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Keywords:

  • Algae;
  • 13C;
  • enrichment;
  • food-chain;
  • fungi;
  • 15N;
  • springtails

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • 1
    Reproduction of Heteromurus nitidus (Collembola) feeding on conidial fungi, ectomycorrhizal fungi and soil algae in single and mixed diets was investigated. Feeding on mixed diets generally increased Collembola reproduction even in combinations of fungi/algae of high food quality with those of low food quality, indicating that Collembola generally benefit from feeding on mixed diets.
  • 2
    The contribution of dietary species in mixed diets to Collembola nutrition was quantified using stable isotope methods. Incorporation of carbon from fungal/algal species into H. nitidus in mixed diets varied with food quality indicating that Collembola are able to adjust the proportion of food materials ingested to maximize fitness.
  • 3
    Fractionation of 13C and 15N in H. nitidus feeding on single and mixed diets varied between diets and differed between juveniles and adults. The trophic structure of fungal feeding soil invertebrates cannot be inferred in a straightforward way from variations in natural abundances of stable isotopes, rather, stable isotope signatures reflect feeding guilds.

Introduction

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

Collembola are polyphagous soil invertebrates that colonize virtually all soils. Owing to their large numbers they constitute an important component of decomposer food webs. Collembola are assumed to feed predominantly on fungi, but their diet also includes algae and detritus (Anderson & Healey 1972; Newell 1984; Ponge 2000). Polyphagous species generally appear to dominate in decomposer systems, which commonly has been ascribed to the lack of coevolution between consumer and resources (Scheu & Setälä 2002). This argument, however, applies only to primary decomposers, which predominantly live on dead organic matter (i.e. plant litter). Fungal feeding species such as Collembola are confronted with living resources and, therefore, one may expect coevolutionary processes similar to those between plants and herbivores to have taken place. Food choice experiments and analyses of gut contents in Collembola indicate that food specialists hardly exist, suggesting that coevolutionary processes in soil differ from those above the ground.

It has been documented that Collembola prefer certain fungal species as food substrate, but preferences appear to differ little between Collembola species, suggesting wide overlap in fungal food resources among Collembola taxa (Klironomos & Kendrick 1995; Maraun et al. 2003). Despite the prevalence of generalist feeders and the use of mixed diets, surprisingly little is known about why mixed diets dominate in Collembola (Hopkin 1997). Fungi arguably contain one of the most complex sets of secondary chemicals (Turner & Aldridge 1983), which should facilitate specialist feeders that are able to detoxify or tolerate fungal toxins. Prevalence of generalist feeding suggests that Collembola benefit from toxin dilution because of the combined ingestion of toxic and high-quality food, and/or from a more balanced food supply owing to the ingestion of food materials complementing each other (Bernays et al. 1994; Bernays & Minkenberg 1997; Hagele & Rowell-Rahier 1999). Indeed, S. Scheu & F. Simmerling (unpublished observations) show that Collembola generally appear to benefit from feeding on mixed diets consisting of different fungal species. The present study complements this work in including a wider range of diets, i.e. conidial fungi (wild type and melanin-deficient mutant of Aspergillus fumigatus), an ectomycorrhizal fungus (Laccaria laccata) and an algae (Chlorococcum infusorium).

Ectomycorrhizal fungi and algae have been assumed to be of high food quality (Wolters 1985; Schultz 1991; Hopkin 1997). However, there are only few studies that experimentally test these assumptions (Shaw 1985; Hiol, Dixon & Curl 1994); most studies on Collembola nutrition are based on food selection and not on animal growth and reproduction, i.e. fitness (Booth & Anderson 1979; Ohlsson & Verhoef 1988). In the present study we analysed the effects of single and mixed diets on animal reproduction. We were particularly interested in evaluating whether including fungal species of low food quality in mixed diets reduces Collembola fitness.

Polyphagous species need to be able to judge the proportion of dietary components that maximize fitness. A precondition for analysing whether Collembola are able to optimize fitness by mixing of food resources in certain proportions is that the proportions of the food materials ingested can be traced, i.e. the extent to which each of the resources contributes to animal nutrition is quantifiable. If two resources are offered, the proportion of carbon incorporated from each of the resources may be determined by using food materials with different stable carbon isotope signatures and then analysing the resulting stable isotope signatures of the Collembola (two source mixing model; Bakonyi, Dobolyi & Thuy 1995; Briones, Ineson & Sleep 1999). In the present study, fungi of different stable carbon isotope signatures were obtained by growing them on agar containing sucrose of either C4 or C3 plant origin.

A difficulty in using mixing models for evaluating incorporation of resources from mixed diets is that consumers may discriminate between different isotopes, i.e. their signature may not exactly correspond to the signature of the food resource. Fractionation is known to be low for 13C but significant in 15N (Petersen & Fry 1987; Michener & Schell 1994). To use stable isotope methodology in food web analyses, the extent of 13C and 15N fractionation in a wide range of taxa of different trophic groups must be evaluated experimentally (Scrimgeour et al. 1995; Gannes, O’Brien & Martinez del Rio 1997; Kelly 2000). Decomposer food webs are a particular problem as saprotrophic bacteria and fungi are one of the main resources for a great number of soil invertebrates. More detailed information on stable isotope fractionation in decomposer food chains is urgently needed. Stable isotope analyses are a very promising tool to understand better the structure and functioning of decomposer food webs (Scheu & Falca 2000; Ponsard & Arditi 2000; Oelbermann & Scheu 2002). In this study we also investigated stable isotope fractionation in decomposer food chain components including carbon resources, fungi/algae and Collembola and evaluated whether feeding on mixed vs single diets affects the fractionation of 15N in Collembola.

Materials and methods

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

fungi, algae and collembola

The fungal species used were taken from laboratory cultures. Cultures of A. fumigatus (wild type (wt) and melanin-deficient form (mdf)) were provided by A. Brakhage (University of Hannover, Germany); cultures of L. laccata were obtained from L. Ruess (University of Darmstadt, Germany); the culture of C. infusorium was obtained from the Algal Collection of the University of Göttingen (Germany). Aspergillus fumigatus is a widespread conidial fungus of soils. Algae of the genus Chlorococcum are ubiquitous in soils. Laccaria laccata is a widespread ectomycorrhizal fungus of forest soils. Aspergillus fumigatus mdf is a laboratory mutant that lacks the pksP gene responsible for pigment biosyntheses and was included because its nutritional quality differs strongly from A. fumigatus wt, but Collembola appear unable to distinguish the two forms (S. Scheu & F. Simmerling, unpublished observations).

Fungi were grown on Czapek-dox agar and algae in liquid culture (Kuhl medium). Cultures of fungi were kept at 15 °C in permanent darkness; algae were grown in a plant growth chamber at 21 °C. Heteromurus nitidus was taken from laboratory cultures; only adult specimens were used in the experiments. Heteromurus nitidus is a widespread Collembola species of forest soils in Germany but usually its density is low. In the laboratory cultures Collembola had been fed with baker's yeast, obtaining stable isotope signatures (δ-values, see below) of −23·04 ± 0·32 and 7·71 ± 0·33 for 13C and 15N, respectively.

single diet experiment

The experiment was established in Perspex vessels (diameter 7 cm, height 5 cm) with a base layer of a mixture of plaster of Paris and activated charcoal (4 : 1) of c. 1 cm. Fungal diets (A. fumigatus wt, A. fumigatus mdf and L. laccata) were offered as agar disks (4 mm diameter). Disks were cut from the edges of the fungal cultures (i.e. young and actively growing hyphae). Algae (C. infusorium) were taken from liquid cultures using a plating eye for microorganisms and offered on coverslips. Three droplets of algae were placed per coverslip. For each diet, two (early in the experiment) to six (later in the experiment) coverslips were placed per vessel, ensuring that the diets were available in excess. Ten adult H. nitidus were added at the start of the experiment. Five replicates were used per treatment. The vessels were serviced at 4-day intervals; diets were replaced, juveniles of H. nitidus were counted and if necessary water was added. Juveniles were counted using a dissecting microscope. The experiment lasted for 48 days. During the experiment the vessels were incubated at 17 °C in permanent darkness.

At the end of the experiment, adult and juvenile Collembola were sampled. Adults from each replicate were analysed for δ13C and δ15N. For stable isotope measurement of juveniles, specimens of replicates one and two, and three and four had to be combined; thus, only three replicates were measured.

mixed diet experiment

The experiment was set up in a similar way to the single diet experiment. The dietary species used in the single diet experiment were offered in two species combinations. From each diet two to four units were offered, ensuring that each diet was available in excess. At the end of the experiment, Collembola were harvested according to the sampling scheme described above and stable isotope signatures of juveniles and adults were determined.

To allow analysis of the contribution of each diet to Collembola nutrition, diets differing in 13C signals were used. For differential labelling of fungi either sucrose from sugar cane (C4 plant; Merck, Darmstadt, Germany; δ13C −10·92) or from sugar beet (C3 plant; Sigma, St. Louis; δ13C −26·03) was used for preparation of Czapek-dox agar media. δ13C signatures of the C4 and C3 agar medium were −22·61 and −12·29, respectively; respective δ15N signatures were 0·44 and 0·46. The following mixed diets were offered: (1) A. fumigatus wt (C3) with A. fumigatus mdf (C4), (2) A. fumigatus wt (C3) with L. laccata (C4), (3) A. fumigatus wt (C4) with C. infusorium, (4) A. fumigatus mdf (C4) with L. laccata (C3) and (5) A. fumigatus mdf (C4) with C. infusorium. For a full factorial statistical design the combination of A. fumigatus wt with A. fumigatus wt, i.e. A. fumigatus wt only (‘mixed’ diet 6), was also included (see below).

Incorporation of carbon from each of the diets into H. nitidus was calculated using a two-source mixing model (Gearing 1991). Signatures of H. nitidus of the two respective single diet treatments (13CK1 and 13CK2) were used to calculate the relative contribution of the first diet to the body carbon of H. nitidus (K1) according to the following formula:

  • K1 (%) = [(13Cmix − 13CK2)/(13CK1 − 13CK2)] × 100.

The contribution of the second diet (K2) is given by the difference of the first from 100.

Based on the contribution of each diet to the body carbon of H. nitidus, fractionation of nitrogen in H. nitidus was calculated. The calculation assumed that nitrogen was assimilated from the two dietary species in the same proportion as carbon. This assumption appears reasonable considering the low specificity of Collembola digestion (Hopkin 1997).

stable isotope measurement

For analysis of stable isotope signatures of sucrose, agar, fungi, algae and H. nitidus dried samples were weighed into tin capsules and stored in a desiccator. Fungi, algae and Collembola were frozen at −60 °C prior to drying at room temperature. For determining fungal stable isotope signatures, hyphae were carefully scratched from the agar surface, avoiding contamination with agar. Algae were passed through a membrane filter (pore size 2 µm) and scratched from the membrane surface. Collembolans containing gut materials were used for measurement of stable isotope signatures.

Isotope ratios were determined using a coupled system of an elemental analyser (NA 1500, Carlo Erba, Milan) and a mass spectrometer (MAT 251, Finnigan, Bremen). The system is computer-controlled, allowing on-line measurement of 15N and 13C. Stable isotope abundance is expressed using the δ notation with

  • δX (‰) = (Rsample − Rstandard)/Rstandard × 1000,

where X represents 15N or 13C, and Rsample and Rstandard represent the 15N/14N or 13C/12C ratios of the sample and standard, respectively. For 15N and 13C, atmospheric nitrogen and Peedee belemnite marine limestone (PDB), respectively, served as the primary standard (Lajtha & Michener 1994). Acetanilide (C8H9NO, Merck, Darmstadt) was used for internal calibration. The mean standard deviation of samples measured containing 10–200 µg N and 20–400 µg C is 0·2‰ (Reineking, Langel & Schikowski 1993).

statistical analysis

Reproduction of H. nitidus was analysed by repeated measures anova. Factors in the single diet experiment were time (days 12, 16, 20, 24, 28, 32, 36, 40, 44, 48) and diet (A. fumigatus wt, A. fumigatus mdf, L. laccata and C. infusorium). In the mixed diet experiment factors were time (as above), A. fumigatus (wild type and melanin-deficient form) and combined dietary species (A. fumigatus wt, L. laccata and C. infusorium). The full factorial design in the mixed diet experiment included the treatment of A. fumigatus wild type only.

Fractionation in 13C and 15N in fungi compared to the agar medium on which they grew was analysed by single factor anova with the factor fungal species/form (A. fumigatus wt, A. fumigatus mdf and L. laccata). Fractionation in 13C and 15N in H. nitidus in the single diet experiment was analysed by two-factor anova with the factors dietary species (A. fumigatus wt, A. fumigatus mdf, L. laccata and C. infusorium) and age of Collembola (juveniles and adults). Fractionation of 15N and incorporation of carbon from each of the dietary species offered in the mixed diet experiment was analysed by three-factor anova; factors were A. fumigatus (wt and mdf), combined dietary species (A. fumigatus wt, A. fumigatus mdf, L. laccata and C. infusorium) and age of Collembola (juveniles and adults). In the A. fumigatus wt only treatment of the mixed diet experiment, the contribution to the nutrition of H. nitidus was half ascribed to the factor A. fumigatus and half to the factor combined dietary species (see Fig. 4).

image

Figure 4. Incorporation of carbon from each of the two dietary species in the mixed diets by (a) juveniles and (b) adults of Heteromurus nitidus as calculated from δ13C values. Note that the treatments include the Aspergillus fumigatus wild-type only diet (see Materials and methods). Means of two to five replicates, for statistical analysis see text.

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Results

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

reproduction

Single diet: reproduction of H. nitidus varied signi-ficantly with diet (F3,21 = 3·46, P = 0·035) but the effect changed with time (F27,189 = 3·35, P < 0·001 for the interaction of time × diet). Generally, the number of juveniles per chamber produced within 48 days was lowest when feeding on the A. fumigatus wt and increased in the order A. fumigatus mdf (+23·4%), C. infusorium (+33·6%) and L. laccata (+54·7%; Fig. 1). The differences between treatments were low until day 24 and then increased, reaching a maximum between days 36 and 44.

image

Figure 1. Reproduction of Heteromurus nitidus (10 individuals per chamber) feeding on single diets for 48 days. Means of six to seven replicates with 1 SD.

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Mixed diet: the number of juveniles per chamber produced within 48 days by H. nitidus was significantly higher in the A. fumigatus mdf than in the A. fumigatus wt treatments (93 vs 109 individuals; F1,29 = 4·33, P = 0·047; Fig. 2). It also depended on the dietary species combined with A. fumigatus (F2,29 = 13·51, P < 0·001), but the effect tended to vary whether these species were combined with A. fumigatus wt or with A. fumigatus mdf (F2,29 = 3·11, P = 0·060). Generally, reproduction was at a maximum in the diets including L. laccata (132 (wt) and 120 (mdf) individuals) and the combined treatment of C. infusorium with A. fumigatus mdf (121 individuals). Reproduction was considerably lower in the combined treatments C. infusorium with A. fumigatus wt (88 individuals), and A. fumigatus wt with A. fumigatus mdf (89 individuals). However, reproduction in these mixed diet treatments was higher than for H. nitidus feeding on A. fumigatus wt only (64 individuals; Fig. 1).

image

Figure 2. Reproduction of Heteromurus nitidus (10 individuals per chamber) feeding on mixed diets for 48 days. Means of five to seven replicates with 1 SD.

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Generally, reproduction increased almost linearly in each of the treatments until day 44 or 48 (Fig. 2). Therefore, compared with the single diet experiment, reproduction of Collembola continued for a longer period of time. The time course of reproduction was similar in the A. fumigatus wt and A. fumigatus mdf treatments (F9,261 = 1·58, P = 0·18 for the time × A. fumigatus interaction), but it varied significantly between the combined dietary species treatments (F18,261 = 6·43, P < 0·001). In the L. laccata treatments Collembola reproduction was similar irrespective of the A. fumigatus form. In contrast, in the C. infusorium with A. fumigatus wt treatment Collembola reproduction was considerably lower than in the C. infusorium with A. fumigatus mdf treatment.

stable isotope fractionation

Stable isotope signatures of the fungi generally resembled that of the agar medium on which they were grown on (Table 1, Fig. 3). The δ13C signatures of A. fumigatus wt grown on C3 agar were significantly higher than that of the agar medium it was grown on. In part, δ15N signatures of the fungi also differed significantly from those of the agar medium (F3,21 = 961·83, P < 0·001), but the differences were less pronounced than those in 13C (F3,21 = 1884·92, P < 0·001).

Table 1.  Stable isotope signatures of the fungi (Aspergillus fumigatus wild type (wt), A. fumigatus melanin-deficient form (mdf), Laccaria laccata) and algae (Chlorococcum infusorium) used in the feeding experiments. For fungi differences in stable isotope signatures between fungal species/forms and the agar medium they were grown on are given; C3 and C4 refer to the type of sucrose added to the agar (see Materials and methods). Means of four replicates with 1 SD
DietSucroseδ13C δ15N
MeanSDΔ13CagarMeanSDΔ15Nagar
A. fumigatus wtC3−18·630·11 4·00 1·540·43 1·10
A. fumigatus wtC4−15·050·15−2·76 1·100·37 0·64
A. fumigatus mdfC3−21·710·11 0·91 2·120·45 1·69
L. laccataC4−13·660·19−1·37−1·040·41−1·50
C. infusorium−18·320·20−7·270·11
image

Figure 3. Signatures of δ13C of the four food chains studied in the single diet experiment. Collembolan diets consisted of Aspergillus fumigatus wild type (C4), A. fumigatus melanin-deficient form (C3), Laccaria laccata (C4) and Chlorococcum infusorium. Fungi were grown on agar medium (see Table 1), C. infusorium in liquid culture. The dotted line represents the signature of Heteromurus nitidus at the start of the experiment. Means of two to five replicates with 1 SD.

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When feeding on single fungal diets, δ15N values within adults of H. nitidus were similar (5·88, 4·66 and 5·73 for A. fumigatus wt, A. fumigatus mdf and L. laccata, respectively). In contrast, δ15N values in juveniles feeding on L. laccata (5·91) exceeded those when feeding on A. fumigatus wt and A. fumigatus mdf (2·78 and 2·93, respectively). Feeding on C. infusorium resulted in considerably lower δ15N values (1·32 and −3·15 for adults and juveniles, respectively), which correspond to lower δ15N values in C. infusorium compared to the fungal diets (Table 1).

Fractionation of 13C in H. nitidus differed markedly between diets (F3,23 = 81·52, P < 0·001) and also between juvenile and adult individuals (F1,23 = 115·38, P < 0·001; Table 2). Furthermore, fractionation of 13C in juveniles and adults depended on diet (F3,23 = 3·89, P = 0·02 for the interaction of diet × age). Fractionation was generally more pronounced in adults (overall mean −2·70) than in juveniles (overall mean −0·91). Similar to 13C, fractionation of 15N also strongly varied with diet (F3,23 = 156·82, P < 0·001), differed between juvenile and adult stages (F1,23 = 131·04, P < 0·001) and this depended on diet (F3,23 = 24·27, P < 0·001 for the interaction of diet × age; Table 2). Overall, fractionation in adults (overall mean 5·67) exceeded that in juveniles (overall mean 3·39) by 2·28 δ units but this difference was less pronounced when feeding on L. laccata, and more pronounced when feeding on C. infusorium. Generally, fractionation was low when feeding on A. fumigatus mdf and high when feeding on L. laccata and C. infusorium.

Table 2.  Fractionation of 13C and 15N in mature and juvenile Heteromurus nitidus feeding on different diets for 48 days (Aspergillus fumigatus wild type (C4), A. fumigatus melanin-deficient form (C3), Laccaria laccata (C4), Chlorococcum infusorium). Means of two to five replicates with 1 SD
Dietδ13C δ15N
JuvenilesAdultsJuvenilesAdults
MeanSDMeanSDMeanSDMeanSD
A. fumigatus wt 0·721·08−1·170·451·670·124·770·32
A. fumigatus mdf−2·590·11−3·450·150·810·192·530·67
L. laccata 0·080·20−1·950·216·950·056·770·13
C. infusorium−1·860·60−4·230·334·130·078·601·04

In the mixed diet experiment δ15N values of H. nitidus varied only between 5·88 (combined diet of A. fumigatus wt and A. fumigatus mdf, adults) and −0·86 (combined diet of A. fumigatus mdf and C. infusorium, juveniles). The lower variation was mainly due to higher δ15N values in juveniles of H. nitidus feeding on the mixed diet of C. infusorium and A. fumigatus mdf compared to the C. infusorium only diet (δ values of −0·86 and −3·15, respectively).

Similar to the single diet experiment, fractionation of 15N differed strongly between adults (overall mean 4·97) and juveniles (overall mean 1·99; F1,34 = 167·09, P < 0·001; Table 3). Furthermore, it differed between the A. fumigatus wt and A. fumigatus mdf treatments (F1,34 = 47·58, P < 0·001) and the combined dietary species treatments (F2,34 = 47·72; P < 0·001) and these were interdependent (F2,34 = 59·44, P < 0·001 for the interaction of A. fumigatus × combined dietary species). Fractionation in the combined treatment of A. fumigatus wt with A. fumigatus mdf (overall mean 3·19) was identical to that in the A. fumigatus wt only treatment (Tables 2 and 3). In addition, fractionation in the combined treatment of A. fumigatus wt and L. laccata (overall mean 1·98) was also similar to that in the treatment of A. fumigatus mdf with L. laccata (overall mean 2·47). Fractionation in the treatment of A. fumigatus wt with C. infusorium (overall mean 7·61) strongly exceeded that of the treatment of A. fumigatus mdf with C. infusorium (overall mean 2·40), particularly in juvenile H. nitidus (F2,34 = 6·29, P = 0·005 for the interaction between age × A. fumigatus × combined dietary species).

Table 3.  Fractionation of 15N in juvenile and mature Heteromurus nitidus feeding on different mixed diets for 48 days (Aspergillus fumigatus wild type and A. fumigatus melanin-deficient form, A. fumigatus wild type and Laccaria laccata, A. fumigatus wild type and Chlorococcum infusorium, A. fumigatus melanin-deficient form and L. laccata, A. fumigatus melanin-deficient form and C. infusorium). Data are calculated assuming that nitrogen was used in the same proportion as carbon from respective resources (see Materials and methods). Means of two to six replicates with 1 SD
DietJuvenilesAdults
MeanSDMeanSD
A. fumigatus wt and A. fumigatus mdf 1·840·184·540·35
A. fumigatus wt and L. laccata 0·690·113·270·65
A. fumigatus wt and C. infusorium 6·751·008·471·55
A. fumigatus mdf and L. laccata 1·130·153·801·04
A. fumigatus mdf and C. infusorium−0·150·644·940·63

carbon incorporation from mixed diets

In each of the mixed diet treatments, H. nitidus incorporated carbon from both of the dietary species offered (Fig. 4). Carbon incorporation was generally similar in adults and juveniles except in the A. fumigatus with C. infusorium diet (F2,33 = 10·55, P < 0·001 for the interaction of age × combined dietary species). Particularly in the A. fumigatus mdf with C. infusorium diet, adults used considerably more carbon from C. infusorium than did juveniles. Generally, the use of carbon varied strongly with the dietary species combined with A. fumigatus (F2,33 = 101·44, P < 0·001) and this variation was different in the A. fumigatus wt and A. fumigatus mdf treatments (F2,33 = 24·11, P < 0·001 for the interaction of A. fumigatus × combined dietary species). Heteromurus nitidus used both forms of A. fumigatus to the same extent. In contrast, in the combined treatments of A. fumigatus with L. laccata or C. infusorium, H. nitidus used less carbon from A. fumigatus and this was most pronounced in the A. fumigatus mdf with L. laccata treatment.

Discussion

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

The dietary species used differed strongly in food quality as measured by Collembola growth and reproduction. Consistent with previous findings, and our expectations, the food quality of A. fumigatus wt was lower than that of A. fumigatus mdf (S. Scheu & F. Simmerling, unpublished observations). Higher food quality of A. fumigatus mdf may have been caused by melanin deficiency because of the lack of the pksP gene, which is known to control melanin synthesis. Furthermore, the content of nitrogen in A. fumigatus wt (1·77%) was lower than that in A. fumigatus mdf (2·13%).

The factors driving food quality of fungi for fungal feeding microarthropods are little understood. Toxic substances reduce and high nutrient content increases the food quality of fungi (Booth & Anderson 1979; Shaw 1985; Sadaka-Laulan et al. 1998). A particularly puzzling phenomenon is that a great variety of fungal feeding soil invertebrates appear to favour similar fungal taxa as food substrate, generally dark pigmented forms (Maraun et al. 2003). Moreover, it is enigmatic why Collembola may generally prefer saprophytic over VA mycorrhizal fungi (Klironomos, Bednarczuk & Neville 1999; Gange 2000) and ectomycorrhizal fungi over other fungal groups (Schultz 1991; Hiol et al. 1994). The lower food quality of A. fumigatus mdf compared with L. laccata in our study was unlikely to be due to toxins (see above). High food quality in L. laccata and C. infusorium was probably a consequence of high nitrogen content (2·45 and 3·39%, respectively).

From an evolutionary perspective, generalist feeding of virtually all Collembola species could be supported if the additional ingestion of a species of low food quality to that of high food quality would increase Collembola fitness. This view, however, appears counterintuitive and is hard to integrate into current theories on the advantages of generalist feeding. Generalist feeding is assumed to be advantageous because it dilutes toxins and/or complements food components (Freeland & Janzen 1974; Pulliam 1975; Rapport 1980; Bernays et al. 1994). Results of the mixed diet experiment of the present study support the suggestion that the addition of low-quality food to that of high quality further increases Collembola fitness. None of the single diets allowed maximum growth of Collembola, suggesting that no single diet was of optimum quality. This conclusion is supported by the fact that in each of the single diets, except A. fumigatus wt, Collembola reproduction stopped after 36–44 days, whereas in the mixed diet treatments it continued for at least 44 days. Presumably, in the single diets essential nutrients necessary for continuous reproduction were lacking.

The results indicate that the prevalence of generalist feeding in Collembola and other microbivorous soil invertebrates might be due to the fact that most species of fungi and algae in soil provide a non-optimum diet. Consequently, to maximize fitness, microbivorous invertebrates have to feed on a variety of diets. A puzzling question in this scenario is why there are no or few species of fungi and algae that allow maximum growth. A possible explanation is the lack of coevolutionary processes that allowed microbivorous soil invertebrates to maximize their fitness when feeding on single species of fungi or algae (cf. Scheu & Setälä 2002).

Microbivorous soil invertebrates live in close contact with their food resources; 1 g of soil may contain several kilometres of hyphae of very different fungal species (Kendrick 1992). It may therefore be almost impossible to specialize on a single fungal species, i.e. ingestion of a mixture of different species of fungi, algae and bacteria, and different detritus substrates may be unavoidable. Presumably, evolutionary processes in microbivorous soil invertebrates resemble those in ungulate herbivores and filter feeders rather than those in above-ground insect herbivores.

As indicated by 13C analyses, H. nitidus differentially fed on the dietary species offered in mixed diets and ingested the two species in a ratio that increased reproduction, i.e. fitness. In both juveniles and adults 10–20% of the animal carbon in the L. laccata with A. fumigatus treatments originated from the species of low food quality (A. fumigatus). Compared with the L. laccata treatments, the admixture of A. fumigatus in the C. infusorium treatments was higher and more variable. As indicated by the results from the single and mixed diet experiment, food quality of C. infusorium was somewhat lower than that of L. laccata. This suggests that ingestion of food materials is more balanced if H. nitidus is confronted with food substrates differing less in quality. Results from the single diet experiment indicated that A. fumigatus mdf was of higher food quality than A. fumigatus wt. Despite this difference, H. nitidus ingested A. fumigatus wt to the same extent as A. fumigatus mdf if offered in combination. This indicates that H. nitidus is unable to distinguish the two forms of A. fumigatus which is consistent with previous results (S. Scheu & F. Simmerling, unpublished observations).

Calculations of the contribution of each of the two dietary species offered as food substrate to H. nitidus nutrition was based on a two-source mixing model. Since we also determined 13C ratios in H. nitidus after feeding on single diets this calculation was independent of differential fractionation of 13C in H. nitidus feeding on different diets. However, for interpreting data on the variation in stable isotope ratios in animals collected in the field it is essential to know to what extent stable isotopes are fractionated in consumers. In the present study, 13C ratios in H. nitidus generally resembled those in the diet. However, compared with juveniles, 13C ratios in adults differed considerably more from those of the diet. In part, this might have been due to incomplete replacement of animal carbon in adult H. nitidus during the 7 weeks of incubation. However, fractionation in adults was also more pronounced in the A. fumigatus mdf treatment in which the animals became more depleted in δ13C during the experiment (Fig. 3). This indicates that fractionation in 13C in adults in fact exceeded that in juveniles. Similar differences in δ15N in juvenile and adult H. nitidus suggest that fractionation in 15N also varies with Collembola age.

Fractionation of 13C in juveniles of H. nitidus was in the range of one to two δ units, which is within the range observed in other animals (Petersen & Fry 1987; Michener & Schell 1994). However, compared with the average increase in 13C ratios per trophic level of 0·4 δ units calculated by Post (2002), fractionation in the present study was high and surprisingly, in certain treatments, H. nitidus was depleted in 13C compared with its diet.

Fractionation of 15N considerably exceeded that of 13C with a minimum in the A. fumigatus mdf diet of 0·81 and a maximum in the L. laccata diet of 6·95. 15N ratios have been documented to increase by 3·4 δ units per trophic level (Wada, Mizutani & Minagawa 1991; Post 2002). Our data are consistent with findings that natural variations in 15N increase per trophic level, but the data indicate that the increase may vary considerably depending on the food materials ingested. Variations in 15N ratios with the quality of the diet has been reported previously (Webb, Hedges & Simpson 1998; Adams & Sterner 2000; Rothe & Gleixner 2000).

For interpreting natural variations in 15N and 13C ratios in microbivorous soil invertebrates it is critical to know the variations in stable isotope signatures in dietary species, i.e. to what extent bacteria, fungi and algae fractionate stable isotopes. It has been documented that 15N and 13C signatures of saprotrophic fungi differ from those of mycorrhizal fungi (Hobbie, Weber & Trappe 2001). Furthermore, 15N signatures in mycorrhizal fungi have been documented to vary significantly between species (Kohzu et al. 1999) and with nitrogen nutrition (Lilleskov, Hobbie & Fahey 2002). However, only sporocarps have been investigated and little is known on stable isotope fractionation in fungal hyphae, the diet of fungal feeding soil invertebrates. Results of the present study indicate that, similar to fungal sporocarps, the fractionation of stable isotopes in fungal hyphae (including conidia) may differ markedly between species. Fractionation in 13C in A. fumigatus wt considerably exceeded the mean trophic fractionation of 0·4 δ units (Post 2002). Interestingly, the fractionation differed between A. fumigatus wt growing on C3 and C4 sucrose, a phenomenon that has been reported previously for sucrose by Henn & Chapela (2000). In contrast to 13C, fractionation in 15N in fungi was considerably lower than the mean trophic fractionation proposed by Wada et al. (1991) and Post (2002). Kohzu et al. (1999) also reported that fractionation of 15N in decomposer fungi (sporocarps of wood-decomposing basidiomycetes) is low. Overall, the results suggest that data on natural variations in stable isotope ratios in fungal-feeding microarthropods collected in the field have to be interpreted with caution. Signatures of 15N may vary considerably between fungal species, i.e. within a single trophic group. Differences in 15N signatures in fungal feeding taxa therefore may reflect feeding guilds rather than the trophic structure of the community as previously hypothesized (Scheu 2002). Presumably, variations in 15N signatures in fungal feeding microarthropods can be used to differentiate, for example, between species feeding on saprotrophic and those feeding on ectomycorrhizal fungi. The differentiation of feeding guilds by stable isotope methodology may therefore contribute substantially to the understanding of the functioning of decomposer systems.

Acknowledgements

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

Financial support by the Deutsche Forschungsgemeinschaft (Graduiertenkolleg 340, TU Darmstadt) is gratefully acknowledged. Comments by two anonymous referees considerably improved the manuscript. We are indebted to Dr A. Reineking and R. Langel (Kompetenzzentrum Stabile Isotope, Forschungszentrum Waldökosysteme, Göttingen) for stable isotope measurements.

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