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Isotope analyses of amino acids in fungi and fungal feeding Diptera larvae allow differentiating ectomycorrhizal and saprotrophic fungi-based food chains

Citation

Pollierer, Melanie Mira; Scheu, Stefan; Tiunov, Alexei V. (2020), Isotope analyses of amino acids in fungi and fungal feeding Diptera larvae allow differentiating ectomycorrhizal and saprotrophic fungi-based food chains, Dryad, Dataset, https://doi.org/10.5061/dryad.vhhmgqnrb

Abstract

1- Both ectomycorrhizal (ECM) and saprotrophic fungi are fundamental to carbon and nutrient dynamics in forest ecosystems; however, the relative importance of these different fungal functional groups for higher trophic levels of the soil food web is virtually unknown. 2- To explore differences between fungal functional groups and their importance for higher trophic levels, we analysed isotopic composition of nitrogen and carbon in amino acids (AAs) and bulk tissue of leaf litter, fungi, and fungal-feeding Diptera larvae. 3- By accounting for isotopic variability of utilized substrates, compound-specific isotope analyses of nitrogen in AAs yielded more realistic results for the trophic position of fungi than bulk isotope analyses, with converging trophic positions of saprotrophic and ECM fungi. 4- Saprotrophic and ECM fungi possessed different AA δ13C signatures separating fungal functional groups and their consumers in fingerprinting approaches, thereby allowing to trace energy fluxes from these basal resources to higher trophic levels. 5- A pronounced isotopic fractionation even in essential/source AAs of fungal-feeding Diptera larvae necessitates further studies on tissue-/compound-specific isotopic differences in fungi and on potential supplementation by gut microorganisms. 6- The results highlight the potential of compound-specific isotope analysis of amino acids to identify and integrate contributions of different fungal functional groups to higher trophic levels in soil food webs.

Methods

Fungal sporocarps (cups/gulls) and Diptera larvae were collected along with fresh leaf litter on September 2016 at Malinki Biological Station, south of Moscow, Russia (55.4595°N, 37.1794°E). Samples were collected within few hectares of an over-ripe coniferous 150- to 160-year-old forest dominated by Norway spruce and Scots pine. Replicate samples of fungi and Diptera larvae were taken from individual sporocarps (Table 1). Leaf litter was collected close to the respective fungi.

We analyzed bulk isotopic composition (δ13C, δ15N) and AA isotopic composition (δ13C, δ15N) of four species of ECM fungi, i.e. Amanita muscaria (L.) Lam., Leccinum scabrum (Bull.) Gray, Lactarius flexuosus (Pers.) Gray, and Gomphidius glutinosus (Schaeff.) Fr., and four species of saprotrophic fungi, including the humus saprotrophic fungi Agaricus arvensis Schaeff. and Macrolepiota procera (Scop.) Singer, and the litter saprotrophic fungi Lycoperdon perlatum Pers. and Mycena pura (Pers.) P. Kumm. Fungal functional groups were assigned according to Agerer (1987), Ingold & Hudson (1993), and Kohzu et al. (1999).

Diptera larvae were collected from within the fungal sporocarps. Larvae of the crane fly Metalimnobia quadrimaculata (Linnaeus, 1760) (Limoniidae) were collected in sporocarps of L. flexuosus. Pegomya sp. Robineau-Desvoidy, 1830 (Anthomyidae) was collected in sporocarps of A. arvensis. The fungus gnat larvae of Mycetophila fungorum (De Geer, 1776) (Mycetophilidae) were collected in sporocarps of A. muscaria. Undetermined Mycetophilidae larvae were collected from sporocarps of M. procera. Depending on dry weight, we used one (M. quadrimaculata) to several individuals (other Diptera larvae) for extraction of AAs. For CSIA of undetermined Mycetophilidae larvae and M. quadrimaculata, we had to use all available individuals and therefore omitted bulk isotope analysis.

For C and N bulk isotope and C-to-N ratio analyses, fungi and leaf litter were dried at 60°C for 24 h and ground to powder. Appropriate amounts (ca. 2 mg for leaf litter and 0.5 to 1.0 mg for fungi) were weighed into tin capsules and stored in a dessicator until analysis. Diptera larvae (between 0.5 and 0.9 mg dry weight) were also weighed into tin capsules and dried at 60°C for 24 h. Stable isotope and C-to-N ratios of fungi, Diptera, and leaf litter were determined using a coupled system of an elemental analyzer and a mass spectrometer (Reineking, Langel, & Schikowski, 1993). Isotopic signatures are expressed using the δ notation as δX (‰) = (Rsample – Rstandard)/Rstandard x 1000, with X  representing the target isotope and R the ratio of heavy to light isotope (13C/12C and 15N/14N). For δ15N and δ13C analyses, N in atmospheric air and Vienna PD Belemnite served as standards, respectively.

For CSIA, dried samples were transferred to Pyrex culture tubes and flushed with N2 gas, sealed and hydrolyzed in 6 N HCl at 110°C in a heating block for 20 h (Larsen et al., 2013). After hydrolysis, lipophilic compounds were removed by adding n-hexane/DCM to the Pyrex tubes that were flushed shortly with N2 gas and sealed before they were vortexed for 30 s. The aqueous phase was then filtered through a Pasteur pipette lined with glass wool that had been pretreated at 450°C . All samples were transferred into 4 ml dram vials before evaporating the samples to dryness under a steam of N2 gas at 110°C in a heating block for 30 min. The samples were then stored at -18°C . To volatize the AAs, we followed the derivatization procedure of Corr, Berstan, & Evershed (2007), methylating the dried samples with acidified methanol and subsequently acetylating them with a mixture of acetic anhydride, trimethylamine, and acetone (1:2:5) to produce N-acetyl methyl ester derivatives. To reduce oxidation of AAs during derivatization, reaction vials were flushed and sealed with N2 prior to the methylation and acetylation reactions.

AA derivatives were injected into a Thermo Trace GC coupled via a GP interface to a Delta Plus mass spectrometer (Thermo, Bremen, Germany). The GC was equipped with an Agilent J&W VF-35ms GC column (30 m x 0.32 mm x 1.00 µm). The temperature program started with 80°C held for 1 min, increased by 20°C per minute to 135°C, then by 5°C per minute to 160°C and held for 3 min, then increased again by 8°C per minute to 300°C and held for 3 min. The injection temperature was 280°C and helium was used as carrier gas. The flow rate of helium was 2 mL min-1. All samples were analyzed in triplicate. To account for C added during derivatization and variability of isotope fractionation during analysis, pure AAs with known δ13C and δ15N values were also derivatized and analyzed. Nor-leucine was used as internal reference. The N isotopic composition of AAs in samples was expressed relative to atmospheric N by normalizing measured values (versus reference gas) using scales derived from known δ15N values of the reference mixture. The C isotopic composition was corrected for carbon added during derivatization following O’Brien, Fogel, & Boggs (2002) and was expressed relative to Vienna PD Belemnite.

 

Usage Notes

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Funding

Deutsche Forschungsgemeinschaft, Award: MA 7145/1-1