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P1: GVG Journal of Chemical Ecology [joec]

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C 2002) Journal of Chemical Ecology, Vol. 28, No. 7, July 2002 (°

EXPERIMENTAL STUDIES OF THE HOST-FINDING BEHAVIOR OF Trogus pennator, A PARASITOID OF SWALLOWTAIL BUTTERFLIES

KAREN R. SIME∗ Department of Entomology Department of Ecology and Evolutionary Biology Cornell University Ithaca, New York (Received July 12, 2001; accepted March 13, 2002)

Abstract—The parasitic wasp Trogus pennator (Hymenoptera: Ichneumonidae) attacks larvae in two genera of Papilionidae, Eurytides and Papilio, on plants in a variety of families. The female wasps’ responses to food plants, feeding damage, and frass were examined in a series of experiments designed to test the hypothesis that parasitic wasps that specialize on host taxa and seek their hosts in a variety of habitats exhibit fixed responses to host-derived cues and more flexible responses to cues associated only with the hosts’ food plants. Naive T. pennator females showed no preferences when offered either a choice between two papilionid food plants or a choice between a food plant and a plant not used for food by Papilionidae. After experience with hosts in the presence of a particular food plant, however, wasps preferred that plant. Naive wasps did prefer plants damaged by host larvae over plants damaged by nonhost (saturniid) larvae and also preferred methylene chloride extracts of host frass over extracts of frass from saturniid larvae fed on the same plant species, results indicating that the responses of T. pennator females to host-derived cues are innate. The chemical compositions of the extracts of frass from several papilionid and one saturniid species were also examined, and the significance of the finding that no host-specific patterns were detected among the major components of the extracts is discussed. Key Words—Ichneumonidae, Papilionidae, host-finding behavior, learning, feeding damage, semiochemicals, frass, flight chamber.

∗

E-mail: [email protected]

1377 C 2002 Plenum Publishing Corporation 0098-0331/02/0700-1377/0 °


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Parasitic wasps in the genus Trogus Panzer (Ichneumonidae) lay their eggs in the larvae of swallowtail butterflies (Lepidoptera: Papilionidae) and emerge as adults from the remains of the host pupae (Mitchell, 1979, 1981). The North American species T. pennator (Fabricius) is widespread in the United States, although it is most common in the southeastern states, and is relatively polyphagous, attacking nearly all the papilionids in its range, including Eurytides marcellus (Cramer) and at least five Papilio species (Heinrich, 1962; Mitchell, 1983; Sime and Wahl, 2002). These hosts feed on a variety of plants, although with the exception of the generalist P. glaucus L., each species tends to specialize within plant families (Feeny, 1991). Trogus pennator has been reared from larvae feeding on eight plant families, suggesting that it does not discriminate among its hosts’ food plants (Sime, 2001). The host-finding behavior of T. pennator has been studied under natural conditions in the Ocala National Forest, Florida, USA, where the usual host is the zebra swallowtail, E. marcellus (Sime, 2001), which feeds on species of pawpaw (Annonaceae: Asimina). By following females as they searched for host larvae, it was established that the wasps discriminate between Asimina and other plants, that they are attracted to plants damaged by hosts, and that they spend more time searching on damaged than on intact plants. While that study provided valuable information about the behavior of undisturbed wasps in a natural environment, it left several important questions unanswered. The evidence indicated that the wasps rely at least in part on vision to identify Asimina plants, but it was not possible to determine whether they also respond to plant odor, as do most parasitic wasps (Vinson, 1984). It was also not possible to establish whether the wasps recognize feeding damage on the basis of odor, or whether they can distinguish the feeding damage of their hosts from that left by other lepidopteran larvae. To discover the nature of the cues involved in orienting to plants, laboratory experiments were conducted to determine whether wasps are attracted to plant odor and whether the sources of useful odors include intact plants as well as plants chewed by host larvae. To establish a chemical basis for their arrestment behavior, the responses of wasps to chemical extracts of host frass were examined and an attempt was made to isolate and identify active compounds. Another shortcoming of the previous field study was that it was not possible to know whether the observed attraction to plants had been learned, as is the case for many parasitic wasps (Vet et al., 1995). No information was available on the life experiences of the wasps observed, although presumably all had encountered hosts, which were abundant at the site. Because the host range of T. pennator and the food plants of its hosts are well known, an investigation of learning and cue specificity in this species provides an opportunity to test ideas concerning the evolution of host range in parasitoids. Gauld (1988) and Vet and Dicke (1992)


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have hypothesized that for parasitoids specializing on particular host taxa, host range is a function of the distribution of chemical compounds used to find and identify hosts. This hypothesis requires, first, that innately attractive cues exist (i.e., the wasps are attracted to them regardless of experience and environment), and second, that the parasitoids are able to use these cues to discriminate hosts from nonhosts. For T. pennator, this hypothesis predicts that the females use compounds characteristic of papilionid larvae to find and recognize these hosts. Responses to hosts are expected to be fixed, but responses to plant-derived cues may be more flexible. This hypothesis was tested in the present study by investigating whether plant- and host-derived chemical cues were innately attractive or learned and by comparing responses to odors produced by papilionid and non-host larvae. METHODS AND MATERIALS

Sources of Insects and Plants. Most of the Trogus pennator females used in these experiments were the offspring of adults caught in the Ocala National Forest, Florida, in late March and early April of 1995, 1997, and 1998. A few were netted at patches of Asimina triloba (L.) (a food plant of E. marcellus) along the James River near Buchanan, Virginia, USA, in July of 1996 and 1998. The wasps were raised on larvae of E. marcellus, also collected in Florida, that had been exposed to female wasps in captivity and maintained on cuttings of A. speciosa Nash and A. triloba. Adult wasps were kept for up to several weeks at room temperature with unlimited access to water and a honey-water solution. They were refrigerated (at 4◦ C) for up to a week at a time when not needed for experiments. Females were intermittently allowed to attack E. marcellus larvae as necessary to maintain the colony. Several other species of Papilionidae were reared for use in experiments. Larvae of Papilio troilus L., taken from a laboratory colony maintained on the Cornell campus (Carter et al., 1999), were reared on foliage clipped from sassafras trees [Sassafras albidum (Nutt.)] in Ithaca, New York, USA. Larvae of Papilio polyxenes Fabricius, also taken from a local colony, were reared on leaves from potted carrot plants (Daucus carota L.) (Brooks, 2000). The offspring of Papilio glaucus L. females caught in the Ithaca area were reared on ash (Fraxinus americana L.). In addition, a nonpapilionid species, Callosamia promethea (Drury) (Saturniidae), was reared for use as a control. This species was chosen because it is readily available and because, like P. troilus and occasionally P. glaucus, it feeds on sassafras, thus introducing a control for the effects of food plant on host odors. The C. promethea larvae were the offspring of moths obtained as pupae from an amateur supplier in New Jersey and were reared on the leaves of local sassafras trees under the same conditions used to rear P. troilus. All insects were bred and reared in a climate-controlled chamber (16L:8D, 30/16◦ C day/night, and 80–85% relative humidity). Wasps used in experiments


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requiring naive females had no contact with hosts or with any plant matter between the time of their emergence and the time of the experiment. The pupae from which they emerged had been cleaned of silk and plant residue and placed on clean paper towels in a nylon-screened cage. Voucher specimens of butterflies, moths, and wasps were deposited in the Cornell University Insect Collection (lot no. 1240). Responses to Plants and Feeding Damage: Flight-Chamber Experiments. The flight chamber was a wooden cage screened with nylon mesh (185 × 92 × 92 cm). Plant samples were placed at one end and separated from each other by a cardboard partition projecting 50 cm into the cage. The wasps were released from vials at the opposite end of the chamber. A 50-cm-diam. fan was placed just outside the plant end of the chamber. Wind speeds were maintained at 1.0 m/sec at the plants, grading down to 0.5 m/sec at the release site, as measured by a handheld anemometer (Davis Instruments, Hayward, California, USA). The assembly was housed in a rooftop greenhouse on the Cornell University campus that was maintained at 24◦ C. Experiments were conducted near midday on sunny days in midsummer, using the sun as the main light source. The plants were placed in the southern end of the flight chamber to take advantage of the wasps’ phototactic responses. Twigs were cut shortly before each trial (except when exposed to host larvae as described below) and were kept in small vials of water during the trials. The positions of the two cuttings were switched with every fifth wasp. A wasp that did not cross into the partitioned area within 10 min was considered nonresponsive and excluded from the data set. The numbers of wasps that chose each plant species or treatment were compared with binomial probability functions (Conover, 1999). The first two flight-chamber experiments (1a and 1b) tested whether the wasps learn the odor of swallowtail food plants by comparing the responses of naive and experienced females to a choice of sassafras (the food plant of a host, P. troilus) and maple (Acer platanoides L.). Maple was chosen as the alternate because it is not fed on by any swallowtails, because it is a common tree in swallowtail habitats in the eastern United States, and because its lobed leaves resemble those of sassafras in gross outline, thus providing a control for visual preferences. Discrimination by naive females would indicate an innate preference. Alternatively, if only wasps with experience attacking hosts in the presence of sassafras favored the food plant, then a learned response would be indicated. In experiment 1a, females were tested before and after they had experience attacking hosts. The wasps were reared from E. marcellus larvae collected in Florida in spring 1996. The naive trials were conducted with 2-week-old mated wasps between June 30 and July 2. After the last trial, the wasps were placed in cages containing P. troilus larvae feeding on sassafras, which they attacked immediately. The experienced trials were conducted July 3–6. Experiment 1b controlled for the age of the wasps. The wasps had been reared from E. marcellus larvae collected in Florida in spring 1997. When they


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were 2 weeks old, the females were divided into two sets, one of which stayed naive while the other—the experienced set—was placed for a few hours on June 27 and 28 in boxes with first- and second-instar P. troilus feeding on sassafras cuttings. The two groups of wasps were tested concurrently from June 29 to July 2. Experiment 2 tested for an innate preference among different swallowtail food plants. Week-old, mated, naive females were offered a choice of sassafras and Asimina triloba, the food plants of P. troilus and E. marcellus, respectively, the most common hosts of T. pennator in the southeastern United States. The wasps were reared from E. marcellus larvae collected in Florida in spring 1997 and raised on Asimina. Any food-plant effects carrying over from the hosts or pupae were, thus, expected to bias the experiment towards Asimina. Experiment 3 tested whether host damage is innately attractive by examining the responses of naive wasps to a choice of sassafras leaves chewed by P. troilus larvae and sassafras leaves that had never been exposed to caterpillars. The naive wasps used in experiment 2 were refrigerated for four days and used again in this experiment. The unexposed sassafras leaves were mechanically damaged with scissors to control for effects of damage unrelated to larval feeding. The “chewed” leaves were exposed to P. troilus larvae overnight; the larvae were removed from the leaves 10 min before the start of the experiment. Experiment 4 expanded on experiment 3 by testing whether the wasps are more attracted to the feeding damage of their hosts than to the damage of other lepidopteran larvae. Naive wasps were offered a choice between sassafras leaves chewed by P. troilus and sassafras leaves chewed by the saturniid C. promethea. As in experiment 3, the larvae fed on the leaves overnight and were removed from them 10 min before the start of the experiment. Fourth and fifth instars of both species were used, so the amount of damage inflicted on the leaves was similar in the two treatments. The female wasps were reared from E. marcellus larvae collected in Florida in spring 1998; they emerged over several days in mid-June and were tested 10 days after emergence. Responses to Frass Extracts. Methylene chloride extracts of frass were prepared from the following species: E. marcellus (35 g frass collected), P. troilus (49 g), P. glaucus (7 g), P. polyxenes (5 g), and C. promethea (60 g). The larvae were reared on plant cuttings inserted into plastic vials and placed in plastic boxes. Each morning, frass that had accumulated in the bottom of the box was transferred to a container of CH2 Cl2 and ground with a glass rod to increase the efficiency of extraction. The extracts were refrigerated at 0◦ C. After collection was complete (usually after several days), the extracts were filtered under low vacuum through glass wool and Whatman No. 1 filter paper. They were then evaporated under reduced pressure at 40◦ C to a concentration of 0.25 g frass equivalent/ml. To test for arrestment responses to these extracts, the time females spent searching in the vicinity of the extract was measured. The extract was presented on a cotton swab in the center of a Petri dish lid (9 cm diam.), supported by a small


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piece of green plastic sponge (2 cm square × 0.5 cm high). The swab contained 2–3 drops of extract, which at the given concentration was roughly equal to the amount of frass produced by a fourth- or fifth-instar caterpillar in a few hours of feeding. The Petri lid was centered on the floor of a plastic box (35 × 27 × 60 cm), and the wasps were released into the box from a glass jar placed at one end of the box. Searching time was recorded as the time spent actively antennating the substrate while within the lid; time spent grooming or resting was excluded. Both experiments were carried out in the climate chamber described above. The first experiment compared the time that naive wasps spent searching at extracts from P. troilus and from the nonhost C. promethea. The trials were paired such that each wasp spent 5 min with one extract and then 5 min with the other (the order of presentation of extracts varied randomly from wasp to wasp). The average times spent at the extracts were compared with a two-tailed paired t test. The wasps originated in Florida and had been reared from E. marcellus larvae collected in spring 1998. To get a better idea of the extent to which these responses were particular to swallowtail frass, a second experiment was conducted which tested the wasps’ responses to extracts from the three Papilio species (P. troilus, P. polyxenes, and P. glaucus) and the C. promethea control. Each wasp spent 4 min with each of the extracts in succession, the order of presentation being varied at random. The wasps used in this experiment had been caught in Virginia at Asimina patches and were assumed to have had experience in the field with E. marcellus and possibly P. troilus. Because the wasps switch readily between host species in the laboratory (K.R.S., personal observation), such experience was not expected to affect the outcome of the experiment. Characterization of Frass Extracts. The preceding experiments compared the arrestant activities of the C. promethea, P. troilus, P. polyxenes, and P. glaucus frass extracts. The E. marcellus extract was simply tested against the solvent with a bioassay identical in design to the paired experiment described above. Following the biosassays, the compositions of the papilionid and nonpapilionid extracts were characterized and compared. Each extract was fractionated on an open column containing 30 g silica gel (Si-60, 32–63 µm). Five fractions were collected, corresponding to five additions of 250 ml solvent: pentane, pentane–methylene chloride (1:1), methylene chloride, methylene chloride–ether (1:1), and ether. The P. troilus extract was analyzed in detail. Each of its five fractions was bioassayed in paired experiments like those described above, comparing the searching activities elicited by the fraction and a solvent control. When the third fraction was identified as active, it was tested against a recombination of the five fractions (controlling for concentration) in order to determine the extent to which activity was limited to that fraction. Fractions of the E. marcellus, P. glaucus, B. philenor, and C. promethea extracts comparable to the third fraction of the P. troilus extract were prepared using the same procedures


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to fractionate the original CH2 Cl2 extracts. The eluting samples were monitored with thin-layer chromatography using a solvent system of equal portions hexane and ethyl acetate. These fractions were analyzed using a Hewlett-Packard HP5890 gas chromatograph coupled to an HP 5970B mass-selective detector. The GC employed helium as carrier gas, with a flow rate of 1 ml/min, and was fitted with an HP-1 capillary column. Injections were splitless. The temperature program started at 60◦ C, rising at a rate of 10◦ /min to 260◦ C, then holding for 5 min at 260◦ C. The major components of the samples were identified by matching EI spectra recorded at 70 eV to spectra in the Wiley library. Finally, to facilitate identification of active compounds, the active fraction of the P. troilus extract was further split into three subfractions (A, B, C). The fractionation procedures were like those described above except that the solvent system consisted of successive additions of 250 ml hexane–chloroform (1:4), chloroform, and chloroform–ethyl acetate (1:1). Each of the three resulting subfractions was bioassayed against chloroform.

RESULTS

Responses to Plants and Feeding Damage: Flight-Chamber Experiments. The outcomes of experiments 1a and 1b were similar (Table 1), with the less conclusive results of 1a possibly indicating effects of age. Naive females did not discriminate between maple and sassafras leaves, but the wasps that had attacked P. troilus larvae feeding on sassafras tended to fly towards sassafras. These results TABLE 1. RESPONSES OF NAIVE AND EXPERIENCED WASPS TO SASSAFRAS AND MAPLE (Acer) LEAVES IN A FLIGHT CHAMBERa Wasps favoring

Experiment 1a Naive Experienced Experiment 1b Naive Experienced a

Sassafras

Maple

P

11 14

13 7

0.42 0.09

16 26

19 14

0.37 0.04

In Experiment 1a, wasps were tested before and after experience with hosts feeding on sassafras. In Experiment 1b, two sets of wasps the same age were tested, one of which was given such experience. Significance levels are based on binomial probability functions.


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SIME

TABLE 2. EXPERIMENTS 2, 3, AND 4: RESPONSES OF NAÏVE WASPS TO VARIOUS CHOICES OF PLANT TREATMENTS IN A FLIGHT CHAMBER Plant choices Experiment 2 Wasps favoring each treatment (N ) Experiment 3 Wasps favoring each treatment (N ) Experiment 4 Wasps favoring each treatment (N )

Sassafras leaves 12 Sassafras damaged by P. troilus 20 Sassafras damaged by P. troilus 14

Asimina leaves 14 Artificially damaged Sassafras 10 Sassafras damaged by C. promethea 6

P

0.42

0.05

0.05

indicate that responses to plants not damaged by hosts are learned. Assuming that differences in visual cues were minimized between the two treatments, these experiments also show that odor plays a role in plant recognition. Because color and shape cues in the leaves were not strictly controlled for, however, the possibility that visual cues contributed to the wasps’ responses cannot entirely be eliminated. Naive wasps did not discriminate between two common swallowtail food plants, Asimina and sassafras (Table 2, experiment 2). Because the wasps used had been reared from E. marcellus larvae feeding on Asimina, this experiment also shows that neither the host nor its food plant biases the preferences of a naive wasp. Experiment 3 indicated that naive wasps favor leaves damaged by hosts over mechanically damaged leaves, while experiment 4 showed that naive wasps prefer leaves damaged by a host over leaves damaged by another lepidopteran species (Table 2). Since there were no differences in the appearances of the leaves used in these treatments, it may be concluded that the wasps were responding to odors associated with host damage. Responses to Frass Extracts. Naive wasps searched longer at a methylene chloride extract of the frass of P. troilus (a host) than at an extract from C. promethea (a nonhost) (Figure 1). Arrestment responses to extracts of the frass of P. troilus, P. glaucus, and P. polyxenes were similar, and all were significantly greater than responses to the extract from C. promethea (Figure 2). Because both the P. troilus and the C. promethea larvae had fed on sassafras, both experiments show that the wasps respond to odors in frass that are peculiar to host larvae. Characterization of Frass Extracts. The preceding experiments showed that the methylene chloride extracts of the frass of P. troilus, P. glaucus, and P. polyxenes larvae elicit an arrestant response in T. pennator. Similarly, an extract from E. marcellus was searched significantly longer than a solvent control (Table 3). Bioassays of the five fractions of the P. troilus extract indicated that


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FIG. 1. Mean searching times of naive female wasps at methylene chloride extracts of frass from a papilionid host, P. troilus, and from the saturniid moth C. promethea (N = 17, P < 0.01). Both species were reared on sassafras foliage.

nearly all of the arrestant activity could be attributed to fraction 3 (Table 3): the wasps searched significantly longer at fraction 3 than at the solvent control, and its activity was comparable to that of the recombined fractions. Of the three subfractions prepared from fraction 3, only the middle one (3B) elicited greater activity than the solvent control. A number of compounds were identified in fraction 3 of frass extracts from C. promethea, P. troilus, E. marcellus, and P. glaucus (Table 4). Concentrations of compounds in fraction 3B of P. troilus proved insufficient to make reliable identifications.

FIG. 2. Mean searching times of female wasps at methylene chloride extracts of frass from three host Papilio species and the saturniid C. promethea (N = 24, P < 0.05).


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SIME TABLE 3. BIOASSAYS OF METHYLENE CHLORIDE EXTRACTS OF FRASS AND FRACTIONS THEREOFa Treatment 1

Treatment 2

N

E. marcellus extract 41.2 ± 9.6∗ P. troilus fraction 1 26.9 ± 7.4 P. troilus fraction 2 18.3 ± 8.0 P. troilus fraction 3 55.3 ± 17.0 P. troilus fraction 4 13.3 ± 5.0 P. troilus fraction 5 4.9 ± 5.0 P. troilus recombined fractions 1–5 41.6 ± 6.0 P. troilus fraction 3A 22.5 ± 5.5 P. troilus fraction 3B 23.3 ± 5.5 P. troilus fraction 3C 13.8 ± 4.1

solvent 18.3 ± 3.3 solvent 14.5 ± 5.6 solvent 4.2 ± 2.0 solvent 28.4 ± 12.9 solvent 4.81 ± 3.2 solvent 2.86 ± 1.0 P. troilus fraction 3 35.6 ± 7.4 solvent 21.8 ± 5.6 solvent 6.2 ± 1.8 solvent 8.7 ± 3.6

11

a

P

0.027 16 0.15 8 0.14 11 0.044 10 0.26 8 0.33 14 0.47 15 0.91 15 0.0047 15 0.16

Mean searching times (seconds) ±SE are presented for each treatment; the means were compared by two-tailed paired t tests. DISCUSSION

The flight-chamber experiments, although limited in the number of plant species tested, provide evidence that T. pennator females emerge without preference for the food plants of swallowtails over other plant species or for particular swallowtail food plants over others. The Ocala National forest is at the southern edge of the geographical range of sassafras (Brown and Kirkman, 1990). The wasps used in these experiments therefore came from a population whose members do not normally encounter sassafras, although they may encounter P. troilus larvae feeding on another lauraceous plant, Persea borbonia L. (Lederhouse et al., 1992). That they can learn to associate the odor of sassafras with the presence of host larvae indicates considerable flexibility in their capacity to recognize plants, as would be expected in a widespread species that attacks hosts feeding on a diverse array of plant families (Vet and Dicke, 1992). The attraction to undamaged plants observed in the flight chamber also helps explain the observation that in the field, female T. pennator approach and search food plants lacking feeding damage significantly more often than they approach damaged plants (by a ratio of about 2:1) (Sime, 2001). While the field observations suggested that vision plays


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TABLE 4. METHYLENE CHLORIDE EXTRACTS OF FRASS COMPARED VIA GC-MSa C. promethea P. troilus P. glaucus E. marcellus Long-chain acids Tetradecanoic acid (16.8)∗ Pentadecanoic acid (17.9) Hexadecanoic acid (18.5) Octadecanoic acid (21.0) Fatty acid esters 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z) (20.3) 9,12-Octadecadienoic acid (Z,Z) methyl ester (20.8) Hexanedioic acid, dioctyl ester (22.5) Terpenoids β-Citronellol (9.3) trans-Geraniol (9.7) Neophytadiene (17.4) (−) - loliolide (15.9) Long-chain alcohol 9,12,15-Octadecatrien-1-ol (12.1) Long-chain aldehyde 9,12,15-Octadecatrienal (20.2) Aromatic aldehydes 1 H-Indole-3-carboxaldehyde (16.7) 4-Hydroxy-3-methoxy-benzeldehyde (11.9) Aromatic esters 1,2-Benzenedicarboxylic acid, bis (2-ethylhexyl) ester (24.7) Aromatic acid benzoic acid (8.7) Benzofuranone, benzopyranone 5,6,7,7a-Tetrahydro-4,4,7a-trimethyl-2(4H)benzofuranone (13.7) 7-Hydroxy-6-methoxy-2H-1-benzopyran2-one (18.5) a

+ +

+ +

+

+ + + + + +

+ + + +

+

+

+

+ + + + +

+ + + + + + +

The compounds listed were identified with a relatively high degree of certainty (>90%) based on library matches. Retention times are given in minutes. ‘+’ = present.

a role in plant recognition, the flight-chamber experiments show that the odor of undamaged plants can also be learned. One consequence of the attraction of naive wasps to odors associated with feeding damage is that they need not land on and search plants at random before their first encounters with host larvae, but instead can rapidly come to focus on the host’s food plant. Field data for female T. pennator indicate that the feeding damage of E. marcellus larvae on Asimina plants is detected at distances of at least 1 m (Sime, 2001); studies of other species, conducted in flight chambers or


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wind tunnels, have also provided evidence that feeding-damage odor is involved in medium-range (on the order of meters) orientation (e.g., Geervliet et al., 1994). The attraction of naive wasps to the so-called plant-host odor complex has been observed in many species of Braconidae (Turlings et al., 1990; Steinberg et al., 1992; Udayagiri and Jones, 1993; Reed et al., 1995; Geervliet et al., 1996) and Ichneumonidae (Lecomte and Thibout, 1986; Vinson and Williams, 1991). Other studies have found that the odor of lepidopteran feeding damage is species specific and that parasitoids can distinguish host odors from those of nonhosts (Blaakmeer et al., 1994; Agelopoulos et al., 1995; DeMoraes and Mescher, 1999), although such discrimination is not always observed (McCall et al., 1993; Geervliet et al., 1996). It has also often been shown that naive wasps are attracted to the frass of their hosts (Lewis and Jones, 1971; Hendry et al., 1973; McKinney and Pass, 1977; Lewis and Tumlinson, 1988; Vet and Dicke, 1992; Suiter et al., 1996). It is usually assumed that the wasps are able to distinguish the frass of their hosts from that of other species, but most studies of frass do not test responses to nonhosts or distinguish compounds of plant origin from those modified by digestion and metabolism in the larvae (Ramachandran et al., 1991). The finding that T. pennator females favored P. troilus frass over C. promethea frass when both species were reared on sassafras indicates that the frass of two species feeding on the same food plant can differ in odor and that the wasps recognize the difference. Furthermore, swallowtail frass is recognized and about equally attractive regardless of food plant. In the four-way experiment, the Papilio species had fed on Lauraceae (P. troilus), Apiaceae (P. polyxenes), and Oleaceae (P. glaucus). To this list may be added Aristolochiaceae: a separate study showed that a methylene chloride extract of the frass of Battus philenor (L.) larvae feeding on Aristolochia was as attractive to T. pennator females as was an extract from P. troilus (Sime, 2002). Whereas the Lauraceae and Apiaceae resemble each other in odor and essential oil content, and the Aristolochiaceae share some of these chemical qualities, the Oleaceae are chemically very different (Feeny, 1991). The chemical basis of frass discrimination is difficult to pinpoint. None of the compounds or classes of compounds identified in the active fraction of the frass extracts (Table 4) is characteristic of swallowtails, and there appears to be as much overlap in frass composition between C. promethea and any given swallowtail as there is among swallowtail species. Even if any of these compounds should prove attractive to T. pennator, it is unlikely that any one compound would provide a basis for distinguishing host from nonhost. Two bases for host discrimination are possible, and neither can be rejected with the available evidence. Critical compounds that characterize swallowtails might be among the minor, unidentified constituents of the extracts. Alternatively, the wasps may respond not to the presence or absence of particular compounds, but instead to a particular blend of compounds; such blends have been found to be critical for recognition of host odors associated with feeding


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damage (Turlings et al., 1990, 1991; Tumlinson et al., 1992, 1993; Agelopoulos and Keller, 1994; Geervliet et al., 1994). The observation that the wasps searched at the C. promethea extracts at all merits mention, for it indicates that, despite their evident preference for swallowtail frass, the wasps may also have generalized responses to the odors of frass. The compounds eliciting the arrestment response would, thus, appear to include a combination of swallowtail-specific cues plus more general cues of either caterpillar or plant origin. Finally, the possibility that C. promethea frass contains deterrent compounds should not be dismissed, as it was not accounted for in the design of these experiments. These results suggest a general caveat for the interpretation of data concerning the semiochemical content of frass. Although many evidently attractive compounds have been identified in the frass of lepidopteran larvae (Tumlinson et al., 1992; Rutledge, 1996), the ecological roles of these compounds have not always been clearly demonstrated. It is usually not known whether any are individually characteristic of the host or if they are simply components of attractive blends used to identify hosts. The distribution of identified attractant compounds within Lepidoptera is not known; in many systems it would be useful to know which appear in the frass of other lepidopteran larvae feeding on the same or similar plants. Compounds widespread in Lepidoptera cannot by themselves permit host recognition. Although the active compounds or blends that permit T. pennator females to distinguish papilionid larvae from nonhosts were not identified, the work reported here provides evidence that hosts differ chemically from nonhosts and that this difference is discerned by wasps with no prior experience of hosts. Odors associated with both host-damaged leaves and host frass were distinguished from odors produced by a saturniid feeding on the same plants. These findings indicate that the host range of T. pennator is a function of attractive compounds shared by and unique to swallowtail larvae, and they are consistent with the hypothesis that the chemical similarity of hosts is an essential parameter of the host range of taxonomic specialists (Gauld, 1988; Vet and Dicke, 1992). One alternative explanation for restricted host ranges is that host range is a function of responses to habitat or food plant (Gauld, 1988). Because T. pennator is not known to attack other Lepidoptera that share food plants with the Papilionidae, this hypothesis cannot provide a complete explanation of its host restriction; moreover, it is invalidated by the finding that responses to plant odors are learned. Another possible explanation is that host range may be a function of physiological compatibility (Brodeur and Vet, 1995). This hypothesis is compatible with the two behavioral hypotheses but would be more compelling had no support been found for the others. Possible physiological mechanisms underlying host range have not been studied in Trogus. However, given the evidence found in this study for behavioral correlates with host range, it is unlikely (at least among the potential host species studied) that physiology is an important determinant of host use.


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Acknowledgments—J. Brooks, J. Carpenter, M. Carter, P. Feeny, C. Heinz, M. McDonald, S. Murphy, C. Tauber, M. Tauber, and S. van Nouhuys helped in the field and laboratory and in the preparation of this manuscript. M. Haribal analyzed GC-MS data, and A. Attygalle (Cornell University Department of Chemistry) provided access to GC-MS instrumentation. J. Clutts (Lake George Ranger District, Silver Springs, Florida) permitted work in the Ocala National Forest. This work was funded by National Science Foundation Research Grants IBN-9119674 and IBN-9420319 to Paul Feeny and IBN-9600094 to K.R.S. and P. Feeny, and by grants from the Rawlins Fund (Department of Entomology, Cornell) and the Andrew Mellon Foundation to K.R.S. The author was also supported by a Liberty Hyde Bailey Fellowship (Cornell University College of Agriculture and Life Sciences) and a National Science Foundation Graduate Research Fellowship. REFERENCES AGELOPOULOS, N. G. and KELLER, M. A. 1994. Plant-natural enemy association in the tritophic system, Cotesia rubecula–Pieris rapae–Brassicaceae (Cruciferae). III. Collection and identification of plant and feces volatiles. J. Chem. Ecol. 20:1955–1967. AGELOPOULOS, N. G., DICKE, M., and POSTHUMUS, M. A. 1995. Role of volatile infochemicals emitted by feces of larvae in host-searching behavior of parasitoid Cotesia rubecula (Hymenoptera: Braconidae): A behavioral and chemical study. J. Chem. Ecol. 21:1789–1811. BLAAKMEER, A., GEERVLIET, J. B. F., VAN LOON, J. J. A., POSTHUMUS, M. A., VAN BEEK, T. A., and DE GROOT, A. 1994. Comparative headspace analysis of cabbage plants damaged by two species of Pieris caterpillars: Consequences for in-flight host location by Cotesia parasitoids. Entomol. Exp. Appl. 73:175–182. BRODEUR, J. and VET, L. E. M. 1995. Relationships between parasitoid host range and host defence: A comparative study of egg encapsulation in two related parasitoid species. Physiol. Entomol. 20:7–12. BROOKS, J. S. 2000. Chemical signals on leaf surface: Keys to recognition by ovipositing insects. PhD dissertation. Cornell University, Ithaca, New York. BROWN, C. L. and KIRKMAN, L. K. 1990. Trees of Georgia and Adjacent States. Timber Press, Portland, Oregon. CARTER, M., FEENY, P., and HARIBAL, M. 1999. An oviposition stimulant for spicebush swallowtail butterfly, Papilio troilus, from leaves of Sassafras albidum. J. Chem. Ecol. 25:1233–1245. CONOVER, W. J. 1999. Practical Non-Parametric Statistics, 3rd ed. Wiley, New York. DEMORAES, C. M. and MESCHER, M. C. 1999. Interactions in entomology: Plant–parasitoid interaction in tritrophic systems. J. Entomol. Sci. 34:31–39. FEENY, P. 1991. Chemical constraints on the evolution of swallowtail butterflies, pp. 315–340, in P. W. Price, T. M. Lewinsohn, G. W. Fernandes, and W. W. Benson (eds.). Plant–Animal Interactions: Evolutionary Ecology in Tropical and Temperate Regions. John Wiley, New York. GAULD, I. D. 1988. Evolutionary patterns of host utilization by ichneumonid parasitoids (Hymenoptera: Ichneumonidae and Braconidae). Biol. J. Linn. Soc. 35:351–377. GEERVLIET, J. B. F., VET, L. E. M., and DICKE, M. 1994. Volatiles from damaged plants as major cues in long-range host-searching by the specialist parasitoid Cotesia rubecula. Entomol. Exp. Appl. 73:289–297. GEERVLIET, J. B. F., VET, L. E. M., and DICKE, M. 1996. Innate responses of the parasitoids Cotesia glomerata and C. rubecula (Hymenoptera: Braconidae) to volatiles from different plant-herbivore complexes. J. Insect Behav. 9:525–538. HEINRICH, G. 1962. Synopsis of the nearctic Ichneumonidae Stenopneusticae with particular reference to the northeastern region (Hymenoptera): Part VII: Synopsis of the Trogini. Can. Entomol. Suppl. 29:807–860.


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