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Keywords:

  • allelochemicals;
  • Brassicaceae;
  • Cotesia glomerata;
  • hyperparasitoid;
  • Lysibia nana;
  • Pieris brassicae

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods and materials
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • 1
    Related plant species with different spatial and/or temporal life-history characteristics often possess differences in secondary chemistry and thus direct defensive capability. These differences are often attributed to a range of divergent selection pressures from herbivores and pathogens. Most studies of insect–plant interactions have examined the effects of plant defence on herbivore performance, with less attention being paid to higher trophic levels, such as parasitoid wasps. Moreover, to date it is not known whether secondary plant compounds may affect organisms in the fourth trophic level.
  • 2
    Here, we study interactions in a four-trophic-level system. The development of a solitary secondary hyperparasitoid, Lysibia nana, and its primary endoparasitoid host, Cotesia glomerata, are compared when reared from a primary herbivore host, Pieris brassicae, which was itself reared on two cruciferous plants with contrasting life histories. Whereas L. nana is known to attack the pupae of a number of primary parasitoids in the genus Cotesia, both C. glomerata and P. brassicae are intimately associated with plants in the family Brassicaceae.
  • 3
    Insects were reared from a feral population of the spring perennial, Brassica oleracea, and a naturally occurring population of a summer annual, B. nigra. Like other cruciferous plants, both species are known to produce glycoside toxins (= glucosinolates) after they are attacked by foliar herbivores. However, concentrations of glucosinolates were more than 3·5 times higher in young shoots of B. nigra than in corresponding shoots of B. oleracea.
  • 4
    Cocoon weight in C. glomerata was unaffected by the foodplant on which P. brassicae was reared, whereas in 24-h-old host cocoons emerging adult hyperparasitoid body mass increased significantly with cocoon size and wasps were significantly larger, and survived better on B. oleracea than on B. nigra. Moreover, body mass in L. nana was typically larger in young (c. 24 h), than in older (c. 72 h) cocoons of C. glomerata. Egg-to-adult development time in L. nana generally increased with host size and age, and wasps on younger hosts completed their development more rapidly on B. nigra.
  • 5
    Our results clearly demonstrate that qualitative differences in herbivore diet can differently affect the performance of interacting organisms across several trophic levels, and suggest that bottom-up forces may also play a role in mediating interactions involving plants–herbivores–parasitoids and hyperparasitoids.

Introduction

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

Many studies have reported that plants play an influential role in mediating a suite of behavioral and physiological interactions amongst the herbivores feeding on them and natural enemies of the herbivores. Plants possess a range of characteristics that may directly affect herbivore–enemy interactions. For example, morphological traits such as hairs, trichomes, or adhesive glands on the leaf surface may inhibit herbivore colonization or movement (Ribeiro, Pimento & Fernandes 1994; van Dam & Hare 1998) whilst simultaneously impeding the searching efficiency of predators or parasitoids (Romeis, Shanower & Zebitz 1998; Sutterlin & van Lenteren 2000; Fordyce & Agrawal 2001).

In addition to morphological defences, many plants produce a range of toxic secondary compounds (= allelochemicals) which may be constitutively expressed, or induced in response to herbivore damage (Karban & Baldwin 1997). These compounds can act as feeding deterrents or significantly alter the physiology and development of some herbivores, through reduced rates of growth, smaller adult size and increased mortality (Giamoustaris & Mithen 1995; van Dam, Hadwich & Baldwin 2000). However, many specialist herbivores are known to exhibit some degree of adaptation and may even require the compounds to be present as feeding stimulants (Renwick & Lopez 1999; Roessingh et al. 2000; Stadler 2000). Plant toxins may be sequestered in the haemolymph or body tissues of resistant herbivores, thus providing them with some degree of protection from their natural enemy complex (Tullberg & Hunter 1996; Wink et al. 2000; Muller et al. 2001).

Because arthropod herbivores derive their nutrition from plants, natural enemies using herbivores as prey or hosts will also obtain their nutrition indirectly from plants (Bottrell, Barbosa & Gould 1998). Several studies have reported that allelochemicals in host or prey diet negatively affects the growth, development, survival and morphology of their predators and parasitoids (Duffey, Bloem & Campbell 1986; Gunasena, Vinson & Williams 1990; Paradise & Stamp 1993; Havill & Raffa 2000). However, in many instances, these effects are also (as in generalist herbivores) more pronounced in generalist natural enemies, whereas many specialists are apparently adapted to plant toxins (Barbosa, Gross & Kemper 1991; Paradise & Stamp 1993; Vinson 1999).

Ultimately, the net effect of secondary plant compounds on plant fitness depends on how each trophic level separately responds to them. Although much attention has been paid to organisms in the third trophic level (e.g. Price et al. 1980; Turlings & Benrey 1998; Dicke 1999; van Loon, de Boer & Dicke 1999; Turlings et al. 2002), foodwebs of course do not stop there (Sullivan & Volkl 1999; Brodeur 2000). Many parasitoids (in the third trophic level) are attacked by one or more species of obligate hyperparasitoids (in the fourth trophic level) which may in turn be attacked by facultative (or tertiary) hyperparasitoids (Brodeur 2000). As pointed out by Sullivan & Volkl (1999), the dynamics of tri-trophic interactions involving plants, herbivores and parasitoids may be profoundly affected by hyperparasitoids. They may exert a significant negative effect on plant-fitness by removing parasitoids or predators of the herbivores, (top-down regulation) or else plant allelochemicals may be transferred vertically through herbivores feeding on plants to the third trophic level and perhaps higher (bottom-up regulation). However, thus far there has been no evidence that plants may be able to affect the fourth trophic level in the latter way (Brodeur 2000).

This study examines the effects of life-history variation in two closely related plant species on the development of an endoparasitoid (in the third trophic level) and its secondary (= pseudo) hyperparasitoid (in the fourth trophic level), when reared from a primary herbivore host. As far as we know, this is the first study to link the performance of an organism in the fourth trophic level to interspecific variation in foodplant quality, as mediated through a herbivore and its primary parasitoid. Conspicuously, hyperparasitoids have been mostly excluded from studies examining multitrophic interactions, even though they may play a profoundly important role in mediating community processes (Sullivan & Volkl 1999).

Cotesia glomerata L. (Hymenoptera: Braconidae) is a fairly host-specific gregarious endoparasitoid that attacks young larvae of several species of pierid butterflies, with its preferred host being Pieris brassicae L. (Lepidoptera: Pieridae). Lysibia nana Gravenhorst (Hymenoptera: Ichneumonidae) is a solitary hyperparasitoid of newly cocooned pre-pupae and pupae of several microgastrine braconids, including C. glomerata. Cotesia glomerata and L. nana are synovigenic parasitoids; adult females emerge with only a small fraction of their eggs fully mature and ready to be laid (Jervis et al. 2001). More eggs are matured over the days immediately following eclosion, although in the case of L. nana potential fecundity is limited because female wasps produce large, yolk-rich anhydropic eggs (as opposed to C. glomerata, which produces smaller, yolk-poor hydropic eggs).

Pieris brassicae is a specialist herbivore that feeds exclusively on plants producing inducible glycoside toxins known as glucosinolates (Renwick & Lopez 1999). Larvae of P. brassicae feed gregariously and are pests of cultivated crucifer species such as cabbage (Brassica oleracea L.), but also readily feed on wild species such as the black mustard (B. nigra L.). Like many gregarious folivores, older larvae of P. brassicae attain brightly contrasting colors which may serve to advertise their possession of repellant chemical defences that are sequestered from the plant (Aplin, d’Arcy-Ward & Rothschild 1975; Hunter 2000).

Although glucosinolates are known to be synthesized by plants in several plant families, they are best studied in the family Brassicaceae. Glucosinolates and their breakdown products play a role in mediating plant–phytophage interactions. (Rask et al. 2000). For example, they have been shown to act as feeding deterrents or to exhibit detrimental effects on the growth and development of herbivores, pathogens and nematodes (Potter et al. 1999; Li et al. 2000; Buskov et al. 2002). Alternatively, they are known to act as oviposition and feeding stimulants for specialist herbivores such as P. brassicae and a related species, P. rapae (van Loon & Schoonhoven 1999; Renwick & Lopez 1999; Stadler 2000). Artificial selection in B. oleracea has resulted in significant morphological variations in the shape and structure of different parts of the plant (Hawkes 1983; Benrey et al. 1998). Several species of cultivated and wild crucifers have also been shown to produce significantly different concentrations of nitrogen (Slansky & Feeny 1977) and glucosinolates (Simmonds 1979).

Thus far, few investigations have correlated the performance of herbivores and their natural enemies to interspecific differences in plant nutritional quality and allelochemistry (but see Karowe & Schoonhoeven 1992, and Benrey et al. 1998). The two crucifer species studied here also have contrasting life-histories: B. nigra is a summer annual whereas B. oleracea is a spring perennial. Different life-histories presumably reflect adaptations to environments with different herbivore and pathogen pressures. Therefore, we may expect these species to exhibit different ecological strategies with regard to induced responses (Karban & Baldwin 1997).

In this study we first compare levels of glucosinolates in young shoots of B. oleracea obtained from seeds of a feral (= established) population, and from a seeds of a naturally occurring population of B. nigra. We then examine the clutch size and pupal mass of C. glomerata when reared from cohorts of P. brassicae that were separately reared on B. oleracea and B. nigra. We lastly compare the survival, adult wasp body mass and development time of L. nana when reared from young (c. 24 h) and old (c. 72 h) pupae of C. glomerata. The aim is to determine if differences in the quality of the host foodplant separately affects the performance of the primary parasitoid and its secondary hyperparasitoid, as mediated through the herbivore host. Lastly, we assess the influence of plant defensive strategies, focusing on the role of allelochemicals, as they affect the development and fitness of herbivores and their natural enemies. The role that plant secondary compounds play in mediating multi-trophic interactions is discussed.

Methods and materials

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

insects

Hosts and parasitoids were reared at 25o± 2 oC with a 16:8 h L:D light regime. Cultures of C. glomerata and P. brassicae were obtained from insects maintained at Wageningen University (WU), The Netherlands, and which were originally collected from agricultural fields close to the University. All P. brassicae larvae used in these experiments had been maintained for many generations on Brassica oleracea var. Titurel (Brussels sprouts) at WU. C. glomerata were reared according to the protocol described in (Harvey 2000). Adult female wasps oviposit approximately 25 eggs into L1–L3 larvae of P. brassicae. During their development parasitoid larvae feed primarily on host haemolymph, and emerge from the host catepillar late during its final instar. After egression they immediately spin cocoons on the host plant adjacent to the still-living host, which perishes within a few days.

Lysibia nana was obtained from cocoons of C. glomerata recovered from leaves of B. oleracea growing in a garden plot adjacent to the Institute of Ecology, Heteren, The Netherlands. Although it is a little studied species, L. nana is known to attack several closely related parasitoids in the higher Microgastrinae, including C. glomerata and C. congregata in Europe. In the laboratory it has also been successfully reared from pupae of C. marginiventris (J.A. Harvey, unpublished).

Like many ectoparasitic idiobionts, adult female wasps perforate the host cocoon with their ovipositor and inject permanently paralysing venom into the pre-pupa or pupa. Following envenomation, a single egg is laid externally upon the moribund host. After the parasitoid egg hatches, the larva perforates the host cuticle with its mandibles and imbibes haemolymph, but as it grows it begins attacking other tissues indiscriminately and eventually consumes the entire host, pupating within the cocoon of C. glomerata. In culture, parasitoids were maintained exclusively on pre-pupae of C. glomerata. Male and female parasitoids were kept together at 10 oC, which enables them to greatly extend their longevity. In order to generate cultures of L. nana, several mated female wasps were periodically placed with approximately 100 C. glomerata cocoons in large Petri dishes (20 cm diam.) for 24 h. Following parasitism, female hyperparasitoids were returned to culture.

plants

Brassica nigra and B. oleracea

Seeds for the two Brassica species were obtained from large, single populations growing within 10 km of the Institute of Ecology. The population of B. oleracea was growing in a roadside hollow and was presumably feral, originating from a local farm, although it was not possible to determine how long it been growing in the wild. Plants were grown in a light- and temperature-controlled ‘phytotron’ facility in pots (20 × 25cm) containing a soil mixture consisting of approximately 30% sand, 5% clay and 65% peat. All plants used in experiments were approximately 4 weeks old.

experimental protocol

Measuring glucosinolate concentrations in Brassica oleracea and B. nigra

For determination of the glucosinolate levels (GLS) in B. olearacea and B. nigra shoots, seedlings were grown on individual 1·2 L pots filled with 1700 g plain river sand. Because Brassicaceae and especially B. nigra are prone to suffer from P-deficiency we supplied the plants every 2–3 days with 0·5 regular Hoagland solution that contained double the amount of P (136·1 mg L−1) for B. oleracea or four times P (272·2 mg L−1) for B. nigra. During the experiment, the pots were weighed at least every 3 days to asses the amount of nutrient solution needed to maintain soil humidity at 15% (w/w). In total, B. oleracea plants received 840 ml and B. nigra received 755 ml nutrient solution over 34 days time, respectively. At day 34, the shoots of five plants of each species were harvested, flash frozen in liquid nitrogen, lyophillized to constant weight, and weighed to determine their dry mass. The dry plant material was ground to a fine powder with a coffee mill and an aliquot of 100 mg was weighed into a 15 ml tube. GLS were extracted and analysed on HPLC following the method described in Graser et al. (2000). Sinigrin was used as an external standard. We used the correction factors in Buchner (1987) to calculate the concentrations of the different types of GLS in both plant species. The total concentration (µmol g−1 DW) of GLS was calculated by summation of all GLS that were detected in either plant species.

parasitoid and hyperparasitoid development

Eggs of P. brassicae were placed in clusters on leaves of the two crucifer species in cages (40 × 70 × 50 cm) and kept in climate room facilities under the conditions described above. Upon hatching, larvae were placed in cages (1 m × 60 cm × 60 cm) that contained two foodplants of each species. Larvae were allowed to feed and develop on their respective foodplants until just after moulting to the third instar. At this stage, they were exposed individually to female parasitoids in plastic vials, which were allowed to parasitize once. In C. glomerata, the threshold between host acceptance and rejection is c. 5 s (J. Brodeur, pers. comm.); therefore, we abandoned hosts into which a female had inserted her ovipositor for less than this time (very few hosts were ultimately rejected by the parasitoid). Following parasitism, hosts were returned to new rearing cages containing four fresh plants. Plants were refreshed every 3 days or earlier if insufficient leaf material remained; to complete development, a total of at least 15 plants of each species were provided to P. brassicae larvae. Within about 24 hours of larval parasitoid emergence, host caterpillars leave the foodplant and crawl to the top of the cage. These caterpillars were removed from cages and reared on excised leaf portions of their respective foodplants in labelled Petri dishes containing moistened filter paper (10 cm diam.).

Pupae of C. glomerata were allowed to develop for c. 24 h after emergence from P. brassicae before being presented to L. nana (this allows cocoon silk to dry but was occurs before C. glomerata pre-pupae void the meconium). A second cohort of host pupae were allowed to develop for a further total of c. 48 h (meaning that they were c. 72 h old, and that the meconium had been voided). Cocoons of C. glomerata were carefully teased apart and groups of 10 per host caterpillar were numbered and weighed individually on a Cahn 33 electrobalance (accuracy 1 µg). The numbered cocoons were then placed in two rows of five in labelled Petri dishes and attached to the plate surface using a tiny drop of honey. A single adult female of the hyperparasitoid L. nana was then given access to each group of 10 cocoons for 24 h. Since the process of oviposition in L. nana may take up to 30 m (pers. obs.), it was assumed that a full day's exposure to 10 pupae would roughly approximate natural parasitism rates for the hyperparasitoid. After 24 h, hyperparasitoids were returned to the culture and the parasitized cocoons were placed individually in labelled plastic vials. Upon eclosion, adults of L. nana were sexed, narcotized using CO2, and weighed on a microbalance. Because insufficient numbers of female wasps emerged from 72-h-old hosts, comparisons with this age cohort were restricted to males.

A subsample of wasps were dry massed for 72 h in an oven at 80 oC, to determine if there was a relationship between fresh weight (FW) and dry weight (DW). Development times (egg-to-adult in days) were also recorded. In order to determine if hyperparasitoid survival varied with the plant upon which the hebivore and primary parasitoid were reared, all cocoons failing to produce C. glomerata or L. nana were carefully opened to verify the presence of either parasitoid species.

statistical analyses

For all comparisons, ‘primary’ host refers to C. glomerata, and ‘secondary host’ refers to P. brassicae. To make the statistical analyses easier to follow, various tests were conducted in the following order: (a) the effect of foodplant species and primary host (cocoon) size on male and female hyperparasitoid size and development time in young (c. 24 h) hosts; (b) the effect of foodplant species and primary host (cocoon) size on male hyperparasitoid size and development time in older (c. 48 h) hosts; (c) the effect of foodplant species, primary host (cocoon) size and age on male hyperparasitoid size and development time (thus comparing (a) and (b) above). For more comprehensive statistical analyses, we decided to include data from all individual adult wasps of L. nana as well as from means of each secondary host (caterpillar), bearing in mind that C. glomerata is gregarious and up to 10 cocoons were used from each secondary host. By comparing individual primary host weights it was possible to generate data over a much broader range of host sizes than when secondary host data were averaged. This allows the effects of host size and plant on hyperparasitoid performance to be more clearly elucidated. However, by also examining the performance of hyperparasitoids from each secondary host (caterpillar), we are thus able to determine the extent (if any) to which the observed effects may be due to variable conditions within the secondary host.

To assess the effects of plant species and pupal age on the fate of L. nana, χ2 analyses were employed. The relationship between adult hyperarasitoid FW and DW was compared by regressing one parameter against the other. To compare clutch sizes and cocoon weights in C. glomerata on the two crucifer species, t-tests were performed.

In order to determine the effect of host size and plant on hyperparasitoid development, it was first necessary to compare separate slopes of regression for male and female wasps on each foodplant. For the 24-h-old host treatment using all emerged wasps as separate data points, the regression slopes for male wasp body mass differed significantly with foodplant (F= 24·87(317), P < 0·001). Moreover, the regression slopes of hyperparasitoid size in hosts of different ages (24 h vs. 72 h) on B. oleracea were also significantly different (F= 3·83(88), P < 0·01). In these analyses, hyperparasitoid size was compared by allocating the cocoon weights of C. glomerata to different size classes and by performing general linear model two-way anovas. Host weight classes were designated as 2·500–2·999 mg, 3·000–3·499 mg, and > 3·500 mg.

However, further comparisons of regression slopes for female body mass in 24-h-old hosts, males in 72-h-old hosts and different-aged hosts in B. nigra revealed no significant differences between the treatments being compared (24 h females: F= 0·36(51), P > 0·05; 72 h males: F= 0·10(141), P > 0·05; 24 h vs. 72 h males for B. nigra: F= 0·61(96), P > 0·05). Furthermore, hyperparasitoid size generated as a mean from each secondary host (caterpillar) revealed that the slopes for regressions did not differ significantly for male (F= 0·27(38), P= 0·27) or female (F= 0·11(12), P > 0·10) wasps. Therefore, in these groups it was possible to compare adult parasitoid size using ancovas.

Results

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

The relationship between FW and DW in L. nana was very highly significant (F= 582·651,29, r2= 0·95, P < 0·001). Thus, FW accurately approximates the body mass of L. nana.

development oflnanaon 24-h-old cocoons ofcglomerata

Both P. brassicae and C. glomerata readily developed on the two crucifer species used in this study. Secondary clutch sizes (emerged cocoons) of the primary parasitoid (C. glomerata) did not differ significantly when reared from P. brassicae reared on either foodplant (t= 0·4235, P = 0·680). Approximately 25–26 wasps emerged from hosts on both of the foodplants (Table 1). Cocoon weights of C. glomerata (24 h) also did not differ significantly with the foodplant upon which the primary host had been reared (t= 1·83362, P= 0·068), although there was a tendency for larger wasps to develop on B. nigra (Table 1). Hyperparasitoid survival was found to be significantly affected by the foodplant upon which P. brassicae, the primary host of C. glomerata, had been reared (Yates’ corrected χ2= 3·941, P < 0·05). Approximately 93% of C. glomerata reared from B. oleracea and parasitized by L. nana successfully producing adult hyperparasitoids, compared with 86% on B. nigra (Table1).

Table 1.  Developmental characteristics of Cotesia glomerata and its secondary hyperparasitoid, Lysibia nana, originating from Pieris brassicae reared on Brassica oleracea and B. nigra
Foodplant (Host age, h)Cotesia glomerata Mean clutch Size (± SE)NMean cocoon weight (± SE)Lysibia nana NPercentage survivalN
  1. Only one clutch size is represented because host cocoons of different ages were obtained from the same clutches of C. glomerata. For statistical analyses and comparisons, refer to results.

Brassica oleracea25·44 (2·48)183·13 (0·04)17893·33180
(24)
Brassica nigra26·76 (1·97)213·22 (0·07)23686·86236
(24)
Brassica oleracea2·96 (0·04)10780·45119
(72)
Brassica nigra3·10 (0·05) 6078·95 76
(72)

Adult male hyperparasitoid size varied significantly with host size (F= 133·512,274, P < 0·001) and foodplant (F= 85·011,286, P < 0·001) and the interaction effect of these parameters on hyperparasitoid size was also significant (F= 6·452,274, P= 0·02). Female hyperparasitoid size covaried significantly with host size (F= 188·811,49, P < 0·001) and foodplant (F= 45·071,49, P < 0·001). Mean data generated from each secondary caterpillar host revealed a similar pattern: adult hyperparasitoid size co-varied significantly with primary host cocoon size for male wasps (F= 320·131,26, P < 0·001) and female wasps (F= 149·271,10, P < 0·001). In both sexes, emerging adult hyperparasitoid size increased markedly with host size and wasps were larger (for a given host mass) when developing on C. glomerata which themselves had been reared from P. brassicae on B. oleracea (Fig. 1).

image

Figure 1. Relationship between 24-h-old host cocoon mass in Cotesia gomerata, and emerging adult body mass in Lysibia nana developing from Brassica oleracea (closed circles, dotted line) and B. nigra (open circles, solid line); (a) male wasps and (b) female wasps. Regression equations for L. nana: males from B. oleracea: y= 0·80x− 0·07, r2= 0·85; males from Bnigra: y= 0·28x+ 0·20, r2= 0·55; females from B. oleracea: y= 0·39x+ 0·04, r2= 0·87; females from B. nigra: y= 0·36x− 0·02, r2= 0·72.

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Two-way anovas comparing hyperparasitoid host size and C. glomerata cocoon size with host plant and offspring sex as factors were also performed. Adult hyperparasitoid weight varied with significantly with host plant (F= 7·171,366, P < 0·01) and offspring sex (F= 5·301,366, P= 0·02) but the interactive effect of these parameters was not significant (F= 2·061,366, P= 0·15). The mass of C. glomerata cocoons producing L. nana varied significantly with host plant (F= 8·641,366, P < 0·01) and with offspring sex in L. nana (F= 22·951,366, P < 0·001). However, the interactive effect of these parameters on host (cocoon) size was not significant (F= 0·181,366, P= 0·67). Overall, male hyperparasitoids developing from B. nigra were the smallest ‘class’ of wasps to emerge, even though C. glomerata cocoons producing males wasps were somewhat larger than C. glomerata cocoons producing both sexes that originated from B. oleracea (Fig. 2a). Female L. nana developing from B. oleracea were larger than wasps from the other three host–plant–offspring sex combinations (Fig. 2a). By contrast, the largest C. glomerata cocoons were those that produced female hyperparasitoids originating from B. nigra (Fig. 2b).

image

Figure 2. Combined developmental parameters for L. nana developing in 24-h-old cocoons of its primary parasitoid host, Cotesia glomerata originating from either Brassica oleracea or B. nigra. (a) Mean adult hyperparasitoid size for male (shaded bars) and female (open bars). (b) Mean cocoon weight of C. glomerata producing male (shaded bars) and female (open bars) hyperparasitoids. Line bars represent standard error of the mean. Bars with different letters differ significantly (P < 0·05; Tukey's pairwise comparisons). Sample sizes: Brassica oleracea (male wasps) = 141, (female wasps) = 25; B. nigra (male wasps) = 177, (female wasps) = 27.

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Development time of male hyperparasitoids varied significantly with host size (F= 11·722,286, P < 0·001) and with the primary host foodplant (F= 9·661,286, P < 0·001), although there was not a significant interaction effect between these parameters on overall development time (F= 1·702,281, P= 0·184). As host size increased, hyperparasitoid development time was typically extended, although wasps developed more rapidly on B. nigra. Comparing overall data, male wasps completed development significantly faster on B. nigra than on B. oleracea (12·10 against 11·89 days). However, comparison of mean data generated from each secondary caterpillar host revealed that there was not a signifiant difference in development time for wasps originating from B. oleracea or B. nigra, although the relationship was close to significant in male wasps (males: t= 1·93(36), P= 0·06; females: t= 1·12(10), P= 0·29).

development oflnanaon 72-h-old cocoons ofcglomerata

As in the 24-h treatment, mean cocoon weight in C. glomerata did not vary significantly with foodplant (t= 1·44141, P= 0·15). Similarly, wasps were slightly larger when reared from B. nigra than from B. oleracea (Table 1). Hyperparasitoid survival was not significantly affected by the primary host foodplant of P. brassicae (Yates’ corrected χ2= 0·011, P > 0·05). Approximately 78–80% of L. nana adults successfully emerged from C. glomerata cocoons originating from the two foodplants (Table 1).

Adult size of male hyperparasitoids covaried significantly with host size (F= 74·60(1,138), P < 0·001) but not with foodplant (F= 0·17(1,138), P= 0·68). The trajectory of parasitoid growth was almost convergent with host size in wasps reared from the two foodplants (Fig. 3), and was much more depressed than in the 24-h-old host treatment. Overall, L. nana reared from B. oleracea and B. nigra attained mean weights of 0·92 and 0·96 mg respectively.

image

Figure 3. Relationship between 72-h-old host cocoon mass in Cotesia glomerata and emerging male adult body mass in Lysibia nana developing from Brassica oleracea (closed circles, dotted line) and B. nigra (open circles, solid line). Regression equations for L. nana: B. oleracea: y= 0·31 ± 0·00, r2= 0·37; B. nigra: y= 0·29x+ 0·08, r2= 0·32.

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In contrast with other treatments, development time of L. nana did not vary significantly with host size (F= 0·20(1,122), P= 0·815) nor with foodplant (F= 0·08(1,122), P= 0·774) and the interaction effect of these two parameters on overall development time was also not significant (F= 0·35(2,122), P= 0·703). Overall, development time of male parasitoids was 13·42 days on B. oleracea and 13·46 days on B. nigra.

developmental comparison oflnanaon 24- and 72-h-old host cocoons

Two-way anova revealed that adult male hyperparasitoid size for wasps originating on B. oleracea varied significantly with host size (F= 47·68(2,196), P < 0·001) and age (F= 96·23(1,196), P < 0·001). However, the interaction effect between these two parameters was not significant (F= 1·04(2,196), P= 0·357). Similarly, adult male hyperparasitoid size when reared from B. nigra co-varied significantly with host size (F= 209·28(1,225), P < 0·001) and host age (F= 24·19(1,225), P < 0·001). Hyperparasitoid male size increased with host size and wasps were typically much larger when developing on younger (24 h) as opposed to older (72 h) hosts (Fig. 4a,b).

image

Figure 4. Relationship between 24-h-old (closed circles, dotted line) and 72-h-old (open circles, solid line) cocoon mass in Cotesia glomerata and emerging adult body mass in Lysibia nana developing from (a) Brassica oleracea and (b) B. nigra. Regression equations derived from Figs 2 & 3.

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Development time of male L. nana reared on B. oleracea did not vary significantly with host cocoon size (F= 0·221,193, P= 0·805) but did vary signifiantly with host age (F= 99·101,193, P < 0·001). However, the interaction effect of these parameters on development time was not significant (F= 0·74(2,193), P= 0·477). Similarly, development time of L. nana on B. nigra did not vary with host size (F= 2·60(2,215), P= 0·076) but was strongly affected by host age (F= 160·32(1,215), P < 0·001), although the interactive effect was not significant (F= 0·48(2,215), P= 0·619). Irrespective of the foodplant from which they had originated, hyperparasitoids typically took 1–2 days longer (or more) to complete development on older host pupae.

glucosinolate analyses forboleraceaandbnigra

Analyses of glucosinolate levels in the shoots of both crucifers revealed that B. nigra produced higher concentrations of secondary compounds than B. oleracea (by a factor of 3·58). Total shoot levels of glucosinolates in B. nigra were 22·60 (± 1·90) µmol g−1 DW (consisting of five different types of glucosinolates, with sinigrin the main compound present). By contrast, total shoot levels of glucosinolates in B. oleracea were 6·30 (± 0·90) µmol g−1 DW (consisting of 13 different types of glucosinolates, with progoitrin being the main compound present).

Discussion

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

The results of this investigation provide the most striking evidence yet that interspecific differences in primary and/or secondary chemistry amongst closely related plant species manifest effects on the performance of organisms several trophic levels higher. Levels of glucosinolates in the shoots of B. nigra were significantly higher than in corresponding shoots of B. oleracea. Several studies have reported an even greater difference in foliar glucosinlate concentrations between the two species (Bradburne & Mithen 2000; Moyes et al. 2000; Francis et al. 2001). However, the differing effects of plant quality in the two crucifers were much more apparent in L. nana than in either C. glomerata or P. brassicae. Hyperparasitoids developing in young cocoons of C. glomerata originating from B. nigra suffered higher mortality and were much smaller than conspecifics developing in B. oleracea. By contrast, in older (c. 72 h) hosts, mortality and body size in L. nana were similar in both Brassica species, although size was depressed and mortality higher compared with younger hosts.

In L. nana, adult wasp size also increased markedly with host size, whereas development time was also extended in larger and older hosts. For many idiobiont species host size determines the size of the resultant parasitoid adult(s), as has been demonstrated in both solitary and gregarious species (Salt 1940; Corrigan & Lashomb 1990; Harvey & Gols 1998; Otto & Mackauer 1998). Since the host represents an ostensibly ‘fixed’ package of resources, larger hosts are expected to provide more resources than smaller hosts, and parasitoids developing on larger hosts are thus expected to benefit in terms of increased size and presumably fitness (Visser 1994). However, larger (and/or older) hosts may take longer to consume (and assimilate), accounting for the increased development time (Mackauer & Sequeira 1993).

As suggested by Benrey et al. (1998) cultivated species and their wild relatives offer considerable promise for examining interactions over three (or more) trophic levels. Artificially selected plants are known to exhibit allometric changes in nutrient allocation and in the potency of allelochemicals which deter herbivores (Evans 1993; Stowe 1998). By contrast, wild plants have potentially evolved for millions of years under natural selection against a range of natural enemies, such as herbivores and pathogens. In line with these observations, some workers have reported that domesticated plants contain lower concentrations of defensive compounds than their wild relatives (Rhoades 1979; Evans 1993). This suggests that artificial selection is frequently incompatible with defence and could help to explain why many herbivores, especially generalists, thrive in agricultural systems (Benrey et al. 1998).

Domestication has potentially reduced the effectiveness of direct plant defences in the Brassicaceae. Sznajder & Harvey (unpublished) found that the development of a generalist herbivore, Spodoptera exigua Hubner, and its solitary endoparasitoid, C. marginiventris Cresson, were much more negatively affected on a wild population of B. nigra, compared with an escaped culitvar of B. oleracea. Both species suffered higher mortality, attained lower body weights, and took longer to complete development when reared on B. nigra. Similarly, Benrey et al. (1998) reported that C. glomerata and its host, P. rapae, developed less successfully on the wild cruficer species Lunaria annua L. than on a cultivar of B. oleracea. By contrast, we found little difference in the performance of P. brassicae and C. glomerata on either B. nigra and B. oleracea. This could be because both plants are much more closely related than those used by Benrey et al. (1998), or because of differences in the degree of adaptation in the two pierids to variation in the phytochemistry (and potential toxicity) of the two plants.

Several other studies have confirmed that artificial selection for reduced levels of allelochemicals, often demonstrated in agricultural systems, can profoundly affect plant–herbivore interactions. For instance, Giamoustaris & Mithen (1995) and Stowe (1998) developed lines in the crucifers Brassica napus L. and Brassica rapa L., respectively, containing quantitative differences in foliar glucosinolate concentration. Both studies revealed that increases in foliar glucosinolates significantly reduced the rate of feeding and damage incurred by generalist herbivores, although the effects on specialists were idiosyncratic. Based on his results, Stowe (1998) argued that herbivores should impose strong selection for increased glucosinolate production in crucifers, something frequently incompatible with the directed selection for the increased production of specific types of crops. Furthermore, if artificial selection can alter the chemical profiles of domesticated plants compared with their wild relatives, it is likely that cultivars will also be more palatable for third and higher trophic levels as well, as has been demonstrated here and by Benrey et al. (1998).

Although it is very little studied, the host range of L. nana is known to include several primary parasitoid species in the higher Microgastrinae. In turn, these parasitoids attack hosts that exhibit differing degrees of dietary specialization. C. glomerata has a very narrow host range, and includes only larvae of pierid butterflies feeding on plants containing glucosinolates (van Loon & Schoonhoven 1999). By contrast, C. congregata Say parasitizes oligophages including Manduca sexta L., a species which feeds on a wide variety of plants containing alkaloid toxins (Yang, Stamp & Osier 1996). Consequently, L. nana must itself be adapted to potentially dramatic differences in host quality related ultimately to the diet of the primary herbivore host.

On the basis of its trophic status and comparatively broad host range, we conclude that L. nana is likely to be less well adapted to the higher toxicity of B. nigra than are the more specialized P. brassicae and C. glomerata. This may be because C. glomerata possesses an efficient regulatory mechanism that diminishes the potential hazard of high chemical loads in the host, or else because both species are able to store allelochemicals in their body tissues, presumably in a defensive capacity. Aplin et al. (1975) investigated the possibility for sequestration of glucosinolates and isothiocyanates in P. brassicae and P. rapae reared from B. nigra. They found evidence for presence of the glucosinolate sinigrin (allylglucosinolate) and allylisothiocyanate in the pupae of both pierid species. Moreover, allyl isothiocyanate was detected in whole-body extracts of larvae and adults of P. brassicae (Aplin et al. 1975). The presence of glucosinolates in haemolymph of the larval sawfly, Athalia rosae L. has been recently demonstrated by Muller et al. (2001). The authors suggest that sequestration also appears to have a defensive function in this species (see also a similar study by Aliabadi, Renwick & Whitman 2002).

Thus far, few studies have found evidence for the transfer of secondary plant compounds from second to higher trophic levels, although it has been inferred (Meiners et al. 1997). In particular, endoparasitoids make excellent candidates for examining the sequestration of secondary plant compounds from the host to the third trophic level. In endoparasitoids, the anal end of the larval alimentary tract is not connected externally until after pupation, which prevents the excretion of faeces that would ultimately lead to contamination and subsequent bacterial infection of internal host tissues and thus reduce host quality (Quicke 1997). Consequently, parasitoid larvae are expected to store plant compounds obtained from feeding on host tissues until at least 24–36 hours after cocoon formation and pupation, when voiding of the meconium (= excretion of wastes) occurs. In the case of C. glomerata, it is therefore likely that allelochemicals are stored in the body tissues during development but may be excreted with frass after pupation. This could explain why the detrimental effects of primary host diet on hyperparasitoid size and survival diminished in older hosts.

Interspecific differences in levels of allelochemicals amongst closely related plants (as mediated through artificial selection) may have a number of ecological consequences. First, specialist herbivores like P. brassicae, which sequester plant compounds and advertise their presence through aposematic coloration, may be at a competitive disadvantage when feeding on less well-defended plants compared with cryptic herbivores. The former species are effectively advertising a toxicity which may be diluted or absent entirely. However, this apparency may benefit the efficacy of generalist predators (and hyperparasitoids like L. nana) which would otherwise avoid them. Second, reduced levels of allelochemicals in cultivated plants enable generalist herbivores and their natural enemies to persist in agroecosystems whereas in natural ecosystems they may be comparatively rare.

It is known that, in agroecosystems, larvae and pupae of C. glomerata suffer heavily from hyperparasitism, often reducing parasitoid survival by 90% or more (J.A. Harvey, pers. obs.). If this is a general rule, then these systems probably represent population ‘sinks’, and parasitoid survival may rest on the availability of hosts feeding on more toxic wild plants which are less attractive to generalist predators and hyperparasitoids. Furthermore, the dynamics (and persistence) of tightly linked multitrophic interactions, such as the system studied here, will be influenced by the ability of herbivores and their antagonists to exploit natural plant communities which are considerably more heterogeneous than simple monocultures that frequently characterize agroecosystems.

Many idiobionts, and in particular idiobiont hyperparasitoids, are known to exhibit reduced fecundity compared with lower trophic levels (Brodeur 2000). In the case of L. nana, lifetime reproductive success under optimal conditions of constant food and unlimited hosts is between 60 and 100 (J.A. Harvey, unpubl.). Moreover, female wasps are able to sequentially parasitize more than 20 hosts in the laboratory, provided these hosts are available. Because L. nana typically exploits gregarious parasitoid hosts (e.g. C. glomerata and C. congregata) that may produce secondary broods of > 20 in nature, it is highly likely that a foraging female hyperparasitoid may encounter and exploit only one or two cocoon ‘clusters’ during her lifetime. This means that her progeny may be generated from a single primary parasitoid mother, a single secondary host and a single foodplant. This raises questions as to the effects of specific or restricted genotypes of lower trophic levels on hyperparasitoid performance, given that most other primary parasitoids may attack many hosts feeding on many individual plants.

In summary, we have reported that plant quality can affect the development of organisms several trophic levels higher, even when the performance of intermediate trophic levels is mostly unaffected. Understanding the many factors influencing the dynamics of multitrophic interactions involving plants, herbivores, parasitoids and hyperparasitoids is a fertile area for future research. Perhaps one of the most challenging areas in contemporary ecology concerns the relative importance of ‘top-down’ and ‘bottom-up’ effects mediating trophic interactions and regulating the structure of communities and food webs. It is known that hyperparasitoids may have a considerable negative influence on plant fitness through reducing the abundance of parasitoids that attack the herbivores feeding on the plants (Rosenheim 1998; Sullivan & Volkl 1999). However, we have demonstrated that plant fitness can be enhanced if secondary compounds more detrimentally affect the fourth trophic level than intermediate levels in food webs. For this to happen, these compounds need to be vertically transferred through herbivores and their parasitoids. It is hoped that further studies will elucidate the relative importance of these forces incorporating measures of plant defence as it affects life-history characteristics of herbivores and their antagonists.

Acknowledgements

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

The authors would like to thank Arjen Biere, Martijn Bezemer, Mark Jervis, Wim van der Putten, and various members of the Department of Multitrophic Interactions for their comments on earlier versions of this manuscript.

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  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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