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Home , Brevicoryne brassicae, Mustard oil bomb, Myrosinase, Sinigrin

Journal of Applied Ecology 2011, 48, 880–887 doi: 10.1111/j.1365-2664.2011.01990.x Chemically mediated tritrophic interactions: opposing effects of on a specialist and its predators

Rebecca Chaplin-Kramer1*, Daniel J. Kliebenstein2, Andrea Chiem3, Elizabeth Morrill1, Nicholas J. Mills1 and Claire Kremen1

1Department of Environmental Science Policy & Management, University of California, Berkeley, 130 Mulford Hall #3114, Berkeley, CA 94720, USA; 2Department of Plant Sciences, University of California, Davis, One Shields Ave., Davis, CA 95616, USA; and 3Department of Integrative Biology, University of California, Berkeley, 3060 Valley Life Sciences Bldg #3140, Berkeley, CA 94720, USA

Summary 1. The occurrence of enemy-free space presents a challenge to the top-down control of agricultural pests by natural enemies, making bottom-up factors such as phytochemistry and plant distributions important considerations for successful pest management. Specialist like the brassicae co-opt the defence system of plants in the by sequestering glucosinolates to utilize in their own defence. The wild nigra,analter- nate host for cabbage , contains more glucosinolates than cultivated Brassica oleracea,and these co-occur in agricultural landscapes. We examined trade-offs between aphid performance and predator impact on these two host plants to test for chemically mediated enemy-free space. 2. content of B. oleracea and mustard B. nigra was measured in plant mat- ter and in cabbage aphids feeding on each food source. Aphid development, aphid fecundity, preda- tion and predator mortality, and field densities of aphids and their natural enemies were also tested for each food source. 3. Cabbage aphids growing on high glucosinolate plants like B. nigra contained more glucosino- lates than aphids on lower glucosinolate B. oleracea. Aphids on B. nigra had shorter generation times and greater daily fecundity, while their predators (Diptera: Syrphidae) had lower feeding and higher mortality rates. Lower syrphid densities were found on B. nigra than on B. oleracea in the field. 4. Synthesis and applications. This study presents physiological and field evidence to suggest that weedy B. nigra may provide enemy-free space from an important predator. Habitat near crops should be examined for its potential to provide enemy-free space and compromise otherwise effec- tive biological control. The issue of pest control must be considered from the bottom up as well as the top down. Key-words: biological control, Brassicaceae, , chemical defences, enemy-free space, parasitism, pest management, physiological trade-off, syrphid

& Lawton 1984). Although the best examples of enemy-free Introduction space have been documented in natural systems and Plants can provide an ecological refuge for herbivores by through experimental host-plant shifts (Denno, Larsson & allowing them to chemically or physically escape their natu- Olmstead 1990; Gratton & Welter 1999; Murphy 2004), the ral enemies, sometimes called an ‘enemy-free space’ (Jeffries concept also has useful application in the context of biologi- cal control for agricultural systems. Our understanding of biological control is generally focused on the top-down *Correspondence author. California Institute for Energy & Envi- ronment, University of California, 2087 Addison Street – 2nd pressures of predators on their prey, but the occurrence Floor, Berkeley, CA 94704, USA. E-mail: [email protected] of enemy-free space reveals important subtleties in the

2011 The Authors. Journal of Applied Ecology 2011 British Ecological Society Chemically mediated tritrophic interactions 881 relationship between these trophic groups. Successful imple- produces volatile toxic just as it does in plants mentation of top-down control by natural enemies may (Francis et al. 2001; Francis, Lognay & Haubruge 2004). hinge on incorporating bottom-up factors such as the distri- The degree to which a plant with high glucosinolate content bution of plants providing refuge to pests. Identifying which like B. nigra serves as a viable predator refuge depends on the plants could provide enemy-free space for different crop trade-off between enemy-free space and herbivore perfor- pests could help guide management to reduce or eliminate mance. Cabbage aphids are completely dependent on their an important potential source of pests to crop fields. food source for the acquisitionofthemustardbomb.They Many wild plants contain higher levels of defence com- cannot synthesize their own glucosinolates (Kazana et al. pounds than their domesticated congeners (Baker 1972; Cole 2007) and therefore can only acquire defences through gluco- 1997a; Gols & Harvey 2009), and specialized herbivores can sinolate consumption (Francis, Haubruge & Gaspar 2000; often utilize those toxins to compile their own chemical arsenal Vanhaelen, Gaspar & Francis 2002; Olmez-Bayhan, Ulusoy & to escape enemies (Nishida 2002; Hopkins, van Dam & van Bayhan 2007; Pratt et al. 2008). Thus, differences in the con- Loon 2009). If this chemical refuge allows pests to escape their centration of glucosinolates within the aphids’ diet may influ- enemies more effectively than when feeding on crops, the ence their ability to deter predation. However, any benefit of occurrence of such plants around farmland could be support- predator refuge derived from glucosinolates may be offset by ing populations of pests that are then unregulated by their slower aphid development or reproduction, because of poten- enemy community. The presence of refugia could allow pests tial energetic costs of sequestering these toxic compounds to build to greater levels than if such refuges were absent from (Cole 1997a). While not framing their study specifically in landscapes. The invasive mustard Brassica nigra (L.) Koch has terms of glucosinolate content, Ulusoy & Olmez-Bayhan much higher glucosinolate levels than the domesticated Bras- (2006) found that aphids grown on another wild mustard, sica oleracea (L.) (Rodman, Kruckeberg & Alshehbaz 1981; Sinapis arvensis (L.), had lower reproductive rates than those Mithen, Raybould & Giamoustairs 1995), and weedy patches reared on domesticated B. oleracea . Aphids on of B. nigra are commonly found in field margins or edges S. arvensis also had shorter generation times, however, result- around B. oleracea fields (cole crops, such as broccoli, cauli- ing in no net difference in intrinsic rate of increase for aphids flower, , cabbage). The co-occurrence of these two Brassica on wild mustard vs. cole crops in the absence of predation. species provides an excellent opportunity to test whether an However, these herbivore development studies have not been invasiveweedcanserveasenemy-freespaceforcroppests. conducted on the same plant species as the studies investigating Plants in the family Brassicaceae are a good study system for effects on their predators, and therefore,norealconclusions chemically mediated enemy-free space, because of their sophis- regarding the potential trade-offs of enemy-free space can be ticated two-part defence system involving a glucosinolate com- drawn from the current literature. pound and protein complex that has been It is possible that wild Brassica species could serve as sources described as a ‘mustard-oil bomb’ (Ratzka et al. 2002). When of pests to nearby crops if they provide enemy-free space with- plant tissue is damaged, glucosinolates come into contact with out compromising the pest’s own population growth. We myrosinase , which remove from the glucosin- simultaneously measured herbivore performance and preda- olates, leading to the formation of toxic products tion risk on two host-plant species to assess the potential for such as isothiocyanates. The cabbage aphid Brevicoryne brassi- chemically mediated trade-offs. Specifically, we quantified the cae (L.) is among a small group of that have found a glucosinolate content of two Brassica species (B. oleracea and strategy for exploiting Brassicaceae, a resource toxic to most B. nigra), measured the glucosinolate content in aphids reared herbivores. Generalist herbivores feeding on brassicaceous on each of the two plant species, assessed aphid development plants, including the lepidopterans Mamestra brassicae (L.), and fecundity while growing on these two food sources, and Spodoptera eridania (Cramer) and Trichoplusia ni (Huebner), evaluated syrphid development and predation of aphids raised tend to rely on direct metabolic detoxification of the glucosino- on each host plant. Subsequently, we addressed the overall lates, receiving little or no predator defence benefit (Li et al. effects of these trade-offs by estimating the abundance of 2000; Lambrix et al. 2001; van Leur et al. 2008). In contrast, aphids, syrphids, and aphid on the two Brassica specialist herbivores, such as the cabbage aphid, the sawfly species at a series of locations in the field. We tested the Athalia rosae (L.), and the harlequin bug Murgantia histrionica hypotheses that (i) high glucosinolate content places a physio- (Hahn), co-opt toxicity from glucosinolates through sequestra- logical burden on herbivores utilizing these compounds for tion, escaping many of their enemies as a result (Francis et al. their own defence, (ii) high glucosinolate content confers an 2001; Muller et al. 2001; Aliabadi, Renwick & Whitman advantage to herbivores in the form of reduced predation and 2002). What makes cabbage aphids relatively unusual among (iii) high glucosinolate content leads to lower densities of aphid these specialists is that they both sequester the glucosinolate to natural enemies on B. nigra in the field. Whether aphid densi- avoid its toxic effects and produce their own myrosinase ties themselves are lower or higher on B. nigra would then , arming themselves with their own mustard bomb, depend on the balance of the bottom-up and top-down forces such that they not only deter but actually harm their predators explored in the first two hypotheses. This research highlights (Bridges et al. 2002; Hopkins, van Dam & van Loon 2009). the impact of on pests and their predators, When the aphid body is damaged, the two compounds are with implications for the role a weed may play in inhibiting mixed and the hydrolysis of glucosinolate by the myrosinase effective biological control in agricultural systems.

2011 The Authors. Journal of Applied Ecology 2011 British Ecological Society, Journal of Applied Ecology, 48, 880–887 882 R. Chaplin-Kramer et al.

rity. The number of G nymphs produced per G aphid each day pro- Materials and methods 2 1 vided a measure of reproductive rate and total fecundity, as the G1

aphids were tracked until their death. The G2 nymphs were removed PLANTS AND INSECTS each day after counting to remove any artefact of crowding from the Broccoli B. oleracea var. italica cv. Gypsy (acquired from Growers effect of the cage.

Transplanting, Salinas, CA, USA) and black mustard B. nigra Forty G1 individuals were tracked for each treatment, but some (acquired from Reimer Seeds, Mount Holly, NC, USA) were grown aphids escaped from the clip cages during the experiment, when leaf in individual pots in potting soil with vermiculite at 18–24 C and 16- surface irregularity prevented a perfect seal. Individuals that escaped h daylength in a greenhouse. All plants were at least 25–30 cm tall before reproduction were excluded from the analysis. Individuals that before being used for the aphid colonies. Cabbage aphids B. brassicae escaped after reproduction were included in the development (time to were reared on these host plants in dense colonies (>100 individuals first reproduction) analysis, but excluded from the analyses on total per leaf) under the same greenhouse conditions for a period of fecundity. This approach yielded 59 G1 aphids (28 in the B. oleracea 3 months prior to the start of the study. treatment and 31 in the B. nigra treatment) for the development anal-

ysis and 32 G1 aphids (17 in the B. oleracea treatment and 15 in the B. nigra treatment) for the fecundity analysis. GLUCOSINOLATE PROFILES

Ten apterous aphids in their penultimate instar were selected from the IMPACTS OF BRASSICA NIGRA ON SYRPHIDS colonies on six different plants in each treatment (B. oleracea and B. nigra) and preserved in 500 lL of 90% methanol for glucosinolate Syrphid larvae are the most common predators of cabbage aphids analysis. Fresh leaf matter totalling 150–250 mg (equivalent to a feeding on B. oleracea in the region where these cabbage aphids were small portion of one leaf) was also collected from each of the 12 indi- obtained (Chaplin-Kramer 2010) and are thought to play an impor- vidual plants (six B. oleracea and six B. nigra) for analysis. tant role in their control for this crop (van Emden 1963; Nieto et al. Glucosinolates were measured via high-performance liquid 2006). Syrphid larvae were reared on aphids from the two different chromatography (HPLC) with diode-array detection (DAD) using Brassica food sources and observed over the course of their larval an Agilent 1100 system (Kliebenstein, Gershenzon & Mitchell-Olds development in a growth chamber at 18 C and a 16-h daylength. 2001). Glucosinolates in both plants and aphids were identified and Syrphid eggs were collected from the field on B. oleracea plants at the quantified in relation to previously purified standards (Reichelt et al. University of California Center for Sustainable Agriculture and Food 2002). All individual aliphatic glucosinolate values within a sample Systems, in Santa Cruz, CA. Brassica nigra was also searched for were summed and standardized to nmol per unit fresh weight for syrphid eggs at the same site, but no eggs were found on these plants. plant matter or per aphid to provide the total aliphatic glucosinolate The syrphid species found at this site included Allograpta obliqua content. (Say), Eupeodes americanus (Wiedemann), Eupeodes volucris (Orsten Sacken), Syrphus opinator (Orsten Sacken), Scaeva pyrastri (L.), Sphaerophoria sulphuripes (Thomson), Toxomerus occidentalis IMPACTS OF BRASSICA NIGRA ON APHIDS (Curran), and Platycheirus stegnus (Say). As species cannot be identi- Small cages were clipped directly onto leaf surfaces of potted B. olera- fied at the egg stage, it was not possible to ensure that an equal number cea or B. nigra plants (10 of each species) in the greenhouse, at 18– of each species was assigned to each treatment, but the distribution of 24 C and 16-h daylength. Each clip cage contained five adult aphids species was assumed to be random across the two treatments.

(denoted hereafter as the zero generation, or G0) originating from the Twenty syrphids were tracked in each treatment from hatching greenhouse colonies on their respective host plants. Each aphid until the died or pupated, and the emerging adults were saved colony had been living on the same host-plant species for several (at for later species identification. The eggs were kept in a moist Petri dish least 8) generations. Aphids from B. nigra colonies were used only on and checked several times per day for hatching. Immediately after experimental B. nigra plants and likewise for B. oleracea. The clip hatching, each syrphid larva was placed in a Petri dish on either a cages allowed individual aphids or offspring cohorts to be tracked on B. oleracea leaf or a B. nigra leaf, with aphids from colonies reared living plants rather than on detached leaves; this is important for sim- on the corresponding plants. Petri dishes with cut leaves were used in ulating true field conditions, because plants will often increase the this case because clip cages could not contain the first-instar syrphid amount of glucosinolates in the leaf when they are attacked (Cui et al. larvae. The senescence of the cut leaves in the Petri dishes may have 2002; Kim & Jander 2007). reduced the glucosinolate content of the food source that the aphids Daily observations were made of the clip cages. On the first day were feeding on immediately prior to the introduction of the syrphid that nymphs were observed on the leaf, all the G0 adults were larva. However, this period represented a very small fraction of the removed and the cohort of between three and six nymphs (G1) aphids’ lifetime of feeding, and any reduction in aphid glucosinolate remained in a cage together until their penultimate instar, at which levels should diminish the difference between treatments, providing a point each of the G1 aphids was moved to its own cage on a separate conservative estimate of the impact of prey food source on predation. leaf on the same plant and tracked individually. This experimental Only prereproductive aphids were selected as prey to ensure that design was necessary to reduce aphid escape from the cages. Solitary no new nymphs were born between feedings. When given a choice, nymphs will often search the plant for other aphids, but keeping a syrphids tend to consume aphids in proportion to their size (younger, cohort of nymphs together in the same cage increased the likelihood smaller syrphids preferentially attack younger, smaller aphids; that they would remain within the cage (R. Chaplin-Kramer, per- R. Chaplin-Kramer, personal observation). However, to standardize sonal observation). Once the G1 adults were in their own individual feedings across the experiment, aphids were selected in their penulti- cages, they continued to be checked on a daily basis for nymph pro- mate instar, the most easily identifiable. Each day, the number of duction. The number of days until the first nymph in the next (G2) aphids remaining was recorded, aphids were replenished to ensure a generation was produced was recorded for each G1 individual as a sufficient amount for each syrphid larva to reach satiation (ranging measure of their development time, from birth to reproductive matu- from 10 to 100 aphids, depending on the age of the syrphid), and the

2011 The Authors. Journal of Applied Ecology 2011 British Ecological Society, Journal of Applied Ecology, 48,880–887 Chemically mediated tritrophic interactions 883 leaf was replaced to maintain freshness. Daily consumption rate and Table 1. Glucosinolate content (in nmol per 10 aphids) of cabbage total lifetime consumption were used as measures of predation by aphids reared on Brassica nigra (black mustard) vs. Brassica oleracea syrphids on the two different aphid sources (fed on B. nigra or (broccoli). F-andP-values denote significance of anova tests B. oleracea). comparing the two host plants, on 1 and 10 degrees of freedom

Glucosinolate B. nigra B. oleracea F P FIELD OBSERVATIONS 3-methylsulfinylpropyl 0Æ34 0Æ49 1Æ14 0Æ316 To determine whether physiological effects detected in laboratory tri- 4-methylsulfinylbutyl 1Æ17 1Æ39 0Æ32 0Æ585 als have ecological consequences, insects were compared on the two Allyl () 16Æ48 0Æ00 11Æ63 0Æ007 different host-plant species in the field. Plant matter was collected 4-hydroxy-indol-3ylmethyl 0Æ55 0Æ22 19Æ44 0Æ001 from B. nigra and B. oleracea on six organic broccoli farms (spaced Indol-3-ylmethyl 0Æ14 0Æ21 1Æ43 0Æ258 >1 km apart) in and around the Salinas Valley, CA. Each farm site 4-methoxy-indol-3ylmethyl 0Æ11 0Æ22 7Æ49 0Æ020 contained a patch of B. nigra growing no more than 25 m from the N-methoxy-indol-3ylmethyl 0Æ03 0Æ15 14Æ23 0Æ004 field edge of the adjacent B. oleracea crop. Between 50 and 150 g of Total aliphatic 17Æ99 1Æ89 11Æ56 0Æ007 leaf material was gathered from 20 B. oleracea plants and 20 B. nigra Total indolic 0Æ82 0Æ79 0Æ03 0Æ842 plants on three dates (19 June, 1 July, and 17 July 2009) at each of the sites. Plants were selected at random every 0Æ5 m along 10-m tran- sects. Leaf and stem materials were selected from the centre of the nearly ten times the aliphatic glucosinolates of aphids reared plants, as the younger plant tissues are more likely to be inhabited by on B. oleracea (F =11Æ56, d.f. = 1, 10, P =0Æ007, Table 1). aphids. The samples were individually bagged and returned to the laboratory for inspection. Each sample was washed over a sieve to count and identify all insects inhabiting the different plants. Aphid IMPACTS OF BRASSICA NIGRA ON APHIDS and syrphid densities were estimated as numbers per unit weight of leaf. Relative per cent parasitism by (McIntosh) Aphids reared on B. nigra reached reproductive maturity 14% was estimated as 100 · the ratio of the number of aphid mummies to faster on average than those reared on B. oleracea (Fig. 1a; the combined number of aphids and mummies. F =18Æ84, d.f. = 1, 57, P <0Æ001). Aphids in the B. nigra treatment also reproduced at a faster rate, producing just over

ANALYSIS three nymphs per day, compared to two nymphs a day for aphids on B. oleracea (Fig. 1b; F =10Æ84, d.f. = 1, 28, r All analyses were performed using the statistical program, (version P =0Æ003). However, aphids survived significantly longer on 2.9.1, http://www.R-project.org). Differences in syrphid mortality B. oleracea than B. nigra (Fig. 1c; F =7Æ46, d.f. = 1, 28, (proportion of larvae that did not survive to pupation) between P =0Æ01), resulting in no significant differences between the B. oleracea and B. nigra treatments were compared using a chi- squared test. All other pairwise comparisons from the laboratory two treatments in overall nymph production per individual experiments were analysed using analysis of variance. In these analy- (Fig. 1d; F =0Æ067, d.f. = 1, 28, P = 0. 79). ses, B. oleracea and B. nigra treatments were the predictor variable for the each of the following response variables: glucosinolate content IMPACTS OF BRASSICA NIGRA ON SYRPHIDS (nmol per 100 mg of leaf material or nmol per 10 aphids), aphid development (days from birth to first reproduction), aphid reproduc- SyrphidlarvalmortalityintheB. nigra treatment was 95%, tive rate (mean nymphs produced per day per adult), aphid fecundity more than double that found in the B. oleracea treatment (total number of nymphs produced per adult), aphid longevity (days (Fig. 2a; v2 =9Æ64, d.f. = 1, P =0Æ002). Syrphids in the from birth to death), syrphid predation rate (mean aphids consumed B. oleracea treatment consumed 65% more aphids per day per day per syrphid larva), and total predation (total number of than syrphids in the B. nigra treatment (Fig. 2b; F =4Æ25, aphids consumed per syrphid larva). Field data on aphid and syrphid d.f. = 1, 38, P =0Æ05). Because of these substantial impacts densities (per unit weight of leaf material) and percentage parasitism were compared for B. nigra and B. oleracea using generalized linear of B. nigra on syrphid larval development, total lifetime aphid mixed effects models with site and sampling date as random effects. consumption by syrphid larvae in the B. oleracea treatment Models for syrphid densities and parasitism included aphid densities was double that of the B. nigra treatment (Fig. 2c; F =4Æ18, as a covariate, as lower prey densities would be expected to result in d.f. = 1, 38, P =0Æ05). Of the 12 larvae that reached pupa- lower enemy densities irrespective of treatment effects. tion (1 on B. nigra,11onB. oleracea), 17% did not emerge from their pupae, 41% were A. obliqua, 25% were S. sulphuri- pes and 17% were E. americanus. Results

GLUCOSINOLATE PROFILES FIELD OBSERVATIONS

Brassica nigra contained several orders of magnitude higher We found no significant differences between aphid densities in total aliphatic glucosinolate concentrations (155 ± 46 nmol B. nigra and the adjacent B. oleracea crop (Fig. 3a; F =2Æ03, per 100 mg fresh plant matter, mean ± standard error) than d.f. = 1, 25, P =0Æ16). Aphid densities were a significant B. oleracea (0Æ68 ± 0Æ15 nmol per 100 mg fresh plant matter). factor in predicting syrphid densities (F =29Æ97, d.f. = 1, 24, Mirroring the endogenous glucosinolate differences between P <0Æ001), but not percentage parasitism by D. rapae. the two food plants, aphids reared on B. nigra sequestered Taking aphid densities into account, syrphid densities were

2011 The Authors. Journal of Applied Ecology 2011 British Ecological Society, Journal of Applied Ecology, 48, 880–887 884 R. Chaplin-Kramer et al.

(a) (b)

(c) (d)

Fig. 1. Impacts of host plant (Brassica oleracea vs. Brassica nigra)oncabbageaphid physiology (means ± SE). (a) Aphid devel- opment time, in days from birth to first reproduction; (b) aphid reproductive rate, in average number of nymphs produced per adult per day; (c) aphid longevity, in number of days from birth until death; (d) total aphid fecundity, in total number of nymphs produced per aphid. orders of magnitude lower on B. nigra than in the crop glucosinolates, but similarly seem much less impacted by low (Fig. 3b; F =15Æ38, d.f. = 1, 24, P <0Æ001), and percentage glucosinolate levels (Vanhaelen, Gaspar & Francis 2002; parasitism was likewise several times lower on B. nigra Olmez-Bayhan, Ulusoy & Bayhan 2007). While the number (Fig. 3c; F =7Æ95, d.f. = 1, 24, P =0Æ01). All other insects and variety of specific glucosinolate compounds prevent a weretooraretoanalyse. direct comparison between this study and previous work, the general trend is congruent with the findings of this study: low glucosinolate plants can pass some of these compounds onto Discussion herbivores without providing the same protection from preda- tors offered by high glucosinolate plants. LABORATORY EXPERIMENTS Surprisingly, we found no trade-off between the use of Cabbage aphids feeding on B. nigra contain more glucosino- B. nigra chemicals as ammunition against their enemies and lates than aphids feeding on B. oleracea, supporting previously the overall performance of cabbage aphids. The sequestration reported differences in glucosinolate sequestration on two of glucosinolates may not impart a significant energy cost upon other plant species (Francis et al. 2001). This suggests that the the aphid as this system only requires a transporter to move aphid’s ability to sequester glucosinolates is not saturated the glucosinolate out of the digestive stream. Furthermore, the when reared upon B. oleracea in spite of the log- differ- cabbage aphid may avoid the myrosinase cells in the brassica- ence in glucosinolate between the two plants. Thus, it appears ceous plant via a behavioural change in stylet penetration, that in this system, the food source can determine the aphid’s reducing or eliminating energetic cost of disabling the myrosin- endogenous defence capacity. Aphids reared on B. oleracea ase (Kim & Jander 2007). While the results presented here may still have detectable levels of glucosinolates, but these concen- not apply to all cultivars of B. oleracea, the fact that aphids trations do not provide as much deterrence against aphid ene- reared on B. nigra in our study reached reproductive maturity mies as that generated by feeding on B. nigra. Similar results faster and had a greater daily reproductive rate suggests that were found for the coccinellid (L.), which the aphid population growth rate would be enhanced on was able to complete its larval development on aphids reared B. nigra compared to broccoli, as these life-history traits are on the low levels of glucosinolates found in Brassica napus (L.), known to translate into faster population growth (Stearns but not on aphids reared on the more concentrated glucosino- 1992). Rather than a physiological cost of living on B. nigra, lates of Sinapis alba (L.) (Francis, Haubruge & Gaspar 2000). there appears to be a slight benefit. To understand the mecha- Other natural enemies show slower growth rates or reduced nisms behind this finding, it may be necessary to consider the reproduction rather than direct mortality with high levels of specific glucosinolate profiles rather than the overall total

2011 The Authors. Journal of Applied Ecology 2011 British Ecological Society, Journal of Applied Ecology, 48,880–887 Chemically mediated tritrophic interactions 885

(a) (b) (c)

Fig. 2. Impacts of cabbage aphid host plant (Brassica oleracea vs. Brassica nigra) on syrphid larvae (means ± SE). (a) Syrphid mortality rate, in proportion of individuals that pupated (grey) vs. died (black); (b) predation rate, in average number of aphids consumed per syrphid per day; (c) total predation, in total number of aphids consumed per syrphid lifetime. content. Harvey et al. (2007) showed that larval development would be found in higher densities on B. nigra.Contraryto time of the lepidopteran Pieris rapae varied dramatically with this expectation, our results showed no difference between plant strains differing in glucosinolate levels, but that total the two food sources. In fact, although high variation in glucosinolate content was not predictive. Instead, larval aphid densities between sites resulted in a nonsignificant dif- growth was slowed by increased concentrations of indolic gluc- ference between the two plant species, the trend was towards osinolate neoglucobrassicin, but showed no relationship to higher not lower aphid densities on B. oleracea.Thisappar- any other specific glucosinolate compound. Several of the ind- ent discrepancy between laboratory-based expectation and olic compounds measured in this study were higher in B. olera- field observations of aphid densities on the two plant species cea than B. nigra, potentially slowing aphid growth on may be attributed to nutrient availability, as aphid popula- B. oleracea relative to B. nigra without enhancing aphid tions on are known to be influenced by amino acid defences, which may be related to other compounds. Cole concentration as well as allelochemicals (Cole 1997a). For (1997a) also found that an indolic glucosinolate suppressed the plants grown in the same soil in a greenhouse, aphid perfor- intrinsic rate of increase in the cabbage aphid, while two ali- mance was greater on B. nigra. In contrast, aphids on culti- phatic glucosinolates enhanced it. Broccoli was not one of the vated B. oleracea plants grown as a commercial fertilized cultivars tested by Cole, but our glucosinolate profile showed crop may well have benefitted from a greater amino acid that it contained markedly less sinigrin, one of the aliphatic concentration than those on the wild B. nigra plants growing glucosinolates he found to be positively correlated with the in the field margins. intrinsicrateofincreaseinthecabbageaphid. Our field data confirm the high syrphid mortality found Our results further show that syrphids are far less effective in our laboratory trials; syrphids were found at far lower predators of cabbage aphids on B. nigra than on B. oleracea. densities in B. nigra patches than in nearby crop. This is in con- They consume fewer aphids per day and substantially fewer trast to Newton, Bullock & Hodgson (2009), who found no aphids on B. nigra in total. Moreover, the dramatic mortality difference between syrphid larval abundance on wild ofsyrphidlarvaeobservedonaphidsfromB. nigra suggests B. oleracea phenotypes differing in the presence and absence that there could be strong selection pressure against syrphid of the glucosinolate sinigrin. As the sinigrin content of these oviposition on B. nigra. The ability of aphids reared upon B. oleracea phenotypes was not estimated, it is possible that B. nigra to compromise the development and survival of their the concentrations in these plants were not high enough to main predator reveals the potential for this weedy mustard to impact enemies to the extent that we saw in B. nigra. In gen- provide a refuge from predation. eral, cultivated B. oleracea has much lower levels of sinigrin than B. nigra (Cole 1997a), and as was previously mentioned, this was certainly the case for the variety of B. oleracea we FIELD OBSERVATIONS studied (broccoli, var. italica). Thus, the much reduced densi- Previous work has suggested that aphids have lower densities ties of natural enemies that we saw on B. nigra compared to on higher glucosinolate food sources in the field, presumably adjacent broccoli fields, beyond what could be expected from because of the higher physiological burden of living on that differences in aphid distributions, suggest that some aspect of food source (Newton, Bullock & Hodgson 2009). However, the glucosinolate profile of B. nigra is far more toxic to syrph- as our laboratory experiments showed that aphid develop- ids than that of B. oleracea. Syrphid host-plant preference ment and reproductive rate were slightly greater on B. nigra should be examined explicitly in the field, to test our hypothesis relative to B. oleracea, and that predation by syrphids was that syrphids avoid ovipositing on B. nigra owing to the much reduced on B. nigra, it may be expected that aphids perceived selection pressure resulting from this toxicity.

2011 The Authors. Journal of Applied Ecology 2011 British Ecological Society, Journal of Applied Ecology, 48, 880–887 886 R. Chaplin-Kramer et al.

(a) (b) (c)

Fig. 3. Field observations of insects on Brassica oleracea and Brassica nigra (means ± SE). (a) Aphid densities per g of leaf; (b) syrphid densities per 100 g of leaf; and (c) percentage parasitism.

It is possible that other natural enemies, in particular parasi- Other areas of exploration for this research include plant- toids, can prey on cabbage aphids on B. nigra in the field. The level variables such as type and floral resources. We evolutionary relationship between herbivores and parasitoids tested only one cultivar of B. oleracea (var. italica), and as may better equip these specialist enemies to manage the higher previously mentioned, glucosinolate concentration as well as glucosinolate concentrations than generalist predators (Gols aphid performance can differ considerably on different culti- et al. 2007; Gols & Harvey 2009). Olmez-Bayhan, Ulusoy & vars (Cole 1997a,b). Therefore, the impact of B. nigra grow- Bayhan (2007) have shown that parasitism rates of the cabbage ing near brassicaceous crop fields may well depend on the aphid are as high in higher glucosinolate plants such as type of cultivar planted, and this could be investigated S. arvensis as on cultivated B. oleracea, indicating that special- through additional trials on other cultivars. Additionally, ist parasitoids continue to provide some measure of pest con- flowering B. nigra such as that found at our field sites could trol in high glucosinolate systems. However, our field results provide nectar and pollen to adult syrphid flies, and such indicate that parasitism by D. rapae was reduced on the higher resources have been shown to extend longevity and enhance glucosinolate B. nigra. This lower rate of parasitism is perhaps reproduction in many natural enemies (Wa¨ ckers & Van Rijn surprising, but the extremely low incidence of parasitism in this 2005). These positive aspects of B. nigra may compensate to system (<5% on B. oleracea,<1%onB. nigra) suggests that some extent for the negative aspect of providing a predator parasitism does not play a major role in top-down control in refuge to pests like the cabbage aphid, although this would any case. Whether specialist parasitoids or other natural ene- be likely to depend on the availability of other floral mies can compensate for lower syrphid predation on B. nigra resources. An energetic analysis of B. nigra pollen and nectar patches probably depends upon geographic locations and and field observations on the frequency of visits of syrphids cropping systems. to these plants could determine the relative importance of B. nigra in providing a resource to syrphids as well as a refuge to aphids. FUTURE DIRECTIONS

While our observations suggest that B. nigra can reduce aphid Conclusion mortality (through its impact on syrphid predators) and enhance aphid production, further work is needed to fully These results provide a new perspective on enemy-free space investigate the implications of these observations. To investi- and underscore the importance of its inclusion in biological gate the potential for B. nigra to serve as a source of pests to control research. The lack of a significant reduction in aphid crop fields, research should address host preference and short- performance on B. nigra compared to B. oleracea indicates distance dispersal patterns in the cabbage aphid. Despite no that there would be no long-term, population-level trade-offs obvious physiological disadvantage of living on B. nigra com- of sequestering glucosinolates even with a near tenfold differ- pared to B. oleracea, aphids may prefer one food source to the ence in glucosinolate concentration in the adult aphid. In fact, other if given a choice, and their preference may determine the shorter aphid generation time and reduced vulnerability whether B. nigra isatrapcroporanaphidexporter.Markand to syrphid predation found on B. nigra suggests that this recapture studies should be implemented to determine whether weedy mustard should be investigated in field studies for its aphids are moving from B. nigra to the crop or vice versa. potential to serve as source of pest colonization to surround- More frequent and longer-term field surveys would also help ing crops. The potential existence of pest refuges from preda- elucidate the relationship between wild B. nigra and B. olera- tors is an important consideration for pest control, and cea crops, revealing whether aphids build up on one food phytochemicalecologycanbeusedtoelucidateandpoten- source earlier in the season and then transfer to the other. tially eliminate the occurrence of such refuges. Food web

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2011 The Authors. Journal of Applied Ecology 2011 British Ecological Society, Journal of Applied Ecology, 48, 880–887