EAG response and behavioral orientation of Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae) to synthetic host-associated volatiles

Dastarcus helophoroides Fairmaire (Coleoptera: Bothrideridae) is an effective predatory beetle of larvae and pupae of several cerambycid beetles including Monochamus alternatus and Anoplophora glabripennis. Electroantennography (EAG) and a dynamic two-choice olfactometer were respectively used to measure the antennal and behavioral responses of both sexes to selected volatile compounds. Female and male D. helophoroides exhibited similar EAG and behavioral responses. Significant dose-dependent EAG responses in both sexes were elicited by nonanal, octanal, cis-3-hexenol, 3-carene, (R)-(+)-α-pinene, (S)-(-)-α-pinene, (R)-(+)-limonene and (S)-(-)-limonene. Female and male beetles were repelled at high concentration by cis-3-hexenol and (S)-(-)-limonene, respectively. Both sexes of D. helophoroides were significantly attracted to nonanal, cis-3-hexenol, 3-carene and (R)-(+)-limonene even at low concentrations. These compounds might be used either individually or in mixtures for developing biological control methods to attract this predatory beetle into forest stands threatened by cerambycid beetles.


Introduction
approximately 40 days after hatching. After each experiment, actively responding beetles were sexed reliably by dissecting the reproductive organs [20]. Ten antennae from different adult beetles and 70-100 adult beetles of each sex were used in EAG and behavioral experiments respectively.

Electroantennogram and odor delivery
The procedure used to prepare the beetles for the electroantennographic recording is described in [21] and was adapted to the special requirements of D. helophoroides. Beetles were starved for 24 h before the experiments. A sharpened tungsten wire prepared by electrolytic etching was used to make a small hole in the thorax region. Later, this hole was used for the insertion of a reference glass electrode filled with Ringer's solution in contact with an Ag/ AgCl wire. The beetle was placed under a high magnification compound microscope (Leica MZ16, Leica Microsystems GmbH, Germany). To stabilize the antenna, a sharpened tungsten wire was used to hold the antennal segment in place. Then, the last antennal segment was punctured with a sharpened tungsten wire using a micromanipulator. This hole was used to insert a recording glass capillary (GB150F-8P, 0.86 × 1.50 × 80 mm with the filament; Science products GmbH, Hofheim-Deutschland) containing Ringer's solution in contact with an Ag/ AgCl wire. The EAG responses were detected through a combi-probe (INR-II; Syntech, the Netherlands). The DC potential was recorded (Universal AC/DC probe), processed and analyzed using EAG 2000 software (Syntech, Hilversum, Netherlands). An air stimulus controller (CS55; Syntech, Hilversum, the Netherlands) was used to deliver purified air and odor. A constant flow (18 l/h) of filtered air was passed over the prepared antenna through the open end of the metal tube positioned close to the antenna. The pulse duration time was 1 s. Each volatile concentration was tested in a 1 min interval. The time interval between each volatile compound was 2 min. Ascending concentrations of each compound (10 −6 to 10 −1 in paraffin mg/mg) were applied to avoid olfactory adaptation. A standard green leaf volatile cis-3-hexenol at 10 −3 (in paraffin oil mg/mg) stimulation was done at the beginning and at the end of each recording to correct for the loss of sensitivity of the antennal preparation. Similarly, a control paraffin oil stimulation was done at the beginning and at the end of each recording to subtract the blank value from the antennal responses. For each compound, EAG responses of ten antennae from different adult beetles of each sex were recorded.

Olfactometer and behavioral test
A custom-built dynamic two-choice olfactometer with 10 parallel tracks was used in the behavior test as described in [18]. A 40 × 180 × 170 mm olfactometer was constructed from PTFE (Polytetrafluoroethylene). The 10 parallel walking chambers were covered with a glass lid. The non-transparent PTFE-walls between tracks isolated the tested beetles from each other as well as both chemical and visual cues. Each walking chamber was 20 mm deep and 150 mm long (Fig 1). The width of each chamber was enlarged to 10 mm to allow D. helophoroides to turn around freely inside the chamber. A white LED plate (200 × 200 mm) was used as a light source beneath the walking tracks. At both ends of each walking chamber, 200 μl filter pipette tips (Sarstedt AG&CO, Nümbrecht, Germany) were inserted: 10 filter tips on one end were drenched with 65 μl of volatile compound, and 10 filter tips on the other end were drenched with pure silicone oil control. Silicone oil was used as a solvent for the behavioral assays, because of the unfavorable viscosity of paraffin oil (used in EAG) tended to obstruct the filter tips of the olfactometer. Filtered and humidified air was fed into both ends of each track at the identical constant flow rate of 0.080 L/min. A live video tracking system, the Etho vision XT 8.0 (Noldus Information Technology, the Netherlands), was used to recognize and record the behavior of the beetles. Three compartment zones of olfactometer were defined as zone 1, zone 2, and the beetle releasing point as "neutral" zone (Fig 1). Ten insects were introduced into the olfactometer at the neutral zone in each trial, with the released beetles unable to contact each other because of the physical separation in the walking arenas. The volatile compound and the silicone oil were placed randomly in the direction of zone 1 or zone 2 in each trial. Each trial began with a 30 s accommodation phase for the beetles and lasted for 15 min. The record was valid as soon as the middle point of a beetle entered zone 1 or zone 2. The olfactometer was cleaned with methanol, and the position of the volatile compound was changed after each trial. To avoid possible influence of asymmetries in illumination on recording data, the olfactometer was turned by 180˚after each true trials.
In behavioral tests, initially, we used all compounds at 10 −1 and 10 −3 doses. Later, those compounds showing significant differences between control and treatment zones at a dose 10 −3 were further tested at a dose of 10 −5 (Table 1). A control experiment with silicone oil vs. silicone oil was conducted to test the behavior of D. helophoroides without stimuli. Fifty replications were conducted for all compounds at selected concentration (10 −1 , 10 −3 , 10 −5 ; Table 1).
To measure the behavioral responses of D. helophoroides beetles responding to both treatment and control zones, we calculated the total distance movement (TDM) of beetles in each trial. Beetles were released in the neutral zone of each track, and the total moving distance for 15 min in any direction in the track (to treatment zone, to control zone, returning to the neutral zone or moving to either of control and treatment zones etc.,) was calculated. Our estimation of TDM assumes that beetles respond behaviorally towards either of the tested zones (control or treatment) in each track, but it does not reveal the kinesis activity of beetles. To evaluate the attraction of beetles to tested stimuli, we calculated the beetle entering frequency (BEF) responses to treatment or control zones within 15 min. Beetles were released in the neutral zone of each track, and scored a single BEF count as the beetle moved into the treatment or control zone (may or may not reach the track end) and return to the neutral zone. As a measure of how beetles show high attraction to chemical stimuli, we calculated the beetle staying duration (BSD) responses at 5, 10, and 15 min intervals in both treatment and control zones. We scored the BSD as beetles that entered the treatment or control zone, and noted how long each beetle stayed in a particular zone across different time durations (5, 10, 15 min). Observations were stopped once the beetle exited of the zone to the releasing point (neutral zone) and resumed when the beetle entered the same zone within the assigned time duration.

Statistical analyses
To correct the EAG responses in comparison to the paraffin oil control, the mean of the blank responses before and after the measurements were subtracted from that to elicited by the volatile compound. All EAG responses data were then normalized to cis-3-hexenol (10 −3 ) as follows: where A is the amplitude (mV) of the EAG response to compound; EAG(ctl1) is the EAG response to control at the beginning of the recording; EAG(ctl2) is the EAG response to control at the end of the recording; EAG(std1) is the EAG response to standard at the beginning of the recording; EAG (std2) is the EAG response to standard at the end of the recording. First, we compared the normalized EAG-responses to selected compounds between the sexes with two-way ANOVA, followed by multiple comparisons corrected with the Bonferroni test (Prism 5, Graphpad Software). Second, the mean EAG responses relative to the standard at different concentrations of each compound were analyzed by two-way ANOVA, followed by multiple comparisons with Fisher's LSD test (Prism 5, Graphpad Software). Third, we checked for significant differences in EAG response among tested compounds at 10 −1 and 10 −2 by Tukey's multiple comparisons test (Prism 5, Graphpad Software).
In the behavioral tests, the time period s D. helophoroides stayed (BSD) in each zone (5, 10 and 15 min) were compared using a multiple t-test (Prism 5, Graphpad Software). Additionally, the frequencies of the beetles entering (BEF) each zone during 15 min of testing were compared using a chi-square test (SPSS Statistics 22, IBM). The TDM, BEF and BSD response measurements by D. helophoroides in the olfactometer were normalized and compared using one-way ANOVA, followed by Tukey's multiple comparisons test (Prism 5, Graphpad Software).
When comparing the normalized EAG responses of both sexes of D. helophoroides to all nine volatile compounds at the two highest doses (10 −1 and 10 −2 ) ( Table 2), the highest EAG response to the aldehydes octanal and nonanal was recorded for female beetles at a dose of 10 −1 (P<0.05, Tukey's multiple comparisons test). In contrast, at a dose of 10 −2 , both sexes showed significantly higher EAG response amplitudes to octanal and nonanal than to the other tested compounds ( Table 2) (P<0.05, Tukey's multiple comparisons test). The EAG response amplitudes of both sexes to trans-β-caryophyllene at 10 −1 and 10 −2 were significantly lower than to all other compounds (  Behavioral responses of D. helophoroides to selected volatile compounds Total distance movement (TDM). We calculated the total distance moved of D. helophoroides as a combined behavioral response to different concentrations (10 −1 , 10 −3 , 10 −5 diluted in silicone oil mg/mg) of all compounds and silicone oil control within 15 min (Table 3). Comparison of TDM for both sexes to all compounds at different doses relative to the control stimulus showed that the female beetles exposed to trans-β-caryophyllene at 10 −1 exhibited a significantly increased TDM. Moreover, the TDM of female beetles was significantly reduced when exposed to a 10 −5 dose of cis-3-hexenol. However, both sexes exposed to 3-carene and (R)-(+)-limonene showed significantly increased TDM at high (10 −1 ) doses compared to low (10 −5 ). Male beetles showed this effect to trans-β-caryophyllene even at a dose of 10 −1 relative to 10 −3 dose (Table 3).
Beetle entering frequency (BEF) into the selected zones. To see the effect of volatile compounds on D. helophoroides behavior, we calculated the sum of entering frequencies for   each treatment-and silicone oil control-zones over a 15 min stimulus period (Fig 3). We found that both sexes exposed to 3-carene and (R)-(+)-limonene showed significantly higher (P < 0.05, chi-square test) BEF than silicone oil alone at nearly all tested doses (10 −1 , 10 −3 , 10 −5 diluted in silicone oil mg/mg) (Fig 3). Similarly, the compound nonanal at 10 −3 , 10 −5 and cis-3-hexenol at a dose of 10 -5 elicited significantly increased BEF for both sexes into the treatment zone compared to the control zone (P < 0.05, chi-square test) (Fig 3). However, only male beetles showed significantly increased BEF into the zones treated with (R)-(+)-α-pinene at 10 −3 and (S)-(-)-α-pinene at 10 −1 and 10 −3 doses (Fig 3) compared to control zones. In contrast, the BEF of male beetles into control zones was significantly higher compared to zones treated with (S)-(-)-limonene and trans-β-caryophyllene at a dose of 10 −1 (Fig 3). Neither sex of D. helophoroides showed any significant differences in BEF when silicone oil was tested versus silicone oil (P > 0.05, chi-square test) (Fig 3). The overall comparisons of BEFs for both sexes over a 15 min interval to all tested compounds at different doses (10 −1 , 10 −3 , 10 −5 diluted in silicone oil mg/mg) are presented in Table 4. Beetle staying duration (BSD) in the selected zones. In order to measure the attractive behavior of D. helophoroides to all tested compounds at different doses (10 −1 , 10 −3 , 10 −5 diluted in silicone oil mg/mg), we compared the mean beetle staying durations at 5, 10 and 15 min stimulus periods in treatment-and silicone oil control-zones (Fig 4). In both sexes at 5 min, the volatile compounds nonanal, cis-3-hexenol, 3-carene, (R)-(+)-limonene and (S)-(-)-α-pinene elicited significantly increased BSD in treatment zones (P< 0.05, t-test) compared to control zones mostly at lower doses (10 −3 , 10 −5 ) (Fig 4). Similarly, beetles exposed to different concentrations of compounds for 10 and 15 min periods revealed that the BSD in zones treated with nonanal, cis-3-hexenol, and (R)-(+)-limonene were significantly increased (P< 0.05, t-test) compared to control zones at a lower dose in both sexes of the beetles (Fig 4). Additionally, higher concentrations (10 −1 ) of (R)-(+)-α-pinene and (S)-(-)-α-pinene elicited significantly increased BSD at 10 min and at 5, 10 and 15 min respectively in treated zones compared to control zones of both sexes (Fig 4). In contrast, female beetles exposed to cis-3-hexenol at a higher dose (10 −1 ) for 10 and 15 min showed significantly higher BSD in the control zones (Fig 4). Neither sex tested in silicone oil versus silicone oil showed any significant difference in BSD at any tested period (Fig 4) (P> 0.05, ttest). The overall comparisons of BSD for both sexes within 5, 10 and 15 min to all compounds at different doses (10 −1 , 10 −3 , 10 −5 diluted in silicone oil mg/mg) are shown in Tables 5-7.

Discussion
Previous results from our laboratory showed that the antenna of D. helophoroides contains different types of sensilla with a porous cuticle, suggesting that these sensilla might be involved in olfaction [11]. In the present study, the EAG responses of D. helophoroides were recorded to explore the antennal detection of their prey-associated volatile compounds [5, 14-16, 9, 10, 22]. To the best of our knowledge, this study is the first to investigate both electrophysiological and behavioral responses of D. helophoroides to their prey-associated volatile compounds at different concentrations. The aldehydes octanal and nonanal eliciting the highest EAG response amplitudes in both sexes of the beetles suggesting that more olfactory chemoreceptors are involved in the detection of these aldehydes [23]. However, in our behavioral tests, both sexes showed statistically significant responses only to nonanal. This shows that highly EAG-active compounds do not always necessarily elicit behavioral responses. Nevertheless, mixtures of these aldehydes might elicit significant behavioral responses by D. helophoroides in an olfactometer as demonstrated by their prey A. glabripennis [24]. Both, female and male antennae showed high EAG responses to cis-3-hexenol. However, different concentrations of this compound have different effects on the behavioral responses of the two sexes. For example, female beetles were significantly repelled when exposed to cis-3-hexenol at a dose of 10 −1 , whereas at a dose of 10 −3 , the same compound was attractive. Even an attractive effect was elicited by cis-3-hexenol at a dose of 10 −5 for both female and male beetles, suggesting a role in finding mating partners and oviposition substrates (Table 8). Moreover, this green leaf volatile is released from damaged deciduous trees, eliciting strong EAG and attractive behavioral responses to B. horsfieldi and A. glabripennis, prey species of D. helophoroides [25][26][27]. Thus, cis-3-hexenol might serve as a kairomone for deciduous trees by providing a clue to the cerambycid herbivores and serve as a synomone by luring predators of these herbivorous beetles.
staying duration in treatment zones, right bars indicate staying duration in control zones. Asterisk indicate significant differences among different treatments (P < 0.05, multiple t-test). The error bars represent the mean standard error (n = 50). Control-silicone oil.

Volatile compounds Beetle staying duration in 5 min (in seconds)
Female The common plant sesquiterpene trans-β-caryophyllene elicited a weak EAG response and a strong repellent behavior in both female and male beetles. This is underlined by significantly increased TDM response of D. helophoroides when exposed to trans-β-caryophyllene suggesting that this compound may contribute to non-host avoidance.

Volatile compounds Beetle staying duration in 10 min (in seconds)
Female In both sexes, we found a strong discrimination between the enantiomers of limonene, both in EAG and behavioral responses of D. helophoroides. (S)-(-)-limonene elicited significantly higher EAG responses in both sexes. However, in behavioral tests only (R)-(+)-limonene elicited an attraction response in both sexes. These results are in agreement with a previous report [10], which demonstrated that D. helophoroides adults were significantly attracted by (R)-(+)-limonene derived from the frass odor of the prey species M. raddei. In addition to these findings, we confirmed significant attractive responses of D. helophoroides to (R)-(+)-limonene over a broad range of concentrations. Similar enantiomeric discrimination has been observed in diverse forest tree pests like Xylotrechus rusticus, Dendroctonus sp., Hylobius abietis [28][29][30][31] as well as prey species of D. helophoroides including A. glabripennis and M. alternatus [32,14,33]. This highlights the importance of enantiomeric discrimination for finding suitable plant and/or prey. Consequently, highly specific enantiomer discrimination of  prey associated compounds might sharpen the searching ability of predators like D. helophoroides to reach their prey. The prey-associated plant volatiles played a crucial role in the prey location process of D. helophoroides, as demonstrated in another coleopteran predator Thanasimus dubius for the forest pest Dendroctonus frontalis [29]. D. helophoroides is an oligophagous predator that preys on at least six forest pest species of cerambycids [2, 10]. Thus, adult D. helophoroides need to adapt to a broad range of prey-associated chemical cues that are not necessarily specific to one prey insect. EAG and behaviorally active compounds in this beetle, such as nonanal, cis-3-hexenol, 3-carene, (R)-(+)-limonene, (R)-(+)-α-pinene and (S)-(-)-α-pinene, are common host volatiles or feeding-induced volatiles of several cerambycid beetle species [2, 10, 34]. For example, nonanal, cis-3-hexenol, 3-carene, (R)-(+)-α-pinene and (S)-(-)-α-pinene were previously reported to generate robust EAG responses and elicit behavioral responses to A. glabripennis and B. horsfieldi [24-27, 35, 32]. Similarly, (R)-(+)-α-pinene and 3-carene were also reported generate robust EAG responses in M. alternatus [14]. This suggests that many cerambycid beetles use host plant volatile cues for finding their host and assessing the suitability for feeding or oviposition. Similarly, an olfactory response of D. helophoroides to prey associated volatile compounds either from their prey host plant or prey frass [9, 10, 26, 32, 35] is viewed as an adaptive strategy to locate the prey. This also suggests that D. helophoroides is mainly relying on different prey associated volatile cues either in individual or in particular ratios to find prey, as well as for mating and oviposition site selection.
In conclusion, the EAG results of this study represent an initial attempt to demonstrate the electrophysiological sensitivity of both sexes of D. helophoroides to prey-associated plant volatiles. In addition to previous behavioral studies [9, 10], we found that the beetles exhibited concentration dependent behavioral responses with different behavioral response affected/ triggered. Most of the selected compounds elicited notable EAG and behavioral responses. The beetle D. helophoroides has 52 odorant binding proteins (OBPs), 19 chemosensory proteins (CSPs),10 olfactory receptors (ORs), 8 ionotropic receptors (IRs), 2 gustatory receptors (GRs), and 5 sensory neuron membrane proteins (SNMPs) in their antennae [13], suggesting a differentiated role in olfaction. Our results can foster future research approaches combining electrophysiological methods with molecular techniques such as RNAi knockdown to understand the olfactory mechanism of this predatory beetle in detail. Our results show that octanal, nonanal, cis-3-hexenol, 3-carene, (S)-(-)-α-pinene and (R)-(+)-limonene are most acutely perceived by D. helophoroides antenna. These compounds might be useful either individually or in mixtures for developing efficient attractants to lure this predatory beetle into forest stands damaged by cerambycid herbivores. For instance, the green leaf volatile cis-3-hexenol is sensitively perceived by D. helophoroides but not by its prey M. alternatus [14]. Therefore, cis-3-hexenol might be used as an allochthonous kairomone [36] to attract the D. helophoroides beetles into the M. alternatus attacked forest stands without attracting more M. alternatus. Additionally, the most effective compounds should be tested in various combinations in the field trapping experiments or in biological control sites to see if the compounds increase the attraction of D. helophoroides and by extension enhance predation rates.