Insect Science (2012) 00, 1–10, DOI 10.1111/j.1744-7917.2012.01575.x
ORIGINAL ARTICLE
Cabbage waxes affect Trissolcus brochymenae response to short-range synomones Francesca Frati, Gianandrea Salerno and Eric Conti Dipartimento di Scienze Agrarie e Ambientali, Universita` di Perugia, Borgo XX Giugno 74, Perugia 06121, Italy
Abstract We show that induced synomones, emitted as a consequence of Murgantia histrionica activity on Brassica oleracea, are adsorbed by the epicuticular waxes of leaves and perceived by the egg parasitoid Trissolcus brochymenae. Leaves were exposed to M. histrionica females placed on the abaxial leaf surface. After 24 h, the leaves were treated mechanically using gum arabic, or chemically using chloroform, on the adaxial surface, and finally the adaxial surface was assayed with T. brochymenae by two-choice tests in a closed arena. Wasp females responded to mechanically dewaxed cabbage leaf portions with feeding punctures and footprints (Ff) and with feeding punctures, oviposition and footprints (FOf), showing no effect of wax removal. In contrast, the removal of the epicuticular waxes from leaf portions close to FOf, and from leaves with oviposition and footprints (Of), determined the lack of responses by T. brochymenae. Solvent extracts of different treatments were bioassayed, but only FOf triggered parasitoid response. Thus the detection of oviposition-induced synomones by the parasitoid depends on their adsorption by the epicuticular waxes. Mechanical wax removal from leaf portions contaminated with host footprints (f) also determined a lack of wasp responses, suggesting that the footprints might trigger the induction of a “footprint-induced synomone” adsorbed onto the epicuticular waxes and exploited by the parasitoid. Leaf portions with the abaxial lamina previously dewaxed and then contaminated by footprints (D+f) of M. histrionica did not affect the parasitoid response, indicating that the abaxial epicuticular waxes are not directly involved in the chemicals induced by M. histrionica footprints. Key words Brassica oleracea, egg parasitoid, induced synomones, Murgantia histrionica, oviposition
Introduction The interface where interactions between herbivorous insects and natural enemies occur is frequently the plant cuticle, and particularly the epicuticular waxes (Eigenbrode & Espelie, 1995; M¨uller & Riederer, 2005; Rost´as et al., 2008). Epicuticular waxes are the outermost layer that cover the higher plant cutin matrix, through
Correspondence: Francesca Frati, Dipartimento di Scienze Agrarie e Ambientali, Universit`a di Perugia, Borgo XX Giugno 74, Perugia 06121, Italy. Email:
[email protected]
a thin film that can be characterized by the presence of aggregate protrusions (Barthlott et al., 1998). These waxes play an essential ecological function as they mediate the interactions with phytophagous insects, presenting the physical and chemical cues involved in the first step of host plant location by herbivores (M¨uller & Riederer, 2005). In particular, the epicuticular layer may adsorb volatile secondary plant metabolites that attract or repel herbivores from a distance (M¨uller & Riederer, 2005). Furthermore, nonvolatile secondary metabolites may be deposited on the plant surface and they can act as kairomones or allomones (M¨uller & Riederer, 2005). 1
C 2012
Institute of Zoology, Chinese Academy of Sciences
2
F. Frati et al.
Epicuticular waxes also mediate some steps of the host location behavior by carnivorous insects, since parasitoids and predators may respond to cues from the plant surface to locate plants with the potential hosts or preys. In addition, the plant surface may interfere with parasitoid attachment and mobility on the substrate (Eigenbrode & Espelie, 1995; Eigenbrode & Kabalo, 1999; Romeis et al., 2003; Rutledge et al., 2003; Chang et al., 2004; Eigenbrode, 2004). Thus, when the foraging success of natural enemies is evaluated, possible effects of the wax layer on kairomone detectability should be considered. In fact, the efficacy of insect parasitoids could be influenced by plants with specific physiochemical properties of leaf waxes that allow the adsorption and release of volatile and/or contact host kairomones (M¨uller & Riederer, 2005). Females of the egg parasitoid Trichogramma evanescens Westwood (Hymenoptera: Trichogrammatidae) can perceive the volatile pheromone produced by their host Mamestra brassicae (L.) (Lepidoptera: Noctuidae), due to pheromone adsorption and subsequent release by the plant waxes of Brussels sprouts for up to 24 h (Noldus et al., 1991). The larval parasitoid Cotesia marginiventris Cresson (Hymenoptera: Braconidae) can detect contact chemical footprints produced by walking caterpillars of its host Spodoptera frugiperda Smith (Lepidoptera: Noctuidae), because they are adsorbed on the wax surface of host plants (Rost´as et al., 2008). Similar to the effect recorded for the above-mentioned system (Rost´as et al., 2008) is the role of broad bean epicuticular waxes on semiochemical communication between Nezara viridula L. (Heteroptera: Pentatomidae) and Trissolcus basalis Wollaston (Hymenoptera: Platygastridae). Chemical footprints left on the substrate by N. viridula females are perceived by T. basalis females as a result of their absorption onto epicuticular waxes (Colazza et al., 2009; Lo Giudice et al., 2010, 2011). During the host location process, egg parasitoids use plant synomones and/or host kairomones, which act both from a distance and from a short range (reviewed by Fatouros et al., 2008; Colazza et al., 2010; Hilker & Meiners, 2010, 2011). Plant synomones play a key role, especially when they are emitted as a consequence of herbivore attack. The emission of plant synomones can be induced by insect oviposition, and these synomones will attract specific egg parasitoids (Meiners & Hilker, 1997, 2000; Hilker & Meiners, 2002; Hilker et al., 2002; Colazza et al., 2004a, 2004b; Fatouros et al., 2005, 2007, 2008, 2009). The egg parasitoids might also respond to short-range induced synomones that are perceived after they have alighted on the plant. In fact, egg deposition can induce changes of the plant surface that arrest a host searching egg parasitoid, thus increasing the probability
of encountering host eggs (Fatouros et al., 2005, 2007; Conti et al., 2010; Hilker & Meiners, 2011; Blenn et al., 2012). In the tri-trophic system Brassica oleracea L. – Murgantia histrionica Hahn (Heteroptera: Pentatomidae) – T. brochymenae Ashmead (Hymenoptera: Platygastridae), the parasitoid responds, increasing its residence time, to host-induced plant synomones which are perceived at a very short distance and on contact (Conti et al., 2010). In all experiments, conducted under controlled laboratory conditions, only the abaxial leaf surface was exposed to the host for the treatments while the adaxial surface was bioassayed. When the leaf portion that had been directly affected by bug activity was assayed, females of T. brochymenae responded to (i) cabbage leaves with feeding damage and footprints; (ii) leaves with feeding, oviposition, and footprints; (iii) leaves with oviposition and footprints; and (iv) leaves with only footprints. When cabbage leaf portions close to the treatments were considered, wasp females reacted to (i) portions close to feeding, oviposition, and footprints, and (ii) close to oviposition and footprints (Conti et al., 2010). These results have conducted to the hypothesis that cabbage compounds, induced as a consequence of M. histrionica activity on cabbage plants, might be partially adsorbed by the leaf epicuticular waxes and then slowly released, thus allowing perception by the parasitoid from a very short distance or through contact. Therefore, in the present study, we tested whether the leaf epicuticular waxes mediate perception by the egg parasitoid T. brochymenae. The parasitoid behaviors after its contact with the plant and its responses to plants subjected to the removal of waxes were investigated. In particular, two techniques were used for wax removal: a mechanical method using an adhesive and a chemical method with a specific solvent (Jetter et al., 2000).
Materials and methods Insect cultures M. histrionica was originally collected from cabbage in the Beltsville area, Maryland, USA in 2000. Adults of T. brochymenae were obtained from M. histrionica eggs laid on Isomeris arborea Nutt. (Capparidaceae) in San Diego, California, in 2000. Both insects were maintained in quarantine conditions at the entomology laboratories of the University of Perugia, Perugia, Italy. The colony of M. histrionica was reared in a controlled condition chamber (25± 1◦ C, 60% ± 5% relative humidity and L : D 15 : 9 h photocycle), inside clear plastic food containers C
2012 Institute of Zoology, Chinese Academy of Sciences, 00, 1–10
Cabbage waxes and oviposition synomones
(300 × 195 × 200 mm high) with 5 cm diameter meshcovered holes. All stages were fed vegetative and reproductive parts of cabbage and broccoli (Brassica oleracea L.). The colony of T. brochymenae was reared on eggs of M. histrionica, and adult wasps were kept in 85 mL glass tubes at controlled conditions in an incubator (25 ± 1◦ C, 80% ± 5% relative humidity and L : D 15 : 9 h photocycle), and fed small drops of Safavi diet (Safavi, 1968). After emergence, male and female parasitoids were kept together to allow mating. In all experiments, female wasps, two or three days old, mated and na¨ıve to the host, were used. Females were individually isolated in small vials 16–17 h before the bioassays, fed small drop of diet and allowed to acclimatize in the bioassay room for at least 30 min before testing. Plants Seeds of savoy cabbage B. oleracea var. sabauda (cv. Salto, kindly provided by Royal Sluis Brand, Seminis, Parma, Italy) were individually planted in pots filled with peat and after seven days were transplanted into plastic pots filled with a mixture of inert substrates such as agriperlite (BPB Vic; Agrimport, Italy) and vermiculite (Sopram, Italy). Plants were kept in a greenhouse under controlled conditions (25 ± 3◦ C, 50%–60% relative humidity and L : D 12 : 12 h photocycle), watered daily and fertilized with a water solution of Flory 9 Hydro (NP-K 15–7-22) (Planta Regenstauf, distributed by Agrimport SPA, Bolzano, Italy), sequestrene (NK 3–15 containing Fe EDDHA, Syngenta) and urea (Hydro Agri Italia Milano). All experiments were carried out using five or six weeks old plants. Treatments The following treated B. oleracea plants were considered for the experiments: plants with M. histrionica feeding punctures and footprints (Ff); plants with feeding punctures, oviposition, and footprints (FOf); plants with oviposition and footprints (Of); plants with M. histrionica footprints (f) (Fig. 1). All treated plants were obtained by exposing them to two mated M. histrionica females (14–16 days old), placed individually on the abaxial leaf surface (Fig. 1), inside a small clip cage. The latter was obtained from two Petri dishes (35 mm diameter, 10 mm height), with the bottoms substituted by a fine nylon mesh and each rim of the opposite side covered with a small foam rubber ring, which was kept tightened to the leaf with the help of a suitable clip. These plant–bug complexes were kept in a controlled C 2012
Institute of Zoology, Chinese Academy of Sciences, 00, 1–10
3
environment cabinet (25◦ C, 60% ± 5% relative humidity and L : D 15 : 9 h photocycle). After 24 h, M. histrionica females were removed and the treated leaves were used for the bioassays. To prevent bug feeding and to obtain plants with oviposition and footprints (Of) or just footprints (f), gravid females with excised stylets were used. Ovipositing females were previously anaesthetized inside a glass tube with CO2 for 4–5 s. Afterward their stylets were drawn from the labium with an entomological pin (no. 000) and amputated half their length with the aid of precision R Stemi SV8) micro-scissors, a stereomicroscope (Zeiss R and optical fiber illumination (Intralux 5000). Treated females were placed inside a plastic dish (12 cm diameter) for 24 h before they were placed on cabbage plants.
Experiment 1 – mechanical removal of epicuticular waxes and leaf bioassays Behavioral assays were performed to test whether shortrange synomones, induced on cabbage plants by M. histrionica activity on the abaxial leaf surface, can be detected by T. brochymenae females on the adaxial leaf surface after mechanical removal of the epicuticular waxes from such surface. Consequently, the bioassays aimed at evaluating whether short-range synomones are adsorbed by the epicuticular waxes. For such purpose, herbivore-treated leaf portions and leaf portions close to the treated areas were tested, focusing on the types of treatments described above [Ff (dewaxed once and three times); FOf; Of and f] (Fig. 1). A different experiment was conducted to evaluate the role of the abaxial epicuticular waxes in the semiochemical communication between M. histrionica and cabbage plants, using cabbage leaves with M. histrionica footprints left on previously dewaxed abaxial surface (D+f) leaving the adaxial leaf surface intact. The epicuticular waxes were mechanically removed from healthy (control) and treated (test) cabbage leaves using an aqueous solution of pretreated gum arabic (Sigma– Aldrich) as described by Jetter and Sch¨affer (2001). A 50% (w/w) aqueous solution of adhesive was applied on the adaxial leaf surface using a small paintbrush and left to air dry at room temperature for about 1 h. Then, the resulting polymeric film was carefully stripped off using forceps. In the case of Ff, three applications of gum arabic were also made to evaluate whether the epicuticular material was exhaustively removed (Wen et al., 2006). Dewaxed leaves from healthy and treated plants were first cut at the petiole level. Then the following leaf
4
F. Frati et al.
Leaf portion of the treated area
Leaf portion close to the treated area
Leaf portions for bioassays on dewaxed adaxial leaf surface
Treatments on the abaxial leaf surface
Feeding (F) Oviposition (O)
Footprints (f) Ff
FOf
Of
f
Fig. 1 A cabbage leaf, exposed to a Murgantia histrionica female placed on the abaxial leaf surface, dewaxed and then tested with Trissolcus brochymenae on the adaxial surface. The following leaf treatments were assayed: M. histrionica feeding punctures and footprints (Ff); feeding punctures, oviposition, and footprints (FOf); oviposition and footprints (Of); footprints (f). Leaf portions of the treated area and leaf portions close to the treated area were used for the bioassays.
squares (200 mm2 ) were cut with a razor and used for the behavioral assays: healthy; directly affected by bug activity, that is, leaf portion on which the host had walked, fed and/or oviposited (local induction); at a close distance from the attacked portion on the same leaf (close-distance induction) (leaf portion that was 8–10 mm away from the treated area). In all experiments, only the adaxial leaf surface was tested (i.e., the surface opposite to that exposed to M. histrionica, as explained above) (Fig. 1) to avoid the presence of perceivable host kairomones on the plant. Moreover, to avoid possible cues originating directly from deposited egg masses, these were removed from the leaf surface before the bioassays.
adaxial surface of cabbage leaves and whether T. brochymenae females respond to solvent extracts of these synomones. Solvent extracts of the cabbage waxes were obtained from the following treated leaves (Fig. 1): Ff, FOf, and Of. Treated leaves were cut at the petiole level and in the case of FOf and Of the egg masses were always removed before solvent extraction. Four milliliter of chloroform (Fluka) were applied to the adaxial leaf surface of a cabbage leaf. The leaf was placed on a flexible rubber mat, a glass cylinder (4 mm diameter) was gently pressed onto the exposed surface, and the solvent mixture was agitated for 30 s by pumping with a Pasteur pipette and then removed. This procedure was repeated four times to treat the entire test area (∼200 mm2 ). The obtained extract was reduced to dryness under a nitrogen stream and rieluited with 1 mL of chloroform. A 500 µL of extract (test) and 500 µL of pure solvent (control), respectively, were applied directly on a half-section of a filter paper disk (5.5 cm diameter; 119 mm2 area; pb ref. 500-A) and left to evaporate for 30–40 min before the bioassays.
Experiment 2 – chemical removal of cuticular waxes and extract bioassays Behavioral tests were carried out to evaluate whether short-range synomones, induced on cabbage plants by M. histrionica activity, can be chemically removed from the
C
2012 Institute of Zoology, Chinese Academy of Sciences, 00, 1–10
Cabbage waxes and oviposition synomones
Bioassay procedures All behavioral, two-choice, tests were performed between 09.00 and 13.00 hours in a climate-controlled room at 25 ± 1◦ C and 55%– 60% relative humidity. In the bioassays with mechanical removal of epicuticular waxes the responses of T. brochymenae females to dewaxed treated leaves (test) and to dewaxed healthy plants (control) were evaluated in a closed arena. The arena was R plate with a circular hole assembled from a Plexiglass in the middle (25 mm diameter and 5 mm height), sandwiched between two glass plates (bottom and cover) and illuminated by two 6 V 20 W halogen lamps (approximately 6500 lux). Expressly made tunnel (3 mm diameter and and 10 mm length) on the external sides of the arena wall was used to introduce the parasitoid female. Two leaf portions, from test and control, were placed on the base of the glass plate, at about 5.0 mm distance from each other, and the whole system was centered on the arena floor. Each portion of cabbage leaves was used for testing about five wasp females. After five bioassays, the entire device was changed and the arena was washed with water and detergent, whereas the glass plates were washed with water and detergent, wiped with acetone, and kept in an oven overnight at 120◦ C. The number of replicates for each bioassay is reported in the figures. The responses of T. brochymenae females to chloroform extracts from treated leaves were performed in a closed arena thinner than that used in the Experiment 1. This arena was utilized to stimulate the parasitoid to walk on the treated surfaces reducing the time spent on the covering glass plate. The arena was assembled from a foam rubber plate (G¨uttermann AG, Landstrasse 1, D79261 Gutach-Breisgau) with a circular hole in the middle (25 mm diameter and 2 mm height) sandwiched between one foam rubber plate (bottom) and one glass plate (cover). Also in this kind of arena, a specifically made tunnel (3 mm diameter and 10 mm length) on the external side of the arena wall was used to introduce the parasitoid. The arena was illuminated as mentioned above. Two half portions of a filter paper treated respectively with solvent extract (test) and pure solvent (control) were placed on the base of foam rubber plate, at about 5.0 mm distance from each other. Each portion of treated paper disk was used for about eight wasps. After eight bioassays the apparatus was changed and the arena and the foam rubber plate were washed with water and detergent, whereas the glass plate was washed with water and detergent, wiped with acetone, and kept in an oven overnight at 120◦ C. The number of replicates for each bioassay is reported in the figures. During all bioassays, a single female was released in the middle of the arena, and observations lasted for the total C 2012
Institute of Zoology, Chinese Academy of Sciences, 00, 1–10
5
duration of 10 min. Parasitoid behavior was recorded using a zoom-equipped (125–175 mm) video camera (JVC KY-M280) connected to a video capture device (Pinnacle Dazzle DVC 100) to allow the digitization of the images (25 frames/s). The behavioral data were collected using a software for behavioral observations (The Observer Video-Pro version 4, Noldus, Information Technology, Wageningen, The Netherlands). Scanning electron microscopy Samples of about 0.5 cm2 of healthy cabbage leaves (with cuticular waxes, dewaxed with gum arabic or treated with chloroform) were slowly allowed to dry overnight at 20–25◦ C. Leaf portions were then mounted on aluminium holders using double-sided adhesive tabs (ProSciTech), taking care to place the treated side upward. All specimens R SCD 040 unit) were sputtered with gold (Balzers Union R and observed with SEM (Philips XL 30). Statistical analysis The time spent by the female wasp searching on the treatment, on the control or on the arena surface was scored and the percentages of residence time (%) on either the treatment or the control, related to total time on leaf surface or treated filter paper, were calculated. The residence time properly indicates the choice of the parasitoid in a two choice test and describes the wasp searching behavior, which is characterized by returning several times to the treated area, followed each time by an examination of the surface around the treated area. The residence time on the treatment was compared to the control time on the test with the Student’s t-test for dependent samples. In the case of the Experiment 1, the comparison, in terms of residence time, between control and treated leaf portion avoided the effect on the results of possible green leaf volatiles emitted by both leaf squares. Before analysis, Box-Cox transformation was used to reduce data heteroscedasticity [Statistica 6.0, Statsoft, 2001, Vigonza (PD), Italy] (Zar, 1999). Results Experiment 1 – mechanical removal of epicuticular waxes and leaf bioassays T. brochymenae females responded both to Ff leaf portions subjected to one (t = 6.926; df = 25; P < 0.000 1) or three (t = 4.696; df = 15; P = 0.000 3) dewaxing procedures, and to FOf (t = 8.174 4; df = 22; P < 0.000 1), as
6
F. Frati et al.
Results with not dewaxed leaves (Conti et al., 2010)
C
