Synthesis of β-damascone derivatives with a lactone ring and their feeding deterrent activity against aphids and lesser mealworms

Article abstract of DOI:10.1039/c4ra03939d

Starting from β-damascone, six new lactones were obtained. The Claisen-Johnson rearrangement of allylic alcohol and halolactonization of γ,δ-unsaturated acid were the key steps of the presented synthesis. The structures of the new derivatives were determined by spectroscopic data. The antifeedant activity of β-damascone towards two insect species with different feeding habits and food preferences, i.e., the peach-potato aphid Myzus persicae (Sulz.) and lesser mealworm, Alphitobius diaperinus Panzer, was studied, as well as the biological consequences of structural modification of the starting substrate. The successive structural modifications of β-damascone that resulted in the greatest antifeedant activity towards M. persicae were the incorporation of a lactone moiety and concomitant presence of bromine in the side chain. All β-damascone derivatives with a lactone moiety deterred the feeding of adults and larvae of A. diaperinus. Halo-δ-lactones were more active than halo-γ-lactones, and A. diaperinus adults were more sensitive to the compounds studied than larvae.

Full text of DOI:10.1039/c4ra03939d

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Synthesis of b-damascone derivatives with a
lactone ring and their feeding deterrent activity
Cite this: RSC Adv., 2014, 4, 39248
against aphids and lesser mealworms
a
A. Gliszczynska, K. Dancewicz,b M. Hnatejko,c M. Szczepanikc and B. Gabrysb
*
´
´
Starting from b-damascone, six new lactones were obtained. The Claisen–Johnson rearrangement of allylic
alcohol and halolactonization of g,d-unsaturated acid were the key steps of the presented synthesis. The
structures of the new derivatives were determined by spectroscopic data. The antifeedant activity of b-
damascone towards two insect species with different feeding habits and food preferences, i.e., the
peach-potato aphid Myzus persicae (Sulz.) and lesser mealworm, Alphitobius diaperinus Panzer, was
studied, as well as the biological consequences of structural modification of the starting substrate. The
successive structural modifications of b-damascone that resulted in the greatest antifeedant activity
towards M. persicae were the incorporation of a lactone moiety and concomitant presence of bromine
in the side chain. All b-damascone derivatives with a lactone moiety deterred the feeding of adults and
larvae of A. diaperinus. Halo-d-lactones were more active than halo-g-lactones, and A. diaperinus adults
were more sensitive to the compounds studied than larvae.
Received 29th April 2014
Accepted 13th August 2014
DOI: 10.1039/c4ra03939d
www.rsc.org/advances
pests and can be useful in the protection of crops. Insects are
responsive to many plant lower terpenoids and their synthetic
Introduction
derivatives. For example, the repellent properties of linalool and
a-terpineol to the peach-potato aphid Myzus persicae were
reported by Hori,12,13 and (S)-limonene restrained phloem sap
ingestion and had other negative effects on the behaviour of
this aphid.14 Citral, linalool, (S)-limonene, a-ionone, and
camphene reduced the total and mean probing time of aphids
and their settling on leaves.15 Following these studies, several
analogues of natural terpenoids, including the lactones, have
been synthetized and their biological activity examined. We
discovered active feeding deterrents among lactones derived
from natural isoprenoids: pulegone,16,17 piperitone,18 and far-
nezol.19 It appeared that the antifeedant activity of synthetic
analogues was highly enantiospecic and depended on various
substituents and functional groups.14,18,20 Chemical trans-
formation of the piperitone molecule by the introduction of a
lactone moiety and a halogen atom strongly increased its anti-
feedant properties against M. persicae and the lesser mealworm
Alphitobius diaperinus.18,21
In the present study, we concentrated on two insect pests
with different feeding habits and food preferences, i.e., the
peach-potato aphid Myzus persicae (Sulz.) (Hemiptera: Aphidi-
dae) and the lesser mealworm, Alphitobius diaperinus Panzer
(Coleoptera: Tenebrionidae). Aphids are responsible for at least
2% of all crop losses attributed to insect feeding.22 Moreover,
the indirect damage caused by aphids due to virus transmission
exceeds their direct impact on crops.23 M. persicae alone can
infest plants of over 40 different families and it is able to
transmit over 100 plant viruses.24 At the same time, it developed
b-Damascone (1) belongs to the group of norisoprenoids
dened as rose ketones, because they were discovered in the
1960s during a quest to identify the characteristic smell of
Bulgarian rose oil.1,2 This compound has been identied not
only as a component of rose oil but it also creates tea aroma and
occurs in some types of tobacco, wine, and whiskey.3–7 b-Dam-
ascone (1) has a pleasant blackcurrant/plum note and is widely
used in perfume compositions. Apart from this commercial
importance, b-damascone has been shown to have a variety of
biological activities. For example, it was discovered that this
compound possesses cancer chemopreventive potential8 and
appeared to be toxic towards three species of mosquitoes, Aedes
aegypti L., Aedes albopictus (Skuse), and Anopheles quad-
rimaculatus Say, the housey, Musca domestica L., the stable y,
Stomoxys calcitrans L., and the sand y, Lutzomyia shannoni
(Dyar).9–11 Apart from that, information on the effect of b-dam-
ascone (1) on arthropods and their behavior is very scarce. Our
interests in the synthesis of isoprenoid lactones with a dam-
ascone skeleton were motivated by the search for new biologi-
cally active compounds that can reduce the population of insect
aDepartment of Chemistry, Wroclaw University of Environmental and Life Sciences,
Norwida 25, 50-375 Wrocław, Poland. E-mail: anna.gliszczynska@wp.pl; Fax: +48
71 3284124; Tel: +48 71 3205257
b
´
Department of Biology and Ecology, University of Zielona Gora, Szafrana 1, 65-516
´
Zielona Gora, Poland
cNicolaus Copernicus University, Department of Invertebrate Zoology, Lwowska 1, 87-
´
100, Torun, Poland
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clones that were resistant to one or more insecticides.23 The aluminium hydride according to standard procedure. The
lesser mealworm is a cosmopolitan pest inhabiting chicken and reaction course and purity of the product were monitored by GC
broiler houses in vast numbers. In poultry houses, the beetles on a capillary column (HP-5). Known dihydro-b-damascone (2),
consume manure, spilled feed, dead birds, and other organic which was identied in tobacco31 and previously synthesized by
materials.25,26 At the same time, the adults and larvae have been Mori et al. in a two-step chemical synthesis from trimethylcy-
reported as potential vectors for the transfer of Campylobacter clohexanone,32 was obtained in high 99% yield. The structure of
jejuni and Salmonella enterica between successive broiler 2 was conrmed by the comparison of its spectral data with
ocks27 and other pathogens.28
previously published data.
Chemicals targeting insect taste receptors are considered
Ketone (2) was next transformed in high 89% yield into the
potential bioinsecticides to protect crops.29 Aphids possess corresponding allylic alcohol, dihydro-b-damascol (3), by treat-
piercing–sucking mouthparts, and they feed on the phloem sap ment with LiAlH4. Preparation of dihydro-b-damascol (3) was
of living plants, while the lesser mealworm is a general stored- patented for the rst time in 1973.33 The crude alcohol (3),
products pest that feeds using chewing mouthparts. Moreover, without further purication (97% purity according to the GC
aphids lack external taste receptors, which makes the pre- analysis), was subjected to the orthoacetate modication of the
ingestional rejection or acceptance of the food impossible.30 In Johnson–Claisen rearrangement.34 The mechanism involves the
contrast, the lesser mealworm's gustatory receptors are located esterication of alcohol followed by subsequent elimination of
on their mouthparts, which allows a preingestional response to ethanol to form ketene acetal. The latter is rearranged to the g,d-
food quality. The different food preferences, modes of food unsaturated esters in a [3.3] sigmatropic shi during heating. In
uptake, and preingestional evaluation ability of food chemistry our case, the reaction afforded the new compound ethyl 2-(2-
may determine different behavioural responses of these insects. butylidene-1,3,3-trimethylcyclohexyl)-acetate (4) in 98% yield.
In the present study, we present the synthesis and structural An absorption band at 1743 cmꢀ1 of the carbonyl group and
modications of b-damascone (1)-derived compounds, characteristic quartet in the 1H NMR spectrum from two
including the lactones, as well as their biological activities protons at 4.05 ppm conrmed the presence of the carboethoxy
expressed as antifeedant properties that affect M. persicae and A. group in the ester (4).
diaperinus.
The ester (4) was subsequently hydrolyzed in ethanolic KOH
solution to the corresponding acid (5) in 85% yield. This
compound has not been obtained before. In the next step, acid
(5) was subjected to halolactonization reactions, giving access to
bromo- and chlorolactones (Scheme 1). The bromolactonization
of acid (5) was carried out with N-bromosuccinimide (NBS) in
tetrahydrofuran, affording a mixture of two new products in a
ratio of 45% : 55% according to GC. We separated them using
column chromatography and established their structure on the
basis of spectroscopic data. The products of cyclization were d-
bromo-g-lactone (6) (minor, 17% yield) and g-bromo-d-lactone
(7) (major, 27% yield). The reaction of g,d-unsaturated acid (5)
with N-chloro-succinimide (NCS) was carried out to obtain
chlorolactones. Using chloride as an electrophilic agent, we
observed a similar situation as in the process of bromolacto-
nization. Two new lactones were formed as products of cycli-
zation – d-chloro-g-lactone (8) and g-chloro-d-lactone (9).
According to GC, the mixture consists of 41% of g-lactone (8)
and 59% d-lactone (9), which were obtained with 18 and 17%
yield respectively. The doublet of doublets protons H-8 in g-
lactone (4.24 ppm) and d-lactone (4.74 ppm) were found in the
1H NMR spectra and also proved that intermolecular cyclization
occurred.
Results and discussion
Chemical synthesis
Six new isoprenoid lactones 6–11 were obtained in a six-step
synthesis (Scheme 1) from the naturally occurring ketone, b-
damascone (1). The damascone carbon skeleton consists of a
trimethyl substituted cyclohexane ring with a four-carbon a,b-
unsaturated ketone side chain. The starting material was
commercially available b-damascone (1).
The rst step of the synthesis was the reduction of the double
bond in the side chain of compound (1) with lithium
The dehydro-halogenation reaction of d-halo-g-lactones
(6),(8) and g-halo-d-lactone (7),(9) with 1,8-diazabicyclo[5.4.0]
undec-7-ene (DBU) gave new cyclic lactones (10),(11) in good
yields (47–57%) as the only products (Scheme 1). The formation
of unsaturated lactone (10) as the only product of the dehy-
drohalogenation of lactones (6),(8) is a result of E2 elimination.
In the IR spectrum of unsaturated lactone (10), absorption
bands for stretching vibrations for the carbonyl group at 1778
cmꢀ1 were observed. The location of a double bond in the side
chain of bicyclic lactone (10) between C-8 and C-9 was
Scheme 1 Synthesis of lactones from b-damascone. Reagents (i)
LiAlH4; (ii) CH3C(OEt)3, CH3COOH, 138 ꢁC; (iii) 1. KOH, EtOH, 2. HCl;
(iv) NBS/NCS, THF; (v) DBU.
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Biological studies
Feeding deterrent activity against Myzus persicae
The potency and mechanism of antifeedant activity of the
compounds studied varied depending on the compound
structure and the duration of exposure of aphids to b-dam-
ascone (1) and its derivatives. On control leaves, aphids did not
leave the substrate (¼leaf) during the no-choice 15 minute
experiment, and the total probing time was approximately 70%
of the time spent on the leaf. The aphids started probing
immediately aer exposure (the delay was 14 seconds on
average), and the average probe was relatively long, i.e.,
approximately 3.5 minutes (Table 1). In the choice-test, there
were no signicant differences in aphid preferences to settle on
either the untreated or the control (i.e., ethanol-treated) leaf,
irrespective of the exposure time (Fig. 2). b-Damascone (1)
appeared to be a weak attractant during the initial contact with
the treated leaves in the no-choice test, as it was observed that
although the rst probe was delayed in comparison to the
control, further probing was rarely interrupted. There were
twice as few probes, and the probes were twice as long in
comparison to aphids on control leaves (Table 1). However, this
effect did not translate into aphid preferences during settling,
as no signicant differences in the number of aphids on treated
Fig. 1 COSY spectrum of lactone 11.
conrmed by the signals from olen protons in the 1H NMR
spectrum. The doublets of triplets from H-8 and H-9 protons, at
5.60 ppm and 5.77 ppm, respectively, indicate the presence of
double bonds. Moreover, the coupling constant J ¼ 15.5 Hz
between these protons proved their trans orientation. The shi
of the absorption band of the C]O band in the lactone moiety
from the range of 1716–1733 cmꢀ1 for d-lactones to a higher
frequency of 1762 cmꢀ1 indicates that in the case of product
(11), the conversion of the d-lactone ring into g-lactone
occurred. In the 1H NMR spectrum, the signal from proton H-8
appeared as a doublet of doublets. It was coupled with protons
at C-9 at 4.25 ppm with coupling constant J ¼ 10.9 and 2.2 Hz.
These couplings are clearly observed in the 2D COSY spectrum
(Fig. 1). This type of cyclization is the result of E2 elimination.
The lack of signal from one of the CH2-3 protons as well as any
signals characteristic for olenic protons and nally the shape
of the signal from the H-8 proton indicated the presence of the
cyclopropane ring. Its formation can be explained by expulsion
of the proton at C-3 by DBU, leading to carbanion stabilization
by the enolate anion. The second step, cyclization, is the result
of the attack of the electron pair as a nucleophile on the C-7a
carbon atom with simultaneous removal of halogen.35
Fig. 2 The effect of b-damascone and its derivatives on settling
preferences of Myzus persicae in the choice test. The data are
expressed as values of indices of deterrence (DI). The standard error is
indicated on the bar. *P < 0.05; ***P < 0.001; (Student's t-test).
Table 1 Modification of Myzus persicae behaviour by b-damascone and its derivatives in nochoice testsa
Compound
Time spent on the leaf (s)
Total probing time (s)
Time to rst probe (s)
Number of probes
Mean probing time (s)
Control
900.0 ꢂ 0.0
892.5 ꢂ 7.5
844.5 ꢂ 47.9
809.5 ꢂ 51.3
867.2 ꢂ 30.0
892.8 ꢂ 7.2
539.0 ꢂ 117.0*
900.0 ꢂ 0.0
799.8 ꢂ 68.6
887.2 ꢂ 9.1
888.6 ꢂ 10.4
608.6 ꢂ 51.8
640.6 ꢂ 44.1
575.8 ꢂ 79.3
457.3 ꢂ 68.0
467.5 ꢂ 78.7
585.8 ꢂ 68.4
256.6 ꢂ 80.8*
707.5 ꢂ 36.4
545.6 ꢂ 76.8
635.4 ꢂ 68.0
640.1 ꢂ 102.1
13.7 ꢂ 6.3
123.1 ꢂ 37.2*
51.5 ꢂ 28.5
14.8 ꢂ 3.0
5.4 ꢂ 0.9
2.4 ꢂ 0.5*
6.2 ꢂ 1.3
7.7 ꢂ 0.9
4.1 ꢂ 0.9
5.8 ꢂ 1.0
4.5 ꢂ 1.0
4.3 ꢂ 0.7
4.3 ꢂ 0.8
5.4 ꢂ 1.0
3.3 ꢂ 0.6
209.8 ꢂ 69.7
435.7 ꢂ 90.1*
194.8 ꢂ 65.9
77.7 ꢂ 22.5*
204.2 ꢂ 66.6
189.6 ꢂ 76.7
43.9 ꢂ 15.8*
249.3 ꢂ 70.8
264.5 ꢂ 93.2
208.9 ꢂ 47.5
335.8 ꢂ 102.7
1
2
3
4
5
6
7
8
9
10
154.2 ꢂ 50.0*
12.5 ꢂ 5.0
104.7 ꢂ 64.4
25.7 ꢂ 7.8
27.4 ꢂ 7.2
22.7 ꢂ 6.1
26.9 ꢂ 11.6
a
Values represent means of n ¼ 12 replicates ꢂSE. An asterisk within a column denotes statistically signicant differences in relation to control
(P < 0.05).
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and untreated leaves were found during the 24 hour experiment reduction of total time spent on the leaf, total probing time (d-
(Fig. 2). bromo-g-lactone (6)), delayed time to the rst probe (b-dam-
The compound synthesized in the rst step in the b-dam- ascone (1) and ester (4)), and the reduction of mean probing
ascone modication, the dihydro-b-damascone (2), did not time (dihydro-b-damascol (3) and d-bromo-g-lactone (6)).
evoke any changes in aphid behaviour during initial contact Considering average durations of probes (0.7–1.2 minutes),
with the treated leaves (no-choice test) as well as in the long- aphid stylets on (3)- and (6)-treated leaves did not penetrate
term experiment showing aphid preferences during settling beyond the epidermis.36 The delayed effects were expressed
(choice-test). Similar results were found aer the application of aer longer times of aphid exposure to the compounds studied.
dihydro-b-damascol (3), except that the probes were signi- The strongest and the most durable effects on aphid settling
cantly four times shorter in comparison to the control. were evoked by the application of ethyl 2-(2-butylidene-1,3,3-
However, aphids did not restrain from probing – the total trimethyl-cyclohexyl)-acetate (4), d-bromo-g-lactone (6), and
probing time was comparable to the control, which nally unsaturated bicyclic g-lactone (10). Moreover, the deterrent
caused no discrimination between treated and untreated leaves effect increased in potency over the course of time. With the
during the 24 hour settling experiment. The b-damascone- exception of d-bromo-g-lactone (6), the effects of ester (4) and
derived ethyl 2-(2-butylidene-1,3,3-trimethylcyclohexyl)-acetate lactone (10) were most likely only post-ingestional, because the
(4) did not cause signicant differences in aphid responses to impediment to settling occurred later than two hours aer
plants during the initial 15 minutes aer exposure, except for exposure. Partial pre-ingestional and ingestional/post-
considerable delay before the rst probe (Table 1).
ingestional deterrent activity was observed with d-bromo-g-
From the second hour aer exposure onwards, the aphids lactone (6), as the aphids were discouraged from probing
showed a signicant preference for untreated leaves. The immediately aer they had gained access to plants but the
indices of deterrence reached relatively high values of 0.5 and probing was not entirely eliminated, which most likely allowed
0.6 aer 2 and 24 hours, respectively (Fig. 2). Further molecular the consumption of plant sap.
modication by the synthesis of 2-(2-butylidene-1,3,3-
trimethylcyclohexyl) acetic acid (5) did not cause any change
in the biological activity towards M. persicae initially, but aer
Feeding deterrent activity against Alphitobius diaperinus
24 hour exposure, aphid settling was signicantly hindered
(ID ¼ 0.2). In contrast, the incorporation of the lactone moiety
and the halogen atoms into the molecule had signicant impact
on aphid behaviour. However, the four halolactones obtained
(6–9) differed in activity depending on the size of the lactone
ring and the kind and position of the halogen atoms in the
molecule. In the behavioural no-choice test, the application of
d-bromo-g-lactone (6) caused a signicant decrease in the total
time spent on the treated leaves and total and mean probing
time, while the g-bromo-d-lactone (7) did not (Table 1). Conse-
quently, the exposure to d-bromo-g-lactone (6) resulted in the
avoidance of treated leaves by freely moving aphids in the
choice-test, from the beginning until the end of the experiment
in contrast to g-bromo-d-lactone (7) (Fig. 2). The replacement of
the bromine atom by a chlorine atom to synthesize the
respective d-chloro-g-lactone (8) and g-chloro-d-lactone (9) did
not cause changes in aphid behaviour during initial contact
with the studied compounds (Table 1). However, long-term
exposure to g-chloro-d-lactone (9) signicantly impeded aphid
settling (Fig. 2). Similar effects on aphid settling and behaviour
were caused by unsaturated bicyclic g-lactone (10) and bicyclic
d-lactone (11): aphid initial responses on treated leaves did not
differ from those on control leaves in the no-choice experiment
(Table 1) but the settling of aphids was signicantly impeded
aer longer exposure times. The settling-deterrent effect of
lactones (10) and (11) increased in potency over the course of
time, with indices of deterrence reaching 0.7 and 0.3 aer 24
hours for lactones (10) and (11), respectively (Fig. 2).
The compounds studied exhibited varying antifeedant activity,
which depended on the structure of the compound and the
developmental stage of the lesser mealworm. Starting b-dam-
ascone (1) was a moderate feeding deterrent for both develop-
mental stages, especially in the no-choice test. The food
consumed by the larvae and adults in this test represented 59.26
and 51.5% of the consumption in the control, respectively
(Fig. 3). The introduction of a lactone moiety into a b-dam-
ascone molecule changed its antifeedant properties.
Unsaturated g-lactone (10) and bicyclic d-lactone (11) were
excellent feeding deterrents, but only against adults. In bioas-
says with larvae, strong activity in the choice test was observed.
Unfortunately, in the no-choice tests, larvae intensively ate the
treated food, as in the case of unsaturated lactone (10) (Table 2).
Fig. 3 The effect of b-damascone and its derivatives on the feeding of
Alphitobius diaperinus in the no-choice test. The data are expressed as
percentages of control consumption. The standard error is indicated
on the bar. *P < 0.05; ***P < 0.001; NS: not significant; (Student's t-
In summary, the antifeedant activity of b-damascone-derived
compounds manifested as both immediate and delayed effects
on aphid behaviour. The immediate effects during initial
contacts with the allelochemical were expressed as the test).
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Table 2 Feeding deterrent activity of the studied compounds in choice and no-choice tests against A. diaperinus
Deterrence coefficients ꢂ SEa
Larvae
Adults
Compound
A
R
T
A
R
T
Dose
1
2
3
4
5
6
7
8
1%
30.70 ꢂ 15.94 abc
25.30 ꢂ 6.80 ab
62.41 ꢂ 9.69 c
33.90 ꢂ 1.20 abc
8.47 ꢂ 4.85 ab
33.58 ꢂ 2.69 c
82.45 ꢂ 11.50 d
67.04 ꢂ 4.36 c
85.71 ꢂ 5.30 d
-2.08 ꢂ 4.74 a
36.01 ꢂ 2.07 b
55.59 ꢂ 3.41 a
86.30 ꢂ 17.37 a
95.99 ꢂ 9.24 a
139.12 ꢂ 6.4 ab
115.64 ꢂ 3.32 ab
98.89 ꢂ 3.84 a
111.38 ꢂ 3.67 ab
162.64 ꢂ 1.83 b
164.81 ꢂ 10.56 b
184 ꢂ 4.29 b
34.74 ꢂ 12.00 a
57.22 ꢂ 2.05 ab
75.47 ꢂ 4.12 b
46.53 ꢂ 15.52 a
50.74 ꢂ 7.85 a
50.34 ꢂ 1.63 a
90.13 ꢂ 7.63 b
72.94 ꢂ 1.29 b
78.70 ꢂ 3.82 b
71.24 ꢂ 2.43 b
72.24 ꢂ 4.60 b
56.94 ꢂ 4.94 ab
63.20 ꢂ 5.93 ab
59.34 ꢂ 9.29 ab
65.59 ꢂ 11.02 b
81.05 ꢂ 17.72 b
23.20 ꢂ 0.30 a
91.05 ꢂ 1.38 b
89.48 ꢂ 10.36 b
64.69 ꢂ 8.23 b
77.45 ꢂ 2.60 b
81.27 ꢂ 4.13 b
91.68 ꢂ 10.52 ab
120.42 ꢂ 6.68 abc
134.81 ꢂ 12.81 bcd
112.13 ꢂ 16.07 abc
131.79 ꢂ 4.59 bcd
73.54 ꢂ 20.70 a
70.69 ꢂ 4.46 b
76.71 ꢂ 3.60 bc
81.74 ꢂ 4.55 bcde
90.42 ꢂ 1.88 cdef
77.80 ꢂ 3.11 bcd
80.19 ꢂ 1.17 bcde
97.77 ꢂ 0.53 f
181.17 ꢂ 1.64 d
162.42 ꢂ 8.72 cd
143.38 ꢂ 10.34 bcd
148.69 ꢂ 9.58 cd
153.5 ꢂ 2.87 cd
9
10
11
98.29 ꢂ 2.96 f
91.43 ꢂ 1.07 def
93.77 ꢂ 2.77 ef
89.35 ꢂ 7.42 a
129.79 ꢂ 5.21 ab
Dose
0.5%
7
8
9
29.78 ꢂ 14.53 a
29.99 ꢂ 6.83 a
57.12 ꢂ 2.47 a
84.61 ꢂ 4.97 b
84.22 ꢂ 6.03 ab
65.62 ꢂ 11.96 a
114.39 ꢂ 16.06 ab
114.21 ꢂ 8.75 a
122.74 ꢂ 12.22 b
82.45 ꢂ 8.84 ab
85.53 ꢂ 1.02 b
64.28 ꢂ 3.10 a
66.95 ꢂ 2.44 a
61.61 ꢂ 8.90 a
63.24 ꢂ 8.86 a
149.40 ꢂ 11.06 a
147.14 ꢂ 9.46 a
127.53 ꢂ 9.43 a
Dose
0.1%
NT
NT
7
8
9
NT
NT
NT
NT
NT
NT
51.91 ꢂ 10.71 a
65.71 ꢂ 24.34 a
47.56 ꢂ 5.07 a
50.67 ꢂ 14.70 a
31.99 ꢂ 8.74 a
62.65 ꢂ 14.87 a
102.58 ꢂ 20.2 a
97.70 ꢂ 27.43 a
110.2 ꢂ 17.46 a
NT
a
Values are the means of the four replicates, each set up with ten larvae or adults (n ¼ 40). A: absolute coefficients; R: relative coefficients; T: total
coefficients. NT: Not tested. Means followed by the same letters within each column and for each concentration are not signicantly different one-
way ANOVA and Tukey’s test (P < 0.05).
A signicant decrease in the feeding of both developmental These particularly active compounds, i.e., g-bromo-d-lactone (7)
stages of A. diaperinus in the presence of halolactones was and both chlorolactones (8, 9), were also very strong anti-
observed. The bromolactone activity was related to the size of feedants against adults at the lower dose of 0.5%. For larvae,
the lactone ring. A much stronger antifeedant for both devel- their activity was poor except for chlorolactone (9) in the no-
opmental stages of the lesser mealworm was g-bromo-d-lactone choice test. These compounds used as 0.1% solutions also
(7) in comparison with d-bromo-g-lactone (6). The bromo- affected the feeding of adults. According to the classication of
lactone with the larger ring was the most potent antifeedant for Nawrot et al., they can be considered as good antifeedants.37
adults among the studied compounds, with only 5.22% Our experiments show that the halogen atoms are responsible
consumption compared with the control. Its activity is compa- for the high antifeedant activity of lactone derivatives of b-
rable with that of the most active known antifeedant, azadir- damascone (1).
achtin.17 This compound also strongly reduced the larval
A dramatic reduction of consumption of food treated with
feeding in relation to the control, which was 9.69% in the no- halolactones obtained from piperitone was also observed. In the
choice test (Fig. 3). When a chlorine atom replaces a bromine presence of the above-mentioned compounds, the food
atom, d-chloro-g-lactone (8) is obtained, which results in an consumed by lesser mealworm adults in the no-choice test
increase of activity. Compared with d-bromo-g-lactone, d- represented only 2.16–2.46% of the consumption of the
chloro-g-lactone with a smaller ring reduced larval feeding by a control.21 Halogen atoms also modify the antifeedant activity of
factor of three and adult feeding by a factor of two (Fig. 3). the other monoterpenes. For instance, the number and kind of
Generally, chlorolactones (8, 9) strongly reduced insect feeding, halogen (Cl or Br) substituents in the cyclohexane ring of cyclic
but for larvae, the best antifeedant was the chlorolactone with a monoterpenes isolated from Plocamium cartilagineum signi-
larger ring. Its activity was similar to the activity of azadirachtin. cantly affect the activity of the obtained derivatives towards the
In the case of adults, this compound (9) was somewhat weaker chrysomelid, Leptinotarsa decemlineata L. and two aphid
as a feeding deterrent in comparison with d-chloro-g-lactone species, Myzus persicae Sulz and Ropalosiphum padi L.38 The
(8), but these differences were not signicant, especially in the menthol derivatives with halogen substituents show a much
no-choice test (Table 2). Considering the results of the no- stronger activity against mosquitoes than the starting product.39
choice tests, the strongest antifeedants for both develop- Among intermediate products of synthesis of lactones from b-
mental stages were halolactones with a larger ring (Fig. 3). damascone (1), a good antifeedant was dihydro-b-damascol (3),
39252 | RSC Adv., 2014, 4, 39248–39256
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ꢁ
ꢁ
which reduced feeding in the no-choice tests by 75.52% for was as follows: injector 250 C, detector (FID) 250 C, column
larvae and 85.83% for adults (Fig. 3). Adult feeding reduction of temperature: 100 ꢁC, 100–300 ꢁC (rate 30ꢁ minꢀ1), 300 ꢁC (hold 2
over 60% was also observed in the presence of the ester (4) and min).
1
1
acid (5). The results showed a signicantly higher sensory
sensitivity of A. diaperinus adults in comparison with larvae. NMR, 13C NMR, DEPT 135, HMQC and 1H–1H COSY spectra
1H NMR, 13C NMR, DEPT 135, HMQC and H–1H COSY. H
¨
Taking into account the total deterrence coefficients among the were recorded in CDCl3 solutions on a Bruker Avance AMX 300
11 compounds, only bromolactone (6) was a clearly weaker spectrometer.
deterrent for adults (Table 2).
IR spectra. IR spectra were determined using a Mattson IR
300 Thermo-Nicolet spectrophotometer with KBr pellets or as
neat.
Melting points. Melting points (uncorrected) were deter-
mined on a Boetius apparatus.
Conclusion
The deterrent activity of individual b-damascone derivatives
varied in potency, time of expression, and duration of effect,
depending on the spatial structure of the lactone and species/
developmental stage of the insect.
The indexes of refraction. The indexes of refraction were
measured on a Carl Zeiss Jena refractometer.
The most successive structural modications of b-dam-
ascone in terms of antifeedant activity towards M. persicae were
the incorporation of a lactone moiety and concomitant presence
of bromine in the side chain. d-Bromo-g-lactone (6) was the
most potent antifeedant at pre-, post- and intraingestional
levels. Ester (4) and unsaturated g-lactone (10) were active at
postingestional levels by impeding aphid settling aer longer
exposure times.
All b-damascone derivatives with a lactone moiety deterred
the feeding of adults and larvae of A. diaperinus. The potency of
the activity was related to the size of the ring and the incorpo-
ration of chlorine or bromine into the molecule. Generally,
halo-d-lactones were more active than halo-g-lactones, and the
adults were more sensitive to the compounds studied than
larvae. Nevertheless, the strongest antifeedants for both devel-
opmental stages of the lesser mealworm were g-bromo-d-
lactone (7), d-chloro-g-lactone (8), and g-chloro-d-lactone (9).
Chemistry
Synthesis and separation of compounds
Synthesis of dihydro-b-damascone (2). A solution of b-dam-
ascone 1 (4 g, 20.8 mmol) in anhydrous diethyl ether (10 mL)
was added dropwise to the stirred solution of LiAlH4 (0.4 g) in
dry diethyl ether (20 mL). Aer 5 h (GC, TLC) water was added,
and the product was extracted with diethyl ether. The organic
layer was washed with brine, dried over anhydrous MgSO4, and
ltered. The solvent was evaporated in vacuo, and pure ketone
(2) was obtained (3.9 g, 99% yield); the physical and spectral
data were consistent with previously published data.32
Synthesis of dihydro-b-damascol (3). Using the same reduction
reaction for the double bond in the side chain of compound (1),
alcohol 3 was obtained (3.6 g, 89% yield) from the ketone (2),
with the physical and spectral data consistent with the literature
report.33
Synthesis of ethyl 2-(2-butylidene-1,3,3-trimethylcyclohexyl)-
acetate (4). A mixture of alcohol (3) (3.6 g, 18.4 mmol), triethy-
lorthoacetate (21.6 mL, 115.2 mmol), and a catalytic amount of
propionic acid (1 drop) was heated at 138 ꢁC for 10 h under the
conditions for distillative removal of ethyl alcohol. When the
reaction was completed (GC, TLC), the mixture was chromato-
graphed on silica gel. Elution with hexane/acetone (19 : 1) gave
the pure ester (4) (4.7 g, 98% yield); (oily liquid); 1H NMR
(CDCl3), d: 0.89 (t, J ¼ 7.3 Hz, 3H, CH3-10), 1.17 (s, 3H, one of the
(CH3)2C<), 1.20 (s, 3H, one of the (CH3)2C<), 1.22 (t, J ¼ 7.4 Hz,
Experimental section
Reagents
b-Damascone (90% purity), N-bromosuccinimide (99% purity),
and N-chlorosuccinimide (98% purity) were purchased from
Aldrich; triethylorthoacetate was purchased from Fluka.
General procedures
Analytical TLC. Analytical TLC was performed on silica gel 3H, CH3-17), 1.27–1.65 (m, 11H, CH2-9, CH2-4, CH2-5, CH2-6,
(Kieselgel 60 F254, Merck) with a mixture of hexane, acetone, CH3-13), 2.14 (m, 2H, CH2-8), 2.37 and 2.48 (two d, J ¼ 13.7 Hz,
and diethyl ether in various ratios as developing systems. 2H, CH2-14), 4.05 (q, J ¼ 7.4 Hz, 2H, CH2-16), 5.20 (t, J ¼ 7.0 Hz,
Compounds were detected by spraying the plates with a solu- 1H, H-7). 13C NMR (CDCl3), d:13.96 and 14.30 (C-10 and C-
tion of Ce(SO4)2 (1 g), H3[P(Mo3O10)4] (2 g) in 10% H2SO4, fol- 17),17.59 (C-5), 23.76 (C-9), 30.51 and 30.64 (C-11 and C-12),
lowed by heating to 120–200 ꢁC.
31.73 (C-13), 32.31 (C-8), 35.33 (C-1), 36.33 (C-6), 39.63 (C-3),
Column chromatography. Column chromatography was 41.21 (C-4), 47.70 (C-14), 59.75 (C-16), 126.82 (C-7), 149.07 (C-
performed on silica gel (Kiesel gel 60, 230–400 mesh ASTM, 2), 172.19 (C-15). IR (lm, cmꢀ1): 2959 (s), 2932 (s), 2870 (m),
Merck) with a mixture of hexane, acetone, and diethyl ether (in 1743 (s), 1193 (w).
various ratios) as eluents.
Synthesis of 2-(2-butylidene-1,3,3-trimethylcyclohexyl)acetic
Gas chromatography. Gas chromatography was performed acid (5). Ester 4 (4 g, 15 mmol) was heated under reux for 3
on an Agilent Technologies 6890N Network GC instrument h in 10% ethanol solution of KOH (40 mL). Aer evaporation of
equipped with autosampler, split injection (20 : 1) and FID ethanol in vacuo, the residue was diluted with water, and
detector using an HP-5 column (30 m ꢃ 0.32 mm ꢃ 0.25 mm) organic impurities were removed by extraction with diethylether
with hydrogen as the carrier gas. The temperature programme (3 ꢃ 40 mL). The water layer was acidied with 0.1 M HCl, and
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the product was extracted with diethyl ether (3 ꢃ 40 mL). The 36.84 (C-9), 37.91 (C-6), 38.82 (C-4), 40.10 (C-3a), 43.96 (C-7),
ethereal fraction was washed with brine and dried over anhy- 45.50 (C-3), 66.74 (C-8), 92.63 (C-7a), 175.95 (C-2). IR (lm,
drous MgSO4. Evaporation of solvent in vacuo afforded pure acid cmꢀ1): 1464 (s), 1775 (s), 2875 (s), 2959 (s).
5 (3 g, 85% yield); (oily liquid); 1H NMR (CDCl3), d: 0.87 (t, J ¼ 6.3
7a-Chloro-3a,7,7-trimethyl-8-propyloctahydroizochromen-2-one (9).
1
Hz, 3H, CH3-10), 1.19 (s, 6H, (CH3)2C<), 1.23 (s, 3H, CH3-13), (0.36 g, 17% yield); (colourless crystal); mp: 81–96 ꢁC; H NMR
1.29–1.62 (m, 9H, CH2-9, CH2-4, CH2-5, CH2-6), 2.11–2.21 (m, (CDCl3), d: 0.97 (t, J ¼ 7.3 Hz, 3H, CH3-11), 1.27, 1.31 and 1.35
2H, CH2-8), 2.41 and 2.55 (two d, J ¼ 13.8 Hz, 2H, CH2-14), 5.26 (three s, 9H, (CH3)2C<, CH3-3a), 1.51–2.16 (m, 10H, CH2-4, CH2-5,
(t, J ¼ 7.0 Hz, 1H, H-7), 9.1 (s, 1H, –COOH). 13C NMR (CDCl3), d: CH2-6, CH2-9, CH2-10), 2.38 and 3.11 (two d, J ¼ 19.5 Hz, 2H, CH2-
13.88 (C-10), 17.55 (C-5), 23.63 (C-9), 30.52 and 30.58 (C-11 and 3), 4.74 (dd, J ¼ 10.3 and 1.6 Hz, 1H, H-8).13 C NMR (CDCl3), d:13.72
C-12), 31.62 (C-13), 32.23 (C-8), 35.29 (C-1), 36.36 (C-6), 39.43 (C- (C-11), 18.15 (C-10), 21.11 (C-5), 24.63 (C-14), 31.10 and 33.02 (C-12
3), 41.29 (C-4), 47.54 (C-14), 127.12 (C-7), 148.88 (C-2), 178.32 (C- and C-13), 34.31 (C-9), 35.07 (C-4), 39.95 (C-6), 40.02 (C-7), 40.22 (C-
15). IR (lm, cmꢀ1): 2958 (s), 1706 (s), 1463 (m).
Preparation of bromolactones (6), (7). A solution of acid (1.86 g, cmꢀ1): 2968 (w), 2931 (w), 2869 (w), 1733 (s), 1463 (s).
7.8 mmol) and N-bromosuccinimide (7.8 mmol) in THF (30 mL) Dehydrohalogenation procedure. 1,8-Diazabicyclo[5.4.0]undec-
3a), 41.58 (C-3), 80.64 (C-7a), 84.57 (C-8), 175.95 (C-2). IR (KBr,
and a drop of acetic acid were stirred at room temperature for 24 7-ene (6 mmol) was added to a solution of the corresponding
h. The mixture was diluted with diethyl ether and then washed halolactone (3 mmol) in dry methylene chloride (20 mL) at room
with saturated NaHCO3 solution and brine. The organic layer temperature. When the reaction was completed (GC, TLC), the
was dried over anhydrous MgSO4, and the solvent was evapo- mixture was concentrated in vacuo to remove methylene chlo-
rated in vacuo. The mixture of products was subjected to column ride. The residue was diluted with diethyl ether. The ethereal
chromatography (hexane/acetone, 3 : 1). The data for isolated solution was washed with brine, dried over anhydrous MgSO4,
lactones are given below.
and evaporated in vacuo. The residue was chromatographed on
7a-(1-Bromobutyl)-3a,7,7-trimethylhexahydrobenzofuran-2-one (6). silica gel. Elution with hexane/acetone (6 : 1) gave lactones (10)
(0.42 g, 17% yield); (oily liquid); 1H NMR (CDCl3), d: 0.94 (t, J ¼ 7.3 and (11) (47–57% yield) with the following data:
Hz, 3H, CH3-11), 1.10 (s, 3H, CH3-14), 1.34 and 1.40 (2s, 6H,
7a-((E)-But-1-enyl)-3a,7,7-trimethylhexahydrobenzofuran-2-one
1
(CH3)2C<), 1.50–2.33 (m, 10H, CH2-4, CH2-5, CH2-6, CH2-9, CH2- (10). (oily liquid); H NMR (CDCl3), d: 0.92 (s, 3H, one of the
10), 2.50 and 2.73 (two d, J ¼ 18.1 Hz, 2H, CH2-3), 4.28 (dd, J ¼ 10.3 (CH3)2C<), 1.01 (t, J ¼ 7.5 Hz, 3H, CH3-11), 1.03 (s, 3H, one of
and 1.2 Hz, 1H, H-8). 13C NMR (CDCl3), d:13.36 (C-11), 17.27 (C-10), the (CH3)2C<), 1.25 (s, 3H, CH3-3a), 1.40–1.62 (m, 6H, CH2-4,
22.62 (C-5), 27.03 and 28.11 (C-12 and C-13), 30.11 (C-14), 36.29 CH2-5, CH2-6), 1.97 and 2.42 (two d, J ¼ 16.3 Hz, 2H, CH2-3),
(C-9), 38.75 (C-4), 38.97 (C-6), 40.88 (C-3a), 44.30 (C-7), 45.27 (C-3), 2.11 (dqd, J ¼ 7.5, 6.3 and 1.3 Hz, 2H, CH2-10), 5.60 (dt, J ¼ 15.5
59.13 (C-8), 91.91 (C-7a), 175.90 (C-2). IR (lm, cmꢀ1): 2958 (m), and 1.3 Hz, 1H, H-8), 5.77 (dt, J ¼ 15.5 and 6.3 Hz, 1H, H-9). 13
C
2874 (m), 1776 (s), 1463 (w), 1226 (w).
NMR (CDCl3), d: 13.80 (C-11), 17.75 (C-5), 21.66 and 24.99 (C-12
7a-Bromo-3a,7,7-trimethyl-8-propyloctahydroisochromen-3-one (7). and C-13), 25.66 (C-10), 26.47 (C-14), 36.25 (C-3a), 36.49 (C-4),
1
ꢁ
(0.67 g, 27% yield); (colourless crystal); mp: 95–104 C; H NMR 36.57 (C-6), 41.75 (C-7), 45.91 (C-3), 91.98 (C-7a), 125.01 (C-9),
(CDCl3), d:0.98 (t, J¼ 7.0 Hz, 3H, CH3-11), 1.16–1.27 (m, 2H, CH2-10), 134.33 (C-8), 176.73 (C-2). IR (lm, cmꢀ1): 2964 (s), 2932 (s),
1.32 (s, 3H, CH3-3a), 1.40 and 1.45 (two s, 6H, (CH3)2C<), 1.52–1.97 1778 (m), 1462 (m), 1289 (m), 1125 (m), 971 (m).
(m, 8H, CH2-6, CH2-5, CH2-4, CH2-9), 2.44 and 3.15 (two d, J ¼ 19.6
3a,7,7-Trimethyl-8-propylhexahydro,cyclopropa[1,2]benzofuran-
Hz, 2H, CH2-3), 4.93 (d, J ¼ 10.6 Hz, 1H, H-8).13 CNMR (CDCl3), 2(3H)-one (11). (oily liquid); 1H NMR (CDCl3), d: 0.96 (t, 3H,
d:13.69 (C-11), 18.24 (C-10), 21.22 (C-5), 24.08 (C-14), 28.48 (C-3a), J ¼ 7.3 Hz, CH3-11), 1.14 and 1.18 (two s, 6H, (CH3)2C<), 1.21 (s,
33.62 and 35.67 (C-12 and C-13), 35.24 (C-9), 35.67 (C-4), 40.70 3H, CH3-3a), 1.29–2.11 (m, 11H, CH2-6, CH2-5, CH2-4, CH2-9,
(C-6), 41.05 (C-3), 84.89 (C-7a), 84.97 (C-8), 170.90 (C-2). IR (KBr, CH2-10 and H-3), 4.25 (dd, J ¼ 10.9 and 2.2 Hz, 1H, H-8).13C
cmꢀ1): 3007 (s), 2918 (s), 1716 (s), 1460 (s).
NMR (CDCl3), d: 13.92 (C-11), 17.73 (C-10), 17.86 (C-14), 20.45
Preparation of chlorolactones (8),(9). A solution of acid (1.87 g, (C-5), 26.13 (C-12), 28.04 (C-3a), 29.50 (C-13), 30.52 (C-7), 33.11
7.8 mmol) and N-chlorosuccinimide (7.8 mmol) in THF (30 mL) (C-9), 34.12 (C-3), 37.27 (C-6), 41.51 (C-4), 45.59 (C-7a), 82.73
and a drop of acetic acid were stirred at room temperature for 24 (C-8), 175.13 (C-2). IR (lm, cmꢀ1): 2936 (s), 2873 (s), 1762 (s),
h. The mixture was diluted with diethyl ether and then washed 1466 (m), 1327 (m), 1188 (s), 976 (s).
with saturated NaHCO3 solution and brine. The organic layer
was dried over anhydrous MgSO4, and the solvent was evapo-
rated in vacuo. The mixture of products was subjected to column
Bioassays
Myzus persicae
chromatography (hexane/diethyl ether, 3 : 1). The data for iso-
lated lactones are given below:
7a-(1-Chlorobutyl)-3a,7,7-trimethylhexahydrobenzofuran-2-one (8).
(0.39 g, 18% yield); (oily liquid); 1H NMR (CDCl3), d: 0.94 (t, J ¼ 7.3
Hz, 3H, CH3-11), 1.11 (s, 3H, CH3-3a), 1.33 and 1.41 (two s, 6H,
(CH3)2C < 7), 1.50–2.24 (m, 10H, CH2-4, CH2-5, CH2-6, CH2-9,
Cultures of aphids and their settling (choice-test) have been
previously described by Grudniewska et al.18
Aphid initial behavioural responses (no-choice test)
CH2-10), 2.52 and 2.62 (two d, J ¼ 18 Hz, 2H, CH2-3), 4.24 (dd, J ¼ Aphid behaviour during the initial contact with the studied
10.6 and 1.6 Hz, 1H, H-8). 13C NMR (CDCl3), d: 13.43 (C-11), 17.18 compound was measured by direct monitoring of the freely
(C-10), 21.90 (C-5), 26.61 and 28.27 (C-12 and C-13), 29.60 (C-14), moving aphids on a treated leaf, using a video recording. This
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bioassay allows studying the preingestional (activating olfactory for calculating the deterrence coefficients (relative R and abso-
receptors) and ingestional (activating gustatory receptors) lute A) according to the formulae:36,41
effects of a chemical, and the results may show the mode of
R ¼ (C ꢀ E)/(C + E) ꢃ 100 (choice test)
action of the compound – whether it is active at the plant
surface or within plant tissues. The experiment was carried out
for 15 min with 12 aphids/compound. The following parameters
A ¼ (CC ꢀ EE)/(CC + EE) ꢃ 100 (no-choice test)
were derived from data obtained in this experiment: total time
where C, CC and E, EE are the weights of the control and the
of aphid presence on the leaf, total time of probing, number of
treated food consumed by the insects in the choice and no-
probes, and mean duration of a probe. The time spent on the
choice tests, respectively.
leaf and the duration of probing were recorded based on the
The total deterrence coefficient, which ranged from ꢀ200 to
relationship between antennal and body movements and
+200, served as an activity index. A compound for which both
penetration of the stylets as described by Hardie et al., who
indices reach a value of 100 and the sum of 200 is called an ideal
associated styled penetration with the position of antennae
deterrent. Activity of the tested compounds was evaluated
parallel to the abdomen and the cessation of body movements.40
according to the classication of Nawrot et al. and Koul.16,42
The compounds with the highest activity (7, 8, 9) were used
for further studies as 0.5 and 0.1% solutions. Because 0.5%
solution moderately decreased larval feeding, the lowest
concentration of these halolactones was used only against
adults. The research methodology was the same as for the case
of 1% solutions. Four replicates of both tests for each
compound were conducted on each insect life stage.
From a practical point of view, it is important to evaluate the
level of insect feeding in a no-choice situation. To estimate and
Statistical analysis
The data derived from the choice-test (aphid settling) were
analysed using Student's t-test. If aphids showed clear prefer-
ence for the leaf treated with the tested compound (P < 0.05), the
compound was described as having attractant properties. If
aphids settled mainly on the control leaf (P < 0.05), the
compound studied in the respective choice-test was classied as
a deterrent. From the data thus obtained, the relative index of
deterrence (DI) was calculated using the equation:
compare larval and adult feeding levels in the no-choice tests,
the amount of treated food consumed was expressed as a
percentage of the consumption in the control according to the
formula:
DI ¼ (C ꢀ T)/(C + T)
where C and T are the numbers of aphids settled on control and
(EE/CC) ꢃ 100
treated leaves, respectively. The value of DI ranged between plus
1 (ideal deterrent) and minus 1 (ideal attractant).
where EE and CC are the weights of the treated and control food
consumed in no-choice tests, respectively.
The data derived from the no-choice-test (aphid behavioural
responses) were analyzed by means of one-way analysis of
variance (ANOVA) followed by comparison of deterrence coef-
Statistical analysis
cients using Tukey's test (P < 0.05).
Antifeedant activity was analyzed by means of one-way analysis
of variance (ANOVA) followed by comparison of deterrence
coefficients with Tukey's test (P < 0.05) using PAST (Paleonto-
logical Statistics) soware.26 Student's t-test (Microso Office
2010, Excel) was used to compare the mean values of
consumption by the larvae and adults in the no-choice test.
Alphitobius diaperinus
The insect culture of Alphitobius diaperinus culture has been
previously described by Szczepanik et al.21
To investigate the antifeedant activity of compounds
studied, choice and no-choice tests were used according to a
procedure previously described.17 The choice test is very sensi-
tive, but insects could easily avoid treated food. Feeding-
deterrent activity of chemical compounds observed in a
choice test thus needs to be conrmed with a no-choice test.
The application of these two types of tests also allowed us to
evaluate the mode of action of the compounds studied: did they
act on the taste organs only (according to the denition of
antifeedants) or were they toxic to the insects?
Acknowledgements
This project was nanced by the European Union from the
European Regional Development Found Grant no.
POIG.01.03.01-00-158/09-03 and partly by Nicolaus Copernicus
University, Rector's grant no. 502-B/2013.
For the feeding assays, 1 mL of acetone solution of the test
compounds at a concentration of 1% was applied to 1 g of oat
akes (Melvit SA Warsaw, Poland) using a micropipette. Aer
evaporation of acetone, weighed akes were placed in Petri
dishes and offered to 10 larvae or 10 unsexed adults during the
following three-day period. All trials were kept in an incubator
at 29 ꢂ 1 ꢁC in the dark. The amount of food eaten was the basis
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´
´
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