Martini et al. Egg cannibalism in A. bipunctata

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Journal of Applied Entomology  1

Original contributions 2

Correspondence: Xavier Martini 3

University of Florida 4

Citrus Research and Education Center 5

Lake Alfred, FL, USA. 6

Phone: 1-863-224-1631 7

Fax: 1-863-956-4631 8

xavier.martini@voila.fr 9

10

Egg-cannibalism by ladybird larvae is less frequent than expected from the nutritional benefit 11

accrued to cannibals 12

MARTINI Xavier1,2, GARRIGUES Jean-François2, HEMPTINNE Jean-Louis2 13

14

1 University of Florida, Citrus Research and Education Center, 700 Experiment Station Road, 15

Lake Alfred, FL, 33850, USA. email: xavier.martini@voila.fr. 16

2 Université Toulouse 3 – CNRS - ENFA, UMR 5174 EDB (Laboratoire Evolution & Diversité 17

Biologique), BP 22687, F-31326, Castanet Tolosan Cedex, France.18

Martini et al. Egg cannibalism in A. bipunctata

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Abstract 19

Egg cannibalism is a widespread phenomenon in predatory arthropods. However, conflicting 20

results have been reported regarding the nutritional value of conspecific eggs. Therefore, 21

evaluation of the value of a mixed diet of conspecific eggs and aphids for survival and growth of 22

Adalia bipunctata (Coccinellidae) larvae was tested. Subsequently, the propensity for 23

cannibalism of naive and experienced larvae was assessed in two experiments in which the 24

relative density and quality of conspecific eggs were manipulated in an experimental arena. It 25

was found that larvae that were fed a mixed diet of conspecific eggs and aphids moulted into 26

larger adults than those fed either aphids or eggs, and those that fed on conspecific eggs lost less 27

mass than those fed only aphids during pupal stage. Additionally, in an experimental arena, 28

attacks on aphids were more frequent than expected when conspecific eggs occupied 50% and 29

75% of the patches. When 50% of patches were occupied by conspecific eggs, the preference for 30

aphids was less marked when larvae had previously experienced cannibalism (76% versus 52% 31

respectively) or when offered in the arena conspecific egg the cuticular hydrocarbons of which 32

had been removed (76% versus 48% respectively). However, cannibalism was not enhanced if 33

larvae experienced heterospecific prey shortages, but were supplemented with an artificial diet. 34

Given that prey choice in A. bipunctata larvae is driven by chemical cues, and that hydrocarbons 35

on the egg surfaces and in larval tracks are very similar, we hypothesize that the naive larvae 36

avoid eggs because of the uncertainty that those hydrocarbons indicate either eggs or conspecific 37

larvae. 38

39

Keywords: Coccinellidae, intraspecific predation, prey choice, cuticular hydrocarbons. 40

41

Martini et al. Egg cannibalism in A. bipunctata

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Introduction 42

Polis (1981) recorded 1,300 different species that practice cannibalism in a wide range of taxa 43

including insects, fish and mammals. The occurrence of cannibalism within a population depends 44

on the balance between benefits and costs. On the one hand, cannibalism is advantageous as it 45

provides energy and eliminates potential competitors (Getto et al. 2005). On the other hand, it 46

facilitates the transmission of pathogens (Saito and Bjornson 2006; Elnagdy et al. 2011) and 47

reduces inclusive fitness if cannibals eat genetically related individuals (Polis 1981; Dixon 48

2000). As a result of this balance between benefits and costs, cannibalism propensity varies 49

among species, and can even differ between populations of particular species (Tayeh et al. 2014). 50

Theories suggest that cannibalism is likely to evolve in species in which the individuals disperse 51

widely because cannibals would then be more likely to attack and eat genetically unrelated 52

conspecifics (Lion and van Baalen 2009; Rudolf et al. 2010). 53

It was hypothesized that conspecific individuals are a better source of essential nutrients than 54

heterospecific prey because of a better match in tissue composition (Pfennig 2000), which could 55

favour the development of a specific taste for conspecifics. Michaud (2003) confirmed this 56

hypothesis by showing that the larvae of the ladybirds Harmonia axyridis Pallas, Olla v-nigrum 57

Mulsant and Cycloneda sanguinea L. developed well on a diet made of conspecific eggs. 58

However, Schausberger and Croft (2000) questioned the benefit of cannibalism by showing that 59

the nutritional quality of conspecific eggs and larvae of different species of predatory mites 60

varies. Similarly, Ware et al. (2009) showed that larvae of the multicolored Asian ladybird, H. 61

axyridis and the two-spotted ladybird Adalia bipunctata (L.) (Coleoptera: Coccinellidae), had a 62

slower development and lower adult size when fed a mix of conspecific eggs and aphids. 63

Martini et al. Egg cannibalism in A. bipunctata

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Aphidophagous ladybirds frequently attack and eat non-sibling eggs both in the field and 64

laboratory (Osawa 1989; Cottrell and Yeargan 1998b, a). In a theoretical study, Martini et al. 65

(2009) proposed that if egg cannibalism is as beneficial as the consumption of prey, then both 66

should occur as frequently, provided the two types of food are encounter at the same rate. The 67

aim of this study was to test the benefit of, and appetence for egg-cannibalism by using third and 68

fourth-instar ladybirds. At this stage, larvae are good models to work on for studying 69

cannibalism, as they are more mobile and less fragile than first and second-instars, more 70

predaceous, and need to accumulate metabolic resources before pupation. Moreover, 71

cannibalism by non-neonate larvae in nature has been reported several times (Mills 1982; 72

Cottrell and Yeargan 1998a, b). 73

Specifically, the benefits of adding conspecific eggs to an aphid diet was evaluated by collecting 74

data on larval mass, mass lost during the pupal stage, adult mass, survival rate and the number of 75

ovarioles. In a second experiment, whether or not ladybird larvae showed a preference for eating 76

conspecifics depending on egg and aphid density was studied. Finally, we also investigated the 77

importance of prior cannibalism experience of the larvae, and the role of cuticular hydrocarbons 78

of the egg surface in cannibalism. 79

80

Materials and Methods 81

Insect cultures. Pea aphids, Acyrthosiphon pisum Harris (Hemiptera: Aphididae) were reared on 82

broad beans, Vicia faba L., grown in compost at 20±1°C under a photoperiod of 16:8 (LD) h. 83

These were collected on 2-wk-old plants and fed to ladybirds. The two-spotted ladybird, A. 84

bipunctata, used in this study came from a laboratory culture of adults collected in the wild near 85

Toulouse, France. This culture was renewed every year after about 12 generations. Each 86

Martini et al. Egg cannibalism in A. bipunctata

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September, about 100 new adults were collected in the field, and reared in laboratory conditions 87

to obtain one generation. To avoid contamination with pathogens, only this new generation was 88

mixed with the existing laboratory culture. During the experimental period the culture consisted 89

of about 120 to 160 adults. Adults were reared at 20 ± 1°C under a photoperiod of 16:8 (L:D) h, 90

in ventilated 5 l plastic boxes. The boxes contained a piece of corrugated filter paper (1745F, 91

Fisher Scientific EU, Illkirch, France) on which the females usually laid eggs. These eggs were 92

either used to perpetuate the laboratory culture or for the experiments. Eggs used to obtain 93

experimental larvae were incubated at 20 ± 1°C and a photoperiod of 16:8 LD. With a fine 94

paintbrush, larvae were isolated just after hatching in 5-cm-diameter Petri dishes and fed A. 95

pisum three times a week until ready for experiments. Eggs used as food in the experiments were 96

placed in plastic boxes and kept in the dark at 4°C ± 1°C to prevent hatching. Eggs were < 6 d 97

old when used in an experiment. To avoid a confounding effect of genetic relatedness, eggs 98

offered to larvae during experiments were from different parents. 99

100

Experiment 1: What is the benefit of adding conspecific eggs to a pure diet of aphids? 101

Newborn larvae were isolated in 60 x 15 mm Petri dishes. Every day when fresh aphids were 102

provided to the larvae, dishes were searched for a shed skin indicating the larva had moulted 103

during the previous 24 h. By this way, we were able to spot larvae that were entering the third 104

instar. We preliminary established that during the 24 h after moulting, third instar body mass was 105

linearly correlated with time after moulting. Using a standard curve (M (mg) = 0.106 hour + 106

1.205, R2=0.92, n=13, Supporting information S1), we determined larvae that had moulted 2 to 107

14 h ago, and those larvae were selected for the experiments. The larvae were randomly sorted 108

into three groups and every day were fed either: (1) 20 adults of A. pisum; (2) 10 adults of A. 109

Martini et al. Egg cannibalism in A. bipunctata

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pisum and 40 (± 4) conspecific eggs or (3) 80 (± 8) conspecific eggs. The quantities provided in 110

the pure diets (1) and (3) ensured that the larvae were fed ad libitum, whereas in treatment (2) 111

there were not enough aphids or eggs to satiate third instar larvae. To be fed ad libitum these 112

larvae were therefore forced to satiate themselves by consuming both types of food. There were 113

32 replicates of each diet. Every day, the larvae were weighed, transferred to clean Petri dishes, 114

and provided fresh food. The emerging adults were weighed before exoskeleton hardened and 115

their sex determined the day they emerged based on the presence or absence of a male-specific 116

pronounced notch in the ventral portion of the last segment of the abdomen (Hodek & Honek, 117

1996). The females were fed A. pisum in excess for 2 wk, then they were dissected and their 118

ovarioles counted. The differences in the average numbers of eggs consumed per day between 119

pure eggs and mixed diet treatment were analysed using Student’s t-test. The proportion of 120

larvae that reached adulthood and the sex ratio were compared using Chi square test . The 121

numbers of ovarioles were compared with one-way analysis of variance (ANOVA). The 122

maximum mass of the larvae, the mass of the adults, and the relative loss of mass during pupal 123

stage were analysed using two-way ANOVA, with diet and sex as the two independent factors. 124

Individuals that died during larval development were excluded from t-test and ANOVA analyses. 125

Model simplification was used by stepwise deletion to assess whether performance on the 126

different diets differed (Crawley 2009). All statistics were conducted using the statistical 127

software R 3.0 (http://www.r-project.org). 128

129

Experiment 2: Effect of egg and aphid density on larval encounters 130

The goal of this experiment was to study if the rate of cannibalism is proportional to the rate of 131

encounter of conspecific prey. The aim was to test, the “meet and eat” hypothesis of Kindlmann 132

Martini et al. Egg cannibalism in A. bipunctata

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and Dixon (2003), according to which “the predator shows no preference for either prey or 133

conspecifics” (Agarwala and Dixon 1992; Dixon 2000). To test this hypothesis, fourth instars 134

that had moulted 24 to 48h ago were selected. The preference of the larvae was tested in an arena 135

placed in a 10 cm diameter Petri dish. The arena consisting of an 8 x 8 cm square divided in 25 136

smaller squares of 1.6 x 1.6 cm. At the centre of each of these, a food patch was presented. Each 137

patch covered approximately 0.4 x 0.4 cm. The larvae were always introduced on the empty 138

central patch at the beginning of the experiment and the remaining 24 patches were randomly 139

assigned to an “egg patch” or an “aphid patch”. An “egg patch” consisted of a cluster of 15 to 20 140

non-sibling conspecific eggs, and an “aphid patch” of two adult A. pisum. Aphids were 141

previously killed by freezing (10 min at -78°C ± 2°C) to avoid the confounding influence of 142

aphid movement on larval behaviour. There were three treatments: (1) 6 egg and 18 aphid 143

patches (ratio egg to aphid patches: 1:3), (2) 12 egg and 12 aphid patches (1:1), and (3) 18 egg 144

and 6 aphid patches (3:1). In a preliminary experiment, it was determined whether dead aphids 145

were eaten as readily by larvae as living aphids. 28 larvae were isolated in 6 cm Petri dishes in 146

the presence of two frozen and two living aphids. The Petri dishes were checked every 30 min 147

until the first aphid was attacked. The proportion of larvae attacking first frozen aphids (16 out of 148

28) did not significantly differ from the proportion attacking first fresh aphids (12 out of 28; 149

Binomial test, P=0.57). 150

At the beginning of the experiment, a fourth-instar larva was placed on the central patch. As it 151

started to forage, we recorded how many times it encountered eggs or aphids before attacking 152

one of these food items. An ‘encounter’ occurred when a larva palpated the eggs or the aphids 153

with its first pair of legs or its mouth parts, and an ‘attack’ when a larva started feeding on the 154

prey. This was determined by a prolonged contact with the prey (> 10s), and subsequently 155

Martini et al. Egg cannibalism in A. bipunctata

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confirmed by observation under a binocular microscope. After a larva attacked a patch, it was 156

removed from the arena. New egg and aphid patches were randomly assigned to the 24 patches 157

and a new larva was placed in the arena. An arena was not used more than five times. This was 158

repeated 29 times for each treatment. A Kruskal-Wallis test was performed to determine whether 159

the number of encounters with the food patches was the same in the three treatments, and a 160

Wilcoxon rank sum test to determine whether the number of encounters with those food patches 161

were the same before attacking eggs or aphids. A Chi square test was performed for goodness of 162

fit (Zar 2010) to see whether each larva encountered egg and aphid patches according to their 163

proportions in the arena. For each treatment, the proportion of larvae attacking eggs was 164

calculated and compared to the proportion of egg patches using a Chi square test. Additionally, 165

we measured the consumer preference for eggs with the Manly’s alpha (Manly 1974) that is 166

given by the following equation: 167

168

𝛼! =𝑓!𝑔!∙

1𝑓! 𝑔! +

𝑓! 𝑔!

169 With fe and fa standing for the number of egg and aphid patches attacked by larvae; and ge and ga 170

for the relative abundance of egg and aphid patches in the arena. A score of 1 indicates an 171

exclusive consumption of eggs whereas a score of 0 denotes a total avoidance of egg patches. 172

173

Experiment 3: Do egg surface hydrocarbons or prior egg consumption affect propensity for 174

egg cannibalism? 175

The experimental design was the same as in the previous experiment with the exception that 50% 176

of the patches were always occupied by eggs. There were four treatments with 25 replicates 177

each: (1) The control: 24h prior to the experiment, the larvae were fed pea aphids ad libitum; (2) 178

Martini et al. Egg cannibalism in A. bipunctata

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The low prey treatment: to simulate a situation where the larvae have food but with a low density 179

of A. pisum, 24h prior to the experiment, the larvae were only fed four adults of A. pisum. To 180

avoid the confounding influence of starvation, this regime was supplemented with an artificial 181

diet rich in proteins and sugars (Dong et al. 2001). A preliminary experiment showed that larvae 182

either daily fed aphids ad libitum or 4 aphids plus the above mentioned artificial diet ad libitum 183

from hatching to pupation did not differ in terms of survival and mass (n=60 for each feeding 184

regime; Mass: 9.90 mg (aphid diet), 10.89 mg (aphid+artificial diet), Mann-Whitney U = 128, P 185

= 0.29; Survival: 80% (aphid diet), 90% (aphids+artificial diet), χ2 = 0.16, P > 0.05). (3)The egg 186

treatment: 24h prior to the experiment, they were isolated in a Petri dish with 15 to 20 eggs of A. 187

bipunctata and were moved back to a Petri dish with A. pisum in excess as soon as the eggs were 188

consumed. (4) The washed eggs treatment: the larvae were treated as in the control but the eggs 189

presented in the arena had been washed with n-hexane to remove the hydrocarbons present on 190

their surface (Hemptinne et al. 2000). To obtain these eggs, the filter paper on which eggs were 191

laid was dampened with distilled water to dissolve the substance that glues them to the substrate. 192

After a few minutes, they were gently removed from the paper using a fine paintbrush. They 193

were then immersed in n-hexane in groups of about 300 in a 200 ml glass beaker for 2 min. We 194

used n-hexane because it is a non-polar solvent suitable for dissolving non-polar molecules such 195

as hydrocarbons. At the end of this period, the solvent was removed and the eggs allow drying 196

for 30 min on filter paper under a fume hood. Finally, groups of 15 to 20 eggs were randomly 197

placed on the egg patches in the experimental arena. Hemptinne et al. (2000) showed that eggs 198

washed as above are only superficially modified. For each of these treatments the proportion of 199

larvae eating eggs was calculated and compared to an expected proportion of 0.50 using a 200

binomial test. 201

Martini et al. Egg cannibalism in A. bipunctata

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202

Results 203

204

Experiment 1: What is the benefit of adding conspecific eggs to a pure diet of aphids? 205

The number of larvae surviving to adulthood did not differ significantly in the different 206

treatments (χ2=1.46, d.f.=2, P=0.46, Table 1). The sex ratio of the survivors was also similar in 207

all the treatments (χ2=0.99, d.f.=2, P=0.61, Table 1). Only five of the larvae given the mixed diet 208

ate eggs during the third instar, but all of them did so during the fourth. These third and fourth-209

instar larvae ate significantly fewer eggs than those in the pure egg treatment (third-instar: 210

t57=7.39, P<0.001; fourth-instar: t57=15.15, P<0.001; Table 2). 211

The interaction between sex and diet did not significantly affect larval mass (F2, 80=0.13, P=0.88; 212

Fig. 1A) and was therefore excluded from the analysis (Crawley 2009). Both sex and diet had a 213

significant effect on larval mass (Sex: F1, 82=46.01, P<0.001; Diet: F2, 82 =3.11, P<0.05, 214

respectively, Fig. 1A). Males were lighter than female, and larvae fed only eggs were lighter than 215

those fed the mixed diet (Fig 1A). 216

For adult mass, there was no interaction between sex and diet (F2, 80=0.19, P=0.82; Fig. 1B), 217

which was therefore excluded from the analysis. Both sex and diet had a significant effect on 218

adult mass (Sex: F1,82=63.83, P<0.001; Diet: F2, 82=5.21, P<0.01; Fig. 1B). Adult males were 219

lighter than adult females. The larvae fed the mixed diet became heavier as adults than those that 220

only ate aphids or eggs. The mass of adults fed either aphids or eggs as larvae was the same (Fig. 221

2B). 222

The percentage of mass lost during pupation was not influenced by the interaction between sex 223

and treatment (F2, 80=1.38, P=0.26), which was therefore excluded from further analyses. Males 224

Martini et al. Egg cannibalism in A. bipunctata

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lost more mass than females (F1, 82=10.13, P<0.01; Fig. 2) and the diets had a significant effect 225

(F2, 82 = 6.66, P<0.01; Fig. 2). Larvae fed aphids lost proportionally more mass than those fed 226

either conspecific eggs or both eggs and aphids, whereas there was no significant difference 227

between the losses of mass when fed the two diets with eggs (Fig. 2). Finally, the number of 228

ovarioles in the females that developed on the three diets were similar (F2, 39=0.59, P=0.56, Table 229

1). 230

231

232

Experiment 2: Effect of egg and aphid density on larval encounters. 233

There was no significant difference in the total number of encounters with food patches in the 234

three treatments (Kruskal Wallis, χ2=1.25, d.f.=2, P=0.54, Fig. 3A). Moreover, there was no 235

significant difference in the total number of encounters with food patches before larvae attacked 236

eggs or aphids (Wilcoxon rank sum, W=664.5, P=0.56, Fig. 3B). With 25 and 50% of the 237

patches in the arena with eggs, the percentage of larvae encountering eggs were not significantly 238

different from the expected frequencies, respectively 25 and 50% (Density 25%: χ2= 27.84, 239

d.f.=28, P=0.47; Density 50%: χ2 = 32.95, d.f.=28, P=0.24, Fig. 3C). However, larvae 240

encountered patches of eggs significantly less frequently than expected when there were 75% of 241

egg patches (Density 75%: χ2= 56.17, d.f.=28, P<0.01, Fig. 3C). Attacks on eggs were less 242

frequent than expected when there were eggs in 50 and 75% of the patches in the arena (Density 243

25%: 5 out of 29, P=0.40; Density 50 %: 6 out of 29, P<0.01; Density 75%: 12 out of 29, 244

P<0.001, Fig. 3C). This was confirmed by the decrease of Manly’s alpha score for eggs when 245

egg density increased in the arena (25%: αe= 0.38, 50%: αe= 0.21, 75% αe= 0.19). 246

247

Martini et al. Egg cannibalism in A. bipunctata

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Experiment 3: Do egg surface hydrocarbons or prior egg consumption affect propensity for 248

egg cannibalism? 249

Nineteen larvae out of 25 attacked aphids in the control treatment compared to 21 out of 25 of 250

those that were supplied with fewer aphids. In these two treatments, larvae displayed a 251

significant preference for aphids (Control: P<0.05; Low prey: P<0.001, Fig. 4), which was 252

confirmed with the low Manly’s alpha score for egg preys (Control: αe=0.24, Low prey: αe = 253

0.16). On the contrary, the larvae that ate eggs before the experiment did not show preference for 254

aphids (P=0.69, Fig. 4), as well as those that were offered washed eggs, (P=0.69, Fig. 4). In this 255

cases Manly’s alpha score were close to 0.5, indicating an absence of preference between eggs 256

and aphids (eggs eaten prior to the experiment: αe=0.48, washed eggs: αe=0.52). 257

258

Discussion 259

We first demonstrated that egg cannibalism by larvae is advantageous even when prey of 260

good quality is abundant. We showed that adding eggs to an aphid diet resulted in heavier adults 261

than ad libitum diets of either aphids or eggs. We also noticed that larvae lost less mass during 262

the pupal stage if they previously ate a pure diet of conspecific eggs or a mixed diet of eggs and 263

aphids rather than only aphids. This result may be due to the fact that eggs are four times richer 264

in lipids than A. pisum (Febvay et al. 1992; Sloggett and Lorenz 2008). Thus, it is not surprising 265

that conspecific eggs appear to be high value food for late instar larvae. Conspecific eggs are an 266

essential food for larvae at hatching, because it provides the very first meal and is highly 267

energetic (Majerus 1994 ; Omkar et al. 2007). Eating eggs at hatching increases survival and 268

reduces larval developmental time (Michaud and Grant 2004). 269

Martini et al. Egg cannibalism in A. bipunctata

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It is likely that egg cannibalism evolved in ladybirds because the costs, mainly pathogen 270

transmission (Saito and Bjornson 2006; Elnagdy et al. 2011) and potential decrease of inclusive 271

fitness, are balanced by benefits (Agarwala and Dixon 1992; Martini et al. 2009). In the case of 272

egg cannibalism by larvae, the probability that eggs and larvae are genetically related is very low 273

because females tend to disperse their eggs among several aphid patches (Hemptinne et al. 274

1992), and oviposition is more inhibited by cues indicating the presence of their own offspring 275

than unrelated larvae (Martini et al. 2013). This behaviour favours cannibalism because the loss 276

of inclusive fitness incurred by the consumption of related eggs is reduced (Rudolf et al. 2010). 277

Since there is no risk of injury associated with egg cannibalism, and apart of the possibility of 278

pathogen transmission (Saito and Bjornson 2006; Elnagdy et al. 2011), larvae should have not 279

avoided conspecific eggs. 280

However, contrary to theories that predict that ladybirds should show no preference for 281

either prey or conspecifics (Agarwala and Dixon 1992; Dixon 2000; Martini et al. 2009), it was 282

observed that egg cannibalism was avoided over aphids particularly when the density of eggs 283

was high. This finding differs from Cottrell (2007) that tested five different species of ladybird, 284

and found that only one species (Cycloneda munda Say) significantly rejected conspecific eggs 285

at the first contact. However, Cottrell’s results were obtained in non-choice tests, and 286

consequently were performed without aphids. Other studies showed that egg cannibalism by A. 287

bipunctata and H. axyridis larvae decreases in the presence of aphids (Burgio et al. 2002). 288

We hypothesized that this relative avoidance of egg cannibalism was likely due to the 289

presence of cuticular hydrocarbons on the egg surface. Indeed, once removed, egg consumption 290

became as frequent as aphid predation. These hydrocarbons may be part of a kind of aposematic 291

signal that indicates the presence of toxic alkaloids in the eggs (Kajita et al. 2010), and might 292

Martini et al. Egg cannibalism in A. bipunctata

14

confer a protection again intra-guild predation (Hemptinne et al. 2000; Omkar et al. 2004; 293

Cottrell 2007; Ware et al. 2008). Our results also suggest that they decrease the palatability of A. 294

bipunctata eggs to naive conspecific larvae. It is interesting to point out that these hydrocarbons, 295

mainly alkanes, are very similar to those present in the tracks left by larvae foraging on plants 296

and that inhibit oviposition by ladybirds from (Table 3 of Hemptinne et al. 2000, and Table 1 of 297

Hemptinne et al. 2001). When foraging for prey, ladybird larvae do not use visual cues and 298

mainly rely on chemoreception and physical contacts (Storch et al. 1976; Stubbs 1980; Hatting 299

and Samways 1995). Larval tracks are also repellent to conspecific larvae, a mechanism that also 300

decrease cannibalism (Meisner et al. 2011). 301

Therefore, we suggest that on perceiving cuticular hydrocarbons naive A. bipunctata 302

larvae are not sure whether they meet eggs or other conspecific larvae. Given that the latter pose 303

a risk of injury, it may be safer to back off. Hydrocarbons on the surface of the eggs might 304

constitute a case of Batesian mimicry. This idea is supported by our finding that larvae develop a 305

taste for eggs after an experience of egg cannibalism”. 306

307

Acknowledgments 308

We thank Anthony Dixon, Natalie Kincy, for their valuable comments on an earlier version of 309

this paper. This work was done in the laboratory “Evolution et Diversité biologique”, which is 310

part of the “Laboratoire d’Excellence” (LABEX) TULIP (ANR -10-LABX-41). 311

312

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Figure legends 412

413

Figure 1: (A) Average (± SE) maximum mass reached by Adalia bipunctata larvae of both sexes 414

fed different diets. (B) Average (± SE) adult mass of A. bipunctata males and females fed 415

different diets during their larval development. Black circles: females, open squares: males. 416

Circles labelled by different capital letters denote significant differences at the α<0.05 level for 417

sex. Circles labelled by different lower case letters are significantly different at the α < 0.05 level 418

for the diet treatment. 419

420

Figure 2: Average (± SE) percent of mass lost during pupal stage by Adalia bipunctata fed on 421

either aphids (black), conspecific eggs (white) or a mixture of both (grey) during their larval 422

development. Bars labelled by different capital letters denote significant differences at the 423

α<0.05 level for sex. Bars labelled by different lower case letters are significantly different at the 424

α < 0.05 level for the diet treatment. 425

426

Figure 3: The total number of encounters with food patches before attacking prey in relation to 427

(A) the proportion of the patches in the experimental arena that contain eggs, (B) the type of prey 428

attacked. (C) The percentages of encounters with eggs (open circles) and attacks on eggs (open 429

triangle) when there were three different percentages (25, 50 and 75%) of the patches in the 430

experimental arena contained eggs. The filled squares correspond to the expected frequencies of 431

encountering and attacking eggs. 432

433

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Figure 4: The percentage of larvae attacking and eating aphids or eggs as a first choice: (A) the 434

control, (B) larvae with experience of cannibalism, (C) larvae fed few aphids but not food 435

deprived and (D) larvae that were presented with washed eggs. 436

437

Supporting information S1: Weight of 3rd instar larvae depending on the time elapsed after the 438

2nd molt. Solid line: linear regression (M (mg) = 0.106 hour + 1.205, R2=0.92), dotted lines: 95% 439

CI. 440

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