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Keywords:

  • agro-ecosystem;
  • dispersal;
  • host-foraging activity;
  • internal marking;
  • parasitoid;
  • stable calcium isotope

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • 1
    Dispersal and host-foraging activity are important behavioural characteristics for parasitoid species used as biological control agents. Appropriate marking techniques are often needed for field investigations into these behaviours because of the small size of parasitoids. This study developed a novel technique using the rare stable calcium isotope 44Ca to mark parasitoids.
  • 2
    As demonstrated with a tri-trophic model system consisting of cabbage plant Brassica oleracea, caterpillar Pieris brassicae and gregarious parasitoid Cotesia glomerata, marking parasitoids with the isotope 44Ca is systemic and non-disruptive. Significant uptake and translocation of 44Ca occurred in cabbage plants after being drenched with 50 mMol aqueous isotope solution, and the isotope was transferred to wasps emerging from the parasitized host caterpillars that fed on the isotope-enriched plant. The isotope enrichment did not adversely influence the survivorship of hosts or the development of parasitoids.
  • 3
    The 44Ca-enriched female wasps could pass the isotope marker further via oviposition into the parasitized host caterpillars, hence allowing the marker to be transferred across parasitoid generations. Greenhouse release experiments validated the transferability of 44Ca from the enriched wasps to hosts through parasitism. The 95% upper confidence limit for the mean 44Ca/40Ca isotope ratio in the hosts without 44Ca enrichment (control) could be used as a statistical marking criterion to identify reliably the individual parasitized hosts within the first 3 days of parasitism.
  • 4
    Synthesis and applications. We have, for the first time, documented the efficient transfer of a stable isotope marker from enriched parasitoids to host insects through parasitism. This isotope technique enables the study of dispersal and host-foraging activity of parasitoids in the field without having to recapture marked individual parasitoids. Information on the dispersal pattern and host-foraging activity of the parasitoid species concerned is obtained from the spatial distribution of the parasitized marked host insects. Furthermore, this isotope technique can be used to quantify the efficacy of parasitism by the parasitoids released in the field.

Introduction

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

Parasitoids are a group of insects whose adults usually live freely and lay eggs in or on the body of their hosts (Godfray 1994). The parasitoids that attack herbivorous insects are known to use plant- and/or host-related cues in their search for hosts (Vet & Dicke 1992; Gu & Dorn 2001; Mattiacci et al. 2001a,b; Dutton et al. 2002). The study of dispersal and host-foraging activity of parasitoids is important for biological control of pest insects and ecological insight into parasitoid–host interactions (Hawkins 1994; Lewis et al. 1998; Umbanhowar, Maron & Harrison 2003). As most parasitoids are small in size, behavioural studies in the field are often very difficult, if not impossible, without appropriate marking techniques.

Various materials have been used to mark insects, including parasitoids (Hagler & Jackson 2001). Marking with tags, paint or fluorescent powder is often used to monitor the dispersal behaviour of herbivorous insects (Toepfer, Gu & Dorn 1999, 2000; Keil, Gu & Dorn 2001; Purse et al. 2003) but these methods are not suitable for the study of trophic interactions between parasitoids and hosts because external markers do not transfer between trophic levels (Steiner 1965). Marking with trace elements (e.g. rubidium and strontium; Hopper & Woolson 1991; Corbett 1996; Fernandes et al. 1997; Gu et al. 2001; Muratori, Perremans & Hance 2005) and proteins (e.g. immunoglobulin G; Hagler 1997; Hagler & Jackson 1998) is internal and transferable between life stages and trophic levels along food chains (Hagler & Jackson 2001). However, neither elemental nor protein markers can be effectively transferred from parasitoids to their hosts via oviposition. Protein markers are only retained on, rather than being incorporated into, the marked insects (Hagler & Miller 2002). The transfer of trace elements is often of insufficient quantity for analytical detection, even though elemental markers are truly incorporated into insect tissues (Akey 1991; Hayes 1991; Jackson 1991; J. Ladner, H. Gu, D. Günther and S. Dorn, unpublished data). With advances in analytical instrumentation, the use of stable isotopes has become a promising alternative to these conventional markers. Stable isotope ratios of carbon (13C/12C) and nitrogen (15N/14N) are most commonly used for studying trophic relationships in various ecosystems (Power et al. 2002; Stapp 2002; Clément et al. 2003). So far, 15N and 13C are the only stable isotopes that have been applied for marking insects (Nienstedt & Poehling 2000; Steffan, Daane & Mahr 2001; Briers et al. 2004). The 15N-enriched bean plant Phaseolus vulgaris was shown to transfer the isotope to the parasitoid Goniozus legneri (Hymenoptera: Bethylidae) via its herbivorous hosts Amyelois transitella (Lepidoptera: Pyralidae) but transfer of the isotope marker from the enriched female parasitoids to progeny was not demonstrated (Steffan, Daane & Mahr 2001).

The aim of the current study was to develop a marking technique using the rare stable calcium isotope 44Ca as an internal marker. Calcium (Ca) is distributed throughout insect bodies (Clark 1958). The isotope 44Ca is commonly used to trace Ca incorporation in mammals (Chandra et al. 1990; Lundgren et al. 1994) and we hypothesized that it can be transferred through the eggs laid by the enriched parasitoids to their hosts. Furthermore, significant uptake of 44Ca in plants (Kuhn, Bauch & Schröder 1995; Kuhn, Schröder & Bauch 2000) can facilitate the self-marking procedure. Recent advances in mass spectrometry technology make it possible to analyse Ca isotopes with very high precision, using inductively coupled plasma mass spectrometry (ICPMS) that provides a high sample throughput and minimizes sample preparation needs.

We report the results from laboratory and greenhouse experiments conducted on a tri-trophic model system consisting of cabbage plant Brassica oleracea L. (Crucifera), caterpillar Pieris brassicae L. (Lepidoptera: Pieridae) and gregarious parasitic wasp Cotesia glomerata (L.) (Hymenoptera: Braconidae). The results are concerned with the systemic and non-disruptive enrichment of parasitoids with 44Ca, the efficiency of the isotope transfer from parasitoids to host caterpillars via oviposition, and the applicability of the technique for tracking the host-foraging activity of parasitoids.

Materials and methods

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

experimental insects and plants

Cotesia glomerata (=Apanteles glomeratus) is a gregarious endoparasitoid that attacks the first to third instar caterpillars of Pieris brassicae and Pieris rapae (Laing & Levin 1982; Bauer, Trenczek & Dorn 1998). The parasitoids and host insects (P. brassicae) came from laboratory colonies that were derived from a Swiss population (Wanner, Gu & Dorn 2006). Host caterpillars were reared on Brussels sprout plants (Brassica oleracea var. geminiferra) in nylon gauze cages (30 × 30 × 30 cm) at 21 ± 1 °C, 60% relative humidity (r.h.) and a 16 light:18 dark cycle. Cabbage plants were grown singly in pots with a standard soil mix (Optima (Otto Hauenstein Samen AE, Rafz, Switzerland); nitrogen 400 mg L−1, phosphorus 200 mg L−1, potassium 370 mg L−1, calcium 220 mg L−1 and magnesium 32 mg L−1) at 24 °C and 12 light:12 dark for approximately 37 days before experimental use. After larval egression and pupation, parasitoid pupa clusters were transferred to new nylon gauze cages and kept at 15 °C, 80% r.h. and 16 light:8 dark. Upon emergence, adult wasps were provided with drops of 1 m sucrose solution (Sigma-Aldrich Chemie GmbH, Munich, Germany; dissolved in ultra-pure water) on small Petri dishes.

uptake of 44ca in cabbage plants

To optimize the systemic and non-disruptive process of enriching parasitoids with the stable isotope 44Ca, we monitored the dynamic uptake of 44Ca in cabbage plants drenched with the isotope solution. The 44CaCO3 powder, with a purity of 98·79% (Oak Ridge National Laboratory, Nashvilk Tennessee, USA), was used as a source of 44Ca. We prepared a 50 mMol 44Ca solution with ultra-pure water and added one drop of sub-boiled HNO3 to solvate 44CaCO3. Three cabbage plants bearing eight fully grown leaves were individually placed in nylon gauze insect cages. Each plant was 44Ca-enriched by pouring 10 mL 44Ca solution over the potting soil every second day. Additionally, the plant was given ultra-pure water to prevent desiccation. Three leaf samples, i.e. circular sections (6 mm in diameter), were taken from the low, middle and top part of each plant 1, 2, 3, 4, 6 and 8 days after the first enrichment, respectively. These samples were used to quantify dynamic changes in the 44Ca/40Ca isotope ratios. The experiment was conducted in insectaries at 21 ± 1 °C, 60% r.h. and 16 light:18 dark. The same conditions were applied to the following experiments, unless otherwise stated.

transfer of 44ca between trophic levels

Five newly parasitized second-instar caterpillars were transferred onto each enriched cabbage plant 2 days after the first enrichment. Meanwhile, three plants drenched with ultra-pure water and exposed to the same number of parasitized caterpillars were used as a control. Three days later, circular sections from two randomly selected cabbage leaves of each plant and two host caterpillars feeding on the same plant were taken for isotope analysis. Thus six plant samples and six host caterpillars were analysed for the treatment group as well as the control. The remaining caterpillars were reared until egression and pupation of parasitoids. The caterpillars were provided either with new 44Ca-enriched plants or un-enriched plants in the control once during this period of time. Parasitoid cocoon clusters were collected and transferred to three new cages for the treatment group and the control. Upon emergence, adult wasps were fed on 1 m sucrose solution. Four days after emergence, two female wasps were randomly chosen from each cage for further experimental use, and the remaining wasps (49 in the treatment and 38 in the control) were used for isotope analysis.

The effects of 44Ca enrichment on both host caterpillars and parasitoids were investigated by comparing the mortality between caterpillars feeding on the enriched cabbage plants (treatment) and those on the un-enriched (control) plants, as well as the development time between parasitoids from the host caterpillars feeding on these enriched and un-enriched plants. The mortality of host caterpillars was measured as the proportion that died before the egression of parasitoids. The developmental time of parasitoids was recorded as the larval period, which was defined as the duration from parasitism in a host caterpillar to the egression and pupation of parasitoids. We did not compare the period of pupal stage between treatment and control because the development time of this stage in C. glomerata is usually less affected by environmental factors (Brodeur, Geervliet & Vet 1998; Soler et al. 2005).

transfer of 44ca to hosts through parasitism

To test for the transferability of 44Ca to hosts through parasitism, 12 isotope-enriched female wasps chosen randomly from different broods were used to parasitize host caterpillars. To avoid multiparasitism (Gu, Wang & Dorn 2003), three second-instar caterpillars were presented to a wasp sequentially in a plastic vial (9 × 35 mm), in which the wasp was allowed to parasitize each caterpillar only once. The parasitized caterpillars were then allowed to feed on un-enriched cabbage plants. Concomitantly, a control was set up under the same conditions, in which the wasps used to parasitize host caterpillars were not enriched with the isotope. After 3 days of feeding, 29 and 28 host caterpillars were taken from those parasitized by 44Ca-enriched wasps and by un-enriched wasps, respectively, for isotope analysis.

retention time

To test the retention time of the 44Ca marker in host caterpillars parasitized by enriched wasps, 20 female wasps were used 2 days after emergence to parasitize second-instar host caterpillars. Each female was used to parasitize two caterpillars, which were offered sequentially in a plastic vial (9 × 35 mm), but allowed to parasitize each caterpillar only once. As a control, 20 un-enriched female wasps were used to parasitize host caterpillars in the same manner.

The parasitized host caterpillars were reared on un-enriched cabbage plants as described above. One, 3, 5 and 7 days after parasitism, 10 caterpillars parasitized by enriched wasps and 10 caterpillars parasitized by control wasps were randomly collected for isotope analysis.

markrelease experiment

We conducted a greenhouse experiment to investigate the potential of the isotope marking technique for tracking the dispersal and host-foraging activity of released C. glomerata wasps. The female wasps released were 44Ca-enriched in the same manner as described above, except for the use of 44CaCO3 with a purity of 96·4% purchased from Isoflex (San Francisco, California, USA). Parasitoid cocoon clusters were collected shortly before they were used in the experiment.

The experiment was carried out in a greenhouse compartment (4 × 2·5 m). Twenty potted cabbage plants were distributed 30 cm apart on a platform in five rows consisting of four plants each. Six second-instar caterpillars were introduced onto each plant. Meanwhile, 60 C. glomerata cocoons, 1 day before the estimated emergence of the first adult wasps, were placed in the middle of the plant patch in a small open plastic box. As the parasitoid colony used in this experiment normally produced a proportion of 30% females (H. Wanner, personal observation), probably because of the effect of inbreeding in the laboratory (Gu & Dorn 2003), approximately 20 female wasps emerged from the cocoons. Three days after the first emerging adult wasps, half of the host caterpillars on each plant were picked at random and used for isotope analysis. The remaining caterpillars were reared on the plants for 2 more days and then dissected under a microscope to determine parasitism. The plant-based parasitism rates determined by isotope analysis and by dissection were compared to test the reliability of the isotope marking technique with 44Ca for tracking the host-foraging activity of parasitoids and evaluating parasitism. Concomitantly, 30 control caterpillars, which were obtained from the same cohort but not exposed to the 44Ca-enriched parasitoids, were also sampled for isotope analysis. The experiment was repeated three times. The environmental conditions were maintained at 21 ± 3 °C and 60% r.h. throughout the experimental period.

isotope analysis

All collections of insect and plant samples were stored in Eppendorf® (Basel, Switzerland) tubes at −80 °C. After being dried at 70 °C for at least 48 h, they were weighed, transformed into a homogeneous liquid phase by adding 200 µL hydrogen peroxide (H2O2) and 400 µL sub-boiled nitric acid (HNO3), and then digested in a microwave autoclave (ultraCLAVE II; MLS GmbH, Leutkirch, Germany). Five C. glomerata samples were digested and analysed together because the quantity of a single wasp was insufficient for analytical detection, while P. brassicae caterpillars were used individually in this process. Two blank samples were prepared for each run of digestion to estimate the background Ca contamination of vessels and chemicals. The digested samples were diluted with ultra-pure water in order to equalize a total mass of 20 g for different plant samples, 10 g for caterpillars and 4 g for wasps. Furthermore, indium (In) was added to all solutions at a concentration of 20 µg L−1 in order to monitor potential non-spectral interferences related to variations in sample uptake and nebulization efficiency during the mass spectrometric analysis.

The accuracy and reproducibility of isotope ratio measurements by ICPMS is improved when the isotopes of highest abundance are used. As 40Ca is the most abundant calcium isotope (96·9%), the ratio 44Ca/40Ca was used to study changes in the abundance of 44Ca with high precision. In this study, the ELAN 6100 DRCplus (Perkin Elmer, Norwalk, Connecticut, USA) was used, which is equipped with a dynamic reaction cell (DRC) to remove spectral interferences selectively by ion molecule reactions with a reactive gas. To remove the dominant 40Ar ± signal, ammonia gas was used; the operating conditions for the ICPMS and the DRC are listed in Table 1. The isotopes 40Ca, 44Ca and 43Ca were measured, the latter used to correct for the temporal mass bias of the instrument. The 44Ca/40Ca isotope ratios in all samples were calculated using the following mass balance equation:

Table 1.  Operating conditions for the ICPMS instrument
ICPMS parameters
Rf-power1450 W
Nebulizer gas flow0·97 L min−1
NebulizerPFA concentric, µ-flow
Spray chamberQuartz cyclonic
Sample uptake160 µL min−1
Auxiliary gas flow0·97 L min−1
Plasma gas flow17 L min−1
AutolensOff
DRC parameters
Reaction gasNH3, 99·96% (Praxxair, B)
Reaction gas flow0·6 mL min−1
RpQ0·6
RpA0
Quadrupole rod offset−7 V
Cell rod offset0 V
Cell path voltage−40 V
Measurement parameters
Dwell time4 ms
No. of sweeps1000
Settling time0·2 ms
Integration time4 s
Readings/replicate1
Replicates10
  • image

where I is recorded intensity (cps), S is I/A and A is the natural abundance of the isotope.

All data were acquired at the centre of the mass peak and with a high scanning frequency to achieve a high signal correlation during the sequential measurements. Ten replicates were measured for each sample and the average 44Ca/40Ca isotope ratios were used for data analysis. However, the samples that had a standard deviation > 0·0003 in this average 44Ca/40Ca isotope ratio (i.e. five out of 168 samples in the experiment on the transfer of 44Ca between trophic levels, seven out of 262 samples in the mark–release experiment and two out of 80 in the retention time experiment) were excluded because of precision considerations.

The dynamic uptake of 44Ca in differently positioned cabbage leaves, as measured by the change of 44Ca/40Ca isotope ratios in relation to enrichment, was examined by regression analysis. Differences in 44Ca/40Ca ratios between treatment and control at the same trophic level were determined by t-test, while those of treated samples among different trophic levels were analysed with one-way anova. Based on Bartlett's test for the homogeneity of variances, a two-sample t-test was applied for comparisons between two heterogeneous sample groups, and a Welch-Satterthwaite t-test was used to compare two homogeneous sample groups. Data on the parasitism rate determined by isotope analysis and by dissection were analysed using Spearman's correlation. All statistical analyses were performed using the software SPSS® 11·0 for Mac OS X (SPSS Inc.).

Results

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

uptake of 44ca in cabbage plants

Significant uptake of 44Ca took place in the cabbage plants that were drenched with 50 mMol 44Ca solution, as shown by the temporal increase of the 44Ca/40Ca isotope ratios in leaves with enrichments (Fig. 1). The isotope uptake depended on the leaf position on a plant (bottom, middle or top). Changes in the 44Ca/40Ca isotope ratio over time could be described by logarithmic regression equations (Fig. 1). The statistical comparison of regression coefficients indicated that the most dramatic increase of the isotope ratios occurred in top leaves and the smallest change in bottom leaves (F2,47 = 87·93, P < 0·001).

image

Figure 1. Temporal changes in 44Ca/40Ca isotope ratios with 44Ca enrichments in cabbage leaves taken from different positions (top, middle and bottom). Mean values are presented with their standard deviations. Lines show logarithmic regression curves [y = 0·0206 + 0·0031ln(x), R2 = 0·599, P = 0·0002 for top leaves; y= 0·0206 + 0·0015ln(x) for middle leaves, R2 = 0·885, P < 0·0001; y= 0·0212 + 0·0004ln(x) for bottom leaves, R2 = 0·689, P < 0·0001].

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transfer of 44ca among trophic levels

The isotope 44Ca was effectively transferred along the food chain, as shown by significant differences in the 44Ca/40Ca isotope ratios between enriched samples and non-enriched controls at each trophic level (t-test, P < 0·01; Fig. 2). On the other hand, mean 44Ca/40Ca isotope ratios in the enriched samples were not significantly different between trophic levels (F3,84 = 2·69, P > 0·05), while those in the control samples were significantly different from plants to parasitoids (F3,70= 11·41, P < 0·001). The isotope ratios of the control cabbage plant samples were lower in comparison with those in both the caterpillars feeding on them (Scheffe's test, P < 0·05) and the caterpillars parasitized by the wasps without 44Ca-enrichment (Scheffe's test, P < 0·01), suggesting that a naturally slight accumulation rather than dilution of the 44Ca concentration occurred in the transfer process from the isotope-enriched cabbage plants to the higher trophic levels.

image

Figure 2. Mean 44Ca/40Ca isotope ratios in the 44Ca-enriched cabbage plants, P. brassicae caterpillars feeding on the plants, adult C. glomerata parasitoids emerging from these caterpillars and the host caterpillars parasitized by these wasps, along with their respective controls. Error bars indicate their standard deviations. Letters above or below bars show statistical differences (Scheffe's test P < 0·05) in the isotope ratios between trophic levels for enriched and control samples, respectively.

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The isotope enrichment did not, however, impose a significant effect on the insects. Both the enriched and control caterpillar samples had the same mortality of 13·33% at the larval stage. The larval duration of parasitoids averaged 14·25 days in parasitoid larvae from the host caterpillars feeding on 44Ca-enriched cabbage plants and 14·00 days in those from hosts on the control plants. The differences between the 44Ca-enriched parasitoid group and the control group were not significant (two-sample t-test, t13 = 0·514, P > 0·6).

transfer of 44ca to hosts through parasitism

The host caterpillars parasitized by the 44Ca-enriched wasps showed significantly higher 44Ca/40Ca isotope ratios, in comparison with those parasitized by the control wasps (without 44Ca-enrichment) (two-sample t-test, t57 = 14·48, P < 0·001). The 44Ca/40Ca isotope ratios in the 28 caterpillars parasitized by the control wasps averaged 0·02086 () with a standard error of 0·00015 (S). The 44Ca/40Ca isotope ratios in 30 out of the 31 samples of caterpillars parasitized by 44Ca-enriched parasitoids were above the 95% upper confidence limit of the control samples (Fig. 3). Therefore, the 95% upper confidence limit for the mean value of 44Ca/40Ca isotope ratios in the control samples could be used as a statistical marking criterion for identification of the caterpillars parasitized by enriched C. glomerata wasps in release experiments. The marking criterion (L2) was calculated as follows:

image

Figure 3. The 44Ca/40Ca isotope ratios in Pieris brassicae caterpillars parasitized by the 44Ca-enriched and control C. glomerata wasps. The horizontal line indicates the marking criterion, defined as the 95% upper confidence limit for the mean background value of the control samples.

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  • L 2 =  + t0·05(2),υ S

where is the mean value of 44Ca/40Ca isotope ratios in the control samples, ν is the degree of freedom, i.e. the number of control samples minus one, and S is the standard error of the mean 44Ca/40Ca isotope ratio in the control samples.

retention time

For each sampling day, the upper 95% confidence limit (L2) of control samples was calculated and used to detect isotope enrichment in samples of the caterpillars parasitized by enriched female wasps (Table 2). The 44Ca marker was detectable in all samples tested on day 1 and in 90% of the sample on day 3. However, the detectability of the 44Ca marker decreased to 66·7% on day 5 and the marker was not detectable from the caterpillars after 7 days of parasitism.

Table 2.  Marking criterion L2, the number of host caterpillars identified as marked and the detectability 1, 3, 5 and 7 days after they were parasitized by 44Ca-enriched female wasps of Cotesia glomerata
Day L 2 n analysed n markedDetectability (%)
10·021781010100
30·0221910 9 90
50·02214 9 6 66·7
70·0276410 0  0

application of marking criterion in release experiments

The 95% upper confidence limit (L2) for the mean 44Ca/40Ca isotope ratios of control samples was calculated for each replicate test to detect the 44Ca enrichment in host larvae samples from the greenhouse experiment (replicate 1, L2 = 0·02176; replicate 2, L2 = 0·02227; replicate 3, L2 = 0·02291). All samples with their 44Ca/40Ca isotope ratios higher than the respective L2 were considered marked, i.e. parasitized. As some of the host caterpillars died during the experimental period (three in replicate 1, six in replicate 2 and five in replicate 3), the number of host caterpillars recovered from each plant was not necessarily the same. Therefore, we calculated a parasitism rate (i.e. the proportion of parasitized caterpillars) for each plant, referring to this as the plant-based parasitism rate. These parasitism rates on individual plants determined by isotope analysis in all three replicates significantly correlated with parasitism rates determined by dissection of caterpillars (Spearman's correlation, R= 0·597, P= 0·005 in replicate 1; R= 0·583, P= 0·007 in replicate 2; R= 0·787, P < 0·001 in replicate 3; Fig. 4). Thus the stable isotope marking technique with 44Ca could be used reliably to identify parasitized host caterpillars because the samples were taken randomly from each plant.

image

Figure 4. Relationships between the parasitism rates of P. brassicae caterpillars identified by isotope analysis and dissection, as shown in three trials of a greenhouse release experiment. Lines show linear regressions (a, y= 0·4516x + 0·2481; b, y= 0·7155x + 0·1767; c, y= 0·6601x + 0·133).

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Discussion

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

These results demonstrate conclusively that cabbage plants are effectively enriched with the stable calcium isotope 44Ca through soil drenching and that the isotope is transferred further along the food chain to the parasitoid C. glomerata via the herbivorous insect P. brassicae. In addition, the isotope is also efficiently transferred from the 44Ca-enriched parasitoid to the host caterpillar through parasitism. This novel feature proves the potential of using this isotope marker to track the dispersal and host-foraging activity of parasitoids and to evaluate the efficacy of parasitism in the field.

The 44Ca/40Ca isotope ratios in all leaf tissues increased with isotope enrichment, especially in new leaves at the top of plants. A similar pattern of changes in the isotopes 15N and 13C has been described for maize plants (Schmidt & Scrimgeour 2001). The developing leaves of cabbage plants are most highly enriched with 44Ca, probably because enlarging plant leaves require a continuous, adequate supply of Ca for formation and expansion of cell walls (Barta & Tibbitts 2000). As the young cabbage leaves are the most preferred by P. brassicae caterpillars (H. Wanner, personal observation), the high 44Ca contents in the young leaves should contribute to the effective transfer of the isotope to the feeding caterpillars.

The efficiency of marking parasitoids with the isotope is achieved in a systemic and non-disruptive process. The development of C. glomerata larvae in the body of herbivorous P. brassicae caterpillars led to the 44Ca-enrichment of the emerged adult wasps. Similarly, a previous study showed the transfer of the stable isotope 15N from herbivorous caterpillars to parasitoids (Steffan, Daane & Mahr 2001). The current study demonstrates, for the first time, the transfer of an isotope marker from the enriched parasitoid to its hosts through parasitism. Approximately 100 femtomoles (10−13 moles) of the rare isotope 44Ca were transferred from the enriched adult female wasp to its host caterpillar through oviposition. It is most likely that the 44Ca transfer occurs through oviposition because the reproductive organs and eggs of insects are known to be rich in calcium (Clark 1958). In this sense, the systemic technique to enrich parasitoids from the beginning of their development with the isotope is essential for subsequent marker transfer to the parasitized hosts.

As an internal marker, the isotope 44Ca is non-toxic, easily applicable and clearly identifiable. The calcium isotope 44Ca is apparently incorporated in body tissues that naturally contain this element, and is therefore effectively transferred between trophic levels. For the same reason, the isotope marker does not adversely influence the marked insects. Both the 44Ca-enriched host caterpillars and parasitoid larvae lived and/or developed comparably to their respective controls, which is similar to the aphids enriched with the stable isotope 15N (Nienstedt & Poehling 2000). It is a totally self-marking procedure, in which herbivorous insects are enriched with 44Ca after feeding on the plants drenched with the isotope solution, and in turn parasitoids developing in the enriched host caterpillars are marked with the isotope. Thus this procedure reduces extra marking effort and avoids direct handling and disturbance of subject insects. Furthermore, as 44Ca is a rare calcium isotope, only accounting for 2·086% of all calcium isotopes existing in nature, enrichment of a subject organism with this isotope can efficiently raise the 44Ca/40Ca isotope ratio above the background level of the organism concerned. Our experimental data have established that the 95% upper confidence limit for the mean 44Ca/40Ca isotope ratio of individual parasitoids emerging from the control host caterpillars (without 44Ca enrichment) can be used as a marking criterion to distinguish reliably the marked from the unmarked counterparts, with an error of < 5%.

The 44Ca marker was found to remain detectable after 3 days in 90% of the host caterpillars parasitized by the isotope-enriched wasps, followed by a decline of detectability to 66·7% 5 days after the hosts were parasitized. This decrease probably resulted from the dilution of the 44Ca marker in the growing body of the host caterpillars. The P. brassicae caterpillars double their weight within this period, covering the moulting from the second to the third larval instar (H. Wanner, personal observations). During this process, the concentration of the 44Ca marker is prone to fall below the level detectable by the equipment used.

As demonstrated in the greenhouse release experiment, the isotope 44Ca can be used as an excellent marker for tracking the host-foraging activity of parasitoids and evaluating parasitism in mark–release experiments. The experimental procedure consists of releasing the 44Ca-enriched wasps and sampling herbivorous hosts from plants at different distances from the release point at regular intervals, but it does not require the direct handling of adult parasitoids because the enriched parasitoid cocoons are placed on plants, simulating the natural situation in the field. For instance, emergent C. glomerata wasps disperse of their own volition away from their release sites, searching for P. brassicae caterpillars in response to the plant-host related cues (Mattiacci et al. 2001a,b). Such parasitized hosts are clearly identified using the marking criterion established as described above.

Marking parasitoids with the isotope 44Ca is more expensive relative to the use of conventional marking materials, but our field studies in a 1-ha cabbage habitat have shown that the cost is not prohibitively high, with material costs for marking c. 8000 wasps and the subsequent analysis of parasitized hosts amounting to approximately US$10 300 (Wanner et al. 2006). Further advances in analytical equipment and technology would be expected to lower the cost of using this technique. The cost of the technique is compensated for by the unique advantages over other methods used in the mark–release–recapture study of parasitoids. Problems in mark–release–recapture experiments are often caused by the lack of efficient recapture techniques or a low recapture rate of released insects (Southwood & Henderson 2000). Using the isotope technique to study dispersal in parasitoids, the recapture of released individuals is not necessary, and hence the recapture rate is no longer a critical issue. Information on the dispersal distance and pattern of the parasitoid species in question is deciphered by the spatial distribution of the parasitized marked hosts. Furthermore, this isotope technique shows promise not only in tracking the host-foraging activity of parasitoids but also in evaluating the efficacy of parasitism in the field.

In conclusion, transferability of 44Ca from the enriched female parasitoids to the hosts through parasitism has been demonstrated in a scaled-up process from laboratory to greenhouse experiments. The novel feature of the isotope marker has potential for use in field investigations into the dispersal and host-foraging activity of parasitoids. However, the isotope marker can only be reliably detected from the host insects within the first 3 days of parasitism, thus a sequential sampling procedure for host insects at an interval of ≤ 3 days is necessary. The application of this technique is particularly promising for cases involving gregarious species because the females of such species deposit egg clutches into their hosts, enhancing the transfer of the marker in quantity.

Acknowledgements

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

We thank Tanja Christoffel for assistance with insect culture and Kathrin Hametner for help with chemical analysis. This study was supported by a TH research grant (ETH Zurich) to H. Gu, D. Günther and S. Dorn. We are grateful to anonymous referees for their helpful comments on an earlier version of the manuscript.

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