discs (Fig. 1). However the responses to treated discs were strongly dependent on the solvents used to extract the scales (G = 16.4, df = 4, p =.2) and were strongest to the water and methanol extracts, and less strong to ethyl acetate, methylene chloride, and hexane extracts (Fig. 1a and Table 1). Water extracts also induced different effects than the methanol extracts in terms of the of the observed behaviors manifested by the females of this species (G =.81, df = 1, p =.1). Metaphycus luteolus responded most strongly to the water extract, showed a weak response to the methanol extract, and no significant response to extracts with the other solvents (G = 23.39, df = 4, p =.1)(Fig. 1b). M. sp. nr. flavus responded more strongly to the water and methanol extracts than M. luteolus (water: G = 33., df = 1, p <.1; methanol: G =.4, df = 1, p =.2). When we extracted soft scales twice with water, the second extract was still slightly active for M. sp. nr. flavus, but inactive for M. luteolus, suggesting that most of the material was removed efficiently during a single 3 minute extraction with water (Fig. 2). The of drums and turns were independent of the two extracts tested (M. sp. nr. flavus: G =.8, df = 1, p =.78; M. luteolus: G =., p =.7). DISCUSSION Our results suggest that Metaphycus sp. nr. flavus and M. luteolus recognize brown soft scale, one of their potential hosts, based on chemical cues on the surface of their hosts. These chemical cues appear to be primarily responsible for acceptance of brown soft scale as a host for these two Metaphycus parasitoids. Wasp behaviors were similar when offered discs treated with water extracts or a brown soft scale. Female wasps inspected the treated discs with their antennae and then commenced drumming/turning and drilling behaviors, in exactly the same manner as they do with a real host. Wasps apparently need only the chemical cue(s) to recognize their hosts, because they responded even to extracts simply spotted on the Petri dish substrate. Brown soft scale kairomones for these two species appear to be polar compounds because they were extractable with water or methanol, and to a lesser extent or not at all with other less polar solvents. The active components must be on the surface of the brown soft scale because behavior is elicited by simply contacting the host cuticle. Metaphycus sp. nr. flavus appears to respond to a wider array of cues relative to M. luteolus, but this needs to be verified once fractionation and/or identification of active compounds has been accomplished. Furthermore, the generally weaker response of M. luteolus relative to that of M. sp. nr. flavus to water extracts could be due to 114

3 differences between the cues used by the two parasitoid species, or simply due to differential sensitivity to concentration of the active component(s) if these species do indeed respond to the same compound(s). Little is known about the chemistry of brown soft scale test covers, but studies on other soft scales such as Ceroplastes spp. (Homoptera: Coccidae) revealed that a high proportion of their cover constituents are water soluble (Tamaki, 1997). In another study, the encyrtid parasitoid Anicetus beneficus Ishi used a mixture of compounds extracted with chloroform but not water from Ceroplastes rubens Maskell covers (Homoptera, Coccidae) to accept this host, but these compounds were active only if applied to paraffin dummies having the same curvature as the natural hosts. The compounds were not active if applied to filter paper discs (Takahashi and Takabayashi, 1984). In the case of two encyrtid parasitoids of cassava mealybug, Phenacoccus herreni William and Cock (Homoptera, Coccidae), a polar compound, O-caffeoylserine, was identified as the host recognition factor for these two parasitoid species. Interestingly, O-caffeoyltyrosine was identified as the host recognition factor of Aphytis melinus DeBach (Hymenoptera, Aphelinidae), a parasitoid of California red scale Aonidiella aurantii Maskell (Homoptera, Diaspididae) (Hare et al. 1993). Comperiella bifasciata Howard (Hymenoptera, Encyrtidae) also responds to water extracts of the scale covers of California red scale (Luck and Uygun, 1986). It would be interesting to determine whether cues utilized by these Metaphycus species for host recognition are similar to those used by other encyrtids and to the aphelinid mentioned above. This in turn could provide insights into how the host ranges of these parasitoids have evolved, or at least what kind of hosts could be potentially included in their host repertoire, given that several Metaphycus parasitoids have been introduced onto several different continents as biological control agents for soft scale pests (Guerierri and Noyes, 2). These compounds also could have implications for screening biological control agents for non-target effects as well. The results presented here represent the preliminary stages of a project whose major objective is to elucidate host acceptance behaviors and identify host recognition factors of a suite of Metaphycus species (both native and exotic) that attack various soft scales in California. Such a comparative study will yield important information on how these parasitoids exploit and utilize hosts. ACKNOWLEDGEMENTS We thank Lisa Forster, Porfirio Pachecco, and Bob Trautman for rearing the soft scales and the parasitoids used in the experiments, and Steve McElfresh for providing help in the lab. This project is funded by a dissertation grant awarded to A.K. REFERENCES Bernal, J.S., Luck, R.F., Morse, J.G., 1999a. Augmentative release trials with Metaphycus spp. (Hymenoptera: Encyrtidae) against citricola scale (Homoptera: Coccidae) in California's San Joaquin Valley. Journal of Economic Entomology. 92, Calatayud, P.A., Auger, J., Thibout, E., Rousset, S., Caicedo, A.M., Calatayud, S., Buschmann, H., Guillaud, J., Mandon, N., Bellotti, A.C., 21. Identification and synthesis of a kairomone mediating host location by two parasitoid species of the cassava mealybug Phenacoccus herreni. Journal of Chemical Ecology 27, Guerrieri, E., Noyes, J.S., 2. Revision of European species of genus Metaphycus Mercet (Hymenoptera: Chalcidoidea: Encyrtidae), parasitoids of scale insects (Homoptera: Coccoidea). Systematic Entomology. 2, Hare, J.D., Millar, J.G., Luck, R.F., A caffeic acid ester mediates host recognition by a parasitic wasp. Naturwissenschaften 8, Kapranas, A., 22. Clutch size, pattern of sex allocation and encapsulation of Metaphycus sp. nr flavus (Hymenoptera: Encyrtidae) eggs, a facultative gregarious endoparasitoid of brown soft scale Coccus hesperidium L. (Homoptera: Coccidae), MS thesis, University of California, Riverside. Luck, R.F., Uygun, N., Host recognition and selection by Aphytis species: Response to California red, oleander, and cactus scale cover extracts. Entomologia Experimentalis et Applicata 4, Schweizer, H., Morse, J.G. Luck, R.F., 23. Evaluation of Metaphycus spp. for suppression of black scale (Homoptera: Coccidae) on southern California citrus. Environmental Entomology 32, Schweizer, H., Morse, J.G. Luck, R.F., Forster, L.D., 22. Augmentative releases of a parasitoid (Metaphycus sp. nr. flavus) against citricola scale (Coccus pseudomagnoliarum) on oranges in the San Joaquin Valley of California. Biological Control. 24, Takabayashi, J., Takahashi, S., Host selection behavior of Anicetus beneficus Ishi et Yasumatsu (Hymenoptera: Encyrtidae) II Bioassay of oviposition stimulants in Ceroplastes rubens (Maskell) (Hemiptera: Coccidae). Applied Entomology and Zoology 19,

4 Tamaki Y., Chemistry of the test cover. pp In Y. Ben-Dov and C. J. Hodgson [eds.], Soft Scale Insects Their Biology, Natural Enemies and Control. Elsevier Science B. V., Amsterdam. Sokal, R. R., Rohlf, F. J Biometry. W. H. Freeman, New York. Fig. 1. Responses of Metaphycus sp. nr. flavus (a) and M. luteolus (b) females to filter paper discs treated with extracts of brown soft scale obtained with different solvents and controls (solvent only), see text for explanation. a H2 H2 ext MeOH MeOH ext EtOAc EtOAc ext CH2Cl2 CH2Cl2 ext Hexane Hexane ext b H2 H2 ext MeOH MeOH ext EtOAc EtOAc ext CH2Cl2 CH2Cl2 ext Hexane Hexane ext 116

5 Fig. 2. Responses of Metaphycus sp. nr. flavus (a) and M. luteolus (b) females to two consecutive water extracts of brown soft scale treat control treat control treat control treat control M. sp. nr. flavus M. sp. nr. flavus M. luteolus M. luteolus 1st extract 2nd extract 1st extract 2nd extract 117

METAPHYCUS SPECIES NEAR FLAVUS RESPONSES TO SEMIOCHEMICALS RELEASED FROM A SCALE HOST, COCCUS HESPERIDUM

METAPHYCUS SPECIES NEAR FLAVUS RESPONSES TO SEMIOCHEMICALS RELEASED FROM A SCALE HOST, COCCUS HESPERIDUM P. Lo Bue, 1,2 L. D. Forster, 2* J. G. Millar, 2 S. Colazza 1, and R. F. Luck 2 1 University of