Introduction

For parasitoids, semiochemicals play an important role in each stage of locating, recognizing, and accepting a potential host (Vinson 1998; Steidle and Van Loon 2002). Finding a host’s microhabitat, e.g., its food plant, is often accomplished by orienting toward herbivore-induced volatile plant compounds (Turlings et al. 1995; Dicke et al. 2003; Heil 2008). Upon landing on a plant, chemical cues of lower volatility that are more closely associated with a host become important. Such chemicals (kairomones) may emanate directly from the host or from host by-products like feces or moth scales (Godfray 1994).

In recent years, it has become clear that small amounts of chemicals remain on the substrate when insects walk over a plant surface. These chemical footprints can be exploited as host location cues by foraging natural enemies, or they may serve other purposes in inter- and intraspecific interactions such as oviposition deterrence or the marking of previously visited flowers in the case of bumblebees (Klomp 1981; Hemptinne et al. 2001; Borges et al. 2003; Conti et al. 2003; Eltz 2006; Colazza et al. 2007; Collatz and Steidle 2008; Rutledge et al. 2008).

The parasitoid Cotesia marginiventris Cresson (Hymenoptera: Braconidae) is distributed from North to South America and has been considered for use in augmentative biological control of vegetable pests. As a solitary koinobiont endoparasitoid, it parasitizes early instar lepidopteran larvae belonging to the Noctuidae family (Riddick 2006). Its host location behavior has been investigated intensively, mainly in studies that address the parasitoids’ long-range attraction to host plants by using herbivore-induced volatiles and in the context of how these signals may be influenced by biotic and abiotic factors (Turlings et al. 1995; Cardoza et al. 2002; Gouinguené and Turlings 2002; Rostás et al. 2006; Winter and Rostás 2008). Earlier research also has assessed the parasitoids’ responses to contact kairomones. By-products of the host such as feces, larval and pupal cutical material, scales, silk, or oral secretion elicit increased klinokinesis, antennal palpation, or ovipositor probing (Loke and Ashley 1984a, b; Dmoch et al. 1985).

In addition to these cues, Rostás et al. (2008) found that naïve C. marginiventris can recognize chemical footprints on the wax surface of leaves produced by walking second-instar caterpillars of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae). In the current study, we addressed the question of how long such caterpillar footprints can be detected by the female parasitoid, and we investigated their chemistry.

Materials and Methods

Insects Eggs of S. frugiperda (Smith) were provided by Bayer CropScience AG (Monheim, Germany) on a weekly basis. After hatching, larvae were reared in plastic boxes (19 × 9 × 5.5 cm) and provided with kidney bean-based artificial diet for noctuids (modified from King and Leppla 1984). Insects were kept in a climate chamber with a 15:9-h L/D photoperiod at 28/25°C (L/D) and 75% humidity. After 5 to 6 days, caterpillars were used either for parasitoid rearing or for experiments. All caterpillars were in their second larval stage in which they are still small and feed gregariously.

A colony of Cotesia marginiventris (Cresson) was maintained in the laboratory. For rearing, about 45 S. frugiperda larvae were offered to three mated C. marginiventris females in a plastic box (20 × 20 × 5.5 cm). Wasps were allowed to oviposit for 24 h and were then removed. Herbivore larvae were kept in the boxes until the emergence of the wasp cocoons. Cocoons were removed from the herbivore boxes and transferred to rearing cages (Bugdorm I, Megaview Science Education Services, Taichung, Taiwan). Cages were checked daily for eclosed imagines. Adult parasitoids were provided with water and honey.

Barley (Hordeum vulgare cv. Bonus) was cultivated in pots (9-cm diameter). Plants were kept in growth chambers at 300–400 μmol photons m−2 s−1 light intensity with a day/night cycle of 16:8 h (24:18°C) and 70% relative humidity.

Behavioral Assays

A 5.5-cm diameter glass Petri dish covered by a glass plate was used as a test arena for observing the wasps’ host location behavior. The Petri dish was surrounded by a 25-cm-high white cardboard cylinder and illuminated by a 25-W light bulb from above. The antennation behavior of single wasps, which had no previous experience with caterpillars, was observed for 10 min using the software Noldus Observer 5.0 (Noldus Information Technologies, Wageningen, Netherlands). Only insects that spent more than 50% of the time in motion were used for analyses. Wasps that showed no antennation behavior throughout the experiment were subjected to a motivation test in which they were provided second-instar host larvae for oviposition. Wasps that laid an egg into at least one caterpillar were included in the analyses as we assumed that they were in oviposition mood even though they did not display antennation behavior. Antennation on the cut edges of the leaves was observed occasionally but not recorded, as C. marginiventris is known to respond to green leaf volatiles released by damaged plant tissue (Hoballah and Turlings 2005). Fifteen wasps were observed in each experiment if not stated otherwise. All dual-choice assays were analyzed by using nonparametric Wilcoxon matched pairs test. Wasp responses to hexane and methylene chloride extracts of caterpillars were compared with Bonferroni-corrected Mann–Whitney U test.

Persistence of Caterpillar Tracks

A clip cage (diameter 20 mm, height 26 mm) covered with gauze on the top and bottom end for ventilation was attached to the upper part of the second leaf of a potted barley plant (H. vulgare cv. Bonus). Clip cages contained four caterpillars, which were left to walk over the leaf surface for 15 min. Clip cages were then removed, and the leaf was examined carefully. Only leaves without bite marks, feces, or silk were used in the experiments. Leaves with any kind of residue were discarded. As controls, leaves were used onto which empty clip cages had been attached. The upper part of the leaf (5 cm length) was cut off at different time points (0, 24, 48, and 72 h after clip cage removal). A test and a control leaf section were placed into the arena for observation of parasitoid antennation behavior (see above). Leaf material and wasps were exchanged after each observation.

Chemistry of Caterpillar Tracks

In a first bioassay, we tested total body extracts of second-instar S. frugiperda assuming that footprint components should be a fragment thereof. Groups of ten caterpillars were shock-frozen in −80°C and then extracted with 1 ml methanol, methylene chloride, and n-hexane (Carl Roth GmbH KG, Karlsruhe, Germany), respectively. After 1 min, extracts were filtered (Schleicher and Schüll 595, Dassel, Germany) and evaporated to dryness under a stream of nitrogen. Residues were dissolved in 100 µl of the appropriate solvent and stored at −80°C until used. Pure solvents were used as controls.

Extracts of chemical footprints were prepared by keeping second-instar S. frugiperda without food overnight to allow for defecation. Thirty-three to 34 larvae were then left to walk on two microscopic glass slides placed side-by-side, since glass has been shown to provide a good surface for isolating and collecting insect footprints. The slides were covered with a small glass Petri dish (diameter 25 mm, height: 11 mm) to prevent larvae from escaping. All glassware was cleaned thoroughly with distilled water, ethanol, acetone, and n-hexane before use. After 1 h, caterpillars were removed, and both glass slides and Petri dish were rinsed with 1 ml n-hexane. This procedure was repeated 9×. Three of the extracts were pooled to obtain the footprints of 100 caterpillars. Each of the three pooled extracts was then concentrated to 80 µl under a gentle flow of nitrogen. Glassware without caterpillars was rinsed with n-hexane as a control. All extracts were kept at −80°C until used for bioassays or GC-MS analyses.

For comparison of cuticular hydrocarbons, the ventral side of the caterpillar body was extracted with n-hexane. Twenty caterpillars were frozen in liquid nitrogen. Then, a 1-µl droplet of n-hexane was placed onto a microscopic glass slide with a gas-tight syringe (Hamilton-Bonadaz, Bonaduz, Switzerland). The drop spread over the glass slide covering it as a thin film. By using soft forceps, a caterpillar was placed quickly into the n-hexane film before the solvent had evaporated completely. By this procedure, the surfaces of the ventral side including legs, pseudopods, and claspers were extracted. Due to the small size of the second instar, it was not feasible to extract only the extremities.

Observations of wasp antennation responses to the total body and chemical footprint extracts were performed as described above. Single C. marginiventris were offered two segments of barley leaves. Each segment was treated with test or control extract (total body 10 µl/leaf, chemical footprints 30 µl/leaf). The solvent was left to evaporate completely before observation started. Wasps were exchanged after each observation. Treated leaves were replaced after five observations (total body) or after each observation (chemical footprints). Fifteen replications were carried out for each treatment.

In another bioassay, wasp antennation responses to the main compounds of the footprint extract were tested. This was accomplished by partially reconstructing the footprint extract with a mixture of standard n-alkanes (nC21–nC32, all Sigma-Aldrich, Taufkirchen, Germany). GC analyses of the reconstructed blend were carried out to verify that quantities and ratios of the alkanes were the same as in the original footprint extract that was previously offered to C. marginiventris.

Gas Chromatography

Hexane extracts were analyzed by gas chromatography and mass spectrometry (Agilent Technologies 6890N Network GC System coupled with a 5973 Network Mass Selective Detector). Three microliters of each sample were injected with an automated injection system in pulsed splitless mode. The column was an Agilent 19091-s933 HP-1 capillary column (length 30 m, diameter 0.25 mm, film thickness 0.25 µm). The oven was held at 150°C for 2 min, and then increased at 5°C min−1 to a final temperature of 320°C, which was held for 20 min. Helium (1.5 ml min−1) was used as carrier gas. Linear alkanes were identified by their characteristic EI-MS fragmentation pattern and in addition by using MSD ChemStation (Agilent Technologies) software with the Wiley 275 and NIST 98 mass spectrum libraries. Identities were confirmed further by comparing retention indices and mass spectra with those of authentic n-alkane standards (nC21–C32, Sigma-Aldrich, Taufkirchen, Germany). In the case of the monomethyl-alkanes, tentative identification was based on the characteristic fragmentation pattern that indicated the position of the methyl-branch and on the retention indices of the closest n-alkanes. Quantities of hydrocarbons were assessed by the external standard method. Calibration curves to determine linearity were obtained from each identified n-alkane at four concentrations (1, 5, 10, 25 ng/µl) with three replications per concentration. Linearity was assumed when the regression coefficient R 2 was >0.998. Quantities of monomethyl-alkanes and unidentified compounds were estimated by comparing peak areas with those of the closest standard n-alkane and, thus, were not as absolute as in the case of n-alkanes.

Results

Bioassays Assessing Kairomone Persistence

The duration of high frequency antennation behavior of C. marginiventris observed at various time points after removal of larvae is summarized in Fig. 1. Choice tests showed that female wasps responded most strongly to leaf segments on which caterpillars had been removed right before the experiment started. All parasitoids responded and displayed prolonged antennal drumming on treated leaves (0 h Z  = 3.408, P < 0.001). Significantly longer sequences of antennation on caterpillar-treated leaves were also observed after a time lag of 24 and 48 h, respectively, following the brief exposure to second-instar S. frugiperda (24 h Z  = 2,981, P  = 0.003; 48 h Z  = 2.293, P  = 0.022). However, 93% of the observed wasps responded in the 24-h test, while 80% displayed antennal palpation in the 48-h test. After a period of 72 h, wasps still showed a tendency for caterpillar-treated leaf segments, but the difference in antennation time compared to control leaves was no longer significant (72 h Z  = 1.820, P  = 0.069). Antennal drumming was observed in 53% of tested wasps.

Fig. 1
figure 1

Antennal drumming of C. marginiventris on caterpillar footprints. Dark boxes represent barley leaves on which caterpillars of S. frugiperda had walked upon for 15 min. White boxes represent controls. Times (0–72 h) above boxes indicate lag time between removal of caterpillars and observation of wasp behavior. Boxes show median (solid line) and mean (dashed line), 25th and 75th percentiles. Whiskers are 5th and 95th percentiles. Dots indicate outliers. Asterisks denote significant differences: ***P < 0.001, *P < 0.05, n.s. not significant

Full size image

Chemical Analyses

Twenty-one out of 26 detected compounds were identified in the hexane extracts of S. frugiperda footprints (Table 1, Fig. 2). All major compounds were odd and even numbered linear alkanes with chain lengths ranging from 21 to 32 carbon atoms (>90% of total). The three most abundant compounds were nC25 (13.5%), nC26 (10.4%), and nC27 (10.3%), representing 34% of the total hydrocarbon extract. Apart from linear alkanes, only monomethyl-branched compounds (4MeC24–4MeC28 and 3MeC25–3MeC28) could be identified. Comparison with hexane extracts of the ventral side of second-instar caterpillars showed that the footprint compounds were present in the same ratios in the body extract, as well.

Fig. 2
figure 2

Chromatograms of footprint and cuticle extracts. Compounds are listed by their numbers in Table 1

Full size image
Table 1 Compounds identified from footprint extracts of Spodoptera frugiperda caterpillars
Full size table

Bioassays that Assessed Kairomone Chemistry

Female C. marginiventris responded equally well to hexane and methylene chloride extracts of whole caterpillars (hexane vs. control, Z  = 2.52, P  = 0.012, methylene chloride, Z  = 2.52, P  = 0.012; hexane vs. methylene chloride, Mann–Whitney U test, U  = 112.5, P  = 0.999; Response: 53% of wasps in both treatments). Virtually, no response was observed when leaves were treated with methanol extracts of the herbivore. One parasitoid showed brief antennal drumming on the test leaf, another individual responded to the control.

In a bioassay that tested hexane extracts of caterpillar footprints, 67% of the observed wasps elicited characteristic antennation behavior (Fig. 3a). The footprint extract was more attractive than the control extract (Z  = 2.803, P  = 0.005). The mix of n-alkanes (C21–32) applied to the barley surface elicited a weaker but still significant response in C. marginiventris (Z  = 1.991, P  = 0.046). Forty percent of the tested individuals responded in the experiment (Fig. 3b).

Fig. 3
figure 3

Antennal drumming of Cotesia marginiventris on a hexane extracts of caterpillar footprints and b n-alkanes found in footprint extract. Dark box represents leaves treated with footprint extract or n-alkanes, respectively. White box represents hexane control. Boxes show median (solid line) and mean (dashed line), 25th and 75th percentiles. Whiskers are 5th and 95th percentiles. Dots indicate outliers. Asterisks denote significant differences: *P < 0.05, **P < 0.01

Full size image

Discussion

The parasitoid C. marginiventris responds to chemical residues (footprints) of its lepidopteran host S. frugiperda with characteristic host recognition behavior, displayed as antennal drumming on the substrate (Rostás et al. 2008). However, the persistence and chemistry of these footprints had not yet been investigated.

Our experiments showed that the host location kairomone was persistent for a relevant period of time. Only 15 min of walking by four small caterpillars left sufficient amounts of infochemicals on the plant surface to induce a clear antennation response 48 h after caterpillars had been removed. Following a lag time of 72 h, antennation time on the treated area was no longer significant. These results suggest that host-derived chemicals are uncharged lipophilic compounds of low volatility, as only such chemicals can be sorbed onto the leaf surface and stay there for some time due to the lipophilic properties of the plant cuticle (Müller and Riederer 2005). Little is known about the persistence of insect walking tracks. Hemptinne et al. (2001) reported that females of the ladybird Adalia bipunctata avoid laying eggs on filter paper contaminated with conspecific larval tracks for at least 10 days. Tracks produced by ladybird larvae were predominantly straight-chain and methyl-branched alkanes in the C21 to C33 range. The bioactivity of footprints is expected to vary with the nature and concentration of deposited chemicals, the substrate, and the perceiving insect species. Most insect tracks reported, so far, consist of long-chain alkanes and alkenes (Kosaki and Yamaoka 1996; Hemptinne et al. 2001; Votsch et al. 2002; Nakashima et al. 2004; Eltz 2006; Colazza et al. 2007). Thus, their persistence can be expected to be in the range of days rather than hours. An exception, however, was found in the tracks of the grain beetle Oryzaephilus surinamensis that can be detected by the parasitoid Cephalonomia tarsalis for only 30 min (Collatz and Steidle 2008). Aldehydes rapidly oxidizing to fatty acids were the active components in the footprints of the beetle (Collatz, personal communication).

Extracts with methylene chloride and hexane, but not methanol, of entire S. frugiperda caterpillars elicited clear antennal drumming responses in female C. marginiventris to equal extents. This shows that wasps use apolar cuticular components for host recognition and suggests that caterpillar footprints should be lipophilic, as well. Further bioassays with hexane extracts of S. frugiperda footprints have confirmed this notion, as wasps responded positively to the offered residue chemicals. The major compounds that account for more than 90% of the footprint extract are a homologous series of saturated alkanes ranging from heneicosane (nC21) to dotriacontane (nC32) with pentacosane (nC25) being the most abundant. Monomethyl-branched alkanes and five unidentified compounds also were present, but occurred only as minor compounds. An almost identical pattern of chemicals was found in the hexane extract of the caterpillar’s ventral cuticle. Thus, we suggest that footprint compounds may be of cuticular origin. Thoracical legs, prolegs, and claspers were observed to be in contact with the substrate (Fig. 4), and it seems that the most likely source for the kairomone should be the caterpillar’s prolegs and claspers. These extremities are covered by a cuticle and represent a larger surface in contact with the leaf than the claws of thoracical legs.

Fig. 4
figure 4

Spodoptera frugiperda walking on a barley leaf. Note that only thoracical legs, prolegs, and claspers touch the substrate

Full size image

With regard to compositions and ratios of cuticular and footprint alkanes, remarkably similar findings have been reported from the pentatomid bug Nezara viridula (Colazza et al. 2007). Trissolcus basalis, a parasitoid of this bug, uses the deposited hydrocarbons to locate host eggs and may even distinguish between male and female residues by the presence or absence of n-nonadecane. In two other species of the genus Cotesia (C. kariyai and C. plutellae), linear, monomethyl- and dimethyl-branched alkanes (C20–C40) from the cuticula of lepidopteran hosts also are known to serve as host recognition cues upon contact with the host (Ohara et al. 1996; Roux et al. 2007). Whether some of these compounds are deposited on the substrate, where they can be exploited as host location kairomones, remains to be investigated.

In a final experiment, we tested the wasps’ response to the main compounds of the footprint extract. Commercially available nC21–nC32 alkanes were used to partially reconstruct the chemical footprint extract in the same amounts and ratios as had been tested in the preceding bioassay. C. marginiventris females showed significant antennal drumming on leaves treated with this n-alkane blend. However, the response was considerably weaker, suggesting that some minor components, such as the methyl-branched alkanes or other unidentified chemicals are important for host recognition, as well. Our observation is consistent with studies on N. viridula and its egg parasitoid T. basalis where a reconstructed blend of straight-chain hydrocarbons extracted from the host also induced only weak arrestment responses in T. basalis females (Colazza et al. 2007). Methyl-branched alkanes and alkenes often occur in insect hydrocarbons, and sometimes, they have behavioral activity as contact pheromones (Ginzel et al. 2003). In plant waxes, however, they can be detected only in trace amounts, if at all. This and the fact that n-alkanes are common plant wax compounds suggests that minor constituents may enable wasps to detect insect hydrocarbons against a background of similar plant hydrocarbons. In the experiments described here, barley leaves were used as a surface for caterpillars to walk on. The epicuticular wax of this plant contains very small proportions of n-alkanes with heptacosane (nC27) as the only overlapping compounds that are also present in the chemical footprint extract (Rostás et al. 2008). At least in this case, it can be assumed that linear alkanes derived from the caterpillars can be recognized as host-specific kairomones. Future research will need to assess whether the degree of overlap between plant and insect hydrocarbons plays a role in the perception of chemical footprints on different waxy surfaces. Depending on the specific wax composition, the detection of chemical footprints on different plant surfaces may be easier or more difficult for hunting parasitoids and predators.

In summary, we confirmed that parasitoids use footprint chemicals deposited on the epicuticular wax layer of plants as low-volatile cues to find their host in its microhabitat. Tracks produced by caterpillars consist mostly of linear alkanes and minor amounts of branched alkanes that presumably originate from the cuticle of prolegs and claspers. On barley leaves, these footprints can be detected by C. marginiventris for a maximum of 2 days. Our findings add to the growing body of literature demonstrating the importance of insect-derived chemicals deposited on plants. These serve as a bridge in time and thus mediate numerous interactions. We expect that future research will establish the significance of residual chemicals in regulating behavior—an aspect of insect ecology that awaits full appreciation.