Identification of 1-octacosanal and 6-methoxy-2-benzoxazolinone from wheat as ovipositional stimulants for Hessian fly, Mayetiola destructor

Article abstract of DOI:10.1023/A:1005499907009

Bioassay-guided fractionations of a dichloromethane extract of wheat epicuticular wax allowed the identification of 1-octacosanal and 6-methoxy-2- benzoxazolinone (MBOA) as the major components that stimulate oviposition by the Hessian fly, Mayetiola destructor. These compounds were identified by their mass spectral fragmentation patterns and by comparison of their gas chromatographic retention times with synthetic samples. Synthetic samples of 1-octacosanal or MBOA stimulated significant oviposition when compared with solvent controls. In combination, these compounds elicited a synergistic effect on the number of eggs laid by females compared to when they were presented alone. In a choice bioassay, a mixture of synthetic 1-octacosanal and MBOA in the approximate concentrations determined to be present in one plant equivalent of crude extract stimulated the same amount of oviposition as one plant equivalent of extract. This showed that together these two compounds appear to be responsible for the major proportion of the ovipositional stimulatory activity of the wheat epicuticular wax extract. Comparison of the activity of five straight-chain primary aldehydes with chain lengths from C₂₂ to C₃₀ revealed a relationship between chain length and the number of eggs laid by female Hessian flies, with 1- hexacosanal and 1-heptacosanal the most active of the aldehydes tested.

Full text of DOI:10.1023/A:1005499907009

Journal of Chemical Ecology, Vol. 26, No. 4, 2000
IDENTIFICATION OF 1-OCTACOSANAL AND
6-METHOXY-2-BENZOXAZOLINONE FROM WHEAT AS
OVIPOSITIONAL STIMULANTS FOR HESSIAN FLY,
Mayetiola destructor
BRUCE D. MORRIS,* STEPHEN P. FOSTER,
and MARION O. HARRIS
The Horticulture and Food Research Institute of New Zealand Ltd.
Private Bag 92169, Auckland, New Zealand
(Received March 30, 1999; accepted December 2, 1999)
Abstract—Bioassay-guided fractionations of
a dichloromethane extract
of wheat epicuticular wax allowed the identification of 1-octacosanal
and 6-methoxy-2-benzoxazolinone (MBOA) as the major components that
stimulate oviposition by the Hessian fly, Mayetiola destructor. These
compounds were identified by their mass spectral fragmentation patterns and
by comparison of their gas chromatographic retention times with synthetic
samples. Synthetic samples of 1-octacosanal or MBOA stimulated significant
oviposition when compared with solvent controls. In combination, these
compounds elicited a synergistic effect on the number of eggs laid by females
compared to when they were presented alone. In a choice bioassay, a mixture
of synthetic 1-octacosanal and MBOA in the approximate concentrations
determined to be present in one plant equivalent of crude extract stimulated
the same amount of oviposition as one plant equivalent of extract. This
showed that together these two compounds appear to be responsible for
the major proportion of the ovipositional stimulatory activity of the wheat
epicuticular wax extract. Comparison of the activity of five straight-chain
primary aldehydes with chain lengths from C22 to C30 revealed a relationship
between chain length and the number of eggs laid by female Hessian flies, with
1-hexacosanal and 1-heptacosanal the most active of the aldehydes tested.
Key Words—Cecidomyiidae, insect behavior, egg-laying, chemoreception,
MBOA, Gramineae.
*To whom correspondence should be addressed.
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0098-0331 00 0400-0859$18.00 0  2000 Plenum Publishing Corporation
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MORRIS, FOSTER, AND HARRIS
INTRODUCTION
The Hessian fly, Mayetiola destructor (Say) (Diptera: Cecidomyiidae), is a major
pest of wheat (Triticum aestivum L.) in North America and North Africa and, to
a lesser extent, in Europe and New Zealand (Barnes, 1956; Ratcliffe and Hatch-
ett, 1997). Hessian fly females oviposit on the surface of wheat leaves and the
larvae move down to feed near the crown of the plant. In addition to wheat,
Hessian flies are known to oviposit and develop on rye (Secale cereale L.), bar-
ley (Hordeum vulgare L.), and a number of wild grasses in the genera Elymus,
Aegilops, Hordeum, and Agropyrum (Barnes, 1956; Harris et al., 1996). How-
ever, females oviposit less on oat (Avena sativa L.) and larvae do not survive on
this grass (Morrill, 1982). As Hessian fly larvae have limited mobility and are
incapable of moving between plants, the choice of oviposition site by the adult
female is critical to larval survival.
Previous studies have shown that the selection of oviposition sites by Hes-
sian flies is influenced by visual and tactile cues and by epicuticular leaf waxes
(Harris and Rose, 1990; Foster and Harris, 1992). In particular, preference for
the host grass wheat over the nonhost grass oat can be largely explained by the
oviposition preferences towards their epicuticular wax extracts. Fractionation of
wheat extracts on silica gel indicated that there were at least two different com-
pounds in the epicuticular wax of wheat that stimulated oviposition by Hessian
fly females (Foster and Harris, 1992).
In this paper we report the identification of two compounds, 1-octacosanal
(1) and 6-methoxy-2-benzoxazolinone (MBOA) (2) (Figure 1), from wheat that
together appear to be responsible for most of the ovipositional stimulatory activ-
ity of wheat leaf wax extract on the Hessian fly.
METHODS AND MATERIALS
Insects. Hessian fly females used in experiments were from a culture in
Auckland maintained on wheat plants. This culture originated from pupae col-
lected in 1994 from a wheat field in Palmerston North, New Zealand. Adults
emerging from these pupae were left to mate, and females subsequently were
FIG. 1. Chemical structures of 1-octacosanal (1) and 6-methoxy-2-benzoxazolinone (2).

OVIPOSITIONAL STIMULANTS FOR HESSIAN FLY
861
allowed to oviposit on wheat plants (cultivar Otane) in the two- to three-leaf
stage. Infested plants were held in a glasshouse (temperatures from 15 to 25 C)
where they were watered and fertilized (Hoagland’s solution) until Hessian fly
puparia were observed (30–50 days after infestation). Plants were then moved
to a controlled environment chamber (21 0.5 C, 50–70% relative humidity,
14L : 10D, with lights on at 07 : 00 hr) until adult flies started to emerge some
2–10 days later. This Hessian fly culture was reared in this manner for four years,
at a rate of five to seven generations a year.
For experiments, mated females were collected daily by aspirator shortly
after the hours of peak female eclosion and mating (07 : 00–10 : 00 hr). Mated
females were distinguished from virgin females by observing whether or not they
exhibited the characteristic calling posture (Bergh et al., 1992). Mated females
were transferred to experimental cages before they began to oviposit (before
11 : 00 hr) (Harris and Rose, 1991).
Wheat Extract. Wheat plants (cultivar Otane) were grown in a glasshouse
(ambient temperature 15–25 C) during September 1998. Plants (approx. 5000)
at the two- to three-leaf stage (11 days after planting) were cut at the base to give
645 g of fresh material. Extracts were prepared by dipping the leaves into cool
(10 C) dichloromethane (500 ml for 1000 plants) for 60 sec after the method of
Bianchi and Figini (1986). The solvent was removed from the combined crude
extracts by rotary evaporation at 37 C in a Buchi Rotavapor-R rotary evaporator,
to give 0.524 g of residue.
Trimethylsilylation of Wheat Extract. Wheat plants (cultivar Otane) were
grown as above during August 1999. Plants (25, 7.0 g fresh weight) were
extracted by dipping the leaves into ethyl acetate (20 ml) for 60 sec. The
solvent was removed by rotary evaporator at 37 C. Dichloromethane (0.5
ml) was added and evaporated (repeated twice) to remove residual water.
Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane
(TMCS) (Sigma Chemical Co., St. Louis, Missouri) (50 ml) was added to the
residue (within 15 min of extraction) and allowed to react for 30 min at 65 C.
This solution was analyzed directly by gas chromatography–mass spectrometry
(GC-MS).
Fractionation. The wheat dichloromethane extract was separated by flash
˚
column chromatography on silica gel (120 g; Merck Kieselgel 60 A, 230–400
mesh) with the following solvent gradient: 240 ml petroleum spirit (fraction 1),
2 × 240 ml petroleum spirit–diethyl ether (9 : 1) (fractions 2 and 3), 2 × 240 ml
petroleum spirit–diethyl ether (4 : 1) (fractions 4 and 5), 2 × 240 ml petroleum
spirit–diethyl ether (1 : 1) (fractions 6 and 7), 240 ml petroleum spirit–diethyl
ether (2 : 3) (fraction 8), 240 ml petroleum spirit–diethyl ether (1 : 4) (fraction
9), 2 × 240 ml diethyl ether (fractions 10 and 11), and 240 ml ethanol + 1%
acetic acid (fraction 12). Fraction 10 (28 mg dry weight) from this column was
further separated by gel permeation chromatography on Sephadex LH-20 (45 g;

862
MORRIS, FOSTER, AND HARRIS
Pharmacia Biotech) with methanol–dichloromethane (1 : 1) as the mobile phase
to give 14 fractions, one each of 50 ml (fraction 1), 30 ml (fraction 2) and 20
ml (fraction 3) and 11 of 10 ml (fractions 4–14). Solvents were analytical grade
(Analar, BDH Laboratory Supplies) and were distilled prior to use.
Synthetic Compounds. Synthetic samples of MBOA (2), 1-docosanol, 1-
hexacosanol, 1-heptacosanol, 1-octacosanol, and 1-triacontanol were obtained
from Sigma Chemical Co. The straight-chain primary alcohols (7–10 mg of
each) were oxidized with pyridinium chlorochromate by the method of Corey
and Suggs (1975) to give the corresponding aldehydes: 1-docosanal, 1-hexa-
cosanal, 1-heptacosanal, 1-octacosanal (1), and 1-triacontanal. These were puri-
fied by passage through a silica gel (1 g) column with a petroleum spirit–diethyl
ether (9 : 1) eluent.
Analytical Methods. Gas chromatography (GC) was performed with a
Hewlett Packard 6890 gas chromatograph with splitless injection and a flame
ionization detector. The column used was a Zebron ZB-1 capillary column (15
m × 0.32 mm ID × 0.10-mm film thickness) (Phenomenex, Torrance, California).
Helium was the carrier gas at a linear velocity of 30 cm sec. The oven was pro-
/
grammed from 80 to 280 C at 20 C min with a 1-min initial delay and a 10-min
/
final hold time. The retention times of compounds in fractions were compared
with the retention times of standard compounds, including long-chain n-alkanes,
alcohols, and aldehydes. A Hewlett Packard 5890 gas chromatograph coupled to
a VG 70-SE mass spectrometer with the same temperature program and column
as above was used for GC-MS analysis of extracts. The mass spectrometer was
run in the electron ionization mode.
Merck silica gel 60 F254 glass-backed plates (E. Merck, Darmstadt, Ger-
many) were used for analytical thin-layer chromatography (TLC). TLC plates
were developed with dichloromethane–ethanol (99 : 1) (Tulloch and Hoffman,
1971). They were sprayed with rhodamine G (Sigma Chemical Co.) and visu-
alized under 254 nm UV light.
Bioassays. The bioassay used was that described by Harris and Rose
(1990). Briefly, an extract, fraction, or synthetic chemical was dissolved in
dichloromethane, and 60 ml of the solution was applied to the top two thirds
of a grass leaf model consisting of 10-cm × 0.5-cm Whatman No. 2 filter paper
strips treated with Hansell’s yellow food coloring (Hansells Ltd., Masterton, New
Zealand). A leaf model treated with 60 ml of dichloromethane served as a con-
trol. Each filter paper strip was attached by its untreated end to a metal clip and
positioned vertically in cages (20 cm diameter × 13 cm high, blue card sides,
screen top, damp sand floor). Treated leaf models were placed in the cage in
random order in a grid formation that maximized the distances between strips
and between strips and the cage wall. Cages were held in a temperature-con-
trolled room (22 0.5 C) under a light source consisting of eight fluorescent
tubes (Biolux-B, NEC). After models were set up (11 : 00–12 : 00 hr), 15 mated

OVIPOSITIONAL STIMULANTS FOR HESSIAN FLY
863
females were placed in each cage. At 15 : 00 hr, the females were removed and
the number of eggs on each model counted by examination under a microscope.
Extracts or fractions from column chromatographic separations were tested at 4
plant equivalents (PE).
Statistical Analysis. Data from each experiment were first checked for
homogeneity of variance and normality by O’Briens test. If they failed to meet
these constraints, data were log-transformed, which in all cases allowed them to
meet the requirements for analysis by ANOVA. Data were analyzed by ANOVA,
and means compared either by Dunnett’s test for comparison with the solvent
control, or by Student’s t test, unless otherwise stated. For the experiments with
a factorial design, the data were log-transformed and analyzed by the computer
program JMP (SAS Institute, 1995), with amounts of the two compounds as the
main effects.
RESULTS
Fractionation of Wheat Extract and Identification of Compounds in Active
Fractions. The wheat leaf extract was separated by flash column chromatogra-
phy on silica gel to give 12 fractions. These were tested in a choice bioassay
to determine the ovipositional stimulatory activity of each. When the log-trans-
formed data were compared with the solvent control by Dunnett’s test, five frac-
tions had significantly greater numbers of eggs laid on them than the control.
These fractions were clustered into two groups: one at fraction 3, and the other
consisting of fractions 8–11. In the second group, fraction 10 received the great-
est number of eggs (Figure 2). GC analysis of fraction 3 (130 mg dry weight)
showed one major peak with a retention time similar to that of 1-octacosanal (1),
and a series of minor peaks corresponding to odd-numbered, C23–C33 straight-
chain hydrocarbons. The identity of compound 1 was confirmed by GC-MS anal-
ysis and comparison of its GC retention time with that of the synthetic compound
as 1-octacosanal (diagnostic ions, m z 408, 390, 380).
/
GC analysis of fractions 9, 10, and 11 showed them to have similar chro-
matographic profiles, with a large peak with a retention time similar to that of
1-octacosanol, along with an unidentified peak at 6.6 min. Fractions 6, 7, and
8 also contained a peak corresponding to 1-octacosanol and a smaller peak cor-
responding to 1-hexacosanol, but no peak at 6.6 min. As the greatest number
of eggs were laid on models treated with fraction 10 out of this group, fur-
ther chemical analysis was confined to this fraction. TLC analysis of fraction 10
showed several spots; one at Rf 0.14 was visible under UV light.
Fraction 10 from the silica gel column separation was further separated by
gel permeation chromatography on Sephadex LH-20 to give 14 fractions. These
were tested in the bioassay, which showed that the activity had been concentrated

864
MORRIS, FOSTER, AND HARRIS
FIG. 2. Mean numbers of eggs laid by female Hessian flies on filter paper leaf models
treated with one of fractions 1–12 from a silica gel column chromatographic separa-
tion of a dichloromethane extract of wheat leaves (4 PE model). Models treated with
/
dichloromethane only were the control. Means with asterisks are significantly different
*
from the control (Dunnett’s test on log-transformed data, 0.05 > P > 0.01, **0.01 > P >
0.001, ***P < 0.001). Each mean was calculated from four replicates. Bars are standard
errors of the means. Total eggs over four replicates 7521.
in fractions 12 and 13 from this column (2.7 mg combined dry weight). When
analyzed by GC, these fractions contained the compound with a retention time
of 6.6 min and again showed a TLC spot at Rf
0.14. Analysis by GC-MS
of fractions 12 and 13 from the Sephadex LH-20 separation showed that the
compound with a retention time of 6.6. min was 6-methoxy-2-benzoxazolinone
(MBOA) (2). The mass spectrum gave ions at m z (%) 165 (100), 150 (48), 122
/
(9), 106 (30), 80 (11), 52 (16), and 39 (12), in agreement with the data of Lyons
et al. (1988). This was confirmed by comparison of the GC retention time and
TLC Rf value with those of a synthetic sample of this compound.
Trimethylsilylated Wheat Extract. The GC-MS chromatogram of the
trimethylsilyl (TMS)-derivatized wheat extract contained prominent peaks for
MBOA–TMS. The mass spectrum showed ions at m z (%) 237 (72), 194 (100),
/
73 (40), and the TMS ether of 1-octacosanol, along with several minor peaks for
trimethylsilylated long-chain carboxylic acids, alcohols, and furanosides. There
were no detectable peaks present for underivatized MBOA or for the TMS
derivative of 2,4-dihydroxy 7-methoxy-2H-1,4-benzoxazin-3-one (DIMBOA-
TMS2) [mass spectrum in Atkinson et al. (1991)].

OVIPOSITIONAL STIMULANTS FOR HESSIAN FLY
865
FIG. 3. Dose–response data for numbers of eggs laid by female Hessian fly on filter
paper leaf models treated with one of five different amounts of 1-octacosanal (1). Models
treated with dichloromethane only were the control. Means with asterisks are significantly
different from the control (Dunnett’s test on log-transformed data, *0.05 > P > 0.01,
***P < 0.001). Each mean was calculated from 10 replicates. Bars are the standard error
of the mean. Total eggs over 10 replicates 3860.
Dose–Response Profiles for Compounds 1 and 2. Compound 1 was bioas-
sayed at 0.3, 3, 15, 30, and 150 mg model (Figure 3). Comparison of log-trans-
/
formed data with the control by Dunnett’s test revealed that the 3-, 15-, 30-,
and 150-mg treatments all had significantly (P < 0.05) more eggs laid on them
than the control. The number of eggs laid on models treated with 15, 30 and
150 mg model were not significantly different from each other (P < 0.05, Stu-
/
dent’s t test). This dose–response pattern showed a leveling off of activity above
a concentration around 15 mg model.
/
Compound 2 was bioassayed at 0.3, 1, 3, 10, and 30 mg model. The data
/
showed a trend of increasing numbers of eggs laid on treatments with higher
dosages of compound 2 (Figure 4), with no apparent plateau over the range
tested. Use of Dunnett’s test showed that the mean numbers of eggs laid on the
3-, 10-, and 30-mg treatments were all significantly (P < 0.05) greater than that
for the solvent control. A linear regression analysis showed a linear relationship
between the mean number of eggs laid ( y) and the amount of compound 2 in
micrograms (x) applied to each treatment [ y 5.64 ( 0.1) x + 21.3 ( 1.4); R2
0.999; F1, 4 2764.1, P < 0.0001].
Combinations of Compounds 1 and 2. It was estimated from the GC peak
areas and the weights of fractions containing compounds 1 and 2 that 1 PE of

866
MORRIS, FOSTER, AND HARRIS
FIG. 4. Dose–response data for number of eggs laid by female Hessian fly on filter paper
leaf models treated with one of five different amounts of 6-methoxy-2-benzoxazolinone
(2). Models treated with dichloromethane only were the control. Means with asterisks
are significantly different from the control (Dunnett’s test on log-transformed data, *0.05
> P > 0.01, ***P < 0.001). Each mean was calculated from 10 replicates. Bars are the
standard error of the mean. Total eggs over 10 replicates 3779.
crude extract contained approximately 35 mg of compound 1 and 1 mg of com-
pound 2. In a three-choice bioassay with a solvent control, a mixture of synthetic
1 and 2 in these amounts, and 1 PE of extract, there was no significant difference
in the mean number of eggs laid on either of the two treatments (Figure 5). Both
had significantly greater numbers of eggs laid on them than the control.
Combinations of Compounds 1 and 2—Factorial. The first factorial-design
bioassay tested combinations with or without compound 1 (15 mg or 0 mg) or
compound 2 (1 or 0 mg) (Figure 6a). The main effect of compound 1 was highly
significant (F 73.8, P < 0.0001), as was the main effect of compound 2 (F
30.5, P < 0.0001). There was a significant interaction between the two main
effects (F 8.08, P 0.007). The second experiment, in which the amount of
compound 1 was the same as in the first experiment (15 or 0 mg) and the amount
of compound 2 was increased (10 or 0 mg), gave a similar result, with both
main effects being highly significant (F 126.6, P < 0.0001 and F 52.3, P <
0.0001, respectively, for compounds 1 and 2), and again a significant interaction
between the two effects (F 11.2, P 0.002) (Figure 6b). Finally, the third
experiment, which had treatments with the higher dosage of compound 2 (10
or 0 mg) and lower dosages of compound 1 (3 or 0 mg), showed a significant
effect for compound 1 (F 19.9, P < 0.0001) and for compound 2 (F 9.45,

OVIPOSITIONAL STIMULANTS FOR HESSIAN FLY
867
FIG. 5. Mean numbers of eggs laid by female Hessian flies on filter paper leaf models
treated with 1 PE of a dichloromethane wheat extract, a mixture of synthetic compounds
1 (35 mg) and 2 (1 mg) or a solvent control. Means with different letters are significantly
different at P < 0.001 when compared using Student’s t test on log-transformed data.
Each mean was calculated from four replicates. Bars show standard errors of the means.
Total eggs over four replicates 3419.
P
0.004) (Figure 6c). However, in this experiment there was no significant
interaction between the two main effects (F 1.65, P 0.2). Thus, in the first
two experiments, the combination of compounds 1 and 2 had a synergistic effect
on egg laying, while in the third experiment, the effect was additive.
Structure–Activity Relationship of Straight Chain Primary Aldehydes. The
primary aldehydes, 1-docosanal, 1-hexacosanal, 1-heptacosanal, 1-octacosanal
(1), and 1-triacontanal were compared in a choice bioassay at a dosage of 30
mg (Figure 7). Relative to the solvent control, all of these compounds elicited
significant numbers of eggs to be laid by females (Student’s t test, P < 0.05). In
this series, the numbers of eggs laid on leaf models treated with 1-hexacosanal
or 1-heptacosanal were greater than the numbers laid on models treated with
(1) or 1-triacontanal. This indicated that the numbers of eggs laid by females
was dependent on aldehyde chain length, with maximum oviposition induced
by aldehydes with chain lengths of C26 and C27.
Activity of 1-Octacosanol. 1-Octacosanol was the most abundant compo-
nent in our wheat leaf wax extract, with each plant containing approximately 70
mg. In a choice bioassay, consisting of models treated with 0 (solvent control), 3,
30, 150, or 300 mg of this compound, there was no significant difference (Dun-

868
MORRIS, FOSTER, AND HARRIS
FIG. 6. Mean numbers of eggs laid by female Hessian flies in factorial-design experiments
with filter paper leaf models treated with compound 1, compound 2, a mixture of compounds
1 and 2, or dichloromethane-only: (a) 15 or 0 mg of 1, 1 or 0 mg of 2; (b) 15 or 0 mg of 1, 10
or 0 mg of 2; (c) 3 or 0 mg of 1, 10 or 0 mg of 2. The analysis of the data is given in the text.
Each mean was calculated from 10 replicates. Bars show standard errors of the means. Total
eggs over 10 replicates: (a) 3074, (b) 6013, (c) 3431.

OVIPOSITIONAL STIMULANTS FOR HESSIAN FLY
869
FIG. 7. Mean numbers of eggs laid by female Hessian flies on filter paper leaf mod-
els treated with one of five straight-chain primary aldehydes. Models treated with
dichloromethane only were the control. Means with different letters are significantly dif-
ferent at P < 0.05 when compared by Student’s t test on log-transformed data. Each mean
was calculated from 10 replicates. Bars show standard errors of the means. Total eggs
over 10 replicates 8457.
nett’s test, P < 0.05) between egg numbers on the solvent control model and
treated models (Figure 8).
DISCUSSION
In this study we identified two compounds from the epicuticular wax of
wheat leaves that stimulate oviposition by Hessian flies: 1-octacosanal (1) and 6-
methoxy-2-benzoxazolinone (MBOA) (2). When applied to leaf models, a mix-
ture of these compounds, containing approximately the amounts in one PE of a
dichloromethane extract of wheat leaves, stimulated female Hessian flies to lay
the same number of eggs as did one PE of crude extract. This suggests that these
two compounds accounted for a high proportion of the stimulatory activity of
the leaf extract.
The leaf waxes of various wheat varieties have been the subject of consid-
erable investigation (Tulloch and Hoffman, 1971, 1973; Bianchi and Corbellini,
1977). In general, alcohols are the most abundant class of compound in wheat
leaf waxes, with octacosanol typically the most abundant alcohol (Tulloch and
Hoffman, 1973; Bianchi and Corbellini, 1977; Bianchi and Figini, 1986; Bianchi

870
MORRIS, FOSTER, AND HARRIS
FIG. 8. Dose–response data for numbers of eggs laid by female Hessian fly on filter paper
leaf models treated with one of four different amounts of 1-octacosanol. Models treated
with solvent only (dichloromethane) served as controls. None of the treatment means
were significantly different from the control mean (Dunnett’s test on log-transformed
data). Each mean was calculated from 10 replicates. Bars show standard errors of the
mean. Total eggs over 10 replicates 860.
et al., 1980). However, octacosanol, at the level found in our wheat extracts did
not stimulate oviposition by Hessian fly. Aldehydes, predominantly 1, are less
abundant than alcohols in wheat waxes (Bianchi and Corbellini, 1977; Bianchi
and Figini, 1986), with the proportion of aldehyde in the wax decreasing as
the plant matures. Moreover, aldehydes are relatively more abundant in younger
wheat leaves than in more mature ones (Bianchi et al., 1979). This corresponds
with the observed preferences of female Hessian flies (Harris and Rose, 1989),
suggesting that levels of 1 in the wax may be a factor in the ovipositional pref-
erence of female flies for young over older leaves.
When we compared the egg-laying responses of females to aldehydes with
different chain lengths, their responses were not greatest to 1, but to 1-hexa-
cosanal and 1-heptacosanal, which we did not detect in our wheat extracts. How-
ever, 1-hexacosanal may comprise up to 20% of the aldehyde complement in the
leaf wax of certain wheat cultivars (Bianchi and Figini, 1986) and is the major
aldehyde of both rye (Bianchi, 1985) and barley (von Wettstein-Knowles, 1974).
From our results, it is apparent that the chain length, as well as the abundance
of the aldehydes present in a leaf wax, influences Hessian fly oviposition pref-
erences for different wheat varieties and grasses.
Benzoxazolinones are the decomposition products of 1,4-benzoxazin-3-

OVIPOSITIONAL STIMULANTS FOR HESSIAN FLY
871
ones, which have been isolated from wheat, maize (Zea mays), rye, and species
in several other genera in the Gramineae, but few species outside this family
(Niemeyer, 1988). In solution, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one
(DIMBOA) decomposes to give MBOA with release of formic acid (Bravo and
Niemeyer, 1985). DIMBOA is the major hydroxamic acid found in wheat tis-
sue (Zuniga and Massardo, 1991), with MBOA, apparently present in 100-fold
´ ˜
lower concentration (Leszczynski and Dixon, 1990). To determine whether the
MBOA identified in our dichloromethane extracts originated from wheat leaf
wax or from decomposition of DIMBOA during storage and fractionation, we
derivatized an ethyl acetate extract with BSTFA to give TMS derivatives. The
BSTFA was added within 15 min of extraction, before significant decomposi-
tion of DIMBOA to MBOA could occur (Bravo and Niemeyer, 1985). GC-MS
analysis of the derivatized extract showed a peak for MBOA–TMS, but lacked a
detectable peak for DIMBOA–TMS2, indicating that MBOA is relatively more
abundant in the leaf wax of wheat.
Following the finding that MBOA is not involved in host location by west-
ern corn rootworm larvae (Bjostad and Hibbard, 1992; Bernklau and Bjostad,
1998), this appears to be the first report of a benzoxazolinone being used in
host identification by an insect.
MBOA (2) and DIMBOA, its precursor in plants, are known to be toxic and
have antifeedant properties to many species of insects (see examples in Bjostad
and Hibbard, 1992). Nothing is known about the toxicity of these compounds
to Hessian fly larvae. MBOA also has some, albeit limited, volatility. The host
selection behavior of the female Hessian fly is influenced by volatile compounds
from wheat leaf wax extracts (Foster and Harris, 1992; Rani and Harris, unpub-
lished). Whether MBOA (2) influences host selection by female Hessian flies
over a distance or only upon contact will be the subject of behavioral studies.
Our results from the wheat cultivar Otane are in accord with a previous,
preliminary investigation into the oviposition stimulants in the epicuticular leaf
wax of the wheat cultivar Newton (Foster and Harris, 1992). Fractionation of the
leaf waxes of Newton on silica gel also resulted in two active fractions, elut-
ing with solvents of similar polarity to those of the Otane extract. Given that
the leaf waxes of different wheat varieties largely contain the same compounds
(Bianchi and Corbellini, 1977; Bianchi et al., 1980; Bianchi and Figini, 1986),
it is reasonable to assume that the activity in the two fractions of Newton was
due, at least in part, to the same two compounds, or class of compounds, as in
Otane. However, in Newton, as opposed to Otane, the later-eluting active frac-
tion had approximately equal activity to the earlier fraction. This suggests that
Newton wax contained relatively higher amounts of benzoxazolinone(s) com-
pared to aldehyde(s) than did Otane. Such variation could be the basis for a
chemical mechanism for the differential preferences of the female Hessian fly
toward different wheat varieties (Harris et al., 1996).

872
MORRIS, FOSTER, AND HARRIS
In the previous study of the cultivar Newton, the combination of the two
most active fractions had a large synergistic effect on egg-laying. In the present
study a similar synergistic effect on Hessian fly egg-laying responses was appar-
ent when compounds 1 and 2 were combined. The magnitude of this effect was
dependent on the amounts of these two compounds applied to leaf models and
was greater for treatments with a larger amount of compound 2 (amount of com-
pound 1 kept constant). However, if the level of compound 1 was below that
which was active when tested alone in the dose–response experiment, a syner-
gistic effect was not apparent. The larger synergistic effect observed with the
combination of fractions of Newton extract may have been due to differing con-
centrations of one or both of these compounds, the presence of other, unidentified
stimulatory compounds, or to differences in host chemical preferences between
the New Zealand and Kansas strains of Hessian fly that were used in the two
studies.
The identification of these compounds should be useful for assessing the
host preferences of female Hessian flies toward different wheat varieties and
could be of value in wheat breeding programs aiming to breed varieties resis-
tant to the Hessian fly. Currently, antibiosis is the major mechanism selected for
when breeding Hessian fly resistant wheat. A possible alternative mechanism
for resistance to Hessian fly is antixenosis, which could be achieved by select-
ing for low production of these two compounds. The levels of these compounds
in different wheat varieties and hosts will be explored in future studies.
Acknowledgments—We wish to thank Manoharie Sandanayake and Dr. Hiroo Kanno for rear-
ing and supplying the insects. We are also grateful to Dr. Lou Bjostad for critical review of the
manuscript and helpful suggestions on MBOA derivatization. Thanks to Drs. Richard Newcomb
and Howard Waring for comments on the draft manuscript. This work was funded in part by the
New Zealand Foundation for Research, Science and Technology (contract number CO 2806).
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