1 Assessment of life history parameters of Aspidiotus nerii (Hemiptera: Diaspididae) 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
-1-
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
-2-
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,
-3-
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
-4-
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 Insect 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 insects 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
-5-
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
-6-
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
-7-
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
-8-
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.)=
-9-
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.
-10-
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,
-11-
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).
-12-
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
-13-
309 (Conselleria de Agricultura of the Generalitat Valenciana), with thanks to José Luis Porcuna,
310 Alberto García and Carmen Laurin.
311
-14-
312 References
313 Beltrán, F.D., Parra, A., Roldán, A., Soler, A., and Vila, E. (2010), 'Pasado, presente y futuro
314 del control integrado de plagas en la provincia de Almería', in Cuadernos de Estudios
315 Agroalimentarios, serial no. 1 (November 2010): Perspectivas del control biológico en
316 agricultura baja plástico, Almeria (España): Fundación Cajamar, pp. 27-43. Available at:
317 http://www.fundacioncajamar.es/publicacion-cea1-271.html.
318 Blom, J.V.d., Robledo, A., Torres, S., and Sánchez, J.A. (2010), 'Control biológico en
319 horticultura en Almería: Un cambio radical, pero racional y rentable', in Cuadernos de
320 Estudios Agroalimentarios, serial no. 1 (November 2010): Perspectivas del control biológico
321 en agricultura baja plástico, Almeria (España): Fundación Cajamar, pp. 45-60. Available at:
322 http://www.fundacioncajamar.es/publicacion-cea1-271.html.
323 DeBach, P., and Fisher, T.W. (1956), 'Experimental evidence for sibling species in the
324 oleander scale, Aspidiotus hederae (Vallot)', Annals of the Entomological Society of America,
325 49, 235-239.
326 DeBach, P., and White, E.B. (1960), 'Commercial mass culture of the California red scale
327 parasite', California Agriculture Experimental Station Bulletin, 770.
328 European Union. (2011), 'Eur-Lex web page', [accesed 07/07 2011], Available at: http://eur-
329 lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:31991L0414:EN:NOT.
330 García-Marí, F. (2003), 'Biología y control del "poll roig" de California en los cítricos
331 valencianos', Phytoma España, 153, 120-123.
-15-
332 Gerson, U., and Hazan, A. (1979), 'A biosystematic study of Aspidiotus nerii Bouché
333 (Homoptera:Diaspididae), with the description of one new species', Journal of Natural
334 History, 13, 275-284.
335 Grafton-Cardwell, E.E. (2006), 'New developments in the San Joaquin Valley California
336 Citrus IPM program', IOBC/WPRS Bulletin, 29 (3), 5-14.
337 Gruwell, M.E., Wu, J., and Normark, B.B. (2009), 'Diversity and phylogeny of Cardinium
338 (Bacteroidetes) in armored scale insects (Hemiptera: Diaspididae)', Annals of the
339 Entomological Society of America, 102 (6), 1050-1061.
340 Hoy, M.A. (2000), 'The David Rosen lecture: biological control in citrus', Crop Protection,
341 19, 657-664.
342 Jacas, J.A., Karamaouna, F., Vercher, R., and Zappalà, L. (2010), 'Citrus Pest Management in
343 the Northern Mediterranean Basin (Spain, Italy and Greece)', in Integrated Management of
344 Arthropods Pests and Insect Borne Diseases, eds. A. Ciancio and K.G. Mukerji, Dordrecht
345 (Netherland): Springer, pp. 3-27.
346 Jacas, J.A., and Urbaneja, A. (2010), 'Biological Control in Citrus in Spain: From Classical to
347 Conservation Biological Control', in Integrated Management of Arthropod Pests and Insect
348 Borne Diseases, eds. A. Ciancio and K.G. Mukerji, Dordrecht (Netherland): Springer, pp. 61-
349 72.
350 Jandel Scientific (1994), TableCurve 2D for Windows, ver. 2.02. AISN Software.
351 Kennet, C.E., McMurtry, J.A., and Beardsley, J.W. (1999), 'Biological control in tropical and
352 subtropical crops', in Handbook of Biological Control, eds. T.S. Bellows and T.W. Fisher,
353 San Diego (California, USA): Academic Press, pp. 713-742.
-16-
354 Manly, B.F.J. (1990), Staged-Structured Populations. Sampling, analysis and simulation,
355 London: Chapman and Hall Ltd.
356 Manly, B.F.J. (1994), Population analysis system: P1f, single species stage frequency
357 analysis, ver. 3.0, Pullman, Washington: Ecological Systems Analysis.
358 Mazih, A. (2008), 'Current situation of citrus pests and the control methods in use in
359 Morocco', IOBC/WPRS Bulletin, 38, 10-16.
360 Moreno, D.S., and Luck, R.F. (1992), 'Augmentative releases of Aphytis melinus
361 (Hymenoptera, Aphelinidae) to suppress California red scale (Homoptera, Diaspididae) in
362 southern California lemon orchards', Journal of Economic Entomology, 85 (4), 1112-1119.
363 Olivas, J., Lucas, A., Calvo, J., and Belda, J.E. (2011), 'Biological control of diaspidid scales
364 Aspidiotus nerii Bouche and Aonidiella aurantii (Maskell) by means of commercial Aphytis
365 melinus DeBach (Hym., Aphelinidae) releases: basis for an IPM strategy in Spanish citrus.',
366 IOBC/WPRS Bulletin, 62, 311-316.
367 Pontius, J.S., Boyer, J.E., and Deaton, M.L. (1989), 'Estimation of stage transition time:
368 application to entomological studies', Annals of the Entomological Society of America, 82,
369 135-148.
370 Pontius, J.S., Boyer, J.E., and Deaton, M.L. (1989), 'Nonparametric estimation of insect stage
371 trasition times', in Estimation and Analysis of Insect Populations, eds. L.L. McDonald, B.F.J.
372 Manly, J.A. Lockwood and J.A. Logan, Berlin: Springer-Verlag, pp. 145-155.
373 Provencher, L.M., Morse, G.E., Weeks, A.R., and Normark, B.B. (2005), 'Parthenogenesis in
374 the Aspidiotus nerii complex (Hemiptera : Diaspididae): A single origin of a worldwide,
-17-
375 polyphagous lineage associated with Cardinium bacteria', Annals of the Entomological
376 Society of America, 98 (5), 629-635.
377 Raciti, E., Saraceno, F., and Siscaro, G. (2003), 'Mass rearing of Aphytis melinus for
378 biological control of Aonidiella aurantii in Sicily', IOBC/WPRS Bulletin, 26 (6), 125-134.
379 Rocha, K.C.G., Adaime da Silva, R., Michelotto, M.D., and Busoli, A.C. (2006), 'Aspectos
380 biológicos, morfológicos e comportamentais de Aspidiotus nerii Bouché, 1833 (Hemiptera:
381 Diaspididae)', Ciência Rural, 36 (2 (marzo-abril)), 363-368.
382 Rose, M. (1990), 'Rearing and mass rearing', in Armored scale insects their biology, natural
383 enemies and control, ed. D. Rosen, Jerusalen (Israel): Elsevier, pp. 357-365.
384 Silva, R.A., Guerreiro, J.C., Michelotto, M.D., and Busoli, A.C. (2003), 'Desenvolvimento e
385 comportamento de predação de Coccidophilus citricola Brèthes, 1905 (Coleoptera:
386 Coccinellidae) sobre Aspidiotus nerii Bouché, 1833 (Hemiptera: Diaspididae)', Boletín de
387 Sanidad Vegetal-Plagas, 29, 9-15.
388 Sorribas, J.J., and García-Marí, F. (2010), 'Comparative efficacy of different combinations of
389 natural enemies for the biological control of California red scale in citrus groves', Biological
390 Control, 55 (1), 42-48.
391 Stat Point Technologies. (2010), Statgraphics Centurion XVI, ver. 16.1.07.
392 University of California. (2011), 'UC Pest Management Guidelines, Citrus: California red
393 scale and yellow scale', [accesed 07/12 2011], Available at:
394 http://www.ipm.ucdavis.edu/PMG/r107301111.html.
-18-
395 Uygun, N., and Elekcioglu, N.Z. (1998), 'Effect of three diaspididae prey species on
396 development and fecundity of the ladybeetle Chilocorus bipustulatus in the laboratory',
397 Biocontrol, 43 (2), 153-162.
398 Vacas, S., Alfaro, C., Navarro-Llopis, V., and Primo, J. (2009), 'The first account of the
399 mating disruption technique for the control of California red scale, Aonidiella aurantii
400 Maskell (Homoptera: Diaspididae) using new biodegradable dispensers', Bulletin of
401 Entomological Research, 99 (4), 415-423.
402 Vacas, S., Alfaro, C., Navarro-Llopis, V., and Primo, J. (2010), 'Mating disruption of
403 California red scale, Aonidiella aurantii Maskell (Homoptera: Diaspididae), using
404 biodegradable mesoporous pheromone dispensers', Pest Management Science, 66 (7), 745-
405 751.
406 Van Lenteren, J.C. (2003), Quality Control and Production of Biological Control Agents:
407 Theory and Testing Procedures, Wallingford, Oxon, GBR: CABI Publishing.
408 Warner, K.D., and Getz, C. (2008), 'A socio-economic analysis of the North American
409 commercial natural enemy industry and implications for augmentative biological control',
410 Biological Control, 45 (1), 1-10.
411 Watson, G.W. (2005), 'World Biodiversity Database: Arthropods of Economic Importance-
412 Diaspididae of the World', [accesed 07/14 2010], Available at:
413 http://nlbif.eti.uva.nl/bis/diaspididae.php?selected=beschrijving&menuentry=soorten&id=91.
414 Zappalà, L. (2010), 'Citrus Integrated Pest Management in Italy', in Integrated Management
415 of Arthropod Pests and Insect Borne Diseases, eds. A. Ciancio and K.G. Mukerji, Dordrecht
416 (Netherland): Springer, pp. 73-100.
-19-
417 Zappalà, L., Campolo, O., Saraceno, F., Grande, S.B., Raciti, E., Siscaro, G., and Palmeri, V.
418 (2008), 'Augmentative releases of Aphytis melinus (Hymenoptera: Aphelinidae) to control
419 Aonidiella aurantii (Hemiptera: Diaspididae) in Sicilian citrus groves', IOBC/WPRS Bulletin,
420 38, 49-54.
421
-20-
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
-21-
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
-22-
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
-23-
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
-24-
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
-25-
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
-26-