Effects of temperature and life stage on the fatty acid composition of Collembola

Fatty acid (FA) analysis is used as a promising tool to investigate trophic interactions in soil food webs. The FA profile of neutral lipids in consumers is affected by the diet, and the occurrence and amount of certain FAs can reflect feeding strategies. We investigated the lipid composition of the Collembola Folsomia candida, Heteromurus nitidus and Protaphorura fimata with the fungus Chaetomium globosum as food source. The impact of environmental temperature and life stage was assessed, with special respect to linoleic acid (18:2ω6,9) as a marker FA for fungal feeding. In all Collembola species the ratio of C16/C18 in neutral lipid fatty acids (NLFAs) increased with decreasing temperature. In the NLFAs of F. candida and H. nitidus the Unsaturation Index and the amount of 18:2ω6,9 decreased with temperature, whereas in P. fimata effects were the opposite. The composition of phospholipid fatty acids (PLFAs) differed between species, but was little affected by temperature. The degree of unsaturation in NLFAs increased with the age of Collembola, mainly due to higher amounts of 18:2ω6,9 and a lower proportion of 18:1ω9. The biomarker linoleic acid represented over 20% of FAs in all fungal feeding Collembola. Despite considerable influence of temperature and life stage on its proportion, the amount was always higher than in individuals reared on other diets. This suggests that linoleic acid can serve as marker for fungal feeding independent of such physiological variations in Collembola.     

Trophic shift of stable isotopes and fatty acids in Collembola on bacterial diets

Fatty acid (FA) analysis is a promising tool to study trophic relationships in soil food webs. We determined FA biomarkers to trace bacterial food sources (Bacillus megaterium, Pseudomonas putida, Enterobacter aerogenes) of Collembola (Heteromurus nitidus, Protaphorura fimata, Folsomia candida). In addition, δ¹⁵N, δ¹³C, C/N ratio, body weight and NLFA/PLFA ratio (neutral lipid/phospholipid fatty acids) of Collembola were assessed. These measures indicated that P. putida ranked first, B. megaterium second and E. aerogenes third in food quality. FAs specific for bacteria were found in the NLFAs of the Collembola reflecting the respective bacterial diet. Biomarker FAs for gram-positive bacteria were methyl branched i14:0, i15:0, a15:0 and i17:0. Consumption of gram-negative bacteria was reflected by the cyclic form cy17:0 (E. aerogenes, P. putida) and by 16:1ω5 (P. putida).     

Population performance of collembolans feeding on soil fungi from different ecological niches

The potential reproductive value of arbuscular mycorrhizal fungi (Glomus intraradices and Glomus invermaium), root pathogenic fungi (Rhizoctonia solani and Fusarium culmorum) and saprotrophic fungi (Penicillium hordei and Trichoderma harzianum) were examined for the collembolans Folsomia candida Willem and Folsomia fimetaria L. Dried baker's yeast (Saccharomyces cerevisiae) was used as a reference standard food in laboratory cultures. Collembolan performance was determined as final size, fecundity and population growth rate after when fed the fungal food sources for 31 days. The mycorrhizal fungi gave the least growth and fecundity compared with the other fungi, but G. intraradices gave good fecundity for F. candida. In terms of growth, Baker's yeast was a high-quality food for both adults and juveniles of both species, but it was a poorer food in terms of fecundity of F. candida. Preference of the fungi in all possible pairwise combinations showed that although F. fimetaria did not perform well on Glomus spp. and F. candida did not grow well on Glomus spp. their preference for these fungi did not reflect this. The highest fecundity was seen with the root pathogen F. culmorum. Different quality indicators such as the C:N ratio of the fungal food sources as well as other biological parameters are discussed in relation to their reproductive value and Collembola preferential feeding.     

Lipid composition of Collembola and their food resources in deciduous forest stands—Implications for feeding strategies

The lipid composition of Collembola and their potential food resources was assessed in three deciduous forest stands, in order to gain insight into food web linkages under field conditions. Fatty acids (FAs) previously assigned as trophic markers in laboratory experiments were used to investigate feeding strategies in situ. As potential food sources soil microbiota and plant debris were characterised by their phospholipid fatty acid (PLFA) composition. Both the amount and the pattern of PLFAs differed between sites and soil depth, in particular the bacterial and plant marker FAs in the upper soil layer. Thus, the availability of resources for micro-detritivores varied due to forest and soil layer. The lipid composition of vital and senescent beech leaves was predominantly influenced by metabolic status and represented a quite homogenous FA resource across forest stands. Comparing Folsomia quadrioculata, Lepidocyrtus lignorum, Neanura muscorum and Pogonognathellus longicornis between the different forests revealed FA profiles to be predominantly affected by site, suggesting a diet shift depending on resources at hand. However, species-specific differences in individual FAs occurred, likely related to feeding strategy and physiological activity. Lipids of Collembola comprised low amounts of bacterial marker FAs, and bacterial consumption may occur to some extent, particularly on Gram-positives. The marker FA for predatory feeding, 20:1ω9, was found in several species, although in low amounts. This contradicts known feeding habits and caution is advisable in using 20:1ω9 as trophic marker. Overall, as indicated by high proportions of oleic (18:1ω9) or linoleic (18:2ω6,9) acid, most species were either plant litter or fungal feeders, with some transitions. The ratio 18:1ω9/18:2ω6,9 is proposed as a tool to distinguish between these two major feeding strategies in Collembola.     

Fatty acid patterns as biomarker for trophic interactions: Changes after dietary switch and starvation

Fatty acid (FA) analysis is becoming increasingly important for investigating trophic interactions in soil food webs. FA profiles of neutral lipids are affected by diet, and the occurrence and amount of certain FAs can reflect feeding strategies. However, to draw conclusions on feeding strategies in the field it is necessary to know physiological parameters of fatty acid metabolism such as the detection time and storage period of FAs. In this study we investigated the chronological change of FA biomarkers in the Collembola Heteromurus nitidus when switched between different food sources: leaves (Tilia europaea), a fungus (Chaetomium globosum) and two bacteria (Stenotrophomonas maltophilia, Bacillus amyloliquefaciens). Additionally, we followed the change of bacterial FA biomarkers during starvation. After 14 days of food deprivation bacterial FAs were still detectable in a sufficient amount to use them as dietary markers. Switching diet experiments demonstrated that FAs typical for a specific diet are already present after one day and are still detectable after 14 days of feeding on a different food source, suggesting that FA analysis can integrate the food choice of Collembola over a longer period of time, in contrast to snapshot methods such as gut content analysis.     

Feeding of the panphytophagous oribatid mite Scheloribates laevigatus (Acari: Oribatida) on cyanobacterial and algal diets in laboratory experiments

The feeding of Scheloribates laevigatus on green algae and cyanobacteria was observed under laboratory conditions using food preference tests, microanatomy of the digestive tract and epifluorescence microscopy. The level of consumption of the algal species studied — Klebsormidium flaccidum, Protosiphon botryoides, Chlorella ellipsoidea and Desmococcus vulgaris — differed. The cyanobacterium Trichormus variabilis was rejected by the mite. The most preferred alga D. vulgaris (syn. Protococcus viridis) was digested only partly, in a manner similar to C. ellipsoidea. Probably, only the contents of cells mechanically damaged by chelicerae were digested, as living cells were observed in faecal pellets on both diets. On the other hand, no living cells of K. flaccidum and P. botryoides were detected after gut passage. Intact cell walls of ruptured cells of P. botryoides, C. ellipsoidea and D. vulgaris were found in the faecal pellets in the rectum. K. flaccidum cell walls were observed in the food boli in the mesenteron, but they were more fragmented in comparison with P. botryoides cell walls. This indicates that the observed algal cell walls were not digested, while the contents were. The starch inside the algal cells seems to be the main energy source for the mites. Extrapolation to natural conditions is discussed. Although S. laevigatus is regarded as a fungivorous grazer, its feeding on algae indicates an alternative feeding guild for this species — algivorous browser.     

Olfactory cues associated with fungal grazing intensity and secondary metabolite pathway modulate Collembola foraging behaviour

Fungal secondary compounds play an important role for springtail food choice and fitness. Little is known, however, on the role of olfactory cues for Collembola foraging behaviour and whether Collembola can olfactorily perceive volatiles associated with fungal secondary metabolite pathways. We investigated the ability of three species of Collembola (Folsomia candida, Heteromurus nitidus and Supraphorura furcifera) to use olfactory cues for discriminating between fungi of different phylogenetic affiliation (Aspergillus nidulans, Cladosporium cladosporioides, Ascomycota; Laccaria bicolor, Basidiomycota) and toxicity using fungal strains of A. nidulans with reduced secondary metabolite production. Furthermore, we studied if olfactory cues from hyphae injured by grazing affect the foraging behaviour of Collembola. We hypothesized that (i) Collembola are able to olfactorily perceive and respond to fungal species/strains with different secondary metabolite pathways and that (ii) Collembola are able to sense fungal mycelia injured by grazing. Each of the Collembola species studied preferentially oriented their foraging towards fungal strains of A. nidulans with suppressed secondary metabolites, and in particular towards the mutant where the global regulator for secondary metabolites (ΔlaeA) has been silenced. Two of the three Collembola species (H. nitidus and S. furcifera) sensed olfactory cues of previously grazed fungi and moved towards ungrazed fungi, however, the response of S. furcifera was restricted to fungi extensively exposed to grazing (5 days) suggesting that the response varies between Collembola species.Overall, the results support the first and second hypothesis indicating that Collembola (1) are able to olfactorily differentiate fungi of different toxicity and move towards more palatable fungi, and (2) showed a lesser preference for fungi previously exposed to grazing.     

Taking it to the next level: Trophic transfer of marker fatty acids from basal resource to predators

Fatty acid (FA) analysis is used increasingly to investigate the trophic structure of soil animal food webs as the technique allows separation of the role of detrital resources such as bacteria, fungi and plant material for consumer nutrition. The applicability of FAs as biomarkers for different diets has been verified for Collembola and Nematoda. However, for the analysis of whole food webs it is crucial to know whether marker FA are valid for different taxa and whether they are transferred along the food chain to higher trophic levels, i.e. predators. Top-predators are integrators of lower level energy fluxes in food webs; therefore analysis of their FAs may allow to identify trophic pathways and to separate bacterial vs. fungal based energy channels. Chilopoda and Arachnida are among the main predators in soil food webs. Our aim was to test the applicability of marker FAs for these two predator taxa and to verify the trophic transfer of marker FAs of different basal resources via first order consumers into predators, i.e. over three trophic levels. Therefore, we investigated the transfer of FAs from different basal resources [fungi (Chaetomium globosum), plant leaf litter (Tilia europaea), Gram-positive (Bacillus amyloliquefaciens) and Gram-negative bacteria (Stenotrophomonas maltophilia)] via Collembola (Heteromurus nitidus) as first order consumers into predators [Lithobius forficatus (Chilopoda) and Pardosa lugubris (Arachnida)]. Fatty acid profiles of predators of food chains with different basal resources differed significantly. Marker FAs of basal resources were clearly detectable in predators, suggesting that FA analysis allows to separate trophic channels of soil food webs. By reflecting basal resources, FAs of predators allow tracking energy/resource fluxes through the food web and thereby clarifying the relative importance of bacterial vs. fungal vs. plant resources for soil animal food webs.     

Contrasting effects of nitrogen limitation and amino acid imbalance on carbon and nitrogen turnover in three species of Collembola

Soil animal detritivores play an important role in facilitating decomposition processes but little information is available on how the quality of dietary resources affects their stoichiometry of carbon (C) nitrogen (N) and phosphorus (P), and turnover of C and N. This study investigated how a fungal diet, Fusarium culmorum, with a low N content and imbalanced amino acid (AA) composition affected the physiology of three soil-dwelling collembolans (Folsomia candida, Protaphorura fimata and Proisotoma minuta) in comparison to a control diet, Saccharomyces cerevisiae, with a high N content and balanced AA composition. We compared the elemental composition of animals, their growth rates and tissue replacement of C and N. We also measured the individual AA δ¹³C to investigate the extent that Collembola may rely on endogenous sources to compensate for scarcity of essential AAs. The results showed that animal's N content tracked closely the composition of their diets, decreasing from around 10 to 7% N from the high to low N diet. They also had a significant increase of C and a decrease of P. P. fimata was less affected than F. candida and P. minuta. The total incorporation of C and N in the animals due to growth and tissue replacement decreased from 11-17 to 6-12% DM d⁻¹ on the high and low N diet respectively with P. fimata experiencing the smallest change. Essential AAs δ¹³C did not always match perfectly between Collembola species and their diets; particularly on the low N diet. Isotope patterns of AAs indicate that bacteria may have been the alternative source of essential AAs. While the results of this study cannot be extrapolated directly to the dynamics of Collembola populations in the field, they serve to demonstrate their flexibility in adapting physiologically to the temporal and spatial patchiness of the soil environment.     

Fatty acid composition and change in Collembola fed differing diets: identification of trophic biomarkers

To assess the potential of fatty acid (FA) compositions to act as biomarkers in the soil food web, two species of Collembola, Folsomia candida and Proisotoma minuta, were switched to four possible diets: Cladosporium cladosporioides (a common soil fungus), Panagrellus redivivus (a bacteria feeding nematode), Zea mays (maize) and Alnus glutinosa (alder). The change in FA content of the Collembola was observed over the following 39 days. The four diets produced significant shifts in the FA compositions of the Collembola, with P. redivivus causing the most extreme changes; Collembola fed P. redivivus gained complex FA compositions similar to those of the nematode diet. Changes in the relative abundances of some FAs were found to follow negative exponential curves, as the components either accumulated in, or were removed from, the FA pool in the Collembola; abundance half-lives varied between 0.5 and 22.4 days, indicating that Collembolan FA compositions changed readily with the input of new exogenous components. The results demonstrate that Collembolan FA compositions are influenced by diet, and that the abundances of FAs such as i15:0, i17:0 and 18:1(n-7) may be used as biomarkers of nematode consumption by Collembola. In contrast, the C₂₀ polyunsaturated FAs cannot be used as biomarkers for nematode predation as Collembola possess the ability to biosynthesise high abundances of these compounds when not provided by the diet.     

Avoidance response of different collembolan species to Betanal

Laboratory avoidance tests were conducted to assess the behavioural reactions of five common collembolan species (Isotoma anglicana, Heteromurus nitidus, Lepidocyrtus violaceus, Folsomia candida, Onychiurus armatus) towards the herbicide Betanal (active ingredient: phenmedipham) in soil. Betanal exerted a dose-dependent repellent action on all tested species. In the case of O. armatus, the sensitivity of the avoidance test exceeded that of mortality and reproduction tests. It is concluded that, in addition to ecotoxicological tests on mortality and reproduction, a test system for assessing behavioural responses could provide more detailed information on the impact of pesticides and other harmful substances on Collembola.     

Impact of quality and quantity of biochar and hydrochar on soil Collembola and growth of spring wheat

The effects of biochar (maize biochar – MBC, wood biochar – WBC) and unfermented or fermented hydrochar (HTC) on the euedaphic Collembola Protaphorura fimata and on spring wheat were investigated in greenhouse experiments. The impact of char type, amount of fermented HTC, and MBC-Collembola interactions were assessed. Generally, shoot and root biomass as well as abundance of P. fimata were not affected by the different chars. However, with increasing amounts of fermented HTC the abundance of P. fimata declined, whereas shoot biomass of wheat increased. Moreover, MBC altered root morphology and resulted in thicker roots with higher volume. The latter was not apparent when Collembola were present.     

Fungal toxins affect the fitness and stable isotope fractionation of Collembola

We investigated the effect of the fungal toxin sterigmatocystin on the fitness and stable isotope fractionation of two Collembola species (Folsomia candida and Heteromurus nitidus) feeding on mixed vs. single diets. Four knock out mutants of Aspergillus nidulans with the sterigmatocystin production blocked at different steps along the biosynthetic pathway were combined in mixed diets with either the high quality fungus Cladosporium cladosporioides or the low quality fungus A. nidulans (wildtype). Using fungi labeled with stable isotopes (¹³C and ¹⁵N) we evaluated the incorporation of carbon and nitrogen from individual fungi. We hypothesised that (i) Collembola fitness decreases with the putative toxicity of the fungi (ii) Collembola benefit from ingestion of mixed diets due to toxin dilution and (iii) fractionation of ¹³C and ¹⁵N is more pronounced in more toxic diets. Mixed diets did not uniformly improve fitness. Toxin dilution, however, played an important role in Collembola fitness. The fractionation of ¹³C and ¹⁵N varied with sterigmatocystin mutant strains, and Collembola species often differed from the expected enrichment per trophic level. The results show that fungal toxin production may affect stable isotope fractionation, presumably by altering consumer excretion rates necessary for detoxification.     

Effects of compost addition on extra-radical growth of arbuscular mycorrhizal fungi in Acacia tortilis ssp. raddiana savanna in a pre-Saharan area

We studied the influence of added compost, consisting of Acacia cyanophylla leaves, on the production of extra-radical mycelia of arbuscular mycorrhizal (AM) fungi in natural stands of Acacia tortilis, which forms a desert savanna. Four different plots with different soil characteristics in terms of nutrient level and water-holding capacity were included in the study. The production of AM fungi was measured as the increase in the amount of the phospholipid fatty acid (PLFA) 16:1ω5 and the neutral lipid fatty acid (NLFA) 16:1ω5 in mesh bags placed in the root zone of A. tortilis trees. The production of AM mycelia was much higher at the site with the highest nutrient level and highest water holding capacity. Principal component analysis revealed that mesh bags from this plot had proportionally more PLFA 16:1ω5 than the other plots, indicating that this plot contained proportionally more AM fungi in the microbial community. Compost addition enhanced the production of AM mycelia in all plots although the response was greatest in the plot with the highest proportion of AM fungi. The beneficial effect of compost addition on growth of the AM fungal biomass found in this study could be one way to improve survival of planted seedlings in arid regions. We suggest that indigenous AM fungi, which are adapted to the limiting conditions in the plots, are the preferable source of inoculum for improving the growth of A. tortilis in plantations in pre-Saharan ecosystems.     

Earthworms and collembola relationships: effects of predatory centipedes and humus forms

Relationships between anecic earthworms (Lumbricus terrestris and Aporrectodea giardi) and the collembolan species Heteromurus nitidus (Templeton, 1835), which is known to be attracted to earthworms, were investigated in an 8-week laboratory experiment. Our aims were (1) to assess whether earthworms influence the population dynamics of H. nitidus, and (2) to study pathways of influence and how earthworm effects are modified by humus forms and predators. Using microcosms with three defaunated humus forms, then provided with earthworms and predators, we intended to demonstrate that, amongst possible favourable effects of earthworms on springtail populations, earthworm activity may provide greater access and more pathways for springtails to explore soil and avoid predation. We expected that the effects of predators (centipedes) on the abundance of H. nitidus would increase from less (calcic mull) to more (moder) compact soil, and we hypothesized that earthworms would reduce predation pressure on H. nitidus by providing escape routes through increased macroporosity. Humus forms and earthworms only affected the population size of H. nitidus under high predation pressure, when collembolan numbers were higher in calcic mull than in moder, and were increased by the presence of earthworms. These results corroborate the hypothesis that earthworms, by increasing soil macroporosity, improve the escape routes for Collembola and thus evade predation. In moder humus earthworms increased the density of H. nitidus whether predators were present or not, so we cannot exclude that earthworms were also directly beneficial to H. nitidus. However, the hypothesis of a functional relationship mediated by soil macroporosity seems relevant since it was supported by differences observed when considering body size. When two size classes were distinguished within populations of H. nitidus (1) the positive effect of earthworms in moder was observed only on larger Collembola (>1 mm), (2) the density of the larger Collembola was decreased by predation only in moder and not in mull, (3) the effects of predators on the smaller individuals were not influenced by the presence of earthworms whatever the humus form, and was not decreased by the presence of earthworms. Nevertheless, factors other than macroporosity may operate as the presence of earthworms in acidic mull led to an unexplained decrease in the abundance of small-sized H. nitidus.     

The impact of earthworms on the abundance of Collembola: improvement of food resources or of habitat?

I assessed the direct influence of earthworm excretions, and the impact of earthworms through their action on the soil structure (increased macroporosity), on the population dynamics of the collembolan species Heteromurus nitidus. The intestinal content of Collembola arising from cultures on different soil types was observed, and two experimental cultures of H. nitidus were run: (1) a culture performed on an inert substrate supplied either with earthworm casts or with soil as food resource, (2) an experiment using microcosms with cores of two humus forms (moder and calcic mull), in the presence or absence of earthworms. The observation of gut contents revealed that H. nitidus feeds on excrements, the composition of which (ratio organic matter/mineral matter) varies according to the humus form where it lived. Slightly aged (10–15 days) organo-mineral casts of earthworms appeared to be a better food than calcic mull aggregates or organic material from moder. Densities of H. nitidus cultured in cores of calcic mull were higher than in moder, except when cores of moder were inhabited by an anecic earthworm for 2 months. The humus form strongly influenced populations of H. nitidus, firstly because densities of predators were higher in moder than in calcic mull, and probably also because of soil macroporosity. It was concluded that earthworms would affect predation on H. nitidus by creating a network of interconnected macropores in which Collembola can move and find shelter.     

Low importance for a fungal based food web in arable soils under mineral and organic fertilization indicated by Collembola grazers

We investigated the Collembola community at an arable field where mineral and organic fertilizers have been applied at low and high rates for 27 years. As food resources for Collembola, the soil microbial community was analyzed using phospholipid fatty acids (PLFAs). A special focus was put on AM fungi, which were estimated by the marker 16:1ω5 in PLFA (viable hyphae) and neutral lipid fatty acid (NLFA – storage fat in spores) fractions. Additionally, whole cellular lipids in crop plant tissues and manure were assessed. Greater Collembola species richness occurred in plots where mineral fertilizer was added. In contrast, soil microbial biomass including AM fungal hyphae increased with addition of organic fertilizer, while the amount of AM fungal spores and biomass of saprotrophic fungi were not affected by fertilizer type. The lipid pattern in wheat roots was altered by fertilizer type, application rate and their interaction, indicating different rhizosphere communities. In sum, the availability and composition of food resources for Collembola changed considerably due to farm management practice. The major diet of three dominant Collembola species, Isotoma viridis, Willemia anophthalma and Polyacanthella schäffer was determined by lipid profiling. Multivariate analysis demonstrated species specific lipid patterns, suggesting greater importance of species than management practice on the diet choice. Nevertheless, feeding strategy was affected by fertilizer type and availability of resources, as trophic biomarker fatty acids indicated feeding on wheat roots (and to some extent saprotrophic fungi) with mineral and a shift to soil organic matter (litter, detritus) with organic fertilization. Although AM fungi dominated the soil fungal community, the AMF marker 16:1ω5 was not detected in Collembola lipids, indicating that these were not consumed. The very low amount of saprotrophic fungi in the soil and the fact that Collembola as major fungal grazers did not feed on AM fungi indicates that the fungal energy channel in the investigated arable field is of little importance to the faunal food web.     

Preference tests with collembolas on isogenic and Bt-maize

Collembolas are important members of belowground food webs. There is little information available on the effects of the plant residues of transgenic maize expressing Bacillus thuringiensis (Bt) toxin on soil animals, including collembola. This is why two questions were addressed in laboratory feeding experiments with three collembolan species: (i) Are collembola equally distributed on residues of isogenic and Bt-maize? and (ii) Do collembola show feeding preference to either of the maize types? Bt-maize (producing Cry1Ab toxin) proved to be a less preferred food source for Folsomia candida than the isogenic one. No similar phenomenon was found in the case of Heteromurus nitidus and Sinella coeca. F. candida reacted to as low as 3.45 (± 0.8 mg g⁻¹) Bt-toxin content of the maize. Our results show that the effect of the Bt-toxin producing maize on the collembolan is species specific.     

Use of C-labelled plant materials and ergosterol, PLFA and NLFA analyses to investigate organic matter decomposition in Antarctic soil

The relationship between organic matter decomposition and changes in microbial community structure were investigated in Antarctic soils using ¹³C-labelled plant materials. Soils with and without labelled Deschampsia antarctica (a native Antarctic grass) were incubated for 42 days and sampled at 0, 7, 14, 21, 28 and 42 days. Changes in microbial community structure were assessed using phospholipid fatty acid analysis (PLFA) and an analysis of the fatty acids associated with the neutral lipid fraction (NLFA). These studies showed that there were no significant changes in PLFA or NLFA profiles over time suggesting no change in microbial community structure during residue decomposition. There was a marked increase however, in ergosterol levels in these soils indicative of growth of the fungal biomass. Analysis of this ergosterol using gas chromatography-mass spectrometry confirmed the transformation of the plant residue by showing the incorporation of ¹³C-plant C into the ergosterol. This incorporation of ¹³C into the ergosterol increased over the incubation period. Importantly, these changes associated with fungal growth were not evident in the analysis of either the PLFA or NLFA fractions thus questioning the reliability of such approaches for studying changes in microbial communities associated with the decomposition of plant residues.