Journal of Chemical Ecology, Vol. 26, No. 3, 2000 RESPONSE OF PARASITOIDS Dendrosoter protuberans AND Cheiropachus quadrum TO ATTRACTANTS OF Phloeotribus scarabaeoides IN AN OLFACTOMETER CARLOS LOZANO,1,* EMILIO GONZALEZ,1 ARANZAZU PENA,˜ 1 MERCEDES CAMPOS,1 M. TERESA PLAZA,2 MIGUEL RODRIGUEZ,2 ISIDORO IZQUIERDO,2 and JUAN TAMAYO2 1Department of Agrobiolog´ıa y Proteccion´ Vegetal Estacion´ Experimental del Zaid´ın (CSIC) Granada, Spain 2Department of Qu´ımica Organica´ Facultad de Farmacia Universidad de Granada Granada Spain (Received May 14, 1999; accepted November 9, 1999) Abstract—The braconid Dendrosoter protuberans and the pteromalid Cheiropachus quadrum are two parasitoids of the olive bark beetle, Phloeotribus scarabaeoides. Several chemicals such as a-pinene, b-pinene, 2-decanone, 2-nonanone, decanal, undecanal, and n-butyl acetate have been identified as attractants in the laboratory for this scolytid. Under red light at 278C in a laboratory olfactometer both parasitoids oriented positively to both enantiomers of a-pinene, and females also responded to 2-decanone. Significant responses did not occur under white light or at lower temperatures. These results suggest that a-pinene and 2-decanone could be involved in the location of P. scarabaeoides by its parasitoids. Consequently an attracticidal control tactic for this scolytid that included these chemicals could eliminate part of the parasitoid population. In an integrated pest management program, this problem should be considered. Key Words—Cheiropachus quadrum, Dendrosoter protuberans, parasitoids, semiochemicals, Phloeotribus scarabaeoides, bioassays. *To whom correspondence should be addressed. 791 0098-0331/ 00/ 0300-0791$18.00/ 0 2000 Plenum Publishing Corporation 792 LOZANO ET AL. INTRODUCTION The first kairomonal response by a coleopteran predator of bark beetles (Coleoptera: Scolytidae) was reported by Wood et al. (1968), who trapped numerous Enoclerus lecontei (Cleridae) on traps baited with the pheromone of Ips paraconfusus. Other examples of kairomonal responses by natural enemies of scolytids are Thanasimus spp. (Cleridae) to the pheromones of Dendroctonus (Vite´ and Williamson, 1970) and Ips (Bakke and Kvamme, 1978), Lasconotus intricatus (Colydiidae) to the pheromone of Polygraphus rufipennis (Bowers and Borden, 1992), and Rhizophagus grandis (Rhizophagidae) to kairomones pro- duced by Dendroctonus micans (Gregoire´ et al., 1992). Hymenopteran parasitoids also use semiochemicals to find a habitat infested by their host or prey (Herald et al., 1988; Fukushima and Kuwahara, 1989; Pettit et al., 1992). These include parasitoids of scolytid beetles in several families such as Pteromalidae, Braconidae, Eurytomidae, or Eupelmidae (Rice, 1969; Camors and Payne, 1972; Lanier et al., 1972; Kennedy, 1979; Dixon and Payne, 1980; Sullivan et al., 1997). The braconid Dendrosoter protuberans (Hymenoptera: Braconidae) and the pteromalid Cheiropachus quadrum (Hymenoptera: Pteromalidae) are common parasitoids of the olive bark beetle, Phloeotribus scarabaeoides in Spanish olive orchards, inflicting up to 15% mortality (Campos and Lozano, 1994). In spring, while developing in logs, larvae and pupae of P. scarabaeoides are vulnerable to attack by these two parasitoids, which deposit eggs on their surface by piercing the bark. Adults of C. quadrum also feed on the host larval fluids, which ooze to the surface through the capillary made by the ovipositor. Several chemicals such as a-pinene, b-pinene, 2-decanone, 2-nonanone, decanal, undecanal, and n-butyl acetate have been identified in the laboratory as attractants for P. scarabaeoides (Rodriguez et al., 1997; Szauman-Szumski et al., 1998), although it is unclear if the parasitoids use these attractants to locate their host larvae. These compounds were identified from both sexes of olive bark bee- tle adults, frass, logs, and leaves by solvent extraction, solid sample injection, and collection of volatiles on an adsorbent in a cryogenic trap. Specifically, the mono- terpenes a-pinene and b-pinene are produced by pruned olive logs and appear to be part of the kairomone used by the olive bark beetle (Szauman-Szumski et al., 1998; Tamayo, 1998). Moreover, these monoterpenes appear also as components of the wood of many forest species attacked by different scolytids, some of which are also parasited by D. protuberans and C. quadrum (Chenier´ and Philogene,` 1989; Landolt and Phillips, 1997; Sullivan et al., 1997). The other compounds were found in bark beetle adults and/ or in the frass from their galleries. The ketone 2- decanone and the aldehyde decanal, found in the pioneer adults and highly attrac- tive in laboratory bioassays, have been proposed as components of the aggregation pheromone for P. scarabaeoides (Szauman-Szumski et al., 1998). SEMIOCHEMICALS FOR PARASITOIDS OF P. scarabaeoides 793 Our objective was to elucidate the response of D. protuberans and C. quadrum to the identified P. scarabaeoides attractants. METHODS AND MATERIALS Parasitoids reared in the laboratory (248C ± 28C, 60 ± 5% relative humidity, 18L : 6D photoperiod) (Gonzalez´ and Campos, 1990; Campos and Lozano, 1994) were sexed just after emerging and maintained for ≥30 min under the conditions used for the bioassays. A horizontally oriented glass Y-tube olfactometer (3 cm diam., 17-cm-long basal arm and 22-cm-long upper arms) was employed. Parasitoids were tested individually, with 25 individuals of each sex tested per sample. The parasitoid was placed in the basal arm and its position was recorded after 10 min. Ten microliters of each sample were added to the glass ball placed in the extreme of one of the upper arms. The other upper arm acted as a control. The bioassays were run under a constant supply of synthetic air flowing into the upper arms at 0.2 m/ sec. Insects were used only once. Blanks were run at the beginning of each set of bioassays, and olfactometers were discarded if contamination was suspected. Results were expressed (Schlyter and Lofqvist,¨ 1986) as the percent of the 25 test individuals that entered either the treatment or control arms, and as the percent of the responding parasitoids that entered the treatment arm. To determine the optimum bioassay conditions, a set of bioassays was car- ried out in red and white light at 17, 22, and 278C with 2-decanone as the test stimulus. Experiments were performed with (±)-a-pinene (98% pure), (+)-a-pinene (98%, 91+% ee), (−)-a-pinene (98%, 81+% ee), b-pinene (99+%), n-butyl acetate (99+%), 2-nonanone (99%), 2-decanone (98%), decanal (95%), and undecanal (97%) at concentrations of 0.1 mg/ ml in dichloromethane. Chemicals were purchased from Aldrich (Madrid, Spain) and were used as received. Responses of parasitoids upwind to either the treatment or control arms were analyzed by the chi-squared test. Differences in responses to the treatment and con- trol arms for each condition or chemical were analyzed with the two-tailed bino- mial test (Martin Andres´ and Luna del Castillo, 1990). In all cases a c 0.05. RESULTS Optimization of Conditions. The percentage of D. protuberans responding to either the 2-decanone-treated or control arm was the same under all six light and temperature conditions (Table 1). The only significant response by females was to the 2-decanone stimulus under red light at 278C (Figure 1). Male D. protuberans did not respond significantly to either the baited or the control arm. Female and male C. quadrum responded upwind to either arm at variable 794 LOZANO ET AL. TABLE 1.PERCENT OF RESPONDING PARASITOIDS TO TREATMENT (2-DECANONE) OR CONTROL WITH DIFFERENT CONDITIONS OF LIGHT AND TEMPERATUREa D. protuberans C. quadrum Conditions Males Females Males Females White light 178C 56 a 28 a 72 a 24 a 228C 56 a 60 a 52 ab 60 ab 278C 40 a 60 a 52 ab 80 b Red light 178C 28 a 36 a 24 a 32 a 228C 56 a 60 a 36 ab 52 ab 278C 56 a 56 a 44 ab 52 ab a Different letters in the same column indicate significant differences among conditions (x 2 test; P < 0.05). levels (Table 1). Again the only significant response by females was to the 2-decanone stimulus under red light at 278C (Figure 1) whereas the male C. quadrum showed the same response as male D. protuberans. Therefore, all fur- ther bioassays for both species were run under red light at 278C. Response to P. scarabaeoides Attractants. Both sexes of D. protuberans responded upwind equally to the test and control odor regardless of the test chemical placed in one of the upper arms of the Y-tube olfactometer (Table 2). Of those individuals that did respond, however, a significantly higher number of both sexes chose the treatment over the control arm, when the test chemical was (±)-, (+)- or (−)-a-pinene (Figure 2). Females also responded significantly to 2-decanone. A 1 : 1 mixture of (±)-a-pinene and 2-decanone did not elicit a higher response than either stimulus alone. When one test arm was baited, more male C. quadrum responded upwind to either arm when the test chemical was (+)-a-pinene than when the test chemical was b-pinene or 2-nonanone (Table 2). Females responded upwind at higher levels when the test stimulus was (±)-, (+)- or (−)-a-pinene, n-butyl acetate, or the mixture of a-pinene + 2-decanone than when the test chemical was b-pinene or 2-nonanone. No differences were observed among the other chemicals (Table 2). Of the individuals that did respond, both sexes of C. quadrum selected the baited arm in a pattern similar to that found with D. protuberans, except that males did not respond significantly to (±)-a-pinene (Figure 2). DISCUSSION The optimal conditions of light and temperature for response in the olfac- tometer by both D. protuberans and C. quadrum (red light and 278C) are similar SEMIOCHEMICALS FOR PARASITOIDS OF SEMIOCHEMICALS FORPARASITOIDS P. scarabaeoides P. FIG. 1. Percent of responding parasitoids to treatment (2-decanone) or control with the different conditions of light and 795 temperature tested.