ReviewThe fat that matters: Soil food web analysis using fatty acids and their carbon stable isotope signature
Graphical abstract
Research highlights
► Insight into soil food web structure by lipids. ► Biomarker fatty acids serve as tool to assign trophic groups. ► Compound specific analysis (13C/12C in fatty acids) allows to verify trophic links.
Introduction
Soils harbour an enormous diversity of organisms in multitrophic food webs that are central to nutrient cycling and ecosystem services (e.g., Bengtsson et al., 1996, Scheu and Setälä, 2002, Mulder, 2006). The soil micro- and mesofauna, in particular the microbial grazers, are key components of the food web, and thus important determinants for energy and carbon flows through terrestrial systems (Mikola and Setälä, 1999, Scheu et al., 2005). Regardless of their significance, trophic relationships in soil are still poorly understood. Most of the soil fauna appear to be generalist feeders, with frequent diet switches related to food availability, but actual resource utilisation remains obscure (Scheu, 2002).
Due to their small size and the cryptic habitat, feeding strategies of soil animals are difficult to address, either experimentally or by direct observation. Diets are commonly assessed by analysis of gut contents and faeces or based on morphological characteristics (Ponge, 2000, Addison et al., 2003, Chauvat et al., 2007). Assigned feeding guilds of the soil fauna therefore often reflect more taxonomic, rather than functional, relationships. Specific preferences may be investigated in food choice experiments (Ruess et al., 2000, Maraun et al., 2003, Newsham et al., 2004, Scheu and Simmerling, 2004). However, such data are restrictive as (i) information is on food consumed during a brief window of time, (ii) discrepancy between the ingested diet and assimilated nutrients can occur, and (iii) feeding preferences observed in the laboratory may not apply under field conditions.
Recent advances in biochemical and molecular methods offer new insights into soil food webs. Over the last decade, stable isotopes have increasingly been used as biomarkers in microbial ecology (Boschker and Middelburg, 2002, Staddon, 2004). The stable isotopic ratios of C (13C/12C) and N (15N/14N) have proven to be most valuable in investigating trophic relationships between organisms (Gannes et al., 1997, Gannes et al., 1998, Post, 2002, Crawford et al., 2008, del Rio et al., 2009). Isotopic ratios in tissues largely reflect the weighted average ratios of the dietary constituents, plus a small amount of change known as fractionation (DeNiro and Epstein, 1978, DeNiro and Epstein, 1981). The stable isotope composition in animal tissues has been employed to reconstruct soil food webs, with nitrogen applied to rank relative trophic levels and carbon to assign food sources (e.g. Ponsard and Arditi, 2000, Scheu and Falca, 2000, Tiunov, 2007). Although useful, such techniques have restrictions, as they cannot distinguish between food sources with similar isotopic ratios, and are limited by the small number of naturally occurring biologically relevant stable isotopes.
Molecular methods may also be applied to unravel belowground food webs. A recent approach is DNA-based gut content analyses of soil invertebrates, which provide information on specific feeding strategies and predator–prey interactions (Juen and Traugott, 2005, Juen and Traugott, 2006). Primers have been developed for multi-species systems (Admassu et al., 2006), offering promising tools for further research. The ingested diet can be detected with a high level of resolution, although this does not necessarily reflect the assimilation of dietary nutrients. More advanced is therefore the combination of stable isotope and molecular techniques to perform nucleic acid-based stable isotope probing (NA-SIP). Using this method trophic connectivity between soil biota and the length of food chains can be analysed (Staddon, 2004, Whiteley et al., 2006). To date studies using NA-SIP have investigated microorganisms actively involved in specific metabolic processes (Radajweski et al., 2000, Manefield et al., 2002) or microbial food webs (Lueders et al., 2004, Lueders et al., 2006) and as yet there has been no application to higher trophic levels. Moreover, the high 13C label necessary makes it difficult to use NA-SIP under field conditions.
The drawbacks associated with stable isotope analysis in animal tissue and in DNA do not apply to fatty acids as biochemical marker molecules. Phospholipid fatty acids (PLFAs) have long been employed as taxonomic markers for the quantification and classification of microorganisms (Tunlid and White, 1992, Frostegård and Bååth, 1996, Zelles, 1997, Zelles, 1999). More recently the lipid pattern in soil biota has successfully been used to assess trophic interactions either by fatty acid profiling solely (Chamberlain et al., 2005, Ruess et al., 2004, Ruess et al., 2005a) or in combination with stable isotope techniques, i.e. compound-specific analysis of the 13C/12C ratio in individual fatty acids (Chamberlain et al., 2004, Chamberlain et al., 2006a, Chamberlain et al., 2006b, Ruess et al., 2005b). Further, PLFA based stable isotope probing (PLFA-SIP) provides quantitative and chemotaxonomic information on resource allocation in soil microbial communities (e.g. Lu et al., 2004, Evershed et al., 2006, Chen et al., 2008).
Within the framework of this review we introduce fatty acid biomarkers and their isotope signatures as tools in assigning feeding strategies to decomposer invertebrates and in determining their diets in situ. We summarize the current understanding of the factors that influence the description of trophic interactions via fatty acids. Five major fields of inquiry are identified: (1) fatty acid profiles of dominant soil fauna groups, (2) dynamics of incorporation of fatty acids into consumers, (3) metabolic modification and isotopic fractionation, (4) pool size of marker fatty acids, (5) analytical concerns. The complexities of these areas are discussed in order to review the current state of knowledge and demonstrate how fatty acids may be used to elucidate below-ground trophic interactions.
Section snippets
Soil organisms and their lipids
Lipids are widely distributed in all living cells (Ratledge and Wilkinson, 1988). They play a vital role in organisms, both as source of energy (i.e. neutral lipids) and as structural components of cell membranes (i.e. phospholipids). Their major components, the fatty acids, consist of carbon chains which can be fully saturated or unsaturated, with fatty acids containing 1 or 2 double bonds as the most common unsaturations. Classification of unsaturated fatty acids is usually based on the
Conclusions
Biochemical ecology has the power to resolve the trophic interactions in cryptic soil food webs. Fatty acid patterns and the δ13C values of individual fatty acid biomarkers provide good indications of true diet histories of the decomposer fauna in situ, given that fatty acid biomarkers and diets with contrasting δ13C values are present. The well developed analytical methods and the ubiquitous occurrence of fatty acids makes them a promising tool. Five major areas are central for the application
Acknowledgements
This manuscript benefited much from the knowledge and experiences we gained when working with colleges holding a great expertise in lipid analysis and metabolism. LR wants to thank Max Häggbloom, Wolfgang Armbruster, and Walter Vetter for their inspirational ideas and many fruitful discussions. PC would like to thank Richard Evershed, Helaina Black and Andy Stott for their help over the years.
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