‘Attract and reward’: Combining a herbivore-induced plant volatile with floral resource supplementation – Multi-trophic level effects

Biological Control 64 (2013) 106–115

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‘Attract and reward’: Combining a herbivore-induced plant volatile with floral resource supplementation – Multi-trophic level effects G.U.S. Orre Gordon a,b,⇑, S.D. Wratten b, M. Jonsson b,c, M. Simpson d, R. Hale e a

Barbara Hardy Institute, University of South Australia, GPO Box 2471, Adelaide, South Australia 5001, Australia Bio-Protection Research Centre, P.O. Box 84, Lincoln University, Lincoln 7647, New Zealand c Department of Ecology, Swedish University of Agricultural Sciences, P.O. Box 7044, SE-750 07 Uppsala, Sweden d EH Graham Centre for Agricultural Innovation, Charles Sturt University, Faculty of Science, School of Agricultural & Wine Sciences, Leeds Parade, Orange, NSW 2800, Australia e Department of Ecology, P.O. Box 84, Lincoln University, Lincoln 7647, New Zealand b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

" Arthropods from two trophic levels

" "

"

"

were affected by elements of ‘attract and reward’. MeSA increased aphid parasitism rate. Different natural enemy species were attracted to either buckwheat (Fagopyrum esculentum) or methyl salicylate (MeSA). Fourth trophic level arthropod species may be attracted to buckwheat. Both MeSA and buckwheat may repel certain species from the third and fourth trophic level.

a r t i c l e

i n f o

Article history: Received 13 June 2012 Accepted 1 October 2012 Available online 16 October 2012 Keywords: Methyl salicylate Buckwheat Conservation biological control

a b s t r a c t Two field experiments were conducted to assess whether a concept termed ‘attract and reward’ (A&R) could enhance conservation biological control (CBC). In A&R, a synthetically-produced herbivore-induced plant volatile (HIPV) (‘attract’) is combined with a floral resource (‘reward’). It is anticipated that the two will work synergistically, attracting natural enemies into the crop (‘attract’) and maintaining them within it (‘reward’). The study was conducted in Canterbury, New Zealand and the system consisted of brassica crop, commonly occurring brassica herbivores, their natural enemies and higher order natural enemies. The HIPV deployed was methyl salicylate (MeSA) and the floral resource was buckwheat Fagopyrum esculentum. The first experiment assessed the abundance of arthropods from three trophic levels and the second evaluated herbivore abundance, parasitism and hyper-parasitism rates. No synergistic effect of ‘attract’ and ‘reward’ was observed in either experiment. Populations of three parasitoids, one hoverfly and one lacewing from the third trophic level and a fourth trophic level lacewing parasitoid increased significantly in treatments with buckwheat. One hoverfly species was significantly more abundant in treatments with MeSA, but less abundant in treatments with buckwheat. The effect of MeSA on

⇑ Corresponding author at: Barbara Hardy Institute, University of South Australia, GPO Box 2471, Adelaide, South Australia 5001, Australia. Fax: +61 8 8302 2252. E-mail addresses: Sofi[email protected] (G.U.S. Orre Gordon), [email protected] (S.D. Wratten), [email protected] (M. Jonsson), [email protected] (R. Hale). 1049-9644/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2012.10.003

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Diadegma semiclausum abundance depended on sex, with fewer males and more females being caught. Treatments with MeSA had significantly higher aphid parasitism rate. Combining MeSA and buckwheat could be beneficial because the two techniques increase the abundance of different natural enemies. Thus, these results indicate that A&R has potential as a CBC technique, as long as any unwanted side effects can be managed. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction The frequent disturbances linked with high-intensity agricultural practices and the modification of the habitat to an environment low in physical and biological resources required by natural enemies of pests often result in a decline in biological control (Chaplin-Kramer et al., 2011). Conservation biological control (CBC) can be used to mitigate those effects by modification of the environment and of existing pesticide practices (Eilenberg et al., 2001; Landis et al., 2000). Habitat manipulation techniques are used within CBC to enhance trophic cascades. These occur when changes in a predatory species’ abundance alters the distribution and abundance of a plant species (Schmitz et al., 2000). One type of habitat manipulation is the provision of floral resources by non-crop plants grown within or around the crop. These provide omnivorous natural enemies with alternative food sources, such as nectar and/or pollen which may enable them to remain in an area with temporarily low prey/host densities (Landis et al., 2000; Olson et al., 2005). Dietary floral supplementation can also increase components of the natural enemies’ ‘ecological fitness’ such as longevity and fecundity (Berndt and Wratten, 2005; Landis et al., 2000; Lavandero et al., 2005). However, the results from field trials with floral resource subsidies have been mixed. For example, Jonsson et al. (2010) found that of the 11 studies that were published between 1998 and 2007, on the effect of floral subsidies on natural enemies of invasive pests in different agricultural systems, seven demonstrated an increase in predation/parasitism rates and only one showed a decrease in pest populations and crop damage. One way to potentially increase the pest-reducing efficiency of natural enemies is by combining provision of floral resources with deployment of substances that attract more enemies into the crop, a new concept termed ‘attract and reward’ (A&R) (Jonsson et al., 2008; Khan et al., 2008; Simpson et al., 2011a). The ‘attract’ concept is based on the use of synthetically produced herbivore-induced plant volatiles (HIPVs). These are a form of induced plant defense that may function both through ‘top-down’, by attracting natural enemies (Dicke and Bruin, 2001) and ‘bottom-up’ mechanisms, repelling the herbivore (Dicke et al., 1990). Production of HIPVs is induced by herbivore feeding damage (Geervliet et al., 1997) or by egg deposition on the plant (Hilker and Meiners, 2002). HIPVs can function as attractants of natural enemies to herbivore-affected plants and as a signal to other plants to produce their own herbivore defenses (inter-plant communication) (Dicke and Bruin, 2001). They can also function as a ‘primer’, signaling to surrounding undamaged plants of an impending herbivore attack without initiating the undamaged plants to produce a full defense response (Engelberth et al., 2004). HIPVs can be synthetically produced and the deployment of some of these substances within agricultural systems has been shown to increase natural enemy numbers near the crop (James and Grasswitz, 2005; Orre et al., 2010; Thaler, 1999). Both HIPVs and floral resources can affect arthropods from second (Dicke and Minkenberg, 1991; Lavandero et al., 2006) and fourth trophic levels (Araj et al., 2008; Jonsson et al., 2009; Orre et al., 2010). Attraction of arthropods from the fourth trophic level may cause an un-wanted trophic cascade, which results in lower

abundance of the lower level natural enemy (third trophic level) and a higher abundance of the mid-level consumers (herbivorous pest) causing a reduction in the abundance of basal producers (crop plants) (Carpenter and Kitchell, 1993). As both ‘attract’ and ‘reward’ may affect arthropods from the untargeted second and fourth trophic levels, the change in abundance of arthropods from these, as well as from the targeted third trophic level needs to be evaluated. Any consequences these changes may have on population and community structure need to be assessed before the A&R-concept can be considered as a potential habitat manipulation strategy within crop pest management. The aim of this work was to examine the extent to which ‘attract and reward’ improves biological control in a brassica crop. The ‘attract’ component was the HIPV methyl salicylate (MeSA) and the ‘reward’ was buckwheat Fagopyrum esculentum (BW). An initial experiment evaluated the effect of ‘attract and reward’ on the abundance of a range of arthropods from three trophic levels, focusing on brassica pests, predators and parasitoids of these pests as well as parasitoids attacking the pests’ predators and parasitoids. A second experiment evaluated any effects these changes may have had on biological control efficacy.

2. Methods 2.1.1. Study system The study system was kale Brassica oleracea L. (Brassicaceae) cv. Sovereign in the first experiment and a mixture of kale and swedes Brassica napus L. (Brassicaceae) in the second as well as the most common herbivores and their associated natural enemies within the crop. Methyl salicylate (MeSA) was used for the ‘attract’ part of this work. MeSA is naturally produced by brassicas in response to herbivore damage (Geervliet et al., 1997; van Poecke et al., 2001) and MeSA produced by herbivore-damaged Brussels sprouts is perceived by parasitoids with hosts on brassicas (Steinberg et al., 1992). Previous experiments in turnip Brassica rapa L. (Brassicaceae) have shown that synthetically produced MeSA can increase the abundance of Diadegma semiclausum Hellén (Hymenoptera: Ichneumonidae) (Orre et al., 2010) a parasitoid of the diamondback moth (DBM) Plutella xylostella L. (Lepidoptera: Plutellidae) one of the most common herbivores reaching pest status in brassicas (Cameron and Walker, 2000; Kok, 2004). Synthetically produced MeSA also increases the abundance of predators in soybean Glycine max L. Merr. (Fabaceae) (Mallanger et al., 2011; Zhu and Park, 2005), cotton Gossypium spp. (Malvaceae) (Yu et al., 2008), apples Malus domestica Borkh. (Rosaceae) (Jones et al., 2011), cherries Prunus spp. (Rosaceae) (Tóth et al., 2009), cranberries Vaccinium spp. (Ericaceae) (Rodriguez-Saona et al., 2011) and strawberries Fragaria spp. (Rosaceae) (Lee, 2010), and predators and parasitoids in hops Humulus spp. (Cannabaceae) and grapes Vitis spp. (Vitaceae) (James and Price, 2004). The flowering plant used for the ‘reward’ part was buckwheat (BW) Fagopyrum esculentum Moench (Polygonaceae) cv. Katowase. Supplementation with BW in the field can contribute to the pest management of DBM (Lavandero et al., 2005; Lee and Heimpel, 2005).

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The study system for the effect of MeSA and/or buckwheat on parasitism rates focussed on cabbage aphids Brevicoryne brassicae (L.) and green peach aphids Myzus persicae (Sulzer) (Hemiptera: Aphididae). Aphids are common pests in brassicas that reduce yield directly via phloem feeding and indirectly, via virus transmission (Ferguson and Blake, 1985). 2.1.2. Study sites and experimental design The study was carried out over two seasons at a different site each season in commercially grown brassica crops. The experiments ran from when the brassica plants were at the cotyledon stage to when they were either sprayed against aphids (first experiment) or the level of aphid parasitism was close to 100% (second experiment). For both experiments a full two factorial randomized block design was used. Each block included one treatment with a slowrelease sachet of MeSA (MeSA) (P-240-lure, ChemTica International, Zeta Industrial Park, La Valencia, Heredia, Costa Rica), one with a 3  3 m flowering buckwheat plot (BW), one with a slow release sachet of MeSA combined with a 3  3 m plot of flowering buckwheat (MeSA + BW) and a control (C) consisting of the crop alone. In buckwheat treatments, buckwheat seeds were sown in 3  3 m squares in the center of each plot within a week after the drilling of the brassica crop. A 30 cm space was left between the lines of buckwheat and new seeds were sown in these to ensure continuous presence of flowering plants during the experimental period (details below). In each replicate of the HIPV treatments, one MeSA sachet was attached 1 m above ground to a string between the wooden stakes placed in the center of the brassica plot (MeSA and C treatments) or the buckwheat plot (BW and MeSA + BW treatments). According to the manufacturers, the volatile would last ‘a season’ but to ensure that the chemical did not run out it was replaced during the experimental period (details below). For both experiments the sampling was done from when the first batch of BW was in bloom. 2.1.2.1. First experiment In the first experiment the effect of the three treatments on the abundance of arthropods from three trophic levels in kale Brassica oleracea L. (Brassicaceae) cv Sovereign was tested. The study was carried out from January 30 to March 12, 2008 at Lincoln University Research Farm, Silverwood, Hororata, Canterbury, New Zealand. The experiment was set up across three adjacent kale fields with four blocks in field 1, three in field 2 and two in field 3. All plots were 3  3 m and each block was located at least 50 m from the field margin. The center of each treatment and control plot was separated from each other by 45 m (east–west) and 55 m (north– south). Aerial brassica herbivores and their natural enemies were sampled at each plot using one yellow sticky trap (24  20 cm) (Trappit, Agrisense-BCS-Ltd., UK, sourced from Fruitfed Supplies Ltd, New Zealand) per plot. Each trap was located in the center of the plot and attached 10 cm above the mean height of the kale plants between two wooden stakes and moved upwards as the plants grew, to maintain the 10 cm distance above the plants. In each replicate of the HIPV treatment, the same two wooden stakes that were used to suspend the MeSA sachet were used to support the traps. This method was used to allow the trap to be located just below the MeSA sachet and 10 cm above the top of the plants. The traps were replaced on February 6, 13, 20 and 27, 2008. The MeSAsachets were replaced twice, once on February 13 and once February 27. The first batch of buckwheat seeds was sown on December 19, 2007. Further batches were sown on January 9 and 30, 2008. The number of individuals of the most common brassica herbivores, natural enemies and hyperparasitoids were counted on each trap (Table 1.). In addition the number of cabbage aphids, green

peach aphids, larvae of the diamondback moth (DBM), cabbage white butterfly (CWB) and leaf mines of Scaptomyza flava were counted on all the leaves of 20 randomly chosen kale plants in a radius of 1.5 m from the trap on March 11, 2008. 2.1.2.2. Second experiment In the second experiment the effect of the same treatments as in the first experiment on aphid density, aphid parasitism and hyperparasitism rates was studied in a field with a mixture of kale Brassica oleracea L. and swedes B. napus. L. (Brassicaceae). The study was carried out from December 5, 2008 to March 31, 2009 in a 170  220 m brassica field near Selwyn Huts, Canterbury, New Zealand. The experiment comprised eight blocks with 3  3 m plots. The centers of plots were separated by 34 m (east– west) and 24 m (north–south). The number of cabbage aphids, green peach aphids and aphid mummies (parasitized aphids) were counted in situ on all the leaves of 12 plants per plot in four directions 1.5 m from the plot center on, January 9, February 25 and March 23, 2009. Aphid mummies were collected on February 26, 2009 to estimate aphid hyper-parasitism rates. A total of 100 mummies per plot were collected within a 0.5 m wide strip 1.5 m from the center of the plot on a randomly chosen plant. Only one colony (aphids clustering together on a leaf) per plant was collected. The leaves with the colony were removed in the field and taken to the laboratory. A maximum of five randomly chosen mummies per colony was then removed and stored in 0.6 ml vials until parasitoid emergence. The emerged parasitoids were identified, counted and the proportion of hyper parasitized aphids was determined. The MeSA-sachets were replaced on January 6 and 22, February 5 and 19 and on March 5 and 19, 2009. The first buckwheat seeds were sown on December 5, 2008. More seeds were sown on December 12, 2008, January 6 and February 24, 2009. 2.1.3. Analysis 2.1.3.1. First experiment The number of D. semiclausum, Diadromus collaris, Cotesia spp., Melanostoma fasciatum, M. tasmaniae and A. zealandica captured on the sticky traps fitted a negative binomial distribution. Consequently, these data were analyzed using a generalized linear mixed model with a logarithm-link. A fixed model was used for the MeSA-treatment, BW-treatment, sex, sampling dates and all the interactions between these and a random model was used for block within sampling dates to account for repeated measures (Glantz and Slinker, 2001). Sex was excluded from the fixed model for Cotesia spp., M. tasmaniae and A. zealandica. For the first two species sex could not be determined for insects on the traps and for A. zealandica too few individuals were caught to include sex in the analysis. Only 7 D. collaris were caught during the last two sampling periods, so to be able to include sex as a variable in the analysis of this species these two sampling periods were excluded. The level of significance was Bonferroni-adjusted to p 6 0.0083 = , p 6 0.0008 =  and p 6 0.0002 =  due to the high number of species analyzed. The numbers of brassica herbivores and damage on the sampled crop plants on March 11, 2008 were too low to be analyzed. 2.1.3.2. Second experiment The effect of the treatments on aphid density and proportion parasitized aphids was analyzed for the data from the herbivore count on February 25, 2009. Block 8, the MeSA + BW treatment in block 1 and the BW treatment in block 4 were excluded from all analysis as treatments with buckwheat did not have flowering buckwheat. The number of aphids was too low on the first herbivore count on January 9 to be analyzed. On the third count, on March 23 the parasitism rate was extremely high with a mean of

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Table 1 Herbivores, natural enemies and higher order natural enemies counted on each trap. Species on the same line indicates host–natural enemy relationships and species in braces indicates multiple host/natural enemy relationships. Herbivore 2nd trophic level Cabbage white butterfly Pieris rapae L. (Lepidoptera: Pieridae) Diamondback moth Plutella xylostella L. (Lepidoptera: Plutellidae)

Natural enemy 3rd trophic level

Higher order natural enemy 4th trophic level

Parasitoid: Cotesia spp. (Hymenoptera: Braconidae)

Hyperparasitoid: Baryscapus galactopus Ratzeburg (Eulophidae: Tetrastichinae)

Diadromus collaris Gravenhorst (Hymenoptera: Ichneumonidae)

Trichomalopsis spp. Hymenoptera: Pteromalidae)

Diadegma semiclausum Hellen (Hymenoptera: Ichneumonidae) Leafmining fly Scaptomyza flava Fallen (Diptera: Drosophilidae)’

Asobara persimilis Papp (Hymenoptera: Braconidae) Proacris spp. (Hymenoptera: Eulophidae) Generalist predator: Brown lacewing Micromus tasmaniae Walker (Neuroptera: Hemerobiidae) Eleven spotted ladybird Coccinella undecimpunctata L. (Coleoptera: Coccinellidae) Seven spotted ladybird Coccinella septempunctata L. (Coleoptera: Coccinellidae) Hoverfly Melangyna novaezealandiae Macquart (Diptera: Syrphidae) Hoverfly Melanostoma fasciatum Macquart (Diptera: Syrphidae)

74% (SE ± 2%) of all the plants having 100% parasitism. Consequently, the data were not analyzed statistically. For aphid abundance the sum of aphids and aphid mummies per plant was used. To analyze the effects on aphid density an unbalanced two-way ANOVA was conducted on mean number of aphids per plant for each plot. To satisfy the assumption of equal variance and normal distribution for the ANOVA model, the analyses were conducted on square-root transformed data (Zar, 1999). Proportion parasitized aphids was calculated as the total number of aphid mummies divided by the total number of live aphids and mummies per plot. A generalized linear model with a binomial distribution and a logit-link function was conducted to analyze mean proportion of aphids parasitized per plot. There was evidence of over-dispersion of the data so the program estimated the dispersal parameter (Glantz and Slinker, 2001). Hyperparasitism rates were calculated based on proportion of hyperparasitized mummies of the 100 mummies collected per plot. An unbalanced two-way ANOVA was conducted on mean proportion of aphids hyperparasitized per plot. To satisfy the assumption of equal variance and normal distribution for the ANOVA model, all analyses p were conducted on arcsine ( (x)) transformed data as this best fitted a normal distribution (Zar, 1999). The data from all experiments were analyzed using GenStat 12th edition. 3. Results 3.1.1. First experiment Of the third trophic level natural enemies expected on the traps, D. semiclausum, D. collaris, Cotesia spp., M. fasciatum and M. tasmaniae were caught in high enough numbers for the data to be analyzed (Table 2). A. zealandica was the only fourth trophic-level natural enemy found. Treatments with buckwheat had a significantly higher abundance of the two DBM parasitoids D. semiclausum (Table 2; Fig. 1A), D. collaris (Table 2; Fig. 1B), the CWB parasitoid Cotesia spp. (Table 2; Fig. 1C), M. tasmaniae (Table 2; Fig. 1D) and its parasitoid A. zealandica (Table 2; Fig. 1E). However, for D. semiclausum the positive effect primarily occurred early in the season (Table 2; Fig. 1A). The abundance of A. zealandica was significantly lower in treatments with MeSA compared to ones without (Table 2). The hoverfly M. fasciatum was significantly more abundant in treatments with MeSA (Table 2) but significantly less

Anacharis zealandica Ashmead (Hymenoptera: Figitidae)

Diplazon laetatorius Fabricius (Hymenoptera: Ichneumonidae)

abundant in treatments with buckwheat (Table 2; Fig. 1F), and there was a near-significant interaction between MeSA and buckwheat, suggesting that the latter may to some extent inhibit the attraction to MeSA. The effect of MeSA on D. semiclausum depended on sex (Table 2), with fewer males and more females being caught in the MeSA treatments (Fig. 2). The brassica herbivore abundance on March 11, 2008 was too low to be statistically analyzed. 3.1.2. Second experiment There was no significant difference in the aphid densities for the different treatments (Fig. 3A). Significantly more aphids were parasitized in treatments with MeSA compared to treatments with no MeSA (p = 0.026, df = 1) (Fig. 3B). The level of hyperparasitism was independent of treatments (Fig. 3C). 4. Discussion Arthropods from two trophic levels were affected by elements of ‘attract and reward’; however, for none of the species was there a synergistic effect between the two techniques. We found that MeSA and buckwheat attracted different species of natural enemies of pests and that MeSA can increase parasitism rates. However, we also found that fourth trophic level species may be attracted to buckwheat and that both techniques may repel certain species from the third and fourth trophic level. 4.1.1. Effects of ‘attract’ In the first experiment the hoverfly M. fasciatum was more abundant in treatments with MeSA than in those without. The attraction of hoverflies to synthetically produced MeSA is in accordance with James’ (2005) findings, where synthetically produced MeSA deployed in a hop yard attracted species of hoverflies and those of Orre et al. (2010), where M. novaezealandiae was attracted to MeSA in a brassica crop. Recently, Rodriguez-Saona et al. (2011) showed similar attraction of hoverflies to MeSA-baited traps in cranberries. However, the nearly significant interaction between MeSA and buckwheat suggests that buckwheat may to some extent inhibit the attraction of hoverflies to MeSA (Table 2; Fig. 1F).

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Table 2 Generalized linear mixed model (GLMM) analysis for the effect of ‘attract’ and ‘reward’ on Diadegma semiclausum, Diadromus collaris, Cotesia spp. Melansotoma fasciatum, Micromus tasmaniae and Anacharis zealandica. MeSA = the effect of treatments with MeSA + yellow sticky trap. BW = the effect of treatments with buckwheat + yellow sticky trap. The significance levels have been Bonferroni-adjusted to p < 0.0083 = , p 6 0.0008 =  and p 6 0.0002 = . Species

Factor

Wald statistic

df

p-value

Level of significance

Diadegma semiclausum (third trophic level)

BW MeSA Sex Time BW  MeSA BW  Sex MeSA  Sex BW  Time MeSA  Time Sex  Time BW  MeSA  Sex BW  MeSA  Time BW  Sex  Time MeSA  Sex  Time BW  MeSA  Sex  Time BW MeSA Sex Time BW  MeSA BW  Sex MeSA  Sex BW  Time MeSA  Time Sex  Time BW  MeSA  Sex BW  MeSA  Time BW  Sex  Time MeSA  Sex  Time BW  MeSA  Sex  Time BW MeSA Time BW x MeSA BW  Time MeSA  Time BW  MeSA  Time BW MeSA Sex Time BW  MeSA BW  Sex MeSA  Sex BW  Time MeSA  Time Sex  Time BW  MeSA  Sex BW  MeSA  Time BW  Sex  Time MeSA  Sex  Time BW  MeSA  Sex  Time BW MeSA Time BW  MeSA BW  Time MeSA  Time BW  MeSA  Time BW MeSA Time BW  MeSA BW  Time MeSA  Time BW  MeSA  Time

9.52 5.06 26.87 32.45 2.83 1.15 8.51 29.13 2.07 33.99 2.39 0.95 0.52 1.41 4.33 81.3 0.19 28.37 21.66 3.75 1.26 4.07 3.75 1.12 17.93 2.46 9.78 0.24 1.27 0 116.36 0.01 46.72 2 6.17 12.91 12.5 47.65 30.49 0.39 17.8 6.13 1.56 0.15 2.29 2.63 12.6 5.66 4.59 1.27 9.34 0.23 23.25 4.37 93.14 5.39 1.91 4.86 4.08 19.96 16.4 3.3 2 9.36 2.28 0.07

1 1 1 4 1 1 1 4 4 4 1 4 4 4 4 1 1 1 2 1 1 1 2 2 2 1 2 2 2 2 1 1 4 1 4 4 4 1 1 1 4 1 1 1 4 4 4 1 4 4 4 4 1 1 4 1 4 4 4 1 1 4 1 4 4 4

0.002 0.025 <0.001 <0.001 0.092 0.283 0.004 <0.001 0.723 <0.001 0.122 0.041 0.972 0.843 0.363 <0.001 0.664 <0.001 0.005 0.054 0.264 0.045 0.156 0.572 <0.001 0.118 0.009 0.885 0.531 1.000 <0.001 0.926 <0.001 0.16 0.195 0.015 0.02 <0.001 <0.001 0.535 <0.001 0.013 0.212 0.696 0.682 0.622 0.013 0.017 0.332 0.866 0.053 0.994 <0.001 0.039 <0.001 0.022 0.752 0.308 0.400 <0.001 <0.001 0.521 0.160 0.059 0.685 0.999

⁄ NS ⁄ ⁄ NS NS ⁄ ⁄ NS ⁄ NS NS NS NS NS ⁄ NS ⁄ ⁄ NS NS NS NS NS ⁄ NS NS NS NS NS ⁄ NS ⁄ NS NS NS NS ⁄ ⁄ NS ⁄ NS NS NS NS NS NS NS NS NS NS NS ⁄ NS ⁄ NS NS NS NS ⁄ ⁄ NS NS NS NS NS

Diadromus collaris (third trophic level)

Cotesia spp. (third trophic level)

Melanostoma fasciatum (third trophic level)

Micromus tasmaniae (third trophic level)

Anacharis zealandica (fourth trophic level)

D. semiclausum females were more abundant in treatments with MeSA while males were less common in such treatments. Previous studies have shown that males and females of natural enemies may respond differently to the same HIPV (Kaplan, 2012). Similarly to the results presented here, D. semiclausum females were more

attracted than males to synthetically produced MeSA in a study by Orre et al. (2010). However, there is also evidence of D. semiclausum being repelled by MeSA (Snoeren et al., 2010). Snoeren et al. (2010) used naïve female D. semiclausum wasps that had neither been exposed to plant material, nor obtained an oviposition experience.

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Fig. 1. The mean number (±SE) of individuals per trap for each species during each sampling period.

Parasitoids can learn to respond to individual compounds following a learning experience with an odor mixture (Meiners et al., 2003). An innate response of D. semiclausum to avoid MeSA-emitting plants could potentially turn into attraction after a learnt experience in the presence of host (Snoeren et al., 2010). However, to be able to interpret what the consequences are of the increase/decrease on CBC,

information on the feeding status and mating status of both sexes are needed. Contrary to the results from Orre et al. (2010), the number of the lacewing parasitoid A. zealandica were significantly lower in treatments with MeSA compared to ones without. A possible reason for this could be differences in MeSA concentrations between

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Fig. 1. (continued)

the studies. The release rate of the MeSA from the slow release sachets is not known. If the release rate was too high it may lead to repellence (Kaplan, 2012). Previous studies have shown that the attraction/repellence effect of MeSA to hymenopterans is concentration dependent (Simpson et al., 2011b; Snoeren et al., 2010). However, it is unclear why the release rate would have been higher in the present study compared to that of Orre et al. (2010). In the second experiment, aphid parasitism rates were significantly higher in treatments with MeSA than in those with none.

This may appear surprising as we in the first experiment did not find any evidence of attraction of parasitoids to MeSA. However, aphid populations were very low in the first experiment and no aphid parasitoids were caught on the sticky traps. The parasitoids that were more abundant in the treatments with buckwheat in the first experiment were parasitoids of chewing insects, while MeSA production has been shown to be induced in plants particularly after phloem sucking herbivores, such as aphids (Stout et al., 2006). Behavioral work on aphid parasitoids has demonstrated

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(A)

(B)

Fig. 2. The mean number (±SE) of females and males of Diadegma semiclausum caught per trap for treatments with MeSA, and treatments without MeSA over the entire sampling period.

the role of plant volatiles in their foraging (Guerrieri et al., 1997; Wickremasinghe and van Emden, 1992) and the aphid parasitoid Aphidius ervi uses HIPVs in the search for its host Acyrthosiphon pisum Harris (Homoptera: Aphididae) under laboratory conditions (Du et al., 1996; Guerrieri et al., 1997). Recently Mallanger et al. (2011) showed increased predation of soybean aphids in response to MeSA-lures. However, results in the present work are the first to show increased parasitism rates in the field in response to synthetically produced MeSA. Overall aphid parasitism was low (Fig. 3B) and aphid hyperparasitism rates were high (Fig. 3C) in the second experiment. The high level of hyperparasitism could explain the overall low aphid parasitism as the majority of aphid parasitoids were parasitized, which would have haltered the aphid parasitoid population growth. The high level of hyperparasitism could have caused a lack of hosts and hence any effects of the treatments on hyperparasitism could have been over-ridden by host limitation. 4.1.2. Effects of ‘reward’ Third trophic level natural enemies that were significantly more abundant in buckwheat treatments in the first experiment were D. semiclausum, D. collaris, Cotesia spp. and the brown lacewing. Both visual aspects and the floral odors of the flowers utilized within CBC can be attractive to natural enemies of pests (Orre et al., 2007) and flowering plants have also shown to ‘attract’ parasitic hymenopterans (Sivinski et al., 2011). Parasitoids have previously been shown to benefit from buckwheat (Lavandero et al., 2005; Lee and Heimpel, 2005; Stephens et al., 1998; Tylianakis et al., 2007). Both Cotesia rubecula (Lee and Heimpel, 2005) and D. semiclausum (Lavandero et al., 2006) have in field studies shown an increased parasitism rate following access to buckwheat. However, in our study, the positive effect of buckwheat on D. semiclausum was not consistent over time, with the effect disappearing towards the end of the experiment. This may be of minor importance as enhanced biological control is likely to have greater benefits early in the season. Under laboratory conditions, buckwheat has been shown to increase the longevity (Robinson et al., 2008) and the

(C)

Fig. 3. (A–C) The mean number (±SE) of aphids per plant (A) and the mean proportion of parasitized (B) and hyperparasitized aphids (C) per plot for the MeSA, buckwheat, MeSA + buckwheat-treatments and the control. The data have been back-transformed from square root-transformation. MeSA = MeSA-only treatment, BW = buckwheat only treatment, MeSA + BW = combined treatments with buckwheat and MeSA.

number of eggs laid by the brown lacewing (Jonsson et al., 2009) when aphid densities were low. Consequently, buckwheat can help lacewings survive periods with low abundance of prey and it can help them maintain a high rate of oviposition as long as some prey are available (Jonsson et al., 2009). The abundance of aphids was low in this field study based on the data from March 11, 2008. Therefore, the brown lacewing’s increased abundance in response to buckwheat could be due to lack of aphids, so the lacewing needed the buckwheat for survival. As mentioned above, the brown lacewing parasitoid A. zealandica, was more abundant in treatments with buckwheat than in ones with none. The parasitoid feeds on floral nectar of buckwheat and under laboratory conditions, both males and females with access to buckwheat lived 7–8 times longer than ones with water only (Jonsson et al., 2009). Consequently, the reason for the parasitoid’s preference for buckwheat could be the presence of both host and adult food resources within these treatments. M. fasciatum was less abundant in treatments with buckwheat. Hoverflies utilize buckwheat flowers as a food resource (Laubertie, 2007). The lower number of hoverflies caught on the traps in the buckwheat treatments was therefore surprising. The result could be due to the hoverflies looking for prey not food resources such as the pollen and/or nectar provided by the buckwheat. Hoverflies lay their eggs close to aphid colonies (Tompkins, 2010). The crop

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itself had aphids on it while the buckwheat had close to no aphids (personal observation) and therefore buckwheat was probably the least preferred oviposition site and consequently might have received the lowest number of hoverflies. 4.1.3. Prospects for biological control using ‘attract and reward’ This work shows that both ‘attract’ and ‘reward’ can increase the abundance of natural enemies and parasitism rates of aphids. However, in none of the cases did the combined deployment of buckwheat and MeSA work synergistically on individual natural enemies rather, the natural enemies ‘preferred’ either buckwheat or MeSA. Overall A&R may work as a habitat manipulation tool as the two components complement each other by increasing the abundance of different species and guilds of natural enemies. However, our results also suggest that buckwheat in some cases may inhibit attraction to MeSA when the two components are deployed together. To avoid this, it may be best to deploy ‘attract and reward’ with some spatial separation between each other. Buckwheat also increased the abundance of a fourth trophiclevel antagonist in this study and Orre et al. (2010) showed that the abundance a leafmining pest, S. flava (Fallén) (Diptera: Drosophilidae) can be increased by MeSA. Consequently, the potential for buckwheat and HIPVs to be deployed together in crop protection will strongly depend on the ability to manage compromising factors such as the unintended attraction of additional herbivores (Orre et al., 2010; Turlings and Ton, 2006) or higher order parasitoids (Baggen et al., 1999; Jonsson et al., 2009; Lavandero et al., 2006). One way of doing this is to search for a combination of HIPVs and floral resources that selectively enhances species from the third trophic level, but not species form the second and fourth. Several studies have reported that the presence of selective food plants (Baggen et al., 1999; Begum et al., 2006; Lavandero et al., 2006), and as different HIPVs are known to attract different natural enemy taxa (Simpson et al., 2011a) there are also prospects for finding selective HIPVs. However, even if A&R is not selectively benefitting natural enemies of pests, the approach may be useful for pest management as long as any benefits to the un-targeted trophic levels is compensated for by an even higher increase in abundance and/or ‘ecological fitness’ of the natural enemy. This study presents data from only one season. Between-year variation by the same species in the same crop in response to MeSA has been observed (Rodriguez-Saona et al., 2011). Further studies on the effect of A&R both on population dynamics and on, when and why annual variations occur is needed before firm conclusions can be drawn whether A&R can be considered as a tool within pest management. Acknowledgments We thank Bruce Carmichael for allowing us to use his brassica field. We also thank Richard Sedcole for statistical support and Andrew Pugh, Anna-Marie Barnes and Hamish Patrick for technical assistance. This work was funded by the Bio-Protection Research Centre, Lincoln University, New Zealand and a New Zealand International Doctoral Research Scholarship. References Araj, S.A., Wratten, S., Lister, A.J., Buckley, H.L., 2008. Floral diversity, parasitoids and hyperparasitoids – a laboratory approach. Basic and Applied Ecology 9, 588– 597. Baggen, L.R., Gurr, G.M., Meats, A., 1999. Flowers in tri-trophic systems: mechanisms allowing selective exploitation by insect natural enemies for conservation biological control. Entomologia Experimentalis et Applicata 91, 155–161.

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