The Role of Semiochemicals in Short-Range Location of Aggregation Sites in Adalia bipunctata (Coleoptera, Coccinellidae) Eline C. Susset, Felipe RamonPortugal, Jean-Louis Hemptinne, Sarah Y. Dewhirst, Michael A. Birkett & Alexandra Magro Journal of Chemical Ecology ISSN 0098-0331 Volume 39 Number 5 J Chem Ecol (2013) 39:591-601 DOI 10.1007/s10886-013-0285-0
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Author's personal copy J Chem Ecol (2013) 39:591–601 DOI 10.1007/s10886-013-0285-0
The Role of Semiochemicals in Short-Range Location of Aggregation Sites in Adalia bipunctata (Coleoptera, Coccinellidae) Eline C. Susset & Felipe Ramon-Portugal & Jean-Louis Hemptinne & Sarah Y. Dewhirst & Michael A. Birkett & Alexandra Magro
Received: 17 November 2012 / Revised: 22 February 2013 / Accepted: 5 April 2013 / Published online: 26 April 2013 # Springer Science+Business Media New York 2013
Abstract To survive unfavorable periods, ladybird beetles form conspicuous aggregations in specific microsites, with these locations remaining the same year after year. This constancy of location leads to the hypothesis that semiochemicals are involved in the attraction and aggregation of ladybirds to the microsite. In this study, we identified two types of semiochemicals that could play key roles in the attraction and aggregation formation of the two-spotted ladybird, Adalia bipunctata. We first isolated and identified three alkylmethoxypyrazines from A. bipunctata and tested the behavioral responses of diapausing ladybirds to these chemicals in a four-way olfactometer. This revealed that 2-isobutyl-3-methoxypyrazine, on its own or as part of a two-component mixture with 2isopropyl-3-methoxypyrazine, elicited a positive behavioral response, causing arrestment of diapausing A. bipunctata. As ladybirds are in contact with each other in aggregations, we investigated the role of cuticular hydrocarbons (CHCs) in driving the cohesion and maintenance of aggregation. When an extract of CHCs from diapausing ladybirds was deposited near an alkylmethoxypyrazine source, ladybirds spent more time in the vicinity of the source. We identified a set of CHCs specific to diapausing A. bipunctata. Alkylmethoxyyrazines and CHCs thus deliver information to diapausing ladybirds E. C. Susset (*) : F. Ramon-Portugal : J.-L. Hemptinne : A. Magro Université de Toulouse, UMR CNRS/EDB/ENFA “Laboratoire Evolution et Diversité Biologique”, 2 route de Narbonne, 31320 Castanet Tolosan, France e-mail:
[email protected] S. Y. Dewhirst : M. A. Birkett Department of Biological Chemistry and Crop Protection, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK
searching for an aggregation site, as well as mediating several other behaviors throughout the ladybird’s life cycle. Chemical parsimony is discussed. Keywords Alkylmethoxypyrazines . Cuticular hydrocarbons . Diapause . Ladybird . Overwintering aggregation . Coleoptera . Coccinellidae
Introduction Many species of insects anticipate and survive unfavorable environmental conditions and low food periods by entering diapause, a series of physiological, morphological, and behavioral changes under hormonal control (Rankin and Rankin, 1980). In a number of species of Coleoptera, Hemiptera, and Lepidoptera, once diapause is induced, they migrate to, and aggregate in, specific locations (Wolda and Denlinger, 1984; Brower, 1996; Toyama et al., 2006). This conspicuous, but ephemeral, phenomenon brings together up to millions of conspecifics. The widespread aggregation behavior of Coleoptera is hypothesized to be adaptive, with benefits including enhanced survival (Mooring and Hartl, 1992; Brower et al., 2008) and increased probability of finding a mate (e.g., Wertheim et al., 2005). Costs include the increased risk of being infected by pathogens, parasitism, or predation (Nalepa and Weir, 2007; Mcclure and Despland, 2011). Aggregations take place at the same sites year after year (Majerus, 1994; Brower, 1996; Sabu et al., 2008). Site selection is based on physical factors, such as exposure, temperature, or topography (Hodek and Honek, 1996). As the majority of Coleoptera, Hemiptera, and Lepidoptera have an annual life history, aggregation behavior is seen as inherited, rather than a
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consequence of learning (e.g., Brower, 1996). However, the information that causes such aggregation behavior at the same places every year is poorly understood. To date, studies have indicated that visual cues play a role in guiding migrants from feeding to aggregation habitats (Nalepa et al., 2005), while olfactory cues are important in short-range attraction to specific aggregation microsites (Majerus, 1997). Insects rely on semiochemicals throughout their life cycle, but few studies have investigated the role of semiochemicals at the time of diapause (Al Abassi et al., 1998). Ladybird beetles are interesting models for the study of aggregation cues at diapause. Although empirical studies are lacking, several authors have described field aggregations for many species (Majerus, 1994; Hodek and Honek, 1996). Moreover, the chemical ecology of ladybirds is relatively well known. Volatile semiochemicals have been identified in the seven spotted, Coccinella septempunctata L., and Harlequin, Harmonia axyridis (Pallas) (Al Abassi et al., 1998; Verheggen et al., 2007) ladybirds, but field experiments to confirm their function in the formation of overwintering aggregations are lacking. Al Abassi et al. (1998) identified the alkylmethoxypyrazine 2-isopropyl-3-methoxypyrazine (IPMP), which was found to fulfil a pheromonal role in attraction among adult C. septempunctata. Other ladybird species, including the two spotted ladybird, Adalia bipunctata (L.), also produce alkylmethoxypyrazines (Moore et al., 1990). Therefore we hypothesized that alkylmethoxypyrazines may play a role in mediating ladybird aggregation behavior. Once an aggregation site has been located, field observations suggest that physical contact among migrants leads to aggregation clusters. This behavior has been observed in ladybirds such as A. bipunctata, Hippodamia undecimnotata Schneider, H. axyridis (Majerus, 1994; Hodek and Honek, 1996), and, more recently, in the brown marmorated stink bug, Halyomorpha halys (Stål) (Toyama et al., 2006). Tactile cues could, therefore, act as arrestants in the establishment and persistence of aggregation. Although several ladybird species may migrate to the same location, they tend to form monospecific clusters (Hodek and Honek, 1996). This suggests that ladybirds use cue-mediated specific recognition. Cuticular hydrocarbons (CHC), synthesized in specialized subcuticle cells under hormonal control, could be well suited to deliver this species-specific information, as they act as recognition cues in a variety of social and non-social insect species (Blomquist and Bagnières, 2010). Moreover, it is known that ladybirds use hydrocarbons to protect their eggs against cannibalism and predation, to recognize conspecifics and to evaluate the quality of an oviposition site (Hemptinne and Dixon, 2000).
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Here, we used the model species A. bipunctata to study the chemical ecology of aggregation in diapausing ladybirds. Distributed across the Holarctic region, A. bipunctata is a generalist aphid predator (Omkar and Pervez, 2005). It aggregates in buildings and crevices, in the bark of trees, or around window frames (Hemptinne, 1985; Majerus, 1994). The aims of this study were to isolate and identify A. bipunctata alkylmethoxypyrazines and CHCs, and to investigate their role in mediating intraspecific attraction. We also determined the differences in alkylmethoxypyrazines and CHC profiles between diapausing and non-diapausing ladybirds.
Methods and Materials Ladybird Cultures Adalia bipunctata adults were collected from lime trees, Tilia cordata Mill., in the vicinity of Toulouse (France) and were maintained in 2 L ventilated plastic boxes in a controlled environment room (20±1 °C, 16:8 L:D). Adults were fed three times a week on an excess of mixedinstar pea aphids, Acyrthosiphon pisum (Harris), which were reared on broad bean plants (Vicia faba L.). Each box contained a piece of corrugated filter paper as an egg-laying substrate. Eggs were collected three times a week. Immediately after egg hatching, a maximum of 10 larvae was placed in ventilated plastic boxes (175 cm3) in controlled environmental conditions known to induce diapause (i.e., 20± 1 °C, 10:14 L:D). These conditions were chosen according to Obrycki et al. (1983): in A. bipunctata, short photoperiods induce diapause, whereas low temperatures have no effect. Ovarian dissection carried out on a set of 30 ladybirds confirmed that they were in reproductive diapause (data not shown). Newly emerged adults were kept in isolation in 5 cm diam. Petri dishes, under the same conditions as the larvae, and transferred to clean dishes once a week. Two days after emergence, the sex of adults was determined under a binocular microscope, following Hodek and Honek, (1996). Couples were formed when ladybirds were 6 d old. Adult ladybirds, larvae, and newly emerged adults were fed three times a week with an excess of mixed-instar pea aphids, A. pisum. Non-diapausing ladybirds were reared as described above, but under a 16:8 L:D photoperiod. Extraction of Alkylmethoxypyrazines A bulk extraction for the olfactometer bioassays was carried out with 8–15 d-old non-diapausing adult A. bipunctata (N=500, sex-ratio 1:1). In addition, other bulk extractions were carried out on three batches of 75 diapausing ladybirds, and on three batches of 75 non-diapausing ladybirds, so as to compare the alkylmethoxypyrazines they produce. In both cases, ladybirds were cooled in liquid nitrogen, ground in a pestle and mortar, and extracted with freshly distilled dichloromethane (200 ml)
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at 25 °C for two consecutive 24-h periods. The extracts were dried with anhydrous magnesium sulphate, filtered, and evaporated under a gentle stream of nitrogen to ca. 5 ml. Volatiles were collected by vacuum distillation (0.03 torr) for 21 h at 25 °C (Al Abassi et al., 1998), and the resulting extracts stored in tightly capped vials in a deep freezer (−20 °C), until required for chemical analysis or bioassays. Analysis of Alkylmethoxypyrazines Extracts of alkylmethoxypyrazines were analyzed by gas chromatography (GC) on both non-polar (HP-1, 50 m×0.32 mm i.d., film thickness 0.52 μm) and polar (DB-WAX, 30 m×0.32 mm i.d., film thickness 0.5 μm) capillary columns, using an HP6890 GC (Agilent Technologies, UK) fitted with a cool-on-column injector, a deactivated retention gap (1 m×0.53 mm i.d.), and a flame ionization detector. The oven temperature was maintained at 30 °C for 1 min. after sample injection (4 μl), and increased by 5 °C.min.−1 to 150 °C, then by 10 °C.min.−1 to 230 °C. The carrier gas was hydrogen. Coupled gas chromatography/mass spectrometry (GC/MS) analysis was performed on a VG Autospec mass spectrometer (Fisons, Manchester, U.K.) coupled to an HP6890 GC fitted with a non-polar column (HP-1, 50 m×0.32 mm i.d., film thickness 0.52 μm), and a cool on—column injector. Ionization was by electron impact (70 eV, source temperature 220 °C). Helium was the carrier gas. The GC oven temperature was maintained at 30 °C for 5 min., and then programmed at 5 °C.min.−1 to 250 °C. Tentative identifications were made by comparison of spectra with mass spectral databases (NIST, 2005). Peak enhancement by co-injection with authentic standards was used to confirm tentative identifications (Pickett, 1990). Quantification of alkylmethoxypyrazines was performed by a single point external standard quantification method with authentic standards (Skelton et al., 2010). Extraction of Cuticular Hydrocarbons (CHCs) Two batches of elytra from 8 to 15 d-old diapausing adult A. bipunctata (N=30, sex-ratio 1:1) were washed x 3 with 1 ml hexane (Merck, Darmstadt, Germany) for the olfactometer bioassays. The extract of each batch of beetles was transferred to a 2-ml autosampler vial and evaporated under a gentle stream of nitrogen. Finally, the dry residue was redissolved in 300 μl of dichloromethane (i.e., 2 beetles equivalent per 10 μl). The two batches were combined and stored in a tightly capped vial at −20 °C until required for the bioassays. In addition, extractions of CHCs were done on three batches of 75 diapausing and on three batches of 75 non-diapausing ladybirds to compare profiles. Analysis of CHCs GC/MS analysis was performed on a mass spectrometer quadrupole detector coupled to a Finnigan Trace 2000 GC (Thermo Fisher Scientific Inc., Illkrich, France), fitted with a capillary column (Restek
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RTX-5MS 30 m×0.25 mm, 0.25 μm film thickness, 5 % diphenyl and 95 % dimethylpolysiloxane) and a splitless injector (280 °C). Ionization was by electron impact (70 eV, source temperature 200 °C). Helium was the carrier gas (1.2 ml.min.−1). The oven temperature was maintained at 50 °C for 1 min. after sample injection (1 μl), then programmed at 20 °C.min. − 1 to 140 °C, then at 3 °C.min.−1 to 300 °C, and held for 3 min. For identification of hydrocarbons in a cuticular extract, selected ion monitoring at m/z=85 was initially carried out. Tentative identifications were made by comparison of spectra with those found in the NIST Standard Reference Database (2005), and by comparison with Kovat’s retention indices (KIs) (Bartle, 1993). Names of the identified compounds were abbreviated according to IUPAC (International Union of Pure and Applied Chemistry) nomenclature. Quantification of CHCs was performed by an external standard quantification method using n-alkanes (n-C12–n-C60) (Skelton et al., 2010). Comparison of Alkylmethoxypyrazines and CHC Composition between Diapausing and Non-Diapausing Ladybirds A comparison of the quality of alkylmethoxypyrazines and the quality and quantities of the CHCs of diapausing and nondiapausing ladybirds also was done. A Mann-Whitney U test was used to test the difference in total amount of CHCs between diapausing and non-diapausing ladybirds and in the differences in absolute amounts (per ladybird) of the CHCs that discriminate most between diapausing and nondiapausing ladybirds. Non-metric multidimensional scaling (nMDS) was used to assess overall similarities among CHC profiles, as implemented in R statistical software version 2.14.1 (R Development Core Team, 2008). Behavioral Bioassays—General Procedure for the Fourway Olfactometer Bioassays The bioassays were done using a Perspex four-way olfactometer (27×27 cm), based on the design of Vet et al. (1983). Air was removed from the center of the olfactometer by a vacuum pump and regulated with a flow meter (600 ml.min.−1). Four equal flows, entering the olfactometer at 150 ml.min−1, were created, and verified through airflow meters. Teflon tubing was used to connect each arm of the olfactometer, separately to a glass vessel (5 ml), to a glass vial (50 ml) of distilled water that humidified the air, to a glass vial (10 ml) filled with activated charcoal to purify the air and, finally, to a flow meter. A piece of filter paper was placed on the arena floor and changed after each replicate. This involved opening the olfactometer and allowing putative volatiles emitted by the ladybird to evaporate. The olfactometer was washed entirely if the ladybird had walked on the Perspex parts of the olfactometer. The walking arena was kept in a dark chamber to avoid experimental bias due to variations in light and to remove any visual stimulation. A 24 W lamp was placed
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underneath the olfactometer to provide uniform light. Behavioral observations were conducted at 20±1 °C. A single diapausing ladybird was introduced to the center of the arena through the central hole. The fourway olfactometer arena was divided into four areas, related to the four regions that correspond to each of the four glass vessels, and was placed on a sheet of paper on which three concentric circles were drawn (10.34 cm, 18.66 cm and 27 cm diam.) to aid data collection. A USB PC-camera (ToUcam, Philips, 1.2 Megapixels) was used to record the ladybird’s behavior every 10 sec. over a 3 min. period. If a ladybird remained stationary for 60 sec., it was considered inactive, and the replicate was repeated with a new ladybird. A ladybird was considered to have chosen a particular arm of the olfactometer when it entered the terminal third of that arm. At the end of the experiment, the ladybird was removed and returned to the diapausing conditions for 24 h. Unless otherwise stated, the first-choice data were analyzed by a Cochran Q test, defined as the extension of the McNemar test for k related samples (Siegel, 1956). Kruskal-Wallis H tests were used to compare the lengths of time spent in the third terminal part of the arms of the olfactometer. Mann-Whitney U tests were used to perform post hoc comparisons. Behavioral Bioassays—The Behavioral Response of Diapausing A. bipunctata to Bulk Alkylmethoxypyrazine Extract of Non-diapausing A. bipunctata The four-way olfactometer was used to measure the behavioral response of diapausing A. bipunctata to bulk extract of non-diapausing A. bipunctata ladybirds. The bulk extract (10 μl; equivalent to 5 ladybirds), or an equivalent amount of solvent (dichloromethane control), was applied by Exmire® microsyringe to a 5 mm2 filter, paper and the solvent was allowed to evaporate for 3 min. (after which the dichloromethane had completely evaporated, but the alkylmethoxypyrazines remained). The filter paper then was placed into one of the glass vessels (5 ml). The three control vessels were treated similarly with the same volume of solvent on filter paper. Twenty replicates were carried out. The first-choice data were recorded and analyzed by χ2 tests (Siegel, 1956). The observed frequencies, related to the first choice of A. bipunctata in the olfactometer bioassays, were compared to the corresponding expected frequencies. If ladybirds are not significantly attracted by chemical stimuli, these expected frequencies are 0.25. Behavioral Bioassays—The Behavioral Response of Diapausing A. bipunctata to Synthetic Alkylmethoxypyrazine Blends Four-way olfactometer bioassays were carried out to investigate the role of akylmethoxypyrazine blends in ladybird aggregation behavior. Three experiments were conducted: 1) a three-component synthetic blend, comprising three
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commercially available alkylmethoxypyrazines [2-isopropyl3-methoxypyrazine (IPMP), 2-isobutyl-3-methoxypyrazine (IBMP), 2-secbutyl-3-methoxypyrazine (SBMP)]; 2) twocomponent blends of the alkylmethoxypyrazines (IPMP + IBMP, IPMP + SBMP, IBMP + SBMP); 3) each alkylmethoxypyrazine presented on its own. The amount of each alkylmethoxypyrazine used was calculated from the bulk distillate extract of non-diapausing A. bipunctata and was equivalent to 5 ladybirds (IPMP: IBMP: SBMP 14.75 ng/μl; 2.4 ng/μl; 1 ng/μl in dichloromethane). The synthetic blend, alkylmethoxypyrazines, or solvent (dichloromethane control) was applied (10 μl) using a Exmire® microsyringe to 5 mm2 filter paper, and the solvent was allowed to evaporate for 3 min. The filter paper then was placed into one of the glass vessels (5 ml). The three control vessels were treated similarly with the same volume of solvent on filter paper. In addition, a blank control, in which no solvent was applied, was performed to investigate any effects of dichloromethane on the searching behavior of ladybirds in the three alkylmethoxypyrazines blend experiment. Twentyone replicates were carried out. Behavioral Bioassays—The Behavioral Response of Diapausing A. bipunctata to Bulk CHC Extract. Four-way olfactometer bioassays were carried out to investigate the role of CHCs in ladybird aggregation behavior. The bulk CHC extract of diapausing ladybirds or solvent (dichloromethane control) was applied (10 μl) to glass beads (2.2 cm diam., similar in size to A. bipunctata medium-sized adults) using an Exmire® microsyringe, and placed 3 cm apart from the entry of one of the olfactometer arms in the walking arena. The threecomponent blend of synthetic alkylmethoxypyrazines (IPMP:IBMP:SBMP 14.75 ng/μl; 2.4 ng/μl; 1.0 ng/μl) was applied (10 μl) with an Exmire® microsyringe to 5 mm2 filter paper, and the solvent was allowed to evaporate for 3 min before the filter paper was placed in the adjacent arm. Untreated glass beads were placed adjacent to the three other arms, and the three control vessels were treated with the same volume of solvent (dichloromethane) on filter paper. In addition to the solvent control, a blank control using untreated beads was also performed. Twenty-one replicates were carried out. Chemical Standards Dichloromethane and synthetic alkylmethoxypyrazines were purchased from Sigma-Aldrich: dichloromethane 99.9 % purity; SBMP 99 % purity; IBMP 99 % purity; IPMP 97 % purity.
Results Identification of Alkylmethoxypyrazines Used for Olfactometer Bioassays Coupled GC/MS and peak enhancement by
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co-injection, on HP-1 and DB-wax columns, confirmed the presence of three alkylmethoxypyrazines: IPMP (29.5 ng/ladybird), IBMP (4.8 ng/ladybird), and SPMP (2 ng/ladybird) in the bulk extract of non-diapausing adult A. bipunctata. The alkylmethoxypyrazines were present in a 14.75:2.4:1 ratio (IPMP:IBMP:SBMP). Comparison of Alkylmethoxypyrazines and CHC Composition between Diapausing and Non-Diapausing Ladybirds Alkylmethoxypyrazines extracts from both diapausing and non-diapausing adult A. bipunctata contained IPMP, IBMP, and SBMP in mean ratios of 3.95:1:2.43 and 6.72:1:1.89, respectively. With regard to CHC composition of elytra, coupled GC/MS analysis and comparison of KIs identified 64 peaks (15 n-alkanes, 35 monomethylalkanes, 5 dimethylalkanes, 1 alkene, and 8 unidentified CHCs) in extracts from diapausing ladybirds, and 56 peaks (6 n-alkanes, 33 monomethylalkanes, 12 dimethylalkanes, no alkenes, and 5 unidentified CHCs) in extracts from non-diapausing ladybirds (Fig. 1). The most prominent CHCs were 9-MeC23 and 7-MeC23, representing 34 % of the total relative amount of CHCs in both diapausing and non-diapausing ladybirds (Table 1). In extracts of diapausing and non-diapausing ladybirds, the CHC
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chain-length ranged from C14–C33. Methylbranched and unsaturated hydrocarbons represented 58 % and 45 % of the total composition of CHCs in diapausing and non-diapausing ladybirds, respectively. The total amount of CHCs was not different between diapausing and non-diapausing ladybirds (U = 1,128, P > 0.05; Table 1). Analysis by nMDS (Fig. 2) revealed that the CHC composition in extracts of diapausing and non-diapausing A. bipunctata elytra was different. Extracts of diapausing ladybirds contained greater amounts of 5,7-diMeC25 (28), 11-MeC28 (40), 5-MeC29 (44), C31:1 (48) and 13MeC31/11-MeC31/9-MeC31 (50), compared to non-diapausing ones, whereas extracts of non-diapausing ladybirds contained greater, but not different, amounts of nC25 (22) and 11-MeC25/9-MeC25 (23) (Fig. 2; Table 1). The Behavioral Response of Diapausing A. bipunctata to Bulk Alkylmethoxypyrazine Extract of Non-Diapausing A. bipunctata Males (χ2 =21.78; df=1; P
