Secondary Predation: Quantification of Food Chain Errors in an Aphid

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Molecular Ecology (2001) 10, 2049–2057

BlackwellSecondary Science, Ltd predation: quantiﬁcation of food chain errors in an aphid−spider−carabid system using monoclonal antibodies

J. D. HARWOOD,* S. W. PHILLIPS,*† K. D. SUNDERLAND† and W. O. C. SYMONDSON* *School of Biosciences, Cardiff University, PO Box 915, Cardiff, CF10 3TL, UK, †Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK

Abstract ‘Secondary predation’ occurs when one predator feeds on a second predator, which has in turn eaten a target prey. Detection of prey remains within predators using monoclonal antibodies cannot distinguish between primary and secondary predation, potentially leading to quantitative and qualitative food chain errors. We report the first fully replicated experiments to measure secondary predation effects, using an aphid–spider–carabid system. Aphids, Sitobion avenae, were fed to spiders, Lepthyphantes tenuis, which were allowed to digest their prey for a range of time intervals. The spiders were then fed to carabids, Poecilus (= Pterostichus) cupreus, which were again allowed to digest their prey for set periods. The anti-aphid monoclonal antibody used to identify S. avenae remains in P. cupreus was one that detected an epitope that increased in availability over the first few hours of digestion, amplifying the signal, extending detection periods and thus increasing the chances of detecting secondary predation. Despite this, and the fact that spiders are known to digest their prey more slowly than many other predators, detection of secondary predation was only possible if the carabids were killed immediately after consuming at least two spiders which were, in turn, eaten immediately after consuming aphids. As this scenario is unlikely to occur frequently in the field it was concluded that secondary predation is unlikely to be a serious source of error during field studies. Keywords: antigen decay rates, food webs, generalist predators, intraguild predation, Lepthyphantes tenuis, Poecilus cupreus Received 6 February 2001; revision received 3 May 2001; accepted 3 May 2001

and (currently the preferred technique) detection of family, Introduction genus, species and even stage-specific protein epitopes Significant advances in our understanding of predator– using monoclonal antibodies (e.g. Greenstone & Morgan 1989; prey interactions have been made by applying ‘molecular’ Hagler et al. 1992; Symondson & Liddell 1996; Symondson techniques to the post mortem analysis of the gut contents et al. 1997, 1999a, 2000; Bacher et al. 1999). Recent experi- of field-caught predators (reviewed in Sunderland ments have further demonstrated that detection of prey 1988; Greenstone 1996; Symondson & Hemingway 1997; DNA from the guts of such predators is a viable option, Sunderland et al. 2001; Symondson 2001). These techniques using prey-specific primers and the polymerase chain have included the detection of prey isoenzymes using reaction (PCR) (Zaidi et al. 1999; Agustí et al. 1999, 2000; electrophoresis (e.g. Murray & Solomon 1978; Giller 1986; Chen et al. 2000; Hoogendoorn & Heimpel 2001). Data Heitmans et al. 1986; Lister et al. 1987; Walrant & Loreau 1995), from field experiments analysed by any of these means can prey proteins using polyclonal antisera (e.g. Coaker & be used to model predation rates (Mills 1997), and hence Williams 1963; Dennison & Hodkinson 1983; Sunderland evaluate different species of predator as biocontrol agents, et al. 1987; Cameron & Reeves 1990; Symondson et al. 1996) or to address fundamental questions in trophic ecology. All of these techniques detect the presence of prey Correspondence: W. O. C. Symondson. Fax: 44-29-20874305; E-mail: remains in the gut of predators but none in fact tells us pre- [email protected] cisely how the material got there. The assumption is almost

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always that direct predation on the prey has taken place. Plaster of Paris and charcoal base to ensure high humidity. Although in some cases other routes are mentioned, par- Following starvation for 2 weeks, 16 spiders (eight male and ticularly scavenging and secondary predation (food chain eight female) were frozen as starved controls. All remaining errors have been reviewed by Sunderland 1996), serious spiders were fed ad libitum on live aphids, Sitobion avenae attempts to quantify these sources of potential error have (F.) (Hemiptera: Aphididae), for 2 h. Any spiders that rarely (if ever) been made. Secondary predation, also failed to eat aphids were excluded from the experiment. In termed hyperpredation (Sabelis 1992), is where a (primary) total, 16 spiders (eight male and eight female) were frozen predator, that has consumed the target prey, is consumed immediately after the feeding period. The remaining by a second predator (or scavenger). The second predator spiders were transferred to clean Petri dishes (prepared as then contains remains of the target prey without directly above). Further batches of spiders (eight male and eight killing that prey itself. female) were killed by freezing at 2, 4, 8, 16, 24, 36, 48, 72, There are many examples in the literature of generalist 96, 120, 144, 168, 192, 216, 240 and 260 h after feeding predators consuming other predators in the field (reviewed and stored at −20 °C. All samples, including starved in Sunderland 1996). Although the problem of secondary controls, were tested by enzyme-linked immunosorbent predation is frequently mentioned (Hagler & Naranjo 1996; assays (ELISA) (see below). Sunderland 1996; Symondson & Hemingway 1997; Morris Poecilus (= Pterostichus) cupreus (L.) (Coleoptera: Carabidae) et al. 1999; Sunderland et al. 2001; Symondson 2001), no were collected from winter wheat fields at Long Ashton previous studies have attempted to quantify its significance. Research Station, Bristol, UK, and maintained under the Tod (1973) developed a polyclonal antiserum against the same environmental conditions as the spiders. Following slug Deroceras reticulatum (Müller) and speculated that collection, P. cupreus were fed for ≈ 1 month on a diet of some of the predators she tested may have given a positive Calliphora vomitoria (L.) larvae that had been killed by freez- reaction due to secondary predation. No experiments to ing. All predators were maintained in separate 90 × 15 mm test this hypothesis were performed. In another study, triple-vented Petri dishes on damp filter paper. Following Dennison & Hodkinson (1983) were apparently unable to starvation for 2 weeks, 16 beetles (eight male and eight detect secondary predation using polyclonal antisera female) were frozen as starved controls. The remaining developed against a range of prey using precipitin tests, beetles were observed to feed ad libitum on freshly killed although no experimental details were provided. (by freezing) S. avenae for 2 h and any non-feeding indi- We report the first fully replicated and quantitative viduals were excluded from the experiment. We were not study of secondary predation using any of the ‘molecular’ interested in predatory behaviour in this experiment and approaches. We used an aphid-specific monoclonal antibody killing the aphids in this way ensured (and co-ordinated) (Symondson et al. 1999b) within a three species system rapid consumption by the beetles. Sixteen beetles (eight (aphid−spider−beetle) to investigate detection limits male and eight female) were frozen immediately after the following secondary predation. Our hypothesis was that if feeding period. All remaining beetles were transferred into beetles ate spiders that had fed recently on aphids, aphid clean Petri dishes and a further eight males and eight proteins would be detectable in the guts of the beetles. Our females were killed by freezing at 2, 4, 8, 16, 24, 36, 48, 72, aim was to quantify the limits of detectability within this 96, 120, 144, 168 and 192 h after the feeding period and system and thus determine whether secondary predation stored at − 20 °C. All samples, including starved controls, is likely to be a significant source of error in subsequent were tested by ELISA (see below). field experiments. Detection of secondary predation Materials and methods The structure of the experiment is described below and summarized in Fig. 1. Predator preparation and antigen decay rates L. tenuis and P. cupreus were collected and maintained Spiders, Lepthyphantes tenuis (Blackwall) (Araneae: Liny- under laboratory conditions for ≈ 1 month (as described phiidae), were collected from winter wheat fields at above). Approximately 1000 female L. tenuis were fed ad Horticulture Research International, Wellesbourne, UK. libitum on live S. avenae for 2 h following a 2-week starva- They were maintained in a controlled environment at 16 °C tion period. Any spiders that failed to feed were excluded, on an 16:8 light/dark cycle, a temperature comparable leaving a total of 808 female L. tenuis that were used for the with average summer temperatures in UK cereal crops quantification of secondary predation. Two hundred and (Harwood et al. 2001). Following collection, spiders were eight spiders were frozen before feeding on S. avenae, eight fed for ≈ 1 month on a diet of live Drosophila melanogaster of which were reserved as starved controls. A further 200 Meigen. Each spider was maintained separately in a were frozen immediately after feeding. All remaining 50 × 15 mm triple-vented Petri dish containing a damp spiders were transferred to clean Petri dishes, prepared as

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Sitobion avenae Lepthyphantes tenuis Poecilus cupreus Fig. 1 Structure of the experiments to quantify Prey First predator Second predator secondary predation, in which aphids were fed to fed to fed to spiders and then the spiders, containing aphid in their guts, were fed to carabids. n = 16 Killed after: Killed after: 0 h 0, 2 or 4 h Poecilus cupreus (eight male and eight female) for each combination of the treatments.

Aphids 2 h 0, 2 or 4 h

4 h 0, 2 or 4 h

Unfed spiders 0, 2 or 4 h

described above, and batches of 200 spiders frozen 2 and 1997). The homogenized samples were diluted to a final 4 h after end of the feeding period. concentration of ×20 000 (w/v) in PBS. Each sample was The 200 spiders, which were frozen immediately after added to two ELISA plate wells, at 200 µL per well, and left feeding on S. avenae, were fed ad libitum to 24 male and 24 to incubate overnight. All wells of the ELISA plate were female P. cupreus (which had been starved for 2 weeks prior washed three times with PBS−Tween (0.05% Tween 20) to the experiment). All beetles were observed to feed on the (Sigma-Aldrich, Poole, UK). The anti-aphid ascites, MdW- spiders and consumed either two or three adult L. tenuis 7(1)G1 (Symondson et al. 1999b), was diluted 1:4000 in (any that ate fewer than two spiders were rejected and PBS−Tween and 200 µL added to one only of the duplicate none ate more than three). Following a 2-h feeding period, wells for each test sample, whereas 200 µL of PBS−Tween 16 beetles (eight male and eight female) were killed by was added to the second well. The plates were allowed to freezing. All remaining beetles were transferred to clean incubate for 2 h to enable binding between the antigen 90 × 15 mm triple-vented Petri dishes on damp filter paper. and antibody. Plates were washed three further times with Further batches of eight male and eight female beetles were PBS-Tween. ImmunoPure® goat antimouse IgG horseradish frozen 2 and 4 h after the feeding period. peroxidase enzyme conjugate (Pierce, Rockford, IL, USA) Three further groups of 48 P. cupreus were fed on either was diluted 1:4000 in PBS−Tween, 200 µL added to all ELISA two or three L. tenuis, which had been killed either 2 or 4 h plate wells and allowed to incubate for 1 h so that the after feeding on S. avenae, or which had been starved. conjugate could bind to the mouse antibodies. Plates were Batches of 16 beetles from each of the groups were then washed a further three times with PBS−Tween, and 200 µL frozen 0, 2 and 4 h after feeding on the spiders. of the enzyme substrate, o-phenylenediamine in a citrate− phosphate buffer, added to wells and placed in the dark for 30 min to allow colour development. The reaction Sample processing and ELISA µ was stopped by adding 50 L per well of 2.5 m H2SO4. All spiders were weighed, homogenized and diluted ×40 Absorbance readings were recorded at 492 nm using an (w/v) in phosphate-buffered saline, pH 7.4 (PBS). The ELISA plate spectrophotometer (Thermomax Plate Reader, homogenate was dispersed for 1 min on a vortex mixer Molecular Devices, CA, USA). Absorbance readings for and centrifuged at 8000 g for 15 min at room temperature. duplicate wells, to which no ascites were added, were The particulate remains were discarded and the supernatants subtracted from readings for wells to which antibody was transferred into clean 0.5 mL Eppendorf tubes and stored added, to eliminate the effects of non-specific binding at −20 °C. (Symondson et al. 2000). Each P. cupreus was allowed to thaw at room temperature Each ELISA plate also included a dilution series (×1.5) and the foregut, or crop, removed as described in detail in of S. avenae standards that provided absorbance readings Symondson et al. (2000). The beetle was carefully teased for aphid protein concentrations between 265.5 and apart between the thorax and abdomen, allowing the 4.6 ng per 200 µL. Aphid protein concentrations were foregut to be pulled away and removed from the body. The calculated following a protein assay using the Bio-Rad foregut was homogenized in PBS to a ×20 dilution (w/v) Protein Assay System (Bio-Rad Laboratories Ltd, GmbH, then dispersed for 1 min on a vortex mixer and centrifuged Munich, Germany). In order to stabilize protein concentra- at 8000 g for 15 min at room temperature. The supernatant tions throughout the dilution series, the aphid standards was transferred to a clean 1.5 mL Eppendorf tube and were diluted with heterologous protein, also diluted 1:20 000 stored at −20 °C. (w/v) in PBS (Symondson & Liddell 1995). In these All invertebrate samples were screened by indirect samples, the heterologous proteins were either starved ELISA at room temperature (Symondson & Hemingway L. tenuis (for the L. tenuis antigen decay experiment) or

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250 (a) Female Results l µ Rate of decay of aphid remains within the gut of 200 Lepthyphantes tenuis The rates of aphid antigen decay in male and female 150 Lepthyphantes tenuis are presented in Fig. 2. The anti-aphid monoclonal antibody MdW-7(1)G1 was shown in an earlier experiment, using the predator Pterostichus melanarius

100 (Illiger), to detect an initial increase in binding to antigen, probably as digestion revealed internal binding sites within target molecules (Symondson et al. 2000). This pattern was repeated here during digestion by L. tenuis. As prey 50 consumption by the predators (both spiders and carabids) could have taken place at any time during the 2 h feeding period, consumption was deemed to have taken place at 0 the mid-point (after 1 h) and thus 1 h has been added to 0 100 200 300 each time interval in Figs 2 and 3 and during regression Time after feeding (hours) analyses (Symondson & Liddell 1993). The rate at which aphid protein within the gut of these spiders decayed showed a linear relationship between 17 250 and 169 h after feeding, for both female and male spiders. (b) Male Using the regression equations, the antigenic half-life l Aphid protein equivalent in ng / 200

µ (the time taken for antigen concentration to decrease to half

200 its original level, which was recorded immediately after feeding) was 101.1 h in females and 105.4 h in males. Analysis of covariance indicated that there was no signiﬁc- ant difference between the rates at which the two slopes 150 declined (F1,14 = 0.13, P > 0.05) or the y-axis intercepts (F1,14 = 0.14, P > 0.05). As the weights of spiders fed ad libitum on aphids differed signiﬁcantly between the sexes 100 [mean (± SE) males, 1.55 ± 0.086 mg; females 3.17 ± 0.363 mg;

F1,14 = 18.96, P < 0.001], but y-axis intercepts did not, the ratios between the mean size of each sex, and the quantity 50 of prey each sex consumed, must have been similar.

Aphid protein equivalent in ng / 200 Mann–Whitney U-tests were used to compare aphid protein equivalents for starved spiders with those that had 0 digested their prey for 169 h or more. Aphid was shown to 0 100 200 300 be detectable in female L. tenuis for up to 193 h (U = 3.0, Time after feeding (hours) n = 16, P < 0.001) and in males for up to 169 h (U = 7.0, Fig. 2 Relationship between Sitobion avenae protein concentration n = 16, P < 0.01). equivalents in the foreguts of (a) female and (b) male Lepthyphantes tenuis and time. Regression lines show linear relationship between 17 and 169 h after feeding. Female regression: y = 226.43 – 1.27x; Rate of decay of aphid remains within the gut of r2 = 0.99. Male regression: y = 224.16 – 1.30x; r2 = 0.99. Poecilus cupreus The rates of aphid antigen decay in male and female P. cupreus are shown in Fig. 3. As with P. melanarius and foreguts of starved P. cupreus (for the secondary predation L. tenuis, detection of antigen increased initially. There and P. cupreus antigen decay experiments). By regress- was a linear rate of antigen decay between 17 and 97 h after ing protein concentrations against absorbance readings, feeding, for both male and female beetles. The half-lives regression equations were obtained from the aphid stand- of the antigen in female (70.8 h) and male (70.8 h) beetles ards on each ELISA plate. These were used to calculate were considerably less than those calculated for the spiders. aphid protein concentration equivalents for predator Analysis of covariance indicated that there was no signiﬁcant samples. difference between the rates at which the antigen decayed

ANTIBODIES TO DETECT SECONDARY PREDATION 2053

250 digested their prey for 145 h or more. Aphid was shown to (a) Female be detectable in female beetles for up to 169 h (U = 9.0, l

µ n = 16, P < 0.05) and in males for up to 145 h (U = 6.0, n = 16, 200 P < 0.01). As there was no signiﬁcant difference between aphid protein decay within the two sexes of L. tenuis or P. cupreus, 150 data were pooled before between-species comparisons were made. Analysis of covariance indicated that there were highly signiﬁcant differences between the rates of antigen decay in the two species (F = 78.66, P < 0.001) 100 1,11 and the y-axis intercepts (F1,11 = 17.73, P < 0.001). Higher concentrations of aphid protein in the dissected guts of the beetles would be expected in comparison with whole- 50 body extracts from the spiders. Aphid protein equivalent in ng / 200

0 Rate of decay of aphid remains following secondary 0 40 80 120 160 200 predation Time after feeding (hours) The rates of aphid antigen decay following secondary predation are shown in Fig. 4. Statistical comparisons using the Mann–Whitney U-test were made with starved L. tenuis 250 controls (Fig. 4). The results show clear detection of aphid (b) Male protein equivalents only in beetles that were frozen imme- l

µ diately after feeding on spiders that were in turn frozen 200 immediately after feeding on Sitobion avenae (P < 0.001). However, signiﬁcant detection was also found for female (but not male) beetles killed 2 h after feeding on spiders

150 that were themselves killed immediately after feeding on aphids (P < 0.05) (Fig. 4a). However, when thresholds for significance in relation to other non-aphid prey, that might be consumed by the beetles in the field, are applied 100 (Symondson et al. 1999b), none of this latter group of beetles would be deemed to contain aphid (see Discussion). If the beetles have 2 h or more to digest the spiders, aphid within 50 those spiders could not be detected, even if the spiders were killed immediately after feeding on aphids (Fig. 4b,c). Aphid protein equivalent in ng / 200 Beetle foreguts containing starved spiders did not react 0 with the monoclonal antibody (Fig. 4d). 0 40 80 120 160 200 The mean foregut weight of P. cupreus fed aphids (mean Time after feeding (hours) weight ± SE = 9.31 ± 1.20 mg) was significantly heavier than that of beetles that were fed spiders (mean weight ± Fig. 3 Relationship between Sitobion avenae protein concentration ± equivalents in the foreguts of (a) female and (b) male Poecilus cupreus SE = 5.87 0.41 mg; t18 = 2.68, P < 0.05). Both groups of and time. Regression lines show linear relationship between 17 and beetles were fed ad libitum with prey (rejecting beetles that 97 h after feeding. Female regression: y = 245.83 – 2.00x; r2 = 0.99. ate fewer than two prey items), and therefore over the 2-h Male regression: y = 255.36 – 2.22x; r2 = 0.99. feeding period a greater quantity of aphid material was consumed.

in male and female beetles (F1,8 = 4.10, P > 0.05) or between the y-axis intercepts (F = 2.35, P > 0.05). The foregut 1,8 Discussion weights of the two sexes, fed ad libitum on aphids, were not signiﬁcantly different either (mean ± SE males 9.06 ± 2.28 mg; Secondary predation has been largely ignored in studies ± females 9.55 1.03 mg; F1,14 = 0.04, P > 0.05) indicating using antibodies to assess predation, despite the obvious that males and females ate the same quantity of aphids. implications in terms of food chain errors (Sunderland 1996). Mann–Whitney U-tests were used to compare aphid protein That food chain errors could lead to misinterpretation of equivalents for starved P. cupreus with those that had results was acknowledged in only 7% of 72 studies reporting

2054 J. D. HARWOOD ET AL.

(a) Fig. 4 Detection of Sitobion avenae proteins 20 20

l (i) Female (ii) Male following secondary predation. Information is µ presented for Poecilus cupreus fed Lepthyphantes 16 1 16 tenuis which were killed (a) 0 h, (b) 2 h, (c) 4 h *** ***3 after feeding on S. avenae, and (d) P. cupreus 12 12 which were fed starved L. tenuis and frozen at set time intervals following feeding. Mean (± SE) are presented for starved P. cupreus (Pc) and 8 8 starved L. tenuis (Lt) (and therefore do not relate to time periods on the x-axis). Signiﬁcance 2 4 * 4 compared against aphid protein equivalent for Pc Lt Pc Lt starved L. tenuis and presented as ***P < 0.001, Aphid protein equivalent in ng / 200 0 0 *P < 0.05. 1Mann–Whitney U = 0.0, n = 16, 024 024 P < 0.001; 2U = 11.0, n = 16, P < 0.05; 3U = 0.0, Time after feeding (hours) n = 16, P < 0.001. (b) 20 20 l

µ (i) Female (ii) Male

16 16

12 12

8 8

4 4

Pc Lt Pc Lt Aphid protein equivalent in ng / 200 0 0 024 024 Time after feeding (hours) (c) 20 20 l (i) Female (ii) Male µ

16 16

12 12

8 8

4 4

Pc Lt Pc Lt

Aphid protein equivalent in ng / 200 0 0 024 024 Time after feeding (hours) (d) 20 20 l

µ (i) Female (ii) Male

16 16

12 12

8 8

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Pc Lt Pc Lt

Aphid protein equivalent in ng / 200 0 0 024 024 Time after feeding (hours)

ANTIBODIES TO DETECT SECONDARY PREDATION 2055

the use of antibody techniques published between 1956 giving the strongest non-specific cross-reaction. During and 1994 (Sunderland 1996). Should secondary predation characterization of the MdW-7(1)G1 antibody this proved be a significant factor, generalist predators collected from to be the whitefly Trialeurodes vaporariorum (Westwood) the field would yield unknown numbers of false positives, (Symondson et al. 1999b). Subsequent screening of potential resulting in both quantitative and qualitative misinter- prey from the field has, to date, revealed no other species pretation of trophic links. Our experiments were designed that gives a stronger reaction (JD Harwood, unpublished to test rigorously the hypothesis that secondary predation data). Based upon this calculation, a beetle gut sample was detectable, using a system that would maximize the would only be considered significant if it gave an aphid possibility of such detection. This was achieved by, first, protein equivalent of 16.5 ng/200 µL at the ×20 000 ELISA using a monoclonal antibody that bound with an epitope dilution. When this is used as the cross-reaction signific- that had been shown, in a previous study, to increase in ance level, only two female and three male P. cupreus gave availability, and hence antibody−antigen binding, over the a positive reaction when killed immediately after feeding first few hours of digestion (Symondson et al. 1999b; Figs 2 on at least two spiders that were killed immediately after and 3). By contrast, most other monoclonal antibodies and feeding on aphids. None of the beetles killed 2 h after feeding polyclonal antisera have demonstrated exponential rates of on spiders that were killed immediately after feeding on antigen decay that are most rapid immediately after prey aphids (Fig. 4a) would have been positive using this criterion. consumption (e.g. Sopp & Sunderland 1989; Symondson & Very few studies have compared the rates at which Liddell 1995; Hagler & Naranjo 1997; Symondson et al. antigens decay in male and female predators. Symondson 1997, 1999a). Second, we chose web-building spiders as et al. (1999a), using a species-specific monoclonal antibody, the first predator. It has been shown in previous work that reported that the detection period for male P. melanarius antigen decay rates in such spiders can be particularly long, was ≈ 30% longer than for females feeding on the slug possibly because they use the branching of the midgut to Arion hortensis (Férussac). This contrasts with the results store, for extended periods, excess food following feeding presented here which showed no evidence for differential on a particularly large meal (Nakamura & Nakamura 1977) antigen decay rates between male and female L. tenuis and/or can vary their metabolic rates in response to or P. cupreus. Given that male spiders were considerably starvation (Anderson 1970). Indeed, the rate of antigenic smaller than the females it appears that both sexes con- decay within the carabid Poecilus cupreus was found to be sumed a similar proportion of aphid prey relative to their ≈ 60% faster than within the spider Lepthyphantes tenuis. size, hence y-axis intercepts and antigen decay rates were Thus, the properties of both the monoclonal antibody and very similar. In the field, however, males of most species the first predator should have maximized overall detection tend to be more active than females (Sunderland 1987; periods, and hence enhanced our ability to detect aphid Topping & Sunderland 1992; Harwood et al. 2001), which remains within the spiders after the latter had been could affect the rates at which antigens decay. Laboratory consumed by carabids. experiments do not take such factors into account. Despite using a system strongly biased in favour of our In conclusion, although it is conceivable that secondary hypothesis, the results suggest that secondary predation predation might be detected within our particular aphid− will rarely be detected in the field using this methodology. spider−carabid system in the field, it is highly unlikely To detect clearly such an effect in our aphid−spider−beetle to be a significant factor. In most other studies using system, a beetle must be caught and killed immediately antibody−antigen systems, antigenic recognition decreases after it has eaten at least two spiders, which had, in turn, exponentially immediately after the predator has fed on just consumed aphids. This is an unlikely scenario. Addi- the prey, making detection of prey after it has gone through tional positive results were obtained from female beetles two predators even less likely. The antigen half-lives and killed within 2 h of feeding on at least two spiders that detection periods recorded in these experiments were were eaten immediately after feeding on aphids. When the considerably longer than those reported in any previous spiders were given 2 h or more to digest their aphid prey study using a monoclonal antibody. Although these results before being ingested by the beetles, aphid proteins could cannot be directly extrapolated to other predator food not be detected. If the beetles had more than 2 h to digest webs, or the use of alternative molecular detection systems the spiders, aphid within those spiders could not be detected (e.g. PCR), they do provide strong evidence that secondary either. In addition, it must be remembered that, in these predation will not a be major source of error in field studies experiments, the beetles were fed spiders containing aphids, using the MdW-7(1)G1 antibody, despite the extended or nothing. In the field the beetles may have eaten other detection periods that it provides. A situation in which non-aphid and non-spider prey. To guard against the secondary predation might, possibly, cause a measurable possibility of false positives, a significance level must be set, food chain error is where a predator species is known to be defined as the mean (plus 2. 5 SD) aphid protein equivalent feeding intensively on another predator species which is, (ng/200 µL) generated in an ELISA by the non-aphid prey in turn, feeding predominantly on the target prey. An

2056 J. D. HARWOOD ET AL.

example would be heteropteran predators consuming Greenstone MH, Morgan CE (1989) Predation on Heliothis zea: the lacewing larvae Chrysoperla carnea (Stephens), that an instar-specific ELISA for stomach analysis. Annals of the were controlling the aphid Aphid gossypii Glover on cotton Entomological Society of America, 84, 457–464. Hagler JR, Cohen AC, Bradley-Dunlop D, Enriquez FJ (1992) Field (Rosenheim et al. 1993). Also, in systems operating at ° evaluation of predation on Lygus hesperus using a species- and temperatures significantly below the 16 C used here stage-specific monoclonal antibody. Environmental Entomology, (Lister et al. 1987), rates of antigen inactivation are slower 21, 896–900. (temperature-dependent in poikilotherms) thus potentially Hagler JR, Naranjo SE (1996) Using gut content immunoassays extending detection periods and the opportunity to detect to evaluate predaceous biological control agents: a case study. secondary predation. If detection of secondary predation is In: The Ecology of Agricultural Pests: Biochemical Approaches (eds found within any food chain it could be eliminated from Symondson WOC, Liddell JE), pp. 383–399. Chapman & Hall, London. analyses either by using antibodies targeted at more labile Hagler JR, Naranjo SE (1997) Measuring the sensitivity of an indirect epitopes or by statistically raising the threshold for signific- predator gut content ELISA: detectability of prey remains in ance following ELISA. The former approach would result relation to predator species, temperature, time and meal size. in extra costs, the latter in a loss of information. It is encour- Biological Control, 9, 112–119. aging therefore that our results suggest that such measures Harwood JD, Sunderland KD, Symondson WOC (2001) Living are unlikely to be necessary. where the food is: web location by linyphiid spiders in relation to prey availability in winter wheat. Journal of Applied Ecology, 38, 88–99. Acknowledgements Heitmans WRB, Overmeer WPJ, van der Geest LPS (1986) The role of Orius vicinus Ribaut (Heteroptera: Anthocoridae) as a predator We would like to thank the Institute of Arable Crops Research, Long of phytophagous and predacious mites in a Dutch orchard. Journal Ashton, Bristol for allowing us to collect P. cupreus from their field of Applied Entomology, 102, 391–402. sites. We thank Cardiff University, Horticulture Research International, Hoogendoorn M, Heimpel GE (2001) PCR-based gut content analysis Wellesbourne, and the Biotechnology and Biological Sciences of insect predators: using ribosomal ITS-1 fragments from prey to Research Council for their financial support. Keith Sunderland was estimate predation frequency. Molecular Ecology, 10, 2059–2067. supported by the UK Ministry of Agriculture, Fisheries and Food. Lister A, Usher MB, Block W (1987) Description and quantification of field attack rates by predatory mites: an example using References electrophoresis method with a species of Antarctic mite. Oecologia, 72, 185–191. Agustí N, de Vicente MC, Gabarra R (1999) Development of sequence Mills N (1997) Techniques to evaluate the efficacy of natural enemies. amplified characterized region (SCAR) markers of Helicoverpa In: Methods in Ecological and Agricultural Entomology (eds Dent DR, armigera: a new polymerase chain reaction-based technique for Walton MP), pp. 271–291. CAB International, Wallingford, UK. predator gut analysis. Molecular Ecology, 8, 1467–1474. Morris TI, Campos M, Kidd NAC, Symondson WOC (1999) What Agustí N, de Vicente MC, Gabarra R (2000) Developing SCAR is consuming Prays oleae (Bernard) (Lep. Yponomeutidae) and markers to study predation on Trialeurodes vaporariorum. Insect when: a serological solution? Crop Protection, 18, 17–22. Molecular Biology, 9, 263–268. Murray RA, Solomon MG (1978) A rapid technique for analysing Anderson JF (1970) Metabolic rates in spiders. Comparative diets of invertebrate predators by electrophoresis. Annals of Applied Biochemistry and Physiology, 33, 51–72. Biology, 90, 7–10. Bacher S, Schenk D, Imboden H (1999) A monoclonal antibody to the Nakamura M, Nakamura K (1977) Population dynamics of the shield beetle Cassida rubiginosa (Coleoptera, Chrysomelidae): a chestnut gall wasp (Cynipidae, Hym.), 5. Estimation of the effect tool for predator gut analysis. Biological Control, 16, 299–309. of predation by spiders on the mortality of imaginal wasps based Cameron EA, Reeves RM (1990) Carabidae (Coleoptera) associated on the precipitin test. Oecologia, 27, 97–116. with Gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), Rosenheim JA, Wilhoit LR, Armer CA (1993) Influence of intraguild populations, subjected to Bacillus thuringiensis Berliner treatments predation among generalist insect predators on the suppression in Pennsylvania. Canadian Entomologist, 122, 123–129. of an herbivore population. Oecologia, 96, 439–449. Chen Y, Giles KL, Payton ME, Greenstone MH (2000) Identifying Sabelis MW (1992) Predatory arthropods. In: Natural Enemies: the key cereal aphid predators by molecular gut analysis. Molecular Population Biology of Predators, Parasites and Diseases (ed. Crawley MJ), Ecology, 9, 1887–1898. pp. 225–264. Blackwell Scientific Publications, Oxford. Coaker TH, Williams DA (1963) The importance of some Carabidae Sopp PI, Sunderland KD (1989) Some factors affecting the detection and Staphylinidae as predators of the cabbage root fly, Erioischia period of aphid remains in predators using ELISA. Entomologia brassicae (Bouché). Entomologia Experimentalis et Applicata, 6, 156 –164. Experimentalis et Applicata, 51, 11–20. Dennison DF, Hodkinson ID (1983) Structure of the predatory Sunderland KD (1987) Spiders and cereal aphids in Europe. Bulletin beetle community in a woodland soil ecosystem. I. Prey selection. SROP/WPRS, 1987/X/1, 82–102. Pedobiologia, 25, 109–115. Sunderland KD (1988) Quantitative methods of detecting inverte- Giller PS (1986) The natural diet of the Notonectidae: field trials brate predation occurring in the field. Annals of Applied Biology, using electrophoresis. Ecological Entomology, 11, 163–172. 112, 201–224. Greenstone MH (1996) Serological analysis of arthropod predation: Sunderland KD (1996) Progress in quantifying predation using anti- past, present and future. In: The Ecology of Agricultural Pests: body techniques. In: The Ecology of Agricultural Pests: Biochemical Biochemical Approaches (eds Symondson WOC, Liddell JE), Approaches (eds Symondson WOC, Liddell JE), pp. 419–455. pp. 265–300. Chapman & Hall, London. Chapman & Hall, London.

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Sunderland KD, Crook NE, Stacey DL, Fuller BJ (1987) A study of Symondson WOC, Liddell JE (1993) Differential antigen decay feeding by polyphagous predators on cereal aphids using ELISA rates during digestion of molluscan prey by carabid predators. and gut dissection. Journal of Applied Ecology, 24, 907–933. Entomologia Experimentalis et Applicata, 69, 277–287. Sunderland KD, Powell W, Symondson WOC (2001) Populations Symondson WOC, Liddell JE (1995) Decay rates for slug antigens and communities. In: Insect Natural Enemies (ed. Jervis M), 2nd edn. within the carabid predator Pterostichus melanarius monitored with Kluwer Academic Publishers, Dordrecht, in press. a monoclonal antibody. Entomologia Experimentalis et Applicata, Symondson WOC (2001) Diagnostic techniques in carabid research. 75, 245–250. In: The Agroecology of Carabid Beetles (ed. Holland J). Intercept Symondson WOC, Liddell JE (1996) A species-specific monoclonal Publishers, Andover, UK, in press. antibody system for detecting the remains of field slugs, Deroceras Symondson WOC, Erickson ML, Liddell JE (1997) Species-specific reticulatum (Müller) (Mollusca: Pulmonata), in carabid beetles detection of predation by Coleoptera on the Milacid slug (Coleoptera: Carabidae). Biocontrol Science and Technology, 6, 91–99. Tandonia budapestensis (Mollusca: Pulmonata). Biocontrol Science Tod ME (1973) Notes on beetle predators of molluscs. Entomologist, and Technology, 7, 457–465. 106, 196–201. Symondson WOC, Erickson ML, Liddell JE (1999a) Development Topping CJ, Sunderland KD (1992) Limitations to the use of pitfall of a monoclonal antibody for the detection and quantification traps in ecological studies exemplified by a study of spiders in a of predation on slugs within the Arion hortensis agg. (Mollusca: field of winter wheat. Journal of Applied Ecology, 29, 485–491. Pulmonata). Biological Control, 16, 274–282. Walrant A, Loreau M (1995) Comparison of iso-enzyme electro- Symondson WOC, Erickson ML, Liddell JE, Jayawardena KGI phoresis and gut content examination for determining the (1999b) Amplified detection, using a monoclonal antibody, of natural diets of the groundbeetle species Abax ater (Coleoptera: an aphid-specific epitope exposed during digestion in the gut Carabidae). Entomologia Generalis, 19, 253–259. of a predator. Insect Biochemstry and Molecular Biology, 29, Zaidi RH, Jaal Z, Hawkes NJ, Hemingway J, Symondson WOC 873–882. (1999) Can multiple-copy sequences of prey DNA be detected Symondson WOC, Glen DM, Erickson ML, Liddell JE, Langdon CJ amongst the gut contents of invertebrate predators? Molecular (2000) Do earthworms help to sustain the predator Pterostichus Ecology, 8, 2081–2087. melanarius (Coleoptera: Carabidae) within crops? Investigations using monoclonal antibodies. Molecular Ecology, 9, 1279–1292. Symondson WOC, Glen DM, Wiltshire CW, Langdon CJ, Liddell JE This work was part of a collaborative research programme into (1996) Effects of cultivation techniques and methods of straw the dynamics of predator–prey interactions within arable crops, disposal on predation by Pterostichus melanarius (Coleoptera: supervised by Bill Symondson at Cardiff University and Keith Carabidae) upon slugs (Gastropoda: Pulmonata) in an arable Sunderland at HRI-Wellesbourne. The experiments reported here field. Journal of Applied Ecology, 33, 741–753. were conducted by James Harwood as part of his PhD, assisted by Symondson WOC, Hemingway J (1997) Biochemical and molecular postdoctoral researcher Sarah Phillips. Our current research seeks techniques. In: Methods in Ecological and Agricultural Entomology to investigate the effects of non-aphid prey, and prey diversity, on (eds Dent DR, Walton MP), pp. 293–350. CAB International, regulation of aphids by generalist predators in cereal crops. Wallingford, UK.