bioRxiv preprint doi: https://doi.org/10.1101/293100; this version posted April 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Plant odour and sex are

2 integral elements of specific mate

3 recognition in an insect herbivore

4 Felipe Borrero-Echeverry - Biological Control Laboratory, Colombian Corporation for 5 Agricultural Research (Corpoica), Mosquera, Colombia - [email protected]

6 Marie Bengtsson - Unit, Department of Plant Protection Biology, 7 Swedish University of Agricultural Sciences, Alnarp, Sweden - [email protected]

8 Peter Witzgall - Chemical Ecology Unit, Department of Plant Protection Biology, Swedish 9 University of Agricultural Sciences, Alnarp, Sweden - [email protected]

10 Correspondence 11 Peter Witzgall (lead author) 12 SLU, Box 102, 23053 Alnarp, Sweden 13 +46 70 2426939, [email protected]

14 Running title:

15 Plant volatiles modulate sex attraction

16 Summary

17 Specific mate recognition strongly relies on the chemical senses in many animals, and

18 especially in nocturnal insects. Two signal types lend to premating olfactory

19 communication in terrestrial habitats: sex signals blend into an atmosphere of habitat

20 odorants, where plant volatiles prevail. We show for the first time that males of the

21 African cotton leafworm Spodoptera littoralis perceive sex pheromone and

22 volatiles of its plant host cotton as a unit, rather than as independent messages. In clean

23 air, S. littoralis males are attracted to flawed pheromone signals, such as single synthetic

24 pheromone components or even the pheromone of a sibling species, Oriental leafworm S.

25 litura. Presence of host plant volatiles, however, strongly reduces the male response to

26 deficient or heterospecific pheromone signals. That plant cues enhance discrimination of bioRxiv preprint doi: https://doi.org/10.1101/293100; this version posted April 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Borrero et al. - p. 2

27 sex pheromone quality confirms the idea that specific mate recognition in noctuid

28 has evolved in concert with host plant . A participation of host plant odour in

29 sexual communication suggests that mate recognition is under natural and sexual

30 selection. Moreover, shifts in either female host preference or sex pheromone

31 biosynthesis give rise to new communication channels that have the potential to initiate

32 or contribute to reproductive isolation.

33 Key words: premating sexual communication, specific mate recognition, reproductive

34 isolation, ecological speciation

35 Introduction

36 Specific mate communication and recognition, which is shaped during adaptation to

37 natural habitats, involves both sex signals and environmental or habitat sensory cues and

38 is under sexual and natural selection (Paterson 1978, 1980; Endler 1992; Blows 2002;

39 Boughman 2002; Scordato et al. 2014; Rosenthal 2017). In nature, of

40 phytophagous insects release sex pheromone into an atmosphere which is filled with plant

41 volatiles.

42 The effect of plant volatiles on the male behavioural response to sex pheromone

43 has long been investigated (Landolt & Phillips 1997; Reddy & Guerrero 2004). Perception

44 of sex and plant volatiles typically employs discrete peripheral input channels, and two

45 different types of insect olfactory receptors, pheromone and general odorant receptors,

46 respectively (Krieger et al. 2004; Sakurai et al. 2004; Zhang & Löfstedt, 2015).

47 Integration of pheromone and plant volatile stimuli has been shown to occur already at

48 the antennal level in some species (Rouyar et al. 2015; Lebreton et al. 2017) and

49 otherwise in the primary olfactory centre, the antennal lobe (Namiki et al. 2008; Trona et

50 al. 2010, 2013; Chaffiol et al. 2012, 2014; Hatano et al. 2015; Ian et al. 2017).

51 Curiously, the behavioural consequences of blending plant volatiles with sex

52 differ widely, between species and according to the plant chemicals investigated: plant

53 volatiles have been shown to both synergize and antagonize the male response to sex bioRxiv preprint doi: https://doi.org/10.1101/293100; this version posted April 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Borrero et al. - p. 3

54 pheromone (Dickens et al. 1990; Light et al. 1993; Yang et al. 2004; Schmidt-Büsser et

55 al. 2009; Trona et al. 2013; Badeke et al. 2016). A tentative explanation is that plant

56 volatiles play different ecological and behavioural roles, they signal adult feeding,

57 or oviposition sites, but also plants that are unsuitable as adult or larval food. Different

58 messages conveyed by plant volatiles would account for discrepant behavioural effects

59 when blended with sex pheromone. This is evidenced by a response modulation according

60 to internal physiological state in males and females of African cotton leafworm,

61 Spodoptera littoralis (Lepidoptera, Noctuidae): unmated female moths are attracted to

62 floral odorants for adult feeding, and soon after mating to cotton leaf volatiles for egg-

63 laying (Saveer et al. 2012). Male moths respond to host leaf volatiles only prior to

64 mating, since these probably signal rendez-vous sites (Kromann et al. 2015). Cotton

65 leafworm moths further discriminate between volatiles of preferred and non-preferred

66 larval food plants (Thöming et al. 2013; Proffit et al. 2015) and between volatiles from

67 healthy and damaged cotton plants (Zakir et al. 2013a,b; Hatano et al. 2015).

68 That deficient plant stimuli, emitted by damaged plants or non-host plants antagonize the

69 male response to pheromone (Hatano et al. 2015; Badeke et al. 2016; Wang et al. 2016)

70 poses the question how deficient pheromone stimuli interact with plant volatiles.

71 Traditionally, the effect of pheromone blend composition and quality on male attraction

72 has been studied in the field, against a uniform plant volatile background, or in the

73 laboratory in charcoal-filtered air, without plant volatiles. We therefore investigated the

74 combined effect of pheromone blend quality and plant volatiles on male sexual attraction.

75 We show that males of S. littoralis best respond to a mixture of conspecific, complete sex

76 pheromone and volatiles of the larval food plant cotton. Attraction to deficient synthetic

77 pheromone, or to heterospecific pheromone of the sibling species S. litura was much

78 reduced in the presence of cotton volatiles. Likewise, the male response to pheromone-

79 releasing conspecific females was reduced by herbivore-damaged cotton volatiles. Our

80 results demonstrate that mate recognition in cotton leafworm is mediated by a

81 combination of plant volatiles and sex pheromone. This finding essentially contributes to

82 our understanding of olfactory-mediated premating communication and has bearings on

83 concepts of phylogenetic diversification in insect herbivores. bioRxiv preprint doi: https://doi.org/10.1101/293100; this version posted April 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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84 Materials and methods

85 Insects

86 An African cotton leafworm Spodoptera littoralis (Lepid., Noctuidea) colony was

87 established with field-collected insects from Alexandria, Egypt and was interbred with

88 insects from Egypt every year. Insects were raised on a semisynthetic agar-based diet

89 (modified from Hinks & Byers 1976) under a 16L:8D photoperiod, at 24oC and 50 to 60%

90 RH. Males and females were separated as pupae into 30 x 30 x 30 cm Plexiglas cages.

91 Three-day-old unmated male moths were used in all bioassays.

92 Oriental cotton leafworm S. litura females were provided by Dr. Kiyoshi Nakamuta of

93 Chiba University. S. litura and S. littoralis share pheromone components and both species

94 show cross-attraction to their respective pheromone blends (Saveer et al., 2014).

95 Plant material

96 Cotton seedlings, Gossypium hirsutum (cv. Delprim DPL 491), were grown singly in pots

97 at 25oC and 70% RH, under daylight and an artificial light source (400 W). Cotton plants

98 used in wind tunnel assays had 8–12 fully developed true leaves. Damaged plants were

99 obtained by letting four, 24-h starved, fifth-instar larvae feed on the plant during 4 h

100 prior to experiments. Larvae were removed before wind tunnel experiments. Plants used

101 for headspace analysis and behavioural tests in previous studies were grown under the

102 same conditions (Saveer et al. 2012; Borrero-Echeverry et al. 2015).

103 Chemicals

104 Antennally and behaviourally active cotton volatiles (Borrero-Echeverry et al., 2015) were

105 used in experiments: β-myrcene (97% chemical purity; CAS #123-35-3; Fluka), (R)-(+)-

106 limonene (95% chemical purity; CAS # 5989-27-5; Aldrich), (E)-β-ocimene (91%

107 chemical purity; CAS #13877-91-3; Fluka), 4,8-dimethyl-1,3(E),7-nonatriene (DMNT)

108 (95% chemical purity; CAS #019945-61-0; a gift from Wittko Francke, Hamburg), (Z)-3-

109 hexenyl acetate (99% chemical purity; CAS #3681-71-8; Aldrich), (R)-(-)-linalool (95% bioRxiv preprint doi: https://doi.org/10.1101/293100; this version posted April 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Borrero et al. - p. 5

110 chemical purity; CAS #126-90-9; Firmenich), (S)-(+)-linalool (95% chemical purity; CAS

111 #126-90-9; Firmenich) and nonanal (90% chemical purity; CAS #124-19-6; Fluka). In

112 addition, α-farnesene (>90% chemical purity; CAS #502-61-4; Bedoukian) and β-

113 farnesene (>90% chemical purity; CAS #18794-84-8; Bedoukian) were included since

114 they occur in maize headspace, another S. littoralis host plant (Bengtsson et al., 2006;

115 Thöming et al., 2013).

116 The S. littoralis pheromone components, (Z,E)-9,11-tetradecadienyl acetate (Z9,E11-

117 14Ac) (main pheromone compound), (Z)-9-tetradecenyl acetate (Z9-14Ac), (Z,E)-9,12-

118 tetradecadienyl acetate (Z9,E12-14Ac), were purchased from Pherobank and (E,E)-10,12-

119 tetradecadienyl acetate (E10,E12-14Ac) was a gift from David Hall (Greenwich, UK).

120 Isomeric purity was >96.3% and for the dienic compounds and >99.1% for Z9-14Ac.

121 These four components were consistently found in pheromone gland extracts (Saveer et

122 al. 2014; El-Sayed 2017). The solvent was redistilled ethanol (Labscan).

123 Wind tunnel bioassay

124 Wind tunnel experiments were performed in a Plexiglas wind tunnel (180 x 90 x 60 cm)

125 following the protocol of Borrero-Echeverry et al. (2015). Briefly, males and females were

126 kept in separate rooms to avoid pre-exposure to pheromone before experiments. One h

127 before experiments, moths were transferred individually to 2.5 x 12.5 cm glass tubes

128 closed with gauze. Tests were carried out between 1 and 4 h after the onset of

129 scotophase. The wind tunnel was illuminated from above and the side (6 lux), moths were

130 flown in a wind speed of 30 cm/s, at 24 ± 2oC air temperature and 60 ± 10% RH.

131 Incoming and outgoing air was filtered with active charcoal. Moths for every treatment

132 (N=50) were released individually from glass tubes at the downwind end of the tunnel.

133 Males were scored for upwind flight over 150 cm, up to ca. 30 cm from the odour source.

134 Synthetic odour blends were delivered from the centre of the upwind end of the wind

135 tunnel from a piezo-electric sprayer (El-Sayed et al. 1999; Becher et al. 2010). Samples

136 were loaded into a 1-ml glass syringe operated by a microinjection pump (CMA

137 Microdialysis AB, Solna, Sweden) that delivered test solutions at a constant rate of 10

138 µl/min through Teflon tubing into a glass capillary with a narrow, elongated tip. The bioRxiv preprint doi: https://doi.org/10.1101/293100; this version posted April 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Borrero et al. - p. 6

139 capillary was attached to a piezo-ceramic disk, which produced an aerosol that was

140 carried downwind. A glass cylinder (95 mm diameter x 100 mm height), covered by a fine

141 metal mesh (pore size 2 mm) was placed in front of the capillary as landing platform. Live

142 plants were placed at the upwind end of the wind tunnel. In experiments with calling,

143 pheromone-releasing females, three calling females were placed downwind from plants in

144 glass tubes covered at both ends with a mesh.

145 We tested the main pheromone compound, Z9,E11-14Ac and a 4-component synthetic

146 pheromone blend of Z9,E11-14Ac, Z9-14Ac, E10,E12-14Ac and Z9,E12-14Ac, in a

147 100:30:20:4 proportion. The release rate of the main compound Z9,E11-14Ac was 100

148 pg/min, corresponding to the amount of pheromone emitted by calling females (Saveer et

149 al. 2014). Males were further tested with pheromone-releasing S. littoralis and S. litura

150 females. Single plant compounds were released at a rate of 10 ng/min, and a 4-

151 component plant volatile blend containing nonanal, (R)-(+)-limonene, (Z)-3-hexenyl

152 acetate, (E)-β-ocimene in a 33:12:33:23 proportion (Borrero-Echeverry et al. 2015) was

153 also released at 10 ng/min. A 5-component plant volatile blend, mimicking herbivore

154 damage, was formulated by adding DMNT at 10 ng/min to the 4-component blend

155 (Hatano et al. 2015). Males were further tested with undamaged cotton plants and plants

156 on which 5th-instar larvae of S. littoralis had been feeding during 4 h. Males were flown

157 to single sources of plant volatiles and pheromones, and to combinations of both.

158 Statistical analysis

159 Generalized linear models (GLM) with a Bernoulli binomial distribution were used to

160 analyse behavioural data. Upwind flight was used as the target effect. Post-hoc Wald

161 pairwise comparison tests were used to identify differences between treatments.

162 Significance was determined at α=95%. All statistical analysis was carried out using R (R

163 Core Team 2013). bioRxiv preprint doi: https://doi.org/10.1101/293100; this version posted April 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Borrero et al. - p. 7

164 Results

165 When cotton volatiles were tested one by one, only α-farnesene elicited significant upwind

166 flight attraction in S. littoralis males (z = 2.066; p = 0.039) (Table 1). All of these plant

167 volatiles significantly reduced male attraction, when added to the main pheromone

168 compound Z9,E11-14Ac. In stark contrast, seven of these plant volatiles did not affect

169 male attraction when mixed with the complete, four-component synthetic sex pheromone.

170 Only three volatiles reduced attraction when mixed with the four-component sex

171 pheromone blend: DMNT was the strongest antagonist (z = 4.602; p < 0.001), followed

172 by (E)-β-ocimene (z = 2.378; p = 0.017) and (Z)-3 hexenyl acetate (z = 1.799; p =

173 0.072) (Table 1). Larval feeding on cotton leaves strongly increases release of DMNT,

174 which has been shown to interfere with perception of the main pheromone compound

175 Z9,E11-14Ac (Hatano et al., 2015).

176 This differential effect of complete vs. incomplete pheromone on male attraction, when

177 mixed with plant compounds, was confirmed by experiments using a 4-component cotton

178 volatile blend, instead of single cotton volatiles. Attraction to a combination of this cotton

179 blend and the main pheromone compound was significantly reduced, compared with

180 attraction to pheromone alone (z = 3.733; p < 0.001), while a combination of the same

181 cotton blend with four-component pheromone did not reduce attraction (Fig. 1A). An

182 undamaged cotton plant produced the same result: male attraction was significantly

183 reduced to the plant in combination with the main pheromone compound (z = 2.208; p =

184 0.027), and not with the complete pheromone blend (Fig. 1B).

185 We next replaced synthetic with authentic sex pheromone, released by live calling

186 conspecific females or by females of the sibling species S. litura. Both species share the

187 main pheromone components, which explains why S. littoralis male attraction to

188 pheromone-releasing females of S. litura and S. littoralis is not significantly different in

189 clean air (Fig. 1C; Saveer et al. 2014). However, these two pheromone blends differ in

190 composition and males are capable of discriminating conspecific from heterospecific

191 pheromone, since they clearly prefer conspecific over heterospecific S. litura females in

192 choice tests (Saveer et al. 2014). Tests with pheromone-releasing females on cotton bioRxiv preprint doi: https://doi.org/10.1101/293100; this version posted April 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Borrero et al. - p. 8

193 plants confirm the results obtained with synthetic pheromone: adding cotton to S. litura

194 females significantly reduced male upwind flights, compared to calling S. litura females

195 alone (z = 1.992; p = 0.046) (Fig. 1C).

196 Lastly, we examined the effect of volatiles from cotton challenged by larval feeding on

197 male sex pheromone attraction. We used a synthetic cotton blend and cotton plants on

198 which S. littoralis larvae had been feeding. The synthetic blend mimicking damaged

199 cotton and a cotton plant damaged by feeding larvae significantly reduced attraction to

200 the 4-component synthetic pheromone, respectively (z = 2.208; p = 0.027 and z =

201 2.953; p = 0.003z = 3.899) (Fig. 2A,B). Damaged cotton plants even reduced attraction

202 to calling S. littoralis females (z = 3.992; p < 0.001) (Fig. 2B).

203 Discussion

204 That mate finding is elicited by an ensemble of sexual and environmental odorants, which

205 mutually effect each other, provides a new perspective of premating communication in

206 moths and has bearings for our understanding of their ecology and phylogenetic

207 diversification.

208 The behavioural role of plant volatiles in male moth sexual behaviour has not been

209 entirely resolved. It has been proposed that host plant volatiles mediate male attraction

210 to mating sites either by themselves, before the onset of pheromone release by females,

211 or by synergizing the response to sex pheromone (Landolt & Phillips 1997; Reddy &

212 Guerrero 2004; Beyaert & Hilker 2014). In some species, host plant volatiles increase

213 male attraction towards sex pheromone (Dickens et al. 1993; Light et al. 1993; Yang et

214 al. 2004; Schmidt-Büsser et al. 2009; Varela et al. 2011; von Arx et al. 2012), whereas

215 they produce an antagonistic effect in other species (Pregitzer et al. 2012; Jung et al.

216 2013; Party et al. 2013; Rouyar et al. 2015). It is conceivable that volatiles from non-

217 host plants (Wang et al. 2016), volatiles from damaged plants, such as DMNT (Fig. 1C;

218 Hatano et al. 2015), or floral odorants, such as β-ocimene, which signal adult food

219 sources (Fig. 1C; Kroman et al. 2015) do not synergize pheromone attraction. It seems

220 counter-intuitive, on the other hand, that volatiles of larval food plants would inhibit male bioRxiv preprint doi: https://doi.org/10.1101/293100; this version posted April 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Borrero et al. - p. 9

221 attraction to conspecific female sex pheromone, since many moths mate on their

222 respective host plants, where females oviposit.

223 Our results offer an explanation for this conundrum. Firstly, volatile signatures from

224 damaged plants, that are less suitable for oviposition, reduce male attraction to

225 conspecific pheromone (Table 1, Fig. 2; Hatano et al. 2015, Zakir et al. 2017). Secondly,

226 host plant volatiles in combination with deficient or heterospecific pheromone reduce male

227 attraction (Table 1, Fig. 1). This compares to findings in grapevine moth Lobesia botrana,

228 where a blend of host plant volatiles increased male attraction to an optimized

229 pheromone blend, but decrased attraction to a single pheromone component (Sans et al.

230 2016).

231 Cotton is a common plant host for the sibling species S. littoralis and S. litura, which are

232 predominantly distributed in Africa and Asia, respectively. Males of the African cotton

233 leafworm S. littoralis are attracted to females of both species, but prefer conspecific

234 females in choice tests; heterospecific with S.litura females are prevented by

235 genital morphology (Saveer et al. 2014). That presence of the plant host diminishes

236 attraction to defective pheromone blends (Fig. 1B,C) accentuates differences between

237 conspecific and heterospecific sex pheromones. This interaction between plant cues and

238 sex signals is consequential, since it facilitates specific mate finding and recognition in

239 closely related species, which frequently use pheromone blends that share compounds

240 and partially overlap in composition (El-Sayed 2017). Sex pheromones typically consist of

241 a blend of several compounds that have been shown to function as a unit, while the single

242 components elicit only an incomplete behavioural response (Linn et al. 1986). Our

243 findings demonstrate that this laboratory-derived concept must be updated to

244 accommodate for an additional role of host plant volatiles in natural environments: it is

245 the ensemble of social signals and environmental cues that acts as a unit to mediate mate

246 finding and recognition.

247 Changes in female pheromone production and the corresponding shift in male preference

248 are driven by . Divergence of sex pheromone blends has been

249 documented in populations of the same species (Cardé et al. 1978; Malausa et al. 2005;

250 Groot et al. 2008; Velasquez-Velez et al. 2011) or in sibling species (Lofstedt & van der bioRxiv preprint doi: https://doi.org/10.1101/293100; this version posted April 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Borrero et al. - p. 10

251 Pers 1985; Bengtsson et al. 2014; Saveer et al. 2014). According to the “asymmetric

252 tracking” hypothesis, males track changes of the female pheromone composition and

253 rather quickly develop a preference for new pheromone blends (Phelan 1992; Heckel

254 2010; Droney et al. 2012). Such changes are believed to enable sympatric speciation in

255 moths through premating behavioural isolation (Smadja & Butlin 2009; M’Gonigle et al.

256 2012).

257 Host plant shifts in females, on the other hand, are driven by natural selection. If shifts in

258 female host plant preference are disruptive, they may lead to speciation with little or no

259 changes in pheromone composition (Lofstedt & van der Pers 1985; Witzgall et al. 1991;

260 Drès & Mallet 2002; Bengtsson et al. 2006; Leppik & Frérot 2012). Matsubayashi et al

261 (2010) suggest that changes in host plant preference may lead to premating isolation

262 based solely on a reduced probability of encounters between populations associated with

263 different hosts. In polyphagous species where populations have generalized diets,

264 individuals may have preferences for a particular host plant and may be subject to

265 selective pressures that may lead to diversification (Bolnick et al. 2003; Rueffler et al.

266 2006). The recent finding that host plant choice in S. littoralis is modified by larval

267 experience or adult learning (Proffit et al. 2015) supports a scenario where individual

268 preference could lead to host plant shifts and initiate divergence.

269 If host plant volatiles and pheromones function as a unit signal, it follows that male moth

270 pheromone detection and mate finding is under combined sexual and natural selection.

271 Traits combining local adaptation and mating decisions have been termed “magic traits”

272 since they facilitate phylogenetic divergence, especially in insect herbivores (Gavrilets

273 2004; Smadja & Butlin 2009; Servedio et al. 2011; Thibert-Plante & Gavrilets 2013;

274 Rebar & Rodríguez 2015). Under sympatric conditions, natural selection alone is unlikely

275 to lead to speciation due to random mating, however, if selection acts on both habitat and

276 mate preference simultaneously, speciation is far more likely to occur.

277 Our system demonstrates a mechanism where the behavioural consequences of shifts in

278 sex pheromone biosynthesis are reinforced by host plant volatiles. New pheromone

279 communication channels may give rise to reproductive isolation, especially in populations

280 diverging onto new host plants. Selection acting on a combination of pheromones and bioRxiv preprint doi: https://doi.org/10.1101/293100; this version posted April 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Borrero et al. - p. 11

281 host plant volatile signatures will reinforce premating barriers when a population

282 undergoes disruptive selection (Ritchie 2007; Butlin et al. 2012; Boughman & Svanbäck

283 2016). This adds further support to the view that sympatric speciation has contributed to

284 shaping the tremendous diversity of phytophagous insects (Tauber & Tauber 1989;

285 Berlocher & Feder 2002; Drès & Mallet 2002; Forbes et al. 2017).

286 Authors' contributions

287 All authors contributed to designing the study and writing the manuscript. FB was

288 responsible for insect rearing and bioassays, MB for chemicals.

289 Acknowledgements

290 We thank Medhat Sadek and Mohamed Khallaf, Assiut, for field-collecting S. littoralis, and

291 Kiyoshi Nakamuta and Saki Matsumoto, Chiba, for providing S. litura. This study was

292 supported by the Linnaeus environment “Insect Chemical Ecology, Ethology and

293 Evolution” IC-E3 (Formas, SLU), the Corporación Colombiana de Investigación

294 Agropecuaria (Corpoica) and the Colombian Administrative Department of Science,

295 Technology, and Innovation (Colciencias).

296 Data accessibility

297 Data will be made available at the Dryad Digital Repository http://dx.doi.org/…

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524 Figures

525 Figure 1. Male S. littoralis upwind flight attraction towards blends of S. littoralis females

526 sex pheromone and cotton volatiles. Main pheromone compound alone, or optimized four-

527 component S. littoralis synthetic pheromone blend (A, B), pheromone-reasing S. littoralis

528 or S. litura females (C), in combination with a synthetic cotton volatile blend (A) or a live

529 cotton plant (B, C). Bars with asterisks are significantly different from attraction to

530 pheromone control (binomial GLM and post-hoc Wald pairwise comparisons; n = 50).

531 Figure 2. Male S. littoralis upwind flight attraction towards blends of S. littoralis

532 pheromone and volatiles of cotton plants damaged by larval feeding. Four-component

533 pheromone blend (A,B), or a pheromone-reasing S. littoralis female (B), combined with a

534 synthetic blend of herbivore-damaged cotton volatiles (A), or a cotton plant damaged by

535 S. littoralis larval feeding (B). Bars with asterisks are significantly different from attraction

536 to pheromone control (binomial GLM and post-hoc Wald pairwise comparisons; n = 50). bioRxiv preprint doi: https://doi.org/10.1101/293100; this version posted April 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Borrero et al. - p. 19

537 Table

538 Table 1. Male cotton leafworm S. littoralis upwind flight attraction to synthetic cotton

539 volatiles (Loughrin et al. 1995; Saveer et al. 2012; Yang et al. 2013) and sex pheromone

540 compounds (Saveer et al. 2014; El-Sayed 2017). Single cotton volatiles were tested

541 alone, in mixtures with the S. littoralis main sex pheromone compound, and with an

542 optimized, four-component synthetic sex pheromone blend. Asterisks show significant

543 differences between attraction to pheromone alone and pheromone blended with single

544 cotton volatile compounds; α-farnesene was the only cotton volatile to elicit significant

545 attraction by itself (binomial GLM and post-hoc Wald pairwise comparison; n = 50).

Male upwind flight attraction [%] Main pheromone 4-Component a b Pheromone added None compound sex pheromone blend c Plant compounds - - 59 80 α-Farnesene 18† 28* 95 Nonanal 0 20* 88 (R)-(+)-Limonene 8 23* 78 (S)-(+)-Linalool 10 18* 78 β-Farnesene 3 25* 73 (R)-(+)-Linalool 0 0* 65 β-Myrcene 10 18* 63 (Z)-3-Hexenylacetate 0 10* 55* (E)-β-Ocimene 5 28* 48* d DMNT 0 3* 20*

546

a 547 Z9,E11-14Ac, release rate 100 pg/min

b 548 100:30:20:4-blend of Z9,E11-14Ac, Z9-14Ac, E10,E12-14Ac and Z9,E12-14Ac, release

549 rate 100 pg/min c 550 release rate 10 ng/min

d 551 4,8-dimethyl-1,3(E),7-nonatriene bioRxiv preprint doi: https://doi.org/10.1101/293100; this version posted April 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/293100; this version posted April 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.