Comparison of Phenylacetates with Benzoates and Phenylpropanoates as Antifeedants for the Pine Weevil, Hylobius abietis

Article abstract of DOI:10.1021/acs.jafc.8b03830

This study concludes an extensive investigation of antifeedants for the pine weevil, Hylobius abietis (Coleoptera: Curculionidae), an economically important pest of planted conifer seedlings. Building on the previously reported antifeedant effects of benzoates and phenylpropanoids (aromatic compounds with one- or three-carbon-atom substituents on the benzene ring), we here report the antifeedant effects of compounds with two-carbon-atom side chains (i.e., phenylacetates). We also present new results; the best antifeedants from the benzoate class were tested at 10-fold lower concentrations in order to find the optimal antifeedants. Generally, for all three compound classes, efficient antifeedants were found to have one or two methyl, chloro, or methoxy substituents on the aromatic ring. For monosubstituted phenylpropanoids, the substituent preferably should be in the para-position. In the search for synergistic antifeedant effects among the three compound classes, combinations of compounds from the three classes were tested in binary and ternary mixtures.

Full text of DOI:10.1021/acs.jafc.8b03830

Subscriber access provided by UNIV OF LOUISIANA
Functional Structure/Activity Relationships
Phenylacetates as Antifeedants for the Pine Weevil, Hylobius
Abietis - Comparison with Benzoates and Phenylpropanoates
C. Rikard Unelius, Bjorn Bohman, and Göran Nordlander
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03830 • Publication Date (Web): 17 Oct 2018
Downloaded from http://pubs.acs.org on October 19, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 40
Journal of Agricultural and Food Chemistry
1
2
3
Phenylacetates as Antifeedants for the Pine Weevil, Hylobius abietis,
Comparison with Benzoates and Phenylpropanoates
4
C. Rikard Unelius,*,† Björn Bohman†,‡ and Göran Nordlander§
5
6
† Department of Chemistry and Biomedical Sciences, Linnaeus University,
7
Kalmar, Sweden
8
‡ Current address: School of Molecular Sciences, the University of Western Australia,
9
Perth, Australia
10
11
12
§ Department of Ecology, Swedish University of Agricultural Sciences (SLU), Uppsala,
Sweden
13
14
15
* Corresponding author: (Tel: +46-708-101514; Email: rikard.unelius@lnu.se)
16
17
18
http://orcid.org/0000-0001-7158-6393
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 40
19
20
Abstract –
21
22
23
24
25
26
27
28
29
30
31
32
33
34
This study concludes an extensive investigation of antifeedants for the pine weevil,
Hylobius abietis (Coleoptera: Curculionidae), an economically important pest of
planted conifer seedlings. Building on previously reported antifeedant effects of
benzoates and phenylpropanoids (aromatic compounds with one or three carbon
atom substituents on the benzene ring) we here report the antifeedant effect of
compounds with a two-carbon atom side chain (i.e. phenylacetates). We also
present new results where the best antifeedants from the benzoate class were
tested at tenfold lower concentrations in order to find the optimal antifeedants.
Generally, for all three compound classes, efficient antifeedants were found to
have one or two methyl, chloro or methoxy substituents on the aromatic ring. For
monosubstituted phenylpropanoids the substituent preferably should be in the
para-position. In search for synergistic antifeedant effects between the three
compound classes, combinations of compounds from the three classes were tested
in binary and ternary mixtures.
35
36
37
Key Words - Pine weevil, Hylobius abietis, synergism, conifer seedling protection,
38
39
feeding deterrent, structure-activity relationships.
2
ACS Paragon Plus Environment

Page 3 of 40
Journal of Agricultural and Food Chemistry
40
INTRODUCTION
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
Killing of planted conifer seedlings by feeding on the bark by the pine weevil
Hylobius abietis (Coleoptera: Curculionidae) is a severe problem for the forestry
industry in large parts of Europe.1 If no countermeasures are taken, seedlings
frequently suffer more than 80% mortality in areas with high population levels.2-3
The likelihood of pine weevil attack can, however, be considerably reduced by
various silvicultural practices,4-6 such as soil scarification providing planting spots
of mineral soil, which the weevils avoid.7-8 Moreover, it is often necessary to
protect the seedlings in the nursery, by insecticide applications or more recently,
by a coating that physically protects the stem.9-10 The use of insecticide treated
seedlings poses health risks for forestry workers,11 and to achieve the goal to
completely abandon insecticides for seedling protection,10 the need for new
alternative methods remains high.12 Furthermore, to ensure high seedling survival
the protective effect needs to last for two seasons.5
An alternative to traditional insecticides is to apply antifeedant compounds. The
strategy of utilizing compounds that deter feeding by specific pest insects without
the intrinsic toxicity of pesticides has been applied for several decades across many
systems. The early work on this approach has been reviewed by Jermy,13 while the
more recent advances in antifeedants and related repellents have been reviewed
Deletre et al.14 Lately, discoveries of antifeedant compounds derived from many
plants, such as Ginkgo biloba against Hyphantria cunea (Lepidoptera: Arctiidae)
larvae15 and Ajuga chamaepitys extract against Tuta absoluta (Lepidoptera:
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 40
62
63
64
65
66
67
68
69
70
Gelechiidae),16 to mention a few, have been reported. Individual compounds have
been proven to be efficient antifeedants in several systems, for example
cinnamaldehyde against the elm pest Ambrostoma quadriimpressum (Coleoptera:
Chrysomelidae).17 Extracts and individual compounds derived from various non-
host plants have been shown to have antifeedant effects also for H. abietis or closely
related species,18-24 although these findings have not yet led to any practical use.
Furthermore, several volatiles produced by bacteria and fungi associated with H.
abietis have antifeedant properties or reduce the attraction of the weevils to host
odors.25-28
71
72
73
74
75
76
77
78
79
80
In H. abietis, antifeedant compounds have specifically been found to be deposited during
ovipositioning.29 The eggs are laid at roots of recently dead conifer trees, where pine
weevils also feed to a large extent (in addition to their feeding on seedlings).30-31 In the
oviposition process, feces are added to the eggs, and it is known that female feces possess
antifeedant properties.29 Bioassay guided fractionation of feces revealed aromatic
compounds with low molecular weight as the main substituents of the most active fraction,
and subsequently benzoate esters and phenylpropanoids with strong antifeedant activity
were isolated.29, 32 Chemically related antifeedants (ethyl cinnamate and ethyl 2,3-dibromo-
3-phenylpropanoate) were also isolated from bark of lodgepole pine (Pinus contorta
Douglas ex Loudon).33
81
82
83
Several synthetic analogues of the isolated active compounds from H. abietis feces
and P. contorta bark have been synthesized and tested for antifeedant activity.29, 32
Furthermore, active phenylpropanoids were derived from the lead compounds in
4
ACS Paragon Plus Environment

Page 5 of 40
Journal of Agricultural and Food Chemistry
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
P. contorta34-36 and benzoate ester analogues were prepared from leads in feces.37
By making systematic variations in the structures, some correlations between
structure and weevil response were revealed. Several structurally related
compounds were found to be active in laboratory antifeedant bioassays.34-35, 37-38
During these previous studies, a small number of phenylacetates were also tested.37
The results from these preliminary trials showed great promise, which encouraged
us to explore the antifeedant activities of this substance class more broadly in this
current study, where the substituents on the aromatic ring were modified based on
our results from our work with phenylpropanoids and benzoates. The overall aim
was to identify new substances useful for practical conifer seedling protection. We
synthesized seven new substituted phenylacetates and subsequently tested and
evaluated these compounds as well as four acetate esters included in previous
studies and two of the parent phenylacetic acids. The antifeedant activity of these
compounds was compared with previously tested benzoates and
phenylpropanoates. Based on our negative experiences with higher benzoates37-38
and phenylpropanoids,34-35 we limited our investigation to methyl esters.
Additionally, for the first time in the series of investigations of H. abietis
antifeedants, we include a test for synergistic effects of binary or ternary blends of
compounds from all three substance classes of benzoate, acetate and propanoate
esters: Three compounds with high antifeedant efficiency were selected and
applied in a matrix on stems of coniferous seedlings.
100
101
102
103
104
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 40
105
MATERIALS AND METHODS
106
107
108
109
110
111
112
113
114
Collection and Maintenance of H. abietis. Both sexes of H. abietis were collected during
spring migration at a sawmill in central Sweden (where they landed in large numbers in
response to massive emissions of attractive conifer volatiles). The weevils were then stored
in darkness at 10 °C and provided with fresh Scots pine (Pinus sylvestris L.) stems with
tender bark as food. These storage conditions interrupted their reproductive development,
so that females did not begin to oviposit until about a week after they had been transferred
to the experimental conditions (a light regime of L18: D6 at 22 °C). This transfer was made
at least 10 d before the weevils were used in bioassays.
Feeding Bioassays. All compounds in Figure 1and 2 were tested for their antifeedant
115
116
117
118
119
120
121
122
123
124
125
126
effect on H. abietis by using a two-choice laboratory bioassay.34 Fresh pieces of
Scots pine twigs (50 mm long, 15 mm diam., and taken from one individual tree)
were split, and each half (=test twig) was wrapped in aluminum foil. In each test
twig, two sharp-edged metal rings (5 mm diam.) were punched 25 mm apart
through the foil and into the bark. The rings and the pieces of aluminum foil inside
them were then removed. The thin outer layers of corky bark inside the two
circular areas on the surface of the twig were also carefully removed with a scalpel.
Thereafter, new rings were fitted into the bark around the two exposed areas.
Next, 100 µL of solvent (methanol or methyl acetate) with a concentration of 5 or
50 mM of the test compound was applied on the bark in one of the two rings. In
the other ring, 100 µL of pure methanol or methyl acetate was added for control
(so that solely the effect of the investigated compound was measured). When the
6
ACS Paragon Plus Environment

Page 7 of 40
Journal of Agricultural and Food Chemistry
127
solvent enclosed by the metal rings had evaporated and/or absorbed into the
wood, the metal rings were removed (Figure 3). Each test twig was placed on
moistened filter paper in a 142-mm-diam. Petri dish, with one weevil in each dish
for 24 h. The assay was replicated 20 times for females and 20 times for males. Each
weevil was used only once. The weevils were all in the reproductive phase of their
life cycle and were starved for 24 h before the test period. The bioassays were
conducted under a light regime of L18: D6 at 22 °C. After the 24 h test period, the
amount of bark that had been removed by weevil feeding within the 20 mm2
treatment and control area of each test twig were recorded by comparison with a
square mm grid. There was generally no significant difference in response
between the sexes, and the data presented were therefore pooled.
128
129
130
131
132
133
134
135
136
137
138
139
The effects of the various treatments are described by two variants of the
antifeedant index, AFI:39 100x(C-T)/(C+T):
140
141
142
143
144
1) In AFIa, C represents the mean area of the control surfaces consumed and T
represents the mean area of the treated surfaces consumed.
2) In AFIn, C represents the number of the control surfaces with any feeding and
T represents the number of the treated surfaces without any feeding.
145
146
147
148
Hence, AFIa tends to be a measure that captures the reduction in feeding, whereas
AFIn is a measure of complete inhibition of the initiation of feeding on the treated
area. The two indices are fairly well correlated, but AFIa tend to be higher than
AFIn because the antifeedant substances generally affect both the initiation of
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 40
149
150
151
152
feeding and the amount of plant material consumed if feeding had started. For
both indices, positive values (up to a maximum of 100) reflect an antifeedant effect,
whereas negative values (down to a minimum of -100) indicate a stimulant effect
on feeding.
153
154
155
156
157
158
Statistical differences in feeding/no feeding between treatment and control (i.e. the
data used for calculating AFIn) were tested for each substance with Fisher´s exact
test of a 2 x 2 table: *P<0.05, **P<0.01, ***P<0.001.
In the experiment to test synergistic effects the single compounds were tested at
15 mM concentration, in two-component lures at 7.5 mM of both components and
in the test of the three-component lure at 5 mM of each component.
159
160
161
162
163
164
165
Test Compounds. The test compounds in Figure 1 and 2; 1, 2, 5, 6, 7, 9, 10, 13, 19, 20, 22,
23, 24, 30, 31, 32, 33, 37, 39, 85, were purchased from Lancaster Synthesis, Lancaster,
England and the test compounds 3, 8, 15, 17, 21, 28, 37 were purchased from (Sigma-
Aldrich, Stockholm, Sweden). The compounds 11 and 35 were obtained from late prof.
Holger Erdtman, KTH, Stockholm, Sweden.
The syntheses of the methyl hydroxy-methoxybenzoates 25, 26, and 29 were executed by
regioselective synthetic sequences reported previously.38 Some of the
166
167
168
169
phenylpropanoids (40 and 41) were synthesized from the corresponding
cinnamates. Methyl 3-(3,4-dimethoxyphenyl)propanoate (42) was obtained by
reacting methyl 3-(3,4-dihydroxyphenyl)propanoate with sodium hydride and
methyl iodide in THF according to the standard procedure.
8
ACS Paragon Plus Environment

Page 9 of 40
Journal of Agricultural and Food Chemistry
170
The rest of the non-commercial test compounds were synthesized from their
corresponding carboxylic acids by acids by refluxing in the alcohol with H2SO4 as a
catalyst. A typical procedure was as follows: The carboxylic acid (2,4-
dimethoxyphenylacetic acid (275 mg, 1.40 mmol) was dissolved in 25 mL
methanol and some drops of sulfuric acid were added. The reaction mixture was
heated at reflux until completion (monitored by TLC, ca 3 h). The solvent was
evaporated (10-20 mm Hg) and the crude product was dissolved in
dichloromethane. The solution was washed twice with brine and once with water.
Drying over magnesium sulfate and concentration gave the ester, in this case, 260
mg (1.24 mmol) of methyl 2,4-dimethoxyphenylacetate in 88% yield.
171
172
173
174
175
176
177
178
179
180
181
182
183
Final purities of all compounds ranged from 96 to 99%, and, if necessary, compounds were
purified by preparative chromatography40 or flash chromatography on silica gel 0.040-
0.063 mm (Merck 60, Darmstadt, Germany).
Gas chromatography-mass spectrometry (GC-MS) and when appropriate, NMR,
184
185
186
187
188
189
190
191
was used to confirm identity and purity of all compounds.
Analysis of Bioassay Results. The following factors were investigated for their
importance for antifeedant activity:
(1) functional groups (carboxylic acid vs methyl ester) (Table 1);
(2) structure of substituents on the aromatic ring (Table 2);
(3) patterns of substituents on the aromatic ring (Table 3).
(4) effect of lowering the concentration of antifeedants (Table 3).
(5) synergy effects by blending compounds from three different substance classes.
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 40
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
Antifeedant activities (AFIa and AFIn) were compiled and compared for the
structural features 1-3 above, for benzoates, phenylacetates and
phenylpropanoids.
Tests for Synergistic Effect of Selected Antifeedants Three selected antifeedants were
tested for synergy effects: (methyl 2,4-dimethoxybenzoate (37), methyl (4-
chlorophenyl)acetate (45), and methyl 3-(4-methylphenyl)propanoate (55). The
selection was based on the antifeedant activity for each substance class, with the
additional criterion of selecting compounds with different substituent types on the
aromatic ring. The 4-chloro-analogue was selected from the phenylacetates, the
2,4-dimethoxy-analogue from the benzoate group and the 4-methyl-analogue
from the phenylpropanoate group. Although a promising antifeedant, the latter
was not the most active phenylpropanoate, but it was found appealing to use a
third type of substituent. If antifeedant activity is a result of multiple interactions
with several receptors, the selection of substances with multiple substituents
would increase the chances of beneficial synergy effects. The experimental design
is presented in Table 4 and 5 together with the results.
RESULTS AND DISCUSSION
212
213
Laboratory Bioassays The selected 13 phenylacetates and two phenylacetic acids
were tested for antifeedant activity and our new results were compared with the
10
ACS Paragon Plus Environment

Page 11 of 40
Journal of Agricultural and Food Chemistry
214
results of our previous studies of benzoate37-38 and phenylpropanoid34-35
antifeedants in Tables 1-3.
215
216
217
218
219
Effect of Functional Groups. From the studies of benzoic and phenylpropanoic
antifeedants we concluded that the carboxylic acids tested were inactive as
antifeedants. This result was confirmed for the two tested phenylacetic acids,
which both showed low antifeedant activity (Table 1, 1-18).
220
221
Effect of Structure of the Substituents on the Aromatic Ring. One or two hydroxy
groups on the aromatic ring seem to reduce the antifeedant activity (Table 2, 19-
24). The negative effect of the hydroxy group on the aromatic ring seems to be
eliminated by an additional methoxy groups for many test compounds (Table 2, 12
+ 25-30). Apparently a methoxy group results in a favorable interaction with the
antifeedant receptors for many substrates, as compounds with exclusively
methoxy substituents, 31–41, are generally strong antifeedants for all substance
222
223
224
225
226
227
228
229
230
231
232
233
234
235
classes
(Table
2).
An
exception
is
the
relatively
long
3,4-
dimethoxyphenylpropanoid 42, for which the antifeedant receptors do not seem
to be able to accommodate both methoxy groups. Methyl and halogen substituents
seem to yield relatively strong antifeedants for all three substance classes,
although very few methylated benzoates were tested (as their relatively high
volatility would make them unsuitable to use as antifeedants in practical
applications) Table 2, 43-61.
11
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 40
236
237
238
239
240
241
Effect of Patterns of Substituents. In order to find general trends, the effect of
aromatic ring substituent pattern on antifeedant activity was studied. It was
difficult to reveal any clear trends. For instance, of the compounds tested, all
dimethoxy
derivatives
except
2,6-dimethoxybenzoate37
and
3,4-
dimethoxyphenylpropanoate derivatives, i.e. 42, exhibited strong antifeedant
activity, Table 2. For five of the 3-chloro and 4-chloro derivatives, AFIa reached
≈100, thus indicating very strong AF activity, Table 2, 44-46 and 52-53.
242
243
244
The Effect of Lowering the Concentration of Antifeedants. It was observed that at 50
mM concentration, maximum or close to maximum antifeedant index was
obtained for several compounds. To differentiate between some of the most
promising antifeedant compounds and understand the relation of antifeedant
activity with concentration, the tests for a subset of compounds were repeated at
5 mM. Monosubstituted benzoates were not tested due to their relatively high
volatility, giving them less potential to be long-lasting antifeedants. All
compounds showed lower antifeedant activity when the concentration was
lowered. In the comparison of ortho- meta- or para-substituted monosubstituted
phenylacetates and phenylpropanoates, no general correlation between
antifeedant activity and substituent position on the aromatic rings could be
revealed (Table 3). Both monomethoxylated phenylacetate 34 and
monochlorinated phenylacetate 45 had much higher AFI than the corresponding
245
246
247
248
249
250
251
252
253
254
255
256
257
monomethoxylated
phenylpropanoate
34
and
monochlorinated
12
ACS Paragon Plus Environment

Page 13 of 40
Journal of Agricultural and Food Chemistry
258
phenylpropanoate 46 (Table 3). Dimethoxy-substituted phenylacetates 14, 39 and
benzoates 2, 37 showed good to excellent antifeedant activity. Some
methylsubstituted phenylacetates 54, 59 and phenylpropanoates 55-57, 60-61 were
top performing antifeedants (Table 3). Phenylpropanoates 46, 49 and 50
halogenated in position 4 were all good antifeedants while chlorination in position
2 or 3 lead to a decrease in activity (47-48 and 53). Interestingly, all three
chlorinated phenylacetates tested 43-45 had high antifeedant activities although
the para-isomer was the most active (Table 3). The 3,4-dichlorophenylacetate 52
also had relatively high AFI at 5 mM.
259
260
261
262
263
264
265
266
267
268
269
270
271
272
To summarize, efficient antifeedants for the pine weevil H. abietis are found among
benzoates, phenylacetates and phenylpropanoates with one or two methyl, chloro
or methoxy substituents on the aromatic rings.
Tests for Synergistic Effect of Selected Antifeedants. Three selected antifeedants were
tested for synergistic effects (Table 4 and 5).
273
274
No synergy effects were found in the tests employing various combinations of
three antifeedants from the different substance classes. All mixtures and single
compounds tested resulted in AFIa values of 60-70, with the exception of the
binary mixture of methyl (4-chlorophenyl)acetate (45) and methyl 3-(4-
methylphenyl)propanoate (55), showing an AFIa as low as 37.
275
276
277
278
13
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 40
279
DISCUSSION
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
Several compounds covered in this study, or closely structurally related analogues
thereof, have been reported previously as biologically active in various systems.
Potentially relevant for this study is the report that methyl 4-
methoxyphenylacetate (34) is emitted by sporulating tree-decaying fungi.41-42 This
compound may function as a signal that the tree stump is infested with fungi and
in a state of decay, thus making it unsuitable for oviposition to H. abietis females.43
Compounds with remotely similar structures have been reported as antifeedants
and oviposition deterrents for other insects. Ethyl 3-(4-nitrophenyl)acrylate was
reported as an oviposition deterrent for the onion fly Delia antiqua (Diptera:
Anthomyiidae),44 although showed no antifeedant activity in H. abietis,35 while
cinnamaldehyde acted as an antifeedant for Tribolium and Sitophilus store product
beetles (Coleoptera: Tenebrionidae and Curculionidae)45 as well as the elm pest
Ambrostoma quadriimpressum (Coleoptera: Chrysomelidae).17 Esters of phenylacetic
acids have been found to act as inhibitors of soybean lipoxygenase46 and were
evaluated as anti-allergenic agents after showing degranulation inhibitory
effects.47 Benzoic- and cinnamic acid esters were recently reported to have strong
antifungal activity against Candida albicans, a relevant fungus for human
infections.48
For H. abietis, over a hundred derivatives of benzoic-, acetic-, phenylpropanoic-
and cinnamic acid have been prepared and tested for antifeedant activity. Despite
the discovery of many active compounds, it has always been difficult to identify
14
ACS Paragon Plus Environment

Page 15 of 40
Journal of Agricultural and Food Chemistry
301
the mode of action of these compounds. Several attempts to correlate physical
properties of compounds with antifeedant activity using both traditional
structure-activity relationship correlations such as the Topliss approach35 and
computational structure activity relationship studies34 have been made on various
subclasses of these small aromatic compounds. None of these investigations has
given any clear indication of which properties are the key to a potent antifeedant.
Substituents with very different electronic and steric properties have shown strong
activity and the optimal substitution pattern has varied between substituent types
as well as type of carboxylic acid.
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
We believe that our current study has merit not only in reporting the activity of
methyl esters of phenylacetic acids as antifeedants, but also serves as a concluding
study comparing the best antifeedants from similar types of compounds in
literature. The results from this study are very well aligned with the results from
35, 37-38
previous studies
and it is interesting that even if no “magic bullet” was
discovered, many active compounds were revealed. From practical considerations,
our results provide many alternatives for forestry protection applications. For such
work, there are several other factors such as volatility, stability and toxicology that
are highly important, and from the array of strongly active antifeedants presented
not only from the phenylacetic acid derivatives, but also from related classes of
compounds, there is a good chance that suitable compounds or combinations of
compounds could be utilized. In our previous studies we did not test for possible
synergistic effects between compounds from the three different substance classes.
15
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 40
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
If there were in fact several taste receptors involved in the interaction with the
antifeedants, this possibility is most likely reality. Therefore, we here compared
three selected representative strong antifeedants. The results were unequivocal:
no relevant synergistic effects were observed. Although arguably possible to
predict, this result is important for improving our understanding of the molecular
interaction between this type of antifeedants and the insect taste receptor. The
antifeedants of these types (esters of benzoic-, phenylacetic- and phenylpropanoic
acid) may act on the same receptor. Based on this prediction, we believe that after
evaluating over a hundred related compounds, the probability to find significantly
more active antifeedants based on the tentative hypothesis of an oviposition
deterrent is slim. In our opinion, further work on practical applications would be
most effective by utilizing best fit candidates from the set of compounds already
tested. Other types of antifeedants, that may signal food quality may however
prove to be important. For example, 2-phenylethanol, an ubiquitous bacterial
metabolite, is a strong antifeedant for H. abietis and has been suggested as a
candidate for use to protect conifer seedlings.25
Since the techniques for application of protective stem coatings for conifer
seedlings have evolved rapidly during the last decade,10, 49 it is a tempting strategy
to combine the flexible coating concept with an effective, non-toxic antifeedant.
Currently used coatings often contain hard particles providing the physical
protection but also making the application process more cumbersome and
costlier. A coating containing an effective antifeedant may therefore offer a less
16
ACS Paragon Plus Environment

Page 17 of 40
Journal of Agricultural and Food Chemistry
345
complicated application process and maybe even an enhanced protective effect.
An urgent task for future research is therefore to find compatible combinations of
coating material and antifeedant that can provide protection against pine weevil
feeding for two seasons without any detrimental effects on the seedling.
346
347
348
349
350
351
352
353
354
355
356
Acknowledgements - Claes Hellqvist, Oskar Ljungqvist and Henrik Nordenhem are
kindly acknowledged for technical assistance.
Funding sources - The Swedish Research Council for Environment, Agricultural
Sciences and Spatial Planning (Formas), the Swedish forestry sector through “The
Swedish Hylobius Research Program”, and Linnaeus University, Kalmar, have
funded the project.
17
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 40
357
358
REFERENCES
359 1. Långström, B.; Day, K. R., Damage control and management of weevil pests,
360
361
362
especially Hylobius abietis. In Bark and wood boring insects in living trees in Europe, a
synthesis, Lieutier, F.; Day, K. R.; Battisti, A.; Gregoire, J. C.; Evans, H. F., Eds.
Kluwer Academic Publisher: Dordrecht, 2004; pp 415–444.
363 2. von Sydow, F., Abundance of pine weevils (Hylobius abietis) and damage to
364
365
conifer seedlings in relation to silvicultural practices. Scand. J. Forest Res. 1997,
12, 157-167.
366 3. Örlander, G.; Nilsson, U., Effect of reforestation methods on pine weevil
367
368
(Hylobius abietis) damage and seedling survival. Scand. J. Forest Res. 1999, 14, 341-
354.
369 4. Petersson, M.; Örlander, G., Effectiveness of combinations of shelterwood,
370
371
scarification, and feeding barriers to reduce pine weevil damage. Can. J. Forest
Res. 2003, 33, 64-73.
372 5. Nordlander, G.; Hellqvist, C.; Hjelm, K., Replanting conifer seedlings after pine
373
374
weevil emigration in spring decreases feeding damage and seedling mortality.
Scand J. Forest Res. 2017, 32, 60-67.
375 6. Nordlander, G.; Hellqvist, C.; Johansson, K.; Nordenhem, H., Regeneration of
376
377
European boreal forests: Effectiveness of measures against seedling mortality
caused by the pine weevil Hylobius abietis. Forest Ecol. Man. 2011, 262, 2354-2363.
378 7. Björklund, N.; Nordlander, G.; Bylund, H., Host-plant acceptance on mineral
379
380
soil and humus by the pine weevil Hylobius abietis (L.). Agr. Forest Entomol. 2003,
5, 61-65.
381 8. Petersson, M.; Örlander, G.; Nordlander, G., Soil features affecting damage to
382 conifer seedlings by the pine weevil Hylobius abietis. Forestry 2005, 78, 83-92.
383 9. Nordlander, G.; Nordenhem, H.; Hellqvist, C., A flexible sand coating
384
385
(Conniflex) for the protection of conifer seedlings against damage by the pine
weevil, Hylobius abietis. Agr. Forest Entomol. 2009, 11, 91-100.
18
ACS Paragon Plus Environment

Page 19 of 40
Journal of Agricultural and Food Chemistry
386 10. Giurca, A.; von Stedingk, H. Forest Stewardship Council pesticides policy in
387 Sweden. http://se.fsc.org/rapporter.289.htm. (Accessed: 12 Oct 2018).
388 11. Kolmodin-Hedman, B.; Akerblom, M.; Flato, S.; Alex, G., Symptoms in forestry
389
390
workers handling conifer plants treated with permethrin. Bull. Environ. Contam.
Tox. 1995, 55, 487-93.
391 12. Zas, R.; Björklund, N.; Sampedro, L.; Hellqvist, C.; Karlsson, B.; Jansson, S.;
392
393
Nordlander, G., Genetic variation in resistance of Norway spruce seedlings to
damage by the pine weevil Hylobius abietis. Tree Genetics Genomes 2017, 13, 111.
394 13. Jermy, T., Prospects of antifeedant approach to pest-control - a critical-review.
395 J. Chem. Ecol. 1990, 16, 3151-3166.
396 14. Deletre, E.; Schatz, B.; Bourguet, D.; Chandre, F.; Williams, L.; Ratnadass, A.;
397
398
Martin, T., Prospects for repellent in pest control: current developments and
future challenges. Chemoecology 2016, 26, 127-142.
399 15. Pan, L.; Ren, L. L.; Chen, F.; Feng, Y. Q.; Luo, Y. Q., Antifeedant activity of
400
401
Ginkgo biloba secondary metabolites against Hyphantria cunea larvae: mechanisms
and applications. Plos One 2016, 11 (5) e0155682.
402 16. Bruce, T. J. A.; Smart, L. E.; Birch, A. N. E.; Blok, V. C.; MacKenzie, K.;
403
404
405
Guerrieri, E.; Cascone, P.; Luna, E.; Ton, J., Prospects for plant defence activators
and biocontrol in IPM - Concepts and lessons learnt so far. Crop. Prot. 2017, 97,
128-134.
406 17. Wang, Y. L.; Xing, X.; Zhao, H. B.; Chen, Q.; Luo, W. Q.; Ren, B. Z., Screening of
407
408
essential oil antifeedants in the elm pest Ambrostoma quadriimpressum
(Coleoptera: Chrysomelidae). Fla Entomol. 2016, 99, 231-238.
409 18. Egigu, M. C.; Ibrahim, M. A.; Yahya, A.; Holopainen, J. K., Cordeauxia edulis
410
411
and Rhododendron tomentosum extracts disturb orientation and feeding behavior
of Hylobius abietis and Phyllodecta laticollis. Entomol. Ex.p Appl. 2011, 138, 162-174.
412 19. Eriksson, C.; Månsson, P. E.; Sjödin, K.; Schlyter, F., Antifeedants and feeding
413
414
stimulants in bark extracts of ten woody non-host species of the pine weevil,
Hylobius abietis. J. Chem. Ecol. 2008, 34, 1290-1297.
19
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 40
415 20. Klepzig, K. D.; Schlyter, F., Laboratory evaluation of plant-derived antifeedants
416
417
against the pine weevil Hylobius abietis (Coleoptera : Curculionidae). J. Econ.
Entomol. 1999, 92, 644-650.
418 21. Luik, A.; Sibul, I.; Voolma, K., On the influence of some plant extracts and
419
420
neem preparations on the maturation feeding of the large pine weevil. Baltic
Forestry 2000, 6, 53-58.
421 22. Månsson, P. E.; Eriksson, C.; Sjödin, K., Antifeedants against Hylobius abietis
422
423
pine weevils: An active compound in extract of bark of Tilia cordata linden. J.
Chem. Ecol. 2005, 31, 989-1001.
424 23. Månsson, P. E.; Schlyter, F.; Eriksson, C.; Sjödin, K., Nonanoic acid, other
425
426
alkanoic acids, and related compounds as antifeedants in Hylobius abietis pine
weevils. Entomol. Exp. Appl. 2006, 121, 191-201.
427 24. Salom, S. M.; Carlson, J. A.; Ang, B. N.; Grosman, D. M.; Day, E. R., Laboratory
428
429
430
evaluation of biologically-based compounds as antifeedants for the pales weevil,
Hylobius pales (Herbst) (Coleoptera, Curculionidae). J. Entomol. Sci. 1994, 29, 407-
419.
431 25. Axelsson, K.; Konstanzer, V.; Rajarao, G. K.; Terenius, O.; Seriot, L.;
432
433
434
Nordenhem, H.; Nordlander, G.; Borg-Karlson, A. K., Antifeedants produced by
bacteria associated with the gut of the pine weevil Hylobius abietis. Microb. Ecol.
2017, 74, 177-184.
435 26. Azeem, M.; Rajarao, G. K.; Nordenhem, H.; Nordlander, G.; Borg-Karlson, A.
436
437
K., Penicillium expansum volatiles reduce pine weevil attraction to host plants. J.
Chem. Ecol. 2013, 39, 120-128.
438 27. Azeem, M.; Rajarao, G. K.; Terenius, O.; Nordlander, G.; Nordenhem, H.;
439
440
441
Nagahama, K.; Norin, E.; Borg-Karlson, A. K., A fungal metabolite masks the
host plant odor for the pine weevil (Hylobius abietis). Fungal. Ecol. 2015, 13, 103-
111.
442 28. Azeem, M.; Terenius, O.; Rajarao, G. K.; Nagahama, K.; Nordenhem, H.;
443
Nordlander, G.; Borg-Karlson, A. K., Chemodiversity and biodiversity of fungi
20
ACS Paragon Plus Environment

Page 21 of 40
Journal of Agricultural and Food Chemistry
444
associated with the pine weevil Hylobius abietis. Fung. Biol.-Uk 2015, 119, 738-746.
445 29. Borg-Karlson, A. K.; Nordlander, G.; Mudalige, A.; Nordenhem, H.; Unelius, C.
446
447
R., Antifeedants in the feces of the pine weevil Hylobius abietis: Identification and
biological activity. J. Chem. Ecol. 2006, 32, 943-957.
448 30. Fedderwitz, F.; Bjorklund, N.; Ninkovic, V.; Nordlander, G., Does the pine
449
450
weevil (Hylobius abietis) prefer conifer seedlings over other main food sources?
Silva Fenn. 2018, 52 (3) doi.org/10.14214/sf.9946.
451 31. Wallertz, K.; Nordlander, G.; Örlander, G., Feeding on roots in the humus layer
452 by adult pine weevil, Hylobius abietis. Agr. Forest Entomol. 2006, 8, 273-279.
453 32. Nordlander, G.; Nordenhem, H.; Borg-Karlson, A.-K.; Unelius, R. Preparation
454
455
of benzoate and benzyl derivatives insect repellents for conifer sapling
protection. WO2000056152A1, 2000.
456 33. Bratt, K.; Sunnerheim, K.; Nordenhem, H.; Nordlander, G.; Långström, B., Pine
457
458
weevil (Hylobius abietis) antifeedants from lodgepole pine (Pinus contorta). J.
Chem. Ecol. 2001, 27, 2253-2262.
459 34. Sunnerheim, K.; Nordqvist, A.; Nordlander, G.; Borg-Karlson, A. K.; Unelius,
460
461
462
C. R.; Bohman, B.; Nordenhem, H.; Hellqvist, C.; Karlen, A., Quantitative
structure-activity relationships of pine weevil antifeedants, a multivariate
approach. J. Agr. Food Chem. 2007, 55, 9365-9372.
463 35. Bohman, B.; Nordlander, G.; Nordenhem, H.; Sunnerheim, K.; Borg-Karlson, A.
464
465
K.; Unelius, C. R., Structure-activity relationships of phenylpropanoids as
antifeedants for the pine weevil Hylobius abietis. J. Chem. Ecol. 2008, 34, 339-352.
466 36. Sunnerheim, K.; Nordlander, G.; Bratt, K.; Nordenhem, H.; Unelius, R.; Borg-
467
468
469
Karlson, A.-K. Compositions containing cinnamate and phenylpropanoate
derivatives for conifer sapling protection against Curculionidae attacks.
WO2002015691A1, 2002.
470 37. Unelius, C. R.; Nordlander, G.; Nordenhem, H.; Hellqvist, C.; Legrand, S.;
471
472
Borg-Karlson, A. K., Structure-activity relationships of benzoic acid derivatives
as antifeedants for the pine weevil, Hylobius abietis. J. Chem. Ecol. 2006, 32, 2191-
21
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 40
473
2203.
474 38. Legrand, S.; Nordlander, G.; Nordenhem, H.; Borg-Karlson, A. K.; Unelius, C.
475
476
R., Hydroxy-methoxybenzoic methyl esters: Synthesis and antifeedant activity
on the pine weevil, Hylobius abietis. Z Naturforsch B 2004, 59, 829-835.
477 39. Blaney, W. M.; Simmonds, M. S.; Evans, S. V.; Fellows, L. E., The role of the
478
479
secondary plant compound 2,5-dihydroxymethyl 3,4-dihydroxypyrrolidine as a
feeding inhibitor for insects. Entomol. Exp. Appl. 1984, 36, 209-216.
480 40. Baeckström, P.; Stridh, K.; Li, L.; Norin, T., Claisen rearrangements with
481
482
483
mesityloxide dimethyl ketal. Synthesis of ipsdienone, E- and Z-ocimenone, 2,6-
dimethyl-2,7-octadien-4-one and 2,6-dimethyl-2,7-octadien-4-ol. Acta. Chem.
Scand. B 1987, 41, 442-447.
484 41. Rosecke, J.; König, W. A., Odorous compounds from the fungus Gloeophyllum
485 odoratum. Flavour Frag. J. 2000, 15, 315-319.
486 42. Rosecke, J.; Pietsch, M.; König, W. A., Volatile constituents of wood-rotting
487 basidiomycetes. Phytochem. 2000, 54, 747-750.
488 43. von Sydow, F., Fungi occurring in the roots and basal parts of one-year-old and
489 2-year-old spruce and pine stumps. Scand. J. Forest Res. 1993, 8, 174-184.
490 44. Cowles, R. S.; Miller, J. R.; Hollingworth, R. M.; Abdel-Aal, M. T.; Szurdoki, F.;
491
492
493
Bauer, K.; Matolcsy, G., Cinnamyl derivatives and monoterpenoids as
nonspecific ovipositional deterrents of the onion fly. J. Chem. Ecol. 1990, 16, 2401-
2428.
494 45. Huang, Y.; Ho, S., Toxicity and antifeedant activities of cinnamaldehyde
495
496
against the grain storage insects, Tribolium castaneum (Herbst) and Sitophilus
zeamais Motsch. J. Stored Prod. Res. 1998, 34, 11-17.
497 46. Sadeghian, H.; Attaran, N.; Jafari, Z.; Saberi, M. R.; Pordel, M.; Riazi, M. M.,
498
499
500
Design and synthesis of 4-methoxyphenylacetic acid esters as 15-lipoxygenase
inhibitors and SAR comparative studies of them. Bioorg. Med. Chem. Lett. 2009,
17, 2327-2335.
501 47. Ishimata, N.; Ito, H.; Tai, A., Structure–activity relationships of vanillic acid
22
ACS Paragon Plus Environment

Page 23 of 40
Journal of Agricultural and Food Chemistry
502
503
ester analogs in inhibitory effect of antigen-mediated degranulation in rat
basophilic leukemia RBL-2H3 cells. Bioorg. Med. Chem. Lett. 2016, 26, 3533-3536.
504 48. Lima, T. C.; Ferreira, A. R.; Silva, D. F.; Lima, E. O.; de Sousa, D. P., Antifungal
505
506
activity of cinnamic acid and benzoic acid esters against Candida albicans strains.
Nat. Prod. Res. 2017, 1-4.
507 49. Skogsstyrelsen. Produktion av skogsplantor 2017, (Production of seedlings
508
509
510
2017) Statistiska Meddelanden JO0313 SM 1801 [Online at
https://www.skogsstyrelsen.se/statistik/statistik-efter-amne/produktion-av-
skogsplantor/], 2018 (Accessed: 12 Oct 2018).
23
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 40
24
ACS Paragon Plus Environment

Page 25 of 40
Journal of Agricultural and Food Chemistry
Figure Captions
Figure 1. Structures of compounds tested for antifeedant activity.
Figure 2. Structures of compounds tested for antifeedant activity.
Figure 3. Scots pine twig with treatment and control area used in the two-choice
feeding bioassay with H. abietis.
25
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 40
Table Legends
Table 1. Effect of Functional Group and the Substituents on the Aromatic Ring on
the Activity of Antifeedant for the Pine Weevil, Hylobius abietis
Table 2. Effect of Aromatic Ring Substituents on Antifeedant Activity for the Pine
Weevil, Hylobius abietis.a
Table 3. Antifeedant Activity at Low Concentration, 5 mM, for the Pine Weevil,
Hylobius abietis sorted after AFIa Rank.
Table 4. Test Setup for Synergetic Effects between Three Selected Antifeedants
from the Three Substance Classes.
Table 5. Antifeedant Activity of the Treatments in the Synergy Experiment for the
Pine Weevil, Hylobius abietis.
a Ranking between all 61 compounds tested at 50 mM concentration.
26
ACS Paragon Plus Environment

Page 27 of 40
Journal of Agricultural and Food Chemistry
Table 1. Effect of Functional Group and the Substituents on the Aromatic Ring on
the Activity of Antifeedant for the Pine Weevil, Hylobius abietis.a
Compound
No.
Rank
AFIa
Rank Fisher
AFIn test b
Compound
AFIa
-4
AFIn
2
1
2
3,5-Dimethoxybenzoic acid
Methyl 3,5-dimethoxybenzoate
3,4-Methylenedioxybenzoic acid
60
17
55
38
54
34
50
17
58
31
57
51
59
10
57
17
48
43
57
32
56
16
57
28
52
49
61
12
ns
***
ns
95
14
57
17
74
22
95
7
84
11
25
2
3
Methyl
4
**
3,4-methylenedioxybenzoate
2-Hydroxy-5-methoxybenzoic
acid
5
ns
Methyl
6
56
3
***
ns
2-hydroxy-5-methoxybenzoate
2-Hydroxy-3-methoxybenzoic
acid
7
Methyl 2-hydroxy-
3-methoxybenzoate
8
85
2
***
ns
9
3,4-Dimethoxybenzoic acid
10
11
12
13
14
Methyl 3,4-dimethoxybenzoate
81
10
21
1
66
5
***
ns
(4-Hydroxy-
3-methoxyphenyl)acetic acid
Methyl (4-hydroxy-
3-methoxyphenyl)acetate
9
ns
3,5-Dimethoxyphenylacetic acid
-4
93
ns
Methyl
98
***
(3,5-dimethoxyphenyl)acetate
a Ranking between all 61 compounds tested at 50 mM concentration.
b * = P<0.05, ** = P<0.01, *** = P<0.001.
27
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 40
15
16
17
18
3-(2-Methylphenyl)propanoic acid
47
91
12
97
44
21
56
13
12
75
4
47
24
53
7
*
Methyl 3-
(2-methylphenyl)propanoate
***
ns
3-(2-Methoxyphenyl)propanoic
acid
Methyl 3-
(2-methoxyphenyl)propanoate
95
***
28
ACS Paragon Plus Environment

Page 29 of 40
Journal of Agricultural and Food Chemistry
Table 2. Effect of Aromatic Ring Substituents on Antifeedant Activity for the Pine
Weevil, Hylobius abietis.a
Compound
No.
Rank
AFIa
Rank Fisher
AFIn
Compound
AFIa
AFIn
13
26
8
test b
19
20
21
22
23
24
25
26
12
27
28
29
30
31
Methyl 2-hydroxybenzoate 21
Methyl 4-hydroxybenzoate 34
51
48
51
45
61
49
46
42
51
39
35
39
37
32
45
*
41
50
50
57
45
53
44
49
38
38
41
33
34
**
ns
Methyl 3-
21
(4-hydroxyphenyl)propanoate
Methyl 2,4-dihydroxybenzoate 46
Methyl 3,4-dihydroxybenzoate -7
Methyl 3,5-dihydroxybenzoate 23
8
ns
2
ns
13
4
ns
Methyl 4-hydroxy-
35
ns
2-methoxybenzoate
Methyl 4-hydroxy-
53
22
9
*
3-methoxybenzoate
Methyl (4-hydroxy-
21
ns
3-methoxyphenyl)acetate
Methyl 3-(4-hydroxy-
54
32
32
26
52
51
***
***
***
***
***
3-methoxyphenyl)propanoate
Methyl 3-hydroxy-
65
4-methoxybenzoate
Methyl 3-hydroxy-
54
5-methoxybenzoate
Methyl 2-hydroxy-
60
4-methoxybenzoate
Methyl 2-methoxybenzoate 80
a Ranking between all 61 compounds tested at 50 mM concentration.
b * = P<0.05, ** = P<0.01, *** = P<0.001.
29
ACS Paragon Plus Environment