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- Materials and methods
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truly incorporated into insect tissues (Akey 1991; Hayes 1991; Jackson 1991; J. Ladner, H. Gu, D. Günther and S. Dorn, unpublished data).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.
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- Materials and methods
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.