Winter is coming: cold hardiness attributes of a field ... · Winter is coming: cold hardiness attributes of a field population of the potato tuberworm Phthorimaea operculella Stefanos

Winter is coming: cold hardiness attributes of a fieldpopulation of the potato tuberworm Phthorimaea operculellaStefanos S Andreadis, Yianna Poulia, Sofia Noukari, Barbara Aslanidou, Matilda Savopoulou-Soultani

The potato tuberworm, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae), is aworldwide pest of solanaceous crops especially devastating to potatoes. In the presentstudy we investigated the cold hardiness profile of short-term acclimated and non-acclimated immature and adult stages of a field population of P. operculella. Late instarsdisplayed the lowest mean supercooling point, for both short-term acclimated and non-acclimated individuals, however, no significant differences were observed amongdevelopmental stages. Unlike supercooling capacity, acclimation at 5 oC for 5 daysenhanced the ability to survive at subzero temperatures after a 2 h exposure. Mean lethaltemperature (LTemp50) of all developmental stages (egg, late instar, pupa and adult)decreased after short-term acclimation, however only adults displayed a significantdifference among acclimated and non-acclimated individuals concerning their LTemp50 (-11.1 and -8.3 oC, respectively). Generally, pupae were the most cold tolerantdevelopmental stage followed in decreasing order by the eggs and adults, whileinterestingly late instars were the least ones. Non-freezing injury above the supercoolingpoint was well documented for all developmental stages indicating a pre-freeze mortalityand suggesting that P. operculella is considered to be chill tolerant rather than freezeintolerant. Nevertheless, given its high degree of cold hardiness, winter mortality of P.operculella due to low temperatures is not likely to occur and potential pest outbreak cantake place following a mild winter.

PeerJ PrePrints | https://dx.doi.org/10.7287/peerj.preprints.1497v1 | CC-BY 4.0 Open Access | rec: 11 Nov 2015, publ: 11 Nov 2015

1 Winter is coming: cold hardiness attributes of a field population of the potato tuberworm

2 Phthorimaea operculella

3

4 Stefanos S. Andreadis1,*, Yianna Poulia1, Sofia Noukari2, Barbara Aslanidou2, and Matilda

5 Savopoulou-Soultani1

6

7 1Laboratory of Applied Zoology and Parasitology, Department of Plant Protection, Faculty of

8 Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece, 2Laboratory of

9 Entomology, Department of Agricultural Technology, Alexander Technological Educational

10 Institute of Thessaloniki, 57400 Sindos, Greece, *Present address: Chemical Ecology Lab,

11 Department of Entomology, Penn State University, University Park, 16802 PA

12

13 *Correspondence: Stefanos S. Andreadis, Chemical Ecology Lab, Department of Entomology,

14 Penn State University, University Park, 16802 PA. USA; email: [email protected]


16 Abstract

17

18 The potato tuberworm, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae), is a

19 worldwide pest of solanaceous crops especially devastating to potatoes. In the present study we

20 investigated the cold hardiness profile of short-term acclimated and non-acclimated immature

21 and adult stages of a field population of P. operculella. Late instars displayed the lowest mean

22 supercooling point, for both short-term acclimated and non-acclimated individuals, however, no

23 significant differences were observed among developmental stages. Unlike supercooling

24 capacity, acclimation at 5 oC for 5 days enhanced the ability to survive at subzero temperatures

25 after a 2 h exposure. Mean lethal temperature (LTemp50) of all developmental stages (egg, late

26 instar, pupa and adult) decreased after short-term acclimation, however only adults displayed a

27 significant difference among acclimated and non-acclimated individuals concerning their

28 LTemp50 (-11.1 and -8.3 oC, respectively). Generally, pupae were the most cold tolerant

29 developmental stage followed in decreasing order by the eggs and adults, while interestingly late

30 instars were the least ones. Non-freezing injury above the supercooling point was well

31 documented for all developmental stages indicating a pre-freeze mortality and suggesting that P.

32 operculella is considered to be chill tolerant rather than freeze intolerant. Nevertheless, given its

33 high degree of cold hardiness, winter mortality of P. operculella due to low temperatures is not

34 likely to occur and potential pest outbreak can take place following a mild winter.

35

36 Keywords Cold hardiness, field population, lower lethal temperature, supercooling point, short-

37 term acclimation, non-freezing injury.

38


39 Key message

40 The potato tuberworm, Phthorimaea operculella, is a major pest of potato both in field

41 and storage

42 In this study we measured the cold hardiness of field-collected immature and adult stages

43 of the potato tuberworm

44 The results of this study indicate that the potato tuberworm due to its enhanced cold

45 tolerance is likely to overwinter successfully in storage houses in high altitudes of

46 Southern Europe

47 Potential pest outbreak can take place following a mild winter


49 Introduction

50

51 The potato tuberworm, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae), is a

52 cosmopolitan pest of solanaceous crops and weeds (Das & Raman, 1994) with a preference for

53 potato (Solanum tuberosum L.) both in field and storage (Trivedi & Rajagopal, 1992). It

54 originates from South America but at present occurs in almost all tropical, subtropical and

55 temperate potato production regions (Cisneros & Gregory, 1994; Rondon, 2010; Saour, Al-

56 Daoude & Ismail, 2012), as a result of its highly adaptability to a wide range of climatic

57 conditions in very different agroecosystems (Kroschel & Koch, 1994; Kroschel et al., 2013).

58 Gravid females lay their eggs on leaf undersurfaces and soil next to the host plant or directly near

59 the eye buds of tubers exposed through soil cracks or when they are kept under storage (Rondon,

60 2010; Navrozidis & Andreadis, 2012; Saour, Al-Daoude & Ismail, 2012). Larvae mine in leaves

61 and stems, and borrow deep tunnels into tubers causing severe damage (Dangles et al., 2008;

62 Rondon, 2010). In addition to direct damage, galleries inside tubers facilitate the entrance of

63 pathogens responsible for further severe losses, which may reach up to 100% under inadequate

64 storage conditions (Rondon, 2010).

65 The potato tuberworm is a multivoltine species with a variable number of generations per

66 year depending on several parameters such as geographical location and weather conditions

67 (Capinera, 2008; Rondon, 2010). Under favorable conditions such as mild winter, locations with

68 year-round crops, potato cull piles, potato tubers left in the soil after harvest and volunteer plants

69 (Coll, Gavish & Dori, 2000; Rondon et al., 2007; Capinera, 2008; Dögramaci, Rondon &

70 DeBano, 2008; Rondon, 2010), it does not undergo diapause to overcome unfavorable

71 environmental conditions, but uses short spells of adequate temperature to continue its


72 development (Kroschel et al., 2013). However, in subtropical regions of Southern Europe, the

73 long, cold winters generally restrict its development and reduce its pest status (Kroschel et al.,

74 2013). Winter populations can be potentially active in storage facilities where optimum

75 temperature for survival is maintained.

76 Low temperature postharvest storage of potatoes is widely used to delay physiological

77 processes and lengthen the marketing season (Singh et al., 2008; Saour, Al-Daoude & Ismail,

78 2012). The exposure temperature during the storage period depends mostly on the desired end

79 use of the tubers (seed tubers, fresh consumption, etc.) (Sonnewald, 2001). Nevertheless, long-

80 term storage to low temperatures, apart from having negative impact on the potato quality due to

81 the cold-induced sweetening of the tubers (Sowokinos et al., 2000; Bhaskar et al., 2010), it also

82 affects the life-history traits of the potato tuberworm by reducing the hatchability of the eggs and

83 the survival of first-instar larvae (Saour, Al-Daoude & Ismail, 2012). Earlier studies

84 demonstrated some survival of potato tuberworm at sub-freezing temperatures, however,

85 prolonged exposure at sub-freezing temperatures eventually proved to be fatal to all stages

86 (Langford, 1934). Potato tuberworm normally cannot withstand freezing, thus in cold climates

87 overwintering survival by immature stages is poor except within potatoes in storage or in cull

88 piles (Capinera, 2008). According to Sporleder et al. (2004) the theoretical lower developmental

89 thresholds estimated by linear regression were 11.0, 13.5, and 11.8o�C for egg, larva, and pupa

90 stages, respectively.

91 The objective of the present work was to study the cold hardiness profile of a field

92 population of P. operculella taking into consideration various parameters, such as insect stage,

93 short-term acclimation to low temperature, mortality at sub zero temperatures and capacity of

94 supercooling. Cold hardiness was evaluated by determining the supercooling point (SCP) and


95 lower lethal temperatures (LTemp) after exposure at sub zero temperatures above the SCP.

96

97 Materials and Methods

98

99 Field collected insects

100

101 In mid September 2011, late instar larvae (fourth to fifth) as well as early pupae (one to three

102 days old) of P. operculella were collected from infested potato tubers (var. Spunda) that were

103 dug the previous day from potato fields in northern Greece (Kato Nevrokopi) (41ο20’Ν,

104 23ο51’Ε). In addition, infested potato tubers were transferred into wooden cages (30 x 30 x 30

105 cm) in the laboratory, where they were maintained at 25 ± 1oC, 70 ± 10 % relative humidity

106 (RH) with a 16:8 h (L:D) photoperiod in order to obtain adults. Eggs were collected by placing

107 emerging adults (4-5 pairs) in truncated transparent plastic cups (13 cm in diameter, 6.5 cm in

108 height) covered with fine mesh (Fig 1). Three holes were punched at the bottom of each cup and

109 were plugged with dental roll wick, which provided the adults with a 20 % sucrose solution in

110 water. Females laid their eggs on the fine mesh, which were daily collected with a fine brush and

111 placed into small plastic boxes (4.5 cm in diameter, 3.0 cm in height).

112

113 Weather data

114

115 Maximum and minimum temperature were obtained from a meteorological station unit (LG48)

116 of National Observatory of Athens (http://penteli.meteo.gr/meteosearch/), located in Nevrokopi

117 municipality (585 m a.s.l.) in the East subarea of the study, at a 2 m high (Fig. 2).


118

119 Short-term acclimation

120

121 To test if short-term acclimation could enhance supercooling capacity and survival of individuals

122 at low temperatures, groups of individuals (three replicates of 10 individuals for each treatment

123 separately) were placed in controlled environmental chambers (Precision Scientific, General

124 Electric, Louisville, KY, USA) at 5 ± 1 oC under a 16:8 h (L:D) photoperiod for a period of 5

125 days. Selection of acclimation temperature was based to the ideal storage for potatoes (Davidson,

126 1958).

127

128 Determination of supercooling points

129

130 The SCP of late instars, early pupae and early adults was determined by cooling individuals from

131 20 to -30 °C at 1.0 °C min−1 using a copper-constantan thermocouple (Digitron 2000T; Kalestead

132 Ltd, U.K.) kept in tight contact with the insect body as described by Andreadis et al. (2008). The

133 lowest temperature reached before an exothermic event occurred due to release of latent heat was

134 taken as the SCP of the individual. Larvae of P. operculella prior to determination of SCP were

135 put individually into plastic boxes and left for 5-6 h without food in order to evacuate their gut.

136 Overall 9-10 replicates were performed for each developmental stage. SCP of egg stage could

137 not be measured due to its small size.

138

139 Determination of lethal temperature

140


141 The temperature, at which 50 % of individuals died, was determined by cooling groups of eggs,

142 larvae, pupae and adults (five replicates of 9-11 individuals for each developmental stage

143 separately) to a range of sub-zero temperatures for 2 h. Exposure temperatures ranged from -10

144 to -16 ºC depending on the developmental stage. Individuals were placed in thin-walled test

145 tubes (1.2 cm in diameter, 10 cm in height) plugged with foam rubber and thereafter were

146 immersed directly from room temperature (25 ± 1 ºC) in a circulating bath (Model 9505,

147 PolyScience, Niles, IL) with a solution of ethylene glycol and water (1:1) where the temperature

148 has been preset to the desired level. Larvae were treated in the same way as mentioned above

149 regarding gut-evacuated condition. Throughout the experiment a copper-constantan

150 thermocouple was placed inside a test tube to continuously measure its ambient temperature.

151 After exposure, individuals were transferred at 25 ± 1 ºC with a 16:8 h (L:D) photoperiod for

152 subsequent mortality assessment. Eggs and pupae were left undisturbed at the room temperature

153 for 10 days and considered to have died if no hatch or emergence was noted by that time. Late

154 instar larvae as well as adults were assumed to be dead if they did not respond to a gentle

155 prodding after 24 h recovery at 25 ºC.

156

157 Statistical analysis

158

159 All statistical tests incorporate a Type I error rate of α = 0.05, and all parametric statistics were

160 carried out using the R software version 2.15.2 (2012). Mean supercooling points were compared

161 using Student’s t-test for comparisons between two groups or one-way ANOVA with multiple

162 comparisons for assessment of more than two groups with insect stage as a fixed effect. If

163 significant differences were detected in ANOVA models, all pairwise comparisons were made


164 among treatment means using Tukey’s HSD test. Results are presented as mean ± standard error

165 (SE). Differences between mortality at each developmental stage were examined in one analysis

166 by a generalized linear model (GLM) with a binomial error distribution followed by Tukey’s

167 HSD (Crawley, 2007). Lethal temperature for 50 and 90 % mortality (LTemp50 and LTemp90,

168 respectively) were calculated by probit analysis after correction for control mortality using

169 Abbott’s formula (Finney, 1952). Differences of LTemp50 and LTemp90 values were based on

170 non-overlapping confidence intervals.

171

172 Results

173

174 Supercooling point determination of field collected individuals

175

176 In general, all tested developmental stages of P. operculella showed an enhanced ability to

177 supercool. However, no significantly differences were observed in SCP mean values between the

178 developmental stages both for non acclimated (F2,26 = 0.797, P = 0.380) and short-term

179 acclimated individuals (F2,27 = 0.811, P = 0.376) (Table 1). Similarly, short-term acclimation did

180 not affect significantly the superccoling capacity of larvae (t16 = 0.023, P = 0.982), pupae (t17

181 =0.591, P = 0.567) and adults (t18 = 0.118, P = 0.907) (Table 1).

182

183 Lethal temperatures

184

185 The temperatures that cause 50 and 90 % mortality (LTemp50 and LTemp90) to short-term

186 acclimated and non-acclimated field collected immature and mature stages after exposure to sub


187 zero temperatures for 2 h are shown in Table 2. In both treatments, pupae displayed the lowest

188 LTemp50 values followed by egg ones. Larval and adult LTemp50 values were significantly

189 higher from the pupal ones, however they did not differ significantly from the corresponding egg

190 ones (based on non-overlapping confidence intervals). In all cases, LTemp50 and LTemp90 values

191 of short-term acclimated stages were lower, however only in the stage of adult acclimation

192 significantly reduced LTemp50 value (based on non-overlapping confidence intervals). Pre-freeze

193 mortality is well addressed since the majority of both non-acclimated and acclimated individuals,

194 apart from pupal stage, died at subzero temperatures well above their supercooling point.

195

196 Mortality at sub-zero temperatures

197

198 Mortality data of each developmental stage exposed to sub zero temperatures are shown in Table

199 3. No complete mortality was achieved for any treatment regardless of the exposure temperature,

200 even after a 2 h exposure to -12 oC. Differences among various stages were significant in all

201 exposure temperatures for both non-acclimated and short-term acclimated treatments (Table 3).

202 At -6 and -8 oC eggs, pupae and adults displayed a similar mortality, which was significantly

203 lower compared to the larvae for both non-acclimated and short-term acclimated treatments.

204 However, at lower exposure temperatures (-10 and -12 oC) pupae were more cold-tolerant

205 followed in decreasing order by eggs, adults and larvae.

206 Mortality at each developmental stage increased significantly with reduction of exposure

207 temperature in most cases, such as in non-acclimated eggs (χ2 = 22.26, df = 3, P < 0.001), non-

208 acclimated larvae (χ2 = 30.69, df = 3, P < 0.001), non-acclimated adults (χ2 = 48.51, df = 3, P <

209 0.001) short-term acclimated larvae (χ2 = 36.36, df = 3, P < 0.001) and short-term acclimated


210 adults: χ2 = 46.06, df = 3, P < 0.001). Reduction of exposure temperature did not affect the

211 mortality of non-acclimated pupae (x2 = 1.73, df = 3, P = 0.630), short-term acclimated eggs (χ2

212 = 7.51, df = 3, P = 0.057) and short-term acclimated pupae (χ2 = 5.13, df = 3, P = 0.163), which

213 may be attributed to the overall enhanced survival rates that were observed for these specific

214 three treatments.

215 Generally, short-term acclimated individuals displayed a lower mortality compared to non-

216 acclimated ones. However, only for eggs exposed to -12 oC (t = 13.602, P < 0.001); pupae

217 exposed to -6 oC (t = 4.949, P < 0.05); adults exposed to -6 oC (t = 9.458, P < 0.01); adults

218 exposed to -10 oC (t = 13.352, P < 0.001); and adults exposed to -12 oC (t = 4.659, P < 0.05) we

219 observed significant differences among short-term acclimated and non-acclimated individuals

220 (Fig. 3).

221

222 Discussion

223

224 In temperate regions, insects are frequently exposed to low and sub-zero temperatures during the

225 winter since ambient temperature drops often below 0 oC. Ice formation within the tissues of

226 insects is at most harmful, as it causes extensive damage either due to perforation of the cells or

227 compression and deformation (Danks, 1996; Storey, 1999). However, even if ice formation does

228 not cause direct damage, the presence of ice within the tissues can lead to extensive dehydration

229 of the cell, since the interior fluids of the cell is absorbed by the growing ice crystal which

230 consequently results in shrinkage and destruction of the cell (Lee, 1989; Block, 1995; Sømme,

231 1999). Moreover, repeated exposure to cold temperatures affects both the behavior and

232 physiology. In the female fruit fly Drosophila melanogaster Meigen (Diptera: Drosophilidae)


233 multiple cold exposures resulted to a change in sex ratio and total number of offspring (Marshall

234 & Sinclair, 2010; MacAlpine, Marshall & Sinclair, 2011). Thus, overwintering insects in order to

235 overcome all of these types of injuries adopt a complex strategy to improve their cold hardiness

236 and enhance their survival under cold environments (Salt, 1961; Bale, 1993; Bale, 1996; Danks,

237 1996). According to the most recent classification of insects related to cold tolerance (Bale,

238 2002), P. opercullela can be classified in the category of insects that are chill tolerant rather than

239 freeze intolerant. This hypothesis is confirmed by the fact that non-freezing injury above the

240 supercooling point was well documented for all developmental stages since exposure to -12 oC

241 for 2h resulted in an enhanced mortality of individuals, thus indicating a pre-freeze mortality. In

242 agreement with our observations, Hemmati, Moharramipour & Talebi (2014) also reported the

243 incapability of a laboratory colony of P. opercullela to tolerate subzero temperatures below their

244 SCPs, though in a lesser extent.

245 In principle, field-collected individuals of potato tuberworm showed an enhanced ability of

246 cold hardiness suggesting that they are well adapted on the environmental conditions of northern

247 Greece and that winter mortality is not likely to occur since temperatures seldom fall below -10

248 oC (Fig. 2). Moreover, given that individuals were collected in early autumn and that SCP of

249 insects in temperate regions is proportional with seasonal air temperature in the wild it is evident

250 that the cold hardiness of the potato tuberworm will further enhance (Carrillo & Cannon, 2005;

251 Ma et al., 2006; Andreadis et al., 2008; Andreadis et al., 2011). This exaggerated ability to

252 tolerate low temperatures might be also explained, to some extent, by parental experience to cold

253 temperatures, which consequently increased cold hardiness in progeny as a result of

254 counterbalancing adaptations against reduced survival in the next generation (Zhou et al., 2013).

255 Adults of Ophraella communa LeSage (Coleoptera: Chrysomelidae) displayed lower


256 supercooling points when parents experienced colder temperatures compared to beetles emerging

257 from parents that experienced room temperatures (Zhou et al., 2013). Likewise, Calliphora

258 vicina Robineau-Desvoidy (Diptera: Calliphoridae) adults exposed to warmer autumn conditions

259 during diapause induction produced larvae with a reduced cold hardiness capacity, which could

260 negatively impact winter survival (Coleman, Bale & Hayward, 2014).

261 Mean SCP was considerably low for all developmental stages that were tested ranging

262 from -19.1 to -20.4 oC. Late instar larvae displayed the lowest mean supercooling point and

263 simultaneously the greatest range, for both short-term acclimated and non-acclimated

264 individuals, such as in the case of another gelechiidae species, the South American tomato

265 leafminer Tuta absoluta (Meyrick) (Van Damme et al., 2014). However, SCP did not differ

266 among developmental stages, which is controversial to what is already known concerning the

267 effect of developmental stage on SCP. In most species supercooling capacity is regarded to be

268 stage specific (Sømme, 1982; Lee, 1991). A possible explanation is that this species does not

269 undergo diapause thus all developmental stages get cold hardy to overcome unfavorable

270 environmental conditions such as exposure to low temperatures (Rondon, 2010; Maharjan &

271 Jung, 2012). Moreover, given that individuals originated from field collected potatoes that were

272 harvest in mid September when already minimum air temperatures in this area dropped below 10

273 oC, it is obvious that both immature stages and adults were triggered in some extend to get cold

274 hardy to cope with severe winter climates. In the European corn borer, Ostrinia nubilalis

275 (Hübner) (Lepidoptera: Crambidae), accumulation of glycerol relates more to exposure to low

276 temperatures than to diapause to withstand harsh temperatures of winter (Nordin, Cui & Yin,

277 1984; Andreadis et al., 2008).

278 Short-term acclimation at 5 oC for 5 days did not affect significantly their ability to


279 supercool. This is in agreement with previous reported studies where acclimation at 5 and 0 oC

280 for 1 week did not alter the supercooling capacity of potato tuberworm (Hemmati,

281 Moharramipour & Talebi, 2014). Similarly, short period of acclimation (1 week at 5 oC) had no

282 influence on the mean SCPs of both diapause and non-diapause larvae of the parasitoid

283 Colpoclypeus florus Walker (Hymenoptera: Eulophidae) (Milonas & Savopoulou-Soultani,

284 2005). One reason could be the inadequate time of acclimation for the production and

285 accumulation of cryoprotectants (i.e. glycerol, trehalose etc.), which plays an important role in

286 the enhancement of the supercooling capacity (Sømme, 1982; Clark & Worland, 2008). For

287 instance, SCP of adults of Cryptolestes ferrugineus (Stephens) (Coleoptera: Cucujidae) was

288 lowered from -17.9 to -20.4 °C when acclimation time at 15 °C increased from 1 to 3 weeks

289 (Smith, 1970). Unlike to supercooling capacity, acclimation at 5 oC for 5 days reduced the

290 LTemp50 values of all developmental stages of the potato tuberworm. However, only adults

291 displayed a significant difference among short-term acclimated and non-acclimated individuals

292 concerning their LTemp50 values, based on non-overlapping confidence intervals, probably due

293 to the same reason as mentioned above for the case of the SCP.

294 Based on the LTemp50 values of the individuals, larvae of the potato tuberworm appear to

295 be the least tolerant developmental stage in contrast to the pupal stage which is the most tolerant

296 followed by the egg and adult stage. Likewise, pupae displayed the lowest mortality when

297 exposed for 2 hours to sub-zero temperatures in both acclimated and non-acclimated treatments.

298 In nearly all temperature regimes, except -12 oC for the acclimated ones, mortality was

299 significantly lower compared to the other stages. This may be partially due to cessation of

300 feeding prior to pupation (Andreadis et al., 2008) which potentially saves energy expenditures on

301 digestion, cause gut regression, and reduce or halt biosynthesis of storage fuels (e.g., glycogen,


302 triglycerides) (Storey & Storey, 2012).

303 In conclusion, winters in Greece, especially in the northern part of the country, which are

304 characterized by relatively low temperatures may cause substantial mortality of potato

305 tuberworm larvae. However, most of the other stages will eventually survive since they present

306 an extremely enhanged cold hardiness, especially pupal ones. Thus, cold temperatures during the

307 winter season are not expected to provide an adequate control of this species, however, it may

308 help to keep populations from building quickly and potato cull piles some time to recover from

309 initial attacks especially if piles are kept as small as possible in order to reduce the possibility to

310 survive the winter and be a source of inoculum early next year. These results have significant

311 implications for predictions of the future range of potato tuberworm and spread rates of the

312 species in areas with a harsh winter climate. Predictions on the future distribution and abundance

313 of the potato tuberworm clearly indicate that the invasiveness of this pest is intimately related to

314 climate-change-caused alterations in global temperatures (Kroschel et al., 2013).

315

316 Competing Interests

317

318 The authors declare there are no competing interests.

319

320 Author Contributions

321

322 SA and MS conceived and designed the experiments. SA, YP, SN and BA performed the

323 experiments. SA analyzed data and wrote the manuscript. All authors read and approved the

324 manuscript.


325

326 Acknowledgements

327

328 We would like to gratefully thank Dr. Zois Zartaloudis for providing us with field-collected

329 individuals of P. operculella.

330

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456 Figure Legends

457 Fig. 1 Schematic draw of the oviposition setting. Adults after emergence were placed in plastic

458 cups covered with fine mesh. Gravid females laid their eggs on the fine mesh as shown. Eggs

459 were collected daily with a fine brush. Adults were provided with 20 % sucrose solution as food.

460

461 Fig. 2 Daily maximum and minimum air temperature 2 m above ground level in the region of

462 Kato Nevrokopi, northern Greece, from 2011 to 2014 (National Observatory of Athens, 2015).

463

464 Fig. 3 Mortality (%) of non-acclimated and short-term acclimated individuals of field-collected

465 immature and mature stages of P. operculella after exposure for 2 hours to sub-zero

466 temperatures. Bars show mean values plus standard error (Student’s test, p < 0.05). Statistical

467 significance is indicated by *** (p < 0.001), ** (p < 0.01) and * (p < 0.05). n.s. stands for not

468 statistical significant.


1Schematic draw of the oviposition setting.

Adults after emergence were placed in plastic cups covered with fine mesh. Gravid females

laid their eggs on the fine mesh as shown. Eggs were collected daily with a fine brush. Adults

were provided with 20 % sucrose solution as food.



2Daily maximum and minimum air temperature 2 m above ground level in the region ofKato Nevrokopi, northern Greece, from 2011 to 2014 (National Observatory of Athens,2015).


3Mortality (%) of non-acclimated and short-term acclimated individuals of field-collectedimmature and mature stages of P. operculella after exposure for 2 hours to sub-zerotemperatures.

Bars show mean values plus standard error (Student’s test, p < 0.05). Statistical significance

is indicated by *** (p < 0.001), ** (p < 0.01) and * (p < 0.05). N.S. stands for not statistical

significant.



Table 1(on next page)

Supercooling points (mean ± SE) of non-acclimated and short-term acclimated fieldcollected immature and mature stages of P. operculella.


1 Table 1 Supercooling points (mean ± SE) of non-acclimated and short-term acclimated field

2 collected immature and mature stages of P. operculella

Physiological stage Developmental stage na Mean SCP (oC ± SE)

Larva 9-20.4 ± 1.5

(-23.4 to -9.8)b

Pupa 9-19.1 ± 0.2

(-19.9 to -18.1)Non-acclimated

Adult 10-19.3 ± 0.4

(-21.6 to -16.4)

Larva 9-20.4 ± 1.2

(-24.0 to -13.9)

Pupa 10-19.5 ± 0.7

(-24.3 to -17.0)Short-term acclimated

Adult 10-19.4 ± 0.3

(-20.5 to -17.9)

3 a number of replicates

4 b range of SCP is given in parenthesis

5



Lethal temperature that causes mortality 50 and 90% (LTemp50 and LTemp90) andconfidence intervals (95%) of field-collected individuals of P. operculella.


1 Table 2 Lethal temperature that causes mortality 50 and 90% (LTemp50 and LTemp90) and confidence intervals (95%) of field-

2 collected individuals of P. operculella.

Physiological stage

Developmental stage n LTemp50 (oC) (95% CI) LTemp90 (oC) (95% CI) x2 df P

Egg 202 -10.9 (-8.9 to -28.4) -24.9 (-15.3 to -1,900.0) 45.521 18 0.001

Larva 197 -6.5 (-5.5 to -7.2) -12.0 (-10.5 to -15.4) 15.192 18 0.649

Pupa 200 -13.3 (-10.8 to -28.0) -41.3 (-22.4 to -631.1) 12.080 18 0.843Non-acclimated

Adult 200 -8.5 (-7.4 to -9.7) -14.2 (-11.8 to -22.3) 37.346 18 0.005

Egg 213 -14.7 (-12.0 to -29.7) -39.7 (-22.9 to -323.6) 6.849 18 0.991

Larva 176 -7.4 (-6.6 to -8.1) -12.7 (-11.2 to -15.9) 14.573 18 0.556

Pupa 200 -17.4 (-13.2 to -54.5) -45.9 (-24.3 to -790.5) 8.352 18 0.973

Short-term acclimated

Adult 200 -11.1 (-10.3 to -12.4) -17.5 (-14.9 to -24.0) 17.485 18 0.490

3 n, number of observations; LTemp50, temperature that causes 50% mortality after exposure to sub zero temperatures for 2h; LTemp90, temperature that causes

4 90% mortality after exposure to sub zero temperatures for 2h; CI, confidence interval



Mortality (%) (mean ± SE) of field-collected individuals of P. operculella after exposurefor 2 hours to sub-zero temperatures.

(n = 5 replicates of 9-11 individuals for each developmental stage and exposure

temperature)


1 Table 3 Mortality (%) (mean ± SE) of field-collected individuals of P. operculella after exposure for 2 hours to sub-zero temperatures

2 (n = 5 replicates of 9-11 individuals for each developmental stage and exposure temperature)

Developmental stagePhysiological stage Temperature

egg larva pupa adultF df P

-6 20.5 ± 8.5aa 44.0 ± 6.8b 24.0 ± 2.4a 20.0 ± 13.0a 9.14 3 *

-8 30.0 ± 8.4a 66.0 ± 6.8b 22.0 ± 7.3a 38.0 ± 10.7a 23.21 3 ***

-10 34.4 ± 8.1a 79.6 ± 5.5b 30.0 ± 3.2a 72.0 ± 4.9b 49.55 3 ***Non-acclimated

-12 68.0 ± 16.2b 92.0 ± 3.7c 32.0 ± 7.3a 78.0 ± 3.7bc 48.02 3 ***

-6 13.0 ± 4.4a 32.5 ± 11.1b 8.0 ± 2.0a 2.0 ± 2.0a 19.30 3 ***

-8 22.9 ± 4.6a 50.0 ± 5.8b 16.0 ± 4.0a 24.0 ± 5.1a 13.58 3 **

-10 25.5 ± 3.4ab 79.9 ± 6.3c 22.0 ± 3.7a 36.0 ± 8.1b 37.85 3 ***

Short-term acclimated

-12 28.7 ± 6.0a 86.4 ± 2.2c 22.0 ± 5.8a 58.0 ± 7.3b 62.37 3 ***

3 a Means in a row followed by the same low case letter are not significantly different (GLM with binomial error distribution followed by Tukey’s HSD, p < 0.05)

4 Statistical significance is indicated by *** (p < 0.001), ** (p < 0.01) and * (p < 0.05)


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Winter is coming: cold hardiness attributes of a field ... · Winter is coming: cold hardiness attributes of a field population of the potato tuberworm Phthorimaea operculella Stefanos