1 Biological Control
2
3 Biological and behavioral effects of a kaolin particle film on larvae and adults of
4 Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae)
5
6 M. Porcel 1* , B.Cotes
1 , M. Campos 1
7
8
9
1
Department of Environmental Protection. Estación Experimental de Zaidín, CSIC,
Profesor Albareda nº 1, 18008, Granada. Spain
10
11 * Corresponding author: Email: mario.porcel@eez.csic.es
; Tlf: +34-958191600 ext.
12 124
13

14 Abstract
15
16
Several laboratory and field experiments were conducted to investigate the effect of the kaolin particle film formulation Surround WP, on the common generalist predator
17 Chrysoperla carnea (Stephens) biology and behaviour. Kaolin 5% (w/v) suspension
18 direct spray did not affect third instar larvae development to adulthood. Recently laid
19 eggs, subjected to the same spraying process, were not altered in their hatchability
20 either. However, third instar larvae coated with particle film after kaolin spray, showed
21 a slightly hampered movement capacity, measured through the variables: Distance
22 moved, mean velocity, angular velocity and time spent in motion, obtained with the use
23 of the computerized system EthoVision. Parameters extracted from recorded larvae
24 movement on a kaolin film surface showed similar decreased mobility results, as well as
25 preference for the clean control surface. Additionally, the larvae had difficulties
26 grasping kaolin treated leaves. C. carnea adult females showed a dominant preference
27 towards kaolin treated leaves in oviposition choice tests. In the field trial a higher
28 number of C. carnea adults were found associate with kaolin treated olive trees. These
29 results indicate that disruption of movement capacity and dislodgment from the plant
30 surface may be the main negative actions of particle films on C.carnea larvae. Adults
31 showed a positive trend in oviposition and abundance towards kaolin treated surfaces.
32
33 Keywords: Ethovision; green lacewing; natural enemies; olive; predator; video tracking
34

35 1. Introduction
36 Kaolin particle technology is a relatively new and promising option for the
37 reduction of pest and disease damage in certain crops. The plants are dusted or sprayed
38 with particles of this non-abrasive, chemically inert, aluminosilicate [Al
4
SI
4
(OH)
18
]
39 mineral creating a film that coats the plant acting as a protective barrier against both
40 pathogens and pest arthropods (Glenn et al., 1999). The use of kaolin technology as a
41 pest management strategy has proved effective, as a broad spectrum compound, against
42 a wide range of pest insects such as the Psylids Cacopsylla pyricola Foerster on pear
43 and Diaphorina citri Kuwayama on citrus (Hall et al., 2007; Puterka et al., 2005), the
44 codling moth, Cydia pomonella (L.) on pear (Unruh et al., 2000), Mediterranean fruit
45 fly, Ceratitis capitata (Wiedemann) on apple (Mazor and Erez, 2004), the thrip Thrips
46 tabaci Lindeman on onions (Larentzaki et al., 2008) and the aphid Myzus persicae
47 (Sulzer) on peach (Karagounis et al., 2006). Specifically in olive farming, recent
48 research has shown particle film effectiveness in suppressing the olive fruit fly,
49 Bactrocera oleae Gmel., key pest in this crop, (Pascual et al., 2010; Saour and Makee,
50 2004) and the black scale, Saissetia oleae (Olivier) (Pascual et al., 2010). These
51 observations point to the feasibility of kaolin particle films as a valid alternative to the
52 extensively used insecticidal control. Particle film technology also has the advantage of
53 being allowed in organic agriculture, standing out, alongside with mass-trapping (Porcel
54 et al., 2009) and natural derived pesticides (Iannotta et al., 2007) as one of the few
55 options available for organic olive growers to control B. oleae damage.
56 Nevertheless, novel pest control methods that inhibit the action of harmful
57 insects must be subjected to evaluation of their effects on beneficial arthropods, in order
58 to improve the knowledge of the impact that their application might cause. This
59 knowledge is useful in joint strategies where alternative pest control methods should not

60 interfere with biological control. In this sense, recent literature has generally focused on
61 field assessment of kaolin effects based on the presence or absence of beneficial
62 arthropods on kaolin treated crops (Karagounis et al., 2006; Marko et al., 2007; Pascual
63 et al., 2010; Sackett et al., 2007). The mechanisms underlying how particle films affect
64 the biology and behaviour of insect pests have been extensively explored by several
65 authors (Cadogan and Scharbach, 2005; Lemoyne et al., 2008; Puterka et al., 2005),
66 describing a variety of effects i.e. direct toxicity and interference with insects ability to
67 settle move or oviposit (Hall et al., 2007). Still, to the best of our knowledge, concrete
68 effects caused by kaolin films to beneficial insects have received little study, and in the
69 case of predators, no information on possible behavioural and biological disruptions is
70 available.
71 The green lacewing, Chrysoperla carnea s. lat . (Stephens) (Neuroptera:
72 Chrysopidae) is one of the most common, naturally occurring, arthropod predator
73 (Duelli, 2001). Its extensive prey range, including almost any soft-bodied arthropod, its
74 wide distribution as well its voracious feeding capacity makes this natural enemy a
75 promising candidate for pest management programs (Tauber et al., 2000). Due to its
76 overall importance as a foliage-dwelling entomophagous predator, this insect has been
77 regarded as one of the relevant test species for the assessment of novel pest management
78 compounds (Mandour, 2009; Medina et al., 2003). In olive orchards, the green lacewing
79 is considered the mayor oophagous predator of the olive moth, Prays oleae Bernard,
80 helping in the reduction of the economic impact of this pest. It is also known to prey
81 upon less harmful insects as the black scale and the olive psylla, Euphylura olivina
82 Costa (Campos, 2001). Studies undertaken in different crops have detected a decreased
83 abundance and alteration of the assemblages of polyphagous predators associate with
84 kaolin treatments (Marko et al., 2007; Pascual et al., 2010; Sackett et al., 2007).

85 Therefore, the aim of the present work is to evaluate both biological and behavioral
86 effects of kaolin particle films on the predator C.carnea
.
87
88 2. Materials and Methods
89 The kaolin-clay based particle film used in the present study was the hydrophilic
90 Surround WP crop protectant manufactured by Englehard Corporation (Iselin, NJ,
91 USA). The compound is based on processed hydrous kaolin particles (1.0 ± 0.5 µm ø)
92 with an incorporated synthetic hydrocarbon spreader-sticker enhancing the particles
93 adhesion to the plant. The compound, hereafter referred to as kaolin, was applied at a
94 rate of 5% in mineral water (50 gram/liter) as recommended by the manufacturer. Plain
95 mineral water was used as control, and both were applied in all laboratory experiments
96 using an airbrush spray gun (Model 350, Badger Air-Brush Co., Franklin Park, IL,
97 USA) connected to an air compressor generating a cone spray pattern. The spray gun
98 was fixed by means of an adjustable height laboratory arm at 33 cm over the table
99 surface preventing from an excessive air flow over the spraying spot position. The spray
100 gun bottle was always in contact with a magnetic stirrer held by the same arm in order
101 to avoid kaolin particles sedimentation during the spraying process.
102
103
In all the laboratory experiments (with the exception of the experiment described in section 2.3), surfaces and larvae themselves were sprayed for a 30 sec period. To quantify kaolin particle retention, preweighed Teflon slides (97 cm
2
) were sprayed 104
105 likewise, and after a 4 h drying process at 25°C, weighed again using an electronic
106
107 analytical balance (AS 220/C/2, RADWAG Balances and Scales, Radom, Poland). The difference between final and initial weight yielded the kaolin film deposited in each

108
109 slide. The process was replicated up to 20 times. Finally the mean kaolin film weight was divided by the total surface obtaining the deposit per surface unit (µg/cm
2
).
110 2.1. Laboratory rearing of C. carnea
111 The larvae used in all the experiments were obtained from C.carnea
eggs
112
113
114 collected from a stock colony established in 2005 with larvae supplied by Koppert
Spain (La Mojonera, Almería) and monthly renewed with new individuals. Larvae were individually reared in petri dishes and fed on eggs of Ephestia kuehniella Zeller
115
116
(Lepidoptera, Pyralidae). Adults were kept in boxes with an ovipositing surface and provided an artificial diet (50% honey and 50% pollen) and mineral water. Both were
117
118 maintained in controlled environment cabinet at 25 ± 1°C, 50 - 60% RH, and a photoperiod of 16:8 (L:D) h.
119
120 2.2. Acute mortality to larvae
121 C.carnea larvae were directly sprayed with the 5% kaolin suspension in order to
122
123 assess any possible effects on mortality . A sufficient number of newly laid eggs were removed from the adults rearing boxes and developed in the same culture conditions
124
125
126 described above. Newly moulted third instar larvae were chosen for the experiment considering that previous observations had revealed that moulting caused an effective removal of the particle layer deposited onto the larva’s dorsal cuticle when spraying
127
128 first and second instars. The fact that third instar larvae most necessarily undergo both cocoon spinning and metamorphosis with an attached kaolin film makes them more
129
130
131 susceptible a priori. The inhibition of these developmental processes, whatever its origin, leads in most cases to the individuals decease (Cadogan and Scharbach, 2005;
Liu and Chen, 2001; Vogt et al., 2001). Recently moulted larvae (less than 12 h),

132
133 homogeneous in age and size, were selected and set aside from the stock. Individuals were transferred by tapping gently the underside of the petri dish, or when necessary,
134
135 using a camel paintbrush, to 4.5 cm ø petri dishes containing a double filter paper layer in order to prevent the formation of large droplets during the spraying process. Larvae
136
137
138 were chilled for 15 min, after which they were sprayed either with kaolin suspension or water. After spraying, the thoroughly wetted larvae typically presented one or more droplets formed onto the insects’ dorsum. Therefore, afterwards the petri dish was
139
140 covered and the individuals were subjected to 1 h drying period after which, in the case of kaolin sprayed larvae, the particle film became visually apparent. Finally, the filter
141
142
143 paper was removed and the larvae were fed and kept in culture conditions. The experiment was a randomized complete block design (RCBD) with replications consisting of 10 insects per treatment replicated five times over time. Tested individuals
144
145 were checked daily for survival until adult emergence recording the developmental stage in which mortality occurred.
146
147 2.3. Acute mortality of eggs
148 Kaolin film applications were tested on C. carnea eggs to determine its effects on egg viability and larval hatching suppression. Two bands (17 × 9 cm) of self 149
150 adhesive green velvety paper (Sadipal Stationary Papers, Girona, Spain ), used as
151
152 ovipositing surface in the C. carnea stock colony, were stuck upside-down onto either sides of the rectangular removable lid (36 × 22 cm) of an adults rearing box containing
153
154
155 an approximate number of 100 mixed male and female adults. After a 12 h period, 30 eggs were selected on each band by discarding the rest. One band was sprayed with the kaolin suspension and the other with the control water. Both treatments were delivered

156
157 hand holding the airbrush at an approximate distance of 10 cm and spraying for 30 seconds, covering the whole band area. Microscope eggs observations allowed detecting
158
159 small droplets on the eggs, which after drying, originated separated fragments of particle film adhered to the egg’s surface. The lid containing the two treated sets of eggs
160
161
162 was kept in a cabinet at culture conditions. After three days, E. kuehniella egg s were sprinkled over the bands in order to feed neonate larvae, preventing thus intraspecific egg predation. From the fourth day onwards, newly hatched C. carnea larvae were daily
163
164 removed from the bands by means of a camel paintbrush and the eggs were checked daily for 8 d to ensure that hatching had ceased. Data regarding viable and non-viable
165
166
167 eggs was recorded under microscope. Non-viable eggs were considered those that desiccated becoming shrivelled in appearance as well as neonate larvae that failed to free themselves from the eggshell, dying in the process. The experimental design
168 corresponded to a RCBD with up to 10 replications made over time.
169
170 2.4. Effect on ability to grasp leaf
171
172
The difference in larvae grasping capacity to leaves surfaces covered by kaolin particle film, compared to untreated leaves was assessed. Olive leaves (cv. Picual) were collected, adequately cut, and stuck side by side to a 100 cm
2
square glass, forming a 173
174 continuous leaf surface platform. Different surfaces were composed with both sides of
175
176 the leaves, pressed by means of a weight and kept at cold temperatures to avoid moisture losses. Following the standard spraying methodology, the platforms were
177
178
179 sprayed firstly with water and left to dry for 2 h. Newly moulted (less than 12 h) third instar individuals were raised and chosen as in section 2.2. The experimental methodology adopted to assess the differential grasping ability is similar to that

180
181 described for psyllids in (Puterka et al., 2005). Leaf platforms were horizontally mounted into a laboratory arm that allowed them to turn 180º. The larvae were
182
183 transferred to the leaf surface and allowed to move for several seconds. When the larva was situated in the central part a leaf (away enough from the border), and always in
184
185
186 motion, the platform was inverted for 30 seconds recording whether the larvae could hold the grip to the surface or fell off the platform. This was repeated using 10 different larvae, after which the platform was sprayed with kaolin, left to dry and used to essay
187
188
10 new larvae on the particle film. The whole process was repeated using the underside of the leaf surface. The experiment followed a RCBD, replicated 5 times over time.
189 Two new leaf platforms (upper and lower surfaces) were composed per replication.
190
191
192
193
2.5. Effect of a particle film covering C. carnea larvae on mobility and behavioural response
Directly sprayed, kaolin film covered larvae were assessed for changes in
194
195
196 behavioural and locomotor parameters. Same third instar size and age larvae were selected as in previous experiments and sprayed either with kaolin or mineral water.
Once delivered the treatment, the larvae were starved for a period of 24 h prior to the
197
198 bioassay. Each individual was taken to a windowless controlled room at 22 ± 2°C, 35 ±
10% RH and 130 lux light intensity, placing the larva containing a 4.5 cm ø petri dish
199
200
201 under a Panasonic CCTV videocamera (Mastsushita Electric Industrial Co., Ltd., Japan) camera. The larva free movement was recorded for a period of 15 min and the track transferred to a computer as part of the Ethovision XT integrated video tracking system
202
203
(Noldus Information Technology, Wageningen, The Netherlands). The Ethovision software allows to automatically determine the location of the individual in the arena
204 calculating several movement parameters derived from changes in position of the

205
206 detected insect. The parameters chosen in this particular experiment were (1) total distance moved (cm), (2) mean velocity (mm/s) (3) mean angular velocity (degrees/cm)
207
208 and (4) moving (%). The variable “moving” was defined as the fraction of time the larvae spent in motion. Parameter descriptions are given in (Noldus et al., 2002) and for
209
210
211 algorithms and calculations see (Noldus et al., 2007). One individual was tested at a time for a period and each larva was used only. The experiment was conducted as a completely randomized design (CRD) during 5 consecutive days, running every day
212
213 from 10 to 15 trials of both treatments until a total number of 30 valid replicates per treatment (60 replicates).
214
215
216
2.6. Effect of a particle film surface on larval mobility and choice
In this experiment we tested both, whether kaolin film covered substrates
217
218 interfered with larval mobility parameters and whether they showed a substrate preference between treated and control surfaces. As in previous experiments newly
219
220
221 moulted third instars were obtained from eggs selected for the purpose. The larvae were submitted to a 24 h starvation period before the bioassay. The 4.5 ø petri dish experimental arena was divided in two equal surface halves. Following the usual
222
223 methodology, one half was sprayed with kaolin and the other half with the control water. A semicircular waterproof plastic covering was used to isolate treatments areas
224
225 during the spraying process never coming in contact with the particle film as this was always delivered after the control area had been sprayed and dried for 2 h. Given that
226
227
228 only the lower part of the petri dish was sprayed, the experimental design was restricted to this area by coating the dish’s circular border with Fluon ®
(AGC Chemicals America
Inc, Moorestown, NJ, USA) preventing the larva from climbing to the lid. The
229 experimental setup and procedures corresponded with those described in section 2.5.

230
231
The parameters extracted from the recorded tracks were: (1) total distance moved (cm),
(2) mean velocity (mm/s) (3) mean angular velocity (degrees/cm), (4) moving (%) and
232
233
(5) time spent in each zone (kaolin and control). All the parameters were calculated for each zone individually for comparison, in a way that, unlike in the precedent experiment
234
235
236
(section 2.5), each individual trial generated the complete set of variables for both treatments. The experiment was carried out in 5 consecutive days in a RCBD with 10 to
13 trials per day until reaching the number of 50 valid replicates. Each block of
237
238 replicates consisted of 10 trials conducted the same day with the same experimental arena, and therefore a new arena was sprayed every day.
239
240
241
2.7. Adults oviposition preference
C. carnea adults were placed in an ovipositing arena and given the choice
242
243 between kaolin film and control surfaces. The arena consisted on an 8.5 cm ø × 2.5 cm high petri dish with two leaf ovipositing surfaces, one upside down attached onto the lid
244
245
246 and the other opposite to the latter attached to the bottom. The 8.5 cm ø surfaces were composed as described in section 2.4. Upper and lower surfaces were composed per arena. Before mounting the surfaces onto the arena, both were divided in equal
247
248 semicircular zones and sprayed with kaolin and water, proceeding as described in section 2.6 by means of a bigger plastic cover. As a result, both leaf surfaces contained
249
250 particle film and control zones of equal area. The upper surface was adhered to the bottom of the dish and the lower to the lid so that the kaolin film always faced the
251
252
253 control zone. Less than 12 h emerged adults were coupled and transferred to small rearing boxes (0.9 L), kept in culture environmental conditions and provided with adult food and water. After seven days, individual couples were transferred to the ovipositing
254 arena to lay eggs for a period of 48 h. Water was supplied by a moistened piece of

255
256 sponge, and food directly, both stuck to the border of the arena, avoiding interference with the surfaces. The eggs deposited in each defined area (upper, lower surfaces, kaolin
257
258 film and control zones) were recorded. Females that did not oviposit were excluded from the experiment. Successively, 10 couples were essayed in the same arena repeating
259
260
261 the process with a total of five different arenas (50 individual replicates). The experiment was designed as a RCBD where each arena constituted a different block.
262
263
2.8. Effect on adults abundance. Field case study
A field experiment was conducted to determine whether a particle film sprayed
264
265
266 on olive trees had an immediate effect on lacewing adults presence. The experiment was conducted in a 258 ha commercial non-irrigated olive orchard (cv. Picual, 90-yr-old) under IPM situated in the province of Granada (37º 17’ 46.7’’ N, 3º 46’ 28.7’’ W),
267
268
Spain. No insecticidal treatment had been performed during the year and a natural regeneration cover was present between trees from the beginning of spring until
269
270
271 naturally drying in June. A 50 g/kaolin solution (treatment) and water (control) were applied to 16 trees (grown 11 × 11 m apart) square plots by means of a tractor pulled
272
273 turbo atomizer (SISTROMATIC AL-TAR 2000N, Máñez y Lozano S.L. Maquinaria
Agrícola, Valencia, Spain) at a rate of 95 L/ha delivering approximately 0.58 kg of kaolin per tree. Treatment and control plots were situated at a minimum distance of 80
274
275 m. The experiment was replicated up to 4 times (8 total plots) following a RCBD with blocks separations of at least 150 m. Applications took place twice, on 15 June 2009
276
277
278 and on 24 September 2009. These dates were chosen due to the known chrysopids flying peaks in southern European olive orchards. The chrysopids were sampled seven days after the applications, in two consecutive days, using an insect aspirator (Modified
279 CDC Backpack Aspirator Model 1412, John W. Hock Co., Gainsville, FL, USA),

280
281 sampling all the trees in the plot. Inner and outer branches of each olive tree were suctioned up to a height of 2 m, and surrounding the tree, with the purpose of covering
282
283 all the orientations, for a period of 2 min. The collected adult chrysopids were counted and identified to species level. Precipitation data obtained from a public agroclimatic
284
285
286 station (IFAPA, Junta de Andalucía) revealed a single rainfall event (5.4 mm) that took place the afternoon after the kaolin treatment was carried out in 15 June and three rainfall events between the 23 September and 1 October adding up a total rainfall of
287
288
10.2 mm.
289 2.9. Statistical analysis
All the statistical tests were performed with the package SPSS statistics 18 for 290
291 windows (SPSS Inc, Chicago, IL, USA). For the analysis of the larvae and eggs acute
292
293 mortality and, larvae leaf grasping experiments (sections 2.2, 2.3 and 2.4) percentages were arcsin-transformed for normalization and compared using Student’s t-test (α =
294
295
296
0.05) for paired comparisons and analysis of variance tests (ANOVA) for multiple comparisons. ANOVA analysis was followed by Tukey tests (α = 0.05) to identify differences between means.
For larvae mobility experiments analysis (sections 2.5 and
297
298
2.6), movement parameters were log
10
(x + 0.5) transformed for normalization and compared by means of a Student’s t-test (α = 0.05). Whenever the data distribution
301
302
303
299
300 failed to satisfy parametric analysis assumptions, untransformed data were subjected to non-parametric Mann-Whitney U tests (α = 0.05). Statistically significant differences at a confidence level α = 0.10 are indicated. In the oviposition preference experiment
(section 2.7) the data set violated parametric analysis assumptions, and therefore treatments were compared using a Kruskal-Wallis test followed by paired Mann-
304 Whitney U tests for individual comparisons. The α value was adjusted by means of the

305
306
Bonferroni-Holm’s correction (α ≤ 0.05) (Holm, 1979). Finally, C. carnea adult captures data from the field case study (section 2.9) were compared using non-
307
308 parametric Mann-Whitney U tests (α = 0.10). In all cases untransformed means are presented.
309
310 3. Results
311 The mean particle density (± SD) deposited on the Teflon slides after drying for
4 h was 290.7 ± 99.7 µg/cm
2
. 312
313
314
315
316
3.1. Acute mortality to larvae
No difference in mortality were observed between the treatment and control individuals (Table 1) neither in the different developmental stages (ANOVA, F = 0.48,
317
318 df = 5, P = 0.788) nor in the percentage of adult emergence ( t -test, t = 0.42, df = 7.97, P
= 0.685). The clearly observable kaolin film particle covering the larvae dorsum did not
319
320 interfere with C. carnea normal development from the third instar state.
321 3.2. Acute mortality to eggs and larval survival
324
325
326
322
323
Eggs hatching and newly emerged first instars early survival were not affected by the kaolin treatment on eggs at the tested environmental conditions. Specifically, an
80.0 ± 8.3% (mean ± SD) particle film sprayed eggs produced normally hatching individuals whilst the hatching rate in water sprayed eggs was 84.3 ± 5.9%. Despite a lower hatching rate effect resulting from the kaolin treatment, no statistical differences
327
328 were found (t-test, t = 0.36 df = 18, P = 0.192) and therefore is to be considered of little biological significance. No cannibalistic egg predation was observed while recording

329 the viable and non-viable eggs, indicating that the recently hatched larvae fed on E.
330 kuehniella eggs supplied for this aim.
331
332 3.3. Effect on ability to grasp leaf
333
334
335
Kaolin particle film covering both sides of olive leaves clearly affected C. carnea larvae ability to grasp the leaf surface. This capacity was altered by the kaolin treatment on both upper and lower side of the leaf (ANOVA, F = 35.8, df = 19, P <
336
337
0.05) and was especially notable in the case of the upper side where the number of larvae that remained on the kaolin treated surface decreased in a 66 %. Concerning
338
339
340 upper and lower parts of the leaf, no difference in grasping performance was observed when comparing water sprayed surfaces, however, the ability to grasp the kaolin treated lower surface was significantly greater than the capacity to grasp the kaolin treated
341 upper surface (Table 2).
342
343
344
3.4: Effect of a particle film covering C. carnea larvae on mobility and behavioural response
345 Observation of the recorded tracks did not reveal the existence of a clear behavioural change resulting from the particle film adhered to the insect’s surface.
346
347 Despite this fact, the numerical variables derived from the tracks provided by the
348
349
Ethovision XT software showed small but significant differences in all of the measured parameters (Fig. 1). Kaolin treated larvae covered a shorter distance within the arena
350
351 than water treated individuals ( t -test, t = 2.60 df = 44.23, P = 0.012). The distance moved reduction can be interpreted as a double effect produced by a decreased mean

352
353 velocity of kaolin treated individuals ( t -test, t = 2.56, df = 44.59, P = 0.014) and an increased frequency of pausing, spending in motion a lower fraction of the total trial
354
355 time (Mann-Whitney U test, Z = -2.61, P = 0.009). Kaolin treatment also affected the shape of the path travelled by the larvae, thus, kaolin covered individuals showed a
356 significantly higher turning rate per unit time ( t -test, t = 2.56, df =58.00, P = 0.001).
357
358 3.5. Effect of a particle film surface on larval mobility and choice
359
360
As above, direct observation did not denote a distinct reaction caused by the presence of a kaolin film. Unlike in the results described section 3.3 (where the larvae
361
362
363 moved freely across both the dish and the lid) the individuals showed a recurrent trend to attempt to climb the dish border from kaolin and control surfaces alike. Analysis of tracks data showed differences in movement parameters induced by the kaolin film (Fig.
364
365
2). The tested individuals covered a shorter distance ( t -test, t = 2.49 df = 98.00, P =
0.014) at a lower velocity (Mann-Whitney U test, Z = - 1.66, P = 0.097) in the kaolin
366
367
368 treated surface. The larvae spent significantly less time on the kaolin film surface ( t -test, t = 3.78 df = 98.00, P = 0.007) and showed a higher stopping frequency ( t -test, t = 3.01 df = 98.00, P = 0.007) as compared to the control surface. No differences were detected
369
370 in the path shape considering that the angular velocity exhibited was similar on both surfaces ( t -test, t = 1.05 df = 82.45, P = 0.295).
371
372 3.6. Adults oviposition preference
373
374
C. carnea female adults laid almost all the eggs upside down on the lower part of the leaves attached to the lid while just a few were deposited on the lower side and

375
376 the lateral border (Table 3). On the lower surface ,where the eggs accounted for a 94.9 ±
6.4% (mean ± SD) of the total, the tested individuals laid more than two-fold number of
377
378 eggs on the kaolin treated semicircular zone than on the control zone (Kruskal-Wallis, F
= 302.88, df = 4, P < 0.05).
379
380 3.7. Effect on adult abundance. Field case study
381 From a total of 256 samples, 111 adult chrysopids were captured belonging to 4
382
383 different species (Table 4). C. carnea adults represented a proportion of 91.9 % of the total individulas, and the only species captured in June. Just few C. carnea adults were
384
385
386 collected in this first sampling dates (Fig. 3), and no significant differences were observed between kaolin treated and control plots (Mann-Whitney U test, Z = -1.33, P =
0.184). However, the total number of C. carnea adults captured increased in nearly
387
388 nine-fold in October sampling dates, and at a confidence level of α < 0.10 a higher abundance was recorded on kaolin film covered trees (Mann-Whitney U test, Z = -1.73,
389 P = 0.084).
390
391 4. Discussion
392
393
Despite the consideration of the kaolin-based particle film as an innocuous compound, its direct spray effect on insects remains unclear. Toxicity results differ
394
395 depending on the tested species. In Psylids, Hall (2007) found no acute toxicity when spraying a 3% Surround WP suspension on Asian Psylid ( Diaphorina citri Kuwayana)
396
397 adults, nymphs and eggs whilst the pear Psylla, subjected to similar tests, suffered significant mortality rates, higher than the control in kaolin sprayed nymphs and adults

398
399
(Puterka et al., 2005). Plutella xylostera (L.) fourth instars mortality was just slightly increased by a particle film residual coating (Barker et al., 2006) and Larentzaki et al.
400
401
(2008) discussed that an interference with the feeding capacity was the most probable cause explaining the higher mortality rates observed in kaolin sprayed Thrips tabaci . In
402
403
404 the case of C.carnea
, direct kaolin application does not appear to cause any direct mortality neither interfering with the feeding capacity nor through other type of hampering effect that may result in reduction of the survival to adulthood. Hall (2007)
405
406 suggested body size as a possible explanation for the differential effect in mortality among insect species. This opens the possibility that early C. carnea first instars
407
408
409
(around 2 mm in lenght), not included in this study, may be affected by residual kaolin coating, however, we have observed that ecdysis removes effectively the kaolin coverage that remains attached to the moult, naturally disposing of the source of a
410
411 possible negative impact. Therefore, we believe that kaolin particles have none, or insignificant acute effect on C.carnea
mortality. As for the ovicidal effect of kaolin, the
412
413
414 results show no significant difference between water and kaolin treated eggs, consistent with experiments carried on moth eggs that resulted unaffected by the kaolin cover
(Barker et al., 2006; Unruh et al., 2000).
415
416
Although the kaolin residual coating did not mortally affect C. carnea larvae with unlimited access to food, the behavioral bioassay showed significant effects on
417
418 larval movement. Thus, kaolin covered larvae presented a reduced general mobility; decreased mean velocity accompanied by a higher stopping frequency resulted in a
419
420
421 reduction of the distance travelled by the larvae. To our best knowledge no such effects of kaolin residual coating have been previously reported for other insect species. The reasons for these differences are not known, but might be related to the accumulation of
422 kaolin particles in the articulations hindering normal walking activity. This fact could

423
424 also help to explain the increase in turning activity detected. The difficulty to keep a straight walking line may have its origin in the kaolin residue deposited on the legs. Due
425
426 to the small difference between means, these results must be taken cautiously. We cannot be sure of an actual consequence of the observed mobility reduction at a field
427
428
429 scale. Chrysopids movement capacity is an essential compound of their foraging efficacy since the prey encounter happens predominantly at random. It is reasonable to think that kaolin particles adhered to the insect’s surface may influence negatively prey
430
431 searching and survival, and therefore the overall biological control performance of the tested species. However, at a field level the deposition of kaolin particles will probably
432
433
434 never reach the density essayed in the laboratory. Highly mobile chrysopid larvae may easily find shelter avoiding partially the kaolin spray. By contrary, small reductions of mobility assessed at laboratory scale in a reduced period of time may translate in an
435
436 important effect in C. carnea larva life span.
The presence of a particle film on olive leaves had a very important influence on
437
438
439 the ability of C. carnea larvae to grasp its surface. Our results also reveal that the olive leaf surface type has a differential effect on the grasping ability. These findings have been described with equal results for the pear psylid. According to Puterka et al. (2005)
440
441 psylla fall off the treated surfaces because the kaolin particles break away at the insects grasping points. Equally, kaolin coated pear leaves underside resulted a better grasping
442
443 surface due to the presence of conspicuous structures such as trichomes and stomates.
Although attachment is expected to differ among insect species depending on their
444
445
446 specific adaptations, C. carnea larvae seem to be affected in the same manner as psyllids. Under field conditions this grasping ability inhibition may result in an increase of the number of larvae falling from the trees canopy to the ground. C. carnea larvae
447 possess a specific adaption to minimize the risk of dropping off the plants. They

448
449 produce an adhesive substance in their malpighian tubules that is applied through the tip of the abdomen. The development of this adaptation is an indication of the danger that
450
451 dislodging from the plant represents, and consequently it has been regarded as a potential source of mortality (Rosenheim et al., 1999). Movement bioassay on the
452
453
454 kaolin film deposited on the petri dish arena revealed differences of the locomotion parameters among kaolin treated and control surfaces. The film presence affected distance covered velocity and induced a higher stopping frequency. Save for the
455
456 increased tortuosity of the trajectory, the overall mobility inhibition results were similar to that of the larvae direct spray. These movement difficulties may also have actuated as
457
458
459 a deterrent inducing the insect to move preferably on the control surface explaining therefore the differences in time spent in each zone. Kaolin films presence have been reported to restrict mobility of adult Psilids (Puterka et al., 2005) and neonate codling
460
461 moth larvae (Unruh et al., 2000). C. carnea movement limitation on kaolin covered surfaces, unlike restrictions due to an insect coating film, can be contemplated as a
462
463
464 probable scenario in orchards using kaolin treatments for pest control management.
Moreover, synergistic effects of both situations, hampering drastically the insect’s ability to forage, may not be discarded. Unruh et al. (2000), based on the perception of
465
466 behavior disturbances caused by kaolin films, predicted that natural enemies disruptions would most likely affect small insects that actively forage on the surface of treated
467
468 plants. Our behavioral results evidence this assertion in the particular case of the predator C. carnea where computerized video tracking technology, used for the first
469
470
471 time in the present study for the assessment of side effects of novel pest control compounds, has proven a valuable methodological tool.
Deterrence of insects oviposition has been acknowledged as one of main actions
472 of particle films on pest insects behavior, contributing to the reduction of the infestation

473
474
(Lapointe et al., 2006; Larentzaki et al., 2008). Our findings suggest that, not only a kaolin film does not deter C.carnea
oviposition on treated surfaces, but by contrary,
475
476 gravid females manifested a preference towards ovipositing on them. This peculiar effect has been previously noted by Medina et al. (2007) that registered an increased
477
478
479 oviposition on kaolin coated twigs compared to the water control. It is clear that females were perfectly able to grasp and oviposit upside-down on treated surfaces, nevertheless the reasons underlying the dominant preference towards kaolin covered leaves are
480
481 unknown. Kaolin particle deposits induce a structural modification of the treated leaf surfaces discouraging certain insects oviposition (Puterka et al., 2005). However, these
482
483
484 modifications might elicit different behavioural responses depending on the species egg type. Consequently, C. carnea females may have chosen the most suitable anchoring substrate for their stalked eggs.
485
486
The literature body addressing the risks of kaolin treatment on beneficial arthropods is scarce and inconclusive as for the concrete effect on chrysopid abundance.
487
488
489
Marko et al.(2007) observed no difference between kaolin and control plots in adults abundances, although the amount of lacewing larvae present was higher in kaolin treated apple trees. Pascual et al.(2010) recorded a significantly lower number of
490
491 chrysopid captures on kaolin sprayed olive trees in one of the three years of study.
However both larvae and adults were grouped together so that particular effects on the
492
493 different lifestages could not be identified. Our field experiment aimed to test the possibility that, given the results on oviposition preference, a kaolin particle film might
494
495
496 exert an attractive influence toward adult C. carnea , increasing the occurrence of the predator at field scale. Even though adult chrysopids are present in olive orchards throughout the complete year (Campos, 2001), the number of captured individuals in
497 the first sampling date, possibly coinciding with a population cyclic decline in summer,

498
499 was insufficient to observe differences. Later on, in October, the captures rose notably, and C. carnea males and females, unlike previously reported from the mentioned
500
501 studies, were more abundant in kaolin plots. In late September and October, the kaolin deposit on olive trees reaches its maximum after several applications performed from
502
503
504 the month of July (Saour and Makee, 2004). As a consequence, the major kaolin effects on adults might take place in these dates as adult populations increase in the field as observed. The results on adult oviposition and abundance presented herein reveal the
505
506 necessity of future research, at field level, aiming to further clarify adult lacewings response to this compound.
507
508
509
Acknowledgements
The authors would like to thank María Luisa Fernández Sierra and Herminia
512
513
514
510
511
Barroso for their laboratory assistance and Dr. Victor Monserrat for his tutelage in lacewings identification. We also thank Juan Castro for his contribution in the field experiment carried out in the experimental orchard provided by Junta de Andalucía.
This study was grant-aided by PO7-AGR-2747 (Junta de Andalucía) and the project
AGL 2005-00932 (Ministry of Science and Education).
515
516
517
518
References
Barker, J.E., Fulton, A., Evans, K.A., Powell, G., 2006. The effects of kaolin particle
519
520
521 film on Plutella xylostella
Science 62 , 498–504.
behaviour and development. Pest Management
Cadogan, B.L., Scharbach, R.D., 2005. Effects of a kaolin-based particle film on
522 oviposition and feeding of gypsy moth (Lep., Lymantriidae) and forest tent

523
524 caterpillar (Lep., Lasiocampidae) in the laboratory. Journal of Applied
Entomology 129 , 498–504.
525
526
Campos, M., 2001. Lacewings in Andalusian olive orchards. In: McEwen, P., New,
T.R., Whittington, A.E., Eds.), Lacewings in the Crop Environment. Cambridge
527
528
529
University Press, Cambridge, pp. 492–497.
Cohen, A.C., Smith, L.K., 1998. A new concept in artificial diets for Chrysoperla rufilabris : The efficacy of solid diets. Biological Control 13 , 49-54.
530
531
Duelli, P., 2001. Lacewings in field crops. In: McEwen, P., New, T.R., Whittington,
A.E., Eds.), Lacewings in the Crop Environment. Cambridge University Press,
532
533
534
Cambridge, pp. 158–171.
Glenn, D.M., Puterka, G.J., Vanderzwet, T., Byers, R.E., Feldhake, C., 1999.
Hydrophobic particle films: A new paradigm for suppression of arthropod pests
535
536 and plant diseases. Journal of Economic Entomology 92 , 759–771.
Hall, D.G., Lapointe, S.L., Wenninger, E.J., 2007. Effects of a particle film on biology
537
538
539 and behavior of Diaphorina citri (Hemiptera: Psyllidae) and its infestations in citrus. Journal of Economic Entomology 100 , 847–854.
Holm, S., 1979. A simple sequentially rejective Bonferroni test procedure. Scandinavian
540
541
Journal of Statistics 6 , 65–70.
Iannotta, N., Belfiore, T., Brandmaya, P., Noce, M.E., Scalercio, S., 2007. Evaluation of
542
543 the impact on entomocoenosis of active agents allowed in organic olive farming against Bactrocera oleae (Gmelin, 1790). Journal of Environmental Science and
544
545
546
Health, Part B 42 783–788
Karagounis, C., Kourdoumbalos, A.K., Margaritopoulos, J.T., Nanos, G.D., Tsitsipis,
J.A., 2006. Organic farming-compatible insecticides against the aphid Myzus

547
548 persicae
154.
(Sulzer) in peach orchards. Journal of Applied Entomology 130 , 150–
549
550
Lapointe, S.L., McKenzie, C.L., Hall, D.G., 2006. Reduced oviposition by Diaprepes abbreviatus (Coleoptera: Curculionidae) and growth enhancement of citrus by
551
552
553 surround particle film. Journal of Economic Entomology 99
Larentzaki, E., Shelton, A.M., Plate, J., 2008. Effect of kaolin particle film on Thrips tabaci
, 109–116.
(Thysanoptera: Thripidae), oviposition, feeding and development on
554
555 onions: a lab and field case study. Crop Protection 27 , 727–734.
Lemoyne, P., Vincent, C., Gaul, S., Mackenzie, K., 2008. Kaolin affects blueberry
556
557
558 maggot behavior on fruit. Journal of Economic Entomology 101
Liu, T.-X., Chen, T.-Y., 2001. Effects of three aphid species (Homoptera: Aphididae) on development, survival and predation of
, 118–125.
Chrysoperla carnea (Neuroptera:
559
560
Chrysopidae). Applied Entomology and Zoology 36 , 361–366.
Mandour, N.S., 2009. Influence of spinosad on immature and adult stages of
561
562
563
Chrysoperla carnea
102.
(Stephens) (Neuroptera: Chrysopidae). BioControl 54 , 93–
Marko, V., Blommers, L.H.M., Bogya, S., Helsen, H., 2007. Kaolin particle films
564
565 suppress many apple pests, disrupt natural enemies and promote woolly apple aphid. Journal of Applied Entomology 132 26–35.
568
569
570
566
567
Mazor, M., Erez, A., 2004. Processed kaolin protects fruits from Mediterranean fruit fly infestations. Crop Protection 23 , 47–51.
Medina, P., Budia, F., Contreras, G., Del Estal, P., González-Núñez, M., García, M.,
Viñuela, E., Adán, A., 2007. Comportamiento reproductor del depredador
Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) y el parasitoide
571 Psyttalia concolor (Szèpligeti) (Hymenoptera: Braconidae) en superficies

572
573 cubiertas con caolín. V Congreso Nacional de Entomología Aplicada. SEdE
Aplicada, Cartagena, Spain, pp. 22–26.
574
575
Medina, P., Smagghe, G., Budia, F., Tirry, L., Viñuela, E., 2003. Toxicity and absorption of azadirachtin, diflubenzuron, pyriproxyfen, and tebufenozide after
576
577
578 topical application in predatory larvae of
Chrysopidae). Environmental Entomology 32 ,
Chrysoperla carnea
196–203.
(Neuroptera:
Noldus, Information, Technology, 2007. Ethovision XT: The next generation of video
579
580 tracking systems. Reference Manual, Version 5, Wageningen, The Netherlands.
Noldus, L.P.J.J., Spink, A.J., Tegelenbosch, R.A.J., 2002. Computerised video tracking,
581
582
583 movement analysis and behaviour recognition in insects. Computers and
Electronics in Agriculture 35 , 201–227.
Pascual, S., Cobos, G., Seris, E., González-Núñez, M., 2010. Effects of processed
584
585 kaolin on pests and non-target arthropods in a Spanish olive grove Journal of
Pest Science 83 , 121–133.
586
587
588
Porcel, M., Ruano, F., Sanllorente, O., Caballero, J.A., Campos, M., 2009. Short communication. Incidence of the OLIPE mass-trapping on olive non-target arthropods. Spanish Journal of Agricultural Research 7 , 660–664.
589
590
Puterka, G.J., Glenn, D.M., Pluta, R.C., 2005. Action of particle films on the biology and behavior of pear psylla (Homoptera: Psyllidae). Journal of Economic
591
592
Entomology 98 , 2079-2088.
Rosenheim, J.A., Limburg, D.D., Colfer, R.G., 1999. Impact of generalist predators on a
593
594
595 biological control agent,
Applications 9 , 409–417.
Chrysoperla carnea : direct observations. Ecological
Sackett, T.E., Buddle, C.M., Vincent, C., 2007. Effects of kaolin on the composition of
596 generalist predator assemblages and parasitism of Choristoneura rosaceana

597
598
(Lep., Tortricidae) in apple orchards. Journal of Applied Entomology 131
485.
, 478–
599
600
Saour, G., Makee, H., 2004. A kaolin-based particle film for suppression of the olive fruit fly Bactrocera oleae Gmelin (Dip., Tephritidae) in olive groves. Journal of
601
602
603
Applied Entomology 128 , 28–31.
Tauber, M.J., Tauber, C.A., Daane, K.M., Hagen, K.S., 2000. Commercialization of predators: recent lessons from green lacewings (Neuroptera: Chrysopidae:
604
605
Chrysoperla ). American Entomologist 46 , 26–38.
Unruh, T.R., Knight, A.L., Upton, J., Glenn, D.M., Puterka, G.J., 2000. Particle films
609
610
611
612
606
607
608 for suppression of codling moth (Lepidoptera: Tortricidae) in apple and pear orchards. Journal of Economic Entomology 93 , 737–743.
Vogt, H., Viñuela, E., Bozsik, A., Hilbeck, A., Bigler, F., 2001. Interactions with plants management strategies. In: McEwen, P., New, T.R., Whittington, A.E., Eds.),
Lacewings in the Crop Environment. Cambridge University Press, Cambridge, pp. 357–379.

613
Table 1. Mean mortality (± SD in different development stages and mean adult
614 emergence (± SD of third instar larvae sprayed either with water (control) or kaolin.
Treatment
615
Kaolin film
Water control
3rd instar
12.0 ± 17.9
14.0 ± 11.4
% mortality a
Prepupa
4.0 ± 8.9
8.0 ± 10.9
Pupa
10.0 ± 7.1
8.0 ± 8.4
616 a
Data arcsin-transformed for statistical analysis. Mean values (± SD) followed by same
617 or no letters are not significantly different (α = 0.05).
% adult a emergence
74.0 ± 15.2
70.0 ± 14.1
618
619
620
Table 2. Mean (± SD) percentage of
C. carnea larvae able to grasp to kaolin treated and control leaf surfaces and therefore not falling within 30 s after inverting them
Treatment
Kaolin film
Water control
Upper
surface
22 ± 11 a
88 ± 11 c
% grasping the leaves a
Lower
surface
62 ± 16 b
92 ± 8 c
621
622
623 a Data arcsin-transformed for statistical analysis. Mean values (± SD) followed by equal letters are not significantly different (Tukey’s test, α
= 0.05).
624

625
626
Table 3. Mean number (± SD) of eggs laid by C. carnea 7 d-old females in a 48 h time period on upper and lower olive leaves surfaces treated either with kaolin particle film
627 or mineral water (control).
628
629
630
631
632
633
Treatment Upper
surface
7.3 ± 6.5 a
17.3 ± 8.7 c
# Eggs laid a
Lower
surface
0.0 ± 0.0 b
0.2 ± 0.7 b
Kaolin film
Water control a Mean values with different letters indicate statically significant differences (Mann-
Whitney U -test, Bonferroni-Holm’s correction α ≤ 0.05).
Lateral border
-
1.2 ± 1.7 d
Table 4. Number of Chrysopidae adults captured by suction sampling in kaolin sprayed and water sprayed (control) olive trees in two different sampling events.
♀
♂
♀
♂
♀
♂
♀
♂
Control
June
Kaolin
5
3
1
2
0
0
0
0
0
0
8
0
0
0
0
0
0
3
October
Control Kaolin
19
17
24
31
1
0
1
1
0
2
41
0
0
0
1
0
3
59
634
635
Species
Chrysoperla carnea
(Stephens)
Dichocrysa prasina
(Burmeister)
Dichocrysa flavifrons
(Brauer)
Chrysopa
Formosa
Brauer
Total

636 Figure caption sheet
637
638
639
Fig. 1. Movement parameters of C.carnea
larvae directly sprayed either with kaolin or mineral water (control). Mean ± SE (a) total distance moved (cm), (b) mean velocity
640
641
(mm/s) (c) mean angular velocity (unsigned degrees/s) and (d) moving (%). Bars with different symbol indicate statistically significant differences ( P < 0.05).
642
643
644
Fig. 2. C.carnea
larvae movement parameters on a circular arena half covered with a kaolin particle film and half kaolin free (control). Mean ± SE (a) Total distance moved
645
646
(cm), (b) mean velocity (cm/s) (c) mean angular velocity (degrees/s), (d) moving (%) and (e) total time spent in zone. Bars with different symbol indicate statistically
647 significant differences **( P < 0.05), *( P < 0.10).
648
649 Fig. 3. Mean (± SE) number of C.carnea
adults captured per tree by suction sampling
650
651 on kaolin treated and water treated (control) olive trees in two different sampling events.
Squares with different symbol indicate statistically significant differences ( P < 0.10)
652 within the same sampling date.
653

654 Fig. 1
655
656

657 Fig. 2
658
659

660 Fig. 3
661