Identication of the Terpenoid Compounds and Behavioral Assays of Alarm Pheromones in the Vetch Aphid Megoura Viciae

Aphids are destructive insect pests worldwide, and alarm pheromones play a key role in their chemical ecology. However, the composition and key active components of alarm pheromone differentiate among aphid species. Here we conducted a detailed analysis of the terpenoid compounds in the vetch aphid Megoura viciae and its host plant Pisum sativum by using gas chromatography-mass spectrometry. The results showed that a variety of terpenoid compounds existed in the aphid, with four major terpene components, i.e., (-)-β-pinene (49.74%), (E)-β-farnesene (32.64%), (-)-α-pinene (9.42%) and (+)-limonene (5.24%), in addition to a trace amount of minor terpenoid components (3.14%). In contrast, the terpenoid compounds were relatively scarce in the host plant, mainly consisting of squalene (66.13%) and its analogue 2,3-epoxysqualene (31.59%) in addition to some minor components. Quantitative analysis of the dynamics of four major terpene components during different developmental stages showed that the monoterpenes increased with continuous development, while the sesquiterpene reached peak at the 3rd -instar; all terpene components remained at a high level in the 4th -instar, with (-)-β-pinene accounting for the highest proportion during all developmental stages. Behavioral assays with single components and mixtures at different concentrations were conducted in a three-compartment olfactometer, revealing that the repellent activities of single components varied in a concentration-dependent manner, but two mixtures (1:44.4:6.5:2.2 and 1:18.4:1.3:0.8) prepared according to the proportions of four major components at the 3rd - and 4th -instar stages maintained a signicant repellent activity at all concentrations tested. Our results suggested that (-)-α-pinene and (-)-β-pinene were the major active components of alarm pheromone in M. viciae, but the mixtures of single components play a key role in the alarm behavior of M. viciae. Our study helps to understand the chemical ecology of insects and design alternative control strategies against aphids. developmental stages of viciae. we conducted a series of behavioral assays with single components and mixtures at different concentrations in a olfactometer. Our study identied a novel set of alarm pheromones in M. viciae. The samples collected from M. viciae and P. sativum were analyzed on an Agilent 6890 gas chromatographer coupled to an Agilent 5973 ion trap mass detector (Agilent Technologies Inc., California, USA). The instrument was equipped with a HP-5 capillary column (300 mm × 0.25 mm × 0.25 µm, Agilent, Santa Clara, USA). The program of GC-MS was set up as described (Huang et al., 2013). Briey, after sample injection, the GC oven temperature was held at 40°C for 1 min, followed by a two-step temperature increase: the rst increase was from 40°C to 130°C at a rate of 4°C/min and maintained for 5 min, and then the temperature was increased at a rate of 10°C/min to 250°C and held for 5 min. The temperatures of injector and ion source were 250°C. The mass spectrometer was operated under the electron impact ionization mode (El, 70 eV) with a m/z scan range of 35–650. Terpenes were identied by comparing their retention time and mass spectra with those of standards (SigmaAldrich, Oakville, Canada) under the same conditions. The quantity of each component was estimated based on the peak area ratio of sample to the chromatographically pure external standard (-)-β-pinene (Magdalena and Henryk, 2016). Three biological replicates were performed for each treatment. The proportions of single components were calculated as their percentages in total terpenoid compounds.


Introduction
Insects use chemical volatiles to communicate in mating, aggregation, predation, alarm and self-defense (Belén et al., 2015). Among them, alarm pheromones as the second largest family of insect pheromones play an important ecological role in insects (Verheggen et al., 2010). Most aphids release alarm pheromones when they are attacked by natural enemies, and both aphid nymphs and adults utilize alarm pheromone to warn con-speci cs of danger (Kunert et al., 2007). Aphids are among the most widespread and harmful agricultural pests in the world (Simon and Peccoud, 2018). Previous studies showed that the major component of alarm pheromone for most aphid species is the sesquiterpene (E)-β-farnesene (Edwards et al., 1973;Pickett and Gri ths, 1980). Francis et al. (2005) tested the composition of volatile molecules in 23 aphid species, nding that (E)-β-farnesene was the main component in 16 species and the minor component in ve species; only two aphid species (Euceraphis punctipennis and Drepanosiphum platanoides) did not release (E)-β-farnesene. They also reported a particular pro le of volatile molecules composed of not only (E)-β-farnesene but also several monoterpenes in the vetch aphid Megoura viciae Buckton (Aphididae: Hemiptera), including (-)-α-pinene, (-)-β-pinene and (+)-limonene, but no behavioral assays were performed. Nevertheless, Bruno et al. (2018) assessed the behavioral response of M. viciae to the compounds identi ed by Francis et al. (2005), indicating that (-)-α-pinene and (+)-limonene were the main active components of alarm pheromone in M. viciae. Moreover, they tested a mixture at the ratio of (E)-β-farnesene (14.2%), (-)-α-pinene (11.8%) and β-pinene (74%) as reported (Table S1), showing a repellent activity against M. viciae. Additionally, molecular studies revealed that the recombinant odorant binding protein MvicOBP3 could bind to all four alarm pheromone components of M. viciae, displaying a much higher a nity for (E)-β-farnesene (K i 0.1 µM) than for β-pinene (K i 2.3 µM), (−)-α-pinene (K i 1.8 µM) and (+)-limonene (K i 2.5 µM) (Northey et al., 2016). It seems that the molecular binding a nity could not re ect the alarm activity of terpene components.
M. viciae feeds exclusively on the Fabaceae (Nuessly et al., 2004), causing serious damage to the broad bean Vicia faba and the pea Pisum sativum (Kunert et al., 2008;Leroy et al., 2011). Its unique composition of terpene components differentiate it from most other aphid species in alarm behavior. It has been shown that alarm pheromone was synthesized by the aphid itself in the cotton aphid Aphis gossypii by rearing aphids with arti cial diets (Sun and Li, 2017). However, it is still unclear whether other aphids also synthesize de novo alarm pheromone. Our group has been working on the biosynthetic mechanisms of aphid alarm pheromone, yet the major component of alarm pheromone in all aphid species that have been investigated was (E)-β-farnesene, including the green peach aphid Myzus persicae (Cheng and Li, 2018;Zhang and Li, 2008;Zhang and Li, 2012), A. gossypii (Ma et al., 2010;Sun and Li, 2017;Sun and Li, 2018) and the bird cherry-oat aphid Rhopalosiphum padi (Sun and Li, 2012; Sun and Li, 2019; Sun and Li, 2020). Thus, M. viciae can provide a good opportunity for comparative study in this line of work. Here we rst analyzed the composition of terpenoid compounds in M. viciae and its host plant Pisum sativum by using gas chromatography-mass spectrometry. Next, the dynamics of the major terpene components was investigated during different developmental stages of M. viciae. Moreover, we conducted a series of behavioral assays with single components and mixtures at different concentrations in a three-compartment olfactometer. Our study identi ed a novel set of alarm pheromones in M. viciae.

Culture of aphids
The aphid M. viciae was provided by the Laboratory of Biological Control led by Dr. Tinghui Liu in Hebei Agricultural University and maintained on P. sativum in the Laboratory of Insect Molecular Ecology in China Agricultural University. The aphids were reared in a climate incubator (RXZ-300B, Ningbo, China) under the conditions of 19 ± 1 ℃, 70 ± 5% relative humidity and a photoperiod of 16L:8D.
Collection of terpenoid compounds fromM. viciaeand its host plantP. sativum M. viciae aphids (overlapping developmental stages, n = 200) were collected and put into a 1.5-mL centrifuge tube containing 500 µL of n-hexane on ice, fully milled, centrifuged at 4 ℃ for 30 min. The supernatant was transferred into a 2-mL vial for gas chromatography-mass spectrometry (GC-MS) analysis.
The same procedure was performed to collect volatile terpenoid compounds from P. sativum seedlings (2.0g) for GC-MS analysis.
Identi cation of terpenoid compounds fromM. viciaeandP. sativumby GC-MS The samples collected from M. viciae and P. sativum were analyzed on an Agilent 6890 gas chromatographer coupled to an Agilent 5973 ion trap mass detector (Agilent Technologies Inc., California, USA). The instrument was equipped with a HP-5 capillary column (300 mm × 0.25 mm × 0.25 µm, Agilent, Santa Clara, USA). The program of GC-MS was set up as described (Huang et al., 2013). Brie y, after sample injection, the GC oven temperature was held at 40°C for 1 min, followed by a two-step temperature increase: the rst increase was from 40°C to 130°C at a rate of 4°C/min and maintained for 5 min, and then the temperature was increased at a rate of 10°C/min to 250°C and held for 5 min. and control (C) were placed in a petri dish (Ф3.0 cm) near the door of the side compartments, respectively, and ve host plants xed in smaller petri dishes (Ф1.0 cm) (covered with 10% agar) were placed in the far side of the lateral compartments. Twenty wingless 3rd -and 4th -instar nymphs were introduced into the petri dish in the middle (A) and allowed to move freely for 30 min. A total of 100 nymphs were used to test their selection preference for each sample compound. All behavioral assays were performed under the same conditions and under a dark environment to avoid light interference. The numbers of aphids crawling close to the host plants in the two lateral compartments were counted. For each sample tested, the behavioral index value (BIV) was calculated according to the following formula: BIV = [(C − T)/ (C + T)] × 100, where C and T are the numbers of aphids in the control and treatment compartments, respectively.

Data analysis
The quantities of (-)-α-pinene, (-)-β-pinene and (+)-limonene and (E)-β-farnesene were statistically analyzed and compared on the GraphPad Statistics version 8.0 (San Diego, USA) using One-way analysis of variance (ANOVA) followed by Tukey's B multiple range test (P < 0.05). The signi cance of differences in the behavioral index values were analyzed on SPSS Statistics version 21 (IBM) using ANOVA followed by Duncan's test (P < 0.05).

Results
Identi cation of the terpenoid compounds from M. viciae and its host plant P. sativum The terpenoid compounds from M. viciae and its host plant P. sativum were identi ed by using GC-MS analysis. The results showed that four major terpene compounds were detected in M. viciae, including the sesquiterpene (E)-β-farnesene and three monoterpenes, i.e., (-)-α-pinene, (-)-β-pinene and (+)-limonene ( Table 1). The compounds (-)-α-pinene, (-)-β-pinene, (+)-limonene and (E)-β-farnesene were corresponding to the peaks nos.1, 2, 3 and 6, respectively, as characterized by GC-MS ( Fig. 2 and Fig. S1). Interestingly, some additional minor peaks were detected, which were identi ed as β-myrcene, (+)-sabinene, camphene and αterpineol, based on comparative analysis with the standard mass spectrometry library NIST17s. Moreover, the proportions of different components were calculated: (-)-α-pinene (9.42%), (-)-β-pinene (49.74%) and (+)limonene (5.24%), and (E)-β-farnesene (32.64%), with the minor components accounting for 3.14% (Fig. 3A). In contrast, the types of terpenoid compounds were relatively scarce in the host plant compared to in the aphid, mainly consisting of squalene (66.13%) and its analogue 2,3-epoxysqualene (31.59%) ( Table 2, Fig. 3B and Fig. S2). Some minor terpenoid components were also identi ed in P. sativum, such as 4isopropyl-5-methylhexa-2,4-dien-1-ol, 2,4-pentadien-1-ol, 3-pentyl-, (2Z)-, limonene and cyclohexanol, 1methyl-4-(1-methylethenyl)-, cis-, based on comparative analysis with the standard library.  Quantitative dynamics of terpene compounds at different developmental stages of M. viciae The contents of four major terpene components were investigated at different developmental stages of aphid: 1st -instar, 2nd -instar, 3rd -instar, 4th -instar and adult. The results showed that the content of (-)-β-pinene increased rapidly from the 1st -to 2nd -instars, and the content of (E)-β-farnesene had a distinct increase from the 2nd -to 3rd -instars, while the contents of (+)-limonene and (-)-α-pinene had a substantial increase from the 3rd -to 4th -instars (Fig. 4 top). All components remained at a high level in the 4th -instar nymph. As a general trend, the contents of all monoterpene components increased with continuous development, while the content of (E)-β-farnesene displayed a different trend. The proportions of different terpenoid components were also calculated for different developmental stages (Fig. 4 bottom), showing that the proportion of (-)-β-pinene was the highest (> 81%) from the 2nd -instar to adult stages. The ratios of the four major terpene components at the 3rd -and 4th -instar stages were used for preparing the two mixtures for behavioral assays.   were composed of winged and wingless forms at different developmental stages in both studies. Thus the difference in the proportions of terpenoid components in M. viciae might be caused by either endogenous or exogenous factors. As a possible exogenous factor, the devices used in two studies were different; as the endogenous factor, the aphids analyzed were different: it was highly probable that the compositions of terpenoid components differentiated in the two different geographic populations of M. viciae. Therefore, the ecological signi cance of our results needs further investigation.
Quantitative analysis of the dynamics of the four major terpene components in M. viciae during different developmental stages revealed different patterns of change trend in the monoterpene and sesquiterpene components: the monoterpenes increased with continuous development, while the latter reached peak at the 3rd -instar. We also found that all terpenoid components remained at a high level in the 4th -instar; the proportion of (-)-β-pinene remained the highest (> 81%) from the 2nd -instar to adult stages. This is the rst report of the temporal dynamics in the composition of terpenoid components in an aphid species. These data formed the basis for the preparation of volatile mixtures for olfactory choice assays.
Behavioral assays revealed that the repellent activities of single components were concentration-dependent but the mixtures not: all single components showed a repellent activity against M. viciae to some extents at 1.0 µg/µL or above, but displayed no or merely weak activity at 0. Last but not least, our results revealed that the terpenoid compounds were relatively scarce in the host plant P. sativum, containing none of the major aphid alarm pheromone components. This result added a biochemical evidence to the notion that alarm pheromone was synthesized de novo in the aphid (Sun and Li, 2017), and it is unlikely that aphid alarm pheromone is taken directly from the host plant.
In summary, we identi ed four major terpene components in addition to some minor terpenoid components in M. viciae. Different types of components exhibited different patterns of change trend across the developmental process of aphid: the monoterpenes increased with continuous development, while the sesquiterpene reached peak at the 3rd -instar; all terpene components remained at a high level in the 4thinstar, with (-)-β-pinene accounting for the highest proportion during all developmental stages. Behavioral assays revealed that the repellent activities of single components varied in a concentration-dependent manner, but the mixtures maintained a signi cant repellent activity at all concentrations tested. Our results suggested that (-)-α-pinene and (-)-β-pinene were the major active components of alarm pheromone in M. viciae, but the mixtures of single components play a key role in the alarm behavior of M. viciae. Our study helps to understand the chemical ecology of insects and design alternative control strategies against aphids.
Declarations Figure 3 Temporal dynamics of terpene compounds at different developmental stages of M. viciae (top). The proportions of four major terpene components at the 1st-instar, 2nd-instar, 3rd-instar, 4th-instar nymphal and adult stages are also shown (bottom). The proportions of different components are calculated based on the percentages of peak areas.

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