1 Assessment of life history parameters of (: ) to

2 improve the mass rearing of Aphytis melinus (Hymenoptera: Aphelinidae)

3

4

5 Author names: J.E. González-Zamora, M.L. Castillo, C. Avilla

6

7 e-mail: [email protected]

8 9 Published version: 10 11 http://dx.doi.org/10.1080/09583157.2012.691157 12

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13 Abstract

14 The biological control of Aonidiella aurantii (Maskell) on citrus can be achieved

15 with periodic releases of the parasitoid Aphytis melinus DeBach. A. melinus is

16 normally reared on parthenogenetic strains of the scale Aspidiotus nerii Bouché.

17 The development, crawler production and survival of A. nerii were studied at two

18 temperatures (25±1 and 30±1 ºC) and two relative humidities (normal 55±5 %,

19 and high 85±5 %) on squashes. The temperature had an important effect on

20 development, but not the relative humidity. At 30 ºC, no development was

21 observed, and therefore, no crawlers were produced. At 25 ºC, the development of

22 the L1, L2, and preoviposition stages required 24.4±0.7, 11.1±0.8, and 13.2±0.3

23 days, respectively. The total times until the appearance of gravid females, the

24 initiation of crawler production and the peak of crawler production were 48.7±0.1,

25 50.1±0.7, and 63.3±1.0 days, respectively. Crawler production lasted 42.6±1.9

26 days, and the 50 % production level was attained on the 13th day. Initial crawler

27 survival was higher at 30 ºC (65.9±2.8 %), whereas at the end of development,

28 survival was much higher at 25 ºC (88.0±2.1%). The relative humidity showed no

29 effect on initial and final survival. Progeny production was similar at the two

30 relative humidities, with an average of 28.0±2.8 crawlers per female at 25 ºC. The

31 relation between the number of crawlers and weight was Number of crawlers =

32 1,031.37 + 835,500.0*weight of crawlers (g), with R2=0.826. The importance of

33 selecting the correct strain of A. nerii is emphasised.

34 Keywords: Aspidiotus nerii; parthenogenetic strain; development; crawler

35 survival; biology.

36

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37 1. Introduction

38 The importance of biological control has increased worldwide for many crops and pests due,

39 among different reasons, to the increasing restrictions on the use and registration of

40 pesticides, e.g., in the European Union with the Council Directive 91/414/EEC (European

41 Union 2011). A clear example of the expansion of this methodology is the extended use in

42 recent years of biological control techniques in commercial greenhouses in southeastern Spain

43 to control pests. The use of these techniques has increased from 1.3 % of the surface in

44 2006/2007 to 44.1 % in 2008/2009. In certain crops, such as sweet pepper or melon, 70 to 90

45 % of the surface is cultivated using integrated pest management criteria (Beltrán, Parra,

46 Roldán, Soler and Vila 2010; Blom, Robledo, Torres and Sánchez 2010).

47 Citrus is a good example for studying the importance of biological control. Since the

48 end of the 19th century, biological control has been implemented against several pests in

49 citrus (Kennet, McMurtry and Beardsley 1999), originally in the USA and later in many other

50 countries. Perhaps one of the most important pests in the citrus industry is Aonidiella aurantii

51 (Maskell) (Hemiptera: Diaspididae) or Californian Red Scale (CRS) (Jacas, Karamaouna,

52 Vercher and Zappalà 2010). Many parasitoids and predators have been considered for use

53 against this pest, but currently, its biological control relies mainly on hymenopteran

54 parasitoids, such as Aphytis africanus Quednau, Aphytis coheni DeBach, Aphytis lingnanensis

55 DeBach, Aphytis melinus DeBach, Comperiella bifasciata Howard, and Encarsia perniciosi

56 (Tower) (Kennet, McMurtry and Beardsley 1999), depending on the citrus region. With an

57 integrated pest management strategy and, of course, with organic management, biological

58 control is the basis of the control of CRS. Such control programmes may also involve the use

59 of several insecticides against this pest or other pests (García-Marí 2003; Grafton-Cardwell

60 2006; Jacas, Karamaouna, Vercher and Zappalà 2010), although mating disruption arises as a

61 promising new complementary strategy (Vacas, Alfaro, Navarro-Llopis and Primo 2009,

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62 2010). Conservation of natural enemies is the most rational approach (Jacas y Urbaneja

63 2010), but an augmentation strategy is used to help control CRS populations and is commonly

64 applied when the size of the parasitoid population is small at the beginning of the first CRS

65 generation (Moreno and Luck 1992; Sorribas and García-Marí 2010; University of California

66 2011). This strategy, based on the mass rearing of A. melinus (one of the most frequently used

67 species of parasitoid), is widely implemented in many citrus regions (Mazih, 2008; Zappalà,

68 Campolo, Saraceno, Grande, Raciti, Siscaro and Palmeri 2008; Zappalá 2010; Olivas, Lucas,

69 Calvo and Belda 2011; University of California, 2011), with subsequent releases of the

70 parasitoid in doses varying between approximately 25,000 and 250,000 individuals per ha and

71 year, depending on the climatic conditions, the densities of CRS and the parasitoids present.

72 Biological control by augmentation requires the regular production of natural enemies

73 by insectaries that seek to maximise production at the lowest cost and with a standard quality

74 (Hoy 2000; Van Lenteren 2003; Warner and Getz 2008). As demand increases under new

75 pest-control scenarios, commercial insectaries must meet these requirements. The production

76 of A. melinus is an appropriate example. The species has been produced in insectaries since

77 the 1950s using a well-known and widespread technology (DeBach and White 1960; Rose

78 1990) based on the use of a parthenogenetic strain of Aspidiotus nerii Bouché (Hemiptera:

79 Diaspididae) as the host and different species of squash as the feeding substrate for the host.

80 The parthenogenetic strain of A. nerii was described first in 1901 and subsequently by

81 several authors for different plants and regions of the world (a brief review of the topic

82 appears in Gerson and Hazan 1979; Provencher, Morse, Weeks and Normark 2005).

83 Parthenogenesis is caused by bacteria of the genus Cardinium (Provencher, Morse, Weeks

84 and Normark 2005; Gruwell, Wu and Normark 2009). The parthenogenetic strain is widely

85 used in laboratories and insectaries for the mass rearing of different parasitoids and predators

86 (Uygun and Elekçioglu 1998; Raciti, Saraceno and Siscaro 2003; Silva, Guerreiro, Michelotto

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87 and Busoli 2003). However, although the demand for natural enemies reared on this host is

88 increasing (as in the case of A. melinus), few recent studies have focused on the influence of

89 various factors on its biology and progeny production (DeBach and Fisher 1956; Gerson and

90 Hazan 1979; Rocha, Silva, Michelotto and Busoli 2006). This information is important for

91 improving the efficiency of the production of parasitoids and predators.

92 For these reasons, the biology of a parthenogenetic strain of A. nerii under different

93 temperatures and relative humidities and the effect of these factors on crawler survival and

94 development on squashes are the primary emphases of this work. Similarly, crawler

95 production and its pattern over time were studied to support the scheduling of production.

96 Finally, a relationship between the weight and the number of crawlers is presented. This

97 relationship is of great interest in studies of mass production.

98

99 2. Material and Methods

100 2.1 origin and mass rearing

101 The uniparental (parthenogenetic) strain of A. nerii was originally supplied by the INRA

102 laboratory of Vallbone (France) in January 2009. The original were received on

103 potatoes, and the crawlers produced were transferred to squashes (Cucurbita moschata Duch

104 ex Lam.). Thereafter, the colony has been continuously reared on squashes in our facilities at

105 25±1 ºC and 55-65 % RH, following the original methodology of DeBach and White (1960)

106 and Rose (1990) with minor adjustments to our local conditions, as is also the case in other

107 insectaries (Raciti, Saraceno and Siscaro 2003).

108 The methodology involves a procedure in which scale-infested squashes are placed on

109 steel shelves. Below, on other shelves, are new, clean squashes, ready to be colonised.

110 Attracted by light, the mobile first-stage nymphs (crawlers) walk towards the tip of the

111 mother squash to search for a place to attach and feed. They fall to the squashes below and

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112 proceed to colonise. We used a variation of the previous methodology (as in Raciti, Saraceno

113 and Siscaro 2003). No squashes were placed on the shelf below. Instead, a sheet of paper was

114 placed on the shelf. The crawlers were collected every day and then distributed onto new

115 squashes using a dispenser resembling a salt shaker. This technique produced a regular

116 distribution of the scale insects on the surface of the newly infested squashes.

117

118 2.2. Survival and development studies

119 Squashes of approximately 15-20 cm in length and 8-12 cm in diameter were selected for the

120 development and fertility studies. The squashes were washed with a sponge soaked in soapy

121 water. They were then rinsed with tap water, thoroughly dried with a paper towel and placed

122 at 25 ºC to reach room temperature prior to infestation with the crawlers. On the day of

123 infestation, the crawlers were collected from the rearing facility and distributed over half of

124 the squash surface. The 2nd or 3rd day following infestation, the squash surface was divided

125 into different areas (between 15 and 20 in total) with an indelible soft pen. This division left at

126 least 50 individuals in each zone.

127 The squashes were maintained at two temperatures (25±1 and 30±1 ºC) and two

128 different relative humidities (normal 55±5 %, high 85±5 %) until crawler production was

129 complete. Two to three squashes were used at each combination of temperature and relative

130 humidity, and the experiment was repeated twice.

131 Initial survival was studied with a stereomicroscope 3 and 7 days after crawler

132 infestation. Dead crawlers and recently attached first-instar nymphs were recorded on two or

133 three areas of each squash. Because the values for both dates were similar, the average value

134 obtained on the two dates for each squash was considered for further analysis. After the 7th

135 day, one area of each squash was observed every 3 or 4 days. The development of all the

136 individuals in the area was recorded. The scale cover was withdrawn to determine whether the

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137 insect was dead or alive. This procedure continued until the first crawlers of the new

138 generation were observed on the squashes. At that point, one of the areas of each squash was

139 surrounded with glue, the number of females were recorded, and the crawlers were counted

140 and removed every two days.

141 Final survival was determined by calculating the average of the last three values of

142 survival in the recording period before crawler production.

143

144 2.3 Relation weight-number of crawlers

145 During the mass rearing process, crawlers produced in a 24 h period were collected twice a

146 week and weighted using an analytical balance (model HR-120, A&D Company, San Jose

147 CA). A subsample was taken on each occasion, and the crawlers were counted using a grid on

148 a Petri dish and a stereomicroscope. A total of seventeen pairs weight-number of crawlers

149 were obtained for further analysis.

150

151 2.4 Data analysis

152 Development durations (first and second instars, preoviposition females and time until the

153 appearance of gravid females) were calculated using Pontius’ method (Pontius, Boyer and

154 Deaton 1989a, 1989b; Manly 1990), a method that is appropriate if mortality does not occur

155 or is negligible. Population size is then constant for all sample times, and the proportions of

156 the population in different stages at different times can be used to estimate the distributions of

157 the stage durations. This method is appropriate for single-cohort analysis. It represents a

158 simple non-parametric approach to the estimation of the mean times required to reach a given

159 developmental stage if samples are taken from the beginning of the development of a cohort

160 to the time at which all individuals are in the last stage of development (Manly 1990). The

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161 calculations were made with the program P1f (Manly 1994), which produces both the mean

162 time for each stage and its standard error.

163 The crawler production from each squash was transformed to relative values and

164 adjusted to a sigmoidal curve with TableCurve 2D for Windows software (Jandel Scientific

165 1994). Because no differences between relative humidities within temperature were found, all

166 values were pooled. One-way and two-way ANOVA were used with the factors temperature

167 and relative humidity to compare development, survival and progeny production. Simple

168 regression was used to study the relationship between weight and number of crawlers. The

169 Statgraphics Centurion XVI package (StatPoint Technologies 2010) was used to analyse the

170 results. The data were transformed using the arcsine of the square root for variables recorded

171 as percentages.

172

173 3. Results

174 3.1 Survival and development studies

175 Initial survival was influenced by temperature (F1,16=6.80, P=0.02), with higher survival at 30

176 ºC (Table 1), but humidity was not relevant (F1,16=0.94, P=0.35). The interaction between

177 temperature and humidity was not significant (F1,16=2.01, P=0.18), but survival at high

178 humidity was higher than at normal humidity at 30 ºC (Table 1). Final survival was also

179 influenced by temperature (F1,20=218.9, P<0.001), with a much higher survival at 25 ºC than

180 at 30 ºC (Table 1). The effect of humidity was not significant (F1,20=0.46, P=0.51), and no

181 interaction occurred between temperature and humidity (F1,20=0.07, P=0.79).

182 The time course of survival following the initial settlement of the crawlers was very

183 similar at 25 ºC and 30 ºC until approximately the 50th day at both humidities (Figure 1a and

184 1b), with average values of approximately 80-90 % (Figure 1c and 1d). At a temperature of 25

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185 ºC, the crawlers started to appear. However, survival decreased rapidly at 30 ºC, and no

186 females were observed.

187 Development was influenced by temperature. At 30 ºC, A. nerii hardly reached the L2

188 instar stage, and further development was not observed. No crawlers were produced at this

189 temperature. The development of A. nerii at 25 ºC differed greatly between instars (Table 2).

190 The duration of L1 (which includes crawlers) was 24.4 days, which was considerably greater

191 than that of L2. The only developmental parameter that varied with humidity was the total

192 time until crawlers were first observed. The mean times for the different instars have been

193 calculated because the values are of interest.

194 Although the relative humidity did not clearly influence development, development

195 appeared shorter at high humidity than at normal humidity, e.g., for L1, the total time until

196 crawlers were first observed (the only parameter that varied significantly with humidity) and

197 for the total time to the maximum production of crawlers (Table 2). The preoviposition period

198 and the total time until gravid females appeared were similar at the two humidities. Specific

199 differences between the two humidities appeared after these stages at approximately the time

200 of crawler production. Crawler production began immediately at high humidity after gravid

201 females appeared, but there was a delay of approximately 3.5 days at normal humidity. This

202 difference was statistically significant (P=0.007, Table 2). The last column of Table 2 shows

203 the time during which crawlers were produced. This time did not differ significantly between

204 the two humidities, although the time was shorter at high humidity than at normal humidity.

205 The number of progeny was similar at the two humidities (F1,10=1,27; P=0.29) (Table

206 3). The average number was 28.0 (±2.8) crawlers per female. The pattern of crawler

207 production was examined at 25 ºC at both values of humidity. Crawler production did not

208 differ between the two values, and all data were pooled (Figure 2). A sigmoidal curve was

209 fitted to the data. The fit was statistically significant, with P<0.0001, R2 (adjusted for d.f.)=

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210 0.926. The standard error of the estimate was 0.092. The production of crawlers reached its

211 peak value on the 11th day, 50 % of the crawler production was reached on the 13th day, and

212 95 % of the crawler production was reached on the 32nd day.

213

214 3.2. Relation weight-number of crawlers

215 The relationship between the number of crawlers and their weight followed the regression

216 Number of crawlers = 1,031.37 + 835,500.0*weight of crawlers (g), with R2=0.826 (n=17)

217 and the standard error of the estimate = 739.4.

218

219 4. Discussion

220 The study of the biology of A. nerii is of interest for understanding and improving the mass

221 production of hymenopterans used in biological control, particularly of the hymenopteran A.

222 melinus, which is raised on A. nerii and used for the control of A. aurantii and even of A. nerii

223 (Olivas, Lucas, Calvo and Belda 2011). The developmental time and associated parameters

224 indicate that the strain of the parthenogenetic A. nerii used in this study differs from the

225 strains used in a number of studies and strongly resembles the strains used in others.

226 Several studies have compared uniparental and biparental strains of A. nerii

227 [Schmutterer 1952 (cited by Gerson and Hazan 1979); DeBach and Fisher 1956; Gerson and

228 Hazan 1979]. Certain biological parameters differ between the two strains. For example,

229 DeBach and Fisher (1956) showed that their uniparental strain had more descendants, tended

230 to live longer and showed higher crawler survival than the uniparental strain. The first and

231 third studies cited immediately above produced the opposite results (Gerson and Hazan 1979).

232 In the Gerson and Hazan (1979) study, the performance of the biparental strain was superior

233 on all the parameters measured compared with the uniparental strain, especially because the

234 biparental strain had shorter development and higher fecundity.

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235 The uniparental strain used in the current study had biological parameters similar to

236 those of the uniparental strain used by Gerson and Hazan (1979). The similarity was

237 especially pronounced for the development of the first and second instar. However, the

238 preoviposition time and the total time from egg to egg were shorter in the current study,

239 compared with the values of approximately 30 and 62 days, respectively, reported by Gerson

240 and Hazan (1979). The value of progeny production reported by these authors was 41.6±17.7

241 crawlers per female at 24 ºC, similar to the value of 28.0±2.8 crawlers per female at 25º C

242 observed in the current study. The developmental times of our strain were also very similar to

243 those found by DeBach and Fisher (1956) in their uniparental strain. However, the progeny

244 production of 94 crawlers per female at 23.9 ºC reported by DeBach and Fisher (1956) in their

245 uniparental strain differs substantially from the progeny production observed in our study.

246 The differences between uniparental and biparental strains resulted in the separation of

247 the strains into sibling species with new scientific names [as in Schmutterer 1952 (cited by

248 Gerson and Hazan 1979) and also in Gerson and Hazan 1979]. Currently, the trend is to treat

249 both strains as a single species, A. nerii (Watson 2011).

250 Rocha et al. (2006) examined a uniparental strain of A. nerii that differs in certain

251 respects from our strain. Differences are evident in developmental time and especially in

252 crawler production. Rocha et al. (2006) reported 175.5±10.29 crawlers per female in their

253 strain at 25º C. Overall, our uniparental strain and the uniparental strains investigated in other

254 studies differ primarily in the production of crawlers. Provencher et al. (2005) found a greater

255 difference between different parthenogenetic stocks of A. nerii than that observed within the

256 biparental lineages.

257 The survival of crawlers is very important to increase the host population on squashes

258 (and thus parasitoid production) and is influenced by the environment (e.g., temperature,

259 humidity). Our results indicate that within the three to seven days after the squash infestation,

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260 survival and attachment to the squash surface are influenced to a certain extent by temperature

261 but not by relative humidity, although at 30 ºC the high humidity produced a greater amount

262 of attachment by the crawlers. The long-term effect of temperature is more interesting and

263 dramatic. Humidity did not influence survival over the period of development examined by

264 the study. Similar values of survival were obtained at high and normal humidity until

265 approximately the 50th day (with 85-90 % survival of the settled scale insects). At that time,

266 crawler production began at 25 ºC. However, a rapid decrease of the population occurred at

267 30 ºC, and the population did not develop beyond the second-instar nymph stage. This result

268 is very similar to the results of DeBach and Fisher (1956), who also found that relative

269 humidity had very little influence on crawler survival but that temperature was very important

270 because at 26.7 ºC, neither the uniparental nor the biparental strains survived 34 days after

271 settlement. As previously mentioned (Provencher, Morse, Weeks and Normark 2005), the

272 different strains used in different studies (and the feeding substrates) must be considered. For

273 example, Gerson and Hazan (1979) obtained crawler production in both uniparental and

274 biparental strains of A. nerii at 28 ºC, although the crawler production of the uniparental strain

275 at this temperature was markedly lower than the production at 24 ºC.

276 The initial mortality of crawlers can be very high, with only 55-65 % of the

277 individuals settling on the squash surface. After the scales have settled, survival was found to

278 be very similar (with high values) at the two humidities tested. These results demonstrate to

279 some degree that the scale cover provides protection against certain environmental factors. In

280 contrast, temperature played an important role in final development. A temperature only a few

281 degrees higher than normal (24 ºC can be considered normal) produced dramatic reductions in

282 fertility, survival or development in the parthenogenetic strain of A. nerii, as shown both here

283 and in other studies (DeBach and Fisher 1956; Gerson and Hazan 1979).

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284 Studying the production of crawlers yields information that allows a schedule for the

285 production of A. nerii on squashes to be defined. The period during which crawlers were

286 produced was relatively high (42.6 days in our study), with peak production on the 11th day

287 (very similar in both aspects to the findings of DeBach and Fisher 1956). Most of the crawlers

288 were produced before the 32nd day (similar to the results of Raciti et al. 2003). However, our

289 results differ substantially from the values obtained by Rocha et al. (2006) with their

290 uniparental strain, which were also reared on squashes.

291 Finally, we studied the relationship between the number and the weight of crawlers.

292 This relationship can be of interest for improving production and for conducting various

293 studies of scale densities on squashes and of crawler production.

294 We have shown that temperature is a very important parameter in the biology of the

295 parthenogenetic strain of A. nerii and that a temperature increase can have highly negative

296 effects on the development of the insect. Relative humidity was shown to be of minor

297 importance even at the crawler stage and at settling. A key conclusion is that the values of

298 crawler production obtained by several different studies differ substantially. It is probable that

299 these discrepancies result from differences between parthenogenetic strains. This conclusion

300 is of primary importance for improving parasitoid production and reducing costs because a

301 commercial strategy must select the most prolific host strain.

302

303 Acknowledgements

304 This work was possible with the financial support of RioTinto Fruits S.A. and the interest of

305 the regional government (Junta de Andalucia). We thank the INRA Vallbonne (France),

306 especially Jean-Claude Malause and Nicolas Ris, for the uniparental strain of Aspidiotus nerii.

307 We also thank Daniel Muñoz for his work and diligence rearing the insect in the laboratory.

308 Finally, we acknowledge all the help and support given by the Servicio de Sanidad Vegetal

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309 (Conselleria de Agricultura of the Generalitat Valenciana), with thanks to José Luis Porcuna,

310 Alberto García and Carmen Laurin.

311

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421

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422 Table 1. Initial and final survival (in percentage) of Aspidiotus nerii at two

423 temperatures and humidities. Numbers within brackets represent standard errors.

424

Initial Survival Final Survival

25 ºC 30ºC Mean 25 ºC 30ºC Mean

HHa 50.4 72.2 61.3 88.7 24.9 62.1

(4.3) (3.4) (4.5) (2.9) (4.3) (9.8) NH 53.0 59.6 56.3 87.3 20.8 59.6

(9.1) (2.0) (4.5) (3.2) (3.3) (10.1)

b 51.7 65.9 ns 88.0 22.9 ns Mean (4.8) (2.8) s (2.1) (2.7) s 425

426 a HH = High humidity (85±5 %); NH = Normal humidity (55±5 %).

427 b ns= not significant; s = significant. Two-way ANOVA with temperature and humidity as the factors;

428 interaction was not significant.

429

430

431

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432 Table 2. Development time (in days) of Aspidiotus nerii at 25ºC and two humidities.

433 Numbers within brackets represent standard errors.

Total to Total to Total to Period of Pre- L1 L2 gravid crawler maximum crawler oviposition femalea productiona crawlera production

HHb 23.3 12.2 13.4 48.9 48.4 61.7 41.1

(0.3) (0.3) (0.2) (0.2) (0.8) (0.9) (2.9) NH 25.4 9.9 13.1 48.4 51.9 64.9 44.0

(1.0) (1.3) (0.6) (0.1) (0.7) (1.6) (2.6)

24.4 11.1 13.2 48.7 50.1 63.3 42.6 Mean (0.7) (0.8) (0.3) (0.1) (0.7) (1.0) (1.9)

Pc=0.10 P=0.15 P=0.59 P=0.08 P=0.007 P=0.12 P=0.30 434

435 a The period began with the inoculation with crawlers at the beginning of the experiment.

436 b HH = High humidity (85±5 %); NH = Normal humidity (55±5 %).

437 c Analysed with a one-way ANOVA.

438

439

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440 Table 3. Progeny production (crawlers per female) of Aspidiotus nerii at 25 ºC and two

441 humidities. Numbers within brackets represent standard errors.

442

Fecundity

HHa 30.1 (4.7)

NH 25.9 (3.1)

Mean 28.0 (2.8)

443

444 a HH = High humidity (85±5 %); NH = Normal humidity (55±5 %).

445

446

447

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448 Figure Legends

449 Figure 1. Time course of Aspidiotus nerii survival reared on squashes at two different

450 temperatures and humidities. (a) and (b) at normal humidity (55±5 %, solid line) and high

451 humidity (85±5 %, dashed line); (c) and (d) average of the two humidities at each

452 temperature.

453

454 Figure 2. Cumulative proportions for crawler production of Aspidiotus nerii reared on

455 squashes at 25 ºC. The average of the high- and normal-humidity conditions is presented.

456

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457 25 ºC 30 ºC

458 100 459 100 (a) (b) 460 80 80

461 60 60 462 40 40

463 Survival % % Survival % 464 20 20 0 465 0 0 10 20 30 40 50 60 70 466 0 10 20 30 40 50 60 70 Days 467 Days 468 469 100 (c) 100 (d) 470 471 80 80 472 60 60

473 40 40 % Survival % 474 Survival % 20 20 475 476 0 0 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 477 Days Days 478 479 480 Figure 1 481

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482 483 1 484 485 486 0.75 487 488 489 490 0.50

491 Proportion 492 493 0.25 494 495 496 497 0 498 0 10 20 30 40 50 60 499 Days 500 501 502 503 504 Figure 2

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