Electroantennogram and Behavioral Responses of the Imported Fire

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Electroantennogram and Behavioral Responses of the Imported Fire Ant, Solenopsis invicta Buren, to an Alarm Pheromone Component and Its Analogues Di Guan,†,‡ Yong-Yue Lu,§ Xiao-Lan Liao,‡ Lei Wang,§ and Li Chen*,† †

State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China ‡ College of Plant Protection, Hunan Agricultural University, Changsha, Hunan 410128, People’s Republic of China § Red Imported Fire Ant Research Centre, South China Agricultural University, Guangzhou, Guangdong 510642, People’s Republic of China S Supporting Information *

ABSTRACT: A characteristic behavior in ants is to move rapidly to emission sources of alarm pheromones. The addition of ant alarm pheromones to bait is expected to enhance its attractiveness. To search for candidate compounds for bait enhancement in fire ant control, 13 related alkylpyrazine analogues in addition to synthetic alarm pheromone component were evaluated for electroantennogram (EAG) and behavioral activities in Solenopsis invicta. Most compounds elicited dose-dependent EAG and behavioral responses. There exists a correlation between the EAG and behavioral responses. Among the 14 tested alkylpyrazines, three compounds, 2-ethyl-3,6(5)-dimethyl pyrazine (1), 2,3,5-trimethylpyrazine (7), and 2,3-diethyl-5-methylpyrazine (12), elicited significant alarm responses at a dose range of 0.1−1000 ng. Further bait discovery bioassay with the three most active alkylpyrazines demonstrated that food bait accompanied by sample-treated filter paper disk attracted significantly more fire ant workers in the first 15 min period. EAG and behavioral bioassays with pure pheromone isomers accumulated by semi-preparative high-performance liquid chromatography demonstrated that 2-ethyl-3,6-dimethylpyrazine was significantly more active than 2ethyl-3,5-dimethylpyrazine. KEYWORDS: Solenopsis invicta, electroantennogram (EAG), 2-ethyl-3,6-dimethyl pyrazine, alarm pheromone, alkylpyrazine analogues, HPLC

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INTRODUCTION Ants are social insects living in large colonies, and many behaviors of ants are mediated by pheromones. Alarm behavior is one of the most obvious ant behaviors, which commonly involves the release of an alarm pheromone that transmits a warning to intraspecific nestmates.1 Alarm pheromones are usually released in response to a disturbance or potential danger,1,2 which have a direct benefit to recipient, such as ensuing dispersal or fast movement of nestmates, and may help them avoid a subsequent attack. Alarm is the most difficult of behavioral responses to define because alarm behaviors include a wide range of responses to danger.1 Wilson and Regnier3 classified alarm behavior into two categories: “panic alarm” characterized by rapid excited bursts of non-directional movement and “aggressive alarm” in which workers run excitedly toward the source of the alarm substance and often attack alien objects at the source of the disturbance. Characteristic ant behaviors displayed during aggressive alarm responses range from increased alertness to, in the most extreme cases, frenzied running and the biting or stinging of alien objects.4,5 Wilson6−8 first defined alarm behavior in the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), as the rapid, erratic movement of workers toward a disturbed worker and suggested that the alarm pheromone was released from the cephalic region of the ant and might be stored in a © XXXX American Chemical Society

mandibular gland as in other myrmicine species. Recently, Vander Meer et al.9 identified an alarm pheromone component, 2-ethyl-3,6-dimethyl pyrazine, from the mandibular gland of S. invicta, which was the first report of this pyrazine functioning as an ant alarm pheromone. Since the introduction to North America in the 1930s, S. invicta has spread throughout the southern United States, affecting agriculture, wildlife, and human well-being, posing a negative ecological impact on biodiversity.10,11 More recently, S. invicta has been introduced to other regions of the world, including the Caribbean, Australia, New Zealand, Taiwan, and south China.12 Many areas around the world, including large portions of Europe, Asia, Africa, and numerous island nations, are at risk for S. invicta infestation.13 This species has diverse detrimental impacts on recipient communities in the infested area.14 Although the establishment and natural spread of Pseudacteon tricuspis and Pseudacteon curvatus have been remarkably successful in the southern United States since their original release and establishment in 1997 and 2000, respectively, the effectiveness of phorid flies in reducing the fire ant populations has not yet been documented to date.15 Baiting Received: September 5, 2014 Revised: November 12, 2014 Accepted: November 13, 2014

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dx.doi.org/10.1021/jf505191s | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

Table 1. Structures of the Tested Alkylpyrazines

a

MF = molecular formula. bMW = molecular weight.

is the most efficient tool for controlling fire ant colonies in large areas, even in phorid fly released areas. In the last several decades, numerous attempts have been made to develop efficient baits. A toxicant (the active ingredient usually less than 1.0%), an attractant, such as soybean oil, and an inert carrier, such as corn cob grits, were used to formulate bait for fire ant control.16,17 A recently developed fire ant bait consisting of dried distillers grains with solubles as a carrier was found to be water-resistant and compatible with a number of pyrethroid insecticides.18 Communication in social insects is based primarily on the use of pheromones, and many of these pheromones have attractive properties. The highly evolved fire ant uses a complex of pheromones, such as trail pheromone, alarm pheromone, and queen-produced recognition pheromone, for colony communications.9 The incorporation of these ant pheromones with attractive properties could enhance the attractiveness and harvest of bait because the efficacy of bait largely depends upon ant foraging activity.19 For instance, alarm pheromonal components from leaf-cutting ants (Atta spp.)

have previously been proven to substantially increase the attractiveness and harvest of the bait.20,21 The potential of alarm pheromones in fire ant control is that they elevate workers to a heightened state of alertness and movement, resulting in quicker location of a bait particle than otherwise possible.22 Previous studies have shown that pheromone homologues and analogues could elicit biological activity in insects comparable to the natural compounds23−26 and may have a high potentiality as alternative material in integrated pest management strategies.27 Thus, the present study aimed to examine both electroantennogram (EAG) and behavioral responses of S. invicta workers to the commercially available 2-ethyl-3,6(5)-dimethyl pyrazine (mixture of the 3,5- and 3,6dimethyl isomers, herein referred to as pheromone isomers) and related alkylpyrazine analogues. Because an alarm response is a rapid reaction and, therefore, requires compounds of low molecular weight (between 100 and 200 with 5−10 carbons) with high volatility, we selected 13 alkylpyrazine analogues B



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was used for HPLC analysis under the same conditions as above at a flow rate of 1 mL/min. A standard curve was calculated by linear regression analysis. The concentration of pyrazine in the 2 mL hexane extract was calculated against the standard curve. The hexane extracts of compounds 1a and 1b were thereafter diluted to 1 μg/μL for EAG study and to 10 ng/μL for behavioral study. A mixture of an equal volume of both compound 1a and 1b solutions at the same concentration was bioassayed to determine the synergistic effect. EAG Experiment. The EAG sensitivity of S. invicta workers was determined by conventional EAG methods.29,30 Briefly, the reference glass electrode was connected to the neck of an isolated head of a major worker, and the recording electrode was connected to the antennal tip. The analogue signal was detected through a PRG-3 probe, amplified with a data acquisition controller IDAC-2, and analyzed with software EAG 2000 (all from Syntech, Kirchzarten, Germany). A 10 μL aliquot of each test compound (in hexane) was applied to a piece of filter paper (4 × 40 mm, Whatman no. 1). After solvent evaporation, the odor-impregnated filter paper strip was placed in a glass Pasteur pipet. Fresh stimulus pipets were prepared before each set of experiments. Hexane was used as the solvent control. Stimuli were delivered as 0.2 s puffs at a humidified flow rate of 1000 mL/min generated by an air stimulus controller CS-55 (Syntech, Kirchzarten, Germany). A test series of the pyrazines of the same dose (0.1, 1, 10, 100, or 1000 μg) was applied to a single antennal preparation, and the presentation order of these compounds was randomized. A blank stimulus (solvent control) was presented before and after the 14 test compounds. EAG recordings were obtained from eight antennal preparations for each dose. For analyses, EAG response to the solvent control (average of two recordings per antennal preparation, less than 0.20 mV) was deducted from the EAG amplitudes elicited by the test compounds. The corrected EAG data were analyzed for each dose using one-way analysis of variance (ANOVA) to establish significant differences among the treatments (test compounds). Means across all 14 compounds for certain doses were compared by the Tukey− Kramer honestly significant difference (HSD) comparison test (p < 0.05).31 Behavioral Bioassay. Behavioral bioassays were conducted as described by Vander Meer et al.,9 with slight modification. At least 2 h prior to a bioassay, 0.1 g of workers (approximately 100−150) from each colony were added to a plastic cup (9 cm tall × 14 cm inner diameter) with the inside wall coated with Fluon to prevent escape. A 1 cm3 sugar-agar block (10% sugar water + 1% agar after boiling and chilling) was placed on the bottom of the cup to allow feeding. Water was provided by filling a 1.5 mL microcentrifuge tube with distilled water and threading a cotton string through a hole in the cap of the tube. A filter paper strip (1 × 3 cm) folded into a triangle was put in an empty space in the bottom of the cup. The experiment was not started until the worker ants had settled into a quiescent group. A total of 10 μL of sample solution was loaded onto the filter paper strip with a micropipet in a gentle movement that would not generate a response. The alarm response of ant workers was observed and recorded. The response to a test compound was evaluated on the number of workers that were running out of the quiescent group during the recording time. After each test, the filter paper strip was replaced with a new strip for the next test. All ant bioassay units (i.e., cups) were tested with the control (10 μL of hexane), and all of the samples were at the same dose. Bioassay units were evaluated in sequence, but the samples presented to the ants were randomized. In this way, workers of a test unit that were alarmed had enough time (more than 30 min) to recover prior to the next test. Ants from a colony were considered as one replicate. A test series of the alkylpyrazines of the same dose (0.1, 1, 10, 100, or 1000 ng) was replicated 8 times. Bioassay data were determined to be normally distributed and then analyzed using oneway ANOVA, followed by the Tukey−Kramer HSD comparison test (p < 0.05), to establish significant differences among the treatments.31 For correlation analysis, behavioral response to the solvent control was deducted from that elicited by the test compounds. It appears that all compounds elicited dose-dependent responses in both EAG experiment and behavioral bioassay. A pilot analysis indicated that the

whose molecular weights range from 94 to 164. For both EAG and behavioral bioassays, a series of dilutions were made to test both EAG and behavioral dose−responses of fire ant workers from various colonies to pheromone isomers and the selected analogues. The pyrazine analogues that proved to be very active in both EAG and behavioral bioassays would potentially be used as bait enhancers for the control of the fire ants in the invaded regions.

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MATERIALS AND METHODS

Insects. Eight mature colonies of S. invicta were collected on the campus of South China Agricultural University (Guangzhou, Guangdong, China). All colonies were considered polygyne because of the presence of multiple queens during collection. Colonies were reared with soil under laboratory conditions and provided daily with purified water, 10% honey solution, and larvae of Tenebrio molitor L. Tests were carried out within 1 month of ant collection. Reagents and Test Chemicals. Acetonitrile (CH3CN) was of high-performance liquid chromatography (HPLC) grade and was used without further purification. Water was purified with a model 7155 Barnstead Nanopure system (Thermo Scientific, Marietta, OH) and subsequently filtered through a 0.45 μm membrane. The alarm pheromone component, 2-ethyl-3,6-dimethylpyrazine (1a), was commercially available as a mixture with the 2-ethyl-3,5-dimethylpyrazine (1b) isomer (Sigma-Aldrich, St. Louis, MO). The mixture consisted of about 45% of compound 1a and 55% of compound 1b [gas chromatography (GC) analysis]. A total of 13 structurally related alkylpyrazine analogues were purchased from Sigma-Aldrich or Acros Organics (Geel, Belgium). The chemical structures of these compounds are shown in Table 1. Each compound was diluted with HPLC-grade n-hexane to make a 100 μg/μL solution. Further serial 10-time dilutions were made to give solutions with different concentrations ranging from 0.01 ng/μL to 10 μg/μL for EAG and behavioral tests. The solutions were kept in a freezer at −20 °C until use. HPLC Analysis. HPLC separation of compound 1 was performed on an Agilent HP 1260 instrument (Agilent Technologies, Palo Alto, CA), using a stainless-steel, 250 × 4.6 mm inner diameter, 5 μm, HC C-18 column (Agilent Technologies, Middelburg, Netherlands). A solution of compound 1 at 10 μg/μL was prepared with CH3CN for both analytical separation and semi-preparation. All chromatographic experiments were performed in the isocratic mode. CH3CN/water was selected as the mobile phase, followed by a previously reported liquid chromatography−mass spectrometry (LC−MS) analysis.28 Various percentages (20−50%) of CH3CN were tested to optimize the separation efficiency of the mobile phase. The flow rate was 0.2 mL/ min, and the detection wavelength was ultraviolet (UV) 278 nm. The injection volume was 1 μL in analytical separations. On the basis of nuclear magnetic resonance (NMR) spectroscopic data obtained by LC−NMR, 2-ethyl-3,5-dimethylpyrazine (1b) was found to be eluted first, followed by 2-ethyl-3,6-dimethylpyrazine (1a).28 In semipreparative separation, a 5 μL sample loop was used. Each peak fraction was manually collected according to the elution profile. After 2 weeks of semi-preparative separation, the collected fractions were combined (1a, 150 mL; 1b, 180 mL) and subjected to sequential liquid−liquid partitions to remove CH3CN and water. The pooled fractions were first partitioned with hexane (50 mL × 3), and then the resultant hexane layer was acidified with 20 mL of 0.1 N HCl. After removal of the hexane layer, the acid layer was neutralized to pH 10 with 15 mL of 0.2 N NaOH and subsequently partitioned with hexane (20 mL × 3). The resultant hexane layers were combined and dried over anhydrous Na2SO4. The hexane extract was finally concentrated to 2 mL under a mild stream of nitrogen and subjected to HPLC quantitation. To quantitate pyrazine 1a or 1b in the hexane extract, 2,3diethylpyrazine (9) was used as an external standard. A stock solution of standard compound 9 (10 μg/μL) was prepared and then diluted to a series of concentrations ranging from 0.25 to 8 μg/μL. All dilutions were transferred to the HPLC autosampler, and 1 μL of each dilution C



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Figure 1. Separation of fire ant alarm pheromone isomers in CH3CN/water mobile phases of various compositions (as indicated in the figure) on an Agilent HC C-18 column. EAG data at five doses correspond well to the behavioral data from the low to high doses. Therefore, the correlation between the corrected EAG data and the corrected behavior data was analyzed using the PROC CORR procedure of SAS statistical software.31 Bait Attraction Bioassay. Three alkylpyrazines with relatively high activity in both EAG and behavior bioassays were selected for further food bait tests. The potential for using these alarm pheromone compounds to enhance the attractiveness of food bait was examined for S. invicta workers. A total of 0.5 g of ant workers was transferred into a plastic basin (55 cm diameter at the bottom) with the inside wall coated with Fluon to prevent escape. A moisturized cotton wad in a Petri dish cover (d = 6 cm) for provision of water was placed at the center of the basin. Holes were made at the side wall of the Petri dish cover for ant movement. Ants were attracted to the water source and left for a 2 h period of acclimatization to settle into a quiescent group. During this period, three aluminum foil disks (5 cm diameter) were mounted on the bottom of the basin. Foil disks were positioned 20 cm away from the center of the basin and at an equal distance from each other. On the foil disk was then placed a filter paper disk (4 cm diameter). Prior to the start of evaluation, a block of ham sausage (0.1 g) used as food bait was placed onto the filter paper disk. A total of 10 μL of hexane was loaded onto both filter paper disk and food block with a micropipet in a gentle movement. For the other two food blocks, 10 μL of sample solution was loaded onto either the filter paper disk or the food block. The number of ants on a filter paper disk was counted at 2, 5, 15, 30, and 45 min after impregnation of the sample solution (or hexane control) onto a filter paper disk or food

block. After each test, ants were discarded and the basin was cleaned with soap water and air-dried for the next test. Each sample solution was repeated 8 times. Two doses (10 and 100 ng) of each compound were tested. Mean numbers of ants for the same compound−dose combination on different filter paper disks were compared using the Tukey−Kramer HSD comparison test (p < 0.05).31

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RESULTS AND DISCUSSION As expected, separation of 2-ethyl-3,6-dimethylpyrazine (1a) and 2-ethyl-3,5-dimethylpyrazine (1b) in the commercial product was accomplished on a reverse-phase C-18 column using CH3CN/water mobile phase. Figure 1 illustrates elution profiles of compound 1 at a flow rate of 0.2 mL/min with variable ratios of CH3CN/water mobile phase. The injection results in two chromatographic peaks, which correspond to the two regio-isomers. The percentage of CH3CN in the mobile phase had a significant influence on separation efficiency. The separation efficiency increases with the decrease of the CH3CN/water ratio. However, when the CH3CN/water ratio decreases, chromatographic retention time increases significantly. At a ratio of 20:80 (v/v), the two regio-isomers, compounds 1a and 1b, are nearly baseline-separated. Because general preparative separations are time-consuming and tedious, we used a ratio of 25:75 (v/v) for scaling-up the preparation of pure isomers at the semi-preparative level. The D



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Figure 2. EAG response [−mV ± standard error (SE)] of S. invicta workers to 14 alkylpyrazines. Means for the same dose having no letter in common are significantly different (p < 0.05; Tukey HSD test).

two peaks derived from preparative compounds 1b and 1a, respectively, were observed at 49.6 and 51.7 min at 278 nm. The external standard method was used to obtain the regression equation, A = 608.6090C + 2.7015, where A is the peak area and C is the concentration of the standard compound (μg/μL). The standard compound showed excellent linearity (r2 = 0.999 98; p < 0.001) in selected concentration ranges. With the obtained calibration curve, the concentrations of 2 mL preparative compound 1a and 1b solutions were determined to be 2.14 and 3.02 μg/μL, respectively. All tested compounds elicited dose-dependent responses from the antennae of S. invicta workers (Figure 2). Significant differences were recorded in the EAG response of ant workers to the various compounds at all tested doses. Statistical analyses (ANOVA) of the corrected EAG data revealed that both the chemical structures and doses of alkylpyrazines influenced electrophysiological responses. Antennal responses to pheromone isomers (1) varied between 0.09 and 1.53 mV across all doses. In comparison to compound 1, EAG responses to 7 compounds of 13 tested analogues were similar or relatively

higher at both the lowest and highest doses. At moderate doses (1, 10, and 100 μg), responses to compounds 7 and 12 were significantly higher than that to compound 1, except for response to compound 7 at a dose of 1 μg. Among the 14 tested alkylpyrazines, compound 12 elicited the highest responses at every dose tested. All alkylpyrazines elicited apparent alarm behavioral responses of S. invicta workers. When exposed to odors of pyrazine analogues, workers were observed to leave the quiescent group rapidly toward the source of odors. The alarm bioassay results for all tested compounds are shown in Figure 3. Among the 14 tested alkylpyrazines, three compounds, 1, 7, and 12, elicited significant alarm responses at every dose tested. Furthermore, the responses to these three compounds increased in a dose-dependent manner. In contrast, the remaining 10 compounds failed to elicit significant behavioral response at low doses. In comparison to compound 1, behavioral responses to the two active alkylpyrazines, compounds 7 and 12, were not significantly different at all tested doses, except that the response to compound 12 was E



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Figure 3. Alarm response of S. invicta workers to 14 alkylpyrazines. Means for the same dose having no letter in common are significantly different (p < 0.05; Tukey HSD test).

Table 2. EAG Response of S. invicta Workers to Alarm Pheromone Isomersa

significantly higher than that to compound 1 at a dose of 100 ng. Generally, the alarm responses induced by the remaining 10 alkylpyrazine analogues were significantly less than that induced by compound 1 at all doses but the lowest dose (0.1 ng). A good correlation between the EAG and behavioral responses was obtained for all 14 alkylpyrazines (y = 6.3857x + 1.3999; r2 = 0.5963; p < 0.0001). This suggests that these alkylpyrazine analogues target the same olfactory neurons on the antennae of fire ant workers. When pure isomers were tested at a dose of 10 μg, compound 1a elicited significantly greater EAG response than compound 1b (Table 2). A mixture of both isomers (1:1) at the same dose elicited intermediate EAG response, suggesting that there exists an addition effect between the two isomers. Furthermore, the EAG response to compound 1a in the second EAG experiment was comparable to responses to compounds 7 and 12 in the first EAG experiment. The data from the behavioral bioassay with pure isomers of compound 1 at a dose of 100 ng showed that the number of workers responding to

compound

EAG (−mV ± SE)

1a 1b 1a + 1b

0.62 ± 0.043 a 0.43 ± 0.034 b 0.54 ± 0.054 ab

Dose = 10 μg. Means followed by different letters are significantly different (p < 0.05; Tukey HSD test). a

odors was significantly more than the number of workers responding to hexane (Table 3). Compound 1a elicited a significantly higher alarm response than compound 1b, and the activity of the mixture of both isomers (1:1) was similar to that of compound 1b. The results of the EAG and behavioral experiments demonstrated the responses of fire ant workers to the commercially available mixture of 2-ethyl-3,6(5)-dimethyl pyrazine and its individual components and support a previous F



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Table 3. Behavioral Response of S. invicta Workers to Alarm Pheromone Isomersa compound CK 1a 1b 1a + 1b

samples. This stimulation may have significant influence on the EAG responsiveness of the isolated head preparation to the same compound 1, especially at low doses, in the following tests. EAG responsiveness of the isolated head preparation to the same stimulus has proven to decrease rapidly, falling below 50% of initial EAG responses after 20 min.33 It seems likely that compound 1 elicits the same level of EAG response of fresh antennal preparation that has not been tested to standard (100 μg of compound 1) as the two active pyrazine analogues 7 and 12. This interpretation is supported by the fact that EAG response to compound 1a is similar to EAG responses to compounds 7 and 12 at a dose of 10 μg. At a dose of 100 ng, the alarm response to compound 12 was significantly greater than that to compounds 1 and 7 but was similar to the alarm response to compound 1a, indicating that compound 12 is equally active as the fire ant alarm pheromone component (1a). Di- and trialkyl-substituted pyrazines are known components of alarm/trail pheromones of ant species, including Linepithema humilis (Mayr), Eutetramorium mocquerysi Emery, and Wasmannia auropunctata (Roger).34−36 It is possible that compound 12 is an unidentified component of the alarm pheromone of S. invicta or the observed response to compound

no. of ants (± SE) 3.2 19.5 16.4 16.7

± ± ± ±

0.25 0.42 0.32 0.53

c a b b

a

Dose = 100 ng. Means followed by different letters are significantly different (p < 0.05; Tukey HSD test).

identification of fire ant alarm pheromone component, 2-ethyl3,6-dimethyl pyrazine (1a).9 Compound 1a was found to be significantly more active than 2-ethyl-3,5-dimethyl pyrazine (1b) in both EAG and behavior bioassays. This is in agreement with previous reports demonstrating that compound 1b was less active than compound 1a in the alarm response experiment with fire ant workers and in the trail following experiment with the Mediterranean ant Pheidole pullidulu.9,32 Interestingly, 2,3diethyl-5-methylpyrazine (12) elicited significantly higher EAG responses compared to pheromone isomers 1 for doses of 1, 10, and 100 μg. In the EAG experiment, we puffed 100 μg of compound 1 to serve as a standard before the tests of all

Figure 4. Responses of S. invicta workers in bait attraction bioassay to the three most active alkylpyrazines at two doses (10 and 100 ng). Means for the same time period having no letter in common are significantly different (p < 0.05; Tukey HSD test). G



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food bait tests. These samples stimulated the same behaviors when incorporated in food baits. The sample loaded onto filter paper disk attracted more workers than loaded onto bait directly in the first 5 min period presumably because of faster recruitment of the sample evaporated in the air from the filter paper disk. Relatively more workers were attracted to bait incorporated with sample in the first 5 min period than to bait incorporated with hexane, but the difference was statistically significant only for compounds 1 and 12 at the high dose. The attraction to food bait by the sample-treated filter paper disk has potential applications. Alkylpyrazines can be used to improve harvest of toxic bait as proven by the alarm pheromone of leaf-cutting ants (Atta spp.), 4-methyl-3heptanone, to significantly increase the attractiveness of enhanced bait sachets and the discovery of nearby unenhanced sachets in the field.20 Future work will test the influence of alkylpyrazines on bait transportation and substantial control of the imported fire ants.

12 may simply be due to their structural similarity to the alarm pheromone component 1a. For methyl-substituted pyrazines, only trimethyl-substituted pyrazine was EAG- and behaviorally active. In contrast, mono-, di-, and tetramethyl-substituted pyrazines turned out to be behaviorally much less active or even inactive, regardless of the position of the methyl substituents. In general, disubstituted pyrazines, for instance, 2-ethyl-3-methylpyrazine (8), 2,3diethylpyrazine (9), 2-methyl-3-propylpyrazine (10), 2-isobutyl-3-methylpyrazine (13), attracted significantly more workers than hexane control but elicited significantly lower response than pheromone isomers 1. The 6 substitution on the pyrazine ring of 2-ethylpyrazine appears to be important because 3methyl- and 3-ethyl-2-ethylpyrazines (8 and 9) were much less behaviorally active than compound 1. Thus, the number of substituents (methyl and ethyl) and the position of substituents in alkylpyrazines exerted a strong influence on the behavioral response. Substitution with propyl, isobutyl, or sec-butyl groups would result in a significant decrease of response. In contrast to the behavioral data, a relatively smaller influence of the chemical structure on the responsiveness was detected by the electrophysiological experiments with the EAG technique. All compounds tested, including the behaviorally inactive compounds, elicited dose-dependent physiological responses from the antennae of ant workers. Three alkylpyrazines, 1, 7, and 12, with both significant EAG and behavior responses were selected for bait discovery bioassays. Immediately after loading samples, ant workers were observed to run toward the odor sources, showing an alarm response. At the beginning of the test, workers ran around the food bait without feeding, then climbed up, and bit the bait intensely. After a few seconds, the workers turned back to their “nest” to recruit nestmates. About 4 min later, a large number of workers ran onto the food bait and started feeding. Workers near the sample-treated food bait appeared more agitated than those in the hexane control and fed on the food bait in a more aggressive manner. The number of workers around food bait increased rapidly over time (Figure 4), and reached the highest level in 45 min for the hexane control and in 15 min for both alkylpyrazine treatments. In general, the sample loaded onto filter paper disk attracted significantly more workers than the sample loaded on food bait and hexane control in 2 and 5 min periods, possibly because of the quick evaporation of the test compound on filter paper disk. In most cases, the number of workers in treatment of the sample loaded onto food bait were not significantly greater than that in the hexane control in a 15 min period. At the end of observation, generally no significant difference was observed in the number of workers among the three treatments of a pyrazine compound and among the six compound × dose combinations (p > 0.05). These results suggest that the three selected alkylpyrazines could significantly enhance bait attractiveness to S. invicta workers. Eusocial insects are generally attracted to emission sources of alarm pheromones and exhibit a collective response to these chemical releasers. Alarm pheromones can also frequently function as recruitment stimuli that recruit large numbers of aggressive individuals to a pheromonal point source with incredible rapidity.37,38 In this study, we observed this characteristic behavior in fire ants. Because excited ant workers would move rapidly to emission sources of alarm pheromones, we used the number of workers on the food bait to evaluate the activity of the three selected alkylpyrazines, 1, 7, and 12, for

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ASSOCIATED CONTENT

S Supporting Information *

HPLC chromatographs of commercial mixture 1 and isolated isomers (Figure S1) and correlation between the EAG and behavioral responses (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Telephone/Fax: +86-10-64807780. E-mail: [email protected]. Funding

This research was supported by the National Basic Research Program of China (Grant 2012CB114105) and the National Natural Science Foundation of China (Grant 30970402). Notes

The authors declare no competing financial interest.

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REFERENCES

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Electroantennogram and Behavioral Responses of the Imported Fire

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