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The Role of Leaf Volatiles of Ludwigia octovalvis (Jacq.) Raven in the

Attraction of Altica cyanea (Weber) (Coleoptera: Chrysomelidae)

Article  in  Journal of Chemical Ecology · July 2017

DOI: 10.1007/s10886-017-0866-4

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AUTHOR'S PROOF JrnlID 10886_ArtID 866_Proof# 1 - 03/07/2017

J Chem Ecol
DOI 10.1007/s10886-017-0866-4
1
3
2

4 The Role of Leaf Volatiles of Ludwigia octovalvis (Jacq.) Raven

5 in the Attraction of Altica cyanea (Weber)
6 (Coleoptera: Chrysomelidae)
7 Saubhik Mitra 1 & Amarnath Karmakar 1 & Abhishek Mukherjee 1 & Anandamay Barik 1
8

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9 Received: 6 February 2017 / Revised: 23 June 2017 / Accepted: 30 June 2017
10 # Springer Science+Business Media, LLC 2017

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11 Abstract Larvae and adults of Altica cyanea (Weber) attractive to A. cyanea females, due to elevated emissions of 36

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12 (Coleoptera: Chrysomelidae) feed on the rice-field weed 3-hexanol, α-pinene, linalool oxide, geraniol, and phytol. 37
13 Ludwigia octovalvis (Jacq.) Raven (Onagraceae), commonly
14 known as willow primrose, which is considered a biocontrol
D Keywords Herbivore induced volatiles . Volatiles . VOCs . 38
15 agent of the weed. Volatile organic compounds from undam- 3-Hexanol . α-Pinene . Linalool oxide . Geraniol . Phytol . 39
16 Coleoptera . Chrysomelidae . Olfactometer bioassay 40
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aged plants, plants after 4, 12, and 36 h of continuous feeding
17 by A. cyanea larvae or adult females and after mechanical
18 damaging were identified by GC-MS and GC-FID analyses.
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19 Twenty nine compounds were identified from undamaged Introduction 41 Q1

20 plants. 2Z–Penten-1-ol, geraniol, and 1-tridecanol were pres-
21 ent in all plants damaged by larvae. In contrast, feeding by Ludwigia octovalvis (Jacq.) Raven (Onagraceae), commonly 42
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22 adults caused the release of 2Z–penten-1-ol only after 12 and known as willow primrose, is an important rice-field weed in 43
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23 36 h; whereas geraniol and 1-tridecanol appeared only after India. The presence of this weed has been recorded from 44
24 36 h. Farnesyl acetone was detected after 12 and 36 h of Pacific islands, South and Central Asia, Australia, over 45
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25 feeding by larvae and after 36 h of feeding by adults. much of Africa, and in warm areas of the Americas (Caton 46
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26 Farnesene was detected after 36 h of feeding by larvae and et al. 2010; Chauhan and Abugho 2012; Mamun 2014; 47
27 48
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adults. Linalool was unique after 36 h of feeding by larvae. In Mesquita et al. 2013; Moody 1989; Raju and Reddy 1986).
28 Y-shaped glass tube olfactometer bioassays, A. cyanea fe- Rapid growth and high seed production of the weed as well as 49
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29 males were attracted to volatiles after 36 h of feeding by larvae its growth from the root stock after cutting, poses a significant 50
30 or adults compared to volatiles released by undamaged plants. threat to rice as the plant competes with rice for resources in 51
31 The insects were attracted to five synthetic compounds: 3- the field. Heavy infestations result in retarded growth of rice, 52
32 hexanol, α-pinene, linalool oxide, geraniol, and phytol. which can lead to substantial economic losses. To address this, 53
33 Synthetic blends were more attractive than individual com- rice-growers usually apply herbicides (bensulfuron, butachlor, 54
34 pounds. Compared to undamaged plants, volatiles released fenoprop, imidazolinone, propanil, MCPA, 2,4-D, quinclorac, 55
35 by plants, damaged by conspecific individuals, were more and thiobencarb) as primary control agents (Chin et al. 2007; 56
Imeokparia 1994; Imeokparia et al. 1992; Kandasamy and 57
Palaniappan 1990; Raju and Reddy 1986). However, applica- 58
Electronic supplementary material The online version of this article tions of synthetic herbicides may be harmful to the users as 59
(doi:10.1007/s10886-017-0866-4) contains supplementary material,
well as to the environment. Furthermore, chemical agents may 60
which is available to authorized users.
destroy or reduce the natural enemies of the weed, which may 61
have an adverse impact on the ecosystem. In this context, it is 62
* Anandamay Barik
anandamaybarik@yahoo.co.in advantageous to use natural enemies to control L. octovalvis in 63
biological control systems. 64
1
Ecology Research Laboratory, Department of Zoology, The The flea beetle, Altica cyanea (Weber) (Coleoptera: 65
University of Burdwan, Burdwan, West Bengal 713 104, India Chrysomelidae) feeds on Ludwigia spp. (Azad et al. 2015; 66
AUTHOR'S PROOF
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J Chem Ecol

67 Nayek and Banerjee 1987), and larvae of the beetle voracious- (CRF), The University of Burdwan, (23°16′ N, 87°54′ E), 117
68 ly consume leaves during three instars to complete larval de- West Bengal, India. The insect colony was maintained on 118
69 velopment, whereas adults feed on weed leaves for 45–55 days L. adscendens leaves in an environmental chamber at 119
70 (Nayek and Banerjee 1987; Xiao-Shui 1990). The beetle oc- 27 ± 1 °C temperature, 65 ± 10% relative humidity, and a 120
71 curs widely in several states of India, indicating its potential as photoperiod of 12 L: 12 D. Between 1- and 2-week-old F2 121
72 a biocontrol agent with strong ecological flexibility. The insect A. cyanea females were used for olfactory bioassays, because 122
73 has also been recorded from Bangladesh, Thailand, Vietnam, they use olfactory cues to locate places both for feeding and 123
74 Pakistan, China, Japan, and Malayasia (Alam and Karim egg laying (Schoonhoven et al. 2005). Before the tests, the 124
75 1980; Azad et al. 2015; Dubey 1981; Maulik 1936; Naples insects were not provided with food for 12 h, but with water. 125
76 and Kessler 2005; Nayek and Banerjee 1987; Xiao-Shui
77 1990).
Plant Uninfested, ca. 8 cm high L. octovalvis plants were 126
78 Insect herbivores use plant volatiles as chemical cues to
handpicked from the CRF, The University of Burdwan and 127
79 recognize suitable host plants as food and for oviposition
planted individually in 12 cm diameter earthen pots contain- 128
80 (Bruce and Pickett 2011; Bruce et al. 2005; Mukherjee et al.
ing ca. 1500 cm3 of soil [organic matter 5.3 ± 0.2% (± 129

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81 2015; Piesik et al. 2011; Schoonhoven et al. 2005; Tasin et al.
Standard Error), pH 7.7]. Plants were grown under natural 130
82 2007). Some of the volatile organic compounds (VOCs) might

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condition during January–February, 2016 at 15–20 °C, 13 h: 131
83 be ubiquitous in volatile blends of different plants growing in
11 h (D:L) photoperiod. Individual whole plants with pot were 132

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84 the habitat, but insects can recognize particular host plants by
covered with a fine mesh nylon net [50 cm (height) × 30 cm 133
85 their specific VOC profile. Furthermore, attraction may

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(diameter)] to prevent insect attacks and infections. Three to 134
86 strongly decrease already upon changes in the pattern of minor
four-week-old plants (about 13–15 cm height) with 10 fully 135
87 constituents of the volatile blends (Najar-Rodriguez et al.
expanded leaves were used for collection of volatiles. 136
88 2010; Padmaja et al. 2010; Malik et al. 2016; Tasin et al.
D
89 2007; Sarkar et al. 2016). So, the specific combination and
90 137
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affixed ratios of VOCs result in species-specific insect attrac- Plant Volatile Collection Ludwigia octovalvis plants were
91 tion (Bruce and Pickett 2011). Hence, identification of corre- placed into environmental chambers (27 ± 1 °C, 65 ± 5% 138
92 sponding behavior mediating VOCs could help develop future RH, and 13 L: 11D) to collect volatiles from undamaged 139
EC

93 biocontrol programs (Padovan et al. 2010; Piesik et al. 2012; (UD), insect damaged (ID), and mechanically damaged 140
94 Smith and Beck 2013; Wheeler and Schaffner 2013). No vol- (MD) plants. Six feeding experiments were conducted with 141
95 atiles released from L. octovalvis leaves to attract A. cyanea ID treatments: (a) plants on which 5 adult females had fed 142
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96 have yet been identified. Furthermore, the induction of VOCs continuously for 4 h or 12 h or 36 h (here after ‘adult feeding’, 143
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97 in plants can vary with herbivore species as well as with de- denoted as ‘AF’), and (b) plants on which 5 s instar larvae had 144
98 velopmental stage (Gouinguene et al. 2003, Piesik et al. fed continuously for 4 h or 12 h or 36 h (here after ‘larval 145
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99 2013a). Hence, it will be interesting to investigate, whether feeding’, denoted as ‘LF’). Immediately after feeding of adults 146
C

100 the composition of volatiles released by L. octovalvis host or larvae, damaged plants were used to collect VOCs. Newly 147
101 148
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plants is different when attacked by either A. cyanea adults emerged second instar larvae were used for all larvae feeding
102 or larvae. To determine this, we conducted experiments to experiments. Volatiles were also collected from UD plants 149
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103 analyze VOCs released by undamaged and mechanically with 10 leaves. For the mechanical damage treatment, 10 150
104 damaged L. octovalvis plants, and from those damaged by leaves of a L. octovalvis plant were wounded once with hole 151
105 adults and larvae. We also studied the behavioral responses punch (each hole 0.5 cm diameter), and volatiles were collect- 152
106 of A. cyanea females to natural volatile blends released from ed right after wounding. Plants with UD, ID, and MD leaves 153
107 undamaged plants, plants damaged by conspecific adults or were placed individually in 4-L closed glass domes with 154
108 larvae, and mechanically damaged plants against each other Teflon bases leaving only a small opening for the stem of 155
109 by using a Y-shaped glass tube olfactometer under laboratory the plant, and the pot was outside the glass dome. Sterilized 156
110 condition. We further studied the attractiveness of individual cotton balls were loosely plugged around the stem of the plant 157
111 synthetic volatiles and synthetic blends equivalent to natural to prevent any abrasion by the Teflon bases. Volatiles from all 158
112 volatiles released by undamaged and differently treated treatments (N = 5 replicates for each treatment) were collected 159
113 L. octovalvis plants. over 6 h during the light phase between 10.00 and 16.00. 160
Charcoal-filtered air was pushed (6 L min−1) into the top of 161
the closed chamber and pulled (1 L min−1) through volatile 162
114 Methods and Materials collector traps (150 mm long × 5 mm o.d.) containing 100 mg 163
of HayeSep Q (80–100 mesh, Sigma Aldrich, Germany) as an 164
115 Insect Adults of A. cyanea were collected by light trap from adsorbent, that were inserted into 4 side sampling ports around 165
116 L. adscendens L. weeds growing in the Crop Research Farm the base of the glass chamber. 166
AUTHOR'S PROOF JrnlID 10886_ArtID 866_Proof# 1 - 03/07/2017

J Chem Ecol

167 Volatiles from each treatment were eluted from the adsor- farnesyl acetone (≥90%) (mixture of isomers), and phytol 217
168 bent with 600 μl methylene chloride and concentrated to (≥97%) were purchased from Sigma Aldrich, Germany. 218
169 150 μl under a flow of nitrogen. From the concentrated
170 150 μl headspace samples of each treatment: (a) 75 μl were Olfactometer Bioassays Behavioral responses of A. cyanea 219
171 used for olfactory bioassays, and (b) 75 μl for chemical anal- females to volatiles were investigated in a Y-shaped glass tube 220
172 yses (GC-MS and GC-FID). For olfactory bioassays, an ali- olfactometer (stem and arms 15 cm long, 0.6 cm radius, 45° Y 221
173 quot of 25 μl (equivalent to volatiles released by a plant during angle) (Karmakar et al. 2016; Mukherjee et al. 2015). The stem 222
174 ca. 1 h) were applied to Whatman No. 41 filter paper (1 cm2). of the olfactometer was connected to a porous glass vial (1 cm 223
175 For quantification through GC, nonyl acetate was added as the radius × 3 cm long) (for passing air through the olfactometer) in 224
176 internal standard (IS) in an amount of 0.4 μg/μl. Solvents which test insects were released. Each arm of the olfactometer 225
177 were purchased from Sigma Aldrich, Germany. was connected to a glass-made micro kit adapter fitted into a 226
glass vial (1 cm radius × 3 cm long). One glass vial contained a 227
178 Volatile Analysis Chemical structures of collected volatiles piece (1 cm2) of Whatman No. 41 filter paper loaded with 25 μl 228
179 were assigned by using gas chromatography coupled with of a headspace sample, while the other glass vial contained a 229

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180 mass spectrometry (GC − MS system: Agilent 6890, coupled filter paper of the same size carrying 25 μl of the control solvent 230

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181 with a 5973 Mass Selective Detector). Separation of individ- (methylene chloride). Charcoal-filtered air was pushed into 231
182 ual compounds was carried out with a SE-30 capillary column each arm of the olfactometer at 125 ml/min. Connections be- 232

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183 (Agilent; Palo Alto, CA, USA; length: tween different parts of the set-up consisted of silicon tubing. 233
234

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184 30 m × 0.25 mm × 0.25-μm film thickness). The oven tem- Reactions of 1–2-week-old A. cyanea females were evalu-
185 perature was initially set to 50 °C held for 3 min, then raised to ated in the laboratory at 27 ± 1 °C, 70 ± 5% RH, and a light 235
186 240 °C at a rate of 3.75 °C/min and finally held for 5 min. intensity of 150 lx. For each experiment, 25 μl of a headspace 236
187 D
Helium was the carrier gas. The MS parameters were 250 °C sample and the control solvent were applied to separate filter 237
188 at the interface, ionization energy 70 eV, and scan speed ap- paper pieces and after evaporation of the solvent introduced 238
TE
189 proximately 1 s. At a split ratio of 1:5, 1 μl of a sample was into the glass vials before the first insect was released into the 239
190 injected (Sarkar et al. 2016), and identities of VOCs were olfactometer. A single adult female was introduced into the 240
191 porous glass vial, which was then connected to the stem of the 241
EC

confirmed by comparison of mass spectra (NIST 2008 library)

192 and GC retention times with those of authentic standards olfactometer and exposed to the test sample. The behavior of 242
193 (Sigma Aldrich). Kovats retention indices (for temperature- each female was observed for 3 min. A female was considered 243
244
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194 programming) were calculated according to Van den Dool to have made a choice when it reached the end of one of the
195 and Kratz (1963) by running C7–C28 straight-chain alkanes two arms, and the choice was recorded as a positive response 245
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196 under the same temperature program. or negative response, respectively. When a female did not 246
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197 Volatiles were quantified by using a Techcomp GC (Em make a choice within 3 min (remained in the common arm 247
198 Macau, Rua De Pequim, Nos. 202A-246, Centro Financeiro of the Y-tube), it was recorded as “no response” and excluded 248
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199 F7, Hong Kong) model 7900 equipped with a SE-30 capillary from the statistical analysis (Adhikary et al. 2015; Karmakar 249
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200 column, which was run under the same temperature condi- et al. 2016). Each experiment with one volatile sample was 250
201 repeated until 90 näive females had responded. After testing 5 251
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tions and split ratio as described for GC-MS analysis. The

202 carrier gas was nitrogen with a flow rate of 18 ml/min, and insects, filter papers containing the control solvent or the test 252
203 the injector port temperature was 280 °C. sample were replaced. The olfactometer set-up was cleaned 253
with hexanes followed by acetone, and the position of the two 254
arms was systematically changed in order to avoid positional 255
204 Chemicals HayeSep Q (80–100 mesh), 2Z–Penten-1-ol bias. After testing 5 individuals, silicon tubes were cleaned 256
205 (95%), 3-hexanol (97%), 2-hexanol (99%), 1-hexanol twice with 70% ethanol and air-dried at room temperature 257
206 (≥99.5%), 2E–hexen-1-ol (96%), 2-heptanone (99%), α-pi- for at least 2 h before further use. 258
207 nene (≥99%), 2E–heptenal (≥95%), benzaldehyde (≥99%),
208 1-heptanol (≥99.5%), sabinene (75%), 1-octen-3-ol (≥98%),
209 3-octanone (≥98%), 3-octanol (99%), 2-octanol (≥97%), Dual Choice Bioassays with A. cyanea Females 259
210 (R)-(+)-limonene (97%), benzyl alcohol (99.8%), ocimene
211 (cis/trans-mixture) (≥90%), linalool oxide (furanoid structure) Bioassay 1 Behavioral responses of A. cyanea to headspace 260
212 (≥97%), 1-octanol (≥99%), nonanal (≥98%), linalool (97%), volatiles from UD, LF, AF, and MD plants were tested against 261
213 limonene oxide (97%), 3Z–nonen-1-ol (95%), 2E–nonenal the solvent control, methylene chloride. 262
214 (97%), 1-nonanol (≥98%), decanal (≥98%), nerol (≥97%),
215 geraniol (98%), 1-undecanol (99%), α-farnesene (mixture of Bioassay 2 Behavioral responses of A. cyanea to headspace 263
216 isomers), 1-tridecanol (97%), methyl jasmonate (≥97%), volatiles of plants on which adults or larvae had been 264
AUTHOR'S PROOF
JrnlID 10886_ArtID 866_Proof# 1 - 03/07/2017

J Chem Ecol

265 continuously feeding (A: 4 h AF, B: 4 h LF, C: 12 h AF, D: synthetic blend consisting of linalool oxide and phytol, equiv- 284
266 12 h LF, E: 36 h AF, F: 36 h LF) were tested against volatiles alent to volatiles collected after 12 h LF during ca. 1 h, was 285
267 of UD or MD plants, and MD plants vs. UD plants. tested against the solvent control. In addition, a synthetic blend 286
consisting of α-pinene, linalool oxide, and phytol, equivalent to 287
268 Bioassay 3 Behavioral responses of A. cyanea to headspace volatiles collected after 12 h AF during ca. 1 h, was tested 288
269 volatiles of ID plants were tested against each other. against the solvent control (Supplementary Table 1a). 289
A synthetic blend of 10 compounds (3-hexanol, α-pinene, 290
270 Bioassay 4 Behavioral responses of A. cyanea to complete benzaldehyde, 3-octanol, linalool oxide, decanal, nerol, gera- 291
271 synthetic blends containing all compounds present in qualita- niol, 1-tridecanol, and phytol), equivalent to volatiles collect- 292
272 tive and quantitative proportions, equivalent to headspace vol- ed after 36 h LF during ca. 1 h, was tested against the solvent 293
273 atiles collected from UD, LF, AF, and MD plants during ca. control. Furthermore, a synthetic blend of 9 compounds 3- 294
274 1 h, were tested against the solvent control. hexanol, α-pinene, benzaldehyde, 3-octanol, linalool oxide, 295
decanal, 1-tridecanol, farnesyl acetone, and phytol, equivalent 296
275 Bioassay 5 Behavioral responses of A. cyanea to individual to volatiles collected after 36 h AF during ca. 1 h, was tested 297

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276 synthetic compounds or synthetic blends (Supplementary against the solvent control. In addition, a synthetic blend 298
277 Table 1), to headspace volatiles of UD, LF, AF, and MD consisting of 3-hexanol, linalool oxide, geraniol, and phytol, 299

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278 plants, released by each type of experimental plant during equivalent to volatiles collected after 36 h LF during ca. 1 h, 300

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279 ca. 1 h, were tested against the solvent control. was tested against the solvent control. Finally, a synthetic 301
280 A synthetic blend of 6 compounds (3-hexanol, α-pinene, blend consisting of 3-hexanol, α-pinene, linalool oxide, and 302

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281 benzaldehyde, 3-octanol, linalool oxide, and phytol), equiva- phytol, equivalent to volatiles collected after 36 h AF during 303
282 lent to volatiles collected after 12 h AF and 12 h LF during ca. ca. 1 h, was tested against the solvent control (Supplementary 304
283 1 h, was tested against the solvent control. Furthermore, a
D Table 1b). 305
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Fig. 1 Principal component
analysis (PCA) of the data set of
volatile organic compounds
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obtained from undamaged

Ludwigia octovalvis plants,
mechanically damaged plants,
and plants after 4, 12 and 36 h of
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continuous feeding by Altica

cyanea larvae and adults
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t1:1 Table 1 Composition (GC-FID Analysis) of volatile organic compounds (VOCs) emitted by Ludwigia octovalvis plants (μg/6 h) under various conditions

t1:2 Compounds Undamaged Larvae feeding on plants (LF) Adults feeding on plants (AF) Mechanically χ2 0.05, 7
J Chem Ecol

t1:3 (UD) 4h 12 h 36 h 4h 12 h 36 h damaged (MD)

t1:4 2Z–Penten-1-ol - 3.24 ± 0.15a 4.93 ± 0.14b 9.31 ± 0.29c - 3.90 ± 0.16d 8.66 ± 0.41c - 38.120
t1:5 3-Hexanol 4.22 ± 0.14a 7.57 ± 0.39b 10.40 ± 0.81c 20.30 ± 0.83d 9.50 ± 0.28c 10.22 ± 0.70c 20.33 ± 1.11d 50.94 ± 3.35e 36.144
t1:6 2-Hexanol 3.82 ± 0.07a 7.34 ± 0.49b 6.70 ± 0.42b 5.24 ± 0.19c 8.75 ± 0.37d 10.53 ± 0.49e 12.23 ± 0.69f 13.25 ± 0.88f 36.205
t1:7 1-Hexanol 10.34 ± 0.38a 14.92 ± 0.70b U18.06 ± 0.62c 18.52 ± 0.55c 20.91 ± 0.93d 21.54 ± 0.95d 25.46 ± 1.31e 39.81 ± 1.73f 35.451
t1:8 2E–Hexen-1-ol 10.20 ± 0.70a 17.61 ± 0.82b 18.81 ± 0.99be 24.21 ± 0.59c 14.29 ± 0.28d 16.85 ± 0.93b 20.99 ± 1.49e 10.20 ± 0.75a 33.773
t1:9 2-Heptanone 22.26 ± 0.78a 33.50 ± 1.75b 48.86 ± 2.89c 50.49 ± 2.46c 47.26 ± 2.05c 49.17 ± 2.78c 51.95 ± 3.82c 127.02 ± 8.90d 29.924
N
t1:10 α-Pinene 7.56 ± 0.34a 10.84 ± 0.95b 16.13 ± 0.79c 16.30 ± 0.53cd 17.40 ± 0.33d 19.52 ± 0.83e 20.64 ± 1.24e 36.08 ± 2.36f 34.756
AUTHOR'S PROOF

t1:11 2E–Heptenal 6.18 ± 0.28a 10.20 ± 0.72b

C
13.94 ± 0.52c 14.52 ± 0.30c 11.33 ± 0.30b 14.48 ± 0.56c 19.90 ± 1.12d 22.38 ± 0.90e 36.108
Benzaldehyde 12.18 ± 0.33a 14.73 ± 0.47b 25.72 ± 1.09c
t1:12
O 28.90 ± 0.87d 26.95 ± 1.02cd 27.26 ± 1.36cd 28.93 ± 1.24d 52.39 ± 3.47e 31.094
t1:13 1-Heptanol 11.96 ± 0.59a 13.87 ± 0.37b 29.07 ± 1.22c
R 31.23 ± 1.40c 46.98 ± 2.52d 47.38 ± 3.73d 57.06 ± 3.12e 70.09 ± 4.17f 36.723
t1:14 Sabinene 10.36 ± 0.67a 15.25 ± 0.71b 21.68 ± 1.13c
R 26.61 ± 0.59d 26.07 ± 0.73d 30.71 ± 1.75e 31.62 ± 1.78e 43.98 ± 2.84f 35.640
t1:15 1-Octen-3-ol 17.63 ± 0.69a 20.29 ± 0.85b 31.61 ± 1.84c 32.90 ± 1.14c 25.68 ± 0.82d 33.33 ± 2.07c 41.41 ± 2.37e 72.12 ± 4.13f 35.497
t1:16 3-Octanone 2.93 ± 0.09a 3.52 ± 0.20b 4.89 ± 0.23c 4.98 ± 0.30cd 4.79 ± 0.13 c 5.23 ± 0.48cd 5.77 ± 0.41d 9.44 ± 0.91e 30.547
t1:17 3-Octanol 3.19 ± 0.12a 4.56 ± 0.24b 5.99 ± 0.49c 6.09 ± 0.32c 5.09 ± 0.15b 6.22 ± 0.61c 6.77 ± 0.44c 8.25 ± 0.61d 28.600
EC
t1:18 2-Octanol 71.52 ± 3.48a 100.78 ± 6.89b 155.51 ± 7.25c 175.80 ± 5.85d 141.71 ± 7.22c 151.98 ± 7.09c 176.74 ± 8.91d 353.50 ± 16.16e 33.679
t1:19 Limonene 9.12 ± 0.46a 9.58 ± 0.51a 11.92 ± 0.78b 16.77 ± 0.43c 12.26 ± 0.32b 12.59 ± 0.69b 15.48 ± 0.86c 20.88 ± 1.23d 33.632
t1:20 Benzyl alcohol 11.01 ± 0.61a 12.50 ± 0.71b 25.92 ± 0.86c 29.61 ± 0.59d 26.71 ± 0.86c 31.81 ± 1.62de 34.34 ± 1.99e 41.73 ± 2.30e 34.522
TE
t1:21 Ocimene 6.25 ± 0.46a 8.22 ± 0.63b 16.26 ± 0.76c 17.56 ± 0.48d 9.95 ± 0.36e 14.17 ± 0.51f 20.49 ± 0.96g 10.62 ± 0.79e 36.070
t1:22 Linalool oxide 5.95 ± 0.35a 9.29 ± 0.81b
D
14.34 ± 0.66c 20.63 ± 1.09d 9.83 ± 0.25b 15.29 ± 1.14c 20.15 ± 1.28d 17.45 ± 0.57e 35.288
t1:23 1-Octanol 5.20 ± 0.34a 8.79 ± 0.74b 10.24 ± 0.62bc 11.17 ± 0.40c 9.75 ± 0.35b 10.22 ± 0.51bc 11.25 ± 0.94c 12.91 ± 0.46e 25.390
t1:24 Nonanal 22.31 ± 0.74a 24.04 ± 1.15a 43.31 ± 3.09b 50.99 ± 2.15c 52.10 ± 2.28c 56.33 ± 3.60cd 59.77 ± 3.18d 65.96 ± 3.85e 30.834
PR
t1:25 Linalool - - - 13.35 ± 0.33 - - - -
t1:26 Limonene oxide 2.11 ± 0.12a 7.50 ± 0.53b 8.70 ± 0.31c 18.31 ± 0.55d 5.42 ± 0.18e 6.03 ± 0.25f 7.68 ± 0.64bc 15.06 ± 0.43g 36.190
O
t1:27 3Z–Nonen-1-ol 5.12 ± 0.35a 5.85 ± 0.48b 10.31 ± 0.62c 14.39 ± 0.57d 5.31 ± 0.15a O9.22 ± 0.49cf 11.97 ± 0.52e 8.61 ± 0.42f 35.093
t1:28 2E–Nonenal 1.52 ± 0.12a 4.96 ± 0.46bd 5.17 ± 0.14b 5.34 ± 0.47b 3.38 ± 0.13c 4.37 ± 0.23d 7.61 ± 0.57e - 34.837
t1:29 1-Nonanol 2.53 ± 0.11a 4.95 ± 0.44b 4.91 ± 0.18b 9.10 ± 0.42c 3.37 ± 0.12d 4.91 ± 0.18b 7.69 ± 0.57e 14.73 ± 0.60f 36.212
F
t1:30 Decanal 9.12 ± 0.30a 9.85 ± 0.46a 14.43 ± 0.81b 25.62 ± 1.08c 16.63 ± 0.58d 19.16 ± 0.98e 20.07 ± 1.15e 31.21 ± 2.28f 36.155
t1:31 Nerol 11.33 ± 0.77a 44.13 ± 3.00b 49.44 ± 2.39be 97.88 ± 2.14c 35.02 ± 1.74d 45.57 ± 2.15b 55.57 ± 3.25e 22.47 ± 1.12f 35.154
t1:32 Gernaniol - 5.90 ± 0.43a 6.31 ± 0.20a 14.17 ± 0.39b - - 7.90 ± 0.71c - 37.321
t1:33 1-Undecanol 3.41 ± 0.14a 7.22 ± 0.69be 7.42 ± 0.28b 11.07 ± 0.54c 4.71 ± 0.18d 6.13 ± 0.44e 10.36 ± 0.27c 3.48 ± 0.18a 36.020
t1:34 α-Farnesene - - - 3.30 ± 0.10a - - 11.65 ± 0.89b - 38.747
t1:35 1-Tridecanol - 3.86 ± 0.41a 4.12 ± 0.23a 5.62 ± 0.19b - - 9.41 ± 0.71c - 38.050
t1:36 Methyl jasmonate 1056.52 ± 54.96a 1505.21 ± 77.04b 1577.98 ± 72.72b 2617.24 ± 116.90c 1310.57 ± 65.96d 1550.65 ± 71.31b 1863.18 ± 82.48e 1666.27 ± 106.59b 30.711
t1:37 Farnesyl acetone - - 2.11 ± 0.12a 2.56 ± 0.17a - - 10.00 ± 0.65b - 38.454
JrnlID 10886_ArtID 866_Proof# 1 - 03/07/2017
AUTHOR'S PROOF
JrnlID 10886_ArtID 866_Proof# 1 - 03/07/2017

J Chem Ecol

χ2 0.05, 7
306

36.799
36.234
Fourteen synthetic compounds (3-hexanol, 1-hexanol, 2-
heptanone, α-pinene, benzaldehyde, 1-heptanol, 1-octen-3- 307
ol, 3-octanol, 2-octanol, limonene, linalool oxide, 1-nonanol, 308

3189.18 ± 172.19g
decanal, and phytol), equivalent to volatiles of MD plants 309
348.33 ± 26.39e
damaged (MD)
310
Mechanically

released during ca. 1 h, were tested against the solvent control.

A synthetic blend consisting of 3-hexanol, α-pinene, and lin- 311
alool oxide in similar amounts and proportions as among vol- 312
atiles of MD plants during ca. 1 h was tested against the 313
solvent control (Supplementary Table 1c). 314
4343.86 ± 187.18f
1630.01 ± 78.60cd

Bioassay 6 Behavioral responses of A. cyanea to headspace 315

volatiles after 36 h LF and 36 h AF (equivalent to volatiles 316
released during ca. 1 h) were tested against individual synthet- 317
36 h

ic compounds or synthetic blends, equivalent to volatiles after 318

F
319
2992.75 ± 145.48bg

36 h LF and 36 h AF.
757.93 ± 49.80b

O
Bioassay 7 The following compounds and amounts were test- 320

O
Adults feeding on plants (AF)

ed at different dosages against the solvent control, 3-hexanol: 321

1.5, 3, 6, and 12 μg/25 μl CH2Cl2; α-pinene: 2, 4, 8, and 322
12 h

PR
16 μg/25 μl CH2Cl2; linalool oxide: 1.5, 3, and 6 μg/25 μl 323
CH2Cl2; geraniol: 2, 4, 8, and 16 μg/25 μl CH2Cl2; phytol: 50, 324
2175.51 ± 90.56e
263.82 ± 10.15e

D 100, 200, and 400 μg/25 μl CH2Cl2. 325

326
TE
Statistical Analyses Data on amounts of total and individual
4h

(Mean ± SE, N = 5). Different superscript letters in the same entry indicate significant differences (P < 0.05)

VOCs from UD, MD, and ID L. octovalvis were not normally 327
distributed as determined by Kolmogorov-Smirnov and 328
5213.24 ± 202.88d

EC
1763.21 ± 78.11d

Shapiro-Wilk tests, and so were analyzed with nonparametric 329

Kruskal-Wallis Test followed by Dunn’s Test with α = 0.05 330
(Zar 1999). The amounts of individual VOCs present in UD, 331
R

MD, and ID plants were subjected to principal component 332

36 h

analysis (PCA) to reduce the redundancy of data and portray 333

the relationship among the variables in a biplot. Further, to 334
O
3721.71 ± 140.30c
1496.63 ± 81.51c

interpret the levels of similarity of the volatile compounds in 335

C

terms of their relative abundance, a cluster analysis (CA) 336

Larvae feeding on plants (LF)

337
N

based on Ward’s method (Legendre and Legendre 1998) was

12 h

carried out, using XL STAT software and SPSS version 16. 338
U

Data on responses of A. cyanea to VOCs were analyzed based 339

2811.09 ± 119.66b

on the null hypothesis that the probability of scores for the test 340
860.99 ± 42.20b

compound(s) or control solvent is equal to 50%, i.e., Chi- 341

square analysis (H 0 : P = 50%) (Adhikary et al. 2015; 342
Magalhães et al. 2012). Insects that did not orient to any arm 343Q2
4h

of the olfactometer were excluded from the analyses (Fig. 1). 344
1406.13 ± 57.35a
60.31 ± 3.06a
Undamaged

Results 345
(UD)

A total of 29, 32, 33, 35, 29, 30, 34, and 28 compounds were 346
detected among volatiles released by UD plants, plants after 347
t1:38 Table 1 (continued)

4 h LF, 12 h LF, 36 h LF, 4 h AF, 12 h AF and 36 h AF, and 348

MD plants, respectively (Table 1, Supplementary material Fig. 349
Compounds

1). 2Z–Penten-1-ol was not detected in UD and MD plants 350

t1:40 Phytol

but, with the exception of 4 h AF, it was found among volatiles 351
t1:41 Total

of all ID plants (least abundant in plants after 4 h LF and 12 h 352

t1:39
AUTHOR'S PROOF JrnlID 10886_ArtID 866_Proof# 1 - 03/07/2017

J Chem Ecol

Fig. 2 The biplot showing the

ordination of volatile organic
compounds

F
O
O
PR
D
TE
EC

353 AF). Linalool was unique among plant volatiles after 36 h LF. was present in higher amounts. Geraniol and 1-tridecanol 355
354 α-Farnesene was detected after 36 h LF and 36 h AF, where it were detected among volatiles of all LF plants and plants after 356
R
R

t2:1 Table 2 Responses of Altica

t2:2 cyanea females to natural volatile Insects responded Non- χ2 P values
O

organic compounds (VOCs)

t2:3 released by undamaged, T1 T2 responders (df = 1)
C

mechanically damaged and insect

t2:4 damaged Ludwigia octovalvis or Volatiles from Ludwigia octovalvis plants (μg in 25 μl CH2Cl2)
N

t2:5 to complete synthetic blends Undamaged (UD) 56 34 7 5.38 0.020

U

t2:6 equivalent to those released by 4 h of continuous feeding by A. cyanea larvae 60 30 5 10 0.002

undamaged, mechanically
t2:7 damaged and insect damaged 4 h of continuous feeding by A. cyanea adults 58 32 6 7.51 0.006
t2:8 plants (T1) vs. the solvent control 12 h of continuous feeding by A. cyanea larvae 64 26 4 16.04 <0.001
t2:9 (T2: CH2Cl2). Each test was 12 h of continuous feeding by A. cyanea adults 62 28 3 12.84 <0.001
carried out with at least 90
t2:10 36 h of continuous feeding by A. cyanea larvae 73 17 3 34.84 <0.001
females
t2:11 36 h of continuous feeding by A. cyanea adults 68 22 3 23.51 <0.001
t2:12 Mechanically damaged (MD) 64 26 5 16.04 <0.001
t2:13 Complete synthetic blends equivalent to volatiles of experimental plants (μg in 25 μl CH2Cl2)
t2:14 Undamaged (UD) 56 34 6 5.38 0.020
t2:15 4 h of continuous feeding by A. cyanea larvae 60 30 4 10 0.002
t2:16 4 h of continuous feeding by A. cyanea adults 58 32 5 7.51 0.006
t2:17 12 h of continuous feeding by A. cyanea larvae 64 26 3 16.04 <0.001
t2:18 12 h of continuous feeding by A. cyanea adults 62 28 4 12.84 <0.001
t2:19 36 h of continuous feeding by A. cyanea larvae 73 17 2 34.84 <0.001
t2:20 36 h of continuous feeding by A. cyanea adults 68 22 3 23.51 <0.001
t2:21 Mechanically damaged (MD) 64 26 5 16.04 <0.001
AUTHOR'S PROOF
JrnlID 10886_ArtID 866_Proof# 1 - 03/07/2017

J Chem Ecol

t3:1 Table 3 Responses of Altica cyanea females to natural volatile organic compounds (VOCs) released by undamaged Ludwigia octovalvis vs. insect
damaged plants, and differently treated insect damaged plants tested against each other. Each test was carried out with at least 90 females

t3:2 Comparison Insects responded Non- χ2 P values

t3:3 T1 T2 T1 T2 responders (df = 1)

t3:4 Plants after continuous feeding by A. cyanea Undamaged plants

t3:5 Larvae for 4 h Undamaged plants 48 42 6 0.4 0.527
t3:6 Adults for 4 h Undamaged plants 47 43 6 0.18 0.673
t3:7 Larvae for 12 h Undamaged plants 52 38 5 2.18 0.140
t3:8 Adults for 12 h Undamaged plants 50 40 6 1.11 0.292
t3:9 Larvae for 36 h Undamaged plants 58 32 4 7.51 0.006
t3:10 Adults for 36 h Undamaged plants 56 34 5 5.38 0.020
t3:11 Plants after continuous feeding by A. cyanea Plants after continuous feeding by A. cyanea
t3:12 Larvae for 4 h Adults for 4 h 48 42 7 0.4 0.527

F
t3:13 Larvae for 12 h Larvae for 4 h 51 39 6 1.6 0.206

O
t3:14 Adults for 12 h Larvae for 4 h 46 44 6 0.04 0.834
t3:15

O
Larvae for 12 h Adults for 4 h 52 38 6 2.18 0.140
t3:16 Larvae for 12 h Adults for 12 h 50 40 7 1.11 0.292

PR
t3:17 Larvae for 36 h Adults for 4 h 56 34 5 5.38 0.020
t3:18 Larvae for 36 h Larvae for 12 h 51 39 5 1.6 0.206
t3:19 Larvae for 36 h Adults for 12 h 54 36 4 3.6 0.058
t3:20 Larvae for 36 h
D Adults for 36 h 46 44 6 0.04 0.834
t3:21 Larvae for 36 h Larvae for 4 h 56 34 5 5.38 0.020
TE
t3:22 Adults for 12 h Adults for 4 h 48 42 7 0.4 0.527
t3:23 Adults for 36 h Larvae for 4 h 55 35 5 4.44 0.035
EC

t3:24 Adults for 36 h Adults for 4 h 56 34 5 5.38 0.020

t3:25 Adults for 36 h Adults for 12 h 48 42 6 0.4 0.527
t3:26 Adults for 36 h Larvae for 12 h 48 42 6 0.4 0.527
R
R

357 36 h AF. Farnesyl acetone was detected after 12 h and 36 h LF, undecanol was least abundant in MD plants. 1-Nonanol and 3- 365
O

358 but four times more after 36 h AF. Except for MD plants, 2E– octanone were least abundant among volatiles of plants after 366
359 nonenal was detected among volatiles of UD and ID plants. 4 h and 36 h AF, respectively (Table 1). The PCA revealed that 367
C

360 Methyl jasmonate was predominant in all samples (Table 1). two factors, F1 and F2, explained more than 82% of the var- 368
N

361 2-Octanol was the second most abundant compound among iation in the observed abundances of VOCs. The biplot repre- 369
362 370
U

volatiles of UD and MD plants, while phytol was the second sented the spatial orientation of variables against two extracted
363 most abundant compound among volatiles of all other treat- factors, and all variables showed loading for the first two 371
364 ments. 2E–Nonenal was least abundant in UD plants, while 1- factors (Supplementary material Table 2, Fig. 1). On the basis 372

t4:1 Table 4 Responses of Altica

t4:2 cyanea females to natural volatile Comparison Insects responded Non- χ2 P values
organic compounds (VOCs)
t4:3 released by mechanically T1 T2 T1 T2 responders (df = 1)
damaged (MD) Ludwigia
t4:4 octovalvis vs. undamaged (UD) MD plants UD plants 52 38 6 2.18 0.140
t4:5 plants, and MD plants vs. insect MD plants Plants after continuous feeding by Altica cyanea
damaged plants. Each test was
t4:6 MD plants Larvae for 4 h 51 39 5 1.6 0.206
carried out with at least 90
t4:7 females MD plants Adults for 4 h 52 38 7 2.18 0.140
t4:8 MD plants Larvae for 12 h 44 46 6 0.04 0.834
t4:9 MD plants Adults for 12 h 50 40 6 1.11 0.292
t4:10 MD plants Larvae for 36 h 39 51 4 1.6 0.206
t4:11 MD plants Adults for 36 h 42 48 5 0.4 0.527

χ2 and P values in italics are for responses of insects to T2

AUTHOR'S PROOF JrnlID 10886_ArtID 866_Proof# 1 - 03/07/2017

J Chem Ecol

373 of relative abundances per sample, CA allowed the classifica- Bioassay 2 Altica cyanea females were attracted to volatiles 389
374 tion of 35 compounds under three central classes (cluster 1: of plants after 36 h AF and 36 h LF compared to volatiles of 390
375 2Z–penten-1-ol, 2E–hexen-1-ol, ocimene, linalool oxide, 3Z– UD plants, but could not distinguish between volatiles from 391
376 nonen-1-ol, 2E–nonenal, nerol, geraniol, 1-undecanol, plants after 4 h AF or 4 h LF or 12 h AF or 12 h LF compared 392
377 farnesene, 1-tridecanol, farnesyl acetone and phytol; cluster to volatiles from UD plants (Table 3). Our observations indi- 393
378 2: linalool, limonene oxide and methyl jasmonate; whereas cate that after 36 h AF and 36 h LF VOCs were most attractive 394
379 cluster 3 contained the remaining 19 compounds) (Fig. 2). to the insects. Altica cyanea females did not prefer volatiles 395
380 Similarities in relative abundances of volatiles were aptly rep- from MD plants compared to volatiles from UD or all ID 396
381 resented in the cluster and the biplot as well. Total amounts of plants (Table 4). 397
382 VOCs were significantly higher in plants after 36 h LF follow-
383 ed by those after 36 h AF, after 12 h LF, MD plants and after
384 12 h AF, after 4 h LF, after 4 h AF, and least in UD plants. Bioassay 3 The insects were attracted to volatiles from plants 398
after 36 h AF or 36 h LF compared to volatiles from plants 399
after 4 h AF or 4 h LF (Table 3). Adult females did not show 400

F
385 Dual Choice Bioassays: attraction to volatiles from plants after 12 h AF or 12 h LF 401
compared to volatiles after 4 h AF or 4 h LF (Table 3). 402

O
386 Bioassay 1 Altica cyanea females were attracted to natural Furthermore, A. cyanea females did not prefer volatiles after 403

O
387 volatiles collected from UD, six types of ID, and MD plants 36 h AF or 36 h LF compared to volatiles after 12 h AF or 12 h 404
388 compared to the solvent control (Table 2). LF (Table 3). Results indicated that VOCs released by plants 405

PR
t5:1 Table 5 Responses of Altica
t5:2 cyanea females to individual Insects responded Non- χ2 P values

t5:3
synthetic compounds and
synthetic blends equivalent to
D T1 T2 responders (df = 1)
TE
those released by insect damaged
t5:4 Ludwigia octovalvis (T1) vs. the Synthetic VOCS in similar amounts released by plants after 36 h of continuous feeding by A. cyanea larvae (μg
solvent control (T2: CH2Cl2). in 25 μl CH2Cl2)
EC

t5:5 Each test was carried out with at a. 3-Hexanol (3.38 μg) 56 34 6 5.38 0.020
least 90 females
t5:6 b. α-Pinene (2.72 μg) 53 37 7 2.84 0.092
t5:7 c. Benzaldehyde (4.82 μg) 51 39 8 1.6 0.206
R

t5:8 d. 3-Octanol (1.02 μg) 51 39 8 1.6 0.206

R

t5:9 e. Linalool oxide (3.44 μg) 62 28 4 12.84 <0.001

t5:10 f. Decanal (4.27 μg)
O

50 40 8 1.11 0.292
t5:11 g. Nerol (16.31 μg) 50 40 7 1.11 0.292
C

t5:12 h. Geraniol (2.36 μg) 56 34 5 5.38 0.020

N

t5:13 i. 1- Tridecanol (0.94 μg) 47 43 8 0.18 0.673

t5:14 j. Phytol (293.87 μg) 62 28 4 12.84 <0.001
U

t5:15 a + b + c + d + e + f + g + h + i + j* 72 18 2 32.4 <0.001

t5:16 a + e + h + j* 69 21 3 25.6 <0.001
t5:17 Synthetic VOCS in similar amounts released by plants after 36 h of continuous feeding by A. cyanea adults (μg
in 25 μl CH2Cl2)
t5:18 a. 3-Hexanol (3.39 μg) 56 34 6 5.38 0.020
t5:19 b. α-Pinene (3.44 μg) 56 34 7 5.38 0.020
t5:20 c. Benzaldehyde (4.82 μg) 51 39 9 1.6 0.206
t5:21 d. 3-Octanol (1.13 μg) 51 39 8 1.6 0.206
t5:22 e. Linalool oxide (3.36 μg) 62 28 4 12.84 <0.001
t5:23 f. Decanal (3.35 μg) 46 44 8 0.04 0.834
t5:24 g. 1-Tridecanol (1.57 μg) 51 39 7 1.6 0.206
t5:25 h. Farnesyl acetone (1.67 μg) 50 40 7 1.11 0.292
t5:26 i. Phytol (271.67 μg) 61 29 4 11.38 <0.001
t5:27 a + b + c + d + e + f + g + h + i* 68 22 3 23.51 <0.001
t5:28 a + b + e + i* 66 24 3 19.6 <0.001

*Indicates compounds as mixture in the amounts given in the different entries

AUTHOR'S PROOF
JrnlID 10886_ArtID 866_Proof# 1 - 03/07/2017

J Chem Ecol

406 after 36 h AF or 36 h LF are preferred by A. cyanea compared compounds (3-hexanol, α-pinene, benzaldehyde, 3-octanol, 432
407 to VOCs from differently treated plants. linalool oxide, decanal, 1-tridecanol, farnesyl acetone, and 433
phytol) in similar amounts present after 36 h AF. Adult females 434
408 Bioassay 4 Altica cyanea females were attracted to synthetic were attracted to the complete blend as well as to a mixture of 435
409 blends equivalent to volatiles of UD or ID or MD plants com- 3-hexanol, α-pinene, linalool oxide, and phytol in similar 436
410 pared to the solvent control (Table 2). amounts and proportions present after 36 h AF (Table 5). 437
The insects displayed behavioral responses to 14 individual 438
411 Bioassay 5 The insects did not react (remained in the common compounds (3-hexanol, 1-hexanol, 2-heptanone, α-pinene, 439
412 arm of the Y-tube) to individual synthetic compounds in sim- benzaldehyde, 1-heptanol, 1-octen-3-ol, 3-octanol, 2-octanol, 440
413 ilar amounts present among volatiles of UD plants, after 4 h limonene, linalool oxide, 1-nonanol, decanal, and phytol) in sim- 441
414 AF, and 4 h LF compared to the solvent control. ilar amounts present among volatiles of MD plants compared to 442
415 The insects displayed behavioral responses to 6 compounds the solvent control. Adult females were attracted to the complete 443
416 (3-hexanol, α-pinene, benzaldehyde, 3-octanol, linalool oxide, blend as well as to a mixture of the three most attractive com- 444
417 and phytol) in similar amounts present among volatiles of plants pounds, 3-hexanol, α-pinene, and linalool oxide in similar 445

F
418 after 12 h AF or 12 h LF compared to the solvent control They amounts present among volatiles of MD plants (Table 6). 446

O
419 were attracted to a corresponding synthetic blend of these vola-
420 tiles, to a mixture of linalool oxide and phytol in similar amounts Bioassay 6 Altica cyanea females were attracted to volatiles 447

O
421 and proportions as after 12 h LF, as well as to a mixture of α- from plants after 36 h LF compared to 10 individual synthetic 448
422 compounds (3-hexanol, α-pinene, benzaldehyde, 3-octanol, 449

PR
pinene, linalool oxide, and phytol in similar amounts and pro-
423 portions as after 12 h AF (Supplementary material Table 3). linalool oxide, decanal, nerol, geraniol, 1-tridecanol, and 450
424 Adult females displayed behavioral responses to 10 individ- phytol) in similar amounts present after 36 h LF (Table 7). 451
425 ual compounds (3-hexanol, α-pinene, benzaldehyde, 3-octanol,
D When the compounds were formulated as a blend in similar 452
426 linalool oxide, decanal, nerol, geraniol, 1-tridecanol, and amounts and proportions present after 36 h LF, the insects 453
TE
427 phytol) in similar amounts as present after 36 h LF compared could not distinguish between the natural sample and the syn- 454
428 to the solvent control. They were attracted to the complete thetic blend. Furthermore, they could not distinguish between 455
429 blend as well as to a mixture of 3-hexanol, linalool oxide, the natural sample and a synthetic blend of 3-hexanol, linalool 456
EC

430 geraniol, and phytol in similar amounts present after 36 h LF oxide, geraniol, and phytol in similar amounts and proportions 457
431 (Table 5). The insects showed behavioral responses to 9 present after 36 h LF (Table 7). 458
R

t6:1 Table 6 Responses of Altica

R

t6:2 cyanea females to individual Insects Non- χ2 P values

responded
O

synthetic compounds and

synthetic blends equivalent to
t6:3 T1 T2 responders (df = 1)
C

those released by mechanically

damaged Ludwigia octovalvis
t6:4 Synthetic VOCs in similar amounts released by mechanically damaged plants (μg in 25 μl CH2Cl2)
N

(T1) vs. the solvent control (T2:

t6:5 CH2Cl2). Each test was carried a. 3-Hexanol (8.49 μg) 60 30 6 10.0 0.002
U

out with at least 90 females

t6:6 b. 1-Hexanol (6.64 μg) 50 40 8 1.11 0.292
t6:7 c. 2-Heptanone (21.17 μg) 52 38 7 2.18 0.140
t6:8 d. α-Pinene (6.01 μg) 58 32 6 7.51 0.006
t6:9 e. Benzaldehyde (8.73 μg) 54 36 7 3.6 0.058
t6:10 f. 1-Heptanol (11.68 μg) 48 42 8 0.4 0.527
t6:11 g. 1-Octen-3-ol (12.02 μg) 52 38 7 2.18 0.140
t6:12 h. 3-Octanol (1.38 μg) 52 38 6 2.18 0.140
t6:13 i. 2-Octanol (58.92 μg) 50 40 8 1.11 0.292
t6:14 j. Limonene (3.48 μg) 50 40 8 1.11 0.292
t6:15 k. Linalool oxide (2.91 μg) 60 30 5 10.0 0.002
t6:16 l. 1-Nonanol (2.46 μg) 48 42 8 0.4 0.527
t6:17 m. Decanal (5.20 μg) 52 38 7 2.18 0.140
t6:18 n. Phytol (58.06 μg) 49 41 8 0.71 0.399
t6:19 a + b + c + d + e + f + g + h + i + j + k + l + m + n* 64 26 4 16.04 <0.001
t6:20 a + d + k* 62 28 4 12.84 <0.001

*Indicates compounds as mixture in the amounts given in the different entries

AUTHOR'S PROOF JrnlID 10886_ArtID 866_Proof# 1 - 03/07/2017

J Chem Ecol

t7:1 Table 7 Responses of Altica cyanea females to natural volatile organic compounds or synthetic blends equivalent to volatiles released from
compounds (VOCs) released by Ludwigia octovalvis after 36 h of plants after 36 h of continuous feeding by A. cyanea larvae or adults.
continuous feeding by A. cyanea larvae or adults vs. synthetic Each test was carried out with at least 90 females Q3

t7:2 Comparison Insects responded Non- χ2 P

t7:3 T1 T2 T1 T2 Responders (df = 1) Values

t7:4 Volatiles from (μg in 25 μl CH2Cl2) plants Synthetic VOCs and mixtures (μg in 25 μl CH2Cl2)
after 36 h of continuous feeding by equivalent to volatiles from plants after 36 h of
A. cyanea larvae continuous feeding by A. cyanea larvae
t7:5 a. 3-Hexanol (3.38 μg) 59 31 5 8.71 0.003
t7:6 b. α-Pinene (2.72 μg) 61 29 3 11.38 <0.001
t7:7 c. Benzaldehyde (4.82 μg) 71 19 2 30.04 <0.001
t7:8 d. 3-Octanol (1.02 μg) 71 19 2 30.04 <0.001
t7:9 e. Linalool oxide (3.44 μg) 55 35 4 4.44 0.035
t7:10 f. Decanal (4.27 μg)

F
69 21 2 25.6 <0.001
t7:11 g. Nerol (16.31 μg) 69 21 2 25.6 <0.001

O
t7:12 h. Geraniol (2.36 μg) 59 31 6 8.71 0.003

O
t7:13 i. 1-Tridecanol (0.94 μg) 71 19 3 30.04 <0.001
t7:14 j. Phytol (293.87 μg) 55 35 5 4.44 0.035

PR
t7:15 a + b + c + d + e + f + g + h + i + j* 46 44 3 0.04 0.834
t7:16 a + e + h + j* 48 42 4 0.4 0.527
t7:17 Volatiles from (μg in 25 μl CH2Cl2) plants Synthetic VOCs and mixtures (μg in 25 μl CH2Cl2)
D
after 36 h of continuous feeding by equivalent to volatiles from plants after 36 h of
A. cyanea adults continuous feeding by A. cyanea adults
TE
t7:18 a. 3-Hexanol (3.39 μg) 56 34 6 5.38 0.020
t7:19 b. α-Pinene (3.44 μg) 56 34 5 5.38 0.020
t7:20 c. Benzaldehyde (4.82 μg)
EC

66 24 3 19.6 <0.001
t7:21 d. 3-Octanol (1.13 μg) 66 24 4 19.6 <0.001
t7:22 e. Linalool oxide (3.36 μg) 49 41 3 0.71 0.399
R

t7:23 f. Decanal (3.35 μg) 66 24 2 19.6 <0.001

t7:24 g. 1-Tridecanol (1.57 μg)
R

64 26 3 16.04 <0.001
t7:25 h. Farnesyl acetone (1.67 μg) 63 27 4 14.4 0.002
O

t7:26 i. Phytol (271.67 μg) 49 41 3 0.71 0.399

C

t7:27 a + b + c + d + e + f + g + h + i* 47 43 3 0.18 0.673

t7:28 a + b + e + j* 48 42 4 0.4 0.527
N

*Indicates compounds as mixture in the amounts given in the different entries

U

459 Except for linalool oxide and phytol, where the insects did not at 12 μg/25 μl (Table 8). Adult females were attracted to α- 473
460 show a clear preference, the insects were attracted to volatiles pinene and geraniol at the minimum amount of 4 μg/25 μl and 474
461 after 36 h AF compared to 7 individual compounds (3-hexanol, displayed highest attraction at 16 μg/25 μl. The insects showed 475
462 α-pinene, benzaldehyde, 3-octanol, decanal, 1-tridecanol, and highest attraction to linalool oxide at 6 μg/25 μl. The insects 476
463 farnesyl acetone) in similar amounts present after 36 h AF were attracted to phytol at the minimum amount of 100 μg/ 477
464 (Table 7). When the compounds were formulated as a blend in 25 μl and displayed highest attraction at 400 μg/25 μl. 478
465 similar amounts and proportions present after 36 h AF, the in-
466 sects could not distinguish between the natural sample and the
467 synthetic blend. Furthermore, the insects could not distinguish Discussion 479
468 between the natural sample and a synthetic blend of 3-hexanol,
469 α-pinene, linalool oxide, and phytol in similar amounts and pro- Plants release a wide range of VOCs, which may act as se- 480
470 portions present after 36 h AF (Table 7). miochemicals in the interaction with insects, pathogens or 481
neighboring plants. The emission pattern of these compounds 482
471 Bioassay 7 The insects were attracted to 3-hexanol at the may change with biotic and abiotic stresses (Dudareva et al. 483
472 minimum amount of 3 μg/25 μl and showed highest attraction 2013; Giorgi et al. 2015; Kessler 2015; Niederbacher et al. 484
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t8:1 Table 8 Dose responses of Altica

t8:2 cyanea females to individual Compounds Dose Insects responded Non- χ2 P values
synthetic compounds (T1) vs. the
t8:3 solvent control (T2: CH2Cl2). (μg/25 μl) T1 T2 responders (df = 1)
Each test was carried out with at
t8:4 least 90 females 3-Hexanol 1.5 46 44 8 0.04 0.834
t8:5 3 56 34 5 5.38 0.020
t8:6 6 58 32 5 7.51 0.006
t8:7 12 66 24 4 19.6 <0.001
t8:8 α-Pinene 2 50 40 7 1.11 0.292
t8:9 4 57 33 5 6.4 0.011
t8:10 8 60 30 4 10 0.002
t8:11 16 64 26 3 16.04 <0.001
t8:12 Linalool oxide 1.5 52 38 7 2.18 0.140
t8:13 3 62 28 4 12.84 <0.001

F
t8:14 6 68 22 3 23.51 <0.001
t8:15 Geraniol 2 51 39 7 1.6 0.206

O
t8:16 4 58 32 6 7.51 0.006

O
t8:17 8 62 28 4 12.84 <0.001
t8:18 16 70 20 3 27.78 <0.001

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t8:19 Phytol 50 48 42 8 0.4 0.527
t8:20 100 56 34 5 5.38 0.020
t8:21 D 200 60 30 4 10 0.002
t8:22 400 66 24 3 19.6 <0.001
TE
EC

485 2015; Niinemets et al. 2013; Piesik et al. 2011; Truong et al. sesquiterpenes and some monoterpene hydrocarbons 512
486 2014). With regard to biotic stresses, infestation by herbivores (Cardoso et al. 2013), however, none of these compounds 513
487 induces plants to increase VOC emissions and even produce were detected in this study. Similarly, myrcene, Z-ocimene, 514
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488 new ones (Copolovici et al. 2011; Mukherjee et al. 2015; 3Z–hexenyl acetate, 3Z–hexenol, bisabolene, (E,E)-α- 515
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489 Niinemets et al. 2013; Sarkar et al. 2016). Our results demon- farnesene, 2-tridecanone and geranyl acetone were detected 516
490 strate that feeding by larvae or adults of A. cyanea on among volatiles of L. hexapetala (Carruthers et al. 2011), 517
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491 L. octovalvis plants induces the release of bouquets of vola- which, except ocimene and farnesene, were absent among 518
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492 tiles that are qualitatively and quantitatively distinct from volatiles of L. octovalvis. 519
493 520
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those released by undamaged plants. Furthermore, different Our olfactometer bioassays demonstrate that A. cyanea pre-
494 studies have shown that emission of VOCs from plants may fer plant released volatiles after 36 h AF or 36 h LF over UD 521
U

495 change qualitatively and quantitatively depending on the type L. octovalvis, suggesting that qualitative and quantitative 522
496 of feeding damage caused by herbivores. Feeding by larvae changes in the emission of VOCs after 36 h herbivory made 523
497 and adults of A. cyanea induced release of geraniol, farnesene, the damaged host more attractive. Emission of higher amounts 524
498 1-tridecanol and farnesyl acetone from L. octovalvis, which of 3-hexanol, α-pinene, linalool oxide, geraniol, and phytol 525
499 were absent in undamaged plants, whereas linalool was from conspecific-damaged L. octovalvis compared to UD 526
500 unique among volatiles of plants after 36 h LF. Furthermore, plants resulted in an increased attraction of A. cyanea. 527
501 released amounts of 2-hexanol, 1-hexanol, α-pinene, 2E– Insects may use a subset of perhaps 3–10 compounds as host 528
502 heptenal, 1-heptanol, sabinene, 1-octen-3-ol, benzyl alcohol, location cues (Bruce and Pickett 2011; Bruce et al. 2005). In 529
503 ocimene, nonanal, 2E–nonenal, farnesene, 1-tridecanol, and this study, we found a synthetic blend of 3-hexanol, linalool 530
504 farnesyl acetone were higher after 36 h AF compared to oxide, geraniol, and phytol (equivalent to volatiles after 36 h 531
505 36 h LF, whereas 2E–hexen-1-ol, limonene oxide, 3Z– LF) or a synthetic blend of 3-hexanol, α-pinene, linalool ox- 532
506 nonen-1-ol, 1-nonanol, decanal, nerol, geraniol, and methyl ide, and phytol (equivalent to volatiles after 36 h AF) to be 533
507 jasmonate were higher after 36 h LF as compared to 36 h attractive to A. cyanea, and the insects were attracted to a 534
508 AF. Our results clearly show that volatile emission patterns synthetic blend of 3-hexanol, α-pinene, and linalool oxide 535
509 of L. octovalvis changed qualitatively and quantitatively dur- (equivalent to volatiles of MD plants). Insects use an array 536
510 ing herbivory by larvae and adults of A. cyanea. Major VOCs of olfactory sensory neurons, which register fluxes and ratios 537
511 in the essential oil of leaves and flowers of L. lagunae oil were of VOCs and modulate behavioral responses to ecologically 538
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539 relevant odors (Couton et al. 2009; Galizia and Rossler 2010; Cardoso CAL, da Rocha CG, Caramão EB (2013) Volatile compounds 592
and free radical scavenging activity of leaf and flower oil of 593
540 Najar-Rodriguez et al. 2010; Piesik et al. 2010; Renou 2014).
Ludwigia lagunae (Onagraceae). J Essent Oil Bear Plants 16:323– 594
541 The current study confirms that natural proportions of vola- 327 595
542 tiles play a crucial role in host selection by A. cyanea. Methyl Carruthers RI, Franc MK, Gee WS, Cossé AA, Grewell BJ, Beck JJ 596
543 jasmonate (MeJa) plays an important role in induced direct (2011) Volatile emissions from the flea beetle Altica litigata 597
544 defense of plants (Devoto and Turner 2005; Okada et al. (Coleoptera: Chrysomelidae) associated with invasive Ludwigia 598
hexapetala. Chemoecology 21:253–259 599
545 2015; Piesik et al. 2013b; Wasternack 2007). Herbivory by Caton BP, Mortimer M, Hill JE, Johnson DE (2010) A practical field 600
546 both larvae and adults of A. cyanea on L. octovalvis causes guide to weeds of rice in Asia, 2nd edn. Los Baños (Philippines), 601
547 an increase of MeJa emission, which may also induce a de- International Rice Research Institute 602
548 fensive reaction in neighboring plants. Here we record elevat- Chauhan BS, Abugho SB (2012) Phenotypic plasticity of spiny amaranth 603
(Amaranthus spinosus) and Longfruited primrose-willow (Ludwigia 604
549 ed emissions of several VOCs including benzyl alcohol and 1- octovalvis) in response to rice interference. Weed Sci 60:411–415 605
550 octen-3-ol in volatiles of L. octovalvis after feeding by either Chin DV, Thien TC, Bi HH, Nhiem NT (2007) Study on weed and weedy 606
551 A. cyanea larvae or adults, which were also present in the rice control by imidazolinone herbicides in CLEARFIELD™ paddy 607
552 natural volatile blend of UD plants. The insects were not grown by imi-tolerance indica rice variety. Omonrice 15:63–67 608
Copolovici L, Kännaste A, Remmel T, Vislap V, Niinemets U (2011) 609

F
553 attracted to synthetic benzyl alcohol and 1-octen-3-ol, which
Volatile emissions from Alnus glutionosa induced by herbivory are 610
554 are known plant defence compounds, induced by herbivory to

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quantitatively related to the extent of damage. J Chem Ecol 37:18– 611
555 attract natural enemies of the insect (De Moraes et al. 1998; 28 612

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556 Kigathi et al. 2009; Xiao et al. 2012). Voracious feeding by Couton L, Minoli S, Kiêu K, Anton S, Rospars JP (2009) Constancy and 613
557 larvae or adults of A. cyanea on L. octovalvis resulted in ele- variability of identified glomeruli in antennal lobes: computational 614

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approach in Spodoptera littoralis. Cell Tissue Res 337:491–511 615
558 vated emissions of 3-hexanol, α-pinene, linalool oxide, gera- De Moraes CM, Lewis WJ, Paré PW, Alborn HT, Tumlinson JH (1998) 616
559 niol, and phytol, which further attracted conspecific Herbivore-infested plants selectively attract parasitoids. Nature 393: 617
560 individuals. D 570–573 618
561 In conclusion, our results demonstrate that A. cyanea fe- Devoto A, Turner JG (2005) Jasmonate-regulated Arabidopsis stress sig- 619
nalling network. Physiol Plant 123:161–172 620
562 males are attracted to a synthetic blend of 3.38 μg 3-hexanol,
TE
Dubey AN (1981) Biological control of weeds in rice fields. Trop Pest 621
563 3.44 μg linalool oxide, 2.36 μg geraniol, and 293.87 μg Manage 27:143–144 622
564 phytol dissolved in 25 μl CH2Cl2 (ratios of 1.4:1.5:1:125), Dudareva N, Klempien A, Muhlemann JK, Kaplan I (2013) Biosynthesis, 623
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565 and a synthetic blend of 3.39 μg 3-hexanol, 3.44 μg α-pinene, function and metabolic engineering of plant volatile organic com- 624
566 3.36 μg linalool oxide, and 271.67 μg phytol dissolved in pounds. New Phytol 198:16–32 625
Galizia CG, Rossler W (2010) Parallel olfactory systems in insects: anat- 626
567 25 μl CH2Cl2 (ratios of 1:1:1:80) which may suggest to use
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omy and function. Annu Rev Entomol 55:399–420 627

568 these blends in the context of biocontrol measurements of the Giorgi A, Manzo A, Nanayakkara NNMC, Giupponi L, Cocucci M, 628
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569 weed in rice fields at the beginning of the vegetation period. Panseri S (2015) Effect of biotic and abiotic stresses on volatile 629
570 Investigations on behavioral responses of natural enemies of emission of Achillea collina Becker ex Rchb. Nat Prod Res 29: 630
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571 A. cyanea larvae and adults to herbivore-induced L. octovalvis
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572 volatiles should be a topic of future research. emissions in maize by different larval instars of Spodoptera 633
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573 Acknowledgements We thank Prof. Wittko Francke and anonymous Imeokparia PO (1994) Weed control in flooded rice with various herbi- 635
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