Identification 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 identified 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 identified as β-myrcene, (+)-sabinene, camphene and α-terpineol, 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 identified in P. sativum, such as 4-isopropyl-5-methylhexa-2,4-dien-1-ol, 2,4-pentadien-1-ol, 3-pentyl-, (2Z)-, limonene and cyclohexanol, 1-methyl-4-(1-methylethenyl)-, cis-, based on comparative analysis with the standard library.
Table 1
GC-MS identification of terpenoid compounds in Megoura viciae
No.
|
Retention time (min)
|
m/z
|
Peak area
|
% Peak area
|
Peak height
|
% Peak height
|
Name of terpenoid compound
|
Molecular formula
|
1
|
10.099
|
TIC
|
1203409
|
0.18
|
456923
|
0.24
|
(-)-α-Pinene
|
C10H16
|
2
|
10.639
|
TIC
|
57434
|
0.01
|
14470
|
0.01
|
Camphene
|
C10H16
|
3
|
11.562
|
TIC
|
75660
|
0.01
|
30386
|
0.02
|
(+)-Sabinene
|
C10H16
|
4
|
11.676
|
TIC
|
6325841
|
0.95
|
2149907
|
1.15
|
(-)-β-Pinene
|
C10H16
|
5
|
12.244
|
TIC
|
229816
|
0.03
|
68879
|
0.04
|
β-Myrcene
|
C10H16
|
6
|
13.665
|
TIC
|
658790
|
0.1
|
195955
|
0.1
|
(+)-Limonene
|
C10H16
|
7
|
19.977
|
TIC
|
40572
|
0.01
|
8983
|
0
|
α-Terpineol
|
C10H180
|
8
|
29.105
|
TIC
|
4102715
|
0.62
|
805659
|
0.43
|
(E)-β-Farnesene
|
C15H24
|
Table 2
GC-MS identification of terpenoid compounds in the host plant Pisum sativum
No.
|
Retention time (min)
|
m/z
|
Peak area
|
% Peak area
|
Peak height
|
% Peak height
|
Name of terpenoid
|
Molecular formula
|
1
|
13.674
|
TIC
|
138863
|
0.02
|
30615
|
0.02
|
Limonene
|
C10H16
|
2
|
22.52
|
TIC
|
42633
|
0.01
|
15660
|
0.01
|
Cyclohexanol, 1-methyl-4-(1-methylethenyl)-, cis-
|
C10H18O
|
3
|
27.43
|
TIC
|
174315
|
0.03
|
83769
|
0.05
|
2,4-Pentadien-1-ol, 3-pentyl-, (2Z)-
|
C10H18O
|
4
|
42.391
|
TIC
|
1057080
|
0.18
|
267448
|
0.15
|
4-Isopropyl-5-methylhexa-2,4-dien-1-ol
|
C10H18O
|
5
|
42.944
|
TIC
|
19187774
|
3.32
|
3786188
|
2.14
|
2,3-Squalene-epoxy
|
C30H50O
|
6
|
43.013
|
TIC
|
13851345
|
2.39
|
3818540
|
2.16
|
Squalene
|
C30H50
|
7
|
43.094
|
TIC
|
26363816
|
4.56
|
5569989
|
3.15
|
Squalene
|
C30H50
|
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.
Behavioral responses of M. viciae to single and mixed terpene compounds
The behavioral responses of M. viciae to single terpene components and their mixture were measured in the three-compartment olfactometer. The two mixtures of (-)-α-pinene, (-)-β-pinene, (+)-limonene and (E)-β-farnesene were prepared in that order at the ratios of 1:44.4:6.5:2.2 (Mix I) and 1:18.4:1.3:0.8 (Mix II) according to the proportions of the four major terpene components at the 3rd - and 4th -instar stages. Behavioral responses were categorized as four types: NR (no response, BIV < 20%), W (weak, 20 < BIV < 40%), M (moderate, 40% < BIV < 60%) and S (strong, BIV > 60%) (Hieu et al., 2014; Khashaveh et al., 2020). The results showed that the repellent activities of the compounds tested varied at different concentrations (Table 3 and Fig. 5). Specifically, (-)-α-pinene, (-)-β-pinene, (+)-limonene and (E)-β-farnesene all displayed repellent activity to some extents at the concentration of 10.0 µg/µL: (-)-α-pinene had the strongest activity with a highest BIV value (Type S); (-)-β-pinene, Mix I and Mix II exhibited moderate repellent activity (M), and (+)-limonene and (E)-β-farnesene showed merely weak activity (W). Similarly, under the concentration of 1.0 µg/µL, (-)-α-pinene, (-)-β-pinene, Mix I and Mix II all showed moderate repellent activity, and (+)-limonene and (E)-β-farnesene had only weak repellent activity. In contrast, under the concentration of 0.1 µg/µL, no behavioral response or merely weak repellent activity were observed for all four single compounds. As a single component, (E)-β-farnesene displayed only weak repellent activity at all tested concentrations. Nevertheless, the two mixtures showed a significant repellent activity against M. viciae (F17, 72=5.748, P < 0.05) at the low concentration of 0.1 µg/µL.
Table 3
Behavioral index values (BIV) of Megoura viciae in response to single and mixed terpene compounds
Compound
|
Concentration (µg/µL)
|
BIV (%)
|
BA
|
(-)-α-Pinene
|
10.0
|
67.85 ± 4.52a
|
Repellent (S)
|
(-)-β-Pinene
|
42.55 ± 9.52 abcde
|
Repellent (M)
|
(+)-Limonene
|
23.49 ± 6.37 cde
|
Repellent (W)
|
(E)-β-farnesene
|
20.33 ± 2.43 cde
|
Repellent (W)
|
Mix I
|
58.19 ± 2.69 ab
|
Repellent (M)
|
Mix II
|
43.55 ± 8.39 abcde
|
Repellent (M)
|
(-)-α-Pinene
|
1.0
|
34.81 ± 12.51 bcde
|
Repellent (M)
|
(-)-β-Pinene
|
38.67 ± 4.94 abcde
|
Repellent (M)
|
(+)-Limonene
|
23.49 ± 6.39 cde
|
Repellent (W)
|
(E)-β-farnesene
|
27.94 ± 6.28 bcde
|
Repellent (W)
|
Mix I
|
43.59 ± 4.80 abcde
|
Repellent (M)
|
Mix II
|
49.00 ± 7.14 abcd
|
Repellent (M)
|
(-)-α-Pinene
|
0.1
|
16.41 ± 8.73 de
|
NR
|
(-)-β-Pinene
|
12.89 ± 6.34 e
|
NR
|
(+)-Limonene
|
13.27 ± 2.45 e
|
NR
|
(E)-β-farnesene
|
22.53 ± 1.56 cd
|
Repellent (W)
|
Mix I
|
51.27 ± 3.94 abc
|
Repellent (M)
|
Mix II
|
40.95 ± 1.90 abcde
|
Repellent (M)
|
Different letters at the same concentration indicate significant difference between different compounds (Tukey’s test, P < 0.05), while the same letters indicate no significant difference between the compounds. BA, behavioral activity. S, strong; M, moderate; W, weak; NR, no response. Mix I: (-)-α-pinene:(-)-β-pinene:(+)-limonene:(E)-β-farnesene = 1:44.4:6.5:2.2; Mix II: (-)-α-pinene:(-)-β-pinene:(+)-limonene:(E)-β-farnesene = 1:18.4:1.3:0.8. |