Behavioral and electrophysiological responses of the parasitic wasp Psyttalia concolor (Szépligeti) (Hymenoptera: Braconidae) to Ceratitis capitata-induced fruit volatiles

Biological Control 64 (2013) 116–124

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Behavioral and electrophysiological responses of the parasitic wasp Psyttalia concolor (Szépligeti) (Hymenoptera: Braconidae) to Ceratitis capitata-induced fruit volatiles Giovanni Benelli a, Santosh Revadi b, Adriano Carpita c, Giulia Giunti a, Alfio Raspi a, Gianfranco Anfora b, Angelo Canale a,⇑ a

Entomology Section, Department of Tree Science, Entomology and Plant Pathology, University of Pisa, Via San Michele degli Scalzi 2, 56124 Pisa, Italy Research and Innovation Centre, Fondazione Edmund Mach, Via Edmund Mach 1, 38010 San Michele all’Adige, Trento, Italy c Department of Chemistry and Industrial Chemistry, University of Pisa, Via Risorgimento 35, 56100 Pisa, Italy b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

" Psyttalia concolor $$ prefer medfly-

"

"

"

"

infested peaches and apples over healthy ones. Twelve volatiles (HIPVs) are characteristic of infested peaches and apples. Except ethanol and ethyl acetate all HIPVs evoke GC-EAD responses in $$ wasps. Among peach HIPVs, ethyl octanoate, decanal and 4-decanolide attract $$ wasps. Among apple HIPVs, butyl butylate and butyl hexanoate attract $$ wasps.

a r t i c l e

i n f o

Article history: Received 12 July 2012 Accepted 9 October 2012 Available online 2 November 2012 Keywords: Opiinae Host location Mediterranean fruit fly Semiochemicals GC-EAD Biological control

a b s t r a c t Psyttalia concolor (Szépligeti) is a koinobiont larval-pupal endoparasitoid of many Tephritidae of great economic importance, such as the medfly, Ceratitis capitata (Wiedemann). In several species of parasitoids it has been demonstrated that the mated females are strongly attracted by specific volatiles from insect-damaged plants. Yet the role of olfactory cues deriving from C. capitata-infested fruits on the female’s decision during the P. concolor host location was poorly investigated. In the present study, the responses of P. concolor females to either healthy or C. capitata-infested fruits was studied through behavioral assays. Volatiles emitted by healthy and infested fruits were SPME-sampled and analyzed by gas chromatography–mass spectrometry (GC–MS). The attractiveness of the identified volatiles was assessed and their electrophysiological activity was analyzed through gas-chromatography coupled with electroantennography (GC-EAD). P. concolor preferred infested peaches and apples over healthy ones, either when visual and olfactory or only olfactory cues were given. Nine compounds were found as exclusive of infested peaches, with respect to healthy ones, and seven of them evoked electrophysiological responses. In apples only quantitative changes in volatile emissions were observed after the medfly infestation. The emissions of 1-butyl butylate, 1-hexyl acetate and 1-butyl esanoate increased in infested apples, whereas 1-hexyl (E)-2-methyl butenoate decreased significantly. Among apple volatiles, 1-butyl butylate, 2-methyl-1-butyl acetate, 1-hexyl acetate, 2-methyl-1-butyl 2-methylbutanoate, 1-butyl hexanoate and 1-hexyl (E)-2-methyl butenoate elicited responses in female antennae. Synthetic blends reproducing the odors emitted by infested peaches and apples elicited strong attraction towards P. concolor

⇑ Corresponding author. Fax: +39 0502216130. E-mail address: [email protected] (A. Canale). 1049-9644/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2012.10.010

G. Benelli et al. / Biological Control 64 (2013) 116–124

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females. For both fruits, the blend attractiveness was mainly due to some specific electrophysiological active chemicals: ethyl octanoate, decanal and 4-decanolide for peach, and 1-butyl butylate and 1-butyl hexanoate for apple. The responses induced by the identified fruit volatiles to P. concolor females allow us to suppose that they play a role as short-range attractants during host location. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction The role of plant volatiles upon the interaction between phytophagous insects and their natural enemies has been first reviewed by Price et al. (1980). They stated the theory on plant insect-interactions can only be realized when the third trophic level is considered. Since the 1990s, it has been demonstrated for many plant species that volatiles induced by insect feeding are used as foraging cues by parasitoids of phytophagous insects (Dicke and Sabelis, 1988; Hare, 2011; Kaplan, 2012). To date, it is widely recognized that many plants have evolved a high diversity of constitutive and induced resistance traits, including the systemic emission of herbivore induced plant volatiles (HIPVs, hereafter) (Hare, 2011; Gols et al., 2011; Lucas-Barbosa et al., 2011). In the latest years, a very large number of laboratory and field trials were performed in order to investigate the HIPV-mediated carnivorous arthropods attraction and their implications in integrated pest management programs (Shiojiri et al., 2010; Kaplan, 2012). A noticeable part of the researches were focused on the trophic interactions between plants, phytophagous insects and parasitic wasps. In several species of parasitoids – including pteromalids (Belda and Riudavets, 2010), eulophids (Rojas et al., 2006; Cusumano et al., 2010), encyrtids (James and Grasswitz, 2005), mymarids (Krugner et al., 2008), ichneumonids (Orre et al., 2010) and braconids (Röse et al., 1997; Gols and Harvey, 2009; Erb et al., 2010; Holopainen and Gershenzon, 2010; Mandour et al., 2011) – it was observed that the mated females are strongly attracted by specific volatiles from insect-damaged plants. For example, among braconids the importance of olfactory cues during host location has been demonstrated in several species that attack larval instars of several tephritid flies (Greany et al., 1977; Messing et al., 1996; Jang et al., 2000; Henneman et al., 2002; Stelinski et al., 2004; Carrasco et al., 2005; Ero et al., 2010). To date, some qualitative and quantitative differences among volatile emissions were found between tephritid-infested and healthy fruits (Henneman et al., 2002; Carrasco et al., 2005; Kendra et al., 2011), and in several cases it was demonstrated that these HIPVs are able to evoke electrophysiological and behavioral responses in braconid females (Carrasco et al., 2005). Psyttalia concolor (Szépligeti) (Hymenoptera: Braconidae) is a koinobiont larval-pupal endoparasitoid of many Tephritidae. It is able to attack at least fourteen tephritids on different wild and/or cultivated plants, including pests of great economic importance, such as the medfly, Ceratitis capitata (Wiedemann), and the olive fruit fly, Bactrocera oleae (Rossi) (Wharton, 1997). In Italy, P. concolor can be found, in late autumn, on B. oleae in Sicily, southern Sardinia, Corsica and in various areas of coastal Tuscany (Raspi et al., 2007). This parasitoid has been used in Italy and other Mediterranean areas for the biological control of B. oleae by inundative and propagative releases, with limited results (for a synthesis see Daane and Johnson, 2010). It has recently been released in Californian olive-groves as part of classical biological control programs (Yokoyama et al., 2008; Wang et al., 2011). Since 1990s, P. concolor has been continuously reared on the medfly in the Entomology laboratory at the University of Pisa. It has been possible to use the parasitoid in experiments aimed at controlling the olive fruit fly (Raspi and Loni, 1994; Loni et al., 2010). Some researches were conducted to describe the courtship and mating behavior of this wasp (Benelli

and Canale, 2012a; Benelli et al., 2012a; Canale et al., 2012), as well as to elucidate the physiological and behavioral interactions that are established between the parasitoid and its hosts (Canale, 1998, 2003; Raspi and Canale, 2000; Canale and Loni, 2006; Benelli and Canale, 2012b; Canale and Benelli, 2012). However, the role of olfactory cues deriving from tephritids-infested fruits on the female’s decision during the P. concolor host location was yet poorly investigated. In a preliminary study conducted in a still-air arena with portions of medfly-infested fruits it was observed that the P. concolor females were more attracted by infested fruits over to both healthy fruits or infested fruits in which medfly larvae were removed immediately before the test (Benelli et al., 2012b). We hypothesize that the HIPVs from host-infested decaying fruits may have a crucial role in affecting the P. concolor host seeking behavior, therefore suggesting the need of a more detailed investigation. Thus, the present research is aimed at determining the nature and the importance of the olfactory cues used by P. concolor females in order to locate the microhabitat (fruit) of its tephritid host C. capitata. Here, (i) the response of P. concolor females to visual and olfactory cues from healthy and medfly-infested apples and peaches was evaluated, (ii) volatiles emitted by healthy and infested fruits were SPME-sampled, analyzed by gas chromatography-mass spectrometry (GC–MS) and compared, (iii) compounds emitted only, or in significantly different amounts, from infested fruits were identified by GC and GC–MS analyses, (iv) the attractiveness of the identified volatiles was evaluated and (v) the electrophysiological responses (GC-EAD) of the HIPVs from both fruits were recorded in order to select the antennally active compounds.

2. Materials and methods 2.1. Parasitoid and host rearing Females of the original parasitoid colony were collected in 1990 from infested olive fruits harvested in Palermo (Sicily, Italy). During the following 22 years of mass-rearing this strain was refreshed three times (in 1995, 1998 and 2004) through the release of wild P. concolor specimens collected from infested olives in Tuscany (1995, 1998) and Calabria (south of the Italy, 2004). Parasitoid P. concolor and host C. capitata were reared as described by Canale and Benelli (2012). In summary, the C. capitata production unit is composed of cylindrical PVC cages, each containing about 2000 adults (sex-ratio 1:1). Adults are fed on a dry diet composed of sugar and yeast extract (10:1). Eggs are collected every 2 days and distributed into plastic bowls (50  15  2 cm) each containing 0.5 kg of artificial culture medium. Cages for P. concolor breeding are made of transparent Plexiglas tubes (diameter: 40 cm, length: 50 cm) into which 300 adults are introduced (6:10 male-to-female sex ratio). Nylon mesh bags, each containing up to 500 medfly fully-grown larvae, are introduced into the cage for the parasitization phase (20 min). After emergence from the host and until the test, parasitoids were stored in cylindrical Plexiglas cages (diameter: 40 cm, height: 50 cm) at a density of 100 specimens (males:females ratio 1:2) per cage [22 ± 1 °C, 45 ± 10% relative humidity and 16:8 (L:D) photoperiod]. A semisolid diet (honey mixed with pollen) and water were

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offered to the wasps. To obtain medfly-infested fruits, apples (cv. ‘‘Golden Delicious’’) and peaches (cv. ‘‘Marygold’’) were collected in the experimental fields of the Department of Tree Science, Entomology and Plant Pathology (University of Pisa) during October and June 2011, respectively, and immediately exposed to C. capitata mated females, inside ovipositional cages that were maintained under the laboratory condition reported above. Fruits were left in the cage for 24 h. Infested fruits were then transferred to plastic boxes with wire-mesh tops. Under these conditions, fruits infested with early third-instar larvae were obtained after 8–10 days of preimmaginal development, and then they were used for behavioral and volatile collection experiments. 2.2. Attractiveness of medfly-infested fruits: effect of combination of cues The preference of P. concolor females for infested or healthy fruits was evaluated through bioassays in a PlexiglasÒ cylindrical arena (diameter: 25 cm, length: 70 cm) (Fig. 1a). In this setup olfactory, visual and contact cues were combined and all available to the wasps. In order to prevent saturation with volatiles, both ends of the arena were made with transparent chiffon fabric, as well as the two little windows (5  3 cm) placed on the top in the central part of the arena. Chiffon fabrics used at the ends of the arena were changed at every bioassay. In each trial, two combinations of cues were simultaneously offered at the opposite ends of the arena. Both for apple and peach fruits, the treatments were carried out as follows: (i) an healthy fruit vs. an odorless fruit dummy (MeschiÒ, Lucca, Italy) and (ii) a medfly-infested fruit vs. an healthy fruit. All tested wasps were 8 days old mated females. After emergence and before testing, the wasps were kept in Pisa laboratories [22 ± 1 °C, 45 ± 10% relative humidity and 16:8 (L:D) photoperiod], and fed on honey and pollen on a cotton wick. For each two-choice bioassay, sixty females were gently transferred to the center of the arena through an entrance hole (diameter: 2 cm) and released inside; six replicates were made. In each replicate, P. concolor females were introduced into the arena in the morning and the number of

wasps landed on each fruit was recorded after 1, 5 and 24 h. Each wasp was used only once. After each trial, the cylindrical arena was cleaned with water, then with pure ethanol and finally with hot water. In each replicate, the arena was rotated clockwise 90° to avoid positional effects and the position of each combination of cues was randomly assigned. Chi-square tests were used to evaluate data from two-choice bioassays (Sokal and Rohlf, 1981). 2.3. Attractiveness of medfly-infested fruits: effect of olfactory cues The role of olfactory cues in P. concolor host seeking behavior was evaluated using the still-air olfactometric arena described in Benelli et al. (2012c), which is schematically represented in Fig. 1b. Both for apple and peach fruits, the treatments to evaluate the role of host-fruit odor stimuli in eliciting the female hostsearching behavior, were carried out as follows (see also Table 1): (i) healthy fruit vs. blank (i.e. an empty plot); (ii) medfly-infested fruit vs. healthy fruit. In each replicate, sixty P. concolor females were gently transferred inside the olfactometer in the morning and the number of wasps present in each chamber was recorded after 24 h. Each wasp was used only once. In each replicate, the two chambers were randomly located in positions A or B, the olfactometer was rotated clockwise 90° to avoid positional effects and the position of each cue was randomly assigned. To prevent visual orientation to the wasps, the plots were covered with a black opaque cloth. After each trial, the glass parts of the olfactometer were dismantled and cleaned with water and then with pure ethanol and hot water. For each two-choice bioassay six replicates were made. Chi-square tests were used to evaluate data from two-choice bioassays (Sokal and Rohlf, 1981). 2.4. SPME sampling of fruit volatiles and chemical analyses Gas chromatography (GC) analyses were performed using a Dani GC 1000 instrument with PTV injectors, equipped with a Dani DDS 1000 data station and two bonded FSOT columns (a Dani DN-5 and a Dani DN-20, both 30 m  0.25 mm i.d.). Gas chromatogra-

Fig. 1. (a) Schematic representation of the still-air cylindrical arena (diameter: 25 cm, length: 70 cm) used to test attraction of Psyttalia concolor females to visual, olfactory and contact cues. A and B: cues were given at opposite ends of the arena, e.g. a healthy fruit (left) and an infested one (right); C and D: the ends of the arena were made with transparent chiffon fabric; E and F: ventilated zone, made of 5  6 cm mesh; G: entrance hole, 30 mm diameter. (b) Schematic representation of the still-air olfactometer used to test attraction of P. concolor females to olfactory cues. A, B: 500-ml glass pot, each containing an odor source; C: PVC connecting black tubes; D: transparent plastic (PET) bottles (300 mm length; 100 mm larger diameter, 25 mm smaller diameter); E: ventilated zone, made of 5x6 cm mesh (F); G: entrance hole, 30 mm diameter (re-adapted from Kaspi, 2000).

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Table 1 Changes in volatile emissions from infested and healthy peaches, and absolute (mV) GC-EAD responses (mean ± SD) of Psyttalia concolor females to synthetic HIPVs at two dosages, 1 and 10 lg/lL (n = 10). In the GC-EAD box, values within the same column followed by the same letter are not statistically different (ANOVA, Tukey HSD test, P < 0.05). For each box’s row, differences in the GC-EAD responses between the two dosages were evaluated using Student’s t-test. The last box reports the number of P. concolor ovipositor probing responses displayed in close proximity (61.5 cm) of filter paper treated with HIPV synthetic chemicals or hexane. Thirty wasps were tested in each experiment. GC–MS R.T. (min)

Healthy fruit*

Infested fruit*

GC-EAD R.T. (min)

GC-EAD ± SD (mV  103 lg) 1 lg/lL

10 lg/lL

Peach blend vs. hexane Ethanol Ethyl acetate Ethyl octanoate

–

– 0 0 0

–

–

1.05 ± 0.36 5.66 ± 1.24 18.59 ± 3.83

– 2.11 1.52 10.45

–

1.65 2.35 15.54

0 0 1.62 ± 0.26 a

0 0 2.31 ± 0.42 a

Decanal

15.91

0

0.77 ± 0.12

12.10

1.80 ± 0.35 a

2.65 ± 0.39 a

Nonanoic acid

17.05

0

0.38 ± 0.13

21.59

4.89 ± 0.89 c

5.09 ± 0.53 c

Decanoic acid

18.11

0

1.03 ± 0.30

23.20

3.26 ± 0.55 b

3.90 ± 1.15 b

(E)-6,10-dimethyl-5,9undecadien-2-one 4-Decanolide

19.15

0

0.87 ± 0.35

17.28

1.93 ± 0.33 a

2.50 ± 0.58 a

19.54

0

0.42 ± 0.19

22.06

2.01 ± 0.40 a

2.19 ± 0.47 a

Dodecanoic acid

20.55

0

2.13 ± 0.57

26.50

2.77 ± 0.57 b

2.90 ± 0.72 b

ANOVA, Tukey’s test

F = 50.2; d.f. = 69; P < 0.001

F = 25.2; d.f. = 69; P < 0.001

Peach volatiles

t-test

Number of probing $$**

– – – t = 4.5; d.f. = 19; P < 0.001 t = 5.1; d.f.=19; P < 0.001 t = 0.7; d.f.=19; P = 0.52 t = 1.3; d.f.=19; P = 0.020 t = 2.7; d.f. = 19; P < 0.05 t = 1.0; d.f. = 19; P = 0.35 t = 0.5; d.f. = 19; P = 0.63

7 0 0 8

vs. vs. vs. vs.

0 0 0 0

5 vs. 0 0 vs. 0 0 vs. 0 0 vs. 0 2 vs. 0 0 vs. 0

GC–MS = gas chromatography-mass spectrometry. GC-EAD = gas chromatography coupled with electroantennography. R.T. = retention time. * For each compound: peak area * 10 2 ± SD * 10 2. ** Tested compound vs. hexane.

phy–electron impact–mass spectrometry (GC–EI–MS) analyses were performed with a mass selective detector 5973 Network interfaced with an Agilent Technologies 6890N Network GC system, equipped with an Agilent HP-5MS bonded FSOT column (30 m  0.25 mm i.d.). Headspace sampling was carried out using a solid phase microextraction (SPME) fibre [poly-dimethyl-siloxane (PDMS) 100-lm film; SupelcoÒ], which was conditioned for 4 min at 280 °C before use and was then, prior to the analysis, placed for 30 min in a 500mL glass crystallizer maintained at 25 °C and containing two infested or healthy fruits. Each analysis was replicated three times. As control, a SPME analysis was carried out in the clean empty crystallizer prior of each fruit headspace sampling, under the conditions above reported; none of the compounds present in these controls supplied signals superimposable to those of the compounds described in this work. GC–MS analysis of sampled volatiles were carried out under splitless conditions, desorbing the SPME fiber for 4 min in the injector at 280 °C, and using helium as carrier gas (1 mL/min). The oven temperature was programmed as follows: 6.10 min at 38 °C, to 250 °C at 10 °C/min, 5 min at 250 °C, to 280 °C at 20 °C/min, 6 min at 280 °C. The retention times, on all three GC columns, and the mass spectra of identified volatiles were identical to those of authentic samples, all purchased at Sigma–Aldrich with only two exceptions, decanoic acid and dodecanoic acid (Fluka Chemie, Buchs, Switzerland). To evaluate differences in volatile emissions between infested and healthy fruits the variance between peak areas was analyzed with Fisher’s F-test, and the Student’s t-test was used to evaluate the statistical differences in the mean values of peak areas between different trials. 2.5. Attractiveness of synthetic compounds from medfly-infested fruits Attractiveness of synthetic single GC-EAD active HIPVs as well as synthetic apple and peach volatile blends were evaluated towards 8–16 days old P. concolor mated females. Two choice bioassays were conducted using a Plexiglas unit (15  15  3 cm) as a

still-air arena (Carpita et al., 2012). In the center of this unit there was a circular chamber (i.e. the specimen release chamber, diameter: 4 cm), connected to two other identical chambers by means of two linear paths (length: 2 cm, width: 1 cm), forming a 90° angle. Each chamber contained a combination of cues. The top of the arena was covered with a removable glass panel. One of the two chambers contained a piece of filter paper (1.5  1.5 cm) treated with 2 lL hexane (control). The other chamber contained an equal filter paper wet with 2 lL hexane solution of 10 lg/lL of HIPVs single compound. Peach and apple blends were formed by an hexane solution of 10 lg/lL of peach and apple HIPVs, respectively. Concentration of HIPVs solutions and dosages were the same used for GC-EAD experiments. HIPVs and hexane filter papers were usually renewed every ten females tested. In each replicate, to begin the tests a specimen was gently transferred to the release chamber using a glass vial and carefully released on the floor of the chamber. Each wasp was observed for 5 min. An individual was considered to have made a choice when it moved to the cue within 4 min after being released and it engaged in searching behaviors on the chosen cue for at least 30 s (i.e. walking inside the chamber followed by arrestment). Specimens that did not operate any choice were discarded. For each experiment, the number of females performing ovipositor probing behavior in close proximity (61.5 cm) of the treated filter paper, was also registered. With each new wasp, the arena was rotated clockwise 90° to avoid positional effects. Moreover the relative position of the cues was randomized at each replicate. For each bioassay 30 replicates were performed. After each bioassay, the odor-cleaning procedure was as follows: the PlexiglasÒ arena and the glass lid were first washed for about 30 s with warm water at 35–40 °C, then cleaned in a water bath with mild soap for about 5 min, rinsed with hot water for about 30 s, and finally rinsed with distilled water at room temperature (Carpita et al., 2012). For each choice-test, a likelihood v2 test with Yates correction was used to compare the number of female wasps landed on each complex of cues (Sokal and Rohlf, 1981). A probability level of P < 0.05 was used for the significance of differences between female choices.

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2.6. Electrophysiological responses of P. concolor to medfly-induced volatiles We tested two blends of compounds, which were chosen based on SPME-GC–MS analyses. The blends contained synthetic compounds diluted in hexane identified as those exclusively emitted by medfly-infested fruits combined with those that were significantly induced by medfly infestation (see Results section, Table 2). Blend from peach fruits contained ethyl acetate, ethanol, ethyl octanoate, decanal, (E)-6,10-dimethyl-5,9-undecadien-2-one, nonanoic acid, 4-decanolide, decanoic acid and dodecanoic acid. Blend form apple fruits contained 1-butyl butylate, 1-hexyl acetate, 1-butyl hexanoate and 1-hexyl (E)-2-methyl butenoate. All chemicals were purchased at Sigma Aldrich (Munich, Germany), with the only exception of nonanoic and decanoic acid (Fluka Chemie, Buchs, Switzerland). The two blends were tested by means of gas chromatography coupled with electroantennography (GC-EAD) at two concentrations, 1 and 10 lg/lL for each diluted compound. Two microliters of the mixtures in hexane were injected into a Hewlett–Packard 5890 gas chromatography (GC) system. This used a polar Innowax column (30 m  0.32 mm; J & W ScientificÒ, Folsom, CA, USA) programmed to increase from 60 °C (hold 3 min) at 8 °C min 1, to 220 °C (hold 7 min), and was interfaced with the EAG apparatus. The outlet of the GC column was split in a 1:1 ratio between the flame ionization detector and an antenna of P. concolor mated female. We used an EAG technique similar to that described by Anfora et al. (2009), using a standard EAG apparatus (SyntechÒ, Hilversum, The Netherlands). A glass capillary indifferent electrode was filled with Kaissling solution (Kaissling, 1987) that contained 5.0 g l 1 polyvinylpyrrolidone K90 (Fluka Chemie, Buchs, Switzerland), and this was inserted into the severed head of the insect. The recording electrode was a similar glass capillary and this was brought into contact with the distal end of the antenna from which the tip was previously cut. Compounds eluting from the capillary column were delivered to the antenna through a glass tube (12 cm  8 mm) in a charcoal-filtered and humidified airstream. The antennal signal and the flame ionization detector signal were amplified and recorded simultaneously using Syntech software. Samples were tested on ten different P. concolor mated females. The mean antennal absolute responses (mV) were calculated and one-way ANOVA followed by the Tukey’s post hoc multiple comparison test (P = 0.05) was used to assess differences in the

response elicited by syntethic compounds for each dosage (StatisticaÒ 9; Statsoft Inc., Tulsa, OK). For each compound, differences in the GC-EAD responses between the two dosages were evaluated using Student’s t-test. Levene’s test was used to verify homogeneity of variances. 3. Results 3.1. Attractiveness of medfly-infested fruits: effect of combination of cues When visual, olfactory and contact cues were provided simultaneously, it was observed that the P. concolor females were more attracted to healthy than to dummy peaches (Fig. 2) (after 1 h: v2 = 29.81, d.f. = 5, P < 0.05; after 5 h: v2 = 36.34, d.f. = 5, P < 0.05; after 24 h: v2 = 28.99, d.f. = 5, P < 0.05), and to infested peaches than to healthy ones (after 1 h: v2 = 33.05, d.f. = 5, P < 0.05; after 5 h: v2 = 28.30, d.f. = 5, P < 0.05; after 24 h: v2 = 26.53, d.f. = 5, P < 0.05). Similarly, it was observed that wasp females were more attracted to healthy apples than to apple dummies (Fig. 2) (after 1 h: v2 = 27.04, d.f. = 5; P < 0.05; after 5 h: v2 = 32.49, d.f. = 5, P < 0.05; after 24 h: v2 = 45.66, d.f. = 5, P < 0.05), and to infested apples than to healthy ones (after 1 h: v2 = 24.61, d.f. = 5, P < 0.05; after 5 h: v2 = 36.52, d.f. = 5, P < 0.05; after 24 h: v2 = 40.52, d.f. = 5, P < 0.05). 3.2. Attractiveness of medfly-infested fruits: effect of olfactory cues Female wasps were more attracted to volatiles emitted by healthy peaches than to the control (v2 = 30.08, d.f. = 5, P < 0.05), and preferred volatiles emitted by infested over those emitted by healthy peaches (v2 = 26.54, d.f. = 5, P < 0.05) (Fig. 3). Similarly, in apple trials was noted that wasp females were more attracted to healthy apples than to the control (Fig. 3) (v2 = 25.42, d.f. = 5, P < 0.05), and to infested apples than to healthy ones (after 24 h: v2 = 23.78, d.f. = 85, P < 0.05). 3.3. SPME sampling of fruit volatiles and GC–MS analysis Nine volatile compounds were identified as exclusive of infested peaches (Table 1). Concerning apple fruits, it was observed that 2-methyl-1-butyl acetate and 2-methyl-1-butyl 2-methylbut-

Table 2 Changes in volatile emissions from infested and healthy apples, and absolute (mV) GC-EAD responses (mean ± SD) of Psyttalia concolor females to synthetic HIPVs at two dosages, 1 lg/lL and 10 lg/lL (n = 10). For each row of the GC–MS box different letters indicate significant differences (Student’s t-test; P < 0.05). In the GC-EAD box, values within the same column followed by the same letter are not statistically different (ANOVA, Tukey HSD test, P < 0.05). For each box’s row, differences in the GC-EAD responses between the two dosages were evaluated using Student’s t-test. The last box reports the number of P. concolor ovipositor probing responses displayed in close proximity (61.5 cm) of filter paper treated with HIPV synthetic chemicals or hexane. Thirty wasps were tested in each experiment. Apple volatiles

Apple blend vs. hexane 2-Methyl-1-butyl acetate 1-Butyl butylate 1-Hexyl acetate 2-Methyl-1-butyl 2methylbutanoate 1-Butyl hexanoate 1-Hexyl (E)-2methylbutenoate

GC–MS R.T. (min)

Healthy fruit*

Infested fruit*

– 7.74 10.20 10.50 12.05

– 18.01 ± 7.82 5.93 ± 1.15 a 33.84 ± 2.91 a 2.15 ± 0.70

– 0 16.09 ± 3.03 b 46.71 ± 5.03 b 0

– 3.59 6.03 7.11 7.20

13.41 15.51

62.11 ± 10.39 a 1.77 ± 0.25 a

161.57 ± 8.25 b 0.27 ± 0.03 b

GC–MS = gas chromatography-mass spectrometry. GC-EAD = gas chromatography coupled with electroantennography. R.T. = retention time. * For each compound: peak area * 10 2 ± SD * 10 2. ** Tested compound vs. hexane.

GC-EAD R.T. (min)

GC-EAD ± SD (mV  103 lg)

t-test

Number of probing $$**

a a b b

– – t = 0.4; d.f. = 19; P = 0.73 t = 0.2; d.f. = 19; P = 0.87 t = 0.3; d.f. = 19; P = 0.79

4 0 4 1 0

2.54 ± 0.48 c 3.32 ± 0.93 d

2.93 ± 0.66 c 3.78 ± 1.02 d

t = 1.5; d.f. = 19; P = 0.14 t = 1.0; d.f. = 19; P = 0.31

6 vs. 0 2 vs. 0

F = 41.1; d.f. = 49; P < 0.001

F = 55.7; d.f. = 59; P < 0.001

1 lg/lL

10 lg/lL

– 0 0.67 ± 0.21 a 1.45 ± 0.27 b 1.49 ± 0.32 b

– 0.54 ± 0.12 0.71 ± 0.23 1.47 ± 0.32 1.45 ± 1.05

9.53 13.49 ANOVA, Tukey’s test

vs. vs. vs. vs. vs.

0 0 0 0 0

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no-choice (%) 24 h, infested peach vs. healthy peach

45.00

24 h, healthy peach vs. peach dummy

32.23

24 h, infested apple vs. healthy apple

16.12

24 h, healthy apple vs. apple dummy

27.23

5 h, infested peach vs. healthy peach

57.78

5 h, healthy peach vs. peach dummy

36.67

5 h, infested apple vs. healthy apple

51.95

5 h, healthy apple vs. apple dummy

63.88

1 h, infested peach vs. healthy peach

63.62

1 h, healthy peach vs. peach dummy

64.72

1 h, infested apple vs. healthy apple

71.67

1 h, healthy apple vs. apple dummy

67.22 40

35

30

25

20

15

10

5

0

5

10

15

20

25

30

35

n. of landing wasps Fig. 2. Attractiveness of Ceratitis capitata-infested fruits towards Psyttalia concolor females: effect of visual, olfactory and contact cues. Two-choice bioassays were conducted in a still-air arena with different combinations of apple and peach fruits, infested or not by medfly larvae. Sixty wasps were tested in each replicate, six replicates were done. For each test, asterisks indicate significant differences in the number of wasps landing on each cue, after 1, 5 and 24 h (v2 test with Yates correction, P < 0.05).

Fig. 3. Attractiveness of Ceratitis capitata-infested fruits towards Psyttalia concolor females: effect of olfactory cues. Two-choice bioassays were conducted in a still-air olfactometer with different combinations of apple and peach fruits, infested or not by medfly larvae. Sixty wasps were tested in each replicate, six replicates were done. For each test, asterisks indicate significant differences in the number of wasps landing on each cue, after 24 h (v2 test with Yates correction, P < 0.05).

anoate were present only in healthy apples (Table 2). The emissions of 1-butyl butylate, 1-hexyl acetate and 1-butyl hexanoate increased significantly in infested apples, with respect to uninfested ones (t = 7.325, d.f. = 2, P < 0.01; t = 5.884, d.f. = 2, P < 0.05, and t = 60.480, d.f. = 2, P < 0.01, respectively). The production of 1-hexyl (E)-2-methylbutenoate decreased consistently in infested apples, with respect to healthy ones (t = 10.73, P < 0.01).

1-butyl hexanoate) elicited considerable P. concolor ovipositor probing responses in close proximity of the filter paper treated with pure chemicals. No female probing behaviors were observed near hexane-treated filter paper, in the control chamber (Table 1 and 2). 3.5. Electrophysiological responses of P. concolor to medfly-induced volatiles

3.4. Attractiveness of synthetic compounds from medfly-infested fruits Both blends of GC-EAD active synthetic chemicals from medflyinfested apples and peaches were shown to exert a significant attractiveness towards naïve P. concolor females (v2 = 5.63, d.f. = 1, P < 0.05; v2 = 7.50, d.f. = 1, P < 0.05, respectively) (Fig. 4). Concerning peach HIPVs, it was observed that three compounds tested singularly – ethyl octanoate, decanal and 4-decanolide – attracted more P. concolor females, with respect to the control (v2 = 9.63, d.f. = 1, P < 0.05; v2 = 5.63, d.f. = 1, P < 0.05; v2 = 5.63, d.f. = 1, P < 0.05, respectively) (Fig. 4). Among apple HIPVs, only 1-butyl butylate and 1-butyl hexanoate were shown to be attractive towards female’s wasps (v2 = 12.03, d.f. = 1, P < 0.05; v2 = 12.03, d.f. = 1, P < 0.05, respectively) (Fig. 4). Some apple and peach HIPVs (e.g. ethyl octanoate, decanal, 1-butyl butylate and

Analyses of the synthetic blends detected seven active compounds for peach and six active compounds for apple to the mated female P. concolor antennae (Table 1 and 2). Concerning infested peach volatiles, the antennal responses were elicited by ethyl octanoate, decanal, (E)-6, 10-dimethyl-5,9-undecadien-2-one, nonanoic acid, 4-decanolide, decanoic acid and dodecanoic acid. As regards to infested apple compounds, the antennal responses were evoked only by 1-butyl butylate, 1-hexil acetate, 2-methyl-1-butyl 2-methylbutanoate, 1-butyl hexanoate and 1-hexil (E)-2-methylbutenoate. Among volatiles exclusive of infested peaches, only ethyl octanoate, decanal and (E)-6, 10-dimethyl-5,9-undecadien-2-one showed significant increase in the antennal responses evoked to P. concolor females with increasing their dosage from 1 to 10 lg/ lL (t = 4.5, d.f. = 19, P < 0.001; t = 5.1, d.f. = 19, P < 0.001; t = 2.7,

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Fig. 4. Behavioral responses of Psyttalia concolor females in two-choice bioassays conducted in a still-air arena with synthetic HIPV chemicals versus hexane. Thirty wasps were tested in each experiment. For each experiment, asterisks indicate significant differences in the number of females landing on the two given cues (v2 test with Yates correction, P < 0.05).

d.f. = 19, P < 0.05, respectively). By contrast, all the compounds from infested apples were able to elicit significant increases in antennal responses by increasing their dosage from 1 to 10 lg/ lL. Only 2-methyl-1-butyl acetate showed an antennal response threshold higher than 1 lg/lL.

4. Discussion The evidence that olfactory cues from infested fruits evoke behavioral responses from mated P. concolor females, the presence of exclusive compounds in medfly-infested peaches, the quantitative variation of some chemicals in medfly-infested apples and their electrophysiological activity on female wasps, all support our hypothesis that the HIPVs identified could be used by P. concolor females as short-range attractants, thus playing a key role during the host location process of this braconid. P. concolor females were more attracted to olfactory cues from infested fruits than healthy fruits. This suggests that the presence of larvae inside the fruit is crucial in the host location behavior of this parasitoid species, at least at short distances. Several authors have stated that olfactory stimuli from host-infested fruit are essential for the host location of many braconids including all hymenopteran wasps that attack phytophagous larval stages (Messing et al., 1996; Eben et al., 2000; Stelinski et al., 2004; Carrasco et al., 2005; Silva et al., 2007; Ero et al., 2010; Segura et al., 2012). However, the finding that P. concolor females were more attracted to healthy fruits over dummy fruit may indicate that some attractive volatiles are common in infested and healthy fruits, as noted in other tri-trophic contexts (Eben et al., 2000; Carrasco et al., 2005). In our opinion, a generalist parasitoid such as P. concolor may be able to perceive volatile chemicals emitted by healthy fruits as cues for host habitat location. Thus the odor bouquet emitted by plant organs where the P. concolor hosts normally feed, might represent a first cue in the linked events of the P. concolor host selection. For host location, females can subsequently use short-range volatiles derived from the host medium and/or excre-

tion of the feeding larvae. We also believe that once the parasitoid has landed on the infested fruit, sounds or vibrations produced by the host larvae feeding or crawling inside the fruit may be important. Indeed, Canale and Raspi (2000) revealed the existence of a different type of tarsal sensilla in P. concolor that could be involved in the perception of host-borne vibrational signals. When visual, olfactory and contact cues were provided simultaneously, the behavioral responses of P. concolor females seem to be stronger compared to experiments where only olfactory cues were given. It is well known that in P. concolor the optimal response during host location was achieved when physical and chemical cues (including contact stimuli) worked synergistically (Canale, 2003). In addition, it was recently proved that visual cues from the host microhabitat also play a key role in parasitoid host-seeking behavior, since P. concolor mated females learn to associate information from the host microhabitat (e.g. the fruit’s color) with ovipositional experiences (Benelli and Canale, 2012b). In all the two-choice bioassays we found a high percentage of females that had not made any choice. However, when visual and contact cues were simultaneously presented, this value appeared to decrease over time inside the arena, probably because the time-increasing production of volatiles from the host-microhabitat elicited a choice in a larger number of observed specimens. The high number of non-responding wasps is not uncommon for P. concolor in laboratory conditions. Indeed, even when stimuli deriving from fresh infested/uninfested fruits were presented to P. concolor females, a consistent percentage of specimens did not make any choice (Benelli et al., 2012b; Mariotti et al., 2012). We hypothesize that uninterrupted massrearing under laboratory conditions may have led to a decrease in the parasitoid ability to search for the host feeding inside the fruit. In addition the extreme artificiality of the tri-trophic context may have increased the number of non-responding females. The volatile blends emitted by uninfested and C. capitata-infested fruits differ mainly in the emission rate of esters, as already reported for other tephritid-infested fruits (Carrasco et al., 2005; Kendra et al., 2011). Nine compounds were found to be exclusive in infested peach fruits over healthy ones, some of which were

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known as the fermentation products of different fruits (Light et al., 1992; Hernández et al., 1999) able to strongly attract Diachasmimorpha longicaudata (Ashmead) females (Greany et al., 1977; Carrasco et al., 2005; Stuhl et al., 2011). The release of most of these HIPVs is likely to be the active response of the plant, because they are only produced after phytophagous feeding and they are not released by intact fruit. We observed mainly quantitative changes in volatile emissions from infested and healthy apples. Indeed, the emissions of 1-butyl butylate, 1-hexyl acetate and 1-butyl esanoate increased in infested apples. In contrast, 1-hexyl (E)-2-methylbutenoate decreased remarkably in infested fruits. Quantitative changes in odour profiles from infested and healthy fruits are not uncommon in similar tri-trophic systems (Hern and Dorn, 2001, 2002). It is well known that several plants respond to insect feeding damage by producing mixtures of metabolites with changes in numbers or in their proportions (Carrasco et al., 2005; Henneman et al., 2002). It is worth noting that among apple fruit volatiles, 1butyl hexanoate is already known as the key attractant for the apple maggot fly, Rhagoletis pomonella (Walsh) (Zhang et al., 1999; Nojima et al., 2003). This would fit with the well-known evidence that the same ubiquitous plant volatile may attract polyphagous insects and their generalist parasitoids in order to locate the host’s microhabitat at a relatively long-range (McCormick et al., 2012 and references therein). The synthetic blends reproducing the odors emitted by C. capitata-infested apples and peaches were shown to be very attractive for P. concolor females. Some of the blend components exhibited a behavioral activity even when tested individually: ethyl octanoate, decanal and 4-decanolide for peach, and 1-butyl butylate and 1butyl hexanoate for apple. Interestingly, such apple and peach synthetic HIPV blends and single compounds elicited P. concolor ovipositor probing responses in close proximity to the treated surface. This finding is quite surprising, since parasitic wasps often need an integration of visual and olfactory cues to initiate host location behavior (Henneman et al., 2002). To our knowledge no similar evidence has been reported in braconids. In our experiments, all the selected volatiles specific to C. capitata infested apples showed significant antennal activity in P. concolor females, whereas among the compounds in infested peaches, only ethanol and ethyl acetate did not elicit GC-EAD responses in female antennae. Similarly, in other braconid wasps it has been shown that different HIPVs elicited considerable electrophysiological responses (Ngumbi et al., 2009, 2010; Sasso et al., 2009; Yu et al., 2010; Seenivasagan and Paul, 2011). Overall, HIPVs are recognized as kairomones for several parasitic wasps (Carrasco et al., 2005; Jumean et al., 2005; Dweck et al., 2010) and, therefore, have the potential to improve biological control programs (Uefune et al., 2011). Indeed, synthetic kairomones for parasitoids have been already tested successfully in field conditions (Colazza et al., 2004; Yu et al., 2008; Uefune et al., 2011). Nevertheless, a deeper knowledge regarding the mechanisms behind HIPVs on the foraging behavior of beneficial arthropods in the field is still needed before any possible commercial application (Kaplan, 2012). Besides field applications, HIPVs could also be used to improve mass-rearing techniques of beneficial insects, since these chemicals may be useful to enhance parasitization rates on alternative hosts. Further research is warranted to assess the role of the identified compounds on P. concolor females in semi-field and field conditions, in order to evaluate their possible use in improving fruit fly IPM programs. Acknowledgments We thank Adrian Wallwork for proofreading the English, Gabriella Bonsignori (The BioRobotics Institute, Sant’Anna School of Advanced Studies, Pisa), Lorenzo Rossi (Institute of Life Science,

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