A hemiterpene glucoside as a probing deterrent of the bean aphid, Megoura crassicauda, from a non-host vetch, Vicia hirsuta

Article abstract of DOI:10.1016/j.phytochem.2005.12.013

The bean aphid, Megoura crassicauda Mordvilko, feeds selectively on plants belonging to the genus Vicia (Fabaceae). However, it never infests the tiny vetch, V. hirsuta (L.) Gray. The aphid appeared to discriminate between host and non-host plants by tasting specific chemicals during penetration of its stylet into the plant tissues. The aphid, after being stimulated by specific probing stimulants, deposited characteristic proteinous stylet sheaths through a parafilm membrane, which has one side in contact with an extract solution of Vicia angustifolia. However, an addition of a V. hirsuta extract to the medium strongly inhibited the salivary sheath formation. A specific probing deterrent was isolated from a V. hirsuta extract by monitoring the inhibitory effect, and identified as (E)-2-methyl-2-butene-1,4-diol 4-O-β-D-glucopyranoside. A mixture of the glycoside and the stimulatory V. angustifolia fraction in the same equivalency found in plants significantly decreased the probing activity in M. crassicauda. Since the stylet insertion process is a crucial step for the aphid's settlement on a plant, the glycoside seems to act as an effective chemical barrier for V. hirsuta.

Full text of DOI:10.1016/j.phytochem.2005.12.013

PHYTOCHEMISTRY
Phytochemistry 67 (2006) 584–588
www.elsevier.com/locate/phytochem
A hemiterpene glucoside as a probing deterrent of the bean aphid,
Megoura crassicauda, from a non-host vetch, Vicia hirsuta
Naohiro Ohta, Naoki Mori, Yasumasa Kuwahara, Ritsuo Nishida *
Laboratory of Chemical Ecology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
Received 15 September 2005; received in revised form 28 October 2005
Available online 24 January 2006
Abstract
The bean aphid, Megoura crassicauda Mordvilko, feeds selectively on plants belonging to the genus Vicia (Fabaceae). However, it
never infests the tiny vetch, V. hirsuta (L.) Gray. The aphid appeared to discriminate between host and non-host plants by tasting specific
chemicals during penetration of its stylet into the plant tissues. The aphid, after being stimulated by specific probing stimulants, depos-
ited characteristic proteinous stylet sheaths through a parafilm membrane, which has one side in contact with an extract solution of Vicia
angustifolia. However, an addition of a V. hirsuta extract to the medium strongly inhibited the salivary sheath formation. A specific prob-
ing deterrent was isolated from a V. hirsuta extract by monitoring the inhibitory effect, and identified as (E)-2-methyl-2-butene-1,4-diol 4-
O-b-D-glucopyranoside. A mixture of the glycoside and the stimulatory V. angustifolia fraction in the same equivalency found in plants
significantly decreased the probing activity in M. crassicauda. Since the stylet insertion process is a crucial step for the aphid’s settlement
on a plant, the glycoside seems to act as an effective chemical barrier for V. hirsuta.
ꢀ
2006 Elsevier Ltd. All rights reserved.
Keywords: Aphid; Megoura crassicauda; Vicia hirsuta; Fabaceae; Probing deterrent; Host selection; Allomone; (E)-2-methyl-2-butene-1,4-diol 4-O-b-D-
glucopyranoside; Hemiterpene glycosides
1
. Introduction
observed on a stretched Parafilm M membrane, which is in
contact with a solution of host plant extract (Mittler and
Dadd, 1965). We have recently characterised two acylated
flavonol glycosides [quercetin 3-O-a-L-arabinopyranosyl-
The bean aphid, Megoura crassicauda Mordvilko, feeds
selectively on a variety of fabaceous plants in the genus Vicia,
such as broad bean (Vicia faba L.) and narrow leaf vetch
00
(1 ! 6)-(2 -O-(E)-p-coumaroyl)-b-D-galactopyranoside and
00
(
Vicia angustifolia L.). The host selectivity of oligophagous
aphids is controlled by specific chemicals in their host plants
Van Emden, 1972; Klingauf, 1987). After landing on a
quercetin 3-O-a-L-arabinopyranosyl-(1 ! 6)-(2 -O-(E)-p-
coumaroyl)-b-D-glucopyranoside] as the specific probing
stimulants of M. crassicauda, to induce the salivary sheath
formation. This suggests that the two compounds are possi-
ble host finding cues prior to ingestion of the phloem sap
(Takemura et al., 2002).
(
plant, the aphid inserts its stylet into the internal tissues
and senses the chemicals contained within (Montllor, 1991
and refs. therein). This information determines whether the
aphid leaves the plant or continues to probe further (Van
Emden, 1972). During the penetration into the host plant tis-
sue towards the phloem, aphids produce proteinous salivary
sheaths (Miles, 1965). The same stylet sheaths can be
In the course of this study, we noticed that the tiny
vetch, Vicia hirsuta (L.) Gray, was never attacked by
M. crassicauda, while dense colonies of the aphids were
commonly observed on a very closely related species,
V. angustifolia. Both the plant species are common vinous
weeds that grow closely together, often tangling with each
other during the spring season (April to early June) in
Japan. Although the seedpods of V. hirsuta are more hairy
*
Corresponding author. Tel.: +81 75 753 6313; fax: +81 75 753 6312.
E-mail address: ritz@kais.kyoto-u.ac.jp (R. Nishida).
0
031-9422/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2005.12.013

N. Ohta et al. / Phytochemistry 67 (2006) 584–588
585
than those of V. angustifolia, the surface structure of the
stem, on which the aphids would colonise, did not appear
different between the two species under a microscope.
When the aphids were exposed to a V. hirsuta plant in
the laboratory, they displayed a short-term probing on the
stem, but eventually left the plant. This suggests that
the aphids were capable of sensing some odd chemicals in
the internal tissues of V. hirsuta. An extract of V. hirsuta
was found to deter the probing behaviour of the aphids
when added to the solution containing the positive stimu-
lants in feeding tests using the parafilm membrane. We
report the isolation and structural elucidation of a specific
probing deterrent in V. hirsuta against M. crassicauda.
ing activity decreased significantly (Table 1). This suggests
the presence of a deterrent (or masking) substance(s) that
interfered with the production of salivary sheaths. The prob-
ing deterrent assay was performed by mixing V. hirsuta frac-
tions (1.0 gle/ml unless otherwise specified) in a stimulative
fraction of V. angustifolia (1.0 gle/ml) as shown in
Table 1.
An aqueous extract of V. hirsuta was applied to an ODS
column with increasing concentrations of methanol in
water as eluant to give six fractions. These were eluted with
water, 10% methanol, 20% methanol, 40% methanol, 60%
methanol and 80% methanol in water, respectively. The
10% methanol eluate exhibited a significant probing deter-
rent activity almost equivalent to that of a crude extract of
V. hirsuta (no significant difference from a mixture of crude
V. hirsuta extract + V. angustifolia extract, Mann–Whitney
U test) (Table 1). The active eluate was further separated
by a reversed-phase HPLC and a single deterrent com-
pound 1 was isolated as an amorphous solid (yield:
370 lg/g leaf). Compound 1 clearly suppressed the stimu-
lant activity of V. angustifolia extract at doses of 1.0 and
2.0 g.l.e, but not at a 0.5 g.l.e. dose (Table 1).
2
. Results and discussion
When freshly cut stems from both V. angustifolia (host)
and V. hirsuta (non-host) were placed together in a cage,
the introduced adult aphids (apterous or alatae) settled
selectively on V. angustifolia and started feeding on the
stems producing a large quantity of honeydew within few
hours. The aphids seemed to conduct some trial probing
on V. hirsuta as well, but they left the plant within a short
period. M. crassicauda actively displayed probing behaviour
toward the crude aqueous extracts of V. angustifolia, depos-
iting a substantial number of thick stylet sheaths (thickness,
approx. 10 lm; length > 100 lm) on a parafilm membrane
at a concentration of 1.0 g fresh leaf equivalent/ml
Compound 1 was deduced to possess the molecular
formula of C H O (mw 264) from the electro-spray
1
1
20
7
ionization (ESI) mass spectra (positive m/z 287
+
+
+
[M + Na] ; 303 [M + K] ), although [M + H] was not
observed. An enzymatic hydrolysis of compound 1 with
b-glucosidase yielded 2-methyl-2-butene-1,4-diol (2) and
D-glucose. Compound 2 was determined to possess the
(E)-configuration by comparing both geometrical isomers
derived from mesaconic acid (trans) and citraconic acid
(
gle/ml) (Takemura et al., 2002). However, the aphid depos-
ited much fewer stylet sheaths on the parafilm in contact
with a crude aqueous extract of V. hirsuta (Table 1). When
a mixture of both V. angustifolia and V. hirsuta extracts
was supplied in an equivalency (1.0 gle/ml each), the prob-
1
(cis), respectively. The H NMR spectrum of 1 (Table 2)
exhibited signals arisen from a methyl (d 1.69), two
methylenes (d 3.96, 2H; d 4.28 and 4.39, 2H) and an ole-
finic methine (d 5.36) of a 2-methyl-2-butene-1,4-diol
moiety. A glucopyranosy moiety was found to be
attached to one of the primary alcohol to form a b-con-
figuration. The linkage of glucose to the hemiterpene diol
Table 1
Probing responses of Megoura crassicauda to solutions of Vicia plant
extracts and mixtures with various fractions and compound 1
Sample
Probing activity (points) (N)
a
Table 2
1
Distilled water
10.5 ± 5.0 (10)
H NMR spectral data for compound 1, (E)- and (Z)-2-methyl-2-butene-
V. angustifolia (Va) extract [host, control] 48.2 ± 6.1 (43)
a
1,4-diols (CD OD, 400 MHz)
V. hirsuta extract [non-host]
Va ext. + V. hirsuta ext.
Va ext. + Fr. 1
9.1 ± 4.3 (10)
7.5 ± 3.3 (10)
45.1 ± 9.0 (9)
3
a
Position
Compound 1
(E)-diol (2)
(Z)-diol
a
Diol
Va ext. + Fr. 2
12.0 ± 5.6 (9)
1
3
4
3.96 s
5.36 br. t (6.8)
4.28 m
3.93 s
5.59 br. t (6.6)
4.13 d (6.3)
4.085 s
5.47 t (6.8)
4.11 d (8.0)
Va ext. + Fr. 3
Va ext. + Fr. 4
Va ext. + Fr. 5
Va ext. + Fr. 6
Va ext. + Compound 1 (0.5 gle/ml)
Va ext. + Compound 1 (1.0 gle/ml)
Va ext. + Compound 1 (2.0 gle/ml)
20.9 ± 5.8 (9)
41.3± 10.4 (9)
34.2 ± 6.3 (9)
35.7 ± 12.3 (9)
22.4 ± 6.9 (10)
17.1 ± 5.0 (10)
13.8 ± 3.4 (10)
4
.39 dd (12.1, 6.2)
5
1.69 s
1.67 s
1.80 s
a
a
Glucosyl
0
1
4.29 d (8.0)
3.17 m
3.34 m
3.25 m
3.31 m
0
0
0
0
2
3
4
5
Probing activity represents the average frequency of stylet sheath
formation.
Dose (gle = gram leaf equivalent): all extracts and fractions were tested at
1
.0 gle/ml, except for compound 1 (0.5–2.0 gle/ml).
Significantly different from control Vicia angustifolia crude extract
0
a
6
3.67 d (11.7, 4.9)
3
.87 br. d (11.7)
(1.0 gle/ml) at p = 0.05 using the Mann–Whitney U test.

586
N. Ohta et al. / Phytochemistry 67 (2006) 584–588
was determined by the heteronuclear multiple bond cor-
relation (HMBC) spectrum, where correlation from H-1
deterrents or toxicants in many cases remains unclear due
to the complexity of host assessment process by aphids plus
the lack of knowledge related to the distribution of the
chemicals within the plant tissues (Montllor, 1991). A sys-
tematic study on the mechanisms of host and non-host rec-
ognition of aphids within closely related plant taxa (or
cultivars) might provide us a new control technique against
pest aphids of economic importance.
0
(
d 4.29) of glucose to the C-4 (d 66.2) of diol, and from
0
H-4 (d 4.39) to the C-1 (d 103.0) of glucose were
observed. This indicated that the glucose moiety was
linked to the C-4 of 2-methyl-2-butene-1,4-diol. Thus,
compound 1 was determined as (E)-2-methyl-2-butene-
1
,4-diol 4-O-b-D-glucopyranoside. Compound 1 appeared
to be a novel compound from plants, although its posi-
tional isomer (E)-2-methyl-2-butene-1,4-diol 1-O-b-D-
glucopyranoside (3) and (Z)-isomer of 3 have been
known from other plants (Nicoletti et al., 1992; Kitajima
et al., 1998; Zhu et al., 1998). Compounds 2 and 3 were
also found in fraction 2 as minor components, but the
compounds did not show any significant deterrent effect
at the equivalent dosage found in the plant.
3. Experimental
3.1. General
Optical rotation was measured with a JASCO DIP-370
spectropolarimeter. The MS data were recorded with a Shi-
madzu LCMS-2010A using a reversed phase column
(
Mightysil RP-18GP, 75 mm · 3 mm i.d.) eluted with 10-
HO
100% MeOH in H O (0.2 ml/min) with an electro-spray
2
1
13
O
HO
HO
ionization (ESI)-positive mode. H and C NMR spectra
were measured with a Bruker AC400 FT-NMR spectrom-
eter (400 MHz) with TMS as an internal standard. The let-
ters s, d, t and m represent singlet, doublet, triplet, and
multiplet, respectively, and coupling constants are given
in Hz.
O
OH
OH
Compound 1
HO
OH
Compound 2
3.2. Plant material
Arial parts of V. hirsuta were collected in Sakyo-ku,
Kyoto, Japan in April 2003, and identified by Dr. Reiichi
Miura of Kyoto University. A voucher specimen is depos-
ited at the Herbarium of Pesticide Research Institute,
Kyoto University.
It is intriguing that a novel hemiterpene glycoside
exhibits probing deterrent activity against the aphid.
Since the stylet insertion process is a crucial step for
the aphid’s settlement on a plant (Klingauf, 1987; Montl-
lor, 1991), the glycoside 1 seems to act as an effective
chemical barrier for V. hirsuta. At the initial step of
probing, M. crassicauda inserted the stylet ‘intercellularly’
through the epidermis and mesophyll of host Vicia plant.
The compound may block the stylet penetration toward
the phloem during gustation, if the substance is detected
along the intercellular path, although we do not know
the distribution of 1 in the plant. Further support for
the feeding preference of the aphids between these two
closely related Vicia species is as follows: firstly, the spe-
cific allelochemical is provided in the absence of the
probing deterrent 1 of the host V. angustifolia and sec-
ondly, the lack of the specific probing stimulants (acyl-
ated flavonol glycosides previously identified from the
host V. angustifolia, Takemura et al., 2002) in the non-
host plant, V. hirsuta.
3.3. Extraction and isolation of deterrent
Fresh leaves and stems of V. hirsuta (300 g) were
extracted with EtOH–H O (9:1, v/v) three times. The
2
combined ethanolic solution was evaporated in vacuo,
and the residue (30.30 g) was dissolved in H O (600 ml)
2
and partitioned four times with hexane (400 ml each).
The crude aqueous extract (14.30 g) was applied to a
reversed phase column (200 g of Cosmosil 140C18-
OPN, Nacalai tesque, 310 · 50 mm i.d.) eluted (2 L each)
in sequence (yields of dry materials given in the paren-
theses) with H O (8.69 g), MeOH–H O (1:9) (0.40 g),
2
2
MeOH–H O (1:4) (0.17 g), MeOH–H O (2:3) (0.30 g),
2
2
MeOH–H O (3:2) (0.41 g) and MeOH–H O (4:1)
2
2
(0.05 g). MeOH–H O (1:9) eluate was subjected to pre-
2
A series of plant secondary metabolites, including terp-
enoids (Asakawa et al., 1988), phenolics (Montgomery and
Arn, 1974; Dreyer and Jones, 1981; Dreyer et al., 1981;
Jones and Klocke, 1987), alkaloids (Corcuera, 1984) and
hydroxamic acid derivatives (Argandona et al., 1983; Givo-
vich and Niemeyer, 1995), have been reported as feeding
deterrents in several aphid species. However, the actual role
of these allelochemicals as probing deterrents, ingestion
parative HPLC using a reverse phase column (YMC-
Pack Pro C18 250 · 10 mm i.d.), eluting with MeOH–
H O (1:9) at flow rate of 2.0 ml/min. Active fraction
2
was found at
a
retention time (Rt) range of
18.4–21.4 min, which was further subjected to the same
column eluting with MeOH–H O (7.5:92.5) at a flow rate
2
of 2.0 ml/min. Compound
1
was isolated at
a
R = 26.0 min. The yield of compound 1 from 100 g of
t

N. Ohta et al. / Phytochemistry 67 (2006) 584–588
587
the leaves was 37 mg. Compound 2 was detected at
3.7. Bioassay
R = 21.5 min in a small quantity under the latter HPLC
t
condition.
Adults of M. crassicauda (apterous viviparae) were
starved for 2 h; and five insects were introduced in a glass
tube fitted with a parafilm membrane in contact with either
3.4. (E)-2-Methyl-2-butene-1,4-diol 4-O-b-D-
glucopyranoside (1)
a test solution (0.4 ml) or distilled H O (control), and
2
allowed to probe through the membrane for 24 h under
laboratory conditions (25 ꢁC, 16L:8D) (Takemura et al.,
2002). The number of stylet sheaths deposited on the para-
film membrane was observed under a microscope after
being stained with a red fuchsin basic solution. The prob-
ing activity was scored by frequency of stylet sheath forma-
tion as described previously (Takemura et al., 2002).
1
D
7
Colourless solid. ½aꢀ ꢁ41.8 (10% MeOH in water; c
1
[
.00); LCMS (ESI-positive) m/z (relative intensity): 287
M + Na] (100), 303 [M + K] (52). For H and
NMR spectroscopic data, see Tables 2 and 3.
+
+
1
13
C
3
.5. Enzymatic hydrolysis of compound 1
Compound
almond, Wako Pure Chemical Industries Ltd., 193 units,
.2 mg) was dissolved in 1 ml of AcOH–NaOAc buffer
1 ml) and incubated for 4 h at 37 ꢁC. The reaction mixture
was acidified with 0.5 N HCl, filtered (syringe filter unit,
Millex), and passed through a reversed phase column
1 (26.4 mg) and b-glucosidase (from
Acknowledgements
5
(
We thank Dr. Reiichi Miura of Kyoto University for
identification of the plant. This study was partly supported
by a Grant-in-Aid for the 21st Century COE Program for
Innovative Food and Environmental Studies Pioneered by
Entomomimetic Sciences, from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
(
10 g, Cosmosil 140C18-OPN, Nacalai tesque) eluted with
water (100 ml). The water eluate was separated by HPLC
Cosmosil AR-II, 200 · 15 mm i.d.) eluting with MeOH–
(
H O (1:4)1 in H O (2.0 ml/min) and D-glucose
2
2
7
ð15:3 mg ½aꢀ ¼ þ43:4Þ and (E)-diol (2) (7.1 mg) were iso-
D
lated at R = 6.6 and 16.8 min, respectively.
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68.0
140.9
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66.2
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75.1
78.2
71.7
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62.8
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