Multifunctional weaponry: The chemical defenses of earwigs

Journal of Insect Physiology 59 (2013) 1186–1193

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Multifunctional weaponry: The chemical defenses of earwigs Tina Gasch a, Matthias Schott a, Christoph Wehrenfennig b, Rolf-Alexander Düring b, Andreas Vilcinskas a,⇑ a b

Institute of Phytopathology and Applied Zoology, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany Institute of Soil Science and Soil Conservation, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany

a r t i c l e

i n f o

Article history: Received 15 May 2013 Received in revised form 19 September 2013 Accepted 20 September 2013 Available online 1 October 2013 Keywords: Dermaptera Chemical ecology Defensive secretion 1,4-Benzoquinones Antimicrobials Nematodes

a b s t r a c t Earwigs protect themselves against predators using pincer-like cerci and/or malodorous exudates secreted from abdominal glands. Little is known about the chemistry of these secretions and their potential functions. However, because earwigs live in aggregations and overwinter in soil, they are exposed to high microbial loads throughout their lifecycle, and we therefore hypothesized that the secretions are used not only to deter predators but also to combat pathogens and parasites in their environment. We analyzed the defensive secretions of the European earwig Forficula auricularia, the short-winged earwig Apterygida media and the woodland earwig Chelidurella guentheri by gas chromatography–mass spectrometry. The secretions of all three species contained 2-methyl-1,4-benzoquinone and 2-ethyl-1,4-benzoquinone, whereas A. media also produced 2,3-dimethyl-1,4-benzoquinone and 2-ethyl-3-methyl-1,4-benzoquinone. The latter has not been identified in the exudates of insects before. The composition and/or quantity of these components were species-specific and partially sex-specific. All secretions showed antimicrobial activity against Gram-positive and Gram-negative bacteria as well as two entomopathogenic fungi. Furthermore, the secretion of F. auricularia displayed nematicidal activity against Caenorhabditis elegans. Our data support the hypothesis that earwig secretions are multifunctional, serving both to deter predators and sanitize the microenvironment. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Many insect orders have evolved exocrine glands to produce substances that protect them against predators (e.g., Cane, 1986; Huth and Dettner, 1990; Pavis et al., 1994; Osborne and Jaffe, 1998). The Dermaptera (earwigs) have evolved two strategies to avoid predation: when disturbed, they initially bend their abdomen forwards to use their pincer-like cerci as a mechanical defense, but if the disturbance persists they can also emit a malodorous defensive fluid from glands located in the third and/or fourth abdominal segments (Vosseler, 1890; Günther and Herter, 1974; Eisner et al., 2000). Such abdominal glands are present in numerous genera representing the families Labiidae, Chelisochidae and Forficulidae (Günther and Herter, 1974) and the glandular exudate is considered to be effective against a broad range of predators. Nevertheless, little is known about earwig chemical defenses. Only two species of earwigs, namely Forficula auricularia and Doru taeniatum, have been investigated in detail to determine the morphology of their defensive glands, the chemistry of the

⇑ Corresponding author. Tel.: +49 641 9937601; fax: +49 641 9937609. E-mail addresses: [email protected] (T. Gasch), matthias.schott@ agrar.uni-giessen.de (M. Schott), [email protected] (C. Wehrenfennig), [email protected] (R.-A. Düring), [email protected] (A. Vilcinskas). 0022-1910/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinsphys.2013.09.006

secretions and their biological functions (Vosseler, 1890; Eisner, 1960; Schildknecht and Weis, 1960; Eisner et al., 2000). Earwigs live in dark and moist crevices, mostly in aggregations (Walker et al., 1993; Hehar et al., 2008). They are positively thigmotactic and therefore seek direct contact with surfaces and conspecifics (Weyrauch, 1929). Female earwigs also hibernate with their eggs in subterranean nests for several months (Günther and Herter, 1974). Groupliving and inter-individual contacts accelerate the transmission of pathogens and therefore of pathogen-mediated diseases (Schmid-Hempel and Schmid-Hempel, 1993; Côte and Poulin, 1995; Wertheim et al., 2005). With soil being the main reservoir for entomopathogenic fungi, bacteria and nematodes (Kung et al., 1990; Picard et al., 1992; Klingen and Haukeland, 2006), the habitat preferences and subsocial behavior of earwigs suggest they need effective protection not only from predators, but also a general defense against pathogenic microorganisms and parasites. Chemical defenses are common among insects and complement their innate immune system (Vilcinskas, 2013). Some species are surrounded by clouds of their allelochemical exudates because gland depletion is not precisely regulated, or the secretion remains on the cuticle for some time after discharge caused by an attack (Dettner et al., 1992). Leaf beetle larvae are known to release antimicrobial substances from exocrine glands into their environment (Gross et al., 2008; Kirsch et al., 2011). The burying beetle Nicrophorus vespilloides produces oral and anal secretions consisting of

T. Gasch et al. / Journal of Insect Physiology 59 (2013) 1186–1193

a cocktail of volatiles that presumably contribute to both sanitation of the microenvironment and chemical preservation of the cadavers used as reproduction and breeding sites (Degenkolb et al., 2011). However, although the typical malodorous scent of the defensive secretion can be perceived wherever several earwigs aggregate (Vossler, 1890), neither the presence of the secretion in the headspace surrounding earwig aggregations nor its effect on potential pathogens or parasites have been evaluated. Here we describe the first comparative survey of the chemical defense of earwigs, including the first compositional analysis of the defensive secretion of Apterygida media and Chelidurella guentheri. In addition, we tested the biological activity of the secretions on microorganisms using inhibition zone assays with Gram-positive and Gram-negative bacteria and two entomopathogenic fungi. The secretion of F. auricularia was also tested against the nematode Caenorhabditis elegans. The headspace surrounding individual specimens and natural aggregations of F. auricularia was analyzed using a needle trap device (NTD) to determine whether the bioactive substances are released into the microhabitat. 2. Materials and methods 2.1. Insects Adult specimens of F. auricularia, A. media and C. guentheri were collected between June and September 2012 in Giessen, Germany. The earwigs were kept in plastic boxes in mixed-sex groups of 10–20 individuals, provided with apple pieces and water ad libitum and maintained for at least 5 days allowing defensive secretions to be replenished (Eisner, 1960). The insects were killed by freezing at 80 °C and were dissected immediately under a stereomicroscope to excise the glandular reservoirs prior to chemical and biological assays. 2.2. GC–MS Analysis Excised glandular sacs from individual insects were transferred to 1.8-ml glass vials (CS – Chromatographie Service GmbH, Langerwehe, Germany) containing 100 ll (200 ll for F. auricularia) n-hexane (Sigma–Aldrich, St. Louis, USA). The sacs were ruptured with a sterile insect needle and sonicated in ice-cold water for 10 min. For quantitation, the solvent was spiked with 10 ng/ll n-octadecane (C18, Sigma–Aldrich) as an internal standard. Five individual insects were analyzed per species and sex. One microliter of each extract was injected splitless into a gas chromatograph-mass spectrometer (GC–MS; CT 1128, Constellation Technology Corporation, Largo, Florida, USA) equipped with a VF-5MS column (30 m  0.25 mm internal diameter, 0.25 lm film thickness; Agilent Technologies, Inc., Santa Clara, USA). The temperature was programmed to rise from 50 °C (isothermic for the first 2 min) to 300 °C at 20 °C/min. The injection port was set to 230 °C and helium was used as carrier gas. Mass spectra were obtained in full scan electron ionization mode at 70 eV. Individual substances were identified by comparing their mass spectra (Supplementary Fig. S1) and retention indices with those of reference compounds (2-methyl-1,4-benzoquinone (MBQ); Sigma–Aldrich) or literature data (2,3-dimethyl-1,4-benzoquinone (DMBQ), 2-ethyl-3–methyl-1,4-benzoquinone (EMBQ); Machado et al., 2005; Föttinger et al., 2010). The defensive secretion of Tribolium castaneum was injected as an authentic reference for 2-ethyl-1,4-benzoquinone (EBQ) (Markarian et al., 1978). For data acquisition and integration of peak areas, the software MSD ChemStation E.02.00.493 (Agilent Technologies) was used. The statistical significance of observed quantitative differences was determined using SigmaStat 11.0 (Systat Software Inc., San Rose, USA). Data were compared pairwise using an unpaired t-test for normal

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distributions with equal variance, or otherwise using a Mann Whitney U test. 2.3. Antibacterial Activity The activity of the exudates against standard Gram-positive and Gram-negative bacteria was assessed using Micrococcus luteus strain DSM 20030 (Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and the lipopolysaccharide-defective, streptomycin/ampicilinresistant mutant of Escherichia coli K-12 strain D31 (Boman et al., 1974). The bacteria were cultured in LB (E. coli) or TSB broth (M. luteus) (Sigma–Aldrich). Fresh broth was inoculated with an aliquot of an overnight culture and grown to mid-log phase (OD600 = 0.5) at 37 °C (E. coli) or 30 °C (M. luteus). For the inhibition zone assays, 50 ml of broth containing 1% agar (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) was inoculated with 200 ll (E. coli) or 500 ll (M. luteus) of the cultured bacteria and 7 ml of the inoculated agar medium was poured into Petri dishes (9 cm diameter). 2.4. Antifungal Activity The antifungal activity of the secretions was evaluated using the entomopathogenic fungi Beauveria bassiana strain FSU 4404 (Jena Microbial Resource Collection, Jena, Germany) and Metarhizium anisopliae ARSEF 2038 (USDA – ARS Collection of Entomopathogenic Fungal Cultures, Ithaca, USA). Both fungi are cosmopolitical and infect a wide range of insect species (Toledo et al., 2006), including earwigs (Günther and Herter, 1974). The fungi were cultured on 1.5% agar plates (Carl Roth) containing PD (B. bassiana) or YPG (M. anisopliae). When fully sporulating, conidia suspensions were prepared in 10 ml 0.9% NaCl, filtered through one layer of sterile Miracloth (Merck KGaA, Darmstadt, Germany) and adjusted with 0.9% NaCl to 1.3  107 spores/ml. For each assay, 70 ml agar was inoculated with 1 ml of the conidia suspension and poured into Petri dishes (9 cm diameter) in 7-ml aliquots. 2.5. General Bioassay Procedure When solidified, seven 3-mm wells were punched into the agar plates and plugs were removed with a vacuum pump. The total quantity of benzoquinones produced by each species, as determined by chemical analysis (Table 1), was used as a guiding value for the concentrations applied in the inhibition zone assays. The defensive glands of seven individuals, separated by species and sex, were therefore pooled and diluted in n-hexane to obtain an average equivalent to one individual. Pure n-hexane was used as a negative control, and dilutions (0.1–12 lg/ll) of 1,4-benzoquinone (Fisher Scientifc GmbH, Schwerte, Germany) in n-hexane were applied as positive controls. We then added 3 ll per sample to each well and incubated the plates for 24 h at 37 °C (E. coli) or 30 °C (M. luteus). The Petri dishes inoculated with fungi were incubated for 72 h at 23 °C for B. bassiana and at 28 °C for M. anisopliae. After incubation the antimicrobial activity of the secretion was determined by measuring the inhibition zone diameter around each well. All tests were carried out in triplicate. 2.6. Nematicidal Activity Due to the rare occurrence of A. media and C. guentheri specimens, nematicidal activity was tested using F. auricularia exudate only. The activity was tested against the model organism C. elegans strain N2 (kindly provided by Prof. Dr. U. Wenzel, Molecular Nutrition Research, Justus Liebig University Giessen, Germany) using two different approaches. The nematodes were cultured at room

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Table 1 Significant differences of the quantitative composition of the defensive glands of male and female F. auricularia, C. guentheri and A. media (mean ± SD in lg/individual). F. auricularia

2-Methyl-1,4-benzoquinone 2-Ethyl-1,4-benzoquinone 2,3-Dimethyl-1,4-benzoquinone 2-Ethyl-3-methyl-1,4-benzoquinone Total

C. guentheri

A. media

$

#

$

#

$

#

9.5 ± 2.7a 21.3 ± 1.2a – – 30.7 ± 3.6a

12.7 ± 2.7a 18.8 ± 6.8a – – 31.5 ± 9.3a

3.1 ± 0.8b 1.3 ± 0.5b – – 4.3 ± 1.3b

4.1 ± 0.7c 1.4 ± 0.3b – – 5.6 ± 1.0b

4.8 ± 0.6d 0.2 ± 0.06c 6.1 ± 1.6a 0.1 ± 0.06a 11.3 ± 2.2c

3.9 ± 0.6c 0.1 ± 0.03d 3.6 ± 1.2b 0.06 ± 0.02b 7.6 ± 1.8b

a-d

Different lowercase letters indicate significant differences (P < 0.05, t-test/Mann Whitney U test, N = 5 per sex and species). All statistical values are provided in Supplementary Tables S2 and S3.

temperature on NGM agar plates with E. coli strain OP50 as a food source (Brenner, 1974). The method of choice to assess nematicidal activity is the motility assay (Gill et al., 2003), in which 96-well plates were filled with 40 ll sterile M9 medium per well (Brenner, 1974). Excised glandular reservoirs from individual earwigs were transferred to each well (N = 6) and ruptured with a sterile insect needle. The depleted cuticular reservoirs were removed after 10 min and 50 worms in M9 medium from a 4-day-old culture representing various developmental stages were added to a total volume of 60 ll per well. Wells containing only medium and worms were used as negative controls (N = 6). To test the influence of adjoining tissue, we added pieces of cuticle from the sixth abdominal segment to the control wells (N = 6). Nematode mortality was recorded using a stereomicroscope after incubation at room temperature for 48 h. The nematodes were considered dead if their bodies were rigid and non-motile. The statistical significance of the observed differences was analyzed using SigmaStat 11.0 (Systat Software Inc.) and since the data were not normally distributed, treatments were compared using the Mann Whitney U test. As some authors have expressed concerns about the subjectivity of the motility test (Gill et al., 2003; Smith et al., 2009), we conducted the microplate thermotolerance assay as an alternative. This assay is used for the high-throughput screening of compounds that affect the nematode lifespan in the presence of thermal stress (Gill et al., 2003). Here, the thermotolerance of the nematodes after treatment with secretion was tested. Worms were prepared as above, incubated for 48 h and washed twice in 10 ml M9 medium containing 1% (v/v) Tween-20. After each wash, the worms were centrifuged for 2 min at 1200 rcf and the supernatant (8 ml) was removed. Forty adult worms per treatment were pipetted individually, in 7.5 ll M9 medium containing 1% (v/v) Tween-20, into each well of a black 384-well plate (Greiner-Bio-One GmbH, Frickenhausen, Germany). As controls, 24 wells were filled with 6.5 ll M9 medium containing 1% (v/v) Tween-20. Finally, 7.5 ll of 2 lM SytoxÒ Green fluorescent dye (Life Technologies GmbH, Darmstadt, Germany) was added to each well. The plate was sealed with VIEWseal film (Greiner Bio-One) to prevent evaporation and covered with a lid (Greiner Bio-One). A lethal heat shock at 37 °C was then applied and fluorescence was measured with a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments GmbH, Bad Friedrichshall, Germany) for 16 h with a total of 33 fluorescence measurements at 30-min intervals. The excitation wavelength was 485 nm and the emission wavelength 525 nm. SytoxÒ Green is a nucleic acid stain that cannot pass through the membrane of intact cells, but it can enter the cell and bind to DNA as a result of thermal stress. The fluorescence intensity therefore scores cellular damage and thus viability of the worms. Given that the secretion has an effect on the worms, the lifespan would be significantly shorter than that of the controls. The data were processed with GraphPAD Prism 5.03 (GraphPAD Software Inc., La Jolla, USA) and displayed as Kaplan–Meier survival curves. Differences in thermotolerance were tested using the Mantel–Haenszel log-rank test implemented in the software.

2.7. Headspace Analysis Because the A. media and C. guentheri specimens were scarce, only F. auricularia was used for headspace analysis. The headspace over individual specimens and natural aggregations of F. auricularia was analyzed using a NTD (for review see Lord et al., 2010) packed with Tenax TA (80/100 mesh) sorbent (PAS Technology Deutschland GmbH, Magdala, Germany). Individual insects (N = 6, three of each sex) were placed carefully, to prevent the provocation of defense behavior, into 10-ml headspace vials, which were closed with screw caps provided with silicone/PTFE seals (CS – Chromatographie Service). A Pasteur pipette filled with charcoal was used as an air filter. The filter and a clean NTD were inserted through the seal and the headspace was sampled 1 cm above the insects for 30 min, 2 h, 6 h or 24 h at a flow rate of 6 ml/min. A second experiment was carried out under field conditions. To promote the formation of natural aggregations, fresh bamboo traps (Huth et al., 2009) were fixed to shrubs located at the biological research station of the Justus Liebig University Giessen, Germany, 30 cm above the ground. After one week, clean NTDs were carefully inserted into the individual traps and the headspace was sampled for 30 min each (6 ml/min flow rate, N = 5). Directly after headspace sampling the number of insects per trap was determined. Subsequently, the NTDs were injected into the GC injector to desorb the trapped analytes. The GC–MS parameters were the same as those used for the analysis of defensive secretions (Section 2.2), but the MS was set to selected ion monitoring (SIM) mode at m/z 122 (characteristic fragment for MBQ) and m/z 136 (characteristic fragment for EBQ) to gain additional sensitivity. The headspace of bamboo traps and vials without F. auricularia was sampled as control. An external standard was used because the sampling technique did not allow the use of an internal standard. For quantification, a calibration curve was established by measuring dilutions of C18 in n-hexane (2–200 ng/ll) with the same parameters desribed above, except that the MS was set to SIM mode at m/z 57 and 254 (characteristic fragments for C18).

3. Results 3.1. GC–MS Analysis The abdominal defensive glands of three species of earwigs were dissected under a stereomicroscope and analyzed by GC– MS. In each species, the glandular reservoirs were located pairwise in the third and fourth abdominal segments. Although smaller in size, the gross morphology of the reservoirs from A. media and C. guentheri was reminiscent of the sac-like reservoirs from F. auricularia and D. taeniatum, which were described in detail by Vosseler (1890) and Eisner et al. (2000). No morphological differences were observed between the sexes. The secretions of all three species possessed the characteristic odor of quinones, but varied in color ranging from pale brown (A. media and C. guentheri) to bright

T. Gasch et al. / Journal of Insect Physiology 59 (2013) 1186–1193

yellow (F. auricularia). Furthermore, when F. auricularia was disturbed, the secretion oozed from the gland orifices and remained on the surrounding cuticle (Fig. 1a), rather than being ejected as a spray, as previously described (Eisner, 1960; Eisner et al., 2000). In total, four different substances were detected in the defensive secretions of the three earwig species (Table 1 and Fig. S2). All substances showed the typical mass fragmentation patterns (Fig. S1) of alkylated 1,4-benzoquinones (Budzikiewicz et al., 1964; Machado et al., 2005). As reported by Eisner et al. (2000) and Schildknecht and Weis (1960), the secretion of F. auricularia qualitatively comprised two components, represented by two distinct peaks in the total ion current chromatogram (TIC). These two peaks were identified by comparison with authentic references as MBQ (peak [1]): m/z (%) 54 (56), 66 (42), 68 (30), 82 (55), 94 (67), 122 (100) and EBQ (peak [2]): m/z (%) 54 (52), 65 (12), 79 (69), 82 (43), 90 (7), 107 (43), 108 (100), 121 (6), 136 (67). Both benzoquinones were also present in the extracts of C. guentheri and A. media, but the TIC of the latter species displayed two additional peaks (Fig. 1b) which were tentatively identified based on their fragmentation pattern only (Fig. S1). Peak [3] shows a molecular ion at m/z 136, a prominent fragment at m/z 54, but no signal at m/z 68, suggesting an alkyl substitution on only one side of the 1,4-benzoquinone ring. The mass spectrum (m/z (%) 54 (48), 65 (4), 77 (10), 79 (44), 82 (46), 90 (8), 107 (46), 108 (46), 136 (100)) is similar to that of EBQ but lacks m/z 121. This characteristic pattern suggested that DMBQ is the only possible candidate. The mass spectrum also matched the published spectrum for DMBQ (Machado et al., 2005). The substance represented by peak [4] has not been detected in insect secretions before and was only found in extracts of A. media. The mass spectrum shows a molecular ion at m/z 150 and fragments at: m/z (%) 54 (26), 67 (14), 79 (37), 82 (27), 107 (73), 121 (19), 122 (30), 135 (11), 150 (100). Again, the intense peak at m/z 54 and the lack of a fragment at m/z 68 suggested an alkyl substitution on only one side of the ring, e.g. a 2,3-substitution. The fragmentation pattern was identical to the mass spectrum of EMBQ found in the defensive secretion of opilionids (Machado et al., 2005; Föttinger et al., 2010). The quantitative analysis of the secretions is summarized in Table 1 and illustrated in Supplementary Fig. S2. Furthermore, the relative compositions are listed in Supplementary Table S1.

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The statistical values of the quantitative analysis are provided in Supplementary Tables S2 and S3. The three species differed in terms of the total benzoquinone content and the ratios of the individual components, with the exception of A. media and C. guentheri males, which showed no significant differences in their total benzoquinone (A. media 7.6 ± 1.8, C. guentheri 5.6 ± 1.0 lg/individual) and MBQ content (A. media 3.9 ± 0.6, C. guentheri 4.1 ± 0.6 lg/ individual). F. auricularia produced the largest quantity of total benzoquinones, with 31.5 ± 9.3 (males) and 30.7 ± 3.6 lg/individual (females). Neither qualitative nor quantitative differences were found between the sexes in F. auricularia, but in C. guentheri the sexes differed in their MBQ content (female 3.1 ± 0.8, male 4.1 ± 0.6 lg/individual). Sex-specific quantitative differences in both the total benzoquinone content and the ratios of the individual components were observed only in A. media (Table 1). 3.2. Antibacterial and Antifungal Activity The antimicrobial activity of the defensive secretions was tested using inhibition zone assays. The solvent control n-hexane showed no inhibitory effect, whereas 1,4-benzoquinone inhibited the growth of all microorganisms at doses P1 lg/ll (Supplementary Table S4). The secretions of all three earwig species inhibited the growth of the selected bacteria and fungi at concentrations equivalent to one individual (Fig. 2) and there was no difference in antimicrobial activity between the sexes. 3.3. Nematicidal Activity The nematicidal activity of the F. auricularia secretion was tested against C. elegans using motility assays and a microplate thermotolerance assay. Both experiments showed that the secretion had a significant impact on nematode survival compared to the corresponding control (Fig. 3a and b). In contrast, the tissue had no impact on nematode survival in either assay (data not shown). In the motility assay, nematode mortality increased by 20-fold in the presence of the secretion (Mann Whitney U test, P < 0.05). In the microplate thermotolerance assay, the nematode

Fig. 1. Abdomen of a female F. auricularia specimen with secretion oozing from the gland orifices (indicated by arrows) (a). Total ion current chromatogram of the defensive secretion of a female A. media specimen, and substances identified therein: [1] 2-methyl-1,4-benzoquinone, [2] 2-ethyl-1,4-benzoquinone, [3] 2,3-dimethyl-1,4benzoquinone, [4] 2-ethyl-3-methyl-1,4-benzoquinone (b). The corresponding mass spectra are provided in Supplementary Fig. S1.

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T. Gasch et al. / Journal of Insect Physiology 59 (2013) 1186–1193 Table 2 Number of earwigs and amount of 2-methyl-1,4-benzoquinone (MBQ) and 2-ethyl1,4-benzoquinone (EBQ) in 180 ml headspace from natural aggregations of F. auricularia as determined by GC–MS analysis.

Fig. 2. Antimicrobial activity of the defensive secretions of F. auricularia, C. guentheri and A. media against selected bacteria and entomopathogenic fungi (mean ± SD in mm inhibition zone diameter, N = 3 each). The solvent control (nhexane) showed no inhibitory effect (Supplementary Table S4).

lifespan was reduced by approximately 50%, compared to untreated worms (Mantel–Haenszel log-rank test, P < 0.0001). 3.4. Headspace Analysis Individual F. auricularia did not release detectable amounts of the two benzoquinones identified in the glandular secretion (Section 3.1) during any of the sampling periods (30 min, 2, 6 or 24 h). This suggests that the sampling method did not evoke defensive behavior and thus the release of secretion. Furthermore, neither of the benzoquinones was detected in the control traps. However, our experiments revealed that both benzoquinones were present in the headspace of natural F. auricularia aggregations (Table 2). The quantities detected in 180 ml of headspace over natural aggregations ranged from 3.0 to 15.8 ng for MBQ and from 1.5 to 14.2 ng for EBQ (Table 2). 4. Discussion Although F. auricularia was one of the first insects to be investigated in terms of chemical defenses, few studies have addressed

Trap No.

No. of earwigs

MBQ [ng]

EBQ [ng]

MBQ + EBQ [ng]

1 2 3 4 5 Mean ± SD

25 37 23 32 16 26.6 ± 8.1

3.7 15.8 3.6 4.8 3.0 6.2 ± 5.4

2.6 14.2 1.5 4.2 2.8 5.1 ± 5.2

6.3 30.0 5.1 9.0 5.8 11.2 ± 10.6

the chemistry or biological activity of earwig defensive secretions. Including the present investigation, chemical data are now available for four species representing the subfamily Forficulinae (Schildknecht and Weis, 1960; Eisner et al., 2000). We identified four alkylated 1,4-benzoquinones in the defensive exudates of F. auricularia, A. media and C. guentheri, including EMBQ, which has not previously been identified in insects, but is found in the exocrine glands of opilionids (Machado et al., 2005; Föttinger et al., 2010; Rocha et al., 2011). Our experiments also demonstrated that the quality and/or quantity of the benzoquinones differed between species, and also between the sexes in A. media and partially in C. guentheri. Many arthropod exudates contain 1,4-benzoquinones (Blum, 1981), often mixed with hydrocarbons as solvents and carriers, but also as surfactants and deterrents (Peschke and Eisner, 1987; Eisner et al., 2000). Earwigs appear to have optimized the formulation of their quinoic secretions to a small number of highly active substances. Eisner et al. (2000) identified MBQ, DMBQ and npentadecane in the glandular reservoirs of D. taeniatum, and we can confirm the presence of MBQ and EBQ in F. auricularia secretions in agreement with previous reports (Schildknecht and Weis, 1960; Walker et al., 1993; Eisner et al., 2000). The corresponding hydroquinones detected by Schildknecht and Weis (1960) using thin-layer chromatography, were not detected in the present study or by Eisner et al. (2000), and could therefore result from different analytical methods. Furthermore, MBQ and EBQ are susceptible to light and temperature and can therefore degrade easily into the respective hydroquinones (Yamada and Hosaka, 1977; Yezerski et al., 2007). Our data show that alkylated benzoquinones are not restricted to F. auricularia and D. taeniatum but may constitute the fundamental principle of forficulid defensive secretions. MBQ

Fig. 3. Impact of the defensive secretion of F. auricularia on the survival of C. elegans. Secretion-induced mortality determined using a motility assay (different lowercase letters indicate significant differences at P < 0.05, Mann Whitney U test, N = 6 each) (a). Secretion-induced reduction of lifespan at 37 °C as determined by the microplate thermotolerance assay (different lowercase letters indicate significant differences at P < 0.0001, Mantel–Haenszel log-rank test, N = 40 each) (b).

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appears to be the common component in all earwig secretions analyzed thus far, and EBQ was absent only in D. taeniatum. Furthermore, D. taeniatum and A. media produce DMBQ, whereas EMBQ was exclusively found in A. media. However, the exudates of all species investigated in this study lacked hydrocarbons. Interestingly, in tenebrionid beetles, only the most primitive species produce quinonic secretions without hydrocarbons, whereas the secretions of morphologically-derived species also contain hydrocarbons (Tschinkel, 1975). The phylogenetic relationships among the Dermaptera remain poorly understood (Jarvis et al., 2005). As in Staphylinidae and Carabidae (Dettner, 1993; Will et al., 2000) the distinctive chemical composition of the secretion may therefore be useful for chemotaxonomic studies. However, chemosystematic inferences cannot be drawn from the current state of research and chemical investigations of additional dermapteran defensive secretions are needed. Chemical variation has been reported for several arthropod secretions and can be influenced by numerous factors including season, age, developmental stage, social caste, predators and physiological status (Brand et al., 1973; Tschinkel, 1975; Blum, 1981; Steidle and Dettner, 1993; Dahbi et al., 1998; Ômura et al., 2002; Pareja et al., 2007). Since defensive secretions need to be effective, but not necessarily specific (Hefetz, 1993), the chemical variation in the three earwig species and the sex-specific differences found in A. media and in part in C. guentheri may reflect that the defensive benzoquinones experienced an adaption to pheromonal communication. For example, 1,4-benzoquinone and MBQ combined with plant volatiles act as sex attractants in Melolontha spp. (Ruther et al., 2001a, 2001b; Reinecke et al., 2002). Because of the sexand stage-unspecific occurrence of these benzoquinones, their ability to act as sex pheromones may have evolved as a secondary function after defense (Ruther et al., 2001b). Thus far, no sex pheromones have been described in the Dermaptera (Ma and Ramaswamy, 2003) and in agreement with Walker et al. (1993) we observed no sexual dimorphism in the defensive secretion of F. auricularia. F. auricularia is known to emit a volatile aggregation pheromone, but its exact origin and chemistry remain unclear (Sauphanor and Sureau, 1993; Walker et al., 1993; Evans and Longépé, 1996; Hehar et al., 2008). According to Evans and Longépé (1996) the abdomen is the source of the aggregation pheromone, whereas Hehar et al. (2008) suggest it may comprise various substances released from several sites, including the glands and their depleted metabolites. Because the main component of the secretion (MBQ) repels conspecifics at concentrations of 0.01 and 0.1 mol/l, it could serve a dual function as a defensive secretion and alarm pheromone (Walker et al., 1993). It is also possible that the benzoquinones exert different effects depending on concentration and context. In Blaps sulcata, low concentrations of benzoquinones promote aggregation, whereas high concentrations defeat predators and repel conspecifics (Kaufmann, 1966). However, it is beyond the scope of our investigation to determine any potential communicative function of earwig secretions. The exocrine secretions of earwigs have been considered as a transient defense strategy, e.g., the benzoquinones secreted by F. auricularia are potent irritants that repel ants and the toad Hyla versicolor (Eisner, 1960). However, the subsocial behavior and habitat preferences of earwigs indicate they also require continuous protection against pathogens and parasites. Defensive compounds used by insects often possess antimicrobial properties (Blum, 1996). Ideally they would be highly volatile, to achieve optimal distribution in habitats such as soil (Chitwood, 2003), and would display non-specific toxicity (Bot et al., 2002; Chitwood, 2003). The benzoquinones produced by earwigs meet these criteria because they are highly reactive, interacting with proteins and inhibiting enzymatic processes by the oxidation of –NH and –SH groups

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(Ladisch, 1966; Ladisch and Suter, 1967). Our results demonstrated that MBQ and EBQ are present in the headspace of natural, undisturbed F. auricularia aggregations, and that the benzoquinones secreted upon challenge remain on the cuticle until they evaporate. Our data also show that all three secretions have a potent antimicrobial effect on Gram-positive and Gram-negative bacteria as well as entomopathogenic fungi at concentrations equivalent to the yield of one earwig. Furthermore, the F. auricularia secretion displayed excellent nematicidal activity against C. elegans. However, the amounts of benzoquinones present in the headspace of aggregations varied and were below the threshold dose required for an antimicrobial effect of 1,4-benzoquinone at 1 lg/ll. The ratio and amount of substances released by earwigs in nature may differ from those extractable from the glands (Hehar et al., 2008). In Tenebrionidae, for example, the external quinone levels are affected by factors such as degradation and deposition into the habitat and are lower than the amounts available internally (Yezerski et al., 2000). Harvestmen have considerable control over the quantities of exudate emitted from their defensive glands and adjust the load in accordance with the intensity of the threat (Machado and Pomini, 2008). Similar secretion-saving mechanisms could be used by earwigs because no benzoquinones were detected in the headspace of individual undisturbed F. auricularia specimens, but this species is known to discharge a secretion up to six times when threatened (Eisner, 1960). A secretion-optimizing regulatory mechanism has been confirmed for D. taeniatum, which ejects only dilute benzoquinone solutions while retaining most of the quinone crystals in the glandular reservoir (Eisner et al., 2000). Insect chemical defense is generally considered beneficial because chemically-protected individuals suffer less from predation. But chemical defense is also associated with certain costs (Skelhorn and Ruxton, 2008). Because the repertoire of substances an organism can produce de novo is limited, there are fitness costs associated with the biosynthesis of toxic compounds (Schmidtberg et al., 2013; Vilcinskas, 2013). These costs may be offset when the secretions are effective against a broad range of potential predators (Prestwich, 1983) or fulfill different purposes (Skelhorn and Ruxton, 2008). Therefore, the multifunctional use of secretions should be favored. In many insects, exocrine secretions serve different functions in different contexts, a phenomenon known as semiochemical parsimony (Blum, 1996). For earwigs there are additional survival costs because higher concentrations of MBQ are repellent to conspecifics (Walker et al., 1993) and the secretion eventually becomes toxic to the producer itself if a certain concentration threshold is exceeded (personal observation). Skelhorn and Rowe (2006) suggested that there may be an optimum strategy allowing the costs and benefits of chemical defense to be balanced, in which insects emit only a proportion of their secretion at a time. The benzoquinone doses we detected in the headspace over earwig aggregations may similarly reflect the balance between the costs of production, the benefits of survival, the potential to repel conspecifics, and the benefits of protection against predators, pathogens and parasites. The three earwig species varied in their ability to suppress microbial growth. This could reflect the total amount of benzoquinones, given that F. auricularia produced the highest amounts and also showed the most potent antimicrobial activity. But the chemical composition could also be important, because it is likely that mixtures exert additive or even synergistic effects (Dettner et al., 1992) as demonstrated for certain ants and beetles (Brand et al., 1973; Dettner and Reissenweber, 1991). In the metapleural secretion of leaf-cutting ants, the individual compounds may even have divergent functions that promote protection against different kinds of infections (Bot et al., 2002). Because only MBQ is commercially available among the substances we identified, we were unable to test this hypothesis for earwigs.

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In summary, the quinoic defensive secretions of earwigs appear to be multifunctional, repelling predators, providing unambiguous antibacterial, antifungal and nematicidal activity, and enveloping earwig aggregations in nature. In this context, the secretions may be beneficial for prophylaxis against co-occurring pathogens. Together with its suggested function as alarm pheromone (Walker et al., 1993) the species-specific and in part sex-specific secretion might also provide an additional mechanism for interspecific and intraspecific communication that is dependent on both context and concentration. Acknowledgments The authors acknowledge funding from the Hessian Ministry of Science and Art via the LOEWE-research projects ‘‘AmbiPobe’’ and ‘‘Insect Biotechnology’’. We thank Dr. H. Schmidtberg, Dr. T. Degenkolb and Dr. M. Rahnamaeian (Institute of Phytopathology and Applied Zoology, Justus Liebig University Giessen, Germany) for technical support and Dr. Claudia Huth (DLR, Neustadt an der Weinstraße, Germany) for providing F. auricularia specimens for analysis. The authors are indebted to Dr. Richard M. Twyman for the careful editing of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jinsphys.2013. 09.006. References Blum, M.S., 1981. Chemical Defenses of Arthropods. Academic Press, New York. Blum, M.S., 1996. Semiochemical parsimony in the arthropoda. Annu. Rev. Entomol. 41, 353–374. Boman, H.G., Nilsson-Faye, I., Paul, K., Rasmuson Jr., T., 1974. Insect immunity, I. Characteristics of an inducible cell-free antibacterial reaction in hemolymph of Samia cynthia pupae. Infect. Immun. 10, 136–145. Bot, A.N.M., Ortius-Lechner, D., Finster, K., Maile, R., Boomsma, J.J., 2002. Variable sensitivity of fungi and bacteria to compounds produced by the metapleural glands of leaf-cutting ants. Insect. Soc. 49, 363–370. Brand, J.M., Blum, M.S., Barlin, M.R., 1973. Fire ant venoms: intraspecific and interspecific variation among castes and individuals. Toxicon 11, 325–331. Brenner, S., 1974. The genetics of Caenorhabditis elegans. Genetics 77, 71–94. Budzikiewicz, H., Djerassi, C., Williams, D.H., 1964. Structure Elucidation of Natural Products by Mass Spectrometry, Volumes 1 & 2, Holden-Day Series in Physical Techniques in Chemistry. Holden-Day Inc., California. Cane, J.H., 1986. Predator deterrence by mandibular gland secretions of bees (Hymenoptera: Apoidea). J. Chem. Ecol. 12, 1295–1309. Chitwood, D.J., 2003. Nematicides. In: Plimmer, J.R. (Ed.), Encyclopedia of Agrochemicals, 3. John Wiley & Sons, New York, pp. 1104–1115. Côte, I.M., Poulin, R., 1995. Parasitism and group size in social animals: a metaanalysis. Behav. Ecol. 6, 159–165. Dahbi, A., Cerdá, X., Lenoir, A., 1998. Ontogeny of colonial hydrocarbon label in callow workers of the ant Cataglyphis iberica. C. R. Acad. Sci. Ser. III 321, 395– 402. Degenkolb, T., Düring, R.-A., Vilcinskas, A., 2011. Secondary metabolites released by the burrying beetle Nicrophorus vespilloides: Chemical analyses and possible ecological functions. J. Chem. Ecol. 37, 724–735. Dettner, K., 1993. Defensive secretions and exocrine glands in free-living staphylinid beetles – their bearing on phylogeny (Coleoptera: Staphylinidae). Biochem. Syst. Ecol. 21, 143–162. Dettner, K., Reissenweber, F., 1991. The defensive secretion of Omaliinae and Proteininae (Coleoptera: Staphylinidae): its chemistry, biological and taxonomic significance. Biochem. Syst. Ecol. 19, 291–303. Dettner, K., Fettköther, R., Ansteeg, O., Deml, R., Liepert, C., Petersen, B., Haslinger, E., Francke, W., 1992. Insecticidal fumigants from defensive glands of insects – a fumigant test with adults of Drosophila melanogaster. J. Appl. Entomol. 113, 128–137. Eisner, T., 1960. Defense mechanisms of arthropods. II. The chemical and mechanical weapons of an earwig. Psyche 67, 62–70. Eisner, T., Rossini, C., Eisner, M., 2000. Chemical defense of an earwig (Doru taeniatum). Chemoecology 10, 81–87. Evans, K.A., Longépé, V., 1996. The European earwig: getting the best of both worlds? In: Wiley, K.B. (Ed.), Proceedings of the Second International Conference on Urban Pests. Exeter Press, UK, pp. 163–167.

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