SEARCH

SEARCH BY CITATION

Keywords:

  • alkaloids;
  • chemical defence;
  • chemical taxonomy;
  • Coccinellidae;
  • intraguild predation;
  • Harmonia axyridis;
  • trophic interactions

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • 1
    Predation can be difficult to measure in the field and immunological and DNA-based gut analyses are now routinely used to identify and quantify prey items consumed by predators. Alternative methods have largely fallen into disuse.
  • 2
    Chromatography has been largely ignored as a method of studying predation on the grounds of low specificity and an inability to provide quantitative results. We demonstrate here that this is not so, using gas chromatography–mass spectrometry of alkaloids of prey ladybird beetles.
  • 3
    The alkaloid hippodamine from a single egg of the ladybird Hippodamia convergens was detected in all ten third instar larvae of another ladybird, Harmonia axyridis, for 12 h after they had consumed a H. convergens egg; in one case of ten, hippodamine was still detected 36 h after egg consumption. Hippodamine was detectable in all 10 second instar larvae of the lacewing Chrysoperla rufilabris sampled 12 h after consuming a H. convergens egg.
  • 4
    The amount of hippodamine in H. axyridis larvae was quantifiable with an internal standard. Larvae that had consumed more eggs exhibited higher levels of hippodamine. The method therefore can be used to estimate the amount of prey biomass consumed.
  • 5
    A comparison of the alkaloids of five ladybird species that co-occur in Kentucky field crops found that, in general, the alkaloids were sufficiently distinct to allow species identification of ladybirds that had been consumed by predators, although there was some overlap between species in alkaloid content.
  • 6
    Especially when combined with mass spectrometry, chromatography is thus a method that potentially can be used to identify multiple prey simultaneously, while also obtaining quantitative information on the prey biomass consumed. This has not been achieved by the commonly-used molecular gut-analyses. We suggest the method is suitable for a wide diversity of prey types possessing endogenous taxon-specific chemicals such as defence compounds or pheromones.
  • 7
    As a secondary consequence of this study, several alkaloids have been identified from new ladybird species. We also have shown that H. axyridis, an invasive intraguild predator of other ladybirds, does not sequester alkaloids from allospecific ladybird prey.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The detection and quantification of natural predation in the field is frequently difficult due to the size, secretive habits or inaccessibility of predators and/or prey. Because long periods of direct observation are generally needed to obtain sufficient data to determine the strength of trophic associations, a series of increasingly ingenious methods for the indirect detection of predation have been utilized by biologists instead (Kiritani & Dempster 1973; Sunderland 1988; Sheppard & Harwood 2005). In particular molecular biological techniques, either immunological or DNA-based in nature, are now widely used for the analysis of gut or faecal content (Greenstone 1996; Symondson 2002; Sheppard & Harwood 2005). These methods are now so widespread that alternative approaches have, in large part, fallen into neglect.

Chromatographic techniques possess potential for the study of trophic associations; however, they have been infrequently used. Studies utilizing chromatography are largely limited to the identification of the diet of aquatic grazers, where the pigments of phototrophic microorganisms are used as taxonomic indicators (e.g. Quiblier-Llobéras et al. 1996; Haug et al. 2003), and for the identification of dietary toxic contaminants (e.g. Negri et al. 1995; Hoekstra et al. 2003). The use of chromatography to deduce predator-prey trophic links has been rare. Work relating to the sequestration of defensive chemicals by predators from prey has been relatively frequent (e.g. Saporito et al. 2007); however, such studies are generally concentrated on determining chemical sources rather than on trophic interactions per se (but see Becerro et al. 2006). Paper chromatography of mite pigments has been used to identify predation of the mites Panonychus ulmi and Bryobia arborea (= B. rubrioculus) (Putman 1965a,b, 1967; Putman & Herne 1966); however, using this method it was not possible to separate the two species when they occurred together (Putman 1965a) nor to separate them from pigments of another mite (Balaustium sp.) (Putman 1967). Multivariate analysis of gas chromatograms of stomach content, predominantly fatty acids, was found to be of use in deducing the prey of lobsters (Homarus gammarus) in laboratory experiments (Knutsen & Vogt 1985a,b); however, changes occurring during digestion and by the mixing of prey in the lobster stomach limited the utility of the approach (Knutsen & Vogt 1985b).

Thus in general chromatography has tended to be dismissed as possessing very limited resolution and no capacity to quantify the contribution of particular prey to a predator's diet (Sunderland 1988; Harwood & Obrycki 2005); however, this is arguably not the case. Semiochemicals such as pheromones or allomones may be more taxon-specific. In the case of defensive chemicals, even if not sequestered they may be more resistant to digestion than the chemical taxonomic markers used thus far. The use of high resolution techniques such as gas or liquid chromatography in combination with mass spectrometry may allow unambiguous identification of even small quantities of these substances. Furthermore, the use of such methods in combination with chemical standards of known quantity may allow the amount of prey biomass consumed to be estimated.

Ladybirds possess endogenously produced alkaloid defences that are present throughout the bodies of all life-history stages (Daloze et al. 1995; King & Meinwald 1996; Laurent et al. 2005). The alkaloids often comprise one or two major types (Daloze et al. 1995; King & Meinwald 1996) which vary phylogenetically: close relatives, such as congeners, often possess similar major alkaloids, although even congeneric species can possess different alkaloid defences (Daloze et al. 1995; Sloggett 2005). Ladybird communities appear to comprise species with a diversity of different alkaloid defences. The alkaloids identified from ladybirds appear to be largely restricted to these beetles. Outside of the Coccinellidae, such alkaloids have been identified from oribatid mites (Takada et al. 2005), and from frogs and toads, which apparently acquire them from their arthropod diet (Daly, Spande & Garraffo 2005).

A variety of generalist predators are reported sometimes consuming ladybirds, including birds and mammals, ants, spiders and other invertebrates (Majerus 1994; Ceryngier & Hodek 1996). However, many such observations are isolated and anecdotal and the extent of such predation in nature remains questionable. In recent years particular attention has focused on intraguild predation (IGP) among aphid consumers, including interspecific predation between aphidophagous ladybirds and predation occurring between ladybirds and other aphidophagous insects (Lucas 2005). This interest has arisen due to the perceived effect of IGP on biological control of aphids (Rosenheim et al. 1995) and because of concerns about how introduced ladybirds affect native species (Harmon et al. 2007; Pell et al. 2008). IGP involving ladybird prey has been well-characterized in the laboratory, varying with factors including size, morphology and defensive chemistry (e.g. Lucas et al. 1998; Sato & Dixon 2004; Ware & Majerus 2008). However, supporting field studies are few in number making the natural prevalence and role of IGP a matter for continuing debate (Cottrell & Yeargan 1998; Kindlmann & Houdková 2006; Harwood et al. 2007). Ladybirds thus comprise a clear example where the development of chromatographic methods to detect their predation is of value in future studies.

In this study, we test whether gas chromatography–mass spectrometry (GC–MS) of prey chemical defence can be used to detect and quantify predation, using ladybird beetles (Coleoptera: Coccinellidae) as model prey (Sloggett et al. 2006). Three specific questions are addressed. First, for how long can the alkaloids of ladybird prey be detected after they have been consumed? Second, is it possible to use alkaloids to estimate the quantity of prey material eaten? Third, within individual communities do ladybird prey differ sufficiently in their chemical defences to allow species-specific identification by GC–MS?

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

insect material

As prey for chemical analyses, we used eggs of the convergent ladybeetle, Hippodamia convergens Guérin-Méneville. This species was chosen as it is easily obtained in large numbers from biological control suppliers. Four alkaloids, hippodamine (Fig. 1 [1]) convergine (Fig. 1 [2]), n-octylamine (Fig. 1 [8]) and harmonine (Fig. 1 [9]) have been found in H. convergens (Tursch et al. 1974; Braconnier et al. 1985a; Braconnier et al. 1985b). Of these alkaloids, hippodamine is easily visualized using GC–MS (Fig. 2).

image

Figure 1. The chemical structures of the ladybird alkaloids discussed in this work.

Download figure to PowerPoint

image

Figure 2. Total ion chromatogram and mass spectrum of hippodamine from Hippodamia convergens. Peaks at the base of the main hippodamine peak, which have different mass spectra than hippodamine, are thermal degradation peaks of convergine, a second H. convergens alkaloid.

Download figure to PowerPoint

Adult H. convergens were purchased from a supplier (Biocontrol Network, Brentwood, TN) and maintained in Petri dishes under standard laboratory conditions. These were used for all adult and larval stocks and experiments [20 °C with a 16:8 h photoperiod; pea aphids, Acyrthosiphon pisum (Harris) daily provided as food with a 0·5 cm3 piece of apple as an additional fluid source]. The resulting eggs were used in experiments. These were typically laid on the sides of the Petri dish, and when required were removed individually using a needle to manipulate them and separate them from the substrate. Eggs up to 2 days old were used in experiments.

Two types of predators were used in investigations. The main predator was another ladybird, Harmonia axyridis (Pallas). This species originates in Asia, but has been introduced and established in North America, Europe and elsewhere (Koch et al. 2006; Brown et al. 2008). Laboratory and field studies both indicate that H. axyridis is a strong intraguild predator of other ladybirds (e.g. Cottrell & Yeargan 1998; Ware & Majerus 2008), leading to concerns about its impact on native ladybirds where it has been introduced (Pell et al. 2008). Adult H. axyridis were collected from the wild in Lexington, KY and eggs obtained in a manner similar to that used for H. convergens. The resulting larvae were reared to the third instar, at which time they were used in experiments. The second predator used was the second instar larva of the chrysopid lacewing Chrysoperla rufilabris (Burmeister). Lacewing larvae are known predators of ladybirds in the wild (Principi & Canard 1984) and C. rufilabris has been shown to be an intraguild predator of ladybirds in the laboratory (Lucas et al. 1998). Eggs of C. rufilabris were obtained from a supplier (Harmony Farm Supply & Nursery, Sebastopol, CA) and the larvae reared to the second instar before being used in experiments.

Other ladybird species used to study alkaloid specificity [Coccinella septempunctata L., Coleomegilla maculata (ssp. lengi Timberlake), Cycloneda munda (Say) and Mulsantina picta (Randall)] were from laboratory cultures originating from ladybirds collected in Lexington.

experiment 1: duration of detection of hippodamine in harmonia axyridis larvae after predation of a single hippodamia convergens egg

Third instar H. axyridis larvae were individually starved for 6 h. During this time they only had access to water, provided in saturated cotton wool balls (c. 0·5 cm3). After the 6 h had elapsed, they were provided with a single H. convergens egg. When the egg had been consumed, larvae were either frozen immediately, at −20 °C or provided again with A. pisum and apple for 3, 6, 12, 18, 24, 30 or 36 h and then frozen. Larvae frozen after 30 or 36 h were given fresh food after c. 24 h. Additionally, negative control larvae were frozen directly after the 6 h starvation, without being further fed eggs or aphids; positive controls were individual frozen H. convergens eggs.

experiment 2: is hippodamine detectable in chrysoperla rufilabris that have been fed a single hippodamia convergens egg?

In order to test whether H. convergens alkaloid is likely to be detected in other predators, a second predator, C. rufilabris was used. In contrast to experiments with H. axyridis we did not attempt to determine the length of time for which H. convergens alkaloid was detectable. Lacewing larvae posses blind-ended guts and do not defaecate until adulthood (Killington 1936); this apparently leads to longer detection times in gut analyses using this group (see Putman 1965a; Fournier et al. 2006). Our experiment was therefore limited to testing whether alkaloid was detectable at all, rather than for how long it was detectable. Second instar C. rufilabris larvae were treated in the same manner as H. axyridis larvae in Experiment 1. Larvae were frozen directly after egg consumption or 12 h after egg consumption. Negative and positive controls were similar to those described for H. axyridis.

experiment 3: quantification of hippodamine from hippodamia convergens eggs consumed by harmonia axyridis larvae

Third instar larvae of H. axyridis were treated as described previously, but were provided with either one or three H. convergens eggs to eat. They were then frozen directly or 3 h after egg consumption. Single eggs or groups of three eggs of H. convergens were also frozen, as controls. Since the experiment aimed to examine alkaloid quantity, rather than alkaloid presence/absence, no (negative) unfed larval controls were used.

preparation of material and gc–ms analysis

Each larval or egg sample was thoroughly homogenized in 100 µL methanol (HPLC grade). After homogenization, the sample was filtered by running 75 µL of the methanol supernatant and 2 × 100 µL methanol through a 60 mg Florisil column (30–60 mesh). The filtrate was then partitioned by adding 400 µL of hexane (HPLC grade, distilled) and shaking vigorously; the hexanic upper phase was discarded. The lower phase was dried under a stream of nitrogen and redissolved in 100 µL methanol. This solution was transferred to an autosampler vial, the methanol was evaporated under a nitrogen stream and the dried extract redissolved in 20 µL methylene chloride (HPLC grade, distilled). For samples from Experiment 3 (quantification), 3 µL of 100 ng µL−1 Z-13-octadecenol in methylene chloride were added to the methanol as an internal standard before homogenization of the sample.

GC–MS analyses were carried out on a Hewlett-Packard 6890 gas chromatograph with a HP 7683 autosampler coupled to a HP 5973 mass spectrometer. A split-splitless injector at 200 °C and a DB5 GC column (0·25 mm diameter; 30 m length; 0·25 µm film thickness) were used. The carrier gas was helium with a flow rate of 1 mL min−1. Mass spectra were obtained using electron ionization mode at 70 eV, scanning was done for the range m/z 35–400. The GC temperature program used was 60 °C for 2 min, then an increase of 10 °C min−1 up to 325 °C, with the final temperature being held for 15 min. The long program and hold time at the end was primarily to eliminate sample contaminants from the column.

Of the final methylene chloride extract, 2 µL was injected, i.e. an extract from 7·5% of each predator or control egg. Samples were injected as randomized blocks comprising one sample of each treatment and controls where used. To avoid carry-over of hippodamine or other contaminants across samples, a methylene chloride blank was injected between them. To ensure that sensitivity remained the same throughout analyses, a hippodamine standard purified from H. convergens eggs was tested between blocks and the electron multiplier detector voltage adjusted to ensure that the hippodamine peak was always of a similar-size (total ion count c. 30 000 for a 0·4% egg extract, easily distinguished above background).

identification and quantification of hippodamine in extracts and analysis of results

Standardized criteria for the detection of hippodamine (and thus predation of H. convergens) were used throughout the study. These were the occurrence of a mass spectrum containing the four characteristic ions of hippodamine (m/z 150, 164, 178 and 192; see Fig. 2) occurring within 0·02 min of a hippodamine peak derived from a standard. Egg hippodamine standards were generally injected at the beginning and end of a block (see above); on occasions (e.g. if peak drift was suspected to be occurring within a block) additional standards were used within a block. In some predators, particularly those that had consumed a H. convergens egg 12 h or more before, it was not possible to distinguish a clear hippodamine peak in total ion chromatograms. In these cases, hippodamine was searched for by using a selected ion count to see if the 192 ion was present, this being the commonest of the four characteristic hippodamine ions. However, only if the other characteristic ions were also present according to the criteria described was the sample considered to contain hippodamine. In samples from Experiment 3, hippodamine was quantified by area comparison to the Z-13-octadecenol peak, which was standardized at 300 ng sample−1.

For Experiment 1 (duration of hippodamine detection) the average time for hippodamine to still be detected after egg consumption by a H. axyridis larva was estimated using a method described by Hoogendoorn & Heimpel (2001). Briefly, the average time span for hippodamine marker detection was calculated using weighted proportions of H. axyridis larvae testing positive for hippodamine, with standard bootstrapping (1000 iterations) used to determine the 95% confidence interval.

For Experiment 3, quantitative data was analyzed using two-way anovas with main effects of time (with or without the egg controls) and egg number. Two anovas were carried out, one including the egg controls and one without the controls included. Data was log transformed to exclude an association between large means and high variances. Analysis was carried out in SPSS 8·0 for Windows, using Type III Sum of Squares and, where applicable, Sidak pairwise multiple comparisons.

experiment 4: the specificity of chemical defences of kentucky ladybirds

To examine whether the chemical defences of ladybirds were sufficiently specific to facilitate species identification of prey ladybirds, the alkaloids of six of the most important species in Kentucky were compared. Four, C. septempunctata, C. maculata, C. munda and H. axyridis comprise the vast majority of ladybirds occurring in Kentucky field crops; the fifth, H. convergens, also used in other experiments, was formerly a common crop species but has declined in recent years (R. T. Bessin pers. commun.; G.C. Brown pers. commun.), while the sixth, M. picta is one of two predominant species on pine trees, the other being H. axyridis.

Extractions and GC–MS analyses were similar to those described above, but were performed using manual injection on a HP 5890A GC coupled to a HP 5972 MS, with a size scan of m/z 35–500. The alkaloids of three of these species (C. septempunctata, H. axyridis, and H. convergens) were already known (Tursch et al. 1971a,b, 1974; Braconnier et al. 1985a,b; Alam et al. 2002) and in these cases GC–MS analysis was concentrated on identifying alkaloids visible using GC–MS that were suitable as taxonomic markers (i.e. species-specific and easily differentiated from other alkaloids). In the other species the alkaloids were unknown (C. munda, M. picta) or were a matter of dispute (C. maculata: Henson et al. 1975; Ayer & Browne 1977): in these cases it was also necessary to identify alkaloids using GC–MS as well as assessing their suitability as markers.

The H. axyridis alkaloid harmonine cannot be detected using our GC–MS method without derivatization. To derivatize harmonine, samples dissolved in 20 µL methylene chloride were heated for 1 h to 100 °C with 10 µL N-methyl-bis (trifluoroacetamide) (MBTFA) (Donike 1973; Attygalle 1998). The resulting harmonine bis-trifluoroacetyl derivative was detectable using the GC–MS methods described above.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

experiment 1: duration of detection of hippodamine in harmonia axyridis larvae after predation of a single hippodamia convergens egg

Hippodamine, and thus predation of an H. convergens egg, was detected in all samples up to 12 h and thereafter in a decreasing number of samples with time (Fig. 3; n = 10 for each treatment and control). In all samples up to 12 h after egg consumption a clear hippodamine GC peak was observable from predators that had consumed eggs (Fig. 4). Peak size declined over time, and from 12 h on, an increasing number of samples in which hippodamine was detected did not exhibit a clear GC peak; however, the mass spectrum of hippodamine was still located using a post hoc selected ion count (Fig. 3). Partial mass spectra (typically ions of m/z 150 and 192) were observed in some samples 18 h or more after egg consumption: these were scored as negative according to our criteria (see above). Clear hippodamine GC peaks and mass spectra were obtained from all H. convergens eggs (positive controls) and no hippodamine was detected in any larvae that had not eaten H. convergens eggs (negative controls).

image

Figure 3. Results of Experiment 1, showing the number of H. axyridis larvae in which hippodamine was detected for different time periods after eating a H. convergens egg, and two controls (a positive one comprising a H. convergens egg and a negative one comprising an unfed H. axyridis larva which was frozen directly after starvation without being given an egg). Ten samples were analyzed for each treatment or control. Black bars indicate that a clear hippodamine peak was visible on the gas chromatogram whereas grey bars indicate that hippodamine was detected using a post hoc selected ion search.

Download figure to PowerPoint

image

Figure 4. Total ion chromatograms of H. axyridis larvae from Experiment 1. (a) An unfed H. axyridis larva. (b) A larva that ate a H. convergens egg 6 h prior to sampling, exhibiting a large hippodamine total ion peak. (c) A larva that ate a H. convergens egg 18 h prior to sampling, with a small hippodamine total ion peak still visible. (d) A larva that ate a H. convergens egg 30 h prior to sampling: no hippodamine total ion peak is visible, but in this larva the occurrence of the four characteristic ions at the correct retention time in post hoc ion sampling meant that the larva could be scored positive for hippodamine.

Download figure to PowerPoint

For field studies it is important to establish the average time span since predation had occurred when a predation event is detected. If only samples in which a clear GC (total ion) peak were scored as positive then the average time span was 8·7 h, with a standard bootstrap 95% confidence interval of 7·2–10·1 h. If samples in which hippodamine was detected using post hoc ion selected ion search were included, the average time span increases to 13·5 h, with confidence interval of 12·2–14·8 h. It should be emphasized that this value is probably specific to the conditions under which the experiment was carried out (i.e. predation of a single egg with larvae maintained at 20 °C).

experiment 2: is hippodamine detectable in chrysoperla rufilabris that have been fed a single hippodamia convergens egg?

Hippodamine was detected in all C. rufilabris larvae that had eaten H. convergens eggs, both directly after predation (0 h) and 12 h after egg consumption; the alkaloid was detected in all positive H. convergens egg controls, and was not detected in any of negative control larvae that had not eaten eggs (n = 10 for each treatment and control). A clear GC peak was observed in all cases where hippodamine was detected, except one 12 h sample in which the characteristic hippodamine ions remained visible as mass spectra.

experiment 3: quantification of hippodamine from hippodamia convergens eggs consumed by harmonia axyridis larvae

Hippodamine was detectable in all samples analyzed for quantification (n = 6 for each treatment or control). The quantity of hippodamine in egg controls and larvae fed one or three eggs is shown in Fig. 5, with results of analyses shown in Table 1. An anova including egg controls as well as the two larval groups differing in time after egg consumption exhibits a highly significant time effect (Table 1anova 1). Sidak pairwise comparisons give no significant difference in hippodamine quantity between H. convergens egg controls and in H. axyridis larvae directly after egg consumption (P = 0·86), but there is significantly less hippodamine in larvae 3 h after egg consumption when compared to either egg controls or larvae directly after egg consumption (P < 0·001 in both cases). Thus the quantity of alkaloid begins to decline after egg ingestion.

image

Figure 5. Means and 95% confidence intervals for measurements of hippodamine quantity in one and three H. convergens eggs (left), and in larvae that have been fed one and three eggs immediately after feeding (middle) and 3 h after feeding (right). The means and confidence intervals here were calculated for log-transformed data and back-transformed.

Download figure to PowerPoint

Table 1.  Results of two-way anovas on log-transformed hippodamine quantity data obtained in Experiment 3 (for a summary of the data, see Fig. 5)
 dfSSFP
anova 1: including egg controls
 Time after consumption (+ controls)21·3526·6<0·001
 Number of eggs13·59141·4<0·001
 Interaction20·173·40·048
 Error300·76  
anova 2: egg controls not included
 Time after consumption11·1275·7<0·001
 Number of eggs11·94131·0<0·001
 Interaction10·106·80·017
 Error200·30  

There is a very strong effect of egg number on the quantity of hippodamine recorded, which was much higher in three-egg than one-egg samples (Fig. 5; Table 1anova 1). This effect persists if data from egg controls is excluded, leaving only data from the egg-eating H. axyridis larvae (Table 1anova 2). A weaker, but nonetheless significant interactive effect between time and egg quantity is also observed after the larvae have eaten eggs (Table 1anova 2). It thus appears that hippodamine declines more rapidly in larvae fed one egg than in those fed three eggs (Fig. 5).

In summary, hippodamine declines with increasing time after egg consumption, but remains, at least for the first 3 h, in greater quantities in predators that have consumed a greater biomass of H. convergens eggs.

experiment 4: the specificity of chemical defences of kentucky ladybirds

A summary of the characteristic alkaloids from the six Kentucky ladybirds examined is shown in Table 2 with ion chromatograms and mass spectra shown in Fig. 6. We found characteristic alkaloids that were not present in any of the other species for four of the five ladybird species of field crop habitats (Table 2). The fifth, H. axyridis, is known to possess two alkaloids, harmonine and 3-hydroxypiperidin-2-one (Alam et al. 2002) and it was possible to visualize harmonine using GC–MS by MBTFA-derivatization (see Materials and methods). However harmonine is also found in H. convergens (Braconnier et al. 1985a,b) and preliminary analysis suggested it also occurred in C. maculata, although in both species it appears to be a minor defence component when compared to H. axyridis. We were unable to detect 2-hydroxypiperidine-2-one using our GC–MS methods, although this alkaloid may well be unique to H. axyridis. Mulsantina picta, which is a species characteristic of pine trees, was found to possess hippodamine like H. convergens, which does not share its habitat. The chemical defence of M. picta is different from H. axyridis, which is the only other species commonly found in pines (Table 2); there was no evidence of harmonine in M. picta.

Table 2.  Species of Kentucky coccinellids, with habitat information, and their alkaloids. All known alkaloids are shown: the alkaloids considered most characteristic for the species in our GC–MS analyses are shown in bold. Numbers in parentheses refer to the chemical structures shown in Fig. 1
Ladybird speciesHabitatAlkaloids
  • To visualize harmonine using GC–MS derivatization with MBTFA was used (see Materials and Methods). Harmonine is considered characteristic of H. axyridis as it is the only H. axyridis alkaloid visible using GC–MS. Hippodamia convergens is known to contain this alkaloid and preliminary analyses suggest that it may also be present in C. maculata.

  • Both coccinelline and convergine thermally degrade with GC and are thus unsuitable for GC analysis; alkaloid peaks consistent with thermal degradation of convergine suggest that M. picta also contains convergine; however, further analyses are necessary to confirm this.

  • §

    We have not been able to visualize 3-hydroxypiperidin-2-one using our GC–MS method, although this may be a unique alkaloid marker for H. axyridis.

  • *

    *New alkaloid identification for this species.

Coleomegilla maculataField cropsmyrrhine[5], harmonine*†[9]
Coccinella septempunctataField cropsprecoccinelline [3], coccinelline[4]
Hippodamia convergensField cropshippodamine [1], convergine[2], n-octylamine [8], harmonine[9]
Cycloneda mundaField cropspsylloborine A (two double bond isomers)*[6,7]
Mulsantina pictaPine treeshippodamine*[1], convergine*‡[2]
Harmonia axyridisField crops/pine treesharmonine[9], 3-hydroxypiperidin-2-one§[10]
image

Figure 6. Selected ion chromatograms and mass spectra for five of the six Kentucky ladybird species analysed (for the sixth, H. convergens, see Fig. 2), showing characteristic alkaloids. For clarity, the gas chromatograms show additive readings of ions of m/z 188, 190, 192, 380, 405 and 474, which are characteristic ions of the alkaloids discussed and their derivatization or degradation products. Inset chromatograms in b, c and d are to facilitate resolution of the different peaks. Note the different retention times of the stereoisomers myrrhine (a), precoccinelline (b) and hippodamine (c), the latter two differing by about 6 s, and the patterns resulting from thermal degradation of coccinelline and convergine, which are consistent across samples. (e) and (f) show samples from H. axyridis without and with MBTFA derivatization, which is necessary to visualize the alkaloid harmonine. The retention time of hippodamine shown here differs slightly from that of Fig. 2 as a consequence of the different GC machines used for the two analyses, although retention times are consistent for individual machines.

Download figure to PowerPoint

The alkaloids analyzed possess sufficiently distinct retention times to allow them to be distinguished, even in cases where a predator has consumed more than one ladybird species. To test this, the two most similar, hippodamine and precoccinelline, which have similar mass spectra and retention times differing by only 6 s, were injected as a mixture. It was still possible to distinguish the peaks of the two alkaloids (Fig. 7).

image

Figure 7. Ion chromatograms of a mixed sample of alkaloids from C. septempunctata and H. convergens, including the stereoisomers precoccinelline and hippodamine, which possess similar mass spectra and retention times differing by only 6 s. The two alkaloids form distinct total ion peaks, although the hippodamine peak overlaps with a thermal degradation peak from another C. septempunctata alkaloid, coccinelline. An ion plot of the 192 ion, characteristic of precoccinelline and hippodamine makes clear that their peaks are distinguishable and do not overlap.

Download figure to PowerPoint

As a consequence of this work, the alkaloids of several species of ladybirds have been characterized for the first time or more fully characterized. Two GC peaks with a molecular ion of m/z = 380 and a base peak of m/z = 188 were observed in C. munda. These mass spectral properties are consistent with the alkaloids psylloborine A and isopsylloborine A, which are double bond isomers of each other (Schröder & Tolasch 1998; LeBrun et al. 1999), although we have not attempted to assign absolute configurations to the two C. munda alkaloids. Hippodamine, which is already described from diverse ladybirds (e.g. Tursch et al. 1974; Daloze et al. 1995; Sloggett 2005), is newly described from M. picta. Peaks surrounding the hippodamine peak in M. picta are also found in H. convergens, in which they have the same mass spectra. In H. convergens these peaks arise from the thermal degradation of convergine (hippodamine N-oxide), therefore we tentatively conclude that M. picta also possesses convergine. The main alkaloid of C. maculata is myrrhine (Ayer & Browne 1977) not precoccinelline (Henson et al. 1975): these stereoisomers possess similar mass spectra but myrrhine has a shorter retention time (see Fig. 6a and 6b). Harmonine also appears to be a component of C. maculata chemical defence.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

There are two common approaches currently used to detect natural predation events, using monoclonal antibodies and DNA (Sheppard & Harwood 2005). Both exhibit a high degree of specificity when required and the two techniques are arguably complementary. Monoclonal antibodies exhibit the highest specificity of all techniques, in insects even differentiating between life-history stages and instars; they can also possess very long detection times, extending to days after a predation event has occurred, and once monoclonal antibodies have been developed, large numbers of samples can be screened rapidly and cheaply (Greenstone 1996; Symondson 2002; Sheppard & Harwood 2005). Furthermore, the method may also be used to estimate how much prey biomass has been consumed (e.g. Crook & Sunderland 1984; Sopp et al. 1992). However, the development of highly specific individual antibodies for multiple prey is both costly and time-consuming (Chen et al. 2000), making this approach problematic in multiple prey systems.

In comparison to monoclonal antibodies, the time for which prey can be detected after predation has occurred is generally shorter for DNA-based techniques (Symondson 2002; Sheppard & Harwood 2005), being measured in hours rather than days after a predation event (e.g. Harwood et al. 2007). Additionally, the number of samples that can be screened using DNA-based techniques may be limited compared to antibody-based techniques due to the higher sample costs and time required for PCR (Sheppard & Harwood 2005). However, DNA-based techniques may potentially be used to rapidly screen for multiple target prey using multiplex-PCR (Harper et al. 2005), which can exhibit high taxon specificity (e.g. Traugott et al. 2006). Furthermore, quantitative PCR may allow the DNA-based estimation of prey biomass consumed (e.g. Weber & Lundgren in press). However, there does not seem to be any immediate prospect of DNA-based analysis of predation that is quantitative and can screen for multiple prey simultaneously.

In contrast to the two molecular techniques, high resolution chromatographic techniques, as illustrated by GC–MS here, would potentially allow both the detection of multiple prey (in this case different ladybird species) and the quantitative assessment of the prey biomass eaten. A single chromatographic run of a predator extract would allow separation of the alkaloids of a number of species and thus the simultaneous detection of multiple prey. Estimation of prey biomass consumed may be achieved by comparison with an internal standard.

For H. convergens alkaloids, the limits of detection are rather similar to those of DNA, being measured in hours rather than days after a predation event (Experiment 1). Nonetheless, for both H. axyridis and C. rufilabris, within 12 h of eating an egg all individuals tested positive for the H. convergens alkaloid hippodamine (Experiments 1 and 2). Using DNA, the signal begins to decline earlier after feeding, even in laboratory tests (e.g. Hoogendoorn & Heimpel 2001; Harwood et al. 2007). The higher initial detection levels with GC–MS may result from the direct measurement of the chemical marker in contrast to molecular detection using a chemical reaction or amplification process. Using post hoc selected ion sampling, distinguishing hippodamine in predators even without a GC peak was possible: the mass spectrum of hippodamine, with four diagnostic ions, is sufficiently distinctive to be distinguishable from background, lengthening the period of detectability. In the field predators are likely to consume a greater prey biomass than a single egg and, as other methods indicate that increasing prey biomass consumed is positively correlated with the length of time for which prey can be detected after ingestion (e.g. Hagler & Naranjo 1997), the experiments described here are probably conservative in their estimate of prey detection limits in field collected samples. Clearly the ability to detect prey may also be affected by a variety of other factors, such as temperature and the type of predators being tested (e.g. Hagler & Naranjo 1997). When ladybird predators are tested for C. septempunctata and C. maculata prey alkaloids, in addition to the H. convergens alkaloid tested here, the method gives results that are qualitatively similar, at least (Sloggett et al. 2006).

The results of Experiment 3 clearly show that the quantity of alkaloid present in a predator indicates the amount of prey biomass the predator has eaten. In consequence, the amount of alkaloid can be used to determine predation rates in the same way as measurements of prey immunoassay response intensity have previously been used (e.g. Sopp et al. 1992). The relationship between alkaloid and biomass consumed will be linear initially, although the interactive effect between time and egg number consumed suggests that the amount of alkaloid may decline exponentially, as can also occur in immunological studies (Sopp et al. 1992). Using either the chemical or immunological method laboratory experiments are necessary to determine the nature of the decay curve for each predator/prey combination; however, the direct measurement of alkaloid and standard rather than reliance on an immunological reaction may increase the stringency of the measurements obtained.

The quantitative use of chemical marker chromatography relies on two assumptions for accurate calculation. The first assumption is that there is limited variation in marker content between prey individuals; minimally any variability must not be discrete or polymodal, which would confound a correlation between prey alkaloid and biomass. The alkaloid concentration in different ladybird life-history stages is apparently similar (Daloze et al. 1995) and although there is some variation between individuals of the same stage, alkaloid concentration seems to be continuous and unimodal (Holloway et al. 1991; de Jong et al. 1991) and unlikely to confound biomass calculations. The second assumption is that predator consumption of the prey includes the parts containing the chemical marker. This is unproblematic in the case of ladybird prey, in which alkaloid is spread throughout the body (e.g. Holloway et al. 1991). However, particularly for prey taxa in which defensive chemicals are concentrated in glands or specific body parts that can be avoided by a predator (e.g. Reitze & Nentwig 1991), detection of predation and particularly quantification of prey biomass might be difficult. These caveats place limitations on the potential of using chromatography to quantify prey biomass consumed; nonetheless, if both assumptions are fulfilled, as ladybirds, the technique described here is expected to provide reliable results.

Earlier studies using chromatography to detect predation were undermined due to a lack of specificity in the markers involved and due to prey mixing and digestion in the stomach. Mixtures of pigments (cf. Putman 1965a) or fatty acids (cf. Knutsen & Vogt 1985b) generate complex results that are difficult to interpret and such problems can be reduced using a single distinctive chemical marker. Ideally the marker chemical should be endogenously produced, to avoid confusion between taxa sequestering chemicals from the same source, such as a host plant. However taxon specificity may still not be guaranteed: diverse groups may evolve to utilize the same chemical independently and close relatives may use identical chemicals by common descent.

Although the alkaloids of the Kentucky ladybirds described here (Experiment 4) included a number of stereoisomers, with similar mass spectra, they are sufficiently distinct that under conditions of mixing they can all be distinguished from each other. Analysis of a mixture of precoccinelline and hippodamine, the two most similar alkaloids in their mass spectra and retention times, indicated that these two can still be distinguished when occurring together. Ladybird species within the both crop field and pine habitats generally possessed at least one distinctive alkaloid, which would allow within-habitat species-level determination of prey ladybirds. However, there is overlap in the occurrence of harmonine in species of crops (H. axyridis, H. convergens and C. maculata). Although the apparent greater quantity of harmonine in H. axyridis and the presence of other alkaloids in the other two species allow separation in most circumstances, this might be problematic in a few cases (e.g. a single predator consuming H. axyridis and another harmonine-bearing species).

Chromatographic techniques are relatively cheap to use if technical facilities for their analysis are already available. The main costs associated with them are for consumables and solvents. However, the very high cost of chromatographs and mass spectrometers for chemical analysis, when compared to PCR machines or immunological plate readers, probably makes chromatography unfeasible if facilities are not already in place. Chromatography is also unsuitable for very large numbers of samples, as the time for preparation and analysis is long. In this study, c. 50 min was required to prepare each sample and each GC run was 44 min long. The latter was doubled by the inclusion of blank runs between samples, although little evidence was found of carry-over of alkaloid from one run to the next and therefore blank runs do not appear to be necessary. Nonetheless, on the basis of sample preparation times alone, it was not feasible to process more than 8–10 samples in a normal working day. This placed a serious limit on study sample sizes, although this is partially mitigated by the ability to simultaneously screen for multiple targets.

There have been two secondary benefits of this study. One has been the identification of a number of alkaloids from new species of ladybird. The second relates to IGP by H. axyridis. It is already clear that this species is a habitual intraguild predator of other ladybirds (Pell et al. 2008) and a potential benefit to H. axyridis of this behaviour would be if it could sequester the chemical defences of its intraguild prey for its own use. It seems clear from this study that H. axyridis does not sequester alkaloids from ladybird prey, as hippodamine is rapidly lost from the ladybird to undetectable levels within 2 days of consumption, as shown in Experiment 1. The presence of prey alkaloids in the gut, even were more prey biomass to be consumed, is too transient to confer much protection, particularly as any release of these alkaloids to a predator would involve severe injury to the ladybird. The absence of long-term sequestration is important in facilitating the use of chemical markers for biomass quantification. If the markers are retained for long periods, then biomass quantification is impossible.

In conclusion, although not suitable in all instances, chromatography of chemical markers exhibits potential as a means to detect and quantify predation. The animal groups in which at least some species possess endogenously produced chemical defences, for example, are diverse: they encompass not only beetles but taxa such as marine invertebrates, mites and other arthropod groups, including insects such as Lepidoptera and Heteroptera (e.g. Dettner et al 1996; Trigo 2000; Paul & Puglisi 2004; Millar 2005; Hoffmann et al. 2006; Saporito et al. 2007). Some other semiochemicals, notably pheromones, exhibit, if anything, higher taxonomic specificity and are also of value as chemical markers. Furthermore searchable internet databases of semiochemicals (e.g. <http://www.pherobase.com>) now provide a rapid means of identifying suitable marker candidates. Chromatography provides a high stringency alternative to conventional molecular techniques, which, unlike them, can potentially be used to simultaneously assess biomass consumed for multiple prey targets. Such a technique is likely to be valuable to researchers in the future.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Authors thank Daniel Bilotta, Susan Moser and Ilja Zeilstra for assistance provided with the collection and care of ladybird stocks and Shelby Stamper for technical assistance with the analyses. This is paper 08-08-050 of the Kentucky Agricultural Experiment Station.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Alam, N., Choi, I.S., Song, K.-S., Hong, J., Lee, C.O. & Jung, J.H. (2002) A new alkaloid from two coccinellid beetles Harmonia axyridis and Aiolocaria hexaspilota. Bulletin of the Korean Chemical Society, 23, 497499.
  • Attygalle, A.A. (1998) Microchemical techniques. Methods in Chemical Ecology. Volume 1. Chemical Methods (eds J.G.Millar & K.F.Haynes), pp. 207294. Kluwer Academic Publishers, Norwell.
  • Ayer, W.A. & Browne, L.M. (1977) The ladybug alkaloids including synthesis and biosynthesis. Heterocycles, 7, 685707.
  • Becerro, M.A, Starmer, J.A. & Paul, V.J. (2006) Chemical defenses of cryptic and aposematic gastropterid molluscs feeding on their host sponge Dysidea granulosa. Journal of Chemical Ecology, 32, 14911500.
  • Braconnier, M.F., Braekman, J.C. & Daloze, D. (1985b) Synthesis of the racemic form of (Z)-1, 17-diaminooctadec-9-ene, an aliphatic diamine from Coccinellidae. Determination of the absolute configuration of the (+)-naturally-occurring antipode. Bulletin des Societes Chimiques Belges, 94, 605613.
  • Braconnier, M.F., Braekman, J.C., Daloze, D. & Pasteels, J.M. (1985a) (Z)-1, 17-diaminooctadec-9-ene, a novel aliphatic diamine from Coccinellidae. Experientia, 41, 519520.
  • Brown, P.M.J., Adriaens, T., Bathon, H., Cuppen, J., Goldarazena, A., Hägg, T., Kenis, M., Klausnitzer, B.E.M., Kovář, I., Loomans, A.J.M., Majerus, M.E.N., Nedved, O., Pedersen, J., Rabitsch, W., Roy, H.E., Ternois, V., Zakharov, I.A. & Roy, D.B. (2008) Harmonia axyridis in Europe: spread and distribution of a non-native coccinellid. BioControl, 53, 521.
  • Ceryngier, P. & Hodek, I. (1996) Enemies of Coccinellidae. Ecology of Coccinellidae (eds I.Hodek & A.Honêk), pp. 319350. Kluwer Academic Publishers, Dordrecht.
  • Chen, Y., Giles, K.L., Payton, M.E. & Greenstone, M.H. (2000) Identifying key cereal aphid predators by molecular gut analysis. Molecular Ecology, 9, 18871898.
  • Cottrell, T.E. & Yeargan, K.V. (1998) Influence of a native weed, Acalypha ostryaefolia (Euphorbiaceae), on Coleomegilla maculata (Coleoptera: Coccinellidae) population density, predation and cannibalism in sweet corn. Environmental Entomology, 27, 13751385.
  • Crook, N.E. & Sunderland, K.D. (1984) Detection of aphid remains in predators by ELISA. Annals of Applied Biology, 105, 413422.
  • Daloze, D., Braekman, J.-C. & Pasteels, J.M. (1995) Ladybird defence alkaloids: structural, chemotaxonomic and biosynthetic aspects (Col.: Coccinellidae). Chemoecology, 5/6, 173183.
  • Daly, J.W., Spande, T.F. & Garraffo, H.M. (2005) Alkaloids from amphibian skin: a tabulation of over eight-hundred compounds. Journal of Natural Products, 68, 15561575.
  • De Jong, P.W., Holloway, G.J., Brakefield, P.M. & De Vos, H. (1991) Chemical defence in ladybird beetles (Coccinellidae). II. Amount of reflex fluid, the alkaloid adaline and individual variation in defence in 2-spot ladybirds (Adalia bipunctata). Chemoecology, 2, 1519.
  • Dettner, K., Scheuerlein, A., Fabian, P., Schulz, S. & Francke, W. (1996) Chemical defense of giant springtail Tetrodontophora bielanensis (Waga) (Insecta: Collembola). Journal of Chemical Ecology, 22, 10511074.
  • Donike, M. (1973) Acylierung mit Bis(acyamiden). N-Methyl-bis(trifluoracetamid) and Bis(trifluoracetamid), zwei neue Reagenzien zur Trifluoracetylierung. Journal of Chromatography, 78, 273278.
  • Fournier, V., Hagler, J.R., Daane, K.M., De León, J.H., Groves, R.L., Costa, H.S. & Henneberry, T.J. (2006) Development and application of a glassy-winged and smoke-tree sharpshooter egg-specific predator gut content ELISA. Biological Control, 37, 108118.
  • Greenstone, M.H. (1996) Serological analysis of arthropod predation: past, present and future. The Ecology of Agricultural Pests: Biochemical Approaches (eds W.O.C.Symondson & J.E.Liddell), pp. 265300. Chapman & Hall, London.
  • Hagler, J.R. & Naranjo, S.E. (1997) Measuring the sensitivity of an indirect predator gut content ELISA: detectability of prey remains in relation to predator species, temperature, time, and meal size. Biological Control, 9, 112119.
  • Harmon, J.P., Stephens, E. & Losey, J. (2007) The decline of native coccinellids (Coleoptera: Coccinellidae) in the United States and Canada. Journal of Insect Conservation, 11, 8594.
  • Harper, G.L., King, R.A., Dodd, C.S., Harwood, J.D., Glen, D.M., Bruford, M.W. & Symondson, W.O.C. (2005) Rapid screening of invertebrate predators for multiple prey DNA targets. Molecular Ecology, 14, 819828.
  • Harwood, J.D. & Obrycki, J.J. (2005) Quantifying aphid predation rates of generalist predators in the field. European Journal of Entomology, 102, 335350.
  • Harwood, J.D., Desneux, N., Yoo, H.J.S., Rowley, D.L., Greenstone, M.H., Obrycki, J.J. & O’Neil, R.J. (2007) Tracking the role of alternative prey in soybean aphid predation by Orius insidiosus: a molecular approach. Molecular Ecology, 16, 43904400.
  • Haug, E., Guillou, M., Connan, S., Goulard, F. & Diouris, M. (2003) HPLC analysis of algal pigments to define diet of sea urchins. Journal of the Marine Biological Association of the United Kingdom, 83, 571573.
  • Henson, R.D., Thompson, A.C., Hedin, P.A., Nichols, P.R. & Neel, W.W. (1975) Identification of precoccinellin in the ladybird beetle, Coleomegilla maculata. Experientia, 31, 145.
  • Hoekstra, P.F., Braune, B.M., Wong, C.S., Williamson, M., Elkin, B. & Muir, D.C.G. (2003) Profile of persistent chlorinated contaminants, including selected chiral compounds, in wolverine (Gulo gulo) livers from the Canadian Arctic. Chemosphere, 53, 551560.
  • Hoffmann, K.H., Dettner, K. & Tomaschko, K.-H. (2006) Chemical signals in insects and other arthropods: from molecular structure to physiological functions. Physiological and Biochemical Zoology, 79, 344356.
  • Holloway, G.J., De Jong, P.W., Brakefield, P.M. & De Vos, H. (1991) Chemical defence in ladybird beetles (Coccinellidae). I. Distribution of coccinelline and individual variation in defence in 7-spot ladybirds (Coccinella septempunctata). Chemoecology, 2, 714.
  • Hoogendoorn, M. & Heimpel, G.E. (2001) PCR-based gut content analysis of insect predators: using ribosomal ITS-1 fragments from prey to estimate predation frequency. Molecular Ecology, 10, 20592067.
  • Killington, F.J. (1936) A Monograph of the British Neuroptera. Volume 1. Ray Society, London.
  • Kindlmann, P. & Houdková, K. (2006) Intraguild predation: fiction or reality? Population Ecology, 48, 317322
  • King, A.G. & Meinwald, J. (1996) Review of the defensive chemistry of coccinellids. Chemical Reviews, 96, 11051122.
  • Kiritani, K. & Dempster, J.P. (1973) Different approaches to the quantitative evaluation of natural enemies. Journal of Applied Ecology, 10, 323330.
  • Knutsen, H. & Vogt, N.B. (1985a) An approach to identifying the feeding patterns of lobsters using chemical analysis and pattern recognition by the method of SIMCA I. Identification of a prey organism Artemza salzna (L.) in the stomachs of juvenile lobsters Homarus gammarus (L.). Journal of Experimental Marine Biology and Ecology, 89, 109119.
  • Knutsen, H. & Vogt, N.B. (1985b) An approach to identifying the feeding patterns of lobsters using chemical analysis and pattern recognition by the method of SIMCA II. Attempts at assigning stomach contents of lobsters Homarus gammarus (L.) to infauna and detritus. Journal of Experimental Marine Biology and Ecology, 89, 121134.
  • Koch, R.L., Venette, R.C. & Hutchison, W.D. (2006) Invasions by Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) in the Western Hemisphere: implications for South America. Neotropical Entomology, 35, 421434.
  • Laurent, P., Braekman, J.-C. & Daloze, D. (2005) Insect chemical defense. The Chemistry of Pheromones and Other Semiochemicals II (ed. S. Schulz). Topics in Current Chemistry, 240, 167229.
  • LeBrun, B., Braekman, J.-C., Daloze, D., Kalushkov, P. & Pasteels, J.M. (1999) Isopsylloborine A, a new dimeric azaphenalene alkaloid from ladybird beetles (Coleoptera: Coccinellidae). Tetrahedron Letters, 40, 81158116.
  • Lucas, É. (2005) Intraguild predation among aphidophagous predators. European Journal of Entomology, 102, 351363.
  • Lucas, É., Coderre, D. & Brodeur, J. (1998) Intraguild predation among aphid predators: characterization and influence of extraguild prey density. Ecology, 79, 10841092.
  • Majerus, M.E.N. (1994) Ladybirds. HarperCollins, London.
  • Millar, J.G. (2005) Pheromones of true bugs. The Chemistry of Pheromones and Other Semiochemicals II (ed. S. Schulz). Topics in Current Chemistry, 240, 3784.
  • Negri, A.P., Jones, G.J. & Hindmarsh, M. (1995) Sheep mortality associated with paralytic shellfish poisons from the cyanobacterium Anabaena circinalis. Toxicon, 33, 13211329.
  • Paul, V.J. & Puglisi, M.P. (2004) Chemical mediation of interactions among marine organisms. Natural Product Reports, 21, 189209.
  • Pell, J.K., Baverstock, J., Roy, H.E., Ware, R.L. & Majerus, M.E.N. (2008) Intraguild predation involving Harmonia axyridis: a review of current knowledge and future perspectives. BioControl, 53, 147168.
  • Principi, M.M. & Canard, M. (1984) Feeding habits. Biology of Chrysopidae (eds M.Canard, Y.Séméria & T.R.New), pp. 7692. Dr W. Junk, The Hague.
  • Putman, W.L. (1965a) Paper chromatography to detect predation on mites. The Canadian Entomologist, 97, 435441.
  • Putman, W.L. (1965b) The predacious thrips Haplothrips faurei Hood (Thysanoptera: Phloeothripidae) in Ontario peach orchards. The Canadian Entomologist, 97, 12081221.
  • Putman, W.L. (1967) Prevalence of spiders and their importance as predators in Ontario peach orchards. The Canadian Entomologist, 99, 160170.
  • Putman, W.L. & Herne, D.H.C. (1966) The role of predators and other biotic agents in regulating the population density of phytophagous mites in Ontario peach orchards. The Canadian Entomologist, 98, 808820.
  • Quiblier-Llobéras, C., Bourdier, G., Amblard, C. & Pepin, D. (1996) A qualitative study of zooplankton grazing in an oligo-mesotrophic lake using phytoplanktonic pigments as organic markers. Limnology and Oceanography, 41, 17671779.
  • Reitze, M. & Nentwig, W. (1991) Comparative investigations into the feeding ecology of six Mantodea species. Oecologia, 86, 568574.
  • Rosenheim, J.A., Kaya, H.K., Ehler, L.E., Marois, J.J. & Jaffee, B.A. (1995) Intraguild predation among biological control agents: theory and evidence. Biological Control, 5, 303335.
  • Saporito, R.A., Donnelly, M.A., Norton, R.A., Garraffo, H.M., Thomas, F., Spande, T.F. & Daly, J.W. (2007) Oribatid mites as a major dietary source for alkaloids in poison frogs. Proceedings of the National Academy of Sciences of the USA, 104, 88858890.
  • Sato, S. & Dixon, A.F.G. (2004) Effect of intraguild predation on the survival and development of three species of aphidophagous ladybirds: consequences for invasive species. Agricultural and Forest Entomology, 6, 2124.
  • Schröder, F.C. & Tolasch, T. (1998) Psylloborine A, a new dimeric alkaloid from a ladybird beetle. Tetrahedron, 54, 1224312248.
  • Sheppard, S.K. & Harwood, J.D. (2005) Advances in molecular ecology: tracking trophic links through predator-prey food webs. Functional Ecology, 19, 751762.
  • Sloggett, J.J. (2005) Are we studying too few taxa? Insights from aphidophagous ladybird beetles (Coleoptera: Coccinellidae). European Journal of Entomology, 102, 391398.
  • Sloggett, J.J., Haynes, K.F. & Obrycki, J.J. (2006) Chemical defense and intraguild predation in aphidophagous ladybird beetles (Coleoptera: Coccinellidae). Available at: http://esa.confex.com/esa/2006/techprogram/paper_25900.htm, accessed 30/9/08.
  • Sopp, P.I., Sunderland, K.D., Fenlon, J.S. & Wratten, S.D. (1992) An improved quantitative method for estimating invertebrate predation in the field using an enzyme-linked immunosorbent assay (ELISA). Journal of Applied Ecology, 29, 295302.
  • Sunderland, K.D. (1988) Quantitative methods for detecting invertebrate predation in the field. Annals of Applied Biology, 112, 201224.
  • Symondson, W.O.C. (2002) Molecular identification of prey in predator diets. Molecular Ecology, 11, 627641
  • Takada, W., Sakata, T., Shimano, S., Enami, Y., Mori, N., Nishida, R. & Kuwahara, Y. (2005) Schlerobatid mites as the source of pumiliotoxins in dendrobatid frogs. Journal of Chemical Ecology, 31, 24032415.
  • Traugott, M., Zangerl, P., Juen, A., Schallhart, N. & Pfiffner, L. (2006) Detecting key parasitoids of lepidopteran pests by multiplex PCR. Biological Control, 39, 3946.
  • Trigo, J.R. (2000) The chemistry of antipredator defense by secondary compounds in neotropical Lepidoptera: facts, perspectives and caveats. Journal of the Brazilian Chemical Society, 11, 551561.
  • Tursch, B., Daloze, D., Braekman, J.C., Hootele, C., Cravador, A., Losman D. & Karlsson R. (1974) Chemical ecology of arthropods. IX. Structure and absolute configuration of hippodamine and convergine, two novel alkaloids from the American ladybug Hippodamia convergens (Coleoptera – Coccinellidae). Tetrahedron Letters, 15, 409412.
  • Tursch, B., Daloze, D., Dupont, M., Hootele, C., Kaisin, M,. Pasteels, J.M. & Zimmermann, D. (1971b) Coccinellin, the defensive alkaloid of the beetle Coccinella septempunctata. Chimia, 25, 307308.
  • Tursch, B., Daloze, D., Dupont, M., Pasteels, J.M. & Tricot, M.-C. (1971a) A defense alkaloid in a carnivorous beetle. Experientia, 27, 13801381.
  • Ware, R.L. & Majerus, M.E.N. (2008) Intraguild predation of immature stages of British and Japanese coccinellids by the invasive ladybird Harmonia axyridis. BioControl, 53, 169188.
  • Weber, D.C. & Lundgren, J.G. (in press) Detection of predation using qPCR: effect of prey quantity, elapsed time, chaser diet, and sample preservation on detectable quantity of prey DNA. Journal of Insect Science.