Role of chemical cues in cabbage stink bug host plant selection

Journal Pre-proofs Role Of Chemical Cues In Cabbage Stink Bug Host Plant Selection Silvana Piersanti, Manuela Rebora, Luisa Ederli, Stefania Pasqualini, Gianandrea Salerno PII: DOI: Reference:

S0022-1910(19)30187-8 https://doi.org/10.1016/j.jinsphys.2019.103994 IP 103994

To appear in:

Journal of Insect Physiology

Received Date: Revised Date: Accepted Date:

16 May 2019 25 November 2019 8 December 2019

Please cite this article as: Piersanti, S., Rebora, M., Ederli, L., Pasqualini, S., Salerno, G., Role Of Chemical Cues In Cabbage Stink Bug Host Plant Selection, Journal of Insect Physiology (2019), doi: https://doi.org/10.1016/ j.jinsphys.2019.103994

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Role Of Chemical Cues In Cabbage Stink Bug Host Plant Selection Silvana Piersanti Conceptualization Methodology Investigation Writing - original draft, Writing

- review & editing, Funding acquisitiona, Manuela Rebora Conceptualization Methodology Investigation Writing - original draft Writing - review & editinga,*,[email protected], Luisa Ederli Conceptualization Writing - review & editinga, Stefania Pasqualini Conceptualization Writing review & editinga, Gianandrea Salerno Conceptualization Methodology Investigation Formal analysis Writing - review & editing Supervisionb aDipartimento

di Chimica, Biologia e Biotecnologie, University of Perugia, Italy

bDipartimento

di Scienze Agrarie, Alimentari e Ambientali, University of Perugia, Italy

author: Dipartimento di Chimica, Biologia e Biotecnologie, University of Perugia, Via Elce di Sotto 8, 06121, Perugia, Italy. *Corresponding

Graphical abstract Highlights

Eurydema oleracea (L.) is an important pest of Brassicacee in Europe Bioassays showed that this stink bug prefers Eruca sativa to Brassica oleracea VOCs Antennae responded to host plant VOCs both from Eruca sativa and Brassica oleracea Single walled and double walled olfactory sensilla were present on antennae These data support a crucial role of olfaction in host plant location of E. oleracea

Abstract The cabbage stink bugs of the genus Eurydema, encompassing several oligophagous species, such as Eurydema oleracea (L.), are known to be important pests of cabbage, broccoli, and other cole crops in Europe. Despite their economic importance, the knowledge regarding the role of chemical cues in host plant selection of these species is very limited. The present investigation on E. oleracea at the adult stage revealed the use of olfaction in host plant selection of this species and demonstrated with behavioural tests that E. oleracea preferred feeding on wild Eruca sativa, rather than on Brassica oleracea. Moreover, ultrastructural data revealed the antennal sensilla of E. oleracea, encompassing single walled and double walled olfactory sensilla, and electroantennographic recordings revealed their sensitivity to several host plant VOCs from E.

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sativa and B. oleracea. The data shown in the present research may be useful in the development of semiochemical-based strategies or trap crops for the control of this pest in the field.

Keywords: Eurydema oleracea, olfactory sensilla, electroantennography, behaviour, Pentatomidae, Brassica oleracea

1. Introduction Chemical ecology plays a key role in interactions between insects and their environment. In particular, volatile chemicals mediate a great range of intra- and interspecific signaling and information in insects, which have evolved richly diverse olfaction-based behaviours (Hansson and Stensmyr, 2011; Martin et al., 2011; Gadenne et al., 2016). Such wide variety of olfaction-based behaviours and diverse olfactory systems can be reflected by the morphological characteristics of antennae, olfactory sensilla type, olfactory receptor repertoire, and antennal lobe architecture (Strausfeld and Hildebrand, 1999; Carey and Carlson, 2011; Martin et al., 2011). During the long period of coevolution with insects, plants developed a wide diversity of features useful to attract pollinators or to defend against herbivores. For this second purpose, plants evolved chemical (rev by Chen, 2008; Mauch-Mani et al., 2017) and physical barriers (Gorb, 2005) heavily affecting insect performance. Simultaneously, phytophagous insects developed the capacity to use volatile organic compounds (VOCs) produced as secondary metabolites by specific plants, to locate and accept them as a suitable host (Finch and Collier, 2000). Insects detect odorants primarily using odorant receptors (OR) housed in the dendritic membrane of olfactory sensory neurons located mainly in the antennal olfactory sensilla. The origin of the OR family seems to be the adaptation of insects to the rapid spread and diversification of vegetation (Missbach et al., 2014; Andersson et al., 2015), confirming the involvement of chemical ecology in the special association between insects and plants. 2

Pentatomidae (Hemiptera, Heteroptera) is a family encompassing numerous pest species; in particular, the genus Eurydema Laporte, 1833 (cabbage stink bugs) is widely distributed in the Palearctic region, with numerous polyphagous species typically feeding on cultivated or wild cabbages (Cruciferae) by sucking sap from leaves and causing damaged areas to become whitish or yellowish. Severe attacks can cause all leaves and pods to turn yellow and may lead to the death of young plants (Hori et al., 1984; Panizzi et al., 2000). Despite the economic importance of the cabbage stink bugs, there is limited knowledge concerning their interaction with plants (Aldrich et al., 1996). For example, no data are available on the antennal sensilla, although some morphological data are available on other Pentatomids (Usha Rani and Medhavendra, 1995; Brézot et al., 1997; Usha Rani and Medhavendra, 2005; Silva et al., 2010; Ahmad et al., 2016). Likewise, behavioural and electrophysiological investigations have never been performed on Eurydema species, except for some bioassays conducted with Eurydema pulchrum Westwood and Eurydema ornatum L. showing a preference for Brassica oleracea var. capitata (Eltez and Karsavuran, 2010; Rather et al., 2010). For other pentatomids, recently Guarino et al. (2018) provided evidence that host plant VOCs were exploited by Bagrada hilaris Burmeister in location and possibly acceptance of Brassica species as host plants. The present study conducted laboratory conditions, investigates the role of chemical cues in host plant selection of Eurydema oleracea (L.), which is an economically important pest of cabbage, broccoli, and other cole crops in Europe (Hori et al., 1984; Rider, 2006; Trdan et al., 2006). In particular, the aims of present study are: 1) to verify the presence of host plant preference based on VOCs between a typical crop host plant, B. oleracea var. capitata (head cabbage), and a wild edible cruciferous plant never reported but frequently personally observed as host plant, Eruca sativa (rocket salad); 2) to perform an electrophysiological screening on male and female antennae, recording the responses to the VOCs blend emitted by the two plants in different developmental stages, and to standard volatiles reported in literature as emitted by cabbage and rocket salad; 3) to

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describe the olfactory antennal sensilla potentially involved in host plant selection by chemical cues in this species. An understanding of the role of olfaction in host plant selection by E. oleracea, together with some knowledge about its host preferences, may provide useful information for the development of nonchemical alternatives for the control of this species, as well as to project integrated pest management programs.

2. Materials and Methods 2.1. Insects Individuals of E. oleracea were collected in the field close to Perugia (Umbria region, Italy) in September 2017 and 2018. Insects were reared in a controlled climate chamber (14:10 light-dark rhythm, at a temperature of 25±1°C and a relative humidity of 70±10 %) inside net cages 30X30X30 cm. Varieties of cabbages (different from that used in the experiments to avoid any bias) were used to feed the insects, according to seasonality. Eggs were collected and kept in separate containers and nymphs were reared separately until the final molt to adults. Only adult insects of both sexes were used in the study.

2.2 Plant material Seedlings. To obtain seedlings for VOCs extraction, commercial seeds (Rosi Sementi, Italy) of B. oleracea var. capitata Brunswick and E. sativa were placed on cotton wool (70 g) soaked with distilled water (300 ml) and held in food metal containers 20X30X5 cm (approximately 1000 seeds for each container, 1.5g for E. sativa and 3.3 g for B. oleracea). The containers were placed in a controlled climate chamber (22±1˚C, 70±10% RH, photoperiod 18L:6D) equipped with lights with a photosynthetic photon fluence rate of 200 μmol m−2 s −1 placed above the foliage. To avoid desiccation, soaked seeds were covered with plastic food film for 2 days. Newly emerged seedlings 4

at the cotyledon stage (4 d later for E. sativa and 7 d later for B. oleracea) were used for VOCs collection. Plants. To obtain plants for VOCs extraction and behavioural experiments, B. oleracea var. capitata and E. sativa were cultivated. In the dual choice experiments aiming to investigate the role of VOCs emitted by cabbage and rocket salad, to have a further plant with identical leaves in both the compartments of the arena, thus resulting in identical visual, tactile and gustatory stimuli, Arabidopsis thaliana was chosen because it is a potential host of E. oleracea and is easily available. Commercial seeds (Rosi Sementi, Italy) of B. oleracea var. capitata cv. Brunswick and E. sativa were sown in sterile soil (Patzer Einheitserde, Manna Italia, Bolzano, Italy) into individual pots (6X6X10 cm). Once the seeds were sown, they were placed in a controlled climate chamber (22± 1˚C, 70±10% RH, photoperiod 18L:6D) equipped with lights with a photosynthetic photon fluence rate of 200 μmol m−2 s −1 placed above the foliage. Water was supplied by sub-irrigation. In both species, plants with six leaves were used both for VOCs collection and behavioural experiments. Seeds of A. thaliana (ecotype Columbia-0; Col-0) wild-type (WT) were obtained from the Nottingham Arabidopsis Stock Center 106 (NASC). The seeds were surface sterilized by soaking them in 70% ethanol for 1 min and disinfected by a 3 min treatment with 15% (v/v) sodium hypochlorite. After disinfection procedure, the seeds were rinsed three times with sterile distilled water and sown in sterile soil (Patzer Einheitserde, Manna Italia, Bolzano, Italy) into individual pots (5.5 cm diam). Once the seeds were sown, they were vernalized at 4 °C for 2 days and moved to a growth chamber under a 12 h photoperiod with a photosynthetic photon fluence rate of 200 μmol m−2 s−1, at 23±2 °C, and 60% to 75% RH. Water was supplied by sub-irrigation. Plants without flowers were used for behavioural experiments.

2.3 VOCs collection

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Collection of volatile compounds from B. oleracea and E. sativa seedlings were carried out according to the procedure described in Conti et al. (2008). In particular, we used clusters (N = 2000) of newly emerged seedling at the cotyledon stage, while collection of volatile compounds from plants of the same species in a later developmental stage (six leaves) were carried out using one plant for each collection. Seedling clusters, with the cotton wool on aluminum foil, or one adult plant, with the pot wrapped by aluminum foil, were placed in a cylindrical glass chamber (5 l volume). An air stream purified by passing through a charcoal filter was pumped through the chamber at 500 ml/ min. A glass cartridge (10X0.5cm) containing a plug of 100 mg of Porapak Q (80–100 mesh; Sigma-Aldrich) was used to collect the VOCs. After collecting for 24 h, the traps were eluted with 0.8 ml of hexane, and the resulting extracts were concentrated to ~200μl under a gentle nitrogen stream. Extracts were stored in a freezer at −18˚C, in glass vials with Teflon cap liners, until used. All replicates were carried out under controlled conditions (25± 2˚C, 50±10% RH and photoperiod 18L: 6D). After each collection, the chambers were washed with water and fragrance-free detergent, rinsed with hexane and acetone, and baked overnight at 150˚C.

2.4. Behaviour To test the existence of a host plant preference between B. oleracea and E. sativa, insects starved for 24 hours were used in dual choice experiments. In each replicate a couple of leaves, one from a six leaf plant of E. sativa and the other from a six leaf plant of B. oleracea, both not excised from the plant, were exposed to one mated E. oleracea female, caged on the surface of the two leaves using a clip-cage constituting a dual choice arena. The clip-cage consisted in a modified plastic Petri dish (3.8 cm i.d.; 1 cm high) with a mesh-covered hole (3 cm i.d.) and the rim covered by a small sponge ring to avoid damage of the leaf. Equal surface of the two leaves was exposed to the test insect inside the clip-cage. Experiments were run under controlled environmental conditions in the growth chamber. The insects were allowed to feed for 24 h, then the arenas with the insects were removed and the leaves were observed with a stereomicroscope (WILD mikroskop M420, 6

Switzerland). The damage due to feeding was measured from digital images using the open source image-processing program ImageJ (Schneider et al., 2012). Thirty-three replicates were performed. To investigate the role of VOCs emitted by cabbage and rocket salad in host plant selection, a dual choice experiment as above described was performed putting two identical leaves of A. thaliana inside the clip-cage, each one associated with a filter paper strip (15 mm x 15 mm, Whatman No.1) used as odour cartridge (Fig. 1). Inside each arena, the paper associated to one leaf was impregnated with 20μl of VOCs extract from E. sativa seedlings, while the other was impregnated with an equal amount of extract from B. oleracea seedlings. Feeding preference was evaluated as damage surface as above reported. Seventeen replicates were performed with mated females. Classical instruments for behavioural investigations on chemical cues, such as Y-tube olfactometers or open vertical olfactometers, successfully used with B. hilaris (Guarino et al., 2018) or E. pulchrum (Rather et al., 2010), were excluded from this research because in preliminary bioassays with these instruments we never succeeded to show a preference of E. oleracea adults for the host plant respect to the empty control. In particular, we performed 30 replicates with the Y-tube olfactometer (see description in Moujahed et al., 2014) and 30 replicates with a vertical open Yshaped olfactometer (see description in Frati et al., 2008). Olfactometer bioassays were carried out as paired choices in which odour source (B. oleracea plants) were always tested versus an empty control. In the Y-tube olfactometer the insect residence time in the two different arms was considered as a parameter while in the vertical olfactometer the first choice was considered. In both experiments the insect choice between the two arms was never statistically different.

2.5. Electroantennography Electroantennography recordings (EAG) were performed to test the responses of male and female antennae to the VOCs blend from plants and seedlings of B. oleracea and E. sativa, collected as reported above, and to investigate the sensitivity of the antennae to different volatiles emitted by Cruciferous plants. 7

Chemicals. The blend of VOCs collected from B. oleracea and E. sativa at seedling and plant stage and eluted in hexane, as reported above, were used as test compounds. Twenty-six substances reported in literature (Geervliet et al., 1997; Jirovetz et al., 2002; Bell et al., 2017; Giron-Calva et al. 2017) as VOCs produced by rocket salad (E. sativa) and head cabbage (B. oleracea), or by both of them, were selected. Odorants were from Sigma Aldrich (St. Louis, MO, USA) and were of the highest grade available (P 98%). In order to prevent rapid evaporation of test compounds before use they were dissolved in paraffin oil to obtain 10% (v/v) solutions. In detail: αpinene, propanoic acid, butanoic acid, hexyl acetate, 1-octanol, decanal, pentanoic acid and dimethyl trisulfide are reported in literature as emitted by B. oleracea; (E)-β-caryophyllene, allyl isotyocianate, (E)-2-hexen-1-ol, (Z)-3 hexenyl butanoate, nonanoic acid, carveol, 2-acetyl thiazol, 2-methyl anisole, hexadecanol, β-citronellol, 2-pentyl furane are reported in literature as emitted by E. sativa; (Z)-3-hexenil acetate, (E)-2-hexenal, octanal, hexanoic acid, hexanol, hexanal, nonanal are reported in literature as emitted by both E. sativa and B. oleracea; To obtain dose-response curves, paraffin oil solutions (0.01, 0.1, 1, 10, 50 and 100% v/v) of octanal and butanoic acid, used as standard compounds, were prepared. To prevent compound degradation, all the test solutions were kept in a freezer at -18°C, in glass vials with Teflon cap liners, until used. Recordings. EAG recordings were carried out according to the procedure described in Rebora et al. (2012). In particular, we used intact males and females of E. oleracea fixed on a small glass slide by dental wax and tape. Antennae were immobilized by paraffin modelled on the pedicel by a cauterizer (WAX Carving PEN Super Max Instant 850F Tool, Max-Wax). Antennal responses were recorded using two glass capillary electrodes with an outer diameter of 1.5 mm and an inner diameter of 1.2 mm filled with Ringer solution (Ephrussi and Beadle, 1936), containing 5 g/l of polyvinylpyrrolidone (Fluka), in contact with a silver wire. The antennal tip has been carefully excised and inserted into the recording electrode. The reference electrode was inserted in the eye. The insect was kept under a constant stream of humidified and filtered air at 200 mL/min. The 8

analog signal was detected through a probe with a high-input impedance preamplifier (10x) (EAG Kombi-probe, Syntech, Germany), and was captured and processed with a data acquisition controller (IDAC-4, Syntech, Germany), and analysed using EAG 2000 software (Syntech, Germany). Stimulations. Stimulations were carried out according to the procedure described in Rebora et al. (2012). In particular, 20 µl of each VOCs extract, as hexane solution, were absorbed on a filter paper strip (15x15 mm, Whatman No.1) placed into a glass Pasteur pipette (150 mm in length, Volac®) to constitute an odour cartridge. Test compounds, as paraffin oil solutions, were delivered as 10 µl samples placed on the filter paper. The control stimuli consisted of similar pipettes containing a strip of filter paper impregnated with the same aliquot of solvent (20 µl of hexane for VOCs extracts and 10 µl of paraffin oil for standard compounds). Fresh stimulus pipettes were prepared every day. The tip of the pipette was placed about 3 mm into a small hole in the wall of a L-shaped glass tube (130 mm long, 12 mm diameter) oriented towards the antennal preparation (~5 mm away from the preparation). The stimuli were provided as 1 sec puffs of purified, charcoalfiltered air into a continuous humidified main air stream at 200 mL/min that was over the flowing antennal preparation at a velocity of 50 cm/sec generated by an air stimulus controller (CS-55, Syntech, Germany). At least 1 min interval was allowed between successive stimulations for antenna recovery. On the basis of preliminary recordings, octanal was chosen as reference standard stimulus and presented to the antenna every 6 stimulations in the recording series, to confirm and to monitor activity of the antennal preparation. Test compounds were presented in a random sequence. Thirty-four female antennae and 21 male antennae belonging to different insects were used for EAG recordings. For dose-response experiments, 13 antennae from different females and 17 antennae from different males were tested with the reference standard stimulus, octanal, and with one compound that showed a relatively large response, butanoic acid; the exposure proceeded from lowest to highest

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concentration for each chemical, with at least 1 min interval between successive stimulations to minimize the effect of olfactory adaptation by strong stimulation.

2.6. Ultrastructure To investigate the antennal olfactory sensilla in E. oleracea, male and female antennae were dissected from anaesthetized adults and fixed for 12 h in 2.5% glutaraldehyde in cacodylate buffer (Electron Microscopy Sciences), pH 7.2, to be observed under scanning (SEM) and transmission (TEM) electron microscopy. SEM analysis. 20 fixed antennae for each sex were repeatedly rinsed in the same buffer, and then dehydrated by using ascending ethanol gradients, followed by critical-point drying in a criticalpoint dryer CPD 030 Bal-Tec (Bal-Tec Union Ltd., Balzers, Liechtenstein). Specimens were mounted on stubs with silver conducting paint, sputter-coated with gold-palladium in an Emitech K550X sputterer (Emitech, Ashford, England), and observed with a SEM Philips XL30 (Philips, Eindhoven, the Netherlands), at an accelerating voltage of 18 kV. Density and distribution of the olfactory sensilla on the last flagellar segment of males and females were evaluated using the software ImageJ (Schneider et al., 2012). TEM analysis. Twenty fixed antennae of E. oleracea were repeatedly rinsed in cacodylate buffer and post-fixed for 1 h at 4 °C in 1% osmium tetroxide in cacodylate buffer (Electron Microscopy Sciences). The samples were then repeatedly washed in the same buffer, dehydrated by using ascending ethanol gradients and finally embedded in an Epon-Araldite mixture resin (SigmaAldrich). Afterward, ultrathin sections were cut on a Leica EM UC6 ultracut (Leica Microsystem GmbH, Wetzlar, Germany) ultramicrotome, collected on Parlodion (Spi-Chem) coated copper grids, stained with uranyl acetate and lead citrate (Electron Microscopy Sciences), and examined with a TEM Philips EM 208 (Philips, Eindhoven, the Netherlands).

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2.7. Statistical analyses For evaluation of EAG responses, the maximum deflection of the recorded EAG signal after stimulation with a test compound was used. Antennal sensitivity to the different chemicals was recorded as a percentage of all recorded EAG responses to the reference standard stimulus (octanal 10% v/v). This allows compensation for antennal sensitivity changes during the experiment and the comparison between experiments performed with antennae of different sensitivity. The responses to plant extracts were analysed using a three-way ANOVA, considering the sex, the host plant species and the developmental stage of each plant species as factors. The responses to standard chemicals were analysed using a two-way ANOVA, considering the sex and the different cues as factors. As post hoc comparison, Dunnett test, was used to compare, separately for males and females, the response to each test compound to those of the control (paraffin oil for standard VOCs and hexane for extracts) (Statistica 6.0, Statsoft Inc., 2001). Data obtained in the dose–response experiments, for each compound, were analysed using a twoway factorial analysis of variance (ANOVA) considering the sex and the compound concentration as the main factors. As post hoc comparison, Dunnett test was used to compare the response to each specific concentration to the control (paraffin oil), for males and females (Statistica 6.0, Statsoft Inc., 2001). In the dual choice behavioural experiments, insect preferences (feeding damage surface in the leaves) were tested for statistical differences using Student’s t-test for dependent samples. Before all the analysis, Box–Cox transformations were used to normalize the data (Sokal and Rohlf, 1998).

3. Results 3.1 Host plant selection bioassays In the dual choice experiments, E. oleracea females showed a clear feeding preference (t= 7.23; d.f.= 32; p < 0.0001) for E. sativa leaves, instead of B. oleracea leaves (Fig 2a). This significant 11

preference was confirmed in the dual choice experiments run with A. thaliana leaves in the arena, associated with “active extracts” of VOCs from cabbage and rocket salad (t= 2.18; d.f.= 16; p = 0. 0446) (Fig.2b).

3.2. EAG responses to extracts of host plant VOCs Both female and male antennae of E. oleracea showed a significant response to the extracts of VOCs collected from E. sativa plants and seedlings, as well as B. oleracea plants and seedlings (Fig. 3), so all of them can be defined as “active extracts”. No significant differences were observed between sexes, but responses were significantly different for extracts from different plant species and different plant developmental stages (Fig. 3).

3.3. EAG responses to standard VOCs Depolarizing EAG responses were recorded in both male and female antennae of E. oleracea when stimulated with most of the 26 test compounds; there was a significant difference between the two sexes and among the stimuli (Fig. 4). For chemicals emitted by B. oleracea, both male and female antennae responded to butanoic acid, hexyl acetate, 1-octanol, decanal and pentanoic acid with EAG activity significantly higher in comparison with that recorded in response to paraffin oil, but only males responded to propanoic acid and neither males nor females responded to α-pinene. For chemicals emitted by both E. sativa and B. oleracea, both male and female antennae responded to all of them ((Z)-3-hexenil acetate, (E)-2-hexenal, octanal, hexanoic acid, hexanol, hexanal and nonanal) with EAG activity significantly higher in comparison with that recorded in response to paraffin oil. For chemicals emitted by E. sativa, both male and female antennae responded to (E)-βcaryophyllene, allyl isotyocianate, (E)-2-hexen-1-ol, 2-acetyl thiazol and 2-methyl anisole with EAG activity significantly higher in comparison with that recorded in response to paraffin oil; while they did not respond to (Z)-3 hexenyl butanoate, nonanoic acid, carveol, hexadecanol, β-citronellol and 2-pentyl furane. In both males and females the magnitude of the responses to butanoic acid, 12

hexanol and (E)-2-hexen-1-ol was particularly high compared with that to the other chemicals (Fig. 4). In the EAG dose-responses tests, males and females showed different responses to increasing concentrations of octanal, with males showing a significant response, in comparison with the solvent (paraffin oil), at 1%, 10%, 20%, 50% concentrations, while females response was significantly higher in comparison to the solvent (paraffin oil) at 10%, 20%, 50% concentrations (Fig. 5). Both males and females significantly responded to higher concentration of octanal, but female responses increased in amplitude with increasing dose of the chemical (20% and 50%), while males’ responses showed the largest amplitude with 10% octanal concentration, and decreased in amplitude with higher concentrations (20% and 50%) (Fig. 5). Increasing doses of butanoic acid elicited similar response in males and females antennae (Fig. 5): the response was significantly higher than the response to the solvent at 10% and 20% concentration, while it was not significant at 50% concentration.

3.4. Olfactory sensilla on the antennal flagellum The antenna of E. oleracea at the adult stage is constituted of a scape, of a long pedicel divided into two subsegments termed pedicellites (Zrzavy, 1992) and a flagellum composed of two segments (Fig. 6a). The two flagellar segments are characterized by the presence of numerous sensilla in the form of hairs (Fig. 6a). The olfactory sensilla located on the two flagellar segments are represented by single–walled multiporous sensilla basiconica (Fig. 6b and inset 1-2), double-walled multiporous sensilla basiconica (Fig. 6c and inset 1-3) and slender porous sensilla trichodea (Fig. 6d and inset 12). The single–walled multiporous sensilla basiconica are unsocketed, slightly curved setae with a blunt apex and measure about 30 µm in length and about 3 µm in width at their base (Fig. 6b). On their whole surface they are characterized by numerous small pores visible under SEM (inset 1 of Fig. 6b) and under TEM (inset 2 of Fig. 6b); cross sections of the sensillum under TEM reveal the presence of numerous dendritic branches inside the sensillar lumen (inset 2 of Fig. 6b). The double13

walled multiporous sensilla basiconica are unsocketed pegs with a rounded apex and a grooved surface (Fig. 6c). They measure about 15 µm in length and about 3 µm in width at their base. Cross sections of the peg under TEM (Fig. 6c inset 1-3) reveal the presence of an outer cuticular wall and an inner cuticular wall surrounding the dendrites. Hollow cuticular spokes (spoke channels) connect the lumen of the sensillum to the external environment (Fig. 6c, inset 1). The slender porous sensilla trichodea are unsocketed, thin setae and measure about 30 µm in length and about 2 µm in width at their base (Fig. 6d). Their surface is smooth on one side and wrinkled on the opposite side (Fig. 6d, inset 1). In correspondence of the wrinkled surface, pores with pore tubules are clearly visible in cross sections under TEM (Fig. 6d, inset 2). Dendrites are visible inside the sensillar lumen (Fig. 6d, inset 2). The morphology and the distribution (Fig. 7) of the different kinds of olfactory sensilla are similar in both sexes of E. oleracea. The most represented olfactory sensilla are the slender porous sensilla trichodea (the density in the last flagellar segment in female is 1.7/mm2 on the dorsal side and 1.4/mm2 on the ventral side; in male 2.1/mm2 on the dorsal side and 2.7/mm2 on the ventral side) followed by the single–walled multiporous sensilla basiconica (the density in the last flagellar segment in female is 0.8/mm2 on the dorsal and 0.9/mm2 on the ventral side; in male 0.8/mm2 on the dorsal and 1.1/mm2 on the ventral side) and by the double-walled multiporous sensilla basiconica (the density in the last flagellar segment in female is 0.4/mm2 both on the dorsal and on the ventral side; in male 0.2/mm2 on the dorsal and 0.6/mm2 on the ventral side). Together with olfactory sensilla, the antenna is characterized by the presence of mechanoreceptors in the form of aporous socketed pointed grooved hairs (sensilla chaetica) (Fig. 6e) and gustatory sensilla in the form of socketed grooved hairs (Fig. 6f) (sensilla chaetica) with a blunt apex bearing an apical pore (Fig. 6f, inset 1). These gustatory sensilla are innervated by six-seven unbranched neurons one of which stops at the base to form a tubular body (Fig. 6f inset 3) (the mechanosensory neuron associated to the chemosensory neurons typical of gustatory sensilla). Occasionally some

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campaniform sensilla (mechanoreceptors) and few coeloconic sensilla (small pegs in pits) of unknown function are visible on the antenna.

4. Discussion The present study investigated the role of volatile chemicals emitted by Cruciferous plants as potential cues used by the cabbage stink bug E. oleracea to locate its host plant. Our behavioural results clearly showed for the first time that E. oleracea adults are able to select host plants using their emitted VOCs. The use of VOCs (obtained in Brassicaceae by hydrolysis of non-volatile glucosinolates) to localise the host plant has been demonstrated in other pests of Brassicaceae, such as the cabbage root flies (Delia radicum L.) and the cabbage butterfly larvae (Pieris rapae L.) (Finch, 1978; Chew, 1980) and, among Pentatomidae, by the Asian stink bug B. hilaris (Guarino et al., 2018).

4.1. Host plant preference between Brassica oleracea and Eruca sativa. In the dual choice experiments E. oleracea strongly preferred E. sativa to B. oleracea. Our results suggested that such a preference is based on chemicals emitted by the plants since the same preference was evident also when only VOCs of the two species are available for selection, associated with identical leaves of A. thaliana in both the compartments of the dual choice arena, thus resulting in identical visual, tactile and gustatory stimuli. It is interesting to consider that in these experiments the insects were allowed to complete the chain of events that typically characterizes the system of host plant selection in many phytophagous insects: the first link is governed by volatile cues, the central link is driven by visual stimuli and the final link is driven by non-volatile chemical cues and completed by feeding (Finch and Collier, 2000). However, in repeated preliminary bioassays using Y-tube olfactometers and open-vertical olfactometers (unpublished data), only olfactory cues were available and feeding was not allowed. Here, E. oleracea did not show any behavioural preference. No data are available on the role of 15

vision in host plant detection by the cabbage stink bugs of the genus Eurydema, but in other pentatomids, such as N. viridula, visual stimuli are known to be relevant (Guarino et al., 2017). The visual aspects of plant detection by insects have been largely disregarded (Prokopy, 1983), but several examples demonstrate that phytophagous insects from different orders use also vision for locating host plants (Reeves, 2011), and in some case it can be particularly relevant, such as aphids preference for yellow colour (Doring and Chittka, 2007). Our results suggest that visual stimuli could combine with chemical ones to drive host plants detection by Eurydema bugs. Further behavioural investigations are needed to clarify the need of these species to associate different kind of stimuli to finalise their feeding behaviour. The preference shown by E. oleracea for E. sativa, never reported in literature as host plant but frequently recorded in our field observations, is particularly relevant. Such a strong selection for E. sativa instead of the typical cabbage crop B. oleracea evidenced in our bioassays suggests that E. sativa could be usefully tested as trap crop for control of E. oleracea in Brassica fields. Indeed, field experiments demonstrated that a border row of mustard reduce harlequin bug injury in collard by roughly 50%, without use of insecticides (Wallingford et al., 2013) and some cabbage stink bugs of the genus Eurydema are successfully controlled by trap crops of oil rape (Bohinc and Trdan, 2012).

4.2. Electrophysiological responses of male and female antennae of E. oleracea Male and female antennae of E. oleracea responded with a significant electroantennographic depolarization to crude extracts of VOCs of E. sativa and of B. oleracea, both at seedling and adult stages. In addition, among the 26 compounds tested and reported in the literature as emitted by E. sativa (Jirovetz et al., 2002; Bell et al., 2017) and /or B. oleracea (Geervliet et al., 1997; GironCalva et al., 2017) females responded to 17 and males to 18 compounds. The strongest responses were recorded to one carboxylic acid (butanoic acid), two alcohols (hexanol and (E)-2-hexen-1-ol), two aldehydes (nonanal and (E)-2-hexenal), one aliphatic alkene (allyl isothiocyanate), and one ketone (2-acetyl thiazol). Considering that electroantennography (EAG) measures the total amount 16

of electrophysiological responses by olfactory neurons in the insect antennae, these results confirm the involvement of olfactory antennal sensilla in host plant selection. Moreover, the EAG responses demonstrate the capability of the peripheral olfactory system of the cabbage stink bugs to perceive a broad range of VOCs emitted by cruciferous plants. This is in agreement with the oligophagous habits of this pest, also considering that EAG-active compounds are frequently of ecological significance (Germinara et al., 2017) as demonstrated by our behavioural results. Among the 26 tested compounds, only one, the propanoic acid, elicited a significant response in males and not in females, thus suggesting a general similarity between sexes in antennal sensitivity. Small differences between the responses of males and females were recorded in stimulations with increasing doses of octanal. Indeed, both males and female responses increased with the ascending dose of the stimulus, but male antennae started to respond at a lower dose (1% instead of 10% in females), and female responses continued to increase their amplitude with increasing doses, while male responses reached the largest amplitude at 10% and decreased in amplitude with higher concentrations. This difference could be related to the different use of the host plant by males and females (feeding in males, feeding and oviposition in females). Responses to increasing doses of butanoic acid are identical for males and females, but probably a more accurate investigation with stimulations at concentrations between 1% and 10% is needed to better describe this dose-response curve. The present results are the first recordings of olfactory responses from the antennae of Eurydema species and confirm their sensitivity to host plant VOCs.

4.3. The olfactory antennal sensilla of E. oleracea potentially involved in host plant selection Antennae are the main chemosensory organ in most insects (Missbach et al., 2011), bearing chemoreceptors of different type, sensitivity and distribution, that play an important role in a number of behaviour such as host recognition, mate location, oviposition, aggregation and defence (Ahmad et al., 2016). As found in other Pentatomid bugs, such as N. viridula (Brèzot et al., 1997), Euschistus heros (F.), Piezodorus guildinii (Westwod) and Edessa meditabunda (F.) (Silva et al., 17

2010), the antenna of E. oleracea presents a scape, a pedicel divided in two pedicellites and a flagellum composed of two segments. Three types of olfactory sensilla are visible on the antennal flagellum: single–walled multiporous sensilla basiconica, slender porous sensilla trichodea and double-walled multiporous sensilla basiconica. The first one shows numerous pores, along the thin cuticle, each enlarging in a pore kettle and branching in numerous pore tubules, as typically described for single walled olfactory sensilla (Altner, 1977; Steinbrecht, 1997). The slender porous sensilla trichoidea are longer, show a thicker cuticle and a reduced number of pores with respect to the typical single walled olfactory sensilla. Double walled sensilla basiconica are very similar to sensilla coeloconica described in the sacculus of Drosophila melanogaster as ancient olfactory sensilla bearing ionotropic receptors (IR) on their sensory neurons (Yao et al., 2005; Benton et al., 2009). Similar grooved or digitated pegs have also been reported in other insects, such as Orthoptera (Altner et al., 1981), Blattoidea (Altner et al., 1977), Lepidoptera (Hill et al., 2010) and Hemiptera (Dihel et al., 2003), were IR sequences have been identified. The specificities of these double walled olfactory sensilla in E. oleracea may reflect ancestral and basic needs, as described for other insects (Croset et al., 2010), and their reduced number compared with that of the other olfactory sensilla could be coherent with a reduced importance of these sensilla in host plant location. The antennal olfactory sensilla of E. oleracea are very similar to those described, mainly by scanning electron microscopy, in N. viridula (Brèzot et al., 1997; Usha Rani and Madhavendra, 2005) and other pentatomids, both phytophagous and predatory (Silva et al., 2010; Ahmad, 2016). Likewise, in other Pentatomidae, (Brézot et al., 1997; Usha Rani and Madhavendra, 2005; Silva et al., 2010; Ahmad et al., 2016), the antennal olfactory sensilla of E. oleracea do not show any evident difference in morphology and distribution in males and females, thus suggesting their crucial involvement in aspects of behaviour different from reproduction.

4.4. Conclusions 18

This study confirmed in the cabbage stink bugs of the genus Eurydema the presence of sensilla whose internal and external morphology suggests an olfactory role, similar to those previously described in other pentatomid species. The EAG recordings provided strong evidence of the functionality of these antennal sensilla and described their large sensitivity to several host plant VOCs, thus confirming the crucial role of antennae in host plant location and selection. Behavioural results confirmed the use of olfaction in host plant selection, alone or in combination with visual and other stimuli. In addition, dual choice chamber results clearly demonstrated for the first time that E. oleracea prefers feeding on E. sativa, instead of a very common cabbage crop such as B. oleracea var. capitata, and suggested that this preference may be based on volatiles emitted by the host plants. Understanding insect pest olfaction from an ecological perspective is crucial to balance insect control and conservation (Haverkamp et al., 2018). In this regard, the results of the present study provide a rich basis for future research including the chemical characterization of extracts obtained from B. oleracea and E. sativa in combination with GC-EAD to identify biologicallyactive compounds, potentially useful for semiochemical-based control strategies of this important cabbage pest. Moreover, behavioural field experiments could be useful to verify the possibility of the use of E. sativa as trap crop in collard.

Acknowledgements We thank Antonello Sotgiu for technical support, Debora Savini, Giovanni Tagliaferri, Federico Di Buccio, Deni Taso and Lorenzo Austeri, for their help in insect rearing and data collection. We are grateful to Rosalba Padula at ARPA UMBRIA, for the use of the scanning electron microscope at “Centro cambiamento climatico e biodiversità in ambienti lacustri e aree umide Isola Polvese”. Funding was provided by the University of Perugia, Department of Chemistry, Biology and Biotechnology, Ricerca di Base 2017 fund.

References 19

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Fig. 1. Schematic drawing of a dual choice experiment setup. Two identical leaf portions of Arabidopsis thaliana were included inside a clip-cage (modified plastic Petri dish with a meshcovered hole and the rim covered by a small sponge ring). A filter paper strip impregnated with VOCs extract from Eruca sativa, or from Brassica oleracea, was placed below each leaf. Fig. 2. Dual choice experiments testing Eurydema oleracea females with Brassica oleracea and Eruca sativa (a) and with Arabidopsis plants associated with “active extracts” from the two plant species. (b). Bars indicate the means ± s.e.m. Columns with different letters are significantly different at P<0.05 (Student’s t-test for dependent samples). 25

Fig. 3. EAG responses of Eurydema oleracea female and male antennae to VOCs extract (eluted in hexane) of Brassica oleracea and Eruca sativa seedlings and plants. Table inset shows the statistical parameters of three-way ANOVA. Bars indicate the means ± s.e.m.. * P<0.05, Dunnett test comparing the responses with those to control (hexane used as solvent). Fig. 4. EAG responses (mean ± s.e.m.), as percentage to the reference standard stimulus (octanal 10% v/v), of females and males antennae of Eurydema oleracea to some synthetic VOCs reported in literature as emitted by Brassica oleracea, Eruca sativa or both of them. Table inset shows the statistical parameters of two-way ANOVA. Bars indicate the means ± s.e.m. * P<0.05, ns not significant; Dunnett test comparing the responses with those to control (paraffin oil used as solvent). Fig. 5. Dose-response relationships for stimulation of male and female antennae of Eurydema oleracea with octanal and butanoic acid solutions in paraffin oil. Table inset shows the statistical parameters of two-way ANOVA. Bars indicate the means ± s.e.m. of absolute responses (mv). * P<0.05, ns not significant; Dunnett test comparing the responses with those to control (paraffin oil used as solvent). Fig. 6. Antenna of the adult of Eurydema oleracea under SEM (a,b and inset 1, c,d and inset 1, e,f and inset 1) and TEM (inset 2 of b, inset 1,2 and 3 of c, inset 2 of d, inset 2 and 3 of f). a, Antenna constituted of a scape (S), a pedicel divided into two pedicellites (P1, P2) and a flagellum composed of two segments (F1, F2); b, Single–walled multiporous sensillum basiconicum. Note in inset 1 the numerous small pores (P) covering the whole cuticular surface; in inset 2 a cross section of the sensillum reveals the numerous pores (P) and the numerous dendritic branches (D) inside the sensillar lumen; c, Double-walled multiporous sensillum basiconicum. Note the grooved surface; inset 1-3, serial cross sections of the peg of a double-walled multiporous sensillum basiconicum from the proximal (3) to the distal (1) peg as reported in c; note the outer (OC) and the inner (IC) cuticular wall surrounding the dendrites (D), and the hollow cuticular spokes (arrow) connecting the lumen of the sensillum to the external environment. C, cuticle; D, dendrites; d, Slender porous 26

sensillum trichodeum. In inset 1 the smooth surface on one side of the hair and the wrinkled (arrows) surface on the opposite side are visible. In correspondence of the wrinkled surface, pores (P) with pore tubules are clearly visible in cross sections under TEM (inset 2). D, dendrites; e, mechanoreceptor in the form of an aporous socketed pointed grooved hair; f, gustatory sensillum in the form of a socketed grooved hair with an apical pore (inset 1); insets 2 and 3 represent serial cross sections of the hair from the proximal (3) to the distal (1) shaft; note the neurons (D), one of which stops at the base to form a tubular body (TB). C, cuticle, S, socket. Fig. 7. Scheme of the second flagellar segment (F2) of Eurydema oleracea (female). The three types of olfactory sensilla are represented separately to show their abundance and distribution on the dorsal and ventral side of F2. The distribution of the olfactory sensilla is similar in both sexes.

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