Biological Control xxx (2013) xxx–xxx

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Biological Control

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Dichotomy of male and female responses to hoverfly-driven cuesand floral competition in the parasitoid wasp Aphidius ervi Haliday

1049-9644/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.biocontrol.2013.08.013

⇑ Corresponding author.E-mail address: david.george@northumbria.ac.uk (D.R. George).

1 These authors contributed equally.2 Present address: Faculty of Health and Life Sciences, Northumbria University,

Newcastle upon Tyne NE1 8ST, UK.

Please cite this article in press as: George, D.R., et al. Dichotomy of male and female responses to hoverfly-driven cues and floral competition in tasitoid wasp Aphidius ervi Haliday. Biological Control (2013), http://dx.doi.org/10.1016/j.biocontrol.2013.08.013

D.R. George a,⇑,1,2, L. King a,1, E. Donkin b,1, C.E. Jones c, P. Croft c, L.A.N. Tilley c

a Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ, UKb Department of Biology, University of York, York YO10 5DD, UKc Stockbridge Technology Centre, Selby YO8 3TZ, UK

h i g h l i g h t s

� Beneficial insects may interact forresources such as prey and floralnectar.� Interactions may be additive or non-

additive, potentially effectingbiocontrol.� Volatile cues from hoverflies were

avoided by female parasitoid wasps.� Female parasitoid wasp longevity was

reduced by high hoverfly presence onflowers.� Responses of male wasps to hoverflies

were more ambiguous.

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

Survivorship curve for female Aphidius ervi when offered water, honey solution and Fagopyrum esculen-tum flowers presented with varying densities of adult female Episyrphus balteatus as a carbohydratesource. Key: BW = Buckwheat; HF = Hoverfly. Pie chart inserts display median female wasp preferencefor varying flower treatments (in terms of time spent in opposing Y-tube arms) in two-way olfactometertests. Flower/Flower refers to the control.

a r t i c l e i n f o

Article history:Received 21 February 2013Accepted 24 August 2013Available online xxxx

Keywords:Biological controlFlower useAntagonismParasitoidHoverfly

a b s t r a c t

The aim of this study was to test the hypothesis that floral visitation and foraging by the hoverfly Episyr-phus balteatus De Geer would have no effect on the attraction to, or use of, flowers by the parasitoid waspAphidius ervi Haliday. Results demonstrated that in two-way choice tests, air-streams emanating fromlive Fagopyrum esculentum Moench (buckwheat) flowers were significantly more attractive than cleanair to both male and female wasps, by more than one and two orders of magnitude, respectively. Whenair-streams from flowers presented alone were compared to those from flowers presented with E. balte-atus, or exposed to E. balteatus prior to use, no preferences were detected. Nevertheless, air that hadpassed through chambers containing only live E. balteatus was significantly repellent to female wasps,with individuals spending 18 times less in these air-streams than clean air. Conversely, air passed overE. balteatus was attractive to male A. ervi, with wasps spending more than six times longer in this vs. cleanair. We argue that hoverfly floral visitation/use should have minimal impact on floral attractiveness to A.ervi based on these results, and that for male wasps greater benefits might be gained by responding pos-itively to hoverfly volatiles in the absence of floral cues.

he par-

2 D.R. George et al. / Biological Control xxx (2013) xxx–xxx

Please cite this article in press as: George, D.R.,asitoid wasp Aphidius ervi Haliday. Biological Co

In a separate experiment, a consistent trend for reduced fitness of female A. ervi on caged F. esculentumwas observed under increasing competitive pressure from E. balteatus. High densities of hoverflies signif-icantly reduced wasp longevity by more than 25%, though lower densities had no significant effect. Con-versely, male A. ervi appeared to survive for longer where higher densities of hoverflies were present,though results were not as clear-cut statistically. We argue that antagonistic interactions seen betweenhoverflies and female A. ervi were the result of behavioural interference and would only be observed inthe field under conditions of extreme competitive pressure.

� 2013 Elsevier Inc. All rights reserved.

1. Introduction

Inclusion of floral resources can be an effective means of pro-moting pest natural enemies and general conservation biologicalcontrol in agricultural landscapes (Landis et al., 2000; Pfiffnerand Wyss, 2004). When appropriate floral resources are providedparasitoids derive multiple benefits in terms of feeding statusand fitness (Heimpel and Jervis, 2005; Wäckers and van Rijn,2012), with some authors reporting benefits to in-field parasitismrates (Baggen and Gurr, 1998; Tylianakis et al., 2004), albeit rela-tively rarely and with no guarantee that these translate to im-proved pest control (Heimpel and Jervis, 2005). FloweringFagopyrum esculentum Moench (buckwheat) is notably beneficialto multiple species (e.g. Lee and Heimpel, 2005; Winkler et al.,2006), including Aphidius ervi Haliday (Araj et al., 2006, 2009). Par-asitoids are not alone in deriving benefit from floral resources,however, with advantages to longevity (Langoya and van Rijn,2008) local abundance and in-field species richness (Haenkeet al., 2009) and pest aphid control (White et al., 1995; Hickmanand Wratten, 1996) also reported for aphidophagous hoverflies,including for Episyrphus balteatus De Geer when given access toF. esculentum flowers (Langoya and van Rijn, 2008; D.R. George,unpublished data).

Simultaneous resource provision to multiple pest natural ene-mies could be considered beneficial. Additive or synergistic effectson pest suppression may result where multiple pest natural ene-mies co-exist (Jonsson et al., 2010; Price et al., 2011), with nichecomplementarity through various forms of resource partitioning(Wilby and Thomas, 2002; Straub and Snyder, 2008) and/or facili-tation (Losey and Denno, 1998) more likely to be encouraged. Fur-thermore, promotion of diverse groups acts as an insurance policy,maintaining biological control in the event of loss or decline in theprimary pest natural enemy (Loreau et al., 2003; Griffiths et al.,2008).

Interactions between multiple pest natural enemies are not al-ways positive. Where diversity of pest natural enemies is in-creased, intra-guild competition can lead to antagonistic effects(Jonsson et al., 2010; Price et al., 2011). Antagonism has been re-ported between ladybirds and parasitoid wasp species, throughboth direct predation (Meyhöfer and Klug, 2002) and a combina-tion of predation and oviposition disruption (Ferguson and Stiling,1996). Larval and adult Coccinella septempuncata L., for example,have been observed to preferentially predate aphids parasitisedby Lysiphlebus fabarum Marshall (Meyhöfer and Klug, 2002). Simi-larly, the predatory bug Orius majusculus Reuter has been shown todisplay a preference for parasitised over unparasitised whiteflythroughout most life-stages of both species (Sohrabi et al., 2013).Though intra-guild competition for prey may have minimal impacton overall pest control, the relationship is highly idiosyncratic(Straub et al., 2008) and numerous examples exist in which pestregulation is adversely affected by increasing natural enemy diver-sity (Jonsson et al., 2010). Thus, it seems logical to avoid antago-nism wherever possible to optimise conservation biologicalcontrol potential, insure against species loss/decline and maximisethe likelihood of additive interactions between groups as reportedabove.

et al. Dichotomy of male and fentrol (2013), http://dx.doi.org/

Work reporting intra-guild antagonism between pest naturalenemies typically considers predation as the determining factor(e.g. Ferguson and Stiling, 1996; Meyhöfer and Klug, 2002), withstudies tending not to consider competition for floral resourcesas extensively, except perhaps for interactions involving pollina-tors (e.g. Reader et al., 2005). Research suggests that E. balteatus fe-males avoid oviposition at sites with high proportions of aphidmummies (Pineda et al., 2007), and that larvae of this species showno preference for parasitised aphids, also being unable to consumemummies (Meyhöfer and Klug, 2002). Though this may suggest atworst relative neutrality with respect to intra-guild predation,studies have reported that hoverfly floral use can have a negativeeffect on the foraging and feeding behaviour of bees (Readeret al., 2005). In field trails employing wild test subjects foragingon bramble, both bumble bees (mixed Bombus spp.) and honeybees (Apis mellifera L.) displayed an aversion to flowers recentlyvisited by hoverflies (mixed spp.) (Reader et al., 2005). Whetheror not E. balteatus flower visitation has a similar negative effecton floral resource use by parasitoids remains to be studied.

The above in mind, the aim of the present study was to test thehypothesis that floral visitation and foraging by the hoverfly E.balteatus would have no effect on the attraction to, or use of, flow-ers by the parasitoid wasp A. ervi. To achieve this aim laboratoryand field-cage experiments were used to assess A. ervi sensoryand life-history responses to combinations of F. esculentum flowersand adult E. balteatus.

2. Materials and methods

2.1. Floral attraction experiment

F. esculentum were sourced from a flowering field margin sownin March 2012 at a commercial carrot farm in Roall, Goole, UK,after glasshouse-sown plants that would have been preferred foruse failed to establish. Flowering plants were transplanted intoindividual pots and maintained at 21 �C, 16:8 light:dark cycle atStockbridge Technology Centre (STC), Selby, UK. A. ervi weresourced from BCP Certis (Cambridgeshire, UK), discarding any indi-viduals that had emerged on arrival. Remaining aphid mummieswere placed within a ventilated 25 � 23 � 38 cm plastic arenaand left for 24 h at 21 �C. Emerged wasps were then removedand placed within a new arena for another 24 h. The resulting24–48 h old wasps were used for study, allowing individuals timeto mate after emergence (Takemoto et al., 2012), but without risk-ing starvation (Wäckers, 1994). Wasps were not fed prior to use inorder to enhance response to foraging cues, though water (on cot-ton wool) was available ad libitum. Adult E. balteatus used in thestudy were wild-caught at STC and stored within a mesh cage(45 cm3) at 21 �C. Whilst in storage hoverflies were provided withwater at all times and, when not being utilised to forage on F. escu-lentum, they were also provided with a 50% sucrose solution (oncotton wool). Any wasp or hoverfly was used only once to avoidpseudo-replication (Rodríguez et al., 2002).

The experiment was conducted in a climate controlled room (c.21 �C) at STC during July and August 2012 and employed a Y-tube

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Fig. 1. Layout of an olfactometer (design adapted from George et al., 2009). Air flow is depicted with lines and arrows. Key: Pump (1 L/min) = 1. Activated carbon = 2.Particulate filter (in-line PTFE 50 mm dia. 0.2 lm, Fisher Scientific, Loughborough, UK) = 3. Distilled water = 4. 3-way connector (to split air-stream) = 5. 1 L conical flaskscontaining nothing, hoverflies, or Fagopyrum esculentum flowers subjected to different treatments = 6. Y-tube (containing wasps and capped with 0.2 mm mesh) = 7. Not toscale.

D.R. George et al. / Biological Control xxx (2013) xxx–xxx 3

olfactometer (Fig. 1) to assess the attractiveness of a range of treat-ments to adult male and female A. ervi. Olfactometers were de-signed according to George et al. (2009) with air pumpedthrough each arm of the Y-tube at a rate of 250 ml/min. All Y-tubesused were of the same design (10 mm internal diameter, two mainarms each 100 mm, third arm of 10 mm) and all stages of the appa-ratus were connected using flexible plastic tubing (5 mm internaldiameter) coupled to hollow rigid glass rods (4 mm internal diam-eter) and plastic bungs of the required size (Fisher Scientific,Loughborough, UK). Y-tubes were placed within a white open-top box and illuminated from above (Rodríguez et al., 2002) tominimise interference from external visual cues whilst still permit-ting observation.

A total of four treatments were tested along with a control, inwhich both arms of the olfactometer contained air passed over un-treated F. esculentum. Treatments used were as follows: Buckwheatonly (clean air vs. air passed over untreated F. esculentum flowers);hoverfly only (clean air vs. air passed over one sexed pair of adult E.balteatus); hoverfly exposed (air passed over untreated F. esculen-tum flowers vs. air passed over F. esculentum flowers exposed toE. balteatus for 24 h); hoverfly presented (air passed over untreatedF. esculentum flowers vs. air passed over F. esculentum flowers pre-sented with one adult pair of E. balteatus). Where F. esculentum wasused, 40–50 individual living flowers from intact potted plantswere placed within flasks (to eliminate effects of excision) by pass-ing stems containing floral sprays along a groove in the bung usedto cap flasks. Any gaps in this groove after adding stems were elim-inated by a Vaseline seal which was employed in all treatments toensure an air-tight seal between bungs and flasks. Plants were cho-sen at random from an experimental population of twelve F. escu-lentum, with any individual plant only used once in any 24 hperiod. This 24 h period was considered sufficient time for anyscent marking by hoverflies to have dissipated (Reader et al.,2005) and for F. esculentum nectar to have replenished itself inplants exposed to hoverflies (Cawoy et al., 2006). For the hoverflyexposed treatment plants were placed within a cage housing threesexed pairs of E. balteatus for 24 h prior to use. Hoverfly gender wasdetermined according to sexual dimorphism and primarily basedon females having dichoptic eyes in contrast to the holoptic eyesof males.

The response of A. ervi to treatment was assessed by placing asingle wasp into the lower arm of the olfactometer and recordingthe time spent by that individual in either arm of the Y tube duringa 5 min observation period. The time taken for wasps to initiallyselect an arm of the olfactometer (from the neutral zone betweenthe two arms and extending 5 mm into each where wasps wereinitially placed) was also recorded, as was the total number of deci-sions made by a given wasp prior to selecting an olfactometer arm(where a ‘decision’ constituted a wasp leaving the neutral zone, butreturning to it). Wasps that did not leave the neutral zoneafter 5 min were considered non-responsive and discarded. During

Please cite this article in press as: George, D.R., et al. Dichotomy of male and feasitoid wasp Aphidius ervi Haliday. Biological Control (2013), http://dx.doi.org/

the course of the experiment only three wasps were non-respon-sive (one female and two males in control and buckwheattreatments).

For each treatment and the control, the response of 20 male and20 female wasps was recorded. The sequence of treatment testingwas randomised and always included complete runs of all treat-ments on any given day to account for any variation in responsebetween the three batches of wasps used in the study. Followingrecommendations by Ramírez et al. (2000), equipment was cleanedafter every use (using ethanol and water) with Y-tube aspect inter-changed to avoid bias (Rodríguez et al., 2002).

The efficacy of the hoverfly exposed treatment was determinedseparately by assessment of floral nectar content of F. esculentumflowers subjected to E. balteatus exposure, as compared to that ofplants subjected to no such exposure as a control. The cumulativenectar content of five open flowers per plant was assessed for eightplants at time zero by measuring the length of nectar drawn intoglass 0.5 ll Drummond micro-capillary tubes (Microcaps�, SigmaAldrich, UK) inserted into flower-heads. Four flowering F. esculen-tum were then placed into mesh cages (45 cm3) with six sexedpairs of E. balteatus (wild caught at STC mimicking the density ofexposure used to treat plants previously) and their nectar contentreassessed 24 h later. The remaining four plants were placed incages without hoverflies following initial assessment and had theirnectar content assessed in the same way 24 h later. The experi-ment was conducted under the same conditions and in the sameclimate controlled facility used previously. F. esculentum usedhad been grown from seed at STC under glasshouse conditions inJohn Innes No. 2 compost. Seed was obtained from E.W. King andCo. Ltd. (Essex, UK) and plants were used at point of optimum flow-ering, approx. 6 weeks post sowing.

The length of time taken for A. ervi to move from the neutralzone of Y-tubes was analysed by Kruskal–Wallis tests because datacould not be made to conform to assumptions of parametric test-ing. Analysis was conducted on males and females separately afterpreliminary exploration of the data suggested an interaction be-tween treatment and sex. Mann–Whitney tests were used to inves-tigate pair-wise differences between treatments. To compare totaltime spent in either arm of the olfactometer for each treatment/sex, Wilcoxon signed rank tests were used following Rodríguezet al. (2002). Total decisions made by both male and female waspswere relatively low (maximum 10 and 12, respectively) and so datawere analysed by Kruskal–Wallis tests, again considering the re-sponses of sexes separately. Where data are presented, median val-ues are displayed with inter-quartile ranges (IQRs). For assessmentof floral nectar content before and after E. balteatus exposure, datawere expressed as nanoliters per plant (i.e. per five flowers) anddisplayed graphically for visual assessment of efficacy. Pre-treat-ment nectar content was compared between control and HFEplants using ANOVA to ensure no pre-existing differences betweengroups. Data were checked for homoscedasticity and Normality

male responses to hoverfly-driven cues and floral competition in the par-10.1016/j.biocontrol.2013.08.013

4 D.R. George et al. / Biological Control xxx (2013) xxx–xxx

prior to analysis using the Levene’s and Kolmogorov–SmirnovTests, respectively.

2.2. A. ervi longevity experiment

F. esculentum were grown under glasshouse conditions at Lan-caster University (Lancaster, UK) from seed obtained from E.W.King and Co. Ltd. Plants were sown as needed in pots of John InnesNo. 2 compost, where from point of sowing flowering heads wereproduced after 6–7 weeks. A. ervi were sourced as pupae from Bio-best (Westerlo, Belgium). Upon receipt, any wasps that hademerged in transit were discarded as their age was unknown.Remaining mummies were then placed in an open Petri-dish with-in a small mesh holding cage at room temperature (c. 20 �C).Emerging adults were provided with a 50:50 solution of honey:-water on cotton wool and used within 24 h of eclosion. E balteatuswere sourced from wild populations and held at 20 �C in a largefine mesh cage prior to use (Exo Terra� 380 L Flexarium measuring760 � 420 � 1220 mm) where they were supplied with floweringF. esculentum plants, finely crushed bee pollen (Holland and Bar-rets, UK) and water ad libitum.

To determine whether or not hoverfly competition effected A.ervi longevity, pairs of wasps were confined with live F. esculentumflowers in chambers containing 0, 1, 2 or 3 adult female E. baltea-tus. Control treatments containing either water only (on cottonwool, also added to all other treatments as a moisture source), ora 50:50 honey:water solution (on cotton wool), but no flowers or

Fig. 2. Initial time taken (secs) by (a) male and (b) female Aphidius ervi to select an airolfactometers. Boxes show median values with IQR; whiskers show data spread withinextreme cases (>3� the IQR). n = 20 in all cases, where all data were included in the anexposed; HFP: Hoverfly presented.

Please cite this article in press as: George, D.R., et al. Dichotomy of male and feasitoid wasp Aphidius ervi Haliday. Biological Control (2013), http://dx.doi.org/

hoverflies were also used. Each treatment was replicated eighttimes. Hoverfly oviposition was not noted in any treatment.

The chambers used housed live floral sprays, simulating meth-ods used by Araj et al. (2006). Chambers were constructed from 2.2L clear polyurethane containers with screw-on plastic lids. A100 mm diameter hole was cut into the lid using a scalpel andfoam mounted over the top face of the lid. Small holes punchedthrough both the foam and the screw top lid allowed securemounting of the foam with flexible garden wire. A small cut inthe centre of the foam allowed access for flowers, insects andwater/sucrose sources.

Chambers were mounted onto garden canes using garden wireand staked into soil-filled pots for stability. Chambers were thenpositioned around growing F. esculentum plants with a stem con-taining a single spray of flowers secured inside each chamber.The entire experiment was conducted under field conditions atthe experimental site in Whalley (Blackburn, UK) during July 2011.

Survival of A. ervi was recorded at 24 h intervals until all waspswere dead. In treatments containing E. balteatus, deceased hover-flies were replaced during wasp observations. Water and sucrosesolutions were replenished as required.

A. ervi longevity data were evaluated by constructing Kaplan–Meier survivorship curves and making pair-wise comparisonsbetween treatments using Mantel-Cox tests. Data for males andfemales were considered separately after preliminary explora-tion of the data suggested an interaction between treatmentand sex.

-stream under different treatments after being introduced to the ‘neutral zone’ of1.5� the IQR; circles show outliers (within 1.5–3� the IQR) and asterisks show

alysis. Key to treatments: BW: Buckwheat only; HF: Hoverfly only: HFE: Hoverfly

male responses to hoverfly-driven cues and floral competition in the par-10.1016/j.biocontrol.2013.08.013

Table 1The olfactometric response of male Aphidius ervi to the five treatments used. Median time in each arm presented in seconds (with lower and upper limits for IQR), where n = 20 inall cases. Key to treatments: BW: Buckwheat only; HF: Hoverfly only; HFE: Hoverfly exposed; HFP: Hoverfly presented.

Treatment Time in treatment air stream (s) Time in clean air stream (s) Z and P value

BW 274 (131/300) 23 (0/87) �2.500; P = 0.012HF 248 (172/300) 40 (0/123) �2.476; P = 0.013

Time in treatment air stream (s) Time in F. esculentum air stream (s)

HFE 183 (60/300) 118 (0/234) �1.093; P = 0.274HFP 173 (20/299) 115 (1/259) �0.545; P = 0.586

Time in left air stream (s) Time in right air stream (s)

Control 142 (43/175) 124 (39/237) �0.243; P = 0.808

Table 2The olfactometric response of female Aphidius ervi to the five treatments used. Median time in each arm presented in seconds (with lower and upper limits for IQRs), where n = 20in all cases. Key to treatments: BW: Buckwheat only; HF: Hoverfly only; HFE: Hoverfly exposed; HFP: Hoverfly presented.

Treatment Time in treatment arm (s) Time in clean air arm (s) Z and P value

BW 298 (236/300) 2 (0/64) �3.396; P = 0.001HF 16 (0/156) 284 (102/300) �1.976; P = 0.048

Time in treatment arm (s) Time in untreated F. esculentum arm (s)

HFE 92 (17/236) 206 (64/283) �0.694; P = 0.488HFP 219 (58/265) 118 (42/295) �0.284; P = 0.776

Time in left air stream (s) Time in right air stream (s)

Control 122 (0/242) 152 (49/300) �0.606; P = 0.544

Table 3The number of decisions made by male and female Aphidius ervi to the five treatmentsused in olfactometers. Median values are presented (with lower and upper limits forIQRs). Key to treatments: BW: Buckwheat only; HF: Hoverfly only; HFE: Hoverflyexposed; HFP: Hoverfly presented.

Treatment Male Female

BW 1 (1/3.25) 2 (1/4)HF 2 (1/3.25) 1.5 (1/3.5)HFE 2 (1/3) 2 (1/4)HFP 2 (1/3.25) 2 (1/4.25)Control 2.5 (1/4.25) 1.5 (1/3.25)v2 and P value 1.563; 0.815 1.926; 0.749

Fig. 3. Mean cumulative nectar content of five Fagopyrum esculentum flowersbefore and after exposure to Episyrphus balteatus at densities representative of thoseused in HFE in olfactometer tests. Means are displayed with ± SE. n = 4 for all means.Key to treatments: HFE: Hoverfly exposed.

D.R. George et al. / Biological Control xxx (2013) xxx–xxx 5

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3. Results

3.1. Floral attraction experiment

Treatment had no significant effect on the time taken by male A.ervi to choose an air stream and leave the neutral zone(v2

(4) = 2.866, P = 0.577) (Fig. 2). Conversely, treatment did signifi-cantly effect female A. ervi decision time (v2

(4) = 9.862, P = 0.043),with wasps selecting an air-stream faster in the hoverfly onlytreatment than all others (P < 0.05 in all cases).

Both male and female A. ervi displayed a preference for un-treated F. esculentum over clean air, spending more time in theair-stream that had passed over flowering plants (Tables 1 and2). Female A. ervi also displayed an aversion to air passed overadult E. balteatus (Table 2), in contrast to male A. ervi which dis-played a preference for this air-stream (Table 1). On no other occa-sion did male or female A. ervi display any preference for one airstream over another (Tables 1 and 2).

Treatment had no significant effect on the number of decisionsmade by either male or female wasps. In both sexes relatively fewdecisions were made during the 5 min observation period(Table 3).

Nectar analysis demonstrated that exposure to E. balteatus over24 h depleted floral nectar in F. esculentum, though not completelyso (Fig. 3). Analysis showed no difference in nectar content be-tween groups prior to treatment (F(1,6) = 1.855, P = 0.222).

3.2. A. ervi longevity experiment

When provided with water alone the longevity of both male andfemale A. ervi was significantly reduced in comparison to all othertreatments (Fig. 4; Table 4). For males, longevity was also reducedwhen provided with F. esculentum without hoverflies, in compari-son to when two or three hoverflies were present or when honeywas provided (Fig. 4; Table 4). Honey provision also increased lon-gevity in female A. ervi in comparison to F. esculentum presentedwith three hoverflies, where longevity was also reduced comparedto when F. esculentum was presented alone (Fig. 4; Table 4). In

male responses to hoverfly-driven cues and floral competition in the par-10.1016/j.biocontrol.2013.08.013

Fig. 4. Survivorship curves for (a) male and (b) female Aphidius ervi when offered water, honey solution and Fagopyrum esculentum flowers presented with varying densitiesof adult female Episyrphus balteatus as a carbohydrate source. Statistical differences between pairs of treatments are displayed in Table 3. Key to treatments:BW = Buckwheat; HF = Hoverfly.

Table 4v2 and P values for pair-wise comparison of survival times (days) of male and female Aphidius ervi when offered water, honey solution and Fagopyrum esculentum flowerspresented with varying densities of adult female Episyrphus balteatus as a carbohydrate source. Key to treatments: BW = Buckwheat; HF = Hoverfly.

Females

Water Honey BW + 0HF BW + 1HF BW + 2HF BW + 3HF

Males Water – 15.86, 0.000 15.86, 0.000 15.86, 0.000 15.86, 0.000 15.86, 0.000Honey 15.06, 0.000 – 1.10, 0.294 3.71, 0.054 3.06, 0.080 8.87, 0.003BW + 0HF 15.06, 0.000 6.83, 0.009 – 2.84, 0.092 1.27, 0.261 8.07, 0.004BW + 1HF 15.06, 0.000 1.14, 0.286 1.89, 0.170 – 0.05, 0.816 3.09, 0.079BW + 2HF 15.06, 0.000 0.33, 0.568 6.56, 0.010 0.37, 0.544 – 0.55, 0.458BW + 3HF 15.06, 0.000 0.98, 0.323 5.21, 0.022 0.02, 0.895 0.30, 0.582 –

6 D.R. George et al. / Biological Control xxx (2013) xxx–xxx

general longevity was higher for females and sexes displayed acontrasting response to hoverflies; longevity of females being

Please cite this article in press as: George, D.R., et al. Dichotomy of male and feasitoid wasp Aphidius ervi Haliday. Biological Control (2013), http://dx.doi.org/

negatively affected at higher hoverfly densities with the oppositeresponse seen for males.

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D.R. George et al. / Biological Control xxx (2013) xxx–xxx 7

4. Discussion

The aim of the current study was to test the hypothesis that flo-ral visitation and foraging by the hoverfly E. balteatus would haveno effect on the attraction to, or use of, flowering F. esculentumby the parasitoid wasp A. ervi. Though both sexes spent more timein olfactometer arms containing air passed over F. esculentum flow-ers than clean air, this response was stronger in female wasps sup-porting a heightened-foraging response over males. In work withTrybliographa rapae Westwood only females were shown to be at-tracted to buckwheat flowers (Nilsson et al., 2012), with similarcontrasts between genders reported for Cotesia glomerata L. withrespect to the effect of adult feeding on flight capacity (Wanneret al., 2006). Such reduced attraction or non-responsiveness to flo-ral cues in male vs. female parasitoids has been attributed to theirlower requirement for energy, resulting from a generally shorterand more sedentary life-style with fewer demands on reproductiveexpenditure (Nilsson et al., 2012).

Gender-based differences were also seen with regard to initialair-stream selection times. Although treatment had no effect onmale A. ervi selection time, female wasps were quicker to leaveneutral zones for olfactometer arms when offered a choice be-tween clean air and air passed over adult E. balteatus, preferentiallyselecting the former. Interestingly this preference was in contrastto that observed for male wasps which were retained for signifi-cantly longer periods in air-streams passed over hoverflies thanclean air-streams. In the absence of floral cues female A. ervi maydisplay avoidance behaviour to hoverfly cues as a means of mini-mising larval predation risk. Although hoverfly larvae have beenfound to avoid mummified aphids, they may nevertheless pose athreat to recently parasitised individuals (Meyhöfer and Klug,2002). As male wasp behaviour need not incorporate mechanismsfor larval predation avoidance, it is suggested that in the absence offloral cues males responded positively to hoverfly volatiles as sur-rogates for the presence of carbohydrate resources (or possibly fe-male A. ervi).

In the presence of F. esculentum any effect of hoverflies on A. erviair-stream selection was nullified, possibly as a result of carbohy-drate-foraging behaviour dominating in the presence of floral cues,as could be expected in wasps that had not fed for at least 24 hsince eclosion. The possibility that wasp responses were drivenby host searching behaviours cannot be entirely discounted basedon the methodology used, though this is perhaps less likely giventheir food-deprived state at the time of use. In support, unfed fe-male Cotesia rubecula Marshall have been observed to respondmore positively to volatiles from buckwheat flowers than to odoursfrom leaves infested with host aphids, though the opposite wastrue of fed wasps (Wäckers, 1994). As nectar generally representsa superior and (with experience) more attractive carbohydratesource than honeydew for A. ervi (Vollhardt et al., 2010), and asneither aphids nor honeydew were present in tests, it can be fur-ther surmised that nectar cues drove responses to flowering F.esculentum. Without further study, however, the possibility thatwasps were responding to plant cues per se (as a surrogate for hon-eydew presence) cannot be completely dismissed. In any event, astrong foraging response was registered in both male and femalewasps, where few non-responding individuals were observed andrelatively few decisions were made during air-stream selection.

For both sexes longevity was shortest when no carbohydratesource was present, with wasps surviving for only a few days atmost when provided with water alone. This adds to the body ofevidence demonstrating the importance of sugar-feeding to para-sitoid wasp survival in this (Azzouz et al., 2004) and other species(Johanowicz and Mitchell, 2000; Siekmann et al., 2001; Wäckers,2004; Winkler et al., 2006). In comparison, wasp survivalwas much increased when individuals were provided with a

Please cite this article in press as: George, D.R., et al. Dichotomy of male and feasitoid wasp Aphidius ervi Haliday. Biological Control (2013), http://dx.doi.org/

sugar-source, with honey being particularly beneficial to longevityin both sexes. This can be attributed to artificial sucrose solutionsbeing especially effective in prolonging parasitoid lifespans, moreso than floral resources in some cases (Witting-Bissinger et al.,2008).

In comparison to controls, longevity was also increased in alltreatments containing F. esculentum with both males and femalessurviving for extended periods even under conditions of high com-petitive pressure from hoverflies. Concealed nectar extractionapparatus (CNEA) are well represented among braconids such asA. ervi (Jervis, 1998). Should such structures be present in A. ervi,this may allow individuals to access nectar from tubular corollas,or similar structures such as the perianth present in F. esculentumflowers. In the present study these small lobe shaped structureswould have been relatively accessible to A. ervi, even in the absenceof CNEA, due to their small body size, but less so to E. balteatus. De-spite this, a general trend was observed for decreasing longevity offemale A. ervi with increasing densities of E. balteatus, with femalewasp longevity significantly reduced (by 25%) at the highest hover-fly density. Though competition for more exposed nectar sourcescould explain this result, incomplete nectar depletion (Fig. 3) bet-ter supports a hypothesis of behavioural interference. Disruption tohoverfly flower feeding by larger pollinators has been observed(Pontin et al., 2006) and instances of its effect on parasitoid waspshave also been reported. In the presence of the ladybird Cyclonedasanguinea (L.), for example, behavioural interference betweenadults is cited as reducing the effectiveness of Aphidius floridaensisSmith (Ferguson and Stiling, 1996).

Differences between male and female responses of A. ervi totreatment were again observed in the longevity experiment. Ingeneral, male longevity was reduced in comparison to that of fe-males, though this was as expected based on the differing life-his-tories of the sexes (Nilsson et al., 2012). Additionally, trends forreduced longevity with increasing hoverfly density were not evi-dent for male A. ervi, presumably reflecting a reduced reliance onfloral resources and consequent lack of effect when in competitionfor these with hoverflies. In contrast to females, male longevitywas actually shorter when provided with F. esculentum alone, sig-nificantly so in comparison to all but one other treatment (F. escu-lentum with one hoverfly). Reasons for this remain unclear, thoughit is possible that without interference from hoverflies, males wereable to mate more vigorously, dying earlier as a result.

The availability of food can impact on several aspects of parasit-oid behaviour and biology not considered in the current study: for-aging, decision making, flight behaviour and multiple aspects offecundity being among them (Olson et al., 2005; Araj et al., 2006,2009; Wanner et al., 2006; Vattala et al., 2006; Witting-Bissingeret al., 2008). Thus, whilst it appears based on the current study thatonly severe hoverfly competition would negatively impact parasit-oid wasp floral resource use, it is possible that reduced competitivepressure could still lead to sub-lethal antagonistic effects. In addi-tion, as F. esculentum is beneficial to other nectar/pollen-feeding in-sects such as lacewings (Neuroptera: Chrysopidae) (Wäckers andvan Rijn, 2012), competition for its floral resources will be morecomplex in the field than tested here. Along with more detailedstudy specifically targeted to investigating differential (and evenconflicting) gender responses of A. ervi to hoverfly cues, these areasrepresent interesting avenues for further research.

The above work outstanding, the current study suggests thatalthough antagonistic interactions between hoverflies and parasit-oid wasps may exist with respect to floral resource use, these per-haps only exert a significant effect on the latter throughbehavioural interference under conditions of extreme competitivepressure. Though hoverflies may be highly attracted to certainflowers and flower mixes (Ambrosino et al., 2006; Pontin et al.,2006) and display a tendency to aggregate around areas of high

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8 D.R. George et al. / Biological Control xxx (2013) xxx–xxx

densities of flowering plants (Sutherland et al., 2001), it is perhapsunlikely that such extreme pressure would be commonplace in thefield. This suggests that hoverflies can be generally considered as‘functionally complementary’ (Jonsson et al., 2010) with parasitoidwasps, at least with respect to floral resource use on F. esculentumfor those species considered. With work elsewhere focusing onparasitised aphid predation by hoverflies, either directly (Meyhöferand Klug, 2002) or via decisions dictating hoverfly ovipositionbehaviour (Pineda et al., 2007), it appears that parasitoid waspsexperience minimal antagonism from hoverflies throughout therespective life-cycles of both groups. The current work suggeststhat this may be further supported by female wasp avoidance ofhoverflies in the absence of floral cues (e.g. when host-searching),with similar avoidance of foot-print chemicals from Harmonia axy-ridis Pallas already reported in female A. ervi during oviposition(Meisner et al., 2011). Assuming that hoverfly species are relativelyunaffected by wasp competition based on their larger size and in-creased mobility (Wäckers and van Rijn, 2012), this suggests thatboth groups could be simultaneously promoted with appropriatehabitat management schemes (George et al., 2010; Wäckers andvan Rijn, 2012).

Acknowledgments

The authors would like to thank Biobest (Westerlo, Belgium) forprovision of Aphidius ervi used in longevity experiments. Aspects ofthis work were conducted in partial fulfilment for an MSc at Lan-caster University (Lancaster, UK) and an MRes at York University(York, UK).

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