1

for Vitis 1 Braunschweig, Germany 2 3

Oviposition behaviour and life-history performance of Epiphyas postvittana 4

(Lepidoptera: Tortricidae) on the leaves of Vitis vinifera (Vitales: Vitaceae) 5

infected with Botrytis cinerea (Helotiales: Sclerotiniaceae) 6 7

S. Z. M. RIZVI and A. RAMAN* 8

Charles Sturt University & Graham Centre for Agricultural Innovation, PO Box 883, Orange, NSW 2800, 9 Australia 10

11 12

Summary 13 14 In three-way interaction systems involving an insect and a plant-pathogenic fungus, 15

both occurring on the same plant, the insects generally gain in terms of their growth and 16

metabolism. In this study we have tested how the infection by Botrytis cinerea on the leaves 17

of Vitis vinifera influences the life-history performance of larvae and the oviposition 18

behaviour of Epiphyas postvittana. We conducted free-choice and two-choice experiments to 19

test the oviposition behaviour of gravid E. postvittana. We also characterized the effects of B. 20

cinerea-infected leaves of V. vinifera on the growth and development of E. postvittana. We 21

found that the oviposition preference of E. postvittana was strongly influenced by the 22

olfactory and tactile cues. Volatiles from B. cinerea-infected plants significantly deterred 23

oviposition and in consequence, adult females laid fewer eggs on B. cinerea-infected leaves of 24

V. vinifera compared with uninfected leaves. The mortality rate of larvae fed on B. cinerea-25

infected leaves were not significantly different from the larvae fed on uninfected leaves of V. 26

vinifera. Whereas, the larvae of E. postvittana fed on B. cinerea-infected leaves had 27

significantly shorter developmental period, attained heavier pupal mass, and on becoming 28

adults they laid more numbers of eggs than the larvae that were enabled to feed on 29

uninfected leaves of V. vinifera. We also reared the larvae of E. postvittana on exclusive-30

fungus diet but all larvae died before pupation indicating that for a better larval performance 31

and adult reproductive output of E. postvittana, the V. vinifera—B. cinerea interacting system 32

is but imperative. 33

34 35 Key words: grapevine, larval development, light-brown apple moth, oviposition deterrence, 36

three-way interaction. 37 38

Running t it le: Effect of grey mould on grapevine moth 39 ___________________________________________________________________________________ 40 *Correspondence to: Anantanarayanan Raman, Charles Sturt University, PO Box 883, Orange, NSW 2800, 41 Australia. Tel. and FAX: +61 2 63657833. E-mail: araman@csu.edu.au 42

2

Introduction 43

Infection by pathogenic fungi alters the chemistry of host plants and influences 44

oviposition preference and larval performance in plant-feeding insects (MONDY and CORIO-COSTET 45

2000; RIZVI et al. 2014a). Fungal infections can interfere with the biosynthetic pathways in the 46

plant in various ways. Some lead to a decrease in the production of insect-attracting volatiles 47

(DÖTTERL et al. 2009), whereas others produce several behaviour-modifying volatiles (TASIN et al. 48

2011). Fungal infection of a plant can suppress its direct defence capability against plant-feeding 49

insects by modifying the plant’s secondary metabolism (THALER et al. 1999) and the nutritional 50

quality of the plant, thus rendering the plant susceptible for insect colonization (CARDOZA et al. 51

2003). On the other hand, fungal infection can deteriorate the nutritional quality of the plant and 52

influence the plant-feeding insect’s development negatively (TASIN et al. 2012). 53

Olfactory cues are critical for searching a suitable host from a distance for oviposition but 54

once the insect lands on the target plant, the post-landing cues, viz., contact, together with the 55

volatiles can influence the insect for an assessment of the plant and can either deter or stimulate 56

oviposition (RENWICK and CHEW 1994; FOSTER et al. 1997; TASIN et al. 2011). Based on the sensory 57

cues of a plant-feeding insect, judgment on the appropriateness of a host plant is essential for the 58

success of progeny performance (GRIPENBERG et al. 2010). Optimization theory of host searching 59

suggests that the gravid female should choose those plants, which maximizes the fitness of her 60

progeny (JAENIKE, 1978), although evidences negating Jaenike (1978) also exist (LARSSON and EKBOM 61

1995; LEYVA et al. 2000). The choice made by juveniles of insects for food can substantially differ 62

from that made by adults, particularly when the larvae and adults feed on different plant parts 63

(MAYHEW 1997). In the natural environment, several organisms attack plants; among these, insects 64

and fungi are associated with a majority of plants, establishing a three-way interacting system. In 65

such contexts, the fungi influence the interaction between the insect and the plant, which can be 66

either mutualistic or antagonistic, and, occasionally, neutral as well (HATCHER 1995). The fungi, in 67

such interacting systems, can induce multiple variations in plant chemistry that can be 68

recognized by plant-feeding insects by their olfactory capacity, which, in turn, can modify the 69

preference behaviour in insects (NAJAR-RODRIGUEZ et al. 2010) 70

Nevertheless, in three-way interacting systems involving an insect and a plant-pathogenic 71

fungus, both developing on the same plant, the insects generally gain in terms of their growth 72

and metabolism. Leaf tissues of Arachis hypogaea (Fabales: Fabaceae)-infected with Sclerotium 73

rolfsii (Atheliales: Atheliaceae) showed significantly greater levels of soluble sugars but lower 74

starch content and total soluble phenolics compared with uninfected leaves of A. hypogaea. 75

3

Spodoptera exigua (Lepidoptera: Noctuidae) showed significantly greater survival rates, produced 76

significantly heavier pupae, and had shorter time span between the last larval instar and 77

pupation, when fed on the foliage of A. hypogaea infected with S. rolfsii (CARDOZA et al. 2003). For 78

instance, in the Botrytis cinerea—Lobesia botrana (Lepidoptera: Tortricidae)—V. vinifera 79

interacting system, L. botrana have been demonstrated to gain from B. cinerea by acquiring 80

sterols, which enhanced their ability to metamorphose rapidly gaining greater biomass (MONDY 81

and CORIO-COSTET 2000). The nutrient composition and levels of fungal infection in plants 82

influence the fitness and reproductive performance of an insect (BIERE and TACK 2013). These 83

findings indicate that the Lepidoptera gain from fungi associated with plants, as demonstrated by 84

accelerated growth rate, although in essence the insect can feed only on plant tissue and still 85

perform. 86

Keeping the above in view, we used a three-way interacting system of Epiphyas 87

postvittana—Vitis vinifera—Botrytis cinerea and proposed that that E. postvittana co-occurs with V. 88

vinifera infected by B. cinerea, and that for a better larval performance and oviposition behaviour 89

of E. postvittana, V. vinifera—B. cinerea interacting system is imperative. Epiphyas postvittana, an 90

Australian native Tortricidae, damages leaves and fruits of V. vinifera grown in Australasia 91

extensively. This species was accidentally introduced into England, Ireland, the Netherlands, 92

Sweden, besides Hawaii and California of USA (SUCKLING and BROCKERHOFF 2010). Field evidences 93

and laboratory studies indicate that the larvae of E. postvittana transmit the conidia of B. cinerea 94

(RIZVI et al. 2014b). Botrytis cinerea is an opportunistic necrotrophic pathogen that induces grey-95

mould disease on the leaves and fruits of V. vinifera (FOURNIER et al. 2013). 96

In this study, we sought answers to the following questions: (1) whether the females of E. 97

postvittana prefer ovipositing on V. vinifera infected by B. cinerea, (2) whether B. cinerea-infected 98

leaves positively influence larval performance of E. postvittana, and (3) whether the larvae of E. 99

postvittana can survive and develop feeding exclusively on B. cinerea. To secure answers, we 100

tested the oviposition preference of E. postvittana using B. cinerea-infected and uninfected leaves 101

of V. vinifera To test the larval performance with or without B. cinerea, we raised the larvae of E. 102

postvittana on uninfected and B. cinerea-infected V. vinifera leaves, using standard plant-nutrient 103

solution (e.g., Knop’s solution) as well as on standard synthetic culture media (viz., potato-104

dextrose agar [PDA], Murashige and Skoog medium [M−S medium]). To verify the survival and 105

development rate of the larvae of E. postvittana, we reared the larvae on B. cinerea cultured on 106

PDA and M−S medium, without V. vinifera. 107

108

4

Material and Methods 109 110 Insect culture 111 112 The larvae of E. postvittana were cultured on a Phaseolus lunatus-based semi-synthetic 113

diet (CUNNINGHAM 2007) and maintained at 21±1°C, 60―80% RH, and 16L: 8D regimen. On 114

emergence, an adult female was placed with a similar-aged adult male to facilitate mating as 115

described previously (RIZVI et al. 2014b). All eggs (5—6 days old) were surface-sterilized using 5% 116

formaldehyde solution and then washed with sterilized water following RAMAN and BEIDERBECK 117

(1992). 118

119 Fungus culture and preparation of conidial suspension 120 121 Botrytis cinerea isolated from infected berries of V. vinifera cv. Chardonnay from the 122

CSU—O’s Chardonnay vineyard in February 2013 was used. Stock culture was maintained on PDA 123

(Fisher Scientific, Inc., Scoresby, Victoria, Australia) at 22°C and 12L: 12D. The conidial suspension 124

was prepared in sterile water following RIZVI et al. (2014a) and adjusted to 106

conidia/ml. 125

126 Inoculation of V. vinifera leaves with B. cinerea 127 128 Sixty plants of V. vinifera cv. Chardonnay (hereafter V. vinifera) were raised in 129

polypropylene pots (17 cm tall, 10 cm Ø) containing commercial potting mix (Osmocote, Plus 130

Organics Vegetable & Herb Mix, Sydney, Australia) in an insect-proof glasshouse at 23±2°C, 60% 131

RH, and 16L:8D. Six-week old plants were used in the trials. The conidial suspension was sprayed 132

using a hand-held atomizer to infect the leaves of V. vinifera. Uninfected (control) leaves were 133

sprayed with sterile water identically. The inoculated and uninoculated plants were covered with 134

a polyethylene bag to enhance humidity and placed in an environmental chamber (Thermoline 135

L+M, Thermoline Scientific, NSW, Australia) (23°C, 70% RH, and 16L: 8D). Those plants which 136

manifested disease symptoms were used in the experiments. The infection level of B. cinerea- was 137

measured in percentage, from the photographs made with a digital camera (D―60, Canon, Tokyo, 138

Japan) and calculating the area of infection using ‘ImageJ’, an open-source image processing 139

software (ABRAMOFF et al. 2004). Those plants which showed 10—30% infection level were used in 140

the oviposition bioassays. 141

142 Oviposition behaviour 143 144 Free-choice experiment 145 146 Three uninfected and three B. cinerea-infected plants were placed in an aluminium-mesh 147

cage (200x80x80 cm3

). The plants were placed 10 cm away from the edge of the cage and were 148

5

distributed in two rows, separated by 40 cm between plants. Uninfected and infected plants were 149

placed alternately (Figure 1). Ten gravid females of E. postvittana were released at the midpoint of 150

the cage allowing the insects to choose either B. cinerea-infected or uninfected plants for 151

oviposition. The setup was left undisturbed. After 72 h, the plants were verified for eggs and the 152

number of eggs was counted under a stereo-binocular microscope (S–20, AIS Instrument Services, 153

Croydon, Victoria, Australia). This experiment was repeated six times. In each replicate new plants 154

were used and the position of the infected and uninfected plants were randomized. 155

156 Two-choice experiment 157 158

One B. cinerea-infected and one uninfected plants were individually placed in two glass 159

containers (60x30x30 cm3

), which had tiny holes on the one side to facilitate the passage of air. 160

The two containers were connected by a horizontal glass tube (30 cm long, 3 cm Ø). At the mid-161

point of the horizontal glass tube, a 2-cm wide circular port was cut to introduce the moths 162

(Figure 2). Five gravid individuals of E. postvittana were introduced through the port, enabling 163

them to choose either B. cinerea-infected or uninfected plant for oviposition. Soon after 164

introduction, the port was closed using a Parafilm® strip. After 72 h, the insects were removed. 165

The choices made by the insects were recorded and the numbers of eggs laid on B. cinerea-166

infected and uninfected V. vinifera were counted under a stereo-binocular microscope. This 167

experiment was repeated six times. In each replicate new plants were used and the position of the 168

infected and uninfected plants were randomized. 169

170

Larval development 171 172 On B. c inerea cultured on synthetic media 173 174 Thirty Petrie plates (9 cm Ø) of PDA were inoculated using a 4 mm

2

PDA block from stock 175

culture of B. cinerea. These plates were incubated at 21°C, 12L: 12D. Thirty sterile PDA plates were 176

used as control (Figure 3). After seven days, ensuring that B. cinerea was growing in the newly 177

introduced plates, the 4 mm2

PDA block was removed. Two neonate larvae (<4 h after emergence) 178

from sterile source were placed in every inoculated and sterile PDA plates, which were sealed 179

immediately using Parafilm. The plates were then incubated at 21°C, 12L: 12D. An identical 180

procedure was applied to raise the larvae of E. postvittana on aseptically cultured B. cinerea on 181

M−S medium (MURASHIGE and SKOOG 1972). All procedures were conducted in the clean-air 182

environment of the horizontal laminar-airflow bench (HWS120, Clyde–Apac, Sydney, Australia). 183

184

6

On B. cinerea - infected and uninfected leaves of V. vinifera maintained on 185 nutrient medium and on synthetic media 186

187 From freshly obtained branches of glasshouse-raised V. vinifera, several 20-cm stem 188

segments bearing 5―6 fully unfurled leaves were excised for use in this experiment. The stem 189

segments were surface-sterilized using 1% NaClO solution for 5 min and rinsed in sterile water 190

(3x). The leaves on these excised stem segments were then sprayed with the spore suspension in a 191

laminar-airflow cabinet. The leaves on stem segments used as control were sprayed with sterile 192

water. The basal tip of each segment was fixed on an Oasis® floral foam (4 cm3

) soaked in Knop’s 193

solution (RYCHTER and MIKULSKA 1990). The inoculated and uninoculated leaf-bearing stem 194

segments were placed in a zip-lock plastic bag (35x40 cm2

), which included a damp-filter paper. 195

The set up was maintained at 22°C and 12L: 12D for 4―5 d. 196

Only those leaves bearing around 10% infection level were used in this experiment. The 197

stem segments bearing either infected or uninfected leaves were placed separately in glass jars 198

(22 cm tall, 15 cm Ø) (Figure 4). Two neonate larvae (<4 h after emergence) were placed on 199

infected and uninfected leaves and maintained at 21±1°C, 60−70% RH, and 16L: 8D. After every 200

four days, the stem segments were changed to maintain the infection level under 30% of leaf area 201

and frass was removed from the plate. Fifty larvae were used for each treatment. Data pertaining 202

to the rate of survival of the larvae were collected every four days. The dates of pupation, number 203

of pupae, and pupal mass were recorded. Pupal mass was used as an indicator of adult-body 204

mass, because adult body mass cannot be obtained without killing them. Each pupa was 205

quarantined to a stoppered glass vial (4 cm tall, 2 cm Ø) until emergence. Adult emergence and sex 206

ratio (%) were recorded. The adults were sexed and paired in a corrugated-wall Dixie cup enabling 207

mating and oviposition. The adults enabled to mate and oviposit were subsequently measured for 208

their performance: fecundity rate, possible delays in oviposition (measured in days), viability 209

(measured as the number of emerged larvae). The eggs were incubated at 21±1°C, 60−70% RH, and 210

16L: 8D for 15 d enabling larval emergence. The number of days from the date of emergence of 211

adults to death was also recorded. 212

Life-history performance of E. postvittana on B. cinerea-infected or uninfected leaf was 213

further verified through using two aseptic nutrient media. Excised surface-sterilized leaf 214

(petioles+laminae) of V. vinifera was fixed onto either the PDA or M−S medium in a test tube (10 215

cm long, 1.5 cm ø, sealed with Parafilm) by pushing the petiole through the solid medium (Figure 216

5). Leaves were sprayed with spore suspension, whereas sterile water sprays were used in control. 217

All work was done on laminar-airflow bench, maintained at 22°C and 12L: 12D. 218

7

Leaves with 10% infection were used for rearing the larvae of E. postvittana. Two larvae 219

were released on each leaf. Every four days, the survival larvae were noted and larvae were 220

shifted onto either a new infected or a new uninfected leaf set up in the PDA or M−S medium as 221

described above. The larvae were allowed to pupate and the life-history traits were noted. Fifty 222

larvae were used for each treatment. Aseptic culture system has provided an environment to 223

experimental verify the life-history performance of many plant feeding insects. One of the 224

offshoots of such trials is raising the plant-feeding arthropods on their host plants in control 225

environment (e.g., FORNECK et al. 1998; FORNECK et al. 1999). 226

Statistical analysis 227 228 In the oviposition bioassay, the numbers of eggs laid on B. cinerea-infected and 229

uninfected leaves in free-choice experiment and two-choice experiment were analyzed using 230

paired sample ‘t’ test. Linear regression was applied to discriminate the significant difference in 231

the mortality rate of larvae applying ‘y=a+bx’, where x is the day and y is the mortality. A 232

contingency table (2

) was used to analyze female sex ratio, survival percentage of larvae from egg 233

hatching to pupae, and survival percentage of pupae from pupation to adult emergence. 234

Independent sample t-test was used to analyse all other life-history traits. Analyses were made 235

with SPSS statistic 17.0 (1993―2007) and GenStat (VSN International 2012). Graphs were 236

generated in MS Excel 2013 and GenStat. 237

Results 238 239

Oviposition behaviour 240 241 Free-choice experiment 242 243 Epiphyas postvittana females deposited significantly more number of eggs on uninfected 244

leaves than on B. cinerea-infected leaves (uninfected vs infected, 732.6±59.44 vs 236±40.5, t= 245

13.91, p<0.001, Figure 6). 246

247 Two-choice Experiment 248 249 Gravid females of E. postvittana showed a significant preference for uninfected leaves 250

(73.7%) as against the infected leaves (26.3%, n=30, χ2

=6.53, p<0.05, Figure 7). The B. cinerea-251

infected leaves significantly inhibited oviposition, compared with the uninfected leaves 252

(uninfected vs infected, 399±64vs 82±34, t= 4.36, p<0.01, Figure 8). 253

254 Larval development 255 256 On B. c inerea cultured on potato-dextrose agar 257 258

8

The mortality rate of the larvae of E. postvittana fed on B. cinerea on PDA 259

(y=120.6−120.4*0.89^

x) was significantly (p=0.039) lesser than the larvae that were fed only on 260

PDA (y=106.61-107.57*0.78^

x, where x represented time (days) and y represented the mortality 261

rate (Figure 9). All larvae were dead at conclusion (maximum15 d). 262

263 On B. c inerea cultured on Murashige and Skoog medium 264 265 The mortality rate of the larvae of E. postvittana fed on B. cinerea cultured (y=119.6-266

119.5*0.87^

x) was significantly (p= 0.027) fewer than the larvae fed on only M−S medium 267

(y=102.69-102.94*0.71^

x, where x = day and y = mortality rate (Figure 10). All larvae were dead at 268

conclusion (15 d). 269

On B. cinerea - infected and uninfected leaves of V. vinifera maintained on 270 Knop’s solution 271

272 Larvae reared on B. cinerea-infected leaves developed faster than larvae reared on 273

uninfected leaves of V. vinifera (Table 1). The mortality rate of larvae fed on B. cinerea-infected 274

leaves (y=29.68-30.55*0.92^

x) was not significantly different than those reared on uninfected 275

leaves of V. vinifera (y=19.65-19.65*0.86^

x), where x = day and y = mortality rate (Figure 11). Pupal 276

masses of female moths reared on B. cinerea-infected leaves of V. vinifera were significantly 277

greater (p<0.05) than larvae reared on uninfected leaves (Table2). Botrytis cinerea infection did 278

not affect pupation and adult emergence percentage, sex ratio of adult and the length of pre-279

oviposition period (Tables3). Botrytis cinerea-infected diet significantly increased the fecundity 280

rates (p<0.05) and viability of eggs (p<0.001) compared with uninfected leaves (Table 3). 281

We found similar effects on larval development and adult performance of E. postvittana, 282

when raised on uninfected or B. cinerea-infected leaves of V. vinifera using PDA and M–S media. 283

284

Discussion 285 286

Gravid E. postvittana prefer to oviposit on uninfected leaves as against that of B. cinerea-infected 287

leaves of V. vinifera. The larvae of E. postvittana which feed on B. cinerea-infected leaves develop 288

quicker, attain a heavier pupal mass particularly in those developing as females, and the adults 289

lay more numbers of eggs than those raised on uninfected leaves of V. vinifera. The viability rate 290

of eggs hatching to neonates too is greater in those adults reared on B. cinerea-infected leaves. 291

292 Females of E. postvittana do not prefer ovipositing on B. cinerea -293

infected leaves of V. vinifera 294 295

In the present study, the gravid individuals of E. postvittana were deterred during oviposition by 296

the olfactory cues arising from V. vinifera leaves infected by B. cinerea. In free-choice experiment 297

9

of this study, when the gravid females of E. postvittana had to choose sites for oviposition among 298

B. cinerea-infected and uninfected leaves of V. vinifera, thus mimicking the natural condition, 299

they deposited significantly more number of eggs on uninfected leaves compared with B. cinerea-300

infected leaves. This observation concurs with our previous finding (RIZVI et al. 2014 a, b), 301

wherein gravid E. postvittana avoided oviposition on the infected leaves and berries of V. vinifera, 302

but laid significantly greater number of eggs on the uninfected leaves and berries of V. vinifera. 303

Preference for oviposition in different species of Lepidoptera has been shown to be regulated 304

generally by either stimulatory or inhibitory cues arising from host plants (RENWICK 1989). Fungus 305

infection can modify the volatiles arising from a plant by interfering with the plant’s biosynthetic 306

pathways. Volatiles, arising during pathogenic interactions between plants and fungi, act either as 307

attractants (COSSÉ et al.1994; CARDOZA et al. 2003) or deterrents (DÖTTERL et al. 2009; Tasin et al. 308

2011) for insects in three-way, three-component interacting systems. Infection by B. cinerea on V. 309

vinifera alters its volatile complexion either by changing their quantity or by producing new 310

compounds, such as alcohols, terpenes, and benzoates (TASIN et al. 2012). Botrytis cinerea-infected 311

berries of V. vinifera include several new alcohols (TASIN et al. 2012), which deter L. botrana. 312

Receptors on the dendritic membrane of sensory neurons of E. postvittana and their specific 313

receptivity reiterate that E. postvittana is capable of recognizing a range of volatiles (e.g., 314

alcohols, terpenoids and benzoates) and volatile-stress signals such as methyl salicylate (JORDAN et 315

al. 2009). 316

A gravid Lepidoptera usually prefers, for oviposition, those sites which maximize its 317

opportunities for a better performance of her progeny (JAENIKE 1978). This builds on the premise 318

that juvenile-life stages have a limited capacity to move on host surfaces and therefore the gravid 319

female chooses the best possible site for oviposition guaranteeing reasonable nutrition to her 320

offspring. In contrast, several arguments and explanations prevail indicating that adult nutrition 321

influences oviposition decision (MAYHEW 1997; SCHEIRS and DE BRUYN 2002). For example, 322

Chromatomyia nigra (Diptera: Agromyzidae) and Altica carduorum (Coleoptera: Chrysomelidae) 323

have been demonstrated to select plants for oviposition that maximize their own fitness rather 324

than that of their progeny, indicating that optimum foraging determines oviposition decisions 325

(SCHEIR et al. 2000; SCHEIRS and De BRUYN 2002). In E. postvittana, the oviposition preference is 326

essentially governed by plant-surface cues, since it does not lay eggs on rough and hairy surfaces 327

(TOMKINS et al. 1991; FOSTER and HOWARD 1999). Incidence of fungal mycelia on the leaf surfaces of 328

V. vinifera, mimicking hairiness precludes oviposition (RIZVI et al. 2014a, b). The lepidopteran 329

larvae are highly mobile and voracious feeders and, therefore, do not connect strongly with 330

10

JAENIKE’s (1978) hypothesis that explains mother’s choice of site for oviposition benefitting the 331

offspring (ROITBERG and MANGEL 1993; SUCKLING and BROCKERHOFF 2010). Under experimental 332

conditions, the larvae of E. postvittana have been established as highly active feeders on plants of 333

various taxa and gravid females lay eggs on those ranges of plants (FOSTER and HOWARD 1999). 334

Botrytis cinerea -infected leaves positively influence larval performance 335 of E. postvittana 336

337 Insect and fungus association ranges from non-specific polyphagy to obligate mutualism (JONSELL 338

and NORDLANDER 2004). In the present study, oviposition choices made by adults of E. postvittana 339

do not synchronize with the best performance of the larvae. This mismatch could be because 340

either the larvae and adults of E. postvittana have differing host requirements or these life forms 341

feed on different organs of different plants. The larvae of E. postvittana feed on leaves and fruits 342

of V. vinifera, M. domestica, and different species of Citrus; whereas their adults feed on floral 343

nectar. Our results suggest a mutualistic relationship between the larvae of E. postvittana and B. 344

cinerea. The larvae develop more rapidly, attain greater pupal mass (particularly in the females), 345

and lay more eggs when fed on B. cinerea-infected leaves. Larval diet plays an important role in 346

determining the size and reproductive performance in the Lepidoptera (MONDY and CORIO-347

COSTET 2000), where the adult females utilize the energy stored by the larvae for egg production 348

(BOGGS, 1997). Botrytis cinerea plays an essential role in the larval diet and the larvae of L. botrana 349

have been demonstrated to disperse the conidia of B. cinerea to adjacent berries of V. vinifera 350

(FERMAUD and LEMENN 1992). Further, the injury caused by the larvae of L. botrana while feeding 351

on V. vinifera tissues facilitates the colonization and establishment of B. cinerea (FERMAUD and 352

LEMENN 1992). CARDOZA et al. (2002) found that leaves of A. hypogaea infected with a necrotrophic 353

fungus S. rolfsii preferably fed by S. exigua and had a positive effect on key life-history traits 354

when fed on S. rolfsii-infected A. hypogaea. 355

Botrytis cinerea is an opportunistic necrotrophic fungus, which, under favourable 356

conditions can kill its hosts. The level and nature of infection by fungi can influence the 357

oviposition behaviour of insects (BIERE and TACK 2013). It is, therefore, possible that over time, the 358

effect of B. cinerea can alter the behaviour of E. postvittana. Nonetheless, the caveat here is that 359

the levels of fungal infection used in the present study have indeed been low inducing a sub-360

lethal infection on V. vinifera leaves. In such a context, the choice of gravid females in avoiding B. 361

cinerea-infected leaves of V. vinifera could be seen as an adaptive strategy because the difference 362

between the time of oviposition and that of egg hatch, which potentially enhances the infection 363

levels on V. vinifera leaves. Therefore, the larval-food quality could have deteriorated with 364

11

accelerated level of infection or the production of a defensive compound could have reached up 365

to the lethal level (TASIN 2012). This could explain why B. cinerea-infected leaves are unattractive 366

for the gravid females for oviposition. Plant-pathogenic fungal infection may influence on the egg 367

survival and its hatchability ratio in insects (VAN BAEL et al. 2009; BITTLESTON et al. 2011). Only 368

further studies can shed light on the survival rate of eggs E. postvittana and its hatchability ratio 369

at different levels of infection by B. cinerea on V. vinifera determining the role played by B. 370

cinerea. 371

372

The larvae of E. postvittana cannot survive and develop by feeding 373 only on B. cinerea 374 375 Lepidopteran larvae require high nutritional levels to match their rapid growth before pupation 376

(SLANSKY and SCRIBER 1985). For example, the host-plant quality has been shown to have a 377

significant, indirect but positive effect on pupation in Manduca sexta (Lepidoptera: Sphingidae) 378

through accelerated growth during its larval stages (DIAMOND and KINGSOLVER 2011). Physiological 379

changes in the larvae in preparation for pupation indicate that later instars switch from protein-380

based diets to lipid-based diets to accumulate more of membrane-bound energy (STOCKHOFF 1993; 381

OJEDA−AVILA et al. 2003). Insects are generally shown to lack the capacity to synthesize sterols that 382

are necessary as precursors to steroids which act as growth regulators. The insects, therefore, 383

acquire sterols from their host plants and/or from associated symbionts (NES et al. 1997; BEHMER 384

and NES 2003). Fungi are an excellent source of sterols and vitamins, which are vital in insect 385

development (MONDY and CORIO-COSTET 2000). Lipids, as principal sources of stored energy and as 386

precursors of steroids are key for insects particularly during metamorphosis (ARRESE and SOULAGES 387

2010). Nevertheless, high levels of lipids and low levels of other essential nutrients appear to 388

stress insects by affecting metabolism and inducing an inability to maintain homeostasis (LORD, 389

2010). One possible reason for the death of larvae reared on exclusive fungus diet in our study 390

could be that the larvae of E. postvittana did not get sufficient energy-containing nutrients, such 391

as carbohydrates and proteins from B. cinerea, although they may have had a rich supply of lipids 392

from B. cinerea. A second possibility is that the concentrated dose of lipidic materials may have 393

induced a dietary imbalance as shown in the larvae of M. sexta parasitized by Cotesia congregata 394

(Hymenoptera: Braconidae) (THOMPSON and RADAK 2010). 395

Conclusion 396 397

Gravid females of E. postvittana use olfactory and volatile cues to ‘evaluate’ infection levels on V. 398

vinifera caused by B. cinerea to oviposit. Larvae of E. postvittana show a mutualistic relationship 399

12

with B. cinerea. On B. cinerea–infected V. vinifera leaves, the larvae have a shorter larval duration, 400

attain heavier pupal mass and the emergent adults lay more eggs than the larvae fed on 401

uninfected V. vinifera leaves. The larvae reared on exclusive-fungus diet died in maximum 15 d 402

indicating that for a better larval performance and oviposition rate of E. postvittana, the V. 403

vinifera—B. cinerea interacting system is but imperative although further chemical-ecological 404

verifications are necessary for confirming this. 405

406 407

Acknowledgment 408 409 We thank Helen Nicol for advice on statistical analysis. 410 411 412 413

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532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 Rizvi and Raman 556 557 Figure 1. Custom-made aluminium cage made for the free-choice experiment of adults of 558 Epiphyas postvittana (not to scale) (am: aluminium mesh, i: infected plant, u: uninfected 559 plant). 560 561 562 563 564 565 566 567 568 569

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am

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Rizvi and Raman 593 594 Figure 2. Custom-made glass device for the two-choice experiment using adults of 595 Epiphyas postvittana (not to scale) (cp: circular port, gj: glass jar, hgt: horizontal glass 596 tube, ip: infected plant, m: moth, p: Parafilm, up: uninfected plant) 597 598 599

gj m

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Rizvi and Raman 610

611 Figure 3. Petri-dish pair for the development of the larvae of E. postvittana on B. cinerea. 612 (not to scale) (l: larva, pd: Petri dish, l: Petri dish lid) 613 614

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Rizvi and Raman 628 Figure 4. Custom-made glass device for the rearing of the larvae of Epiphyas postvittana 629 (not to scale) (ff: floral foam, il: infected leaf, l: larva, ps: parafilm seal, ul: uninfected 630 leaf) 631

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Rizvi and Raman 645

Figure 5. Custom-made glass device for the rearing of the larvae of Epiphyas postvittana 646 (not to scale) (l: larva, gt: glass tube, il: infected leaf, ms: medium slant, ps: Parafilm seal, 647 ul: uninfected leaf) 648

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Rizvi and Raman 666 667

Figure 6. Mean number of eggs laid by E. postvittana in a ‘free-choice’ experiment with 668 uninfected (control) leaves ( ) and B. cinerea-infected leaves ( ) of V. vinifera. 669

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684 Rizvi and Raman 685 686 Figure 7. Response of females E. postvittana in a ‘two-choice’ experiment towards 687 uninfected (control) leaves ( ) and B. cinerea-infected leaves ( ) of V. vinifera. 688

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Rizvi and Raman 705 706 Figure 8. Mean number of eggs laid by E. postvittana in a ‘two-choice’ experiment with 707 uninfected (control) leaves ( ) and B. cinerea-infected leaves ( ) of V. vinifera. 708

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Rizvi and Raman 726 727 Figure 9. Mortality rate of E. postvittana larvae reared solely on B. cinerea ( ) 728 and control (or PDA, ). 729

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Rizvi and Raman 746 747 Figure 10. Mortality rate of larvae reared on B. cinerea ( ) and control (or M−S 748 medium, ). 749 750

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Rizvi and Raman 767 768 Figure 11. Mortality rate of larvae reared on B. cinerea-leaves ( ) or uninfected 769 ( ) leaves of V. vinifera using Knop’s solution 770

26

771

772

773

774

Rizvi and Raman 775

776

Table 1. Larval developmental time, pupal duration and adult life span (mean ± s.e) of E. 777 postvittana reared on B. cinerea-infected or uninfected leaves of V. vinifera using Knop’s solution 778

779

780

781

782

t = Independent sample t-test 783 784

785

786

787

788

789

790

791

792 793 794 795 796 797

Treatment Larval developmental time (d) Pupal duration (d) Adult life span(d)

Male Female Male Female Male Female

Uninfected

leaves 32.5±0.9 34.0±0.8 10.2±0.3 9.3±0.1 8.2±0.7 8.8±0.9

B. cinerea

Infected leaves 27.9±0.5 30.9±0.5 10.0 ±0.2 9.2±0.1 7.5±0.9 9.6±0.7

Statistic t = 2.0 t = 2.8 t = 0.6 t = 1.7 t = 1.9 t = 0.4

p 0.047 0.007 0.50 0.89 0.06 0.69

27

798 799 800 801 802 803 804 805 806 Rizvi and Raman 807 808 Table 2. Pupal mass, percentage of larvae surviving from hating to pupae, percentage of 809 larvae surviving from hating to adult and female sex ratio (mean ± s.e) of E. postvittana 810 reared on B. cinerea-infected or uninfected leaves of V. vinifera using Knop’s solution: 811 812

813

814

815

t = Independent sample t-test 816 χ2

= Contingency table (χ2

) 817

Treatment

Pupal mass (mg) % surviving from

hatching to pupa

% surviving from

hatching to adult

Female sex

ratio (%)

Male Female

Uninfected

leaves 20.9±0.7 27.5±1.1 78 66 46

B. cinerea

Infected

leaves

19.7±0.7 30.6±0.9 76 71 48

Statistic

t = 1.1 t = 2.1 χ2

=0.11 χ2

= 0.58 χ2

=0.08

p 0.25 0.04 0.737 0.44 0.77

28

818 819 820 821 822 823 824 825 826 827 828 829 830 831 Rizvi and Raman 832 833 834 Table 3. Adult performance (mean ± s.e) of E. postvittana reared on B. cinerea-infected 835 and uninfected leaves of V. vinifera using Knop’s solution: 836 837

838 839

t = Independent sample t-test 840 χ2

= Contingency table (χ2

) 841 842

Treatment

Delay in egg laying

(days)

Fecundity

(no. eggs per female)

No. of larvae emerged

per female

Uninfected

leaves 2.3±0.1 80.0±10.9 67.2±5.8

B. cinerea

Infected leaves 2.1±0.1 115.1±13.2 106.7±12.3

Statistic

t = 0.98 t = 2.5 t = 2.98

p 0.86 0.01 0.006