Biological and behavioral effects of a kaolin particle film on larvae and adults of Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae)
M. Porcel1*, B.Cotes1, M. Campos1
1 Department of Environmental Protection. Estación Experimental de Zaidín, CSIC, Profesor Albareda nº 1, 18008, Granada. Spain
* Corresponding author: Email: firstname.lastname@example.org; Tlf: +34-958191600 ext. 124
Several laboratory and field experiments were conducted to investigate the effect of the kaolin particle film formulation Surround WP, on the common generalist predator Chrysoperla carnea (Stephens) biology and behaviour. Kaolin 5% (w/v) suspension direct spray did not affect third instar larvae development to adulthood. Recently laid eggs, subjected to the same spraying process, were not altered in their hatchability either. However, third instar larvae coated with particle film after kaolin spray, showed a slightly hampered movement capacity, measured through the variables: Distance moved, mean velocity, angular velocity and time spent in motion, obtained with the use of the computerized system EthoVision. Parameters extracted from recorded larvae movement on a kaolin film surface showed similar decreased mobility results, as well as preference for the clean control surface. Additionally, the larvae had difficulties grasping kaolin treated leaves. C. carnea adult females showed a dominant preference towards kaolin treated leaves in oviposition choice tests. In the field trial a higher number of C. carnea adults were found associate with kaolin treated olive trees. These results indicate that disruption of movement capacity and dislodgment from the plant surface may be the main negative actions of particle films on C.carnea larvae. Adults showed a positive trend in oviposition and abundance towards kaolin treated surfaces.
Keywords: Ethovision; green lacewing; natural enemies; olive; predator; video tracking
Kaolin particle technology is a relatively new and promising option for the reduction of pest and disease damage in certain crops. The plants are dusted or sprayed with particles of this non-abrasive, chemically inert, aluminosilicate [Al4SI4(OH)18] mineral creating a film that coats the plant acting as a protective barrier against both pathogens and pest arthropods (Glenn et al., 1999). The use of kaolin technology as a pest management strategy has proved effective, as a broad spectrum compound, against a wide range of pest insects such as the Psylids Cacopsylla pyricola Foerster on pear and Diaphorina citri Kuwayama on citrus (Hall et al., 2007; Puterka et al., 2005), the codling moth, Cydia pomonella (L.) on pear (Unruh et al., 2000), Mediterranean fruit fly, Ceratitis capitata (Wiedemann) on apple (Mazor and Erez, 2004), the thrip Thrips tabaci Lindeman on onions (Larentzaki et al., 2008) and the aphid Myzus persicae (Sulzer) on peach (Karagounis et al., 2006). Specifically in olive farming, recent research has shown particle film effectiveness in suppressing the olive fruit fly, Bactrocera oleae Gmel., key pest in this crop, (Pascual et al., 2010; Saour and Makee, 2004) and the black scale, Saissetia oleae (Olivier) (Pascual et al., 2010). These observations point to the feasibility of kaolin particle films as a valid alternative to the extensively used insecticidal control. Particle film technology also has the advantage of being allowed in organic agriculture, standing out, alongside with mass-trapping (Porcel et al., 2009) and natural derived pesticides (Iannotta et al., 2007) as one of the few options available for organic olive growers to control B. oleae damage.
Nevertheless, novel pest control methods that inhibit the action of harmful insects must be subjected to evaluation of their effects on beneficial arthropods, in order to improve the knowledge of the impact that their application might cause. This knowledge is useful in joint strategies where alternative pest control methods should not interfere with biological control. In this sense, recent literature has generally focused on field assessment of kaolin effects based on the presence or absence of beneficial arthropods on kaolin treated crops (Karagounis et al., 2006; Marko et al., 2007; Pascual et al., 2010; Sackett et al., 2007). The mechanisms underlying how particle films affect the biology and behaviour of insect pests have been extensively explored by several authors (Cadogan and Scharbach, 2005; Lemoyne et al., 2008; Puterka et al., 2005), describing a variety of effects i.e. direct toxicity and interference with insects ability to settle move or oviposit (Hall et al., 2007). Still, to the best of our knowledge, concrete effects caused by kaolin films to beneficial insects have received little study, and in the case of predators, no information on possible behavioural and biological disruptions is available.
The green lacewing, Chrysoperla carnea s. lat. (Stephens) (Neuroptera: Chrysopidae) is one of the most common, naturally occurring, arthropod predator (Duelli, 2001). Its extensive prey range, including almost any soft-bodied arthropod, its wide distribution as well its voracious feeding capacity makes this natural enemy a promising candidate for pest management programs (Tauber et al., 2000). Due to its overall importance as a foliage-dwelling entomophagous predator, this insect has been regarded as one of the relevant test species for the assessment of novel pest management compounds (Mandour, 2009; Medina et al., 2003). In olive orchards, the green lacewing is considered the mayor oophagous predator of the olive moth, Prays oleae Bernard, helping in the reduction of the economic impact of this pest. It is also known to prey upon less harmful insects as the black scale and the olive psylla, Euphylura olivina Costa (Campos, 2001). Studies undertaken in different crops have detected a decreased abundance and alteration of the assemblages of polyphagous predators associate with kaolin treatments (Marko et al., 2007; Pascual et al., 2010; Sackett et al., 2007). Therefore, the aim of the present work is to evaluate both biological and behavioral effects of kaolin particle films on the predator C.carnea.
2. Materials and Methods
The kaolin-clay based particle film used in the present study was the hydrophilic Surround WP crop protectant manufactured by Englehard Corporation (Iselin, NJ, USA). The compound is based on processed hydrous kaolin particles (1.0 ± 0.5 µm ø) with an incorporated synthetic hydrocarbon spreader-sticker enhancing the particles adhesion to the plant. The compound, hereafter referred to as kaolin, was applied at a rate of 5% in mineral water (50 gram/liter) as recommended by the manufacturer. Plain mineral water was used as control, and both were applied in all laboratory experiments using an airbrush spray gun (Model 350, Badger Air-Brush Co., Franklin Park, IL, USA) connected to an air compressor generating a cone spray pattern. The spray gun was fixed by means of an adjustable height laboratory arm at 33 cm over the table surface preventing from an excessive air flow over the spraying spot position. The spray gun bottle was always in contact with a magnetic stirrer held by the same arm in order to avoid kaolin particles sedimentation during the spraying process.
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 cm2) were sprayed likewise, and after a 4 h drying process at 25°C, weighed again using an electronic 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 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/cm2).
2.1. Laboratory rearing of C. carnea
The larvae used in all the experiments were obtained from C.carnea eggs 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 (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 maintained in controlled environment cabinet at 25 ± 1°C, 50 - 60% RH, and a photoperiod of 16:8 (L:D) h.
2.2. Acute mortality to larvae
C.carnea larvae were directly sprayed with the 5% kaolin suspension in order to 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 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 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 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), 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, 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 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 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 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 were checked daily for survival until adult emergence recording the developmental stage in which mortality occurred.
2.3. Acute mortality of eggs
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 adhesive green velvety paper (Sadipal Stationary Papers, Girona, Spain ), used as 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 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 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 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 was kept in a cabinet at culture conditions. After three days, E. kuehniella eggs 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 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 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 corresponded to a RCBD with up to 10 replications made over time.
2.4. Effect on ability to grasp leaf
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 cm2 square glass, forming a continuous leaf surface platform. Different surfaces were composed with both sides of 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 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 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 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 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 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. Two new leaf platforms (upper and lower surfaces) were composed per replication.
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 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 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 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 (Noldus Information Technology, Wageningen, The Netherlands). The Ethovision software allows to automatically determine the location of the individual in the arena calculating several movement parameters derived from changes in position of the 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) 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 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 from 10 to 15 trials of both treatments until a total number of 30 valid replicates per treatment (60 replicates).
2.6. Effect of a particle film surface on larval mobility and choice
In this experiment we tested both, whether kaolin film covered substrates interfered with larval mobility parameters and whether they showed a substrate preference between treated and control surfaces. As in previous experiments newly 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 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 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 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 experimental setup and procedures corresponded with those described in section 2.5. 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 (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 (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 replicates consisted of 10 trials conducted the same day with the same experimental arena, and therefore a new arena was sprayed every day.
2.7. Adults oviposition preference
C. carnea adults were placed in an ovipositing arena and given the choice 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 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 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 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 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 arena to lay eggs for a period of 48 h. Water was supplied by a moistened piece of 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 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 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.
2.8. Effect on adults abundance. Field case study
A field experiment was conducted to determine whether a particle film sprayed 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), 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 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 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 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 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 CDC Backpack Aspirator Model 1412, John W. Hock Co., Gainsville, FL, USA), 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 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 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 10.2 mm.
2.9. Statistical analysis
All the statistical tests were performed with the package SPSS statistics 18 for windows (SPSS Inc, Chicago, IL, USA). For the analysis of the larvae and eggs acute 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 = 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 2.6), movement parameters were log10 (x + 0.5) transformed for normalization and compared by means of a Student’s t-test (α = 0.05). Whenever the data distribution 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-Whitney U tests for individual comparisons. The α value was adjusted by means of the 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-parametric Mann-Whitney U tests (α = 0.10). In all cases untransformed means are presented.
The mean particle density (± SD) deposited on the Teflon slides after drying for 4 h was 290.7 ± 99.7 µg/cm2.
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, 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 interfere with C. carnea normal development from the third instar state.
3.2. Acute mortality to eggs and larval survival
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 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 the viable and non-viable eggs, indicating that the recently hatched larvae fed on E. kuehniella eggs supplied for this aim.
3.3. Effect on ability to grasp leaf
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 < 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 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 upper surface (Table 2).
3.4: Effect of a particle film covering C. carnea larvae on mobility and behavioural response
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. Despite this fact, the numerical variables derived from the tracks provided by the 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 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 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 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 significantly higher turning rate per unit time (t-test, t = 2.56, df =58.00, P = 0.001).
3.5. Effect of a particle film surface on larval mobility and choice
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 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. 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 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 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).
3.6. Adults oviposition preference
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 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 eggs on the kaolin treated semicircular zone than on the control zone (Kruskal-Wallis, F = 302.88, df = 4, P < 0.05).
3.7. Effect on adult abundance. Field case study
From a total of 256 samples, 111 adult chrysopids were captured belonging to 4 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 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 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, P = 0.084).